Pyrrolo[2,3-b]pyridine-3-carboxamide compositions and methods for ameliorating hearing loss

Abstract

Disclosed are N-(3-substituted thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamides (I) and (II), which are N-(3-substituted oxazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamides. The compounds activate Yap and inhibit Lats kinase. Therefore, the compounds are useful for the treatment of hearing loss.

Description

Detailed Description of the Invention

[0001] <Government Rights Notice> This invention was made with government support under Grant No. T32GM007739 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.

[0002] [Background of the invention] <Technical field> This application relates generally to N-(3-substituted thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamides and N-(3-substituted oxazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamides that inhibit Lats kinase and thereby increase Yap activity. The compounds are useful for inducing proliferation of supporting cells in the inner ear and thus potentially treating hearing loss.

[0003] <Background information> Regeneration, initiated in response to injury, is a complex process that can restore the structure and function of damaged tissues. Some mature mammalian tissues have a gradually decreased regenerative capacity after completing development. Regeneration occurs either by activation and proliferation of resident stem cells, as in the skin and intestinal epithelium, or by dedifferentiation and proliferation of cells, as in the liver. In other cases, such as central nervous tissue and cardiac muscle tissue, cells show little or no regenerative potential after injury.

[0004] Given its fundamental roles in development, proliferation, stem cell maintenance, and dedifferentiation, Hippo signaling is an intriguing target for driving regeneration. The regenerative potential of the Hippo pathway has been well-defined in multiple organs, including the heart, retina, liver, and intestine. Hippo signaling limits the size of the developing murine utricle, a sensory organ in the vestibular portion of the inner ear, and the Yap-Tead complex is active during and required for regenerative proliferation in the neonatal utricle. These observations suggest that chemical activation of Yap signaling may result in a critical yet undiscovered step in mammalian inner ear regeneration: proliferation of supporting cells in adult tissues.

[0005] In an effort to identify an activator of Yap, we performed a small molecule screen on cultured cells. We identified a compound that we found to function as an inhibitor of Lats kinase. To test our own hypothesis, we treated explanted utricles from adult mice with the substance and found that a few days of exposure caused supporting cells to re-enter the cell cycle, a crucial step in the robust regeneration of hair cells.

[0006] [Summary of the Invention] The present invention relates to N-(3-substituted azol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamides, pharmaceutical compositions and methods for inhibiting Lats or activating Yap and thereby stimulating the regeneration of target cells, particularly hair cells.

[0007] In a first aspect, the present invention provides a compound of formula I: [ka] (In the formula, R 1 is (C1-C6) alkyl, carboxy, (C3-C7) carbomonocyclyl, (C9-C 11)carbobicyclyl, heteromonocyclyl, and heterobicyclyl, wherein the (C1-C6)alkyl, (C3-C7)carbomonocyclyl, (C9-C 11 )carbobicyclyl, heteromonocyclyl, and heterobicyclyl are optionally selected from halogen, cyano, hydroxy, nitro, amino, acetoxy, carboxy, (C1-C7)hydrocarbyl, halo(C1-C6)alkyl, (C1-C3)alkoxy, halo(C1-C3)alkoxy, (C1-C6)acyl, (C1-C3)alkoxy(C1-C3)alkyl, hydroxy(C1-C3)alkyl, heteroaryl, benzenesulfonyl, (C1-C3)alkoxycarbonyl, aminocarbonyl, (C1-C3)alkylaminocarbo nyl, di(C1-C3)alkylaminocarbonyl, (C1-C3)alkylamino, di(C1-C3)alkylamino, amino(C1-C3)alkyl, (C1-C3)alkylamino(C1-C3)alkyl, (C1-C3)dialkylamino(C1-C3)alkyl, (C1-C3)alkylthio, (C1-C3)alkylsulfonylamino, (C1-C3)alkylsulfinyl, (C1-C3)alkylsulfonyl, phenoxy, and benzyloxy; R 2 is hydrogen, halogen, (C1-C7)hydrocarbyl, halo(C1-C6)alkyl, (C1-C6)acyl, hydroxy(C1-C3)alkyl, -C(=O)O(C1-C6)alkyl, -C(=O)NR 20 R 21 and (C1-C6)oxaalkyl; R 3 is selected from the group consisting of hydrogen, halogen, (C1-C6)hydrocarbyl, halo(C1-C6)alkyl, (C1-C6)acyl, and (C1-C3)alkoxy; R 4 is selected from the group consisting of hydrogen, halogen, (C1-C6)hydrocarbyl, halo(C1-C6)alkyl, (C1-C6)acyl, and (C1-C3)alkoxy; R 10is independently selected in each occurrence from the group consisting of hydrogen and methyl; R 20 is selected from the group consisting of hydrogen and (C1-C6)hydrocarbyl; R 21 is hydrogen, (C1-C6)hydrocarbyl, (C1-C6)oxaalkyl, amino(C1-C6)alkyl, (C1-C3)alkylamino(C1-C6)alkyl, di(C1-C3)alkylamino(C1-C6)alkyl, and -(CH2) m -Het, where Het is selected from the group consisting of hydroxy, amino, acetoxy, carboxy, (C1-C7)hydrocarbyl, halo(C1-C6)alkyl, (C 1‐ an aliphatic monocyclic or bicyclic heterocycle optionally substituted with a substituent selected from the group consisting of (C3)alkoxy, halo(C1-C3)alkoxy, (C1-C6)acyl, (C1-C3)alkoxy(C1-C3)alkyl, hydroxy(C1-C3)alkyl, aminocarbonyl, (C1-C3)alkylaminocarbonyl, di(C1-C3)alkylaminocarbonyl, (C1-C3)alkylamino, and di(C1-C3)alkylamino; Or R 20 and R 21 is R 20 and R 21 forms an aliphatic heterocycle together with the nitrogen to which it is attached, n is 0, 1 or 2; m is 0, 1 or 2, and X is S or n is 1 and R 1 When is optionally substituted phenyl, X may further be O; However, R 1 is phenyl the law of nature , X but Sulfur and n but 1 If so, R 2 , R 3 , R 4 and R 10 At least one of these is not hydrogen. The present invention relates to a compound represented by the formula:

[0008] In another aspect, the invention relates to a pharmaceutical composition comprising a pharma- ceutically acceptable carrier and a compound as described herein.

[0009] In another aspect, the invention relates to a method of activating YAP in a cell that expresses YAP, comprising exposing the cell to a compound as described herein.

[0010] In another aspect, the invention relates to a method for the inhibition of LATS in a cell population expressing LATS comprising exposing the cell population to a compound as described herein.

[0011] In another aspect, the invention relates to a method for LATS inhibition in a cell population comprising exposing the cell population to a compound as described herein.

[0012] In another aspect, the invention relates to a method of stimulating the regeneration of hair cells comprising exposing a supporting cell population to a compound as described herein.

[0013] [Detailed Description of the Invention] Formula I [ka] It has been found that compounds represented by I are useful for inhibiting Lats or activating Yap and are therefore potential therapeutic agents for stimulating the regeneration of target cells, particularly hair cells. Such compounds may be useful for the treatment of hearing loss. The group of compounds represented by I can be divided into two subgroups.

[0014] In a first subgroup, X is sulfur and the compound has formula II: [ka] The compound is thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide, represented by the formula:

[0015] In a second subgroup, X is oxygen and the compound has formula III: [ka] (wherein n is 1 and R 1 is optionally substituted phenyl. The compound is oxazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide represented by the formula:

[0016] In some embodiments of Formulas I and II, n can be 0. In other embodiments of Formulas I-III, n can be 1. In these embodiments, R 10 can be hydrogen.

[0017] In some embodiments, R 1 is optionally substituted (C1-C4)alkyl, carboxy, phenyl, cyclohexyl, 5-membered heterocyclyl, 6-membered heterocyclyl or heterobicyclyl. In particular, R 1 can be methyl, ethyl, aminobutyl, and carboxyethyl. In other embodiments, R 1 is optionally substituted cyclohexyl, or R 1 is optionally substituted phenyl, or R 1 is optionally substituted heterocyclyl, such as pyridinyl, pyrazolyl, piperidinyl, tetrahydropyranyl, tetrahydrofuranyl, or tetrahydroisoquinolinyl. 1 When R is an optionally substituted phenyl, the phenyl may bear one or two substituents independently selected from halogen, cyano, hydroxy, amino, carboxy, (C1-C6)hydrocarbyl, trifluoromethyl, methoxy, acetyl, formyl, hydroxy(C1-C3)alkyl, methoxycarbonyl [-C(=O)OCH3], carboxamido [-C(=O)NH2], methanesulfonylamino, and amino(C1-C3)alkyl. 1When is pyridinyl, pyrazolyl, piperidinyl, tetrahydropyranyl, or tetrahydroisoquinolinyl, each heterocycle is optionally substituted with 1 or 2 substituents independently selected from amino, hydroxy, and (C1-C6)hydrocarbyl.

[0018] In some embodiments, R 1 is selected from the group consisting of carboxy and optionally substituted (C1-C4)alkyl, phenyl, cyclohexyl, 5-membered heterocyclyl, 6-membered heterocyclyl, and heterobicyclyl. 1 is selected from the group consisting of methyl, ethyl, aminobutyl, and carboxyethyl. 1 is optionally substituted cyclohexyl. In some of the above, R 1 is an optionally substituted heterocyclyl. The heterocycle may be pyridinyl, pyrazolyl, piperidinyl, tetrahydropyranyl, tetrahydrofuranyl, and tetrahydroisoquinolinyl, each of which is optionally substituted. The optional substituents include one or two substituents independently selected from the group consisting of amino, hydroxy, and (C1-C6)hydrocarbyl.

[0019] In some embodiments, R 1 is an optionally substituted phenyl, which may be substituted with one or two substituents independently selected from the group consisting of halogen, cyano, hydroxy, amino, carboxy, (C1-C6)hydrocarbyl, trifluoromethyl, methoxy, acetyl, formyl, hydroxy(C1-C3)alkyl, methoxycarbonyl, carboxamido, methanesulfonylamino, and amino(C1-C3)alkyl. In some of the above embodiments, R 1 is phenyl substituted at the ortho position and n is 0; in other embodiments, R 1 is optionally substituted phenyl and n is 1.

[0020] In some embodiments, R 2 is -C(=O)O(C1-C6)alkyl, -C(=O)NR 20 R 21 In some of these embodiments, R 20 is selected from hydrogen and methyl, and R 21 is hydrogen, methyl, (C1-C6) oxaalkyl, dimethylamino(C1-C6) alkyl, and -(CH2) m In other cases, R 20 and R 21 is R 20 and R 21 Together with the nitrogen to which it is attached, it forms a 4- to 7-membered aliphatic heterocycle. Typical aliphatic heterocycles include piperidine, piperazine, morpholine, pyrrolidine, azetidine, and azepine.

[0021] In some embodiments, R 2 is selected from hydrogen, methyl, ethyl, propyl, cyclopropyl, hydroxymethyl, and trifluoromethyl.

[0022] In some embodiments, R 3 and R 4 is selected from hydrogen, chloro and methyl.

[0023] Of course, in various embodiments, the pharmaceutical compositions of the present invention include one or more pharma- ceutically acceptable additives, such as, but not limited to, one or more binders, bulking agents, buffers, stabilizers, surfactants, wetting agents, lubricants, excipients, disintegrants, thickening or thinning agents, emulsifying agents, suspending agents, preservatives, antioxidants, opacifying agents, flow agents, processing aids, colorants, sweeteners, flavoring agents, fragrances, flavoring agents, excipients, polishing agents, polymer matrix systems, plasticizers, and other known additives that provide a drug presentation or aid in the manufacture of a pharmaceutical or drug product comprising the composition of the present invention. Examples of carriers and additives well known to those skilled in the art are described in detail, for example, in Ansel, Howard C. et al., "Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems", (Philadelphia, USA), Lippincott, Williams and Wilkins, 2004; Gennaro, Alfonso R. et al., "Remington: The Science and Practice of Pharmacy", (Philadelphia, USA), Lippincott, Williams and Wilkins, 2000; and Rowe, Raymond C., "Handbook of Pharmaceutical Excipients", (Chicago, USA), Pharmaceutical Press, 2005.

[0024] In various embodiments, non-limiting examples of additives include, but are not limited to, corn starch, potato starch, or other starches, gelatin, natural and synthetic gums, such as gum arabic, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, calcium carboxymethylcellulose, sodium carboxymethylcellulose), polyvinylpyrrolidone, methylcellulose, pregelatinized starch, hydroxypropyl methylcellulose (e.g., 2208, 2906, 2910), hydroxypropyl cellulose, titanium dioxide, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, silicic acid, sorbitol, starch, pregelatinized starch, cellulose acetate ... cellulose, agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, other starches, pregelatinized starch, other starches, clay, other algins, other celluloses, gums, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oils (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laurate, agar, syloid® silica gel (AEROSIL 200, available from W. R. Grace, Inc., Baltimore, MD, USA). Grace Co.), synthetic silica agglomerated aerosol (sold by Degussa Co., Plano, Texas, USA), CAB-O-SIL® (a pyrogenic silicon dioxide product sold by Cabot Co., Boston, Massachusetts, USA), colorants, and mixtures thereof.

[0025] The terms "subject" or "subject in need thereof" are used interchangeably herein. These terms refer to a patient who has been diagnosed with the underlying disease to be treated. Typically, the patient will be a human. The subject may currently be experiencing symptoms associated with the disease or may have experienced symptoms in the past. In addition, a "subject in need thereof" may be a patient who is at risk of developing a particular disease or who is suffering from one or more of the physiological systems of the disease, even if the disease has not yet been diagnosed.

[0026] As used herein, the terms "treatment" or "treating" are used interchangeably. These terms refer to an approach to obtain a beneficial or desired result, such as, but not limited to, a therapeutic benefit. A therapeutic benefit includes eradication or amelioration of the underlying disease being treated; and also includes eradication or amelioration of one or more symptoms associated with the underlying disease, such that the patient observes an improvement, even if the patient is still afflicted by the underlying disease.

[0027] As used herein, the term "optionally substituted" can be used interchangeably with "unsubstituted or substituted." The term "substituted" refers to one or more hydrogen atoms in a specified group being replaced with a specified radical. For example, substituted aryl, heterocyclyl, etc. means that one or more H atoms of each residue are replaced with a halogen, haloalkyl, alkyl, (C 1‐8) hydrocarbyl, acyl, alkoxyalkyl, hydroxy lower alkyl, carbonyl, phenyl, heteroaryl, benzenesulfonyl, hydroxy, lower alkoxy, haloalkoxy, oxaalkyl, carboxy, alkoxycarbonyl [i.e. -C(=O)O-alkyl], carboxamido [i.e. -C(=O)NH2], alkylaminocarbonyl [i.e. -C(=O)NH-alkyl], cyano, acetoxy, nitro, amino, alkylamino, dialkylamino, dialkylaminoalkyl, dialkylaminoalkoxy, heterocyclylalkoxy, arylalkyl, (cycloalkyl) It refers to aryl or heterocyclyl substituted by alkyl, heterocyclyl, heterocyclylalkyl, alkylaminoalkyl, heterocyclylaminoalkyl, heterocyclylalkylaminoalkyl, cycloalkylaminoalkyl, cycloalkylalkylaminoalkyl, arylaminoalkyl, and arylalkylaminoalkyl, mercapto, alkylthio, alkylsulfinyl, benzyl, heterocyclyl, phenoxy, benzyloxy, heteroaryloxy, aminosulfonyl, amidino, guanidino, ureido, -SO2alkyl, -SO2NH2, or -SO2NHalkyl.Preferred subsitutents are halogen, cyano, hydroxy, nitro, amino, acetoxy, carboxy, (C1-C7)hydrocarbyl, halo(C1-C6)alkyl, (C1-C3)alkoxy, halo(C1-C3)alkoxy, (C1-C6)acyl, (C1-C3)alkoxy(C1-C3)alkyl, hydroxy(C1-C3)alkyl, heteroaryl, benzenesulfonyl, (C1-C3)alkoxycarbonyl [i.e. -C(=O)O(C1-C3)alkyl], carboxamide [i.e. -C (=O)NH2], (C1-C3)alkylaminocarbonyl [i.e. -C(=O)NH-(C1-C3)alkyl], (C1-C3)alkylamino, di(C1-C3)alkylamino, amino(C1-C3)alkyl, (C1-C3)alkylamino(C1-C3)alkyl, (C1-C3)dialkylamino(C1-C3)alkyl, (C1-C3)alkylthio, (C1-C3)alkylsulfonylamino, (C1-C3)alkylsulfinyl, (C1-C3)alkylsulfonyl, phenoxy, and benzyloxy.

[0028] Unless otherwise specified, alkyl is linear or branched hydrocarbyl. Unless otherwise specified, unsubstituted alkyl has 1 to 20 carbon atoms (e.g., 1 to 6 carbon atoms). Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, s- and t-butyl, and the like.

[0029] Hydrocarbon or hydrocarbyl (as a substituent) includes alkyl, cycloalkyl, polycycloalkyl, alkenyl, alkynyl, aryl, and combinations thereof. Examples include cyclopropylmethyl, benzyl, phenethyl, cyclohexylmethyl, camphoryl, and naphthylethyl. Hydrocarbon refers to any substituent composed solely of hydrogen and carbon as elemental moieties. Cycloalkyl is a subset of hydrocarbyl and includes cyclic hydrocarbon groups of 3 to 8 carbon atoms. Examples of cycloalkyl groups include c-propyl, c-butyl, c-pentyl, norbornyl, and the like.

[0030] Unless otherwise specified, the term "carbocycle" is a ring system in which the ring atoms are all carbon of any oxidation state. Thus, (C3-C8) carbocycle refers to both non-aromatic and aromatic systems, e.g., systems such as cyclopropane, benzene, and cyclohexene; (C8-C 12 ) Carbopolycycle refers to systems such as norbornane, decalin, indane and naphthalene. Carbocycle refers to monocyclic, bicyclic and polycyclic rings, unless otherwise limited.

[0031] Oxaalkyl refers to an alkyl residue in which one or more carbons (and their associated hydrogens) have been replaced by oxygen. Examples include methoxypropoxy, 3,6,9-trioxadecyl, and the like. The term oxaalkyl has the meaning as it is understood in the art [see "Naming and Indexing of Chemical Substances for Chemical Abstracts", 196, published by the American Chemical Society, except as provided in 127(a)], i.e., the term refers to compounds in which oxygen is bonded to its adjacent atom by a single bond (forming an ether bond); it does not refer to a double-bonded oxygen such as that found in a carbonyl group. Alkoxy or alkoxyl is a subset of oxaalkyl, referring to groups of 1 to 8 carbon atoms of straight or branched configuration attached to the parent structure through an oxygen. Examples include methoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy, cyclohexyloxy, and the like. Lower alkoxy refers to groups containing 1 to 4 carbons. For the purposes of this application, alkoxy and lower alkoxy include methylenedioxy and ethylenedioxy.

[0032] Unless otherwise specified, acyl refers to formyl and groups of 1, 2, 3, 4, 5, 6, 7, and 8 carbon atoms of linear, branched, cyclic configuration that are saturated, unsaturated, and aromatic and combinations thereof, attached to the parent structure by a carbonyl functionality. One or more carbons in the acyl residue may be replaced by nitrogen, oxygen, or sulfur as long as the point of attachment to the parent is at the carbonyl. Examples include formyl, acetyl, benzoyl, propionyl, isobutyryl, t-butoxycarbonyl, benzyloxycarbonyl, and the like. Lower acyl refers to groups containing 1-4 carbons. The double bonded oxygen is referred to as "oxo" when referring to itself as a substituent.

[0033] Aryl and heteroaryl refer to (i) a phenyl group (or benzene) or a monocyclic 5- or 6-membered heteroaromatic ring containing 1-4 heteroatoms selected from O, N, or S; (ii) a bicyclic 9- or 10-membered aromatic or heteroaromatic ring structure containing 0-4 heteroatoms selected from O, N, or S; or (iii) a tricyclic 13- or 14-membered aromatic or heteroaromatic ring structure containing 0-5 heteroatoms selected from O, N, or S. Examples of 6- to 14-membered aromatic carbocycles include benzene, naphthalene, indane, tetralin, and fluorene, and examples of 5- to 10-membered aromatic heterocycles include imidazole, pyridine, indole, thiophene, benzopyranone, thiazole, furan, benzimidazole, quinoline, isoquinoline, quinoxaline, pyrimidine, pyrazine, tetrazole, and pyrazole. As used herein, aryl and heteroaryl refer to moieties in which one or more rings are aromatic, but not necessarily all of the rings are aromatic.

[0034] Arylalkyl refers to a substituent in which an aryl residue is attached to the parent structure by an alkyl. Examples include benzyl, phenethyl, etc. Heteroarylalkyl refers to a substituent in which a heteroaryl residue is attached to the parent structure by an alkyl. In one embodiment, the alkyl group of the arylalkyl or heteroarylalkyl is an alkyl group having 1 to 6 carbons. Examples include, for example, pyridinylmethyl, pyrimidinylethyl, etc.

[0035] Heterocycle means a cycloalkyl or aryl carbocyclic moiety in which one to four carbons are replaced by a heteroatom selected from the group consisting of N, O, and S. The nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized. Unless otherwise specified, a heterocycle may be non-aromatic (i.e., aliphatic) or aromatic. Examples of heterocycles include pyrrolidine, pyrazole, pyrrole, indole, quinoline, isoquinoline, tetrahydroisoquinoline, benzofuran, benzodioxane, benzodioxole (commonly referred to as methylenedioxyphenyl when present as a substituent), tetrazole, morpholine, thiazole, pyridazine, pyrimidine, thiophene, furan, oxazole, oxazoline, isoxazole, dioxane, tetrahydrofuran, and the like. Note that heteroaryl is a subset of heterocycle in which the heterocycle is aromatic. Examples of heteroaromatic rings include furan, benzofuran, isobenzofuran, pyrrole, indole, isoindole, thiophene, benzothiophene, imidazole, benzimidazole, purine, pyrazole, indazole, oxazole, benzoxazole, isoxazole, benzisoxazole, thiazole, benzothiazole, triazole, tetrazole, pyridine, quinoline, isoquinoline, pyrazine, quinoxaline, acridine, pyrimidine, quinazoline, pyridazine, cinnoline, phthalazine, and triazine.Further examples of heterocyclyl moieties include piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxo-pyrrolidinyl, 2-oxoazepinyl, azepinyl, 4-piperidinyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyrazinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolyl, quinuclidinyl, isothiazolidinyl, benzimidazolyl, thiadiazolyl, benzopyranyl, benzothiazolyl, tetrahydrofuryl, tetrahydropyranyl, thienyl, benzothienyl, thiamorpholinyl, thiamorpholinylsulfoxide, thiamorpholinylsulfone, oxadiazolyl, triazolyl and tetrahydroquinolinyl.

[0036] Oxygen heterocycles are heterocycles that contain at least one oxygen in the ring; they may contain additional oxygen and other heteroatoms. Sulfur heterocycles are heterocycles that contain at least one sulfur in the ring; they may contain additional sulfur and other heteroatoms. Oxygen heteroaryls are a subset of oxygen heterocycles; examples include furan and oxazole. Sulfur heteroaryls are a subset of sulfur heterocycles; examples include thiophene and thiazine. Nitrogen heterocycles are heterocycles that contain at least one nitrogen in the ring; they may contain additional nitrogen and other heteroatoms. Aliphatic nitrogen heterocycles include piperidine, piperazine, morpholine, pyrrolidine, thiomorpholine, azetidine, azepine, and oxazepine. Nitrogen heteroaryls are a subset of nitrogen heterocycles; examples include pyridine, pyrrole, and thiazole.

[0037] As used herein, and as would be understood by one of ordinary skill in the art, a reference to a "compound" is intended to include salts of that compound, unless further limited is apparent. Thus, for example, reference to a "compound of formula I" as shown above includes T32 as the free base and as a salt thereof: [ka] (wherein Z is any counter ion). In certain embodiments, the term "compound of formula I" refers to the compound or its pharma- ceutically acceptable salt. The term "pharma- ceutically acceptable salt" refers to a salt prepared from a pharma- ceutically acceptable non-toxic acid or base, such as inorganic and organic acids and bases. When the compound of the present invention is basic, as shown above in this paragraph, salts can be prepared from pharma- ceutically acceptable non-toxic acids, including inorganic and organic acids. Suitable pharma- ceutically acceptable acid addition salts for the compounds of the present invention include those of acetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid (besylic acid), benzoic acid, boric acid, butyric acid, camphoric acid, camphorsulfonic acid, carbonic acid, citric acid, ethanedisulfonic acid, ethanesulfonic acid, ethylenediaminetetraacetic acid, formic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydrazine acid, and the like. Examples of such acids include addition salts of R, isethionic, lactic, lactobionic, laurylsulfonic, maleic, malic, mandelic, methanesulfonic, mucic, naphthylenesulfonic, nitric, oleic, pamoic, pantothenic, phosphoric, pivalic, polygalacturonic, salicylic, stearic, succinic, sulfuric, tannic, tartaric, teoclatic, and p-toluenesulfonic acids. When the compound contains an acidic side chain, e.g., T34, R 1When is COOH, suitable pharma- ceutically acceptable base addition salts for the compounds of the invention include, but are not limited to, metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc, or organic salts made from lysine, arginine, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine. Further pharma- ceutically acceptable salts include non-toxic ammonium cations, and carboxylate, sulfonate, and phosphonate anions, attached, where appropriate, to alkyls having 1 to 20 carbon atoms.

[0038] <Treatment method> The compositions described herein can be administered to subjects having or at risk of developing hearing loss (e.g., sensorineural hearing loss) and / or vestibular dysfunction by a variety of routes, including, for example, local administration to the middle or inner ear (e.g., administration to or through the vestibular window, round window, or semicircular canal (e.g., the horizontal canal), or by transtympanic or intratympanic injection), intravenous, parenteral, intradermal, transdermal, intramuscular, intranasal, subcutaneous, transdermal, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, perfusion, irrigation, and oral administration. The most suitable route of administration in any given case will vary depending on the particular composition being administered, the patient, how the pharmaceutical is formulated, the method of administration (e.g., time and route of administration), the age, weight, sex of the patient, the severity of the disease being treated, the patient's diet, and the patient's excretion rate. The compositions can be administered once, or more than once (eg, once yearly, twice yearly, three times yearly, bimonthly, monthly, or biweekly).

[0039] Subjects treatable as described herein include those who have or are at risk of developing hearing loss and / or vestibular dysfunction (e.g., those who have or are at risk of developing hearing loss, vestibular dysfunction, or both). The compositions and methods described herein are useful for treating subjects who have or are at risk of developing cochlear hair cell damage (e.g., damage related to acoustic trauma, disease or infection, head trauma, ototoxic drugs, or aging), subjects who have or are at risk of developing vestibular hair cell damage (e.g., damage related to disease or infection, head trauma, ototoxic drugs, or aging), subjects who have or are at risk of developing sensorineural hearing loss, hearing loss, or auditory neuropathy, ... vestibular hair cell damage (e.g., damage related to disease or infection, head trauma, ototoxic drugs, or aging), subjects who have or are at risk of developing vestibular hair cell damage (e.g., damage related to disease or infection, head trauma, ototoxic drugs, or aging), subjects who have or are at risk of developing vestibular hair cell damage (e.g., damage related to The compositions described herein can be used to treat subjects with or at risk of developing hearing loss and / or vestibular dysfunction (e.g., dizziness, vertigo, loss of balance, bilateral vestibular hypofunction, oscillopsia, or balance disorders), subjects with tinnitus (e.g., tinnitus alone or tinnitus associated with sensorineural hearing loss or vestibular dysfunction), subjects with a genetic mutation associated with hearing loss and / or vestibular dysfunction, or subjects with a family history of hereditary hearing loss, hearing loss, auditory neuropathy, tinnitus, or vestibular dysfunction. In some embodiments, the subject has or is at risk of developing hearing loss and / or vestibular dysfunction associated with or resulting from loss of hair cells (e.g., cochlear or vestibular hair cells). The methods described herein can include screening the subject for one or more mutations in genes known to be associated with hearing loss and / or vestibular dysfunction prior to treatment with or administration of a composition described herein. Subjects can be screened for genetic mutations using standard methods known to those skilled in the art (e.g., genetic testing). The methods described herein can further include a step of evaluating the hearing and / or vestibular function of the subject prior to treatment with or administration of the compositions described herein. Hearing can be evaluated using standard tests, such as audiometry, auditory brainstem response (ABR), electrocochleography (ECOG), and otoacoustic emissions.Vestibular function can be evaluated using standard tests, such as eye movement tests (e.g., electronystagmography (ENG) or video nystagmography (VNG)), tests of the vestibulo-ocular reflex (VOR) (e.g., head impulse test (Halmagyi-Curthoys test), which can be performed at the bedside or using video head impulse test (VHIT), or caloric reflex test), posturography, rotary chair test, ECOG, vestibular evoked myogenic potential (VEMP), and specialized clinical balance function tests, such as those described in Mancini and Horak, Eur J Phys Rehabil Med, (Italy), 2010, Vol. 46, p. 239. These tests can also be used to evaluate the hearing and / or vestibular function of a subject after treatment with or administration of the compositions described herein. The compositions and methods described herein may be used as a preventative treatment for patients at risk of developing hearing loss and / or vestibular dysfunction, for example, patients with a family history of hearing loss or vestibular dysfunction (e.g., hereditary hearing loss or vestibular dysfunction), patients who have not yet exhibited hearing loss or vestibular dysfunction but have a genetic mutation associated with hearing loss or vestibular dysfunction, or patients who have been exposed to risk factors for acquired hearing loss (e.g., acoustic trauma, disease or infection, head trauma, ototoxic drugs, or aging) or vestibular dysfunction (e.g., disease or infection, head trauma, ototoxic drugs, or aging).

[0040] The compositions and methods described herein can be used to induce or enhance hair cell regeneration in a subject, such as regeneration of cochlear and / or vestibular hair cells. Subjects who may benefit from compositions that induce or enhance hair cell regeneration include subjects suffering from hearing loss or vestibular dysfunction as a result of hair cell loss (e.g., hair cell loss associated with trauma (e.g., acoustic trauma or head trauma), disease or infection, ototoxic drugs, or aging), and subjects with abnormal hair cells (e.g., hair cells that do not function properly compared to normal hair cells), damaged hair cells (e.g., hair cell damage associated with trauma (e.g., acoustic trauma or head trauma), disease or infection, ototoxic drugs, or aging), or a reduced number of hair cells due to genetic mutations or congenital abnormalities.

[0041] The compositions and methods described herein can also be used to prevent or reduce hearing loss and / or vestibular dysfunction caused by hair cell damage or death (e.g., cochlear and / or vestibular hair cell damage or death) caused by ototoxic drugs in subjects who have been treated with an ototoxic drug or who are currently undergoing or soon to begin treatment with an ototoxic drug. Ototoxic drugs are toxic to cells of the inner ear and can cause sensorineural hearing loss, vestibular dysfunction (e.g., vertigo, floating dizziness, imbalance, bilateral vestibular hypofunction, oscillopia, or balance disorders), tinnitus, or a combination of these conditions. Drugs that have been found to have ototoxic effects include aminoglycoside antibiotics (e.g., gentamicin, neomycin, streptomycin, tobramycin, kanamycin, vancomycin, and amikacin), viomycin, antineoplastic agents (e.g., platinum-containing chemotherapy agents such as cisplatin, carboplatin, and oxaliplatin), loop diuretics (e.g., ethacrynic acid and furosemide), salicylates (e.g., aspirin, especially in high doses), and quinine. In some embodiments, the methods and compositions described herein can be used to treat bilateral vestibular hypofunction or oscillopsia. Bilateral vestibular hypofunction and oscillopsia can be caused by aminoglycosides (e.g., the methods and compositions described herein can be used to promote or increase hair cell regeneration in subjects with or at risk of developing aminoglycoside-induced bilateral vestibular hypofunction or oscillopsia).

