Synthesis and application of bifunctional molecules targeting HDAC / PD-L1 based on o-phenylenediamine and hydrazide

Abstract

The present invention relates to the field of medicinal chemistry, specifically to a bifunctional molecule targeting HDAC / PD-L1 using o-phenylenediamine and hydrazide as ZBGs, its preparation method, and application. The bifunctional molecule is a compound of formula (I) and (II). This compound can simultaneously inhibit HDAC3 activity and degrade PD-L1 protein in melanoma cells, thereby inhibiting cancer cell proliferation, and is expected to be used in cancer immunotherapy. #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the field of medicinal chemistry, and specifically relates to an o-phenylenediamine and hydrazide-based bifunctional molecule targeting HDAC / PD-L1 and its use. Background Art

[0002] The occurrence of cancer is associated with genomic changes and epigenetic modifications. Among them, histone modifications are considered promising targets for epigenetic drug discovery. Among different histone modifications, histone acetylation is the most common epigenetic dysregulation process in cancer. Histone acetylation is regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Generally speaking, HDACs can tightly bind to negatively charged DNA, thereby affecting various cellular processes such as transcription, cell cycle and cellular metabolism. So far, eighteen HDACs subtypes have been identified and divided into four major categories.

[0003] To date, five HDAC inhibitors have been approved for marketing. However, most current HDAC inhibitors are pan-inhibitors, and adverse reactions such as bone marrow suppression and cardiotoxicity have limited their use to a certain extent. Therefore, the development of subtype-selective HDAC inhibitors is expected to overcome the shortcomings of existing drugs. HDAC3 is a unique class I HDAC located in the cell nucleus and cytoplasm. Accumulating evidence indicates that abnormal HDAC3 expression plays a key role in many diseases, such as liver cancer, gastric cancer, acute myeloid leukemia, and breast cancer. In addition to cancer, HDAC3 also plays a key role in the development and progression of inflammatory, metabolic, and neurodegenerative diseases. In addition, studies have shown that HDAC3 can regulate the expression of the PD-L1 protein, thereby playing an important role in promoting tumor immunity. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a bifunctional molecule targeting HDAC / PD-L1, comprising structures represented by formulas (I) and (II), with o-phenylenediamine and hydrazide as ZBGs, as well as a preparation method and application thereof. This compound can simultaneously inhibit HDAC3 activity and degrade PD-L1 protein in melanoma cells, thereby inhibiting cancer cell proliferation, and is expected to be used in cancer immunotherapy.

[0005] In the first aspect of the present invention, there is provided a compound represented by formula (I) or (II) or a pharmaceutically acceptable salt thereof:

[0006]

[0007] Linker can be selected from -,

[0008] In a second aspect, the present invention provides the use of the compounds represented by the above formula (I) and (II) or their pharmaceutically acceptable salts, or the above drugs in treating diseases related to histone deacetylase (HDAC) activity or expression.

[0009] In some embodiments of the present invention, the HDAC activity or expression level includes at least one of HDAC1, HDAC3, HDAC6, and HDAC8, preferably HDAC3.

[0010] The third aspect of the present invention provides the use of the compounds represented by the above formula (I) and (II) or their pharmaceutically acceptable salts, or the above drugs in the treatment and prevention of cancer.

[0011] The compounds represented by formula (I) and (II) of the present invention, especially the compound 5-(phenylamino)methyl-N-(4-(2-propylhydrazine-1-carbonyl)benzyl)thiazole-2-carboxamide, can effectively reduce PD-L1 protein expression and enhance lymphocyte infiltration in tumor tissues, thereby enhancing anti-tumor activity.

[0012] In some embodiments of the present invention, the cancer is colon cancer, breast cancer, T-cell lymphoma, melanoma and liver cancer; preferably melanoma.

[0013] In some embodiments of the present invention, the condensation is carried out in the presence of a solvent, and the solvent is N,N-dimethylformamide.

[0014] In some embodiments of the present invention, the condensation is carried out in the presence of a condensing agent and an organic base.

[0015] In some preferred embodiments of the present invention, the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole or 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, and the organic base is triethylamine.

[0016] In some preferred embodiments of the present invention, the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to 1-hydroxybenzotriazole is 1:1.

[0017] In some preferred embodiments of the present invention, the molar ratio of compound A:compound B:condensing agent:organic base is 1:1-1.2:2-3:3-4.

[0018] In some embodiments of the present invention, the condensation temperature is 20-30° C., and the condensation time is 2-4 hours.

[0019] The fifth aspect of the present invention provides a method for preparing the compound represented by formula (I) or (II), or a pharmaceutically acceptable salt thereof.

[0020] The compound according to the embodiment of the present invention has at least the following beneficial effects:

[0021] The present invention provides an o-phenylenediamine and hydrazide-based bifunctional molecule targeting HDAC / PD-L1, containing the structures represented by formulas (I) and (II). The eight compounds exhibited certain anti-proliferative effects against human colon cancer cells HCT116, mouse melanoma B16-F10, human breast cancer cells MCF-7, human liver cancer cells HepG2, and human peripheral blood leukemia Jurkat-T cells. In particular, the compound 5-(phenylamino)methyl-N-(4-(2-propylhydrazine-1-carbonyl)benzyl)thiazole-2-carboxamide effectively inhibited the proliferation of mouse melanoma B16-F10 in mice. Further studies have shown that the compound can effectively inhibit HDAC3 activity, reduce PD-L1 protein expression, and enhance lymphocyte infiltration in tumor tissues, thus showing promise for cancer immunotherapy.

[0022] The term "pharmaceutically acceptable salts" herein includes conventional salts formed with pharmaceutically acceptable inorganic or organic acids, or inorganic or organic bases. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The present invention will be further described below with reference to the accompanying drawings and examples, in which:

[0024] Figure 1 The hydrogen nuclear magnetic resonance spectrum of the compound (E)-N-(2-aminophenyl)-3-(4-(5-(phenylamino)methyl)thiazol-2-yl)phenyl)acrylamide (HQ-1) represented by formula (I) of the present invention is shown.

