Alkyl diamine-substituted bis-aromatic heterocyclic thioether compound, preparation thereof, and application thereof in preparation of drugs for treating and / or preventing tumors

Abstract

The present invention relates to an alkyl diamine-substituted bis-aromatic heterocyclic thioether compound, a preparation thereof, and an application thereof in the preparation of drugs for treating and / or preventing tumors. The structural formula of the compound is as shown in formula (I): the compound has a clear growth-inhibitory effect on various human tumor cells, with good inhibitory activity against breast cancer, liver cancer, pancreatic cancer, kidney cancer, lung cancer, gastric cancer, glioma, ovarian cancer, prostate cancer, esophageal cancer, melanoma, nasopharyngeal carcinoma, colon cancer, cervical cancer, lymphoma, leukemia and other such tumors, wherein the effective IC50 concentration of the compound against tumor cells, such as breast cancer, pancreatic cancer, lung cancer, glioma, ovarian cancer, esophageal cancer, melanoma, colon cancer, cervical cancer, is lower than cisplatin, and experiments show that the compound has broad-spectrum anti-tumor activity. Experiments have further detected that the compound exhibits the effect of degrading PD-L1 proteins, and is a PD-L1 immunomodulator. The present invention provides a new approach for using alkyl diamine-substituted bis-aromatic heterocyclic thioether compounds in the preparation of drugs for treating and / or preventing tumors.

Description

An alkyldiamine-substituted bis-aromatic heterocyclic sulfide compound, its preparation, and use in preparing drugs for treating and / or preventing tumors Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an alkyldiamine-substituted bis-aromatic heterocyclic sulfide compound and its application in preparing drugs for treating and / or preventing tumors. Background Art

[0002] Cancer poses a serious threat to human health. The World Health Organization's International Agency for Research on Cancer (IARC) released its latest report on the global cancer burden: an estimated 20 million new cancer cases and 9.7 million cancer deaths will occur worldwide in 2022. In China, there will be approximately 4.8247 million new cancer cases and 2.5742 million cancer deaths in 2022. It is projected that by 2050, new cancer cases worldwide will exceed 35 million, a 77% increase compared to 2022.

[0003] The results of the third national survey on causes of death conducted by the Ministry of Health of my country show that cancer has become the second leading cause of death in my country after cardiovascular and cerebrovascular diseases, accounting for 22.32% of the total number of deaths, and has become the leading cause of death in my country's cities, accounting for 1 / 4 of the total number of deaths in my country's cities.

[0004] Currently, drug therapy has become an important means of clinical cancer treatment. However, for solid tumors, which pose the greatest threat to human health and account for over 90% of malignant tumors, there remains a lack of highly effective and specific drugs. With the application of new concepts, technologies, and methods, many key molecules and genes involved in tumor development and progression have been discovered, driving the continuous development of anti-tumor drugs. The continuous emergence of new anti-tumor drugs has brought hope of survival to some cancer patients.

[0005] Literature search has so far revealed no reports on the anti-tumor effects of alkyldiamine-substituted bis-heteroaromatic sulfide compounds. The present invention designs and synthesizes alkyldiamine-substituted bis-heteroaromatic sulfide compounds and explores their anti-tumor effects. Summary of the Invention

[0006] The purpose of the present invention is to provide a new compound having good anti-tumor effect, an alkyldiamine-substituted bis-aromatic heterocyclic sulfide compound, and its preparation and use in preparing drugs for treating and / or preventing tumors.

[0007] The purpose of the present invention can be achieved by the following technical solutions:

[0008] The present invention designs a new compound, namely an alkyldiamine-substituted bis-heteroaromatic sulfide compound, the structural formula of which is as follows:

[0009] Where:

[0010] R1 is halogen, hydrogen, cyano, hydroxyl, mercapto, nitro, C 1-6 -alk(ene / ynyl) group, C 1-6 -alk(en / yn)yloxy, C 1-6 -Alkyl (ene / ynyl) thio group, C 1-6 -Alkyl (ene / ynyl) sulfonyl, halogenated C 1-6 -Alkyl (ene / ynyl) group, halogenated C 1-6 -Alkyl (ene / ynyl) oxy, halogenated C 1-6 -Alkyl (ene / ynyl) thio, halogenated C 1-6 -Alk(en / yn)ylsulfonyl.

[0011] R2 is a six-membered ring such as benzene, cyclohexane, cyclohexene, cyclohexadiene, or a six-membered heterocyclic ring such as pyridine, piperidine, piperazine, pyridazine, pyrimidine, pyrazine, pyran; or a five-membered ring such as cyclopentane, cyclopentene, cyclopentadiene, or a five-membered heterocyclic ring such as thiophene, furan, thiazole, pyrrole, imidazole, pyrazole, oxazole.

[0012] R3 is halogen, hydrogen, cyano, hydroxyl, mercapto, nitro, C 1-6 -alk(ene / ynyl) group, C 1-6 -alk(en / yn)yloxy, C 1-6 -Alkyl (ene / ynyl) thio group, C 1-6 -Alkyl (ene / ynyl) sulfonyl, halogenated C 1-6 -Alkyl (ene / ynyl) group, halogenated C 1-6 -Alkyl (ene / ynyl) oxy, halogenated C 1-6 -Alkyl (ene / ynyl) thio, halogenated C 1-6 -Alk(en / yn)ylsulfonyl.

[0013] R4 is a substituted chain alkyl group, including -CH2-, -CH2-CH2-, and -CH2-CH2-CH2-.

[0014] R5 is a substituted chain alkyl group, including -CH2-, -CH2-CH2-, and -CH2-CH2-CH2-.

[0015] R6 is halogen, hydrogen, cyano, hydroxyl, mercapto, nitro, C 1-6 -alk(ene / ynyl) group, C 1-6 -alk(en / yn)yloxy, C 1-6 -Alkyl (ene / ynyl) thio group, C 1-6 -Alkyl (ene / ynyl) sulfonyl, halogenated C 1-6 -Alkyl (ene / ynyl) group, halogenated C 1-6 -Alkyl (ene / ynyl) oxy, halogenated C 1-6-Alkyl (ene / ynyl) thio, halogenated C 1-6 -alk(en / yn)ylsulfonyl, or two R6 connected to the same carbon atom can form a 3-6 membered spiro-connected cycloalkyl.

[0016] X, Y, Z, and W each independently represent a carbon or nitrogen atom, and when selected from nitrogen atoms, only one of them is nitrogen;

[0017] m represents 0, 1, 2, 3, 4 or 5.

[0018] n represents 0, 1, 2, 3 or 4.

[0019] p represents 0, 1, 2, 3, 4, 5 or 6.

[0020] Halogen includes F, Cl, Br or I.

[0021] Halogenated C 1-6 -Alkyl (ene / ynyl) is a halogenated C 1-6 -alk(ene / ynyl), dihalogenated C 1-6 -alk(ene / ynyl) or polyhalogenated C 1-6 -alk(en / yn)yl.

[0022] R1 is prioritized over CF3.

[0023] R3 is prioritized over CF3.

[0024] R4 is preferably -CH2-CH2-.

[0025] R5 is preferably -CH2-CH2-.

[0026] X, Y, Z, and W are preferably carbon atoms.

[0027] Priority is

[0028] Furthermore, the alkyldiamine-substituted bis-heteroaromatic sulfide compound is preferably the following compound:

[0029] Furthermore, the chemical structure of the above compound is as follows:

[0030] Furthermore, the alkyldiamine-substituted bis-heteroaromatic sulfide compounds include, but are not limited to, their tautomers, mesomers, racemates, enantiomers, diastereomers, or possible derivatives based on their structures, or mixtures thereof.

[0031] The present invention also provides the use of an alkyldiamine-substituted bis-heteroaromatic sulfide compound, a pharmaceutical composition containing the compound, or a derivative of the alkyldiamine-substituted bis-heteroaromatic sulfide compound in preparing drugs for preventing and / or treating tumors.

[0032] Furthermore, the alkyldiamine-substituted bis-heteroaromatic sulfide compound or its pharmaceutical composition includes but is not limited to its pharmaceutically acceptable salts, ethers, esters, prodrugs, metabolites, solvates or crystals.

[0033] Furthermore, the alkyldiamine-substituted bis-heteroaromatic sulfide compound or its pharmaceutical composition includes a pharmaceutically acceptable salt thereof; preferably, the pharmaceutically acceptable salt includes but is not limited to hydrochloride, bromate, fumarate, acetate, citrate, sulfate, methanesulfonate, formate or trifluoroacetate.

[0034] Furthermore, the alkyldiamine-substituted bis-heteroaromatic sulfide compound or its pharmaceutical composition includes but is not limited to tablets, injections, capsules, oral solutions, pills, granules, powders, aerosols, patches, ointments, paints or suppositories.

[0035] Furthermore, the alkyldiamine-substituted bis-heteroaromatic sulfide compound or its pharmaceutical composition also includes conventional anti-tumor drugs; preferably, including but not limited to chemotherapeutic drugs, biological targeted therapy drugs, metabolic therapy drugs or immunotherapy drugs.

[0036] Furthermore, the alkyldiamine-substituted bis-heteroaromatic sulfide compound or its pharmaceutical composition is used for, but not limited to, surgical resection, chemotherapy or radiotherapy.

[0037] Furthermore, the alkyldiamine-substituted bis-heteroaromatic sulfide compound or its pharmaceutical composition is used for, but not limited to, the preparation of PD-L1 immunomodulator-related drugs.

[0038] Furthermore, the effects of the alkyldiamine-substituted bis-heteroaromatic sulfide compound or its pharmaceutical composition include but are not limited to inhibiting tumor growth and / or metastasis.

[0039] Furthermore, the alkyldiamine-substituted bis-heteroaromatic sulfide compound or its pharmaceutical composition is used for the prevention and / or treatment of diseases including but not limited to breast cancer, liver cancer, pancreatic cancer, kidney cancer, lung cancer, gastric cancer, glioma, ovarian cancer, prostate cancer, esophageal cancer, melanoma, nasopharyngeal cancer, colon cancer, cervical cancer, lymphoma, and leukemia.

[0040] Furthermore, the preventive and / or therapeutic effects on breast cancer include but are not limited to preventive and / or therapeutic effects on breast cancer cells MDA-MB-231, MDA-MB-231LM2, and MCF7.

[0041] Furthermore, the preventive and / or therapeutic effects on liver cancer include but are not limited to preventive and / or therapeutic effects on liver cancer cells MHCC-97L.

[0042] Furthermore, the preventive and / or therapeutic effects on pancreatic cancer include but are not limited to preventive and / or therapeutic effects on pancreatic cancer cells Panc-1.

[0043] Furthermore, the preventive and / or therapeutic effects on renal clear cell adenocarcinoma include but are not limited to preventive and / or therapeutic effects on renal clear cell adenocarcinoma cell line 786-O.

[0044] Furthermore, the preventive and / or therapeutic effects on lung cancer include but are not limited to preventive and / or therapeutic effects on lung cancer cells A549.

[0045] Furthermore, the preventive and / or therapeutic effects on gastric cancer include but are not limited to preventive and / or therapeutic effects on gastric cancer cell SGC-7901.

[0046] Furthermore, the preventive and / or therapeutic effects on glioma include but are not limited to preventive and / or therapeutic effects on glioma cells U251.

[0047] Furthermore, the preventive and / or therapeutic effects on ovarian cancer include but are not limited to preventive and / or therapeutic effects on ovarian cancer cell A2780.