[0042] Treatment may include administration of various unit doses of a composition containing a compound described herein. Each unit dose will usually contain a predetermined amount of the therapeutic composition. The amount administered, as well as the specific route of administration and dosage form, are within the skill of those skilled in the art of clinical treatment. The unit dose need not be administered as a single injection, but may include continuous infusion over a set period of time. Dosing may be performed using a syringe pump to control the rate of infusion to minimize damage to the inner ear (e.g., the cochlea and / or vestibular system).

[0043] The compositions described herein are administered in an amount sufficient to improve hearing, improve vestibular function (e.g., improve balance function or reduce dizziness or vertigo), reduce tinnitus, treat bilateral vestibular hypofunction, treat oscillopia, treat balance disorders, increase or induce hair cell regeneration (e.g., cochlear and / or vestibular hair cell regeneration), increase hair cell number, activate YAP, and / or inhibit LATS. Hearing can be assessed using standard hearing tests (e.g., audiometry, ABR, electrocochleography (ECOG), and otoacoustic emissions), and may be improved compared to audiometric results obtained prior to treatment. Vestibular function can be assessed using standard tests for balance and vertigo (e.g., oculomotor testing (e.g., ENG or VNG), posturography, VOR testing (e.g., head impulse testing (Halmagyi-Curthoys test, e.g., VHIT), or caloric reflex testing), rotation chair testing, ECOG, VEMP, specialized clinical balance testing), and may be improved compared to measurements obtained before treatment. In some embodiments, the composition is administered in an amount sufficient to improve the subject's ability to understand speech. The compositions described herein can also be administered in an amount sufficient to delay or prevent the onset or progression of sensorineural hearing loss and / or vestibular dysfunction (e.g., in subjects who have a genetic mutation associated with hearing loss or vestibular dysfunction, who have a family history of hearing loss or vestibular dysfunction (e.g., genetic hearing loss or vestibular dysfunction), who have been exposed to risk factors associated with hearing loss or vestibular dysfunction (e.g., ototoxic drugs, head trauma, disease or infection, or acoustic trauma) but who do not exhibit hearing loss or vestibular dysfunction (e.g., vertigo, dizziness, or disequilibrium), or who exhibit mild to moderate hearing loss or vestibular dysfunction). The above effects can occur within, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, or more after administration of the compositions described herein.The patient may be evaluated 1, 2, 3, 4, 5, 6 or more months after administration of the composition, depending on the dose and route of administration used for the treatment. Depending on the results of the evaluation, the patient may receive additional treatment.

[0044] <Preparation of Compounds> The following abbreviations are used: THF (tetrahydrofuran), MeOH (methanol), DCM (dichloromethane), DMF (N,N-dimethylformamide), ACN (acetonitrile), EtOH (ethanol), EtOAc (ethyl acetate), IPA (2-propanol), DMSO (dimethylsulfoxide), MTBE (methyl tert-butyl ether), TEA (triethylamine), DIPEA (N,N-diisopropylethylamine), TMEDA (tetramethylethylenediamine), DMAP (N,N-dimethylpyridin-4-amine), EDCI (N-(3-dimethylphenyl)-2-pyridine), EDTA (N-dimethylphenyl)-2-pyridine, ... The synthetic routes used include N-methylaminopropyl-N'-ethylcarbodiimide hydrochloride), HOBt (1-hydroxybenzotriazole hydrate), HBTU ((2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate), T3P (propanephosphonic anhydride), TBAI (tetrabutylammonium iodide), LAH (lithium aluminum hydride), XPhos (2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl), and TFA (trifluoroacetic acid).

[0045] Preparative HPLC purification refers to the use of a water / acetonitrile gradient using a suitable hydrophobic stationary phase, optionally with additives such as HCl, formic acid, TFA, or NH4HCO3.

[0046] The compounds of the present invention can be prepared as shown below in general Schemes I-IV and more specifically in Schemes 1-69. Detailed descriptions of the synthesis of intermediates and exemplary compounds are also disclosed below.

[0047] [ka] As shown in Scheme I, when a compound of formula (Ia) containing an imine group is treated with a compound of formula (Ib) (wherein X is chloro, bromo or -OH) under coupling conditions well known to one skilled in the art, a compound of formula I is obtained. Typical conditions for the reaction of a compound of formula (Ib) (wherein X is chloro) with a compound of formula (Ia) include, but are not limited to, stirring an equimolar mixture of the compounds in a solvent such as chloroform or dichloromethane in the presence of a base, such as, but not limited to, triethylamine and N,N-dimethylpyridin-4-amine (DMAP), at 5-25° C. for 1-12 hours. Acid coupling conditions for compounds of formula (Ib) (wherein X is -OH) with compounds of formula (Ia) include, but are not limited to, stirring an equimolar mixture of the compounds with a coupling reagent such as, but not limited to, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole hydrate (HOBt), ((2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), propanephosphonic anhydride (T3P) in the presence of a base such as, but not limited to, N,N-diisopropylethylamine (DIPEA) in a solvent such as, but not limited to, DMF, EtOAc, and pyridine. Typical reactions can be carried out at 25-110°C for 1-12 hours.

[0048] [ka] As shown in Scheme II, compounds of formula (IIa) may be converted to compounds of formula I, which are representative compounds of the present invention. Exemplary conditions include, but are not limited to, treating compounds of formula (IIa) with potassium carbonate (K2CO3) in DMF at 25° C., followed by the addition of a reagent such as R1-Y, where R1 is defined in formula I and Y is chloro, bromo, iodo, mesyl, or tosylate. Exemplary reactions can be carried out in a microwave reactor at 100° C. to facilitate alkylation.

[0049] [ka] Compounds of formula (Ia) in Scheme I can be prepared according to the method outlined in Scheme III. Compounds of formula (IIIa) can be treated with potassium carbonate (K2CO3) in DMF at 25°C, followed by addition of a reagent such as R1-Y (wherein R1 is defined in formula I and Y is chloro, bromo, iodo, mesyl or tosylate) to give compounds of formula (IIIb / Ia). A typical reaction can be carried out in a microwave reactor at 100°C to facilitate alkylation.

[0050] [ka] Scheme IV describes another alternative preparation of compound of formula (Ia) of Scheme I. Compound of formula (IVa) is treated with ethyl N-(thioxomethylene)carbamate and tetramethylethylenediamine (TMEDA) to give compound of formula (IVb). Compound of formula (IVb) is treated with compound of formula (IVc) in the presence of cesium carbonate (Cs2CO3) in acetonitrile to give compound of formula (IVd). Compound of formula (IVd) is treated with sulfonyl chloride (SOCl2) in the presence of N,N-diisopropylethylamine (DIPEA) to give compound of formula (IVe). Compound of formula (IVe) is treated with sodium hydroxide (NaOH) to give compound of formula (IVf / Ia).

[0051] The acids Ib used in Scheme I are shown in Table A. For known acids, the CAS numbers are given in the Reference column. For all other acids, the synthetic schemes are given in the present application. [Table 1]

[0052] Intermediates Ia / IIIb used in Scheme I and prepared by the methods outlined in Scheme III are shown in Table B. [Table 2] TIFF0007678595000012.tif204131TIFF0007678595000013.tif91135

[0053] Intermediates Ia / IVf used in Scheme I and prepared by the methods outlined in Scheme IV are shown in Table C. [Table 3] TIFF0007678595000015.tif210131TIFF0007678595000016.tif205131TIFF0007678595000017.tif215131

[0054] The preparation of intermediates in Table D is shown in Schemes 51-52. [Table 4]

[0055] <Preparation of intermediate B1> [ka] To a solution of 2-thiazolamine (10.00 g, 99.86 mmol, 1.0 equiv) in DMF (200 mL) was added chloromethylbenzene (18.96 g, 149.8 mmol, 1.5 equiv). The reaction mixture was heated to 80° C. and stirred for 12 h. The reaction mixture was concentrated under reduced pressure. The resulting residue was diluted with EtOAc (200 mL). Aqueous NaOH (100 mL, 40% (wt%)) was added at 0° C. and stirred at 0° C. for 1 h. The mixture was extracted with EtOAc (3×100 mL). The combined organic layers were washed with brine (3×100 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue which was purified by silica gel column chromatography (petroleum ether / EtOAc=5:1→1:1) to give 3-benzylthiazol-2(3H)-imine B1 (13.00 g, crude) as a yellow oil.

[0056] <Alternative method for preparing intermediate B1> [ka] <Ethyl N-(benzylcarbamothioyl)carbamate> To a solution of phenylmethanamine (5.00 g, 46.7 mmol, 1.0 equiv.) and O-ethyl carbonisothiocyanatidate (6.43 g, 49.0 mmol, 1.05 equiv.) in ethyl acetate (40 mL) was added TMEDA (0.54 g, 4.67 mmol, 0.1 equiv.) under nitrogen atmosphere at 25° C. The mixture was stirred at 25° C. for 6 h and then concentrated under reduced pressure. The residue was triturated twice with a solution of petroleum ether and ethyl acetate (v / v=10 / 1, 55 mL) to give 10.0 g of ethyl N-(benzylcarbamothioyl)carbamate as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 11.04 (br. s, 1H), 10.22 (br. t, J = 5.6 Hz, 1H), 7.39 - 7.23 (m, 5H), 4.81 (d, J = 5.6 Hz, 2H), 4.14 (q, J = 7.2 Hz, 2H), 1.21 (t, J = 7.2 Hz, 3H).

[0057] <(Z)-Ethyl ((benzylamino)((2-oxoethyl)thio)methylene)carbamate> To a mixture of ethyl N-(benzylcarbamothioyl)carbamate (4.00 g, 16.8 mmol, 1.0 equiv.) and cesium carbonate (13.7 g, 42.0 mmol, 2.5 equiv.) in acetonitrile (50 mL) was added 2-chloroacetaldehyde (13.2 g, 67.2 mmol, 4.0 equiv.). The reaction mixture was stirred at 25° C. for 12 h, poured into ice water (20 mL), and then extracted with MTBE (2×200 mL). The combined organic phase was washed with brine (2×20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=100:1→3:1) to give 5.00 g of (Z)-ethyl ((benzylamino)((2-oxoethyl)thio)methylene)carbamate as a yellow solid. LCMS (m / z [M+H] + ): 280.9.

[0058] <(Z)-ethyl (3-benzylthiazol-2(3H)-ylidene)carbamate> To a mixture of (Z)-ethyl ((benzylamino)((2-oxoethyl)thio)methylene)carbamate (5.00 g, 12.4 mmol, 1.0 equiv) and diisopropylethylamine (4.81 g, 37.2 mmol, 3.0 equiv) in tetrahydrofuran (50 mL) was added thionyl chloride (1.48 g, 12.4 mmol, 1.0 equiv). The mixture was stirred at 25° C. for 16 h, then poured into ice water (20 mL) and extracted with ethyl acetate (3×100 mL). The combined organic phase was washed with brine (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=100:1→4:1) to give 2.80 g of (Z)-ethyl (3-benzylthiazol-2(3H)-ylidene)carbamate as a yellow solid. 1 H NMR (400 MHz, CD3OD) δ 7.36 - 7.28 (m, 5H), 7.22 (d, J = 4.8 Hz, 1H), 6.84 (d, J = 4.8 Hz, 1H), 5.31 (s, 2H), 4.20 (q, J = 7.2 Hz, 2H), 1.30 (t, J = 7.2Hz, 3H). LCMS (m / z [M+H] + ): 262.9.

[0059] <Intermediate B1> A mixture of (Z)-ethyl (3-benzylthiazol-2(3H)-ylidene)carbamate (2.80 g, 9.06 mmol, 1.0 equiv) and sodium hydroxide (7.25 g, 181 mmol, 20 equiv) in ethanol (20 mL) was stirred at 50° C. for 1.5 h. The mixture was poured into ice water (20 mL) and extracted with ethyl acetate (5×100 mL). The combined organic phase was washed with brine (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give 1.60 g of 3-benzylthiazol-2(3H)-imine B1 as a yellow gum.

[0060] <Preparation of intermediate B2> [ka] To a solution of 2-thiazolamine (1.00 g, 9.99 mmol, 1.0 equiv) in EtOH (10 mL) was added MeI (1.84 g, 13.0 mmol, 808 μL, 1.3 equiv). The reaction mixture was stirred at 90° C. for 12 h. The suspension was filtered and the solid was washed with EtOH (60 mL) and dried under reduced pressure. The solid was collected, diluted with 40% aqueous NaOH (10 mL) and extracted with EtOAc (3×10 mL). The combined organic layers were washed with 40% aqueous NaOH (3×10 mL) and brine (3×10 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue that afforded 160 mg of 3-methyl-thiazol-2-amine B2.

[0061] <Preparation of intermediate B3> [ka] A mixture of 2-thiazolamine (3.00 g, 30.0 mmol, 1.0 equiv) and ethyl iodide EtI (6.07 g, 38.9 mmol, 1.3 equiv) in EtOH (30 mL) was degassed and purged with N2 three times and stirred under N2 atmosphere at 90 °C for 12 h. The reaction mixture was diluted with 40% aqueous NaOH (50 mL) and extracted with EtOAc (2 x 30 mL). The combined organic layers were washed with 40% aqueous NaOH (3 x 20 mL) and brine (3 x 20 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give 2.80 g of 3-ethylthiazol-2(3H)-imine B3 as a dark red oil.

[0062] <Preparation of intermediate B4> [ka] A mixture of 2-thiazolamine (120 mg, 1.20 mmol, 1.0 equiv), tert-butyl (4-bromobutan-2-yl)carbamate (302 mg, 1.20 mmol, 1.0 equiv) and DIPEA (170 mg, 1.32 mmol, 230 L, 1.1 equiv) was stirred in a 5 mL sealed tube at 90° C. for 4 h. The reaction mixture was diluted with MeOH (1 mL) and the mixture was purified by silica gel column chromatography (petroleum ether / ethyl acetate=1 / 1→0 / 1, then ethyl acetate / ammonium hydroxide (25%)=50 / 1) to give 30 mg of tert-butyl (4-(2-iminothiazol-3(2H)-yl)butan-2-yl)carbamate B4 as a yellow oil.

[0063] <Preparation of Intermediate B5> [ka] A mixture of 2-thiazolamine (1.00 g, 10.0 mmol, 1.0 equiv) and 4-(bromomethyl)benzonitrile (1.96 g, 10.0 mmol, 1.0 equiv) in i-PrOH (20 mL) was stirred at 60° C. for 12 h. The reaction mixture was filtered to collect the solid, which was washed with 20 mL of EtOAc and dried in vacuum to give 2.1 g of 2-amino-3-(4-cyanobenzyl)thiazol-3-ium B5 as a yellow solid.

[0064] <Preparation of intermediate B6> [ka] To a solution of 2-thiazolamine (1.00 g, 9.99 mmol, 1.0 equiv) and (bromomethyl)cyclohexane (1.77 g, 9.99 mmol, 1.39 mL, 1.0 equiv) in DMF (10 mL) was added TBAI (1.84 g, 4.99 mmol, 0.5 equiv) at room temperature. The reaction mixture was then stirred at 85° C. for 12 h under N2. An additional 1.0 equiv of (bromomethyl)cyclohexane was added to the mixture and the reaction mixture was stirred at 100° C. for an additional 24 h under N2. The reaction mixture was diluted with saturated aqueous NaHCO3 (100 mL) and extracted with EtOAc (3×50 mL). The combined organic layers were washed with brine (3×50 mL), dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate / MeOH=0:1:0→0:1:1) to give 428 mg of 3-(cyclohexylmethyl)thiazol-2(3H)-imine B6 as a yellow solid.

[0065] <Preparation of intermediate B7> [ka] 5-(chloromethyl)-1-methyl-1H-pyrazole To a stirred solution of (1-methyl-1H-pyrazol-5-yl)methanol (200 mg, 1.78 mmol, 1.0 equiv) in DCM (4 mL) was added SOCl2 (0.32 mL) dropwise at 25°C. The reaction mixture was then stirred under N2 at 25°C for 2 h. The reaction mixture was diluted with water (20 mL) and adjusted to pH 8 with saturated aqueous NaHCO3. The solution was extracted with EtOAc (3 x 20 mL) and the combined organic layers were washed with brine (3 x 10 mL), dried over Na2SO4, and filtered. The filtrate was concentrated under reduced pressure to give 200 mg of crude 5-(chloromethyl)-1-methyl-1H-pyrazole as a yellow oil. 1 H NMR: (400MHz, CHLOROFORM-d) δ 7.41 (d, J = 1.8 Hz, 1H), 6.28 (d, J = 1.8 Hz, 1H), 4.62 (s, 2H), 3.93 (s, 3H).

[0066] <Intermediate B7> To a solution of 5-(chloromethyl)-1-methyl-1H-pyrazole (120 mg, 919 μmol, 1.0 equiv.) in i-PrOH (1 mL), 2-thiazolamine (74 mg, 735 μmol, 0.8 equiv.) was added at room temperature and then stirred at 60° C. under N2 for 12 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate / MeOH=1:1:0→0:1:0→0:1:9) to give 76 mg of 3-((1-methyl-1H-pyrazol-5-yl)methyl)thiazol-2(3H)-imine B7 as a yellow solid.

[0067] <Preparation of intermediate B8> [ka] To a solution of ethyl 2-aminothiazole-4-carboxylate (2.00 g, 11.6 mmol, 1.0 equiv) in i-PrOH (20 mL) was added (bromomethyl)benzene (1.38 mL, 11.6 mmol, 1.0 equiv) at 25° C. The mixture was heated to 80° C. and stirred for 6 h. The reaction mixture was cooled to room temperature and allowed to stand for 3 days. The precipitate was collected by filtration and the filter cake was dried under high vacuum to give 0.80 g of ethyl 3-benzyl-2-imino-2,3-dihydrothiazole-4-carboxylate B8 as a white solid.

[0068] <Preparation of intermediate B9> [ka] To a solution of 5-methyl-2-thiazolamine (1.00 g, 8.76 mmol, 1.0 equiv) in acetone (15 mL) was added bromomethylbenzene (1.65 g, 9.63 mmol, 1.1 equiv) at 25° C. The reaction mixture was heated to 70° C. and stirred for 5 h. The reaction mixture was cooled to 25° C. and aqueous NaOH (2N) was added to adjust the pH to >7.0. The reaction mixture was filtered and the filter cake was washed with acetone (5 mL), MTBE (10 mL) and dried under reduced pressure to give 1.1 g of crude 3-benzyl-5-methylthiazol-2(3H)-imine B9 as a brown solid. The structure was confirmed by NOE.

[0069] <Preparation of intermediate B10> [ka] A mixture of 2-thiazolamine (100 mg, 998 μmol, 1.0 equiv), 2-(bromomethyl)pyridine (278 mg, 1.10 mmol, 1.1 equiv, HBr) and K2CO3 (152 mg, 1.10 mmol, 1.1 equiv) in acetone (5 mL) was degassed and purged with N2 three times, then the reaction mixture was stirred at 60° C. under N2 atmosphere for 5 h. The resulting mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC to give 100 mg of compound 3-(pyridin-2-ylmethyl)thiazol-2(3H)-imine B10 as a white solid.

[0070] <Preparation of intermediate B11> [ka] To a solution of 2-thiazolamine (0.30 g, 3.0 mmol, 1.0 equiv) in i-PrOH (5 mL) was added 2-(bromomethyl)benzonitrile (587 mg, 3.00 mmol, 1.0 equiv). The reaction mixture was stirred at 25° C. for 12 h and then heated to 60° C. and stirred for an additional 4 h. The reaction mixture was directly concentrated under reduced pressure to give a residue which was purified by silica gel flash column chromatography (ethyl acetate / MeOH) to give 0.41 g of 2-((2-iminothiazol-3(2H)-yl)methyl)benzonitrile B11 as a yellow solid.

[0071] <Preparation of intermediate B12> [ka] To a solution of 2-bromoethylbenzene (3.70 g, 20.0 mmol, 1.0 equiv) in i-PrOH (35 mL) was added 2-thiazolamine (2.00 g, 20.0 mmol, 1.0 equiv). The reaction mixture was stirred at 80° C. for 12 h. The reaction mixture was cooled to room temperature and then directly concentrated to obtain a residue, which was purified by reverse phase column chromatography to obtain 2.00 g of compound 3-phenethylthiazol-2(3H)-imine B12 as a pale yellow solid.

[0072] <Preparation of intermediate B13> [ka] <(Tetrahydro-2H-pyran-4-yl)methyl 4-methylbenzenesulfonate> To a solution of (tetrahydro-2H-pyran-4-yl)methanol (2.00 g, 17.2 mmol, 1.0 equiv.) in 2-methyltetrahydrofuran (20 mL) was added NaOH (4.13 g, 51.6 mmol, 50% purity, 3.0 equiv.), followed by the dropwise addition of a solution of 4-methylbenzenesulfonyl chloride (5.91 g, 31.0 mmol, 1.8 equiv.) in 2-methyltetrahydrofuran (6 mL) at 0° C. The reaction mixture was then stirred at 25° C. for 12 h. The reaction mixture was diluted with aqueous HCl (6 M) and then 3.0 mL of cyclohexane was added. The solution was stirred at 0° C. for 0.5 h, but no solid precipitated. The mixture was then extracted with EtOAc (3×60 mL) and the combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give 4.2 g of crude (tetrahydro-2H-pyran-4-yl)methyl 4-methylbenzenesulfonate as a white solid. LCMS (m / z [M+H] + ): 271.1.

[0073] <Intermediate B13> To a solution of (tetrahydro-2H-pyran-4-yl)methyl 4-methylbenzenesulfonate (2.70 g, 9.99 mmol, 1.0 equiv.) and 2-thiazolamine (1.00 g, 9.99 mmol, 1.0 equiv.) in DMF (10 mL) was added TBAI (1.84 g, 4.99 mmol, 0.5 equiv.) at room temperature. The reaction mixture was stirred at 80° C. under N2 for 12 h. The reaction mixture was diluted with water (80 mL) and extracted with EtOAc (3×60 mL), the combined organic layers were discarded, and the aqueous phase was adjusted to pH=9 with NaOH (50% pure). The aqueous solution was extracted with EtOAc (3×50 mL), the combined organic layers were washed with brine (100 mL), dried over Na2SO4, and filtered. The filtrate was concentrated under reduced pressure to give 500 mg of 3-((tetrahydro-2H-pyran-4-yl)methyl)thiazol-2(3H)-imine B13 as a yellow solid.

[0074] <Preparation of intermediate B14> [ka] To a solution of 2-thiazolamine (0.60 g, 5.99 mmol, 1.0 equiv) in i-PrOH (10 mL) was added methyl 3-(bromomethyl)benzoate (1.37 g, 5.99 mmol, 1.0 equiv). The reaction mixture was stirred at 60° C. for 12 h. The reaction mixture was cooled to 25° C. and diluted with MTBE. The suspension was decanted and dried under reduced pressure to give 1.5 g of crude product methyl 3-((2-iminothiazol-3(2H)-yl)methyl)benzoate B14 as a brown solid.

[0075] <Preparation of intermediate B15> [ka] A mixture of 2-thiazolamine (100 mg, 1.00 mmol, 1.0 equiv), 2-(bromomethyl)pyridine (278 mg, 1.10 mmol, 1.1 equiv, HBr) and K2CO3 (152 mg, 1.10 mmol, 1.1 equiv) in acetone (5 mL) was degassed and purged with N2 three times. The reaction mixture was stirred at 60 °C under N2 atmosphere for 5 h. The resulting mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC to give 100 mg of 3-(pyridin-2-ylmethyl)thiazol-2(3H)-imine B15 as a white solid.

[0076] <Preparation of intermediate B16> [ka] To a solution of 2-thiazolamine (1.00 g, 9.99 mmol, 1.0 equiv) in EtOAc (20 mL) was added 3-(bromomethyl)benzonitrile (1.96 g, 9.99 mmol, 1.0 equiv) at 25° C. The reaction mixture was then stirred at 25° C. for 12 h. The reaction mixture was diluted with EtOAc (100 mL) and then NaOH (100 mL, 40%) was added at 0° C. The resulting mixture was stirred at 0° C. for 1 h. The solution was extracted with EtOAc (3×100 mL). The combined organic layers were washed with brine (3×100 mL), dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether / EtOAc = 5:1 → EtOAc:NH3·H2O (25%) = 100:1) to give 500 mg of 3-((2-iminothiazol-3(2H)-yl)methyl)benzonitrile B16 as a yellow oil.

[0077] <Preparation of intermediate B17> [ka] 4-(Chloromethyl)-1-methyl-1H-pyrazole To a stirred solution of (1-methyl-1H-pyrazol-4-yl)methanol (500 mg, 4.46 mmol, 1.0 equiv) in DCM (1.6 mL) was added SOCl2 (2.39 g, 20.1 mmol, 1.46 mL, 4.5 equiv) dropwise at 25 °C. The reaction mixture was stirred at 25 °C under N2 for 2 h. The reaction mixture was concentrated under reduced pressure to give 500 mg of 4-(chloromethyl)-1-methyl-1H-pyrazole as a white solid. 1 H NMR (400MHz, DMSO-d6) δ 7.79 (s, 1H), 7.48 (s, 1H), 4.68 (s, 2H), 3.81 (s, 3H).

[0078] <Intermediate B17> To a solution of 4-(chloromethyl)-1-methyl-1H-pyrazole (450 mg, 3.45 mmol, 1.0 equiv) in DMF (4.5 mL) was added 2-thiazolamine (345 mg, 3.45 mmol, 1.0 equiv), followed by K2CO3 (953 mg, 6.89 mmol, 2.0 equiv) at room temperature. The mixture was then stirred at 80° C. under N2 for 12 h. The reaction mixture was diluted with water (30 mL) and extracted with EtOAc (3×20 mL), the organic layer was washed with brine (30 mL), dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate / MeOH=1:1:0→0:1:1) to give 50 mg of 3-((1-methyl-1H-pyrazol-4-yl)methyl)thiazol-2(3H)-imine B17 as a yellow solid.

[0079] <Preparation of intermediate B18> [ka] To a solution of 2-oxazolamine (0.50 g, 5.95 mmol, 1.0 equiv) in acetone (10 mL) was added bromomethylbenzene (1.12 g, 6.54 mmol, 777 μL, 1.1 equiv) and the mixture was stirred for 5 h at 60° C. The reaction mixture was concentrated under reduced pressure to give 0.70 g of 3-benzyloxazol-2(3H)-imine B18 as a yellow gelatinous oil.

[0080] <Preparation of intermediate B19> [ka] To a solution of 4-methyl-2-oxazolamine (0.400 g, 3.26 mmol, 1.0 equiv) in i-PrOH (2 mL) was added (bromomethyl)benzene (0.387 mL 3.26 mmol, 1.0 equiv) dropwise at 20° C. The reaction mixture was heated to 80° C. and stirred for 3 h. The mixture was concentrated under reduced pressure and the residue was purified by preparative HPLC to give 0.20 g of 3-benzyl-4-methyloxazol-2(3H)-imine 2,2,2-trifluoroacetate B19 as a yellow oil.

[0081] <Preparation of intermediate B20> [ka] To a solution of ethyl 2-imino-3H-oxazole-4-carboxylate (1.35 g, 8.65 mmol) in isopropyl alcohol (14 mL) was added bromomethylbenzene (1.03 mL, 8.65 mmol) at 25° C. and the mixture was stirred at 80° C. for 12 hours. The reaction mixture was cooled to room temperature and the precipitate was collected by filtration. The filter cake was dried under reduced pressure to give 1.20 g of ethyl 3-benzyl-2-imino-2,3-dihydrooxazole-4-carboxylate B20 as a white solid.

[0082] <Preparation of intermediate C1> [ka] <Ethyl N-(phenylcarbamothioyl)carbamate> To a solution of aniline (2.00 g, 21.48 mmol, 1.0 equiv) in EtOAc (20 mL) was added ethyl N-(thioxomethylene)carbamate (2.82 g, 21.48 mmol, 1.0 equiv) and TMEDA (250 mg, 2.15 mmol, 0.1 equiv). The reaction mixture was stirred at 25° C. for 5 h. The reaction mixture was directly concentrated under reduced pressure to give a yellow solid. The solid was redissolved in EtOH (10 mL), stirred for 0.5 h, and then filtered. The filter cake was washed with EtOH (2×5 mL) and dried in vacuum to give 3.70 g of ethyl N-(phenylcarbamothioyl)carbamate as a white solid.

[0083] <(Z)-Ethyl (((2-oxoethyl)thio)(phenylamino)methylene)carbamate> To a solution of ethyl N-(phenylcarbamothioyl)carbamate (2.70 g, 12.0 mmol, 1.0 equiv.) and Cs2CO3 (6.67 g, 20.5 mmol, 1.7 equiv.) in CH3CN (40 mL) was added 2-chloroacetaldehyde (2.95 g, 15.0 mmol, 1.3 equiv.) while maintaining the temperature below 25° C. After the addition, the reaction mixture was stirred at 25° C. for 12 h. The reaction mixture was diluted with MTBE (80 mL) at 20° C., washed with saturated aqueous NaHCO3 (75 ml), brine (75 mL), dried over Na2SO4, and filtered. The filtrate was concentrated under reduced pressure to give 0.32 g of crude product (Z)-ethyl (((2-oxoethyl)thio)(phenylamino)methylene)carbamate as a brown oil.

[0084] <(Z)-ethyl (3-phenylthiazol-2(3H)-ylidene)carbamate> To a solution of ethyl (Z)-ethyl(((2-oxoethyl)thio)(phenylamino)methylene)carbamate (3.20 g, 12.0 mmol, 1.0 equiv.) in THF (40 mL) was added DIPEA (4.66 g, 36.0 mmol, 3.0 equiv.) and SOCl2 (1.43 g, 12.0 mmol, 1.0 equiv.) at 0° C. The reaction mixture was stirred below 15° C. for 4 h. The reaction mixture was concentrated under reduced pressure. The residue was redissolved in MTBE (100 mL) and adjusted to pH 8.0 by addition of saturated aqueous NaHCO3. The organic layer was washed with brine, dried over Na2SO4, and concentrated under vacuum. The residue was purified by flash silica gel chromatography (ethyl acetate / petroleum ether) to give 1.40 g of (Z)-ethyl(3-phenylthiazol-2(3H)-ylidene)carbamate as a yellow solid.

[0085] <Intermediate C1> To a solution of (Z)-ethyl (3-phenylthiazol-2(3H)-ylidene)carbamate (0.80 g, 3.22 mmol, 1.0 equiv) in EtOH (10 mL) was added 6N NaOH (10 mL, 60.0 mmol, 19 equiv). The reaction mixture was stirred at 90° C. for 4 h. The reaction mixture was diluted with water (25 mL) and extracted with EtOAc (2×25 mL). The combined organic layers were dried over Na2SO4 and concentrated to give 0.60 g of crude 3-phenylthiazol-2-imine C1 as a brown solid. The crude product was purified by reverse phase MPLC to give 0.23 g of pure 3-phenylthiazol-2(3H)-imine C1 as a brown oil.

