AN ANTIBODY-DRUG CONJUGATE HAVING TWO OR MORE DIFFERENT FUNCTIONAL SMALL MOLECULES FOR ENHANCED TREATMENT OF REFRACTORY DISEASES

The present invention relates to an antibody-drug conjugate containing two or more functional small molecules for enhancement of targeted treatment of cancers and refractory diseases. The invention also relates to preparation of such conjugate, pharmaceutical compositions, and methods in treatment of cancers and refractory diseases.

BACKGROUND

Antibody-drug conjugates (ADCs) that combine the target specificity of a monoclonal antibody (mAb) with the potent cytotoxic drugs are a rapidly expanding class of therapeutic agents against cancers. There are 15 ADCs so far approved as monotherapy or combinational therapy for several solid and liquid cancers. Due to the spectacular success of 9 of the 15 ADC approved in the past 5 years for some highly treatment-refractory diseases, over a 200 new ADCs are now in clinical trials encompassing a wide variety of tumor types according to https://www.clinicaltrials.gov. Despite the explosion of interest in ADCs, challenges remain to expand their therapeutic index (with greater efficacy and less toxicity) and applications in treatment of various cancers (Dean, A. Q., et al, mAbs, 2021, 13 (1), 1951427). Thus, more and more ADCs are combined with chemotherapeutical drugs, immunotherapy compounds and others for the expansion in the treatment (Li, Y, et al, Am J Cancer Res. 2023, 13 (1): 161-17; Ceci, C., et al, Pharmacol. 2022, 236:108106; Gerber, H. P, et al, Biochem Pharmacol. 2016, 102:1-6). However, the half-lives of chemotherapeutical drugs are normally quite short and they have to be given more frequently to patients, therefore leading to more toxicity in synergistic use with ADCs (in comparison with application of the monotherapy of ADCs) (Fuentes-Antrás, J., et al, Trends Cancer 2023, 9 (4): 339-354). Here in this application, we disclose an ADC containing one or more chemical drugs or functional small molecules covalently linked to the antibody, which can expand the half-lives of the conjugated small molecules, resulting in enhancement of the targeted treatment of various cancers and refractory diseases. Further disclosed are preparation of the conjugate, pharmaceutical compositions, screening, and medical treatment methods.

BRIEF SUMMARY OF THE INVENTION

The present invention provides an antibody-drug conjugate (ADC) with a branch linked chemical drugs or functional small molecules for the enhancement of targeted treatment of cancers and refractory diseases. The preferred formulas of the ADCs are represented as (I), (II), (III) and (IV) below:

The present invention also provides an antibody-drug conjugate (ADC), comprising a monoclonal antibody, or an antigen-binding fragment thereof, a cytotoxin, and a linker having a functional small molecule, and/or an affinity ligand, such as for bombesin receptors/neurotensin receptors (including neuropeptide-Y receptors), and/or a cell-penetrating peptide, and or an affinity peptide, such as the programmed death ligand-1 (PD-L1, or CD274), expressed on tumor cells and tumor-infiltrating immune cells, resulting in enhancement of targeted treatment of cancers and refractory diseases. In a further embodiment the antigen binding proteins are conjugated to a potent toxin such as a tubulysin analog, a camptothecin (CPT) analog, a PBD dimer, an eribulin, an auristatin analog, a duocarmycin analog, or an anthracycline analog, or the other cytotoxic agent or its analogs that described in the present invention.

In addition, the invention provides compositions comprising the foregoing antibody-drug conjugate, and a pharmaceutically acceptable carrier, and methods of the targeted treatment of various cancers and refractory diseases.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

“Alkyl” refers to an aliphatic hydrocarbon group or univalent groups derived from alkane by removal of one or two hydrogen atoms from carbon atoms. It may be straight or branched having C1-C8 (1 to 8 carbon atoms) in the chain. “Branched” means that one or more lower C numbers of alkyl groups such as methyl, ethyl or propyl are attached to a linear alkyl chain. Exemplary alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, 3-pentyl, octyl, nonyl, decyl, cyclopentyl, cyclohexyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl, 3-methyl-hexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 3,5-dimethylhexyl, 2,4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl, n-heptyl, isoheptyl, n-octyl, and isooctyl. A C1-C8 alkyl group can be unsubstituted or substituted with one or more groups including, but not limited to, —C1-C8alkyl, —O—(C1-C8 alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH2, —C(O)NHR′, —C(O)N(R′)2, —NHC(O)R′, —SR′, —S(O)2R′, —S(O)R′, —OH, -halogen, —N3, —NH2, —NH(R′), —N(R′)2 and —CN; where each R′ is independently selected from —C1-C8alkyl and aryl. When the alkyl group is inserted in the middle of a group, such as in a middle of a linker, thus the “alkyl” means “alkylene” group in this application.

“Halogen” refers to fluorine, chlorine, bromine or iodine atom; preferably fluorine and chlorine atom.

“Heteroalkyl” refers to C2-C8 alkyl in which one to four carbon atoms are independently replaced with a heteroatom from the group consisting of O, S and N.

“Alkenyl” refers to an aliphatic hydrocarbon group containing a carbon-carbon double bond which may be straight or branched having 2 to 8 carbon atoms in the chain. Exemplary alkenyl groups include ethenyl, propenyl, n-butenyl, i-butenyl, 3-methylbut-2-enyl, n-pentenyl, hexylenyl, heptenyl, octenyl.

“Alkynyl” refers to an aliphatic hydrocarbon group containing a carbon-carbon triple bond which may be straight or branched having 2 to 8 carbon atoms in the chain. Exemplary alkynyl groups include ethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, 5-pentynyl, n-pentynyl, hexylynyl, heptynyl, and octynyl.

“Alkylene” refers to a saturated, branched or straight chain or cyclic hydrocarbon radical of 1-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkane. Typical alkylene radicals include, but are not limited to: methylene (—CH2—), 1,2-ethyl (—CH2CH2—), 1,3-propyl (—CH2CH2CH2—), 1,4-butyl (—CH2CH2CH2CH2—), and the like.

“Alkenylene” refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical of 2-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkene. Typical alkenylene radicals include, but are not limited to: 1,2-ethylene (—CH═CH—).

“Alkynylene” refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical of 2-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkyne. Typical alkynylene radicals include, but are not limited to: acetylene, propargyl and 4-pentynyl.

“Aryl” or Ar refers to an aromatic or hetero aromatic group, composed of one or several rings, comprising three to fourteen carbon atoms, preferentially six to ten carbon atoms. The term of “hetero aromatic group” refers one or several carbon on aromatic group, preferentially one, two, three or four carbon atoms are replaced by O, N, Si, Se, P or S, preferentially by O, S, and N. The term aryl or Ar also refers to an aromatic group, wherein one or several H atoms are replaced independently by —R′, -halogen, —OR′, or —SR′, —NR′R″, —N═NR′, —N═R′, —NR′R″, —NO2, —S(O)R′, —S(O)2R′, —S(O)2OR′, —OS(O)2OR′, —PR′R″, —P(O)R′R″, —P(OR′)(OR″), —P(O)(OR′)(OR″) or —OP(O)(OR′)(OR″) wherein R′, R″ are independently H, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, arylalkyl, carbonyl, or pharmaceutical salts.

“Heterocycle” refers to a ring system in which one to four of the ring carbon atoms are independently replaced with a heteroatom from the group of O, N, S, Se, B, Si and P. Preferable heteroatoms are O, N and S. Heterocycles are also described in The Handbook of Chemistry and Physics, 78th Edition, CRC Press, Inc., 1997-1998, p. 225 to 226, the disclosure of which is hereby incorporated by reference. Preferred nonaromatic heterocyclic include epoxy, aziridinyl, thiiranyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxiranyl, tetrahydrofuranyl, dioxolanyl, tetrahydropyranyl, dioxanyl, dioxolanyl, piperidyl, piperazinyl, morpholinyl, pyranyl, imidazolinyl, pyrrolinyl, pyrazolinyl, thiazolidinyl, tetrahydrothiopyranyl, dithianyl, thiomorpholinyl, dihydropyranyl, tetrahydropyranyl, dihydropyranyl, tetrahydropyridyl, dihydropyridyl, tetrahydropyrimidinyl, dihydrothiopyranyl, azepanyl, as well as the fused systems resulting from the condensation with a phenyl group.

“Alkyl”, “cycloalkyl”, “alkenyl”, “alkynyl”, “aryl”, “heteroaryl”, “heterocyclic” and the like refer also to the corresponding “alkylene”, “cycloalkylene”, “alkenylene”, “alkynylene”, “arylene”, “heteroarylene”, “heterocyclene” and the likes which are formed by the removal of two hydrogen atoms.

“Arylalkyl” refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp3 carbon atom, is replaced with an aryl radical. Typical arylalkyl groups include, benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and the like.

“Heteroarylalkyl” refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp3 carbon atom, is replaced with a heteroaryl radical. Examples of heteroarylalkyl groups are 2-benzimidazolylmethyl, 2-furylethyl.

“Leaving group” refers to a functional group that can be substituted by another functional group. Such leaving groups are well known in the art, and examples include, a halide (e.g., chloride, bromide, and iodide), methanesulfonyl (mesyl), p-toluenesulfonyl (tosyl), trifluoro-methylsulfonyl (triflate), and trifluoromethylsulfonate. A preferred leaving group is selected from nitrophenol; N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol; difluorophenol; monofluorophenol; pentachlorophenol; triflate; imidazole; dichlorophenol; tetrachlorophenol; 1-hydroxybenzotriazole; tosylate; mesylate; 2-ethyl-5-phenylisoxazolium-3′-sulfonate, anhydrides formed its self, or formed with the other anhydride, e.g. acetyl anhydride, formyl anhydride; or an intermediate molecule generated with a condensation reagent for peptide coupling reactions or for Mitsunobu reactions.

“Pharmaceutically” or “pharmaceutically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate.

“Pharmaceutically acceptable solvate” or “solvate” refer to an association of one or more solvent molecules and a disclosed compound. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid and ethanolamine.

“Pharmaceutically acceptable excipient” includes any carriers, diluents, adjuvants, or vehicles, such as preserving or antioxidant agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions as suitable therapeutic combinations.

As used herein, “pharmaceutical salts” refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, tartaric, citric, methanesulfonic, benzenesulfonic, glucuronic, glutamic, benzoic, salicylic, toluenesulfonic, oxalic, fumaric, maleic, lactic and the like. Further addition salts include ammonium salts such as tromethamine, meglumine, epolamine, etc., metal salts such as sodium, potassium, calcium, zinc or magnesium.

“Administering” or “administration” refers to any mode of transferring, delivering, introducing or transporting a pharmaceutical drug or other agent to a subject. Such modes include oral administration, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intranasal, subcutaneous or intrathecal administration. Also contemplated by the present invention is utilization of a device or instrument in administering an agent. Such device may utilize active or passive transport and may be slow-release or fast-release delivery device.

The term “antibody” is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity and fusion proteins comprising an antibody, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site. An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments. A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization. The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W. H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

As used herein, “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, Nature 256:495, 1975, or may be made by recombinant DNA methods such as described in U.S. Pat. No. 4,816,567. The monoclonal antibodies may also be isolated from phage libraries generated using the techniques described in McCafferty et al., Nature 348:552-554, 1990, for example.

As used herein, “humanized” antibody refers to forms of non-human (e.g. murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other antigen binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Preferably, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Preferred are antibodies having Fc regions modified as described in WO 99/58572. Other forms of humanized antibodies have one or more CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, or CDR H3) which are altered with respect to the original antibody, which are also termed one or more CDRs “derived from” one or more CDRs from the original antibody.

As used herein, “human antibody” means an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and/or which has been made using any of the techniques for making human antibodies known to those skilled in the art or disclosed herein. This definition of a human antibody includes antibodies comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody comprising murine light chain and human heavy chain polypeptides. Human antibodies can be produced using various techniques known in the art. In one embodiment, the human antibody is selected from a phage library, where that phage library expresses human antibodies (Vaughan et al., Nature Biotechnology, 14:309-314, 1996; Sheets et al., Proc. Natl. Acad. Sci. (USA) 95:6157-6162, 1998; Hoogenboom and Winter, J. Mol. Biol., 227:381, 1991; Marks et al., J. Mol. Biol., 222:581, 1991). Human antibodies can also be made by immunization of animals into which human immunoglobulin loci have been transgenically introduced in place of the endogenous loci, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016. Alternatively, the human antibody may be prepared by immortalizing human B lymphocytes that produce an antibody directed against a target antigen (such B lymphocytes may be recovered from an individual or from single cell cloning of the cDNA, or may have been immunized in vitro). See, e.g., Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985; Boerner et al., J. Immunol., 147 (1):86-95, 1991; and U.S. Pat. No. 5,750,373.

The term “chimeric antibody” is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

The terms “polypeptide”, “oligopeptide”, “peptide” and “protein” are used interchangeably herein to refer to chains of amino acids of any length, preferably, relatively short (e.g., 10-100 amino acids). The chain may be linear or branched, it may comprise modified amino acids, and/or may be interrupted by non-amino acids. The terms also encompass an amino acid chain that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that the polypeptides can occur as single chains or associated chains.

A “monovalent antibody” comprises one antigen binding site per molecule (e.g., IgG or Fab). In some instances, a monovalent antibody can have more than one antigen binding sites, but the binding sites are from different antigens.

A “monospecific antibody” comprises two identical antigen binding sites per molecule (e.g. IgG) such that the two binding sites bind identical epitope on the antigen. Thus, they compete with each other on binding to one antigen molecule. Most antibodies found in nature are monospecific. In some instances, a monospecific antibody can also be a monovalent antibody (e.g. Fab).

A “bivalent antibody” comprises two antigen binding sites per molecule (e.g., IgG). In some instances, the two binding sites have the same antigen specificities. However, bivalent antibodies may be bispecific.

A “bispecific” or “dual-specific” is a hybrid antibody having two different antigen binding sites. The two antigen binding sites of a bispecific antibody bind to two different epitopes, which may reside on the same or different protein targets.

A “bifunctional” is antibody is an antibody having identical antigen binding sites (i.e., identical amino acid sequences) in the two arms but each binding site can recognize two different antigens.

A “heteromultimer”, “heteromultimeric complex”, or “heteromultimeric polypeptide” is a molecule comprising at least a first polypeptide and a second polypeptide, wherein the second polypeptide differs in amino acid sequence from the first polypeptide by at least one amino acid residue. The heteromultimer can comprise a “heterodimer” formed by the first and second polypeptide or can form higher order tertiary structures where polypeptides in addition to the first and second polypeptide are present.

A “heterodimer”, “heterodimeric protein”, “heterodimeric complex,” or “heteromultimeric polypeptide” is a molecule comprising a first polypeptide and a second polypeptide, wherein the second polypeptide differs in amino acid sequence from the first polypeptide by at least one amino acid residue.

The “hinge region”, “hinge sequence”, and variations thereof, as used herein, includes the meaning known in the art, which is illustrated in, for example, Janeway et al., ImmunoBiology: the immune system in health and disease, (Elsevier Science Ltd., NY) (4th ed., 1999); Bloom et al., Protein Science (1997), 6:407-415; Humphreys et al., J. Immunol. Methods (1997), 209:193-202.

The “immunoglobulin-like hinge region”, “immunoglobulin-like hinge sequence,” and variations thereof, as used herein, refer to the hinge region and hinge sequence of an immunoglobulin-like or an antibody-like molecule (e.g., immunoadhesins). In some embodiments, the immunoglobulin-like hinge region can be from or derived from any IgG1, IgG2, IgG3, or IgG4 subtype, or from IgA, IgE, IgD or IgM, including chimeric forms thereof, e.g., a chimeric IgG1/2 hinge region.

Since the disulfide bonds in different IgG forms of antibody are various, thus drug/antibody ratios (DARs) with the thiol-ether conjugation (such as through the Michael addition reaction of a maleimide of a drug/linker complex and a cysteine in an antibody) can be various. For instance, the DARs (or “n” in this application) can be up to 30 for IgG2, IgG3 or IgG4 form of an ADC.

The term “immune effector cell” or “effector cell” as used herein refers to a cell within the natural repertoire of cells in the human immune system which can be activated to affect the viability of a target cell. The viability of a target cell can include cell survival, proliferation, and/or ability to interact with other cells.

Antibodies of the invention can be produced using techniques well known in the art, e.g., recombinant technologies, phage display technologies, synthetic technologies or combinations of such technologies or other technologies readily known in the art (see, for example, Jayasena, S. D., Clin. Chem., 45:1628-50, 1999 and Fellouse, F. A., et al, J. Mol. Biol., 373 (4): 924-40, 2007).

The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At211, I131, I125, Y90, In111, Re186, Re188, Sm153, Bi212, P32, Pb212, Zr89, F18, and radioactive isotopes of Lu, e.g. Lu177); chemotherapeutic agents or drugs (e.g., tubulysin, maytansin, auristatin, DNA minor groove binders (such as PBD dimers), duocarmycin, topoisomerase inhibitor I or II (such as camptothecins or etoposides), RNA polymerase inhibitors, DNA alkylators, methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and the various antitumor or anticancer agents disclosed throughout this patent application.

“Linker” refers to a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches an antibody to a drug moiety. In various embodiments, linkers include a divalent radical such as an alkyldiyl, an aryldiyl, a heteroaryldiyl, moieties such as: —(CR2)nO(CR2)n—, repeating units of alkyloxy (e.g. polyethylenoxy, PEG, poly(2-methyl)ethylenoxy, polymethyleneoxy, polypropyleneoxy) and alkylamino (e.g. polyethyleneamino); and diacid ester and amides including succinate, succinamide, diglycolate, malonate, and caproamide. In various embodiments, linkers can comprise one or more amino acid residues, such as valine, phenylalanine, lysine, and homolysine.

The words “comprise”, “comprising”, “include”, “including” and “includes” when used in this specification and claims are intended to specify the presence of stated features, integers, components, or steps, but they do not preclude the presence or addition of one or more other features, integers, components, steps, or groups thereof. The novel conjugates disclosed herein are the antibody conjugates targeting tumor specific antigens. Examples of the conjugates and their synthesis are shown in the examples 1-482 below of the specification.

the Antibody Drug Conjugate of the Invention.

The invention provides an antibody-drug conjugate that have enhancement of killing of tumor cells. The antibody-drug conjugate (ADC) contains a branch (side chain) linker wherein a group of an affinity small molecule or/and an affinity peptide (such as neurotensin peptide), or a small molecule chemotherapeutic drug at the terminal of the side chain linker that have synergies of cytotoxicities, resulting in enhanced treatment of the tumors and refractory diseases. The formulas of the ADC of the present invention are represented as:

wherein  is a site that links a drug or a site of linker L1 or L2; “#” is a site that links a S (thiol), O (phenol), NH (amino), CHO (aldehyde), C(═O) (ketone), C(O)(NH) (amide) and C(O)(OH) (carboxylate) of an antibody; wherein R1 and R2 are H, C1-C6 of alkyl or a peptide containing 1˜4 units of aminoacids; X1′, X2′ and X are O, NH, S, CH2; the connecting bond “—” in the middle of the two atoms means it can link either one of the two atoms, Ar is an aromatic group; A1, A2, A3, A4, A5 and A6 are independently an affinity ligand including affinity peptide and cell-penetrating peptide (CPP), a ligand for bombesin receptors/neurotensin receptors (including neuropeptide-Y receptors), a chemotherapeutic drug, a stimulating agent, and/or a nucleoside antimetabolite/analogs, which have either the synergy with D1 or D2, or enhanced affinity for mAb.

In some embodiments, the affinity ligand/peptide including the cell-penetrating peptide (CPP) to a receptor on a faulty cell is EC50<100 nM. The CPP is a linear or cyclo peptide having less than 50 amino acids and containing one, two, or several arginines or lysines and enables to internalize (trafficking) over 40% of the ligand bound on a cell or help to internalize 40% of ADCs bound on a cell to cross the cell membrane in 2 hours.

In some embodiments, the affinity ligand/peptide including the cell-penetrating peptide (CPP) is independently selected from:

wherein Dap is(S)-2,3-Diaminopropanoic acid, Nle is L-Norleucine, Anon is(S)-2-Aminononanoic acid, Cha is L-Cyclohexylalanine. The others are natural amino acids.

and its analogs as the structures shown below:

In another embodiments, A1, A2, A3, A4, A5 and A6 are preferably Nucleoside analogues, which have synergy with D1 or/and D2. The nucleoside analogues are molecules that act like nucleosides in DNA synthesis. They include a range of antiviral products used to prevent viral replication in infected cells. Nucleoside analogues can be used against hepatitis B virus, hepatitis C virus, herpes simplex, and HIV. Once they are phosphorylated, they work as antimetabolites by being similar enough to nucleotides to be incorporated into growing DNA strands. Less selective nucleoside analogues are used as chemotherapy agents to treat cancer, e.g. gemcitabine and 5-FU. Antimetabolite is a chemical that inhibits the use of a metabolite, which is another chemical that is part of normal metabolism. Such substances are often similar in structure to the metabolite that they interfere with, such as the antifolates that interfere with the use of folic acid. The presence of antimetabolites can have toxic effects on cells, such as halting cell growth and cell division, so these compounds are used as chemotherapy for cancer. Some conjugatable structures of nucleoside analogues for this invention are illustrated below:

the Antibody

The antibody (mAb) used for the conjugation process is preferred a cell-binding antibody or antibody-like protein molecule that binds to, complexes with, or reacts with a moiety of a cell population sought to be therapeutically or otherwise biologically modified.

For convenience in this section and elsewhere, “antibody” should be understood to include “antibody-like protein and peptide” except where the context requires otherwise. Suitable antibody-like proteins which may be present in the conjugates of the invention include for example peptides, polypeptides, antibodies, antibody fragments, enzymes, cytokines, chemokines, receptors, blood factors, peptide hormones, toxin, transcription antibody-like proteins, or multimeric antibody-like proteins, wherein they have interchain disulfide bonds structurally.

Other antibody-like proteins of interest are allergen antibody-like proteins disclosed by Dreborg et al Crit. Rev. Therap. Drug Carrier Syst. (1990) 6 315-365 as having reduced allergenicity when conjugated with a polymer such as poly(alkylene oxide) and consequently are suitable for use as tolerance inducers. Among the allergens disclosed are Ragweed antigen E, honeybee venom, mite allergen and the like.

Glycopolypeptides such as immunoglobulins, ovalbumin, lipase, glucocerebrosidase, lectins, tissue plasminogen activator and glycosylated interleukins, interferons and colony stimulating factors are of interest, as are immunoglobulins such as IgG, IgE, IgM, IgA, IgD and fragments thereof. Of particular interest are receptor and ligand binding antibody-like proteins and antibodies and antibody fragments which are used in clinical medicine for diagnostic and therapeutic purposes.

The antibody herein is preferred (A): the group consisting of an antibody, an antibody-like protein molecule, probody, nanobody, peptides, an antibody coating on polymeric micelle, an antibody-liposome, a lipoprotein-based drug carrier, an antibody coating on nano-particle, an antibody-dendrimer, and a particle said above coated or linked with an antibody-like protein (antibody), or a combination of said above thereof;

In general, a monoclonal antibody is preferred as a cell-surface binding agent if an appropriate one is available. And the antibody may be murine, human, humanized, chimeric, or derived from other species.

Production of antibodies used in the present invention involves in vivo or in vitro procedures or combinations thereof. Methods for producing polyclonal anti-receptor peptide antibodies are well-known in the art, such as in U.S. Pat. No. 4,493,795 (to Nestor et al). A monoclonal antibody is typically made by fusing myeloma cells with the spleen cells from a mouse that has been immunized with the desired antigen (Köhler, G.; Milstein, C. (1975). Nature 256:495-7). The detailed procedures are described in “Antibodies—A Laboratory Manual”, Harlow and Lane, eds., Cold Spring Harbor Laboratory Press, New York (1988), which is incorporated herein by reference. Particularly monoclonal antibodies are produced by immunizing mice, rats, hamsters or any other mammal with the antigen of interest such as the intact target cell, antigens isolated from the target cell, whole virus, attenuated whole virus, and viral proteins. Splenocytes are typically fused with myeloma cells using polyethylene glycol (PEG) 6000. Fused hybrids are selected by their sensitivity to HAT (hypoxanthine-aminopterin-thymine). Hybridomas producing a monoclonal antibody useful in practicing this invention are identified by their ability to immunoreact specified receptors or inhibit receptor activity on target cells.

A monoclonal antibody used in the present invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules of the appropriate antigen specificity. The culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the antibody molecules into the medium. The antibody-containing medium is then collected. The antibody molecules can then be further isolated by well-known techniques, such as using protein-A affinity chromatography; anion, cation, hydrophobic, or size exclusive chromatographies (particularly by affinity for the specific antigen after protein A, and sizing column chromatography); centrifugation, differential solubility, or by any other standard technique for the purification of proteins.

Media useful for the preparation of these compositions are both well-known in the art and commercially available and include synthetic culture media. An exemplary synthetic medium is Dulbecco's minimal essential medium (DMEM; Dulbecco et al., Virol. 8, 396 (1959)) supplemented with 4.5 gm/l glucose, 0˜20 mM glutamine, 0˜20% fetal calf serum, several ppm amount of heavy metals, such as Cu, Mn, Fe, or Zn, etc, or/and the other heavy metals added in their salt forms, and with an anti-foaming agent, such as polyoxyethylene-polyoxypropylene block copolymer.

In addition, antibody-producing cell lines can also be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with an oncovirus, such as Epstein-Barr virus (EBV, also called human herpesvirus 4 (HHV-4)) or Kaposi's sarcoma-associated herpesvirus (KSHV). See, U.S. Pat. Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917; 4,472,500; 4,491,632; 4,493,890. A monoclonal antibody may also be produced via an anti-receptor peptide or peptides containing the carboxyl terminal as described well-known in the art. See Niman et al., Proc. Natl. Acad. Sci. USA, 80:4949-53 (1983); Geysen et al., Proc. Natl. Acad. Sci. USA, 82:178-82 (1985); Lei et al. Biochemistry 34 (20): 6675-88, (1995). Typically, the anti-receptor peptide or a peptide analog is used either alone or conjugated to an immunogenic carrier, as the immunogen for producing anti-receptor peptide monoclonal antibodies.

There are also a number of other well-known techniques for making monoclonal antibodies as binding molecules in this invention. Particularly useful are methods of making fully human antibodies. One method is phage display technology which can be used to select a range of human antibodies binding specifically to the antigen using methods of affinity enrichment. Phage display has been thoroughly described in the literature and the construction and screening of phage display libraries are well known in the art, see, e.g., Dente et al, Gene. 148(1):7-13 (1994); Little et al, Biotechnol Adv. 12(3): 539-55 (1994); Clackson et al., Nature 352:264-8 (1991); Huse et al., Science 246:1275-81 (1989).

Monoclonal antibodies derived by hybridoma technique from another species than human, such as mouse, can be humanized to avoid human anti-mouse antibodies when infused into humans. Among the more common methods of humanization of antibodies are complementarity-determining region grafting and resurfacing. These methods have been extensively described, see e.g. U.S. Pat. Nos. 5,859,205 and 6,797,492; Liu et al, Immunol Rev. 222:9-27 (2008); Almagro et al, Front Biosci. 13:1619-33 (2008); Lazar et al, Mol Immunol. 44(8): 1986-98 (2007); Li et al, Proc. Natl. Acad. Sci. USA. 103(10): 3557-62 (2006) each incorporated herein by reference. Fully human antibodies can also be prepared by immunizing transgenic mice, rabbits, monkeys, or other mammals, carrying large portions of the human immunoglobulin heavy and light chains, with an immunogen. Examples of such mice are: the Xenomouse. (Abgenix/Amgen), the HuMAb-Mouse (Medarex/BMS), the VelociMouse (Regeneron), see also U.S. Pat. Nos. 6,596,541, 6,207,418, 6,150,584, 6,111,166, 6,075,181, 5,922,545, 5,661,016, 5,545,806, 5,436,149 and 5,569,825. In human therapy, murine variable regions and human constant regions can also be fused to construct called “chimeric antibodies” that are considerably less immunogenic in man than murine mAbs (Kipriyanov et al, Mol Biotechnol. 26:39-60 (2004); Houdebine, Curr Opin Biotechnol. 13:625-9 (2002) each incorporated herein by reference). In addition, site-directed mutagenesis in the variable region of an antibody can result in an antibody with higher affinity and specificity for its antigen (Brannigan et al, Nat Rev Mol Cell Biol. 3:964-70, (2002)); Adams et al, J Immunol Methods. 231:249-60 (1999)) and exchanging constant regions of a mAb can improve its ability to mediate effector functions of binding and cytotoxicity.

Antibodies immunospecific for a malignant cell antigen can also be obtained commercially or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques. The nucleotide sequence encoding antibodies immune-specific for a malignant cell antigen can be obtained commercially, e.g., from the GenBank database or a database like it, the literature publications, or by routine cloning and sequencing.

Apart from an antibody, an antibody like peptide or protein that bind/block/target or in some other way interact with the epitopes or corresponding receptors on a targeted cell can be used as a binding molecule. These antibody-like peptides or proteins could be any random peptide or proteins that have an affinity for the epitopes or corresponding receptors and they don't necessarily have to be of the immune-globulin family. These peptides can be isolated by similar techniques as for phage display antibodies (Szardenings, J Recept Signal Transduct Res. 2003, 23(4): 307-49). The use of peptides from such random peptide libraries can be similar to antibodies and antibody fragments. The binding molecules of antibody like peptides or proteins may be conjugated on or linked to a large molecules or materials, such as, but is not limited, an albumin, a polymer, a liposome, a nano particle, a dendrimer, as long as such attachment permits the peptide or protein to retain its antigen binding specificity.

Many of tumor-associated antigens (TAA) or tumor cell receptors are known in the art, and can be prepared for use in generating antibodies using methods and information which are well known in the art. In attempts to discover effective cellular targets for cancer diagnosis and therapy, researchers have sought to identify transmembrane or otherwise tumor-associated polypeptides or glycoproteins that are specifically expressed on the surface of one or more particular type(s) of cancer cell as compared to on one or more normal non-cancerous cell(s). Often, such tumor-associated polypeptides are more abundantly expressed on the surface of the cancer cells as compared to on the surface of the non-cancerous cells. The identification of such tumor-associated cell surface antigen polypeptides has given rise to the ability to specifically target cancer cells for destruction via the ADCs of this application. Examples of the TAA and cognate antibodies with their known in art are:

In certain preferred embodiments, the binding molecule for the conjugate in the present invention, can bind to both a receptor and a receptor complex expressed on an activated lymphocyte which is associated with an autoimmune disease. The receptor or receptor complex can comprise an immunoglobulin gene superfamily member (e.g. CD2, CD3, CD4, CD8, CD19, CD20, CD22, CD28, CD30, CD33, CD37, CD38, CD56, CD70, CD79, CD79b, CD90, CD125, CD137, CD138, CD147, CD152/CTLA-4, PD-1, or ICOS), a TNF receptor superfamily member (e.g. CD27, CD40, CD95/Fas, CD134/OX40, CD137/4-1BB, INF-R1, TNFR-2, RANK, TAC1, BCMA, osteoprotegerin, Apo2/TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, and APO-3), an integrin, a cytokine receptor, a chemokine receptor, a major histocompatibility protein, a lectin (C-type, S-type, or I-type), or a complement control protein.

In another specific embodiment, useful cell binding ligands that are immunospecific for a viral or a microbial antigen are humanized or human monoclonal antibodies. As used herein, the term “viral antigen” includes, but is not limited to, any viral peptide, polypeptide protein (e.g. HIV gp120, HIV nef, RSV F glycoprotein, influenza virus neuramimi-dase, influenza virus hemagglutinin, HTLV tax, herpes simplex virus glycoprotein (e.g. gB, gC, gD, and gE) and hepatitis B surface antigen) that is capable of eliciting an immune response. As used herein, the term “microbial antigen” includes, but is not limited to, any microbial peptide, polypeptide, protein, saccharide, polysaccharide, or lipid molecule (e.g., bacteria, fungi, pathogenic protozoa, or yeast polypeptides including, e.g., LPS and capsular polysaccharide 5/8) that is capable of eliciting an immune response. Examples of antibodies available 1 for the viral or microbial infection include, but are not limited to, Palivizumab which is a humanized anti-respiratory syncytial virus monoclonal antibody for the treatment of RSV infection; PRO542 which is a CD4 fusion antibody for the treatment of HIV infection; Ostavir which is a human antibody for the treatment of hepatitis B virus; PROTVIR which is a humanized IgG.sub.1 antibody for the treatment of cytomegalovirus; and anti-LPS antibodies.

According to a further object, the present invention also concerns pharmaceutical compositions comprising the conjugate of the invention together with a pharmaceutically acceptable carrier, diluent, or excipient for treatment of cancers, infections or autoimmune disorders. The method for treatment of cancers, infections and autoimmune disorders can be practiced in vitro, in vivo, or ex vivo. Examples of in vitro uses include treatments of cell cultures in order to kill all cells except for desired variants that do not express the target antigen; or to kill variants that express undesired antigen. Examples of ex vivo uses include treatments of hematopoietic stem cells (HSC) prior to the performance of the transplantation (HSCT) into the same patient in order to kill diseased or malignant cells. For instance, clinical ex vivo treatment to remove tumour cells or lymphoid cells from bone marrow prior to autologous transplantation in cancer treatment or in treatment of autoimmune disease, or to remove T cells and other lymphoid cells from allogeneic bone marrow or tissue prior to transplant in order to prevent graft-versus-host disease, can be carried out as follows. Bone marrow is harvested from the patient or other individual and then incubated in medium containing serum to which is added the conjugate of the invention, concentrations range from about 1 pM to 0.1 mM, for about 30 minutes to about 48 hours at about 37° C. The exact conditions of concentration and time of incubation (=dose) are readily determined by the skilled clinicians. After incubation, the bone marrow cells are washed with medium containing serum and returned to the patient by i.v. infusion according to known methods. In circumstances where the patient receives other treatment such as a course of ablative chemotherapy or total-body irradiation between the time of harvest of the marrow and reinfusion of the treated cells, the treated marrow cells are stored frozen in liquid nitrogen using standard medical equipment.

In further preferred embodiments, the present invention also provides an antibody-drug conjugate (ADC) comprising a monoclonal antibody, or an antigen-binding fragment thereof, conjugated with a cytotoxin, via a linker containing a glutamate urea small molecule, such as 2-[3-(1,3-dicarboxypropyl) ureido]-pentanedioic acid (DUPA), urea-based glutamate heterodimers, 2-(phosphonomethyl)-pentanedioic acid (PMPA), phosphoramidates, glu-urea-lys, or 2-(phosphinylmethyl) pentanedioic acids analog group to direct against prostate antigen (PSA) of a tumor cell, and/or an affinity ligand for bombesin receptors (Gastrin releasing peptide receptor (GRPR), neurotensin receptors (including Neurotensin receptor 1 (NTR1) and neuropeptide-Y receptors), and/or a cell-penetrating peptide, and/or an affinity peptide that can bind with a protein called programmed death ligand-1 (PD-L1, or CD274) which is expressed on tumor cells and tumor-infiltrating immune cells, blocking its interactions with both PD-1 and B7.1 receptors. The affinity to the receptors are at least EC50<10 μM, preferably EC50<100 nM, and more preferably EC50<50 nM. In a further embodiment the antigen binding proteins are conjugated to a cytotoxin, such as, but not limited, a tubulysin analog, a camptothecin (CPT) analog, a PBD dimer, an anthracycline, or an auristatin analog.

In some embodiments, the cell-penetrating peptide (CPP) used in this invention can be seleted from CPP database (http://crdd.osdd.net/raghava/cppsite) or from known publications with less than 100 amino acids of sequences or from amendment of known peptide sequences with replacement of one or several amino acids, and then is subjected to redundancy check. The preferred CPP is a linear or cyclo-peptide having less than 50 amino acids, preferably less than 20 natural or unnatural amino acids, more preferably less than 15 amino acids and containing one, two, or several arginines and/or lysines. The CPP is more preferably a cyclopeptide, in particular CPP is a cyclopeptide having less than 8 amino acids. The selected peptides are normally further analyzed to filter out the ambiguous peptides with undesirable chemical modifications. The amphipathicity prediction can be through the online server AMPHIPASEEK (https://npsa-prabi.ibcp.fr/cgibin/npsa_automat.pl?page=/NPSA/npsa_amphipaseek.html). AMPHIPASEEK provides a score for every residue between a range of 0 and 5 for the given peptide sequences. Higher scores imply high amphipathic nature and vice versa (0=low and 5=high). Hydropathy values were (K-Me3) calculated using the online server (https://www.peptide2.com/N_peptide_hydrophobicity_hydrophilicity.php). The CPPs are normally required to pass through the criteria of peptide solubility and the cell-penetrating property using Innovagen peptide solubility calculator (https://pepcalc.com/) and CPPpred (http://bioware.ucd.ie/˜compass/biowareweb/Server_pages/cpppred.php), respectively. The CPP score is given within the range of 0-1, wherein the peptides with the score of >0.5 are suggestive of better cell penetration. The efficiency of CPP penetration of a cell can be measured in several different methods (Lee, H-M, et al, Nature Communications Biology 2021, 4:205; Penedo, M. et al, Scientific Reports, 2021, 11:7756 and the references they incorporated). In general, the preferable CPP should enable to internalize (trafficking) over 40% of the ligand bound on a cell or help to internalize 40% of ADCs bound on a cell to cross the cell membrane in 2 hours.

In some embodiments, the present invention provides antigen binding antibody-drug conjugates which bind to membrane bound targets and wherein the antigen binding ADC is capable of internalisation. In a further embodiment there is provided an immunoconjugate comprising the antigen binding protein of the present invention and a cytotoxic agent. In a further embodiment the antigen binding protein has ADCC effector function for example the antigen binding protein has enhanced ADCC effector function. In one such embodiment there is provided antigen binding antibodies/proteins or fragments of the antibodies used for ADCs against various cancers thereof.

In one aspect of the invention, the provided an antibody/protein used for the antibody-drug conjugate of this invention is preferably selected from an antibody having affinity to an antigen of highly expressed on tumor cells The information including the sequences of the provided antibody can be found in the known public domains, such as in the databases of patents in WIPO, USPTO, Espacenet, CNIPA, JPO, etc.

The antigen binding antibodies/proteins of the present invention may comprise heavy chain variable regions and light chain variable regions of the invention which may be formatted into the structure of a natural antibody or functional fragment or equivalent thereof. An antigen binding protein of the invention may therefore comprise the VH regions of the invention formatted into a full-length antibody, a (Fab′)2 fragment, a Fab fragment, or equivalent thereof (such as scFV, bi- tri- or tetra-bodies, Tandabs etc.), when paired with an appropriate light chain. The antibody may be an IgG1, IgG2, IgG3, or IgG4; or IgM; IgA, IgE or IgD or a modified variant thereof. The constant domain of the antibody heavy chain may be selected accordingly. The light chain constant domain may be a kappa or lambda constant domain. Furthermore, the antigen binding protein may comprise modifications of all classes e.g. IgG dimers, Fc mutants that no longer bind Fc receptors or mediate C1q binding. The antigen binding protein may also be a chimeric antibody of the type described in WO86/001533 which comprises an antigen binding region and a non-immunoglobulin region.

The constant region is selected according to any functionality required e.g. an IgG1 may demonstrate lytic ability through binding to complement and/or will mediate ADCC (antibody dependent cell cytotoxicity).

In one aspect the antigen binding protein is an antibody or antigen binding fragment thereof comprising one or more CDR's according to the invention described herein, or one or both of the heavy or light chain variable domains according to the invention described herein. The antigen binding protein is selected from the group consisting of a dAb, Fab, Fab′, F(ab′)2, Fv, diabody, triabody, tetrabody, miniantibody, and a minibody.

In one aspect of the present invention the antigen binding protein is a humanized or chimeric antibody, in a further aspect the antibody is humanized.

In another aspect, the antibody is a monoclonal antibody or a bispecific antibody.

In another aspect the antigen binding protein binds to human antigens with high affinity for example when measured by Biacore or ForteBio, the antigen binding protein binds to human antigens with an affinity of 20 nM or less or an affinity of 15 nM or less or an affinity of 5 nM or less or an affinity of 1000 pM or less or an affinity of 500 pM or less or an affinity of 400 pM or less, or 300 pM or less or for example about 120 pM. In a further embodiment the antigen binding protein binds to human antigens when measured by Biacore of between about 100 pM and about 500 pM or between about 100 pM and about 400 pM, or between about 100 pM and about 300 pM. In one embodiment of the present invention the antigen binding protein binds antigens with an affinity of less than 150 pM.

In one such embodiment, this is measured by Biacore or ForteBio.

In another aspect the antigen binding protein/antibody binds to human antigens in a cell neutralisation assay wherein the antigen binding protein has an IC50 of between about 1 nM and about 500 nM, or between about 1 nM and about 100 nM, or between about 1 nM and about 50 nM, or between about 1 nM and about 25 nM, or between about 5 nM and about 15 nM. In a further embodiment of the present invention the antigen binding protein binds antigens and neutralizes antigens in a cell neutralization assay wherein the antigen binding protein has an IC50 of about 10 nM.

The antigen binding proteins, for example antibodies of the present invention may be produced by transfection of a host cell with an expression vector comprising the coding sequence for the antigen binding protein of the invention. An expression vector or recombinant plasmid is produced by placing these coding sequences for the antigen binding protein in operative association with conventional regulatory control sequences capable of controlling the replication and expression in, and/or secretion from, a host cell. Regulatory sequences include promoter sequences, e.g., CMV promoter, and signal sequences which can be derived from other known antibodies. Similarly, a second expression vector can be produced having a DNA sequence which encodes a complementary antigen binding protein light or heavy chain. In certain embodiments this second expression vector is identical to the first except insofar as the coding sequences and selectable markers are concerned, so to ensure as far as possible that each polypeptide chain is functionally expressed. Alternatively, the heavy and light chain coding sequences for the antigen binding protein may reside on a single vector.

A selected host cell is co-transfected by conventional techniques with both the first and second vectors (or simply transfected by a single vector) to create the transfected host cell of the invention comprising both the recombinant or synthetic light and heavy chains. The transfected cell is then cultured by conventional techniques to produce the engineered antigen binding protein of the invention. The antigen binding protein which includes the association of both the recombinant heavy chain and/or light chain is screened from culture by appropriate assay, such as ELISA or RIA. Similar conventional techniques may be employed to construct other antigen binding proteins.

Suitable vectors for the cloning and subcloning steps employed in the methods and construction of the compositions of this invention may be selected by one of skill in the art. For example, the conventional pUC series of cloning vectors may be used. One vector, pUC19, is commercially available from supply houses, such as Amersham Bioscience (Buckinghamshire, United Kingdom) or GenScript (Nanjing, China). Additionally, any vector which is capable of replicating readily, has an abundance of cloning sites and selectable genes (e.g., antibiotic resistance), and is easily manipulated may be used for cloning. Thus, the selection of the cloning vector is not a limiting factor in this invention.

The expression vectors may also be characterized by genes suitable for amplifying expression of the heterologous DNA sequences, e.g., the mammalian dihydrofolate reductase gene (DHFR). Other vector sequences include a poly A signal sequence, such as from bovine growth hormone (BGH) and the betaglobin promoter sequence (betaglopro). The expression vectors useful herein may be synthesized by techniques well known to those skilled in this art.

The components of such vectors, e.g. replicons, selection genes, enhancers, promoters, signal sequences and the like, may be obtained from commercial or natural sources or synthesized by known procedures for use in directing the expression and/or secretion of the product of the recombinant DNA in a selected host. Other appropriate expression vectors of which numerous types are known in the art for mammalian, bacterial, insect, yeast, and fungal expression may also be selected for this purpose.

The present invention also encompasses a cell line transfected with a recombinant plasmid containing the coding sequences of the antigen binding proteins of the present invention. Host cells useful for the cloning and other manipulations of these cloning vectors are also conventional. However, cells from various strains of E. Coli may be used for replication of the cloning vectors and other steps in the construction of antigen binding proteins of this invention.

Suitable host cells or cell lines for the expression of the antigen binding proteins of the invention include mammalian cells such as NS0, Sp2/0, CHO (e.g. DG44), COS, HEK, a fibroblast cell (e.g., 3T3), and myeloma cells, for example it may be expressed in a CHO or a myeloma cell. Human cells may be used, thus enabling the molecule to be modified with human glycosylation patterns.

Alternatively, other eukaryotic cell lines may be employed. The selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening and product production and purification are known in the art. See, e.g., Sambrook et al., (1989). Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

Bacterial cells may prove useful as host cells suitable for the expression of the recombinant Fabs or other embodiments of the present invention (see, e.g., Pluckthun, A., Immunol. Rev., 130:151-188 (1992)). However, due to the tendency of proteins expressed in bacterial cells to be in an unfolded or improperly folded form or in a non-glycosylated form, any recombinant Fab produced in a bacterial cell would have to be screened for retention of antigen binding ability. If the molecule expressed by the bacterial cell was produced in a properly folded form, that bacterial cell would be a desirable host, or in alternative embodiments the molecule may express in the bacterial host and then be subsequently re-folded. For example, various strains of E. Coli used for expression are well-known as host cells in the field of biotechnology. Various strains of B. Subtilis, Streptomyces, other bacilli and the like may also be employed in this method.

