Tiparp inhibitor compounds

Abstract

To provide TIPARP inhibitor compounds.SOLUTION: Provided are compounds of Formula (I) that inhibit the activity of TIPARP, and pharmaceutically acceptable salts thereof, the variables having any of the values defined in the specification.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to compounds that inhibit the activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced poly[adenosine diphosphate (ADP)-ribose] polymerase (TIPARP). [Background technology]

[0002] The poly(ADP-ribose) polymerase (PARP) family of enzymes regulates fundamental cellular processes, including transcription, metabolism, and numerous cellular stress responses, via ADP-ribosylation. TIPARP, also known as PARP7, is a PARP enzyme that negatively regulates type I interferon (IFN) signaling. The gene encoding TIPARP is located on chromosome 3q, a region frequently exhibiting copy number gain in some tumors, resulting in increased expression and suppression of antitumor immune responses. It has been hypothesized that inhibition of TIPARP in such tumors could restore type I IFN signaling, selectively activating antitumor immune responses in the tumor microenvironment and avoiding systemic cytokine production. (Joseph M. Gozgit et al., PARP7 Negatively Regulates the Type I Interferon Response in Cancer Cells and Its Inhibition Triggers Antitumor Immunity, 39 Cancer Cell, Vol. 1214 (2021)). However, off-target effects may prevent some compounds that inhibit TIPARP from fully exerting their immune-mediated mechanisms of action. Compounds with insufficient selectivity for TIPARP may also functionally inhibit other PARP enzymes (eg, PARP1) at concentrations relevant for inhibiting TIPARP, resulting in application-limiting side effects. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Joseph M. Gozgit et al., PARP7 Negatively Regulates the Type I Interferon Response in Cancer Cells and Its Inhibition Triggers Antitumor Immunity, 39 Cancer Cell, Volume 1214 (2021) Summary of the Invention [Problem to be solved by the invention]

[0004] There remains a need in the art for improved compounds that selectively inhibit TIPARP. In particular, there remains a need in the art for compounds that exhibit high potency and high selectivity for TIPARP inhibition while also exhibiting low inhibition of other PARP enzymes. [Means for solving the problem]

[0005] (Summary of the Invention) In one aspect, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0006] [ka] [In the formula, L is selected from the group consisting of a bond and CH; R 1 , R 2 , R 3 and R 4 is independently selected from the group consisting of H and CH3; R 1 , R 2 , R 3 and R 4 at least one and not more than two of Z is selected from the group consisting of CF3 and C(CH3)2OH.

[0007] Another aspect of the present invention provides a compound of the first aspect, or a pharmaceutically acceptable salt thereof, wherein L is a bond.

[0008] Another aspect of the present invention is R 1 or R 4 One of the groups is CH3 or R 1 and R 4 are both CH3 or R 2 or R 3 One of the groups is CH3 or R 2 and R 3 and R are both CH3, or a pharmaceutically acceptable salt thereof.

[0009] Another aspect of the present invention provides the compound of the third aspect, or a pharmaceutically acceptable salt thereof, wherein Z is CF3.

[0010] Another aspect of the present invention provides a compound of the first aspect, or a pharmaceutically acceptable salt thereof, wherein L is CH2.

[0011] Another aspect of the present invention is R 1 or R 4 One of the groups is CH3 or R 1 and R 4 are both CH3 or R 2 or R 3 One of the groups is CH3 or R 2 and R 3 and R are both CH3, or a pharmaceutically acceptable salt thereof.

[0012] Another aspect of the present invention provides a compound of the sixth aspect, or a pharmaceutically acceptable salt thereof, wherein Z is C(CH3)2OH.

[0013] Another aspect of the invention is a compound of the first aspect, 5-[(1S,3s)-3-{(2R)-2-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one, 5-[(1s,3s)-3-{(2R,6S)-2,6-dimethyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one, 5-[(1S,3s)-3-{(3R)-3-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one, 5-[(1s,3s)-3-{(3R,5S)-3,5-dimethyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one, 5-[(1s,3s)-3-{(3R,5S)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-3,5-dimethylpiperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one, 5-[(1R,3s)-3-{(2S)-2-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one, 5-[(1S,3s)-3-{(3R)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-3-methylpiperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one, 5-[(1R,3s)-3-{(3S)-3-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one, 5-[(1S,3s)-3-{(2R)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-2-methylpiperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one, 5-[(1R,3s)-3-{(3S)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-3-methylpiperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one, 5-[(1R,3s)-3-{(2S)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-2-methylpiperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one, 5-{[(1S,3s)-3-{(2R)-2-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one, 5-{[(1s,3s)-3-{(2R,6S)-2,6-dimethyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one, 5-{[(1s,3s)-3-{(3R,5S)-3,5-dimethyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one, 5-{[(1S,3s)-3-{(3R)-3-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one, 5-{[(1R,3s)-3-{(3S)-3-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one, 5-{[(1R,3s)-3-{(2S)-2-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one, 5-{[(1S,3s)-3-{(3R)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-3-methylpiperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one, 5-{[(1S,3s)-3-{(2R)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-2-methylpiperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one, 5-{[(1R,3s)-3-{(3S)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-3-methylpiperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one, 5-{[(1R,3s)-3-{(2S)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-2-methylpiperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one and 5-{[(1s,3s)-3-{(3R,5S)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-3,5-dimethylpiperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one The present invention provides a compound selected from the group consisting of:

[0014] Another aspect of the present invention provides 5-[(1S,3s)-3-{(2R)-2-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one or a pharmaceutically acceptable salt thereof.

[0015] Another aspect of the present invention provides 5-[(1S,3s)-3-{(2R)-2-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one.

[0016] Another aspect of the present invention provides pharmaceutically acceptable salts of 5-[(1S,3s)-3-{(2R)-2-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one.

[0017] Another aspect of the present invention provides a pharmaceutical composition comprising a compound of the first aspect, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0018] Another aspect of the invention provides a method for treating head and neck squamous cell carcinoma (HNSCC), comprising administering to a human patient in need thereof a compound of the first aspect, or a pharmaceutically acceptable salt thereof. [Brief explanation of the drawings]

[0019] [Figure 1] Example 1 demonstrates in vivo efficacy in reducing tumor volume in the MC-38 (Ker) syngeneic model. [Figure 2] 1 shows the induction of IFNβ in plasma and tumors after administration of Example 1 in an MC-38 (Ker) syngeneic model. [Figure 3] Example 1 demonstrates in vivo efficacy in reducing tumor volume in the NCI-H1373 xenograft model. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention provides compounds that inhibit the activity of TIPARP.

[0021] The compounds disclosed herein, including any intermediates, may contain one or more variables that occur more than once in any substituent or formula herein. The definition of a variable at each occurrence is independent of its definition at other occurrences.

[0022] The compounds disclosed herein, including any intermediates, have been named using the ACD / Name 2023.1.2 (File Version N25E41, Build 134315, July 12, 2023) software program and / or by using the Struct=Name naming algorithm as part of CHEMDRAW® Professional v.20.1.1.125.

[0023] The compounds disclosed herein, including any intermediates, can possess multiple tautomeric forms and exist as equilibrium mixtures thereof. The formulas and structures found herein represent only one of the possible tautomeric forms, but should be considered to encompass both the individual tautomeric forms and mixtures thereof.

[0024] definition As used in this specification and the appended claims, unless specified to the contrary, the following terms have the meanings indicated: The phrase "pharmaceutical composition" refers to a composition suitable for administration in medical use.

[0025] The phrase "pharmaceutically acceptable salt" refers to a salt that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, or the like, commensurate with a reasonable benefit / risk balance.

[0026] The phrase "therapeutically effective amount" refers to the amount of a compound or a pharmaceutically acceptable salt thereof that, when administered therapeutically to a particular human patient or population of human patients, is sufficient to prevent the onset of, or alleviate to some extent, one or more of the symptoms of the condition or disorder being treated.

[0027] The terms "treat," "treating," and "treatment," as used herein, refer to a method of alleviating or inhibiting a disease and / or its associated symptoms.

[0028] compound The present disclosure relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0029] [ka] [In the formula, L is selected from the group consisting of a bond and CH; R 1 , R 2 , R 3 and R 4 is independently selected from the group consisting of H and CH3; R 1 , R 2 , R 3 and R 4 at least one and not more than two of Z is selected from the group consisting of CF3 and C(CH3)2OH.

[0030] Exemplary compounds of formula (I) are set forth below in Table 1. It should be understood that if there is a discrepancy between the name of any compound disclosed herein and a structure found in Table 1, the structure in Table 1 shall control.

[0031] [Table 1] TIFF2025169211000004.tif208162TIFF2025169211000005.tif154162

[0032] The compounds of formula (I) can be used in the form of pharmaceutically acceptable salts. Such compounds may contain either basic or acidic functional groups, or both, and can be converted into pharmaceutically acceptable salts, if desired, by using an appropriate acid or base.

[0033] The compounds of formula (I) exhibit a combination of functional properties, including, but not limited to, high potency for inhibiting TIPARP, high selectivity for inhibiting TIPARP, and low inhibition of PARP1 and / or PARP2 compared to other PARP family enzymes. The compounds of formula (I) can exhibit high potency and selectivity for inhibiting TIPARP. Furthermore, the compounds of formula (I) can exhibit low inhibition of PARP enzymes other than TIPARP. As used herein, low inhibition of PARP enzymes other than TIPARP means that the compounds of formula (I) have insufficient potency for inhibiting PARP enzymes other than TIPARP. For example, the compounds of formula (I) can exhibit low inhibition of PARP1, PARP2, or a combination thereof.

[0034] Methods of Making Exemplary Compounds The compounds of the present disclosure can be better understood in connection with the following synthetic schemes and methods, which illustrate means by which the compounds can be prepared. The compounds of the present disclosure can be prepared by a variety of synthetic procedures. Representative synthetic procedures are shown in Schemes 1-8. The variables have any of the values ​​defined herein, for example, in the Summary of the Invention.

[0035] Synthesis scheme

[0036] [ka]

[0037] As shown in Scheme 1, methyl 3-(6-methoxy-5-(trifluoromethyl)pyridin-3-yl)cyclobutanecarboxylate can be prepared by reacting 5-bromo-2-methoxy-3-(trifluoromethyl)pyridine with methyl 3-bromocyclobutanecarboxylate. The reaction can be carried out in the presence of a cross-coupling catalyst, such as (4,4'-dtbbpy)NiCl, a photocatalyst, such as (Ir[dF(CF)ppy](dtbpy))PF, 1,1,1,3,3,3-hexamethyl-2-(trimethylsilyl)trisilane, 2,6-lutidine, and LED blue light. The reaction can be carried out under an inert atmosphere in a solvent, such as 1,2-dimethoxyethane. The trans isomer can be separated from the cis isomer via flash chromatography.

[0038] [ka]

[0039] As shown in Scheme 2, 5-bromo-2-methoxy-3-(trifluoromethyl)pyridine can be reacted with methyl 3-((4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)methylene)cyclobutane-1-carboxylate under Suzuki coupling conditions in the presence of a catalyst such as Pd(dppf)Cl·CHCl and a base such as potassium carbonate to provide methyl 3-((6-methoxy-5-(trifluoromethyl)pyridin-3-yl)methylene)cyclobutane-1-carboxylate. The reaction is typically carried out under an inert atmosphere at elevated temperature and in a solvent such as dioxane, water, or a mixture thereof.

[0040] Methyl 3-((6-methoxy-5-(trifluoromethyl)pyridin-3-yl)methylene)cyclobutane-1-carboxylate can be treated with hydrogen gas in the presence of a catalyst such as Pd / C to give methyl 3-((6-methoxy-5-(trifluoromethyl)pyridin-3-yl)methyl)cyclobutane-1-carboxylate, which can contain a mixture of separable cis and trans isomers. The trans isomer can be separated from the cis isomer via flash chromatography. The reaction is typically carried out at ambient temperature in a solvent such as tetrahydrofuran.

[0041] [ka]

[0042] Scheme 3 illustrates the synthesis of a compound of formula (3), 1 , R 2 , R 3 , R 4 and Z are as described herein. A compound of formula (1) can be reacted with a compound of formula (2) in the presence of a base, such as triethylamine, to give a compound of formula (3). The reaction is typically carried out in a solvent, such as acetonitrile.

[0043] [ka]

[0044] Scheme 4 illustrates the synthesis of a compound of formula (3), where R 1 , R 2 , R 3 , R 4and Z are as described herein. A compound of formula (4) (wherein PG is a protecting group, e.g., a BOC group) can be reacted with a compound of formula (2) in the presence of a base, e.g., triethylamine or N,N-diisopropylethylamine, to give a compound of formula (5). The reaction is typically carried out in a solvent, e.g., acetonitrile or dimethylacetamide. When PG is a BOC group, the compound of formula (5) can be treated with an acid, e.g., trifluoroacetic acid or hydrogen chloride, in a solvent, e.g., dichloromethane, acetonitrile, dioxane, or a mixture thereof, to give a compound of formula (3).

[0045] [ka]

[0046] Scheme 5 illustrates the synthesis of a compound of formula (8) 1 , R 2 , R 3 and R 4 is as described herein. The compound of formula (4) can be reacted with methyl 2-chloropyrimidine-5-carboxylate in the presence of a base, such as potassium carbonate, to give a compound of formula (6). The reaction is typically carried out at elevated temperature in a solvent, such as N,N-dimethylformamide.

[0047] Alternatively, the compound of formula (4) can be reacted with methyl 2-chloropyrimidine-5-carboxylate in N-methyl-2-pyrrolidinone to give the compound of formula (6), which is typically carried out at elevated temperatures.

