Combination therapies with pikfyve kinase inhibitors

Abstract

The present invention relates to methods for treating, preventing, or reducing a disease or disorder in a subject in need thereof, the method comprising co-administering to the subject an effective amount of a phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) inhibitor and a p-glycoprotein (PGP) (also known as multidrug resistance protein 1 (MDR1)) inhibitor. The present invention also relates to pharmaceutical compositions comprising a PIKfyve inhibitor and a PGP inhibitor.

Description

[0001] Docket No. 4390.3002 WO

[0002] Combination Therapies with PIKfyve Kinase Inhibitors

[0003] RELATED APPLICATION

[0004] This application claims the benefit of U.S. Provisional Application No. 63 / 734,506 filed December 16, 2024. The entire contents of the above-referenced application are incorporated by reference herein.

[0005] BACKGROUND OF THE INVENTION

[0006] Phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) is named after its function and domain structure (phosphoinositide kinase for five position containing a FYVE finger) is a lipid kinase that phosphorylates phosphatidylinositol-3-phosphate (PI(3)P), producing PI(3,5)P2, which is involved in cellular processes including membrane trafficking and cytoskeletal reorganization. Inhibiting PIKfyve can alter both lipid levels and TFEB-driven transcription leading to changes autophagy and resultant increases in autophagic flux and exocytosis and downstream degradation / clearance of protein aggregates (Hung et al., Staats et al., Shi et al., Soares et al., Lucus-Del-Pozo et aL). Inhibiting PIKfyve may be useful in the treatment of various diseases or conditions such as viral infections, autoimmune diseases, inflammatory diseases, cancer, pain, or many neurological diseases.

[0007] However, despite advancements in the area of various drug delivery systems, many drugs are prone to poor oral bioavailability due to biological barriers at the enterocyte level, termed “intestinal first pass metabolism”. In addition, the poor oral bioavailability is attributed to efflux transporters e.g. permeability-glycoprotein (P-gp) at the enterocyte luminal membrane.

[0008] P-gp can efflux out a variety of drugs from cells which ultimately lead to unsuccessful drug therapy. It also affects various pharmacokinetic parameters of drugs, like absorption, distribution, metabolism and excretion from the body which leads to modified bioavailability and possible adverse drug reactions. Thus, it is believed that the P-glycoprotein efflux pump prevents certain pharmaceutical compounds from transversing the mucosal cells of the small intestine and, therefore, from being absorbed into the systemic circulation. Thus, there is a need for compositions, combinations, and / or methods for improved bioavailability of drugs.

[0009] SUMMARY OF THE INVENTION

[0010] The present invention relates to methods for treating, preventing, or reducing a disease or disorder in a subject in need thereof, the method comprising co-administering to the subject an effective amount of a phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) Docket No. 4390.3002 WO inhibitor and a p-glycoprotein (PGP) (also known as multidrug resistance protein 1 (MDR1)) inhibitor. The present invention also relates to pharmaceutical compositions comprising a PIKfyve inhibitor and a PGP inhibitor.

[0011] BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.

[0013] Fig. 1 demonstrates that compound Al (Example Al) achieves significantly higher brain concentrations when co-administered with elacridar.

[0014] Fig. 2 demonstrates that compound Al (Example Al) covalently modifies PIKfyve in a mouse brain after a single dose.

[0015] Fig. 3 demonstrates that compound Al (Example Al) achieves PIKfyve occupancy in the central nervous system (CNS) when co-administered with elacridar.

[0016] DETAILED DESCRIPTION OF THE INVENTION

[0017] Definitions

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0019] The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting. Docket No. 4390.3002 WO

[0020] As used herein, the term “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. As used herein when referring to a measurable value such as an amount, a temporal duration, and the like, the term “about” is meant to encompass variations of ±10%, including ±5%, ±1%, and ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0021] The term “treat,” “treated,” “treating,” or “treatment” refers to an approach for obtaining beneficial or desired results including but not limited to therapeutic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying state, disease, or disorder being treated. Also, a therapeutic benefit includes the diminishment or alleviation of at least one symptom associated or caused by the state, disorder or disease being treated.

[0022] As used herein, the term “prevent” or “prevention” means no disorder or disease development if none had occurred, or no further disorder or disease development if there had already been development of the disorder or disease. Also considered is the ability of one to prevent one, some, or all of the symptoms associated with the disorder or disease.

[0023] As used herein, the term “patient,” “individual” or “subject” includes a mammal. The mammal can be e.g., any mammal, e.g., a human, a primate, a mouse, a rat, a dog, a cat, a cow, a horse, a goat, a camel, a sheep, or a pig. Preferably, the mammal is a human.

[0024] As used herein, the terms “effective amount,” “pharmaceutically effective amount,” and “therapeutically effective amount” refer to a nontoxic but sufficient amount of an agent to provide the desired biological result. That result may be reduction or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system.

[0025] As used herein, the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0026] As used herein, the term “pharmaceutically acceptable salt” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts of the present invention include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present invention can be synthesized from the parent Docket No. 4390.3002 WO compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. The phrase “pharmaceutically acceptable salt” is not limited to a mono, or 1 : 1, salt. For example, “pharmaceutically acceptable salt” also includes bis-salts, such as a bis-hydrochloride salt. Lists of suitable salts are found in Remington’s Pharmaceutical Sciences, 17thed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety.

[0027] As used herein, the term “composition” or “pharmaceutical composition” refers to a mixture of at least one compound useful within the invention with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient or subject. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary, and topical administration.

[0028] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the invention within or to the patient such that it may perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the invention, and not injurious to the patient. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer Docket No. 4390.3002 WO solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.

[0029] As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the invention and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions. The “pharmaceutically acceptable carrier” may further include a pharmaceutically acceptable salt of the compound useful within the invention. Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the invention are known in the art and described, for example in Remington’s Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

[0030] As used herein, the term “administration” of a compound or a composition to a subject includes any route of introducing or delivering the agent to a subject to perform its intended function. Administration can be carried out by any suitable oral or non-oral route as described herein. Administration includes self-administration and the administration by another.

[0031] The term "co-administration," "administered in combination with," and their grammatical equivalents, as used herein, encompasses administration of two or more agents to an animal so that both agents and / or their metabolites are present in the animal at the same time. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which both agents are present.

[0032] The term “alkyl”, unless otherwise specified, refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to eight carbon atoms, and which is attached to the rest of the molecule by a single bond, e.g. methyl, ethyl, n-propyl, 1 -methylethyl (isopropyl), n-butyl, n- pentyl, and 1,1 -dimethylethyl (t-butyl). The term “Ci-6 alkyl” refers to an alkyl group as defined above having 1 to 6 carbon atoms. The term “C1-3 alkyl” refers to an alkyl group as defined above having 1 to 3 carbon atoms. In appropriate circumstances, the term “alkyl” refers to a hydrocarbon chain radical as mentioned above which is bivalent.

[0033] The term “alkenyl”, unless otherwise specified, refers to an aliphatic hydrocarbon group containing one or more carbon-carbon double bonds and which may be a straight or branched or branched chain having about 2 to about 10 carbon atoms, e.g., ethenyl, 1- Docket No. 4390.3002 WO propenyl, 2-propenyl (allyl), iso-propenyl, 2-methyl-l -propenyl, 1-butenyl, and 2-butenyl. The term “C2-6 alkenyl” refers to an alkenyl group as defined above having 2 to 6 carbon atoms. In appropriate circumstances, the term “alkenyl” refers to a hydrocarbon group as mentioned above which is bivalent.

[0034] The term “alkynyl”, unless otherwise specified, refers to a straight or branched chain hydrocarbyl radical having at least one carbon-carbon triple bond, and having in the range of 2 to up to 12 carbon atoms (with radicals having in the range of 2 to up to 10 carbon atoms presently being preferred) e.g., ethynyl, propynyl, and butnyl. The term “C2-6 alkynyl” refers to an alkynyl group as defined above having 2 to 6 carbon atoms. In appropriate circumstances, the term “alkynyl” refers to a hydrocarbyl radical as mentioned above which is bivalent.

[0035] The term "alkoxy" unless otherwise specified, denotes an alkyl, cycloalkyl, or cycloalkylalkyl group as defined herein attached via an oxygen linkage to the rest of the molecule. The term “substituted alkoxy” refers to an alkoxy group where the alkyl constituent is substituted (i.e., -©-(substituted alkyl)). For example, “alkoxy” refers to the group -O- alkyl, including from 1 to 8 carbon atoms of a straight, branched, cyclic configuration and combinations thereof attached to the parent structure through an oxygen atom. Examples include methoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy, and cyclohexyloxy. In appropriate circumstances, the term “alkoxy” refers to a group as mentioned above which is bivalent.

[0036] The term “cycloalkyl”, unless otherwise specified, denotes a non-aromatic mono or multi cyclic ring system of about 3 to 12 carbon atoms such as cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Examples of multi cyclic cycloalkyl groups include perhydronaphthyl, adamantyl and norbornyl groups, bridged cyclic groups, and sprirobicyclic groups, e.g., spiro[4.4]non-2-yl. The term “C3-6 cycloalkyl” refers to a cycloalkyl group as defined above having 3 to 6 carbon atoms.

[0037] The term “cycloalkylalkyl”, unless otherwise specified, refers to a cyclic ringcontaining radical containing in the range of about 3 up to 8 carbon atoms directly attached to an alkyl group which is then attached to the main structure at any carbon from the alkyl group, such as cyclopropylmethyl, cyclobutylethyl, and cyclopentylethyl.

[0038] The term “cycloalkenyl”, unless otherwise specified, refers to cyclic ring-containing radicals containing in the range of about 3 to 8 carbon atoms with at least one carbon-carbon double bond such as cyclopropenyl, cyclobutenyl, and cyclopentenyl. Docket No. 4390.3002 WO

[0039] The term “cycloalkenylalkyl” refers to a cycloalkenyl group directly attached to an alkyl group which is then attached to the main structure at any carbon from the alkyl group.

[0040] The term “aryl”, unless otherwise specified, refers to aromatic radicals having in the range of 6 to 20 carbon atoms such as phenyl, naphthyl, tetrahydronaphthyl, indanyl, and biphenyl.

[0041] The term “arylalkyl”, unless otherwise specified, refers to an aryl group as defined above directly bonded to an alkyl group as defined above, e.g., -CEECeEE and -C2H5C6H5.

[0042] The term “heterocyclic ring”, unless otherwise specified, refers to a non-aromatic 3 to 15 member ring radical which consists of carbon atoms and at least one heteroatom selected from nitrogen, phosphorus, oxygen and sulfur. For purposes of this invention, the heterocyclic ring radical may be a mono-, bi-, tri- or tetracyclic ring system, which may include fused, bridged or spiro ring systems, and the nitrogen, phosphorus, carbon, oxygen or sulfur atoms in the heterocyclic ring radical may be optionally oxidized to various oxidation states. In addition, the nitrogen atom may be optionally quaternized. The heterocyclic ring radical may be attached to the main structure at any heteroatom or carbon atom. Examples of such heterocyclic radicals include, but are not limited to, piperidinyl, piperazinyl, 2- oxopiperazinyl, 2-oxopiperidinyl, 2- oxopyrrolidinyl, 2-oxoazepinyl, azepinyl, 4-piperidonyl, pyrrolidinyl, oxazolinyl, morpholinyl, quinuclidinyl, octahydroindolyl, octahydroisoindolyl, decahydroisoquinolyl, tetrahydrofuryl, tetrahydropyranyl, thiamopholinyl, thiamorpholinyl sulfoxide, thiamopholinyl sulfone, and di oxaphospholanyl.

[0043] The term “heterocyclyl”, unless otherwise specified, refers to a heterocylic ring radical as defined above. The heterocylcyl ring radical may be attached to the main structure at any heteroatom or carbon ring atom. In appropriate circumstances, the term “heterocyclyl” refers to a hydrocarbon chain radical as mentioned above which is bivalent. Examples of such heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl [1,3 ]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2- oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo- thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl.

[0044] The term “heterocyclylalkyl”, unless otherwise specified, refers to a radical of the formula -Rw-heterocyclyl where Rwis an alkylene chain as defined herein. If the heterocyclyl is a nitrogen-containing heterocyclyl, the heterocyclyl is optionally attached to the alkyl radical at the nitrogen atom. The alkylene chain of the heterocyclylalkyl radical is optionally Docket No. 4390.3002 WO substituted as defined herein for an alkylene chain. The heterocyclyl part of the heterocyclylalkyl radical is optionally substituted as defined herein for a heterocyclyl group.

[0045] The term “heteroaryl”, unless otherwise specified, refers to an optionally substituted 5 to 14 member aromatic ring having one or more heteroatoms selected from N, O, and S as ring atoms. The heteroaryl may be a mono-, bi- or tricyclic ring system. Examples of such “heteroaryl” radicals include, but are not limited to, oxazolyl, thiazolyl, imidazolyl, pyrrolyl, furanyl, pyridinyl, pyrimidinyl, pyrazinyl, benzofuranyl, indolyl, benzothiazolyl, benzoxazolyl, carbazolyl, quinolyl, isoquinolyl, azetidinyl, acridinyl, benzodi oxolyl, benzodi oxanyl, benzofuranyl, carbazolyl, cinnolinyl, dioxolanyl, indolizinyl, naphthyridinyl, perhydroazepinyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, quinazolinyl, quinoxalinyl, tetrazoyl, tetrahydroisoquinolyl, pyridazinyl, oxazolidinyl, triazolyl, indanyl, isoxazolyl, isoxazolidinyl, thiazolinyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, isoindolyl, indolinyl, isoindolinyl, benzimidazolyl, thiadiazolyl, benzopyranyl, thienyl, benzothienyl, oxadiazolyl, chromanyl, and isochromanyl. The term “5 or 6-membered heteroaryl” refers to a heteroaryl having 5- or 6-ring atoms. The term “5-6 or 6-5 membered bicyclic heteroaryl” refers to a bicyclic heteroaryl with a five- membered ring fused to a six-membered ring, where the 5-membered ring is bound to the rest of the molecule (referred to as a “5-6 membered bicyclic heteroaryl”) or the 6-membered ring is bound to the rest of the molecule (referred to as a “6-5 membered bicyclic heteroaryl”). The term “6-6 membered bicyclic heteroaryl” refers to a bicyclic heteroaryl with a six membered ring fused to a another six-membered ring, where one of the 6-membered rings is bound to the rest of the molecule. The heteroaryl ring radical may be attached to the main structure at any heteroatom or carbon atom. The term “substituted heteroaryl” also includes ring systems substituted with one or more oxide (-O-) substituents, such as pyridinyl N-oxides.

[0046] The term “heteroarylalkyl”, unless otherwise specified, refers to a radical of the formula -Rw-heteroaryl, where Rwis an alkylene chain as defined above. If the heteroaryl is a nitrogen-containing heteroaryl, the heteroaryl is optionally attached to the alkyl radical at the nitrogen atom. The alkylene chain of the heteroarylalkyl radical is optionally substituted as defined herein for an alkylene chain. The heteroaryl part of the heteroarylalkyl radical is optionally substituted as defined herein for a heteroaryl group.

[0047] The term “cyclic ring” refers to a cyclic ring containing 3 to 10 carbon atoms.

[0048] The term “substituted” unless otherwise specified, refers to substitution with any one or any combination of the following substituents which may be the same or different and are independently selected from hydrogen, hydroxy, halogen, carboxyl, cyano, nitro, oxo (=0), Docket No. 4390.3002 WO thio (=S), substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted cycloalkenylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heterocyclic ring, substituted heterocyclylalkyl ring, substituted or unsubstituted guanidine, -COORa, -C(O)Ra, -C(S)Ra, - C(O)NRaRb, -C(O)NRa(ORb), -NRbRc, -NRaCONRbRc, -N(Ra)SORb, -N(Ra)SO2Rb, =N- NRaRb, - NRaC(O)ORb, -NRaRb, -NRaC(O)Rb, -NRaC(S)Rb-NRaC(S)NRbRc, -SONRaRb, - SO2NRaRb, - ORa, -ORaC(O)NRbRc, -ORaC(O)ORb, -OC(O)Ra, -OC(O)NRaRb, - RaNRbC(O)Rc, -R0Rb, - RaC(O)ORb, -RaC(O)NRbRc, -RaC(O)Ra, -ROC(O)Rb, -SRa, -SORa, -SO2Ra, and -ONO2, wherein Ra, Rband Rcin each of the above groups can be hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted amino, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heterocyclic ring, or substituted heterocyclylalkyl ring, or any two of Ra, Rband Rcmay be joined to form a substituted or unsubstituted saturated or unsaturated 3-10 membered ring, which may optionally include heteroatoms which may be the same or different and are selected from O, NRa(e.g., Racan be hydrogen or Ci-6 alkyl) or S. Substitution or the combinations of substituents envisioned by this invention are preferably those that result in the formation of a stable or chemically feasible compound. The term stable as used herein refers to the compounds or the structure that are not substantially altered when subjected to conditions to allow for their production, detection and preferably their recovery, purification and incorporation into a pharmaceutical composition. The substituents in the aforementioned “substituted” groups cannot be further substituted. For example, when the substituent on “substituted alkyl” is “substituted aryl”, the substituent on “substituted aryl” cannot be “substituted alkenyl”.

[0049] The term “halo”, “halide”, or, alternatively, “halogen” means fluoro, chloro, bromo or iodo.

[0050] The terms “haloalkyl”, “haloalkenyl”, “haloalkynyl”, and “haloalkoxy” include alkyl, alkenyl, alkynyl and alkoxy structures that are substituted with one or more halogen groups Docket No. 4390.3002 WO or with combinations thereof. For example, the terms “fluoroalkyl” and “fluoroalkoxy” include haloalkyl and haloalkoxy groups, respectively, in which the halogen is fluorine.

[0051] Certain of the compounds described herein contain one or more asymmetric centers and can thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined, in terms of absolute stereochemistry, as (R)- or (S)-. The present chemical entities, pharmaceutical compositions and methods are meant to include all such possible isomers, including racemic mixtures, optically pure forms and intermediate mixtures.

[0052] Additionally, the instant invention also includes the compounds which differ only in the presence of one or more isotopically enriched atoms for example replacement of hydrogen with deuterium or tritium, the replacement of a carbon by13C- or14C-enriched carbon, or the replacement of a nitrogen by15N. The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium, iodine-125 (125I) or carbon-14 (14C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.

[0053] “Pharmaceutically acceptable salt” includes both acid and base addition salts. A pharmaceutically acceptable salt of any one of the compounds described herein is intended to encompass any and all pharmaceutically suitable salt forms. Preferred pharmaceutically acceptable salts of the compounds described herein are pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0054] “Pharmaceutically acceptable acid addition salt” refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid, hydrofluoric acid, phosphorous acid, and the like. Also included are salts that are formed with organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and. aromatic sulfonic acids, etc. and include, for example, acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Exemplary salts thus include sulfates, pyrosulfates, bi sulfates, sulfites, bi sulfites, nitrates, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, Docket No. 4390.3002 WO chlorides, bromides, iodides, acetates, trifluoroacetates, propionates, caprylates, isobutyrates, oxalates, malonates, succinate suberates, sebacates, fumarates, maleates, mandelates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, phthalates, benzenesulfonates, toluenesulfonates, phenylacetates, citrates, lactates, malates, tartrates, methanesulfonates, and the like. Also contemplated are salts of amino acids, such as arginates, gluconates, and galacturonates (see, for example, Berge S. M. et al., “Pharmaceutical Salts,” Journal of Pharmaceutical Science, 66: 1-19 (1997)). Acid addition salts of basic compounds are, in some embodiments, prepared by contacting the free base forms with a sufficient amount of the desired acid to produce the salt according to methods and techniques with which a skilled artisan is familiar.

[0055] “Pharmaceutically acceptable base addition salt” refers to those salts that retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Pharmaceutically acceptable base addition salts are, in some embodiments, formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, for example, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, N,N-dibenzylethylenediamine, chloroprocaine, hydrabamine, choline, betaine, ethylenediamine, ethylenedianiline, N-methylglucamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N- ethylpiperidine, polyamine resins and the like. See Berge et al., supra.

[0056] The invention provides a method for treating, preventing, or reducing a disease or disorder in a subject in need thereof. The methods of the present invention can be employed for the treatment of conditions where the inhibition of PIKfyve would be beneficial. As such, in one aspect, the compounds and compositions provided herein are useful in inhibiting PIKfyve and / or for treating a disease or disorder associated with PIKfyve in a subject (such as a human subject) in need thereof. For example, the methods of the invention may be used for the treatment of various diseases or conditions such as viral infections, autoimmune Docket No. 4390.3002 WO diseases, inflammatory diseases, cancer, pain, or many neurological diseases as described herein.

[0057] In particular, the present invention relates to a method for treating, preventing, or reducing a disease or disorder in a subject in need thereof, the method comprising coadministering to the subject an effective amount of a phosphatidylinositol-3-phosphate 5- kinase (PIKfyve) inhibitor and a p-glycoprotein (PGP) inhibitor.

[0058] In accordance with the methods described herein, a "subject in need of' is a subject having a disease, disorder or condition, or a subject having an increased risk of developing a disease, disorder or condition relative to the population at large. The subject in need thereof can be one that is "non-responsive" or "refractory" to a currently available therapy for the disease or disorder, for example cancer. In this context, the terms "non-responsive" and "refractory" refer to the subject's response to therapy as not clinically adequate to relieve one or more symptoms associated with the disease or disorder.

[0059] In one embodiment, the invention provides a method of treating a subject having a neurological disease. The method comprises administering to the subject an effective amount of a PIKfyve inhibitor as described herein (or a pharmaceutical composition thereof) and a P- gp inhibitor (or a pharmaceutical composition thereof) to the subject. In embodiments, the PIKfyve inhibitor and the PGP inhibitor are part of separate pharmaceutical compositions. In embodiments, the PIKfyve inhibitor and the PGP inhibitor are part of the same pharmaceutical composition.

[0060] A “neurological disease” is any disease that causes electrical, biochemical, or structural abnormalities in the brain, spine, or neurons. For example, a neurological disease may be a neurodegenerative disease. The neurodegenerative disease may result in motor neuron degeneration, for example. The neurological disease may be amyloid lateral sclerosis, Huntington’s disease, Alzheimer’s disease, or frontotemporal dementia, for example. Further examples of neurological diseases include, but are not limited to, Parkinson’s disease, multiple sclerosis, peripheral myopathy, Rasmussen’s encephalitis, attention deficit hyperactivity disorder, autism, central pain syndromes, anxiety, and / or depression, for example. Neurodegenerative diseases result in the progressive destruction of neurons that affects neuronal signaling. For example, a neurodegeneration may be amyotrophic lateral sclerosis, Alzheimer’s disease, Huntington’s disease, Friedreich’s ataxia, Lewy body disease, Parkinson’s disease, spinal muscle atrophy, primary lateral sclerosis, progressive muscle atrophy, progressive bulbar palsy, and pseudobulbar palsy. Docket No. 4390.3002 WO

[0061] The neurological disease may be one that has neuronal death generated by intracellular aggregates. In certain embodiments, the method includes treating amyotrophic lateral sclerosis (ALS). In certain embodiments, the method includes treating frontotemporal dementia (FTD). In certain embodiments, the method includes treating a neurological disease that is associated with aberrant endosomal trafficking. In certain embodiments, the method includes treating a neurological disease that is associated with aberrant lysosomal trafficking.

[0062] In further embodiments, the method includes treating a subject who has a (GGGGCC)n repeat expansion in the C9ORF72 gene. The C9ORF72 gene (also known as chromosome 9 open reading frame 72, FTDALS1, ALSFTD, and FTD ALS) is located on the short (p) arm of chromosome 9 at position 21.2. C9ORF72 is a 481 amino acid protein with a molecular mass of 54328 Da, which may undergo post-translational modifications of ubiquitination and phosphorylation. The expression levels of C9ORF72 may be highest in the central nervous system and the protein localizes in the cytoplasm of neurons as well as in presynaptic terminals. C9ORF72 plays a role in endosomal and lysosomal trafficking regulation and has been shown to interact with RAB proteins that are involved in autophagy and endocytic transport. C9ORF72 activates RAB5, a GTPase that mediates early endosomal trafficking. Mutations in C9ORF72 have been associated with ALS and FTD. The GGGGCC repeat expansion ((GGGGCC)n) in C9ORF72 may be present in subjects suffering from a neurological disease. For example, (GGGGCC)n hexanucleotide expansion is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), accounting for about 10% of each worldwide. The (GGGGGCC)n hexanucleotide expansion may be located between exons la and lb of C9ORF72. The (GGGGCC)n hexanucleotide expansion may be present in a neurological disease, wherein n is greater than 25, wherein n is greater than 30, wherein n is greater than 35, wherein n is greater than 40, wherein n is greater than 45, wherein n is greater than 50, wherein n is greater than 55, wherein n is greater than 60, wherein n is greater than 65, or wherein n is greater than 70, for example, n may be between 25 and 100, between 29 and 95, between 30 and 90, between 35 and 85, between 40 and 80, between 45 and 75, between 50 and 70, between 55 and 65, or between 55 and 60, for example.

[0063] In further embodiments, the subject is haploinsufficient for C9ORF72. In further embodiments, the method includes treating patients who have a 50% or greater reduction in C9ORF72 protein activity. In further embodiments, the method includes a C9ORF72 gene product that comprises a dipeptide repeat resulting from the (GGGGCC)n expansion. In further embodiments, the method includes a gain-of-function or loss of function mutation Docket No. 4390.3002 WO resulting from the (GGGGCC)n expansion. In further embodiments, the neurological disease is associated with neuronal hyperexcitability.

[0064] In one embodiment, the invention provides a method of treating a subject having amyotrophic lateral sclerosis (ALS) comprising administering to the subject an effective amount of a PIKfyve inhibitor as described herein (or a pharmaceutical composition thereof) and a P-gp inhibitor (or a pharmaceutical composition thereof) to the subject. In embodiments, the PIKfyve inhibitor and the PGP inhibitor are part of separate pharmaceutical compositions. In embodiments, the PIKfyve inhibitor and the PGP inhibitor are part of the same pharmaceutical composition.

[0065] In one embodiment, the invention provides a method of treating a subject having frontotemporal dementia (FTD) comprising administering to the subject an effective amount of a PIKfyve inhibitor as described herein (or a pharmaceutical composition thereof) and a P- gp inhibitor (or a pharmaceutical composition thereof) to the subject. In embodiments, the PIKfyve inhibitor and the PGP inhibitor are part of separate pharmaceutical compositions. In embodiments, the PIKfyve inhibitor and the PGP inhibitor are part of the same pharmaceutical composition.

[0066] In one embodiment, the invention provides a method of treating a subject having Alzheimer's disease comprising administering to the subject an effective amount of a PIKfyve inhibitor as described herein (or a pharmaceutical composition thereof) and a P-gp inhibitor (or a pharmaceutical composition thereof) to the subject. In embodiments, the PIKfyve inhibitor and the PGP inhibitor are part of separate pharmaceutical compositions. In embodiments, the PIKfyve inhibitor and the PGP inhibitor are part of the same pharmaceutical composition.

[0067] In one embodiment, the invention provides a method of treating a subject having Parkinson's disease comprising administering to the subject an effective amount of a PIKfyve inhibitor as described herein (or a pharmaceutical composition thereof) and a P-gp inhibitor (or a pharmaceutical composition thereof) to the subject. In embodiments, the PIKfyve inhibitor and the PGP inhibitor are part of separate pharmaceutical compositions. In embodiments, the PIKfyve inhibitor and the PGP inhibitor are part of the same pharmaceutical composition.

[0068] In one embodiment, the invention provides a method of treating a subject having Huntington's disease comprising administering to the subject an effective amount of a PIKfyve inhibitor as described herein (or a pharmaceutical composition thereof) and a P-gp inhibitor (or a pharmaceutical composition thereof) to the subject. In embodiments, the Docket No. 4390.3002 WO

[0069] PIKfyve inhibitor and the PGP inhibitor are part of separate pharmaceutical compositions. In embodiments, the PIKfyve inhibitor and the PGP inhibitor are part of the same pharmaceutical composition.

[0070] In one embodiment, the invention provides a method of treating a subject having Charcot-Marie-Tooth disease (CMT) comprising administering to the subject an effective amount of a PIKfyve inhibitor as described herein (or a pharmaceutical composition thereof) and a P-gp inhibitor (or a pharmaceutical composition thereof) to the subject. In embodiments, the PIKfyve inhibitor and the PGP inhibitor are part of separate pharmaceutical compositions. In embodiments, the PIKfyve inhibitor and the PGP inhibitor are part of the same pharmaceutical composition.

[0071] The invention also provides methods for treating a cell proliferative disease, a cancer, or a viral infection in a subject, preferably a human subject, in need of such treatment, by administering an effective amount of a compound of the present invention or a pharmaceutical composition comprising the same, to the subject.

[0072] In one embodiment, the invention provides a method of treating a subject having a viral infection. The method comprises administering to the subject an effective amount of a PIKfyve inhibitor as described herein (or a pharmaceutical composition thereof) and a P-gp inhibitor (or a pharmaceutical composition thereof) to the subject. In embodiments, the PIKfyve inhibitor and the PGP inhibitor are part of separate pharmaceutical compositions. In embodiments, the PIKfyve inhibitor and the PGP inhibitor are part of the same pharmaceutical composition.

[0073] Viral infections include, but are not limited to, infections such as those caused by a virus selected from measles, Ebola virus (EboV), Marburg virus (MarV), borna disease, human immunodeficiency virus (HIV), severe acute respiratory system virus (SARS), middle east respiratory syndrome virus (MERS), JC polyomavirus (JC), BK polyomavirus (BK), Herpes Simplex Virus (HSV), Venezuelan equine encephalitis virus (VEEV) and Lymphocytic choriomeningitis virus (LCMV). The viral infection can be caused by any type of virus such as RNA and DNA viruses. In one embodiment, the virus is Ebola virus. In another embodiment, the virus is middle east respiratory syndrome virus (MERS). In yet another embodiment, the virus is JC polyomavirus (JC). In yet another embodiment, the virus is BK polyomavirus (BK). In yet another embodiment, the virus is Herpes Simplex Virus (HSV). In yet another embodiment, the virus is Marburg virus (MarV). In yet another embodiment, the virus is Venezuelan equine encephalitis virus (VEEV). In yet another embodiment, the virus is Lymphocytic choriomeningitis virus (LCMV). Docket No. 4390.3002 WO

[0074] In one embodiment, the invention provides a method of treating a subject having pain. The method comprises administering to the subject an effective amount of a PIKfyve inhibitor as described herein (or a pharmaceutical composition thereof) and a P-gp inhibitor (or a pharmaceutical composition thereof) to the subject. In embodiments, the PIKfyve inhibitor and the PGP inhibitor are part of separate pharmaceutical compositions. In embodiments, the PIKfyve inhibitor and the PGP inhibitor are part of the same pharmaceutical composition.

[0075] Examples of conditions to be treated include, but are not limited to, nociceptive pain (pain transmitted across intact neuronal pathways), neuropathic pain (pain caused by damage to neural structures), pain from nerve injury (neuromas and neuromas in continuity), pain from neuralgia (pain originating from disease and / or inflammation of nerves), pain from myalgias (pain originating from disease and / or inflammation of muscle), pain associated with painful trigger points, pain from tumors in soft tissues, pain associated with neurotransmitter- dysregulation syndromes (disruptions in quantity / quality of neurotransmitter molecules associated with signal transmission in normal nerves) and pain associated with orthopedic disorders such as conditions of the foot, knee, hip, spine, shoulders, elbow, hand, head and neck.

[0076] In one embodiment, the invention provides a method of treating a subject having an inflammatory disease. The method comprises administering to the subject an effective amount of a PIKfyve inhibitor as described herein (or a pharmaceutical composition thereof) and a P-gp inhibitor (or a pharmaceutical composition thereof) to the subject. In embodiments, the PIKfyve inhibitor and the PGP inhibitor are part of separate pharmaceutical compositions. In embodiments, the PIKfyve inhibitor and the PGP inhibitor are part of the same pharmaceutical composition.

[0077] “Inflammatory disease” in the context of the present teachings encompasses, without limitation, any disease, as defined herein, resulting from the biological response of vascular tissues to harmful stimuli, including but not limited to such stimuli as pathogens, damaged cells, irritants, antigens and, in the case of autoimmune disease, substances and tissues normally present in the body. Examples of inflammatory disease include rheumatoid arthritis (RA), atherosclerosis, asthma, autoimmune diseases, chronic inflammation, chronic prostatitis, glomerulonephritis, hypersensitivities, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, transplant rejection, celiac disease, colitis, irritable bowel syndrome, intestinal hyperplasia, metabolic syndrome, obesity, diabetes, liver disease, Docket No. 4390.3002 WO hepatic steatosis, fatty liver disease, non-alcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH), and vasculitis.

[0078] In one embodiment, the invention provides a method of treating a subject having an autoimmune disease. The method comprises administering to the subject an effective amount of a PIKfyve inhibitor as described herein (or a pharmaceutical composition thereof) and a P- gp inhibitor (or a pharmaceutical composition thereof) to the subject. In embodiments, the PIKfyve inhibitor and the PGP inhibitor are part of separate pharmaceutical compositions. In embodiments, the PIKfyve inhibitor and the PGP inhibitor are part of the same pharmaceutical composition.

[0079] “Autoimmune disease” encompasses any disease, as defined herein, resulting from an immune response against substances and tissues normally present in the body. Examples of suspected or known autoimmune diseases include rheumatoid arthritis, juvenile idiopathic arthritis, seronegative spondyloarthropathies, ankylosing spondylitis, psoriatic arthritis, antiphospholipid antibody syndrome, autoimmune hepatitis, Behcet's disease, bullous pemphigoid, coeliac disease, Crohn's disease, dermatomyositis, Goodpasture's syndrome, Graves' disease, Hashimoto's disease, idiopathic thrombocytopenic purpura, IgA nephropathy, Kawasaki disease, systemic lupus erythematosus, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, polymyositis, primary biliary cirrhosis, psoriasis, scleroderma, Sjogren's syndrome, ulcerative colitis, vasculitis, Wegener's granulomatosis, temporal arteritis, Takayasu's arteritis, Henoch-Schonlein purpura, leucocytoclastic vasculitis, polyarteritis nodosa, Churg-Strauss Syndrome, and mixed cryoglobulinemic vasculitis.

[0080] In one embodiment, the invention provides a method of treating a subject having a lysosomal storage disorder. The method comprises administering to the subject an effective amount of a PIKfyve inhibitor as described herein (or a pharmaceutical composition thereof) and a P-gp inhibitor (or a pharmaceutical composition thereof) to the subject. In embodiments, the PIKfyve inhibitor and the PGP inhibitor are part of separate pharmaceutical compositions. In embodiments, the PIKfyve inhibitor and the PGP inhibitor are part of the same pharmaceutical composition.

[0081] Lysosomal storage diseases / disorders (LSDs) includes over forty metabolic disorders, many of which involve genetic defects in various lysosomal hydrolases. Representative lysosomal storage diseases and the associated defective enzymes (in parenthesis) include, but are not limited to, Aspartylglucosaminuria (Aspartylglucosaminidase), Fabry (alpha. - Galactosidase A), Infantile Batten Disease* (CNL1) (Palmitoyl Protein Thioesterase), Classic Late Infantile Batten Disease* (CNL2) (Tripeptidyl Peptidase), Juvenile Batten Disease* Docket No. 4390.3002 WO

[0082] (CNL3) (Lysosomal Transmembrane Protein), Batten, other forms* (CNL4-CNL8) (Multiple gene products), Cystinosis (Cysteine transporter), Farber (Acid ceramidase), Fucosidosis (Acid.alpha.-L-fucosidase), Galactosidosialidosis (Protective protein / cathepsin A), Gaucher types 1, 2*, and 3* (Acid .beta. -glucosidase, G.sub.Ml gangliosidosis* (Acid .beta.- galactosidase), Hunter* (Iduronate-2-sulfatase), Hurl er- Scheie* (alpha. -L-Iduronidase), Krabbe* (Galactocerebrosidase), alpha.-Mannosidosis* (Acid. alpha. -mannosidase), beta.- Mannosidosis* (Acid.beta. -mannosidase), Maroteaux-Lamy (Arylsulfatase B), Metachromatic leukodystrophy* (Aryl sulfatase A), Morquio A (N-Acetylgalactosamine-6- sulfate), Morquio B (Acid .beta. -galactosidase), Mucolipidosis II / III* (N-Acetylglucosamine- 1-phoshpate transferase) Niemann-Pick A*, B (Acid sphingomyelinase), Niemann-Pick C* (NPC-1), Pompe* (Acid.alpha.-glucosidase), Sandhoff* (.beta.-Hexosaminidase B), Sanfilippo A* (Heparan N-sulfatase), Sanfilippo B* (. alpha. -N-Acetylglucosaminidase), Sanfilippo C* (Acetyl-CoA: alpha. -glucosaminide), Sanfilippo D* (N-Acetylglucosamine-6- sulfate), Schindler Disease* (. alpha. -N-Acetylgalactosaminidase), Schindler-Kanzaki (alpha. - N-Acetylgalactosaminidase), Sialidosis (. alpha. -Neuramidase), Sly* (.beta. -Glucuronidase), Tay-Sachs* (.beta.-Hexosaminidase A), and Wolman* (Acid Lipase). (*CNS involvement).

