CN114539110B

CN114539110B - HDAC inhibitor containing RAS/RAF protein interfering group and preparation method thereof

Info

Publication number: CN114539110B
Application number: CN202011345547.7A
Authority: CN
Inventors: 苟少华; 夏升进; 刘执坤
Original assignee: Southeast University
Current assignee: Southeast University
Priority date: 2020-11-26
Filing date: 2020-11-26
Publication date: 2023-02-14
Anticipated expiration: 2040-11-26
Also published as: WO2022110958A1; CN114539110A

Abstract

The invention relates to an HDAC inhibitor containing RAS/RAF protein interfering groups and a preparation method thereof, wherein the HDAC inhibitor is based on known 2,4, 6-trimethoxyphenyl-4-methoxy-3-aminobenzyl sulfone taking RAS/RAF as a target point as a cap group, hydroximic acid or benzamide structural units taking Histone Deacetylase (HDAC) as a target point are zinc ion binding groups, and are connected through linear groups to obtain the HDAC inhibitor containing the RAS/RAF protein interfering groups so as to obtain high-efficiency and low-toxicity antitumor drugs; the invention also discloses a preparation method of the compounds and application of the compounds in preparing antitumor drugs.

Description

HDAC inhibitor containing RAS/RAF protein interfering group and preparation method thereof

Technical Field

The invention specifically relates to a method for preparing an HDAC inhibitor containing RAS/RAF protein interfering groups by using a 2,4, 6-trimethoxyphenyl-4-methoxy-3-aminobenzyl sulfone structural unit taking RAS/RAF as a target as a capping group and hydroximic acid or benzamide structural unit taking Histone Deacetylase (HDAC) as a target as a zinc ion binding group through linear group connection, so as to obtain an anti-tumor drug with high efficiency and low toxicity; the invention belongs to a preparation method of the compounds in medicinal chemistry and the application field of the compounds in the preparation of antitumor drugs.

Background

As a member of guanine nucleotide binding proteins, RAS proteins and related proteins play key roles in numerous cell regulatory processes, such as cell proliferation and differentiation, intracellular intercellular transport, intracellular oxidase production, cytoskeleton regulation, and transmembrane transmission of extracellular information. RAS, with the help of extracellular signals, acts as a molecular switch regulating GDP/GTP conversion, leading to the activation of several downstream cascades including the RAF-MEK-ERK pathway. Upon extracellular stimulation, RAS switches from an active GTP-binding signal state to an inactive GTP-binding signal state using its inherent GTPase function. Since mutations frequently occur in the three subtypes of the RAS family (HRAS, NRAS and KRAS), mutations in the RAS gene decrease GTPase activity or increase the insensitivity of GAPs, which in turn activate RAS proteins, leading to uncontrolled cell growth leading to carcinogenesis. These facts indicate that RAS plays an important role in the development and progression of human cancers, and is an important target for cancer treatment.

HDACs are a key enzyme catalyzing the deacetylation of lysine. Research shows that it is closely related to the biological processes of gene transcription, cell proliferation, angiogenesis, migration, differentiation, metastasis and the like, and has an over-expression phenomenon in various tumor cells of rectal cancer, gastric cancer, liver cancer, breast cancer, lung cancer and the like. Therefore, HDACs have become important targets in the design of antitumor drugs. Currently, there are 18 HDACs known, which can be subdivided into four categories based on their sequence homology: class I HDACs, including HDACs 1, 2, 3, and 8; class II HDACs, comprising HDACs 4, 5, 7 and 9 of class IIa and HDACs 6 and 10 of class IIb; class III HDACs comprising sir2 associated enzymes (sirtuins); class IV HDACs, comprising HDAC11. Generally, the HDAC inhibitor pharmacophore can be divided into three parts: (1) a cap group for identifying the HDAC active pocket; (2) A zinc ion binding group, generally divided into two types, namely benzamide and hydroxamic acid, for chelating zinc ions at the bottom of the HDAC catalytic pocket; (3) A linking group for linking the zinc ion binding group to the capping group. To date, several HDAC inhibitors have been marketed for cancer therapy, such as Vorinostat (SAHA), belinostat (pxd-101), panobinostat (LBH 589), and Tacedinaline (CI 994), among others. However, the marketed HDAC inhibitors have not ideal therapeutic effect on solid tumors and generate various side effects, limiting their clinical application scope. Therefore, the development of novel HDAC inhibitors is very beneficial as antitumor agents.

Disclosure of Invention

The technical problem is as follows: the invention aims to obtain an HDAC inhibitor containing RAS/RAF protein interfering groups by using a 2,4, 6-trimethoxyphenyl-4-methoxy-3-aminobenzyl sulfone structural unit which takes RAS/RAF as a target and is reported in the literature as a cap group and a benzamide or hydroximic acid structural unit which takes HDAC as a target as a zinc ion binding group through linear group connection; the invention also discloses a preparation method of the compound.

The technical scheme is as follows: the invention relates to an HDAC inhibitor containing RAS/RAF protein interfering group, which is based on known 2,4, 6-trimethoxyphenyl-4-methoxy-3-aminobenzyl sulfone (9) taking RAS/RAF as a target point as a cap group, and has the structure shown in formula 1,

introducing a benzamide or hydroximic acid structural unit into the structure of the compound through a connecting group to obtain a compound shown as a formula 2, namely an HDAC inhibitor which is divided into 4 compounds of I-A, I-B, I-C and I-D which are representative below,

in formula 2, n =1-5.

The present invention provides a method for preparing an HDAC inhibitor containing RAS/RAF protein interfering group, wherein HDAC inhibitor I-A is prepared according to the following reaction formula 3-1:

wherein TBTU represents 2- (1H-benzotriazol L-1-yl) -1, 3-tetramethyluronium tetrafluoroborate, et ₃ N represents triethylamine, and DMF represents N, N-dimethylformamide.

The synthesis of the HDAC inhibitor I-A specifically adopts the following steps:

dissolving compound 13, 14 or 15 and 1.0-1.5 equivalent of TBTU in anhydrous DMF, stirring at room temperature for 5-10min, adding 1.0-1.5 equivalent of Et ₃ N, adding 1.0-2.0 equivalent of o-phenylenediamine, stirring the reaction solution at 25-35 ℃ for 2-4h under the protection of nitrogen, then removing the solvent under reduced pressure, and separating the concentrated solution by silica gel column chromatography, wherein the eluent is DCM and CH ₃ And mixing OH with the solvent to respectively obtain the compound 16, 17 or 18 as a yellowish solid product.

HDAC inhibitors I-B are prepared according to the following reaction scheme 3-2:

The synthesis of HDAC inhibitors I-B specifically employs the following steps:

dissolving compound 13, 14 or 15 and 1.0-1.5 equivalent of TBTU in anhydrous DMF, stirring at room temperature for 5-10min, adding 1.0-1.5 equivalent of Et ₃ N, adding 1.0-2.0 equivalent of hydroxylamine hydrochloride, stirring the reaction solution at 25-35 ℃ for 2-4h under the protection of nitrogen, then removing the solvent under reduced pressure, and separating the concentrated solution by silica gel column chromatography, wherein the eluent is DCM and CH ₃ And mixing OH with a solvent to obtain a light yellow solid product of the compound 19, 20 or 21 respectively.

HDAC inhibitors I-C are prepared according to the following reaction scheme 3-3:

wherein TBTU represents 2- (1H-benzotriazol L-1-yl) -1, 3-tetramethyluronium tetrafluoroborate, et ₃ N represents triethylamine and DCM represents dichloromethane.

The synthesis of HDAC inhibitors I-C specifically employs the following steps:

dissolving compound 28, 29 or 30 and 1.0-1.5 equivalent of HATU in anhydrous DCM, stirring at room temperature for 5-10min, adding 1.0-1.5 equivalent of Et ₃ N, adding 1.0-2.0 equivalent of o-phenylenediamine, stirring the reaction solution at 25-35 ℃ for 2-4h under the protection of nitrogen, then removing the solvent under reduced pressure, and separating the concentrated solution by silica gel column chromatography, wherein the eluent is DCM and CH ₃ OH mixed solvent to give 31, 32 or 33 yellowish solid product.