Where desired, strains of yeast cells known to those skilled in the art are also available as host cells, as well as insect cells, e.g. Drosophila and Lepidoptera and viral expression systems. See, e.g. Miller et al., Genetic Engineering, 8:277-298, Plenum Press (1986) and McGuire, S. et al, Trends Genet. (2004) 20, 384-391 and references cited therein.

The general methods by which the vectors may be constructed, the transfection methods required to produce the host cells of the invention, and culture methods necessary to produce the antigen binding protein of the invention from such host cell may all be conventional techniques. Typically, the culture method of the present invention is a serum-free culture method, usually by culturing cells serum-free in suspension. Likewise, once produced, the antigen binding proteins of the invention may be purified from the cell culture contents according to standard procedures of the art, including ammonium precipitation, affinity columns, column chromatography, gel electrophoresis and the like. Such techniques are within the skill of the art and do not limit this invention. For example, preparations of altered antibodies are described in WO 99/058679 and WO 96/016990. Yet another method of expression of the antigen binding proteins may utilize expression in a transgenic animal, such as described in U.S. Pat. No. 4,873,316. This relates to an expression system using the animals casein promoter which when transgenically incorporated into a mammal permits the female to produce the desired recombinant protein in its milk.

In a further embodiment of the invention there is provided a method of producing an antibody of the invention which method comprises the step of culturing a host cell transformed or transfected with a vector encoding the light and/or heavy chain of the antibody of the invention and recovering the antibody thereby produced.

In accordance with the present invention there is provided a method of producing an antibody of the present invention which binds to and neutralises the activity of human ANTIGENS which method comprises the steps of; providing a first vector encoding a heavy chain of the antibody; providing a second vector encoding a light chain of the antibody; transforming a mammalian host cell (e.g. CHO) with said first and second vectors; culturing the host cell of step (c) under conditions conducive to the secretion of the antibody from said host cell into said culture media; recovering the secreted antibody of step (d).

Once expressed by the desired method, the antibody is then examined for in vitro activity by use of an appropriate assay. Presently conventional ELISA assay formats are employed to assess qualitative and quantitative binding of the antibody to ANTIGENS. Additionally, other in vitro assays may also be used to verify neutralizing efficacy prior to subsequent human clinical studies performed to evaluate the persistence of the antibody in the body despite the usual clearance mechanisms.

The dose and duration of treatment relates to the relative duration of the molecules (the antibody and the antibody-drug conjugate) of the present invention in the human circulation, and can be adjusted by one of skill in the art depending upon the condition being treated and the general health of the patient. It is envisaged that repeated dosing (e.g. once a week or once every two weeks or once every 3 weeks or once every 4 weeks) over an extended time period (e.g. four to six months) may be required to achieve maximal therapeutic efficacy.

In one embodiment of the present invention there is provided a recombinant transformed, transfected or transduced host cell comprising at least one expression cassette, for example where the expression cassette comprises a polynucleotide encoding a heavy chain of an antigen binding protein according to the invention described herein and further comprises a polynucleotide encoding a light chain of an antigen binding protein according to the invention described herein or where there are two expression cassettes and the 1.sup.st encodes the light chain and the second encodes the heavy chain. For example in one embodiment the first expression cassette comprises a polynucleotide encoding a heavy chain of an antigen binding protein comprising a constant region or antigen binding fragment thereof which is linked to a constant region according to the invention described herein and further comprises a second cassette comprising a polynucleotide encoding a light chain of an antigen binding protein comprising a constant region or antigen binding fragment thereof which is linked to a constant region according to the invention described herein for example the first expression cassette comprises a polynucleotide encoding a heavy chain and a second expression cassette comprising a polynucleotide encoding a light chain.

In another embodiment of the invention there is provided a stably transformed host cell comprising a vector comprising one or more expression cassettes encoding a heavy chain and/or a light chain of the antibody comprising a constant region or antigen binding fragment thereof which is linked to a constant region as described herein. For example, such host cells may comprise a first vector encoding the light chain and a second vector encoding the heavy chain, for example the first vector encodes a heavy chain and a second vector encoding a light chain.

In another embodiment of the present invention there is provided a host cell according to the invention described herein wherein the cell is eukaryotic, for example where the cell is mammalian. Examples of such cell lines include CHO or NSO.

In another embodiment of the present invention there is provided a method for the production of an antibody comprising a constant region or antigen binding fragment thereof which is linked to a constant region according to the invention described herein which method comprises the step of culturing a host cell in a culture media, for example serum-free culture media.

In another embodiment of the present invention there is provided a method according to the invention described herein wherein said antibody is further purified to at least 95% or greater (e.g. 98% or greater) with respect to said antibody containing serum-free culture media.

In yet another embodiment there is provided a pharmaceutical composition comprising an antigen binding protein and a pharmaceutically acceptable carrier.

In another embodiment of the present invention there is provided a kit-of-parts comprising the composition according to the invention described herein described together with instructions for use.

The mode of administration of the therapeutic agent of the invention may be any suitable route which delivers the agent to the host. The antigen binding proteins, and pharmaceutical compositions of the invention are particularly useful for parenteral administration, i.e., subcutaneously (s.c.), intrathecally, intraperitoneally, intramuscularly (i.m.) or intravenously (i.v.). In one such embodiment the antigen binding proteins of the present invention are administered intravenously or subcutaneously.

Therapeutic agents of the invention may be prepared as pharmaceutical compositions containing an effective amount of the antigen binding protein of the invention as an active ingredient in a pharmaceutically acceptable carrier. In one embodiment the prophylactic agent of the invention is an aqueous suspension or solution containing the antigen binding protein in a form ready for injection. In one embodiment the suspension or solution is buffered at physiological pH. In one embodiment the compositions for parenteral administration will comprise a solution of the antigen binding protein of the invention or a cocktail thereof dissolved in a pharmaceutically acceptable carrier. In one embodiment the carrier is an aqueous carrier. A variety of aqueous carriers may be employed, e.g., 0.9% saline, 0.3% glycine, and the like. These solutions may be made sterile and generally free of particulate matter. These solutions may be sterilized by conventional, well known sterilization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, etc. The concentration of the antigen binding protein of the invention in such pharmaceutical formulation can vary widely, i.e., from less than about 0.5%, usually at or at least about 1% to as much as about 15 or 20% by weight and will be selected primarily based on fluid volumes, viscosities, etc., according to the particular mode of administration selected.

Thus, a pharmaceutical composition of the invention for intravenous infusion could be made up to contain about 250 ml of sterile Ringer's solution, and about 1 to about 30 or 5 mg to about 25 mg of an antigen binding protein of the invention per ml of Ringer's solution. Actual methods for preparing parenterally administrable compositions are well known or will be apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science, 15.sup.th ed., Mack Publishing Company, Easton, PA, USA. For the preparation of intravenously administrable antigen binding protein formulations of the invention see Parkins D. and Lasmar U. “The formulation of Biopharmaceutical products”, Pharm. Sci. Tech. Today, 3 (2000) 129-137; Wang, W “Instability, stabilisation and formulation of liquid protein pharmaceuticals”, Int. J. Pharm 185 (1999) 129-188; Jorgensen, L. et al, “Recent trends in stabilising peptides and proteins in pharmaceutical formulation-considerations in the choice of excipients” Expert Opin Drug Deliv. 6 (2009) 1219-1230; Akers, M. J. “Excipient-Drug interactions in Parenteral Formulations”, J. Pharm Sci 91 (2002) 2283-2300; Imamura, K et al “Effects of types of sugar on stabilization of Protein in the dried state”, J Pharm Sci 92 (2003) 266-274; Izutsu, Kkojima, S. “Excipient crystallinity and its protein-structure-stabilizing effect during freeze-drying”, J. Pharm. Pharmacol, 54 (2002) 1033-1039; Johnson, R, et al “Mannitol-sucrose mixtures—versatile formulations for protein lyophilization”, J. Pharm. Sci, 91 (2002) 914-922; Kerwin B. “Polysorbates 20 and 80 used in the formulation of protein biotherapeutics: structure and degradation pathways” J. Pharm Sci. 97 (2008) 2924-2935; Ha, E., et al “Peroxide formation in polysorbate 80 and protein stability”, J. Pharm Sci, 91 (2002), 2252-2264, and He, F., et al, “Effect of sugar molecules on the viscosity of high concentration monoclonal antibody solutions” Pharm Res. 28 (2011) 1552-1560; and the entire contents of which are incorporated herein by reference and to which the reader is specifically referred.

In one embodiment the antibody of the invention, when in a pharmaceutical preparation, is present in unit dose forms. The appropriate therapeutically effective dose will be determined readily by those of skill in the art. Suitable doses may be calculated for patients according to their weight, for example suitable doses may be in the range of about 0.1 to about 200 mg/kg, for example about 1 to about 20 mg/kg, for example about 10 to about 20 mg/kg or for example about 1 to about 15 mg/kg, for example about 5 to about 15 mg/kg. To effectively treat conditions such as Multiple myeloma, SLE or IPT in a human, suitable doses may be within the range of about 0.1 to about 2000 mg, for example about 0.1 to about 500 mg, for example about 500 mg, for example about 0.1 to about 150 mg, or about 0.1 to about 80 mg, or about 0.1 to about 60 mg, or about 0.1 to about 40 mg, or for example about 1 to about 100 mg, or about 1 to about 50 mg, of an antigen binding protein of this invention, which may be administered parenterally, for example subcutaneously, intravenously or intramuscularly. Such dose may, if necessary, be repeated at appropriate time intervals selected as appropriate by a physician.

The antigen binding proteins described herein can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immunoglobulins and art-known peroxidise and reconstitution techniques can be employed.

In another aspect of the invention there is provided an antigen binding protein as herein described for use in a medicament.

In one aspect of the present invention there is provided an antigen binding protein according to the invention as herein described for use in the treatment of rheumatoid arthitis, Type 1 Diabetes Mellitus, multiple sclerosis or psoriasis wherein said method comprises the step of administering to said patient a therapeutically effective amount of the antigen binding protein as described herein.

In one embodiment of the present invention, methods are provided for treating cancer in a human comprising administering to said human an antigen binding protein that specifically binds to antigens on the tumor cells. In some instances, the antigen binding protein is part of an immunoconjugate.

The term “antibody-drug conjugate (ADC),” as used herein, refers to a compound comprising a monoclonal antibody (mAb) attached to a cytotoxic agent (generally a small molecule drug with a high systemic toxicity) via chemical linkers. The ADC of this invention is represented as the formula of:

wherein D1 and D2 are a small molecule cytotoxin or a functional small molecule, in general called payload; L1 and L2 are a function linker that has an affinity ligand; and mAb is a monoclonal antibody. In some embodiments, an ADC may comprise a small molecule cytotoxin that has been chemically modified to contain a linker with an affinity ligand, or a linker containing an affinity ligand is part of payload which is called a traceless linker. The linker is generally used to conjugate the cytotoxin to the antibody, or antigen-binding fragment thereof. Upon binding to the target antigen on the surface of a cell, the ADC is internalized and trafficked to the lysosome where the cytotoxin is released by either proteolysis of a cleavable linker (e.g., by cathepsin B found in the lysosome) or by proteolytic degradation of the antibody, if attached to the cytotoxin via a non-cleavable linker. The cytotoxin then translocates out of the lysosome and into the cytosol or nucleus, where it can then bind to its target, depending on its mechanism of action.

The antibody-drug conjugate described herein may comprise a whole antibody or an antibody fragment. A whole antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each of the heavy chains contains one N-terminal variable (VH) region and three C-terminal constant (CH1, CH2 and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen binding site of an antibody. The VH and VL regions have the same general structure, with each region comprising four framework regions, whose sequences are relatively conserved. The framework regions are connected by three complementarity determining regions (CDRs). The three CDRs, known as CDR1, CDR2, and CDR3, form the “hypervariable region” of an antibody, which is responsible for antigen binding.

The ADC may comprise an antigen-binding fragment of an antibody. The terms “antibody fragment,” “antigen-binding fragment,” “functional fragment of an antibody,” and “antigen-binding portion” are used interchangeably herein and refer to one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen. The antibody fragment may comprise, for example, one or more CDRs, the variable region (or portions thereof), the constant region (or portions thereof), or combinations thereof. Examples of antibody fragments include, but are not limited to, (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CHI domains; (ii) a F(ab′)2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (iv) a single chain Fv (scFv), which is a monovalent molecule consisting of the two domains of the Fv fragment (i.e., VL and VH) joined by a synthetic linker which enables the two domains to be synthesized as a single polypeptide chain (see, e.g., Kabat E A, Wu T T., J Immunol. 1991, 147(5): 1709-19) and (v) a diabody, which is a dimer of polypeptide chains, wherein each polypeptide chain comprises a VH connected to a VL by a peptide linker that is too short to allow pairing between the VH and VL on the same polypeptide chain, thereby driving the pairing between the complementary domains on different VH-VL polypeptide chains to generate a dimeric molecule having two functional antigen binding sites (see, e.g. Hudson P J, Kortt A A, J Immunol Methods. 1999, 231(1-2): 177-89; Holliger P, Winter G. Cancer Immunol Immunother. 1997, 45(3-4):128-30).

The monoclonal antibody, or an antigen-binding fragment thereof, directed against a certain antigen may comprise any suitable binding affinity to the antigen or an epitope thereof. The term “affinity” refers to the equilibrium constant for the reversible binding of two agents and is expressed as the dissociation constant (KD). The affinity of an antibody or antigen-binding fragment thereof for an antigen or epitope of interest can be measured using any method known in the art. Such methods include, for example, fluorescence activated cell sorting (FACS), surface plasmon resonance (e.g., Biacore™, ProteOn™), biolayer interferometry (BLI, e.g. Octet), kinetics exclusion assay (e.g. KinExA™), separable beads (e.g., magnetic beads), antigen panning, and/or ELISA (see, e.g., J R Crowther, Methods Mol Biol. 2000, 149: III-IV, 1-413). It is known in the art that the binding affinity of a particular antibody will vary depending on the method that is used to analyze the binding affinity.

Affinity of a binding agent to a ligand, such as affinity of an antibody for an epitope, can be, for example, from about 1 picomolar (pM) to about 1 micromolar (1 μM) (e.g., from about 1 picomolar (pM) to about 1 nanomolar (nM), or from about 1 nM to about 1 micromolar (μM)). In one embodiment, the monoclonal antibody or an antigen-binding fragment thereof may bind to a certain antigen with a Kd less than or equal to 100 nanomolar (e.g., 100 nM, about 90 nM, about 80 nM, about 70 nM, about 60 nM, about 50 nM, about 40 nM, about 30 nM, about 20 nM, or about 10 nM, or a range defined by any two of the foregoing values).

In another embodiment, the monoclonal antibody may bind to a certain antigen with a Kd less than or equal to 10 nanomolar (e.g., about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.9 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, about 0.4 nM, about 0.3 nM, about 0.2 nM, about 0.1 nM, about 0.05 nM, about 0.02 nM, about 0.01 nM, about 0.001 nM, or a range defined by any two of the foregoing values).

In another embodiment, the monoclonal antibody may bind to A CERTAIN ANTIGEN with a Kd less than or equal to 200 pM (e.g., about 190 pM, about 175 pM, about 150 pM, about 125 pM, about 110 pM, about 100 pM, about 90 pM, about 80 pM, about 70 pM, about 60 pM, about 50 pM, about 40 pM, about 30 pM, about 25 pM, about 20 pM, about 15 pM, about 10 pM, about 5 pM, about 1 pM, or a range defined by any two of the foregoing values).

In one embodiment, the affinity of the antibody or antigen-binding fragment thereof, as measured by surface plasmon resonance (SPR), is about 90 nM, about 80 nM, about 70 nM, about 60 nM, about 50 nM, about 40 nM, about 30 nM, or a range defined by any two of the foregoing values, for example, about 50 nM to about 70 nM, about 55 nM to about 65 nM, or about 58 nM to about 62 nM.

In one embodiment, the affinity of the antibody or antigen-binding fragment thereof to membrane-bound antigens, as measured by FACS, is less than or equal to 10 nanomolar (e.g., about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.9 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, about 0.4 nM, about 0.3 nM, about 0.2 nM, about 0.1 nM, about 0.05 nM, about 0.02 nM, about 0.01 nM, about 0.001 nM, or a range defined by any two of the foregoing values).

In one embodiment, the antibody-drug conjugate comprises a variable region of a monoclonal antibody. In this respect, the ADC may comprise a light chain variable region, a heavy chain variable region, or both a light chain variable region and a heavy chain variable region of a monoclonal antibody.

The monoclonal antibody, or antigen-binding fragment thereof, may be conjugated to a cytotoxin using any suitable method known in the art, including site-specific or non-site specific conjugation methods. Conventional conjugation strategies for antibodies typically rely on randomly (i.e., non-specifically) conjugating the payload to the antibody, antigen-binding fragment thereof, through lysines or cysteines. Accordingly, in some aspects the antibody or antigen-binding fragment thereof is randomly conjugated to a cytotoxic agent, for example, by partial reduction of the antibody or antibody fragment, followed by reaction with a desired agent with or without a linker moiety attached. For example, the antibody or antigen-binding fragment thereof may be reduced using dithiothreitol (DTT), TCEP, thiolethenol or a similar reducing agent. The cytotoxic agent, with or without a linker moiety attached thereto, can then be added at a molar excess to the reduced antibody or antibody fragment in the presence of dimethyl sulfoxide (DMSO), or DMA. After conjugation, excess free cysteine may be added to quench unreacted agent. The cytotoxic agent, with or without a linker moiety having an amino-reactivable, or phenol-reactivable, or the others reactivable group (e.g. NHS, PFP) thereto, can be added directly at a molar excess to the antibody or antibody fragment in the presence of DMSO, or DMA to form a conjugate. The reaction mixture may then be purified through chromatography or buffer-exchanged into a suitable buffer, such as phosphate buffered saline (PBS), citrate buffer, or histidine buffer.

The terms “cytotoxin” and “cytotoxic agent” refer to any molecule that inhibits or prevents the function of cells and/or causes destruction of cells (cell death), and/or exerts anti-proliferative effects. A cytotoxin or cytotoxic agent of an ADC also is referred to in the art as the “payload” of the ADC. A number of classes of cytotoxic agents are known in the art to have potential utility in ADC molecules and can be used in the ADC described herein. Such classes of cytotoxic agents include, for example, anti-microtubule agents (e.g., tubulysins, auristatins and maytansinoids), DNA minor groove binders (e.g. pyrrolobenzodiazepines (PBDs) or indolinobenzodiazepines (IGN) and their dimers), RNA polymerase II inhibitors (e.g., amatoxins), inhibitor of DNA topoisomerase I (e.g., camptothecins) and DNA alkylating agents (e.g., duocarmycin, CC-1065, pyrrolobenzodiazepine dimers or pseudodimers or indolinobenzodiazepine pseudodimers). Examples of specific cytotoxic agents that may be used in the ADC described herein include, but are not limited to, tubulysins, amanitins, auristatins, calicheamicin, camptothecins, daunomycins, doxorubicins, duocarmycins, dolastatins, enediynes, lexitropsins, taxanes, puromycins, maytansinoids, vinca alkaloids, and pyrrolobenzodiazepines (PBDs). More specifically, the cytotoxic agent may be, for example tubulysins, auristatins (AFP, MMAF, MMAE, AEB, AEVB, E), paclitaxels, docetaxels, CC-1065 (duocarmysin, DC1, DC4, CBI-dimers), camptothecins (SN-38, topotecan, exatecan), morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combretatstatin, chalicheamicin, maytansine (DM1, DM4, DM21), vinblastine, methotrexate, netropsin, or derivatives or analogs thereof. Cytotoxins suitable for use in ADCs are also described in, for example, International Patent Application Publication No. PCT/CN2021/128453.

In general, a chemotherapeutic agent or a functional compound can also be conjugated to the antibody of this invention. A chemotherapeutic agent or a functional compound is selected from the group consisting of:

(8). The pharmaceutically acceptable salts, acids, derivatives, hydrate or hydrated salt; or a crystalline structure; or an optical isomer, racemate, diastereomer or enantiomer of any of the above drugs.

In another embodiment, the drug D can be polyalkylene glycols that are used for extending the half-life of the cell-binding antibody, or antibody molecule when administered to a mammal. Polyalkylene glycols include, but are not limited to, poly(ethylene glycols) (PEGs), poly(propylene glycol) and copolymers of ethylene oxide and propylene oxide; particularly preferred are PEGs, and more particularly preferred are monofunctionally activated hydroxyPEGs (e.g., hydroxyl PEGs activated at a single terminus, including reactive esters of hydroxyPEG-monocarboxylic acids, hydroxy PEG-monoaldehydes, hydroxy PEG-monoamines, hydroxyPEG-monohydrazides, hydroxy PEG-monocarbazates, hydroxyl PEG-monoiodoacetamides, hydroxyl PEG-monomaleimides, hydroxyl PEG-monoorthopyridyl disulfides, hydroxyPEG-monooximes, hydroxyPEG-monophenyl carbonates, hydroxyl PEG-monophenyl glyoxals, hydroxyl PEG-monothiazolidine-2-thiones, hydroxyl PEG-monothioesters, hydroxyl PEG-monothiols, hydroxyl PEG-monotriazines and hydroxyl PEG-monovinylsulfones).

In certain such embodiments, the polyalkylene glycol has a molecular weight of from about 10 Daltons to about 200 kDa, preferably about 88 Da to about 40 kDa; two branches each with a molecular weight of about 88 Da to about 40 kDa; and more preferably two branches, each of about 88 Da to about 20 kDa. In one particular embodiment, the polyalkylene glycol is poly(ethylene)glycol and has a molecular weight of about 10 kDa; about 20 kDa, or about 40 kDa. In specific embodiments, the PEG is a PEG 10 kDa (linear or branched), a PEG 20 kDa (linear or branched), or a PEG 40 kDa (linear or branched). A number of US patents have disclosed the preparation of linear or branched “non-antigenic” PEG polymers and derivatives or conjugates thereof, e.g., U.S. Pat. Nos. 5,428,128; 5,621,039; 5,622,986; 5,643,575; 5,728,560; 5,730,990; 5,738,846; 5,811,076; 5,824,701; 5,840,900; 5,880,131; 5,900,402; 5,902,588; 5,919,455; 5,951,974; 5,965,119; 5,965,566; 5,969,040; 5,981,709; 6,011,042; 6,042,822; 6,113,906; 6,127,355; 6,132,713; 6,177,087, and 6,180,095.

In yet another embodiment, D is more preferably a potent cytotoxic agent, selected from a tubulysin and its analogs, a maytansinoid and its analogs, a taxanoid (taxane) and its analogs, a CC-1065 and its analogs, a daunorubicin or doxorubicin and its analogs, an amatoxin and its analogs, a benzodiazepine dimer (e.g., dimers of pyrrolobenzodiazepine (PBD), tomaymycin, anthramycin, indolinobenzodiazepines, imidazobenzothiadiazepines, or oxazolidinobenzo-diazepines) and their analogs, a calicheamicin and the enediyne antibiotic and their analogs, an actinomycin and its analogs, an azaserine and its analogs, a bleomycin and its analogs, an epirubicin and its analogs, a tamoxifen and its analogs, an idarubicin and its analogs, a dolastatin and its analogs, an auristatin (including monomethyl auristatin E (MMAE), MMAF, auristatin PYE, auristatin TP, Auristatins 2-AQ, 6-AQ, EB (AEB), and EFP (AEFP)) and its analogs, a combretastatin, a duocarmycin and its analogs, a camptothecin, a geldanamycin and its analogs, a methotrexate and its analogs, a thiotepa and its analogs, a vindesine and its analogs, a vincristine and its analogs, a hemiasterlin and its analogs, a nazumamide and its analogs, a spliceostatin, a pladienolide, a microginin and its analogs, a radiosumin and its analogs, an alterobactin and its analogs, a microsclerodermin and its analogs, a theonellamide and its analogs, an esperamicin and its analogs, PNU-159682 and its analogs, a protein kinase inhibitor, a MEK inhibitor, a KSP inhibitor, a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, an immunotoxin, a cell receptor agonist, a cell stimulating molecule or intracellular signaling molecule, one, two or more DNA, RNA, mRNA, small interfering RNA (siRNA), microRNA (miRNA), and PIWI interacting RNAs (piRNA), and stereoisomers, isosteres, analogs, or derivatives thereof.

Tubulysin analog having the following formula (IV′):

In certain such embodiments, cryptophycin or their derivatives are peferably the cytotoxic agent of the present invention. The cryptophycins are a family of 16-membered macrolide antimitotic agents isolated from the cyanobacteria Nostoc sp. The mechanism of anticancer activity of the cryptophycins has been associated with their destabilization of microtubules and induction of bcl-2 phosphorylation leading to apoptosis. Cryptophycins demonstrated activity against the wide spectrum of solid tumors including those that overexpress the multidrug resistance efflux pump P-glycoprotein.

The structures of cryptophycins of this invention are preferred the following formula:

The structure of Camptothecin (CPT) used for the patent is illustrated below formula:

The structures of camptothecins are preferred the following formula:

In certain such embodiments, Combretastatins are natural phenols with vascular disruption properties in tumors and are preferably the cytotoxic agent of the invention. Exemplary combretastatins and their derivatives include, but are not limited to, combretastatin A-4 (CA-4), CA4-βGals, CA-4PD, CA4-NPs and ombrabulin, having the following formula:

In certain such embodiments, Anthracyclines are preferably the cytoxic agents of the invention. Anthracyclines are mammalian DNA topoisomerases II inhibitors that are able to stabilize enzyme-DNA complexes wherein DNA strands are cut and covalently linked to the antibody. These anticancer agents maintain a prominent role in treating many forms of solid tumors and acute leukemias during the last several decades. However, anthracyclines cause cardiovascular morbidity and mortality (Sagi, J. C., et al, Pharmacogenomics. 2016, 17(9), 1075-87; McGowan, J. V., et al, Cardiovasc Drugs Ther. 2017, 31(1), 63-75). Thus, to enhance specific activity of such molecules while reducing the cardiotoxicity, reasearchers actively are using the conjugation of anthracyclines to a cell-binding antibody, or antibody molecule as a general approach for improving the therapeutic index of these drugs, (Mollaev, M. et al, Int J Pharm. 2018 Dec. 29. pii: S0378-5173(18) 30991-8; Rossin, R., et al, Bioconjug Chem. 2016, 27(7):1697-706; Dal Corso, A., et al, J Control Release. 2017, 264:211-218). Exemplary anthracyclines include, but are not limited to, daunorubicin, doxorubicin (i.e., adriamycin), epirubicin, idarubicin, valrubicin, and mitoxantrone. The structures of anthracyclines used for the present application are preferred the following formula;

In certain such embodiments, Vinca alkaloids are preferably the cytoxic agents of the invention. Vinca alkaloids are a set of anti-mitotic and anti-microtubule alkaloid agents that work by inhibiting the ability of cancer cells to divide. Vinca alkaloids include vinblastine, vincristine, vindesine, leurosine, vinorelbine, catharanthine, vindoline, vincaminol, vineridine, minovincine, methoxyminovincine, minovincinine, vincadifformine, desoxyvincaminol, vincamajine, vincamine, vinpocetine, and vinburnine. The structures of vinca alkaloids are preferred vinblastine, vincristine having the following formula:

In certain such embodiments, Hemiasterlin and its analogues (e.g., HTI-286) are preferably the cytoxic agents of the invention. They bind to the tubulin, disrupt normal microtubule dynamics, and, at stoichiometric amounts, depolymerize microtubules. The structures of hemiasterlins are preferred the following formula:

In certain such embodiments, Eribulin is preferably the cytoxic agents of the invention. Eribulin is binding predominantly to a small number of high affinity sites at the plus ends of existing microtubules and has both cytotoxic and non-cytotoxic mechanisms of action. Its cytotoxic effects are related to its antimitotic activities, wherein apoptosis of cancer cells is induced following prolonged and irreversible mitotic blockade (Kuznetsov, G. et al, Cancer Research. 2004, 64 (16): 5760-6. Towle, M. J, et al, Cancer Research. 2010, 71 (2): 496-505). In addition to its cytotoxic, antimitotic-based mechanisms, preclinical studies in human breast cancer models have shown that eribulin also exerts complex effects on the biology of surviving cancer cells and residual tumors that appear unrelated to its antimitotic effects. Eribulin has been approved by US FDA for the treatment of metastatic breast cancer who have received at least two prior chemotherapy regimens for late-stage disease, including both anthracycline- and taxane-based chemotherapies, as well as for the treatment of liposarcoma (a specific type of soft tissue sarcoma) that cannot be removed by surgery (unresectable) or is advanced (metastatic). Eribulin has been used as payload for ADC conjugates (US20170252458). The structure of Eribulin is preferred the following formula, Eb01:

In certain such embodiments, spliceostatins and pladienolides are anti-tumor compounds which inhibit splicing and interacts with spliceosome, SF3b, and are also preferred as cytotoxic agents of the present patent. Examples of spliceostatins include, but are not limited to, spliceostatin A, FR901464, and (2S, 3Z)-5-{[(2R, 3R, 5S, 6S)-6-{(2E, 4E)-5-[(3R, 4R, 5R, 7S)-7-(2-hydrazinyl-2-oxoethyl)-4-hydroxy-1, 6-dioxaspiro[2.5] oct-5-yl]-3-methylpenta-2, 4-dien-1-y-1}-2,5-dimethyltetrahydro-2H-pyran-3-yl]amino}-5-oxopent-3-en-2-yl acetate having the core structure of:

In certain such embodiments, protein kinase inhibitors are also preferred as cytotoxic agents of the present patent. Protein kinase inhibitors block the action of an enzyme to add a phosphate (PO4) group to serine, threonine, or tyrosine amino acids on a protein, and can modulate the protein function. The protein kinase inhibitors can be used to treat diseases due to hyperactive protein kinases (including mutant or overexpressed kinases) in cancer or to modulate cell functions to overcome other disease drivers. The structures of protein kinase inhibitors are preferred to selected from Adavosertib, Afatinib, Axitinib, Bafetinib, Bosutinib, Cobimetinib, Crizotinib, Cabozantinib, Dasatinib, Entrectinib, Erdafitinib, Erlotinib, Erlotinib, Fostamatinib, Gefitinib, Ibrutinib, Imatinib, Lapatinib, Lenvatinib, Mubritinib, Nilotinib, Pazopanib, Pegaptanib, Ponatinib, Rebastinib, Regorafenib, Ruxolitinib, Sorafenib, Sunitinib, SU6656, Tofacitinib, Vandetanib, Vemurafenib, Entrectinib, Palbociclib, Ribociclib, Abemaciclib, Dacomitinib, Neratinib, Rociletinib (CO-1686), Osimertinib, AZD3759, Nazartinib (EGF816), having the following formula, PK01˜PK40:

In certain such embodiments, proteinase inhibitors are also preferred as cytotoxic agents of the present patent. The proteinase inhibitors are selected from Carfilzomib, Clindamycin, Retapamulin, Indibulin, having the following formulae:

In certain such embodiments, an immunotoxin is also preferred as cytotoxic agents of the present patent. The immunotoxin is selected from Diphtheria toxin (DT), Cholera toxin (CT), Trichosanthin (TCS), Dianthin, Pseudomonas exotoxin A (ETA), Erythrogenic toxins, Diphtheria toxin, AB toxins, Type III exotoxins, proaerolysin, and topsalysin;

In certain such embodiments, an immunotoxin herein can be a cytotoxic agent of the invention. The immunotoxin herein is a macromolecular drug which is usually a cytotoxic protein derived from a bacterial or plant protein, such as Diphtheria toxin (DT), Cholera toxin (CT), Trichosanthin (TCS), Dianthin, Pseudomonas exotoxin A (ETA′), Erythrogenic toxins, Diphtheria toxin, AB toxins, Type III exotoxins, etc. It also can be a highly toxic bacterial pore-forming protoxin that requires proteolytic processing for activation. An example of this protoxin is proaerolysin and its genetically modified form, topsalysin. Topsalysin is a modified recombinant protein that has been engineered to be selectively activated by an enzyme in the prostate, leading to localized cell death and tissue disruption without damaging neighboring tissue and nerves; An immunotoxin herein is preferably conjugated via the process of the application through an amino acid having free amino, thiol or carboxyl acid group; and more preferably through N-terminal amino acid.

In addition, a certain cell receptor agonist, a cell stimulating molecule or intracellular signalling molecule can be as a chemotherapeutic/cytotoxic agent conjugated to the antibody of the invention.

In certain embodiments, one, two or more DNA, RNA, mRNA, small interfering RNA (siRNA), microRNA (miRNA), and PIWI interacting RNAs (piRNA) can be as a chemotherapeutic/function compound conjugated to the antibody of the invention:

In certain such embodiments, a MEK inhibitor can be the cytotoxic agent of the invention. A MEK inhibitor inhibits the mitogen-activated protein kinases MEK1 and/or MEK2 which is often overactive in some cancers. MEK inhibitors are especially used for treatment of BRAF-mutated melanoma, and KRAS/BRAF mutated colorectal cancer, breast cancer, and non-small cell lung cancer (NSCLC). MEK inhibitors are selected from PD0325901, selumetinib (AZD6244), cobimetinib (XL518), refametinib, trametinib (GSK1120212), pimasertib, Binimetinib (MEK162), AZD8330, RO4987655, RO5126766, WX-554, E6201, GDC-0623, PD-325901 and TAK-733. The preferred MEK inhibitors are selected from Trametinib (GSK1120212), Cobimetinib (XL518), Binimetinib (MEK162), selumetinib having the following formula:

In a further embodiment, either one or both of Drug 1 or/and Drug 2 of the present patent application can be independently selected from a chemotherapeutical drug as following:

or hydrated salts; or the polymorphic crystalline structures of these compounds; or the optical isomers, racemates, diastereomers or enantiomers; wherein  is the linkage site.

or hydrated salts; or the polymorphic crystalline structures of these compounds; or the optical isomers, racemates, diastereomers or enantiomers; wherein  is the linkage site.

Preferably, the self-immolative linker component has one of the following structures:

The non-self-immolative linker component is one of the following structures:

Wherein the (*) atom is the point of attachment of additional spacer R1 or releasable linkers, the cytotoxic agents, and/or the binding molecules; X1, Y1, U1, R1, R5, R5′ are defined as above; r is 0˜100; m and n are 0˜6 independently.

Further preferably, L1, L2, La1, La2, Lb1, Lb2, Lc1 and Lc2 may independently be a releasable linker. The term releasable linker refers to a linker that includes at least one bond that can be broken under physiological conditions, such as a pH-labile, acid-labile, base-labile, oxidatively labile, metabolically labile, biochemically labile, or enzyme-labile bond. It is appreciated that such physiological conditions resulting in bond breaking do not necessarily include a biological or metabolic process, and instead may include a standard chemical reaction, such as a hydrolysis or substitution reaction, for example, an endosome having a lower pH than cytosolic pH, and/or disulfide bond exchange reaction with a intracellular thiol, such as a millimolar range of abundant of glutathione inside the malignant cells.

Examples of the releasable linkers (L, L1 or L2) include, but not limited:

Example structures of the components of the linker L1, L2, La1, La2, Lb1, Lb2, Lc1 and Lc2 may independently contain one or several of the following structures:

In another embodiments, the L1, L2, La1, La2, Lb1, Lb2, E1, E2, Lv1′, Lv2′, Lc1 and Lc2 that are jointly constructed in the structures of Formula (I), (II) and (III) are (IV) are accordingly selected from the following preferences:

Wherein “” is a site that links a drug or a site of linker L1 or L2; “#” is a site that links a S (thiol), O (phenol), NH (amino), CHO (aldehyde), C(═O) (ketone), C(O) (NH) (amide) and C(O)(OH) (carboxylate) of an antibody; Aa is L- or D-natural or unnatural amino acids; “@” is a site that links Lc1 or Lc2 described in the formula (I), (II), (III) and (IV);

More preferably, the following core linker structure (L1″) having an affinity ligand in Formula (I) (or L1″ in Formula (Ib′)) as:

Wherein Aa is L- or D-natural or unnatural amino acids; A1 is the affinity ligand defined the same above;

Or the following core structure (called L1″ and L2″ fused jointly) in Formula (III) (or the L1″ and I2″ fused in Formula (IIIb′) accordingly);

Which is preferably the following formula (Ib′) and (Ic′):

In certain embodiments, the examples of the conjugates of formula (I), (II), (III) and (IV) are illustrated below:

In certain embodiments, the conjugates of Formula (I), (II) and (III) are prepared readily via conjugation reaction of the antibody with compounds having the following formula (V), (VI) and (VII) respectively:

Or the conjugate of Formula (IV) is prepared through sequential conjugation reaction of Formula (V) and Formula (V′) to an antibody:

Lv1 and Lv2 are a reactive group and are independently or jointly selected from:

In the formula (VI) and formula (VII) wherein

can be accordingly selected from:

wherein Lv3, Lv3′, X1′, and X2′, are described above; the connecting bond “—” in the middle of the two atoms means it can link either one of the two atoms.

Examples of Formula (V), (VI), (VII) and (V′) are illustrated below:

In some embodiments, the antibody drug conjugates are preferably prepared via a homogenous conjugation process, which comprises the following three key steps:

The Zinc cation-amino chelate/complex, Zn(NR1R2R3)m12+, used in step (a) is 0.01 mM-1.0 mM in concentration, or 0.5˜20 equivalents in moles of the protein used, and it can be added to the reaction solution with a water-soluble organic solvent, selected from, ethanol, methanol, propanol, propandiol, DMA, DMF, DMSO, THF, CH3CN.

The reductant is an organic phosphine, preferably selected from Tris(2-carboxyethyl)-phosphine (TECP) or Tris(hydroxypropyl) phosphine and its use in the reaction solution is 0.02 mM-1.0 mM in concentration, or 1.0-20 equivalents in moles of the protein used. The oxidant to be added in step (c) may be DHAA, Fe3+, I2, Cu2+, Mn3+, MnO2, or mixture of Fe3+/I−. The oxidant used in the reaction solution is 0.02 mM-1.0 mM in concentration, or 0.2-100 equivalents in moles of the protein used. The optimum pH in the conjugation reaction is typically between about 5.0 to 8.0, and preferably, about 5.5 to 7.5. The optimum temperature in the conjugation reaction is typically between about-5 to about 40° C., and preferably, about 0 to 37° C.; more preferably about 2 to 8° C.; further preferably about 2 to 6° C. The optimum time of the conjugation reaction is typically between about 15 min to about 48 hours and preferably, about 30 min to overnight (10˜16 h), more preferably about 2 h˜6 h. The optimal reaction conditions (e.g. pH, temperature, buffer, concentrations of the reactants) of course are depended upon specifically an antibody-like protein, a payload/linker complex, a reductant and/or Zn(NR1R2R3)m12+ used.

In further embodiments, under the homogenous conjugation process, the resulted conjugates of formula (I), (II), (III) or (IV) are over 75% linked to the cysteine sites between heavy-light chains of an antibody, and are less than 15% linked to the cysteine sites between heavy-heavy chains (hinge region) of an antibody. Typically, for formula (I), (II) (III) or (IV), when drug/antibody ratio (DAR) is set to be 4 and a drug linked to the linker is at one to one ratio, the distributions in percentage of the numbers of drugs in the antibody are: D0<1%, D2<10%, D4>65%, D6<10%, D8<10%; for formula (III). If the drug to linker ratio is above to 1, such as, 2, 3, 4, 5, or 6 drugs per linker (when a side chain or a multiple branched linker is used), the distributions in percentages of numbers of drugs in the antibody or antibody-like protein are increased by timing the ratios of drug/linker accordingly. The DAR can also be set up to around 6, with majority D6>65%, using both more equivalents of reducing agents, such as TCEP and more equivalents of one drug per linker payload/linker complex of formula (V), (VI), (VII) and (V′), wherein the drugs are mainly conjugated to the sites of disulfide bonds between heavy-light chains and the disulfide bonds of the upper hinge region of IgG antibodies.

In general, two steps of reaction is chosen to run conjugation of two types of payload/linker complexes containing similar maleimide groups or the other thiol reactable groups, such as making formula (IV), the first step reaction can use the homogenous conjugation reaction to conjugate the first functional payload, then the second step is to use Traut's regent or a thiol lactone to introduce a thiol through its reaction with a lysine of an antibody and then simultaneously conjugate the second functional payload/linker complex. It also can be first performed Traut's regent reaction or a thiol lactone reaction to conjugate the first functional payload/linker complex, then to conduct the second conjugation of the other functional payload/linker complex through the homogeneous conjugation reaction.

The resulted conjugates may be purified by standard biochemical means, such as gel filtration on a Sephadex G25 or Sephacryl S300 column, adsorption chromatography, ion (cation or anion) exchange chromatography, affinity chromatography (e.g. protein A column) or by dialysis (ultrafiltration or hyperfiltration (UF) and diafiltration (DF)). In some cases, a small size molecule of antibody (e.g. <100 KD) conjugated with a small molecular drugs can be purified by a chromatography such as by (reverse phase) HPLC or FPLC, size-exclusion chromatography, medium pressure column chromatography, ion exchange chromatography, or hydroxylapatite chromatography.

In further embodiments, for preparation of the conjugate of Formula (IV), the reaction of the cytotoxic drug/cytotoxic drug-linker complex of Formula (V) and (V′) to a amino acid in the antibody can be conducted at simultaneously or sequentially at the same or different conditions in the same pot. If the conjugation reactions are preformed simultaneously at the same condition, the Lv1 of Formula (V) and Lv2 of formula (V′) are normally differentiated. For instance, a thiol reactive group (e.g. maleimido, vinylsulfonyl, haloacetyl, acrylic, substituted propiolic) is selected for Lv1, then an amino reactive group of N-hydoxylsuccinimidyl (NHS) ester, pentfluorophenyl ester, dinitrophenyl ester, or carboxylic acid chloride group can be chosen for Lv2. A clickable chemistry group (e.g. azide, alkyne, dibenzocyclooctyne, BCN ((1R, 8S, 9s)-bicyclo[6.1.0]non-4-yn-9-ylmethanol)) can be selected for either Lv1 or Lv2 if the conjugated antibody is introduced a clickable reactive group ahead of the click chemistry reaction. Lv1 and Lv2 can be selected from many pairs of different function/reactive groups, such as: Amine-to-Sulfhydryl (succinimidyl (NHS) ester/maleimide, NHS ester/pyridyldithiol, NHS esters/haloacetyl), diazirine (SDA)-to-Sulfhydryl, Azide-to-Sulfhydryl, Alkyne-to-Sulfhydryl, Sulfhydryl-to-Carbohydrate (Maleimide/Hydrazide, Pyridyldithiol/Hydrazide, haloacetyl/Hydrazide), Hydroxyl-to-Sulfhydryl (Isocyanate/Maleimide), Sulfhydryl-to-DNA (Maleimide/Psoralen, Pyridyldithiol/Psoralen, haloacetyl/Psoralen), Sulfhydryl-to-Carboxyl (Carbodiimide). The conjugation reactions of Formula (V) and (V′) can also be preformed sequentially, as long as Lv1 and Lv2 have different reactive ability to the amino acids in the antibody/antibody like protein. For example, when both Lv1 and Lv2 are selected for reacting to thiols (cysteines) in the antibody/protein, Lv1 can be maleimido, which can react to a thiol group as fast as a few seconds at pH 5,0˜ 7.5, 2.0˜37° C.; Lv2 is then selected from slow reactive vinylsulfonyl or haloacetyl group in which the conjugation reaction with a thiol group in antibody has to be at pH>7.0, temperature over 30° C. for over 6 h.

If both Lv1 and Lv2 have the same (terminal) conjugatable groups, e.g., maleimido, haloacetyl or pyridyldithiol, the first conjugation with Formula (V) can be performed according to the above homogenous conjugation process, wherein the payload/linker complex is conjugated to the disulfide sites between heavy-light chains (of the Fab region) of a IgG antibody, then the Formula (V′) is adding sequentially to the reaction mixture to be conjugated at the disulfide bonds of hinge region of the IgG antibody. The first step of conjugation reaction can be conducted at low temperature, and the second step of the conjugation reaction with Formula (V′) can then be performed at room (25° C.) or higher temperature without purification of the first step reaction product as long as the compound of Formula (V′) is added in much more (3 or more) equivalents than the compound of Formula (V). The second step can also be through the reaction of Traut's reagent (2-Iminothiolane or 2-IT) with primary amines (—NH2, normally lysine) at neutral pH (˜7.0˜7,5) to introduce sulfhydryl (—SH) groups while maintaining charge properties similar to the original amino group. Thus the introduced sulfhydryl (—SH) groups can react to a payload/linker complex containing maleimido, haloacetyl or pyridyldithiol group to generate the conjugates of Formula (IV).

