CN112442091B - Replication protein A targeted platinum compound - Google Patents

Abstract

The invention discloses a replication protein A targeted platinum compound, which is a series of platinum anti-tumor compounds synthesized by combining tetravalent cisplatin with an RPA (replication protein A) small molecule inhibitor through an axial hydroxyl position and introducing an aliphatic hydrocarbon chain. The platinum anti-tumor compound has the advantages of inhibiting RPA activity and tumor specificity selection, thereby showing anti-tumor activity superior to that of the classical platinum drugs and potential for overcoming clinical platinum drug resistance.

Description

Platinum compounds targeting replication protein A

technical field

The invention belongs to the technical field of platinum compounds, and more specifically relates to a class of DNA damage repair-based platinum compound anti-tumor activity, reducing toxic and side effects and overcoming clinical platinum drug resistance.

Background technique

Malignant tumors are always a threat to human health and life safety, and are of great value and significance to the treatment and research of malignant tumors. Improving the targeting, selectivity and safety of chemotherapy drugs is the current research and development trend of new anti-tumor drugs. Platinum drugs represented by cisplatin (CDDP) play a vital role in clinical tumor treatment (such as ovarian cancer, non-small cell lung cancer), and more than 50% of tumor chemotherapy requires the participation of platinum drugs. However, tumor drug resistance greatly limits the development of such drugs. More than 80% of patients with epithelial ovarian cancer have tumor recurrence due to platinum drug resistance, and chemotherapy fails.

From the molecular mechanism of platinum-based drugs, we know that since the target of platinum-based drugs is DNA, inter-strand or intra-strand cross-links can occur with DNA to form DNA adducts, resulting in DNA damage, hindering DNA replication, transcription, and ultimately cause apoptosis. Therefore, DNA damage repair is closely related to the chemotherapy effect of platinum-based drugs and tumor drug resistance. Studies have shown that nucleoside excision repair (NER) and homologous recombination (HR) can greatly reduce the chemotherapy effect of platinum-based drugs, therefore, selective inhibition of both can further enhance tumor Chemosensitivity of cells to platinum-based drugs.

Replication protein A (replication protein A, RPA) is the main single-chain binding protein in eukaryotic cells, including three subunits RPA1, RPA2 and RPA3, and plays an important role in the process of DNA replication and damage repair. During DNA replication, RPA has the functions of melting, binding single-stranded templates and maintaining continuous DNA replication; when DNA is damaged, RPA, as an important DNA damage repair protein, plays a role in nucleotide excision repair (NER), DNA mismatch repair (MMR ), homologous recombination repair (HR) and other repair processes play an important role. At the same time, RPA and proteins with chromosome structure maintenance, protection, and repair functions gather at DNA damage sites to jointly complete the detection and repair of DNA damage. At the same time, DNA damage homologous repair proteins Rad51, Rad52 cooperate with RPA-ssDNA to complete DNA damage repair. The effect of RPA on repair methods such as NER and HR reduces the efficacy of platinum-based chemotherapy drugs and enhances the drug resistance of tumor cells (Shuck S C, Turchi J J. Targeted inhibition of RPA reveals cytotoxic activity, synergy with chemotherapeutic DNA damaging agents and insight into cellular function [J]. Cancer Research, 2010, 70(8): 3189.). In recent years, the discovery of RPA inhibitors has brought new light to patients with malignant tumors. The combination of RPA inhibitors and platinum drugs will be of great significance for improving anti-tumor activity and anti-tumor drug resistance.

Contents of the invention

The object of the present invention is to overcome the deficiency of existing platinum drugs, and provide the preparation method and application of novel platinum (IV) RPA inhibitor prodrugs, compared with clinical drug cisplatin, platinum antitumor compound of the present invention (i.e. novel platinum Class (IV) RPA inhibitor prodrugs) show significantly better antitumor activity than cisplatin in vivo and in vitro, and have the advantages of reducing systemic toxicity and overcoming cisplatin resistance.

Combining RPA small molecule inhibitors with platinum drugs to form new platinum (IV) RPA small molecule inhibitor prodrugs has the following advantages: (1) From the perspective of chemical structure, the new platinum compounds change the polarity of the previous compounds, log P, transmembrane transport rate and other physical and chemical properties, and then change the uptake mode of platinum drugs to increase the platinum content in cells; at the same time, use the chemical inertness of Pt(IV) octahedral chemical configuration to reduce the platinum drug reactivity and toxicity, and reduce and release synergistic molecules in cells, which improves the curative effect of platinum-based chemotherapy. (2) From the perspective of the target, the RPA inhibitor blocks the RPA-DNA interaction. In DNA replication, RPA inhibitors take advantage of the highly proliferative nature of cancer cells to arrest cells in S phase and cause apoptosis. Therefore, the targeted and synergistic characteristics of small molecule inhibitors of replication protein A can be used to reduce the repair of platinum drugs by the DNA repair mechanism and overcome the resistance of tumor cells to platinum drugs.