[0048] Compounds of formula (6) can be treated with methylmagnesium bromide solution to give compounds of formula (7). The reaction is usually carried out under nitrogen at low temperature in a solvent such as tetrahydrofuran. When PG is a BOC group, compounds of formula (7) can be treated with an acid such as trifluoroacetic acid or hydrogen chloride in a solvent such as dichloromethane, ethyl acetate, acetonitrile, dioxane, or a mixture thereof to give compounds of formula (8).

[0049] [ka]

[0050] As shown in Scheme 6, treatment of compounds of formula (9) (which can be prepared as described in Schemes 1-2) with lithium hydroxide or sodium hydroxide in water can provide compounds of formula (10). The reaction is typically carried out at ambient temperature in a solvent such as methanol, tetrahydrofuran, or a mixture thereof.

[0051] [ka]

[0052] As shown in Scheme 7, compounds of formula (10) can be treated with para-toluenesulfonic acid monohydrate and lithium chloride to give compounds of formula (11). The reaction is typically carried out at elevated temperature in a solvent such as N,N-dimethylformamide.

[0053] Compounds of formula (I) can be prepared by reacting a compound of formula (11), where L is a bond or CH, with a compound of formula (3) in the presence of propanephosphonic anhydride and a base such as N,N-diisopropylethylamine. The reaction is typically carried out at ambient temperature in a solvent such as N,N-dimethylformamide.

[0054] Alternatively, when L is a bond or CH, compounds of formula (I) can be prepared by reacting a compound of formula (11) with a compound of formula (3) in the presence of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate and a base such as N,N-diisopropylethylamine. The reaction is typically carried out at ambient or elevated temperature in a solvent such as N,N-dimethylformamide.

[0055] [ka]

[0056] As shown in Scheme 8, compounds of formula (12) can be prepared by reacting a compound of formula (10), where L is a bond or CH, with a compound of formula (3) in the presence of propanephosphonic anhydride and a base such as N,N-diisopropylethylamine. The reaction is typically carried out at ambient temperature in a solvent such as N,N-dimethylformamide.

[0057] Compounds of formula (12) can be treated with para-toluenesulfonic acid monohydrate and lithium chloride to give compounds of formula (I). The reaction is typically carried out under an inert atmosphere at elevated temperature in a solvent such as N,N-dimethylformamide.

[0058] Specific procedures are also provided in the Synthetic Examples section. Unless otherwise noted, starting materials and reagents are commercially available or may be prepared from commercially available materials by one of ordinary skill in the art using methods known in the art.

[0059] Pharmaceutical Composition When employed as pharmaceuticals, the compounds of the present disclosure can be administered in the form of pharmaceutical compositions, which can include a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, together with one or more pharmaceutically acceptable excipients.

[0060] How to use The compound of formula (I) or a pharmaceutically acceptable salt thereof and pharmaceutical compositions comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof can be administered to a human patient suffering from head and neck squamous cell carcinoma (HNSCC). The term "administering" refers to the method of contacting a human patient with a compound.

[0061] The compounds of formula (I) or pharmaceutically acceptable salts thereof may also be used in the preparation of a medicament, which may be used in the treatment of HNSCC. [Example]

[0062] Synthesis Examples

[0063] [Example 1] 5-[(1S,3s)-3-{(2R)-2-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one

[0064] Example 1A (1s,3s)-Methyl 3-(6-methoxy-5-(trifluoromethyl)pyridin-3-yl)cyclobutanecarboxylate In Example 1A, 5-bromo-2-methoxy-3-(trifluoromethyl)pyridine (5.12 g, 20.0 mmol), methyl 3-bromocyclobutanecarboxylate (4.63 g, 24.0 mmol), (4,4'-dtbbpy)NiCl (398 mg, 1.00 mmol, CAS 1034901-50-2), (Ir[dF(CF)ppy](dtbpy))PF (224 mg, 0.200 mmol, CAS 870987-63-6), 1,1,1,3,3,3-hexamethyl-2-(trimethylsilyl)trisilane (5.97 g, 24.0 mmol), and 2,6-lutidine (5.82 mL, 50.0 mmol) were combined with 1,2-dimethoxyethane (80 mL). The reaction mixture was purged with nitrogen for 5 minutes and stirred under irradiation (450 nm LED blue light) for 16 hours. The reaction mixture was partitioned between ethyl acetate and water. The organic layer was washed with saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by flash chromatography (silica gel, 0-30% ethyl acetate in heptane) to give two fractions. The first fraction to elute was the stereoisomer with the trans configuration. The second fraction to elute was the title compound with the cis configuration. MS (ESI) m / z 290 (M+H) + .

[0065] Example 1B (1s,3s)-3-(6-methoxy-5-(trifluoromethyl)pyridin-3-yl)cyclobutanecarboxylic acid To a solution of Example 1A (900 mg, 3.11 mmol) in a mixture of tetrahydrofuran (6.0 mL) / methanol (6.0 mL) / water (2.0 mL) was added sodium hydroxide (622 mg, 15.6 mmol). The reaction mixture was stirred at room temperature for 1 hour and diluted with water. The pH was adjusted to 4 by the addition of 1 M aqueous HCl and extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated in vacuo to give the title compound. MS (ESI) m / z 276 (M+H) + .

[0066] Example 1C (1s,3s)-3-(6-oxo-5-(trifluoromethyl)-1,6-dihydropyridin-3-yl)cyclobutanecarboxylic acid Example 1B (730 mg, 2.65 mmol), para-toluenesulfonic acid monohydrate (1.01 g, 5.30 mmol), and lithium chloride (562 mg, 13.3 mmol) were combined in N,N-dimethylformamide (15 mL). The reaction mixture was stirred at 100° C. for 10 hours, cooled to room temperature, and partitioned between ethyl acetate and water. The organic layer was washed with saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated in vacuo to give the title compound. MS (ESI) m / z 262 (M+H) + .

[0067] Example 1D (R)-2-(3-methylpiperazin-1-yl)-5-(trifluoromethyl)pyrimidine In Example 1D, a solution of (R)-2-methylpiperazine (2.50 g, 25.0 mmol) and triethylamine (6.96 mL, 49.9 mmol) in acetonitrile (80 mL) was cooled to 0° C. in an ice bath and treated with 2-chloro-5-(trifluoromethyl)pyrimidine (4.56 g, 25.0 mmol). The reaction mixture was stirred overnight at room temperature. The insoluble salt material was filtered and washed with a small amount of acetonitrile. The filtrate was concentrated under vacuum. The corresponding residue was triturated with diethyl ether (25 mL), filtered, and dried under vacuum to give the title compound. The filtrate was concentrated under vacuum to approximately 8 mL and placed in a refrigerator overnight, and the mixture was filtered. The material was dried under vacuum to give additional title compound. MS (APCI) m / z 288 (M+CH3CN+H). + .

[0068] Example 1E 5-[(1S,3s)-3-{(2R)-2-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one To a mixture of Example 1C (20.9 mg, 0.0800 mmol), Example 1D (22.6 mg, 0.0920 mmol), and N,N-diisopropylethylamine (0.0840 mL, 0.480 mmol) in N,N-dimethylformamide (1.0 mL) was added 50% propanephosphonic anhydride in N,N-dimethylformamide (102 mg, 0.160 mmol). The reaction mixture was stirred at room temperature for 1 hour and partitioned between ethyl acetate and water. The organic layer was washed with saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash chromatography to provide the title compound. 1 H NMR (400 MHz, DMSO-d6, 90℃) δ ppm 11.84 (s, br, 1H), 8.64 (d, J = 0.8 Hz, 2H), 7.77 (d, J = 2.6 Hz, 1H), 7.42 (d, J = 2.6 Hz, 1H), 4.64 - 4.29 (m, 3H), 3.96 (s, br, 1H), 3.38 - 3.24 (m, 3H), 3.23 - 3.04 (m, 2H), 2.54 - 2.45 (m, 2H), 2.25 - 2.09 (m, 2H), 1.08 (d, J = 6.7 Hz, 3H). MS (ESI) m / z 490 (M+H) + .

[0069] [Example 2] 5-[(1s,3s)-3-{(2R,6S)-2,6-dimethyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one Example 2 was prepared according to the procedure used for the preparation of Example 13F, substituting Example 1C for Example 13D to give the title compound. 1H NMR (400 MHz, DMSO-d6, 90℃) δ ppm 11.84 (s, br, 1H), 8.65 (d, J = 0.8 Hz, 2H), 7.79 (d, J = 2.6 Hz, 1H), 7.43 (d, J = 2.6 Hz, 1H), 4.60 - MS (ESI) m / z 504 (M+H) + .

[0070] [Example 3] 5-[(1S,3s)-3-{(3R)-3-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one

[0071] Example 3A (R)-tert-butyl 3-methyl-4-(5-(trifluoromethyl)pyrimidin-2-yl)piperazine-1-carboxylate In Example 3A, a mixture of 2-chloro-5-(trifluoromethyl)pyrimidine (5.00 g, 27.4 mmol), (R)-tert-butyl 3-methylpiperazine-1-carboxylate (5.49 g, 27.4 mmol), and triethylamine (7.64 mL, 54.8 mmol) in acetonitrile (100 mL) was stirred at 80° C. for 3 hours and cooled to room temperature. The insoluble salt material was filtered and washed with a small amount of acetonitrile. The filtrate was concentrated in vacuo. Water was added to the residue, and the mixture was extracted twice with ethyl acetate. The combined organic layers were washed with saturated aqueous sodium chloride, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated in vacuo. The residue was purified by flash chromatography (silica gel, 0-25% ethyl acetate in heptane) to give the title compound. MS (APCI) m / z 347 (M+H) + .

[0072] Example 3B (R)-2-(2-methylpiperazin-1-yl)-5-(trifluoromethyl)pyrimidine A solution of Example 3A (8.02 g, 23.2 mmol) in dioxane (50 mL) was treated with 4 M hydrogen chloride in dioxane (57.9 ml, 232 mmol) at 0° C. The reaction mixture was stirred at room temperature overnight and filtered. The filter cake was washed with diethyl ether and dried under vacuum to give the title compound as the hydrochloride salt. MS (ESI) m / z 247 (M+H) + .

[0073] Example 3C 5-[(1S,3s)-3-{(3R)-3-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one To a mixture of Example 1C (20 mg, 0.077 mmol), Example 3B (22 mg, 0.077 mmol), and N,N-diisopropylethylamine (0.080 mL, 0.46 mmol) in N,N-dimethylformamide (1.0 mL) was added 50% propanephosphonic anhydride in N,N-dimethylformamide (97 mg, 0.15 mmol). The reaction mixture was stirred at room temperature for 1 hour and purified by reverse-phase HPLC (C18, 20-100% acetonitrile in water with 0.1% trifluoroacetic acid). The acetonitrile was removed by evaporation, and the mixture was neutralized with saturated sodium bicarbonate water. The mixture was extracted with ethyl acetate, and the organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated in vacuo to give the title compound. 1 H NMR (400 MHz, DMSO-d 6,90℃) δ ppm 11.84 (s, 1H), 8.66 (d, J = 0.8 Hz, 2H), 7.78 (d, J = 2.6 Hz, 1H), 7.43 (d, J = 2.6 Hz, 1H), 4.94 - 4.85 (m, 1H), 4.52 - 4.43 (m, 1H), 4.14 (s, br, 1H), 3.85 (s, br, 1H), 3.39 - 3.23 (m, 3H), 3.07 (s, br, 2H), 2.55 - 2.49 (m, 2H), 2.24 - 2.14 (m, 2H), 1.14 (d, J = 6.7 Hz, 3H). MS (ESI) m / z 490 (M+H) + .

[0074] [Example 4] 5-[(1s,3s)-3-{(3R,5S)-3,5-dimethyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one

[0075] Example 4A (3R,5S)-tert-butyl 3,5-dimethyl-4-(5-(trifluoromethyl)pyrimidin-2-yl)piperazine-1-carboxylate To a solution of tert-butyl (3R,5S)-3,5-dimethylpiperazine-1-carboxylate (2.00 g, 9.33 mmol) in dimethylacetamide (18.7 mL) was added N,N-diisopropylethylamine (2.45 mL, 14.0 mmol) and 2-chloro-5-(trifluoromethyl)pyrimidine (1.70 g, 9.33 mmol). The reaction mixture was stirred at 90 °C for 18 hours, cooled to room temperature, diluted with water, and extracted with ethyl acetate. The organic layer was washed twice with saturated aqueous sodium chloride, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated in vacuo. The residue was purified by flash chromatography (silica gel, 1-20% ethyl acetate in heptane) to give the title compound. MS (ESI) m / z 305 (M-56+H). + .

[0076] Example 4B 2-((2R,6S)-2,6-dimethylpiperazin-1-yl)-5-(trifluoromethyl)pyrimidine A solution of Example 4A (2.18 g, 6.05 mmol) in dioxane (20.2 mL) was treated with 4 M hydrogen chloride in dioxane (15.1 ml, 60.4 mmol). The reaction mixture was stirred overnight at room temperature, filtered, and the filter cake was dried under vacuum to give the title compound as the hydrochloride salt. MS (ESI) m / z 261 (M+H). + .

[0077] Example 4C 5-[(1s,3s)-3-{(3R,5S)-3,5-dimethyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one Example 4C was prepared according to the procedure used for the preparation of Example 3C, substituting Example 4B for Example 3B to provide the title compound. 1 H NMR (400 MHz, DMSO-d 6, 90℃) δ ppm 11.82 (s, 1H), 8.68 (s, 2H), 7.80 (d, J = 2.6 Hz, 1H), 7.44 (d, J = 2.6 Hz, 1H), 4.89 - 4.79 (m, 2H), 4.05 (s, br, 2H), 3.45 - 3.24 (m, 2H), 3.11 (s, br, 2H), 2.57 - 2.50 (m, 2H), 2.29 - 2.16 (m, 2H), 1.19 (d, J = 6.9 Hz, 6H). MS (ESI) m / z 504 (M+H) + .