[0086] <Preparation of intermediate C2> [ka] <Ethyl N-[(2-bromo-4-pyridyl)carbamothioyl]carbamate> A mixture of 2-bromopyridin-4-amine (5.00 g, 28.9 mmol, 1.0 equiv), ethyl N-(thioxomethylene)carbamate (3.79 g, 28.9 mmol, 3.41 mL, 1.0 equiv) and TMEDA (336 mg, 2.89 mmol, 436 uL, 0.1 equiv) in EtOAc (50 mL) was degassed and purged with N2 three times at room temperature, then the reaction mixture was stirred at 25° C. under N2 atmosphere for 12 h. The mixture was directly concentrated under reduced pressure to give a yellow solid. The solid was redissolved in EtOH (30 mL), stirred for 10 min, then filtered. The filter cake was washed with EtOH (2×5 mL) and then concentrated under reduced pressure to give 4.2 g of ethyl N-[(2-bromo-4-pyridyl)carbamothioyl]carbamate as a pale yellow solid.

[0087] <(Z)-Ethyl (((2-bromopyridin-4-yl)amino)((2-oxoethyl)thio)methylene)carbamate> To a solution of ethyl N-[(2-bromo-4-pyridyl)carbamothioyl]carbamate (1.50 g, 4.93 mmol, 1.0 equiv) and Cs2CO3 (2.73 g, 8.38 mmol, 1.7 equiv) in MeCN (30 mL) was added 2-chloroacetaldehyde (484 mg, 6.16 mmol, 397 L, 1.2 equiv) at room temperature. After addition, the reaction mixture was stirred at 25° C. for 12 h. The mixture was diluted with water (50 mL) and extracted with ethyl acetate (3×30 mL). The combined organic phase was washed with brine (30 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. (Z)-Ethyl (((2-bromopyridin-4-yl)amino)((2-oxoethyl)thio)methylene)carbamate (1.60 g, crude) was obtained as a brown oil.

[0088] (Z)-Ethyl (3-(2-bromopyridin-4-yl)thiazol-2(3H)-ylidene)carbamate To a solution of (Z)-ethyl (((2-bromopyridin-4-yl)amino)((2-oxoethyl)thio)methylene)carbamate (1.60 g, 4.62 mmol, 1.0 equiv) in THF (20 mL) was added DIPEA (1.79 g, 13.9 mmol, 2.42 mL, 3.0 equiv) and SOCl2 (550 mg, 4.62 mmol, 335□L, 1.0 equiv) at 0° C. The reaction mixture was stirred below 20° C. for 12 h. The mixture was quenched with saturated aqueous NaHCO3 (50 mL) and extracted with dichloromethane (3×20 mL). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated to give 1.40 g of (Z)-ethyl (3-(2-bromopyridin-4-yl)thiazol-2(3H)-ylidene)carbamate as a brown oil.

[0089] (Z)-Ethyl (3-(2-((tert-butoxycarbonyl)amino)pyridin-4-yl)thiazol-2(3H)-ylidene)carbamate A mixture of (Z)-ethyl (3-(2-bromopyridin-4-yl)thiazol-2(3H)-ylidene)carbamate (700 mg, 2.13 mmol, 1.0 equiv), NH2Boc (750 mg, 6.40 mmol, 3.0 equiv), K3PO4 (1.81 g, 8.53 mmol, 4.0 equiv), Pd2(dba)3 (195 mg, 213 μmol, 0.1 equiv) and Xantphos (123 mg, 213 □mol, 0.1 equiv) in dioxane (10 mL) was degassed and purged with N2 three times, then the reaction mixture was stirred at 110° C. under N2 atmosphere for 12 h. The mixture was poured into water (30 mL) and extracted with ethyl acetate (3×20 mL). The combined organic layers were washed with brine (30 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 50 / 1 → 1 / 1) to give 140 mg of (Z)-ethyl (3-(2-((tert-butoxycarbonyl)amino)pyridin-4-yl)thiazol-2(3H-ylidene)carbamate as a yellow solid. 1H NMR (400 MHz, METHANOL-d4) δ 8.37 (d, J= 5.50 Hz, 1H), 8.09 (s, 1H), 7.43 (d, J= 4.75 Hz, 1H), 7.29 (dd, J = 5.50, 1.75 Hz, 1H), 7.01 (d, J = 4.88 Hz, 1H), 4.18 (q, J = 7.13 Hz, 2H), 1.54 (s, 9H), 1.26 (t, J = 7.07 Hz, 3H).

[0090] <Intermediate C2> To a solution of (Z)-ethyl (3-(2-((tert-butoxycarbonyl)amino)pyridin-4-yl)thiazol-2(3H)-ylidene)carbamate (110 mg, 302 μmol, 1.0 equiv) in EtOH (4 mL) was added NaOH (6 M, 4.0 mL, 80 equiv). The reaction mixture was stirred at 60° C. for 2 h. The mixture was diluted with water (30 mL) and extracted with THF (2×20 mL). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give intermediate C2, tert-butyl (4-(2-iminothiazol-3(2H)-yl)pyridin-2-yl)carbamate (100 mg, crude) as a brown solid.

[0091] <Preparation of intermediate C3> [ka] <Ethyl N-(o-tolylcarbamothioyl)carbamate> To a solution of o-toluidine (2.00 g, 18.7 mmol, 1.0 equiv.) in ethyl acetate (20 mL) was added TMEDA (0.217 g, 1.87 mmol, 0.1 equiv.) and O-ethyl carbomethane isothiocyanatidate (2.45 g, 18.7 mmol, 1.0 equiv.) at 25° C. The mixture was stirred at 25° C. for 2 h and then evaporated under reduced pressure. The residue was triturated in ethanol (10 mL) to give 3.90 g of ethyl N-(o-tolylcarbamothioyl)carbamate as a white solid. 1H NMR (400 MHz, CDCl3) δ 11.18 (br. s, 1H), 8.36 (br. s, 1H), 7.71 - 7.58 (m, 1H), 7.38 - 7.20 (m, 3H), 4.31 (q, J = 7.2 Hz, 2H), 2.34 (s, 3H), 1.37 (t, J = 7.2 Hz, 3H). LCMS (m / z [M+H] + ):239.1.

[0092] <(Z)-Ethyl (((2-oxoethyl)thio)(o-tolylamino)methylene)carbamate> To a solution of ethyl N-(o-tolylcarbamothioyl)carbamate (3.90 g, 16.3 mmol, 1.0 equiv.) and Cs2CO3 (9.05 g, 27.8 mmol, 1.7 equiv.) in acetonitrile (40 mL) was added 2-chloroacetaldehyde (3.85 g, 19.6 mmol, 40% aqueous solution, 1.2 equiv.) while maintaining the temperature below 25° C. After the addition, the mixture was stirred at 25° C. for 12 h, then diluted with MTBE (100 mL), washed with saturated aqueous NaHCO3 (100 ml), followed by brine (50 mL), dried over Na2SO4, and filtered. The filtrate was concentrated under reduced pressure to give 5.80 g of crude (Z)-ethyl (((2-oxoethyl)thio)(o-tolylamino)methylene)carbamate as a brown gum. 1 H NMR (400 MHz, CDCl3) δ 7.40 - 7.27 (m, 5H), 5.39 (d, J= 5.2 Hz, 1H), 4.16 (q, J = 7.2 Hz, 2H), 3.65 (dd, J = 6.0, 12.4 Hz, 1H), 3.28 (d, J = 12.4 Hz, 1H), 2.20 (s, 3H), 1.26 (t, J= 7.2 Hz, 3H). LCMS (m / z [M+H] + ): 281.1.

[0093] <(Z)-Ethyl (3-(o-tolyl)thiazol-2(3H)-ylidene)carbamate To a solution of (Z)-ethyl (((2-oxoethyl)thio)(o-tolylamino)methylene)carbamate (5.80 g, 12.1 mmol, 1.0 equiv.) and DIEA (6.32 mL, 36.3 mmol, 3.0 equiv.) in THF (60 mL) was added SOCl2 (0.878 mL, 12.1 mmol, 1.0 equiv.) dropwise at 0° C. The mixture was stirred at 0° C. for 4 h, then quenched with water (100 mL) and extracted with ethyl acetate (30 mL×3). The combined organic layers were dried over sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate=10:1→3:1) to give 2.70 g of (Z)-ethyl (3-(o-tolyl)thiazol-2(3H)-ylidene)carbamate as a yellow oil. 1 H NMR (400 MHz, CDCl3) δ 7.41 - 7.28 (m, 3H), 7.25 - 7.19 (m, 1H), 6.85 (d, J = 4.8 Hz, 1H), 6.69 (d, J = 4.8 Hz, 1H), 4.21 (q, J = 7.2 Hz, 2H), 2.14 (s, 3H), 1.29 (t, J = 7.2 Hz, 3H). LCMS (m / z[M+H] + ): 263.1.

[0094] <Intermediate C3> A solution of (Z)-ethyl (3-(o-tolyl)thiazol-2(3H)-ylidene)carbamate (0.500 g, 1.79 mmol, 1.0 equiv.) and NaOH (1.43 g, 35.8 mmol, 20 equiv.) in ethanol (6 mL) was stirred at 50° C. for 1 h. The mixture was cooled to room temperature, diluted with ethyl acetate (20 mL), washed with water (20 mL), followed by brine (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane:methanol=20:1) to give 0.23 g of 3-(o-tolyl)thiazol-2(3H)-imine C3 as a yellow oil.

[0095] <Preparation of intermediate C4> [ka] <Ethyl N-[(2-methoxyphenyl)carbamothioyl]carbamate> To a solution of 2-methoxyaniline (2.00 g, 16.2 mmol, 1.0 equiv) in ethyl acetate (20 mL) was added O-ethylcarbonyl isothiocyanatidate (1.92 mL, 16.2 mmol, 1.0 equiv) and tetramethylethylenediamine (0.245 mL, 1.62 mmol, 0.1 equiv) at 25° C. The mixture was stirred at this temperature for 5 h. The mixture was concentrated under reduced pressure to remove ethyl acetate. The residue was triturated twice with ethanol (5 mL) to give 3.40 g of ethyl N-[(2-methoxyphenyl)carbamothioyl]carbamate as an off-white solid. 1 H NMR (400 MHz, DMSO-d6) δ 11.90 (br. s, 1H), 11.25 (br. s, 1H), 8.48 (dd, J = 8.0, 1.2 Hz, 1H), 7.19 (dt, J = 8.4, 1.6 Hz, 1H), 7.10 (dd, J = 8.0, 1.2 Hz, 1H), 6.96 (dt, J = 8.0, 1.2 Hz, 1H), 4.22 (q, J = 7.2 Hz, 2H), 3.85 (s, 3H), 1.25 (t, J = 7.2 Hz, 3H).

[0096] <(Z)-Ethyl(((2-methoxyphenyl)amino)((2-oxoethyl)thio)methylene)carbamate> To a suspension of ethyl N-[(2-methoxyphenyl)carbamothioyl]carbamate (3.40 g, 13.4 mmol, 1.0 equiv) and cesium carbonate (7.41 g, 22.7 mmol, 1.7 equiv) in acetonitrile (40 mL) was added 2-chloroacetaldehyde (2.69 mL, 16.7 mmol, 1.2 equiv) dropwise at 25° C. The resulting mixture was stirred at this temperature for 12 h. Additional 2-chloroacetaldehyde (1.08 mL, 6.68 mmol, 0.5 equiv) was added dropwise to the mixture at 25° C. and stirred for an additional 2 h. The mixture was poured into water (150 mL) and extracted with MTBE (100 mL×3), and the combined organic layers were washed with brine (75 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give 4.60 g of (Z)-ethyl (((2-methoxyphenyl)amino)((2-oxoethyl)thio)methylene)carbamate as a brown oil. 1 H NMR (400 MHz, DMSO-d6): δ 7.37 (dt, J = 8.4, 1.6 Hz, 1H), 7.19 (dd, J= 7.6, 1.6 Hz, 1H), 7.13 (dd, J = 8.4, 0.8 Hz, 1H), 7.00 (dt, J = 7.6, 1.2 Hz, 1H), 6.75 (d, J = 7.2 Hz, 1H), 5.46 (dt, J =7.6, 2.4 Hz, 1H), 3.93 (q, J = 7.2 Hz, 2H), 3.77 (s, 3H), 3.59 (dd, J = 12.4, 6.4Hz, 1H), 1.12 - 1.10 (m, 3H). LCMS (m / z [M+H] + ): 296.8.

[0097] <(Z)-ethyl (3-(2-methoxyphenyl)thiazol-2(3H)-ylidene)carbamate> To a solution of (Z)-ethyl (((2-methoxyphenyl)amino)((2-oxoethyl)thio)methylene)carbamate (2.60 g, 6.10 mmol, 1.0 equiv) in THF (20 mL) was added diisopropylethylamine (3.19 mL, 18.3 mmol, 3.0 equiv) followed by SOCl2 (0.442 mL, 6.10 mmol, 1.0 equiv) at 0° C. The mixture was stirred below 15° C. for 4 h. The mixture was added to saturated aqueous sodium bicarbonate (20 mL), stirred for 5 min, and extracted with ethyl acetate (100 mL×3). The combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=10 / 1→5 / 1) to give 1.00 g of (Z)-ethyl (3-(2-methoxyphenyl)thiazol-2(3H)-ylidene)carbamate as an off-white solid. 1 H NMR (400 MHz, DMSO-d6): δ 7.51 - 7.47 (m, 1H), 7.35 (dd, J = 6.8, 1.6 Hz, 1H), 7.32 (d, J= 4.8 Hz, 1H), 7.25 (dd, J = 8.4, 0.8 Hz, 1H), 7.10 - 7.06 (m, 1H), 6.99 (d, J= 4.8 Hz, 1H), 3.98 (q, J = 7.2 Hz, 2H), 3.76 (s, 3H), 1.13 (t, J = 7.2 Hz, 3H). LCMS (m / z [M+H] + ): 278.8.

[0098] <Intermediate C4> To a solution of ethyl (3-(2-methoxyphenyl)thiazol-2(3H)-ylidene)carbamate (0.500 g, 1.80 mmol, 1.0 equiv) in ethanol (5 mL) was added sodium hydroxide (1.44 g, 35.9 mmol, 20 equiv) in one portion at 25° C. and the mixture was stirred for 1 h. The mixture was poured into water (20 mL) and extracted with ethyl acetate (50 mL×3). The combined organic layers were washed with brine (25 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give 0.350 g of 3-(2-methoxyphenyl)thiazol-2(3H)-imine C4 as a brown oil.

[0099] <Preparation of intermediate C5> [ka] <Ethyl N-[(2-chlorophenyl)carbamothioyl]carbamate> To a solution of 2-chloroaniline (2.20 g, 17.3 mmol, 1.0 equiv.), O-ethylcarbonyl isothiocyanatidate (2.26 g, 17.3 mmol, 1.0 equiv.) in ethyl acetate (20 mL) was added tetramethylethylenediamine (0.200 g, 1.72 mmol, 0.1 equiv.) at 0° C. The resulting mixture was stirred at 25° C. for 2 h and then concentrated under reduced pressure. The residue was triturated with methanol (30 mL) to give 3.00 g of ethyl N-[(2-chlorophenyl)carbamothioyl]carbamate as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 11.60 (s, 1H), 11.48 (s, 1H), 7.94 (dd, J = 8.0, 1.6 Hz, 1H), 7.56 (dd, J = 8.0, 1.6 Hz, 1H), 7.40 - 7.36 (m, 1H), 7.33 - 7.29 (m, 1H), 4.23 (q, J = 7.2 Hz, 2H), 1.26 (t, J =7.2 Hz, 3H).

[0100] <(Z)-Ethyl (((2-chlorophenyl)amino)((2-oxoethyl)thio)methylene)carbamate> To a suspension of ethyl N-[(2-chlorophenyl)carbamothioyl]carbamate (3.00 g, 11.6 mmol, 1.0 equiv.) and Cs2CO3 (6.42 g, 19.7 mmol, 12 equiv.) in acetonitrile (40 mL) was added 2-chloroacetaldehyde (2.84 g, 14.5 mmol, 1.2 equiv.) while maintaining the temperature below 25° C. The reaction mixture was stirred at 25° C. for 12 h. The mixture was poured into water (100 mL) and extracted with methyl TBME (150 mL×2), the combined organic layers were washed with brine (200 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 3.80 g of crude (Z)-ethyl (((2-chlorophenyl)amino)((2-oxoethyl)thio)methylene)carbamate as a brown gum. 1 H NMR (400 MHz, DMSO-d6) δ 7.66 - 7.58 (m, 1H), 7.49 - 7.35 (m, 3H), 6.99 (d, J = 6.4 Hz, 1H), 5.53- 5.42 (m, 1H), 4.00 - 3.90 (m, 2H), 3.70 - 3.65 (m, 1H), 3.23 - 3.15 (m, 1H), 1.11 (t, J = 7.2 Hz, 3H).

[0101] (Z)-Ethyl (3-(2-chlorophenyl)thiazol-2(3H)-ylidene)carbamate To a solution of (Z)-ethyl (((2-chlorophenyl)amino)((2-oxoethyl)thio)methylene)carbamate (1.80 g, 5.98 mmol, 1.0 equiv) in THF (20 mL) was added diisopropylethylamine (2.32 g, 17.9 mmol, 3.0 equiv) and thionyl chloride (0.710 g, 5.98 mmol, 1.0 equiv) at 0° C. The mixture was stirred at 15° C. for 4 h, then poured into water (100 mL) and extracted with ethyl acetate (150 mL×4). The combined organic phase was washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=1 / 0→10 / 1) to give 1.101 g of (Z)-ethyl (3-(2-chlorophenyl)thiazol-2(3H)-ylidene)carbamate as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.73 - 7.71 (m, 1H), 7.60 - 7.53 (m, 3H), 7.45 (d, J = 4.8 Hz, 1H), 7.09 (d, J= 4.8 Hz, 1H), 4.00 (q, J = 6.8 Hz, 2H), 1.14 (t, J = 7.2 Hz, 3H). LCMS (m / z [M+H] + ): 282.9.

[0102] <Intermediate C5> To a solution of (Z)-ethyl (3-(2-chlorophenyl)thiazol-2(3H)-ylidene)carbamate (0.80 g, 2.75 mmol, 1.0 equiv) in ethanol (10 mL) was added sodium hydroxide (2.34 g, 58.4 mmol, 21 equiv) at 25° C. The mixture was heated to 50° C. and stirred for 1 h, then poured into water (80 mL) and extracted with ethyl acetate (120 mL×4). The combined organic layers were washed with brine (100 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give 0.54 g of 3-(2-chlorophenyl)thiazol-2(3H)-imine C5 as a yellow solid.

[0103] <Preparation of intermediate C6> [ka] <Ethyl N-(m-tolylcarbamothioyl)carbamate> To a solution of m-toluidine (2.00 g, 18.6 mmol, 1.0 equiv) in ethyl acetate (20 mL) was added O-ethylcarbonyl isothiocyanatidate (2.21 mL, 18.6 mmol, 1.0 equiv) and tetramethylethylenediamine (0.282 mL, 1.87 mmol, 0.1 equiv). The mixture was stirred at 25° C. for 5 h. The mixture was concentrated under reduced pressure to remove ethyl acetate. The residue was triturated twice with ethanol (5 mL) to give 3.00 g of ethyl N-(m-tolylcarbamothioyl)carbamate as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 11.51 (br. s, 1H), 11.23 (br. s, 1H), 7.43 - 7.38 (m, 2H), 7.26 (t, J = 7.6 Hz, 1H), 7.05 (d, J = 7.6 Hz, 1H), 4.20 (q, J = 8.0, 7.2 Hz, 2H), 2.30 (s, 3H), 1.25 (t, J = 6.8 Hz, 3H).

[0104] <(Z)-Ethyl (((2-oxoethyl)thio)(m-tolylamino)methylene)carbamate> To a suspension of ethyl N-(m-tolylcarbamothioyl)carbamate (3.00 g, 12.6 mmol, 1.0 equiv.) and cesium carbonate (6.97 g, 21.4 mmol, 1.7 equiv.) in acetonitrile (40 mL), 2-chloroacetaldehyde (2.53 mL, 15.7 mmol, 1.25 equiv.) was added dropwise at 25° C. and the mixture was stirred for 12 h. The mixture was poured into water (150 mL), extracted with methyl tert-butyl ether (100 mL×3), the combined organic layers were washed with brine (75 mL), dried over sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give 4.00 g of (Z)-ethyl (((2-oxoethyl)thio)(m-tolylamino)methylene)carbamate as a brown oil. 1 H NMR (400 MHz, DMSO-d6) δ 7.34 - 7.30 (m, 1H), 7.16 - 7.13 (m, 3H), 6.85 (d, J = 7.2 Hz, 1H), 5.60 (dt, J=7.2, 1.6 Hz, 1H), 3.96 (q, J = 7.2 Hz, 2H), 2.32 (s, 3H), 1.14 - 1.10 (m, 3H). LCMS (m / z [M+H] + ): 280.9.

[0105] <(Z)-ethyl (3-(m-tolyl)thiazol-2(3H)-ylidene)carbamate> To a solution of (Z)-ethyl (((2-oxoethyl)thio)(m-tolylamino)methylene)carbamate (2.00 g, 5.58 mmol, 1.0 equiv.) in THF (20 mL) was added diisopropylethylamine (2.92 mL, 16.8 mmol, 3.0 equiv.) followed by SOCl2 (0.405 mL, 5.58 mmol, 1.0 equiv.) under nitrogen atmosphere at 0° C. The mixture was stirred below 15° C. for 4 h. The mixture was poured into saturated aqueous sodium bicarbonate to adjust pH=8, then extracted with ethyl acetate (100 mL×3). The combined organic layers were washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=10 / 1) to give 1.00 g of (Z)-ethyl (3-(m-tolyl)thiazol-2(3H)-ylidene)carbamate as a yellow oil. 1 H NMR (400 MHz, DMSO-d6) δ 7.49 (d, J = 4.8 Hz, 1H), 7.42 (t, J = 8.0 Hz,1H), 7.30 - 7.28 (m, 3H), 7.05 (d, J = 4.8 Hz, 1H), 4.04 - 3.99 (m, 2H), 2.37 (s, 3H), 1.15 (t, J= 7.2 Hz, 3H). LCMS (m / z [M+H] + ): 262.8.

[0106] <Intermediate C6> To a solution of (Z)-ethyl (3-(m-tolyl)thiazol-2(3H)-ylidene)carbamate (0.200 g, 0.762 mmol, 1.0 equiv) in ethanol (2 mL) was added sodium hydroxide (0.610 g, 15.2 mmol, 20 equiv) in one portion at 25° C., then the mixture was heated to 50° C. and stirred for 1 h. The mixture was poured into water (50 mL) and extracted with ethyl acetate (50 mL×4). The combined organic layers were washed with brine (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give 140 mg of 3-(m-tolyl)thiazol-2(3H)-imine C6 as a brown oil.

[0107] <Preparation of intermediate C7> [ka] <Ethyl N-[(3-methoxyphenyl)carbamothioyl]carbamate> To a solution of 3-methoxyaniline (2.00 g, 16.2 mmol, 1.0 equiv.) in ethyl acetate (10 mL) was added TMEDA (0.189 g, 1.62 mmol, 0.1 equiv.) and O-ethyl carbomethane isothiocyanatidate (2.13 g, 16.2 mmol, 1.0 equiv.) at 25° C. The mixture was stirred at 25° C. for 2 h and then evaporated under reduced pressure. The residue was crystallized from petroleum ether / ethyl acetate (v / v=5:1, 10 mL) to give 3.70 g of ethyl N-[(3-methoxyphenyl)carbamothioyl]carbamate as a yellow solid. 1 H NMR (400 MHz, CDCl3) δ 11.48 (br. s, 1H), 8.11 (br. s, 1H), 7.41 (t, J = 2.0 Hz, 1H), 7.30 (t, J = 8.0 Hz, 1H), 7.14 (dd, J = 1.6, 8.0 Hz, 1H), 6.82 (ddd, J = 0.8, 2.4, 8.4 Hz, 1H), 4.30 (q, J = 7.2 Hz, 2H), 3.83 (s, 3H), 1.36 (t, J = 7.2 Hz, 3H). LCMS (m / z [M+H] + ): 255.1.

[0108] <(Z)-Ethyl(((3-methoxyphenyl)amino)((2-oxoethyl)thio)methylene)carbamate> To a solution of ethyl N-[(3-methoxyphenyl)carbamothioyl]carbamate (3.70 g, 14.5 mmol, 1.0 equiv.) and Cs2CO3 (8.01 g, 24.6 mmol, 1.7 equiv.) in acetonitrile (40 mL) was added 2-chloroacetaldehyde (3.40 g, 17.3 mmol, 40% aqueous solution, 1.2 equiv.) while maintaining the temperature below 25° C. The mixture was stirred at 25° C. for 12 h, then diluted with MTBE (100 mL), washed with saturated aqueous NaHCO3 (100 ml), followed by brine (50 mL), dried over Na2SO4, and filtered. The filtrate was concentrated under reduced pressure to give 5.00 g of (Z)-ethyl (((3-methoxyphenyl)amino)((2-oxoethyl)thio)methylene)carbamate as a yellow oil. 1 H NMR (400 MHz, CDCl3) δ 7.33 (t, J = 8.0 Hz, 1H), 7.04 - 6.95 (m, 2H), 6.87 (ddd, J = 0.8, 2.4, 8.4 Hz, 1H), 5.61 (d, J = 5.2 Hz, 1H), 4.18 LCMS (m / z) 297.1 [M+H] + ): 297.1.

[0109] (Z)-Ethyl (3-(3-methoxyphenyl)thiazol-2(3H)-ylidene)carbamate To a solution of (Z)-ethyl (((3-methoxyphenyl)amino)((2-oxoethyl)thio)methylene)carbamate (5.00 g, 11.3 mmol, 1.0 equiv.) and DIEA (4.37 g, 33.8 mmol, 3.0 equiv.) in THF (60 mL) was added SOCl2 (0.818 mL, 11.3 mmol, 1.0 equiv.) dropwise at 0° C. The mixture was stirred at 0° C. for 4 h, then quenched with water (100 mL) and extracted with ethyl acetate (30 mL×3). The combined organic layers were dried over sodium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=10:1→3:1) to give 2.70 g of (Z)-ethyl (3-(3-methoxyphenyl)thiazol-2(3H)-ylidene)carbamate as a yellow oil. 1 H NMR (400 MHz, CDCl3) δ 7.38 (t, J = 8.0 Hz, 1H), 7.07 - 6.98 (m, 3H), 6.94 (dd, J= 2.0, 8.0 Hz, 1H), 6.66 (d, J = 4.8 Hz, 1H), 4.22 (q, J = 7.2 Hz, 2H), 3.83 (s, 3H), 1.30 (t, J = 7.2 Hz, 3H). LCMS (m / z [M+H] + ): 279.1.

[0110] <Intermediate C7> A solution of (Z)-ethyl(3-(3-methoxyphenyl)thiazol-2(3H)-ylidene)carbamate (0.500 g, 1.70 mmol, 1.0 equiv) and NaOH (1.36 g, 34.1 mmol, 20 equiv) in ethanol (6 mL) was stirred at 50° C. for 1 h. The mixture was cooled to room temperature, diluted with ethyl acetate (20 mL), washed with water (20 mL) followed by brine (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane:methanol=20:1) to give 230 mg of 3-(3-methoxyphenyl)thiazol-2(3H)-imine C7 as a yellow oil.

[0111] <Preparation of intermediate C8> [ka] <Ethyl N-[(3-chlorophenyl)carbamothioyl]carbamate> To a solution of 3-chloroaniline (2.00 g, 15.7 mmol, 1.0 equiv.) in ethyl acetate (20 mL) was added O-ethylcarbonyl isothiocyanatidate (1.85 mL, 15.7 mmol, 1.0 equiv.) and tetramethylethylenediamine (0.237 mL, 1.57 mmol, 0.1 equiv.). The mixture was stirred at 25° C. for 5 h. The mixture was concentrated under reduced pressure. The residue was triturated twice with petroleum ether / ethyl acetate (v / v=10 / 1, 11 mL) to give 3.20 g of ethyl N-[(3-chlorophenyl)carbamothioyl]carbamate as a yellow solid. 1 H NMR (400 MHz, DMSO-d6): δ 11.55 (br. s, 1H), 11.35 (br. s, 1H), 7.84 (s, 1H), 7.49 - 7.47 (m, 1H), 7.41 (t, J = 7.6 Hz, 1H), 7.32 - 7.30 (m, 1H), 4.21 (q, J= 7.6 Hz, 2H), 1.25 (t, J = 7.2 Hz, 3H).

[0112] <(Z)-Ethyl (((3-chlorophenyl)amino)((2-oxoethyl)thio)methylene)carbamate> To a suspension of ethyl N-[(3-chlorophenyl)carbamothioyl]carbamate (3.20 g, 12.4 mmol, 1.0 equiv.) and cesium carbonate (6.85 g, 21.0 mmol, 1.7 equiv.) in acetonitrile (40 mL) was added 2-chloroacetaldehyde (2.49 mL, 15.4 mmol, 1.25 equiv.) dropwise at 25° C. The resulting mixture was stirred at this temperature for 12 h. The mixture was poured into water (150 mL) and extracted with MTBE (100 mL×3) at 25° C. The combined organic layers were washed with brine (75 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 3.80 g of (Z)-ethyl (((3-chlorophenyl)amino)((2-oxoethyl)thio)methylene)carbamate as a brown oil. 1 H NMR (400 MHz, DMSO-d6): δ 7.50 - 7.46 (m, 2H), 7.43 - 7.40 (m, 1H), 7.38 - 7.36 (m, 1H), 6.95 (br. s, 1H), 5.69 - 5.68 (m, 1H), 3.98 (t, J= 7.6Hz, 2H), 3.68 - 3.55 (m, 1 H), 3.10 - 3.05 (m, 1 H), 1.14 (t, J = 7.6Hz, 3H). LCMS (m / z [M+H] + ): 300.8.

[0113] (Z)-Ethyl (3-(3-chlorophenyl)thiazol-2(3H)-ylidene)carbamate To a solution of (Z)-ethyl (((3-chlorophenyl)amino)((2-oxoethyl)thio)methylene)carbamate (1.80 g, 3.68 mmol, 1.0 equiv.) in THF (20 mL) was added diisopropylethylamine (1.92 mL, 11.0 mmol, 3.0 equiv.) followed by SOCl2 (0.267 mL, 3.68 mmol, 1.0 equiv.) under nitrogen atmosphere at 0° C. The mixture was stirred below 15° C. for 4 h. To the mixture was added SOCl2 (0.134 mL, 1.84 mmol, 0.5 equiv.) dropwise at 0° C. and the reaction mixture was stirred at 15° C. for 2 h. The mixture was added into saturated aqueous sodium bicarbonate solution to adjust pH=8 and extracted with ethyl acetate (100 mL×3). The combined organic layers were washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1 → 5 / 1) to give 0.95 g of (Z)-ethyl (3-(3-chlorophenyl)thiazol-2(3H)-ylidene)carbamate as an off-white solid. 1 H NMR (400 MHz, DMSO-d6): δ 7.70 (s, 1H), 7.58 - 7.54 (m, 3H), 7.53 - 7.51 (m, 1H), 7.07 (d, J = 4.8 Hz, 1H), 4.03 (q, J = 7.2 Hz, 2H), 1.16 (t, J = 7.2 Hz, 3H). LCMS (m / z [M+H] + ): 282.8.