Technical purpose of the present invention is achieved through the following technical solutions:

Platinum compounds based on RPA inhibitors (i.e. platinum compounds targeting replication protein A) have the structure shown in the following chemical formula:

Wherein R ₁ is I or Br; R ₂ is a hydrogen atom, butyryl, hexanoyl, octanoyl, dodecanoyl, hexadecanoyl; n is 2 or 3. Preferably R ₁ is I; R ₂ is a hydrogen atom or hexanoyl; n is 2 or 3.

As far as this application is concerned, the tetravalent cisplatin prodrug is used as the parent, and the RPA small molecule inhibitor and fatty chain are combined in the axial position to synthesize the target compound.

TDRL-505: R=Br, n=2; TDRL-550: R=I, n=2

TDRL-543: R=Br, n=3; TDRL-551: R=1, n=3

The preparation method of the above-mentioned platinum compound is prepared according to the following conditions: 1. Preparation of substance 3-substance 3 is obtained by reacting substance 2 and substance TDRL (such as TDRL-551, TDRL-550, TDRL-505 and TDRL-543) , wherein relative to substance 2, substance TDRL is in excess, the molar ratio of substance TDRL to substance 2 is (1.1-1.2):1, the reaction temperature is 50-60°C, preferably 55-60°C, and the reaction time is 12-36h, Preferably 18-24h, the reaction process is carried out under the protection of an inert protective gas (such as argon, nitrogen, helium) in a light-proof environment, select magnetic stirring, 200-300 revolutions per minute, and select an organic solvent to provide the reaction for substance 2 and substance TDRL Environment, the solubility and dispersibility of substance 2 and substance TDRL in organic solvents need to be considered, such as dimethyl sulfoxide, N,N'-dimethylformamide, tetrahydrofuran; after the reaction is completed, the product in the reaction solution is extracted and removed Remove solvent and unreacted raw materials, catalysts and impurities. The dichloromethane layer was collected, dried over anhydrous sodium sulfate, suction filtered, rotary evaporated, and purified by thin layer chromatography (developing solvent [CH ₂ Cl ₂ :CH ₃ OH]=10:1) to obtain substance 3

2. Preparation of substances 3a-3e—substances 3a-3e are obtained by reacting substance 3 with a series of corresponding fatty acid anhydrides, and the molar ratio of substance 3 and fatty acid anhydrides is 1:(2-2.5). The reaction temperature is 45-55°C, preferably 50-55°C, the reaction time is 4-8h, preferably 6-8h, and the reaction is protected from light under the protection of an inert protective gas (such as argon, nitrogen, helium), and magnetic stirring is selected. 200-300 revolutions per minute, choose an organic solvent to provide a reaction environment for substance 3 and a series of corresponding fatty acid anhydrides, the solubility and dispersibility of substance 3 and a series of corresponding fatty acid anhydrides in organic solvents need to be considered, such as dimethyl sulfoxide, N , N'-dimethylformamide, tetrahydrofuran (Muhammad N, Sadia N, Zhu C, et al.Biotin-tagged platinum(IV) complexes atargeted cytostatic agents against breast cancer cells[J].Chem.Commun.2017:10.1039 .C7CC05311H.). After the reaction, the solvent DMF was removed by rotary evaporation, and the final products 3a-3e were purified by thin-layer chromatography (developing solvent [CH ₂ Cl ₂ :CH ₃ OH]=20:1).

In the above reaction, the catalyst O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroboric acid (TBTU) and triethylamine (TEA) can be optionally used, and the moles of substances TDRL and TBTU The ratio is 1:2, the molar ratio of substance TDRL and triethylamine is 1:4.

In the above technical solution, substance 2 is produced by reacting cisplatin and 30% by volume H ₂ O ₂ aqueous solution at 70-80° C. under reflux and stirring in the dark, and the reaction time is 4-6 hours. The substance TDRL is formed by stirring the intermediate and glutaric anhydride in chloroform solution at 60-70°C under reflux, and the reaction time is 3-4h. (Mishra AK, Dormi SS, Turchi AM, et al.Chemical inhibitor targeting the replication protein A–DNA interaction increases the efficacy of Pt-based chemotherapy in lung and ovarian cancer[J].BiochemicalPharmacology,2015,93(1):25-33 .).

