Anti-tumor drug based on Thiolutin targeting PSMD14 / HDAC and preparation method thereof

Abstract

The present invention belongs to the field of medicine, and specifically discloses an anti-tumor drug based on a thiolutin-targeted PSMD14 / HDAC dual-target inhibitor, its preparation method and application. Using the natural product thiolutin as the lead compound, three types of Zn 2+ By combining these groups, we designed and synthesized three types of PSMD14 / HDAC dual-target inhibitors. These three types of dual-target inhibitors showed excellent inhibitory effects in lung cancer A549 and esophageal squamous cell carcinoma (ESCC) KYSE30, and have the potential to be used as dual-target anti-solid tumor drugs.

Description

Technical Field

[0001] The present invention relates to the field of medicinal chemistry, and in particular to an anti-tumor drug with Thiolutin as a lead compound, and a preparation method and application thereof. Background Art

[0002] Natural products are a treasure trove of structural diversity and various biological activities that can be developed directly or as lead compounds for new drugs. In 1952, Celmer and his colleagues first isolated and identified the natural product Thiolutin from a strain of Streptomyces albus. [1] ( Figure 1 Thiolutin has a broad spectrum of biological activities against bacteria, fungi and tumors, and its excellent biological activity has attracted widespread attention from pharmaceutical chemists. In 1953, Pfizer [2] Thiolutin was first prepared by fermentation and its MIC was determined in several bacterial strains. Despite such excellent biological activity, the large-scale preparation of thiolutin has hindered the development of thiolutin. [3] A synthesis method of Thiolutin was reported by Welichem [4] Drawing on their synthesis method, they synthesized a series of Thiolutin and its derivatives, and conducted anti-proliferative tests in colon cancer, cervical cancer, and breast cancer. They then further optimized it in another patent. [5] , and screened out several highly active compounds. All synthesized compounds were submitted to the National Cancer Institute (NCI) for bioassays on more than 60 cancer cells, but the NCI comparative calculation method did not find any significant activity correlation with the compounds in the NCI database. Welichem's results show that dithiopyrrolidine compounds exert anti-tumor effects through a unique mechanism of action. Current research has confirmed that Thiolutin is a new type of PSMD14 inhibitor, and its anti-cancer mechanism is explained as follows: Thiolutin first enters the cell as a prodrug, and the thioether is reduced to an active form [6] , dithiol and Zn of PSMD14 in cells 2+ Chelation inhibits PSMD14 activity, thereby inhibiting tumor growth [7-10] PSMD14, also known as RPN11 or POH1, has a JAMM domain and is activated by Zn 2+ It can only exert its deubiquitinating enzyme activity under dependent conditions and is highly expressed in a variety of tumor cells.

[11] PSMD14 affects the occurrence and development of tumors by regulating protein deubiquitination and stability, and regulates various malignant biological behaviors such as tumor proliferation, invasion and metastasis, and chemotherapy resistance.

[0003] Histone deacetylase (HDAC) plays an important role in the post-translational modification of amino acid side chains and is widely and highly expressed in tumor cells.

[12] Therefore, the research and development of HDAC inhibitors is of great significance for the treatment of tumors. HDAC inhibitors can be divided into three parts: cap group, linker and Zn 2+ Chelated active site (ZBG). HDAC inhibitors are based on their interaction with Zn 2+ The chemical structure characteristics of the combination can be divided into four categories: hydroxamic acids, cyclic tetrapeptides, short-chain fatty acids and phenylamides

[13] ( Figure 3 ). Hydroxamic acid HDACs inhibitor SAHA (Vorinostat, 1)

[14] , mainly used to treat cutaneous T-cell lymphoma. Romidepsin (2) is a cyclic tetrapeptide HDACs inhibitor launched by Celgene.

[15] , only used for the treatment of T lymphocyte tumors. O-amino HDACs inhibitor RG2833 (3)

[16] , mainly used for T lymphocyte tumors, breast cancer, bladder cancer, etc. Azumamide E (4) is the only short-chain fatty carboxylic acid inhibitor

[17] , which has not yet been used in clinical trials.

[0004] Studies have shown that HDACs inhibitors have a synergistic anti-tumor effect when used in combination with PSMD14 inhibitors ( Figure 4 ), and its interaction mechanism is as follows: 1. Thiolutin disrupts the interaction between PSMD14 and Snail, promotes the ubiquitination and degradation of Snail, thereby inhibiting epithelial-mesenchymal transition (EMT) and weakening the migration ability of tumor cells. 2. HDACs inhibitors can activate the p53 tumor suppressor function by regulating the acetylation process of Lys382. p53 activates miRNA-34 targeting Snail, thereby inhibiting the activity of Snail. 3. Snail causes deacetylation of p53K382 by binding to p53 and histone deacetylase (HDAC1).

[0005] In the previous research, the research team screened a strain of Streptomyces populiradicis TRM70303 from the local soil and isolated its metabolite Thiolutin.

[18] Thiolutin has certain anti-tumor cell activity in different tumor cells. 2+Binding group (ZBG): hydroxamic acid, o-phenylenediamine and short-chain fatty carboxylic acid. Through the pharmacophore fusion strategy, a series of PSMD14 / HDAC dual-target inhibitors were designed and synthesized for the first time and their activity against esophageal cancer in vivo and in vitro was explored.

[0006] References:

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[0013] [7]Jing C,Li X,Zhou M,et al.The Psmd14 Inhibitor Thiolutin as a NovelTherapeutic Approach for Esophageal Squamous Cell Carcinoma throughFacilitating Snail Degradation[J].Theranostics,2021,11(12):5847.

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[16] Testa G, Cavallo F, Troglio F, et al.Histone Deacetylase Inhibitorsand Uses Thereof for Treating Autism Spectrum Disorder and IntellectualDisability,WO2022058405[P / OL].2022-.

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[0024]

[18] Zhang Lili, Liu Zhanwen, Wan Chuanxing, et al. Preparation method and application of thiolutin from a new species, CN115261422A[P / OL]. 2022-11-01.

[0025]

[19] Zhu R, Liu Y, Zhou H, et al. Deubiquitinating Enzyme Psmd14 Promotes Tumor Metastasis through Stabilizing Snail in Human Esophageal Squamous CellCarcinoma[J]. Cancer letters, 2018, 418(125-134. Summary of the Invention

[0026] This paper uses the natural product Thiolutin as the lead compound and introduces three types of Zn through different alkyl diacid linkers. 2+ By combining these groups, three types of PSMD14 / HDAC dual-target inhibitors were designed and synthesized, and the present invention was completed through activity verification.

[0027] The present invention first provides a PSMD14 / HDAC dual-target inhibitor, the structural formula of which is as follows:

[0028]

[0029] Among them, n is 5-12, preferably 5, 6, 7, 8, 10, 12, and most preferably 6.

[0030] Preferably, the structure is one of the following:

[0031] Among them, n is 5-12, preferably 5, 6, 7, 8, 10, or 12.

[0032] More preferably, the structure is as follows:

[0033]

[0034] The present invention also provides a method for preparing the PSMD14 / HDAC dual-target inhibitor, which comprises the following steps:

[0035] S1, Thiolutin is hydrolyzed by 12M hydrochloric acid in 1,4-dioxane at high temperature to give 7-aminothioglucanin hydrochloride;

[0036] S2. Alkyl diacid (pimelic acid, suberic acid, azelaic acid, sebacic acid, dodecyl diacid, tetradecyl diacid) is prepared by adding tert-butanol to a dichloromethane system with EDCI = 1:1 and catalyzing with DMAP to obtain mono-tert-butyl esters of alkyl diacids (pimelic acid, suberic acid, azelaic acid, sebacic acid, dodecyl diacid, tetradecyl diacid);

[0037] S3, condensing a mixed anhydride of alkyl diacid mono-tert-butyl ester and isobutyl chloroformate with 7-aminothiogarcinol hydrochloride in an alkaline solution of dichloromethane to obtain 7-aminothiogarcinol alkyl diacid (pimelic acid, suberic acid, azelaic acid, sebacic acid, dodecyl diacid, tetradecyl diacid) mono-tert-butyl ester;

[0038] S4, trifluoroacetic acid de-tert-butylation to obtain 7-aminothiolutein-alkyl monocarboxylic acid (Formula I);

[0039] S5, 7-aminothioglucose-alkyl monocarboxylic acid reacts with hydroxylamine or o-phenylenediamine through a mixed acid anhydride method or an active ester method to obtain a compound of formula II or formula III.

[0040] Preferably, the specific method is:

[0041]

[0042] Synthesis route, reagents and conditions of the compound: (I) 1,4-dioxane, hydrochloric acid, reaction at room temperature to heating reflux; (II) dichloromethane, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine, reaction at 0℃ to room temperature; (III) dichloromethane, isobutyl chloroformate, compound 2,4-methylmorpholine, reaction at 0℃ to room temperature; (IV) dichloromethane, trifluoroacetic acid, reaction at 0℃ to room temperature; (V) NN-dimethylformamide, isobutyl chloroformate, hydroxylamine hydrochloride, 4-methylmorpholine, reaction at 0℃ to room temperature (VI) tetrahydrofuran, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, 4-pyrrolidinylpyridine, reaction at room temperature.

[0043] The present invention also provides use of the PSMD14 / HDAC dual-target inhibitor in the preparation of anti-tumor drugs.

[0044] Specifically, the tumor refers to esophageal cancer or lung cancer.

[0045] The present invention also provides an anti-tumor drug containing the PSMD14 / HDAC dual-target inhibitor as an active ingredient.

[0046] Specifically, it further contains pharmaceutically acceptable excipients, and more specifically, it is an injection dosage form and an oral dosage form.