[0114] <Intermediate C8> To a solution of (Z)-ethyl (3-(3-chlorophenyl)thiazol-2(3H)-ylidene)carbamate (0.200 g, 0.707 mmol, 1.0 equiv) in ethanol (2 mL) was added sodium hydroxide (0.566 g, 14.2 mmol, 20 equiv) in one portion at 25° C., and the mixture was stirred at this temperature for 1 h. The mixture was added into water (50 mL) and extracted with ethyl acetate (50 mL×4). The combined organic layers were washed with brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 130 mg of 3-(3-chlorophenyl)thiazol-2(3H)-imine C8 as a brown solid.

[0115] <Preparation of intermediate C9> [ka] To a solution of p-toluidine (2.00 g, 18.7 mmol, 1.0 equiv.) in ethyl acetate (10 mL) was added TMEDA (0.217 g, 1.87 mmol, 0.1 equiv.) and O-ethylcarbonyl isothiocyanatidate (2.45 g, 18.7 mmol, 1.0 equiv.) at 25° C. The mixture was stirred at 25° C. for 2 h and then evaporated under reduced pressure. The residue was triturated with ethanol (10 mL) to give 3.70 g of ethyl N-(p-tolylcarbamothioyl)carbamate as a yellow solid. 1 H NMR (400 MHz, CDCl3) δ 11.35 (br s, 1H), 8.21 (br s, 1H), 7.48 (d, J = 8.4 Hz, 2H), 7.21 (d, J = 8.0 Hz, 2H), 4.29 (q, J = 7.2 Hz, 2H), 2.37 (s, 3H), 1.35 (t, J = 7.2 Hz, 3H). LCMS (m / z [M+H] + ): 239.1.

[0116] <(Z)-Ethyl (((2-oxoethyl)thio)(p-tolylamino)methylene)carbamate> To a solution of ethyl N-(p-tolylcarbamothioyl)carbamate (3.70 g, 15.5 mmol, 10 equiv.) and Cs2CO3 (8.59 g, 26.4 mmol, 1.7 equiv.) in acetonitrile (40 mL) was added 2-chloroacetaldehyde (3.65 g, 18.6 mmol, 40% aqueous solution, 1.2 equiv.) while maintaining the temperature below 25° C. After addition, the mixture was stirred at 25° C. for 12 h, then diluted with MTBE (100 mL), washed with saturated aqueous NaHCO3 (100 ml), followed by brine (50 mL), dried over Na2SO4, and filtered. The filtrate was concentrated under reduced pressure to give 5.30 g of crude (Z)-ethyl (((2-oxoethyl)thio)(p-tolylamino)methylene)carbamate as a yellow oil. 1 H NMR (400 MHz, CDCl3) δ 7.39 - 7.18 (m, 5H), 5.56 (dd, J = 0.5, 5.6 Hz, 1H), 4.16 (q, J = 7.2 Hz, 2H), 3.57 (dd, J = 5.6, 12.4 Hz, 1H), 3.19 (dd, J = 1.2, 12.0 Hz, 1H), 2.35 (s, 3H), 1.26(t, J = 7.2 Hz, 3H). LCMS (m / z [M+H] + ): 281.1.

[0117] <(Z)-ethyl (3-(p-tolyl)thiazol-2(3H)-ylidene)carbamate> To a solution of (Z)-ethyl (((2-oxoethyl)thio)(p-tolylamino)methylene)carbamate (5.30 g, 10.8 mmol, 1.0 equiv.) and DIEA (4.19 g, 32.4 mmol, 3.0 equiv.) in THF (60 mL) was added SOCl2 (0.783 mL, 10.8 mmol, 1.0 equiv.) dropwise at 0° C. The mixture was stirred at 0° C. for 4 h, then quenched with water (100 mL) and extracted with ethyl acetate (30 mL×3). The combined organic layers were dried over sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=10:1→3:1) to give 2.20 g of (Z)-ethyl (3-(p-tolyl)thiazol-2(3H)-ylidene)carbamate as a yellow oil. 1 H NMR (400 MHz, CDCl3) δ 7.26 (d, J = 8.4 Hz, 2H), 7.21 (d, J = 8.4 Hz, 2H), 6.93 (d, J = 4.8 Hz, 1H), 6.58 (d, J = 4.8 Hz, 1H), 4.14 (q, J = 7.2 Hz, 2H), 2.32 (s, 3H), 1.22 (t, J = 7.2 Hz, 3H). LCMS o(m / z [M+H] + ): 263.1.

[0118] <Intermediate C9> A solution of (Z)-ethyl (3-(p-tolyl)thiazol-2(3H)-ylidene)carbamate (0.500 g, 1.86 mmol, 1.0 equiv.) and NaOH (1.49 g, 37.2 mmol, 20 equiv.) in ethanol (6 mL) was stirred at 50° C. for 1 h. The mixture was cooled to room temperature, diluted with ethyl acetate (20 mL), washed with water (20 mL), followed by brine (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol=20:1) to give 230 mg of 3-(p-tolyl)thiazol-2(3H)-imine C9 as a yellow oil.

[0119] <Preparation of intermediate C10> [ka] <Ethyl N-[(4-methoxyphenyl)carbamothioyl]carbamate> To a solution of 4-methoxyaniline (2.00 g, 16.2 mmol, 1.0 equiv.) and O-ethylcarbonisothiocyanatidate (2.13 g, 16.2 mmol, 1.0 equiv.) in ethyl acetate (20 mL) was added tetramethylethylenediamine (0.189 g, 1.62 mmol, 0.1 equiv.) at 25° C., and the mixture was stirred for 2 h at 25° C. The mixture was concentrated under vacuum, the residue triturated with ethanol (8 mL), and the precipitate was dried under vacuum to give 3.50 g of ethyl N-[(4-methoxyphenyl)carbamothioyl]carbamate as a white solid. 1 H NMR (400 MHz, CDCl3): δ 11.37 (br. s, 1H), 11.18 (br. s, 1H), 7.44 (d, J =8.8 Hz, 2H), 6.94 (d, J =8.8 Hz, 2H), 4.20 (q, J = 7.2 Hz, 2H), 3.76 (s, 1H), 1.26 (t, J = 6.8 Hz, 3H).

[0120] <(Z)-Ethyl(((4-methoxyphenyl)amino)((2-oxoethyl)thio)methylene)carbamate> To a solution of ethyl N-[(4-methoxyphenyl)carbamothioyl]carbamate (3.50 g, 13.7 mmol, 1.0 equiv.) and cesium carbonate (7.62 g, 23.4 mmol, 1.7 equiv.) in acetonitrile (40 mL) was added 2-chloroacetaldehyde (3.38 g, 17.2 mmol, 1.25 equiv.) while maintaining the temperature below 25° C. After addition, the mixture was stirred at 25° C. for 12 h. The mixture was poured into water (150 mL) and extracted with TBME (150 mL×2). The combined organic layers were washed with brine (200 mL), dried over sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give 3.80 g of (Z)-ethyl (((4-methoxyphenyl)amino)((2-oxoethyl)thio)methylene)carbamate as a brown gum. 1H NMR (400 MHz, DMSO-d6): δ 7.28 - 7.20 (m, 2H), 7.02 - 6.95 (m, 2H), 6.92 (d, J= 7.2 Hz, 1H), 5.59 - 5.54 (m, 1H), 3.95 (q, J = 7.2 Hz, 2H), 3.78 (s, 3H), 1.12 (t, J = 7.2 Hz, 3H). LCMS (m / z [M +H] + ): 297.0.

[0121] (Z)-Ethyl (3-(4-methoxyphenyl)thiazol-2(3H)-ylidene)carbamate To a solution of (Z)-ethyl (((4-methoxyphenyl)amino)((2-oxoethyl)thio)methylene)carbamate (1.80 g, 6.07 mmol, 1.0 equiv) in tetrahydrofuran (20 mL) was added DIEA (2.36 g, 18.2 mmol, 3.0 equiv) followed by thionyl chloride (0.723 g, 6.07 mmol, 1.0 equiv) at 0° C. After addition, the mixture was warmed to 15° C. and stirred for 4 h. The mixture was directly concentrated under reduced pressure. The residue was dissolved in ethyl acetate (100 mL) and adjusted to pH=8.0 by addition of saturated aqueous sodium bicarbonate. The organic layer was separated, washed with brine (20 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=10 / 1→5 / 1) to give 1.20 g of (Z)-ethyl (3-(4-methoxyphenyl)thiazol-2(3H)-ylidene)carbamate as a yellow solid. 1 H NMR (400 MHz, CDCl3) δ 7.47 (d, J= 4.8 Hz, 1H), 7.41 (d, J = 5.2 Hz, 2H), 7.07 (d, J = 4.8 Hz, 2H), 7.04 (d, J = 4.8 Hz, 1H), 4.01 (q, J = 6.8 Hz, 2H), 3.82 (s, 3H), 1.16 (t, J = 7.2 Hz, 3H).

[0122] <Intermediate C10> To a solution of ethyl (3-(4-methoxyphenyl)thiazol-2(3H)-ylidene)carbamate (0.200 g, 0.718 mmol, 1.0 equiv) in ethanol (2 mL) was added sodium hydroxide (0.575 g, 14.4 mmol, 20 equiv). The mixture was stirred at 50° C. for 2 h. The mixture was diluted with water (30 mL) and extracted with ethyl acetate (15 mL×2). The combined organic layers were dried over sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol=10 / 1) to give 100 mg of 3-(4-methoxyphenyl)thiazol-2(3H)-imine C10 as a yellow oil.

[0123] <Preparation of intermediate C11> [ka] <Ethyl N-[(4-chlorophenyl)carbamothioyl]carbamate> To a solution of 4-chloroaniline (2.00 g, 15.7 mmol, 1.0 equiv.) and O-ethylcarbonyl isothiocyanatidate (2.06 g, 15.7 mmol, 1.0 equiv.) in ethyl acetate (20 mL) was added tetramethylethylenediamine (0.182 g, 1.57 mmol, 0.1 equiv.) at 25° C., and the resulting mixture was stirred at 25° C. for 2 h. The mixture was concentrated under reduced pressure. The residue was triturated with ethanol (8 mL) to give 3.00 g of ethyl N-[(4-chlorophenyl)carbamothioyl]carbamate as a white solid. 1 H NMR (400 MHz, CDCl3-d): δ 11.51 (br. s, 1H), 11.31 (br. s, 1H), 7.62 (d, J =8.8 Hz, 2H), 7.44 (d, J =8.8 Hz, 2H), 4.21 (q, J = 7.2 Hz, 2H), 1.26 (t, J = 6.8 Hz, 3H).

[0124] <(Z)-Ethyl (((4-chlorophenyl)amino)((2-oxoethyl)thio)methylene)carbamate> To a suspension of ethyl ethyl N-[(4-chlorophenyl)carbamothioyl]carbamate (3.00 g, 11.60 mmol, 1.0 equiv.) and cesium carbonate (6.42 g, 19.7 mmol, 1.7 equiv.) in acetonitrile (40 mL) was added 2-chloroacetaldehyde (2.84 g, 14.5 mmol, 1.25 equiv.) dropwise while maintaining the temperature below 25° C. After addition, the mixture was stirred at 25° C. for 12 h. The mixture was diluted with tert-butyl methyl ether (150 mL×2), washed with brine (50 mL), dried over sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give 3.50 g of (Z)-ethyl (((4-chlorophenyl)amino)((2-oxoethyl)thio)methylene)carbamate as a brown gum. 1 H NMR (400 MHz, DMSO-d6) δ 7.57 - 7.46 (m, 2H), 7.43 - 7.33 (m, 2H), 6.92(d, J = 7.2 Hz, 1H), 5.70 - 5.61 (m, 1H), 3.98 (q, J=6.8 Hz, 2H), 1.13 (t, J = 7.2 Hz, 3H). LCMS (m / z [M +H] + ): 301.0.

[0125] (Z)-Ethyl (3-(4-chlorophenyl)thiazol-2(3H)-ylidene)carbamate To a solution of (Z)-ethyl(((4-chlorophenyl)amino)((2-oxoethyl)thio)methylene)carbamate (1.80 g, 5.78 mmol, 1.0 equiv) in tetrahydrofuran (20 mL) was added N-ethyl-N-isopropylpropan-2-amine (2.24 g, 17.3 mmol, 3.0 equiv) and thionyl chloride (0.687 g, 5.78 mmol, 1.0 equiv) at 0° C. The mixture was stirred at 25° C. for 4 h. The mixture was directly concentrated under reduced pressure. The residue was dissolved in ethyl acetate (100 mL) and adjusted to pH=8.0 by addition of saturated aqueous sodium bicarbonate. The organic layer was separated, washed with brine (20 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=10 / 1→5 / 1) to give 1.10 g of (Z)-ethyl (3-(4-chlorophenyl)thiazol-2(3H)-ylidene)carbamate as a yellow solid. 1 H NMR (400 MHz, CDCl3-d): δ 7.65 - 7.55 (m, 4H), 7.54 (d, J = 4.4 Hz, 1H), 7.07 (d, J = 4.4 Hz, 1H), 4.03 (q, J = 7.2 Hz, 2H), 1.16 (t, J = 7.2Hz, 3H).

[0126] <Intermediate C11> To a solution of (Z)-ethyl (3-(4-chlorophenyl)thiazol-2(3H)-ylidene)carbamate (0.200 g, 0.707 mmol, 1.0 equiv) in ethanol (2 mL) was added sodium hydroxide (0.569 g, 14.2 mmol, 20 equiv) at 25° C. The reaction mixture was then heated to 50° C. and stirred for 1.5 h. The mixture was diluted with water (80 mL) and extracted with ethyl acetate (50 mL×2). The combined organic layers were dried over sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 120 mg of 3-(4-chlorophenyl)thiazol-2(3H)-imine C11 as a yellow oil.

[0127] <Preparation of intermediate C12> [ka] <Ethyl N-(3-pyridylcarbamothioyl)carbamate> To a solution of pyridin-3-amine (1.00 g, 10.6 mmol, 1.0 equiv.) and O-ethylcarbonyl isothiocyanatidate (1.39 g, 10.6 mmol, 1.0 equiv.) in ethyl acetate (10 mL) was added tetramethylethylenediamine (0.123 g, 1.06 mmol, 0.1 equiv.) at 25° C., and the mixture was then stirred at this temperature for 1 h. The mixture was concentrated, the residue was triturated with ethanol (10 mL), and the precipitate was collected by filtration and dried under reduced pressure to give 1.80 g of ethyl N-(3-pyridylcarbamothioyl)carbamate as a white solid. 1 H NMR (400MHz, CDCl3) δ 11.56 (br. s, 1H), 8.70 (d, J=2.4 Hz, 1H), 8.52 (dd, J = 4.8 Hz, 1.6 Hz, 1H), 8.30 (br. s, 1H), 8.28 - 8.23 ​​(m, 1H), 7.39 - 7.33 (m, 1H), 4.33 (q, J = 7.2 Hz, 2H), 1.38 (t, J = 7.2 Hz, 3H).

[0128] <(Z)-Ethyl(((2-oxoethyl)thio)(pyridin-3-ylamino)methylene)carbamate> To a suspension of ethyl N-(3-pyridylcarbamothioyl)carbamate (0.60 g, 2.66 mmol, 1.0 equiv.) and cesium carbonate (1.48 g, 4.53 mmol, 1.7 equiv.) in acetonitrile (10 mL) was added 2-chloroacetaldehyde (1.05 g, 5.33 mmol, 2.0 equiv.) dropwise while maintaining the temperature at 25° C. After addition, the mixture was stirred at this temperature for 12 h. The mixture was poured into water (100 mL) and extracted with ethyl acetate (50 mL×3). The combined organic layers were washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 0.40 g of (Z)-ethyl (((2-oxoethyl)thio)(pyridin-3-ylamino)methylene)carbamate as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 8.58 (d, J = 2.0 Hz , 1H), 8.52 (dd, J = 1.6 Hz , 4.8Hz, 1H), 7.83-7.80 (m, 1H), 7.50 (q, J = 4.8 Hz , 1H),7.00 (d, J = 7.2 Hz , 1H), 5.72 (t, J = 6.0 Hz , 1H), 4.04-3.96 (m, 2H), 3.66 - 3.61 (m, 1H), 3.15 (dd, J= 12.4 Hz , 1.2 Hz, 1H), 1.13 (t, J = 6.8 Hz, 3H).LCMS (m / z [M+H] + ): 268.1.

[0129] <(Z)-ethyl (3-(pyridin-3-yl)thiazol-2(3H)-ylidene)carbamate> To a solution of (Z)-ethyl (((2-oxoethyl)thio)(pyridin-3-ylamino)methylene)carbamate (0.570 g, 2.13 mmol, 1.0 equiv.) in tetrahydrofuran (6 mL), diisopropylethylamine (0.830 g, 6.40 mmol, 3.0 equiv.) and thionyl chloride (0.250 g, 2.13 mmol, 1.0 equiv.) were added at 0° C. The mixture was stirred at 25° C. for 4 h. The mixture was directly concentrated under reduced pressure. The residue was dissolved in ethyl acetate (100 mL) and adjusted to pH=8.0 with saturated aqueous sodium bicarbonate solution. The organic layer was separated, washed with brine (20 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure to give 0.40 g of (Z)-ethyl (3-(pyridin-3-yl)thiazol-2(3H)-ylidene)carbamate as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 8.77 (d, J = 2.4 Hz, 1H), 8.65 (dd, J = 4.8 Hz, 1.6 Hz, 1H), 8.07 - 8.00 (m, 1H), 7.63 (d, J = 4.8 Hz, 1H), 7.63 - 7.59 (m, 1H), 7.12 (d, J = 4.8 Hz, 1H), 4.03 (q, J = 7.2 Hz, 2H), 1.15 (t, J = 7.2 Hz, 3H).

[0130] <Intermediate C12> To a solution of ethyl (3-(pyridin-3-yl)thiazol-2(3H)-ylidene)carbamate (0.200 g, 0.800 mmol, 1.0 equiv) in ethanol (2 mL) was added sodium hydroxide (0.640 g, 16.1 mmol, 20 equiv) at 25° C., then the mixture was heated to 50° C. and stirred for 1 h. The mixture was diluted with water (50 mL) and extracted with ethyl acetate (50 mL×2). The combined organic layers were dried over sodium sulfate, filtered and concentrated to give 140 mg of 3-(pyridin-3-yl)thiazol-2(3H)-imine C12 as a brown oil. 1H NMR (400 MHz, DMSO-d6, 400 MHz) δ 8.80 (d, J = 2.0 Hz, 1H), 8.45 (d, J = 4.0 Hz, 1H), 8.28 (br. s, 1H), 8.06 (d, J = 7.6 Hz, 1H), 7.51 - 7.43 (m, 1H), 7.12 (d, J = 5.2 Hz, 1H), 6.25 (d, J = 5.2 Hz, 1H).

[0131] <Preparation of intermediate C13> [ka] <Ethyl N-(2-pyridylcarbamothioyl)carbamate> To a solution of pyridin-2-amine (1.00 g, 10.6 mmol, 1.0 equiv.) in ethyl acetate (10 mL) was added O-ethylcarbonyl isothiocyanatidate (1.26 mL, 10.6 mmol, 1.0 equiv.) and N1,N1,N2,N2-tetramethylethane-1,2-diamine (0.160 mL, 1.06 mmol, 0.1 equiv.) at 25° C. The mixture was then stirred at this temperature for 5 h. The mixture was concentrated under reduced pressure. The residue was triturated three times with ethanol (10 mL) to give 0.95 g of ethyl N-(2-pyridylcarbamothioyl)carbamate as an off-white solid. 1 H NMR (400 MHz, CDCl3) δ 12.06 (br.s, 1H), 8.80 (d, J = 8.0 Hz, 1H), 8.41 (d, J = 4.0 Hz, 1H), 8.07 (br. s, 1H), 7.79 - 7.74 (m, 1H), 7.16 - 7.13 (m, 1H), 4.31 (q, J = 7.2 Hz, 2H), 1.35 (t, J= 7.2 Hz, 3H). LCMS (m / z [M+H] + ): 225.8.

[0132] <(Z)-Ethyl(((2-oxoethyl)thio)(pyridin-2-ylamino)methylene)carbamate> To a suspension of ethyl N-(2-pyridylcarbamothioyl)carbamate (0.800 g, 3.55 mmol, 1.0 equiv.) and cesium carbonate (1.97 g, 6.04 mmol, 1.7 equiv.) in acetonitrile (8 mL), 2-chloroacetaldehyde (0.714 mL, 4.44 mmol, 1.25 equiv.) was added dropwise at 25° C. After addition, the mixture was stirred for 5 h. Then, 2-chloroacetaldehyde (0.285 mL, 1.78 mmol, 0.5 equiv.) was added to the mixture and stirred for another 2 h. The mixture was poured into water (20 mL) and extracted with ethyl acetate (70 mL×3). The combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give (Z)-ethyl (((2-oxoethyl)thio)(pyridin-2-ylamino)methylene)carbamate (1.00 g) as a brown oil. 1 H NMR (400 MHz, CDCl3): δ 8.34 - 8.32 (m, 1H), 8.06 (d, J = 8.4 Hz, 1H), 7.93 - 7.79 (m, 1H), 7.17 - 7.14 (m, 1H), 6.19 (d, J = 6.4 Hz, 1H), 5.44 (s, 1H), 4.28 - 4.22 (m, 2H), 3.51 - 3.47 (m, 1H), 3.28 - 3.24 (m, 1H), 1.33 (t, J= 7.2 Hz, 3H).

[0133] <(Z)-ethyl (3-(pyridin-2-yl)thiazol-2(3H)-ylidene)carbamate> To a solution of (Z)-ethyl (((2-oxoethyl)thio)(pyridin-2-ylamino)methylene)carbamate (0.500 g, 1.87 mmol, 1.0 equiv) in THF (5 mL) was added N-ethyl-N-isopropylpropan-2-amine (0.977 mL, 5.61 mmol, 3.0 equiv), followed by SOCl2 (136 μL, 1.87 mmol, 1.0 equiv) at 0° C. The mixture was stirred below 15° C. for 4 h. The mixture was poured into water (10 mL) and extracted with ethyl acetate (40 mL×3). The combined organic layers were washed with brine (25 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=20 / 1→10 / 1) to give 210 mg of (Z)-ethyl (3-(pyridin-2-yl)thiazol-2(3H)-ylidene)carbamate as a brown solid. 1 H NMR (400 MHz, CDCl3) δ 8.51 - 8.49 (m, 1H), 8.42 (d, J = 8.4 Hz, 1H), 7.92 - 7.88 (m, 1H), 7.80 (d, J = 5.2 Hz, 1H), 7.31 - 7.28 (m, 1H), 0.65 (d, J = 5.2Hz, 1H), 4.28 (q, J = 7.2 Hz, 2H), 1.36 (t, J = 7.2 Hz, 3H). LCMS (m / z[M+H] + ): 249.8.

[0134] <Intermediate C13> To a solution of sodium hydroxide (0.674 g, 16.8 mmol, 20.0 equiv) in ethanol (3 mL) was added (Z)-ethyl (3-(pyridin-2-yl)thiazol-2(3H)-ylidene)carbamate (0.210 g, 0.842 mmol, 1.0 equiv) at 25° C. The mixture was then heated to 50° C. and stirred for 1 h. The mixture was poured into water (15 mL), stirred for 3 min, and extracted with ethyl acetate (40 mL×3). The combined organic phase was washed with brine (15 mL×2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 130 mg of 3-(pyridin-2-yl)thiazol-2(3H)-imine C13 as a brown oil. 1 H NMR (400 MHz, DMSO-d6) δ 8.82 (s, 1H), 8.62 - 8.60 (m, 1H), 8.41 (d, J= 4.0 Hz, 1H), 7.89 - 7.86 (m, 1H), 7.60 (s, 1H), 7.24 - 7.21 (m, 1H), 7.26 (d, J = 5.2 Hz, 1H). LCMS (m / z [M+H] + ): 177.9.

[0135] <Preparation of intermediate C14> [ka] <Ethyl N-(tetrahydropyran-4-ylcarbamothioyl)carbamate> To a mixture of tetrahydro-2H-pyran-4-amine (0.800 g, 7.91 mmol, 1.0 equiv.) and O-ethylcarbonyl isothiocyanatidate (1.09 g, 8.30 mmol, 1.05 equiv.) in ethyl acetate (10 mL) was added TMEDA (92 mg, 0.791 mmol, 0.1 equiv.). The mixture was stirred at 25° C. for 6 h and then concentrated under reduced pressure. The residue was triturated twice with a solution of petroleum ether / ethanol (v / v=20 / 1, 21 mL) to give 1.80 g of ethyl N-(tetrahydropyran-4-ylcarbamothioyl)carbamate as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 10.99 (br. s, 1H), 9.83 (d, J = 7.2 Hz, 1H), 4.40 - 4.26 (m, 1H), 4.12 (q, J = 7.2 Hz, 2H), 3.87 - 3.73 (m, 2H), 3.48 - 3.36 (m, 2H), 1.97 - 1.86 (m, 2H), 1.59 - 1.45 (m, 2H), 1.21(t, J= 7.2 Hz, 3H).

[0136] <(Z)-Ethyl(((2-oxoethyl)thio)((tetrahydro-2H-pyran-4-yl)amino)methylene)carbamate> To a mixture of ethyl N-(tetrahydropyran-4-ylcarbamothioyl)carbamate (0.800 g, 3.44 mmol, 1.0 equiv.) and cesium carbonate (1.91 g, 5.85 mmol, 1.7 equiv.) in acetonitrile (10 mL) was added 2-chloroacetaldehyde (2.09 g, 10.66 mmol, 3.1 equiv.). The resulting mixture was stirred at 25° C. for 9 h, then poured into ice water (10 mL) and extracted with ethyl acetate (100 mL×2). The combined organic phase was washed with brine (20 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=100 / 1→0 / 1) to give 0.30 g of (Z)-ethyl (((2-oxoethyl)thio)((tetrahydro-2H-pyran-4-yl)amino)methylene)carbamate as a yellow solid. LCMS (m / z [M+H] + ): 274.9.

[0137] (Z)-Ethyl (3-(tetrahydro-2H-pyran-4-yl)thiazol-2(3H)-ylidene)carbamate To a mixture of (Z)-ethyl(((2-oxoethyl)thio)((tetrahydro-2H-pyran-4-yl)amino)methylene)carbamate (0.30 g, 0.52 mmol, 1.0 equiv) and diisopropylethylamine (133 mg, 1.03 mmol, 2.0 equiv) in tetrahydrofuran (10 mL) was added thionyl chloride (61 mg, 0.52 mmol, 1.0 equiv). The mixture was stirred at 25° C. for 2 h, then poured into ice water (20 mL) and extracted with ethyl acetate (100 mL×3). The combined organic layers were washed with brine (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=100 / 1→0 / 1) to give 120 mg of (Z)-ethyl (3-(tetrahydro-2H-pyran-4-yl)thiazol-2(3H)-ylidene)carbamate as a yellow solid. 1 H NMR (400 MHz, CD3OD) δ 7.41 (d, J =4.8Hz, 1H), 6.87 (d, J = 4.8 Hz, 1H), 5.02 - 4.91 (m, 1H), 4.20 (q, J = 7.2 Hz, 2H), 4.09 - 4.04 (m, 2H), 3.62 - 3.58 (m, 2H), 1.99 - 1.89 (m, 4H), 1.31 (t, J = 7.2 Hz, 3H). LCMS (m / z [M+H] + ): 256.9.

[0138] <Intermediate C14> A mixture of (Z)-ethyl (3-(tetrahydro-2H-pyran-4-yl)thiazol-2(3H)-ylidene)carbamate (0.120 g, 0.468 mmol, 1.0 equiv.) and sodium hydroxide (0.374 g, 9.36 mmol, 20 equiv.) in ethanol (1 mL) was stirred at 50° C. for 1 h. The mixture was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (dichloromethane / methanol=100 / 1→10 / 1) to give 80 mg of 3-(tetrahydro-2H-pyran-4-yl)thiazol-2(3H)-imine C14 as a yellow solid.

[0139] <Preparation of intermediate C15> [ka] <Ethyl N-(phenylcarbamothioyl)carbamate> To a solution of aniline (1.96 mL, 21.5 mmol, 1.0 equiv) in ethyl acetate (20 mL) was added O-ethylcarbonyl isothiocyanatidate (2.54 mL, 21.5 mmol, 1.0 equiv) and tetramethylethylenediamine (0.324 mL, 2.15 mmol, 0.1 equiv). The mixture was stirred at 25° C. for 4 h and then directly concentrated under reduced pressure. The residue was triturated with petroleum ether (8 ml) to give 4.20 g of ethyl N-(phenylcarbamothioyl)carbamate as a yellow oil. 1 H NMR (400 MHz, DMSO-d6) δ 11.55 (br. s, 1H), 11.25 (br. s, 1H), 7.61 -7.59 (m, 2H), 7.41 - 7.37 (m, 2H), 7.26 -7.22 (m, 1H), 4.21 (q, J = 7.2 Hz, 2H), 1.26 (t, J = 7.2 Hz, 3H).

[0140] <(Z)-Ethyl (((2-oxopropyl)thio)(phenylamino)methylene)carbamate> To a solution of N-(phenylcarbamothioyl)carbamate (4.20 g, 18.7 mmol, 1.0 equiv.) and cesium carbonate (10.4 g, 31.8 mmol, 1.7 equiv.) in acetonitrile (50 mL) was added 1-chloropropan-2-one (2.17 g, 23.4 mmol, 1.25 equiv.) while maintaining the temperature below 25° C. After addition, the mixture was stirred at 25° C. for 2 h, then diluted with MTBE (120 mL), washed with saturated aqueous sodium bicarbonate (100 ml), followed by brine (100 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=50:1→4:1) to give 1.80 g of (Z)-ethyl (((2-oxopropyl)thio)(phenylamino)methylene)carbamate as a brown oil. 1 H NMR (400 MHz, CDCl3) δ 7.46 -7.42 (m, 2H), 7.40 -7.36 (m, 1H), 7.28 - 7.27 (m, 1H), 7.27 - 7.26(m, 1H), 4.14 (q, J = 7.2.0 Hz, 2H), 3.47(d, J = 12.0 Hz, 1H), 3.65 (d, J = 12.0 Hz, 1H), 1.47 (s, 3H), 1.26 (t, J = 7.6 Hz, 4H). LCMS (m / z [M+H] + ): 280.9.

[0141] <(Z)-ethyl (4-methyl-3-phenylthiazol-2(3H)-ylidene)carbamate> To a solution of (Z)-ethyl (((2-oxopropyl)thio)(phenylamino)methylene)carbamate (0.600 g, 2.09 mmol, 1.0 equiv) in THF (6 mL) was added diisopropylethylamine (0.810 g, 6.27 mmol, 3.0 equiv) and thionyl chloride (0.167 mL, 2.30 mmol, 1.1 equiv) at 0° C. The mixture was stirred at 20° C. for 4 h and then concentrated under reduced pressure. The residue was diluted with saturated aqueous sodium bicarbonate (40 mL) and extracted with ethyl acetate (30 mL×3). The combined organic phase was washed with brine (60 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by reverse phase HPLC to give 0.32 g of (Z)-ethyl (4-methyl-3-phenylthiazol-2(3H)-ylidene)carbamate as a yellow oil. 1 H NMR (400 MHz, CDCl3) δ 7.68(s, 3H), 7.45 (s, 2H), 6.92(s, 1H), 4.34(q, J = 7.2 Hz, 2H), 2.08(s, 3H), 1.34 (t, J = 7.2 Hz, 3H). LCMS (m / z [M+H] + ): 263.3.