[0048] Furthermore, the preventive and / or therapeutic effects on prostate cancer include but are not limited to preventive and / or therapeutic effects on prostate cancer cells PC-3.

[0049] Furthermore, the preventive and / or therapeutic effects on esophageal cancer include but are not limited to preventive and / or therapeutic effects on esophageal cancer cells EC109.

[0050] Furthermore, the preventive and / or therapeutic effects on melanoma include but are not limited to preventive and / or therapeutic effects on melanoma cells A375.

[0051] Furthermore, the preventive and / or therapeutic effects on nasopharyngeal carcinoma include but are not limited to preventive and / or therapeutic effects on nasopharyngeal carcinoma cells 5-8F.

[0052] Furthermore, the preventive and / or therapeutic effects on intestinal cancer include but are not limited to preventive and / or therapeutic effects on intestinal cancer cells HCT116.

[0053] Furthermore, the preventive and / or therapeutic effects on cervical cancer include but are not limited to preventive and / or therapeutic effects on cervical cancer cell SiHa.

[0054] Furthermore, the preventive and / or therapeutic effects on lymphoma include but are not limited to preventive and / or therapeutic effects on lymphoma cell line MINO.

[0055] Furthermore, the preventive and / or therapeutic effects on leukemia include but are not limited to preventive and / or therapeutic effects on leukemia cells HL60.

[0056] Compared with the prior art, the present invention has the following beneficial effects:

[0057] The present invention synthesizes an alkyldiamine-substituted bis-heteroaromatic sulfide compound, explores its anti-tumor effect and its new use in preparing drugs for treating and / or preventing cancer, and there are no related reports at home and abroad.

[0058] The alkyldiamine-substituted bis-aromatic heterocyclic sulfide compounds of the present invention have obvious and broad-spectrum anti-tumor activity and have inhibitory effects on a variety of human tumor cells cultured in vitro, such as breast cancer, liver cancer, pancreatic cancer, kidney cancer, lung cancer, gastric cancer, glioma, ovarian cancer, prostate cancer, esophageal cancer, melanoma, nasopharyngeal cancer, colon cancer, cervical cancer, lymphoma, leukemia and other tumor cells; some compounds have IC 200 activity against breast cancer, pancreatic cancer, lung cancer, glioma, ovarian cancer, esophageal cancer, melanoma, colon cancer, cervical cancer and other tumor cells. 50 The alkyldiamine-substituted bis(heteroaromatic) sulfide compounds of the present invention can be used to prepare anti-tumor drugs and prevent the occurrence and metastasis of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] FIG1 shows the effect of alkyldiamine-substituted bis-heteroaromatic sulfide compounds on the degradation of PD-L1 protein. DETAILED DESCRIPTION

[0060] The principles and features of the present invention are described below. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, where specific conditions are not specified, conventional conditions or manufacturer-recommended conditions were used. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially.

[0061] Example 1:

[0062] Compound T01 (Target 1) was prepared by the following method:

[0063] 1-Bromo-2-iodobenzene (5.00 g, 17.6 mmol, 1 equiv) and tert-butylpiperazine-1-carboxylate (3.29 g, 17.6 mmol, 1 equiv) were placed in a 100 mL single-necked flask containing toluene (50.0 mL). Sodium tert-butoxide (2.55 g, 26.5 mmol, 1.5 equiv), Pd2(dba)3 (809 mg, 883 μmol, 0.05 equiv), and Xantphos (1.02 g, 1.77 mmol, 0.1 equiv) were added sequentially. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 85°C for 2 hours. LC-MS / MS indicated the reaction was complete. TLC monitoring showed the presence of multiple new spots indicating complete consumption of the starting material. After cooling, the reaction mixture was added to distilled water (200 mL) and extracted with ethyl acetate (100 mL x 2). The combined organic phases were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure to yield the crude product. The crude product was purified by column chromatography (gradient: (SiO2, Petroleum ether / Ethyl acetate = 1 / 0 to 1 / 1)) to afford tert-butyl 4-(2-bromophenyl)piperazine-1-carboxylate (4.60 g, 13.4 mmol, 76.2% yield) as a yellow oil.

[0064] Tert-butyl 4-(2-bromophenyl)piperazine-1-carboxylate (200 mg, 586 μmol, 1 equiv) and 4-methylbenzene-1-thiol (80.0 mg, 644 μmol, 1.1 equiv) were placed in a 10.0 mL single-necked vial containing toluene (3.00 mL). Sodium tert-butoxide (84.4 mg, 879 μmol, 1.5 equiv), Pd2(dba)3 (26.8 mg, 29.3 μmol, 0.05 equiv), and Xantphos (33.9 mg, 58.6 μmol, 0.1 equiv) were added sequentially. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 100°C for 16 hours. LC-MS / MS indicated the reaction was complete. TLC monitoring revealed two new spots indicating incomplete consumption of the starting material. After cooling, the reaction mixture was added to distilled water (30.0 mL) and extracted with ethyl acetate (15.0 mL x 2). The combined organic phases were washed with saturated brine (15.0 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to yield a crude product. The crude product was purified by chromatography (SiO2, petroleum ether / ether acetate = 5 / 1) to yield tert-butyl 4-{2-[(4-methylphenyl)mercapto]phenyl}piperazine-1-carboxylate (120 mg, 312 μmol, 53.2% yield) as a yellow oil.

[0065] In a 10.0 mL single-necked bottle, tert-butyl 4-{2-[(4-methylphenyl)mercapto]phenyl}piperazine-1-carboxylate (120 mg, 312 μmol, 1 equivalent) was dissolved in dioxane hydrochloride solution (3.00 mL). The resulting reaction solution was stirred at 25°C for 2 hours. LC-MS / MS showed that the reaction was complete. The reaction solution was directly concentrated under reduced pressure to obtain a crude product. The crude product was purified by high performance liquid chromatography (column model: Phenomenex luna C 18 150*25mm*10um; mobile phase: [water (HCl)-acetonitrile]; B%: 15%-45%, 10 min) was used for purification to obtain 1-{2-[(4-methylphenyl)mercapto]phenyl}piperazine (25.5 mg, 79.4 μmol, 25.46% yield, 99.84% purity, hydrochloride) as an off-white solid.

[0066] 1 H NMR (400 MHz, deuterated dimethyl sulfoxide) δ = 9.13 (br s, 2H), 7.39-7.32 (m, 2H), 7.31-7.25 (m, 2H), 7.22-7.12 (m, 2H), 7.04-6.97 (m, 1H), 6.71-6.66 (m, 1H), 3.18 (s, 8H), 2.35 (s, 3H).

[0067] Example 2:

[0068] Compound T02 (Target 2) was prepared by the following method:

[0069] Tert-butyl 4-(2-bromophenyl)piperazine-1-carboxylate (200 mg, 586 μmol, 1 equiv) and 2-methoxybenzene-1-thiol (82.1 mg, 586 μmol, 1 equiv) were placed in a 10.0 mL single-necked vial containing toluene (3.00 mL). Sodium tert-butoxide (84.4 mg, 879 μmol, 1.5 equiv), Pd2(dba)3 (26.8 mg, 29.3 μmol, 0.05 equiv), and Xantphos (33.9 mg, 58.6 μmol, 0.1 equiv) were added sequentially. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 100°C for 16 hours. LC-MS / MS indicated the reaction was complete. TLC showed two new spots. After cooling, the reaction mixture was added to distilled water (30.0 mL) and extracted with ethyl acetate (15.0 mL x 2). The combined organic phases were washed with saturated brine (15.0 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to yield a crude product. The crude product was purified by chromatography (SiO2, petroleum ether / ether acetate = 5 / 1) to afford tert-butyl 4-{2-[(2-methoxyphenyl)mercapto]phenyl}piperazine-1-carboxylate (190 mg, 474 μmol, 80.9% yield) as a yellow oil.

[0070] In a 10.0 mL single-necked flask, tert-butyl 4-{2-[(2-methoxyphenyl)mercapto]phenyl}piperazine-1-carboxylate (190 mg, 474 μmol, 1 equivalent) was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (1.54 g, 13.4 mmol, 1.00 mL, 28.3 equivalents) was added. The resulting reaction solution was stirred at 25°C for 30 minutes. LC / MS / MS indicated the reaction was complete. After cooling, the reaction mixture was added to saturated aqueous sodium bicarbonate (30.0 mL) and extracted with ethyl acetate (15.0 mL x 2). The combined organic phases were washed with saturated brine (15.0 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by HPLC (column model: Phenomenex luna C 18 The product was placed in a 50.0 mL single-necked flask containing ethyl acetate hydrochloride (10.0 mL) and stirred at 25°C for 30 minutes. The reaction solution was then concentrated under reduced pressure to obtain 1-{2-[(2-methoxyphenyl)mercapto]phenyl}piperazine (25.3 mg, 75.1 μmol, 15.8% yield, 99.7% purity, hydrochloride) as a white solid.

[0071] 1H NMR (400 MHz, deuterated dimethyl sulfoxide) δ = 9.28 (br s, 2H), 7.46-7.38 (m, 1H), 7.25 (dd, J = 1.6, 7.6 Hz, 1H), 7.22-7.10 (m, 3H), 7.05-6.93 (m, 2H), 6.69 (dd, J = 1.2, 7.6 Hz, 1H), 3.77 (s, 3H), 3.20 (br d, J = 5.2 Hz, 4H), 3.15 (br s, 4H).

[0072] Example 3:

[0073] Compound T03 (Target 3) was prepared by the following method:

[0074] Tert-Butyl 4-(2-bromophenyl)piperazine-1-carboxylate (200 mg, 586 μmol, 1 eq) and 3,4-dimethylbenzene-1-thiol (89.1 mg, 644 μmol, 1.1 eq) were placed in a 10.0 mL single-necked flask containing toluene (3.00 mL). Sodium tert-butoxide (112 mg, 1.17 mmol, 2 eq) and RuPhos Pd G3 (49.0 mg, 58.6 μmol, 0.1 eq) were added sequentially. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 100°C for 16 hours. LC-MS / MS indicated the reaction was complete. TLC monitoring revealed two new spots indicating complete consumption of the starting material. After cooling, the reaction mixture was added to distilled water (30.0 mL) and extracted with ethyl acetate (15.0 mL x 2). The combined organic phases were washed with saturated brine (15.0 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure to obtain the crude product. The crude product was purified by plate chromatography (separation gradient: (SiO2, Petroleum ether / Ethyl acetate = 5 / 1) to give tert-butyl 4-{2-[(3,4-dimethylphenyl)mercapto]phenyl}piperazine-1-carboxylate (180 mg, 451 μmol, 76.9% yield) as a yellow solid.

[0075] In a 10.0 mL single-necked bottle, tert-butyl 4-{2-[(3,4-dimethylphenyl)mercapto]phenyl}piperazine-1-carboxylate (180 mg, 451 μmol, 1 equivalent) was dissolved in ethyl acetate hydrochloride (10.0 mL). The resulting reaction solution was stirred at 25°C for 2 hours. LC-MS / MS showed that the reaction was complete. The reaction solution was directly concentrated under reduced pressure to obtain a crude product. The crude product was purified by high performance liquid chromatography (column model: Phenomenex luna C 18150*25mm*10um; mobile phase: [water (hydrochloric acid)-acetonitrile]; B%: 20%-50%, 18 min) to give 1-{2-[(3,4-dimethylphenyl)mercapto]phenyl}piperazine (25.8 mg, 77.1 μmol, 17.0% yield, 99.9% purity, hydrochloride) as a white solid.

