A compound capable of inhibiting the proliferation of cervical cancer, its preparation method and application

Abstract

The present invention belongs to the field of medicinal chemistry, and specifically discloses a compound capable of inhibiting the proliferation of cervical cancer, and a preparation method and application thereof. The compound has a structural formula shown in formula (1) and is formed by the condensation reaction of compound A and compound B. The compound can effectively degrade the NAT10 protein in cancer cells, and has the ability to inhibit the proliferation and invasion of cervical cancer cells, can promote the inhibitory effect of the anti-PD-L1 immunotherapeutic agent on cervical cancer and change the immune microenvironment of cervical cancer. The combined action with the anti-PD-L1 immunotherapeutic agent can make the immune microenvironment develop in the direction of cervical cancer remission, and is suitable for the immunotherapy of cervical cancer.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry, and particularly relates to a compound capable of inhibiting the proliferation of cervical cancer, and its preparation method and application. Background Art

[0002] As an effective protein degradation technology, Proteolysis Targeting Chimeras (PROTACs) have attracted extensive attention. PROTACs are bifunctional molecules composed of three parts: an E3 ubiquitin ligase ligand, a target protein ligand (POI), and a linker in the middle. PROTACs can bind to POI and E3 ubiquitin ligase to form a ternary complex, and then promote the degradation of target proteins through the ubiquitin-proteasome system. Currently, PROTACs have been applied to the degradation of many disease-related proteins, such as protein receptors like AR, BET, and BTK.

[0003] Compared with traditional small molecule inhibitors, the advantages of PROTACs are mainly reflected in stronger targeting, being able to play a role under trace conditions, and higher safety. Based on the above research, it is of great significance to develop and find an effective PROTAC molecule that can overcome the defects of traditional small molecule drugs and obtain better therapeutic effects. 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. For this reason, the present invention provides a compound capable of inhibiting the proliferation of cervical cancer, and its preparation method and application. This compound can effectively degrade the NAT10 protein in cancer cells, and has the ability to inhibit the proliferation and invasion of cervical cancer cells, can promote the inhibitory effect of anti-PD-L1 immunotherapeutic agents on cervical cancer and change the immune microenvironment of cervical cancer. The combined action with anti-PD-L1 immunotherapeutic agents can make the immune microenvironment develop in the direction of cervical cancer remission, and is applicable to the immunotherapy of cervical cancer.

[0005] To solve the above technical problems, the first aspect of the present invention provides a compound represented by the structural formula as shown in formula (1) or a pharmaceutically acceptable salt thereof:

[0006]

[0007] Wherein: L represents

[0008] Any one of

[0009] The "pharmaceutically acceptable salts" referred to in the present invention include conventional salts formed with pharmaceutically acceptable inorganic acids, organic acids, inorganic bases, or organic bases.

[0010] As a further improvement of the above solution, the compound is selected from one of the following structural formulas:

[0011]

[0012] The second aspect of the present invention provides a preparation method of the above compound or its pharmaceutically acceptable salt. The compound is formed by the condensation reaction of compound A and compound B. The structural formulas of compound A and compound B are shown in formulas (2) and (3) respectively:

[0013]

[0014] As a further improvement of the above solution, the condensation reaction is carried out in the presence of a condensing agent and an organic base.

[0015] Preferably, the condensing agent includes at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, and 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate.

[0016] Preferably, when the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole, the molar ratio of the two is 1.1:1.1.

[0017] Preferably, the organic base includes triethylamine.

[0018] As a further improvement of the above solution, the condensation reaction is carried out in the presence of a solvent.

[0019] Preferably, the solvent includes N,N-dimethylformamide.

[0020] As a further improvement of the above solution, the molar ratio of compound A, compound B, the condensing agent, and the organic base is 1:(1 - 1.2):(1.2 - 2.2):(3 - 4).

[0021] As a further improvement of the above solution, the temperature of the condensation reaction is 20 - 30 °C, and the time of the condensation reaction is 4 - 6 hours.

[0022] The third aspect of the present invention provides a pharmaceutical composition, which includes the compound or its pharmaceutically acceptable salt described in the first aspect of the present invention.

[0023] The "pharmaceutical composition" referred to in the present invention includes products containing a therapeutically effective amount of the compound of the present invention or its pharmaceutically acceptable salt, and any products directly or indirectly produced by the combination of the compound of the present invention or its pharmaceutically acceptable salt.

[0024] As a further improvement of the above-mentioned solution, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient or carrier.

[0025] Specifically, the compound or its pharmaceutically acceptable salt, or the pharmaceutical composition can be administered in unit dosage form, and the administration route can be oral, intramuscular, subcutaneous, nasal, buccal mucosa, skin, peritoneal or rectal. The dosage forms can be tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, or lyophilized powder injections. It can be a conventional preparation, a sustained-release preparation, a controlled-release preparation and various particulate drug delivery systems.

[0026] As a further improvement of the above-mentioned solution, the pharmaceutical composition contains 0.1-99.9% by mass of the compound or its pharmaceutically acceptable salt.

[0027] As a further improvement of the above-mentioned solution, the pharmaceutical composition further comprises other active pharmaceutical ingredients.

[0028] Specifically, the administration form of the pharmaceutical composition includes simultaneous, separate or sequential administration of the compound or its pharmaceutically acceptable salt and other active pharmaceutical ingredients.

[0029] As a further improvement of the above-mentioned solution, the other active pharmaceutical ingredient is a PD-L1 immunotherapeutic agent.