[0078] [Example 5] 5-[(1s,3s)-3-{(3R,5S)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-3,5-dimethylpiperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one Example 5 was prepared according to the procedure used for the preparation of Example 1E, substituting Example 22C for Example 1D. Purification by flash chromatography afforded the title compound. 1 H NMR (400 MHz, DMSO-d 6, 90℃) δ ppm 11.84 (s, br, 1H), 8.44 (s, 2H), 7.79 (d, J = 2.6 Hz, 1H), 7.44 (d, J = 2.6 Hz, 1H), 4.84 - 4.67 (m, 3H), 3.97 (s, br, MS (ESI) m / z 476 (M-H2O+H) + .

[0079] [Example 6] 5-[(1R,3s)-3-{(2S)-2-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one

[0080] Example 6A (S)-tert-butyl 2-methyl-4-(5-(trifluoromethyl)pyrimidin-2-yl)piperazine-1-carboxylate In Example 6A, (S)-tert-butyl 2-methylpiperazine-1-carboxylate (4.00 g, 20.0 mmol), 2-chloro-5-(trifluoromethyl)pyrimidine (3.65 g, 20.0 mmol), and triethylamine (5.57 mL, 39.9 mmol) were combined in acetonitrile (80 mL). The reaction mixture was stirred at room temperature overnight. The insoluble salt material was filtered. The filtrate was concentrated under vacuum. The residue was triturated with water, filtered, rinsed with water, and dried under vacuum to give the title compound. MS (ESI) m / z 291 (M-56+H). + .

[0081] Example 6B (S)-2-(3-methylpiperazin-1-yl)-5-(trifluoromethyl)pyrimidine To a solution of Example 6A (6.20 g, 17.9 mmol) in acetonitrile (50 mL) was added 4 M hydrogen chloride in dioxane (22.4 mL, 89.6 mmol). The reaction mixture was stirred at room temperature for 4 hours, diluted with tert-butyl methyl ether, and filtered. The material was dried under vacuum to give the title compound as the hydrochloride salt. MS (ESI) m / z 247 (M+H) + .

[0082] Example 6C 5-[(1R,3s)-3-{(2S)-2-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one Example 6C was prepared according to the procedure used for the preparation of Example 1E, substituting Example 6B for Example 1D to provide the title compound. 1 H NMR (400 MHz, DMSO-d6, 90℃) δ ppm 11.84 (s, br, 1H), 8.64 (d, J = 0.9 Hz, 2H), 7.77 (d, J = 2.6 Hz, 1H), 7.42 (d, J = 2.6 Hz, 1H), 4.62 - 4.32 (m, 3H), 3.97 (s, br, 1H), 3.37 - 3.24 (m, 3H), 3.21 - 3.06 (m, 2H), 2.54 - 2.44 (m, 2H), 2.26 - 2.11 (m, 2H), 1.08 (d, J = 6.7 Hz, 3H). MS (ESI) m / z 490 (M+H) + .

[0083] [Example 7] 5-[(1S,3s)-3-{(3R)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-3-methylpiperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one

[0084] Example 7A Methyl (R)-2-(4-(tert-butoxycarbonyl)-2-methylpiperazin-1-yl)pyrimidine-5-carboxylate To a solution of methyl 2-chloropyrimidine-5-carboxylate (3.00 g, 17.4 mmol) in N,N-dimethylformamide (30 mL) were added potassium carbonate (7.21 g, 52.2 mmol) and (R)-tert-butyl 3-methylpiperazine-1-carboxylate (3.48 g, 17.4 mmol). The reaction mixture was stirred at 80° C. for 12 hours and filtered. The filtrate was slowly poured into ice water (500 mL). The formed material was collected via filtration. The filter cake was washed with water and dried under vacuum to give the title compound.

[0085] Example 7B tert-Butyl (R)-4-(5-(2-hydroxypropan-2-yl)pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate To a solution of Example 7A (3.50 g, 10.4 mmol) in tetrahydrofuran (35 mL) was added dropwise over 30 minutes at -78°C under nitrogen, methylmagnesium bromide solution (31.2 mL, 31.2 mmol). The reaction mixture was allowed to warm slowly to 25°C overnight. The reaction mixture was quenched with saturated aqueous ammonium chloride and extracted three times with ethyl acetate. The combined organic layers were washed with saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated in vacuo. The residue was purified by flash chromatography (silica gel, petroleum ether / ethyl acetate = 100:0 to 35:65) to give the title compound. MS (ESI) m / z 337 (M+H) + .

[0086] Example 7C (R)-2-(2-(2-methylpiperazin-1-yl)pyrimidin-5-yl)propan-2-ol A solution of Example 7B (2.60 g, 7.73 mmol) in dichloromethane (30 mL) was treated with trifluoroacetic acid (10 mL). The reaction mixture was stirred at room temperature for 30 minutes, quenched by the addition of saturated aqueous sodium bicarbonate, and concentrated to remove the organic solvent. The aqueous residue was lyophilized to give the crude product, which was purified by reverse-phase HPLC (C18, 5-45% acetonitrile in water containing 10 mM ammonium bicarbonate) to give the title compound. MS (ESI) m / z 237 (M+H) + .

[0087] Example 7D 5-[(1S,3s)-3-{(3R)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-3-methylpiperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one Example 7D was prepared according to the procedure used for the preparation of Example 1E, substituting Example 7C for Example 1D. Purification by flash chromatography afforded the title compound. 1 H NMR (400 MHz, DMSO-d 6, 90℃) δ ppm 11.83 (s, br, 1H), 8.43 (s, 2H), 7.78 (d, J = 2.6 Hz, 1H), 7.42 (d, J = 2.6 Hz, 1H), 4.87 - 4.77 (m, 1H), 4.73 (s, 1H), 4.42 - 4.35 (m, 1H), 3.97 (s, br, 3H), 3.40 - 3.06 (m, 4H), 2.55 - 2.47 (m, 2H), 2.24 - 2.13 (m, 2H), 1.43 (s, 6H), 1.07 (d, J = 6.6 Hz, 3H). MS (ESI) m / z 462 (M-H2O+H) + .

[0088] [Example 8] 5-[(1R,3s)-3-{(3S)-3-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one

[0089] Example 8A (S)-tert-butyl 3-methyl-4-(5-(trifluoromethyl)pyrimidin-2-yl)piperazine-1-carboxylate In Example 8A, (S)-tert-butyl 3-methylpiperazine-1-carboxylate (4.00 g, 20.0 mmol), 2-chloro-5-(trifluoromethyl)pyrimidine (3.65 g, 20.0 mmol), and triethylamine (5.57 mL, 39.9 mmol) were combined in acetonitrile (80 mL). The reaction mixture was stirred at 80° C. for 4 hours and cooled to room temperature. The insoluble salt material was filtered. The filtrate was concentrated under vacuum. The residue was purified by flash chromatography (silica gel, 0-50% ethyl acetate in heptane) to give the title compound. MS (ESI) m / z 347 (M+H) + .

[0090] Example 8B (S)-2-(2-methylpiperazin-1-yl)-5-(trifluoromethyl)pyrimidine To a solution of Example 8A (5.80 g, 16.8 mmol) in acetonitrile (50 mL) was added 4 M hydrogen chloride in dioxane (21 mL, 84 mmol). The reaction mixture was stirred at room temperature for 4 hours and filtered. The filter cake was rinsed with a small amount of acetonitrile and dried under vacuum to give the title compound as the hydrochloride salt. The filtrate was concentrated and dried under vacuum to give additional title compound as the hydrochloride salt. MS (ESI) m / z 247 (M+H) + .

[0091] Example 8C 5-[(1R,3s)-3-{(3S)-3-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one Example 8C was prepared according to the procedure used for the preparation of Example 3C, substituting Example 8B for Example 3B to provide the title compound. 1 H NMR (400 MHz, DMSO-d 6, 90℃) δ ppm 11.83 (s, 1H), 8.66 (d, J = 0.8 Hz, 2H), 7.78 (d, J = 2.6 Hz, 1H), 7.43 (d, J = 2.6 Hz, 1H), 4.94 - 4.84 (m, 1H), 4.53 - 4.43 (m, 1H), 4.14 (s, br, 1H), 3.82 (s, br, 1H), 3.39 - 3.23 (m, 3H), 3.08 (s, br, 2H), 2.56 - 2.49 (m, 2H), 2.24 - 2.14 (m, 2H), 1.14 (d, J = 6.7 Hz, 3H). MS (ESI) m / z 490 (M+H) + .

[0092] [Example 9] 5-[(1S,3s)-3-{(2R)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-2-methylpiperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one

[0093] Example 9A Methyl (R)-2-(4-(tert-butoxycarbonyl)-3-methylpiperazin-1-yl)pyrimidine-5-carboxylate Example 9A was prepared according to the procedure used for the preparation of Example 7A, using tert-butyl (R)-2-methylpiperazine-1-carboxylate instead of (R)-tert-butyl 3-methylpiperazine-1-carboxylate to obtain the title compound. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.79 (s, 2H), 4.58 - 4.45 (m, 2H), 4.28 - 4.19 (m, 1H), 3.80 (s, 3H), 3.29 (br dd, J = 4.0, 13.4 Hz, 2H), 3.13 - 3.07 (m, 2H), 1.41 (s, 9H), 1.02 (d, J = 6.7 Hz, 3H).

[0094] Example 9B tert-Butyl (R)-4-(5-(2-hydroxypropan-2-yl)pyrimidin-2-yl)-2-methylpiperazine-1-carboxylate Example 9B was prepared according to the procedure used for the preparation of Example 7B, substituting Example 9A for Example 7A to give the title compound. MS (ESI) m / z 337 (M+H) + .

[0095] Example 9C (R)-2-(2-(3-methylpiperazin-1-yl)pyrimidin-5-yl)propan-2-ol Example 9C was prepared according to the procedure used for the preparation of Example 7C, substituting Example 9B for Example 7B to give the title compound. MS (ESI) m / z 237 (M+H) + .

[0096] Example 9D 5-[(1S,3s)-3-{(2R)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-2-methylpiperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one Example 9D was prepared according to the procedure used for the preparation of Example 1E, substituting Example 9C for Example 1D. Purification by flash chromatography afforded the title compound. 1 H NMR (400 MHz, DMSO-d 6,90℃) δ ppm 11.83 (s, br, 1H), 8.42 (s, 2H), 7.77 (d, J = 2.6 Hz, 1H), 7.42 (d, J = 2.6 Hz, 1H), 4.73 (s, 1H), 4.57 - 4.29 (m, 3H), MS (ESI) m / z 462 (M-H2O+H) + .

[0097] [Example 10] 5-[(1R,3s)-3-{(3S)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-3-methylpiperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one

[0098] Example 10A Methyl (S)-2-(4-(tert-butoxycarbonyl)-2-methylpiperazin-1-yl)pyrimidine-5-carboxylate Example 10A was prepared according to the procedure used for the preparation of Example 7A, using tert-butyl (S)-3-methylpiperazine-1-carboxylate instead of (R)-tert-butyl 3-methylpiperazine-1-carboxylate to give the title compound. MS (ESI) m / z 281 (M-56+H). + .

[0099] Example 10B tert-Butyl (S)-4-(5-(2-hydroxypropan-2-yl)pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate Example 10B was prepared according to the procedure used for the preparation of Example 7B, substituting Example 10A for Example 7A to give the title compound. MS (ESI) m / z 337 (M+H) +.

[0100] Example 10C (S)-2-(2-(2-methylpiperazin-1-yl)pyrimidin-5-yl)propan-2-ol Example 10C was prepared according to the procedure used for the preparation of Example 7C, substituting Example 10B for Example 7B to give the title compound. MS (ESI) m / z 237 (M+H). + .

[0101] Example 10D 5-[(1R,3s)-3-{(3S)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-3-methylpiperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one Example 10D was prepared according to the procedure used for the preparation of Example 1E, substituting Example 10C for Example 1D. Purification by flash chromatography afforded the title compound. 1 H NMR (400 MHz, DMSO-d 6, 90℃) δ ppm 11.84 (s, br, 1H), 8.43 (s, 2H), 7.78 (d, J = 2.6 Hz, 1H), 7.42 (d, J = 2.6 Hz, 1H), 4.88 - 4.78 (m, 1H), 4.73 (s, 1H), 4.43 - 4.35 (m, 1H), 3.97 (s, br, 3H), 3.40 - 3.04 (m, 4H), 2.55 - 2.45 (m, 2H), 2.25 - 2.12 (m, 2H), 1.43 (s, 6H), 1.07 (d, J = 6.6 Hz, 3H). MS (ESI) m / z 462 (M-H2O+H) + .

[0102] [Example 11] 5-[(1R,3s)-3-{(2S)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-2-methylpiperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one

[0103] Example 11A Methyl (S)-2-(4-(tert-butoxycarbonyl)-3-methylpiperazin-1-yl)pyrimidine-5-carboxylate Example 11A was prepared according to the procedure used for the preparation of Example 7A, using tert-butyl (S)-2-methylpiperazine-1-carboxylate instead of (R)-tert-butyl 3-methylpiperazine-1-carboxylate to obtain the title compound. 1 H NMR (400 MHz, DMSO-d6) δ ppm 8.79 (s, 2H), 4.59 - 4.45 (m, 2H), 4.24 (br d, J = 3.0 Hz, 1H), 3.80 (m, 4H), 3.31 - 3.26 (m, 1H), 3.09 (d, J = 9.5 Hz, 2H), 1.41 (s, 9H), 1.02 (d, J = 6.7 Hz, 3H).

[0104] Example 11B tert-Butyl (S)-4-(5-(2-hydroxypropan-2-yl)pyrimidin-2-yl)-2-methylpiperazine-1-carboxylate Example 11B was prepared by following the procedure used for the preparation of Example 7B, substituting Example 11A for Example 7A to give the title compound. MS (ESI) m / z 337 (M+H). + .