When the same pH and/or temperature conditions are chosen for thioether linked conjugates under the above homogenous conjugation process, the over four times equivalents of the cytotoxic drug-linker complex containing dual terminal thiol reactive are used for the cross conjugation of disulfide bonds of heavy-light chains of an antibody. It should be noted that a preferred method of synthesis of the disulfide or thiol-ether linked conjugates can be through the first chemical synthesis the drug-linker complex having disulfide or thiol-reactive compounds of the formula (V), (VI), (VII) or (V′); following by reaction with the thiols in the protein (antibody) according the process of the invention. Synthesis of conjugates bearing an acid labile hydrazone linkage can be achieved by reaction of a carbonyl group with the hydrazide moiety in the linker, by methods known in the art (see, for example, P. Hamann et al., Cancer Res. 53, 3336-34, 1993; B. Laguzza et al., J. Med. Chem., 32; 548-55, 1959; P. Trail et al., Cancer Res., 57; 100-5, 1997). Synthesis of conjugates bearing triazole linkage can be achieved by reaction of a 1-yne group of the cytotoxic drug/cytotoxic drug-linker complex or a binding ligand/binding ligand-linker complex with the azido moiety in the linker of formula (V), (VI), (VII), or (V′), through the click chemistry (Huisgen cycloaddition) (Lutz, J-F. et al, 2008, Adv. Drug Del. Rev. 60, 958-70; Sletten, E. M. et al 2011, AccChem. Research 44, 666-76). Synthesis of the conjugates linked via oxime is achieved by reaction of a modified antibody containing a ketone or aldehyde and a cytotoxic drug/cytotoxic drug-linker complex or a binding ligand/binding ligand-linker complex containing oxyamine group. A cytotoxic drug/cytotoxic drug-, or a binding ligand/binding ligand-linker complex containing an amino group can condensate with a carboxyl ester of NHS, imidazole, nitrophenoxyl; N-hydroxysuccinimide (NHS); methylsufonyl-phenoxyl; dinitrophenoxyl; pentafluorophenoxyl; tetrafluorophenoxyl; difluorophenoxyl; monofluo-rophenoxyl; pentachlorophenoxyl; triflate; imidazole; dichlorophenoxyl; tetrachlorophenoxyl; 1-hydroxybenzotriazole; tosylate; mesylate; 2-ethyl-5-phenylisoxazolium-3′-sulfonate in an antibody-linker complex to give a conjugate via amide bond linkage of Formula (I), (II), (III) or (IV). Many regular chemical and biochemical processes of the antibody-drug conjugation are known in the art (see, e.g. Matsuda, Y. and Mendelsohn, B. A., Expert Opin Biol Ther. 2021, 21 (7): 963-975; Puthenveetil, S., Methods Mol Biol. 2020, 2078:99-112; van Delft, F., and Lambert, J. M., ed. “Chemical Linkers in Antibody-Drug Conjugates (ADCs)”, Royal Soc. Chem. Pub., 22, December 2021, ISBN 978-1-83916-263-3, doi: 10.1039/9781839165153; Tumey, L. N., ed. “Antibody-Drug Conjugates, Methods and Protocols”, Springer Pub., 2020, ISBN: 978-1-4939-9929-3; Khongorzul, P. et al, Mol Cancer Res. 2020, 18(1):3-19; and many references incorporated in these books and papers). In over all, it is more preferable that the conjugates of Formula (I), (II), (III) and (IV) are directly conjugated with Formula (V), (VI), (VII), and (V) plus (V′) accordingly to an antibody in water based solution, and the compounds of Formula (V), (VI), (VII) and (V′) are constructed by chemical synthesis.

In general, the conjugate of Formula (I), (II), (III) or (IV) is preferably generated from a drug/linker complex of Formula (V), (VI), (VII), or (V) plus (V′), as in a one pot reaction. When a thiol reduced from an antibody reacts a thiol reactive group in the terminal of drug/linker complex of Formula (V), (VI), (VII), or (V′), the Ellman reagent can be optionally used to monitor the efficient reduction of the disulfide bonds and conjugation of the thiols through measurement of the numbers of the free thiols during the reactions. A UV spectrometry at wavelength of range 190-390 nm, preferably at 240-380 nm, more preferably at 240-370 nm is preferred to be used in assisting the reaction (via monitoring the conjugation). The conjugation reaction can be thus measured or conducted in a quartz cell or Pyrex flask in temperature control environment. The drug/protein (antibody) ratios (DAR) of the conjugates can also be measured by UV at wavelength of range 240-380 nm via calculation of the concentrations of the drug and the protein, by Hydrophobic Interaction Chromatography (HIC-HPLC) or Reverse Phase Chromatography (RP-HPLC) via measurement of the integration areas of each drug/protein fragment, or by Capilary electrophoresis (CE), and/or by LC-MS or LC-MS/MS or CE-MS (the combination of liquid chromatography (LC) or CE with mass spectrometry (MS) via measurement of both the integration areas of LC or CE and Peak intensity of MS for each drug/protein fragment). It is also noted in the conjugation process of the present invention, when a drug or a drug/linker complex is not well soluble in a water-based buffer solution, up to 30% of water mixable (miscible) organic solvents, such as DMA, DMF, ethanol, methanol, acetone, acetonitrile, THF, isopropanol, dioxane, propylene glycol, or ethylene diol can be added as the co-solvent in water based buffer solution.

The aqueous solutions for the modification of the antibody are buffered between pH 4 and 9, preferably between 6.0 and 7.5 and can contain any non-nucleophilic buffer salts useful for these pH ranges. Typical buffers include phosphate, acetate, triethanolamine HCl, HEPES, and MOPS buffers, which can contain additional components, such as cyclodextrins, sucrose and salts, for examples, NaCl and KCl. Other biological buffers that are used for the conjugation process are listed in the definition section. The progress of the reaction can be monitored by measuring the decrease in the absorption at a certain UV wavelength, such as at 254 nm, or increase in the absorption at a certain UV wavelength, such as 280 nm, or the other appropriate wavelength. After the reaction is complete, isolation of the modified cell-binding antibody agent can be performed in a routine way, using for example gel filtration chromatography, or adsorptive chromatography.

When disulfide exchange reaction is used for modification of the antibody, the extent of the modification can be assessed by measuring the absorbance of the nitropyridine thione, dinitropyridine dithione, pyridine thione, carboxylamidopyridine dithione and dicarboxyl-amidopyridine dithione group released via UV spectra. For the conjugation without a chromophore group, the modification or conjugation reaction can be monitored by LC-MS, preferably by UPLC-QTOF mass spectrometry, or Capilary electrophoresis-mass spectrometry (CE-MS). The linker compounds have diverse functional groups that can react with drugs, preferably cytotoxic agents that possess a suitable substituent. For examples, the modified antibody bearing an amino or hydroxyl substituent can react with drugs bearing an N-hydroxysuccinimide (NHS) ester, the modified antibody bearing a thiol substituent can react with drugs bearing a maleimido or haloacetyl group. Additionally, the modified antibody bearing a carbonyl (ketone or aldehyde) substituent can react with drugs bearing a hydrazide or an alkoxyamine. One skilled in the art can readily determine which linker to use based on the known reactivity of the available functional group on the linkers.

Formulation and Application

The antibody drug conjugates of the patent application are formulated to liquid, or suitable to be lyophilized and subsequently be reconstituted to a liquid formulation. The conjugate in a liquid formula or in the formulated lyophilized powder may take up 0.01%-99% by weight as major gradient in the formulation. In general, a liquid formulation comprising 0.1 g/L ˜300 g/L of concentration of the conjugate active ingredient for delivery to a patient without high levels of antibody aggregation may include one or more polyols (e.g. sugars), a buffering agent with pH 4.5 to 7.5, a surfactant (e.g. polysorbate 20 or 80), an antioxidant (e.g. ascorbic acid and/or methionine), a tonicity agent (e.g. mannitol, sorbitol or NaCl), chelating agents such as EDTA; metal complexes (e.g. Zn-protein complexes); biodegradable polymers such as polyesters; a preservative (e.g. benzyl alcohol) and/or a free amino acid.

Suitable buffering agents for use in the formulations include, but are not limited to, organic acid salts such as sodium, potassium, ammounium, or trihydroxyethylamino salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid or phtalic acid; Tris, tromethamine hydrochloride, sulfate or phosphate buffer. In addition, amino acid cationic components can also be used as buffering agent. Such amino acid component includes without limitation arginine, glycine, glycylglycine, and histidine. The arginine buffers include arginine acetate, arginine chloride, arginine phosphate, arginine sulfate, arginine succinate, etc. In one embodiment, the arginine buffer is arginine acetate. Examples of histidine buffers include histidine chloride-arginine chloride, histidine acetate-arginine acetate, histidine phosphate-arginine phosphate, histidine sulfate-arginine sulfate, histidine succinate-argine succinate, etc. The formulations of the buffers have a pH of 4.5 to pH 7.5, preferably from about 4.5 to about 6.5, more preferably from about 5.0 to about 6.2. In some embodiments, the concentration of the organic acid salts in the buffer is from about 10 mM to about 500 mM.

A “polyol” that may optionally be included in the formulation is a substance with multiple hydroxyl groups. Polyols can be used as stabilizing excipients and/or isotonicity agents in both liquid and lyophilized formulations. Polyols can protect biopharmaceuticals from both physical and chemical degradation pathways. Preferentially excluded co-solvents increase the effective surface tension of solvent at the protein interface whereby the most energetically favorable structural conformations are those with the smallest surface areas. Polyols include sugars (reducing and nonreducing sugars), sugar alcohols and sugar acids. A “reducing sugar” is one which contains a hemiacetal group that can reduce metal ions or react covalently with lysine and other amino groups in proteins and a “nonreducing sugar” is one which does not have these properties of a reducing sugar. Examples of reducing sugars are fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose and glucose. Nonreducing sugars include sucrose, trehalose, sorbose, melezitose and raffinose. Sugar alcohols are selected from mannitol, xylitol, erythritol, maltitol, lactitol, erythritol, threitol, sorbitol and glycerol. Sugar acids include L-gluconate and metallic salts thereof. The polyol in the liquid formula or in the formulated lyophilized solid can be 0.0%-20% by weight. Preferably, a nonreducing sugar, sucrose or trehalose at a concentration of about from 0.1% to 15% is chosen in the formulation, wherein trehalose being preferred over sucrose, because of the solution stability of trehalose.

A “preservative” optionally in the formulations is a compound that essentially reduces bacterial action therein. Examples of potential preservatives include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenoxyl, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. The preservative in the liquid formula or in the formulated lyophilized powder can be 0.0%-5.0% by weight. In one embodiment, the preservative herein is benzyl alcohol.

Suitable free amino acids as a bulky material, or tonicity agent, or osmotic pressure adjustment in the formulation, is selected from, but are not limited to, one or more of arginine, cystine, glycine, lysine, histidine, ornithine, isoleucine, leucine, alanine, glycine glutamic acid or aspartic acid. The inclusion of a basic amino acid is preferred i.e. arginine, lysine and/or histidine. If a composition includes histidine then this may act both as a buffering agent and a free amino acid, but when a histidine buffer is used it is typical to include a non-histidine free amino acid e.g. to include histidine buffer and lysine. An amino acid may be present in its D- and/or L-form, but the L-form is typical. The amino acid may be present as any suitable salt e.g. a hydrochloride salt, such as arginine-HCl. The amino acid in the liquid formula or in the formulated lyophilized powder can be 0.0%-30% by weight.

The formulations can optionally comprise methionine, glutathione, cysteine, cystine or ascorbic acid as an antioxidant at a concentration of about up to 5 mg/ml in the liquid formula or 0.0%-5.0% by weight in the formulated lyophilized powder; The formulations can optionally comprise metal chelating agent, e.g., EDTA, EGTA, etc., at a concentration of about up to 2 mM in the liquid formula or 0.0%-0.3% by weight in the formulated lyophilized powder.

The final formulation can be adjusted to the preferred pH with a buffer adjusting agent (e.g. an acid, such as HCl, H2SO4, acetic acid, H3PO4, citric acid, etc, or a base, such as NaOH, KOH, NH4OH, ethanolamine, diethanolamine or triethanol amine, sodium phosphate, potassium phosphate, trisodium citrate, tromethamine, etc) and the formulation should be controlled “isotonic” which is meant that the formulation of interest has essentially the same osmotic pressure as human blood. Isotonic formulations will generally have an osmotic pressure from about 250 to 350 mOsm. Isotonicity can be measured using a vapor pressure or ice-freezing type osmometer, for example. The isotonic agent is selected from mannitol, sorbitol, sodium acetate, potassium chloride, sodium phosphate, potassium phosphate, trisodium citrate, or NaCl. In general, both the buffer salts and the isotonic agent may take up to 30% by weight in the formulation.

Other contemplated excipients, which may be utilized in the aqueous pharmaceutical compositions of the patent application include, for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids such as phospholipids or fatty acids, steroids such as cholesterol, protein excipients such as serum albumin (human serum albumin), recombinant human albumin, gelatin, casein, salt-forming counterions such sodium and the like. These and additional known pharmaceutical excipients and/or additives suitable for use in the formulations of the invention are known in the art, e.g., as listed in “The Handbook of Pharmaceutical Excipients, 4th edition, Rowe et al., Eds., American Pharmaceuticals Association (2003); and Remington: the Science and Practice of Pharmacy, 21th edition, Gennaro, Ed., Lippincott Williams & Wilkins (2005).

A pharmaceutical container or vessel is used to hold the pharmaceutical formulation of any of conjugates of the patent application. The vessel is a vial, bottle, pre-filled syringe, pre-filled or auto-injector syringe. The liquid formula can be freeze-dried or drum-dryed to a form of cake or powder in a borosilicate vial or soda lime glass vial. The solid powder can also be prepared by efficient spray drying, and then packed to a vial or a pharmaceutical container for storage and distribution.

In a further embodiment, the invention provides a method for preparing a formulation comprising the steps of: (a) lyophilizing the formulation comprising the conjugates, excipients, and a buffer system; and (b) reconstituting the lyophilized mixture of step (a) in a reconstitution medium such that the reconstituted formulation is stable. The formulation of step (a) may further comprise a stabilizer and one or more excipients selected from a group comprising bulking agent, salt, surfactant and preservative as hereinabove described. As reconstitution media, several diluted organic acids or water, i.e. sterile water, bacteriostatic water for injection (BWFI) or may be used. The reconstitution medium may be selected from water, i.e. sterile water, bacteriostatic water for injection (BWFI) or the group consisting of acetic acid, propionic acid, succinic acid, sodium chloride, magnesium chloride, acidic solution of sodium chloride, acidic solution of magnesium chloride and acidic solution of arginine, in an amount from about 10 to about 250 mM.

A liquid pharmaceutical formulation of the conjugates of the patent application should exhibit a variety of pre-defined characteristics. One of the major concerns in liquid drug products is stability, as the antibodies tend to form soluble and insoluble aggregates during manufacturing and storage. In addition, various chemical reactions can occur in solution (deamidation, oxidation, clipping, isomerization etc.) leading to an increase in degradation product levels and/or loss of bioactivity. Preferably, a conjugate in either liquid or lyophilizate formulation should exhibit a shelf life of more than 6 months at 25° C. More preferred a conjugate in either liquid or lyophilizate formulation should exhibit a shelf life of more than 12 months at 25° C. Most preferred liquid formulation should exhibit a shelf life of about 24 to 36 months at 2-8° C. and the lyophilizate formulation should exhibit a shelf life of about preferably up to 60 months at 2-8° C. Both liquid and lyophilizate formulations should exhibit a shelf life for at least two years at −20° C., or −70° C.

In certain embodiments, the formulation is stable following freezing (e.g., −20° C., or −70° C.) and thawing of the formulation, for example following 1, 2 or 3 cycles of freezing and thawing. Stability can be evaluated qualitatively and/or quantitatively in a variety of different ways, including evaluation of drug/antibody ratio and aggregate formation (for example using UV, size exclusion chromatography, by measuring turbidity, and/or by visual inspection); by assessing charge heterogeneity using cation exchange chromatography, image capillary isoelectric focusing (icIEF) or capillary zone electrophoresis; amino-terminal or carboxy-terminal sequence analysis; mass spectrometric analysis, or matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF MS), or HPLC-MS/MS; SDS-PAGE analysis to compare reduced and intact antibody; peptide map (for example tryptic or LYS—C) analysis; evaluating biological activity or antigen binding function of the antibody; etc. Instability may involve any one or more of: aggregation, deamidation (e.g. Asn deamidation), oxidation (e.g. Met oxidation), isomerization (e.g. Asp isomeriation), clipping/hydrolysis/fragmentation (e.g. hinge region fragmentation), succinimide formation, unpaired cysteine(s), N-terminal extension, C-terminal processing, glycosylation differences, etc.

A stable conjugate should also “retains its biological activity” in a pharmaceutical formulation, if the biological activity of the conjugate at a given time, e.g. 24 month, within about 20%, preferably about 10% (within the errors of the assay) of the biological activity exhibited at the time the pharmaceutical formulation was prepared as determined in an antigen binding assay, and/or in vitro, cytotoxic assay, for example.

For clinical in vivo use, the conjugate of the invention will be supplied as solutions or as a lyophilized solid that can be redissolved in sterile water for injection. Examples of suitable protocols of conjugate administration are as follows. Conjugates are given daily, weekly, biweekly, triweekly, once every four weeks or monthly for 8˜108 weeks as an i.v. bolus. Bolus doses are given in 50 to 1000 ml of normal saline to which human serum albumin (e.g. 0.5 to 1 mL of a concentrated solution of human serum albumin, 100 mg/mL) can optionally be added. Dosages will be about 50 μg to 20 mg/kg of body weight per week, i.v. (range of 10 μg to 200 mg/kg per injection). 4˜108 weeks after treatment, the patient may receive a second course of treatment. Specific clinical protocols with regard to route of administration, excipients, diluents, dosages, times, etc., can be determined by the skilled clinicians.

Examples of medical conditions that can be treated according to the in vivo or ex vivo methods of killing selected cell populations include malignancy of any types of cancer, autoimmune diseases, graft rejections, and infections (viral, bacterial or parasite).

The amount of a conjugate which is required to achieve the desired biological effect, will vary depending upon a number of factors, including the chemical characteristics, the potency, and the bioavailability of the conjugates, the type of disease, the species to which the patient belongs, the diseased state of the patient, the route of administration, all factors which dictate the required dose amounts, delivery and regimen to be administered.

In general terms, the conjugates of this invention may be provided in an aqueous physiological buffer solution containing 0.1 to 10% w/v conjugates for parenteral administration. Typical dose ranges are from 1 μg/kg to 0.1 g/kg of body weight daily; weekly, biweekly, triweekly, or monthly, a preferred dose range is from 0.01 mg/kg to 25 mg/kg of body weight weekly, biweekly, triweekly, or monthly, an equivalent dose in a human. The preferred dosage of drug to be administered is likely to depend on such variables as the type and extent of progression of the disease or disorder, the overall health status of the particular patient, the relative biological efficacy of the compound selected, the formulation of the compound, the route of administration (intravenous, intramuscular, or other), the pharmacokinetic properties of the conjugates by the chosen delivery route, and the speed (bolus or continuous infusion) and schedule of administrations (number of repetitions in a given period of time).

In some embodiment, when the reconstituted conjugates are injected under the skin, into a muscle, or into other tissues of the body, a hyaluronidase (HAase) is preferably administered together with the conjugates. The hyaluronidase here is used as an aid in helping patient body absorb the injected conjugates. The hyaluronidase is synergistically used 20-200 unit doses, preferably in 40-160 unit doses.

The conjugates of the present invention are also capable of being administered in unit dose forms, wherein the term “unit dose” means a single dose which is capable of being administered to a patient, and which can be readily handled and packaged, remaining as a physically and chemically stable unit dose comprising either the active conjugate itself, or as a pharmaceutically acceptable composition, as described hereinafter. As such, typical total daily/weekly/biweekly/triweekly/monthly dose ranges are from 0.01 to 100 mg/kg of body weight. By way of general guidance, unit doses for humans range from 1 mg to 3000 mg per day, or per week, per two weeks (biweekly), triweekly, or per month. Preferrably the unit dose range is from 1 to 500 mg administered one to four times a month and even more preferably from 1 mg to 100 mg, once a week, or once a biweek, or once a triweek. Conjugatess provided herein can be formulated into pharmaceutical compositions by admixture with one or more pharmaceutically acceptable excipients. Such unit dose compositions may be prepared for use by oral administration, particularly in the form of tablets, simple capsules or soft gel capsules; or intranasally, particularly in the form of powders, nasal drops, or aerosols; or dermally, for example, topically in ointments, creams, lotions, gels or sprays, or via trans-dermal patches. The compositions may conveniently be administered in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical art, for example, as described in Remington: The Science and Practice of Pharmacy, 21th ed.; Lippincott Williams & Wilkins: Philadelphia, PA, 2005.

The formulations include pharmaceutical compositions in which a compound of the present invention is formulated for oral or parenteral administration. For oral administration, tablets, pills, powders, capsules, troches and the like can contain one or more of any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, or gum tragacanth; a diluent such as starch or lactose; a disintegrant such as starch and cellulose derivatives; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, or methyl salicylate. Capsules can be in the form of a hard capsule or soft capsule, which are generally made from gelatin blends optionally blended with plasticizers, as well as a starch capsule. In addition, dosage unit forms can contain various other materials that modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or enteric agents. Other oral dosage forms syrup or elixir may contain sweetening agents, preservatives, dyes, colorings, and flavorings. In addition, the active compounds may be incorporated into fast dissolve, modified-release or sustained-release preparations and formulations, and wherein such sustained-release formulations are preferably bi-modal. Preferred tablets contain lactose, cornstarch, magnesium silicate, croscarmellose sodium, povidone, magnesium stearate, or talc in any combination.

Liquid preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. The liquid compositions may also include binders, buffers, preservatives, chelating agents, sweetening, flavoring, and coloring agents, and the like. Non-aqueous solvents include alcohols, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic esters such as ethyl oleate. Aqueous carriers include mixtures of alcohols and water, buffered media, and saline. In particular, biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be useful excipients to control the release of the active compounds. Intravenous vehicles can include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Other potentially useful parenteral delivery systems for these active compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.

Alternative modes of administration include formulations for inhalation, which include such means as dry powder, aerosol, or drops. They may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally. Formulations for buccal administration include, for example, lozenges or pastilles and may also include a flavored base, such as sucrose or acacia, and other excipients such as glycocholate. Formulations suitable for rectal administration are preferably presented as unit-dose suppositories, with a solid based carrier, such as cocoa butter, and may include a salicylate. Formulations for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which can be used include petroleum jelly, lanolin, polyethylene glycols, alcohols, or their combinations. Formulations suitable for transdermal administration can be presented as discrete patches and can be lipophilic emulsions or buffered, aqueous solutions, dissolved and/or dispersed in a polymer or an adhesive.

In yet another embodiment, a pharmaceutical composition comprising a therapeutically effective amount of the conjugate of Formula (I), (II), (III), (IV) or any conjugates described through the present patent can be administered with the other therapeutic agents such as the chemotherapeutic agent, the radiation therapy, immunotherapy agents, autoimmune disorder agents, anti-infectious agents or the other conjugates for synergistically effective treatment or prevention of a cancer, or an autoimmune disease, or an infectious disease. The term “coadministered,” as used herein, refers to administering one or more additional therapeutic agents and the antibody or ADC described herein, or the antibody or ADC-containing composition, sufficiently close in time such that the antibody or ADC can enhance the effect of one or more additional therapeutic agents, or vice versa. In this regard, the antibody or ADC or the composition containing the same may be administered first, and the one or more additional therapeutic agents may be administered second, or vice versa. For example, the antibody or ADC or composition containing the same may be administered in combination with other agents (e.g., as an adjuvant) for the treatment or prevention of multiple myeloma. In this respect, the antibody or ADC or antibody or ADC-containing composition can be used in combination with at least one other anticancer agent including, for example, any suitable chemotherapeutic agent known in the art, ionization radiation, small molecule anticancer agents, cancer vaccines, biological therapies (e.g., other monoclonal antibodies, cancer-killing viruses, gene therapy, and adoptive T-cell transfer), and/or surgery. The synergistic drugs or radiation therapy can be administered prior or subsequent to administration of a conjugate, in one aspect at least an hour, 12 hours, a day, a week, biweeks, triweeks, a month, in further aspects several months, prior or subsequent to administration of a conjugate of the invention.

In some embodiments, the disclosure also provides a composition comprising the above-described antibody or antibody-drug conjugate and a pharmaceutically acceptable (e.g., physiologically acceptable) carrier. Any suitable carrier known in the art can be used within the context of the invention. The choice of carrier will be determined, in part, by the particular site to which the composition may be administered and the particular method used to administer the composition. The composition optionally may be sterile. The compositions can be generated in accordance with conventional techniques described in, e.g., Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, Philadelphia, Pa. (2001).

The composition of this invention desirably comprises the antibody or ADCs in an amount that is effective to treat or prevent cancers. As used herein, the terms “treatment,” “treating,” and the like refer to obtaining a desired pharmacologic and/or physiologic effect. Preferably, the effect is therapeutic, i.e., the effect partially or completely cures a disease and/or adverse symptom attributable to the disease. To this end, the inventive method comprises administering a “therapeutically effective amount” of the antibody or ADC or the composition comprising the antibody or ADC and a pharmaceutically acceptable carrier. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or ADC to elicit a desired response in the individual. For example, a therapeutically effective amount of the ADC of the invention is an amount which binds to a certain antigen on cancer cells and destroys them.

A pharmacologic and/or physiologic effect of treatment may be prophylactic, i.e., the effect completely or partially prevents a disease or symptom thereof. In this respect, the inventive method comprises administering a “prophylactically effective amount” of the ADC or a composition comprising the ADC to a mammal that is predisposed to multiple myeloma. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic result (e.g., prevention of disease onset). Therapeutic or prophylactic efficacy can be monitored by periodic assessment of treated patients. In one embodiment, the ADC described herein inhibits or suppresses proliferation of prostate cancer cells by at least about 10% (e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%). Cell proliferation can be measured using any suitable method known in the art, such as measuring incorporation of labeled nucleosides (e.g., 3H-thymidine or bromodeoxyuridine Brd(U)) into genomic DNA (see, e.g., Madhavan, H. N., J. Stem Cells Regen. Med., 3 (1): 12-14 (2007)).

The invention of the ADCs further provides a method of treating a patient having or at risk of having an immune disorder mediated by immune cells expressing the antigens comprising administering to the patient an effective regime of any of the above described ADCs. Optionally, the disorder is a B cell mediated disorder. Optionally, the immune disorder is rheumatoid arthritis, systemic lupus E (SLE), Type I diabetes, asthma, atopic dermitus, allergic rhinitis, thrombocytopeni purpura, multiple sclerosis, psoriasis, Sjorgren's syndrome, Hashimoto's thyroiditis, Grave's disease, primary biliary cirrhosis, Wegener's granulomatosis, tuberculosis, and graft versus host disease.

In some embodiments, the invention of the ADCs further provides a method of treating a patient having or at risk of having a cancer, an autoimmune disease, an infectious disease, viral disease or a pathogenic infection, through administering to the patient an effective regime of any of the above ADCs, or any of the above described ADCs concurrently with the other therapeutic agents such as the chemotherapeutic agent, the radiation therapy, immunotherapy agents, autoimmune disorder agents, anti-infectious agents or the other conjugates.

According to a further object, the present invention also concerns pharmaceutical compositions comprising the ADCs of the invention together with a pharmaceutically acceptable carrier, diluent, or excipient for treatment of cancers, infections or autoimmune disorders. The method for treatment of cancers, infections and autoimmune disorders can be practiced in vitro, in vivo, or ex vivo. Examples of in vitro uses include treatments of cell cultures in order to kill all cells except for desired variants that do not express the target antigen; or to kill variants that express undesired antigen. Examples of ex vivo uses include treatments of hematopoietic stem cells (HSC) prior to the performance of the transplantation (HSCT) into the same patient in order to kill diseased or malignant cells. For instance, clinical ex vivo treatment to remove tumour cells or lymphoid cells from bone marrow prior to autologous transplantation in cancer treatment or in treatment of autoimmune disease, or to remove T cells and other lymphoid cells from allogeneic bone marrow or tissue prior to transplant in order to prevent graft-versus-host disease, can be carried out as follows. Bone marrow is harvested from the patient or other individual and then incubated in medium containing serum to which is added the conjugate of the invention, concentrations range from about 1 pM to 0.1 mM, for about 30 minutes to about 48 hours at about 37° C. The exact conditions of concentration and time of incubation (=dose) are readily determined by the skilled clinicians. After incubation, the bone marrow cells are washed with medium containing serum and returned to the patient by i.v. infusion according to known methods. In circumstances where the patient receives other treatment such as a course of ablative chemotherapy or total-body irradiation between the time of harvest of the marrow and reinfusion of the treated cells, the treated marrow cells are stored frozen in liquid nitrogen using standard medical equipment.

Examples

The invention is further described in the following examples, which are not intended to limit the scope of the invention. Cell lines described in the following examples were maintained in culture according to the conditions specified by the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany (DMSZ), The Shanghai Cell Culture Institute of Chinese Acadmy of Science, or Nanjing Cobioer Biosciences Co., unless otherwise specified. Cell culture reagents were obtained from Invitrogen Corp., unless otherwise specified. All anhydrous solvents were commercially obtained and stored in Sure-seal bottles under nitrogen. PEG compounds were purchased from Biomatrik Inc, Jiaxing, China. Some chemical compounds, when were not referred synthesis from, were provided by CROs (e.g. Wuxi Apptec, Chemexpress, Raybow Pharma, GL Biochem, Asymchem, and Medicilin (in Hangzhou) in China. Dxd-GGFG payload/linker complex which was used for comparison with the payload/ligand/linker complexes of the present invention was purchased from Chemexpress (Shanghai). Experimental animals were purchased from National Resource Center of Model Mice via GemPharmatech. Co., Ltd, Najing, China and Shanghai SLAC Laboratory Animal Co., Ltd., Shanghai, China. All other reagents and solvents were purchased as the highest grade available and used without further purification. The preparative HPLC separations were performed with Varain PreStar HPLC. HPLC analysis was conducted on Agilent 1260. The mass spectral data were acquired on a Waters Xevo QTOF mass spectrum equipped with Waters Acquity UPLC separations module and Acquity TUV detector. NMR spectra were recorded on Zhongke-niujin WNMR-I 400 MHz instrument at the Department of Chemistry of Zhejiang Sci-Tech University. Chemical shifts (8) are reported in parts per million (ppm) referenced to tetramethylsilane at 0.00 and coupling constants (J) are reported in Hz. The elemental analysis of C, H, and/or N was provided by the Department of Chemistry of Zhejiang Sci-Tech University and conducted on Elementar UNICUBE. Quantitative analysis of metal atoms was performed on Agilent ICPOES 730 ICP-MS.

A solution of compound 1 (6.00 g, 33.500 mmol) and compound 2 (25.60 g, 67.000 mmol) in MeOH (50 mL) was stirred at room temperature for 6 h. The mixture was diluted with DCM (100 mL), washed with 5% HCl, dried over Na2SO4 and concentrated under vacuum to give 3 (18.40 g, 96% yield) as a yellow oil. ESI MS m/z: calcd for C25H42N2O12 [M+H]+: 563.27; found 563.28.

To a solution of compound 3 (18.20 g, 32.300 mmol) and chlorotriisopropylsilane (12.50 g, 64.700 mmol) in DCM (50 mL) was added imidazole (6.60 g, 97.100 mmol). After stirring for 3 h, the mixture was concentrated under vacuum. The residue was purified by flash column chromatography to give 4 (18.40 g, 79% yield) as a yellow oil. ESI MS m/z: calcd for C34H62N2O12Si [M+H]+: 719.41; found 719.50.

A solution of compound 4 (18.40 g, 25.600 mmol) and Pd/C (2.0 g) in ethyl acetate (200 mL) were stirred at room temperature for 20 h under H2 protection. The mixture was filtered and the filtrate was concentrated to give 5 (17.00 g, 96% yield) as a yellow oil. ESI MS m/z: calcd for C34H64N2O10Si [M+H]+: 689.41; found 689.45.

To a solution of compound 5 (17.00 g, 24.700 mmol) and compound 6 (4.70 g, 27.200 mmol) in THF (200 mL) was added EEDQ (8.00 g, 32.100 mmol). After stirred at room temperature for 30 h, the mixture was concentrated under vacuum. The residue was purified by flash column chromatography to give 7 (20.00 g, 96% yield) as a yellow oil. ESI MS m/z: calcd for C41H73N3O13Si [M+H]+: 844.49; found 844.52.

To a solution of compound 7 (9.58 g, 11.600 mmol) and pyrrolidine (5.30 g, 23.200 mmol) in DCM (100 mL) was added Pd(PPh3)4 (1.34 g, 1.200 mmol) under N2 protection. After stirred at room temperature for 1 h, the mixture was concentrated under vacuum. The residue was purified by flash column chromatography to give 8 (8.50 g, 96% yield) as a yellow oil. ESI MS m/z: calcd for C37H69N3O11Si [M+H]+: 760.47; found 760.51.

To a solution of compound 9 (10.00 g, 29.641 mmol) in DMF (100 mL) at room temperature were added benzyl L-valinate (7.22 g, 29.623 mmol), HATU (11.27 g, 29.639 mmol) and DIPEA (7.66 g, 59.265 mmol). After stirred at room temperature for 1 h, the mixture was concentrated under vacuum. The residue was purified by flash column chromatography to give 10 (18.20 g, 100% yield) as a yellow oil. ESI MS m/z: calcd for C29H38N2O7 [M+H]+: 527.27; found 527.27.

To a solution of compound 10 (9.50 g, 18.039 mmol) in isopropyl alcohol (100 mL) at room temperature was added Pd/C (1.20 g, 10 wt %). The resulting mixture was stirred at room temperature under hydrogen atmosphere overnight before it was filtered through a pad of Celite. The filtrate was concentrated under vacuum. Flash column chromatography purification afforded 11 (1.80 g, 33% yield) as a yellow oil. ESI MS m/z: calcd for C14H26N2O5 [M+H]+: 303.18; found 303.18.

To a solution of compound 12 (6.00 g, 6.937 mmol) in DCM (8 mL) at room temperature were added NHS (0.88 g, 7.646 mmol) and EDCI (2.66 g, 13.876 mmol). The resulting mixture was stirred at room temperature for 1 h, washed with water, concentrated under vacuum, and purified by flash column chromatography to afford 13 (3.46 g, 65% yield) as a yellow oil. ESI MS m/z: calcd for C43H71N5O19 [M+H]+: 962.47; found 962.52.

To a solution of compound 14 (2.27 g, 1.975 mmol) in DMF (2 mL) was added HATU (0.83 g, 2.173 mmol). The mixture was stirred at RT for 30 min and then compound 8 (1.50 g, 1.975 mmol) and DIPEA (1.02 g, 7.900 mmol) were added to the reaction. The mixture was stirred at RT for 0.5 h, and then purified by prep-HPLC to afford 15 (0.50 g, 13% yield) as a yellow oil. ESI MS m/z: calcd for C90H160N9O31Si [M+H]+: 1891.1; found 1892.0.

To a solution of compound 15 (350 mg, 0.185 mmol) in DCM (5 mL) was added TFA (105 mg, 0.925 mmol). The mixture was stirred at RT for 1 h, and used for the next step without purification. ESI MS m/z: calcd for C67H112N9O24 [M+H]+: 1735.0; found 1735.7

To a solution of compound 18 (76 mg, 0.035 mmol) in DCM (3 mL) was added TFA (2 mL). The mixture was stirred at RT for 0.5 h, and then concentrated in vacuo to afford 19 (74 mg, crude) as a yellow oil. ESI MS m/z: calcd for C102H151FN12O36 [M+H]+: 2140.04; found 2140.23.

To a solution of compound 19 (1000 mg, 0.047 mmol) in DCM (10 mL) at 0° C. were added PFP—OH (100 mg, 0.056 mmol) and EDCI (18 mg, 0.09 3 mmol). The resulting mixture was stirred at 0° C. for 20 min and warmed to r.t. The mixture was stirred at room temperature for 2 h before it was quenched with H2O. The layers were separated, and the aqueous layer was extracted with DCM. The combined organic layers were dried over Na2SO4 and concentrated in vacuo to afford 20 (108 mg, 100% yield) as a yellow oil. ESI MS m/z: calcd for C108H150F6N12O36 [M+H]+: 2305.02; found 2306.43.

To a solution of compound 21 (750 mg, 1.113 mmol) in DMF (10 mL) at room temperature were added K2CO3 (461 mg, 3.339 mmol), BnBr (209 mg, 1.224 mmol) and KI (92 mg, 0.556 mmol). The mixture was stirred at room temperature for 3 h before it was quenched with H2O. The layers were separated, and the aqueous layer was extracted with EA. The organic phases were concentration, purified by flash column chromatography to afford 22 (720 mg, 85% yield) as a yellow oil. ESI MS m/z: calcd for C42H61N5O6S [M+H]+: 764.43; found 764.04.

To a solution of compound 23 (550 mg, 0.588 mmol) in DCM (5 mL) at room temperature was added 4M HCl/EA (3 mL). The resulting mixture was stirred at room temperature for 3 h before it was concentrated in vacuo to afford 24 (562 mg, 100% yield) as a yellow oil. ESI MS m/z: calcd for C45H66N6O7S [M+H]+: 835.47; found 835.12

To a solution of compound 24 (562 mg, 0.588 mmol) in DCM (10 mL) at 0° C. were added N-Boc-L-Alanine (111 mg, 0.588 mmol), HATU (246 mg, 0.647 mmol) and DIEA (52 mg, 1.176 mmol). The resulting mixture was stirred at 0° C. for 20 min, warmed to RT, stirred for 1 h and quenched with H2O. The layers were separated, and the aqueous layer was extracted with DCM. The organic phase was concentrated and purified by prep-HPLC to afford 25 (562 mg, 100% yield) as a yellow oil. ESI MS m/z: calcd for C53H79N7O10S [M+H]+: 1006.56; found 1006.31

To a solution of compound 25 (220 mg, 0.219 mmol) in MeOH (20 mL) at room temperature was added Pd(OH)2 (40 mg, 10 wt %). The resulting mixture was stirred at room temperature under hydrogen atmosphere for 18 h before it was filtered through a pad of Celite. The filtrate was concentrated in vacuo, purified by prep-HPLC to afford 26 (88 mg, 44% yield) as a white solid. ESI MS m/z: calcd for C46H73N7O10S [M+H]+: 916.51; found: 916.19.

To a solution of compound 26 (80 mg, 0.088 mmol) in DCM (5 mL) at room temperature was added TFA (1 mL). The resulting mixture was stirred at room temperature for 1 h before it was concentrated in vacuo to afford 27 (76 mg, 100% yield) as a white solid. ESI MS m/z: calcd for C41H65N7O8S [M+H]+: 816.46; found 816.07.

To a solution of compound 13 (108 mg, 0.047 mmol) in DMF (3 mL) at 0° C. were added compound 28 (36 mg, 0.044 mmol) and DIEA (16.0 μL, 0.094 mmol). The mixture was stirred at 0° C. for 20 min and warmed to r.t. The mixture was stirred at room temperature for 30 min before it was purified by prep-HPLC to afford 29 (71 mg, 51% yield) as a white solid. ESI MS m/z: calcd for C143H214FN19O43S [M+2H]2+: 1469.24; found 1469.35.

To a solution of compound 21 (227 mg, 0.430 mmol) in DCM (10 mL) at 0° C. were added PFP—OH (118 mg, 0.650 mmol) and EDCI (165 mg, 0.860 mmol). The resulting mixture was stirred at 0° C. for 20 min and warmed to r.t. The mixture was stirred at room temperature for 2 h before it was quenched with H2O. The layers were separated, and the aqueous layer was extracted with CH2Cl2. The combined organic layers were dried over Na2SO4 and concentrated in vacuo to afford 31 (300 mg, 51% yield) as a white solid. ESI MS m/z: calcd for C31H41F5N4O6S [M+H]+: 693.27; found 693.27.

To a solution of compound 32 (155 mg, 0.433 mmol) in CH3CN (10 mL) was added 31 (300 mg, 0.433 mmol). The mixture was stirred at 80° C. for 72 h and then concentrated in vacuo to afford 33 (420 mg, crude) as a white solid. ESI MS m/z: calcd for C46H62F5N6O9S [M]+: 969.40; found 968.91.

To a solution of compound 35 (43 mg, 0.043 mmol) in DCM (4 mL) was added TFA (1 mL). The mixture was stirred for 0.5 hours at RT, concentrated in vacuo to afford 36 (38 mg, crude) as a white solid. ESI MS m/z: calcd for C47H70N7O8S [M]+: 892.51; found 891.92.

To a solution of (tert-butoxycarbonyl)-L-alanine (2.00 g, 10.570 mmol) and PFP—OH (1.95 g, 10.570 mmol) in DCM (20 mL) was added EDCI (2.03 g, 10.570 mmol) at room temperature. The mixture was stirred at room temperature for 1.0 h, washed with brine (10 mL×2), then concentrated under reduced pressure to give 37 (3.50 g, 93% yield) as a white solid. ESI MS m/z: calcd for C14H14F5NO4 [M+H]+: 356.08; found 355.26

To a solution of compound 38 (390 mg, 0.366 mmol) in DCM (6 mL) was added TFA (3 mL). The mixture was stirred at room temperature for 1 h, and then concentrated under reduced pressure. The resulting residue was purified by prep-HPLC to afford 39 (225 mg, 64% yield) as a white solid. ESI MS m/z: calcd for C50H75N8O9 S [M]+: 963.54; found 963.75.

A peptide synthesizer glass vessel (Chemgalss, 100-mL) were charged with 2-chlorotrityl chloride resin (3.30 g, 1.5 g/mmol), 45 (3.08 g, 9.900 mmol) in 50 mL of anhydrous DCM and DIPEA (1.92 g, 14.850 mmol). The reaction was run overnight and the resin was washed with DCM (3×50 mL), followed by DMF (2×50 mL) to give compound 46.

A peptide synthesizer glass vessel (Chemgalss, 100-mL) was charged with compound 46. Fmoc deprotection was performed using 10% piperidine/DMF solution (40 mL). The resin was then washed with DMF (3×50 mL). And then (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine (2.31 g, 7.425 mmol) dissolved in 50 mL of anhydrous DMF, HOBt (1.00 g, 7.425 mmol) and DIC (0.94 g, 7.425 mmol) were added. The reaction was run overnight and the resin was washed with DMF (3×50 mL) to give 47.

A peptide synthesizer glass vessel (Chemgalss, 100-mL) was charged with compound 47. Fmoc deprotection was performed using 10% piperidine/DMF solution (40 mL). The resin was then washed with DMF (3×50 mL). And then compound 42 (3.16 g, 7.425 mmol) dissolved in 50 mL of anhydrous DMF, HOBt (1.00 g, 7.425 mmol), and DIC (0.94 g, 7.425 mmol) were added. The reaction was run overnight and the resin was washed with DMF (3×50 mL) to give compound 48.

A peptide synthesizer glass vessel (Chemgalss, 100-mL) was charged with compound 48. Fmoc deprotection was performed using 10% piperidine/DMF solution (40 mL). The resin was then washed with DMF (3×50 mL). And then compound 12 (5.14 g, 5.940 mmol) dissolved in 50 mL of anhydrous DMF, HOBt (0.80 g, 5.940 mmol), and DIC (0.75 g, 5.940 mmol) were added. The reaction was run overnight and the resin was washed with DMF (3×50 mL) to give compound 49.

A peptide synthesizer glass vessel (Chemgalss, 100-mL) was charged with compound 49. The resin cleavage was performed using 1,1,1,3,3,3-hexafluoropropan-2-ol (15 mL) and DCM (35 mL), followed by a DCM wash (3×50 mL). The deprotection eluent was evaporated under reduced pressure to give 50 (5.90 g, 100% yield) as a white oil. ESI MS m/z: calcd for C54H93N7O22 [M+H]+: 1192.64; found 1192.73.

The reaction mixture was stirred at room temperature for 1.5 h, and then concentrated under reduced pressure to give 51 (3.32 g, 100% yield) as a yellow oil. ESI MS m/z: calcd for C81H118FN11O26 [M+H]+: 1680.82; found 1681.07.

To a solution of compound 51 (3.32 g, 1.975 mmol) in DCM (10 mL) was added TFA (8 mL). The mixture was stirred at room temperature for 1 h, and then concentrated under reduced pressure. The resulting residue was purified by prep-HPLC to give 52 (1.37 g, 42% yield) as a yellow solid. ESI MS m/z: calcd for C77H110FN11O26 [M+H]+: 1624.76; found 1623.78.

To a solution of compound 52 (200 mg, 0.123 mmol) and PFP—OH (27 mg, 0.148 mmol) in DCM (3 mL) was added EDCI (35 mg, 0.185 mmol) at room temperature. The mixture was stirred at room temperature for 1.0 h, washed with brine (10 mL×2), then concentrated under reduced pressure to give 53 (220 mg, 100% yield) as a yellow solid. ESI MS m/z: calcd for C83H109F6N11O26 [M+H]+: 1790.74 found 1789.86.

To a solution of compound 59 (1.40 g, 2.040 mmol) in DCM (20 mL) at room temperature was added diethylamine (10 mL). The reaction mixture was stirred at room temperature for 1 h, concentrated to afford 60 (0.95 g, 100% yield) as a yellow oil. ESI: m/z: calcd for C24H39N3O6 [M+H]+: 466.28; found 466.35.

To a solution of compound 60 (0.95 g, 2.040 mmol) in THF (20 mL) at room temperature were added Fmoc-L-Valine (0.69 g, 2.040 mmol), HATU (1.01 g, 2.650 mmol) and DIEA (0.53 g, 4.080 mmol). The reaction mixture was stirred at room temperature for 0.5 h and diluted with EA (150 mL), washed with 5% Na2CO3 (50 mL), 1 N HCl (50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The resulting residue was purified by silica gel column, eluted with ethyl acetate and petroleum ether, concentrated to afford 61 (1.00 g, 67% yield) as a white solid. ESI: m/z: calcd for C44H58N4O9 [M+H]+: 787.42; found 787.36.

To a solution of compound 61 (0.90 g, 1.140 mmol) in DCM (5 mL) at room temperature was added TFA (5 mL). The reaction mixture was stirred at room temperature for 1.5 h, concentrated to afford 62 (0.72 g, 100% yield) as a yellow oil. ESI: m/z: calcd for C35H42N4O7 [M+H]+: 631.31; found 631.58.

To a solution of compound 63 (1.30 g, 1.140 mmol) in DCM (10 mL) at room temperature was added diethylamine (5 mL). The reaction mixture was stirred at room temperature for 0.5 h, concentrated to afford 64 (0.46 g, 44% yield) as a white solid. ESI: m/z: calcd for C45H72N8O10S [M+H]+: 917.51; found 917.25.

To a solution of compound 67 (1.40 g, 3.550 mmol) in DCM (100 mL) at room temperature were added Fmoc-GABA-OH (1.27 g, 3.900 mmol), HATU (1.75 g, 4.610 mmol) and DIEA (0.92 g, 7.100 mmol). The reaction mixture was stirred at room temperature for 1 h. The mixture was washed with 1 N HCl (50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The resulting residue was purified by silica gel column, eluted with ethyl acetate and petroleum ether, concentrated to afford 68 (1.70 g, 68% yield) as a white solid. ESI: m/z: calcd for C40H51N3O8 [M+H]+: 702.37; found 702.54.

To a solution of compound 68 (1.70 g, 2.410 mmol) in DCM (10 mL) at room temperature was added diethylamine (10 mL). The reaction mixture was stirred at room temperature for 2 h, concentrated to afford 69 (1.16 g, 100% yield) as a yellow oil. ESI: m/z: calcd for C25H41N3O6 [M+H]+: 480.30; found 480.21.

To a solution of compound 69 (1.16 g, 2.410 mmol) in DCM (30 mL) at room temperature were added compound 70 (0.99 g, 2.410 mmol), HATU (1.10 g, 2.900 mmol) and DIEA (0.63 g, 4.820 mmol). The reaction mixture was stirred at room temperature for 0.5 h. The mixture was washed with 1 N HCl (50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The resulting residue was purified by silica gel column, eluted with ethyl acetate and petroleum ether, concentrated to afford 71 (2.11 g, 100% yield) as a white solid. ESI: m/z: calcd for C48H65N5O10 [M+H]+: 872.47; found 872.54.

To a solution of compound 71 (1.00 g, 1.140 mmol) in DCM (5 mL) at room temperature was added TFA (10 mL). The reaction mixture was stirred at room temperature for 1 h, concentrated to afford 72 (0.82 g, 100% yield) as a yellow oil. ESI: m/z: calcd for C39H49N5O8 [M+H]+: 716.36; found 716.26.

To a solution of compound 73 (1.40 g, 1.140 mmol) in DCM (10 mL) at room temperature was added diethylamine (10 mL). The reaction mixture was stirred at room temperature for 0.5 h, concentrated to afford 74 (0.50 g, 43% yield) as a yellow oil. ESI: m/z: calcd for C49H79N9O11S [M+H]+: 1002.56; found 1002.89.

To a solution of compound 77 (2.89 g, 6.900 mmol) in DMF (60 mL) at room temperature were added 2 (1.36 g, 6.900 mmol) and DMTMM (3.07 g, 10.400 mmol). The reaction mixture was stirred at room temperature for 2 days. The reaction mixture was resolved in EA (300 mL), then washed with 1 N HCl (100 mL), brine (100 mL), dried over Na2SO4, filtered and concentrated under reduced pressure, purified by silica gel column, eluted with DCM and methanol, concentrated to afford 78 (2.30 g, 59% yield) as a white solid. ESI: m/z: calcd for C28H35N3O9 [M+H]+: 558.24; found 558.32.

To a solution of compound 78 (2.10 g, 3.800 mmol) in DMF (30 mL) at room temperature were added HATU (2.86 g, 7.500 mmol) and TEA (0.76 g, 7.500 mmol). The reaction mixture was stirred at room temperature for 1 day. The reaction mixture was diluted with EA (300 mL), then washed with 1 N HCl (100 mL), brine (100 mL), dried over Na2SO4, filtered and concentrated under reduced pressure, purified by silica gel column, eluted with DCM and methanol, concentrated to afford 79 (1.40 g, 69% yield) as a white solid. ESI: m/z: calcd for C28H33N3O8 [M+H]+: 540.23; found 540.12.

To a solution of compound 79 (1.40 g, 5.000 mmol) in isopropanol (40 mL) at 70° C. was added 10% Pd/C (0.60 g). The mixture was stirred overnight under hydrogen balloon at 70° C. The mixture was filtered with Celite. The filtrated was concentrated to give 80 (0.80 g, 63% yield) as a brown oil. ESI: m/z: calcd for C12H21N3O4 [M+H]+: 272.15; found 272.11.

To a solution of compound 80 (0.80 g, 2.900 mmol) in DCM (20 mL) at room temperature were added exo-3,6-Epoxy-1,2,3,6-tetrahydrophthalic Anhydride (0.98 g, 5.900 mmol) and TEA (0.60 g, 5.900 mmol). The reaction mixture was stirred at room temperature for 1 h, and then concentrated to give 81 (1.78 g, 100% yield) as a white solid. ESI: m/z: calcd for C28H33N3O12 [M+H]+: 604.21; found 604.58.

To a solution of compound 83 (0.90 g, 2.000 mmol) in CH2Cl2 (10 mL) at room temperature was added TFA (5 mL). The reaction mixture was stirred at room temperature for 1 h, concentrated to afford 84 (0.83 g, 100% yield) as a yellow oil. ESI: m/z: calcd for C16H13N3O8 [M+H]+: 376.07; found 376.57.

To a solution of compound 84 (0.20 g, 0.500 mmol) in DCM (15 mL) at room temperature were added EDCI (0.20 g, 1.100 mmol), PFP (0.11 g, 0.600 mmol). The reaction mixture was stirred at room temperature for 1 h, then washed with brine (20 mL×2), dried over Na2SO4, filtered and concentrated under reduced pressure to afford 85 (0.29 g, 100% yield) as a white solid. ESI: m/z: calcd for C22H12F5N3O8 [M+H]+: 542.05; found 542.15.

To a solution of compound 86 (0.25 g, 0.150 mmol) in DCM (10 mL) at room temperature were added EDCI (57 mg, 0.180 mmol), PFP—OH (57 mg, 0.300 mmol). The reaction mixture was stirred at room temperature for 2 h, then washed with brine (20 mL×2), dried over Na2SO4, filtered and concentrated under reduced pressure to afford 87 (0.27 g, 100% yield) as a yellow solid. ESI: m/z: calcd for C90H112F6N10O25 [M+H]+: 1847.77; found 1847.25.

To a solution of compound 88 (350 mg, 0.140 mmol) in DCM (10 mL) at room temperature was added diethylamine (5 mL). The reaction mixture was stirred at room temperature for 2 h, concentrated, purified by prep-HPLC, lyophilized to afford 89 (140 mg, 44% yield) as a white solid. ESI: m/z: calcd for C114H173FN18O32S [M+H]+: 2358.22; found 2358.58.

To a solution of compound 91 (22.00 g, 45.215 mmol) in DCM (220 mL) were added oxalyl chloride (11.48 g, 90.431 mmol) and DMF (0.2 mL). The mixture was stirred at room temperature for 3 h, and then concentrated under reduced pressure to give 92 (22.83 g, 100% yield) as a yellow oil. ESI MS m/z: calcd for C21H41ClO11 [M+H]+: 505.23 found 504.27.

To a solution of sodium carbonate (11.98 g, 113.020 mmol) and sodium hydroxide (2.71 g, 67.812 mmol) in water (250 mL) were added compound 93 (28.32 g, 76.853 mmol) and compound 92 (22.83 g, 45.208 mmol). The mixture was stirred at room temperature for 3 h, and then concentrated under reduced pressure. The resulting residue was purified by prep-HPLC to give 94 (11.28 g, 29% yield) as a yellow oil. ESI MS m/z: calcd for C42H64N2O15 [M+H]+: 837.52 found 837.80.

A peptide synthesizer glass vessel (Chemgalss, 100-mL) was charged with compound 48. Fmoc deprotection was performed using 10% piperidine/DMF solution (40 mL). The resin was then washed with DMF (3×50 mL). And then compound 95 (2.93 g, 9.000 mmol) dissolved in 50 mL of anhydrous DMF, HOBt (1.22 g, 9.000 mmol), and DIC (1.14 g, 9.000 mmol) were added. The reaction was run overnight and the resin was washed with DMF (3×50 mL) to give compound 96.

A peptide synthesizer glass vessel (Chemgalss, 100-mL) was charged with compound 96. Fmoc deprotection was performed using 10% piperidine/DMF solution (40 mL) The resin was then washed with DMF (3×50 mL). And then compound 94 (4.02 g, 4.800 mmol) dissolved in 50 mL of anhydrous DMF, HOBt (0.65 g, 4.800 mmol), and DIC (0.65 g, 4.800 mmol) were added. The reaction was run overnight and the resin was washed with DMF (3×50 mL) to give compound 97.

A peptide synthesizer glass vessel (Chemgalss, 100-mL) was charged with compound 97. The resin cleavage was performed using 1,1,1,3,3,3-hexafluoropropan-2-ol (15 mL) and DCM (35 mL), followed by a DCM wash (3×50 mL). The deprotection eluent was evaporated under reduced pressure to give 98 (7.25 g, 96% yield) as a white oil. ESI MS m/z: calcd for C61H96N6O21 [M+H]+: 1249.66; found 1249.81.

To a solution of compound 99 (2.74 g, 1.577 mmol) in DCM (15 mL) was added TFA (8 mL). The mixture was stirred at room temperature for 3 h, and then concentrated under reduced pressure. The resulting residue was purified by prep-HPLC to give 100 (1.50 g, 56% yield) as a yellow solid. ESI MS m/z: calcd for C84H113FN10O25 [M+H]+: 1681.79; found 1681.17.

To a solution of compound 100 (1000 mg, 0.595 mmol) and PFP—OH (131 mg, 0.714 mmol) in DCM (10 mL) was added EDCI (171 mg, 0.892 mmol) at room temperature. The mixture was stirred at room temperature for 1.0 h, washed with brine (10 mL×2), then concentrated under reduced pressure. The resulting residue was purified by prep-HPLC to give 101 (880 mg, 80% yield) as a yellow solid. ESI MS m/z: calcd for C90H112F6N10O25 [M+H]+: 1847.77; found 1848.13.

To a solution of compound 103 (570 mg, 0.217 mmol) in DMF (10 mL) was added piperidine (1 mL). The mixture was stirred at room temperature for 0.5 h, and then concentrated under reduced pressure. The resulting residue was purified by prep-HPLC to give 103 (380 mg, 72% yield) as a yellow solid. ESI MS m/z: calcd for C119H176FN8O31 S [M+H]2+: 1202.12; found 1202.22.

A peptide synthesizer glass vessel (Chemgalss, 100-mL) was charged with 2-chlorotrityl chloride resin (4.00 g, 1.5 g/mmol), compound 109 (4.07 g, 12.000 mmol) dissolved in 50 mL of anhydrous DCM and DIPEA (3.10 g, 24.000 mmol). The reaction was run overnight and the resin was washed with DCM (3×50 mL), followed by DMF (2×50 mL) to give compound 110.

A peptide synthesizer glass vessel (Chemgalss, 100-mL) was charged with compound 110. Fmoc deprotection was performed using 10% piperidine/DMF solution (40 mL). The resin was then washed with DMF (3×50 mL) and then compound 111 (3.83 g, 9.000 mmol) dissolved in 50 mL of anhydrous DMF, HOBt (1.22 g, 9.000 mmol), and DIC (1.14 g, 9.000 mmol) were added. The reaction was run overnight and the resin was washed with DMF (3×50 mL) to give compound 112.

A peptide synthesizer glass vessel (Chemgalss, 100-mL) was charged with compound 112. Fmoc deprotection was performed using 10% piperidine/DMF solution (40 mL). The resin was then washed with DMF (3×50 mL). And then compound 95 (2.93 g, 9.000 mmol) dissolved in 50 mL of anhydrous DMF, HOBt (1.22 g, 9.000 mmol), and DIC (1.14 g, 9.000 mmol) were added. The reaction was run overnight and the resin was washed with DMF (3×50 mL) to give compound 113.

A peptide synthesizer glass vessel (Chemgalss, 100-mL) was charged with compound 113. Fmoc deprotection was performed using 10% piperidine/DMF solution (40 mL). The resin was then washed with DMF (3×50 mL). And then compound 94 (5.02 g, 6.000 mmol) was dissolved in 50 mL of anhydrous DMF, and HOBt (0.81 g, 6.000 mmol), DIC (0.76 g, 6.000 mmol) were added. The reaction was run overnight and the resin was washed with DMF (3×50 mL) to give compound 115.

A peptide synthesizer glass vessel (Chemgalss, 100-mL) was charged with compound 115. The resin cleavage was performed using 1,1,1,3,3,3-hexafluoropropan-2-ol (15 mL) and DCM (35 mL), followed by a DCM wash (3×50 mL). The deprotection eluent was evaporated under reduced pressure to give 116 (6.15 g, 84% yield) as a yellow oil. ESI MS m/z: calcd for C60H95N5O20 [M+H]+: 1206.66; found 1205.82.

To a solution of compound 118 (1.60 g, 0.900 mmol), 4-nitrophenyl carbonochloridate (0.73 g, 3.600 mmol) in DCM (20 mL) was added pyridine (0.57 g, 7.200 mmol). The reaction mixture was stirred at room temperature for 1.0 h, concentrated under reduced pressure. The resulting residue was purified by flash column chromatography to give 119 (0.94 g, 53% yield) as a white solid. ESI MS m/z: calcd for C95H145N9O34 [M+H]+: 1956.99; found 1956.23.

To a solution of compound 119 (0.94 g, 0.480 mmol) in DCM (4 mL) was added TFA (2 mL). The mixture was stirred at room temperature for 3 h, and then concentrated under reduced pressure. The resulting residue was purified by prep-HPLC to give 120 (738 mg, 81% yield) as a yellow solid. ESI MS m/z: calcd for C91H137N9O34 [M+H]+: 1900.93; found 1900.34.

To a solution of compound 121 (270 mg, 0.123 mmol) and PFP—OH (270 mg, 0.147 mmol) in DCM (10 mL) was added EDCI (35 mg, 0.184 mmol) at room temperature. The mixture was stirred at room temperature for 1.0 h, washed with brine (10 mL×2), then concentrated under reduced pressure. The resulting residue was purified by prep-HPLC to give 122 (249 mg, 85% yield) as a yellow solid. ESI MS m/z: calcd for C115H153F6N11O25 [M+H]+: 2363.04; found 2363.02.

To a solution of compound 123 (330 mg, 0.106 mmol) in DMF (10 mL) was added piperidine (1 mL). The mixture was stirred at room temperature for 0.5 h, and then concentrated under reduced pressure. The resulting residue was purified by prep-HPLC to give 124 (210 mg, 67% yield) as a yellow solid. ESI MS m/z: calcd for C144H217FN19O41S [M+H]2+: 1459.76; found 1459.28.

To a solution of compound 129 (295 mg, 0.130 mmol) and PFP—OH (28 mg, 0.156 mmol) in DCM (10 mL) was added EDCI (37 mg, 0.195 mmol) at room temperature. The mixture was stirred at room temperature for 1.0 h, washed with brine (10 mL×2), then concentrated under reduced pressure. The resulting residue was purified by prep-HPLC to give 130 (220 mg, 68% yield) as a yellow solid. ESI MS m/z: calcd for C118H158F6N12O36 [M+H]+: 2434.08; found 2434.09.

To a solution of compound 131 (280 mg, 0.087 mmol) in DMF (10 mL) was added piperidine (1 mL). The mixture was stirred at room temperature for 0.5 h, and then concentrated under reduced pressure. The resulting residue was purified by prep-HPLC to give 132 (130 mg, 49% yield) as a yellow solid. ESI MS m/z: calcd for C147H222FN20O42S [M]2+: 1495.28; found 1495.72.

To solution of compound 135 (500 mg, 0.578 mmol) in DCM (5 mL) were added PFP—OH (138.32 mg, 0.075 mmol) and EDCI (166.22 mg, 0.087 mmol), and then stirred at room temperature for an hour. The organic phase was concentrated under reduced pressure. The resulting residue was purified by prep-HPLC to afford 136 (200 mg, 33% yield) as a white solid. ESI MS m/z: calcd for C45H67F5N4O17 [M+H]+: 1031.44; found 1031.25.

To solution of compound 137 (120 mg, 0.077 mmol) in DCM (1.2 mL) were added PFP—OH (15.7 mg, 0.085 mmol) and EDCI (17.83 mg, 0.093 mmol), and then stirred at room temperature for an hour. The organic phase was concentrated under reduced pressure. The resulting residue was purified by prep-HPLC to afford 138 (100 mg, 76% yield) as a yellow solid. ESI MS m/z: calcd for C84H93F6N7O25 [M+H]+: 1715.61; found 1716.15.

To solution of compound 138 (100 mg, 0.058 mmol) and compound 39 (59 mg, 0.061 mmol) in DMF (0.1 mL) was added DIEA (15.08 mg, 0.117 mmol). The reaction solution was stirred at room temperature for 1 h. The organic phase was concentrated under reduced pressure to give a crude product 139 (110 mg, 75% yield) as a yellow oil-like product. ESI MS m/z: calcd for C128H167FN15O33S [M]+2495.16; found 2494.52.

To solution of compound 139 (100 mg, 0.040 mmol) in DMF (0.1 mL) was added piperidine (18.6 mg, 0.220 mmol). The reaction solution was stirred at room temperature for half 1 h. The organic phase was concentrated under reduced pressure. The resulting residue was purified by prep-HPLC to afford 140 (52 mg, 57% yield) as a yellow solid. ESI MS m/z: calcd for C113H157FN15O31S [M]+: 2273.09; found 2272.73.

To solution of compound 140 (50 mg, 0.022 mmol) and compound 136 (24 mg, 0.023 mmol) in DMF (0.5 mL) was added DIEA (5.69 mg, 0.044 mmol). The reaction solution was stirred at room temperature for 1 h. The organic phase was concentrated under reduced pressure. The resulting residue was purified by prep-HPLC to afford 141 (17 mg, 24% yield) as light yellow solid. ESI MS m/z: calcd for C152H223FN19O47 [M+H]2+1560.21; found 1560.41.

To a stirring solution of compound 146 (0.35 g, 0.186 mmol) in CH2Cl2 (5 mL) at room temperature was added TFA (5 mL). The reaction mixture was stirred at room temperature for 1 h, concentrated to afford 147 (0.32 g, 97% yield). ESI MS m/z: [M+H]+ calcd for C83H119FN12O29: 1767.82; found 1767.82.

To a solution of sodium carbonate (16.12 g, 152.078 mmol) and sodium hydroxide (3.55 g, 88.712 mmol) in water (65 mL) were added compound 158 (26.64 g, 95.050 mmol) and compound 92 (32.00 g, 63.366 mmol). The mixture was stirred at room temperature for 3 h, and then concentrated under reduced pressure. The resulting residue was purified by prep-HPLC to give 159 (33.00 g, 69% yield) as a colorless oil. ESI MS m/z: calcd for C35H60N2O15 [M+H]+: 749.40; found 749.00.

To a solution of compound 161 (7.24 g, 8.008 mmol) in DCM (50 mL) was added TFA (25 mL). The mixture was stirred at room temperature for 2 h, and then concentrated under reduced pressure. The resulting residue was purified by prep-HPLC to give 162 (6.50 g, 95% yield) as a colorless oil. ESI MS m/z: calcd for C40H69N3O16 [M+H]+: 848.47; found 848.17.

To a solution of compound 164 (1.10 g, 0.767 mmol) and 4-nitrophenyl carbonochloridate (0.31 g, 1.535 mmol) in DCM (20 mL) was added pyridine (0.24 g, 3.069 mmol). The reaction mixture was stirred at room temperature for 1.0 h, concentrated under reduced pressure. The resulting residue was purified by flash column chromatography to give 166 (817 mg, 66% yield) as a white solid. ESI MS m/z: calcd for C75H119N7O30 [M+H]+: 1598.80; found 1598.33.

A solution of compound 168 (970 mg, 0.512 mmol) and Pd/C (55 mg) in methanol (10 mL) was stirred at room temperature for 2 h under H2 protection. The mixture was filtered and the filtrate was concentrated and purified by prep-HPLC to give 169 (620 mg, 68% yield) as a yellow solid. ESI MS m/z: calcd for C85H130FN9O29 [M+H]+: 1760.90; found 1760.16.

To a solution of compound 175 (3.27 g, 4.672 mmol) in DCM (40 mL) was added TFA (30 mL). The reaction mixture was stirred at room temperature for 1.0 h, concentrated under reduced pressure. The resulting residue was purified by prep-HPLC to afford 6 (2.10 g, 82% yield) as a colorless oil. ESI MS m/z: calcd for C25H42N3O10 [M+H]+: 544.28; found 543.89

To a solution of compound 181 (125 mg, 0.058 mmol) and compound 39 (58 mg, 0.058 mmol) in DMF (2 mL) was added DIEA (7.51 mg, 0.058 mmol). The reaction solution was stirred at room temperature for 1 h and then purified by prep-HPLC to afford 182 (67 mg, 39% yield) as a white solid. ESI MS m/z: calcd for C148H208FN20O40S [M+H]2+: 1478.23 found 1478.7.

A peptide synthesizer glass vessel (Chemgalss, 100-mL) was charged with 2-chlorotrityl chloride resin (3.00 g, 1.5 mmol/g), compound 201 (5.37 g, 9.000 mmol) in 50 mL of anhydrous DCM and DIEA (2.33 g, 18.000 mmol). The reaction was run overnight and the resin was washed with DCM (3×50 mL), followed by DMF (2×50 mL) to give compound 202.

A peptide synthesizer glass vessel (Chemgalss, 100-mL) was charged with compound 202. Fmoc deprotection was performed using 10% piperidine/DMF solution (50 mL). The resin was then washed with DMF (3×50 mL) to give compound 203.

A peptide synthesizer glass vessel (Chemgalss, 100-mL) were charged with compound 203, compound 204 (4.03 g, 6.750 mmol) in 50 mL of anhydrous DMF and HOBt (0.91 g, 6.750 mmol), DIC (0.85 g, 6.750 mmol). The reaction was run overnight and the resin was washed with DMF (3×50 mL) to give compound 205.

A peptide synthesizer glass vessel (Chemgalss, 100-mL) was charged with compound 205. Fmoc deprotection was performed using 10% piperidine/DMF solution (50 mL). The resin was then washed with DMF (3×50 mL) to give compound 206.

A peptide synthesizer glass vessel (Chemgalss, 100-mL) were charged with compound 206, compound 207 (2.10 g, 6.750 mmol) in 50 mL of anhydrous DMF, HOBt (0.91 g, 6.750 mmol) and DIC (0.85 g, 6.750 mmol). The reaction was run overnight and the resin was washed with DMF (3×50 mL) to give compound 208.

A peptide synthesizer glass vessel (Chemgalss, 100-mL) was charged with compound 208. Fmoc deprotection was performed using 10% piperidine/DMF solution (50 mL). The resin was then washed with DMF (3×50 mL) to give compound 209.

A peptide synthesizer glass vessel (Chemgalss, 100-mL) was charged with compound 209.

A peptide synthesizer glass vessel (Chemgalss, 100-mL) was charged with compound 211. Fmoc deprotection was performed using 10% piperidine/DMF solution (50 mL). The resin was then washed with DMF (3×50 mL) to give compound 212.

A peptide synthesizer glass vessel (Chemgalss, 100-mL) were charged with compound 212, compound 213 (2.39 g, 6.750 mmol) dissolved in 50 mL of anhydrous DMF, HOBt (0.91 g, 6.750 mmol), and DIC (0.85 g, 6.750 mmol). The reaction was run overnight and the resin was washed with DMF (3×50 mL) to give compound 214.

A peptide synthesizer glass vessel (Chemgalss, 100-mL) was charged with compound 214. Fmoc deprotection was performed using 10% piperidine/DMF solution (50 mL). The resin was then washed with DMF (3×50 mL) to give compound 215.

A peptide synthesizer glass vessel (Chemgalss, 100-mL) were charged with compound 215, compound 216 (4.12 g, 6.750 mmol) in 50 mL of anhydrous DMF, HOBt (0.91 g, 6.750 mmol), and DIC (0.85 g, 6.750 mmol). The reaction was run overnight and the resin was washed with DMF (3×50 mL) to give compound 217.

A peptide synthesizer glass vessel (Chemgalss, 100-mL) was charged with compound 217. Fmoc deprotection was performed using 10% piperidine/DMF solution (50 mL) The resin was then washed with DMF (3×50 mL) to give compound 218.

A peptide synthesizer glass vessel (Chemgalss, 100-mL) was charged with compound 218. Compound 219 (2.78 g, 6.750 mmol) dissolved in 50 mL of anhydrous DMF and HOBt (0.91 g, 6.750 mmol), DIC (0.85 g, 6.750 mmol) were added. The reaction was run overnight and the resin was washed with DMF (3×50 mL) to give compound 220.

A peptide synthesizer glass vessel (Chemgalss, 100-mL) was charged with compound 220. Fmoc deprotection was performed using 10% piperidine/DMF solution (50 mL). The resin was then washed with DMF (3×50 mL) to give compound 221.

A peptide synthesizer glass vessel (Chemgalss, 100-mL) was charged with compound 221. TFA (20 mL) and DCM (40 mL) and triisopropylsilane (1 mL) were added. The reaction was run for 4 h and the resin was washed with DCM (3×50 mL). The mixture was concentrated under reduced pressure. The resulting residue was purified by prep-HPLC to give compound 222 (500 mg) as a white solid. ESI MS m/z: calcd for C32H54N10O13 [M+H]+: 787.39; found 786.89.

To a solution of compound 223 (800 mg, 0.507 mmol) and compound 202 (204 mg, 1.013 mmol) in DCM (10 mL) was added Pyridine (62 mg, 1.013 mmol) and DIEA (4.45 g, 34.426 mmol). The reaction mixture was stirred at room temperature for 1 h, and then concentrated under reduced pressure. The resulting residue was purified by flash column chromatography to give 224 (703 mg, 79% yield) as a colorless oil. ESI MS m/z: calcd for C81H130N8O33 [M+H]+: 1743.87; found 1743.40.

To a solution of compound 224 (703 mg, 0.403 mmol) in DCM (9 mL) was added TFA (3 mL). The mixture was stirred at room temperature for 1 h, and then concentrated under reduced pressure to give 225 (680 mg, 100% yield) as a colorless oil. ESI MS m/z: calcd for C77H121N7O34 [M+H]+: 1688.80; found 1687.49.

To a solution of compound 227 (150 mg, 0.076 mmol) and NHS (11 mg, 0.091 mmol) in DCM (3 mL) was added EDCI (22 mg, 0.113 mmol). The reaction mixture was stirred at room temperature for 2 h, and then concentrated under reduced pressure to give 228 (160 mg, 100% yield) as a yellow oil. ESI MS m/z: calcd for C99H142FN11O36 [M+H]+: 2080.96; found 2081.13.

To a solution of compound 228 (129 mg, 0.080 mmol) and compound 222 (63 mg, 0.080 mmol) in DMF (3 mL) was added DIEA (21 mg, 0.160 mmol). The reaction mixture was stirred at room temperature for 1 h, and then concentrated under reduced pressure. The resulting residue was purified by prep-HPLC to give 229 (130 mg, 72% yield) as white solid. ESI MS m/z: calcd for C102H171N17O41 [M+2H]2+: 1375.67; found 1375.71.

To a solution of compound 231 (187 mg, 1.043 mmol) in DMF (2 mL) was added compound 232 (200 mg, 0.522 mmol). The mixture was stirred at RT for 24 h and extracted with DCM/MeOH (v/v=19/1) (10 mL×3). The combined organic layers were washed with H2O (10 mL×3), dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluted with DCM/MeOH=19/1) to afford 233 (130 mg, 45% yield) as a light yellow oil. ESI: m/z: calcd for C25H43N2O12 [M+H]+: 563.3; found 563.21.

To a solution of compound 233 (52.4 g, 93.137 mmol) in MeOH (250 mL) was added Pd/C (5.24 g, 10 wt %). The mixture was stirred at RT for 3 h under H2 and then filtered over Celite. The filtrate was concentrated in vacuo to afford 234 (49.61 g, crude) as a yellow oil. ESI: m/z: calcd for C25H45N2O10 [M+H]+: 533.34; found 533.83.

To a solution of compound 234 (49.61 g, 93.142 mmol) and compound 35 (37.7 g, 121.084 mmol) in THF (250 mL) was added EEDQ (29.94 g, 121.084 mmol). The mixture was stirred at RT overnight and then concentrated in vacuo. The residue was purified by silica gel column chromatography (eluted with DCM/MeOH=20/1) to afford 236 (66.8 g, 87% yield in two steps) as a light yellow oil. ESI: m/z: calcd for C43H60N3O13 [M+H]+: 826.41; found 826.88.

To a solution of compound 236 (1.00 g, 1.211 mmol) in DMF (50 mL) was added piperidine (150 mg, 1.816 mmol). The mixture was stirred at RT for 0.5 h, and then concentrated in vacuo to afford 237 (1.05 g, crude) as a white solid. ESI: m/z: calcd for C28H50N3O11 [M+H]+: 604.34; found 605.25.

To a solution of compound 239 (375 mg, 0.405 mmol) in DCM (5 mL) was added compound 240 (246 mg, 0.810 mmol), then added DIEPA (375 mg, 0.405 mmol). The mixture was stirred at RT overnight and used for the next step without work-up. ESI: m/z: calcd for C55H72N5O18 [M+H]+: 1090.58; found 1090.87.

To a solution of previous step (241, 441 mg, 0.405 mmol) in DMF (5 mL) were added compound 242 (200 mg, 0.37 mmol) and HOBT (54 mg, 0.405 mmol). The mixture was stirred at RT for 1 h and concentrated in vacuo. The residue was purified by prep-HPLC (eluted with CH3CN/H2O=65/35) to afford 243 (154 mg, 30% yield in two steps) as a yellow oil. ESI: m/z: calcd for C73H89FN7O19 [M+H]+: 1386.64; found 1387.45.

To a solution of compound 243 (154 mg, 0.111 mmol) in DMF (2 mL) was added piperidine (14 mg, 0.167 mmol). The mixture was stirred at RT for 0.5 h, and then concentrated in vacuo to afford 244 (129 mg, crude) as a white solid. ESI: m/z: calcd for C58H79FN7O17 [M+H]+: 1164.52; found 1164.32.

To a solution of compound 244 (129 mg, 0.111 mmol) and compound 245 (66 mg, 0.144 mmol) in DMF (2 mL) were added HATU (55 mg, 0.144 mmol) and DIPEA (29 mg, 0.222 mmol). The mixture was stirred at RT for 10 min and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluted with DCM/MeOH=20/1) to afford 246 (152 mg, 85% yield in two steps) as a yellow solid. ESI: m/z: calcd for C85H102FN8O22 [M+H]+: 1605.71; found 1605.52.

To a solution of compound 246 (152 mg, 0.095 mmol) in MeOH (5 mL) was added Pd/C (15 mg, 10% Pd). The mixture was stirred at RT under H2 overnight and then filtered with Celite. The filtrate was concentrated in vacuo to afford 247 (138 mg, 96% yield) as a white solid. ESI: m/z: calcd for C78H96FN8O22 [M+H]+: 1515.71; found 1515.32.

To a solution of compound 237 (730 mg, 1.211 mmol) and compound 248 (365 mg, 1.816 mmol) in DMF (10 mL) were added HATU (691 mg, 1.816 mmol) and DIPEA (313 mg, 2.421 mmol). The mixture was stirred at RT for 10 min and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluted with DCM/MeOH=20/1) to afford 249 (851 mg, 90% yield in two steps) as a white solid. ESI: m/z: calcd for C37H63N4O14 [M+H]+: 787.44; found 787.41.

To a solution of compound 250 (152 mg, 0.31 mmol) in THF (5 mL) was added LiHMDS (1 mol/L in THF) (0.388 mL, 0.388 mmol) at −60° C. dropwise. The mixture was warmed to RT for 0.5 h. Compound 251 (246 mg, 0.258 mmol) was added and the reaction mixture was stirred for 0.5 h, quenched with saturated NH4Cl solution, then extracted with DCM (10 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and concentrated in vacuo to afford 252 (336 mg, crude) as a yellow oil. ESI: m/z: calcd for C59H100F2N7O19Si2 [M+H]+: 1304.72; found 1304.62.

To a solution of compound 253 (315 mg, 0.258 mmol) in THF (5 mL) was added TBAF (1 mol/L in THF) (0.516 mL, 0.516 mmol). The mixture was stirred at RT for 0.5 h, and then concentrated in vacuo. The residue was purified by column chromatography on silica (eluted with DCM/MeOH=10/1) to afford 254 (112 mg, 44% yield in three steps) as a yellow foam. ESI: m/z: calcd for C43H68F2N7O17 [M+H]+: 992.50; found 992.30.

To a solution of compound 239 (1.46 g, 1.578 mmol) in DMF (15 mL) was added piperidine (1.5 mL). The mixture was stirred at room temperature for 10 min and concentrated under reduced pressure to give 263 (1.11 g, 100% yield) as a white solid. ESI: m/z: calcd for C33H58N4O12 [M+H]+: 703.41; found 703.26.

To a solution of compound 267 (1.83 g, 1.541 mmol) in DCM (10 mL) was added TFA (10 mL). The mixture was stirred at room temperature for 1 h, and then concentrated under reduced pressure. The resulting residue was purified by prep-HPLC to give 268 (1.69 g, 100% yield) as a white solid. ESI: m/z: calcd for C53H74N6O21 [M+H]+: 1131.49; found 1131.50.

To a solution of compound 272 (98 mg, 0.038 mmol) in DMF (5 mL) was added Pd/C (10 mg, 10 wt %). The mixture was stirred at RT for 6 h under H2 and then filtered over Celite. The filtrate was purified by prep-HPLC (eluted with CH3CN/H2O=40/60) to afford 273 (57 mg, 61% yield) as a white solid. ESI: m/z: calcd for C107H141F7N19O38 [M+H]+: 2432.95; found 2432.17.

To a solution of compound 277 (5.00 g, 22.398 mmol) and compound 276 (3.75 g, 22.398 mmol) and HATU (9.37 g, 24.638 mmol) in DCM (60 mL) was added DIEA (7.24 g, 55.996 mmol). The reaction mixture was stirred at room temperature for 1 h and quenched by H2O (10 mL). The organic phase was separated then washed with saturated Na2CO3, dried over Na2SO4 and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography to afford 278 (1.93 g, 87% yield) as a white solid. ESI: m/z: calcd for C17H25N2O5 [M+H]+: 337.17; found 337.11.

To a solution of compound 278 (6.80 g, 20.215 mmol) in DCM (60 mL) was added TFA (35 mL). The mixture was stirred at room temperature for 16 h, and then concentrated under reduced pressure. The resulting residue was triturated with petroleum ether to afford 279 (5.67 g, 20.215 mmol, 100% yield) as a white gum. ESI: m/z: calcd for C13H17N2O5 [M+H]+: 281.11; found 281.12.

To a solution of compound 279 (2.84 g, 10.133 mmol) in THF (30 mL) were added lead tetraacetate (10.79 g, 10.133 mmol) and pyridine (0.80 g, 10.133 mmol). The mixture was stirred at 70° C. for 1 h before it was filtered through a pad of Celite. The filtrate was concentrated under reduced pressure. The resulting residue was purified by flash column chromatography to afford 280 (1.52 g, 51% yield) as a white solid. ESI: m/z: calcd for C14H18N2O5Na [M+Na]+: 317.11; found 316.82.

To a solution of compound 281 (5.00 g, 9.568 mmol) in DCM (50 mL) was added TFA (10 mL). The mixture was stirred at room temperature for 16 h, and then concentrated under reduced pressure. The resulting residue was washed with MTBE to afford 282 (4.04 g, 100% yield) as a colorless oil. ESI: m/z: calcd for C17H31N2O10 [M+H]+: 423.19; found 423.00.

To a solution of compound 282 (4.04 g, 9.564 mmol) in THF (40 mL) was added 1 N NaOH (50 mL). Then Cbz-Cl (6.52 g, 38.254 mmol) was added. The mixture was stirred at room temperature for 30 min before it was diluted with EA (30 mL). The aqueous phase was separated and adjusted pH to about 3 with 1 N HCl, concentrated and purified by prep-HPLC to afford 283 (1.50 g, 28% yield) as a white solid. ESI: m/z: calcd for C25H37N2O12 [M+H]+: 557.23; found 556.71.

To a solution of compound 284 (1.00 g, 1.882 mmol) and compound 280 (1.11 g, 3.763 mmol) in THF (10.0 mL) was added PPTS (0.19 g, 0.753 mmol). The reaction mixture was stirred at room temperature for 5 h, and then concentrated under reduced pressure. The resulting residue was purified by flash column chromatography to afford 285 (872 mg, 60% yield) as white foam. ESI MS m/z: calcd for C37H38F2N5O11 [M+H]+: 766.25; found 765.61.

To a solution of compound 285 (872 mg, 1.139 mmol) in MeOH (20 mL) at room temperature was added Pd/C (90 mg, 10 wt %). The resulting mixture was stirred at room temperature under a H2 balloon for 1 h before it was filtered through a pad of Celite. The filtrate was concentrated under reduced pressure to afford 286 (413 mg, 99% yield) as a colorless oil. ESI MS m/z: calcd for C13H20F2N5O5 [M+H]+: 364.14; found 363.81.

To a solution of compound 286 (413 mg, 1.137 mmol) and compound 277 (254 mg, 1.137 mmol) and HATU (454 mg, 1.194 mmol) in DMF (5 mL) was added DIEA (220.4 mg, 1.705 mmol). The reaction mixture was stirred at room temperature for 4 h and then concentrated under reduced pressure. The resulting residue was purified by flash column chromatography to afford 287 (445 mg, 68% yield) as light yellow oil. ESI MS m/z: calcd for C24H31F2N6O8 [M+H]+: 569.21; found 568.81.

To a solution of compound 287 (445 mg, 0.783 mmol) in MeOH (10 mL) at room temperature was added Pd/C (45 mg, 10 wt %). The resulting mixture was stirred at room temperature under a H2 balloon for 7 h before it was filtered through a pad of Celite. The filtrate was concentrated under reduced pressure to afford 288 (340 mg, 100% yield) as a colorless oil. ESI MS m/z: calcd for C16H25F2N6O6 [M+H]+: 435.17; found 434.81.

To a solution of compound 288 (128 mg, 0.230 mmol) and compound 283 (340 mg, 0.783 mmol) and HATU (272 mg, 0.714 mmol) in DMF (5 mL) was added DIEA (220.4 mg, 1.705 mmol). The reaction mixture was stirred at room temperature for 10 min and then concentrated under reduced pressure. The resulting residue was purified by flash column chromatography to afford 289 (150 mg, 36% yield) as a white solid. ESI MS m/z: calcd for C73H103F6N2O027 [M+H]+: 1805.71; found 1805.73.

To a solution of compound 289 (90 mg, 0.050 mmol) in MeOH (5 mL) at room temperature was added Pd/C (11 mg, 10 wt %). The resulting mixture was stirred at 50° C. under a H2 balloon for 4 h before it was filtered through a pad of Celite. The filtrate was concentrated under reduced pressure to afford 290 (83 mg, 99% yield) as light yellow oil. ESI MS m/z: calcd for C65H97F6N2O025 [M+H]+: 1671.68; found 1671.21.

To a solution of compound 290 (106 mg, 0.050 mmol) in DMF (3 mL) was added HATU (20 mg, 0.055 mmol) at room temperature. The mixture was stirred at room temperature for 10 min. Then compound 291 (83 mg, 0.050 mmol) and DIEA (10 mg, 0.075 mmol) were added. The reaction mixture was stirred at room temperature for 0.5 h and then quenched by formic acid (0.5 mL), purified by prep-HPLC to afford 292 (51 mg, 27% yield) as a white solid. ESI MS m/z: calcd for C167H248F6N32059 [M+2H]2+: 1880.87 found 1880.87.

To a solution of compound 294 (2.00 g, 3.763 mmol) in DCM (5 mL) were added 1H-tetrazole (0.53 g, 7.526 mmol) and compound 295 (2.09 g, 7.526 mmol). The mixture was stirred at RT for 1 h, and used for the next step without work-up. MS ESI (m/z): calcd for C33H40F2N3O10P [M+H−56]+: 708.24; found 652.61.

To a solution of compound 296 (2.66 g, 3.763 mmol) was added m-CPBA (710 mg, 4.139 mmol) at −50° C. The mixture was stirred at −50° C. for 0.5 h and quenched with saturated Na2SO3 solution and extracted with DCM (30 mL×3). The organic layers were combined and dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluted with DCM/MeOH=20/1) to afford 297 (2.60 g, 95% yield) as a colorless oil. MS ESI (m/z): calcd for C33H40F2N3O11P [M+H]+: 724.23; found 723.61.

To a solution of compound 297 (2.60 g, 3.674 mmol) in MeOH (20 mL) was added Pd/C (260 mg, 10 wt % Pd). The mixture was stirred at RT under H2 for 2 h and filtered over Celite. The filtrate was concentrated to afford 298 (1.61 g, crude) as a white solid. MS ESI (m/z): calcd for C17H28F2N3O7P [M+H]+: 456.16; found 455.81.

To a solution of compound 302 (300 mg, 0.527 mmol) in THF (5 mL) was added LiHMDS (1 mol/L in THF) (0.79 mL, 0.790 mmol) at −60° C. dropwise. The mixture was warmed to RT for 0.5 h and compound 299 (502 mg, 0.527 mmol) was added. After stirring at RT for 0.5 h, the reaction mixture was quenched with saturated NH4Cl solution, then extracted with DCM (20 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and concentrated in vacuo to afford 303 (728 mg, crude) as a yellow oil. MS ESI (m/z): calcd for C61H102F2N7O22PSi [M+H]+: 1382.66; found 1381.94.

To a solution of compound 304 (683 mg, 0.526 mmol) in THF (10 mL) was added TBAF (1 mol/L in THF) (1.315 mL, 1.315 mmol). The mixture was stirred at RT for 0.5 h, and then concentrated in vacuo to afford 305 (300 mg, 48% yield in three steps) as a white solid. MS ESI (m/z): calcd for C51H84F2N7O20P [M+H]+: 1184.55; found 1183.83.

To a solution of compound 305 (35 mg, 0.030 mmol) was added HCl/dioxane (5 mL, 4 mol/L). The mixture was stirred at RT for 0.5 h, and then filtered. The collected cake was washed with DCM and dried to afford 306 (31 mg, crude) as a white solid. MS ESI (m/z): calcd for C43H68F2N7O20P [M+H]+: 1072.42; found 1072.21.

To a solution of compound 309 (243 mg, 0.150 mmol) in DMF (3 mL) was added HATU (63 mg, 0.165 mmol) at room temperature. The mixture was stirred at room temperature for 10 min. Then compound 290 (250.0 mg, 0.150 mmol) and DIEA (29 mg, 0.225 mmol) were added. The reaction mixture was stirred at room temperature for 0.5 h, and then quenched by formic acid (0.5 mL), purified by prep-HPLC to afford 313 (105 mg, 21% yield) as a white solid. ESI MS m/z: calcd for C142H205F7N31O50 [M+2H]2+: 1639.22; found 1639.35.

To a solution of compound 315 (220 mg, 0.139 mmol) in DMF (3.0 mL) was added piperidine (24 mg, 0.277 mmol). The reaction mixture was stirred at room temperature for 0.5 h, and then concentrated under reduced pressure to afford 316 (190 mg, 100% yield) as a yellow oil. ESI MS m/z: calcd for C66H91FN9O21 [M+H]+: 1364.62; found 1364.51.