[0083] In one embodiment, the invention provides a method of treating a subject having cancer. The method comprises administering to the subject an effective amount of a PIKfyve inhibitor as described herein (or a pharmaceutical composition thereof) and a P-gp inhibitor (or a pharmaceutical composition thereof) to the subject. In embodiments, the PIKfyve inhibitor and the PGP inhibitor are part of separate pharmaceutical compositions. In embodiments, the PIKfyve inhibitor and the PGP inhibitor are part of the same pharmaceutical composition.

[0084] In one aspect of the methods described here, the subject in need thereof is a subject having cancer whose cancer is refractory to standard therapy or whose cancer has recurred following standard treatment.

[0085] In one embodiment, the cancer is brain cancer, glioma, sarcoma, breast cancer, lung cancer, non- small-cell lung cancer, mesothelioma, appendiceal cancer, genitourinary cancers, renal cell carcinoma, prostate cancer, bladder cancer, testicular cancer, penile cancer, cervical cancer, ovarian cancer, von Hippel Lindau disease, head and neck cancer, gastrointestinal cancer, hepatocellular carcinoma, gallbladder cancer, esophageal cancer, gastric cancer, colorectal cancer, pancreatic cancer, neuroendocrine tumors, thyroid tumor, pituitary tumor, adrenal tumor, hematological malignancy, or leukemia. In another embodiment, the cancer is a melanoma. Docket No. 4390.3002 WO

[0086] In one embodiment the cancer is a lymphoma. In one embodiment, the lymphoma is a B cell lymphoma. In one embodiment, the B cell lymphoma is selected from the group consisting of a Hodgkin's B cell lymphoma and a non-Hodgkin's B cell lymphoma. In one embodiment, the B cell lymphoma is a non-Hodgkin's B cell lymphoma selected from the group consisting of DLBCL, follicular lymphoma, marginal zone lymphoma (MZL) or mucosa associated lymphatic tissue lymphoma (MALT), small cell lymphocytic lymphoma (overlaps with chronic lymphocytic leukemia) and mantle cell lymphoma. In one embodiment, the B cell lymphoma is a non-Hodgkin's B cell lymphoma selected from the group consisting of Burkitt's lymphoma, Primary mediastinal (thymic) large B-cell lymphoma, Lymphoplasmacytic lymphoma, which may manifest as Waldenstrom macroglobulinemia, Nodal marginal zone B cell lymphoma (NMZL), Splenic marginal zone lymphoma (SMZL), Intravascular large B-cell lymphoma, Primary effusion lymphoma, Lymphomatoid granulomatosis, T cell / histiocyte-rich large B-cell lymphoma, Primary central nervous system lymphoma, Primary cutaneous diffuse large B-cell lymphoma, leg type (Primary cutaneous DLBCL, leg type), EBV positive diffuse large B-cell lymphoma of the elderly, Diffuse large B-cell lymphoma associated with inflammation, Intravascular large B- cell lymphoma, ALK -positive large B-cell lymphoma, and Plasmablastic lymphoma.

[0087] In a preferred embodiment of any of the methods described herein, the subject is a human subject.

[0088] In any of the methods described herein, the method may further comprise also administering an effective amount of a potassium channel activator, an inhibitor of a glutamate receptor (such as the receptor NMD A, AMPA, or kainite) (e.g., AP5, CNQX, and NBQX), or any combination of any of the foregoing.

[0089] In any of the methods described herein, the method may further comprise administering an effective amount of one or more pharmaceutically active agents such as immunostimulatory agents, anti-viral agents, antibiotics, anti-fungal agents, anti-parasitic agents, anti-tumor agents, cytokines, chemokines, growth factors, anti -angiogenic factors, chemotherapeutic agents, antibodies and gene silencing agents. Preferably, the pharmaceutically active agent is selected from the group consisting of an immunostimulatory agent, an anti-bacterial agent, an anti-viral agent, an anti-inflammatory agent and an antitumor agent. The more than one pharmaceutically active agents may be of the same or different category. Docket No. 4390.3002 WO

[0090] In certain embodiments, the one or more pharmaceutically active agents includes but are not limited to, an antigen, an anti-viral vaccine, an anti-bacterial vaccine, and / or an antitumor vaccine, wherein the vaccine can be prophylactic and / or therapeutic.

[0091] According to some of any of the embodiments described herein, in any of the methods and uses as described herein, the method may further comprise an additional therapy for treating the disease associated with a tumor (e.g., cancer).

[0092] According to some of any of the embodiments described herein, in any of the methods and uses as described herein, the additional therapy for treating the disease associated with a tumor is an anti-cancer therapy.

[0093] Suitable anti-cancer therapy includes, for example, chemotherapy, radiotherapy, phototherapy and / or photodynamic therapy, surgery, nutritional therapy, ablative therapy, combined radiotherapy and chemotherapy, brachiotherapy, proton beam therapy, immunotherapy, cellular therapy and photon beam radiosurgical therapy, and any combination of the foregoing.

[0094] Chemotherapeutic drugs (e.g., anti-cancer drugs) that may optionally be coadministered to the subject prior to, concomitant with and / or subsequent to one or more compounds as described herein in any of the respective embodiments include, but are not limited to acivicin, aclarubicin, acodazole, acronine, adozelesin, aldesleukin, altretamine, ambomycin, ametantrone, aminoglutethimide, amsacrine, anastrozole, anthramycin, asparaginase, asperlin, azacitidine, azetepa, azotomycin, batimastat, benzodepa, bicalutamide, bisantrene, bisnafide, bizelesin, bleomycin, brequinar, bropirimine, busulfan, cactinomycin, calusterone, caracemide, carbetimer, carboplatin, carmustine, carubicin, carzelesin, cedefingol, chlorambucil, cirolemycin, cisplatin, cladribine, crisnatol, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, decitabine, dexormaplatin, dezaguanine, diaziquone, docetaxel, doxorubicin, droloxifene, dromostanolone, duazomycin, edatrexate, eflomithine, elsamitrucin, enloplatin, enpromate, epipropidine, epirubicin, erbulozole, esorubicin, estramustine, etanidazole, etoposide, etoprine, fadrozole, fazarabine, fenretinide, floxuridine, fludarabine, fluorouracil, flurocitabine, fosquidone, fostriecin, gemcitabine, hydroxyurea, idarubicin, ifosfamide, ilmofosine, interferon alfa-2a, interferon alfa-2b, interferon alfa-nl, interferon alfa-n3, interferon beta-la, interferon gamma-Ib, iproplatin, irinotecan, lanreotide, letrozole, leuprolide, liarozole, lometrexol, lomustine, losoxantrone, masoprocol, maytansine, mechlorethamine, megestrol, melengestrol, melphalan, menogaril, mercaptopurine, methotrexate, metoprine, meturedepa, mitindomide, mitocarcin, mitocromin, mitogillin, mitomalcin, mitomycin, mitosper, mitotane, Docket No. 4390.3002 WO mitoxantrone, mycophenolic acid, nocodazole, nogalamycin, ormaplatin, oxisuran, paclitaxel, pegaspargase, peliomycin, pentamustine, peplomycin, perfosfamide, pipobroman, piposulfan, piroxantrone, plicamycin, plomestane, porfimer, porfiromycin, prednimustine, procarbazine, puromycin, pyrazofurin, riboprine, rogletimide, safingol, semustine, simtrazene, sparfosate, sparsomycin, spirogermanium, spiromustine, spiroplatin, streptonigrin, streptozocin, sulofenur, talisomycin, tecogalan, tegafur, teloxantrone, temoporfin, teniposide, teroxirone, testolactone, thiamiprine, thioguanine, thiotepa, tiazofurin, tirapazamine, topotecan, toremifene, trestolone, triciribine, trimetrexate, triptorelin, tubulozole, uracil mustard, uredepa, vapreotide, verteporfin, vinblastine, vincristine, vindesine, vinepidine, vinglycinate, vinleurosine, vinorelbine, vinrosidine, vinzolidine, vorozole, zeniplatin, zinostatin, zorubicin, and any pharmaceutically acceptable salts thereof.

[0095] In some embodiments, the anti-cancer therapy comprises immunotherapy, including, for example, checkpoint inhibitors, CAR-T cell therapy, and / or vaccine adjuvants (e.g., interferon or saponin), and immune-adjuvants, such as aluminum salts, organic adjuvants, and genomic material-based adjuvants, such as CpG. Anti-tumor immunity can be further augmented by inhibition of immune suppressor cells.

[0096] According to some embodiments, the anti-cancer therapy comprises administration of an anti-cancer immune modulator agent. As used herein, the term “anti-cancer immune modulator agent” refers to an agent capable of eliciting an immune response (e.g. T cell, NK cell) against a cancerous cell. Exemplary anti -cancer immune modulator agents include, but are not limited to, a cancer antigen, a cancer vaccine, an anti -cancer antibody, a cytokine capable of inducing activation and / or proliferation of a T cell and an immune-check point regulator.

[0097] Alternatively or additionally, such modulators may be immune stimulators such as immune-check point regulators which are of specific value in the treatment of cancer. As used herein the term “immune-check point regulator” refers to a molecule that modulates the activity of one or more immune-check point proteins in an agonistic or antagonistic manner resulting in activation of an immune cell. As used herein the term “immune-check point protein” refers to a protein that regulates an immune cell activation or function. Immune check-point proteins can be either co-stimulatory proteins (i.e. transmitting a stimulatory signal resulting in activation of an immune cell) or inhibitory proteins (i.e. transmitting an inhibitory signal resulting in suppressing activity of an immune cell). According to some embodiments, the immune check-point protein regulates activation or function of a T cell. Numerous checkpoint proteins are known in the art and include, but not limited to, PD1, Docket No. 4390.3002 WO

[0098] PDL-1, B7H2, B7H4, CTLA-4, CD80, CD86, LAG-3, TIM-3, KIR, IDO, CD19, 0X40, 4- 1BB (CD137), CD27, CD70, CD40, GITR, CD28 and ICOS (CD278).

[0099] According to some embodiments, the anti-cancer therapy comprises a surgical procedure, for example, resection or excision of at least a portion of the tumor.

[0100] In any of the methods described herein, administration of the PIKfyve inhibitor and the p-glycoprotein inhibitor may, independently, be by any route by which the PIKfyve inhibitor and the p-glycoprotein inhibitor will be bioavailable in effective amounts including oral and parenteral routes. Although oral administration is preferred, the PIKfyve inhibitor and the p-glycoprotein inhibitors may also be administered intravenously, topically, subcutaneously, intranasally, rectally, intramuscularly, or by other parenteral routes. When administered orally, the PIKfyve inhibitor and the p-glycoprotein inhibitor may be administered in any convenient dosage form including, for example, capsule, tablet, liquid, suspension, and the like.

[0101] Co-Administration

[0102] The term “co-administration” generally refers to the administration of at least two different substances, for example, a PIKfyve inhibitor and a PGP inhibitor, sufficiently close in time to provide the desired response. Co-administration includes sequential, simultaneous or separate administration of the active compound in a way that the therapeutical effects of the first administered one is not entirely disappeared when the subsequent is administered.

[0103] The term “in combination with” generally means in the course of treating the same disease in the same patient and includes administering two or more agents in any order, including co-administration, as well as temporally spaced order from a few seconds up to several days apart. Such combination treatment may also include more than a single administration of one or more of the agents. The administration of the two or more agents may be by the same or different routes.

[0104] In embodiments, the PIKfyve inhibitor is administered to the subject at the same time as the PGP inhibitor. In some embodiments, the PIKfyve inhibitor and the PGP inhibitor are administered concurrently but on different schedules.

[0105] In embodiments, the PIKfyve inhibitor is administered to the subject prior to the PGP inhibitor. For example, the PIKfyve inhibitor can administered to the subject 1 minute to about 60 minutes, 0.5, 1, 2, 3, 4, 5, 10, 12, or 18 hours, 1, 2, 3, 4, 5, or 6 days, or 1, 2, 3, or 4 weeks prior to the administration of the PGP inhibitor. Docket No. 4390.3002 WO

[0106] In embodiments, the PIKfyve inhibitor is administered to the subject after the PGP inhibitor. For example, the PIKfyve inhibitor can administered to the subject 1 minute to about 60 minutes, 0.5, 1, 2, 3, 4, 5, 10, 12, or 18 hours, 1, 2, 3, 4, 5, or 6 days, or 1, 2, 3, or 4 weeks after the administration of the PGP inhibitor.

[0107] PIKfyve Inhibitors

[0108] The present invention relates to compounds useful as inhibitors of phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) as well as their use for treating diseases and disorders associated with PIKfyve. In particular, the PIKfyve inhibitors of the invention comprise a functional group modification that allows for a covalent interaction with PIKfyve. In some embodiments, a compound of the invention forms a covalent bond with an amino acid residue of the PIKfyve protein.

[0109] One of ordinary skill in the art will recognize that certain reactive functional groups can act as “warheads.” As used herein, the term “warhead” or “warhead group” refers to a functional group present on a compound of the present invention wherein that functional group is capable of covalently binding to an amino acid residue (such as cysteine, lysine, histidine, or other residues capable of being covalently modified), present in or near the binding pocket of the PIKfyve target protein, thereby inhibiting the PIKfyve protein. In some embodiments, the covalent binding of the compound of the invention to the amino acid of the PIKfyve protein is reversible. In some embodiments, the covalent binding of the compound of the invention to the amino acid of the PIKfyve protein is irreversible.

[0110] It will be appreciated that in some embodiments the Linker- Warhead group ((Q)p-W), as defined and described herein, provides such warhead groups for covalently, and reversibly, inhibiting the PIKfyve protein.

[0111] It will be appreciated that in some embodiments the Linker- Warhead group ((Q)p-W), as defined and described herein, provides such warhead groups for covalently, and irreversibly, inhibiting the PIKfyve protein.

[0112] In some embodiments, a compound of the invention forms a covalent bond with a sulfur atom of a cysteine residue of the PIKfyve protein. In embodiments, the covalent bonding between the compound of the invention and the cysteine residue of PIKfyve is irreversible.

[0113] Any suitable method for determining a reversible or an irreversible (e.g., covalently reversible or irreversible) interaction between a compound of the invention and the PIKfyve Docket No. 4390.3002 WO protein can be used. Methods for identifying if a compound is acting as a reversible or an irreversible inhibitor are known to one of ordinary skill in the art.

[0114] In one aspect, the invention provides a PIKfyve inhibitor of formula (I): or a pharmaceutically acceptable salt thereof, wherein Ring A is a 5- or 6-membered aromatic ring having 0-3 heteroatoms selected from N, O, or S, preferably 1-3 heteroatoms, preferably 2 heteroatoms, preferably 3 heteroatoms, preferably wherein the heteroatom is N; T is selected from halogen, Ci-Ce alkyl, or a 6-membered aryl having 0-3 heteroatoms selected from N, O, or S, preferably 0, wherein the Ci-Ce alkyl, or the 6-membered aryl is optionally substituted; V is a C1-C3 alkyl, wherein the C1-C3 alkyl is optionally substituted; each Y is independently CRi, N, O, or S, provided at least one Y is N, O, or S (preferably, N), wherein Ri is H, D, halogen (preferably Cl, F), or substituted or unsubstituted Ci-Ce alkyl; X is a 5- to 8-membered heterocyclic ring comprising at least one heteroatom selected from N or O, wherein the ring is optionally substituted, wherein the ring is optionally fused or bridged (e.g., forming a bicyclic ring); L is -Xi-(CR3R4)k-, -(CR3R4)k-, -Xi-(CR3R4)k-Xi-, -(CR3R4)k- Xi-, -(CR3R4)k-Xi-(CR3R4)k-; each Xi is independently selected from -O-, -C(O)-, -C(O)O-, - OCO-, -S-, -S(O)2-, -S(O)-, -S(O)(NR2)-, -NR2-, -NR2C(O)-, and -C(O)NR2-; each occurrence of R3 and R4 are independently H, halogen, nitro, hydroxy, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, or substituted or unsubstituted alkoxy, R2 is hydrogen, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 cycloalkyl, or substituted or unsubstituted C1-6 heterocycloalkyl; alternatively, at least one occurrence of R3 and R4 forms an optionally substituted cycloalkyl or heterocycloalkyl (preferably, oxetane, azetidine, pyrrolidine, or piperidine); alternatively, when Xi comprises -NR2-, any two of one R3, one R4, and R2 forms an optionally substituted heterocycloalkyl; Ring B is a substituted or unsubstituted 5- or 6- membered aromatic ring having 0-3 heteroatoms selected from N, O, or S, preferably 1-3 heteroatoms, preferably 2 heteroatoms, preferably 3 heteroatoms, preferably wherein the heteroatom is N; alternatively, when L comprises -(CR3R4)k, B and one adjacent occurrence Docket No. 4390.3002 WO of R3 forms a bicyclic ring; Q is selected from -C(O)-, -CR3R4-, -O-, -S(O)2-, -NR2-, -C(O)O- , -OC(O)-, C(O)NR2-, -NR2C(O)-, -S(O)-, -S(O)(NR2)-, -OC(O)NR2-, NR2C(O)O-; W is a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, wherein the heteroaryl comprises one or more heteroatoms selected from N, O, or S; m is 0 or 1; j is 0 or 1; p is 0 or 1; and k is 1, 2, 3, 4, 5, or 6.

[0115] In one embodiment, ring A is a 5-membered aromatic ring having 1-3 heteroatoms selected from N, O, or S. Preferably, ring A comprises 2-3 heteroatoms. Preferably, ring A comprises 2 heteroatoms. Preferably, ring A comprises 3 heteroatoms. Preferably, the heteroatom is N. In embodiments, ring A is selected from: squiggly lines indicate the point of attachment to the rest of the molecule).

[0116] In embodiments, ring A is selected from: Docket No. 4390.3002 WO

[0117] In embodiments, Ring A is selected from: In embodiments, Ring A is selected from:

[0118] In some of any of the embodiments described herein, m is 1 and T is substituted or unsubstituted phenyl or substituted or unsubstituted pyridine.

[0119] In some of any of the embodiments described herein, T is phenyl, a halogensubstituted phenyl, an alkyl-substituted phenyl, a halogenated alkyl -substituted phenyl, or an alkoxy-substituted phenyl.

[0120] In some of any of the embodiments described herein, T is selected from phenyl, 3- methylphenyl, 3-fluorophenyl, and 3 -chlorophenyl.

[0121] In some of any of the embodiments described herein, T is pyridine, a halogensubstituted pyridine, an alkyl-substituted pyridine, a halogenated alkyl-substituted pyridine, or an alkoxy-substituted pyridine. Docket No. 4390.3002 WO

[0122] In some of any of the embodiments described herein, T is selected from pyridine, 3- methylpyridine, 3 -fluoro pyridine, and 3 -chloro pyridine.

[0123] In some of any of the embodiments described herein, the ring A moiety, e.g., moiety is selected from: Docket No. 4390.3002 WO

[0124] In some of any of the embodiments described herein, the moiety is selected from: Docket No. 4390.3002 WO

[0125] In some of any of the embodiments described herein, X is selected from: wherein each of the rings above is optionally substituted, and the substituents are independently selected from halogen, -OH, a Ci-4 alkyl, and a Ci-4 alkoxy.

[0126] In some of any of the embodiments described herein, X is selected from: Docket No. 4390.3002 WO

[0127] In some of any of the embodiments described herein, X is: ' /

[0128] In some of any of the embodiments described herein, L is -Xi-(CR3R4)k-.

[0129] In embodiments, L is -O-(CR3R4)I-4-, wherein each R3 and R4 are independently selected from H, D, halogen, Ci-4 alkyl (preferably L is -O-CH2CH2-, or - O-CH2-). In embodiments L is -NR2-(CR3R4)I-4-, wherein each R3 and R4 are independently selected from H, D, halogen, Ci-4 alkyl (preferably L is -NHCH2CH2-, or - NHCH2-).

[0130] In embodiments, L is -O-(CH2)I-4-.

[0131] In some of any of the embodiments described herein, the PIKfyve inhibitor of

[0132] Formula I is a PIKfyve inhibitor of Formula Ila or lib : ), or a pharmaceutically acceptable salt thereof.

[0133] In some of any of the embodiments described herein, Ring B is a 5-membered aromatic ring having 1-3 heteroatoms selected from N, O, or S. Preferably, Ring B comprises 2-3 heteroatoms. Preferably, Ring B comprises 2 heteroatoms. Preferably, Ring B comprises

[0134] 3 heteroatoms. Preferably, the heteroatom is N.

[0135] In embodiments, ring B can be optionally substituted. In embodiments, the substituents are independently selected from halogen, -OH, a Ci-4 alkyl, and a Ci-4 alkoxy.

[0136] In some of any of the embodiments described herein, Ring B is selected from: Docket No. 4390.3002 WO ring B binds to L and wherein where ring B binds to “(Q)p-W”.

[0137] In some of any of the embodiments described herein, Ring B is selected from: Docket No. 4390.3002 WO

[0138] In some of any of the embodiments described herein, Ring B is selected from:

[0139] Docket No. 4390.3002 WO

[0140] In some of any of the embodiments described herein, Ring B is selected from:

[0141] In some of any of the embodiments described herein, Ring B is:

[0142] In some of any of the embodiments described herein, the PIKfyve inhibitor of Formula I is a PIKfyve inhibitor of Formula III: formula (III) or a pharmaceutically acceptable salt thereof; wherein Xi is -NR2-, or O (preferably, -O-); k is 1, 2, 3, or 4; each R3 and R4 are independently selected from H, D, halogen, Ci-4 alkyl; p is 0, or p is 1, Q is -S(O)2-; Yi, Y2, Y3, are each independently selected from CRi, N, O, or S (preferably from CRi, N), provided that two of Yi, Y2 and Y3 are N; RY is halogen, nitrile, alkyl, hydroxyl, or alkoxy; or RY and one of the adjacent R3 and R4 with the atoms to which they are attached form a bicyclic ring system; q is 0, 1, or 2. In some of any of the embodiments described herein, p is 0.

[0143] In some of any of the embodiments described herein, p is 1, Q is -S(O)2-. Docket No. 4390.3002 WO

[0144] In some of any of the embodiments described herein, the PIKfyve inhibitor of formula l is a PIKfyve inhibitor of formula IV: formula (IV) or a pharmaceutically acceptable salt thereof; wherein Zi, Z2, Z3, Z4, and Z5 are each independently selected from CRi, N, O, or S (preferably from CRi, N), provided at least three of Zi, Z2, Z3, Z4, and Z5 are CRi; and Rz is halogen, nitrile, alkyl, or -G-Rx, wherein G is selected from -C(O)-, -CR3R4-, -O-, -S(O)2-, -NR2-, -C(O)O-, -OC(O)-, C(O)NR2-, - NR2C(O)-, -S(O)-, -S(O)(NR2)-, -OC(O)NR2-, NR2C(O)O-; RX is H or a C1-C4 alkyl; o is 0, 1, 2, 3, 4, or 5.

[0145] In some of any of the embodiments described herein, the PIKfyve inhibitor of formula l is a PIKfyve inhibitor of formula Va or Vb:

[0146] (formula Va); Docket No. 4390.3002 WO

[0147] (formula Vb), or a pharmaceutically acceptable salt thereof.

[0148] In some of any of the embodiments described herein, wherein the PIKfyve inhibitor of formula I is a PIKfyve inhibitor of formula Via or VIb: ), or a pharmaceutically acceptable salt thereof. Docket No. 4390.3002 WO

[0149] In some of any of the embodiments described herein, wherein the PIKfyve inhibitor of formula l is a PIKfyve inhibitor of formula Vic or Vid: or a pharmaceutically acceptable salt thereof.

[0150] In some of any of the embodiments described herein, wherein the PIKfyve inhibitor of formula I is PIKfyve inhibitor of formula Vila or Vllb: (formula Vila) Docket No. 4390.3002 WO (formula Vllb), or a pharmaceutically acceptable salt thereof.

[0151] In some of any of the embodiments described herein, wherein the PIKfyve inhibitor of formula l is a PIKfyve inhibitor of formula Villa, Vlllb, VIIIc, or Vllld: u ), Docket No. 4390.3002 WO u ld) or a pharmaceutically acceptable salt thereof; wherein Xi is -NR2-, or O (preferably, -O-); k is 1, 2, 3, or 4; each R3 and R4 are independently selected from H, D, halogen, Ci-4 alkyl; and wherein W is substituted or unsubstituted phenyl, substituted or unsubstituted pyridine, or substituted or unsubstituted pyrimidine.

[0152] Docket No.4390.3002 WO

[0153] In some of any of the embodiments described herein, wherein the PIKfyve inhibitor of formula lisa PIKfyve inhibitor of formula IXa, IXb, IXc, or IXd: ua c Docket No. 4390.3002 WO pharmaceutically acceptable salt thereof; wherein Xi is -NR2- or -O- (preferably, -O-), and Ar is a substituted or unsubstituted aryl ring, preferably a substituted or unsubstituted phenyl or a substituted or unsubstituted pyridinyl, wherein preferred substituents are selected from fluorine, chlorine, and methyl; and wherein W is substituted phenyl, substituted pyridine, or substituted pyrimidine; wherein the phenyl, pyridine, or pyrimidine is substituted with 1-5 substituents selected from halogen, nitrile, -S(O)2-Rx, wherein Rx is H or a C1-C4 alkyl (preferably, methyl).

[0154] In another aspect, the PIKfyve inhibitor is a compound of formula (X): or a pharmaceutically acceptable salt thereof, wherein R2 is independently selected from hydrogen, D, halogen, a substituted or unsubstituted alkyl, a substituted or unsubstituted alkoxy, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, and a substituted or unsubstituted heteroalkyl; R3 is selected from substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein the aryl or heteroaryl can be optionally fused or bridged; R4 and Rs are each independently selected from hydrogen, D, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted fluruoalkyl, and -CN; or R4 and Rs can be taken together with the carbons to which they are Docket No. 4390.3002 WO attached to form a substituted or unsubstituted 4, 5, 6, 7, or 8 membered ring; Q is selected from absent, -C(O)-, -CRBRC-, -S(O)2-, -S(O)-, -NRA-, -C(O)O-, -NRAC(O)-, -C(O)-CRBRC-, and -C(O)NRA-; RB and Rc are independently H, halogen, a substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, or substituted or unsubstituted alkoxy, and RA is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted haloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl; or RB and Rc can be taken together with the atom to which they are attached to form a substituted or unsubstituted 4, 5, 6, 7, or 8 membered ring; W is a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted aryl or a substituted or unsubstituted heteroaryl; each Rz is independently selected from D, halogen, -CN, a substituted or unsubstituted alkyl, a substituted or unsubstituted alkoxy, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, and a substituted or unsubstituted heteroalkyl; alternatively, (Rz)n, together with the heterocyclic ring to which Rzis attached, forms a bridged or fused ring structure; and n is 0, 1, 2, 3, or 4.

[0155] In embodiments of the PIKfyve inhibitor of Formula X, R.2 is hydrogen, a substituted or unsubstituted alkyl, a substituted or unsubstituted alkoxyl, a substituted or unsubstituted heterocycloalkyl, or a substituted or unsubstituted heteroalkyl.

[0156] In embodiments of the PIKfyve inhibitor of Formula X, R2is H, a Ci-6 alkyl, a substituted or unsubstituted heterocycloalkyl (e.g., oxetane, azetidine), -Ci-6 alkyl-ORA, -Ci-6 alkyl-NRsRc, or -Ci-6 alkyl-C(O)NRBRc; wherein RAIS hydrogen, Ci-4 alkyl, Ci-4 fluoroalkyl, a C3-8 cycloalkyl, a heterocycloalkyl (e.g., oxetane, azetidine), an aryl; RB and Rc are independently selected from H, Ci-4 alkyl, Ci-4 fluoroalkyl; or RB and Rc can be taken together with the nitrogen to which they are attached to form a substituted or unsubstituted 4, 5, 6, 7, or 8 membered ring.

[0157] In embodiments of the PIKfyve inhibitor of Formula X, R2is -C1-4 alkyl-ORA; wherein RAIS H, -CH3, -CF3, -C2H5, -C3H7, -C(CH )3, -CHF2, or a C3-6 cycloalkyl.

[0158] In embodiments of the PIKfyve inhibitor of Formula X, R2is -C1-6 alkyl-NRsRc, RB and Rc are independently selected from H, Ci-4 alkyl, Ci-4 fluoroalkyl, or RB and Rc forms a Docket No. 4390.3002 WO substituted or unsubstituted azetidine ring, wherein the substituents are selected from Ci-4 alkyl and halogen.

[0159] In any of the preceding embodiments of the PIKfyve inhibitor of Formula X, R3 is a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl.

[0160] In any of the preceding embodiments of the PIKfyve inhibitor of Formula X, R3 is a phenyl. In any of the preceding embodiments, R3 is a substituted phenyl, selected from halogen (preferably F, Cl), Ci-4 alkyl (preferably methyl or ethyl), Ci-4 fluoroalkyl, -ORA, - NRBRC, -NRAC(O)RB, -C(O)NRBRC, wherein RA, RB, and Rc are independently selected from H and a Ci-4 alkyl.

[0161] In any of the preceding embodiments of the PIKfyve inhibitor of Formula X, R3 is selected from one of the following by removal of a hydrogen atom: wherein each of these groups is unsubstituted, or substituted with one to three groups selected from halogen (preferably F, Cl), Ci-4 alkyl (preferably methyl or ethyl), Ci-4 fluoroalkyl, - ORA, -NRBRC, -NRAC(O)RB, -C(O)NRBRC, a C3-8 cycloalkyl, a heterocycloalkyl; wherein RA, RB, and Rc are independently selected from H and a Ci-4 alkyl.

[0162] In any of the preceding embodiments of the PIKfyve inhibitor of Formula X, R3 is substituted or unsubstituted pyridine, substituted or unsubstituted indole, substituted or unsubstituted pyrrol opyrr ole, substituted or unsubstituted thiophene, wherein the substituents are selected from F, Cl, methyl, ethyl, -CF3, -OH, -NH2, -OC1-4 alkyl, -N(CI-4 alkyl)2, - NHC(O)CH3, -C(O)NHCH3, -C(O)NH2, a 5- or 6-membered heterocycloalkyl comprising at least one nitrogen ring atom, such as: Docket No. 4390.3002 WO

[0163] In any of the preceding embodiments of the PIKfyve inhibitor of Formula X, R4 is selected from hydrogen or halogen. In any of the preceding embodiments, Rs is selected from hydrogen or halogen.

[0164] In any of the preceding embodiments of the PIKfyve inhibitor of Formula X, Q is absent, -C(O)-, -S(O)2-, -S(O)-.

[0165] In any of the preceding embodiments of the PIKfyve inhibitor of Formula X, W is a substituted or unsubstituted aryl or a substituted or unsubstituted heteroaryl.

[0166] In any of the preceding embodiments of the PIKfyve inhibitor of Formula X, W is a substituted or unsubstituted phenyl or a substituted or unsubstituted 5- or 6-membered heteroaryl, wherein the heteroaryl comprises at least one nitrogen ring atom.

[0167] In any of the preceding embodiments of the PIKfyve inhibitor of Formula X, W is a substituted or unsubstituted phenyl, a substituted or unsubstituted pyridine, a substituted or unsubstituted pyridazine, a substituted or unsubstituted pyrimidine, a substituted or unsubstituted pyrazine, a substituted or unsubstituted 1,2,4-triazine, or a substituted or unsubstituted 1,3,5-triazine.

[0168] In any of the preceding embodiments of the PIKfyve inhibitor of Formula X, W is a substituted or unsubstituted phenyl, a substituted or unsubstituted pyridine, or a substituted or unsubstituted pyrimidine, which, when substituted, comprises one, two, three, four, or five substituents selected from halogen (preferably, F, Cl), a haloalkyl (preferably a fluoroalkyl such as -CF3), a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkoxy, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heterocycloalkyl, -CN, -CONRDRE, -COORF, -NRDC(O)RF, -C(O)NRD-CI-S alkyl-NRDC(O)RF, -NRDC(O)-CI-8alkyl-CONRoRs, -C(O)NRD-CI-8 alkyl-CONRoRs, - NRDC(O)-CI-8 alkyl-NRoC(O)RF, a substituted or unsubstituted (PEG)I-2O, -C(O)NRD-CI-S alkyl-NRDC(0)-(PEG)i-2o-NRDC(0)RF, -NRDC(O)-CI-8 alkyl-C(0)NRD-(PEG)i-2o- CONRDRE; wherein RD, RE, and RF are independently selected from H, D, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted (PEG)I-2O; or RD and RE can be taken together with the nitrogen to which they are attached to form a substituted or unsubstituted 4, 5, 6, 7, or 8 membered ring; wherein (PEG)i -20 represents a polyethylene glycol that comprises 1 to 20 repeat unit (-CH2CH2O-).

[0169] In any of the preceding embodiments of the PIKfyve inhibitor of Formula X, W is a substituted phenyl, a substituted pyridine, or a substituted pyrimidine, comprising one, two, Docket No. 4390.3002 WO three, four, or five substituents selected from F, Cl, -Ci-6 alkyl, -C3-8 cycloalkyl, -3- to 8- membered heterocycloalkyl, -C1-6 alkenyl, -C1-6 alkoxy, -C1-6 fluoroalkyl, -C3-8 fluorosubstituted cycloalkyl, a 3- to 8-membered fluoro-substituted heterocycloalkyl, -CONRDRE, - CN, -C1-6 fluoro-substituted alkoxy, -C(O)NRD-CI-4 alkyl-NRDC(O)-(PEG)i-8-NRDC(O)Rr, RD, RE, and RF are independently H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl.

[0170] In any of the preceding embodiments of the PIKfyve inhibitor of Formula X, W is a substituted phenyl, substituted by one to five substituents selected from halogen (preferably F, Cl), Ci-4 alkyl (preferably methyl or ethyl), Ci-4 fluoroalkyl, -CF3, -OCF3, -CN, -CH=CH2, -CONRDRE, -C3-6 cycloalkyl (such as cyclopropyl), -3- to 6-membered heterocycloalkyl (such as oxetane), a fluoro-substituted -C3-6 cycloalkyl (such as fluoro-substituted cyclopropyl), a fluoro-substituted 3- to 6-membered heterocycloalkyl (such as a fluoro- substituted oxetane); wherein RD and RE are independently H, Ci-4 alkyl, or Ci-4 fluoroalkyl.

[0171] In any of the preceding embodiments of the PIKfyve inhibitor of Formula X, W is a substituted pyridine, substituted by one to five substituents selected from halogen (preferably F, Cl), Ci-4 alkyl (preferably methyl or ethyl), Ci-4 fluoroalkyl, -CF3, -OCF3, -CN, -CH=CH2, -CONRDRE, -C3-6 cycloalkyl (such as cyclopropyl), -3- to 6-membered heterocycloalkyl (such as oxetane), a fluoro-substituted -C3-6 cycloalkyl (such as fluoro-substituted cyclopropyl), a fluoro-substituted 3- to 6-membered heterocycloalkyl (such as a fluoro- substituted oxetane); wherein RD and RE are independently H, Ci-4 alkyl, or Ci-4 fluoroalkyl.

[0172] In any of the preceding embodiments of the PIKfyve inhibitor of Formula X, W is a substituted pyrimidine, substituted by one to five substituents selected from halogen (preferably F, Cl), Ci-4 alkyl (preferably methyl or ethyl), Ci-4 fluoroalkyl, -CF3, -OCF3, -CN, -CH=CH2, -CONRDRE, -C3-6 cycloalkyl (such as cyclopropyl), -3- to 6-membered heterocycloalkyl (such as oxetane), a fluoro-substituted -C3-6 cycloalkyl (such as fluoro- substituted cyclopropyl), a fluoro-substituted 3- to 6-membered heterocycloalkyl (such as a fluoro-substituted oxetane); wherein RD and RE are independently H, Ci-4 alkyl, or Ci-4 fluoroalkyl.

[0173] In any of the preceding embodiments of the PIKfyve inhibitor of Formula X, at least one of the substituents is -CONH-C1-4 alkyl-NHC(O)RF-(PEG)i-8-NHC(O)RF, wherein RF comprises a fused ring structure.

[0174] In any of the preceding embodiments of the PIKfyve inhibitor of Formula X, each Rz is independently selected from halogen, -CN, a substituted or unsubstituted alkyl, a substituted or unsubstituted alkoxy; and n is 1, 2, 3, or 4. Docket No. 4390.3002 WO

[0175] In any of the preceding embodiments of the PIKfyve inhibitor of Formula X, each Rz is independently selected from halogen (preferably F, Cl) and Ci-4 alkyl (preferably methyl or ethyl).

[0176] In any of the preceding embodiments of the PIKfyve inhibitor of Formula X, n is 0.

[0177] In any of the preceding embodiments, the PIKfyve inhibitor forms a covalent bond with an amino acid residue in the PIKfyve protein.

[0178] In any of the preceding embodiments, the PIKfyve inhibitor forms a covalent bond with a sulfur atom of a cysteine residue in the PIKfyve protein.

[0179] In any of the preceding embodiments, the PIKfyve inhibitor is an irreversible inhibitor of the PIKfyve protein.

[0180] Permeability-glycoprotein (also referred to herein as “p-glycoprotein”, “PGP” or “P- gP”)

[0181] P-glycoprotein ((also known as multidrug resistance protein 1 (MDR1) or ATP - binding cassette sub-family B member 1 (ABCBl)) plays a crucial role in determining response against medications, including cancer therapeutics by acting as an ATP dependent pump that pumps out small molecules from cells. PGP is widely expressed in a variety of organs including the liver, kidney and gastrointestinal tract, and Upregulation of PGP is closely linked with increased chemoresistance in cancer.

[0182] As used herein, the term “p-glycoprotein (PGP) inhibitor” refers to compounds which inhibit the activity of the p-glycoprotein mediated active transport system present in the gut. This transport system actively transports drugs which have been absorbed from the intestinal lumen and into the gut epithelium back out into the lumen. Inhibition of this p-glycoprotein mediated active transport system will cause less drug to be transported back into the lumen and will thus increase the net drug transport across the gut epithelium and will increase the amount of drug ultimately available in the blood.

[0183] A number of known non-cytotoxic pharmacological agents have been shown to inhibit PGP are disclosed in, for example, U.S. Pat. Nos. 6,451,815, 6,469,022, and 6,171,786.

[0184] These patents are incorporated herein by reference.

[0185] PGP inhibitors include water soluble forms of vitamin E, polyethylene glycol, pol oxamers including Pluronic F-68, polyethylene oxide, polyoxyethylene castor oil derivatives including Cremophor EL and Cremophor RH 40, Chrysin, (+)-Taxifolin, Naringenin, Diosmin, Quercetin, cyclosporin A (also known as cyclosporine), verapamil, tamoxifen, quinidine, zosuquidar, tariquidar, phenothiazines, and 9,10-dihydro-5-methoxy-9- Docket No. 4390.3002 WO oxo-N-[4-[2-(l,2,3,4-tetrahydro-6,7,-dimethoxy-2-isoquinolinyl)ethyl]phenyl]-4- acridinecarboxamide or a salt thereof.

[0186] Polyethylene glycols (PEGs) are liquid and solid polymers of the general formula H(OCH2CH2)nOH, where n is greater than or equal to 4, having various average molecular weights ranging from about 200 to about 20,000. In embodiments, the polyethylene glycols for use in the present invention are PEG 200, PEG 400, PEG 600, PEG 1000, PEG 1450, PEG 1500, PEG 4000, PEG 4600, and PEG 8000.

[0187] Polysorbate 80 is an oleate ester of sorbitol and its anhydrides copolymerized with approximately 20 moles of ethylene oxide for each mole of sorbitol and sorbitol anhydrides. Polysorbate 80 is made up of sorbitan mono-9-octadecanoate poly(oxy-l,2-ethanediyl) derivatives. Polysorbate 80, also known as Tween 80, is well known and appreciated in the pharmaceutical arts and is readily available.

[0188] Water-soluble vitamin E, also known as d-alpha-tocopheryl polyethylene glycol 1000 succinate [TPGS], is a water-soluble derivative of natural -source vitamin E. TPGS may be prepared by the esterification of the acid group of crystalline d-alpha-tocopheryl acid succinate by polyethylene glycol 1000. This product is well known and appreciated in the pharmaceutical arts and is readily available. For example, a water-soluble vitamin E product is available commercially from Eastman Corporation as Vitamin E TPGS.

[0189] Naringenin is the bioflavonoid compound 2,3 -dihydro-5, 7-dihydroxy-2-(4- hydroxyphenyl)-4H-l-benzopyran-4-one and is also known as 4',5,7-trihydroxyflavanone. Naringenin is the aglucon of naringen which is a natural product found in the fruit and rind of grapefruit. Naringenin is readily available to the public from commercial sources.

[0190] Quercetin is the bioflavonoid compound 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy- 4H-l-benzopyran-4-one and is also known as 3,3 ',4', 5,7-pentahydroxyflavone. Quercetin is the aglucon of quercitrin, of rutin and of other glycosides. Quercetin is readily available to the public from commercial sources.

[0191] Diosmin is the naturally occurring flavonic glycoside compound 7-[[6-O-6-deoxy- alpha-L-mannopyranosyl)-beta-D-glucopyranosyl]oxy]-5-hydroxy-2-(3-hydroxy-4- methoxyphenyl)-4H-l-benzopyran-4-one. Diosmin can be isolated from various plant sources including citrus fruits. Diosmin is readily available to the public from commercial sources.

[0192] Chrysin is the naturally occurring compound 5,7-dihydroxy-2-phenyl-4H-l- benzopyran-4-one which can be isolated from various plant sources. Chrysin is readily available to the public from commercial sources. Docket No. 4390.3002 WO

[0193] Poloxamers are alpha-hydro-omega-hydroxypoly(oxyethylene)poly (oxypropylene)poly(oxyethylene) block copolymers. Poloxamers are a series of closely related block copolymers of ethylene oxide and propylene oxide conforming to the general formula HO(C2H4O)a(C3HeO)b(C2H4O)aH. For example, poloxamer 124 is a liquid with “a” being 12, “b” being 20, and having an average molecular weight of from about 2090 to about 2360. Poloxamers are well known and appreciated in the pharmaceutical arts and are readily available commercially.

[0194] Polyethylene oxide is a nonionic homopolymer of ethylene oxide conforming to the general formula (OCH2CH2)n in which n represents the average number of oxy ethylene groups. Polyethylene oxides are available in various grades which are well known and appreciated by those in the pharmaceutical arts and several different types of material are commercially available. The preferred grade of polyethylene oxide is NF and the like which are commercially available.

[0195] (+)-Taxifolin is (2R-trans)-2-(3,4-dihydroxyphenyl)-2,3-dihydro-3,5,7-trihydroxy-4H- 1 -benzo pyran-4-one. Other common names for (+)-taxifolin are (+)-dihydroquercetin; 3,3 ',4', 5, 7-pentahydroxy -flavanone; diquertin; taxifoliol; and distylin. (+)-Taxifolin is well known and appreciated in the art of pharmaceutical arts and is readily available commercially.

[0196] 9,10-dihydro-5-methoxy-9-oxo-N-[4-[2-(l,2,3,4-tetrahydro-6,7,-dimethoxy-2- isoquinolinyl)ethyl]phenyl]-4-acridinecarboxamide is also known as GF120918 and Elacridar. Elacridar is sometimes written as N-4-[2-(l ,2,3,4-tetrahydro-6,7-dimethoxy-2- isoquinolinyl)ethyl]-phenyl)-9,10- dihydro-5-methoxy- 9-oxo-4-acridine carboxamide. Elacridar was originally described as a P-gp selective inhibitor but is now recognized as a dual P-gp / BCRP inhibitor. (Matsson P, Pedersen JM, Norinder U, Bergstrom CA, and Artursson P 2009 Identification of novel specific and general inhibitors of the three major human ATP -binding cassette transporters P-gp, BCRP and MRP2 among registered drugs. Pharm Res 26: 1816-1831). Elacridar has the structure: Docket No. 4390.3002 WO

[0197] Zosuquidar (also known as zosuquidar trihydrochloride or LY335979) was developed as a potent and selective inhibitor against p-gp, with minimal inhibitory effect on other MDR proteins (MRP1, MRP2). Zosuquidar has the structure:

[0198] Tariquidar (also referred to as XR9576) is a potent and specific inhibitor of p-gp, acting as an ATPase inhibitor. Tariquidar has the structure:

[0199] In certain embodiments, the P-GP inhibitor selected from the group consisting of alfentanil, amiloride, amiodarone, amitriptyline, astemizole, atovaquone, atorvastatin, azelastine, azidopine, azithromycin, bepidil, biricodar, bromocriptine, carbamazepine, Docket No. 4390.3002 WO carvedilol, chloroquine, chlorpromazine, clarithromycin, cyclosporin, cyproheptadine, darunavir, desethylamiodarone, desipramine, dexniguldipine, dexrazoxane, diltiazem, dipyridamole, disulfiram, doxazosin, elacridqr, emetine, erythromycin, felodipine, fenofibrate, fentanyl, flavonoids, fluoxetine, fluphenazine, fluvoxamine, fucidin, gallpamil, glyburide, gramicidin D, grapefruit juice, garlic, green tea (catechins), haloperidol, hydrocortisone, hyroxyzine, josamycin, ketoconazole, imipramine, itraconazole, ivermectin, ketoconazole, laniquidar, lansoprazole, levothyroxin, lidocaine, loperamide, lopinavir-acute, loratadine, lovastatin, maprotiline, mefloquine, methadone, mibefradil, midazolam, mitomycin C, nefazodone, nelfinavir, nicardipine, nitrendipine, nobilitin, norverapamil, omeprazole, orange juice-Seville, ofloxacin, paroxetine, phenothiazines, piperine, pimozide, probenecid, progesterone, promethazine, propafenone, propranolol, quercetin, quinacrine, quinidine, quinine, reserpine, ritonavir, saquinavir, sertraline, simvastatin, spironolactone, sufentanil, tacrolimus, tamoxifen, tariquidar, telithromycin, terfenadine, testosterone, tetrabenzine, thioridazine, trifluoperazine, trifluopromazine, trimipramine, valinomycin, vanadate, venlafaxine, verapamil, vinblastine, FK506, RU486 (mifepristone), Valspodar (PSC 833), zosuquidar, 2npropylquinoline, and ONT-093.

[0200] In certain embodiments, the efflux inhibitor is a dual BCRP and P-GP inhibitor. In certain embodiments, the efflux inhibitor is selected from the group consisting of elacridar, biricodar, pantoprazole, and tariquidar. In certain embodiments, the efflux inhibitor is elacridar.

[0201] Pharmaceutical Composition

[0202] One or more compounds according to the invention can be part of a pharmaceutical composition suitable for use in a subject, such as a human. The pharmaceutical composition may comprise at least one pharmaceutically acceptable excipient or carrier. The pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally. Docket No. 4390.3002 WO

[0203] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals with toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0204] The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.

[0205] The pharmaceutical composition may also include at least one additional active agent or therapy. Such agent or therapy may be useful for treating or preventing the disease or condition and does not diminish the activity of the compound according to the invention. Agent(s) useful for treating or preventing the disease or condition include, but are not limited to, small molecules, peptides vaccines, antigens, antibodies, preferably monoclonal antibodies, cytotoxic agents, kinase inhibitors, allergens, antibiotics, siRNA molecules, antisense oligonucleotides, TLR antagonist (e.g. antagonists of TLR3 and / or TLR7 and / or antagonists of TLR8 and / or antagonists of TLR9), chemotherapeutic agents (both traditional chemotherapy and modem targeted therapies), targeted therapeutic agents, activated cells, peptides, proteins, gene therapy vectors, peptide vaccines, protein vaccines, DNA vaccines, Docket No. 4390.3002 WO adjuvants, and co-stimulatory molecules (e.g. cytokines, chemokines, protein ligands, transactivating factors, peptides or peptides comprising modified amino acids), or combinations thereof. In embodiments, the agent can be an alkylating agent, an intercalating agent, a tubulin binding agent, a corticosteroid, or any combination of any of the foregoing. Examples of additional active agents include, but are not limited to, ibrutinib, rituximab, doxorubicin, prednisolone, vincristine, velcade, and everolimus, In one embodiment, the at least one additional active agent is a therapeutic agent selected from cyclophosphamide, hydroxydaunorubicin (also referred to as doxorubicin) vincristine, prednisone, prednisolone, and any combination of any of the foregoing.

[0206] A pharmaceutical composition can be provided as a dosage unit form, such as an ampoule, a vial, a suppository, a dragee, a tablet, or a capsule. The pharmaceutical compositions can take any suitable form (e.g., liquids, aerosols, solutions, inhalants, mists, sprays; or solids, powders, ointments, pastes, creams, lotions, gels, patches and the like) for administration by any desired route (e.g., pulmonary, inhalation, intranasal, oral, buccal, sublingual, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, intrapleural, intrathecal, transdermal, transmucosal, rectal, and the like). For example, a pharmaceutical composition of the invention may be in the form of an aqueous solution or powder for aerosol administration by inhalation or insufflation (either through the mouth or the nose), in the form of a tablet or capsule for oral administration, in the form of a sterile aqueous solution or dispersion suitable for administration by either direct injection or by addition to sterile infusion fluids for intravenous infusion, or in the form of a lotion, cream, foam, patch, suspension, solution, or suppository for transdermal or transmucosal administration.

[0207] A pharmaceutical composition can be in the form of an orally acceptable dosage form including, but not limited to, capsules, tablets, buccal forms, troches, lozenges, and oral liquids in the form of emulsions, aqueous suspensions, dispersions or solutions. Capsules may contain mixtures of a compound of the present invention with inert fillers and / or diluents such as the pharmaceutically acceptable starches (e.g., com, potato or tapioca starch), sugars, artificial sweetening agents, powdered celluloses, such as crystalline and microcrystalline celluloses, flours, gelatins, gums, etc. In the case of tablets for oral use, carriers which are commonly used include lactose and com starch. Lubricating agents, such as magnesium stearate, can also be added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions and / or emulsions are administered orally, the compound of the present invention may be suspended or dissolved in an oily phase Docket No. 4390.3002 WO is combined with emulsifying and / or suspending agents. If desired, certain sweetening and / or flavoring and / or coloring agents may be added.

[0208] A pharmaceutical composition can be in the form of a tablet. The tablet can comprise a unit dosage of a compound of the present invention together with an inert diluent or carrier such as a sugar or sugar alcohol, for example lactose, sucrose, sorbitol or mannitol. The tablet can further comprise a non-sugar derived diluent such as sodium carbonate, calcium phosphate, calcium carbonate, or a cellulose or derivative thereof such as methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, and starches such as corn starch. The tablet can further comprise binding and granulating agents such as polyvinylpyrrolidone, disintegrants (e.g. swellable crosslinked polymers such as crosslinked carboxymethylcellulose), lubricating agents (e.g. stearates), preservatives (e.g. parabens), antioxidants (e.g. BHT), buffering agents (for example phosphate or citrate buffers), and effervescent agents such as citrate / bicarbonate mixtures.

[0209] Dosage

[0210] Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0211] In particular, the selected dosage level will depend upon a variety of factors including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well, known in the medical arts.

[0212] A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could begin administration of the pharmaceutical composition to dose the disclosed compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0213] In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of the disclosed compound calculated Docket No. 4390.3002 WO to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the disclosed compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding / formulating such a disclosed compound for the treatment of a patient in need thereof.

[0214] Examples

[0215] Preparation of the Compounds

[0216] Compounds of the present invention can be prepared in a variety of ways using commercially available starting materials, compounds known in the literature, or from readily prepared intermediates, by employing standard synthetic methods and procedures either known to those skilled in the art, or which will be apparent to the skilled artisan in light of the teachings herein. Standard synthetic methods and procedures for the preparation of organic molecules and functional group transformations and manipulations can be obtained from the relevant scientific literature or from standard textbooks in the field. The following descriptions of synthetic methods are designed to illustrate, but not to limit, general procedures for the preparation of compounds of the present invention.

[0217] Compounds of the present invention can be conveniently prepared by a variety of methods familiar to those skilled in the art. The compounds of this invention with each of the formulae described herein may be prepared according to the following procedures from commercially available starting materials or starting materials which can be prepared using literature procedures. These procedures show the preparation of representative compounds of this invention.

[0218] The synthetic processes of the invention can tolerate a wide variety of functional groups; therefore various substituted starting materials can be used. The processes generally provide the desired final compound at or near the end of the overall process, although it may be desirable in certain instances to further convert the compound to a pharmaceutically acceptable salt, polymorph, hydrate, solvate or co-crystal thereof.

[0219] Synthesis of PIKfyve inhibitors of Formulas I through IX Docket No. 4390.3002 WO

[0220] Abbreviations

[0221] DCM - dichloromethane

[0222] EtOAc - ethyl acetate h - hour(s) MeOH - methanol min - minute(s)

[0223] LCMS Methods

[0224] Method C Docket No. 4390.3002 WO

[0225] Method-C Fast

[0226] Method H Docket No. 4390.3002 WO

[0227] Method J Docket No. 4390.3002 WO

[0228] Example 1: 4-(2-(2-(l-(phenylsulfonyl)-lH-pyrazol-4-yl)ethoxy)-6-(3-(m-tolyl)-lH- pyrazol-l-yl)pyrimidin-4-yl)morpholine

[0229] To a stirred solution of 4-(2-(2-(lH-pyrazol-4-yl)ethoxy)-6-(3-(m-tolyl)-lH-pyrazol-l- yl)pyrimidin-4-yl)morpholine (WO2021163727 Al) (0.100 g, 0.232 mmol) in DCM (2 mL) was added triethylamine (0.1 mL, 0.464 mmol) at 0 °C under nitrogen atmosphere. After 5 min benzenesulfonyl chloride (0.082 g, 0.696 mmol) was added at 0 °C. The reaction mixture was stirred at room temperature for 2 h. After reaction completion, solvent was removed in vacuum to obtain crude product. The resulting crude was purified by silica gel flash column chromatography. The desired product was eluted at 40 % EtOAc in hexanes to obtain title compound as off white solid (0.056 g, 0.098 mmol, 42 %).

[0230] 'H NMR (400 MHz, DMSO-d6): 82.38 (s, 3H), 2.90 (t, 2H), 3.55-3.80 (m, 8H), 4.48 (t, 2H), 6.88 (s, 1H), 7.06 (d, 1H), 7.20-7.25 (m, 1H), 7.30-7.40 (m, 1H), 7.62-7.66 (m, 2H), 7.75-7.81 (m, 3H), 7.93-7.95 (m, 3H), 8.38 (s, 1H), 8.60 (d, 1H).

[0231] LCMS (Method-C): Retention time: 2.073 min, ES(+ve): 572.3 [M+H]+

[0232] Example 2: 4-(2-(2-(l-((perfluorophenyl)sulfonyl)-lH-pyrazol-4-yl)ethoxy)-6-(3-(m- tolyl)-lH-pyrazol-l-yl)pyrimidin-4-yl)morpholine

[0233] To a stirred solution of 4-(2-(2-(lH-pyrazol-4-yl)ethoxy)-6-(3-(m-tolyl)-lH-pyrazol-l- yl)pyrimidin-4-yl)morpholine (WO2021163727 Al) (0.100 g, 0.232 mmol) in DCM (3 mL), was added pyridine (0.1 mL, 1.159 mmol) at 0 °C under nitrogen atmosphere. After 5 min, 2,3,4,5,6-pentafluorobenzenesulfonyl chloride (0.123 g, 0.464 mmol) was added at 0°C. The reaction mixture was stirred at room temperature for 1 h. After completion of reaction, the reaction mixture was quenched with 17V HC1 (10 mL) and extracted with DCM (3 x 30 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered and the solvent Docket No. 4390.3002 WO was removed in vacuum to obtain crude product. The resulting crude was purified by silica gel flash column chromatography. The desired product was eluted at 50 % EtOAc in hexanes to obtain title compound as off white solid (0.056 g, 0.085 mmol, 37 %).

[0234] 'H NMR (400 MHz, DMSO-d6): 6 2.39 (s, 3H), 2.97 (t, 2H), 3.63-3.77 (m, 8H), 4.53 (t, 2H), 6.90 (s, 1H), 7.06 (d, 1H), 7.22 (d, 1H), 7.35 (t, 1H), 7.78 (d, 1H), 7.82 (s, 1H), 8.08 (s, 1H), 8.50 (s, 1H), 8.60 (d, 1H).

[0235] 19F NMR (400 MHz, DMSO-d6): 6 - 158.25 - 158.39 (2F), -141.26_-141.38 (IF), -136.40_- 136.48 (2F).

[0236] LCMS (Method-C): Retention time: 2.181 min, ES(+ve): 662.3 [M+H]+

[0237] Example 3: 4-((4-(2-((4-morpholino-6-(3-(m-tolyl)-lH-pyrazol-l-yl)pyrimidin-2- yl)oxy)ethyl)-lH-pyrazol-l-yl)sulfonyl)benzonitrile

[0238] Prepared by the method of Example 2 using 4-cyanobenzenesulfonyl chloride (0.093 g, 0.464 mmol). Yield (0.066 g, 0.110 mmol, 48 %).

[0239] 'H NMR (400 MHz, DMSO-d6): 8 2.33 (s, 3H), 2.91 (t, 2H), 3.60-3.68 (m, 8H), 4.49 (t, 2H), 6.88 (s, 1H), 7.06 (d, 1H), 7.21 (d, 1H), 7.35 (t, 1H), 7.78 (d, 1H), 7.82 (s, 1H), 7.96 (s, 1H), 8.11 (s, 4H), 8.44 (s, 1H), 8.59 (d, 1H).

[0240] LCMS (Method-C): Retention time: 2.053 min, ES(+ve): 597.3 [M+H]+

[0241] Example 4: 2,6-difluoro-4-((4-(2-((4-morpholino-6-(3-(m-tolyl)-lH-pyrazol-l- yl)pyrimidin-2-yl)oxy)ethyl)-lH-pyrazol-l-yl)sulfonyl)benzonitrile

[0242] Step 1: Synthesis of 4-cyano-3,5-difluorobenzenesulfonyl chloride Docket No. 4390.3002 WO

[0243] Thionyl chloride (0.48 mL, 6.481 mmol) was added to water (10 mL) at 0 °C, keeping the temperature at 0 °C (very exothermic, slow and careful addition with dropping funnel). The mixture was allowed to warm to room temperature for 1 h after which CuCl (16 mg, 0.161 mmol, 0.05 eq) was added and the solution was again cooled to 0 °C (reaction mixture-1). Separately, concentrated hydrochloric acid (5 mL, 36 % w / w) was added to 4-amino-2,6- difluorobenzonitrile (500 mg, 3.244 mmol) keeping the temperature below 0 °C. A solution of sodium nitrite (223 mg, 3.240 mmol) in water (5 mL) was added dropwise keeping the temperature between -5 and 0 °C and the mixture stirred for an additional 15 min (reaction mixture-2). The solution of reaction mixture-2 was added dropwise to the reaction mixture- 1 solution while keeping the temperature of both solutions between 0 and -5 °C and this mixture was stirred for 1 h. After completion, the reaction mixture was diluted with water (60 mL) and the aqueous layer was extracted with EtOAc (3 x 20 mL). The combined organic phase was dried over sodium sulfate and evaporated under reduced pressure to get crude product. The crude residue was purified by silica gel flash chromatography and pure compound was eluted at 10 % EtOAc in hexanes to obtain title compound as a yellow liquid (230 mg, 0.967 mmol, 29 %).

[0244] 'H NMR (400 MHz, CDC13): 8 7.77-7.80 (m, 2H).

[0245] 19F NMR (400 MHz, CDCI3): 6 -96 22 (2F)

[0246] Step 2: Synthesis of 2,6-difluoro-4-((4-(2-((4-morpholino-6-(3-(m-tolyl)-lH-pyrazol-l- yl)pyrimidin-2-yl)oxy)ethyl)-lH-pyrazol-l-yl)sulfonyl)benzonitrile

[0247] Prepared by the method of Example 1 using 4-cyano-3,5-difluorobenzenesulfonyl chloride (85 mg, 0.362 mmol). Yield (28 mg, 0.044 mmol, 24 %).

[0248] 'H NMR (400 MHz, DMSO-d6): 6 2.39 (s, 3H), 2.95 (t, 2H), 3.63-3.76 (m, 8H), 4.51 (t, 2H), 6.89 (s, 1H), 7.07 (d, 1H), 7.21-7.24 (m, 1H), 7.34-7.37 (m, 1H), 7.77-7.79 (m, 1H), 7.81-7.83 (m, 1H), 8.04 (s, 1H), 8.10 (d, 2H), 8.45 (s, 1H), 8.59 (d, 1H).

[0249] 19F NMR (400 MHz, DMSO-d6): 6 -100.12 (2F).

[0250] LCMS (Method-H): Retention time: 4.060 min, ES(+ve): 633.2 [M+H]+

[0251] Example 5: 3,5-Difluoro-4-((4-(2-((4-morpholino-6-(3-(m-tolyl)-lH-pyrazol-l- yl)pyrimidin-2-yl)oxy)ethyl)-lH-pyrazol-l-yl)sulfonyl)benzonitrile Docket No. 4390.3002 WO

[0252] Step 1: Synthesis of 4-(benzylthio)-3,5-difluorobenzonitrile

[0253] To a stirred solution of 4-bromo-3,5-difluorobenzonitrile (600 mg, 2.752 mmol) and benzyl mercaptan (0.3 mL, 2.752 mmol) in toluene (10 mL) was added DIPEA (2.43 mL, 13.762 mmol) at room temperature under nitrogen atmosphere. The reaction mixture was purged with nitrogenfor 10 min. Pd2(dba)3 (125 mg, 0.137 mmol) and Xantphos (CAS: 161265-03-8) (156 mg, 0.275 mmol) were added, and the reaction mixture was again purged with nitrogen gas for 5 min. The reaction mixture was heated at 110 °C for 2 h. After completion, the reaction mixture was evaporated under reduced pressure to get crude product. The crude residue was purified by silica gel flash chromatography and pure compound was eluted at 10 % EtOAc in hexanes to obtain title compound as off white solid (700 mg, 2.68 mmol, 97 %).

[0254] 'H NMR (400 MHz, DMSO-d6): 84.24 (s, 2H), 7.20-7.33 (m, 5H), 7.79-7.83 (m, 2H).

[0255] 19F NMR (400 MHz, DMSO-d6): 6 -102 22 (2F)

[0256] Step 2: Synthesis of 4-cyano-2,6-difluorobenzenesulfonyl chloride

[0257] To a stirred solution 4-(benzylthio)-3,5-difluorobenzonitrile (700 mg, 2.681 mmol) in acetonitrile (10 mL) at 0 °C was added a mixture of acetic acid (1 mL and water (1 mL). After 5 min, tri chloroisocyanuric acid (622 mg, 2.681 mmol) was added to reaction mixture and reaction mixture was stirred at 0 °C for 30 min. After completion, the reaction mixture was quenched with 10 % aq. NaHCCh Solution (10 mL). The aqueous layer was extracted with DCM (2 x 30 mL). The combined DCM layers were dried over sodium sulphate and evaporated under reduced pressure to obtain crude title compound as white solid (600 mg, 2.53 mmol, 94 %). The crude product was used in the next step without purification or further analysis. Docket No. 4390.3002 WO

[0258] Step 3: Synthesis of 3,5-difluoro-4-((4-(2-((4-morpholino-6-(3-(m-tolyl)-lH-pyrazol-l- yl)pyrimidin-2-yl)oxy)ethyl)-lH-pyrazol-l-yl)sulfonyl)benzonitrile

[0259] Prepared by the method of Example 1 using 4-cyano-2,6-difluorobenzenesulfonyl chloride (87 mg, 0.372 mmol). Yield (17 mg, 0.027 mmol, 15 %).

[0260] 'H NMR (400 MHz, DMSO-d6): 6 2.39 (s, 3H), 2.96 (t, 2H), 3.62-3.80 (m, 8H), 4.52 (t, 2H), 6.90 (s, 1H), 7.07 (d, 1H), 7.21-7.23 (m, 1H), 7.34-7.37 (m, 1H), 7.77-7.82 (m, 2H), 8.03 (s, 1H), 8.11 (d, 2H), 8.49 (s, 1H), 8.60 (d, 1H).

[0261] 19F NMR (400 MHz, DMSO-d6): 6 -104.00 (2F).

[0262] LCMS (Method-C): Retention time: 2.111 min, ES(+ve): 633.3 [M+H]+

[0263] Example 6: 4-(2-(2-(l-(4-(methylsulfonyl)phenyl)-lH-pyrazol-4-yl)ethoxy)-6-(3-(m- tolyl)-lH-pyrazol-l-yl)pyrimidin-4-yl)morpholine

[0264] To a stirred solution of 4-(2-(2-(lH-pyrazol-4-yl)ethoxy)-6-(3-(m-tolyl)-lH-pyrazol-l- yl)pyrimidin-4-yl)morpholine (WO2021163727 Al) (0.120 g, 0.278 mmol) in DMSO (2 mL), was added Cui (0.026 g, 0.139 mmol), K2CO3 (0.077 g, 0.417 mmol), L-Proline (0.032 g, 0.278 mmol) and l-bromo-4-(methylsulfonyl)benzene (0.098 g, 0.583 mmol) at room temperature under nitrogen atmosphere. The reaction mixture was stirred at 130 °C for 16 h. After completion, the reaction mixture was quenched with water (20 mL) and extracted with EtOAc (3 x 30 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered and the solvent was removed in vacuo to obtain crude. The resulting crude was purified by silica gel flash column chromatography. The desired product was eluted at 60-80 % EtOAc in hexanes to obtain title compound as off white solid (0.064 g, 0.109 mmol, 39 %).

[0265] 'H NMR (400 MHz, DMSO-d6): 82.38 (s, 3H), 2.99 (t, 2H), 3.24 (s, 3H), 3.64-3.75 (m, 8H), 4.54 (t, 2H), 6.90 (s, 1H), 7.06 (d, 1H), 7.21 (d, 1H), 7.35 (t, 1H), 7.76-7.83 (m, 3H), 8.01-8.09 (m, 4H), 8.60-8.61 (m, 2H).

[0266] LCMS (Method-C): Retention time: 2.015 min, ES(+ve): 586.3 [M+H]+ Docket No. 4390.3002 WO

[0267] Example 7 : 4-(2-(2-(l-(2,3,5,6-tetrafluoro-4-(methylsulfonyl)phenyl)-lH-pyrazol-4- yl)ethoxy)-6-(3-(m-tolyl)-lH-pyrazol-l-yl)pyrimidin-4-yl)morpholine

[0268] Step 1: Synthesis of l,2,3,4,5-pentafluoro-6-(methylsulfonyl)benzene

[0269] In a 1 L two neck dried round bottom flask under nitrogen atmosphere, Oxone (194 g, 315 mmol) solution in water (140 mL) was introduced and cooled to 0 °C. To this reaction mixture methyl(perfluorophenyl)sulfane (15 g, 70.0 mmol) in acetone (140 mL) was added dropwise at 0 °C under nitrogen atmosphere. Reaction mixture was stirred at 25 °C for 16 h. Progress of the reaction was monitored by TLC (20 % EtOAc in hexane, 0.37 Rf). After the completion of reaction, reaction mixture was diluted with water (100 mL) and extracted with DCM (2 x 100 mL). The combined organic layers were washed water (100 mL) and brine solution (50 mL), dried over sodium sulphate and concentrated under reduced pressure to get crude product. Thus, obtained crude was purified by silica gel column chromatography using 15-20 % EtOAc in hexane to get pure l,2,3,4,5-pentafluoro-6-(methylsulfonyl)benzene (14 g, 56.9 mmol, 81 % yield) as white solid.

[0270] 'II NMR (400 MHz, CDC13): 6 3 36 (s, 3H)

[0271] 19F NMR (376 MHz, CDCI3): 6 -136.02 -136.15 (2F), -143.15 -143.3 (IF), -157.47_-157.64

[0272] (2F).

[0273] Step 2: Synthesis of 4-(2-(2-(l-(2,3,5,6-Tetrafluoro-4-(methylsulfonyl)phenyl)-lH- pyrazol-4-yl)ethoxy)-6-(3-(m-tolyl)-lH-pyrazol-l-yl)pyrimidin-4-yl)morpholine

[0274] To a stirred solution of 4-(2-(2-(lH-pyrazol-4-yl)ethoxy)-6-(3-(m-tolyl)-lH-pyrazol-l- yl)pyrimidin-4-yl)morpholine (WO2021163727 Al) (0.100 g, 0.231 mmol) in DMF (3 mL), was added potassium carbonate (0.064 g, 0.463 mmol) and l,2,3,4,5-pentafluoro-6- (methylsulfonyl)benzene (0.086 g, 0.347 mmol) at room temperature under nitrogen atmosphere. The reaction mixture was stirred at 60 °C for 2 h. After completion, the reaction mixture was quenched with water (20 mL) and extracted with EtOAc (3 x 30 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered and the solvent was Docket No. 4390.3002 WO removed in vacuum to obtain crude. The resulting crude was purified by silica gel flash column chromatography. The desired product was eluted at 60-80 % EtOAc in hexanes to obtain title compound as off white solid (0.036 g, 0.055 mmol, 24 %).

[0275] 'H NMR (400 MHz, DMSO-d6): 62.38 (s, 3H), 3.01 (t, 2H), 3.56 (s, 3H), 3.65-3.77 (m, 8H), 4.54 (t, 2H), 6.90 (s, 1H), 7.06 (d, 1H), 7.21 (d, 1H), 7.35 (t, 1H), 7.77 (d, 1H), 7.82 (s, 1H), 7.96 (s, 1H), 8.15 (s, 1H), 8.60 (d, 1H).

[0276] 19F NMR (400 MHz, DMSO-d6): 6 -146.58_-146.66 (2F), -138.02_-138.07 (2F).

[0277] LCMS (Method-C): Retention time: 2.065 min, ES(+ve): 658.3 [M+H]+

[0278] Example 8: 4-(2-(2-(l-(2-bromo-3,5,6-trifluoro-4-(methylsulfonyl)phenyl)-lH-pyrazol-4- yl)ethoxy)-6-(3-(m-tolyl)-lH-pyrazol-l-yl)pyrimidin-4-yl)morpholine

[0279] Step 1: Synthesis of (3-bromo-2,4,5,6-tetrafluorophenyl)(methyl)sulfane

[0280] To a stirred solution of l,3-dibromo-2,4,5,6-tetrafluorobenzene (10 g, 0.0324 mol) in dry THF (100 mL) was added iPrMgCl.LiCI (1.3 M in THF) (24.9 mL, 0.0324 mol) dropwise at -78 °C under argon gas atmosphere. The reaction mixture was stirred at -78 °C for 30 min. A solution of -S-Methyl methanethiosulfonate (4.09 g, 0.0324 mol) in dry THF (10 mL) was added drop wise into the reaction mixture at -78 °C. The reaction mixture was stirred for 1 h. After completion, the reaction was quenched by dil. HC1 (100 mL) and aqueous layer was extracted with hexanes (100 mL), dried over sodium sulphate and evaporated under reduced pressure at 30 °C to obtain crude product. The crude residue was purified by silica gel flash column chromatography. The pure compound was eluted in hexanes (100 %) as colorless liquid (7 g, 0.0254 mol, 78 %).

[0281] ’ll NMR (400 MHz, CDCh): 8 2.48 (s, 3H) Docket No. 4390.3002 WO

[0282] Step 2: Synthesis of l-bromo-2,3,4,6-tetrafluoro-5-(methylsulfonyl)benzene

[0283] To a stirred solution of (3-bromo-2,4,5,6-tetrafluorophenyl)(methyl)sulfane (13 g, 0.0472 mol) in MeOH (90 mL) and water (40 mL) was added Oxone (72.63 g, 0.2363 mol) in portion wise manner at 0 °C under nitrogen atmosphere. The reaction mixture was warmed to room temperature and stirred at room temperature for 16 h. After completion, MeOH was evaporated under vacuum. The aqueous layer was extracted with DCM (2 x 250 mL). The combined DCM layers were dried over sodium sulphate and evaporated under reduced pressure to obtain crude product. The crude residue was purified by silica gel flash column chromatography at a gradient of 0-20 % EtOAc in hexanes to obtain title compound as a white solid (10.0 g, 0.0325 mol, 69 %).

[0284] 'II NMR (400 MHz, CDC13): 8 3.36 (s, 3H)

[0285] 19F NMR (400 MHz, CDCI3): 6 -104.08_-104.12 (IF), -113.73 -113.83 (IF), -129.54_- 129.64 (IF), -157.31 -157.46 (IF).

[0286] Step 3: Synthesis of 4-(2-(2-(l-(2-bromo-3,5,6-trifluoro-4-(methylsulfonyl)phenyl)-lH- pyrazol-4-yl)ethoxy)-6-(3-(m-tolyl)-lH-pyrazol-l-yl)pyrimidin-4-yl)morpholine

[0287] To a stirred solution of 4-(2-(2-(lH-pyrazol-4-yl)ethoxy)-6-(3-(m-tolyl)-lH-pyrazol-l- yl)pyrimidin-4-yl)morpholine (WO2021163727 Al) (0.100 g, 0.231 mmol) in DMF (3 mL), was added K2CO3 (0.064 g, 0.463 mmol) and l-bromo-2,3,4,6-tetrafluoro-5- (methylsulfonyl)benzene (0.106 g, 0.345 mmol) at room temperature under nitrogen atmosphere. The reaction mixture was stirred at 60 °C for 2 h. After completion, the reaction mixture was quenched with water (20 mL) and extracted with EtOAc (3 x 30 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered and the solvent was removed in vacuo to obtain crude product. The resulting crude product was purified by preparative HPLC. Fractions containing the pure compound were combined and lyophilized to obtain title compound as off white solid (0.010 g, 0.001 mmol, 6 %).

[0288] Preparative HPLC Purification Method: The compound was purified on Shimadzu LC- 20AP and UV detector. The column used was X-B RIDGE Cl 8, 250 mm x 19 mm, 5 pm. Column flow rate was 13.0 mL / min. Mobile phases used were: (A) 0.1 % NH3 in water and (B) 100 % MeCN. The gradient expressed as % solvent (B) was 40 % over 0.01 min, 70 % over 22 min, 70 % over 35 min, 100 % over 35.01 min, 100 % over 37 min, 40 % over 37.01 min, and 40 % over 43 min. The UV spectra were recorded at 220 nm and 254 nm Lambdamax. Docket No. 4390.3002 WO

[0289] 'H NMR (400 MHz, DMSO-d6): 62.38 (s, 3H), 3.01 (t, 2H), 3.52 (s, 3H), 3.65-3.75 (m, 8H), 4.54 (t, 2H), 6.90 (s, 1H), 7.04 (d, 1H), 7.23 (d, 1H), 7.35 (t, 1H), 7.76 (d, 1H), 7.80 (s, 1H), 7.89 (s, 1H), 8.02 (s, 1H), 8.60 (d, 1H).

[0290] 19F NMR (400 MHz, DMSO-d6): 6 -143.84_-143.94 (IF), -131.91_-131.97 (IF), -103.33 - 103.36 (IF).

[0291] LCMS (Method-C): Retention time: 2.129 min, ES(+ve): 718.3 / 720.3 [M+H]+

[0292] Example 9: 4-(2-(2-(l-(3-(methylsulfonyl)phenyl)-lH-pyrazol-4-yl)ethoxy)-6-(3-(m- tolyl)-lH-pyrazol-l-yl)pyrimidin-4-yl)morpholine

[0293] Prepared by the method of Example 6 using l-bromo-3-(methylsulfonyl)benzene (0.137 g, 0.583 mmol). Yield (0.034 g, 0.058 mmol, 21 %).

[0294] 'H NMR (400 MHz, DMSO-d6): 82.38 (s, 3H), 2.99 (t, 2H), 3.25 (s, 3H), 3.62-3.75 (m, 8H), 4.54 (t, 2H), 6.90 (s, 1H), 7.06 (d, 1H), 7.21 (d, 1H), 7.35 (t, 1H), 7.74-7.82 (m, 5H), 8.15-8.17 (m, 1H), 8.32 (s, 1H), 8.58-8.64 (m, 2H).

[0295] LCMS (Method-C): Retention time: 2.008 min, ES(+ve): 586.3 [M+H]+

[0296] Example 10: 4-(2-(2-(l-(2,3,4,6-tetrafluoro-5-(methylsulfonyl)phenyl)-lH-pyrazol-4- yl)ethoxy)-6-(3-(m-tolyl)-lH-pyrazol-l-yl)pyrimidin-4-yl)morpholine

[0297] Step 1: Synthesis of methyl(2,3,5,6-tetrafluoro-4-nitrophenyl)sulfane Docket No. 4390.3002 WO

[0298] To a stirred solution of l,2,3,4,5-pentafluoro-6-nitrobenzene (5.00 g, 0.023 mmol) in tetrahydrofuran (50 mL), was added sodium thiomethoxide as a ~ 15 % in H2O (10 mL, 0.023 mmol) at 0 °C. The reaction mixture was warmed to room temperature and stirred for 16 h. The reaction mixture was quenched with cold water (100 mL) and the aqueous layer was extracted with DCM (3 x 100 mL). Combined organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the crude product. The crude residue was purified by silica gel flash chromatography. The desired product was eluted with 40 - 60 % EtOAc in hexane to obtain title compound as brown liquid (1.500 g, 0.006 mmol, 26 %).

[0299] 'll NMR (400 MHz, DMSO-d6): 82 66 (m, 3H)

[0300] 19F NMR (400 MHz, DMSO-d6): 6 -146.86 -146.91 (2F), -134.00_-134.04 (2F).

[0301] Step 2: Synthesis of 4-(6-(3-(m-tolyl)-lH-pyrazol-l-yl)-2-(2-(l-(2,4,5-trifluoro-3- (methylthio)-6-nitrophenyl)-lH-pyrazol-4-yl)ethoxy)pyrimidin-4-yl)morpholine

[0302] To a stirred solution of 4-(2-(2-(lH-pyrazol-4-yl)ethoxy)-6-(3-(m-tolyl)-lH-pyrazol-l- yl)pyrimidin-4-yl)morpholine (WO2021163727 Al) (0.250 g, 0.580 mmol) and methyl(2,3,5,6-tetrafluoro-4-nitrophenyl)sulfane (0.279 g, 1.157 mmol) in 1,4-dioxane (3 mL) was added DIPEA (0.3 mL, 0.580 mmol). The reaction mixture was heated at 100 °C for 4 h. After completion, the reaction mixture was evaporated under reduced pressure to give crude product. The crude residue was purified by silica gel flash chromatography. The desired product was eluted with 20 - 40 % EtOAc in hexanes to obtain title compound as brown solid (0.240 g, 0.368 mmol, 35 %).

[0303] LCMS (Method-C): Retention time: 2.357 min, ES(+ve): 653.3 [M+H]+

[0304] Step 3: Synthesis of 4-(2-(2-(l-(2,3,4,6-tetrafluoro-5-(methylthio)phenyl)-lH-pyrazol-4- yl)ethoxy)-6-(3-(m-tolyl)-lH-pyrazol-l-yl)pyrimidin-4-yl)morpholine

[0305] To a stirred solution of 4-(6-(3-(m-tolyl)-lH-pyrazol-l-yl)-2-(2-(l-(2,4,5-trifluoro-3- (methylthio)-6-nitrophenyl)-lH-pyrazol-4-yl)ethoxy)pyrimidin-4-yl)morpholine (0.150 g, 0.230 mmol) in DMSO (2.5 mL) was added 18-Crown-6 (0.03 g, 0.113mmol) and KF (0.040 g, 0.689 mmol) at room temperature. The resulting reaction mixture was stirred at 130 °C for 2 h. The reaction mixture was quenched with cold water (20 mL) and aqueous layer was extracted with EtOAc (3 x 20 mL). Combined organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the crude product. The crude Docket No. 4390.3002 WO product was purified by silica gel flash chromatography. The desired product was eluted with 30 - 50 % EtOAc in hexanes to obtain title compound as yellow solid (0.097 g, 0.155 mmol, 67 %).

[0306] 'H NMR (400 MHz, DMSO-d6): 82.38 (s, 3H), 2.49 (s, 3H), 2.99 (t, 2H), 3.65-3.75 (m, 8H), 4.52 (t, 2H), 6.90 (s, 1H), 7.05 (d, 1H), 7.21 (d, 1H), 7.34 (t, 1H), 7.75-7.83 (m, 3H), 8.03 (s, 1H), 8.61 (d, 1H).

[0307] LCMS (Method-C): Retention time: 2.378 min, ES(+ve): 626.3 [M+H]+

[0308] Step 4: Synthesis of 4-(2-(2-(l-(2,3,4,6-Tetrafluoro-5-(methylsulfonyl)phenyl)-lH- pyrazol-4-yl)ethoxy)-6-(3-(m-tolyl)-lH-pyrazol-l-yl)pyrimidin-4-yl)morpholine

[0309] To a stirred solution of 4-(2-(2-(l-(2,3,4,6-tetrafluoro-5-(methylthio)phenyl)-lH-pyrazol-4- yl)ethoxy)-6-(3-(m-tolyl)-lH-pyrazol-l-yl)pyrimidin-4-yl)morpholine (0.087 g, 0.139 mmol) in MeOH (5.0 mL) was added ammonium molybdate tetrahydrate (0.100 g, 0.080 mmol) and 30 % w / v aqueous H2O2 (1.0 mL) at 0 °C under nitrogen atmosphere. The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was quenched with water (20 mL) and aqueous layer was extracted with DCM (3 x 20 mL). Combined organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the crude product. The crude residue was purified by silica gel flash chromatography. The desired product was eluted with 30-50 % EtOAc in hexanes to obtain title compound as brown solid (0.010 g, 0.132 mmol, 11 %).

[0310] ' H NMR (400 MHz, DMSO-d6): 62.39 (s, 3H), 3.01 (t, 2H), 3.54 (s, 3H), 3.65-3.77 (m, 8H), 4.54 (t, 2H), 6.92 (s, 1H), 7.06 (d, 1H), 7.22 (d, 1H), 7.36 (t, 1H), 7.78 (d, 1H), 7.82(s, 1H), 7.89 (s, 1H), 8.07 (s, 1H), 8.61 (d, 1H).

[0311] 19F NMR (400 MHz, DMSO-d6): 6 -160.23_-160.38 (IF), -132.09 -132.18 (IF), -130.56_- 130.65 (IF), -121.87_-121.89 (IF).

[0312] LCMS (Method-C): Retention time: 1.893 min, ES(+ve): 658.3 [M+H]+

[0313] Example 11: 4-(2-(2-(l-(pyridin-4-yl)-lH-pyrazol-4-yl)ethoxy)-6-(3-(m-tolyl)-lH- pyrazol-l-yl)pyrimidin-4-yl)morpholine

[0314] Example 11 Docket No. 4390.3002 WO

[0315] Prepared by a method generally analogous to Example 6 using 4-iodopyridine (71 mg, 0.348 mmol). The crude product was purified using reversed phase column chromatography eluting with 70 % acetonitrile in water. Fractions containing pure compound were combined and lyophilized to obtain the title compound. Yield (43 mg, 0.084 mmol, 36 %).

[0316] 'H NMR (400 MHz, DMSO-d6): 6 2.37 (s, 3H), 2.98 (t, 2H), 3.62-3.77 (m, 8H), 4.53 (t, 2H), 6.89 (s, 1H), 7.06 (t, 1H), 7.20 (d, 1H), 7.34 (t, 1H), 7.75-7.88 (m, 5H), 8.59-8.63 (m, 4H).

[0317] LCMS (Method-C): Retention time: 1.762 min, ES(+ve): 509.3[M+H]+

[0318] Example 12: 4-(2-(2-(l-(2-fluoropyridin-4-yl)-lH-pyrazol-4-yl)ethoxy)-6-(3-(m-tolyl)- lH-pyrazol-l-yl)pyrimidin-4-yl)morpholine

[0319] To a stirred solution of 4-(2-(2-(lH-pyrazol-4-yl)ethoxy)-6-(3-(m-tolyl)-lH-pyrazol-l- yl)pyrimidin-4-yl)morpholine (WO2021163727 Al) (0.100 g, 0.232 mmol) in dioxane (5 mL), was added Cui (0.022g, 0.116 mmol), K3PO4 (0.098 g, 0.462 mmol), trans-1,2- diaminocyclohexane (0.099 g, 0.462 mmol) and 4-bromo-2-fluoropyridine (0.122 g, 0.693 mmol) at room temperature under nitrogen atmosphere. The reaction mixture was stirred at 90 °C for 4 h. After completion, the reaction mixture was quenched with water (20 mL) and extracted with EtOAc (3 x 30 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered and the solvent was removed in vacuo to obtain crude. The resulting crude was purified by RP -flash chromatography. Product was eluted at 70 % acetonitrile in water. Fractions containing pure compound were combined and lyophilized to obtain title compound as off white solid (0.037 g, 0.070 mmol, 30 %).

[0320] 'H NMR (400 MHz, DMSO-d6): 8 2.38 (s, 3H), 2.99 (t, 2H), 3.62-3.75 (m, 8H), 4.54 (t, 2H), 6.90 (s, 1H), 7.05-7.07 (m, 1H), 7.20 (d, 1H), 7.34 (t, 1H), 7.58 (d, 1H), 7.76-7.81 (m, 3H), 7.89 (s, 1H), 8.28-8.29 (m, 1H), 8.59-8.60 (m, 1H), 8.68 (s, 1H).

[0321] 19F NMR (400 MHz, DMSO-d6): 6 -67 01 (IF)

[0322] LCMS (Method-C): Retention time: 2.164 min, ES(+ve): 527.3 [M+H]+ Docket No. 4390.3002 WO

[0323] Example 13: 4-(2-(2-(l-(perfluoropyridin-4-yl)-lH-pyrazol-4-yl)ethoxy)-6-(3-(m-tolyl)- lH-pyrazol-l-yl)pyrimidin-4-yl)morpholine

[0324] Prepared by the method of Example 7, Step 2 using perfluoropyridine (70 mg, 0.417 mmol). After completion, the reaction mixture was quenched with ice cold water. The aqueous layer was extracted with EtOAc (3 x 30 mL). The combined EtOAc layer was dried over sodium sulfate and evaporated under reduced pressure to obtain crude product. The crude residue was purified by RP-flash chromatography by eluting with acetonitrile in water. After lyophilization title compound was obtained as a white solid (90 mg, 0.155 mmol, 55 %).

[0325] 'H NMR (400 MHz, DMSO-d6): 6 2.39 (s, 3H), 3.03 (t, 2H), 3.65-3.75 (m, 8H), 4.55 (t, 2H), 6.91 (s, 1H), 7.06 (d, 1H), 7.22 (d, 1H), 7.35 (t, 1H), 7.78 (d, 1H), 7.82 (s, 1H), 8.03 (s, 1H), 8.23 (s, 1H), 8.60 (d, 1H).

[0326] 19F NMR (400 MHz, DMSO-d6): 6 -90.72_ -90.81 (2F), -149.41_ -149.51 (2F).

[0327] LCMS (Method-C): Retention time:2.205 min, ES(+ve): 581.3 [M+H]+

[0328] Example 14: 4-(2-(2-(l-(6-fluoropyridin-3-yl)-lH-pyrazol-4-yl)ethoxy)-6-(3-(m-tolyl)- lH-pyrazol-l-yl)pyrimidin-4-yl)morpholine

[0329] Step 1: Synthesis of 4-(2-(2-(l-(6-nitropyridin-3-yl)-lH-pyrazol-4-yl)ethoxy)-6-(3-(m- tolyl)-lH-pyrazol-l-yl)pyrimidin-4-yl)morpholine Docket No. 4390.3002 WO

[0330] Prepared by a method generally analogous to Example 12 using 5-bromo-2-nitropyridine (0.282 g, 1.389 mmol). After completion, the reaction mixture was quenched with water (20 mL) and extracted with DCM (3 x 30 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered and the solvent was removed in vacuo to obtain crude. The crude residue was purified by silica gel flash chromatography. The desired product was eluted with 60-65 % EtOAc in hexanes to obtain title compound as yellow solid (0.060 g, 0.108 mmol, 47 %).

[0331] LCMS (Method-C): Retention time: 2.109 min, ES(+ve): 554.3 [M+H]+

[0332] Step 2: Synthesis of 4-(2-(2-(l-(6-fluoropyridin-3-yl)-lH-pyrazol-4-yl)ethoxy)-6-(3-(m- tolyl)-lH-pyrazol-l-yl)pyrimidin-4-yl)morpholine

[0333] Prepared by a method generally analogous to Example 10, Step 3, using 4-(2-(2-(l-(6- nitropyridin-3-yl)-lH-pyrazol-4-yl)ethoxy)-6-(3-(m-tolyl)-lH-pyrazol-l-yl)pyrimidin-4- yl)morpholine (0.060 g, 0.108 mmol). The crude product was purified by RP -flash chromatography. Product was eluted at 45 % acetonitrile in water. Fractions containing pure compound were combined and lyophilized to obtain title compound as off white solid (0.019 g, 0.070 mmol, 30 %).

[0334] 'H NMR (400 MHz, DMSO-d6): 8 2.39 (s, 3H), 2.99 (t, 2H), 3.65-3.75 (m, 8H), 4.54 (t, 2H), 6.91 (s, 1H), 7.06 (d, 1H), 7.22 (d, 1H), 7.34-7.37 (m, 2H), 7.77-7.82 (m, 3H), 8.38-842 (m, 1H), 8.51 (s, 1H), 8.61 (d, 1H), 8.70-8.71 (m, 1H).

[0335] 19F NMR (400 MHz, DMSO-d6): 6 -72 39 (IF)

[0336] LCMS (Method-J): Retention time: 4.194 min, ES(+ve): 527.2 [M+H]+

[0337] Example 15: 4-(2-(2-(l-(2-fluoropyrimidin-4-yl)-lH-pyrazol-4-yl)ethoxy)-6-(3-(m-tolyl)- lH-pyrazol-l-yl)pyrimidin-4-yl)morpholine

[0338] To a stirred solution of 4-(2-(2-(lH-pyrazol-4-yl)ethoxy)-6-(3-(m-tolyl)-lH-pyrazol-l- yl)pyrimidin-4-yl)morpholine (WO2021163727 Al) (0.100 g, 0.231 mmol) in DMSO (2 mL), was added DIPEA (0.2 mL, 1.390 mmol) and 2,4-difluoropyrimidine (0.100 g, 0.926 mmol) Docket No. 4390.3002 WO at room temperature under nitrogen atmosphere. The reaction mixture was stirred at 60 °C for 16 h. After completion, the reaction mixture was quenched with cold water (20 mL) and extracted with EtOAc (3 x 20 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered and the solvent was removed in vacuum to obtain crude product. The resulting crude product was purified by RP-flash column chromatography. The product was eluted at 70 % acetonitrile in water. Fractions containing pure compound were combined and lyophilized to obtain title compound as white solid (0.025 g, 0.047 mmol, 20 %).

[0339] 'H NMR (400 MHz, DMSO-d6): 8 2.38 (s, 3H), 3.00 (t, 2H), 3.63-3.76 (m, 8H), 4.55 (t, 2H), 6.89 (s, 1H), 7.05 (d, 1H), 7.20 (d, 1H), 7.34 (t, 1H), 7.76 (d, 1H), 7.81 (s, 1H), 7.83-7.86 (m, 1H), 7.99 (s, 1H), 8.60 (d, 2H), 8.78-8.79 (m, 1H).

[0340] 19F NMR (400 MHz, DMSO-d6): 6 -45 62 (IF)

[0341] LCMS (Method-C): Retention time: 2.091 min, ES(+ve): 528.4 [M+H]+

[0342] Example 16: 4-(2-(2-(l-(2,5-difluoropyrimidin-4-yl)-lH-pyrazol-4-yl)ethoxy)-6-(3-(m- tolyl)-lH-pyrazol-l-yl)pyrimidin-4-yl)morpholine

[0343] To a stirred solution of 4-(2-(2-(lH-pyrazol-4-yl)ethoxy)-6-(3-(m-tolyl)-lH-pyrazol-l- yl)pyrimidin-4-yl)morpholine (WO2021163727 Al) (0.100 g, 0.231 mmol) in DMSO (3 mL), was added DIPEA (0.12 mL, 0.696 mmol) and 2,4,5-trifluoropyrimidine (0.062 g, 0.462 mmol) at room temperature under nitrogen atmosphere. The reaction mixture was stirred at room temperature for 16 h. After completion, the reaction mixture was quenched with water (20 mL) and extracted with EtOAc (3 x 20 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered and the solvent was removed in vacuum to obtain crude product. The crude residue was purified by silica gel flash chromatography. The desired product was eluted with 70 % EtOAc in hexanes to obtain title compound as an off-white solid (0.035 g, 0.064 mmol, 27 %).

[0344] 'H NMR (400 MHz, DMSO-d6): 6 2.38 (s, 3H), 3.02 (t, 2H), 3.64-3.76 (m, 8H), 4.55 (t, 2H), 6.90 (s, 1H), 7.05 (d, 1H), 7.21 (d, 1H), 7.35 (t, 1H), 7.77 (d, 1H), 7.81 (s, 1H), 8.04 (s, 1H), 8.58-8.60 (m, 2H), 8.95 (d, 1H).

[0345] 19F NMR (400 MHz, DMSO-d6): 6 -145.67_-145.74 (IF), -50.92_-51.00 (IF). Docket No. 4390.3002 WO

[0346] LCMS (Method-C): Retention time: 2.094 min, ES(+ve): 546.5 [M+H]+

[0347] Example 17: 4-(2-(2-(l-(6-fluoropyrimidin-4-yl)-lH-pyrazol-4-yl)ethoxy)-6-(3-(m-tolyl)- lH-pyrazol-l-yl)pyrimidin-4-yl)morpholine

[0348] Prepared by the method of Example 16 using 4,6-difluoropyrimidine (0.065 g, 0.560 mmol). The crude product was purified by RP-flash column chromatography. The product was eluted at 80 % acetonitrile in water. Fractions containing pure compound were combined and lyophilized to obtain title compound as white solid (0.015 g, 0.028 mmol, 10 %).

[0349] 'H NMR (400 MHz, DMSO-d6): 8 2.38 (s, 3H), 3.00 (t, 2H), 3.63-3.75 (m, 8H), 4.55 (t, 2H), 6.89 (s, 1H), 7.05 (d, 1H), 7.20 (d, 1H), 7.34 (t, 1H), 7.59 (s, 1H), 7.76 (d, 1H), 7.81 (s, 1H), 7.99 (s, 1H), 8.60 (d, 1H), 8.63 (s, 1H), 8.87 (s, 1H).

[0350] 19F NMR (400 MHz, DMSO-d6): 6 -59 53 (IF)

[0351] LCMS (Method-J): Retention time: 4.026 min, ES(+ve): 528.4 [M+H]+

[0352] Example 18: 4-(2-(2-(l-(4-fluoropyrimidin-2-yl)-lH-pyrazol-4-yl)ethoxy)-6-(3-(m-tolyl)- lH-pyrazol-l-yl)pyrimidin-4-yl)morpholine

[0353] A solution of 4-(2-(2-(lH-pyrazol-4-yl)ethoxy)-6-(3-(m-tolyl)-lH-pyrazol-l-yl)pyrimidin-4- yl)morpholine (WO2021163727 Al) (1.0 g, 2.317 mmol) in THF (15 mL) was stirred for 5 min at room temperature. To the resulting solution, CS2CO3 (2.259 g, 6.952 mmol) and 2,4- difluoropyrimidine (1.075 g, 9.269 mmol) were added at room temperature under nitrogen atmosphere. The reaction mixture was stirred at room temperature for 16 h. After completion, the reaction mixture was quenched with cold water (100 mL) and extracted with EtOAc (3 x 100 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered and the solvent was removed in vacuo to obtain crude. The resulting crude was purified by RP- Flash column chromatography. Product was eluted at 45-65% MeCN in water. Fractions Docket No. 4390.3002 WO containing pure compound were combined and lyophilized to obtain title compound as a white solid (0.061 g, 0.115 mmol, 5 %).

[0354] 'H NMR (400 MHz, DMSO-d6): 8 2.38 (s, 3H), 3.00 (t, 2H), 3.69 (s, 8H), 4.54 (t, 2H), 6.89 (s, 1H), 7.05 (d, 1H), 7.21 (d, 1H), 7.30-7.36 (m, 2H), 7.77 (d, 1H), 7.81 (s, 1H), 7.89 (s, 1H), 8.58-8.61 (m, 2H), 8.92-8.96 (m, 1H).

[0355] 19F NMR (400 MHz, DMSO-d6): 6 -58 596 (IF).

[0356] LCMS (Method-C): Retention time: 2.011 min, ES(+ve): 528.4 [M+H]+

[0357] Example 19: N-(2-(l-(4-fluoropyridin-2-yl)-lH-pyrazol-4-yl)ethyl)-N-methyl-4- morpholino-6-(3-phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine

[0358] Step 1: Synthesis of N-(2-(lH-pyrazol-4-yl)ethyl)-N-methyl-4-morpholino-6-(3-phenyl- lH-pyrazol-l-yl)pyrimidin-2-amine

[0359] To a stirred solution of 4-(2-iodo-6-(3-phenyl-lH-pyrazol-l-yl)pyrimidin-4-yl)morpholine (WO2021163727 Al) (2.0 g, 4.616 mmol) and N-methyl-2-(lH-pyrazol-4-yl)ethan-l-amine (0.86 g, 6.927 mmol) in dioxane (20 mL) was added CS2CO3 (6.0 g, 18.472 mmol) at room temperature under nitrogen atmosphere. The reaction mixture was purged with nitrogen gas for 10 min. Pd2(dba)3(0.634 g, 0.692 mmol) and XPhos (CAS: 564483-18-7) (1.1 g, 2.309 mmol) were added and the reaction mixture was heated at 100 °C for 4 h. After completion, the reaction mixture was diluted with water (50 mL) and the aqueous layer was extracted with EtOAc (3 x 100 mL). The combined organic phases were dried over sodium sulfate and evaporated under reduced pressure. The crude residue was purified by silica gel flash chromatography and pure compound was eluted at 30 % EtOAc in hexanes to obtain title compound as a brown solid (1.40 g, 3.251 mmol, 70 %).

[0360] 'H NMR (400 MHz, DMSO-d6): 6 2.73 (t, 2H), 3.11 (s, 3H), 3.57-3.75 (m, 10H), 6.56 (s, 1H), 7.04 (d, 1H), 7.37-7.40 (m, 2H), 7.44-7.48 (m, 3H), 7.96-7.98 (m, 2H), 8.60 (s, 1H), 12.54 (s, 1H). Docket No. 4390.3002 WO

[0361] Step 2: Synthesis of N-(2-(l-(4-fluoropyridin-2-yl)-lH-pyrazol-4-yl)ethyl)-N-methyl-4- morpholino-6-(3-phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine

[0362] To a stirred solution of N-(2-(lH-pyrazol-4-yl)ethyl)-N-methyl-4-morpholino-6-(3-phenyl- lH-pyrazol-l-yl)pyrimidin-2-amine (0.150 g, 0.348 mmol) in DMSO (1 mL), was added DIPEA (0.24 mL, 1.394 mmol) and 2,4-difluoropyridine (0.080 g, 0.697 mmol) at room temperature under nitrogen atmosphere. The reaction mixture was stirred at room temperature for 3 h. After completion, the reaction mixture was quenched by water (40 mL) and extracted with EtOAc (2 x 50 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered and the solvent was removed in vacuum to obtain crude product. The resulting crude product was purified by column chromatography. The product was eluted at 25 % EtOAc in hexanes to obtain title compound as white solid (0.080 g, 0.152 mmol, 43 %).

[0363] 'H NMR (400 MHz, DMSO-d6): 8 2.85 (t, 2H), 3.15 (s, 3H), 3.55-3.75 (m, 8H), 3.83-3.95 (m, 2H), 6.55 (s, 1H), 7.03 (d, 1H), 7.24-7.28 (m, 1H), 7.37-7.41 (m, 1H), 7.45-7.49 (m, 2H), 7.62-7.65 (m, 1H), 7.77 (s, 1H), 7.96-7.98 (m, 2H), 8.46-8.51 (m, 2H), 8.63 (d, 1H).

[0364] 19F NMR (400 MHz, DMSO-d6): 6 -99 75 (IF).

[0365] LCMS (Method-H): Retention time: 4.743 min, ES(+ve): 526.4 [M+H]+

[0366] Example 20: N-(2-(l-(2-fluoropyrimidin-4-yl)-lH-pyrazol-4-yl)ethyl)-N-methyl-4- morpholino-6-(3-phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine and Example 21: N-(2-(l- (4-fluoropyrimidin-2-yl)-lH-pyrazol-4-yl)ethyl)-N-methyl-4-morpholino-6-(3-phenyl- lH-pyrazol-l-yl)pyrimidin-2-amine

[0367] To a stirred solution of N-(2-(lH-pyrazol-4-yl)ethyl)-N-methyl-4-morpholino-6-(3-phenyl- lH-pyrazol-l-yl)pyrimidin-2-amine (Example 19, Step 1) (0.100 g, 0.232 mmol) in DMSO (1 mL), was added DIPEA (0.16 mL, 0.929 mmol) and 2,4-difluoropyrimidine (0.05 g, 0.464 mmol) at room temperature under nitrogen atmosphere. The reaction mixture was stirred at room temperature for 4 h. After completion, the reaction mixture was quenched by water and extracted with EtOAc (2 x 50 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered and solvent was removed in vacuum to obtain crude product. The resulting crude product was purified by column chromatography. Elution with 8 % EtOAc in Docket No. 4390.3002 WO hexanes afforded N-(2-(l-(2-fluoropynmidin-4-yl)-lH-pyrazol-4-yl)ethyl)-N-methyl-4- morpholino-6-(3-phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine (Example 20) (0.019 g, 0.036 mmol, 15 %) as a white solid.

[0368] 'H NMR (400 MHz, DMSO-d6): 6 2.85 (t, 2H), 3.14 (s, 3H), 3.58-3.65 (m, 4H), 3.66-3.72 (m, 4H), 3.86-3.94 (m, 2H), 6.54 (s, 1H), 7.02 (d, 1H), 7.37-7.41 (m, 1H), 7.45-7.48 (m, 2H), 7.79-7.81 (m, 1H), 7.93-7.97 (m, 3H), 8.54 (s, 1H), 8.62 (d, 1H), 8.75 (d, 1H).

[0369] 19F NMR (400 MHz, DMSO-d6): 6 -45 62 (IF).

[0370] LCMS (Method-C): Retention time: 2.301 min, ES(+ve): 527.6 [M+H]+

[0371] Further elution with 21 % EtOAc in hexanes afforded N-(2-(l-(4-fluoropyrimidin-2-yl)-lH- pyrazol-4-yl)ethyl)-N-methyl-4-morpholino-6-(3-phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine (Example 21) (0.025 g, 0.047 mmol, 20 %) as a white solid.

[0372] 'H NMR (400 MHz, DMSO-d6): 6 2.85 (t, 2H), 3.15 (s, 3H), 3.60-3.65 (m, 4H), 3.66-3.73 (m, 4H), 3.85-3.93 (m, 2H), 6.55 (s, 1H), 7.03 (d, 1H), 7.27-7.29 (m, 1H), 7.37-7.40 (m, 1H), 7.45-7.48 (m, 2H), 7.83 (s, 1H), 7.96-7.98 (m, 2H), 8.52 (s, 1H), 8.62 (d, 1H), 8.89-8.93 (m, 1H).

[0373] 19F NMR (400 MHz, DMSO-d6): 6 -58 63 (IF).

[0374] LCMS (Method-C): Retention time: 2.150 min, ES(+ve): 527.6 [M+H]+

[0375] Example 22: N-(2-(l-(2,5-difluoropyrimidin-4-yl)-lH-pyrazol-4-yl)ethyl)-N-methyl-4- morpholino-6-(3-phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine

[0376] Prepared by a method generally analogous to Example 16 using N-(2-(lH-pyrazol-4-yl)ethyl)- N-methyl-4-morpholino-6-(3-phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine (Example 19, Step 1) (0.100 g, 0.232 mmol). The reaction mixture was stirred for 1 h. The crude product was purified by column chromatography. The product was eluted at 10 % EtOAc in hexanes to obtain title compound as a white solid (0.065 g, 0.119 mmol, 51 %).

[0377] 'H NMR (400 MHz, DMSO-d6): 8 2.87 (t, 2H), 3.14 (s, 3H), 3.58-3.65 (m, 4H), 3.67-3.73 (m, 4H), 3.86-3.95 (m, 2H), 6.55 (s, 1H), 7.02 (d, 1H), 7.37-7.40 (m, 1H), 7.47 (t, 2H), 7.95- 7.99 (m, 3H), 8.52 (s, 1H), 8.62 (d, 1H), 8.92 (d, 1H). Docket No. 4390.3002 WO

[0378] 19F NMR (400 MHz, DMSO-d6): 6 -145.74_-145.82 (IF), -50.90_-50.98 (IF).

[0379] LCMS (Method-C): Retention time: 2.237 min, ES(+ve): 545.6 [M+H]+

[0380] Example 23: N-(2-(l-(6-fluoropyrimidin-4-yl)-lH-pyrazol-4-yl)ethyl)-N-methyl-4- morpholino-6-(3-phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine

[0381] Prepared the method of Example 20 using 4,6-difluoropyrimidine (0.064 g, 0.557 mmol). The crude product was purified by column chromatography. The product was eluted at 20 % EtOAc in hexanes to obtain title compound as white solid (0.080 g, 0.152 mmol, 54 %).

[0382] 'H NMR (400 MHz, DMSO-d6): 8 2.85 (t, 2H), 3.13 (s, 3H), 3.55-3.71 (m, 8H), 3.82-3.93 (m, 2H), 6.53 (s, 1H), 7.02 (d, 1H), 7.36-7.40 (m, 1H), 7.44-7.48 (m, 2H), 7.53 (s, 1H), 7.92- 7.97 (m, 3H), 8.57 (s, 1H), 8.62 (d, 1H), 8.83 (s, 1H).

[0383] 19F NMR (400 MHz, DMSO-d6): 6 -59 51 (IF)

[0384] LCMS (Method-C): Retention time: 2.379 min, ES(+ve): 527.7 [M+H]+

[0385] Example 24: N-(2-(l-(6-fluoro-2-methylpyrimidin-4-yl)-lH-pyrazol-4-yl)ethyl)-N- methyl-4-morpholino-6-(3-phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine

[0386] Prepared by the method of Example 19, Step 2 using N-(2-(lH-pyrazol-4-yl)ethyl)-N-methyl- 4-morpholino-6-(3-phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine (Example 19, Step 1) (0.120 g, 0.278 mmol) and 4,6-difluoro-2-methylpyrimidine (0.072 g, 0.557 mmol). The resulting crude product was purified by column chromatography. The product was eluted at 20 % EtOAc in hexane to obtain title compound as white solid (0.083 g, 0.153 mmol, 55 %).

[0387] 'H NMR (400 MHz, DMSO-d6): 62.57 (s, 3H), 2.86 (t, 2H), 3.14 (s, 3H), 3.59-3.64 (m, 4H), 3.66-3.72 (m, 4H), 3.86-3.95 (m, 2H), 6.54 (s, 1H), 7.03 (d, 1H), 7.33 (s, 1H), 7.37-7.41 (m, 1H), 7.45-7.49 (m, 2H), 7.90 (s, 1H), 7.96-7.98 (m, 2H), 8.54 (s, 1H), 8.63 (d, 1H). Docket No. 4390.3002 WO

[0388] 19F NMR (400 MHz, DMSO-d6): 6 -60.08 (IF).

[0389] LCMS (Method-H): Retention time: 4.823 min, ES(+ve): 541.4 [M+H]+

[0390] Example 25: N-(2-(l-(6-chloropyrimidin-4-yl)-lH-pyrazol-4-yl)ethyl)-N-methyl-4- morpholino-6-(3-phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine

[0391] Prepared by a method generally analogous to Example 15 using N-(2-(lH-pyrazol-4-yl)ethyl)- N-methyl-4-morpholino-6-(3-phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine (Example 19, Step 1) (0.100 g, 0.232 mmol) and 4, 6-di chloropyrimidine (0.069 g, 0.464 mmol). The crude product was purified by column chromatography. The desired product was eluted at 15 % EtOAc in hexanes to obtain title compound as off-white solid. (0.035 g, 0.064 mmol, 27 %).

[0392] 'H NMR (400 MHz, DMSO-d6): 8 2.86 (t, 2H), 3.14 (s, 3H), 3.60-3.64 (m, 4H), 3.66-3.72 (m, 4H), 3.85-3.95 (m, 2H), 6.54 (s, 1H), 7.02 (d, 1H), 7.37-7.41 (m, 1H), 7.47 (t, 2H), 7.88- 7.98 (m, 4H), 8.57 (s, 1H), 8.62 (d, 1H), 8.90 (d, 1H).

[0393] LCMS (Method-C): Retention time: 2.421 min, ES(+ve): 543.1 / 545.1 [M+H]+

[0394] Example 26: N-(2-(l-(2,5-difluoropyrimidin-4-yl)-lH-pyrazol-4-yl)ethyl)-N-methyl-4- morpholino-6-(lH-pyrazol-l-yl)pyrimidin-2-amine and Example 27: N-(2-(l-(4,5- difluoropyrimidin-2-yl)-lH-pyrazol-4-yl)ethyl)-N-methyl-4-morpholino-6-(lH-pyrazol- l-yl)pyrimidin-2-amine Docket No. 4390.3002 WO

[0395] Step 1: Synthesis of 4-morpholino-6-(lH-pyrazol-l-yl)pyrimidin-2-amine

[0396] To a stirred solution of 4-chloro-6-morpholinopyrimidin-2-amine (10.0 g, 46.587 mmol) in DMF (100 mL), was added CS2CO3 (30.28 g, 93.174 mmol) and IH-pyrazole (8.10 g, 51.246 mmol) at room temperature under nitrogen atmosphere. The reaction mixture was heated at 120 °C and stirred for 10 h. After completion, the reaction mixture was quenched with water (300 mL) and extracted with EtOAc (3 x 400 mL). The combined organic phase was dried over sodium sulfate and evaporated under reduced pressure to get crude product. The crude was purified by silica gel flash chromatography. Pure compound was eluted at a gradient of 20-30% EtOAc in hexanes to obtain title compound as a solid (5.7 g, 23.145 mmol, 49 %). LCMS (Method-C): Retention time: 1.373 min, ES(+ve): 247.3 [M+H]+

[0397] Step 2: Synthesis of 4-(2-iodo-6-(lH-pyrazol-l-yl)pyrimidin-4-yl)morpholine

[0398] To a solution of 4-morpholino-6-(lH-pyrazol-l-yl)pyrimidin-2-amine (1.00 g, 4.060 mmol) in THF (10 mL) were added Cui (0.85 g, 4.466), diiodomethane (5.43 g, 20.302 mmol) and isoamyl nitrite (0.95 g, 8.121 mmol) under nitrogen atmosphere and the mixture was stirred at 100 °C for 30 min. After completion, the reaction solvent was evaporated under reduced pressure to obtain crude compound. The crude was purified by silica gel flash chromatography. Pure compound was eluted at a gradient of 10-15 % EtOAc in hexanes to obtain title compound as an off-white solid (0.430 g, 1.203 mmol, 29 %).

[0399] 'H NMR (400 MHz, CDCI3): 8 3.62-3.83 (m, 8H), 6.44-6.46 (m, 1H), 7.04 (s, 1H), 7.74 (d, 1H), 8.49 (d, 1H).

[0400] LCMS (Method-C): Retention time: 1.825 min, ES(+ve): 358.2 [M+H]+

[0401] Step 3: Synthesis of N-(2-(lH-pyrazol-4-yl)ethyl)-N-methyl-4-morpholino-6-(lH- pyrazol-l-yl)pyrimidin-2-amine

[0402] CS2CO3 (1.45 g, 4.479 mmol) was added to a stirred solution of 4-(2-iodo-6-(lH-pyrazol-l- yl)pyrimidin-4-yl)morpholine (0.40 g, 1.119 mmol) and N-methyl-2-(lH-pyrazol-4-yl)ethan- 1-amine (0.21 g, 1.679 mmol) in dioxane (4.0 mL) at room temperature under nitrogen atmosphere. The reaction mixture was purged with nitrogen gas for 10 min. Pd2(dba)3 (0.153 g, 0.167 mmol) and XPhos (CAS: 564483-18-7) (0.266 g, 0.559 mmol) were added and the reaction mixture was heated at 100 °C for 4 h. After completion, the reaction mixture was diluted with water (10 mL) and the aqueous layer was extracted with EtOAc (3 x 20 mL). The combined organic phases were dried over sodium sulfate and evaporated under reduced pressure. The crude residue was purified by silica gel flash chromatography and pure Docket No. 4390.3002 WO compound was eluted at 2-4 % MeOH in DCM to obtain title compound as a yellow solid (0.23 g, 0.648 mmol, 57 %).

[0403] 'H NMR (400 MHz, DMSO-d6): 62.66-2.73 (m, 2H), 3.09 (s, 3H), 3.50-3.80 (m, 10H), 6.45 (s, 1H), 6.52 (s, 1H), 7.30-7.65 (m, 2H), 7.77 (s, 1H), 8.53 (s, 1H), 12.55 (s, 1H).

[0404] LCMS (Method-H): Retention time: 2.838 min, ES(+ve): 355.2 [M+H]+

[0405] Step 4: Synthesis of N-(2-(l-(2,5-difluoropyrimidin-4-yl)-lH-pyrazol-4-yl)ethyl)-N- methyl-4-morpholino-6-(lH-pyrazol-l-yl)pyrimidin-2-amine and N-(2-(l-(4,5- difluoropyrimidin-2-yl)-lH-pyrazol-4-yl)ethyl)-N-methyl-4-morpholino-6-(lH-pyrazol- l-yl)pyriniidin-2-aniine

[0406] To a stirred solution of N-(2-(lH-pyrazol-4-yl)ethyl)-N-methyl-4-morpholino-6-(lH-pyrazol- l-yl)pyrimidin-2-amine (0.100 g, 0.282 mmol) in DMSO (1 mL), was added DIPEA(0.19 mL, 1.128 mmol) and 2,4,5-trifluoropyrimidine (0.075 g, 0.564 mmol) at room temperature under nitrogen atmosphere. The reaction mixture was stirred at room temperature for 16 h. After completion, the reaction mixture was quenched by water (5 mL) and extracted with EtOAc (2 x 30 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered and solvent was removed in vacuum to obtain crude product. The resulting crude product was purified by column chromatography. Elution with 10 % EtOAc in hexanes afforded N-(2-(l- (2,5-difluoropyrimidin-4-yl)-lH-pyrazol-4-yl)ethyl)-N-methyl-4-morpholino-6-(lH-pyrazol- l-yl)pyrimidin-2-amine as a white solid (0.048 g, 0.102 mmol, 36 %).

[0407] Hl NMR (400 MHz, DMSO-d6): 8 2.85 (t, 2H), 3.11 (s, 3H), 3.55-3.60 (m, 4H), 3.63-3.68 (m, 4H), 3.83-3.91 (m, 2H), 6.43 (s, 1H), 6.50 (s, 1H), 7.75 (s, 1H), 7.96 (s, 1H), 8.49 (s, 1H), 8.55 (d, 1H), 8.92 (d, 1H).

[0408] 19F NMR (400 MHz, DMSO-d6): 6 -145.75 -145.83 (IF), -50.92_-50.99 (IF).

[0409] LCMS (Method-H): Retention time: 3.539 min, ES(+ve): 469.0 [M+H]+

[0410] Further elution with 20 % EtOAc in hexane afforded N-(2-(l-(4,5-difluoropyrimidin-2-yl)-lH- pyrazol-4-yl)ethyl)-N-methyl-4-morpholino-6-(lH-pyrazol-l-yl)pyrimidin-2-amine as an off- white solid (0.009 g, 0.019 mmol, 7 %).

[0411] 'H NMR (400 MHz, DMSO-d6): 6 2.82 (t, 2H), 3.11 (s, 3H), 3.54-3.60 (m, 4H), 3.63-3.68 (m, 4H), 3.82-3.88 (m, 2H), 6.43 (s, 1H), 6.51 (s, 1H), 7.76 (s, 1H), 7.79 (s, 1H), 8.44 (s, 1H), 8.55 (s, 1H), 9.02-9.08 (m, 1H).

[0412] 19F NMR (400 MHz, DMSO-d6): 6 -158.34 -158.41 (IF), -78.41_-78.48 (IF).

[0413] LCMS (Method-H): Retention time: 3.436 min, ES(+ve): 469.0 [M+H]+ Docket No. 4390.3002 WO

[0414] Example 28: N-(2-(l-(2-fluoropyrimidin-4-yl)-lH-pyrazol-4-yl)ethyl)-N-methyl-4- morpholino-6-(lH-pyrazol-l-yl)pyrimidin-2-amine and Example 29: N-(2-(l-(4- fluoropyrimidin-2-yl)-lH-pyrazol-4-yl)ethyl)-N-methyl-4-morpholino-6-(lH-pyrazol-l- yl)pyrimidin-2-amine

[0415] Prepared by a method generally analogous to Examples 20 and 21 using N-(2-(lH-pyrazol-4- yl)ethyl)-N-methyl-4-morpholino-6-(lH-pyrazol-l-yl)pyrimidin-2-amine (Examples 26 and 27, Step 3) (0.200 g, 0.564 mmol). The reaction mixture was stirred at room temperature for 16 h. The crude product was purified by column chromatography. Elution with 20 % EtOAc in hexanes afforded N-(2-(l-(2-fluoropyrimidin-4-yl)-lH-pyrazol-4-yl)ethyl)-N-methyl-4- morpholino-6-(lH-pyrazol-l-yl)pyrimidin-2-amine (Example 28) (0.055 g, 0.122 mmol, 21 %) as an off-white solid.

[0416] 'H NMR (400 MHz, DMSO-d6): 8 2.84 (t, 2H), 3.12 (s, 3H), 3.54-3.61 (m, 4H), 3.63-3.71 (m, 4H), 3.85-3.89 (m, 2H), 6.43 (s, 1H), 6.50-6.51 (m, 1H), 7.76 -7.81 (m, 2H), 7.92 (s, 1H), 8.53-8.56 (m, 2H), 8.76-8.78 (m, 1H).

[0417] 19F NMR (400 MHz, DMSO-d6): 6 -45 63 (IF).

[0418] LCMS (Method-C): Retention time: 1.929 min, ES(+ve): 451.4 [M+H]+

[0419] Further elution with 2 % MeOH in DCM afforded N-(2-(l-(4-fluoropyrimidin-2-yl)-lH- pyrazol-4-yl)ethyl)-N-methyl-4-morpholino-6-(lH-pyrazol-l-yl)pyrimidin-2-amine (Example 29) (0.035 g, 0.0776 mmol, 14 %) as an off-white solid.

[0420] 'H NMR (400 MHz, DMSO-d6): 6 2.83 (t, 2H), 3.11 (s, 3H), 3.53-3.61 (m, 4H), 3.62-3.69 (m, 4H), 3.72-3.91 (m, 2H), 6.43 (s, 1H), 6.50 (s, 1H), 7.27 -7.29 (m, 1H), 7.76 -7.80 (m, 2H), 8.50- 8.55 (m, 2H), 8.88-8.92 (m, 1H).

[0421] 19F NMR (400 MHz, DMSO-d6): 6 -58 64 (IF).

[0422] LCMS (Method-C): Retention time: 1.770 min, ES(+ve): 451.4 [M+H]+ Docket No. 4390.3002 WO

[0423] Example 30: N-(2-(l-(6-fluoropyrimidin-4-yl)-lH-pyrazol-4-yl)ethyl)-N-methyl-4- morpholino-6-(lH-pyrazol-l-yl)pyrimidin-2-amine

[0424] Example 30

[0425] Prepared by a method generally analogous to Example 17 using N-(2-(lH-pyrazol-4- yl)ethyl)-N-methyl-4-morpholino-6-(lH-pyrazol-l-yl)pyrimidin-2-amine (Examples 26 and 27, Step 3) (0.120 g, 0.338 mmol). The reaction mixture was stirred at room temperature for 5 h. The crude product was purified by column chromatography. The product was eluted at 25 % EtOAc in hexanes to obtain title compound as a white solid (0.055 g, 0.122 mmol, 36 %). 'H NMR (400 MHz, DMSO-d6): 8 2.85 (t, 2H), 3.13 (s, 3H), 3.55-3.62 (m, 4H), 3.64-3.70 (m, 4H), 3.83-3.92 (m, 2H), 6.44 (s, 1H), 6.51-6.52 (m, 1H), 7.54 (s, 1H), 7.77 (d, H), 7.91 (s, 1H), 8.56 (d, 2H), 8.84 (d, 1H).

[0426] 19F NMR (400 MHz, DMSO-d6): 6 -59 66 (IF).

[0427] LCMS (Method-C): Retention time: 1.977 min, ES(+ve): 451.4 [M+H]+

[0428] Example 31 : N-(2-(l-(2-fluoropyrimidin-4-yl)-lH-pyrazol-4-yl)ethyl)-4-morpholino-6- (3-phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine and Example 32: N-(2-(l-(4- fluoropyrimidin-2-yl)-lH-pyrazol-4-yl)ethyl)-4-morpholino-6-(3-phenyl-lH-pyrazol-l- yl)pyrimidin-2-amine

[0429] Example 31 Example 32 Docket No. 4390.3002 WO

[0430] Step 1: Synthesis of N-(2-(lH-pyrazol-4-yl)ethyl)-4-morpholino-6-(3-phenyl-lH-pyrazol- l-yl)pyrimidin-2-amine

[0431] CS2CO3 (0.90 g, 2.769 mmol) was added to a stirred solution of 4-(2-iodo-6-(3-phenyl-lH- pyrazol-l-yl) pyrimidin-4-yl) morpholine (WO2021163727 Al) (0.30 g, 0.692 mmol) and 2- (lH-pyrazol-4-yl) ethan-l-amine (0.154 g, 1.384 mmol) in dioxane (3.0 mL) at room temperature under nitrogen atmosphere. The reaction mixture was purged with nitrogen gas for 10 min. Pd2(dba)3(0.095 g, 0.103 mmol) and XPhos (CAS: 564483-18-7) (0.165 g, 0.346 mmol) were added and the reaction mixture was heated at 100 °C for 16 h. After completion, the reaction mixture was diluted with water (10 mL) and aqueous layer was extracted with EtOAc (3 x 30 mL). The combined organic phases were dried over sodium sulfate and evaporated under reduced pressure. The crude residue was purified by silica gel flash chromatography and pure compound was eluted at 2-4 % MeOH in DCM to obtain title compound as a yellow solid (0.12 g, 0.288 mmol, 42 %).

[0432] 'H NMR (400 MHz, DMSO-d6): 82.66-2.72 (m, 2H), 3.37-3.39 (m, 2H), 3.56-3.73 (m, 8H),

[0433] 6.56 (s, 1H), 6.92-6.99 (m, 1H), 7.02-7.03 (m, 1H), 7.36-7.38 (m. 2H), 7.44-7.47 (m, 2H), 7.52-

[0434] 7.57 (m, 1H), 7.95-7.97 (m, 2H), 8.47-8.54 (m, 1H), 12.54 (s, 1H).

[0435] LCMS (Method-C): Retention time: 1.695 min, ES(+ve): 417.4 [M+H]+

[0436] Step 2: Synthesis of N-(2-(l-(2-fluoropyrimidin-4-yl)-lH-pyrazol-4-yl)ethyl)-4- morpholino-6-(3-phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine and N-(2-(l-(4- fluoropyrimidin-2-yl)-lH-pyrazol-4-yl)ethyl)-4-morpholino-6-(3-phenyl-lH-pyrazol-l- yl)pyrimidin-2-amine

[0437] Prepared by a method generally analogous to Examples 20 and 21 using N-(2-(lH-pyrazol-4- yl)ethyl)-4-morpholino-6-(3-phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine (0.120 g, 0.288 mmol). The reaction mixture was stirred at room temperature for 16 h. The resulting crude product was purified by column chromatography. Elution with 20 % EtOAc in hexanes afforded N-(2-(l-(2-fluoropyrimidin-4-yl)-lH-pyrazol-4-yl)ethyl)-4-morpholino-6-(3-phenyl- lH-pyrazol-l-yl)pyrimidin-2-amine (Example 31) (0.008 g, 0.016 mmol, 5 %) as an off- white solid.

[0438] 'H NMR (400 MHz, DMSO-d6): 6 2.82 (t, 2H), 3.53-3.72 (m, 10H), 6.55 (s, 1H), 7.01-7.12 (m, 2H), 7.36-7.39 (m, 1H), 7.43-7.47 (m, 2H), 7.81-7.83 (m, 1H), 7.94-7.96 (m, 3H), 8.43- 8.65 (m, 2H), 8.76-8.77 (m, 1H).

[0439] 19F NMR (400 MHz, DMSO-d6): 6 -45 57 (IF)

[0440] LCMS (Method-C): Retention time: 1.980 min, ES(+ve): 513.2 [M+H]+ Docket No. 4390.3002 WO

[0441] Further elution with 70 % EtOAc in hexanes afforded N-(2-(l-(4-fluoropyrimidin-2-yl)-lH- pyrazol-4-yl)ethyl)-4-morpholino-6-(3-phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine (Example 32) (0.050 g, 0.097 mmol, 34 %) as an off-white solid.

[0442] 'H NMR (400 MHz, DMSO-d6): 6 2.81 (t, 2H), 3.52-3.72 (m, 10H), 6.57 (s, 1H), 7.02-7.08 (m, 2H), 7.28-7.32 (m, 1H), 7.38-7.40 (m, 1H), 7.45-7.48 (m, 2H), 7.84 (s, 1H), 7.95-7.97 (m, 2H), 8.45-8.65 (m, 2H), 8.90-8.95 (m, 1H).

[0443] 19F NMR (400 MHz, DMSO-d6): 6 -58 63 (IF).

[0444] LCMS (Method-C): Retention time: 1.879 min, ES(+ve): 513.5 [M+H]+

[0445] Example 33: N-(2-(l-(2,5-dichloropyrimidin-4-yl)-lH-pyrazol-4-yl)ethyl)-N-methyl-4- morpholino-6-(3-phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine and Example 34: N-(2-(l- (4,5-dichloropyrimidin-2-yl)-lH-pyrazol-4-yl)ethyl)-N-methyl-4-morpholino-6-(3- phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine

[0446] Example 33 Example 34

[0447] Prepared by a method generally analogous to Examples 20 and 21 using 2,4,5- trichloropyrimidine (0.170 g, 0.929 mmol). The reaction mixture was stirred at room temperature for 16 h. The resulting crude product was purified by column chromatography. Elution with 15 % EtOAc in hexanes afforded N-(2-(l-(2,5-dichloropyrimidin-4-yl)-lH- pyrazol-4-yl)ethyl)-N-methyl-4-morpholino-6-(3-phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine (Example 33) (0.050 g, 0.086 mmol, 18 %) as a pale yellow solid.

[0448] 'H NMR (400 MHz, DMSO-d6): 8 2.82-2.90 (m, 2H), 3.14 (s, 3H), 3.58-3.72 (m, 8H), 3.82- 3.93 (m, 2H), 6.54 (s, 1H), 7.02 (s, 1H), 7.38-7.39 (m, 1H), 7.44-7.48 (m, 2H), 7.95-7.96 (m, 3H), 8.47 (s, 1H), 8.60-8.63 (m, 1H), 8.94 (s, 1H).

[0449] LCMS (Method-H): Retention time: 4.635 min, 577.4 / 579.3 [M+H]+

[0450] Further elution with 30 % EtOAc in hexanes afforded N-(2-(l-(4,5-dichloropyrimidin-2-yl)- lH-pyrazol-4-yl)ethyl)-N-methyl-4-morpholino-6-(3-phenyl-lH-pyrazol-l-yl)pyrimidin-2- amine (Example 34) (0.050 g, 0.086 mmol, 18 %) as a pale yellow solid. Docket No. 4390.3002 WO

[0451] 'H NMR (400 MHz, DMSO-d6): 6 2.80-2.88 (m, 2H), 3.13 (s, 3H), 3.54-3.72 (m, 8H), 3.80- 3.92 (m, 2H), 6.53 (s, 1H), 7.02 (s, 1H), 7.38-7.39 (m, 1H), 7.44-7.46 (m, 2H), 7.84 (m, 1H), 7.95-7.97 (m, 2H), 8.49 (s, 1H), 8.59-8.64 (m, 1H), 8.99 (s, 1H).

[0452] LCMS (Method-H): Retention time: 4.430 min, ES(+ve): 577.1 / 579.2 [M+H]+

[0453] Example 35: N-(2-(l-(5-chloro-2-fluoropyrimidin-4-yl)-lH-pyrazol-4-yl)ethyl)-N- methyl-4-morpholino-6-(3-phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine and Example 36:

[0454] N-(2-(l-(5-chloro-4-fluoropyrimidin-2-yl)-lH-pyrazol-4-yl)ethyl)-N-methyl-4- morpholino-6-(3-phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine

[0455] Example 35 Example 36

[0456] Prepared by a method generally analogous to Examples 20 and 21 using 5-chloro-2,4- difluoropyrimidine (0.105 g, 0.697 mmol). The reaction mixture was stirred at room temperature for 1 h. The resulting crude product was purified by column chromatography. Elution with 20 % EtOAc in hexanes afforded N-(2-(l-(5-chloro-2-fluoropyrimidin-4-yl)-lH- pyrazol-4-yl)ethyl)-N-methyl-4-morpholino-6-(3-phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine (Example 35) (0.063 g, 0.112 mmol, 32 %) as an off-white solid.

[0457] 'H NMR (400 MHz, DMSO-d6): 8 2.87 (t, 2H), 3.15 (s, 3H), 3.60-3.65 (m, 4H), 3.66-3.72 (m, 4H), 3.85-3.93 (m, 2H), 6.55 (s, 1H), 7.02 (d, 1H), 7.37-7.40 (m, 1H), 7.45-7.48 (m, 2H), 7.95-7.97 (m, 3H), 8.51 (s, 1H), 8.62 (d, 1H), 8.97 (s, 1H).

[0458] 19F NMR (400 MHz, DMSO-d6): 6 -49 22 (IF).

[0459] LCMS (Method-H): Retention time: 4.419 min, ES(+ve): 561.2 / 563.0 [M+H]+

[0460] Further elution with 35 % EtOAc in hexanes afforded N-(2-(l-(5-chloro-4-fluoropyrimidin-2- yl)-lH-pyrazol-4-yl)ethyl)-N-methyl-4-morpholino-6-(3-phenyl-lH-pyrazol-l-yl)pyrimidin- 2-amine (Example 36) (0.015 g, 0.027 mmol, 8 %) as an off-white solid.

[0461] 'H NMR (400 MHz, DMSO-d6): 6 2.85 (t, 2H), 3.14 (s, 3H), 3.60-3.65 (m, 4H), 3.67-3.72 (m, 4H), 3.84-3.92 (m, 2H), 6.55 (s, 1H), 7.03 (d, 1H), 7.37-7.40 (m, 1H), 7.45-7.48 (m, 2H), 7.85 (s, 1H), 7.95-7.98 (m, 2H), 8.49 (s, 1H), 8.62 (d, 1H), 9.03-9.06 (m, 1H).

[0462] 19F NMR (400 MHz, DMSO-d6): 6 -63 26 (IF). Docket No. 4390.3002 WO

[0463] LCMS (Method-H): Retention time: 4.218 min, ES(+ve): 561.2 / 563.1 [M+H]+

[0464] Example 37: N-(2-(l-(6-chloro-2-fluoropyrimidin-4-yl)-lH-pyrazol-4-yl)ethyl)-N- methyl-4-morpholino-6-(3-phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine

[0465] Example 37

[0466] Prepared by the method of Example 19, Step 2 using 4,6-dichloro-2-fluoropyrimidine (0.116 g, 0.697 mmol). The resulting crude product was purified by column chromatography. The product was eluted at 20 % EtOAc in hexanes to obtain title compound as an off-white solid (0.018 g, 0.032 mmol, 9 %).

[0467] 'H NMR (400 MHz, DMSO-d6): 8 2.84 (t, 2H), 3.12 (s, 3H), 3.56-3.63 (m, 4H), 3.64-3.71 (m, 4H), 3.83-3.92 (m, 2H), 6.53 (s, 1H), 7.01 (d, 1H), 7.35-7.39 (m, 1H), 7.44-7.47 (m, 2H), 7.84 (s, 1H), 7.94-7.96 (m, 3H), 8.52 (s, 1H), 8.61 (d, 1H).

[0468] 19F NMR (400 MHz, DMSO-d6): 6 -44 60 (IF)

[0469] LCMS (Method-C): Retention time: 2.486 min, ES(+ve): 561.4 / 563.4 [M+H]+

[0470] Example 38: N-(2-(l-(4,6-dichloropyrimidin-2-yl)-lH-pyrazol-4-yl)ethyl)-N-methyl-4- morpholino-6-(3-phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine and Example 39: N-(2-(l- (2,6-dichloropyrimidin-4-yl)-lH-pyrazol-4-yl)ethyl)-N-methyl-4-morpholino-6-(3- phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine

[0471] To a stirred solution of N-(2-(lH-pyrazol-4-yl)ethyl)-N-methyl-4-morpholino-6-(3-phenyl- lH-pyrazol-l-yl)pyrimidin-2-amine (Example 19, Step 1) (0.150 g, 0.348 mmol) in THF (1.5 mL), was added DIPEA(0.15 mL, 0.861 mmol) and 2,4,6-trichloropyrimidine (0.15 mL, 0.512 mmol) at room temperature under nitrogen atmosphere. The reaction mixture was stirred at 60 °C for 16 h. After completion, the reaction mixture was quenched with water and extracted Docket No. 4390.3002 WO with EtOAc (2 x 50 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered and solvent was removed in vacuum to obtain crude product. The resulting crude was purified by reverse phase column chromatography. Elution with 45 % MeCN in water afforded N-(2-(l-(4,6-dichloropyrimidin-2-yl)-lH-pyrazol-4-yl)ethyl)-N-methyl-4-morpholino-6-(3- phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine (Example 38) (0.020 g, 0.034 mmol, 10 %) as an off-white solid.

[0472] 'H NMR (400 MHz, DMSO-d6): 8 2.80-2.87 (m, 2H), 3.13 (s, 3H), 3.58-3.64 (m, 4H), 3.65- 3.71 (m, 4H), 3.84-3.93 (m, 2H), 6.54 (s, 1H), 7.01 (d, 1H), 7.35-7.41 (m, 1H), 7.44-7.47 (m, 2H), 7.86 (s, 2H), 7.94-7.96 (m, 2H), 8.47 (s, 1H), 8.61-8.62 (m, 1H).

[0473] LCMS (Method-C): Retention time: 2.381 min, ES(+ve): 577.2 / 579.2 [M+H]+

[0474] Further elution with 56 % MeCN in water afforded N-(2-(l-(2,6-dichloropyrimidin-4-yl)-lH- pyrazol-4-yl)ethyl)-N-methyl-4-morpholino-6-(3-phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine (Example 39) (0.020 g, 0.034 mmol, 10 %) as an off-white solid.

[0475] 'H NMR (400 MHz, DMSO-d6): 6 2.81-2.89 (m, 2H), 3.13 (s, 3H), 3.57-3.64 (m, 4H), 3.65- 3.71 (m, 4H), 3.84-3.92 (m, 2H), 6.53 (s, 1H), 7.01 (d, 1H), 7.35-7.41 (m, 1H), 7.43-7.47 (m, 2H), 7.86 (s, 1H), 7.94-7.96 (m, 3H), 8.52 (s, 1H), 8.61-8.62 (m, 1H).

[0476] LCMS (Method-C): Retention time: 2.603 min, ES(+ve): 577.2 / 579.3 [M+H]+

[0477] Example 40: N-(2-(l-(4,5-difluoropyrimidin-2-yl)-lH-pyrazol-4-yl)ethyl)-N-methyl-4- morpholino-6-(3-phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine

[0478] To a stirred mixture of A-(2-(lH-pyrazol-4-yl)ethyl)-A-methyl-4-morpholino-6-(3-phenyl-lH- pyrazol-l-yl)pyrimidin-2-amine (Example 19, Step 1) (0.200 g, 0.464 mmol) and CS2CO3 (0.408 g, 1.254 mmol) was added a solution of 2,4,5-trifluoropyrimidine (0.199 g, 1.486 mmol) in THF (2 mL) at room temperature under nitrogen atmosphere. The reaction mixture was stirred at room temperature for 10 min. After completion, the reaction mixture was quenched with water (100 mL) and extracted with EtOAc (2 x 100 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered and solvent was removed in vacuum to obtain crude product. The resulting crude product was purified by column chromatography. Docket No. 4390.3002 WO

[0479] The product was eluted at 30% EtO Ac in hexanes to obtain title compound as an off-white solid (0.027 g, 0.049 mmol, 10 %).

[0480] 'H NMR (400 MHz, DMSO-d6): 6 2.83 (t, 2H), 3.13 (s, 3H), 3.53-3.75 (m, 8H), 3.82-3.93 (m, 2H), 6.54 (s, 1H), 7.02 (d, 1H), 7.34-7.41 (m, 1H), 7.44-7.48 (m, 2H), 7.81(s, 1H), 7.95- 7.97 (m, 2H), 8.45 (s, 1H), 8.61 (d, 1H), 9.03-9.06 (m, 1H).

[0481] 19F NMR (400 MHz, DMSO-d6): 6 -158.41 -158.34 (IF), -78.47_-78.40 (IF).

[0482] LCMS (Method-H): Retention time: 4.121 min, ES(+ve): 545.2 [M+H]+

[0483] Example 41: N-(2-(l-(6-fluoro-5-methylpyrimidin-4-yl)-lH-pyrazol-4-yl)ethyl)-N- methyl-4-morpholino-6-(3-phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine

[0484] Prepared by the method of Example 16 using A-(2-(lH-pyrazol-4-yl)ethyl)-A-methyl-4- morpholino-6-(3-phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine (Example 19, Step 1) (0.120 g, 0.278 mmol) and 4,6-difluoro-5-methylpyrimidine (0.072 g, 0.557 mmol). The resulting crude product was purified by column chromatography. The product was eluted at 15 % EtO Ac in hexanes to obtain title compound as an off-white solid (0.043 g, 0.079 mmol, 28 %)

[0485] 'H NMR (400 MHz, DMSO-d6): 8 2.43 (s, 3H), 2.81-2.88 (m, 2H), 3.15 (s, 3H), 3.58-3.64 (m, 4H), 3.65-3.72 (m, 4H), 3.83-3.92 (m, 2H), 6.53 (s, 1H), 7.01 (s, 1H), 7.35-7.42 (m, 1H), 7.44-7.48 (m, 2H), 7.88 (s, 1H), 7.95-7.96 (m, 2H), 8.55 (s, 1H), 8.61 (s, 1H), 8.66 (s, 1H).

[0486] 19F NMR (400 MHz, DMSO-d6): 6 -64 21 (IF).

[0487] LCMS (Method-H): Retention time: 4.811 min, ES(+ve): 541.4 [M+H]+

[0488] Docket No. 4390.3002 WO

[0489] Example 42: N-(2-(l-(2-fluoropyrimidin-4-yl)-lH-pyrazol-3-yl)ethyl)-N-methyl-4- morpholino-6-(3-phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine and Example 43: N-(2-(l- (4-fluoropyrimidin-2-yl)-lH-pyrazol-5-yl)ethyl)-N-methyl-4-morpholino-6-(3-phenyl- lH-pyrazol-l-yl)pyrimidin-2-amine

[0490] Step 1: Synthesis of N-(2-(lH-pyrazol-3-yl)ethyl)-N-methyl-4-morpholino-6-(3-phenyl- lH-pyrazol-l-yl)pyrimidin-2-amine

[0491] To a stirred solution of 4-(2-iodo-6-(3-phenyl-lH-pyrazol-l-yl)pyrimidin-4-yl)morpholine (WO2021163727 Al) (0.3 g, 0.693 mmol) and N-methyl-2-(lH-pyrazol-3-yl)ethan-l-amine (0.130 g, 1.039 mmol) in dioxane (3 mL) was added CS2CO3 (0.903 g, 2.770 mmol) at room temperature under nitrogen atmosphere. The reaction mixture was purged with nitrogen gas for 10 min. Pd2(dba)3(0.095 g, 0.104 mmol) and XPhos (CAS: 564483-18-7) (0.165 g, 0.346 mmol) were added and the reaction mixture was heated at 100 °C for 2 h. After completion, the reaction mixture was diluted with water (50 mL) and the aqueous layer was extracted with EtOAc (2 x 100 mL). The combined organic phases were dried over sodium sulfate and evaporated under reduced pressure. The crude residue was purified by silica gel flash chromatography and pure compound was eluted at 1.5 % MeOH in DCM to obtain title compound as an off-white solid (0.18 g, 0.418 mmol, 40 %).

[0492] LCMS (Method-H): Retention time: 3.612 min, ES(+ve): 431.6 [M+H]+

[0493] Step 2: Synthesis of N-(2-(l-(2-fluoropyrimidin-4-yl)-lH-pyrazol-3-yl)ethyl)-N-methyl- 4-morpholino-6-(3-phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine and N-(2-(l-(4- Docket No. 4390.3002 WO fluoropyrimidin-2-yl)-lH-pyrazol-5-yl)ethyl)-N-methyl-4-morpholino-6-(3-phenyl-lH- pyrazol-l-yl)pyrimidin-2-amine

[0494] To a stirred solution of N-(2-(lH-pyrazol-3-yl)ethyl)-N-methyl-4-morpholino-6-(3-phenyl- lH-pyrazol-l-yl)pyrimidin-2-amine (0.180 g, 0.418 mmol) in DMSO (1.8 mL), was added DIPEA (0.29 mL, 1.672 mmol) and 2,4-difluoropyrimidine (0.097 g, 0.836 mmol) at room temperature under nitrogen atmosphere. The reaction mixture was stirred at room temperature for 16 h. After completion, the reaction mixture was quenched by water (20 mL) and extracted with EtOAc (2 x 50 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered and solvent was removed in vacuum to obtain crude product. The resulting crude product was purified by column chromatography. Elution with 10 % EtOAc in hexanes afforded N-(2-(l-(2-fluoropyrimidin-4-yl)-lH-pyrazol-3-yl)ethyl)-N-methyl-4-morpholino-6- (3-phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine (Example 42) as an off-white solid (0.023 g, 0.044 mmol, 10 %).

[0495] 1H NMR (400 MHz, DMSO-d6): 8 3.01 (t, 2H), 3.15 (s, 3H), 3.58-3.71 (m, 8H), 3.90-4.00 (m, 2H), 6.54 (s, 1H), 6.62 (s, 1H), 7.01 (s, 1H), 7.36-7.40 (m, 1H), 7.44-7.48 (m, 2H), 7.79 (s, 1H), 7.95 (d, 2H), 8.54 (s, 1H), 8.60 (s, 1H), 8.75-8.81 (m, 1H).

[0496] 19F NMR (400 MHz, DMSO-d6): 6 -45 56 (IF)

[0497] LCMS (Method-C Fast): Retention time: 2.312 min, ES(+ve): 527.8 [M+H]+

[0498] Further elution with 40 % EtOAc in hexane afforded N-(2-(l-(4-fluoropyrimidin-2-yl)-lH- pyrazol-5-yl)ethyl)-N-methyl-4-morpholino-6-(3-phenyl-lH-pyrazol-l-yl)pyrimidin-2-amine (Example 43) as an off-white solid (0.014 g, 0.026 mmol, 6 %).

[0499] 'H NMR (400 MHz, DMSO-d6): 62.91-3.07 (m, 3H), 3.42-3.74 (m, 10H), 3.86-3.97 (m, 2H), 6.38-6.57 (m, 1H), 6.51 (s, 1H), 7.00 (s, 1H), 7.28-7.33 (m, 1 H), 7.36-7.40 (m, 1H), 7.44-7.48 (m, 2H), 7.71 (s, 1H), 7.96 (d, 2H), 8.27-8.63 (br. m, 1H), 8.86-8.97 (m, 1H).

[0500] 19F NMR (400 MHz, DMSO-d6): 6 -58 38 (IF)

[0501] LCMS (Method-C): Retention time: 2.109 min, ES(+ve): 527.5 [M+H]+ Docket No. 4390.3002 WO

[0502] Example 44: N-(2-(l-(4-fluoropyrimidin-2-yl)-lH-pyrazol-4-yl)ethyl)-4-morpholino-6- (lH-pyrazol-l-yl)pyrimidin-2-amine

[0503] Step 1: Synthesis of N-(2-(lH-pyrazol-4-yl)ethyl)-4-morpholino-6-(lH-pyrazol-l- yl)pyrimidin-2-amine

[0504] Prepared by the method of Example 26, Step 3 using 2-(lH-pyrazol-4-yl) ethan-l-amine (0.155 g, 1.399 mmol). The resulting crude product was purified by silica gel flash chromatography eluted with 2-4 % MeOH in DCM to obtain title compound as a yellow solid (0.10 g, 0.293 mmol, 42 %).

[0505] LCMS (Method-C): Retention time: 1.409 min, ES(+ve): 341.4 [M+H]+

[0506] Step 2: Synthesis of N-(2-(l-(4-fluoropyrimidin-2-yl)-lH-pyrazol-4-yl)ethyl)-4- morpholino-6-(lH-pyrazol-l-yl)pyrimidin-2-amine

[0507] Prepared by the method of Example 28 and Example 29 using N-(2-(lH-pyrazol-4-yl)ethyl)- 4-morpholino-6-(lH-pyrazol-l-yl)pyrimidin-2-amine (0.10 g, 0.293 mmol). The crude product was purified by column chromatography eluted with 70 % EtOAc in hexanes to obtain the title product as off white solid (0.020 g, 0.045 mmol, 15 %).

[0508] 'H NMR (400 MHz, DMSO-d6): 82.77-2.83 (m, 2H), 3.51-3.61 (m, 6H), 3.63-3.69 (m, 4H), 6.45 (s, 1H), 6.51-6.52 (m, 1H), 6.97-7.11 (m, 1H), 7.29 -7.31 (m, 1H), 7.77 (d, 1H), 7.82 (s,lH) 8.37-8.63 (br. s, 1H), 8.51 (s, 1H), 8.90-8.94 (m, 1H).

[0509] 19F NMR (400 MHz, DMSO-d6): 6 -58 63 (IF)

[0510] LCMS (Method-J): Retention time: 3.155 min, ES(+ve): 437.0 [M+H]+ Docket No. 4390.3002 WO

[0511] Example 45: N-(2-(l-(2-fluoropyrimidin-4-yl)-lH-pyrazol-4-yl)ethyl)-N-methyl-4-(3- methyl-lH-pyrazol-l-yl)-6-morpholinopyrimidin-2-amine

[0512] Step 1: Synthesis of 4-(3-methyl-lH-pyrazol-l-yl)-6-morpholinopyrimidin-2-amine

[0513] Prepared by a method analogous to Example 26, Step 1 using 3-methyl-lH-pyrazole (1.0 g, 12.178 mmol). The reaction mixture was heated at 120 °C for 16 h. The crude product was purified by silica gel flash chromatography. The title product was eluted with 40-50 % EtOAc in hexane to obtain a white solid (1.90 g, 7.30 mmol, 54 %).

[0514] LCMS (Method-C): Retention time: 1.377 min, ES(+ve): 261.0 [M+H]+

[0515] Step 2: Synthesis of 4-(2-iodo-6-(3-methyl-lH-pyrazol-l-yl)pyrimidin-4-yl)morpholine

[0516] Prepared by a method analogous to Example 26, Step 2 using 4-(3-methyl-lH-pyrazol-l-yl)- 6-morpholinopyrimidin-2-amine (1.90 g, 7.307 mmol). The reaction mixture was heated at 100 °C for 16 h. The title compound was obtained as a brown solid (0.450 g, 1.21 mmol, 16 %).

[0517] LCMS (Method-C): Retention time: 2.043 min, ES(+ve): 372.2 [M+H]+

[0518] Step 3: Synthesis of N-(2-(lH-pyrazol-4-yl)ethyl)-N-methyl-4-(3-methyl-lH-pyrazol-l- yl)-6-morpholinopyrimidin-2-amine

[0519] Prepared by a method analogous to Example 26, Step 3 using 4-(2-iodo-6-(3-methyl-lH- pyrazol-l-yl)pyrimidin-4-yl)morpholine (0.400 g, 0.538 mmol). The reaction mixture was heated at 100 °C for 16 h. The crude product was purified by silica gel flash chromatography. The title product was eluted with 70 % EtOAc in hexane to obtain a brown solid (0.300 g, 0.815 mmol, 75 %). Docket No. 4390.3002 WO

[0520] LCMS (Method-C): Retention time: 1.621 min, ES(+ve): 369.4 [M+H]+

[0521] Step 4: Synthesis of N-(2-(l-(2-fluoropyrimidin-4-yl)-lH-pyrazol-4-yl)ethyl)-N-methyl- 4-(3-methyl-lH-pyrazol-l-yl)-6-morpholinopyrimidin-2-amine

[0522] Prepared by the method of Example 28 and Example 29 using N-(2-(lH-pyrazol-4-yl)ethyl)- N-methyl-4-(3 -methyl- lH-pyrazol-l-yl)-6-morpholinopyrimidin-2-amine (0.300 g, 0.814 mmol). The crude product was purified by silica gel flash chromatography. The title product was eluted with 40-70 % EtOAc in hexane to obtain an off-white solid (0.012 g, 0.025 mmol, 3 %).

[0523] 'H NMR (400 MHz, DMSO-d6): 82.26 (s, 3H), 2.83 (t, 2H), 3.11 (s, 3H), 3.52-3.59 (m, 4H), 3.64-3.70 (m, 4H), 3.83-3.90 (m, 2H), 6.31-6.34 (m, 2H), 7.78-7.83 (m, 1H), 7.91 (s, 1H), 8.43 (d, 1H), 8.53 (s, 1H), 8.75-8.79 (m, 1H).

[0524] 19F NMR (400 MHz, DMSO-d6): 6 -45 65 (IF)

[0525] LCMS (Method-C): Retention time: 1.929 min, ES(+ve): 465.4 [M+H]+.

[0526] Example 46: 4-(3-chloro-lH-pyrazol-l-yl)-N-(2-(l-(2-fluoropyrimidin-4-yl)-lH-pyrazol- 4-yl)ethyl)-N-methyl-6-morpholinopyrimidin-2-amine

[0527] Prepared by a synthetic sequence directly analogues to Example 45 using 3-chloro-lH- pyrazole (2.3 g, 10.731 mmol) in Step 1.

[0528] Step 1: 4-(3-chloro-lH-pyrazol-l-yl)-6-morpholinopyrimidin-2-amine

[0529] Yield (2.9 g, crude). Off-white solid.

[0530] LCMS (Method-C): Retention time: 1.528 min, ES(+ve): 281.2 / 283.2 [M+H]

[0531] Step 2: 4-(6-(3-chloro-lH-pyrazol-l-yl)-2-iodopyrimidin-4-yl)morpholine Docket No. 4390.3002 WO

[0532] Yield (0.8 g, 2.042 mmol, 38 %). Brown solid.

[0533] LCMS (Method-C): Retention time: 2.161 min, ES(+ve): 392.2 / 394.2 [M+H]+

[0534] Step 3: N-(2-(lH-pyrazol-4-yl)ethyl)-4-(3-chloro-lH-pyrazol-l-yl)-N-methyl-6- morpholinopyrimidin-2-amine

[0535] Yield (0.140 g, 0.360 mmol, 33 %). Brown oil.

[0536] 'H NMR (400 MHz, DMSO-d6): 62.65-2.75 (m, 2H), 3.09 (s, 3H), 3.53-3.82 (m, 10H), 6.34

[0537] (s, 1H), 6.64 (s, 1H), 7.28-7.63 (m, 2H), 8.59 (s, 1H), 12.53 (s, 1H).

[0538] LCMS (Method-C): Retention time: 1.756 min, ES(+ve): 389.4 / 391.3 [M+H]+

[0539] Step 4: 4-(3-chloro-lH-pyrazol-l-yl)-N-(2-(l-(2-fluoropyrimidin-4-yl)-lH-pyrazol-4- yl)ethyl)-N-methyl-6-morpholinopyrimidin-2-amine

[0540] Yield (0.060 g, 1.237 mmol, 34 %). White solid.

[0541] Hl NMR (400 MHz, DMSO-d6): 8 2.82 (t, 2H), 3.10 (s, 3H), 3.53-3.60 (m, 4H), 3.61-3.67 (m, 4H), 3.82-3.90 (m, 2H), 6.30 (s, 1H), 6.62 (d, 1H), 7.76-7.81 (m, 1H), 7.90 (s, 1H), 8.52 (s, 1H), 8.61(d, 1H), 8.76 (d, 1H).

[0542] 19F NMR (400 MHz, DMSO-d6): 6 -45 64 (IF)

[0543] LCMS (Method-C): Retention time: 2.114 min, ES(+ve): 485.4 / 487.4 [M+H]+

[0544] Example 47: 4-(3-bromo-lH-pyrazol-l-yl)-N-(2-(l-(4-fluoropyrimidin-2-yl)-lH-pyrazol- 4-yl)ethyl)-N-methyl-6-morpholinopyrimidin-2-amine

[0545] Prepared by a synthetic sequence very similar to Example 45 using 3-bromo-lH-pyrazole (1.80 g, 12.814 mmol) in Step 1, and stirring Step 4 for only 2 h to give the alternative fluoropyrimidine regio-isomer. Docket No. 4390.3002 WO

[0546] Step 1: 4-(3-bromo-lH-pyrazol-l-yl)-6-morpholinopyrimidin-2-amine

[0547] Yield (3.5 g, 11.692 mmol, 92 %). Off-white solid.

[0548] 'H NMR (400 MHz, DMSO-d6): 63.53-3.69 (m, 8H), 6.36 (s, 1H), 6.45 (s, 2H), 6.67 (d, 1H), 8.37 (d, 1H).

[0549] Step 2: 4-(6-(3-bromo-lH-pyrazol-l-yl)-2-iodopyrimidin-4-yl)morpholine

[0550] Yield (1.8 g, 4.128 mmol, 38 %). Brown solid.

[0551] LCMS (Method-J): Retention time: 3.665 min, ES(+ve): 436.1 / 438.1 [M+H]+

[0552] Step 3: N-(2-(lH-pyrazol-4-yl)ethyl)-4-(3-bromo-lH-pyrazol-l-yl)-N-methyl-6- morpholinopyrimidin-2-amine

[0553] Yield (0.110 g, 0.253 mmol, 36 %). Brown solid.

[0554] LCMS (Method-H): Retention time: 3.311 min, ES(+ve): 433.4 / 435.4 [M+H]+

[0555] Step 4: 4-(3-bromo-lH-pyrazol-l-yl)-N-(2-(l-(4-fluoropyrimidin-2-yl)-lH-pyrazol-4- yl)ethyl)-N-methyl-6-morpholinopyrimidin-2-amine

[0556] (0.025 g, 0.047 mmol, 20 %). White solid.

[0557] Hl NMR (400 MHz, DMSO-d6): 6 2.81 (t, 2H), 3.10 (s, 3H), 3.53-3.71 (m, 8H), 3.80-3.90 (m, 2H), 6.32 (s, 1H), 6.68 (t, 1H), 7.28 (d, 1H), 7.80 (s, 1H), 8.49 (s, 1H), 8.56 (t, 1H), 8.87- 8.91 (m, 1H).

[0558] 19F NMR (400 MHz, DMSO-d6): 6 -53 887 (IF)

[0559] LCMS (Method-H): Retention time: 3.697 min, ES(+ve): 529.4 / 531.4 [M+H]+

[0560] Example 48: N-(2-(l-(2-fluoropyrimidin-4-yl)-lH-pyrazol-3-yl)ethyl)-N-methyl-4- morpholino-6-(lH-pyrazol-l-yl)pyrimidin-2-amine

[0561] Step 1: Synthesis of N-(2-(lH-pyrazol-3-yl)ethyl)-N-methyl-4-morpholino-6-(lH-pyrazol- l-yl)pyrimidin-2-amine Docket No. 4390.3002 WO

[0562] 4-(2-Iodo-6-(lH-pyrazol-l-yl)pyrimidin-4-yl)morpholine (Examples 26 and 27, Step 2) (0.3 g, 0.839 mmol) and N-methyl-2-(lH-pyrazol-3-yl)ethan-l-amine (0.157 g, 1.259 mmol) were added to dioxane (3 mL). To the above solution, CS2CO3 (1.094 g, 3.359 mmol) was added at room temperature under nitrogen atmosphere. The reaction mixture was purged with nitrogen gas for 10 min. Pd2(dba)3 (0.115 g, 0.130 mmol) and XPhos (CAS: 564483-18-7) (0.200 g, 0.419 mmol) were added, and the reaction mixture was heated at 100 °C for 3 h. After completion, the reaction mixture was diluted with water (50 mL) and the aqueous layer was extracted with EtOAc (2 x 100 mL). The combined organic phases were dried over sodium sulfate and evaporated under reduced pressure. The crude residue was purified by silica gel flash chromatography and pure compound was eluted at 3 % MeOH in DCM to obtain title compound as an off white solid (0.160 g, 0.451 mmol, 36 %).

[0563] LCMS (Method-H): Retention time: 2.832 min, ES(+ve): 355.5 [M+H]+

[0564] Step 2: N-(2-(l-(2-fluoropyrimidin-4-yl)-lH-pyrazol-3-yl) ethyl)-N-methyl-4- morpholino-6-(lH-pyrazol-l-yl) pyrimidin-2-amine

[0565] To a stirred solution of N-(2-(lH-pyrazol-3-yl)ethyl)-N-methyl-4-morpholino-6-(lH-pyrazol- l-yl)pyrimidin-2-amine (0.15 g, 0.423 mmol) in DMSO (1.5 mL), was added DIPEA (0.29 mL, 1.693 mmol) and 2,4-difluoropyrimidine (0.098 g, 0.846 mmol) at room temperature under nitrogen atmosphere. The reaction mixture was stirred at room temperature for 16 h. After completion, the reaction mixture was quenched with water and extracted with EtOAc (2 x 50 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain crude. The resulting crude was purified by column chromatography. The product was eluted in 30 % EtOAc in hexane to obtain the title compound (0.024 g, 0.053 mmol, 13 %) as a white solid.

[0566] 'H NMR (400 MHz, DMSO-d6): 82.99 (t, 2H), 3.13 (s, 3H), 3.53-3.70 (m, 8H), 3.88-3.97 (m, 2H), 6.43 (s, 1H), 6.49 (s, 1H), 6.61 (s, 1H), 7.73-7.82 (m, 2H), 8.53 (s, 2H), 8.73-8.80 (m, 1H).

[0567] 19F NMR (400 MHz, DMSO-d6): 6 -45 57 (IF).

[0568] LCMS (Method-C): Retention time: 1.946 min, ES(+ve): 451.4 [M+H]+ Docket No. 4390.3002 WO

[0569] Synthesis of PIKfyve inhibitors of Formula X

[0570] LCMS Methods

[0571] Method 1

[0572] Instrument: Waters Acquity UPLC I class Series with SQD 2 Mass detector, Column: Zorbax Eclipse Plus C18; 2. lx 50 mm, 1.8 p. Gradient [time (min) / solvent B (%)]: 0.0 / 5, 0.2 / 5,2.5 / 98, 3.5 / 98, 3.6 / 5, 4.0 / 5. (Solvent A= 0.1% Formic acid in H2O; Solvent B = 0.05% Formic acid in ACN; Injection volume 1 pL (may vary); UV detection 210 to 400 nm; Column temperature 40°C; 0.8 mL / min.

[0573] Method 2

[0574] Instrument: Agilent Technologies 1290 Infinity II Series with MSD 6135B mass detector, Column: Zorbax Eclipse Plus C18;2.1x 50 mm 1.8 p. Gradient [time (min) / solvent B (%)]: 0.0 / 5, 0.2 / 5,2.5 / 98, 3.5 / 98, 3.6 / 5, 4.0 / 5. (Solvent A= 0.1% Formic acid in H2O; Solvent B = 0.05% Formic acid in ACN; Injection volume 1 pL (may vary); UV detection 210 to 400 nm; Column temperature 40°C; 0.8 mL / min.

[0575] Method 3

[0576] Instrument: Shimadzu N series UPLC with LCMS 2020 Mass Detector, Column: X bridge C8; 3.5p, 4.6x 50 mm. Gradient [time (min) / solvent B (%)]: 0.0 / 10, 0.2 / 10,2.5 / 98, 3.5 / 98, 3.6 / 10, 4.0 / 10. (Solvent A= 5mM Ammonium Bicarbonate in H2O; Solvent B = ACN; Injection volume 1 pL (may vary); UV detection 210 to 400 nm; Column temperature 40°C; 1.0 mL / min.

[0577] Method 4 Instrument: Waters Acquity UPLC I class Series with SQD 2 Mass detector, Column: Acquity UPLC BEH C18;1.7p, 2. lx 50 mm. Gradient [time (min) / solvent B (%)]: 0.0 / 5, 0.2 / 5,2.5 / 98, 3.5 / 98, 3.6 / 5, 4.0 / 5. (Solvent A= 0.1% Formic acid in H2O; Solvent B = 0.05% Formic acid in ACN; Injection volume 1 pL (may vary); UV detection 210 to 400 nm; Column temperature 35°C; 0.8 mL / min. Alternatively, for UV inactive compounds, an ELSD detector was employed with the above instrument to visualize compounds.

[0578] Method 5 Instrument: Shimadzu N series UPLC with LCMS 2020 Mass Detector, Column: : Acquity UPLC BEHC18(2.1x50)mm,1.7p. Gradient [time (min) / solvent B (%)]: 0.0 / 5, 0.2 / 5, 2.5 / 98, 3.5 / 98, 3.6 / 5, 4.0 / 5. (SolventA= 5 mM Ammonium Acetate in H2O; Solvent B = 100% ACN; Injection volume 1 pL (may vary); UV detection 210 to 400 nm; Column temperature 35°C; 0.6 mL / min.

[0579] UPLC-MS Methods

[0580] Method 6 (4 minutes) Instrument: Waters Acquity UPLC I class Series with SQD 2 Mass detector, Column: Zorbax Eclipse Plus Cl 8; 2. lx 50 mm, 1.8 p, 2. lx 50 mm. Gradient [time Docket No. 4390.3002 WO

[0581] (min) / solvent B (%)]: 0.0 / 5, 0.2 / 5,2.5 / 98, 3.5 / 98, 3.6 / 5, 4.0 / 5. (Solvent A= 0.1% Formic acid in H2O; Solvent B = 0.05% Formic acid in ACN; Injection volume 1 pL; UV detection 210 to 400 nm; Column temperature 40°C; 0.8 mL / min.

[0582] Method 7 (3 minutes) Instrument: Waters Acquity UPLC I class Series with SQD 2 Mass detector, Column: Acquity UPLC BEH C18;1.7p, 2.1x 30 mm. Gradient [time (min) / solvent B (%)]: 0.0 / 5, 0.4 / 5,1.8 / 100, 2.4 / 100, 2.6 / 5, 3.0 / 5. (Solvent A= 0.1% Formic acid in H2O; Solvent B = 0.05% Formic acid in ACN; Injection volume 1 pL; UV detection 210 to 400 nm; Column temperature 30°C; 1.0 mL / min.

[0583] Method 8 Instrument: Shimadzu N series UPLC with LCMS 2020 Mass Detector, Column: Phenomenex kinetex EVO C18 2.6p, 3. Ox 50 mm. Gradient [time (min) / solvent B (%)]: 0.0 / 5, 0.2 / 5,2.50 / 95, 3.55 / 95, 3.6 / 5, 4.0 / 5. (Solvent A= 5mM Ammonium bicarbonate in H2O; Solvent B = ACN; Injection volume 1 pL (may vary); UV detection 210 to 400 nm; Column temperature 40°C; 0.8 mL / min.

[0584] Method 9 Instrument: Agilent Technologies 1290 Infinity II Series with MSD 6135B mass detector, Column: Zorbax Eclipse Plus C18;2.1x 50 mm 1.8 p. Gradient [time (min) / solvent B (%)]: 0.0 / 5, 0.2 / 5,2.5 / 95, 3.5 / 95, 3.6 / 5, 4.0 / 5. (Solvent A= 0.1% Formic acid in H2O; Solvent B = 0.05% Formic acid in ACN; Injection volume 1 pL (may vary); UV detection 210 to 400 nm; Column temperature 40°C; 0.8 mL / min.

[0585] HPLC Methods

[0586] Method A: Instrument: Shimadzu LC; Prominence-I Series LC-2050C, Column: X-Select CSHC18, 4.6xl50mm;3.5p. Gradient [time (min) / solvent B (%)]:0.0 / 10, 2.0 / 10, 12 / 100,17.0 / 100,18.0 / 10, 20.0 / 10 (Solvent A=lmL of FA in 1000 mL of Milli-Q Water; Solvent B=ACN); Injection volume IpL (may vary); UV detection 210 to 400 nm; Column temperature 35°C; 1.2 mL / min.

[0587] Method B: Instrument: Shimadzu LC; Prominence-I Series LC-2050C, Column: Atlantis T3 C18, 4.6xl50mm;3.0p. Gradient [time (min) / solvent B (%)]:0.0 / 10, 2.0 / 10, 12 / 100,17.0 / 100,18.0 / 10, 20.0 / 10 (Solvent A=lmL of FA in 1000 mL of Milli-Q Water;

[0588] Solvent B=ACN); Injection volume IpL (may vary); UV detection 210 to 400 nm; Column temperature 35°C; 1.2 mL / min.

[0589] Method C: Instrument: Shimadzu LC; Prominence-I Series LC-2050C, Column: X- bridgeC18, 4.6xl50mm;5.0p. Gradient [time (min) / solvent B (%)]:0.0 / 5, 2.0 / 5, 12 / 100,17.0 / 100,18.0 / 5, 20.0 / 5. (Solvent A=5mM Ammonium Bicarbonate in Milli-Q Water; Solvent B=ACN); Injection volume IpL (may vary); UV detection 210 to 400 nm; Column temperature 35°C; 1.2 mL / min. Docket No. 4390.3002 WO

[0590] Method D: Instrument: Shimadzu LC; Prominence-I Series LC-2050C, Column: X- bridgeC18, 4.6xl50mm;3.5p. Gradient [time (min) / solvent B (%)]:0.0 / 5, 2.0 / 5, 12 / 100,17.0 / 100,18.0 / 5, 20.0 / 5. (Solvent A=5mM Ammonium Bicarbonate in Milli-Q Water; Solvent B=ACN); Injection volume IpL (may vary); UV detection 210 to 400 nm; Column temperature 35°C; 1.2 mL / min.

[0591] Benzyl(2,3,5,6-tetrafluorophenyl)sulfane (2):

[0592] In a 20 mL vial, 3-bromo-l,2,4,5-tetrafluorobenzene (0.3 g, 1.310 mmol) in DIPEA (0.685 ml, 3.93 mmol) and dioxane (3.0 ml) were added under a nitrogen atmosphere. Reaction mixture was degassed and benzyl mercaptan (0.146 g, 1.179 mmol) followed by XantPhos (0.152 g, 0.262 mmol) and Pd2(dba)3 (0.120 g, 0.131 mmol) were introduced at rt. The resulting reaction mixture was heated at 110 °C for 15 h. The reaction was monitored by TLC. After completion of the reaction, volatiles were removed under reduced pressure to get the crude. The obtained crude compound was purified by isolera column chromatography using 100-200 mesh silicagel and eluted with 100% Hexane to get compound benzyl(2,3,5,6-tetrafluorophenyl)sulfane (220 mg, 0.808 mmol, 61.7%). 1H-NMR (400 MHz, DMSO-d6): 8 7.30-7.24 (m, 5H), 7.00 (s, 1H), 4.15 (s, 2H).

[0593] Benzyl(4-chloro-2,3,5,6-tetrafluorophenyl)sulfane (3):

[0594] To a stirred solution of benzyl(2,3,5,6-tetrafluorophenyl)sulfane (0.39 g, 1.432 mmol) in THF (4 ml) was added BuLi (1.6 M in Hexane) (0.985 ml, 1.576 mmol) at -78 °C under nitrogen, followed by stirring for Ih at -78 °C. A solution of hexachloroethane (1.695 g, 7.16 mmol) in

[0595] 2 mL of THF was introduced at -78° C then the reaction mixture was allowed to stir for 1 h at Docket No. 4390.3002 WO

[0596] 0 °C . The reaction was monitored by TLC, quenched with ice water (3 mL) and extracted with ethyl acetate (2 x 50 mL). The combined organic layers were dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure affording crude product. The crude product was purified column chromatography using 100-200 mesh silica gel in 100% Hexane 5 as eluent to get benzyl(4-chloro-2,3,5,6-tetrafluorophenyl)sulfane (215 mg, 0.701 mmol, 48.9%).LCMS: No ionization. 'H-NMR (400 MHz, DMSO-d6): 8 7.28 - 7.23 (m, 5H), 4.12 (s, 2H).

[0597] 4-chloro-2,3,5,6-tetrafluorobenzenesulfonyl chloride (4):

[0598] 10 To a stirred solution of benzyl(4-chloro-2,3,5,6-tetrafluorophenyl)sulfane (215 mg, 0.701 mmol) in mixture of acetic acid (4.5 mL) and water (0.5 mL), 1,3 -di chi oro-5, 5- dimethylimidazolidine-2, 4-dione (276 mg, 1.402 mmol) was added at 0°C. The resulting reaction mixture was stirred for Ih at rt. The reaction was monitored by TLC: quenched with ice water (3 mL) and extracted with hexane (3 x 50 mL). The combined organic layers were 15 dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure affording 4-chloro-2,3,5,6-tetrafluorobenzenesulfonyl chloride (175 mg, 0.618 mmol, 88%). LCMS:47%, RT: 1.14, m / z: 263 (M-H+) [corresponding sulfonic acid mass]. Docket No. 4390.3002 WO

[0599] Synthesis of tert-butyl 4-((2-chloropyrimidin-4-yl)amino)piperidine-l -carboxylate (3)

[0600] In an oven-dried 500 mL two-neck round-bottom flask equipped with N2 balloon and a rubber septum, 2, 4-di chloropyrimidine (15 g, 101 mmol) and EtOH (200 ml) were added. The reaction mixture was cooled to 0 °c, tert-butyl 4-aminopiperidine-l -carboxylate (20.17 g, 101 mmol) and DIPEA (13.01 g, 101 mmol) were introduced at the same temperature then the resulting reaction mixture was stirred for 16 h at 25 °C. The reaction was monitored by TLC. After completion of the reaction volatiles were removed under reduced pressure. The crude mass was purified by flash column chromatography using 40% Ethyl acetate in hexane to get tert-butyl 4-((2-chloropyrimidin-4-yl)amino)piperidine-l -carboxylate (20 g, 62.0 mmol, 61.6 % yield) as an off white solid.

[0601] LCMS: 95.5%, RT: 1.67 min, m / z = 313.1 (M+H)+; 'H-NMR (400 MHz, DMSO-< 6 7.89 - 7.85 (m, 2H), 6.41 (d, J = 4.4 Hz, 1H), 4.04 - 3.84 (m, 3H), 2.91 (brs, 2H), 1.83 (d, J = 11.2 Hz, 2H), 1.40 (s, 9H), 1.35 - 1.24 (m, 2H).

[0602] Synthesis of tert-butyl 4-((2-chl oropyrimidin-4-yl)(2 -methoxy ethyl)amino)piperi dine- 1- carb oxy late (5)

[0603] To an oven dried 250 mL two-neck round-bottom flask equipped with N2 balloon and a rubber septum, tert-butyl 4-((2-chloropyrimidin-4-yl)amino)piperidine-l -carboxylate (6 g, 19.18 mmol) and DMF (100 mL) were added. The reaction mixture was cooled to 0 °C, sodium hydride (2.30 g, 57.5 mmol) was added portion wise at the same temperature, stirred for 20 min, then DMF solution of l-bromo-2-methoxy ethane (9.01 mL, 96 mmol) in (10 mL) was introduced. The resulting reaction mixture was stirred for 16 h at 25 °C. The reaction was quenched with ice water (100 mL) and extracted with Ethyl acetate (2 x 100 mL). The combined organic layers were dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure to afford crude product. The crude mass was purified by flash column chromatography using 50-60% Ethyl acetate in hexanes to afford tert-butyl 4- Docket No. 4390.3002 WO

[0604] ((2-chloropyrimidin-4-yl)(2-methoxyethyl)amino)piperidine-l-carboxylate (6.0 g, 16.21 mmol, 84.6% yield) as an off-white solid.

[0605] LCMS : 92.4%, RT: 1.912, m / z = 371.1 (M+H)+; 'H-NMR (400 MHz, DMSO-tfc): 6 8.07 (d, J = 6.0 Hz, 1H), 6.79 (brs, 1H), 4.07 - 4.01 (m, 2H), 3.55 - 3.48 (m, 3H), 3.45 - 3.40 (m, 2H), 3.26 (s, 3H), 2.81 (brs, 2H), 1.64 (s, 4H), 1.41 (s, 9H).

[0606] Synthesis of tert-butyl 4-((2-methoxyethyl)(2-(phenylamino)pyrimidin-4- yl)amino)piperidine-l -carboxylate (7)

[0607] To an oven dried 250 mL two-neck round-bottom flask equipped with N2 balloon, tert-butyl 4- ((2-chloropyrimidin-4-yl)(2-methoxyethyl)amino)piperidine-l-carboxylate (10.5 g, 28.3 mmol), isobutanol (100 mL), aniline (2.58 mL, 28.3 mmol) and TFA(2.4 mL, 31.1 mmol) were added at room temperature. The reaction mixture was stirred for 2h at 80 °C. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was concentrated under reduced pressure. The obtained crude was purified by flash column chromatography using 75 % Ethyl acetate in hexane to afford tert-butyl 4-((2 -methoxy ethyl)(2- (phenylamino)pyrimidin-4-yl)amino)piperidine-l -carboxylate (6.3 g, 14.29 mmol, 50.5% yield).

[0608] LCMS: 97.05%, RT: 1.405, m / z = 428.2 (M+H)+;‘H-NMR (400 MHz, DMSO-tfc): 6 10.03 (s, 1H), 7.98 (d, 7.2 Hz, 1H), 7.55 (d, J = 8.0 Hz, 2H), 7.39 (t, J = 7.2 Hz, 2H), 7.15 (t, J = 7.6 Hz, 1H), 6.50 (brs, 1H), 4.09 - 4.00 (m, 2H), 3.66 - 3.63 (m, 2H), 3.51 - 3.41 (m, 3H), 3.24 (s, 3H), 2.82 (brs, 2H), 1.70 -1.63 (m, 4H), 1.41 (s, 9H).

[0609] Synthesis of A4-(2-methoxyethyl)-A2-phenyl-A4-(piperidin-4-yl)pyrimidine-2,4-diamine hydrochloride (Intermediate A)

[0610] To an oven dried 50 mL round-bottom flask equipped with N2 balloon, tert-butyl 4-((2- methoxyethyl)(2-(phenylamino)pyrimidin-4-yl)amino)piperidine-l-carboxylate (1 g, 2.339 Docket No. 4390.3002 WO mmol) and DCM (10 mL) were added. 4M HC1 in dioxane (5 mL, 165 mmol) at 25 °C was added and the reaction mixture was stirred for 2h. The reaction was monitored by TLC. After completion of the reaction volatiles were removed under reduced pressure to get crude. Thus obtained crude was triturated with MTBE to afford N4-(2-methoxyethyl)-N2-phenyl-N4- (piperidin-4-yl)pyrimidine-2,4-diamine hydrochloride (810 mg, 2.226 mmol, 95% yield) as an off white solid.

[0611] LCMS: 98.16%, RT: 1.728, m / z = 328.1 [(M-HC1)+H]+

[0612] EXAMPLE Al Synthesis of 2,3,5,6-tetrafluoro-4-((4-((2-methoxyethyl)(2- (phenylamino)pyrimidin-4-yl)amino)piperidin-l-yl)sulfonyl)-N,N-dimethylbenzamide

[0613] To an oven dried 50 mL two-neck round-bottom flask equipped with N2 balloon, N4-(2- methoxyethyl)-N2-phenyl-N4-(piperidin-4-yl)pyrimidine-2,4-diamine hydrochloride (350 mg, 0.827 mmol) in DCM (15 mL) were added TEA (251 mg, 2.480 mmol) and stirred for 10 minutes. The resulted solution was cooled to -78 °cfollowed by 4-(dimethylcarbamoyl)- 2,3,5,6-tetrafluorobenzenesulfonyl chloride (317 mg, 0.992 mmol) solution in DCM (5 mL) was added at the same temperature and further stirred for 1 h at -78 °C. The reaction was monitored by TLC; quenched with aqueous saturated NaHCCh solution (15 mL) and extracted with DCM (2 x 15 mL). The combined organic layers were dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure affording crude product. The crude mass was purified by prep HPLC (Method C) to afford 2,3,5,6-tetrafluoro-4-((4-((2- methoxyethyl)(2-(phenylamino)pyrimidin-4-yl)amino)piperidin-l-yl)sulfonyl)-N,N-dimethyl benzamide (275 mg, 0.446 mmol, 53.9% yield) as an off white solid. Docket No. 4390.3002 WO

[0614] The compounds in the following table were prepared in the same manner as Al from the appropriate starting materials.

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[0644] EXAMPLE A28 Synthesis of N4-(l-(2,5-difluoropyrimidin-4-yl)piperidin-4-yl)-N4-(2- methoxyethyl)-N2-phenylpyrimidine-2,4-diamine

[0645] In an oven-dried 8 mL pressure vial, N4-(2-methoxyethyl)-N2-phenyl-N4-(piperidin-4- yl)pyrimidine-2,4-diamine hydrochloride (200 mg, 0.611 mmol), diisopropylamine (0.532 ml, 3.05 mmol), 2,4,5-trifluoropyrimidine (73.7 mg, 0.550 mmol) and DMF (10 mL) were added at room temperature and further stirred for 3h at the same temperature. The reaction was monitored by TLC and LCMS, quenched with cold water (15 ml) and extracted with ethyl acetate (2 x 15 ml). The combined organic layers were washed with water (2 x 20 mL) and dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure affording crude product. The crude mass was purified by prep HPLC (Method-A) and product containing fraction was lyophilized to get N4-(l-(2,5-difluoropyrimidin-4-yl)piperidin-4-yl)- N4-(2-methoxyethyl)-N2-phenylpyrimidine-2,4-diamine (27 mg, 0.060 mmol, 9.81% yield)) as an off white solid.

[0646] EXAMPLE A29 was prepared in the same manner as A28 from the appropriate starting materials.