[0076] 1 H NMR (400 MHz, deuterated dimethyl sulfoxide) δ = 9.31 (br s, 2H), 7.27 (s, 1H), 7.26-7.21 (m, 1H), 7.21-7.17 (m, 1H), 7.17-7.12 (m, 2H), 7.03-6.95 (m, 1H), 6.71-6.61 (m, 1H), 3.18 (s, 8H), 2.25 (s, 3H), 2.23 (s, 3H).

[0077] Example 4:

[0078] Compound T04 (Target 4) was prepared by the following method:

[0079] In a 250 mL single-necked flask, 1-fluoro-2-nitrobenzene (10.0 g, 70.9 mmol, 7.49 mL, 1 equivalent) and tert-butylpiperazine-1-carboxylate (13.9 g, 74.4 mmol, 1.05 equivalent) were dissolved in N,N-dimethylformamide (70.0 mL). Potassium carbonate (11.7 g, 85.0 mmol, 1.2 equivalents) was added at 20°C. The resulting reaction solution was stirred at 50°C for 10 hours. LC / MS / MS indicated the reaction was complete. After cooling, the reaction mixture was poured into 300 mL of water and extracted with ethyl acetate (100 mL x 3). The combined organic phases were washed with saturated brine (200 mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to yield tert-butyl 4-(2-nitrophenyl)piperazine-1-carboxylate (21.0 g, crude) as a light yellow oil.

[0080] tert-Butyl 4-(2-nitrophenyl)piperazine-1-carboxylate (16.0 g, 52.0 mmol, 1 equivalent) was placed in a 250 mL hydrogenation bottle containing ethanol (100 mL). Wet palladium on carbon (1.60 g, 1.50 mmol, 10% purity, 2.89 e-2 equivalent) was added. After argon replacement three times, the mixture was stirred at 50°C under a hydrogen atmosphere (50 PSI) for 5 hours. TLC indicated the reaction was complete. The reaction solution was filtered through celite and rinsed with methanol (100 mL x 3). After rinsing, the filtrate was concentrated under reduced pressure to yield tert-butyl 4-(2-aminophenyl)piperazine-1-carboxylate (16.0 g, crude) as a gray solid.

[0081] Tert-butyl 4-(2-aminophenyl)piperazine-1-carboxylate (18.0 g, 64.9 mmol, 1 eq) was placed in a 500 mL three-necked flask containing ethanol (90.0 mL) and water (90.0 mL). Concentrated hydrochloric acid (12 M, 10.8 mL, 2 eq) was added at 0°C. A solution of sodium nitrite (4.52 g, 65.5 mmol, 1.01 eq) in water (30 mL) was then slowly added dropwise to the reaction mixture at 0°C. After the addition was complete, the mixture was stirred at 0°C for 0.5 hours. This solution was then added dropwise to a solution of ethoxy(potassium heterocationic mercapto)methanone (20.8 g, 130 mmol, 2 eq) in water (90.0 mL) at 40°C. LC-MS / MS indicated the reaction was complete. TLC showed two new spots. The reaction mixture was adjusted to pH 8-9 with aqueous sodium bicarbonate and extracted with ethyl acetate (200 mL x 3). The combined organic phases were washed with saturated brine (200 mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure to yield a crude product. The crude product was purified by silica gel flash column separation (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 1 / 1) to yield tert-butyl 4-{2-[(ethoxymethylthioyl)mercapto]phenyl}piperazine-1-carboxylate (14.0 g, 36.6 mmol, 56.4% yield) as a brown oil.

[0082] Tert-butyl 4-{2-[(ethoxymethylthioyl)mercapto]phenyl}piperazine-1-carboxylate (1.00 g, 2.61 mmol, 1 equivalent) was placed in a 50.0 mL single-necked flask containing ethanol (5.00 mL). A solution of potassium hydroxide (631 mg, 11.3 mmol, 4.31 eq) in ethanol (5.00 mL) was added at 20°C. The resulting reaction solution was stirred at 20°C for 1 hour. LC / MS analysis indicated the reaction was complete. The reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was dissolved in water and ethyl acetate and separated by extraction. The aqueous phase was adjusted to pH 2-3 with 1 M HCl and extracted with ethyl acetate (20.0 mL x 3). The combined organic phases were washed with saturated brine (20.0 mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by reverse phase purification (0.1% HCl system) and lyophilized to give tert-butyl 4-(2-mercaptophenyl)piperazine-1-carboxylate (400 mg, 1.21 mmol, 46.5% yield, hydrochloride) as a white solid.

[0083] tert-Butyl 4-(2-mercaptophenyl)piperazine-1-carboxylate (50.0 mg, 151 μmol, 1 equiv, hydrochloride) and 2-bromo-3,5-dimethylthiophene (34.6 mg, 181 μmol, 1.2 equiv) were placed in a 10.0 mL single-necked vial containing toluene (0.300 mL). Sodium tert-butoxide (43.6 mg, 453 μmol, 3 equiv), BINAP (18.8 mg, 30.2 μmol, 0.2 equiv), and Pd(dba)2 (8.69 mg, 15.1 μmol, 0.1 equiv) were added sequentially. After three replacements, the mixture was stirred at 90°C for 10 hours. LC / MS analysis indicated the reaction was complete. TLC monitoring showed the formation of multiple new spots after the starting material was consumed. After cooling, the mixture was added to distilled water (5.00 mL) and extracted with ethyl acetate (5.00 mL x 3). The combined organic phases were washed with saturated brine (10.0 mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure to yield the crude product. The crude product was then separated and purified using preparative chromatography (SiO2, petroleum ether / ether acetate = 5 / 1) to afford tert-butyl 4-{2-[(3,5-dimethylthiophen-2-yl)mercapto]phenyl}piperazine-1-carboxylate (60.0 g, 148 μmol, 98.1% yield) as a yellow oil.

[0084] Tert-butyl 4-{2-[(3,5-dimethylthiophen-2-yl)mercapto]phenyl}piperazine-1-carboxylate (60.0 mg, 148 μmol, 1 eq) was placed in a 10.0 mL single-necked vial containing ethyl acetate (0.500 mL). Ethyl acetate hydrochloride (2 M, 0.500 mL, 6.74 eq) was added, and the mixture was stirred at 20°C for 2 hours. LC-MS / MS indicated the reaction was complete. The reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was purified by HPLC (Phenomenex luna C18 column, 150 x 25 mm x 10 μm; mobile phase: water (hydrochloric acid)-acetonitrile; gradient: 20% to 50% B over 10 minutes). 1-{2-[(3,5-dimethylthiophen-2-yl)mercapto]phenyl}piperazine (24.5 mg, 71.8 μmol, 48.4% yield, 100% purity, hydrochloride) was obtained as a white solid.

[0085] 1H NMR (400 MHz, deuterated dimethyl sulfoxide) δ = 9.05-8.54 (m, 2H), 7.23-7.13 (m, 2H), 7.09-7.03 (m, 1H), 6.90 (s, 1H), 6.51 (d, J = 7.6 Hz, 1H), 3.26 (br d, J = 4.4 Hz, 4H), 3.16 (br d, J = 4.4 Hz, 4H), 2.46 (s, 3H), 2.11 (s, 3H).

[0086] Example 5:

[0087] Compound T05 (Target 5) was prepared by the following method:

[0088] Tert-Butyl 4-(2-mercaptophenyl)piperazine-1-carboxylate (100 mg, 302 μmol, 1 equiv, hydrochloride) and 5-bromo-2,4-dimethyl-1,3-thiazole (69.7 mg, 363 μmol, 1.2 equiv) were placed in a 10.0 mL single-necked vial containing toluene (0.700 mL). Sodium tert-butoxide (87.1 mg, 907 μmol, 3 equiv), BINAP (37.6 mg, 60.4 μmol, 0.2 equiv), and Pd(dba)2 (17.4 mg, 30.2 μmol, 0.1 equiv) were added sequentially. After nitrogen was purged three times, the mixture was stirred at 90°C for 10 hours. LC / MS analysis indicated the reaction was complete. TLC monitoring showed the presence of multiple new spots indicating complete consumption of the starting material. After cooling, the reaction mixture was added to distilled water (5.00 mL) and extracted with ethyl acetate (5.00 mL x 3). The combined organic phases were washed with saturated brine (10.0 mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure to yield the crude product. The crude product was purified by post-preparative chromatography (SiO2, petroleum ether / ether acetate = 5 / 1) to afford tert-butyl 4-{2-[(2,4-dimethyl-1,3-thiazol-5-yl)mercapto]phenyl}piperazine-1-carboxylate (60.0 mg, 148 μmol, 48.9% yield) as a yellow oil.

[0089] Tert-butyl 4-{2-[(2,4-dimethyl-1,3-thiazol-5-yl)mercapto]phenyl}piperazine-1-carboxylate (60.0 mg, 148 μmol, 1 eq) was placed in a 10.0 mL single-necked vial containing ethyl acetate (0.500 mL). Ethyl acetate hydrochloride (2 M, 0.500 mL, 6.76 eq) was added, and the mixture was stirred at 20°C for 2 hours. LC-MS / MS indicated the reaction was complete. The reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was purified by HPLC (Phenomenex luna C18 column, 150 x 25 mm x 10 μm; mobile phase: water (hydrochloric acid)-acetonitrile; gradient: 5% to 35% B over 10 minutes). 1-{2-[(2,4-dimethyl-1,3-thiazol-5-yl)mercapto]phenyl}piperazine (30.5 mg, 88.8 μmol, 60.0% yield, 99.5% purity, hydrochloride) was obtained as a white solid.

[0090] 1 H NMR (400 MHz, deuterated dimethyl sulfoxide) δ = 9.26-8.79 (m, 2H), 7.29-7.16 (m, 2H), 7.14-7.01 (m, 1H), 6.68-6.39 (m, 1H), 3.26 (br d, J = 2.0 Hz, 4H), 3.20-3.10 (m, 4H), 2.68 (s, 3H), 2.31 (s, 3H).

[0091] Example 6:

[0092] Compound T06 (Target 6) was prepared by the following method:

[0093] Tert-butyl 4-(2-mercaptophenyl)piperazine-1-carboxylate (100 mg, 302 μmol, 1 equiv, hydrochloride) and 1-bromo-2,4-bis(trifluoromethyl)benzene (106 mg, 363 μmol, 61.1 μL, 1.2 equiv) were placed in a 10.0 mL single-necked vial containing DMF (0.700 mL). Cesium carbonate (295 mg, 907 μmol, 3 equiv) was added. The mixture was stirred at 90°C for 10 hours. LC-MS / MS indicated the reaction was complete. TLC monitoring showed the formation of two new spots after the starting material was consumed. After cooling, the reaction mixture was added to distilled water (5.00 mL) and extracted with ethyl acetate (5.00 mL x 3). The combined organic phases were washed with saturated brine (10.0 mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure to obtain the crude product. The crude product was purified by post-preparative chromatography (SiO2, Petroleum ether / Ethyl acetate = 5 / 1) to give tert-butyl 4-(2-{[2,4-bis(trifluoromethyl)phenyl]mercapto}phenyl)piperazine-1-carboxylate (90.0 mg, 179 μmol, 58.8% yield) as a colorless oil.

[0094] Tert-butyl 4-(2-{[2,4-bis(trifluoromethyl)phenyl]mercapto}phenyl)piperazine-1-carboxylate (90.0 mg, 178 μmol, 1 equivalent) was placed in a 10.0 mL single-necked vial containing ethyl acetate (0.500 mL). Ethyl acetate hydrochloride (2 M, 0.500 mL, 5.63 eq) was added, and the mixture was stirred at 20°C for 2 hours. LC / MS / MS indicated the reaction was complete. The reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was purified by HPLC (Phenomenex luna C18 column, 150 x 25 mm x 10 μm; mobile phase: water (hydrochloric acid)-acetonitrile; gradient: 25% to 55% B over 10 minutes). 1-(2-{[2,4-bis(trifluoromethyl)phenyl]mercapto}phenyl)piperazine (25.5 mg, 57.6 μmol, 32.4% yield, 100% purity, hydrochloride) was obtained as a white solid.

[0095] 1H NMR (400 MHz, deuterated dimethyl sulfoxide) δ = 8.82-8.61 (m, 2H), 8.08 (s, 1H), 7.90 (d, J = 8.0 Hz, 1H), 7.53 (dt, J = 1.6, 7.7 Hz, 1H), 7.40 (dd, J = 1.2, 7.7 Hz, 1H), 7.34 (d, J = 7.6 Hz, 1H), 7.29-7.21 (m, 1H), 7.19 (d, J = 8.4 Hz, 1H), 3.15 (br d, J = 4.4 Hz, 4H), 2.95 (br s, 4H).

[0096] 13 F NMR (377 MHz, deuterated dimethyl sulfoxide) δ = -60.65 (s, 3F), -61.10 (s, 3F)

[0097] Example 7:

[0098] Compound T07 (Target 7) was prepared by the following method:

[0099] In a 10.0 mL single-necked vial, tert-butyl 4-(2-mercaptophenyl)piperazine-1-carboxylate (200 mg, 604 μmol, 1 eq, hydrochloride) was dissolved in DMF (3.00 mL). Cesium carbonate (393 mg, 1.21 mmol, 2 eq) and 4-bromo-3-methylbenzonitrile (118 mg, 604 μmol, 1 eq) were added. The resulting reaction mixture was stirred at 90°C for 16 hours. LC-MS / MS indicated the reaction was complete. TLC monitoring revealed multiple new spots after the starting material was consumed. After cooling, the reaction mixture was added to distilled water (30.0 mL) and extracted with ethyl acetate (15.0 mL x 2). The combined organic phases were washed with saturated brine (15.0 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure to obtain the crude product. The crude product was purified by separation on a chromatography plate (SiO2, Petroleum ether / Ethyl acetate = 5 / 1) to give tert-butyl 4-{2-[(4-cyano-2-methylphenyl)mercapto]phenyl}piperazine-1-carboxylate (130 mg, 317 μmol, 52.5% yield) as a yellow oil.

[0100] In a 10.0 mL single-necked bottle, tert-butyl 4-{2-[(4-cyano-2-methylphenyl)mercapto]phenyl}piperazine-1-carboxylate (130 mg, 317 μmol, 1 equivalent) was dissolved in ethyl acetate hydrochloride (10.0 mL). The resulting reaction solution was stirred at 25°C for 1 hour. LC-MS / MS showed that the reaction was complete. The reaction solution was directly concentrated under reduced pressure to obtain a crude product. The crude product was purified by high performance liquid chromatography (column model: Phenomenex luna C 18150*25mm*10um; mobile phase: [water (hydrochloric acid)-acetonitrile]; B%: 15%-45%, 10 min) to give 3-methyl-4-{[2-(piperazin-1-yl)phenyl]mercapto}benzonitrile (25.7 mg, 74.1 μmol, 23.3% yield, 99.7% purity, hydrochloride) as an off-white solid.

[0101] 1 H NMR (400 MHz, deuterated dimethyl sulfoxide) δ = 9.13 (br s, 2H), 7.78 (d, J = 1.2 Hz, 1H), 7.56 (dd, J = 1.6, 8.4 Hz, 1H), 7.46-7.39 (m, 1H), 7.27 (d, J = 7.6 Hz, 1H), 7.20-7.12 (m, 2H), 6.99 (d, J = 8.3 Hz, 1H), 3.21-3.15 (m, 4H), 2.99 (br d, J = 4.4 Hz, 4H), 2.36 (s, 3H).

[0102] Example 8:

[0103] Compound T08 (Target 8) was prepared by the following method:

[0104] Tert-butyl 4-(2-bromophenyl)piperazine-1-carboxylate (200 mg, 586 μmol, 1 equiv) and 4-fluoro-2-methylbenzene-1-thiol (83.3 mg, 586 μmol, 1 equiv) were placed in a 10.0 mL single-necked vial containing toluene (3.00 mL). Sodium tert-butoxide (84.4 mg, 879 μmol, 1.5 equiv), Pd2(dba)3 (26.8 mg, 29.3 μmol, 0.05 equiv), and Xantphos (33.9 mg, 58.6 μmol, 0.1 equiv) were added sequentially. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 100°C for 16 hours. LC-MS / MS indicated the reaction was complete. TLC monitoring revealed two new spots indicating complete consumption of the starting material. After cooling, the reaction mixture was added to distilled water (30.0 mL) and extracted with ethyl acetate (15.0 mL x 2). The combined organic phases were washed with saturated brine (15.0 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to yield a crude product. The crude product was purified by chromatography (SiO2, petroleum ether / ether acetate = 5 / 1) to afford tert-butyl 4-{2-[(4-fluoro-2-methylphenyl)mercapto]phenyl}piperazine-1-carboxylate (110 mg, 273 μmol, 46.5% yield) as a yellow oil.

[0105] In a 10.0 mL single-necked flask, tert-butyl 4-{2-[(4-fluoro-2-methylphenyl)mercapto]phenyl}piperazine-1-carboxylate (110 mg, 273 μmol, 1 equivalent) was dissolved in dichloromethane (2.00 mL), and trifluoroacetic acid (767 mg, 6.73 mmol, 0.50 mL, 24.6 equivalents) was added. The resulting reaction solution was stirred at 25°C for 30 minutes. LC-MS / MS indicated the reaction was complete. After cooling, the reaction mixture was added to saturated aqueous sodium bicarbonate (20.0 mL) and extracted with ethyl acetate (10 mL x 2). The combined organic phases were washed with saturated brine (10.0 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by HPLC (column model: Phenomenex luna C 18 The product was placed in a 50.0 mL single-necked flask containing ethyl acetate hydrochloride (10.0 mL) and stirred at 25°C for 30 minutes. The reaction solution was then concentrated under reduced pressure to obtain 1-{2-[(4-fluoro-2-methylphenyl)mercapto]phenyl}piperazine (25.0 mg, 73.1 μmol, 26.7% yield, 98.8% purity, hydrochloride) as a bright yellow solid.

[0106] 1 H NMR (400 MHz, deuterated dimethyl sulfoxide) δ = 9.18 (br s, 2H), 7.48 (dd, J = 6.0, 8.4 Hz, 1H), 7.33 (dd, J = 2.8, 9.6 Hz, 1H), 7.20-7.11 (m, 3H), 7.03-6.96 (m, 1H), 6.44 (d, J = 7.6 Hz, 1H), 3.20 (br s, 8H), 2.29 (s, 3H).

[0107] Example 9:

[0108] Compound T09 (Target 9) was prepared by the following method:

[0109] tert-Butyl 4-(2-mercaptophenyl)piperazine-1-carboxylate (100 mg, 302 μmol, 1 equiv, hydrochloride) and 1-bromo-4-chloro-2-methylbenzene (74.5 mg, 363 μmol, 48.3 μL, 1.2 equiv) were placed in a 10.0 mL single-necked vial containing toluene (0.700 mL). Sodium tert-butoxide (87.1 mg, 907 μmol, 3 equiv), BINAP (37.6 mg, 60.4 μmol, 0.2 equiv), and Pd(dba)2 (17.4 mg, 30.2 μmol, 0.1 equiv) were added sequentially. After nitrogen purging three times, the mixture was stirred at 90°C for 10 hours. LC / MS analysis indicated the reaction was complete. TLC monitoring showed the formation of three new spots after the starting material was consumed. After cooling, the reaction mixture was added to distilled water (5.00 mL) and extracted with ethyl acetate (5.00 mL x 3). The combined organic phases were washed with saturated brine (10.0 mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure to yield a crude product. The crude product was purified by post-preparative chromatography (SiO2, petroleum ether / ethyl acetate = 5 / 1) to yield tert-butyl 4-{2-[(4-chloro-2-methylphenyl)mercapto]phenyl}piperazine-1-carboxylate (90.0 mg, 215 μmol, 71.1% yield) as a colorless oil.

[0110] Tert-butyl 4-{2-[(4-chloro-2-methylphenyl)mercapto]phenyl}piperazine-1-carboxylate (90.0 mg, 215 μmol, 1 equiv) was placed in a 10.0 mL single-necked vial containing ethyl acetate (0.500 mL). Ethyl acetate hydrochloride (2 M, 0.500 mL, 4.66 equiv) was added, and the mixture was stirred at 20°C for 2 hours. LC-MS / MS indicated the reaction was complete. The reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was purified by HPLC (Phenomenex luna C18 column, 150 x 25 mm x 10 μm; mobile phase: [water (hydrochloric acid)-acetonitrile]; gradient: 23% to 53% B over 10 minutes). 1-{2-[(4-chloro-2-methylphenyl)mercapto]phenyl}piperazine (25.1 mg, 70.5 μmol, 32.8% yield, 99.7% purity, hydrochloride) was obtained as a white solid.

[0111] 1 H NMR (400 MHz, deuterated dimethyl sulfoxide) δ = 9.04 (br s, 2H), 7.53 (s, 1H), 7.34 (s, 2H), 7.27-7.18 (m, 2H), 7.08-7.01 (m, 1H), 6.70-6.57 (m, 1H), 3.19 (s, 8H), 2.30 (s, 3H).

[0112] Example 10:

[0113] Compound T10 (Target 10) was prepared by the following method:

[0114] In a 50.0 mL single-necked flask, tert-butyl 4-(2-bromophenyl)piperazine-1-carboxylate (200 mg, 586 μmol, 1 eq) and 2,4-difluorobenzene-1-thiol (85.6 mg, 586 μmol, 1 eq) were dissolved in toluene (2.00 mL). Sodium tert-butoxide (90.12 mg, 937.7 μmol, 1.6 eq), BINAP (14.6 mg, 23.44 μmol, 0.04 eq), and Pd2(dba)3 (10.7 mg, 11.7 μmol, 0.02 eq) were added sequentially. The resulting reaction solution was heated at 100°C and stirred under nitrogen for 10 hours. LC-MS analysis indicated residual tert-butyl 4-(2-bromophenyl)piperazine-1-carboxylate, and the product, tert-butyl 4-{2-[(2,4-difluorophenyl)mercapto]phenyl}piperazine-1-carboxylate, was detected. TLC indicated residual tert-butyl 4-(2-bromophenyl)piperazine-1-carboxylate, along with the generation of numerous new spots. The reaction mixture was concentrated under reduced pressure to yield the crude product. The crude product was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 0 / 1). Tert-butyl 4-{2-[(2,4-difluorophenyl)mercapto]phenyl}piperazine-1-carboxylate (151 mg, 371 μmol, 63.3% yield) was obtained as a yellow solid.

[0115] In a 10.0 mL single-necked vial, tert-butyl 4-{2-[(2,4-difluorophenyl)mercapto]phenyl}piperazine-1-carboxylate (151 mg, 371 μmol, 1 eq) was dissolved in dichloromethane (1.50 mL), and trifluoroacetic acid (2.30 g, 20.1 mmol, 1.5 mL, 54 eq) was slowly added dropwise. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 20°C for 1 hour. LC-MS / MS indicated the reaction was complete. The pH of the reaction solution was adjusted to 9 with sodium bicarbonate and extracted with dichloromethane (10.0 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by HPLC (column model: Waters Xbridge 150 x 25 mm x 5 μm; mobile phase: [water (sodium bicarbonate)-acetonitrile]; gradient: 34% to 64% B over 9 minutes). The crude product obtained by HPLC was purified by slurrying with ethyl acetate hydrochloride (4.00 mL) at 20°C for one hour, and the filter cake was concentrated under reduced pressure to yield 1-{2-[(2,4-difluorophenyl)mercapto]phenyl}piperazine (25.3 mg, 73.8 μmol, 19.8% yield, 100% purity, hydrochloride) as a light yellow solid.

[0116] 1 H NMR (400 MHz, deuterated dimethyl sulfoxide) δ = 9.20 (br s, 2H), 7.60 (dt, J = 6.4, 8.4 Hz, 1H), 7.49 (dt, J = 2.4, 9.2 Hz, 1H), 7.32-7.16 (m, 3H), 7.13-6.99 (m, 1H), 6.63 (d, J = 7.6 Hz, 1H), 3.19 (br s, 8H).

[0117] 19 F NMR (400 MHz, deuterated dimethyl sulfoxide) δ = -102.40 (d, J = 4.8 Hz, 1F), -105.31--109.30 (m, 1F).

[0118] Example 11:

[0119] Compound T11 (Target 11) was prepared by the following method:

[0120] In a 50.0 mL single-necked vial, tert-butyl 4-(2-mercaptophenyl)piperazine-1-carboxylate hydrochloride (200 mg, 604 μmol, 1 eq, hydrochloride) and 1-bromo-2-fluoro-4-methylbenzene (114 mg, 604 μmol, 1 eq) were dissolved in toluene (2.00 mL). Sodium tert-butoxide (92.9 mg, 967 μmol, 1.6 eq), BINAP (15.0 mg, 24.1 μmol, 0.04 eq), and Pd2(dba)3 (11.0 mg, 12.0 μmol, 0.02 eq) were added sequentially. After nitrogen purging three times, the resulting reaction solution was heated at 90°C and stirred under nitrogen for 10 hours. LC / MS / MS indicated the reaction was complete. TLC analysis indicated complete consumption of the tert-butyl 4-(2-mercaptophenyl)piperazine-1-carboxylate hydrochloride, with the generation of numerous new spots. The reaction mixture was cooled to room temperature, and water (4.00 mL) was added, followed by extraction with dichloromethane (3.00 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by pre-thin-layer chromatography (silica, petroleum ether / ethyl acetate = 10 / 1). Tert-butyl 4-{2-[(2-fluoro-4-methylphenyl)mercapto]phenyl}piperazine-1-carboxylate (172 mg, 427 μmol, 70.6% yield) was obtained as a yellow oil.

[0121] In a 10.0 mL single-necked vial, tert-butyl 4-{2-[(2-fluoro-4-methylphenyl)mercapto]phenyl}piperazine-1-carboxylate (172 mg, 427 μmol, 1 eq) was dissolved in ethyl acetate (2.00 mL). Ethyl acetate hydrochloride (2 M, 4.00 mL) was slowly added dropwise. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 20°C for 1 hour. LC / MS / MS indicated the reaction was complete. The reaction mixture was concentrated under reduced pressure to obtain the crude product. Purification was performed by beating with ethyl acetate hydrochloride (2.00 mL) at 20°C for one hour, and the filter cake was then concentrated under reduced pressure. This afforded 1-{2-[(2-fluoro-4-methylphenyl)mercapto]phenyl}piperazine (25.2 mg, 72.6 μmol, 17.0% yield, 97.7% purity, hydrochloride) as a gray solid.

[0122] 1 H NMR (400 MHz, deuterated dimethyl sulfoxide) δ = 9.16 (br s, 2H), 7.41 (t, J = 7.6 Hz, 1H), 7.28-7.10 (m, 4H), 7.06-6.96 (m, 1H), 6.59 (d, J = 8.0 Hz, 1H), 3.20 (br d, J = 3.2 Hz, 8H), 2.38 (s, 3H).

[0123] 19F NMR (400 MHz, deuterated dimethyl sulfoxide) δ = -108.32 (s, 1F).

[0124] Example 12:

[0125] Compound T12 (Target 12) was prepared by the following method:

[0126] tert-Butyl 4-(2-mercaptophenyl)piperazine-1-carboxylate (200 mg, 604 μmol, 1 equiv, hydrochloride) and 1-bromo-4-methyl-2-(trifluoromethyl)benzene (173 mg, 725 μmol, 1.2 equiv) were placed in a 10.0 mL single-necked flask containing toluene (1.00 mL). Sodium tert-butoxide (174 mg, 1.81 mmol, 3 equiv), BINAP (75.3 mg, 121 μmol, 0.2 equiv), and Pd(dba)2 (34.8 mg, 60.5 μmol, 0.1 equiv) were added sequentially. After nitrogen purging three times, the mixture was stirred at 90°C for 10 hours. LC-MS / MS indicated the reaction was complete. TLC monitoring showed the formation of multiple new spots following the complete consumption of the starting material. After cooling, the reaction mixture was added to distilled water (5.00 mL) and extracted with ethyl acetate (5.00 mL x 3). The combined organic phases were washed with saturated brine (10.0 mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to yield a crude product. The crude product was purified by silica gel flash column separation (SiO2, petroleum ether / ether acetate = 1 / 0 to 1 / 1) to yield tert-butyl 4-(2-{[4-methyl-2-(trifluoromethyl)phenyl]mercapto}phenyl)piperazine-1-carboxylate (240 mg, 530 μmol, 87.7% yield) as a yellow oil.

[0127] Tert-butyl 4-(2-{[4-methyl-2-(trifluoromethyl)phenyl]mercapto}phenyl)piperazine-1-carboxylate (240 mg, 530 μmol, 1 equivalent) was placed in a 10.0 mL single-necked vial containing ethyl acetate (1.00 mL). Ethyl acetate hydrochloride (2 M, 2.00 mL, 7.54 eq) was added, and the mixture was stirred at 20°C for 2 hours. LC / MS / MS indicated the reaction was complete. The reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was purified by HPLC (Phenomenex luna C18 column, 150 x 25 mm x 10 μm; mobile phase: water (hydrochloric acid)-acetonitrile; gradient: 13% to 43% B over 10 minutes). 1-(2-{[4-methyl-2-(trifluoromethyl)phenyl]mercapto}phenyl)piperazine (25.3 mg, 65.0 μmol, 12.2% yield, 100% purity, hydrochloride salt) was obtained as an off-white solid.

[0128] 1 H NMR (400 MHz, deuterated dimethyl sulfoxide) δ = 9.13-8.87 (m, 2H), 7.73 (s, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.38 (d, J = 8.0 Hz, 1H), 7.30-7.17 (m, 2H), 7.10-7.00 (m, 1H), 6.68 (dd, J = 1.2, 7.8 Hz, 1H), 3.17 (br d, J = 2.0 Hz, 8H), 2.42 (s, 3H).

[0129] 19 F NMR (377 MHz, deuterated dimethyl sulfoxide) δ = -59.32 (s, 3F).

[0130] Example 13:

[0131] Compound T13 (Target 13) was prepared by the following method:

[0132] Tert-butyl 4-(2-mercaptophenyl)piperazine-1-carboxylate (200 mg, 604 μmol, 1 equiv, hydrochloride) and 1-bromo-2-methyl-4-(trifluoromethyl)benzene (173 mg, 725 μmol, 1.2 equiv) were placed in a 10.0 mL single-necked flask containing DMF (1.40 mL). Cesium carbonate (492 mg, 1.51 mmol, 2.5 equiv) was added. The mixture was stirred at 100°C for 10 hours. LC-MS / MS indicated the reaction was complete. TLC monitoring showed the formation of two new spots after the starting material was consumed. After cooling, the reaction mixture was added to distilled water (5.00 mL) and extracted with ethyl acetate (5.00 mL x 3). The combined organic phases were washed with saturated brine (10.0 mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column flash separation (SiO2, Petroleum ether / Ethyl acetate = 1 / 0 to 1 / 1) to give tert-butyl 4-(2-{[2-methyl-4-(trifluoromethyl)phenyl]mercapto}phenyl)piperazine-1-carboxylate (100 mg, 221 μmol, 36.6% yield) as a yellow solid.

[0133] Tert-butyl 4-(2-{[2-methyl-4-(trifluoromethyl)phenyl]mercapto}phenyl)piperazine-1-carboxylate (100 mg, 221 μmol, 1 equiv) was placed in a 10.0 mL single-necked vial containing ethyl acetate (1.00 mL). Ethyl acetate hydrochloride (2 M, 1.00 mL, 9.05 equiv) was added, and the mixture was stirred at 20°C for 2 hours. LC / MS / MS indicated the reaction was complete. The reaction mixture was concentrated under reduced pressure to obtain the crude product. The crude product was purified by HPLC (Phenomenex luna C18 column, 150 x 25 mm x 10 μm; mobile phase: water (hydrochloric acid)-acetonitrile; gradient: 23% to 53% B over 10 minutes). 1-(2-{[2-methyl-4-(trifluoromethyl)phenyl]mercapto}phenyl)piperazine (25.8 mg, 65.9 μmol, 29.8% yield, 99.2% purity, hydrochloride) was obtained as a white solid.

[0134] 1 H NMR (400 MHz, deuterated dimethyl sulfoxide) δ = 9.02 (br s, 2H), 7.72 (s, 1H), 7.53 (br d, J = 8.4 Hz, 1H), 7.42-7.34 (m, 1H), 7.29-7.19 (m, 2H), 7.12 (dt, J = 1.2, 7.5 Hz, 1H), 7.00 (dd, J = 1.2, 7.8 Hz, 1H), 3.26-3.16 (m, 4H), 3.08 (br d, J = 4.4 Hz, 4H), 2.41 (s, 3H).

[0135] 19 F NMR (377 MHz, deuterated dimethyl sulfoxide) δ = -60.85 (s, 3F).

[0136] Example 14:

[0137] Compound T14 (Target 14) was prepared by the following method:

[0138] In a 50.0 mL single-necked vial, 2,4-dimethylbenzene-1-thiol (711 mg, 5.14 mmol, 695 μL, 1.1 eq) was dissolved in DMF (9.00 mL). Potassium carbonate (969 mg, 7.02 mmol, 1.5 eq) and 3-bromo-4-chloropyridine (900 mg, 4.68 mmol, 1 eq) were added sequentially. After nitrogen substitution three times, the resulting reaction solution was heated at 100°C and stirred under nitrogen for 10 hours. LC-MS / MS indicated the reaction was complete. TLC analysis showed complete consumption of 3-bromo-4-chloropyridine, with the formation of a new spot. The reaction mixture was filtered and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (silica, petroleum ether / ethyl acetate = 0 / 1 to 1 / 0). 3-Bromo-4-[(2,4-dimethylphenyl)mercapto]pyridine (1.19 g, 4.04 mmol, 86.4% yield) was obtained as a white solid.

[0139] In a 50.0 mL single-necked flask, 3-bromo-4-[(2,4-dimethylphenyl)mercapto]pyridine (200 mg, 679 μmol, 1 eq) and tert-butylpiperazine-1-carboxylate (126 mg, 679 μmol, 1 eq) were dissolved in toluene (2.00 mL). Sodium tert-butoxide (104 mg, 1.09 mmol, 1.6 eq), Pd2(dba)3 (12.4 mg, 13.6 μmol, 0.02 eq), and BINAP (16.9 mg, 27.1 μmol, 0.04 eq) were then added in sequence. The resulting reaction mixture was heated at 85°C under nitrogen and stirred for 5 hours. Liquid chromatography-mass spectrometry (LC-MS) revealed the presence of 3-bromo-4-[(2,4-dimethylphenyl)mercapto]pyridine and the product, tert-butyl 4-{4-[(2,4-dimethylphenyl)mercapto]pyridin-3-yl}piperazine-1-carboxylate. TLC revealed residual 3-bromo-4-[(2,4-dimethylphenyl)mercapto]pyridine and the generation of numerous new spots. The reaction mixture was filtered and the filtrate concentrated under reduced pressure to yield the crude product. The crude product was purified by pre-thin-layer chromatography (SiO2, petroleum ether / ethyl acetate = 5 / 1). Tert-butyl 4-{4-[(2,4-dimethylphenyl)mercapto]pyridin-3-yl}piperazine-1-carboxylate (141 mg, 352 μmol, 51.9% yield) was obtained as a yellow solid.

[0140] In a 50.0 mL single-necked vial, tert-butyl 4-{4-[(2,4-dimethylphenyl)mercapto]pyridin-3-yl}piperazine-1-carboxylate (141 mg, 352 μmol, eq) was dissolved in dichloromethane (1.50 mL), and trifluoroacetic acid (2.30 g, 20.1 mmol, 1.50 mL, 57.2 eq) was slowly added dropwise. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 20°C for 1 hour. LC-MS / MS indicated the reaction was complete. The pH of the reaction solution was adjusted to 9 with sodium bicarbonate and extracted with dichloromethane (10.0 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by HPLC (column model: Waters Xbridge 150 x 25 mm x 5 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 30% to 60% B over 9 minutes). The crude product obtained by HPLC was purified by slurrying with ethyl acetate hydrochloride (4.00 mL) at 20°C for one hour, and the filter cake was concentrated under reduced pressure to yield 1-{4-[(2,4-dimethylphenyl)mercapto]pyridin-3-yl}piperazine (25.3 mg, 75.2 μmol, 21.3% yield, 99.7% purity, hydrochloride) as a yellow solid.

[0141] 1 H NMR (400 MHz, deuterated dimethyl sulfoxide) δ = 9.60 (br s, 2H), 8.55 (s, 1H), 8.25 (d, J = 6.4 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.40 (s, 1H), 7.25 (br d, J = 8.0 Hz, 1H), 6.67 (d, J = 6.0 Hz, 1H), 3.47-3.36 (m, 4H), 3.29 (br s, 4H), 2.38 (s, 3H), 2.27 (s, 3H).

[0142] Example 15:

[0143] Compound T15 (Target 15) was prepared by the following method:

[0144] 2-Bromobenzene-1-thiol (1.00 g, 5.29 mmol, 621 μL, 1 equiv) and 1-bromo-2-methyl-4-(trifluoromethyl)benzene (1.52 g, 6.35 mmol, 1.2 equiv) were placed in a 50.0 mL single-necked flask containing toluene (7.00 mL). Potassium tert-butoxide (1.19 g, 10.6 mmol, 2 equiv), BINAP (659 mg, 1.06 mmol, 0.2 equiv), and Pd(dba)2 (304 mg, 529 μmol, 0.1 equiv) were added sequentially. After nitrogen was purged three times, the mixture was stirred at 100°C for 10 hours. LC-MS / MS indicated the reaction was complete. TLC monitoring showed the formation of multiple new spots following complete consumption of the starting material. After cooling, the reaction mixture was added to distilled water (20.0 mL) and extracted with ethyl acetate (20.0 mL x 3). The combined organic phases were washed with saturated brine (20.0 mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel flash column separation (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 2 / 1) to obtain 1-[(2-bromophenyl)mercapto]-2-methyl-4-(trifluoromethyl)benzene (800 mg, crude) as a colorless oil.

[0145] 1-[(2-Bromophenyl)mercapto]-2-methyl-4-(trifluoromethyl)benzene (800 mg, 2.30 mmol, 1 eq) and tert-butyl 2,6-diazaspiro[3.3]heptane-2-carboxylate (548 mg, 2.77 mmol, 1.2 eq) were placed in a 50.0 mL single-necked flask containing toluene (5.50 mL). Sodium tert-butoxide (443 mg, 4.61 mmol, 2 eq), BINAP (287 mg, 461 μmol, 0.2 eq), and Pd(dba)2 (132 mg, 230 μmol, 0.1 eq) were added sequentially. After nitrogen was purged three times, the mixture was stirred at 100°C for 10 hours. LC / MS analysis indicated the reaction was complete. TLC monitoring showed the formation of two new spots after the starting material was consumed. After the reaction mixture cooled, it was added to distilled water (10.0 mL) and extracted with ethyl acetate (5.00 mL*3). The combined organic phases were washed with saturated brine (10.0 mL*3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified on a silica gel column (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 1 / 1) to obtain tert-butyl 6-(2-{[2-methyl-4-(trifluoromethyl)phenyl]mercapto}phenyl)-2,6-diazaspiro[3.3]heptane-2-carboxylate (900 mg, 1.94 mmol, 84.1% yield) as a yellow oil.

[0146] Tert-butyl 6-(2-{[2-methyl-4-(trifluoromethyl)phenyl]mercapto}phenyl)-2,6-diazaspiro[3.3]heptane-2-carboxylate (500 mg, 1.08 mmol, 1 equiv) was placed in a 10.0 mL single-necked flask containing dichloromethane (2.50 mL). Trifluoroacetic acid (3.84 g, 33.7 mmol, 2.50 mL, 31.3 equiv) was added, and the mixture was stirred at 20°C for 1 hour. LC / MS / MS indicated the reaction was complete. The reaction solution was adjusted to pH 8-9 with saturated aqueous sodium bicarbonate and extracted with dichloromethane (5.00 mL x 3). The combined organic phases were washed with saturated brine (10.0 mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by normal-phase HPLC (column type: Welch Ultimate XB-SiOH 250*50mm*10um; mobile phase: [n-hexane-ethanol]; gradient: 10%-50% B over 15 minutes) to afford 2-(2-{[2-methyl-4-(trifluoromethyl)phenyl]mercapto}phenyl)-2,6-diazaspiro[3.3]heptane (21.2 mg, 57.6 μmol, 5.35% yield, 98.6% purity) as a white solid.

[0147] 1 H NMR (400 MHz, deuterated dimethyl sulfoxide) δ = 7.58 (s, 1H), 7.41 (br d, J = 8.0 Hz, 1H), 7.39-7.33 (m, 1H), 7.28 (dd, J = 1.2, 7.6 Hz, 1H), 6.81 (t, J = 7.6 Hz, 1H), 6.73-6.56 (m, 2H), 3.99 (s, 4H), 3.47 (s, 4H), 2.42 (s, 3H). 19 F NMR (400 MHz, deuterated dimethyl sulfoxide) δ = -53.61--64.26 (s, 3F).

[0148] Example 16:

[0149] Compound T16 (Target 16) was prepared by the following method:

[0150] 1-[(2-Bromophenyl)mercapto]-2-methyl-4-(trifluoromethyl)benzene (300 mg, 864 μmol, 1 eq) and tert-butyl octahydropyrrolo[3,4-c]pyrrole-2-carboxylate (220 mg, 1.04 mmol, 1.2 eq) were placed in a 10.0 mL single-necked vial containing toluene (2.00 mL). Sodium tert-butoxide (166 mg, 1.73 mmol, 2 eq), BINAP (107 mg, 173 μmol, 0.2 eq), and Pd(dba)2 (150 mg, 86.4 μmol, 0.1 eq) were added sequentially. After nitrogen was purged three times, the mixture was stirred at 100°C for 10 hours. LC / MS analysis indicated the reaction was complete. TLC monitoring showed the formation of two new spots after the starting material was consumed. After the reaction mixture cooled, distilled water (5.00 mL) was added and extracted with ethyl acetate (5.00 mL*3). The combined organic phases were washed with saturated brine (10.0 mL*3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel flash column separation (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 1 / 1) to obtain tert-butyl 5-(2-{[2-methyl-4-(trifluoromethyl)phenyl]mercapto}phenyl)-octahydropyrrolo[3,4-c]pyrrole-2-carboxylate (220 mg, 456 μmol, 53.2% yield) as a yellow oil.

[0151] Tert-butyl 5-(2-{[2-methyl-4-(trifluoromethyl)phenyl]mercapto}phenyl)-octahydropyrrolo[3,4-c]pyrrole-2-carboxylate (170 mg, 355 μmol, 1 equiv) was placed in a 10.0 mL single-necked vial containing dioxane hydrochloride (2 M, 3.00 mL, 16.9 equiv). The mixture was stirred at 20°C for 1 hour. LC / MS / MS indicated the reaction was complete. The reaction mixture was concentrated under reduced pressure to obtain the crude product. The crude product was purified by HPLC (Phenomenex luna C18 column, 150 x 25 mm x 10 μm; mobile phase: water (hydrochloric acid)-acetonitrile; gradient: 25% to 55% B over 10 minutes). 2-(2-{[2-methyl-4-(trifluoromethyl)phenyl]mercapto}phenyl)-octahydropyrrolo[3,4-c]pyrrole (25.3 mg, 60.4 μmol, 17.0% yield, 99.0% purity, hydrochloride) was obtained as a white solid.

[0152] 1H NMR (400 MHz, deuterated dimethyl sulfoxide) δ = 9.15-8.81 (m, 2H), 7.67 (s, 1H), 7.49 (br d, J = 8.0 Hz, 1H), 7.44-7.34 (m, 1H), 7.25-7.17 (m, 1H), 7.15-7.10 (m, 1H), 7.09-6.99 (m, 2H), 3.29 (d, J = 9.6 Hz, 2H), 3.07 (dd, J = 6.0, 9.6 Hz, 2H), 2.98-2.87 (m, 2H), 2.80-2.70 (m, 2H), 2.40 (s, 3H).

[0153] 19 F NMR (377 MHz, deuterated dimethyl sulfoxide) δ = -60.73 (s, 3F).

[0154] Example 17:

[0155] Compound T17 (Target 17) was prepared by the following method:

[0156] 1-Bromo-2-iodobenzene (500 mg, 1.77 mmol, 1 equiv) and tert-butyl 2,6-diazaspiro[3.3]heptane-2-carboxylate (350 mg, 1.77 mmol, 1 equiv) were placed in a 50.0 mL single-necked flask containing toluene (6.00 mL). Sodium tert-butoxide (254 mg, 2.65 mmol, 1.5 equiv), Pd2(dba)3 (80.9 mg, 88.3 μmol, 0.05 equiv), and BINAP (110 mg, 176 μmol, 0.1 equiv) were added sequentially. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 85°C for 2 hours. LC-MS / MS indicated the reaction was complete. TLC monitoring showed multiple new spots indicating complete consumption of the starting material. After cooling, the reaction mixture was added to distilled water (60.0 mL) and extracted with ethyl acetate (30.0 mL x 2). The combined organic phases were washed with saturated brine (30.0 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (gradient: (SiO2, Petroleum ether / Ethyl acetate = 1 / 0 to 1 / 1) to obtain tert-butyl 6-(2-bromophenyl)-2,6-diazaspiro[3.3]heptane-2-carboxylate (380 mg, 1.08 mmol, 60.8% yield) as a yellow solid.

[0157] Tert-butyl 6-(2-bromophenyl)-2,6-diazaspiro[3.3]heptane-2-carboxylate (150 mg, 424 μmol, 1 equiv) and 2,4-dimethylbenzene-1-thiol (58.7 mg, 424 μmol, 1 equiv) were placed in a 10.0 mL single-necked vial containing toluene (2.00 mL). Sodium tert-butoxide (81.6 mg, 849 μmol, 2 equiv) and RuPhos Pd G3 (35.5 mg, 42.4 μmol, 0.1 equiv) were added sequentially. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 100°C for 15 hours. LC-MS / MS indicated the reaction was complete. TLC showed two new spots. After cooling, the reaction mixture was added to distilled water (30.0 mL) and extracted with ethyl acetate (15.0 mL x 2). The combined organic phases were washed with saturated brine (15.0 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was separated and purified by chromatography (SiO2, petroleum ether / ethyl acetate = 5 / 1) to give tert-butyl 6-{2-[(2,4-dimethylphenyl)mercapto]phenyl}-2,6-diazaspiro[3.3]heptane-2-carboxylate (140 mg, 340 μmol, 80.3% yield) as a yellow solid.

[0158] In a 10.0 mL single-necked vial, tert-butyl 4-{2-[(4-fluoro-2-methylphenyl)mercapto]phenyl}piperazine-1-carboxylate (110 mg, 273 μmol, 1 equivalent) was dissolved in dichloromethane (2.00 mL), and trifluoroacetic acid (767 mg, 6.73 mmol, 0.50 mL, 19.7 equivalents) was added. The resulting reaction mixture was stirred at 25°C for 30 minutes. LC / MS / MS indicated the reaction was complete. After cooling, the reaction mixture was added to saturated aqueous sodium bicarbonate (20.0 mL) and extracted with ethyl acetate (10.0 mL x 2). The combined organic phases were washed with saturated brine (10.0 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by high performance liquid chromatography (column model: Welch Ultimate XB-SiOH 250*50mm*10um; mobile phase: [n-hexane-ethanol]; B%: 20%-60%, 15 minutes) to give 2-{2-[(2,4-dimethylphenyl)mercapto]phenyl}-2,6-diazaspiro[3.3]heptane (25.9 mg, 77.2 μmol, 22.6% yield, 92.3% purity) as a yellow colloid.

[0159] 1H NMR (400 MHz, deuterated dimethyl sulfoxide) δ = 7.25-7.15 (m, 1H), 7.07 (s, 1H), 6.96 (dd, J = 1.2, 7.6 Hz, 1H), 6.94-6.89 (m, 1H), 6.75-6.69 (m, 1H), 6.67 (d, J = 7.6 Hz, 1H), 6.56 (dd, J = 1.2, 8.4 Hz, 1H), 4.02 (s, 4H), 3.61 (s, 4H), 2.26 (s, 3H), 2.23 (s, 3H).

[0160] Example 18:

[0161] Compound T18 (Target 18) was prepared by the following method:

[0162] 1-Bromo-2-iodobenzene (500 mg, 1.77 mmol, 1 equiv) and tert-butyl octahydropyrrolo[3,4-c]pyrrole-2-carboxylate (375 mg, 1.77 mmol, 1 equiv) were placed in a 50.0 mL single-necked flask containing toluene (5.00 mL). Sodium tert-butoxide (254 mg, 2.65 mmol, 1.5 equiv), Pd2(dba)3 (80.9 mg, 88.3 μmol, 0.05 equiv), and Xantphos (102 mg, 176 μmol, 0.1 equiv) were added sequentially. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 85°C for 2 hours. LC-MS / MS indicated the reaction was complete. TLC monitoring showed multiple new spots indicating complete consumption of the starting material. After cooling, the reaction mixture was added to distilled water (30.0 mL) and extracted with ethyl acetate (15.0 mL x 2). The combined organic phases were washed with saturated brine (15.0 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to yield a crude product. The crude product was purified by column chromatography (gradient: (SiO2, Petroleum ether / Ethyl acetate = 1 / 0 to 1 / 1)) to afford tert-butyl 5-(2-bromophenyl)-octahydropyrrolo[3,4-c]pyrrole-2-carboxylate (450 mg, 1.23 mmol, 69.3% yield) as a yellow oil.

[0163] Tert-butyl 5-(2-bromophenyl)-octahydropyrrolo[3,4-c]pyrrole-2-carboxylate (150 mg, 408 μmol, 1 equiv) and 2,4-dimethylbenzene-1-thiol (56.4 mg, 408 μmol, 1 equiv) were placed in a 10.0 mL single-necked vial containing toluene (3.00 mL). Sodium tert-butoxide (78.5 mg, 816 μmol, 2 equiv) and RuPhos Pd G3 (34.1 mg, 40.8 μmol, 0.1 equiv) were added sequentially. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 100°C for 16 hours. LC-MS / MS indicated the reaction was complete. TLC showed two new spots. After cooling, the reaction mixture was added to distilled water (30.0 mL) and extracted with ethyl acetate (15.0 mL x 2). The combined organic phases were washed with saturated brine (15.0 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to yield a crude product. The crude product was purified by chromatography (SiO2, petroleum ether / ether acetate = 5 / 1) to afford tert-butyl 5-{2-[(2,4-dimethylphenyl)mercapto]phenyl}-octahydropyrrolo[3,4-c]pyrrole-2-carboxylate (150 mg, 353 μmol, 86.5% yield) as a yellow oil.

[0164] In a 10.0 mL single-necked bottle, tert-butyl 5-{2-[(2,4-dimethylphenyl)mercapto]phenyl}-octahydropyrrolo[3,4-c]pyrrole-2-carboxylate (150 mg, 353 μmol, 1 equivalent) was dissolved in ethyl acetate hydrochloride (10.0 mL). The resulting reaction solution was stirred at 25°C for 1 hour. LC-MS showed that the reaction was complete. The reaction solution was directly concentrated under reduced pressure to obtain a crude product. The crude product was purified by high performance liquid chromatography (column model: Phenomenex luna C 18 The reaction mixture was stirred at 4 ℃ for 10 minutes (150*25mm*10um; mobile phase: [water (hydrochloric acid)-acetonitrile]; B%: 23%-53%, 10 min) to obtain 2-{2-[(2,4-dimethylphenyl)mercapto]phenyl}-octahydropyrrolo[3,4-c]pyrrole (25.2 mg, 69.9 μmol, 19.8% yield, 100% purity, hydrochloride) as an off-white solid.

[0165] 1H NMR (400 MHz, deuterated dimethyl sulfoxide) δ = 9.25-8.99 (m, 2H), 7.26 (d, J = 7.6 Hz, 1H), 7.22 (s, 1H), 7.17-7.11 (m, 2H), 7.08 (d, J = 7.6 Hz, 1H), 6.95-6.89 (m, 1H), 6.50 (d, J = 7.6 Hz, 1H), 3.59-3.50 (m, 2H), 3.29 (d, J = 8.8 Hz, 2H), 3.04-2.88 (m, 6H), 2.31 (s, 3H), 2.24 (s, 3H).

[0166] Example 19:

[0167] Compound T19 (Target 19) was prepared by the following method:

[0168] In a 50.0 mL single-necked vial, 2,4-dimethylbenzene-1-thiol (790 mg, 5.72 mmol, 773 μL, 1.1 eq) was dissolved in DMF (10.0 mL). Potassium carbonate (1.08 g, 7.79 mmol, 1.5 eq) and 3-bromo-2-chloropyridine (1.00 g, 5.20 mmol, 1 eq) were added sequentially. After nitrogen substitution three times, the resulting reaction solution was heated at 100°C and stirred under nitrogen for 10 hours. LC-MS / MS indicated the reaction was complete. TLC revealed incomplete consumption of 3-bromo-2-chloropyridine, with a new spot generated. The reaction mixture was filtered and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 0 / 1 to 1 / 0). 3-Bromo-2-[(2,4-dimethylphenyl)mercapto]pyridine (1.08 g, 3.67 mmol, 70.6% yield) was obtained as a colorless oil.

[0169] In a 50.0 mL single-necked vial, 3-bromo-2-[(2,4-dimethylphenyl)mercapto]pyridine (200 mg, 679 μmol, 1 eq) and tert-butylpiperazine-1-carboxylate (126 mg, 679 μmol, 1 eq) were dissolved in toluene (2.00 mL). Sodium tert-butoxide (104 mg, 1.09 mmol, 1.6 eq), Pd2(dba)3 (12.4 mg, 13.6 μmol, 0.02 eq), and BINAP (16.9 mg, 27.1 μmol, 0.04 eq) were added sequentially. The resulting reaction solution was heated at 100°C and stirred under nitrogen for 10 hours. LC-MS / MS indicated the reaction was complete. TLC revealed complete consumption of 3-bromo-4-[(2,4-dimethylphenyl)mercapto]pyridine, with the generation of numerous new spots. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by pre-thin layer chromatography (SiO2, petroleum ether / ethyl acetate = 5 / 1) to give tert-butyl 4-{2-[(2,4-dimethylphenyl)mercapto]pyridin-3-yl}piperazine-1-carboxylate (163 mg, 407 μmol, 60.0% yield) as a yellow oil.

[0170] In a 50.0 mL single-necked vial, tert-butyl 4-{2-[(2,4-dimethylphenyl)mercapto]pyridin-3-yl}piperazine-1-carboxylate (163 mg, 407.96 μmol, 1 eq) was dissolved in dichloromethane (1.50 mL), and trifluoroacetic acid (2.30 g, 20.1 mmol, 1.50 mL, 57.2 eq) was slowly added dropwise. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 20°C for 1 hour. LC-MS / MS indicated the reaction was complete. The pH of the reaction solution was adjusted to 9 with sodium bicarbonate and extracted with dichloromethane (10.0 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by HPLC (column model: Waters Xbridge 150 x 25 mm x 5 μm; mobile phase: [water (sodium bicarbonate)-acetonitrile]; gradient: 2% to 62% B over 9 minutes). The crude product obtained by HPLC was purified by slurrying with ethyl acetate hydrochloride (4.00 mL) at 20°C for one hour, and the filter cake was concentrated under reduced pressure to obtain 1-{2-[(2,4-dimethylphenyl)mercapto]pyridin-3-yl}piperazine hydrochloride (25.1 mg, 74.4 μmol, 18.2% yield, 99.6% purity, HCl) as a yellow solid.

[0171] 1H NMR (400 MHz, deuterated dimethyl sulfoxide) δ = 9.43 (br s, 2H), 7.99 (dd, J = 1.2, 4.8 Hz, 1H), 7.50 (dd, J = 1.2, 8.0 Hz, 1H), 7.34 (d, J = 7.6 Hz, 1H), 7.18 (s, 1H), 7.11 (dd, J = 4.8, 8.0 Hz, 1H), 7.04 (br d, J = 7.6 Hz, 1H), 3.25 (br s, 8H), 2.31 (s, 3H), 2.19 (s, 3H).

