Conjugates comprising antifungal agents and casein kinase (CK1) inhibitors and methods of use thereof

Abstract

The present application relates to conjugated compounds of formula (I): A-L1-B (I) wherein: A is a moiety that increases fungal cell uptake and / or fungal cell permeability; b is a CK1 suppression part; and L1 is a linker; the present invention relates to compounds of formula (I), or pharmaceutically acceptable salts, solvates and / or prodrugs thereof, compositions comprising these compounds or pharmaceutically acceptable salts, solvates and / or prodrugs thereof, processes for their preparation, and their use in treatments such as the treatment or prevention of fungal related diseases, disorders or conditions.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to co-pending U.S. Provisional Patent Application No. 63 / 434,607, filed on December 22, 2022, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present application relates to conjugate compounds, methods for their preparation, compositions comprising them, and their use in therapy. More specifically, the present invention relates to conjugates comprising a moiety that increases fungal cell uptake and / or fungal cell permeability and a type 1 casein kinase inhibitor moiety, which can be used to treat or prevent fungal-related diseases, disorders, or conditions. Background Art

[0004] Fungal pathogens have a huge impact on global human health (Fisher et al., Science 360, 739-742 (2018)). The poor clinical outcomes for most invasive fungal infections can be attributed to the extremely limited number of effective antifungal agents available for systemic infections and the emergence of clinical resistance to each of the three major modes of action they target (Brown et al., Sci Transl Med B, 165rv13 (2012)). The demand for new, more effective, and less resistant antifungal agents is rapidly increasing (Banumathy et al., Science 425, 407-410 (2012)).

[0005] Protein kinases have emerged as a particularly promising, yet so far clinically underexplored, target area for the development of new antifungal drugs. Protein kinases are core signaling molecules crucial for the regulation of proliferation, survival, and stress responses. Consequently, they have been extensively investigated as potential targets for a variety of therapeutic indications, most notably cancer (Elkins et al., Nat Biotechnol 34, 95-103 (2016); Zhang et al., Nat Rev Cancer 9, 28-39 (2009)). However, kinase inhibitors also hold considerable promise as antimicrobial agents against a variety of pathogenic organisms, including those that cause tuberculosis (Carette et al., mBio 9, e02333-17 (2018)), malaria (Derbyshire et al., Chembiochem 15, 1920-30 (2014)), and the most common systemic fungal diseases (Mattos et al., mBio 11, e02962-19 (2020); Lafayette et al., PLoS Pathog 6, e1001069 (2010); Lee et al., Nat Commun 11(1):1521 (2020)). In fungi, kinases are involved in the regulation of multiple biological processes required for major pathogenic species to cause life-threatening systemic diseases (Mattos et al., mBio11, e02962-19 (2020); LaFayette et al., PLoS Pathog 6, e1001069 (2010); Lee et al., Nat Commun 11, 1521 (2020); Fu et al., Nat Commun 12, 6497 (2021); O'Meara et al., mBio 9, e01581-18 (2018)), but to date, no kinase inhibitors have been approved as antifungal agents for clinical use (Perfect JR., Nat Rev Drug Discov 16, 603-616 (2017)).

[0006] Members of the type 1 casein kinase (CK1) family of serine / threonine kinases are expressed in most eukaryotes (Cheong et al., Int. J. Biochem. Cell Biol 43, 465-469 (2011); Knippschild et al., Cell Signal 17, 675-89 (2005)), but there is still considerable opportunity in developing isoform-specific inhibitors (Fulcher et al., Biochem J 477, 4603-4621 (2020); Monastyrskyi et al., Bioorg Med Chem 26, 590-602 (2018); Wager et al., ACS Chem Neurosci 5, 1253-65 (2014)). In the model fungus Saccharomyces cerevisiae, CK1 family members are involved in cell cycle progression, cell morphology and cell wall integrity, as well as endocytosis, cell morphogenesis, mRNA localization and nutrient sensing (Robinson et al., Mol Biol Cell 10, 1077–1092 (1999); Robinson et al., Mol Cell Biol 13, 2870–2881 (1993); Snowdon et al., Mol Biol Cell 27, 3369–3375 (2016); Hoekstra et al., Science 253, 1031–4 (1991); Ho et al., Proc Natl Acad Sci USA, 94, 581–6 (1997)).

[0007] In a variety of human fungal pathogens, CK1 family members Yck2 and Hrr25 are known to play key roles in cell wall integrity, genome maintenance, virulence, and drug resistance (Wang et al., Eukaryot Cell 10, 1455–1464 (2011); Caplan et al., Cell Chem Biol 27, 1-14 (2020)). In Candida albicans, the most common cause of invasive fungal infections in humans, the kinase domains of Yck2 and Hrr25 are extremely highly conserved, but the two proteins themselves play very different biological roles. Yck2 is non-essential under rich laboratory growth conditions but is required for virulence and antifungal resistance (Park et al., Eukaryot Cell 8, 1498-510 (2009); Jung et al., Plos one 12, e0187721 (2017); Liboro et al., Front Cell Infect Microbiol 11, 636834 (2021)).

[0008] On the contrary, Hrr25 has been shown to be essential for fungal viability even under rich laboratory culture conditions (Segal et al., mBio 9, e02048-18 (2018)). In Candida albicans (C.albicans), the role of Yck2 in virulence has been most extensively studied using genetic techniques and small molecule inhibitors (Caplan et al., Cell Chem Biol 27, 1-14 (2020)). Genetic depletion or inhibitor treatment of YCK2 disrupts the morphogenesis program of Candida albicans, induces cell wall stress response, and sensitizes resistant isolates to conventional antifungal agents in culture. As a proof of concept, using genetic techniques to inhibit YCK2 expression has also been shown to significantly reduce fungal load and prolong the survival of Candida albicans-infected mice (Caplan et al., Cell Chem Biol 27, 1-14 (2020)).

[0009] New therapeutic approaches are needed to eliminate fungal virulence and enhance the efficacy of conventional antifungal agents. Summary of the Invention

[0010] The present application includes a conjugate compound of formula (I), or a pharmaceutically acceptable salt, solvate and / or prodrug thereof,

[0011] A–L 1 –B

[0012] (I)

[0013] wherein: A is a moiety that increases fungal cell uptake and / or fungal cell permeability; B is a casein kinase type 1 (CK1) inhibitory moiety; and L 1 is a linker comprising at least one first complementary functional group to covalently link to a functional group in A, and at least one second complementary functional group to covalently link to a functional group in B, thereby forming a conjugate of formula (I).

[0014] In some embodiments, the compound of Formula I optionally comprises one or more isotopic variant atoms. In some embodiments, the isotopic variant is deuterium, and the compound of Formula I comprises one or more deuterium atoms.

[0015] Also included is a method of treating or preventing a fungus-related disease, disorder, or condition comprising administering to a subject in need thereof a therapeutically effective amount of one or more conjugated compounds of the present application.

[0016] In some embodiments, the present application includes a method of inhibiting or preventing fungal growth, comprising administering to an individual in need thereof a therapeutically effective amount of one or more conjugated compounds of the present application.

[0017] In some embodiments, the present application includes a method of inhibiting fungal CK1 activity, comprising administering to an individual in need thereof a therapeutically effective amount of one or more conjugated compounds of the present application.

[0018] In some embodiments, the present application includes a method of selectively inhibiting fungal CK1 activity, comprising administering to an individual in need thereof a therapeutically effective amount of one or more conjugated compounds of the present application.

[0019] In some embodiments, the present application includes a method of treating or preventing fungal disease, comprising administering to an individual in need thereof a therapeutically effective amount of one or more conjugated compounds of the present application.

[0020] In some embodiments, the present application includes a method for treating or preventing a fungal-related disease, disorder, or condition, comprising administering to an individual in need thereof a therapeutically effective amount of one or more conjugated compounds of the present application in combination with another known agent for treating or preventing a fungal-related disease, disorder, or condition.

[0021] The present application also includes a pharmaceutical composition comprising one or more conjugated compounds of the present application or pharmaceutically acceptable salts and / or solvates thereof, and a pharmaceutically acceptable carrier and / or diluent.

[0022] In some embodiments, the present application includes an agricultural composition comprising one or more conjugated compounds of the present application or salts and / or solvates thereof, and an agriculturally acceptable carrier and / or diluent.

[0023] Other features and advantages of the present application will become apparent from the following specific embodiments. However, it should be understood that although the specific embodiments and specific examples illustrate embodiments of the present application, they are given only in an illustrative manner, and the scope of the claims should not be limited by these embodiments, but should be given the broadest interpretation consistent with the entire specification. DETAILED DESCRIPTION

[0024] I. Definition

[0025] Unless otherwise stated, the definitions and embodiments described in this section and elsewhere are intended to apply to all applicable embodiments and aspects of the application described herein, as will be understood by those skilled in the art.

[0026] As used in this application and claims, the words "comprising" (and any forms of comprising, such as "comprise" and "comprises"), "having" (and any forms of having, such as "have" and "has"), "including" (and any forms of including, such as "include" and "includes"), or "containing" (and any forms of containing, such as "contain" and "contains") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0027] As used herein, the term "consisting of" and its derivatives are intended to serve as closed terms that specify the presence of stated features, elements, components, groups, integers and / or steps, and exclude the presence of other unstated features, elements, components, groups, integers and / or steps.

[0028] As used herein, the term “consisting essentially of is intended to specify the stated features, elements, components, groups, integers, and / or steps, as well as the presence of those features, elements, components, groups, integers, and / or steps that do not materially affect the basic and novel characteristics of such features, elements, components, groups, integers, and / or steps.

[0029] As used herein, the terms "about," "substantially," and "approximately" mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be interpreted as including a deviation of at least ±5% of the modified term, provided that the deviation does not negate the meaning of the word it modifies, or unless the context suggests otherwise to a person skilled in the art.

[0030] As used in this application, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. For example, an embodiment including "a compound" should be understood to present certain aspects involving one compound or two or more additional compounds.

[0031] In embodiments comprising an "additional" or "second" component or effect, such as an additional or second compound, the second compound, as used herein, is different from the other compounds or the first compound. The "third" compound is different from the additional first and second compounds, and further listed or "additional" compounds are likewise different.

[0032] As used herein, the term "and / or" means the presence or use of the listed items, alone or in combination. In practice, the term means the use or presence of "at least one" or "one or more" of the listed items. The term "and / or" with respect to enantiomers, prodrugs, salts, and / or solvates thereof means that the compounds of the present application are present as a combination of individual enantiomers, prodrugs, salts, and hydrates, as well as salts of solvates of the compounds of the present application, for example.

[0033] As used herein, the term "compound of the present application" or "conjugated compound of the present application" refers to a conjugated compound of formula (I), or a pharmaceutically acceptable salt, solvate and / or prodrug thereof.

[0034] As used herein, the term "composition of the application" or "composition of the present application" and the like refers to a composition comprising one or more conjugated compounds of the present application.

[0035] As used herein, the term "suitable" means that the selection of particular compounds or conditions will depend on the specific synthetic procedure to be performed, the identity of the molecule to be transformed, and / or the specific use of the compound, but will be well within the capabilities of one trained in the art.

[0036] As used herein, the term "protecting group" or "PG" refers to a chemical moiety that protects or masks a reactive portion of a molecule to prevent side reactions in these reactive portions of the molecule while the other portions of the molecule are being manipulated or reacted. After the manipulation or reaction is complete, the protecting group is removed without degrading or decomposing the rest of the molecule. Suitable protecting groups can be selected by those skilled in the art. Many conventional protecting groups are known in the art, such as those described in "Protective Groups in Organic Chemistry" by McOmie, JFW, ed., Plenum Press, 1973; Greene, TW and Wuts, PGM, "Protective Groups in Organic Synthesis", John Wiley & Sons, 3rd edition, 1999; and Kocienski, P. Protecting Groups, 3rd edition, 2003, Georg Thieme Verlag (The Americas).

[0037] As used herein, the term "inert organic solvent" refers to a solvent that is generally considered to be non-reactive with the functional groups present in the compounds to be combined, and thus will not interfere with or inhibit the desired synthetic transformations in any given reaction. Organic solvents are generally non-polar and dissolve compounds that are insoluble in aqueous solutions.

[0038] As used herein, the term "alkyl", whether used alone or as part of another group, means a straight or branched chain saturated alkyl group. The number of carbon atoms in the alkyl group is indicated by the prefix "C n1-n2 " means. For example, the term C 1-10 Alkyl means an alkyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.

[0039] The term "alkylene", whether used alone or as part of another group, means a straight or branched saturated alkylene, i.e., a saturated carbon chain containing substituents at both ends. The number of carbon atoms possible in the alkylene mentioned is indicated by the prefix "C n1-n2 " means. For example, the term C 1-6 Alkylene means an alkylene group having 1, 2, 3, 4, 5 or 6 carbon atoms.

[0040] As used herein, the term "alkenyl", whether used alone or as part of another group, means a straight or branched unsaturated alkyl group containing at least one double bond. The number of carbon atoms possible in an alkylene group is indicated by the prefix "C n1-n2 " means. For example, the term C 2-6Alkenyl means an alkenyl group having 2, 3, 4, 5 or 6 carbon atoms and at least one double bond.

[0041] As used herein, the term "alkynyl", whether used alone or as part of another group, means a straight-chain or branched unsaturated alkynyl group containing at least one triple bond. The number of carbon atoms in a referenced alkyl group is indicated by the prefix "C n1-n2 " means. For example, the term C 2-6 Alkynyl means an alkynyl group having 2, 3, 4, 5 or 6 carbon atoms.

[0042] As used herein, the term "cycloalkyl", whether used alone or as part of another group, means a saturated carbocyclic group containing from 3 to 20 carbon atoms and one or more rings. The number of carbon atoms possible in a cycloalkyl group is indicated by the numerical prefix "C n1-n2 " means. For example, the term C 3-10 Cycloalkyl means a cycloalkyl group having 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.

[0043] As used herein, the term "aryl," whether used alone or as part of another group, refers to a carbocyclic group containing at least one aromatic ring and containing from 6 to 20 carbon atoms.

[0044] As used herein, the term "heterocycloalkyl", whether used alone or as part of another group, refers to a cyclic group containing at least one non-aromatic ring and containing 3 to 20 atoms, wherein one or more of the atoms is a heteroatom selected from O, S and N, and the remaining atoms are C. Heterocycloalkyl is saturated or unsaturated (i.e., contains one or more double bonds). When heterocycloalkyl contains the prefix C n1-n2 When , the prefix indicates the number of carbon atoms in the corresponding carbocyclic group, wherein one or more (suitably 1 to 5) of the ring atoms are substituted by heteroatoms selected from O, S and N, and the remaining atoms are C. The heterocycloalkyl group is optionally benzo-fused.

[0045] As used herein, the term "heteroaryl", whether used alone or as part of another group, refers to a cyclic group containing at least one heteroaromatic ring containing 5 to 20 atoms, one or more of which is a heteroatom selected from O, S and N, and the remaining atoms are C. When the heteroaryl group contains the prefix C n1-n2 When , the prefix indicates the number of carbon atoms in the corresponding carbocyclic group, wherein one or more (suitably 1 to 5) of the ring atoms are substituted by heteroatoms as defined above. The heteroaryl group is optionally benzo-fused.

[0046] All cyclic groups, including aryl, heteroaryl, heterocycloalkyl and cycloalkyl, contain one ring or more than one ring (i.e., are polycyclic). When a cyclic group contains more than one ring, the rings may be fused, bridged, spirofused or connected by a bond.

[0047] As used herein, the term "benzo-fused" refers to a polycyclic group in which a benzene ring is fused to another ring.

[0048]

[0026] When a first ring is "fused" to a second ring, it means that the first ring and the second ring share two adjacent atoms therebetween.

[0049]

[0046] A first ring is "bridged" to a second ring if the first and second rings share two non-adjacent atoms therebetween.

[0050] When a first ring is "spiro-fused" to a second ring, it means that the first ring and the second ring share one atom therebetween.

[0051] The term "fluoro-substituted" means that one or more (including all) available hydrogens in the referenced group are replaced by fluoro.

[0052]

[0046] As used herein, the term "halo" or "halogen," whether used alone or as part of another group, refers to a halogen atom, including fluorine, chlorine, bromine, and iodine.

[0053] As used herein, the term "isotopic variant" refers to an atom having the same atomic number as the isotope predominantly found in nature, but an atomic mass or mass number different from the atomic mass or mass number of the isotope predominantly found in nature.

[0054] The term "available," as in "available hydrogen atom" or "available atom," refers to an atom known to those skilled in the art to be capable of being replaced by a substituent.

[0055] As used herein, the term "cross-coupling" refers to a chemical reaction in which two different starting materials (each of which typically carries an activating group) react together under the action of a metal catalyst. The result is the loss of two activating groups and the formation of new covalent bonds between the remaining fragments.

[0056] As used herein, the term "cell" refers to a single cell or a plurality of cells, and includes cells in cell culture or cells in an individual.

[0057] As used herein, the term "subject" includes all members of the animal kingdom, including mammals, and the plant kingdom. Thus, the methods and uses of the present application are suitable for human therapy, veterinary medicine, and agricultural applications.

[0058] The term "pharmaceutically acceptable" means compatible with treatment of a subject (eg, a human).

[0059] The term "pharmaceutically acceptable carrier" means a non-toxic solvent, dispersant, excipient, adjuvant or other material with which the active ingredient is mixed to allow for formation of a pharmaceutical composition, ie, a dosage form capable of administration to a subject.

[0060] The term "pharmaceutically acceptable salt" means an acid addition salt or a base addition salt that is suitable or compatible with the treatment of the subject.

[0061] As used herein, the term "solvate" means a compound or salt and / or prodrug of a compound in which molecules of a suitable solvent are incorporated into the crystal lattice. Suitable solvents are physiologically tolerable at the administered dose.

[0062] As used herein, the term "prodrug" means a compound or a salt and / or solvate of a compound that is converted to an active drug after administration.

[0063] As used herein, the term "moiety" refers to a portion of a molecule to which a name is given, typically used to describe a larger and characteristic portion of an organic molecule.

[0064] As used herein and as fully understood in the art, the term "treating" or "treatment" means a method for obtaining a beneficial or desired result (including a clinical result). Beneficial or desired clinical results may include, but are not limited to, alleviating or improving one or more symptoms or symptoms, alleviating the extent of the disease, stabilizing (i.e., no longer worsening) the disease state, preventing the spread of the disease, delaying or slowing the progression of the disease, improving or alleviating the disease state, reducing disease recurrence, and alleviating (whether partial or total), whether detectable or undetectable. "Treatment" and "treatment" may also mean extending survival compared to the expected survival without treatment. As used herein, "treating" and "treatment" also include preventive treatment. For example, an individual with early-stage cancer can be treated to prevent progression, or alternatively, an individual in remission can be treated with a compound or composition of the present application to prevent recurrence. Methods of treatment include administering a therapeutically effective amount of one or more compounds of the present application to an individual, and optionally consisting of a single administration, or alternatively comprising a series of administrations.

[0065] By "alleviating" a disease or disorder, it is meant that the extent and / or undesirable clinical manifestations of the disorder or disease state are reduced and / or the time course of progression is slowed or prolonged, as compared to not treating the disorder.

[0066] As used herein, the term "prevention" or "prophylaxis" or synonyms thereof refers to reducing the risk or likelihood that a patient will develop a fungal-related disease, disorder or condition, or exhibit symptoms associated with a fungal-related disease, disorder or condition.

[0067] As used herein, the term "effective amount" or "therapeutically effective amount" means an amount of a compound or one or more compounds of the present application effective, at dosages and for periods of time necessary, to achieve the desired result.

[0068] As used herein, the expression "inhibit CK1" refers to inhibiting, blocking and / or disrupting CK1 activity in fungal cells, whether directly or indirectly. Such inhibition, blocking and / or disruption may result in a therapeutic effect in the cells.

[0069] "Inhibit, block and / or disrupt" means any detectable inhibition, blocking and / or disruption in the presence of a compound compared to otherwise identical conditions except that the compound is absent.

[0070] The term "fungal-related disease, disorder, or condition" means that the disease, disorder, or condition to be treated is directly or indirectly affected by, mediated by, and / or has some biological basis that includes fungal activity. When the fungal activity associated with the disease, disorder, or condition is inhibited by one or more compounds or compositions of the present application, the disease will respond favorably.

[0071] The term "CK1" as used herein refers to casein kinase type 1.

[0072] As used herein, the term "fungal cell uptake" or "fungal cell permeability" refers to the regulation or control of the exchange of molecules between a cell and its environment, eg, increased cell permeability means an increased ability of a molecule to be internalized into a cell.

[0073] As used herein, the term "administering" means administering a therapeutically effective amount of a compound, or one or more compounds, or a composition of the present application to cells in cell culture or cells in a subject.

[0074] As used herein, the term "linker moiety" refers to any molecular structure that joins two or more other molecular structures together.

[0075] As used herein, the term "complementary functional group" refers to a group of atoms or a single atom that will react with another group of atoms or a single atom to form a covalent bond between the two groups or atoms.

[0076] As used herein, the term "react with" generally means that there is a flow of electrons or a transfer of electrostatic charge, resulting in the formation of a covalent bond.

[0077] The term "conjugate" as used herein means the joining together of two molecules via a covalent bond.

[0078] This specification refers to many chemical terms and abbreviations used by those skilled in the art. However, for the sake of clarity and consistency, definitions of selected terms are provided.

[0079] As used herein, the term "aq." refers to an aqueous solution.

[0080] The term "rt" as used herein refers to room temperature.

[0081] The term "Me" as used herein refers to a methyl group.

[0082] The term "Et" as used herein refers to ethyl.

[0083] The term "Pr" as used herein refers to propyl.

[0084] The term "Bu" as used herein refers to butyl.

[0085] The term "Ac" as used herein refers to acetyl.

[0086] The term "Ph" as used herein refers to a phenyl group.

[0087] The term "Ts" as used herein refers to tosyl.

[0088] The term "Ms" as used herein means methanesulfonyl.

[0089] The term "THF" as used herein refers to tetrahydrofuran.

[0090] As used herein, the term "HATU" refers to azabenzotriazole tetramethyluronium hexafluorophosphate.

[0091] As used herein, the term "DIPEA" or "DIEA" refers to N,N-diisopropylethylamine.

[0092] The term "TEA" as used herein refers to triethylamine.

[0093] As used herein, the term "TBME" or "MTBE" refers to methyl tert-butyl ether.

[0094] The term "DMF" as used herein refers to dimethylformamide.

[0095] The term "DCM" as used herein refers to dichloromethane.

[0096] As used herein, the term "DME" refers to dichloroethane.

[0097] As used herein, the term "DMAP" refers to 4-dimethylaminopyridine.

[0098] The term "DMA" as used herein refers to dimethylacetamide.

[0099] As used herein, the term "DIB" refers to diisobutylene.

[0100] As used herein, the term "DEA" refers to diethanolamine.

[0101] As used herein, the term "DBU" refers to 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0102] The term "TFA" as used herein refers to trifluoroacetic acid.

[0103] As used herein, the term "ACN" refers to acetonitrile.

[0104] The term "TMS" as used herein refers to trimethylsilyl.

[0105] As used herein, the term "SEM" refers to trimethylsilylethoxymethyl.

[0106] The term "BOC" as used herein refers to tert-butyloxycarbonyl.

[0107] The term "DMSO" as used herein refers to dimethyl sulfoxide.

[0108] As used herein, the term "NBS" refers to N-bromosuccinimide.

[0109] As used herein, the term "IPA" refers to isopropyl alcohol.

[0110] As used herein, the term "DMP" refers to Dess-Martin periodinane.

[0111] As used herein, the term "CDI" refers to 1,1'-carbonyldiimidazole.

[0112] As used herein, the term "HBPIN" refers to pinacol borane or 4,4,5,5-tetramethyl-1,3,2-dioxaborolane.

[0113] As used herein, the term "TEMPO" refers to a (2,2,6,6-tetramethylpiperidin-1-yl)oxy or (2,2,6,6-tetramethylpiperidin-1-yl)oxy free radical.

[0114] The term "CTAB" as used herein refers to cetrimonium bromide or cetyltrimethylammonium bromide or cetyltrimethylammonium bromide.

[0115] The term "MW" as used herein refers to microwaves.

[0116] As used herein, the term "ee" refers to enantiomeric excess.

[0117] The term "HPLC" as used herein refers to high performance liquid chromatography.

[0118] The term "NMR" as used herein refers to nuclear magnetic resonance.

[0119] The term "MS" as used herein refers to mass spectrometry.

[0120] The term "LCMS" as used herein refers to liquid chromatography-mass spectrometry.

[0121] As used herein, the term "IC 50 ” refers to the half-maximal inhibitory concentration.

[0122] The term "ATP" as used herein refers to adenosine 5'-triphosphate.

[0123] The term "PCR" as used herein refers to polymerase chain reaction.

[0124] As used herein, the term "MIC" refers to minimum inhibitory concentration.

[0125] As used herein, the term "TEV" refers to tobacco etch virus.

[0126] II. Compounds and Compositions

[0127] This application describes a new class of conjugated compounds.

[0128] Therefore, the present application includes a conjugate compound of formula (I) or a pharmaceutically acceptable salt, solvate and / or prodrug thereof,

[0129] A–L 1 –B

[0130] (I)

[0131] in:

[0132] A is a moiety that increases fungal cell uptake and / or fungal cell permeability;

[0133] B is a CK1 inhibitory moiety; and

[0134] L 1 is a linker comprising at least one first complementary functional group to covalently link to a functional group in A, and at least one second complementary functional group to covalently link to a functional group in B.

[0135] In some embodiments, the compound of Formula I optionally comprises one or more isotopic variant atoms. In some embodiments, the isotopic variant is deuterium, and the compound of Formula I comprises one or more deuterium atoms.

[0136] In some embodiments, A is an antifungal moiety. In some embodiments, A comprises one or more of the following: an alkylene moiety, an azole moiety, an indole moiety, a phenyl moiety, a carbazole moiety, a pyridoindole moiety, a tetrahydropyridoindole moiety, a polyamine moiety, a fatty acid alkyl chain moiety, a flucytosine (5-FC) moiety, and a triphenylphosphonium moiety, each of which is optionally substituted with one or more groups independently selected from the group consisting of halogen, OH, C 1-4 Alkyl, C 1-4 Halogenated alkyl, OC 1-4 Alkyl, C(O)OH, C(O)NH2, C(O)C 1-4 Alkyl, C(O)OC 1-4 Alkyl, C(O)NHC 1-4 Alkyl, C(O)N(C 1-4 Alkyl)C 1-4 alkyl), C(NH)NH2, C(NC 1-4 Alkyl)NH2、C(NC 1-4 alkyl)NHC 1-4 Alkyl, C(NC 1-4 alkyl)N(C 1-4 Alkyl)(C 1-4 alkyl), C(NH)NHC 1-4 Alkyl and C(NH)N(C 1-4 Alkyl)(C 1-4 alkyl), wherein each alkyl is optionally substituted by halogen, suitably substituted by fluorine. In some embodiments, A optionally comprises one or more isotopic variant atoms. In some embodiments, the isotopic variant is deuterium, and A comprises one or more deuterium atoms. In some embodiments, the azole moiety refers to a five-membered heterocycle containing nitrogen and at least one other non-carbon atom having the following general structure: wherein X, Y and Z are independently selected from CH, N, O and S, and optionally their reduced analogs and / or optionally substituted forms thereof, wherein the optional substituents are as defined above. The point of attachment represented by may be at any atom in the ring with a valence permitting covalent attachment.In some embodiments, the azole moiety is selected from an imidazole moiety, a triazole moiety, a tetrazole moiety, and a thiazole moiety.

[0137] In some embodiments, the indole moiety has the following general structure: and its optionally substituted forms, wherein the optional substituents are as defined above. The indicated points of attachment may be at any atom in the two rings having valencies permitting covalent attachment.

[0138] In some embodiments, the carbazole moiety has the following general structure: and optionally substituted forms thereof, wherein the optional substituents are as defined above. The indicated points of attachment may be at any atom in the three rings with a valence permitting covalent attachment.

[0139] In some embodiments, the pyridoindole moiety has the following general structure: and optionally substituted forms thereof, wherein the optional substituents are as defined above. The indicated points of attachment may be at any atom in the three rings with a valence permitting covalent attachment.

[0140] In some embodiments, the tetrahydropyridoindole moiety has the following general structure: and optionally substituted forms thereof, wherein the optional substituents are as defined above. The indicated points of attachment may be at any atom in the three rings with a valence permitting covalent attachment.