HDAC inhibitors I-D are prepared according to the following reaction scheme:

wherein TBTU represents the condensing agent 2- (1H-benzotriazol-L-1-yl) -1, 3-tetramethyluronium tetrafluoroborate, et ₃ N represents triethylamine and DCM represents dichloromethane.

The synthesis of HDAC inhibitors I-D specifically employs the following steps:

dissolving compound 28, 29 or 30 and 1.0-1.5 equivalent of TBTU in anhydrous DCM, stirring at room temperature for 5-10min, adding 1.0-1.5 equivalent of Et ₃ N, adding 1.0-2.0 equivalent of hydroxylamine hydrochloride, stirring the reaction solution at 25-35 ℃ for 2-4h under the protection of nitrogen, then removing the solvent under reduced pressure, and separating the concentrated solution by silica gel column chromatography, wherein the eluent is DCM and CH ₃ OH is mixed with the solvent to obtain 34, 35 or 36 light yellow solid products.

Further, the preparation of compound 9 and its derivatives 13, 14, 15, 28, 29 and 30 in formula 1 was carried out according to the reaction formula shown in formula 4.

In formula 4, meOH represents methanol, etOH represents ethanol, et ₃ N represents triethylamine, DMF represents N, N-dimethylformamide, DCM represents dichloromethane, and THF represents tetrahydrofuran; n =1-5 unless otherwise specified.

Compound 9 and its derivatives 13, 14, 15, 28, 29 and 30 were prepared as follows:

1) Compounds 2-9 were synthesized according to literature procedures (j.med.chem.2020, 63, 186-204);

2) Synthesis of compound 10, 11 or 12: monoethyl succinate, monomethyl glutarate or monomethyl adipate were dissolved in DCM and oxalyl chloride was added at 0 ℃ and stirred at rt overnight. Distilling under reduced pressure to remove solvent, dissolving with DCM, adding compound 9, stirring at room temperature for 15min, and dropwise adding Et ₃ N, stirring is continued for 2h. After completion of the reaction, the solution was extracted with DCM, and the organic phase was washed three times with saturated brine, dried over anhydrous sodium sulfate and concentrated to give yellow solid 10, 11 or 12.

3) Synthesis of compound 13, 14 or 15: dissolving Compound 10, 11 or 12 in EtOH H ₂ To the O (2 ₂ O, then stirred overnight. After completion of the reaction, the solvent was distilled off under reduced pressure to obtain a pale yellow solid, which was washed with a small amount of water and filtered to obtain 13, 14 or 15 as a pale yellow solid.

4) Synthesis of compound 25, 26 or 27: dissolving compound 9 in DMF, adding compound bromoethyl acetate (22), bromoethyl propionate (23) or bromoethyl hexanoate (24) at room temperature, and adding K ₂ CO ₃ And KI, and stirring the reaction mixed solution at 70 ℃ for 3 hours under the protection of nitrogen. After completion of the reaction, the solution was extracted with DCM, and the organic phase was washed three times with saturated brine, dried over anhydrous sodium sulfate, and then the solvent was distilled off under reduced pressure to obtain a solid as PE (petroleum ether): EA (ethyl acetate): DCM (dichloromethane) (10.

5) Synthesis of compound 28, 29 or 30: dissolving Compound 25, 26 or 27 in THF H ₂ To the O (2 ₂ O, and then stirred for 24h. After completion of the reaction, the solvent was distilled off under reduced pressure to obtain a pale yellow solid, which was washed with a small amount of water and filtered to obtain 28, 29 or 30 pale yellow solids.

The structures of some representative compounds described herein are shown in Table 1, but these representative compounds do not limit the scope of the present invention.

TABLE 1 Structure of representative Compounds

Has the advantages that: the MTT method is adopted to firstly determine the anticancer activity of the representative compounds on six cancer cells A549, H1975, MCF-7, SGC7901, hepG2 and A431, and the specific result is shown in Table 2. The target compounds 16-21 and 31-36 all have anticancer activity. Of note are compounds 19 and 34 that exhibit strong anti-cancer activity against the cancer cells tested, where the IC of compound 19 ₅₀ Value between 0.084 and 1.68. Mu.M, IC of Compound 34 ₅₀ Values between 0.045 and 0.99. Mu.M are clearly higher than the activity of the parent compound 9. With positive control SAHA (IC) ₅₀ Values between 4.64 and 7.56 μ M) the anticancer activity of compounds 19 and 34 was also significantly higher. Of the six cancer cells tested, compounds 19 and 34 were most cytotoxic to non-small cell lung carcinoma H1975 cells. The structure-activity relationship shows that the anticancer activity of the compound obtained by adopting the longer carbon chain connecting group is weaker than that of the compound obtained by adopting the shorter carbon chain connecting group. In addition, compounds 19-21 and 34-36, which have hydroxamic acid as the zinc ion binding group, have greater antiproliferative activity than compounds 16-18 and 31-33, which have benzamide as the zinc ion binding group. This is probably due to the structural molecular structure of the hydroxamic acidsHigher affinity, more readily binds to zinc ions, resulting in higher cytotoxicity. In addition, the toxicity of these compounds on human normal liver cells LO2 and umbilical vein endothelial cells HUVEC was also determined. While compounds 19 and 34 were more toxic to either cancer cell tested than the positive control drug SAHA to cancer cells, they were less toxic to normal cells than SAHA. This indicates that the obtained HDAC inhibitor has reduced toxicity to normal cells while increasing anti-tumor activity.

We also tested compounds 19 and 34 for their anti-cancer activity against four cancer cells PANC-1, HT29, K562, MV4-11, with the parent compound 9 and HDAC inhibitor SAHA as a positive control, and the results are shown in table 3. Compound 34 exhibited potent anti-cancer activity against cells of the cancer tested, and its IC ₅₀ The value is between 0.022-0.80. Mu.M, which is significantly higher than the activity of parent compound 9 and drug SAHA, and also superior to compound 19.

Based on the above results, we tested compounds 19 and 34 for HDAC activity and the results are shown in table 4. Compounds 19 and 34 have good inhibitory effects on HDAC1, 2, 3, 4,6 and 8, where IC is on HDAC1, 2 and 3 ₅₀ Values in the 8-20nM region, and IC for HDAC4, 6 and 8 ₅₀ Values are in the 60-90nM region. Compounds 19 and 34 showed significantly higher inhibition of HDACs 1, 2 and 3 than HDACs 4 and 6, indicating that they selectively inhibit class I HDACs. Compounds 19 and 34 showed significant inhibitory effects on HDAC3, the IC thereof ₅₀ Values were 9.61nM and 8.97nM, respectively. It is noted that SAHA inhibits HDAC8 activity on a micromolar scale with little inhibition, while compounds 19 and 34 still have moderate inhibition of HDAC8 with IC ₅₀ Values were 89.76nM and 88.64nM, respectively. In addition, compounds 19 and 34 also showed significantly higher inhibitory effects on class II HDACs 4 and 6 than SAHA. In conclusion, the introduction of highly reactive cap-based structural groups contributes to the enhancement of inhibition of HDAC.

We tested the effect of compounds 19 and 34 on RAS and RAF protein expression using co-immunoprecipitation experiments and immunoblot analysis, and the results are shown in figure 1. In the input group, cells treated with 19 and 34 showed no significant change in RAS protein expression, whereas P-RAF (C-RAFS) ^ser338 ) The expression of the protein is obviously down-regulated. In the IP group, cells treated with 19 and 34 had only slight down-regulation of RAS protein expression, but P-RAF (C-RAFS) ^ser338 ) The expression of the protein is obviously reduced, the down-regulation effect is better than that of the positive control compound 9, and SAHA has no influence on the expression of RAS and RAF proteins. The results indicate that RAS and RAF are targeting proteins for target compounds 19 and 34, which can block signal transduction between RAS and RAF proteins, resulting in down-regulation of P-RAF protein expression.