Application of the above-mentioned platinum compounds in the preparation of antitumor drugs. Compared with the prior art, the anti-tumor activity of the platinum compound of the present invention in HeLa, A549, NCI-H460 and other cell lines is obviously better than that of classic platinum drugs such as cisplatin. The platinum compound of the present invention still maintains excellent anti-tumor activity in the NCI-H460/cis cisplatin-resistant cell line. The novel platinum (IV) RPA small molecule inhibitor prodrug of the present invention can inhibit DNA repair by two repair modes of NER (nucleotide excision repair) and HRR (homologous recombination repair) while causing DNA damage. In the anti-tumor experiment in vivo, the invention has good anti-tumor activity and anti-tumor drug resistance characteristics, and simultaneously reduces the toxicity of platinum drugs.

Description of drawings

Fig. 1 is the proton nuclear magnetic resonance spectrum of substance 3 prepared in the embodiment of the present invention.

Fig. 2 is a carbon nuclear magnetic resonance spectrum of substance 3 prepared in the example of the present invention.

Fig. 3 is a liquid-phase purity detection spectrum of substance 3 prepared in the example of the present invention.

Fig. 4 is a high-resolution mass spectrum of substance 3 prepared in the example of the present invention.

Fig. 5 is the H NMR spectrum of the substance 3a prepared in the example of the present invention.

Fig. 6 is a carbon nuclear magnetic resonance spectrum of substance 3a prepared in the example of the present invention.

Fig. 7 is a liquid-phase purity detection spectrum of the substance 3a prepared in the example of the present invention.

Fig. 8 is the H NMR spectrum of the substance 3b prepared in the example of the present invention.

Fig. 9 is a carbon nuclear magnetic resonance spectrum of substance 3b prepared in the example of the present invention.

Fig. 10 is a high-resolution mass spectrum of substance 3b prepared in the example of the present invention.

Fig. 11 is a liquid-phase purity detection spectrum of the substance 3b prepared in the example of the present invention.

Fig. 12 is the H NMR spectrum of substance 3c prepared in the example of the present invention.

Fig. 13 is a carbon nuclear magnetic resonance spectrum of substance 3c prepared in the example of the present invention.

Fig. 14 is a liquid-phase purity detection spectrum of the substance 3c prepared in the example of the present invention.

Fig. 15 is a high-resolution mass spectrum of substance 3c prepared in the example of the present invention.

Fig. 16 is a 3d proton nuclear magnetic resonance spectrum of the substance prepared in the example of the present invention.

Fig. 17 is a 3d carbon nuclear magnetic resonance spectrum of the substance prepared in the example of the present invention.

Fig. 18 is a 3d liquid phase purity detection spectrum of the substance prepared in the example of the present invention.

Fig. 19 is a proton nuclear magnetic resonance spectrum of substance 3e prepared in the example of the present invention.

Fig. 20 is a carbon nuclear magnetic resonance spectrum of substance 3e prepared in the example of the present invention.

Fig. 21 is a liquid-phase purity detection spectrum of the substance 3e prepared in the example of the present invention.

Fig. 22 is the proton nuclear magnetic resonance spectrum of substance 4 prepared in the example of the present invention.

Fig. 23 is a carbon nuclear magnetic resonance spectrum of substance 4 prepared in the example of the present invention.

Fig. 24 is a high-resolution mass spectrum of substance 4 prepared in the example of the present invention.

Fig. 25 is the proton nuclear magnetic resonance spectrum of substance 5 prepared in the example of the present invention.

Fig. 26 is a carbon nuclear magnetic resonance spectrum of substance 5 prepared in the example of the present invention.

Fig. 27 is a high-resolution mass spectrum of substance 5 prepared in the example of the present invention.

Fig. 28 is the proton nuclear magnetic resonance spectrum of substance 6 prepared in the example of the present invention.

Fig. 29 is a carbon nuclear magnetic resonance spectrum of substance 6 prepared in the example of the present invention.

Fig. 30 is a high-resolution mass spectrum of substance 6 prepared in the example of the present invention.

Fig. 31 is a schematic diagram of the results of 3b prepared in the embodiment of the present invention on cell cycle and apoptosis.

Figure 32 is a schematic diagram of the results of in vivo anti-tumor experiments on 3b prepared in the examples of the present invention.