To a solution of compound 260 (2.00 g, 2.312 mmol) in DCM (20 mL) were added PFP—OH (0.55 g, 3.006 mmol) and EDCI (0.66 g, 3.468 mmol). The mixture was stirred at room temperature for 2 h, and then diluted with DCM (20 mL), washed with brine (5 mL×2). The solution was concentrated under reduced pressure to afford 317 (1.50 g, 63% yield) as a white solid. ESI MS m/z: calcd for C45H68F5N4O17 [M+H]+: 1031.44; found 1031.02.

To a solution of compound 316 (190 mg, 0.139 mmol) in DMF (3 mL) were added compound 317 (287 mg, 0.278 mmol) and DIEA (45 mg, 0.348 mmol). The mixture was stirred at room temperature for 1 h and concentrated under reduced pressure. The resulting residue was purified by prep-HPLC to afford 318 (100 mg, 32% yield) as a white solid. ESI MS m/z: calcd for C105H157FN13O37 [M+H]+: 2211.07; found 2211.68.

To a solution of compound 321 (300 mg, 0.462 mmol) and compound 245 (213 mg, 0.462 mmol) in DMF (4 mL) were added HATU (193 mg, 0.509 mmol) and DIPEA (179 mg, 1.387 mmol). The mixture was stirred at RT for 10 min and then concentrated in vacuo. The residue was purified by silica gel column chromatography (eluted with DCM/MeOH=20/1) to afford 323 (328 mg, 65% yield) as a white solid. MS ESI (m/z): calcd for C60H60FN7O12 [M+H]+: 1090.41; found 1089.72.

To a solution of compound 323 (150 mg, 0.138 mmol) in DMF (5 mL) was added Pd/C (15 mg, 10% Pd). The mixture was stirred at RT overnight under H2 and then filtered over Celite. The filtrate was concentrated to afford 324 (137 mg, crude) as a white solid. MS ESI (m/z): calcd for C53H54FN7O12 [M+H]+: 999.41; found 999.62.

To a solution of compound 324 (100 mg, 0.101 mmol) and compound 310 (111 mg, 0.111 mmol) in DMF (4 mL) were added HATU (46 mg, 0.121 mmol) and DIPEA (26 mg, 0.202 mmol). The mixture was stirred at RT for 10 min and then concentrated in vacuo. The residue was purified by silica gel column chromatography (eluted with DCM/MeOH=7/1) to afford 325 (156 mg, 65% yield) as a yellow solid. MS ESI (m/z): calcd for C96H119F3N14O28 [M+H]+: 1973.82; found 1973.81.

To a solution of compound 325 (156 mg, 0.079 mmol) in DMF (5 mL) was added piperidine (13 mg, 0.158 mmol). The mixture was stirred at RT for 1 h and then concentrated in vacuo. The residue was purified by silica gel column chromatography (DCM/MeOH=3/1) to afford 326 (92 mg, 66% yield) as a yellow solid. MS ESI (m/z): calcd for C81H109F3N14O26 [M+H]+: 1751.81; found 1751.25.

To a solution of compound 329 (600 mg, 1.280 mmol) in DCM (6 mL) were added triphosgene (228 mg, 0.760 mmol) and DMAP (782 mg, 6.400 mmol). After stirring at room temperature for 5 min, compound 330 (1.00 g, 1.280 mmol) in 6 mL DCM was added into the mixture. The reaction solution was stirred at room temperature for 20 min under N2 atmosphere, and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography to afford 331 (1.00 g, 65% yield) as a yellow solid. ESI MS m/z: calcd for C62H81FN6O22 [M+H]+: 1281.35; found 1281.58.

To a solution of compound 331 (1.00 g, 0.780 mmol) in DCM (6 mL) were added Pd(PPh3)4 (45 mg, 0.040 mmol) and tetrahydropyrrole (111 mg, 1.560 mmol). The reaction solution was stirred at room temperature for 1 h under N2 atmosphere and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography to afford 332 (830 mg, 88% yield) as a yellow oil product. ESI MS m/z: calcd for C58H77FN6O20 [M+H]+: 1198.27; found 1198.18.

To a solution of compound 332 (830 mg, 0.690 mmol), compound 133 (312 mg, 0.760 mmol) and HATU (290 mg, 0.760 mmol) in DCM (10 mL) was added DIEA (134 mg, 1.030 mmol). The reaction solution was stirred at room temperature for 1 h and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography to afford 334 (950 mg, 86% yield) as a yellow solid. ESI MS m/z: calcd for C81H98FN7O25 [M+H]+: 1588.66; found 1588.74.

To a solution of compound 334 (200 mg, 0.125 mmol) in DCM (3 mL) were added Pd(PPh3)4 (7 mg, 0.006 mmol) and tetrahydropyrrole (18 mg, 0.250 mmol). The reaction solution was stirred at room temperature for 30 min under N2 atmosphere and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography to afford 335 (140 mg, 71% yield) as a yellow oil product. ESI MS m/z: calcd for C78H94FN7O25 [M+H]+: 1548.63; found 1547.73.

To a solution of compound 335 (140 mg, 0.090 mmol), compound 310 (90 mg, 0.090 mmol) and HATU (38 mg, 0.100 mmol) in DCM (2 mL) was added DIEA (18 mg, 0.140 mmol). The reaction solution was stirred at room temperature for 1 h and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography to afford 336 (200 mg, 86% yield) as a yellow solid. ESI MS m/z: calcd for C121H159F3N14O41 [M+2H]2+: 1261.54; found 1261.48.

To a solution of compound 336 (200 mg, 0.080 mmol) in DMF (2.5 mL) was added piperidine (7 mg, 0.080 mmol). The reaction solution was stirred at room temperature for half an hour, and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography to afford 337 (108 mg, 59% yield) as a yellow solid. ESI MS m/z: calcd for C106H149F3N14O39 [M+2H]2+: 1150.52; found 1150.23.

To a solution of compound 340 (478 mg, 0.502 mmol) and compound 341 (254 mg, 0.502 mmol) in DMF (5 mL) were added HOBt (68 mg, 0.502 mmol) and DIPEA (195 mg, 1.506 mmol). The mixture was stirred at RT for 2 h and concentrated in vacuo. The resulting residue was purified by silica gel column chromatography (eluted with DCM/MeOH=20/1) to afford 342 (660 mg, 99% yield) as a yellow solid. MS ESI (m/z): calcd for C65H87FN8O20 [M+H]+: 1319.61; found 1318.77.

To a solution of compound 342 (660 mg, 0.500 mmol) in DCM (5 mL) were added Pd(PPh3)4 (29 mg, 0.025 mmol) and pyrrolidine (71 mg, 1.000 mmol). The mixture was stirred at RT for 0.5 h, and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluted with DCM/MeOH=10/1) to afford 343 (617 mg, 99% yield) as a yellow solid. MS ESI (m/z): calcd for C61H83FN8O18 [M+H]+: 1235.59; found 1234.81.

To a solution of compound 344 (588 mg, 0.351 mmol) in DMF (5 mL) was added Pd/C (60 mg, 10 wt %). The mixture was stirred at RT overnight under H2 and filtered over Celite. The filtrate was concentrated in vacuo to afford 345 (556 mg, crude) as a yellow solid. MS ESI (m/z): calcd for C81H100FN9O23 [M+H]+: 1586.70; found 1585.46.

To a solution of compound 346 (181 mg, 0.071 mmol) in DMF (5 mL) was added piperidine (9 mg, 0.106 mmol). The mixture was stirred at RT for 1 h and concentrated in vacuo to afford 347 (165 mg, crude) as a yellow solid. MS ESI (m/z): calcd for C109H155F3N16O37 [M+H]+: 2338.08; found 2337.84.

To a solution of compound 350 (101 mg, 0.032 mmol) in DMF (4 mL) was added piperidine (5 mg, 0.063 mmol). The mixture was stirred at RT for 1 h and then concentrated in vacuo to afford 351 (94 mg, crude) as a yellow solid. MS ESI (m/z): calcd for C128H178F7N27O47 [M+2H]2+: 1490.12; found 1489.82.

To a solution of compound 355 (187 mg, 0.110 mmol) in DCM (5 mL) was added Pd (PPh3)4 (6 mg, 0.006 mmol), then pyrrolidine (16 mg, 0.221 mmol). The mixture was stirred at RT for 15 min, and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluted with DCM/MeOH=15/1) to afford 356 (100 mg, 55% yield) as a yellow solid. MS ESI (m/z): calcd for C81H102FN9O25S [M+H]+: 1652.68; found 1652.83.

To a solution of compound 357 (200 mg, 0.060 mmol) in DMF (4 mL) was added piperidine (10 mg, 0.121 mmol). The mixture was stirred at RT for 1 h and then concentrated in vacuo to afford 358 (186 mg, crude) as a yellow solid. MS ESI (m/z): calcd for C131H186F7N29O47S [M+2H]2+: 1542.14; found 1542.13.

To a solution of compound 361 (400 mg, 0.710 mmol) in DCM (20 mL) were added triphosgene (126 mg, 0.426 mmol) and DMAP (347 mg, 2.840 mmol). The mixture was stirred at RT for 20 min and then compound 330 (1098 mg, 1.420 mmol) was added. After stirring at RT for 1 h, the reaction mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (eluted with DCM/MeOH=20/1) to afford 362 (850 mg, 87% yield) as a white solid. MS ESI (m/z): calcd for C68H90FN7O22 [M+H]+: 1376.62; found 1376.96.

To a solution of compound 362 (850 mg, 0.618 mmol) in DCM (5 mL) was added Pd (PPh3)4 (36 mg, 0.031 mmol), followed by pyrrolidine (279 mg, 1.235 mmol). The mixture was stirred at RT for 0.5 h, and then concentrated in vacuo. The residue was purified by silica gel column chromatography (eluted with DCM/MeOH=15/1) to afford 363 (405 mg, 51% yield) as a white solid. MS ESI (m/z): calcd for C64H86FN7O20 [M+H]+: 1292.59; found 1292.47.

To a solution of compound 363 (405 mg, 0.313 mmol) in DMF (5 mL) was added HATU (131 mg, 0.345 mmol). After stirring at RT for 5 min, compound 133 (141 mg, 0.345 mmol) and DIPEA (81 mg, 0.627 mmol) were added. The mixture was stirred at RT for 10 min and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluted with DCM/MeOH=94/6) to afford 364 (530 mg, 100% yield) as a white solid. MS ESI (m/z): calcd for C87H107FN8O25 [M+H]+: 1683.73; found 1685.15.

To a solution of compound 285 (2.70 g, 7.431 mmol) and 202 (1.87 g, 7.431 mmol) and HATU (2.97 g, 7.803 mmol) in DMF (20 mL) was added DIEA (1.44 g, 11.147 mmol). The reaction mixture was stirred at room temperature for 20 min and then concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (DCM:MeOH=85:15) to afford 370 (3.60 g, 81% yield) as a yellow solid. ESI MS m/z: calcd for C26H35F2N6O8 [M+H]+: 597.24; found 597.15.

To a solution of compound 370 (3.60 g, 6.034 mmol) in MeOH (40 mL) at room temperature was added Pd/C (0.36 g, 10 wt %). The resulting mixture was stirred at room temperature under a H2 balloon for 7 h before it was filtered through a pad of Celite. The filtrate was concentrated under reduced pressure to afford 371 (3.00 g, crude) as a yellow solid. ESI MS m/z: calcd for C18H29F2N6O6 [M+H]+: 463.20; found 463.09.

To a solution of compound 283 (95 mg, 0.171 mmol) and compound 371 (300 mg, 0.649 mmol) and HATU (208 mg, 0.546 mmol) in DMF (3 mL) was added DIEA (88 mg, 0.683 mmol). The reaction mixture was stirred at room temperature for 30 min and concentrated under reduced pressure. The resulting residue was purified by prep-HPLC to afford 372 (160 mg, 49% yield) as a white solid. ESI MS m/z: calcd for C79H115F6N20O27 [M+H]+: 1889.81; found 1889.81.

To a solution of compound 372 (160 mg, 0.085 mmol) in MeOH (4 mL) at room temperature were added Pd/C (18 mg, 10 wt %) and NH3/MeOH (0.5 mL). The resulting mixture was stirred at 50° C. under a H2 balloon for 1.5 h before it was filtered through a pad of Celite. The filtrate was concentrated under reduced pressure to afford 373 (128 mg, 86% yield) as a white solid. ESI MS m/z: calcd for C71H109F6N20O25 [M+H]+: 1755.77; found 1756.06.

To a solution of compound 373 (112 mg, 0.053 mmol) in DMF (3 mL) was added HATU (21 mg, 0.055 mmol) at room temperature. The mixture was stirred at room temperature for 10 min and then compound 291 (250 mg, 0.150 mmol) and DIEA (10 mg, 0.079 mmol) were added. The reaction mixture was stirred at room temperature for 0.5 h, and quenched by formic acid (0.5 mL), purified by prep-HPLC to afford 374 (109 mg, 45% yield) as a white solid. ESI MS m/z: calcd for C173H257F7N32O60 [M/2+H]+: 1938.90; found 1939.73.

To a solution of compound 283 (1.75 g, 3.144 mmol), compound 376 (3.54 g, 11.005 mmol) and HATU (3.95 g, 10.376 mmol) in DMF (20 mL) was added DIEA (1.63 g, 12.557 mmol). The reaction mixture was stirred at room temperature for 1 h and then concentrated under reduced pressure. The resulting residue was diluted with DCM (40 mL), and then washed with saturated Na2CO3 (10 mL), concentrated under reduced pressure to afford 377 (4.65 g, 100% yield) as a yellow oil. ESI MS m/z: calcd for C70H124N5O27 [M+H]+: 1466.84; found 1466.97.

To a solution of compound 377 (4.65 g, 3.170 mmol) in DCM (40 mL) at room temperature was added TFA (30 mL). The resulting mixture was stirred at room temperature for 2 h before it was concentrated under reduced pressure. The resulting residue was purified by prep-HPLC to afford 378 (2.49 g, 60% yield) as a colorless oil. ESI MS m/z: calcd for C58H100N5O27 [M+H]+: 1298.65; found 1298.31.

To a solution of compound 379 (1.53 g, 0.582 mmol) in MeOH (20 mL) at room temperature were added Pd/C (0.30 g, 10 wt %) and NH3/MeOH (2.0 mL). The resulting mixture was stirred at 50° C. under a H2 balloon for 1.5 h before it was filtered through a pad of Celite. The filtrate was concentrated under reduced pressure to afford 380 (1.40 g, 96% yield) as a white solid. ESI MS m/z: calcd for C104H171F6N23O40 [M+2H]2+: 1249.10 found 1249.05.

To a solution of compound 291 (112 mg, 0.053 mmol) in DMF (3 mL) was added HATU (21 mg, 0.055 mmol) at room temperature. The mixture was stirred at room temperature for 10 min and then compound 380 (131.0 mg, 0.052 mmol) and DIEA (10 mg, 0.079 mmol) were added. The reaction mixture was stirred at room temperature for 0.5 h, quenched by formic acid (0.5 mL), and purified by prep-HPLC to afford 381 (109 mg, 45% yield) as a white solid. ESI MS m/z: calcd for C206H320F7N35O75 [M+2H]2+: 2309.61 found 2309.55.

To a solution of compound 383 (215 mg, 0.084 mmol) in DMF (3 mL) was added HATU (34 mg, 0.0884 mmol) at room temperature. The mixture was stirred at room temperature for 10 min. Then compound 380 (209 mg, 0.084 mmol) and DIEA (16 mg, 0.126 mmol) were added and the reaction mixture was stirred at room temperature for 0.5 h, quenched by formic acid (0.5 mL), then purified by prep-HPLC to afford 384 (188 mg, 44% yield) as a white solid. ESI MS m/z: calcd for C225H357F7N36O84 [M+4H]4+: 1261.12; found 1261.17.

To a solution of compound 309 (340 mg, 0.209 mmol) in DCM (8 mL) were added PFP-OH (50 mg, 0.272 mmol) and EDCI (64 mg, 0.335 mmol). The mixture was stirred at room temperature for 2.0 h, and then washed with brine (5 mL×2), dried over Na2SO4, concentrated under reduced pressure to afford 389 (370 mg, 100% yield) as a yellow solid. ESI MS m/z: calcd for C83H109F6N11O26 [M+H]+: 1790.74; found 1791.01.

To a solution of compound 391 (173 mg, 0.085 mmol) in DMF (3 mL) was added HATU (34 mg, 0.089 mmol) at room temperature. The mixture was stirred at room temperature for 10 min. And then compound 380 (180 mg, 0.085 mmol) and DIEA (34 mg, 0.089 mmol) were added. The reaction mixture was stirred at room temperature for 0.5 h, quenched by formic acid (0.5 mL), and purified by prep-HPLC to afford 392 (140 mg, 36% yield) as a white solid. ESI MS m/z: calcd for C200H316F7N35O74 [M+3H]3+: 1509.40; found 1509.39.

To a solution of compound 383 (280 mg, 0.109 mmol) in DMF (3 mL) was added HATU (44 mg, 0.114 mmol) at room temperature. The mixture was stirred at room temperature for 10 min. And then compound 371 (51 mg, 0.109 mmol) and DIEA (21 mg, 0.164 mmol) were added. The reaction mixture was stirred at room temperature for 0.5 h, quenched by formic acid (0.5 mL), and purified by prep-HPLC to afford 394 (65 mg, 19% yield) as a white solid. ESI MS m/z: calcd for C139H214F3N19O50 [M+2H]2+: 1504.24; found 1504.21.

To a solution of compound 251 (300 mg, 0.610 mmol) and compound 396 (115 mg, 0.610 mmol) in CH3CN (5 mL) were added TCFH (205 mg, 0.732 mmol) and NMI (100 mg, 1.22 mmol). The mixture was stirred at RT for 30 min and then concentrated in vacuo. The residue was purified by silica gel column chromatography (eluted with DCM/MeOH=50/1) to afford 397 (400 mg, 100% yield) as a white solid. MS ESI (m/z): calcd for C29H52F2N4OSi2 [M+H]+: 663.34; found 662.89.

To a solution of compound 402 (36 mg, 0.021 mmol) in CH3CN (5 mL) was added TMSI (25 mg, 0.126 mmol) dropwise. The mixture was stirred at RT for 1.5 h and then quenched with H2O (1 mg, 0.042 mmol), diluted with EA (20 mL). The mixture was filtered and the cake was washed with EA to afford 403 (33 mg, crude) as a yellow solid. MS ESI (m/z): calcd for C65H93F6N17O25 [M+H]+: 1626.65; found 1626.18.

A solid phase peptide synthesis vessel (Chemgalss, 500-mL) were charged with 2-chlorotrityl chloride resin (8.00 g, 1.5 mmol/g) and DCM (80 mL). The mixture was stirred at RT for 2 h and then filtered. The resin was washed with DCM (80 mL), then a solution of compound 407 (4.07 g, 12.000 mmol) and DIPEA (3.10 g, 24.000 mmol) in anhydrous DCM (80 mL) was added. The reaction mixture was run overnight and the resin was washed with DCM (3×50 mL) to afford 408.

A solid phase peptide synthesis vessel (Chemgalss, 500-mL) was charged with 408. Fmoc deprotection was performed using piperidine (4.08 g, 48 mmol) in DMF (80 mL) for 2 h. The resin was then washed with DMF (3×80 mL), a solution of compound 409 (4.79 g, 12.000 mmol), HOBt (2.43 g, 18.000 mmol) and DIC (2.27 g, 18.000 mmol) in DMF (80 mL) was added. The reaction mixture was run for 2 h and the resin was washed with DMF (3×80 mL) to afford 410.

A solid phase peptide synthesis vessel (Chemgalss, 500-mL) was charged with 410. Fmoc deprotection was performed using piperidine (4.08 g, 48.000 mmol) in DMF (80 mL) for 2 h. The resin was then washed with DMF (3×80 mL), a solution of compound 411 (5.11 g, 12.000 mmol), HOBt (2.43 g, 18.000 mmol) and DIC (2.27 g, 18.000 mmol) in DMF (80 mL) was added. The reaction mixture was run for 2 h and the resin was washed with DMF (3×80 mL) to afford 412.

A solid phase peptide synthesis vessel (Chemgalss, 100-mL) was charged with 412 (6 mmol). Fmoc deprotection was performed using piperidine (2.04 g, 24.000 mmol) in DMF (40 mL) for 2 h. The resin was then washed with DMF (3×40 mL), and a solution of compound 409 (2.55 g, 6.000 mmol), HOBt (1.22 g, 9.000 mmol) and DIC (1.14 g, 9.000 mmol) in DMF (40 mL) was added. The reaction mixture was run for 2 h and the resin was washed with DMF (3×40 mL) to afford 413.

A solid phase peptide synthesis vessel (Chemgalss, 100-mL) was charged with 413. Fmoc deprotection was performed using piperidine (2.04 g, 24.000 mmol) in DMF (40 mL) for 2 h. The resin was then washed with DMF (3×40 mL), and a solution of compound 411 (2.55 g, 6.000 mmol), HOBt (1.22 g, 9.000 mmol) and DIC (1.14 g, 9.000 mmol) in DMF (40 mL) was added. The reaction mixture was run for 2 h and the resin was washed with DMF (3×40 mL) to afford 414.

A solid phase peptide synthesis vessel (Chemgalss, 100-mL) was charged with 414. Fmoc deprotection was performed using piperidine (2.04 g, 24.000 mmol) in DMF (40 mL) for 2 h. The resin was then washed with DMF (3×40 mL), and a solution of compound 409 (2.55 g, 6.000 mmol), HOBt (1.22 g, 9.000 mmol) and DIC (1.14 g, 9.000 mmol) in DMF (40 mL) was added. The reaction mixture was run for 2 h and the resin was washed with DMF (3×40 mL) to afford 415.

A solid phase peptide synthesis vessel (Chemgalss, 100-mL) was charged with 415. Fmoc deprotection was performed using piperidine (2.04 g, 24.000 mmol) in DMF (40 mL) for 2 h. The resin was then washed with DMF (3×40 mL), and a solution of compound 416 (1.1 g, 6.000 mmol), HOBt (1.22 g, 9.000 mmol) and DIC (1.14 g, 9.000 mmol) in DMF (40 mL) was added. The reaction mixture was run for 2 h and the resin was washed with DMF (3×40 mL) to afford 417.

A solid phase peptide synthesis vessel (Chemgalss, 100-mL) were charged with 417, compound 418 (17 mL) and DCM (40 mL). The reaction mixture was run for 0.5 h and the resin was washed with DCM (3×40 mL), and the wash was concentrated in vacuo to afford 419 (3.47 g, 51% yield) as a colorless oil. MS ESI (m/z): calcd for C52H87N7O20 [M+H]+: 1130.61; found 1130.25.

To a solution of compound 421 (4.86 g, 18.601 mmol) and in DCM (50 mL) was added TFA (20 mL). The mixture was stirred at RT for 1 h and concentrated in vacuo to afford 422 (3 g, crude) as a colorless oil. MS ESI (m/z): calcd for C6H1NO4 [M+H]+: 162.08; found 161.88.

To a solution of compound 422 (3.00 g, 18.615 mmol) in THF (30 mL) was added a solution of LiOH (3.12 g, 74.460 mmol) in H2O (74 mL), and then a solution of Cbz-C1 (4.89 g, 28.667 mmol) in THF (30 mL) was added dropwise at 0° C. The mixture was stirred at 0° C. for 30 min and concentrated in vacuo. The residue was acidified with HCl (2 mol/L) to pH=1, and then extracted with DCM (30 mL×2). The aqueous layer was saturated with solid NaCl, and extracted with DCM/MeOH (10/1) (40 mL×4). The combined organic layers were dried over anhydrous Na2SO4 and concentrated in vacuo to afford 423 (2.98 g, 54% yield) as a white solid. MS ESI (m/z): calcd for C14H17NO6 [M+H]+: 296.11; found 295.87.

To a solution of compound 426 (2.02 g, 2.638 mmol) in MeOH (20 mL) were added Pd/C (200 mg, 10 wt %) and NH3/MeOH (0.5 mL, 7 mol/L). The mixture was stirred at RT for 5 h under H2 and then filtered over Celite, and the filtrate was concentrated in vacuo to afford 427 (0.96 g, crude) as a white solid. MS ESI (m/z): calcd for C13H19F2N5O5 [M+H]+: 364.14; found 363.85.

To a solution of compound 429 (1.46 g, 2.447 mmol) in MeOH (15 mL) were added Pd/C (150 mg, 10 wt %) and NH3/MeOH (0.5 mL, 7 mol/L). The mixture was stirred at RT for 4 h under H2 and then filtered over Celite. The filtrate was concentrated in vacuo to afford 430 (1.13 g, crude) as a white solid. MS ESI (m/z): calcd for C18H28F2N6O6 [M+H]+: 463.21; found 462.94.

To a solution of compound 431 (180 mg, 0.852 mmol) in MeOH (10 mL) were added Pd/C (18 mg, 10 wt %) and NH3/MeOH (0.5 mL, 7 mol/L). The mixture was stirred at 50° C. for 2 h under H2 and then filtered over Celite. The filtrate was concentrated in vacuo to afford 432 (159 mg, crude) as a white solid. MS ESI (m/z): calcd for C42H63F4N13O14 [M+H]+: 1050.47; found 1049.82.

To a solution of compound 419 (3.47 g, 3.070 mmol) in DMF (20 mL) was added HATU (1.28 g, 3.377 mmol). The mixture was stirred at RT for 10 min, and compound 433 (1.85 g, 3.070 mmol) and DIPEA (1.19 g, 9.210 mmol) were added to the reaction. The mixture was stirred at RT for 10 min and diluted with water (40 mL). After extraction with DCM (40 mL×3), the combined organic layers were washed with H2O (40 mL×3), dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluted with DCM/MeOH=15/1˜10/1) to afford 434 (2.91 g, 55% yield) as a yellow oil. MS ESI (m/z): calcd for C80H134N10O30 [M+H]+: 1715.94; found 1716.74.

To a solution of compound 434 (2.91 g, 1.696 mmol) and 4-nitrophenyl carbonochloridate (0.68 g, 3.392 mmol) in DCM (20 mL) was added pyridine (0.27 g, 3.392 mmol). The mixture was stirred at RT for 5 min and concentrated in vacuo. The residue was purified by prep-HPLC (eluted with CH3CN/H2O=50/50) to afford 435 (2.32 g, 73% yield) as a yellow solid. MS ESI (m/z): calcd for C87H137N11O34 [M+H]+: 1880.94; found 1880.46.

To a solution of compound 435 (2.32 g, 1.233 mmol) in DCM (30 mL) was added TFA (20 mL). The mixture was stirred at RT for 1 h. The reaction mixture was concentrated in vacuo to afford 436 (2.18 g, crude) as a colorless oil. MS ESI (m/z): calcd for C79H121N11O34 [M+H]+: 1768.82; found 1768.19.

To a solution of compound 437 (180 mg, 0.076 mmol) in DCM (5 mL) was added TFA (2 mL). The mixture was stirred at RT for 1 h and then concentrated in vacuo to afford 438 (171 mg, crude) as a colorless oil. MS ESI (m/z): calcd for C101H163N13O44 [M+H]+: 2263.10; found 2263.10.

To a solution of 294 (3.00 g, 5.645 mmol, 1.0 eq) and (2-(((benzyloxy)carbonyl)amino)-acetamido) methyl acetate (3.16 g, 11.289 mmol, 2.0 eq) in THF (10.0 mL) was added PPTS (0.57 g, 2.258 mmol, 0.4 eq). The reaction mixture was stirred at room temperature for 12 h and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC to afford 442 (1.00 g, 23.6% yield) as a white solid. ESI MS m/z: calcd for C36H35F2N5O11 [M+H]+ 752.23 found 752.67.

To a solution of 442 (1.00 g, 1.330 mmol) in DMF (20 mL) at room temperature was added Pd/C (140.0 mg, 10 wt %). The resulting mixture stirred at room temperature under a H2 balloon for 10 h before it was filtered through a pad of celite. The filtrate was concentrated under reduced pressure to afford 443 (460.0 mg, 100.0% yield) as brown oil. ESI MS m/z: [M+H]+ calcd for C12H17F2N5O5 350.12 found 350.20.

To a solution of 444 (800.0 mg, 1.245 mmol, 1.0 eq) in DMF (10 mL) was added piperidine (171 μL, 1.868 mmol, 1.5 eq). The mixture was stirred at room temperature for 1 h and concentrated under reduced pressure to afford 445 (520 mg, 100.0% yield) as a yellow solid. ESI MS m/z: [M+H]+ calcd for C15H22F2N6O6 421.16 found 420.79.

To a solution of 446 (581.0 mg, 0.783 mmol, 1.0 eq) in DMF (5 mL) was added piperidine (108 μL, 1.175 mmol, 1.5 eq). The mixture was stirred at room temperature for 1 h and concentrated under reduced pressure. The residue was purified by prep-HPLC to afford 447 (162.0 mg, 39.5% yield) as a colorless oil. ESI MS m/z: [M+H]+ calcd for C20H31F2N7O7 520.23 found 519.90.

To a solution of compound 20 (1.00 eq) in DMF (1000% v/w) at 0° C. were added L-valine (1.20 eq) and DIEA (2.00 eq). The mixture was stirred at room temperature for 30 minutes and then purified by prep-HPLC, followed by lyophilization to afford 450. MS ESI (m/z): calcd for C107H160FN13O37 [M+H]+: 2239.10; found 2239.48.

To a solution of compound 450 (1.00 eq) in DCM (1000% v/w) at 0° C. were added PFP-OH (1.20 eq) and EDCI (1.30 eq). The resulting mixture was stirred at 0° C. for 20 min, warmed to RT and stirred for 2 h before it was quenched with H2O. The layers were separated, and the aqueous layer was extracted with CH2Cl2. The combined organic layers were dried over Na2SO4 and concentrated in vacuo to afforded 451. MS ESI (m/z): calcd for C113H159F6N13O37 [M+H]+: 2405.09; found 2405.18.

To a solution of compound 451 (1.00 eq) in DMF (1000% v/w) at 0° C. were added L-valine (1.20 eq) and DIEA (2.00 eq). The mixture was stirred at 0° C. for 20 min, warmed to RT and stirred at room temperature for 30 min before it was purified by prep-HPLC, followed by lyophilization to afforded 452. MS ESI (m/z): calcd for C110H165FN14O38 [M+H]+: 2310.14; found 2310.56.

To a solution of compound 20 (1.00 eq) in DMF (1000% v/w) at 0° C. were added tert-butyl (2-aminoethyl)carbamate (1.20 eq) and DIEA (2.00 eq). The mixture was stirred at room temperature for 30 minutes and then purified by prep-HPLC, followed by lyophilization to afford 454. MS ESI (m/z): calcd for C109H165FN14O37 [M+H]+: 2282.14; found 2282.16.

To a solution of compound 454 (1.00 eq) in DCM (500% v/w) was added TFA (500% vol/w). The mixture was stirred at RT for 1 h, and then concentrated in vacuo to afford 455 (2.18 g, crude) as a colorless oil. MS ESI (m/z): calcd for C104H157FN14O35 [M+H]+: 2182.09; found 2182.56.

To a solution of Boc-L-alanyl-L-valine (1.50 eq) in DMF (1000% v/w) was added HATU (1.50 eq). After stirring for 10 min, compound 455 (1.00 eq) and DIPEA (2.00 eq) were added. The mixture was stirred at RT for 10 min, and then concentrated in vacuo. The residue was purified by prep-HPLC to afford 456. MS ESI (m/z): calcd for C117H179FN16O39 [M+H]+: 2452.25; found 2452.25.

To a solution of compound 456 (1.00 eq) in DCM (500% v/w) was added TFA (500% vol/w). The mixture was stirred at RT for 1 h, and then concentrated in vacuo to afford 457 (2.18 g, crude) as a colorless oil. MS ESI (m/z): calcd for C112H171FN16O37 [M+H]+: 2352.20; found 2352.8.

To a solution of compound 452 (1.00 eq), 2-aminoethan-1-ol (1.50 eq) in DMF (1000% v/w) were added HATU (1.50 eq) and DIPEA (2.00 eq). The mixture was stirred at RT for 1 h, and then concentrated in vacuo. The residue was purified by prep-HPLC to afford 459. MS ESI (m/z): calcd for C112H170FN15O38 [M+H]+: 2353.18; found 2352.94.

A peptide synthesis glass vessel (Chemgalss, 100-mL) were charged with 2-chlorotrityl chloride resin (3.00 g, 1.5 g/mmol), 480 (5.27 g, 9.900 mmol) dissolved in 50 mL of anhydrous DCM and DIEA (3.48 g, 14.850 mmol). The reaction was run overnight and the resin was washed with DCM (3×50 mL), followed by DMF (2×50 mL) to give compound 481.

The resulting resin-supported Fmoc-protected dipeptide was then sequentially deprotected and coupled with the third amino acid and so forth in an iterative fashion to give the desired resin-supported product 482.

A peptide synthesis glass vessel (Chemgalss, 100-mL) was charged with compound 482. Fmoc deprotection was performed using 10% piperidine/DMF solution (1000% v/w). The resin was then washed with DMF (1000% v/w) and then 452 (1.65 eq) dissolved in anhydrous DMF (1000% v/w), HOBt (1.65 eq) and DIC (1.65 eq) were added. The reaction was run for 4 h and the resin was then washed with DMF (3×1000% v/w). LCMS analysis was performed on a peptide aliquot, which was cleaved from the resin (analytical amount was treated with a TFA/TIS (96:4) solution (600% v/w) at room temperature). The peptide-resin was washed with DCM (3×1000% v/w). The wash was concentrated under reduced pressure to give a residue, which was purified by prep-HPLC to give 483, MS ESI (m/z): calcd for C145H222FN27O51S2 [M+4H]4+: 827.89; found 827.89.

To a solution of compound 483 in MeOH (1000% v/w) was added water (1000% v/w). The mixture was stirred at room temperature overnight, and then concentrated under reduced pressure to give a residue, which was purified by prep-HPLC to give 484, MS ESI (m/z): calcd for C145H220FN27O51S2 [M+4H]4+: 810.62; found 810.62.

A peptide synthesis glass vessel (Chemgalss, 100-mL) was charged with Sieber resin (3.00 g, 1.5 g/mmol). The resin was treated with 20% piperidine/DMF solution (2×50 mL) and DMF (3×50 mL). A solution of 480 (5.27 g, 9.900 mmol), HATU (3.80 g, 9.900 mmol) and 4-Methylmorpholine (2.00 g, 9.900 mmol) in DMF (50 mL) was added. The reaction was run overnight and the resin was washed with DMF (2×50 mL) to give compound 485.

The resulting resin-supported Fmoc-protected dipeptide was then sequentially deprotected and coupled with the third amino acid and so forth in an iterative fashion to give the desired resin-supported product 486.

A peptide synthesis glass vessel (Chemgalss, 100-mL) was charged with compound 486. Fmoc deprotection was performed using 10% piperidine/DMF solution (1000% v/w). The resin was then washed with DMF (1000% v/w). 452 (1.65 eq) dissolved in 50 mL of anhydrous DMF and HOBt (1.65 eq), DIC (1.65 eq) were added. The reaction was run for 4 h and the resin was then washed with DMF (3×1000% v/w). LCMS analysis was performed on a peptide aliquot, which was cleaved from the resin (analytical amount was treated with a TFA/TIS (96:4) solution (600% v/w) at room temperature). The peptide-resin was washed with DCM (3×1000% v/w). The wash was concentrated under reduced pressure to give a residue, which was purified by prep-HPLC to give 487. MS ESI (m/z): calcd for C145H220FN27O51S2 [M+4H]4+: 810.88; found 810.88.

To a solution of compound 487 in MeOH (1000% v/w) was added water (1000% v/w). The mixture was stirred at room temperature overnight, and then concentrated under reduced pressure to give a residue, which was purified by prep-HPLC to give 488. MS ESI (m/z): calcd for C145H221FN28O50S2 [M+4H]4+: 810.38; found 810.38.

A peptide synthesis glass vessel (Chemgalss, 100-mL) was charged with compound 482. LCMS analysis was performed on a peptide aliquot, which was cleaved from the resin (analytical amount was treated with a TFA/TIS (96:4) solution (1000% v/w) at room temperature). The peptide-resin was washed with DCM (3×1000% v/w). The wash was concentrated under reduced pressure to give a residue, which was purified by prep-HPLC to give linear peptide. To the linear peptide in MeOH (1000% v/w) was added water (1000% v/w). The mixture was stirred at room temperature overnight and then concentrated under reduced pressure to give a residue, which was purified by prep-HPLC to give 489.

To a solution of 489 in DMF (1000% v/w) at room temperature were added 452 (1.00 eq), HATU (1.00 eq) and DIEA (2.00 eq). After stirring at room temperature for 1 h, the mixture was concentrated under reduced pressure to give a residue. Fmoc deprotection was performed using 10% piperidine/DMF solution (1000% v/w). After stirring at room temperature for 1 h, the mixture was concentrated under reduced pressure to give a residue, which was purified by prep-HPLC to give 490. MS ESI (m/z): calcd for C145H220FN27O51S2 [M+4H]4+: 810.62; found 810.62.

To a solution of compound 490 in DCM (1000% v/w) at room temperature were added pyridine (4.00 eq) and acetyl chloride (2.00 eq) in sequence. The mixture was concentrated under reduced pressure to give a residue, which was purified by prep-HPLC to give 491. MS ESI (m/z): calcd for C147H222FN27O52S2 [M+4H]4+: 821.12; found 821.11.

To a solution of compound 457 (1.00 eq), compound 460 (1.50 eq) in DMF (1000% v/w) were added HATU (1.50 eq) and DIPEA (2.00 eq). The mixture was stirred at RT for 1 h, and then concentrated in vacuo. The residue was purified by prep-HPLC to afford 461.

The resulting resin-supported Fmoc-protected dipeptide was then sequentially deprotected and coupled with the third amino acid and so forth in an iterative fashion to give the desired resin-supported product 462.

A peptide synthesis glass vessel (Chemgalss, 100-mL) was charged with compound 482. Fmoc deprotection was performed using 10% piperidine/DMF solution (1000% v/w). The resin was then washed with DMF (1000% v/w). LCMS analysis was performed on a peptide aliquot, which was cleaved from the resin (analytical amount was treated with a TFA/TIS (96:4) solution (600% v/w) at room temperature). The peptide-resin was washed with DCM (3×1000% v/w). The wash was concentrated under reduced pressure to give a residue, which was purified by prep-HPLC to give 463.

To a solution of compound 463 in MeOH (1000% v/w) was added water (1000% v/w). The mixture was stirred at room temperature overnight, and then concentrated under reduced pressure to give a residue, which was purified by prep-HPLC to give 497. MS ESI (m/z): calcd for C147H226FN29O50S2 [M+2H]2+: 1641.28: found 1641.11.

A peptide synthesis glass vessel (Chemgalss, 100-mL) was charged with compound 482. LCMS analysis was performed on a peptide aliquot, which was cleaved from the resin (analytical amount was treated with 10% 1,1,1,3,3,3-hexafluoropropan-2-ol/DCM (1000% v/w) at room temperature). The peptide-resin was washed with DCM (3×1000% v/w). The wash was concentrated under reduced pressure to give a residue, which was purified by prep-HPLC to give 493.

To a solution of peptide 493 in DMF (1000% v/w) at room temperature were added 457 (1.00 eq), HATU (1.00 eq) and DIEA (2.00 eq). After stirring at room temperature for 1 h, the mixture was concentrated under reduced pressure to give a residue. Fmoc deprotection was performed using 10% piperidine/DMF solution (1000% v/w). After stirring at room temperature for 1 h, the mixture was concentrated under reduced pressure to give a residue. And then MeOH (1000% v/w) and water (1000% v/w) were added. The mixture was stirred at room temperature overnight, and then concentrated under reduced pressure to give a residue, which was purified by prep-HPLC to give 495.

A peptide synthesis glass vessel (Chemgalss, 100-mL) were charged with 2-chlorotrityl chloride resin (3.00 g, 1.5 g/mmol), 560 (6.13 g, 9.900 mmol) dissolved in 50 mL of anhydrous DCM and DIEA (3.48 g, 14.850 mmol). The reaction was run overnight and the resin was washed with DCM (3×50 mL), followed by DMF (2×50 mL) to give compound 561.

The resulting resin-supported Fmoc-protected dipeptide was then sequentially deprotected and coupled with the third amino acid and so forth in an iterative fashion to give the desired resin-supported product 562.

A peptide synthesis glass vessel (Chemgalss, 100-mL) was charged with compound 562. LCMS analysis was performed on a peptide aliquot, which was cleaved from the resin (analytical amount was treated with a TFA/TIS (96:4) solution (600% v/w) at room temperature). The peptide-resin was washed with DCM (3×1000% v/w). The wash was concentrated under reduced pressure to give a residue. And then 459 (2.00 eq) dissolved in acetone (1000% v/w) and K2CO3 (1.65 eq) were added. The reaction was run overnight at 60° C. Fmoc deprotection was performed using 10% piperidine/DMF solution (1000% v/w). The mixture was concentrated under reduced pressure to give a residue, which was purified by prep-HPLC to give 563. MS ESI (m/z): calcd for C176H256FN41O51 [M+4H]4+: 945.72; found 945.72.

A peptide synthesis glass vessel (Chemgalss, 100-mL) was charged with compound 562. Fmoc deprotection was performed using 10% piperidine/DMF solution (1000% v/w). The resin was then washed with DMF (1000% v/w). And then 452 (1.65 eq) dissolved in anhydrous DMF (1000% v/w), HOBt (1.65 eq) and DIC (1.65 eq) were added. The reaction was run for 4 h, and the resin was then washed with DMF (3×1000% v/w). LCMS analysis was performed on a peptide aliquot, which was cleaved from the resin (analytical amount was treated with a TFA/TIS (96:4) solution (600% v/w) at room temperature). The peptide-resin was washed with DCM (3×1000% v/w). The wash was concentrated under reduced pressure to give a residue, which was purified by prep-HPLC to give 564. MS ESI (m/z): calcd for C174H251FN40O51 [M+4H]4+: 934.96; found 934.96.

A peptide synthesis glass vessel (Chemgalss, 100-mL) was charged with compound 562. LCMS analysis was performed on a peptide aliquot, which was cleaved from the resin (analytical amount was treated with 10% 1,1,1,3,3,3-hexafluoropropan-2-ol/DCM (1000% v/w) at room temperature). The peptide-resin was washed with DCM (3×1000% v/w). The wash was concentrated under reduced pressure to give a residue, which was purified by prep-HPLC to give 565. To the linear peptide in DMF (1000% v/w) at room temperature were added 457 (1.00 eq), HATU (1.00 eq) and DIEA (2.00 eq). After stirring at room temperature for 1 h, the mixture was concentrated under reduced pressure to give a residue. Fmoc deprotection was performed using 10% piperidine/DMF solution (1000% v/w). After stirring at room temperature for 1 h, the mixture was concentrated under reduced pressure to give a residue, which was purified by prep-HPLC to give 566. MS ESI (m/z): calcd for C174H252FN41O51 [M+4H]4+: 819.71; found 819.71.

A peptide synthesis glass vessel (Chemgalss, 100-mL) was charged with Sieber resin (3.00 g, 1.5 g/mmol). The resin was treated with 20% piperidine/DMF solution (2×50 mL) and then washed with DMF (3×50 mL). A solution of 567 (6.04 g, 9.900 mmol), HATU (3.80 g, 9.900 mmol) and 4-Methylmorpholine (2.00 g, 9.900 mmol) in DMF (50 mL) was added. The reaction was run overnight and the resin was washed with DMF (2×50 mL) to give compound 568.

The resulting resin-supported Fmoc-protected dipeptide was then sequentially deprotected and coupled with the third amino acid and so forth in an iterative fashion to give the desired resin-supported product 569.

A peptide synthesis glass vessel (Chemgalss, 100-mL) was charged with compound 569. Fmoc deprotection was performed using 10% piperidine/DMF solution (1000% v/w). The resin was then washed with DMF (1000% v/w). And 452 (1.65 eq) dissolved in 50 mL of anhydrous DMF, HOBt (1.65 eq) and DIC (1.65 eq) were added. The reaction was run for 4 h, and the resin was then washed with DMF (3×1000% v/w). LCMS analysis was performed on a peptide aliquot, which was cleaved from the resin (analytical amount was treated with a TFA/TIS (96:4) solution (600% v/w) at room temperature). The peptide-resin was washed with DCM (3×1000% v/w). The wash was concentrated under reduced pressure to give a residue, which was purified by prep-HPLC to give 570. MS ESI (m/z): calcd for C180H295FN5O052 [M+4H]4+: 1003.05; found 1003.05.

A peptide synthesis glass vessel (Chemgalss, 100-mL) were charged with 2-chlorotrityl chloride resin (3.00 g, 1.5 g/mmol), 463 (4.21 g, 9.900 mmol) dissolved in 50 mL of anhydrous DCM and DIEA (3.48 g, 14.850 mmol). The reaction was run overnight and the resin was washed with DCM (3×50 mL), followed by DMF (2×50 mL) to give compound 464.