The results of in vitro anticancer activity and HDAC activity tests, co-immunoprecipitation and immunoblotting experiments show that the target compound has a remarkable inhibitory effect on some cancer cells, and can be used for preparing medicaments.

Drawings

FIG. 1. Effect of compounds on RAS-RAF protein.

Detailed Description

The invention takes hydroximic acid or benzamide structure units which take Histone Deacetylase (HDAC) as targets as zinc ion binding groups, and the HDAC inhibitors containing RAS/RAF protein interference groups are obtained by linear group connection, so as to obtain high-efficiency and low-toxicity antitumor drugs; the invention also discloses a preparation method of the compounds and application of the compounds in preparing antitumor drugs.

The invention is further illustrated by the following examples, which are not intended to limit the invention. Unless otherwise indicated, some compounds such as compounds 2-9 were prepared using literature methods. The molecular structure of the compound prepared by the method is determined by nuclear magnetic hydrogen spectrum, carbon spectrum and high-resolution mass spectrum.

Preparation of compound 9 and its derivatives 13, 14, 15, 28, 29 and 30

(1) Preparation of Compound 2

Compound 1 (3.0g, 18.0mmol) was dissolved in DMF (30.0 mL), and CH was added ₃ I (3.8g, 26.7mmol) and anhydrous K ₂ CO ₃ (6.2g, 45.0 mmol), and the mixture was stirred at room temperature overnight. The reaction was monitored by TLC. After the reaction was completed, the solvent was distilled off under reduced pressure to obtain a crude product.The crude product was dissolved with DCM and washed three times with saturated brine solution (100 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo to afford a white solid. The yield was 91.5%.

¹ H NMR(600MHz,CDCl ₃ )δ9.95(s,1H),8.36(d,J＝2.1Hz,1H),8.10(dd,J＝8.7,2.1Hz,1H),7.25(d,J＝8.7Hz,1H),4.07(s,3H).

(2) Preparation of Compound 3

Compound 2 (1.01g, 5.58mmol) is dissolved in 20mL MeOH and NaBH is added gradually at 0 deg.C ₄ (0.27g, 7.25mmol) and stirred at the same temperature for 20 minutes. The progress of the reaction was monitored by TLC. After the reaction was completed, 1M HCl solution was added dropwise at 0 ℃ and the solvent was distilled off under reduced pressure to obtain a crude product as a white solid. DCM (300 mL) was then added to dissolve the crude product and washed three times with saturated brine solution (100 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo to afford a white solid. Yield: 100 percent.

¹ H NMR(600MHz,CDCl ₃ )δ7.85(d,J＝2.1Hz,1H),7.54(dd,J＝8.6,2.1Hz,1H),7.08(d,J＝8.6Hz,1H),4.68(s,2H),3.96(s,3H),1.99(s,1H).

(3) Preparation of Compound 4

Compound 3 (600.0 mg, 3.28mmol) was dissolved in 20mL DCM, and PBr was gradually added at 0 deg.C ₃ (1.32g, 4.92mmol) and stirred at the same temperature for 3h, the progress of the reaction was monitored by TLC. After the reaction was complete, 1M NaHCO was added dropwise at 0 deg.C ₃ And (4) reacting the solution. The resulting solid product was dissolved in DCM, washed three times with saturated brine solution (100 mL), and the organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo to give a white solid. The yield was 99.3%.

¹ H NMR(600MHz,CDCl ₃ )δ7.90(d,J＝2.3Hz,1H),7.58(dd,J＝8.6,2.3Hz,1H),7.07(d,J＝8.7Hz,1H),4.47(s,2H),3.97(s,3H).

(4) Preparation of Compound 5

Compound 4 (1g, 4mmol) was dissolved in 20mL MeOH, TGA (467.92mg, 5mmol), and NaOH (337.79mg, 8.53mmol) were added at 0 deg.C, the reaction mixture was stirred at room temperature for 3h, and the progress of the reaction was monitored by TLC. After the reaction was completed, 1M HCl solution was added dropwise at 0 ℃, and the solvent was removed by distillation under reduced pressure to obtain a solid crude product. Washed with a small amount of water and filtered to give a pale yellow solid. The yield was 90.1%.

¹ H NMR(600MHz,DMSO)δ12.64(s,1H),7.83(d,J＝2.2Hz,1H),7.61(dd,J＝8.6,2.3Hz,1H),7.34(d,J＝8.7Hz,1H),3.92(s,3H),3.83(s,2H),3.14(s,2H).

HR-MS(m/z)(ESI):calcd for C ₁₀ H ₁₁ NO ₂ S,[M-H] ^- :256.04；found:256.0498.

(5) Preparation of Compound 6

Compound 5 (514.6mg, 3.8mmol) was dissolved in 20mL of AcOH, and 30% H was added dropwise at room temperature ₂ O ₂ Solution (1.322g, 38.8mmol) and the reaction mixture was stirred at 50 ℃ for 6h and the progress of the reaction was monitored by TLC. After the reaction is completed, the solvent is distilled off under reduced pressure to obtain a crude product. Silica gel chromatography was performed with DCM: meOH (80. The yield was 71.4%.

¹ H NMR(600MHz,DMSO)δ7.92(d,J＝2.2Hz,1H),7.68(dd,J＝8.7,2.2Hz,1H),7.43(d,J＝8.8Hz,1H),4.69(s,2H),4.22(s,2H),3.95(s,3H).

HR-MS(m/z)(ESI):calcd for C ₁₀ H ₁₁ NO ₇ S,[M-H] ^- :288.03；found:288.0324.

(6) Preparation of Compound 8

Compound 6 (514.6mg, 3.8mmol) was dissolved in 30mL of toluene, and piperidine (412. Mu.L, 4.2 mmol), benzoic acid (650mg, 5.2mmol), and compound 7 (1.1g, 5.7mmol) were added in this order, and the mixture was refluxed overnight at 40 ℃ with stirring, and the progress of the reaction was monitored by TLC. After the reaction was complete, the solution was cooled to room temperature and filtered to give the crude product which was recrystallized from MeOH to give a yellow solid. The yield was 36.3%.

¹ H NMR(600MHz,DMSO)δ7.88(d,J＝1.9Hz,1H),7.64(dd,J＝8.7,1.9Hz,1H),7.50(d,J＝15.6Hz,1H),7.39(d,J＝8.7Hz,1H),7.09(d,J＝15.6Hz,1H),6.29(s,2H),4.55(s,2H),3.93(s,3H),3.85(s,9H).

(7) Preparation of Compound 9

Will combine withSubstance 8 (3.60g, 8.6mmol) was dissolved in 20mL EtOH, and H was added sequentially ₂ O(10mL)，NH ₄ Cl (2.27g, 5.2mmol), fe powder (2.86g, 50.1mmol), the reaction mixture was stirred at 70 ℃ for 6h and the progress of the reaction was monitored by TLC. After the reaction was completed, the solid residue was removed by filtration, and the obtained filtrate was distilled under reduced pressure to remove the solvent to obtain a crude product, which was recrystallized from DCM to obtain a yellow solid. The yield was 91.1%.

¹ H NMR(600MHz,DMSO)δ7.59(d,J＝15.7Hz,1H),7.09(d,J＝15.7Hz,1H),6.74(d,J＝8.2Hz,1H),6.63(d,J＝2.1Hz,1H),6.49(dd,J＝8.2,2.1Hz,1H),6.29(s,2H),4.77(s,2H),4.21(s,2H),3.85(s,6H),3.84(s,3H),3.74(s,3H).

HR-MS(m/z)(ESI):calcd for C ₁₉ H ₂₃ NO ₆ S,[M+H] ⁺ :394.13；found:394.1315.

