Benzisothiazolinone compound as well as pharmaceutical composition and application thereof

Abstract

The invention discloses synthesis of a benzisothiazolinone compound with indoleamine 2, 3-dioxygenase IDO1 (indoleamine 2, 3-dioxygenase) inhibitory activity and application of the benzisothiazolinone compound in preparation of antitumor drugs. The structural general formula of the inhibitor is as shown in formula (1). Pharmacological experiments show that the compound provided by the invention not only has low cytotoxicity, but also shows good IDO1 inhibitory activity, and has relatively good inhibitory activity on lung cancer paclitaxel drug-resistant cell strains. In addition, an AhR activation experiment shows that the compound disclosed by the invention is not strong in AhR activation activity. In-vivo experiments show that the compound has no obvious acute toxicity, is safe to take orally and can be applied to preparation of antitumor drugs for inhibiting IDO1-mediated tryptophan metabolic pathways.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, in particular to the field of pharmaceutical chemical synthesis and pharmacology, and specifically to the synthesis and pharmacological testing of benzisothiazolinone compounds. Background Art

[0002] Indoleamine 2,3-dioxygenase (IDO) is a heme monomer enzyme and the only enzyme outside the liver that catalyzes the degradation of tryptophan, converting it to kynurenine and its derivatives via the kynurenine pathway. IDO comprises three enzymes: two distinct isoforms, IDO1 and IDO2, and tryptophan 2,3-dioxygenase (TDO).

[0003] IDO1 catalyzes the first step in the kynurenic acid metabolic pathway—the oxidative cleavage of the indole ring from L-Trp to N-formylkynurenine (NFK). It is one of the rate-limiting enzymes in the kynurenic acid metabolic pathway. IDO1 is widely distributed and expressed in various tissues and cells besides the liver, including the small intestine, placenta, thymus, spleen, pancreas, kidney tissue, and microglia.

[0004] Tumor immunotherapy, which inhibits tumor growth and even kills tumor cells by activating the body's anti-tumor immune response, is considered the most promising approach for treating cancer. Immune checkpoints are receptors expressed by immune cells that regulate T lymphocytes. However, this regulatory pathway can be affected by tumors, preventing the immune system from attacking tumor cells and leading to immune escape. Therefore, immunotherapy checkpoint blockade is undoubtedly a new breakthrough in cancer treatment.

[0005] IDO1 is currently considered a hotly researched immune checkpoint due to its role as a key rate-limiting enzyme in the kynurenic acid pathway of tryptophan metabolism, which plays a major role in tumor immune evasion. Numerous studies have demonstrated that IDO1 could serve as a potential diagnostic biomarker for various cancers, including breast, colon, prostate, and gastric cancers. Therefore, the development of IDO1 inhibitors is of great significance.

[0006] Isothiazolinone compounds have a five-membered isothiazolinone ring structure and possess antimicrobial, antibacterial, antifungal, antiviral, anticancer, and anti-inflammatory properties. Currently, some isothiazolinone compounds have been detected to have anti-tumor activity. Summary of the Invention

[0007] The main purpose of the present invention is to address the above problems and provide a benzisothiazolinone compound with anti-tumor activity, a pharmaceutical composition thereof and its application.

[0008] To achieve the above objectives, the present invention provides, in a first aspect, a benzisothiazolinone compound or a pharmaceutically acceptable salt thereof, which has good IDO inhibitory activity and provides a new therapeutic strategy for tumor treatment. The main feature is that the structural formula of the compound is shown in the following general formula (1):

[0009]

[0010] Wherein, ring A is triazole or oxadiazole;

[0011] R1 is selected from substituted or unsubstituted C1-C6 alkyl, C1-C6 alkoxy, hydroxy-, halogen;

[0012] R2 is selected from substituted or unsubstituted aryl, and the substituent of the substituted aryl is selected from one or more of linear or branched C1-C6 alkyl, C1-C6 alkoxy, hydroxyl, and halogen;

[0013] L is selected from amide, substituted or unsubstituted C1-C6 alkyl, C1-C6 alkoxy;

[0014] The substituent of the substituted C1-C6 alkyl group is selected from halogen, which is selected from F, Cl, and Br.

[0015] Preferably, the benzisothiazolinone compound is selected from

[0016]

[0017] The second aspect of the present invention provides a pharmaceutical composition, the main feature of which is that it comprises the benzisothiazolinone compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier and / or excipient.

[0018] The third aspect of the present invention provides use of the pharmaceutical composition in the preparation of an indoleamine 2,3-dioxygenase IDO1 inhibitor.

[0019] The third aspect of the present invention provides the use of the pharmaceutical composition in the preparation of anti-tumor drugs.

[0020] Preferably, the tumor is a tumor with high expression of indoleamine 2,3-dioxygenase IDO1.

[0021] Preferably, the tumor is cervical cancer or non-small cell lung cancer.

[0022] The benzisothiazolinone compounds can inhibit the expression of IDO1, thereby reducing the production of kynurenine. IDO1 is the key rate-limiting enzyme in the conversion of tryptophan to kynurenine. Kynurenine is the first stable metabolite produced after tryptophan is oxidized by indoleamine 2,3-dioxygenase IDO1 and tryptophan 2,3-dioxygenase TDO2. Under IDO1 catalysis, the double bond in the indole ring of tryptophan is oxidatively cleaved to produce N-formylkynurenine. Interferon IFN-γ can induce the expression of IDO1 and the production of kynurenine Kyn.

[0023] Compared with the prior art, the present invention has the following beneficial technical effects:

[0024] The synthesis and application of benzisothiazolinone compounds with IDO1 inhibitory activity provided by the present invention provide a new therapeutic strategy for tumor treatment. Experimental results demonstrate that the compounds of the present invention not only have low cytotoxicity but also exhibit good IDO1 inhibitory activity, especially against paclitaxel-resistant lung cancer cell lines. In vivo experiments have shown that the compounds of the present invention have no significant acute toxicity and are safe for oral administration. They can be used to prepare antitumor drugs that inhibit the IDO1-mediated tryptophan metabolic pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1a 、 Figure 1b The cellular IC values ​​of L-13 and 1-MT are 50 Result graph.

[0026] Figure 2 IC of enzyme levels of L-1, L-13, and L-23 50 Result graph.

[0027] Figure 3 This is the test result of the aryl hydrocarbon receptor activation level. DETAILED DESCRIPTION

[0028] In order to more clearly understand the technical content of the present invention, the following embodiments are given in detail. However, it should be noted that these descriptions are only for further illustrating the features and advantages of the present invention, and are not intended to limit the claims of the invention.

[0029] Unless otherwise specified, the reagents and methods involved in the examples are commonly used in the art.

[0030] Through sustained and long-term research, the inventors of the present application discovered a benzisothiazolinone-based IDO1 inhibitor that not only has low cytotoxicity but also exhibits good IDO1 inhibitory activity and drug-resistant A549R cells. Based on this, the present invention was completed.

[0031] Example 1

[0032] Synthesis of N-((1-(Substituted Phenyl)-1H-1,2,3-Triazol-4-yl)methyl)-Substituted-3-Oxobenzo[d]isothiazole-2(3H)-carboxamides (L-1 to L-25)

[0033]

[0034] 1.1 Synthesis of (substituted)benzo[d]isothiazol-3(2H)-ones (1-1)

[0035] 2-Bromo-(substituted)benzene nitrile (10 mmol) was added to a two-necked flask. Under N2 protection, sodium ethanethiolate (15 mmol), Xantphos (0.1 mol), and PD2(dba)3 (0.05 mmol) were added. 45 mL of toluene and 20 mmol of DIPEA were added to dissolve the above compounds. After the addition was complete, the temperature was raised to 95°C and the reaction was allowed to react for 12 hours. TLC was used to monitor the reaction. Upon confirmation of completion, the reaction was terminated and the temperature was lowered to room temperature. The pH was adjusted to 7-8 with 1N HCl. The resulting solution was extracted three times with ethyl acetate. The organic phase was washed with saturated NaCl, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Silica gel column chromatography (PE:EA = 4:1) yielded a yellow liquid. 5 mmol of the yellow liquid was dissolved in 4.2 mL of PhCl. SO2Cl2 (6.5 mmol) and H2O (7 mmol) were added dropwise under an ice bath. After the addition was complete, the temperature was raised to 75°C and the reaction was allowed to proceed for 1 hour. The reaction was monitored by TLC. A yellow solid was precipitated, which was filtered and the filter cake was washed with an appropriate amount of PhCl to obtain a yellow solid with a yield of about 65%.