The resulting resin-supported Fmoc-protected dipeptide was then sequentially deprotected and coupled with the third amino acid and so forth in an iterative fashion to give the desired resin-supported product 465.

Compound 465 was treated with 10% 1,1,1,3,3,3-hexafluoropropan-2-ol/DCM (1000% v/w) at room temperature. The peptide-resin was washed with DCM (3×1000% v/w). And the wash was concentrated under reduced pressure to give a residue. Fmoc deprotection was performed using 10% piperidine/DMF solution (1000% v/w). After stirring at room temperature for 1 h, the mixture was concentrated under reduced pressure to give a residue, which was purified by prep-HPLC to give 498.

To the linear peptide 498 in DMF (10000% v/w) at room temperature were added HATU (1.00 eq) and DIEA (2.00 eq). After stirring at room temperature for 1 h, the mixture was concentrated under reduced pressure to give a residue, to which TFA/TIS (96:4) solution (600% v/w) was added at room temperature. The mixture was stirred for 30 min and concentrated under reduced pressure to give a residue, which was purified by prep-HPLC to give 499. MS ESI (m/z): calcd for C150H232FN27O53 [M+2H]2+: 1641.31; found 1641.05.

To a solution of compound 457 (1.00 eq), 2-chloroacetic acid (1.50 eq) in DMF (1000% v/w) were added HATU (1.50 eq) and DIEA (2.00 eq). The mixture was stirred at RT for 1 h, and then concentrated in vacuo. The residue was purified by prep-HPLC to afford 553.

The resulting resin-supported Fmoc-protected dipeptide was then sequentially deprotected and coupled with the third amino acid and so forth in an iterative fashion to give the desired resin-supported product 554.

A peptide synthesis glass vessel (Chemgalss, 100-mL) was charged with compound 554. LCMS analysis was performed on a peptide aliquot, which was cleaved from the resin (analytical amount was treated with a TFA/TIS (96:4) solution (600% v/w) at room temperature). The peptide-resin was washed with DCM (3×1000% v/w). And the wash was concentrated under reduced pressure to give a residue, which was purified by prep-HPLC to give 602. MS ESI (m/z): calcd for C136H210FN23O46S [M+2H]2+: 1477.23; found 1477.58.

Example 329:501, 508, 516 were prepared following the general synthetic sequence described in SCHEME 5.

Example 331:515, 517, 524, 539 were prepared following the general synthetic sequence described in SCHEME 7.

Example 333:644 were prepared following the general synthetic sequence described in SCHEME 9.

Example 336:576, 577, 578, 579, 580, 581 were prepared following the general synthetic sequence described in SCHEME 12.

Example 337:546, 548, 549, 550 were prepared following the general synthetic sequence described in SCHEME 13.

Example 338:513, 552, 627, 690 were prepared following the general synthetic sequence described in SCHEME 14.

Example 350: Synthesis of A001

To a stirring solution of triphosgene (33.43 g, 112.677 mmol) in CH2Cl2 (300 mL) at −78° C. were added a solution of S2 (100.00 g, 338.066 mmol) in CH2Cl2 (400 mL) and a solution of DIPEA (235.55 mL, 1352.265 mmol) in CH2Cl2 (100 mL) in sequence. The resulting mixture was stirred at −78° C. for 2 h before a mixture solution of S1 (126.06 g, 338.066 mmol) and DIPEA (58.89 mL, 338.066 mmol) in CH2Cl2 (400 mL) were added. The resulting mixture was naturally warmed to room temperature and stirred for 5 h before it was quenched with water (1 L). The layers were separated, and the aqueous layer was extracted with CH2Cl2 (3× 250 mL). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S3 was obtained as a colorless oil (207.86 g, 99%) after flash column chromatography purification (silica gel, hexane:EtOAc=2:1). HRMS (ESI): calcd for C32H52N3O9+ [M+H]+ 622.3698, found 622.3704.

To a stirring solution of S3 (311.80 g, 501.472 mmol) in MeOH (2 L) at room temperature was added Pd/C (31.20 g, 10% wt/wt). The resulting mixture was stirred at room temperature under H2 atmosphere for 12 h before it was filtered through a pad of celite. The filtrate was collected and concentrated in vacuo. The intermediate S4 was obtained as a yellow oil (244.54 g, 100%) and used without further purification. HRMS (ESI): calcd for C24H46N3O7+ [M+H]+ 488.3330, found 488.3331.

To a stirring solution of S4 (244.54 g, 501.472 mmol) and S5 (189.02 g, 752.215 mmol) in CH2Cl2 (2.5 L) at 0° C. was added EDCI (144.20 g, 752.215 mmol). The resulting mixture was warmed to room temperature and stirred for 2 h before it quenched with citric acid (10% wt/wt, aq., 500 mL). The layers were separated, and the aqueous layer was washed with NaCl (sat. aq., 200 mL). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S5 was obtained as a white solid (240.20 g, 66%) by recrystallization (CH2Cl2:hexane=1:1, room temperature). HRMS (ESI): calcd for C37H61N4O10+ [M+H]+ 721.4382, found 721.4385.

To a stirring solution of S5 (80.27 g, 111.347 mmol) in MeOH (800 mL) at room temperature was added Pd/C (8.03 g, 10% wt/wt). The resulting mixture was stirred at room temperature under H2 atmosphere for 3 h before it was filtered through a pad of celite. The filtrate was collected and concentrated in vacuo. The intermediate 5 was obtained as a yellow oil (65.34 g, 100%) and used without further purification. HRMS (ESI): calcd for C29H55N4O8+ [M+H]+ 587.4014, found 587.4022.

To a stirring solution of S8 (100.00 g, 262.854 mmol) in CH2Cl2 (1.6 L) at 0° C. were added HATU (104.94 g, 275.996 mmol), S9 (67.27 g, 275.996 mmol) and DIPEA (84.93 g, 657.134 mmol) in sequence. The resulting mixture was warmed to room temperature and stirred for 4 h before it was quenched with citric acid (10% wt/wt, aq., 500 mL). The layers were separated, and the aqueous layer was washed with NaCl (sat. aq., 200 mL). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S10 was obtained as a white solid (135.00 g, 90%) by recrystallization (CH2Cl2:hexane=1:20, room temperature). HRMS (ESI): calcd for C31H44N3O7+ [M+H]+ 570.3174, found 570.3175.

To a stirring solution of S10 (151.50 g, 265.929 mmol) in CH2Cl2 (1 L) at room temperature was added TFA (330 mL). The resulting mixture was stirred at room temperature for 2 h before it was concentrated in vacuo directly. The intermediate S11 was obtained as a yellow oil (124.88 g, 100%) and used without further purification. HRMS (ESI): calcd for C26H36N3O5+ [M+H]+ 470.2649, found 470.2653.

To a stirring solution of S12 (73.75 g, 265.940 mmol) in THF (1 L) at 0° C. was added HATU (106.18 g, 279.2374 mmol). The resulting mixture was stirred at 0° C. for 20 min before addition of a solution of S11 (124.88 g, 265.929 mmol) in THF (500 mL) and DIPEA (103.12 g, 797.820 mmol) in sequence. The resulting mixture was warmed to room temperature and stirred for further 2 h before it was quenched with citric acid (10% wt/wt, aq., 1 L). The layers were separated, and the aqueous layer was extracted with CH2Cl2 (3× 300 mL). The combined organic layers were washed with NaCl (sat. aq., 500 mL), dried (Na2SO4) and concentrated in vacuo. The intermediate S13 was obtained as a yellow oil (193.84 g, 100%) after flash column chromatography (silica gel, CH2Cl2:hexane=10:1). HRMS (ESI): calcd for C38H57N4O10+ [M+H]+ 729.4069, found 729.4074.

To a stirring solution of S13 (193.84 g, 265.929 mmol) in CH2Cl2 (1 L) at room temperature was added TFA (500 mL). The resulting mixture was stirred at room temperature for 2 h before it was concentrated in vacuo directly. The intermediate S14 was obtained as a yellow oil (167.22 g, 100%) and used without further purification. HRMS (ESI): calcd for C33H49N4O8+ [M+H]+ 629.3545, found 629.3545.

To a stirring solution of S14 (167.22 g, 265.948 mmol) in CH2Cl2 (1.5 L) at 0° C. were added S15 (59.10 g, 265.948 mmol) and EDCI (76.47 g, 398.922 mmol) in sequence. The resulting mixture was warmed to room temperature and stirred for 4 h before it quenched with citric acid (10% wt/wt, aq., 500 mL). The layers were separated, and the aqueous layer was washed with Na2CO3 (5% wt/wt, aq., 200 mL). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S16 was obtained as a yellow oil (221.53, 100%) and used without further purification. HRMS (ESI): calcd for C45H61N4O11+ [M+H]+ 833.4331, found 833.4333.

To a stirring solution of S16 (221.53 g, 265.948 mmol) in CH2Cl2 (600 mL) at 40° C. was added HCO2H (1.2 L). The resulting mixture was stirred at 40° C. for 12 h before it was concentrated in vacuo directly. The residue was dissolved with water (1 L) and extracted with EtOAc (1 L×3). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S17 was obtained as a white solid (162.00 g, 78% over 5 steps) by recrystallization (hexane:EtOAc=1:1, room temperature). HRMS (ESI): calcd for C41H53N4O11+ [M+H]+ 777.3705, found 777.3706.

To a stirring solution of S17 (49.20 g, 63.330 mmol) in CH2Cl2 (500 mL) at room temperature were added HATU (28.90 g, 75.996 mmol) and DIPEA (12.28 g, 94.995 mmol) in sequence. The resulting mixture was stirred at room temperature for 0.5 h before addition of a solution of 7 (37.16 g, 63.330 mmol) in CH2Cl2 (200 mL). The resulting mixture was stirred at room temperature for further 1 h before it was quenched with HCl (1 M aq, 200 mL). The layers were separated and the aqueous layer was extracted with CH2Cl2 (3× 100 mL). The combined organic layers were washed with NaCl (sat. aq., 500 mL), dried (Na2SO4) and concentrated in vacuo. The intermediate S18 was obtained as a yellow oil (75.23 g, 88%) after flash column chromatography purification (silica gel, CH2Cl2:hexane=10:1). HRMS (ESI): calcd for C70H105N8O18+ [M+H]+ 1345.7541, found 1345.7543.

To a stirring solution of S18 (100.00 g, 74.314 mmol) in MeOH (1 L) at room temperature was added Pd/C (10.00 g, 10% wt/wt). The resulting mixture was stirred at room temperature under H2 atmosphere for 12 h before it was filtered through a pad of celite. The filtrate was collected and concentrated in vacuo. The intermediate S19 was obtained as a yellow solid (79.89 g, 96%) after trituration with EtOAc (500 mL). HRMS (ESI): calcd for C55H93N8O16+ [M+H]+ 1121.6704, found 1121.6704.

To a stirring solution of S19 (208.10 g, 185.575 mmol) in DMF (3 L) at room temperature were added S20 (77.77 g, 222.690 mmol) and DIPEA (59.96 g, 463.937 mmol). The resulting mixture was stirred at room temperature for 3 h before it was concentrated in vacuo directly. The intermediate S21 was obtained as a light yellow solid (165.00 g, 69%) after flash column chromatography purification (silica gel, CH2Cl2:hexane=10:1). HRMS (ESI): calcd for C63H100N9O19+ [M+H]+ 1286.7130, found 1286.7136.

To a stirring solution of S22 (22.00 g, 178.629 mmol) and S23 (33.80 g, 178.629 mmol) in THF (200 mL) at room temperature was added EEDQ (48.59 g, 196.492 mmol). The resulting mixture was stirred at room temperature for 15 h before it was concentrated in vacuo directly. The intermediate S24 was obtained as a light yellow solid (39.13 g, 74%) after trituration with hexane/EtOAc (50% V/V). HRMS (ESI): calcd for C15H23N2O4+ [M+H]+ 295.1652, found 295.1654.

To a stirring solution of S24 (20 g, 67.946 mmol) in THF (300 mL) at 0° C. were added PPh3 (35.64 g, 135.893 mmol) and NBS (24.19 g, 135.893 mmol) in sequence. The resulting mixture was stirred at 0° C. for 1 h before it was quenched with water (100 mL). The layers were separated and the aqueous layer was extracted with EtOAc (3×50 mL). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S25 was obtained as a white solid (17.23 g, 72%) after flash column chromatography purification (silica gel, hexane:EtOAc=2:1). HRMS (ESI): calcd for C15H22BrN2O3+ [M+H]+ 357.0808, found 357.0806.

To a stirring solution of S26 (30 g, 163.773 mmol) and S27 (43 g, 163.773 mmol) in toluene (600 mL) at room temperature was added PPTS (2.06 g, 8.189 mmol). The resulting mixture was heated to reflux and stirred overnight before it was cooled to room temperature and filtered. The filter cake was collected and stirred in toluene at reflux for 2 h before it was cooled and filtered. The filter cake was collected and dried in vacuo. The intermediate S28 was obtained as a dark yellow solid (61 g, 91%). HRMS (ESI): calcd for C22H20FN2O5+ [M+H]+ 411.1351, found 411.1355.

To a stirring solution of S28 (4.14 g, 10.088 mmol) and S29 (7.56 g, 60.526 mmol) in acetone (150 mL) at room temperature were added K2CO3 (4.18 g, 30.263 mmol) and KI (1.51 g, 10.088 mmol) in sequence. The resulting mixture was heated to 60° C. and stirred for 20 h before it was cooled to room temperature and concentrated in vacuo directly. The intermediate S30 was obtained as a dark yellow solid (2.6 g, 57%) after flash column chromatography purification (silica gel, DCM:MeOH=10:1). HRMS (ESI): calcd for C24H24FN2O6+ [M+H]+ 455.1613, found 455.1614.

To a stirring solution of S30 (2.00 g, 4.401 mmol) in THF (50 mL) at 0° C. was added NaH (264 mg, 6.601 mmol, 60% dispersion in mineral oil). The resulting mixture was stirred at 0° C. for 0.5 h before addition of S25 (1.57 g, 4.401 mmol). The resulting mixture was stirred at 0° C. for 1 h before it was quenched with NH4Cl (sat. aq., 30 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (3×30 mL). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S31 was prepared after RP-HPLC purification and lyophilization as a yellow solid (2.88 g, 89%). HRMS (ESI): calcd for C39H44FN4O9+ [M+H]+ 731.3087, found 731.3090.

To a stirring solution of S31 (2.00 g, 2.737 mmol) in 1,4-dioxane (20 mL) at 0° C. was added HCl (3.42 mL, 13.684 mmol, 4.0 M in 1,4-dioxane). The resulting mixture was stirred at 0° C. for 1 h before it was concentrated in vacuo directly. The intermediate S32 was obtained as a yellow oil (1.92 g, 105%) and used without further purification. HRMS (ESI): calcd for C34H36FN4O7+ [M+H]+ 631.2563, found 631.2565.

To a stirring solution of S21 (4.22 g, 3.284 mmol) in DMF (50 mL) at room temperature was added HATU (1.25 g, 3.284 mmol). The resulting mixture was stirred at room temperature for 15 min before addition of S32 (1.92 g, 2.737 mmol) and DIPEA (1.06 g, 3.284 mmol) in sequence. The resulting mixture was stirred at room temperature for 30 min before it was concentrated in vacuo directly. The intermediate S33 was obtained as a yellow oil (8.08 g, 284%) and used without further purification. HRMS (ESI): calcd for C97H133FN13O25+ [M+H]+ 1898.9514, found 1898.9514.

To a stirring solution of S32 (8.08 g, 2.737 mmol) in CH2Cl2 (50 mL) at room temperature was added TFA (50 mL). The resulting mixture was stirred at room temperature for 4 h before it was concentrated in vacuo. The product A001 was obtained as a yellow solid (1.02 g, 21% over 3 steps) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C85H109FN13O25+ [M+H]+ 1730.7636, found 1730.7636.

Example 351: Synthesis of A002

To a stirring solution of S23 (7.84 g, 41.420 mmol) in DMF (400 mL) at room temperature were added HATU (15.75 g, 41.420 mmol) and DIPEA (12.17 g, 94.135 mmol) in sequence. The resulting mixture was stirred at room temperature for 30 min before addition of S34 (20.00 g, 37.654 mmol). The resulting mixture was stirred at room temperature for further 1 h before it was quenched with EtOAc (2 L) and filtered. The filter cake was collected and dried in vacuo. The intermediate S35 was obtained as a yellow solid (22.80 g, 99%) and used without further purification. HRMS (ESI): calcd for C32H36FN4O7+ [M+H]+ 607.2563, found 607.2568.

To a stirring solution of S35 (10.00 g, 16.484 mmol) in CH2Cl2 (150 mL) at room temperature was added TFA (75 mL). The resulting mixture was stirred at room temperature for 2 h before it was concentrated in vacuo directly. The intermediate S36 was obtained as a yellow solid (9.22 g, 91%) after trituration with MTBE (1 L). HRMS (ESI): calcd for C27H28FN4O5+ [M+H]+ 507.2038, found 507.2044.

To a stirring solution of S21 (8.18 g, 6.358 mmol) in DMF (150 mL) at room temperature was added HATU (2.54 g, 6.676 mmol). The resulting mixture was stirred at room temperature for 10 min before addition of S36 (4.14 g, 6.676 mmol) and DIPEA (2.05 g, 15.895 mmol) in sequence. The resulting mixture was stirred at room temperature for further 1 h before it was concentrated in vacuo directly. The intermediate S37 was obtained as a dark yellow oil (11.29 g, 100%) and used without further purification. HRMS (ESI): calcd for C90H125FN13O23+ [M+H]+ 1774.8990, found 1774.8990.

To a stirring solution of S37 (11.29 g, 6.360 mmol) in CH2Cl2 (50 mL) at room temperature was added TFA (100 mL). The resulting mixture was stirred at room temperature for 2 h before it was concentrated in vacuo directly. The product A002 was obtained as a yellow solid (5.68 g, 55% over 3 steps) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C78H101FN13O23+ [M+H]+ 1606.7112, found 1606.7113.

Example 352: Synthesis of A003

To a stirring solution of S30 (700 mg, 1.540 mmol) and S23 (292 mg, 1.540 mmol) in CH2Cl2 (10 mL) at were room temperature added DCC (636 mg, 3.081 mmol) and DMAP (188.18 mg, 1.540 mmol) in sequence. The resulting mixture was stirred at room temperature for 1 h before it was concentrated in vacuo directly. The intermediate S38 was obtained as a yellow solid (840 mg, 87%) after flash column chromatography purification (silica gel, CH2Cl2:MeOH=10:1). HRMS (ESI): calcd for C32H37FN3O9+ [M+H]+ 626.2508, found 626.2516.

To a stirring solution of S38 (150 mg, 0.240 mmol) in CH2Cl2 (4 mL) at room temperature was added TFA (2 mL). The resulting mixture was stirred at room temperature for 0.5 h before it was concentrated in vacuo directly. The intermediate S39 was obtained as a yellow oil (153 mg, 100%) and used without further purification. HRMS (ESI): calcd for C27H29FN3O7+ [M+H]+ 526.1984, found 526.1983.

To a stirring solution of S21 (280 mg, 0.218 mmol) in DMF (3 mL) at room temperature was added HATU (87 mg, 0.229 mmol). The resulting mixture was stirred at room temperature for 10 min before addition of S39 (153 mg, 0.240 mmol) and DIPEA (70 mg, 0.545 mmol) in sequence. The resulting mixture was stirred at room temperature for further 1 h before it was concentrated in vacuo directly. The intermediate S40 was obtained as a yellow oil (391 mg, 100%) and used without further purification. HRMS (ESI): calcd for C90H126FN12O25+ [M+H]+ 1793.8936, found 1794.2627.

To a stirring solution of S40 (391 mg, 0.218 mmol) in CH2Cl2 (3 mL) at room temperature was added TFA (3 mL). The resulting mixture was stirred at room temperature for 1 h before it was concentrated in vacuo directly. The product A003 was obtained as a yellow solid (38.0 mg, 10% over 3 steps) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C78H102FN12O25+ [M+H]+ 1625.7058, found 1625.7060.

Example 353: Synthesis of A004

To a stirring solution of S41 (3.00 g, 9.305 mmol) in MeCN (30 mL) at room temperature was added S25 (3.32 g, 9.305 mmol). The resulting mixture was stirred at room temperature for 24 h before it was quenched with henane/EtOAc (1:1) and filtered. The filter cake was collected and dried in vacuo. The intermediate S42 was obtained as a yellow solid (4.20 g, 75%) and used without further purification. HRMS (ESI): calcd for C35H43N4O5+ M+ 599.3228, found 599.3226.

To a stirring solution of S42 (4.10 g, 6.836 mmol) in DMF (30 mL) at room temperature was added piperidine (3 mL). The resulting mixture was stirred at room temperature for 15 min before it was concentrated in vacuo directly. The intermediate S43 was obtained as a white solid (2.58 g, 100%) after trituration with hexane (100 mL). HRMS (ESI): calcd for C20H33N4O3+ M+ 377.2547, found 377.2548.

To a stirring solution of S34 (500 mg, 0.941 mmol) in DMF (10 mL) at 0° C. were added S44 (190 mg, 0.941 mmol) and DIPEA (486 mg, 0.941 mmol) in sequence. The resulting mixture was stirred at 0° C. for 30 min before addition of S43 (355 mg, 0.941 mmol). The resulting mixture was stirred at 0° C. for 1 h before it was quenched with EtOAc (50 mL) and filtered. The filter cake was collected and dried in vacuo. The intermediate S45 was obtained as a yellow solid (790 mg, 100%) without further purification. HRMS (ESI): calcd for C45H53FN7O8+ M+ 838.3934, found 838.3942.

To a stirring solution of S45 (790 mg, 0.941 mmol) in CH2Cl2 (6 mL) at room temperature was added TFA (3 mL). The resulting mixture was stirred at room temperature for 1 h before it was concentrated in vacuo directly. The intermediate S46 was obtained as a yellow oil (787 mg, 100%) and used without further purification. HRMS (ESI): calcd for C40H45FN7O6+ M+ 738.3410, found 738.3416.

To a stirring solution of S21 (1.00 g, 0.784 mmol) in DMF (15 mL) at room temperature was added HATU (358 mg, 0.941 mmol). The resulting mixture was stirred at room temperature for 15 min before addition of S46 (787 mg, 0.941 mmol) and DIPEA (304 mg, 2.353 mmol) in sequence. The resulting mixture was stirred at room temperature for further 1 h before it was concentrated in vacuo directly. The intermediate S47 was obtained as a dark yellow oil (1.57 g, 100%) and used without further purification. HRMS (ESI): calcd for C103H142FN16O24+ [M+H]+ 2006.0361, found 2006.0364.

To a stirring solution of S47 (1.57 g, 0.784 mmol) in CH2Cl2 (20 mL) at room temperature was added TFA (10 mL). The resulting mixture was stirred at room temperature for 1 h before it was concentrated in vacuo directly. The product A004 was obtained as a yellow solid (350 mg, 20% over 4 steps) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C91H118FN16O24+ [M+H]+ 1837.8483, found 1837.8482.

Example 354: Synthesis of A005

To a stirring solution of S48 (2.00 g, 9.847 mmol) and HNMe (OMe)·HCl (1.15 g, 11.817 mmol) in CH2Cl2 (20 mL) at room temperature were added HATU (4.49 g, 11.817 mmol) and DIPEA (3.82 g, 29.541 mmol). The resulting mixture was stirred at room temperature for 1 h before it was quenched with Na2CO3 (5% wt/wt aq., 10 mL). The layers were separated, and the organic layer was washed with HCl (1 M aq., 10 mL) and water (10 mL). The organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S49 was obtained as a yellow solid (2.42 g, 100%) and used without further purification. HRMS (ESI): calcd for C9H9F2N2O4+ [M+H]+ 247.0525, found 247.0528.

To a stirring solution of S49 (2.42 g, 9.847 mmol) and S50 (2.47 g, 14.746 mmol) in MeCN (20 mL) at room temperature was added K2CO3 (4.08 g, 29.492 mmol). The resulting mixture was heated to 80° C. and stirred for 2 h before it was cooled to room temperature and quenched with water (50 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (50 mL×3). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S51 was obtained as a yellow solid (3.87 g, 100%) and used without further purification. HRMS (ESI): calcd for C18H21FN3O6+ [M+H]+ 394.1409, found 394.1414.

To a stirring solution of S51 (3.87 g, 9.847 mmol) in EtOH/H2O (30 mL, 50% V/V) at room temperature were added NH4Cl (5.26 g, 98.381 mmol) and Fe powder (3.85 g, 68.866 mmol). The resulting mixture was heated to 80° C. and stirred for 2 h before it was cooled to room temperature and filtered through a pad of celite. The filtrate was collected and concentrated in vacuo. The intermediate S52 was obtained as a yellow solid (3.58 g, 100%) and used without further purification. HRMS (ESI): calcd for C18H23FN3O4+ [M+H]+ 364.1667, found 364.1672.

To a stirring solution of S52 (3.58 g, 9.847 mmol) in 1,4-dioxane/H2O (100 mL, 70% V/V) at 0° C. were added Na2CO3 (2.09 g, 19.694 mmol) and FmocCl (6.37 g, 24.618 mmol). The resulting mixture was warmed to room temperature and stirred for 12 h before it was concentrated in vacuo. The residual aqueous layer was extracted with EtOAc (50 mL×3). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S53 was obtained as a white solid (5.00 g, 87% over 4 steps) after flash column chromatography purification (silica gel, hexane:EtOAc=3:1). HRMS (ESI): calcd for C33H33FN3O6+ [M+H]+ 586.2348, found 586.2351.

To a stirring solution of S53 (53.00 g, 65.604 mmol) in THF (500 mL) at 0° C. was added EtMgBr (154.4 mL, 3.4 mol/L in 2-Me-THF, 524.831 mmol). The resulting mixture was warmed to room temperature and stirred for 6 h before it was quenched with NH4Cl (sat. aq., 800 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (500 mL×3). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S54 was obtained as a yellow oil (50.19 g, 100%) and used without further purification. HRMS (ESI): calcd for C33H32FN2O5+ [M+H]+ 555.2290, found 555.2298.

To a stirring solution of S54 (50.19 g, 65.604 mmol) in DMF (500 mL) at room temperature was added piperidine (50 mL). The resulting mixture was stirred at room temperature for 30 min before it was concentrated in vacuo directly. The intermediate S55 was obtained as a yellow solid (21.48 g, 71% over 2 steps) flash column chromatography (silica gel, hexane:EtOAc=8:1). HRMS (ESI): calcd for C18H22FN2O3+ [M+H]+ 333.1609, found 333.1618.

To a stirring solution of S55 (25.30 g, 76.118 mmol) and S27 (20.04 g, 76.118 mmol) in toluene (500 mL) at room temperature was added PPTS (1.91 g, 7.612 mmol). The resulting mixture was heated to reflux and stirred overnight before it was cooled to room temperature and filtered. The filter cake was collected and stirred in refluxing toluene for 2 h before it was cooled and filtered. The filter cake was collected and dried in vacuo. The intermediate S56 was obtained as a dark yellow solid (35.54 g, 88%). HRMS (ESI): calcd for C31H31FN3O6+ [M+H]+ 560.2191, found 560.2200.

To a stirring solution of S56 (21 g, 37.527 mmol) in CH2Cl2 (40 mL) at room temperature was added TFA (20 mL). The resulting mixture was stirred at room temperature for 1 h before it was concentrated in vacuo directly. The intermediate S57 was obtained as a yellow solid (10.20 g, 66%) after flash column chromatography purification (silica gel, CH2Cl2:hexane=20:1). HRMS (ESI): calcd for C22H21FN3O4+ [M+H]+ 410.1511, found 410.1512.

To a stirring solution of S58 (1.00 g, 4.061 mmol) in DMF (20 mL) at room temperature were added HATU (1.70 g, 4.467 mmol) and DIPEA (1.57 g, 12.183 mmol) in sequence. The resulting mixture was stirred at room temperature for 15 min before addition of 57 (1.83 g, 4.467 mmol). The resulting mixture was stirred at room temperature for further 1 h before it was concentrated in vacuo directly. The intermediate S59 was obtained as a dark yellow oil (1.31 g, 92%) after flash column chromatography purification (silica gel, CH2Cl2:hexane=20:1). HRMS (ESI): calcd for C32H37FN5O8+ [M+H]+ 638.2621, found 638.2626.

To a stirring solution of S59 (1.31 g, 2.054 mmol) in CH2Cl2 (10 mL) at room temperature was added TFA (5 mL). The resulting mixture was stirred at room temperature for 1 h before it was concentrated in vacuo directly. The residue was dispersed with MTBE (50 mL) and stirred for 30 min before it was filtered. The filter cake was collected and dried in vacuo. The intermediate S60 was obtained as a yellow solid (1.31 g, 100%) and used without further purification. HRMS (ESI): calcd for C27H29FN5O6+ [M+H]+ 538.2096, found 538.2100.

To a stirring solution of S21 (2.64 g, 2.054 mmol) in DMF (50 mL) at room temperature was added HATU (781 mg, 2.054 mmol). The resulting mixture was stirred at room temperature for 15 min before addition of S60 (1.31 g, 2.054 mmol) and DIPEA (797 mg, 6.162 mmol) in sequence. The resulting mixture was stirred at room temperature for further 1 h before it was concentrated in vacuo directly. The intermediate S61 was obtained as a dark yellow oil (3.71 g, 100%) and used without further purification. HRMS (ESI): calcd for C90H126FN14O24+ [M+H]+ 1805.9048, found 1805.9057.

To a stirring solution of S61 (3.71 g, 2.054 mmol) in CH2Cl2 (50 mL) at room temperature was added TFA (25 mL). The resulting mixture was stirred at room temperature for 1 h before it was concentrated in vacuo directly. The product A005 was obtained as a yellow solid (1.51 g, 45% over 3 steps) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C78H102FN14O24+ [M+H]+ 1637.7170, found 1637.7172.

Example 355: Synthesis of A006

To a stirring solution of S62 (300 mg, 2.605 mmol) in MeCN (10 mL) at room temperature was added S25 (930 mg, 2.605 mmol). The resulting mixture was stirred at room temperature for 12 h before it was concentrated in vacuo directly. The residue was dispersed with MTBE (50 mL) and stirred for 30 min before it was filtered. The filter cake was collected and dried in vacuo. The intermediate S63 was obtained as a white solid (1.19 g, 97%) and used without further purification. HRMS (ESI): calcd for C21H34N3O4+ M+ 392.2544, found 392.2547.

To a stirring solution of S63 (1.19 g, 2.514 mmol) in MeCN (30 mL) at 0° C. were added S44 (608 mg, 3.016 mmol) and DIPEA (975 mg, 7.541 mmol) in sequence. The resulting mixture was stirred at 0° C. for 2 h before it was concentrated in vacuo directly. The residue was dispersed with MTBE (50 mL) and stirred for 30 min before it was filtered. The filter cake was collected and dried in vacuo The intermediate S64 was obtained as a white solid (1.41 g, 90%) and used without further purification. HRMS (ESI): calcd for C28H37N4O7+ M+ 541.2657, found 541.2660.

To a stirring solution of S64 (1.41 g, 2.265 mmol) in DMF (30 mL) at room temperature were added S34 (1.20 g, 2.265 mmol) and DIPEA (879 mg, 6.795 mmol) in sequence. The resulting mixture was stirred at room temperature for 2 h before it was concentrated in vacuo directly. The residue was dispersed with MTBE (50 mL) and stirred for 30 min before it was filtered. The intermediate S65 was obtained as a yellow solid (2.11 g, 100%) and used without further purification. HRMS (ESI): calcd for C46H54FN6O9+ M+ 853.3931, found 853.3935.

To a stirring solution of S65 (2.11 g, 2.259 mmol) in CH2Cl2 (50 mL) at room temperature was added TFA (25 mL). The resulting mixture was stirred at room temperature for 1 h before it was concentrated in vacuo directly. The residue was dispersed with MTBE (50 mL) and stirred for 30 min before it was filtered. The filter cake was collected and dried in vacuo The intermediate S66 was obtained as a yellow solid (2.10 g, 99%) and used without further purification. HRMS (ESI): calcd for C41H46FN6O7+ M+ 753.3407, found 753.3407.

To a stirring solution of S21 (2.90 g, 2.256 mmol) in DMF (50 mL) at room temperature was added HATU (858 mg, 2.256 mmol). The resulting mixture was stirred at room temperature for 15 min before addition of S66 (2.10 g, 2.256 mmol) and DIPEA (875 mg, 6.768 mmol) in sequence. The resulting mixture was stirred at room temperature for further 1 h before it was concentrated in vacuo directly. The intermediate S67 was obtained as a dark yellow oil (4.74 g, 100%) and used without further purification. HRMS (ESI): calcd for C104H143FN15O25+ M+ 2021.0358, found 2021.0360.

To a stirring solution of S67 (4.74 g, 2.256 mmol) in CH2Cl2 (50 mL) at room temperature was added TFA (25 mL). The resulting mixture was stirred at room temperature for 1 h before it was concentrated in vacuo directly. The product A006 was obtained as a yellow solid (1.95 g, 46% over 2 steps) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C92H119FN15O25+ [M+H] 1852.8480, found 1852.8484.

Example 356: Synthesis of A007

To a stirring solution of S68 (300 mg, 2.908 mmol) in MeCN (10 mL) at room temperature was added S25 (1.04 g, 2.908 mmol). The resulting mixture was stirred at room temperature for 12 h before it was concentrated in vacuo directly. The residue was dispersed with MTBE (50 mL) and stirred for 30 min before it was filtered. The filtrate was collected and concentrated in vacuo. The intermediate S69 was obtained as a white solid (1.33 g, 99%) and used without further purification. HRMS (ESI): calcd for C20H34N3O4+ M+ 380.2544, found 380.2549.

To a stirring solution of S69 (1.33 g, 2.889 mmol) in MeCN (30 mL) at 0° C. were added S44 (699 mg, 3.466 mmol) and DIPEA (1.17 g, 8.666 mmol) in sequence. The resulting mixture was stirred at 0° C. for 2 h before it was concentrated in vacuo directly. The residue was dispersed with MTBE (50 mL) and stirred for 30 min before it was filtered. The filter cake was collected and dried in vacuo The intermediate S70 was obtained as a white solid (1.68 g, 95%) and used without further purification. HRMS (ESI): calcd for C27H37N4O7+ M+ 529.2657, found 529.2658.

To a stirring solution of S70 (1.68 g, 2.756 mmol) in DMF (30 mL) at room temperature were added S34 (1.46 g, 2.756 mmol) and DIPEA (1.07 g, 8.269 mmol) in sequence. The resulting mixture was stirred at room temperature for 2 h before it was concentrated in vacuo directly. The residue was dispersed with MTBE (50 mL) and stirred for 30 min before it was filtered. The intermediate S71 was obtained as a yellow solid (2.20 g, 88%) and used without further purification. HRMS (ESI): calcd for C44H52FN6O9+ M+ 827.3774, found 827.3778.

To a stirring solution of S71 (2.20 g, 2.423 mmol) in CH2Cl2 (40 mL) at room temperature was added TFA (20 mL). The resulting mixture was stirred at room temperature for 1 h before it was concentrated in vacuo directly. The residue was dispersed with MTBE (50 mL) and stirred for 30 min before it was filtered. The filter cake was collected and dried in vacuo The intermediate S72 was obtained as a yellow solid (2.19 g, 99%) and used without further purification. HRMS (ESI): calcd for C39H45BrFN6O7+ M+ 807.2512, found 807.2514.

To a stirring solution of S21 (3.11 g, 2.418 mmol) in DMF (50 mL) at room temperature was added HATU (920 mg, 2.418 mmol). The resulting mixture was stirred at room temperature for 15 min before addition of S72 (2.19 g, 2.418 mmol) and DIPEA (938 mg, 7.254 mmol) in sequence. The resulting mixture was stirred at room temperature for further 1 h before it was concentrated in vacuo directly. The intermediate S73 was obtained as a dark yellow oil (5.02 g, 100%) and used without further purification. HRMS (ESI): calcd for C102H141FN15O25+ M+ 1995.0202, found 1995.0208.

To a stirring solution of S73 (5.02 g, 2.418 mmol) in CH2Cl2 (50 mL) at room temperature was added TFA (25 mL). The resulting mixture was stirred at room temperature for 1 h before it was concentrated in vacuo directly. The product A007 was obtained as a yellow solid (2.52 g, 55% over 2 steps) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C89H119FN15O25+ [M+H]+ 1816.8480, found 1816.8487.

Example 357: Synthesis of A018

To a stirring solution of S74 (1.50 g, 7.386 mmol) in THF (50 mL) at 0° C. was added NaH (650 mg, 16.248 mmol, 60% dispersion in mineral oil). The resulting mixture was stirred at 0° C. for 0.5 h before addition of BnOH (2.00 g, 18.4638 mmol). The resulting mixture was stirred at 0° C. for 1 h before it was quenched with NH4Cl (sat. aq., 30 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (3×30 mL). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S75 was obtained as a yellow solid (1.45 g, 67%) after flash column chromatography purification (silica gel, CH2Cl2:MeOH=20:1). HRMS (ESI): calcd for C14H11FNO5+ [M+H]+ 292.0616, found 292.0620.

To a stirring solution of S75 (1.45 g, 4.977 mmol) and HNMe (OMe)·HCl (583 mg, 5.972 mmol) in CH2Cl2 (20 mL) at room temperature were added HATU (2.27 g, 5.972 mmol) and DIPEA (1.93 g, 14.931 mmol). The resulting mixture was stirred at room temperature for 1 h before it was quenched with Na2CO3 (5% wt/wt aq., 10 mL). The layers were separated, and the organic layer was washed with HCl (1 M aq., 10 mL) and water (10 mL). The organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S76 was obtained as a yellow solid (1.60 g, 96%) and used without further purification. HRMS (ESI): calcd for C16H16FN2O5+ [M+H]+ 335.1038, found 335.1039.

To a stirring solution of S76 (1.60 g, 4.786 mmol) in EtOH/H2O (30 mL, 50% V/V) at room temperature were added NH4Cl (2.56 g, 47.861 mmol) and Fe powder (1.87 g, 33.503 mmol). The resulting mixture was heated to 80° C. and stirred for 2 h before it was cooled to room temperature and filtered through a pad of celite. The filtrate was collected and concentrated in vacuo. The intermediate S77 was obtained as a yellow solid (1.45 g, 100%) and used without further purification. HRMS (ESI): calcd for C16H18FN2O3+ [M+H]+ 305.1296, found 305.1300.

To a stirring solution of S77 (1.45 g, 4.765 mmol) in 1,4-dioxane/H2O (50 mL, 70% V/V) at 0° C. were added Na2CO3 (1.51 g, 14.294 mmol) and FmocCl (3.08 g, 11.9118 mmol). The resulting mixture was warmed to room temperature and stirred for 12 h before it was concentrated in vacuo. The residual aqueous layer was extracted with EtOAc (20 mL×3). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S78 was obtained as a white solid (2.50 g, 100%) after flash column chromatography purification (silica gel, hexane:EtOAc=2:1). HRMS (ESI): calcd for C31H28FN2O5+ [M+H]+ 527.1977, found 527.1982.

To a stirring solution of S78 (2.50 g, 65.604 mmol) in THF (30 mL) at 0° C. was added S79 (9.50 mL, 1.0 M in THF, 9.496 mmol). The resulting mixture was warmed to room temperature and stirred for 6 h before it was quenched with NH4Cl (sat. aq., 20 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (20 mL×3). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S80 was obtained as a yellow oil (2.00 g, 78%) after flash column chromatography purification (silica gel, hexane:EtOAc=2:1). HRMS (ESI): calcd for C33H31FNO5+ [M+H]+ 540.2181, found 540.2182.

To a stirring solution of S80 (2.00 g, 3.706 mmol) in DMF (20 mL) at room temperature was added piperidine (2 mL). The resulting mixture was stirred at room temperature for 30 min before it was concentrated in vacuo directly. The intermediate S81 was obtained as a yellow solid (1.10 g, 94%) after flash column chromatography purification (silica gel, hexane:EtOAc=10:1). HRMS (ESI): calcd for C18H21FNO3+ [M+H]+ 318.1500, found 318.1500.

To a stirring solution of S81 (1.10 g, 3.466 mmol) and 27 (913 mg, 3.466 mmol) in toluene (30 mL) at room temperature was added PPTS (87 mg, 0.347 mmol). The resulting mixture was heated to reflux and stirred overnight before it was cooled to room temperature and filtered. The filter cake was collected and stirred in toluene at reflux for 2 h before it was cooled and filtered. The filter cake was collected and dried in vacuo. The intermediate S82 was obtained as a yellow solid (1.45 g, 77%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C31H30FN2O6+ [M+H]+ 545.2082, found 545.2088.

To a stirring solution of S82 (3.00 g, 5.509 mmol) in CH2Cl2 (40 mL) at room temperature was added TFA (20 mL). The resulting mixture was stirred at room temperature for 1 h before it was concentrated in vacuo directly. The intermediate S83 was obtained as a yellow solid (2.20 g, 87%) after flash column chromatography purification (silica gel, CH2Cl2:hexane=20:1). HRMS (ESI): calcd for C24H24FN2O6+ [M+H]+ 455.1613, found 455.1614.

To a stirring solution of S83 (2.20 g, 4.841 mmol) and S29 (3.63 g, 29.046 mmol) in acetone (50 mL) at room temperature were added K2CO3 (2.01 g, 14.523 mmol) and KI (804 mg, 4.841 mmol) in sequence. The resulting mixture was heated to 60° C. and stirred for 20 h before it was cooled to room temperature and concentrated in vacuo directly. The intermediate S84 was obtained as a dark yellow solid (1.33 g, 55%) after flash column chromatography purification (silica gel, DCM:MeOH=10:1). HRMS (ESI): calcd for C26H28FN2O7+ [M+H]+ 499.1875, found 499.1875.

To a stirring solution of S84 (1.00 g, 2.006 mmol) and 85 (1.53 g, 4.012 mmol) in DMF (30 mL) at room temperature was added p-TsOH·H2O (191 mg, 1.003 mmol). The resulting mixture was heated to 60° C. and stirred for 2 h before it was cooled to room temperature and concentrated in vacuo directly. The intermediate S86 was obtained as a white solid (1.22 g, 74%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C45H46FN4O10+ [M+H]+ 821.3192, found 821.3195.

To a stirring solution of S86 (1.22 g, 1.486 mmol) in DMF (10 mL) at room temperature was added piperidine (1 mL). The resulting mixture was stirred at room temperature for 30 min before it was concentrated in vacuo directly. The intermediate S87 was obtained as a yellow solid (823 mg, 92%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C30H36FN4O8+ [M+H]+ 599.2512, found 599.2513.

To a stirring solution of S21 (1.77 g, 1.375 mmol) in DMF (30 mL) at room temperature was added HATU (523 mg, 1.375 mmol). The resulting mixture was stirred at room temperature for 15 min before addition of S86 (823 mg, 1.375 mmol) and DIPEA (533 mg, 4.124 mmol) in sequence. The resulting mixture was stirred at room temperature for further 1 h before it was concentrated in vacuo directly. The intermediate S87 was obtained as a dark yellow oil (2.57 g, 100%) and used without further purification. HRMS (ESI): calcd for C93H133FN13O26+ [M+H]+ 1866.9463, found 1866.9464.

To a stirring solution of S87 (2.57 g, 1.375 mmol) in CH2Cl2 (30 mL) at room temperature was added TFA (15 mL). The resulting mixture was stirred at room temperature for 1 h before it was concentrated in vacuo directly. The product A018 was obtained as a yellow solid (880 mg, 38% over 2 steps) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C81H109FN13O26+ [M+H]+ 1698.7585, found 1698.7592.

Example 358: Synthesis of A019

To a stirring solution of 78 (2.00 g, 3.800 mmol) in THF (30 mL) at 0° C. was added 88 (5.70 mL, 1.0 M in THF, 5.697 mmol). The resulting mixture was warmed to room temperature and stirred for 6 h before it was quenched with NH4Cl (sat. aq., 20 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (20 mL×3). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate 89 was obtained as a yellow oil (1.46 g, 60%) after flash column chromatography purification (silica gel, hexane:EtOAc=2:1). HRMS (ESI): calcd for C38H43FNO5Si+ [M+H]+ 640.2889, found 640.2896.

To a stirring solution of 89 (1.46 g, 2.282 mmol) and 27 (601 mg, 2.282 mmol) in toluene (30 mL) at room temperature was added PPTS (58 mg, 0.228 mmol). The resulting mixture was heated to reflux and stirred overnight before it was cooled to room temperature and filtered. The filter cake was collected and stirred in toluene at reflux for 2 h before it was cooled and filtered. The filter cake was collected and dried in vacuo. The intermediate 90 was obtained as a yellow solid (1.09 g, 74%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C36H42FN2O6Si+ [M+H]+ 645.2791, found 645.2793.