(8) Preparation of Compound 10

Ethyl succinate (919.8mg, 6.3mmol) was dissolved in 20mL of DCM solution, oxalyl chloride (2 mL) was added at 0 deg.C, and the reaction mixture was stirred at room temperature overnight. The solvent was distilled off under reduced pressure to give a colorless oil, which was dissolved in DCM (5 mL), added to a solution of Compound 9 (2.25g, 5.7 mmol) in DCM (15 mL) at 0 ℃ and stirred for 15min, and Et was added dropwise ₃ N (761.7mg, 1048.5. Mu.L), stirring was continued for a further 2h and the progress of the reaction was monitored by TLC. After completion of the reaction, the solution was extracted with DCM (300 mL), and the organic phase was washed three times with saturated aqueous sodium chloride solution (100 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo to give a yellow solid. The yield was 86.2%.

¹ H NMR(600MHz,CDCl ₃ )δ8.35(s,1H),7.87(s,1H),7.84(d,J＝15.6Hz,1H),7.15(dd,J＝8.3,1.8Hz,1H),7.09(d,J＝15.6Hz,1H),6.87(d,J＝8.4Hz,1H),6.08(s,2H),4.20(s,2H),4.16–4.13(m,2H),3.88(s,3H),3.84(s,3H),3.82(s,6H),2.69(dd,J＝7.8,4.4Hz,4H),1.26(t,J＝7.1Hz,3H).

HR-MS(m/z)(ESI):calcd for C ₂₅ H ₃₁ NO ₉ S,[M+H] ⁺ :522.17；found:522.1800.

(9) Preparation of Compound 11

Synthesis of Compound 10 as described aboveOxalyl chloride (2 mL), DCM (20 mL), monomethyl glutarate (6.3 mmol), et ₃ N (1048.5. Mu.L) and compound 9 (5.7 mmol) gave compound 11. Yellow solid, yield 88.3%.

¹ H NMR(600MHz,CDCl ₃ )δ8.37(s,1H),7.84(d,J＝15.6Hz,1H),7.72(s,1H),7.15(dd,J＝8.4,2.0Hz,1H),7.09(s,1H),6.87(d,J＝8.4Hz,1H),6.08(s,2H),4.21(s,2H),3.88(s,3H),3.84(s,3H),3.83(s,6H),3.68(s,3H),2.43(dd,J＝13.7,7.0Hz,4H),2.02(p,J＝7.2Hz,2H).

HR-MS(m/z)(ESI):calcd for C ₂₅ H ₃₁ NO ₉ S,[M-H] ^- :520.17；found:520.1772

(10) Preparation of Compound 12

Synthesis of Compound 10 above, using oxalyl chloride (2 mL), DCM (20 mL), monomethyl adipate (6.3 mmol), et ₃ N (1048.5. Mu.L) and compound 9 (5.7 mmol) gave compound 12. Yellow solid, yield 85.4%.

¹ H NMR(600MHz,CDCl ₃ )δ8.37(s,1H),7.84(d,J＝15.6Hz,1H),7.70(s,1H),7.15(dd,J＝8.4,1.8Hz,1H),7.11(d,J＝15.6Hz,1H),6.87(d,J＝8.4Hz,1H),6.08(s,2H),4.21(s,2H),3.88(s,3H),3.84(s,3H),3.83(s,6H),3.67(s,3H),2.39–2.34(m,4H),1.71(dd,J＝6.9,4.1Hz,4H).

HR-MS(m/z)(ESI):calcd for C ₂₆ H ₃₃ NO ₉ S,[M+H] ⁺ :536.20；found:536.2076.

(11) Preparation of Compound 13

Compound 10 (1773.1mg, 3.4mmol) was dissolved in EtOH: h ₂ To O (2 ₂ O (357mg, 8.5mmol), then stirred overnight at room temperature and the progress of the reaction monitored by TLC. After the reaction was completed, the solvent was distilled off under reduced pressure to obtain a solid crude product. Washed with a small amount of water and filtered to give a pale yellow solid. The yield was 97.0%.

¹ H NMR(600MHz,DMSO)δ12.14(s,1H),9.16(s,1H),8.03(s,1H),7.59(d,J＝15.6Hz,1H),7.11(d,J＝15.6Hz,1H),7.04–6.99(m,2H),6.29(s,2H),4.34(s,2H),2.63(t,J＝6.6Hz,2H),2.47(d,J＝6.6Hz,2H).

HR-MS(m/z)(ESI):calcd for C ₂₃ H ₂₇ NO ₉ S,[M-H] ^- :492.16；found:492.1654.

(12) Preparation of Compound 14

According to the above synthesis method of compound 13, compound 11 was used instead of compound 10 to react, yielding a pale yellow solid with a yield of 91.0%.

¹ H NMR(600MHz,DMSO)δ12.09(s,1H),9.09(s,1H),7.97(s,1H),7.59(d,J＝15.6Hz,1H),7.12(d,J＝15.6Hz,1H),7.04(dd,J＝8.5,1.3Hz,1H),7.01(d,J＝8.5Hz,1H),6.29(s,2H),4.35(s,2H),3.85(s,6H),3.84(s,3H),3.82(s,3H),2.40(t,J＝7.3Hz,2H),2.26(t,J＝7.4Hz,2H),1.81–1.74(m,2H).

HR-MS(m/z)(ESI):calcd for C ₂₄ H ₂₉ NO ₉ S,[M-H] ^- :506.18；found:506.1856.

(13) Preparation of Compound 15

Following the above synthesis of compound 13, using compound 12 instead of compound 10, the reaction gave a pale yellow solid with a yield of 90.5%.

¹ H NMR(600MHz,DMSO)δ12.02(s,1H),9.06(s,1H),7.98(s,1H),7.59(d,J＝15.6Hz,1H),7.12(d,J＝15.7Hz,1H),7.04(dd,J＝8.5,1.2Hz,1H),7.01(d,J＝8.5Hz,1H),6.29(s,2H),4.34(s,2H),3.85(s,6H),3.84(s,3H),3.82(s,3H),2.37(t,J＝6.7Hz,2H),2.23(t,J＝7.0Hz,2H),1.58–1.50(m,4H).

HR-MS(m/z)(ESI):calcd for C ₂₅ H ₃₁ NO ₉ S,[M+H] ⁺ :522.18；found:522.1822.

(14) Preparation of Compound 25

Compound 9 (393.4mg, 1mmol) was dissolved in DMF (30 mL), and Compound 22 (664.0mg, 4mmol), K, was added sequentially at room temperature ₂ CO ₃ (552.0mg, 4mmol) and KI (66.4mg, 0.4mmol), the reaction mixture solution was stirred at 70 ℃ for 3 hours under a nitrogen blanket, and the progress of the reaction was monitored by TLC. After completion of the reaction, the solution was extracted with DCM (300 mL), and the organic phase was washed three times with a saturated saline solution (100 mL), dried over anhydrous sodium sulfate, and distilled under reduced pressure to obtain a solid which was washed with PE: EA: DCM (10Removing agent, separating and purifying by silica gel chromatography to obtain yellow solid. The yield was 80.0%.

¹ H NMR(600MHz,CDCl ₃ )δ7.80(d,J＝15.7Hz,1H),7.26(s,1H),7.04(d,J＝15.7Hz,1H),6.73(d,J＝8.1Hz,1H),6.71(dd,J＝8.1,1.9Hz,1H),6.50(d,J＝1.8Hz,1H),6.08(s,2H),4.22(q,J＝7.1Hz,2H),4.16(s,2H),3.86(s,2H),3.85(s,3H),3.84(s,3H),3.82(s,6H),1.28(t,J＝7.1Hz,3H).

HR-MS(m/z)(ESI):calcd for C ₂₃ H ₂₉ NO ₈ S,[M+H] ⁺ :480.16；found:480.1698.

(15) Preparation of Compound 26

Compound 26 was obtained by the above-mentioned synthesis method of compound 25, using compound 23 instead of compound 22. Yellow solid, yield 82.1%.