[0105] Example 11C (S)-2-(2-(3-methylpiperazin-1-yl)pyrimidin-5-yl)propan-2-ol Example 11C was prepared according to the procedure used for the preparation of Example 7C, substituting Example 11B for Example 7B to give the title compound. MS (ESI) m / z 237 (M+H). + .

[0106] [Example 11D] 5-[(1R,3s)-3-{(2S)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-2-methylpiperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one Example 11D was prepared according to the procedure used for the preparation of Example 1E, substituting Example 11C for Example 1D. Purification by flash chromatography afforded the title compound. 1 H NMR (400 MHz, DMSO-d 6, 90℃) δ ppm 11.85 (s, br, 1H), 8.42 (s, 2H), 7.77 (d, J = 2.6 Hz, 1H), 7.42 (d, J = 2.6 Hz, 1H), 4.73 (s, 1H), 4.57 - 4.27 (m, 3H), 3.97 (s, br, 2H), 3.51 - 3.41 (m, 1H), 3.35 - 3.08 (m, 3H), 2.53 - 2.44 (m, 2H), 2.25 - 2.10 (m, 2H), 1.43 (s, 6H), 1.09 (d, J = 6.6 Hz, 3H). MS (ESI) m / z 462 (M-H2O+H) + .

[0107] [Example 12] 5-{[(1S,3s)-3-{(2R)-2-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one Example 12 was prepared according to the procedure used for the preparation of Example 1E, substituting Example 13D for Example 1C to give the title compound. 1H NMR (400 MHz, DMSO-d6, 90℃) δ ppm 11.79 (s, br, 1H), 8.64 (d, J = 0.9 Hz, 2H), 7.71 (d, J = 2.6 Hz, 1H), 7.39 (d, J = 2.6 Hz, 1H), 4.59 - 4.34 (m, 3H), 3.95 (s, br, 1H), 3.32 - 3.03 (m, 4H), 2.45 (d, J = 7.0 Hz, 2H), 2.42 - 2.34 (m, 1H), 2.25 - 2.15 (m, 2H), 1.93 - 1.79 (m, 2H), 1.06 (d, J = 6.7 Hz, 3H). MS (ESI) m / z 504 (M+H) + .

[0108] [Example 13] 5-{[(1s,3s)-3-{(2R,6S)-2,6-dimethyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one

[0109] Example 13A Methyl 3-((6-methoxy-5-(trifluoromethyl)pyridin-3-yl)methylene)cyclobutanecarboxylate In Example 13A, 5-bromo-2-methoxy-3-(trifluoromethyl)pyridine (1.02 g, 4.00 mmol), methyl 3-((4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)methylene)cyclobutanecarboxylate (1.01 g, 4.00 mmol), Pd(dppf)ClCHCl ([1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II), dichloromethane complex, 163 mg, 0.200 mmol), and potassium carbonate (1.11 g, 8.00 mmol) were combined in a mixture of dioxane (9.0 mL) and water (3.0 mL). The reaction mixture was purged with nitrogen for 5 minutes, stirred at 90° C. for 16 hours, cooled to room temperature, and partitioned between ethyl acetate and water. The organic layer was dried over anhydrous sodium sulfate, treated with 3-mercaptopropyl-functionalized silica gel, filtered, and the filtrate was concentrated in vacuo. The residue was purified by flash chromatography (silica gel, 0-20% ethyl acetate in heptane) to give the title compound. MS (ESI) m / z 302 (M+H) + .

[0110] Example 13B (1r,3s)-Methyl 3-((6-methoxy-5-(trifluoromethyl)pyridin-3-yl)methyl)cyclobutanecarboxylate Example 13A (740 mg, 2.46 mmol) and tetrahydrofuran (10 mL) were added to 210 mg of wet 5% Pd / C in a pressure reactor. The reaction mixture was stirred under hydrogen (50 psi) at 25° C. for 8 hours, filtered, washed with tetrahydrofuran, and concentrated in vacuo. The residue was purified by flash chromatography (silica gel, heptane in 0-20% ethyl acetate) to give the title compound, which contained approximately 20% of the corresponding trans isomer. MS (ESI) m / z 304 (M+H). + .

[0111] Example 13C (1r,3s)-3-((6-methoxy-5-(trifluoromethyl)pyridin-3-yl)methyl)cyclobutanecarboxylic acid To a solution of Example 13B (710 mg, 2.34 mmol) in a mixture of tetrahydrofuran (6.0 mL) / methanol (6.0 mL) / water (2.0 mL) was added sodium hydroxide (468 mg, 11.7 mmol). The reaction mixture was stirred at room temperature for 1 hour and diluted with water. The pH was adjusted to 4 by the addition of 1 M aqueous HCl and extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated in vacuo to give the title compound. This compound contained approximately 20% of the corresponding trans isomer. MS (ESI) m / z 290 (M+H) + .

[0112] Example 13D (1r,3s)-3-((6-oxo-5-(trifluoromethyl)-1,6-dihydropyridin-3-yl)methyl)cyclobutanecarboxylic acid Example 13C (676 mg, 2.34 mmol), para-toluenesulfonic acid monohydrate (889 mg, 4.67 mmol), and lithium chloride (495 mg, 11.7 mmol) were combined in N,N-dimethylformamide (10 mL). The reaction mixture was stirred at 100° C. for 10 hours, cooled to room temperature, and partitioned between ethyl acetate and water. The organic layer was washed with saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under vacuum to give the title compound, which contained approximately 20% of the corresponding trans isomer. The material was further purified by chiral SFC (Chiralpak® AD-H, 5 μm, 30 × 250 mm; mobile phase A: carbon dioxide; mobile phase B: isopropanol; flow rate: 80 g / min). The first eluting fractions were collected to give the title compound. MS (ESI) m / z 276 (M+H) + .

[0113] Example 13E 2-((3R,5S)-3,5-dimethylpiperazin-1-yl)-5-(trifluoromethyl)pyrimidine In Example 13E, a mixture of (2R,6S)-2,6-dimethylpiperazine (1.00 g, 8.76 mmol), 2-chloro-5-(trifluoromethyl)pyrimidine (1.60 g, 8.76 mmol), and triethylamine (4.88 mL, 35.0 mmol) was combined in acetonitrile (35 mL). The reaction mixture was stirred at room temperature overnight. The insoluble salt material was filtered and washed with a small amount of acetonitrile. The combined filtrate was concentrated in vacuo to give the title compound. MS (ESI) m / z 261 (M+H) + .

[0114] [Example 13F] 5-{[(1s,3s)-3-{(2R,6S)-2,6-dimethyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one Example 13D (27.5 mg, 0.100 mmol), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (76.0 mg, 0.200 mmol), and N,N-diisopropylethylamine (0.070 mL, 0.400 mmol) were combined in N,N-dimethylformamide (1.0 mL), and the reaction mixture was stirred at room temperature for 5 minutes. Example 13E (26.0 mg, 0.100 mmol) was added to the reaction mixture, and the resulting mixture was stirred at 50 °C for 24 hours, cooled to room temperature, and purified by reverse-phase HPLC (C18, 20-100% acetonitrile / water with 0.1% trifluoroacetic acid). The acetonitrile was removed by evaporation, and the mixture was neutralized with saturated sodium bicarbonate water. The mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated in vacuo to give the title compound. 1H NMR (400 MHz, DMSO-d6, 90℃) δ ppm 11.79 (s, br, 1H), 8.64 (d, J = 0.8 Hz, 2H), 7.71 (d, J = 2.6, 1H), 7.39 (d, J = 2.6 Hz, 1H), 4.54 (d, J = 13.7 Hz, 2H), 4.36 (s, br, 2H), 3.26 - 3.12 (m, 3H), 2.46 (d, J = 7.3 Hz, 2H), 2.43 - 2.33 (m, 1H), 2.24 - 2.15 (m, 2H), 1.95 - 1.84 (m, 2H), 1.14 (d, J = 6.9 Hz, 6H). MS (ESI) m / z 518 (M+H) + .

[0115] [Example 14] 5-{[(1s,3s)-3-{(3R,5S)-3,5-dimethyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one Example 14 was prepared according to the procedure used for the preparation of Example 1E, substituting Example 13D and Example 4B for Example 1C and Example 1D, respectively, to give the title compound. 1 H NMR (400 MHz, DMSO-d 6, 90℃) δ ppm 11.65 (s, br, 1H), 8.68 (d, J = 0.9 Hz, 2H), 7.71 (d, J = 2.6 Hz, 1H), 7.40 (d, J = 2.6 Hz, 1H), 4.87 - 4.78 (m, 2H), 3.98 (s, br, 2H), 3.42 - 2.90 (m, 3H), 2.47 (d, J = 7.5 Hz, 2H), 2.45 - 2.35 (m, 1H), 2.27 - 2.18 (m, 2H), 1.96 - 1.85 (m, 2H), 1.17 (d, J = 6.8 Hz, 6H). MS (ESI) m / z 518 (M+H) + .

[0116] [Example 15] 5-{[(1S,3s)-3-{(3R)-3-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one Example 15 was prepared according to the procedure used for the preparation of Example 1E, substituting Example 13D and Example 3B for Example 1C and Example 1D, respectively, to give the title compound. 1 H NMR (400 MHz, DMSO-d 6, 90℃) δ ppm 11.76 (s, br, 1H), 8.65 (d, J = 0.9 Hz, 2H), 7.71 (d, J = 2.6 Hz, 1H), 7.39 (d, J = 2.6 Hz, 1H), 4.93 - 4.81 (m, 1H), 4.51 - 4.41 (m, 1H), 3.97 (s, br, 2H), 3.38 - 3.00 (m, 4H), 2.45 (d, J = 7.7 Hz, 2H), 2.43 - 2.34 (m, 1H), 2.28 - 2.15 (m, 2H), 1.93 - 1.81 (m, 2H), 1.12 (d, J = 6.7 Hz, 3H). MS (ESI) m / z 504 (M+H) + .

[0117] [Example 16] 5-{[(1R,3s)-3-{(3S)-3-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one Example 16 was prepared according to the procedure used for the preparation of Example 1E, substituting Example 13D and Example 8B for Example 1C and Example 1D, respectively, to give the title compound. 1 H NMR (400 MHz, DMSO-d 6,90℃) δ ppm 11.78 (s, br, 1H), 8.65 (d, J = 0.9 Hz, 2H), 7.71 (d, J = 2.6 Hz, 1H), 7.39 (d, J = 2.6 Hz, 1H), 4.92 - 4.82 (m, 1H), 4.49 - 4.42 (m, 1H), 3.98 (s, br, 2H), 3.35 - 3.01 (m, 4H), 2.45 (d, J = 7.5 Hz, 2H), 2.43 - 2.34 (m, 1H), 2.28 - 2.15 (m, 2H), 1.93 - 1.82 (m, 2H), 1.12 (d, J = 6.7 Hz, 3H). MS (ESI) m / z 504 (M+H) + .

[0118] [Example 17] 5-{[(1R,3s)-3-{(2S)-2-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one Example 17 was prepared according to the procedure used for the preparation of Example 1E, substituting Example 13D and Example 6B for Example 1C and Example 1D, respectively, to give the title compound. 1 H NMR (400 MHz, DMSO-d 6, 90℃) δ ppm 11.78 (s, br, 1H), 8.64 (d, J = 0.9 Hz, 2H), 7.70 (d, J = 2.6 Hz, 1H), 7.39 (d, J = 2.6 Hz, 1H), 4.60 - 4.30 (m, 3H), 3.95 (s, br, 1H), 3.32 - 3.04 (m, 4H), 2.45 (d, J = 7.6 Hz, 2H), 2.42 - 2.33 (m, 1H), 2.24 - 2.16 (m, 2H), 1.93 - 1.79 (m, 2H), 1.05 (d, J = 6.7 Hz, 3H). MS (ESI) m / z 504 (M+H) + .

[0119] [Example 18] 5-{[(1S,3s)-3-{(3R)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-3-methylpiperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one Example 18 was prepared according to the procedure used for the preparation of Example 1E, substituting Example 13D and Example 7C for Example 1C and Example 1D, respectively. Purification by flash chromatography afforded the title compound. 1 H NMR (400 MHz, DMSO-d 6, 90℃) δ ppm 11.79 (s, br, 1H), 8.42 (s, 2H), 7.71 (d, J = 2.6 Hz, 1H), 7.39 (d, J = 2.6 Hz, 1H), 4.86 - 4.66 (m, 2H), 4.41 - 4.33 (m, 1H), 3.94 (s, br, 2H), 3.29 - 3.02 (m, 4H), 2.45 (d, J = 7.2 Hz, 2H), 2.43 - 2.34 (m, 1H), 2.28 - 2.14 (m, 2H), 1.92 - 1.82 (m, 2H), 1.42 (s, 6H), 1.05 (d, J = 6.7 Hz, 3H). MS (ESI) m / z 476 (M-H2O+H) + .

[0120] [Example 19] 5-{[(1S,3s)-3-{(2R)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-2-methylpiperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one Example 19 was prepared according to the procedure used for the preparation of Example 1E, substituting Example 13D and Example 9C for Example 1C and Example 1D, respectively. Purification by flash chromatography afforded the title compound. 1 H NMR (400 MHz, DMSO-d 6,90℃) δ ppm 11.78 (s, br, 1H), 8.41 (s, 2H), 7.70 (d, J = 2.6 Hz, 1H), 7.39 (d, J = 2.6 Hz, 1H), 4.73 (s, 1H), 4.55 - 4.21 (m, 3H), 3.95 (s, br, 1H), 3.22 - 2.85 (m, 4H), 2.45 (d, J = 7.4 Hz, 2H), 2.42 - 2.33 (m, 1H), 2.24 - 2.15 (m, 2H), 1.93 - 1.79 (m, 2H), 1.42 (s, 6H), 1.06 (d, J = 6.7 Hz, 3H). MS (ESI) m / z 476 (M-H2O+H) + .

