Provided herein is a compound of Formula (I):   or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, or deuterated analog thereof, a pharmaceutical composition comprising a compound of the present disclosure, together with a pharmaceutically acceptable excipient thereof, and a method of treating cancer with the same.

BACKGROUND

Adenosine diphosphate (ADP)-ribosylation is a well conserved post-translational modification found in viruses, bacteria, and eukaryotes. It is catalyzed by members of the ART superfamily of proteins, which transfer ADPr from nicotinamide adenine dinucleotide (NAD+) onto substrates via N—, O—, or S— glycosidic linkages on target molecules. One subset of ART's is the poly(adenosine diphosphate-ribose) polymerases (PARPs), that are members of a family of seventeen known enzymes that regulate fundamental cellular processes including gene expression, protein degradation, and multiple cellular stress responses (M. S. Cohen, P. Chang, Insights into the biogenesis, function, and regulation of ADP-ribosylation. (Nat. Chem Biol 14, 236-243 (2018)). The ability of cancer cells to survive under stress is a fundamental cancer mechanism and an emerging approach for novel therapeutics.

Of particular interest is 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly(ADP ribose) polymerase (TIPARP), a CCCH-type zinc finger domain-containing protein. (Proc. Nat. Acad. Sci. 114 (10) 2681-2686 (2017)). TIPARP is also called PARP7 and ARTD14. PARP7 acts as negative regulator of certain aryl hydrocarbon receptor (AHR) transcriptional targets. AHR, in turn, is activated by many substrates, including cigarette smoke. PARP7 inhibitors have been shown to restore type I interferon (IFN) signaling responses to nucleic acids and causes tumor regression in a CT26 tumor-bearing, immunocompetent BALB/c mouse model. (Gozgit, et al., Cancer Cell 39, 1214-1226 (2021)).

There are currently no approved PARP7 inhibiting pharmaceuticals. Therefore, it would be useful to provide a PARP7 inhibiting compound with properties suitable for administration as a pharmaceutical agent to a mammal, particularly a human.

Thus, there is a need for improved PARP7 inhibitors for the treatment of cancer.

BRIEF SUMMARY

Provided herein are compounds and pharmaceutical compositions useful as inhibitors of PARP7. Some compounds of the disclosure may find use in pharmaceutical compositions, together with at least one pharmaceutically acceptable excipient, for treating a subject in need thereof.

Provided herein is a compound of Formula (I):

or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, or deuterated analog thereof, wherein J is:

X1is N, CR4, or CR4R4;X2is CR5;X3is CR5;X4is N or CR5;X5is CR5;X6is CR5;A is O or NH;R1is selected from H, C1-6alkyl, and C3-12cycloalkyl, wherein the C1-6alkyl is optionally substituted with one or more R6;R2is selected from H, halo, C1-6alkyl, —OH, —O(C1-6alkyl), and cyclopropyl;R3aand R3bare each independently selected from H, halo, —OH, C1-6alkyl, and —O(C1-6alkyl), wherein each C1-6alkyl is independently optionally substituted with one or more R6;Z is selected from 5-12 membered heteroaryl optionally substituted with one or more R7; C6-10aryl optionally substituted with one or more R7; C3-12cycloalkyl optionally substituted with one or more R7; and 4-12 membered heterocyclyl optionally substituted with one or more R7; wherein the 5-12 membered heteroaryl or C6-10aryl is monocyclic or bicyclic; and the C3-12cycloalkyl or 4-12 membered heterocyclyl is monocyclic, bicyclic, fused bicyclic, spirocyclic, or bridged;each R4is independently selected from H, halo, C1-6alkyl, C3-12cycloalkyl, and —O(C1-6alkyl);each R5is independently selected from H, halo, C1-6alkyl, C4-10cycloalkyl, —OH, and —O(C1-6alkyl);each R6is independently selected from H, halo, —OH, —O(C1-6haloalkyl), and —O(C1-6alkyl); andeach R7is independently selected from halo, C1-6alkyl, —NH2, —SO2(C1-6alkyl), —O(C1-6alkyl), and C3-12cycloalkyl, wherein each C1-6alkyl is optionally substituted with one or more halo or —OH or CN.

Provided herein is a compound of Formula (I′):

or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, or deuterated analog thereof, wherein J is:

X1is N, CR4, or CR4R4;X2is CR5;X3is CR5;X4is N or CR5;X5is CR5;X6is CR5;A is O or NH;R1is selected from H, C1-6alkyl, and C3-12cycloalkyl, wherein the C1-6alkyl is optionally substituted with one or more R6;R2is selected from H, halo, C1-6alkyl, —OH, —O(C1-6alkyl), and cyclopropyl;R3aand R3bare each independently selected from H, halo, —OH, C1-6alkyl, and —O(C1-6alkyl), wherein each C1-6alkyl is independently optionally substituted with one or more R6;Z is selected from 5-12 membered heteroaryl optionally substituted with one or more R7; C6-10aryl optionally substituted with one or more R7; C3-12cycloalkyl optionally substituted with one or more R7; and 4-12 membered heterocyclyl optionally substituted with one or more R7; wherein the 5-12 membered heteroaryl or C6-10aryl is monocyclic or bicyclic; and the C3-12cycloalkyl or 4-12 membered heterocyclyl is monocyclic, bicyclic, fused bicyclic, spirocyclic, or bridged;each R4is independently selected from H, halo, C1-6alkyl, C3-12cycloalkyl, and —O(C1-6alkyl);each R is independently selected from H, halo, C1-6alkyl, C4-10cycloalkyl, —OH, and —O(C1-6alkyl);each R6is independently selected from H, halo, —OH, —O(C1-6haloalkyl), and —O(C1-6alkyl); andeach R7is independently selected from halo, C1-6alkyl, —NH2, —SO2(C1-6alkyl), —O(C1-6alkyl), and C3-12cycloalkyl, wherein each C1-6alkyl is optionally substituted with one or more halo or —OH.

There is also provided a pharmaceutical composition comprising a compound, or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, or deuterated analog thereof, of the present disclosure, together with a pharmaceutically acceptable excipient.

Further, there is provided a method of treating cancer, in a subject in need thereof, comprising administering to said patient a compound of the present disclosure, or a pharmaceutical composition comprising a compound of the present disclosure.

DETAILED DESCRIPTION

A dash (“—”) that is not between two letters or symbols is used to indicate a point of attachment for a substituent. For example, —C(O)NH2is attached through the carbon atom. A dash at the front or end of a chemical group is a matter of convenience; chemical groups may be depicted with or without one or more dashes without losing their ordinary meaning. A wavy line drawn through a line in a structure indicates a point of attachment of a group. Unless chemically or structurally required, no directionality is indicated or implied by the order in which a chemical group is written or named.

A wavy line

indicates a point of attachment.

The prefix “Cu-v” indicates that the following group has from u to v carbon atoms. For example, “C1-6alkyl” indicates that the alkyl group has from 1 to 6 carbon atoms.

Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. In certain embodiments, the term “about” includes the indicated amount ±10%. In other embodiments, the term “about” includes the indicated amount ±5%. In certain other embodiments, the term “about” includes the indicated amount 1%. Also, to the term “about X” includes description of “X”. Also, the singular forms “a” and “the” include plural references unless the context clearly dictates otherwise. Thus, e.g., reference to “the compound” includes a plurality of such compounds and reference to “the assay” includes reference to one or more assays and equivalents thereof known to those skilled in the art.

“Alkenyl” refers to an alkyl group containing at least one carbon-carbon double bond and having from 2 to 20 carbon atoms (i.e., C2-20alkenyl), 2 to 8 carbon atoms (i.e., C2-8alkenyl), 2 to 6 carbon atoms (i.e., C2-6alkenyl), or 2 to 4 carbon atoms (i.e., C2-4alkenyl). Examples of alkenyl groups include ethenyl, propenyl, butadienyl (including 1,2-butadienyl and 1,3-butadienyl).

“Alkynyl” refers to an alkyl group containing at least one carbon-carbon triple bond and having from 2 to 20 carbon atoms (i.e., C2-20alkynyl), 2 to 8 carbon atoms (i.e., C2-8alkynyl), 2 to 6 carbon atoms (i.e., C2-6alkynyl), or 2 to 4 carbon atoms (i.e., C2-4alkynyl). The term “alkynyl” also includes those groups having one triple bond and one double bond.

“Haloalkoxy” refers to an alkoxy group as defined above, wherein one or more hydrogen atoms are replaced by a halogen.

“Alkylthio” refers to the group “alkyl-S—”.

“Amido” refers to both a “C-amido” group which refers to the group —C(O)NRyRzand an “N-amido” group which refers to the group —NRyC(O)Rz, wherein Ryand RZare independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroalkyl, or heteroaryl; each of which may be optionally substituted, as defined herein, or Ryand Rzare taken together to form a cycloalkyl or heterocyclyl; each of which may be optionally substituted, as defined herein.

“Amino” refers to the group —NRyRywherein each Ryis independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, cycloalkyl or heteroaryl, each of which is optionally substituted, as defined herein.

“Aryl” refers to an aromatic carbocyclic group having a single ring (e.g. monocyclic) or multiple rings (e.g. bicyclic or tricyclic) including fused systems. As used herein, aryl has 6 to 20 ring carbon atoms (i.e., C6-20aryl), 6 to 12 carbon ring atoms (i.e., C6-10aryl), or 6 to 10 carbon ring atoms (i.e., C6-10aryl). Examples of aryl groups include phenyl, naphthyl, fluorenyl, and anthryl. Aryl, however, does not encompass or overlap in any way with heteroaryl defined below. If one or more aryl groups are fused with a heteroaryl, the resulting ring system is heteroaryl. If one or more aryl groups are fused with a heterocyclyl, the resulting ring system is heterocyclyl.

“Cyano” refers to the group —CN.

“Keto” refers to a group C═O.

“Carbamoyl” refers to both an “O-carbamoyl” group which refers to the group —O—C(O)NRyRzand an “N-carbamoyl” group which refers to the group —NRyC(O)ORz, wherein Ryand Rzare independently selected from the group consisting of hydrogen, alkyl, aryl, haloalkyl, or heteroaryl; each of which may be optionally substituted.

“Ester” (i.e. carboxyl ester) refers to both —OC(O)R and —C(O)OR, wherein R is a substituent; each of which may be optionally substituted, as defined herein.

“Cycloalkyl” refers to a saturated or partially unsaturated cyclic alkyl group having a single ring or multiple rings including fused, bridged, and spiro ring systems. The term “cycloalkyl” includes cycloalkenyl groups (i.e. the cyclic group having at least one double bond). As used herein, cycloalkyl has from 3 to 20 ring carbon atoms (i.e., C3-20cycloalkyl), 3 to 12 ring carbon atoms (i.e., C3-12cycloalkyl), 3 to 10 ring carbon atoms (i.e., C3-10cycloalkyl), 3 to 8 ring carbon atoms (i.e., C3-8cycloalkyl), or 3 to 6 ring carbon atoms (i.e., C3-6cycloalkyl). Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

“Halogen” or “halo” includes fluoro, chloro, bromo, and iodo. “Haloalkyl” refers to an unbranched or branched alkyl group as defined above, wherein one or more hydrogen atoms are replaced by a halogen. For example, where a residue is substituted with more than one halogen, it may be referred to by using a prefix corresponding to the number of halogen moieties attached. Dihaloalkyl and trihaloalkyl refer to alkyl substituted with two (“di”) or three (“tri”) halo groups, which may be, but are not necessarily, the same halogen. Examples of haloalkyl include difluoromethyl (—CHF2) and trifluoromethyl (—CF3).

“Heteroalkyl” refers to an alkyl group in which one or more of the carbon atoms (and any associated hydrogen atoms) are each independently replaced with the same or different heteroatomic group. The term “heteroalkyl” includes unbranched or branched saturated chain having carbon and heteroatoms. By way of example, 1, 2 or 3 carbon atoms may be independently replaced with the same or different heteroatomic group. Heteroatomic groups include, but are not limited to, —NR—, —O—, —S—, —S(O)—, —S(O)2—, and the like, where R is H, alkyl, aryl, cycloalkyl, heteroalkyl, heteroaryl or heterocyclyl, each of which may be optionally substituted. Examples of heteroalkyl groups include —OCH3, —CH2OCH3, —SCH3, —CH2SCH3, —NRCH3, and —CH2NRCH3, where R is hydrogen, alkyl, aryl, arylalkyl, heteroalkyl, or heteroaryl, each of which may be optionally substituted. As used herein, heteroalkyl include 1 to 10 carbon atoms, 1 to 8 carbon atoms, or 1 to 4 carbon atoms; and 1 to 3 heteroatoms, 1 to 2 heteroatoms, or 1 heteroatom.

“Heteroaryl” refers to an aromatic group having a single ring, multiple rings, or multiple fused rings, with one or more ring heteroatoms independently selected from nitrogen, oxygen, and sulfur. As used herein, heteroaryl includes 1 to 20 ring carbon atoms (i.e., C1-20heteroaryl), 3 to 12 ring carbon atoms (i.e., C3-12heteroaryl), or 3 to 8 carbon ring atoms (i.e., C3-8heteroaryl); and 1 to 5 heteroatoms, 1 to 4 heteroatoms, 1 to 3 ring heteroatoms, 1 to 2 ring heteroatoms, or 1 ring heteroatom independently selected from nitrogen, oxygen, and sulfur. Examples of heteroaryl groups include pyrimidinyl, purinyl, pyridyl, pyridazinyl, benzothiazolyl, and pyrazolyl. Examples of the fused-heteroaryl rings include, but are not limited to, benzo[d]thiazolyl, quinolinyl, isoquinolinyl, benzo[b]thiophenyl, indazolyl, benzo[d]imidazolyl, pyrazolo[1,5-a]pyridinyl, and imidazo[1,5-a]pyridinyl, where the heteroaryl can be bound via either ring of the fused system. Any aromatic ring, having a single or multiple fused rings, containing at least one heteroatom, is considered a heteroaryl regardless of the attachment to the remainder of the molecule (i.e., through any one of the fused rings). Heteroaryl does not encompass or overlap with aryl as defined above.

“Heterocyclyl” or “heterocycle” refers to a saturated or unsaturated cyclic alkyl group, with one or more ring heteroatoms independently selected from nitrogen, oxygen and sulfur. The term “heterocyclyl” includes heterocycloalkenyl groups (i.e. the heterocyclyl group having at least one double bond), bicyclic heterocyclyl groups, bridged-heterocyclyl groups, fused-heterocyclyl groups, and spiro-heterocyclyl groups. A heterocyclyl may be a single ring or multiple rings wherein the multiple rings may be fused, bridged, or spiro. Any non-aromatic ring containing at least one heteroatom is considered a heterocyclyl, regardless of the attachment (i.e., can be bound through a carbon atom or a heteroatom). Further, the term heterocyclyl is intended to encompass any non-aromatic ring containing at least one heteroatom, which ring may be fused to an aryl or heteroaryl ring, regardless of the attachment to the remainder of the molecule. As used herein, heterocyclyl has 2 to 20 ring atoms (i.e., 4-20 membered heterocyclyl), 2 to ring atoms (i.e., 4-12 membered heterocyclyl), 4 to 10 ring atoms (i.e., 4-10 membered heterocyclyl), 4 to 8 ring atoms (i.e., 4-8 membered heterocyclyl), or 4 to 6 ring carbon atoms (i.e., 4-6 membered heterocyclyl); having 1 to 5 ring heteroatoms, 1 to 4 ring heteroatoms, 1 to 3 ring heteroatoms, 1 to 2 ring heteroatoms, or 1 ring heteroatom independently selected from nitrogen, sulfur or oxygen. A heterocyclyl may contain one or more C═O and/or thiC═O groups. Examples of heterocyclyl groups include pyrrolidinyl, piperidinyl, piperazinyl, oxetanyl, diC═Olanyl, azetidinyl, azetidinyl, morpholinyl, thiomorpholinyl, 4-7 membered sultam, 4-7 membered cyclic carbamate, 4-7 membered cyclic carbonate, 4-7 membered cyclic sulfide and morpholinyl. As used herein, the term “bridged-heterocyclyl” refers to a four- to ten-membered cyclic moiety connected at two non-adjacent atoms of the heterocyclyl with one or more (e.g. 1 or 2) four- to ten-membered cyclic moiety having at least one heteroatom where each heteroatom is independently selected from nitrogen, oxygen, and sulfur. As used herein, bridged-heterocyclyl includes bicyclic and tricyclic ring systems. Also used herein, the term “spiro-heterocyclyl” refers to a ring system in which a three- to ten-membered heterocyclyl has one or more additional ring, wherein the one or more additional ring is three- to ten-membered cycloalkyl or three- to ten-membered heterocyclyl, where a single atom of the one or more additional ring is also an atom of the three- to ten-membered heterocyclyl. Examples of the spiro-heterocyclyl rings include bicyclic and tricyclic ring systems, such as 2-oxa-7-azaspiro[3.5]nonanyl, 2-oxa-6-azaspiro[3.4]octanyl, and 6-oxa-1-azaspiro[3.3]heptanyl. Examples of the fused-heterocyclyl rings include, but are not limited to, 1,2,3,4-tetrahydroisoquinolinyl, 1-C═O-1,2,3,4-tetrahydroisoquinolinyl, 1-C═O-1,2-dihydroisoquinolinyl, 4,5,6,7-tetrahydrothieno[2,3-c]pyridinyl, indolinyl, and isoindolinyl, where the heterocyclyl can be bound via either ring of the fused system. As used herein, a bicyclic heterocyclyl group is a heterocyclyl group attached at two points to another cyclic group, wherein the other cyclic group may itself be a heterocyclic group, or a carbocyclic group.

As used herein, the term “nitrogen or sulfur containing heterocyclyl” means a heterocyclyl moiety that contains at least one nitrogen atom or at least one sulfur atom, or both a nitrogen atom and a sulfur atom within the ring structure. It is to be understood that other heteroatoms, including oxygen, may be present in addition to the nitrogen, sulfur, or combinations thereof. Examples of nitrogen or sulfur containing heterocyclyls include morpholinyl, thiomorpholinyl, thiazolyl, isothiazolyl, oxazolidinone 1,2 dithiolyl, piperidinyl, piperazinyl, and the like.

“Hydroxy” or “hydroxyl” refers to the group —OH. “Hydroxyalkyl” refers to an unbranched or branched alkyl group as defined above, wherein one or more hydrogen atoms are replaced by a hydroxyl.

“Nitro” refers to the group —NO2.

“Sulfonyl” refers to the group —S(O)2R, where R is a substituent, or a defined group.

“Alkylsulfonyl” refers to the group —S(O)2R, where R is a substituent, or a defined group.

“Alkylsulfinyl” refers to the group —S(O)R, where R is a substituent, or a defined group.

“Thiocyanate” refers to the group —SCN.

“Thiol” refers to the group —SR, where R is a substituent, or a defined group.

“ThiC=O” or “thione” refer to the group C(═S) or C(S).

Certain commonly used alternative chemical names may be used. For example, a divalent group such as a divalent “alkyl” group, a divalent “aryl” group, etc., may also be referred to as an “alkylene” group or an “alkylenyl” group, an “arylene” group or an “arylenyl” group, respectively. Also, unless indicated explicitly otherwise, where combinations of groups are referred to herein as one moiety, e.g. arylalkyl, the last mentioned group contains the atom by which the moiety is attached to the rest of the molecule.

The terms “optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. Also, the term “optionally substituted” refers to any one or more hydrogen atoms on the designated atom or group may or may not be replaced by a moiety other than hydrogen. “Optionally substituted” may be zero to the maximum number of possible substitutions, and each occurrence is independent. When the term “substituted” is used, then that substitution is required to be made at a substitutable hydrogen atom of the indicated substituent. An optional substitution may be the same or different from a (required) substitution.

When a moiety is “optionally substituted,” and reference is made to a general term, such as any “alkyl,” “alkenyl,” “alkynyl,” “haloalkyl,” “cycloalkyl,” “aryl,” or “heteroaryl,” then the general term can refer to any antecedent specifically recited term, such as (C1-3alkyl), (C4-6alkyl), —O(C1-4alkyl), (C3-10cycloalkyl), O—(C3-10cycloalkyl) and the like. For example, “any aryl” includes both “aryl” and “—O(aryl) as well as examples of aryl, such as phenyl or naphthyl and the like. Also, the term “any heterocyclyl” includes both the terms “heterocyclyl” and O-(heterocyclyl),” as well as examples of heterocyclyls, such as oxetanyl, tetrahydropyranyl, morpholino, piperidinyl and the like. In the same manner, the term “any heteroaryl” includes the terms “heteroaryl” and “O-(heteroaryl),” as well as specific heteroaryls, such as pyridine and the like.

Some of the compounds exist as tautomers. Tautomers are in equilibrium with one another. For example, amide containing compounds may exist in equilibrium with imidic acid tautomers. Regardless of which tautomer is shown, and regardless of the nature of the equilibrium among tautomers, the compounds are understood by one of ordinary skill in the art to comprise both amide and imidic acid tautomers. Thus, the amide containing compounds are understood to include their imidic acid tautomers. Likewise, the imidic acid containing compounds are understood to include their amide tautomers.

Any formula or structure given herein, is also intended to represent unlabeled forms as well as isotopically labeled forms of the compounds. Isotopically labeled compounds have structures depicted by the formulas given herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into compounds of the disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as, but not limited to2H (deuterium, D),3H (tritium),11C,13C,14C,15N,18F,31P,32P,35S,36Cl and125I. Various isotopically labeled compounds of the present disclosure, for example those into which radioactive isotopes such as3H and14C, are incorporated. Such isotopically labelled compounds may be useful in metabolic studies, reaction kinetic studies, detection or imaging techniques, such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays or in radioactive treatment of patients.

The disclosure also includes “deuterated analogues” or “deuterated analogs” of compounds of Formula (I) in which from 1 to n hydrogens attached to a carbon atom is/are replaced by deuterium, in which n is the number of hydrogens in the molecule. Such compounds may exhibit increased resistance to metabolism and are thus useful for increasing the half-life of any compound of Formula (I) when administered to a mammal, particularly a human. See, for example, Foster, “Deuterium Isotope Effects in Studies of Drug Metabolism,” Trends Pharmacol. Sci. 5(12):524-527 (1984). Such compounds are synthesized by means well known in the art, for example by employing starting materials in which one or more hydrogens have been replaced by deuterium.

Deuterium labelled or substituted therapeutic compounds of the disclosure may have improved DMPK (drug metabolism and pharmacokinetics) properties, relating to distribution, metabolism and excretion (ADME). Substitution with heavier isotopes such as deuterium may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life, reduced dosage requirements and/or an improvement in therapeutic index. An18F labeled compound may be useful for PET or SPECT studies. Isotopically labeled compounds of this disclosure and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the schemes or in the examples and preparations described below by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent. It is understood that deuterium in this context is regarded as a substituent in the compound of Formula (I).

The concentration of such a heavier isotope, specifically deuterium, may be defined by an isotopic enrichment factor. In the compounds of this disclosure any atom not specifically designated as a particular isotope is meant to represent any stable isotope of that atom. Unless otherwise stated, when a position is designated specifically as “H” or “hydrogen”, the position is understood to have hydrogen at its natural abundance isotopic composition. Accordingly, in the compounds of this disclosure any atom specifically designated as a deuterium (D) is meant to represent deuterium.

In many cases, the compounds of this disclosure are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto.

Provided are also pharmaceutically acceptable salts, hydrates, solvates, tautomeric forms, polymorphs, and prodrugs of the compounds described herein. “Pharmaceutically acceptable” or “physiologically acceptable” refer to compounds, salts, compositions, dosage forms and other materials which are useful in preparing a pharmaceutical composition that is suitable for veterinary or human pharmaceutical use.

The term “pharmaceutically acceptable salt” of a given compound refers to salts that retain the biological effectiveness and properties of the given compound, and which are not biologically or otherwise undesirable. “Pharmaceutically acceptable salts” or “physiologically acceptable salts” include, for example, salts with inorganic acids and salts with an organic acid. In addition, if the compounds described herein are obtained as an acid addition salt, the free base can be obtained by basifying a solution of the acid salt. Conversely, if the product is a free base, an addition salt, particularly a pharmaceutically acceptable addition salt, may be produced by dissolving the free base in a suitable organic solvent and treating the solution with an acid, in accordance with conventional procedures for preparing acid addition salts from base compounds. Those skilled in the art will recognize various synthetic methodologies that may be used to prepare nontoxic pharmaceutically acceptable addition salts. Pharmaceutically acceptable acid addition salts may be prepared from inorganic and organic acids. Salts derived from inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Salts derived from organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid, salicylic acid, and the like. Likewise, pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases. Salts derived from inorganic bases include, by way of example only, sodium, potassium, lithium, ammonium, calcium and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, such as alkyl amines (i.e., NH2(alkyl)), dialkyl amines (i.e., HN(alkyl)2), trialkyl amines (i.e., N(alkyl)3), substituted alkyl amines (i.e., NH2(substituted alkyl)), di(substituted alkyl) amines (i.e., HN(substituted alkyl)2), tri(substituted alkyl) amines (i.e., N(substituted alkyl)3), alkenyl amines (i.e., NH2(alkenyl)), dialkenyl amines (i.e., HN(alkenyl)2), trialkenyl amines (i.e., N(alkenyl)3), substituted alkenyl amines (i.e., NH2(substituted alkenyl)), di(substituted alkenyl) amines (i.e., HN(substituted alkenyl)2), tri(substituted alkenyl) amines (i.e., N(substituted alkenyl)3, mono-, di- or tri-cycloalkyl amines (i.e., NH2(cycloalkyl), HN(cycloalkyl)2, N(cycloalkyl)3), mono-, di- or tri-arylamines (i.e., NH2(aryl), HN(aryl)2, N(aryl)3), or mixed amines, etc. Specific examples of suitable amines include, by way of example only, isopropylamine, trimethyl amine, diethyl amine, tri(iso-propyl) amine, tri(n-propyl) amine, ethanolamine, 2-dimethylaminoethanol, piperazine, piperidine, morpholine, N-ethylpiperidine, and the like.

The term “substituted” means that any one or more (e.g, 1 to 5, 1 to 4, 1 to 3, and the like) hydrogen atoms on the designated atom or group is replaced with one or more (e.g, 1 to 5, 1 to 3, and the like) substituents other than hydrogen, provided that the designated atom's normal valence is not exceeded. The one or more substituents include, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy, acyl, amino, amido, amidino, aryl, azido, carbamoyl, carboxyl, carboxyl ester, cyano, guanidino, halo, haloalkyl, haloalkoxy, heteroalkyl, heteroaryl, heterocyclyl, hydroxy, hydrazino, imino, C═O, nitro, alkylsulfinyl, sulfonic acid, alkylsulfonyl, thiocyanate, thiol, thione, or combinations thereof. Polymers or similar indefinite structures arrived at by defining substituents with further substituents appended ad infinitum (e.g., a substituted aryl having a substituted alkyl which is itself substituted with a substituted aryl group, which is further substituted by a substituted heteroalkyl group, etc.) are not intended for inclusion herein. Unless otherwise noted, the maximum number of serial substitutions in compounds described herein is three. For example, serial substitutions of substituted aryl groups with two other substituted aryl groups are limited to ((substituted aryl) substituted aryl) substituted aryl. Similarly, the above definitions are not intended to include impermissible substitution patterns (e.g., methyl substituted with 5 fluorines or heteroaryl groups having two adjacent oxygen ring atoms). Such impermissible substitution patterns are well known to the skilled artisan. When used to modify a chemical group, the term “substituted” may describe other chemical groups defined herein. Unless specified otherwise, where a group is described as optionally substituted, any substituents of the group are themselves unsubstituted. For example, in some embodiments, the term “substituted alkyl” refers to an alkyl group having one or more substituents including hydroxyl, halo, alkoxy, cycloalkyl, heterocyclyl, aryl, and heteroaryl. In other embodiments, the one or more substituents may be further substituted with halo, alkyl, haloalkyl, hydroxyl, alkoxy, cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is substituted. In other embodiments, the substituents may be further substituted with halo, alkyl, haloalkyl, alkoxy, hydroxyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is unsubstituted. One skilled in the art will recognize that substituents and other moieties of the compounds of the generic formula herein should be selected in order to provide a compound which is sufficiently stable to provide a pharmaceutically useful compound which can be formulated into an acceptably stable pharmaceutical composition. Compounds which have such stability are contemplated as falling within the scope of the present disclosure. It should be understood by one skilled in the art that any combination of the definitions and substituents described above should not result in an inoperable species or compound.

A “solvate” is formed by the interaction of a solvent and a compound. Solvates of salts of the compounds described herein are also provided. Hydrates of the compounds described herein are also provided.

In the present context, the term “therapeutically effective” or “effective amount” indicates that the materials or amount of material is effective to prevent, alleviate, or ameliorate one or more symptoms of a disease or medical condition, and/or to prolong the survival of the subject being treated. The therapeutically effective amount will vary depending on the compound, the disorder or condition and its severity and the age, weight, etc., of the mammal to be treated. For example, an effective amount is an amount sufficient to effectuate a beneficial or desired clinical result. The effective amounts can be provided all at once in a single administration or in fractional amounts that provide the effective amount in several administrations. The precise determination of what would be considered an effective amount may be based on factors individual to each subject, including their size, age, injury, and/or disease or injury being treated, and amount of time since the injury occurred or the disease began. One skilled in the art will be able to determine the effective amount for a given subject based on these considerations which are routine in the art.

Provided herein is a compound of Formula (I):

or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, or deuterated analog thereof, wherein J is:

Provided herein is a compound of Formula (I′):

or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, or deuterated analog thereof,wherein J is:

In some embodiments, R3ais H and R3bis selected from halo, —OH, C1-6alkyl, and —O(C1-6alkyl), wherein each C1-6alkyl is independently optionally substituted with one or more R6. In some embodiments, R3ais H and R3bis selected from halo, —OH, C1-6alkyl, and —O(C1-6alkyl), wherein each C1-6alkyl is independently optionally substituted with one to five R6. In some embodiments, R3ais H and R3bis selected from halo, —OH, and —OMe. In some embodiments, R3ais H and R3bis F. In some embodiments, R3ais H and R3bis —OH or methyl. In some embodiments, R3ais H and R3bis —OH.

In some embodiments, provided is a compound of Formula (I-A):

or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, or deuterated analog thereof, wherein A, J, R1, and R2are as described herein.

In some embodiments, provided is a compound of Formula (I-B):

or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, or deuterated analog thereof, wherein A, J, R1, and R2are as described herein.

In some embodiments, the compound is not 7-fluoro-2-[(4S)-2-hydroxy-4-[[6-oxo-5-(trifluoromethyl)-1H-pyridazin-4-yl]amino]pentyl]-6-[5-(trifluoromethyl)pyrimidin-2-yl]isoquinolin-1-one, 7-fluoro-2-[(2S,4S)-2-hydroxy-4-[[6-oxo-5-(trifluoromethyl)-1H-pyridazin-4-yl]amino]pentyl]-6-[5-(trifluoromethyl)pyrimidin-2-yl]isoquinolin-1-one, or 7-fluoro-2-[(2R, 4S)-2-hydroxy-4-[[6-oxo-5-(trifluoromethyl)-1H-pyridazin-4-yl]amino]pentyl]-6-[5-(trifluoromethyl)pyrimidin-2-yl]isoquinolin-1-one.

In some embodiments, provided is a compound of Formula (I-C):

or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, or deuterated analog thereof, wherein A, J, R1, and R2are as described herein.

In some embodiments, provided is a compound of Formula (I-D):

or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, or deuterated analog thereof, wherein A, J, R1, and R2are as described herein.

In some embodiments, A is NH. In some embodiments, A is O.

In some embodiments, provided is a compound of Formula (II-A):

or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, or deuterated analog thereof, wherein J, R1, and R2are as described herein.

In some embodiments, provided is a compound of Formula (II-B):

or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, or deuterated analog thereof, wherein J, R1, and R2are as described herein.

In some embodiments, the compound is not 7-fluoro-2-[(4S)-2-hydroxy-4-[[6-oxo-5-(trifluoromethyl)-1H-pyridazin-4-yl]amino]pentyl]-6-[5-(trifluoromethyl)pyrimidin-2-yl]isoquinolin-1-one, 7-fluoro-2-[(2S,4S)-2-hydroxy-4-[[6-oxo-5-(trifluoromethyl)-1H-pyridazin-4-yl]amino]pentyl]-6-[5-(trifluoromethyl)pyrimidin-2-yl]isoquinolin-1-one, or 7-fluoro-2-[(2R, 4S)-2-hydroxy-4-[[6-oxo-5-(trifluoromethyl)-1H-pyridazin-4-yl]amino]pentyl]-6-[5-(trifluoromethyl)pyrimidin-2-yl]isoquinolin-1-one.

In some embodiments, provided is a compound of Formula (II-C):

or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, or deuterated analog thereof, wherein J, R1, and R2are as described herein.

In some embodiments, provided is a compound of Formula (II-D):

or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, or deuterated analog thereof, wherein J, R1, and R2are as described herein.

In some embodiments, provided is a compound of Formula (III-A):

or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, or deuterated analog thereof, wherein J, R1, and R2are as described herein.

In some embodiments, provided is a compound of Formula (III-B):

or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, or deuterated analog thereof, wherein J, R1, and R2are as described herein.

In some embodiments, provided is a compound of Formula (III-C):

or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, or deuterated analog thereof, wherein J, R1, and R2are as described herein.

In some embodiments, provided is a compound of Formula (III-D):

or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, or deuterated analog thereof, wherein J, R1, and R2are as described herein.

In some embodiments, provided is a compound of Formula (IV-A):

or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, or deuterated analog thereof, wherein J is as described herein.

The dashed line of Ring J indicates a single or double bond (i.e. X1is ═N—, ═C(R4)—, or —C(R4)2—).

In some embodiments, J is

In some embodiments, J is

In some embodiments, J is

In some embodiments, J is:

In some embodiments, each R5is independently selected from H and halo. In some embodiments, each R5is F. In some embodiments, each R5is H. In some embodiments, R5is either F or H.

In some embodiments, R4is F and each R5is H. In some embodiments, R4is F and each R5is F. In some embodiments, R4is H, and one or two of R is F, and the remaining R5is H. In some embodiments, R4is H and R is either F or H.

In some embodiments, J is selected from the group consisting of:

In some embodiments, J is selected from the group consisting of:

In some embodiments, J is:

In some embodiments, J is:

In some embodiments, J is:

In some embodiments, J is:

In some embodiments, J is:

In some embodiments, J is:

In some embodiments, Z is 4-12 membered heterocyclyl optionally substituted with one to three R7or 5-12 membered heteroaryl optionally substituted with one to three R7, and each R7is independently selected from halo, C1-6alkyl, —NH2, —SO2(C1-3alkyl), —O(C1-3alkyl), and C3-6cycloalkyl, wherein each C1-6alkyl optionally substituted with one to four halo or —OH or CN.

In some embodiments, Z is 4-12 membered heterocyclyl optionally substituted with one or more R7. In some embodiments, Z is 4-12 membered heterocyclyl optionally substituted with one to five R7. In some embodiments, Z is 4-12 membered heterocyclyl optionally substituted with one to three R7. In some embodiments, Z is 4-12 membered heterocyclyl optionally substituted with one to two R7.

In some embodiments, Z is 5-12 membered heteroaryl optionally substituted with one or more R7. In some embodiments, Z is 5-12 membered heteroaryl optionally substituted with one to five R7. In some embodiments, Z is 5-12 membered heteroaryl optionally substituted with one to three R7. In some embodiments, Z is 5-12 membered heteroaryl optionally substituted with one to two R7.

In some embodiments, Z is a 6-membered heteroaryl optionally substituted with one or more R7. In some embodiments, Z is a 6-membered heteroaryl optionally substituted with one to three R7. In some embodiments, Z is an unsubstituted 6-membered heteroaryl. In some embodiments, Z is a 6-membered heteroaryl substituted with one R7. In some embodiments, Z is a 6-membered heteroaryl substituted with two R7. In some embodiments, Z is a 9-membered heteroaryl optionally substituted with one or more R7. In some embodiments, Z is an unsubstituted 9-membered heteroaryl.

In some embodiments, Z is:

wherein w is zero to three, inclusive; and t is zero to four, inclusive, and wherein R7is bound to any substitutable position on Z.

In some embodiments, w is zero. In some embodiments, w is one. In some embodiments, w is three.

In some embodiments, t is zero. In some embodiments, t is one. In some embodiments, t is two. In some embodiments, t is three. In some embodiments, t is four.

In some embodiments, each R7is independently selected from halo, C1-6alkyl, —NH2, —SO2(C1-6alkyl), —O(C1-6alkyl), and C3-12cycloalkyl, wherein each C1-6alkyl optionally substituted with one or more halo or —OH or CN. In some embodiments, each R7is independently selected from halo, C1-6alkyl, —NH2, —SO2(C1-6alkyl), —O(C1-6alkyl), and C3-12cycloalkyl, wherein each C1-6alkyl optionally substituted with one to five halo or —OH or CN. In some embodiments, each R7is independently selected from halo, C1-6alkyl, —NH2, —SO2(C1-6alkyl), —O(C1-6alkyl), and C3-12cycloalkyl, wherein each C1-6alkyl optionally substituted with one to four halo or —OH or CN. In some embodiments, each R7is independently selected from halo, C1-6alkyl, —NH2, —SO2(C1-3alkyl), —O(C1-3alkyl), and C30.6 cycloalkyl, wherein each C1-6alkyl optionally substituted with one to four halo or —OH or CN.

In some embodiments, each R7is independently selected from halo, C1-6alkyl, —NH2, —SO2(C1-6alkyl), —O(C1-6alkyl), and C3-12cycloalkyl, wherein each C1-6alkyl optionally substituted with one or more halo or —OH. In some embodiments, each R7is independently selected from halo, C1-6alkyl, —NH2, —SO2(C1-6alkyl), —O(C1-6alkyl), and C3-12cycloalkyl, wherein each C1-6alkyl optionally substituted with one to five halo or —OH. In some embodiments, each R7is independently selected from halo, C1-6alkyl, —NH2, —SO2(C1-6alkyl), —O(C1-6alkyl), and C3-12cycloalkyl, wherein each C1-6alkyl optionally substituted with one to four halo or —OH.

In some embodiments, an C1-6alkyl of R7is deuterated.

In some embodiments, R7is C1-6alkyl optionally substituted with one, two, or three halo. In some embodiments, R7is C1-6alkyl optionally substituted with one, two, or three F. In some embodiments, R7is C1-6alkyl optionally substituted with one, two, or three —OH. In some embodiments, R7is C1-6alkyl optionally substituted with one, two, three, or four halo or —OH. In some embodiments, R7is C1-6alkyl optionally substituted with one, two, or three halo or —OH. In some embodiments, R7is C1-6alkyl optionally substituted with one, two, or three F or —OH.

In some embodiments, R7is —O(C1-6alkyl), wherein the C1-6alkyl is unsubstituted. In some embodiments, R7is —O(C1-6alkyl), wherein the C1-6alkyl is optionally substituted with one, two, or three halo. In some embodiments, R7is —O(C1-6alkyl), wherein the C1-6alkyl is optionally substituted with one, two, or three —OH. In some embodiments, R7is —O(C1-6alkyl), wherein the C1-6alkyl is optionally substituted with one, two, or three CN.

In some embodiments, Z is

In some embodiments, Z is:

In some embodiments, provided is a compound, or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, or deuterated analog thereof, selected from:

Some embodiments provide for a compound, or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, or deuterated analog thereof, selected from Table 1 or Table 2.

In some embodiments, provided is a compound selected from Table 1, or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, or deuterated analog thereof:

In some embodiments, provided is a compound selected from Table 2, or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, or deuterated analog thereof:

III. Method of Treatment

Disclosed herein are methods of treatment of a disease in which inhibition of PARP is beneficial, the method comprising administering a compound disclosed herein. Also disclosed herein are methods of treatment of a disease in which inhibition of PARP7 is beneficial, the method comprising administering a compound disclosed herein. In some embodiments, the disease is cancer. In some embodiments, the cancer is breast cancer, ovarian cancer, pancreatic cancer, prostate cancer, a hematological cancer, a gastrointestinal cancer such as gastric cancer and colorectal cancer, or lung cancer. In some embodiments, the cancer is breast cancer, ovarian cancer, pancreatic cancer, or prostate cancer. In some embodiment, the cancer is leukemia, colon cancer, glioblastoma, lymphoma, melanoma, triple-negative breast cancer, urothelial cancer, or cervical cancer.

In some embodiments, the cancer comprises a BRCA1 and/or a BRCA2 mutation.

Provided herein is a method of treating cancer, comprising administering to a patient in need thereof a compound disclosed herein, or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, or deuterated analog thereof, or a pharmaceutical composition comprising a compound disclosed herein.

Some embodiments provide for a method of treating cancer, comprising administering to a patient in need thereof an effective amount of compound disclosed herein, or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, or deuterated analog thereof, or a pharmaceutical composition comprising a compound disclosed herein.

Patients being treated by administration of the PARP7 inhibitors of the disclosure often exhibit diseases or conditions that benefit from treatment with other therapeutic agents. These diseases or conditions can be of an oncology nature or can be related to inflammation, metabolic disorders, gastrointestinal disorders and the like. Thus, one aspect of the disclosure is a method of treating cancer, comprising administering a compound of the disclosure in combination with one or more compounds useful for the treatment of such diseases to a subject, particularly a human subject, in need thereof.

In some embodiments, a compound of the present disclosure is co-formulated with the additional one or more active ingredients. In some embodiments, the other active ingredient is administered at approximately the same time, in a separate dosage form. In some embodiments, the other active ingredient is administered sequentially, and may be administered at different times in relation to a compound of the present disclosure.

Illustrative Targets

Illustrative Mechanisms of Action

Immune Checkpoint Modulators

In some embodiments, a compound disclosed herein or an antibody and/or fusion protein provided herein is administered with one or more blockers or inhibitors of inhibitory immune checkpoint proteins or receptors and/or with one or more stimulators, activators or agonists of one or more stimulatory immune checkpoint proteins or receptors. Blockade or inhibition of inhibitory immune checkpoints can positively regulate T-cell or NK cell activation and prevent immune escape of cancer cells within the tumor microenvironment. Activation or stimulation of stimulatory immune check points can augment the effect of immune checkpoint inhibitors in cancer therapeutics. In some embodiments, the immune checkpoint proteins or receptors regulate T cell responses (e.g., reviewed in Xu, et al.,J Exp Clin Cancer Res. (2018) 37:110). In some embodiments, the immune checkpoint proteins or receptors regulate NK cell responses (e.g., reviewed in Davis, et al.,Semin Immunol. (2017) 31:64-75 and Chiossone, et al.,Nat Rev Immunol. (2018) 18(11):671-688). Inhibition of regulatory T-cells (Treg) or Treg depletion can alleviate their suppression of antitumor immune responses and have anticancer effects (e.g., reviewed in Plitas and Rudensky,Annu. Rev. Cancer Biol. (2020) 4:459-77; Tanaka and Sakaguchi,Eur. J. Immunol. (2019) 49:1140-1146).

In some embodiments, a compound disclosed herein or an antibody and/or fusion protein provided herein is administered with one or more blockers or inhibitors of one or more T-cell inhibitory immune checkpoint proteins or receptors. Illustrative T-cell inhibitory immune checkpoint proteins or receptors include CD274 (CD274, PDL1, PD-L1); programmed cell death 1 ligand 2 (PDCD1LG2, PD-L2, CD273); programmed cell death 1 (PDCD1, PD1, PD-1); cytotoxic T-lymphocyte associated protein 4 (CTLA4, CD152); CD276 (B7H3); V-set domain containing T cell activation inhibitor 1 (VTCN1, B7H4); V-set immunoregulatory receptor (VSIR, B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, VSIG3); TNFRSF14 (HVEM, CD270), TNFSFI4 (HVEML); CD272 (B and T lymphocyte associated (BTLA)); PVR related immunoglobulin domain containing (PVRIG, CD 112R); T cell immunoreceptor with Ig and ITIM domains (TIGIT); lymphocyte activating 3 (LAG3, CD223); hepatitis A virus cellular receptor 2 (HAVCR2, TIMD3, TIM3); galectin 9 (LGALS9); killer cell immunoglobulin like receptor, three Ig domains and long cytoplasmic tail 1 (KIR, CD158E1); killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1); killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2); killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 3 (KIR2DL3); and killer cell immunoglobulin like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1). In some embodiments, a compound disclosed herein or the antibody and/or fusion protein provided herein is administered with one or more agonist or activators of one or more T-cell stimulatory immune checkpoint proteins or receptors. Illustrative T-cell stimulatory immune checkpoint proteins or receptors include without limitation CD27, CD70; CD40, CD40LG; inducible T cell costimulator (ICOS, CD278); inducible T cell costimulator ligand (ICOSLG, B7H2); TNF receptor superfamily member 4 (TNFRSF4, OX40); TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF9 (CD137), TNFSF9 (CD137L); TNFRSF18 (GITR), TNFSF18 (GITRL); CD80 (B7-1), CD28; nectin cell adhesion molecule 2 (NECTIN2, CD112); CD226 (DNAM-1); CD244 (2B4, SLAMF4), Poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155). See, e.g., Xu, et al.,JExp Clin Cancer Res. (2018) 37:110.

In some embodiments, a compound disclosed herein or the antibody and/or fusion protein provided herein is administered with one or more blockers or inhibitors of one or more NK-cell inhibitory immune checkpoint proteins or receptors. Illustrative NK-cell inhibitory immune checkpoint proteins or receptors include killer cell immunoglobulin like receptor, three Ig domains and long cytoplasmic tail 1 (KIR, CD158E1); killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1); killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2); killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 3 (KIR2DL3); killer cell immunoglobulin like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1); killer cell lectin like receptor C1 (KLRC1, NKG2A, CD159A); killer cell lectin like receptor D1 (KLRD1, CD94), killer cell lectin like receptor G1 (KLRG1; CLEC15A, MAFA, 2F1); sialic acid binding Ig like lectin 7 (SIGLEC7); and sialic acid binding Ig like lectin 9 (SIGLEC9). In some embodiments, a compound disclosed herein or the antibody and/or fusion protein provided herein is administered with one or more agonist or activators of one or more NK-cell stimulatory immune checkpoint proteins or receptors. Illustrative NK-cell stimulatory immune checkpoint proteins or receptors include CD16, CD226 (DNAM-1); CD244 (2B4, SLAMF4); killer cell lectin like receptor K1 (KLRK1, NKG2D, CD314); SLAM family member 7 (SLAMF7). See, e.g., Davis, et al.,Semin Immunol. (2017) 31:64-75; Fang, et al.,Semin Immunol. (2017) 31:37-54; and Chiossone, et al.,Nat Rev Immunol. (2018) 18(11):671-688.

In some embodiments, the one or more immune checkpoint inhibitors comprises a proteinaceous (e.g., antibody or fragment thereof, or antibody mimetic) inhibitor of PD-L1 (CD274), PD-1 (PDCD1), CTLA4, or TIGIT. In some embodiments, the one or more immune checkpoint inhibitors comprises a small organic molecule inhibitor of PD-L1 (CD274), PD-1 (PDCD1), CTLA4, or TIGIT. In some embodiments, the one or more immune checkpoint inhibitors comprises a proteinaceous (e.g., antibody or fragment thereof, or antibody mimetic) inhibitor of LAG3.

Examples of inhibitors of LAG3 that can be co-administered include leramilimab (LAG525).

Inhibition of regulatory T-cell (Treg) activity or Treg depletion can alleviate their suppression of antitumor immune responses and have anticancer effects. See, e.g., Plitas and Rudensky,Annu. Rev. Cancer Biol. (2020) 4:459-77; Tanaka and Sakaguchi,Eur. J Immunol. (2019) 49:1140-1146. In some embodiments, a compound disclosed herein or an antibody and/or fusion protein provided herein is administered with one or more inhibitors of Treg activity or a Treg depleting agent. Treg inhibition or depletion can augment the effect of immune checkpoint inhibitors in cancer therapeutics.

In some embodiments, a compound disclosed herein or an antibody and/or fusion protein provided herein is administered with one or more Treg inhibitors. In some embodiments, the Treg inhibitor can suppress the migration of Tregs into the tumor microenvironment. In some embodiments, the Treg inhibitor can reduce the immunosuppressive function of Tregs. In some embodiments, the Treg inhibitor can modulate the cellular phenotype and induce production of proinflammatory cytokines. Exemplary Treg inhibitors include without limitation, CCR4 (NCBI Gene ID: 1233) antagonists and degraders of Ikaros zinc-finger proteins (e.g., Ikaros (IKZF1; NCBI Gene ID: 10320), Helios (IKZF2; NCBI Gene ID: 22807), Aiolos (IKZF3; NCBI Gene ID: 22806), and Eos (IKZF4; NCBI Gene ID: 64375).

Examples of Helios degraders that can be co-administered include without limitation I-57 (Novartis) and compounds disclosed in WO2019038717, WO2020012334, WO20200117759, and WO2021101919.

In some embodiments, a compound disclosed herein or an antibody and/or fusion protein provided herein is administered with one or more Treg depleting agents. In some embodiments, the Treg depleting agent is an antibody. In some embodiments, the Treg depleting antibody has antibody-dependent cytotoxic (ADCC) activity. In some embodiments, the Treg depleting antibody is Fc-engineered to possess an enhanced ADCC activity. In some embodiments, the Treg depleting antibody is an antibody-drug conjugate (ADC). Illustrative targets for Treg depleting agents include without limitation CD25 (IL2RA; NCBI Gene ID: 3559), CTLA4 (CD152; NCBI Gene ID: 1493); GITR (TNFRSF18; NCBI Gene ID: 8784); 4-1BB (CD137; NCBI Gene ID: 3604), OX-40 (CD134; NCBI Gene ID: 7293), LAG3 (CD223; NCBI Gene ID: 3902), TIGIT (NCBI Gene ID: 201633), CCR4 (NCBI Gene ID: 1233), and CCR8 (NCBI Gene ID: 1237).

Examples of Treg depleting anti-CCR4 antibodies that can be administered include mogamulizumab.

Inhibiting, depleting, or reprogramming of non-stimulatory myeloid cells in the tumor microenvironment can enhance anti-cancer immune responses (see, e.g., Binnewies et al.,Nat. Med. (2018) 24(5): 541-550; WO2016049641). Illustrative targets for depleting or reprogramming non-stimulatory myeloid cells include triggering receptors expressed on myeloid cells, TREM-1 (CD354, NCBI Gene ID: 54210) and TREM-2 (NCBI Gene ID: 54209). In some embodiments, a compound disclosed herein or an antibody and/or fusion protein provided herein is administered with one or more myeloid cell depleting or reprogramming agents, such as an anti-TREM-1 antibody (e.g. PY159; antibodies disclosed in WO2019032624) or an anti-TREM-2 antibody (e.g., PY314; antibodies disclosed in WO2019118513).

Cluster of Differentiation Agonists or Activators

Cluster of Differentiation 47 (CD47) Inhibitors

SIRPa Targeting Agents

In some embodiments, a compound disclosed herein or the antibody and/or fusion protein provided herein is administered with a FLT3R agonist. In some embodiments, a compound disclosed herein or the antibody and/or fusion protein provided herein is administered with a FLT3 ligand. In some embodiments, a compound disclosed herein or the antibody and/or fusion protein provided herein is administered with a FLT3L-Fc fusion protein, e.g., as described in WO2020263830. In some embodiments, a compound disclosed herein or the antibody and/or fusion protein provided herein is administered with GS-3583 or CDX-301. In some embodiments, a compound disclosed herein or the antibody and/or fusion protein provided herein is administered with GS-3583.

TNF Receptor Superfamily (TNFRSF) Member Agonists or Activators

Example anti-TNFRSF5 (CD40) antibodies that can be co-administered include RG7876, SEA-CD40, APX-005M, and ABBV-428.

In some embodiments, the anti-TNFRSF17 (BCMA) antibody GSK-2857916 is co-administered.

In some embodiments, a compound disclosed herein or antibody and/or fusion protein provided herein is administered with an inhibitor of protein tyrosine phosphatase non-receptor type 11 (PTPN11; BPTP3, CFC, JMML, METCDS, NS1, PTP-1D, PTP2C, SH-PTP2, SH-PTP3, SHP2; NCBI Gene ID: 5781). Examples of SHP2 inhibitors include TNO155 (SHP-099), RMC-4550, JAB-3068, RMC-4630, and those described in WO2018172984 and WO2017211303.

In some embodiments, a compound disclosed herein or the antibody and/or fusion protein provided herein is administered with an inhibitor of mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1, HPK1; NCBI Gene ID: 11184). Examples of Hematopoietic Progenitor Kinase 1 (HPK1) inhibitors include without limitation, those described in WO2020092621, WO2018183956, WO2018183964, WO2018167147, WO2018049152, WO2020092528, WO2016205942, WO2016090300, WO2018049214, WO2018049200, WO2018049191, WO2018102366, WO2018049152, and WO2016090300.

In some embodiments, a compound disclosed herein or the antibody and/or fusion protein provided herein is administered with an ASK inhibitor, e.g., mitogen-activated protein kinase kinase kinase 5 (MAP3K5; ASK1, MAPKKK5, MEKK5; NCBI Gene ID: 4217). Examples of ASK1 inhibitors include those described in WO2011008709 (Gilead Sciences) and WO 2013112741 (Gilead Sciences).

In some embodiments, a compound disclosed herein or the antibody and/or fusion protein provided herein is administered with a targeted E3 ligase ligand conjugate. Such conjugates have a target protein binding moiety and an E3 ligase binding moiety (e.g., an inhibitor of apoptosis protein (IAP) (e.g., XIAP, c-IAP1, c-IAP2, NIL-IAP, Bruce, and surviving) E3 ubiquitin ligase binding moiety, Von Hippel-Lindau E3 ubiquitin ligase (VHL) binding moiety, a cereblon E3 ubiquitin ligase binding moiety, mouse double minute 2 homolog (MDM2) E3 ubiquitin ligase binding moiety), and can be used to promote or increase the degradation of targeted proteins, e.g., via the ubiquitin pathway. In some embodiments, the targeted E3 ligase ligand conjugates comprise a targeting or binding moiety that targets or binds a protein described herein, and an E3 ligase ligand or binding moiety. In some embodiments, the targeted E3 ligase ligand conjugates comprise a targeting or binding moiety that targets or binds a protein selected from Cbl proto-oncogene B (CBLB; Cbl-b, Nbla00127, RNF56; NCBI Gene ID: 868) and hypoxia inducible factor 1 subunit alpha (HIF1A; NCBI Gene ID: 3091). In some embodiments, the targeted E3 ligase ligand conjugates comprise a kinase inhibitor (e.g., a small molecule kinase inhibitor, e.g., of BTK and an E3 ligase ligand or binding moiety. See, e.g., WO2018098280. In some embodiments, the targeted E3 ligase ligand conjugates comprise a binding moiety targeting or binding to Interleukin-1 (IL-1) Receptor-Associated Kinase-4 (IRAK-4); Rapidly Accelerated Fibrosarcoma (RAF, such as c-RAF, A-RAF and/or B-RAF), c-Met/p38, or a BRD protein; and an E3 ligase ligand or binding moiety. See, e.g., WO2019099926, WO2018226542, WO2018119448, WO2018223909, WO2019079701. Additional targeted E3 ligase ligand conjugates that can be co-administered are described, e.g., in WO2018237026, WO2019084026, WO2019084030, WO2019067733, WO2019043217, WO2019043208, and WO2018144649.

RAS and RAS Pathway Inhibitors

Chemotherapeutic Agents

In some embodiments, a compound disclosed herein or the antibody and/or fusion protein provided herein is administered with a chemotherapeutic agent or anti-neoplastic agent.

Also included in the definition of “chemotherapeutic agent” are anti-hormonal agents such as anti-estrogens and selective estrogen receptor modulators (SERMs), inhibitors of the enzyme aromatase, anti-androgens, and pharmaceutically acceptable salts, acids or derivatives of any of the above that act to regulate or inhibit hormone action on tumors.

An example progesterone receptor antagonist includes onapristone. Additional progesterone targeting agents include TRI-CYCLEN LO (norethindrone+ethinyl estradiol), norgestimate+ethinylestradiol (Tri-Cyclen) and levonorgestrel.

In some embodiments, a compound disclosed herein or the antibody and/or fusion protein provided herein is administered with an anti-fibrotic agent. Anti-fibrotic agents that can be co-administered include the compounds such as beta-aminoproprionitrile (BAPN), as well as the compounds disclosed in U.S. Pat. No. 4,965,288 relating to inhibitors of lysyl oxidase and their use in the treatment of diseases and conditions associated with the abnormal deposition of collagen and U.S. Pat. No. 4,997,854 relating to compounds which inhibit LOX for the treatment of various pathological fibrotic states, which are herein incorporated by reference. Further exemplary inhibitors are described in U.S. Pat. No. 4,943,593 relating to compounds such as 2-isobutyl-3-fluoro-, chloro-, or bromo-allylamine, U.S. Pat. Nos. 5,021,456, 5,059,714, 5,120,764, 5,182,297, 5,252,608 relating to 2-(1-naphthyloxymemyl)-3-fluoroallylamine, and US 20040248871, which are herein incorporated by reference.

Exemplary anti-fibrotic agents also include the primary amines reacting with the carbonyl group of the active site of the lysyl oxidases, and more particularly those which produce, after binding with the carbonyl, a product stabilized by resonance, such as the following primary amines: emylenemamine, hydrazine, phenylhydrazine, and their derivatives; semicarbazide and urea derivatives; aminonitriles such as BAPN or 2-nitroethylamine; unsaturated or saturated haloamines such as 2-bromo-ethylamine, 2-chloroethylamine, 2-trifluoroethylamine, 3-bromopropylamine, and p-halobenzylamines; and selenohomocysteine lactone.

Other anti-fibrotic agents are copper chelating agents penetrating or not penetrating the cells. Exemplary compounds include indirect inhibitors which block the aldehyde derivatives originating from the oxidative deamination of the lysyl and hydroxylysyl residues by the lysyl oxidases. Examples include the thiolamines, particularly D-penicillamine, and its analogs such as 2-amino-5-mercapto-5-methylhexanoic acid, D-2-amino-3-methyl-3-((2-acetamidoethyl)dithio)butanoic acid, p-2-amino-3-methyl-3-((2-aminoethyl)dithio)butanoic acid, sodium-4-((p-1-dimethyl-2-amino-2-carboxyethyl)dithio)butane sulphurate, 2-acetamidoethyl-2-acetamidoethanethiol sulphanate, and sodium-4-mercaptobutanesulphinate trihydrate.

Examples of inhibitors of prostaglandin-endoperoxide synthase 1 (PTGS1, COX-1; NCBI Gene ID: 5742) that can be co-administered include mofezolac, GLY-230, and TRK-700.

Examples of inhibitors of arachidonate 5-lipoxygenase (ALOX5, 5-LOX; NCBI Gene ID: 240) that can be co-administered include meclofenamate sodium, zileuton.

Examples of inhibitors of soluble epoxide hydrolase 2 (EPHX2, SEH; NCBI Gene ID: 2053) that can be co-administered include compounds described in WO2015148954. Dual inhibitors of COX-2/SEH that can be co-administered include compounds described in WO2012082647. Dual inhibitors of SEH and fatty acid amide hydrolase (FAAH; NCBI Gene ID: 2166) that can be co-administered include compounds described in WO2017160861.

Tumor Oxygenation Agents

In some embodiments, a compound disclosed herein or the antibody and/or fusion protein provided herein is administered with an agent that promotes or increases tumor oxygenation or reoxygenation, or prevents or reduces tumor hypoxia. Illustrative agents that can be co-administered include, e.g., Hypoxia inducible factor-1 alpha (HIF-1α) inhibitors, such as PT-2977, PT-2385; VEGF inhibitors, such as bevasizumab, IMC-3C5, GNR-011, tanibirumab, LYN-00101, ABT-165; and/or an oxygen carrier protein (e.g., a heme nitric oxide and/or oxygen binding protein (HNOX)), such as OMX-302 and HNOX proteins described in WO2007137767, WO2007139791, WO2014107171, and WO2016149562.

Immunotherapeutic Agents

The exemplified therapeutic antibodies can be further labeled or combined with a radioisotope particle such as indium-111, yttrium-90 (90Y-clivatuzumab), or iodine-131.

Illustrative therapeutic agents (e.g., anticancer or antineoplastic agents) that can be conjugated to the drug-conjugated antibodies, fragments thereof, or antibody mimetics include without limitation monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), a calicheamicin, ansamitocin, maytansine or an analog thereof (e.g., mertansine/emtansine (DM1), ravtansine/soravtansine (DM4)), an anthracyline (e.g., dC=Orubicin, daunorubicin, epirubicin, idarubicin), pyrrolobenzodiazepine (PBD) DNA cross-linking agent SC-DR002 (D6.5), duocarmycin, a microtubule inhibitors (MTI) (e.g., a taxane, avincaalkaloid, an epothilone), a pyrrolobenzodiazepine (PBD) or dimer thereof, a duocarmycin (A, B1, B2, C1, C2, D, SA, CC-1065), and other anticancer or anti-neoplastic agents described herein. In some embodiments, the therapeutic agent conjugated to the drug-conjugated antibody is a topoisomerase I inhibitor (e.g., a camptothecin analog, such as irinotecan or its active metabolite SN38). In some embodiments, the therapeutic agents (e.g., anticancer or antineoplastic agents) that can be conjugated to the drug-conjugated antibodies, fragments thereof, or antibody mimetics include an immune checkpoint inhibitor. In some embodiments, the conjugated immune checkpoint inhibitor is a conjugated small molecule inhibitor of CD274 (PDL1, PD-L1), programmed cell death 1 (PDCD1, PD1, PD-1) or CTLA4. In some embodiments, the conjugated small molecule inhibitor of CD274 or PDCD1 is selected from the group consisting of GS-4224, GS-4416, INCB086550 and MAX10181. In some embodiments, the conjugated small molecule inhibitor of CTLA4 comprises BPI-002.

In some embodiments, the ADCs that can be co-administered include an antibody targeting carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1; CD66a; NCBI Gene ID: 634). In some embodiments, the CEACAM1 antibody is hMN-14 (e.g., as described in WO1996011013). In some embodiments, the CEACAM1-ADC is as described in WO2010093395 (anti-CEACAM-1-CL2A-SN38). In some embodiments, a compound disclosed herein or the antibody and/or fusion protein provided herein is administered with the CEACAM1-ADC IMMU-130.

In some embodiments, the ADCs that can be co-administered include an antibody targeting MHC class II cell surface receptor encoded by the human leukocyte antigen complex (HLA-DR). In some embodiments, the HLA-DR antibody is hL243 (e.g., as described in WO2006094192). In some embodiments, the HLA-DR-ADC is as described in WO2010093395 (anti-HLA-DR-CL2A-SN38). In some embodiments, a compound disclosed herein or the antibody and/or fusion protein provided herein is administered with the HLA-DR-ADC IMMU-140.

Cancer Gene Therapy and Cell Therapy

In some embodiments, a compound disclosed herein or the antibody and/or fusion protein provided herein is administered with a cancer gene therapy and cell therapy. Cancer gene therapies and cell therapies include the insertion of a normal gene into cancer cells to replace a mutated or altered gene; genetic modification to silence a mutated gene; genetic approaches to directly kill the cancer cells; including the infusion of immune cells designed to replace most of the patient's own immune system to enhance the immune response to cancer cells, or activate the patient's own immune system (T cells or Natural Killer cells) to kill cancer cells, or find and kill the cancer cells; genetic approaches to modify cellular activity to further alter endogenous immune responsiveness against cancer.

Cellular Therapies

In some embodiments, a compound disclosed herein or the antibody and/or fusion protein provided herein is administered with one or more cellular therapies. Illustrative cellular therapies include without limitation co-administration of one or more of a population of natural killer (NK) cells, NK-T cells, T cells, cytokine-induced killer (CIK) cells, macrophage (MAC) cells, tumor infiltrating lymphocytes (TILs) and/or dendritic cells (DCs). In some embodiments, the cellular therapy entails a T cell therapy, e.g., co-administering a population of alpha/beta TCR T cells, gamma/delta TCR T cells, regulatory T (Treg) cells and/or TRuC™ T cells. In some embodiments, the cellular therapy entails a NK cell therapy, e.g., co-administering NK-92 cells. As appropriate, a cellular therapy can entail the co-administration of cells that are autologous, syngeneic or allogeneic to the subject.

In some embodiments, the cellular therapy entails co-administering cells comprising chimeric antigen receptors (CARs). In such therapies, a population of immune effector cells engineered to express a CAR, wherein the CAR comprises a tumor antigen-binding domain. In T cell therapies, the T cell receptors (TCRs) are engineered to target tumor derived peptides presented on the surface of tumor cells.

With respect to the structure of a CAR, in some embodiments, the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular domain comprises a primary signaling domain, a costimulatory domain, or both of a primary signaling domain and a costimulatory domain. In some embodiments, the primary signaling domain comprises a functional signaling domain of one or more proteins selected from the group consisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCERIG), FcR beta (Fc Epsilon Rlb), CD79a, CD79b, Fcgamma RIIa, DAP10, and DAP12.

Exemplified Combination Therapies

Lymphoma or Leukemia Combination Therapy

One modified approach is radioimmunotherapy, wherein a monoclonal antibody is combined with a radioisotope particle, such as indium-111, yttrium-90, and iodine-131. Examples of combination therapies include, but are not limited to, iodine-131 tositumomab (BEXXAR®), yttrium-90 ibritumomab tiuxetan (ZEVALIN®), and BEXXAR® with CHOP.

The abovementioned therapies can be supplemented or combined with stem cell transplantation or treatment. Therapeutic procedures include peripheral blood stem cell transplantation, autologous hematopoietic stem cell transplantation, autologous bone marrow transplantation, antibody therapy, biological therapy, enzyme inhibitor therapy, total body irradiation, infusion of stem cells, bone marrow ablation with stem cell support, in vitro-treated peripheral blood stem cell transplantation, umbilical cord blood transplantation, immunoenzyme technique, low-LET cobalt-60 gamma ray therapy, bleomycin, conventional surgery, radiation therapy, and nonmyeloablative allogeneic hematopoietic stem cell transplantation.

Non-Hodgkin's Lymphomas Combination Therapy

Treatment of non-Hodgkin's lymphomas (NHL), especially those of B cell origin, includes using monoclonal antibodies, standard chemotherapy approaches (e.g., CHOP (cyclophosphamide, dC=Orubicin, vincristine, and prednisone), CVP (cyclophosphamide, vincristine, and prednisone), FCM (fludarabine, cyclophosphamide, and mitoxantrone), MCP (Mitoxantrone, Chlorambucil, Prednisolone), all optionally including rituximab (R) and the like), radioimmunotherapy, and combinations thereof, especially integration of an antibody therapy with chemotherapy.

Mantle Cell Lymphoma Combination Therapy

Therapeutic treatments for mantle cell lymphoma (MCL) include combination chemotherapies such as CHOP, hyperCVAD, and FCM. These regimens can also be supplemented with the monoclonal antibody rituximab to form combination therapies R—CHOP, hyperCVAD-R, and R-FCM. Any of the abovementioned therapies may be combined with stem cell transplantation or ICE in order to treat MCL.

An alternative approach to treating MCL is immunotherapy. One immunotherapy uses monoclonal antibodies like rituximab. Another uses cancer vaccines, such as GTOP-99, which are based on the genetic makeup of an individual patient's tumor.

A modified approach to treat MCL is radioimmunotherapy, wherein a monoclonal antibody is combined with a radioisotope particle, such as iodine-131 tositumomab (BEXXAR®) and yttrium-90 ibritumomab tiuxetan (ZEVALIN®). In another example, BEXXAR® is used in sequential treatment with CHOP.

Other approaches to treating MCL include autologous stem cell transplantation coupled with high-dose chemotherapy, administering proteasome inhibitors such as bortezomib (VELCADE® or PS-341), or administering antiangiogenesis agents such as thalidomide, especially in combination with rituximab.

Another treatment approach is administering drugs that lead to the degradation of Bcl-2 protein and increase cancer cell sensitivity to chemotherapy, such as oblimersen, in combination with other chemotherapeutic agents.

A further treatment approach includes administering mTOR inhibitors, which can lead to inhibition of cell growth and even cell death. Non-limiting examples are sirolimus, temsirolimus (TORISEL®, CCI-779), CC-115, CC-223, SF-1126, PQR-309 (bimiralisib), voxtalisib, GSK-2126458, and temsirolimus in combination with RITUXAN@, VELCADE®, or other chemotherapeutic agents.

Examples of therapeutic procedures used to treat WM include peripheral blood stem cell transplantation, autologous hematopoietic stem cell transplantation, autologous bone marrow transplantation, antibody therapy, biological therapy, enzyme inhibitor therapy, total body irradiation, infusion of stem cells, bone marrow ablation with stem cell support, in vitro-treated peripheral blood stem cell transplantation, umbilical cord blood transplantation, immunoenzyme techniques, low-LET cobalt-60 gamma ray therapy, bleomycin, conventional surgery, radiation therapy, and nonmyeloablative allogeneic hematopoietic stem cell transplantation.

Diffuse Large B-cell Lymphoma (DLBCL) Combination Therapy

Chronic Lymphocytic Leukemia Combination Therapy

Therapeutic agents used to treat chronic lymphocytic leukemia (CLL) include chlorambucil, cyclophosphamide, fludarabine, pentostatin, cladribine, dC=Orubicin, vincristine, prednisone, prednisolone, alemtuzumab, many of the agents listed for WM, and combination chemotherapy and chemoimmunotherapy, including the following common combination regimens: CVP, R—CVP, ICE, R-ICE, FCR, and FR.

High Risk Myelodysplastic Syndrome (HR MDS) Combination Therapy

Low Risk Myelodysplastic Syndrome (LR MDS) Combination Therapy

Therapeutic agents used to treat LR MDS include lenalidomide, azacytidine, and combinations thereof. In some embodiments, therapeutic agents used to treat LR MDS include roxadustat, luspatercept, imetelstat, LB-100, or rigosertib.

Acute Myeloid Leukemia (AML) Combination Therapy

Multiple Myeloma (MM) Combination Therapy

Breast Cancer Combination Therapy

Triple Negative Breast Cancer (TNBC) Combination Therapy

Bladder Cancer Combination Therapy

Colorectal Cancer (CRC) Combination Therapy

Esophageal and Esophagogastric Junction Cancer Combination Therapy

Therapeutic agents used to treat esophageal and esophagogastric junction cancer include capecitabine, carboplatin, cisplatin, docetaxel, epirubicin, fluoropyrimidine, fluorouracil, irinotecan, leucovorin, oxaliplatin, paclitaxel, ramucirumab, trastuzumab, and any combinations thereof. In some embodiments, therapeutic agents used to treat gastroesophageal junction cancer (GEJ) include herceptin, cisplatin, 5-FU, ramicurimab, or paclitaxel. In some embodiments, therapeutic agents used to treat GEJ cancer include ALX-148, AO-176, or IBI-188.

Gastric Cancer Combination Therapy

Head and Neck Cancer Combination Therapy

Non-Small Cell Lung Cancer Combination Therapy

Small Cell Lung Cancer Combination Therapy

Ovarian Cancer Combination Therapy

Pancreatic Cancer Combination Therapies

Therapeutic agents used to treat pancreatic cancer include 5-FU, leucovorin, oxaliplatin, irinotecan, gemcitabine, nab-paclitaxel (Abraxane®), FOLFIRINOX, and combinations thereof. In some embodiments, therapeutic agents used to treat pancreatic cancer include 5-FU+leucovorin+oxaliplatin+irinotecan, 5-FU+nanoliposomal irinotecan, leucovorin+nanoliposomal irinotecan, and gemcitabine+nab-paclitaxel.

Prostate Cancer Combination Therapies

Additional Exemplified Combination Therapies

In some embodiments, a compound disclosed herein or the antibody and/or fusion protein provided herein is administered with one or more therapeutic agents selected from a PI3K inhibitor, a Trop-2 binding agent, CD47 antagonist, a SIRPa antagonist, a FLT3R agonist, a PD-1 antagonist, a PD-L1 antagonist, an MCL1 inhibitor, a CCR8 binding agent, an HPK1 antagonist, a DGKa inhibitor, a CISH inhibitor, a PARP-7 inhibitor, a Cbl-b inhibitor, a KRAS inhibitor (e.g., a KRAS G12C or G12D inhibitor), a KRAS degrader, a beta-catenin degrader, a helios degrader, a CD73 inhibitor, an adenosine receptor antagonist, a TIGIT antagonist, a TREM1 binding agent, a TREM2 binding agent, a CD137 agonist, a GITR binding agent, an OX40 binding agent, and a CAR-T cell therapy.

While it is possible for the active ingredients to be administered alone it may be preferable to present them as pharmaceutical formulations (compositions). The formulations, both for veterinary and for human use, of the disclosure comprise at least one active ingredient, as above defined, together with one or more acceptable carriers therefor and optionally other therapeutic ingredients. The camer(s) must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and physiologically innocuous to the recipient thereof.

In certain embodiments, formulations suitable for oral administration are presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient.

The amount of active ingredient that is combined with the inactive ingredients to produce a dosage form will vary depending upon the host treated and the particular mode of administration. For example, in some embodiments, a dosage form for oral administration to humans contains approximately 1 to 1000 mg of active material formulated with an appropriate and convenient amount of carrier material (e.g., inactive ingredient or excipient material). In certain embodiments, the carrier material varies from about 5 to about 95% of the total compositions (weight: weight). In some embodiments, the pharmaceutical compositions described herein contain about 1 to 800 mg, 1 to 600 mg, 1 to 400 mg, 1 to 200 mg, 1 to 100 mg or 1 to 50 mg of the compound of Formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutical compositions described herein contain no more than about 400 mg of the compound of Formula (I). In some embodiments, the pharmaceutical compositions described herein contain about 100 mg of the compound of Formula (I), or a pharmaceutically acceptable salt thereof.

Veterinary compositions comprising at least one active ingredient as above defined together with a veterinary carrier are further provided.

Veterinary carriers are materials useful for the purpose of administering the composition and may be solid, liquid or gaseous materials which are otherwise inert or acceptable in the veterinary art and are compatible with the active ingredient. These veterinary compositions may be administered orally, parenterally or by any other desired route.

Effective dose of active ingredient depends at least on the nature of the condition being treated, toxicity, whether the compound is being used prophylactically (lower doses), the method of delivery, and the pharmaceutical formulation, and will be determined by the clinician using conventional dose escalation studies.

VI. Routes of Administration

One or more compounds of Formula (I) (herein referred to as the active ingredients), or a pharmaceutically acceptable salt thereof, are administered by any route appropriate to the condition to be treated. Suitable routes include oral, rectal, nasal, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural), and the like. It will be appreciated that the preferred route may vary with for example the condition of the recipient. An advantage of the compounds of this disclosure is that they are orally bioavailable and can be dosed orally. Accordingly, in one embodiment, the pharmaceutical compositions described herein are oral dosage forms. In certain embodiments, the pharmaceutical compositions described herein are oral solid dosage forms.

Formulation Example 1

Hard gelatin capsules containing the following ingredients are prepared:

The above ingredients are mixed and filled into hard gelatin capsules.

Formulation Example 2

A tablet Formula is prepared using the ingredients below:

The components are blended and compressed to form tablets.

Formulation Example 3

A dry powder inhaler formulation is prepared containing the following components:

The active ingredient is mixed with the lactose and the mixture is added to a dry powder inhaling appliance.

Formulation Example 4

Tablets, each containing 30 mg of active ingredient, are prepared as follows:

The active ingredient, starch and cellulose are passed through a No. 20 mesh U.S. sieve and mixed thoroughly. The solution of polyvinylpyrrolidone is mixed with the resultant powders, which are then passed through a 16 mesh U.S. sieve. The granules so produced are dried at 50° C. to 60° C. and passed through a 16 mesh U.S. sieve. The sodium carboxymethyl starch, magnesium stearate and talc, previously passed through a No. 30 mesh U.S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets each weighing 120 mg.

Formulation Example 5

Suppositories, each containing 25 mg of active ingredient are made as follows:

The active ingredient is passed through a No. 60 mesh U.S. sieve and suspended in the saturated fatty acid glycerides previously melted using the minimum heat necessary. The mixture is then poured into a suppository mold of nominal 2.0 g capacity and allowed to cool.

Formulation Example 6

Suspensions, each containing 50 mg of active ingredient per 5.0 mL dose are made as follows:

The active ingredient, sucrose and xanthan gum are blended, passed through a No. 10 mesh U.S. sieve and then mixed with a previously made solution of the microcrystalline cellulose and sodium carboxymethyl cellulose in water. The sodium benzoate, flavor and color are diluted with some of the water and added with stirring. Sufficient water is then added to produce the required volume.

Formulation Example 7

A subcutaneous formulation may be prepared as follows:

Formulation Example 8

An injectable preparation is prepared having the following composition:

Formulation Example 9

A topical preparation is prepared having the following composition:

All of the above ingredients, except water, are combined and heated to 60° C. with stirring. A sufficient quantity of water at 60° C. is then added with vigorous stirring to emulsify the ingredients and water then added q.s. 100 g.

Formulation Example 10

Sustained Release Composition

Sustained release formulations of this disclosure may be prepared as follows: compound and pH-dependent binder and any optional excipients are intimately mixed(dry-blended). The dry-blended mixture is then granulated in the presence of an aqueous solution of a strong base which is sprayed into the blended powder. The granulate is dried, screened, mixed with optional lubricants (such as talc or magnesium stearate) and compressed into tablets. Preferred aqueous solutions of strong bases are solutions of alkali metal hydroxides, such as sodium or potassium hydroxide, preferably sodium hydroxide, in water (optionally containing up to 25% of water-miscible solvents such as lower alcohols). The resulting tablets may be coated with an optional film-forming agent, for identification, taste-masking purposes and to improve ease of swallowing. The film forming agent will typically be present in an amount ranging from between 2% and 4% of the tablet weight. Suitable film-forming agents are well known to the art and include hydroxypropyl methylcellulose, cationic methacrylate copolymers (dimethylaminoethyl methacrylate/methyl-butyl methacrylate copolymers—Eudragit® E—Röhm. Pharma) and the like. These film-forming agents may optionally contain colorants, plasticizers and other supplemental ingredients.

The compressed tablets preferably have a hardness sufficient to withstand 8 Kp compression. The tablet size will depend primarily upon the amount of compound in the tablet. The tablets will include from 300 to 1100 mg of compound free base. Preferably, the tablets will include amounts of compound free base ranging from 400-600 mg, 650-850 mg and 900-1100 mg.

In order to influence the dissolution rate, the time during which the compound containing powder is wet mixed is controlled. Preferably the total powder mix time, i.e. the time during which the powder is exposed to sodium hydroxide solution, will range from 1 to 10 minutes and preferably from 2 to 5 minutes. Following granulation, the particles are removed from the granulator and placed in a fluid bed dryer for drying at about 60° C.

Formulation Example 11

A tablet Formula is prepared using the ingredients below:

The components are blended and compressed to form tablets.

EXAMPLES

In the general schemes shown below, compounds of the present disclosure may be prepared by the synthetic route shown, wherein R3b′is selected from H, F, optionally protected OH, or OMe, PG is a suitable protecting group, and the remaining variables are as defined herein.

Stereoisomers described herein were prepared via an asymmetric synthesis as described herein or separated as described herein. For compounds separated via chiral separation, stereocenters were arbitrarily assigned.

Example 1: Preparation of 7-fluoro-2-[(4S)-2-fluoro-4-[[6-oxo-5-(trifluoromethyl)-1H-pyridazin-4-yl]amino]pentyl]-6-[5-(trifluoromethyl)pyrimidin-2-yl]isoquinolin-1-one

Step 2. In a flask were placed tert-butyl N-[(1S)-4-(6-bromo-7-fluoro-1-oxo-2-isoquinolyl)-1-methyl-3-oxo-butyl]carbamate (9.12 g, 20.7 mmol) and THF (200 mL) and NaBH4(1.17 g, 31.0 mmol) was added. After stirring overnight, the solution is carefully quenched with water (20 mL) and the pH is adjusted to ˜4 with 1N HCl. The mixture was diluted with water, extracted with EtOAc, and concentrated in vacuo to give tert-butyl N-[(1S)-4-(6-bromo-7-fluoro-1-oxo-2-isoquinolyl)-3-hydroxy-1-methyl-butyl]carbamate (Intermediate 1). ES/MS m/z: 444.9 [M+H]+.

Step 3. In a flask were placed tert-butyl N-[(1S)-4-(6-bromo-7-fluoro-1-oxo-2-isoquinolyl)-3-hydroxy-1-methyl-butyl]carbamate (9.16 g, 20.7 mmol) and TFA (10 mL) in DCM (10 mL). The mixture was stirred at room temperature for 2 hours and concentrated to give 2-[(4S)-4-amino-2-hydroxy-pentyl]-6-bromo-7-fluoro-isoquinolin-1-one. ES/MS m/z: 345.4 [M+2H]++.

Step 5. In a flask were placed 6-bromo-7-fluoro-2-[(4S)-2-hydroxy-4-[[6-oxo-5-(trifluoromethyl)-1-(2-trimethylsilylethoxymethyl)pyridazin-4-yl]amino]pentyl]isoquinolin-1-one (4.96 g, 7.80 mmol), (diethylamino)difluorosulfonium tetrafluoroborate (2.68 g, 11.7 mmol), triethylamine trihydrofluoride (3.8 mL, 23.4 mmol), and DCE (78 mL). The mixture was heated to 75° C. and stirred overnight. The solution was cooled to room temperature and quenched with saturated aq. NaHCO3and diluted with DCM and water. Brine was added to the organic layer to break down the emulsion. The layers were separated and filtered over a pad of Celite®. The remaining organic and aqueous layers were separated again, concentrated in vacuo, and purified by flash chromatography eluting with EtOAc in hexanes 0-100% to afford 6-bromo-7-fluoro-2-[(4S)-2-fluoro-4-[[6-oxo-5-(trifluoromethyl)-1-(2-trimethylsilylethoxymethyl)pyridazin-4-yl]amino]pentyl]isoquinolin-1-one. ES/MS m/z: 638.9 [M+H]+.

Step 6. A flask was charged with 6-bromo-7-fluoro-2-[(4S)-2-fluoro-4-[[6-oxo-5-(trifluoromethyl)-1-(2-trimethylsilylethoxymethyl)pyridazin-4-yl]amino]pentyl]isoquinolin-1-one (2.26 g, 3.55 mmol), 1,1′-Bis(diphenylphosphino)ferrocene-palladium(II)dichloride (579 mg, 0.71 mmol), potassium acetate (1.39 g, 14.2 mmol), and bis(pinacolato)diboron (2.70 g, 10.6 mmol) and flushed with nitrogen gas. Dioxane (35 mL) was added and the solution was heated to 100° C. for 4 h. After cooling, 2-iodo-5-(trifluoromethyl)pyrimidine (1.46 g, 5.32 mmol), CataCXium Pd G4 (132 mg, 0.18 mmol), cesium carbonate (3.47 g, 10.6 mmol), and water (8.75 mL) were added. The solution was then stirred at 80° C. overnight. The solution is cooled and filtered through a pad of Celite® with EtOAc. The filtrate is diluted with EtOAc and water and the layers are stirred vigorously stirred for 10 minutes. The organic layer is concentrated in vacuo and purified by column chromatography eluting with EtOAc in hexanes 0-100% to afford 7-fluoro-2-[(4S)-2-fluoro-4-[[6-oxo-5-(trifluoromethyl)-1-(2-trimethylsilylethoxymethyl)pyridazin-4-yl]amino]pentyl]-6-[5-(trifluoromethyl)pyrimidin-2-yl]isoquinolin-1-one. ES/MS: m/z 705.3 [M+H]+.

Example 2 and Example 3: Preparation of 7-fluoro-2-((2R,4S)-2-fluoro-4-((6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl)amino)pentyl)-6-(5-(trifluoromethyl)pyrimidin-2-yl)isoquinolin-1(2H)-one and 7-fluoro-2-((2S,4S)-2-fluoro-4-((6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl)amino)pentyl)-6-(5-(trifluoromethyl)pyrimidin-2-yl)isoquinolin-1(2H)-one

Step 1. Examples 2 and Example 3 were separated via chiral SFC (AD-H, 5 μm, 21×250 mm column; 35% EtOH as co-solvent; 100 bar; 40° C.). The first eluting peak was arbitrarily assigned as the (R)-configuration (Example 2), and the second eluting peak was arbitrarily assigned as the (S)-configuration (Example 3).

Example 10: Preparation of 7-fluoro-2-((4S)-2-methoxy-4-((6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl)amino)pentyl)-6-(5-(trifluoromethyl)pyrimidin-2-yl)isoquinolin-1(2H)-one

The title compound was synthesized as described in Example 1, starting with Intermediate 1 and with the following modification in step 1.

Example 11: Preparation of 7-fluoro-2-((4S)-2-fluoro-4-((6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl)oxy)pentyl)-6-(5-(trifluoromethyl)pyrimidin-2-yl)isoquinolin-1(2H)-one

Step 1. In a round-bottomed flask 1-methoxy-4-[[(1S)-1-methylbut-3-enoxy]methyl]benzene (4.00 g, 19.4 mmol) was dissolved in DCM (20 mL, 1M) and cooled to 0° C. Triethylamine trihydrofluoride (7.89 mL, 48.5 mmol) was added, followed by N-iodosuccinimide (4.8 g, 21.3 mmol) and the solution was warmed room temperature and stirred for 8 h. The solution is quenched by addition of saturated aq. NaHCO3(50 ml), and the aqueous layer was extracted with DCM (3×75 mL). The combined organic layers were washed with saturated aq. Na2S2O3(100 mL), and the organic layer was dried over MgSO4, filtered, and concentrated by rotary evaporation to afford a crude oil which was purified by flash chromatography (0-20% EtOAc in Hexane) to give 1-[[(1S)-3-fluoro-4-iodo-1-methyl-butoxy]methyl]-4-methoxy-benzene. ES/MS: m z 353.1 [M+H]+.

Step 2. In a round-bottomed flask was added −[[(1S)-3-fluoro-4-iodo-1-methyl-butoxy]methyl]-4-methoxy-benzene (0.695 g, 1.97 mmol), 6-bromo-7-fluoro-2H-isoquinolin-1-one (0.525 g, 2.17 mmol), and cesium carbonate (1.29 g, 3.95 mmol) in DMF (10 mL). The solution was stirred for 48 h at room temperature, and then quenched with water (100 mL) and extracted with EtOAc (3×75 mL). The combined organic extracts were then washed with 10% LiCl(aq)solution (100 mL) and the organic layer was dried over MgSO4, filtered, and concentrated by rotary evaporation to afford a crude oil which was purified by flash chromatography (0-100% EtOAc in hexane) to give 6-bromo-7-fluoro-2-[(4S)-2-fluoro-4-[(4-methoxyphenyl)methoxy]pentyl]isoquinolin-1-one. ES/MS: m z 467.1 [M+H]+.

Step 3. In a round-bottomed flask, 6-bromo-7-fluoro-2-[(4S)-2-fluoro-4-[(4-methoxyphenyl)methoxy]pentyl]isoquinolin-1-one (0.624 g, 1.34 mmol) was dissolved in DCM/Water (27 mL, 19:1 DCM/water) and cooled to 0° C. 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (1.22 g, 5.35 mmol) was added in one portion, and the solution was allowed to warm to room temperature and stirred for 2 h at room temperature. The solution was filtered over Celite©, diluted with DCM (50 mL), and washed with sat. aq. NaHCO3(3×25 mL). The combined organic layer were dried over MgSO4, filtered, and concentrated by rotary evaporation to afford a crude oil which was purified by flash chromatography (0-100% EtOAc in hexane) to give 6-bromo-7-fluoro-2-[(4S)-2-fluoro-4-hydroxy-pentyl]isoquinolin-1-one. ES/MS: m z 347.1 [M+H]+.

Step 4. In a flask were placed 6-bromo-7-fluoro-2-[(4S)-2-fluoro-4-hydroxy-pentyl]isoquinolin-1-one (0.435 g, 1.26 mmol), 5-chloro-4-(trifluoromethyl)-2-(2-trimethylsilylethoxymethyl)pyridazin-3-one (0.689 g, 1.88 mmol), DIPEA (0.66 mL, 3.77 mmol), and DMF (7 mL). The mixture was heated to 85° C. for 48 h, then quenched with water (100 mL) and extracted with EtOAc (3×75 mL). The combined organic layers were washed with 10% LiCI solution(aq) (100 mL) and the organic layer was dried over MgSO4, filtered, and concentrated by rotary evaporation to afford a crude oil which was purified by flash chromatography (0-100% EtOAc in hexane) to give 6-bromo-7-fluoro-2-[(4S)-2-fluoro-4-[6-oxo-5-(trifluoromethyl)-1-(2-trimethylsilylethoxymethyl)pyridazin-4-yl]oxy-pentyl]isoquinolin-1-one. ES/MS: m z 638.1 [M+H]+.

Step 6. A vial was charged with 7-fluoro-2-((4S)-2-fluoro-4-((6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl)oxy)pentyl)-6-(5-(trifluoromethyl)pyrimidin-2-yl)isoquinolin-1(2H)-one (136 mg, 0.194 mmol), TFA (5 mL), and DCM (15 mL). The solution was stirred for 1 h and concentrated in vacuo. The residue is then treated with 2 N ammonia in methanol (3 ml), stirred for 15 minutes, concentrated in vacuo and directly purified by reverse phase prep-HPLC (5-100% MeCN in water, 0.1% TFA) to afford Example 11, 7-fluoro-2-((4S)-2-fluoro-4-((6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl)oxy)pentyl)-6-(5-(trifluoromethyl)pyrimidin-2-yl)isoquinolin-1(2H)-one. ES/MS: m z 576.1 [M+H]+.

Example 12 and Example 13: Preparation of 7-fluoro-2-((2R,4S)-2-fluoro-4-((6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl)oxy)pentyl)-6-(5-(trifluoromethyl)pyrimidin-2-yl)isoquinolin-1(2H)-one and 7-fluoro-2-((2S,4S)-2-fluoro-4-((6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl)oxy)pentyl)-6-(5-(trifluoromethyl)pyrimidin-2-yl)isoquinolin-1(2H)-one

Step 1. Example 12 and Example 13 were separated via chiral SFC (AD-H, 5 μm, 4.6×100 mm column; 30% EtOH as co-solvent; 100 bar; 40° C.). The first eluting peak was arbitrarily assigned as the (R)-configuration (Example 12), and the second eluting peak was arbitrarily assigned as the (S)-configuration (Example 13).

Example 16: Preparation of 7-fluoro-2-(4S)-2-hydroxy-4-((6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl)oxy)pentyl)-6-(5-(trifluoromethyl)pyrimidin-2-yl)isoquinolin-1(2H)-one

Step 1. In a round-bottomed flask, 1-methoxy-4-[[(1S)-1-methylbut-3-enoxy]methyl]benzene (4.70 g, 22.8 mmol) was dissolved in DCM (100 mL). mCPBA (8.43 g, 34.2 mmol) was added in one portion and stirred for 8 h. The gel-like suspension was filtered to remove excess benzoic acid, and washed with 50 mL of 2 N Na2CO3(aq) solution. The organic layer was dried over MgSO4, filtered, and concentrated by rotary evaporation to afford a crude oil which was purified by flash chromatography (0-30% EtOAc in hexane) to give 2-[(2S)-2-[(4-methoxyphenyl)methoxy]propyl]oxirane.

Step 2. In a round-bottomed flask was added 2-[(2S)-2-[(4-methoxyphenyl)methoxy]-propyl]oxirane (1.38 g, 6.23 mmol), 6-bromo-7-fluoro-2H-isoquinolin-1-one (1.66 g, 6.85 mmol), and cesium carbonate (4.06 g, 12.5 mmol) in DMF (31 mL). The solution was stirred for 48 h at room temperature, and then quenched with water (100 mL) and extracted with EtOAc (3×75 mL). The organic extracts were then washed with 10% LiCl solution (100 mL) and the organic layer was dried over MgSO4, filtered, and concentrated by rotary evaporation to afford a crude oil which was purified by flash chromatography (0-100% EtOAc in hexane) to give 6-bromo-7-fluoro-2-[(4S)-2-hydroxy-4-[(4-methoxyphenyl)methoxy]pentyl]isoquinolin-1-one.

Step 3. In a round bottomed flask, 6-bromo-7-fluoro-2-[(4S)-2-hydroxy-4-[(4-methoxyphenyl)methoxy]pentyl]isoquinolin-1-one (1.07 g, 2.30 mmol) was dissolved in THF (15 mL, 0.15 M) followed by DIPEA (2.01 mL, 11.5 mmol), and bromomethyl methyl ether (0.564 mL, 6.91 mmol) was added at room temperature. The solution is monitored by LCMS, and when the reaction no longer proceeds, the suspension is filtered over Celite®, concentrated, and resubjected to the reaction conditions. A total of five cycles was required for reaction completion. Upon completion, the solution is filtered over Celite®, concentrated by rotary evaporation, and directly purified by flash chromatography (0-100% EtOAc in hexane) to give 6-bromo-7-fluoro-2-[(4S)-2-(methoxymethoxy)-4-[(4-methoxyphenyl)methoxy]pentyl]isoquinolin-1-one.

Step 4. In a round-bottomed flask, 6-bromo-7-fluoro-2-[(4S)-2-(methoxymethoxy)-4-[(4-methoxyphenyl)methoxy]pentyl]isoquinolin-1-one (0.950 g, 1.87 mmol) was dissolved in DCM/Water (40 mL, 19:1 DCM/water) and cooled to 0° C. 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (1.70 g, 7.47 mmol) was added in one portion, and the solution was allowed to warm to room temperature and stirred for 2 h at room temperature. The solution was filtered over Celite®, diluted with DCM (100 mL), and washed with saturated aq. NaHCO3(3×50 mL). The combined organic fractions were dried over MgSO4, filtered, and concentrated by rotary evaporation to afford a crude oil which was purified by flash chromatography (0-100% EtOAc in hexane) to give 6-bromo-7-fluoro-2-[(4S)-4-hydroxy-2-(methoxymethoxy)pentyl]isoquinolin-1-one.

Step 5. In a flask were placed 6-bromo-7-fluoro-2-[(4S)-4-hydroxy-2-(methoxymethoxy)pentyl]isoquinolin-1-one (0.624 g, 1.61 mmol), 5-chloro-4-(trifluoromethyl)-2-(2-trimethylsilylethoxymethyl)pyridazin-3-one (0.881 g, 2.41 mmol), DIPEA (0.84 mL, 4.82 mmol), and DMF (8 mL). The mixture was heated to 85° C. for 48 h and then quenched with water (100 mL) and extracted with EtOAc (3×75 mL). The combined organic extracts were then washed with 10% LiCI solution (100 mL) and the organic layer was dried over MgSO4, filtered, and concentrated by rotary evaporation to afford a crude oil which was purified by flash chromatography (0-100% EtOAc in hexane) to give 6-bromo-7-fluoro-2-[(4S)-2-(methoxymethoxy)-4-[6-oxo-5-(trifluoromethyl)-1-(2-trimethylsilylethoxymethyl)pyridazin-4-yl]oxy-pentyl]isoquinolin-1-one.

Example 17 and Example 18: Preparation of 7-fluoro-2-((2R,4S)-2-hydroxy-4-((6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl)oxy)pentyl)-6-(5-(trifluoromethyl)pyrimidin-2-yl)isoquinolin-1(2H)-one and 7-fluoro-2-((2R,4S)-2-hydroxy-4-((6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl)oxy)pentyl)-6-(5-(trifluoromethyl)pyrimidin-2-yl)isoquinolin-1(2H)-one

Step 1. Examples 17 and Example 18 were separated via chiral SFC (AD-H, 5 mm, 4.6×100 mm column; 30% EtOH as co-solvent; 100 bar; 40° C.). The first eluting peak was arbitrarily assigned as the (R)-configuration (Example 17), and the second eluting peak was arbitrarily assigned as the (S)-configuration (Example 18).

Example 21: Preparation of 6-([1,2,4]triazolo[1,5-a]pyridin-2-yl)-7-fluoro-2-((4S)-2-hydroxy-4-((6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl)amino)pentyl)isoquinolin-1(2H)-one

Step 1. A flask was charged with 6-bromo-7-fluoro-2-((4S)-2-hydroxy-4-((6-oxo-5-(trifluoromethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1,6-dihydropyridazin-4-yl)amino)pentyl)isoquinolin-1(2H)-one (330 mg, 0.493 mmol), 1,1′-bis(diphenylphosphino)ferrocene-palladium(II)dichloride (376 mg, 1.5 mmol), potassium acetate (194 mg, 1.97 mmol), and bis(pinacolato)diboron (376 mg, 1.48 mmol) and flushed with nitrogen gas. Dioxane (10.0 mL) was added and the solution was heated to 100° C. for 4 h. After cooling, 2-bromo-[1,2,4]triazolo[1,5-a]pyridine (371 mg, 1.88 mmol), CataCXium Pd G4 (17.4 mg, 0.024 mmol), 2 M Na2CO3(aq) (0.70 mL, 1.4 mmol) was added. The solution was then stirred at 80° C. overnight. The solution is cooled and filtered through a pad of Celite® and washed with EtOAc. The filtrate is diluted with EtOAc and water and the layers are stirred vigorously for 10 minutes. The organic layer is concentrated in vacuo and purified by column chromatography (hexanes/EtOAc) to give 7-fluoro-2-[(4S)-2-fluoro-4-[[6-oxo-5-(trifluoromethyl)-1-(2-trimethylsilylethoxymethyl)pyridazin-4-yl]amino]pentyl]-6-[5-(trifluoromethyl)pyrimidin-2-yl]isoquinolin-1-one. ES/MS: m/z 573.5 [M+H]+.

Example 22 and Example 23: Preparation of 6-([1,2,4]triazolo[1,5-a]pyridin-2-yl)-7-fluoro-2-((2R,4S)-2-hydroxy-4-((6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl)amino)pentyl)isoquinolin-1(2H)-one and 6-([1,2,4]triazolo[1,5-a]pyridin-2-yl)-7-fluoro-2-((2S,4S)-2-hydroxy-4-((6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl)amino)pentyl)isoquinolin-1(2H)-one

Step 1. Examples 22 and Example 23 were separated via chiral SFC (AD-H, 5 μm, 4.6×100 mm column; 30% EtOH as co-solvent; 100 bar; 40° C.). The first eluting peak was arbitrarily assigned as the (R)-configuration (Example 22), and the second eluting peak was arbitrarily assigned as the (S)-configuration (Example 23).

Example 24: Preparation of 6-(4-amino-5-(difluoromethyl)pyrimidin-2-yl)-4,7-difluoro-2-((2R,4S)-2-fluoro-4-((6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl)amino)pentyl)isoquinolin-1(2H)-one

Step 1. Methyl (4S)-4-(tert-butoxycarbonylamino)pentanoate (5200 mg, 22.5 mmol) in THF (100 mL) was added to a solution of LDA (45 ml, 2 M in THF, 89.9 mmol) in THF (100 mL) at −78° C. After 45 minutes, the solution was transferred via cannula to a solution of NFSI (19.7 g, 112 mmol) in THF (100 mL) at −78° C. After 30 minutes, the solution mixture was quenched with saturated aq. NH4Cl (25 mL), followed by the addition of dimethyl sulfide (9.0 mL, 135 mmol). The mixture was diluted with EtOAc, washed with saturated aq. NH4Cl, saturated aq. NaHCO3(2×), water, and brine. The combined organic layers were dried over MgSO4, concentrated, and the resulting residue was purified via column chromatography (0-100% EtOAc in hexanes) to afford methyl (2R,4S)-4-(tert-butoxycarbonylamino)-2-fluoro-pentanoate. ES/MS: m/z 250.0 [M+H]+.

Step 2. NaBH4(3375 mg, 89.2 mmol) was added to methyl (2R,4S-4-(tert-butoxycarbonylamino)-2-fluoro-pentanoate in THE (100 ml) at 0° C. The solution was allowed to warm to room temperature and stirred at this temperature for 16 h. Upon completion, the solution was cooled to 0° C., and slowly quenched with saturated aq. NH4Cl and extracted with EtOAc. Organic layer was dried over MgSO4, concentrated, and the resulting residue was purified via column chromatography (0-100% EtOAc in hexanes) to afford tert-butyl N-[(1S,3R)-3-fluoro-4-hydroxy-1-methyl-butyl]carbamate. ES/MS: m/z 222.0 [M+H]+.

Step 3. Iodine (1.74 g, 6.87 mmol) was added to a solution of triphenylphosphine (1.80 g, 6.87 mmol) and imidazole (0.468 g, 6.87 mmol) in DCM (50 ml). The mixture was stirred at room temperature for 30 minutes and then tert-butyl N-[(1S,3R)-3-fluoro-4-hydroxy-1-methyl-butyl]carbamate (1.22 g, 5.50 mmol) was added. The solution was then heated at 40° C. for 18 h. Upon completion, the mixture was cooled to room temperature and the mixture was filtered and then concentrated. The residue is dissolved in EtOAc and washed with saturated aq. sodium bisulfite and then brine. The organic phase is dried over MgSO4and the solvent is removed under reduced pressure. The residue is purified by flash chromatography using a gradient of EtOAc/hexane (0 to 100%) as eluent to give tert-butyl N-[(1S,3R)-3-fluoro-4-iodo-1-methyl-butyl]carbamate. ES/MS: m/z 331.9 [M+H]+.

Step 5. To a solution of tert-butyl N-[(1S,3R)-4-(6-bromo-4,7-difluoro-1-oxo-2-isoquinolyl)-3-fluoro-1-methyl-butyl]carbamate (503 mg, 1.09 mmol) in dichloromethane (10.0 mL) was added trifluoroacetic acid (2.0 mL) at room temperature and the mixture was stirred for 1 h. Upon completion, the solvent was removed under reduced pressure to afford 2-[(2R,4S)-4-amino-2-fluoro-pentyl]-6-bromo-4,7-difluoro-isoquinolin-1-one which was used without further purification. ES/MS: m/z 364.218 [M+H]+.

Step 6. A mixture of 2-[(2R,4S)-4-amino-2-fluoro-pentyl]-6-bromo-4,7-difluoro-isoquinolin-1-one (394 mg, 1.08 mmol), 5-chloro-4-(trifluoromethyl)-2-(2-trimethylsilylethoxymethyl)pyridazin-3-one (764 mg, 1.63 mmol), and N,N-diisopropylethylamine (1.89 mL, 10.8 mmol) in DMF (20 mL) was stirred at room temperature for 2 h. Upon completion, the solution was diluted with EtOAc, washed with water, washed with brine, dried over MgSO4and concentrated in vacuo to give the crude product. The crude residue was purified using column chromatography eluting with EtOAc in hexanes 0-100% to provide 6-bromo-4,7-difluoro-2-[(2R,4S)-2-fluoro-4-[[6-oxo-5-(trifluoromethyl)-1-(2-trimethylsilylethoxymethyl)pyridazin-4-yl]amino]pentyl]isoquinolin-1-one. ES/MS: m/z 657.0 [M+H]+.

Step 7. In a flask was placed 6-bromo-4,7-difluoro-2-[(2R,4S)-2-fluoro-4-[[6-oxo-5-(trifluoromethyl)-1-(2-trimethylsilylethoxymethyl)pyridazin-4-yl]amino]pentyl]isoquinolin-1-one (100 mg, 0.153 mmol), 1,1′-bis(diphenylphosphino)ferrocene-palladium(II)dichloride dichloromethane complex (12.5 mg, 0.015 mmol), potassium acetate (44.9 mg, 0.334 mmol), and bis(pinacolato)diboron (58.1 mg, 0.229 mmol) in dioxane (3 mL). The mixture was purged with nitrogen gas for 5 minutes and then stirred at 80° C. for 18 h. The solution was cooled to room temperature and cataCXium Pd G4 (11.3 mg, 0.015 mmol), 2 M aqueous sodium carbonate (0.16 mL, 0.334 mmol), and 2-chloro-5-(difluoromethyl)pyrimidin-4-amine (41.1 mg, 0.229 mmol) were added. The mixture was then stirred at 90° C. for 3 h. Upon completion, the mixture was cooled to room temperature, diluted with EtOAc and filtered through a plug of Celite®. The volatiles were evaporated in vacuo and the residue was purified via flash chromatography using MeOH in DCM 0-20% to give 6-[4-amino-5-(difluoromethyl)pyrimidin-2-yl]-4,7-difluoro-2-[(2R,4S)-2-fluoro-4-[[6-oxo-5-(trifluoromethyl)-1-(2-trimethylsilylethoxymethyl)pyridazin-4-yl]amino]pentyl]isoquinolin-1-one. ES/MS m/z: 720.1 [M+H].

The following Examples were synthesized as described in Example 24 or a modification of the procedure above from the corresponding intermediates.

Example 33: Preparation of 2-(5-(difluoromethoxy)-4-((6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl)oxy)pentyl)-7-fluoro-6-(5-(2-hydroxypropan-2-yl)pyrimidin-2-yl)isoquinolin-1(2H)-one

Step 1. To a stirred solution of 3-(2-(4-methoxyphenyl)-1,3-dioxolan-4-yl)propan-1-ol (2.93 g, 12.3 mmol) and triethylamine (3.43 mL, 24.6 mmol) in dichloromethane (50 mL) was added p-toluenesulfonyl chloride (3.51 g, 18.4 mmol) followed by DMAP (75 mg, 0.62 mmol). The mixture was stirred at room temperature for 16 h. Upon completion, the mixture was quenched with saturated aqueous NaHCO3and extracted with dichloromethane. The combined organic layers were washed with brine, dried over MgSO4and concentrated in vacuo to give the crude product. The crude residue was purified using column chromatography eluting with EtOAc in hexanes 5-100% to afford 3-(2-(4-methoxyphenyl)-1,3-dioxolan-4-yl)propyl 4-methylbenzenesulfonate. ES/MS: m/z 393.0 [M+H]+.

Step 2. To a mixture of 6-bromo-7-fluoroisoquinolin-1(2H)-one (3.4 g, 14.0 mmol) and 3-(2-(4-methoxyphenyl)-1,3-dioxolan-4-yl)propyl 4-methylbenzenesulfonate (5.51 g mg, 14.0 mmol) in DMF (50 mL) was added Cs2CO3(5.49 g, 16.9 mmol) and the solution was stirred at room temperature for 2 h. Upon completion, the mixture was diluted with EtOAc, washed with water, washed with brine, dried over MgSO4and concentrated in vacuo to give the crude product. The crude residue was purified using column chromatography eluting with EA in hexanes 5-100% to afford 6-bromo-7-fluoro-2-(3-(2-(4-methoxyphenyl)-1,3-dioxolan-4-yl)propyl)isoquinolin-1(2H)-one. ES/MS: m/z 344.0 [M-C8H6O+H]+.

Step 3. To a solution of 6-bromo-7-fluoro-2-(3-(2-(4-methoxyphenyl)-1,3-dioxolan-4-yl)propyl)isoquinolin-1(2H)-one (3.5 g, 7.57 mmol) in DCM (80 mL) at −78° C. was added diisobutylaluminum hydride (1.0 M in DCM, 23 mL, 22.7 mmol). The solution was stirred at −78° C. for 2 h and then quenched slowly with MeOH (40 mL). The mixture was allowed to warm up to room temperature and 100 mL of saturated aqueous Rochelle salt was added. The mixture was vigorously stirred until two clear phases were obtained. After separation, the aqueous phase was extracted with EtOAc. The combined organic layers were washed with water, washed with brine, dried over MgSO4and concentrated in vacuo to give the crude product. The crude residue was purified using column chromatography eluting with EA in hexanes 5-100% to afford 6-bromo-7-fluoro-2-(5-hydroxy-4-((4-methoxybenzyl)oxy)pentyl)isoquinolin-1(2H)-one. ES/MS: m/z 486.1 [M+Na]+.

Step 4. To a mixture of 6-bromo-7-fluoro-2-(5-hydroxy-4-((4-methoxybenzyl)oxy)pentyl)isoquinolin-1(2H)-one (1.8 g, 3.9 mmol) and KHF2(908 mg, 11.6 mmol) in DCM (7 mL) and water (7 mL) was added [bromo(difluoro)methyl]-trimethyl-silane (1.2 g, 5.8 mmol) dropwise. The solution was stirred at room temperature for 2 h. Additional equivalence of [bromo(difluoro)methyl]-trimethyl-silane (1.2 g, 5.8 mmol) was added and the solution was stirred at room temperature for 2 h. Upon completion, the mixture was diluted with DCM, washed with water, washed with brine, dried over MgSO4and concentrated in vacuo to give the crude product. The crude residue was purified using column chromatography eluting with EA in hexanes 5-100% to afford 6-bromo-2-(5-(difluoromethoxy)-4-((4-methoxybenzyl)oxy)pentyl)-7-fluoroisoquinolin-1(2H)-one. ES/MS: m/z 536.1 [M+Na]+.

Step 5. To a solution of 6-bromo-2-(5-(difluoromethoxy)-4-((4-methoxybenzyl)oxy)pentyl)-7-fluoroisoquinolin-1(2H)-one (1.48 g, 2.9 mmol) in DCM (40 mL) and water (2 mL) at 0° C. was added DDQ (2.6 g, 11.5 mmol). The solution was stirred at room temperature for 2 h. The mixture was filtered, and the mother liquor was washed with saturated aqueous NaHCO3. After separation, the aqueous phase was extracted with DCM. The combined organic layers were washed with water, washed with brine, dried over MgSO4and concentrated in vacuo to give the crude product. The crude residue was purified using column chromatography eluting with EA in hexanes 5-100% to afford 6-bromo-2-(5-(difluoromethoxy)-4-hydroxypentyl)-7-fluoroisoquinolin-1(2H)-one. ES/MS m/z: 394.1 [M+H]+.

Step 6. A solution of 6-bromo-2-(5-(difluoromethoxy)-4-hydroxypentyl)-7-fluoroisoquinolin-1(2H)-one (1.02 g, 2.59 mmol), 5-chloro-4-(trifluoromethyl)-2-(2-trimethylsilylethoxymethyl)pyridazin-3-one (1.7 g, 5.18 mmol), and N,N-diisopropylethylamine (1.35 mL, 7.76 mmol) in DMF (10 mL) was stirred at 85° C. for 24 h. Upon cooling, the mixture was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with water, washed with brine, dried over MgSO4and concentrated in vacuo to give the crude product. The crude residue was purified using column chromatography eluting with EA in hexanes 0-100% to afford 6-bromo-2-(5-(difluoromethoxy)-4-((6-oxo-5-(trifluoromethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1,6-dihydropyridazin-4-yl)oxy)pentyl)-7-fluoroisoquinolin-1(2H)-one. ES/MS: m/z 686.1 [M+H]+.

Step 7. Using 6-bromo-2-(5-(difluoromethoxy)-4-((6-oxo-5-(trifluoromethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1,6-dihydropyridazin-4-yl)oxy)pentyl)-7-fluoroisoquinolin-1(2H)-one and 2-(2-chloropyrimidin-5-yl)propan-2-ol, 2-(5-(difluoromethoxy)-4-((6-oxo-5-(trifluoromethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1,6-dihydropyridazin-4-yl)oxy)pentyl)-7-fluoro-6-(5-(2-hydroxypropan-2-yl)pyrimidin-2-yl)isoquinolin-1(2H)-one was synthesized following the procedure described in the step 6 of Example 1. ES/MS: m/z 744.3 [M+H]+.

Step 8. Using 2-(5-(difluoromethoxy)-4-((6-oxo-5-(trifluoromethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1,6-dihydropyridazin-4-yl)oxy)pentyl)-7-fluoro-6-(5-(2-hydroxypropan-2-yl)pyrimidin-2-yl)isoquinolin-1(2H)-one, 2-(5-(difluoromethoxy)-4-((6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl)oxy)pentyl)-7-fluoro-6-(5-(2-hydroxypropan-2-yl)pyrimidin-2-yl)isoquinolin-1(2H)-one was synthesized following the procedure described in the step 7 of Example 1.

Example 34 and Example 35: Preparation of (S)-2-(5-(difluoromethoxy)-4-((6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl)oxy)pentyl)-7-fluoro-6-(5-(2-hydroxypropan-2-yl)pyrimidin-2-yl)isoquinolin-1(2H)-one and (R)-2-(5-(difluoromethoxy)-4-((6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl)oxy)pentyl)-7-fluoro-6-(5-(2-hydroxypropan-2-yl)pyrimidin-2-yl)isoquinolin-1(2H)-one

Examples 34 and Example 35 were separated via chiral SFC (AD-H, 5 μm, 21×250 mm column; 45% EtOH as co-solvent; 100 bar; 40° C.). The first eluting peak was arbitrarily assigned as the (S)-configuration (Example 34), and the second eluting peak was arbitrarily assigned as the (R)-configuration (Example 35).

The following Examples were synthesized as described in the reference examples noted in the table or a modification of the procedure from the corresponding intermediates (stereochemistry was arbitrarily assigned).

Example 68: Preparation of 4-bromo-7-fluoro-2-((2R,4S)-2-fluoro-4-((6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl)oxy)pentyl)-6-(5-(2-hydroxypropan-2-yl)pyrimidin-2-yl)isoquinolin-1(2H)-one

Intermediate 3: Preparation of 2-(2-bromopyrimidin-5-yl)oxy-2,2-difluoro-ethanol

Step 1. NaI (3.8 g, 25 mmol) and LiBr (610 mg, 7.0 mmol) were heated with a heat gun under vacuum and then cooled under a stream of dry N2. 2-chloropyrimidine-5-carbaldehyde (1.0 g, 7.0 mmol), [bromo(difluoro)methyl]-trimethyl-silane (2.9 mL, 18 mmol), and DME (10 mL) were added to the dried inorganics. The reaction was heated at 80° C. for 4 hours before additional [bromo(difluoro)methyl]-trimethyl-silane (2.9 mL, 18 mmol) was charged. After an additional 16 hours at 80° C., the reaction was cooled to ambient temperature. TFA (1.0 mL, 14 mmol) and EtOH (5 mL) were added and the reaction was stirred an additional 90 minutes before the reaction was diluted with EtOAc and filtered through a pad of Celite®. The filtrate was evaporated, and the crude residue was purified via flash chromatography (100% hex→70% EtOAc/Hex) to afford a mixture of 1-(2-chloropyrimidin-5-yl)-2,2-difluoro-2-iodo-ethanol and 1-(2-iodopyrimidin-5-yl)-2,2-difluoro-2-iodo-ethanol, which was carried forward without further purification.

Step 2. The mixture 1-(2-chloropyrimidin-5-yl)-2,2-difluoro-2-iodo-ethanol and 1-(2-iodopyrimidin-5-yl)-2,2-difluoro-2-iodo-ethanol (˜0.75 mmol) was taken up in toluene (4.7 mL) and tri-n-butyltin hydride (0.40 mL, 1.5 mmol) was added. The reaction was heated to 80° C. before the addition of 2,2′-azobis(isobutyronitrile) (12 mg, 0.07 mmol). After 30 minutes, the reaction was cooled, diluted with EtOAc, and filtered through a pad of Celite®. The filtrate was evaporated, and the crude residue was purified via flash chromatography (100% hex→100% EtOAc) to afford 1-(2-chloropyrimidin-5-yl)-2,2-difluoro-ethanol. ES/MS m/z: 194.8 [M]+.

Intermediate 4: Preparation of 2-(2-chloropyrimidin-5-yl)-1,1-difluoro-propan-2-ol

2-chloro-5-iodo-pyrimidine (1.0 g, 4.2 mmol) was dissolved in THF and cooled to −78° C. in a dry ice/acetone bath. A solution of n-butyllithium (2.5 M, 5.2 mmol) was added slowly and the reaction was maintained in the cooling bath. After 40 minutes, 1,1-difluoroacetone (0.51 mL, 6.2 mmol) was then added dropwise. The reaction was stirred for an additional 2 hours in the cooling bath at which, a solution of 10% aq. KHSO4was added, and the mixture was allowed to warm to room temperature. The mixture was extracted (3x) with EtOAc. The combined organic layers were dried over MgSO4, filtered, and evaporated. The crude residue was purified via flash chromatography (100% hex→70% EtOAc/Hex) to afford 2-(2-chloropyrimidin-5-yl)-1,1-difluoro-propan-2-ol. ES/MS m/z: 209.4 [M+H]+.

Intermediate 5: Preparation of 2-(2-bromopyrimidin-5-yl)oxy-2,2-difluoro-ethanol

Step 1. 2-bromopyrimidin-5-ol (200 mg, 1.1 mmol) was dissolved in THF (7.2 mL) and stirred at ambient temperature. Sodium hydride (60% in oil, 48 mg, 1.3 mmol) was then added followed by Pd(OAc)2(2.6 mg, 0.01 mmol) and triphenylphosphine (12 mg, 0.04 mmol). The reaction was then cooled in an ice/water bath. 3-bromo-3,3-difluoro-prop-1-ene (0.17 mL, 1.7 mmol) was added as a solution in THF (1.2 mL). The reaction was then warmed to 40° C. for 1.5 hours before being cooled and quenched with addition of saturated bicarbonate and extracted (3x) with EtOAc. The combined organic layers were dried over MgSO4, filtered, and evaporated. The crude residue was purified via flash chromatography (100% hex→60% EtOAc/Hex) to afford 2-bromo-5-(1,1-difluoroallyloxy)pyrimidine. ES/MS m/z: 250.9/252.8 [M]+.

Step 2. 2-bromo-5-(1,1-difluoroallyloxy)pyrimidine (250 mg, 0.96 mmol) was dissolved in dioxane (8.8 mL) and water (8.8 mL) and stirred at ambient temperature. Potassium osmate dihydrate (18 mg, 0.05 mmol) and NaIO4(620 mg, 2.9 mmol) were then added. After 4 hours, LC/MS indicated incomplete conversion and 2,6-lutidine (0.22 mL, 1.9 mmol) was added. After stirring for 84 hours, the reaction was quenched via addition of saturated sodium thiosulfate and extracted (3x) with EtOAc. The combined organic layers were washed with saturated sodium thiosulfate and brine before being dried over Na2SO4, filtered, and evaporated. The crude residue was then dissolved in MeOH (8.0 mL) and stirred at ambient temperature. NaBH4(180 mg, 4.8 mmol) was added, and the reaction stirred for 2.5 hours at which point it was quenched with 10% KHSO4. The mixture was extracted (3x) with EtOAc and the combined organics dried over MgSO4, filtered, and evaporated. The crude residue was purified via flash chromatography (100% hex→100% EtOAc) to afford 2-(2-bromopyrimidin-5-yl)oxy-2,2-difluoro-ethanol. ES/MS m/z: 254.9/256.8 [M]+.

Intermediate 6: Preparation of 3-(2-bromopyrimidin-5-yl)oxy-1,1,1-trifluoro-propan-2-ol

2-bromopyrimidin-5-ol (250 mg, 1.4 mmol) and K2CO3(490 mg, 3.6 mmol) were taken up in DMF (3.0 mL) and stirred at ambient temperature. 2-(trifluoromethyl)oxirane (0.12 mL, 1.4 mmol) was then added. After 2.5 hours, additional 2-(trifluoromethyl)oxirane (24 μL, 0.28 mmol) was added and the reaction was stirred for an additional 96 hours at which point it was diluted with water and extracted (3x) with EtOAc. The combined organics were washed with brine, dried over MgSO4, filtered, and evaporated. The crude residue was purified via flash chromatography (100% hex→100% EtOAc) to afford 3-(2-bromopyrimidin-5-yl)oxy-1,1,1-trifluoro-propan-2-ol. ES/MS m/z: 286.9/288.8 [M]+.

Intermediate 7: Preparation of 6-[5-(1,1-difluoro-2-hydroxy-ethyl)pyrimidin-2-yl]-7-fluoro-2-[(2R,4S)-2-fluoro-4-[[6-oxo-5-(trifluoromethyl)-1-(2-trimethylsilylethoxymethyl)pyridazin-4-yl]amino]pentyl]isoquinolin-1-one

Step 2. A vial was charged with 6-bromo-7-fluoro-2-[(2R,4S)-2-fluoro-4-[[6-oxo-5-(trifluoromethyl)-1-(2-trimethylsilylethoxymethyl)pyridazin-4-yl]amino]pentyl]isoquinolin-1-one (370 mg, 0.56 mmol), 1,1′-Bis(diphenylphosphino)ferrocene-palladium(II) dichloride (20 mg, 0.03 mmol), potassium acetate (160 mg, 1.7 mmol), and bis(pinacolato)diboron (190 mg, 0.72 mmol) and flushed with nitrogen gas. Dioxane (4.5 mL) was added, and the reaction was heated to 100° C. for 80 minutes. After cooling, 2-iodo-5-(trifluoromethyl)pyrimidine (0.56 mmol) was charged as a solution in dioxane (2.0 mL) followed by [di(adamantan-1-yl)(butyl)phosphine](methanesulfonato-κO)[2′-(methylamino)-2-biphenylyl]palladium (CataCXium Pd G4, 20 mg, 0.03 mmol), and aqueous sodium carbonate (2.0 M, 0.83 mL). The mixture was bubbled with nitrogen gas briefly. The reaction was then stirred at 80° C. for 90 minutes before being diluted with EtOAc and filtered through a plug of Celite®. The filtrate was evaporated and the crude purified via flash chromatography (100% heptanes→100% [3:1 EtOAc:EtOH]) to afford 2,2-difluoro-2-[2-[7-fluoro-2-[(2R,4S)-2-fluoro-4-[[6-oxo-5-(trifluoromethyl)-1-(2-trimethylsilylethoxymethyl)pyridazin-4-yl]amino]pentyl]-1-oxo-6-isoquinolyl]pyrimidin-5-yl]acetic acid. ES/MS: m/z 731.2 [M+H]+.

Step 3. 2,2-difluoro-2-[2-[7-fluoro-2-[(2R,4S)-2-fluoro-4-[[6-oxo-5-(trifluoromethyl)-1-(2-trimethylsilylethoxymethyl)pyridazin-4-yl]amino]pentyl]-1-oxo-6-isoquinolyl]pyrimidin-5-yl]acetic acid (190 mg, 0.25 mmol) was taken up in THF (4.0 mL) and treated with TEA (53 μL, 0.38 mmol) before being cooled to 0° C. Ethyl chloroformate (36 μL, 0.38 mmol) was added and the reaction stirred for 30 minutes. The mixture was then filtered through a frit directly into a stirred solution of NaBH4(38 mg, 1.0 mmol) in THF (3.5 mL) and water (1.0 mL) at 0° C. The frit was rinsed with a small amount of additional THF. After 50 minutes, additional NaBH4b (150 mg, 4 mmol) was added before allowing to stir an additional 2 hours at which point the reaction was quenched by addition of 1000 KHSO4. The mixture was extracted 2x with EtOAc and the combined organic layers were dried over MgSO4, filtered, and evaporated. The crude purified via flash chromatography (100% heptanes→80% [3:1 EtOAc:EtOH]/heptanes) to afford 6-[5-(1,1-difluoro-2-hydroxy-ethyl)pyrimidin-2-yl]-7-fluoro-2-[(2R,4S)-2-fluoro-4-[[6-oxo-5-(trifluoromethyl)-1-(2-trimethylsilylethoxymethyl)pyridazin-4-yl]amino]pentyl]isoquinolin-1-one. ES/MS: m/z 717.3 [M+H]+.

The following Examples were synthesized as described in Example 24 or a modification of the procedure from the corresponding intermediates.

The title compound was synthesized as described in Example 24, with the following modification in step 4.

Example 119 and Example 120: (R)-2-(5-(difluoromethoxy)-4-((6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl)oxy)pentyl)-7-fluoro-6-(5-(trifluoromethyl)pyrimidin-2-yl)isoquinolin-1(2H)-one and (S)-2-(5-(difluoromethoxy)-4-((6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl)oxy)pentyl)-7-fluoro-6-(5-(trifluoromethyl)pyrimidin-2-yl)isoquinolin-1(2H)-one

The title compounds were synthesized as described in Examples 33-35, using 2-chloro-5-(trifluoromethyl)pyrimidine in place of 2-(2-chloropyrimidin-5-yl)propan-2-ol. Examples 119 and Example 120 were separated via chiral SFC (AD-H, 5 μm, 21×250 mm column; 45% EtOH as co-solvent; 100 bar; 40° C.). The first eluting peak was arbitrarily assigned as the (R)-configuration (Example 119), and the second eluting peak was arbitrarily assigned as the (S)-configuration (Example 120).

Example 121 and Example 122: 2-((2S,4R)-5-(difluoromethoxy)-2-fluoro-4-((6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl)oxy)pentyl)-7-fluoro-6-(5-(2-hydroxypropan-2-yl)pyrimidin-2-yl)isoquinolin-1(2H)-one and 2-((2R,4R)-5-(difluoromethoxy)-2-fluoro-4-((6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl)oxy)pentyl)-7-fluoro-6-(5-(2-hydroxypropan-2-yl)pyrimidin-2-yl)isoquinolin-1(2H)-one

Step 1. (4R)-4-(2-fluoro-3-iodopropyl)-2-(4-methoxyphenyl)-1,3-dioxolane was synthesized as described in Step 1 of Example 11, using (4R)-4-allyl-2-(4-methoxyphenyl)-1,3-dioxolane. ES/MS: m/z 366.5 [M+H]+.

Step 9. Examples 121 and Example 122 were separated via chiral SFC (AD-H, 5 μm, 21×250 mm column; 45% EtOH as co-solvent; 100 bar; 40° C.) of 2-((4R)-5-(difluoromethoxy)-2-fluoro-4-((6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl)oxy)pentyl)-7-fluoro-6-(5-(2-hydroxypropan-2-yl)pyrimidin-2-yl)isoquinolin-1(2H)-one. The first eluting peak was arbitrarily assigned as the (2R,4R)-configuration (Example 121), and the second eluting peak was arbitrarily assigned as the (2S,4R)-configuration (Example 122).

Intermediate 8: Preparation of 2-[(2R,4R)-4-amino-2-fluoro-5-hydroxy-pentyl]-6-bromo-7-fluoro-isoquinolin-1-one

Step 1. To a solution of triethyl 2-fluoro-2-phosphonoacetate (1.0 g, 4.36 mmol) in THF (20.0 mL) at −78° C. was added a solution of n-butyllithium (4.8 mmol, 1.9 mL) and the mixture was stirred for 10 minutes. The cooling bath was then removed, and the mixture was stirred for 20 minutes, and then cooled back down to −78° C. A solution of tert-butyl (4S)-4-formyl-2,2-dimethyl-oxazolidine-3-carboxylate (1.00 g, 4.36 mmol) in THF (20 mL) was then added slowly and the mixture was stirred for 1 h at 78° C. and quenched with a solution of saturated NH4Cl(aq)and allowed to warm to room temperature. The mixture was extracted 3× with EtOAc, dried over MgSO4, and evaporated. The crude residue was purified via flash chromatography (100% hexanes→35% EtOAc) to afford tert-butyl (4R)-4-[€-3-ethoxy-2-fluoro-3-oxo-prop-1-enyl]-2,2-dimethyl-oxazolidine-3-carboxylate. ES/MS: m/z 339.6 [M+Na]+.

Step 2. Tert-butyl 4-(3-ethoxy-2-fluoro-3-oxo-prop-1-enyl)-2,2-dimethyl-oxazolidine-3-carboxylate (1.2 g, 3.47 mmol) and 10% Pd/C (185 mg, 0.17 mmol) were taken up in EtOH (29.0 mL) and stirred at ambient temperature. The reaction headspace was evacuated/backfilled with nitrogen gas four times then with hydrogen gas four times. The reaction was then stirred under a balloon of hydrogen gas for 1 hour before the catalyst was filtered off through a plug of Celite® and washed with additional EtOH. The filtrate was evaporated to provide tert-butyl 4-(3-ethoxy-2-fluoro-3-oxo-propyl)-2,2-dimethyl-oxazolidine-3-carboxylate, which was carried forward without purification. ES/MS: m/z 342.0 [M+H+Na]+.

Step 3. tert-butyl 4-(3-ethoxy-2-fluoro-3-oxo-propyl)-2,2-dimethyl-oxazolidine-3-carboxylate (1.1 g, 3.47 mmol) was dissolved in THF (14.5 mL) and cooled to 0° C. LiBH4(2.0 M, 6.94 mmol) was added slowly. Thirty minutes after complete addition, the cooling bath was removed, and the reaction was stirred a further 16 hours at ambient temperature. The reaction was then cooled again to 0° C. and quenched with 10% KHSO4. The mixture was extracted 3× with EtOAc. The combined organics were dried over MgSO4, filtered, and evaporated to afford tert-butyl 4-(2-fluoro-3-hydroxy-propyl)-2,2-dimethyl-oxazolidine-3-carboxylate, which was carried forward without purification. ES/MS: m/z 300.0 [M+H+Na]+.

Step 4. Tert-butyl 4-(2-fluoro-3-hydroxy-propyl)-2,2-dimethyl-oxazolidine-3-carboxylate (941 mg, 3.22 mmol) was dissolved in DCM (24 mL) and TEA (1.1 mL, 8.1 mmol). The mixture was then cooled to 0° C. before the addition of toluene-4-sulfonyl chloride (799 mg, 4.19 mmol) and DMAP (33 mg, 0.27 mmol). The cooling bath was then removed, and the mixture was stirred for 18 h. The reaction was quenched with 10% KHSO4and extracted 3× with EtOAc. The combined organics were dried over MgSO4, filtered, and evaporated. The crude residue was purified via flash chromatography (100% hexanes+35% EtOAc) to afford tert-butyl 4-[2-fluoro-3-(p-tolylsulfonyloxy)propyl]-2,2-dimethyl-oxazolidine-3-carboxylate. ES/MS: m/z 433.2 [M+H]+.

Step 6. To a solution of tert-butyl (4R)-4-[(2R)-3-(6-bromo-7-fluoro-1-oxo-2-isoquinolyl)-2-fluoro-propyl]-2,2-dimethyl-oxazolidine-3-carboxylate (1.2 g mg, 2.31 mmol) in dichloromethane (16.5 mL) was added trifluoroacetic acid (3.5 mL) at room temperature and the mixture was stirred for 1 h. The reaction was then diluted with water and trifluoroacetic acid (3.5 mL) and stirred at room temperature for 18 h. The solvent was removed under reduced pressure and the excess solvent was removed by forming an azeotrope with toluene to afford 2-[(2R,4S)-4-amino-2-fluoro-pentyl]-6-bromo-4,7-difluoro-isoquinolin-1-one, which was used without further purification. ES/MS: m/z 361.218 [M+H]+.

Step 7. In a flask were placed 2-[(2R,4R)-4-amino-2-fluoro-5-hydroxy-pentyl]-6-bromo-7-fluoro-isoquinolin-1-one (835 mg, 2.31 mmol), 5-chloro-4-(trifluoromethyl)-2-(2-trimethylsilylethoxymethyl)pyridazin-3-one (1.41 g, 3.01 mmol), DIPEA (4.0 mL, 2.31 mmol), and DMF (16.4 mL). The mixture was heated to 60° C. for 3 h, then quenched with water and extracted with EtOAc (3X). The combined organic layers were washed with brine and dried over MgSO4, filtered, and concentrated by rotary evaporation to afford a crude oil which was purified by flash chromatography (0-100% EtOAc in hexane) to give 6-bromo-7-fluoro-2-[(2R,4R)-2-fluoro-5-hydroxy-4-[[6-oxo-5-(trifluoromethyl)-1-(2-trimethylsilylethoxymethyl)pyridazin-4-yl]amino]pentyl]isoquinolin-1-one, Intermediate 8. ES/MS: m z 655.1 [M+H]+.

The following Examples were synthesized as described in Example 123 or a modification of the procedure above from the corresponding reagents and Intermediate 8.

Example 126: Preparation of 2-[(2R)-5-(difluoromethoxy)-2-fluoro-4-[[6-oxo-5-(trifluoromethyl)-1H-pyridazin-4-yl]amino]pentyl]-7-fluoro-6-[5-(trifluoromethyl)pyrimidin-2-yl]isoquinolin-1-one

Step 1. Triethyl 2-fluoro-2-phosphonoacetate (8.7 g, 36 mmol) was dissolved in DCM (150 mL) and stirred at room temperature, followed by the addition of DBU (5.7 ml, 38.0 mL). After 50 minutes, tert-butyl (4S)-4-formyl-2,2-dimethyl-oxazolidine-3-carboxylate (5.0 g, 22 mmol) was added via an addition funnel as a solution in DCM (100 mL) over the course of 60 minutes. The reaction was stirred for an additional 3 hours, at which point it was quenched by the slow addition of pre-chilled 10% KHSO4(aq). The mixture was then extracted 3× with DCM, dried over MgSO4, and evaporated. The crude residue was purified via flash chromatography (100% hexanes→35% EtOAc) to afford tert-butyl 4-(3-ethoxy-2-fluoro-3-oxo-prop-1-enyl)-2,2-dimethyl-oxazolidine-3-carboxylate as a mixture of isomers. Note that the C—N stereocenter was racemized in this reaction. ES/MS: m/z 339.6 [M+Na]+.

Step 2. tert-butyl 4-(3-ethoxy-2-fluoro-3-oxo-prop-1-enyl)-2,2-dimethyl-oxazolidine-3-carboxylate (950 mg, 2.8 mmol) and 10% Pd/C (160 mg, 0.15 mmol) were taken up in EtOH (25.0 mL) and stirred at room temperature. The reaction headspace was evacuated/backfilled with nitrogen gas four times and then filled with hydrogen gas four times. The reaction was then stirred under a balloon of hydrogen gas for 1 hour before the catalyst was filtered off through a plug of Celite® and washed with additional EtOH. The filtrate was evaporated to provide tert-butyl 4-(3-ethoxy-2-fluoro-3-oxo-propyl)-2,2-dimethyl-oxazolidine-3-carboxylate, which was carried forward without purification. ES/MS: m/z 342.0 [M+H+Na]+.

Step 3. Tert-butyl 4-(3-ethoxy-2-fluoro-3-oxo-propyl)-2,2-dimethyl-oxazolidine-3-carboxylate (2.8 mmol) was dissolved in THF (20.0 mL) and cooled to 0° C. LiBH4(2.0 M, 5.7 mmol) was added slowly. 30 minutes after complete addition, the cooling bath was removed, and the reaction was stirred for 16 hours at room temperature. The reaction was then cooled to 0° C. and quenched with 10% KHSO4(aq). The mixture was extracted 3× with EtOAc. The combined organic layers were dried over MgSO4, filtered, and evaporated to afford tert-butyl 4-(2-fluoro-3-hydroxy-propyl)-2,2-dimethyl-oxazolidine-3-carboxylate, which was carried forward without purification. ES/MS: m/z 300.0 [M+H+Na]+.

Step 4. tert-butyl 4-(2-fluoro-3-hydroxy-propyl)-2,2-dimethyl-oxazolidine-3-carboxylate (2.8 mmol) was dissolved in DCM (20.0 mL) and TEA (0.94 mL, 6.8 mmol). The reaction was then cooled to 0° C. before the addition of toluene-4-sulfonyl chloride (670 mg, 3.5 mmol) and DMAP (33.0 mg, 0.27 mmol). The cooling bath was removed, and the reaction was stirred for 2 hours before an additional portion of TEA (0.94 mL, 6.8 mmol) was added. The reaction was stirred for 18 hours, and then was quenched with 10% KHSO4(aq) and extracted 3× with DCM. The combined organic layers were dried over MgSO4, filtered, and evaporated to afford tert-butyl 4-[2-fluoro-3-(p-tolylsulfonyloxy)propyl]-2,2-dimethyl-oxazolidine-3-carboxylate, which was carried forward without purification. ES/MS: m/z 432.0 [M+H]+.

Step 6. Tert-butyl 4-[3-(6-bromo-7-fluoro-1-oxo-2-isoquinolyl)-2-fluoro-propyl]-2,2-dimethyl-oxazolidine-3-carboxylate (810 mg, 1.5 mmol) was taken up in CAN (15 mL) and stirred at room temperature, followed by the addition of BiBr3(69 mg, 0.15 mmol). The reaction was stirred for 18 hours, followed by the addition of another portion of BiBr3(69 mg, 0.15 mmol). After 24 hours, the reaction was quenched with sat. NaHCO3(aq)and extracted 3× w/EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and evaporated to afford tert-butyl 4-[3-(6-bromo-7-fluoro-1-oxo-2-isoquinolyl)-2-fluoro-propyl]-2,2-dimethyl-oxazolidine-3-carboxylate, which was carried forward without purification. ES/MS: m/z 501.0/502.9 [M]+.

Step 7. tert-butyl N-[4-(6-bromo-7-fluoro-1-oxo-2-isoquinolyl)-3-fluoro-1-(hydroxymethyl)butyl]carbamate (1.5 mmol) was taken up in DCM (4.0 mL) and water (3.5 mL) and stirred at room temperature. Potassium acetate (1.2 g, 12 mmol) was charged followed by [bromo(difluoro)methyl]-trimethyl-silane (0.96 mL, 6.1 mmol). After stirring for 3 hours, an additional portion of [bromo(difluoro)methyl]-trimethyl-silane (0.96 mL, 6.1 mmol) was added. After an additional 1 hour, the reaction was cooled to 0° C. and quenched by slow addition of sat. NaHCO3(aq). The mixture was extracted 3× w/DCM. The combined organic layers were dried over MgSO4, filtered, and evaporated. The crude residue was purified via flash chromatography (100% hexanes→80% EtOAc) to afford tert-butyl N-[4-(6-bromo-7-fluoro-1-oxo-2-isoquinolyl)-1-(difluoromethoxymethyl)-3-fluoro-butyl]carbamate. ES/MS: m/z 511.0/512.9 [M]+.

The following Examples were synthesized as described in Example 126 or a modification of the procedure above from the corresponding reagents and Intermediate 9.

Example 132: Preparation of 7-fluoro-2-[(2R,4S)-2-fluoro-4-[[6-oxo-5-(trifluoromethyl)-1H-pyridazin-4-yl]amino]pentyl]-6-[5-(trifluoromethyl)pyrimidin-2-yl]-3,4-dihydroisoquinolin-1-one

Step 1. tert-butyl N-[(1S,3R)-3-(iodomethyl)cyclohexyl]carbamate (544.0 mg, 1.56 mmol), 6-bromo-7-fluoro-1,2,3,4-tetrahydroisoquinoline hydrochloride (499.0 mg, 1.87 mmol), and Cs2CO3(2.03 g, 6.24 mmol) were taken up in DMF (1.4 mL) and stirred at ambient temperature for 16 hours at which point the reaction was heated to 60° C. After an additional 5 hr, the reaction was deemed complete by LC/MS analysis and was cooled to ambient temperature. The mixture was then diluted with water and extracted 3× with EtOAc The combined organics were washed with brine, dried over Na2SO4, filtered, and evaporated to afford tert-butyl N-[(1S,3R)-3-[(6-bromo-7-fluoro-3,4-dihydro-1H-isoquinolin-2-yl)methyl]cyclohexyl]carbamate. ES/MS m/z: 434.3/436.4 [M]+.

Step 3. tert-butyl N-[(1S,3R)-3-[(6-bromo-7-fluoro-1-oxo-3,4-dihydroisoquinolin-2-yl)methyl]cyclohexyl]carbamate (91.0 mg, 0.193 mmol) was dissolved in DCM (2.0 mL) and the solution was stirred at room temperature, followed by the addition of TFA (0.15 mL, 1.93 mmol). After 1 hour, the mixture was evaporated to dryness in vacuo and the crude 2-[(2R,4S)-4-amino-2-fluoro-pentyl]-6-bromo-7-fluoro-3,4-dihydroisoquinolin-1-one was carried forward without purification. ES/MS m/z: 347.0/349.0 [M]+.

Step 4. 2-[(2R,4S)-4-amino-2-fluoro-pentyl]-6-bromo-7-fluoro-3,4-dihydroisoquinolin-1-one (67.0 mg, 0.193 mmol) and 5-chloro-4-(trifluoromethyl)-2-(2-trimethylsilylethoxymethyl)pyridazin-3-one (118 mg, 0.251 mmol) were dissolved in DMF (2.50 mL). DIPEA (0.34 mL, 1.93 mmol) was then added and the reaction was stirred at 60° C. for 3 hours, at which point it was cooled to room temperature, diluted w/10% aq. KHSO4, and extracted with EtOAc (3×). The combined organic layers were washed with brine, dried over MgSO4, filtered, and evaporated. The crude material was purified via flash chromatography (100% hex→100% EtOAc) to afford 6-bromo-7-fluoro-2-[(2R,4S)-2-fluoro-4-[[6-oxo-5-(trifluoromethyl)-1-(2-trimethylsilylethoxymethyl)pyridazin-4-yl]amino]pentyl]-3,4-dihydroisoquinolin-1-one, Intermediate 9. ES/MS m/z: 639.1/641.1 [M]+.

v. Biological Data

LANCE Ultra phospho-STAT1 (Tyr701) kits are designed for the detection of phosphorylated STAT1 in cell lysates using a simple, homogeneous LANCE Ultra sandwich assay (Cat. #TRF4028M). This assay is intended for assessing compound induction of endogenous levels of cellular STAT1 (phosphorylated at Tyr701) in NCI-H1373 cells. The NCI-H1373 cells are cultured in RPMI 1640 media containing 10% heat inactivated FBS, GlutaMAX, 1% Penicillin-Streptomycin. An Echo acoustic liquid handler is used to transfer 60 nanoliters of compound dilutions using the Echo Qualified, 384-well polypropylene microplate clear flat bottom source plates into a Greiner (#781080) cell culture microplate. NCI-H1373 cells are seeded into these compound-spotted culture plates at 30,000 cells/well in a 60 uL volume in growth media. The plates are incubated in a 5% CO2humidified incubator at 37° C. for 48 hours. The media is removed and the cells are processed according to the manufacturer's suggested protocol. Briefly, 20 mL of supplemented lysis buffer is added to each well and allowed to shake for 1 hour at 400 rpm. Next, 5 ul of remixed antibody solutions (vol/vol) prepared in detection buffer are added to each well and allowed to incubate at room temperature overnight. After spinning the plate down at 300 rpm for 1 min, the plate is read on an EnVision plate reader set up for Eu3+Cryptate and fluorescence emission is measured at two different wavelengths (665 nm and 620 nm). The HTRF ratio is then calculated (665 nM/620 nM) for each well to determine the amount of pSTAT1 in the cell lysate, and the data is then normalized to 10 uM (S)-5-((1-(3-oxo-3-(4-(5-(trifluoromethyl)pyrimidin-2-yl)piperazin-1-yl)propoxy)propan-2-yl)amino)-4-(trifluoromethyl)pyridazin-3(2H)-one positive and DMSO negative controls. The values were then plotted as a function of compound concentration and a 4-parameter fit was applied to derive the EC50values.