¹ H NMR(600MHz,CDCl ₃ )δ7.84(d,J＝15.7Hz,1H),7.06(d,J＝15.7Hz,1H),6.72(d,J＝8.1Hz,1H),6.69(dd,J＝8.1,1.8Hz,1H),6.62(d,J＝1.6Hz,1H),6.09(s,2H),4.17(s,2H),4.16–4.12(m,2H),3.84(s,3H),3.83(s,3H),3.82(s,6H),3.41(t,J＝6.5Hz,2H),2.57(d,J＝6.5Hz,2H),1.26(d,J＝7.1Hz,3H).

HR-MS(m/z)(ESI):calcd for C ₂₄ H ₃₁ NO ₈ S,[M+H] ⁺ :494.18；found:494.1865.

(16) Preparation of Compound 27

Compound 27 was obtained by following the above synthesis procedure for compound 25, substituting compound 24 for compound 22. Yellow solid, yield 84.2%.

¹ H NMR(600MHz,CDCl ₃ )δ7.83(d,J＝15.7Hz,1H),7.05(d,J＝15.7Hz,1H),6.70(d,J＝8.1Hz,1H),6.65(dd,J＝8.1,2.0Hz,1H),6.58(d,J＝1.9Hz,1H),6.08(s,2H),4.22(s,1H),4.16(s,2H),4.14–4.10(m,2H),3.84(s,3H),3.82(s,3H),3.81(s,6H),3.11(t,J＝6.9Hz,2H),2.36(t,J＝7.3Hz,2H),1.91(dd,J＝14.3,7.1Hz,2H),1.24(d,J＝7.1Hz,3H).

HR-MS(m/z)(ESI):calcd for C ₂₇ H ₃₇ NO ₈ S,[M+H] ⁺ :536.23；found:536.2331

(17) Preparation of Compound 28

Compound 25 (719.1mg, 1.5mmol) was dissolved in THF: H ₂ To a mixed solvent of O (2 ₂ O (126mg, 3mmol) and then stirred for 24h, the progress of the reaction was monitored by TLC. After the completion of the reaction, the solution was distilled under reduced pressure to remove the solvent to obtain a pale yellow crude product. The crude product was washed with a small amount of water and filtered to give a pale yellow solid in 89.4% yield.

¹ H NMR(600MHz,DMSO)δ7.55(d,J＝15.7Hz,1H),7.11(d,J＝15.7Hz,1H),6.80(d,J＝8.2Hz,1H),6.58(dd,J＝8.1,1.9Hz,1H),6.42(d,J＝2.0Hz,1H),6.29(s,2H),5.75(s,1H),4.26(s,2H),3.85(s,6H),3.84(s,3H),3.79(s,3H),3.72(s,2H).

HR-MS(m/z)(ESI):calcd for C ₂₁ H ₂₅ NO ₈ S,[2M+H] ⁺ :903.28；found:903.2828

(18) Preparation of Compound 29

Following the above synthetic procedure for compound 28, using compound 26 instead of compound 25, the reaction was carried out to give a pale yellow solid in 87.6% yield.

HR-MS(m/z)(ESI):caled for C ₂₂ H ₂₇ NO ₈ S,[M+H] ⁺ :466.16；found:466.1568

(19) Preparation of Compound 30

Following the above synthetic procedure for compound 28, using compound 27 instead of compound 25, the reaction was carried out to give a pale yellow solid in 88.4% yield.

1H NMR(600MHz,DMSO)δ12.01(s,1H),7.53(d,J＝15.7Hz,1H),7.11(d,J＝15.7Hz,1H),6.75(d,J＝8.2Hz,1H),6.52(dd,J＝8.1,1.9Hz,1H),6.47(d,J＝1.9Hz,1H),6.29(s,2H),4.73(s,1H),4.28(s,2H),3.84(s,9H),3.76(s,3H),2.91(t,J＝6.8Hz,2H),2.17(t,J＝7.4Hz,2H),1.50–1.42(m,4H),1.24(dd,J＝10.3,4.9Hz,2H).

HR-MS(m/z)(ESI):calcd for C ₂₅ H ₃₃ NO ₈ S,[M+H] ⁺ :508.20；found:508.2035.

(II) preparation of target Compound

EXAMPLE 1 preparation of Compound 16

Compound 13 (250.0mg, 0.51mmol) was dissolved in DMF (30 mL), and TBTU (245.6mg, 0.76mmol) and Et were added in this order at room temperature ₃ N (106.4. Mu.L, 0.765 mmol) and o-phenylenediamine (108.0 mg, 1.01mmol) were stirred at 35 ℃ for 2h under nitrogen. After completion of the reaction, the solution was extracted with DCM (300 mL), and the organic phase was washed three times with saturated aqueous sodium chloride solution (100 mL), dried over anhydrous sodium sulfate, distilled under reduced pressure, and the resulting solid was isolated and purified by silica gel chromatography with DCM: meOH (60.

¹ H NMR(600MHz,DMSO)δ9.20(s,1H),9.19(s,1H),8.02(s,1H),7.60(d,J＝15.6Hz,1H),7.15(dd,J＝7.7,0.9Hz,1H),7.12(d,J＝15.6Hz,1H),7.06–7.03(m,1H),7.01(d,J＝8.4Hz,1H),6.89(td,J＝8.0,1.4Hz,1H),6.71(dd,J＝8.0,1.2Hz,1H),6.53(td,J＝7.7,1.2Hz,1H),6.29(s,2H),4.98(s,2H),4.35(s,2H),3.85(s,6H),3.84(s,3H),3.82(s,3H),2.73(t,J＝6.9Hz,2H),2.63(t,J＝7.0Hz,2H). ¹³ C NMR(151MHz,DMSO)δ171.12,170.91,164.11,161.43(C×2),149.88,142.49,133.38,127.72,127.45,126.31,126.00,124.78,124.01,123.84,121.10,116.56,116.15,111.26,103.05,91.40(C×2),60.34,56.53(C×2),56.22,56.09,32.01,31.36.

HR-MS(m/z)(ESI):calcd for C ₂₉ H ₃₃ N ₃ O ₈ S,[M+H] ⁺ :584.20；found:584.2095.

EXAMPLE 2 preparation of Compound 17

The above synthesis of compound 16 was followed, using compound 14 instead of compound 13, to give a pale yellow solid in 38.4% yield.

¹ H NMR(600MHz,DMSO)δ9.13(s,1H),9.12(s,1H),8.03(s,1H),7.60(d,J＝15.6Hz,1H),7.19-7.17(m,1H),7.13(d,J＝15.6Hz,1H),7.05(dd,J＝8.4,1.2Hz,1H),7.01(d,J＝8.5Hz,1H),6.91–6.88(m,1H),6.72(dd,J＝8.0,1.2Hz,1H),6.55(td,J＝7.8,1.2Hz,1H),6.29(s,2H),4.91(s,2H),4.35(s,2H),3.86(s,6H),3.84(s,3H),3.82(s,3H),2.45(t,J＝7.2Hz,2H),2.38(t,J＝7.4Hz,2H),1.88(dd,J＝14.6,7.3Hz,2H). ¹³ C NMR(151MHz,DMSO)δ171.45,171.26,164.12,161.43(C×2),150.00,142.32,133.41,127.61,127.70,126.20,125.87,124.99,124.03,124.02,121.08,116.65,116.35,111.26,103.05,91.41(C×2),60.33,56.53(C×2),56.22,56.08,35.91,35.52,21.84.

HR-MS(m/z)(ESI):calcd for C ₃₀ H ₃₅ N ₃ O ₈ S,[M+H] ⁺ :598.20；found:598.2320.

EXAMPLE 3 preparation of Compound 18

The above synthesis of compound 16 was followed, using compound 15 instead of compound 13, to give a pale yellow solid in 37.6% yield.