[0121] [Example 20] 5-{[(1R,3s)-3-{(3S)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-3-methylpiperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one Example 20 was prepared according to the procedure used for the preparation of Example 1E, substituting Example 13D and Example 10C for Example 1C and Example 1D, respectively. Purification by flash chromatography afforded the title compound. 1 H NMR (400 MHz, DMSO-d 6,90℃) δ ppm 11.79 (s, br, 1H), 8.42 (s, 2H), 7.71 (d, J = 2.6, 1H), 7.39 (d, J = 2.6 Hz, 1H), 4.86 - 4.66 (m, 2H), 4.41 - 4.33 (m, 1H), 3.95 (s, br, 2H), 3.29 - 3.00 (m, 4H), 2.45 (d, J = 7.2 Hz, 2H), 2.42 - 2.34 (m, 1H), 2.28 - 2.14 (m, 2H), 1.93 - 1.82 (m, 2H), 1.42 (s, 6H), 1.05 (d, J = 6.6 Hz, 3H). MS (ESI) m / z 476 (M-H2O+H) + .

[0122] [Example 21] 5-{[(1R,3s)-3-{(2S)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-2-methylpiperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one Example 21 was prepared according to the procedure used for the preparation of Example 1E, substituting Example 13D and Example 11C for Example 1C and Example 1D, respectively. Purification by flash chromatography afforded the title compound. 1 H NMR (400 MHz, DMSO-d 6,90℃) δ ppm 11.78 (s, br, 1H), 8.41 (s, 2H), 7.71 (d, J = 2.6 Hz, 1H), 7.39 (d, J = 2.6 Hz, 1H), 4.73 (s, 1H), 4.54 - 4.23 (m, 3H), 3.96 (s, br, 1H), 3.22 - 2.85 (m, 4H), 2.45 (d, J = 7.3 Hz, 2H), 2.42 - 2.33 (m, 1H), 2.24 - 2.15 (m, 2H), 1.94 - 1.79 (m, 2H), 1.42 (s, 6H), 1.06 (d, J = 6.7 Hz, 3H). MS (ESI) m / z 476 (M-H2O+H) + .

[0123] [Example 22] 5-{[(1s,3s)-3-{(3R,5S)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-3,5-dimethylpiperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one

[0124] Example 22A Methyl 2-((2S,6R)-4-(tert-butoxycarbonyl)-2,6-dimethylpiperazin-1-yl)pyrimidine-5-carboxylate In Example 22A, methyl 2-chloropyrimidine-5-carboxylate (1.50 g, 8.69 mmol) and tert-butyl (3S,5R)-3,5-dimethylpiperazine-1-carboxylate (3.73 g, 17.4 mmol) were combined in N-methyl-2-pyrrolidinone (20 mL). The reaction mixture was stirred at 140 °C for 12 hours, cooled to room temperature, diluted with water, and extracted three times with ethyl acetate. The combined organic layers were concentrated in vacuo. The residue was purified by flash chromatography (silica gel, 0-100% ethyl acetate in petroleum ether) to give the title compound. MS (ESI) m / z 351 (M+H) + .

[0125] Example 22B tert-Butyl (3S,5R)-4-(5-(2-hydroxypropan-2-yl)pyrimidin-2-yl)-3,5-dimethylpiperazine-1-carboxylate To a solution of Example 22A (1.60 g, 4.57 mmol) in tetrahydrofuran (15 mL) under nitrogen at −78° C. was added 3.0 M methylmagnesium bromide (7.61 mL, 22.8 mmol). The reaction mixture was stirred at room temperature for 2 hours, quenched with aqueous ammonium chloride, diluted with water, and extracted three times with ethyl acetate. The combined organic layers were washed with saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated in vacuo. The residue was purified by flash chromatography (silica gel, 0-50% ethyl acetate in petroleum ether) to give the title compound. MS (ESI) m / z 351 (M+H) + .

[0126] [Example 22C] 2-(2-((2S,6R)-2,6-dimethylpiperazin-1-yl)pyrimidin-5-yl)propan-2-ol A mixture of Example 22B (800 mg, 2.28 mmol) in 4 M HCl in ethyl acetate (2 mL) was stirred at room temperature for 2 hours and concentrated in vacuo. The pH was adjusted to approximately 8 by slow addition of saturated aqueous sodium bicarbonate. The mixture was purified by preparative reverse-phase HPLC (C18, 5-95% acetonitrile in water) to give the title compound. MS (ESI) m / z 251 (M+H). + .

[0127] [Example 22D] 5-{[(1s,3s)-3-{(3R,5S)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-3,5-dimethylpiperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one Example 22D was prepared according to the procedure used for the preparation of Example 1E, substituting Example 13D and Example 22C for Example 1C and Example 1D, respectively. Purification by flash chromatography afforded the title compound.1 H NMR (400 MHz, DMSO-d6, 90℃) δ ppm 11.78 (s, br, 1H), 8.44 (s, 2H), 7.71 (d, J = 2.6 Hz, 1H), 7.40 (d, J = 2.6 Hz, 1H), 4.80 - 4.67 (m, 3H), 3.95 (s, br, 2H), 3.34 - 3.02 (m, 3H), 2.46 (d, J = 7.5 Hz, 2H), 2.44 - 2.35 (m, 1H), 2.27 - 2.18 (m, 2H), 1.96 - 1.86 (m, 2H), 1.43 (s, 6H), 1.12 (d, J = 6.8 Hz, 6H). MS (ESI) m / z 490 (M-H2O+H) + .

[0128] [Example 23] 3-(Trifluoromethyl)-5-[(1s,3s)-3-{4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]pyridin-2(1H)-one

[0129] Example 23A Methyl 3-(6-methoxy-5-(trifluoromethyl)pyridin-3-yl)cyclobutanecarboxylate Example 23A was prepared according to the procedure used for the preparation of Example 1A. Purification afforded the title compound as a mixture of stereoisomers having both trans and cis configurations. MS (ESI) m / z 290 (M+H) + .

[0130] Example 23B 3-(6-methoxy-5-(trifluoromethyl)pyridin-3-yl)cyclobutanecarboxylic acid Example 23B was prepared according to the procedure used for the preparation of Example 1B, substituting Example 23A for Example 1A to give the title compound. MS (ESI) m / z 276 (M+H). + .

[0131] Example 23C tert-Butyl 4-(5-(trifluoromethyl)pyrimidin-2-yl)piperazine-1-carboxylate In Example 23C, a four-neck, 1 L jacketed reactor was equipped with overhead stirring, a Huber temperature probe, and a nitrogen inlet. Under a nitrogen atmosphere, the reactor was charged with 2-chloro-5-(trifluoromethyl)pyrimidine (59.6 g, 326 mmol), which was dissolved in acetonitrile (600 mL). The interior of the solution was cooled to 5°C, and then tert-butyl piperazine-1-carboxylate (60.8 g, 326 mmol) was added, followed by N,N-diisopropylethylamine (142 mL, 816 mmol). The reaction mixture was stirred at room temperature for 1 hour, transferred to a round-bottom flask, and concentrated under vacuum. The residue was dissolved in dichloromethane, washed with saturated aqueous ammonium chloride, washed with saturated aqueous sodium chloride, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated under vacuum to give the title compound. MS (APCI) m / z 277 (M-56+H). + .

[0132] [Example 23D] 2-(Piperazin-1-yl)-5-(trifluoromethyl)pyrimidine In Example 23D, a four-neck, 2 L jacketed reactor was equipped with overhead stirring, a Huber temperature probe, and a nitrogen inlet. Under a nitrogen atmosphere, the reactor was charged with Example 23C (108 g, 325 mmol), followed by dioxane (1000 mL) and 4 M HCl (300 mL, 1200 mmol). The reaction mixture was stirred at room temperature for 18 hours, filtered, rinsed with tert-butyl methyl ether, and dried under vacuum. The residue was suspended in dichloromethane and cooled in an ice bath. Saturated aqueous sodium bicarbonate was added slowly until a neutral pH was reached, and the mixture was separated. The aqueous layer was extracted twice with dichloromethane. The combined organic layers were washed with saturated aqueous sodium chloride, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated under vacuum to give the title compound. NaOH pellets were added to the aqueous layer to adjust the pH to >10, and the mixture was extracted three times with dichloromethane. The combined organic layers were washed with saturated aqueous sodium chloride, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated in vacuo to give additional title compound. MS (APCI) m / z 233 (M+H). + .

[0133] Example 23E (3-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)cyclobutyl)(4-(5-(trifluoromethyl)pyrimidin-2-yl)piperazin-1-yl)methanone To a mixture of Example 23B (84.0 mg, 0.305 mmol), Example 23D (70.9 mg, 0.305 mmol), and N,N-diisopropylethylamine (0.320 mL, 1.83 mmol) in N,N-dimethylformamide (2.0 mL) was added 50% propanephosphonic anhydride in N,N-dimethylformamide (388 mg, 0.610 mmol). The reaction mixture was stirred at room temperature for 1 hour and partitioned between ethyl acetate and water. The organic layer was washed with saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated in vacuo to give the title compound. MS (ESI) m / z 490 (M+H) + .

[0134] [Example 23F] 3-(Trifluoromethyl)-5-[(1s,3s)-3-{4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]pyridin-2(1H)-one Example 23E (134 mg, 0.274 mmol), para-toluenesulfonic acid monohydrate (104 mg, 0.548 mmol), and lithium chloride (58.0 mg, 1.37 mmol) were combined in N,N-dimethylformamide (2.0 mL). The reaction mixture was stirred at 100°C for 10 hours, cooled to room temperature, and purified by reverse-phase HPLC (C18, 10-100% acetonitrile / water with 0.1% trifluoroacetic acid). The acetonitrile was removed by evaporation under vacuum, and the resulting mixture was neutralized with saturated aqueous sodium bicarbonate. The resulting mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under vacuum. The residue was further purified by flash chromatography to obtain two fractions. The first eluted fraction contained the stereoisomer with the trans configuration. The second eluted fraction contained the title compound with the cis configuration. 1 H NMR (500 MHz, DMSO-d6) δ ppm 12.21 (s, 1H), 8.73 (d, J = 0.8 Hz, 2H), 7.83 (d, J = 2.6 Hz, 1H), 7.50 (d, J = 2.6 Hz, 1H), 3.86 - 3.79 (m, MS (ESI) m / z 476 (M+H) + .

[0135] [Example 24] 5-[(1r,3r)-3-{(3R,5S)-3,5-dimethyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one Example 24 was prepared according to the procedure used for the preparation of Example 1E, substituting Example 25C and Example 4B for Example 1C and Example 1D, respectively, to give the title compound. 1 H NMR (400 MHz, DMSO-d 6, 90℃) δ ppm 11.61 (s, 1H), 8.68 (d, J = 0.9 Hz, 2H), 7.85 (d, J = 2.6 Hz, 1H), 7.51 (d, J = 2.6 Hz, 1H), 4.89 - 4.79 (m, 2H), 3.86 (s, MS (ESI) m / z 504 (M+H) + .

[0136] [Example 25] 5-[(1R,3r)-3-{(2R)-2-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one

[0137] Example 25A (1r,3r)-Methyl 3-(6-methoxy-5-(trifluoromethyl)pyridin-3-yl)cyclobutanecarboxylate Example 25A was prepared as described in Example 1A. The first eluting fraction contained the title compound in the trans configuration. MS (ESI) m / z 290 (M+H) + .

[0138] Example 25B (1r,3r)-3-(6-methoxy-5-(trifluoromethyl)pyridin-3-yl)cyclobutanecarboxylic acid Example 25B was prepared according to the procedure used for the preparation of Example 1B, substituting Example 25A for Example 1A to give the title compound. MS (ESI) m / z 276 (M+H) + .

[0139] Example 25C (1r,3r)-3-(6-oxo-5-(trifluoromethyl)-1,6-dihydropyridin-3-yl)cyclobutanecarboxylic acid Example 25C was prepared according to the procedure used for the preparation of Example 1C, substituting Example 25B for Example 1B to give the title compound. MS (ESI) m / z 262 (M+H). + .

[0140] Example 25D 5-[(1R,3r)-3-{(2R)-2-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one Example 25D was prepared according to the procedure used for the preparation of Example 1E, substituting Example 25C for Example 1C to give the title compound. 1 H NMR (400 MHz, DMSO-d6, 90℃) δ ppm 11.87 (s, 1H), 8.65 (d, J = 0.8 Hz, 2H), 7.84 (d, J = 2.6 Hz, 1H), 7.49 (d, J = 2.7 Hz, 1H), 4.69 - 4.27 (m, 3H), 3.82 (s, br, 1H), 3.42 - 3.28 (m, 3H), 3.26 - 3.03 (m, 2H), 2.64 - 2.50 (m, 2H), 2.31 - 2.20 (m, 2H), 1.08 (d, J = 6.7 Hz, 3H). MS (ESI) m / z 490 (M+H) + .

[0141] [Example 26] 3-(Trifluoromethyl)-5-[(1r,3r)-3-{4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]pyridin-2(1H)-one Example 26 was prepared as described in Example 23F. The first fraction to elute contained the title compound in the trans configuration. 1 H NMR (600 MHz, DMSO-d6) δ ppm 12.22 (s, 1H), 8.73 (d, J = 0.9 Hz, 2H), 7.93 (d, J = 2.6 Hz, 1H), 7.58 (d, J = 2.6 Hz, 1H), 3.86 - 3.81 (m, MS (ESI) m / z 476 (M+H) + .

[0142] [Example 27] 5-{[(1s,3s)-3-{4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one

[0143] Example 27A 2-(2-(piperazin-1-yl)pyrimidin-5-yl)propan-2-ol In Example 27A, 2-(2-chloropyrimidin-5-yl)propan-2-ol (1.00 g, 5.79 mmol), piperazine (1.50 g, 17.4 mmol), and N,N-diisopropylethylamine (5.06 mL, 29.0 mmol) were combined in acetonitrile (10 mL). The reaction mixture was stirred at 80° C. for 2 hours, cooled to room temperature, and concentrated in vacuo. The residue was purified by flash chromatography to give the title compound. MS (ESI) m / z 223 (M+H) + .

[0144] Example 27B 5-{[(1s,3s)-3-{4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one Example 27B was prepared according to the procedure used for the preparation of Example 1E, substituting Example 13D and Example 27A for Example 1C and Example 1D, respectively. Purification by flash chromatography afforded the title compound. 1 H NMR ppm (400 MHz, DMSO-d6) δ 12.15 (s, 1H), 8.44 (s, 2H), 7.80 (d, J = 2.5 Hz, 1H), 7.48 (d, J = 2.5 Hz, 1H), 5.08 (s, 1H), 3.71 - 3.63 (m, 4H), 3.53 - 3.46 (m, 2H), 3.43 - 3.36 (m, 2H), 3.24 - 3.11 (m, 1H), 2.43 (d, J = 7.3 Hz, 2H), 2.40 - 2.30 (m, 1H), 2.20 - 2.10 (m, 2H), 1.89 - 1.79 (m, 2H), 1.41 (s, 6H). MS (ESI) m / z 462 (M-H2O+H) + .

[0145] [Example 28] 5-[(1s,3s)-3-{4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one Example 28 was prepared according to the procedure used for the preparation of Example 1E, substituting Example 27A for Example 1D. The reaction mixture was purified by reverse-phase HPLC (C18, 20-100% acetonitrile / water with 0.1% trifluoroacetic acid). The acetonitrile was removed by evaporation under vacuum, and the resulting mixture was neutralized with saturated aqueous sodium bicarbonate, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under vacuum. The residue was further purified by flash chromatography to provide the title compound.1 H NMR (400 MHz, DMSO-d6) δ ppm 12.20 (s, 1H), 8.45 (s, 2H), 7.83 (d, J = 2.6 Hz, 1H), 7.50 (d, J = 2.6 Hz, 1H), 5.08 (s, 1H), 3.75 - 3.65 MS (ESI) m / z 448 (M-H2O+H) + .

[0146] [Example 29] 3-(Trifluoromethyl)-5-{[(1s,3s)-3-{4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]methyl}pyridin-2(1H)-one Example 29 was prepared according to the procedure used for the preparation of Example 1E, substituting Example 13D and Example 23D for Example 1C and Example 1D, respectively, to give the title compound. 1 H NMR (600 MHz, DMSO-d6) δ ppm 12.15 (s, br, 1H), 8.72 (d, J = 0.8 Hz, 2H), 7.80 (d, J = 2.5 Hz, 1H), 7.49 (d, J = 2.5 Hz, 1H), 3.83 - 3.77 (m, 4H), 3.56 - 3.51 (m, 2H), 3.47 - 3.41 (m, 2H), 3.22 - 3.15 (m, 1H), 2.43 (d, J = 7.5 Hz, 2H), 2.40 - 2.30 (m, 1H), 2.20 - 2.12 (m, 2H), 1.89 - 1.80 (m, 2H). MS (ESI) m / z 490 (M+H) + .

[0147] TIPARP GST-(TEV) fusion protein expression and purification Expression A full-length cDNA encoding the 657-amino acid human TIPARP (NP_001171647.1, UniProtKB-Q7Z3E1) was used as a template to generate an expression construct. A codon-optimized nucleotide sequence was synthesized for expression in Sf9 insect cells. TIPARP fusion proteins contain GST, glutathione S-transferase, a 26 kDa protein, and their DNA sequences are frequently incorporated into expression vectors for recombinant protein production.

[0148] A truncated TIPARP protein sequence consisting of amino acids 441–657 was cloned with an N-terminal GST tag followed by a TEV protease site into the vector pFastBac™1 (Thermo Fisher Scientific) to generate the TIPARP fusion protein, TIPARP GST-(TEV).

[0149] TIPARP fusion proteins were heterologously expressed in Sf9 insect cell cultures seeded with baculovirus-infected insect cells (BIICs) infected with pFastBac™1 recombinant baculovirus. Baculovirus-infected cell cultures were grown in a Wave Bioreactor (Cytiva, Marlborough, MA, USA) at a 20-liter scale. Sf9 insect cells were grown in Sf-900™ II SFM medium (Thermo Fisher Scientific, Waltham, MA, USA) at a density of 2.4 × 10 6 Cells were grown at 1000 x g / mL and then infected with baculovirus. BIIC was added for infection, and after 72 h of incubation, cells were harvested by centrifugation at 4,000 x g for 10 min at 5°C and then transferred to -80°C until further processing.

[0150] TIPARP fusion proteins expressed in insect cells were purified using the following protocol: cells were thawed, homogenized, and lysed in 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.5, 250 mM NaCl, 5% glycerol (w / v), 0.1% Triton X100, 0.5 mM MgCl2, and 0.5 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) using a Dounce homogenizer. Benzonase® Nuclease (MilliporeSigma, Burlington, MA, USA) and SIGMAFAST™ Protease Inhibitor Cocktail Tablets (MilliporeSigma) were added to the lysate according to the manufacturer's recommendations. The cell lysate was clarified by centrifugation at 35,000 × g for 60 minutes at 5°C. The supernatant was sterile filtered through a 0.45 μm filter.

[0151] purification The TIPARP fusion protein was purified from the supernatant in batch mode using Glutathione Sepharose 4 Fast Flow GST-tagged protein purification resin (Cytiva) equilibrated with column buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol (v / v), 0.5 mM TCEP). The supernatant and resin were gently mixed and allowed to settle into the column by gravity flow. The resin was washed with 20 column volumes of column buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol, 0.5 mM TCEP). The protein was eluted from the column using column buffer with the addition of 10 mM glutathione. The protein was further diluted 3-fold with 20 mM MES (2-(N-morpholino)ethanesulfonic acid) pH 6.1, 5% glycerol (v / v), and 0.5 mM TCEP buffer and loaded onto HiTrap® SP Sepharose™ Fast Flow resin (MilliporeSigma) equilibrated with 20 mM MES pH 6.1, 50 mM NaCl, 5% glycerol, and 0.5 mM TCEP. The TIPARP fusion protein was eluted with a 0.05 M to 1 M sodium chloride gradient over 10 column volumes, and the fusion protein was eluted at 400 mM NaCl. The molecular weight of the purified fusion protein was confirmed by mass spectrometry.

[0152] Synthesis of probe 1 Probe 1 N-{2-[2-(2-{[1-(5-cyanopyridin-2-yl)-4-{3-[(4-oxo-3,4-dihydrophthalazin-1-yl)methyl]benzoyl}piperazin-2-yl]methoxy}ethoxy)ethoxy]ethyl}-2',7'-difluoro-3',6'-dihydroxy-3-oxo-3H-spiro[[2]benzofuran-1,9'-xanthene]-5-carboxamide

[0153] Probe 1A 3-[(E)-(3-oxo-2-benzofuran-1(3H)-ylidene)methyl]benzonitrile To a mixture of isobenzofuran-1(3H)-one (5 g, 37.3 mmol) and 3-formylbenzoic acid (4.89 g, 37.3 mmol) in ethyl acetate (20 mL) was added dropwise sodium methoxide (30.2 g, 168 mmol) at 0° C. Methanol (10 mL) was added and the mixture was stirred at 80° C. for 1 hour. LCMS showed that the starting material was consumed. The solvent was evaporated and the residue was diluted with water (1.5 L). The mixture was acidified at 20° C. using 2 N aqueous hydrochloric acid to obtain a pH of 1-2. The mixture was filtered, and the filter cake was washed with water (500 mL) and dried under high vacuum to give the title compound. 1 H NMR (400 MHz, DMSO-d6) δ ppm 7.45 - 7.61 (m, 7H) 8.24 - 8.36 (m, 2H). ESI-MS m / z 246.0 (MH).

[0154] Probe 1B 3-[(4-oxo-3,4-dihydrophthalazin-1-yl)methyl]benzoic acid To a mixture of 3-[(E)-(3-oxo-2-benzofuran-1(3H)-ylidene)methyl]benzonitrile (6 g, 24.27 mmol) in water (36 mL) was added a solution of sodium hydroxide (5.82 g, 146 mmol) in water (7.2 mL) at 20 °C. The reaction mixture was warmed to 90 °C and stirred for 1 h. The reaction mixture was cooled to 70 °C, and hydrazine (10.88 mL, 340 mmol) was added. The resulting mixture was stirred at 70 °C for another 18 h. Thin layer chromatography (tetrahydrofuran / petroleum ether = 1 / 1) showed that the starting material was consumed and a new spot was formed. The mixture was extracted with ethyl acetate (3 × 60 mL). The organic phase was discarded, and the aqueous phase was acidified to pH = 4 with 1 N aqueous hydrochloric acid at 20 °C. The precipitate was collected by filtration. The filter cake was washed with water (60 mL) and petroleum ether (60 mL) and dried under high vacuum to give the title compound. 1H NMR (400 MHz, DMSO-d6) δ ppm 4.38 (s, 2H) 7.42 (t, J=7.70 Hz, 1H) 7.58 (br d, J=7.58 Hz, 1H) 7.80 (br dd, J=16.44, 7.89 Hz, 2H) 7.85 - 7.93 (m, 2H) 7.93 - 7.99 (m, 1H) 8.26 (d, J=7.58 Hz, 1H) 12.60 (s, 1H) 12.67 - 13.07 (m, 1H). ESI-MS m / z 281.1 (M+H) + .

[0155] Probe 1C tert-Butyl 4-(5-cyanopyridin-2-yl)-3-(hydroxymethyl)piperazine-1-carboxylate For probe 1C, tert-butyl 3-(hydroxymethyl)piperazine-1-carboxylate (300 mg, 1.387 mmol) was combined with 6-chloronicotinonitrile (192 mg, 1.387 mmol) in dimethylacetamide (4 mL). N,N-Diisopropylethylamine (0.727 mL, 4.16 mmol) was added, and the reaction was heated at 90°C for 2.25 hours. The reaction mixture was cooled, diluted with water, and extracted with ethyl acetate. The organic layer was dried over sodium sulfate and filtered. The filtrate was concentrated, and the residue was purified using a silica gel cartridge, eluting with 0-40% ethyl acetate / heptane to give the title compound. 1H NMR (600 MHz, CDCl3) δ ppm 8.40 (dd, J = 2.4, 0.8 Hz, 1H), 7.65 (dd, J = 9.0, 2.3 Hz, 1H), 6.65 (d, J = 9.0 Hz, 1H), 4.60 (s, 2H), 4.27 (s, 1H), 4.06 (d, J = 12.6 Hz, 1H), 3.98 (s, 1H), 3.63 (s, 1H), 3.49 (s, 2H), 3.33 (s, 1H), 3.27 (td, J = 12.2, 3.7 Hz, 1H), 3.16 (s, 1H), 1.50 (s, 8H). ESI-MS m / z 219.67 (M+H-Boc) + .

[0156] Probe 1D tert-Butyl 3-({2-[2-(2-azidoethoxy)ethoxy]ethoxy}methyl)-4-(5-cyanopyridin-2-yl)piperazine-1-carboxylate For probe 1D, tert-butyl 4-(5-cyanopyridin-2-yl)-3-(hydroxymethyl)piperazine-1-carboxylate (58.3 mg, 0.183 mmol) and 2-(2-(2-azidoethoxy)ethoxy)ethyl methanesulfonate (55.7 mg, 0.220 mmol) were combined in anhydrous N,N-dimethylformamide (1 mL). Sodium hydride (60% in mineral oil, 11 mg, 0.275 mmol) was added, and the mixture was stirred at ambient temperature for 1 hour, then at 50° C. for 2 hours. Additional 2-(2-(2-azidoethoxy)ethoxy)ethyl methanesulfonate (55.7 mg, 0.220 mmol) was added, and the reaction mixture was stirred at 50° C. for 2 hours. The reaction mixture was diluted to 3 mL with 90% DMSO / water and purified in a single injection on a Gilson® RP-HPLC running uniPoint software using a Waters™ Deltapak C18 200 x 25 mm column (15 μm particle size, 100 Å porosity). The column was eluted with a gradient of 5% A (0.1% TFA-water):B (acetonitrile) to 100% A:B [0-5 min: 5% A; 5-45 min: linear gradient to 100% B, 2.375% / min gradient] at a flow rate of 20 mL / min. Fractions containing the title compound were combined and lyophilized to give the title compound as the trifluoroacetate salt. ESI-MS m / z 476.1 (M+H) + .

[0157] Probe 1E 6-[2-({2-[2-(2-azidoethoxy)ethoxy]ethoxy}methyl)piperazin-1-yl]pyridine-3-carbonitrile For probe 1E, tert-butyl 3-({2-[2-(2-azidoethoxy)ethoxy]ethoxy}methyl)-4-(5-cyanopyridin-2-yl)piperazine-1-carboxylate trifluoroacetate (47.2 mg, 0.080 mmol) was dissolved in 1 mL of trifluoroacetic acid and immediately evaporated to dryness under a stream of dry nitrogen gas to give the title compound as the bistrifluoroacetate salt. ESI-MS m / z 376.16 (M+H) + .

[0158] Probe 1F 6-[2-({2-[2-(2-azidoethoxy)ethoxy]ethoxy}methyl)-4-({3-[(4-oxo-3,4-dihydrophthalazin-1-yl)methyl]phenyl}methyl)piperazin-1-yl]pyridine-3-carbonitrile For probe 1F, 6-[2-({2-[2-(2-azidoethoxy)ethoxy]ethoxy}methyl)piperazin-1-yl]pyridine-3-carbonitrile bistrifluoroacetate (48.2 mg, 0.080 mmol) and 3-[(4-oxo-3,4-dihydrophthalazin-1-yl)methyl]benzoic acid (24.63 mg, 0.088 mmol) were combined in anhydrous N,N-dimethylformamide (1 mL). PyAOP ((7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, 45.8 mg, 0.088 mmol) and N,N-diisopropylethylamine (69.7 μL, 0.399 mmol) were added, and the reaction was shaken at ambient temperature for 1 hour. An additional 24 mg of PyAOP was added, and the reaction mixture was shaken at ambient temperature for an additional hour. The reaction mixture was diluted to 3 mL with 90% DMSO / water and purified in a single injection on a Gilson® RP-HPLC running uniPoint software using a Waters™ Deltapak C18 200 × 25 mm column (15 μm particle size, 100 Å porosity). The eluate was eluted with a gradient of 5% A (0.1% TFA-water):B (acetonitrile) to 100% A:B [0–5 min: 5% A; 5–45 min: linear gradient to 100% B, 2.375% / min gradient] at a flow rate of 20 mL / min. Fractions containing the title compound were combined and lyophilized to give the title compound as the trifluoroacetate salt. 1H NMR (600 MHz, DMSO-d6) δ ppm 12.61 (s, 1H), 8.51 (dd, J = 2.4, 0.7 Hz, 1H), 8.27 (dd, J = 7.8, 1.4 Hz, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.92 - 7.80 (m, 4H), 7.44 (d, J = 7.8 Hz, 1H), 7.39 (t, J = 7.6 Hz, 3H), 7.28 (s, 2H), 6.90 (d, J = 9.2 Hz, 1H), 4.80 (s, 1H), 4.50 (s, 1H), 4.36 (s, 3H), 4.31 (s, ESI-MS m / z 637.81 (M+H) + .

[0159] Probe 1G 6-[2-({2-[2-(2-aminoethoxy)ethoxy]ethoxy}methyl)-4-{3-[(4-oxo-3,4-dihydrophthalazin-1-yl)methyl]benzoyl}piperazin-1-yl]pyridine-3-carbonitrile To 6-[2-({2-[2-(2-azidoethoxy)ethoxy]ethoxy}methyl)-4-({3-[(4-oxo-3,4-dihydrophthalazin-1-yl)methyl]phenyl}methyl)piperazin-1-yl]pyridine-3-carbonitrile trifluoroacetate (31 mg, 0.049 mmol) in tetrahydrofuran was added palladium on carbon (25.9 mg, 0.024 mmol), and the reaction mixture was stirred under H at 1 atm for 16 h. The reaction mixture was filtered, and the filtrate was purified on a silica gel cartridge eluted with NHOH / CHOH / CHCl to give the title compound. Analytical LCMS TFA method [a gradient of 5–100% acetonitrile (A) and 0.1% trifluoroacetic acid in water (B) at a flow rate of 1.5 mL / min (0–0.05 min: 5% A, 0.05–1.2 min: 5–100% A, 1.2–1.4 min: 100% A, 1.4–1.5 min: 100–5% A; 0.25 min post-run delay] was used: R t =0.73 min, ESI-MS m / z 612.5(M+H) + .

[0160] Probe 1H N-{2-[2-(2-{[1-(5-cyanopyridin-2-yl)-4-{3-[(4-oxo-3,4-dihydrophthalazin-1-yl)methyl]benzoyl}piperazin-2-yl]methoxy}ethoxy)ethoxy]ethyl}-2',7'-difluoro-3',6'-dihydroxy-3-oxo-3H-spiro[[2]benzofuran-1,9'-xanthene]-5-carboxamide For probe 1H, 6-[2-({2-[2-(2-aminoethoxy)ethoxy]ethoxy}methyl)-4-{3-[(4-oxo-3,4-dihydrophthalazin-1-yl)methyl]benzoyl}piperazin-1-yl]pyridine-3-carbonitrile and 2,5-dioxopyrrolidin-1-yl 2′,7′-difluoro-3′,6′-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-5-carboxylate (8.79 mg, 0.017 mmol) were combined in anhydrous N,N-dimethylformamide containing 2% N,N-diisopropylethylamine (v / v), and the mixture was shaken overnight at ambient temperature. The reaction mixture was diluted to 3 mL with 90% dimethyl sulfoxide / water and purified in a single injection on a Gilson® RP-HPLC running uniPoint software using a Waters™ Deltapak C18 200 × 25 mm column (15 μm particle size, 100 Å porosity) eluted with a gradient of 5% A (0.1% TFA-water):B (acetonitrile) to 100% A:B [0–5 min: 5% A; 5–45 min: linear gradient to 100% B, 2.375% / min gradient] at a flow rate of 20 mL / min. Fractions containing the title compound were combined and lyophilized to give the title compound as the trifluoroacetate salt. Analytical LCMS FA method [5–100% acetonitrile (A) and water:acetonitrile (98:2) with 0.1% formic acid (B) gradient was used at a flow rate of 1.5 mL / min (0–0.05 min: 0% A, 0.05–2.8 min: 0–100% A, 2.8–3.0 min: 100–0% A, 160–1500 amu positive / negative ESI-MS ionization)]: R t =1.493 min, ESI-MS m / z 503.8(M+2H) 2+ , 1006.4(M+H) + , 1005.0(MH) - Analytical LCMS AA method [5–100% acetonitrile (A) and water:acetonitrile (98:2) with 10 mM ammonium acetate (B) gradient was used at a flow rate of 1.5 mL / min (0–0.05 min: 0% A, 0.05–2.8 min: 0–100% A, 2.8–3.0 min: 100–0% A, 160–1500 amu positive / negative ESI-MS ionization)]: Rt =1.016 min, ESI-MS m / z 503.4(M+2H) 2+ , 1028.0(M+Na) + , 1003.8(MH) - .

[0161] Synthesis of probe 2 Probe 2 2',7'-Difluoro-N-[2-(2-{2-[{[4-(8-fluoro-6-oxo-3,4,5,6-tetrahydro-1H-azepino[5,4,3-cd]indol-2-yl)phenyl]methyl}(methyl)amino]ethoxy}ethoxy)ethyl]-3',6'-dihydroxy-3-oxo-3H-spiro[[2]benzofuran-1,9'-xanthene]-5-carboxamide

[0162] Probe 2A tert-Butyl [2-(2-{2-[{[4-(8-fluoro-6-oxo-3,4,5,6-tetrahydro-1H-azepino[5,4,3-cd]indol-2-yl)phenyl]methyl}(methyl)amino]ethoxy}ethoxy)ethyl]carbamate For probe 2A, 8-fluoro-2-{4-[(methylamino)methyl]phenyl}-1,3,4,5-tetrahydro-6H-azepino[5,4,3-cd]indol-6-one hydrochloride (100.45 mg, 0.253 mmol) was combined with 2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl methanesulfonate (Julie Moreau and Jacqueline Marchand-Brynaert, Eur. J. Org. Chem., 2011, pp. 1641-1644; 124 mg, 0.380 mmol) in 1 mL of anhydrous N,N-dimethylformamide. Potassium carbonate (105 mg, 0.760 mmol) was added, and the reaction mixture was stirred at 70 °C for 16 h. The reaction mixture was diluted with 25 mL of saturated aqueous NH4Cl and extracted with 3 x 15 mL of dichloromethane. The combined organic layers were dried over MgSO4, filtered, and concentrated. The residue was diluted to 4 mL with 90% DMSO / water and purified by Gilson® RP-HPLC running uniPoint software using a Waters™ Deltap C18 200 x 25 mm column (15 μm particle size, 100 Å porosity) in two injections. The eluate was eluted with a gradient of 5% A (0.1% TFA-water):B (acetonitrile) to 100% A:B [0-5 min: 5% A; 5-45 min: linear gradient to 100% B, 2.375% / min gradient] at a flow rate of 20 mL / min. Fractions containing the title compound were combined and lyophilized to give the title compound as the trifluoroacetate salt. 1H NMR (600 MHz, DMSO-d6) δ ppm 11.77 (s, 1H), 9.71 (s, 1H), 8.28 (t, J = 5.8 Hz, 1H), 7.76 - 7.72 (m, 2H), 7.69 - 7.64 (m, 2H), 7.45 (dd, J = 10.9, 2.4 Hz, 1H), 7.36 (dd, J = 9.1, 2.5 Hz, 1H), 6.76 (t, J = 5.7 Hz, 1H), 4.52 - 4.45 (m, 1H), 4.34 (dd, J = 12.8, 5.3 Hz, 1H), 3.80 (t, J = 5.3 Hz, 2H), 3.63 - 3.53 (m, 8H), 3.40 (q, J = 5.0 Hz, 4H), 3.34 (s, 1H), 3.28 - 3.22 (m, 1H), 3.07 (q, J = 6.0 Hz, 4H), 2.78 (d, J = 4.4 Hz, 3H), 1.37 (d, J = 1.9 Hz, 1H), 1.35 (s, 8H). ESI-MS m / z 555.51 (M+H) + .

[0163] Probe 2B 2-(4-{[{2-[2-(2-aminoethoxy)ethoxy]ethyl}(methyl)amino]methyl}phenyl)-8-fluoro-1,3,4,5-tetrahydro-6H-azepino[5,4,3-cd]indol-6-one For probe 2B, tert-butyl [2-(2-{2-[{[4-(8-fluoro-6-oxo-3,4,5,6-tetrahydro-1H-azepino[5,4,3-cd]indol-2-yl)phenyl]methyl}(methyl)amino]ethoxy}ethoxy)ethyl]carbamate trifluoroacetate (21.63 mg, 0.032 mmol) was dissolved in trifluoroacetic acid (1 mL) and immediately evaporated to dryness to give the title compound as the trifluoroacetate salt. ESI-MS m / z 455.04 (M+H) + .

[0164] Probe 2C 2',7'-Difluoro-N-[2-(2-{2-[{[4-(8-fluoro-6-oxo-3,4,5,6-tetrahydro-1H-azepino[5,4,3-cd]indol-2-yl)phenyl]methyl}(methyl)amino]ethoxy}ethoxy)ethyl]-3',6'-dihydroxy-3-oxo-3H-spiro[[2]benzofuran-1,9'-xanthene]-5-carboxamide For probe 2C, 2-(4-{[{2-[2-(2-aminoethoxy)ethoxy]ethyl}(methyl)amino]methyl}phenyl)-8-fluoro-1,3,4,5-tetrahydro-6H-azepino[5,4,3-cd]indol-6-one bistrifluoroacetate (22 mg, 0.032 mmol) and 2,5-dioxopyrrolidin-1-yl 2',7'-difluoro-3',6'-dihydroxy-3-oxo-3H-spiro[[2]benzofuran-1,9'-xanthene]-5-carboxylate (ThermoFisher Scientific, 18.06 mg, 0.035 mmol) were combined in 1 mL of anhydrous dimethylformamide containing 2% diisopropylethylamine (v / v) and shaken at ambient temperature for 24 h. The reaction mixture was diluted to 3 mL with 90% dimethyl sulfoxide / water and purified in a single injection time-preparative manner on a Phenomenex® Gemini® 5 μM NX-C18 110 Å 250 × 21.2 mm column, eluting with a gradient of A (0.1% TFA-water):B (CH3CN) [0–5 min: 5% A; 5–35 min: linear gradient to 100% B, 3.16% / min gradient] at a flow rate of 20 mL / min. Fractions containing the title compound were combined and lyophilized to afford the title compound as the trifluoroacetate salt. Analytical LCMS TFA method [a gradient of 5–100% acetonitrile (A) and 0.1% trifluoroacetic acid in water (B) at a flow rate of 1.5 mL / min (0–0.05 min: 5% A, 0.05–1.2 min: 5–100% A, 1.2–1.4 min: 100% A, 1.4–1.5 min: 100–5% A; 0.25 min post-run delay] was used: R t =0.68 min, ESI-MS m / z 425.4(M+2H) 2+ , 849.6(M+H) + .

[0165] Time-resolved fluorescence resonance energy transfer (TR-FRET) assay The TR-FRET assay was used to measure the binding of compounds to TIPARP and PARP1 and characterize their biochemical selectivity. Compound stock solutions (10 mM) were serially diluted in 3-fold dilutions in dimethyl sulfoxide (DMSO). The diluted compounds were dispensed (30 nL) into white ProxiPlate 384-well plates (PerkinElmer, Waltham, MA, USA) using an ECHO® 550 acoustic Labcyte dispenser (Beckman-Coulter Life Sciences, Indianapolis, IN, USA) to achieve final starting concentrations in the assay ranging from 30 μM to 0.5 nM. Following this, 5 μL of 2× protein / probe mix (Table 2) prepared in assay buffer (Tris-HCl 50 mM pH 8.0; 5 mM MgCl2; 5 μM ZnCl2; 1 mM DTT (dithiothreitol); 0.15% BSA (bovine serum albumin) (w / v); and 0.01% Triton X-100 (w / v)) at the final concentrations listed in Table 2 was added. Subsequently, 5 μL of 2× antibody (Tb-labeled anti-GST antibody, Thermo Fisher Scientific, Waltham, MA, USA) in HEPES-buffered saline was added to each well at a final concentration of 1 nM. The samples were then incubated for 2 hours under ambient conditions. Fluorescence was measured and the TR-FRET ratio was determined using an EnVision multimode plate reader (PerkinElmer) using a 520 nanometer (nm) excitation wavelength and collecting the fluorescence emission at 495 nm. The results are shown in Table 3. As shown in Table 3, compounds according to formula (I) described herein exhibited high potency for inhibiting TIPARP and relatively low inhibition of PARP1 (i.e., high selectivity for TIPARP inhibition).

[0166] [Table 2]

[0167] [Table 3]

[0168] CellTiter-Glo® Cellular Assay for TIPARP Inhibition NCI-H1373 cells have been shown to undergo cell cycle arrest following TIPARP inhibition. The NCI-H1373 CellTiter-Glo® Assay was used to determine cell number following TIPARP inhibition-induced cell cycle arrest, allowing compounds to be screened for TIPARP inhibitory potency in cells.

[0169] Compound stock solutions (10 mM) were serially diluted in 3-fold dilutions in dimethyl sulfoxide. Sample aliquots (120 nL) were dispensed into Corning® 384-well Flat Clear Bottom White Polystyrene TC-treated Microplates (Corning Life Sciences, Corning, NY, USA) using an ECHO® 550 acoustic Labcyte dispenser (Beckman-Coulter Life Sciences, Indianapolis, IN, USA) to achieve final concentrations between 30 μM and 0.5 nM in the assay. Following this, 40 μL of 1000 NCI-H1373 cells (ATCC®, Manassas, VA, USA) were cultured in complete growth medium RPMI 1640 growth medium (Thermo Fisher Scientific) supplemented with 20 mg / L L-glutamate, 25 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 10% (v / v) heat-inactivated FBS (fetal bovine serum), 1% (w / v) penicillin-streptomycin, and 100 nM 10-Cl-BBQ (10-chloro-7H-benzimidazo[2,1-a]benz[de]isoquinolin-7-one, (Tocris, Minneapolis, MN, USA)). Replicate cell plates containing 40 μL cells / well with various concentrations of compound were kept in Nunc™ Square BioAssay Dishes (Thermo Fisher Scientific) at 37°C in a 5% CO2 incubator with 98% relative humidity for 6 days. On day 7, the plates were transferred to ambient laboratory conditions for 10 minutes to allow the temperature to equilibrate. After equilibration, CellTiter-Glo® Luminescent Cell Viability Assay Reagent (Promega™, Madison, WI, USA) was added (15 μL) to each well of the plate. After 20 minutes of incubation, luminescence was detected using a ViewLux™ microplate imager (PerkinElmer, Waltham, MA, USA).Data analysis was performed using GraphPad Prism (Dotmatics, Boston, MA, USA), and the results are shown in Table 4.

[0170] [Table 4]

[0171] CellTiter-Glo® DLD-1 BRCA2(- / -) and DLD-1 Parent Cell Assay for PARP1 / 2 Inhibition The CellTiter-Glo® assay for DLD-1 BRCA2(- / -) mice measures the synthetic lethality of PARP1 / 2 inhibitors and was used as a surrogate for measuring both PARP1 and PARP2 activity in cells. When PARP1 and PARP2 are inhibited and trapped in chromatin during DNA replication, replication forks stall and double-strand breaks accumulate. In BRCA wild-type cells, the resulting double-strand breaks are repaired via homologous recombination. In cells with BRCA1 or BRCA2 mutations, homologous recombination repair of DNA double-strand breaks is defective, resulting in cell death after treatment with PARP1 / 2 inhibitors (Lord CJ, Ashworth A. PARP inhibitors: synthetic lethality in the clinic. Science. 2017 March 17;355(6330):1152-1158). Testing compounds in viability assays using DLD-1-BRCA2(- / -) and parental DLD-1 cells allowed for evaluation of PARP1 / 2-dependent (cytotoxicity only in BRCA2(- / -) cells) versus non-selective cytotoxicity in both cell lines. Thus, viability assays using parental DLD-1 and DLD-1 BRCA2 cells demonstrated that compounds of formula (I) have low PARP1 / 2 inhibitory activity.

[0172] Compound stock solutions (10 mM) were serially diluted in 3-fold dilutions in dimethyl sulfoxide (DMSO). Sample aliquots (120 nL) were dispensed into Corning 384-well Flat Clear Bottom White Polystyrene TC-treated Microplates (Corning Life Sciences, Corning, NY, USA) using an ECHO® 550 acoustic Labcyte dispenser (Beckman-Coulter Life Sciences, Indianapolis, IN, USA) to achieve concentrations between 30 μM and 0.5 nM in the assay. In the assay, 40 μL of 500 DLD-1 parental cells (HORIZON® Therapeutics, Deerfield, IL, USA) or 40 μL of 1200 DLD-1-BRCA2(− / −) cells (HORIZON® Therapeutics) were added to complete growth medium (ThermoFisher Scientific, Waltham, MA, USA) containing RPMI 1640 growth medium supplemented with 20 mg / L L-glutamate, 25 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 10% (v / v) heat-inactivated FBS (fetal bovine serum), and 1% (w / v) penicillin-streptomycin.

[0173] Replicate cell plates containing 40 μL cells / well with various concentrations of compound were kept in Nunc™ Square BioAssay Dishes (Thermo Fisher Scientific) in a 98% relative humidity, 5% CO2 incubator at 37°C for 6 days. On day 7, the plates were transferred to ambient laboratory conditions for 10 minutes to allow the temperature to equilibrate. After equilibration, CellTiter-Glo® Luminescent Cell Viability Assay Reagent (Promega™, Madison, WI, USA) was added (15 μL) to each well of the plate. After a 20-minute incubation, luminescence was detected using a ViewLux™ microplate imager (PerkinElmer, Waltham, MA, USA). Data analysis was performed using GraphPad Prism (Dotmatics, Boston, MA, USA). The results are shown in Table 5. As shown in Table 5, DLD-1 parental line and DLD-1BRCA2(- / -) cells showed good viability (i.e., low inhibition of PARP1 / 2) after incubation with compounds according to formula (I) described herein.

[0174] [Table 5]

[0175] MC-38 (Ker) isogeneic model validity The murine cell line MC-38, derived from C57BL6 murine colon adenocarcinoma cells, was obtained from Kerafast (Boston, MA, USA). 2.5 × 10 cells were cultured in culture medium mixed with Matrigel® (Corning Life Sciences, Lowell, MA, USA; 1:1, volume:volume). 5 The cell suspension was injected subcutaneously into the right hind flank of female C57BL / 6 mice to form tumors. The flank tumors were approximately 120 mm in size. 3Treatment began when the vehicle and four different doses of Example 1 were administered to each of eight mice once daily at each dose level for up to 21 days. Each administration consisted of a single 0.2 mL oral (PO) dose in a vehicle formulation (10% ethanol, 30% PEG 300, and 60% PHOSAL® 50PG).

[0176] The results are shown in Figure 1. As shown, administration of Example 1 inhibited the growth of MC-38 (Ker) tumors compared to vehicle. The maximum tumor growth inhibition (TGI) achieved at doses A to D was 1.25. Max ) were 46%, 77%, 98% and 99%, respectively.

[0177] Tumor-selective induction of IFN-β in the MC-38(Ker) syngeneic model The murine cell line MC-38, derived from C57BL / 6 murine colon adenocarcinoma cells, was obtained from Kerafast (Boston, MA, USA). 2.5 × 10 cells were cultured in culture medium mixed with Matrigel® (Corning Life Sciences, Lowell, MA, USA; 1:1, volume:volume). 5 The cell suspension was injected subcutaneously into the right hind flank of female C57BL / 6 mice to form tumors. Flank tumors were approximately 500 mm in size. 3 Treatment was initiated at this time. Vehicle and three different doses of Example 1 were administered to four to five mice each at each dose level once daily for two days. Each administration consisted of a single 0.2 mL PO dose in a vehicle formulation (10% ethanol, 30% PEG 300, and 60% PHOSAL® 50PG).

[0178] Three hours after the last dose, blood and tumors were collected. Blood was transferred to a microtube containing EDTA and centrifuged at 2,000 × g for 8 minutes at 4 °C. The upper plasma layer was transferred to a new tube and frozen at -80 °C. Tumors were weighed, and 200–500 mg of tumor tissue was transferred to a microtube, which was then centrifuged at 15,000 × g for 10 minutes at 4 °C. The supernatant was transferred to a fresh 96-well plate. 20 μL of cold Dulbecco's phosphate-buffered saline (DPBS) was added to the tumor-containing tube, and the tumor was removed with a pipette tip. The tube was then centrifuged at 500 × g for 10 minutes at 4 °C. The supernatant was combined with the corresponding supernatant from the previous step in a 96-well plate. The plate containing the combined supernatants (tumor fluid) was centrifuged at 500 × g for 3 min at 4 °C, and the clear supernatant was transferred to a new 96-well plate, which was then frozen at −80 °C. The concentration of IFNβ in the thawed plasma and tumor fluid was determined using the Milliplex MAP Mouse IFNβ Assay (EMD Millipore, Burlington, MA, USA).

[0179] The results are shown in Figure 2. As shown, administration of Example 1 induced IFNβ in MC-38(Ker) tumors but not in plasma compared to vehicle, indicating selective induction of the cytokine within the tumor microenvironment.

[0180] Efficacy in NCI-H1373 xenograft tumor models The human xenograft line NCI-H1373 was obtained from ATCC (American Type Culture Collection, Manassas, Virginia, USA). 5 × 10 cells were cultured in culture medium mixed with Matrigel® (Corning Life Sciences, Lowell, MA, USA; 1:1, volume:volume). 6 The cell suspension was injected subcutaneously into the right hind flank of female SCID beige mice to form tumors. The flank tumors were approximately 120 mm in size. 3Treatment was initiated when the TIPARP inhibitory activity of Example 1 was assessed by dosing five separate treatment groups with vehicle and four different doses of Example 1 administered twice daily (BID) for 14 days. Each dose consisted of a 0.2 mL PO dose in a vehicle formulation (10% ethanol, 30% PEG 300, and 60% PHOSAL® 50).

[0181] The results are shown in Figure 3. As shown, administration of Example 1 inhibited the growth of NCI-H1373 tumors compared to vehicle. TGI achieved at doses A to D Max were 26%, 46%, 73% and 92%, respectively.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof 【Chemistry 1】 [In the formula, L is a bond and CH ２ is selected from the group consisting of R １ , R ２ , R ３ and R ４ are independently H and CH ３ and R １ , R ２ , R ３ and R ４ At least one and not more than two of ３ and Z is CF ３ and C(CH ３ ) ２ OH].

2. 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein L is a bond.

3. R １ Or R ４ One of them is CH ３ Or R １ and R ４ Both are CH ３ Or R ２ Or R ３ One of them is CH ３ or R ２ and R ３ Both are CH ３ 3. The compound of claim 2, wherein:

4. Z is CF ３ 4. The compound of claim 3, wherein:

5. L is CH ２ 2. The compound of claim 1, wherein:

6. R １ Or R ４ One of them is CH ３ Or R １ and R ４ Both are CH ３ Or R ２ Or R ３ One of them is CH ３ or R ２ and R ３ Both are CH ３ 6. The compound of claim 5, wherein:

7. Z is C(CH ３ ) ２ 7. The compound of claim 6, or a pharmaceutically acceptable salt thereof, wherein R is 0 or 1;

8. 5-[(1S,3s)-3-{(2R)-2-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one, 5-[(1s,3s)-3-{(2R,6S)-2,6-dimethyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one, 5-[(1S,3s)-3-{(3R)-3-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one, 5-[(1s,3s)-3-{(3R,5S)-3,5-dimethyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one, 5-[(1s,3s)-3-{(3R,5S)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-3,5-dimethylpiperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one, 5-[(1R,3s)-3-{(2S)-2-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one, 5-[(1S,3s)-3-{(3R)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-3-methylpiperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one, 5-[(1R,3s)-3-{(3S)-3-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one, 5-[(1S,3s)-3-{(2R)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-2-methylpiperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one, 5-[(1R,3s)-3-{(3S)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-3-methylpiperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one, 5-[(1R,3s)-3-{(2S)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-2-methylpiperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one, 5-{[(1S,3s)-3-{(2R)-2-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one, 5-{[(1s,3s)-3-{(2R,6S)-2,6-dimethyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one, 5-{[(1s,3s)-3-{(3R,5S)-3,5-dimethyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one, 5-{[(1S,3s)-3-{(3R)-3-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one, 5-{[(1R,3s)-3-{(3S)-3-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one, 5-{[(1R,3s)-3-{(2S)-2-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one, 5-{[(1S,3s)-3-{(3R)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-3-methylpiperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one, 5-{[(1S,3s)-3-{(2R)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-2-methylpiperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one, 5-{[(1R,3s)-3-{(3S)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-3-methylpiperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one, 5-{[(1R,3s)-3-{(2S)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-2-methylpiperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one, and 5-{[(1s,3s)-3-{(3R,5S)-4-[5-(2-hydroxypropan-2-yl)pyrimidin-2-yl]-3,5-dimethylpiperazine-1-carbonyl}cyclobutyl]methyl}-3-(trifluoromethyl)pyridin-2(1H)-one 2. The compound of claim 1, selected from the group consisting of: or a pharmaceutically acceptable salt thereof.

9. 9. The compound of claim 8, which is 5-[(1S,3s)-3-{(2R)-2-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one or a pharmaceutically acceptable salt thereof.

10. The compound of claim 9, which is 5-[(1S,3s)-3-{(2R)-2-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one.

11. 10. The compound of claim 9, which is a pharmaceutically acceptable salt of 5-[(1S,3s)-3-{(2R)-2-methyl-4-[5-(trifluoromethyl)pyrimidin-2-yl]piperazine-1-carbonyl}cyclobutyl]-3-(trifluoromethyl)pyridin-2(1H)-one.

12. 10. A pharmaceutical composition comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

13. 10. A method for treating head and neck squamous cell carcinoma (HNSCC), comprising administering to a human patient in need thereof a compound of claim 1 or a pharmaceutically acceptable salt thereof.

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