[0141] In some embodiments, the polyamine moiety is a saturated or unsaturated, branched or linear alkyl chain and contains 4 to 20 carbon atoms and two or more amino groups of the formula NR y an amino group, and optionally an amino group of the formula N(R y )2 terminal amino group, wherein R y Selected from H, C 1-6 Alkyl and C 1-6 Halogenated alkyl.

[0142] In some embodiments, the fatty acid alkyl chain portion refers to an aliphatic chain comprising 4 to 28 carbons, which is saturated or unsaturated, branched or straight-chain, and is optionally substituted with halogen, suitably substituted with fluorine. In some embodiments, when the fatty acid is an unsaturated fatty acid, the fatty acid is a monounsaturated fatty acid or a polyunsaturated fatty acid containing two or more carbon-carbon double bonds. In some embodiments, the fatty acid contains two, three or four double bonds. In some embodiments, the fatty acid contains three double bonds.

[0143] In some embodiments, the flucytosine (5-FC) moiety is of the formula Compounds and analogs thereof, wherein The indicated point of attachment may be at any atom in the moiety with a valence permitting covalent attachment.

[0144] In some embodiments, the triphenylphosphonium moiety refers to a moiety having the following general formula: Among them - It is the associated anion.

[0145] In some embodiments, A is any antifungal agent known in the art. In some embodiments, A is selected from fluconazole, itraconazole, clotrimazole, ketoconazole, voriconazole, posaconazole, isavuconazole, miconazole, flucytosine, olorofim, manogepix, erefungin, caspofungin, micafungin, anidulafungin, rezafungin, amphotericin B, and VT-1161, each of which is bound to L via any reactive functional group or atom in the molecule. 1 combination.

[0146] In some embodiments, A is selected from:

[0147]

[0148]

[0149] wherein each n is independently an integer from 0 to 4, and Indicates that L 1 The attachment point of the invention includes all stereoisomers thereof.

[0150] In some embodiments, A is selected from:

[0151]

[0152]

[0153] wherein each n is independently an integer from 0 to 4, and Indicates that L 1 includes all stereoisomers thereof; and

[0154] wherein A optionally comprises one or more isotopic variant atoms, for example, wherein the isotopic variant is deuterium and A comprises one or more deuterium atoms.

[0155] In some embodiments, L 1 Contains one or more independently selected groups selected from C(O), C(=NR 1 ), O, NR 1 、C 1-12 Alkylene, C 6-20 Arylene, C 3-10 Cycloalkylene, C 2-20 Heteroarylene and C 3-10 Heterocycloalkylene, wherein each of alkylene, arylene, cycloalkylene, heteroarylene and heterocycloalkylene is optionally replaced by one or more R 2 Replace, and each R2 Independently selected from C 1-12 Alkyl, NR 3 C(O)R 4 NR 3 R 4 , halogen, C(O)NR 3 R 4 OR 3 ; and R 1 、R 3 and R 4 Independently selected from H, C 1-12 Alkyl and C 1-12 In some embodiments, L 1 Optionally contains one or more isotopic variant atoms. In some embodiments, the isotopic variant is deuterium, and L 1 Contains one or more deuterium atoms.

[0156] In some embodiments, L 1 Contains 6 independently selected from C(O), C(=NR 1 ), O, NR 1 、C 1-12 Alkylene, C 6-20 Arylene, C 3-10 Cycloalkylene, C 2-20 Heteroarylene and C 3-10 In some embodiments, L 1 Contains 5 independently selected from C(O), C(=HR 1 ), O, NR 1 、C 1-12 Alkylene, C 6-20 Arylene, C 3-10 Cycloalkylene, C 2-20 Heteroarylene and C 3-10 In some embodiments, L 1 Contains 4 independently selected from C(O), C(=R 1 ), O, NR 1 、C 1-12 Alkylene, C 6-20 Arylene, C 3-10 Cycloalkylene, C 2-20 Heteroarylene and C 3-10 In some embodiments, L 1 Contains 3 independently selected from C(O), C(=NR 1 ), O, NR 1 、C 1-12 Alkylene, C 6-20 Arylene, C 3-10 Cycloalkylene, C2-20 Heteroarylene and C 3-10 In some embodiments, L 1 Contains 2 selected from C(O), C(=NR1), O, NR 2 、C 1-12 Alkylene, C 6-20 Arylene, C 3-10 Cycloalkylene, C 2-20 Heteroarylene and C 3-10 In some embodiments, each of the alkylene, arylene, cycloalkylene, heteroarylene, and heterocycloalkylene groups is optionally and independently replaced by one or more, one to six, one to five, one to four, one to three, one or two, or one R 2 In some embodiments, L 1 Contains C 1-6 Alkylene, C 6-10 Arylene, C 3-8 Cycloalkylene, C 2-10 Heteroarylene and / or C 3-8 One or more, one to six, one to five, one to four, one to three, one or two, or one of heterocycloalkylene, each of which is optionally and independently replaced by one or more, one to six, one to five, one to four, one to three, one or two, or one R 2 replace.

[0157] In some embodiments, L 1 Selected from C 1-12 Alkylene-NR 1 -C(O), C 1-6 Alkylene-C 6-10 Arylene-NR 1 -C(O), C 1-6 Alkylene-NR 1 -C(O)-C 2-10 Heteroarylene, C 1-6 Alkylene-C 2-10 Heteroarylene-NR 1 -C(O), C 2-10 Heteroarylene-NR 1 -C(O), C 1-6 Alkylene-C 3-10 Heterocycloalkylene-NR 1 -C(O)-C 2-10 Heteroarylene, C 1-6 Alkylene-C 3-10 Heterocycloalkylene-C 2-10 Heteroarylene, C 1-6 Alkylene-C 3-10 Heterocycloalkylene-NR1 -C(O), C 1-6 Alkylene-C 3-10 Heterocycloalkylene-C 1-6 Alkylene-NR 1 -C(O), C 1-6 Alkylene-C 3-10 Heterocycloalkylene-C 6-10 Arylene-NR 1 -C(O), C 1-6 Alkylene-C 3-10 Heterocycloalkylene-C 1-6 Alkylene-C 6-10 Arylene-NR 1 -C(O), C 1-6 Alkylene-C(O)-C 3-10 Heterocycloalkylene-NR 1 -C(O), C 1-6 Alkylene-C(O)-C 3-10 Heterocycloalkylene-NR 1 -C 1-6 Alkylene, C 1-6 Alkylene-OC(O)-C 1-6 Alkylene-C 3-10 Heterocycloalkylene-NR 1 -C(O), C 1-6 Alkylene-C 3-10 Heterocycloalkylene-NR 1 -C 1-6 Alkylene, C 1-6 Alkylene-C 3-10 Heterocycloalkylene-C 1-6 Alkylene-NR 1 -C 1-6 Alkylene, C 1-6 Alkylene-C 3-10 Heterocycloalkylene-C 1-6 Alkylene, C 1-6 Alkylene-NR 1 -C 1-6 Alkylene, OC 1-12 Alkylene, C 1-6 Alkylene-C 3-10 Heterocycloalkylene-OC 1-6 Alkylene-C 6-10 Arylene-NR 1 -C(O), C 1-6 Alkylene-C 3-10 Heterocycloalkylene-OC 1-6 Alkylene-OC 6-10 Arylene-NR 1 -C(O) and NR 1 -C1-6 Alkylene-OC 1-6 Alkylene-OC 1-6 Alkylene-NR 1 Each L defined above 1 Optionally, one or more R 2 Those skilled in the art will appreciate that each of A and B is covalently bonded to L as defined above. 1 either end of .

[0158] In some embodiments, each R 2 Independently selected from C 1-6 Alkyl, NR 3 C(O)R 4 NR 3 R 4 、C(O)NR 3 R 4 OR 3 ; and R 3 and R 4 Independently selected from H, C 1-6 Alkyl and C 1-6 In some embodiments, each R 2 Independently selected from methyl, ethyl, propyl, NH2, NHC(O)C 1-6 alkyl, C(O)NH2 and OH. In some embodiments, each R 2 In some embodiments, R 3 and R 4 Independently selected from H, methyl, ethyl and propyl.

[0159] In some embodiments, L 1 Selected from:

[0160]

[0161]

[0162]

[0163] wherein each of m, p, q, r and s is independently an integer from 0 to 6, and represents the point of attachment to A or B, including all stereoisomers thereof.

[0164] In some embodiments, L 1 Selected from:

[0165]

[0166]

[0167]

[0168]

[0169] wherein each of m, p, q, r and s is independently an integer from 0 to 6, and represents the point of attachment to A or B, including all stereoisomers thereof, and

[0170] Among them L 1 optionally comprising one or more isotopic variant atoms, for example, wherein the isotopic variant is deuterium, and L 1 Contains one or more deuterium atoms.

[0171] In some embodiments, B is C containing 2 to 6 N heteroatoms. 4-20 In some embodiments, B comprises at least one fluorophenyl group. In some embodiments, B comprises one or more of the following: a pyridine group, a pyrimidine group, a pyrazole group, a piperazine group, and an imidazole group. In some embodiments, B comprises a pyrazolopyridine group or a pyrrolopyridone group. In some embodiments, B comprises any compound known to inhibit CK1, such as the compounds listed in Table 1 below, including all stereoisomers thereof. The compound represented by B can be reacted with L via any reactive functional group present in the molecule. 1 In some embodiments, B optionally comprises one or more isotopic variant atoms. In some embodiments, the isotopic variant is deuterium, and B comprises one or more deuterium atoms.

[0172] Table 1: CK1 inhibitors

[0173]

[0174]

[0175] In some embodiments, B is selected from:

[0176]

[0177]

[0178] in Indicates that L 1 connection point.

[0179] In some embodiments, B optionally comprises one or more isotopic variant atoms, for example, wherein the isotopic variant is deuterium, and B comprises one or more deuterium atoms.

[0180] In some embodiments, the conjugated compound of formula (I) is selected from the compounds listed in Table 2:

[0181] Table 2

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230] or an enantiomer thereof (if applicable), or a pharmaceutically acceptable salt, solvate and / or prodrug thereof, or an isotopic variant thereof, for example wherein the isotopic variant is deuterium and the compound of formula I contains one or more deuterium atoms.

[0231] The present application also includes a pharmaceutical composition comprising one or more conjugated compounds of the present application, or a pharmaceutically acceptable salt, solvate and / or prodrug thereof, and a pharmaceutically acceptable carrier and / or diluent. In some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent.

[0232] In embodiments of the present application, the compounds described herein may have at least one asymmetric center. In the case where a compound has more than one asymmetric center, they may exist as diastereomers. It should be understood that all of these isomers and mixtures of any ratio thereof are included within the scope of the present application. It should also be understood that although the stereochemistry of the compound may be as shown in any given compound listed herein, such compounds may also contain a certain amount (e.g., less than 20%, suitably less than 10%, more suitably less than 5%) of the present application compound with alternative stereochemistry. The present application is intended to include any optical isomer such as isolated, pure or partially purified optical isomers or their racemic mixtures within the scope of protection.

[0233] The compounds of the present application may also exist in different tautomeric forms, and the present application intends to include any tautomeric forms formed by the compounds and mixtures thereof within the scope of protection.

[0234] The compounds of the present application may also exist in different polymorphic forms, and any polymorphs formed or mixtures thereof are intended to be included within the scope of the present application.

[0235] In the compounds of the present application and their pharmaceutically acceptable salts, solvates and / or prodrugs, atoms may exhibit their natural isotopic abundance, or one or more of the atoms may be artificially enriched in a particular isotope having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number primarily found in nature. The present application is intended to include all suitable isotopic variants of the compounds of the present application. For example, different isotopic forms of hydrogen (H) include protium ( 1 H), deuterium ( 2 H) and tritium ( 3 H). Protium is the major isotope of hydrogen found in nature. In some embodiments, the compounds of the present application contain one or more deuterium atoms.

[0236] The compounds of the present application may also be radiolabeled, and therefore all radiolabeled forms of the compounds of the present application are included within the scope of the present application. Therefore, the compounds of the present application also include those compounds that incorporate one or more radioactive atoms into their structure.

[0237] In one embodiment, the pharmaceutically acceptable salt is an acid addition salt or a base addition salt. Suitable salts can be selected by one skilled in the art (see, for example, SM Berge et al., "Pharmaceutical Salts," J. Pharm. Sci. 1977, 66, 1-19).

[0238] Acid addition salts suitable for individual treatment or compatible with individual treatment are any nontoxic organic or inorganic acid addition salts of any basic compound. Basic compounds forming acid addition salts include, for example, compounds containing an amine group. Exemplary inorganic acids forming suitable salts include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid and phosphoric acid, as well as acidic metal salts, such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Exemplary organic acids forming suitable salts include monocarboxylic acids, dicarboxylic acids and tricarboxylic acids. Examples of such organic acids are, for example, acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, hydroxymaleic acid, benzoic acid, hydroxybenzoic acid, phenylacetic acid, cinnamic acid, mandelic acid, salicylic acid, 2-phenoxybenzoic acid, p-toluenesulfonic acid and other sulfonic acids, such as methanesulfonic acid, ethanesulfonic acid and 2-hydroxyethanesulfonic acid. In one embodiment, monobasic acid salt or dibasic acid salt is formed, and this type of salt exists in the form of hydrate, solvate or substantially anhydrous. Usually, acid addition salt is more soluble in water and multiple hydrophilic organic solvents, and compared with the free base form, they usually show higher fusing point. The selection criteria of suitable salt is well known to those skilled in the art. Other non-pharmaceutically acceptable salts can be used, such as but not limited to oxalate, for example, for laboratory use in the separation of the present application's compound, or for being converted into pharmaceutically acceptable acid addition salts subsequently.

[0239] Suitable for individual treatment or with individual treatment compatible base addition salt is any nontoxic organic or inorganic base addition salt of any acidic compound.The acidic compound that forms base addition salt includes, for example, compound comprising carboxylic acid group.The exemplary inorganic base that forms suitable salt includes lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide or barium hydroxide and ammonia.The exemplary organic base that forms suitable salt includes aliphatic, alicyclic or aromatic organic amine, for example isopropylamine, methylamine, trimethylamine, picoline, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrazine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resin etc. Exemplary organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. The selection of an appropriate salt may be useful, for example, so that an ester functional group (if any) elsewhere in the compound is not hydrolyzed. The selection criteria for an appropriate salt are well known to those skilled in the art.

[0240] Solvates of the compounds of the present invention include, for example, those prepared with pharmaceutically acceptable solvents. Examples of such solvents include water (the resulting solvate is referred to as a hydrate) and ethanol.

[0241] Prodrugs of the compounds of the present invention can be conventional esters formed, for example, with available hydroxyl, thiol, amino or carboxyl groups. Some common esters that have been used as prodrugs are phenyl esters, aliphatic (C1-C 24 ) esters, acyloxymethyl esters, carbamates and amino acid esters.

[0242] The compounds of the present application can be suitably formulated into compositions using one or more carriers in a conventional manner. Therefore, the present application also includes a composition comprising one or more compounds of the present application and a carrier. The compounds of the present application can be suitably formulated into pharmaceutical compositions for administration to an individual in a biocompatible form suitable for in vivo administration. Therefore, the present application also includes a pharmaceutical composition comprising one or more compounds of the present application and a pharmaceutically acceptable carrier.

[0243] The compounds of the present application (including salts and / or solvates thereof) can be used alone as appropriate, but are generally administered in the form of a composition in which one or more compounds of the present application (active ingredients) are combined with an acceptable carrier. Depending on the mode of administration, the composition will contain from about 0.05 wt % to about 99 wt % or from about 0.10 wt % to about 70 wt % of the active ingredient, and from about 1 wt % to about 99.95 wt % or from about 30 wt % to about 99.90 wt % of an acceptable carrier, all weight percentages being based on the total composition.

[0244] The compound of the present application can be applied to individuality in various forms, and this depends on the selected route of administration, as understood by those skilled in the art. The compound of the present application can be applied, for example, by oral, parenteral, buccal, sublingual, nose, rectum, patch, pump or transdermal administration, and accordingly prepare pharmaceutical composition. Administration can be carried out regularly or continuously by means of pump. For selecting and preparing the conventional procedure and composition of suitable composition, for example, Remington's Pharmaceutical Sciences (2000 the 20th edition) and The United States Pharmacopeia:The National Formulary (USP 24NF19) published in 1999 are described.

[0245] Parenteral administration includes intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary (e.g., by use of an aerosol), intrathecal, rectal, and topical (including use of a patch or other transdermal delivery device) administration. Parenteral administration can be performed by continuous infusion over a selected period of time.

[0246] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to ensure easy syringability.

[0247] The compounds of the present application can be administered orally, for example, with an inert diluent or with an absorbable edible carrier, or can be encapsulated in a hard or soft shell gelatin capsule, or can be compressed into tablets, or can be directly mixed with the food of the diet. For oral therapeutic administration, the compound can be mixed with an excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, caplets, pills, granules, lozenges, chewing gums, powders, syrups, elixirs, wafers, aqueous solutions and suspensions. In the case of tablets, the carriers used include lactose, corn starch, sodium citrate and phosphates. Pharmaceutically acceptable excipients include binders (e.g., pregelatinized corn starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc or silicon dioxide); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets may be coated by methods well known in the art. In the case of tablets, capsules, caplets, pills or granules for oral administration, a pH-sensitive enteric coating such as Eudragits designed to control the release of the active ingredient is optionally used. TM. Oral dosage forms also include modified release formulations, such as immediate release formulations and timed release formulations. Examples of modified release formulations include, for example, sustained release (SR), extended release (ER, XR or XL), timed release (time-release or timed-release), controlled release (CR) or continuous release (CR or Contin), which are used, for example, in the form of coated tablets, osmotic delivery devices, coated capsules, microencapsulated microspheres, agglomerated particles (e.g., molecular sieve type particles) or fine hollow permeable fiber bundles or short chopped hollow permeable fibers (agglomerated or held in fiber packages). Timed release compositions can be formulated, such as liposomes or those in which the active compound is protected by a differentially degradable coating, such as by microencapsulation, multiple coatings, etc. Liposomal delivery systems include, for example, small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholine. For oral administration in capsule form, useful carriers or diluents include lactose and dry corn starch.

[0248] Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they are suitably present as dry products and are formulated with water or other suitable vehicles before use. When aqueous suspensions and / or emulsions are administered orally, the compound of the present application is suitably suspended or dissolved in the oil phase in combination with an emulsifier and / or suspending agent. If necessary, certain sweeteners and / or flavorings and / or coloring agents can be added. Such liquid preparations for oral administration can be prepared by conventional means with pharmaceutically acceptable additives, such as suspending agents (for example, sorbitol syrup, methylcellulose or hydrogenated edible fats), emulsifiers (for example, lecithin or gum arabic), non-aqueous vehicles (for example, almond oil, oily esters or ethanol) and preservatives (for example, methylparaben or propylparaben or sorbic acid). Useful diluents include lactose and high molecular weight polyethylene glycol.

[0249] It is also possible to lyophilize the compounds of the present application and use the lyophilizate obtained, for example, for the preparation of injection products.

[0250] The compounds of the present application can also be administered parenterally. Solutions of the compounds of the present application can be prepared in water suitably mixed with surfactants such as hydroxypropyl cellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, DMSO, and mixtures thereof with or without alcohol, as well as in oils. Under common storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms. Those skilled in the art will know how to prepare suitable preparations. For parenteral administration, sterile solutions of the compounds of the present application are generally prepared, and the pH of the solution is appropriately adjusted and buffered. For intravenous use, the total concentration of the solute should be controlled to make the preparation isotonic. For ophthalmic administration, ointments or dropperable liquids can be delivered by ophthalmic delivery systems known in the art such as applicators or eye drops. Such compositions can include mucus mimetics such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose, or polyvinyl alcohol, preservatives such as sorbic acid, EDTA, or benzyl chromium chloride, and conventional amounts of diluents or carriers. For pulmonary administration, appropriate diluents or carriers will be selected to allow aerosol formation.

[0251] The compound of the present application can be formulated for parenteral administration, including the use of conventional catheter insertion techniques or infusions. Injection preparations can exist in unit dosage form, for example, in ampoules or multi-dose containers, and preservatives are added. The composition can take the form of a sterile suspension, solution or emulsion in an oily or aqueous vehicle, and can contain preparatons, such as suspending agents, stabilizers and / or dispersants. In all cases, the form must be sterile and must be fluid to ensure the presence of easy injection. Alternatively, the compound of the present application is suitably in a sterile powder form for combination with a suitable vehicle (e.g., sterile pyrogen-free water) before use.

[0252] Compositions for nasal administration may conveniently be formulated as aerosols, drops, gels and powders.

[0253] For intranasal administration or inhalation administration, the compound of the present application is conveniently delivered in the form of a solution, dry powder formulation or suspension from a pump spray container squeezed or pumped by the patient, or presented as an aerosol spray and delivered from a pressurized container or a nebulizer. Aerosol formulations generally comprise a solution or fine suspension of active substance in a physiologically acceptable aqueous or non-aqueous solvent, and are generally present in a sealed container in a sterile form of a single dose or multiple doses, which can be used in the form of a cartridge or a refill together with an atomizing device. Alternatively, the sealed container can be an integrated dispensing device, such as a single dose nasal inhaler or an aerosol dispenser equipped with a metering valve that is discarded after use. When the dosage form includes an aerosol dispenser, it will contain a propellant, which can be a compressed gas, such as compressed air, or an organic propellant, such as a fluorochlorocarbon. Suitable propellants include but are not limited to dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, heptafluoroalkane, carbon dioxide or other suitable gases. In the case of a pressurized aerosol, the dosage unit is suitably determined by providing a valve to deliver a metered amount. The pressurized container or sprayer may contain a solution or suspension of the active compound. Capsules and cartridges (e.g., made of gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of the compound of the present application and a suitable powder base such as lactose or starch. Aerosol formulations may also be in the form of a pump-sprayer.

[0254] Compositions suitable for buccal or sublingual administration include tablets, lozenges and pastilles, in which the active ingredient is formulated with a carrier such as sugar, acacia, tragacanth or gelatin and glycerin. Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base, such as cocoa butter.

[0255] The suppository form of the present invention compound is used for vaginal, urethral and rectal administration. Such suppositories are generally composed of a mixture of substances that is solid at room temperature but melts at body temperature. The materials commonly used to produce such vehicles include, but are not limited to, cocoa butter (also known as cocoa butter), glycerinated gelatin, other glycerides, hydrogenated vegetable oils, polyethylene glycol mixtures of various molecular weights, and fatty acid esters of polyethylene glycol. See, for example: Remington's Pharmaceutical Sciences, 16th edition, Mack Publishing, Easton, PA, 1980, for further discussion of suppository dosage forms on pages 1530-1533.

[0256] The compound of the present application can also be coupled with a soluble polymer as a targetable drug carrier. Such polymers can include polyvinyl pyrrolidone, pyran copolymer, polyhydroxypropyl methacrylamide-phenol, polyhydroxyethyl asparagine-phenol, or the polyethylene oxide-polylysine substituted with palmitoyl residues. In addition, the compound of the present application can be coupled with a class of biodegradable polymers for realizing controlled drug release, such as cross-linked or amphiphilic block copolymers of polylactic acid, polyglycolic acid, polylactic acid and polyglycolic acid, poly-ε caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and hydrogels.

[0257] In one embodiment, the compounds of the present application can be coupled to viral vectors, non-viral vectors or other vectors. Viral vectors can include retroviruses, lentiviruses, adenoviruses, herpes viruses, pox viruses, alphaviruses, vaccinia viruses or adeno-associated viruses. Non-viral vectors can include nanoparticles, cationic lipids, cationic polymers, metal nanoparticles, nanorods, liposomes, micelles, microbubbles, cell penetrating peptides or lipid spheres. Nanoparticles can include silica, lipids, carbohydrates or other pharmaceutically acceptable polymers.

[0258] In some embodiments, depending on the mode of administration, the pharmaceutical composition comprises from about 0.05 wt % to about 99 wt % or from about 0.10 wt % to about 70 wt % of the active ingredient (one or more compounds of the present application), and from about 1 wt % to about 99.95 wt % or from about 30 wt % to about 99.90 wt % of one or more pharmaceutically acceptable carriers, all weight percentages being based on the total composition.

[0259] In one embodiment, the compound of the present application and another therapeutic agent are administered simultaneously or sequentially in a separate unit dosage form, or are administered together in a single unit dosage form. Therefore, the present application provides a single unit dosage form comprising one or more compounds of the present application (e.g., a compound of formula (I)), another therapeutic agent, and a pharmaceutically acceptable carrier.

[0260] It is to be understood that, in the above, the term "compound" also includes embodiments in which one or more compounds are mentioned.

[0261] III. Methods and Uses of the Present Application

[0262] The compounds of the present application have been shown to be capable of inhibiting fungal activity.

[0263] Therefore, the present application also includes a method for treating or preventing fungus-related diseases, disorders or conditions, which comprises administering a therapeutically effective amount of one or more conjugated compounds of the present application to an individual in need thereof.

[0264] The present application also includes a method of inhibiting or preventing fungal growth, comprising administering to an individual in need thereof a therapeutically effective amount of one or more conjugated compounds of the present application.

[0265] Also provided is a method of inhibiting fungal CK1 activity, comprising administering to an individual in need thereof a therapeutically effective amount of one or more conjugated compounds of the present application. In some embodiments, the method comprises selectively inhibiting fungal CK1 activity.

[0266] The present application also provides a method for treating fungal-related diseases, disorders or conditions that can be treated by inhibiting fungal CK1, which comprises administering to an individual in need thereof a therapeutically effective amount of one or more conjugated compounds of the present application, in combination with the conjugated compound, another known agent for treating fungal-related diseases, disorders or conditions that can be treated by inhibiting fungal CK1.

[0267] The present application also includes the use of one or more conjugated compounds of the present application for treating or preventing fungal-related diseases, disorders, or conditions, and the use of one or more conjugated compounds of the present application in the preparation of a medicament for treating or preventing fungal-related diseases, disorders, or conditions. The present application also includes one or more conjugated compounds of the present application for treating or preventing fungal-related diseases, disorders, or conditions.

[0268] Since the conjugated compounds of the present application have been shown to inhibit fungal activity, the conjugated compounds of the present application can be used to treat or prevent fungal-related diseases, disorders, or conditions by inhibiting fungal activity. Therefore, the conjugated compounds of the present application can be used as drugs. Therefore, the present application includes a conjugated compound of the present application for use as a drug.

[0269] In some embodiments, fungal-related diseases, disorders or conditions include mycoses, including superficial mycoses, subcutaneous mycoses and systemic mycoses. In some embodiments, fungal-related diseases, disorders or conditions include dermatophytes, candidiasis, pneumocystis, pityriasis versicolor, aspergillosis, mucormycosis, talaromycosis marneffei, frog dung mold, blastomycosis, chromoblastomycosis, cryptococcosis, coccidioidomycosis, otomycosis, mycetoma, histoplasmosis, lobocytosis, paracoccidioidomycosis, phaeohyphomycosis, scedosporisis, sporotrichosis and emmonsiosis.

[0270] Therefore, the present application provides a method for treating or preventing fungal infection or fungal disease, comprising administering a therapeutically effective amount of one or more conjugated compounds of the present application to an individual in need thereof. In some embodiments, the fungal disease is candidiasis.

[0271] In some embodiments, the fungi causing the infection include yeasts, molds, and / or dimorphic fungi that cause the infections described herein.

[0272] When used in conjunction with other agents or therapies for treating fungal-related diseases, disorders or conditions, one embodiment is that the conjugated compounds of the present application are administered simultaneously with those agents or therapies. As used herein, "simultaneous administration" of two substances or therapies to an individual means providing each of two substances or therapies so that they are both biologically active in the individual at the same time. The exact details of administration will depend on the pharmacokinetics of the two substances or therapies in the presence of each other, and may include administering the two substances or therapies successively within a few hours, or even administering one substance or therapies within 24 hours of administering another substance or therapies, if the pharmacokinetics are suitable. The design of a suitable dosing regimen is conventional for those skilled in the art. In a specific embodiment, the substances or therapies will be administered substantially simultaneously, that is, administered successively within a few minutes, or administered with a single composition when two substances are administered. Another embodiment of the application is to administer a combination of medicaments or therapies to an individual in a non-simultaneous manner.

[0273] In some embodiments, the subject is a mammal. In some embodiments, the subject is livestock, such as cattle, sheep, goats, poultry, etc. In some embodiments, the subject is a human. In some embodiments, fungal diseases in humans affect the skin, mucous membranes such as the mouth and vagina, nails, organs such as the lungs and brain, eyes, nose, sinuses, bones, joints, etc.