[0172] Performance testing

[0173] The performance of the alkyldiamine-substituted bis-heteroaromatic sulfide compounds prepared in the above examples was tested:

[0174] (1) Detection of the inhibitory effect of alkyldiamine-substituted bis-heteroaryl sulfide compounds on the growth of two tumor cells

[0175] The CCK8 colorimetric assay was used to compare the cytotoxicity of 19 alkyldiamine-substituted bis(heteroaryl)sulfide compounds and cisplatin against human breast cancer MDA-MB-231LM2 cell line and lung adenocarcinoma A549 cell line.

[0176] 19-alkyldiamine-substituted bis-heteroaryl sulfide compounds or cisplatin were diluted to concentrations of 0, 0.01, 0.1, 1, 5, 10, 20, 40, 80, and 160 μM. Breast cancer MDA-MB-231LM2 cells or lung adenocarcinoma A549 cells were seeded at a density of 8,000 cells / well in 96-well plates. Twenty-four hours after seeding, the medium in the original wells was discarded, and 100 μL of the diluted drug solution was added to each well, with four replicates per group. At 48 hours of treatment, 10 μL of CCK8 was added to each well and incubated for 3 hours. The OD value at A450 was measured using a multi-function microplate reader, and the inhibition rate was calculated.

[0177] Table 1 IC values ​​of two tumor cell lines treated with alkyldiamine-substituted bis-heteroaryl sulfide compounds and cisplatin for 48 h 50

[0178] The results showed that most of the alkyldiamine-substituted bicyclic heterocyclic sulfide compounds had inhibitory effects on both tumor cells. Among them, compounds T03, T04, T06, T09, T12, T13, T15, T16, and T18 had an IC 50 Less than 20μM (see Table 1 for details).

[0179] (2) Detection of the inhibitory effect of alkyldiamine-substituted bis(heteroaromatic) sulfide compounds on the growth of various tumor cells

[0180] The CCK8 colorimetric assay was used to detect the cytotoxicity of alkyldiamine-substituted bis(heteroaromatic)sulfide compounds (T03, T06, T09, T12, T13, T15, T16, T18) and cisplatin on human breast cancer cell lines MDA-MB-231, MDA-MB-231LM2, MCF-7, liver cancer cell line MHCC-97L, pancreatic cancer cell line Panc-1, renal clear cell adenocarcinoma cell line 786-O, lung cancer cell line A549, gastric cancer cell line SGC-7901, glioma cell line U251, ovarian cancer cell line A2780, prostate cancer cell line PC-3, esophageal cancer cell line EC109, melanoma cell line A375, nasopharyngeal cancer cell line 5-8F, colon cancer cell line HCT116, cervical cancer cell line SiHa, lymphoma cell line MINO, and leukemia cell line HL60.

[0181] After counting cells, cells were seeded at a density of 8,000 cells / well in 96-well plates. After overnight cell attachment, cisplatin (CDDP) was used as a control drug. Compounds T03, T06, T09, T12, T13, T15, T16, and T18 were diluted to 0, 0.01, 0.1, 1, 5, 10, 20, 40, 80, and 160 μM, respectively. The culture medium was discarded, and 100 μL of each drug was added to the corresponding wells, with four replicates per group. After 48 hours of treatment, 10 μL of CCK8 was added to each well and incubated for 2 hours. The OD value at A450 was measured using a multi-function microplate reader, and the inhibition rate was calculated.

[0182] Table 2 IC values ​​of some alkyldiamine-substituted bis-heteroaryl sulfide compounds in treating various tumor cell lines for 48 h 50

[0183] The results showed that 8-alkyldiamine-substituted bis(heteroaromatic)sulfide compounds and cisplatin had inhibitory effects on various tumor cells.

[0184] In various cell lines, the IC values ​​of 8 alkyldiamine-substituted bis(heteroaryl)sulfide compounds were 50 The range is between 2.41 and 21.86 μM, and in most tumor cells, the IC 50 The IC values ​​of eight alkyldiamine-substituted bis(heteroaryl)sulfide compounds were lower than 10 μM in breast cancer MCF-7, MDA-MB-231, MDA-MB-231LM2, pancreatic cancer Panc-1, lung cancer A549, glioma U251, ovarian cancer A2780, esophageal cancer EC109, melanoma A375, colon cancer HCT116, and cervical cancer Siha cells. 50 It is significantly lower than cisplatin and has a more significant inhibitory effect on these cells (see Table 2 for details).

[0185] (3) The role of alkyldiamine-substituted bicyclic heterocyclic sulfide compounds in degrading PD-L1 protein.

[0186] Take the MDA-MB-231 cell suspension in the logarithmic growth phase and adjust the cell density to 4×10 5 After the cells adhered to the wall, the culture medium was replaced with the medium containing compounds T03, T06, T09, T12, T13, T15, T16, and T18 (the concentrations of each compound were the IC values ​​of MDA-MB-231 cells, respectively). 50 The concentrations of the proteins were half of the control group (i.e., T03: 7.5 μM, T06: 9 μM, T09: 6 μM, T12: 7 μM, T13: 6 μM, T15: 6.5 μM, T16: 5 μM, T18: 9 μM). After incubation for 48 h, proteins were collected and analyzed by Western blot (see Figure 5).

[0187] Compared to the untreated group (blank control group), compounds T03, T06, T09, T12, T13, T15, T16, and T18 all significantly reduced PD-L1 protein. This result demonstrates that alkyldiamine-substituted bis-heteroaryl sulfide compounds can effectively reduce PD-L1 protein levels and are PD-L1 immunomodulators.

[0188] It should be noted that the above description is merely an embodiment of the present invention and does not limit the scope of the present invention. Any direct or indirect application of the present invention in other related technical fields shall be included in the scope of protection of the present invention. Therefore, any technical solution that can be obtained by a person skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of the existing technology shall be within the scope of protection determined by the claims.

Claims

1. An alkyldiamine-substituted bis-heteroaromatic sulfide compound, whose chemical structure is shown in (I): Where: R1 is halogen, hydrogen, cyano, hydroxyl, mercapto, nitro, C 1-6 -alk(ene / ynyl) group, C 1-6 -alk(en / yn)yloxy, C 1-6 -Alkyl (ene / ynyl) thio group, C 1-6 -Alkyl (ene / ynyl) sulfonyl, halogenated C 1-6 -Alkyl (ene / ynyl) group, halogenated C 1-6 -Alkyl (ene / ynyl) oxy, halogenated C 1-6 -Alkyl (ene / ynyl) thio, halogenated C 1-6 -alk(en / yn)ylsulfonyl. R2 is a six-membered ring such as benzene, cyclohexane, cyclohexene, cyclohexadiene, or a six-membered heterocyclic ring such as pyridine, piperidine, piperazine, pyridazine, pyrimidine, pyrazine, pyran; or a five-membered ring such as cyclopentane, cyclopentene, cyclopentadiene, or a five-membered heterocyclic ring such as thiophene, furan, thiazole, pyrrole, imidazole, pyrazole, oxazole. R3 is halogen, hydrogen, cyano, hydroxyl, mercapto, nitro, C 1-6 -alk(ene / ynyl) group, C 1-6 -alk(en / yn)yloxy, C 1-6 -Alkyl (ene / ynyl) thio group, C 1-6 -Alkyl (ene / ynyl) sulfonyl, halogenated C 1-6 -Alkyl (ene / ynyl) group, halogenated C 1-6 -Alkyl (ene / ynyl) oxy, halogenated C 1-6 -Alkyl (ene / ynyl) thio, halogenated C 1-6 -Alk(en / yn)ylsulfonyl. R4 is a substituted chain alkyl group, including -CH2-, -CH2-CH2-, and -CH2-CH2-CH2-. R5 is a substituted chain alkyl group, including -CH2-, -CH2-CH2-, and -CH2-CH2-CH2-. R6 is halogen, haloalkyl, hydrogen, cyano, hydroxyl, mercapto, nitro, C 1-6 -alk(ene / ynyl) group, C 1-6 -alk(en / yn)yloxy, C 1-6 -Alkyl (ene / ynyl) thio group, C 1-6 -Alkyl (ene / ynyl) sulfonyl, halogenated C 1-6 -Alkyl (ene / ynyl) group, halogenated C 1-6 -Alkyl (ene / ynyl) oxy, halogenated C 1-6 -Alkyl (ene / ynyl) thio, halogenated C 1-6 -alk(en / yn)ylsulfonyl, or two R6 connected to the same carbon atom can form a 3-6 membered spiro-connected cycloalkyl. X, Y, Z, and W each independently represent a carbon or nitrogen atom, and when selected from nitrogen atoms, only one of them is nitrogen; m represents 0, 1, 2, 3, 4 or 5. n represents 0, 1, 2, 3 or 4. p represents 0, 1, 2, 3, 4, 5 or 6.

2. The alkyldiamine-substituted bis-heteroaromatic sulfide compound according to claim 1, wherein: Halogen includes F, Cl, Br or I. Halogenated C 1-6 -Alkyl (ene / ynyl) is a halogenated C 1-6 -Alkyl (ene / ynyl) group, dihalogenated C 1-6 -alk(ene / ynyl) or polyhalogenated C 1-6 -alk(en / yn)yl. R1 is prioritized over CF3. R3 is prioritized over CF3. R4 is preferably -CH2-CH2-. R5 is preferably -CH2-CH2-. X, Y, Z, and W are preferably carbon atoms. Priority is 3. The alkyldiamine-substituted bis-heteroaromatic sulfide compound according to claim 1 or 2, characterized in that: The compounds include:

4. The alkyldiamine-substituted bis-heteroaromatic sulfide compound according to claim 1 or 2, characterized in that: The compounds are in the form of their tautomers, mesomers, racemates, enantiomers, diastereomers or possible derivatives based on their structures or mixtures thereof.

5. An application of the alkyldiamine-substituted bis-heteroaromatic sulfide compound as claimed in claim 1, characterized in that: Use of the compound, a pharmaceutical composition containing the compound, or a derivative of the compound in preparing drugs for preventing and / or treating tumors.

6. The use according to claim 5, characterized in that The compound is a pharmaceutically acceptable salt, ether, ester, prodrug, metabolite, solvate or crystal thereof; The pharmaceutically acceptable salts include hydrochloride, bromate, fumarate, acetate, citrate, sulfate, methanesulfonate, formate or trifluoroacetate.

7. The use according to claim 5 or 6, characterized in that The medicine is in the form of tablets, injections, capsules, oral solutions, pills, granules, powders, aerosols, patches, ointments, paints or suppositories.

8. The use according to claim 5 or 6, characterized in that The drugs are conventional anti-tumor drugs, including chemotherapy drugs, biological targeted therapy drugs, metabolic therapy drugs or immunotherapy drugs.

9. The use according to claim 5 or 6, characterized in that: The application of the drug in the preparation of PD-L1 immunomodulator-related drugs.

10. [Corrected 23.03.2025 under Rule 26] The use according to claim 5 or 6, characterized in that The effects of the drug include inhibiting tumor growth and / or metastasis.

11. The compound or its composition is used as a drug for the prevention and / or treatment of breast cancer, liver cancer, pancreatic cancer, kidney cancer, lung cancer, gastric cancer, glioma, ovarian cancer, prostate cancer, esophageal cancer, melanoma, nasopharyngeal cancer, colon cancer, cervical cancer, lymphoma, and leukemia.

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