¹ H NMR(600MHz,DMSO)δ9.14(s,1H),9.07(s,1H),8.01(s,1H),7.60(d,J＝15.6Hz,1H),7.16(dd,J＝7.8,0.9Hz,1H),7.13(d,J＝15.6Hz,1H),7.05(d,J＝8.2Hz,1H),7.01(d,J＝8.5Hz,1H),6.91–6.88(m,1H),6.73(dd,J＝7.9,1.0Hz,1H),6.57–6.53(m,1H),6.29(s,2H),4.92(s,2H),4.35(s,2H),3.85(s,6H),3.84(s,3H),3.82(s,3H),2.42(s,2H),2.34(d,J＝6.0Hz,2H),1.63(s,4H). ¹³ C NMR(151MHz,DMSO)δ171.67,171.52,164.11,161.42(C×2),150.02,142.21,133.39,127.69,127.52,126.19,125.78,125.02,124.10,124.02,121.09,116.77,116.44,111.25,103.05,91.40(C×2),60.31,56.52(C×2),56.21,56.08,36.36,36.12,25.52.25.43

HR-MS(m/z)(ESI):calcd for C ₃₁ H ₃₇ N ₃ O ₈ S,[M+H] ⁺ :612.20；found:612.2515.

EXAMPLE 4 preparation of Compound 19

Compound 13 (208.0mg, 0.42mmol) was dissolved in DMF (30 mL), and TBTU (175.3mg, 0.55mmol) and Et were added in this order at room temperature ₃ N (350.6. Mu.L, 2.52 mmol) and NH ₂ OH & HCl (116.7mg, 1.68mmol), the reaction mixture was stirred at 35 ℃ for 2h under nitrogen and the progress of the reaction was monitored by TLC. After completion of the reaction, the solution was extracted with DCM (300 mL), and the organic phase was washed three times with a saturated saline solution (100 mL), dried over anhydrous sodium sulfate, and evaporated under reduced pressureThe resulting solid was further isolated and purified by silica gel chromatography using DCM: meOH (50. The yield was 60.9%.

¹ H NMR(600MHz,DMSO)δ10.43(s,1H),9.17(s,1H),8.74(s,1H),8.03(s,1H),7.59(d,J＝15.6Hz,1H),7.11(d,J＝15.6Hz,1H),7.01(t,J＝7.5Hz,2H),6.29(s,2H),4.34(s,2H),3.85(s,6H),3.84(s,3H),3.82(s,3H),2.63(t,J＝7.1Hz,2H),2.26(t,J＝7.1Hz,2H). ¹³ C NMR(151MHz,DMSO)δ170.77,168.91,164.11,161.42(C×2),149.77,133.38,127.73,127.40,124.62,123.99,121.07,111.22,103.03,91.40(C×2),60.35,56.53(C×2),56.22,56.08,31.82,28.01.

HR-MS(m/z)(ESI):calcd for C ₂₃ H ₂₈ N ₂ O ₉ S,[M+H] ⁺ :509.16；found:509.1625.

EXAMPLE 5 preparation of Compound 20

Following the above synthesis of compound 19, using compound 14 instead of compound 13, a yellow solid was obtained in 62.3% yield.

¹ H NMR(600MHz,DMSO)δ10.40(s,1H),9.08(s,1H),8.71(d,J＝1.1Hz,1H),8.00(s,1H),7.59(d,J＝15.6Hz,1H),7.12(d,J＝15.6Hz,1H),7.04(dd,J＝8.5,1.1Hz,1H),7.01(d,J＝8.5Hz,1H),6.29(s,2H),4.35(s,2H),3.85(s,6H),3.84(s,3H),3.82(s,3H),2.37(t,J＝7.3Hz,2H),2.00(t,J＝7.4Hz,2H),1.79–1.76(m,2H). ¹³ C NMR(151MHz,DMSO)δ171.30,169.24,164.12,161.43(C×2),150.00,133.39,127.90–127.69,127.60,124.98,124.03,121.07,111.26,103.04,91.41(C×2),60.30,56.53(C×2),56.23,56.09,35.86,32.15,21.83.

HR-MS(m/z)(ESI):calcd for C ₂₄ H ₃₀ N ₂ O ₉ S,[M+H] ⁺ :523.16；found:523.1737

EXAMPLE 6 preparation of Compound 21

Following the above synthesis of compound 19, using compound 15 instead of compound 13, the reaction gave a yellow solid in 63.6% yield.

¹ H NMR(600MHz,DMSO)δ10.38(s,1H),9.05(s,1H),8.70(s,1H),7.99(s,1H),7.59(d,J＝15.6Hz,1H),7.12(d,J＝15.6Hz,1H),7.02(dd,J＝19.4,8.3Hz,2H),6.29(s,2H),4.34(s,2H),3.85(s,6H),3.84(s,3H),3.82(s,3H),2.37(s,2H),1.97(s,2H),1.52(s,4H). ¹³ C NMR(151MHz,DMSO)δ171.63,169.46,164.12,161.42(C×2),150.01,133.39,127.60,125.00,124.01,121.08,111.25,103.04,91.40(C×2),60.30,56.53(C×2),56.16,36.26,32.64,25.35.

HR-MS(m/z)(ESI):calcd for C ₂₅ H ₃₂ N ₂ O ₉ S,[M+H] ⁺ :537.16；found:537.1925.

EXAMPLE 7 preparation of Compound 31

Compound 28 (225.6 mg,0.5 mmol) was dissolved in DMF (30 mL), and HATU (247.1mg, 0.65mmol), et and then added successively at room temperature ₃ N (90.3. Mu.L, 0.65 mmol) and o-phenylenediamine (162.15mg, 1.5mmol) were stirred at 35 ℃ for 2h under nitrogen protection, and the progress of the reaction was monitored by TLC. After completion of the reaction, the solution was extracted with DCM (300 mL), and the organic phase was washed three times with saturated aqueous sodium chloride solution (100 mL), dried over anhydrous sodium sulfate, distilled under reduced pressure, and the resulting solid was further isolated and purified by silica gel chromatography with DCM: meOH (100. The yield was 37.0%.

¹ H NMR(600MHz,DMSO)δ9.18(s,1H),7.60(d,J＝15.7Hz,1H),7.18(dd,J＝7.8,1.2Hz,1H),7.11(d,J＝15.7Hz,1H),6.92–6.89(m,1H),6.83(d,J＝8.2Hz,1H),6.72(dd,J＝8.0,1.2Hz,1H),6.61(dd,J＝8.1,1.8Hz,1H),6.54(dd,J＝7.5,1.2Hz,1H),6.52(d,J＝1.9Hz,1H),6.29(s,2H),5.33(s,1H),4.87(s,2H),4.29(s,2H),3.86(s,2H),3.85(s,6H),3.84(s,3H),3.82(s,3H). ¹³ C NMR(151MHz,DMSO)δ169.00,164.08,161.40(C×2),147.10,142.66,137.68,133.22,126.56,126.18,124.15,119.90,116.61,116.18,112.72,109.95,91.44(C×2),60.93,56.52(C×2),56.08,55.95,47.03.

HR-MS(m/z)(ESI):calcd for C ₂₇ H ₃₁ N ₃ O ₇ S,[M+H] ⁺ :542.20；found:542.2022

EXAMPLE 8 preparation of Compound 32

The above synthesis of compound 31 was followed using compound 29 instead of compound 28 to give a pale yellow solid in 38.4% yield.

¹ H NMR(600MHz,DMSO)δ9.19(s,1H),7.59(d,J＝15.7Hz,1H),7.14(dd,J＝15.8,8.2Hz,2H),6.92–6.89(m,1H),6.78(d,J＝8.2Hz,1H),6.72(d,J＝7.9Hz,1H),6.61(d,J＝1.5Hz,1H),6.58–6.53(m,2H),6.29(s,2H),4.94(d,J＝40.8Hz,3H),4.30(s,2H),3.85(s,6H),3.83(s,3H),3.77(s,3H),3.29(t,J＝6.4Hz,2H),2.62(t,J＝6.7Hz,2H). ¹³ C NMR(151MHz,DMSO)δ170.46,164.05,161.38(C×2),147.02,142.46,138.00,133.14,126.39,126.00,124.12,123.66,121.90,119.27,116.60,116.26,112.62,109.82,103.04,91.38(C×2),60.90,56.50(C×2),56.06,55.85,39.97,35.67.