[0142] <Intermediate C15> To a solution of (Z)-ethyl (4-methyl-3-phenylthiazol-2(3H)-ylidene)carbamate (0.240 g, 0.891 mmol, 1.0 equiv) in ethanol (4 mL) was added sodium hydroxide (0.713 g, 17.8 mmol, 20 equiv) at 25° C. The mixture was heated to 60° C. and stirred for 1 h, then diluted with water (40 mL) and extracted with ethyl acetate (30 mL×3). The combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give 180 mg of 4-methyl-3-phenylthiazol-2(3H)-imine C15 as a yellow solid.

[0143] <Preparation of intermediate C16> [ka] <Ethyl N-(benzylcarbamothioyl)carbamate> To a solution of phenylmethanamine (5.00 g, 46.7 mmol, 1.0 equiv.) and O-ethylcarbonyl isothiocyanatidate (6.43 g, 49.0 mmol, 1.05 equiv.) in ethyl acetate (40 mL) was added TMEDA (0.542 g, 4.67 mmol, 1.0 equiv.) under nitrogen atmosphere at 25° C. The mixture was stirred at 25° C. for 6 h and then concentrated under reduced pressure. The residue was triturated twice from a solution of petroleum and ethyl acetate (v / v=10 / 1, 55 mL) to give 10.0 g of ethyl N-(benzylcarbamothioyl)carbamate as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 11.04 (br. s, 1H), 10.22 (br. t, J = 5.6 Hz, 1H), 7.39 - 7.23 (m, 5H), 4.81 (d, J = 5.6 Hz, 2H), 4.14 (q, J = 7.2 Hz, 2H), 1.21 (t, J = 7.2 Hz, 3H).

[0144] <(Z)-Ethyl ((benzylamino)((2-oxobutyl)thio)methylene)carbamate> To a suspension of ethyl N-(benzylcarbamothioyl)carbamate (3.00 g, 12.6 mmol, 1.0 equiv) and Cs2CO3 (6.97 g, 21.4 mmol, 1.7 equiv) in acetonitrile (45 mL) was added 1-bromobutan-2-one (2.38 g, 15.7 mmol, 1.25 equiv) while maintaining the temperature below 25° C. After addition, the mixture was stirred at 25° C. for 5 h, then poured into water (80 mL) and extracted with ethyl acetate (120 mL×3). The combined organic layers were washed with brine (100 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=1:0→5:1) to give 2.50 g of (Z)-ethyl ((benzylamino)((2-oxobutyl)thio)methylene)carbamate as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 7.32 - 7.27 (m, 3H), 7.25 - 7.22 (m, 2H), 4.96 (d, J = 15.6 Hz, 1H), 4.71 (d, J= 15.6 Hz, 1H), 4.18 (q, J = 7.2 Hz, 2H), 3.28 (d, J = 12.0 Hz, 1H), 3.11 (d, J = 12.0 Hz, 1H), 1.97 - 1.92(m, 1H) ,1.67 - 1.62 (m, 1H), 1.31 - 1.28 (m, 3H), 0.88 (t, J = 7.6 Hz, 3H ).LCMS (m / z [M+H] + ): 308.9.

[0145] <(Z)-ethyl (3-benzyl-4-ethylthiazol-2(3H)-ylidene)carbamate> To a solution of ethyl (Z)-ethyl ((benzylamino)((2-oxobutyl)thio)methylene)carbamate (2.50 g, 8.11 mmol, 1.0 equiv.) in THF (38 mL) was added diisopropylethylamine (3.14 g, 24.3 mmol, 3.0 equiv.) and SOCl2 (0.964 g, 8.11 mmol, 1.0 equiv.) at 0° C. The mixture was stirred at 25° C. for 16 h, then poured into water (80 mL) and extracted with ethyl acetate (100 mL×3). The combined organic layers were washed with brine (100 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give 2.20 g of (Z)-ethyl (3-benzyl-4-ethylthiazol-2(3H)-ylidene)carbamate as a dark brown oil. 1H NMR (400 MHz, DMSO-d6) δ 7.37 - 7.33 (m, 2H), 7.29 - 7.27 (m, 1H), 7.09 (d, J = 7.2 Hz, 2H), 6.61 (s, 1H), 5.36 - 5.34 (m, 2H), 4.07- 4.03 (m, 2H), 2.46 (dd, J = 2.0 Hz ,7.6 Hz , 2H), 1.17 (t, J = 7.2 Hz , 3H), 1.11 (t, J = 7.2 Hz , 3H). LCMS (m / z [M+H] + ): 291.1.

[0146] <Intermediate C16> To a solution of (Z)-ethyl (3-benzyl-4-ethylthiazol-2(3H)-ylidene)carbamate (1.00 g, 3.44 mmol, 1.0 equiv) in ethanol (10 mL) was added 6N NaOH (2.75 g, 68.9 mmol, 20 equiv) at 25° C. The mixture was heated to 50° C. and stirred for 1 h, then poured into water (80 mL) and extracted with ethyl acetate (100 mL×3). The combined organic layers were washed with brine (100 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=1:0→5:1) to give 0.49 g of 3-benzyl-4-ethylthiazol-2(3H)-imine C16 as a dark brown oil.

[0147] <Preparation of intermediate C17> [ka] (Z)-Ethyl ((benzylamino)((3-methyl-2-oxobutyl)thio)methylene)carbamate> To a mixture of ethyl N-(benzylcarbamothioyl)carbamate (0.500 g, 2.10 mmol, 1.0 equiv) and cesium carbonate (1.16 g, 3.57 mmol, 1.7 equiv) in acetonitrile (5 mL) was added 1-bromo-3-methylbutan-2-one (0.519 g, 3.15 mmol, 1.5 equiv) at 25° C. The mixture was stirred for 5 h, then poured into water (10 mL) and extracted with MTBE (100 mL×2). The combined organic phase was washed with brine (20 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give 0.67 g of crude (Z)-ethyl ((benzylamino)((3-methyl-2-oxobutyl)thio)methylene)carbamate as a yellow solid. LCMS (m / z [M+H] + ): 323.0.

[0148] <Ethyl (Z)-ethyl (3-benzyl-4-isopropylthiazol-2(3H)-ylidene)carbamate> To a solution of (Z)-ethyl ((benzylamino)((3-methyl-2-oxobutyl)thio)methylene)carbamate (0.670 g, 2.08 mmol, 1.0 equiv) and diisopropylethylamine (1.09 mL, 6.23 mmol, 3.0 equiv) in THF (10 mL) was added thionyl chloride (0.247 g, 2.08 mmol, 1.0 equiv). The mixture was stirred at 25° C. for 16 h, then poured into water (20 mL) and extracted with ethyl acetate (100 mL×3). The combined organic layers were washed with brine (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=100 / 1→4 / 1) to give 0.53 g of ethyl (Z)-ethyl (3-benzyl-4-isopropylthiazol-2(3H)-ylidene)carbamate as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 7.38 - 7.24 (m, 3H), 7.04(d, J=7.2 Hz, 2H), 6.70 (s, 1H), 5.39 (s, 2H), 4.04 (q, J = 7.2 Hz, 2H), 2.87 - 2.77 (m, 1H), 1.16 (t, J =7.2 Hz, 3H), 1.08 (d, J= 6.8 Hz, 6H). LCMS (m / z: 305.1[M+H] + ): 305.1.

[0149] <Intermediate C17> A mixture of ethyl (Z)-ethyl (3-benzyl-4-isopropylthiazol-2(3H)-ylidene)carbamate (0.300 g, 0.830 mmol) and sodium hydroxide (0.664 g, 16.61 mmol) in ethanol (5 mL) was stirred at 50° C. for 1 h. The mixture was poured into water (10 mL) and extracted with ethyl acetate (100 mL×3). The combined organic layers were washed with brine (10 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol=100 / 1→10 / 1) to give 150 mg of 3-benzyl-4-isopropylthiazol-2(3H)-imine C17 as a yellow solid.

[0150] <Preparation of intermediate C18> [ka] <(Z)-Ethyl((benzylamino)((2-cyclopropyl-2-oxoethyl)thio)methylene)carbamate> To a mixture of ethyl N-(benzylcarbamothioyl)carbamate (0.500 g, 2.10 mmol, 1.0 equiv.) and cesium carbonate (1.16 g, 3.57 mmol, 1.7 equiv.) in acetonitrile (5 mL) was added 2-bromo-1-cyclopropyl-ethanone (0.513 g, 3.15 mmol, 1.5 equiv.). The resulting mixture was stirred at 25° C. for 5 h, then poured into ice water (10 mL) and extracted with MTBE (100 mL×2). The combined organic layers were washed with brine (20 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give (Z)-ethyl ((benzylamino)((2-cyclopropyl-2-oxoethyl)thio)methylene)carbamate (0.670 g, crude) as a yellow solid. LCMS (m / z [M+H] + ): 321.1.

[0151] <Ethyl (Z)-ethyl (3-benzyl-4-cyclopropylthiazol-2(3H)-ylidene)carbamate> To a mixture of (Z)-ethyl((benzylamino)((2-cyclopropyl-2-oxoethyl)thio)methylene)carbamate (0.670 g, 2.08 mmol, 1.0 equiv) and diisopropylethylamine (0.810 g, 6.27 mmol, 3.0 equiv) in tetrahydrofuran (10 mL) was added thionyl chloride (0.248 g, 2.09 mmol, 1.2 equiv). The mixture was stirred at 25° C. for 16 h, then poured into ice water (20 mL) and extracted with ethyl acetate (100 mL×3). The combined organic layers were washed with brine (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=100 / 1→4 / 1) to give 0.25 g of ethyl (Z)-ethyl (3-benzyl-4-cyclopropylthiazol-2(3H)-ylidene)carbamate as a yellow gum. 1H NMR (400 MHz, DMSO-d6): δ 7.40 - 7.24 (m, 3H), 7.14 (d, J = 7.2Hz, 2H), 6.59 (s, 1H), 5.48(s, 2H), 4.03 (q, J = 7.2Hz, 2H), 1.69 - 1.60 (m, 1H), 1.17 (t, J = 7.2 Hz, 3H), 0.82 - 0.75 (m, 2H), 0.60 - 0.54 (m, 2H). LCMS (m / z [M+H] + ): 305.1.

[0152] <Intermediate C18> A mixture of ethyl (Z)-ethyl (3-benzyl-4-cyclopropylthiazol-2(3H)-ylidene)carbamate (0.200 g, 0.661 mmol, 1.0 equiv) and 6N sodium hydroxide (0.529 g, 13.23 mmol, 20 equiv) in ethanol (4 mL) was stirred at 50° C. for 1 h. The mixture was poured into ice water (10 mL) and extracted with ethyl acetate (100 mL×3). The combined organic layers were washed with brine (10 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give 160 mg of 3-benzyl-4-cyclopropylthiazol-2(3H)-imine C18 as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.57 (br. s, 1H), 7.36 - 7.30 (m, 2H), 7.27 - 7.20 (m, 3H), 5.64 (s, 1H), 5.04 (s, 2H), 1.48 - 1.36 (m, 1H), 0.72 - 0.64 (m, 2H), 0.52 - 0.42 (m, 2H). LCMS (m / z [M+H] + ): 233.2.

[0153] <Preparation of intermediate C19> [ka] <(Z)-Ethyl((benzylamino)((3,3,3-trifluoro-2-oxopropyl)thio)methylene)carbamate> To a suspension of ethyl N-(benzylcarbamothioyl)carbamate (0.500 g, 2.10 mmol, 1.0 equiv.) and cesium carbonate (1.16 g, 3.57 mmol, 1.7 equiv.) in acetonitrile (8 mL) was added 3-bromo-1,1,1-trifluoropropan-2-one (0.654 mL, 6.30 mmol, 3.0 equiv.) while maintaining the temperature below 25° C. After addition, the mixture was stirred at 25° C. for 12 h, then poured into water (40 mL) and extracted with MTBE (30 mL×3). The combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=50:1:→10:1) to give 0.75 g of (Z)-ethyl ((benzylamino)((3,3,3-trifluoro-2-oxopropyl)thio)methylene)carbamate as a yellow oil. 1 H NMR (400 MHz, CDCl3) δ 7.35 - 7.28 (m, 5H), 5.37 (d, J = 15.6 Hz,1H), 4.62 (d, J = 16.0 Hz, 1H), 4.27 - 4.18 (m, 2H), 3.79 (br. s, 1H), 3.69 (d, J = 12.8 Hz, 1H), 3.25 (dd, J = 12.0 Hz, 0.8 Hz, 1H), 1.35 - 1.31 (m, 3H). 19 F NMR (400 MHz, CDCl3) δ-79.04 (s, 3F). LCMS (m / z [M+H] + ): 348.9.

[0154] <(Z)-ethyl (3-benzyl-4-(trifluoromethyl)thiazol-2(3H)-ylidene)carbamate> To a solution of (Z)-ethyl ((benzylamino)((3,3,3-trifluoro-2-oxopropyl)thio)methylene)carbamate (0.550 g, 1.44 mmol, 1.0 equiv) in THF (20 mL) was added diisopropylethylamine (0.754 mL, 4.33 mmol, 3.0 equiv) and thionyl chloride (0.115 mL, 1.59 mmol, 1.1 equiv) at 0° C. The mixture was heated to 60° C. and stirred for 2 h, then added to saturated aqueous sodium bicarbonate (40 mL) and extracted with ethyl acetate (30 mL×3). The combined organic layers were washed with brine (60 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=50:1→10:1) to give 0.40 g of (Z)-ethyl (3-benzyl-4-(trifluoromethyl)thiazol-2(3H)-ylidene)carbamate as a brown oil. 1 H NMR (400 MHz, CDCl3): δ 7.34 - 7.28 (m, 3H), 7.18 - 7.13 (m, 3H), 5.48 (s, 2H), 4.26 (q, J= 7.2 Hz, 2H), 1.34 (t, J = 6.8 Hz, 3H). LCMS (m / z [M+H] + ): 330.9.

[0155] <Intermediate C19> To a solution of (Z)-ethyl (3-benzyl-4-(trifluoromethyl)thiazol-2(3H)-ylidene)carbamate (0.300 g, 0.884 mmol, 1.0 equiv) in ethanol (5 mL) was added sodium hydroxide (0.707 g, 17.7 mmol, 20 equiv) at 25° C. The mixture was heated to 60° C. and stirred for 1 h, then poured into water (40 mL) and extracted with ethyl acetate (30 mL×3). The combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=20:1) to give 150 mg of 3-benzyl-4-(trifluoromethyl)thiazol-2(3H)-imine C19 as a brown oil.

[0156] <Preparation of intermediate C20> [ka] <Ethyl N-[(3-cyanophenyl)carbamothioyl]carbamate> To a stirred mixture of 3-aminobenzonitrile (2.50 g, 21.16 mmol, 1.0 equiv) in EtOAc (25 mL) was added O-ethyl carboisothiocyanatidate (2.78 g, 21.16 mmol, 1.0 equiv) and TMEDA (246 mg, 2.12 mmol, 319 uL, 0.1 equiv) at 25° C. The reaction mixture was then stirred at 25° C. for 5 h under N2. The reaction mixture was concentrated under reduced pressure. EtOH (50 mL) was added to the mixture and the resulting mixture was stirred at 25° C. for 0.5 h. The mixture was filtered and the filter cake was dried under reduced pressure to give 2.80 g of ethyl N-[(3-cyanophenyl)carbamothioyl]carbamate as a yellow solid.

[0157] <(Z)-Ethyl(((3-cyanophenyl)amino)((2-oxoethyl)thio)methylene)carbamate> To a mixture of ethyl N-[(3-cyanophenyl)carbamothioyl]carbamate (2.80 g, 11.2 mmol, 1.0 equiv) and Cs2CO3 (6.22 g, 19.1 mmol, 1.7 equiv) in MeCN (40 mL) was added 2-chloroacetaldehyde (2.76 g, 14.0 mmol, 2.26 mL, 1.2 equiv) at 25° C. The reaction mixture was stirred at 25° C. for 12 h under N2. An additional 1.0 equiv of 2-chloroacetaldehyde was added and the reaction mixture was stirred at 25° C. for another 12 h. The reaction mixture was diluted with MTBE (100 mL) and washed with NaHCO3 solution (2×50 mL). The organic layer was washed with brine (50 mL), dried over Na2SO4, and filtered. The filtrate was concentrated under reduced pressure to give 2.80 g of (Z)-ethyl (((3-cyanophenyl)amino)((2-oxoethyl)thio)methylene)carbamate as a brown solid.

[0158] (Z)-Ethyl (3-(3-cyanophenyl)thiazol-2(3H)-ylidene)carbamate To a mixture of (Z)-ethyl (((3-cyanophenyl)amino)((2-oxoethyl)thio)methylene)carbamate (2.80 g, 9.61 mmol, 1.0 equiv) in THF (30 mL) was added DIPEA (3.73 g, 28.8 mmol, 5.02 mL, 3.0 equiv) and SOCl2 (1.14 g, 9.61 mmol, 697 μL, 1.0 equiv) at 0 °C. The reaction mixture was then stirred at 25 °C under N2 for 15 h. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in MTBE (200 mL) and the mixture was adjusted to pH = 8 with saturated aqueous NaHCO3 and the organic layer was discarded. The aqueous phase was extracted with EtOAc (3 x 100 mL), the organic layer was washed with brine (100 mL), dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure. To the mixture was added MTBE (10 mL) and the mixture was stirred for 0.5 h at 25° C. The mixture was then filtered and the filter cake was dried under reduced pressure to give 1.30 g of ethyl (Z)-ethyl (3-(3-cyanophenyl)thiazol-2(3H)-ylidene)carbamate as a brown solid. 1H NMR: (400 MHz, DMSO-d6) δ 8.12 (s, 1H), 7.99-7.91 (m, 2H), 7.79-7.74 (m, 1H), 7.61 (d, J = 4.8 Hz, 1H), 7.11 (d, J = 4.8 Hz, 1H), 4.09-3.98 (m, 2H), 1.17 (t, J = 7.1 Hz, 3H).

[0159] <Intermediate C20> To a mixture of (Z)-ethyl (3-(3-cyanophenyl)thiazol-2(3H)-ylidene)carbamate (400 mg, 1.46 mmol, 1.0 equiv) in EtOH (4 mL) was added NaOH (6 M, 4.00 mL, 16.4 equiv) at 25° C. The reaction mixture was then stirred at 25° C. for 12 h and at 60° C. for 2 h under N2. The reaction mixture was adjusted to pH=7 with concentrated HCl at 0° C., then the resulting solution was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC to give 130 mg of 3-(2-iminothiazol-3(2H)-yl)benzamide C20 as a brown solid.

[0160] <Preparation of intermediate C21> [ka] <tert‐ブチル7‐ニトロ‐3,4‐ジヒドロイソキノリン‐2(1H)‐カルボキシラート> To a solution of 7-nitro-1,2,3,4-tetrahydroisoquinoline (4.00 g, 22.4 mmol, 1.0 equiv) in DCM (40 mL) was added TEA (4.54 g, 44.9 mmol, 6.25 mL, 2.0 equiv) and di-tert-butyl dicarbonate (5.39 g, 24.7 mmol, 5.67 mL, 1.1 equiv) at 25° C. The reaction mixture was stirred at 25° C. for 2 h. The reaction mixture was diluted with water (100 mL) and extracted with EtOAc (4×200 mL). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (0-17% ethyl acetate / petroleum ether gradient eluent, 100 mL / min) to give 5.30 g of tert-butyl 7-nitro-3,4-dihydroisoquinoline-2(1H)-carboxylate as a yellow oil. 1 H NMR (400 MHz, DMSO-d6) δ 8.12 (s, 1H), 8.02 (dd, J = 1.9, 8.4 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 4.63 (br s, 2H), 3.64-3.52 (m, 2H), 2.97-2.80 (m, 2H), 1.43 (s, 9H).

[0161] <tert‐ブチル7‐アミノ‐3,4‐ジヒドロイソキノリン‐2(1H)‐カルボキシラート> To a solution of tert-butyl 7-nitro-3,4-dihydroisoquinoline-2(1H)-carboxylate (5.9 g, 21.2 mmol, 1.0 equiv) in MeOH (50 mL) was added Pd / C (2.5 g, 10% purity) under N2 at 25°C. The suspension was degassed under vacuum and purged with H2 three times. The reaction mixture was stirred under H2 (15 psi) at 25°C for 12 h. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give tert-butyl 7-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate (4.5 g, crude) as a red oil.

[0162] <tert‐ブチル7‐(3‐(エトキシカルボニル)チオウレイド)‐3,4‐ジヒドロイソキノリン‐2(1H)‐カルボキシラート> To a mixture of tert-butyl 7-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate (1.00 g, 4.03 mmol, 1.0 equiv) in EtOAc (10 mL) was added O-ethylcarbonisothiocyanatidate (528 mg, 4.03 mmol, 1.0 equiv) and TMEDA (47 mg, 0.40 mmol, 61 μL, 0.1 equiv) at 25° C. The reaction mixture was then stirred at 25° C. for 5 h under N2. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was dissolved in EtOH (10 mL) and the mixture was stirred at 25° C. for 0.5 h. The mixture was filtered and the filter cake was dried under reduced pressure to give 1.20 g of tert-butyl 7-(3-(ethoxycarbonyl)thioureido)-3,4-dihydroisoquinoline-2(1H)-carboxylate as a white solid. LCMS (m / z [M+Na] + ): 402.2.

[0163] (Z)-tert-Butyl 7-((((ethoxycarbonyl)imino)((2-oxoethyl)thio)methyl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate To a mixture of tert-butyl 7-(3-(ethoxycarbonyl)thioureido)-3,4-dihydroisoquinoline-2(1H)-carboxylate (1.80 g, 4.74 mmol, 1.0 equiv) in MeCN (20 mL) was added Cs2CO3 (2.63 g, 8.06 mmol, 1.7 equiv) and 2-chloroacetaldehyde (1.16 g, 5.93 mmol, 954 μL, 1.25 equiv) at 25° C. The reaction mixture was then stirred under N2 at 25° C. for 12 h. The reaction mixture was diluted with MTBE (100 mL) and then the mixture was washed with saturated aqueous NaHCO3 (2×50 mL). The organic layer was washed with brine (50 mL), dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to give (Z)-tert-butyl 7-((((ethoxycarbonyl)imino)((2-oxoethyl)thio)methyl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate (2.4 g, crude) as a brown oil.

[0164] <tert‐ブチル7‐[(2Z)‐2‐エトキシカルボニルイミノチアゾール‐3‐イル]‐3,4‐ジヒドロ‐1H‐イソキノリン‐2‐カルボキシラート> To a mixture of (Z)-tert-butyl 7-((((ethoxycarbonyl)imino)((2-oxoethyl)thio)methyl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate (2.40 g, 5.69 mmol, 1.0 equiv) in THF (30 mL) was added DIPEA (2.21 g, 17.1 mmol, 2.98 mL, 3.0 equiv) and SOCl2 (677 mg, 5.69 mmol, 413 μL, 1.0 equiv) at 0 °C. The reaction mixture was then stirred at 25 °C under N2 for 12 h. The reaction mixture was diluted with water (100 mL) and extracted with EtOAc (3 x 50 mL). The organic layer was washed with brine (100 mL), dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® silica flash column, elution with a 0-40% ethyl acetate / petroleum ether gradient at 75 mL / min) to give 1.0 g of (Z)-tert-butyl 7-((((ethoxycarbonyl)imino)((2-oxoethyl)thio)methyl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate as a brown oil.

[0165] <Intermediate C21> To a mixture of (Z)-tert-butyl 7-((((ethoxycarbonyl)imino)((2-oxoethyl)thio)methyl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate (800 mg, 1.98 mmol, 1.0 equiv) in EtOH (8 mL) was added NaOH (6 M, 8.00 mL, 24 equiv) at room temperature. The reaction mixture was then stirred at 60° C. for 2 h under N2. The reaction mixture was filtered, the filter cake was washed with EtOAc (3×20 mL), and the filtrate was then concentrated under reduced pressure. The residue was purified by reverse phase column to give 170 mg of tert-butyl 7-(2-iminothiazol-3(2H)-yl)-3,4-dihydroisoquinoline-2(1H)-carboxylate C21 as a yellow solid.

[0166] <Preparation of intermediate C22> [ka] <Ethyl N-[(2-bromophenyl)carbamothioyl]carbamate> To a solution of 2-bromoaniline (10.00 g, 58.1 mmol, 1.0 equiv) in EtOAc (100 mL) was added ethyl N-(thioxomethylene)carbamate (7.62 g, 58.1 mmol, 1.0 equiv) and TMEDA (675 mg, 5.81 mmol, 877 μL, 0.1 equiv) at room temperature. The mixture was stirred at 20° C. for 12 h. The reaction mixture was concentrated under reduced pressure. The residue was triturated with EtOH (50 mL) for 0.5 h. The mixture was filtered and the filter cake was washed with EtOH (2×30 mL). The filter cake was collected to give 16.0 g of ethyl N-[(2-bromophenyl)carbamothioyl]carbamate as a white solid.

[0167] <(Z)-Ethyl(((2-bromophenyl)amino)((2-oxoethyl)thio)methylene)carbamate> To a solution of ethyl N-[(2-bromophenyl)carbamothioyl]carbamate (16.0 g, 52.8 mmol, 1.0 equiv) in ACN (200 mL) was added Cs2CO3 (31.0 g, 95.0 mmol, 1.8 equiv) and 2-chloroacetaldehyde (20.7 g, 106 mmol, 17.0 mL, 2.0 equiv) at room temperature. The mixture was stirred at 20° C. for 12 h. The reaction mixture was quenched by addition of water (500 mL) and extracted with ethyl acetate (3×500 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give 24.0 g of (Z)-ethyl (((2-bromophenyl)amino)((2-oxoethyl)thio)methylene)carbamate as a pale yellow solid.

[0168] (Z)-Ethyl (3-(2-bromophenyl)thiazol-2(3H)-ylidene)carbamate To a solution of (Z)-ethyl (((2-bromophenyl)amino)((2-oxoethyl)thio)methylene)carbamate (24.0 g, 69.5 mmol, 1.0 equiv) in THF (240 mL) was added DIPEA (27.0 g, 209 mmol, 36.3 mL, 3.0 equiv) and SOCl2 (8.27 g, 69.5 mmol, 5.0 mL, 1.0 equiv) at 0 °C. The mixture was stirred at 15 °C for 4 h. The reaction mixture was quenched by the addition of saturated NaHCO3 solution (400 mL) and extracted with ethyl acetate (3 x 400 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=3 / 1→1 / 1) to give 13.43 g of (Z)-ethyl (3-(2-bromophenyl)thiazol-2(3H)-ylidene)carbamate. LCMS: (m / z [M+H + ]): 329.0.

[0169] <Intermediate C22> To a solution of (Z)-ethyl (3-(2-bromophenyl)thiazol-2(3H)-ylidene)carbamate (2.00 g, 6.11 mmol, 1.0 equiv) in EtOH (20 mL) was added 6N NaOH (4.89 g, 122 mmol, 20 equiv) at room temperature. The mixture was stirred at 50° C. under N2 for 1.5 h. The reaction mixture was quenched by addition of water (40 mL) and extracted with ethyl acetate (3×40 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give 1.56 g of 3-(2-bromophenyl)thiazol-2(3H)-imine C22 as a yellow oil. LCMS (m / z [M+H] + ): 255.1.

[0170] <Preparation of intermediate C23> [ka] <tert‐ブチル2‐アミノベンジルカルバマート> To a solution of 2-(aminomethyl)aniline (5.00 g, 40.9 mmol, 1.0 equiv) in DCM (50 mL) was added a solution of BOCO (8.93 g, 40.9 mmol, 9.40 mL, 1.0 equiv) in DCM (20 mL). The reaction mixture was stirred at 25° C. under N2 atmosphere for 12 h. The reaction mixture was extracted with H2O (25 mL). The combined organic layers were washed with brine (25 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give 8.36 g of tert-butyl 2-aminobenzylcarbamatenyl)methyl]carbamate as a pale yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.18 (s, 1H), 7.19-7.18 (m, 1H), 7.05-7.00 (m, 1H), 7.02 (dt, J = 1.3, 7.6 Hz, 1H), 6.95 (d, J = 7.3 Hz, 1H), 6.65-6.53 (m, 2H), 4.19-4.14 (m, 1H), 4.19-4.11 (m, 1H), 4.16 (d, J = 6.4 Hz, 2H), 1.36-1.36 (m, 1H), 1.37 (s, 9H). LCMS(m / z [M+H] + ): 166.9.

[0171] <Ethyl N-[[2-[(tert-butoxycarbonylamino)methyl]phenyl]carbamothioyl]carbamate> To a solution of tert-butyl 2-aminobenzylcarbamatenyl)methyl]carbamate (5.00 g, 22.5 mmol, 1.0 equiv) in EtOAc (50 mL) was added TMEDA (2.61 g, 22.5 mmol, 3.39 mL, 1.0 equiv) and ethyl N-(thioxomethylene)carbamate (4.43 g, 33.7 mmol, 1.5 equiv). The reaction mixture was stirred at 25° C. for 5 h. The mixture was directly concentrated under reduced pressure to give a yellow oil. This oil was dissolved in MTBE (20 mL) and stirred for 0.5 h, then filtered. The filter cake was washed with MTBE (2×10 mL) and dried in vacuum to give 7.75 g of ethyl N-[[2-[(tert-butoxycarbonylamino)methyl]phenyl]carbamothioyl]carbamate as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δ 11.18 (br s, 1H), 11.21-11.15 (m, 1H), 11.24-11.09 (m, 1H), 11.24-11.09 (m, 1H), 7.35-7.31 (m, 1H), 7.29-7.19 (m, 3H), 7.12-7.03 (m, 1H), 7.12-7.02 (m, 1H), 7.13-7.00 (m, 1H), 4.62 (s, 1H), 4.65-4.55 (m, 1H), 4.29-4.12 (m, 2H), 4.10-4.02 (m, 2H), 1.37 (s, 9H), 1.28-1.19 (m, 1H), 1.23 (td, J = 7.2, 16.0 Hz, 3H). LCMS (m / z [M+H] + ): 298.1.

[0172] <(Z)-Ethyl (((2-(((tert-butoxycarbonyl)amino)methyl)phenyl)amino)((2-oxoethyl)thio)methylene)carbamate> To a solution of ethyl N-[[2-[(tert-butoxycarbonylamino)methyl]phenyl]carbamothioyl]carbamate (3.70 g, 10.5 mmol, 1.0 equiv) in MeCN (50 mL) was added Cs2CO3 (5.80 g, 17.8 mmol, 1.7 equiv) followed by 2-chloroacetaldehyde (2.57 g, 13.1 mmol, 2.10 mL, 1.25 equiv) at 10° C. The reaction mixture was stirred at 20° C. for 12 h. The reaction mixture was filtered and concentrated under reduced pressure. The residue was diluted with water (50 mL) and extracted with EtOAc (3×20 mL). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=1 / 0→1 / 1) to give 3.0 g of (Z)-ethyl (((2-(((tert-butoxycarbonyl)amino)methyl)phenyl)amino)((2-oxoethyl)thio)methylene)carbamate as a yellow solid. LCMS (m / z [M+H] + ):396.1.

[0173] (Z)-Ethyl (3-(2-(((tert-butoxycarbonyl)amino)methyl)phenyl)thiazol-2(3H)-ylidenecarbamate To a solution of (Z)-ethyl(((2-(((tert-butoxycarbonyl)amino)methyl)phenyl)amino)((2-oxoethyl)thio)methylene)carbamate (2.00 g, 5.06 mmol, 1.0 equiv) in THF (40 mL) was added DIPEA (1.96 g, 15.2 mmol, 2.64 mL, 3.0 equiv) and SOCl2 (602 mg, 5.06 mmol, 367 μL, 1.0 equiv) at 0 °C. The reaction mixture was stirred at 15 °C under N2 atmosphere for 1 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was diluted with saturated aqueous NaHCO3 (25 mL) and extracted with MTBE (3 x 25 mL). The combined organic layers were washed with brine (25 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=1 / 0→2 / 1) to give 1.06 g of (Z)-ethyl (3-(2-(((tert-butoxycarbonyl)amino)methyl)phenyl)thiazol-2(3H)-ylidene)carbamate as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δ 7.55-7.47 (m, 1H), 7.42 (br s, 1H), 7.46-7.39 (m, 1H), 7.37 (d, J = 4.6 Hz, 1H), 7.28 (d, J = 7.7 Hz, 1H), 7.24 (br t, J = 5.6 Hz, 1H), 7.08 (d, J = 4.9 Hz, 1H), 4.07-3.93 (m, 2H), 3.88 (br d, J = 6.0 Hz, 2H), 1.40-1.21 (m, 9H), 1.20-1.09 (m, 3H).LCMS (m / z [M+H] + ): 378.3.

[0174] <Intermediate C23> To a solution of (Z)-ethyl (3-(2-(((tert-butoxycarbonyl)amino)methyl)phenyl)thiazol-2(3H)-ylidene)carbamate (340 mg, 901 μmol, 1.0 equiv) in EtOH (3.4 mL) was added NaOH (831 mg, 20.8 mmol, 23 equiv). The reaction mixture was stirred at 50° C. under N2 atmosphere for 1.5 h. The reaction mixture was diluted with water (20 mL) and extracted with EtOAc (3×20 mL). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC to give 133 mg of tert-butyl 2-(2-iminothiazol-3(2H)-yl)benzylcarbamate C23 as a pale yellow solid.

[0175] <Preparation of intermediate C24> [ka] <tert‐ブチル3‐アミノベンジルカルバマート> To a solution of 3-(aminomethyl)aniline (5.00 g, 40.9 mmol, 1.0 equiv) in MeCN (50 mL) was added dropwise a solution of Boc2O (8.13 g, 37.2 mmol, 8.56 mL, 0.9 equiv) in MeCN (50 mL) at 25° C. The reaction mixture was then stirred at 25° C. for 12 h under N2. The reaction mixture was diluted with ethyl acetate (100 mL) and filtered through a pad of silica gel, which was then washed with ethyl acetate (500 mL). The filtrate was concentrated under reduced pressure to give 9.4 g of tert-butyl 3-aminobenzylcarbamate as a yellow oil. 1 H NMR (400MHz, DMSO-d6) δ 7.23 (br t, J = 5.9 Hz, 1H), 6.95 (t, J = 7.6 Hz, 1H), 6.48-6.38(m, 3H), 4.99 (s, 2H), 4.00 (br d, J= 6.1 Hz, 2H), 1.42 (s, 9H).

[0176] <Ethyl N-[[3-[(tert-butoxycarbonylamino)methyl]phenyl]carbamothioyl]carbamate> To a solution of tert-butyl 3-aminobenzylcarbamate (5.00 g, 22.5 mmol, 1.0 equiv) in EtOAc (50 mL) was added ethyl O-ethylcarbonylisothiocyanatidate (2.95 g, 22.5 mmol, 1.0 equiv) and TMEDA (261 mg, 2.25 mmol, 339 μL, 0.1 equiv) at 25° C. The reaction mixture was stirred at 25° C. for 12 h under N2. The reaction mixture was concentrated under reduced pressure. The residue was diluted with MTBE (50 mL) and stirred at 25° C. for 0.5 h, which was filtered to recover 4.3 g of ethyl N-[[3-[(tert-butoxycarbonylamino)methyl]phenyl]carbamothioyl]carbamate as a white solid. 1 H NMR (400MHz, DMSO-d6) δ 11.55 (s, 1H), 11.22 (br s, 1H), 7.53 (br d, J = 7.8 Hz, 1H), 7.46-7.38 (m, 2H), 7.33 (t, J = 7.8 Hz, 1H), 7.11 (d, J= 7.6 Hz, 1H), 4.22 (q, J = 7.1 Hz, 2H), 4.13 (br d, J = 6.1 Hz, 2H), 1.40 (s, 9H), 1.27 (t, J = 7.1 Hz, 3H).

[0177] <(Z)-Ethyl (((3-(((tert-butoxycarbonyl)amino)methyl)phenyl)amino)((2-oxoethyl)thio)methylene)carbamate> To a solution of ethyl N-[[3-[(tert-butoxycarbonylamino)methyl]phenyl]carbamothioyl]carbamate (4.30 g, 12.2 mmol, 1.0 equiv) and Cs2CO3 (6.74 g, 20.7 mmol, 1.7 equiv) in MeCN (44 mL) was added 2-chloroacetaldehyde (2.98 g, 15.2 mmol, 2.45 mL, 1.25 equiv) while maintaining the temperature below 25° C. After addition, the reaction mixture was stirred at 25° C. under N2 for 12 h. The reaction mixture was diluted with MTBE (80 mL), washed with saturated aqueous NaHCO3 (75 ml), brine (75 mL), dried over Na2SO4, and filtered. The filtrate was concentrated under reduced pressure to give 4.7 g of (Z)-ethyl (((3-(((tert-butoxycarbonyl)amino)methyl)phenyl)amino)((2-oxoethyl)thio)methylene)carbamate as a brown solid.

[0178] <(Z)-ethyl (3-(3-(((tert-butoxycarbonyl)amino)methyl)phenyl)thiazol-2(3H)-ylidene)carbamate> To a solution of (Z)-ethyl(((3-(((tert-butoxycarbonyl)amino)methyl)phenyl)amino)((2-oxoethyl)thio)methylene)carbamate (4.70 g, 11.9 mmol, 1.0 equiv) in THF (47 mL) was added DIPEA (4.61 g, 35.6 mmol, 6.21 mL, 3.0 equiv) and SOCl2 (1.41 g, 11.9 mmol, 862 μL, 1.0 equiv) at 0 °C. The reaction mixture was stirred at 25 °C under N2 for 1 h. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in EtOAc (20 mL), diluted with saturated aqueous NaHCO3 (60 mL) and extracted with EtOAc (3 x 80 mL). The combined organic layers were washed with brine (80 mL), dried over Na2SO4 and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (eluting with a 0-40% ethyl acetate / petroleum ether gradient at 75 mL / min) to give 2.4 g of (Z)-ethyl (3-(3-(((tert-butoxycarbonyl)amino)methyl)phenyl)thiazol-2(3H)-ylidene)carbamate as a white solid. 1 H NMR (400MHz, DMSO-d6) δ 7.53-7.47 (m, 2H), 7.41-7.33 (m, 3H), 7.08 (d, J = 4.8 Hz, 1H), 4.21 (br d, J = 6.1 Hz, 2H), 4.06-4.00 (m, 2H), 1.40(s, 9H), 1.16 (t, J= 7.1 Hz, 3H).

[0179] <Intermediate C24> To a solution of (Z)-ethyl (3-(3-(((tert-butoxycarbonyl)amino)methyl)phenyl)thiazol-2(3H)-ylidene)carbamate (1.00 g, 2.65 mmol, 1.0 equiv) in EtOH (10 mL) was added 6N NaOH (2.40 g, 60.0 mmol, 23.0 equiv) at room temperature. The reaction mixture was stirred at 60° C. under N2 for 5 h. The reaction mixture was adjusted to pH 7 with aqueous HCl (6 M). The neutralized solution was then filtered and the filter cake was washed with EtOH (200 mL). The filtrate was concentrated under reduced pressure. The crude product was purified by reverse phase column to give 300 mg of tert-butyl 3-(2-iminothiazol-3(2H)-yl)benzylcarbamate C24 as a yellow solid.

[0180] <Preparation of intermediate C25> [ka] <Ethyl N-[(4-bromophenyl)carbamothioyl]carbamate> To a solution of 4-bromoaniline (5.00 g, 29.1 mmol, 1.0 equiv) in EtOAc (50 mL) was added ethyl N-(thioxomethylene)carbamate (3.81 g, 29.1 mmol, 1.0 equiv) and TMEDA (338 mg, 2.91 mmol, 439 μL, 0.1 equiv) at room temperature. The reaction mixture was stirred at 20° C. for 12 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was triturated with EtOH (25 mL) for 0.5 h. The mixture was filtered and the filter cake was washed with EtOH (2×25 mL) and dried to give 7.5 g of ethyl N-[(4-bromophenyl)carbamothioyl]carbamate as a white solid.

[0181] <(Z)-Ethyl (((4-bromophenyl)amino)((2-oxoethyl)thio)methylene)carbamate> To a solution of ethyl N-[(4-bromophenyl)carbamothioyl]carbamate (7.50 g, 24.7 mmol, 1.0 equiv) in CH3CN (100 mL) was added Cs2CO3 (13.70 g, 42.0 mmol, 1.7 equiv) and 2-chloroacetaldehyde (6.07 g, 30.9 mmol, 4.97 mL, 1.25 equiv) at room temperature. The reaction mixture was stirred at 20° C. under N2 for 12 h. Cs2CO3 (8.06 g, 24.7 mmol, 1.0 equiv) and 2-chloroacetaldehyde (3.64 g, 18.6 mmol, 2.98 mL, 0.75 equiv) were added to the reaction solution. The reaction mixture was stirred at 20° C. under N2 for 4 h. The reaction mixture was quenched by the addition of water (300 mL) and extracted with ethyl acetate (3 x 300 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give 10 g of (Z)-ethyl (((4-bromophenyl)amino)((2-oxoethyl)thio)methylene)carbamate as a pale yellow solid. LCMS (m / z[M+H] + ): 345.0.

[0182] (Z)-Ethyl (3-(4-bromophenyl)thiazol-2(3H)-ylidene)carbamate To a solution of (Z)-ethyl (((4-bromophenyl)amino)((2-oxoethyl)thio)methylene)carbamate (10.00 g, 28.97 mmol, 1.0 equiv) in THF (120 mL) was added DIPEA (11.23 g, 86.90 mmol, 15.14 mL, 3.0 equiv) and SOCl2 (3.45 g, 28.97 mmol, 2.10 mL, 1.0 equiv) at 0 °C. The reaction mixture was stirred at 15 °C for 4 h. The reaction mixture was quenched by the addition of saturated NaHCO3 solution (200 mL) and extracted with ethyl acetate (3 x 200 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=3 / 1→1 / 1) to obtain 3.60 g of (Z)-ethyl (3-(4-bromophenyl)thiazol-2(3H)-ylidene)carbamate compound as a yellow solid.1 H NMR (400 MHz, DMSO-d6) δ 7.75 (d, J=8.63 Hz, 2H), 7.47-7.56 (m, 3H), 7.08 (d, J=4.88 Hz, 1H), 4.03 (q, J=7.09 Hz, 2H), 1.16 (t, J=7.07 Hz, 3H).

[0183] <Intermediate C25> To a solution of (Z)-ethyl (3-(4-bromophenyl)thiazol-2(3H)-ylidene)carbamate (1.00 g, 3.06 mmol, 1.0 equiv) in EtOH (10 mL) was added 6N NaOH (2.44 g, 61.1 mmol, 20 equiv). The reaction mixture was stirred under N2 at 50°C for 1.5 hours. The reaction was combined with a previous batch (100 mg starting material was used). The reaction mixture was quenched by addition of water (100 mL) and extracted with ethyl acetate (3 x 100 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give 750 mg of 3-(4-bromophenyl)thiazol-2(3H)-imine C25 as a yellow oil.

[0184] <Preparation of intermediate C26> [ka] <Ethyl N-((cyclohexylmethyl)carbamothioyl)carbamate> To a solution of cyclohexylmethanamine (2.00 g, 17.7 mmol, 1.0 equiv.) in ethyl acetate (20 mL) was added O-ethylcarbonyl isothiocyanatidate (2.09 mL, 17.7 mmol, 1.0 equiv.) and tetramethylethylenediamine (0.267 mL, 1.77 mmol, 0.1 equiv.) at 25° C. The mixture was then stirred at this temperature for 5 h. The mixture was concentrated under reduced pressure and the residue was triturated twice with ethanol (10 mL) to give 3.50 g of ethyl N-((cyclohexylmethyl)carbamothioyl)carbamate as an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ 10.94 (br. s, 1H), 9.92 (br. t, J = 5.2 Hz, 1H), 4.14 (q, J = 7.2 Hz, 2H), 3.42 - 3.39 (m, 2H), 2.83 (s, 1H), 1.69 - 1.60 (m, 6H), 1.22 - 1.10 (m, 5H), 0.98 - 0.90 (m, 2H). LCMS (m / z [M+H] + ): 244.9.

[0185] <(Z)-ethyl (3-(cyclohexylmethyl)-4-methylthiazol-2(3H)-ylidene)carbamate> To a solution of ethyl N-((cyclohexylmethyl)carbamothioyl)carbamate (1.00 g, 4.09 mmol, 1.0 equiv.) and cesium carbonate (2.27 g, 6.96 mmol, 1.7 equiv.) in acetonitrile (10 mL) was added 1-chloropropan-2-one (0.568 g, 6.14 mmol, 1.5 equiv.) dropwise at 25° C. After addition, the mixture was stirred at this temperature for 2 h. The mixture was poured into water (30 mL) and extracted with ethyl acetate (50 mL×3). The combined organic layers were washed with brine (25 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=10 / 1→5 / 1) to give 0.80 g of (Z)-ethyl (3-(cyclohexylmethyl)-4-methylthiazol-2(3H)-ylidene)carbamate as a yellow oil. 1 H NMR (400 MHz, DMSO-d6) δ 6.44 (s, 1H), 4.01 - 3.99 (m, 2H), 3.22 - 3.09 (m, 2H), 3.08 - 3.05 (m, 1H), 1.65 - 1.61 (m, 6H), 1.48 (s, 3H), 1.15 - 1.14 (m, 3H), 1.12 - 1.10 (m, 2H), 0.90 - 0.88 (m, 2H). LCMS (m / z [M+H] + ): 282.9.

[0186] <Intermediate C26> To a solution of (Z)-ethyl (3-(cyclohexylmethyl)-4-methylthiazol-2(3H)-ylidene)carbamate (0.350 g, 1.08 mmol, 1.0 equiv) in ethanol (3 mL) was added 6N sodium hydroxide (0.862 g, 21.5 mmol, 20 equiv) in one portion at 25° C. The reaction mixture was heated to 50° C. and stirred for 1 h. The mixture was added into water (15 mL) and extracted with ethyl acetate (30 mL×3). The combined organic phase was washed with brine (25 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=10 / 1→0 / 1) to give 150 mg of 3-(cyclohexylmethyl)-4-methylthiazol-2(3H)-imine C26 as a yellow oil.

[0187] <Preparation of intermediate C27> [ka] <Ethyl N-(cyclohexylcarbamothioyl)carbamate> To a mixture of cyclohexanamine (0.800 g, 8.07 mmol, 1.0 equiv.) and O-ethylcarbonyl isothiocyanatidate (1.11 g, 8.47 mmol, 1.05 equiv.) in ethyl acetate (10 mL) was added TMEDA (93 mg, 0.806 mmol, 1.0 equiv.) at 25° C. The mixture was stirred at this temperature for 6 h and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate=100 / 1→3 / 1) to give 1.80 g of ethyl N-(cyclohexylcarbamothioyl)carbamate as a yellow gum. 1H NMR (400 MHz, CD3Cl) δ 9.64 (br. s, 1H), 7.96 (br. s, 1H), 4.29 - 4.22 (m, 2H), 2.09 - 2.01 (m, 2H), 1.77 - 1.68 (m, 2H), 1.62-1.63 (m, 1H), 1.49 - 1.33 (m, 4H), 1.33 - 1.19 (m, 5H). LCMS (m / z [M+H] + ): 231.1.

[0188] <(Z)-Ethyl((cyclohexylamino)((2-oxopropyl)thio)methylene)carbamate> To a mixture of ethyl N-(cyclohexylcarbamothioyl)carbamate (0.800 g, 3.47 mmol, 1.0 equiv.) and cesium carbonate (1.92 g, 5.90 mmol, 1.7 equiv.) in acetonitrile (10 mL) was added 1-chloropropan-2-one (0.353 g, 3.82 mmol, 1.1 equiv.) at 25° C. The resulting mixture was stirred at this temperature for 4 h, then poured into ice water (10 mL) and extracted with MTBE (100 mL×2). The combined organic phase was washed with brine (20 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give 900 mg of crude (Z)-ethyl ((cyclohexylamino)((2-oxopropyl)thio)methylene)carbamate as a yellow solid, which was used directly in the next step without purification. LCMS (m / z [M+H] + ): 287.1.

[0189] <(Z)-ethyl(3-cyclohexyl-4-methylthiazol-2(3H)-ylidene)carbamate> To a mixture of (Z)-ethyl((cyclohexylamino)((2-oxopropyl)thio)methylene)carbamate (0.900 g, 3.14 mmol, 1.0 equiv) and diisopropylethylamine (0.812 g, 6.29 mmol, 2.2 equiv) in tetrahydrofuran (20 mL) was added thionyl chloride (0.224 g, 1.89 mmol, 0.6 equiv) at 0° C. The mixture was stirred at 25° C. for 2 h, then poured into ice water (20 mL) and extracted with ethyl acetate (100 mL×3). The combined organic phase was washed with brine (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=100 / 1→4 / 1) to give 0.50 g of (Z)-ethyl (3-cyclohexyl-4-methylthiazol-2(3H)-ylidene)carbamate as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 6.54 (br. s, 1H), 4.09-4.03 (m, 2H), 2.29 - 2.25 (m, 3H), 1.82 - 1.62 (m, 6H), 1.36 - 1.23 (m, 3H), 1.21 - 1.19 (m, 5H).LCMS (m / z [M+H] + ): 268.9.

[0190] <Intermediate C27> To a mixture of (Z)-ethyl (3-cyclohexyl-4-methylthiazol-2(3H)-ylidene)carbamate (0.200 g, 0.745 mmol, 1.0 equiv) in ethanol (4 mL) was added NaOH (0.596 g, 14.90 mmol, 20 equiv) at 25° C. The mixture was heated to 50° C. and stirred for 1 h, then poured into ice water (50 mL) and extracted with ethyl acetate (50 mL×5). The combined organic phase was washed with brine (10×2 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give 120 mg of 3-cyclohexyl-4-methylthiazol-2(3H)-imine C27 as a yellow solid.

[0191] <Preparation of intermediate C28> [ka] <Ethyl N-[(2-chlorophenyl)carbamothioyl]carbamate> To a solution of 2-chloroaniline (8.26 mL, 78.4 mmol) in ethyl acetate (100 mL) was slowly added O-ethylcarbonisothiocyanatidate (10.3 g, 78.4 mmol) and tetramethylethylenediamine (1.18 mL, 7.84 mmol) at 0° C., and then the mixture was stirred at 50° C. for 5 h. The reaction mixture was cooled to room temperature and quenched by dropwise addition of 100 mL of methanol. The precipitated solid was collected by filtration and then triturated with methanol (100 mL) at 25° C. for 10 min to give 16.5 g of ethyl N-[(2-chlorophenyl)carbamothioyl]carbamate as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 11.60 (br. s, 1H), 11.47 (br. s, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.57 - 7.55 (m, 1H), 7.41 - 7.36 (m, 1H), 7.33 - 7.29 (m, 1H), 4.23 (q, J = 7.2 Hz, 2H), 1.27 (t, J = 7.2 Hz, 3H). LCMS (m / z [M+H] + ): 258.9.

[0192] <(Z)-Methyl 3-((N-(2-chlorophenyl)-N'-(ethoxycarbonyl)carbamimidoyl)thio)-2-oxopropanoate> To a solution of ethyl N-[(2-chlorophenyl)carbamothioyl]carbamate (5.00 g, 19.3 mmol) and cesium carbonate (7.56 g, 23.2 mmol) in acetonitrile (100 mL) was added methyl 3-bromo-2-oxopropanoate (2.67 mL, 25.1 mmol) at 25° C., and the mixture was stirred at 25° C. for 2 h. The reaction mixture was quenched with water (100 mL) at 25° C. and extracted with ethyl acetate (200 mL×3). The combined organic layers were washed with brine (200 mL×2), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate=10 / 1-2 / 1) to give 2.80 g of (Z)-methyl 3-((N-(2-chlorophenyl)-N'-(ethoxycarbonyl)carbamimidoyl)thio)-2-oxopropanoate as a brown oil. 1 H NMR (CDCl3, 400 MHz) δ 7.53 - 7.49 (m, 1H), 7.47 - 7.44 (m, 1H), 7.35 - 7.31 (m, 2H), 4.90 (br. s, 1H), 4.16 (q, J = 7.2 Hz, 2H), 4.00 (d, J = 11.6 Hz, 1H), 3.75 (s, 3H), 3.47 - 3.42 (m, 1H), 1.26 (t, J = 7.2 Hz, 3H). LCMS (m / z [M+H] + ): 358.9.

[0193] (Z)-Methyl 3-(2-chlorophenyl)-2-((ethoxycarbonyl)imino)-2,3-dihydrothiazole-4-carboxylate To a mixture of (Z)-methyl 3-((N-(2-chlorophenyl)-N'-(ethoxycarbonyl)carbamimidoyl)thio)-2-oxopropanoate (2.80 g, 7.80 mmol) and DIPEA (2.04 mL, 11.7 mmol) in THF (15 mL) was added thionyl chloride (0.85 mL, 11.7 mmol) at 0°C. The mixture was stirred at 25°C under nitrogen atmosphere for 2 h. The reaction mixture was quenched with water (100 mL) at 25°C and extracted with ethyl acetate (200 mL x 3). The combined organic layer was washed with brine (200 mL x 2), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure to give 2.20 g of (Z)-methyl 3-(2-chlorophenyl)-2-((ethoxycarbonyl)imino)-2,3-dihydrothiazole-4-carboxylate as a yellow oil, which was used directly in the next step without further purification. 1 H NMR (CDCl3, 400 MHz) δ 7.64 (s, 1H), 7.54 - 7.51 (m, 1H), 7.46 - 7.41 (m, 2H), 7.40 - 7.37 (m, 1H), 4.20 (q, J = 7.2 Hz, 2H), 3.74 (s, 3H), 1.29 (t, J = 7.2 Hz, 3H). LCMS (m / z [M+H] + ): 340.9.

[0194] (Z)-Ethyl (3-(2-chlorophenyl)-4-(methylcarbamoyl)thiazol-2(3H)-ylidene)carbamate To a mixture of (Z)-methyl 3-(2-chlorophenyl)-2-((ethoxycarbonyl)imino)-2,3-dihydrothiazole-4-carboxylate (1.5 g, 4.40 mmol) in THF (15 mL) was added methylamine (2 M, 22 mL) dropwise and 3,4,6,7,8,9-hexahydro-2H-pyrimido[1,2-a]pyrimidine (0.306 g, 2.20 mmol) was added at 25° C. The mixture was stirred at 60° C. under nitrogen atmosphere for 2 h, then poured slowly into ice water (50 mL), followed by extraction with ethyl acetate (100 mL×3). The combined organic layer was washed with brine (200 mL×2), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure to give 1.55 g of (Z)-ethyl (3-(2-chlorophenyl)-4-(methylcarbamoyl)thiazol-2(3H)-ylidene)carbamate as a yellow solid. 1 H NMR (DMSO-d6, 400 MHz) δ8.63 (br. d, J = 4.8 Hz, 1H), 7.59 (d, J = 7.6 Hz, 1H), 7.54 (s, 1H), 7.49 - 7.43 (m, 3H), 4.02 (q, J = 7.2 Hz, 2H), 2.57 (d, J = 4.8 Hz, 3H), 1.19 - 1.04 (t, J = 7.2 Hz, 3H). LCMS (m / z [M+H] + ): 339.9.

[0195] <Intermediate C28> To a solution of (Z)-ethyl (3-(2-chlorophenyl)-4-(methylcarbamoyl)thiazol-2(3H)-ylidene)carbamate (0.360 g, 1.06 mmol) in DMF (7 mL) was added bromotrimethylsilane (0.687 mL, 5.30 mmol) at 25 °C. The reaction mixture was stirred at 80 °C for 1 h and then poured slowly into ice water (20 mL). The mixture was neutralized with saturated aqueous sodium bicarbonate. The mixture was directly purified by reverse phase flash (0.1% NH3·H2O / MeCN / water) to give 80 mg of 3-(2-chlorophenyl)-2-imino-N-methyl-2,3-dihydrothiazole-4-carboxamide C28 as a yellow solid.

[0196] <Preparation of intermediate C29> [ka] <Preparation of intermediate C29> <Ethyl (Z)-(3-(2-chlorophenyl)-4-(dimethylcarbamoyl)thiazol-2(3H)-ylidene)carbamate> To a mixture of methyl (Z)-3-(2-chlorophenyl)-2-((ethoxycarbonyl)imino)-2,3-dihydrothiazole-4-carboxylate (0.380 g, 1.12 mmol) and dimethylamine (2M, 5.6 mL) in THF (1 mL) was added 3,4,6,7,8,9-hexahydro-2H-pyrimido[1,2-a]pyrimidine (0.078 g, 0.560 mmol) in one portion at 25° C. under nitrogen atmosphere. The reaction mixture was stirred at 60° C. for 2 h and then carefully poured into 100 mL of ice water. The aqueous phase was extracted with ethyl acetate (50 mL×3) and then washed with brine (10 mL). The organic layer was dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure to a residue which was purified by reverse phase flash (0.1% trifluoroacetic acid / MeCN / water) to give 140 mg of ethyl (Z)-[3-(2-chlorophenyl)-4-(dimethylcarbamoyl)thiazol-2-ylidene]carbamate as a yellow solid. 1H NMR (DMSO-d6, 400 MHz) δ 7.63 - 7.61 (m, 1H), 7.52 - 7.43 (m, 3H), 7.36 (s, 1H), 4.04 (m, 2H), 3.11 (s, 3H), 2.76 (s, 3H), 1.14 (t, J = 7.2 Hz, 3H). LCMS (m / z[M+H] + ): 353.9.

[0197] <Intermediate C29> To a solution of ethyl (Z)-[3-(2-chlorophenyl)-4-(dimethylcarbamoyl)thiazol-2-ylidene]carbamate (0.390 g, 1.10 mmol) in ethyl alcohol (4 mL) was added solid sodium hydroxide (0.960 g, 24.0 mmol). The mixture was stirred at 50° C. for 2 h. The crude product was directly purified by reverse phase flash (0.1% ammonium hydroxide / MeCN / water) without workup to give 90 mg of 3-(2-chlorophenyl)-2-imino-N,N-dimethyl-thiazole-4-carboxamide C29 as a yellow solid.

[0198] <Preparation of intermediate D1> [ka] <2-Phenylacetyl chloride> To a solution of 2-phenylacetic acid (5.00 g, 36.7 mmol, 4.63 mL, 1.0 equiv) in DCM (50 mL) was added DMF (268 mg, 3.67 mmol, 283 μL, 0.1 equiv), and then SOCl2 (8.74 g, 73.4 mmol, 5.33 mL, 2.0 equiv) was added to the mixture at 0° C. The reaction mixture was stirred at 25° C. for 1 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. Compound 2-phenylacetyl chloride (5.68 g, crude) was obtained as a colorless oil.

[0199] <1-Diazo-3-phenylpropan-2-one> To a solution of 2-phenylacetyl chloride (5.68 g, 36.7 mmol, 4.90 mL, 1.0 equiv) in MeCN (50 mL) was added TMSCHN2 (2 M, 36.74 mL, 2 equiv) at 0° C. The reaction mixture was then stirred at 25° C. for 2 h. The resulting yellow solution was used directly in the next step without further purification.

[0200] <1-Bromo-3-phenylpropan-2-one> To a solution of 1-diazo-3-phenylpropan-2-one in MeCN was added HBr (13.5 g, 55.1 mmol, 9.06 mL, 1.5 equiv) (33% in AcOH). The mixture was stirred at 25° C. for 12 h. The mixture was poured into water (50 mL) and extracted with EtOAc (3×30 mL). The organic phase was washed with brine (50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=0 / 1→6 / 1) to give 2.30 g of 1-bromo-3-phenylpropan-2-one as a red oil.

[0201] <Intermediate D1> To a solution of thiourea (715 mg, 9.39 mmol, 1.0 equiv) in refluxing EtOH (20 mL) was added 1-bromo-3-phenylpropan-2-one (2.00 g, 9.39 mmol, 1.0 equiv) and pyridine (742 mg, 9.39 mmol, 758 μL, 1.0 equiv). The reaction mixture was stirred at 70° C. for 4 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=50 / 1→1 / 1) to give 0.6 g of crude product. 490 mg of crude product was purified by preparative HPLC to give 160 mg of 4-benzylthiazol-2(3H)-imine D1 as a white solid. 1 H NMR: (400 MHz, DMSO-d6) δ 7.31-7.12 (m, 5H), 6.83 (s, 2H), 6.12 (s, 1H), 3.71 (s, 2H). The structure was confirmed by HMBC and HSQC.

[0202] <Preparation of intermediate D3> [ka] To a solution of benzylthiourea (1.00 g, 6.02 mmol, 1.0 equiv) in acetone (15 mL) was added HCl (1.20 g, 12.0 mmol, 1.18 mL, 36% purity, 2.0 equiv) and DMSO (939 mg, 12.0 mmol, 939 μL, 2.0 equiv) at 40° C. The reaction mixture was stirred for 48 h. The reaction mixture was directly concentrated under reduced pressure. The residue was purified by reverse phase MPLC to give 0.80 g of 3-benzyl-4-methylthiazol-2(3H)-imine D3 as a white solid.

[0203] <Preparation of Acid D> [ka] 4-Methyl-1H-pyrrolo[2,3-b]pyridine To a solution of 4-chloro-1H-pyrrolo[2,3-b]pyridine (10.00 g, 65.54 mmol, 1.0 equiv) and Pd(dppf)Cl2 (1.20 g, 1.64 mmol, 0.025 equiv) in toluene (200 mL) was added MeMgBr (3 M, 109 mL, 5.0 equiv) dropwise at 25 °C. After the addition, the reaction mixture was stirred at 80 °C for 12 h, then cooled to 25 °C, quenched by the addition of ice water (300 mL) at 0 °C, and extracted with EtOAc (2 x 200 mL). The combined organic layers were washed with saturated aqueous NH4Cl (400 mL), brine (200 mL), dried over Na2SO4, and filtered. The filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash chromatography (0-100% ethyl acetate / petroleum ether gradient eluted at 100 mL / min) to give 7.00 g of 4-methyl-1H-pyrrolo[2,3-b]pyridine as a white solid. LCMS (m / z [M+H] + ): 133.1.

[0204] 2,2,2-trichloro-1-(4-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)ethanone To a solution of 4-methyl-1H-pyrrolo[2,3-b]pyridine (1.00 g, 7.57 mmol, 1.0 equiv) in DCM (20 mL) was added AlCl3 (2.52 g, 18.9 mmol, 2.5 equiv) at 25° C. The reaction mixture was stirred at 25° C. for 10 min, then 2,2,2-trichloroacetyl chloride (2.06 g, 11.4 mmol, 1.5 equiv) was added. The reaction mixture was stirred at 25° C. for another 3 h 50 min. The mixture was poured into ice water (30 mL) and the precipitate was filtered. The filtrate was extracted with DCM (2×30 mL) and concentrated under reduced pressure to give 2.0 g of crude 2,2,2-trichloro-1-(4-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)ethanone as a pale yellow solid.

[0205] 4-Methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxylic acid (acid D) A mixture of 2,2,2-trichloro-1-(4-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)ethanone (2.00 g, 7.21 mmol, 1.0 equiv) in aqueous NaOH (3 M, 30 mL, 12.5 equiv) was stirred at 25° C. for 3 h under N2 atmosphere. The mixture was cooled to 0° C. and adjusted to pH<6.0 by addition of concentrated aqueous HCl. The precipitate was collected by filtration, and the filter cake was washed with H2O (2×5 mL) and dried under vacuum to give 1.2 g of crude 4-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxylic acid (acid D) as an off-white solid.

[0206] <Preparation of Example T01> [ka] To a mixture of 3-benzylthiazol-2(3H)-imine B1 (1.20 g, 6.31 mmol, 1.0 equiv.), 1H-pyrrolo[2,3-b]pyridine-3-carboxylic acid A (0.818 g, 5.05 mmol, 0.8 equiv.), HOBt (1.28 g, 9.46 mmol, 1.5 equiv.) and diisopropylethylamine (2.45 g, 18.9 mmol, 3.0 equiv.) in dimethylformamide (10 mL) was added EDCI (1.81 g, 9.46 mmol, 1.5 equiv.). The mixture was stirred at 25° C. for 1.5 h and then poured into ice water (200 mL). The precipitate was collected by filtration and the filter cake was washed with acetonitrile (3×30 mL) and then triturated with acetonitrile / methanol=5:1 (5×30 mL), followed by reverse phase HPLC and recrystallization from methanol (2×250 mL) to give 352 mg of (Z)-N-(3-benzylthiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T01 as a white solid. 1 H-NMR (DMSO-d6, 400 MHz) δ12.18 (br. s, 1H), 8.53 - 8.50 (m, 1H), 8.25 - 8.10 (m, 2H), 7.60 (d, J= 4.8 Hz, 1H), 7.42 - 7.40 (m, 5H), 7.36 (d, J = 7.2 Hz, 1H), 6.96 (d, J= 4.8 Hz, 1H), 5.52 (s, 2H). LCMS (m / z [M+H] + ) = 335.1.

[0207] The following examples in Table E were prepared in a similar manner as shown above in Scheme 54 using Intermediate B1 and the appropriate acid from Table A. Table E [Table 5]

[0208] The examples in Table F are prepared in a similar manner as shown above in Scheme 54 using the appropriate intermediates in Tables B / C and Acid A in Table A. Table F [Table 6] TIFF0007678595000075.tif239164TIFF0007678595000076.tif255163TIFF0007678595 000077.tif234164TIFF0007678595000078.tif251164TIFF0007678595000079.tif87164

[0209] The preparation of further examples of Table G is shown in Schemes 55-71. Table G [Table 7] TIFF0007678595000081.tif214141TIFF0007678595000082.tif188141TIFF0007678595000083.tif187141TIFF0007678595000084.tif45128

[0210] <Preparation of Examples T37 and T38> [ka] <Example T37> To a solution of 1H-pyrrolo[2,3-b]pyridine-3-carboxylic acid A (199 mg, 1.23 mmol, 1.0 equiv) in DCM (10 mL) was added oxalyl chloride (COCl)2 (156 mg, 1.23 mmol, 107 μL, 1.0 equiv) and DMF (1 μL, 12.3 μmol, 0.01 equiv) at 0° C. The reaction mixture was stirred at 15° C. for 2 h, then TEA (187 mg, 1.84 mmol, 1.5 equiv) and 3-methylthiazol-2-imine B2 (140 mg, 1.23 mmol, 1.0 equiv) were added. The resulting mixture was stirred at 15° C. for 12 h. The reaction mixture was quenched by the addition of H2O (20 mL) and extracted with EtOAc (3×15 mL). The combined organic layers were washed with brine (3×10 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC to give 21 mg of (Z)-N-(3-methylthiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T37 as a white solid.

[0211] <Example T38> To a solution of 1H-pyrrolo[2,3-b]pyridine-3-carboxylic acid A (379 mg, 2.34 mmol, 1.0 equiv) in DCM (10 mL) was added oxalyl chloride (COCl)2 (297 mg, 2.34 mmol, 1.0 equiv) and DMF (2 μL, 23.4 μmol, 0.01 equiv) at 0° C. The reaction mixture was stirred at 15° C. for 2 h, then TEA (355 mg, 3.51 mmol, 1.5 equiv) and 3-ethylthiazol-2-imine B3 (300 mg, 2.34 mmol, 1.0 equiv) were added. The resultant was stirred at 15° C. for 12 h. The reaction mixture was quenched by the addition of water (30 mL) and extracted with EtOAc (3×20 mL). The combined organic layers were washed with brine (2×20 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC to give 24 mg of (Z)-N-(3-ethylthiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T38 as a white solid.

[0212] <Preparation of Example T39> [ka] (Z)-N-(thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide To a solution of 1H-pyrrolo[2,3-b]pyridine-3-carboxylic acid A (0.60 g, 3.70 mmol, 1.0 equiv) in pyridine (5.88 g, 74.3 mmol, 20 equiv) was added HBTU (2.81 g, 7.40 mmol, 2.0 equiv) and thiazol-2-amine (556 mg, 5.55 mmol, 1.5 equiv). The reaction mixture was heated to 100° C. and stirred at 100° C. for 12 h. The reaction mixture was cooled to 0° C. and poured into ice water (15 mL). The solid was collected by filtration. The filter cake was washed with water (2×5 mL) and dried under reduced pressure to give the crude product. The crude product was recrystallized from MeOH / EtOH (4 mL / 4 mL) by refluxing at 60° C. for 0.5 h, then cooled to 25° C. and filtered. The solid was washed with cold EtOH (5 mL) and dried in vacuum to give 0.41 g of (Z)-N-(thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide as a brown solid.

[0213] <(Z)-N-(3-(1-phenylethyl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T39> To a solution of (Z)-N-(thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide (0.20 g, 819 umol, 1.0 equiv) in DMF (3 mL) was added K2CO3 (226 mg, 1.64 mmol, 2.0 equiv) and 1-chloroethylbenzene (173 mg, 1.23 mmol, 1.5 equiv). The reaction mixture was stirred at 25° C. for 12 hours. The reaction mixture was heated to 60° C. for 18 hours. The reaction mixture was cooled to room temperature and filtered to give a yellow solution in DMF. The yellow solution was purified by preparative HPLC to give 25 mg of (Z)-N-(3-(1-phenylethyl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T39 as a pale yellow solid.

[0214] <Preparation of Example T41> [ka] (Z)-tert-butyl (4-(2-((1H-pyrrolo[2,3-b]pyridine-3-carbonyl)imino)thiazol-3(2H)-yl)butan-2-yl)carbamate To a solution of 1H-pyrrolo[2,3-b]pyridine-3-carboxylic acid A (30 mg, 184 μmol, 1.0 equiv), HOBt (37 mg, 276 μmol, 1.5 equiv), EDCI (53 mg, 276 μmol, 1.5 equiv) and DIPEA (71 mg, 553 μmol, 96 μL, 3.0 equiv) in DMF (1 mL) was added tert-butyl (4-(2-iminothiazol-3(2H)-yl)butan-2-yl)carbamate B4 (50 mg, 184 μmol, 1.0 equiv). The reaction mixture was stirred at 25° C. for 12 h. The reaction mixture was diluted with 20 mL of water and extracted with EtOAc (3×10 mL). The combined organic layers were washed with 20 mL of brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel preparative TLC (petroleum ether / ethyl acetate) to give 40 mg of (Z)-tert-butyl (4-(2-((1H-pyrrolo[2,3-b]pyridine-3-carbonyl)imino)thiazol-3(2H)-yl)butan-2-yl)carbamate as a white solid.

[0215] <(Z)-N-(3-(3-aminobutyl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T41> A mixture of (Z)-tert-butyl (4-(2-((1H-pyrrolo[2,3-b]pyridine-3-carbonyl)imino)thiazol-3(2H)-yl)butan-2-yl)carbamate (40 mg, 96 μmol, 1.0 equiv) in HCl / EtOAc (4 M, 2 mL) was stirred at 25° C. for 1 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC to give 28 mg of (Z)-N-(3-(3-aminobutyl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T41 as a white solid.

[0216] <Preparation of Examples T42 and T43> [ka] <(Z)-N-(3-(2-cyanobenzyl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T42> To a solution of 1H-pyrrolo[2,3-b]pyridine-3-carboxylic acid A (369 mg, 2.28 mmol, 1.0 equiv) in DMF (6 mL) was added EDCI (524 mg, 2.73 mmol, 1.2 equiv), HOBt (369 mg, 2.73 mmol, 1.2 equiv), TEA (691 mg, 6.83 mmol, 3.0 equiv) and 2-((2-iminothiazol-3(2H)-yl)methyl)benzonitrile B11 (0.49 g, 2.28 mmol, 1.0 equiv). The reaction mixture was stirred at 25 °C for 12 h. The reaction mixture was quenched by the addition of water (15 mL) at 0° C., and the precipitate was collected by filtration, washed with water (2×5 mL), and dried in vacuum to give 0.60 g of crude product (Z)-N-(3-(2-cyanobenzyl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T42 as a grey solid. 180 mg of crude product T42 was further purified by preparative HPLC to give 92 mg of pure (Z)-N-(3-(2-cyanobenzyl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T42 as a pale yellow solid.

[0217] <(Z)-N-(3-(2-(aminomethyl)benzyl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T43> To a solution of LAH (48 mg, 1.25 mmol, 3.0 equiv) in THF (4 mL) was added (Z)-N-(3-(2-cyanobenzyl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T42 (0.15 g, 417 μmol, 1.0 equiv) in small portions at 0° C. After addition, the reaction mixture was gradually warmed to 25° C. and stirred at 25° C. for 4 h. The reaction mixture was quenched by the addition of aqueous HCl (2N, 0.5 mL) at 0° C. and stirred for 0.5 h. The mixture was diluted with THF / DMF (20 mL / 4 mL) and filtered. The filter cake was washed with MeOH (2×5 mL). The combined filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC to give 8.3 mg of (Z)-N-(3-(2-(aminomethyl)benzyl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T43 as an off-white solid.

[0218] <Preparation of Examples T44 and T45> [ka] <(Z)-N-(3-(3-cyanobenzyl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T44> To a solution of 1H-pyrrolo[2,3-b]pyridine-3-carboxylic acid A (264 mg, 1.63 mmol, 1.0 equiv), HOBt (330 mg, 2.44 mmol, 1.5 equiv), EDCI (468 mg, 2.44 mmol, 1.5 equiv) and DIPEA (630 mg, 4.88 mmol, 850 μL, 3.0 equiv) in DMF (8 mL) was added 3-((2-iminothiazol-3(2H)-yl)methyl)benzonitrile B17 (350 mg, 1.63 mmol, 1.0 equiv) at 25° C. The reaction mixture was then stirred at 25° C. for 12 h. The reaction mixture was diluted with 50 mL of water and extracted with EtOAc (3×30 mL). The combined organic layers were washed with 50 mL of brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was diluted with MeOH (20 mL) and stirred for 20 min; the suspension was filtered to collect the solid. The collected solid was washed with MeOH (10 mL) and then dried in vacuum to give 500 mg of (Z)-N-(3-(3-cyanobenzyl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T44 as a yellow solid. 100 mg of the crude product was purified by preparative HPLC to give 62 mg of pure (Z)-N-(3-(3-cyanobenzyl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T44 as a white solid.

[0219] <(Z)-N-(3-(3-(aminomethyl)benzyl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T45> To a suspension of (Z)-N-(3-(3-cyanobenzyl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T44 (200 mg, 556 μmol, 1.0 equiv.) in THF (5 mL) under N2 at 0° C. was added LAH (63 mg, 1.67 mmol, 3.0 equiv.). The reaction mixture was then stirred at 25° C. for 3 h. The reaction mixture was quenched by the addition of water (5 mL) and acidified to pH=3 with HCl (4N) until the mixture became clear. The mixture was directly purified by preparative HPLC to give 76 mg of (Z)-N-(3-(3-(aminomethyl)benzyl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T45 as a white solid.

[0220] <Preparation of Examples T46, T47, and T48> [ka] <(Z)-N-(3-(4-cyanobenzyl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T46> To a solution of 1H-pyrrolo[2,3-b]pyridine-3-carboxylic acid A (1.09 g, 6.75 mmol, 1.0 equiv), HOBt (1.37 g, 10.1 mmol, 1.5 equiv), EDCI (1.94 g, 10.1 mmol, 1.5 equiv) and DIPEA (2.62 g, 20.3 mmol, 3.5 mL, 3.0 equiv) in DMF (20 mL) was added 4-((2-iminothiazol-3(2H)-yl)methyl)benzonitrile B5 (2.00 g, 6.80 mmol, 1.0 equiv) at 25° C. The reaction mixture was stirred at 25° C. for 12 h. The reaction mixture was diluted with 150 mL of water and extracted with EtOAc (3×100 mL). The combined organic layers were washed with 200 mL of brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was diluted with MeOH (50 mL) and stirred for 20 min; the suspension was filtered to collect the solid. The filter cake was washed with MeOH (20 mL) and dried in vacuum to give 1.8 g of (Z)-N-(3-(4-cyanobenzyl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T46 as a yellow solid. 200 mg of the crude product T46 was purified by preparative HPLC to give 132 mg of pure (Z)-N-(3-(4-cyanobenzyl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T46 as a white solid.

[0221] <(Z)-N-(3-(4-(aminomethyl)benzyl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T47> <(Z)-N-(3-(4-formylbenzyl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T48> To a mixture of (Z)-N-(3-(4-cyanobenzyl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T46 (300 mg, 835 μmol, 1.0 equiv.) in THF (7 mL) under N2 at 0° C. was added LAH (95 mg, 2.5 mmol, 3.0 equiv.). The mixture was stirred at 25° C. for 3 h. The reaction mixture was quenched by the addition of water (5 mL) and acidified to pH=3 with 4N HCl. The mixture was purified by preparative HPLC to give 108 mg of (Z)-N-(3-(4-(aminomethyl)benzyl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T47 as a white solid and 31 mg of (Z)-N-(3-(4-formylbenzyl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T48 as a white solid. Preparation of Examples T54 and T64

[0222] To a solution of the appropriate imine (1.0 equiv.) in pyridine was added HBTU (2.0 equiv.) and 1H-pyrrolo[2,3-b]pyridine-3-carboxylic acid A (1.0 equiv.). The reaction mixture was stirred at 100° C. for 12 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC to give T54 and T64.

[0223] <Preparation of Example T51> [ka] <N‐(チアゾール‐2‐イル)‐1H‐ピロロ[2,3‐b]ピリジン‐3‐カルボキサミド> To a solution of 1H-pyrrolo[2,3-b]pyridine-3-carboxylic acid A (0.60 g, 3.70 mmol, 1.0 equiv) in pyridine (6 mL) was added HBTU (2.81 g, 7.40 mmol, 2.0 equiv) and 2-thiazolamine (556 mg, 5.55 mmol, 1.5 equiv) at 0° C. The reaction mixture was gradually heated to 100° C. and stirred at 100° C. for 12 h. The reaction mixture was poured into ice water (12 mL). The solid was collected by filtration, washed with H2O (5×2 mL), and dried under reduced pressure. The crude product was recrystallized by refluxing in MeOH / EtOH (4 mL / 4 mL) at 70° C. for 0.5 h, then cooled to room temperature and filtered to give 0.41 g of N-(thiazol-2-yl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide as a brown solid.

[0224] <Methyl 3-[(2Z)-2-(1H-pyrrolo[2,3-b]pyridine-3-carbonylimino)thiazol-3-yl]propanoate> To a solution of N-(thiazol-2-yl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide (0.29 g, 1.19 mmol, 1.0 equiv) in DMF (5 mL) was added K2CO3 (656 mg, 4.75 mmol, 4.0 equiv) and methyl 3-bromopropanoate (595 mg, 3.56 mmol, 3.0 equiv). After addition, the reaction mixture was transferred to a sealed microwave reactor and stirred at 100 °C for 2 h in a microwave reactor. The reaction mixture was quenched by the addition of H2O (10 mL) at 0 °C and extracted with EtOAc (2 x 10 mL). The combined organic layers were washed with H2O (20 mL), brine (20 mL), dried over Na2SO4, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel preparative TLC (petroleum ether / ethyl acetate=1:1) to give 0.30 g of methyl 3-[(2Z)-2-(1H-pyrrolo[2,3-b]pyridine-3-carbonylimino)thiazol-3-yl]propanoate as a brown solid. A portion of the residue (0.10 g) was further purified twice by silica gel preparative TLC (petroleum ether / ethyl acetate=2:1) ​​to give 20 mg of methyl 3-[(2Z)-2-(1H-pyrrolo[2,3-b]pyridine-3-carbonylimino)thiazol-3-yl]propanoate as a white solid.

[0225] <Example T51> To a solution of (Z)-methyl 3-(2-((1H-pyrrolo[2,3-b]pyridine-3-carbonyl)imino)thiazol-3(2H)-yl)propanoate (0.20 g, 605 μmol, 1.0 equiv) in dioxane (4 mL) / HO (1 mL) was added LiOH.HO (127 mg, 3.03 mmol, 5.0 equiv). The reaction mixture was stirred at 25° C. for 12 h. The mixture was concentrated. The residue was diluted with water (5 mL) and adjusted to pH<5.0 by addition of concentrated HCl. The suspension was filtered. The filter cake was washed with HO (5 mL) and dried under reduced pressure. The residue was purified by preparative HPLC to give 52 mg of (Z)-3-(2-((1H-pyrrolo[2,3-b]pyridine-3-carbonyl)imino)thiazol-3(2H)-yl)propanoic acid as a white solid.

[0226] <Preparation of Example T52> [ka] A mixture of N-(thiazol-2-yl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide (100 mg, 409.38 μmol, 1.0 equiv.), 4-(bromomethyl)pyridine (104 mg, 409 μmol, 1.0 equiv., HBr), K2CO3 (170 mg, 1.23 mmol, 3.0 equiv.) in DMF (1 mL) was degassed and purged with N2 three times, then the reaction mixture was stirred under N2 atmosphere at 30° C. for 12 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC to give 37 mg of (Z)-N-(3-(pyridin-4-ylmethyl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T52 as a white solid.

[0227] <Preparation of Example T53> [ka] A mixture of N-(thiazol-2-yl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide (100 mg, 409 μmol, 1.0 equiv.), 3-(bromomethyl)pyridine (104 mg, 409 μmol, 1.0 equiv., HBr), K2CO3 (170 mg, 1.23 mmol, 3.0 equiv.) in DMF (1 mL) was degassed and purged with N2 three times, then the reaction mixture was stirred under N2 atmosphere at 30° C. for 12 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC to give 32 mg of (Z)-N-(3-(pyridin-3-ylmethyl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T53 as a white solid.

[0228] Preparation of Example T57 [ka] To a solution of N-(thiazol-2-yl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide (200 mg, 819 μmol, 1.0 equiv) in DMF (2 mL) was added K2CO3 (226 mg, 1.64 mmol, 2.0 equiv), followed by bromocyclohexane (2.67 g, 16.4 mmol, 2.02 mL, 20 equiv) at room temperature. The reaction mixture was then stirred at 100° C. for 12 h. An additional 10 equiv of bromocyclohexane was added to the reaction mixture, and the mixture was then stirred at 100° C. under N2 for an additional 12 h. The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (50 mL×3), the combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC to give 7.3 mg of (Z)-N-(3-cyclohexylthiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T57 as a white solid. The structure was confirmed by NOE.

[0229] <Preparation of Example T61> [ka] (Z)-tert-butyl 3-(2-((1H-pyrrolo[2,3-b]pyridine-3-carbonyl)imino)thiazol-3(2H)-yl)benzylcarbamate To a solution of 1H-pyrrolo[2,3-b]pyridine-3-carboxylic acid A (80 mg, 491 μmol, 1.0 equiv.) in DMF (2 mL) was added EDCI (141 mg, 737 μmol, 1.5 equiv.), HOBt (100 mg, 737 μmol, 1.5 equiv.), DIPEA (190 mg, 1.47 mmol, 257 μL, 3.0 equiv.) and tert-butyl 3-(2-iminothiazol-3(2H)-yl)benzylcarbamate C24 (150 mg, 491 μmol, 1.0 equiv.) at 25° C. The reaction mixture was then stirred under N2 at 25° C. for 2 h. The reaction mixture was diluted with water (5 mL) causing a solid to precipitate. The solid was collected by filtration, washed with water (5 mL) and dried under residue pressure to give 170 mg of (Z)-tert-butyl 3-(2-((1H-pyrrolo[2,3-b]pyridine-3-carbonyl)imino)thiazol-3(2H)-yl)benzylcarbamate as a white solid.

[0230] <(Z)-N-(3-(3-(aminomethyl)phenyl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T61> To a solution of (Z)-tert-butyl 3-(2-((1H-pyrrolo[2,3-b]pyridine-3-carbonyl)imino)thiazol-3(2H)-yl)benzylcarbamate (170 mg, 378 μmol, 1.0 equiv.) in EtOAc (1 mL) was added HCl / EtOAc (378 μmol, 3 mL, 1.0 equiv.) at 25° C. The solution was then stirred under N2 at 25° C. for 1 h. The reaction mixture was filtered and the filter cake was washed with EtOAc (30 mL). The filter cake was concentrated under reduced pressure and purified by preparative HPLC to give 52 mg of (Z)-N-(3-(3-(aminomethyl)phenyl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T61 as a white solid.

[0231] Preparation of Example T62 [ka] (Z)-tert-Butyl 7-(2-((1H-pyrrolo[2,3-b]pyridine-3-carbonyl)imino)thiazol-3(2H)-yl)-3,4-dihydroisoquinoline-2(1H)-carboxylate To a mixture of 1H-pyrrolo[2,3-b]pyridine-3-carboxylic acid A (80 mg, 493 μmol, 1.0 equiv.) in DMF (3 mL), HOBt (100 mg, 740 μmol, 1.5 equiv.), EDCI (142 mg, 740 μmol, 1.5 equiv.), DIPEA (191 mg, 1.48 mmol, 258 μL, 3.0 equiv.) and tert-butyl 7-(2-iminothiazol-3(2H)-yl)-3,4-dihydroisoquinoline-2(1H)-carboxylate C21 (164 mg, 493 μmol, 1.0 equiv.) were added at 25° C. The reaction mixture was then stirred under N2 at 25° C. for 12 h. The reaction mixture was diluted with water (20 mL) and filtered to collect the solid. The collected solid was dried under reduced pressure to give 200 mg of (Z)-tert-butyl 7-(2-((1H-pyrrolo[2,3-b]pyridine-3-carbonyl)imino)thiazol-3(2H)-yl)-3,4-dihydroisoquinoline-2(1H)-carboxylate as a brown solid. The crude product was used directly in the next step.

[0232] <(Z)-N-(3-(1,2,3,4-tetrahydroisoquinolin-7-yl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T62> To a mixture of (Z)-tert-butyl 7-(2-((1H-pyrrolo[2,3-b]pyridine-3-carbonyl)imino)thiazol-3(2H)-yl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (170 mg, 357 μmol, 1.0 equiv.) in EtOAc (1 mL) was added HCl / EtOAc (4 M, 3 mL, 33 equiv.) at 25° C. Then the reaction mixture was stirred at 25° C. for 2 h under N2. The solid was collected by filtration and purified by preparative HPLC to give 88 mg of (Z)-N-(3-(1,2,3,4-tetrahydroisoquinolin-7-yl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T62 as a white solid.

[0233] Preparation of Example T72 [ka] (Z)-Ethyl 2-((1H-pyrrolo[2,3-b]pyridine-3-carbonyl)imino)-3-benzyl-2,3-dihydrothiazole-4-carboxylate To a solution of 1H-pyrrolo[2,3-b]pyridine-3-carboxylic acid (0.238 g, 1.47 mmol, 1.0 equiv.) in DMF (5 mL), EDCI (0.422 g, 2.20 mmol, 1.5 equiv.), HOBT (0.298 g, 2.20 mmol, 1.5 equiv.), diisopropylethylamine (0.758 mL, 4.41 mmol, 3.0 equiv.) and ethyl 3-benzyl-2-imino-2,3-dihydrothiazole-4-carboxylate (0.400 g, 1.47 mmol, 1.0 equiv.) were added at 25° C. The mixture was then stirred at 25° C. for 1 h. The reaction mixture was then poured into ice water (15 mL) to precipitate a solid, which was collected by filtration. The filter cake was triturated three times with ethanol (15 mL) at 25° C. to give 0.40 g of (Z)-ethyl 2-((1H-pyrrolo[2,3-b]pyridine-3-carbonyl)imino)-3-benzyl-2,3-dihydrothiazole-4-carboxylate as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 12.27 (br. s, 1H), 8.45 - 8.42 (m, 1H), 8.26 (dd, J = 4.8, 1.2 Hz, 1H), 8.21 - 8.20 (m, 1H), 7.99 (s, 1H), 7.35 - 7.31 (m, 2H), 7.27 - 7.2 (m, 3H), 7.14 (dd, J = 8.0, 4.8 Hz, 1H), 5.93 (s, 2H), 4.27 (q, J = 7.2 Hz, 2H), 1.25 (t, J = 7.2 Hz, 3H).LCMS (m / z [M+H] + ): 407.0.

[0234] <(Z)-N-(3-benzyl-4-(hydroxymethyl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T72> To a suspension of (Z)-ethyl 2-((1H-pyrrolo[2,3-b]pyridine-3-carbonyl)imino)-3-benzyl-2,3-dihydrothiazole-4-carboxylate (0.200 g, 0.407 mmol) in THF (5 mL) was added a solution of lithium borohydride in THF (2 M, 0.4 mL, 0.8 mmol) dropwise under nitrogen atmosphere at 0° C. After addition, the reaction mixture was warmed to 25° C. and stirred for 1 h. The mixture was then added dropwise into water (5 mL) at 25° C., followed by saturated aqueous sodium carbonate (5 mL). The aqueous phase was extracted with ethyl acetate (50 mL×3). The combined organic layers were washed with brine (15 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC to give 32 mg of (Z)-N-(3-benzyl-4-(hydroxymethyl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T72 as a white solid.

[0235] <Preparation of Example T75> [ka] (Z)-tert-Butyl 4-(2-((1H-pyrrolo[2,3-b]pyridine-3-carbonyl)imino)thiazol-3(2H)-yl)piperidine-1-carboxylate To a solution of (Z)-N-(thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide (20 mg, 82 μmol, 1.0 equiv.) in DMF (1 mL) was added K2CO3 (57 mg, 246 μmol, 3.0 equiv.) and tert-butyl 4-bromopiperidine-1-carboxylate (216 mg, 819 μmol, 10.0 equiv.). The reaction mixture was transferred to a microwave reaction tube. The sealed tube was heated at 100° C. in a microwave reactor for 2 h. The reaction mixture was quenched by the addition of water (5 mL) resulting in the formation of a brown precipitate. The solid was collected by filtration and washed with water (2 mL). The filtrate was dried under reduced pressure to give the crude product (Z)-tert-butyl 4-(2-((1H-pyrrolo[2,3-b]pyridine-3-carbonyl)imino)thiazol-3(2H)-yl)piperidine-1-carboxylate (30 mg, crude) as a brown solid.

[0236] <(Z)-N-(3-(piperidin-4-yl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide hydrochloride T75> To a solution of (Z)-tert-butyl 4-(2-((1H-pyrrolo[2,3-b]pyridine-3-carbonyl)imino)thiazol-3(2H)-yl)piperidine-1-carboxylate (0.03 g, 70 umol, 1.0 equiv.) in EtOAc (1 mL) was slowly added dropwise HCl / EtOAc (4 M, 1.2 mL, 68.4 equiv.). The reaction mixture was stirred at 25° C. for 2 h. The reaction mixture was concentrated under dunder reduced pressure. The residue was purified by preparative HPLC to give 5 mg of (Z)-N-(3-(piperidin-4-yl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide hydrochloride salt T75 as a white solid.

[0237] Preparation of Example T77 [ka] To a solution of 3-benzyl-4-methyloxazole-2(3H)-imine 2,2,2-trifluoroacetate B19 (0.100 g, 0.285 mmol, 1.0 equiv.) and triethylamine (0.198 mL, 1.43 mmol, 5.0 equiv.) in chloroform (1 mL) was added a solution of N,N-dimethylpyridin-4-amine (3 mg, 0.03 mmol, 0.1 equiv.) and 1H-pyrrolo[2,3-b]pyridine-3-carbonyl chloride hydrochloride A (62 mg, 0.28 mmol, 1.0 equiv.) in chloroform (2 mL) at 0° C. The reaction mixture was warmed to 25° C. and stirred for 1 h. The mixture was concentrated under reduced pressure and the residue was dissolved in methanol (2 mL) and then poured into water (10 mL) to precipitate a solid. The precipitate was collected by filtration and purified by preparative HPLC to give 8 mg of (Z)-N-(3-benzyl-4-methyloxazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T77 as a white solid.

[0238] Preparation of Example T78 [ka] Ethyl (Z)-2-((1H-pyrrolo[2,3-b]pyridine-3-carbonyl)imino)-3-benzyl-2,3-dihydrooxazole-4-carboxylate To a solution of ethyl 3-benzyl-2-imino-2,3-dihydrooxazole-4-carboxylate B20 (0.450 g, 1.83 mmol) and 1H-pyrrolo[2,3-b]pyridine-3-carboxylic acid (acid A) (0.356 g, 2.19 mmol) in DMF (6 mL) was added DIPEA (1.59 mL, 9.14 mmol), EDCI (0.525 g, 2.74 mmol) and HOBt (0.247 g, 1.83 mmol) at 25° C. The reaction mixture was stirred at 50° C. for 2 h. The reaction was quenched with water (50 mL) and then extracted with ethyl acetate (30 mL×3). The combined organic layers were washed with saturated aqueous sodium bicarbonate (50 mL×3) and brine (50 mL×3), dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure. The crude product was purified by reverse phase HPLC (0.1% NH3·H2O condition) to give 0.220 g of (Z)-ethyl 2-((1H-pyrrolo[2,3-b]pyridine-3-carbonyl)imino)-3-benzyl-2,3-dihydrooxazole-4-carboxylate as a yellow solid. 1 H NMR (DMSO-d6, 400 MHz) δ12.18 (br. s, 1H), 8.52 (s, 1H), 8.34 (dd, J = 1.6, 6.4 Hz, 1H), 8.24 (dd, J = 1.6, 3.2 Hz, 1H), 8.07 (d, J = 2.0 Hz, 1H), 7.37 - 7.36 (m, 4H), 7.33 - 7.29 (m, 1H), 7.10 (dd, J = 3.2, 4.8 Hz, 1H), 5.37 (s, 2H), 4.28 (q, J = 7.2 Hz, 2H), 1.25 (t, J = 7.2 Hz, 3H). + ): 391.0.

[0239] <(Z)-N-(3-benzyl-4-(hydroxymethyl)oxazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide T78> To a solution of (Z)-ethyl 2-((1H-pyrrolo[2,3-b]pyridine-3-carbonyl)imino)-3-benzyl-2,3-dihydrooxazole-4-carboxylate (0.100 g, 0.231 mmol) in THF (3.0 mL) was added dropwise lithium borohydride solution (0.348 mL, 2 M in THF) at 0° C. under nitrogen atmosphere. The reaction mixture was stirred at 0° C. for 1 h. The mixture was poured into water (20 mL) at 25° C., and then saturated aqueous ammonium chloride solution (20 mL) was added. The aqueous phase was extracted with ethyl acetate (30 mL×3). The combined organic phase was washed with brine (50 mL), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure. The mixture was purified by preparative HPLC (column: Gemini®-NX C from Phenomenex). 18 75*30mm*3um; mobile phase: [water (10mM NH4HCO3)-ACN]; B%: 17%-47%, 8min) to give 9mg of (Z)-N-(3-benzyl-4-(hydroxymethyl)oxazol-2(3H)-ylidene)-1-pyrrolo[2,3-b]pyridine-3-carboxamide T78 as a white solid.

[0240] Preparation of Examples T79 and T80 [ka] <Ethyl (2Z)-3-benzyl-2-(1H-pyrrolo[2,3-b]pyridine-3-carbonylimino)thiazole-4-carboxylate T79> To a solution of 1H-pyrrolo[2,3-b]pyridine-3-carboxylic acid (acid A) (4.62 g, 28.4 mmol) in DMF (100 mL) was added HOBt (3.08 g, 22.8 mmol), EDCI (8.20 g, 42.8 mmol), DIPEA (14.9 mL, 85.6 mmol) and ethyl 3-benzyl-2-imino-thiazole-4-carboxylate hydrobromide B8 (10.0 g, 28.5 mmol) at 25° C. The resulting mixture was stirred at 25° C. for 1 h. The reaction was poured into ice water (200 mL). The precipitate was collected by filtration and the filter cake was triturated with ethanol (150 mL) to give 10.0 g of ethyl (2Z)-3-benzyl-2-(1H-pyrrolo[2,3-b]pyridine-3-carbonylimino)thiazole-4-carboxylate T79 as a white solid.

[0241] <(Z)-3-benzyl-N-[2-(dimethylamino)ethyl]-2-(1H-pyrrolo[2,3-b]pyridine-3-carbonylimino)thiazole-4-carboxamide T80> To a solution of ethyl (2Z)-3-benzyl-2-(1H-pyrrolo[2,3-b]pyridine-3-carbonylimino)thiazole-4-carboxylate T79 (0.050 g, 0.123 mmol) and N',N'-dimethylethane-1,2-diamine (0.108 g, 1.23 mmol, 0.134 mL) in THF (1 mL) was added 3,4,6,7,8,9-hexahydro-2H-pyrimido[1,2-a]pyrimidine (0.009 g, 0.062 mmol). The mixture was stirred at 60° C. for 1 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (Phenomenex Waters Xbridge™ 150*25mm*5um; mobile phase: [water (10mM NH4HCO3)-acetonitrile]; B%: 24%-54%, 10min) to give 54mg of (Z)-3-benzyl-N-[2-(dimethylamino)ethyl]-2-(1H-pyrrolo[2,3-b]pyridine-3-carbonylimino)thiazole-4-carboxamide T80 as a white solid.

[0242] <General method> LCMS conditions [Table 8] Method for IVKA assay at 10uM:

[0243] An in vitro kinase assay (HTRF KinEASE™-STK S1, 62ST1PEB from CisBio) was optimized for the linear reaction range of the Lats1 enzyme (01-123 from Carna). Reactions were performed with 10 μM STK1 substrate and 10 μM ATP or 2 mM ATP as indicated below. DMSO concentration was maintained at 0.5% for all experiments, and compounds dissolved in DMSO were added to the reaction using a Janus® 384MDT (PerkinElmer) equipped with a 50 nL pin tool (V and P Scientific, Inc.). Enzyme, substrate, ATP, and test compounds of the invention were combined in a low volume 384-well plate and stirred at room temperature for 50 minutes unless otherwise specified. The reaction was stopped by adding detection reagent prepared as a biotin:streptavidin ratio of 8:1 and stirring for 60 minutes at room temperature. All reactions were performed in triplicate and the signal was detected using Synergy™ NEO (Biotek).

[0244] Reagents for IVKA at 2 mM ATP, final concentrations in assay: Kit: HTRFKinEASE-STKS1, 62ST1PEB from Sysbio); 1x Kinase Buffer (KB); 5 mM MgCl2; 1 mM DTT; ATP 2 mM; STK1, 2.5 μM; 50 pg / μL (Lats1, 01-123 from Karna); BSA 1 mg / mL; Detection Reagent Streptavidin-XL665 with a biotin:streptavidin ratio of 8:1: 156 nM for a final reaction volume of 10 μL and a final detection volume of 20 μL. The assay was performed in a 384-well plate. Dose curves of drugs were generated as 16 points, each diluted 3-fold in 100% DMSO, in triplicate. Lats1 activity measured as described above is shown in the following table:

[0245] [Table 9] TIFF0007678595000104.tif203145 Compound T01 was studied in detail to investigate its mechanism of action and utility in regenerating ear hair cells. The effects of the compound were tested on utricles isolated from 8-12 week old mice. Inner ears were excised from mice euthanized with fluothane, placed in ice-cold Hank's balanced salt solution, and cultured as previously described by Gnedeva, K. and Hudspeth [Proc. Natl. Acad. Sci., (US), 2015, Vol. 112, p. 14066-14071]. For proliferation assays, utricles were incubated with 10 μM 5-ethynyl-2'-deoxyuridine (EdU) detected by click chemistry.

[0246] Immunohistochemical analysis demonstrated that T01 promoted robust Yap nuclear translocation in supporting cells after 24 h of treatment at a concentration of 10 μM (quantified as a ratio to the constitutively expressed protein Sall2; control=0.6; T01-treated=1.0; p<0.0001 by unpaired two-tailed t-test, n=570 (control) and 680 (treated) nuclei), and T01 caused a significant reduction in Yap phosphorylation levels as detected by Western blot. After 5 days of treatment, T01 induced robust re-entry of adult utricle supporting cells into the cell cycle, giving rise to hundreds of EdU+ daughter supporting cells (control=20 EdU+ supporting cells; T01-treated=250 EdU+ supporting cells; p=0.021 by unpaired one-tailed t-test, control n=2, T01 n=3).

Claims

1. Formula I: 【Chemistry 1】 (In the formula, R １ (C １ -C ６ ) alkyl, carboxy, (C ３ -C ７ ) carbomonocyclyl, (C ９ -C １１ ) selected from the group consisting of carbobicyclyl, heteromonocyclyl, and heterobicyclyl, １ -C ６ ) alkyl, (C ３ -C ７ ) carbomonocyclyl, (C ９ -C １１ ) Carbobicyclyl, heteromonocyclyl, and heterobicyclyl are optionally selected from halogen, cyano, hydroxy, nitro, amino, acetoxy, carboxy, (C １ -C ７ ) hydrocarbyl, halo(C １ -C ６ ) alkyl, (C １ -C ３ ) alkoxy, halo (C １ -C ３ ) alkoxy, (C １ -C ６ ) acyl, (C １ -C ３ ) Alkoxy (C １ -C ３ ) alkyl, hydroxy (C １ -C ３ ) alkyl, heteroaryl, benzenesulfonyl, (C １ -C ３ ) alkoxycarbonyl, aminocarbonyl, (C １ -C ３ ) alkylaminocarbonyl, di(C １ -C ３ ) alkylaminocarbonyl, (C １ -C ３ ) alkylamino, di(C １ -C ３ ) alkylamino, amino (C １ -C ３ ) alkyl, (C １ -C ３ ) alkylamino (C １ -C ３ ) alkyl, (C １ -C ３ ) Dialkylamino (C １ -C ３ ) alkyl, (C １ -C ３ ) alkylthio, (C １ -C ３ ) alkylsulfonylamino, (C １ -C ３ ) alkylsulfinyl, (C １ -C ３ ) optionally substituted with 1 to 3 substituents independently selected from the group consisting of alkylsulfonyl, phenoxy, and benzyloxy; R ２ is hydrogen, halogen, (C １ -C ７ ) hydrocarbyl, halo(C １ -C ６ ) alkyl, (C １ -C ６ ) acyl, hydroxy (C １ -C ３ ) alkyl, -C(=O)O(C １ -C ６ ) alkyl, -C(=O)NR ２０ R ２１ , and (C １ -C ６ ) oxaalkyl; R ３ is hydrogen, halogen, (C １ -C ６ ) hydrocarbyl, halo(C １ -C ６ ) alkyl, (C １ -C ６ ) acyl, and (C １ -C ３ ) alkoxy; R ４ is hydrogen, halogen, (C １ -C ６ ) hydrocarbyl, halo(C １ -C ６ ) alkyl, (C １ -C ６ ) acyl, and (C １ -C ３ ) alkoxy; R １０ is independently selected in each occurrence from the group consisting of hydrogen and methyl; R ２０ is hydrogen and (C １ -C ６ ) hydrocarbyl; R ２１ is hydrogen, (C １ -C ６ ) hydrocarbyl, (C １ -C ６ ) oxaalkyl, amino (C １ -C ６ ) alkyl, (C １ -C ３ ) alkylamino (C １ -C ６ ) alkyl, di(C １ -C ３ ) alkylamino (C １ -C ６ ) alkyl, and -(CH ２ ) ｍ -Het, where Het is selected from the group consisting of hydroxy, amino, acetoxy, carboxy, (C １ -C ７ ) hydrocarbyl, halo(C １ -C ６ ) alkyl, (C １‐ C ３ ) alkoxy, halo (C １ -C ３ ) alkoxy, (C １ -C ６ ) acyl, (C １ -C ３ ) Alkoxy (C １ -C ３ ) alkyl, hydroxy (C １ -C ３ ) alkyl, aminocarbonyl, (C １ -C ３ ) alkylaminocarbonyl, di(C １ -C ３ ) alkylaminocarbonyl, (C １ -C ３ ) alkylamino, and di(C １ -C ３ ) an aliphatic monocyclic or bicyclic heterocycle optionally substituted with a substituent selected from the group consisting of alkylamino; Or, R ２０ and R ２１ is R ２０ and R ２１ forms an aliphatic heterocycle together with the nitrogen to which it is attached, n is 0, 1 or 2; m is 0, 1 or 2; and X is S or n is 1 and R １ When is optionally substituted phenyl, X is S or O; However, R １ When is phenyl, X is sulfur, and n is 1, R ２ , R ３ , R ４ and R １０ At least one of is other than hydrogen. A compound represented by the formula:

2. R ２ is -C(=O)O(C １ -C ６ ) alkyl, -C(=O)NR ２０ R ２１ , and (C １ -C ６ 2. The compound of claim 1, wherein said compound is selected from the group consisting of:

3. R ２０ is selected from hydrogen and methyl, and R ２１ is hydrogen, methyl, (C １ -C ６ ) oxaalkyl, dimethylamino (C １ -C ６ ) alkyl, and -(CH ２ ) ｍ The compound according to claim 2 , wherein the group is selected from -Het.

4. R ２０ and R ２１ is R ２０ and R ２１ The compound according to claim 1, wherein, together with the nitrogen to which it is attached, forms a 4- to 7-membered aliphatic heterocycle.

5. Below formula: 【Chemistry 2】 (In the formula, R １ (C １ -C ６ ) alkyl, carboxy, (C ３ -C ７ ) carbomonocyclyl, (C ９ -C １１ ) selected from the group consisting of carbobicyclyl, heteromonocyclyl, and heterobicyclyl, １ -C ６ ) alkyl, (C ３ -C ７ ) carbomonocyclyl, (C ９ -C １１ ) Carbobicyclyl, heteromonocyclyl, and heterobicyclyl are optionally selected from halogen, cyano, hydroxy, nitro, amino, acetoxy, carboxy, (C １ -C ７ ) hydrocarbyl, halo(C １ -C ６ ) alkyl, (C １ -C ３ ) alkoxy, halo (C １ -C ３ ) alkoxy, (C １ -C ６ ) acyl, (C １ -C ３ ) Alkoxy (C １ -C ３ ) alkyl, hydroxy (C １ -C ３ ) alkyl, heteroaryl, benzenesulfonyl, (C １ -C ３ ) alkoxycarbonyl, aminocarbonyl, (C １ -C ３ ) alkylaminocarbonyl, (C １ -C ３ ) alkylamino, di(C １ -C ３ ) alkylamino, amino (C １ -C ３ ) alkyl, (C １ -C ３ ) alkylamino (C １ -C ３ ) alkyl, (C １ -C ３ ) Dialkylamino (C １ -C ３ ) alkyl, (C １ -C ３ ) alkylthio, (C １ -C ３ ) alkylsulfonylamino, (C １ -C ３ ) alkylsulfinyl, (C １ -C ３ ) optionally substituted with 1 to 3 substituents independently selected from the group consisting of alkylsulfonyl, phenoxy, and benzyloxy; R ２ is hydrogen, halogen, (C １ -C ６ ) hydrocarbyl, halo(C １ -C ６ ) alkyl, (C １ -C ６ ) acyl, hydroxy (C １ -C ３ ) alkyl, and (C １ -C ３ ) alkoxy; R ３ is hydrogen, halogen, (C １ -C ６ ) hydrocarbyl, halo(C １ -C ６ ) alkyl, (C １ -C ６ ) acyl, and (C １ -C ３ ) alkoxy; R ４ is hydrogen, halogen, (C １ -C ６ ) hydrocarbyl, halo(C １ -C ６ ) alkyl, (C １ -C ６ ) acyl, and (C １ -C ３ ) alkoxy; R １０ is independently selected in each occurrence from the group consisting of hydrogen and methyl; n is 0, 1 or 2, and X is O or S; However, R １ When is phenyl, X is sulfur, and n is 1, R ２ , R ３ , R ４ and R １０ At least one of is other than hydrogen. The compound according to claim 1 ,

6. The compound according to any one of claims 1 to 5, wherein n is 0.

7. The compound according to any one of claims 1 to 5, wherein n is 1.

8. R １０ The compound of claim 7 , wherein is hydrogen.

9. R １ is carboxy as well as optionally substituted (C １ -C ４ 6. The compound according to any one of claims 1 to 5, wherein the aryl group is selected from the group consisting of alkyl, phenyl, cyclohexyl, 5-membered heterocyclyl, 6-membered heterocyclyl, and heterobicyclyl.

10. R １ 10. The compound of claim 9, wherein is selected from the group consisting of methyl, ethyl, aminobutyl, and carboxyethyl.

11. R １ The compound of claim 9 , wherein is optionally substituted cyclohexyl.

12. R １ The compound of claim 9 , wherein is optionally substituted phenyl.

13. R １ The compound of claim 9 , wherein is optionally substituted heterocyclyl.

14. R １ 14. The compound of claim 13, wherein is selected from the group consisting of pyridinyl, pyrazolyl, piperidinyl, tetrahydropyranyl, tetrahydrofuranyl, and tetrahydroisoquinolinyl, each of which is optionally substituted.

15. R １ is phenyl or halogen, cyano, hydroxy, amino, carboxy, (C １ -C ６ ) hydrocarbyl, trifluoromethyl, methoxy, acetyl, formyl, hydroxy (C １ -C ３ ) alkyl, methoxycarbonyl, carboxamido, methanesulfonylamino, and amino (C １ -C ３ 13. The compound of claim 12, wherein said phenyl is substituted with one or two substituents independently selected from the group consisting of: alkyl, aryl, phenyl, phenyl substituted with one or two substituents independently selected from the group consisting of: alkyl, aryl, aryl substituted with one or two substituents independently selected from the group consisting of: alkyl ...

16. R １ 16. The compound of claim 15, wherein is phenyl substituted at the ortho position and n is 0.

17. R １ are each an amino group, a hydroxy group, and a (C １ -C ６ 15. The compound of claim 14, wherein the aryl group is selected from the group consisting of pyridinyl, pyrazolyl, piperidinyl, tetrahydropyranyl, and tetrahydroisoquinolinyl, optionally substituted with 1 or 2 substituents independently selected from the group consisting of hydrocarbyl.

18. 10. The compound of claim 9, wherein X is S.

19. 10. The compound of claim 9, wherein X is O.

20. R ３ and R ４ 10. The compound of claim 9, wherein is independently selected from the group consisting of hydrogen, chloro and methyl.

21. R ２ 10. The compound of claim 9, wherein is selected from the group consisting of hydrogen, methyl, ethyl, propyl, cyclopropyl, hydroxymethyl, and trifluoromethyl.

22. A compound according to claim 1 selected from the compounds in the table below. 【Chemistry 3】 【change】 【change】 【change】 【change】 23. The compound of claim 1, which is a compound of the following formula: 【Chemistry 4】

24. A pharmaceutical composition comprising a pharma- ceutically acceptable carrier and a compound according to any one of claims 1 to 23.

25. Below formula: 【Chemistry 5】 (In the formula, R １ (C １ -C ６ ) alkyl, carboxy, (C ３ -C ７ ) carbomonocyclyl, (C ９ -C １１ ) selected from the group consisting of carbobicyclyl, heteromonocyclyl, and heterobicyclyl, １ -C ６ ) alkyl, (C ３ -C ７ ) carbomonocyclyl, (C ９ -C １１ ) Carbobicyclyl, heteromonocyclyl, and heterobicyclyl are optionally selected from halogen, cyano, hydroxy, nitro, amino, acetoxy, carboxy, (C １ -C ７ ) hydrocarbyl, halo(C １ -C ６ ) alkyl, (C １ -C ３ ) alkoxy, halo (C １ -C ３ ) alkoxy, (C １ -C ６ ) acyl, (C １ -C ３ ) Alkoxy (C １ -C ３ ) alkyl, hydroxy (C １ -C ３ ) alkyl, heteroaryl, benzenesulfonyl, (C １ -C ３ ) alkoxycarbonyl, aminocarbonyl, (C １ -C ３ ) alkylaminocarbonyl, di(C １ -C ３ ) alkylaminocarbonyl, (C １ -C ３ ) alkylamino, di(C １ -C ３ ) alkylamino, amino (C １ -C ３ ) alkyl, (C １ -C ３ ) alkylamino (C １ -C ３ ) alkyl, (C １ -C ３ ) Dialkylamino (C １ -C ３ ) alkyl, (C １ -C ３ ) alkylthio, (C １ -C ３ ) alkylsulfonylamino, (C １ -C ３ ) alkylsulfinyl, (C １ -C ３ ) optionally substituted with 1 to 3 substituents independently selected from the group consisting of alkylsulfonyl, phenoxy, and benzyloxy; R ２ is hydrogen, halogen, (C １ -C ６ ) hydrocarbyl, halo(C １ -C ６ ) alkyl, (C １ -C ６ ) acyl, hydroxy (C １ -C ３ ) alkyl, -C(=O)O(C １ -C ６ ) alkyl, -C(=O)NR ２０ R ２１ , and (C １ -C ６ ) oxaalkyl; R ３ is hydrogen, halogen, (C １ -C ６ ) hydrocarbyl, halo(C １ -C ６ ) alkyl, (C １ -C ６ ) acyl, and (C １ -C ３ ) alkoxy; R ４ is hydrogen, halogen, (C １ -C ６ ) hydrocarbyl, halo(C １ -C ６ ) alkyl, (C １ -C ６ ) acyl, and (C １ -C ３ ) alkoxy; R １０ is independently selected in each occurrence from the group consisting of hydrogen and methyl; R ２０ is hydrogen and (C １ -C ６ ) hydrocarbyl; R ２１ is hydrogen, (C １ -C ６ ) hydrocarbyl, (C １ -C ６ ) oxaalkyl, amino (C １ -C ６ ) alkyl, (C １ -C ３ ) alkylamino (C １ -C ６ ) alkyl, di(C １ -C ３ ) alkylamino (C １ -C ６ ) alkyl, and -(CH ２ ) ｍ -Het, where Het is selected from the group consisting of hydroxy, amino, acetoxy, carboxy, (C １ -C ７ ) hydrocarbyl, halo(C １ -C ６ ) alkyl, (C １ -C ３ ) alkoxy, halo (C １ -C ３ ) alkoxy, (C １ -C ６ ) acyl, (C １ -C ３ ) Alkoxy (C １ -C ３ ) alkyl, hydroxy (C １ -C ３ ) alkyl, aminocarbonyl, (C １ -C ３ ) alkylaminocarbonyl, di(C １ -C ３ ) alkylaminocarbonyl, (C １ -C ３ ) alkylamino, and di(C １ -C ３ ) an aliphatic monocyclic or bicyclic heterocycle optionally substituted with a substituent selected from the group consisting of alkylamino; Or, R ２０ and R ２１ is R ２０ and R ２１ forms an aliphatic heterocycle together with the nitrogen to which it is attached, n is 0, 1 or 2; m is 0, 1 or 2; and X is S or n is 1 and R １ When is optionally substituted phenyl, X is S or O. A composition for activating YAP in a cell expressing YAP, comprising a compound represented by the formula: The composition, wherein the cell is exposed to the compound.

26. Below formula: 【Chemistry 6】 (In the formula, R １ (C １ -C ６ ) alkyl, carboxy, (C ３ -C ７ ) carbomonocyclyl, (C ９ -C １１ ) selected from the group consisting of carbobicyclyl, heteromonocyclyl, and heterobicyclyl, １ -C ６ ) alkyl, (C ３ -C ７ ) carbomonocyclyl, (C ９ -C １１ ) Carbobicyclyl, heteromonocyclyl, and heterobicyclyl are optionally selected from halogen, cyano, hydroxy, nitro, amino, acetoxy, carboxy, (C １ -C ７ ) hydrocarbyl, halo(C １ -C ６ ) alkyl, (C １ -C ３ ) alkoxy, halo (C １ -C ３ ) alkoxy, (C １ -C ６ ) acyl, (C １ -C ３ ) Alkoxy (C １ -C ３ ) alkyl, hydroxy (C １ -C ３ ) alkyl, heteroaryl, benzenesulfonyl, (C １ -C ３ ) alkoxycarbonyl, aminocarbonyl, (C １ -C ３ ) alkylaminocarbonyl, di(C １ -C ３ ) alkylaminocarbonyl, (C １ -C ３ ) alkylamino, di(C １ -C ３ ) alkylamino, amino (C １ -C ３ ) alkyl, (C １ -C ３ ) alkylamino (C １ -C ３ ) alkyl, (C １ -C ３ ) Dialkylamino (C １ -C ３ ) alkyl, (C １ -C ３ ) alkylthio, (C １ -C ３ ) alkylsulfonylamino, (C １ -C ３ ) alkylsulfinyl, (C １ -C ３ ) optionally substituted with 1 to 3 substituents independently selected from the group consisting of alkylsulfonyl, phenoxy, and benzyloxy; R ２ is hydrogen, halogen, (C １ -C ６ ) hydrocarbyl, halo(C １ -C ６ ) alkyl, (C １ -C ６ ) acyl, hydroxy (C １ -C ３ ) alkyl, -C(=O)O(C １ -C ６ ) alkyl, -C(=O)NR ２０ R ２１ , and (C １ -C ６ ) oxaalkyl; R ３ is hydrogen, halogen, (C １ -C ６ ) hydrocarbyl, halo(C １ -C ６ ) alkyl, (C １ -C ６ ) acyl, and (C １ -C ３ ) alkoxy; R ４ is hydrogen, halogen, (C １ -C ６ ) hydrocarbyl, halo(C １ -C ６ ) alkyl, (C １ -C ６ ) acyl, and (C １ -C ３ ) alkoxy; R １０ is independently selected in each occurrence from the group consisting of hydrogen and methyl; R ２０ is hydrogen and (C １ -C ６ ) hydrocarbyl; R ２１ is hydrogen, (C １ -C ６ ) hydrocarbyl, (C １ -C ６ ) oxaalkyl, amino (C １ -C ６ ) alkyl, (C １ -C ３ ) alkylamino (C １ -C ６ ) alkyl, di(C １ -C ３ ) alkylamino (C １ -C ６ ) alkyl, and -(CH ２ ) ｍ -Het, where Het is selected from the group consisting of hydroxy, amino, acetoxy, carboxy, (C １ -C ７ ) hydrocarbyl, halo(C １ -C ６ ) alkyl, (C １ -C ３ ) alkoxy, halo (C １ -C ３ ) alkoxy, (C １ -C ６ ) acyl, (C １ -C ３ ) Alkoxy (C １ -C ３ ) alkyl, hydroxy (C １ -C ３ ) alkyl, aminocarbonyl, (C １ -C ３ ) alkylaminocarbonyl, di(C １ -C ３ ) alkylaminocarbonyl, (C １ -C ３ ) alkylamino, and di(C １ -C ３ ) an aliphatic monocyclic or bicyclic heterocycle optionally substituted with a substituent selected from the group consisting of alkylamino; Or, R ２０ and R ２１ is R ２０ and R ２１ forms an aliphatic heterocycle together with the nitrogen to which it is attached, n is 0, 1 or 2; m is 0, 1 or 2; and X is S or n is 1 and R １ When is optionally substituted phenyl, X is S or O. A composition for inhibiting LATS in a cell expressing LATS, comprising a compound represented by the formula: The composition, wherein the cell is exposed to the compound.

27. Below formula: 【Chemistry 7】 (In the formula, R １ (C １ -C ６ ) alkyl, carboxy, (C ３ -C ７ ) carbomonocyclyl, (C ９ -C １１ ) selected from the group consisting of carbobicyclyl, heteromonocyclyl, and heterobicyclyl, １ -C ６ ) alkyl, (C ３ -C ７ ) carbomonocyclyl, (C ９ -C １１ ) Carbobicyclyl, heteromonocyclyl, and heterobicyclyl are optionally selected from halogen, cyano, hydroxy, nitro, amino, acetoxy, carboxy, (C １ -C ７ ) hydrocarbyl, halo(C １ -C ６ ) alkyl, (C １ -C ３ ) alkoxy, halo (C １ -C ３ ) alkoxy, (C １ -C ６ ) acyl, (C １ -C ３ ) Alkoxy (C １ -C ３ ) alkyl, hydroxy (C １ -C ３ ) alkyl, heteroaryl, benzenesulfonyl, (C １ -C ３ ) alkoxycarbonyl, aminocarbonyl, (C １ -C ３ ) alkylaminocarbonyl, di(C １ -C ３ ) alkylaminocarbonyl, (C １ -C ３ ) alkylamino, di(C １ -C ３ ) alkylamino, amino (C １ -C ３ ) alkyl, (C １ -C ３ ) alkylamino (C １ -C ３ ) alkyl, (C １ -C ３ ) Dialkylamino (C １ -C ３ ) alkyl, (C １ -C ３ ) alkylthio, (C １ -C ３ ) alkylsulfonylamino, (C １ -C ３ ) alkylsulfinyl, (C １ -C ３ ) optionally substituted with 1 to 3 substituents independently selected from the group consisting of alkylsulfonyl, phenoxy, and benzyloxy; R ２ is hydrogen, halogen, (C １ -C ６ ) hydrocarbyl, halo(C １ -C ６ ) alkyl, (C １ -C ６ ) acyl, hydroxy (C １ -C ３ ) alkyl, -C(=O)O(C １ -C ６ ) alkyl, -C(=O)NR ２０ R ２１ , and (C １ -C ６ ) oxaalkyl; R ３ is hydrogen, halogen, (C １ -C ６ ) hydrocarbyl, halo(C １ -C ６ ) alkyl, (C １ -C ６ ) acyl, and (C １ -C ３ ) alkoxy; R ４ is hydrogen, halogen, (C １ -C ６ ) hydrocarbyl, halo(C １ -C ６ ) alkyl, (C １ -C ６ ) acyl, and (C １ -C ３ ) alkoxy; R １０ is independently selected in each occurrence from the group consisting of hydrogen and methyl; R ２０ is hydrogen and (C １ -C ６ ) hydrocarbyl; R ２１ is hydrogen, (C １ -C ６ ) hydrocarbyl, (C １ -C ６ ) oxaalkyl, amino (C １ -C ６ ) alkyl, (C １ -C ３ ) alkylamino (C １ -C ６ ) alkyl, di(C １ -C ３ ) alkylamino (C １ -C ６ ) alkyl, and -(CH ２ ) ｍ -Het, where Het is selected from the group consisting of hydroxy, amino, acetoxy, carboxy, (C １ -C ７ ) hydrocarbyl, halo(C １ -C ６ ) alkyl, (C １ -C ３ ) alkoxy, halo (C １ -C ３ ) alkoxy, (C １ -C ６ ) acyl, (C １ -C ３ ) Alkoxy (C １ -C ３ ) alkyl, hydroxy (C １ -C ３ ) alkyl, aminocarbonyl, (C １ -C ３ ) alkylaminocarbonyl, di(C １ -C ３ ) alkylaminocarbonyl, (C １ -C ３ ) alkylamino, and di(C １ -C ３ ) an aliphatic monocyclic or bicyclic heterocycle optionally substituted with a substituent selected from the group consisting of alkylamino; Or, R ２０ and R ２１ is R ２０ and R ２１ forms an aliphatic heterocycle together with the nitrogen to which it is attached, n is 0, 1 or 2; m is 0, 1 or 2; and X is S or n is 1 and R １ When is optionally substituted phenyl, X is S or O. A composition for stimulating hair cell regeneration comprising a compound represented by the formula: The composition, wherein a feeder cell population is exposed to the compound.

28. The composition of any one of claims 25 to 27, wherein the cell, cell population, or support cell population is in vivo in a subject.

29. 30. The composition of claim 28, wherein the subject has or is at risk of developing hearing loss.

30. 30. The composition of claim 28, wherein the subject has or is at risk of developing vestibular dysfunction.

31. A composition for treating a subject having or at risk of developing hearing loss, comprising a compound according to any one of claims 1 to 23, wherein an effective amount of the compound is administered to the subject.

32. 32. The composition of claim 29 or 31, wherein the hearing loss is hereditary hearing loss.

33. 33. The composition of claim 32, wherein the hereditary hearing loss is autosomal dominant hearing loss, autosomal recessive hearing loss, or X-linked hearing loss.

34. 32. The composition of claim 29 or 31, wherein the hearing loss is acquired hearing loss.

35. 35. The composition of claim 34, wherein the acquired hearing loss is noise-induced hearing loss, age-related hearing loss, disease or infection-related hearing loss, head trauma-related hearing loss, or ototoxic drug-induced hearing loss.

36. A composition for treating a human subject having or at risk of developing tinnitus, comprising a compound according to any one of claims 1 to 23, wherein an effective amount of the compound is administered to the subject.

37. A composition for treating a subject having or at risk of developing a vestibular dysfunction, comprising a compound described in any one of claims 1 to 23, wherein an effective amount of the compound is administered to the subject.

38. 40. The composition of claim 30 or 37, wherein the vestibular dysfunction comprises vertigo, dizziness, disequilibrium, bilateral vestibular hypofunction, oscillopsia, or balance disorders.

39. 40. The composition of any one of claims 30, 37 or 38, wherein the vestibular dysfunction is age-related vestibular dysfunction, head trauma-related vestibular dysfunction, disease or infection-related vestibular dysfunction, or ototoxic drug-induced vestibular dysfunction.

40. 40. The composition of claim 35 or 39, wherein the ototoxic drug is an aminoglycoside, an antineoplastic agent, ethacrynic acid, furosemide, a salicylate, or quinine.

41. 40. The composition of any one of claims 30, 37 and 38, wherein the vestibular dysfunction is associated with a genetic mutation.

42. 40. The composition of any one of claims 30, 37 and 38, wherein the vestibular dysfunction is idiopathic.

43. The composition of any one of claims 28 to 42, which is administered topically.

44. 44. The composition of claim 43, administered to the inner ear or middle ear.

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