[0274] In some embodiments, the subject is a crop plant such as rice, wheat, barley, oats, rye, sugarcane and other sugar crops, maize (corn), potato, palm, canola, flax, safflower, cassava, legumes such as beans, soybeans, peas, chickpeas, peanuts, lentils, lupine, mesquite, carob, tamarind, alfalfa and clover, sunflower, rapeseed, mustard, sorghum, millet, hemp, sugar beets, peanuts, sweet potatoes, bananas, cotton, yams, various nuts, or other vegetables or fruits.

[0275] In the context of treating a fungal-related disease, disorder, or condition, an effective amount is an amount that inhibits fungal activity, for example, as compared to inhibition without administration of one or more conjugated compounds. The effective amount can vary depending on factors such as the individual's disease state, age, sex, and / or weight, fungal type, and the like. The amount of a given compound corresponding to such an amount will vary depending on various factors, such as the given drug or compound, the pharmaceutical formulation, the route of administration, the condition, the type of disease or disorder, the identity of the individual being treated, and the like, but can be routinely determined by one skilled in the art. An effective amount is an amount that results in an improvement or alleviation of any disease symptom following treatment therewith.

[0276] The dosage of the compounds of the present invention can vary according to many factors, such as the pharmacodynamic properties of the compound, the mode of administration, the age, health and weight of the recipient, the nature and extent of the symptoms, the frequency of treatment and the type of simultaneous treatment (if any), and the clearance rate of the compound in the individual to be treated. Those skilled in the art can determine the appropriate dosage based on the above factors. The compounds of the present invention can initially be administered at a suitable dosage, which can be adjusted as needed based on the clinical response. The dosage will generally be selected to maintain a serum level of the compound of the present invention of about 0.01 μg / cc to about 1000 μg / cc, or about 0.1 μg / cc to about 100 μg / cc. As a representative example, for adults, the oral dosage of one or more compounds of the present invention will be in the range of about 1 mg / day to about 1000 mg / day, suitably about 1 mg / day to about 500 mg / day, and more suitably about 1 mg / day to about 200 mg / day. For parenteral administration, a representative amount is about 0.001 mg / kg to about 10 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.01 mg / kg to about 1 mg / kg, or about 0.1 mg / kg to about 1 mg / kg. For oral administration, a representative amount is about 0.001 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 0.01 mg / kg to about 1 mg / kg, or about 0.1 mg / kg to about 1 mg / kg. For administration in the form of a suppository, a representative amount is about 0.1 mg / kg to about 10 mg / kg, or about 0.1 mg / kg to about 1 mg / kg. In one embodiment of the present application, the composition is formulated for oral administration, and the compound is suitably in tablet form, each tablet containing 0.25, 0.5, 0.75, 1.0, 5.0, 10.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 75.0, 80.0, 90.0, 100.0, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 mg of active ingredient. The compound of the present application can be administered in a daily, weekly or monthly dosage, or the total daily dose can be divided into two, three or four daily doses.

[0277] In some embodiments, the compound of the present application is administered at least once a week. However, in another embodiment, the compound is administered to an individual every two weeks, three weeks, or one month. In another embodiment, the compound is administered from about once a week to about once a day. In another embodiment, the compound is administered 2 times, 3 times, 4 times, 5 times, or 6 times a day. The length of the treatment period depends on a variety of factors, such as the severity of the disease, disorder, or condition, the age of the individual, the concentration and / or activity of the compound of the present application, and / or a combination thereof. It should also be understood that the effective dose of the compound for treatment can be increased or decreased during a specific treatment regimen. The change in dosage can be caused and become apparent by standard diagnostic tests known in the art. In some cases, long-term administration is required. For example, the compound is administered to the individual in an amount and duration sufficient to treat the individual.

[0278] IV. Methods for preparing the compounds of the present application

[0279] The compounds of the present application can be prepared by various synthetic methods. The choice of specific structural features and / or substituents may influence the selection of one method over another. It is within the ability of those skilled in the art to select a specific method to prepare a given conjugated compound of formula (I). Some of the raw materials used to prepare the compounds of the present application can be obtained from commercial chemical sources. Other raw materials are easily prepared from available precursors using simple transformation reactions well known in the art.

[0280] In some embodiments, the compound of formula I can be assembled as follows: the linking group is connected to one of A or B, and then the other of A or B is connected. Standard chemical methods known in the art can be used to assemble the compound of formula I, including but not limited to nucleophilic displacement, cross coupling, Michael reaction and / or activated group strategy. Since many A and B groups are known, or based on known compounds, compounds that can be used to react with the linking group are easy to obtain from commercial sources or using synthetic methods known in the art. For example, many such compounds have at least one functional group in their structure that can form a covalent bond with the linking group. Examples include amines, thiols, halogens, hydroxyls, alkoxyls, carboxyls, esters, amides and / or oxo groups.

[0281] In some embodiments, the compound of Formula (I) has the structure of Formula (IA):

[0282]

[0283] In some embodiments, the compound of formula (IA) is synthesized by reacting intermediate 1a with amine 1b in a solvent according to known reactions to obtain intermediate Ic, which is then reduced under known reduction conditions to obtain the compound of formula (IA), as shown in Scheme 1. In some embodiments, RA' 、R A” and R A”' independently selected from H, halogen, C 1-6 Alkyl and C 1-6 In some embodiments, R A' 、R A” and R A”' is independently selected from H, F, CH3, CH2CH3, CF2H, CF3, CFH2, CH2CF2H and CH2CF3. A' 、R A” and R A”' In some embodiments, R A' 、R A” 、R A”' 、L 1 and A have the meanings of the corresponding groups in compounds I-1, I-6 to I-13, I-25, I-188, I-191 to I-209, I-230, I-237, I-238 and I-240 to I-242 shown in Table 2.

[0284]

[0285] Route 1

[0286] In some embodiments, the compound of Formula (I) has the structure of Formula (IB):

[0287]

[0288] In some embodiments, compound (I) of formula (IB) is synthesized as follows: according to known reactions, intermediate 2a with a protecting group PG is reacted with amine 2b in a solvent to obtain intermediate 2c. Intermediate 2c can then be reduced to obtain intermediate 2d. The protecting group PG can be removed using known deprotection methods to obtain intermediate 2e. 2e is reacted with compound 2f with a leaving group LG under known coupling conditions to obtain formula (IB), as shown in Scheme 2. In some embodiments, R B' 、R B” and R B”' independently selected from H, halogen, C 1-6 Alkyl and C 1-6 In some embodiments, R B' 、R B” and R B”' is independently selected from H, F, CH3, CH2CH3, CF2H, CF3, CFH2, CH2CF2H and CH2CF3. B' 、R B”and R B”' In some embodiments, R B' 、R B” and R B”' 、L 1 and A have the meanings of the corresponding groups in compounds I-2, I-18 to I-22, and I-26 to I-31 shown in Table 2.

[0289]

[0290] Route 2

[0291] In some embodiments, the compound of Formula (I) has the structure of Formula (IC):

[0292]

[0293] In some embodiments, the compound of formula (IC) is synthesized as follows: according to known reactions, intermediate 3a is reacted with amine 3b in a solvent to obtain intermediate 3c. Intermediate 3c can then be reacted with halide 3d through alkylation conditions to obtain intermediate 3e. Intermediate 3e can then be halogenated according to known conditions to obtain 3f and boronated to obtain 3g, and then subjected to Suzuki coupling conditions in the presence of compound 3h to obtain intermediate 3i. The functional group (FG) on intermediate 3i can then be substituted according to various known methods to obtain intermediate 3j and the final compound of formula (IC), as shown in Scheme 3. In some embodiments, R C' 、R C” and R C”' independently selected from H, halogen, C 1-6 Alkyl and C 1-6 In some embodiments, R C' 、R C” and R C”' is independently selected from H, F, CH3, CH2CH3, CF2H, CF3, CFH2, CH2CF2H and CH2CF3. C' 、R C” and R C”' is independently selected from H, F and CH3. In some embodiments, the linker L 1 and A are as defined in formula (I). In some embodiments, R C' 、R C” 、R C”' 、L 1and A are as defined by the corresponding groups in compounds I-3 to I-5, I-14 to I-17, I-23, I-24, I-89, I-90, I-189, I-190, I-233, I-235, I-236, I-239 and I-244 to I-246 in Table 2.

[0294]

[0295] Route 3

[0296] In some embodiments, the compound of Formula (I) has the structure of Formula (ID):

[0297]

[0298] In some embodiments, the compound of formula (ID) is synthesized as follows: according to known reactions, in the presence of a base, intermediate 4a is reacted with amine 4b or its protected form in a solvent to obtain intermediate 4c. Intermediate 4c can then be coupled with compound 4d to obtain intermediate 4e. If necessary, any protecting group in intermediate 4e can be removed using known deprotection methods and then subjected to known condensation conditions to obtain a compound of formula (ID), as shown in Scheme 4. In some embodiments, R D' 、R D” and R D”' independently selected from H, halogen, C 1-6 Alkyl, C 1-6 haloalkyl and optionally C 1-6 Alkyl or C 3-6 Cycloalkyl substituted C 3-10 In some embodiments, R D' 、R D” and R D”' is independently selected from H, F, CH3, CH2CH3, CF2H, CF3, CFH2, CH2CF2H, CH2CF3 and pyrazole. D' 、R D” and R D”' Independently selected from H, F, CH3 and In some embodiments, L 1 and A are as defined in formula (I). In some embodiments, R D' 、R D” and R D”' 、L 1 and A are as defined for the corresponding groups in compounds I-32 to I-86, I-91 to I-99, I-210 to I-229, I-231, I-232, and I-234 shown in Table 2.

[0299]

[0300] Route 4

[0301] In some embodiments, the compound of Formula (I) has the structure of Formula (IE), wherein R E” and R E”' One of them is L 1 -A:

[0302]

[0303] In some embodiments, compounds of formula (IE) are synthesized by reacting intermediate 5a with a suitable protecting group (PG) with base 5b to provide intermediate 5c. Intermediate 5c can then be reacted with compound 5d to provide intermediate 5e, which can be subjected to known hydrolysis conditions to provide intermediate 5f. Intermediate 5f can then be coupled with boronate compound 5g under known Suzuki coupling conditions to provide compounds of formula (IE), as shown in Scheme 5.

[0304]

[0305] Route 5

[0306] For compounds wherein the pyridine group is pyrimidine, such as formula (IF) As shown, or a compound in which pyridine is connected at position 4 instead of position 3, such as formula (IG) As shown, a similar approach can be performed.

[0307] In some embodiments, R E' Selected from H, halogen, C 1-6 Alkyl and C 1-6 In some embodiments, R E” and R E”' One of H, halogen, C 1-6 Alkyl, C 1-6 haloalkyl and optionally C 1-6 Alkyl or C 3-6 Cycloalkyl substituted C 3-10 heteroaryl, and the other is L 1 -A. In some embodiments, R E' , and R E” and R E”' In some embodiments, R E' , and R E” and R E”'One of which is independently selected from H, F, CH3 and In some embodiments, R E' 、R E” 、R E”' 、L 1 and A are as defined for the corresponding groups in compounds I-87, I-88, I-100 to I-187 and I-243 shown in Table 2.

[0308] In the whole method, it should be understood that, where appropriate, suitable protecting groups will be added to various reactants and intermediates in a manner readily understood by those skilled in the art, and subsequently removed from various reactants and intermediates. The conventional procedures using such protecting groups and examples of suitable protecting groups are described in, for example, "Protective Groups in Organic Synthesis", TW Green, PGM Wuts, Wiley-Interscience, New York, (1999). It should also be understood that by chemical operations, a group or substituent is converted into another group or substituent and can be carried out on any intermediate or final product on the path of the final product, where possible conversion types are only limited to the inherent incompatibility of other functional groups carried by the molecule at this stage with the conditions or reagents used in the conversion. Those skilled in the art will readily understand such inherent incompatibility, and the way to circumvent them by carrying out appropriate conversion and synthesis steps in an appropriate order. Examples of conversions are provided herein, and it should be understood that the conversions described are not limited to the universal groups or substituents of the conversions exemplified. References and descriptions of other suitable transformations are given in "Comprehensive Organic Transformations—A Guide to Functional Group Preparations" RC Larock, VHC Publishers, Inc. (1989). References and descriptions of other suitable reactions are described in organic chemistry textbooks, for example, "Advanced Organic Chemistry", March, 4th edition McGraw Hill (1992) or "Organic Synthesis", Smith, McGraw Hill, (1994).

[0309] Isotopic variations of the compounds of the present invention can be prepared using methods known in the art, for example, using isotopic variant isotopically enriched reagents, including, for example, deuterated aluminum-based reducing agents such as lithium aluminum deuteride, deuterated alkylating agents, and the like.

[0310] Techniques for purification of intermediates and final products include, for example, normal and reverse phase chromatography on columns or spinning plates, recrystallization, distillation, and liquid-liquid or solid-liquid extraction, as will be readily appreciated by those skilled in the art.

[0311] Salts of the compounds of the present invention are generally formed by dissolving the neutral compound in an inert organic solvent, adding the desired acid or base, and isolating the resulting salt by filtration or other known methods.

[0312] The formation of the solvate of the present application compound will vary according to the compound and solvate. Generally, the solvate is formed by dissolving the compound in a suitable solvent and separating the solvate by cooling or using an anti-solvent. The solvate is usually dried or azeotropic under ambient conditions. The suitable conditions for forming a specific solvate can be selected by those skilled in the art. The example of a suitable solvent is ethanol, water, etc. When water is the solvent, the molecule is referred to as a "hydrate."

[0313] Prodrugs of the compounds of the present invention can be, for example, conventional esters formed with available hydroxyl, thiol, amino or carboxyl groups. For example, available hydroxyl or amino groups can be acylated using an activated acid in the presence of a base and optionally in an inert solvent (e.g., an acyl chloride in pyridine). Some common esters that have been used as prodrugs are phenyl esters, aliphatic (C1-C 24 ) esters, acyloxymethyl esters, carbamates and amino acid esters.

[0314] The following non-limiting examples serve to illustrate the present application.

[0315] Example

[0316] General Methods

[0317] All raw materials used herein are either commercially available or previously described in the literature. 1 H and 13 CNMR spectra were recorded on Bruker 300, Bruker DPX400 or Varian +400 spectrometers. 1 H NMR, operating at 300, 400, and 400 MHz, respectively, using TMS or residual solvent signal as internal standard, and deuterated chloroform as solvent. All reported chemical shifts are in ppm, using the δ scale, and the fine splitting of the signals appearing in the recording is usually indicated, for example, as s: singlet, br s: broad singlet, d: doublet, t: triplet, q: quartet, m: multiplet. Unless otherwise stated, in the following tables, the wavelengths were obtained at 400 MHz using CDCl3 as solvent. 1 H NMR data.

[0318] Purification of the product was performed using Chem Elut extraction cartridges (Varian, cat. #1219-8002), Mega BE-SI (BondElut silica) SPE cartridges (Varian, cat. #12256018; 12256026; 12256034) or by flash chromatography in silica gel-filled glass columns.

[0319] Preparation of intermediates and specific examples

[0320] Example 1: Synthesis of compound 6-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-imidazol-1-yl)propyl)-4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one (I-1)

[0321]

[0322] The procedure for synthesizing 6-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-imidazol-1-yl)propyl)-4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one is shown in Scheme 6:

[0323]

[0324] Route 6

[0325] 3-(4-Fluorophenyl)-1H-pyrazole(1-2)

[0326]

[0327] DMF-DMA (100 g, 847 mmol) was added to 1-(4-fluorophenyl)ethan-1-one 1-1 (100 g, 725 mmol). The reaction solution was stirred at 100°C for 16 hours under nitrogen. The reaction solution was then concentrated and evaporated to dryness to obtain a reddish-brown solid. The product was redissolved in 500 mL of ethanol, and 40 mL of hydrazine monohydrate was added thereto, and heated at 70°C for 4 hours. The reaction solution was poured into water, extracted with ethyl acetate, dried over sodium sulfate, and the solvent was evaporated. The product was recrystallized from ethyl acetate and hexane to obtain 110 g of intermediate 1-2 as light yellow crystals. 1 H-NMR (CDCl3) δ: 6.57 (d, J = 2.4Hz, 1H), 7.05-7.12 (m, 2H), 7.60 (d, J = 2.4Hz, 1H), 7.70-7.76 (m, 2H).

[0328] 3-(4-Fluorophenyl)-1-methyl-1H-pyrazole (1-3)

[0329]

[0330] At 0 ° C, Cs2CO3 (453 g, 1358 mmol) was added to a solution of 3- (4-fluorophenyl) pyrazole 1-2 (110 g, 679 mmol) in anhydrous DMF (1000 mL) and the reaction was continued for 15 min. Iodomethane (50 mL, 815 mmol) was added and the temperature was slowly raised to room temperature. The reaction solution was stirred at room temperature for 2 hours. The reaction mixture was diluted with water (1000 mL), extracted with EtOAc (3 × 1000 mL), dried (Na2SO4), concentrated, and purified by flash column (dichloromethane: methanol 50: 1) to give intermediate 1-3 (50 g, 41.9% yield) as a white solid. MS (ESi+): 177 (M+H)+.

[0331] 4-Bromo-3-(4-fluorophenyl)-1-methyl-1H-pyrazole(1-4)

[0332]

[0333] To a solution of 3-(4-fluorophenyl)-1-methyl-1H-pyrazole (50 g, 284 mmol) 1-3 in DMF (500 ml) was added NBS (50 g, 284 mmol). The reaction mixture was stirred at room temperature for 2 hours, quenched with water (500 ml), and extracted with EtOAc (500 ml * 3). The organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 42 g of Intermediate 1-4 as a white solid in a 58.4% yield.

[0334] 3-(4-Fluorophenyl)-1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole(1-5)

[0335]

[0336] Under nitrogen, at -78 ° C, n-BuLi (hexane, 2.5M, 35 mL, 87 mmol) was added to a solution of 4-bromo-3-(4-fluorophenyl)-1-methyl-1H-pyrazole 1-4 (20 g, 79 mmol) in THF (200 mL) over 10 min. After 45 min, 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (17.8 mL, 87 mmol) was added. After 1 h 45 min, saturated NH4Cl aqueous solution and EtOAc were added. The cooling bath was removed and the reaction mixture was stirred to room temperature. The layers were separated, and the organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. Silica gel chromatography using hexanes:EtOAc (3:1) as eluent afforded 3-(4-fluorophenyl)-1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole 1-5 (8 g, 33.5% yield) as a white solid.

[0337] 3-Fluoro-4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)picolinonitrile (1-6)

[0338]

[0339] 3-(4-Fluorophenyl)-1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole 1-5 (8 g, 26.5 mmol) and 3-fluoro-4-iodopyridine carbonitrile (5.5 g, 21 mmol) were combined in 100 mL DMF and treated with Cs2CO3 (13.2 g, 40.8 mmol). The resulting suspension was purged with nitrogen for 20 min and treated once with tris(dibenzylideneacetone)dipalladium(0) (1 g, 1.1 mmol). Nitrogen purging was continued for another 20 min, and the dark suspension was stirred at room temperature for 30 min. The reaction solution was heated to 50 ° C for 6 hours and then cooled to room temperature. The thick slurry was added to 150 mL EtOAc and filtered through Celite®. TM ) was filtered, and the organic layer was washed with 3 x 30 mL of brine. The organic layer was dried over anhydrous NaSO and concentrated in vacuo to yield 12 g of a crude solid. This solid was purified by flash column with a 5-80% EtOAc / heptane gradient to yield 5.8 g of Intermediate 1-6 as a white solid in 74% yield. MS m / z 297.0 (M+1); 1H NMR (400MHz, CDCl3) δ8.28 (d, J = 5.1, 1H), 7.84 (d, J = 2.9, 1H), 7.40 (dd, J = 5. 3,2.2,2H),7.25(dd,J=5.8,4.9,1H),7.09(dd,J=8.6,8.6,2H),4.02(s,3H).

[0340] 4-(3-(4-Fluorophenyl)-1-methyl-1H-pyrazol-4-yl)pyridine-2,3-dicarbonitrile (1-7)

[0341]

[0342] To a solution of 3-fluoro-4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)picolinonitrile 1-6 (5.8 g, 19.6 mmol) in DMSO (60 mL) was added potassium cyanide (1.36 g, 20.3 mmol) under gentle heating, heated to 50° C., and stirred. After 2 hours, the mixture was cooled in an ice bath and 0.1N aqueous NaOH solution (50 mL) was added. The resulting slurry was stirred for 5 minutes, and the solid was collected, rinsed with water, and air-dried to afford 4 g of compound 1-7 as a light cream solid in a 67% yield. 1 H NMR (400MHz, CDCl3) δ 8.56 (d, J = 5.2, 1H), 8.02 (s, 1H), 7.33 (dd, J = 8.9, 5.2, 2H), 7.24 (d, J = 5.5, 1H), 7.06 (dd, J = 8.6, 8.6, 2H), 4.02 (s, 3H).

[0343] 4-(3-(4-Fluorophenyl)-1-methyl-1H-pyrazol-4-yl)pyridine-2,3-dicarboxylic acid dipotassium salt (1-8)

[0344]

[0345] 4-(3-(4-Fluorophenyl)-1-methyl-1H-pyrazol-4-yl)pyridine-2,3-dicarbonitrile 1-7 (4 g, 13.2 mmol) and aqueous KOH solution (14.7 g, 262 mmol) were dissolved in 100 mL of water and heated at 100° C. for 24 hours. The mixture was cooled in ice to precipitate a thick white solid. The material was warmed to room temperature, filtered, and air-dried to give 5 g (91%) of compound 1-8 as a cream-colored solid: LCMS m / z 342.1 (M+1).

[0346] 4-(3-(4-Fluorophenyl)-1-methyl-1H-pyrazol-4-yl)furo[3,4-b]pyridine-5,7-dione(1-9)

[0347]

[0348] 4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)pyridine-2,3-dicarboxylic acid dipotassium salt (1.00 g, 2.4 mmol) was slurried with acetic acid (7 mL) and acetic anhydride (2.5 mL) and heated to 110° C. for 4 hours. The resulting homogeneous mixture of compound 1-9 was cooled and used directly in the next step.

[0349] 6-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-imidazol-1-yl)propyl)-4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)-5H-pyrrolo[3,4-b]pyridine-5,7(6H)-dione (1-11)

[0350]

[0351] To the reaction mixture containing compound 1-9 from the previous step was added a solution of 1-amino-2-(2,4-difluorophenyl)-3-(1H-imidazol-1-yl)propan-2-ol 1-10 (0.167 mL, 1.53 mmol) in THF (4.7 mL). The mixture was heated at reflux for 16 hours, cooled, and then concentrated to a brown solid. The substance was purified by reverse phase column with H2O / MeOH 5%-95% to give 110 mg of product 1-11 as a yellow solid, 8.2% yield in 2 steps: LCMSm / z 559 (M+1).

[0352] 6-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-imidazol-1-yl)propyl)-4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one (I-1)

[0353]

[0354] To a flask equipped with an overhead stirring apparatus was added 6-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-imidazol-1-yl)propyl)-4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)-5H-pyrrolo[3,4-b]pyridine-5,7(6H)-dione 1-11 (110 mg, 0.2 mmol) and zinc powder (<10 microns) (111 mg, 2 mmol), followed by acetic acid (3 mL). The mixture was heated to 105 ° C, stirred for 4.5 hours, and then cooled to room temperature. Celite was added to the reaction mixture and filtered to remove zinc. The filter pad was rinsed with EtOAc, and the filtrate was concentrated and purified by reverse phase column with H2O / MeOH 5%-95% to give 10 mg of compound I-1 as a white solid in a yield of 9.2%. LCMSm / z 545 (M+1); 1 H NMR (400MHz, methanol-d4) δ8.46(d,J=5.3Hz,1H),8.03(s,1H),7.45–7.37(m,2H),7.36–7.32(m,2H),7.09(d,J=5.3Hz,1H),7.03(dd,J=9.9,7.7Hz,3H ),6.87(s,1H),6.84(d,J=2.6Hz,1H),6.74(s,1H),4.55(q,J=8.2,6.8Hz ,4H), 4.28(dd,J=14.5,7.1Hz,2H), 4.00(s,3H),3.91(d,J=14.6Hz,1H).

[0355] Example 2: Synthesis of Compound 4-(1-(3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butyl)-3-(4-fluorophenyl)-1H-pyrazol-4-yl)-6-methyl-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one (I-2)

[0356]

[0357] The procedure for synthesizing compound 4-(1-(3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butyl)-3-(4-fluorophenyl)-1H-pyrazol-4-yl)-6-methyl-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one (I-2) is shown in the following Scheme 7:

[0358]

[0359] Route 7

[0360] 3-(4-Fluorophenyl)-1H-pyrazole(2-2)

[0361]

[0362] DMF-DMA (100 g, 847 mmol) was added to 1-(4-fluorophenyl)ethan-1-one 2-1 (100 g, 725 mmol). The reaction solution was stirred at 100 ° C for 16 hours under nitrogen protection. The reaction solution was then concentrated and evaporated to dryness to obtain a reddish-brown solid. The product was redissolved in 500 mL of ethanol, and 40 mL of hydrazine monohydrate was added thereto and heated at 70 ° C for 4 hours. The reaction solution was poured into water, extracted with ethyl acetate, dried over sodium sulfate, and the solvent was evaporated. The product was recrystallized from ethyl acetate and hexane to obtain 110 g of compound 2-2 as light yellow crystals. 1 H-NMR (CDCl3) δ: 6.57 (d, J = 2.4Hz, 1H), 7.05-7.12 (m, 2H), 7.60 (d, J = 2.4Hz, 1H), 7.70-7.76 (m, 2H).

[0363] 3-(4-Fluorophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole(2-3)

[0364]

[0365] To a solution of 3-(4-fluorophenyl)-1H-pyrazole (10 g, 61.7 mmol) in anhydrous N, N-dimethylformamide (100 ml) was slowly added NaH (3.2 g, 67.9 mmol, 60% in mineral oil) at room temperature. The suspension was stirred for 15 minutes, and 2-(trimethylsilyl)ethoxymethyl chloride (12.8 ml, 61.7 mmol) was added. The mixture was stirred at room temperature for 3 hours. The reaction mixture was diluted with water (250 mL) and extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with water (50 mL), brine, dried over MgSO4, filtered, and concentrated in vacuo to give product 2-3 and its isomers (15.3 g) as yellow waxy solids with a yield of 85%.

[0366] 4-Bromo-3-(4-fluorophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole(2-4)

[0367]

[0368] To a solution of 3-(4-fluorophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole 2-3 (15.3 g, 52.4 mmol) in DMF (100 ml) was added NBS (9.2 g, 52.4 mmol). The reaction mixture was stirred at room temperature for 2 hours, quenched with water (100 mL), and extracted with EtOAc (100 mL*3). The organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give 18 g of compound 2-4 as a white solid with a yield of 92.8%.

[0369] 3-(4-Fluorophenyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole(2-5)

[0370]

[0371] Under nitrogen, to a solution of 4-bromo-3-(4-fluorophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole 2-4 (18 g, 48.6 mmol) in THF (200 mL) was added isopropylmagnesium chloride lithium chloride complex (1.3 M, 49 mL, 63.2 mmol) at -78 ° C over 10 min. After 45 min, 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (12 mL, 58.3 mmol) was added. After 1 h 45 min, saturated aqueous NH4Cl solution and EtOAc were added. The cooling bath was removed and the reaction mixture was stirred to room temperature. The layers were separated, and the organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. Silica gel chromatography using hexane:EtOAc (3:1) as eluent gave compound 2-5 (10 g, 49.2% yield) as a white solid.

[0372] 3-Fluoro-4-(3-(4-fluorophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)picolinonitrile (2-6)

[0373]

[0374] 3-(4-Fluorophenyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole 2-5 (10 g, 23.9 mmol) and 3-fluoro-4-iodopicolinonitrile (5.0 g, 19 mmol) were combined in 100 mL of DMF and treated with Cs2CO3 (12.0 g, 37.1 mmol). The resulting suspension was purged with nitrogen for 20 min and treated with tris(dibenzylideneacetone)dipalladium(0) (0.91 g, 1.0 mmol) in one portion. Nitrogen purging was continued for another 20 min, and the dark suspension was stirred at room temperature for another 30 min. The reaction solution was heated to 50°C for 6 hours and then cooled to room temperature. The thick slurry was added to 150 mL of EtOAc, filtered through celite, and the organic layer was washed with 3 x 30 mL of brine. The organic layer was dried over anhydrous NaSO and concentrated in vacuo to yield 12 g of a crude solid. This solid was purified by flash column with a 5-80% EtOAc / heptane gradient to yield 9.0 g of compound 2-6 as a white solid in 91% yield. MS m / z 413.0 (M+1).