HR-MS(m/z)(ESI):calcd for C ₂₈ H ₃₃ N ₃ O ₇ S,[M+H] ⁺ :556.22；found:556.2161.

EXAMPLE 9 preparation of Compound 33

Following the above synthesis of compound 31, using compound 30 instead of compound 28, the reaction was carried out to give a pale yellow solid in 39.3% yield.

¹ H NMR(600MHz,DMSO)δ9.08(s,1H),7.55(d,J＝15.7Hz,1H),7.16(dd,J＝7.8,1.1Hz,1H),7.12(d,J＝15.7Hz,1H),6.89(t,J＝7.6Hz,1H),6.75(d,J＝8.1Hz,1H),6.72(d,J＝9.1Hz,1H),6.55–6.52(m,2H),6.50(d,J＝1.6Hz,1H),6.29(s,2H),4.79(d,J＝76.4Hz,3H),4.28(s,2H),3.84(s,6H),3.84(s,3H),3.76(s,3H),2.95(t,J＝7.0Hz,2H),2.30(t,J＝7.4Hz,2H),1.58–1.53(m,4H),1.34–1.30(m,2H). ¹³ C NMR(151MHz,DMSO)δ171.57,164.05,161.39(C×2),146.79,142.29,138.24,133.19,126.15,125.72,124.14,124.09,121.97,118.76,116.69,116.39,112.23,109.74,103.10,91.40(C×2),60.97,56.49(C×2),56.07,55.80,43.08,36.26,28.80,26.83,25.59.

HR-MS(m/z)(ESI):calcd for C ₃₁ H ₃₉ N ₃ O ₇ S,[M+H] ⁺ :598.26；found:598.2642.

EXAMPLE 10 preparation of Compound 34

Compound 28 (189.6mg, 0.42mmol) was dissolved in DCM (30 mL) and TBTU (175.3mg, 0.55mmol) and Et were added sequentially at room temperature ₃ N (350.6. Mu.L, 2.52 mmol) and NH ₂ OHHCl (116.7mg, 1.68mmol) and the reaction mixture was stirred at 35 ℃ for 2h under nitrogen and the progress of the reaction was monitored by TLC. After the reaction was completed, the solution was extracted with DCM (300 mL), and the organic phase was washed three times with saturated aqueous saline solution (100 mL), dried over anhydrous sodium sulfate, distilled under reduced pressure, and the resulting solid was further separated and purified by silica gel chromatography using DCM: meOH (60. The yield was 52.6%.

¹ H NMR(600MHz,DMSO)δ10.63(s,1H),8.90(s,1H),7.60(s,1H),7.10(d,J＝15.6Hz,1H),6.81(d,J＝8.2Hz,1H),6.60(d,J＝8.0Hz,1H),6.46(d,J＝23.1Hz,1H),6.29(s,2H),5.09(s,1H),4.27(s,2H),3.85(s,6H),3.85(s,3H),3.80(s,3H),3.55(s,2H). ¹³ C NMR(151MHz,DMSO)δ166.93,164.08,161.41(C×2),147.10,137.63,133.20,124.10,121.75,119.93,112.76,109.89,103.05,91.43(C×2),60.90,56.51(C×2),56.08,55.92,44.40.

HR-MS(m/z)(ESI):calcd for C ₂₁ H ₂₆ N ₂ O ₈ S,[M+H] ⁺ :467.14；found:467.1579.

EXAMPLE 11 preparation of Compound 35

Following the above synthesis of compound 31, using compound 29 instead of compound 28, the reaction was carried out to give a pale yellow solid in 51.2% yield.

¹ H NMR(600MHz,DMSO)δ10.49(s,1H),8.82(s,1H),7.58(d,J＝15.7Hz,1H),7.13(d,J＝15.7Hz,1H),6.78(d,J＝8.0Hz,1H),6.56(d,J＝9.2Hz,2H),6.30(s,2H),4.92(s,1H),4.30(s,2H),3.86(d,J＝3.0Hz,9H),3.77(s,3H),3.19(t,J＝6.6Hz,2H),2.26(t,J＝6.8Hz,2H). ¹³ C NMR(151MHz,DMSO)δ168.29,164.04,161.37(C×2),146.92,137.81,133.10,124.08,121.86,119.25,112.47,109.77,103.00,91.37(C×2),60.78,56.50(C×2),56.07,55.81,39.87,32.30.

HR-MS(m/z)(ESI):calcd for C ₂₂ H ₂₈ N ₂ O ₈ S,[M+H] ⁺ :481.16；found:481.1663.

EXAMPLE 12 preparation of Compound 36

Following the above synthesis of compound 31, using compound 30 instead of compound 28, the reaction gave a pale yellow solid with 50.2% yield.

¹ H NMR(600MHz,DMSO)δ10.36(s,1H),8.70(s,1H),7.55(d,J＝15.7Hz,1H),7.12(d,J＝15.7Hz,1H),6.75(d,J＝8.2Hz,1H),6.52(dd,J＝8.1,1.7Hz,1H),6.48(d,J＝1.7Hz,1H),6.29(s,2H),4.72(s,1H),4.28(s,2H),3.85(s,9H),3.76(s,3H),2.92(dd,J＝12.7,6.6Hz,2H),1.93(t,J＝7.4Hz,2H),1.47(ddt,J＝22.9,15.2,7.4Hz,4H),1.24–1.20(m,2H). ¹³ C NMR(151MHz,DMSO)δ169.53,164.05,161.38(C×2),146.75,138.20,133.16,124.09,121.94,118.74,112.17,109.69,103.04,91.36(C×2),60.90,56.48(C×2),56.08,55.77,43.05,32.76,28.63,26.76,25.42.

HR-MS(m/z)(ESI):calcd for C ₂₅ H ₃₄ N ₂ O ₈ S,[M+H] ⁺ :523.21；found:523.2129.

(III) in vitro cytotoxic Activity assay of Compounds

The experimental method comprises the following steps: the cells in logarithmic growth phase are counted and inoculated into 96-well culture plates with about 8000-10000 cells per well. Culturing overnight, and administering after cell adherence, wherein an administration group and a control group are respectively arranged. The target compounds to be tested were formulated as stock solutions in DMSO and diluted to a range of concentrations in cell culture medium just prior to use, with the final DMSO concentration not exceeding 4% o (the same experiment below). Each concentration was provided with 3 multiple wells. After adding the drug, the mixture is cultured for 72h, 20 mu L of MTT with the concentration of 5mg/mL is added, the mixture is incubated for 4h at 37 ℃, the supernatant is discarded, and 150 mu L of DMSO is added for dissolution. Measuring the OD value of each hole by using an enzyme-labeling instrument at 490 nm wavelength, calculating the inhibition rate, and calculating the IC by using a concentration-inhibition rate curve ₅₀ The value is obtained.

Experimental example 1.

The antiproliferative activity of the target compounds on non-small cell lung cancer cells A549 and H1975, gastric cancer cell SGC-7901, liver cancer cell HepG2, breast cancer cell MCF-7 and epidermal cancer cell A431 and their toxicity to human normal liver cell LO2 and umbilical vein endothelial cell HUVEC were tested, and the parent compound 9 and HDAC inhibitor SAHA were used as positive controls. Observing the inhibition of the compound on the growth of tumor cells under different concentrations, and calculating the inhibition rate and IC thereof ₅₀ The cytotoxic activity of the drug was evaluated and the results are shown in table 2.

TABLE 2 in vitro anticancer Activity and toxicity to Normal cells of Compounds

Experimental example 2.