[0647] 19F NMR(376 MHZ, DMSO-d6): 8 -120.83 (IF), -133.00 (IF), -141.51 (IF).1HNMR(400 MHz, DMSO-d6): 6 9.05 (s, 1H), 7.97 (d, J = 6.0 Hz, 1H), 7.74 (d, J = 7.6 Hz, 2H), 7.24 (t, J = 7.6 Hz, 2H), 6.89 (t, J = 7.2 Hz, 1H), 6.23 (brs, 1H), 3.59 - 3.48 (m, 9H), 3.39 - 3.34 (m, 3H), 3.29 (s, 3H), 1.91 - 1.80 (m, 4H). LCMS (Method 2): RT: 1.55, m / z = 561.2 (M+H)+ Docket No. 4390.3002 WO

[0648] EXAMPLE A30 (4-((2-methoxyethyl)(2-(phenylamino)pyrimidin-4-yl)amino)piperidin- 1 yl)(perfluorophenyl)methanone :

[0649] To an oven dried 50 mL two-neck round-bottom flask equipped with N2 balloon, 2, 3, 4,5,6- pentafluorobenzoic acid (117 mg, 0.550 mmol), DMF (10 mL), PyBOP (358 mg, 0.687 mmol), N4-(2-methoxyethyl)-N2-phenyl-N4-(piperidin-4-yl)pyrimidine-2,4-diamine hydrochloride (150 mg, 0.458 mmol) and DIPEA (0.240 ml, 1.374 mmol) were added at 0 °c. The resulting reaction mixture was stirred for 12 h at room temperature. The reaction was monitored by TLC; quenched with ice cold water (30 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic layers were dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure to afford crude product. The crude mass was purified by prep HPLC (Method C) to give (4-((2-methoxyethyl)(2-(phenylamino)pyrimidin-4-yl)amino)piperidin-l- yl)(perfluorophenyl)methanone (40.01 mg, 0.076 mmol, 16.63 % yield) as an off-white solid.

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[0651] The compounds in the following table were prepared in the same manner as A30 from the appropriate starting materials.

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[0674] Docket No. 4390.3002 WO tert-butyl 4-((2,5-dichloropyrimidin-4-yl)amino)piperidine-l -carboxylate (3):

[0675] In an oven-dried, 500 mL, two-neck round-bottom flask equipped with a N2 balloon and a rubber septum, 2,4,5-trichloropyrimidine (10.0 g, 54.5 mmol) and ethanol (100 ml) were added. The solution was cooled to 0 °c. and tert-butyl 4-aminopiperidine-l -carboxylate (16.38 g, 82 mmol) was added. The reaction mixture was stirred under nitrogen atmosphere for 16 h at 50 °c. The reaction was monitored by LCMS. After completion of the reaction, ice water (100 ml) was added and extracted with ethyl acetate (2 x 80 ml). The combined organic portions were washed with water (2 x 50 ml) followed by brine solution (50 ml) and then dried over anhydrous Na2SC>4, filtered and concentrated under reduced pressure. The obtained crude material was purified by flash column chromatography using 30-40% ethyl acetate in hexanes to provide tert-butyl 4-((2,5-dichloropyrimidin-4-yl)amino)piperidine-l -carboxylate (8.1 g, 22.63 mmol, 41.5%) as off white solid. LC-MS (ES+): 97.02%, RT: 2.55 min, m / z 347.2 [M + H]+; 1 -(4-((5-chloro-2-((4-(4-methylpiperazin- 1 -yl)phenyl)amino)pyrimidin-4-yl)amino)- 114- piperidin- 1 -yl)-2,2,2-trifluoroethan- 1 -one (5) :

[0676] In an oven dried 25 mL two-neck round-bottom flask equipped with N2 balloon, a solution of tert-butyl 4-((2, 5-di chi oropyrimidin-4-yl)amino)piperi dine- 1 -carboxylate (0.2 g, 0.576 mmol) Docket No. 4390.3002 WO and 4-(4-methylpiperazin-l-yl)aniline (0.132 g, 0.691 mmol) was dissolved in 1,4 -Dioxane (2 mL). TFA (2.399 ml, 31.1 mmol) was added. The resulting reaction mixture was stirred at 100 °C for 16 h. The reaction was monitored by TLC. After completion of the reaction volatiles were removed under reduced pressure to afford l-(4-((5-chloro-2-((4-(4-methylpiperazin-l- yl)phenyl)amino)pyrimidin-4-yl)amino)-ll4-piperidin-l-yl)-2,2,2-trifluoroethan-l-one (0.260 g, 0.647 mmol, 90%). LC-MS (ES+): 76%, RT: 0.28 min, m / z 402.2 [M + H]+;

[0677] EXAMPLE A65 5 -chloro-N2-(4-(4-methylpiperazin- 1 -yl)phenyl)-N4-( 1 - ((perfluorophenyl)sulfonyl)piperidin-4-yl)pyrimidine-2,4-diamine:

[0678] To an oven dried 50 mL two-neck round-bottom flask equipped with N2 balloon, l-(4-((5- chloro-2-((4-(4-methylpiperazin-l-yl)phenyl)amino)pyrimidin-4-yl)amino)-ll4-piperidin-l- yl)-2,2,2-trifluoroethan-l-one (0.250 g, 0.622 mmol), triethylamine (0.874 mL, 6.22 mmol) and DCM (3.0 mL) were added and stirred for 10 minutes at 0 °c. The resulting reaction mixture was cooled to -78 °c. A solution of 2,3,4,5,6-pentafluorobenzenesulfonyl chloride (0.166 g, 0.622 mmol) in DCM (3 mL) was introduced and stirring continued for 1 h at -78 °C. The reaction was monitored by TLC and LCMS. Apon completion, the reaction was quenched with ice water (3 mL) and extracted with DCM (2 x 10 mL). The combined organic layers were dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure affording crude product. Thus obtained crude product was purified by prep HPLC to give 5- chloro-N2-(4-(4-methylpiperazin-l-yl)phenyl)-N4-(l-((perfluorophenyl)sulfonyl)piperi din-4- yl)pyrimidine-2,4-diamine (58.9 mg, 0.091 mmol, 14.68%) as off white solid.

[0679] Docket No. 4390.3002 WO

[0680] The compounds in the following table were prepared in the same manner as A65 from the appropriate starting materials.

[0681] 155

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[0690] Synthesis of tert-butyl 4-((2-((tert-butyldimethylsilyl)oxy)ethyl)(2-chloropyrimidin-4- yl)amino)piperidine-l -carboxylate (3) To an oven dried 50 mL two-neck round-bottom flask equipped with N2 balloon and a rubber septum, tert-butyl 4-((2-chloropyrimidin-4-yl)amino)piperidine-l -carboxylate (500 mg, 1.598 mmol) and DMF (3 mL) were added. The reaction mixture was cooled to 0 °c, sodium hydride (320 mg, 7.99 mmol) was added by portion-wise at the same temperature and stirred for 20 minutes. A DMF (2 mL) solution of (2-bromoethoxy)(tert-butyl)dimethylsilane (1.372 mL, 6.39 mmol) was added and the resulting reaction mixture was stirred for 16 h at

[0691] 25 °C. The reaction was monitored by TLC; quenched with ice water (100 mL) and extracted with ethyl acetate (2 x 100 mL). The combined organic layers were dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure affording crude product. The crude mass was purified by flash column chromatography using 30-40% ethyl acetate in hexane to afford tert-butyl 4-((2-((tert-butyldimethylsilyl)oxy)ethyl)(2-chloropyrimidin-4- yl)amino)piperidine-l -carboxylate (300 mg, 0.630 mmol, 39.4%) as a colorless gum. LCMS: 98.23%, RT: 2.67, m / z = 471.2 (M+H)+. Docket No. 4390.3002 WO

[0692] Synthesis of tert-butyl 4-((2-hydroxyethyl)(2-(phenylamino)pyrimidin-4- yl)amino)piperidine-l -carboxylate (5)

[0693] To an oven dried 25 mL two-neck round-bottom flask equipped with N2 balloon, tert-butyl 4- ((2-((tert-butyldimethylsilyl)oxy)ethyl)(2-chloropyrimidin-4-yl)amino)piperidine-l- carboxylate (280 mg, 0.594 mmol), isobutanol (4 mL), aniline (83 mg, 0.892 mmol) and TFA (0.364 mL, 4.75 mmol) were added at room temperature. Then the reaction mixture was stirred for 16h at 80 °C. The reaction was monitored by TLC. After completion, the reaction mixture was concentrated under reduced pressure to give crude tert-butyl 4-((2-hydroxyethyl)(2- (phenylamino)pyrimidin-4-yl)amino)piperidine-l -carboxylate (380 mg, 0.242 mmol, 40.8%) as a dark liquid. LCMS: 77.68%, RT: 1.42, m / z = 414.2 (M+H)+.

[0694] Synthesis of 2,2,2-trifluoro-l-(4-((2-hydroxyethyl)(2-(phenylamino)pyrimidin-4-yl)amino)- 114-piperidin- 1 -yl)ethan- 1 -one (6)

[0695] To an oven dried 25 mL round-bottom flask equipped with N2 balloon, tert-butyl 4- ((2-hydroxyethyl)(2-(phenylamino)pyrimidin-4-yl)amino)piperidine-l -carboxylate (380 mg, 0.242 mmol), 1,2-Dichloroethane (4 mL) and TFA (314 mg, 2.76 mmol) were added at room temperature. The resulting reaction mixture was stirred for 3 h at 80 °C. The reaction was monitored by TLC. After completion of the reaction volatiles were removed under reduced pressure to afford 2,2,2-trifluoro-l-(4-((2-hydroxyethyl)(2-(phenylamino)pyrimidin-4- yl)amino)-114-piperidin-l-yl)ethan-l-one (350 mg, 0.558 mmol, 60.8%) as a black gum. LCMS: 50%, RT: 0.36 min, m / z=314.1 [(M-TFA)+H]+. Docket No. 4390.3002 WO

[0696] EXAMPLE A79 Synthesis of 2-((l-((perfluorophenyl)sulfonyl)piperidin-4-yl)(2- (phenylamino)pyrimidin-4-yl)amino)ethan-l-ol.

[0697] To an oven dried 25 mL two-neck round-bottom flask equipped with N2 balloon, 2,2,2-trifluoro-l-(4-((2-hydroxyethyl)(2-(phenylamino)pyrimidin-4-yl)amino)-114-piperidin- l-yl)ethan-l-one (200 mg, 0.638 mmol) in DCM (5 mL) were added TEA (0.356 mL, 2.55 mmol) and stirred for 10 minutes. The resulting solution was cooled to -78 °cfollowed by addition of ,3,4,5, 6-pentafluorobenzenesulfonyl chloride (187 mg, 0.702 mmol) solution in DCM (2 mL) at the same temperature and further stirring for 1 h at -78 °C. The reaction was monitored by TLC; quenched with aqueous saturated NaHCOs solution (15 mL) and extracted with DCM (2 x 15 mL). The combined organic layers were dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure affording crude product. The crude mass was purified by prep HPLC (Method C) to afford 2-((l- ((perfluorophenyl)sulfonyl)piperidin-4-yl)(2-(phenylamino)pyrimidin-4-yl)amino)ethan-l-ol (42.8 mg, 0.076 mmol, 11.90%) as an off white solid.

[0698] Docket No. 4390.3002 WO

[0699] The compounds in the following table were prepared in the same manner as A79 from the appropriate starting materials.

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[0704] Synthesis of tert-butyl 4-((2-chloropyrimidin-4-yl)(2-cyclopropoxyethyl)amino)piperidine-l- carboxylate

[0705] To an oven dried 50 mb two-neck round-bottom flask equipped with N2 balloon and a rubber septum, tert-butyl 4-((2-chloropyrimidin-4-y l)amino)piperi dine- 1 -carboxylate (300 mg, 0.959 mmol) and DMF (15 mL) were added. The reaction mixture was cooled to 0 °cand sodium hydride (115 mg, 2.88 mmol) was added by portion wise at the same temperature. After stirring for 20 min, a DMF (2 mL) solution of 2-cyclopropoxyethyl 4-methylbenzenesulfonate (492 mg, 1.918 mmol) was introduced. The resulting reaction mixture was stirred for 16 h at 25 °C. The reaction was monitored by TLC then it was quenched with ice water (100 mL) and extracted with ethyl acetate (2 x 30 mL). The combined organic layers were dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure affording crude product. The crude mass was purified by flash column chromatography using 30-40% ethyl acetate in hexane to afford tert-butyl 4-((2-chloropyrimidin-4-yl)(2- cyclopropoxyethyl)amino)piperidine-l -carboxylate (100 mg, 0.227 mmol, 23.64 % yield) as an off white solid. LCMS: 87.68%, RT: 2.16, m / z = 397.28 (M+H)+. Docket No. 4390.3002 WO

[0706] Synthesis of tert-butyl 4-((2-(methylamino)-2-oxoethyl)amino)piperidine-l -carboxylate (3)

[0707] To an oven dried 25 mb two-neck round-bottom flask equipped with N2 balloon, tertbutyl 4-aminopiperidine-l -carboxylate (1 g, 4.99 mmol), DMF (10 mL), K2CO3 (1.380 g, 9.99 mmol) and 2-chloro-N-methylacetamide (0.537 g, 4.99 mmol) were added. The resulting reaction mixture was stirred under nitrogen atmosphere for 16 h at 50 °C. The reaction was monitored by TLC and ELSD-LCMS. After complete conversion, the reaction mixture was diluted with ice cold water (30 mL), extracted with ethyl acetate (2 x 20 mL). The combined organic layers were dried over Na2SO4 and evaporated under reduced pressure to get crude tertbutyl 4-((2-(methylamino)-2-oxoethyl)amino)piperidine-l -carboxylate (520 mg, 4.99 mmol, 34.5%) as a brown liquid. LCMS(ELSD): 91.64%, RT: 0.91 mm, m / z = 272.2 (M+H)+. Synthesis of tert-butyl 4-((2-chloropyrimidin-4-yl)(2-(methylamino)-2- oxoethyl)amino)piperidine-l -carboxylate (5).

[0708] In a 20 mL microwave vial tert-butyl 4-((2-(methylamino)-2-oxoethyl)amino)piperidine- 1 -carboxylate (250 mg, 0.921 mmol), 2,4-dichloropyrimidine (165 mg, 1.106 mmol), DIPEA (0.642 mL, 3.69 mmol) and were combined in DMF (5 mL). The reaction mixture was irradiated at 120 °C for 1 h in a microwave reactor. The reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was quenched with ice cold water (15 mL) and extracted with ethyl acetate (2 x 15 mL). The combined organics were washed with water (2 x 15 mL) followed by brine (20 mL), dried over Na2SO4 and concentrated under reduced pressure. The obtained crude compound was purified by column chromatography using neutral alumina and eluting with 10% MeOH in / DCM to afford tert-butyl 4-((2-chloropyrimidin-4-yl)(2- (methylamino)-2-oxoethyl)amino)piperidine-l -carboxylate (280 mg, 0.715 mmol, 78%) as an off white solid.

[0709] LCMS: 98.43%, RT: 1.519 mm, m / z = 484.1 (M+H)+ Docket No. 4390.3002 WO

[0710] Synthesis of tert-butyl 4-((2-(methylamino)-2-oxoethyl)(2-(phenylamino)pyrimidin-4- yl)amino)piperidine- 1 -carboxylate (7)

[0711] To an oven dried 25 mL two-neck round-bottom flask equipped with N2 balloon, tert-butyl 4-((2- chloropyrimidin-4-yl)(2-(methylamino)-2-oxoethyl)amino)piperidine- 1 -carboxylate (392 mg, 1.020 mmol), Isobutanol (5 mL), aniline (95 mg, 1.020 mmol) and TFA (0.156 mL, 2.040 mmol) were added at room temperature. Then the reaction mixture was stirred for 2h at 60 °C. The reaction was monitored by TLC. After completion of the reaction, reaction mixture was concentrated under reduced pressure to get the crude. Thus obtained crude was purified by reverse phase column chromatography by using method [Column:18C 50 gm column, Mobile phase A: 0.1% FA in milli-Q water; Mobile phase B: CH3CN] and fraction was lyophilized to afford tert-butyl 4-((2-(methylamino)-2-oxoethyl)(2-(phenylamino)pyrimidin-4- yl)amino)piperidine-l -carboxylate (375 mg, 0.843 mmol, 83%) as an off white solid. LCMS: 99.16%, RT: 1.353, m / z = 44.3 (M+H)+.

[0712] N-methyl-2-((2-(phenylamino)pyrimidin-4-yl)(l-(2,2,2-trifluoroacetyl)-114-piperidin-4- yl)amino)acetamide (8):

[0713] To an oven dried 25 mL round-bottom flask equipped with N2 balloon, tert-butyl 4-((2- (methylamino)-2-oxoethyl)(2-(phenylamino)pyrimidin-4-yl)amino)piperidine-l -carboxylate (375 mg, 0.843 mmol), DCM (5 mL) and TFA (165 mg, 1.70 mmol) were added at room temperature. The resulting reaction mixture was stirred for 2 h at rt. The reaction was monitored by TLC. After completion of the reaction, volatiles were removed under reduced pressure to afford N-methyl-2- ((2-(phenylamino)pyrimidin-4-yl)( 1 -(2,2,2-trifluoroacetyl)- 114-piperidin-4-yl)amino)acetamide (150 mg, 0.342 mmol, 40%) as a black gum. Docket No. 4390.3002 WO

[0714] EXAMPLE A86 Synthesis of 2-((l-((4-(difhioromethoxy)-2,3,5,6- tetrafluorophenyl)sulfonyl)piperidin-4-yl)(2-(phenylamino)pyrimidin-4-yl)amino)-N-

[0715] To an oven dried 25 mL two-neck round-bottom flask equipped with N2 balloon, N-methyl-2-((2- (pheny lamino)pyrimidin-4-y 1)( 1 -(2,2,2-trifluoroacety 1)- 114-piperidin-4-yl)amino)acetamide (75 mg, 0.220 mmol) in THF (5 mL) were added DIPEA (0.269 mL, 1.542 mmol) and the mixture was stirred for 10 minutes. The resultant solution was cooled to -78 °cand a 4-

[0716] (difluoromethoxy)-2,3,5,6-tetrafluorobenzenesulfonyl chloride (104 mg, 0.330 mmol) solution in DCM (2 mL) was added at the same temperature and further stirred for 10 min at -78 °C. The reaction was monitored by TLC, quenched with aqueous saturated NaHCOs solution (15 mL) and extracted with DCM (2 x 15 mL). The combined organic layers were dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure affording crude product. The crude mass was purified by prep HPLC (Method C) to afford 2-((l-((4-(difluoromethoxy)- 2,3,5,6-tetrafluorophenyl)sulfonyl)piperidin-4-yl)(2-(phenylamino)pyrimidin-4-yl)amino)-N- methylacetamide (29.3 mg, 0.047 mmol, 21.38%) as an off white solid.

[0717] EXAMPLE A872-((l-((4-chloro-2,3,5,6-tetrafluorophenyl)sulfonyl)piperidin-4-yl)(2- (phenylamino)pyrimidin-4-yl)amino)-N-methylacetamide A87 was prepared a similar method to that used to prepare A86.

[0718] 19F NMR (376 MHz, DMSO-d6): -138.83 to -138.787 (2F), -135.26 to -135.22 (2F).1H-VT-NMR

[0719] (400 MHz, DMSO-d6): 8 8.93 (s, 1H), 7.95 (d, J = 6.00 Hz, 1H), 7.61 (d, J = 7.60 Hz, 3H), 7.21 Docket No. 4390.3002 WO

[0720] (t, J = 8.40 Hz, 2H), 6.93 (t, J = 7.20 Hz, 1H), 6.14 (d, J = 6.00 Hz, 1H), 4.34-4.32 (m, 1H), 4.04 (s, 2H), 3.88 (d, J = 12.40 Hz, 2H), 2.91 (t, J = 12.00 Hz, 2H), 2.62 (d, J = 4.80 Hz, 3H), 1.93-1.90 Synthesis of tert-butyl 4-((2-chloropyrimidin-4-yl)(2-hydroxyethyl)amino)piperidine-l- carboxylate (2)

[0721] To an oven dried 100 mb two-neck round-bottom flask equipped with N2 balloon and a rubber septum, tert-butyl 4-((2-((tert-butyldimethylsilyl)oxy)ethyl)(2-chloropyrimidin-4- yl)amino)piperidine-l -carboxylate (1.7 g, 3.61 mmol) and THF (20 ml) were added. The resulting solution was cooled to 0 °C followed by dropwise addition of TBAF (5.41 ml, 5.41 mmol) in THF (IM). The reaction was allowed to stir for 4 h at RT. The reaction was monitored by TLC and LCMS, quenched with ice water (3 mL) and extracted with ethyl acetate (2 x 20 mL). The combined organic layers were dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure affording crude product. The crude product was triturated with 77-pentane to afford tert-butyl 4-((2-chloropyrimidin-4-yl)(2- hydroxyethyl)amino)piperidine-l -carboxylate (1.2 g, 3.26 mmol, 90%) as an off white solid. LCMS: 97.02%, RT: 1.64, m / z = 357.1 (M+H)+. Docket No. 4390.3002 WO

[0722] Synthesis of tert-butyl 4-((2-chloropyrimidin-4-yl)(2-(difluoromethoxy)ethyl)amino)piperidine- 1 -carboxylate (4):

[0723] To an oven dried 40 mb vail, tert-butyl 4-((2-chloropyrimidin-4-yl)(2- hydroxyethyl)amino)piperidine-l -carboxylate (900 mg, 2.52 mmol), acetonitrile (12 mL), and copper iodide (96 mg, 0.504 mmol) were added. The resulting reaction mixture was stirred for 10 min at 50 °C. To this mixture a solution of 2,2-difluoro-2-(fluorosulfonyl)acetic acid (898 mg, 5.04 mmol) in acetonitrile (2 mL) was added at 50 °cand stirred for 2 h at the same temperature. The reaction was monitored by TLC. After completion the reaction, volatiles were removed under reduced pressure to get the crude which was purified by flash column chromatography using silica gel and eluted with 20% ethyl acetate in hexane giving tert-butyl 4-((2- chloropyrimidin-4-yl)(2-(difluoromethoxy)ethyl)amino)piperidine-l-carboxylate (100 mg, 0.211 mmol, 8.38%) as a brown thick liquid. LCMS: 86.36%, RT: 2.04 min, m / z = 407.1 (M+H)+. Intermediate tert-butyl 4-((2-chloropyrimidin-4-yl)(2-(difluoromethoxy)ethyl)amino)piperidine- 1 -carboxylate was used to prepare compounds A88 and A89 using a similar protocol as used for

[0724] Al

[0725] Synthesis of (5-bromopyridin-3-yl)(4-((2-methoxyethyl)(2-(phenylamino)pyrimidin-4- yl)amino)piperidin- 1 -yl)methanone (3 ) : Docket No. 4390.3002 WO

[0726] To an oven dried 50 mL two-neck round-bottom flask equipped with N2 balloon, 5- bromonicotinic acid (139 mg, 0.687 mmol), DMF (8 mL), PyBOP (536 mg, 1.031 mmol), N4-(2- methoxyethyl)-N2-phenyl-N4-(piperidin-4-yl)pyrimidine-2,4-diamine hydrochloride (250 mg, 0.687 mmol) and DIPEA (266 mg, 2.061 mmol) were added at 0 °C. The resulting reaction mixture stirred for 4 h at room temperature. The reaction was monitored by TLC, quenched with ice cold water (30 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic layers were dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure to afford crude product. The crude mass was purified by column chromatography by using silica gel 100- 200 and eluted with 100% ethyl acetate to afford (5-bromopyridin-3-yl)(4-((2-methoxyethyl)(2- (phenylamino)pyrimidin-4-yl)amino)piperidin-l-yl)methanone (270 mg, 0.327 mmol, 47.6%) as a light brown gum. LCMS: 62.45%, RT: 1.24 min, m / z = 513.1 (M+2)+.

[0727] EXAMPLE A91 Synthesis of (4-((2-methoxyethyl)(2-(phenylamino)pyrimidin-4- yl)amino)piperidin-l -yl)(5-vinylpyridin-3-yl)methanone A91.

[0728] In a 40 mL reaction vessel, (5-bromopyridin-3-yl)(4-((2-methoxyethyl)(2- (phenylamino)pyrimidin-4-yl)amino)piperidin-l-yl)methanone (200 mg, 0.391 mmol), trifluoro(vinyl)-14-borane, potassium salt (105 mg, 0.782 mmol), Na2COs (124 mg, 1.173 mmol) and dioxane (4 mL) were added at 25 °C. After degassing the reaction mixture with nitrogen for 5 min, PdC12(dppf).CH2C12 adduct (15.97 mg, 0.020 mmol) was added and further degassed for 5 min. The resulting reaction mixture stirred for 4 h at 80 °c. The reaction was monitored by TLC and LCMS. After completion, the volatiles were removed under reduced pressure. The obtained residue was diluted with water (20 mL), extracted with Ethyl acetate (2 x 15 mL) and the combined organic layers were washed with water (20 mL) followed by brine (20 mL), dried over Na2SO4, concentrated under reduced pressure providing crude product. The obtained crude was purified by prep HPLC to afford (4-((2-methoxyethyl)(2-(phenylamino)pyrimidin-4- yl)amino)piperidin-l-yl)(5-vinylpyridin-3-yl)methanone (32 mg, 0.069 mmol, 17.67%) as an off white solid. Docket No. 4390.3002 WO tert-butyl 4-((2-methoxyethyl)(2-((l-methyl-lH-indol-5-yl)amino)pyrimidin-4- yl)amino)piperidine- 1 -carboxylate (2) : An oven-dried 50 mb two-neck round-bottom flask equipped with N2 balloon and a rubber septum was charged with a solution of l-methyl-lH-indol-5-amine (197 mg, 1.348 mmol), tertbutyl 4-((2-chloropyrimidin-4-yl)(2-methoxyethyl)amino)piperidine-l -carboxylate (500 mg, 1.348 mmol) in iBuOH (5 mL) and TFA (154 mg, 1.348 mmol) was added and the reaction stirred for 2h at 80 °C. The reaction was monitored by LCMS, quenched with ice water (10 mL) and extracted with ethyl acetate (2 x 20 mL). The combined organic layers were dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure afforded the crude product. Thus obtained crude product was purified by flash column chromatography using 70% ethyl acetate / hexane as eluent to provide tert-butyl 4-((2-methoxyethyl)(2-((l -methyl- IH-indol- 5-yl)amino)pyrimidin-4-yl)amino)piperidine-l -carboxylate (300 mg, 0.610 mmol, 45.2%) as white solid. LCMS (Method): 97.65%, RT: 1.57, m / z = 481.3 (M+H)+.

[0729] 2,2,2-trifluoro-l-(4-((2-methoxyethyl)(2-((l-methyl-lH-indol-5-yl)amino)pyrimidin-4- yl)amino)- 114-piperidin- 1 -yl)ethan- 1 -one (3): Docket No. 4390.3002 WO

[0730] In a 10 mL Round bottom flask a mixture of tert-butyl 4-((2-methoxyethyl)(2-((l-methyl-lH- indol-5-yl)amino)pyrimidin-4-yl)amino)piperidine-l -carboxylate (100 mg, 0.208 mmol) in DCM (5 ml) was combined with TEA (0.016 ml, 0.208 mmol) at 0 °C. The resulting reaction mixture was stirred for 1 h at 25 °C. The reaction was monitored by TLC. After completion of the reaction, volatiles were removed under reduced pressure to afford 2,2,2-trifluoro-l-(4-((2- methoxyethyl)(2-((l -methyl-lH-indol-5-yl)amino)pyrimidin-4-yl)amino)-l 14-piperidin- 1 - yl)ethan-l-one (60 mg, 0.069 mmol, 33.3%). LCMS: 44.6%, RT: 0.87, m / z = 381.2 (M+H)+ EXAMPLE A952,6-difluoro-4-((4-((2-methoxyethyl)(2-((l-methyl-lH-indol-5- yl)amino)pyrimidin-4-yl)amino)piperidin-l-yl)sulfonyl)benzonitrile:

[0731] In an oven-dried 10 mL two-neck round-bottom flask equipped with N2 balloon and a rubber septum, 2,2,2-trifluoro-l-(4-((2-methoxyethyl)(2-((l-methyl-lH-indol-5-yl)amino)pyrimidin-4- yl)amino)- 114-piperidin- 1 -yl)ethan- 1 -one (84 mg, 0.221 mmol) in DCM (5 mL) was combined with TEA (44.7 mg, 0.442 mmol) and stirred for 5 min. The resulting reaction mixture was cooled to -78 °C followed by addition of 4-cyano-3,5-difluorobenzenesulfonyl chloride (52.5 mg, 0.221 mmol) . The reaction was stirred for 30 min at the same temperature. The reaction was monitored by LCMS, quenched with ice water (10 mL) and extracted with DCM (2 x 20 mL). The combined organic layers were dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure. Thus obtained crude was purified by prep-HPLC to get 2,6- difhioro-4-((4-((2-methoxyethyl)(2-((l-methyl-lH-indol-5-yl)amino)pyrimidin-4- yl)amino)piperidin-l-yl)sulfonyl)benzonitrile (5.5 mg, 0.093 mmol, 42.1%) as white solid. 19E NMR (376 MHz, DMSO-d6): 8 -101.37 (2E). 1H-NMR (400 MHz, DMSO-d6):8 8.63 (s, 1H), 7.94 (d, J = 7.2 Hz, 2H), 7.85 (d, J = 6.0 Hz, 1H), 7.43 (s, 1H), 7.18 (d, J = 7.6 Hz, 1H), 6.36 (d, J = 8.0 Hz, 1H), 6.01 (d, J = 6.0 Hz, 1H), 4.26 (bs, 1H), 3.86 (d, J = 10.8 Hz, 2H), 3.43 - 3.40 (m, 4H), 3.27 (s, 3H), 3.11 (t, J = 8.0 Hz, 2H), 2.70-2.69 (m, 1H), 2.62 (s, 3H), 2.55 - 2.51 (m, 1H), 1.82 - 1.75 (m, 4H). LCMS (Method 2): RT: 1.63, m / z = 584.2 (M+H)+. Docket No. 4390.3002 WO tert-butyl 4-((2-methoxyethyl)(2-((l-methylindolin-5-yl)amino)pyrimidin-4-yl)amino)piperidine-

[0732] 1 -carboxylate (1)

[0733] In a 10 mL vial a mixture of tert-butyl 4-((2-methoxyethyl)(2-((l-methyl-lH-indol-5- yl)amino)pyrimidin-4-yl)amino)piperidine-l -carboxylate (280 mg, 0.583 mmol) were combined in acetic acid (5 ml) and NaCNBIL (110 mg, 1.748 mmol) was added at 0 °C. The resulting reaction mixture was stirred for 2 h at 25 °C. The reaction was monitored by TLC & LCMS, quenched with ice water (10 mL) and extracted with ethyl acetate (3 x 20 mL). The combined organic layers were dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure affording crude product (130 mg, 0.269 mmol, 46%) as yellow solid. LCMS: 84.59%, RT: 1.58, m / z = 483.3 (M+H)+;

[0734] Docket No. 4390.3002 WO

[0735] 2,2,2-trifhjoro-l-(4-((2-methoxyethyl)(2-((l-methylindolin-5-yl)amino)pyrimidin-4-yl)amino)-

[0736] 114-piperidin- 1 -y l)ethan- 1 -one (2) :

[0737] In a 10 mL Round bottom flask tert-butyl 4-((2-methoxyethyl)(2-((l-methylindolin-5- yl)amino)pyrimidin-4-yl)amino)piperidine-l -carboxylate (130 mg, 0.269 mmol) was dissolved in DCM (5 mL). TFA (52 mg, 0.528 mmol) was added at 0 °C. The resulting reaction mixture was stirred for 2 h at 25 °C. The reaction was monitored by TLC & LCMS. After completion of the reaction volatiles were removed under reduced pressure to afford 2,2,2-trifluoro-l-(4-((2- methoxyethyl)(2-((l-methylindolin-5-yl)amino)pyrimidin-4-yl)amino)-ll4-piperidin-l-yl)ethan- 1-one (90 mg, 0.207 mmol, 70%); LCMS: 87.89%, RT: 0.73, m / z = 383.2 (M+H)+;

[0738] EXAMPLE A962,3,5,6-tetrafluoro-4-((4-((2-methoxyethyl)(2-((l-methylindolin-5- yl)amino)pyrimidin-4-yl)amino)piperidin-l-yl)sulfonyl)-N,N-dimethylbenzamide:

[0739] In a 10 mL vial, 2,2,2-trifhioro-l-(4-((2-methoxyethyl)(2-((l-methylindolin-5- yl)amino)pyrimidin-4-yl)amino)-ll4-piperidin-l-yl)ethan-l-one (90 mg, 0.207 mmol) was dissolved in DCM (5 mL) and TEA was added (53.4 mg, 0.528 mmol) at 0 °C followed by 4- (dimethylcarbamoyl)-2,3,5,6-tetrafluorobenzenesulfonyl chloride (84 mg, 0.264 mmol) at the same temperature. The resulting reaction mixture was stirred for 30 min at rt. The reaction was monitored by TLC & LCMS, quenched with ice water (10 mL) and extracted with DCM (2 x 10 mL). The combined organic layers were dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure affording crude product. The crude product was purified by prep HPLC to afford 2,3,5,6-tetrafluoro-4-((4-((2-methoxyethyl)(2-((l-methylindolin-5- yl)amino)pyrimidin-4-yl)amino)piperidin-l-yl)sulfonyl)-N,N-dimethylbenzamide (20 mg, 0.030 mmol, 11.22%) as a white solid.19F NMR (376 MHz, DMSO-d6): 8 -134.570 (2F), -139.889 to - Docket No. 4390.3002 WO

[0740] 139.980 (2F). 'H-NMR (400 MHz, DMSO-d6): 8 8.62 (s, 1H), 7.87 (d, J = 6.00 Hz, 1H), 7.44 (s, 1H), 7.21 (d, J = 8.00 Hz, 1H), 6.39 (d, J = 8.40 Hz, 1H), 6.05 (d, J = 6.00 Hz, 1H), 3.86 (d, J = 11.20 Hz, 2H), 3.51 (bs, 2H), 3.43 (t, J = 6.00 Hz, 2H), 3.27 (s, 3H), 3.13 (t, J = 8.00 Hz, 2H), 3.07 (s, 3H), 2.98 - 2.92 (m, 5H), 2.76 (t, J = 7.20 Hz, 2H), 2.62 (s, 3H), 2.53 (s, 1H), 1.86-1.79 (m, 4H). LCMS (Method 4): RT: 1.46, m / z = 666.3 (M+H)+.

[0741] EXAMPLE A972,6-difluoro-4-((4-((2-methoxyethyl)(2-((l -methylindolin-5- yl)amino)pyrimidin-4-yl)amino)piperidin-l-yl)sulfonyl)benzonitrile:

[0742] The title compound was prepared in a manner similar to A96. 19F NMR (376 MHz, DMSO-d6): 8 -101.37 (2F). 1H-NMR (400 MHz, DMSO-d6):8 8.63 (s, 1H), 7.94 (d, J = 7.2 Hz, 2H), 7.85 (d, J = 6.0 Hz, 1H), 7.43 (s, 1H), 7.18 (d, J = 7.6 Hz, 1H), 6.36 (d, J = 8.0 Hz, 1H), 6.01 (d, J = 6.0 Hz, 1H), 4.26 (bs, 1H), 3.86 (d, J = 10.8 Hz, 2H), 3.43 - 3.40 (m, 4H), 3.27 (s, 3H), 3.11 (t, J = 8.0 Hz, 2H), 2.70-2.69 (m, 1H), 2.62 (s, 3H), 2.55 - 2.51 (m, 1H), 1.82 - 1.75 (m, 4H). LCMS (Method 2): RT: 1.63, m / z = 584.2 (M+H)+.