[0025] Figure 2 The carbon nuclear magnetic resonance spectrum of the compound (E)-N-(2-aminophenyl)-3-(4-(5-(phenylamino)methyl)thiazol-2-yl)phenyl)acrylamide (HQ-1) represented by formula (I) of the present invention is shown.

[0026] Figure 3 This is the hydrogen nuclear magnetic resonance spectrum of the compound N-(2-aminophenyl)-4-(5-(phenylamino)methyl)thiazol-2-yl)benzamide (HQ-2) represented by formula (I) of the present invention.

[0027] Figure 4 The carbon nuclear magnetic resonance spectrum of the compound N-(2-aminophenyl)-4-(5-(phenylamino)methyl)thiazol-2-yl)benzamide (HQ-2) represented by formula (I) of the present invention is shown.

[0028] Figure 5 The hydrogen nuclear magnetic resonance spectrum of the compound N-(2-aminophenyl)-4-(4-(5-(phenylaminomethyl)thiazol-2-yl)benzamidomethyl)benzamide (HQ-3) represented by formula (I) of the present invention is shown.

[0029] Figure 6 The carbon nuclear magnetic resonance spectrum of the compound N-(2-aminophenyl)-4-(4-(5-(phenylaminomethyl)thiazol-2-yl)benzamidomethyl)benzamide (HQ-3) represented by formula (I) of the present invention is shown.

[0030] Figure 7 The hydrogen nuclear magnetic resonance spectrum of the compound N-(2-aminophenyl)-4-(4-(5-(phenylamino)methyl)thiazol-2-yl)benzamide)benzamide (HQ-4) represented by formula (I) of the present invention is shown.

[0031] Figure 8 The carbon NMR spectrum of the compound N-(2-aminophenyl)-4-(4-(5-(phenylamino)methyl)thiazol-2-yl)benzamide)benzamide (HQ-4) represented by formula (I) of the present invention is shown.

[0032] Figure 9 This is the hydrogen nuclear magnetic resonance spectrum of the compound (E)-N-(4-(3-((2-aminophenyl)amino)-3-oxopropyl-1-en-1-yl)benzyl)-4-(5-(phenylamino)methyl)thiazol-2-yl)benzamide (HQ-5) represented by formula (I) of the present invention.

[0033] Figure 10 The carbon NMR spectrum of the compound (E)-N-(4-(3-((2-aminophenyl)amino)-3-oxopropyl-1-en-1-yl)benzyl)-4-(5-(phenylamino)methyl)thiazol-2-yl)benzamide (HQ-5) represented by formula (I) of the present invention is shown.

[0034] Figure 11 The hydrogen nuclear magnetic resonance spectrum of the compound N1-(2-aminophenyl)-N7-(4-(5-((phenylamino)methyl)thiazol-2-yl)phenyl)pimelanediamide (HQ-10) represented by formula (I) of the present invention is shown.

[0035] Figure 12 The carbon NMR spectrum of the compound N1-(2-aminophenyl)-N7-(4-(5-((phenylamino)methyl)thiazol-2-yl)phenyl)pimelanediamide (HQ-10) represented by formula (I) of the present invention is shown.

[0036] Figure 13This is the hydrogen nuclear magnetic resonance spectrum of the compound N-(4-((2-aminophenyl)carbamoyl)benzyl)-5-((phenylamino)methyl)thiazole-2-carboxamide (HQ-19) represented by formula (II) of the present invention.

[0037] Figure 14 The carbon nuclear magnetic resonance spectrum of the compound N-(4-((2-aminophenyl)carbamoyl)benzyl)-5-((phenylamino)methyl)thiazole-2-carboxamide (HQ-19) represented by formula (II) of the present invention is shown.

[0038] Figure 15 This is the hydrogen nuclear magnetic resonance spectrum of the compound 5-(phenylamino)methyl-N-(4-(2-propylhydrazine-1-carbonyl)benzyl)thiazole-2-carboxamide (HQ-30) represented by formula (II) of the present invention.

[0039] Figure 16 This is the carbon nuclear magnetic resonance spectrum of the compound represented by formula (II) of the present invention, 5-(phenylamino)methyl-N-(4-(2-propylhydrazine-1-carbonyl)benzyl)thiazole-2-carboxamide (HQ-30).

[0040] Figure 17 This is a liquid phase diagram of the compound represented by formula (II) of the present invention, 5-(phenylamino)methyl-N-(4-(2-propylhydrazine-1-carbonyl)benzyl)thiazole-2-carboxamide (HQ-30).

[0041] Figure 18 The mass spectrum of the compound 5-(phenylamino)methyl-N-(4-(2-propylhydrazine-1-carbonyl)benzyl)thiazole-2-carboxamide (HQ-30) represented by formula (II) of the present invention is shown.

[0042] Figure 19 The effects of HQ-2, a compound represented by formula (I), and HQ-30, a compound represented by formula (II), on the expression level of PD-L1 protein in melanoma cells are shown in Figure 3. A: Western Blot analysis of the effects of HQ-2, a compound represented by formula (I), and HQ-30, a compound represented by formula (II), on the expression level of PD-L1 protein; B: Quantification of the effects of HQ-2, a compound represented by formula (I), and HQ-30, a compound represented by formula (II), on the expression level of PD-L1 protein. * P<0.05.

[0043] Figure 20 The compound HQ-30 shown in formula (II) degrades PD-L1 DC 50A: Western Blot verification of the effect of HQ-30, a compound represented by formula (II), on the expression of PD-L1 protein with varying concentrations; B: HQ-30, a compound represented by formula (II), degrades PD-L1 protein DC 50 C: Western Blot analysis of the degradation mechanism of PD-L1 protein by HQ-30, a compound of formula (II); D: Quantification of the effect of HQ-30, a compound of formula (II), on PD-L1 protein expression. ** P<0.01, *** P<0.001.

[0044] Figure 21 In vivo antitumor activity evaluation of the compound HQ-30 of formula (II), including tumor tissue images (A), tumor volumes (B), tumor weights (C), and mouse body weights (D) of mice in the blank control group (Control), PD-L1 inhibitor group (NP19), positive control group (MS-275), and the compound HQ-30 group of formula (II).

[0045] Figure 22 The effect of the compound HQ-30 shown in formula (II) on tumor immune activity, including the infiltrating lymphocytes (CD3 + CD4 + ,CD3 + CD8 + ) ratio. * P<0.05, ** P<0.01, *** P<0.001.

[0046] Figure 23 The effect of the compound HQ-30 represented by formula (II) on the expression level of PD-L1 protein in melanoma tissues, A: Western Blot detection of the effect of the compound HQ-30 represented by formula (II) on the expression level of PD-L1 protein; B: Quantitative results of the effect of the compound HQ-30 represented by formula (II) on the expression level of PD-L1 protein. Compared with the blank control group, * P<0.05, *** P<0.001. DETAILED DESCRIPTION

[0047] The following examples further illustrate the specific implementation of the present invention, but the implementation and protection of the present invention are not limited thereto. It should be noted that if there are any processes not specifically described below, they can be implemented or understood by those skilled in the art with reference to the existing technology.

[0048] The reagents or instruments used without indicating the manufacturer are deemed to be conventional products that can be purchased commercially. The known starting materials of the present invention can be synthesized by methods known in the art, or can be purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd., Anaiji Chemical, Shanghai Haohong Biopharmaceutical Technology Co., Ltd., etc.; the thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate, the silica gel plate used for thin layer chromatography (TLC) uses a specification of 0.15mm-0.20mm, and the column chromatography uses Yantai Huanghai silica gel 100-200 mesh silica gel as a carrier; the structure of the compound is determined by nuclear magnetic resonance (NMR). NMR shifts are given in units of (ppm). NMR was measured using a (Bruker Avance III400) nuclear magnetometer, and the measuring solvents were deuterated dimethyl sulfoxide (DMSO-de), deuterated chloroform (CDC13), and the internal standard was tetramethylsilane (TMS).

[0049] Example

[0050] Example 1 Synthesis method:

[0051]

[0052] Intermediate: Preparation of 4-bromobenzenethioamide 2

[0053] Dissolve 4-bromobenzonitrile (1.0 g, 5.49 mmol) in methanol, add 20% aqueous ammonium sulfide (0.56 g, 8.24 mmol), and reflux the resulting solution at 60°C for 6 hours. After the reaction, pour the mixture into ice water, and filter the precipitate to obtain Intermediate 2 (1.5 g) as a yellow solid. Yield: 96%.

[0054] Intermediate: Preparation of 2-(4-bromophenyl)thiazole-5-carboxaldehyde 3

[0055] Intermediate 2 (1.5 g, 6.94 mmol) and 2-bromomalondialdehyde (1.05 g, 6.94 mmol) were dissolved in 20 mL of ethylene glycol dimethyl ether. The resulting reaction solution was stirred at room temperature for 10 hours. After completion of the reaction, the reaction solution was diluted with water and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography using petroleum ether / ethyl acetate (5:1) to obtain Intermediate 3 (376 mg) as a white solid. Yield: 25.1%.

[0056] Intermediate: Preparation of N-(2-(4-bromophenyl)thiazol-5-ylmethyl)aniline 4

[0057] Intermediate 3 (376.0 mg, 1.40 mmol) and aniline (195.8 mg, 2.10 mmol) were dissolved in dichloromethane. Two drops of glacial acetic acid and sodium cyanoborohydride (440.6 mg, 7.01 mmol) were added. The reaction mixture was stirred at room temperature for 3 hours. After completion of the reaction, dichloromethane was added for extraction. The organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. This was purified by column chromatography using petroleum ether / ethyl acetate (10:1) to obtain Intermediate 4 as a yellow solid. Yield: 79.8%.

[0058] Intermediate: Preparation of (E)-3-(4-(5-(phenylamino)methyl)thiazol-2-yl)phenylacrylate 5

[0059] Compound 4 (300 mg, 0.87 mmol), methyl acrylate (89.8 mg, 1.04 mmol), palladium acetate (19.5 mg, 86.9 μmol), triphenylphosphine (68.4 mg, 0.26 mmol), and triethylamine were dissolved in DMF. The reaction mixture was reacted at 110°C under a nitrogen atmosphere for 12 hours. After completion of the reaction, the reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product, which was purified by column chromatography using petroleum ether / ethyl acetate (10:1) to obtain intermediate 5 as a white solid. Yield: 52.1%.

[0060] Intermediate: Preparation of (E)-3-(4-(5-(phenylamino)methyl)thiazol-2-yl)phenyl)acrylic acid 6

[0061] Compound 5 was dissolved in methanol, and saturated aqueous sodium hydroxide solution was added. The reaction solution was stirred at room temperature for 2 h. After the reaction was complete, the methanol was removed under reduced pressure, and 3 M HCl was added to adjust the pH of the solution to 4. The precipitate was collected by filtration and dried to obtain 6 as a white solid with a yield of 78.7%.

[0062] Preparation of the compound of formula I (Compound HQ-1) 4(E)-N-(2-aminophenyl)-3-(4-(5-(phenylamino)methyl)thiazol-2-yl)phenyl)acrylamide:

[0063] Compound 6 (15 mg, 44.6 μmol) and o-phenylenediamine (5.8 mg, 53.5 μmol) were dissolved in DMF. Triethylamine (13.5 mg, 0.13 mmol), EDCI (9.4 mg, 49.1 μmol) and HOBT (6.6 mg, 49.1 μmol) were added to the reaction solution. The resulting reaction solution was stirred at room temperature for 4 hours. After the reaction was completed, water was added, the precipitate was collected by filtration, and dried to obtain an off-white solid. Yield: 57.1%. The specific identification results are: 1 H NMR (400MHz, DMSO-d6) δ9.44(s,1H),7.91(d,J=32.2Hz,3H),7.72(s,2H),7.58(d,J=15.3Hz,1H),7.36(s,1H ),7.08(d,J=9.4Hz,2H),7.05–6.85(m,2H),6.80–6.51(m,5H),6.42–6.29(m,1H),4.97(s,2H),4.54(s,2H). 13 C NMR (101MHz, DMSO-d6) δ165.7,163.7,148.3,142.0,140.6,139.0,136.7,134. 4,129.3,128.8,126.8,125.1,123.7,116.9,116.7,116.4,113.0,39.8.LC-MS m / z:[M+Na] + calculated for C 25 H 22 N4OS:449.15,found:449.62.Purity:96.2%by HPLC(t R =17.34min).

[0064] Example 2 Synthesis method:

[0065]

[0066] Intermediate: Preparation of methyl 4-(5-((phenylamino)methyl)thiazol-2-yl)benzoate 10

[0067] Compound 10 was prepared similarly to compound 4, except that 4-bromobenzonitrile was replaced with methyl 4-cyanobenzoate to afford a yellow solid in a yield of 62.8%.

[0068] Intermediate: Preparation of 4-(5-((phenylamino)methyl)thiazol-2-yl)benzoic acid 11

[0069] The preparation method of compound 11 is similar to that of compound 6, except that compound 10 is used as the starting material instead of compound 5 to obtain a white solid with a yield of 85.1%.

[0070] Preparation of Formula I compound (Compound HQ-2) N-(2-aminophenyl)-4-(5-(phenylamino)methyl)thiazol-2-yl)benzamide: Compound HQ-2 was synthesized using the same method as Compound HQ-1, substituting Compound 11 for Compound 6 to obtain an off-white solid. Yield: 58.6%. Specific identification results: 1 H NMR (400MHz, DMSO-d6) δ9.78 (s, 1H), 8.13–8.05 (m, 2H), 8.01 (d, J = 8.4Hz, 2H),7.91(s,1H),7.17(d,J=7.3Hz,1H),7.14–7.05(m,2H),6.98(td,J=7. 7,1.7Hz,1H),6.79(dd,J=8.0,1.5Hz,1H),6.73–6.64(m,2H),6.59(q,J=7 .6,7.1Hz,2H),6.37(t,J=6.1Hz,1H),4.94(s,2H),4.56(d,J=6.1Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ165.4,165.0,148.3,143.6,142.0,141.2,136.0,1 29.4,129.1,127.2,126.0,123.5,117.0,116.6,116.5,113.0,39.8.LC-MS m / z:[M+Na] + calculated for C 23 H 20 N4OS:423.14,found:423.54.Purity:95.8%by HPLC(t R =16.19min).

[0071] Intermediate: Preparation of Compound 12

[0072] Compound 11 (65 mg, 0.21 mmol) and methyl 4-(aminomethyl)benzoate (38.1 mg, 0.23 mmol) were dissolved in DMF. Triethylamine (63.6 mg, 0.63 mmol) and HATU (95.6 mg, 0.25 mmol) were added to the reaction solution. The resulting reaction solution was stirred at room temperature for 4 hours. After the reaction was completed, water was added to the reaction solution, and the precipitate was collected by filtration and dried to obtain a white solid. Yield: 65.4%.

[0073] Intermediates: Preparation of Compounds 13-14

[0074] The preparation method of compound 13-14 is similar to that of compound 12, except that 4-aminobenzoic acid methyl ester and (4-bromophenyl)methylamine are used as the raw materials instead of 4-(aminomethyl)benzoic acid methyl ester to obtain a white solid.

[0075] Intermediate: Preparation of compound (E)-3-(4-(4-(5-(phenylamino)methyl)thiazol-2-yl)benzamidomethyl)phenylacrylate 15

[0076] The preparation method of compound 15 is similar to that of 5, except that compound 14 is used as the starting material instead of compound 4 to obtain a yellow solid with a yield of 42.9%.

[0077] Preparation of the compound of formula I (compound HQ-3) N-(2-aminophenyl)-4-(4-(5-(phenylaminomethyl)thiazol-2-yl)benzamidomethyl)benzamide: Compound HQ-3 was synthesized with reference to compound HQ-1, with compound 12 replacing compound 5 as the starting material. Specific identification results are: 1 H NMR(400MHz,DMSO-d6)δ9.64(s,1H),9.26(s,1H),8.07(t,J=8.6Hz,2H),8.00(s,3H ),7.97–7.92(m,2H),7.89(s,1H),7.45(d,J=8.0Hz,2H),7.17(d,J=7.8Hz,1H),7.0 9(t,J=7.6Hz,1H),6.97(t,J=7.8Hz,1H),6.78(d,J=8.1Hz,1H),6.67(d,J=7.9Hz,2 H), 6.59 (q, J = 7.9Hz, 2H), 6.36 (t, J = 6.0Hz, 1H), 4.90 (s, 2H), 4.56 (t, J = 6.4Hz, 4H). 13 C NMR(101MHz,DMSO-d6)δ166.0,165.3,148.2,143.5,142.0,141.1,136.0,135.6,129.3,128 .6,128.3,127.4,127.1,126.9,126.2,123.7,117.0,116.7,116.5,113.0,42.9,39.8.LC-MS m / z:[M+Na] + calculated for C 31 H 27 N5O2S:556.19,found:556.61.Purity:95.4%by HPLC(t R =16.32min).

[0078] Preparation of the compound of formula I (compound HQ-4) N-(2-aminophenyl)-4-(4-(5-(phenylamino)methyl)thiazol-2-yl)benzamide)benzamide: Compound HQ-4 was synthesized with reference to compound HQ-1, with compound 13 replacing compound 5 as the starting material. The specific identification results are: 1 H NMR (400MHz, DMSO-d6) δ10.16 (s, 1H), 9.77 (s, 1H), 8.08 (d, J = 8.2Hz, 3H), 8.01(d,J=8.4Hz,2H),7.91(s,1H),7.17(d,J=8.1Hz,1H),7.14–7.05(m,3H ),7.02–6.95(m,1H),6.79(dd,J=8.1,1.5Hz,1H),6.70–6.64(m,3H),6.59( q,J=7.5Hz,3H),6.37(t,J=6.2Hz,1H),4.94(s,2H),4.56(d,J=6.1Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ165.4,148.3,143.6,142.0,141.2,136.0,129.4,1 29.1,127.2,127.0,126.0,123.5,117.0,116.6,116.5,113.0,39.8.LC-MS m / z:[M+Na] + calculated for C 30 H 25 N5O2S:542.17,found:542.57.Purity:97.8%by HPLC(t R =16.17min).

[0079] Preparation of the compound of formula I (compound HQ-5) (E)-N-(4-(3-((2-aminophenyl)amino)-3-oxopropyl-1-en-1-yl)benzyl)-4-(5-(phenylamino)methyl)thiazol-2-yl)benzamide: Compound HQ-5 was synthesized with reference to compound HQ-1, except that compound 15 was used as the starting material instead of compound 5. The specific identification results were: 1H NMR (400MHz, DMSO-d6) δ9.40 (s, 1H), 9.21 (t, J = 6.0Hz, 1H), 8.00 (s, 4H), 7.89 (s, 1H), 7. 60(d,J=7.9Hz,2H),7.54(d,J=15.7Hz,1H),7.40(d,J=7.8Hz,2H),7.34(d,J=7.4Hz,1H) ,7.14–7.04(m,2H),6.96–6.85(m,2H),6.75(dd,J=8.0,1.5Hz,1H),6.67(d,J=7.9Hz,2H ), 6.58 (t, J = 7.4Hz, 2H), 6.36 (t, J = 6.2Hz, 1H), 4.96 (s, 2H), 4.54 (dd, J = 9.8, 6.0Hz, 5H). 13 C NMR(101MHz,DMSO-d6)δ165.9,165.3,163.9,148.2,142.0,141.6,141.1,139.8,136.0,135.5,133.9, 129.3,128.6,128.2,128.1,126.1,125.1,123.9,122.2,117.0,116.7,116.4,113.0,42.9,39.8.LC-MS m / z:[M+Na] + calculated for C 33 H 29 N5O2S:582.20,found:582.61.Purity:95.9%by HPLC(t R =16.98min).

[0080] Example 3 Synthesis method

[0081]

[0082] Intermediate: Preparation of N-(2-(4-nitrophenyl)thiazol-5-ylmethyl)aniline 19

[0083] Compound 19 was prepared similarly to compound 4, except that 4-nitrobenzonitrile was used instead of 4-bromobenzonitrile to give a yellow solid with a yield of 66.7%.

[0084] Intermediate: Preparation of N-(2-(4-aminophenyl)thiazol-5-ylmethyl)aniline 20

[0085] Compound 19 (0.3 g, 0.96 mmol) and ammonium chloride (0.10 g, 1.93 mmol) were dissolved in 20 mL of ethanol. 4 mL of water was added, and reduced iron powder (0.54 g, 9.64 mmol) was added to the reaction mixture. The mixture was refluxed at 80°C for 2 hours. After completion of the reaction, the reaction mixture was filtered, and the filtrate was decompressed to remove the remaining solvent to obtain the crude product. The crude product was purified by column chromatography using petroleum ether / ethyl acetate (2:1) to obtain intermediate 20 as a yellow solid. Yield: 80.6%.

[0086] Intermediate: Preparation of Compound 21

[0087] The preparation method of compound 21 is similar to that of 12, except that ethyl hydrogen pimelate is used as the starting material instead of compound 11 to obtain a white solid with a yield of 54.7%.

[0088] Preparation of the compound of formula I (compound HQ-10) N1-(2-aminophenyl)-N7-(4-(5-((phenylamino)methyl)thiazol-2-yl)phenyl)pimelane diamide: Compound HQ-10 was synthesized with reference to compound HQ-1, with compound 21 replacing compound 5 as the starting material. Specific identification results are: 1 H NMR (400MHz, DMSO-d6) δ10.12(s,1H),9.10(s,1H),7.90–7.74(m,3H),7.70(d,J=8.5 Hz,2H),7.20–7.10(m,1H),7.07(d,J=7.7Hz,2H),6.89(t,J=7.6Hz,1H),6.69(dd,J= 20.1,8.0Hz,3H),6.55(dt,J=18.0,7.4Hz,2H),6.29(t,J=6.1Hz,1H),4.82(s,2H),4 .50(d,J=6.1Hz,2H),2.38–2.26(m,4H),1.64(h,J=7.2Hz,4H),1.36(p,J=7.6Hz,2H). 13 CNMR(101MHz,DMSO-d6)δ171.9,171.5,166.3,148.3,142.3,141.5,141.3,139.1,129.3,128.4,126. 9,126.1,125.7,123.9,119.6,116.9,116.6,116.3,113.0,39.8,36.8,36.0,28.7,25.5,25.3.LC-MS m / z:[M+Na] + calculated for C 29 H 31N5O2S:536.22,found:536.67.Purity:95.3%by HPLC(t R =16.84min).

[0089] Example 4 Synthesis Method

[0090]

[0091] Intermediate: Preparation of 5-((phenylamino)methyl)thiazole-2-carboxylic acid 25

[0092] Compound 25 was prepared similarly to compound 11, except that ethyl thiooxamate was used instead of methyl 4-cyanobenzoate to give a yellow solid with a yield of 78.4%.

[0093] Intermediate: Preparation of methyl 4-(((phenylamino)methyl)thiazole-2-carboxamido)methyl)benzoate 26

[0094] The preparation method of compound 26 is similar to that of 12, except that compound 25 is used as the starting material instead of compound 11 to obtain a yellow solid with a yield of 57.4%.

[0095] Preparation of Formula II compound (Compound HQ-19) N-(4-((2-aminophenyl)carbamoyl)benzyl)-5-((phenylamino)methyl)thiazole-2-carboxamide: Compound HQ-19 was synthesized with reference to Compound HQ-1, with Compound 26 replacing Compound 5 as the starting material. Specific identification results are: 1 H NMR(400MHz,DMSO-d6)δ9.63(d,J=7.7Hz,1H),9.43(t,J=6.6Hz,1H),7.92(d, J=7.8Hz,3H),7.40(t,J=8.9Hz,2H),7.16(d,J=7.6Hz,1H),7.09(t,J=7.6Hz,2 H),6.97(t,J=7.8Hz,1H),6.83–6.75(m,1H),6.62(dd,J=21.7,7.7Hz,4H),6. 35(t,J=6.2Hz,1H),4.89(s,2H),4.56(d,J=6.2Hz,2H),4.50(d,J=6.4Hz,2H). 13C NMR(101MHz,DMSO-d6)δ162.5,159.8,148.1,145.8,143.5,143.0,141.6,133.6,129.3, 128.2,128.2,127.4,127.1,126.9,123.7,117.1,116.6,116.5,113.1,42.7,39.9.LC-MS m / z:[M+Na] + calculated for C 25 H 23 N5O2S:480.16,found:458.57.Purity:97.4%by HPLC(t R =14.82min).

[0096] Example 5 Synthesis Method

[0097]

[0098] Intermediate: Preparation of tert-butyl 2-(4-((phenylamino)methyl)thiazole-2-carboxamido)methyl)benzoyl)hydrazine-1-carboxylate 27

[0099] Compound 27 was prepared similarly to compound 12, except that tert-butyl carbazate was used instead of methyl 4-(aminomethyl)benzoate to give a yellow-brown solid. Yield: 58.6%.

[0100] Preparation of the compound of Formula II (Compound HQ-30) 5-(phenylamino)methyl-N-(4-(2-propylhydrazine-1-carbonyl)benzyl)thiazole-2-carboxamide: Compound 27 (0.1 mmol) was dissolved in dichloromethane, and a 4M hydrogen chloride / dioxane solution (0.8 mmol) was added. The reaction was allowed to react at room temperature for 4 hours. After completion of the reaction, the remaining solvent was removed under reduced pressure to yield a tan solid. The resulting solid (100 mg, 0.24 mmol), propionaldehyde (13.9 mg, 0.24 mmol), and triethylamine (48.43 mg, 0.48 mmol) were dissolved in dichloromethane / methanol (1 / 1). Two drops of acetic acid and sodium cyanoborohydride (75.2 mg, 1.2 mmol) were added, and the mixture was stirred at room temperature for 3 hours. The reaction mixture was extracted with dichloromethane. The organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography using dichloromethane / methanol (30:1) to obtain compound HQ-30 as a white solid. Yield: 38.6%. Specific identification results were: 1H NMR (400MHz, DMSO-d6) δ9.96 (s, 1H), 9.39 (t, J = 6.4Hz, 1H), 7.92 (s, 1H), 7.76 (d, J=8.2Hz,2H),7.35(d,J=8.2Hz,2H),7.17–6.99(m,2H),6.69–6.61(m,2H),6.58(t ,J=7.3Hz,1H),6.34(t,J=6.3Hz,1H),5.08(s,1H),4.55(d,J=6.2Hz,2H),4.46(d, J=6.4Hz,2H),2.74(t,J=7.1Hz,2H),1.46(q,J=7.3Hz,2H),0.90(t,J=7.4Hz,3H). 13 C NMR(101MHz,Chloroform-d)δ162.3,159.5,146.6,144.6,141.6,140.8,132.1 ,129.4,127.9,127.9,127.3,118.8,113.2,54.0,43.1,41.1,21.2,11.5.LC-MS m / z:[M+Na] + calculated for C 22 H 25 N5O2S:446.17,found:446.65.Purity:96.1%by HPLC(t R =15.26min).

[0101] Example 6 In vitro antitumor activity study

[0102] The in vitro anti-tumor activity of the compounds of the present invention was demonstrated using the following test methods. These results indicate that the compounds of the present invention are useful for treating cancer. The specific test methods are as follows:

[0103] Tumor cell lines were purchased from ATCC (USA). The antiproliferative activity of the test compounds against human colorectal cancer cell line HCT-116, human breast cancer cell line MCF-7, melanoma cell line B16-F10, and human hepatocellular carcinoma cell line HepG2 was determined using the standard MTT assay. The cytotoxicity of the human lymphoma Jurkat-T cell line was evaluated using the CCK-8 assay. All cancer cell lines were cultured in RPMI-1640 supplemented with 10% fetal bovine serum. The cells were cultured at 37°C and 5% CO2.

[0104] Plating: Add 50 μL of cell suspension (containing 5,000 cells) to each well of a 96-well plate and incubate overnight in an incubator. Dosing: Add 50 μL of compound at varying concentrations to each well, with three replicates for each concentration. After dosing, incubate the 96-well plate in an incubator for 48 hours.

[0105] Cell viability assay: After 48 h, add 10 μL of MTT or CCK8 to each well and continue incubating in the incubator for 4 h. Discard the supernatant and add 100 μL of DMSO to each well. Incubate at 37°C for 15 min and measure the absorbance at a wavelength of 570 nM using a microplate reader.

[0106] Data processing: Graphpad Prism software was used to calculate the IC of compounds for inhibiting cell proliferation. 50 value.

[0107] The test results are shown in Table 1:

[0108] Table 1 Antitumor activity of the compounds described in the examples

[0109]

[0110] The above in vitro experimental results show that the compounds of the present invention have effective anti-proliferative activity against five tumor cells: human T lymphocytes Jurkat, human colon cancer cells HCT-116, mouse melanoma cells B16-F10, human breast cancer cells MCF-7, and human liver cancer cells HepG2. In particular, the effects of compounds HQ-2 and HQ-30 are significantly better than the inhibitory activity of the positive control (MS-275).

[0111] Example 7 In vitro inhibition of HDAC activity

[0112] The specific test method for the in vitro inhibition of HDAC activity by the compounds of the present invention is as follows:

[0113] The enzyme inhibitory activity of compounds G3 and G12 was determined using a fluorescence assay. HDAC1 (#ab101661) and HDAC6 (#ab42632) enzymes were purchased from Abcam, HDAC3 (#BML-SE515-0050) from Enzo, and HDAC8 (#H90-30H-05) from SignalChem. The buffer contained 25 mmol / L Tris (pH 8.0), 1 mmol / L MgCl2, 0.1 mg / mL BSA, 137 mmol / L NaCl, and 2.7 mmol / L KCl. HDACs (HDAC1, 7.2 ng / well; HDAC3, 3.4 ng / well; HDAC6, 15 ng / well; HDAC8, 22 ng / well) were added in a total volume of 40 μL. Test compounds (10 dilutions: 500 μM, 125 μM, 31.25 μM, 7.81 μM, 1.95 μM, 0.49 μM, 0.12 μM, 0.03 μM, 7.6 nM, and 1.88 nM, with three replicates per well) were diluted in 10% dimethyl sulfoxide (DMSO). 5 μL of the dilution was added and pre-incubated. Purified recombinant HDAC was added and incubated for 5 minutes at room temperature before adding substrate. Finally, enzyme substrates (Ac-Leu-Gly-Lys(Ac)-AMC, 10 μM for HDACs 1, 3, and 6; Ac-Leu-Gly-Lys(Tfa)-AMC, 2 μM for HDAC 8) were added and incubated at 37°C in a final volume of 50 μL for 30 minutes. The reaction was quenched with 50 μL of HDAC assay developer (1 mg / mL trypsin and 2 μmol / L TSA in assay buffer) for 30 min at room temperature. The amount of fluorescent product in the mixture was measured using a microplate reader. Fluorescence intensity was then read on a TECAN microplate reader at excitation wavelengths of 350-360 nm and emission wavelengths of 450-460 nm. 50 The values were calculated by nonlinear regression and normalized dose-response fitting using Prism-GraphPad software. All experiments were performed at least three times independently.

[0114] The activity results are shown in Table 2:

[0115] Table 2 Anti-HDAC activity of the compounds described in the examples

[0116]

[0117]

[0118] The above in vitro experimental results show that the compounds of the present invention have a strong inhibitory effect on HDAC3, which is better than the positive control (MS-275), and have no inhibitory activity or weak inhibitory activity on HDAC6 and HDAC8, especially compound HQ-2 (IC 50 =0.066 μM), HQ-4 (IC 50 =0.094 μM) and HQ-30 (IC 50 =0.089 μM) and their inhibitory activities against HDAC3 were all below 0.1 μM.

[0119] Example 8 Study on in vitro degradation activity of PD-L1

[0120] 2 × 10 5 B16-F10 cells were incubated overnight at 37°C. Different concentrations of HQ-30 (0.5, 1, 2, 4, 6, 8, and 10 μM) were added to each well and incubated at 37°C for 24 hours. The culture medium was removed and the cells were washed with PBS. The cells were lysed on ice for half an hour using RIPA cell lysis buffer and centrifuged at 12,000 g for 10 minutes at 4°C. The supernatant was collected and the protein concentration was determined by BCA protein assay. Subsequently, the protein sample was heat denatured at 100°C for 10 minutes. 10 μL of the prepared protein sample was added to the SDS-PAGE gel loading wells for electrophoresis. The gel was placed in a glass dish containing electrotransfer buffer and transferred to a PVDF membrane. After transfer, the membrane was blocked with 5% skim milk powder for 2 hours at room temperature. The PVDF membrane was washed with TBST solution for 5 minutes x 5 and incubated with an anti-antibody overnight at 4°C. The primary antibody was removed, and the PVDF membrane was washed with TBST solution for 5 min × 5 times. The secondary antibody was added in proportion to the dilution and incubated at room temperature for 1 hour. For color development, ECL developer was evenly applied to the PVDF membrane and placed in an imaging analysis system for visual analysis of protein expression and protein degradation mechanism. The images obtained from the Western Blot experiment were processed using Image J software, the grayscale value was calculated, and the DC value of the compound was calculated using GraphPad Prism 7 software. 50 . “DC 50 " refers to the dose when 50% of the protein is degraded. Figure 19 、 20 The compound HQ-30 can reduce PD-L1 expression in a dose-dependent manner, DC 50 Further studies found that HQ-30 degraded PD-L1 through the lysosomal pathway rather than the proteasomal pathway.

[0121] Example 9 In vivo pharmacokinetic study

[0122] All experimental procedures and protocols were reviewed and approved by the National Institutional Animal Care and Ethics Committee of Southern Medical University. Male Sprague-Dawley rats (250-260 g) were injected orally (20 mg / kg) or intravenously (2 mg / kg) with compound HQ-30 and blood samples (0.5 mL) were collected from the tail vein at 0.0833, 0.25, 0.5, 1, 2, 3, 4, 6, 8, and 12 h. The drug concentrations in the samples were determined using liquid chromatography-mass spectrometry (LC-MS / MS). The chromatography system was a Waters ACQUITY UPLC I-Class / Xevo TQD, and the column was a CORTECS UPLC C18 column (2.1 mm × 100 mm, 1.6 μm). The column temperature was 40°C. Acetonitrile served as mobile phase A, and 0.1% formic acid in water served as mobile phase B. The flow rate was set at 0.4 mL / min. HQ-30 and the internal standard (Midazolam) were separated using a C18 column with a 5 μL injection volume and a 3-min run time. The mobile phase consisted of a mixture of solvent A (acetonitrile) and solvent B (formic acid / ultrapure water, 1:1000, v / v). The mobile phase flow rate was 0.4 mL / min, and the injection volume was 5 μL. Mass spectrometry was performed using an electrospray ionization (ESI) source in positive ion mode, with ion fragmentation generated by multiple reaction monitoring (MRM). The capillary voltage was 1 kV, the desolvation temperature was 600°C, the source temperature was 150°C, the sheath gas flow rate was 50 L / H, and the desolvation gas flow rate was 1000 L / H.

[0123] The measured results are shown in Table 3:

[0124]

[0125] The above pharmacokinetic experimental results show that the compound HQ-30 of the present invention has a suitable half-life and excellent oral properties.

[0126] Example 10 In vivo anticancer activity study

[0127] The specific test method for the in vivo anti-tumor activity of the compound HQ-30 of the present invention is as follows:

[0128] This study used 7-week-old C57 mice purchased from the Experimental Animal Center of Southern Medical University. The experiment was approved by the National Institutional Animal Care and Ethics Committee of Southern Medical University to study the inhibitory effect of compound HQ-30 on the subcutaneous transplantation model of melanoma cells. B16-F10 cells (1.0×10 6 / mL) was suspended in PBS, and then 200 μL was injected into mice to establish a tumor model. Mice were randomly divided into four groups (n=8). The test compound was dissolved in a solution of dimethyl sulfoxide:polyoxyethylene castor oil:normal saline (3:22:75) to produce the desired concentration. Mice in the blank group were intraperitoneally injected with an equal volume of the blank solvent, while mice in the other groups were treated with 200 μL of the blank solvent administered orally once daily for 16 days. Specific groups and drug dosages are shown in Table 4.

[0129] Table 4 Groups and drug composition and dosage

[0130]

[0131] The body weight of mice was monitored throughout the experiment to assess drug toxicity, and the tumor volume was measured with a vernier caliper and calculated using the formula a × b 2 × 0.5 to calculate the tumor volume, where a and b represent the major diameter and minor diameter, respectively. 16 days after the start of the experiment, the experimental mice were dissected and the tumor tissues were removed. The tumors were weighed and the tumor growth inhibition rate (TGI) was calculated: TGI (%) = [1-W t / W v ]×100%, where Wt and Wv are the average tumor weights of the treatment and control groups.

[0132] The activity results were as follows Figure 21 As shown, compound HQ-30 (TGI = 67%) exhibited significantly greater antitumor activity than the positive control drug MS-275 (TGI = 37%). Measurement of mouse body weight revealed that the positive control drug MS-275 caused a significant decrease in weight, whereas the HQ-30 treatment group did not experience this effect, demonstrating the favorable in vivo safety profile of HQ-30.

[0133] Example 11 Study on enhancing tumor immune activity

[0134] The specific test method for the study of the tumor immunity enhancement activity of the compound HQ-30 of the present invention is as follows:

[0135] 16 days after the start of the above experiment, the experimental mice were killed, and their tumor tissues were obtained after dissection. They were placed on a 200-mesh stainless steel mesh in a culture dish and crushed with a syringe needle. The stainless steel mesh was rinsed with PRMI 1640 culture medium to collect the cell suspension. The cells were washed once with PBS (1500r / min, 5min). The red blood cells were lysed with 3-5 times the volume of red blood cell lysis buffer, reacted at room temperature for 2min, centrifuged at 500g for 5min, the supernatant was removed, and the cell pellet was washed twice with PBS (1500r / min, 5min). After washing, the cells were resuspended for testing. The sample was placed in a centrifuge at 1000 rpm for 5min to collect the cells, the supernatant was discarded, and the cells were washed twice with pre-cooled PBS. Then PBS was added to resuspend the cells so that the cell concentration was 1.0×10 6 Pipette 100 μL of the above cell suspension into separate centrifuge tubes: no antibody was added to the blank control tube; 1 μL of FITC-CD3 antibody was added to the CD3 single-staining tube; 1 μL of PE-CD4 antibody was added to the CD4 single-staining tube; and 1 μL of APC-CD8 antibody was added to the CD8 single-staining tube. 1 μL of FITC-CD3, 1 μL of PE-CD4, and 1 μL of APC-CD8 antibody were added to the sample tube simultaneously. The isotype control tubes were also added with the corresponding isotype control antibodies (1 μL of FITC-ISO, 1 μL of APC-ISO, and 1 μL of PE-ISO). After gentle mixing, the cells were incubated at room temperature (25°C) in the dark for 30 min. Then, 1 mL of PBS was added to each tube to resuspend the cells. The cells were centrifuged at 1000 rpm for 5 min to collect the cells, the supernatant was discarded, and the cells were washed twice with pre-chilled PBS. Finally, 300 μL of PBS was added to each tube and the cells were analyzed by flow cytometry. Select appropriate channels (FL1 channel to detect FITC, FL2 channel to detect PE, and FL4 channel to detect APC) and simultaneously detect the expression of PD-L1 protein in tumor tissue.

[0136] The activity results were as follows Figure 22 、 23 Compound HQ-30 can enhance lymphocyte infiltration in tumor tissues. In addition, compound HQ-30 can significantly reduce PD-L1 protein expression in tumor tissues, thereby participating in the tumor immune regulation process and effectively increasing anti-tumor activity.

[0137] The present invention discloses a bifunctional molecule targeting HDAC / PD-L1 using o-phenylenediamine and hydrazide as ZBG, as well as its preparation method and application. The bifunctional molecule is a compound represented by the following formula, or a pharmaceutically acceptable salt or cocrystal thereof:

[0138]

[0139]

[0140] The compound provided by the present invention has a novel structure, and test results show that it has excellent anti-tumor activity, HDAC3 inhibitory activity and PD-L1 protein degradation effect; the preparation method of the compound is simple, rapid, green and safe, and the process route is mature; the compound or its pharmaceutically acceptable salt or cocrystal can be widely used in the preparation of drugs for treating diseases related to HDAC and PD-L1 activity or expression.

Claims

1. A compound of general formula (I), wherein: Linker can be selected from single key, 2. A compound or a pharmaceutically acceptable salt thereof, the compound being selected from one of the following structures:

3. Use of the compound of formula (I) according to claim 1, or the compound or pharmaceutically acceptable salt thereof according to any one of claims 2, in the preparation of a medicament for treating diseases related to HDAC and PD-L1 activity or expression; the related diseases are colon cancer, breast cancer, T-cell lymphoma, melanoma, and liver cancer.

4. The use according to claim 3, characterized in that The HDAC activity or expression level includes at least one of HDAC1, HDAC3, HDAC6, and HDAC8.

5. The use according to claim 4, characterized in that The HDAC activity or expression level is HDAC3.

6. The use according to claim 3, characterized in that The related disease is melanoma.

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