[0036] 1.2 Synthesis of 1-azido-(substituted)-benzene (1-2)

[0037] Dissolve (substituted) aniline (5 mmol) in 10 mL of acetonitrile, cool in an ice bath, and add tert-butyl nitrite (7.5 mmol) with stirring. Once the reaction mixture is thoroughly mixed, add trimethylsilyl azide (7.5 mmol) dropwise. After complete addition, allow to react at room temperature for 1 hour (due to the significant heat released during the reaction, the reaction must be open). Monitor the reaction by TLC. Once complete, extract three times with ethyl acetate. The organic layer is washed with saturated brine and saturated sodium bicarbonate, dried over anhydrous sodium sulfate, and then rotary evaporated at low temperature. Since the product has a low boiling point and is easily removed by rotary evaporation, do not remove the solvent completely. Pass the solution through a column using a 10:1 ratio of petroleum ether to ethyl acetate as the eluent to obtain the final product in an approximately 70% yield.

[0038] 1.3 Synthesis of 3-oxo-N-(prop-2-yn-1-yl)-(substituted)-benzisothiazole-2(3H)-carboxamide (1-3)

[0039] Propargylamine (5 mmol) was suspended in 10 mL of dichloromethane. 10 mL of a dichloromethane solution containing 1.6 mmol of triphosgene was slowly added under an ice bath. Then, 10 mL of a dichloromethane solution containing 10 mmol of triethylamine was slowly added dropwise. After the addition was complete, the temperature was raised to 50°C and the reaction mixture was allowed to react for 2 hours. The reaction mixture was then dried by rotary evaporation to obtain a crude intermediate product. (Substituted) BIT (1.65 mmol), 10 mL of tetrahydrofuran, and 10 mL of dichloromethane were added, and the temperature was raised to 65°C and the reaction mixture was allowed to react for 8 hours. The reaction was monitored by TLC. After confirming the reaction was complete, the product was rotary evaporated and the resulting solid was filtered using a 1:1 ratio of BT:H2O. The filter cake was washed with a 1:1 ratio of BT:H2O to obtain a yellow solid with a yield of approximately 50%.

[0040] 1.4 Synthesis of compounds L-1 to L-25

[0041] Compound 1-1 (1.5 mmol) prepared in step 1 and compound 1-2 (1 mmol) prepared in step 2 were added to a two-necked flask and dissolved in 10 mL of methanol. A mixed aqueous solution (0.8 mL) of copper sulfate (0.07 mmol) and sodium ascorbate (0.14 mmol) was added under nitrogen. After the addition was complete, the reaction was carried out at room temperature under nitrogen for 12 hours. The reaction was monitored by TLC and the reaction was terminated after confirmation of completion. The solvent was removed by swirl, and the mixture was extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, and filtered with 1:1 BT:H20. The resulting solid was passed through a column with petroleum ether:ethyl acetate = 1:1 as the eluent to obtain a solid product, which was filtered with suction. The yield was approximately 50%.

[0042] N-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)-3-oxobenzo[d]isothiazole-2(3H)-carboxamide (L-1), yellow flaky solid, yield 9.9%.

[0043] 1 H NMR (400MHz, Chloroform-d) δ9.42 (s, 1H), 8.11–7.99 (m, 2H), 7.72 (t, J = 7.1Hz, 3H), 7.62–7.39 (m, 6H), 4.82 (d, J = 5.9Hz, 2H); 13 C NMR (151MHz, CDCl3) δ165.15,151.47,140.79,137.03,134.02,129.77,128 .86,127.44,125.96,124.82,120.95,120.68,120.52,36.16; HRMS(ESI)m / z caled for C 17 H 13 N5O2S[(M+H) +]:352.0868,found:352.0869.

[0044] N-((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-3-oxobenzo[d]isothiazole-2(3H)-carboxamide (L-2), white flaky solid, yield 44%.

[0045] 1 H NMR(400MHz,Chloroform-d)δ9.41(s,1H),8.07–7.99(m,2H),7.75–7.64(m,3H),7.58(d, J=8.1Hz,1H),7.53–7.46(m,2H),7.43(ddd,J=8.0,7.1,0.9Hz,1H),4.81(d,J=6.0Hz,2H); 13 C NMR (101MHz, CDCl3) δ165.15,151.48,140.76,135.50,134.63,134.05,129 .95,127.43,125.98,124.77,121.77,120.83,120.52,36.10; HRMS(ESI)m / z caled forC 17 H 13 ClN5O2S[(M+H) + ]:386.0478,found:386.0479.

[0046] N-((1-(3-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-3-oxobenzo[d]isothiazole-2(3H)-carboxamide (L-3), orange solid, yield 9.5%.

[0047] 1 H NMR(400MHz,Chloroform-d)δ9.43(s,1H),8.10(s,1H),8.03(d,J=7.9Hz,1H),7.79(t,J=2.0Hz,1H),7.75 –7.68(m,1H),7.64(dt,J=7.8,1.7Hz,1H),7.58(d,J=8.1Hz,1H),7.48–7.40(m,3H),4.81(d,J=5.2Hz,2H); 13C NMR (101MHz, CDCl3) δ165.16,151.49,140.77,135.64,134.05,130.87,128 .90,127.43,125.98,124.77,120.88,120.53,118.59,36.08; HRMS(ESI)m / z caled for C 17 H 13 ClN5O2S[(M+Na) + ]:408.0298,found:408.0297.

[0048] N-((1-(2-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-3-oxobenzo[d]isothiazole-2(3H)-carboxamide (L-4), orange solid, yield 40.9%.

[0049] 1 H NMR(400MHz,Chloroform-d)δ9.42(t,J=5.7Hz,1H),8.06(s,1H),8.02(d,J=8.0Hz,1H),7.7 0(ddd,J=8.3,7.1,1.3Hz,1H),7.66–7.52(m,3H),7.50–7.38(m,3H),4.84(d,J=5.8Hz,2H); 13 C NMR (101MHz, CDCl3) δ165.14,151.39,140.81,134.88,134.00,130.82,130.79,128 .69,127.92,127.86,127.41,125.93,124.84,124.63,120.51,36.10; HRMS(ESI)m / z caled for C 17 H 13 ClN5O2S[(M+Na) + ]:408.0298,found:408.0296.

[0050] N-((1-(3-Fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-3-oxobenzo[d]isothiazole-2(3H)-carboxamide (L-5), orange-red solid, yield 25.4%.

[0051] 1H NMR(400MHz,Chloroform-d)δ9.43(s,1H),8.07(s,1H),8.02(d,J=7.9Hz,1H),7.71(ddd,J=8.3,7.1,1.2Hz,1H ),7.61–7.50(m,3H),7.50–7.46(m,1H),7.46–7.39(m,1H),7.14(tt,J=8.3,1.9Hz,1H),4.81(d,J=5.8Hz,2H); 13 C NMR (101MHz, CDCl3) δ165.16,164.34,161.86,151.49,140.78,134.05,131.26,131.17, 127.43,125.98,124.78,120.52,115.89,115.65,108.52,108.26,36.10; HRMS(ESI)m / z caled for C 17 H 13 FN5O2S[(M+H) + ]:370.0774,found:370.0777.

[0052] N-((1-(2-Fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-3-oxobenzo[d]isothiazole-2(3H)-carboxamide (L-6), yellow solid, yield 46.5%.

[0053] 1 H NMR(400MHz,Chloroform-d)δ9.42(s,1H),8.15(d,J=2.7Hz,1H),8.02(dd,J=8.0,1.1Hz,1H),7.93(td,J=7.7,1.7Hz,1H ),7.70(ddd,J=8.2,7.1,1.2Hz,1H),7.57(d,J=8.1Hz,1H),7.52–7.38(m,2H),7.36–7.24(m,3H),4.84(d,J=5.8Hz,2H); 13 C NMR (101MHz, CDCl3) δ165.13,154.68,152.18,151.38,140.82,133.99,130.34,127.41,12 5.92,125.27,125.23,125.00,124.84,120.51,117.14,116.95,36.05; HRMS(ESI)m / zcaled for C 17 H 13 FN5O2S[(M+H)+ ]:370.0774,found:370.0773.

[0054] N-((1-(4-Bromophenyl)-1H-1,2,3-triazol-4-yl)methyl)-3-oxobenzo[d]isothiazole-2(3H)-carboxamide (L-7), white flocculent solid, yield 41%.

[0055] 1 H NMR(400MHz,Chloroform-d)δ9.42(s,1H),8.08–7.99(m,2H),7.71(ddd,J=8.3,7.1,1.2Hz,1H),7 .67–7.60(m,4H),7.58(d,J=8.1Hz,1H),7.43(ddd,J=8.0,7.1,0.9Hz,1H),4.81(d,J=5.7Hz,2H); 13 C NMR (101MHz, CDCl3) δ165.16,151.49,140.77,135.98,134.06,132.93,127 .44,125.98,124.77,122.51,122.01,120.76,120.53,36.11; HRMS(ESI)m / z caled for C17H13BrN5O2S[(M+Na)+]:453.9772, found:453.9772.

[0056] N-((1-(3-Bromophenyl)-1H-1,2,3-triazol-4-yl)methyl)-3-oxobenzo[d]isothiazole-2(3H)-carboxamide (L-8), yellow solid, yield 38%.

[0057] 1 H NMR (400MHz, Chloroform-d) δ9.43 (s, 1H), 8.07 (s, 1H), 8.05–7.99 (m, 1H), 7.93 (t, J = 2. 0Hz,1H),7.75–7.62(m,2H),7.61–7.53(m,2H),7.47–7.34(m,2H),4.81(d,J=5.9Hz,2H); 13 C NMR (151MHz, CDCl3) δ165.16,151.50,140.78,137.91,134.06,131.86,131.09,12 7.44,125.98,124.77,123.69,123.33,120.53,119.08,36.11; HRMS(ESI)m / zcaled for C17 H 13 BrN5O2S[(M+H) + ]:429.9973,found:429.9971.

[0058] N-((1-(2-Methylphenyl)-1H-1,2,3-triazol-4-yl)methyl)-3-oxobenzo[d]isothiazole-2(3H)-carboxamide (L-9) was obtained as a white solid in 35.8% yield.

[0059] 1 H NMR(400MHz,Chloroform-d)δ9.43(t,J=5.8Hz,1H),8.02(dt,J=8.0,1.0Hz,1H),7.81(s,1H),7.70(ddd,J=8.3, 7.1,1.2Hz,1H),7.57(d,J=8.1Hz,1H),7.47–7.35(m,3H),7.35–7.27(m,2H),4.84(d,J=5.8Hz,2H),2.22(s,3H); 13 C NMR (151MHz, CDCl3) δ165.15,151.42,144.16,140.80,136.42,134.01,131.51 ,129.91,126.85,126.02,124.85,124.08,120.52,36.16,17.94; HRMS(ESI)m / z caled for C 18 H 15 N5O2S[(M+H) + ]:366.1025,found:366.1026.

[0060] N-((1-(3-Methylphenyl)-1H-1,2,3-triazol-4-yl)methyl)-3-oxobenzo[d]isothiazole-2(3H)-carboxamide (L-10), orange flaky solid, yield 29%.

[0061] 1 H NMR(400MHz,Chloroform-d)δ9.35(t,J=5.8Hz,1H),8.02–7.93(m,2H),7.64(td,J=7.7,7.1,1.3Hz,1H),7.58–7.47(m ,2H),7.44(dd,J=8.1,2.1Hz,1H),7.34(dt,J=16.1,7.7Hz,2H),7.23–7.15(m,2H),4.75(d,J=5.8Hz,2H),2.38(s,3H);13 C NMR (151MHz, CDCl3) δ165.13,151.45,140.86,140.01,136.95,134.01,129.60,129.54,127 .61,127.42,126.25,124.82,121.30,120.94,120.56,118.04,36.16,21.40; HRMS(ESI)m / z caled forC 18 H 15 N5O2S[(M+H) + ]:366.1025,found:366.1023.

[0062] N-((1-(4-Methylphenyl)-1H-1,2,3-triazol-4-yl)methyl)-3-oxobenzo[d]isothiazole-2(3H)-carboxamide (L-11), white solid, yield 5.7%.

[0063] 1 H NMR(400MHz,Chloroform-d)δ9.41(s,1H),8.02(d,J=7.5Hz,2H),7.70(ddd,J=8.3,7.1,1.3Hz,1H),7 .59(t,J=8.3Hz,3H),7.42(t,J=7.6Hz,1H),7.30(d,J=8.1Hz,2H),4.81(d,J=5.8Hz,2H),2.41(s,3H); 13 C NMR(151MHz, CDCl3)δ165.14,151.45,140.79,138.99,134.01,130.26,127.44,125.95,124.84,120.58,36.16,21.11; HRMS(ESI)m / zcaled for C 18 H 15 N5O2S[(M+Na) + ]:388.0844,found:388.0843.

[0064] N-((1-(2-Methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-3-oxobenzo[d]isothiazole-2(3H)-carboxamide (L-12) was obtained as a white solid in 22.3% yield.

[0065] 1H NMR (400MHz, Chloroform-d) δ9.37(d,J=7.3Hz,1H),8.16(s,1H),8.02(dt,J=8.0,1.0Hz,1H),7.75(dd,J=7.9,1.7Hz,1H),7.69(ddd,J =8.3,7.1,1.2Hz,1H),7.57(d,J=8.1Hz,1H),7.42(ddd,J=9.2,7.8,1.6Hz,2H),7.13–7.04(m,2H),4.83(d,J=5.8Hz,2H),3.89(s,3H); 13 C NMR (151MHz, CDCl3) δ164.06,150.29,150.18,139.76,126.38,125.23,124.88 ,124.60,123.87,123.77,120.18,119.49,111.18,55.00,35.22; HRMS(ESI)m / z caled for C 18 H 15 N5O3S[(M+H) + ]:382.0974,found:382.0976.

[0066] N-((1-(4-Isopropylphenyl)-1H-1,2,3-triazol-4-yl)methyl)-3-oxobenzo[d]isothiazole-2(3H)-carboxamide (L-13), white flaky solid, yield 32.5%.

[0067] 1 H NMR(400MHz,Chloroform-d)δ9.41(s,1H),8.06–7.98(m,2H),7.70(ddd,J=8.3,7.2,1.3Hz,1H),7.66–7.60(m,2H),7.57(dt,J=8.1, 0.9Hz,1H),7.42(ddd,J=8.0,7.1,0.9Hz,1H),7.39–7.31(m,2H),4.81(d,J=5.8Hz,2H),2.96(h,J=6.9Hz,1H),1.28(d,J=6.9Hz,6H); 13CNMR (151MHz, CDCl3) δ180.26,165.13,150.87,143.92,140.62,133.39,129.76,127.6 7,125.25,124.65,121.91,119.39,118.66,103.69,98.07,35.47,18.04; HRMS(ESI)m / z caled for C 20 H 19 N5O2S[(M+Na) + ]:416.1157,found:416.1158.

[0068] N-((1-(3-Bromo-4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-3-oxobenzo[d]isothiazole-2(3H)-carboxamide (L-14), gray solid, yield 47%.

[0069] 1 H NMR(400MHz,Chloroform-d)δ9.43(s,1H),8.06–7.95(m,3H),7.75–7.64(m,2H),7.62–7 .53(m,1H),7.43(ddd,J=8.1,7.1,1.0Hz,1H),7.30–7.27(m,1H),4.80(d,J=5.9Hz,2H); 13 C NMR (151MHz, CDCl3) δ165.19,159.87,158.22,151.53,145.52,140.77,134.10,127 .45,126.01,125.90,124.75,121.00,120.53,117.54,117.38,36.04; HRMS(ESI)m / z caled for C 17 H 11 BrFN5O2S[(M+H) + ]:447.9879,found:447.9877.

[0070] N-((1-(4-Fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-5-methyl-3-oxobenzo[d]isothiazole-2(3H)-carboxamide (L-15), yellow solid, yield 36%.

[0071] 1H NMR(400MHz,Chloroform-d)δ9.44(s,1H),8.02(s,1H),7.82(dt,J=1.8,0.8Hz,1H),7.73–7.67(m,2H),7 .53(dd,J=8.3,1.8Hz,1H),7.46(d,J=8.3Hz,1H),7.24–7.17(m,2H),4.80(d,J=5.9Hz,2H),2.46(s,3H); 13 C NMR (151MHz, CDCl3) δ165.18,151.57,145.30,137.84,135.65,127.08,124.87 ,122.65,122.60,121.07,120.21,116.80,116.65,36.08,21.06; HRMS(ESI)m / z caled for C 18 H 14 FN5O2S[(M+H) + ]:384.0930,found:384.0931.

[0072] N-((1-(4-Fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-6-methyl-3-oxobenzo[d]isothiazole-2(3H)-carboxamide (L-16), orange solid, yield 52.6%.

[0073] 1 H NMR(400MHz,Chloroform-d)δ9.42(s,1H),8.02(s,1H),7.90(d,J=8.2Hz,1H),7.7 4–7.66(m,2H),7.36(s,1H),7.25–7.17(m,3H),4.80(d,J=5.9Hz,2H),2.50(s,3H); 13 C NMR (151MHz, CDCl3) δ165.10,163.30,161.64,151.61,145.51,141.06,133.33,127.65,127 .08,122.66,122.60,122.42,121.04,120.30,116.80,116.65,36.08,22.23; HRMS(ESI)m / z caled for C 18 H 14 FN5O2S[(M+Na) + ]:406.0750,found:406.0748.

[0074] N-((1-(4-Fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-7-methyl-3-oxobenzo[d]isothiazole-2(3H)-carboxamide (L-17), yellow solid, yield 16.5%.

[0075] 1 H NMR(400MHz,DMSO-d6)δ9.53(t,J=5.8Hz,1H),8.91(s,1H),8.17–8.05(m,3H),7.94(s, 1H),7.80(dd,J=8.4,1.8Hz,1H),7.67–7.56(m,2H),4.87(d,J=5.8Hz,2H),2.59(s,3H); 13 C NMR (151MHz, CDCl3) δ165.71,163.30,161.65,151.58,145.25,140.74,134.15,133.29,130.2 2,126.58,124.82,124.63,122.65,121.06,116.80,116.65,36.10,18.49; HRMS(ESI)m / zcaled for C 18 H 14 FN5O2S[(M+Na) + ]:406.0750,found:406.0748.

[0076] N-((1-(4-Fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-trifluoromethyl-3-oxobenzo[d]isothiazole-2(3H)-carboxamide (L-18) was obtained as a white solid in 25.6% yield.

[0077] 1 H NMR (400MHz, Chloroform-d) δ9.40 (s, 1H), 8.00 (s, 1H), 7.82–7.74 (m, 3H), 7.73–7.67 (m, 2H), 7.25–7.14 (m, 2H), 4.80 (d, J = 5.9Hz, 2H); 13 C NMR (151MHz, CDCl3) δ163.32,162.28,161.66,151.05,144.89,143.57,133.26,133.06, 124.66,124.41,124.37,122.68,122.62,121.02,116.81,116.67,36.19; HRMS(ESI)m / z caled for C 18 H 11F4N5O2S[(M+H) + ]:438.0650,found:438.0648.

[0078] N-((1-(4-Fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-bromo-3-oxobenzo[d]isothiazole-2(3H)-carboxamide (L-19), yellow flaky solid, yield 32%.

[0079] 1 H NMR(400MHz,Chloroform-d)δ9.40(t,J=5.7Hz,1H),8.00(s,1H),7.74–7.66(m,2H),7. 60(dd,J=7.0,1.6Hz,1H),7.55–7.47(m,2H),7.25–7.17(m,2H),4.81(d,J=5.8Hz,2H); 13 C NMR (151MHz, CDCl3) δ163.35,161.66,151.15,145.00,143.67,134.17,133.26,131.16, 122.94,122.67,122.61,121.51,120.96,119.71,116.82,116.67,36.17; HRMS(ESI)m / z caled for C 17 H 11 FBrN5O2S[(M+H) + ]:449.9859,found:449.9850.

[0080] N-((1-(4-Fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-methoxy-3-oxobenzo[d]isothiazole-2(3H)-carboxamide (L-20), pale yellow solid, yield 8.8%.

[0081] 1 H NMR (400MHz, DMSO-d6) δ9.37(t,J=5.7Hz,1H),8.74(s,1H),7.99–7.90(m,2H),7.69(t,J= 8.1Hz,1H),7.51–7.39(m,3H),6.98(d,J=8.1Hz,1H),4.68(d,J=5.7Hz,2H),3.90(s,3H); 13C NMR (151MHz, CDCl3) δ163.99,160.64,151.53,143.18,135.51,122.68,122.62,12 0.89,116.79,116.65,113.03,112.26,106.86,56.10,36.15; HRMS(ESI)m / zcaled for C 18 H 14 FN5O3S[(M+Na) + ]:422.0699,found:422.0702.

[0082] N-((1-(4-Fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-hydroxy-3-oxobenzo[d]isothiazole-2(3H)-carboxamide (L-21) was obtained as a white solid in 31% yield.

[0083] 1 H NMR(400MHz,Chloroform-d)δ9.09(s,2H),8.01(s,1H),7.75–7.66(m,2H),7.57(t,J=8.1Hz,1H), 7.25–7.18(m,2H),7.02(dd,J=7.9,0.6Hz,1H),6.81(dd,J=8.1,0.6Hz,1H),4.80(d,J=5.8Hz,2H); 13 C NMR (151MHz, CDCl3) δ166.70,163.34,159.32,150.71,144.89,140.53,136.54,133.25, 122.65,122.59,120.99,116.85,116.69,111.72,111.08,110.97,36.10; HRMS(ESI)m / z caled for C 17 H 12 FN5O3S[(M) - ]:384.0567,found:384.0564.

[0084] N-((1-(2-Hydroxy-5-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-methyl-3-oxobenzo[d]isothiazole-2(3H)-carboxamide (L-22), a red solid, yield 24.6%.

[0085] 1H NMR (400MHz, DMSO-d6) δ10.93(s,1H),9.39(t,J=5.7Hz,1H),8.48(s,1H),7.79(d,J=8.1Hz,1H),7.70(dd,J=10.9,2.5Hz,1H),7. 61(t,J=7.7Hz,1H),7.39(dd,J=8.8,2.7Hz,1H),7.22(d,J=7.3Hz,1H),7.12(d,J=8.8Hz,1H),4.69(d,J=5.7Hz,2H),2.67(s,3H); 13 CNMR(151MHz,DMSO-d6)δ180.26,165.13,150.87,143.92,140.62,133.39,129.76,127. 67,125.25,124.65,121.91,119.39,118.66,103.69,98.07,35.47,18.04; HRMS(ESI)m / z caled for C 18 H 15 ClN5O3S[(M+H) + ]:416.0584,found:416.0583.

[0086] N-((1-(3-Hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-5-methyl-3-oxobenzo[d]isothiazole-2(3H)-carboxamide (L-23), yellow solid, yield 12.5%.

[0087] 1 H NMR (400MHz, DMSO-d6) δ9.34(t,J=5.8Hz,1H),8.68(s,1H),7.92(d,J=8.3Hz,1H),7.77(s,1H),7.63(dd,J=8. 4,1.8Hz,1H),7.35(t,J=8.0Hz,1H),7.32–7.24(m,2H),6.91–6.84(m,1H),4.68(d,J=5.8Hz,2H),2.42(s,3H); 13 C NMR(151MHz,DMSO)δ164.86,159.00,151.41,138.44,138.07,136.36,135.97,131.20, 126.50,125.22,122.37,121.64,116.09,110.81,107.42,36.07,20.92; HRMS(ESI)m / z caled for C 18 H15 N5O3S[(M+Na) + ]:404.0793,found:404.0791.

[0088] N-((1-(2-Methylphenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-methyl-3-oxobenzo[d]isothiazole-2(3H)-carboxamide (L-24), yellow solid, yield 27.9%.

[0089] 1 H NMR(400MHz,Chloroform-d)δ9.50(t,J=5.8Hz,1H),7.81(s,1H),7.52(t,J=7.7Hz,1H),7 .42–7.30(m,5H),7.14(d,J=7.4Hz,1H),4.83(d,J=5.9Hz,2H),2.74(s,3H),2.22(s,3H); 13 C NMR (151MHz, CDCl3) δ165.92,151.63,144.24,142.00,141.53,136.45,133.70,133.38,131.50 ,129.88,127.77,126.83,126.01,124.01,122.29,117.97,36.08,18.51,17.95; HRMS(ESI)m / z caled for C 19 H 17 N5O2S[(M+H) + ]:380.1181,found:380.1183.

[0090] N-((1-(2-Fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-methyl-3-oxobenzo[d]isothiazole-2(3H)-carboxamide (L-25), brown solid, yield 53.9%.

[0091] 1 H NMR(400MHz,Chloroform-d)δ9.49(t,J=5.9Hz,1H),8.14(d,J=2.8Hz,1H),7.93(td,J=7.7,1.7Hz,1H),7.52(t, J, =. 7.7Hz, 1H), 7.48–7.39 (m, 1H), 7.39–7.28 (m, 3H), 7.13 (d, J = 7.4Hz, 1H), 4.82 (d, J = 5.8Hz, 2H), 2.74 (s, 3H); 13C NMR (151MHz, CDCl3) δ165.92,154.25,152.59,151.59,142.00,141.54,133.36,130.31,130 .26,127.75,125.25,125.23,124.99,122.27,117.96,116.97,35.97,18.50; HRMS(ESI)m / z caled forC 18 H 14 FN5O2S[(M+H) + ]:384.0930,found:384.0931.

[0092] Example 2

[0093] Synthesis of N-((4-(3-hydroxyphenyl)-1,2,5-oxadiazol-3-yl)methyl)-3-oxo-4-methyl-benzo[d]isothiazole-2(3H)-carboxamide (Q2)

[0094]

[0095] 2.1 Synthesis of 4-methylbenzo[d]isothiazol-3(2H)-one (2-1)

[0096] Synthesis steps are the same as 1.1

[0097] 2.2 Synthesis of 2-(propan-4-yn-1-yl)benzisothiazol-3(2H)-one (2-2) and 3-(propan-3-yn-1-yloxy)benzisothiazole (2-3).

[0098] Compound 2-1 (2 mmol) and potassium carbonate (4 mmol) were weighed into a round-bottom flask, dissolved in 20 mL of acetonitrile, and stirred at room temperature for 30 minutes. After stirring, propargyl(butyl)amine (3 mmol) was added dropwise. The mixture was allowed to react at room temperature for 4.5 hours and monitored by TLC. After completion of the reaction, the mixture was extracted three times with ethyl acetate, washed with water, and dried over anhydrous sodium sulfate. The organic layer was concentrated and filtered through a column using a 1:1 ratio of PE:DCM to obtain the purified product in a 67% yield.

[0099] 2.3 Synthesis of compounds L-26 to L-29

[0100] The synthesis steps are the same as in Example 1.4.

[0101] 2-(2-(1-(3-Hydroxyphenyl)-1H-1,2,3-triazol-4-yl)ethyl)-4-methylbenzisothiazol-3(2H)-one (L-26), pale yellow solid, yield 48.9%.

[0102] 1 H NMR (400MHz, DMSO-d6) δ10.03(s,1H),8.61(s,1H),7.71(d,J=8.1Hz,1H),7.49(t,J=7.7Hz,1H),7.36(t,J=8.0Hz,1H), 7.28–7.20(m,2H),7.16(d,J=7.3Hz,1H),6.89–6.82(m,1H),4.13(t,J=7.0Hz,2H),3.10(t,J=7.0Hz,2H),2.69(s,3H); 13 C NMR (151MHz, DMSO) δ165.58,158.95,144.93,141.69,139.49,138.17,131.63,131.25,127.5 8,121.56,121.51,119.71,115.91,110.64,107.20,43.10,25.72,17.99; HRMS(ESI)m / zcaled for C 18 H 15 N5O3S[(M) - ]:351.0916,found:351.0928.

[0103] 3-((1-(3-Hydroxyphenyl)-1H-1,2,3-triazol-4-yl)ethoxy)-4-methylbenzisothiazole (L-27), orange solid, yield 32.1%.

[0104] 1 H NMR (400MHz, DMSO-d6) δ10.04(s,1H),8.66(s,1H),7.83(d,J=8.1Hz,1H),7.44(t,J=7.6Hz,1H),7.35(t,J=8.0Hz,1H), 7.30–7.22(m,2H),7.17(d,J=7.1Hz,1H),6.90–6.83(m,1H),4.79(t,J=6.4Hz,2H),3.29(t,J=6.4Hz,2H),2.55(s,3H); 13 C NMR (151MHz, DMSO) δ163.97,158.95,152.42,145.18,138.22,135.91,131.23,129.35,126.8 6,123.74,121.44,118.85,115.89,110.71,107.27,67.86,25.75,19.51; HRMS(ESI)m / zcaled for C18H15N5O3S[(M)- ]:351.0916,found:351.0915.

[0105] 2-(2-(1-(3-hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-methylbenzisothiazol-3(2H)-one (L-28), khaki solid, yield 23.8%.

[0106] 1 H NMR (400MHz, DMSO-d6) δ10.04(s,1H),8.80(s,1H),7.72(d,J=8.1Hz,1H),7.51(t,J=7.7Hz,1H),7.36(t,J= 8.3Hz,1H),7.29(dd,J=4.9,2.5Hz,2H),7.18(d,J=7.3Hz,1H),6.91–6.84(m,1H),5.13(s,2H),2.72(s,3H); 13 C NMR (151MHz, DMSO) δ165.39,158.95,143.94,141.83,139.66,137.95,134.94,131.79,131.26,127.66,122 .58,121.48,119.77,116.19,112.95,110.89,110.13,107.43,106.23,38.29,21.10,18.02; HRMS(ESI)m / z caled for C18H15N5O3S[(M) - ]:337.0759,found:337.0753.

[0107] 3-((1-(3-Hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-methylbenzisothiazole (L-29), white solid, yield 33.7%.