[0030] Preferably, the mass ratio of the compound or its pharmaceutically acceptable salt to the PD-L1 immunotherapeutic agent is (2-8):1.

[0031] The fourth aspect of the present invention provides the use of the pharmaceutical composition described in the third aspect of the present invention in the preparation of a drug for treating and / or preventing cancer.

[0032] As a further improvement of the above-mentioned solution, the drug is a drug for inhibiting tumor growth.

[0033] Preferably, the drug includes NAT10-PROTAC.

[0034] Preferably, the cancer includes cervical cancer.

[0035] The above technical solution of the present invention has at least the following technical effects or advantages compared with the prior art:

[0036] The present invention provides a compound with the structural formula shown in formula (1). This compound can effectively degrade the NAT10 protein in cancer cells, has the ability to inhibit the proliferation and invasion of cervical cancer cells, can promote the inhibitory effect of anti-PD-L1 immunotherapeutic agents on cervical cancer and change the immune microenvironment of cervical cancer. The combined action with anti-PD-L1 immunotherapeutic agents can make the immune microenvironment develop in the direction of cervical cancer remission, and is applicable to the immunotherapy of cervical cancer.

[0037] The preparation method of the compound with the structural formula shown in formula (1) of the present invention has the advantages of high yield, simple post-treatment, and good economy. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 1H NMR spectrum of compound 1161;

[0039] Figure 2 13C NMR spectrum of compound 1161;

[0040] Figure 3 1H NMR spectrum of compound 1163;

[0041] Figure 4 13C NMR spectrum of compound 1163;

[0042] Figure 5 1H NMR spectrum of compound 1172;

[0043] Figure 6 13C NMR spectrum of compound 1172;

[0044] Figure 7 1H NMR spectrum of compound 1192;

[0045] Figure 8 13C NMR spectrum of compound 1192;

[0046] Figure 9 1H NMR spectrum of compound 1193;

[0047] Figure 10 13C NMR spectrum of compound 1193;

[0048] Figure 11 Effect diagram of different compounds degrading NAT10;

[0049] Figure 12 Effect diagram of compound 1192 degrading NAT10;

[0050] Figure 13 CCK experiment result diagram;

[0051] Figure 14 It is the result graph of the colony formation experiment;

[0052] Figure 15 It is the result graph of the transwell migration assay;

[0053] Figure 16 It is the graph of lactic acid absorption capacity;

[0054] Figure 17 It is the graph of ATP absorption capacity;

[0055] Figure 18 It is the graph of the absorption degree of glucose analogues;

[0056] Figure 19 It is the growth curve graph of subcutaneous cervical cancer in C57 mice;

[0057] Figure 20 It is the physical picture of subcutaneous cervical cancer tumor in C57 mice;

[0058] Figure 21 It is the in vivo imaging graph of subcutaneous cervical cancer tumor in C57 mice;

[0059] Figure 22 It is the comparison graph of the change in the number of CD8+ cells;

[0060] Figure 23 It is the comparison graph of the change in the number of M1 cells;

[0061] Figure 24 It is the comparison graph of the change in the number of M2 macrophages;

[0062] Figure 25 It is the comparison graph of the change in the number of MDSC cells;

[0063] Figure 26 It is the comparison graph of the change in the number of Treg cells. Detailed implementation manners

[0064] The present invention will be specifically described below in conjunction with embodiments to facilitate the understanding of those skilled in the art. It is necessary to specifically point out here that the embodiments are only used to further illustrate the present invention and cannot be understood as a limitation on the protection scope of the present invention. Non-essential improvements and adjustments made by those skilled in the art to the present invention based on the above invention content should still fall within the protection scope of the present invention.

[0065] The known starting materials of the present invention can be adopted or synthesized according to methods known in the art; for thin-layer chromatography silica gel plates, Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates are used, and the specification of the silica gel plates used in thin-layer chromatography (TLC) is 0.15 mm - 0.20 mm. For column chromatography, Yantai Huanghai silica gel with a mesh size of 100 - 200 is used as the carrier; the structure of the compound is determined by nuclear magnetic resonance (NMR), and the NMR chemical shifts are given in units of (ppm); the NMR measurement is performed using a (Bruker Avance III 400) nuclear magnetic resonance instrument, and the solvents for measurement are deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and the internal standard is tetramethylsilane (TMS).

[0066] Example 1: Preparation of Intermediate

[0067] Synthesis Method 1:

[0068]

[0069] 1. Preparation of Intermediate: 2-Cyclopentylhydrazine-1-carbothioamide (2)

[0070] Thiosemicarbazide (1) (10.97 mmol) and cyclopentanone (10.97 mmol) were refluxed in 20 mL of isopropanol at 82 °C for 12 hours. The resulting precipitate was cooled and filtered to obtain the product 2-cyclopentylhydrazine-1-carbothioamide (2), with a yield of 75%.

[0071] 2. Preparation of Intermediate: 4-(2-(2-Cyclopentylhydrazino)thiazol-4-yl)benzonitrile (3)

[0072] 2-Cyclopentylhydrazine-1-carbothioamide (2) (22.26 mmol) and 2-bromo-4'-cyanoacetophenone (22.26 mmol) were dissolved in 30 mL of isopropanol and stirred at room temperature for 12 hours. After precipitation, the product 4-(2-(2-cyclopentylhydrazino)thiazol-4-yl)benzonitrile (3) was obtained by suction filtration, with a yield of 45%.

[0073] 3. Intermediate: Preparation of N-(4-(4-cyanophenyl)thiazol-2-yl)-N-(cyclopentylimino)glycine (4) Dissolve 4-(2-(2-cyclopentylhydrazono)thiazol-4-yl)benzonitrile (3), methyl bromoacetate and potassium carbonate in DMF (eq = 1:1.2:2), stir and react at 80 °C for 2 hours. After the reaction, add water to the reaction solution, extract with ethyl acetate, and back-extract with saturated brine. Purify on a silica gel column using petroleum ether / ethyl acetate = 8:1 as the eluent, then dissolve the obtained product in 20 mL of methanol, add 10 equivalents of sodium hydroxide, stir at room temperature for 2 hours. After the reaction, dry under reduced pressure, extract with ethyl acetate, back-extract with saturated brine, and evaporate the solvent under reduced pressure to obtain the product N-(4-(4-cyanophenyl)thiazol-2-yl)-N-(cyclopentylimino)glycine (4), yield: 65%.

[0074] 4. Intermediate: Preparation of 4-(1-(4-(4-cyanophenyl)thiazol-2-yl)-2-cyclopentylhydrazono)butanoic acid (5)

[0075] The preparation method of 4-(1-(4-(4-cyanophenyl)thiazol-2-yl)-2-cyclopentylhydrazono)butanoic acid (5) refers to that of N-(4-(4-cyanophenyl)thiazol-2-yl)-N-(cyclopentylimino)glycine (4), using methyl 4-bromobutyrate instead of methyl bromoacetate, yield: 71%.

[0076] 5. Intermediate: Preparation of 7-(1-(4-(4-cyanophenyl)thiazol-2-yl)-2-cyclopentylhydrazono)heptanoic acid (6)

[0077] The preparation method of 7-(1-(4-(4-cyanophenyl)thiazol-2-yl)-2-cyclopentylhydrazono)heptanoic acid (6) refers to that of N-(4-(4-cyanophenyl)thiazol-2-yl)-N-(cyclopentylimino)glycine (4), using methyl 7-bromoheptanoate instead of methyl bromoacetate, yield: 68%.

[0078] Synthesis Method Two:

[0079]

[0080] 6. Intermediate: Preparation of 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (8)

[0081] Dissolve 3-fluorophthalic anhydride (7), 3-aminopiperidine-2,6-dione hydrochloride and potassium acetate in glacial acetic acid, reflux at 120 °C for 5 hours (eq = 1:1:3). After the reaction, pour the reaction solution into ice water, filter to generate a precipitate to obtain the product 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (8), yield: 65%.

[0082] 7. Intermediate: Preparation of 4-((4-aminobutyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione hydrochloride (10)

[0083] Dissolve 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (8) (1.0 g, 3.62 mmol) and N-Boc-1,4-butanediamine (0.82 mL, 4.34 mmol) in 5 mL of DMF, add DIPEA (1.26 mL, 7.24 mmol), and react at 90 °C for 4 hours. After the reaction is completed, add water to the reaction system and extract with ethyl acetate (25 mL × 3). The organic layer is washed with water, saturated brine, dried over anhydrous sodium sulfate, and the solvent is evaporated under reduced pressure. Flash column chromatography (0.5%-2% methanol / dichloromethane) gives 0.36 g of a yellow solid (9) with a yield of 36%. Dissolve the obtained yellow solid in 20 mL of dichloromethane, add 1 mL of 4.0 M hydrogen chloride / dioxane solution dropwise under ice bath, and react at room temperature for 1 hour. After the reaction is completed, a solid precipitates. Filter by suction and dry to obtain 0.32 g of a yellow-green solid, 4-((4-aminobutyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione hydrochloride (10), with a yield of 91%.

[0084] 8. Intermediate: Preparation of 2-(2,6-dioxopiperidin-3-yl)-4-(piperazin-1-yl)isoindoline-1,3-dione hydrochloride (13)

[0085] The preparation method of 2-(2,6-dioxopiperidin-3-yl)-4-(piperazin-1-yl)isoindoline-1,3-dione hydrochloride (13) refers to that of 4-((4-aminobutyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione hydrochloride (10), using N-Boc piperazine (11) instead of N-Boc-1,4-butanediamine, and the yield is 82%.

[0086] 9. Intermediate: Preparation of 2-(2,6-dioxopiperidin-3-yl)-4-(4-oxopiperidin-1-yl)isoindoline-1,3-dione (16a)

[0087] 2-(2,6-Dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (8) (1.0 g, 3.62 mmol) and 4-hydroxypiperidine (14a) (439.42 mg, 4.34 mmol) were dissolved in 5 mL of DMF. DIPEA (1.26 mL, 7.24 mmol) was added, and the reaction was carried out at 90 °C for 4 hours. After the reaction was completed, water and ethyl acetate (25 mL × 3) were added to the reaction system for extraction. The organic layer was washed with water, saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain 1.1 g of a yellow solid (15a) with a yield of 78.6%. The obtained yellow solid was dissolved in 20 mL of dichloromethane, and Dess-Martin periodinane (2.37 g, 5.60 mmol) was slowly added under an ice bath. The reaction was carried out at room temperature for 2 hours. After the reaction was completed, saturated sodium bicarbonate and saturated sodium thiosulfate solution (v / v = 1 / 1) were added to the reaction solution to adjust the pH to 7 - 8. Dichloromethane (25 mL × 3) was added to the reaction system for extraction. The organic layer was washed with water, saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain 1.0 g of a yellow solid, 2-(2,6-dioxopiperidin-3-yl)-4-(4-oxopiperidin-1-yl)isoindoline-1,3-dione (16a), with a yield of 90.9%.

[0088] 10. Intermediate: Preparation of 2-(2,6-dioxopiperidin-3-yl)-4-(4-(piperazin-1-yl)piperidin-1-yl)isoindoline-1,3-dione hydrochloride (18)

[0089] 2-(2,6-Dioxopiperidin-3-yl)-4-(4-oxopiperidin-1-yl)isoindoline-1,3-dione (16a) (1.0 g, 2.81 mmol) and 1-Boc-piperazine (2.1 g, 11.26 mmol) were dissolved in 20 mL of dichloromethane. 2 drops of glacial acetic acid were added, and sodium cyanoborohydride (0.88 g, 14.07 mmol) was slowly added portionwise under an ice bath. The mixture was stirred at room temperature for 2 hours. After the reaction was completed, water and dichloromethane (25 mL × 3) were added to the reaction system for extraction. The organic layer was washed with water, saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. Flash column chromatography (0.5% - 2% methanol / dichloromethane) was carried out to obtain 0.71 g of a yellow solid (17a) with a yield of 71%. The obtained yellow solid was dissolved in 15 mL of dichloromethane, and 1 mL of 4.0 M hydrogen chloride / dioxane solution was added dropwise under an ice bath. The reaction was carried out at room temperature for 1 hour, and a solid precipitated after the reaction was completed. The solid was filtered by suction and dried to obtain 0.65 g of a yellow solid, 2-(2,6-dioxopiperidin-3-yl)-4-(4-(piperazin-1-yl)piperidin-1-yl)isoindoline-1,3-dione hydrochloride (18), with a yield of 91.5%

[0090] 1. Preparation of Intermediate: 2-(2,6-Dioxopiperidin-3-yl)-4-(4-(piperazin-1-ylmethyl)piperidin-1-yl)isoindole-1,3-dione Hydrochloride (19)

[0091] The preparation method of 2-(2,6-dioxopiperidin-3-yl)-4-(4-(piperazin-1-ylmethyl)piperidin-1-yl)isoindole-1,3-dione hydrochloride (19) refers to that of 2-(2,6-dioxopiperidin-3-yl)-4-(4-(piperazin-1-yl)piperidin-1-yl)isoindoline-1,3-dione hydrochloride (18), using 4-(hydroxymethyl)piperidine (14b) instead of 4-hydroxypiperidine (14a), with a yield of 55%.

[0092] Example 2: Preparation of Compounds

[0093] Synthetic Method 3:

[0094]

[0095] 1. Preparation of Compound (1161) of Formula (1): 4-(2-(2-Cyclopentyl-1-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperazin-1-yl)-2-oxoethyl)hydrazino)thiazol-4-yl)benzonitrile

[0096] Dissolve N-(4-(4-Cyanophenyl)thiazol-2-yl)-N-(cyclopentylimino)glycine (4) (40.0 mg, 0.12 mmol) and 2-(2,6-dioxopiperidin-3-yl)-4-(piperazin-1-yl)isoindoline-1,3-dione hydrochloride (13) (49.0 mg, 0.13 mmol) in DMF, add HATU (53.6 mg, 0.14 mmol) and triethylamine (0.065 mL, 0.47 mmol), and stir at room temperature for 4 hours. After the reaction is completed, add water to the reaction system and extract with dichloromethane (25 mL × 3): The organic layer is washed with water, saturated brine, dried over anhydrous sodium sulfate, the solvent is evaporated under reduced pressure, and flash column chromatography (0.5% - 2% methanol / dichloromethane) is performed to obtain 41 mg of yellow solid (1161) with a yield of 46%.

[0097] The characterization results of the 1H NMR and 13C NMR spectra of Compound 1161 are as follows:

[0098] 1 H NMR (400 MHz, DMSO-d 6)δ11.11(s,1H),7.99(d,J = 8.9Hz,2H),7.80(d,J = 8.4Hz,2H),7.74(d,J = 7.6Hz,1H),7.48(s,1H),7.42(d,J = 7.0Hz,1H),7.37(d,J = 8.8Hz,1H),5.18(d,J = 5.7Hz,1H),5.15–5.07(m,1H),4.69(d,J = 12.5Hz,1H),3.70(d,J = 23.6Hz,4H),3.52(d,J = 33.2Hz,3H),3.39(s,5H),3.28(s,3H),2.95–2.81(m,1H),2.66–2.53(m,2H),2.46(s,1H),2.04(dd,J = 12.6,6.1Hz,1H), as specifically shown in Figure 1 shown below.

[0099] 13 C NMR(101MHz,Chloroform-d)δ176.38,175.14,171.07,168.32,167.07,167.03,166.64,149.72,149.61,140.63,135.90,132.28,126.17,123.31,116.67,110.30,107.73,54.20,51.75,49.20,41.69,34.81,31.36,30.13,29.63,29.25,22.61, as specifically shown in Figure 2 shown below.

[0100] 2. Preparation of the compound (1163) of formula (1): 4-(2-(2-Cyclopentyl-1-(2-(4-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperidin-4-yl)piperazin-1-yl)-2-oxoethyl)hydrazino)thiazol-4-yl)benzonitrile:

[0101] The preparation method of 4-(2-(2-Cyclopentyl-1-(2-(4-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperidin-4-yl)piperazin-1-yl)-2-oxoethyl)hydrazino)thiazol-4-yl)benzonitrile (1163) refers to (1161), using 2-(2,6-dioxopiperidin-3-yl)-4-(4-(piperazin-1-yl)piperidin-1-yl)isoindoline-1,3-dione hydrochloride (18) instead of 2-(2,6-dioxopiperidin-3-yl)-4-(piperazin-1-yl)isoindoline-1,3-dione hydrochloride (13), yield: 41%.

[0102] The characterization results of the 1H NMR and 13C NMR spectra of Compound 1163 are as follows:

[0103] 1 H NMR (400 MHz, Chloroform-d) δ 8.68 (s, 1H), 7.87 (d, J = 8.2 Hz, 2H), 7.65 (d, J = 8.0 Hz, 2H), 7.59 (t, J = 8.0 Hz, 1H), 7.40 (d, J = 7.2 Hz, 1H), 7.18 (d, J = 8.4 Hz, 1H), 7.00 (s, 1H), 5.02 (dd, J = 12.2, 5.3 Hz, 1H), 4.75 (s, 2H), 4.63 (s, 2H), 3.82 (t, J = 12.0 Hz, 2H), 3.65 (s, 2H), 3.55 (d, J = 4.9 Hz, 2H), 2.85 (dp, J = 37.0, 13.7, 12.5 Hz, 6H), 2.70 (d, J = 6.2 Hz, 2H), 2.63 (d, J = 5.4 Hz, 2H), 2.53 (d, J = 11.7 Hz, 1H), 2.18–2.10 (m, 1H), 1.92 (d, J = 11.2 Hz, 6H), 1.86–1.75 (m, 3H), as specifically shown in Figure 3 shown below.

[0104] 13 C NMR (101 MHz, DMSO-d 6 ) δ 175.41, 173.25, 170.45, 167.52, 166.75, 166.58, 150.20, 149.21, 139.47, 136.19, 134.05, 132.93, 126.46, 124.34, 119.51, 116.81, 114.99, 109.64, 108.47, 60.92, 54.11, 50.81, 49.21, 48.96, 45.04, 42.15, 31.37, 29.42, 28.18, 22.48, as specifically shown in Figure 4 shown below.

[0105] 3. Preparation of the compound of formula (1) (1172): 4-(1-(4-(4-cyanophenyl)thiazol-2-yl)-2-cyclopentylidenehydrazono)-N-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)butyl)butanamide:

[0106] 4-(1-(4-(4-Cyanophenyl)thiazol-2-yl)-2-cyclopentylidenehydrazino)butanoic acid (5) (40.0 mg, 0.11 mmol) and 4-((4-aminobutyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione hydrochloride (10) (45.5 mg, 0.12 mmol) were dissolved in DMF, and EDCI (22.9 mg, 0.12 mmol), HOBT (16.1 mg, 0.12 mmol), and triethylamine (0.06 mL, 0.43 mmol) were added. The mixture was stirred at room temperature for 4 hours. After the reaction was completed, water and dichloromethane (25 mL × 3) were added to the reaction system for extraction. The organic layer was washed with water, saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. Flash column chromatography (0.5%-2% methanol / dichloromethane) gave 42 mg of a yellow solid (1172) with a yield of 49%.

[0107] The characterization results of the 1H NMR and 13C NMR spectra of compound 1172 are as follows:

[0108] 1 H NMR (400 MHz, Chloroform-d) δ 8.63 (s, 1H), 7.91 (d, J = 8.0 Hz, 2H), 7.64 (d, J = 8.0 Hz, 2H), 7.48 (t, J = 7.9 Hz, 1H), 7.08 (d, J = 7.1 Hz, 1H), 6.97 (s, 1H), 6.84 (d, J = 8.6 Hz, 1H), 6.18 (d, J = 6.0 Hz, 1H), 6.00 (d, J = 6.3 Hz, 1H), 4.93 (dd, J = 11.8, 5.3 Hz, 1H), 3.98 (t, J = 7.1 Hz, 2H), 3.23 (q, J = 6.7 Hz, 4H), 2.94–2.71 (m, 3H), 2.58 (d, J = 7.1 Hz, 2H), 2.47 (d, J = 7.3 Hz, 2H), 2.24 (t, J = 7.3 Hz, 2H), 2.16–2.08 (m, 1H), 2.04–1.98 (m, 2H), 1.86 (d, J = 5.4 Hz, 4H), 1.64–1.58 (m, 2H), 1.52 (t, J = 7.7 Hz, 2H), as specifically shown Figure 5 as follows.

[0109] 1313C NMR (101 MHz, Chloroform-d) δ 183.23, 172.42, 172.32, 171.26, 169.46, 168.57, 167.53, 149.74, 146.73, 139.15, 136.15, 132.33, 126.29, 116.58, 111.49, 110.49, 109.88, 106.86, 52.02, 48.83, 42.06, 38.92, 33.80, 33.66, 31.49, 31.36, 26.99, 26.45, 24.95, 24.20, 22.82, 22.71, specifically as Figure 6 shown.

[0110] Compound (1192) of formula (1): Preparation of 4-(2-cyclopentyl-1-(7-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperazin-1-yl)-7-oxoheptyl)hydrazino)thiazol-4-yl)benzonitrile:

[0111] The preparation method of 5-(2-cyclopentyl-1-(7-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperazin-1-yl)-7-oxoheptyl)hydrazino)thiazol-4-yl)benzonitrile (1192) refers to (1161), using 7-(1-(4-(4-cyanophenyl)thiazol-2-yl)-2-cyclopentylhydrazono)heptanoic acid (6) instead of N-(4-(4-cyanophenyl)thiazol-2-yl)-N-(cyclopentylimino)glycine (4), yield: 36%.

[0112] The characterization results of the 1H NMR and 13C NMR of Compound 1192 are as follows:

[0113] 11H NMR (400 MHz, Chloroform-d) δ 8.37 (s, 1H), 7.92 (d, J = 8.2 Hz, 2H), 7.67–7.62 (m, 3H), 7.46 (d, J = 7.2 Hz, 1H), 7.15 (d, J = 8.3 Hz, 1H), 6.94 (s, 1H), 4.99 (dd, J = 12.1, 5.4 Hz, 1H), 4.20 (s, 2H), 3.95–3.80 (m, 2H), 3.74 (dd, J = 9.4, 5.0 Hz, 2H), 3.67 (q, J = 7.4, 6.3 Hz, 2H), 3.27 (t, J = 5.1 Hz, 2H), 2.95–2.74 (m, 3H), 2.38 (t, J = 7.5 Hz, 2H), 2.14 (dd, J = 11.7, 5.5 Hz, 1H), 1.87 (p, J = 4.6 Hz, 2H), 1.81–1.74 (m, 4H), 1.69 (s, 4H), 1.46 (qt, J = 9.1, 4.5 Hz, 6H), specifically as Figure 7 shown.

[0114] 13 13C NMR (101 MHz, Chloroform-d) δ 174.57, 171.67, 170.87, 168.15, 167.11, 166.60, 149.83, 139.32, 135.82, 134.09, 132.31, 126.20, 123.31, 119.18, 117.98, 116.42, 110.30, 106.17, 54.21, 51.95, 50.17, 49.17, 45.65, 41.34, 33.05, 31.35, 29.63, 28.98, 26.31, 25.76, 24.93, 22.61, specifically as Figure 8 shown.

[0115] 5. Preparation of the compound (1193) of formula (1): 4-(2-(2-Cyclopentyl-1-(7-(4-((1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperidin-4-yl)methyl)piperazin-1-yl)-7-oxoheptyl)hydrazino)thiazol-4-yl)benzonitrile:

[0116] The preparation method of 4-(2-(2-cyclopentyl-1-(7-(4-((1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperidin-4-yl)methyl)piperazin-1-yl)-7-oxoheptyl)hydrazino)thiazol-4-yl)benzonitrile (1193) refers to (1192), using 2-(2,6-dioxopiperidin-3-yl)-4-(4-(piperazin-1-ylmethyl)piperidin-1-yl)isoindole-1,3-dione hydrochloride (19) instead of 2-(2,6-dioxopiperidin-3-yl)-4-(piperazin-1-yl)isoindoline-1,3-dione hydrochloride (13), yield: 31%.

[0117] The characterization results of the 1H NMR and 13C NMR of compound 1193 are as follows:

[0118] 1 H NMR(400MHz,Chloroform-d)δ8.22(s,1H),7.93(d,J=8.2Hz,2H),7.66(d,J=8.2Hz,2H),7.61–7.55(m,1H),7.38(d,J=7.2Hz,1H),7.19(d,J=8.4Hz,1H),6.95(s,1H),4.98(dd,J=12.1,5.4Hz,1H),4.19(s,2H),3.79–3.71(m,4H),3.62(d,J=5.4Hz,2H),3.45(t,J=5.0Hz,2H),3.00–2.84(m,4H),2.84–2.70(m,2H),2.40(q,J=4.9Hz,4H),2.33(t,J=7.5Hz,2H),2.27(d,J=7.2Hz,2H),2.17–2.10(m,1H),1.91(dt,J=13.2,6.6Hz,3H),1.81–1.74(m,4H),1.72–1.64(m,4H),1.51–1.42(m,7H), specifically as Figure 9 shown.

[0119] 1313C NMR (101 MHz, Chloroform-d) δ 174.55, 171.39, 170.97, 168.22, 167.38, 166.65, 150.83, 149.92, 139.35, 135.44, 134.08, 132.31, 126.21, 123.60, 119.18, 117.13, 115.25, 110.30, 106.15, 64.26, 54.28, 53.98, 53.12, 51.86, 51.53, 49.05, 45.51, 41.51, 32.98, 32.94, 31.36, 30.81, 29.63, 29.02, 26.36, 25.79, 24.97, 22.63, specifically as Figure 10 shown.

[0120] Example 3: Effect of the compound on cervical cancer

[0121] 1. Degradation effect of compounds 1161, 1163, 1172, 1192 and 1193 on NAT10

[0122] (1) Experimental procedure:

[0123] ① Cell lines and cell culture: SiHa, U14 and human cervical squamous cell carcinoma (HFF) cells were purchased from the Cell Bank of the Chinese Academy of Sciences. All cells (2×10 5 cells per well) were seeded in 6-well plates with DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. The temperature of the incubator was maintained at 37 °C and the carbon dioxide level was 5%. After the cells adhered, 20 μmol of compounds 1161, 1163, 1172, 1192 and 1193 were added respectively, and the cells were collected after 36 hours.

[0124] ② Western blot (WB) analysis: SiHa, U14 and HFF cells were homogenized in RIPA lysis buffer, and the protein lysates were normalized using a bicinchoninic acid (BCA) protein assay kit. 20 μg of protein lysate was loaded onto acrylamide gels and electrophoretically separated under denaturing conditions, and then transferred to PVDF membranes by wet electrophoresis; the membranes were blocked with PBS containing 5% non-fat milk and 0.05% surfactant Tween-20 for 1 hour at room temperature and incubated overnight at 4 °C: two antibodies, NAT10 and β-tubulin, were incubated for 1 hour at room temperature. After antibody incubation, the membranes were washed with PBS containing 0.05% Tween-20, washed 3 times at 15-minute intervals.

[0125] (2) Experimental results

[0126] Figure 11The effect diagrams of different compounds degrading NAT10 are shown as follows. Figure 11 It can be seen that NAT10 is an important regulatory enzyme for acetylation and promotes the progression of cervical cancer. Compounds 1161, 1163, 1172, 1192, and 1193 can all inhibit the protein of NAT10, and among them, compound 1192 has the best inhibitory effect.

[0127] Figure 12 The effect diagram of compound 1192 degrading NAT10 is shown as follows. Figure 12 It can be seen that NAT10 is an important regulatory enzyme for acetylation and promotes the progression of cervical cancer. Compound 1192 can inhibit the protein of NAT10 and has no degrading effect on normal cells.

[0128] 2. The phenotype of compound 1192 on inhibiting cervical cancer cell lines

[0129] (1) Experimental procedures:

[0130] ① CCK8 assay: SiHa cells (2×105 cells per well) were seeded in 6-well plates with DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. The temperature of the incubator was maintained at 37°C and the carbon dioxide level was 5%. After the cells adhered, 20 μmol of compound 1192 was added. After 36 hours, the cells and RPMI 1640 medium containing 10% fetal bovine serum were seeded in 96-well plates (3000 cells per well). After the cells adhered to the bottom of the well, 10 μL of CCK-8 solution was added, and the plates were returned to the incubator for 2 hours. The absorbance at a wavelength of 450 nm (Related450nmAbsorbtlon) was measured using a microplate reader.

[0131] ② Colony formation assay: SiHa cells (2×10 5 cells per well) were seeded in 6-well plates and cultured in RPMI1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin compound. The temperature of the incubator was maintained at 37°C and the carbon dioxide level was 5%. After the cells adhered, 20 μmol of compound 1192 was added. After 36 hours, the cells were seeded in 96-well plates (3000 cells per well) and RPMI 1640 medium containing 10% fetal bovine serum. After at least 50 colonies formed, they were fixed with methanol for 20 minutes, stained with 0.1% gentian violet for 10 minutes, washed 3 times with PBS, and finally imaged and counted. All analyses were performed using GraphPad Prism9, and the data were expressed as the mean ± SEM.

[0132] ③ Transwell migration assay: SiHa cells (2×10 5(2×10⁵ cells) were seeded in 6-well plates and cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin compound. The temperature of the incubator was maintained at 37 °C and the carbon dioxide level was 5%. After the cells adhered, 20 μmol of compound 1192 was added. After 36 hours, co-culture experiments were performed with SiHa and SiHa + 1192 cells. Migration was stimulated by the concentration gradient between the upper and lower chambers. Cells in serum-free medium were seeded in the upper chamber, and exosome-free complete medium was added to the lower chamber. After incubation for 18 hours, the membranes were fixed with methanol for 20 minutes, stained with 0.1% gentian violet solution for 10 minutes, and washed with PBS. Finally, cell migration was calculated. All analyses were performed using GraphPad Prism 9, and the data were expressed as the mean ± SEM.

[0133] (2) Experimental results

[0134] Figure 13 and Figure 14 are the experimental result graphs of CCK and colony formation respectively. As can be seen from Figure 13 - 14 compound 1192 can effectively inhibit the proliferation of cervical cancer cells.

[0135] Figure 15 is the result graph of the transwell migration assay. As can be seen from Figure 15 compound 1192 can effectively inhibit the invasive ability of cervical cancer cells and prevent the progression of cervical cancer.

[0136] 3. Absorption capacity of compound 1192 for lactate / ATP / glucose

[0137] (1) Experimental procedures:

[0138] ① Lactate determination: SiHa cells (2×10⁵ cells per well) were seeded in 6-well plates and cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin compound. The temperature of the incubator was maintained at 37 °C and the carbon dioxide level was 5%. After the cells adhered, 20 μmol of compound 1192 was added. After 36 hours, the operation was carried out according to the lactate detection kit. All analyses were performed using GraphPad Prism 9, and the data were expressed as the mean ± SEM.

[0139] ② ATP assay: SiHa cells (2×10⁵ cells per well) were seeded in 6-well culture dishes and cultured in 10% fetal bovine serum and 1% penicillin-streptomycin compound. The temperature of the incubator was maintained at 37 °C and the carbon dioxide level was 5%. After the cells adhered, 20 μmol of compound 1192 was added. After 36 hours, the operation was carried out according to the ATP detection kit, and all analyses were performed using GraphPad Prism 9, and the data were expressed as the mean ± SEM.

[0140] ③ Glucose absorption assay: SiHa cells (2×10⁵ cells per well) were seeded in 6-well culture dishes containing 10% fetal bovine serum and 1% penicillin-streptomycin compound. The temperature of the incubator was maintained at 37 °C and the carbon dioxide level was 5%. After the cells adhered, 20 μmol of compound 1192 was added. After 36 hours, the cells were processed according to the 2-NBDG protocol, and all analyses were performed using GraphPad Prism 9. The data were expressed as the mean ± SEM.

[0141] (2) Experimental results

[0142] Figure 16 - 17 They are the graph of the lactic acid absorption capacity content (Related lactic acid level) and the graph of the ATP absorption capacity content (Related ATP concentration), Figure 18 and the graph of the absorption degree of glucose analogs. It can be seen from Figure 16 - 18 that with the addition of compound 1192, the lactic acid production of tumor cells decreased, the ATP increased, and the glycolysis level decreased.

[0143] 4. Effect of compound 1192 on the immune microenvironment of cervical cancer

[0144] (1) Experimental procedures:

[0145] Six-week-old female C57 mice were purchased from the Guangdong Medical Experimental Animal Center. The animal handling and experimental procedures were approved by the Institutional Animal Research Ethics Committee of Southern Medical University.

[0146] Experiment 1 (Growth curve and tumor): U14 cells (10⁷ cells per mouse, approximately 100 - 150 μL cell suspension) were implanted subcutaneously into the lower dorsal lateral side of the mice. When the tumor volume reached 50 mm 3 , the tumor growth was evaluated to determine exosomes from different cells (Group 1: U14 cells; Group 2: U14 + Remodelin cells; Group 3: U14 + anti-PDL1 cells; Group 4: U14 + 1192 cells; Group 5: U14 + anti-PDL1 + 1192 cells). The tumor volume was measured every 3 days, and the calculation formula was as follows: Volume = (Width) 2 × Length / 2. Remodelin and 1192 were injected into the peritumoral area every 2 days at a dose of 25 mg / kg, and the anti-mouse PD-L1 in vivo antibody with low endotoxin was injected every 2 days at a dose of 2.5 mg / kg. One week later, these mice were euthanized, the tumors were excised and weighed, and then the immune microenvironment tests were performed on the tumor tissues and peritumoral tissues.

[0147] Experiment 2 (in vivo imaging): Lentivirus expressing firefly luciferase was transfected into U14 cells. After screening and amplification with puromycin, U14 cells with stable firefly luciferase were generated. C57 mice were randomly divided into 5 groups for further experiments: Group 1: U14 cells; Group 2: U14 + Remodelin cells; Group 3: U14 + anti-PDL1 cells; Group 4: U14 + 1192 cells; Group 5: U14 + anti-PDL1 + 1192 cells, and each group was experimented 3 times. After the tumors were stable, the luciferase substrate was injected into the mice every 5 days, and the fluorescence values were obtained using the in vivo imaging system Maestro.

[0148] Experiment 3 (immunofluorescence flow cytometry): After the mice were euthanized, immunofluorescence flow cytometry was performed on tumor tissues and peritumoral tissues using an immunofluorescence flow kit. The immunofluorescence flow antibodies included CD11b-BV711, F4 / 80-AF647, CD206-PE, CD86-BV605, CD4-CD4-FITC IL-17-17-CY7 / CY7, CD25-PE, FOXP3-AF647, CD4-BV605, CD45-PE / CY7, CD11b-BV711, and LY-6G / LY-6C-BV421 anti-human / mouse antibodies. All analyses were performed using GraphPad Prism 9, and the data were expressed as the mean ± SEM.

[0149] (2) Experimental results

[0150] Figure 19 - 21 They are respectively the growth curve graph, the physical picture of the tumor, and the in vivo imaging graph of the subcutaneous cervical cancer volume (Tumor volume) of C57 mice. It can be seen from Figure 19 - 21 that compound 1192 can effectively inhibit the proliferation of cervical cancer in mice, prevent the progression of cervical cancer, and enhance the anti-cancer effect of the tumor immune inhibitor anti-PDL1.

[0151] Figure 22 - 26 They are respectively the comparison graphs of the changes in the numbers of CD8+ cells, M1 cells, M2 macrophages, MDSC cells, and Treg cells. It can be seen from Figure 22 - 26 that compound 1192 can effectively change the immune microenvironment of cervical cancer in mice, increase the number of CD8+ cells, reduce the number of M2 macrophages, reduce the number of MDSC cells, and reduce the number of Treg cells; and the combined effect with anti-PDL1 can increase the number of CD8+ and M1 cells more and reduce the number of M2 macrophages, MDSC cells, and Treg cells less. The combined effect can make the immune microenvironment develop in the direction of cervical cancer remission and is suitable for the immunotherapy of cervical cancer.

[0152] For those of ordinary skill in the art to which the present invention pertains, without departing from the concept of the present invention, several simple deductions or substitutions can be made without the need for creative labor. Therefore, any simple improvements made by those skilled in the art based on the disclosure of the present invention should fall within the protection scope of the present invention. The above embodiments are the preferred embodiments of the present invention, and all processes similar to the present invention and equivalent changes made thereto shall fall within the protection scope of the present invention.

Claims

1. A compound of the structural formula shown in formula (1) or a pharmaceutically acceptable salt thereof: Wherein: L represents any one of them.

2. The compound or a pharmaceutically acceptable salt thereof according to claim 1, Characterized in that The compound is selected from one of the following structural formulas:

3. A method for preparing a compound, Characterized in that The synthetic route of the compound is as follows:

4. A pharmaceutical composition, Characterized in that The pharmaceutical composition comprises the compound or a pharmaceutically acceptable salt thereof according to claim 1 or 2.

5. The pharmaceutical composition according to claim 4, Characterized in that The pharmaceutical composition further comprises a PD-L1 immunotherapeutic agent, and the mass ratio of the compound or a pharmaceutically acceptable salt thereof to the PD-L1 immunotherapeutic agent is (2-8):

1.

6. Use of the pharmaceutical composition according to claim 4 or 5 in the preparation of a drug for treating and / or preventing cervical cancer.

7. The use according to claim 6, Characterized in that The drug is selected from NAT10-PROTAC.

Images