[0047] Using the natural product Thiolutin as the lead compound, three types of Zn were introduced through different alkyl diacid linkers. 2+ By combining these groups, we designed and synthesized three classes of PSMD14 / HDAC dual-target inhibitors. These three classes of dual inhibitors showed excellent inhibitory effects in lung cancer A549 and esophageal squamous cell carcinoma (ESCC) KYSE30, and have the potential to develop dual-target anti-solid tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 、(A)Structure of Thiolutin.

[0049] Figure 2 , the mechanism of antitumor activity of Thiolutin.

[0050] Figure 3 、 Four Structures of major classes of HDAC inhibitors.

[0051] Figure 4 Synergistic anti-tumor mechanism of PSMD14 inhibitors and HDAC inhibitors

[0052] Figure 5Effects of compound 8b on PSMD14 and HDAC. KYSE30 cells were treated with SAHA (0.1 μM), Capzimin (3 μM), and 8b (0.1 μM) for 24 h, respectively. The expression levels of PSMD14, Snail, acetyl-H3 (Ac-H3), and acetyl-H4 (Ac-H4) proteins were detected by western blot (A). Western blotting was performed using ImageJ for quantitative analysis (B). Data are expressed as mean ± SEM, n = 3. *P < 0.05, **P < 0.01 compared with the control group.

[0053] Figure 6 、 8 b Effects on KYSE30 cell motility. (A) Migration assay, scale bar 100 μm, (B) Gap closure rate, (C) Transwell assay, scale bar 200 μm, (D) Cell number. (Data in this figure are mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001.)

[0054] Figure 7 Molecular docking models of compound 8b with PSMD14 and HDAC protein (PDB code 1C3R). (A) Surface docking model of compound 8b with PSMD14. (B) Docking model of compound 8b with the PSMD14 catalytic domain. (C) Surface docking model of compound 8b with the HDAC catalytic domain (PDB code 1C3R). (D) Docking model of compound 8b with the HDAC catalytic domain (PDB code 1C3R).

[0055] Figure 8 In vivo antitumor activity of compound 8b. (A) Antitumor activity of compound 14d compared with CAP and SAHA in a nude mouse KYSE30 xenograft model. Tumor inhibition index (TGI) = (1 - tumor volume / control group tumor volume) × 100%. Data are presented as mean ± SD. (B) Changes in mouse body weight during treatment. (C) Anatomical image of KYSE30 tumor tissue. *P < 0.05, ***P < 0.001, tested using one-way analysis of variance.

[0056] Figure 9 Design of dual-target PSMD14-HDAC inhibitors. (A) PSMD14 protein was obtained from the 26S deubiquitinating enzyme (PDB 5GJR). (B) Binding mode of thiolutin and PSMD14. (C) Sequestration of thiolutin and the PSMD14 active site. (D) Design strategy for dual-target PSMD14-HDAC inhibitors.

[0057] Figure 10 , Thiolutin fermentation and separation and purification

[0058] Figure 11 , synthetic routes, conditions and reagents of dual-target inhibitors. DETAILED DESCRIPTION

[0059] The present invention is described below through specific embodiments in order to better understand the present invention, but it does not constitute a limitation of the present invention.

[0060] Example 1. Structural design of novel PSMD14 / HDAC dual inhibitors

[0061] 1.1 Construction of PSMD14 protein

[0062] PSMD14 was taken from chain 9 of PDB ID: 5GJR, and the missing residues 1-27 and 164-189 were constructed by Modeller 10.4; the catalytic core Zn 2+ The residue deletion sites of the domain (beyond Ser224) were obtained from another deubiquitinase CSN5 (protein subunit of the COPS signalosome; PDB ID: 5JOG), and the final structure was obtained by aligning the respective catalytic sites (HIS-138, HIS-140, ASP-151) and (HIS-113, HIS-115, ASP-126). Figure 5 Middle A).

[0063] 1.2 Structural design of PSMD14 / HDAC dual inhibitors

[0064] At present, the research on biological activity is mainly focused on Thiolutin, and there are few reports on the research of Thiolutin derivatives. On the one hand, the chemical synthesis steps of Thiolutin are lengthy, difficult and low in yield; on the other hand, the cost of obtaining Thiolutin by microbial fermentation and separation is high and the yield is low. According to the authorized patent of Weilichem, the derivatives of Thiolutin after aminoacylation at the N7 position often have stronger anti-cancer activity. Considering that the disulfide bond is the active site of Thiolutin, the steric hindrance and electronegativity caused by the addition of the C3 position may affect the function of Thiolutin in the body. On the other hand, the docking of the above-constructed PSMD14 protein structure (5-A) with Thiolutin revealed the binding mode of the two: the enedithiol of Thiolutin directly binds to the Zn in PSMD14. 2+ Chelation, the side chain of the substituent at position N7 of Thiolutin is not bound to the protein and is exposed to the solvent region ( Figure 5 Middle B and Figure 5 Middle C).

[0065] Based on the binding mode of Thiolutin and PSMD14, the physicochemical properties of Thiolutin and the characteristics of HDAC inhibitors, we adopted a pharmacophore fusion strategy to connect the HDAC inhibitor ZBG at the N7 position of Thiolutin through an alkyl linker to design and synthesize a dual-target inhibitor of PSMD14 and HDAC ( Figure 5 Middle D).

[0066] Example 2. Extraction and separation of Thiolutin

[0067] The fermentation broth of Streptomyces populiradicis TRM70303 was dried to obtain a yellow powder. The powder was purified by beating, centrifuging, and drying to obtain a crude product, which was purified by column chromatography to obtain Thiolutin ( Figure 6 ).

[0068] Example 3. Synthesis of the chemical compound of the present invention

[0069] Its synthetic route is Figure 7 Thiolutin 1 was hydrolyzed with amide and recrystallized to obtain the amino salt of thiolutin 2. Compounds 4a-4f and compound 2 were successfully synthesized to form 5a-5f, which were then deprotected to obtain the carboxylic acids of thiolutin 6a-6f. Finally, the carboxylic acids of thiolutin were condensed with amide to obtain the target compounds 7a-7f and 8a-8f. The structures of 6a-6f, 7a-7f, and 8a-8f Figure 7 As shown, the 18 target compounds synthesized were characterized by NMR and HR-ESI-MS, and HPLC>95%.

[0070] 3.1. 6-amino-4-methyl-[1,2]dithio[4,3-b]pyrrol-5(4H)-one hydrochloride or (7-aminothioglucanin hydrochloride) (Compound 2)

[0071] A mixture of 1.4-dixone (500 mL) and thiolutin (12 g, 52.6 mmol) was added to a three-necked flask and heated to reflux. HCl was slowly added to the reaction system. The reaction continued, and the system gradually turned dark green. The reaction system was cooled to room temperature and filtered under reduced pressure to obtain a black solid. The black solid was recrystallized from hot HCl to obtain dark green prismatic crystals 2 (5.9 g, 50.4% yield). Due to the instability of compound 2 in DMSO-d6, it was not further characterized by NMR.

[0072] 3.2. Mono-tert-butyl pimelate (Compound 4a)

[0073] Pimelic acid 3a (10 g, 62.43 mmol) was dissolved in CH2Cl2 (300 mL) and stirred rapidly. EDCI (11.97 g, 1 eq), DMAP (7.63 g, 1 eq), and tert-butanol (46.3 g, 10 eq) were added to the suspension at 0°C, returned to room temperature, and allowed to react overnight. The reaction solution was added to 1M HCl and extracted three times with CH2Cl2. The organic phases were combined and washed with saturated brine. The organic phase was dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography to obtain a colorless liquid 4a (4.1 g, 18.96 mmol, 30.4% yield). 1 H NMR (500MHz, DMSO-d6) δ11.98(s,1H),2.17(dd,J=16.4,7.5Hz,4H),1.50–1.44(m,4H),1.38(s,9H),1.29–1.22(m,2H). 13 C NMR(125MHz,DMSO-d6)δ174.43,172.24,79.40,34.67,33.53,27.97,27.78,24.38,24.21.HRMS(ESI)forC 11 H 19 O4[MH] - calcd215.1289,found215.1277.

[0074] 3.3. Mono-tert-butyl suberate (Compound 4b)

[0075] The synthesis process was similar to that of compound 4a, except that suberic acid 3b was used as the starting material. Suberic acid 3b (10 g, 57.41 mmol), EDCI (11.01 g, 1 eq), DMAP (7.01 g, 1 eq), and tert-butanol (42.6 g, 10 eq) were added to afford 4b as a colorless liquid (5.5 g, 23.88 mmol) in a yield of 41.6%. 1 H NMR (500MHz, DMSO-d6) δ11.97(s,1H),2.17(dd,J=17.0,7.5Hz,4H),1.47(m,4H),1.38(s,9H),1.24(m,8H). 13 C NMR(125MHz,DMSO-d6)δ174.51,172.31,79.33,34.78,33.67,28.63,28.58,28.51,28.37,27.78,24.62,24.49.HRMS(ESI)forC 12 H 21 O4[MH] -calcd229.1445,found 229.1450.

[0076] 3.4. Mono-tert-butyl azelate (Compound 4c)

[0077] The synthesis process was similar to that of compound 4a, except that azelaic acid 3c was used as the starting material. Azelaic acid 3c (10 g, 53.13 mmol), EDCI (10.18 g, 1 eq), DMAP (6.49 g, 1 eq), and tert-butanol (39.4 g, 10 eq) were added to afford 4c as a colorless liquid (5.2 g, 21.28 mmol) in a 40.1% yield. 1 H NMR (500MHz, DMSO-d6) δ11.97(s,1H),2.17(dd,J=9.9,7.4Hz,4H),1.47(m,4H),1.38(s,9H),1.24(m,6H). 13 C NMR(125MHz,DMSO-d6)δ174.52,172.32,79.35,34.76,33.64,28.42,28.28,27.78,24.59,24.44.HRMS(ESI)forC 13 H 23 O4[MH] - calcd243.1602,found 243.1596.

