Compounds and compositions useful for treating disorders related to NTRK

This disclosure relates to inhibitors of NTRK that are active against wild-type NTRK and its resistant mutants, such as compounds of Formula (I):

BACKGROUND

Neurotrophic Tyrosine Receptor Kinase (NTRK) 1, 2 and 3 are receptor tyrosine kinases (RTKs) that activate multiple downstream pathways involved in cell proliferation and survival. Various genetic fusions, arising from aberrant chromosomal translocations of the genes coding for these RTKs, are implicated in the etiology of multiple cancers including high and low grade glioma, cholangiocarcinoma, papillary thyroid carcinoma, colon cancer and non-small cell lung cancer. A genomics analysis on the landscape of kinase fusions identified NTRK fusions in a wide array of additional cancer types including head and neck squamous cell carcinoma, pancreatic adenocarcinoma, sarcoma and melanoma, thereby providing further therapeutic rationale for deploying inhibitors of these kinases to treat multiple oncologic indications.

The identification of NTRK fusions as the underlying cause of certain cancers prompted the discovery and clinical development of several NTRK kinase inhibitors to treat tumors that harbor an NTRK fusion protein. Early clinical data support the viability of this approach in providing benefit to patients with specific human malignancies. Ultimately however, despite clear signs of clinical activity, most patients' cancers will become resistant to kinase inhibitor therapy leading to relapse and progression of the disease. Kinase reactivation via an intrinsic mutation is a frequent mechanism of resistance. When resistance occurs, the patient's treatment options are often very limited. There is thus a need for compounds that inhibit NTRK, as well as its resistant mutants.

SUMMARY OF THE INVENTION

The invention features compounds and pharmaceutical compositions comprising compounds of Formula (I) or pharmaceutically acceptable salts thereof, wherein:

Rings A and B are each independently selected from aryl, heteroaryl, cycloalkyl and heterocyclyl;

each R′ is independently selected from C1-C6alkyl, C1-C6heteroalkyl, halo, hydroxyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, cycloalkyl and cyano; or 2 R′ together with the atom(s) to which they are attached form a cycloalkyl or heterocyclyl ring;

Any of the compounds disclosed herein may be used, alone or in combination with another therapeutic agent, to treat any of the diseases disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, the terms a “patient,” “subject,” “individual,” and “host” refer to either a human or a non-human animal suffering from or suspected of suffering from a disease or disorder associated with aberrant NTRK expression (i.e., increased NTRK activity caused by signaling through NTRK) or biological activity.

“Treat” and “treating” such a disease or disorder refers to ameliorating at least one symptom of the disease or disorder. These terms, when used in connection with a condition such as a cancer, refer to one or more of: impeding growth of the cancer, causing the cancer to shrink by weight or volume, extending the expected survival time of the patient, inhibiting tumor growth, reducing tumor mass, reducing size or number of metastatic lesions, inhibiting the development of new metastatic lesions, prolonging survival, prolonging progression-free survival, prolonging time to progression, and/or enhancing quality of life.

The term “preventing” when used in relation to a condition or disease such as cancer, refers to a reduction in the frequency of, or delay in the onset of, symptoms of the condition or disease. Thus, prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic treatment relative to an untreated control population, and/or delaying the appearance of detectable cancerous growths in a treated population versus an untreated control population, e.g., by a statistically and/or clinically significant amount.

The term “therapeutic effect” refers to a beneficial local or systemic effect in animals, particularly mammals, and more particularly humans, caused by administration of a compound or composition of the invention. The phrase “therapeutically-effective amount” means that amount of a compound or composition of the invention that is effective to treat a disease or condition caused by over expression of NTRK or aberrant NTRK biological activity at a reasonable benefit/risk ratio.

The therapeutically effective amount of such substance will vary depending upon the subject and disease condition being treated, 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 skill in the art.

As used herein, “developing resistance” means that when a drug is first administered to the patient, the patient's symptoms improve, whether measured by decrease in tumor volume, a decrease in the number of new lesions, or some other means that a physician uses to judge disease progression; however, those symptoms stop improving, or even worsen at some point. At that time, the patient is said to have developed resistance to the drug.

“Aliphatic group” means a straight-chain, branched-chain, or cyclic hydrocarbon group and includes saturated and unsaturated groups, such as an alkyl group, an alkenyl group, and an alkynyl group.

“Alkylene” refers to a divalent radical of an alkyl group, e.g., —CH2—, —CH2CH2—, and CH2CH2CH2—.

“Alkenyl” means an aliphatic group containing at least one double bond.

“Alkoxyl” or “alkoxy” means an alkyl group having an oxygen radical attached thereto.

Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. The term “haloalkoxy” refers to an alkoxyl in which one or more hydrogen atoms are replaced by halo, and includes alkoxyl moieties in which all hydrogens have been replaced by halo (e.g., perfluoroalkoxy).

“Alkenylene” refers to an alkenyl group having two connecting points. For example, “ethenylene” represents the group —CH═CH—. Alkenylene groups can also be in an unsubstituted form or substituted form with one or more substituents.

“Alkynyl” refers to a straight or branched hydrocarbon chain containing 2-12 carbon atoms and characterized in having one or more triple bonds. Examples of alkynyl groups include, but are not limited to, ethynyl, propargyl, and 3-hexynyl. One of the triple bond carbons may optionally be the point of attachment of the alkynyl substituent.

“Alkynylene” refers to an alkynyl having two connecting points. For example, “ethynylene” represents the group —C≡C—. Alkynylene groups can also be in an unsubstituted form or substituted form with one or more substituents.

“Hydroxyalkylene” or “hydroxyalkyl” refers to an alkylene or alkyl moiety in which an alkylene or alkyl hydrogen atom is replaced by a hydroxyl group. Hydroxyalkylene or hydroxyalkyl includes groups in which more than one hydrogen atom has been replaced by a hydroxyl group.

“Aromatic ring system” is art-recognized and refers to a monocyclic, bicyclic or polycyclic hydrocarbon ring system, wherein at least one ring is aromatic.

“Aryl” refers to a monovalent radical of an aromatic ring system. Representative aryl groups include fully aromatic ring systems, such as phenyl, naphthyl, and anthracenyl, and ring systems where an aromatic carbon ring is fused to one or more non-aromatic carbon rings, such as indanyl, phthalimidyl, naphthimidyl, or tetrahydronaphthyl, and the like.

“Arylalkyl” or “aralkyl” refers to an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. Aralkyl includes groups in which more than one hydrogen atom has been replaced by an aryl group. Examples of “arylalkyl” or “aralkyl” include benzyl, 2-phenylethyl, 3-phenylpropyl, 9-fluorenyl, benzhydryl, and trityl groups.

“Aryloxy” refers to —O-(aryl), wherein the heteroaryl moiety is as defined herein.

“Halo” refers to a radical of any halogen, e.g., —F, —Cl, —Br, or —I.

“Haloalkyl” and “haloalkoxy” refers to alkyl and alkoxyl structures that are substituted with one or more halo groups or with combinations thereof. For example, the terms “fluoroalkyl” and “fluoroalkoxy” include haloalkyl and haloalkoxyl groups, respectively, in which the halo is fluorine.

“Haloalkylene” refers to a divalent alkyl, e.g., —CH2—, —CH2CH2—, and —CH2CH2CH2—, in which one or more hydrogen atoms are replaced by halo, and includes alkyl moieties in which all hydrogens have been replaced by halo.

“Heteroalkyl” refers to an optionally substituted alkyl, which has one or more skeletal chain atoms selected from an atom other than carbon, e.g., oxygen, nitrogen, sulfur, phosphorus or combinations thereof. A numerical range may be given, e.g. C1-C6heteroalkyl which refers to the number of carbons in the chain, which in this example includes 1 to 6 carbon atoms. For example, a —CH2OCH2CH3radical is referred to as a “C3” heteroalkyl. Connection to the rest of the molecule may be through either a heteroatom or a carbon in the heteroalkyl chain. “Heteroalkylene” refers to a divalent optionally substituted alkyl, which has one or more skeletal chain atoms selected from an atom other than carbon, e.g., oxygen, nitrogen, sulfur, phosphorus or combinations thereof.

“Carbocyclic ring system” refers to a monocyclic, bicyclic or polycyclic hydrocarbon ring system, wherein each ring is either completely saturated or contains one or more units of unsaturation, but where no ring is aromatic.

“Carbocyclyl” refers to a monovalent radical of a carbocyclic ring system. Representative carbocyclyl groups include cycloalkyl groups (e.g., cyclopentyl, cyclobutyl, cyclopentyl, cyclohexyl and the like), and cycloalkenyl groups (e.g., cyclopentenyl, cyclohexenyl, cyclopentadienyl, and the like).

“Cycloalkyl” refers to a cyclic, bicyclic, tricyclic, or polycyclic non-aromatic hydrocarbon groups having 3 to 12 carbons. Any substitutable ring atom can be substituted (e.g., by one or more substituents). The cycloalkyl groups can contain fused or spiro rings. Fused rings are rings that share a common carbon atom. Examples of cycloalkyl moieties include, but are not limited to, cyclopropyl, cyclohexyl, methylcyclohexyl, adamantyl, and norbornyl. In some embodiments, the cycloalkyl is bicyclo[3.1.0]hexanyl.

“Cycloalkylalkyl” refers to a -(cycloalkyl)-alkyl radical where cycloalkyl and alkyl are as disclosed herein. The “cycloalkylalkyl” is bonded to the parent molecular structure through the cycloalkyl group.

“Heteroaromatic ring system” is art-recognized and refers to monocyclic, bicyclic or polycyclic ring system wherein at least one ring is both aromatic and comprises at least one heteroatom (e.g., N, O or S); and wherein no other rings are heterocyclyl (as defined below). In certain instances, a ring which is aromatic and comprises a heteroatom contains 1, 2, 3, or 4 ring heteroatoms in such ring.

“Heteroaryl” refers to a monovalent radical of a heteroaromatic ring system. Representative heteroaryl groups include ring systems where (i) each ring comprises a heteroatom and is aromatic, e.g., imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrrolyl, furanyl, thiophenyl pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl; (ii) each ring is aromatic or carbocyclyl, at least one aromatic ring comprises a heteroatom and at least one other ring is a hydrocarbon ring or e.g., indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, pyrido[2,3-b]-1,4-oxazin-3-(4H)-one, 5,6,7,8-tetrahydroquinolinyl and 5,6,7,8-tetrahydroisoquinolinyl; and (iii) each ring is aromatic or carbocyclyl, and at least one aromatic ring shares a bridgehead heteroatom with another aromatic ring, e.g., 4H-quinolizinyl.

“Heterocyclic ring system” refers to monocyclic, bicyclic and polycyclic ring systems where at least one ring is saturated or partially unsaturated (but not aromatic) and comprises at least one heteroatom. A heterocyclic ring system can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted.

“Heterocyclyl” refers to a monovalent radical of a heterocyclic ring system. Representative heterocyclyls include ring systems in which (i) every ring is non-aromatic and at least one ring comprises a heteroatom, e.g., tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl; (ii) at least one ring is non-aromatic and comprises a heteroatom and at least one other ring is an aromatic carbon ring, e.g., 1,2,3,4-tetrahydroquinolinyl, 1,2,3,4-tetrahydroisoquinolinyl; and (iii) at least one ring is non-aromatic and comprises a heteroatom and at least one other ring is aromatic and comprises a heteroatom, e.g., 3,4-dihydro-1H-pyrano[4,3-c]pyridine, and 1,2,3,4-tetrahydro-2,6-naphthyridine.

“Heterocyclylalkyl” refers to an alkyl group substituted with a heterocyclyl group.

“Cyano” refers to a —CN radical.

“Hydroxy” or “hydroxyl” refers to —OH.

“Hydroxyalkylene” refers to a divalent alkyl, e.g., —CH2—, —CH2CH2—, and —CH2CH2CH2—, in which one or more hydrogen atoms are replaced by a hydroxy, and includes alkyl moieties in which all hydrogens have been replaced by hydroxy.

“Substituted”, whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at each position. Combinations of substituents envisioned under this invention are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable”, as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.

Unless otherwise indicated, when a disclosed compound is named or depicted by a structure without specifying the stereochemistry and has one or more chiral centers, it is understood to represent all possible stereoisomers of the compound, as well as enantiomeric mixtures thereof.

The “enantiomeric excess” or “% enantiomeric excess” of a composition can be calculated using the equation shown below. In the example shown below a composition contains 90% of one enantiomer, e.g., the S enantiomer, and 10% of the other enantiomer, i.e., the R enantiomer.
ee=(90−10)/100=80%.

Thus, a composition containing 90% of one enantiomer and 10% of the other enantiomer is said to have an enantiomeric excess of 80%.

The compounds or compositions described herein may contain an enantiomeric excess of at least 50%, 75%, 90%, 95%, or 99% of one form of the compound, e.g., the S-enantiomer. In other words such compounds or compositions contain an enantiomeric excess of the S enantiomer over the R enantiomer.

The compounds described herein may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example deuterium (2H), tritium (3H), carbon-13 (13C), or carbon-14 (14C). All isotopic variations of the compounds disclosed herein, whether radioactive or not, are intended to be encompassed within the scope of the present invention. In addition, all tautomeric forms of the compounds described herein are intended to be within the scope of the invention.

Compounds

The invention features compounds of Formula (I), or a stereoisomer, enantiomer, tautomer, or isotopically labeled form thereof, or a pharmaceutically acceptable salt of any of the foregoing, wherein:

Rings A and B are each independently selected from aryl, heteroaryl, cycloalkyl and heterocyclyl;

each R′ is independently selected from C1-C6alkyl, C1-C6heteroalkyl, halo, hydroxyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, cycloalkyl and cyano; or 2 R′ together with the atom(s) to which they are attached form a cycloalkyl or heterocyclyl ring; and

In some embodiments, Ring A is cycloalkyl. In some embodiments, Ring A is a 5-membered or 6-membered cycloalkyl ring. In some embodiments, Ring A is cyclopentyl or cyclohexyl. In some embodiments, Ring A is heterocyclyl. In some embodiments, Ring A is a 5-membered or 6-membered heterocyclyl. In some embodiments, Ring A is tetrahydropyranyl, tetrahydrofuranyl, or pyrrolidinyl. In some embodiments, Ring A is a cycloalkenyl ring. In some embodiments, Ring A is cyclopentenyl.

In some embodiments, Ring B is aryl. In some embodiments, Ring B is phenyl. In some embodiments, Ring B is heteroaryl. In some embodiments, Ring B is pyridyl. In some embodiments, Ring B is heterocyclyl. In some embodiments, Ring B is pyrrolidinyl.

wherein “*” represents a portion of L2bound to ring B. In some embodiments, L1is —NH—, L2is —C(O)— and ring B is pyrrolidinyl. In some embodiments, L1is —NH—, L2is —C(O)— and ring B is pyrrolidin-1-yl.

In some embodiments, each R1is independently selected from hydrogen and C1-C6alkyl substituted with 0-5 occurrences of Rb. In some embodiments, each R1is independently selected from hydrogen and —CH3.

In some embodiments, each RAis independently selected from hydroxyl, C1-C6alkyl, C1-C6alkoxyl, halo, —C(O)—N(R1)(R1), —C(O)OR1, —S(O)2R1, and C1-C6haloalkyl. In some embodiments, each RAis additionally and independently selected from —CN, oxetanyl, and C1-C6hydroxyalkyl, or two RAbound to adjacent ring carbon atoms on ring A are taken together to form a C3-C6cycloalkyl fused to ring A. In some embodiments each RAis independently selected from hydroxyl, fluoro, oxetan-3-yl, —CHF2, —CH2CH3, —C(CH3)2OH, —OCH3, —C(O)N(CH3)2, —C(O)OCH3, —S(O)2CH3; or two RAbound to adjacent ring carbon atoms on ring A are taken together to form a cyclopropyl fused to ring A.

In some embodiments, each RBis independently selected from halo, C1-C6alkyl, cyano, C1-C6alkoxyl, aryl, heteroaryl, and C1-C6haloalkoxy. In some embodiments, each RBis additionally selected from oxo.

In some embodiments, Ring B is pyrrolidinyl and at least one RBis optionally substituted aryl or heteroaryl. In some embodiments, Ring B is pyrrolidinyl and at least one RBis optionally substituted phenyl or pyridyl.

In some embodiments, Ring B is pyrrolidinyl and one additional RB, if present, is fluoro.

In some embodiments, Ring B is other than pyrrolidinyl, and each RBis independently selected from chloro, fluoro, oxo, —CH3, —CF3, —CN, —OCH3, —OCF3, and —OCHF2.

In another aspect, the invention features compounds of Formula (Ia):

or a stereoisomer, enantiomer, tautomer, or isotopically labeled form thereof, or a pharmaceutically acceptable salt of any of the foregoing, wherein:

Ring A is selected from aryl, heteroaryl, cycloalkyl and heterocyclyl;

each R′ is independently selected from C1-C6alkyl, C1-C6heteroalkyl, halo, hydroxyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, cycloalkyl and cyano; or 2 R′ together with the atom(s) to which they are attached form a cycloalkyl or heterocyclyl ring; and

In some embodiments, Ring A is cycloalkyl. In some embodiments, Ring A is a 5-membered or 6-membered cycloalkyl ring. In some embodiments, Ring A is cyclopentyl or cyclohexyl. In some embodiments, Ring A is heterocyclyl. In some embodiments, Ring A is a 5-membered or 6-membered heterocyclyl. In some embodiments, Ring A is tetrahydropyran, tetrahydrofuran, or pyrrolidinyl. In some embodiments, Ring A is a cycloalkenyl ring. In some embodiments, Ring A is cyclopentenyl.

In some embodiments, each R1is independently selected from hydrogen and C1-C6alkyl substituted with 0-5 occurrences of Rb. In some embodiments, each R1is independently selected from hydrogen and —CH3.

In some embodiments, each RAis independently selected from hydroxyl, C1-C6alkyl, C1-C6alkoxyl, halo, —C(O)—N(R1)(R1), —C(O)OR1, —S(O)2R1, and C1-C6haloalkyl. In some embodiments, each RAis additionally and independently selected from —CN, oxetanyl, and C1-C6hydroxyalkyl, or two RAbound to adjacent ring carbon atoms on ring A are taken together to form a C3-C6cycloalkyl fused to ring A. In some embodiments each RAis independently selected from hydroxyl, fluoro, oxetan-3-yl, —CHF2, —CH2CH3, —C(CH3)2OH, —OCH3, —C(O)N(CH3)2, —C(O)OCH3, —S(O)2CH3; or two RAbound to adjacent ring carbon atoms on ring A are taken together to form a cyclopropyl fused to ring A.

In some embodiments, each RBis independently selected from halo, C1-C6alkyl, cyano, C1-C6alkoxyl, aryl, heteroaryl, and C1-C6haloalkoxy. In some embodiments, each RBis additionally selected from oxo.

In some embodiments, when ring B is pyrrolidinyl one additional RB, if present, is fluoro.

In some embodiments, p is 0, 1 or 2.

In some embodiments, q is 1, 2 or 3.

In another aspect, the invention features compounds of Formula (II):

or a stereoisomer, enantiomer, tautomer, or isotopically labeled form thereof, or a pharmaceutically acceptable salt of any of the foregoing, wherein:

Rings A and B are each independently selected from aryl, heteroaryl, cycloalkyl and heterocyclyl;

R1bis selected from hydrogen and C1-C6alkyl;

each R′ is independently selected from C1-C6alkyl, C1-C6heteroalkyl, halo, hydroxyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, cycloalkyl and cyano; or 2 R′ together with the atom(s) to which they are attached form a cycloalkyl or heterocyclyl ring; and

In some embodiments, Ring A is cycloalkyl. In some embodiments, Ring A is a 5-membered or 6-membered cycloalkyl ring. In some embodiments, Ring A is cyclopentyl or cyclohexyl. In some embodiments, Ring A is heterocyclyl. In some embodiments, Ring A is a 5-membered or 6-membered heterocyclyl. In some embodiments, Ring A is tetrahydropyran, tetrahydrofuran, or pyrrolidinyl. In some embodiments, Ring A is a cycloalkenyl ring. In some embodiments, Ring A is cyclopentenyl.

In some embodiments, Ring B is aryl. In some embodiments, Ring B is phenyl. In some embodiments, Ring B is heteroaryl. In some embodiments, Ring B is pyridyl.

In some embodiments, each R1is independently selected from hydrogen and C1-C6alkyl substituted with 0-5 occurrences of Rb.

In some embodiments, each RAis independently selected from hydroxyl, C1-C6alkyl, C1-C6alkoxyl, halo, —C(O)—N(R1)(R1), —C(O)OR1, —S(O)2R1, and C1-C6haloalkyl. In some embodiments, each RAis additionally and independently selected from —CN, oxetanyl, and C1-C6hydroxyalkyl, or two RAbound to adjacent ring carbon atoms on ring A are taken together to form a C3-C6cycloalkyl fused to ring A. In some embodiments each RAis independently selected from hydroxyl, fluoro, oxetan-3-yl, —CHF2, —CH2CH3, —C(CH3)2OH, —OCH3, —C(O)N(CH3)2, —C(O)OCH3, —S(O)2CH3; or two RAbound to adjacent ring carbon atoms on ring A are taken together to form a cyclopropyl fused to ring A.

In some embodiments, each R′ is independently selected from C1-C6alkyl, C1-C6haloalkyl and C1-C6hydroxyalkyl; or 2 R′ together with the atom(s) to which they are attached form a cycloalkyl or heterocyclyl ring. In some embodiments one R′ is hydrogen, and the other R′ is selected from hydrogen, C1-C6alkyl, C1-C6haloalkyl and C1-C6hydroxyalkyl; or 2 R′ together with the atom(s) to which they are attached form a cycloalkyl ring. In some embodiments one R′ is hydrogen, and the other R′ is selected from hydrogen, —CH2OH, —CH3, or —CF3, or 2 R′ together with the atom(s) to which they are attached form a cycloprop-1,1-diyl ring.

In some embodiments, p is 0, 1 or 2.

In some embodiments, q is 0, 1, 2 or 3.

Although, as indicated above, various embodiments and aspects thereof for a variable in Formula (I), (Ia), or (II), may be selected from a group of chemical moieties, the invention also encompasses as further embodiments and aspects thereof situations where such variable is: a) selected from any subset of chemical moieties in such a group; and b) any single member of such a group.

Although various embodiments and aspects thereof are set forth (or implied, as discussed in the preceding paragraph) individually for each variable in Formula (I), (Ia) and (II), the invention encompasses all possible combinations of the different embodiments and aspects for each of the variables in Formula (I), (Ia), and (II).

The structures, as well as the NMR and LCMS data of exemplary compounds of the invention are shown inFIG. 1. In certain embodiments, the compound of the invention is selected from the group consisting of any one of the compounds inFIG. 1and pharmaceutically acceptable salts, solvates, hydrates, tautomers, stereoisomers, and isotopically labeled derivatives thereof.

The invention also features pharmaceutical compositions containing a pharmaceutically acceptable carrier and any compound of Formulas (I), (Ia) and (II).

Pharmaceutically acceptable salts of these compounds are also contemplated for the uses described herein.

Pharmaceutical Compositions

The pharmaceutically acceptable compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs. Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topically-transdermal patches may also be used.

For topical applications, the pharmaceutically acceptable compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers.

Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutically acceptable compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

Dosages

Toxicity and therapeutic efficacy of compounds of the invention, including pharmaceutically acceptable salts and deuterated variants, can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The LD50is the dose lethal to 50% of the population. The ED50is the dose therapeutically effective in 50% of the population. The dose ratio between toxic and therapeutic effects (LD50/ED50) is the therapeutic index. Compounds that exhibit large therapeutic indexes are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

Treatment

NTRK fusions have been implicated in several types of cancers. These fusions harbor an intact NTRK kinase domain that is identical to the native or wild-type form of the receptor; therefore, as used herein, any NTRK protein (NTRK1, 2 or 3) with the same kinase domain as wild-type NTRK will be referred to as “wild-type NTRK.” Mutations can occur in the NTRK kinase domain, leading to mutants that are resistant to kinase inhibitor therapy. These resistance mutations can be predicted using structural biology and computational analyses, as well as by examining codon sequences in which a sequence change gives rise to a codon for a different amino acid. Alternatively, resistance mutations for a given inhibitor can be identified experimentally by administration of that inhibitor (e.g., a known NTRK wild-type inhibitor) and exposing cells to a mutation-promoting agent, such as ENU. The cells are washed, then plated with increasing concentrations (2-100× proliferation IC50) of the compound of choice. The wells with cellular outgrowth are then collected after 3-4 weeks. In particular, a mutation at amino acid position 595 within the NTRK fusion (NTRK1 wt numbering), effecting a change from a glycine to an arginine residue (heretofore designated ‘G595R’) was identified via both methods. This mutation was subsequently demonstrated to confer significant resistance to two NTRK inhibitors that are being clinically evaluated (shown in the table below). As shown in the table, the compounds are active against the wild-type NTRK, but are markedly less active against the G595R mutant form of the NTRK fusion.

Accordingly, in another aspect the invention provides a method for treating a subject suffering from a condition mediated by aberrant neurotrophic tyrosine receptor kinase (NTRK) activity, comprising administering to the subject a therapeutically effective amount of a compound or pharmaceutical composition of a compound described herein.

The invention provides compounds that inhibit both wild-type NTRK and resistant G595R mutants of NTRK.

In another aspect, the invention provides a method for treating a subject who has developed resistance to a cancer treatment, comprising administering to the subject a therapeutically effective amount of a compound or pharmaceutical composition of a compound described herein.

Furthermore, the inhibitors can be selective for wild-type NTRK, over other kinases, thus leading to reduced toxicities associated with inhibiting other kinases. Because of their activity against wild-type and mutant NTRK, the compounds described herein can be used to treat a patient with a condition associated with aberrant NTRK activity. They can also be used to treat various cancers. In some embodiments, the cancer is selected from non-small cell lung cancer, breast cancer, melanoma, low and high grade glioma, glioblastoma, pediatric astrocytoma, colorectal cancer, papillary thyroid carcinoma, pancreatic adenocarcinoma, head and neck cancer, cholangiocarcinoma, acute myelogenous leukemia, secretory breast cancer, salivary cancer and spitzoid neoplasms.

The compounds can also be used to treat a patient who has developed resistance to a wild-type NTRK inhibitor, or a patient with a mutant form of NTRK, such as the G595R mutant. The method includes the step of administering a compound or composition of the invention that is active against the NTRK resistant mutant. By “active” is meant that a compound has an IC50of less than 1 μM, 500 nM, 250 nM, 100 nM, 75 nM, 50 nM, 25 nM, 10 nM, or 5 nM when measured in a biochemical assay, against at least one resistant mutant.

The compounds and compositions described herein can be administered alone or in combination with other compounds, including other NTRK-modulating compounds, or other therapeutic agents. In some embodiments, the compound or composition of the invention may be administered in combination with one or more compounds selected from Cabozantinib (COMETRIQ), Vandetanib (CALPRESA), Sorafenib (NEXAVAR), Sunitinib (SUTENT), Regorafenib (STAVARGA), Ponatinib (ICLUSIG), Bevacizumab (AVASTIN), Crizotinib (XALKORI), or Gefitinib (IRESSA). The compound or composition of the invention may be administered simultaneously or sequentially with the other therapeutic agent by the same or different routes of administration. The compound of the invention may be included in a single formulation with the other therapeutic agent or in separate formulations.

Synthesis

Compounds of the invention, including salts and N-oxides thereof, can be prepared using known organic synthesis techniques and can be synthesized according to any of numerous possible synthetic routes, such as those in the Schemes below. The reactions for preparing compounds of the invention can be carried out in suitable solvents which can be readily selected by one of skill in the art of organic synthesis. Suitable solvents can be substantially non-reactive with the starting materials (reactants), the intermediates, or products at the temperatures at which the reactions are carried out, e.g., temperatures which can range from the solvent's freezing temperature to the solvent's boiling temperature. A given reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the particular reaction step, suitable solvents for a particular reaction step can be selected by the skilled artisan.

Preparation of compounds of the invention can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Wuts and Greene, Protective Groups in Organic Synthesis, 4th ed., John Wiley & Sons: New Jersey, (2006), which is incorporated herein by reference in its entirety.

Reactions can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance (NMR) spectroscopy (e.g.,1H or13C), infrared (IR) spectroscopy, spectrophotometry (e.g., UV-visible), mass spectrometry (MS), or by chromatographic methods such as high performance liquid chromatography (HPLC) or thin layer chromatography (TLC). Analytical instruments and methods for compound characterization:

Unless otherwise indicated, all liquid chromatography-mass spectrometry (LC-MS) data (sample analyzed for purity and identity) were obtained with an Agilent model-1260 LC system using an Agilent model 6120 mass spectrometer utilizing ES-API ionization fitted with an Agilent Poroshel 120 (EC-C18, 2.7 um particle size, 3.0×50 mm dimensions) reverse-phase column at 22.4 degrees Celsius. The mobile phase consisted of a mixture of solvent 0.1% formic acid in water and 0.1% formic acid in acetonitrile. A constant gradient from 95% aqueous/5% organic to 5% aqueous/95% organic mobile phase over the course of 4 minutes was utilized. The flow rate was constant at lmL/min.

Preparative HPLC was performed on a Shimadzu Discovery VP® Preparative system fitted with a Luna 5u C18(2) 100A, AXIA packed, 250×21.2 mm reverse-phase column at 22.4 degrees Celsius. The mobile phase consisted of a mixture of solvent 0.1% formic acid in water and 0.1% formic acid in acetonitrile. A constant gradient from 95% aqueous/5% organic to 5% aqueous/95% organic mobile phase over the course of 25 minutes was utilized. The flow rate was constant at 20 mL/min. Reactions carried out in a microwave were done so in a Biotage Initiator microwave unit.

Preparative HPLC to resolve chiral mixtures was performed on a Thar SFC Pre-80 instrument fitted with a Chiralpak AS-H column (5 mm, 3.0 cm id×25 cm L). The mobile phases consisted of SFC CO2(A) and MeOH/0.1% NH4OH(B). A constant gradient from 67% to 33% (B) was maintained at a flow rate of 65 g/min, with a system back pressure of 100 bar. The separation progress was monitored by UV detection at a wavelength of 220 nm.

Silica gel chromatography was performed on either a Teledyne Isco CombiFlash® Rf unit or a Biotage® Isolera Four unit.

Unless otherwise indicated, all 1H NMR spectra were obtained with a Varian 400 MHz Unity Inova 400 MHz NMR instrument (acquisition time=3.5 seconds with a 1 second delay; 16 to 64 scans). Where characterized, all protons were reported in DMSO-d6solvent as parts-per million (ppm) with respect to residual DMSO (2.50 ppm).

EXAMPLES

The following examples are intended to be illustrative, and are not meant in any way to be limiting.

The below Schemes are meant to provide general guidance in connection with preparing the compounds of the invention. One skilled in the art would understand that the preparations shown in the Schemes can be modified or optimized using general knowledge of organic chemistry to prepare various compounds of the invention.

General Synthesis 1:

For certain compounds, the general synthesis begins with appropriate nitrogen-protection (P) of a di-halide substituted 1H-pyrazolo[3,4-d]pyrimidine. The nitrogen-protected bicycle can be substituted at the halide on the pyrimidine ring with an appropriately substituted Ring A under appropriate conditions, for example, nucleophilic aromatic substitution reaction conditions, using a base, such as diisopropylethylamine (DIPEA), in a polar solvent such as dioxane to provide the bicycle substituted with Ring A. The halide of the pyrazole ring can be substituted under Palladium-mediated carbonyl insertion reaction conditions followed by hydrolysis to provide the resultant carboxylic acid. The carboxylic acid can be reacted with Ring B under appropriate coupling conditions, for example amide coupling reaction conditions, to afford the nitrogen-protected compound substituted with Rings A and B. The removal of the protecting group can afford compounds of Formula I.

A slightly more specific version of General Synthesis scheme 1 is shown above in Synthetic Protocol 1. The synthetic protocol begins with SEM-group protection of 3-bromo-4-chloro-1H-pyrazolo[3,4-d]pyrimidine 1. The SEM-protected heterocycle 2 can be substituted with an amino alcohol under nucleophilic aromatic substitution reaction conditions using a base such as diisopropylethylamine (DIPEA) in a polar solvent such as dioxane to provide the amine-substituted heterocycle 3. The 3-bromo pyrazolo pyrimidine 3 is subjected to a palladium-mediated carbonyl insertion reaction in a DMF-MeOH solvent mixture to afford the methyl ester 4. Following the hydrolysis of the ester with NaOH treatment, the carboxylic acid is 5 reacted with a benzyl amine or a pyrrolidine under amide coupling reaction conditions to afford the SEM-protected compound 6. The SEM-protecting group can be removed using TBAF or under acidic conditions to afford the final compound 7. The compounds described below were prepared using General Synthesis 1, 2 or 3, as further detailed in Synthetic Protocol 1, 2, or 3, respectively.

General Synthesis 2:

For certain compounds, the general synthesis begins with appropriate nitrogen-protection (P) of 4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylic acid. The nitrogen-protected bicycle can be chlorinated and coupled with an amine in the presence of a chlorinating reagent such as thionyl chloride. The resulting compound can be substituted at the halide on the pyrimidine ring with an appropriately substituted Ring A under appropriate conditions, for example, nucleophilic aromatic substitution reaction conditions, using a base, such as diisopropylethylamine (DIPEA), in a polar solvent such as dioxane to provide the bicycle substituted with Ring A. The removal of the protecting group can afford compounds of Formula I. Compounds described below can be prepared using this general synthesis. Further, chiral HPLC can be employed to resolve chiral mixtures of compounds of Formula I, (Ia), (Ia-1), (Ia-2), (Ib), (Ib-1, (Ib-2), II, (IIa), (IIb), (IIc).

A slightly more specific version of General Synthesis scheme 2 is shown above in Synthetic Protocol 2. The synthetic protocol begins with SEM-protected 4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylic acid 1 which can be chlorinate with thionyl chloride/DMF and then coupled with a benzyl amine or a pyrrolidine under mild heat to afford the SEM-protected compound 2. The SEM-protected heterocycle 2 can be substituted with an amino alcohol under nucleophilic aromatic substitution reaction conditions using a base such as diisopropylethylamine (DIPEA) in a polar solvent such as dioxane to provide the amine-substituted heterocycle 3. The SEM-protecting group can be removed using TBAF or under acidic conditions to afford the final compound 4.

General Synthesis 3

For certain compounds, the general synthesis begins with appropriate nitrogen-protection (P) of 4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylic acid. The carboxylic acid can be coupled to an amine using amide coupling conditions. The resulting compound can be chlorinated using thionyl chloride followed by substitution at the chloride on the pyrimidine ring with an appropriately substituted Ring A under appropriate conditions, for example, nucleophilic aromatic substitution reaction conditions, using a base, such as diisopropylethylamine (DIPEA), in a polar solvent such as dioxane to provide the bicycle substituted with Ring A. The removal of the protecting group can afford compounds of Formula I. Compounds described below can be prepared using this general synthesis. Further, chiral HPLC can be employed to resolve chiral mixtures of compounds of Formula I, (Ia), (Ia-1), (Ia-2), (Ib), (Ib-1, (Ib-2), II, (IIa), (IIb), (IIc).

A slightly more specific version of General Synthesis scheme 3 is shown above in Synthetic Protocol 3. The synthetic protocol begins with SEM-protected 4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylic acid 1 which can be coupled with a benzyl amine or a pyrrolidine under amide coupling conditions. The SEM-protected heterocycle 2 can be chlorinated with thionyl chloride/DMF and then substituted with an amino alcohol under nucleophilic aromatic substitution reaction conditions using a base such as diisopropylethylamine (DIPEA) in a polar solvent such as dioxane to provide the amine-substituted heterocycle 3. The SEM-protecting group can be removed using TBAF or under acidic conditions to afford the final compound 4.

All of the compounds set forth inFIG. 1, as well as other compounds of the invention were prepared using one of three general synthesis schemes and protocols depicted above. Further, chiral HPLC can be employed to resolve chiral mixtures of compounds of Formula I, (Ia), (Ia-1), (Ia-2), (Ib), (Ib-1, (Ib-2), II, (IIa), (IIb), (IIc). Certain specific examples of synthesis are set forth in the Examples.

Example 1. Synthesis of Compound 45

Step 1: Synthesis of 3-bromo-4-chloro-1-((2-(trimethylsilyl)ethoxy)methyl)-H-pyrazolo[3,4-d]pyrimidine

Step 2: Synthesis of (1R,2R)-2-((3-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino)cyclopentan-1-ol

Step 3: Synthesis of methyl 4-(((1R,2R)-2-hydroxycyclopentyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylate

Step 5: Synthesis of ((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)(4-(((1R,2R)-2-hydroxycyclopentyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)methanone

Step 6: Synthesis of ((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)(4-(((1R,2R)-2-hydroxycyclopentyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)methanone

To a solution of ((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)(4-(((1R,2R)-2-hydroxycyclopentyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)methanone (20.00 mg, 35.80 umol) in dioxane (20 mL) was added TBAF (80.61 mg, 358.00 umol) at 20° C., and the reaction was heated at 80° C. for 16 hrs. After TLC (EtOAc, Rf=0.1) showed the reaction was complete, the solution was concentrated And 10 mL of H2O was added to the residue. The solution was extracted with EtOAc (10 mL*3), and the organic layer was dried over Na2SO4and concentrated. The residue was purified by acidic preparative HPLC (MeOH/H2O/TFA solvent system) to give ((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)(4-(((1R,2R)-2-hydroxycyclopentyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)methanone (11.10 mg, yield: 72.37%) as a brown solid.

Example 2. Synthesis of Compound 97 and Compound 98

Step 1: Synthesis of (1R,2R,4R)-2-((3-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino)-4-(methylsulfonyl)cyclopentan-1-ol

Step 2: Synthesis of methyl 4-(((1R,2R,4R)-2-hydroxy-4-(methylsulfonyl)cyclopentyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylate

To a mixture of (1R,2R,4R)-2-((3-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino)-4-(methylsulfonyl)cyclopentan-1-ol (350.00 mg, 691.03 umol) in MeOH (10.00 mL)/DMF (2.00 mL) was added Et3N (139.85 mg, 1.38 mmol) and Pd(dppf)Cl2(25.28 mg, 34.55 umol). After addition, the mixture was stirred at 75° C. for 16 hrs under CO (50 Psi). Once LCMS showed the reaction was complete, the mixture was concentrated to give the crude product, which was purified by preparative TLC (PE:EtOAc=0:1) to give methyl 4-(((1R,2R,4R)-2-hydroxy-4-(methylsulfonyl)cyclopentyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylate (250.00 mg, yield: 74.50%) as a red solid.

Step 3: Synthesis of 4-(((1R,2R,4R)-2-hydroxy-4-(methylsulfonyl)cyclopentyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylic acid

Step 4: Synthesis of N-(2,5-difluorobenzyl)-4-(((1R,2R,4R)-2-hydroxy-4-(methylsulfonyl)cyclopentyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide

Step 5: Synthesis of N-(2,5-difluorobenzyl)-4-(((1R,2R,4R)-2-hydroxy-4-(methylsulfonyl)cyclopentyl)amino)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide

N-(2,5-difluorobenzyl)-4-(((1R,2R,4R)-2-hydroxy-4-(methylsulfonyl)cyclopentyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide (50.00 mg, 83.79 umol) in DCM (5.00 mL) was stirred in a mixture of TFA (5.00 mL) at 20° C. for 16 hrs, after which LCMS showed the reaction was complete. The mixture was concentrated to give the crude product, which was purified by preparative HPLC (TFA) and chiral HPLC (retention times of the resolved isomers were 7.46 min and 9.20 min, respectively). N-(2,5-difluorobenzyl)-4-(((1R,2R,4R)-2-hydroxy-4-(methylsulfonyl)cyclopentyl)amino)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide (2.80 mg, yield: 7.16%) and N-(2,5-difluorobenzyl)-4-(((1S,2S,4S)-2-hydroxy-4-(methylsulfonyl)cyclopentyl)amino)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide (4.00 mg, yield: 10.23%) were obtained as white solids. LC-MS conditions for these compounds were as follows: flow rate=0.8 mL·min, mobile phase: from 99% [water+0.375% c v/v TFA] and 1% [CH3CN+0.188% c v/v TFA], under this condition for 0.4 min, then changed to 10% [water+0.375% c v/v TFA] and 90% [CH3CN+0.188% c v/v TFA] in 3.0 min, then changed to 100% [CH3CN+0.188‰ v/v TFA] in 0.45 min, finally changed to 99% [water+0.375% c v/v TFA] and 1% [CH3CN+0.188% c v/v TFA] in 0.01 min, then under this condition for 0.64 min; 98.887% purity and 96.551%, respectively.

Example 3. Synthesis of Compound 20 and Compound 21

To a mixture of 3-bromo-4-chloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidine (600.00 mg, 1.65 mmol) and 2-amino-5-fluoro-cyclopentanol (196.58 mg, 1.65 mmol) in dioxane (15 mL) was added DIPEA (426.49 mg, 3.30 mmol). The mixture was stirred at 110° C. for 16 hrs, after which TLC (PE/EtOAc=1:1) showed the reaction was complete. The mixture was cooled to 25° C. and concentrated in reduced pressure at 50° C. To the residue was added EtOAc (50 mL), and the organic phase was washed with H2O (20 mL*3), dried over Na2SO4, filtered and concentrated in vacuum to afford (1S,2R,5S)-2-((3-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino)-5-fluorocyclopentan-1-ol (600.00 mg, crude). The residue was used directly in next step without further purification.

To a solution of (1S,2R,5S)-2-((3-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino)-5-fluorocyclopentan-1-ol (600.00 mg, 1.34 mmol) in MeOH/DMF (20 mL, v:v=2/1) was added Pd(dppf)Cl2(49.17 mg, 67.21 umol) and Et3N (408.03 mg, 4.03 mmol) under N2. The suspension was degassed under vacuum and purged with CO several times. The mixture was stirred under CO (50 psi) at 70° C. for 16 hrs, after which TLC (PE/EtOAc=1:1) showed the starting material was consumed completely. The reaction mixture was filtered and the filtrate was concentrated to afford methyl 4-(((1R,2S,3S)-3-fluoro-2-hydroxycyclopentyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylate (700.00 mg, crude). The crude product was used directly without purification.

To a solution of methyl 4-(((1R,2S,3S)-3-fluoro-2-hydroxycyclopentyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylate (700.00 mg, 1.65 mmol) in MeOH/H2O (15 mL, v/v=2/1) was added NaOH (132.00 mg, 3.30 mmol) in one portion, which was stirred at 25° C. for 2 hrs. After LCMS showed the reaction was complete, the mixture was concentrated in reduced pressure at 40° C. The aqueous phase was adjusted to pH=4 and filtered to afford 4-(((1R,2S,3S)-3-fluoro-2-hydroxycyclopentyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylic acid (700.00 mg, crude) as a white solid.

To a mixture of 4-(((1R,2S,3S)-3-fluoro-2-hydroxycyclopentyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylic acid (100.00 mg, 243.01 umol) and T3P (231.97 mg, 729.04 umol) was added Et3N (49.18 mg, 486.03 umol) in DMF (2.00 mL) at 25° C., followed by the addition of (2,5-difluorophenyl)methanamine (69.57 mg, 486.03 umol) in one portion after 10 min. The mixture was stirred at 25° C. for 16 hrs. After LCMS showed the reaction was complete, the mixture was concentrated under reduced pressure at 60° C. to afford N-(2,5-difluorobenzyl)-4-(((1R,2S,3S)-3-fluoro-2-hydroxycyclopentyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide (200 mg, crude). The residue was not purified and used directly.

A mixture of N-(2,5-difluorobenzyl)-4-(((1R,2S,3S)-3-fluoro-2-hydroxycyclopentyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide (200.00 mg, 372.70 umol) in TFA/DCM (15.00 mL, v/v=1/1) was stirred at 25° C. for 3 hrs, then concentrated under reduced pressure at 30° C. To the residue was added MeOH (20 mL) and KOAc (100 mg), and the mixture was stirred for 16 hrs at 25° C. Once LCMS showed the reaction was complete, the mixture was concentrated under reduced pressure at 30° C. The residue was purified by acidic preparative HPLC followed by chiral preparative HPLC to afford N-(2,5-difluorobenzyl)-4-(((1R,2S,3 S)-3-fluoro-2-hydroxycyclopentyl)amino)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide (25.00 mg, yield: 16.51%) as a white solid and N-(2,5-difluorobenzyl)-4-(((1S,2R,3R)-3-fluoro-2-hydroxycyclopentyl)amino)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide (30.00 mg, yield: 19.81%) as a grey solid. LC-MS conditions for these compounds were as follows: flow rate=0.8 mL·min−1, mobile phase: from 95% [water+10 mM NH4HCO3] and 5% CH3CN, under this condition for 0.4 min, then changed to 10% [water+10 mM NH4HCO3] and 90% CH3CN in 2.6 min, then changed to 100% CH3CN in 0.85 min, finally changed to 95% [water+10 mM NH4HCO3] and 5% CH3CN in 0.01 min, then under this condition for 0.64 min. 97.125% purity and 97.690% purity, respectively.

Synthesis of Amine Intermediates

Example 4. Synthesis of (1R,2S,3R)-3-aminocyclopentane-1,2-diol

Example 5. Synthesis of (1R,2R,3S,4R,5S)-4-Aminobicyclo[3.1.0]hexane-2,3-diol

To a solution of (1S,2S,4R)-2-azido-4-(benzyloxy)cyclopentyl acetate (8.80 g, 31.97 mmol) in EtOH (100.00 mL) was added Pd(OH)2(4.42 g, 31.97 mmol) under N2. The suspension was degassed under vacuum and purged with H2several times. The mixture was stirred under H2(50 psi) at 70° C. for 32 hrs. TLC (PE:EtOAc=2:1) showed the starting material was consumed completely. The reaction mixture was filtered and the filtrate was concentrated. The crude product was purified by silica gel chromatography (PE:EtOAc=10:1, 2:1) to give (1S,2S,4R)-2-((tert-butoxycarbonyl)amino)-4-hydroxycyclopentyl acetate (3.80 g, yield: 45.84%) as a yellow solid.

To a mixture of (1S,2S,4S)-2-((tert-butoxycarbonyl)amino)-4-fluorocyclopentyl acetate (700.00 mg, 2.68 mmol) in MeOH (20.00 mL) was added NaOH (160.80 mg, 4.02 mmol) in one portion. The mixture was stirred at 25° C. for 1 hr. TLC (PE:EtOAc=3:1) showed the reaction was complete. The mixture was concentrated under reduced pressure at 30° C. to afford tert-butyl ((1S,2S,4S)-4-fluoro-2-hydroxycyclopentyl)carbamate (650.00 mg, crude) as a white solid.

Step 1: Preparation of (1R,2R,4S)-2-((tert-butoxycarbonyl)Amino)-4-((methylsulfonyl)oxy)cyclopentyl acetate

To a mixture of (1R,2R,4S)-2-((tert-butoxycarbonyl)amino)-4-hydroxycyclopentyl acetate (3.00 g, 11.57 mmol) and Et3N (4.68 g, 46.28 mmol) in DCM (50.00 mL) was added dropwise MsCl (3.98 g, 34.71 mmol) at 0° C., then the mixture was stirred at 20° C. for 3 hrs. TLC (PE:EtOAc=1:1) showed the reaction was complete. The mixture was washed with water (100 mL*3), then the organic layer was dried over Na2SO4and concentrated to give (1R,2R,4S)-2-((tert-butoxycarbonyl)amino)-4-((methylsulfonyl)oxy)cyclopentyl acetate (3.5 g, crude: 100%) as a yellow solid.

To a mixture of (1R,2R,4S)-2-((tert-butoxycarbonyl)amino)-4-((methylsulfonyl)oxy)cyclopentyl acetate (3.50 g, 10.37 mmol) in DMF (30.00 mL) was added NaSMe (4.36 g, 12.45 mmol). The mixture was then stirred at 90° C. for 2 hrs, and TLC (PE:EtOAc=2:1) showed the reaction was complete. The mixture was concentrated to give the crude (1R,2R,4R)-2-((tert-butoxycarbonyl)amino)-4-(methylthio)cyclopentyl acetate (3.00 g, crude 100%) as a yellow solid.

To a mixture of CeCl3.7H2O (24.00 g, 64.42 mmol) in MeOH (120.00 mL) was added cyclopent-2-en-1-one (5.00 g, 60.90 mmol) at 15° C. After 5 min, NaBH4(4.61 g, 121.80 mmol) was added into the mixture in portions at 0° C. The resulting mixture was stirred at 25° C. for 1 hr, after which TLC (PE:EtOAc=5:1) showed several spots were generated and a part of the starting material was remained. The reaction was quenched by H2O (100 mL) and the organic solvent was concentrated in vacuum. To the residue was added H2O (300 mL), followed by extraction with MTBE (200 mL*3). The combined organic layers were dried over Na2SO4and concentrated under vacuum to give the crude product cyclopent-2-en-1-ol (3.00 g, crude) as a brown oil. It was used directly to the next step without further purification.1H-NMR (400 MHz, CDCl3) δ ppm 5.91 (d, 1H, J=4.8 Hz), 5.77-5.76 (m, 1H), 4.79 (d, 1H, J=3.6 Hz), 2.47-2.42 (m, 1H), 2.21-2.15 (m, 2H), 1.64-1.59 (m, 1H).

Example 10. Synthesis of 3-Fluoro-5-((2R,4S)-4-fluoropyrrolidin-2-yl)pyridine

To the mixture of tert-butyl ((2R)-2-((tert-butyldimethylsilyl)oxy)-4-(5-fluoropyridin-3-yl)-4-hydroxybutyl)carbamate (8.70 g, 20.98 mmol, 1.00 eq) and Et3N (31.84 g, 314.70 mmol, 15.00 eq) in DCM (500.00 mL) was added dropwise MsCl (31.24 g, 272.74 mmol, 13.00 eq) at −60° C. over 0.5 hr. The mixture was then stirred at −60° C. for 1 hr, and the reaction mixture was allowed to warm to 25° C. and stirred for 18 hrs. LCMS showed the starting material was consumed completely. The mixture was then washed with H2O (200 mL*3), and the aqueous phase was extracted with DCM (200 mL*4). The combined organic layers were dried over Na2SO4and concentrated in vacuo to give crude product tert-butyl (4R)-4-((tert-butyldimethylsilyl)oxy)-2-(5-fluoropyridin-3-yl)pyrrolidine-1-carboxylate (8.30 g, crude) as a black/brown oil, which was used directly without purification.

Example 11. Synthesis of (3R,4S,5R)-5-aminotetrahydro-2H-pyran-3,4-diol

(S)-3,6-dihydro-2H-pyran-3-yl acetate (1.9 g, 13.37 mmol) was taken up in MeOH (30 ml) and Water (20 ml). Triethylamine (7 ml, 50.2 mmol) was added and stirred at room temperature for 30 min. The solvent was removed under reduced pressure. The residual water was then extracted with EtOAc three times. The organic layers were combined, dried over sodium sulfate and the solvent was removed. (S)-3,6-dihydro-2H-pyran-3-ol (1.1 g, 10.99 mmol, 82% yield) was recovered as a clear oil. The crude product was carried on without further purification.

(S)-3,6-dihydro-2H-pyran-3-ol (1.1 g, 10.99 mmol) was taken up in CH2Cl2(20 ml) and cooled to 0° C. mCPBA (4.55 g, 13.18 mmol) was added portion wise. The reaction mixture was stirred while warming to room temperature, overnight. The white precipitate of the reaction mixture was filtered off, the elutant was retained, the solvent was removed and triturated with diethyl ether. This step was repeated. The residue, (1S,5R,6R)-3,7-dioxabicyclo[4.1.0]heptan-5-ol (1.2 g, 100% yield) was carried on without further purification.

(3R,4S,5R)-5-(((R)-1-phenylethyl)amino)tetrahydro-2H-pyran-3,4-diol (0.350 g, 1.475 mmol) was taken up in EtOH (3 ml) and Pd—C (0.031 g, 0.295 mmol) was added. The reaction mixture was stirred under H2balloon overnight. The reaction mixture was filtered through Celite and the solvent was removed to give (3R,4S,5R)-5-aminotetrahydro-2H-pyran-3,4-diol (0.190 g, 1.427 mmol, 97% yield) as an off white solid. The crude product was carried on without further purification. LCMS (M+H) 134.

To a mixture of tert-butyl (2R,4R)-4-((tert-butyldimethylsilyl)oxy)-2-(5-fluoropyridin-3-yl)pyrrolidine-1-carboxylate (2.40 g, 6.05 mmol) in THF (60.00 mL) was added TBAF (3.16 g, 12.10 mmol) in one portion at 25° C. The mixture was concentrated under reduced pressure at 50° C.

To a mixture of tert-butyl (2R,4R)-2-(5-fluoropyridin-3-yl)-4-hydroxypyrrolidine-1-carboxylate (1.30 g, 4.60 mmol) and trichloroisocyanuric acid (1.10 g, 4.60 mmol) was added TEMPO (72.41 mg, 460.49 umol) at −10° C. The mixture was stirred at −10° C. for 15 min, then warmed to 25° C. and stirred for 1 hr. TLC (EtOAc) showed the reaction was complete. The organic phase was washed with NaHCO3(20 mL*2), dried over Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate=50/1, 10/1) to afford tert-butyl (R)-2-(5-fluoropyridin-3-yl)-4-oxopyrrolidine-1-carboxylate (1.10 g, yield: 85.32%) as a brown oil.

To a mixture of tert-Butyl (2R,4R)-2-(2,5-difluorophenyl)-4-((methylsulfonyl)oxy)pyrrolidine-1-carboxylate (3.60 g, 9.54 mmol, 1.00 eq) in DMSO (20.00 mL) was added KCN (745.49 mg, 11.45 mmol, 1.20 eq) in one portion. The mixture was stirred at 90° C. for 3 hrs. 80 mL of H2O was added to the mixture, and the mixture was extracted by EtOAc (80 mL*4). The combined organic layer was concentrated under reduced pressure. The residue was purified by silica gel chromatography (PE/EtOAc=40:1, 30:1, 10:1). tert-Butyl (2R,4S)-4-cyano-2-(2,5-difluorophenyl)pyrrolidine-1-carboxylate (1.60 g, 5.19 mmol, yield: 54.40%) was obtained as light green liquid.

3-Fluoro-5-((2R,4S)-4-fluoropyrrolidin-2-yl)benzonitrile (0.050 g, 0.240 mmol) (Prepared as in WO 2012/034095) was taken up in TFA (0.800 ml, 10.38 mmol) and H2SO4(0.200 ml, 3.75 mmol) and stirred overnight at room temperature. The reaction mixture was diluted with ice water (3 ml) and the solid was isolated by filtration, and used directly.

2-chloro-5-fluoronicotinaldehyde (20 g, 125 mmol) was taken up in THF (150 ml) at 0° C. (R)-2-Methylpropane-2-sulfinamide (16.71 g, 138 mmol) was added followed by dropwise addition of titaniumtetraethanolate (22.88 ml, 150 mmol). The reaction mixture was stirred while warming to RT. After 3 hours the reaction mixture was cooled to 0° C., and 150 ml of brine was added and stirred for 20 minutes. The mixture was filtered through Celite. The aqueous layer was separated and discarded. The organic layer with dried over Na2SO4and the solvent was removed to give (S,Z)—N-((2-chloro-5-fluoropyridin-3-yl)methylene)-2-methylpropane-2-sulfinamide (32 g, 122 mmol, 97% yield), which was carried on without further purification. LCMS: 263 M+H.

(R,E)-N-((2-chloro-5-fluoropyridin-3-yl)methylene)-2-methylpropane-2-sulfinamide (32.9 g, 125 mmol) was dissolved in HMPA (100 ml) and cooled to 0° C. Zinc (16.37 g, 250 mmol), allyl bromide (21.67 ml, 250 mmol) and water (2.256 ml, 125 mmol) were added at 0° C. and the reaction mixture was allowed to warm to RT overnight. LCMS showed complete conversion to desired product. 100 ml of water was added at RT and stirred for 30 minutes. 30 ml of MBTE was added followed by 60 ml of 10% citric acid and the reaction mixture was stirred for 30 minutes. The mixture was filtered through Celite and washed with MTBE. The organic layer was washed with 10% citric acid, water and brine. The solvent was removed under vacuum to give (R)—N—((R)-1-(2-chloro-5-fluoropyridin-3-yl)but-3-en-1-yl)-2-methylpropane-2-sulfinamide (14.5 g, 47.6 mmol, 38.0% yield) as an orange oil. LCMS: 305 M+H.

(R)—N—((R)-1-(2-chloro-5-fluoropyridin-3-yl)but-3-en-1-yl)-2-methylpropane-2-sulfinamide (7.5 g, 24.61 mmol) was taken up in 10 ml MeOH. HCl (4M in dioxane) (30.8 ml, 123 mmol) was added and stirred at RT for 1 h. The solvent was removed under vacuum and the residue was diluted in DCM and washed with saturated aqueous NaHCO3. The layers were separated and the organic layer was dried with Na2SO4and the solvent was removed under vacuum. Recovered (R)-1-(2-chloro-5-fluoropyridin-3-yl)but-3-en-1-amine, HCl (5.83 g, 24.59 mmol, 100% yield) as a solid. LCMS: 201 M+H.

To (R)-1-(2-chloro-5-fluoropyridin-3-yl)but-3-en-1-amine.HCl (5.83 g, 24.59 mmol) in DCM (70.3 ml) at 0° C. was added TEA (4.11 ml, 29.5 mmol) and acetic anhydride (2.320 ml, 24.59 mmol). The mixture was stirred for 2 hours. The reaction mixture was poured into saturated aqueous NaHCO3and extracted with DCM. The organic layer was washed with brine, dried over MgSO4, and evaporated under reduced pressure. Recovered (R)—N-(1-(2-chloro-5-fluoropyridin-3-yl)but-3-en-1-yl)acetamide (5.97 g, 24.60 mmol, 100% yield) and was carried on without further purification. LCMS: 243 M+H.

(R)—N-(1-(2-chloro-5-fluoropyridin-3-yl)but-3-en-1-yl)acetamide (5.97 g, 24.60 mmol) was taken up in THF (56.2 ml) and water (14.06 ml), followed by addition of I2(18.73 g, 73.8 mmol) and stirred overnight at RT. The crude reaction was diluted with saturated NaHCO3and Na2S2O3solutions and extracted twice with EtOAc. Aqueous layer was basified with saturated aqueous NaHCO3and extracted with EtOAc to obtain (5R)-5-(2-chloro-5-fluoropyridin-3-yl)pyrrolidin-3-yl acetate (5.9 g, 22.81 mmol, 93% yield) as a light yellow oil. LCMS: 259 M+H.

To a solution of (5R)-5-(2-chloro-5-fluoropyridin-3-yl)pyrrolidin-3-yl acetate (5.9 g, 22.81 mmol) in dioxane (76 ml) and water (76 ml) was added BOC-anhydride (7.94 ml, 34.2 mmol) followed by careful addition of 2N NaOH (7 ml) to achieve pH ˜9. The reaction mixture was stirred for 1 hour at RT. The reaction mixture was diluted with water and extracted with EtOAc three times. The organic layer was dried over Na2SO4and the solvent was removed under vacuum to give (2R)-tert-butyl 4-acetoxy-2-(2-chloro-5-fluoropyridin-3-yl)pyrrolidine-1-carboxylate (3.5 g, 9.75 mmol, 42.8% yield), which was carried on without further purification. LCMS: 359 M+H.

(2R)-tert-butyl 4-acetoxy-2-(2-chloro-5-fluoropyridin-3-yl)pyrrolidine-1-carboxylate (3.5 g, 9.75 mmol) was taken up in MeOH (48.8 ml) followed by addition of 2M NaOH (5.37 ml, 10.73 mmol) and the reaction mixture was stirred at RT for 2 hours. The solvent was removed under vacuum and the aqueous layer was neutralized with 1N HCl, and extracted with EtOAc three times. The combined organic layers were dried over Na2SO4. The solvent was removed under vacuum and the residue was purified via silica gel chromatography (0-70% Hex/EtOAc) to give (2R)-tert-butyl 2-(2-chloro-5-fluoropyridin-3-yl)-4-hydroxypyrrolidine-1-carboxylate (2.1 g, 6.63 mmol, 68.0% yield). LCMS: 317 M+H.

(R)-tert-butyl 2-(2-chloro-5-fluoropyridin-3-yl)-4-oxopyrrolidine-1-carboxylate (1.6 g, 5.08 mmol) was suspended in ethanol (33.9 ml) and cooled to 0° C. NaBH4was added portionwise (0.096 g, 2.54 mmol) and stirred for 45 minutes at 0° C. The reaction was quenched slowly with saturated NH4Cl and allowed to warm to RT, and the solution was extracted with DCM×3. The organic layers were combined and dried over Na2SO4. The residue was purified via flash chromatography (0-70% Hex/EtOAc) to give (2R,4R)-tert-butyl 2-(2-chloro-5-fluoropyridin-3-yl)-4-hydroxypyrrolidine-1-carboxylate (1.446 g, 4.57 mmol, 90% yield). LCMS: 317 M+H.

(2R,4R)-tert-butyl 2-(2-chloro-5-fluoropyridin-3-yl)-4-hydroxypyrrolidine-1-carboxylate (1.0 g, 3.16 mmol) was taken up in DCM (25 ml) and cooled to −78° C. TEA-HF (1.098 ml, 9.47 mmol) was added and stirred for 10 minutes. XtalFluor-E (1.446 g, 6.31 mmol) was added and after 10 minutes the reaction mixture was transferred to an ice bath and allowed to warm to 0° C. After 2 hours the reaction mixture was diluted with DCM and quenched with saturated aqueous NaHCO3. The organic layers were separated, and the solvent was removed under vacuum. The residue was purified via ISCO (0-50% Hex/EtOAc; 12 g column) to give (2R,4S)-tert-butyl 2-(2-chloro-5-fluoropyridin-3-yl)-4-fluoropyrrolidine-1-carboxylate (0.805 g, 2.53 mmol, 80% yield) as a white solid. LCMS: 319 M+H.

5-Fluoro-3-((2R,4S)-4-fluoropyrrolidin-2-yl)-2-methoxypyridine was prepared in the same way as 3-fluoro-5-((2R,4S)-4-fluoropyrrolidin-2-yl)benzamide, substituting for 5-fluoro-2-methoxynicotinaldehyde for 2-chloro-5-fluoronicotinaldehyde.

Example 18. Synthesis of methyl (1R,3R,4R)-3-amino-4-hydroxycyclopentane-1-carboxylate

To the mixture of (3aS,4S,6aR)-2,2-dimethyl-3a,6a-dihydro-4H-cyclopenta[d][1,3]dioxol-4-ol (8.00 g, 51.22 mmol, 1.00 eq) and isoindoline-1,3-dione (9.04 g, 61.46 mmol, 1.20 eq) in toluene (250.00 mL) was added PPh3(20.15 g, 76.83 mmol, 1.50 eq) at 20° C. Then DIAD (15.54 g, 76.83 mmol, 1.50 eq) was added dropwise to the mixture at 0° C. After addition, the mixture was allowed to 80° C. and stirred for 16 hrs. TLC (PE/EtOAc=5/1) showed the reaction was complete. The mixture was concentrated. The residue was purified by column chromatography on silica gel (PE/EtOAc=25/1 to 15/1). The obtained product was crude as yellow oil with some polar spots on TLC. So 80 mL of MeOH was added and the white precipitate was generated and collected by filtration. 2-((3aS,4R,6aR)-2,2-dimethyl-3a,6a-dihydro-4H-cyclopenta[d][1,3]dioxol-4-yl)isoindoline-1,3-dione (9.60 g, 33.65 mmol, yield: 65.70%) was obtained as a white solid.

To the mixture of 2-((3aS,4R,6aR)-2,2-dimethyl-3a,6a-dihydro-4H-cyclopenta[d][1,3]dioxol-4-yl)isoindoline-1,3-dione (9.52 g, 33.37 mmol, 1.00 eq) in EtOH (300.00 mL) was added ethane-1,2-diamine (4.01 g, 66.74 mmol, 2.00 eq). The resulting mixture was stirred at 80° C. for 16 hrs. Lots of white precipitate was generated. TLC (PE/EtOAc=5/1) showed the starting material was consumed completely. The precipitate was filtered. To the filtrate was added 300 mL of NaOH (0.5 M). The mixture was extracted with DCM (200 mL*5), dried over Na2SO4and concentrated. (3aS,4R,6aR)-2,2-dimethyl-3a,6a-dihydro-4H-cyclopenta[d][1,3]dioxol-4-amine (4.90 g, 31.57 mmol, yield: 94.62%) was obtained as a yellow oil.

A mixture of (3aR,4S,6R,6aS)-6-amino-2,2-dimethyltetrahydro-4H-cyclopenta[d][1,3]dioxol-4-ol (0.43 g, 2.46 mmol, 1.00 eq), Phthalic anhydride (0.36 g, 2.46 mmol, 1 eq) and DIEA (0.65 mL, 3.7 mmol, 1.5 eq) in Toluene (6.2 mL) was stirred at 100° C. for 9 hrs. LCMS showed the reaction was complete. EtOAc was added to the reaction mixture and then washed with aqueous saturated sodium bicarbonate solution (15 mL). The combined organic layers were washed with saturated brine solution, dried over Na2SO4and concentrated in vacuo. The residue was purified by column chromatography on silica gel (Hexanes/EtOAc) to get the product 2-((3aS,4R,6S,6aR)-6-hydroxy-2,2-dimethyltetrahydro-4H-cyclopenta[d][1,3]dioxol-4-yl)isoindoline-1,3-dione (0.62 g, 83%) as a white solid.

To a solution of 2-((3aS,4R,6S,6aR)-6-hydroxy-2,2-dimethyltetrahydro-4H-cyclopenta[d][1,3]dioxol-4-yl)isoindoline-1,3-dione (0.20 g, 0.68 mmol, 1.00 eq) in DCM (4.5 mL) was added PCC (0.29 g, 1.35 mmol, 2 eq) and the solution was stirred at 23° C. for 16 hrs. Another aliquot of PCC (0.15 g, 0.67 mmol) was added and the reaction continued for another 16 hours. LCMS showed the reaction was complete. EtOAc was added to the reaction mixture and then filtered through a celite pad. The residue was concentrated and then purified by column chromatography on silica gel (Hexanes/EtOAc) to get the product 2-((3aS,4R,6aS)-2,2-dimethyl-6-oxotetrahydro-4H-cyclopenta[d][1,3]dioxol-4-yl)isoindoline-1,3-dione (0.19 g, 94%) as an off-white solid.

To a solution of 2-((3aS,4R,6aS)-2,2-dimethyl-6-oxotetrahydro-4H-cyclopenta[d][1,3]dioxol-4-yl)isoindoline-1,3-dione (0.16 g, 0.53 mmol, 1.00 eq) in DCM (3.5 mL) was added DAST (0.42 g, 2.64 mmol, 5 eq) and the solution was stirred at reflux for 16 hrs. Another aliquot of DAST (0.42 g, 2.64 mmol, 5 eq) was added and the reaction continued for another 16 hours at 23° C. The reaction mixture was diluted with DCM and then washed with aqueous saturated sodium bicarbonate solution. The combined organic layers were washed with saturated brine solution, dried over Na2SO4and concentrated in vacuo. The residue was purified by column chromatography on silica gel (Hexanes/EtOAc) to get the product 2-((3aS,4R,6aS)-6,6-difluoro-2,2-dimethyltetrahydro-4H-cyclopenta[d][1,3]dioxol-4-yl)isoindoline-1,3-dione (0.065 g, 38%).

To a solution of 2-((3aS,4R,6aS)-6,6-difluoro-2,2-dimethyltetrahydro-4H-cyclopenta[d][1,3]dioxol-4-yl)isoindoline-1,3-dione (0.065 g, 0.2 mmol, 1.00 eq) in Ethanol (1.8 mL) was added Hydrazine monohydrate (0.015 mL, 0.3 mmol, 1.5 eq) and the solution was stirred at 50° C. for 2 hrs and then at 70° C. for another 2 hours. The heterogeneous reaction mixture was filtered using minimum volume of Ethanol. The filtrate was then concentrated and the isolated crude product (3aS,4R,6aS)-6,6-difluoro-2,2-dimethyltetrahydro-4H-cyclopenta[d][1,3]dioxol-4-amine was used without further purification in the next step.

Example 22. Synthesis of (1R,3R,4R)-4-aminocyclohexane-1,3-diol

To an ice-bath cooled solution of 4-(benzyloxy)cyclohexanone (31.0 g, 152 mmol) in 500 mL methanol, sodium borohydride (5.78 g, 153 mmol) was added in several potions during a period of 10 min, then the solution was stirred at 20° C. for 2 h. Then the mixture was quenched by saturated aqueous solution of ammonium chloride (50 mL), concentrated and the residue was dissolved in 200 mL water and extracted with ethyl acetate (200 mL×3), the combined organic phase was dried over sodium sulfate, then concentrated under vacuo to give title product (1r,4r)-4-(benzyloxy)cyclohexanol and (1s,4s)-4-(benzyloxy)cyclohexanol as a pale yellow oil (31.0 g, crude) which was used to next step directly without further purification, MS (ES+) C13H18O2requires: 206. found: 207[M+H]+.

To an ice-bath cooled solution of (1r,4r)-4-(benzyloxy)cyclohexanol and (1s,4s)-4-(benzyloxy)cyclohexanol (30.0 g, 145 mmol) and N,N-Diisopropylethylamine (28.1 g, 218 mmol) in 1200 mL dichloromethane, trifluoromethanesulfonic anhydride (30.7 g, 109 mmol) was added dropwise during a period of 30 min, then the solution was stirred at 25° C. for 18 h. Then the mixture was concentrated under vacuo and the residue was purified with silica gel column chromatography, eluting with petroleum ether:ethyl acetate=12:1 to give the title compound (28.0 g, yield 100%) as a yellow oil. MS (ES+) C13H16O requires: 188. found: 189 [M+H]+.

A solution of ((cyclohex-3-enyloxy)methyl)benzene (12.0 g, 63.7 mmol) in dichloromethane (200 mL) was treated at 0° C. with meta-chloroperoxybenzoic acid (21.9 g, 127 mmol). The reaction mixture was stirred 2 h at 0° C. and then 15 min at room temperature. Evaporation of the washed (10% aqueous solution of sodium sulfite, 5% aqueous sodium hydroxide solution and then water) organic solution afforded a liquid residue, which was separated with silica gel column chromatography, eluting with hexane:isopropyl ether:ethyl acetate=65:28:7 to give the title compound (4.18 g, yield 32%) as a yellow oil. The trans-(1S,3R,6R)-3-(benzyloxy)-7-oxa-bicyclo[4.1.0]heptane showed a little less polarity on TLC and eluted firstly. The cis-(1R,3R,6S)-3-(benzyloxy)-7-oxa-bicyclo[4.1.0]heptane eluted secondly. MS (ES+) C13H16O2requires: 204. found: 205 [M+H]+.

Lithium perchlorate (7.27 g, 68.4 mmol) was added to an ice-bath cooled stirred solution of (1R,3R,6S)-3-(benzyloxy)-7-oxa-bicyclo[4.1.0]heptane (7.0 g, 34.2 mmol) in 120 mL 4A-MS dried acetonitrile, the bath was removed and (S)-1-phenylethanamine (5.58 g, 46.1 mmol) was added dropwise during a period of 15 min, then the solution was stirred at 25° C. for 18 h. Then the mixture was diluted in 200 mL water and extracted with ethyl acetate (200 mL×3), the combined organic phase was dried over sodium sulfate, then concentrated and the residue was purified with silica gel column chromatography, eluting with petroleum ether:ethyl acetate:triethylamine=98:0:2˜49:49:2 to give the title compound (3.5 g, yield 31%) as a yellow oil. The (1S,2S,5S)-5-(benzyloxy)-2-((S)-1-phenylethylamino)cyclohexanol showed a little less polarity on TLC, and eluted firstly. The (1R,2R,5R)-5-(benzyloxy)-2-((S)-1-phenylethylamino)cyclohexanol eluted secondly. MS (ES+) C21H27NO2requires: 325. found: 326[M+H]+.

tert-Butyldimethylsilyl trifluoromethanesulfonate (13.0 g, 49.5 mmol) was added to an ice-bath cooled, stirred solution of (1R,2R,5R)-5-(benzyloxy)-2-((S)-1-phenylethylamino)cyclohexanol (5.4 g, 16.5 mmol) and triethylamine (5.0 g, 49.5 mmol) in 100 mL dried dichloromethane. After 30 min, this was washed with a saturated aqueous solution of sodium bicarbonate, dried over sodium sulfate. Removal of the solvent, and the residue was purified with silica gel column chromatography, eluting with petroleum ether:ethyl acetate=100:0-70:30 to give the title compound (5.4 g, yield 71%) as a yellow oil. MS (ES+) C27H41NO2Si requires: 439. found: 440 [M+H]+.

Tetrabutylammonium fluoride (2.66 g, 10.2 mmol) was added to a stirred solution of (1R,2R,4R)-4-(benzyloxy)-2-(tert-butyldimethylsilyloxy)-N—((S)-1-phenylethyl)cyclohexanamine (1.5 g, 3.41 mmol) in 50 mL dried oxolane at room temperature. Then this solution was stirred at 65° C. for 2 h. Then the mixture was concentrated under vacuo and the residue was diluted in 200 mL water and extracted with ethyl acetate (200 mL×3), the combined organic phase was washed with water and saturated aqueous solution of sodium chloride, dried over sodium sulfate. Removal of the solvent, and the residue was purified with silica gel column chromatography, eluting with petroleum ether:ethyl acetate=100:0-70:30 to give the title compound (0.75 g, yield 68%) as a colorless oil. MS (ES+) C21H27NO2requires: 325. found: 326[M+H]+.

10% Palladium hydroxide in activated carbon (1.9 g, catalyst) was added to a solution of (1R,2R,4R)-4-(benzyloxy)-2-(tert-butyldimethylsilyloxy)-N—((S)-1-phenylethyl)cyclohexanamine from the previous example (2.0 g, 4.54 mmol) and di-tert-butyl dicarbonate (3.95 g, 18.1 mmol) in 60 mL ethanol at room temperature. Then this solution was stirred at 50° C. for 20 h under hydrogen. Then the mixture was cooled and filtered through celite, the filter-cake was washed with methanol:dichloromethane=1:10, the filtrate was concentrated under vacuo and the residue was diluted in 20 mL methanol:dichloromethane=1:10 solution and concentrated, dried under high-vacuo to give the title compound (1.2 g, yield 77%) as a colorless oil. MS (ES+) C17H35NO4Si requires: 345. found: 346 [M+H]+.

Example 23. Synthesis of tert-butyl (1R,2R)-2-(tert-butyldimethylsilyloxy)-4-oxocyclohexylcarbamate and (1R,3R,4R)-4-aminocyclohexane-1,3-diol

To an ice-bath cooled solution of 4-(benzyloxy)cyclohexanone (31.0 g, 152 mmol) in 500 mL methanol, sodium borohydride (5.78 g, 153 mmol) was added in several portions during a period of 10 min, then the solution was stirred at 20° C. for 2 h. Then the mixture was quenched by saturated aqueous solution of ammonium chloride (50 mL), concentrated and the residue was dissolved in 200 mL water and extracted with ethyl acetate (200 mL×3), the combined organic phase was dried over sodium sulfate, then concentrated under vacuo to give title product (1R,4R)-4-(benzyloxy)cyclohexanol and (1S,4S)-4-(benzyloxy)cyclohexanol as a pale yellow oil (31.0 g, crude) which was used to next step directly without further purification. MS (ES+) C13H18O2requires: 206. found: 207[M+H]+.

To an ice-bath cooled solution of (1R,4R)-4-(benzyloxy)cyclohexanol and (1S,4S)-4-(benzyloxy)cyclohexanol (30.0 g, 145 mmol) and N,N-Diisopropylethylamine (28.1 g, 218 mmol) in 1200 mL dichloromethane, trifluoromethanesulfonic anhydride (30.7 g, 109 mmol) was added dropwise during a period of 30 min, then the solution was stirred at 25° C. for 18 h. Then the mixture was concentrated under vacuo and the residue was purified with silica gel column chromatography, eluting with petroleum ether:ethyl acetate=12:1 to give the title compound (28.0 g, yield 100%) as a yellow oil. MS (ES+) C13H16O requires: 188. found: 189 [M+H]+.

A solution of ((cyclohex-3-enyloxy)methyl)benzene (12.0 g, 63.7 mmol) in dichloromethane (200 mL) was treated at 0° C. with meta-chloroperoxybenzoic acid (21.9 g, 127 mmol). The reaction mixture was stirred 2 h at 0° C. and then 15 min at room temperature. Evaporation of the washed (10% aqueous solution of sodium sulfite, 5% aqueous sodium hydroxide solution and then water) organic solution afforded a liquid residue, which was separated with silica gel column chromatography, eluting with hexane:isopropyl ether:ethyl acetate=65:28:7 to give the title compound (4.18 g, yield 32%) as a yellow oil. The trans-(1S,3R,6R)-3-(benzyloxy)-7-oxa-bicyclo[4.1.0]heptane showed a little less polarity on TLC and eluted firstly. The cis-(1R,3R,6S)-3-(benzyloxy)-7-oxa-bicyclo[4.1.0]heptane eluted secondly. MS (ES+) C13H16O2requires: 204. found: 205 [M+H]+.

Lithium perchlorate (7.27 g, 68.4 mmol) was added to an ice-bath cooled stirred solution of (1R,3R,6S)-3-(benzyloxy)-7-oxa-bicyclo[4.1.0]heptane (7.0 g, 34.2 mmol) in 120 mL 4A-MS dried acetonitrile, the bath was removed and (S)-1-phenylethanamine (5.58 g, 46.1 mmol) was added dropwise during a period of 15 min, then the solution was stirred at 25° C. for 18 h. Then the mixture was diluted in 200 mL water and extracted with ethyl acetate (200 mL×3), the combined organic phase was dried over sodium sulfate, then concentrated and the residue was purified with silica gel column chromatography, eluting with petroleum ether:ethyl acetate:triethylamine=98:0:2˜49:49:2 to give the title compound (3.5 g, yield 31%) as a yellow oil. The (1S,2S,5S)-5-(benzyloxy)-2-((S)-1-phenylethylamino)cyclohexanol showed a little less polarity on TLC, and eluted firstly. The (1R,2R,5R)-5-(benzyloxy)-2-((S)-1-phenylethylamino)cyclohexanol eluted secondly. MS (ES+) C21H27NO2requires: 325. found: 326[M+H]+.

Step 4: Synthesis of (1R,2R,4R)-4-(benzyloxy)-2-(tert-butyldimethylsilyloxy)-N—((S)-1-phenylethyl)cyclohexanamine

tert-Butyldimethylsilyl trifluoromethanesulfonate (13.0 g, 49.5 mmol) was added to an ice-bath cooled, stirred solution of (1R,2R,5R)-5-(benzyloxy)-2-((S)-1-phenylethylamino)cyclohexanol (5.4 g, 16.5 mmol) and triethylamine (5.0 g, 49.5 mmol) in 100 mL dried dichloromethane. After 30 min, this was washed with a saturated aqueous solution of sodium bicarbonate, dried over sodium sulfate. Removal of the solvent, and the residue was purified with silica gel column chromatography, eluting with petroleum ether:ethyl acetate=100:0-70:30 to give the title compound (5.4 g, yield 71%) as a yellow oil. MS (ES+) C27H41NO2Si requires: 439. found: 440 [M+H]+.

Tetrabutylammonium fluoride (2.66 g, 10.2 mmol) was added to a stirred solution of (1R,2R,4R)-4-(benzyloxy)-2-(tert-butyldimethylsilyloxy)-N—((S)-1-phenylethyl)cyclohexanamine (1.5 g, 3.41 mmol) in 50 mL dried oxolane at room temperature. Then this solution was stirred at 65° C. for 2 h. Then the mixture was concentrated under vacuo and the residue was diluted in 200 mL water and extracted with ethyl acetate (200 mL×3), the combined organic phase was washed with water and saturated aqueous solution of sodium chloride, dried over sodium sulfate. Removal of the solvent, and the residue was purified with silica gel column chromatography, eluting with petroleum ether:ethyl acetate=100:0-70:30 to give the title compound (0.75 g, yield 68%) as a colorless oil. MS (ES+) C21H27NO2requires: 325. found: 326[M+H]+.

10% Palladium hydroxide in activated carbon (697 mg, catalyst) was added to a solution of (1R,2R,5R)-5-(benzyloxy)-2-((S)-1-phenylethylamino)cyclohexanol (650 mg, 1.99 mmol) in 15 mL ethanol at room temperature. Then this solution was stirred at 50° C. for 20 h under hydrogen. Then the mixture was cooled and filtered though celite, the filter-cake was washed with methanol:dichloromethane=1:10, the filtrate was concentrated under vacuo and the residue was diluted in 20 mL methanol:dichloromethane=1:10 solution and concentrated, dried under high-vacuo, then cooled at −20° C. to give the title compound (240 mg, yield 92%) as a white crystal. MS (ES+) C6H13NO2requires: 131. found: 132[M+H]+.

Example 24. Synthesis of Compound 232

To a solution of methyl (1R,3R,4R)-3-((3-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidine-1-carbonyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino)-4-hydroxycyclopentane-1-carboxylate (120.00 mg, 189.06 umol, 1.00 eq) in DCM (3.00 mL) was added TFA (3.00 mL) at 15° C. The reaction was stirred at 15° C. for 16 hrs. LCMS showed the starting material was consumed. Little of (1R,3R,4R)-methyl 3-((3-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidine-1-carbonyl)-1-(hydroxymethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino)-4-hydroxycyclopentanecarboxylate was detected. The reaction was concentrated. The residue was dissolved in MeOH (20.00 mL). KOAc (185.54 mg, 1.89 mmol, 10.00 eq) was added to the reaction. The reaction was heated at 50° C. for 16 hrs. LCMS showed the reaction was complete. The solution was concentrated. The residue was dissolved in EtOAc (20 mL) and washed with brine (10 mL), dried over Na2SO4and concentrated to give methyl (1R,3R,4R)-3-((3-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidine-1-carbonyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino)-4-hydroxycyclopentane-1-carboxylate (80.00 mg, crude) as a red solid which was used in the next step without purification.

To a solution of methyl (1R,3R,4R)-3-((3-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidine-1-carbonyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino)-4-hydroxycyclopentane-1-carboxylate (80.00 mg, 158.59 umol, 1.00 eq) in THF (20.00 mL) was added MeMgBr (3 M, 1.59 mL, 30.00 eq) at −70° C. The reaction was slowly warmed to 15° C. and stirred for 2 hrs. TLC (EtOAc, Rf=0.24) and LCMS showed the reaction was complete. The solution was neutralized with 1N aq. HCl to pH=7. The reaction mixture was concentrated. The residue was purified by neutral prep-HPLC. to give ((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)(4-(((1R,2R,4R)-2-hydroxy-4-(2-hydroxypropan-2-yl)cyclopentyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)methanone (17.40 mg, yield: 21.75%) as a yellow solid.

Example 25. Synthesis of Compound 229

A solution of 3-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidine-1-carbonyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (0.085 g, 0.17 mmol, 1 eq) was stirred with thionyl chloride (0.031 mL, 0.43 mmol, 2.5 eq) and few drops of DMF in DCM (0.7 mL) at 50° C. for 3 hours. LCMS indicated complete consumption of SM to the chloro-heterocycle intermediate. The reaction mixture was cooled on ice and added Dioxane (0.7 mL) followed by DIEA (0.21 mL, 1.21 mmol, 7 eq) and tert-butyl (3R,4R)-3-amino-4-hydroxypyrrolidine-1-carboxylate (0.05 g, 0.26 mmol, 1.5 eq). The reaction mixture was then stirred at 70° C. for 3 hours. LCMS indicated reaction was complete. The reaction mixture was then diluted with DCM and washed with aqueous saturated sodium bicarbonate solution. The combined organic layers were washed with saturated brine solution, dried over Na2SO4and concentrated in vacuo. The residue was purified by column chromatography on silica gel (Hexanes/EtOAc) to get the product tert-butyl (3R,4R)-3-((3-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidine-1-carbonyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino)-4-hydroxypyrrolidine-1-carboxylate (0.084 g, 72%).

A solution of (3R,4R)-3-((3-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidine-1-carbonyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino)-4-hydroxypyrrolidine-1-carboxylate (0.084 g, 0.12 mmol, 1 eq) in EtOAc (1.25 mL) was treated with HCl in Dioxane (4M, 0.9 mL, 3.72 mmol, 30 eq). After stirring at 23° C. for 4 hours, LCMS indicated reaction was complete. The reaction mixture was diluted with EtOAc and washed with aqueous saturated sodium bicarbonate solution. The combined organic layers were washed with saturated brine solution, dried over Na2SO4and concentrated in vacuo. The crude product ((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)(4-(((3R,4R)-4-hydroxypyrrolidin-3-yl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)methanone was used without further purification in the next step.

To a solution of ((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)(4-(((3R,4R)-4-hydroxypyrrolidin-3-yl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)methanone (0.036 g, 0.062 mmol, 1 eq) in DCE (0.5 mL) was added few drops of Acetic acid (2 μL, 0.031 mmol, 0.5 eq) followed by oxetan-3-one (0.015 mL, 0.21 mmol, 3.3 eq) and the reaction mixture was heated at 60° C. for 2 hours. Added sodium triacetoxyborohydride (0.033 g, 0.16 mmol, 2.5 eq) and the solution was stirred at 23° C. for 24 hours. LCMS indicated the reaction was complete. The reaction mixture was diluted with EtOAc and washed with aqueous saturated sodium bicarbonate solution. The combined organic layers were washed with saturated brine solution, dried over Na2SO4and concentrated in vacuo. The residue was then purified by column chromatography on silica gel (DCM/MeOH) to isolate the product ((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)(4-(((3R,4R)-4-hydroxy-1-(oxetan-3-yl)pyrrolidin-3-yl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)methanone (0.030 g, 75%).

A solution of ((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)(4-(((3R,4R)-4-hydroxy-1-(oxetan-3-yl)pyrrolidin-3-yl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)methanone (0.060 g, 0.095 mmol, 1 eq) in DCM (1 mL) was treated with TFA (0.73 mL, 9.5 mmol, 100 eq) for 16 hours. The reaction mixture was diluted with DCM and washed with aqueous saturated sodium bicarbonate solution. The combined organic layers were washed with saturated brine solution, dried over Na2SO4and concentrated in vacuo. To the intermediate in DCM/MeOH (1/1, 1 mL) was added sodium acetate (0.016 g, 0.19 mmol, 2 eq) and the reaction was stirred at 23° C. for 2 hours. The reaction mixture was diluted with DCM and then washed with aqueous saturated sodium bicarbonate solution. The combined organic layers were washed with saturated brine solution, dried over Na2SO4and concentrated in vacuo. The residue was then purified first by column chromatography on silica gel (DCM/MeOH containing 10% NH4OH) and then by preparative-TLC to isolate the product ((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)(4-(((3R,4R)-4-hydroxy-1-(oxetan-3-yl)pyrrolidin-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)methanone (0.034 g, 70%).

Example 26. Synthesis of Compound 156

A solution of 1-bromo-4-chloro-2-methylbenzene (3.00 g, 14.60 mmol, 1.00 eq), NBS (2.34 g, 13.14 mmol, 0.90 eq) and AIBN (239.75 mg, 1.46 mmol, 0.10 eq) in CCl4(20.00 mL) was stirred at 90° C. for 12 hrs. The solution was concentrated under vacuum to give a crude product 1-bromo-2-(bromomethyl)-4-chlorobenzene (5.40 g, crude) as a yellow solid which was used directly in the next step without further purification.

To the mixed solvents TFA (10.00 mL) and DCM (10.00 mL) was added N-(5-chloro-2-cyanobenzyl)-4-(((1R,2S,3R)-2,3-dihydroxycyclopentyl)amino)-N-methyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide (520.00 mg, 908.88 umol, 1.00 eq), the resulting mixture was stirred at 25° C. for 1 hr. The solvent was evaporated by N2to give a crude product. The crude product was dissolved in MeOH (15.00 mL), adjusted to pH=7-8 by NaHCO3, and KOAc (178.39 mg, 1.82 mmol, 2.00 eq) was added, it was stirred at 50° C. for 2 hrs. When the reaction was complete, the mixture was concentrated under vacuum to give a crude product which was dissolved in EtOAc (50 mL), washed by H2O (15 mL*3). The organic layer was concentrated under vacuum to give a crude product which was purified by acidic prep-HPLC (TFA) to obtain N-(5-chloro-2-cyanobenzyl)-4-(((1R,2S,3R)-2,3-dihydroxycyclopentyl)amino)-N-methyl-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide (171.00 mg, yield: 33.85%, TFA) as a white solid.

Example 27. Synthesis of Compound 226

A solution of (4-(((1R,2R,4R)-2-((tert-butyldiphenylsilyl)oxy)-4-(difluoromethyl)cyclopentyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)methanone (38.00 mg, 43.93 umol, 1.00 eq) in TBAF/THF (5.00 mL) was heated at 50° C. for 2 hrs. LCMS showed (4-(((1R,2R,4R)-4-(difluoromethyl)-2-hydroxycyclopentyl)amino)-1-(hydroxymethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)methanone was remained. The reaction mixture was concentrated. The residue was dissolved in EtOAc (20 mL) and washed with brine (10 mL*2). The organic layer was concentrated and dissolved in MeOH (20.00 mL). KOAc (21.56 mg, 219.65 umol, 5.00 eq) was added to the reaction. The reaction was heated at 50° C. for 16 hrs. LCMS showed the reaction was complete. The solution was concentrated. The residue was purified by prep-HPLC (MeOH/TFA system). (13.50 mg, yield: 50.34%, TFA) of (4-(((1R,2R,4R)-4-(difluoromethyl)-2-hydroxycyclopentyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)methanone as a yellow solid was obtained.

Example 28. Synthesis of Other Compounds

Additional compounds of the invention were synthesized using similar techniques to those set forth in the above examples. The table below indicates the specific example (“Example”) upon which the synthesis of each compound (“Cmpd”) was based, as well as the appropriate amino alcohol and amine that were used to synthesize each specific compound.

TABLE 1Protocol and Intermediates Used for Synthesizing Exemplary Compounds.CmpdAmino AlcoholAmineExample1121314151617181921021111221321431531631731831932032132212312412512612712812913013113213313423523613713813914014114214314414514614714814915015123521531541551561571581591601613623631641651662672681691702712721731741753763771781791801811821831842285186287288189190291292193194195396397298299110011011102231031104110511061107110811092311011112311211132311411152116211711181119112011211122112331242125212611271128112911301131113211331134113524136113711381139114024141241421143241441145114624147114811491150115131522415311542415511562415711581159116011611162116311641165241661167241681169117011711172117311741175117611771178317911801181118211832418411851186318731881189119011911192119311941195119611971198119912001201120212031204120512061207320832091210222111212121312141215121612171218121912201221122212231224122512262722712281229232301231123222233123412351236123712381239124012411242124312441

The NMR and LC MS data obtained for compounds disclosed herein are shown inFIG. 1.

NTRK1 Wild Type Assay at 1 mM ATP

In each well of a 384-well plate, 1 nM-1.5 nM of wild type NTRK1 enzyme (BPS Bioscience; 40280) was incubated in a total of 12.5 μL of buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 10 mM MgCl2, 1 mM DTT) with 1-2 μM CSKtide (Tuft's University or Anaspec; FITC-AHA-KKKKD DIYFFFG-NH2) and 1 mM ATP at 25° C. for 60 minutes in the presence or absence of a dosed concentration series of compound (1% DMSO final concentration). The reaction was stopped by the addition of 70 μL of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 35 mM EDTA and 0.2% of Coating Reagent 3 (Caliper Lifesciences)). The plate was then read on a Caliper EZReader 2 (protocol settings: −1.7 psi, upstream voltage −500, downstream voltage −3000, post sample sip 35s). Data was normalized to 0% and 100% inhibition controls and the IC50calculated using a 4-parameter fit in the CORE LIMS.

NTRK Wild Type and G595R Mutant Cellular Assays Protocol

KM12 wild type colon carcinoma cell line harboring the TPM3-NTRK1 fusion protein was obtained from the National Cancer Institute (NCI). This line has been previously shown to be dependent upon the NTRK activity derived from the NTRK fusion protein for growth and survival. The KM12 Cliff (G595R) cell line was generated by mutagenizing the wild type KM12 line with a DNA methylating agent and subsequently selecting for clones that were resistant to chronic exposure to high concentration of a known NTRK inhibitor (Crizotinib). Cells were first plated in 384-well plates at 1000 cells/well in complete media (10% FBS and 1% pen/strep) and incubated overnight at 37° C. Cells were then dosed with test articles at varying concentrations using the Bravo liquid handling system. Concentrations ranged from 25 uM down to 9.5 pM (4-fold dilutions, 10 concentrations total). Each compound was run in duplicate per plate. DMSO and staurosporine (25 uM) were included on each plate as negative and positive controls for growth inhibition. 72 hr after dosing, assay plates were developed using CellTiter-Glo (Promega) and resultant luminescence was read on the Envision plate reader. IC50determinations were calculated using a 4-parameter curve fitting algorithm

The table below summarizes the results from the biological assays described above. The following designations are used to indicate IC50in each assay:

INCORPORATION BY REFERENCE

EQUIVALENTS