[0743] The following intermediates were used in the preparation of A54

[0744] N,N-dimethyl-5-nitropyridin-2-amine (3):

[0745] To a 25 mL round bottom flask solution of 2-chloro-5-nitropyridine (1.5 g, 9.46 mmol) in Isobutanol (5 mL), triethyl amine (3.96 mL, 28.4 mmol) followed by dimethylamine hydrochloride (0.849 g, 10.41 mmol) were added. The resulting reaction mixture was heated in a microwave reactor at 100 °C for 2 h. The reaction was monitored by LCMS, quenched with ice water (3 mL) and extracted with ethyl acetate (2 x 50 mL). The combined organic layers were dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure Docket No. 4390.3002 WO affording N,N-dimethyl-5-nitropyridin-2-amine (1.3 g, 7.7 mmol, 81%) as pale yellow gum;

[0746] LCMS: 99.67%, RT: 1.33, m / z = 168 (M+H)+

[0747] N2,N2-dimethylpyridine-2,5-diamine (4):

[0748] N,N-dimethyl-5-nitropyridin-2-amine (1.4 g, 8.37 mmol) was dissolved in methanol (20 mL) and purged with nitrogen for 2 min. Pd / C (0.446 g, 4.19 mmol) was added and the reaction mixture was stirred under H2 (1 atm) pressure overnight. The reaction was monitored by LCMS, filtered through celite bed and volatiles were removed under reduced pressure to give N2,N2- dimethylpyridine-2, 5 -diamine (900 mg 5.97 mmol, 71.3%) as white solid. LCMS: 91.94%, RT:

[0749] O.21, m / z = 138.06 (M+H)+. l-(4-((2-((6-(dimethylamino)pyridin-3-yl)amino)pyrimidin-4-yl)(2-methoxyethyl)amino)-ll4- piperidin-1 -yl)-2,2,2-trifluoroethan-l -one (5):

[0750] In an oven-dried 100 mL two-neck round-bottom flask equipped with N2 balloon and a rubber septum, tert-butyl 4-((2-chloropyrimidin-4-yl)(2-methoxyethyl)amino)piperidine- 1 -carboxylate (616 mg, 1.660 mmol) and N2,N2-dimethylpyridine-2,5-diamine (253 mg, 1.844 mmol) where dissolved in isobutanol (5 mL). TFA (0.565 mL, 7.38 mmol) was added and the reaction was stirred for 4 h at 80 °C. The reaction was monitored by LCMS. After completion of the reaction, volatiles were removed under reduced pressure. The crude was purified by reverse phase by using RediSep Rr Gold® silica(100g column) [Mobile phase AMO mM ammonium bicarbonate in milli-Q water; Mobile phase B: CH3CN] while the desire product was eluted atl0-20% of the mobile phase; and the fractions were lyophilized to afford l-(4-((2-((6-(dimethylamino)pyridin- 3-yl)amino)pyrimidin-4-yl)(2-methoxyethyl)amino)-l □4-piperidin-l-yl)-2,2,2-trifluoroethan-l- Docket No. 4390.3002 WO one (500 mg, 1.252 mmol, 67.9%) as a brown solid. LCMS: 93.9%, RT: 1.68, m / z = 372.2 (M+H)+. l-(4-((2-((6-(dimethylamino)-5-methylpyridin-3-yl)amino)pyrimidin-4-yl)(2- methoxyethyl)amino)-114-piperidin-l-yl)-2,2,2-trifluoroethan-l-one (5) was prepared using a similar strategy and intermediates and used to make A55:

[0751] In an oven-dried 100 mL two-neck round-bottom flask equipped with N2 balloon and a rubber septum, tert-butyl 4-((2-chloropyrimidin-4-yl)(2-methoxyethyl)amino)piperidine- 1 -carboxylate (845 mg, 2.280 mmol) and N2,N2,3-trimethylpyridine-2,5-diamine ((450 mg, 2.59 mmol) were dissolved in isobutanol (5 mL), TFA (0.814 ml, 10.13 mmol) was added and the reaction stirred for 4 h at 80 °C. The reaction was monitored by LCMS. After completion of the reaction, volatiles were removed under reduced pressure. Thus obtained crude was purified by reverse phase by using RediSep Rr Gold® silica gel(100 g column) [Mobile phase AMO mM ammonium bicarbonate in milli-Q water; Mobile phase B: CH3CN] while the desire product was eluted atlO- 20% of the mobile phase; and the fractions were lyophilized to afford l-(4-((2-((6-

[0752] (dimethylamino)-5-methylpyridin-3-yl)amino)pyrimidin-4-yl)(2-methoxyethyl)amino)-114- piperidin-l-yl)-2,2,2-trifluoroethan-l-one (550 mg, 1.063 mmol, 42.0%) as a brown solid. Docket No. 4390.3002 WO methyl 2-vinylnicotinate (3):

[0753] In a 40 mL reaction vail, methyl 2-bromonicotinate (500 mg, 2.31 mmol), trifluoro(vinyl)-14-borane, potassium salt (620 mg, 4.63 mmol), Na2COs (735 mg, 6.94mmol) and dioxane (8 mL) were added at 25 °C. After degassing the reaction mixture with nitrogen for 5 min, PdC12(dppf).CH2C12 adduct (15.97 mg, 0.020 mmol) was added and further degassed for 5 min. The resulting reaction mixture stirred for 4 h at 80 °C. The progress of the reaction was monitored by TLC and LCMS. After completion reaction, volatiles were removed under reduced pressure. The obtained residue was diluted with water (20 mL), extracted with EtOAc (2 x 15 mL), combined organic layers were washed with water (20 mL) followed by brine (20 mL), dried over Na2SO4, concentrated under reduced pressure afforded crude product. The obtained crude was purified by silica gel chromatography with 30% Ethyl acetate in hexane as eluent to afford (methyl 2-vinylnicotinate (300 mg, 1.84 mmol, 80%) as an off white solid. LCMS: 96%, RT: 1.11 mm, m / z = 164.0 (M+H)+.

[0754] 2-vinylnicotinic acid (4):

[0755] In a 50 ml single necked round bottom flask methyl 2-vinylisonicotinate (300 mg, 1.84 mmol) was dissolved in Tetrahydrofuran (10 mL) and the reaction mixture cooled to 0 °C. To this reaction mixture NaOH (IM), MeOH, H2O (1 mL, 0.25mL, 1 mL) were introduced at the same temperature and the mixture was stirred for 30 min. The progress of the reaction was monitored by TLC and LCMS. Volatiles were removed under reduced pressure and reaction mixture was acidified with 1.5 N HC1 a pH of 3 to 4 and the solid collected by filtration. The obtained solid was dried under vacuum to afford 2-vinylisonicotinic acid (220 mg, 1.48 mmol, 80%). LCMS: 95%, RT: 0.42 min, m / z: 149.9 (M+H+). Docket No. 4390.3002 WO

[0756] EXAMPLE A114 (4-((2-methoxyethyl)(2-(phenylamino)pyrimidin-4-yl)amino)piperidin-l- yl)(2-vinylpyridin-3-yl)methanone

[0757] To an oven dried 50 mb two-neck round-bottom flask equipped with N2 balloon, 2-vinylnicotinic acid (220 mg, 1.48 mmol), DMF (6 mL), PyBOP (536 mg, 1.62 mmol), N4-(2-methoxyethyl)- N2-phenyl-N4-(piperidin-4-yl)pyrimidine-2,4-diamine hydrochloride (530 mg, 1.48 mmol) and DIPEA (266 mg, 2.061 mmol) were added at 0 °C. The resulting reaction mixture stirred for 4 h at room temperature. The progress of the reaction was monitored by TLC, quenched with ice cold water (30 mL) and extracted with EtOAc (3 x 50 mL). The combined organic layers were dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure to afford crude product. The crude mass was purified by column chromatography by using silica gel 100- 200 and eluted with 100% EtOAc to afford (4-((2-methoxyethyl)(2-(phenylamino)pyrimidin-4- yl)amino)piperidin-l-yl)(2-vinylpyridin-3-yl)methanone (60 mg, 0.130 mmol, 9%) as an off white solid.

[0758] 1H-NMR (400 MHz, DMSO-d6): 8 8.70 (s, 1H), 8.63-8.61 (m, 1H), 7.96 (d, J = 6.00 Hz, 1H), 7.74 (d, J = 7.2 Hz, 1H), 7.71 (d, J = 7.60 Hz, 2H), 7.36 (dd, J = 8.00.4.80 Hz, 1H), 7.24 (t, J = 8.40 Hz, 2H), 6.90 (t, J = 7.60 Hz, 1H), 6.84-6.77 (m, 1H), 6.39 (dd, J = 17.2, 2.4 Hz, 1H), 6.19 (d, J = 6.00 Hz, 1H), 5.55 (dd, J = 10.8, 2.4 Hz, 1H), 4.74 (bs, 1H), 4.50 (bs, 1H), 3.58-3.47 (m, 4H), 3.38-3.35 (m, 1H), 3.31 (s, 3H), 3.17-3.14 (m, 1H), 2.93-2.90 (m, 1H), 1.84-1.65 (m, 4H) ppm. LCMS (Method 2): RT: 1.22, m / z = 459.2 (M+H)+. Docket No. 4390.3002 WO

[0759] EXAMPLE A115 (4-((2-methoxyethyl)(2-(phenylamino)pyrimidin-4-yl)amino)piperidin-l- yl)(4-vinylpyridin-2-yl)methanone

[0760] The title compound was prepared in a similar manner to A114. 1H-NMR (400 MHz, DMSO-d6): 89.05 (s, 1H), 8.56 (d, J = 4.80 Hz, 1H), 7.96 (d, J = 6.00 Hz, 1H), 7.71 (t, J = 7.60 Hz, 3H), 7.56- 7.55 (m, 1H), 7.24 (t, J = 8.00 Hz, 2H), 6.90-6.78 (m, 2H), 6.23 (d, J = 18.00 Hz, 2H), 5.61 (d, J = 11.20 Hz, 1H), 4.62 (d, J = 70.80 Hz, 2H), 3.80 (d, J = 12.80 Hz, 1H), 3.54 - 3.47 (m, 4H), 3.28 (s, 3H), 3.20 - 3.14 (m, 1H), 2.91 - 2.85 (m, 1H), 1.84 - 1.63 (m, 4H). LCMS (Method 3): RT: 2.23, m / z = 459.2 (M+H)+.

[0761] A121 (4-((2-methoxyethyl)(2-(phenylamino)pyrimidin-4-yl)amino)piperidin-l-yl)(6- vinylpyridin-2-yl)methanone

[0762] The title compound was prepared in a similar manner to A114. LCMS (Method 2): RT: 2.25, m / z = 459.2[M+H]+.

[0763] A122 (4-((2-methoxyethyl)(2-(phenylamino)pyrimidin-4-yl)amino)piperidin-l-yl)(4- vinylpyri din-3 -yl)methanone

[0764] The title compound was prepared in a similar manner to A114. LCMS (Method 4): RT: 1.12, m / z = 459.2[M+H]+.

[0765] A124 (4-((2-methoxyethyl)(2-(phenylamino)pyrimidin-4-yl)amino)piperidin-l-yl)(6- vinylpyri din-3 -yl)methanone Docket No. 4390.3002 WO

[0766] The title compound was prepared in a similar manner to A114. LCMS (Method 1): RT: 1.26, m / z

[0767] = 459.2[M+H]+.

[0768] A133 (4-((2-methoxyethyl)(2-(phenylamino)pyrimidin-4-yl)amino)piperidin-l-yl)(2- vinylphenyl)methanone

[0769] The title compound was prepared in a similar manner to A114. LCMS (Method 1): RT: 1.48, m / z = 458.2[M+H]+.

[0770] 2,3,5,6-tetrafluoro-4-(methylthio)benzoyl chloride (2):

[0771] To a stirred solution of 2,3,5, 6-tetrafluoro-4-(methylthio)benzoic acid (500 mg, 2.082 mmol) and DCM (5 mL) was added oxalyl chloride (0.357 mL, 4.16 mmol) and 1 drop of DMF (0.016 mL, Docket No. 4390.3002 WO

[0772] 0.208 mmol) at 0 °C. Then the reaction mixture was stirred at 25 °C or 1 h. Progress of the reaction was monitored by TLC. Volatiles were removed under reduced pressure to afford the desired product 2,3,5,6-tetrafluoro-4-(methylthio)benzoyl chloride (538 mg, 2.080 mmol, 100%) as colorless liquid which was used for further reaction without further purification.

[0773] (4-((2-methoxyethyl)(2-(phenylamino)pyrimidin-4-yl)amino)piperidin-l-yl)(2,3,5,6-tetrafluoro-

[0774] 4-(methylthio)phenyl)methanone (3):

[0775] To a stirred solution of 2,2,2-trifhioro- 1 -(4-((2-methoxyethyl)(2-(phenylamino)pyrimidin-4- yl)amino)-114-piperidin-l-yl)ethan-l-one (905 mg, 2.049 mmol) in DCM (10 mL) was added triethylamine (1.428 mL, 10.25 mmol) at 0 °C and the mixture was stirred for 5 min followed by addition of a 2,3,5,6-tetrafluoro-4-(methylthio)benzoyl chloride (530 mg, 2.049 mmol) solution in DCM (2 mL). The reaction mixture was stirred at the same temperature for 1 h. The progress of the reaction was monitored by LCMS, quenched with ice cold water (5 mL) and extracted with DCM (3 x 50 mL). The combined organic layers were dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure to get crude product. The obtained crude product was purified by chromatography using 100-200 mesh silica gel with 0-100% EtOAc in hexane to get (4-((2-methoxyethyl)(2-(phenylamino)pyrimidin-4-yl)amino)piperidin- l-yl)(2,3,5,6-tetrafhioro-4-(methyhhio)phenyl)methanone (380 mg, 0.636 mmol, 31.0%). LCMS: 92.2%, RT: 2.58, m / z = 550.2 (M+H)+.

[0776] Docket No. 4390.3002 WO

[0777] EXAMPLES A116 and A117 (4-((2-methoxyethyl)(2-(phenylamino)pyrimidin-4- yl)amino)piperidin-l-yl)(2,3,5,6-tetrafluoro-4-(methylsulfinyl)phenyl)methanone

[0778] To a stirred solution of (4-((2-methoxyethyl)(2-(phenylamino)pyrimidin-4-yl)amino)piperidin-l- yl)(2,3,5,6-tetrafluoro-4-(methylthio)phenyl)methanone (330 mg, 0.600 mmol) in mixture of acetone (5 mL) and water (5 mL) was added Oxone (240 mg, 0.781 mmol). Then the reaction mixture stirred at rt for 15 h. Progress of the reaction was monitored by TLC, quenched with ice cold water and extracted with EtOAc (3 x 50 mL).The combined organic layers were dried over Na2SO4 and concentrated at reduced pressure. The crude residue was purified by chromatography using 100-200 mesh silica gel with 0-50% EtOAc in hexane as eluent to get 270 mg of pure compound. Then the enantiomers were separated by chiral HPLC using column IZ- [250*30] mm, 5um and mobile phase: CO2: Methanol. First eluting isomer A116-(4-((2- methoxyethyl)(2-(phenylamino)pyrimidin-4-yl)amino)piperidin-l-yl)(2,3,5,6-tetrafluoro-4- (methylsulfinyl)phenyl)methanone (90 mg, 0.158 mmol, 26.2%) and second eluting isomer A117 (R)-(4-((2-methoxyethyl)(2-(phenylamino)pyrimidin-4-yl)amino)piperidin-l-yl)(2, 3,5,6- tetrafluoro-4-(methylsulfinyl)phenyl)methanone (100 mg, 0.175 mmol, 29.2%) were isolated as off white solids.

[0779] 19F NMR (376 MHz, DMSO-d6): 8 -139.27 to -138.91 (IF), -140.25 to -139.81 (IF), -141.29 to -141.03 (2F) 1H-VT-NMR (400 MHz, DMSO-d6): 8 8.71 (s, 1H), 7.97 (d, J = 6.00 Hz, 1H), 7.70 (d, J = 7.60 Hz, 2H), 7.25 (t, J = 8.40 Hz, 2H), 6.91 (t, J = 7.20 Hz, 1H), 6.21 (d, J = 6.00 Hz, 1H), 4.67 (d, J = 13.20 Hz, 1H), 4.54-4.51 (m, 1H), 3.72 (d, J = 13.20 Hz, 1H), 3.57-3.48 (m, 4H), 3.32-3.29 (m, 4H), 3.22 (s, 3H), 2.99 (t, J = 10.80 Hz, 1H), 1.92-1.88 (m, 1H), 1.80-1.65 (m, 3H), LCMS(Method 2): RT: 2.23, m / z = 566.2 (M+H)+. Docket No. 4390.3002 WO

[0780] Synthesis of 4,4'-disulfanediylbis(2,3,5,6-tetrafluorobenzoic acid) (2):

[0781] To a stirred solution of 2,3,5,6-tetrafluoro-4-mercaptobenzoic acid (1 g, 4.42 mmol) in acetic acid (15 mL) and water (6 mL), sodium perborate tetrahydrate (1.361 g, 8.84 mmol) was added. The resulting reaction mixture was stirred at RT for 16h. The progress of the reaction was monitored by UPLC. After completion, the reaction mixture was diluted with water (50 mL) and extracted with Ethyl acetate (50 mL X 3); the combined organic extracts were washed with brine (20 mL), dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure (bath temperature: 45 °C) to afford 4,4'-disulfanediylbis(2,3,5,6-tetrafluorobenzoic acid) (850 mg, 1.850 mmol, 41.8%) as an off white solid. LCMS: 98.57%, RT: 1.37 mm, m / z = 448.97 (M-H)' Synthesis of 4,4'-disulfanediylbis(2,3,5,6-tetrafhioro-N,N-dimethylbenzamide) (4):

[0782] To a stirred solution of 4,4'-disulfanediylbis(2,3,5,6-tetrafluorobenzoic acid) (1.2 g, 2.67 mmol) and dimethylamine hydrochloride (0.456 g, 5.60 mmol) in DME (20 mL) DIPEA (1.857 mL, 10.66 mmol) was added followed by PyBOP (3.33 g, 6.40 mmol) at 0 °C. The resulting reaction mixture was stirred at 0 °C for 15 min. The progress of the reaction was monitored by Docket No. 4390.3002 WO

[0783] LCMS. After completion, the reaction was quenched with water and extracted with EtOAc (2 x 30 mL), the combined organics were dried over anhydrous Na2SO4, filtered and concentrated to afford a crude residue. The obtained crude was purified by neutral alumina using 30% EtOAc / hexane as eluent to afford 4,4'-disulfanediylbis(2,3,5,6-tetrafluoro-N,N-dimethylbenzamide) (270 mg, 0.401 mmol, 15.06%) as a pale yellow gum. LCMS: 75.67%, RT: 2.31, m / z = 505.0 (M+H)+Synthesis of 2, 3 , 5 , 6-tetrafluor o-4- ((4- ((2-methoxy ethy 1)(2- (pheny lamino)py rimidin-4- yl)amino)piperidin-l -yl)thio)-N,N-dimethylbenzamide (6):

[0784] To a solution of N4-(2-methoxyethyl)-N2-phenyl-N4-(piperidin-4-yl)pyrimidine-2,4- diamine (234 mg, 0.714 mmol) and 4,4'-disulfanediylbis(2,3,5,6-tetrafluoro-N,N- dimethylbenzamide) (240 mg, 0.476 mmol) in MeOH (2.5 mL) was added silver acetate (159 mg, 0.952 mmol) and the mixture was stirred at rt for 1.5 h. The progress of the reaction was monitored by LCMS After completion, the reaction was diluted with MeOH (5 mL), filtered and concentrated under reduced pressure. The crude was purified by neutral alumina using 40%EtOAc / hexane as eluent to afford 2,3,5,6-tetrafluoro-4-((4-((2-methoxyethyl)(2- (phenylamino)pyrimidin-4-yl)amino)piperidin-l-yl)thio)-N,N-dimethylbenzamide (160 mg, 0.235 mmol, 49.4%) as a pale yellow gum. LCMS: 85.58%, RT: 1.75, m / z = 579.32 (M+H)+

[0785] EXAMPLE A118 2,3,5,6-tetrafluoro-4-((4-((2-methoxyethyl)(2-(phenylamino)pyrimidin-4- yl)amino)piperidin-l-yl)sulfinyl)-N,N-dimethylbenzamide

[0786] To a solution of 2,3,5,6-tetrafluoro-4-((4-((2-methoxyethyl)(2-(phenylamino)pyrimidin-4- yl)amino)piperidin-l-yl)thio)-N,N-dimethylbenzamide (90 mg, 0.156 mmol) in DCM (5 mL) was added mCPBA (53.7 mg, 0.311 mmol) at -5 °C and the reaction was stirred at the same temperature for 30 min. The progress of the reaction was monitored by LCMS. After completion, Docket No. 4390.3002 WO the reaction was quenched with water and extracted with DCM (2 x 20 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated. The crude was purified by prep-HPLC using 0.1%ABC in water / ACN as buffer to afford 2,3,5,6-tetrafluoro-4- ((4-((2-methoxyethyl)(2-(phenylamino)pyrimidin-4-yl)amino)piperidin-l-yl)sulfinyl)-N,N- dimethylbenzamide (9 mg, 0.015 mmol, 9.63%).

[0787] 19F NMR (376 MHz, DMSO-d6): 8 -141.51 to -140.56 (4F). 'H-VT-NMR (400 MHz, DMSO- d6): 8 8.72 (s, 1H), 7.95 (d, J = 6.00 Hz, 1H), 7.68 (d, J = 8.00 Hz, 2H), 7.16 (t, J = 8.00 Hz, 2H), 6.88 (t, J = 7.20 Hz, 1H), 6.18 (d, J = 6.00 Hz, 1H), 4.378-4.34 (m, 1H), 3.86 (d, J = 11.20 Hz,

[0788] 1H), 3.66 (d, J = 12.80 Hz, 1H), 3.52-3.47 (m, 4H), 3.29 (s, 3H), 3.09 (s, 3H), 2.97 (s, 3H), 2.88- 2.75 (m, 2H), 1.92-1.82 (m, 4H). LCMS (Method 2): RT: 1.40, m / z = 595.2 (M+H)+.

[0789] Synthesis of N4-(l-(6-chloropyrimidin-4-yl)piperidin-4-yl)-N4-(2-methoxyethyl)-N2- pheny lpyrimidine-2,4-diamine (2) :

[0790] In an oven dried 40 mL reaction vial, N4-(2-methoxyethyl)-N2'phenyl-N4-(piperidin-4- yl)pyrimidine-2,4-diamine Hydrochloride (100 mg, 0.275 mmol), ethanol (5 mL), 2,4- dichloropyrimidine (40.9 mg, 0.275 mmol) and triethylamine (0.077 mL, 0.550 mmol) were combined at 0 °C and stirred for 3h at rt. The progress of the reaction was monitored by TLC and LCMS. After completion, the volatiles were removed under reduced pressure, the residue was diluted with water (10 mL) and extracted with EtOAc (2 x 10 mL). The combined organics were dried over Na2SO4 and concentrated under reduced pressure to afford N4-(l-(2-chloropyrimidin- Docket No. 4390.3002 WO

[0791] 4-yl)piperidin-4-yl)-N4-(2-methoxyethyl)-N2-phenylpyrimidine-2,4-diamine (90 mg, 0.170 mmol, 61.8%) as an off white solid. LCMS: 83.88%, RT: 1.325 min, m / z = 440.1 (M+H)+. EXAMPLE A119 N4-(2-methoxyethyl)-N2-phenyl-N4-(l -(6-vinylpyrimidin-4-yl)piperidin-4- yl)pyrimidine-2,4-diamine

[0792] To a solution of N4-(l-(6-chloropyrimidin-4-yl)piperidin-4-yl)-N4-(2-methoxyethyl)-N2- phenylpyrimidine-2,4-diamine (200 mg, 0.455 mmol) and potassium vinyltrifluoroborate (122 mg, 0.909 mmol) in 1,4-Dioxane (5 mL) Na2COs (145 mg, 1.364 mmol) was added. The reaction mixture was degassed with nitrogen for 2 min. To this solution, PdC12(dppf)-CH2C12 adduct (37.1 mg, 0.045 mmol) was added and the mixture was heated at 100 °C for 2h in microwave reactor. The progress of the reaction was monitored by LCMS, quenched with water and extracted with EtOAc (2 x 20 mL), The combined organic portions were dried over Na2SO4, filtered and concentrated under reduced pressure.. The obtained crude was purified by Prep-HPLC to afford N4-(2-methoxyethyl)-N2-phenyl-N4-(l-(6-vinylpyrimidin-4-yl)piperidin-4-yl)pyrimidine-2,4- diamine (35.5 mg, 0.082 mmol, 18.08%) as an off-white solid.

[0793] 1H-VT-NMR (400 MHz, DMSO-d6): 8 8.71 (s, 1H), 8.47 (s, 1H), 7.96 (d, J = 6.00 Hz, 1H), 7.72 (d, J = 8.00 Hz, 2H), 7.23 (t, J = 8.00 Hz, 2H), 6.91-6.87 (m, 2H), 6.65 (q, J = 10.40 Hz, 1H), 6.37 (dd, J = 17.20, 1.60 Hz, 1H), 6.21 (d, J = 6.40 Hz, 1H), 5.54 (dd, J = 2.00, 10.60 Hz, 1H), 4.63- 4.56 (m, 3H), 3.53-3.46 (m, 4H), 3.26 (s, 3H), 3.01-2.98 (m, 2H), 1.81-1.69 (m, 4H) LCMS(Method 2): RT: 1.32, m / z = 432.2 (M+H)+.

[0794] EXAMPLE A126 N4-(2-methoxyethyl)-N2-phenyl-N4-(l -(4-vinylpyridin-2-yl)piperidin-4- yl)pyrimidine-2,4-diamine Docket No. 4390.3002 WO

[0795] EXAMPLE A127 N4-(2-methoxyethyl)-N2-phenyl-N4- (l-(2-vinylpyridin-4-yl)piperidin-4- yl)pyrimidine-2,4-diamine

[0796] EXAMPLE A129 N4-(2-methoxyethyl)-N2-phenyl-N4- (l-(2-vinylpyrimidin-4-yl)piperidin-4- yl)pyrimidine-2,4-diamine

[0797] EXAMPLE A130 N4-(2-methoxyethyl)-N2-phenyl-N4- (l-(4-vinylpyrimidin-2-yl)piperidin-4- yl)pyrimidine-2,4-diamine EXAMPLE A131 N4-(l -(5-fluoro-2-vinylpyridin-4-yl)piperidin-4-yl)-N4-(2-methoxyethyl)-N2- phenylpyrimidine-2,4-diamine

[0798] EXAMPLE A134 N4-(2-methoxyethyl)-N2-phenyl-N4- (l-(6-vinylpyridin-3-yl)piperidin-4- yl)pyrimidine-2,4-diamine Docket No. 4390.3002 WO

[0799] Example 49: PIKfyve Protein Expression and Purification:

[0800] Full length wild type PIKfyve (aa 1-2098; UniProt ID: Q9Y2I7-1) construct with N-terminal 3xFLAG and C-terminal His tag was cloned into pcDNA3.1 vector and transfected into Expi293F cells (Thermo, Cat. No. Al 4527). Protein was expressed for 72 hours at 30°C after transfection. The cells were harvested by centrifugation and flash-frozen for storage.

[0801] Frozen cell pellets were re-suspended in ice-cold lysis buffer (50 mM HEPES, 500 mM NaCl, 10% Glycerol, 0.1% Tween 20, 1 mM TCEP, 1 / 200 (v / v) Halt™ protease inhibitor cocktail (Thermo, Cat. No. 78429), 0.1 mg Benzonase / IL culture, pH 8.0) and transferred into a Dounce homogenizer and lysed with 10 strokes. The supernatant after centrifugation was incubated with Anti-FLAG-Gl affinity resin (GenScript, Cat. No. L00432) for 1.5 hours. Then, the resin was washed 3 times with lOx bed volumes of wash buffer (50 mM HEPES, 300 mM NaCl, 10% Glycerol, 1 mM TCEP, pH 8.0) and eluted 3 times with 2x bed volume of elution buffer (50 mM HEPES, 300 mM NaCl, 10% Glycerol, 1 mM TCEP, 300 pg / mL FLAG peptide [GenScript, Cat. No. RP10586], pH 8.0). The elution fractions were pooled and further purified using a HiLoad 16 / 600 Superose™ 6 pg (Cytiva, Cat. No. 29323952) gel filtration column pre-equilibrated with gel filtration buffer (50 mM HEPES, 600 mM NaCl, 5% Glycerol, 0.5 mM TCEP, pH 8.0). Peak fractions of interest were collected and flash frozen in liquid nitrogen for storage at -80°C.

[0802] Quality control after one freeze-thaw cycle was performed to confirm protein was a monodisperse sample with no indication of aggregation or oligomerization using a Superose™ 6 Increase 5 / 150 GL (Cytiva, Cat. No. 29091597) gel filtration column pre-equilibrated with gel filtration buffer.

[0803] Example 50: Enzymatic Assay for inhibition of PIKfyve

[0804] The inhibition of Recombinant PIKfyve protein [hPIKfyve( 1-2098) or commercial hPIKfyve (Carna, cat no. 11-118)] was measured using ADP-Glo kinase-assay kit (Promega). 300 nl of compounds (dissolved in DMSO) in serial dilution were transferred to 384-well Optip lates (Perkin Elmer) using Echo 650 series liquid handler (Beckman Coulter). 5 pl of Recombinant Plkfyve in assay buffer (25 mM HEPES pH 7.5, 10 mM MgCh, 1 pM CaCh, 2 mM DTT, 0.05% BSA, and 0.002% Triton-XlOO) is added to the compounds and preincubated for 120 minutes at room temperature. To initiate the reaction, 5 ul of ATP and PI(3)P:PS substrate are added. For Docket No. 4390.3002 WO hPIKfyve( 1-2098) the final concentration of recombinant protein, ATP, and Substrate are 5 nM, 20 uM, and 62.5:500 uM, respectively while for hPIKfyve (Carna), the final concentration of recombinant protein, ATP, and Substrate are 5 nM, 50 uM, and 62.5:500 uM, respectively. The plate was incubated for 90 minutes at room temperature then 10 ul of the ADP-GLO™ reagent was added and incubated for 60 minutes. 20 ul of the kinase detection reagent was added and incubated for an additional 30 minutes then luminescence (LUM) was detected using the Pherastar microplate reader (BMG Labtech). The enzyme activity was normalized using control wells containing negative control (0% inhibition) of 3% DMSO or positive control (100% inhibition) of 10 pM YM-201636 (MedChemExpress).

[0805] % Inhibition = 100 x [l-(LUMCmPd - LUMpOs) / (LUMneg - LUMpos)]

[0806] Where LUMCmPd, LUMpos, and LUMneg are the relative luminescence units of compound, YM- 201636, and DMSO treated wells respectively. The %inhibition values were plotted as a function of compound concentration and the IC50 values of compounds were determined using a four- parameter logistic fit Y = Min + [(Max-Min) / (1 +(X / IC5o)H111 slope)] or three-parameter fit if Max or Min of curves were fixed to 100% or 0% respectively.

[0807] Table 1 shows the activity of exemplar PIKfyve inhibitors of Formula I against two forms of the PIKfyve enzyme using the ADP-Glo assay format described above, where ‘A’, ‘B’ and ‘C’ have the following meaning: ‘A’: IC5o < O.Ol pM

[0808] ‘B’: ICso > 0.01 pM and < 0.10 pM

[0809] ‘C’: IC5o > O.lO pM and < 0.50 pM

[0810] Table 1 Docket No. 4390.3002 WO Docket No. 4390.3002 WO

[0811] Table 2 shows the activity of exemplar PIKfyve inhibitors of Formula X against the PIKfyve enzyme using the ADP-Glo assay format described above expressed as the concentration required to inhibit 50% of the enzymatic activity: IC50 = A < 10 nM < B < 100 nM < C < 1000 nM Docket No. 4390.3002 WO

[0812] Example 51: Concentration of PIKfyve inhibitor Plasma and Brain Animals

[0813] Male C57BL / 6 mice (25±5 g body weight) were obtained from Hylasco Biosciences, Hyderabad, India. Upon arrival at the facility, mice were quarantined and acclimatized in the laboratory for one week. They were maintained under standard environmental conditions with a 12 h light / dark cycle with free access to rodent chow and filtered water. All animal experiments were approved by the Institutional Animal Ethics Committee (SYNGENE / IAEC / 1632 / 10-2024) and were in agreement with the Committee for Control and Supervision of Experiments on Animals (CCSEA), Ministry of Fisheries, Animal Husbandry and Dairying, Department of Animal Husbandry and Dairying, Government of India.

[0814] Formulation:

[0815] The oral formulations of Compound Al (Example Al) comprised of 15% (v / v) DMSO, 25% (v / v) of Polyethylene glycol (PEG) 400, and 60% (v / v) of 15% (w / v) Hydroxypropyl beta cyclodextrin in water. The oral formulation of elacridar comprised 20% (v / v) DMSO, 20% (v / v) PEG400, 40% (v / v) PG, and 20% (v / v) 25% Captisol. Compound Al and elacridar were administered at a 10 mL / kg dose volume. The elacridar was administered as a 30-minute pretreatment to Compound Al administration in 50 mg / kg dose group animals (group-1). Docket No. 4390.3002 WO

[0816] Study design:

[0817] Before the start of experiments, animals were fasted for 4-6 hours with water supplied ad libitum. Feed was provided 4-h post-dose. The study consists of two groups, containing twelve animals in each group. The animals from group-1 received Compound Al at the dose of 50 mg / kg by oral administration whereas group-2 animals received Compound Al at 100 mg / kg by oral gavage administration. Elacridar (100 mg / kg) was administered by oral gavage to group-1 animals, 30 min before Compound Al administration. Group-2 animals received blank vehicles 30 min before Compound Al administration. Plasma and brain samples were collected from both groups at 0.5, 1, 2, and 4 hours post-Compound Al administration. At each time point, animals were anesthetized using light isoflurane, and approximately 100 pL of blood was collected from the retro-orbital sinus into K2EDTA (0.2% v / v) containing Eppendorf tubes. Following blood collection, animals were again anesthetized with deep isoflurane. The abdominal and thoracic cavity was cut open with a mid-line incision to expose the heart. Intra-cardial perfusion was performed with 20 mL of K2EDTA and the abdominal aorta was cut to drain the blood. After intracardial perfusion, the cranial cavity was cut open and the whole brain was collected. The collected brain was blotted dry on filter paper, weighed, and stored in labeled tubes. Blood samples were centrifuged at 13,000 rpm for 10 minutes at 4°C. Plasma and brain samples were stored at dry ice / -80 °C until bioanalysis. The whole brain was homogenized with phosphate buffer. For 1 g of brain tissue weight, 2X volume of PBS was added for homogenization. A protein precipitation method was used for the extraction of analyte from the plasma and brain homogenate samples. The study samples were analyzed by fit-for-purpose LC-MS / MS method for estimation of Compound Al in plasma and brain homogenate samples.

[0818] The brain-to-plasma ratios were calculated at each interval by dividing total brain concentrations by that of plasma concentrations. Fig. 1 demonstrates that the brain concentration of Compound Al was significantly increased in brain tissue in elacridar pre-treated animals. Fig. 2 demonstrates that oral administration of Compound Al (single dose, 50 mg / kg) in elacridar pre-treated animals results in >50% PIKfyve occupancy in mouse brains for > 4h and is well tolerated. Fig. 3 demonstrates that Compound Al (single oral dose, 100 mg / kg) administered alone provides >50% peripheral target occupancy (TO) but no occupancy in the brain. In contrast, A single oral dose (50 mg / kg) of Compound Al combined with elacridar provides >50% target occupancy in the brain. Docket No. 4390.3002 WO

[0819] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

Docket No. 4390.3002 WOCLAIMSWhat is claimed:

1. A method for treating, preventing, or reducing a disease or disorder in a subject in need thereof, the method comprising co-administering to the subject an effective amount of a PIKfyve inhibitor and an effective amount of a p-glycoprotein (PGP) inhibitor.

2. The method according to claim 1 , wherein the PIKfyve inhibitor is administered to the subject at the same time as the PGP inhibitor; wherein the PIKfyve inhibitor is administered to the subject prior to the PGP inhibitor; or wherein the PIKfyve inhibitor is administered to the subject after the PGP inhibitor.

3. The method according to claim 2, wherein the PGP inhibitor is administered to the subject prior to the PIKfyve inhibitor.

4. The method according to claim 3, wherein the PGP inhibitor is administered to the subject about 1 minute to about 60 minutes prior to the PIKfyve inhibitor.

5. The method according to any one of claims 1-4, wherein the PIKfyve inhibitor is a compound of formula I.

6. The method according to any one of claims 1-5, wherein the PIKfyve inhibitor is a compound of formula X.

7. The compound according to any preceding claim, wherein the PIKfyve inhibitor forms a covalent bond with an amino acid residue in the PIKfyve protein.

8. The compound according to any preceding claim, wherein the PIKfyve inhibitor forms a covalent bond with a sulfur atom of a cysteine residue in the PIKfyve protein.

9. The compound according to any preceding claim, wherein the PIKfyve inhibitor is an irreversible inhibitor of the PIKfyve protein.

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