[0078] 3.5, Mono-tert-butyl sebacate 4d

[0079] The synthesis process was similar to that of compound 4a, except that sebacic acid 3d was used as the starting material. Sebacic acid 3d (10 g, 49.44 mmol), EDCI (9.5 g, 1 eq), DMAP (6.0 g, 1 eq), and tert-butanol (36.7 g, 10 eq) were added to afford 4d as a colorless liquid (5.8 g, 22.50 mmol) in a 45.4% yield. 1 H NMR (500MHz, DMSO-d6) δ11.97(s,1H),2.17(dd,J=17.0,7.5Hz,4H),1.47(m,4H),1.38(s,9H),1.24(m,8H). 13 C NMR(125MHz,DMSO-d6)δ174.51,172.31,79.33,34.78,33.67,28.63,28.58,28.51,28.37,27.78,24.62,24.49.HRMS(ESI)forC 14 H 25 O4[MH] -calcd257.1758,found 257.1751.

[0080] 3.6. Mono-tert-butyl dodecyl diacid ester (Compound 4e)

[0081] The synthesis process was similar to that of compound 4a, except that the starting material was changed to dodecyl diacid 3e. Dodecyl diacid 3e (10 g, 43.42 mmol), EDCI (8.3 g, 1 eq), DMAP (5.3 g, 1 eq), and tert-butanol (32.2 g, 10 eq) were added to afford compound 4d as a white solid (5.5 g, 19.20 mmol) in a 44.2% yield. 1 H NMR (500MHz, DMSO) δ11.93(s,1H),2.19–2.16(m,2H),2.16–2.12(m,2H),1.47m,4H),1.38(s,9H),1.24(m,12H). 13 C NMR (125MHz, DMSO) δ174.41,172.20,79.23,34.78,33.66,28.87,28.76,28.69,28.60,28.41,27.73,24.62,24.51.HRMS(ESI)forC 16 H 29 O4[MH] - calcd285.2071,found 285.2077.

[0082] 3.7. Tetradecyldioic acid tert-butyl ester (compound 4f)

[0083] The synthesis process was similar to that of compound 4a, except that tetradecanoic acid 3f was used as the starting material. Tetradecanoic acid 3f (10 g, 38.71 mmol), EDCI (7.4 g, 1 eq), DMAP (4.7 g, 1 eq), and tert-butanol (28.7 g, 10 eq) were added to afford compound 4d as a white solid (5.5 g, 17.49 mmol) in a 45.2% yield. 1 H NMR (500MHz, DMSO) δ11.93(s,1H),2.17(t,J=5.6Hz,2H),2.16–2.13(m,2H),1.50–1.44(m,4H),1.38(s,9H),1.24(d,J=7.7Hz,16H). 13CNMR(125MHz,DMSO)δ174.41,172.18,79.22,34.77,33.65,29.00,28.97,28 .95,28.90,28.79,28.69,28.59,28.40,27.72,24.61,24.51.HRMS(ESI)forC 18 H 33 O4[MH] - calcd313.2384,found 313.2388.

[0084] 3.8. 7-((4-methyl-5-oxo-4,5-dihydro-[1,2]dithio[4,3-b]pyrrol-6-yl)amino)-7-oxoheptanoic acid tert-butyl ester or (7-aminothioluteolin-pimeloyl monotert-butyl ester) (Compound 5a)

[0085] Under argon protection, CH2Cl2, 4a (971 mg, 2 eq), and 4-methylmorpholine (908 mg, 4 eq) were added to a three-necked flask. At 0°C, a solution of isobutyl chloroformate (460 mg, 1.5 eq) in CH2Cl2 (2 mL) was slowly added dropwise, and the mixture was allowed to return to room temperature and react for 20 minutes. The reaction system was cooled to 0°C, compound 2 (0.5 g, 3.37 mmol) was added, and the mixture was allowed to return to room temperature and react for 6 hours. Saturated NaHCO3 solution was added, and the mixture was extracted three times with CH2Cl2. The organic phases were combined and washed twice with saturated NaCl solution. The organic phases were dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography to obtain a yellow solid 5a (680 mg, 1.77 mol) with a yield of 78.8%. 1 H NMR(500MHz,DMSO)δ9.95(s,1H),7.33(s,1H),3.25(s,3H),2.33(t,J=7.3Hz,2H),2 .17(t,J=7.3Hz,2H),1.50(dp,J=15.0,7.4Hz,4H),1.38(s,9H),1.28–1.21(m,2H). 13 C NMR(125MHz,DMSO)δ172.28,171.76,166.19,135.97,132.39,114.78,110.91,79.39,34.67,34.48,27.95,27.77,27.53,24.78,24.36.HRMS(ESI)forC 17 H 25 N2O4S2[MH] -Calcd 383.1105, found 383.1102. HPLC purity calculated to be 99.4%. Retention time: 16.182 min, eluted with 50% methanol / 50% water.

[0086] 3.9, 8-((4-methyl-5-oxo-4,5-dihydro-[1,2]dithio[4,3-b]pyrrol-6-yl)amino)-8-oxooctanoic acid tert-butyl ester or (7-aminothiolutein-suberoyl monotert-butyl ester) (Compound 5b)

[0087] The synthesis process was similar to that of compound 5a, except that the starting material was changed to mono-tert-butyl suberate 4b. 4b (1030 mg, 2 eq), 4-methylmorpholine (908 mg, 4 eq), isobutyl chloroformate (460 mg, 1.5 eq), and compound 2 (0.5 g, 3.37 mmol) were added to afford product 5b as a yellow solid (711 mg, 1.78 mmol) in a yield of 79.5%. 1 H NMR(500MHz,DMSO)δ9.94(s,1H),7.33(s,1H),3.25(s,3H),2.33(t,J=7.4Hz,2H ),2.16(t,J=7.3Hz,2H),1.52–1.45(m,4H),1.38(d,J=2.1Hz,9H),1.25(m,4H). 13 C NMR(125MHz,DMSO)δ172.29,171.81,166.20,135.98,132.41,114.78,110.88, 79.37,34.71,34.62,28.24,28.11,27.78,27.53,24.94,24.47.HRMS(ESI)forC 18 H 27 N2O4S2[MH] - Calcd 397.1256, found 397.1259. HPLC purity calculated to be 97.7%. Retention time: 18.273 min, eluted with 50% methanol / 50% water.

[0088] 3.10, 9-((4-methyl-5-oxo-4,5-dihydro-[1,2]dithio[4,3-b]pyrrol-6-yl)amino)-9-oxononanoic acid tert-butyl ester or (7-aminothiolutein-azelayl mono-tert-butyl ester) (Compound 5c)

[0089] The synthesis process was similar to that of compound 5a, except that the starting material was changed to mono-tert-butyl azelate 4c. 4c (1100 mg, 2 eq), 4-methylmorpholine (908 mg, 4 eq), isobutyl chloroformate (460 mg, 1.5 eq), and compound 2 (0.5 g, 3.37 mmol) were added to afford product 5c as a yellow solid (703 mg, 1.70 mmol) in a yield of 75.9%. 1 H NMR (500MHz, DMSO) δ9.94(s,1H),7.33(s,1H),3.25(s,3H),2.33(t,J=7.4Hz,2H),2.16(t,J=7.3Hz,2H),1.48(m,4H),1.38(s,9H),1.25(m,6H). 13 C NMR (126MHz, DMSO) δ172.33,171.85,166.21,135.98,132.42,114.78,110.89,79. 35,34.75,34.62,28.40,28.38,28.27,27.78,27.54,25.01,24.60.HRMS(ESI)forC 19 H 29 N2O4S2[MH] - Calcd 411.1418, found 411.1421. HPLC purity calculated to be 96.8%, retention time: 20.22 min, eluted with 50% methanol / 50% water.

[0090] 3.11. 10-((4-methyl-5-oxo-4,5-dihydro-[1,2]dithio[4,3-b]pyrrol-6-yl)amino)-10-oxodecanoic acid tert-butyl ester or (7-aminothiolutein-sebacoyl monotert-butyl ester) (Compound 5d)

[0091] The synthesis process was similar to that of compound 5a, except that the starting material was changed to azelayl mono-tert-butyl ester 4d. 4d (1160 mg, 2 eq), 4-methylmorpholine (908 mg, 4 eq), isobutyl chloroformate (460 mg, 1.5 eq), and compound 2 (0.5 g, 3.37 mmol) were added to afford product 5d as a yellow solid (768 mg, 1.80 mmol) in a yield of 80.2%. 1 H NMR(500MHz,DMSO)δ9.94(s,1H),7.34(s,1H),3.25(s,3H),2.34(t,J=7.4H z,2H),2.16(t,J=7.3Hz,2H),1.56–1.45(m,4H),1.39(s,9H),1.25(m,8H). 13C NMR (125MHz, DMSO) δ172.31,171.85,166.20,135.99,135.97,132.40,114.81,110.87,79. 33,34.78,34.66,34.64,28.59,28.50,28.38,27.78,27.52,25.04,24.62.HRMS(ESI)forC 20 H 31 N2O4S2[MH] - Calcd 425.1574, found 425.1573. Calculated purity by HPLC: 98.3%. Retention time: 21.028 min, eluted with 50% methanol / 50% water.

[0092] 3.12, 12-((4-methyl-5-oxo-4,5-dihydro-[1,2]dithio[4,3-b]pyrrol-6-yl)amino)-12-oxododecanoic acid tert-butyl ester or (7-aminothioluteolin-dodecyl diacyl mono-tert-butyl ester) (Compound 5e)

[0093] The synthesis process was similar to that of compound 5a, except that the starting material was changed to mono-tert-butyl dodecyl diacid 4e. 4e (1290 mg, 2 eq), 4-methylmorpholine (908 mg, 4 eq), isobutyl chloroformate (460 mg, 1.5 eq), and compound 2 (0.5 g, 3.37 mmol) were added to afford product 5e as a yellow solid (727 mg, 1.60 mmol) in a yield of 71.2%. 1 H NMR (500MHz, DMSO) δ9.91 (s, 1H), 7.33 (s, 1H), 3.25 (s, 3H), 2.33 (t, J = 7.4Hz, 2H), 2.1 5(t,J=7.3Hz,2H),1.48(dt,J=22.6,4.9Hz,4H),1.38(s,9H),1.24(d,J=5.0Hz,12H). 13 C NMR (126MHz, DMSO) δ172.27,171.83,166.18,135.96,132.36,114.79,110.77,79.28,34.76, 34.64,28.83,28.81,28.66,28.63,28.53,28.37,27.75,27.49,25.05,24.60.HRMS(ESI)forC 22 H 35 N2O4S2[M+H] +Calcd 455.2033, found 455.2034. HPLC purity calculated to be 97.5%. Retention time: 25.656 min, eluted with 50% methanol / 50% water.

[0094] 3.13, 14-((4-methyl-5-oxo-4,5-dihydro-[1,2]dithio[4,3-b]pyrrol-6-yl)amino)-14-oxotetradecanoic acid tert-butyl ester or (7-aminothioluteolin-tetradecyl diacyl mono-tert-butyl ester) (Compound 5f)

[0095] The synthesis process was similar to that of compound 5a, except that the starting material was changed to tetradecyl diacid mono-tert-butyl ester 4f. 4f (1410 mg, 2 eq), 4-methylmorpholine (908 mg, 4 eq), isobutyl chloroformate (460 mg, 1.5 eq), and compound 2 (0.5 g, 3.37 mmol) were added to afford product 5f as a yellow solid (703 mg, 1.46 mmol) in a yield of 64.9%. 1 H NMR (500MHz, DMSO) δ9.91 (s, 1H), 7.33 (s, 1H), 3.25 (s, 3H), 2.33 (t, J = 7.4Hz, 2H), 2.1 5(t,J=7.3Hz,2H),1.48(dt,J=22.6,4.9Hz,4H),1.38(s,9H),1.24(d,J=5.0Hz,16H). 13 C NMR (126MHz, DMSO) δ172.27,171.83,166.18,135.96,132.36,114.79,110.77,79.28,34.76, 34.64,28.83,28.81,28.66,28.63,28.53,28.37,27.75,27.49,25.05,24.60.HRMS(ESI)forC 24 H 39 N2O4S2[M+H] + Calcd 483.2346, found 483.2343. HPLC purity calculated to be 96.3%. Retention time: 28.538 min, eluted with 50% methanol / 50% water.

[0096] 3.14. 7-((4-methyl-5-oxo-4,5-dihydro-[1,2]dithio[4,3-b]pyrrol-6-yl)amino)-7-oxoheptanoic acid or (7-aminothiolutein-1-amide-7-heptanoic acid) (Compound 6a)

[0097] Compound 5a (500 mg, 1.3 mmol) was dissolved in 20 mL:10 mL CH₂Cl₂:CF₃COOH and added to a single-necked flask with rapid stirring. After 1 h of reaction, the mixture was concentrated under reduced pressure. Toluene (5 mL x 2) was added and the mixture was further concentrated to afford 6a (413 mg, 1.26 mmol) as a yellow solid in a 96.8% yield. 1 H NMR(500MHz,DMSO-d6)δ12.00(s,1H),9.95(s,1H),7.33(s,1H),3.25(s,3H),2.3 4(t,J=7.4Hz,2H),2.19(t,J=7.4Hz,2H),1.50(m,4H),1.25(p,J=7.2,6.2Hz,2H). 13 CNMR(125MHz,DMSO-d6)δ174.53,171.79,166.21,159.58,135.98,132.41,114.78,110.93,34.53,33.57,28.13,27.54,24.84,24.24.HRMS(ESI)forC 13 H 17 N2O4S2[MH] - Calcd 327.0479, found 327.0474. HPLC purity calculated to be 98.4%. Retention time: 10.051 min, eluted with 10% methanol / 90% water.

[0098] 3.15, 8-((4-methyl-5-oxo-4,5-dihydro-[1,2]dithio[4,3-b]pyrrol-6-yl)amino)-8-oxooctanoic acid or (7-aminothiolutein-1-amide-8-octanoic acid) (Compound 6b)

[0099] The synthesis process was similar to that of compound 6a, except that the starting material was 7-aminothioluteolin-suberoyl mono-tert-butyl ester 5b. Compound 5b (500 mg, 1.25 mmol) was dissolved in 20 mL:10 mL CH2Cl2:CF3COOH to afford product 6b as a yellow solid (419 mg, 1.22 mmol) in a 97.5% yield. 1 H NMR(500MHz,DMSO)δ11.97(s,1H),9.94(s,1H),7.33(s,1H),3.25(s,3H),2 .33(t,J=7.3Hz,2H),2.18(t,J=7.3Hz,2H),1.48(m,4H),1.31–1.21(m,4H). 13CNMR(125MHz,DMSO)δ174.51,171.83,166.21,135.98,132.42,114.78,110.90,34.63,33.64,28.31,28.28,27.54,24.96,24.38.HRMS(ESI)forC 14 H 19 N2O4S2[MH] - Calcd 341.0635, found 341.0624. HPLC purity calculated to be 99.9%. Retention time: 12.204 min, eluted with 10% methanol / 90% water.

[0100] 3.16, 9-((4-methyl-5-oxo-4,5-dihydro-[1,2]dithio[4,3-b]pyrrol-6-yl)amino)-9-oxononanoic acid or (7-aminothiolutein-1-amide-nonanoic acid) (Compound 6c)

[0101] The synthesis process was similar to that of compound 6a, except that the starting material was 7-aminothioluteolin-azelayl mono-tert-butyl ester 5c. Compound 5c (500 mg, 1.21 mmol) was dissolved in 20 mL:10 mL CH2Cl2:CF3COOH to afford product 6c as a yellow solid (419 mg, 1.21 mmol) in a 97.0% yield. 1 H NMR(500MHz,DMSO)δ11.97(s,1H),9.94(s,1H),7.33(s,1H),3.25(s,3H),2 .33(t,J=7.3Hz,2H),2.18(t,J=7.3Hz,2H),1.48(m,4H),1.31–1.21(m,6H). 13 CNMR(125MHz,DMSO)δ174.51,171.83,166.21,135.98,132.42,114.78,110.90,34.63,33.64,28.31,27.54,24.96,24.38.HRMS(ESI)forC 15 H 21 N2O4S2[MH] - Calcd 355.0792, found 355.0787. HPLC purity calculated to be 96.8%. Retention time: 15.053 min, eluted with 10% methanol / 90% water.

[0102] 3.17 10-((4-methyl-5-oxo-4,5-dihydro-[1,2]dithio[4,3-b]pyrrol-6-yl)amino)-10-oxodecanoic acid or (7-aminothiolutein-1-amide-10-decanoic acid) (Compound 6d)

[0103] The synthesis process was similar to that of compound 6a, except that the starting material was 7-aminothioglucose sebacoyl mono-tert-butyl ester 5d. Compound 5d (500 mg, 1.17 mmol) was dissolved in 20 mL:10 mL CH2Cl2:CF3COOH to afford product 6d as a yellow solid (434 mg, 1.17 mmol) in 100% yield. 1 H NMR(500MHz,DMSO)δ11.97(s,1H),9.93(s,1H),7.33(s,1H),3.25(s,3H),2 .33(t,J=7.3Hz,2H),2.18(t,J=7.3Hz,2H),1.56–1.43(m,4H),1.24(m,8H). 13 C NMR (126MHz, DMSO) δ174.52,171.85,166.19,135.97,132.40,114.78,110.88, 34.66,33.68,28.66,28.64,28.57,28.55,27.54,25.07,24.51.HRMS(ESI)forC 16 H 23 N2O4S2[MH] - Calcd 369.0948, found 369.0947. HPLC purity calculated to be 98.7%. Retention time: 17.829 min, eluted with 10% methanol / 90% water.

[0104] 3.18, 12-((4-methyl-5-oxo-4,5-dihydro-[1,2]dithio[4,3-b]pyrrol-6-yl)amino)-12-oxododecanoic acid or (7-aminothiolutein-1-amide-12-dodecanoic acid) (Compound 6e)

[0105] The synthesis process was similar to that of compound 6a, except that the starting material was 7-aminothioluteolin-dodecyl diacyl mono-tert-butyl ester 5e. Compound 5e (500 mg, 1.10 mmol) was dissolved in 20 mL:10 mL CH2Cl2:CF3COOH to afford product 6e as a yellow solid (435 mg, 1.09 mmol) in a 99% yield. 1H NMR (500MHz, DMSO) δ11.94(s,1H),9.92(s,1H),7.33(s,1H),3.25(s,3H),2.33(t,J=7.4Hz,2H),2.17(t,J=7.4Hz,2H),1.49(m,4H),1.23(m,12H). 13 C NMR (125MHz, DMSO) δ174.96,172.33,166.66,136.44,132.86,115.26,111.28,35.12, 34.13,29.45,29.36,29.33,29.20,29.16,29.03,27.98,25.52,24.97.HRMS(ESI)forC 18 H 27 N2O4S2[M+H] + Calcd 399.1407, found 399.1407. HPLC calculated purity: 95.5%. Retention time: 25.39 min, eluted with 8% methanol / 92% water.

[0106] 3.19 14-((4-methyl-5-oxo-4,5-dihydro-[1,2]dithio[4,3-b]pyrrol-6-yl)amino)-14-oxotetradecanoic acid 6 or (7-aminothiolutein-1-amide-14-tetradecanoic acid) (Compound 6f)

[0107] The synthesis process was similar to that of compound 6a, except that the starting material was 7-aminothioluteolin-tetradecyl diacyl mono-tert-butyl ester 5f. Compound 5f (500 mg, 1.04 mmol) was dissolved in 20 mL:10 mL CH2Cl2:CF3COOH to afford product 6f as a yellow solid (442 mg, 1.04 mmol) in 100% yield. 1 H NMR (500MHz, DMSO) δ11.95(s,1H),9.91(s,1H),7.32(s,1H),3.25(s,3H),2.33(t,J=7.4Hz,2H),2.17(t,J=7.4Hz,2H),1.49(m,4H),1.23(m,16H). 13 C NMR (125MHz, DMSO) δ174.95,172.32,166.66,136.45,132.85,115.27,111.24,35.12,34.12, 29.48,29.46,29.42,29.39,29.23,29.17,29.04,29.01,27.97,25.53,24.97.HRMS(ESI)forC20 H 31 N2O4S2[M+H] + Calcd 427.1720, found 427.1713. HPLC purity calculated to be 98.7%. Retention time: 25.185 min, eluted with 10% methanol / 90% water.

[0108] 3.20N 1 -Hydroxy-N 7 -(4-methyl-5-oxo-4,5-dihydro-[1,2]dithio[4,3-b]pyrrol-6-yl)pimelanediamide or (7-aminothiolutein-1-amide-7-heptylhydroxamic acid) (Compound 7a)

[0109] Compound 6a (100 mg, 304.5 μmol) was dissolved in DMF (5 mL), and 4-methylmorpholine (123 mg, 3.5 eq) was then added to the vial with rapid stirring. The reaction system was placed at -20°C, and a solution of isobutyl chloroformate (125 mg, 3 eq) in DMF (0.5 mL) was slowly added dropwise. After the addition was complete, the reaction system was warmed to 0°C and the reaction was continued for 10 minutes. Finally, a suspension of freshly prepared hydroxylamine hydrochloride (127 mg, 6 eq), 4-methylmorpholine (185 mg, 6 eq), and DMF (3 mL) was added dropwise. The reaction system was returned to room temperature and reacted. After 30 minutes, the starting material was detected to be completely consumed. 20 mL of ice water was added, and after all the yellow solid precipitated, the crude product was filtered under reduced pressure to obtain the crude product. The crude product was purified by column chromatography to obtain a yellow solid 7a (52 mg, 151.4 μmol) with a yield of 49.8%. 1 HNMR(500MHz,DMSO)δ10.41–10.28(m,1H),9.95(s,1H),8.67(d,J=1.7Hz,1H),7.34(s,1H),3.2 5(s,3H),2.33(t,J=7.4Hz,2H),1.93(t,J=7.4Hz,2H),1.49(h,J=7.8Hz,4H),1.30–1.17(m,2H). 13 C NMR (125MHz, DMSO) δ171.78,169.05,166.20,135.98,132.39,114.78,110.94,34.54,32.16,28.17,27.54,24.91,24.82.HRMS(ESI)forC 13 H 18 N3O4S2[MH] -Calcd 342.0588, found 342.0583. HPLC purity calculated to be 95.2%. Retention time: 15.388 min, eluted with 8% methanol / 92% water.

[0110] 3.21, N 1 -Hydroxy-N 8 -(4-methyl-5-oxo-4,5-dihydro-[1,2]dithio[4,3-b]pyrrol-6-yl)octanediamide or (7-aminothiolutein-1-amide-8-octanedihydroxamic acid) (Compound 7b)

[0111] The synthesis process was similar to that of compound 7a, except that the starting material was changed to 7-aminothiolutein-1-amide-8-octanoic acid 6b. Compound 6b (100 mg, 292.0 μmol), 4-methylmorpholine (103 mg, 3.5 eq), isobutyl chloroformate (120 mg, 3 eq), and a freshly prepared solution of hydroxylamine hydrochloride (122 mg, 6 eq) and 4-methylmorpholine (177 mg, 6 eq) were added to afford product 7b as a yellow solid (61 mg, 170.6 μmol) in a 58.4% yield. 1 H NMR(500MHz,DMSO)δ10.33(s,1H),9.94(s,1H),8.66(s,1H),7.33(s,1H),3.25(s, 3H),2.33(t,J=7.3Hz,2H),1.92(t,J=7.3Hz,2H),1.54–1.42(m,4H),1.23(m,4H). 13 CNMR(125MHz,DMSO)δ171.83,169.11,166.21,135.98,132.43,114.78,110.92,34.65,32.26,28.37,28.34,27.54,25.05,25.00.HRMS(ESI)forC 14 H 20 N3O4S2[MH] - Calcd 356.0744, found 356.0739. HPLC purity calculated to be 97.2%. Retention time: 17.019 min, eluted with 8% methanol / 92% water.

[0112] 3.22, N 1 -Hydroxy-N 9 -(4-methyl-5-oxo-4,5-dihydro-[1,2]dithio[4,3-b]pyrrol-6-yl)azelazone or (7-aminothioluteolin-1-amide-9-nonanohydroxamic acid) (Compound 7c)

[0113] The synthesis process was similar to that of compound 7a, except that the starting material was changed to 7-aminothiolutein-1-amide-9-nonanoic acid 6c. Compound 6c (100 mg, 280.5 μmol), 4-methylmorpholine (99 mg, 3.5 eq), isobutyl chloroformate (115 mg, 3 eq), and a freshly prepared solution of hydroxylamine hydrochloride (117 mg, 6 eq) and 4-methylmorpholine (170 mg, 6 eq) were added to afford product 7c as a yellow solid (59 mg, 158.8 μmol) in a 56.6% yield. 1 H NMR(500MHz,DMSO)δ10.33(s,1H),9.94(s,1H),8.66(s,1H),7.34(s,1H),3.25 (s,3H),2.33(t,J=7.3Hz,2H),1.92(t,J=7.3Hz,2H),1.49(m,4H),1.23(m,6H). 13 C NMR (125MHz, DMSO) δ171.86,169.12,166.21,135.98,132.44,114.77,110.93,34.65,32.27,28.48,27.55,25.13,25.05.HRMS(ESI)forC 15 H 22 N3O4S2[MH] - Calcd 370.0901, found 370.0896. HPLC calculated purity: 97.3%. Retention time: 18.789 min, eluted with 8% methanol / 92% water.

[0114] 3.23, N 1 -Hydroxy-N 10 -(4-methyl-5-oxo-4,5-dihydro-[1,2]dithio[4,3-b]pyrrol-6-yl)decanediamide or (7-aminothiolutein-1-amide-10-decanedihydroxamic acid) (Compound 7d)

[0115] The synthesis process was similar to that of compound 7a, except that the starting material was changed to 7-aminothiolutein-1-amide-10-decanoic acid 6d. Compound 6d (100 mg, 269.9 μmol), 4-methylmorpholine (96 mg, 3.5 eq), isobutyl chloroformate (115 mg, 3 eq), and a freshly prepared solution of hydroxylamine hydrochloride (113 mg, 6 eq) and 4-methylmorpholine (164 mg, 6 eq) were added to afford product 7d as a yellow solid (72 mg, 186.8 μmol) in a 69.2% yield. 1H NMR(500MHz,DMSO)δ10.33(s,1H),9.94(s,1H),8.66(s,1H),7.34(s,1H),3.25(s,3H), 2.34(t,J=7.3Hz,2H),1.93(t,J=7.4Hz,2H),1.49(dt,J=20.9,7.2Hz,4H),1.24(s,8H). 13 C NMR (125MHz, DMSO) δ172.32,169.57,166.66,136.43,132.87,115.23,111.36, 35.11,32.71,29.14,29.10,29.04,29.03,27.99,25.58,25.53.HRMS(ESI)forC 16 H 24 N3O4S2[MH] - Calcd 384.1057, found 384.1047. HPLC purity calculated to be 96.1%. Retention time: 20.873 min, eluted with 8% methanol / 92% water.

[0116] 3.24, N 1 -Hydroxy-N 12 -(4-methyl-5-oxo-4,5-dihydro-[1,2]dithio[4,3-b]pyrrol-6-yl)dodecanediamide or (7-aminothiolutein-1-amide-12-dodecanehydroxamic acid) (Compound 7e)

[0117] The synthesis process was similar to that of compound 7a, except that the starting material was changed to 7-aminothioluteolin-1-amide-12-dodecanoic acid 6e. Compound 6e (100 mg, 250.9 μmol), 4-methylmorpholine (89 mg, 3.5 eq), isobutyl chloroformate (103 mg, 3 eq), and a freshly prepared solution of hydroxylamine hydrochloride (105 mg, 6 eq) and 4-methylmorpholine (152 mg, 6 eq) were added to afford product 7e as a yellow solid (79 mg, 191.0 μmol) in a yield of 76.1%. 1 H NMR(500MHz,DMSO)δ10.30(s,1H),9.92(s,1H),8.63(s,1H),7.33(s,1H),3.25(s, 3H),2.33(t,J=7.3Hz,2H),1.92(t,J=7.3Hz,2H),1.48(m,4H),1.28–1.19(m,12H). 13CNMR(125MHz,DMSO)δ172.33,169.58,166.67,136.44,132.87,115.26,111.29,35.11,32. 74,29.48,29.45,29.38,29.23,29.17,29.07,29.01,27.99,25.59,25.52.HRMS(ESI)forC 18 H 28 N3O4S2[M+H] + Calcd 414.1516, found 414.1532. HPLC purity calculated to be 98.1%. Retention time: 23.95 min, eluted with 8% methanol / 92% water.

[0118] 3.25N1-Hydroxy-N14-(4-methyl-5-oxo-4,5-dihydro-[1,2]dithio[4,3-b]pyrrol-6-yl)tetradecanediamide or (7-aminothiolutein-1-amide-14-tetradecanehydroxamic acid) (Compound 7f)

[0119] The synthesis process was similar to that of compound 7a, except that the starting material was changed to 7-aminothiolutein-1-amide-1,4-dimethyl-1-tetradecanoic acid 6f. Compound 6f (100 mg, 234 μmol), 4-methylmorpholine (83 mg, 3.5 eq), isobutyl chloroformate (96 mg, 3 eq), and a freshly prepared solution of hydroxylamine hydrochloride (97 mg, 6 eq) and 4-methylmorpholine (142 mg, 6 eq) were added to afford product 7f as a yellow solid (85 mg, 192.5 μmol) in an 82.1% yield. 1 H NMR(500MHz,DMSO)δ10.30(s,1H),9.92(s,1H),8.63(s,1H),7.33(s,1H),3.25(s, 3H),2.33(t,J=7.3Hz,2H),1.92(t,J=7.3Hz,2H),1.48(m,4H),1.28–1.19(m,16H). 13 CNMR(125MHz,DMSO)δ172.33,169.58,166.67,136.44,132.87,115.26,111.29,35.11,32. 74,29.48,29.45,29.38,29.23,29.17,29.07,29.01,27.99,25.59,25.52.HRMS(ESI)forC 20 H 32 N3O4S2[M+H] +Calcd 442.1829, found 442.1827. HPLC purity calculated to be 96.9%. Retention time: 28.731 min, eluted with 8% methanol / 92% water.

[0120] 3.26, N 1 -(2-aminophenyl)-N 7 -(4-methyl-5-oxo-4,5-dihydro-[1,2]dithio[4,3-b]pyrrol-6-yl)pimelamide 8 or (7-aminothiolutein-1,7-pimelamide-7-o-aminobenzene) (Compound 8a)

[0121] Compound 7a (100 mg, 304.5 μmol) and 1,2-phenylenediamine (99 mg, 3 eq) were dissolved in THF (10 mL). EDCI (88 mg, 1.5 eq), HOBt (62 mg, 1.5 eq), and 4-pyrrolidinylpyridine (4.5 mg, 0.1 eq) were added to the suspension at 0°C and allowed to react at room temperature. The reaction solution was added to water (20 mL) and extracted three times with ethyl acetate. The combined organic phases were washed twice with saturated brine. The organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography to afford 8a (104 mg, 248.5 μmmol) as a colorless liquid in an 81.6% yield. 1 H NMR (500MHz, DMSO) δ9.94(s,1H),9.08(s,1H),7.33(s,1H),7.14(d,J=7.8Hz,1H),6.88(t,J=7.6Hz,1H),6.71(d,J=8.0Hz,1 H),6.53(t,J=7.5Hz,1H),4.83(s,2H),3.25(s,3H),2.35(t,J=7.4Hz,2H),2.30(t,J=7.5Hz,2H),1.56(m,4H),1.31(m,2H). 13 C NMR (125MHz, DMSO) δ171.82,171.12,166.19,141.81,135.97,132.41,125.66,125.28,123.6 0,116.19,115.88,114.78,110.82,35.74,34.66,28.44,28.40,27.51,25.01.HRMS(ESI)forC 19 H 23 N4O3S2[M+H] +Calcd 419.1206, found 419.1203. HPLC purity calculated to be 95.1%. Retention time: 19.628 min, eluted with 8% methanol / 92% water.

[0122] 3.27, N 1 -(2-aminophenyl)-N 8 -(4-methyl-5-oxo-4,5-dihydro-[1,2]dithio[4,3-b]pyrrol-6-yl) suberamide or (7-aminothiolutein-1,8-suberamide-8-o-aminobenzene) (Compound 8b)

[0123] The synthesis process was similar to that of compound 8a, except that the starting material was changed to 7-aminothiolutein-1-amide-8-octanoic acid 6b. Compound 6b (100 mg, 292.0 μmol) was added along with 1.2-phenylenediamine (95 mg, 3 eq), EDCI (84 mg, 1.5 eq), HOBt (59 mg, 1.5 eq), and 4-pyrrolidinylpyridine (4.3 mg, 0.1 eq) to afford product 8b as a yellow solid (101 mg, 233.5 μmol) in a yield of 79.6%. 1 HNMR(500MHz,DMSO)δ9.94(s,1H),9.08(s,1H),7.33(s,1H),7.14(d,J=7.8Hz,1H),6.88(t,J=7.6Hz,1H),6.71(d,J=8.0Hz, 1H),6.53(t,J=7.5Hz,1H),4.83(s,2H),3.25(s,3H),2.35(t,J=7.4Hz,2H),2.30(t,J=7.5Hz,2H),1.56(m,4H),1.31(m,4H). 13 C NMR (125MHz, DMSO) δ171.82,171.12,166.19,141.81,135.97,132.41,125.66,125.28,123.60,1 16.19,115.88,114.78,110.82,35.74,34.66,28.44,28.40,27.51,25.20,25.01.HRMS(ESI)forC 20 H 25 N4O3S2[M+H] + Calcd 433.1363, found 433.1358. HPLC purity calculated to be 98.3%. Retention time: 21.058 min, eluted with 8% methanol / 92% water.

[0124] 3.28, N 1-(2-aminophenyl)-N 9 -(4-methyl-5-oxo-4,5-dihydro-[1,2]dithio[4,3-b]pyrrol-6-yl)azelazone or (7-aminothioluteolin-1,9-azelazone-9-o-aminobenzene) (Compound 8c)

[0125] The synthesis process was similar to that of compound 8a, except that the starting material was changed to 7-aminothiolutein-1-amide-9-nonanoic acid 6c. Compound 6c (100 mg, 280.5 μmol) was added along with 1.2-phenylenediamine (90 mg, 3 eq), EDCI (81 mg, 1.5 eq), HOBt (57 mg, 1.5 eq), and 4-pyrrolidinylpyridine (4.2 mg, 0.1 eq) to afford product 8c as a yellow solid (108 mg, 241.8 μmol) in 86.2% yield. 1 HNMR(500MHz,DMSO)δ9.93(s,1H),9.08(s,1H),7.33(s,1H),7.14(d,J=7.8Hz,1H),6.98–6.84(m,1H),6.70(d, J=8.1Hz,1H),6.60–6.49(m,1H),4.80(s,2H),3.25(s,3H),2.32(m,4H),1.65–1.49(m,4H),1.40–1.24(m,6H). 13 C NMR (125MHz, DMSO) δ171.86,171.18,166.21,141.88,135.98,132.43,125.69,125.28,123.60,116. 20,115.90,114.78,110.85,35.78,34.67,28.57,28.53,28.50,27.52,25.30,25.07.HRMS(ESI)forC 21 H 27 N4O3S2[M+H] + Calcd 447.1519, found 447.1512. HPLC purity calculated to be 96.7%. Retention time: 22.401 min, eluted with 8% methanol / 92% water.

[0126] 3.29N 1 -(2-aminophenyl)-N 10 -(4-methyl-5-oxo-4,5-dihydro-[1,2]dithio[4,3-b]pyrrol-6-yl)decanamide or (7-aminothiolutein-1,10-decanamide-10-o-aminobenzene) (Compound 8d)

[0127] The synthesis process was similar to that of compound 8a, except that the starting material was changed to 7-aminothiolutein-1-amide-10-decanoic acid 6d. Compound 6d (100 mg, 269.9 μmol) was added along with 1.2-phenylenediamine (88 mg, 3 eq), EDCI (78 mg, 1.5 eq), HOBt (55 mg, 1.5 eq), and 4-pyrrolidinylpyridine (4 mg, 0.1 eq) to afford product 8d as a yellow solid (110 mg, 238.4 μmol) in 88.5% yield. 1 HNMR (500MHz, DMSO) δ9.92(s,1H),9.08(s,1H),7.33(s,1H),7.14(d,J=7.7Hz,1H),6.88(t,J=7.6Hz,1H),6.71(d,J=8.0Hz,1H),6.53(t ,J=7.6Hz,1H),4.84(s,2H),3.25(s,3H),2.34(t,J=7.4Hz,2H),2.30(t,J=7.5Hz,2H),1.55(dt,J=29.8,7.1Hz,4H),1.35–1.22(m,8H). 13 C NMR (125MHz, DMSO) δ171.85,171.15,166.19,141.81,135.97,132.39,125.66,125.25,123.61,1 16.21,115.90,114.78,110.82,35.77,34.66,28.69,28.57,27.51,25.30,25.07.HRMS(ESI)forC 22 H 29 N4O3S2[M+H] + Calcd 461.1676, found 461.1665. HPLC purity calculated to be 95.5%. Retention time: 20.921 min, eluted with 20% methanol / 80% water.

[0128] 3.30N 1 -(2-aminophenyl)-N 12 -(4-methyl-5-oxo-4,5-dihydro-[1,2]dithio[4,3-b]pyrrol-6-yl)dodecanediamide or (7-aminothiolutein-1,12-dodecanediamide-12-o-aminobenzene) (Compound 8e)

[0129] The synthesis process was similar to that of compound 8a, except that the starting material was changed to 7-aminothiolutein-1-amide-12-dodecanoic acid 6e. Compound 6e (100 mg, 250.9 μmol) was added along with 1.2-phenylenediamine (81 mg, 3 eq), EDCI (72 mg, 1.5 eq), HOBt (51 mg, 1.5 eq), and 4-pyrrolidinylpyridine (3.7 mg, 0.1 eq) to afford product 8e as a yellow solid (107 mg, 218.9 μmol) in 87.3% yield. 1 H NMR (500MHz, DMSO) δ9.92(s,1H),9.07(s,1H),7.32(s,1H),7.14(d,J=7.7Hz,1H),6.88(t,J=7.6Hz,1H),6.70( d,J=8.0Hz,1H),6.53(t,J=7.5Hz,1H),4.79(s,2H),3.25(s,3H),2.31(m,4H),1.62–1.46(m,4H),1.27m,12H). 13 C NMR (125MHz, DMSO) δ171.85,171.15,166.18,141.85,135.97,132.40,125.65,125.23,123.59,116.16, 115.88,114.78,110.80,35.77,34.66,28.94,28.79,28.70,28.57,27.50,25.31,25.06.HRMS(ESI)forC 24 H 33 N4O3S2[M+H] + Calcd 489.1989, found 489.1982. HPLC purity calculated to be 98.1%. Retention time: 26.214 min, eluted with 8% methanol / 92% water.

[0130] 3.31N 1 -(2-aminophenyl)-N 14 -(4-methyl-5-oxo-4,5-dihydro-[1,2]dithio[4,3-b]pyrrol-6-yl)tetradecanediamide or (7-aminothiolutein-1,14-tetradecanediamide-14-o-aminobenzene) (Compound 8f)

[0131] The synthesis process was similar to that of compound 8a, except that the starting material was changed to 7-aminothiolutein-1-amide-14-tetradecanoic acid 6f. Compound 6f (100 mg, 234.0 μmol) was added along with 1.2-phenylenediamine (76 mg, 3 eq), EDCI (67 mg, 1.5 eq), HOBt (48 mg, 1.5 eq), and 4-pyrrolidinylpyridine (3.5 mg, 0.1 eq) to afford product 8f as a yellow solid (107 mg, 207.1 μmol) in 88.3% yield. 1 H NMR(500MHz,DMSO)δ9.92(s,1H),9.07(s,1H),7.33(s,1H),7.14(d,J=7.8Hz,1H),6.96–6.81(m,1H),6.7 0(d,J=7.9Hz,1H),6.59–6.44(m,1H),4.79(s,2H),3.25(s,3H),2.31(m,4H),1.54(m,4H),1.26(m,16H). 13 C NMR (125MHz, DMSO) δ171.89,171.14,166.19,141.83,135.96,132.18,125.65,125.23,124.26,116.16,115 .87,114.60,110.79,35.76,34.64,29.00,28.91,28.81,28.69,28.53,27.50,25.31,25.04.HRMS(ESI)forC 26 H 37 N4O3S2[M+H] + Calcd 517.2302, found 517.2294. HPLC purity calculated to be 99.4%. Retention time: 28.271 min, eluted with 8% methanol / 92% water.

[0132] Example 4. Inhibitory activity of compounds against HDAC1 and HDAC6

[0133] HDAC1 (H83-30G) and HDAC6 (H85-30G) were purchased from Signal Chem, and HDAC Substrate (Cat: 50037) was purchased from BPS. HDAC Assay Buffer (25 mM Tris, pH 8.0, 1 mM MgCl2, 0.1 mg / mL BSA, 37 mM NaCl, 2.7 mM KCl) was prepared. The test compound (100 μM) was diluted 2-fold in the buffer to obtain 10 different test concentrations, with each concentration tested in triplicate. To each well of a 96-well plate, 34 μL of HDAC Assay Buffer, 5 μL of compound, 5 μL of HDAC Substrate (10 μM), and 5 μL of Trypsin (50 μg / mL) were added. Finally, 1 mL of HDAC1 or HDAC3 was added and mixed thoroughly. No compound was added to the negative control, and no HDAC1 or HDAC3 was added to the positive control. The 96-well plate was incubated at 37°C in the dark with shaking for 30 minutes. The color was measured on a full-wavelength microplate reader (Ex / Em 360 / 460 nm) and fitted in GraphPad 10.0 software to obtain the IC value of the compound. 50 value.

[0134] Table 1. Inhibitory activity of target compounds against HDAC1 and HDAC6 a

[0135]

[0136]

[0137] a IC50 values ​​are the average of at least three independent determinations and are expressed as mean ± SD

[0138] The 18 compounds showed stronger inhibitory activity against HDAC1 than against HDAC6, but showed poor inhibitory activity against HDAC6, indicating that these compounds selectively inhibited HDAC1 (Table 1). Initially, carboxylic acids were used as ZBGs (6a-6f), which showed poor inhibitory activity against HDAC1 (IC 50 >5μM). The compounds (7a-7f) in which the carboxylic acid was replaced by hydroxamic acid showed significantly enhanced inhibitory activity in the HDAC1 activity test. Among them, the inhibitory activity was the best when the linker length was 8 (IC 50=72.5nM). In addition, when the number of linker carbons is even, its activity is slightly stronger than that of odd-numbered carbons (7b>7d>7c>7a). When the linker length is greater than 10, the inhibitory activity of HDAC1 begins to decline. These results indicate that when the linker length is 8, hydroxamic acid compounds are most conducive to HDAC1 binding. Compounds whose carboxylic acid is substituted with 1.2-phenylenediamine (8a-8f) have slightly weaker inhibitory activity against HDAC1 than hydroxamic acid compounds. 1.2-phenylenediamino compounds 8a-8f are similar to hydroxamic acids 7a-7f. When the number of linker carbons is 8, 8b has the best inhibitory activity against HDAC1, IC 50 =141.2 nM. It is noteworthy that the activity of the 1,2-phenylenediamino compound decreased significantly when the number of linkers exceeded 10.

[0139] In summary, in the HDAC1 activity test of the three types of compounds, hydroxamic acid was the most preferred ZBG, followed by 1,2-phenylenediamine, and carboxylic acid. Linker selection was generally 7-10, with the best effect achieved when Linker = 8.

[0140] Example 5. In vitro antiproliferative activity and structural relationship

[0141] Human cancer cell lines KYSE30 and A549 were purchased from Wuhan Punosai Life Science Technology Co., Ltd. Compound preparation: The compound was dissolved in DMSO to a storage concentration of 200 mM, and then diluted with DMSO to a maximum dose of 50 μM, and 2-fold dilution was used to obtain 10 doses. Relative cell growth medium was added to obtain the final maximum dose (10 μM, 1% DMSO). Cell culture and detection: The cell density was 4500 cells / well and incubated overnight. 1 μL of compound was added and incubated for 24 hours. Finally, 10 μL CCK-8 was added and detected at 450 nm after 30 minutes. IC was calculated using Prism 10.0 (GraphPad Software, San Diego, CA, USA) 50 value.

[0142] Table 2. Antiproliferative activity of target compounds against KYSE30 (esophageal cancer) and A549 (lung cancer) b

[0143] Compound <![CDATA[KYSE30(IC 50 nM)]]> <![CDATA[A549(IC 50 nM))]]> 6a 15646±387.5 4872±255 6b 17570±424.4 3716±268 6c 2664±886.5 155.5±7.1 6d 9017±492.5 139.6±7.2 6e 2841±119 223.2±14.1 6f 2571±86 389.1±33.9 7a 554.7±58.5 626±37 7b 588.1±40.6 313.9±12.1 7c 398±22.2 208.1±5.9 7d 390.6±14 76.22±2.4 7e 550.3±15.7 99.43±5.5 7f 2358±76 192.3±15.6 8a 477.3±29.8 114.2±5 8b 29.94±2.22 29.85±4.68 8c 407±15.1 37.88±5.8 8d 805±42.9 40.38±3.97 8e 3012±121.5 99.21±3.08 8f 12887±111.5 1402±71 SAHA 8077±259 3659±469 Capzimin 3126±137.6 553.3±17.9 Thiolutin 625.1±22.4 370.4±35

[0144] b IC50 values ​​are the average of at least three independent determinations and are expressed as mean ± SD

[0145] As shown in Table 2, most compounds exhibited strong inhibitory activity against both human cancer cell lines. The antiproliferative activity of the 8-series compounds, which use o-phenylenediamine as the ZBG, was generally stronger than that of the 7-series compounds, which use hydroxamic acid as the ZBG. Generally speaking, the relationship between cellular antiproliferative activity and ZBG is carboxylic acid < hydroxamic acid < 1,2-phenylenediamine; for example, compound 8b > 7b > 6b. Notably, the longer the linker between the ZBG and thiolutin units (exceeding 8) resulted in weaker activity against KYSE30, presumably due to the RO5-like property rule (see Table 1). For example, compound 8f (which does not conform to the RO5 rule) exhibited over 100-fold lower activity than 8b in both cancer cell lines. The in vitro antitumor activity was generally consistent with the inhibitory activity against both targets. For example, compound 8b exhibited superior HDAC1 inhibitory activity and was more potent than compounds 7b and 6b. Compound 8b exhibited the strongest inhibitory activity against the KYSE30 cell line (IC 50 =29.94nM), which is much stronger than the three positive drugs. In summary, ZBG has the best activity when selected with 1,2-phenylenediamine, and the linker selection is generally 7-10. When the linker is 8, the effect is the best. When the linker is greater than 10, the activity of the compound begins to decline.

[0146] Taking into account the cell activity and enzyme activity, we selected compound 8b for further biological activity evaluation.

[0147] Example 6. Effects of Compounds on PSMD14, HDAC and Their Substrates in KYSE30 Cells

[0148] KYSE30 cells (1.5 million cells per well) were plated in flat-bottom six-well plates and cultured for 24 hours before being treated with 8b, SAHA, and capzimin. After 24 hours, the cells were washed with PBS (×2) and lysed with RIPA buffer on ice for 30 minutes. The cells were harvested and centrifuged at 14,000 rpm for 15 minutes at 4°C. The supernatant was collected and protein concentration was determined by BCA assay. SDS-PAGE electrophoresis, transfer, blocking, and washing of the membranes were performed, followed by incubation with primary antibodies overnight at 4°C. The membranes were rinsed twice with TBST buffer and incubated with secondary antibodies for 1 hour. Images were acquired using a Tanon 5200. Data were analyzed using one-way analysis of variance, with P levels < 0.05 considered statistically significant.

[0149] PSMD14 regulates Snail expression in KYSE30 cells [7,19], inhibiting PSMD14 activity and accelerating Snail degradation, inhibiting cell migration and proliferation. Based on this, we tested the effects of compound 8b, SAHA, and Capzimin on PSMD14 and Snail in KYSE30 cells. Western blot results showed that 8b and Capzimin did not affect the expression of PSMD14, but at the same time reduced the expression of snail. This shows that 8b and Capzimin have the same effect, they inhibit the function of PSMD14 but do not affect its expression. Figure 5 A and Figure 5 B). To explore the effect of 8b on HDAC, we used Western blotting to measure the acetylation levels of histones H3 and H4, two common HDAC substrates, in KYSE30 cells ( Figure 5 ). Similar to HDAC1 inhibitors, compound 8b increased the acetylation levels of H3 and H4 in cells. These results indicate that inhibition of HDAC1 activity in KYSE30 cells is one of the mechanisms of compound 8b's biological activity ( Figure 5 A and Figure 5 B).

[0150] Example 7. Effect of compound 8b on KYSE30 cell motility

[0151] KYSE 30 cells were seeded into 6-well plates. When the cells were close to 100% confluence, they were scratched with a 10 μL pipette tip. Then, the cells were washed and cultured in fresh medium without FBS for 48 hours. Each image of the gap was collected at the beginning (0 hours) and end (48 hours) of the experiment. In the in vitro invasion assay, KYSE30 cells (1×10 5 Cells were seeded into transwell inserts pre-coated with Matrigel (BD, Franklin Lakes, NJ, USA). The bottom chamber was filled with 650 μl of culture medium supplemented with 20% FBS. After incubation at 37°C for 24 hours, the invading cells were fixed with methanol and stained with 0.1% crystal violet. Three randomly selected fields of view were photographed and counted using an inverted microscope. Data were analyzed using one-way analysis of variance, and P levels < 0.05 were considered statistically significant.

[0152] Snail regulates EMT transcription, which is a key factor in cancer metastasis. Therefore, we further studied the effects of compound 8b and Capzinmin on the migration and invasion of KYSE30 cells. Transwell and scratch assay results showed that 8b significantly reduced the migration and invasion of KYSE 30 cells, which was better than the positive drug Capzinmin ( Figure 6 ).

[0153] Example 8. Molecular docking studies

[0154] HDAC protein (PDB: 1C3R) was retrieved from the Protein Data Bank. Compound 8b was prepared using the Ligand Preparation Wizard in Maestro using standard settings. HDAC1 and PSMD14 proteins were preprocessed according to the standard Autodock vina protocol, and meshes for the HDAC protein and PSMD14 were generated using Vina (version 11.5). Flexible docking of conformational groups was performed using Glide in standard and ultraprecision modes with standard settings. Only poses with low-energy conformations and good hydrogen bonding geometry were considered. Small molecule ligand-protein docking results were plotted using PyMO.

[0155] We selected compounds 6e, 7e, and 8b for molecular docking studies with PSMD14, and elucidated the binding modes of compounds 6e, 7e, and 8b on HDAC and PSMD14. Figure 7 -A, the heterocyclopentadiene thiol of 8b acts on the active site cavity of PSMD14. The ene dithiol of compounds 6e, 7e, and 8b forms hydrogen bonds with amino acids Val82, Glu52, and His83. These hydrogen bonding forces position a suitable spatial conformation to directly bind to Zn 2+ Chelation ( Figure 7 The molecular docking score of compound 8b is lower than that of compounds 7e and 8e, which may be one of the reasons why compound 8e has better anti-tumor proliferation activity. Compound 8b interacts with HDAC (PDBID: 1C3R), and the hydrophobic alkyl side chain of 8b can be inserted into the cavity of the active site ( Figure 7 In C), the pyrrole planar structure acts as a cap to form a pp stacking interaction with the amino acid Tyr 91 residue, the primary amino group of 1,2-phenylenediamine forms a hydrogen bond with Asp 168, and the H atom of the amide also forms a hydrogen bond with the carbonyl group of Gly140. These interactions make compound 8b adjacent to the amino group, carbonyl oxygen and Zn 2+ Direct contact ( Figure 7 Middle D).

[0156] Example 9. ADMET studies

[0157] The compound structure SDF format file was imported into Schrodinger software, and the ADME (absorption, distribution, metabolism and elimination) characteristics of the compound were predicted using the Qikprop module, and the calculated results were exported as a table file.

[0158] Table 3. ADMET of target compounds

[0159]

[0160] a QlogPo / w: predicted octanol / water partition coefficient (recommended: -2.0 to 6.5).

[0161] b QlogS: predicted aqueous solubility, log SS in mol.dm-3 is the concentration of the solute in a saturated solution in equilibrium with the crystalline solid (recommended: -6.5 to 0.5).

[0162] c QlogBB: predicted brain / blood partition coefficient (recommended: range -3.0 to 1.2).

[0163] d QlogKhsa: predicted binding to human serum albumin (recommended: range -1.5 to 1.5).

[0164] e% Human Oral Absorption: Predicted human absorption ranges from 0 to 100% (recommendation: >80% is good, <25% is poor).

[0165] f Ro5: Number of violations of the Lipinski Rule (recommendation: maximum 4 violations).

[0166] The drugability of 18 target compounds was evaluated and the RO 5 and ADME (absorption, distribution, metabolism, and elimination) properties. Among all compounds, compound 8f (molecular mass >500, water solubility QPlogS = -6.580) violated the RO5 principle and was less than the theoretical (QPlogS) value. The compounds' lipophilic octanol / water partition coefficients (QPlogPo / w) ranged from -0.482 to 4.494, suggesting good absorption. Brain / blood coefficients (QPlogBB) for all compounds were within an acceptable range (-2.549 to -1.307). Human serum binding capacity coefficients (QPlogKhsa) ranged from -1.136 to 0.349, indicating high bioavailability. Compounds 8b-8f exhibited oral absorption rates exceeding 80% in humans, while the other compounds exhibited moderate oral absorption rates (Table 3).

[0167] Example 10. In vivo activity study of compound 8b

[0168] BALB / C nude female mice (certificate SYXK-2018-0003, weight 18-20 g) were obtained from the Second Hospital of Lanzhou University. KYSE30 tumor xenograft model was used for in vivo efficacy experiments. KYSE30 cells (5 × 10 6 The drug was injected subcutaneously into the abdominal region of 6-week-old female nude mice until subcutaneous tumors were formed (>100 mm 3Mice were randomly divided into six groups (4 mice per group). Mice were administered with saline, compound 8b (0.8, 1.2, and 1.6 mg / kg), SAHA (24 mg / kg), and capzimin (8 mg / kg) every 3 days. Tumor volume and body weight were measured every 5 days, and mice were sacrificed 21 days later. Data were analyzed using one-way analysis of variance, and P levels < 0.05 were considered statistically significant.

[0169] Given that the PSMD14 / HDAC dual-target compound 8b exhibited the best in vitro antitumor activity and had a clear mechanism of action, compound 8b was selected for in vivo antitumor activity evaluation against KYSE30 cell xenografts in nude mice, with SAHA (experimentally synthesized) and Capzimin (purchased from TargetMol) as positive control drugs. Figure 8 As shown in Figure A, compared with the positive control groups SAHA (TGI = 54%) and Capzimin (TGI = 50), compound 8b exhibited better in vivo antitumor activity. When compound 8b was administered at a dose of 0.8 mg / kg every 3 days, the TGI was 77%, and when it was increased to 1.6 mg / kg, the TGI was 86.5%. The in vivo tumor inhibition rate was dose-dependent. In addition, no significant changes were observed in the body weight of the test mice after administration, indicating that compound 8b has relatively low toxicity and side effects ( Figure 8 Middle B). The in vivo antitumor activity results indicate that PSMD14 / HDAC dual-target inhibitors have significant advantages and are worthy of further study.

Claims

1. A PSMD14 / HDAC dual-target inhibitor, characterized in that: Its structural formula is one of the following: ； Wherein, n is 5-12.

2. The PSMD14 / HDAC dual-target inhibitor according to claim 1, characterized in that n is 5, 6, 7, 8, 10 or 12.

3. The PSMD14 / HDAC dual-target inhibitor according to claim 2, characterized in that Its structural formula is as follows: 。 4. A method for preparing the PSMD14 / HDAC dual-target inhibitor according to claim 3, characterized in that: The reaction formula is as follows: ； The reaction reagents and conditions are as follows: (I) 1,4-dioxane, hydrochloric acid, reaction at room temperature to reflux; (II) dichloromethane, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine, reaction at 0 °C to room temperature; (III) dichloromethane, isobutyl chloroformate, compound 2, 4-methylmorpholine, reaction at 0 °C to room temperature; (IV) dichloromethane, trifluoroacetic acid, reaction at 0 °C to room temperature; (V) tetrahydrofuran, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, 4-pyrrolidinylpyridine, reaction at room temperature.

5. Use of the PSMD14 / HDAC dual-target inhibitor according to claim 1, 2 or 3 in the preparation of anti-tumor drugs.

6. The use according to claim 5, characterized in that The tumor refers to esophageal cancer or lung cancer.

7. An anti-tumor drug, characterized in that: It contains the PSMD14 / HDAC dual-target inhibitor according to claim 1, 2 or 3 as an active ingredient.

8. The antitumor drug according to claim 7, characterized in that It also contains pharmaceutically acceptable excipients.

9. The antitumor drug according to claim 8, characterized in that It is available in injection and oral dosage forms.

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