[0108] 1 H NMR (400MHz, DMSO-d6) δ10.06(s,1H),8.92(s,1H),7.87(dt,J=8.1,0.9Hz,1H),7.47(dd,J=8.3,7.1Hz,1H),7.42–7.35(m,1H ),7.32(tdd,J=3.3,2.0,1.1Hz,2H),7.21(dt,J=7.1,0.9Hz,1H),6.89(ddd,J=8.0,2.3,1.2Hz,1H),5.67(s,2H),2.65(s,3H); 13C NMR (151MHz, DMSO) δ163.50,158.98,152.68,143.89,137.98,135.92,131.28,129.51,12 6.98,123.61,123.44,118.98,116.22,110.99,107.54,62.14,19.62; HRMS(ESI)m / zcaled for C18H15N5O3S[(M) - ]:337.0759,found:337.0745.

[0109] Example 3

[0110] Synthesis of N-((4-(3-hydroxyphenyl)-1,2,5-oxadiazol-3-yl)methyl)-3-oxo-4-methylbenzo[d]isothiazole-2(3H)-carboxamide (L-30).

[0111]

[0112] 3.1 Synthesis of 3-(chloromethyl)-4-(3-hydroxyphenyl)-1,2,5-oxadiazole (3-1).

[0113] Hydroxylamine hydrochloride (1.7 g, 25 mmol) was suspended in 25 mL of ethanol, and triethylamine (2.45 g, 24.2 mmol) was added. After stirring at room temperature for 30 minutes, m-hydroxybenzonitrile (1.19 g, 10 mmol) was added and the temperature was raised to 50°C for 12 hours. The ethanol was removed by concentration, and the mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, and concentrated under low pressure. The resulting crude product was dissolved in 20 mL of DCM, and DIPEA (2.58 g, 20 mmol) was added under ice-cooling. Chloroacetyl chloride (2.2610 mmol) was then added dropwise. The mixture was heated to room temperature and stirred for 4 hours. The reaction was monitored by TLC. After completion of the reaction, the mixture was extracted three times with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate and concentrated under low pressure. Column chromatography (PE:EA = 1:1) yielded a yellow liquid in a 72% yield.

[0114] 3.2 Synthesis of 2-(4-(3-hydroxyphenyl)-1,2,5-oxadiazol-3-yl)methyl)isoindole-1,3-dione (3-2).

[0115] Compound 3-1 (2.1 g, 10 mmol) was weighed and suspended in 16 mL of DMF. Potassium phthalimide (1.85 g, 10 mmol) was slowly added in batches. The temperature was raised to 60°C for 4 hours and the reaction was monitored by TLC. After confirming that the reaction was complete, the mixture was cooled to room temperature, and about 50 mL of ice water was added. The mixture was sonicated and filtered. The filter cake was washed with a small amount of water and dried to obtain a yellow solid with a yield of 57%.

[0116] 3.3 Synthesis of (4-(3-hydroxyphenyl)-1,2,5-oxadiazol-3-yl)methanamine (3-3).

[0117] Compound 3-2 (321.3 mg, 1 mmol) was dissolved in 4.6 mL of ethanol, and 80% hydrazine hydrate (60 mg, 1.2 mmol) was added. The temperature was raised to 70°C and refluxed for 4 hours. The reaction was monitored by TLC. After the reaction was complete, the mixture was cooled to room temperature, the solvent was removed by low pressure, water was added, the pH was adjusted to alkaline, and the mixture was extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, concentrated under low pressure, and filtered through a column using PE:EA = 1:1 as the eluent to obtain a white solid with a yield of 37%.

[0118] 3.4 Synthesis of compound L-30.

[0119] The synthesis method is the same as that of Examples 1.3 and 1.4

[0120] N-((4-(3-Hydroxyphenyl)-1,2,5-oxadiazol-3-yl)methyl)-3-oxo-4-methylbenzo[d]isothiazole-2(3H)-carboxamide as a white solid in 52% yield.

[0121] 1 H NMR(400MHz, DMSO-d6)δ9.69(s,1H),9.45(t,J=5.8Hz,1H),7.65(d,J=8.1Hz,1H),7.48(t,J=7.7Hz,1H),7.30–7.22(m,2H) ,7.18(t,J=7.8Hz,1H),7.09(dt,J=7.4,1.0Hz,1H),6.80(ddd,J=8.1,2.6,1.2Hz,1H),4.76(d,J=5.8Hz,2H),2.55(s,3H); 13 CNMR(151MHz,DMSO)δ176.49,167.12,164.51,157.27,150.66,140.99,140.17,132.98,129 .90,127.19,126.44,121.12,118.92,118.10,117.12,112.91,36.29,17.46; HRMS(ESI)m / z caled for C 18 H 14 N4O4S[(M) - ]:381.0658,found:381.0614.

[0122] Example 4

[0123] Test of the inhibitory activity of the compounds of the present invention on IDO1 at the cellular level

[0124] Cell culture

[0125] The human cervical cancer cell line CL-0101 used in this example was purchased from Pusino. HeLa cells were cultured in complete DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution. All operations were performed in a clean bench. The positive control in this example was INCB024360.

[0126] Cell recovery: Remove cells from liquid nitrogen and thaw in a 37°C water bath. After thawing, transfer cells to a 15 mL centrifuge tube, add 10 mL of DMEM complete medium, centrifuge at 2500 rpm for 3 minutes, remove the supernatant, add 2 mL of culture medium, pipette, resuspend, transfer to a culture flask, add 5 mL of culture medium, and culture in a 37°C incubator with 5% CO2.

[0127] Cell digestion: When the cells have grown to 90%, discard the old culture medium in the culture flask, add 2 ml of PBS to rinse the cells, aspirate the PBS and add 2 mL of trypsin. Place the culture flask in a 37°C incubator for digestion. After 3 minutes, add 2 mL of culture medium to terminate the digestion.

[0128] Cell passaging: Transfer the digested cells to a 15 mL centrifuge tube using a pipette, centrifuge at 2500 rpm for 3 minutes, discard the supernatant, add 2 mL of culture medium, pipette, resuspend, transfer to a culture flask, add 5 mL of culture medium, and culture in a 37°C incubator with 5% CO2.

[0129] Seed board

[0130] Take Hela cell suspension in the logarithmic growth phase, take 10 μL of cell suspension with a pipette, pipette it into a cell counting plate at an angle, count it on a cell counter, and dilute it to 5×10 3 The diluted cell suspension was inoculated into a 96-well plate, 100 μL per well, and cultured in a 37°C incubator containing 5% CO2 for 24 hours.

[0131] Dosing

[0132] Remove the 96-well plate and add DMEM medium containing human IFN-γ (final concentration 100 ng / mL), L-Trp (final concentration 15 μg / mL), and various concentrations of the compound. Each compound is plated in triplicate wells, 200 μL per well. Additionally, a blank control, a blank control containing IFN-γ, and a positive control group are set up. The 96-well plate is incubated at 37°C in a 5% CO2 incubator for 48 hours.

[0133] Measure absorbance

[0134] After 48 hours, remove the 96-well plate. Pipette 140 μL of the supernatant from each well, add 10 μL of 6,1N trichloroacetic acid solution, and incubate in a 50°C water bath for 30 minutes. At this time, N'-formylkynurenine produced by IDO-catalyzed oxidative cleavage of the indole ring of tryptophan will be hydrolyzed to kynurenine. The resulting mixture was centrifuged at 4000 rpm and 4°C for 20 minutes to separate the supernatant from the precipitate. Pipette 100 μL of the supernatant and mix it with 100 μL of acetic acid solution containing 2% p-dimethylaminobenzaldehyde (w / v). React at room temperature for 5 minutes and measure the absorbance at 480 nm using a microplate reader.

[0135] Calculation of inhibition rate

[0136] The inhibition rate was calculated as follows: Cell level inhibition rate = (OD value of drug treatment group - OD value of blank control group) / (OD value of blank control group containing IFN-γ - OD value of blank control group) × 100%

[0137] Experimental results analysis

[0138] The experimental results are shown in Table 1, which show that most of the compounds of the present invention have good inhibitory activity on cellular IDO1 at a concentration of 10 μM, and also have certain inhibitory activity on cellular IDO1 at a concentration of 1 μM.

[0139] Table 1

[0140]

[0141]

[0142] IC analysis of compound L-13, which has outstanding activity 50 Tested and compared with 1-MT, the results are as follows Figure 1a 、 Figure 1b The results showed that the activity of L-13 was particularly outstanding, and HelaIDO1 IC 50 It reached the nanomolar level of 690±36nM, which was significantly better than 1-MT (HelaIDO1 IC 50 =123.55±1.48 μM), indicating that it is a promising IDO1 inhibitor.

[0143] Example 5

[0144] Test of the inhibitory activity of the compounds of the present invention on IDO1 at the enzyme level

[0145] Establishment of enzyme system

[0146] The human IDO1 enzyme HY-P70679 used in this example was purchased from MedChemExpress. Catalase S25259, potassium phosphate buffer (pH 6.5) R27370, and methylene blue S19043 were all purchased from source leaves. All operations were performed in a clean bench. The positive control in this example was INCB024360.

[0147] The system was established as follows: 50 mM potassium phosphate buffer, 20 mM sodium ascorbate, 20 nM human IDO1 enzyme, 0.2 mg / mL catalase, 2 mM L-tryptophan, and 10 μM methylene blue.

[0148] Pipette 25 mL of potassium phosphate buffer into a 50 mL centrifuge tube. Add methylene blue, sodium ascorbate, L-tryptophan, and catalase, weighed using a precision balance, to a concentration of 10 μM, 20 mM, 2 mM, and 0.2 mg / mL, respectively. Vortex the tube to fully dissolve the compounds and ensure the solution is evenly mixed.

[0149] Dosing

[0150] Take the solution of the established enzyme system and add the synthesized compound to prepare it to the desired concentration. Take a new 96-well plate and add 50mM potassium phosphate buffer, 20mM sodium ascorbate, 20nM human IDO1 enzyme, 0.2mg / mL catalase, 2mM L-tryptophan, 10μM methylene blue. And DMEM culture medium with different concentrations of compounds, three replicates for each compound, 180μL per well. In addition, set up a blank control combination, a blank control group containing IFN-γ, and a positive control group. Place the 96-well plate in a 37°C incubator with 5% CO2 and incubate for 1 hour.

[0151] Measure absorbance

[0152] After 1 hour, remove the 96-well plate. Pipette 140 μL of the supernatant from each well of the 96-well plate, add 10 μL of 30% 6,1N trichloroacetic acid solution, and incubate in a 60°C water bath for 30 minutes. Centrifuge the resulting mixture at 2500 rpm and 4°C for 10 minutes to separate the supernatant from the precipitate. Pipette 100 μL of the supernatant and mix it with 100 μL of acetic acid solution containing 2% p-dimethylaminobenzaldehyde (w / v). Incubate at room temperature for 5 minutes. Measure the absorbance at 480 nm using a microplate reader.

[0153] Calculation of inhibition rate

[0154] The inhibition rate was calculated as follows: enzyme level inhibition rate = (OD value of drug treatment group - OD value of blank control group) / (OD value of blank control group containing IFN-γ - OD value of blank control group) × 100%

[0155] Experimental results analysis

[0156] The experimental results are shown in Table 2, which show that most of the compounds of the present invention have good inhibitory activity against IDO enzyme at a concentration of 10 μM, and also have certain inhibitory activity against IDO enzyme at a concentration of 1 μM, and are all better than 1-MT.

[0157] Table 2

[0158]

[0159]

[0160] Enzyme IC was performed on the compounds L-13 and L-23 with outstanding activity and the compound L-1 without any substituents. 50 Test results as follows Figure 2 As shown, IC of L-1 50 The IC value of L-13 was 6.79±0.02μM. 50 The IC value of L-23 was 3.07±5.67μM. 50 It is 4.74±0.72 μM, indicating that the transformation of the present invention is successful.

[0161] Example 6

[0162] Cytotoxicity test of the compounds of the present invention on Hela cells.

[0163] Cell culture

[0164] The in vitro cytotoxicity of compounds is measured using the MTS assay. Viable cells metabolize the MTS reagent, producing a colored formazan compound. The color intensity of this compound is proportional to the number of viable cells, allowing cell viability to be quantified by measuring absorbance at a specific wavelength. Since dead cells lack the ability to metabolize formazan and do not produce formazan, this method effectively distinguishes live from dead cells. In this example, the positive control is paclitaxel.

[0165] The cell culture method is the same as that in Example 4.

[0166] Seed board

[0167] Take Hela cell suspension in the logarithmic growth phase, take 10 μL of cell suspension with a pipette, pipette it into a cell counting plate at an angle, count it on a cell counter, and dilute it to 3×10 3 The diluted cell suspension was inoculated into a 96-well plate, 90 μL per well, and cultured in a 37°C incubator containing 5% CO2 for 24 hours.

[0168] Dosing

[0169] Add 1 mL of DMEM complete medium to an Eppendorf tube. Add 1 μL of the prepared 10 μM compound using a pipette and shake thoroughly. Remove the plate and add 10 μL of DMEM medium containing various concentrations of compound to each well, using six replicates for each compound. Additionally, set up a blank control and a positive control group. Incubate the 96-well plate in a 37°C incubator with 5% CO₂ for 48 hours.

[0170] Measure absorbance

[0171] After 48 hours, remove the 96-well plate. Add 20 μL of MTS / PMS solution to each well and incubate in a 37°C incubator with 5% CO₂ for 30 minutes. Remove the 96-well plate and measure the absorbance at 490 nm using a microplate reader.

[0172] Calculation of inhibition rate

[0173] The inhibition rate was calculated as follows: Cytotoxicity inhibition rate = 1-(OD value of drug-treated group / OD value of blank control group) × 100% Experimental results analysis

[0174] The experimental results are shown in Table 3, which show that the compounds of the present invention have very low cytotoxicity, indicating that the compounds with this structure have almost no damage to cells.

[0175] Table 3

[0176]

[0177]

[0178] Example 7

[0179] Anti-proliferation test of the compound of the present invention on A549 paclitaxel-resistant cell line Cell culture

[0180] The paclitaxel-resistant lung cancer strain KG3503 used in this example was purchased from Keytruda. HeLa cells were cultured in F12K medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution. F12K medium KGL1905-500 and 0.25% trypsin (containing EDTA and phenol red) KGL2102-100 were also purchased from Keytruda. Cells were cultured at 37°C in a 5% CO2 incubator. All operations in this example were performed in a cleanroom. The positive control used in this example was INCB024360.

[0181] Cell digestion: When the cells have grown to 90%, discard the old medium in the culture flask and add 2 mL of PBS to rinse the cells. After aspirating the PBS with a pipette, add 2 mL of 0.25% trypsin (containing EDTA and phenol red). Place the culture flask in a 37°C incubator for digestion. After 5 minutes, add 2 mL of culture medium to terminate the digestion.

[0182] Cell passaging: Transfer the digested cells to a 15 mL centrifuge tube using a pipette, centrifuge at 2500 rpm for 3 minutes, discard the supernatant, add 2 mL of culture medium, pipette, resuspend, transfer to a culture flask, add 5 mL of culture medium, and culture in a 37°C incubator with 5% CO2.

[0183] Seed board

[0184] Take the A549 / R cell suspension in the logarithmic growth phase, take 10 μL of cell suspension with a pipette, pipette it into a cell counting plate at an angle, count it on a cell counter, and dilute it to 2×10 3 The diluted cell suspension was inoculated into a 96-well plate, 100 μL per well, and cultured in a 37°C incubator containing 5% CO2 for 24 hours.

[0185] Dosing

[0186] Remove the 96-well plate, aspirate the old medium, and add 100 μL of F12K medium containing various compound concentrations. In triplicate wells, perform each compound test. Additionally, establish a blank control and a positive control group. Incubate the 96-well plate in a 37°C incubator with 5% CO₂ for 48 hours.

[0187] Measure absorbance

[0188] After 48 hours, remove the 96-well plate. Use a dispenser to add 10 μL of CCK8 solution to each well. After addition, shake the 96-well plate on a microplate shaker for 1 minute. Then, incubate the plate in a 37°C incubator with 5% CO₂ for 2 hours. Remove the 96-well plate and measure the absorbance at 450 nm using a microplate reader.

[0189] Experimental results analysis

[0190] The experimental results are shown in Table 4, which show that the IC 50 lower, indicating that these compounds have stronger anti-proliferative activity against A549 paclitaxel-resistant cell line.

[0191] Table 4

[0192]

[0193]

[0194] Example 8

[0195] Aryl hydrocarbon receptor activation test of the compounds of the present invention

[0196] The CT-26 cells, TRIzol reagent, HiScript RT SuperMix cDNA Synthesis Kit, and AceQ qPCR SYBR Green Master Mix reagent used in this example were all purchased from Vazyme Biotech. The positive control in this example was FICZ.

[0197] RNA extraction

[0198] Mouse colon cancer cells were cultured to the logarithmic growth phase and then seeded into six-well plates at a density of 5×10 4 Different concentrations of compounds, blank controls, and positive controls were incubated with CT-26 for 24 hours, and then total RNA was extracted using TRIzol reagent.

[0199] Discard the culture medium and wash once with PBS. 2 Add 1 mL of RNA isolater to the cells in the culture area, ensuring that the cell surface is fully covered. Then, dislodge the cells with a pipette. Transfer the lysate to a 1.5 mL centrifuge tube and repeatedly pipette until fully lysed. Incubate on ice for 5 minutes. Add 1 / 5 volume of chloroform to the lysate. Shake vigorously for 15 seconds to form an emulsion. Incubate at 4°C for 5 minutes. Centrifuge at 11,200 rpm for 15 minutes at 4°C. Remove the centrifuge tube. The solution will now separate into three layers: a colorless aqueous phase, a white middle layer, and a red organic layer (lower layer). Carefully transfer the upper aqueous phase to a new centrifuge tube. Add an equal volume of pre-chilled isopropanol, mix by inversion, and incubate at 4°C for 10 minutes. Centrifuge at 11,200 rpm for 15 minutes at 4°C. Carefully discard the supernatant, add 1 mL of 75% ethanol, gently flick the bottom of the tube to resuspend the pellet, and invert several times. Incubate at room temperature for 3-5 minutes, centrifuge, and discard the supernatant. Dry the precipitate in a clean environment at room temperature for 2-5 minutes. Add an appropriate amount of RNase-free ddH2O to dissolve the precipitate. Once completely dissolved, take a small amount for testing and store the remaining amount at -85--65°C.

[0200] Reverse transcription

[0201] The extracted RNA was reverse transcribed into cDNA using the HiScript RT SuperMix cDNA Synthesis Kit. First, the RNA template was denatured. After preparing the mixture in a centrifuge tube, heat it at 65°C for 5 minutes, quickly chill it on ice, and let it sit on ice for 2 minutes. A first-strand cDNA synthesis reaction solution was prepared and gently pipetted to mix thoroughly. The first-strand cDNA synthesis reaction was then performed. The reverse transcription system is shown in Table 5:

[0202] Table 5: Reverse transcription reaction system

[0203]

[0204] The reverse transcription conditions are shown in Table 6:

[0205] Table 6: Reverse transcription reaction conditions

[0206]

[0207] PCR amplification

[0208] After reverse transcription, the cDNA template was added to a PCR reaction containing sequence-specific primers and AceQqPCR SYBR Green Master Mix reagent. qPCR was performed using the MyiQ2 detection system. Thermal cycling consisted of an initial denaturation at 95°C for 5 minutes, followed by 40 cycles of 95°C for 10 seconds and 60°C for 30 seconds. The Cyp1A1 primer sequences were: forward 5′-TGGAGC CTC ATG TAC CTG GTA ACC-3′ and reverse 5′-CTG CCGTC TCT GCC AAT CAC TG-3′; the β-actin primer sequences were: forward 5′-ATC ACT ATT GG CAA C GCG GTT C-3′ and reverse 5′-CAG CAC TGTGTT GGC ATA GAG GTC-3′. The PCR reaction system is shown in Table 7.

[0209] Table 7: PCR reaction system

[0210]

[0211] The PCR reaction process is shown in Table 8:

[0212] Table 8: PCR reaction process

[0213]

[0214] Result Analysis

[0215] Experimental results using 2 －△△Ct Method evaluation. Set the internal reference gene β-actin Ct value, subtract the target gene Ct value from the β-actin Ct value, and get the △Ct. Subtract the △Ct value of each target gene in the experimental group from the △Ct value of each gene in the control group, and take the negative value of the difference, that is, -△△Ct. Among them, the expression level of each target gene in the experimental group relative to the internal reference gene in the control group is 2 －△△Ct The results are as follows Figure 3 By calculating the mRNA expression level of CYP1A1, it was found that compound L-13 had no significant effect on the CYP1A1 gene in CT-26 cells, indicating that it had no agonist activity on AhR.

[0216] Example 9

[0217] Study on acute toxicity of the compounds of the present invention to mice

[0218] Animal experiments

[0219] The BALB / c mice used in this embodiment were purchased from Shanghai JST Experimental Animal Co., Ltd. and subsequently raised in an SPF-level environment. The feed and bedding used were also purchased from JST Experimental Animal Co., Ltd., and the drinking water for the mice was ultrapure water.

[0220] Twenty six-week-old BALB / c mice, half male and half female, were divided into two groups according to sex and weight: a blank control group (normal saline) and a treatment group. The treatment group was gavage-injected with 600 mg / kg of compound L-13, while the blank group was injected with an equal dose of normal saline. The activity status and mortality of the animals were observed within 14 days of injection.

[0221] Experimental results analysis

[0222] The experimental results are shown in Tables 9 and 10. Over the 14 days, mice in both the blank and treatment groups ate and drank normally, exhibited no toxic reactions, and had no deaths. This suggests that compound L-13 has low oral toxicity, a single dose tolerable dose exceeding 600 mg / kg, and is safe for use.

[0223] Table 9

[0224]

[0225] Table 10

[0226]

[0227] In this specification, the present invention has been described with reference to specific embodiments thereof. However, it will be apparent that various modifications and variations may be made without departing from the spirit and scope of the present invention. Therefore, the description is to be regarded as illustrative rather than restrictive.

Claims

1. A benzisothiazolinone compound or a pharmaceutically acceptable salt thereof, characterized in that: The structural formula of the compound is shown in the following general formula (1): Wherein, ring A is triazole or oxadiazole; R1 is selected from substituted or unsubstituted C1-C6 alkyl, C1-C6 alkoxy, hydroxy-, halogen; R2 is selected from substituted or unsubstituted aryl, and the substituent of the substituted aryl is selected from one or more of linear or branched C1-C6 alkyl, C1-C6 alkoxy, hydroxyl, and halogen; L is selected from amide, substituted or unsubstituted C1-C6 alkyl, C1-C6 alkoxy; The substituents substituted on the C1-C6 alkyl group are selected from halogen.

2. The benzisothiazolinone compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein: The compound is selected from:

3. A pharmaceutical composition, characterized in that The invention comprises the benzisothiazolinone compound or a pharmaceutically acceptable salt thereof according to claim 1 or 2 and a pharmaceutically acceptable carrier and / or excipient.

4. Use of the pharmaceutical composition according to claim 3 in the preparation of an indoleamine 2,3-dioxygenase (IDO1) inhibitor.

5. Use of the pharmaceutical composition according to claim 3 in the preparation of anti-tumor drugs.

6. The use according to claim 5, characterized in that The tumor is a tumor with high expression of indoleamine 2,3-dioxygenase IDO1.

7. The use according to claim 5, characterized in that The tumors are cervical cancer and non-small cell lung cancer.

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