To a stirring solution of 90 (1.09 g, 1.690 mmol) in THF (30 mL) at room temperature was added TBAF (2.54 mL, 1.0 M in THF, 2.536 mmol). The resulting mixture was stirred at temperature and for 30 min before it was quenched with NH4Cl (sat. aq., 20 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (20 mL×3). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate 91 was obtained as a yellow solid (724 mg, 80%) after flash column chromatography purification (silica gel, hexane:EtOAc=1:1). HRMS (ESI): calcd for C30H28FN2O6+ [M+H]+ 531.1926, found 531.1934.

To a stirring solution of 91 (724 mg, 1.365 mmol) in CH2Cl2 (10 mL) at room temperature were added Et3N (553 mg, 5.460 mmol) and Ac2O (279 mg, 2.730 mmol). The resulting mixture was stirred at room temperature for 2 h before it was quenched with NaHCO3 (sat. aq., 5 mL). The layers were separated, and the aqueous layer was extracted with CH2Cl2 (10 mL×3). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate 92 was obtained as a yellow oil (655 mg, 84%) after flash column chromatography purification (silica gel, hexane:EtOAc=1:1). HRMS (ESI): calcd for C32H30FN2O7+ [M+H]+ 573.2032, found 573.2035.

To a stirring solution of 92 (665 mg, 1.144 mmol) in CH2Cl2 (10 mL) at room temperature was added TFA (5 mL). The resulting mixture was stirred at room temperature for 1 h before it was concentrated in vacuo directly. The intermediate 93 was obtained as a yellow solid (550 mg, 100%) after flash column chromatography purification (silica gel, CH2Cl2:hexane=20:1). HRMS (ESI): calcd for C25H24FN2O7+ [M+H]+ 483.1562, found 483.1563.

To a stirring solution of 84 (1.00 g, 5.230 mmol) and 94 (650 mg, 10.460 mmol) in THF (30 mL) at room temperature was added p-TsOH·H2O (498 mg, 2.615 mmol). The resulting mixture was heated to 60° C. and stirred for 2 h before it was cooled to room temperature and concentrated in vacuo directly. The intermediate 95 was obtained as a colorless oil (1.20 g, 60%) after flash column chromatography purification (silica gel, hexane:EtOAc=5:1). HRMS (ESI): calcd for C21H25N2O5+ [M+H]+ 385.1758, found 385.1765.

To a stirring solution of 95 (439 mg, 1.144 mmol) in THF (10 mL) at 0° C. was added PPh3 (359 mg, 1.373 mmol) and DEAD (240 mg, 1.373 mmol). The resulting mixture was stirred at 0° C. for 15 min before addition of 93 (550 mg, 1.144 mmol). The resulting mixture was stirred at 0° C. for further 1 h before it was with NaCl (sat. aq., 5 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (10 mL×3). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate 96 was obtained as a yellow oil (760 mg, 78%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C46H46FN4O11+ [M+H]+ 849.3142, found 849.3144.

To a stirring solution of 96 (760 mg, 0.895 mmol) in DMF (10 mL) at room temperature was added piperidine (1 mL). The resulting mixture was stirred at room temperature for 30 min before it was concentrated in vacuo directly. The intermediate 97 was obtained as a yellow solid (400 mg, 71%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C31H36FN4O9+ [M+H]+ 627.2461, found 627.2461.

To a stirring solution of 21 (820 mg, 0.638 mmol) in DMF (10 mL) at room temperature was added HATU (243 mg, 0.638 mmol). The resulting mixture was stirred at room temperature for 15 min before addition of 97 (400 mg, 0.638 mmol) and DIPEA (248 mg, 1.914 mmol) in sequence. The resulting mixture was stirred at room temperature for further 1 h before it was concentrated in vacuo directly. The intermediate 98 was obtained as a dark yellow oil (1.20 g, 100%) and used without further purification. HRMS (ESI): calcd for C94H133FN13O27+ [M+H]+ 1894.9412, found 1894.9418.

To a stirring solution of 87 (1.20 g, 0.638 mmol) in CH2Cl2 (20 mL) at room temperature was added TFA (10 mL). The resulting mixture was stirred at room temperature for 1 h before it was concentrated in vacuo directly. The product A019 was obtained as a yellow solid (330 mg, 30% over 2 steps) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C82H109FN13O27+ [M+H]+ 1726.7534, found 1726.7534.

Example 359: Synthesis of A020

To a stirring solution of S78 (5.00 g, 9.496 mmol) in THF (50 mL) at 0° C. was added MeMgBr (4.75 mL, 3.0 mol/L in Et2O, 14.243 mmol). The resulting mixture was warmed to room temperature and stirred for 1 h before it was quenched with NH4Cl (sat. aq., 20 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (20 mL×3). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S99 was obtained as a yellow solid (4.23 g, 93%) after flash column chromatography purification (silica gel, hexane:EtOAc=5:1). HRMS (ESI): calcd for C30H25FNO4+ [M+H]+ 482.1762, found 482.1765.

To a stirring solution of S100 (2.50 g, 4.244 mmol) in CH2Cl2 (50 mL) at 0° C. were added Et3N (1.72 g, 16.975 mmol) and MsCl (973 mg, 8.488 mmol) in sequence. The resulting mixture stirred at 0° C. for 1 h before it was quenched with NaHCO3 (sat. aq., 10 mL). The layers were separated, and the aqueous layer was extracted with CH2Cl2 (10 mL×3). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S101 was obtained as a yellow solid (2.65 g, 99%) after flash column chromatography purification (silica gel, hexane:EtOAc=4:1). HRMS (ESI): calcd for C35H36FN2O6S+ [M+H]+ 631.2273, found 631.2276.

To a stirring solution of S101 (2.65 g, 4.201 mmol) in DMF (30 mL) at room temperature was added piperidine (3 mL). The resulting mixture was stirred at room temperature for 30 min before it was concentrated in vacuo directly. The intermediate S102 was obtained as a yellow solid (1.69 g, 98%) after flash column chromatography purification (silica gel, hexane:EtOAc=2:1). HRMS (ESI): calcd for C20H26FN2O4S+ [M+H]+ 409.1592, found 409.1595.

To a stirring solution of S102 (1.50 g, 3.672 mmol) and S27 (967 mg, 3.672 mmol) in toluene (30 mL) at room temperature was added PPTS (185 mg, 0.734 mmol). The resulting mixture was heated to reflux and stirred overnight before it was cooled to room temperature and filtered. The filter cake was collected and stirred in refluxing toluene for 2 h before it was cooled and filtered. The filter cake was collected and dried in vacuo. The intermediate S103 was obtained as a yellow solid (1.05 g, 45%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C33H35FN3O7S+ [M+H]+ 636.2174, found 636.2172.

To a stirring solution of S103 (1.00 g, 1.573 mmol) in CH2Cl2 (20 mL) at room temperature was added TFA (10 mL). The resulting mixture was stirred at room temperature for 1 h before it was concentrated in vacuo directly. The intermediate S104 was obtained as a yellow solid (858 mg, 100%) after flash column chromatography purification (silica gel, CH2Cl2:hexane=20:1). HRMS (ESI): calcd for C26H29FN3O7S+ [M+H]+ 546.1705, found 546.1706.

To a stirring solution of S95 (353 mg, 0.917 mmol) in THF (10 mL) at 0° C. was added PPh3 (289 mg, 1.100 mmol) and DEAD (192 mg, 1.100 mmol). The resulting mixture was stirred at 0° C. for 15 min before addition of S104 (500 mg, 0.917 mmol). The resulting mixture was stirred at 0° C. for further 1 h before it was with NaCl (sat. aq., 5 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (10 mL×3). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S105 was obtained as a yellow solid (433 mg, 52%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C47H51FN5O11S+ [M+H]+ 912.3284, found 912.3285.

To a stirring solution of S105 (433 mg, 0.475 mmol) in DMF (10 mL) at room temperature was added piperidine (1 mL). The resulting mixture was stirred at room temperature for 30 min before it was concentrated in vacuo directly. The intermediate S106 was obtained as a yellow solid (300 mg, 92%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C32H41FN5O9S+ [M+H]+ 690.2604, found 690.2609.

To a stirring solution of S21 (466 mg, 0.362 mmol) in DMF (10 mL) at room temperature was added HATU (138 mg, 0.362 mmol). The resulting mixture was stirred at room temperature for 15 min before addition of S107 (250 mg, 0.362 mmol) and DIPEA (141 mg, 1.087 mmol) in sequence. The resulting mixture was stirred at room temperature for further 1 h before it was concentrated in vacuo directly. The intermediate S108 was obtained as a dark yellow oil (709 mg, 100%) and used without further purification. HRMS (ESI): calcd for C95H138FN14O27S+ [M+H]+ 1957.9555, found 1957.9561. STEP 10:

To a stirring solution of S108 (709 mg, 0.362 mmol) in CH2Cl2 (3 mL) at room temperature was added TFA (1.5 mL). The resulting mixture was stirred at room temperature for 1 h before it was concentrated in vacuo directly. The product A020 was obtained as a yellow solid (250 mg, 39% over 2 steps) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C83H114FN14O27S+ [M+ H]+ 1789.7677, found 1789.7686.

Example 360: Synthesis of A021

Step 1˜STEP 8: Intermediate S108˜S114 and product A021 were prepared according to the procedures in A020. A021: HRMS (ESI): calcd for C82H113FN15O27S+ [M+H]+ 1790.7630, found 1790.7638.

Example 361: Synthesis of A023

To a stirring solution of S100 (5.00 g, 8.488 mmol) in CH2Cl2 (100 mL) at 0° C. were added Et3N (1.72 g, 16.975 mmol) and CbzCl (2.17 g, 12.731 mmol) in sequence. The resulting mixture was stirred at 0° C. for 2 h before it was quenched with NaHCO3 (sat. aq., 20 mL). The layers were separated, and the aqueous layer was extracted with CH2Cl2 (20 mL×3). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S115 was obtained as a yellow solid (5.80 g, 99%) after flash column chromatography purification (silica gel, hexane:EtOAc=5:1). HRMS (ESI): calcd for C42H40FN2O6+ [M+H]+ 687.2865, found 687.2870.

To a stirring solution of S115 (5.80 g, 8.445 mmol) in DMF (30 mL) at room temperature was added piperidine (3 mL). The resulting mixture was stirred at room temperature for 30 min before it was concentrated in vacuo directly. The intermediate S116 was obtained as a yellow solid (3.90 g, 99%) after flash column chromatography purification (silica gel, hexane:EtOAc=2:1). HRMS (ESI): calcd for C27H30FN2O4+ [M+H]+ 465.2184, found 465.2185.

To a stirring solution of S116 (3.90 g, 8.395 mmol) and S27 (2.21 g, 8.395 mmol) in toluene (100 mL) at room temperature was added PPTS (422 mg, 1.679 mmol). The resulting mixture was heated to reflux and stirred overnight before it was cooled to room temperature and filtered. The filter cake was collected and stirred in refluxing toluene for 2 h before it was cooled and filtered. The filter cake was collected and dried in vacuo. The intermediate S117 was obtained as a yellow solid (4.17 g, 72%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C40H39FN3O7+ [M+H]+ 692.2767, found 692.2767.

To a stirring solution of S117 (4.17 g, 6.08 mmol) in CH2Cl2 (50 mL) at room temperature was added TFA (25 mL). The resulting mixture was stirred at room temperature for 1 h before it was concentrated in vacuo directly. The intermediate S118 was obtained as a yellow solid (3.50 g, 97%) after flash column chromatography purification (silica gel, CH2Cl2:hexane=20:1). HRMS (ESI): calcd for C33H33FN3O7+ [M+H]+ 602.2297, found 602.2298.

To a stirring solution of S118 (3.50 g, 5.818 mmol) and S119 (4.85 g, 34.908 mmol) in acetone (50 mL) at room temperature were added K2CO3 (2.41 g, 17.44 mmol) and KI (966 mg, 5.818 mmol) in sequence. The resulting mixture was heated to 60° C. and stirred for 20 h before it was cooled to room temperature and concentrated in vacuo directly. The intermediate S120 was obtained as a dark yellow solid (3.20 g, 83%) after flash column chromatography purification (silica gel, DCM:MeOH=10:1). HRMS (ESI): calcd for C36H39FN3O8+ [M+H]+ 660.2716, found 660.2723.

To a stirring solution of S121 (3.00 g, 4.547 mmol) in MeOH (30 mL) at room temperature was added Pd/C (3.00 g, 10% wt/wt). The resulting mixture was stirred at room temperature under H2 atmosphere for 1 h before it was filtered through a pad of celite. The filtrate was collected and concentrated in vacuo. The intermediate S121 was obtained as a yellow oil (2.39 g, 100%) and used without further purification. HRMS (ESI): calcd for C28H33FN3O6+ [M+H]+ 526.2348, found 526.2348.

To a stirring solution of S24 (5.00 g, 16.987 mmol) in MeCN (100 mL) at 0° C. were added DIPEA (3.29 g, 25.480 mmol) and S44 (4.11 g, 20.384 mmol) in sequence. The resulting mixture was stirred at 0° C. for 3 h before it was quenched with NaHCO3 (sat. aq., 20 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (20 mL×3). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S122 was obtained as a yellow solid (6.33 g, 81%) after flash column chromatography purification (silica gel, hexane:EtOAc=1:1). HRMS (ESI): calcd for C22H26N3O8+ [M+H]+ 460.1714, found 460.1720.

To a stirring solution of S121 (2.39 g, 4.547 mmol) in DMF (50 mL) at room temperature were added DIPEA (882 mg, 6.821 mmol) and S122 (2.51 g, 5.547 mmol) in sequence. The resulting mixture was stirred at room temperature for 2 h before it was cooled to room temperature and concentrated in vacuo directly. The intermediate S123 was obtained as a dark yellow solid (3.45 g, 89%) after flash column chromatography purification (silica gel, DCM:MeOH=20:1). HRMS (ESI): calcd for C44H53FN5O11+ [M+H]+ 846.3720, found 846.3721.

To a stirring solution of S123 (3.45 g, 4.078 mmol) in CH2Cl2 (40 mL) at room temperature was added TFA (20 mL). The resulting mixture was stirred at room temperature for 1 h before it was concentrated in vacuo directly. The intermediate S124 was obtained as a yellow solid (3.50 g, 100%) after trituration with MTBE (100 mL). HRMS (ESI): calcd for C39H45FN5O9+ [M+H]+ 746.3196, found 746.3196.

To a stirring solution of S21 (5.24 g, 4.071 mmol) in DMF (100 mL) at room temperature was added HATU (1.55 g, 4.071 mmol). The resulting mixture was stirred at room temperature for 15 min before addition of S124 (3.50 g, 4.071 mmol) and DIPEA (1.58 g, 12.212 mmol) in sequence. The resulting mixture was stirred at room temperature for further 1 h before it was concentrated in vacuo directly. The intermediate S125 was obtained as a dark yellow oil (8.20 g, 100%) and used without further purification. HRMS (ESI): calcd for C102H142FN14O27+ [M+H]+ 2014.0147, found 2014.0152.

To a stirring solution of S125 (8.20 g, 4.071 mmol) in CH2Cl2 (100 mL) at room temperature was added TFA (50 mL). The resulting mixture was stirred at room temperature for 1 h before it was concentrated in vacuo directly. The product A023 was obtained as a yellow solid (3.36 g, 45% over 2 steps) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C89H116FN14O27+ [M+H]+ 1831.8113, found 1831.8120.

Example 362: Synthesis of A022

To a stirring solution of S126 (5.00 g, 25.616 mmol) and S27 (6.74 g, 25.616 mmol) in toluene (100 mL) at room temperature was added PPTS (3.22 g, 12.808 mmol). The resulting mixture was heated to reflux and stirred overnight before it was cooled to room temperature and filtered. The filter cake was collected and stirred in refluxing toluene for 3 h before it was cooled and filtered. The filter cake was collected and dried in vacuo. The intermediate S127 was obtained as a yellow solid (7.11 g, 66%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C23H20FN2O5+ [M+H]+ 423.1351, found 423.1355.

To a stirring solution of S128 (10.00 g, 111.012 mmol) and S94 (84.91 g, 222.025 mmol) in THF (500 mL) at room temperature was added p-TsOH·H2O (10.56 g, 55.506 mmol). The resulting mixture was heated to 60° C. and stirred for 2 h before it was cooled to room temperature and concentrated in vacuo directly. The intermediate S95 was obtained as a colorless oil (12.12 g, 24%) after flash column chromatography purification (silica gel, hexane:EtOAc=5:1). HRMS (ESI): calcd for C22H25N2O6+ [M+H]+ 413.1707, found 413.1713.

To a stirring solution of S129 (1.95 g, 4.735 mmol) in THF (50 mL) at 0° C. was added PPh3 (1.49 g, 5.682 mmol) and DEAD (990 mg, 5.682 mmol). The resulting mixture was stirred at 0° C. for 15 min before addition of 127 (2.00 g, 4.735 mmol). The resulting mixture was stirred at 0° C. for further 1 h before it was with NaCl (sat. aq., 20 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (20 mL×3). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S130 was obtained as a yellow oil (1.45 g, 38%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C44H42FN4O10+ [M+H]+ 805.2879, found 805.2880.

To a stirring solution of S130 (1.45 g, 1.802 mmol) in DMF (20 mL) at room temperature was added piperidine (2 mL). The resulting mixture was stirred at room temperature for 30 min before it was concentrated in vacuo directly. The intermediate S131 was obtained as a yellow solid (620 mg, 59%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C29H32FN4O8+ [M+H]+ 583.2199, found 583.2204.

To a stirring solution of S21 (1.37 g, 1.064 mmol) in DMF (20 mL) at room temperature was added HATU (405 mg, 1.064 mmol). The resulting mixture was stirred at room temperature for 15 min before addition of S131 (620 mg, 1.064 mmol) and DIPEA (413 mg, 3.192 mmol) in sequence. The resulting mixture was stirred at room temperature for further 1 h before it was concentrated in vacuo directly. The intermediate S132 was obtained as a dark yellow oil (1.97 g, 100%) and used without further purification. HRMS (ESI): calcd for C92H129FN13O26+ [M+H]+ 1850.9150, found 1850.9151.

To a stirring solution of S131 (1.97 g, 1.064 mmol) in CH2Cl2 (30 mL) at room temperature was added TFA (15 mL). The resulting mixture was stirred at room temperature for 1 h before it was concentrated in vacuo directly. The product A022 was obtained as a yellow solid (448 mg, 25% over 2 steps) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C80H105FN13O26+ [M+H]+ 1682.7272, found 1682.7277.

Example 363: Synthesis of A026

To a stirring solution of S99 (5.00 g, 10.384 mmol) and S133 (8.32 g, 51.919 mmol) and (HCHO) n (1.56 g, 51.919 mmol) in i-PrOH (120 mL) at room temperature was added conc. HCl (1.50 mL). The resulting mixture was heated to reflux and stirred for 12 h before it was cooled to room temperature and quenched with MTBE (200 mL). The resulting mixture was filtered and the filter cake was collected, stirred in refluxing MTBE (50 mL) for 2 h before it was cooled and filtered. The filter cake was collected and dried in vacuo. The intermediate S134 was obtained as a yellow solid (1.56 g, 22%). HRMS (ESI): calcd for C38H41FN3O6+ [M+H]+ 654.2974, found 654.2975.

To a stirring solution of S134 (1.56 g, 2.260 mmol) in CH2Cl2 (50 mL) at 0° C. were added Et3N (915 mg, 9.041 mmol) and MsCl (518 mg, 4.520 mmol) in sequence. The resulting mixture stirred at 0° C. for 1 h before it was quenched with NaHCO3 (sat. aq., 10 mL). The layers were separated, and the aqueous layer was extracted with CH2Cl2 (10 mL×3). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S135 was obtained as a yellow solid (1.11 g, 67%) after flash column chromatography purification (silica gel, hexane:EtOAc=2:1). HRMS (ESI): calcd for C39H43FN3O8S+ [M+H]+ 732.2749, found 732.2755.

To a stirring solution of S135 (1.11 g, 1.517 mmol) in CH2Cl2 (40 mL) at room temperature was added TFA (20 mL). The resulting mixture was stirred at room temperature for 1 h before it was concentrated in vacuo directly. The residue was dispersed with MTBE (100 mL) and stirred for 30 min before it was filtered. The filter cake was collected and dried in vacuo. The intermediate S136 was obtained as a yellow solid (1.12 g, 99%) and used without further purification. HRMS (ESI): calcd for C34H35FN3O6S+ [M+H]+ 632.2225, found 632.2228.

To a stirring solution of S23 (341 mg, 1.802 mmol) in DMF (15 mL) at room temperature were added HATU (686 mg, 1.802 mmol) and DIPEA (583 mg, 4.506 mmol) in sequence. The resulting mixture was stirred at room temperature for 30 min before addition of S136 (1.12 g, 1.502 mmol). The resulting mixture was stirred at room temperature for further 1 h before it was quenched with NaHCO3 (sat. aq., 10 mL). The mixture layers was extracted with CH2Cl2 (20 mL×3). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S137 was obtained as a yellow oil (1.02 mg, 85%) after flash column chromatography purification (silica gel, hexane:EtOAc=1:1). HRMS (ESI): calcd for C42H48FN4O9S+ [M+H]+ 803.3121, found 803.3122.

To a stirring solution of S137 (1.02 g, 1.270 mmol) in DMF (10 mL) at room temperature was added piperidine (1 mL). The resulting mixture was stirred at room temperature for 30 min before it was concentrated in vacuo directly. The intermediate S138 was obtained as a yellow solid (688 mg, 93%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C27H38FN4O7S+ [M+H]+ 581.2440, found 581.2440.

To a stirring solution of S138 (680 mg, 1.171 mmol) and S27 (309 mg, 1.171 mmol) in toluene (10 mL) at room temperature was added PPTS (69 mg, 0.234 mmol). The resulting mixture was heated to reflux and stirred overnight before it was cooled to room temperature and filtered. The filter cake was collected and stirred in refluxing toluene for 3 h before it was cooled and filtered. The filter cake was collected and dried in vacuo. The intermediate S139 was obtained as a white solid (500 mg, 52%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C40H47FN5O10S+ [M+H]+ 808.3022, found 808.3026.

To a stirring solution of S139 (500 mg, 0.619 mmol) in MeOH (10 mL) at room temperature was added Pd/C (50 mg, 10% wt/wt). The resulting mixture was stirred at room temperature under H2 atmosphere for 5 h before it was filtered through a pad of celite. The filtrate was collected and concentrated in vacuo. The intermediate S140 was obtained as a yellow solid (383 mg, 86%) after flash column chromatography purification (silica gel, CH2Cl2:hexane=10:1). HRMS (ESI): calcd for C33H41FN5O10S+ [M+H]+ 718.2553, found 718.2554.

To a stirring solution of S140 (380 mg, 0.529 mmol) and S119 (442 mg, 3.177 mmol) in acetone (10 mL) at room temperature were added K2CO3 (220 mg, 1.588 mmol) and KI (88 mg, 0.529 mmol) in sequence. The resulting mixture was heated to 60° C. and stirred for 20 h before it was cooled to room temperature and concentrated in vacuo directly. The intermediate S141 was obtained as a yellow solid (190 mg, 46%) after flash column chromatography purification (silica gel, DCM:MeOH=10:1). HRMS (ESI): calcd for C36H47FN5O11S+ [M+H]+ 776.2971, found 776.2978.

To a stirring solution of S141 (190 mg, 0.245 mmol) in CH2Cl2 (5 mL) at room temperature was added TFA (2 mL). The resulting mixture was stirred at room temperature for 1 h before it was concentrated in vacuo directly. The intermediate S142 was obtained as a yellow solid (150 mg, 78%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C31H39FN5O9S+ [M+H]+ 676.2447, found 676.2455.

To a stirring solution of S21 (244 mg, 0.190 mmol) in DMF (10 mL) at room temperature was added HATU (72 mg, 0.190 mmol). The resulting mixture was stirred at room temperature for 15 min before addition of S142 (150 mg, 0.190 mmol) and DIPEA (74 mg, 0.570 mmol) in sequence. The resulting mixture was stirred at room temperature for further 1 h before it was concentrated in vacuo directly. The intermediate S143 was obtained as a dark yellow oil (369 mg, 100%) and used without further purification. HRMS (ESI): calcd for C94H136FN14O27S+ [M+H]+ 1943.9399, found 1943.9402.

To a stirring solution of S143 (369 mg, 0.190 mmol) in CH2Cl2 (5 mL) at room temperature was added TFA (2 mL). The resulting mixture was stirred at room temperature for 1 h before it was concentrated in vacuo directly. The product A026 was obtained as a yellow solid (124 mg, 36% over 2 steps) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C82H112FN14O27S+ [M+H]+ 1775.7521, found 1775.7530.

Example 364: Synthesis of A027

To a stirring solution of S99 (10.00 g, 20.768 mmol) and S144 (12.17 g, 103.838 mmol) and (HCHO) n (3.12 g, 103.838 mmol) in i-PrOH (250 mL) at room temperature was added conc. HCl (3 mL). The resulting mixture was heated to reflux and stirred for 12 h before it was cooled to room temperature and quenched with MTBE (200 mL). The resulting mixture was filtered and the filter cake was collected, stirred in refluxing MTBE (100 mL) for 2 h before it was cooled and filtered. The filter cake was collected and dried in vacuo. The intermediate S145 was obtained as a yellow solid (2.09 g, 15%). HRMS (ESI): calcd for C37H40FN2O5+ [M+H]+ 611.2916, found 611.2917.

To a stirring solution of S145 (2.00 g, 3.090 mmol) in CH2Cl2 (50 mL) at 0° C. were added Et3N (1.25 g, 12.361 mmol) and MsCl (708 mg, 6.181 mmol) in sequence. The resulting mixture stirred at 0° C. for 1 h before it was quenched with NaHCO3 (sat. aq., 10 mL). The layers were separated, and the aqueous layer was extracted with CH2Cl2 (10 mL×3). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S146 was obtained as a yellow solid (1.74 g, 81%) after flash column chromatography purification (silica gel, hexane:EtOAc=1:1). HRMS (ESI): calcd for C38H42FN2O7S+ [M+H]+ 689.2691, found 689.2693.

To a stirring solution of S146 (1.50 g, 2.178 mmol) in DMF (15 mL) at room temperature was added piperidine (1.5 mL). The resulting mixture was stirred at room temperature for 30 min before it was concentrated in vacuo directly. The intermediate S147 was obtained as a yellow solid (980 mg, 87%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C23H32FN2O5S+ [M+H]+ 467.2010, found 467.2012.

To a stirring solution of S147 (890 mg, 1.908 mmol) and 27 (503 mg, 1.908 mmol) in toluene (20 mL) at room temperature was added PPTS (96 mg, 0.382 mmol). The resulting mixture was heated to reflux and stirred overnight before it was cooled to room temperature and filtered. The filter cake was collected and stirred in refluxing toluene for 3 h before it was cooled and filtered. The filter cake was collected and dried in vacuo. The intermediate S148 was obtained as a white solid (650 mg, 49%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C36H41FN3O8S+ [M+H]+ 694.2593, found 694.2600.

To a stirring solution of S148 (650 mg, 0.619 mmol) in MeOH (10 mL) at room temperature was added Pd/C (65 mg, 10% wt/wt). The resulting mixture was stirred at room temperature under H2 atmosphere for 6 h before it was filtered through a pad of celite. The filtrate was collected and concentrated in vacuo. The intermediate S149 was obtained as a yellow solid (535 mg, 95%) after flash column chromatography purification (silica gel, CH2Cl2:hexane=10:1). HRMS (ESI): calcd for C29H35FN3O8S+ [M+H]+ 604.2123, found 604.2130.

To a stirring solution of S149 (500 mg, 0.828 mmol) and S119 (576 mg, 0.828 mmol) in acetone (10 mL) at room temperature were added K2CO3 (573 mg, 4.141 mmol) and KI (138 mg, 4.141 mmol) in sequence. The resulting mixture was heated to 60° C. and stirred for 20 h before it was cooled to room temperature and concentrated in vacuo directly. The intermediate S150 was obtained as a yellow solid (230 mg, 42%) after flash column chromatography purification (silica gel, DCM:MeOH=10:1). HRMS (ESI): calcd for C32H41FN3O9S+ [M+H]+ 662.2542, found 662.2546.

To a stirring solution of S150 (230 mg, 0.348 mmol) in CH2Cl2 (4 mL) at room temperature was added TFA (2 mL). The resulting mixture was stirred at room temperature for 1 h before it was concentrated in vacuo directly. The product intermediate S151 was obtained as a yellow solid (152 mg, 72%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C28H33FN3O9S+ [M+H]+ 606.1916, found 606.1918.

To a stirring solution of S151 (152 mg, 0.251 mmol) and 85 (228 mg, 0.753 mmol) in DMF (5 mL) at room temperature was added p-TsOH·H2O (24 mg, 0.125 mmol). The resulting mixture was heated to 60° C. and stirred for 2 h before it was cooled to room temperature and concentrated in vacuo directly. The intermediate S152 was obtained as a white solid (89 mg, 38%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C47H51FN5O12S+ [M+H]+ 928.3233, found 928.3237.

To a stirring solution of S152 (150 mg, 0.162 mmol) in DMF (2 mL) at room temperature was added piperidine (0.2 mL). The resulting mixture was stirred at room temperature for 30 min before it was concentrated in vacuo directly. The intermediate S153 was obtained as a yellow oil (74 mg, 65%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C32H41FN5O10S+ [M+H] 706.2553, found 706.2555.

To a stirring solution of S21 (135 mg, 0.105 mmol) in DMF (10 mL) at room temperature was added HATU (40 mg, 0.105 mmol). The resulting mixture was stirred at room temperature for 15 min before addition of S153 (74 mg, 0.105 mmol) and DIPEA (41 mg, 0.315 mmol) in sequence. The resulting mixture was stirred at room temperature for further 1 h before it was concentrated in vacuo directly. The intermediate S154 was obtained as a dark yellow oil (207 mg, 100%) and used without further purification. HRMS (ESI): calcd for C95H138FN14O28S+ [M+H]+ 1973.9504, found 1973.9505.

To a stirring solution of S154 (207 mg, 0.105 mmol) in CH2Cl2 (4 mL) at room temperature was added TFA (2 mL). The resulting mixture was stirred at room temperature for 1 h before it was concentrated in vacuo directly. The product A027 was obtained as a yellow solid (99 mg, 52% over 2 steps) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C83H114FN14O28S+ [M+H]+ 1805.7626, found 1805.7630.

Example 365: Synthesis of A024

To a stirring solution of S155 (20.00 g, 96.530 mmol) and HNMe (OMe)·HCl (11.30 g, 115.836 mmol) in CH2Cl2 (250 mL) at room temperature were added HATU (44.05 g, 115.836 mmol) and DIPEA (37.43 g, 289.589 mmol). The resulting mixture was stirred at room temperature for 1 h before it was quenched with Na2CO3 (5% wt/wt aq., 100 mL). The layers were separated, and the organic layer was washed with HCl (1 M aq., 50 mL) and water (100 mL). The organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S156 was obtained as a yellow solid (22.00 g, 91%) after flash column chromatography purification (silica gel, hexane:EtOAc=2:1). HRMS (ESI): calcd for C12H15N2O4+ [M+H]+ 251.1026, found 251.1033.

To a stirring solution of S156 (20.00 g, 79.920 mmol) in EtOH/H2O (300 mL, 50% V/V) at room temperature were added NH4Cl (42.75 g, 799.200 mmol) and Fe powder (31.24 g, 159.840 mmol). The resulting mixture was heated to 80° C. and stirred for 2 h before it was cooled to room temperature and filtered through a pad of celite. The filtrate was collected and concentrated in vacuo. The intermediate S157 was obtained as a yellow solid (13.40 g, 71%) after flash column chromatography purification (silica gel, hexane:EtOAc=2:1). HRMS (ESI): calcd for C12H17N2O2+ [M+H]+ 221.1285, found 221.1290.

To a stirring solution of S157 (13.00 g, 59.018 mmol) in 1,4-dioxane/H2O (500 mL, 70% V/V) at 0° C. were added Na2CO3 (18.77 g, 177.092 mmol) and FmocCl (38.17 g, 147.545 mmol). The resulting mixture was warmed to room temperature and stirred for 12 h before it was concentrated in vacuo. The residual aqueous layer was extracted with EtOAc (200 mL×3). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S158 was obtained as a white solid (25.12 g, 96%) after flash column chromatography purification (silica gel, hexane:EtOAc=4:1). HRMS (ESI): calcd for C27H27N2O4+ [M+H]+ 443.1965, found 443.1966.

Step 4: To a stirring solution of S158 (20.00 g, 45.196 mmol) in THF (300 mL) at 0° C. was added MeMgBr (22.60 mL, 3.0 mol/L in Et2O, 67.794 mmol). The resulting mixture was warmed to room temperature and stirred for 1 h before it was quenched with NH4Cl (sat. aq., 100 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (100 mL×3). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S159 was obtained as a yellow solid (19.52 g, 86%) after flash column chromatography purification (silica gel, hexane:EtOAc=5:1). HRMS (ESI): calcd for C26H24NO3+ [M+H]+ 398.1751, found 398.1760.

To a stirring solution of S159 (15.00 g, 37.739 mmol) and i-Pr2NH·HCl (18.03 g, 188.693 mmol) and (HCHO) n (5.67 g, 188.693 mmol) in i-PrOH (300 mL) at room temperature was added conc. HCl (3 mL). The resulting mixture was heated to reflux and stirred for 12 h before it was cooled to room temperature and quenched with MTBE (500 mL). The resulting mixture was filtered and the filter cake was collected, stirred in refluxing MTBE (50 mL) for 2 h before it was cooled and filtered. The filter cake was collected and dried in vacuo. The intermediate S160 was obtained as a yellow solid (9.85 g, 51%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C30H33N2O3+ [M+H]+ 469.2486, found 469.2486.

To a stirring solution of S160 (9.00 g, 17.820 mmol) in CH2Cl2 (150 mL) at 0° C. were added Et3N (7.21 g, 71.279 mmol) and MsCl (4.08 mg, 35.639 mmol) in sequence. The resulting mixture stirred at 0° C. for 1 h before it was quenched with NaHCO3 (sat. aq., 20 mL). The layers were separated, and the aqueous layer was extracted with CH2Cl2 (30 mL×3). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S161 was obtained as a yellow solid (8.33 g, 85%) after flash column chromatography purification (silica gel, hexane:EtOAc=1:1). HRMS (ESI): calcd for C31H35N2O5S+ [M+H]+ 547.2261, found 547.2263.

To a stirring solution of S161 (8.00 g, 14.634 mmol) in DMF (100 mL) at room temperature was added piperidine (10 mL). The resulting mixture was stirred at room temperature for 30 min before it was concentrated in vacuo directly. The intermediate S162 was obtained as a yellow solid (3.78 g, 79%) after flash column chromatography purification (silica gel, CH2Cl2:hexane=10:1). HRMS (ESI): calcd for C16H25N2O3S+ [M+H]+ 325.1580, found 325.1581.

To a stirring solution of S162 (3.50 g, 10.788 mmol) and S27 (3.41 g, 12.945 mmol) in toluene (200 mL) at room temperature was added PPTS (1.36 g, 5.394 mmol). The resulting mixture was heated to reflux and stirred overnight before it was cooled to room temperature and filtered. The filter cake was collected and stirred in refluxing toluene for 3 h before it was cooled and filtered. The filter cake was collected and dried in vacuo. The intermediate S163 was obtained as a white solid (1.60 g, 26%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C29H34N3O6S+ [M+H]+ 552.2163, found 552.2166.

To a stirring solution of S163 (1.50 g, 2.719 mmol) in CH2Cl2 (50 mL) at room temperature were added triphosgene (275 mg, 0.924 mmol) The resulting mixture was stirred at room temperature for 2 h before addition of S24 (881 mg, 2.991 mmol) and DIPEA (1.05 g, 8.157 mmol) in sequence. The resulting mixture was stirred at room temperature for further 2 h before it was quenched with NaHCO3 (sat. aq., 10 mL). The layers were separated, and the aqueous layer was extracted with CH2C12 (10 mL×3). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S164 was obtained as a yellow solid (1.02 g, 43%) after flash column chromatography purification (CH2Cl2:hexane=20:1). HRMS (ESI): calcd for C45H54N5OnS+ [M+H]+ 872.3535, found 872.3539.

To a stirring solution of S164 (200 mg, 0.229 mmol) in CH2Cl2 (4 mL) at room temperature was added TFA (2 mL). The resulting mixture was stirred at room temperature for 1 h before it was concentrated in vacuo directly. The intermediate S165 was obtained as a yellow solid (130 mg, 64%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C40H46N5O9S+ [M+H]+ 772.3011, found 772.3012.

To a stirring solution of S21 (189 mg, 0.147 mmol) in DMF (10 mL) at room temperature was added HATU (56 mg, 0.147 mmol). The resulting mixture was stirred at room temperature for 15 min before addition of S165 (130 mg, 0.147 mmol) and DIPEA (57 mg, 0.440 mmol) in sequence. The resulting mixture was stirred at room temperature for further 1 h before it was concentrated in vacuo directly. The intermediate S166 was obtained as a dark yellow oil (299 mg, 100%) and used without further purification. HRMS (ESI): calcd for C103H143N14O27S+ [M+H]+ 2039.9962, found 2039.9969.

To a stirring solution of S166 (299 mg, 0.147 mmol) in CH2Cl2 (4 mL) at room temperature was added TFA (2 mL). The resulting mixture was stirred at room temperature for 1 h before it was concentrated in vacuo directly. The product A024 was obtained as a yellow solid (100 mg, 36% over 2 steps) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C91H119N14O27S+ [M+H]+ 1871.8084, found 1871.8088.

Example 366: Synthesis of A025

Step 1˜STEP 7: Intermediate S167˜S172 and product A025 were prepared according to the procedures in A024. A025: HRMS (ESI): calcd for C90H118N15O27S+ [M+H]+ 1872.8037, found 1872.8037.

Example 367: Synthesis of A063

To a stirring solution of S173 (10.00, 55.825 mmol) in DMF (100 mL) at room temperature was added S174 (16.55 g, 58.617 mmol). The resulting mixture was stirred at room temperature for 24 h before it was concentrated in vacuo directly. The intermediate S175 was obtained as a yellow oil (22.22 g, 86%) after flash column chromatography purification (hexane:EtOAc=2:1). HRMS (ESI): calcd for C25H24N3O6+ [M+H]+ 462.1660, found 462.1661.

To a stirring solution of S175 (20.00 g, 43.340 mmol) in DMF (200 mL) at room temperature was added piperidine (20 mL). The resulting mixture was stirred at room temperature for 30 min before it was concentrated in vacuo directly. The intermediate S176 was obtained as a yellow solid (9.66 g, 93%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C10H14N3O4+ [M+H]+ 240.0979, found 240.0980.

To a stirring solution of S177 (10.00 g, 66.565 mmol) and S178 (58.43 g, 299.541 mmol) in MeCN (300 mL) at room temperature were added K2CO3 (41.40 g, 299.541 mmol). The resulting mixture was heated to 60° C. and stirred for 24 h before it was cooled to room temperature and concentrated in vacuo directly. The intermediate S179 was obtained as a yellow oil (27.45 g, 84%) after flash column chromatography purification (silica gel, hexane:EtOAc=5:1). HRMS (ESI): calcd for C27H45N2O6+ [M+H]+ 493.3272, found 493.3272.

To a stirring solution of S179 (25.00 g, 50.745 mmol) in MeOH (500 mL) at room temperature was added Pd/C (2.50 g, 10% wt/wt). The resulting mixture was stirred at room temperature under H2 atmosphere for 2 h before it was filtered through a pad of celite. The filtrate was collected and concentrated in vacuo. The intermediate S180 was obtained as a yellow oil (20.42 g, 100%) and used without further purification. HRMS (ESI): calcd for C20H39N2O6+ [M+H]+ 403.2803, found 403.2803.

To a stirring solution of S180 (20.42 g, 50.729 mmol) and S181 (17.43 g, 76.094 mmol) in MeCN (300 mL) at room temperature were added K2CO3 (10.52 g, 76.094 mmol). The resulting mixture was heated to 60° C. and stirred for 24 h before it was cooled to room temperature and concentrated in vacuo directly. The intermediate S182 was obtained as a yellow oil (25.66 g, 92%) after flash column chromatography purification (silica gel, hexane:EtOAc=4:1). HRMS (ESI): calcd for C29H47N2O8+ [M+H]+ 551.3327, found 551.3328.

To a stirring solution of S182 (23.66 g, 42.964 mmol) in CH2Cl2 (150 mL) at room temperature was added TFA (300 mL). The resulting mixture was stirred at room temperature for 2 h before it was concentrated in vacuo directly. The residue was dispersed with MTBE (250 mL) and stirred for 30 min before it was filtered. The filter cake was collected and dried in vacuo. The intermediate S183 was obtained as a yellow solid (13.00 g, 79%) and used without further purification. HRMS (ESI): calcd for C17H23N2O8+ [M+H]+ 383.1449, found 383.1452.

To a stirring solution of S183 (10.00 g, 26.153 mmol) and S184 (33.10 g, 86.304 mmol) in THF (500 mL) at room temperature were added HOBt (11.66 g, 86.304 mmol) and EDCI·HCl (16.54 g, 86.304 mmol) in sequence. The resulting mixture was stirred at room temperature for 5 h before it was quenched with HCl (1.0 M, aq., 200 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (100 mL×3). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S185 was obtained as a yellow oil (27.85 g, 72%) after flash column chromatography purification (CH2Cl2:MeOH=10:1). HRMS (ESI): calcd for C68H128N5O29+ [M+H]+ 1478.8689, found 1478.8693.

To a stirring solution of S185 (5.00 g, 3.38 mmol) in MeOH (100 mL) at room temperature was added Pd/C (0.50 g, 10% wt/wt). The resulting mixture was stirred at room temperature under H2 atmosphere for 2 h before it was filtered through a pad of celite. The filtrate was collected and concentrated in vacuo. The intermediate S186 was obtained as a yellow solid (4.70 g, 100%) and used without further purification. HRMS (ESI): calcd for C61H122N5O29+ [M+H]+ 1388.8220, found 1388.8225.

To a stirring solution of S186 (4.70 g, 3.385 mmol) in DMF (100 mL) at room temperature was added HATU (1.42 g, 3.723 mmol). The resulting mixture was stirred at room temperature for 15 min before addition of S176 (891 mg, 3.723 mmol) and DIPEA (1.31 g, 10.154 mmol) in sequence. The resulting mixture was stirred at room temperature for further 1 h before it was concentrated in vacuo directly. The intermediate S187 was obtained as a yellow solid (4.11 g, 75%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C71H133N8O32+ [M+H]+ 1609.9020, found 1609.9022.

To a stirring solution of S187 (1.00 g, 0.621 mmol) in MeOH (20 mL) at room temperature was added Pd/C (0.10 g, 10% wt/wt). The resulting mixture was stirred at room temperature under H2 atmosphere for 5 h before it was filtered through a pad of celite. The filtrate was collected and concentrated in vacuo. The intermediate S188 was obtained as a yellow solid (980 mg, 100%) and used without further purification. HRMS (ESI): calcd for C71H135N8O30+ [M+H]+ 1579.9279, found 1579.9284.

To a stirring solution of S189 (232 mg, 0.744 mmol) in DMF (10 mL) at room temperature were added HATU (283 mg, 0.744 mmol) and DIPEA (120 mg, 0.930 mmol). The resulting mixture was stirred at room temperature for 15 min before addition of S188 (980 mg, 0.620 mmol). The resulting mixture was stirred at room temperature for further 1 h before it was concentrated in vacuo directly. The intermediate S190 was obtained as a yellow solid (800 mg, 69%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C89H150N9O33+ [M+H]+ 1873.0331, found 1873.0333.

To a stirring solution of S190 (800 mg, 0.427 mmol) in DMF (10 mL) at room temperature was added piperidine (1 mL). The resulting mixture was stirred at room temperature for 30 min before it was concentrated in vacuo directly. The intermediate S191 was obtained as a yellow solid (520 mg, 74%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C74H140N9O31+ [M+H]+ 1650.9650, found 1650.9658.

To a stirring solution of S21 (446 mg, 0.346 mmol) in DMF (10 mL) at room temperature was added HATU (132 mg, 0.346 mmol). The resulting mixture was stirred at room temperature for 15 min before addition of S191 (520 mg, 0.315 mmol) and DIPEA (122 mg, 0.945 mmol) in sequence. The resulting mixture was stirred at room temperature for further 1 h before it was concentrated in vacuo directly. The intermediate S192 was obtained as a yellow solid (600 mg, 65%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C137H237N18O49+ [M+H]+ 2918.6601, found 2918.6605.

To a stirring solution of S192 (500 mg, 0.171 mmol) in CH2Cl2 (10 mL) at 0° C. were added pyridine (28 mg, 0.343 mmol) and S44 (52 mg, 0.257 mmol) in sequence. The resulting mixture was stirred at 0° C. for 2 h before it was quenched with NaHCO3 (sat. aq., 5 mL). The layers were separated, and the aqueous layer was extracted with CH2Cl2 (10 mL×3). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S193 was obtained as a yellow oil (425 mg, 80%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C144H240N19O53+ [M+H] 3083.6663, found 3083.6666.

To a stirring solution of S193 (400 mg, 0.130 mmol) in DMF (10 mL) at room temperature were added HOBt (21 mg, 0.156 mmol) and S34 (83 mg, 0.156 mmol). The resulting mixture was stirred at room temperature for 6 h before it was concentrated in vacuo directly. The intermediate S194 was obtained as a dark yellow oil (428 mg, 100%) and used without further purification. HRMS (ESI): calcd for C162H257FN21O54+ [M+H]+ 3379.7988, found 3379.7988.

To a stirring solution of S194 (428 mg, 0.127 mmol) in CH2Cl2 (5 mL) at room temperature was added TFA (2.5 mL). The resulting mixture was stirred at room temperature for 1 h before it was concentrated in vacuo directly. The product A063 was obtained as a yellow solid (125 mg, 31% over 2 steps) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C150H233FN21O54+ [M+H]+ 3211.6110, found 3211.6111.

Example 368: Synthesis of A037

To a stirring solution of S195 (5.00 g, 14.821 mmol) in CH2Cl2 (100 mL) at 0° C. were added HATU (5.92 g, 15.562 mmol), S9 (3.79 g, 15.562 mmol) and DIPEA (4.79 g, 37.053 mmol) in sequence. The resulting mixture was warmed to room temperature and stirred for 2 h before it was quenched with citric acid (10% wt/wt, aq., 20 mL). The layers were separated, and the aqueous layer was washed with NaCl (sat. aq., 20 mL). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S196 was obtained as a white solid (7.70 g, 98%) by recrystallization (hexane:CH2Cl2=20:1, room temperature). HRMS (ESI): calcd for C29H39N2O7+ [M+H]+ 527.2752, found 527.2755.

To a stirring solution of S196 (7.50 g, 14.241 mmol) in CH2Cl2 (100 mL) at room temperature was added TFA (33 mL). The resulting mixture was stirred at room temperature for 2 h before it was concentrated in vacuo directly. The intermediate S197 was obtained as a yellow oil (6.70 g, 100%) and used without further purification. HRMS (ESI): calcd for C25H31N2O7+ [M+H]+ 471.2126, found 471.2131.

To a stirring solution of S198 (5.00 g, 21.079 mmol) and S12 (12.85 g, 46.374 mmol) in CH2Cl2 (100 mL) at 0° C. was added EDCI (8.89 g, 46.374 mmol). The resulting mixture was warmed to room temperature and stirred for 2 h before it quenched with citric acid (10% wt/wt, aq., 20 mL). The layers were separated, and the aqueous layer was washed with NaCl (sat. aq., 20 mL). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S199 was obtained as a white solid (15.20 g, 95%) by recrystallization (hexane:CH2Cl2=5:1, room temperature). HRMS (ESI): calcd for C36H62N5O12+ [M+H]+ 756.4389, found 756.4396.

To a stirring solution of S199 (7.50 g, 19.844 mmol) in CH2Cl2 (100 mL) at room temperature was added TFA (50 mL). The resulting mixture was stirred at room temperature for 2 h before it was concentrated in vacuo directly. The intermediate S200 was obtained as a yellow solid (14.60 g, 94%) with stirred with MTBE (100 mL). HRMS (ESI): calcd for C26H46N5O8+ [M+H]+ 556.3341, found 556.3342.

To a stirring solution of S200 (10.00 g, 12.760 mmol) and S15 (6.24 g, 28.071 mmol) in CH2Cl2 (150 mL) at 0° C. was added EDCI (5.38 g, 28.071 mmol). The resulting mixture was warmed to room temperature and stirred for 2 h before it quenched with citric acid (10% wt/wt, aq., 20 mL). The layers were separated, and the aqueous layer was washed with NaCl (sat. aq., 20 mL). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S201 was obtained as a yellow oil (11.30 g, 92%) after flash column chromatography purification (CH2Cl2:MeOH=10:1). HRMS (ESI): calcd for C50H70N5O14+ [M+H]+ 964.4914, found 964.4918.

To a stirring solution of S201 (11.30 g, 11.721 mmol) in CH2Cl2 (100 mL) at 40° C. was added HCO2H (200 mL). The resulting mixture was stirred at 40° C. for 12 h before it was concentrated in vacuo directly. The residue was dissolved with water (100 mL) and extracted with EtOAc (100 mL×3). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S202 was obtained as a white solid (7.65 g, 76%) by recrystallization (hexane:EtOAc=1:1, room temperature). HRMS (ESI): calcd for C42H54N5O14+ [M+H]+ 852.3662, found 852.3663.

To a stirring solution of S202 (5.00 g, 5.869 mmol) in CH2Cl2 (150 mL) at room temperature were added HATU (4.69 g, 12.325 mmol) and DIPEA (2.28 g, 17.607 mmol) in sequence. The resulting mixture was stirred at room temperature for 0.5 h before addition of a solution of 7 (7.23 g, 12.325 mmol) in CH2C12 (80 mL). The resulting mixture was stirred at room temperature for further 1 h before it was quenched with HCl (1 M aq, 50 mL). The layers were separated and the aqueous layer was extracted with CH2Cl2 (3× 50 mL). The combined organic layers were washed with NaCl (sat. aq., 50 mL), dried (Na2SO4) and concentrated in vacuo. The intermediate S203 was obtained as a yellow oil (7.77 g, 67%) after flash column chromatography purification (silica gel, CH2Cl2:MeOH=10:1). HRMS (ESI): calcd for C100H158N13O28+ [M+H]+ 1989.1334, found 1989.1335.

To a stirring solution of S203 (7.77 g, 3.906 mmol) in MeOH (150 mL) at room temperature was added Pd/C (0.77 g, 10% wt/wt). The resulting mixture was stirred at room temperature under H2 atmosphere for 5 h before it was filtered through a pad of celite. The filtrate was collected and concentrated in vacuo. The intermediate S204 was obtained as a yellow solid (5.15 g, 71%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C92H152N13O26+ [M+H]+ 1855.0966, found 1855.0970.

To a stirring solution of S197 (500 mg, 1.063 mmol) in DMF (10 mL) at room temperature was added HATU (425 mg, 1.116 mmol). The resulting mixture was stirred at room temperature for 15 min before addition of S204 (2.07 g, 1.116 mmol) in DMF (10 mL) and DIPEA (344 mg, 2.657 mmol) in sequence. The resulting mixture was stirred at room temperature for further 1 h before it was concentrated in vacuo directly. The intermediate S205 was obtained as a dark yellow oil (2.45 g, 100%) and used without further purification. HRMS (ESI): calcd for C117H180N15O32+ [M+H]+ 2307.2913, found 2307.2914.

To a stirring solution of S204 (2.45 g, 1.062 mmol) in MeOH (100 mL) at room temperature was added Pd/C (245 mg, 10% wt/wt). The resulting mixture was stirred at room temperature under H2 atmosphere for 2 h before it was filtered through a pad of celite. The filtrate was collected and concentrated in vacuo. The intermediate S205 was obtained as a dark yellow oil (2.21 g, 100%) and used without further purification. HRMS (ESI): calcd for C102H168N15O30+ [M+H]+ 2083.2076, found 2083.2077.

To a stirring solution of S206 (2.21 g, 1.061 mmol) in DMF (50 L) at room temperature were added S20 (556 mg, 1.591 mmol) and DIPEA (275 mg, 2.121 mmol). The resulting mixture was stirred at room temperature for 3 h before it was concentrated in vacuo directly. The intermediate S207 was obtained as a light yellow solid (655 mg, 27% over 3 steps) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C110H175N16O33+ [M+H]+ 2248.2502, found 2248.2504.

To a stirring solution of S207 (655 mg, 0.291 mmol) in DMF (10 mL) at room temperature was added HATU (116 mg, 0.306 mmol). The resulting mixture was stirred at room temperature for 15 min before addition of S36 (190 mg, 0.306 mmol) and DIPEA (113 mg, 0.874 mmol) in sequence. The resulting mixture was stirred at room temperature for further 1 h before it was concentrated in vacuo directly. The intermediate S208 was obtained as a dark yellow oil (797 mg, 100%) and used without further purification. HRMS (ESI): calcd for C137H200FN20O37+ [M+H]+ 2736.4362, found 2736.4364.

To a stirring solution of S208 (797 mg, 0.291 mmol) in CH2Cl2 (5 mL) at room temperature was added TFA (10 mL). The resulting mixture was stirred at room temperature for 4 h before it was concentrated in vacuo directly. The product A037 was obtained as a yellow solid (225 mg, 36% over 2 steps) with RP-HPLC and lyophilization. HRMS (ESI): calcd for C113H152FN20O37+ [M+H]+ 2400.0606, found 2400.0609.

To a stirring solution of S19 (200 mg, 0.178 mmol) in DMF (2 mL) at room temperature were added S220 (116 mg, 0.214 mmol) and DIPEA (35 mg, 0.214 mmol). The resulting mixture was stirred at room temperature for 3 h before it was concentrated in vacuo directly. The intermediate S221 was obtained as a light yellow solid (232 mg, 88%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C71H104N11O23+ [M+H]+ 1478.7301, found 1478.7302.

To a stirring solution of S221 (232 mg, 0.157 mmol) in DMF (10 mL) at room temperature was added HATU (63 mg, 0.165 mmol). The resulting mixture was stirred at room temperature for 15 min before addition of S36 (103 mg, 0.165 mmol) and DIPEA (61 mg, 0.471 mmol) in sequence. The resulting mixture was stirred at room temperature for further 1 h before it was concentrated in vacuo directly. The intermediate S222 was obtained as a dark yellow oil (304 mg, 100%) and used without further purification. HRMS (ESI): calcd for C96H125FN15O27+ [M+H]+ 1938.8848, found 1938.8852.

To a stirring solution of S222 (304 mg, 0.157 mmol) in CH2Cl2 (2 mL) at room temperature was added TFA (4 mL). The resulting mixture was stirred at room temperature for 4 h before it was concentrated in vacuo directly. The product A028 was obtained as a yellow solid (142 mg, 51% over 2 steps) with RP-HPLC and lyophilization. HRMS (ESI): calcd for C84H101FN15O27+ [M+H]+ 1770.6970, found 1770.6971.

A029 and A030 were prepared according to procedures in A028 using corresponding component part (S223 and S226).

Example 383˜385: Synthesis of A059, A062 and A064

Example 386: Synthesis of A065

To a stirring solution of S23 (183 mg, 0.967 mmol) in DMF (10 mL) at room temperature were added HATU (368 mg, 0.967 mmol) and DIPEA (313 mg, 2.417 mmol) in sequence. The resulting mixture was stirred at room temperature for 30 min before addition of S34 (500 mg, 0.806 mmol). The resulting mixture was stirred at room temperature for further 1 h before it was quenched with EtOAc (50 mL) and filtered. The filter cake was collected and dried in vacuo. The intermediate S235 was obtained as a yellow solid (513 mg, 93%) and used without further purification. HRMS (ESI): calcd for C35H41FN5O8+ [M+H]+ 678.2934, found 678.2935.

To a stirring solution of S235 (513 mg, 0.757 mmol) in CH2Cl2 (10 mL) at room temperature was added TFA (5 mL). The resulting mixture was stirred at room temperature for 2 h before it was concentrated in vacuo directly. The intermediate S236 was obtained as a yellow solid (480 mg, 91%) after trituration with MTBE (80 mL). HRMS (ESI): calcd for C30H33FN5O6+ [M+H]+ 578.2409, found 578.2410.

Intermediate S238˜S246 were prepared according to the procedures in A001 using 237 as corresponding component part.

To a stirring solution of S246 (200 mg, 0.159 mmol) in DMF (10 mL) at room temperature was added HATU (64 mg, 0.167 mmol). The resulting mixture was stirred at room temperature for 15 min before addition of S236 (116 mg, 0.167 mmol) and DIPEA (52 mg, 0.397 mmol) in sequence. The resulting mixture was stirred at room temperature for further 1 h before it was concentrated in vacuo directly. The intermediate S247 was obtained as a dark yellow oil (288 mg, 100%) and used without further purification. HRMS (ESI): calcd for C91H126FN14O24+ [M+H]+ 1817.9048, found 1817.9050.

To a stirring solution of S247 (288 mg, 0.158 mmol) in CH2Cl2 (2 mL) at room temperature was added TFA (4 mL). The resulting mixture was stirred at room temperature for 4 h before it was concentrated in vacuo directly. The product A065 was obtained as a yellow solid (126 mg, 48% over 2 steps) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C79H102FN14O24+ [M+H]+ 1649.7170, found 1649.7174.

Example 387: Synthesis of A066

To a stirring solution of S173 (1.00, 5.583 mmol) in DMF (20 mL) at room temperature was added S184 (2.26 g, 5.893 mmol). The resulting mixture was stirred at room temperature for 24 h before it was concentrated in vacuo directly. The intermediate S248 was obtained as a yellow oil (2.99 g, 95%) after flash column chromatography purification (hexane:EtOAc=2:1). HRMS (ESI): calcd for C25H43N2O12+ [M+H]+ 563.2811, found 563.2812.

To a stirring solution of S248 (2.99 g, 5.315 mmol) in MeOH (50 mL) at room temperature was added Pd/C (300 mg, 10% wt/wt). The resulting mixture was stirred at room temperature under H2 atmosphere for 12 h before it was filtered through a pad of celite. The filtrate was collected and concentrated in vacuo. The intermediate S249 was obtained as a yellow oil (2.83 g, 100%) and used without further purification. HRMS (ESI): calcd for C25H45N2O10+ [M+H]+ 533.3069, found 533.3070.

To a stirring solution of S189 (1.99 g, 6.376 mmol) in DMF (200 mL) at room temperature were added HATU (2.42 g, 6.376 mmol) and DIPEA (1.37 g, 10.627 mmol) in sequence. The resulting mixture was stirred at room temperature for 30 min before addition of S249 (2.83 g, 5.313 mmol). The resulting mixture was stirred at room temperature for further 1 h before it was concentrated in vacuo directly. The residue was dissolved in CH2Cl2 (200 mL) again and washed with Na2CO3 (5% wt/wt aq., 50 mL) and HCl (1 M aq., 50 mL) in sequence. The organic layer was dried (Na2SO4) and concentrated in vacuo. The intermediate S250 was obtained as a yellow oil (4.39 g, 100%) and used without further purification. HRMS (ESI): calcd for C43H60N3O13+ [M+H]+ 826.4121, found 826.4122.

To a stirring solution of S250 (4.39 g, 5.315 mmol) in DMF (100 mL) at room temperature was added piperidine (10 mL). The resulting mixture was stirred at room temperature for 15 min before it was concentrated in vacuo directly. The intermediate S251 was obtained as a yellow oil (2.15 g, 67%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C28H50N3O11+ [M+H]+ 604.3440, found 604.3441.

To a stirring solution of S197 (2.08 g, 4.430 mmol) in DMF (100 mL) at room temperature were added HATU (1.68 g, 4.430 mmol). The resulting mixture was stirred at room temperature for 30 min before addition of S252 (1.00 g, 4.027 mmol) and DIPEA (1.30 g, 10.068 mmol) in sequence. The resulting mixture was stirred at room temperature for further 1 h before it was concentrated in vacuo directly. The residue was dissolved in CH2Cl2 (100 mL) again and washed with Na2CO3 (5% wt/wt aq., 20 mL) and HCl (1 M aq., 20 mL) in sequence. The organic layer was dried (Na2SO4) and concentrated in vacuo. The intermediate S253 was obtained as a yellow oil (2.50 g, 88%) after flash column chromatography purification (silica gel, CH2Cl2:MeOH=10:1). HRMS (ESI): calcd for C36H53N4O10+ [M+H]+ 701.3756, found 701.3760.

Intermediate S254˜ S260 were prepared according to the procedures in A001 with 255 as corresponding component part.

To a stirring solution of S260 (2.13 g, 1.739 mmol) in DMF (100 mL) at room temperature were added HATU (662 mg, 1.739 mmol). The resulting mixture was stirred at room temperature for 30 min before addition of S251 (1.00 g, 1.656 mmol) and DIPEA (536 mg, 4.141 mmol) in sequence. The resulting mixture was stirred at room temperature for further 1 h before it was concentrated in vacuo directly. The residue was dissolved in CH2Cl2 (100 mL) again and washed with Na2CO3 (5% wt/wt aq., 20 mL) and HCl (1 M aq., 20 mL) in sequence. The organic layer was dried (Na2SO4) and concentrated in vacuo. The intermediate S253 was obtained as a yellow oil (3.00 g, 100%) after flash column chromatography purification (silica gel, CH2Cl2:MeOH=10:1). HRMS (ESI): calcd for C85H143N120301 [M+H]+ 1812.0028, found 1812.0029.

To a stirring solution of S253 (3.00 g, 1.656 mmol) in CH2Cl2 (50 mL) at 0° C. were added pyridine (262 mg, 3.311 mmol) and S44 (500 mg, 2.483 mmol) in sequence. The resulting mixture was stirred at 0° C. for 2 h before it was quenched with NaHCO3 (sat. aq., 10 mL). The layers were separated, and the aqueous layer was extracted with CH2Cl2 (3× 20 mL). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S262 was obtained as a white solid (2.44 g, 74% over 3 steps) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C92H146N13O34+ [M+H]+ 1977.0090, found 1977.0092.

To a stirring solution of S262 (500 mg, 0.253 mmol) in DMF (10 mL) at room temperature were added HOBt (41 mg, 0.303 mmol), S34 (162 mg, 0.303 mmol) and DIPEA (99 mg, 0.759 mmol) in sequence. The resulting mixture was stirred at room temperature for 5 h before it was concentrated in vacuo directly. The intermediate S263 was obtained as a dark yellow oil (575 mg, 100%) and used without further purification. HRMS (ESI): calcd for C110H163FN15O35+ [M+H]+ 2273.1415, found 2273.1417.

To a stirring solution of S263 (575 mg, 0.253 mmol) in CH2Cl2 (2 mL) at room temperature was added TFA (4 mL). The resulting mixture was stirred at room temperature for 4 h before it was concentrated in vacuo directly. The product A066 was obtained as a yellow solid (110 mg, 20% over 2 steps) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C98H139FN15O35+ [M+H] 2104.9537, found 2104.9538.

Example 388: Synthesis of A067

Intermediate S264˜S271 and A067 were prepared according to the procedures in A066 using S15 as corresponding component part.

Example 389: Synthesis of A068

Intermediate S273˜S276 and A068 were prepared according to the procedures in A039 with S272 as corresponding component part.

Intermediate S288˜S294 and product A058, A060 and A061 were prepared according to the procedures in A028 with corresponding component part (S220, S223 and S226).

Example 406: Synthesis of A069

To a stirring solution of S295 (334 mg, 0.808 mmol) and S14 (500 mg, 0.673 mmol) in CH2C12 (20 mL) at room temperature were added EDCI (156 mg, 0.808 mmol) and DIPEA (174 mg, 1.346 mmol) in sequence. The resulting mixture was stirred at room temperature for 3 h before it was quenched with Na2CO3 (5% wt/wt aq., 5 mL). The layers were separated, and the aqueous layer was extracted with CH2Cl2 (3×5 mL). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S296 was obtained as a yellow oil (688 mg, 100%) and used without further purification. HRMS (ESI): calcd for C56H75N6O12+ [M+H]+ 1023.5437, found 1023.5438.

To a stirring solution of S296 (388 mg, 0.673 mmol) in CH2Cl2 (10 mL) at room temperature was added TFA (5 mL). The resulting mixture was stirred at room temperature for 1 h before it was concentrated in vacuo directly. The intermediate S297 was obtained as a yellow solid (400 mg, 57% over 2 steps) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C51H67N6O10+ [M+H]+ 923.4913, found 923.4913.

To a stirring solution of S298 (249 mg, 1.230 mmol) and S4 (500 mg, 1.025 mmol) in CH2Cl2 (20 mL) at room temperature was added EDCI (238 mg, 1.230 mmol). The resulting mixture was stirred at room temperature for 3 h before it was quenched with Na2CO3 (5% wt/wt aq., 5 mL). The layers were separated, and the aqueous layer was extracted with CH2Cl2 (3× 5 mL). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The intermediate S299 was obtained as a yellow oil (723 mg, 100%) and used without further purification. HRMS (ESI): calcd for C37H60N3O10+ [M+H]+ 706.4273, found 706.4276.

To a stirring solution of S299 (723 mg, 1.025 mmol) in MeOH (10 mL) at room temperature was added Pd/C (73 mg, 10% wt/wt). The resulting mixture was stirred at room temperature under H2 atmosphere for 2 h before it was filtered through a pad of celite. The filtrate was collected and concentrated in vacuo. The intermediate S300 was obtained as a yellow oil (550 mg, 87%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C30H54N3O10+ [M+H]+ 616.3804, found 616.3808.

To a stirring solution of S300 (200 mg, 0.325 mmol) in DMF (10 mL) at room temperature were added HATU (130 mg, 0.341 mmol) and DIPEA (126 mg, 0.974 mmol) in sequence. The resulting mixture was stirred at room temperature for 20 min before addition of S297 (354 mg, 0.341 mmol). The resulting mixture was stirred at room temperature for further 1 h before it was concentrated in vacuo directly. The residue was dissolved in CH2Cl2 (20 mL) again and washed with Na2CO3 (5% wt/wt aq., 5 mL) and HCl (1 M aq., 5 mL) in sequence. The organic layer was dried (Na2SO4) and concentrated in vacuo. The intermediate S301 was obtained as a dark yellow oil (494 mg, 100%) and used without further purification. HRMS (ESI): calcd for C81H118N9O19+ [M+H]+ 1520.8538, found 1520.8540.

To a stirring solution of S301 (494 mg, 0.325 mmol) in MeOH (10 mL) at room temperature was added Pd/C (50 mg, 10% wt/wt). The resulting mixture was stirred at room temperature under H2 atmosphere for 5 h before it was filtered through a pad of celite. The filtrate was collected and concentrated in vacuo. The intermediate S302 was obtained as a yellow solid (380 mg, 90%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C66H106N9O17+ [M+H]+ 1296.7701, found 1296.7701.

To a stirring solution of S302 (300 mg, 0.231 mmol) in DMF (5 mL) at room temperature were added S20 (122 mg, 0.347 mmol) and DIPEA (60 mg, 0.463 mmol) in sequence. The resulting mixture was stirred at room temperature for 2 h before it was concentrated in vacuo. The intermediate S303 was obtained as a white solid (250 mg, 74%) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C74H113N10O20+ [M+H]+ 1461.8127, found 1461.8130.

To a stirring solution of S303 (200 mg, 0.137 mmol) in DMF (10 mL) at room temperature was added HATU (55 mg, 0.144 mmol). The resulting mixture was stirred at room temperature for 15 min before S36 (92 mg, 0.144 mmol) and DIPEA (54 mg, 0.410 mmol) were added in sequence. The resulting mixture was stirred at room temperature for further 1 h before it was concentrated in vacuo directly. The intermediate S304 was obtained as a dark yellow oil (266 mg, 100%) and used without further purification. HRMS (ESI): calcd for C101H138FN14O24+ [M+H]+ 1949.9987, found 1949.9990.

To a stirring solution of S305 (266 mg, 0.137 mmol) in CH2Cl2 (2 mL) at room temperature was added TFA (4 mL). The resulting mixture was stirred at room temperature for 4 h before it was concentrated in vacuo directly. The product A069 was obtained as a yellow solid (135 mg, 56% over 2 steps) after RP-HPLC purification and lyophilization. HRMS (ESI): calcd for C89H114FN14O24+ [M+H]+ 1781.8109, found 1781.8110.

Example 407: Synthesis of A070

SPPS synthetic procedures were operated according to general literature guideline. S312. HRMS (ESI): calcd for C71H123N8O27+ [M+H]+ 1519.8492, found 1519.8498. STEP 6:

To a stirring solution of S312 (400 mg, 0.263 mmol) in DMF (10 mL) at room temperature was added HATU (110 mg, 0.289 mmol). The resulting mixture was stirred at room temperature for 30 min before it were added S313 (52 mg, 0.263 mmol) and DIPEA (68 mg, 0.526 mmol) in sequence. The resulting mixture was stirred at room temperature for further 1 h before it was concentrated in vacuo directly. The residue was dissolved in CH2Cl2 (20 mL) again and washed with Na2CO3 (5% wt/wt aq., 5 mL) and HCl (1 M aq., 5 mL) in sequence. The organic layer was dried (Na2SO4) and concentrated in vacuo. The intermediate S314 was prepared as a dark yellow oil (446 mg, 100%) and used without further purification. HRMS (ESI): calcd for C81H135N10O28+ [M+H]+ 1695.9442, found 1695.9444.

To a stirring solution of S314 (446 mg, 0.263 mmol) in DMF (10 mL) at room temperature were added S315 (96 mg, 0.316 mmol), DMAP (32 mg, 0.263 mmol) and DIPEA (170 mg, 1.315 mmol). The resulting mixture was stirred at room temperature for 2 h before it were added exatecane meslyate (210 mg, 0.394 mmol) and HOBt (54 mg, 0.394 mmol) in sequence. The resulting mixture was stirred at room temperature for further 6 h before it was concentrated in vacuo directly. The intermediate S317 was prepared as a dark yellow oil (142 mg, 25%) and used without further purification. HRMS (ESI): calcd for C106H155FN13O33+ [M+H]+ 2157.0829, found 2157.0833.

To a stirring solution of S317 (142 mg, 0.066 mmol) in CH2Cl2 (1 mL) at room temperature was added TFA (1 mL). The resulting mixture was stirred at room temperature for 2 h before it was concentrated in vacuo directly. The residue was dispersed with MTBE (20 mL) and slurrying for 30 min before it was filtered. The filter cake was collected. The intermediate S318 was prepared as a yellow solid (120 mg, 91%) through RP-HPLC and lyophilization. HRMS (ESI): calcd for C94H131FN13O33+ [M+H]+ 1988.8951, found 1988.8952.

To a stirring solution of 318 (120 mg, 0.060 mmol) in DMF (10 mL) at room temperature was added HATU (83 mg, 0.217 mmol). The resulting mixture was stirred at room temperature for 30 min before it were added gemcitabine (64 mg, 0.241 mmol) and DIPEA (56 mg, 0.434 mmol) in sequence. The resulting mixture was stirred at room temperature for further 1 h before it was concentrated in vacuo directly. The product A070 was prepared as a dark yellow oil (90 mg, 54%) and used without further purification. HRMS (ESI): calcd for C121H158F7N22O42+ [M+H]+ 2724.0787, found 2724.0787.

Example 408. The structures of the conjugates that were prepared by both the traditional conjugation process and the homogeneous conjugation process are illustrated below:

Experiment 409: Procedure for Recombinant protein/antibody expression, production and purification:

Expression vector construction: DNA sequences encoding the HC and LC of the Steap-1 antibodies were synthesized in General BioL (Anhui, China), and then subcloned into plasmids GS2U and modified pTT5 expression vectors. For transient expression, modified pTT5 vectors containing HC and LC genes were co-transfected into CHO-K1 cells. Cells were cultured for 5 days and supernatant was collected for protein purification using Protein A column or Protein G column. For stable expression, GS2U vectors containing HC and LC genes were co-transfected into CHO-K1 cells. At day 2, cells were diluted to 1×105 cells/mL, and MSX was added to final concentration 25 μM, followed by culture for 10-15 days then subcloned in 96 well plates. Clones with high and stable expression were selected for antibody production at 1 L, and then 5 L, 10 L, 25 L, 200 L or 500 L through various steps of optimization controls of pH, and gases, as well of adjustment of addition of medias, vitamins, metal ions and sugars. After production, the antibody was filtered, purified by Protein A affinity chromatography, anion exchange chromatography and cation exchange chromatography to afford >98% pure, with overall >70% yield of the Steap-1 antibodies.

Experiment 410: Procedure for Conjugation

Traditional Conjugation: A monoclonal antibody was conjugated to a cytotoxin/linker complex having a terminal of maleimido group. Specifically, purified antibody was incubated with a 2.0-12.0 equivalents of the reducing agent TCEP (Tris(2-carboxyethyl) phosphine) in PBS pH 6.2-7.5, 1 mM EDTA (Ethylenediamine tetraaceticacid) for 1 hours at 37° C. Subsequently, 5.0-12.0 equivalents of the payload of a cytotoxin/linker complex having a terminal of maleimido group from a stock solution in 10% (v/v) DMA or DMSO was added, followed by incubation at room temperature for 1 hour to 3 hours under gentle rotation. The conjugation reaction was optionally quenched by the addition of 4 equivalents (over the payload) of N-acetyl cysteine, and the TCEP was optionally quenched by the addition of 1 or 2 equivalents of 4-azidomethylbenzoic acid or 4-azidobenzoic acid. After incubation, the reducing agent and the excess payload/linker complexes were removed by 2-10 times of dialysis in PBS pH 5.0-7.2, at 4° C. using 20,000 MWCO dialysis cassettes or purified by ion exchange chromatography. For the payload/linker complex containing a disulfide bond, the reduced antibody was isolated through a chromatography (with ion exchange or size exclusion column) or dialysis prior to run conjugation reaction. For Dxd-GGFG conjugation, the conjugates with DAR >7.2 were purified against formulated buffers (normally, 0.02% Tween-20 or Tween-80, 6-7% sugar, 20-50 mM histine, pH=5.5-7.0).

The conjugation process may result in 0.1 to 10% of aggregate formation (e.g. Steap1-GGFG-Dxd ADC). Thus, macromolecular aggregates, conjugation reagents, including payloads quenched by cysteine, and other added regents can be removed using ceramic hydroxyapatite Type II chromatography (CHT) as described in e.g. Thompson et al., J. Control Release, 236:100-116 (2016) or by ion exchange chromatography. The ADCs were optionally formulated in 25 mM Histidine-HCl, or citrate buffer containing 6-7% sucrose, 0.02% Tween-20 or Tween-80, and 0.1% methionine, at pH 5.0-6.5.

Experiment 411: Preparation of ADC of the Present Invention Via the Homogeneous Conjugation Reaction:

A zinc amino complex (e.g. Zinc 2-methylpropane-1,2-diamine chloride complex) (in 10-60 mM, 1.0-5.0 eq. of an antibody used) and TCEP (in 100 mM, 2.5-7.5 eq. of an antibody used) were added in sequence to a solution containing the antibody (10-50 mg/mL, in 20 mM PBS, pH 5.5-7.5) at 2-8° C. After incubation at 2-8° C. for 12-20 h (overnight), a payload/linker complex (100-200 mM, 2.0-15.0 eq) was introduced and the incubation was continued for further 2-4 h. For the payload/linker contain a disulfide bond, the excess of reductant, such as TCEP, is removed from the reaction mixture through column (affinity, anion or cation exchange) chromatography or UF/DF prior to addition of the payload/linker complex. After the incubation, cystine or 4-(azidomethyl)benzoic acid or 4-azidobenzoic acid (100-200 mM, 4.0-10.0 eq) was added to deplete the excess TCEP; cysteine (100-200 mM, 2.0-10.0 eq) was added to deplete the excess payload/linker complex; EDTA (100-200 mM, 4.0-6.0 eq) was added to trap zinc ion; and DHAA (100-200 mM, 8.0-30.0 eq) was added to oxidize the free thiol groups in the antibody. The reaction mixture was finally purified using a de-salting column (Zeba Spin Desalting Columns, 40K MWCO), or UF/DF, or ion exchange chromatography, and drug/antibody ratios (DAR) were analyzed using HIC-HPLC or HPLC-MS. For the payload/linker complex containing a disulfide bond, the reduced antibody was isolated at 2-8° C. through a chromatography (with ion exchange or size exclusion column) or dialysis prior to running conjugation reaction. In general, average DARs of the invention conjugates by either UV or HIC-HPLC were controlled either 4.0=0.4 or 6.0±0.5. For the two steps of conjugation of two types of payload/linker complexes containing the similar maleimide group or the other thiol reactable groups, the first step reaction can use the homogenous conjugation reaction to conjugate the first functional payload, then the second step is chosen to use Traut's regent to introduce thiols through reaction of a lysine of an antibody and then simultaneously conjugate the second functional payload. It can be performed Traut's regent reaction with the first functional payload, then conducted conjugation of the second functional payload with the homogeneous conjugation reaction.

Experiment 412: General Formulation of the Conjugates.

In a liquid formulation of 80 mg of each conjugate of the invention in a 10 mL of borosilicate vial containing 240 mg of sucrose and 0.8 mg of Tween-80, 24 mg of sodium citrate in 4 mL of sterile water were adjusted with citric acid to pH 5.5. Then each of the conjugate solution was lyophilized at temperature from −65° C. to 0° C., and to RT at reduced pressure (5˜10 torr) to form a dryness cake. The conjugate cakes were stored at 2˜ 8° C., and then reconstituted with 4 mL of sterile water for further application.

For PC-3-4H7, parent cell line PC-3 from Nanjing Cobioer Biosciences Co., was introduced with pasmid co-expressing both human PSMA and RFP-Neomycin fusion protein and plasmid co-expressing human Steap1 and GFP-Blasticidin fusion protein. The high expression cells were selected by neomycin and Blasticidin, and the selected clone 4H7 having high expression of both PSMA and Steap1 was verified by FACs and chosen for further in vitro and in vivo studies.

For PC-3-4G5, parent cell line PC-3 was introduced with pasmid expressing RFP-Neomycin fusion protein and plasmid co-expressing human Steap1 and GFP-Blasticidin fusion protein. The high expression cells were selected by neomycin and Blasticidin, and the selected clone 4G5 having high expression of Steap1 was verified by FACs and chosen for further in vitro and in vivo studies.

For GUCY2C-9C9, parent cell line GUCY2C was introduced with pasmid expressing RFP-Neomycin fusion protein and plasmid co-expressing human GUCY2C and GFP-Blasticidin fusion protein. The high expression cells were selected by neomycin and Blasticidin, and the selected clone 9C9 having high expression of GUCY2C was verified by FACs and choosen for further in vitro and in vivo studies.

Experiment 414: Affinity Measurement of the Antibody and ADCs by EELISA.

Antigens were immobilized on the surface of polystyrene microplate wells at a concentration of 1 μg/mL and incubated overnight at 4° C. For blocking, 200 μL of 5% BSA in PBS was added to each well and incubated for 1 hour at 37° C. Subsequently, the wells were washed three times with 300 μL of PBST. The antibody or the ADC was then diluted to the starting concentration, followed by serial dilutions, and added to the microplate wells. The immobilized antigens were incubated with antigen-specific primary antibodies that affinity-bound to the antigens. Afterward. a HRP-conjugated secondary antibody was added and incubated for 1 hour. The microplate wells were then washed three times with 300 μL of PBST. Following TMB color development, the absorbances were measured using a microplate reader.

Experiment 415: Characterization of the Antibody and the ADC Conjugate.

To determine monomeric content, aggregates, and fragments of ADCs, analytical size-exclusion chromatography (SEC-HPLC) was performed using 100 μg (100 μL volume) of antibodies or ADCs, which were loaded into a TSKgel® G3000WXL column (Tosoh Bioscience, Tokyo, Japan). The mobile phase was composed of 0.1 M sodium sulfate, 0.1 M sodium phosphate, and 10% isopropanol, pH 6.0˜7.0. The flow rate was 1 mL/min, and each analysis was carried out for 10-45 minutes at room temperature.

Experiment 416: Reduced Molecular Weight and DAR Analysis for the Deglycosylated ADCs by LC-MS.

Sample preparation: Reduction of an ADC with 5 mM dithiothreitol at 37° C. for about 2 h, followed by a deglycosylation step with PNGase F at 37° C. overnight generated six or more fragments. HC and LC existed as naked or conjugated forms carrying some payloads. The masses of each ADC fragments and the average DARs of the ADC can be detected. The following equation was used for average DAR calculation for conventional conjugated ADC.

Experiment 417: DAR analysis by HIC-HPLC:

DAR was analyzed by using HIC-HPLC, and the HPLC parameters are as follow Table 1:

The condition for DAR analysis by HIC-HPLC.

Phase B
100 mM NaH2PO4,

Sample
Dilute with buffer A to about 2 mg/mL, injection volume

Experiment 418: Drug Conjugation Site Analysis by MS

Sample Preparation

A 500 μg sample was dissolved in Urea (to a final concentration of approximately 5.9 mol/L), followed by the addition of an appropriate amount of DTT (final concentration 9.8 mmol/L). The reaction system was placed in a water bath at 56° C. for 40 minutes to denature and reduce the sample. After denaturation and reduction, the sample was removed from the water bath and allowed to cool to room temperature. An appropriate amount of IAM (to a final concentration of approximately 29 mmol/L) was added, and the sample was reacted at room temperature in the dark for 40 minutes to alkylate and block free thiol groups. Following the alkylation reaction, the sample was diluted with six volumes of 50 mmol/L Tris buffer (pH 7.0). The sample was then mixed with Trypsin enzyme at a 50:1 (w/w) ratio and incubated at 37° C. for 4 hours to perform the enzymatic digestion. The digestion was quenched by adding formic acid to a final concentration of approximately 0.5% (v/v) for subsequent analysis.

Data acquisition was performed using a Waters ACQUITY UPLC ultra-performance liquid chromatography system interfaced with a Waters Xevo-G2XS Q-TOF mass spectrometer. A Waters Acquity UPLC Protein BEH C18, 1.7 μm, 2.1×100 mm column was used. A volume of 5 μL of the digested ADC solution was injected onto the column with a 0.2 mL/min flow rate and set the column temperature at 60° C. The mobile phase system was composed of the following: mobile phase A was HPLC-grade water with 0.1% formic acid, mobile phase B was HPLC-grade acetonitrile with 0.1% formic acid. The gradient program consisted of a 95 min linear gradient from 1% to 40% B, followed by an increase to 80% B in 10 min, then a 5 min hold at 80% B, next a decrease to 1% B in 1 min, finally re-equilibrating at 1% B for 9 min.

The Waters Xevo-G2XS Q-TOF mass spectrometer is operated in sensitive mode with a capillary voltage of 3.0 kV. The sample cone was set to 40 V. Source and desolvation temperature were set at 100 and 300° C., respectively. Desolvation and cone gas flows were set at 500 and 50 L/h, respectively. Mass range was from 100 m/z to 2500 m/z. Low energy CE and High energy CE were set at 6 and 15-45 V, respectively.

The mass raw data analysis was conducted using the UNIFI software.

As data indicated in FIGS. 1 and 2, and in Table 2, by using liquid chromatography-mass spectrometry (LC-MSF), the drug conjugation sites in vandortuzumab-ADCs were identified. The primary drug conjugation sites of the Steap1-ADC were almost exclusively located at the cysteine residues SC (227) DK of the heavy and GEC (220) of the light chain, when DAR was control at ˜4.0 by homogeneous conjugation.

As data indicated in FIGS. 3 and 4, and in Table 3, when DAR was control at ˜6.0 by homogeneous conjugation, the drug conjugation sites in Steap1-A002 can be identified through using liquid chromatography-mass spectrometry (LC-MSE). The primary drug conjugation sites of Steap1-A002 ADC were almost exclusively located on the cysteine residues SC (227) DK of the heavy and GEC (220) of the light chain and the cysteine residue THTC(233)PPCPAPELLGGPSVFLFPPKPK in (the upper level of) the hinge region peptide segment.

Mass Information for the Identification of Drug-Conjugated Sites

in a Steap1-ADC at DAR = 4.0

Observed 
Observed 
Expected 
Mass error

indicates data missing or illegible when filed

Mass Information for the Identification of Drug-Conjugated

Sites in a Steap1-ADC at DAR = 5.95

Observed RT
Observed mass
Expected 
Mass

Experiment 419: In Vitro Efficacy of Antibody-Drug Conjugates

The cell lines used in the cytotoxicity assays were: C4-2B Cells which was obtained from ATCC through license agreement with MD Anderson Cancer Center, Houston, TX, USA. 22RV1, A549, U87, NCI-N87, MKN-7, BxPC-3, JIMT-1, MDA-MB-231, SU86.86, SW1990, OVCAR-3, Calu-3, A431, AsPC-1, CFPAC-1, DMS53, HCC827 and NCI-H1975 cells were purchased from ATCC, Nanjing Cobioer and The Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, and the others. C4-2B and PC-3-4H7 are both Steap1 and PSMA antigen high express cells, but medium expressed of both B7H3 and DLL3 antigens. 22RV1 is low express cells for Steap1, PSMA, B7H3 and DLL3 antigens. U87 is glioblastoma (GBM) cells, NCI-N87 and MKN-7 cell line are stable models of the gastric epithelium. A431 is human epidermoid carcinoma. JIM-1 is trastuzumab-resistant Her2 breast tumor cells. SU86.86, SW1990, CFPAC-1 and AsPC-1 are pancreatic cancer cell lines. OVCAR-3 is ovarian cancer cell lines. Calu-3, HCC827 and NCI-H1975 are a human lung adenocarcinoma. All these cells were grown according to the provider manuals. To run the assay, the cells (180 μl, 6000 cells) were added to each well in a 96-well plate and incubated for 24 hours at 37° C. with 5% CO2. Next, the cells were treated with test compounds (20 μl) at various concentrations in appropriate cell culture medium (total volume, 0.2 mL). The control wells contain cells and the medium but lack the test compounds. The plates were incubated for 120 hours at 37° C. with 5% CO2. MTT (5 mg/mL) was then added to the wells (20 μl) and the plates were incubated for 1.5 hr at 37° C. The medium was carefully removed and DMSO (180 μl) was added afterward. After it was shaken for 15 min, the absorbance was measured at 490 nm and 570 nm with a reference filter of 620 nm. The inhibition % was calculated according to the following equation:

Proliferation-inhibiting effects of Anti-STEAP1 ADCs in C4-2B cell line.

Experiment 420: In Vivo Efficacy and Safety of Antibody-Drug Conjugates

The in vivo efficacy of conjugates of against tumor cells, in xenograft models were exampled in FIGS. 11-24. Five-week-old female BALB/c Nude mice (6 animals per group) were inoculated subcutaneously in the area under the right shoulder with respective carcinoma cells (5×106 cells/mouse) in 0.1-0.2 mL of serum-free medium. The tumors were grown for 6-35 days to an average size of 150 mm3, or 8-40 days to an average size of 180 mm3. The animals were then randomly divided into different groups (6 animals per group). The first group of mice served as the control group and was treated with the phosphate-buffered saline (PBS) vehicle. The other groups were treated with conjugates at doses 1.0˜8.0 mg/Kg (some of them were described in the figures), administered only once intravenously. If a control of paclitaxel was used, it was administrated at dose of 15 mg/Kg, once a week for three weeks intravenously. Three dimensions of the tumor were measured every 3 or 4 days (twice a week) and the tumor volumes were calculated using the formula tumor volume=1/2×(length×width×height). The weight of the animals was also measured at the same time. A mouse was sacrificed when any one of the following criteria was met: (1) loss of body weight of more than 20% from pretreatment weight, (2) tumor volume larger than 1500 mm3, (3) too sick to reach food and water, or (4) skin necrosis. A mouse was considered to be tumor-free if no tumor was palpable.

The results of the examples were plotted in FIGS. 11-24. All the conjugates did not cause the animal body weight loss at the administrated doses or up to 300 mg/Kg. All conjugates demonstrated antitumor activity as comparison with PBS buffer, and most of the conjugates of invention showed better antitumor activities than the conjugates with the payload/linker complexes of GGFG-Dxd.

Example 421. Pharmacokinetic Study in Mice

CD-1 mice were randomly assigned to each group (n=6) and were administered intravenously with each ADC at a dose of 10 mg kg. Blood samples (50 μL) were collected from each animal via the tail vein at each time point (5 min, 4 h, 1 day, 2 days, 3 days, 4 days, 7 days, 10 days, 14 days, 21 days, and 28 days). After removal of cells by centrifugation (10 min at 2200×g at 4° C.), Serum samples were stored at −80° C. until used for subsequent sandwich ELISA.

Example 422. ELISA Assay Method to Measure Drug Concentration in Mouse Serum

For determination of the total antibody concentration (both conjugated and unconjugated), a 96-well plate (Corning) was coated with target protein (2 μg/ml, 100 μl per well) diluted in PBS. After overnight incubation at 4° C., the plate was blocked with 100 μL of 1% BSA in PBS containing 0.05% Tween 20 (PBS-T) at room temperature for 2 h. Subsequently, the solution was removed and each diluted serum sample was added to each well, and the plate was incubated at room temperature for 1 h. After each well was washed three times with PBS-T, 100 μL of goat anti-human IgG Fc-HRP conjugate (1:50,000) was added. After being incubated at room temperature for 1 h, the plate was washed and color development was performed by adding 100 μL per well TMB substrate to the plate. For determination of ADC concentration (conjugated only), assays were performed in the same manner using mouse anti-payload antibody for plate coating, biotinylated target protein (1:5000), and SA-HRP conjugate (1:120,000) as secondary and tertiary detection antibodies, respectively. All assays were performed in duplicate. Concentrations were calculated based on a standard curve.