# Patent

## Title
Peptidomimetic macrocycles

## Abstract

The present invention provides novel peptidomimetic macrocycles and methods of using such macrocycles for the treatment of disease.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a National Stage Entry of International Application No. PCT/US11/47692, filed Aug. 13, 2011, which claims the priority benefit of U.S. Provisional Application Ser. Nos. 61/373,701, filed Aug. 13, 2010, 61/373,638, filed Aug. 13, 2010, and 61/374,163, filed Aug. 16, 2010, each of which is incorporated herein by reference in their entirety for all purposes.


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 6, 2013, is named 35224-757.831_SL.txt and is 586,346 bytes in size.
BACKGROUND OF THE INVENTION
The human transcription factor protein p53 induces cell cycle arrest and apoptosis in response to DNA damage and cellular stress, and thereby plays a critical role in protecting cells from malignant transformation. The E3 ubiquitin ligase HDM2 negatively regulates p53 function through a direct binding interaction that neutralizes the p53 transactivation activity, leads to export from the nucleus of p53 protein, and targets p53 for degradation via the ubiquitylation-proteasomal pathway. Loss of p53 activity, either by deletion, mutation, or HDM2 overexpression, is the most common defect in human cancers. Tumors that express wild type p53 are vulnerable to pharmacologic agents that stabilize or increase the concentration of active p53. In this context, inhibition of the activities of HDM2 has emerged as a validated approach to restore p53 activity and resensitize cancer cells to apoptosis in vitro and in vivo. HDMX (HDM4) has more recently been identified as a similar negative regulator of p53, and studies have revealed significant structural homology between the p53 binding interfaces of HDM2 and HDMX.
The p53-HDM2 and p53-HDMX protein-protein interactions are mediated by the same 15-residue alpha-helical transactivation domain of p53, which inserts into hydrophobic clefts on the surface of HDM2 and HDMX. Three residues within this domain of p53 (F19, W23, and L26) are essential for binding to HDM2 and HDMX. The present invention provides p53-based peptidomimetic macrocycles that modulate the activities of p53 by inhibiting the interactions between p53 and HDM2, p53 and HDMX, or p53 and both HDM2 and HDMX proteins, and that may be used for treating diseases including but not limited to cancer and other hyperproliferative diseases.
SUMMARY OF THE INVENTION
Described below are stably cross-linked peptides related to a portion of human p53 (“p53 peptidomimetic macrocycles”). These cross-linked peptides contain at least two modified amino acids that together form an intramolecular cross-link that can help to stabilize the alpha-helical secondary structure of a portion of p53 that is thought to be important for binding of p53 to HDM2 and for binding of p53 to HDMX. Accordingly, a cross-linked polypeptide described herein can have improved biological activity relative to a corresponding polypeptide that is not cross-linked. The p53 peptidomimetic macrocycles are thought to interfere with binding of p53 to HDM2 and/or of p53 to HDMX, thereby liberating functional p53 and inhibiting its destruction. The p53 peptidomimetic macrocycles described herein can be used therapeutically, for example to treat cancers and other disorders characterized by an undesirably low level or a low activity of p53, and/or to treat cancers and other disorders characterized by an undesirably high level of activity of HDM2 or HDMX. The p53 peptidomimetic macrocycles may also be useful for treatment of any disorder associated with disrupted regulation of the p53 transcriptional pathway, leading to conditions of excess cell survival and proliferation such as cancer and autoimmunity, in addition to conditions of inappropriate cell cycle arrest and apoptosis such as neurodegeneration and immunedeficiencies. In some instances, the p53 peptidomimetic macrocycles bind to HDM2 (e.g., GenBank® Accession No.: 228952; GI:228952) and/or HDMX (also referred to as HDM4; GenBank® Accession No.: 88702791; GI:88702791).
In one aspect, the present invention provides a peptidomimetic macrocycle comprising an amino acid sequence which is at least about 60%, 80%, 90%, or 95% identical to an amino acid sequence chosen from the group consisting of the amino acid sequences in Table 1, 2, 3, or 4. Alternatively, an amino acid sequence of said peptidomimetic macrocycle is chosen from the group consisting of the amino acid sequences in Table 1. Alternatively, an amino acid sequence of said peptidomimetic macrocycle is chosen as above, and further wherein the macrocycle does not include a thioether or a triazole. In some embodiments, the peptidomimetic macrocycle comprises a helix, such as an α-helix. In other embodiments, the peptidomimetic macrocycle comprises an α,α-disubstituted amino acid. A peptidomimetic macrocycle of the invention may comprise a crosslinker linking the α-positions of at least two amino acids. At least one of said two amino acids may be an α,α-disubstituted amino acid.
In some embodiments, the peptidomimetic macrocycle has the formula:




wherein:

    
    
        each A, C, D, and E is independently a natural or non-natural amino acid, and the terminal D and E independently optionally include a capping group;
        B is a natural or non-natural amino acid, amino acid analog,
    
    






    
    
         [—NH-L3-CO-], [—NH-L3-SO2—], or [—NH-L3-];
        R1 and R2 are independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-;
        R3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R5;
        L is a macrocycle-forming linker of the formula -L1-L2-;
        L1 and L2 are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [—R4—K—R4—]n, each being optionally substituted with R5;
        each R4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;
        each K is O, S, SO, SO2, CO, CO2, or CONR3;
        each R5 is independently halogen, alkyl, —OR6, —N(R6)2, —SR6, —SOR6, —SO2R6, —CO2R6, a fluorescent moiety, a radioisotope or a therapeutic agent;
        each R6 is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;
        R7 is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R5, or part of a cyclic structure with a D residue;
        R8 is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R5, or part of a cyclic structure with an E residue;
        v and w are independently integers from 1-1000;
        u is an integer from 1-10;
        x, y and z are independently integers from 0-10; and
        n is an integer from 1-5.
    
    


In various embodiments, the peptidomimetic macrocycle includes L1 and L2 wherein L1 and L2 either alone or in combination do not include a thioether or a triazole.
In other embodiments, the peptidomimetic macrocycle may comprise a crosslinker linking a backbone amino group of a first amino acid to a second amino acid within the peptidomimetic macrocycle. For example, the invention provides peptidomimetic macrocycles of the formula (IV) or (IVa):




wherein:

    
    
        each A, C, D, and E is independently a natural or non-natural amino acid, and the terminal D and E independently optionally include a capping group;
        B is a natural or non-natural amino acid, amino acid analog,
    
    






    
    
         [—NH-L3-CO—], [—NH-L3-SO2—], or [—NH-L3-];
        R1 and R2 are independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-, or part of a cyclic structure with an E residue;
        R3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R5;
        L1 and L2 are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [—R4—K—R4—]n, each being optionally substituted with R5;
        each R4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;
        each K is O, S, SO, SO2, CO, CO2, or CONR3;
        each R5 is independently halogen, alkyl, —OR6, —N(R6)2, —SR6, —SOR6, —SO2R6, —CO2R6, a fluorescent moiety, a radioisotope or a therapeutic agent;
        each R6 is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;
        R7 is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R5;
        v and w are independently integers from 1-1000;
        u is an integer from 1-10;
        x, y and z are independently integers from 0-10; and
        n is an integer from 1-5.
    
    


Additionally, the invention provides a method of treating cancer in a subject comprising administering to the subject a peptidomimetic macrocycle of the invention. Also provided is a method of modulating the activity of p53 or HDM2 or HDMX in a subject comprising administering to the subject a peptidomimetic macrocycle of the invention, or a method of antagonizing the interaction between p53 and HDM2 and/or HDMX proteins in a subject comprising administering to the subject such a peptidomimetic macrocycle.
INCORPORATION BY REFERENCE
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.



BRIEF DESCRIPTION OF THE DRAWINGS
The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
FIG. 1 describes the synthesis of Fmoc-Me-6-Chloro-Tryptophan & Fmoc-6-Chloro-Tryptophan.
FIG. 2 shows an LC-MS trace of Me-6-Chloro-(Boc)Tryptophan-Ni—S—BPB.
FIG. 3 shows a 1H-NMR spectrum of Me-6-Chloro-(Boc)Tryptophan-Ni—S—BPB.
FIG. 4 shows an LC-MS trace of Fmoc-Me-6-Chloro-(Boc)Tryptophan.
FIG. 5 shows a 1H-NMR spectrum of Fmoc-Me-6-Chloro-(Boc)Tryptophan.
FIGS. 6a-f describe the results of a cell viability assay, a competition ELISA assay, GRIP assay, Kd data, p21 activation assay, fluorescence polarization competition binding and circular helicity data for exemplary peptidomimetic macrocycles of the invention (SEQ ID NOS 38-178, respectively, in order of appearance).
FIGS. 7A-D provide data from a variety of macrocycles (FIGS. 7A-7B disclose SEQ ID NOS 42, 163, 177, 214, 217, 344, 289-290, 383, 533, 529, 543, 601, 544, 594, 279, 374 and 660, respectively in order of appearance, and FIGS. 7C-7D disclose SEQ ID NOS 702, 699, 704, 706, 689, 507, 624, 703, 716, 606, 605, 642, 691, 731, 375, 727 662 587 and 714 respectively in order of appearance).
FIGS. 8A-B provide data from a variety of macrocycles.



DETAILED DESCRIPTION OF THE INVENTION
As used herein, the term “macrocycle” refers to a molecule having a chemical structure including a ring or cycle formed by at least 9 covalently bonded atoms.
As used herein, the term “peptidomimetic macrocycle” or “crosslinked polypeptide” refers to a compound comprising a plurality of amino acid residues joined by a plurality of peptide bonds and at least one macrocycle-forming linker which forms a macrocycle between a first naturally-occurring or non-naturally-occurring amino acid residue (or analog) and a second naturally-occurring or non-naturally-occurring amino acid residue (or analog) within the same molecule. Peptidomimetic macrocycle include embodiments where the macrocycle-forming linker connects the α carbon of the first amino acid residue (or analog) to the α carbon of the second amino acid residue (or analog). The peptidomimetic macrocycles optionally include one or more non-peptide bonds between one or more amino acid residues and/or amino acid analog residues, and optionally include one or more non-naturally-occurring amino acid residues or amino acid analog residues in addition to any which form the macrocycle. A “corresponding uncrosslinked polypeptide” when referred to in the context of a peptidomimetic macrocycle is understood to relate to a polypeptide of the same length as the macrocycle and comprising the equivalent natural amino acids of the wild-type sequence corresponding to the macrocycle.
As used herein, the term “stability” refers to the maintenance of a defined secondary structure in solution by a peptidomimetic macrocycle of the invention as measured by circular dichroism, NMR or another biophysical measure, or resistance to proteolytic degradation in vitro or in vivo. Non-limiting examples of secondary structures contemplated in this invention are α-helices, β-turns, and β-pleated sheets.
As used herein, the term “helical stability” refers to the maintenance of a helical structure by a peptidomimetic macrocycle of the invention as measured by circular dichroism or NMR. For example, in some embodiments, the peptidomimetic macrocycles of the invention exhibit at least a 1.25, 1.5, 1.75 or 2-fold increase in α-helicity as determined by circular dichroism compared to a corresponding uncrosslinked macrocycle.
The term “α-amino acid” or simply “amino acid” refers to a molecule containing both an amino group and a carboxyl group bound to α carbon which is designated the α-carbon. Suitable amino acids include, without limitation, both the D- and L-isomers of the naturally-occurring amino acids, as well as non-naturally occurring amino acids prepared by organic synthesis or other metabolic routes. Unless the context specifically indicates otherwise, the term amino acid, as used herein, is intended to include amino acid analogs.
The term “naturally occurring amino acid” refers to any one of the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V.
The term “amino acid analog” or “non-natural amino acid” refers to a molecule which is structurally similar to an amino acid and which can be substituted for an amino acid in the formation of a peptidomimetic macrocycle. Amino acid analogs include, without limitation, compounds which are structurally identical to an amino acid, as defined herein, except for the inclusion of one or more additional methylene groups between the amino and carboxyl group (e.g., α-amino β-carboxy acids), or for the substitution of the amino or carboxy group by a similarly reactive group (e.g., substitution of the primary amine with a secondary or tertiary amine, or substitution of the carboxy group with an ester). Non-natural amino acids include structures according to the following:































A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of a polypeptide without abolishing or substantially altering its essential biological or biochemical activity (e.g., receptor binding or activation). An “essential” amino acid residue is a residue that, when altered from the wild-type sequence of the polypeptide, results in abolishing or substantially abolishing the polypeptide's essential biological or biochemical activity.
A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., K, R, H), acidic side chains (e.g., D, E), uncharged polar side chains (e.g., G, N, Q, S, T, Y, C), nonpolar side chains (e.g., A, V, L, I, P, F, M, W), beta-branched side chains (e.g., T, V, I) and aromatic side chains (e.g., Y, F, W, H). Thus, a predicted nonessential amino acid residue in a polypeptide, for example, is preferably replaced with another amino acid residue from the same side chain family. Other examples of acceptable substitutions are substitutions based on isosteric considerations (e.g. norleucine for methionine) or other properties (e.g. 2-thienylalanine for phenylalanine).
The term “capping group” refers to the chemical moiety occurring at either the carboxy or amino terminus of the polypeptide chain of the subject peptidomimetic macrocycle. The capping group of a carboxy terminus includes an unmodified carboxylic acid (ie —COOH) or a carboxylic acid with a substituent. For example, the carboxy terminus may be substituted with an amino group to yield a carboxamide at the C-terminus. Various substituents include but are not limited to primary and secondary amines, including pegylated secondary amines. Representative secondary amine capping groups for the C-terminus include:




The capping group of an amino terminus includes an unmodified amine (ie —NH2) or an amine with a substituent. For example, the amino terminus may be substituted with an acyl group to yield a carboxamide at the N-terminus. Various substituents include but are not limited to substituted acyl groups, including C1-C6 carbonyls, C7-C30 carbonyls, and pegylated carbamates. Representative capping groups for the N-terminus include:




The term “member” as used herein in conjunction with macrocycles or macrocycle-forming linkers refers to the atoms that form or can form the macrocycle, and excludes substituent or side chain atoms. By analogy, cyclodecane, 1,2-difluoro-decane and 1,3-dimethyl cyclodecane are all considered ten-membered macrocycles as the hydrogen or fluoro substituents or methyl side chains do not participate in forming the macrocycle.
The symbol “” when used as part of a molecular structure refers to a single bond or a trans or cis double bond.
The term “amino acid side chain” refers to a moiety attached to the α-carbon in an amino acid. For example, the amino acid side chain for alanine is methyl, the amino acid side chain for phenylalanine is phenylmethyl, the amino acid side chain for cysteine is thiomethyl, the amino acid side chain for aspartate is carboxymethyl, the amino acid side chain for tyrosine is 4-hydroxyphenylmethyl, etc. Other non-naturally occurring amino acid side chains are also included, for example, those that occur in nature (e.g., an amino acid metabolite) or those that are made synthetically (e.g., an α,α di-substituted amino acid).
The term “α,α di-substituted amino” acid refers to a molecule or moiety containing both an amino group and a carboxyl group bound to α carbon (the α-carbon) that is attached to two natural or non-natural amino acid side chains.
The term “polypeptide” encompasses two or more naturally or non-naturally-occurring amino acids joined by a covalent bond (e.g., an amide bond). Polypeptides as described herein include full length proteins (e.g., fully processed proteins) as well as shorter amino acid sequences (e.g., fragments of naturally-occurring proteins or synthetic polypeptide fragments).
The term “macrocyclization reagent” or “macrocycle-forming reagent” as used herein refers to any reagent which may be used to prepare a peptidomimetic macrocycle of the invention by mediating the reaction between two reactive groups. Reactive groups may be, for example, an azide and alkyne, in which case macrocyclization reagents include, without limitation, Cu reagents such as reagents which provide a reactive Cu(I) species, such as CuBr, CuI or CuOTf, as well as Cu(II) salts such as Cu(CO2CH3)2, CuSO4, and CuCl2 that can be converted in situ to an active Cu(I) reagent by the addition of a reducing agent such as ascorbic acid or sodium ascorbate. Macrocyclization reagents may additionally include, for example, Ru reagents known in the art such as Cp*RuCl(PPh3)2, [Cp*RuCl]4 or other Ru reagents which may provide a reactive Ru(II) species. In other cases, the reactive groups are terminal olefins. In such embodiments, the macrocyclization reagents or macrocycle-forming reagents are metathesis catalysts including, but not limited to, stabilized, late transition metal carbene complex catalysts such as Group VIII transition metal carbene catalysts. For example, such catalysts are Ru and Os metal centers having a +2 oxidation state, an electron count of 16 and pentacoordinated. Additional catalysts are disclosed in Grubbs et al., “Ring Closing Metathesis and Related Processes in Organic Synthesis” Acc. Chem. Res. 1995, 28, 446-452, and U.S. Pat. No. 5,811,515. In yet other cases, the reactive groups are thiol groups. In such embodiments, the macrocyclization reagent is, for example, a linker functionalized with two thiol-reactive groups such as halogen groups.
The term “halo” or “halogen” refers to fluorine, chlorine, bromine or iodine or a radical thereof.
The term “alkyl” refers to a hydrocarbon chain that is a straight chain or branched chain, containing the indicated number of carbon atoms. For example, C1-C10 indicates that the group has from 1 to 10 (inclusive) carbon atoms in it. In the absence of any numerical designation, “alkyl” is a chain (straight or branched) having 1 to 20 (inclusive) carbon atoms in it.
The term “alkylene” refers to a divalent alkyl (i.e., —R—).
The term “alkenyl” refers to a hydrocarbon chain that is a straight chain or branched chain having one or more carbon-carbon double bonds. The alkenyl moiety contains the indicated number of carbon atoms. For example, C2-C10 indicates that the group has from 2 to 10 (inclusive) carbon atoms in it. The term “lower alkenyl” refers to a C2-C6 alkenyl chain. In the absence of any numerical designation, “alkenyl” is a chain (straight or branched) having 2 to 20 (inclusive) carbon atoms in it.
The term “alkynyl” refers to a hydrocarbon chain that is a straight chain or branched chain having one or more carbon-carbon triple bonds. The alkynyl moiety contains the indicated number of carbon atoms. For example, C2-C10 indicates that the group has from 2 to 10 (inclusive) carbon atoms in it. The term “lower alkynyl” refers to a C2-C6 alkynyl chain. In the absence of any numerical designation, “alkynyl” is a chain (straight or branched) having 2 to 20 (inclusive) carbon atoms in it.
The term “aryl” refers to a 6-carbon monocyclic or 10-carbon bicyclic aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring are substituted by a substituent. Examples of aryl groups include phenyl, naphthyl and the like. The term “arylalkyl” or the term “aralkyl” refers to alkyl substituted with an aryl. The term “arylalkoxy” refers to an alkoxy substituted with aryl.
“Arylalkyl” refers to an aryl group, as defined above, wherein one of the aryl group's hydrogen atoms has been replaced with a C1-C5 alkyl group, as defined above. Representative examples of an arylalkyl group include, but are not limited to, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-ethylphenyl, 3-ethylphenyl, 4-ethylphenyl, 2-propylphenyl, 3-propylphenyl, 4-propylphenyl, 2-butylphenyl, 3-butylphenyl, 4-butylphenyl, 2-pentylphenyl, 3-pentylphenyl, 4-pentylphenyl, 2-isopropylphenyl, 3-isopropylphenyl, 4-isopropylphenyl, 2-isobutylphenyl, 3-isobutylphenyl, 4-isobutylphenyl, 2-sec-butylphenyl, 3-sec-butylphenyl, 4-sec-butylphenyl, 2-t-butylphenyl, 3-t-butylphenyl and 4-t-butylphenyl.
“Arylamido” refers to an aryl group, as defined above, wherein one of the aryl group's hydrogen atoms has been replaced with one or more —C(O)NH2 groups. Representative examples of an arylamido group include 2-C(O)NH2-phenyl, 3-C(O)NH2-phenyl, 4-C(O)NH2-phenyl, 2-C(O)NH2-pyridyl, 3-C(O)NH2-pyridyl, and 4-C(O)NH2-pyridyl,
“Alkylheterocycle” refers to a C1-C5 alkyl group, as defined above, wherein one of the C1-C5 alkyl group's hydrogen atoms has been replaced with a heterocycle. Representative examples of an alkylheterocycle group include, but are not limited to, —CH2CH2-morpholine, —CH2CH2-piperidine, —CH2CH2CH2-morpholine, and —CH2CH2CH2-imidazole.
“Alkylamido” refers to a C1-C5 alkyl group, as defined above, wherein one of the C1-C5 alkyl group's hydrogen atoms has been replaced with a —C(O)NH2 group. Representative examples of an alkylamido group include, but are not limited to, —CH2—C(O)NH2, —CH2CH2—C(O)NH2, —CH2CH2CH2C(O)NH2, —CH2CH2CH2CH2C(O)NH2, —CH2CH2CH2CH2CH2C(O)NH2, —CH2CH(C(O)NH2)CH3, —CH2CH(C(O)NH2)CH2CH3, —CH(C(O)NH2)CH2CH3, —C(CH3)2CH2C(O)NH2, —CH2—CH2—NH—C(O)—CH3, —CH2—CH2—NH—C(O)—CH3—CH3, and —CH2—CH2—NH—C(O)—CH═CH2.
“Alkanol” refers to a C1-C5 alkyl group, as defined above, wherein one of the C1-C5 alkyl group's hydrogen atoms has been replaced with a hydroxyl group. Representative examples of an alkanol group include, but are not limited to, —CH2OH, —CH2CH2OH, —CH2CH2CH2OH, —CH2CH2CH2CH2OH, —CH2CH2CH2 CH2CH2OH, —CH2CH(OH)CH3, —CH2CH(OH)CH2CH3, —CH(OH)CH3 and —C(CH3)2CH2OH.
“Alkylcarboxy” refers to a C1-C5 alkyl group, as defined above, wherein one of the C1-C5 alkyl group's hydrogen atoms has been replaced with a—COOH group. Representative examples of an alkylcarboxy group include, but are not limited to, —CH2COOH, —CH2CH2COOH, —CH2CH2CH2COOH, —CH2CH2CH2CH2COOH, —CH2CH(COOH)CH3, —CH2CH2CH2CH2CH2COOH, —CH2CH(COOH)CH2CH3, —CH(COOH)CH2CH3 and —C(CH3)2CH2COOH.
The term “cycloalkyl” as employed herein includes saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons, preferably 3 to 8 carbons, and more preferably 3 to 6 carbons, wherein the cycloalkyl group additionally is optionally substituted. Some cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.
The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring are substituted by a substituent. Examples of heteroaryl groups include pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, and the like.
The term “heteroarylalkyl” or the term “heteroaralkyl” refers to an alkyl substituted with a heteroaryl. The term “heteroarylalkoxy” refers to an alkoxy substituted with heteroaryl.
The term “heteroarylalkyl” or the term “heteroaralkyl” refers to an alkyl substituted with a heteroaryl. The term “heteroarylalkoxy” refers to an alkoxy substituted with heteroaryl.
The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3 atoms of each ring are substituted by a substituent. Examples of heterocyclyl groups include piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and the like.
The term “substituent” refers to a group replacing a second atom or group such as a hydrogen atom on any molecule, compound or moiety. Suitable substituents include, without limitation, halo, hydroxy, mercapto, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, thioalkoxy, aryloxy, amino, alkoxycarbonyl, amido, carboxy, alkanesulfonyl, alkylcarbonyl, and cyano groups.
In some embodiments, the compounds of this invention contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of these compounds are included in the present invention unless expressly provided otherwise. In some embodiments, the compounds of this invention are also represented in multiple tautomeric forms, in such instances, the invention includes all tautomeric forms of the compounds described herein (e.g., if alkylation of a ring system results in alkylation at multiple sites, the invention includes all such reaction products). All such isomeric forms of such compounds are included in the present invention unless expressly provided otherwise. All crystal forms of the compounds described herein are included in the present invention unless expressly provided otherwise.
As used herein, the terms “increase” and “decrease” mean, respectively, to cause a statistically significantly (i.e., p<0.1) increase or decrease of at least 5%.
As used herein, the recitation of a numerical range for a variable is intended to convey that the invention may be practiced with the variable equal to any of the values within that range. Thus, for a variable which is inherently discrete, the variable is equal to any integer value within the numerical range, including the end-points of the range. Similarly, for a variable which is inherently continuous, the variable is equal to any real value within the numerical range, including the end-points of the range. As an example, and without limitation, a variable which is described as having values between 0 and 2 takes the values 0, 1 or 2 if the variable is inherently discrete, and takes the values 0.0, 0.1, 0.01, 0.001, or any other real values ≧0 and ≦2 if the variable is inherently continuous.
As used herein, unless specifically indicated otherwise, the word “or” is used in the inclusive sense of “and/or” and not the exclusive sense of “either/or.”
The term “on average” represents the mean value derived from performing at least three independent replicates for each data point.
The term “biological activity” encompasses structural and functional properties of a macrocycle of the invention. Biological activity is, for example, structural stability, alpha-helicity, affinity for a target, resistance to proteolytic degradation, cell penetrability, intracellular stability, in vivo stability, or any combination thereof.
The details of one or more particular embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
In some embodiments, the peptide sequences are derived from the p53 protein.
A non-limiting exemplary list of suitable p53 peptides for use in the present invention is given below.







TABLE 1







(SEQ ID NOS 1-18, respectively, in order of appearance)








Sequence (bold = critical residue; X = cross-linked amino acid)
Design Notes





























Ac-

Gln
Ser
Gln
Gln
Thr
Phe
Ser
Asn
Leu
Trp
Arg
Leu
Leu
Pro
Gln
Asn
—NH2
linear


 


Ac-
X
Gln
Ser
Gln
X
Thr
Phe
Ser
Asn
Leu
Trp
Arg
Leu
Leu
Pro
Gln
Asn
—NH2
i--> i + 4 x-link





















#1


 


Ac-

X
Ser
Gln
Gln
X
Phe
Ser
Asn
Leu
Trp
Arg
Leu
Leu
Pro
Gln
Asn
—NH2
i--> i + 4 x-link





















#2


 


Ac-

Gln
Ser
X
Gln
Thr
Phe
X
Asn
Leu
Trp
Arg
Leu
Leu
Pro
Gln
Asn
—NH2
i--> i + 4 x-link





















#3


 


Ac-

Gln
Ser
Gln
X
Thr
Phe
Ser
X
Leu
Trp
Arg
Leu
Leu
Pro
Gln
Asn
—NH2
i--> i + 4 x-link





















#4


 


Ac-

Gln
Ser
Gln
Gln
X
Phe
Ser
Asn
X
Trp
Arg
Leu
Leu
Pro
Gln
Asn
—NH2
i--> i + 4 x-link





















#5


 


Ac-

Gln
Ser
Gln
Gln
Thr
Phe
X
Asn
Leu
Trp
X
Leu
Leu
Pro
Gln
Asn
—NH2
i--> i + 4 x-link





















#6


 


Ac-

Gln
Ser
Gln
Gln
Thr
Phe
Ser
X
Leu
Trp
Arg
X
Leu
Pro
Gln
Asn
—NH2
i--> i + 4 x-link





















#7


 


Ac-

Gln
Ser
Gln
Gln
Thr
Phe
Ser
Asn
Leu
Trp
X
Leu
Leu
Pro
X
Asn
—NH2
i--> i + 4 x-link





















#8


 


Ac-

Gln
Ser
Gln
Gln
Thr
Phe
Ser
Asn
Leu
Trp
Arg
X
Leu
Pro
Gln
X
—NH2
i--> i + 4 x-link





















#9


 


Ac-
X
Gln
Ser
Gln
Gln
Thr
Phe
X
Asn
Leu
Trp
Arg
Leu
Leu
Pro
Gln
Asn
—NH2
i--> i + 7 x-link





















#1


 


Ac-

X
Ser
Gln
Gln
Thr
Phe
Ser
X
Leu
Trp
Arg
Leu
Leu
Pro
Gln
Asn
—NH2
i--> i + 7 x-link





















#2


 


Ac-

Gln
X
Gln
Gln
Thr
Phe
Ser
Asn
X
Trp
Arg
Leu
Leu
Pro
Gln
Asn
—NH2
i--> i + 7 x-link





















#3


 


Ac-

Gln
Ser
Gln
X
Thr
Phe
Ser
Asn
Leu
Trp
X
Leu
Leu
Pro
Gln
Asn
—NH2
i--> i + 7 x-link





















#4


 


Ac-

Gln
Ser
Gln
Gln
X
Phe
Ser
Asn
Leu
Trp
Arg
X
Leu
Pro
Gln
Asn
—NH2
i--> i + 7 x-link





















#5


 


Ac-

Gln
Ser
Gln
Gln
Thr
Phe
X
Asn
Leu
Trp
Arg
Leu
Leu
X
Gln
Asn
—NH2
i--> i + 7 x-link





















#6


 


Ac-

Gln
Ser
Gln
Gln
Thr
Phe
Ser
X
Leu
Trp
Arg
Leu
Leu
Pro
X
Asn
—NH2
i--> i + 7 x-link





















#7


 


Ac-

Gln
Ser
Gln
Gln
Thr
Phe
Ser
Asn
X
Trp
Arg
Leu
Leu
Pro
Gln
X
—NH2
i--> i + 7 x-link





















#8
















TABLE 2







(SEQ ID NOS 19-31, respectively, in order of appearance)








Sequence (bold = critical residue; X = cross-linked amino acid)
Design Notes


























Ac-

Leu
Thr
Phe
Glu
His
Tyr
Trp
Ala
Gln
Leu
Thr
Ser

—NH2
linear


 


Ac-
X
Leu
Thr
Phe
X
His
Tyr
Trp
Ala
Gln
Leu
Thr
Ser

—NH2
i--> i + 4 x-link


















#1


 


Ac-

X
Thr
Phe
Glu
X
Tyr
Trp
Ala
Gln
Leu
Thr
Ser

—NH2
i--> i + 4 x-link


















#2


 


Ac-

Leu
X
Phe
Glu
His
X
Trp
Ala
Gln
Leu
Thr
Ser

—NH2
i--> i + 4 x-link


















#3


 


Ac-

Leu
Thr
Phe
X
His
Tyr
Trp
X
Gln
Leu
Thr
Ser

—NH2
i--> i + 4 x-link


















#4


 


Ac-

Leu
Thr
Phe
Glu
X
Tyr
Trp
Ala
X
Leu
Thr
Ser

—NH2
i--> i + 4 x-link


















#5


 


Ac-

Leu
Thr
Phe
Glu
His
Tyr
Trp
X
Gln
Leu
Thr
X

—NH2
i--> i + 4 x-link


















#6


 


Ac-

Leu
Thr
Phe
Glu
His
Tyr
Trp
Ala
X
Leu
Thr
Ser
X
—NH2
i--> i + 4 x-link


















#7


 


Ac-

X
Thr
Phe
Glu
His
Tyr
Trp
X
Gln
Leu
Thr
Ser

—NH2
i--> i + 7 x-link


















#1


 


Ac-

Gln
X
Phe
Glu
His
Tyr
Trp
Ala
X
Leu
Thr
Ser

—NH2
i--> i + 7 x-link


















#2


 


Ac-

Gln
Thr
Phe
X
His
Tyr
Trp
Ala
Gln
Leu
X
Ser

—NH2
i--> i + 7 x-link


















#3


 


Ac-

Gln
Thr
Phe
Glu
X
Tyr
Trp
Ala
Gln
Leu
Thr
X

—NH2
i--> i + 7 x-link


















#4


 


Ac-

Gln
Thr
Phe
Glu
His
X
Trp
Ala
Gln
Leu
Thr
Ser
X
—NH2
i--> i + 7 x-link


















#5
















TABLE 3







(SEQ ID NOS 32-37, respectively, in order of appearance)








Sequence bold = critical residue; X = cross-linked amino acid
Design Notes
























Ac-

Phe
Met
Aib/His/Asn
Tyr
6-ClTrp
Glu
Ac3c/Gln/Leu
Leu



—NH2
linear


 


Ac-
X
Phe
Met
Aib/His/Asn
X
6-ClTrp
Glu
Ac3c/Gln/Leu
Leu



—NH2
i--> i + 4 x-link
















#1


 


Ac-

Phe
X
Aib/His/Asn
Tyr
6-ClTrp
X
Ac3c/Gln/Leu
Leu



—NH2
i--> i + 4 x-link
















#2


 


Ac-

Phe
Met
X
Tyr
6-ClTrp
Glu
X
Leu



—NH2
i--> i + 4 x-link
















#3


 


Ac-
X
Phe
Met
Aib/His/Asn
Tyr
6-ClTrp
Glu
X
Leu



—NH2
i--> i + 7 x-link
















#1


 


Ac-

Phe
X
Aib/His/Asn
Tyr
6-ClTrp
Glu
Ac3c/Gln/Leu
Leu
X


—NH2
i--> i + 7 x-link
















#2









In Table 3 and elsewhere, “Aib” represents a 2-aminoisobutyric acid residue, while “Ac3c” represents a aminocyclopropane carboxylic acid residue.
Peptidomimetic Macrocycles
In some embodiments, a peptidomimetic macrocycle of the invention has the Formula (I):




wherein:

    
    
        each A, C, D, and E is independently a natural or non-natural amino acid, and the terminal D and E independently optionally include a capping group;
        B is a natural or non-natural amino acid, amino acid analog,
    
    






    
    
         [—NH-L3-CO—], [—NH-L3-SO2—], or [—NH-L3-];
        R1 and R2 are independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-;
        R3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R5;
        L is a macrocycle-forming linker of the formula -L1-L2-;
        L1 and L2 are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [—R4—K—R4—]n, each being optionally substituted with R5;
        each R4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;
        each K is O, S, SO, SO2, CO, CO2, or CONR3;
        each R5 is independently halogen, alkyl, —OR6, —N(R6)2, —SR6, —SOR6, —SO2R6, —CO2R6, a fluorescent moiety, a radioisotope or a therapeutic agent;
        each R6 is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;
        R7 is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R5, or part of a cyclic structure with a D residue;
        R8 is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R5, or part of a cyclic structure with an E residue;
        v and w are independently integers from 1-1000;
        u is an integer from 1-10;
        x, y and z are independently integers from 0-10; and
        n is an integer from 1-5.
    
    


In one embodiment, L1 and L2, either alone or in combination, do not form a triazole or a thioether.
In one example, at least one of R1 and R2 is alkyl, unsubstituted or substituted with halo-. In another example, both R1 and R2 are independently alkyl, unsubstituted or substituted with halo-. In some embodiments, at least one of R1 and R2 is methyl. In other embodiments, R1 and R2 are methyl.
In some embodiments of the invention, x+y+z is at least 3. In other embodiments of the invention, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Each occurrence of A, B, C, D or E in a macrocycle or macrocycle precursor of the invention is independently selected. For example, a sequence represented by the formula [A]x, when x is 3, encompasses embodiments where the amino acids are not identical, e.g. Gln-Asp-Ala as well as embodiments where the amino acids are identical, e.g. Gln-Gln-Gln. This applies for any value of x, y, or z in the indicated ranges. Similarly, when u is greater than 1, each compound of the invention may encompass peptidomimetic macrocycles which are the same or different. For example, a compound of the invention may comprise peptidomimetic macrocycles comprising different linker lengths or chemical compositions.
In some embodiments, the peptidomimetic macrocycle of the invention comprises a secondary structure which is an α-helix and R8 is —H, allowing intrahelical hydrogen bonding. In some embodiments, at least one of A, B, C, D or E is an α,α-disubstituted amino acid. In one example, B is an α,α-disubstituted amino acid. For instance, at least one of A, B, C, D or E is 2-aminoisobutyric acid. In other embodiments, at least one of A, B, C, D or E is




In other embodiments, the length of the macrocycle-forming linker L as measured from a first Cα to a second Cα is selected to stabilize a desired secondary peptide structure, such as an α-helix formed by residues of the peptidomimetic macrocycle including, but not necessarily limited to, those between the first Cα to a second Cα.
In one embodiment, the peptidomimetic macrocycle of Formula (I) is:




wherein each R1 and R2 is independently independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-.

In related embodiments, the peptidomimetic macrocycle of Formula (I) is:




In other embodiments, the peptidomimetic macrocycle of Formula (I) is a compound of any of the formulas shown below:










wherein “AA” represents any natural or non-natural amino acid side chain and “” is [D]v, [E]w as defined above, and n is an integer between 0 and 20, 50, 100, 200, 300, 400 or 500. In some embodiments, n is 0. In other embodiments, n is less than 50.

Exemplary embodiments of the macrocycle-forming linker L are shown below.




In other embodiments, D and/or E in the compound of Formula I are further modified in order to facilitate cellular uptake. In some embodiments, lipidating or PEGylating a peptidomimetic macrocycle facilitates cellular uptake, increases bioavailability, increases blood circulation, alters pharmacokinetics, decreases immunogenicity and/or decreases the needed frequency of administration.
In other embodiments, at least one of [D] and [E] in the compound of Formula I represents a moiety comprising an additional macrocycle-forming linker such that the peptidomimetic macrocycle comprises at least two macrocycle-forming linkers. In a specific embodiment, a peptidomimetic macrocycle comprises two macrocycle-forming linkers. In an embodiment, u is 2.
In the peptidomimetic macrocycles of the invention, any of the macrocycle-forming linkers described herein may be used in any combination with any of the sequences shown in Tables 1-4 and also with any of the R— substituents indicated herein.
In some embodiments, the peptidomimetic macrocycle comprises at least one α-helix motif For example, A, B and/or C in the compound of Formula I include one or more α-helices. As a general matter, α-helices include between 3 and 4 amino acid residues per turn. In some embodiments, the α-helix of the peptidomimetic macrocycle includes 1 to 5 turns and, therefore, 3 to 20 amino acid residues. In specific embodiments, the α-helix includes 1 turn, 2 turns, 3 turns, 4 turns, or 5 turns. In some embodiments, the macrocycle-forming linker stabilizes an α-helix motif included within the peptidomimetic macrocycle. Thus, in some embodiments, the length of the macrocycle-forming linker L from a first Cα to a second Cα is selected to increase the stability of an α-helix. In some embodiments, the macrocycle-forming linker spans from 1 turn to 5 turns of the α-helix. In some embodiments, the macrocycle-forming linker spans approximately 1 turn, 2 turns, 3 turns, 4 turns, or 5 turns of the α-helix. In some embodiments, the length of the macrocycle-forming linker is approximately 5 Å to 9 Å per turn of the α-helix, or approximately 6 Å to 8 Å per turn of the α-helix. Where the macrocycle-forming linker spans approximately 1 turn of an α-helix, the length is equal to approximately 5 carbon-carbon bonds to 13 carbon-carbon bonds, approximately 7 carbon-carbon bonds to 11 carbon-carbon bonds, or approximately 9 carbon-carbon bonds. Where the macrocycle-forming linker spans approximately 2 turns of an α-helix, the length is equal to approximately 8 carbon-carbon bonds to 16 carbon-carbon bonds, approximately 10 carbon-carbon bonds to 14 carbon-carbon bonds, or approximately 12 carbon-carbon bonds. Where the macrocycle-forming linker spans approximately 3 turns of an α-helix, the length is equal to approximately 14 carbon-carbon bonds to 22 carbon-carbon bonds, approximately 16 carbon-carbon bonds to 20 carbon-carbon bonds, or approximately 18 carbon-carbon bonds. Where the macrocycle-forming linker spans approximately 4 turns of an α-helix, the length is equal to approximately 20 carbon-carbon bonds to 28 carbon-carbon bonds, approximately 22 carbon-carbon bonds to 26 carbon-carbon bonds, or approximately 24 carbon-carbon bonds. Where the macrocycle-forming linker spans approximately 5 turns of an α-helix, the length is equal to approximately 26 carbon-carbon bonds to 34 carbon-carbon bonds, approximately 28 carbon-carbon bonds to 32 carbon-carbon bonds, or approximately 30 carbon-carbon bonds. Where the macrocycle-forming linker spans approximately 1 turn of an α-helix, the linkage contains approximately 4 atoms to 12 atoms, approximately 6 atoms to 10 atoms, or approximately 8 atoms. Where the macrocycle-forming linker spans approximately 2 turns of the α-helix, the linkage contains approximately 7 atoms to 15 atoms, approximately 9 atoms to 13 atoms, or approximately 11 atoms. Where the macrocycle-forming linker spans approximately 3 turns of the α-helix, the linkage contains approximately 13 atoms to 21 atoms, approximately 15 atoms to 19 atoms, or approximately 17 atoms. Where the macrocycle-forming linker spans approximately 4 turns of the α-helix, the linkage contains approximately 19 atoms to 27 atoms, approximately 21 atoms to 25 atoms, or approximately 23 atoms. Where the macrocycle-forming linker spans approximately 5 turns of the α-helix, the linkage contains approximately 25 atoms to 33 atoms, approximately 27 atoms to 31 atoms, or approximately 29 atoms. Where the macrocycle-forming linker spans approximately 1 turn of the α-helix, the resulting macrocycle forms a ring containing approximately 17 members to 25 members, approximately 19 members to 23 members, or approximately 21 members. Where the macrocycle-forming linker spans approximately 2 turns of the α-helix, the resulting macrocycle forms a ring containing approximately 29 members to 37 members, approximately 31 members to 35 members, or approximately 33 members. Where the macrocycle-forming linker spans approximately 3 turns of the α-helix, the resulting macrocycle forms a ring containing approximately 44 members to 52 members, approximately 46 members to 50 members, or approximately 48 members. Where the macrocycle-forming linker spans approximately 4 turns of the α-helix, the resulting macrocycle forms a ring containing approximately 59 members to 67 members, approximately 61 members to 65 members, or approximately 63 members. Where the macrocycle-forming linker spans approximately 5 turns of the α-helix, the resulting macrocycle forms a ring containing approximately 74 members to 82 members, approximately 76 members to 80 members, or approximately 78 members.
In other embodiments, the invention provides peptidomimetic macrocycles of Formula (IV) or (IVa):




wherein:

    
    
        each A, C, D, and E is independently a natural or non-natural amino acid, and the terminal D and E independently optionally include a capping group;
        B is a natural or non-natural amino acid, amino acid analog
    
    






    
    
         [—NH-L3-CO—], [—NH-L3-SO2—], or [—NH-L3-];
        R1 and R2 are independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-, or part of a cyclic structure with an E residue;
        R3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R5;
        L is a macrocycle-forming linker of the formula -L1-L2-;
        L1 and L2 are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [—R4—K—R4—]n, each being optionally substituted with R5;
        each R4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;
        each K is O, S, SO, SO2, CO, CO2, or CONR3;
        each R5 is independently halogen, alkyl, —OR6, —N(R6)2, —SR6, —SOR6, —SO2R6, —CO2R6, a fluorescent moiety, a radioisotope or a therapeutic agent;
        each R6 is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;
        R7 is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R5;
        V and w are independently integers from 1-1000;
        u is an integer from 1-10;
        x, y and z are independently integers from 0-10; and
        n is an integer from 1-5.
    
    


In one example, L1 and L2, either alone or in combination, do not form a triazole or a thioether.
In one example, at least one of R1 and R2 is alkyl, unsubstituted or substituted with halo-. In another example, both R1 and R2 are independently alkyl, unsubstituted or substituted with halo-. In some embodiments, at least one of R1 and R2 is methyl. In other embodiments, R1 and R2 are methyl.
In some embodiments of the invention, x+y+z is at least 1. In other embodiments of the invention, x+y+z is at least 2. In other embodiments of the invention, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Each occurrence of A, B, C, D or E in a macrocycle or macrocycle precursor of the invention is independently selected. For example, a sequence represented by the formula [A]x, when x is 3, encompasses embodiments where the amino acids are not identical, e.g. Gln-Asp-Ala as well as embodiments where the amino acids are identical, e.g. Gln-Gln-Gln. This applies for any value of x, y, or z in the indicated ranges.
In some embodiments, the peptidomimetic macrocycle of the invention comprises a secondary structure which is an α-helix and R8 is —H, allowing intrahelical hydrogen bonding. In some embodiments, at least one of A, B, C, D or E is an α,α-disubstituted amino acid. In one example, B is an α,α-disubstituted amino acid. For instance, at least one of A, B, C, D or E is 2-aminoisobutyric acid. In other embodiments, at least one of A, B, C, D or E is




In other embodiments, the length of the macrocycle-forming linker L as measured from a first Cα to a second Cα is selected to stabilize a desired secondary peptide structure, such as an α-helix formed by residues of the peptidomimetic macrocycle including, but not necessarily limited to, those between the first Cα to a second Cα.
Exemplary embodiments of the macrocycle-forming linker -L1-L2- are shown below.




Preparation of Peptidomimetic Macrocycles
Peptidomimetic macrocycles of the invention may be prepared by any of a variety of methods known in the art. For example, any of the residues indicated by “X” in Tables 1, 2, 3, or 4 may be substituted with a residue capable of forming a crosslinker with a second residue in the same molecule or a precursor of such a residue.
Various methods to effect formation of peptidomimetic macrocycles are known in the art. For example, the preparation of peptidomimetic macrocycles of Formula I is described in Schafineister et al., J. Am. Chem. Soc. 122:5891-5892 (2000); Schafmeister & Verdine, J. Am. Chem. Soc. 122:5891 (2005); Walensky et al., Science 305:1466-1470 (2004); U.S. Pat. No. 7,192,713 and PCT application WO 2008/121767. The α,α-disubstituted amino acids and amino acid precursors disclosed in the cited references may be employed in synthesis of the peptidomimetic macrocycle precursor polypeptides. For example, the “S5-olefin amino acid” is (S)-α-(2′-pentenyl) alanine and the “R8 olefin amino acid” is (R)-α-(2′-octenyl) alanine. Following incorporation of such amino acids into precursor polypeptides, the terminal olefins are reacted with a metathesis catalyst, leading to the formation of the peptidomimetic macrocycle. In various embodiments, the following amino acids may be employed in the synthesis of the peptidomimetic macrocycle:




In other embodiments, the peptidomimetic macrocyles of the invention are of Formula IV or IVa. Methods for the preparation of such macrocycles are described, for example, in U.S. Pat. No. 7,202,332.
Additional methods of forming peptidomimetic macrocycles which are envisioned as suitable to perform the present invention include those disclosed by Mustapa, M. Firouz Mohd et al., J. Org. Chem. (2003), 68, pp. 8193-8198; Yang, Bin et al. Bioorg Med. Chem. Lett. (2004), 14, pp. 1403-1406; U.S. Pat. Nos. 5,364,851; 5,446,128; 5,824,483; 6,713,280; and 7,202,332. In such embodiments, aminoacid precursors are used containing an additional substituent R— at the alpha position. Such aminoacids are incorporated into the macrocycle precursor at the desired positions, which may be at the positions where the crosslinker is substituted or, alternatively, elsewhere in the sequence of the macrocycle precursor. Cyclization of the precursor is then effected according to the indicated method.
Assays
The properties of the peptidomimetic macrocycles of the invention are assayed, for example, by using the methods described below. In some embodiments, a peptidomimetic macrocycle of the invention has improved biological properties relative to a corresponding polypeptide lacking the substituents described herein.
Assay to Determine α-helicity.
In solution, the secondary structure of polypeptides with α-helical domains will reach a dynamic equilibrium between random coil structures and α-helical structures, often expressed as a “percent helicity”. Thus, for example, alpha-helical domains are predominantly random coils in solution, with α-helical content usually under 25%. Peptidomimetic macrocycles with optimized linkers, on the other hand, possess, for example, an alpha-helicity that is at least two-fold greater than that of a corresponding uncrosslinked polypeptide. In some embodiments, macrocycles of the invention will possess an alpha-helicity of greater than 50%. To assay the helicity of peptidomimetic macrocyles of the invention, the compounds are dissolved in an aqueous solution (e.g. 50 mM potassium phosphate solution at pH 7, or distilled H2O, to concentrations of 25-50 μM). Circular dichroism (CD) spectra are obtained on a spectropolarimeter (e.g., Jasco J-710) using standard measurement parameters (e.g. temperature, 20° C.; wavelength, 190-260 nm; step resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1 nm; path length, 0.1 cm). The α-helical content of each peptide is calculated by dividing the mean residue ellipticity (e.g. [Φ]222obs) by the reported value for a model helical decapeptide (Yang et al. (1986), Methods Enzymol. 130:208)).
Assay to Determine Melting Temperature (Tm).
A peptidomimetic macrocycle of the invention comprising a secondary structure such as an α-helix exhibits, for example, a higher melting temperature than a corresponding uncrosslinked polypeptide. Typically peptidomimetic macrocycles of the invention exhibit Tm of >60° C. representing a highly stable structure in aqueous solutions. To assay the effect of macrocycle formation on melting temperature, peptidomimetic macrocycles or unmodified peptides are dissolved in distilled H2O (e.g. at a final concentration of 50 μM) and the Tm is determined by measuring the change in ellipticity over a temperature range (e.g. 4 to 95° C.) on a spectropolarimeter (e.g., Jasco J-710) using standard parameters (e.g. wavelength 222 nm; step resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1 nm; temperature increase rate: 1° C./min; path length, 0.1 cm).
Protease Resistance Assay.
The amide bond of the peptide backbone is susceptible to hydrolysis by proteases, thereby rendering peptidic compounds vulnerable to rapid degradation in vivo. Peptide helix formation, however, typically buries the amide backbone and therefore may shield it from proteolytic cleavage. The peptidomimetic macrocycles of the present invention may be subjected to in vitro trypsin proteolysis to assess for any change in degradation rate compared to a corresponding uncrosslinked polypeptide. For example, the peptidomimetic macrocycle and a corresponding uncrosslinked polypeptide are incubated with trypsin agarose and the reactions quenched at various time points by centrifugation and subsequent HPLC injection to quantitate the residual substrate by ultraviolet absorption at 280 nm Briefly, the peptidomimetic macrocycle and peptidomimetic precursor (5 mcg) are incubated with trypsin agarose (Pierce) (S/E  ̃125) for 0, 10, 20, 90, and 180 minutes. Reactions are quenched by tabletop centrifugation at high speed; remaining substrate in the isolated supernatant is quantified by HPLC-based peak detection at 280 nm. The proteolytic reaction displays first order kinetics and the rate constant, k, is determined from a plot of ln [S] versus time (k=−1× slope).
Ex Vivo Stability Assay.
Peptidomimetic macrocycles with optimized linkers possess, for example, an ex vivo half-life that is at least two-fold greater than that of a corresponding uncrosslinked polypeptide, and possess an ex vivo half-life of 12 hours or more. For ex vivo serum stability studies, a variety of assays may be used. For example, a peptidomimetic macrocycle and a corresponding uncrosslinked polypeptide (2 mcg) are incubated with fresh mouse, rat and/or human serum (2 mL) at 37° C. for 0, 1, 2, 4, 8, and 24 hours. To determine the level of intact compound, the following procedure may be used: The samples are extracted by transferring 100 μl of sera to 2 ml centrifuge tubes followed by the addition of 10 μL of 50% formic acid and 500 μL acetonitrile and centrifugation at 14,000 RPM for 10 min at 4±2° C. The supernatants are then transferred to fresh 2 ml tubes and evaporated on Turbovap under N2<10 psi, 37° C. The samples are reconstituted in 100 μL of 50:50 acetonitrile:water and submitted to LC-MS/MS analysis.
In Vitro Binding Assays.
To assess the binding and affinity of peptidomimetic macrocycles and peptidomimetic precursors to acceptor proteins, a fluorescence polarization assay (FPA) isused, for example. The FPA technique measures the molecular orientation and mobility using polarized light and fluorescent tracer. When excited with polarized light, fluorescent tracers (e.g., FITC) attached to molecules with high apparent molecular weights (e.g. FITC-labeled peptides bound to a large protein) emit higher levels of polarized fluorescence due to their slower rates of rotation as compared to fluorescent tracers attached to smaller molecules (e.g. FITC-labeled peptides that are free in solution).
For example, fluoresceinated peptidomimetic macrocycles (25 nM) are incubated with the acceptor protein (25-1000 nM) in binding buffer (140 mM NaCl, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room temperature. Binding activity is measured, for example, by fluorescence polarization on a luminescence spectrophotometer (e.g. Perkin-Elmer LS50B). Kd values may be determined by nonlinear regression analysis using, for example, Graphpad Prism software (GraphPad Software, Inc., San Diego, Calif.). A peptidomimetic macrocycle of the invention shows, in some instances, similar or lower Kd than a corresponding uncrosslinked polypeptide.
In Vitro Displacement Assays to Characterize Antagonists of Peptide-Protein Interactions.
To assess the binding and affinity of compounds that antagonize the interaction between a peptide and an acceptor protein, a fluorescence polarization assay (FPA) utilizing a fluoresceinated peptidomimetic macrocycle derived from a peptidomimetic precursor sequence is used, for example. The FPA technique measures the molecular orientation and mobility using polarized light and fluorescent tracer. When excited with polarized light, fluorescent tracers (e.g., FITC) attached to molecules with high apparent molecular weights (e.g. FITC-labeled peptides bound to a large protein) emit higher levels of polarized fluorescence due to their slower rates of rotation as compared to fluorescent tracers attached to smaller molecules (e.g. FITC-labeled peptides that are free in solution). A compound that antagonizes the interaction between the fluoresceinated peptidomimetic macrocycle and an acceptor protein will be detected in a competitive binding FPA experiment.
For example, putative antagonist compounds (1 nM to 1 mM) and a fluoresceinated peptidomimetic macrocycle (25 nM) are incubated with the acceptor protein (50 nM) in binding buffer (140 mM NaCl, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room temperature. Antagonist binding activity is measured, for example, by fluorescence polarization on a luminescence spectrophotometer (e.g. Perkin-Elmer LS50B). Kd values may be determined by nonlinear regression analysis using, for example, Graphpad Prism software (GraphPad Software, Inc., San Diego, Calif.).
Any class of molecule, such as small organic molecules, peptides, oligonucleotides or proteins can be examined as putative antagonists in this assay.
Assay for Protein-Ligand Binding by Affinity Selection-Mass Spectrometry
To assess the binding and affinity of test compounds for proteins, an affinity-selection mass spectrometry assay is used, for example. Protein-ligand binding experiments are conducted according to the following representative procedure outlined for a system-wide control experiment using 1 μM peptidomimetic macrocycle plus 5 μM hMDM2. A 1 μL DMSO aliquot of a 40 μM stock solution of peptidomimetic macrocycle is dissolved in 19 μL of PBS (Phosphate-buffered saline: 50 mM, pH 7.5 Phosphate buffer containing 150 mM NaCl). The resulting solution is mixed by repeated pipetting and clarified by centrifugation at 10 000 g for 10 min. To a 4 μL aliquot of the resulting supernatant is added 4 μL at of 10 μM hMDM2 in PBS. Each 8.0 μL at experimental sample thus contains 40 pmol (1.5 μg) of protein at 5.0 μM concentration in PBS plus 1 μM peptidomimetic macrocycle and 2.5% DMSO. Duplicate samples thus prepared for each concentration point are incubated for 60 min at room temperature, and then chilled to 4° C. prior to size-exclusion chromatography-LC-MS analysis of 5.0 μL injections. Samples containing a target protein, protein-ligand complexes, and unbound compounds are injected onto an SEC column, where the complexes are separated from non-binding component by a rapid SEC step. The SEC column eluate is monitored using UV detectors to confirm that the early-eluting protein fraction, which elutes in the void volume of the SEC column, is well resolved from unbound components that are retained on the column. After the peak containing the protein and protein-ligand complexes elutes from the primary UV detector, it enters a sample loop where it is excised from the flow stream of the SEC stage and transferred directly to the LC-MS via a valving mechanism. The (M+3H)3+ ion of the peptidomimetic macrocycle is observed by ESI-MS at the expected m/z, confirming the detection of the protein-ligand complex.
Assay for Protein-Ligand Kd Titration Experiments.
To assess the binding and affinity of test compounds for proteins, a protein-ligand Kd titration experiment is performed, for example. Protein-ligand Kd titrations experiments are conducted as follows: 2 μL DMSO aliquots of a serially diluted stock solution of titrant peptidomimetic macrocycle (5, 2.5, . . . , 0.098 mM) are prepared then dissolved in 38 μL of PBS. The resulting solutions are mixed by repeated pipetting and clarified by centrifugation at 10 000 g for 10 min. To 4.0 μL aliquots of the resulting supernatants is added 4.0 μL of 10 μM hMDM2 in PBS. Each 8.0 μL experimental sample thus contains 40 pmol (1.5 μg) of protein at 5.0 μM concentration in PBS, varying concentrations (125, 62.5, . . . , 0.24 μM) of the titrant peptide, and 2.5% DMSO. Duplicate samples thus prepared for each concentration point are incubated at room temperature for 30 min, then chilled to 4° C. prior to SEC-LC-MS analysis of 2.0 μL injections. The (M+H)1+, (M+2H)2+, (M+3H)3+, and/or (M+Na)1+ ion is observed by ESI-MS; extracted ion chromatograms are quantified, then fit to equations to derive the binding affinity Kd as described in “A General Technique to Rank Protein-Ligand Binding Affinities and Determine Allosteric vs. Direct Binding Site Competition in Compound Mixtures.” Annis, D. A.; Nazef, N.; Chuang, C. C.; Scott, M. P.; Nash, H. M. J. Am. Chem. Soc. 2004, 126, 15495-15503; also in “ALIS: An Affinity Selection-Mass Spectrometry System for the Discovery and Characterization of Protein-Ligand Interactions” D. A. Annis, C.-C. Chuang, and N. Nazef. In Mass Spectrometry in Medicinal Chemistry. Edited by Wanner K, Höfner G: Wiley-VCH; 2007:121-184. Mannhold R, Kubinyi H, Folkers G (Series Editors): Methods and Principles in Medicinal Chemistry.
Assay for Competitive Binding Experiments by Affinity Selection-Mass Spectrometry
To determine the ability of test compounds to bind competitively to proteins, an affinity selection mass spectrometry assay is performed, for example. A mixture of ligands at 40 μM per component is prepared by combining 2 μL aliquots of 400 μM stocks of each of the three compounds with 14 μL of DMSO. Then, 1 μL at aliquots of this 40 μM per component mixture are combined with 1 μL at DMSO aliquots of a serially diluted stock solution of titrant peptidomimetic macrocycle (10, 5, 2.5, . . . , 0.078 mM). These 2 μL samples are dissolved in 38 μL of PBS. The resulting solutions were mixed by repeated pipetting and clarified by centrifugation at 10 000 g for 10 min. To 4.0 μL aliquots of the resulting supernatants is added 4.0 μL of 10 μM hMDM2 protein in PBS. Each 8.0 μL experimental sample thus contains 40 pmol (1.5 μg) of protein at 5.0 μM concentration in PBS plus 0.5 μM ligand, 2.5% DMSO, and varying concentrations (125, 62.5, . . . , 0.98 μM) of the titrant peptidomimetic macrocycle. Duplicate samples thus prepared for each concentration point are incubated at room temperature for 60 min, then chilled to 4° C. prior to SEC-LC-MS analysis of 2.0 μL injections. Additional details on these and other methods are provided in “A General Technique to Rank Protein-Ligand Binding Affinities and Determine Allosteric vs. Direct Binding Site Competition in Compound Mixtures.” Annis, D. A.; Nazef, N.; Chuang, C. C.; Scott, M. P.; Nash, H. M. J. Am. Chem. Soc. 2004, 126, 15495-15503; also in “ALIS: An Affinity Selection-Mass Spectrometry System for the Discovery and Characterization of Protein-Ligand Interactions” D. A. Annis, C.-C. Chuang, and N. Nazef. In Mass Spectrometry in Medicinal Chemistry. Edited by Wanner K, Höfner G: Wiley-VCH; 2007:121-184. Mannhold R, Kubinyi H, Folkers G (Series Editors): Methods and Principles in Medicinal Chemistry.
Binding Assays in Intact Cells.
It is possible to measure binding of peptides or peptidomimetic macrocycles to their natural acceptors in intact cells by immunoprecipitation experiments. For example, intact cells are incubated with fluoresceinated (FITC-labeled) compounds for 4 hrs in the absence of serum, followed by serum replacement and further incubation that ranges from 4-18 hrs. Cells are then pelleted and incubated in lysis buffer (50 mM Tris [pH 7.6], 150 mM NaCl, 1% CHAPS and protease inhibitor cocktail) for 10 minutes at 4° C. Extracts are centrifuged at 14,000 rpm for 15 minutes and supernatants collected and incubated with 10 μl goat anti-FITC antibody for 2 hrs, rotating at 4° C. followed by further 2 hrs incubation at 4° C. with protein A/G Sepharose (50 μl of 50% bead slurry). After quick centrifugation, the pellets are washed in lysis buffer containing increasing salt concentration (e.g., 150, 300, 500 mM). The beads are then re-equilibrated at 150 mM NaCl before addition of SDS-containing sample buffer and boiling. After centrifugation, the supernatants are optionally electrophoresed using 4%-12% gradient Bis-Tris gels followed by transfer into Immobilon-P membranes. After blocking, blots are optionally incubated with an antibody that detects FITC and also with one or more antibodies that detect proteins that bind to the peptidomimetic macrocycle.
Cellular Penetrability Assays.
A peptidomimetic macrocycle is, for example, more cell penetrable compared to a corresponding uncrosslinked macrocycle. Peptidomimetic macrocycles with optimized linkers possess, for example, cell penetrability that is at least two-fold greater than a corresponding uncrosslinked macrocycle, and often 20% or more of the applied peptidomimetic macrocycle will be observed to have penetrated the cell after 4 hours. To measure the cell penetrability of peptidomimetic macrocycles and corresponding uncrosslinked macrocycle, intact cells are incubated with fluoresceinated peptidomimetic macrocycles or corresponding uncrosslinked macrocycle (10 μM) for 4 hrs in serum free media at 37° C., washed twice with media and incubated with trypsin (0.25%) for 10 min at 37° C. The cells are washed again and resuspended in PBS. Cellular fluorescence is analyzed, for example, by using either a FACSCalibur flow cytometer or Cellomics' KineticScan® HCS Reader.
Cellular Efficacy Assays.
The efficacy of certain peptidomimetic macrocycles is determined, for example, in cell-based killing assays using a variety of tumorigenic and non-tumorigenic cell lines and primary cells derived from human or mouse cell populations. Cell viability is monitored, for example, over 24-96 hrs of incubation with peptidomimetic macrocycles (0.5 to 50 μM) to identify those that kill at EC50<10 μM. Several standard assays that measure cell viability are commercially available and are optionally used to assess the efficacy of the peptidomimetic macrocycles. In addition, assays that measure Annexin V and caspase activation are optionally used to assess whether the peptidomimetic macrocycles kill cells by activating the apoptotic machinery. For example, the Cell Titer-glo assay is used which determines cell viability as a function of intracellular ATP concentration.
In Vivo Stability Assay.
To investigate the in vivo stability of the peptidomimetic macrocycles, the compounds are, for example, administered to mice and/or rats by IV, IP, PO or inhalation routes at concentrations ranging from 0.1 to 50 mg/kg and blood specimens withdrawn at 0′, 5′, 15′, 30′, 1 hr, 4 hrs, 8 hrs and 24 hours post-injection. Levels of intact compound in 25 μL of fresh serum are then measured by LC-MS/MS as above.
In Vivo Efficacy in Animal Models.
To determine the anti-oncogenic activity of peptidomimetic macrocycles of the invention in vivo, the compounds are, for example, given alone (IP, IV, PO, by inhalation or nasal routes) or in combination with sub-optimal doses of relevant chemotherapy (e.g., cyclophosphamide, doxorubicin, etoposide). In one example, 5×106 RS4;11 cells (established from the bone marrow of a patient with acute lymphoblastic leukemia) that stably express luciferase are injected by tail vein in NOD-SCID mice 3 hrs after they have been subjected to total body irradiation. If left untreated, this form of leukemia is fatal in 3 weeks in this model. The leukemia is readily monitored, for example, by injecting the mice with D-luciferin (60 mg/kg) and imaging the anesthetized animals (e.g., Xenogen In Vivo Imaging System, Caliper Life Sciences, Hopkinton, Mass.). Total body bioluminescence is quantified by integration of photonic flux (photons/sec) by Living Image Software (Caliper Life Sciences, Hopkinton, Mass.). Peptidomimetic macrocycles alone or in combination with sub-optimal doses of relevant chemotherapeutics agents are, for example, administered to leukemic mice (10 days after injection/day 1 of experiment, in bioluminescence range of 14-16) by tail vein or IP routes at doses ranging from 0.1 mg/kg to 50 mg/kg for 7 to 21 days. Optionally, the mice are imaged throughout the experiment every other day and survival monitored daily for the duration of the experiment. Expired mice are optionally subjected to necropsy at the end of the experiment. Another animal model is implantation into NOD-SCID mice of DoHH2, a cell line derived from human follicular lymphoma, that stably expresses luciferase. These in vivo tests optionally generate preliminary pharmacokinetic, pharmacodynamic and toxicology data.
Clinical Trials.
To determine the suitability of the peptidomimetic macrocycles of the invention for treatment of humans, clinical trials are performed. For example, patients diagnosed with cancer and in need of treatment are selected and separated in treatment and one or more control groups, wherein the treatment group is administered a peptidomimetic macrocycle of the invention, while the control groups receive a placebo or a known anti-cancer drug. The treatment safety and efficacy of the peptidomimetic macrocycles of the invention can thus be evaluated by performing comparisons of the patient groups with respect to factors such as survival and quality-of-life. In this example, the patient group treated with a peptidomimetic macrocyle show improved long-term survival compared to a patient control group treated with a placebo.
Pharmaceutical Compositions and Routes of Administration
The peptidomimetic macrocycles of the invention also include pharmaceutically acceptable derivatives or prodrugs thereof A “pharmaceutically acceptable derivative” means any pharmaceutically acceptable salt, ester, salt of an ester, pro-drug or other derivative of a compound of this invention which, upon administration to a recipient, is capable of providing (directly or indirectly) a compound of this invention. Particularly favored pharmaceutically acceptable derivatives are those that increase the bioavailability of the compounds of the invention when administered to a mammal (e.g., by increasing absorption into the blood of an orally administered compound) or which increases delivery of the active compound to a biological compartment (e.g., the brain or lymphatic system) relative to the parent species. Some pharmaceutically acceptable derivatives include a chemical group which increases aqueous solubility or active transport across the gastrointestinal mucosa.
In some embodiments, the peptidomimetic macrocycles of the invention are modified by covalently or non-covalently joining appropriate functional groups to enhance selective biological properties. Such modifications include those which increase biological penetration into a given biological compartment (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism, and alter rate of excretion.
Pharmaceutically acceptable salts of the compounds of this invention include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, benzoate, benzenesulfonate, butyrate, citrate, digluconate, dodecylsulfate, formate, fumarate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, tosylate and undecanoate. Salts derived from appropriate bases include alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl)4+ salts.
For preparing pharmaceutical compositions from the compounds of the present invention, pharmaceutically acceptable carriers include either solid or liquid carriers. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances, which also acts as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. Details on techniques for formulation and administration are well described in the scientific and patent literature, see, e.g., the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton Pa.
In powders, the carrier is a finely divided solid, which is in a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.
Suitable solid excipients are carbohydrate or protein fillers include, but are not limited to sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; and gums including arabic and tragacanth; as well as proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents are added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water/propylene glycol solutions. For parenteral injection, liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.
The pharmaceutical preparation is preferably in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
When the compositions of this invention comprise a combination of a peptidomimetic macrocycle and one or more additional therapeutic or prophylactic agents, both the compound and the additional agent should be present at dosage levels of between about 1 to 100%, and more preferably between about 5 to 95% of the dosage normally administered in a monotherapy regimen. In some embodiments, the additional agents are administered separately, as part of a multiple dose regimen, from the compounds of this invention. Alternatively, those agents are part of a single dosage form, mixed together with the compounds of this invention in a single composition.
Methods of Use
In one aspect, the present invention provides novel peptidomimetic macrocycles that are useful in competitive binding assays to identify agents which bind to the natural ligand(s) of the proteins or peptides upon which the peptidomimetic macrocycles are modeled. For example, in the p53/HDMX system, labeled peptidomimetic macrocycles based on p53 can be used in a HDMX binding assay along with small molecules that competitively bind to HDMX. Competitive binding studies allow for rapid in vitro evaluation and determination of drug candidates specific for the p53/HDMX system. Such binding studies may be performed with any of the peptidomimetic macrocycles disclosed herein and their binding partners.
The invention further provides for the generation of antibodies against the peptidomimetic macrocycles. In some embodiments, these antibodies specifically bind both the peptidomimetic macrocycle and the precursor peptides, such as p53, to which the peptidomimetic macrocycles are related. Such antibodies, for example, disrupt the native protein-protein interaction, for example, binding between p53 and HDMX.
In other aspects, the present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant (e.g., insufficient or excessive) expression or activity of the molecules including p53, HDM2 or HDMX.
In another embodiment, a disorder is caused, at least in part, by an abnormal level of p53 or HDM2 or HDMX, (e.g., over or under expression), or by the presence of p53 or HDM2 or HDMX exhibiting abnormal activity. As such, the reduction in the level and/or activity of p53 or HDM2 or HDMX, or the enhancement of the level and/or activity of p53 or HDM2 or HDMX, by peptidomimetic macrocycles derived from p53, is used, for example, to ameliorate or reduce the adverse symptoms of the disorder.
In another aspect, the present invention provides methods for treating or preventing a disease including hyperproliferative disease and inflammatory disorder by interfering with the interaction or binding between binding partners, for example, between p53 and HDM2 or p53 and HDMX. These methods comprise administering an effective amount of a compound of the invention to a warm blooded animal, including a human. In some embodiments, the administration of the compounds of the present invention induces cell growth arrest or apoptosis.
As used herein, the term “treatment” is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease.
In some embodiments, the peptidomimetics macrocycles of the invention is used to treat, prevent, and/or diagnose cancers and neoplastic conditions. As used herein, the terms “cancer”, “hyperproliferative” and “neoplastic” refer to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. Hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of breast, lung, liver, colon and ovarian origin. “Pathologic hyperproliferative” cells occur in disease states characterized by malignant tumor growth. Examples of non-pathologic hyperproliferative cells include proliferation of cells associated with wound repair. Examples of cellular proliferative and/or differentiative disorders include cancer, e.g., carcinoma, sarcoma, or metastatic disorders. In some embodiments, the peptidomimetics macrocycles are novel therapeutic agents for controlling breast cancer, ovarian cancer, colon cancer, lung cancer, metastasis of such cancers and the like.
Examples of cancers or neoplastic conditions include, but are not limited to, a fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer, rectal cancer, pancreatic cancer, ovarian cancer, prostate cancer, uterine cancer, cancer of the head and neck, skin cancer, brain cancer, squamous cell carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular cancer, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, or Kaposi sarcoma.
Examples of proliferative disorders include hematopoietic neoplastic disorders. As used herein, the term “hematopoietic neoplastic disorders” includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof. Preferably, the diseases arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia. Additional exemplary myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus (1991), Crit. Rev. Oncol./Hemotol. 11:267-97); lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphomas include, but are not limited to non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Sternberg disease.
Examples of cellular proliferative and/or differentiative disorders of the breast include, but are not limited to, proliferative breast disease including, e.g., epithelial hyperplasia, sclerosing adenosis, and small duct papillomas; tumors, e.g., stromal tumors such as fibroadenoma, phyllodes tumor, and sarcomas, and epithelial tumors such as large duct papilloma; carcinoma of the breast including in situ (noninvasive) carcinoma that includes ductal carcinoma in situ (including Paget's disease) and lobular carcinoma in situ, and invasive (infiltrating) carcinoma including, but not limited to, invasive ductal carcinoma, invasive lobular carcinoma, medullary carcinoma, colloid (mucinous) carcinoma, tubular carcinoma, and invasive papillary carcinoma, and miscellaneous malignant neoplasms. Disorders in the male breast include, but are not limited to, gynecomastia and carcinoma.
Examples of cellular proliferative and/or differentiative disorders of the lung include, but are not limited to, bronchogenic carcinoma, including paraneoplastic syndromes, bronchioloalveolar carcinoma, neuroendocrine tumors, such as bronchial carcinoid, miscellaneous tumors, and metastatic tumors; pathologies of the pleura, including inflammatory pleural effusions, noninflammatory pleural effusions, pneumothorax, and pleural tumors, including solitary fibrous tumors (pleural fibroma) and malignant mesothelioma.
Examples of cellular proliferative and/or differentiative disorders of the colon include, but are not limited to, non-neoplastic polyps, adenomas, familial syndromes, colorectal carcinogenesis, colorectal carcinoma, and carcinoid tumors.
Examples of cellular proliferative and/or differentiative disorders of the liver include, but are not limited to, nodular hyperplasias, adenomas, and malignant tumors, including primary carcinoma of the liver and metastatic tumors.
Examples of cellular proliferative and/or differentiative disorders of the ovary include, but are not limited to, ovarian tumors such as, tumors of coelomic epithelium, serous tumors, mucinous tumors, endometrioid tumors, clear cell adenocarcinoma, cystadenofibroma, Brenner tumor, surface epithelial tumors; germ cell tumors such as mature (benign) teratomas, monodermal teratomas, immature malignant teratomas, dysgerminoma, endodermal sinus tumor, choriocarcinoma; sex cord-stomal tumors such as, granulosa-theca cell tumors, thecomafibromas, androblastomas, hill cell tumors, and gonadoblastoma; and metastatic tumors such as Krukenberg tumors.
In other or further embodiments, the peptidomimetics macrocycles described herein are used to treat, prevent or diagnose conditions characterized by overactive cell death or cellular death due to physiologic insult, etc. Some examples of conditions characterized by premature or unwanted cell death are or alternatively unwanted or excessive cellular proliferation include, but are not limited to hypocellular/hypoplastic, acellular/aplastic, or hypercellular/hyperplastic conditions. Some examples include hematologic disorders including but not limited to fanconi anemia, aplastic anemia, thalaessemia, congenital neutropenia, and myelodysplasia.
In other or further embodiments, the peptidomimetics macrocycles of the invention that act to decrease apoptosis are used to treat disorders associated with an undesirable level of cell death. Thus, in some embodiments, the anti-apoptotic peptidomimetics macrocycles of the invention are used to treat disorders such as those that lead to cell death associated with viral infection, e.g., infection associated with infection with human immunodeficiency virus (HIV). A wide variety of neurological diseases are characterized by the gradual loss of specific sets of neurons. One example is Alzheimer's disease (AD). Alzheimer's disease is characterized by loss of neurons and synapses in the cerebral cortex and certain subcortical regions. This loss results in gross atrophy of the affected regions. Both amyloid plaques and neurofibrillary tangles are visible in brains of those afflicted by AD Alzheimer's disease has been identified as a protein misfolding disease, due to the accumulation of abnormally folded A-beta and tau proteins in the brain. Plaques are made up of β-amyloid. β-amyloid is a fragment from a larger protein called amyloid precursor protein (APP). APP is critical to neuron growth, survival and post-injury repair. In AD, an unknown process causes APP to be cleaved into smaller fragments by enzymes through proteolysis. One of these fragments is fibrils of β-amyloid, which form clumps that deposit outside neurons in dense formations known as senile plaques. Plaques continue to grow into insoluble twisted fibers within the nerve cell, often called tangles. Disruption of the interaction between β-amyloid and its native receptor is therefore important in the treatment of AD. The anti-apoptotic peptidomimetics macrocycles of the invention are used, in some embodiments, in the treatment of AD and other neurological disorders associated with cell apoptosis. Such neurological disorders include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS) retinitis pigmentosa, spinal muscular atrophy, and various forms of cerebellar degeneration. The cell loss in these diseases does not induce an inflammatory response, and apoptosis appears to be the mechanism of cell death.
In addition, a number of hematologic diseases are associated with a decreased production of blood cells. These disorders include anemia associated with chronic disease, aplastic anemia, chronic neutropenia, and the myelodysplastic syndromes. Disorders of blood cell production, such as myelodysplastic syndrome and some forms of aplastic anemia, are associated with increased apoptotic cell death within the bone marrow. These disorders could result from the activation of genes that promote apoptosis, acquired deficiencies in stromal cells or hematopoietic survival factors, or the direct effects of toxins and mediators of immune responses. Two common disorders associated with cell death are myocardial infarctions and stroke. In both disorders, cells within the central area of ischemia, which is produced in the event of acute loss of blood flow, appear to die rapidly as a result of necrosis. However, outside the central ischemic zone, cells die over a more protracted time period and morphologically appear to die by apoptosis. In other or further embodiments, the anti-apoptotic peptidomimetics macrocycles of the invention are used to treat all such disorders associated with undesirable cell death.
Some examples of neurologic disorders that are treated with the peptidomimetics macrocycles described herein include but are not limited to Alzheimer's Disease, Down's Syndrome, Dutch Type Hereditary Cerebral Hemorrhage Amyloidosis, Reactive Amyloidosis, Familial Amyloid Nephropathy with Urticaria and Deafness, Muckle-Wells Syndrome, Idiopathic Myeloma; Macroglobulinemia-Associated Myeloma, Familial Amyloid Polyneuropathy, Familial Amyloid Cardiomyopathy, Isolated Cardiac Amyloid, Systemic Senile Amyloidosis, Adult Onset Diabetes, Insulinoma, Isolated Atrial Amyloid, Medullary Carcinoma of the Thyroid, Familial Amyloidosis, Hereditary Cerebral Hemorrhage With Amyloidosis, Familial Amyloidotic Polyneuropathy, Scrapie, Creutzfeldt-Jacob Disease, Gerstmann Straussler-Scheinker Syndrome, Bovine Spongiform Encephalitis, a prion-mediated disease, and Huntington's Disease.
In another embodiment, the peptidomimetics macrocycles described herein are used to treat, prevent or diagnose inflammatory disorders. Numerous types of inflammatory disorders exist. Certain inflammatory diseases are associated with the immune system, for example, autoimmune diseases. Autoimmune diseases arise from an overactive immune response of the body against substances and tissues normally present in the body, i.e. self antigens. In other words, the immune system attacks its own cells. Autoimmune diseases are a major cause of immune-mediated diseases. Rheumatoid arthritis is an example of an autoimmune disease, in which the immune system attacks the joints, where it causes inflammation (i.e. arthritis) and destruction. It can also damage some organs, such as the lungs and skin. Rheumatoid arthritis can lead to substantial loss of functioning and mobility. Rheumatoid arthritis is diagnosed with blood tests especially the rheumatoid factor test. Some examples of autoimmune diseases that are treated with the peptidomimetics macrocycles described herein include, but are not limited to, acute disseminated encephalomyelitis (ADEM), Addison's disease, ankylosing spondylitis, antiphospholipid antibody syndrome (APS), autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, Bechet's disease, bullous pemphigoid, coeliac disease, Chagas disease, Churg-Strauss syndrome, chronic obstructive pulmonary disease (COPD), Crohn's disease, dermatomyositis, diabetes mellitus type 1, endometriosis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's disease, Hidradenitis suppurativa, idiopathic thrombocytopenic purpura, inflammatory bowl disease (IBD), interstitial cystitis, lupus erythematosus, morphea, multiple sclerosis, myasthenia gravis, narcolepsy, neuromyotonia, pemphigus vulgaris, pernicious anaemia, Polymyositis, polymyalgia rheumatica, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, schizophrenia, scleroderma, Sjögren's syndrome, temporal arteritis (also known as “giant cell arteritis”), Takayasu's arteritis, Vasculitis, Vitiligo, and Wegener's granulomatosis.
Some examples of other types of inflammatory disorders that are treated with the peptidomimetics macrocycles described herein include, but are not limited to, allergy including allergic rhinitis/sinusitis, skin allergies (urticaria/hives, angioedema, atopic dermatitis), food allergies, drug allergies, insect allergies, and rare allergic disorders such as mastocytosis, asthma, arthritis including osteoarthritis, rheumatoid arthritis, and spondyloarthropathies, primary angitis of the CNS, sarcoidosis, organ transplant rejection, fibromyalgia, fibrosis, pancreatitis, and pelvic inflammatory disease.
Examples of cardiovascular disorders (e.g., inflammatory disorders) that are treated or prevented with the peptidomimetics macrocycles of the invention include, but are not limited to, aortic valve stenosis, atherosclerosis, myocardial infarction, stroke, thrombosis, aneurism, heart failure, ischemic heart disease, angina pectoris, sudden cardiac death, hypertensive heart disease; non-coronary vessel disease, such as arteriolosclerosis, small vessel disease, nephropathy, hypertriglyceridemia, hypercholesterolemia, hyperlipidemia, xanthomatosis, asthma, hypertension, emphysema and chronic pulmonary disease; or a cardiovascular condition associated with interventional procedures (“procedural vascular trauma”), such as restenosis following angioplasty, placement of a shunt, stent, synthetic or natural excision grafts, indwelling catheter, valve or other implantable devices. Preferred cardiovascular disorders include atherosclerosis, myocardial infarction, aneurism, and stroke.
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
EXAMPLES
Example 1
Synthesis of 6-chlorotryptophan Fmoc Amino Acids







Tert-butyl 6-chloro-3-formyl-1H-indole-1-carboxylate, 1. To a stirred solution of dry DMF (12 mL) was added dropwise POCl3 (3.92 mL, 43 mmol, 1.3 equiv) at 0° C. under Argon. The solution was stirred at the same temperature for 20 min before a solution of 6-chloroindole (5.0 g, 33 mmol, 1 eq.) in dry DMF (30 mL) was added dropwise. The resulting mixture was allowed to warm to room temperature and stirred for an additional 2.5 h. Water (50 mL) was added and the solution was neutralized with 4M aqueous NaOH (pH  ̃8). The resulting solid was filtered off, washed with water and dried under vacuum. This material was directly used in the next step without additional purification. To a stirred solution of the crude formyl indole (33 mmol, 1 eq.) in THF (150 mL) was added successively Boc2O (7.91 g, 36.3 mmol, 1.1 equiv) and DMAP (0.4 g, 3.3 mmol, 0.1 equiv) at room temperature under N2. The resulting mixture was stirred at room temperature for 1.5 h and the solvent was evaporated under reduced pressure. The residue was taken up in EtOAc and washed with 1N HCl, dried and concentrated to give the formyl indole 1 (9 g, 98% over 2 steps) as a white solid. 1H NMR (CDCl3) δ: 1.70 (s, Boc, 9H); 7.35 (dd, 1H); 8.21 (m, 3H); 10.07 (s, 1H).
Tert-butyl 6-chloro-3-(hydroxymethyl)-1H-indole-1-carboxylate, 2. To a solution of compound 1 (8.86 g, 32 mmol, 1 eq.) in ethanol (150 mL) was added NaBH4 (2.4 g, 63 mmol, 2 eq.). The reaction was stirred for 3 h at room temperature. The reaction mixture was concentrated and the residue was poured into diethyl ether and water. The organic layer was separated, dried over magnesium sulfate and concentrated to give a white solid (8.7 g, 98%). This material was directly used in the next step without additional purification. 1H NMR (CDCl3) δ: 1.65 (s, Boc, 9H); 4.80 (s, 2H, CH2); 7.21 (dd, 1H); 7.53 (m, 2H); 8.16 (bs, 1H).
Tert-butyl 3-(bromomethyl)-6-chloro-1H-indole-1-carboxylate, 3. To a solution of compound 2 (4.1 g, 14.6 mmol, 1 eq.) in dichloromethane (50 mL) under argon was added a solution of triphenylphosphine (4.59 g, 17.5 mmol, 1.2 eq.) in dichloromethane (50 mL) at −40° C. The reaction solution was stirred an additional 30 min at 40° C. Then NBS (3.38 g, 19 mmol, 1.3 eq.) was added. The resulting mixture was allowed to warm to room temperature and stirred overnight. Dichloromethane was evaporated, Carbon Tetrachloride (100 mL) was added and the mixture was stirred for 1 h and filtrated. The filtrate was concentrated, loaded in a silica plug and quickly eluted with 25% EtOAc in Hexanes. The solution was concentrated to give a white foam (3.84 g, 77%). 1H NMR (CDCl3) δ: 1.66 (s, Boc, 9H); 4.63 (s, 2H, CH2); 7.28 (dd, 1H); 7.57 (d, 1H); 7.64 (bs, 1H); 8.18 (bs, 1H).
αMe-6Cl-Trp(Boc)-Ni—S—BPB, 4. To S-Ala-Ni—S—BPB (2.66 g, 5.2 mmol, 1 eq.) and KO-tBu (0.87 g, 7.8 mmol, 1.5 eq.) was added 50 mL of DMF under argon. The bromide derivative compound 3 (2.68 g, 7.8 mmol, 1.5 eq.) in solution of DMF (5.0 mL) was added via syringe. The reaction mixture was stirred at ambient temperature for 1 h. The solution was then quenched with 5% aqueous acetic acid and diluted with water. The desired product was extracted in dichloromethane, dried and concentrated. The oily product 4 was purified by flash chromatography (solid loading) on normal phase using EtOAc and Hexanes as eluents to give a red solid (1.78 g, 45% yield). αMe-6Cl-Trp(Boc)-Ni—S—BPB, 4: M+H calc. 775.21, M+H obs. 775.26; 1H NMR (CDCl3) δ: 1.23 (s, 3H, αMe); 1.56 (m, 11H, Boc+CH2); 1.82-2.20 (m, 4H, 2CH2); 3.03 (m, 1H, CHα); 3.24 (m, 2H, CH2); 3.57 and 4.29 (AB system, 2H, CH2 (benzyl), J=12.8 Hz); 6.62 (d, 2H); 6.98 (d, 1H); 7.14 (m, 2H); 7.23 (m, 1H); 7.32-7.36 (m, 5H); 7.50 (m, 2H); 7.67 (bs, 1H); 7.98 (d, 2H); 8.27 (m, 2H).
Fmoc-αMe-6Cl-Trp(Boc)-OH, 6. To a solution of 3N HCl/MeOH (1/3, 15 mL) at 50° C. was added a solution of compound 4 (1.75 g, 2.3 mmol, 1 eq.) in MeOH (5 ml) dropwise. The starting material disappeared within 3-4 h. The acidic solution was then cooled to 0° C. with an ice bath and quenched with an aqueous solution of Na2CO3 (1.21 g, 11.5 mmol, 5 eq.). Methanol was removed and 8 more equivalents of Na2CO3 (1.95 g, 18.4 mmol) were added to the suspension. The Nickel scavenging EDTA disodium salt dihydrate (1.68 g, 4.5 mmol, 2 eq.) was then added and the suspension was stirred for 2 h. A solution of Fmoc-OSu (0.84 g, 2.5 mmol, 1.1 eq.) in acetone (50 mL) was added and the reaction was stirred overnight. Afterwards, the reaction was diluted with diethyl ether and 1N HCl. The organic layer was then dried over magnesium sulfate and concentrated in vacuo. The desired product 6 was purified on normal phase using acetone and dichloromethane as eluents to give a white foam (0.9 g, 70% yield). Fmoc-αMe-6Cl-Trp(Boc)-OH, 6: M+H calc. 575.19, M+H obs. 575.37; 1H NMR (CDCl3) 1.59 (s, 9H, Boc); 1.68 (s, 3H, Me); 3.48 (bs, 2H, CH2); 4.22 (m, 1H, CH); 4.39 (bs, 2H, CH2); 5.47 (s, 1H, NH); 7.10 (m, 1H); 7.18 (m, 2H); 7.27 (m, 2H); 7.39 (m, 2H); 7.50 (m, 2H); 7.75 (d, 2H); 8.12 (bs, 1H).
6Cl-Trp(Boc)-Ni—S—BPB, 5. To Gly-Ni—S—BPB (4.6 g, 9.2 mmol, 1 eq.) and KO-tBu (1.14 g, 10.1 mmol, 1.1 eq.) was added 95 mL of DMF under argon. The bromide derivative compound 3 (3.5 g, 4.6 mmol, 1.1 eq.) in solution of DMF (10 mL) was added via syringe. The reaction mixture was stirred at ambient temperature for 1 h. The solution was then quenched with 5% aqueous acetic acid and diluted with water. The desired product was extracted in dichloromethane, dried and concentrated. The oily product 5 was purified by flash chromatography (solid loading) on normal phase using EtOAc and Hexanes as eluents to give a red solid (5 g, 71% yield). 6Cl-Trp(Boc)-Ni—S—BPB, 5: M+H calc. 761.20, M+H obs. 761.34; 1H NMR (CDCl3) δ: 1.58 (m, 11H, Boc+CH2); 1.84 (m, 1H); 1.96 (m, 1H); 2.24 (m, 2H, CH2); 3.00 (m, 1H, CHα); 3.22 (m, 2H, CH2); 3.45 and 4.25 (AB system, 2H, CH2 (benzyl), J=12.8 Hz); 4.27 (m, 1H, CHα); 6.65 (d, 2H); 6.88 (d, 1H); 7.07 (m, 2H); 7.14 (m, 2H); 7.28 (m, 3H); 7.35-7.39 (m, 2H); 7.52 (m, 2H); 7.96 (d, 2H); 8.28 (m, 2H).
Fmoc-6Cl-Trp(Boc)-OH, 7. To a solution of 3N HCl/MeOH (1⁄3, 44 mL) at 50° C. was added a solution of compound 5 (5 g, 6.6 mmol, 1 eq.) in MeOH (10 ml) dropwise. The starting material disappeared within 3-4 h. The acidic solution was then cooled to 0° C. with an ice bath and quenched with an aqueous solution of Na2CO3 (3.48 g, 33 mmol, 5 eq.). Methanol was removed and 8 more equivalents of Na2CO3 (5.57 g, 52 mmol) were added to the suspension. The Nickel scavenging EDTA disodium salt dihydrate (4.89 g, 13.1 mmol, 2 eq.) and the suspension was stirred for 2 h. A solution of Fmoc-OSu (2.21 g, 6.55 mmol, 1.1 eq.) in acetone (100 mL) was added and the reaction was stirred overnight. Afterwards, the reaction was diluted with diethyl ether and 1N HCl. The organic layer was then dried over magnesium sulfate and concentrated in vacuo. The desired product 7 was purified on normal phase using acetone and dichloromethane as eluents to give a white foam (2.6 g, 69% yield). Fmoc-6Cl-Trp(Boc)-OH, 7: M+H calc. 561.17, M+H obs. 561.37; 1H NMR (CDCl3) 1.63 (s, 9H, Boc); 3.26 (m, 2H, CH2); 4.19 (m, 1H, CH); 4.39 (m, 2H, CH2); 4.76 (m, 1H); 5.35 (d, 1H, NH); 7.18 (m, 2H); 7.28 (m, 2H); 7.39 (m, 3H); 7.50 (m, 2H); 7.75 (d, 2H); 8.14 (bs, 1H).
Example 2
Peptidomimetic Macrocycles of the Invention
Peptidomimetic macrocycles were synthesized, purified and analyzed as previously described and as described below (Schafmeister et al., J. Am. Chem. Soc. 122:5891-5892 (2000); Schafmeister & Verdine, J. Am. Chem. Soc. 122:5891 (2005); Walensky et al., Science 305:1466-1470 (2004); and U.S. Pat. No. 7,192,713). Peptidomimetic macrocycles were designed by replacing two or more naturally occurring amino acids with the corresponding synthetic amino acids. Substitutions were made at i and i+4, and i and i+7 positions. Peptide synthesis was performed either manually or on an automated peptide synthesizer (Applied Biosystems, model 433A), using solid phase conditions, rink amide AM resin (Novabiochem), and Fmoc main-chain protecting group chemistry. For the coupling of natural Fmoc-protected amino acids (Novabiochem), 10 equivalents of amino acid and a 1:1:2 molar ratio of coupling reagents HBTU/HOBt (Novabiochem)/DIEA were employed. Non-natural amino acids (4 equiv) were coupled with a 1:1:2 molar ratio of HATU (Applied Biosystems)/HOBt/DIEA. The N-termini of the synthetic peptides were acetylated, while the C-termini were amidated.
Purification of cross-linked compounds was achieved by high performance liquid chromatography (HPLC) (Varian ProStar) on a reverse phase C18 column (Varian) to yield the pure compounds. Chemical composition of the pure products was confirmed by LC/MS mass spectrometry (Micromass LCT interfaced with Agilent 1100 HPLC system) and amino acid analysis (Applied Biosystems, model 420A).
Table 4 shows a list of peptidomimetic macrocycles of the invention prepared.












TABLE 4





SEQ




Observed


ID


Exact

mass


NO:
SP
Seq
Mass
M + 2
(m/e)




















38
SP-1
Ac-LSQETF$r8DLWKLL$EN-NH2
2068.13
1035.07
1035.36


39
SP-2
Ac-LSQETF$r8NLWKLL$QN-NH2
2066.16
1034.08
1034.31


40
SP-3
Ac-LSQQTF$r8NLWRLL$QN-NH2
2093.18
1047.59
1047.73


41
SP-4
Ac-QSQQTF$r8NLWKLL$QN-NH2
2080.15
1041.08
1041.31


42
SP-5
Ac-QSQQTF$r8NLWRLL$QN-NH2
2108.15
1055.08
1055.32


43
SP-6
Ac-QSQQTA$r8NLWRLL$QN-NH2
2032.12
1017.06
1017.24


44
SP-7
Ac-QAibQQTF$r8NLWRLL$QN-NH2
2106.17
1054.09
1054.34


45
SP-8
Ac-QSQQTFSNLWRLLPQN-NH2
2000.02
1001.01
1001.26


46
SP-9
Ac-QSQQTF$/r8NLWRLL$/QN-NH2
2136.18
1069.09
1069.37


47
SP-10
Ac-QSQAibTF$r8NLWRLL$QN-NH2
2065.15
1033.58
1033.71


48
SP-11
Ac-QSQQTF$r8NLWRLL$AN-NH2
2051.13
1026.57
1026.70


49
SP-12
Ac-ASQQTF$r8NLWRLL$QN-NH2
2051.13
1026.57
1026.90


50
SP-13
Ac-QSQQTF$r8ALWRLL$QN-NH2
2065.15
1033.58
1033.41


51
SP-14
Ac-QSQETF$r8NLWRLL$QN-NH2
2109.14
1055.57
1055.70


52
SP-15
Ac-RSQQTF$r8NLWRLL$QN-NH2
2136.20
1069.10
1069.17


53
SP-16
Ac-RSQQTF$r8NLWRLL$EN-NH2
2137.18
1069.59
1069.75


54
SP-17
Ac-LSQETFSDLWKLLPEN-NH2
1959.99
981.00
981.24


55
SP-18
Ac-QSQ$TFS$LWRLLPQN-NH2
2008.09
1005.05
1004.97


56
SP-19
Ac-QSQQ$FSN$WRLLPQN-NH2
2036.06
1019.03
1018.86


57
SP-20
Ac-QSQQT$SNL$RLLPQN-NH2
1917.04
959.52
959.32


58
SP-21
Ac-QSQQTF$NLW$LLPQN-NH2
2007.06
1004.53
1004.97


59
SP-22
Ac-RTQATF$r8NQWAibANle$TNAibTR-NH2
2310.26
1156.13
1156.52


60
SP-23
Ac-QSQQTF$r8NLWRLL$RN-NH2
2136.20
1069.10
1068.94


61
SP-24
Ac-QSQRTF$r8NLWRLL$QN-NH2
2136.20
1069.10
1068.94


62
SP-25
Ac-QSQQTF$r8NNleWRLL$QN-NH2
2108.15
1055.08
1055.44


63
SP-26
Ac-QSQQTF$r8NLWRNleL$QN-NH2
2108.15
1055.08
1055.84


64
SP-27
Ac-QSQQTF$r8NLWRLNle$QN-NH2
2108.15
1055.08
1055.12


65
SP-28
Ac-QSQQTY$r8NLWRLL$QN-NH2
2124.15
1063.08
1062.92


66
SP-29
Ac-RAibQQTF$r8NLWRLL$QN-NH2
2134.22
1068.11
1068.65


67
SP-30
Ac-MPRFMDYWEGLN-NH2
1598.70
800.35
800.45


68
SP-31
Ac-RSQQRF$r8NLWRLL$QN-NH2
2191.25
1096.63
1096.83


69
SP-32
Ac-QSQQRF$r8NLWRLL$QN-NH2
2163.21
1082.61
1082.87


70
SP-33
Ac-RAibQQRF$r8NLWRLL$QN-NH2
2189.27
1095.64
1096.37


71
SP-34
Ac-RSQQRF$r8NFWRLL$QN-NH2
2225.23
1113.62
1114.37


72
SP-35
Ac-RSQQRF$r8NYWRLL$QN-NH2
2241.23
1121.62
1122.37


73
SP-36
Ac-RSQQTF$r8NLWQLL$QN-NH2
2108.15
1055.08
1055.29


74
SP-37
Ac-QSQQTF$r8NLWQAmlL$QN-NH2
2094.13
1048.07
1048.32


75
SP-38
Ac-QSQQTF$r8NAmlWRLL$QN-NH2
2122.17
1062.09
1062.35


76
SP-39
Ac-NlePRF$r8DYWEGL$QN-NH2
1869.98
935.99
936.20


77
SP-40
Ac-NlePRF$r8NYWRLL$QN-NH2
1952.12
977.06
977.35


78
SP-41
Ac-RF$r8NLWRLL$Q-NH2
1577.96
789.98
790.18


79
SP-42
Ac-QSQQTF$r8N2ffWRLL$QN-NH2
2160.13
1081.07
1081.40


80
SP-43
Ac-QSQQTF$r8N3ffWRLL$QN-NH2
2160.13
1081.07
1081.34


81
SP-44
Ac-QSQQTF#r8NLWRLL#QN-NH2
2080.12
1041.06
1041.34


82
SP-45
Ac-RSQQTA$r8NLWRLL$QN-NH2
2060.16
1031.08
1031.38


83
SP-46
Ac-QSQQTF%r8NLWRLL%QN-NH2
2110.17
1056.09
1056.55


84
SP-47
HepQSQ$TFSNLWRLLPQN-NH2
2051.10
1026.55
1026.82


85
SP-48
HepQSQ$TF$r8NLWRLL$QN-NH2
2159.23
1080.62
1080.89


86
SP-49
Ac-QSQQTF$r8NL6cIWRLL$QN-NH2
2142.11
1072.06
1072.35


87
SP-50
Ac-QSQQTF$r8NLMe6cIwRLL$QN-NH2
2156.13
1079.07
1079.27


88
SP-51
Ac-LTFEHYWAQLTS-NH2
1535.74
768.87
768.91


89
SP-52
Ac-LTF$HYW$QLTS-NH2
1585.83
793.92
794.17


90
SP-53
Ac-LTFE$YWA$LTS-NH2
1520.79
761.40
761.67


91
SP-54
Ac-LTF$zr8HYWAQL$zS-NH2
1597.87
799.94
800.06


92
SP-55
Ac-LTF$r8HYWRQL$S-NH2
1682.93
842.47
842.72


93
SP-56
Ac-QS$QTFStNLWRLL$s8QN-NH2
2145.21
1073.61
1073.90


94
SP-57
Ac-QSQQTASNLWRLLPQN-NH2
1923.99
963.00
963.26


95
SP-58
Ac-QSQQTA$/r8NLWRLL$/QN-NH2
2060.15
1031.08
1031.24


96
SP-59
Ac-ASQQTF$/r8NLWRLL$/QN-NH2
2079.16
1040.58
1040.89


97
SP-60
Ac-$SQQ$FSNLWRLLAibQN-NH2
2009.09
1005.55
1005.86


98
SP-61
Ac-QS$QTF$NLWRLLAibQN-NH2
2023.10
1012.55
1012.79


99
SP-62
Ac-QSQQ$FSN$WRLLAibQN-NH2
2024.06
1013.03
1013.31


100
SP-63
Ac-QSQQTF$NLW$LLAibQN-NH2
1995.06
998.53
998.87


101
SP-64
Ac-QSQQTFS$LWR$LAibQN-NH2
2011.06
1006.53
1006.83


102
SP-65
Ac-QSQQTFSNLW$LLA$N-NH2
1940.02
971.01
971.29


103
SP-66
Ac-$/SQQ$/FSNLWRLLAibQN-NH2
2037.12
1019.56
1019.78


104
SP-67
Ac-QS$/QTF$/NLWRLLAibQN-NH2
2051.13
1026.57
1026.90


105
SP-68
Ac-QSQQ$/FSN$/WRLLAibQN-NH2
2052.09
1027.05
1027.36


106
SP-69
Ac-QSQQTF$/NLW$/LLAibQN-NH2
2023.09
1012.55
1013.82


107
SP-70
Ac-QSQ$TFS$LWRLLAibQN-NH2
1996.09
999.05
999.39


108
SP-71
Ac-QSQ$/TFS$/LWRLLAibQN-NH2
2024.12
1013.06
1013.37


109
SP-72
Ac-QS$/QTFSt//NLWRLL$/s8QN-NH2
2201.27
1101.64
1102.00


110
SP-73
Ac-$r8SQQTFS$LWRLLAibQN-NH2
2038.14
1020.07
1020.23


111
SP-74
Ac-QSQ$r8TFSNLW$LLAibQN-NH2
1996.08
999.04
999.32


112
SP-75
Ac-QSQQTFS$r8LWRLLA$N-NH2
2024.12
1013.06
1013.37


113
SP-76
Ac-QS$r5QTFStNLW$LLAibQN-NH2
2032.12
1017.06
1017.39


114
SP-77
Ac-$/r8SQQTFS$/LWRLLAibQN-NH2
2066.17
1034.09
1034.80


115
SP-78
Ac-QSQ$/r8TFSNLW$/LLAibQN-NH2
2024.11
1013.06
1014.34


116
SP-79
Ac-QSQQTFS$/r8LWRLLA$/N-NH2
2052.15
1027.08
1027.16


117
SP-80
Ac-QS$/r5QTFSt//NLW$/LLAibQN-NH2
2088.18
1045.09
1047.10


118
SP-81
Ac-QSQQTFSNLWRLLAibQN-NH2
1988.02
995.01
995.31


119
SP-82
Hep/QSQ$/TF$/r8NLWRLL$/QN-NH2
2215.29
1108.65
1108.93


120
SP-83
Ac-ASQQTF$r8NLRWLL$QN-NH2
2051.13
1026.57
1026.90


121
SP-84
Ac-QSQQTF$/r8NLWRLL$/Q-NH2
2022.14
1012.07
1012.66


122
SP-85
Ac-QSQQTF$r8NLWRLL$Q-NH2
1994.11
998.06
998.42


123
SP-86
Ac-AAARAA$r8AAARAA$AA-NH2
1515.90
758.95
759.21


124
SP-87
Ac-LTFEHYWAQLTSA-NH2
1606.78
804.39
804.59


125
SP-88
Ac-LTF$r8HYWAQL$SA-NH2
1668.90
835.45
835.67


126
SP-89
Ac-ASQQTFSNLWRLLPQN-NH2
1943.00
972.50
973.27


127
SP-90
Ac-QS$QTFStNLW$r5LLAibQN-NH2
2032.12
1017.06
1017.30


128
SP-91
Ac-QSQQTFAibNLWRLLAibQN-NH2
1986.04
994.02
994.19


129
SP-92
Ac-QSQQTFNleNLWRLLNleQN-NH2
2042.11
1022.06
1022.23


130
SP-93
Ac-QSQQTF$/r8NLWRLLAibQN-NH2
2082.14
1042.07
1042.23


131
SP-94
Ac-QSQQTF$/r8NLWRLLNleQN-NH2
2110.17
1056.09
1056.29


132
SP-95
Ac-QSQQTFAibNLWRLL$/QN-NH2
2040.09
1021.05
1021.25


133
SP-96
Ac-QSQQTFNleNLWRLL$/QN-NH2
2068.12
1035.06
1035.31


134
SP-97
Ac-QSQQTF%r8NL6cIWRNleL%QN-NH2
2144.13
1073.07
1073.32


135
SP-98
Ac-QSQQTF%r8NLMe6cIWRLL%QN-NH2
2158.15
1080.08
1080.31


136
SP-101
Ac-FNle$YWE$L-NH2
1160.63
—
1161.70


137
SP-102
Ac-F$r8AYWELL$A-NH2
1344.75
—
1345.90


138
SP-103
Ac-F$r8AYWQLL$A-NH2
1343.76
—
1344.83


139
SP-104
Ac-NlePRF$r8NYWELL$QN-NH2
1925.06
963.53
963.69


140
SP-105
Ac-NlePRF$r8DYWRLL$QN-NH2
1953.10
977.55
977.68


141
SP-106
Ac-NlePRF$r8NYWRLL$Q-NH2
1838.07
920.04
920.18


142
SP-107
Ac-NlePRF$r8NYWRLL$-NH2
1710.01
856.01
856.13


143
SP-108
Ac-QSQQTF$r8DLWRLL$QN-NH2
2109.14
1055.57
1055.64


144
SP-109
Ac-QSQQTF$r8NLWRLL$EN-NH2
2109.14
1055.57
1055.70


145
SP-110
Ac-QSQQTF$r8NLWRLL$QD-NH2
2109.14
1055.57
1055.64


146
SP-111
Ac-QSQQTF$r8NLWRLL$S-NH2
1953.08
977.54
977.60


147
SP-112
Ac-ESQQTF$r8NLWRLL$QN-NH2
2109.14
1055.57
1055.70


148
SP-113
Ac-LTF$r8NLWRNleL$Q-NH2
1635.99
819.00
819.10


149
SP-114
Ac-LRF$r8NLWRNleL$Q-NH2
1691.04
846.52
846.68


150
SP-115
Ac-QSQQTF$r8NWWRNleL$QN-NH2
2181.15
1091.58
1091.64


151
SP-116
Ac-QSQQTF$r8NLWRNleL$Q-NH2
1994.11
998.06
998.07


152
SP-117
Ac-QTF$r8NLWRNleL$QN-NH2
1765.00
883.50
883.59


153
SP-118
Ac-NlePRF$r8NWWRLL$QN-NH2
1975.13
988.57
988.75


154
SP-119
Ac-NlePRF$r8NWWRLL$A-NH2
1804.07
903.04
903.08


155
SP-120
Ac-TSFAEYWNLLSP-NH2
1467.70
734.85
734.90


156
SP-121
Ac-QTF$r8HWWSQL$S-NH2
1651.85
826.93
827.12


157
SP-122
Ac-FM$YWE$L-NH2
1178.58
—
1179.64


158
SP-123
Ac-QTFEHWWSQLLS-NH2
1601.76
801.88
801.94


159
SP-124
Ac-QSQQTF$r8NLAmwRLNle$QN-NH2
2122.17
1062.09
1062.24


160
SP-125
Ac-FMAibY6cIWEAc3cL-NH2
1130.47
—
1131.53


161
SP-126
Ac-FNle$Y6cIWE$L-NH2
1194.59
—
1195.64


162
SP-127
Ac-F$zr8AY6cIWEAc3cL$z-NH2
1277.63
639.82
1278.71


163
SP-128
Ac-F$r8AY6cIWEAc3cL$A-NH2
1348.66
—
1350.72


164
SP-129
Ac-NlePRF$r8NY6cIWRLL$QN-NH2
1986.08
994.04
994.64


165
SP-130
Ac-AF$r8AAWALA$A-NH2
1223.71
—
1224.71


166
SP-131
Ac-TF$r8AAWRLA$Q-NH2
1395.80
698.90
399.04


167
SP-132
Pr-TF$r8AAWRLA$Q-NH2
1409.82
705.91
706.04


168
SP-133
Ac-QSQQTF%r8NLWRNleL%QN-NH2
2110.17
1056.09
1056.22


169
SP-134
Ac-LTF%r8HYWAQL%SA-NH2
1670.92
836.46
836.58


170
SP-135
Ac-NlePRF%r8NYWRLL%QN-NH2
1954.13
978.07
978.19


171
SP-136
Ac-NlePRF%r8NY6cIWRLL%QN-NH2
1988.09
995.05
995.68


172
SP-137
Ac-LTF%r8HY6cIWAQL%S-NH2
1633.84
817.92
817.93


173
SP-138
Ac-QS%QTF%StNLWRLL%s8QN-NH2
2149.24
1075.62
1075.65


174
SP-139
Ac-LTF%r8HY6cIWRQL%S-NH2
1718.91
860.46
860.54


175
SP-140
Ac-QSQQTF%r8NL6cIWRLL%QN-NH2
2144.13
1073.07
1073.64


176
SP-141
Ac-%r8SQQTFS%LWRLLAibQN-NH2
2040.15
1021.08
1021.13


177
SP-142
Ac-LTF%r8HYWAQL%S-NH2
1599.88
800.94
801.09


178
SP-143
Ac-TSF%r8QYWNLL%P-NH2
1602.88
802.44
802.58


179
SP-147
Ac-LTFEHYWAQLTS-NH2
1535.74
768.87
769.5


180
SP-152
Ac-F$er8AY6cIWEAc3cL$e-NH2
1277.63
639.82
1278.71


181
SP-153
Ac-AF$r8AAWALA$A-NH2
1277.63
639.82
1277.84


182
SP-154
Ac-TF$r8AAWRLA$Q-NH2
1395.80
698.90
699.04


183
SP-155
Pr-TF$r8AAWRLA$Q-NH2
1409.82
705.91
706.04


184
SP-156
Ac-LTF$er8HYWAQL$eS-NH2
1597.87
799.94
800.44


185
SP-159
Ac-CCPGCCBaQSQQTF$r8NLWRLL$QN-NH2
2745.30
1373.65
1372.99


186
SP-160
Ac-CCPGCCBaQSQQTA$r8NLWRLL$QN-
2669.27
1335.64
1336.09




NH2





187
SP-161
Ac-CCPGCCBaNlePRF$r8NYWRLL$QN-NH2
2589.26
1295.63
1296.2


188
SP-162
Ac-LTF$/r8HYWAQL$/S-NH2
1625.90
813.95
814.18


189
SP-163
Ac-F%r8HY6cIWRAc3cL%-NH2
1372.72
687.36
687.59


190
SP-164
Ac-QTF%r8HWWSQL%S-NH2
1653.87
827.94
827.94


191
SP-165
Ac-LTA$r8HYWRQL$S-NH2
1606.90
804.45
804.66


192
SP-166
Ac-Q$r8QQTFSN$WRLLAibQN-NH2
2080.12
1041.06
1041.61


193
SP-167
Ac-QSQQ$r8FSNLWR$LAibQN-NH2
2066.11
1034.06
1034.58


194
SP-168
Ac-F$r8AYWEAc3cL$A-NH2
1314.70
658.35
1315.88


195
SP-169
Ac-F$r8AYWEAc3cL$S-NH2
1330.70
666.35
1331.87


196
SP-170
Ac-F$r8AYWEAc3cL$Q-NH2
1371.72
686.86
1372.72


197
SP-171
Ac-F$r8AYWEAibL$S-NH2
1332.71
667.36
1334.83


198
SP-172
Ac-F$r8AYWEAL$S-NH2
1318.70
660.35
1319.73


199
SP-173
Ac-F$r8AYWEQL$S-NH2
1375.72
688.86
1377.53


200
SP-174
Ac-F$r8HYWEQL$S-NH2
1441.74
721.87
1443.48


201
SP-175
Ac-F$r8HYWAQL$S-NH2
1383.73
692.87
1385.38


202
SP-176
Ac-F$r8HYWAAc3cL$S-NH2
1338.71
670.36
1340.82


203
SP-177
Ac-F$r8HYWRAc3cL$S-NH2
1423.78
712.89
713.04


204
SP-178
Ac-F$r8AYWEAc3cL#A-NH2
1300.69
651.35
1302.78


205
SP-179
Ac-NlePTF%r8NYWRLL%QN-NH2
1899.08
950.54
950.56


206
SP-180
Ac-TF$r8AAWRAL$Q-NH2
1395.80
698.90
699.13


207
SP-181
Ac-TSF%r8HYWAQL%S-NH2
1573.83
787.92
787.98


208
SP-184
Ac-F%r8AY6cIWEAc3cL%A-NH2
1350.68
676.34
676.91


209
SP-185
Ac-LTF$r8HYWAQI$S-NH2
1597.87
799.94
800.07


210
SP-186
Ac-LTF$r8HYWAQNle$S-NH2
1597.87
799.94
800.07


211
SP-187
Ac-LTF$r8HYWAQL$A-NH2
1581.87
791.94
792.45


212
SP-188
Ac-LTF$r8HYWAQL$Abu-NH2
1595.89
798.95
799.03


213
SP-189
Ac-LTF$r8HYWAbuQL$S-NH2
1611.88
806.94
807.47


214
SP-190
Ac-LTF$er8AYWAQL$eS-NH2
1531.84
766.92
766.96


215
SP-191
Ac-LAF$r8HYWAQL$S-NH2
1567.86
784.93
785.49


216
SP-192
Ac-LAF$r8AYWAQL$S-NH2
1501.83
751.92
752.01


217
SP-193
Ac-LTF$er8AYWAQL$eA-NH2
1515.85
758.93
758.97


218
SP-194
Ac-LAF$r8AYWAQL$A-NH2
1485.84
743.92
744.05


219
SP-195
Ac-LTF$r8NLWANleL$Q-NH2
1550.92
776.46
776.61


220
SP-196
Ac-LTF$r8NLWANleL$A-NH2
1493.90
747.95
1495.6


221
SP-197
Ac-LTF$r8ALWANleL$Q-NH2
1507.92
754.96
755


222
SP-198
Ac-LAF$r8NLWANleL$Q-NH2
1520.91
761.46
761.96


223
SP-199
Ac-LAF$r8ALWANleL$A-NH2
1420.89
711.45
1421.74


224
SP-200
Ac-A$r8AYWEAc3cL$A-NH2
1238.67
620.34
1239.65


225
SP-201
Ac-F$r8AYWEAc3cL$AA-NH2
1385.74
693.87
1386.64


226
SP-202
Ac-F$r8AYWEAc3cL$Abu-NH2
1328.72
665.36
1330.17


227
SP-203
Ac-F$r8AYWEAc3cL$Nle-NH2
1356.75
679.38
1358.22


228
SP-204
Ac-F$r5AYWEAc3cL$s8A-NH2
1314.70
658.35
1315.51


229
SP-205
Ac-F$AYWEAc3cL$r8A-NH2
1314.70
658.35
1315.66


230
SP-206
Ac-F$r8AYWEAc3cI$A-NH2
1314.70
658.35
1316.18


231
SP-207
Ac-F$r8AYWEAc3cNle$A-NH2
1314.70
658.35
1315.66


232
SP-208
Ac-F$r8AYWEAmlL$A-NH2
1358.76
680.38
1360.21


233
SP-209
Ac-F$r8AYWENleL$A-NH2
1344.75
673.38
1345.71


234
SP-210
Ac-F$r8AYWQAc3cL$A-NH2
1313.72
657.86
1314.7


235
SP-211
Ac-F$r8AYWAAc3cL$A-NH2
1256.70
629.35
1257.56


236
SP-212
Ac-F$r8AYWAbuAc3cL$A-NH2
1270.71
636.36
1272.14


237
SP-213
Ac-F$r8AYWNleAc3cL$A-NH2
1298.74
650.37
1299.67


238
SP-214
Ac-F$r8AbuYWEAc3cL$A-NH2
1328.72
665.36
1329.65


239
SP-215
Ac-F$r8NleYWEAc3cL$A-NH2
1356.75
679.38
1358.66


240
SP-216
5-FAM-BaLTFEHYWAQLTS-NH2
1922.82
962.41
962.87


241
SP-217
5-FAM-BaLTF%r8HYWAQL%S-NH2
1986.96
994.48
994.97


242
SP-218
Ac-LTF$r8HYWAQhL$S-NH2
1611.88
806.94
807


243
SP-219
Ac-LTF$r8HYWAQTle$S-NH2
1597.87
799.94
799.97


244
SP-220
Ac-LTF$r8HYWAQAdm$S-NH2
1675.91
838.96
839.09


245
SP-221
Ac-LTF$r8HYWAQhCha$S-NH2
1651.91
826.96
826.98


246
SP-222
Ac-LTF$r8HYWAQCha$S-NH2
1637.90
819.95
820.02


247
SP-223
Ac-LTF$r8HYWAc6cQL$S-NH2
1651.91
826.96
826.98


248
SP-224
Ac-LTF$r8HYWAc5cQL$S-NH2
1637.90
819.95
820.02


249
SP-225
Ac-LThF$r8HYWAQL$S-NH2
1611.88
806.94
807


250
SP-226
Ac-LTIgl$r8HYWAQL$S-NH2
1625.90
813.95
812.99


251
SP-227
Ac-LTF$r8HYWAQChg$S-NH2
1623.88
812.94
812.99


252
SP-228
Ac-LTF$r8HYWAQF$S-NH2
1631.85
816.93
816.99


253
SP-229
Ac-LTF$r8HYWAQIgl$S-NH2
1659.88
830.94
829.94


254
SP-230
Ac-LTF$r8HYWAQCba$S-NH2
1609.87
805.94
805.96


255
SP-231
Ac-LTF$r8HYWAQCpg$S-NH2
1609.87
805.94
805.96


256
SP-232
Ac-LTF$r8HhYWAQL$S-NH2
1611.88
806.94
807


257
SP-233
Ac-F$r8AYWEAc3chL$A-NH2
1328.72
665.36
665.43


258
SP-234
Ac-F$r8AYWEAc3cTle$A-NH2
1314.70
658.35
1315.62


259
SP-235
Ac-F$r8AYWEAc3cAdm$A-NH2
1392.75
697.38
697.47


260
SP-236
Ac-F$r8AYWEAc3chCha$A-NH2
1368.75
685.38
685.34


261
SP-237
Ac-F$r8AYWEAc3cCha$A-NH2
1354.73
678.37
678.38


262
SP-238
Ac-F$r8AYWEAc6cL$A-NH2
1356.75
679.38
679.42


263
SP-239
Ac-F$r8AYWEAc5cL$A-NH2
1342.73
672.37
672.46


264
SP-240
Ac-hF$r8AYWEAc3cL$A-NH2
1328.72
665.36
665.43


265
SP-241
Ac-Igl$r8AYWEAc3cL$A-NH2
1342.73
672.37
671.5


266
SP-243
Ac-F$r8AYWEAc3cF$A-NH2
1348.69
675.35
675.35


267
SP-244
Ac-F$r8AYWEAc3cIgl$A-NH2
1376.72
689.36
688.37


268
SP-245
Ac-F$r8AYWEAc3cCba$A-NH2
1326.70
664.35
664.47


269
SP-246
Ac-F$r8AYWEAc3cCpg$A-NH2
1326.70
664.35
664.39


270
SP-247
Ac-F$r8AhYWEAc3cL$A-NH2
1328.72
665.36
665.43


271
SP-248
Ac-F$r8AYWEAc3cL$Q-NH2
1371.72
686.86
1372.87


272
SP-249
Ac-F$r8AYWEAibL$A-NH2
1316.72
659.36
1318.18


273
SP-250
Ac-F$r8AYWEAL$A-NH2
1302.70
652.35
1303.75


274
SP-251
Ac-LAF$r8AYWAAL$A-NH2
1428.82
715.41
715.49


275
SP-252
Ac-LTF$r8HYWAAc3cL$S-NH2
1552.84
777.42
777.5


276
SP-253
Ac-NleTF$r8HYWAQL$S-NH2
1597.87
799.94
800.04


277
SP-254
Ac-VTF$r8HYWAQL$S-NH2
1583.85
792.93
793.04


278
SP-255
Ac-FTF$r8HYWAQL$S-NH2
1631.85
816.93
817.02


279
SP-256
Ac-WTF$r8HYWAQL$S-NH2
1670.86
836.43
836.85


280
SP-257
Ac-RTF$r8HYWAQL$S-NH2
1640.88
821.44
821.9


281
SP-258
Ac-KTF$r8HYWAQL$S-NH2
1612.88
807.44
807.91


282
SP-259
Ac-LNleF$r8HYWAQL$S-NH2
1609.90
805.95
806.43


283
SP-260
Ac-LVF$r8HYWAQL$S-NH2
1595.89
798.95
798.93


284
SP-261
Ac-LFF$r8HYWAQL$S-NH2
1643.89
822.95
823.38


285
SP-262
Ac-LWF$r8HYWAQL$S-NH2
1682.90
842.45
842.55


286
SP-263
Ac-LRF$r8HYWAQL$S-NH2
1652.92
827.46
827.52


287
SP-264
Ac-LKF$r8HYWAQL$S-NH2
1624.91
813.46
813.51


288
SP-265
Ac-LTF$r8NleYWAQL$S-NH2
1573.89
787.95
788.05


289
SP-266
Ac-LTF$r8VYWAQL$S-NH2
1559.88
780.94
780.98


290
SP-267
Ac-LTF$r8FYWAQL$S-NH2
1607.88
804.94
805.32


291
SP-268
Ac-LTF$r8WYWAQL$S-NH2
1646.89
824.45
824.86


292
SP-269
Ac-LTF$r8RYWAQL$S-NH2
1616.91
809.46
809.51


293
SP-270
Ac-LTF$r8KYWAQL$S-NH2
1588.90
795.45
795.48


294
SP-271
Ac-LTF$r8HNleWAQL$S-NH2
1547.89
774.95
774.98


295
SP-272
Ac-LTF$r8HVWAQL$S-NH2
1533.87
767.94
767.95


296
SP-273
Ac-LTF$r8HFWAQL$S-NH2
1581.87
791.94
792.3


297
SP-274
Ac-LTF$r8HWWAQL$S-NH2
1620.88
811.44
811.54


298
SP-275
Ac-LTF$r8HRWAQL$S-NH2
1590.90
796.45
796.52


299
SP-276
Ac-LTF$r8HKWAQL$S-NH2
1562.90
782.45
782.53


300
SP-277
Ac-LTF$r8HYWNleQL$S-NH2
1639.91
820.96