The antiproliferative activity of the compounds of interest 19 and 34 on pancreatic cancer cells PANC-1, colon cancer cells HT29, chronic myelogenous leukemia cancer cells K562 and human myelomonocytic leukemia cells MV4-11 were tested, the parent compound 9 and the HDAC inhibitor SAHA as positive controls. Observing the inhibition of the compound on the growth of tumor cells under different concentrations, and calculating the inhibition rate and IC thereof ₅₀ The cytotoxic activity of the drugs was evaluated and the results are shown in Table 3.

TABLE 3 IC of Compounds 19 and 34 on some cancer cells ₅₀ Value of

(IV) detection of inhibitory Activity of Compounds on HDAC

The experimental method comprises the following steps: the levels of human histone deacetylases (HDAC 1, 2, 3, 4,6, 8) in the samples were determined using a double antibody sandwich method according to the human histone deacetylase HDAC kit instructions (manufactured by shanghai seoul industries ltd.). And blank holes (the blank reference holes are not added with the sample and the enzyme labeling reagent, and the rest steps are operated in the same way), standard holes and sample holes to be detected are respectively arranged. Different concentrations of drugs and HDAC subtype proteins were mixed and applied to the bottom of the wells of the ELISA plate, and the plate was sealed with a sealing plate and incubated at 37 ℃ for 30min. After washing with the washing solution 5 times, 50. Mu.L of the enzyme-labeled reagent was added to each well except for blank wells. The plates were then sealed and incubated at 37 ℃ for 30min and washed 5 times. Then adding 50 mu L of color developing agent A into each hole, then adding 50 mu L of color developing agent B, lightly shaking and uniformly mixing, and developing in a dark place at 37 DEG CColor 10min. Finally, 50. Mu.L of stop solution was added to each well to stop the reaction. The blank wells were zeroed and the absorbance (OD) of each well was measured sequentially at a wavelength of 450nm using a microplate reader. And calculating the inhibition rate, and making concentration-inhibition rate curve to calculate IC ₅₀ The value is obtained.

Experimental example 3.

The inhibition of six HDAC isoforms, HDAC1, HDAC2, HDAC3, HDAC4, HDAC6, HDAC8, by the target compounds 19 and 34 was tested. Using HDAC inhibitor SAHA as positive control, observing the inhibition of the compound on HDAC enzyme under different concentrations, and calculating the inhibition rate and IC thereof ₅₀ The enzyme activity of the compounds was evaluated and the results are shown in Table 4.

TABLE 4 inhibitory Activity of Compounds on human Histone deacetylase HDAC

(V) detection of the Effect of Compounds on RAS and RAF proteins

The experimental method comprises the following steps: the effect of compounds on RAS and RAF proteins was tested using experimental methods of co-immunoprecipitation and immunoblot detection. Counting H1975 cells in logarithmic growth phase, inoculating into 6-well culture plate with about 100000-1000000 cells per well, culturing overnight, and administering for 24 hr after cell attachment. Digesting cells, transferring the cells into a centrifuge tube, adding a precooled cell lysate, placing the cell lysate on a shaking table platform at 4 ℃, and carrying out mild oscillation for 15min; then, the mixture was centrifuged at 10,000rpm at 4 ℃ for 5min, and the supernatant was transferred to a new centrifugal tube. At this time, the supernatants were divided into two groups, one group was IP group, RAS antibody was added to the supernatant, and the mixture was shaken slowly at 4 ℃ overnight. Add well resuspended protein G agarose, 4 ℃ and shake slowly for 4h. Centrifuge at 2500rpm for 5min, carefully aspirate off the supernatant and wash the pellet 5 times with PBS. After washing, removing supernatant, adding 1xSDS-PAGE electrophoresis loading buffer for resuspension, mixing uniformly, carrying out water bath at 95 ℃ for 10min to obtain a sample protein, and detecting by an immunoblotting method. And the other group is used as an input group, 1xSDS-PAGE electrophoresis loading buffer solution is directly added into the supernatant for resuspension, after uniform mixing, water bath at 95 ℃ is carried out for 10min to obtain a sample protein, and then the detection is carried out by an immunoblotting experimental method.

Firstly, preparing separation gel and concentrated gel, after the gel is formed, sucking a proper amount of sample protein and adding the sample protein into sample holes, adding a pre-dyed protein Marker into the holes beside the sample, and adding 1x SDS loading buffer into the holes without adding sample supernatant to keep the gel surface balance. The power supply is turned on, the voltage is set to 60V, electrophoresis is started, and after the protein sample enters the separation gel, the voltage can be increased to 120V. After the electrophoresis is finished, the PVDF membrane is started to be rotated, the current flow is set to be 1.1A, the voltage is set to be 25V, and the half-dry rotation is carried out for 30min. After the membrane transfer was complete, the PVDF membrane was removed and labeled, and washed 3 times with TBST solution for 10min each. Then putting the PVDF membrane into a plate, adding a confining liquid, and oscillating for 1.5-2h by a shaking table. After blocking, washing with TBST solution. The membrane was then placed in an incubation cassette containing the primary antibody and incubated overnight at 4 ℃ with shaking. The next day, shake at room temperature for 30min, remove primary antibody by suction, and wash with TBST 3 times. And (5) incubating the mixture with the second antibody, and oscillating the mixture at room temperature in a shaking table for reaction for 2 hours. After the secondary antibody reaction was completed, the reaction mixture was washed with TBST. Mixing A and B liquids in the EcL chemiluminescence kit according to l: l equal volume, and preparing into working solution for later use. Taking out the PVDF film from TBST, dripping a proper amount of working solution, and developing and imaging by using G, BOX chemiXR5

Experimental example 4.

The effect of compounds 19 and 34 on RAS and RAF proteins was tested with compound 9 and SAHA as positive controls. The effect of different compounds on RAS and RAF protein expression at equivalent concentrations was observed and the results are shown in figure 1.

Claims

1. An HDAC inhibitor containing RAS/RAF protein interfering group is characterized in that the HDAC inhibitor is based on known 2,4, 6-trimethoxyphenyl-4-methoxy-3-aminobenzyl sulfone (9) taking RAS/RAF as a target as a cap group, and the structure of the HDAC inhibitor is shown as formula 1,

in formula 2, n =1-5.

2. A method for preparing an HDAC inhibitor comprising RAS/RAF protein interfering group as claimed in claim 1, wherein HDAC inhibitor I-a is prepared according to the following reaction scheme 3-1:

3. The method of claim 2, wherein the step of synthesizing HDAC inhibitor I-a comprises the steps of:

4. A method for preparing an HDAC inhibitor comprising RAS/RAF protein interfering group according to claim 1, wherein HDAC inhibitor I-B is prepared according to the following reaction scheme 3-2:

5. The method of claim 4, wherein the step of synthesizing HDAC inhibitor I-B comprises:

6. A method of preparing an HDAC inhibitor comprising RAS/RAF protein interfering group as claimed in claim 1, wherein HDAC inhibitor I-C is prepared according to the following reaction scheme 3-3:

7. A method according to claim 6, wherein the HDAC inhibitor I-C is synthesized by the steps of:

dissolving compound 28, 29 or 30 with 1.0-1.5 equivalents of TBTU in anhydrous DCMStirring at room temperature for 5-10min, adding Et 1.0-1.5 equivalent ₃ N, adding 1.0-2.0 equivalent of o-phenylenediamine, stirring the reaction solution at 25-35 ℃ for 2-4h under the protection of nitrogen, then removing the solvent under reduced pressure, and separating the concentrated solution by silica gel column chromatography, wherein the eluent is DCM and CH ₃ OH is mixed with the solvent to obtain 31, 32 or 33 light yellow solid products.

8. A method of preparing an HDAC inhibitor comprising RAS/RAF protein interfering groups as claimed in claim 1, wherein HDAC inhibitors I-D are prepared according to the following reaction scheme:

9. The method of claim 8, wherein the step of synthesizing HDAC inhibitors I-D comprises the steps of: