Certain chemical compositions and methods of use thereof

The present disclosure provides CDK9 inhibitors. Also provided are methods of treating a disease or a disorder comprising administering to a subject in need of treatment one of the CDK9 inhibitors disclosed herein. In some embodiments, the disease or disorder to be treated is cancer. In some embodiments, the disease or disorder is liver cancer.

BACKGROUND OF THE INVENTION

Despite advances, treatment of cancer remains relatively difficult. Systemic treatments such as chemotherapies may be toxic and have negative side effects on patients. While CDK9 inhibitors have shown promise as small molecule cancer therapeutics, their potential utility has been limited by poor targeting to cancerous tissue and resulting peripheral exposure. Accordingly, there is a need for the development of CDK9 inhibitors as small molecule cancer therapeutics with improved targeting.

SUMMARY OF THE INVENTION

Provided herein, in one aspect, is a compound of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein:

Ring A is selected from C3-6cycloalkyl, 3- to 10-membered heterocycloalkyl, C6-10aryl, and 6- to 10-membered heteroaryl;

X1is selected from N and CR11;

X2is selected from N and CR12;

X3is selected from N and CR13;

X4is selected from N and CR14;

R4, R4′, and R4″are taken together, along with the carbon atom to which they are attached, to form —C(O)R18or 6- to 10-membered heteroaryl;

R6and R7, along with the nitrogen atom to which they are attached, are taken together to form a 3- to 10-membered heterocycloalkyl optionally substituted with one or more substituents selected from oxo, halo, —OR18, C1-4alkyl, C1-4alkoxy, C1-4heteroalkyl, C1-4haloalkyl, —CN, and —NR20R21;

an R16and an R17may be taken together along with the nitrogen atom to which they are attached to form a 3- to 10-membered heterocycloalkyl;

an R20and an R21may be taken together along with the nitrogen atom to which they are attached to form a 3- to 10-membered heterocycloalkyl; and

In some embodiments, Ring A is selected from C3-6cycloalkyl and 3- to 10-membered heterocycloalkyl. In some embodiments, Ring A is C3-6cycloalkyl. In some embodiments, Ring A is selected from the group consisting of:

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

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

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

In some embodiments, one of X1, X2, X3, and X4is N. In some embodiments, none of X1, X2, X3, and X4is N.

In some embodiments, R2is Me and R3is H.

In some embodiments,

R4′and R4″are each independently selected from H, C1-6alkyl, C1-6haloalkyl, and C3-6cycloalkyl; wherein each alkyl and cycloalkyl is independently optionally substituted with one or more substituents selected from halo, —OR18, —CN, and —NR16R17; or

R4, R4′, and R4″are taken together, along with the carbon atom to which they are attached, to form —C(O)R18or 6- to 10-membered heteroaryl.

In some embodiments,

R4, R4′, and R4″are taken together, along with the carbon atom to which they are attached, to form —C(O)R18or 6- to 10-membered heteroaryl.

In some embodiments,

In some embodiments,

R4, R4′, and R4″are taken together, along with the carbon atom to which they are attached, to form —C(O)R18or 6- to 10-membered heteroaryl.

In some embodiments,

R6and R7, along with the nitrogen atom to which they are attached, are taken together to form a 3- to 10-membered heterocycloalkyl optionally substituted with one or more substituents selected from oxo, halo, —OR18, C1-4alkyl, C1-4alkoxy, C1-4heteroalkyl, C1-4haloalkyl, —CN, and —NR20R21.

In some embodiments, R6and R7are each independently selected from H and —(C1-4alkyl)(3- to 10-membered heterocycloalkyl); wherein each alkyl and heterocycloalkyl is independently optionally substituted with one or more substituents selected from halo, —OR18, —CN, and —NR20R21.

In some embodiments, one of R6and R7is H and the other is

In some embodiments, the compound of Formula (I) is represented by Formula (I-A):

In some embodiments, the compound of Formula (I) is represented by Formula (I-B):

In some embodiments, the compound of Formula (I) is represented by Formula (I-C), Formula (I-D), Formula (I-E), or Formula (I-F):

Provided herein, in another aspect, is a pharmaceutical composition comprising a compound described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

Provided herein, in another aspect, is a method of treating a disease or disorder in a patient in need thereof, comprising administering to the subject a therapeutically effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein.

In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is selected from leukemia, breast cancer, prostate cancer, ovarian cancer, colon cancer, cervical cancer, lung cancer, lymphoma, and liver cancer. In some embodiments, the cancer is liver cancer.

INCORPORATION BY REFERENCE

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, the singular form “a”, “an” and “the” includes plural references unless the context clearly dictates otherwise.

The term “Cx-y” when used in conjunction with a chemical moiety, such as alkyl, alkenyl, or alkynyl is meant to include groups that contain from x to y carbons in the chain. For example, the term “C1-6alkyl” refers to substituted or unsubstituted saturated hydrocarbon groups, including straight-chain alkyl and branched-chain alkyl groups that contain from 1 to 6 carbons. The term —Cx-yalkylene-refers to a substituted or unsubstituted alkylene chain with from x to y carbons in the alkylene chain. For example —C1-6alkylene-may be selected from methylene, ethylene, propylene, butylene, pentylene, and hexylene, any one of which is optionally substituted.

“Alkyl” refers to substituted or unsubstituted saturated hydrocarbon groups, including straight-chain alkyl and branched-chain alkyl groups. An alkyl group may contain from one to twelve carbon atoms (e.g., C1-12alkyl), such as one to eight carbon atoms (C1-8alkyl) or one to six carbon atoms (C1-6alkyl). Exemplary alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, septyl, octyl, nonyl, and decyl. An alkyl group is attached to the rest of the molecule by a single bond. Unless stated otherwise specifically in the specification, an alkyl group is optionally substituted by one or more substituents such as those substituents described herein.

“Haloalkyl” refers to an alkyl group that is substituted by one or more halogens. Exemplary haloalkyl groups include trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl, and 1,2-dibromoethyl.

“Alkenyl” refers to substituted or unsubstituted hydrocarbon groups, including straight-chain or branched-chain alkenyl groups containing at least one double bond. An alkenyl group may contain from two to twelve carbon atoms (e.g., C2-12alkenyl). Exemplary alkenyl groups include ethenyl (i.e., vinyl), prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1,4-dienyl, and the like. Unless stated otherwise specifically in the specification, an alkenyl group is optionally substituted by one or more substituents such as those substituents described herein.

“Alkynyl” refers to substituted or unsubstituted hydrocarbon groups, including straight-chain or branched-chain alkynyl groups containing at least one triple bond. An alkynyl group may contain from two to twelve carbon atoms (e.g., C2-12alkynyl). Exemplary alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. Unless stated otherwise specifically in the specification, an alkynyl group is optionally substituted by one or more substituents such as those substituents described herein.

“Heteroalkyl”, “heteroalkenyl” and “heteroalkynyl” refer to substituted or unsubstituted alkyl, alkenyl and alkynyl groups which respectively have one or more skeletal chain atoms selected from an atom other than carbon. Exemplary skeletal chain atoms selected from an atom other than carbon include, e.g., O, N, P, Si, S, or combinations thereof, wherein the nitrogen, phosphorus, and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. If given, a numerical range refers to the chain length in total. For example, a 3- to 8-membered heteroalkyl has a chain length of 3 to 8 atoms. Connection to the rest of the molecule may be through either a heteroatom or a carbon in the heteroalkyl, heteroalkenyl or heteroalkynyl chain. Unless stated otherwise specifically in the specification, a heteroalkyl, heteroalkenyl, or heteroalkynyl group is optionally substituted by one or more substituents such as those substituents described herein.

“Aryl” refers to an aromatic ring wherein each of the atoms forming the ring is a carbon atom. Aryl groups can be optionally substituted. Examples of aryl groups include, but are not limited to, phenyl and naphthyl. In some embodiments, the aryl is phenyl. Depending on the structure, an aryl group can be a monoradical or a diradical (i.e., an arylene group). Unless stated otherwise specifically in the specification, the term “aryl” or the prefix “ar-” (such as in “aralkyl”) is meant to include aryl radicals that are optionally substituted.

The term “cycloalkyl” refers to a monocyclic or polycyclic non-aromatic radical, wherein each of the atoms forming the ring (i.e. skeletal atoms) is a carbon atom. In some embodiments, cycloalkyls are saturated or partially unsaturated. In some embodiments, cycloalkyls are spirocyclic or bridged compounds. In some embodiments, cycloalkyls are fused with an aromatic ring (in which case the cycloalkyl is bonded through a non-aromatic ring carbon atom). Cycloalkyl groups include groups having from 3 to 10 ring atoms. Representative cycloalkyls include, but are not limited to, cycloalkyls having from three to ten carbon atoms, from three to eight carbon atoms, from three to six carbon atoms, or from three to five carbon atoms. Monocyclic cycloalkyl radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic radicals include, for example, adamantyl, 1,2-dihydronaphthalenyl, 1,4-dihydronaphthalenyl, tetrainyl, decalinyl, 3,4-dihydronaphthalenyl-1(2H)-one, spiro[2.2]pentyl, norbornyl and bicycle[1.1.1]pentyl. Unless otherwise stated specifically in the specification, a cycloalkyl group may be optionally substituted.

The term “heterocycloalkyl” refers to a cycloalkyl group that includes at least one heteroatom selected from nitrogen, oxygen, and sulfur. Unless stated otherwise specifically in the specification, the heterocycloalkyl radical may be a monocyclic, or bicyclic ring system, which may include fused (when fused with an aryl or a heteroaryl ring, the heterocycloalkyl is bonded through a non-aromatic ring atom) or bridged ring systems. The nitrogen, carbon or sulfur atoms in the heterocyclyl radical may be optionally oxidized. The nitrogen atom may be optionally quaternized. The heterocycloalkyl radical may be partially or fully saturated. Examples of heterocycloalkyl radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, 1,1-dioxo-thiomorpholinyl. The term heterocycloalkyl also includes all ring forms of carbohydrates, including but not limited to monosaccharides, disaccharides and oligosaccharides. Unless otherwise noted, heterocycloalkyls have from 2 to 12 carbons in the ring. It is understood that when referring to the number of carbon atoms in a heterocycloalkyl, the number of carbon atoms in the heterocycloalkyl is not the same as the total number of atoms (including the heteroatoms) that make up the heterocycloalkyl (i.e. skeletal atoms of the heterocycloalkyl ring). Unless stated otherwise specifically in the specification, a heterocycloalkyl group may be optionally substituted.

It will be understood by those skilled in the art that substituents can themselves be substituted, if appropriate. Unless specifically stated as “unsubstituted,” references to chemical moieties herein are understood to include substituted variants. For example, reference to a “heteroaryl” group or moiety implicitly includes both substituted and unsubstituted variants.

“Optional” or “optionally” means that the subsequently described event of circumstances may or may not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. For example, “optionally substituted aryl” means that the aryl group may or may not be substituted and that the description includes both substituted aryl groups and aryl groups having no substitution.

Compounds of the present disclosure also include crystalline and amorphous forms of those compounds, pharmaceutically acceptable salts, and active metabolites of these compounds having the same type of activity, including, for example, polymorphs, pseudopolymorphs, solvates, hydrates, unsolvated polymorphs (including anhydrates), conformational polymorphs, and amorphous forms of the compounds, as well as mixtures thereof.

The compounds described herein may exhibit their natural isotopic abundance, or one or more of the atoms may be artificially enriched in a particular isotope having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature. All isotopic variations of the compounds of the present disclosure, whether radioactive or not, are encompassed within the scope of the present disclosure. For example, hydrogen has three naturally occurring isotopes, denoted1H (protium),2H (deuterium), and3H (tritium). Protium is the most abundant isotope of hydrogen in nature. Enriching for deuterium may afford certain therapeutic advantages, such as increased in vivo half-life and/or exposure, or may provide a compound useful for investigating in vivo routes of drug elimination and metabolism. Isotopically-enriched compounds may be prepared by conventional techniques well known to those skilled in the art.

“Isomers” are different compounds that have the same molecular formula. “Stereoisomers” are isomers that differ only in the way the atoms are arranged in space. “Enantiomers” are a pair of stereoisomers that are non superimposable mirror images of each other. A 1:1 mixture of a pair of enantiomers is a “racemic” mixture. The term “(±)” is used to designate a racemic mixture where appropriate. “Diastereoisomers” or “diastereomers” are stereoisomers that have at least two asymmetric atoms but are not mirror images of each other. The absolute stereochemistry is specified according to the Cahn-Ingold-Prelog R-S system. When a compound is a pure enantiomer, the stereochemistry at each chiral carbon can be specified by either R or S. Resolved compounds whose absolute configuration is unknown can be designated (+) or (−) depending on the direction (dextro- or levorotatory) in which they rotate plane polarized light at the wavelength of the sodium D line. Certain compounds described herein contain one or more asymmetric centers and can thus give rise to enantiomers, diastereomers, and other stereoisomeric forms, the asymmetric centers of which can be defined, in terms of absolute stereochemistry, as (R)- or (S)-. The present chemical entities, pharmaceutical compositions and methods are meant to include all such possible stereoisomers, including racemic mixtures, optically pure forms, mixtures of diastereomers and intermediate mixtures. Optically active (R)- and (S)-isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. The optical activity of a compound can be analyzed via any suitable method, including but not limited to chiral chromatography and polarimetry, and the degree of predominance of one stereoisomer over the other isomer can be determined.

Chemical entities having carbon-carbon double bonds or carbon-nitrogen double bonds may exist in Z- or E-form (or cis- or trans-form). Furthermore, some chemical entities may exist in various tautomeric forms. Unless otherwise specified, chemical entities described herein are intended to include all Z-, E- and tautomeric forms as well.

Isolation and purification of the chemical entities and intermediates described herein can be effected, if desired, by any suitable separation or purification procedure such as, for example, filtration, extraction, crystallization, column chromatography, thin-layer chromatography or thick-layer chromatography, or a combination of these procedures. Specific illustrations of suitable separation and isolation procedures can be had by reference to the examples herein below. However, other equivalent separation or isolation procedures can also be used.

When stereochemistry is not specified, certain small molecules described herein include, but are not limited to, when possible, their isomers, such as enantiomers and diastereomers, mixtures of enantiomers, including racemates, mixtures of diastereomers, and other mixtures thereof, to the extent they can be made by one of ordinary skill in the art by routine experimentation. In those situations, the single enantiomers or diastereomers, i.e., optically active forms, can be obtained by asymmetric synthesis or by resolution of the racemates or mixtures of diastereomers. Resolution of the racemates or mixtures of diastereomers, if possible, can be accomplished, for example, by conventional methods such as crystallization in the presence of a resolving agent, or chromatography, using, for example, a chiral high-pressure liquid chromatography (HPLC) column. Furthermore, a mixture of two enantiomers enriched in one of the two can be purified to provide further optically enriched form of the major enantiomer by recrystallization and/or trituration. In addition, such certain small molecules include Z- and E-forms (or cis- and trans-forms) of certain small molecules with carbon-carbon double bonds or carbon-nitrogen double bonds. Where certain small molecules described herein exist in various tautomeric forms, the term “certain small molecule” is intended to include all tautomeric forms of the certain small molecule.

The term “salt” or “pharmaceutically acceptable salt” refers to salts derived from a variety of organic and inorganic counter ions well known in the art. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, specifically such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In some embodiments, the pharmaceutically acceptable base addition salt is chosen from ammonium, potassium, sodium, calcium, and magnesium salts.

The term “effective amount” or “therapeutically effective amount” refers to that amount of a compound described herein that is sufficient to affect the intended application, including but not limited to disease treatment, as defined below. The therapeutically effective amount may vary depending upon the intended treatment application (in vivo), or the subject and disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will induce a particular response in target cells, e.g., reduction of platelet adhesion and/or cell migration. The specific dose will vary depending on the particular compounds chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.

As used herein, “treatment” or “treating” refers to an approach for obtaining beneficial or desired results with respect to a disease, disorder, or medical condition including but not limited to a therapeutic benefit and/or a prophylactic benefit. A therapeutic benefit can include, for example, the eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit can include, for example, the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. In certain embodiments, for prophylactic benefit, the compositions are administered to a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.

A “therapeutic effect,” as that term is used herein, encompasses a therapeutic benefit and/or a prophylactic benefit as described above. A prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.

The term “co-administration,” “administered in combination with,” and their grammatical equivalents, as used herein, encompass administration of two or more agents to an animal, including humans, so that both agents and/or their metabolites are present in the subject at the same time. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which both agents are present.

The terms “antagonist” and “inhibitor” are used interchangeably, and they refer to a compound having the ability to inhibit a biological function (e.g., activity, expression, binding, protein-protein interaction) of a target protein or enzyme. Accordingly, the terms “antagonist” and “inhibitor” are defined in the context of the biological role of the target protein. While preferred antagonists herein specifically interact with (e.g., bind to) the target, compounds that inhibit a biological activity of the target protein by interacting with other members of the signal transduction pathway of which the target protein is a member are also specifically included within this definition. A preferred biological activity inhibited by an antagonist is associated with the development, growth, or spread of a tumor.

Compounds

Provided herein, in one aspect, is a compound of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein:

Ring A is selected from C3-6cycloalkyl, 3- to 10-membered heterocycloalkyl, C6-10aryl, and 6- to 10-membered heteroaryl;

X1is selected from N and CR11;

X2is selected from N and CR12;

X3is selected from N and CR13;

X4is selected from N and CR14;

R4, R4′, and R4″are taken together, along with the carbon atom to which they are attached, to form —C(O)R18or 6- to 10-membered heteroaryl;

R6and R7, along with the nitrogen atom to which they are attached, are taken together to form a 3- to 10-membered heterocycloalkyl optionally substituted with one or more substituents selected from oxo, halo, —OR18, C1-4alkyl, C1-4alkoxy, C1-4heteroalkyl, C1-4haloalkyl, —CN, and —NR20R21;

an R16and an R17may be taken together along with the nitrogen atom to which they are attached to form a 3- to 10-membered heterocycloalkyl;

an R20and an R21may be taken together along with the nitrogen atom to which they are attached to form a 3- to 10-membered heterocycloalkyl; and

In some embodiments, Ring A is selected from C3-6cycloalkyl, 3- to 10-membered heterocycloalkyl, C6-10aryl, and 6- to 10-membered heteroaryl. In some embodiments, Ring A is selected from C3-6cycloalkyl, 3- to 10-membered heterocycloalkyl, and C6-10aryl. In some embodiments, Ring A is selected from C3-6cycloalkyl and 3- to 10-membered heterocycloalkyl. In some embodiments, Ring A is 3- to 10-membered heterocycloalkyl. In some embodiments, Ring A is C6-10aryl. In some embodiments, Ring A is 6- to 10-membered heteroaryl. In some embodiments, Ring A is C3-6cycloalkyl. In some embodiments, Ring A is selected from the group consisting of:

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is selected from the group consisting of

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

In some embodiments, Ring A is selected from the group consisting of

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, R2is Me and R3is H.

In some embodiments,

R4′and R4″are each independently selected from H, C1-6alkyl, C1-6haloalkyl, and C3-6cycloalkyl; wherein each alkyl and cycloalkyl is independently optionally substituted with one or more substituents selected from halo, —OR18, —CN, and —NR16R17; or

R4, R4′, and R4″are taken together, along with the carbon atom to which they are attached, to form —C(O)R18or 6- to 10-membered heteroaryl.

In some embodiments,

R4, R4′, and R4″are taken together, along with the carbon atom to which they are attached, to form —C(O)R18or 6- to 10-membered heteroaryl.

In some embodiments,

In some embodiments,R3is H; andR4, R4′, and R4″are taken together, along with the carbon atom to which they are attached, to form —C(O)R18or 6- to 10-membered heteroaryl.

In some embodiments,R3is H; andR4, R4′, and R4″are taken together, along with the carbon atom to which they are attached, to form —C(O)R18or 6- to 10-membered heteroaryl.

In some embodiments,R3is H; andR4, R4′, and R4″are taken together, along with the carbon atom to which they are attached, to form 6- to 10-membered heteroaryl.

In some embodiments,

R6and R7, along with the nitrogen atom to which they are attached, are taken together to form a 3- to 10-membered heterocycloalkyl optionally substituted with one or more substituents selected from oxo, halo, —OR18, C1-4alkyl, C1-4alkoxy, C1-4heteroalkyl, C1-4haloalkyl, —CN, and —NR20R21.

In some embodiments, R6and R7are each independently selected from H and —(C1-4alkyl)(3- to 10-membered heterocycloalkyl); wherein each alkyl and heterocycloalkyl is independently optionally substituted with one or more substituents selected from halo, —OR18, —CN, and —NR20R21.

In some embodiments, one of R6and R7is H and the other is

In some embodiments, R6is H and R7is

In some embodiments, R7is H and R6is

In some embodiments, n is 0, 1, 2, 3, or 4. In some embodiments, n is 0, 1, 2, or 3. In some embodiments, n is 0, 1, or 2. In some embodiments, n is 0 or 1. In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4.

In some embodiments, the compound of Formula (I) is represented by Formula (I-A):

In some embodiments, the compound of Formula (I) is represented by Formula (I-B):

In some embodiments, the compound of Formula (I) is represented by Formula (I-C), Formula (I-D), Formula (I-E), or Formula (I-F):

In some embodiments, the compound of Formula (I) is represented by Formula (I-C):

In some embodiments, the compound of Formula (I) is represented by Formula (I-D):

In some embodiments, the compound of Formula (I) is represented by Formula (I-E):

In some embodiments, the compound of Formula (I) is represented by Formula (I-F):

In some embodiments, the compound is

In some embodiments, the compound is

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In some embodiments, the compound is

Provided herein, in another aspect, is a pharmaceutical composition comprising a compound described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

Provided herein, in another aspect, is a method of treating a disease or disorder in a patient in need thereof, comprising administering to the subject a therapeutically effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein.

In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is selected from leukemia, breast cancer, prostate cancer, ovarian cancer, colon cancer, cervical cancer, lung cancer, lymphoma, and liver cancer. In some embodiments, the cancer is leukemia. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is cervical cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is lymphoma. In some embodiments, the cancer is liver cancer.

Methods of Use

In one aspect, the present invention provides a method for treating a proliferative disorder in a subject in need thereof, comprising administering to said subject a compound having Formula (I), as disclosed further herein. In some embodiments, the method for treating the proliferative disorder comprises administering to said subject a CDK9 inhibitor. In some embodiments, the compound having Formula (I) is the CDK9 inhibitor. In some embodiments, the proliferative disorder is a cancer condition. In some further embodiments, said cancer condition is a cancer selected from the group consisting of leukemia, breast cancer, prostate cancer, ovarian cancer, colon cancer, cervical cancer, lung cancer, lymphoma, and liver cancer. In some embodiments, said cancer condition is liver cancer.

In some embodiments, the CDK9 inhibitors disclosed herein are highly targeted to the liver. In some embodiments, the CDK9 inhibitors disclosed herein have superior liver targeting as compared with known CDK9 inhibitors. In some embodiments, the CDK9 inhibitors disclosed herein accumulate in the liver while avoiding peripheral exposure to nearby tissues. In some embodiments, the CDK9 inhibitors disclosed herein have reduced peripheral exposure to nearby tissues as compared with known CDK9 inhibitors. In some embodiments, the CDK9 inhibitors disclosed herein have reduced toxicity as compared with known CDK9 inhibitors.

In a further embodiment, the present invention provides a method of treating a cancer condition, wherein the compound having Formula (I) (e.g., a CDK9 inhibitor) is effective in one or more method of inhibiting proliferation of cancer cells, inhibiting metastasis of cancer cells, reducing severity or incidence of symptoms associated with the presence of cancer cells, and promoting an immune response to tumor cells. In some embodiments, said method comprises administering to the cancer cells a therapeutically effective amount of a compound having Formula (I). In some embodiments, the compound having Formula (I) is a CDK9 inhibitor. In some embodiments, the administration takes place in vitro. In other embodiments, the administration takes place in vivo.

As used herein, a therapeutically effective amount of a CDK9 inhibitor refers to an amount sufficient to effect the intended application, including but not limited to, disease treatment, as defined herein. Also contemplated in the subject methods is the use of a sub-therapeutic amount of a CDK9 inhibitor for treating an intended disease condition.

The amount of the CDK9 inhibitor administered may vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.

Measuring inhibition of biological effects of CDK9 can comprise performing an assay on a biological sample, such as a sample from a subject. Any of a variety of samples may be selected, depending on the assay. Examples of samples include, but are not limited to, blood samples (e.g. blood plasma or serum), exhaled breath condensate samples, bronchoalveolar lavage fluid, sputum samples, urine samples, and tissue samples.

A subject being treated with a CDK9 inhibitor may be monitored to determine the effectiveness of treatment, and the treatment regimen may be adjusted based on the subject's physiological response to treatment. For example, if inhibition of a biological effect of CDK9 degradation is above or below a threshold, the dosing amount or frequency may be decreased or increased, respectively. The methods can further comprise continuing the therapy if the therapy is determined to be efficacious. The methods can comprise maintaining, tapering, reducing, or stopping the administered amount of a compound in the therapy if the therapy is determined to be efficacious. The methods can comprise increasing the administered amount of a compound in the therapy if it is determined not to be efficacious. Alternatively, the methods can comprise stopping therapy if it is determined not to be efficacious. In some embodiments, treatment with a CDK9 inhibitor is discontinued if inhibition of the biological effect is above or below a threshold, such as in a lack of response or an adverse reaction. The biological effect may be a change in any of a variety of physiological indicators.

In general, a CDK9 inhibitor is a compound that inhibits one or more biological effects of CDK9. Such biological effects may be inhibited by about or more than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.

In some other embodiments, the subject methods are useful for treating a disease condition associated with CDK9. Any disease condition that results directly or indirectly from an abnormal activity or expression level of CDK9 can be an intended disease condition. In some embodiments, the disease condition is a proliferative disorder, such as described herein, including but not limited to cancer. In some embodiments, the disease condition is cancer. A role of CDK9 in tumorigenesis and tumor progression has been implicated in many human cancers. Consequently, agents that target CDK9 have therapeutic value.

The data presented in the Examples herein below demonstrate the anti-cancer effects of a CDK9 inhibitor. As such, the subject method is particularly useful for treating a proliferative disorder, such as a neoplastic condition.

In some embodiments, the methods of administering a CDK9 inhibitor described herein are applied to the treatment of cancers of the blood, breast, prostate, ovaries, colon, cervix, lungs, lymph nodes, liver, or any combination thereof.

Therapeutic Efficacy

In some embodiments, therapeutic efficacy is measured based on an effect of treating a proliferative disorder, such as cancer. In general, therapeutic efficacy of the methods and compositions of the invention, with regard to the treatment of a proliferative disorder (e.g. cancer, whether benign or malignant), may be measured by the degree to which the methods and compositions promote inhibition of tumor cell proliferation, the inhibition of tumor vascularization, the eradication of tumor cells, the reduction in the rate of growth of a tumor, and/or a reduction in the size of at least one tumor. Several parameters to be considered in the determination of therapeutic efficacy are discussed herein. The proper combination of parameters for a particular situation can be established by the clinician. The progress of the inventive method in treating cancer (e.g., reducing tumor size or eradicating cancerous cells) can be ascertained using any suitable method, such as those methods currently used in the clinic to track tumor size and cancer progress. The primary efficacy parameter used to evaluate the treatment of cancer by the inventive method and compositions preferably is a reduction in the size of a tumor. Tumor size can be figured using any suitable technique, such as measurement of dimensions, or estimation of tumor volume using available computer software, such as FreeFlight software developed at Wake Forest University that enables accurate estimation of tumor volume. Tumor size can be determined by tumor visualization using, for example, CT, ultrasound, SPECT, spiral CT, MRI, photographs, and the like. In embodiments where a tumor is surgically resected after completion of the therapeutic period, the presence of tumor tissue and tumor size can be determined by gross analysis of the tissue to be resected, and/or by pathological analysis of the resected tissue.

In some desirable embodiments, the growth of a tumor is stabilized (i.e., one or more tumors do not increase more than 1%, 5%, 10%, 15%, or 20% in size, and/or do not metastasize) as a result of the inventive method and compositions. In some embodiments, a tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks. In some embodiments, a tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months. In some embodiments, a tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years. Preferably, the inventive method reduces the size of a tumor at least about 5% (e.g., at least about 10%, 15%, 20%, or 25%). More preferably, tumor size is reduced at least about 30% (e.g., at least about 35%, 40%, 45%, 50%, 55%, 60%, or 65%). Even more preferably, tumor size is reduced at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, or 95%). Most preferably, the tumor is completely eliminated, or reduced below a level of detection. In some embodiments, a subject remains tumor free (e.g. in remission) for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks following treatment. In some embodiments, a subject remains tumor free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months following treatment. In some embodiments, a subject remains tumor free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years after treatment.

In some embodiments, the efficacy of the inventive method in reducing tumor size can be determined by measuring the percentage of necrotic (i.e., dead) tissue of a surgically resected tumor following completion of the therapeutic period. In some further embodiments, a treatment is therapeutically effective if the necrosis percentage of the resected tissue is greater than about 20% (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%), more preferably about 90% or greater (e.g., about 90%, 95%, or 100%). Most preferably, the necrosis percentage of the resected tissue is 100%, that is, no tumor tissue is present or detectable.

The efficacy of the inventive method can be determined by a number of secondary parameters. Examples of secondary parameters include, but are not limited to, detection of new tumors, detection of tumor antigens or markers (e.g., CEA, PSA, or CA-125), biopsy, surgical downstaging (i.e., conversion of the surgical stage of a tumor from unresectable to resectable), PET scans, survival, disease progression-free survival, time to disease progression, quality of life assessments such as the Clinical Benefit Response Assessment, and the like, all of which can point to the overall progression (or regression) of cancer in a human. Biopsy is particularly useful in detecting the eradication of cancerous cells within a tissue. Radioimmunodetection (RAID) is used to locate and stage tumors using serum levels of markers (antigens) produced by and/or associated with tumors (“tumor markers” or “tumor-associated antigens”), and can be useful as a pre-treatment diagnostic predicate, a post-treatment diagnostic indicator of recurrence, and a post-treatment indicator of therapeutic efficacy. Examples of tumor markers or tumor-associated antigens that can be evaluated as indicators of therapeutic efficacy include, but are not limited to, carcinembryonic antigen (CEA), prostate-specific antigen (PSA), CA-125, CA19-9, ganglioside molecules (e.g., GM2, GD2, and GD3), MART-1, heat shock proteins (e.g., gp96), sialyl Tn (STn), tyrosinase, MUC-1, HER-2/neu, c-erb-B2, KSA, PSMA, p53, RAS, EGF-R, VEGF, MAGE, and gp100. Other tumor-associated antigens are known in the art. RAID technology in combination with endoscopic detection systems also can efficiently distinguish small tumors from surrounding tissue (see, for example, U.S. Pat. No. 4,932,412).

In additional desirable embodiments, the treatment of cancer in a human patient in accordance with the inventive method is evidenced by one or more of the following results: (a) the complete disappearance of a tumor (i.e., a complete response), (b) about a 25% to about a 50% reduction in the size of a tumor for at least four weeks after completion of the therapeutic period as compared to the size of the tumor before treatment, (c) at least about a 50% reduction in the size of a tumor for at least four weeks after completion of the therapeutic period as compared to the size of the tumor before the therapeutic period, and (d) at least a 2% decrease (e.g., about a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% decrease) in a specific tumor-associated antigen level at about 4-12 weeks after completion of the therapeutic period as compared to the tumor-associated antigen level before the therapeutic period. While at least a 2% decrease in a tumor-associated antigen level is preferred, any decrease in the tumor-associated antigen level is evidence of treatment of a cancer in a patient by the inventive method. For example, with respect to unresectable, locally advanced pancreatic cancer, treatment can be evidenced by at least a 10% decrease in the CA19-9 tumor-associated antigen level at 4-12 weeks after completion of the therapeutic period as compared to the CA19-9 level before the therapeutic period. Similarly, with respect to locally advanced rectal cancer, treatment can be evidenced by at least a 10% decrease in the CEA tumor-associated antigen level at 4-12 weeks after completion of the therapeutic period as compared to the CEA level before the therapeutic period.

With respect to quality of life assessments, such as the Clinical Benefit Response Criteria, the therapeutic benefit of the treatment in accordance with the invention can be evidenced in terms of pain intensity, analgesic consumption, and/or the Karnofsky Performance Scale score. The treatment of cancer in a human patient alternatively, or in addition, is evidenced by (a) at least a 50% decrease (e.g., at least a 60%, 70%, 80%, 90%, or 100% decrease) in pain intensity reported by a patient, such as for any consecutive four week period in the 12 weeks after completion of treatment, as compared to the pain intensity reported by the patient before treatment, (b) at least a 50% decrease (e.g., at least a 60%, 70%, 80%, 90%, or 100% decrease) in analgesic consumption reported by a patient, such as for any consecutive four week period in the 12 weeks after completion of treatment as compared to the analgesic consumption reported by the patient before treatment, and/or (c) at least a 20 point increase (e.g., at least a 30 point, 50 point, 70 point, or 90 point increase) in the Karnofsky Performance Scale score reported by a patient, such as for any consecutive four week period in the 12 weeks after completion of the therapeutic period as compared to the Karnofsky Performance Scale score reported by the patient before the therapeutic period.

The treatment of a proliferative disorder (e.g. cancer, whether benign or malignant) in a human patient desirably is evidenced by one or more (in any combination) of the foregoing results, although alternative or additional results of the referenced tests and/or other tests can evidence treatment efficacy.

In some embodiments, tumor size is reduced as a result of the inventive method preferably without significant adverse events in the subject. Adverse events are categorized or “graded” by the Cancer Therapy Evaluation Program (CTEP) of the National Cancer Institute (NCI), with Grade 0 representing minimal adverse side effects and Grade 4 representing the most severe adverse events. Desirably, the inventive method is associated with minimal adverse events, e.g. Grade 0, Grade 1, or Grade 2 adverse events, as graded by the CTEP/NCI. However, as discussed herein, reduction of tumor size, although preferred, is not required in that the actual size of tumor may not shrink despite the eradication of tumor cells. Eradication of cancerous cells is sufficient to realize a therapeutic effect. Likewise, any reduction in tumor size is sufficient to realize a therapeutic effect.

Detection, monitoring and rating of various cancers in a human are further described in Cancer Facts and Figures 2001, American Cancer Society, New York, N.Y., and International Patent Application WO 01/24684. Accordingly, a clinician can use standard tests to determine the efficacy of the various embodiments of the inventive method in treating cancer. However, in addition to tumor size and spread, the clinician also may consider quality of life and survival of the patient in evaluating efficacy of treatment.

In some embodiments, administration of a CDK9 inhibitor provides improved therapeutic efficacy. Improved efficacy may be measured using any method known in the art, including but not limited to those described herein. In some embodiments, the improved therapeutic efficacy is an improvement of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 100%, 110%, 120%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 1000% or more, using an appropriate measure (e.g. tumor size reduction, duration of tumor size stability, duration of time free from metastatic events, duration of disease-free survival). Improved efficacy may also be expressed as fold improvement, such as at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 1000-fold, 10000-fold or more, using an appropriate measure (e.g. tumor size reduction, duration of tumor size stability, duration of time free from metastatic events, duration of disease-free survival).

Pharmaceutical Compositions

A composition of the present disclosure may be formulated in any suitable pharmaceutical formulation. A pharmaceutical composition of the present disclosure typically contains an active ingredient (e.g., a compound of Formula (I), or a pharmaceutically acceptable salt and/or coordination complex thereof), and one or more pharmaceutically acceptable excipients or carriers, including but not limited to: inert solid diluents and fillers, diluents, sterile aqueous solution and various organic solvents, permeation enhancers, solubilizers, and adjuvants. A composition of the present disclosure may be formulated in any suitable pharmaceutical formulation. In some embodiments, the pharmaceutical acceptable carriers or excipients are selected from water, alcohol, glycerol, chitosan, alginate, chondroitin, Vitamin E, mineral oil, and dimethyl sulfoxide (DMSO).

Pharmaceutical formulations may be provided in any suitable form, which may depend on the route of administration. In some embodiments, the pharmaceutical composition disclosed herein can be formulated in dosage form for administration to a subject. In some embodiments, the pharmaceutical composition is formulated for oral, intravenous, intraarterial, aerosol, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, intranasal, intrapulmonary, transmucosal, inhalation, and/or intraperitoneal administration. In some embodiments, the dosage form is formulated for oral administration. For example, the pharmaceutical composition can be formulated in the form of a pill, a tablet, a capsule, an inhaler, a liquid suspension, a liquid emulsion, a gel, or a powder. In some embodiments, the pharmaceutical composition can be formulated as a unit dosage in liquid, gel, semi-liquid, semi-solid, or solid form.

The amount of each compound administered will be dependent on the mammal being treated, the severity of the disorder or condition, the rate of administration, the disposition of the compound and the discretion of the prescribing physician. However, an effective dosage may be in the range of about 0.001 to about 100 mg per kg body weight per day, in single or divided doses. In some instances, dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect, e.g., by dividing such larger doses into several small doses for administration throughout the day. In some embodiments, an effective dosage may be provided in pulsed dosing (i.e., administration of the compound in consecutive days, followed by consecutive days of rest from administration).

In some embodiments, the composition is provided in one or more unit doses. For example, the composition can be administered in 1, 2, 3, 4, 5, 6, 7, 14, 30, 60, or more doses. Such amount can be administered each day, for example in individual doses administered once, twice, or three or more times a day. However, dosages stated herein on a per day basis should not be construed to require administration of the daily dose each and every day. For example, if one of the agents is provided in a suitably slow-release form, two or more daily dosage amounts can be administered at a lower frequency, e.g., as a depot injection or oral prodrug administered every second day to once a month or even longer. Most typically and conveniently for the subject, a CDK9 inhibitor can be administered once a day, for example in the morning, in the evening or during the day.

The unit doses can be administered simultaneously or sequentially. The composition can be administered for an extended treatment period. Illustratively, the treatment period can be at least about one month, for example at least about 3 months, at least about 6 months or at least about 1 year. In some cases, administration can continue for substantially the remainder of the life of the subject.

In some embodiments, the CDK9 inhibitor can be administered as part of a therapeutic regimen that comprises administering one or more second agents (e.g. 1, 2, 3, 4, 5, or more second agents), either simultaneously or sequentially with the CDK9 inhibitor. When administered sequentially, the CDK9 inhibitor may be administered before or after the one or more second agents. When administered simultaneously, the CDK9 inhibitor and the one or more second agents may be administered by the same route (e.g. injections to the same location; tablets taken orally at the same time), by a different route (e.g. a tablet taken orally while receiving an intravenous infusion), or as part of the same combination (e.g. a solution comprising a CDK9 inhibitor and one or more second agents).

A combination treatment according to the invention may be effective over a wide dosage range. For example, in the treatment of adult humans, dosages from 0.01 to 1000 mg, from 0.5 to 100 mg, from 1 to 50 mg per day, and from 5 to 40 mg per day are examples of dosages that may be used. The exact dosage will depend upon the agent selected, the route of administration, the form in which the compound is administered, the subject to be treated, the body weight of the subject to be treated, and the preference and experience of the attending physician.

Pharmaceutical Composition for Oral Administration

In some embodiments, the disclosure provides a pharmaceutical composition for oral administration containing at least one compound of the present disclosure and a pharmaceutical excipient suitable for oral administration. The composition may be in the form of a solid, liquid, gel, semi-liquid, or semi-solid. In some embodiments, the composition further comprises a second agent.

In some embodiments, the invention provides a solid pharmaceutical composition for oral administration containing: (i) a CDK9 inhibitor; and (ii) a pharmaceutical excipient suitable for oral administration. In some embodiments, the composition further contains: (iii) a third agent or even a fourth agent. In some embodiments, each compound or agent is present in a therapeutically effective amount. In other embodiments, one or more compounds or agents is present in a sub-therapeutic amount, and the compounds or agents act synergistically to provide a therapeutically effective pharmaceutical composition.

Pharmaceutical compositions of the disclosure suitable for oral administration can be presented as discrete dosage forms, such as hard or soft capsules, cachets, troches, lozenges, or tablets, or liquids or aerosol sprays each containing a predetermined amount of an active ingredient as a powder or in granules, a solution, or a suspension in an aqueous or non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil liquid emulsion, or dispersible powders or granules, or syrups or elixirs. Such dosage forms can be prepared by any of the methods of pharmacy, which typically include the step of bringing the active ingredient(s) into association with the carrier. In general, the composition are prepared by uniformly and intimately admixing the active ingredient(s) with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation. For example, a tablet can be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared by compressing in a suitable machine the active ingredient(s) in a free-flowing form such as powder or granules, optionally mixed with an excipient such as, but not limited to, a binder, a lubricant, an inert diluent, and/or a surface active or dispersing agent. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

This disclosure further encompasses anhydrous pharmaceutical composition and dosage forms comprising an active ingredient, since water can facilitate the degradation of some compounds. For example, water may be added (e.g., 5%) in the pharmaceutical arts as a means of simulating long-term storage in order to determine characteristics such as shelf-life or the stability of formulations over time. Anhydrous pharmaceutical compositions and dosage forms of the disclosure can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. Pharmaceutical compositions and dosage forms of the disclosure which contain lactose can be made anhydrous if substantial contact with moisture and/or humidity during manufacturing, packaging, and/or storage is expected. An anhydrous pharmaceutical composition may be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions may be packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastic or the like, unit dose containers, blister packs, and strip packs.

An active ingredient can be combined in an intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier can take a wide variety of forms depending on the form of preparation desired for administration. In preparing the composition for an oral dosage form, any of the usual pharmaceutical media can be employed as carriers, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like in the case of oral liquid preparations (such as suspensions, solutions, and elixirs) or aerosols; or carriers such as starches, sugars, micro-crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents can be used in the case of oral solid preparations, in some embodiments without employing the use of lactose. For example, suitable carriers include powders, capsules, and tablets, with the solid oral preparations. If desired, tablets can be coated by standard aqueous or nonaqueous techniques.

Examples of suitable fillers for use in the pharmaceutical composition and dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.

Disintegrants may be used in the composition of the disclosure to provide tablets that disintegrate when exposed to an aqueous environment. Too much of a disintegrant may produce tablets which may disintegrate in the bottle. Too little of a disintegrant may be insufficient for disintegration to occur and may alter the rate and extent of release of the active ingredient(s) from the dosage form. A sufficient amount of disintegrant that is neither too little nor too much to detrimentally alter the release of the active ingredient(s) may be used to form the dosage forms of the compounds disclosed herein. The amount of disintegrant used may vary based upon the type of formulation and mode of administration, and may be readily discernible to those of ordinary skill in the art. About 0.5 to about 15 weight percent of disintegrant, or about 1 to about 5 weight percent of disintegrant, may be used in the pharmaceutical composition. Disintegrants that can be used to form pharmaceutical composition and dosage forms of the disclosure include, but are not limited to, agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, other starches, pre-gelatinized starch, other starches, clays, other algins, other celluloses, gums or mixtures thereof.

Lubricants which can be used to form pharmaceutical composition and dosage forms of the disclosure include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethylaureate, agar, or mixtures thereof. Additional lubricants include, for example, a syloid silica gel, a coagulated aerosol of synthetic silica, or mixtures thereof. A lubricant can optionally be added, in an amount of less than about 1 weight percent of the pharmaceutical composition.

When aqueous suspensions and/or elixirs are desired for oral administration, the active ingredient therein may be combined with various sweetening or flavoring agents, coloring matter or dyes and, if so desired, emulsifying and/or suspending agents, together with such diluents as water, ethanol, propylene glycol, glycerin and various combinations thereof.

The tablets can be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed. Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin or olive oil.

Surfactant which can be used to form pharmaceutical composition and dosage forms of the disclosure include, but are not limited to, hydrophilic surfactants, lipophilic surfactants, and mixtures thereof. That is, a mixture of hydrophilic surfactants may be employed, a mixture of lipophilic surfactants may be employed, or a mixture of at least one hydrophilic surfactant and at least one lipophilic surfactant may be employed.

A suitable hydrophilic surfactant may generally have an HLB value of at least 10, while suitable lipophilic surfactants may generally have an HLB value of or less than about 10. An empirical parameter used to characterize the relative hydrophilicity and hydrophobicity of non-ionic amphiphilic compounds is the hydrophilic-lipophilic balance (“HLB” value). Surfactants with lower HLB values are more lipophilic or hydrophobic, and have greater solubility in oils, while surfactants with higher HLB values are more hydrophilic, and have greater solubility in aqueous solutions. Hydrophilic surfactants are generally considered to be those compounds having an HLB value greater than about 10, as well as anionic, cationic, or zwitterionic compounds for which the HLB scale is not generally applicable. Similarly, lipophilic (i.e., hydrophobic) surfactants are compounds having an HLB value equal to or less than about 10. However, HLB value of a surfactant is merely a rough guide generally used to enable formulation of industrial, pharmaceutical and cosmetic emulsions.

Hydrophilic surfactants may be either ionic or non-ionic. Suitable ionic surfactants include, but are not limited to, alkylammonium salts; fusidic acid salts; fatty acid derivatives of amino acids, oligopeptides, and polypeptides; glyceride derivatives of amino acids, oligopeptides, and polypeptides; lecithins and hydrogenated lecithins; lysolecithins and hydrogenated lysolecithins; phospholipids and derivatives thereof; lysophospholipids and derivatives thereof; camitine fatty acid ester salts; salts of alkylsulfates; fatty acid salts; sodium docusate; acylactylates; mono- and di-acetylated tartaric acid esters of mono- and di-glycerides; succinylated mono- and di-glycerides; citric acid esters of mono- and di-glycerides; and mixtures thereof.

Within the aforementioned group, ionic surfactants include, by way of example: lecithins, lysolecithin, phospholipids, lysophospholipids and derivatives thereof; carnitine fatty acid ester salts; salts of alkylsulfates; fatty acid salts; sodium docusate; acylactylates; mono- and di-acetylated tartaric acid esters of mono- and di-glycerides; succinylated mono- and di-glycerides; citric acid esters of mono- and di-glycerides; and mixtures thereof.

Hydrophilic non-ionic surfactants may include, but not limited to, alkylglucosides; alkylmaltosides; alkylthioglucosides; lauryl macrogolglycerides; polyoxyalkylene alkyl ethers such as polyethylene glycol alkyl ethers; polyoxyalkylene alkylphenols such as polyethylene glycol alkyl phenols; polyoxyalkylene alkyl phenol fatty acid esters such as polyethylene glycol fatty acids monoesters and polyethylene glycol fatty acids diesters; polyethylene glycol glycerol fatty acid esters; polyglycerol fatty acid esters; polyoxyalkylene sorbitan fatty acid esters such as polyethylene glycol sorbitan fatty acid esters; hydrophilic transesterification products of a polyol with at least one member of the group of glycerides, vegetable oils, hydrogenated vegetable oils, fatty acids, and sterols; polyoxyethylene sterols, derivatives, and analogues thereof; polyoxyethylated vitamins and derivatives thereof, polyoxyethylene-polyoxypropylene block copolymers; and mixtures thereof, polyethylene glycol sorbitan fatty acid esters and hydrophilic transesterification products of a polyol with at least one member of the group of triglycerides, vegetable oils, and hydrogenated vegetable oils. The polyol may be glycerol, ethylene glycol, polyethylene glycol, sorbitol, propylene glycol, pentaerythritol, or a saccharide.

Suitable lipophilic surfactants include, by way of example only: fatty alcohols; glycerol fatty acid esters; acetylated glycerol fatty acid esters; lower alcohol fatty acids esters; propylene glycol fatty acid esters; sorbitan fatty acid esters; polyethylene glycol sorbitan fatty acid esters; sterols and sterol derivatives; polyoxyethylated sterols and sterol derivatives; polyethylene glycol alkyl ethers; sugar esters; sugar ethers; lactic acid derivatives of mono- and di-glycerides; hydrophobic transesterification products of a polyol with at least one member of the group of glycerides, vegetable oils, hydrogenated vegetable oils, fatty acids and sterols; oil-soluble vitamins/vitamin derivatives; and mixtures thereof. Within this group, preferred lipophilic surfactants include glycerol fatty acid esters, propylene glycol fatty acid esters, and mixtures thereof, or are hydrophobic transesterification products of a polyol with at least one member of the group of vegetable oils, hydrogenated vegetable oils, and triglycerides.

In one embodiment, the composition may include a solubilizer to ensure good solubilization and/or dissolution of the compound of the present disclosure and to minimize precipitation of the compound of the present disclosure. This can be especially important for composition for non-oral use, e.g., composition for injection. A solubilizer may also be added to increase the solubility of the hydrophilic drug and/or other components, such as surfactants, or to maintain the composition as a stable or homogeneous solution or dispersion.

The amount of solubilizer that can be included is not particularly limited. The amount of a given solubilizer may be limited to a bioacceptable amount, which may be readily determined by one of skill in the art. In some circumstances, it may be advantageous to include amounts of solubilizers far in excess of bioacceptable amounts, for example to maximize the concentration of the drug, with excess solubilizer removed prior to providing the composition to a patient using conventional techniques, such as distillation or evaporation. If present, the solubilizer can be in a weight ratio of 10%, 25%, 50%, 100%, or up to about 200% by weight, based on the combined weight of the drug, and other excipients. If desired, very small amounts of solubilizer may also be used, such as 5%, 2%, 1% or even less. Typically, the solubilizer may be present in an amount of about 1% to about 100%, more typically about 5% to about 25% by weight.

EXAMPLES

All chemicals, reagents, and solvents were purchased from commercial sources when available and used without further purification.

To a solution of 1-bromo-2-(2-bromoethoxy)ethane (INT-1, 20 g, 86.24 mmol) and propanedinitrile (6.27 g, 94.86 mmol) in DMF (30 mL) was added DBU (26.26 g, 172.48 mmol). The reaction mixture was stirred at 85° C. for 3 hours, cooled to ambient temperature, diluted with water (100 mL), and extracted with ethyl acetate (100 mL×3). The combined organic phase was washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to provide tetrahydro-4H-pyran-4,4-dicarbonitrile (INT-2, 12.61 g, crude) as a brown solid. The crude product was used directly in the next step without further purification.1H NMR (400 MHz, methanol-d4) δ=3.86-3.77 (m, 4H), 2.32-2.21 (m, 4H).

To a solution of tetrahydro-4H-pyran-4,4-dicarbonitrile (INT-2, 9.0 g, 66.10 mmol) in EtOH (270 mL) was added NaBH4(7.50 g, 198.31 mmol) in portions. The reaction mixture was stirred at 20° C. for 4 hours, quenched by water (200 mL), and extracted with ethyl acetate (200 mL×3). The combined organic phase was washed with brine (150 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to provide 4-(aminomethyl)tetrahydro-2H-pyran-4-carbonitrile (INT-3, 7.23 g, 78% yield) as a brown oil. The crude product was used directly in the next step without further purification.

To a solution of 4-(((5′-chloro-2′-fluoro-[2,4′-bipyridin]-6-yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile (INT-5, 4.33 g, 12.49 mmol) and cyclohexane-1,4-diamine (2.14 g, 18.73 mmol) in DMSO (50 mL) was added TEA (2.53 g, 24.97 mmol). The reaction mixture was stirred at 110° C. for 16 hours, diluted with water (40 mL), and extracted with ethyl acetate (100 mL×3). The combined organic phase was washed with brine (60 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The resulting residue was dissolved in ethyl acetate (100 mL), added dropwise to HCl/dioxane (50 mL), filtered, and washed with ethyl acetate. The resulting solid was dissolved in water (150 mL), basified with NaHCO3to pH 9, and extracted with ethyl acetate (100 mL×3). The combined organic phase was washed with brine (60 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to provide 4-(((2′-(((1R,4R)-4-aminocyclohexyl)amino)-5′-chloro-[2,4′-bipyridin]-6-yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile (Intermediate 1, 4.1 g, 61.11% yield) as a brown solid. MS (ESI) m/z=441.3 [M+H]+.

To a solution of AlCl3(3.67 g, 27.55 mmol) in THF (50 mL) was added NaN3(4.87 g, 74.97 mmol). The reaction mixture was stirred at 60° C. for 2 hours and cooled to 20° C. 2-chloroacetonitrile (INT-6, 2.0 g, 26.49 mmol) was added and the reaction mixture was heated to 70° C. and stirred for 24 hours. The reaction mixture was concentrated and the resulting residue was acidified with aq. HCl (37%) to pH 2 and extracted with EtOAc (100 mL×3). The combined organic layers were dried over Na2SO4, filtered, and concentrated to provide 5-(chloromethyl)-2H-tetrazole (INT-7, 3.0 g, crude) as a white solid. The crude product was used directly in the next step without further purification.1H NMR (400 MHz, DMSO-d6): δ=5.08 (s, 2H).

To a solution of 4-chloro-6-methyl-pyrimidine (INT-12, 2.5 g, 19.45 mmol) in dioxane (30 mL) was added NH2NH2.H2O (1.67 g, 33.45 mmol) and K2CO3(2.74 g, 19.84 mmol). The reaction mixture was stirred at 100° C. for 6 hr, diluted with H2O (20 mL), and extracted with EtOAc (50 mL×3). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered, and concentrated in vacuo to provide 4-hydrazine-6-methylpyrimidine (INT-13, 2 g, 16.11 mmol, 82.85% yield) as a yellow solid. The crude product was used directly in the next step without further purification.

To a solution of 4-hydrazine-6-methylpyrimidine (INT-13, 0.9 g, 7.25 mmol) in H2O (2 mL) were added HCl (2 M, 3.62 mL) and NaNO2(500.19 mg, 7.25 mmol). The reaction mixture was stirred at 0° C. for 8 hr and filtered, and the filter cake was concentrated under reduced pressure to provide (Z)—N-(1-(1H-tetrazol-5-yl)prop-1-en-2-yl)formamide (INT-14, 0.6 g, 3.92 mmol, 54.04% yield) as a brown solid. The crude product was used directly in the next step without further purification. MS (ESI) m/z=296.1 [M+H]+.

Step 3: Preparation of 5-(2-oxopropyl)-1H-tetrazol-1-ium chloride (INT-15)

To a solution of (Z)—N-(1-(1H-tetrazol-5-yl)prop-1-en-2-yl)formamide (INT-14, 0.8 g, 5.22 mmol) in H2O (4 mL) was added HCl (1 mL). The reaction mixture was stirred at 80° C. for 12 hr and concentrated under reduced pressure to provide 5-(2-oxopropyl)-1H-tetrazol-1-ium chloride (INT-15, 0.7 g, 4.31 mmol, 82.42% yield) as a yellow solid. The crude product was used directly in the next step without further purification.

To a solution of 4-[[[6-[2-[(4-aminocyclohexyl)amino]-5-chloro-4-pyridyl]-2-pyridyl]amino]methyl]tetrahydropyran-4-carbonitrile (Intermediate 1, 100 mg, 0.227 mmol), isopropyl 2-acetonyloxyacetate (Intermediate 5, 43 mg, 0.249 mmol) and AcOH (41 mg, 0.680 mmol) in DCE (2 mL) was added NaBH(OAc)3(67 mg, 0.317 mmol) at 0° C. The mixture was stirred at 20° C. for 20 hr. The mixture was quenched with H2O (5 mL). The resulting mixture was extracted with EtOAc (20 mL×3). The organic phases were combined and washed with brine (50 mL) and dried over anhydrous Na2SO4, filtered and concentrated in vacuo, which was purified by chromatography on silica gel (15-20% MeOH in DCM) to give the isopropyl 2-[2-[[4-[[5-chloro-4-[6-[(4-cyanotetrahydropyran-4-yl)methylamino]-2-pyridyl]-2-pyridyl]amino]cyclohexyl]amino]propoxy]acetate (Compound 14, 50 mg, 12% yield) as a black brown solid.

To a solution of 4-[[[6-[2-[(4-aminocyclohexyl)amino]-5-chloro-4-pyridyl]-2-pyridyl]amino]methyl]tetrahydropyran-4-carbonitrile (Intermediate 1, 200 mg, 0.454 mmol), tert-butyl 2-acetonyloxyacetate (94 mg, 0.499 mmol) and AcOH (82 mg, 1.36 mmol) in DCE (2 mL) was added NaBH(OAc)3(Intermediate 6, 135 mg, 0.635 mmol) at 0° C. The mixture was stirred at 20° C. for 20 hr. The mixture was quenched with H2O (5 mL). The resulting mixture was extracted with EtOAc (20 mL×3). The organic phases were combined and washed with brine (50 mL) and dried over anhydrous Na2SO4, filtered and concentrated in vacuo, which was purified by chromatography on silica gel (15-20% MeOH in DCM) to give the tert-butyl 2-[2-[[4-[[5-chloro-4-[6-[(4-cyanotetrahydropyran-4-yl)methylamino]-2-pyridyl]-2-pyridyl]amino]cyclohexyl]amino]propoxy]acetate (Compound 17, 110 mg, 25% yield) as a black brown solid.

To a solution of 5-(2-methylallyloxymethyl)-2H-tetrazole (INT-20, 600 mg, 3.89 mmol) in DMF (6 mL) was added DIEA (5.03 g, 38.92 mmol) and 1-chloroethyl ethyl carbonate (5.94 g, 38.92 mmol). The mixture was stirred at 70° C. for 16 hours. The mixture was diluted with ethyl acetate (100 mL), which was washed with brine (100 mL×3). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give the residue, which was purified by flash column chromatography (silica gel, 100-200 mesh, 0-10% ethyl acetate in petroleum ether) to afford ethyl 1-[5-(2-methylallyloxymethyl)tetrazol-1-yl]ethyl carbonate (INT-21, 326 mg, 31% yield) as yellow oil and ethyl 1-[5-(2-methylallyloxymethyl)tetrazol-2-yl]ethyl carbonate (INT-22, 464 mg, 44% yield) as yellow oil.

To a solution of ethyl 1-[5-(2-methylallyloxymethyl)tetrazol-2-yl]ethyl carbonate (INT-21, 464 mg, 1.72 mmol) in THF (10 mL) and H2O (10 mL) was added K2OsO4.2H2O (32 mg, 0.086 mmol) and NaIO4(845 mg, 3.95 mmol). The mixture was stirred at 15° C. for 4 hours. The mixture was diluted with water (50 mL), which was extracted with ethyl acetate (50 mL×3). The combined organic phase was washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give the residue, which was purified by flash column chromatography (silica gel, 100-200 mesh, 0-20% ethyl acetate in petroleum ether) to afford 1-[5-(acetonyloxymethyl)tetrazol-2-yl]ethyl ethyl carbonate (INT-23, 400 mg, 86% yield) as yellow oil.

To a solution of ethyl 1-[5-(2-methylallyloxymethyl)tetrazol-1-yl]ethyl carbonate (INT-22, 326 mg, 1.21 mmol) in THF (7 mL) and H2O (7 mL) was added K2OsO4.2H2O (22 mg, 0.060 mmol) and NaIO4(593 mg, 2.77 mmol). The mixture was stirred at 15° C. for 4 hours. The reaction mixture was diluted with water (50 mL), which was extracted with ethyl acetate (50 mL×3). The combined organic phase was washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give the residue, which was purified by flash column chromatography (silica gel, 100-200 mesh, 0-25% ethyl acetate in petroleum ether) followed by chiral SFC to afford [(1R)-1-[5-(acetonyloxymethyl)tetrazol-1-yl]ethyl] ethyl carbonate (Intermediate 9, 142 mg, 43% yield) as yellow oil and [(1S)-1-[5-(acetonyloxymethyl)tetrazol-1-yl]ethyl] ethyl carbonate (Intermediate 10, 195 mg, 59% yield) as yellow oil.

To a solution of [(1R)-1-[5-(acetonyloxymethyl)tetrazol-2-yl]ethyl] ethyl carbonate (Intermediate 7, 123 mg, 0.45 mmol) and 4-[[[6-[2-[(4-aminocyclohexyl)amino]-5-chloro-4-pyridyl]-2-pyridyl]amino]methyl]tetrahydropyran-4-carbonitrile (Intermediate 1, 166 mg, 0.38 mmol) in DCE (2 mL) was added HOAc (68 mg, 1.13 mmol) and NaBH(OAc)3(112 mg, 0.53 mmol) at 0° C. The mixture was stirred at 20° C. for 16 hours. The mixture was quenched by water (50 mL) at 0° C., which was extracted with DCM (50 mL×3). The combined organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give the residue, which was purified by RP-HPLC (16 to 46% acetonitrile in water and 0.225% formic acid) to afford [(1R)-1-[5-[2-[[4-[[5-chloro-4-[6-[(4-cyanotetrahydropyran-4-yl)methylamino]-2-pyridyl]-2-pyridyl]amino]cyclohexyl]amino]propoxymethyl]tetrazol-2-yl]ethyl] ethyl carbonate (Compound 20, 168 mg, 63% yield) as yellow oil.

To a solution of [(1S)-1-[5-(acetonyloxymethyl)tetrazol-2-yl]ethyl] ethyl carbonate (Intermediate 8, 270 mg, 0.99 mmol) and 4-[[[6-[2-[(4-aminocyclohexyl)amino]-5-chloro-4-pyridyl]-2-pyridyl]amino]methyl]tetrahydropyran-4-carbonitrile (Intermediate 1, 364 mg, 0.83 mmol) in DCE (3 mL) was added HOAc (149 mg, 2.48 mmol) and NaBH(OAc)3(245 mg, 1.16 mmol) at 0° C. The mixture was stirred at 20° C. for 16 hours. The mixture was quenched by water (50 mL) at 0° C., which was extracted with DCM (50 mL×3). The combined organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give the residue, which was purified by RP-HPLC (16 to 46% acetonitrile in water and 0.225% formic acid) to afford [(1S)-1-[5-[2-[[4-[[5-chloro-4-[6-[(4-cyanotetrahydropyran-4-yl)methylamino]-2-pyridyl]-2-pyridyl]amino]cyclohexyl]amino]propoxymethyl]tetrazol-2-yl]ethyl] ethyl carbonate (Compound 23, 260 mg, 44% yield) as yellow oil.

To a solution of [(1R)-1-[5-(acetonyloxymethyl)tetrazol-1-yl]ethyl] ethyl carbonate (Intermediate 9, 122 mg, 0.45 mmol) and 4-[[[6-[2-[(4-aminocyclohexyl)amino]-5-chloro-4-pyridyl]-2-pyridyl]amino]methyl]tetrahydropyran-4-carbonitrile (Intermediate 1, 165 mg, 0.37 mmol) in DCE (2 mL) was added HOAc (67 mg, 1.12 mmol) and NaBH(OAc)3(111 mg, 0.52 mmol) at 0° C. The mixture was stirred at 20° C. for 16 hours under N2. The mixture was quenched by water (50 mL) at 0° C., which was extracted with DCM (50 mL×3). The combined organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give the residue, which was purified by prep-HPLC (45 to 75% acetonitrile in water and 0.225% formic acid) to afford [(1R)-1-[5-[2-[[4-[[5-chloro-4-[6-[(4-cyanotetrahydropyran-4-yl)methylamino]-2-pyridyl]-2-pyridyl]amino]cyclohexyl]amino]propoxymethyl]tetrazol-1-yl]ethyl] ethyl carbonate (Compound 26, 73 mg, 27% yield) as yellow oil.

To a solution of [(1S)-1-[5-(acetonyloxymethyl)tetrazol-1-yl]ethyl] ethyl carbonate (Intermediate 10, 175 mg, 0.64 mmol) and 4-[[[6-[2-[(4-aminocyclohexyl)amino]-5-chloro-4-pyridyl]-2-pyridyl]amino]methyl]tetrahydropyran-4-carbonitrile (Intermediate 1, 236 mg, 0.54 mmol) in DCE (2 mL) was added HOAc (97 mg, 1.61 mmol) and NaBH(OAc)3(159 mg, 0.75 mmol) at 0° C. The mixture was stirred at 20° C. for 16 hours under N2. The mixture was quenched by water (50 mL) at 0° C., which was extracted with DCM (50 mL×3). The combined organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give the residue, which was purified by RP-HPLC (15 to 45% acetonitrile in water and 0.225% formic acid) to afford [(1S)-1-[5-[2-[[4-[[5-chloro-4-[6-[(4-cyanotetrahydropyran-4-yl)methylamino]-2-pyridyl]-2-pyridyl]amino]cyclohexyl]amino]propoxymethyl]tetrazol-1-yl]ethyl] ethyl carbonate (Compound 29, 204 mg, 54% yield) as yellow oil.

To a solution of tert-butyl N-[2-[2-[[4-[[5-chloro-4-[6-[(4-cyanotetrahydropyran-4-yl)methylamino]-2-pyridyl]-2-pyridyl]amino]cyclohexyl]amino]propoxy]ethyl]carbamate (Compound 38, 200 mg, 0.31 mmol) in DCM (5 mL) was added TFA (710.17 mg, 6.23 mmol). The mixture was stirred at 20° C. for 2 hours. The mixture was concentrated and then extracted with DCM (30 mL×3). The combined organic phases were washed with saturated aq. Na2CO3(30 mL), brine (30 mL), dried over anhydrous sodium sulfate, filtered and concentrated to give 4-[[[6-[2-[[4-[[2-(2-aminoethoxy)-1-methyl-ethyl]amino]cyclohexyl]amino]-5-chloro-4-pyridyl]-2-pyridyl]amino]methyl]tetrahydropyran-4-carbonitrile (Compound 39, 210 mg, crude) as a yellow solid.

Chloromethanesulfonyl chloride (INT-30) is converted to 1-(2-oxopropoxy)-N-(2,2,2-trifluoroethyl)methanesulfonamide (Intermediate 14) in five synthetic steps.

To a solution of 4-[[[6-[2-[(4-aminocyclohexyl)amino]-5-chloro-4-pyridyl]-2-pyridyl]amino]methyl]tetrahydropyran-4-carbonitrile (Intermediate 1) and 1-(2-oxopropoxy)-N-(2,2,2-trifluoroethyl)methanesulfonamide (Intermediate 14) in DCE is added HOAc and NaBH(OAc)3. The mixture is stirred at 20° C. for 36 hours. The mixture is quenched by water at 0° C., which is extracted with DCM. The combined organic phase is washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue is purified by prep-HPLC to afford 1-(2-(((1r,4r)-4-((5′-chloro-6-(((4-cyanotetrahydro-2H-pyran-4-yl)methyl)amino)-[2,4′-bipyridin]-2′-yl)amino)cyclohexyl)amino)propoxy)-N-(2,2,2-trifluoroethyl)methanesulfonamide (Compound 42).

To a solution of ethyl 1-hydroxycyclopropane-1-carboxylate (INT-31) in DMF is added NaH and the reaction mixture is stirred at 0° C. for 0.5 hours. 1-bromo-2-methyl-2-propene is added and the reaction mixture is stirred at 25° C. for 2 hours, quenched with brine, and extracted with EtOAc. The combined organic layers are washed with brine, dried over Na2SO4, filtered, and concentrated to provide ethyl 1-((2-methylallyl)oxy)cyclopropane-1-carboxylate (INT-32). The crude product is used directly in the next step without further purification.

To a solution of ethyl 1-((2-methylallyl)oxy)cyclopropane-1-carboxylate (INT-32) in THF and H2O is added K2OsO4.2H2O and NaIO4. The mixture is stirred at 15° C. for 4 hours. The mixture is diluted with water and extracted with ethyl acetate. The combined organic phases are washed with aq. Na2S2O3, brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give the residue which is purified by silica gel chromatography to afford ethyl 1-(2-oxopropoxy)cyclopropane-1-carboxylate (Intermediate 15).

To a solution of 4-[[[6-[2-[(4-aminocyclohexyl)amino]-5-chloro-4-pyridyl]-2-pyridyl]amino]methyl]tetrahydropyran-4-carbonitrile (Intermediate 1) and ethyl 1-(2-oxopropoxy)cyclopropane-1-carboxylate (Intermediate 15) in DCE is added HOAc and NaBH(OAc)3. The mixture is stirred at 20° C. for 36 hours. The mixture is quenched by water at 0° C., which is extracted with DCM. The combined organic phase is washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue is purified by prep-HPLC to afford ethyl 1-(2-(((1r,4r)-4-((5′-chloro-6-(((4-cyanotetrahydro-2H-pyran-4-yl)methyl)amino)-[2,4′-bipyridin]-2′-yl)amino)cyclohexyl)amino)propoxy)cyclopropane-1-carboxylate (Compound 45).

To a solution of ethyl 2-hydroxy-2-methylpropanoate (INT-33) in DMF is added NaH and the reaction mixture is stirred at 0° C. for 0.5 hours. 1-bromo-2-methyl-2-propene is added and the reaction mixture is stirred at 25° C. for 2 hours, quenched with brine, and extracted with EtOAc. The combined organic layers are washed with brine, dried over Na2SO4, filtered, and concentrated to provide ethyl 2-methyl-2-((2-methylallyl)oxy)propanoate (INT-34). The crude product is used directly in the next step without further purification.

To a solution of ethyl 2-methyl-2-((2-methylallyl)oxy)propanoate (INT-34) in THF and H2O is added K2OsO4.2H2O and NaIO4. The mixture is stirred at 15° C. for 4 hours. The mixture is diluted with water and extracted with ethyl acetate. The combined organic phases are washed with aq. Na2S2O3, brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give the residue which is purified by silica gel chromatography to afford ethyl 2-methyl-2-(2-oxopropoxy)propanoate (Intermediate 16).

To a solution of 4-[[[6-[2-[(4-aminocyclohexyl)amino]-5-chloro-4-pyridyl]-2-pyridyl]amino]methyl]tetrahydropyran-4-carbonitrile (Intermediate 1) and ethyl 2-methyl-2-(2-oxopropoxy)propanoate (Intermediate 16) in DCE is added HOAc and NaBH(OAc)3. The mixture is stirred at 20° C. for 36 hours. The mixture is quenched by water at 0° C., which is extracted with DCM. The combined organic phase is washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue is purified by prep-HPLC to afford ethyl 2-(2-(((1r,4r)-4-((5′-chloro-6-(((4-cyanotetrahydro-2H-pyran-4-yl)methyl)amino)-[2,4′-bipyridin]-2′-yl)amino)cyclohexyl)amino)propoxy)-2-methylpropanoate (Compound 48).

To a solution of ethyl (R)-2-hydroxypropanoate (INT-35) in DMF is added NaH and the reaction mixture is stirred at 0° C. for 0.5 hours. 1-bromo-2-methyl-2-propene is added and the reaction mixture is stirred at 25° C. for 2 hours, quenched with brine, and extracted with EtOAc. The combined organic layers are washed with brine, dried over Na2SO4, filtered, and concentrated to provide ethyl (R)-2-((2-methylallyl)oxy)propanoate (INT-36). The crude product is used directly in the next step without further purification.

To a solution of ethyl (R)-2-((2-methylallyl)oxy)propanoate (INT-36) in THF and H2O is added K2OsO4.2H2O and NaIO4. The mixture is stirred at 15° C. for 4 hours. The mixture is diluted with water and extracted with ethyl acetate. The combined organic phases are washed with aq. Na2S2O3, brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give the residue which is purified by silica gel chromatography to afford ethyl (R)-2-(2-oxopropoxy)propanoate (Intermediate 17).

To a solution of 4-[[[6-[2-[(4-aminocyclohexyl)amino]-5-chloro-4-pyridyl]-2-pyridyl]amino]methyl]tetrahydropyran-4-carbonitrile (Intermediate 1) and ethyl (R)-2-(2-oxopropoxy)propanoate (Intermediate 17) in DCE is added HOAc and NaBH(OAc)3. The mixture is stirred at 20° C. for 36 hours. The mixture is quenched by water at 0° C., which is extracted with DCM. The combined organic phase is washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue is purified by prep-HPLC to afford ethyl (2R)-2-(2-(((1r,4R)-4-((5′-chloro-6-(((4-cyanotetrahydro-2H-pyran-4-yl)methyl)amino)-[2,4′-bipyridin]-2′-yl)amino)cyclohexyl)amino)propoxy)propanoate (Compound 51).

To a solution of ethyl (S)-2-hydroxypropanoate (INT-37) in DMF is added NaH and the reaction mixture is stirred at 0° C. for 0.5 hours. 1-bromo-2-methyl-2-propene is added and the reaction mixture is stirred at 25° C. for 2 hours, quenched with brine, and extracted with EtOAc. The combined organic layers are washed with brine, dried over Na2SO4, filtered, and concentrated to provide ethyl (S)-2-((2-methylallyl)oxy)propanoate (INT-38). The crude product is used directly in the next step without further purification.

To a solution of ethyl (S)-2-((2-methylallyl)oxy)propanoate (INT-38) in THF and H2O is added K2OsO4.2H2O and NaIO4. The mixture is stirred at 15° C. for 4 hours. The mixture is diluted with water and extracted with ethyl acetate. The combined organic phases are washed with aq. Na2S2O3, brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give the residue which is purified by silica gel chromatography to afford ethyl (S)-2-(2-oxopropoxy)propanoate (Intermediate 18).

To a solution of 4-[[[6-[2-[(4-aminocyclohexyl)amino]-5-chloro-4-pyridyl]-2-pyridyl]amino]methyl]tetrahydropyran-4-carbonitrile (Intermediate 1) and ethyl (S)-2-(2-oxopropoxy)propanoate (Intermediate 18) in DCE is added HOAc and NaBH(OAc)3. The mixture is stirred at 20° C. for 36 hours. The mixture is quenched by water at 0° C., which is extracted with DCM. The combined organic phase is washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue is purified by prep-HPLC to afford ethyl (2S)-2-(2-(((1r,4S)-4-((5′-chloro-6-(((4-cyanotetrahydro-2H-pyran-4-yl)methyl)amino)-[2,4′-bipyridin]-2′-yl)amino)cyclohexyl)amino)propoxy)propanoate (Compound 54).

To a solution of (6-nitropyridin-2-yl)methanol (INT-39) in DMF is added NaH and the reaction mixture is stirred at 0° C. for 0.5 hours. 1-bromo-2-methyl-2-propene is added and the reaction mixture is stirred at 25° C. for 2 hours, quenched with brine, and extracted with EtOAc. The combined organic layers are washed with brine, dried over Na2SO4, filtered, and concentrated to provide 2-(((2-methylallyl)oxy)methyl)-6-nitropyridine (INT-40). The crude product is used directly in the next step without further purification.

To a solution of 2-(((2-methylallyl)oxy)methyl)-6-nitropyridine (INT-40) in THF and H2O is added K2OsO4.2H2O and NaIO4. The mixture is stirred at 15° C. for 4 hours. The mixture is diluted with water and extracted with ethyl acetate. The combined organic phases are washed with aq. Na2S2O3, brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give the residue which is purified by silica gel chromatography to afford ethyl (S)-2-(2-oxopropoxy)propanoate (Intermediate 19).

To a solution of 4-[[[6-[2-[(4-aminocyclohexyl)amino]-5-chloro-4-pyridyl]-2-pyridyl]amino]methyl]tetrahydropyran-4-carbonitrile (Intermediate 1) and ethyl (S)-2-(2-oxopropoxy)propanoate (Intermediate 19) in DCE is added HOAc and NaBH(OAc)3. The mixture is stirred at 20° C. for 36 hours. The mixture is quenched by water at 0° C., which is extracted with DCM. The combined organic phase is washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue is purified by prep-HPLC, reduced with iron and acetic acid, and mesylated with methanesulfonyl chloride and triethylamine to afford N-(6-((2-(((1r,4r)-4-((5′-chloro-6-(((4-cyanotetrahydro-2H-pyran-4-yl)methyl)amino)-[2,4′-bipyridin]-2′-yl)amino)cyclohexyl)amino)propoxy)methyl)pyridin-2-yl)methanesulfonamide (Compound 57).

To a solution of (4-methoxypyrimidin-2-yl)methanol (INT-41) in DMF is added NaH and the reaction mixture is stirred at 0° C. for 0.5 hours. 1-bromo-2-methyl-2-propene is added and the reaction mixture is stirred at 25° C. for 2 hours, quenched with brine, and extracted with EtOAc. The combined organic layers are washed with brine, dried over Na2SO4, filtered, and concentrated to provide 4-methoxy-2-(((2-methylallyl)oxy)methyl)pyrimidine (INT-42). The crude product is used directly in the next step without further purification.

To a solution of 4-methoxy-2-(((2-methylallyl)oxy)methyl)pyrimidine (INT-42) in THF and H2O is added K2OsO4.2H2O and NaIO4. The mixture is stirred at 15° C. for 4 hours. The mixture is diluted with water and extracted with ethyl acetate. The combined organic phases are washed with aq. Na2S2O3, brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give the residue which is purified by silica gel chromatography to afford 1-((4-methoxypyrimidin-2-yl)methoxy)propan-2-one (Intermediate 20).

To a solution of 4-[[[6-[2-[(4-aminocyclohexyl)amino]-5-chloro-4-pyridyl]-2-pyridyl]amino]methyl]tetrahydropyran-4-carbonitrile (Intermediate 1) and 1-((4-methoxypyrimidin-2-yl)methoxy)propan-2-one (Intermediate 20) in DCE is added HOAc and NaBH(OAc)3. The mixture is stirred at 20° C. for 36 hours. The mixture is quenched by water at 0° C., which is extracted with DCM. The combined organic phase is washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue is purified by prep-HPLC, silylated with TMSI, and fluorinated with SelectFluor to afford 4-(((5′-chloro-2′-(((1r,4r)-4-((1-((5-fluoro-6-oxo-1,6-dihydropyrimidin-2-yl)methoxy)propan-2-yl)amino)cyclohexyl)amino)-[2,4′-bipyridin]-6-yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile (Compound 76).

To a solution of tert-butyl 1-hydroxycyclopropane-1-carboxylate (INT-43) in DMF is added NaH and the reaction mixture is stirred at 0° C. for 0.5 hours. 1-bromo-2-methyl-2-propene is added and the reaction mixture is stirred at 25° C. for 2 hours, quenched with brine, and extracted with EtOAc. The combined organic layers are washed with brine, dried over Na2SO4, filtered, and concentrated to provide tert-butyl 1-((2-methylallyl)oxy)cyclopropane-1-carboxylate (INT-44). The crude product is used directly in the next step without further purification.

To a solution of tert-butyl 1-((2-methylallyl)oxy)cyclopropane-1-carboxylate (INT-44) in THF and H2O is added K2OsO4.2H2O and NaIO4. The mixture is stirred at 15° C. for 4 hours. The mixture is diluted with water and extracted with ethyl acetate. The combined organic phases are washed with aq. Na2S2O3, brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give the residue which is purified by silica gel chromatography to afford tert-butyl 1-(2-oxopropoxy)cyclopropane-1-carboxylate (Intermediate 21).

To a solution of 4-[[[6-[2-[(4-aminocyclohexyl)amino]-5-chloro-4-pyridyl]-2-pyridyl]amino]methyl]tetrahydropyran-4-carbonitrile (Intermediate 1) and tert-butyl 1-(2-oxopropoxy)cyclopropane-1-carboxylate (Intermediate 21) in DCE is added HOAc and NaBH(OAc)3. The mixture is stirred at 20° C. for 36 hours. The mixture is quenched by water at 0° C., which is extracted with DCM. The combined organic phase is washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue is purified by prep-HPLC to afford tert-butyl 1-(2-(((1r,4r)-4-((5′-chloro-6-(((4-cyanotetrahydro-2H-pyran-4-yl)methyl)amino)-[2,4′-bipyridin]-2′-yl)amino)cyclohexyl)amino)propoxy)cyclopropane-1-carboxylate (Compound 79).

To a solution of tert-butyl 2-hydroxy-2-methylpropanoate (INT-45) in DMF is added NaH and the reaction mixture is stirred at 0° C. for 0.5 hours. 1-bromo-2-methyl-2-propene is added and the reaction mixture is stirred at 25° C. for 2 hours, quenched with brine, and extracted with EtOAc. The combined organic layers are washed with brine, dried over Na2SO4, filtered, and concentrated to provide tert-butyl 2-methyl-2-((2-methylallyl)oxy)propanoate (INT-46). The crude product is used directly in the next step without further purification.

To a solution of tert-butyl 2-methyl-2-((2-methylallyl)oxy)propanoate (INT-46) in THF and H2O is added K2OsO4.2H2O and NaIO4. The mixture is stirred at 15° C. for 4 hours. The mixture is diluted with water and extracted with ethyl acetate. The combined organic phases are washed with aq. Na2S2O3, brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give the residue which is purified by silica gel chromatography to afford tert-butyl 2-methyl-2-(2-oxopropoxy)propanoate (Intermediate 22).

To a solution of 4-[[[6-[2-[(4-aminocyclohexyl)amino]-5-chloro-4-pyridyl]-2-pyridyl]amino]methyl]tetrahydropyran-4-carbonitrile (Intermediate 1) and tert-butyl 2-methyl-2-(2-oxopropoxy)propanoate (Intermediate 22) in DCE is added HOAc and NaBH(OAc)3. The mixture is stirred at 20° C. for 36 hours. The mixture is quenched by water at 0° C., which is extracted with DCM. The combined organic phase is washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue is purified by prep-HPLC to afford tert-butyl 2-(2-(((1r,4r)-4-((5′-chloro-6-(((4-cyanotetrahydro-2H-pyran-4-yl)methyl)amino)-[2,4′-bipyridin]-2′-yl)amino)cyclohexyl)amino)propoxy)-2-methylpropanoate (Compound 84).

To a solution of tert-butyl (R)-2-hydroxypropanoate (INT-47) in DMF is added NaH and the reaction mixture is stirred at 0° C. for 0.5 hours. 1-bromo-2-methyl-2-propene is added and the reaction mixture is stirred at 25° C. for 2 hours, quenched with brine, and extracted with EtOAc. The combined organic layers are washed with brine, dried over Na2SO4, filtered, and concentrated to provide tert-butyl (R)-2-((2-methylallyl)oxy)propanoate (INT-48). The crude product is used directly in the next step without further purification.

To a solution of tert-butyl (R)-2-((2-methylallyl)oxy)propanoate (INT-48) in THF and H2O is added K2OsO4.2H2O and NaIO4. The mixture is stirred at 15° C. for 4 hours. The mixture is diluted with water and extracted with ethyl acetate. The combined organic phases are washed with aq. Na2S2O3, brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give the residue which is purified by silica gel chromatography to afford tert-butyl (R)-2-(2-oxopropoxy)propanoate (Intermediate 23).

To a solution of 4-[[[6-[2-[(4-aminocyclohexyl)amino]-5-chloro-4-pyridyl]-2-pyridyl]amino]methyl]tetrahydropyran-4-carbonitrile (Intermediate 1) and tert-butyl (R)-2-(2-oxopropoxy)propanoate (Intermediate 23) in DCE is added HOAc and NaBH(OAc)3. The mixture is stirred at 20° C. for 36 hours. The mixture is quenched by water at 0° C., which is extracted with DCM. The combined organic phase is washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue is purified by prep-HPLC to afford tert-butyl (2R)-2-(2-(((1r,4R)-4-((5′-chloro-6-(((4-cyanotetrahydro-2H-pyran-4-yl)methyl)amino)-[2,4′-bipyridin]-2′-yl)amino)cyclohexyl)amino)propoxy)propanoate (Compound 89).

To a solution of tert-butyl (S)-2-hydroxypropanoate (INT-49) in DMF is added NaH and the reaction mixture is stirred at 0° C. for 0.5 hours. 1-bromo-2-methyl-2-propene is added and the reaction mixture is stirred at 25° C. for 2 hours, quenched with brine, and extracted with EtOAc. The combined organic layers are washed with brine, dried over Na2SO4, filtered, and concentrated to provide tert-butyl (S)-2-((2-methylallyl)oxy)propanoate (INT-50). The crude product is used directly in the next step without further purification.

To a solution of tert-butyl (S)-2-((2-methylallyl)oxy)propanoate (INT-50) in THF and H2O is added K2OsO4.2H2O and NaIO4. The mixture is stirred at 15° C. for 4 hours. The mixture is diluted with water and extracted with ethyl acetate. The combined organic phases are washed with aq. Na2S2O3, brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give the residue which is purified by silica gel chromatography to afford tert-butyl (S)-2-(2-oxopropoxy)propanoate (Intermediate 24).

To a solution of 4-[[[6-[2-[(4-aminocyclohexyl)amino]-5-chloro-4-pyridyl]-2-pyridyl]amino]methyl]tetrahydropyran-4-carbonitrile (Intermediate 1) and tert-butyl (S)-2-(2-oxopropoxy)propanoate (Intermediate 24) in DCE is added HOAc and NaBH(OAc)3. The mixture is stirred at 20° C. for 36 hours. The mixture is quenched by water at 0° C., which is extracted with DCM. The combined organic phase is washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue is purified by prep-HPLC to afford tert-butyl (2S)-2-(2-(((1r,4S)-4-((5′-chloro-6-(((4-cyanotetrahydro-2H-pyran-4-yl)methyl)amino)-[2,4′-bipyridin]-2′-yl)amino)cyclohexyl)amino)propoxy)propanoate (Compound 94).

To a solution of 1-(2-trityl-2H-tetrazol-5-yl)cyclopropan-1-ol (INT-51) in DMF is added NaH and the reaction mixture is stirred at 0° C. for 0.5 hours. 1-bromo-2-methyl-2-propene is added and the reaction mixture is stirred at 25° C. for 2 hours, quenched with brine, and extracted with EtOAc. The combined organic layers are washed with brine, dried over Na2SO4, filtered, and concentrated to provide 5-(1-((2-methylallyl)oxy)cyclopropyl)-2-trityl-2H-tetrazole (INT-52). The crude product is used directly in the next step without further purification.

To a solution of 5-(1-((2-methylallyl)oxy)cyclopropyl)-2-trityl-2H-tetrazole (INT-52) in THF and H2O is added K2OsO4.2H2O and NaIO4. The mixture is stirred at 15° C. for 4 hours. The mixture is diluted with water and extracted with ethyl acetate. The combined organic phases are washed with aq. Na2S2O3, brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give the residue which is purified by silica gel chromatography to afford 1-(1-(2-trityl-2H-tetrazol-5-yl)cyclopropoxy)propan-2-one (Intermediate 25).

To a solution of 4-[[[6-[2-[(4-aminocyclohexyl)amino]-5-chloro-4-pyridyl]-2-pyridyl]amino]methyl]tetrahydropyran-4-carbonitrile (Intermediate 1) and 1-(1-(2-trityl-2H-tetrazol-5-yl)cyclopropoxy)propan-2-one (Intermediate 25) in DCE is added HOAc and NaBH(OAc)3. The mixture is stirred at 20° C. for 36 hours. The mixture is quenched by water at 0° C., which is extracted with DCM. The combined organic phase is washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue is purified by prep-HPLC and deprotected with HCl to afford 4-(((2′-(((1r,4r)-4-((1-(1-(2H-tetrazol-5-yl)cyclopropoxy)propan-2-yl)amino)cyclohexyl)amino)-5′-chloro-[2,4′-bipyridin]-6-yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile (Compound 99).

To a solution of 2-(2-trityl-2H-tetrazol-5-yl)propan-2-ol (INT-53) in DMF is added NaH and the reaction mixture is stirred at 0° C. for 0.5 hours. 1-bromo-2-methyl-2-propene is added and the reaction mixture is stirred at 25° C. for 2 hours, quenched with brine, and extracted with EtOAc. The combined organic layers are washed with brine, dried over Na2SO4, filtered, and concentrated to provide 5-(2-((2-methylallyl)oxy)propan-2-yl)-2-trityl-2H-tetrazole (INT-54). The crude product is used directly in the next step without further purification.

To a solution of 5-(2-((2-methylallyl)oxy)propan-2-yl)-2-trityl-2H-tetrazole (INT-54) in THF and H2O is added K2OsO4.2H2O and NaIO4. The mixture is stirred at 15° C. for 4 hours. The mixture is diluted with water and extracted with ethyl acetate. The combined organic phases are washed with aq. Na2S2O3, brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give the residue which is purified by silica gel chromatography to afford 1-((2-(2-trityl-2H-tetrazol-5-yl)propan-2-yl)oxy)propan-2-one (Intermediate 26).

To a solution of 4-[[[6-[2-[(4-aminocyclohexyl)amino]-5-chloro-4-pyridyl]-2-pyridyl]amino]methyl]tetrahydropyran-4-carbonitrile (Intermediate 1) and 1-((2-(2-ttyl-2H-tetrazol-5-yl)propan-2-yl)oxy)propan-2-one (Intermediate 26) in DCE is added HOAc and NaBH(OAc)3. The mixture is stirred at 20° C. for 36 hours. The mixture is quenched by water at 0° C., which is extracted with DCM. The combined organic phase is washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue is purified by prep-HPLC and deprotected with HCl to afford 4-(((2′-(((1r,4r)-4-((1-((2-(2H-tetrazol-5-yl)propan-2-yl)oxy)propan-2-yl)amino)cyclohexyl)amino)-5′-chloro-[2,4′-bipyridin]-6-yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile (Compound 102).

To a solution of 1-(2-trityl-2H-tetrazol-5-yl)ethan-1-ol (INT-55) in DMF is added NaH and the reaction mixture is stirred at 0° C. for 0.5 hours. 1-bromo-2-methyl-2-propene is added and the reaction mixture is stirred at 25° C. for 2 hours, quenched with brine, and extracted with EtOAc. The combined organic layers are washed with brine, dried over Na2SO4, filtered, and concentrated to provide 5-(1-((2-methylallyl)oxy)ethyl)-2-trityl-2H-tetrazole (INT-56). The crude product is used directly in the next step without further purification.

To a solution of 5-(1-((2-methylallyl)oxy)ethyl)-2-trityl-2H-tetrazole (INT-56) in THF and H2O is added K2OsO4.2H2O and NaIO4. The mixture is stirred at 15° C. for 4 hours. The mixture is diluted with water and extracted with ethyl acetate. The combined organic phases are washed with aq. Na2S2O3, brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give the residue which is purified by silica gel chromatography to afford 1-(1-(2-trityl-2H-tetrazol-5-yl)ethoxy)propan-2-one (Intermediate 27).

To a solution of 4-[[[6-[2-[(4-aminocyclohexyl)amino]-5-chloro-4-pyridyl]-2-pyridyl]amino]methyl]tetrahydropyran-4-carbonitrile (Intermediate 1) and 1-(1-(2-trityl-2H-tetrazol-5-yl)ethoxy)propan-2-one (Intermediate 27) in DCE is added HOAc and NaBH(OAc)3. The mixture is stirred at 20° C. for 36 hours. The mixture is quenched by water at 0° C., which is extracted with DCM. The combined organic phase is washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue is purified by prep-HPLC and deprotected with HCl to afford 4-(((2′-(((1r,4r)-4-((1-((2-(2H-tetrazol-5-yl)propan-2-yl)oxy)propan-2-yl)amino)cyclohexyl)amino)-5′-chloro-[2,4′-bipyridin]-6-yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile (Compound 102).

Hydrazinecarboxamide hydrochloride (INT-57) is converted to 5-(hydroxymethyl)-2,4-dihydro-3H-1,2,4-triazol-3-one (INT-58) in three synthetic steps.

To a solution of 4-[[[6-[2-[(4-aminocyclohexyl)amino]-5-chloro-4-pyridyl]-2-pyridyl]amino]methyl]tetrahydropyran-4-carbonitrile (Intermediate 1) and 5-((2-oxopropoxy)methyl)-2,4-dihydro-3H-1,2,4-triazol-3-one (Intermediate 28) in DCE is added HOAc and NaBH(OAc)3. The mixture is stirred at 20° C. for 36 hours. The mixture is quenched by water at 0° C., which is extracted with DCM. The combined organic phase is washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue is purified by prep-HPLC to afford 4-(((5′-chloro-2′-(((1r,4r)-4-((1-((5-oxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)methoxy)propan-2-yl)amino)cyclohexyl)amino)-[2,4′-bipyridin]-6-yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile (Compound 110).

To each well of a 96-well plate was added 5× kinase assay buffer with 10 mM DTT (6 μL), 500 μM ATP (1 μL), 5×CDK substrate (10 μL), and water (8 μL). 5 μL of compound were added to the test and positive control groups, while 5 μL of solvent was added to the blank groups. 100 ng of CDK9/CyclinT in 20 μL water was added to the test and positive control groups, while 20 μL of 1× kinase assay buffer was added to the blank groups. The reaction mixtures were incubated at 30° C. for 45 minutes, and 50 μL of Kinase-Glo® Max was added to each well and the plates were shielded from light and incubated for 15 minutes at room temperature. Luminescence was measured on a microplate reader and IC50values were calculated using Prism 9 software.

The IC50values obtained according to the above procedure are summarized in Table 1:

Example 73: Cancer Cell Viability Assay

Human liver cancer cell lines HepG2, Hep3B, Huh7, and SK-HEP-1 cells were rinsed and trypsinized with 0.25% trypsin (Corning #25-053-CI) in an incubator at 37° C. until detached. The cells were resuspended and seeded in 96-well plates to a density of 5,000 cells per well. Following cell adhesion, compounds were added to a final concentration of 2× dilution. 72 hours following compound treatment, CellTiter-Glo® 2.0 was added to the well in a 2:1 ratio of media/CellTiter-Glo® 2.0. The plates were shielded from light, shaken for 2 minutes, and incubated for 10 minutes. Luminescence was measured on a microplate reader and EC50values were calculated using Prism 9 software.

The EC50values obtained according to the above procedure are summarized in Table 2:

Example 74: Pharmacokinetic and Tissue Distribution Study

For mice experiments, the liver and blood pharmacokinetics of compounds were analyzed in CD-1 mice following a single oral administration dose of 5 mg/kg compound suspension.

Liver and blood samples for determination of compound concentration were obtained at 2 hours following administration of compounds (n=3 for each compound). The ratios of compound concentrations in liver versus blood in CD-1 mice following a single oral administration dose of 5 mg/kg compound suspension are summarized inFIG.1. The data inFIG.1show that Compounds 18, 40, and 33 have higher liver/blood ratios than NVP-2, demonstrating that these compounds have improved liver selectivity.

For rat experiments, the pharmacokinetics of compounds in blood collected from jugular vein and portal vein were analyzed in Sprague-Dawley (SD) rats following a single oral administration dose of 5 mg/kg compound suspension. Blood samples for determination of compound concentration were obtained from jugular vein and portal vein at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h following administration of compounds (n=3 for each compound). Collected liver and blood samples were analyzed using an LC-MS/MS method to quantify compound concentrations. The ratios of compound concentrations in blood collected from jugular vein versus portal vein in Sprague-Dawley (SD) rats following a single oral administration dose of 5 mg/kg compound suspension are summarized inFIG.2. The data inFIG.2show that Compounds 6 and 18 have a lower liver efflux/influx ratio than NVP-2, demonstrating that these compounds have improved liver selectivity.

Example 75: Tolerability and Safety Assessment Study

Based on the body weight, BALB/c nude mice were randomly assigned to respective groups using a computer-generated randomization procedure. Body weights of all animals were measured daily to record weight changes (relative to day 1) of BALB/c nude mice treated with vehicle and compounds. For routine monitoring, all study animals were monitored for behavior such as mobility, food and water consumption, body weight, eye/hair matting and any other abnormal effect. Any mortality and/or abnormal clinical signs were recorded. The animals were euthanized when they lost significant body mass (emaciated, obvious body weight loss >20%). Mean weight changes (relative to day 1) of BALB/c nude mice treated with vehicle and compounds are summarized inFIG.3. The data inFIG.3show that Compound 18 displays lower toxicity than NVP-2.

It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.