820.98


301
SP-278
Ac-LTF$r8HYWVQL$S-NH2
1625.90
813.95
814.03


302
SP-279
Ac-LTF$r8HYWFQL$S-NH2
1673.90
837.95
838.03


303
SP-280
Ac-LTF$r8HYWWQL$S-NH2
1712.91
857.46
857.5


304
SP-281
Ac-LTF$r8HYWKQL$S-NH2
1654.92
828.46
828.49


305
SP-282
Ac-LTF$r8HYWANleL$S-NH2
1582.89
792.45
792.52


306
SP-283
Ac-LTF$r8HYWAVL$S-NH2
1568.88
785.44
785.49


307
SP-284
Ac-LTF$r8HYWAFL$S-NH2
1616.88
809.44
809.47


308
SP-285
Ac-LTF$r8HYWAWL$S-NH2
1655.89
828.95
829


309
SP-286
Ac-LTF$r8HYWARL$S-NH2
1625.91
813.96
813.98


310
SP-287
Ac-LTF$r8HYWAQL$Nle-NH2
1623.92
812.96
813.39


311
SP-288
Ac-LTF$r8HYWAQL$V-NH2
1609.90
805.95
805.99


312
SP-289
Ac-LTF$r8HYWAQL$F-NH2
1657.90
829.95
830.26


313
SP-290
Ac-LTF$r8HYWAQL$W-NH2
1696.91
849.46
849.5


314
SP-291
Ac-LTF$r8HYWAQL$R-NH2
1666.94
834.47
834.56


315
SP-292
Ac-LTF$r8HYWAQL$K-NH2
1638.93
820.47
820.49


316
SP-293
Ac-Q$r8QQTFSN$WRLLAibQN-NH2
2080.12
1041.06
1041.54


317
SP-294
Ac-QSQQ$r8FSNLWR$LAibQN-NH2
2066.11
1034.06
1034.58


318
SP-295
Ac-LT2Pal$r8HYWAQL$S-NH2
1598.86
800.43
800.49


319
SP-296
Ac-LT3Pal$r8HYWAQL$S-NH2
1598.86
800.43
800.49


320
SP-297
Ac-LT4Pal$r8HYWAQL$S-NH2
1598.86
800.43
800.49


321
SP-298
Ac-LTF2CF3$r8HYWAQL$S-NH2
1665.85
833.93
834.01


322
SP-299
Ac-LTF2CN$r8HYWAQL$S-NH2
1622.86
812.43
812.47


323
SP-300
Ac-LTF2Me$r8HYWAQL$S-NH2
1611.88
806.94
807


324
SP-301
Ac-LTF3CI$r8HYWAQL$S-NH2
1631.83
816.92
816.99


325
SP-302
Ac-LTF4CF3$r8HYWAQL$S-NH2
1665.85
833.93
833.94


326
SP-303
Ac-LTF4tBu$r8HYWAQL$S-NH2
1653.93
827.97
828.02


327
SP-304
Ac-LTF5F$r8HYWAQL$S-NH2
1687.82
844.91
844.96


328
SP-305
Ac-LTF$r8HY3BthAAQL$S-NH2
1614.83
808.42
808.48


329
SP-306
Ac-LTF2Br$r8HYWAQL$S-NH2
1675.78
838.89
838.97


330
SP-307
Ac-LTF4Br$r8HYWAQL$S-NH2
1675.78
838.89
839.86


331
SP-308
Ac-LTF2CI$r8HYWAQL$S-NH2
1631.83
816.92
816.99


332
SP-309
Ac-LTF4CI$r8HYWAQL$S-NH2
1631.83
816.92
817.36


333
SP-310
Ac-LTF3CN$r8HYWAQL$S-NH2
1622.86
812.43
812.47


334
SP-311
Ac-LTF4CN$r8HYWAQL$S-NH2
1622.86
812.43
812.47


335
SP-312
Ac-LTF34CI2$r8HYWAQL$S-NH2
1665.79
833.90
833.94


336
SP-313
Ac-LTF34F2$r8HYWAQL$S-NH2
1633.85
817.93
817.95


337
SP-314
Ac-LTF35F2$r8HYWAQL$S-NH2
1633.85
817.93
817.95


338
SP-315
Ac-LTDip$r8HYWAQL$S-NH2
1673.90
837.95
838.01


339
SP-316
Ac-LTF2F$r8HYWAQL$S-NH2
1615.86
808.93
809


340
SP-317
Ac-LTF3F$r8HYWAQL$S-NH2
1615.86
808.93
809


341
SP-318
Ac-LTF4F$r8HYWAQL$S-NH2
1615.86
808.93
809


342
SP-319
Ac-LTF4I$r8HYWAQL$S-NH2
1723.76
862.88
862.94


343
SP-320
Ac-LTF3Me$r8HYWAQL$S-NH2
1611.88
806.94
807.07


344
SP-321
Ac-LTF4Me$r8HYWAQL$S-NH2
1611.88
806.94
807


345
SP-322
Ac-LT1Nal$r8HYWAQL$S-NH2
1647.88
824.94
824.98


346
SP-323
Ac-LT2Nal$r8HYWAQL$S-NH2
1647.88
824.94
825.06


347
SP-324
Ac-LTF3CF3$r8HYWAQL$S-NH2
1665.85
833.93
834.01


348
SP-325
Ac-LTF4NO2$r8HYWAQL$S-NH2
1642.85
822.43
822.46


349
SP-326
Ac-LTF3NO2$r8HYWAQL$S-NH2
1642.85
822.43
822.46


350
SP-327
Ac-LTF$r82ThiYWAQL$S-NH2
1613.83
807.92
807.96


351
SP-328
Ac-LTF$r8HBipWAQL$S-NH2
1657.90
829.95
830.01


352
SP-329
Ac-LTF$r8HF4tBuWAQL$S-NH2
1637.93
819.97
820.02


353
SP-330
Ac-LTF$r8HF4CF3WAQL$S-NH2
1649.86
825.93
826.02


354
SP-331
Ac-LTF$r8HF4CIWAQL$S-NH2
1615.83
808.92
809.37


355
SP-332
Ac-LTF$r8HF4MeWAQL$S-NH2
1595.89
798.95
799.01


356
SP-333
Ac-LTF$r8HF4BrWAQL$S-NH2
1659.78
830.89
830.98


357
SP-334
Ac-LTF$r8HF4CNWAQL$S-NH2
1606.87
804.44
804.56


358
SP-335
Ac-LTF$r8HF4NO2WAQL$S-NH2
1626.86
814.43
814.55


359
SP-336
Ac-LTF$r8H1NalWAQL$S-NH2
1631.89
816.95
817.06


360
SP-337
Ac-LTF$r8H2NalWAQL$S-NH2
1631.89
816.95
816.99


361
SP-338
Ac-LTF$r8HWAQL$S-NH2
1434.80
718.40
718.49


362
SP-339
Ac-LTF$r8HY1NalAQL$S-NH2
1608.87
805.44
805.52


363
SP-340
Ac-LTF$r8HY2NalAQL$S-NH2
1608.87
805.44
805.52


364
SP-341
Ac-LTF$r8HYWAQI$S-NH2
1597.87
799.94
800.07


365
SP-342
Ac-LTF$r8HYWAQNle$S-NH2
1597.87
799.94
800.44


366
SP-343
Ac-LTF$er8HYWAQL$eA-NH2
1581.87
791.94
791.98


367
SP-344
Ac-LTF$r8HYWAQL$Abu-NH2
1595.89
798.95
799.03


368
SP-345
Ac-LTF$r8HYWAbuQL$S-NH2
1611.88
806.94
804.47


369
SP-346
Ac-LAF$r8HYWAQL$S-NH2
1567.86
784.93
785.49


370
SP-347
Ac-LTF$r8NLWANleL$Q-NH2
1550.92
776.46
777.5


371
SP-348
Ac-LTF$r8ALWANleL$Q-NH2
1507.92
754.96
755.52


372
SP-349
Ac-LAF$r8NLWANleL$Q-NH2
1520.91
761.46
762.48


373
SP-350
Ac-F$r8AYWAAc3CI$A-NH2
1256.70
629.35
1257.56


374
SP-351
Ac-LTF$r8AYWAAL$S-NH2
1474.82
738.41
738.55


375
SP-352
Ac-LVF$r8AYWAQL$S-NH2
1529.87
765.94
766


376
SP-353
Ac-LTF$r8AYWAbuQL$S-NH2
1545.86
773.93
773.92


377
SP-354
Ac-LTF$r8AYWNleQL$S-NH2
1573.89
787.95
788.17


378
SP-355
Ac-LTF$r8AbuYWAQL$S-NH2
1545.86
773.93
773.99


379
SP-356
Ac-LTF$r8AYWHQL$S-NH2
1597.87
799.94
799.97


380
SP-357
Ac-LTF$r8AYWKQL$S-NH2
1588.90
795.45
795.53


381
SP-358
Ac-LTF$r8AYWOQL$S-NH2
1574.89
788.45
788.5


382
SP-359
Ac-LTF$r8AYWRQL$S-NH2
1616.91
809.46
809.51


383
SP-360
Ac-LTF$r8AYWSQL$S-NH2
1547.84
774.92
774.96


384
SP-361
Ac-LTF$r8AYWRAL$S-NH2
1559.89
780.95
780.95


385
SP-362
Ac-LTF$r8AYWRQL$A-NH2
1600.91
801.46
801.52


386
SP-363
Ac-LTF$r8AYWRAL$A-NH2
1543.89
772.95
773.03


387
SP-364
Ac-LTF$r5HYWAQL$s8S-NH2
1597.87
799.94
799.97


388
SP-365
Ac-LTF$HYWAQL$r8S-NH2
1597.87
799.94
799.97


389
SP-366
Ac-LTF$r8HYWAAL$S-NH2
1540.84
771.42
771.48


390
SP-367
Ac-LTF$r8HYWAAbuL$S-NH2
1554.86
778.43
778.51


391
SP-368
Ac-LTF$r8HYWALL$S-NH2
1582.89
792.45
792.49


392
SP-369
Ac-F$r8AYWHAL$A-NH2
1310.72
656.36
656.4


393
SP-370
Ac-F$r8AYWAAL$A-NH2
1244.70
623.35
1245.61


394
SP-371
Ac-F$r8AYWSAL$A-NH2
1260.69
631.35
1261.6


395
SP-372
Ac-F$r8AYWRAL$A-NH2
1329.76
665.88
1330.72


396
SP-373
Ac-F$r8AYWKAL$A-NH2
1301.75
651.88
1302.67


397
SP-374
Ac-F$r8AYWOAL$A-NH2
1287.74
644.87
1289.13


398
SP-375
Ac-F$r8VYWEAc3cL$A-NH2
1342.73
672.37
1343.67


399
SP-376
Ac-F$r8FYWEAc3cL$A-NH2
1390.73
696.37
1392.14


400
SP-377
Ac-F$r8WYWEAc3cL$A-NH2
1429.74
715.87
1431.44


401
SP-378
Ac-F$r8RYWEAc3cL$A-NH2
1399.77
700.89
700.95


402
SP-379
Ac-F$r8KYWEAc3cL$A-NH2
1371.76
686.88
686.97


403
SP-380
Ac-F$r8ANleWEAc3cL$A-NH2
1264.72
633.36
1265.59


404
SP-381
Ac-F$r8AVWEAc3cL$A-NH2
1250.71
626.36
1252.2


405
SP-382
Ac-F$r8AFWEAc3cL$A-NH2
1298.71
650.36
1299.64


406
SP-383
Ac-F$r8AWWEAc3cL$A-NH2
1337.72
669.86
1338.64


407
SP-384
Ac-F$r8ARWEAc3cL$A-NH2
1307.74
654.87
655


408
SP-385
Ac-F$r8AKWEAc3cL$A-NH2
1279.73
640.87
641.01


409
SP-386
Ac-F$r8AYWVAc3cL$A-NH2
1284.73
643.37
643.38


410
SP-387
Ac-F$r8AYWFAc3cL$A-NH2
1332.73
667.37
667.43


411
SP-388
Ac-F$r8AYWWAc3cL$A-NH2
1371.74
686.87
686.97


412
SP-389
Ac-F$r8AYWRAc3cL$A-NH2
1341.76
671.88
671.94


413
SP-390
Ac-F$r8AYWKAc3cL$A-NH2
1313.75
657.88
657.88


414
SP-391
Ac-F$r8AYWEVLSA-NH2
1330.73
666.37
666.47


415
SP-392
Ac-F$r8AYWEFL$A-NH2
1378.73
690.37
690.44


416
SP-393
Ac-F$r8AYWEWL$A-NH2
1417.74
709.87
709.91


417
SP-394
Ac-F$r8AYWERL$A-NH2
1387.77
694.89
1388.66


418
SP-395
Ac-F$r8AYWEKL$A-NH2
1359.76
680.88
1361.21


419
SP-396
Ac-F$r8AYWEAc3cL$V-NH2
1342.73
672.37
1343.59


420
SP-397
Ac-F$r8AYWEAc3cL$F-NH2
1390.73
696.37
1392.58


42T
SP-398
Ac-F$r8AYWEAc3cL$W-NH2
1429.74
715.87
1431.29


422
SP-399
Ac-F$r8AYWEAc3cL$R-NH2
1399.77
700.89
700.95


423
SP-400
Ac-F$r8AYWEAc3cL$K-NH2
1371.76
686.88
686.97


424
SP-401
Ac-F$r8AYWEAc3cL$AV-NH2
1413.77
707.89
707.91


425
SP-402
Ac-F$r8AYWEAc3cL$AF-NH2
1461.77
731.89
731.96


426
SP-403
Ac-F$r8AYWEAc3cL$AW-NH2
1500.78
751.39
751.5


427
SP-404
Ac-F$r8AYWEAc3cL$AR-NH2
1470.80
736.40
736.47


428
SP-405
Ac-F$r8AYWEAc3cL$AK-NH2
1442.80
722.40
722.41


429
SP-406
Ac-F$r8AYWEAc3cL$AH-NH2
1451.76
726.88
726.93


430
SP-407
Ac-LTF2NO2$r8HYWAQL$S-NH2
1642.85
822.43
822.54


431
SP-408
Ac-LTA$r8HYAAQL$S-NH2
1406.79
704.40
704.5


432
SP-409
Ac-LTF$r8HYAAQL$S-NH2
1482.82
742.41
742.47


433
SP-410
Ac-QSQQTF$r8NLWALL$AN-NH2
1966.07
984.04
984.38


434
SP-411
Ac-QAibQQTF$r8NLWALL$AN-NH2
1964.09
983.05
983.42


435
SP-412
Ac-QAibQQTF$r8ALWALL$AN-NH2
1921.08
961.54
961.59


436
SP-413
Ac-AAAATF$r8AAWAAL$AA-NH2
1608.90
805.45
805.52


437
SP-414
Ac-F$r8AAWRAL$Q-NH2
1294.76
648.38
648.48


438
SP-415
Ac-TF$r8AAWAAL$Q-NH2
1310.74
656.37
1311.62


439
SP-416
Ac-TF$r8AAWRAL$A-NH2
1338.78
670.39
670.46


440
SP-417
Ac-VF$r8AAWRAL$Q-NH2
1393.82
697.91
697.99


441
SP-418
Ac-AF$r8AAWAAL$A-NH2
1223.71
612.86
1224.67


442
SP-420
Ac-TF$r8AAWKAL$Q-NH2
1367.80
684.90
684.97


443
SP-421
Ac-TF$r8AAWOAL$Q-NH2
1353.78
677.89
678.01


444
SP-422
Ac-TF$r8AAWSAL$Q-NH2
1326.73
664.37
664.47


445
SP-423
Ac-LTF$r8AAWRAL$Q-NH2
1508.89
755.45
755.49


446
SP-424
Ac-F$r8AYWAQL$A-NH2
1301.72
651.86
651.96


447
SP-425
Ac-F$r8AWWAAL$A-NH2
1267.71
634.86
634.87


448
SP-426
Ac-F$r8AWWAQL$A-NH2
1324.73
663.37
663.43


449
SP-427
Ac-F$r8AYWEAL$-NH2
1231.66
616.83
1232.93


450
SP-428
Ac-F$r8AYWAAL$-NH2
1173.66
587.83
1175.09


451
SP-429
Ac-F$r8AYWKAL$-NH2
1230.72
616.36
616.44


452
SP-430
Ac-F$r8AYWOAL$-NH2
1216.70
609.35
609.48


453
SP-431
Ac-F$r8AYWQAL$-NH2
1230.68
616.34
616.44


454
SP-432
Ac-F$r8AYWAQL$-NH2
1230.68
616.34
616.37


455
SP-433
Ac-F$r8HYWDQL$S-NH2
1427.72
714.86
714.86


456
SP-434
Ac-F$r8HFWEQL$S-NH2
1425.74
713.87
713.98


457
SP-435
Ac-F$r8AYWHQL$S-NH2
1383.73
692.87
692.96


458
SP-436
Ac-F$r8AYWKQL$S-NH2
1374.77
688.39
688.45


459
SP-437
Ac-F$r8AYWOQL$S-NH2
1360.75
681.38
681.49


460
SP-438
Ac-F$r8HYWSQL$S-NH2
1399.73
700.87
700.95


461
SP-439
Ac-F$r8HWWEQL$S-NH2
1464.76
733.38
733.44


462
SP-440
Ac-F$r8HWWAQL$S-NH2
1406.75
704.38
704.43


463
SP-441
Ac-F$r8AWWHQL$S-NH2
1406.75
704.38
704.43


464
SP-442
Ac-F$r8AWWKQL$S-NH2
1397.79
699.90
699.92


465
SP-443
Ac-F$r8AWWOQL$S-NH2
1383.77
692.89
692.96


466
SP-444
Ac-F$r8HWWSQL$S-NH2
1422.75
712.38
712.42


467
SP-445
Ac-LTF$r8NYWANleL$Q-NH2
1600.90
801.45
801.52


468
SP-446
Ac-LTF$r8NLWAQL$Q-NH2
1565.90
783.95
784.06


469
SP-447
Ac-LTF$r8NYWANleL$A-NH2
1543.88
772.94
773.03


470
SP-448
Ac-LTF$r8NLWAQL$A-NH2
1508.88
755.44
755.49


471
SP-449
Ac-LTF$r8AYWANleL$Q-NH2
1557.90
779.95
780.06


472
SP-450
Ac-LTF$r8ALWAQL$Q-NH2
1522.89
762.45
762.45


473
SP-451
Ac-LAF$r8NYWANleL$Q-NH2
1570.89
786.45
786.5


474
SP-452
Ac-LAF$r8NLWAQL$Q-NH2
1535.89
768.95
769.03


475
SP-453
Ac-LAF$r8AYWANleL$A-NH2
1470.86
736.43
736.47


476
SP-454
Ac-LAF$r8ALWAQL$A-NH2
1435.86
718.93
719.01


477
SP-455
Ac-LAF$r8AYWAAL$A-NH2
1428.82
715.41
715.41


478
SP-456
Ac-F$r8AYWEAc3cL$AAib-NH2
1399.75
700.88
700.95


479
SP-457
Ac-F$r8AYWAQL$AA-NH2
1372.75
687.38
687.78


480
SP-458
Ac-F$r8AYWAAc3cL$AA-NH2
1327.73
664.87
664.84


481
SP-459
Ac-F$r8AYWSAc3cL$AA-NH2
1343.73
672.87
672.9


482
SP-460
Ac-F$r8AYWEAc3cL$AS-NH2
1401.73
701.87
701.84


483
SP-461
Ac-F$r8AYWEAc3cL$AT-NH2
1415.75
708.88
708.87


484
SP-462
Ac-F$r8AYWEAc3cL$AL-NH2
1427.79
714.90
714.94


485
SP-463
Ac-F$r8AYWEAc3cL$AQ-NH2
1442.76
722.38
722.41


486
SP-464
Ac-F$r8AFWEAc3cL$AA-NH2
1369.74
685.87
685.93


487
SP-465
Ac-F$r8AWWEAc3cL$AA-NH2
1408.75
705.38
705.39


488
SP-466
Ac-F$r8AYWEAc3cL$SA-NH2
1401.73
701.87
701.99


489
SP-467
Ac-F$r8AYWEAL$AA-NH2
1373.74
687.87
687.93


490
SP-468
Ac-F$r8AYWENleL$AA-NH2
1415.79
708.90
708.94


491
SP-469
Ac-F$r8AYWEAc3cL$AbuA-NH2
1399.75
700.88
700.95


492
SP-470
Ac-F$r8AYWEAc3cL$NleA-NH2
1427.79
714.90
714.86


493
SP-471
Ac-F$r8AYWEAibL$NleA-NH2
1429.80
715.90
715.97


494
SP-472
Ac-F$r8AYWEAL$NleA-NH2
1415.79
708.90
708.94


495
SP-473
Ac-F$r8AYWENleL$NleA-NH2
1457.83
729.92
729.96


496
SP-474
Ac-F$r8AYWEAibL$Abu-NH2
1330.73
666.37
666.39


497
SP-475
Ac-F$r8AYWENleL$Abu-NH2
1358.76
680.38
680.39


498
SP-476
Ac-F$r8AYWEAL$Abu-NH2
1316.72
659.36
659.36


499
SP-477
Ac-LTF$r8AFWAQL$S-NH2
1515.85
758.93
759.12


500
SP-478
Ac-LTF$r8AWWAQL$S-NH2
1554.86
778.43
778.51


501
SP-479
Ac-LTF$r8AYWAQI$S-NH2
1531.84
766.92
766.96


502
SP-480
Ac-LTF$r8AYWAQNle$S-NH2
1531.84
766.92
766.96


503
SP-481
Ac-LTF$r8AYWAQL$SA-NH2
1602.88
802.44
802.48


504
SP-482
Ac-LTF$r8AWWAQL$A-NH2
1538.87
770.44
770.89


505
SP-483
Ac-LTF$r8AYWAQI$A-NH2
1515.85
758.93
759.42


506
SP-484
Ac-LTF$r8AYWAQNle$A-NH2
1515.85
758.93
759.42


507
SP-485
Ac-LTF$r8AYWAQL$AA-NH2
1586.89
794.45
794.94


508
SP-486
Ac-LTF$r8HWWAQL$S-NH2
1620.88
811.44
811.47


509
SP-487
Ac-LTF$r8HRWAQL$S-NH2
1590.90
796.45
796.52


510
SP-488
Ac-LTF$r8HKWAQL$S-NH2
1562.90
782.45
782.53


511
SP-489
Ac-LTF$r8HYWAQL$W-NH2
1696.91
849.46
849.5


512
SP-491
Ac-F$r8AYWAbuAL$A-NH2
1258.71
630.36
630.5


513
SP-492
Ac-F$r8AbuYWEAL$A-NH2
1316.72
659.36
659.51


514
SP-493
Ac-NlePRF%r8NYWRLL%QN-NH2
1954.13
978.07
978.54


515
SP-494
Ac-TSF%r8HYWAQL%S-NH2
1573.83
787.92
787.98


516
SP-495
Ac-LTF%r8AYWAQL%S-NH2
1533.86
767.93
768


517
SP-496
Ac-HTF$r8HYWAQL$S-NH2
1621.84
811.92
811.96


518
SP-497
Ac-LHF$r8HYWAQL$S-NH2
1633.88
817.94
818.02


519
SP-498
Ac-LTF$r8HHWAQL$S-NH2
1571.86
786.93
786.94


520
SP-499
Ac-LTF$r8HYWHQL$S-NH2
1663.89
832.95
832.38


521
SP-500
Ac-LTF$r8HYWAHL$S-NH2
1606.87
804.44
804.48


522
SP-501
Ac-LTF$r8HYWAQL$H-NH2
1647.89
824.95
824.98


523
SP-502
Ac-LTF$r8HYWAQL$S-NHPr
1639.91
820.96
820.98


524
SP-503
Ac-LTF$r8HYWAQL$S-NHsBu
1653.93
827.97
828.02


525
SP-504
Ac-LTF$r8HYWAQL$S-NHiBu
1653.93
827.97
828.02


526
SP-505
Ac-LTF$r8HYWAQL$S-NHBn
1687.91
844.96
844.44


527
SP-506
Ac-LTF$r8HYWAQL$S-NHPe
1700.92
851.46
851.99


528
SP-507
Ac-LTF$r8HYWAQL$S-NHChx
1679.94
840.97
841.04


529
SP-508
Ac-ETF$r8AYWAQL$S-NH2
1547.80
774.90
774.96


530
SP-509
Ac-STF$r8AYWAQL$S-NH2
1505.79
753.90
753.94


531
SP-510
Ac-LEF$r8AYWAQL$S-NH2
1559.84
780.92
781.25


532
SP-511
Ac-LSF$r8AYWAQL$S-NH2
1517.83
759.92
759.93


533
SP-512
Ac-LTF$r8EYWAQL$S-NH2
1589.85
795.93
795.97


534
SP-513
Ac-LTF$r8SYWAQL$S-NH2
1547.84
774.92
774.96


535
SP-514
Ac-LTF$r8AYWEQL$S-NH2
1589.85
795.93
795.9


536
SP-515
Ac-LTF$r8AYWAEL$S-NH2
1532.83
767.42
766.96


537
SP-516
Ac-LTF$r8AYWASL$S-NH2
1490.82
746.41
746.46


538
SP-517
Ac-LTF$r8AYWAQL$E-NH2
1573.85
787.93
787.98


539
SP-518
Ac-LTF2CN$r8HYWAQL$S-NH2
1622.86
812.43
812.47


540
SP-519
Ac-LTF3CI$r8HYWAQL$S-NH2
1631.83
816.92
816.99


541
SP-520
Ac-LTDip$r8HYWAQL$S-NH2
1673.90
837.95
838.01


542
SP-521
Ac-LTF$r8HYWAQTle$S-NH2
1597.87
799.94
800.04


543
SP-522
Ac-F$r8AY6cIWEAL$A-NH2
1336.66
669.33
1338.56


544
SP-523
Ac-F$r8AYdI6brWEAL$A-NH2
1380.61
691.31
692.2


545
SP-524
Ac-F$r8AYdI6fWEAL$A-NH2
1320.69
661.35
1321.61


546
SP-525
Ac-F$r8AYdI4mWEAL$A-NH2
1316.72
659.36
659.36


547
SP-526
Ac-F$r8AYdI5cIWEAL$A-NH2
1336.66
669.33
669.35


548
SP-527
Ac-F$r8AYdI7mWEAL$A-NH2
1316.72
659.36
659.36


549
SP-528
Ac-LTF%r8HYWAQL%A-NH2
1583.89
792.95
793.01


550
SP-529
Ac-LTF$r8HCouWAQL$S-NH2
1679.87
840.94
841.38


551
SP-530
Ac-LTFEHCouWAQLTS-NH2
1617.75
809.88
809.96


552
SP-531
Ac-LTA$r8HCouWAQL$S-NH2
1603.84
802.92
803.36


553
SP-532
Ac-F$r8AYWEAL$AbuA-NH2
1387.75
694.88
694.88


554
SP-533
Ac-F$r8AYWEAI$AA-NH2
1373.74
687.87
687.93


555
SP-534
Ac-F$r8AYWEANle$AA-NH2
1373.74
687.87
687.93


556
SP-535
Ac-F$r8AYWEAmIL$AA-NH2
1429.80
715.90
715.97


557
SP-536
Ac-F$r8AYWQAL$AA-NH2
1372.75
687.38
687.48


558
SP-537
Ac-F$r8AYWAAL$AA-NH2
1315.73
658.87
658.92


559
SP-538
Ac-F$r8AYWAbuAL$AA-NH2
1329.75
665.88
665.95


560
SP-539
Ac-F$r8AYWNleAL$AA-NH2
1357.78
679.89
679.94


561
SP-540
Ac-F$r8AbuYWEAL$AA-NH2
1387.75
694.88
694.96


562
SP-541
Ac-F$r8NleYWEAL$AA-NH2
1415.79
708.90
708.94


563
SP-542
Ac-F$r8FYWEAL$AA-NH2
1449.77
725.89
725.97


564
SP-543
Ac-LTF$r8HYWAQhL$S-NH2
1611.88
806.94
807


565
SP-544
Ac-LTF$r8HYWAQAdm$S-NH2
1675.91
838.96
839.04


566
SP-545
Ac-LTF$r8HYWAQIgl$S-NH2
1659.88
830.94
829.94


567
SP-546
Ac-F$r8AYWAQL$AA-NH2
1372.75
687.38
687.48


568
SP-547
Ac-LTF$r8ALWAQL$Q-NH2
1522.89
762.45
762.52


569
SP-548
Ac-F$r8AYWEAL$AA-NH2
1373.74
687.87
687.93


570
SP-549
Ac-F$r8AYWENleL$AA-NH2
1415.79
708.90
708.94


571
SP-550
Ac-F$r8AYWEAibL$Abu-NH2
1330.73
666.37
666.39


572
SP-551
Ac-F$r8AYWENleL$Abu-NH2
1358.76
680.38
680.38


573
SP-552
Ac-F$r8AYWEAL$Abu-NH2
1316.72
659.36
659.36


574
SP-553
Ac-F$r8AYWEAc3cL$AbuA-NH2
1399.75
700.88
700.95


575
SP-554
Ac-F$r8AYWEAc3cL$NleA-NH2
1427.79
714.90
715.01


576
SP-555
H-LTF$r8AYWAQL$S-NH2
1489.83
745.92
745.95


577
SP-556
mdPEG3-LTF$r8AYWAQL$S-NH2
1679.92
840.96
840.97


578
SP-557
mdPEG7-LTF$r8AYWAQL$S-NH2
1856.02
929.01
929.03


579
SP-558
Ac-F$r8ApmpEt6cIWEAL$A-NH2
1470.71
736.36
788.17


580
SP-559
Ac-LTF3CI$r8AYWAQL$S-NH2
1565.81
783.91
809.18


581
SP-560
Ac-LTF3CI$r8HYWAQL$A-NH2
1615.83
808.92
875.24


582
SP-561
Ac-LTF3CI$r8HYWWQL$S-NH2
1746.87
874.44
841.65


583
SP-562
Ac-LTF3CI$r8AYWWQL$S-NH2
1680.85
841.43
824.63


584
SP-563
Ac-LTF$r8AYWWQL$S-NH2
1646.89
824.45
849.98


585
SP-564
Ac-LTF$r8HYWWQL$A-NH2
1696.91
849.46
816.67


586
SP-565
Ac-LTF$r8AYWWQL$A-NH2
1630.89
816.45
776.15


587
SP-566
Ac-LTF4F$r8AYWAQL$S-NH2
1549.83
775.92
776.15


588
SP-567
Ac-LTF2F$r8AYWAQL$S-NH2
1549.83
775.92
776.15


589
SP-568
Ac-LTF3F$r8AYWAQL$S-NH2
1549.83
775.92
785.12


590
SP-569
Ac-LTF34F2$r8AYWAQL$S-NH2
1567.83
784.92
785.12


591
SP-570
Ac-LTF35F2$r8AYWAQL$S-NH2
1567.83
784.92
1338.74


592
SP-571
Ac-F3CI$r8AYWEAL$A-NH2
1336.66
669.33
705.28


593
SP-572
Ac-F3CI$r8AYWEAL$AA-NH2
1407.70
704.85
680.11


594
SP-573
Ac-F$r8AY6cIWEAL$AA-NH2
1407.70
704.85
736.83


595
SP-574
Ac-F$r8AY6cIWEAL$-NH2
1265.63
633.82
784.1


596
SP-575
Ac-LTF$r8HYWAQLSt/S-NH2
16.03
9.02
826.98


597
SP-576
Ac-LTF$r8HYWAQL$S-NHsBu
1653.93
827.97
828.02


598
SP-577
Ac-STF$r8AYWAQL$S-NH2
1505.79
753.90
753.94


599
SP-578
Ac-LTF$r8AYWAEL$S-NH2
1532.83
767.42
767.41


600
SP-579
Ac-LTF$r8AYWAQL$E-NH2
1573.85
787.93
787.98


601
SP-580
mdPEG3-LTF$r8AYWAQL$S-NH2
1679.92
840.96
840.97


602
SP-581
Ac-LTF$r8AYWAQhL$S-NH2
1545.86
773.93
774.31


603
SP-583
Ac-LTF$r8AYWAQCha$S-NH2
1571.88
786.94
787.3


604
SP-584
Ac-LTF$r8AYWAQChg$S-NH2
1557.86
779.93
780.4


605
SP-585
Ac-LTF$r8AYWAQCba$S-NH2
1543.84
772.92
780.13


606
SP-586
Ac-LTF$r8AYWAQF$S-NH2
1565.83
783.92
784.2


607
SP-587
Ac-LTF4F$r8HYWAQhL$S-NH2
1629.87
815.94
815.36


608
SP-588
Ac-LTF4F$r8HYWAQCha$S-NH2
1655.89
828.95
828.39


609
SP-589
Ac-LTF4F$r8HYWAQChg$S-NH2
1641.87
821.94
821.35


610
SP-590
Ac-LTF4F$r8HYWAQCba$S-NH2
1627.86
814.93
814.32


611
SP-591
Ac-LTF4F$r8AYWAQhL$S-NH2
1563.85
782.93
782.36


612
SP-592
Ac-LTF4F$r8AYWAQCha$S-NH2
1589.87
795.94
795.38


613
SP-593
Ac-LTF4F$r8AYWAQChg$S-NH2
1575.85
788.93
788.35


614
SP-594
Ac-LTF4F$r8AYWAQCba$S-NH2
1561.83
781.92
781.39


615
SP-595
Ac-LTF3CI$r8AYWAQhL$S-NH2
1579.82
790.91
790.35


616
SP-596
Ac-LTF3CI$r8AYWAQCha$S-NH2
1605.84
803.92
803.67


617
SP-597
Ac-LTF3CI$r8AYWAQChg$S-NH2
1591.82
796.91
796.34


618
SP-598
Ac-LTF3CI$r8AYWAQCba$S-NH2
1577.81
789.91
789.39


619
SP-599
Ac-LTF$r8AYWAQhF$S-NH2
1579.84
790.92
791.14


620
SP-600
Ac-LTF$r8AYWAQF3CF3$S-NH2
1633.82
817.91
818.15


621
SP-601
Ac-LTF$r8AYWAQF3Me$S-NH2
1581.86
791.93
791.32


622
SP-602
Ac-LTF$r8AYWAQ1Nal$S-NH2
1615.84
808.92
809.18


623
SP-603
Ac-LTF$r8AYWAQBip$S-NH2
1641.86
821.93
822.13


624
SP-604
Ac-LTF$r8FYWAQL$A-NH2
1591.88
796.94
797.33


625
SP-605
Ac-LTF$r8HYWAQL$S-NHAm
1667.94
834.97
835.92


626
SP-606
Ac-LTF$r8HYWAQL$S-NHiAm
1667.94
834.97
835.55


627
SP-607
Ac-LTF$r8HYWAQL$S-NHnPr3Ph
1715.94
858.97
859.79


628
SP-608
Ac-LTF$r8HYWAQL$S-NHnBu3,3Me
1681.96
841.98
842.49


629
SP-610
Ac-LTF$r8HYWAQL$S-NHnPr
1639.91
820.96
821.58


630
SP-611
Ac-LTF$r8HYWAQL$S-NHnEt2Ch
1707.98
854.99
855.35


631
SP-612
Ac-LTF$r8HYWAQL$S-NHHex
1681.96
841.98
842.4


632
SP-613
Ac-LTF$r8AYWAQL$S-NHmdPeg2
1633.91
817.96
818.35


633
SP-614
Ac-LTF$r8AYWAQL$A-NHmdPeg2
1617.92
809.96
810.3


634
SP-615
Ac-LTF$r8AYWAQL$A-NHmdPeg4
1705.97
853.99
854.33


635
SP-616
Ac-F$r8AYdI4mWEAL$A-NH2
1316.72
659.36
659.44


636
SP-617
Ac-F$r8AYdI5cIWEAL$A-NH2
1336.66
669.33
669.43


637
SP-618
Ac-LThF$r8AYWAQL$S-NH2
1545.86
773.93
774.11


638
SP-619
Ac-LT2Nal$r8AYWAQL$S-NH2
1581.86
791.93
792.43


639
SP-620
Ac-LTA$r8AYWAQL$S-NH2
1455.81
728.91
729.15


640
SP-621
Ac-LTF$r8AYWVQL$S-NH2
1559.88
780.94
781.24


641
SP-622
Ac-LTF$r8HYWAAL$A-NH2
1524.85
763.43
763.86


642
SP-623
Ac-LTF$r8VYWAQL$A-NH2
1543.88
772.94
773.37


643
SP-624
Ac-LTF$r8IYWAQL$S-NH2
1573.89
787.95
788.17


644
SP-625
Ac-FTF$r8VYWSQL$S-NH2
1609.85
805.93
806.22


645
SP-626
Ac-ITF$r8FYWAQL$S-NH2
1607.88
804.94
805.2


646
SP-627
Ac-2NalTF$r8VYWSQL$S-NH2
1659.87
830.94
831.2


647
SP-628
Ac-ITF$r8LYWSQL$S-NH2
1589.89
795.95
796.13


648
SP-629
Ac-FTF$r8FYWAQL$S-NH2
1641.86
821.93
822.13


649
SP-630
Ac-WTF$r8VYWAQL$S-NH2
1632.87
817.44
817.69


650
SP-631
Ac-WTF$r8WYWAQL$S-NH2
1719.88
860.94
861.36


651
SP-632
Ac-VTF$r8AYWSQL$S-NH2
1533.82
767.91
768.19


652
SP-633
Ac-WTF$r8FYWSQL$S-NH2
1696.87
849.44
849.7


653
SP-634
Ac-FTF$r8IYWAQL$S-NH2
1607.88
804.94
805.2


654
SP-635
Ac-WTF$r8VYWSQL$S-NH2
1648.87
825.44
824.8


655
SP-636
Ac-FTF$r8LYWSQL$S-NH2
1623.87
812.94
812.8


656
SP-637
Ac-YTF$r8FYWSQL$S-NH2
1673.85
837.93
837.8


657
SP-638
Ac-LTF$r8AY6cIWEAL$A-NH2
1550.79
776.40
776.14


658
SP-639
Ac-LTF$r8AY6cIWSQL$S-NH2
1581.80
791.90
791.68


659
SP-640
Ac-F$r8AY6cIWSAL$A-NH2
1294.65
648.33
647.67


660
SP-641
Ac-F$r8AY6cIWQAL$AA-NH2
1406.72
704.36
703.84


661
SP-642
Ac-LHF$r8AYWAQL$S-NH2
1567.86
784.93
785.21


662
SP-643
Ac-LTF$r8AYWAQL$S-NH2
1531.84
766.92
767.17


663
SP-644
Ac-LTF$r8AHWAQL$S-NH2
1505.84
753.92
754.13


664
SP-645
Ac-LTF$r8AYWAHL$S-NH2
1540.84
771.42
771.61


665
SP-646
Ac-LTF$r8AYWAQL$H-NH2
1581.87
791.94
792.15


666
SP-647
H-LTF$r8AYWAQL$A-NH2
1473.84
737.92
737.29


667
SP-648
Ac-HHF$r8AYWAQL$S-NH2
1591.83
796.92
797.35


668
SP-649
Ac-aAibWTF$r8VYWSQL$S-NH2
1804.96
903.48
903.64


669
SP-650
Ac-AibWTF$r8HYWAQL$S-NH2
1755.91
878.96
879.4


670
SP-651
Ac-AibAWTF$r8HYWAQL$S-NH2
1826.95
914.48
914.7


671
SP-652
Ac-fWTF$r8HYWAQL$S-NH2
1817.93
909.97
910.1


672
SP-653
Ac-AibWWTF$r8HYWAQL$S-NH2
1941.99
972.00
972.2


673
SP-654
Ac-WTF$r8LYWSQL$S-NH2
1662.88
832.44
832.8


674
SP-655
Ac-WTF$r8NleYWSQL$S-NH2
1662.88
832.44
832.6


675
SP-656
Ac-LTF$r8AYWSQL$a-NH2
1531.84
766.92
767.2


676
SP-657
Ac-LTF$r8EYWARL$A-NH2
1601.90
801.95
802.1


677
SP-658
Ac-LTF$r8EYWAHL$A-NH2
1582.86
792.43
792.6


678
SP-659
Ac-aTF$r8AYWAQL$S-NH2
1489.80
745.90
746.08


679
SP-660
Ac-AibTF$r8AYWAQL$S-NH2
1503.81
752.91
753.11


680
SP-661
Ac-AmfTF$r8AYWAQL$S-NH2
1579.84
790.92
791.14


681
SP-662
Ac-AmwTF$r8AYWAQL$S-NH2
1618.86
810.43
810.66


682
SP-663
Ac-NmLTF$r8AYWAQL$S-NH2
1545.86
773.93
774.11


683
SP-664
Ac-LNmTF$r8AYWAQL$S-NH2
1545.86
773.93
774.11


684
SP-665
Ac-LSarF$r8AYWAQL$S-NH2
1501.83
751.92
752.18


685
SP-667
Ac-LGF$r8AYWAQL$S-NH2
1487.82
744.91
745.15


686
SP-668
Ac-LTNmF$r8AYWAQL$S-NH2
1545.86
773.93
774.2


687
SP-669
Ac-TF$r8AYWAQL$S-NH2
1418.76
710.38
710.64


688
SP-670
Ac-ETF$r8AYWAQL$A-NH2
1531.81
766.91
767.2


689
SP-671
Ac-LTF$r8EYWAQL$A-NH2
1573.85
787.93
788.1


690
SP-672
Ac-LT2Nal$r8AYWSQL$S-NH2
1597.85
799.93
800.4


691
SP-673
Ac-LTF$r8AYWAAL$S-NH2
1474.82
738.41
738.68


692
SP-674
Ac-LTF$r8AYWAQhCha$S-NH2
1585.89
793.95
794.19


693
SP-675
Ac-LTF$r8AYWAQChg$S-NH2
1557.86
779.93
780.97


694
SP-676
Ac-LTF$r8AYWAQCba$S-NH2
1543.84
772.92
773.19


695
SP-677
Ac-LTF$r8AYWAQF3CF3$S-NH2
1633.82
817.91
818.15


696
SP-678
Ac-LTF$r8AYWAQ1Nal$S-NH2
1615.84
808.92
809.18


697
SP-679
Ac-LTF$r8AYWAQBip$S-NH2
1641.86
821.93
822.32


698
SP-680
Ac-LT2Nal$r8AYWAQL$S-NH2
1581.86
791.93
792.15


699
SP-681
Ac-LTF$r8AYWVQL$S-NH2
1559.88
780.94
781.62


700
SP-682
Ac-LTF$r8AWWAQL$S-NH2
1554.86
778.43
778.65


701
SP-683
Ac-FTF$r8VYWSQL$S-NH2
1609.85
805.93
806.12


702
SP-684
Ac-ITF$r8FYWAQL$S-NH2
1607.88
804.94
805.2


703
SP-685
Ac-ITF$r8LYWSQL$S-NH2
1589.89
795.95
796.22


704
SP-686
Ac-FTF$r8FYWAQL$S-NH2
1641.86
821.93
822.41


705
SP-687
Ac-VTF$r8AYWSQL$S-NH2
1533.82
767.91
768.19


706
SP-688
Ac-LTF$r8AHWAQL$S-NH2
1505.84
753.92
754.31


707
SP-689
Ac-LTF$r8AYWAQL$H-NH2
1581.87
791.94
791.94


708
SP-690
Ac-LTF$r8AYWAHL$S-NH2
1540.84
771.42
771.61


709
SP-691
Ac-aAibWTF$r8VYWSQL$S-NH2
1804.96
903.48
903.9


710
SP-692
Ac-AibWTF$r8HYWAQL$S-NH2
1755.91
878.96
879.5


711
SP-693
Ac-AibAWTF$r8HYWAQL$S-NH2
1826.95
914.48
914.7


712
SP-694
Ac-fWTF$r8HYWAQL$S-NH2
1817.93
909.97
910.2


713
SP-695
Ac-AibWWTF$r8HYWAQL$S-NH2
1941.99
972.00
972.7


714
SP-696
Ac-WTF$r8LYWSQL$S-NH2
1662.88
832.44
832.7


715
SP-697
Ac-WTF$r8NleYWSQL$S-NH2
1662.88
832.44
832.7


716
SP-698
Ac-LTF$r8AYWSQL$a-NH2
1531.84
766.92
767.2


717
SP-699
Ac-LTF$r8EYWARL$A-NH2
1601.90
801.95
802.2


718
SP-700
Ac-LTF$r8EYWAHL$A-NH2
1582.86
792.43
792.6


719
SP-701
Ac-aTF$r8AYWAQL$S-NH2
1489.80
745.90
746.1


720
SP-702
Ac-AibTF$r8AYWAQL$S-NH2
1503.81
752.91
753.2


721
SP-703
Ac-AmfTF$r8AYWAQL$S-NH2
1579.84
790.92
791.2


722
SP-704
Ac-AmwTF$r8AYWAQL$S-NH2
1618.86
810.43
810.7


723
SP-705
Ac-NmLTF$r8AYWAQL$S-NH2
1545.86
773.93
774.1


724
SP-706
Ac-LNmTF$r8AYWAQL$S-NH2
1545.86
773.93
774.4


725
SP-707
Ac-LSarF$r8AYWAQL$S-NH2
1501.83
751.92
752.1


726
SP-708
Ac-TF$r8AYWAQL$S-NH2
1418.76
710.38
710.8


727
SP-709
Ac-ETF$r8AYWAQL$A-NH2
1531.81
766.91
767.4


728
SP-710
Ac-LTF$r8EYWAQL$A-NH2
1573.85
787.93
788.2


729
SP-711
Ac-WTF$r8VYWSQL$S-NH2
1648.87
825.44
825.2


730
SP-713
Ac-YTF$r8FYWSQL$S-NH2
1673.85
837.93
837.3


731
SP-714
Ac-F$r8AY6cIWSAL$A-NH2
1294.65
648.33
647.74


732
SP-715
Ac-ETF$r8EYWVQL$S-NH2
1633.84
817.92
817.36


733
SP-716
Ac-ETF$r8EHWAQL$A-NH2
1563.81
782.91
782.36


734
SP-717
Ac-ITF$r8EYWAQL$S-NH2
1589.85
795.93
795.38


735
SP-718
Ac-ITF$r8EHWVQL$A-NH2
1575.88
788.94
788.42


736
SP-719
Ac-ITF$r8EHWAQL$S-NH2
1563.85
782.93
782.43


737
SP-720
Ac-LTF4F$r8AYWAQCba$S-NH2
1561.83
781.92
781.32


738
SP-721
Ac-LTF3CI$r8AYWAQhL$S-NH2
1579.82
790.91
790.64


739
SP-722
Ac-LTF3CI$r8AYWAQCha$S-NH2
1605.84
803.92
803.37


740
SP-723
Ac-LTF3CI$r8AYWAQChg$S-NH2
1591.82
796.91
796.27


741
SP-724
Ac-LTF3CI$r8AYWAQCba$S-NH2
1577.81
789.91
789.83


742
SP-725
Ac-LTF$r8AY6cIWSQL$S-NH2
1581.80
791.90
791.75


743
SP-726
Ac-LTF4F$r8HYWAQhL$S-NH2
1629.87
815.94
815.36


744
SP-727
Ac-LTF4F$r8HYWAQCba$S-NH2
1627.86
814.93
814.32


745
SP-728
Ac-LTF4F$r8AYWAQhL$S-NH2
1563.85
782.93
782.36


746
SP-729
Ac-LTF4F$r8AYWAQChg$S-NH2
1575.85
788.93
788.35


747
SP-730
Ac-ETF$r8EYWVAL$S-NH2
1576.82
789.41
788.79


748
SP-731
Ac-ETF$r8EHWAAL$A-NH2
1506.79
754.40
754.8


749
SP-732
Ac-ITF$r8EYWAAL$S-NH2
1532.83
767.42
767.75


750
SP-733
Ac-ITF$r8EHWVAL$A-NH2
1518.86
760.43
760.81


751
SP-734
Ac-ITF$r8EHWAAL$S-NH2
1506.82
754.41
754.8


752
SP-735
Pam-LTF$r8EYWAQL$S-NH2
1786.07
894.04
894.48


753
SP-736
Pam-ETF$r8EYWAQL$S-NH2
1802.03
902.02
902.34


754
SP-737
Ac-LTF$r8AYWLQL$S-NH2
1573.89
787.95
787.39


755
SP-738
Ac-LTF$r8EYWLQL$S-NH2
1631.90
816.95
817.33


756
SP-739
Ac-LTF$r8EHWLQL$S-NH2
1605.89
803.95
804.29


757
SP-740
Ac-LTF$r8VYWAQL$S-NH2
1559.88
780.94
781.34


758
SP-741
Ac-LTF$r8AYWSQL$S-NH2
1547.84
774.92
775.33


759
SP-742
Ac-ETF$r8AYWAQL$S-NH2
1547.80
774.90
775.7


760
SP-743
Ac-LTF$r8EYWAQL$S-NH2
1589.85
795.93
796.33


761
SP-744
Ac-LTF$r8HYWAQL$S-NHAm
1667.94
834.97
835.37


762
SP-745
Ac-LTF$r8HYWAQL$S-NHiAm
1667.94
834.97
835.27


763
SP-746
Ac-LTF$r8HYWAQL$S-NHnPr3Ph
1715.94
858.97
859.42


764
SP-747
Ac-LTF$r8HYWAQL$S-NHnBu3,3Me
1681.96
841.98
842.67


765
SP-748
Ac-LTF$r8HYWAQL$S-NHnBu
1653.93
827.97
828.24


766
SP-749
Ac-LTF$r8HYWAQL$S-NHnPr
1639.91
820.96
821.31


767
SP-750
Ac-LTF$r8HYWAQL$S-NHnEt2Ch
1707.98
854.99
855.35


768
SP-751
Ac-LTF$r8HYWAQL$S-NHHex
1681.96
841.98
842.4


769
SP-752
Ac-LTF$r8AYWAQL$S-NHmdPeg2
1633.91
817.96
855.35


770
SP-753
Ac-LTF$r8AYWAQL$A-NHmdPeg2
1617.92
809.96
810.58


771
SP-754
Ac-LTF$r5AYWAAL$s8S-NH2
1474.82
738.41
738.79


772
SP-755
Ac-LTF$r8AYWCouQL$S-NH2
1705.88
853.94
854.61


773
SP-756
Ac-LTF$r8CouYWAQL$S-NH2
1705.88
853.94
854.7


774
SP-757
Ac-CouTF$r8AYWAQL$S-NH2
1663.83
832.92
833.33


775
SP-758
H-LTF$r8AYWAQL$A-NH2
1473.84
737.92
737.29


776
SP-759
Ac-HHF$r8AYWAQL$S-NH2
1591.83
796.92
797.72


777
SP-760
Ac-LT2Nal$r8AYWSQL$S-NH2
1597.85
799.93
800.68


778
SP-761
Ac-LTF$r8HCouWAQL$S-NH2
1679.87
840.94
841.38


779
SP-762
Ac-LTF$r8AYWCou2QL$S-NH2
1789.94
895.97
896.51


780
SP-763
Ac-LTF$r8Cou2YWAQL$S-NH2
1789.94
895.97
896.5


781
SP-764
Ac-Cou2TF$r8AYWAQL$S-NH2
1747.90
874.95
875.42


782
SP-765
Ac-LTF$r8ACou2WAQL$S-NH2
1697.92
849.96
850.82


783
SP-766
Dmaac-LTF$r8AYWAQL$S-NH2
1574.89
788.45
788.82


784
SP-767
Hexac-LTF$r8AYWAQL$S-NH2
1587.91
794.96
795.11


785
SP-768
Napac-LTF$r8AYWAQL$S-NH2
1657.89
829.95
830.36


786
SP-769
Pam-LTF$r8AYWAQL$S-NH2
1728.06
865.03
865.45


787
SP-770
Ac-LT2Nal$r8HYAAQL$S-NH2
1532.84
767.42
767.61


788
SP-771
Ac-LT2Nal$/r8HYWAQL$/S-NH2
1675.91
838.96
839.1


789
SP-772
Ac-LT2Nal$r8HYFAQL$S-NH2
1608.87
805.44
805.9


790
SP-773
Ac-LT2Nal$r8HWAAQL$S-NH2
1555.86
778.93
779.08


791
SP-774
Ac-LT2Nal$r8HYAWQL$S-NH2
1647.88
824.94
825.04


792
SP-775
Ac-LT2Nal$r8HYAAQW$S-NH2
1605.83
803.92
804.05


793
SP-776
Ac-LTW$r8HYWAQL$S-NH2
1636.88
819.44
819.95


794
SP-777
Ac-LT1Nal$r8HYWAQL$S-NH2
1647.88
824.94
825.41









In the sequences shown above and elsewhere, the following abbreviations are used: “Nle” represents norleucine, “Aib” represents 2-aminoisobutyric acid, “Ac” represents acetyl, and “Pr” represents propionyl. Amino acids represented as “$” are alpha-Me S5-pentenyl-alanine olefin amino acids connected by an all-carbon i to i+4 crosslinker comprising one double bond. Amino acids represented as “$r5” are alpha-Me R5-pentenyl-alanine olefin amino acids connected by an all-carbon i to i+4 crosslinker comprising one double bond. Amino acids represented as “$s8” are alpha-Me S8-octenyl-alanine olefin amino acids connected by an all-carbon i to i+7 crosslinker comprising one double bond. Amino acids represented as “$r8” are alpha-Me R8-octenyl-alanine olefin amino acids connected by an all-carbon i to i+7 crosslinker comprising one double bond. “Ahx” represents an aminocyclohexyl linker. The crosslinkers are linear all-carbon crosslinker comprising eight or eleven carbon atoms between the alpha carbons of each amino acid Amino acids represented as “$/” are alpha-Me S5-pentenyl-alanine olefin amino acids that are not connected by any crosslinker. Amino acids represented as “$/r5” are alpha-Me R5-pentenyl-alanine olefin amino acids that are not connected by any crosslinker. Amino acids represented as “$/s8” are alpha-Me S8-octenyl-alanine olefin amino acids that are not connected by any crosslinker. Amino acids represented as “$/r8” are alpha-Me R8-octenyl-alanine olefin amino acids that are not connected by any crosslinker. Amino acids represented as “Amw” are alpha-Me tryptophan amino acids. Amino acids represented as “Aml” are alpha-Me leucine amino acids. Amino acids represented as “2ff” are 2-fluoro-phenylalanine amino acids. Amino acids represented as “3ff” are 3-fluoro-phenylalanine amino acids. Amino acids represented as “St” are amino acids comprising two pentenyl-alanine olefin side chains, each of which is crosslinked to another amino acid as indicated. Amino acids represented as “St//” are amino acids comprising two pentenyl-alanine olefin side chains that are not crosslinked. Amino acids represented as “% St” are amino acids comprising two pentenyl-alanine olefin side chains, each of which is crosslinked to another amino acid as indicated via fully saturated hydrocarbon crosslinks.
For example, the compounds represented as SP-72, SP-56 and SP-138 have the following structures(SEQ ID NOS 109, 93 and 173, respectively, in order of apperance):




For example, additional compounds have the following structures(SEQ ID NOS 83, 177, 303, 163, 225, 273, 366, 217, 214, 387 and 184, respectively, in order of appearance):










Example 3
Competition Binding ELISA (HDM2 & HDMX)
p53-His6 (“His6” disclosed as SEQ ID NO: 796) protein (30 nM/well) is coated overnight at room temperature in the wells of a 96-well Immulon plates. On the day of the experiment, plates are washed with 1× PBS-Tween 20 (0.05%) using an automated ELISA plate washer, blocked with ELISA Micro well Blocking for 30 minutes at room temperature; excess blocking agent is washed off by washing plates with 1× PBS-Tween 20 (0.05%). Peptides are diluted from 10 mM DMSO stocks to 500 μM working stocks in sterile water, further dilutions made in 0.5% DMSO to keep the concentration of DMSO constant across the samples. The peptides are added to wells at 2× desired concentrations in 50 μl volumes, followed by addition of diluted GST-HDM2 or GST-HMDX protein (final concentration: 10 nM). Samples are incubated at room temperature for 2 h, plates are washed with PBS-Tween 20 (0.05%) prior to adding 100 μl of HRP-conjugated anti-GST antibody [Hypromatrix, INC] diluted to 0.5 μg/ml in HRP-stabilizing buffer. Post 30 min incubation with detection antibody, plates are washed and incubated with 100 μl per well of TMB-E Substrate solution up to 30 minutes; reactions are stopped using 1M HCL and absorbance measured at 450 nm on micro plate reader. Data is analyzed using Graph Pad PRISM software.
Example 4
SJSA-1 Cell Viability Assay
SJSA1 cells are seeded at the density of 5000 cells/100 μl/well in 96-well plates a day prior to assay. On the day of study cells are washed once with Opti-MEM Media and 90 μL of the Opti-MEM Media is added to cells. Peptides are diluted from 10 mM DMSO stocks to 500 μM working stocks in sterile water, further dilutions made in 0.5% DMSO to keep the concentration of DMSO constant across the samples. The final concentration range μM will be 50, 25, 12.5, 6.25, 3.1, 1.56, 0.8 and 0 μM in 100 μL final volume per well for peptides. Final highest DMSO concentration is 0.5% and will be used as the negative control. Cayman Chemicals Cell-Based Assay (−)-Nutlin-3 (10 mM) is used as positive control. Nutlin was diluted using the same dilution scheme as peptides 10 μl of 10× desired concentrations is added to the appropriate well to achieve the final desired concentrations. Cells are then incubated with peptides for 20-24 h at 37° C. in humidified 5% CO2 atmosphere. Post-incubation period, cell viability is measured using Promega Cell Titer-Glo reagents according to manufacturer' instructions.
Example 5
SJSA-1 p21 Up-Regulation Assay
SJSA1 cells are seeded at the density of 0.8 million cells/2 ml/well in 6-well plates a day prior to assay. On the day of study cells are washed once with Opti-MEM Media and 1350 μL of the Opti-MEM Media is added to cells. Peptides are diluted from 10 mM DMSO stocks to 500 μM working stocks in sterile water, further dilutions made in 0.5% DMSO to keep the concentration of DMSO constant across the samples. Final highest DMSO concentration is 0.5% and is used as the negative control. Cayman Chemicals Cell-Based Assay (−)-Nutlin-3 (10 mM) is used as positive control. Nutlin is diluted using the same dilution scheme as peptides 150 μl of 10× desired concentrations is added to the appropriate well to achieve the final desired concentrations. Cells are then incubated with peptides for 18-20 h at 37° C. in humidified 5% CO2 atmosphere. Post-incubation period, cells are harvested, washed with 1× PBS (without Ca++/Mg++) and lysed in 1× Cell lysis buffer (Cell Signaling technologies 10× buffer diluted to 1× and supplemented with protease inhibitors and Phosphatase inhibitors) on ice for 30 min. Lysates are centrifuged at 13000 rpm speed in a microfuge at 40 C for 8 min; clear supernatants are collected and stored at −80° C. till further use. Total protein content of the lysates is measured using BCA protein detection kit and BSA standards from Thermofisher. 25 μg of the total protein is used for p21 detection ELISA assay. Each condition is set in triplicate for ELISA plate. The ELISA assay protocol is followed as per the manufacturer's instructions. 25 μg total protein used for each well, and each well is set up in triplicate. Data is analyzed using Graph Pad PRISM software.
Example 6
p53 GRIP Assay
Thermo Scientific* Biolmage p53-Hdm2 Redistribution Assay monitors the protein interaction with Hdm2 and cellular translocation of GFP-tagged p53 in response to drug compounds or other stimuli. Recombinant CHO-hIR cells stably express human p53(1-312) fused to the C-terminus of enhanced green fluorescent protein (EGFP) and PDE4A4-Hdm2(1-124), a fusion protein between PDE4A4 and Hdm2(1-124). They provide a ready-to-use assay system for measuring the effects of experimental conditions on the interaction of p53 and Hdm2. Imaging and analysis is performed with a HCS platform.
CHO-hIR cells are regularly maintained in Ham's F12 media supplemented with 1% Penicillin-Streptomycin, 0.5 mg/ml Geneticin, 1 mg/ml Zeocin and 10% FBS. Cells seeded into 96-well plates at the density of 7000 cells/100 μl per well 18-24 hours prior to running the assay using culture media. The next day, media is refreshed and PD177 is added to cells to the final concentration of 3 μM to activate foci formation. Control wells are kept without PD-177 solution. 24 h post stimulation with PD177, cells are washed once with Opti-MEM Media and 50 μL of the Opti-MEM Media supplemented with PD-177(6 μM) is added to cells. Peptides are diluted from 10 mM DMSO stocks to 500 μM working stocks in sterile water, further dilutions made in 0.5% DMSO to keep the concentration of DMSO constant across the samples. Final highest DMSO concentration is 0.5% and is used as the negative control. Cayman Chemicals Cell-Based Assay (−)-Nutlin-3 (10 mM) is used as positive control. Nutlin was diluted using the same dilution scheme as peptides. 50 μl of 2× desired concentrations is added to the appropriate well to achieve the final desired concentrations. Cells are then incubated with peptides for 6 h at 37° C. in humidified 5% CO2 atmosphere. Post-incubation period, cells are fixed by gently aspirating out the media and adding 150 μl of fixing solution per well for 20 minutes at room temperature. Fixed cells are washed 4 times with 200 μl PBS per well each time. At the end of last wash, 100 μl of 1 μM Hoechst staining solution is added. Sealed plates incubated for at least 30 min in dark, washed with PBS to remove excess stain and PBS is added to each well. Plates can be stored at 4° C. in dark up to 3 days. The translocation of p53/HDM2 is imaged using Molecular translocation module on Cellomics Arrayscan instrument using 10× objective, XF-100 filter sets for Hoechst and GFP. The output parameters was Mean−CircRINGAveIntenRatio (the ratio of average fluorescence intensities of nucleus and cytoplasm, (well average)). The minimally acceptable number of cells per well used for image analysis was set to 500 cells.
Example 7
Direct Binding Assay hDM2 with Fluorescence Polarization (FP)
The assay was performed according to the following general protocol:
1. Dilute hDM2 (In-house, 41 kD) into FP buffer (High salt buffer-200 mM Nacl, 5 mM CHAPS, pH 7.5) to make 10 μM working stock solution.
2. Add 30 μl of 10 μM of protein stock solution into A1 and B1 well of 96-well black HE microplate (Molecular Devices).
3. Fill in 30 μl of FP buffer into column A2 to A12, B2 to B12, C1 to C12, and D1 to D12.
4. 2 or 3 fold series dilution of protein stock from A1, B1 into A2, B2; A2, B2 to A3, B3; . . . to reach the single digit nM concentration at the last dilution point.
5. Dilute 1 mM (in 100% DMSO) of FAM labeled linear peptide with DMSO to 100 μM (dilution 1:10). Then, dilutefrom 100 μM to 10 μM with water (dilution 1:10) and then dilute with FP buffer from 10 μM to 40 nM (dilution 1:250). This is the working solution which will be a 10 nM concentration in well (dilution 1:4). Keep the diluted FAM labeled peptide in the dark until use 6. Add 10 μl of 10 nM of FAM labeled peptide into each well and incubate, and read at different time points. Kd with 5-FAM-BaLTFEHYWAQLTS-NH2 (SEQ ID NO: 795) is  ̃13.38 nM.
Example 8
Competitive Fluorescence Polarization Assay for hDM2
The assay was performed according to the following general protocol:
1. Dilute hDM2 (In-house, 41 kD) into FP buffer (High salt buffer-200 mM Nacl, 5 mM CHAPS, pH 7.5) to make 84 nM (2×) working stock solution.
2. Add 20 μl of 84 nM (2×) of protein stock solution into each well of 96-well black HE microplate (Molecular Devices)
3. Dilute 1 mM (in 100% DMSO) of FAM labeled linear peptide with DMSO to 100 μM (dilution 1:10). Then, dilute from 100 μM to 10 μM with water (dilution 1:10) and then dilute with FP buffer from 10 μM to 40 nM (dilution 1:250). This is the working solution which will be a 10 nM concentration in well (dilution 1:4). Keep the diluted FAM labeled peptide in the dark until use.
4. Make unlabeled peptide dose plate with FP buffer starting with 1 μM (final) of peptide and making 5 fold serial dilutions for 6 points using following dilution scheme.
Dilute 10 mM (in 100% DMSO) with DMSO to 5 mM (dilution 1:2). Then, dilute from 5 mM to 500 μM with H2O (dilution 1:10) and then dilute with FP buffer from 500 μM to 20 μM (dilution 1:25). Making 5 fold serial dilutions from 4 μM (4×) for 6 points.
5. Transfer 10 μl of serial diluted unlabeled peptides to each well which is filled with 20 μl of 84 nM of protein.
6. Add 10 μl of 10 nM (4×) of FAM labeled peptide into each well and incubate for 3 hr to read.
Results of Examples 7 and 8 are provided in HDM2 data in FIGS. 7A-D.
Example 9
Direct Binding Assay hDMX with Fluorescence Polarization (FP)
The assay was performed according to the following general protocol:
1. Dilute hDMX (In-house, 40 kD) into FP buffer (High salt buffer-200 mM Nacl, 5 mM CHAPS, pH 7.5) to make 10 μM working stock solution.
2. Add 30 μl of 10 μM of protein stock solution into A1 and B1 well of 96-well black HE microplate (Molecular Devices).
3. Fill in 30 μl of FP buffer into column A2 to A12, B2 to B12, C1 to C12, and D1 to D12.
4. 2 or 3 fold series dilution of protein stock from A1, B1 into A2, B2; A2, B2 to A3, B3; . . . to reach the single digit nM concentration at the last dilution point.
5. Dilute 1 mM (in 100% DMSO) of FAM labeled linear peptide with DMSO to 100 μM (dilution 1:10). Then, dilute from 100 μM to 10 μM with water (dilution 1:10) and then dilute with FP buffer from 10 μM to 40 nM (dilution 1:250). This is the working solution which will be a 10 nM concentration in well (dilution 1:4). Keep the diluted FAM labeled peptide in the dark until use.
6. Add 10 μl of 10 nM of FAM labeled peptide into each well and incubate, and read at different time points.
Kd with 5-FAM-BaLTFEHYWAQLTS-NH2 (SEQ ID NO: 795)is  ̃51 nM.
Example 10
Competitive Fluorescence Polarization Assay for hDMX
The assay was performed according to the following general protocol:
1. Dilute hDMX (In-house, 40 kD) into FP buffer (High salt buffer-200 mM Nacl, 5 mM CHAPS, pH 7.5.) to make 300 nM (2×) working stock solution.
2. Add 20 μl of 300 nM (2×) of protein stock solution into each well of 96-well black HE microplate (Molecular Devices)
3. Dilute 1 mM (in 100% DMSO) of FAM labeled linear peptide with DMSO to 100 μM (dilution 1:10). Then, dilute from 100 μM to 10 μM with water (dilution 1:10) and then dilute with FP buffer from 10 μM to 40 nM (dilution 1:250). This is the working solution which will be a 10 nM concentration in well (dilution 1:4). Keep the diluted FAM labeled peptide in the dark until use.
4. Make unlabeled peptide dose plate with FP buffer starting with 5 μM (final) of peptide and making 5 fold serial dilutions for 6 points using following dilution scheme.
5. Dilute 10 mM (in 100% DMSO) with DMSO to 5 mM (dilution 1:2). Then, dilute from 5 mM to 500 μM with H2O (dilution 1:10) and then dilute with FP buffer from 500 μM to 20 μM (dilution 1:25). Making 5 fold serial dilutions from 20 μM (4×) for 6 points.
6. Transfer 10 μl of serial diluted unlabeled peptides to each well which is filled with 20 μl of 300 nM of protein.
7. Add 10 μl of 10 nM (4×) of FAM labeled peptide into each well and incubate for 3 hr to read.
Results of Examples 9 and 10 are provided in HDMX data in FIGS. 7A-D.
Example 11
Cell Viability Assay
The assay was performed according to the following general protocol:
Cell Plating: Trypsinize, count and seed cells at the pre-determined densities in 96-well plates a day prior to assay. Following cell densities are used for each cell line in use:

    
    
        SJSA-1: 7500 cells/well
        RKO: 5000 cells/well
        RK0-E6: 5000 cells/well
        HCT-116: 5000 cells/well
        SW-480: 2000 cells/well
        MCF-7: 5000 cells/well
    
    


On the day of study, replace media with fresh media with 11% FBS (assay media) at room temperature. Add 180 μL of the assay media per well. Control wells with no cells, receive 200 μl media.
Peptide dilution: all dilutions are made at room temperature and added to cells at room temperature.

    
    
        Prepare 10 mM stocks of the peptides in DMSO. Serially dilute the stock using 1:3 dilution scheme to get 10, 3.3, 1.1, 0.33, 0.11, 0.03, 0.01 mM solutions using DMSO as diluents. Dilute the serially DMSO-diluted peptides 33.3 times using sterile water. This gives range of 10× working stocks. Also prepare DMSO/sterile water (3% DMSO) mix for control wells.
        Thus the working stocks concentration range μM will be 300, 100, 30, 10, 3, 1, 0.3 and 0 μM. Mix well at each dilution step using multichannel.
        Row H has controls. H1-H3 will receive 20 ul of assay media. H4-H9 will receive 20 ul of 3% DMSO-water vehicle. H10-H12 will have media alone control with no cells.
        Positive control: HDM2 small molecule inhibitor, Nutlin-3a (10 mM) is used as positive control. Nutlin was diluted using the same dilution scheme as peptides.
    
    


Addition of working stocks to cells:

    
    
        Add 20 μl of 10× desired concentration to appropriate well to achieve the final concentrations in total 200 μl volume in well. (20 μl of 300 μM peptide+180 μl of cells in media=30 μM final concentration in 200 μl volume in wells). Mix gently a few times using pipette. Thus final concentration range used will be 30, 10, 3, 1, 0.3, 0.1, 0.03 & 0 μM (for potent peptides further dilutions are included).
        Controls include wells that get no peptides but contain the same concentration of DMSO as the wells containing the peptides, and wells containing NO CELLS.
        Incubate for 72 hours at 37° C. in humidified 5% CO2 atmosphere.
        The viability of cells is determined using MTT reagent from Promega. Viability of SJSA-1, RKO, RKO-E6, HCT-116 cells is determined on day 3, MCF-7 cells on day 5 and SW-480 cells on day 6. At the end of designated incubation time, allow the plates to come to room temperature. Remove 80 μl of assay media from each well. Add 15 μl of thawed MTT reagent to each well.
        Allow plate to incubate for 2 h at 37° C. in humidified 5% CO2 atmosphere and add 100 μl solubilization reagent as per manufacturer's protocol. Incubate with agitation for 1 h at room temperature and read on Synergy Biotek multiplate reader for absorbance at 570 nM.
        Analyze the cell viability against the DMSO controls using GraphPad PRISM analysis tools.
    
    


Reagents:

    
    
        Invitrogen cell culture Media
        
            i. Falcon 96-well clear cell culture treated plates (Nunc 353072)
        
        
        DMSO (Sigma D 2650)
        RPMI 1640 (Invitrogen 72400)
        MTT (Promega G4000)
    
    


Instruments: Multiplate Reader for Absorbance readout (Synergy 2)
Results of Example 11 are provided in SJSA-1 EC50 data in FIGS. 7A-D.
Example 12
P21 ELISA Assay
The assay was performed according to the following general protocol:
Cell Platimg:

    
    
        Trypsinize, count and seed SJSA1 cells at the density of 7500 cells/100 μl/well in 96-well plates a day prior to assay.
        On the day of study, replace media with fresh RPMI-11% FBS (assay media). Add 90 μL of the assay media per well. Control wells with no cells, receive 100 μl media.
    
    


Peptide Dilution:

    
    
        Prepare 10 mM stocks of the peptides in DMSO. Serially dilute the stock using 1:3 dilution scheme to get 10, 3.3, 1.1, 0.33, 0.11, 0.03, 0.01 mM solutions using DMSO as diluents. Dilute the serially DMSO-diluted peptides 33.3 times using sterile water This gives range of 10× working stocks. Also prepare DMSO/sterile water (3% DMSO) mix for control wells.
        Thus the working stocks concentration range μM will be 300, 100, 30, 10, 3, 1, 0.3 and 0 μM. Mix well at each dilution step using multichannel.
        Row H has controls. H1-H3 will receive 10 ul of assay media. H4-H9 will receive 10 ul of 3% DMSO-water vehicle. H10-H12 will have media alone control with no cells.
        Positive control: HDM2 small molecule inhibitor, Nutlin-3a (10 mM) is used as positive control. Nutlin was diluted using the same dilution scheme as peptides.
    
    


Addition of Working Stocks to Cells:

    
    
        Add 10 μl of 10× desired concentration to appropriate well to achieve the final concentrations in total 100 μl volume in well. (10 μl of 300 μM peptide+90 μl of cells in media=30 μM final concentration in 100 μl volume in wells). Thus final concentration range used will be 30, 10, 3, 1, 0.3& 0 μM.
        Controls will include wells that get no peptides but contain the same concentration of DMSO as the wells containing the peptides, and wells containing NO CELLS.
        20 h-post incubation, aspirate the media; wash cells with 1× PBS (without Ca++/Mg++) and lyse in 60 μl of 1× Cell lysis buffer (Cell Signaling technologies 10× buffer diluted to 1× and supplemented with protease inhibitors and Phosphatase inhibitors) on ice for 30 min.
        Centrifuge plates in at 5000 rpm speed in at 4° C. for 8 min; collect clear supernatants and freeze at −80° C. till further use.
    
    


Protein Estimation:

    
    
        Total protein content of the lysates is measured using BCA protein detection kit and BSA standards from Thermofisher. Typically about 6-7 μg protein is expected per well.
        Use 50 μl of the lysate per well to set up p21 ELISA.
    
    


Human Total p21 ELISA: The ELISA assay protocol is followed as per the manufacturer's instructions. 50 μl lysate is used for each well, and each well is set up in triplicate.
Reagents:

    
    
        —Cell-Based Assay (−)-Nutlin-3 (10 mM): Cayman Chemicals, catalog #600034
        —OptiMEM, Invitrogen catalog #51985
        —Cell Signaling Lysis Buffer (10×), Cell signaling technology, Catalog #9803
        —Protease inhibitor Cocktail tablets(mini), Roche Chemicals, catalog #04693124001
        —Phosphatase inhibitor Cocktail tablet, Roche Chemicals, catalog #04906837001
        —Human total p21 ELISA kit, R&D Systems, DYC1047-5
        —STOP Solution (1M HCL), Cell Signaling Technologies, Catalog #7002
    
    


Instruments: Micro centrifuge—Eppendorf 5415D and Multiplate Reader for Absorbance readout (Synergy 2)
Results of Example 12 are provided in p21 data in FIGS. 7A-D.
Example 13
Caspase 3 Detection Assay
The assay was performed according to the following general protocol:
Cell Platimg: Trypsinize, count and seed SJSA1 cells at the density of 7500 cells/100 μl/well in 96-well plates a day prior to assay. On the day of study, replace media with fresh RPMI-11% FBS (assay media). Add 180 μL of the assay media per well. Control wells with no cells, receive 200 μl media.
Peptide Dilution:

    
    
        Prepare 10 mM stocks of the peptides in DMSO. Serially dilute the stock using 1:3 dilution scheme to get 10, 3.3, 1.1, 0.33, 0.11, 0.03, 0.01 mM solutions using DMSO as diluents. Dilute the serially DMSO-diluted peptides 33.3 times using sterile water This gives range of 10× working stocks. Also prepare DMSO/sterile water (3% DMSO) mix for control wells.
        Thus the working stocks concentration range μM will be 300, 100, 30, 10, 3, 1, 0.3 and 0 μM. Mix well at each dilution step using multichannel. Add 20 ul of 10× working stocks to appropriate wells.
        Row H has controls. H1-H3 will receive 20 ul of assay media. H4-H9 will receive 20 ul of 3% DMSO-water vehicle. H10-H12 will have media alone control with no cells.
        Positive control: HDM2 small molecule inhibitor, Nutlin-3a (10 mM) is used as positive control. Nutlin was diluted using the same dilution scheme as peptides.
    
    


Addition of Working Stocks to Cells:

    
    
        Add 10 μl of 10× desired concentration to appropriate well to achieve the final concentrations in total 100 μl volume in well. (10 μl of 300 μM peptide+90 μl of cells in media=30 μM final concentration in 100 μl volume in wells). Thus final concentration range used will be 30, 10, 3, 1, 0.3& 0 μM.
        Controls will include wells that get no peptides but contain the same concentration of DMSO as the wells containing the peptides, and wells containing NO CELLS.
        48 h-post incubation, aspirate 80 μl media from each well; add 100 μl Caspase 3/7 Glo assay reagent (Promega Caspase 3/7 glo assay system, G8092)per well, incubate with gentle shaking for 1 h at room temperature.
        read on Synergy Biotek multiplate reader for luminescence.
        Data is analyzed as Caspase 3 activation over DMSO-treated cells.
    
    


Results of Example 13 are provided in p21 data in FIGS. 7A-D.

## Claims

1. A peptidomimetic macrocycle comprising an amino acid sequence which is at least 60% identical to an amino acid sequence of SEQ ID NO. 689.

2. The peptidomimetic macrocycle of claim 1, wherein the peptidomimetic macrocycle comprises an amino acid sequence which is at least 80% identical to an amino acid sequence of SEQ ID NO. 689.

3. The peptidomimetic macrocycle of claim 1, wherein the peptidomimetic macrocycle comprises an amino acid sequence which is at least 90% identical to an amino acid sequence of SEQ ID NO. 689.

4. The peptidomimetic macrocycle of claim 1, wherein the peptidomimetic macrocycle comprises an amino acid sequence which is at least 95% identical to an amino acid sequence of SEQ ID NO. 689.

5. The peptidomimetic macrocycle of claim 1, wherein the peptidomimetic macrocycle comprises an amino acid sequence which is SEQ ID NO. 689.

6. The peptidomimetic macrocycle of claim 1, wherein the peptidomimetic macrocycle comprises a helix.

7. The peptidomimetic macrocycle of claim 1, wherein the peptidomimetic macrocycle comprises an α-helix.

8. The peptidomimetic macrocycle of claim 1, wherein the peptidomimetic macrocycle comprises an α,α-disubstituted amino acid.

9. The peptidomimetic macrocycle of claim 1, wherein the peptidomimetic macrocycle comprises a crosslinker linking the α-positions of at least two amino acids.

10. The peptidomimetic macrocycle of claim 9, wherein at least one of said two amino acids is an α,α-disubstituted amino acid.

11. The peptidomimetic macrocycle of any one of claims 1-10, wherein the peptidomimetic macrocycle has the formula:



wherein:
each A, C, D, and E is independently a natural or non-natural amino acid, and D and E independently optionally include a capping group;
B is a natural or non-natural amino acid, amino acid analog,




 [—NH-L3-CO—], [—NH-L3-SO2—], or [—NH-L3-];
R1 and R2 are independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-;
R3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R5;
L is a macrocycle-forming linker of the formula -L1-L2-;
L1 and L2 are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [—R4—K—R4—]n, each being optionally substituted with R5;
each R4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;
each K is O, S, SO, SO2, CO, CO2, or CONR3;
each R5 is independently halogen, alkyl, —OR6, —N(R6)2, —SR6, —SOR6, —SO2R6, —CO2R6, a fluorescent moiety, a radioisotope or a therapeutic agent;
each R6 is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;
R7 is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R5, or part of a cyclic structure with a D residue;
R8 is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R5, or part of a cyclic structure with an E residue;
v and w are independently integers from 1-1000;
u is an integer from 1-10;
x, y and z are independently integers from 0-10; and
n is an integer from 1-5.

12. The peptidomimetic macrocycle of claim 11, wherein L does not include a thioether or a triazole.

13. The peptidomimetic macrocycle of claim 1, wherein the peptidomimetic macrocycle comprises a cross linker linking a backbone amino group of a first amino acid to a second amino acid within the peptidomimetic macrocycle.

14. The peptidomimetic macrocycle of claim 13, wherein the peptidomimetic macrocycle has the formula (IV) or (IVa):



wherein:
each A, C, D, and E is independently a natural or non-natural amino acid, and D and E independently optionally include a capping group;
B is a natural or non-natural amino acid, amino acid analog,




 [—NH-L3-CO—], [—NH-L3-SO2], or [—NH-L3];
R1 and R2 are independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-, or part of a cyclic structure with an E residue;
R3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R5;
L1 and L2 are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [—R4—K—R4—]n, each being optionally substituted with R5;
each R4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;
each K is O, S, SO, SO2, CO, CO2, or CONR3;
each R5 is independently halogen, alkyl, —OR6, —N(R6)2, —SR6, —SOR6, —SO2R6, —CO2R6, a fluorescent moiety, a radioisotope or a therapeutic agent;
each R6 is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;
R7 is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R5;
v and ware independently integers from 1-1000;
u is an integer from 1-10;
x, y and z are independently integers from 0-10; and
n is an integer from 1-5.

15. The peptidomimetic macrocycle of claim 14, wherein L1 and L2 either alone or in combination do not include a thioether or a triazole.

16. The peptidomimetic macrocycle of claim 11, wherein L1 and L2 are independently alkylene, alkenylene or alkynylene.

17. The peptidomimetic macrocycle of claim 11, wherein L1 and L2 are independently C3-C10 alkylene or alkenylene.

18. The peptidomimetic macrocycle of claim 17, wherein L1 and L2 are independently C3-C6 alkylene or alkenylene.

19. The peptidomimetic macrocycle of claim 11, wherein R1 and R2 are H.

20. The peptidomimetic macrocycle of claim 11, wherein R1 and R2 are independently alkyl.

21. The peptidomimetic macrocycle of claim 11, wherein R1 and R2 are methyl.

22. A method of treating cancer in a subject comprising administering to the subject a peptidomimetic macrocycle of any one of claims 1-10.

23. A method of modulating the activity of a p53 protein, a HDM2 protein, a HDMX protein, or a combination thereof, in a subject comprising administering to the subject a peptidomimetic macrocycle of any one of claims 1-10.

24. A method of antagonizing the interaction between a p53 protein and a HDM2 proteins, between a p53 protein and a HDMX protein, or a combination thereof, in a subject comprising administering to the subject a peptidomimetic macrocycle of any one of claims 1-10.