[0375] 4-(3-(4-Fluorophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyridine-2,3-dicarbonitrile (2-7)

[0376]

[0377] To a solution of 3-fluoro-4-(3-(4-fluorophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)picolinonitrile 2-6 (9 g, 21.8 mmol) in DMSO (50 mL) was added potassium cyanide (1.52 g, 22.6 mmol) under gentle heating, and the mixture was heated to 50° C. and stirred. After 2 hours, the mixture was cooled in an ice bath and 0.1 N NaOH aqueous solution (50 mL) was added. The resulting slurry was stirred for 5 minutes, and the solid was collected, rinsed with water, and air-dried to give 7.3 g of compound 2-7 as a light cream solid with a yield of 80%.

[0378] 4-(3-(4-Fluorophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyridine-2,3-dicarboxylic acid dipotassium salt (2-8)

[0379]

[0380] 4-(3-(4-fluorophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyridine-2,3-dicarbonitrile 2-7 (7 g, 17.4 mmol) and aqueous KOH solution (9.8 g, 174 mmol) were dissolved in 100 mL of water and heated at 100 ° C for 24 hours. The mixture was cooled in ice to precipitate a thick white solid. The material was warmed to room temperature, filtered, and air-dried to give 4.2 g (45.3%) of the product 2-8 as a cream-colored solid: LCMS m / z 456 (M-1).

[0381] 4-(3-(4-Fluorophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)furo[3,4-b]pyridine-5,7-dione (2-9)

[0382]

[0383] 4-(3-(4-fluorophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyridine-2,3-dicarboxylic acid dipotassium salt 2-8 (4.2 g, 7.9 mmol) was slurried with acetic acid (7 mL) and acetic anhydride (2.5 mL) and heated to 110 ° C for 4 hours. The resulting homogeneous mixture containing intermediate 2-9 was cooled and used directly in the next step.

[0384] 4-(3-(4-Fluorophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)-6-methyl-5H-pyrrolo[3,4-b]pyridine-5,7(6H)-dione (2-10)

[0385]

[0386] To the reaction mixture containing compound 2-9 from the previous step was added a THF solution of methylamine (4 mL, 2.0 M, 7.9 mmol), and the mixture was heated at reflux for 16 hours, cooled, and then concentrated to a brown solid. The material was purified by reverse phase column with H2O / MeOH 5%-95% to give 1.2 g of compound 2-10 as a yellow solid, 33.6% yield over 2 steps: LCMSm / z 453 (M+1).

[0387] 4-(3-(4-Fluorophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)-6-methyl-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one (2-11)

[0388]

[0389] To a flask equipped with an overhead stirring apparatus was added 4-(3-(4-fluorophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)-6-methyl-5H-pyrrolo[3,4-b]pyridine-5,7(6H)-dione 2-10 (1.2 g, 2.7 mmol) and zinc powder (<10 microns) (877 mg, 13.5 mmol), followed by acetic acid (20 mL). The mixture was heated to 105 ° C, stirred for 4 hours, and then cooled to room temperature. Celite was added to the reaction mixture and filtered to remove zinc. The filter pad was rinsed with EtOAc, and the filtrate was concentrated and purified by reverse phase column with H2O / MeOH 5%-95% to give 300 mg of compound 2-11 as a white solid in 25% yield. LCMSm / z 439 (M+1).

[0390] 4-(3-(4-Fluorophenyl)-1H-pyrazol-4-yl)-6-methyl-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one (2-12)

[0391]

[0392] A stirred solution of 4-(3-(4-fluorophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)-6-methyl-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one 2-11 (300 mg, 0.68 mmol) in DCM (2.0 ml) was treated with TFA (2.0 ml) at ambient temperature, and the resulting mixture was stirred for 4 hours. The mixture was concentrated in vacuo, and the residue was purified by reverse phase column with H2O / MeOH 5%-95% to give 200 mg of compound 2-12 as a white solid in 95% yield.

[0393] 4-(1-(3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butyl)-3-(4-fluorophenyl)-1H-pyrazol-4-yl)-6-methyl-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one (I-2)

[0394]

[0395] A solution of 4-(3-(4-fluorophenyl)-1H-pyrazol-4-yl)-6-methyl-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one 2-12 (100 mg, 0.32 mmol) in DMF (3 ml) was treated with 3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butyl methanesulfonate 2-13 (174 mg, 0.5 mmol) and cesium carbonate (325 mg, 1 mmol). The mixture was stirred at room temperature for 16 hours until the reaction was complete. The reaction solution was directly purified by reverse phase column with H2O / MeOH 5%-95% to give 54 mg of compound (I-2) as a white solid with a yield of 30.2%. 1 H NMR (400MHz, methanol-d4) δ8.32(d,J=5.4Hz,1H),8.27(s,1H),8.03(s,1H),7.76(s,1 H),7.36(td,J=9.1,6.7Hz,1H),7.23–7.18(m,2H),7.12(t,J=8.7Hz,2H),6.87– 6.77(m,3H),4.63–4.49(m,2H),4.45(s,2H),4.15(ddd,J=13.9,10.4,5.7Hz,1H ),3.81–3.73(m,1H),3.16(s,3H),2.53(td,J=12.7,11.1,5.0Hz,1H),2.35–2.2 7 (m, 1H). Example 3: (R) -6- (2- (1- (2- (2,4-difluorophenyl) -2-hydroxy-3- (1H-1,2,4-triazol-1-yl) propyl) piperidin-4-yl) ethyl) -4- (3- (4-fluorophenyl) -1-methyl -1H-pyrazol-4-yl) -6,7-dihydro -5H- pyrrolo [3,4-b] pyridin-5-one (I -247) and (S)-6-(2-(1-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)piperidin-4-yl)ethyl)-4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one (I-248)

[0396] Compounds I-247 and I-248 were prepared by chiral separation, as shown in Scheme 8 below:

[0397]

[0398] Route 8

[0399] (R)-6-(2-(1-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)piperidin-4-yl)ethyl)-4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one (I-247) and (S)-6-(2-(1-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)piperidin-4-yl)ethyl)-4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one (I-248)

[0400]

[0401] Chiral separation of 6-(2-(1-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)piperidin-4-yl)ethyl)-4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one (I-240) was performed using an SFC-150 mg / min (waters) instrument. Column: Daicel AD-3 (25*250 mm, 10 μm); mobile phase: CO₂ / MeOH [0.2% NH₃ (7 M in MeOH)] = 50 / 50; flow rate: 100 ml / min; back pressure: 100 bar; detection wavelength: 214 nm; cycle time: 15 min; sample solution: 350 mg dissolved in 9 ml of MeOH; injection volume: 4 ml.

[0402] Peak 1 (the first peak) is compound (R)-6-(2-(1-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)piperidin-4-yl)ethyl)-4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one (I-247) (1.04 min, 100% ee).

[0403] LCMS: exact calculated mass = 656.3, found [M+H]+ (ESI+) = 657.2.

[0404] 1H NMR (400MHz, DMSO) δ8.50(d,J=5.2Hz,1H),8.30(s,1H),8.15(s,1H),7.74(s,1H),7.40(dd,J=15.9 ,8.9Hz,1H),7.34(dd,J=8.7,5.6Hz,2H),7.20–7.10(m,3H),7.01–6.92(m,2H),5.59(s,1H),4.60–4 .49(m,2H),4.45(s,2H),3.94(s,3H),3.47(t,J=7.4Hz,2H),2.82(d,J=14.2Hz,1H),2.65(d,J=13.7 Hz,2H),2.56(d,J=11.5Hz,1H),2.13–1.96(m,2H),1.63–1.39(m,4H),1.05(dd,J=20.8,8.9Hz,3H).

[0405] Peak 2 (the second peak) is compound (S)-6-(2-(1-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)piperidin-4-yl)ethyl)-4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one (I-248) (2.73 min, 100% ee).

[0406] LCMS: exact calculated mass = 656.3, found [M+H]+ (ESI+) = 657.2.

[0407] 1 H NMR (400MHz, DMSO) δ8.50(d,J=5.2Hz,1H),8.30(s,1H),8.15(s,1H),7.74(s,1H),7.40(dd,J=15.9 ,8.9Hz,1H),7.34(dd,J=8.7,5.6Hz,2H),7.20–7.10(m,3H),7.01–6.92(m,2H),5.59(s,1H),4.60–4 .49(m,2H),4.45(s,2H),3.94(s,3H),3.47(t,J=7.4Hz,2H),2.82(d,J=14.2Hz,1H),2.65(d,J=13.7 Hz,2H),2.56(d,J=11.5Hz,1H),2.13–1.96(m,2H),1.63–1.39(m,4H),1.05(dd,J=20.8,8.9Hz,3H).

[0408] Example 4: Synthesis of 2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)propan-2-yl 2-(4-((4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)-5-oxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)methyl)piperidin-1-yl)acetate (I-250)

[0409] The procedure for synthesizing compound 2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)propan-2-yl 2-(4-((4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)-5-oxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)methyl)piperidin-1-yl)acetate (I-250) is shown in the following Scheme 9:

[0410]

[0411] Route 9

[0412] Tert-butyl (2-(1-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,3-triazol-1-yl)propyl)piperidin-4-yl)ethyl)carbamate (3-2)

[0413]

[0414] 1-((2-(2,4-difluorophenyl)oxirane-2-yl)methyl)-1H-1,2,3-triazole 3-1 (500 mg, 2.1 mmol) was dissolved in 5 mL of EtOH with NEt3 (638 mg, 6.3 mmol) and tert-butyl (2-(piperidin-4-yl)ethyl)carbamate (718 mg, 3.2 mmol). The reaction mixture was stirred at 80 ° C for 16 hours. The reaction mixture was then concentrated, diluted with 30 mL of EtOAc, washed twice with 20 mL of water, and washed with 30 mL of brine. The organic layer was dried over NaSO, concentrated, and purified by flash chromatography (DCM / MeOH = 10 / 1) to afford 900 mg of tert-butyl (2-(1-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,3-triazol-1-yl)propyl)piperidin-4-yl)ethyl)carbamate 3-2 as a white solid in 91% yield. LCMS: exact calculated mass = 465.2, observed [M+H](ESI) = 466.1.

[0415] 1-(4-(2-aminoethyl)piperidin-1-yl)-2-(2,4-difluorophenyl)-3-(1H-1,2,3-triazol-1-yl)propan-2-ol (3-3)

[0416]

[0417] Tert-butyl (2-(1-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,3-triazol-1-yl)propyl)piperidin-4-yl)ethyl)carbamate 3-2 (900 mg, 1.93 mmol) was dissolved in 10 mL of DCM and 2 mL of TFA was added at 0 °C. The mixture was stirred at room temperature for 2 hours. Upon completion of the reaction, the solvent was evaporated to give the crude product 3-3. LCMS: calculated mass = 365.2, found [M+H]+(ESI+) = 366.1.

[0418] 6-(2-(1-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,3-triazol-1-yl)propyl)piperidin-4-yl)ethyl)-4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)-5H-pyrrolo[3,4-b]pyridine-5,7(6H)-dione (3-5)

[0419]

[0420] A solution of 4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)furo[3,4-b]pyridine-5 (150 mg, 0.46 mmol) 3-4 and 1-(4-(2-aminoethyl)piperidin-1-yl)-2-(2,4-difluorophenyl)-3-(1H-1,2,3-triazol-1-yl)propan-2-ol 3-3 (252 mg, 0.69 mmol) in THF (10 mL) was heated under reflux for 16 hours, cooled, and then concentrated to a brown solid. The substance was purified by reverse phase column with H2O / MeOH 5%-95% to give 123 mg of product 3-5 as a yellow solid in 40% yield. LCMS: exact calculated mass = 670.3, found [M+H]+(ESI+) = 671.1.

[0421] 2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)propan-2-yl 2-(4-((4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)-5-oxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)methyl)piperidin-1-yl)acetate (I-250)

[0422]

[0423] A solution of 6-(2-(1-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,3-triazol-1-yl)propyl)piperidin-4-yl)ethyl)-4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)-5H-pyrrolo[3,4-b]pyridine-5,7(6H)-dione 3-5 (123 mg, 0.18 mmol) and zinc powder (<10 microns) (119 mg, 1.8 mmol) in acetic acid (3 mL) was heated to 105° C., stirred for 4 hours, and then cooled to room temperature. The reaction mixture was filtered to remove zinc, the filter pad was rinsed with EtOAc (50 mL), and the filtrate was concentrated and purified by reverse phase column with HO / MeOH 5%-95% to afford 2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)propan-2-yl 2-(4-((4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)-5-oxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)methyl)piperidin-1-yl)acetate I-250 (39 mg) as a white solid in 33% yield.

[0424] LCMS: exact calculated mass = 656.3, found [M+H]+ (ESI+) = 657.2.

[0425] 1 H NMR (400MHz, DMSO) δ8.49(d,J=5.2Hz,1H),8.14(s,1H),7.86(d,J=0.8Hz,1H),7.56(s,1H),7.35(ddd,J= 10.7,8.8,5.5Hz,3H),7.20–7.09(m,3H),6.98(d,J=5.2Hz,1H),6.93(td,J=8.5,2.4Hz,1H),5.67(s,1H) ,4.74(dd,J=51.0,14.1Hz,2H),4.44(s,2H),3.94(s,3H),3.46(t,J=7.2Hz,2H),2.83(d,J=13.8Hz,1H), 2.66(t,J=14.4Hz,2H),2.56(s,1H),2.06(dt,J=27.3,10.5Hz,2H),1.60–1.41(m,4H),1.18–0.97(m,3H).

[0426] Example 5: Synthesis of 2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)propan-2-yl 2-(4-((4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)-5-oxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)methyl)piperidin-1-yl)acetate (I-249)

[0427] The procedure for synthesizing compound 2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)propan-2-yl 2-(4-((4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)-5-oxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)methyl)piperidin-1-yl)acetate (I-249) is shown in the following Scheme 10:

[0428]

[0429] Route 10

[0430] 2-(2,4-Difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)propan-2-yl 2-chloroacetate (4-2)

[0431]

[0432] 2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)propan-2-ol 4-1 (9.2 g, 30 mmol) was dissolved in 100 mL of THF, followed by the addition of NaH (60%, 3.6 g, 90 mmol) at 0 ° C under nitrogen protection. The reaction solution was then stirred at 70 ° C for 1 hour. 2-Chloroacetyl chloride (10 g, 90 mmol) was then added at room temperature. The reaction mixture was stirred at room temperature for 16 hours. The reaction solution was slowly poured into 300 mL of saturated NH4Cl aqueous solution and extracted three times with 100 mL of EtOAc. The combined organic layers were washed with 200 mL of brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by flash column chromatography to give 2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)propan-2-yl 2-chloroacetate 4-2 (2.8 g) as a yellow solid in 24% yield. LCMS: exact calculated mass = 382.1, found [M+H]+ (ESI+) = 383.2.

[0433] 2-(2,4-Difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)propan-2-yl 2-(4-(((tert-butoxycarbonyl)amino)methyl)piperidin-1-yl)acetate (4-3)

[0434]

[0435] 2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)propan-2-yl 2-chloroacetate 4-2 (2.6 g, 6.8 mmol) and tert-butyl (piperidin-4-ylmethyl)carbamate (1.6 g, 7.5 mmol) were dissolved in 30 mL of MeCN, followed by the addition of K2CO3 (1.9 g, 13.6 mmol) at room temperature. The reaction mixture was stirred at the same temperature for 1 hour. The reaction mixture was then concentrated in vacuo, diluted in 100 mL of EtOAc, washed twice with 50 mL of H2O, and washed with 100 mL of brine. The organic layer was dried over Na2SO4 and concentrated in vacuo. The residue was purified by flash chromatography (DCM / MeOH = 10:1) to give 2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)propan-2-yl 2-(4-(((tert-butoxycarbonyl)amino)methyl)piperidin-1-yl)acetate 4-3 as a white solid (800 mg) in 21% yield. LCMS: exact calculated mass = 560.3, found [M+H]+ (ESI+) = 561.2.

[0436] 2-(2,4-Difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)propan-2-yl 2-(4-(aminomethyl)piperidin-1-yl)acetate (4-4)

[0437]

[0438] 2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)propan-2-yl 2-(4-(((tert-butoxycarbonyl)amino)methyl)piperidin-1-yl)acetate 4-3 (800 mg, 1.43 mmol) was dissolved in 5 mL of DCM, and 1 mL of TFA was added at 0 ° C. The reaction solution was stirred at 0 ° C for 1 hour. The mixture was then concentrated in vacuo and purified by flash chromatography (DCM / MeOH=10 / 1) to give 2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)propan-2-yl 2-(4-(aminomethyl)piperidin-1-yl)acetate 4-4 (200 mg) as a white solid in a 30% yield. LCMS: accurate calculated mass = 460.2, found [M+H] + (ESI +) = 461.2.

[0439] 2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)propan-2-yl 2-(4-((4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)-5,7-dioxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)methyl)piperidin-1-yl)acetate (4-6)

[0440]

[0441] A solution of 4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)furo[3,4-b]pyridine-5 (150 mg, 0.46 mmol) 4-5 and 2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)propan-2-yl 2-(4-(aminomethyl)piperidin-1-yl)acetate 4-4 (318 mg, 0.69 mmol) in THF (10 mL) was heated at reflux for 16 hours, cooled, and then concentrated to a brown solid. The material was purified by reverse phase column with H2O / MeOH 5%-95% to give 2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)propan-2-yl 2-(4-((4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)-5,7-dioxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)methyl)piperidin-1-yl)acetate 4-6 (100 mg) as a yellow solid in 28% yield. LCMS: exact calculated mass = 765.3, found [M+H]+ (ESI+) = 766.2.

[0442] 2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)propan-2-yl 2-(4-((4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)-5-oxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)methyl)piperidin-1-yl)acetate (I-249)

[0443]

[0444] To a flask equipped with an overhead stirrer was added 2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)propan-2-yl 2-(4-((4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)-5,7-dioxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)methyl)piperidin-1-yl)acetate 4-6 (100 mg, 0.13 mmol) and zinc powder (<10 microns) (130 mg, 2 mmol), followed by acetic acid (3 mL). The mixture was heated to 105° C. and stirred for 4 hours. The reaction mixture was filtered to remove zinc, the filter pad was rinsed with EtOAc (50 mL), and the filtrate was concentrated and purified by reverse phase column with HO / MeOH 5%-95% to afford 2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)propan-2-yl 2-(4-((4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)-5-oxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)methyl)piperidin-1-yl)acetate I-249 (7 mg) as a white solid in 7% yield.

[0445] LCMS: exact calculated mass = 751.3, found [M+H]+ (ESI+) = 752.2.

[0446] 1 H NMR (400MHz, DMSO) δ8.51(d,J=5.2Hz,1H),8.31(s,2H),8.17(s,1H),7.93(s,2H),7.32(dd,J=8.7,5 .6Hz,2H),7.29–7.21(m,1H),7.13(t,J=8.9Hz,2H),7.07(d,J=6.9Hz,1H),7.02–6.94(m,2H),5.17(d ,J=14.2Hz,2H),5.02(d,J=14.9Hz,2H),4.48(s,2H),3.94(s,3H),3.37(s,2H),3.21(s,2H),2.80(d ,J=10.8Hz,2H),2.12(t,J=11.5Hz,2H),1.68(s,1H),1.52(d,J=10.2Hz,2H),1.15(d,J=12.4Hz,2H).

[0447] Example 6: Synthesis of 2-(2,4-difluorophenyl)-4-(((4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)methyl)amino)-1-(1H-1,2,4-triazol-1-yl)butan-2-ol (I-3) and analogs (I-4) and (I-5)

[0448]

[0449] The procedure for synthesizing the common intermediate 4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)nicotinaldehyde (5-9) is shown in Scheme 11:

[0450]

[0451] Route 11

[0452] Intermediates 5-2, 5-3, 5-4 and 5-5 were obtained according to the procedure described in Example 1 to obtain intermediates 1-2, 1-3, 1-4 and 1-5.

[0453] 4-(3-(4-Fluorophenyl)-1-methyl-1H-pyrazol-4-yl)nicotinonitrile (5-6)

[0454]

[0455] 3-(4-Fluorophenyl)-1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole 5-5 (25 g, 67 mmol) and 4-chloronicotinonitrile (18.5 g, 134 mmol) were combined in 400 mL of DMF and treated with LiOH (4.8 g, 201 mmol). The resulting suspension was purged with nitrogen for 20 min and treated with Pd(dppf)Cl2 (4.9 g, 6.7 mmol) in one go. Nitrogen purging was continued for another 20 min, and the dark suspension was stirred at room temperature for another 30 min. The reaction solution was heated to 80 ° C for 16 hours and then cooled to room temperature. The thick slurry was added to 250 mL of EtOAc and filtered through celite. The organic layer was washed with 3×300 mL of brine. The solid was purified by flash column with a 5-80% EtOAc / heptane gradient to afford 10 g of compound 5-6 as a white solid in 53.7% yield. MS m / z 279.0 (M+1).

[0456] 4-(3-(4-Fluorophenyl)-1-methyl-1H-pyrazol-4-yl)nicotinic acid (5-7)

[0457]

[0458] To a solution of 4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)nicotinonitrile 5-6 (10 g, 36 mmol) in EtOH (120 mL) was added 10 N NaOH (0.5 mL) under gentle heating and stirred at 90° C. for 2 hours. The reaction mixture was then concentrated, diluted with 200 mL EtOAc, and extracted with 200 mL H O. The aqueous layer was acidified to pH = 3 with 1 M HCl. 10 g of solid intermediate 5-7 was collected by suction filtration in a 93.5% yield.

[0459] (4-(3-(4-Fluorophenyl)-1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)methanol (5-8)

[0460]

[0461] To a solution of 4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)nicotinic acid 5-7 (5.0 g, 16.8 mmol) in THF (40 mL) was added LiAlH4 (1272 mg, 33.6 mmol) at 0°C. The resulting mixture was stirred at 0°C for 5 min, then warmed to room temperature for 1 hour and quenched with 0.5 mL of water, 0.5 mL of 15% NaOH solution, and 2 mL of water. After stirring at room temperature for 15 min, sodium sulfate was added to the mixture, stirred for another 15 min, and then filtered. The resulting solution was concentrated to give 1.6 g of crude product 5-8, which was used directly in the next step without further purification. LCMS (ESI) m / z = 284 (M+1).

[0462] 4-(3-(4-Fluorophenyl)-1-methyl-1H-pyrazol-4-yl)nicotinaldehyde(5-9)

[0463]

[0464] To a solution of (4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)methanol (400 g, 1.4 mmol) 5-8 in DCM (5 mL) was added DIB (700 mg, 8.6 mmol) and TEMPO (45 mg, 0.28 mmol). The reaction mixture was stirred at room temperature overnight, and then the solvent was concentrated. The residue was purified by flash column chromatography to give 30 mg of the desired product 5-9 as a white solid in 7.5% yield. LCMS(ESI)m / z=282.2(M+1)1H NMR (400MHz, DMSO) δ9.88(s,1H),8.89(s,1H),8.73(d,J=5.2Hz,1H),8.16(s,1H),7.32(td,J=5.2,2.3Hz,3H),7.18–7.13(m,2H),3.97(s,3H).

[0465] The procedure for synthesizing compound 2-(2,4-difluorophenyl)-4-(((4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)methyl)amino)-1-(1H-1,2,4-triazol-1-yl)butan-2-ol (I-3) is shown in Scheme 12:

[0466]

[0467] Route 12

[0468] 2-(2,4-difluorophenyl)-4-(((4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)methyl)amino)-1-(1H-1,2,4-triazol-1-yl)butan-2-ol (I-3)

[0469]

[0470] 380 mg of 4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)nicotinaldehyde 5-9 (1.5 mmol) was mixed with 4-amino-2-(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)butan-2-ol 5-10 (2 mmol, 536 mg) and a catalytic amount of Ti(OiPr) in 4 mL of EtOH. The reaction was stirred in a microwave at 80°C for 2 hours. NaBH (170 mg, 4.5 mmol) was then added at room temperature, and the reaction was stirred for an additional 2 hours. The reaction mixture was concentrated and purified by reverse phase column chromatography using H2O / MeOH 5%-95% to yield 80 mg of the desired product (I-3) as a white solid in a 10% yield. MS (ESI+): 534 (M+H). 1H NMR(400MHz,DMSO-d6)δ8.52(s,1H),8.40(d,J=5.0Hz,1H),8.25(s,1H),7.88(s,1 H),7.74(s,1H),7.26(dd,J=8.8,6.0Hz,3H),7.15(d,J=2.8Hz,1H),7.13–7.05(m, 4H),6.91(d,J=2.6Hz,1H),4.41(s,2H),3.93(s,3H),3.38(d,J=13.3Hz,1H),3.27 (s,1H),2.36–2.31(m,1H),2.16(s,1H),1.99(d,J=14.0Hz,1H),1.75–1.68(m,1H).

[0471] The procedure for synthesizing compound 2-(2,4-difluorophenyl)-1-(4-(((4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)methyl)amino)piperidin-1-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-4) is shown in Scheme 13:

[0472]

[0473] Route 13

[0474] 2-(2,4-difluorophenyl)-1-(4-(((4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)methyl)amino)piperidin-1-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-4)

[0475]

[0476] 480 mg of 4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)nicotinaldehyde 5-9 (1.7 mmol) was mixed with 1-(4-aminopiperidin-1-yl)-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol 5-11 (2.6 mmol, 864 mg) and a catalytic amount of Ti(OiPr) in 4 mL of EtOH. The reaction was stirred in a microwave at 80°C for 2 hours. NaBH (193 mg, 5.1 mmol) was then added at room temperature, and the reaction was stirred for an additional 2 hours. The reaction mixture was concentrated and purified by reverse phase column chromatography using H2O / MeOH 5%-95% to yield 75 mg of the desired product (I-4) as a white solid in a 7.3% yield. MS (ESI+): 603 (M+H). 1H NMR(400MHz, DMSO-d6)δ8.61(s,1H),8.37(d,J=5.0Hz,1H),8.27(s,1H),7.95(s,1H),7.73(s,1H),7.37(t d,J=9.0,6.8Hz,1H),7.28(ddd,J=8.7,5.5,2.6Hz,2H),7.17–7.07(m,3H),7.04(d,J=5.0Hz,1H),6.94(td, J=8.5,2.6Hz,1H),5.55(s,1H),4.51(d,J=4.0Hz,2H),3.91(s,3H),3.44(s,2H),2.77(dd,J=13.7,1.8Hz, 1H), 2.59 (d, J = 13.9Hz, 2H), 2.08 (d, J = 4.3Hz, 1H), 2.00–1.93 (m, 2H), 1.39 (s, 2H), 0.99 (q, J = 11.1Hz, 2H).

[0477] The procedure for synthesizing compound 2-(2,4-difluorophenyl)-1-(((4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)methyl)amino)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-5) is shown in Scheme 14:

[0478]

[0479] Route 14

[0480] 2-(2,4-difluorophenyl)-1-(((4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)methyl)amino)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-5)

[0481]

[0482] 120 mg of 4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)nicotinaldehyde 5-9 (0.67 mmol) and 1-amino-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol 5-12 (1.0 mmol, 254 mg) were mixed in 4 mL of DCM, followed by the addition of NaBH(OAc) (282 mg, 1.33 mmol). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated and purified by reverse phase column chromatography using HO / MeOH (5%-95%) to afford 56.4 mg of the desired product (I-5) as a white solid in a 16.2% yield. MS (ESI+): 520 (M+H).1 H NMR (400MHz, DMSO-d6) δ8.55 (s, 1H), 8.36 (d, J = 5.0Hz, 1H), 8.22 (s, 1H), 7.8 5(s,1H),7.71(s,1H),7.34–7.28(m,1H),7.27–7.23(m,2H),7.13–7.06(m,3H ),7.01(d,J=5.0Hz,1H),6.93–6.86(m,1H),5.67(s,1H),4.46(d,J=3.0Hz,2 H), 3.91 (s, 3H), 3.46 (s, 2H), 2.82 (d, J = 12.3Hz, 1H), 2.71 (d, J = 12.3Hz, 1H).

[0483] Example 7: Synthesis of 1-(4-(5-(2-aminopyrimidin-4-yl)-4-(4-fluorophenyl)-1H-imidazol-1-yl)piperidin-1-yl)-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-32), its enantiomers (I-33) and (I-34), and analogs (I-35) and (I-54)

[0484]

[0485] The procedure for synthesizing the common intermediate 4-(4-(4-fluorophenyl)-1-(piperidin-4-yl)-1H-imidazol-5-yl)pyrimidin-2-amine 6-9 is shown in Scheme 15:

[0486]

[0487] Route 15

[0488] N-((4-Fluorophenyl)(tosyl)methyl)formamide (6-3)

[0489]

[0490] 20 g (161.3 mmol) of 4-fluorobenzaldehyde 6-1, 16 mL (2.5 eq, 403 mmol) of formamide, and 22.5 mL (177.4 mmol) of trimethylsilyl chloride were dissolved in 55 mL of acetonitrile and 55 mL of toluene. The reaction solution was stirred at 50°C for 4 hours, 37.7 g (241.7 mmol) of p-toluenesulfinic acid 6-2 was added, and heating was continued for 4 hours. The solution was cooled to room temperature, 55 mL of TBME was added, followed by 250 mL of water. The resulting mixture was cooled to 0°C, kept warm for 1 hour, and the precipitated white solid was collected by filtration. The reaction flask was washed with 35 mL of TBME, and the rinse was poured onto the filter cake. The filter cake was dried to yield 30 g (56%) of the desired product 6-3, which did not require further purification.

[0491] 1-Fluoro-4-(isocyanato(tosyl)methyl)benzene(6-4)

[0492]

[0493] A solution of N-((4-fluorophenyl)(tosyl)methyl)formamide 6-3 (20 g, 65.1 mmol) in DME (200 mL) was cooled to -10 ° C. POCl (18.2 mL, 195.3 mmol) was added, followed by a solution of triethylamine (55.2 mL, 387.6 mmol) in DME (300 mL) was added dropwise, maintaining the internal temperature below -5 ° C. The mixture was gradually warmed to ambient temperature over 1 hour, poured into H2O, and extracted with EtOAc. The organic phase was washed with saturated NaHCO3 aqueous solution, dried (Na2SO4), and concentrated. The resulting residue was triturated with petroleum ether and filtered to give compound 6-4 (10 g, 53.2% yield): 1 H NMR(CDCl3)δ7.63(d,2H),7.33(m,4H),7.10(t,2H),5.60(s,1H),2.50(s,3H).

[0494] Benzyl (4-(((1-benzylpiperidin-4-yl)imino)methyl)pyrimidin-2-yl)carbamate (6-7)

[0495]

[0496] 45% KOH aqueous solution (8.56g, 152.5mmol) is added to ice-cold HCl aqueous solution (38.2ml, 145.2mmol) of (4-formylpyrimidin-2-yl) benzylcarbamate 6-5 (5.5g, 21.4mmol), while maintaining the temperature below 15 ℃. In the neutralized solution, CH2Cl2 (100mL) and K2CO3 (3.0g, 21.4mmol) are added, followed by 4-amino-1-benzylpiperidine 6-6 (2.69g, 16mmol). The reaction mixture is gradually warmed to room temperature, and continued to stir for 2 hours, until no longer having raw material remaining. The reaction mixture is diluted with CH2Cl2, washed with water, dried (Na2SO4), filtered, and concentrated. The crude product 6-7 is used for the next step without purification.

[0497] Benzyl (4-(1-(1-benzylpiperidin-4-yl)-4-(4-fluorophenyl)-1H-imidazol-5-yl)pyrimidin-2-yl)carbamate (6-8)

[0498]

[0499] 1-Fluoro-4-(isocyanato(tosyl)methyl)benzene 6-4 (6.8 g, 23.5 mmol) and 20% aqueous KCO (6.5 g, 47 mmol) were added directly to a solution of crude (benzyl 4-(((1-benzylpiperidin-4-yl)imino)methyl)pyrimidin-2-yl)carbamate 6-7 (21.4 mmol) from the previous step in CHCl at 20°C and stirred for 16 hours. Additional imine (2.14 mmol) and KCO (2.27 g, 15.6 mmol) were added, and the reaction mixture was stirred at 30°C overnight. The reaction mixture was diluted with CHCl, washed with water, dried (MgSO), filtered, and concentrated. Flash chromatography using (DCM / MeOH / EtOAc 20:1) gave the desired product 6-8 (7.5 g, 56.8%) as a light yellow solid. 1 H NMR(DMSO-d6,400MHz)δ1.91-1.94(4H,m),2.01-2.08(2H,m),2.82(2H,br d,J=10.8Hz),3.45(2H,s),4.84^1.88(1H,m),5.20(2H,s),6.86(1H,d,J=5.2Hz),7. 13-7.17(2H,m),7.22-7.45(2H,m),8.14(1H,s),8.51(1H,d,J=5.2Hz),10.73(1H,s).

[0500] 4-(4-(4-Fluorophenyl)-1-(piperidin-4-yl)-1H-imidazol-5-yl)pyrimidin-2-amine (6-9)

[0501]

[0502] 10% Pd / C (0.75 g) and ammonium formate (8.36 g, 133 mmol) were added to a solution of benzyl (4-(1-(1-benzylpiperidin-4-yl)-4-(4-fluorophenyl)-1H-imidazol-5-yl)pyrimidin-2-yl)carbamate 6-8 (7.5 g, 13.3 mmol) in methanol (100 mL). The resulting reaction mixture was heated to 50° C. and stirred overnight. The reaction mixture was filtered through celite and concentrated under reduced pressure to give the residue 6-9 without purification (4 g, 89%). 1 H NMR(MeOH-d4,400MHz)δ2.08-2.17(2H,m),2.37-2.40(2H,m),3.02-3.09(2 H,m),3.41-3.47(2H,m),4.85-4.94(1H,m),6.40(1H,d,J=5.2Hz),7.06-7.1 1(2H,m),7.39-7.43(2H,m),8.03(1H,s),8.12(1H,d,J=5.2Hz),8.53(1H,s,NH).

[0503] The procedure for synthesizing compound 1-(4-(5-(2-aminopyrimidin-4-yl)-4-(4-fluorophenyl)-1H-imidazol-1-yl)piperidin-1-yl)-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-32) is shown in Scheme 16:

[0504]

[0505] Route 16

[0506] 1-(4-(5-(2-aminopyrimidin-4-yl)-4-(4-fluorophenyl)-1H-imidazol-1-yl)piperidin-1-yl)-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-32)

[0507]

[0508] 4-(4-(4-fluorophenyl)-1-(piperidin-4-yl)-1H-imidazol-5-yl)pyrimidin-2-amine 6-9 (250 mg, 0.74 mmol) and 1-((2-(2,4-difluorophenyl)oxirane-2-yl)methyl)-1H-1,2,4-triazole 6-10 (526 mg, 2.2 mmol) were dissolved in 4 mL of EtOH, followed by the addition of Et3N (0.3 mL, 2.2 mmol). The reaction solution was stirred in a microwave at 80 ° C for 4 hours. The reaction mixture was then concentrated and purified by a reverse phase column with H2O / MeOH 5%-95% to give 101 mg of compound (I-32) as a white solid in a yield of 23.7%. 1 H NMR(400MHz,DMSO-d6)δ8.29(s,1H),8.19(d,J=5.0Hz,1H),7.95(s,1H),7.74(s,1H), 7.42(ddd,J=8.8,5.6,2.7Hz,3H),7.20–7.09(m,3H),6.99–6.94(m,1H),6.79(s,2H),6 .39 (d, J = 5.0 Hz, 1H), 5.66 (s, 1H), 4.57 (s, 2H), 4.18 (d, J = 6.7 Hz, 1H), 2.88 (d, J = 13.6 Hz, 1H), 2.82 (d, J = 11.6 Hz, 1H), 2.72 (d, J = 14.4 Hz, 2H), 2.26–2.16 (m, 2H), 1.82 (m, 4H). LCMS: exact calculated mass = 576.2, found [M+H]+ (ESI+) = 576.3.

[0509] The procedure for separating compounds (S)-1-(4-(5-(2-aminopyrimidin-4-yl)-4-(4-fluorophenyl)-1H-imidazol-1-yl)piperidin-1-yl)-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-33) and (R)-1-(4-(5-(2-aminopyrimidin-4-yl)-4-(4-fluorophenyl)-1H-imidazol-1-yl)piperidin-1-yl)-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-34) is shown in Scheme 17:

[0510]

[0511] Route 17

[0512] (S)-1-(4-(5-(2-aminopyrimidin-4-yl)-4-(4-fluorophenyl)-1H-imidazol-1-yl)piperidin-1-yl)-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-33) and (R)-1-(4-(5-(2-aminopyrimidin-4-yl)-4-(4-fluorophenyl)-1H-imidazol-1-yl)piperidin-1-yl)-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-34)

[0513]

[0514] Chiral separation of 1-(4-(5-(2-aminopyrimidin-4-yl)-4-(4-fluorophenyl)-1H-imidazol-1-yl)piperidin-1-yl)-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-32) was performed by chiral HPLC. Two isomers were obtained. Chiral AD-H column (0.46 cm ID x 15 cm), CO2:IPA (0.05% DEA) = 70:30; flow rate 2.5 mL / min, monitoring at 254 nm.

[0515] Peak 1 (the first peak) is compound (R)-1-(4-(5-(2-aminopyrimidin-4-yl)-4-(4-fluorophenyl)-1H-imidazol-1-yl)piperidin-1-yl)-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-34) (5.79 min, 100% ee).

[0516] LCMS: calculated mass = 576.2, found [M+H] + (ESI+)=576.2.

[0517] 1H NMR(400MHz,DMSO-d6)δ8.29(s,1H),8.19(d,J=5.0Hz,1H),7.95(s,1H),7.74(s,1H), 7.42(ddd,J=8.7,5.5,2.6Hz,3H),7.21–7.08(m,3H),6.97(td,J=8.5,2.6Hz,1H),6.79 (s,2H),6.39(d,J=5.0Hz,1H),5.66(s,1H),4.58(s,2H),4.25–4.14(m,1H),2.85(dd, J=25.1,12.7Hz,2H),2.77–2.69(m,2H),2.27–2.13(m,2H),1.83(q,J=8.6,6.5Hz,4H).

[0518] Peak 2 (the second peak) is compound (S)-1-(4-(5-(2-aminopyrimidin-4-yl)-4-(4-fluorophenyl)-1H-imidazol-1-yl)piperidin-1-yl)-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-33) (6.25 min, 99.8% ee).

[0519] LCMS: calculated mass = 576.2, found [M+H] + (ESI+)=576.2.

[0520] 1 H NMR (400MHz, DMSO-d6) δ8.29(s,1H),8.19(d,J=5.0Hz,1H),7.95(s,1H),7.74(s,1H),7.42( ddd,J=8.7,5.6,2.6Hz,3H),7.20–7.09(m,3H),6.97(td,J=8.5,2.6Hz,1H),6.79(s,2H),6.3 9(d,J=5.0Hz,1H),5.66(s,1H),4.58(s,2H),4.19(dt,J=15.6,9.3Hz,1H),2.88(d,J=14.0H z,1H),2.82(d,J=11.5Hz,1H),2.72(d,J=14.1Hz,2H),2.28–2.16(m,2H),1.88–1.75(m,4H).

[0521] The procedure for synthesizing compound 1-(4-(5-(2-aminopyrimidin-4-yl)-4-(4-fluorophenyl)-1H-imidazol-1-yl)piperidin-1-yl)-2-(2,4-difluorophenyl)-3-(1H-1,2,3-triazol-1-yl)propan-2-ol (I-35) is shown in Scheme 18:

[0522]

[0523] Route 18

[0524] 1-(2,4-Difluorophenyl)-2-(1H-1,2,3-triazol-1-yl)ethan-1-one (6-12)

[0525]

[0526] A mixture of 2-chloro-2',4'-difluoroacetophenone 6-11 (5 g, 26 mmol), 1,2,3-triazole (1.85 mL, 32 mmol), and sodium bicarbonate (2.68 g, 32 mmol) in toluene (25 mL) was refluxed for 4 hours. The reaction mixture was poured into crushed ice and extracted with EtOAc (2 × 25 mL). The combined organic layers were washed with H2O (2 × 10 mL) and brine (20 mL) and dried over Na2SO4. The product was then purified by flash column chromatography using petroleum ether / ethyl acetate (50:50) to give the desired product 6-12 (3.3 g, 61%). 1 H NMR (400MHz, CDCl3) δ 8.06 (m, 1H), 7.81 (d, J = 0.7Hz, 1H), 7.71 (d, J = 0.9Hz, 1H), 7.07 (m, 1H), 6.99 (m, 1H), 5.81 (d, J = 3.5Hz, 2H).

[0527] 1-((2-(2,4-difluorophenyl)oxiran-2-yl)methyl)-1H-1,2,3-triazole(6-13)

[0528]

[0529] To a solution of 1-(2,4-difluorophenyl)-2-(1H-1,2,3-triazol-1-yl)ethan-1-one 6-12 (1 g, 4.4 mmol) in toluene (20 mL) was added trimethylsulfoxide iodide (1.18 g, 5.2 mmol), followed by 20% sodium hydroxide solution (1 mL). The reaction mixture was then heated at 60 ° C for 4 hours. The mixture was diluted with EtOAc (10 mL) and poured into an ice-water mixture. The organic layer was washed with H2O (2×10 mL) and brine (20 mL) and dried over Na2SO4. The product was then purified by flash column chromatography using petroleum ether / ethyl acetate (40:60) to give compound 6-13 (810 mg, 78%). 1 H NMR (400MHz CDCl3) δ7.62 (d, J = 1.7 Hz, 2H), 7.11 (m, 1H), 6.78 (m, 2H), 5.05 (d, J = 14.7 Hz, 1H), 4.65 (d, J = 14.7 Hz, 1H), 2.88 (dd, J = 12.6, 4.7 Hz, 2H).

[0530] 1-(4-(5-(2-aminopyrimidin-4-yl)-4-(4-fluorophenyl)-1H-imidazol-1-yl)piperidin-1-yl)-2-(2,4-difluorophenyl)-3-(1H-1,2,3-triazol-1-yl)propan-2-ol (I-35)

[0531]

[0532] 4-(4-(4-fluorophenyl)-1-(piperidin-4-yl)-1H-imidazol-5-yl)pyrimidin-2-amine 6-9 (100 mg, 0.3 mmol) and 1-((2-(2,4-difluorophenyl)oxirane-2-yl)methyl)-1H-1,2,3-triazole 6-13 (210 mg, 0.88 mmol) were dissolved in 4 mL of EtOH, followed by the addition of Et3N (0.12 mL, 0.88 mmol). The reaction solution was stirred in a microwave at 80 ° C for 4 hours. The reaction mixture was then concentrated and purified by a reverse phase column with H2O / MeOH 5%-95% to give 39.4 mg of compound (I-35) as a white solid in a yield of 22.8%. 1H NMR (400MHz, DMSO-d6) δ8.20(d,J=5.0Hz,1H),7.95(s,1H),7.86(d,J=1.0Hz,1H),7.57(d,J=1.0Hz,1H), 7.45–7.40(m,2H),7.40–7.35(m,1H),7.18(ddd,J=11.8,9.1,2.5Hz,1H),7.15–7.09(m,2H),6.95(td,J=8 .4,2.6 Hz,1H),6.79(s,2H),6.39(d,J=5.0 Hz,1H),5.75(s,1H),4.85–4.68(m,2H),4.19(t,J=5.2 Hz,1H),2.92–2.87(m,1H),2.83(d,J=11.6 Hz,1H),2.73(d,J=13.7 Hz,2H),2.28–2.14(m,2H),1.93–1.72(m,4H). LCMS: exact calculated mass = 576.2, found [M+H]+(ESI+) = 576.3.

[0533] The procedure for synthesizing compound 1-(4-(5-(2-aminopyrimidin-4-yl)-4-(4-fluorophenyl)-1H-imidazol-1-yl)piperidin-1-yl)-2-(2,4-difluorophenyl)-3-(1H-1,2,3-triazol-1-yl)propan-2-ol (I-54) is shown in the following Scheme 19:

[0534]

[0535] Route 19

[0536] 3-(2,4-Difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butanal (6-15)

[0537]

[0538] To a solution of 3-(2,4-difluorophenyl)-4-(1H-1,2,4-triazol-1-yl)butane-1,3-diol 6-14 (200 mg, 0.74 mmol) in CH2Cl2 (5 mL) was added Dess-Martin periodinane (0.62 g, 1.47 mmol) in several batches (over ~10 min) and cooled with water / ice bath to maintain the temperature below 5°C. After the reaction was complete, the mixture was poured into saturated aqueous sodium bicarbonate solution (5 mL). The layers were separated and the aqueous layer was extracted with DCM (10 mL). The combined organic extracts were washed with brine (10 mL), dried over sodium sulfate, concentrated, and purified by flash column to give 160 mg of intermediate 6-15 as a white solid in 81% yield.

[0539] 1-(4-(5-(2-aminopyrimidin-4-yl)-4-(4-fluorophenyl)-1H-imidazol-1-yl)piperidin-1-yl)-2-(2,4-difluorophenyl)-3-(1H-1,2,3-triazol-1-yl)propan-2-ol (I-54)

[0540]

[0541] 4-(4-(4-Fluorophenyl)-1-(piperidin-4-yl)-1H-imidazol-5-yl)pyrimidin-2-amine 6-9 (135 mg, 0.4 mmol) and 3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butanal (160 mg, 0.6 mmol) 6-15 were mixed in 4 mL of THF, followed by the addition of NaBH(OAc) (127 mg, 0.6 mmol). The reaction was stirred at room temperature for an additional 2 hours. The reaction mixture was concentrated and purified by reverse phase column chromatography using H2O / MeOH 5%-95% to afford the target compound (I-54) (139 mg, 59% yield) as a white solid. MS (ESI+): 590 (M+H)+. 1 H NMR(400MHz, DMSO-d6)δ8.29(s,1H),8.19(d,J=5.0Hz,1H),8.04(s,1H),7.76(s,1H),7.47–7.41(m ,3H),7.21(ddd,J=11.8,9.1,2.6Hz,1H),7.15–7.09(m,2H),7.00(td,J=8.5,2.6Hz,1H),6.87(s,1H ),6.78(s,2H),6.40(d,J=5.0Hz,1H),4.51(s,2H),4.24(s,1H),2.92(d,J=7.4Hz,1H),2.64(d,J=7. 6Hz, 1H), 2.30–2.24 (m, 1H), 2.18–2.07 (m, 2H), 1.93 (dd, J = 12.8, 6.4Hz, 5H), 1.84 (d, J = 8.3Hz, 2H).

[0542] Example 8: Synthesis of N-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)-2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridine-6-carboxamide (I-115) and N-(1-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)piperidin-4-yl)-2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridine-6-carboxamide (I-100)

[0543]

[0544] The procedure for synthesizing the common intermediate 2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridine-6-carboxylic acid 7-10 is shown in Scheme 20:

[0545]

[0546] Route 20

[0547] 1-Amino-3-(trifluoromethyl)pyridin-1-ium 2,4,6-trimethyl-benzylsulfonate (7-2)

[0548]

[0549] At 0 ° C, TFA (20 mL) was added portionwise to N-tert-butoxycarbonyl-O-(mesitylenesulfonyl)hydroxylamine 7-1 (10 g, 31.7 mmol) over 15 min. The solution was stirred at room temperature for 15 minutes. The solution was poured into an ice-water mixture and the resulting precipitate was collected by filtration. The solid was redissolved in DCM and dried over Na2SO4. Na2SO4 was removed by filtration, and the filtrate was added to a DCM solution of 3-(trifluoromethyl)pyridine (4.6 g, 31.7 mmol). The mixture was stirred for 45 minutes and then filtered. MTBE was added to the filtrate and the product was precipitated. Solid 7-2 was collected by filtration, washed with METB, and used directly in the next step.

[0550] 2-(4-Fluorophenyl)-6-(trifluoromethyl)pyrazolo[1,5-a]pyridine-3-carboxylic acid methyl ester (7-4)

[0551]

[0552] To 3- (4- fluorophenyl) propiolic acid methyl ester 7-3 (1.78g, 10mmol) and 1- amino -3- (trifluoromethyl) pyridin -1- ium 2,4,6- trimethyl - benzyl sulfonate 7-2 (31.7mmol, from the previous step) in anhydrous acetonitrile (20mL) was added dropwise an acetonitrile solution (5mL) of DBU (4.7mL, 31.7mmol). The mixture was stirred at room temperature for 16 hours. The solvent was evaporated in vacuo, and the residue was dissolved in EtOAc and washed with water. The organic layer was dried, concentrated, and flash column chromatography was performed with PE: EA 10: 1 to obtain 700mg of intermediate 7-4 as a white solid in a yield of 20.7%.

[0553] 2-(4-Fluorophenyl)-6-(trifluoromethyl)pyrazolo[1,5-a]pyridine-3-carboxylic acid (7-5)

[0554]

[0555] A solution of 2-(4-fluorophenyl)-6-(trifluoromethyl)pyrazolo[1,5-a]pyridine-3-carboxylic acid methyl ester 7-4 (700 mg, 2 mmol) in 2N NaOH aqueous solution (10 mL) and MeOH (5 mL) was refluxed for 3 hours. The mixture was filtered, the filtrate was washed with PE (10 mL), and then concentrated in vacuo to about half the volume. Concentrated HCl was added to adjust the pH to 2, and the precipitate was collected by filtration, washed with water, and dried to give 470 mg of intermediate 7-5 as a white solid with a yield of 70%.

[0556] 3-Bromo-2-(4-fluorophenyl)-6-(trifluoromethyl)pyrazolo[1,5-a]pyridine(7-6)

[0557]

[0558] To a solution of 2-(4-fluorophenyl)-6-(trifluoromethyl)pyrazolo[1,5-a]pyridine-3-carboxylic acid 7-5 (470 mg, 1.45 mmol) in anhydrous DMF (10 mL) was added sodium bicarbonate (365 mg, 4.35 mmol), followed by NBS (258 mg, 1.45 mmol), and the mixture was stirred at room temperature under nitrogen for 2 hours. The mixture was diluted with EtOAc (20 mL), washed with water (10 mL*2), dried, and concentrated. Flash column chromatography with DCM:MeOH 20:1 afforded the desired product 7-6 (470 mg, 90% yield) as a tan solid.

[0559] 2-(4-Fluorophenyl)-3-(pyridin-4-yl)-6-(trifluoromethyl)pyrazolo[1,5-a]pyridine(7-7)

[0560]

[0561] A solution of 3-bromo-2-(4-fluorophenyl)-6-(trifluoromethyl)pyrazolo[1,5-a]pyridine 7-6 (479 mg, 1.31 mmol), 2-fluoro-4-pyridylboronic acid (177 mg, 1.44 mmol), sodium carbonate (208 mg, 1.97 mmol) and dichlorobis(triphenylphosphine) palladium (455 mg, 0.66 mmol) in DMF (5 mL) was stirred in microwave at 110 ° C for 2 hours. The reaction solution was then cooled to room temperature, filtered through a celite pad, and rinsed with EtOAc (50 mL). The filtrate was washed with water (20 mL*2), brine (20 mL), dried, and concentrated. Reverse phase column chromatography was performed with H2O / MeOH 5%-95% to obtain 300 mg of product 7-7 as a white solid with a yield of 64%.

[0562] 2-(4-Fluorophenyl)-3-(pyridin-4-yl)-6-(triethoxymethyl)pyrazolo[1,5-a]pyridine(7-8)

[0563]

[0564] To an ethanol solution of sodium ethoxide (575 mg, 8.4 mmol) was added 2-(4-fluorophenyl)-3-(pyridin-4-yl)-6-(trifluoromethyl)pyrazolo[1,5-a]pyridine 7-7 (300 mg, 0.84 mmol), and the reaction mixture was stirred at 80 ° C for 12 hours. The reaction mixture was cooled and concentrated in vacuo to about a quarter of the original volume. The resulting mixture was diluted with water and extracted with ethyl acetate. The organic layer was washed with water and brine, and then dried over sodium sulfate. Filtered and concentrated to give 300 mg of intermediate 7-8 as an off-white solid product with a yield of 82%.

[0565] Ethyl 2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridine-6-carboxylate (7-9)

[0566]

[0567] To a solution of 2-(4-fluorophenyl)-3-(pyridin-4-yl)-6-(triethoxymethyl)pyrazolo[1,5-a]pyridine 7-8 (300 mg, 0.69 mmol) in acetone (4 mL) and water (1 mL) was added p-toluenesulfonic acid monohydrate (328 mg, 1.73 mmol). The reaction mixture was stirred at 40°C for 6 hours. The pH of the reaction mixture was adjusted to slightly alkaline using saturated aqueous sodium bicarbonate solution. The reaction mixture was concentrated and purified by reverse phase column with H2O / MeOH 5%-95% to give the desired product 7-9 (200 mg, 80% yield).

[0568] 2-(4-Fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridine-6-carboxylic acid (7-10)

[0569]

[0570] To a solution of ethyl 2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridine-6-carboxylate 7-9 (200 mg, 0.55 mmol) in dioxane (4 mL) and water (1 mL) was added lithium hydroxide monohydrate (120 mg, 5 mmol). The reaction mixture was heated at 95 ° C for 5 hours. The reaction mixture was concentrated in vacuo and purified by reverse phase column with H2O / MeOH 5%-95% to give a white solid product 7-10 (38.4 mg, 21% yield).

[0571] The procedure for synthesizing N-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)-2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridine-6-carboxamide (I-115) is shown in the following Scheme 21:

[0572]

[0573] Route 21

[0574] N-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)-2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridine-6-carboxamide (I-115)

[0575]

[0576] 2-(4-Fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridine-6-carboxylic acid 7-10 (20 mg, 0.06 mmol), 1-amino-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol 7-11 (18.3 mg, 0.072 mmol), HATU (34.2 mg, 0.09 mmol), and DIPEA (31.5 μL, 0.18 mmol) were dissolved in 2 mL of DMF. The solution was stirred at room temperature for 2 hours. The mixture was then purified by reverse phase column with H2O / MeOH 5%-95% to give the product (I-115) as a white solid (15.5 mg, 45.4% yield). LCMS: calculated mass = 570.2, found [M+H]+ (ESI+) = 570.4. 1H NMR(400MHz, DMSO-d6)δ9.28(t,J=1.3Hz,1H),8.75(t,J=6.1Hz,1H),8.62–8.55(m,2H),8 .34(s,1H),7.78(dd,J=9.4,0.9Hz,1H),7.76(s,1H),7.68(dd,J=9.4,1.6Hz,1H),7.58–7 .53(m,2H),7.43(td,J=9.0,6.8Hz,1H),7.35–7.26(m,4H),7.20(ddd,J=11.9,9.2,2.6Hz ,1H),6.94(td,J=8.5,2.6Hz,1H),6.20(s,1H),4.79–4.59(m,2H),3.83(t,J=5.5Hz,2H).

[0577] The procedure for synthesizing N-(1-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)piperidin-4-yl)-2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridine-6-carboxamide (I-100) is shown in Scheme 22:

[0578]

[0579] Route 22

[0580] N-(1-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)piperidin-4-yl)-2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridine-6-carboxamide (I-100)

[0581]

[0582] 2- (4- fluorophenyl) -3- (pyridin-4-yl) pyrazolo [1,5-a] pyridine -6- carboxylic acid 7-10 (20 mg, 0.06 mmol), 1- (4- aminopiperidin-1-yl) -2- (2,4- difluorophenyl) -3- (1H-1,2,4- triazole -1- base) propan-2-ol 7-12 (24.3 mg, 0.072 mmol), HATU (34.2 mg, 0.09 mmol) and DIPEA (31.5 μ L, 0.18 mmol) were dissolved in 2 mL of DMF. The solution was stirred at room temperature for 2 hours. The mixture was then purified by reverse phase column with HO / MeOH 5%-95% to give 10 mg of compound (I-100) as a white solid product in a yield of 25.6%. LCMS: exact calculated mass = 653.3, found [M+H]+ (ESI+) = 653.4.1 H NMR (400MHz, DMSO-d6) δ9.30(d,J=1.4Hz,1H),8.59(d,J=5.0Hz,2H),8.40(d,J=7.6Hz,1H),8.31(s,1H),7 .77(s,3H),7.60–7.51(m,2H),7.47–7.40(m,1H),7.35–7.25(m,4H),7.19–7.12(m,1H),6.98(td,J=8.5,2. 6Hz,1H),5.70(s,1H),4.57(s,2H),3.71(q,J=4.7Hz,1H),2.93(d,J=13.8Hz,1H),2.81(d,J=11.4Hz,1H),2 .69(t,J=14.7Hz,2H), 2.21(td,J=13.1,10.3Hz,2H), 1.68(d,J=12.1Hz,2H), 1.46(q,J=12.8,12.2Hz,2H).

[0583] Example 9: Synthesis of 2-(2,4-difluorophenyl)-1-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)-3-(1H-1,2,3-triazol-1-yl)propan-2-ol (I-251)

[0584] The procedure for synthesizing compound 2-(2,4-difluorophenyl)-1-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)-3-(1H-1,2,3-triazol-1-yl)propan-2-ol (I-251) is shown in the following Scheme 23.

[0585]

[0586] Route 23

[0587] 1-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)piperidin-4-one (8-2)

[0588]

[0589] 1-((2-(2,4-difluorophenyl)oxirane-2-yl)methyl)-1H-1,2,3-triazole 8-1 (500mg, 2.1mmol) and NEt3 (638mg, 6.3mmol) and piperidine-4-one hydrochloride (432mg, 3.2mmol) are dissolved in 5mL EtOH. The reaction solution is stirred in microwave at 90 ° C for 16 hours. The reaction solution is then concentrated, diluted with 30mL EtOAc, washed twice with 20mL water, and washed with 30mL brine. The organic layer is dried with Na2SO4, concentrated, and purified by flash chromatography (DCM / MeOH=10 / 1) to give 1-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)piperidin-4-one 8-2 (500mg) as a white solid with a yield of 70%. LCMS: exact calculated mass = 336.1, found [M+H]+ (ESI+) = 337.3.

[0590] 2-(2,4-difluorophenyl)-1-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)-3-(1H-1,2,3-triazol-1-yl)propan-2-ol (I-251)

[0591]

[0592] (2-(4-Fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methanamine 8-3 (100 mg, 0.314 mmol) and 1-((2-(2,4-difluorophenyl)oxiran-2-yl)methyl)-1H-1,2,3-triazole 8-2 (149 mg, 0.628 mmol) were dissolved in 15 mL of EtOH, followed by the addition of Et3N (0.09 mL, 0.628 mmol). The reaction was stirred in a microwave at 90°C for 6 hours. The mixture was concentrated in vacuo and flash column chromatography was performed using DCM / MeOH 0-5% to afford 120 mg (0.216 mmol) of the desired product as a yellow solid in a 68% yield. Purification by reverse phase column with H2O / MeOH 5%-95% afforded 2-(2,4-difluorophenyl)-1-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)-3-(1H-1,2,3-triazol-1-yl)propan-2-ol I-251 (52 mg, 0.093 mmol) as a white solid in 29.7% yield.

[0593] LCMS: exact calculated mass = 555.2, found [M+H]+ (ESI+) = 556.2.

[0594] 1 H NMR (400MHz, DMSO) δ8.64(s,1H),8.56(dd,J=4.5,1.6Hz,2H),7.84(d,J=0.9H z,1H),7.69(d,J=9.4Hz,1H),7.57–7.51(m,3H),7.39–7.25(m,6H),7.12(ddd ,J=11.9,9.2,2.5Hz,1H),6.92(td,J=8.5,2.5Hz,1H),5.87(s,1H),4.78(dd, J=29.8,14.2Hz,2H),3.75(s,2H),3.29(s,1H),2.96(dd,J=31.3,12.5Hz,2H).

[0595] Example 10: Preparation of (R)-2-(2,4-difluorophenyl)-1-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-252) and (S)-2-(2,4-difluorophenyl)-1-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-253)

[0596] Compounds I-252 and I-253 were prepared by chiral separation, as shown in Scheme 24 below:

[0597]

[0598] Route 24

[0599] Chiral separation of 2-(2,4-difluorophenyl)-1-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-152) was performed by chiral HPLC. Two isomers (I-252 and I-253) were obtained. Chiral IG column (4.6 x 250 mm 5 μm); mobile phase: n-hexane (0.1% DEA): EtOH (0.1% DEA) = 50:50; flow rate: 1 mL / min; temperature: 40°C; wavelength: 254 nm.

[0600] Peak 1 (first peak) is compound (R)-2-(2,4-difluorophenyl)-1-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-252) (12.29 min, 99% ee). LCMS: exact calculated mass = 555.6, observed [M+H]+ (ESI+) = 556.2. 1 H NMR (400MHz, DMSO) δ8.63 (s, 1H), 8.59–8.53 (m, 2H), 8.28 (d, J = 6.3Hz, 1H), 7.72 (s, 1H), 7.69 (d,J=9.2Hz,1H),7.54(dd,J=8.7,5.5Hz,2H),7.38(dd,J=16.0,9.0Hz,1H),7.31(dd,J=4.9,3 .4Hz,3H),7.27(t,J=8.9Hz,2H),7.11(ddd,J=11.8,9.2,2.4Hz,1H),6.94(td,J=8.4,2.4Hz, 1H), 5.79 (s, 1H), 4.65–4.51 (m, 2H), 3.74 (s, 2H), 2.94 (dd, J = 27.8, 12.6Hz, 2H), 2.38 (s, 1H).

[0601] Peak 2 (second peak) is compound (s)-2-(2,4-difluorophenyl)-1-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-253) (14.10 min, 99% ee). LCMS: exact calculated mass = 555.6, observed [M+H]+ (ESI+) = 556.2. 1H NMR (400MHz, DMSO) δ8.63 (s, 1H), 8.59–8.53 (m, 2H), 8.28 (d, J = 6.3Hz, 1H), 7.72 (s, 1H), 7.69 (d,J=9.2Hz,1H),7.54(dd,J=8.7,5.5Hz,2H),7.38(dd,J=16.0,9.0Hz,1H),7.31(dd,J=4.9,3 .4Hz,3H),7.27(t,J=8.9Hz,2H),7.11(ddd,J=11.8,9.2,2.4Hz,1H),6.94(td,J=8.4,2.4Hz, 1H), 5.79 (s, 1H), 4.65–4.51 (m, 2H), 3.74 (s, 2H), 2.94 (dd, J = 27.8, 12.6Hz, 2H), 2.38 (s, 1H).

[0602] Example 11: Synthesis of Compound 2-(2,4-difluorophenyl)-1-(4-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)piperidin-1-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-254)

[0603] The procedure for synthesizing compound 2-(2,4-difluorophenyl)-1-(4-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)piperidin-1-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-254) is shown in the following Scheme 25:

[0604]

[0605] Route 25

[0606] 2-(2,4-difluorophenyl)-1-(4-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)piperidin-1-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-254)

[0607]

[0608] (2-(4-Fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methanamine (100 mg, 0.314 mmol) 9-1 and 1-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)piperidin-4-one (158 mg, 0.471 mmol) 9-2 were dissolved in 10 mL of EtOH, followed by the addition of tetraisopropyl titanate (two drops). The reaction solution was stirred in a microwave at 90°C for 2 hours. NaBH4 (60 mg, 1.57 mmol) was then added and stirred at room temperature for 15 minutes. The reaction solution was quenched with water (30 mL) and extracted with AcOEt (10 mL*3). The organic layer was washed with brine (20 mL), dried over Na2SO4, filtered, concentrated under reduced pressure, and purified by reverse phase column with H2O / MeOH 5%-95% to afford 2-(2,4-difluorophenyl)-1-(4-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)piperidin-1-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol I-254 (17.5 mg, 0.027 mmol) as a white solid in 8.7% yield.

[0609] LCMS: exact calculated mass = 638.2, found [M+H]+(ESI+) = 639.2.

[0610] 1 H NMR (400MHz, DMSO) δ8.66(s,1H),8.55(d,J=6.0Hz,2H),8.29(s,1H),7.73(s,1H),7.69(d,J=9.1Hz, 1H),7.53(dd,J=8.7,5.5Hz,2H),7.38(dd,J=9.2,4.1Hz,2H),7.31–7.24(m,4H),7.18–7.12(m,1H), 6.97–6.92(m,1H),5.60(s,1H),4.58–4.50(m,2H),3.73(s,2H),3.29(s,1H),2.82(d,J=14.8Hz,1H) ,2.66(t,J=14.4Hz,3H),2.30(s,1H),2.09(dd,J=20.3,9.0Hz,2H),1.67(s,2H),1.23–1.13(m,2H).

[0611] Example 12: Synthesis of Compound 3-(2,4-difluorophenyl)-N-((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butanamide (I-255)

[0612] The procedure for synthesizing compound 3-(2,4-difluorophenyl)-N-((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butanamide (I-255) is shown in the following Scheme 26:

[0613]

[0614] Route 26

[0615] 3-(2,4-Difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butanoic acid (10-2)

[0616]

[0617] Methyl 3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butanoate 10-1 (500 mg, 1.68 mmol) was dissolved in MeOH (10 ml), followed by the addition of 1 M NaOH (8.42 ml, 8.42 mmol), and the mixture was stirred at room temperature for 2 hours. The majority of the MeOH was concentrated, and the pH was adjusted to 4 with 1 N HCl. The filtered solid was concentrated to afford the desired product 10-2 (450 mg, 1.59 mmol) as a white solid in a 94.7% yield. LCMS: calculated mass = 283, observed [M+H]+ (ESI+) = 284.

[0618] 3-(2,4-Difluorophenyl)-N-((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butanamide (I-255)

[0619]

[0620] A solution of (2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methanamine 10-3 (280 mg, 0.88 mmol), 3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butanoic acid 10-2 (25 mg, 0.88 mmol), HATU (501 mg, 1.32 mmol) and Et3N (0.25 mL, 1.76 mmol) in DCM (10 mL) was stirred at room temperature for 4 hours. The reaction solution was quenched with water (50 ml) and extracted with DCM (20 mL*3). The organic layer was washed with brine (20 mL), dried over Na2SO4, filtered, concentrated under reduced pressure, and purified by flash column with DCM / MeOH 0-10% to afford 3-(2,4-difluorophenyl)-N-((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butanamide I-255 (300 mg, 0.514 mmol) as a yellow solid in 58.4% yield.

[0621] LCMS: exact calculated mass = 583.1, found [M+H]+ (ESI+) = 584.2.

[0622] 1 H NMR (400MHz, DMSO) δ8.72(t,J=5.9Hz,1H),8.57(dd,J=4.5,1.5Hz,2H),8.46(s,1H),8.36(s,1H),7.81(s ,1H),7.55(ddd,J=10.5,8.7,5.7Hz,3H),7.42(dd,J=15.9,9.0Hz,1H),7.31–7.25(m,4H),7.18(ddd,J=11 .7,9.1,2.5Hz,1H),6.94(dd,J=9.2,1.2Hz,1H),6.92–6.87(m,1H),6.49(s,1H),4.56(q,J=14.3Hz,2H),4 .30(dd,J=15.2,6.1Hz,1H), 4.15(dd,J=15.4,5.5Hz,1H), 2.97(d,J=14.9Hz,1H), 2.62(d,J=14.7Hz,1H).

[0623] Example 13: Synthesis of Compound 2-(2,4-difluorophenyl)-4-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)-1-(1H-1,2,4-triazol-1-yl)butan-2-ol (I-258)

[0624] The procedure for synthesizing compound 2-(2,4-difluorophenyl)-4-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)-1-(1H-1,2,4-triazol-1-yl)butan-2-ol (I-258) is shown in the following Scheme 27:

[0625]

[0626] Route 27

[0627] 2-(2,4-difluorophenyl)-4-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)-1-(1H-1,2,4-triazol-1-yl)butan-2-ol (I-258)

[0628]

[0629] Under N2, borane-tetrahydrofuran (20 ml) was added to 3-(2,4-difluorophenyl)-N-((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butanamide I-255 (250 mg, 0.429 mmol) at 0°C. The reaction solution was stirred at 80°C for 3 hours. The reaction solution was quenched with saturated ammonium chloride solution (50 mL) and extracted with AcOEt (30 mL*3). The organic layer was washed with brine (30 mL), dried over Na2SO4, filtered, concentrated under reduced pressure, and purified by preparative HPLC (0.05% NH3 in ACN / H2O) to afford 2-(2,4-difluorophenyl)-4-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)-1-(1H-1,2,4-triazol-1-yl)butan-2-ol I-258 as a white solid (3.7 mg) in 1.52% yield.

[0630] LCMS: exact calculated mass = 569.2, found [M+H]+ (ESI+) = 570.2.

[0631] 1H NMR(400MHz,DMSO)δ8.59(s,1H),8.57(dd,J=4.5,1.6Hz,2H),8.28(s,1H),7.77–7.69(m,2 H),7.56–7.50(m,2H),7.34–7.29(m,4H),7.27(d,J=8.9Hz,3H),7.12(ddd,J=11.8,9.1,2.5 Hz,1H),6.66(td,J=8.5,2.4Hz,1H),4.49–4.39(m,2H),3.69(d,J=13.3Hz,1H),3.50(d,J= 13.5Hz,1H),2.91(s,1H),2.33(d,J=1.8Hz,1H),2.12(d,J=15.6Hz,1H),1.89–1.80(m,1H).

[0632] Example 14: Synthesis of 1-(trans-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)-N-((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)methanamine (I-256) and 1-(cis-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)-N-((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)methanamine (I-257)

[0633] The procedure for synthesizing compounds I-256 and I-257 is shown in the following Scheme 28:

[0634]

[0635] Route 28

[0636] trans-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-carbaldehyde (11-2)

[0637]

[0638] (trans-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methanol 11-1 (500 mg, 1.69 mmol) and NaHCO3 (710 mg, 8.45 mmol) were dissolved in DCM (20 ml), followed by the addition of Dess-Martin periodinane (1.433 g, 3.38 mmol) at 0°C. The reaction solution was stirred at room temperature for 2 hours. The reaction solution was then filtered and rinsed with DCM (50 mL). The filtrate was washed with saturated sodium thiosulfate solution (50 mL*3), dried over Na2SO4, filtered, concentrated under reduced pressure, and purified by flash column with DCM / MeOH 0-10% to give the target product 11-2 (350 mg, 1.19 mmol) as a white solid with a yield of 70.7%. LCMS: exact calculated mass = 583.1, found [M+H]+ (ESI+) = 584.2.

[0639] 1-(cis-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)-N-((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)methanamine (I-256) and 1-(trans-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)-N-((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)methanamine (I-257)

[0640]

[0641] (2-(4-Fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methanamine 11-3 (271 mg, 0.853 mmol) and trans-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-carbaldehyde 11-2 (250 mg, 0.853 mmol) were dissolved in 15 mL of EtOH, followed by the addition of tetraisopropyl titanate (4 drops). The reaction solution was stirred in a microwave at 90 ° C for 2 hours. NaBH4 (162 mg, 4.26 mmol) was then added and stirred at room temperature for 30 minutes. The reaction solution was quenched with water (50 mL) and extracted with AcOEt (20 mL * 3). The organic layer was washed with brine (30 mL), dried over Na2SO4, filtered, concentrated under reduced pressure, and purified by preparative HPLC (containing 0.05% The reaction mixture was purified by adding 1-(trans-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)-N-((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)methanamine I-256 (60 mg) as a white solid. Yield 11.8%, and 1-(cis-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)-N-((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)methanamine I-257 (53 mg) as a white solid in 10.4% yield.

[0642] 1-(trans-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)-N-((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)methanamine I-256. LCMS: exact calculated mass = 595.2, found [M+H]+ (ESI+) = 596.2

[0643] 1H NMR (400MHz, DMSO) δ8.59(s,1H),8.56(dd,J=4.5,1.6Hz,2H),8.32(s,1H),7.80(s,1H),7.69(d, J=9.1Hz,1H),7.56–7.50(m,2H),7.34–7.23(m,6H),7.19(ddd,J=11.6,9.2,2.5Hz,1H),6.87(td ,J=8.4,2.3Hz,1H),4.43(dd,J=43.5,14.3Hz,2H),4.08–4.02(m,1H),3.64(s,2H),3.53(t,J=8. 0Hz, 1H), 2.69–2.60 (m, 1H), 2.39–2.25 (m, 3H), 2.16–2.04 (m, 1H), 1.80 (dd, J = 11.4, 7.9Hz, 1H).

[0644] 1-(cis-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)-N-((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)methanamine I-257. LCMS: exact calculated mass = 595.2, found [M+H]+ (ESI+) = 596.2.

[0645] 1 H NMR(400MHz,DMSO)δ8.66(s,1H),8.56(dd,J=4.5,1.6Hz,2H),8.30(s,1H),7.75(s,1H) ,7.71(d,J=9.2Hz,1H),7.56–7.51(m,2H),7.37(dd,J=9.2,1.3Hz,1H),7.32–7.21(m,6 H),6.96(td,J=8.5,2.4Hz,1H),4.53(s,2H),4.02(t,J=7.9Hz,1H),3.69(s,2H),3.54( t,J=8.2Hz,1H),2.45–2.30(m,4H),2.22(d,J=7.5Hz,1H),1.91(dd,J=12.7,9.0Hz,1H).

[0646] Example 14: Synthesis of Compound 2-(2,4-difluorophenyl)-1-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)(methyl)amino)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol I-259

[0647] The procedure for synthesizing compound 2-(2,4-difluorophenyl)-1-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)(methyl)amino)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol I-259 is shown in the following Scheme 29:

[0648]

[0649] Route 29

[0650] 2-(2,4-difluorophenyl)-1-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)(methyl)amino)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-259)

[0651]

[0652] 2-(2,4-difluorophenyl)-1-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol I-152 (140 mg, 0.25 mmol) was dissolved in EtOH (5 mL), followed by the addition of formaldehyde (0.46 mL, 1.25 mmol), and the reaction was stirred at room temperature for 8 hours. NaBH4 (95 mg, 2.5 mmol) was then added, and the reaction was stirred at 60°C for 24 hours. The reaction solution was quenched with water (30 mL) and extracted with AcOEt (20 mL*3). The organic layer was washed with brine (30 mL), dried over Na2SO4, filtered, concentrated under reduced pressure, and purified by preparative HPLC (ACN / H2O with 0.05% NH3) to afford 2-(2,4-difluorophenyl)-1-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)(methyl)amino)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol I-259 (20 mg, 0.035 mmol) as a white solid in 13.9% yield.

[0653] LCMS: exact calculated mass = 569.2, found [M+H]+ (ESI+) = 570.2.

[0654] 1H NMR (400MHz, DMSO) δ8.59–8.52(m,3H),8.29(s,1H),7.73(s,1H),7.61(d,J=9.2Hz,1H),7.56–7.50 (m,2H),7.43(dd,J=15.9,8.9Hz,1H),7.28(dd,J=16.7,7.5Hz,4H),7.12(ddd,J=11.8,9.1,2.5Hz,1 H),7.04(d,J=9.3Hz,1H),6.96(td,J=8.5,2.5Hz,1H),5.82(s,1H),4.54(q,J=14.3Hz,2H),3.66(d, J=13.5Hz,1H),3.46(d,J=13.5Hz,1H),3.10(d,J=15.0Hz,1H),2.80(d,J=13.7Hz,1H),2.15(s,3H).

[0655] Example 15: Synthesis of Compound 2-(2,4-difluorophenyl)-1-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)-3-(1H-tetrazol-1-yl)propan-2-ol (I-260)

[0656] The procedure for synthesizing compound 2-(2,4-difluorophenyl)-1-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)-3-(1H-tetrazol-1-yl)propan-2-ol (I-260) is shown in the following Scheme 30:

[0657]

[0658] Route 30

[0659] 1-(2,4-Difluorophenyl)-2-(1H-tetrazol-1-yl)ethan-1-one (12-2)

[0660]

[0661] A solution of 2-chloro-1-(2,4-difluorophenyl)ethan-1-one 12-1 (4.5 g, 23.7 mmol), 1H-tetrazole (1.66 g, 23.7 mmol), and Et3N (6.6 ml, 47.4 mmol) in EtOH (50 mL) was stirred at 70°C for 8 hours. The reaction solution was quenched with water (100 ml) and extracted with AcOEt (50 mL*3). The organic layer was washed with brine (100 mL), dried over Na2SO4, filtered, concentrated under reduced pressure, and purified by flash column chromatography with DCM / MeOH 0-5% to give the desired product 12-2 (2.1 g, 9.37 mmol) as a yellow solid in a 39.6% yield. LCMS: calculated mass = 224, found [M+H]+ (ESI+) = 225.

[0662] 1-((2-(2,4-difluorophenyl)oxiran-2-yl)methyl)-1H-tetrazole(12-3)

[0663]

[0664] A solution of 1-(2,4-difluorophenyl)-2-(1H-tetrazol-1-yl)ethan-1-one 12-2 (2.1 g, 9.3 mmol), TMSOI (8.25 g, 37.5 mmol), KOH (2.13 g, 37.5 mmol) and CTAB (339 mg, 0.93 mmol) in DCM (20 mL) and H2O (2 mL) was stirred at 50 ° C for 24 hours. The reaction solution was quenched with water (150 mL) and extracted with AcOEt (50 mL*3). The organic layer was washed with brine (100 mL), dried over Na2SO4, filtered, concentrated under reduced pressure, and purified by preparative HPLC (ACN / H2O containing 0.05% NH3) to give the target product 12-3 (1.1 g, 4.62 mmol) as a white solid in 50% yield. LCMS: exact calculated mass=238, found [M+H]+(ESI+)=239.

[0665] 2-(2,4-difluorophenyl)-1-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)-3-(1H-tetrazol-1-yl)propan-2-ol (I-260)

[0666]

[0667] 1-((2-(2,4-difluorophenyl)oxiran-2-yl)methyl)-1H-tetrazole 12-3 (224 mg, 0.94 mmol) and (2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methanamine 12-4 (150 mg, 0.47 mmol) were dissolved in 10 mL of EtOH, followed by the addition of Et3N (0.13 mL, 0.94 mmol). The reaction solution was stirred in a microwave at 100 ° C for 3 hours. The reaction mixture was then concentrated and purified by preparative HPLC (0.05% NH in ACN / H2O) to afford 2-(2,4-difluorophenyl)-1-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)-3-(1H-tetrazol-1-yl)propan-2-ol I-260 (31 mg) as a white solid in 11.8% yield.

[0668] LCMS: exact calculated mass = 556.2, found [M+H]+ (ESI+) = 557.2.

[0669] 1 H NMR(400MHz,)δ9.12(s,1H),8.67(s,1H),8.57(d,J=6.0Hz,2H),7.70(d,J=9.2Hz,1H),7.54(dd,J=8.7,5.6Hz,2H),7.30( dt,J=17.8,9.6Hz,6H),7.18(s,1H),6.94(s,1H),6.02(s,1H),4.88(s,2H),3.77(s,2H),2.99(s,2H),2.49–2.43(m,1H).

[0670] Example 16: Synthesis of Compound 2-(2,4-difluorophenyl)-1-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl-d2)amino)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I261)

[0671] The procedure for synthesizing compound 2-(2,4-difluorophenyl)-1-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl-d2)amino)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-261) is shown in the following Scheme 31:

[0672]

[0673] Route 31

[0674] (2-(4-Fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methane-d2-amine 13-2

[0675]

[0676] 2-(4-Fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridine-6-carbonitrile 13-1 (250 mg, 0.79 mmol) and AlCl₃ (525 mg, 3.95 mmol) were dissolved in Et₂O (10 mL), followed by the addition of lithium aluminum deuteride (165.8 mg, 3.95 mmol) at 0°C. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was then diluted with water (50 mL) and extracted with EA (30 mL*3). The organic layer was washed with brine (30 mL), dried over Na₂SO₄, filtered, concentrated under reduced pressure, and purified by flash column chromatography using DCM / MeOH 0-20% to afford the desired product 13-2 (100 mg, 0.31 mmol) as a white solid in a 39.3% yield. LCMS: calculated mass = 320, observed [M+H]⁺ (ESI⁺) = 321.

[0677] 2-(2,4-difluorophenyl)-1-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl-d2)amino)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-261)

[0678]

[0679] (2-(4-Fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methan-d2-amine 13-2 (70 mg, 0.218 mmol) and 1-((2-(2,4-difluorophenyl)oxiran-2-yl)methyl)-1H-1,2,4-triazole 13-3 (78 mg, 0.328 mmol) were dissolved in 5 mL of EtOH, followed by the addition of Et3N (0.06 mL, 0.436 mmol). The reaction solution was stirred in a microwave at 90 ° C for 2 hours. The reaction mixture was then concentrated and purified by preparative HPLC (0.05% NH in ACN / H2O) to afford 2-(2,4-difluorophenyl)-1-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl-d2)amino)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol I-261 (19.5 mg) as a white solid in 16.1% yield.

[0680] 1H NMR (400MHz, DMSO) δ8.64 (s, 1H), 8.56 (dd, J = 4.5, 1.5Hz, 2H), 8.28 (s, 1H), 7.74 –7.67(m,2H),7.57–7.51(m,2H),7.38(dd,J=15.9,9.0Hz,1H),7.30(ddd,J=20. 7,11.4,6.2Hz,5H),7.12(ddd,J=11.9,9.2,2.5Hz,1H),6.94(td,J=8.5,2.5Hz, 1H), 5.80 (s, 1H), 4.63–4.52 (m, 2H), 2.94 (dd, J = 27.7, 12.5Hz, 2H), 2.36 (s, 1H).

[0681] Example 16: Synthesis of Compound 2-(2,4-difluorophenyl)-1,1-difluoro-1-(5-(4-((((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)methyl)phenyl)pyridin-2-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-262)

[0682] The procedure for synthesizing compound 2-(2,4-difluorophenyl)-1,1-difluoro-1-(5-(4-((((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)methyl)phenyl)pyridin-2-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-262) is shown in the following Scheme 32:

[0683]

[0684] Route 32

[0685] 4-(6-(2-(2,4-difluorophenyl)-1,1-difluoro-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)pyridin-3-yl)benzaldehyde (14-2)

[0686]

[0687] A solution of 1-(5-bromopyridin-2-yl)-2-(2,4-difluorophenyl)-1,1-difluoro-3-(1H-1,2,4-triazol-1-yl)propan-2-ol 14-1 (synthesized according to the following patent: WO2015 / 143172, A1) (550 mg, 1.28 mmol), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzaldehyde (594 mg, 2.56 mmol), tetrakis(triphenylphosphine)palladium(0) (148 mg, 0.13 mmol) and potassium carbonate (353.5 mg, 9.51 mmol) in dioxane (10 mL) and H2O (2.5 mL) was stirred in a microwave at 100°C for 2 hours. The reaction solution was then cooled to room temperature, filtered through a celite pad, and rinsed with EtOAc (50 mL). The filtrate was washed with water (30 mL x 2) and brine (30 mL), dried over NaSO, filtered, concentrated under reduced pressure, and purified by flash column chromatography using DCM / MeOH 0-5% to afford the desired product 14-2 (450 mg, 0.98 mmol) as a yellow solid in a 77.1% yield. LCMS: calculated mass = 456.1, observed [M+H](ESI) = 457.1.

[0688] 2-(2,4-Difluorophenyl)-1,1-difluoro-1-(5-(4-((((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)methyl)phenyl)pyridin-2-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-262)

[0689]

[0690] A solution of 4-(6-(2-(2,4-difluorophenyl)-1,1-difluoro-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)pyridin-3-yl)benzaldehyde 14-2 (150 mg, 0.328 mmol), (2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methanamine 14-3 (156 mg, 0.482 mmol), and tetraisopropyl titanate (2 drops) in EtOH (10 mL) was stirred in a microwave at 90° C. for 2 hours. The reaction solution was then cooled to room temperature, and sodium borohydride (62 mg, 1.64 mmol) was added to the mixture, followed by stirring at room temperature for 30 minutes. The mixture was concentrated and then purified by preparative HPLC (0.05% NH in ACN / H2O) to afford 2-(2,4-difluorophenyl)-1,1-difluoro-1-(5-(4-((((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)methyl)phenyl)pyridin-2-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol I-262 (54.3 mg) as a white solid in 21.8% yield.

[0691] LCMS: exact calculated mass = 758.2, found [M+H]+ (ESI+) = 759.1.

[0692] 1 H NMR(400MHz,DMSO)δ8.91(d,J=1.9Hz,1H),8.70(s,1H),8.59–8.52(m,2H),8.36(s,1 H),8.20(d,J=8.3Hz,1H),7.77–7.69(m,4H),7.56–7.49(m,5H),7.45(d,J=9.2Hz,1H) ,7.33–7.23(m,5H),7.13(ddd,J=11.9,9.2,2.5Hz,1H),7.05(s,1H),6.86(td,J=8.6, 2.4Hz, 1H), 5.39 (d, J = 14.7Hz, 1H), 4.86 (d, J = 14.6Hz, 1H), 3.79 (s, 4H), 2.98 (s, 1H).

[0693] Example 17: Synthesis of (R)-2-(2,4-difluorophenyl)-1-(((S)-1-(2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)ethyl)amino)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (relative stereochemistry) (I-263) and (S)-2-(2,4-difluorophenyl)-1-(((S)-1-(2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)ethyl)amino)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (relative stereochemistry) (I-264)

[0694] The procedure for synthesizing compounds I-263 and I-264 is shown in the following Scheme 33:

[0695]

[0696] Route 33

[0697] 1-(2-(4-Fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)ethan-1-one(15-2)

[0698]

[0699] 2-(4-Fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridine-6-carbonitrile 15-1 (350 mg, 1.11 mmol) was dissolved in THF (3 mL), followed by the addition of MeMgBr (11.1 ml, 11.1 mmol) at 0°C. The reaction mixture was stirred at 70°C for 16 hours. The reaction mixture was then diluted with saturated ammonium chloride solution (50 mL) and extracted with EA (30 mL*3). The organic layer was washed with brine (30 mL), dried over Na2SO4, filtered, concentrated under reduced pressure, and purified by flash column chromatography using DCM / MeOH 0-10% to afford the desired product 15-2 (40 mg) as a yellow solid in a 10.8% yield. LCMS: calculated mass = 331, observed [M+H]+(ESI+) = 332.

[0700] (R)-2-(2,4-difluorophenyl)-1-(((S)-1-(2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)ethyl)amino)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (relative stereochemistry) (I-263) and (S)-2-(2,4-difluorophenyl)-1-(((S)-1-(2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)ethyl)amino)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (relative stereochemistry) (I-264)

[0701]

[0702] A solution of 1-(2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)ethan-1-one 15-2 (40 mg, 0.12 mmol), 1-amino-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol 15-3 (46 mg, 0.18 mmol) and tetraisopropyl titanate (2 drops) in EtOH (10 mL) was stirred at 90 ° C. for 8 hours in a microwave. The reaction solution was then cooled to room temperature, sodium borohydride (46 mg, 1.2 mmol) was added to the mixture, and stirred at 50 ° C. for 16 hours. The mixture was filtered and concentrated, and then analyzed by preparative HPLC (containing 0.05% Purification with 1% NH3 in ACN / H2O) afforded (R)-2-(2,4-difluorophenyl)-1-(((S)-1-(2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)ethyl)amino)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (relative stereochemistry) I-263 (1.8 mg) as a white solid. , yield 2.6%, and (S)-2-(2,4-difluorophenyl)-1-(((S)-1-(2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)ethyl)amino)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (relative stereochemistry) I-264 (1.5 mg) as a white solid in 2.2% yield.

[0703] (R)-2-(2,4-Difluorophenyl)-1-(((S)-1-(2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)ethyl)amino)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (relative stereochemistry) (I-263) LCMS: exact calculated mass = 569.2, found [M+H]+ (ESI+) = 570.2.

[0704] 1 H NMR (400MHz, DMSO) δ8.59–8.54(m,3H),8.28(s,1H),7.73(s,1H),7.66(d,J=9.2Hz,1 H),7.54(dd,J=8.7,5.6Hz,2H),7.37(dd,J=16.0,9.0Hz,1H),7.32–7.24(m,5H),7.0 7(ddd,J=11.8,9.2,2.5Hz,1H),6.94(td,J=8.5,2.5Hz,1H),5.80(s,1H),4.57–4.48 (m,2H),3.83–3.74(m,1H),2.84(d,J=6.8Hz,2H),2.23(s,1H),1.28(d,J=6.5Hz,3H).

[0705] (S)-2-(2,4-Difluorophenyl)-1-(((S)-1-(2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)ethyl)amino)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (relative stereochemistry) (I-264) LCMS: exact calculated mass = 569.2, found [M+H]+ (ESI+) = 570.2.

[0706] 1 H NMR (400MHz, DMSO) δ8.64(s,1H),8.57(d,J=6.0Hz,2H),8.26(s,1H),7.73(d,J=9.3Hz,1H),7.67(s,1 H),7.54(dd,J=8.7,5.6Hz,2H),7.41(d,J=9.4Hz,1H),7.36(d,J=7.0Hz,1H),7.33–7.25(m,4H),7.11( ddd,J=11.9,9.3,2.4Hz,1H),6.94(td,J=8.6,2.5Hz,1H),5.79(s,1H),4.62(d,J=14.3Hz,1H),4.51( d,J=14.3Hz,1H),3.76–3.68(m,1H),2.84(dd,J=14.6,6.6Hz,2H),2.38(s,1H),1.30(d,J=6.6Hz,3H). Example 18: Synthesis of Compound 2-(2,4-difluorophenyl)-1,1-difluoro-1-(5-(4-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)phenyl)pyridin-2-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-265)

[0707] The procedure for synthesizing compound 2-(2,4-difluorophenyl)-1,1-difluoro-1-(5-(4-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)phenyl)pyridin-2-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-265) is shown in the following Scheme 34:

[0708]

[0709] Route 34

[0710] 4-(6-(2-(2,4-difluorophenyl)-1,1-difluoro-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)pyridin-3-yl)phenol(16-2)

[0711]

[0712] A solution of 1-(5-bromopyridin-2-yl)-2-(2,4-difluorophenyl)-1,1-difluoro-3-(1H-1,2,4-triazol-1-yl)propan-2-ol 16-1 (550 mg, 1.28 mmol), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (563 mg, 2.56 mmol), tetrakis(triphenylphosphine)palladium(0) (148 mg, 0.13 mmol) and potassium carbonate (353.5 mg, 9.51 mmol) in dioxane (10 mL) and H2O (2.5 mL) was stirred in a microwave at 100° C. for 2 hours. The reaction solution was then cooled to room temperature, filtered through a pad of celite, and rinsed with EtOAc (50 mL). The filtrate was washed with water (30 mL x 2) and brine (30 mL), dried over NaSO, filtered, concentrated under reduced pressure, and purified by flash column chromatography using DCM / MeOH 0-5% to afford the desired product 16-2 (450 mg) as a yellow solid in 79% yield. LCMS: calculated mass = 444.1, observed [M+H](ESI) = 445.1.

[0713] 4-(6-(2-(2,4-difluorophenyl)-1,1-difluoro-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)pyridin-3-yl)phenyl trifluoromethanesulfonate (16-3)

[0714]

[0715] 4-(6-(2-(2,4-difluorophenyl)-1,1-difluoro-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)pyridin-3-yl)phenol 16-2 (250 mg, 0.56 mmol) and pyridine (222.5 mg, 2.81 mmol) were dissolved in DCM (5 mL), followed by the addition of trifluoromethanesulfonic anhydride (317.4 mg, 1.13 mmol) at 0°C. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was then diluted with water (30 mL) and extracted with DCM (20 mL*2). The organic layer was washed with brine (20 mL), dried over Na2SO4, filtered, concentrated under reduced pressure, and purified by flash column with DCM / MeOH 0-5% to give the target product 16-3 (200 mg) as a yellow solid in a 62% yield. LCMS: exact calculated mass=576, found [M+H]+(ESI+)=577.

[0716] 2-(2,4-Difluorophenyl)-1,1-difluoro-1-(5-(4-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)phenyl)pyridin-2-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-265)

[0717]

[0718] A solution of 4-(6-(2-(2,4-difluorophenyl)-1,1-difluoro-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)pyridin-3-yl)phenyl trifluoromethanesulfonate 16-3 (145 mg, 0.25 mmol), (2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methanamine 16-4 (80 mg, 0.25 mmol), tris(dibenzylideneacetone)dipalladium (46 mg, 0.05 mmol), tripotassium phosphate (106.8 mg, 0.5 mmol) and X-Phos (24 mg, 0.05 mmol) in dioxane (10 mL) was stirred in a microwave at 100° C. for 6 hours. The reaction solution was then cooled to room temperature, filtered through a pad of celite, and rinsed with EtOAc (30 mL). The organic layer was washed with brine (20 ml), dried over Na2SO4, filtered, concentrated under reduced pressure, and purified by preparative HPLC (ACN / H2O with 0.05% NH3) to afford 2-(2,4-difluorophenyl)-1,1-difluoro-1-(5-(4-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)phenyl)pyridin-2-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol I-265 (11.3 mg) as a white solid in 6.1% yield.

[0719] LCMS: calculated mass = 744.2, found [M+H]+(ESI+) = 745.1

[0720] 1 H NMR (400MHz, DMSO) δ8.82–8.76(m,2H),8.56(dd,J=4.5,1.5Hz,2H),8.35(s,1H),8.04(dd,J=8.3,2.2Hz,1 H),7.76(d,J=9.2Hz,1H),7.69(s,1H),7.57–7.49(m,4H),7.44(dd,J=9.2,1.2Hz,1H),7.39(d,J=8.3Hz,1 H),7.30(dd,J=4.5,1.6Hz,2H),7.29–7.22(m,3H),7.11(ddd,J=12.0,9.2,2.6Hz,1H),7.01(s,1H),6.88– 6.82 (m, 1H), 6.80 (d, J = 8.7Hz, 3H), 5.36 (d, J = 14.7Hz, 1H), 4.83 (d, J = 14.7Hz, 1H), 4.44 (d, J = 6.0Hz, 2H).

[0721] Example 19: Synthesis of Compound (2R,3R)-2-(2,4-difluorophenyl)-3-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)-1-(1H-1,2,4-triazol-1-yl)butan-2-ol (I-266)

[0722] The procedure for synthesizing compound (2R,3R)-2-(2,4-difluorophenyl)-3-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)-1-(1H-1,2,4-triazol-1-yl)butan-2-ol (I-266) is shown in the following Scheme 35:

[0723]

[0724] Route 35

[0725] (2R,3R)-2-(2,4-difluorophenyl)-3-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)-1-(1H-1,2,4-triazol-1-yl)butan-2-ol (I-266)

[0726]

[0727] A solution of 1-(((2R,3S)-2-(2,4-difluorophenyl)-3-methyloxiran-2-yl)methyl)-1H-1,2,4-triazole 17-1 (200 mg, 0.796 mmol), (2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methanamine 17-2 (506 mg, 1.593 mmol) and LiClO4 (338 mg, 3.184 mmol) in IPA (20 mL) was stirred in microwave at 120°C for 20 hours. The mixture was concentrated and then purified by preparative HPLC (0.05% NH in ACN / H2O) to afford (2R,3R)-2-(2,4-difluorophenyl)-3-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)-1-(1H-1,2,4-triazol-1-yl)butan-2-ol I-266 (26.5 mg) as a white solid in 5.84% yield.

[0728] LCMS: calculated mass = 569.2, found [M+H]+(ESI+) = 570.2

[0729] 1H NMR (400MHz, DMSO) δ8.88(s,1H),8.57(dd,J=4.5,1.6Hz,2H),8.25(s,1H),7.77(d,J=9.2Hz,1H),7 .61(s,1H),7.59–7.50(m,3H),7.33(dd,J=4.5,1.6Hz,2H),7.31–7.22(m,3H),7.08(ddd,J=11.9,9. 2,2.5Hz,1H),6.86(td,J=8.5,2.5Hz,1H),5.53(s,1H),4.97(d,J=14.4Hz,1H),4.63(d,J=14.7Hz,1 H), 4.05 (d, J = 13.3Hz, 1H), 3.83 (d, J = 11.7Hz, 1H), 3.21 (s, 1H), 2.11 (s, 1H), 0.81 (d, J = 6.5Hz, 3H).

[0730] Example 20: Synthesis of Compound 2-(2,4-difluorophenyl)-1,1-difluoro-1-(5-((4-((((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)methyl)phenyl)ethynyl)pyridin-2-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-267)

[0731] The procedure for synthesizing compound 2-(2,4-difluorophenyl)-1,1-difluoro-1-(5-((4-((((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)methyl)phenyl)ethynyl)pyridin-2-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-267) is shown in the following Scheme 36:

[0732]

[0733] Route 36

[0734] 4-(6-(2-(2,4-difluorophenyl)-1,1-difluoro-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)pyridin-3-yl)phenol(18-2)

[0735]

[0736] A solution of 1-(5-bromopyridin-2-yl)-2-(2,4-difluorophenyl)-1,1-difluoro-3-(1H-1,2,4-triazol-1-yl)propan-2-ol 18-1 (270 mg, 0.63 mmol), 4-ethynylbenzaldehyde (163.3 mg, 1.26 mmol), bis(triphenylphosphine)palladium(II) chloride (44 mg, 0.06 mmol) and N,N-diisopropylethylamine (162.3 mg, 1.26 mmol) in THF (10 mL) was stirred in a microwave at 80° C. for 2 hours. The reaction mixture was then cooled to room temperature, filtered through a pad of celite, and rinsed with EtOAc (30 mL). The organic layer was washed with brine (20 mL), dried over Na2SO4, filtered, concentrated under reduced pressure, and purified by flash column chromatography using DCM / MeOH 0-5% to afford the desired product 18-2 (200 mg) as a brown solid in 66% yield. LCMS: calculated exact mass = 480.1, observed [M+H]+ (ESI+) = 481.1.

[0737] 2-(2,4-Difluorophenyl)-1,1-difluoro-1-(5-((4-((((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)methyl)phenyl)ethynyl)pyridin-2-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-267)

[0738]

[0739] A solution of 4-(6-(2-(2,4-difluorophenyl)-1,1-difluoro-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)pyridin-3-yl)phenol 18-2 (200 mg, 0.42 mmol), (2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methanamine 18-3 (100 mg, 0.31 mmol) and tetraisopropyl titanate (2 drops) in EtOH (10 mL) was stirred in a microwave at 90° C. for 2 hours. The reaction solution was then cooled to room temperature, and sodium borohydride (60 mg, 1.57 mmol) was added to the mixture, followed by stirring at room temperature for 30 minutes. The mixture was concentrated and then purified by preparative HPLC (0.05% NH in ACN / H2O) to afford 2-(2,4-difluorophenyl)-1,1-difluoro-1-(5-((4-((((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)methyl)phenyl)ethynyl)pyridin-2-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol I-267 (19 mg) as a white solid in 7.8% yield.

[0740] LCMS: calculated mass = 782.2, found [M+H]+(ESI+) = 783.1

[0741] 1 H NMR (400MHz, DMSO) δ8.70 (d, J=5.1Hz, 2H), 8.58–8.54 (m, 2H), 8.36 (s, 1H), 8.07 (dd, J=8.2, 2.0Hz,1H),7.76–7.70(m,2H),7.59–7.52(m,4H),7.50–7.42(m,4H),7.32(dd,J=4.5,1.5Hz, 2H),7.27(t,J=8.9Hz,2H),7.19(d,J=7.0Hz,1H),7.17–7.11(m,1H),7.08(s,1H),6.85(dd,J =13.7, 5.5Hz, 1H), 5.35 (d, J = 14.7Hz, 1H), 4.85 (d, J = 14.8Hz, 1H), 3.77 (s, 4H), 2.97 (s, 1H).

[0742] Example 21: Synthesis of Compound 2-(2,4-difluorophenyl)-1,1-difluoro-1-(5-((4-((((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)methyl)phenyl)ethynyl)pyridin-2-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-268)

[0743] The procedure for synthesizing compound 2-(2,4-difluorophenyl)-1,1-difluoro-1-(5-((4-((((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)methyl)phenyl)ethynyl)pyridin-2-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-268) is shown in the following Scheme 37:

[0744]

[0745] Route 37

[0746] 1-(5-((4-((tert-Butyldimethylsilyl)oxy)phenyl)ethynyl)pyridin-2-yl)-2-(2,4-difluorophenyl)-1,1-difluoro-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (19-2)

[0747]

[0748] A solution of 1-(5-bromopyridin-2-yl)-2-(2,4-difluorophenyl)-1,1-difluoro-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (850 mg, 1.98 mmol), tert-butyl(4-ethynylphenoxy)dimethylsilane 19-1 (688.3 mg, 2.97 mmol), bis(triphenylphosphine)palladium(II) chloride (138.7 mg, 0.2 mmol) and N,N-diisopropylethylamine (511.6 mg, 3.95 mmol) in THF (10 mL) was stirred in a microwave at 80° C. for 2 hours. The reaction mixture was then cooled to room temperature, filtered through a pad of celite, and rinsed with EtOAc (50 mL). The organic layer was washed with brine (20 mL), dried over Na2SO4, filtered, concentrated under reduced pressure, and purified by flash column chromatography using DCM / MeOH 0-5% to afford the desired product 19-2 (1.05 g) as a white solid in 91% yield. LCMS: exact calculated mass = 582.2, found [M+H]+ (ESI+) = 583.2.

[0749] 4-((6-(2-(2,4-difluorophenyl)-1,1-difluoro-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)pyridin-3-yl)ethynyl)phenol(19-3)

[0750]

[0751] A solution of 1-(5-((4-((tert-butyldimethylsilyl)oxy)phenyl)ethynyl)pyridin-2-yl)-2-(2,4-difluorophenyl)-1,1-difluoro-3-(1H-1,2,4-triazol-1-yl)propan-2-ol 19-2 (1.05 g, 1.8 mmol) and tetrabutylammonium fluoride (2.7 mL, 1 M in THF, 2.7 mmol) in THF (10 mL) was stirred at room temperature for 2 hours. The reaction solution was then diluted with water (100 mL) and extracted with EtOAc (50 mL*3). The organic layer was washed with brine (20 mL), dried over Na2SO4, filtered, concentrated under reduced pressure, and purified by flash column chromatography with DCM / MeOH 0-10% to give the desired product 19-3 (800 mg) as a white solid in 95% yield. LCMS: exact calculated mass = 468.1, found [M+H]+ (ESI+) = 469.1.

[0752] 4-((6-(2-(2,4-difluorophenyl)-1,1-difluoro-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)pyridin-3-yl)ethynyl)phenyl trifluoromethanesulfonate (19-4)

[0753]

[0754] 4-(6-(2-(2,4-difluorophenyl)-1,1-difluoro-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)pyridin-3-yl)phenol 19-3 (270 mg, 0.58 mmol) and pyridine (228 mg, 2.88 mmol) were dissolved in DCM (3 mL), followed by the addition of trifluoromethanesulfonic anhydride (179 mg, 0.63 mmol) at 0°C. The reaction mixture was stirred at 0°C for 1 hour. The reaction mixture was then diluted with water (30 mL) and extracted with DCM (20 mL*2). The organic layer was washed with brine (20 mL), dried over Na2SO4, filtered, concentrated under reduced pressure, and purified by flash column with DCM / MeOH 0-5% to give the target product 19-4 (200 mg) as a white solid in a 57% yield. LCMS: exact calculated mass=732, found [M+H]+(ESI+)=733.

[0755] 2-(2,4-Difluorophenyl)-1,1-difluoro-1-(5-((4-((((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)methyl)phenyl)ethynyl)pyridin-2-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (I-268)

[0756]

[0757] A solution of 4-((6-(2-(2,4-difluorophenyl)-1,1-difluoro-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)pyridin-3-yl)ethynyl)phenyl trifluoromethanesulfonate 19-4 (170 mg, 0.28 mmol), (2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methanamine 19-5 (90 mg, 0.28 mmol), tris(dibenzylideneacetone)dipalladium (52 ​​mg, 0.06 mmol), tripotassium phosphate (120 mg, 0.57 mmol) and X-Phos (27 mg, 0.06 mmol) in dioxane (10 mL) was stirred in a microwave at 100° C. for 6 hours. The reaction solution was then cooled to room temperature, filtered through a pad of celite, and rinsed with EtOAc (30 mL). The organic layer was washed with brine (20 mL), dried over Na2SO4, filtered, concentrated under reduced pressure, and purified by preparative HPLC (0.05% NH3 in ACN / H2O) to afford 2-(2,4-difluorophenyl)-1,1-difluoro-1-(5-(4-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)phenyl)pyridin-2-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol I-268 (29.2 mg) as a white solid in 13.6% yield.

[0758] LCMS: calculated mass = 768.2, found [M+H]+ (ESI+) = 769.1

[0759] 1 H NMR (400MHz, DMSO) δ8.78(s,1H),8.61(d,J=1.8Hz,1H),8.56(dd,J=4.5,1.6Hz,2H),8.35(s,1H),7.95(dd ,J=8.2,2.1Hz,1H),7.76(d,J=9.2Hz,1H),7.70(s,1H),7.55–7.50(m,2H),7.41(d,J=8.0Hz,2H),7.31(dd ,J=9.1,4.8Hz,4H),7.29–7.24(m,2H),7.22–7.16(m,1H),7.13(s,1H),7.04(s,1H),6.98(s,1H),6.85(d, J=2.5Hz,1H),6.72(d,J=8.8Hz,2H),5.34(d,J=14.7Hz,1H),4.83(d,J=14.7Hz,1H),4.42(d,J=6.0Hz,2H).

[0760] Example 22: Synthesis of Compound 2-(2,4-difluorophenyl)-1,1-difluoro-1-(5-(4-((((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)methyl)phenyl)pyridin-2-yl)-3-(1H-tetrazol-1-yl)propan-2-ol (I-269)

[0761] The procedure for synthesizing compound 2-(2,4-difluorophenyl)-1,1-difluoro-1-(5-(4-((((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)methyl)phenyl)pyridin-2-yl)-3-(1H-tetrazol-1-yl)propan-2-ol (I-269) is shown in the following Scheme 38:

[0762]

[0763] Route 38

[0764] 4-(6-(2-(2,4-difluorophenyl)-1,1-difluoro-2-hydroxy-3-(1H-tetrazol-1-yl)propyl)pyridin-3-yl)benzaldehyde (20-2)

[0765]

[0766] A solution of 1-(5-bromopyridin-2-yl)-2-(2,4-difluorophenyl)-1,1-difluoro-3-(1H-1,2,4-triazol-1-yl)propan-2-ol 20-1 (300 mg, 0.7 mmol), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzaldehyde (323 mg, 1.4 mmol), tetrakis(triphenylphosphine)palladium(0) (148 mg, 0.7 mmol) and potassium carbonate (192 mg, 1.4 mmol) in dioxane (10 mL) and H O (2.5 mL) was stirred in a microwave at 100° C. for 2 hours. The reaction mixture was then cooled to room temperature, filtered through a pad of celite, and rinsed with EtOAc (30 mL). The organic layer was washed with brine (20 mL), dried over Na2SO4, filtered, concentrated under reduced pressure, and purified by flash column chromatography using DCM / MeOH 0-5% to afford the desired product 20-2 (250 mg) as a brown solid in 78% yield. LCMS: calculated exact mass = 457.1, observed [M+H]+ (ESI+) = 458.1.

[0767] 2-(2,4-Difluorophenyl)-1,1-difluoro-1-(5-(4-((((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)methyl)phenyl)pyridin-2-yl)-3-(1H-tetrazol-1-yl)propan-2-ol (I-269)

[0768]

[0769] A solution of 4-(6-(2-(2,4-difluorophenyl)-1,1-difluoro-2-hydroxy-3-(1H-tetrazol-1-yl)propyl)pyridin-3-yl)benzaldehyde 20-2 (158 mg, 0.35 mmol), (2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methanamine 20-3 (110 mg, 0.35 mmol) and tetraisopropyl titanate (2 drops) in EtOH (10 mL) was stirred in a microwave at 90 ° C for 2 hours. The reaction solution was then cooled to room temperature, sodium borohydride (65.7 mg, 1.73 mmol) was added to the mixture, and stirred at room temperature for 1 hour. The mixture was concentrated and then purified by preparative HPLC (0.05% NH in ACN / H2O) to afford 2-(2,4-difluorophenyl)-1,1-difluoro-1-(5-(4-((((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)methyl)phenyl)pyridin-2-yl)-3-(1H-tetrazol-1-yl)propan-2-ol I-269 (113 mg) as a white solid in 40% yield.

[0770] LCMS: calculated mass = 759.2, found [M+H]+(ESI+) = 760.1

[0771] 1 H NMR(400MHz,DMSO)δ9.16(s,1H),8.94(d,J=1.9Hz,1H),8.71(s,1H),8.56(d,J =6.0Hz,2H),8.23(dd,J=8.3,2.0Hz,1H),7.75(t,J=6.5Hz,3H),7.56–7.50(m, 5H),7.45(d,J=9.2Hz,1H),7.35–7.25(m,6H),7.24–7.17(m,1H),6.95–6.86(m ,1H),5.68(d,J=14.7Hz,1H),5.12(d,J=14.8Hz,1H),3.80(s,4H),3.03(s,1H).

[0772] Example 23: Synthesis of Compound 2-(2,4-difluorophenyl)-1,1-difluoro-1-(5-(4-((((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)methyl)phenyl)pyridin-2-yl)-3-(1H-tetrazol-1-yl)propan-2-ol (I-270)

[0773] The procedure for synthesizing compound 2-(2,4-difluorophenyl)-1,1-difluoro-1-(5-(4-((((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)methyl)phenyl)pyridin-2-yl)-3-(1H-tetrazol-1-yl)propan-2-ol (I-270) is shown in the following Scheme 39:

[0774]

[0775] Route 39

[0776] 2-(2,4-difluorophenyl)-1,1-difluoro-1-(5-((4-(2-hydroxyethoxy)phenyl)ethynyl)pyridin-2-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (21-2)

[0777]

[0778] 4-((6-(2-(2,4-difluorophenyl)-1,1-difluoro-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)pyridin-3-yl)ethynyl)phenol 21-1 (500 mg, 1.07 mmol) and 2-bromoethan-1-ol (265 mg, 2.14 mmol) were dissolved in 10 mL of DMF, followed by the addition of potassium carbonate (295 mg, 2.14 mmol). The reaction mixture was stirred at 70°C for 48 hours. The reaction mixture was then diluted with water (100 mL) and extracted with EtOAc (50 mL*2). The organic layer was washed with brine (100 mL*3), dried over Na2SO4, filtered, concentrated under reduced pressure, and purified by flash column chromatography with DCM / MeOH 0-5% to give the desired product 21-2 (400 mg) as a white solid in a 73% yield. LCMS: exact calculated mass = 512.1, found [M+H]+ (ESI+) = 513.1.

[0779] 2-(4-((6-(2-(2,4-difluorophenyl)-1,1-difluoro-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)pyridin-3-yl)ethynyl)phenoxy)acetaldehyde (21-3)

[0780]

[0781] 2-(2,4-difluorophenyl)-1,1-difluoro-1-(5-((4-(2-hydroxyethoxy)phenyl)ethynyl)pyridin-2-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol 21-2 (250 mg, 0.49 mmol) was dissolved in 10 mL of DCM, followed by the addition of Dess-Martin periodinane (414 mg, 0.98 mmol). The reaction mixture was stirred at room temperature for 48 hours. The reaction mixture was then filtered and rinsed with DCM (20 mL). The organic layer was washed with saturated sodium thiosulfate (30 mL*3), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the crude product 21-3 (140 mg). LCMS: calculated mass = 510, found [M+H]+(ESI+) = 543.

[0782] 2-(2,4-Difluorophenyl)-1,1-difluoro-1-(5-(4-((((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)methyl)phenyl)pyridin-2-yl)-3-(1H-tetrazol-1-yl)propan-2-ol (I-270)

[0783]

[0784] 2-(4-((6-(2-(2,4-difluorophenyl)-1,1-difluoro-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)pyridin-3-yl)ethynyl)phenoxy)acetaldehyde 21-3 (165 mg, 0.32 mmol) and (2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methanamine 21-4 (103 mg, 0.32 mmol) were dissolved in 10 mL of EtOH, followed by the addition of sodium cyanoborohydride (61.5 mg, 0.98 mmol) and acetic acid (2 drops). The reaction was stirred in a microwave at 70 ° C for 2 hours. The reaction was then concentrated and purified by preparative HPLC (0.05% NH in ACN / H2O) to afford 2-(2,4-difluorophenyl)-1,1-difluoro-1-(5-(4-((((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)methyl)phenyl)pyridin-2-yl)-3-(1H-tetrazol-1-yl)propan-2-ol I-270 (62.6 mg) as a white solid in 24% yield.

[0785] LCMS: calculated mass = 812.2, found [M+H]+ (ESI+) = 813.2

[0786] 1H NMR (400MHz, DMSO) δ8.74(s,1H),8.68(s,1H),8.56(d,J=5.4Hz,2H),8.36(s,1H),8.03(d,J=8.5Hz,1H), 7.72(d,J=10.0Hz,2H),7.54(t,J=8.1Hz,4H),7.44(dd,J=13.2,8.7Hz,2H),7.31(d,J=5.7Hz,2H),7.27(t ,J=8.9Hz,2H),7.22–7.11(m,2H),7.08(s,1H),7.02(d,J=8.7Hz,2H),6.85(t,J=8.5Hz,1H),5.35(d,J=14 .5Hz, 1H), 4.85 (d, J = 14.6Hz, 1H), 4.11 (t, J = 5.3Hz, 2H), 3.85 (s, 2H), 2.91 (t, J = 5.3Hz, 2H), 2.60 (s, 1H).

[0787] Example 24: Synthesis of Compound 2-(2,4-difluorophenyl)-5-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)-1-(1H-1,2,4-triazol-1-yl)pentan-2-ol (I-279)

[0788] The procedure for synthesizing compound 2-(2,4-difluorophenyl)-5-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)-1-(1H-1,2,4-triazol-1-yl)pentan-2-ol (I-279) is shown in the following Scheme 40:

[0789]

[0790] Route 40

[0791] 5-((tert-Butyldimethylsilyl)oxy)-2-(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)pent-3-yn-2-ol (22-2)

[0792]

[0793] Dissolve tert-butyldimethyl(prop-2-yn-1-yloxy)silane (11.4 g, 67.2 mmol) in THF (50 mL). Add n-butyllithium in THF (2.5 M, 30.5 mL, 76.1 mmol) dropwise at -78°C in a dry ice acetone bath. Stir the reaction mixture at -78°C for 1 hour. Then, add a solution of 1-(2,4-difluorophenyl)-2-(1H-1,2,4-triazol-1-yl)ethan-1-one 22-1 (10 g, 44.8 mmol) in THF (10 mL) dropwise at -78°C. Stir the mixture at -78°C until it reaches room temperature for 6 hours. The mixture is quenched with NH4Cl (100 mL), extracted with EtOAc (100 mL x 2), and washed with brine (100 mL). The organic layer was dried over NaSO, concentrated, and purified by flash chromatography (PE / EA = 10 / 1 to 1:1) to afford 5-((tert-butyldimethylsilyl)oxy)-2-(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)pent-3-yn-2-ol 22-2 (6 g) as a white solid in 34% yield. LCMS: exact calculated mass = 393.2, observed [M+H] (ESI) = 394.1.

[0794] 4-(2,4-Difluorophenyl)-5-(1H-1,2,4-triazol-1-yl)pent-2-yne-1,4-diol (22-3)

[0795]

[0796] At room temperature, to a 20mL THF solution of 5-((tert-butyldimethylsilyl)oxy)-2-(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)pent-3-yn-2-ol 22-2 (6g, 15.2mmol) was added a THF solution of TBAF (1M, 45.6mL, 45.6mmol). The reaction solution was stirred for 6 hours. The reaction solution was then concentrated, diluted with 100mL EtOAc, and washed with 100mL brine. The organic layer was dried with Na2SO4, concentrated, and purified by flash chromatography (DCM / MeOH=10 / 1) to give 4-(2,4-difluorophenyl)-5-(1H-1,2,4-triazol-1-yl)pent-2-yn-1,4-diol 22-3 (4g) as a white solid in 95% yield. LCMS: exact calculated mass = 279.1, found [M+H]+ (ESI+) = 280.2.

[0797] 4-(2,4-Difluorophenyl)-5-(1H-1,2,4-triazol-1-yl)pentane-1,4-diol (22-4)

[0798]

[0799] To a solution of 4-(2,4-difluorophenyl)-5-(1H-1,2,4-triazol-1-yl)pentane-2-yn-1,4-diol 22-3 (2 g, 7.1 mmol) in 20 mL of MeOH was added 10% Pd / C (400 mg, 20% wt) at room temperature. The reaction was stirred for 4 hours. The reaction was then concentrated to afford 4-(2,4-difluorophenyl)-5-(1H-1,2,4-triazol-1-yl)pentane-1,4-diol 22-4 (2 g) as a white solid in 98% yield. LCMS: calculated mass = 283.1, found [M+H]+ (ESI+) = 284.2.

[0800] 4-(2,4-Difluorophenyl)-4-hydroxy-5-(1H-1,2,4-triazol-1-yl)pentanal (22-5)

[0801]

[0802] To a solution of 4-(2,4-difluorophenyl)-5-(1H-1,2,4-triazol-1-yl)pentane-1,4-diol 22-4 (2 g, 7.0 mmol) in 20 mL of DCM was added NaHCO₃ (0.8 g, 10.5 mmol) and Dess-Martin periodinane (4.4 g, 10.5 mmol) at room temperature. The reaction mixture was stirred for 16 hours. The reaction mixture was then filtered and washed with Na₂SO₃ solution (30 mL). The organic layer was concentrated to give the crude product 4-(2,4-difluorophenyl)-4-hydroxy-5-(1H-1,2,4-triazol-1-yl)pentanal 22-5 (1.8 g).

[0803] 2-(2,4-difluorophenyl)-5-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)-1-(1H-1,2,4-triazol-1-yl)pentan-2-ol (I-279)

[0804]

[0805] To a solution of 4-(2,4-difluorophenyl)-4-hydroxy-5-(1H-1,2,4-triazol-1-yl)pentanal 22-5 (176 mg, 0.62 mmol) and (2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methanamine 22-6 (100 mg, 0.31 mmol) in 8 mL of EtOH was added NaBH3CN (98 mg, 1.5 mmol) and a drop of tetraisopropyl titanate. The reaction was stirred in a microwave at 70°C for 2 hours. The mixture was concentrated in vacuo, purified by flash column with DCM / MeOH 0-5%, and purified by preparative HPLC (0.05% NH in ACN / H2O) to afford 2-(2,4-difluorophenyl)-5-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)-1-(1H-1,2,4-triazol-1-yl)pentan-2-ol I-280 as a white solid (66.3 mg) in 36% yield.

[0806] LCMS: exact calculated mass = 583.2, found [M+H]+(ESI+) = 584.2.

[0807] 1 H NMR(400MHz,DMSO)δ8.66(s,1H),8.57(dd,J=4.5,1.6Hz,2H),8.28(s,1H),7.77–7.69(m,2H), 7.58–7.50(m,2H),7.38(ddd,J=13.2,6.6,4.0Hz,2H),7.33–7.24(m,4H),7.15(ddd,J=11.8,9 .1,2.5Hz,1H),6.95(td,J=8.5,2.5Hz,1H),6.77(s,1H),4.47(q,J=14.1Hz,2H),3.72–3.60(m ,2H),2.51(s,1H),2.45–2.32(m,2H),2.23–2.12(m,1H),1.75–1.59(m,1H),1.43–1.12(m,2H).

[0808] Example 25: Synthesis of Compound 2-(2,4-difluorophenyl)-5-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)-1-(1H-1,2,4-triazol-1-yl)pent-3-yn-2-ol (I-280)

[0809] The procedure for synthesizing compound 2-(2,4-difluorophenyl)-5-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)-1-(1H-1,2,4-triazol-1-yl)pent-3-yn-2-ol (I-280) is shown in the following Scheme 41:

[0810]

[0811] Route 41

[0812] 4-(2,4-Difluorophenyl)-4-hydroxy-5-(1H-1,2,4-triazol-1-yl)pent-2-ynal (23-2)

[0813]

[0814] To a solution of 4-(2,4-difluorophenyl)-5-(1H-1,2,4-triazol-1-yl)pent-2-yn-1,4-diol 23-1 (500 mg, 1.8 mmol) in 10 mL of DCM was added NaHCO₃ (226 mg, 2.7 mmol) and Dess-Martin periodinane (1.1 g, 2.7 mmol) at room temperature. The reaction mixture was stirred for 16 hours. The reaction mixture was then filtered and washed with Na₂SO₃ solution (30 mL). The organic layer was concentrated to give the crude product 4-(2,4-difluorophenyl)-4-hydroxy-5-(1H-1,2,4-triazol-1-yl)pent-2-ynal 23-2 (300 mg).

[0815] 2-(2,4-Difluorophenyl)-5-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)-1-(1H-1,2,4-triazol-1-yl)pent-3-yn-2-ol (I-280)

[0816]

[0817] To a solution of 4-(2,4-difluorophenyl)-4-hydroxy-5-(1H-1,2,4-triazol-1-yl)pent-2-ynal 23-2 (171 mg, 0.62 mmol) and (2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methanamine 23-3 (100 mg, 0.31 mmol) in 8 mL of EtOH was added NaBH3CN (98 mg, 1.5 mmol) and a drop of tetraisopropyl titanate. The reaction was stirred in a microwave at 70 ° C for 2 hours. The mixture was concentrated in vacuo, purified by flash column with DCM / MeOH 0-5%, and purified by preparative HPLC (0.05% NH in ACN / HO) to afford 2-(2,4-difluorophenyl)-5-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)-1-(1H-1,2,4-triazol-1-yl)pent-3-yn-2-ol I-280 as a white solid (21.6 mg) in 11% yield.

[0818] LCMS: exact calculated mass = 579.2, found [M+H]+ (ESI+) = 580.2.

[0819] 1 H NMR (400MHz, DMSO) δ8.67(s,1H),8.57(d,J=6.0Hz,2H),8.38(s,1H),7.78(s,1H),7.73(d,J=9.2Hz,1H),7.54(dd,J=8.6,5.6Hz,2 H),7.41(dd,J=16.7,9.1Hz,2H),7.33–7.23(m,5H),7.02–6.93(m,2H),4.72–4.58(m,2H),3.80(s,2H),3.40(s,2H),2.71(s,1H).

[0820] Example 26: Synthesis of Compound 4-(3-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)propoxy)benzamidine HCl Salt (I-281)

[0821] The procedure for synthesizing compound 4-(3-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)propoxy)benzimidamide HCl salt (I-281) is shown in the following Scheme 42:

[0822]

[0823] Route 42

[0824] tert-Butyl ((4-hydroxyphenyl)(imino)methyl)carbamate (24-2)

[0825]

[0826] To a solution of 4-hydroxybenzamidine hydrochloride 24-1 (9.0 g, 52.2 mmol), DMAP (630 mg, 5.2 mmol) and DIPEA (34.2 mL, 208.8 mmol) in anhydrous DMF (100 mL) was added Boc2O (22.5 g, 104.4 mmol) at 0°C, and the resulting suspension was stirred at room temperature overnight. The reaction mixture was diluted with water (200 mL) to form an off-white precipitate and filtered. The solid was dissolved in 200 mL of MeOH, 200 mL of 10% NaOH solution was added, and the reaction mixture was stirred at room temperature for 1.5 h. The mixture was concentrated, diluted with water (200 mL), and extracted with AcOEt (200 mL*3). The combined organic layers were then washed with brine (200 mL) and dried over anhydrous Na2SO4. The mixture was concentrated under reduced pressure to give tert-butyl ((4-hydroxyphenyl)(imino)methyl)carbamate 24-2 (9 g), which was used directly without further purification. LCMS: exact calculated mass = 236.1, found [M+H]+ (ESI+) = 237.2.

[0827] tert-Butyl ((4-(3-bromopropyloxy)phenyl)(imino)methyl)carbamate (24-3)

[0828]

[0829] Tert-butyl ((4-hydroxyphenyl)(imino)methyl)carbamate 24-2 (1g, 4.2mmol) and K2CO3 (2.3g, 16.8mmol) were dissolved in 12mL MeCN. 1,3-dibromopropane (2.31mL, 21mmol) was added in batches, and the mixture was refluxed for 6h. After cooling to room temperature, the resulting mixture was quenched with water (20mL) and extracted with AcOEt (20mL*3). The combined organic layer was washed with brine (20mL), dried over Na2SO4, and evaporated in vacuo to give a crude product, which was purified by flash chromatography (DCM / MeOH=10:1) to give tert-butyl ((4-(3-bromopropoxy)phenyl)(imino)methyl)carbamate 24-3 (1.0g) as a white solid in a 66% yield. LCMS: exact calculated mass = 356.1, found [M+H]+ (ESI+) = 357.2.

[0830] tert-Butyl ((4-(3-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)propyloxy)phenyl)(imino)methyl)carbamate (24-5)

[0831]

[0832] To a 10 mL DMF solution of tert-butyl ((4-(3-bromopropoxy)phenyl)(imino)methyl)carbamate 24-3 (400 mg, 1.1 mmol) and (2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methanamine 24-4 (357 mg, 1.1 mmol) was added KCO (607 mg, 4.4 mmol) and KI (90 mg, 0.55 mmol). The reaction solution was stirred at room temperature for 16 hours. The mixture was diluted with EtOAc (100 mL), washed with brine (100 mL), and concentrated. The crude product was purified by silica gel column (DCM / MeOH=10:1) to give tert-butyl ((4-(3-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)propoxy)phenyl)(imino)methyl)carbamate 24-5 (200 mg) as a white solid in 30% yield.

[0833] LCMS: exact calculated mass = 579.2, found [M+H]+ (ESI+) = 580.2.

[0834] 1 H NMR(400MHz,DMSO)δ8.94(s,2H),8.70(s,1H),8.59–8.51(m,2H),7.93(d,J=8 .8Hz,2H),7.69(d,J=9.2Hz,1H),7.58–7.49(m,2H),7.40(d,J=9.2Hz,1H),7.2 8(dd,J=16.8,7.6Hz,4H),6.97(d,J=8.9Hz,2H),4.12(t,J=6.3Hz,2H),3.78( s, 2H), 2.67 (t, J = 6.7Hz, 2H), 2.44 (s, 1H), 1.90 (p, J = 6.4Hz, 2H), 1.44 (s, 9H).

[0835] 4-(3-(((2-(4-Fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)propoxy)benzamidine HCl salt (I-281)

[0836]

[0837] To a solution of tert-butyl ((4-(3-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)propyloxy)phenyl)(imino)methyl)carbamate 24-5 (100 mg, 0.16 mmol) in 2 mL of MeOH was added a 4 M HCl solution in MeOH (10 mL). The reaction solution was stirred at 50 ° C for 16 hours. The mixture was concentrated and basified with NaHCO solution (20 mL). The precipitate was collected and the crude product was purified by preparative HPLC (ACN / HO containing 0.05% HCl) to give 4-(3-(((2-(4-fluorophenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)methyl)amino)propyloxy)benzamidine HCl salt I-281 (9.4 mg) as a white solid in 10% yield.

[0838] LCMS: exact calculated mass = 494.2, found [M+H]+ (ESI+) = 495.2.

[0839] 1 H NMR (400MHz, MeOD) δ9.02(s,1H),8.69(d,J=5.8Hz,2H),8.13(d,J=9.2Hz,1H),7.94(d,J=5.9Hz,2H),7.80(t,J=5.9Hz,3H),7.62(dd ,J=8.5,5.4Hz,2H),7.24(t,J=8.7Hz,2H),7.18(d,J=8.9Hz,2H),4.45(s,2H),4.27(t,J=5.7Hz,2H),3.47–3.34(m,2H),2.33(s,2H). 1 H NMR (400MHz, DMSO) δ9.91(d,J=55.9Hz,2H),9.21(dd,J=25.6,11.1Hz,3H),8.97(d,J=32.9Hz,2H),8.76(d,J=5.6Hz,2H),8.05(t,J=8.0Hz,1H), 7.95–7.69(m,5H),7.67–7.54(m,2H),7.34(t,J=8.8Hz,2H),7.15(d,J=8 .8Hz,2H),4.33(s,2H),4.23(t,J=5.8Hz,2H),3.12(s,2H),2.24(s,2H).

[0840] B: Biological Assays

[0841] (a) Hotspot kinase assay

[0842] Using an in vitro hotspot kinase assay (purified enzyme, 33 IC50 values ​​were determined using 3-ATP, appropriate substrates, and 1 μM ATP. For enzyme inhibition assays, purified recombinant Yck2 was used and compounds were tested at 10 concentrations ranging from 10 μM to 0.0005 μM. Reaction conditions were 1 μM ATP, one hour incubation with inhibitors, and phosphorylation of appropriately selected peptide substrates using 3-ATP to detect kinase activity.

[0843] Recombinant Yck2 protein

[0844] Yck2 residues 37-345 were PCR amplified from Candida albicans genomic DNA and subcloned into the vector pMCSG53, which encodes an N-terminal His6 tag, a TEV protease site, and then the Yck2 protein. The plasmid was transformed into Escherichia coli BL21(DE3)-Gold competent cells and the Yck2 protein was purified using a previously described method (Stogios et al., ACS Chem. Biol. 13, 1322-1332 (2018)).

[0845] Table 3: Hotspot kinase assay results

[0846] Compound ID IC50 (IC50-nM) I-77 0.4 I-237 1.14 I-148 1.61 I-232 2.61 I-230 2.93 I-58 3.19 I-80 3.33 I-152 3.37 I-115 3.67 I-231 4.13 I-240 4.15 I-117 5.08 I-35 6.8 I-238 7.84 I-116 7.91 I-54 8.12 I-234 8.12 I-55 8.41 I-71 9.33 I-53 9.52 I-33 12.29 I-242 15.34 I-229 16.38 I-100 32.94 I-147 36.8 I-1 46.26 I-102 46.57 I-90 47.59 I-153 48.69 I-103 49.48 I-233 65.93 I-224 72.26 I-6 72.71 I-2 106.8 I-235 107.4 I-236 168.9 I-154 176.3 I-4 177.5 I-3 179.2 I-239 184.8 I-5 334.8 I-151 764.6 I-248 4.69 I-252 2.07 I-253 5.95 I-254 3.09

[0847] (b) Antifungal susceptibility testing

[0848] Minimum inhibitory concentrations (MICs) were determined in flat-bottom 96-well plates using a modified microbroth dilution protocol described previously (Singh et al., PLoS Pathog. 5, e1000532 (2009); LaFayette et al., PLoS Pathog. 6, 79–80 (2010)). Compounds were formulated in dimethyl sulfoxide (DMSO, Sigma-Aldrich Co.). Each compound was tested in duplicate in at least two independent experiments.

[0849] Table 4: Antifungal susceptibility test results

[0850] Compound ID CaCi2 MIC (MIC50-uM) I-103 0.0488 I-100 0.0651 I-147 0.0977 I-117 0.195 I-239 0.195 I-154 0.195 I-90 0.586 I-238 0.781 I-155 0.781 I-115 1.27 I-153 1.56 I-230 1.88 I-236 2.34 I-102 3.12 I-116 6.25 I-148 6.25 I-224 25 I-231 >25 I-232 >25 I-58 >25 I-32 >50 I-253 0.137 I-257 0.295 I-261 0.195 I-262 0.293 I-266 0.195 I-270 0.586 I-268 1.17 I-255 1.56

[0851] (c) Mammalian cytotoxicity

[0852] HepG2 cells were seeded at 500 cells / well in 384-well plates overnight and then incubated with compounds (10 concentration gradients) for 48 h. Reagent (ThermoFisher Scientific) was added to each well, and after incubation at room temperature for 10 minutes, the luminescent signal was measured using an Envision microplate reader (Perkin Elmer).

[0853] Table 5: Mammalian cytotoxicity results

[0854]

[0855]

[0856] While the present application has been described with reference to what are presently considered to be preferred embodiments, it is to be understood that the application is not limited to the disclosed embodiments, but is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0857] All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. In the event that a definition of a term in this application differs from that in a document incorporated by reference, the definition provided herein shall govern that term.

Claims

1. A conjugated compound of formula (I) or an enantiomer thereof or a pharmaceutically acceptable salt, solvate and / or prodrug thereof, A–L 1 –B (I) in: A is a moiety that increases fungal cell uptake and / or fungal cell permeability; B is a CK1 inhibitory moiety; and L 1 is a linker comprising at least one first complementary functional group to be covalently linked to a functional group in A, and at least one second complementary functional group to be covalently linked to a functional group in B, thereby forming the conjugate of formula (I), wherein the compound of formula I optionally contains one or more isotopic variant atoms.

2. The conjugate compound of claim 1, wherein A is an antifungal moiety.

3. The conjugate compound according to claim 1 or 2, wherein A comprises one or more of the following: an alkylene moiety, an azole moiety, an indole moiety, a phenyl moiety, a carbazole moiety, a pyridoindole moiety, a tetrahydropyridoindole moiety, a polyamine moiety, a fatty acid alkyl chain moiety, a flucytosine (5-FC) moiety, and a triphenylphosphonium moiety, each of which is optionally substituted with one or more groups independently selected from the group consisting of halogen, OH, C 1-4 Alkyl, C 1-4 Halogenated alkyl, OC 1-4 Alkyl, C(O)OH, C(O)NH2, C(O)C 1-4 Alkyl, C(O)OC 1-4 Alkyl, C(O)NHC 1-4 Alkyl, C(O)N(C 1-4 Alkyl)C 1-4 alkyl), C(NH)NH2, C(NC 1-4 Alkyl)NH2、C(NC 1-4 alkyl)NHC 1-4 Alkyl, C(NC 1-4 alkyl)N(C 1-4 Alkyl)(C 1-4 alkyl), C(NH)NHC 1-4 Alkyl and C(NH)N(C 1-4 Alkyl)(C 1-4 alkyl), wherein each alkyl is optionally substituted by halogen, suitably substituted by fluorine. The conjugate compound according to claim 3 , wherein the azole moiety is selected from the group consisting of an imidazole moiety, a triazole moiety, and a tetrazole moiety.

5. The conjugate according to claim 1 or 2, wherein A is selected from fluconazole, itraconazole, clotrimazole, ketoconazole, voriconazole, posaconazole, isavuconazole, miconazole, flucytosine, olorofim, manogepix, erefungin, caspofungin, micafungin, anidulafungin, rezafungin, amphotericin B and VT-1161.

6. The conjugate according to claim 1 or 2, wherein A is selected from: wherein each n is independently an integer from 0 to 4, and Indicates that L 1 The attachment point of the invention includes all stereoisomers thereof.

7. The conjugate according to claim 1 or 2, wherein A is selected from: wherein each n is independently an integer from 0 to 4, and Indicates that L 1 includes all stereoisomers thereof; and wherein A optionally comprises one or more isotopic variant atoms, for example, wherein the isotopic variant is deuterium and A comprises one or more deuterium atoms.

8. The conjugate compound according to any one of claims 1 to 7, wherein B is a substituted C containing 2 to 6 N heteroatoms. 4-20 Heteroaryl moiety. The conjugate compound according to claim 8 , wherein B comprises at least one fluorophenyl group.

10. The conjugate compound according to claim 8 or 9, wherein B comprises at least one of the following: a pyridine moiety, a pyrimidine moiety, a pyrazole moiety, a piperazine moiety, and an imidazole moiety. The conjugate compound according to claim 8 , wherein B comprises a pyrazolopyridine moiety or a pyrrolopyridinone moiety.

12. The conjugate compound according to any one of claims 1 to 7, wherein B comprises a compound listed in Table 1.

13. The conjugate compound according to any one of claims 1 to 7, wherein B is selected from: in Indicates that L 1 connection point.

14. The conjugated compound according to any one of claims 1 to 13, wherein L 1 Contains one or more independently selected groups selected from C(O), C(=NR 1 ), O, NR 1 、C 1-12 Alkylene, C 6-20 Arylene, C 3-10 Cycloalkylene, C 2-20 Heteroarylene and C 3-10 Heterocycloalkylene, wherein each of the alkylene, arylene, cycloalkylene, heteroarylene and heterocycloalkylene is optionally substituted by one or more R 2 Replace, and each R 2 Independently selected from C 1-12 Alkyl, NR 3 C(O)R 4 NR 3 R 4 , halogen, C(O)NR 3 R 4 OR 3 ; and R 1 、R 3 and R 4 Independently selected from H and C 1-12 alkyl.

15. The conjugated compound according to any one of claims 1 to 13, wherein L 1 Selected from C 1-12 Alkylene-NR 1 -C(O), C 1-6 Alkylene-C 6-10 Arylene-NR 1 -C(O), C 1-6 Alkylene-NR 1 -C(O)-C 2-10 Heteroarylene, C 1-6 Alkylene-C 2-10 Heteroarylene-NR 1 -C(O), C 2-10 Heteroarylene-NR 1 -C(O), C 1-6 Alkylene-C 3-10 Heterocycloalkylene-NR 1 -C(O)-C 2-10 Heteroarylene, C 1-6 Alkylene-C 3-10 Heterocycloalkylene-C 2-10 Heteroarylene, C 1-6 Alkylene-C 3-10 Heterocycloalkylene-NR 1 -C(O), C 1-6 Alkylene-C 3-10 Heterocycloalkylene-C 1-6 Alkylene-NR 1 -C(O), C 1-6 Alkylene-C 3-10 Heterocycloalkylene-C 6-10 Arylene-NR 1 -C(O), C 1-6 Alkylene-C 3-10 Heterocycloalkylene-C 1-6 Alkylene-C 6-10 Arylene-NR 1 -C(O), C 1-6 Alkylene-C(O)-C 3-10 Heterocycloalkylene-NR 1 -C(O), C 1-6 Alkylene-C(O)-C 3-10 Heterocycloalkylene-NR 1 -C 1-6 Alkylene, C 1-6 Alkylene-OC(O)-C 1-6 Alkylene-C 3-10 Heterocycloalkylene-NR 1 -C(O), C 1-6 Alkylene-C 3-10 Heterocycloalkylene-NR 1 -C 1-6 Alkylene, C 1-6 Alkylene-C 3-10 Heterocycloalkylene-C 1-6 Alkylene-NR 1 -C 1-6 Alkylene, C 1-6 Alkylene-C 3-10 Heterocycloalkylene-C 1-6 Alkylene, C 1-6 Alkylene-NR 1 -C 1-6 Alkylene, OC 1-12 Alkylene, C 1-6 Alkylene-C 3-10 Heterocycloalkylene-OC 1-6 Alkylene-C 6-10 Arylene-NR 1 -C(O), C 1-6 Alkylene-C 3-10 Heterocycloalkylene-OC 1-6 Alkylene-OC 6-10 Arylene-NR 1 -C(O) and NR 1 -C 1-6 Alkylene-OC 1-6 Alkylene-OC 1-6 Alkylene-NR 1 , each L 1 Optionally, one or more R 2 substituted, and A and B are covalently bonded to L 1 either end of .

16. The conjugate according to claim 14 or 15, wherein each R 2 Independently selected from C 1-6 Alkyl, NR 3 C(O)R 4 NR 3 R 4 、C(O)NR 3 R 4 OR 3 ; and R 3 and R 4 Independently selected from H and C 1-6 alkyl.

17. The conjugate according to claim 15, wherein each R 2 independently selected from methyl, ethyl, propyl, F, Cl, NH2, NHC(O)C 1-6 Alkyl, C(O)NH2 and OH.

18. The conjugate according to claim 15, wherein R 3 and R 4 Independently selected from H, methyl, ethyl and propyl.

19. The conjugated compound according to any one of claims 1 to 13, wherein L 1 Selected from: wherein each of m, p, q, r and s is independently an integer from 0 to 6, and represents the point of attachment to A or B, including all stereoisomers thereof.

20. The conjugated compound according to any one of claims 1 to 13, wherein L 1 Selected from: wherein each of m, p, q, r and s is independently an integer from 0 to 6, and represents the point of attachment to A or B, including all stereoisomers thereof, and Among them L 1 optionally comprising one or more isotopic variant atoms, for example, wherein the isotopic variant is deuterium, and L 1 Contains one or more deuterium atoms.

21. The conjugate compound according to claim 1, wherein the compound of formula (I) is selected from the compounds listed in Table 2 or salts and / or solvates thereof.

22. A method of treating or preventing a fungus-related disease, disorder or condition comprising administering to a subject in need thereof a therapeutically effective amount of one or more conjugate compounds according to any one of claims 1 to 21.

23. A method of inhibiting or preventing fungal growth comprising administering to a subject in need thereof a therapeutically effective amount of one or more conjugate compounds according to any one of claims 1 to 21.

24. A method of inhibiting fungal CK1 activity, comprising administering to a subject in need thereof a therapeutically effective amount of one or more conjugated compounds according to any one of claims 1 to 21.

25. A method for selectively inhibiting fungal CK1 activity, comprising administering to a subject in need thereof a therapeutically effective amount of one or more conjugate compounds according to any one of claims 1 to 21.

26. A method of treating or preventing fungal disease, comprising administering to a subject in need thereof a therapeutically effective amount of one or more conjugated compounds according to any one of claims 1 to 21.

27. A method for treating or preventing a fungus-related disease, disorder or condition, comprising administering to a subject in need thereof a therapeutically effective amount of one or more conjugate compounds according to any one of claims 1 to 21 in combination with another known agent for treating or preventing a fungus-related disease, disorder or condition.

28. A pharmaceutical composition comprising one or more conjugated compounds according to any one of claims 1 to 21, or pharmaceutically acceptable salts and / or solvates thereof, and a pharmaceutically acceptable carrier and / or diluent.

29. The pharmaceutical composition of claim 28, further comprising an additional therapeutic agent.

30. An agricultural composition comprising one or more conjugated compounds according to any one of claims 1 to 21, or salts and / or solvates thereof, and an agriculturally acceptable carrier and / or diluent.

Images