Detailed ways

The technical solutions of the present invention will be further described below in conjunction with specific embodiments. The experimental equipment is as follows: nuclear magnetic resonance analyzer, AVANCE III 400MHz of Bruker Company, Switzerland; high-resolution mass spectrometry, Agilent 6520Q-TOF LC/MS of Agilent Company of the United States; high performance liquid chromatography, SPD-20A of Shimadzu Company of Japan; confocal laser microscope, Japan Olympus FV-1000; electronic analytical balance, Germany Sartorius BP211D; rotary evaporator, Shanghai Shensheng Technology Co., Ltd. R204; constant temperature magnetic stirrer, Henan Gongyi Yuhua Instrument Co., Ltd. 85-2. The raw materials and reagents are as follows: cisplatin, Shandong Boyuan Pharmaceutical Co., Ltd., chemically pure; TBTU, Tianjin Xiens Biochemical Technology Co., Ltd., chemically pure; 30% H ₂ O ₂ , Tianjin Beilian Chemical Co., Ltd., chemically pure; Ultra-dry N,N'-dimethylformamide and ultra-dry N,N'-dimethyl sulfoxide, Bailingwei Technology Co., Ltd., chemically pure; dichloromethane, Tianjin Jindong Tianzheng Fine Chemical Reagent Factory, analytically pure ; anhydrous ether, Tianjin Concord Technology Co., Ltd., analytically pure; methanol, Tianjin Concord Technology Co., Ltd., chromatographically pure; human non-small cell lung cancer cell line A549, human non-small cell lung cancer cell line NCI-H460 and human cervical cancer cell line HeLa, American ATCC; human non-small cell lung cancer cell line NCI-H460cisR, Peking Union Medical College Cell Resource Center; DMEM medium and RPMI 1640 medium, American Gibco Company; fetal bovine serum, American Hyclone Company.

Example 1—Synthesis and characterization of prodrugs based on novel platinum-based Pt(IV) RPA small molecule inhibitors, as shown in Figures 1-30

Accurately weigh cisplatin (100 mg, 0.34 mmol) in a 10 mL round bottom flask, slowly add 2 mL of 30% H ₂ O ₂ dropwise, and reflux the reaction solution at 75° C. °C overnight. After standing still, the suspension was separated by centrifugation to obtain a yellow solid precipitate, which was washed with distilled water, ethanol and ether, and then vacuum-dried to obtain 89 mg of yellow solid powder of substance 2, with a yield of 88.28%.

Accurately weigh substance 2 (50.11 mg, 0.15 mmol) into a 25 mL round bottom flask, add DMSO to dissolve, and stir the reaction solution at 60° C. under the protection of argon for 30 min in the dark until the solution is clear. In addition, accurately weigh the substances TDRL-551 (0.15mmol) and TBTU (0.30mmol), add DMSO to dissolve, add 70μL triethylamine (0.6mmol) to the reaction solution, ultrasonically react for 30min at room temperature, and add the reaction solution to the substance 2 React in a round bottom flask. The reaction solution was stirred at 60° C. under argon protection for 12 h in the dark to obtain a reddish-brown clear liquid. The reaction solution was washed with water and centrifuged to obtain a pale yellow solid precipitate. The precipitate was purified by thin-layer chromatography using dichloromethane-methanol (15:1) as the developing solvent, and substance 3 was obtained as light yellow solid powder. Yield: 45.5 mg, Yield: 33.4%, Purity: 97.16%. ¹ H NMR (400MHz, DMSO-d ₆ , ppm), δ7.96(d, J=8.9Hz, 1H), 7.83(d, J=8.4Hz, 1H), 7.59(d, J=8.4Hz, 1H ), 7.33(d, J=2.1Hz, 1H), 7.25(dd, J=9.0, 2.3Hz, 1H), 6.29-5.75(m, 4H), 4.19(q, J=6.9Hz, 1H), 3.97 (dd,J=18.0,12.1Hz,1H),3.21(dd,J=28.3,5.2Hz,1H),2.86(dtd,J=30.5,15.4,7.5Hz,1H),2.26(t,J=7.3 Hz, 1H), 1.91-1.71 (m, 1H), 1.39 (t, J=6.9Hz, 1H); ¹³ C NMR (101MHz, DMSO-d ₆ , ppm): δppm 181.21, 170.98, 160.97, 154.22, 148.77 ,138.18,131.13,130.58,129.97,129.24,122.66,120.86,107.24,97.89,64.30,41.06,36.46,33.58,21.63,15.02.HR-MS(ESI,positive-ion mode), m/z: calcd for _C H ₂₉ Cl ₃ IN ₅ O ₅ Pt[M+H] ⁺ :905.9927; found:905.9911.

Accurately weigh substance 3 (30mg, 0.033mmol) into a 25mL round bottom flask, add DMF to dissolve until clear, add butyric anhydride (10.80μL, 0.066mmol) to the solution, and react in the dark at 45°C under the protection of argon 24h, a light yellow solution was obtained. After the reaction was completed, DMF was removed by rotary evaporation, and purified by thin-layer chromatography to obtain substance 3a as a pale yellow solid powder. Yield: 20.10 mg, Yield: 59.11%, Purity: 96.14%. ¹ H NMR (400MHz, DMSO-d ₆ , ppm) δ7.95 (d, J = 9.0Hz, 1H), 7.84 (d, J = 8.3Hz, 1H), 7.59 (d, J = 8.3Hz, 1H) ,7.34(d,J=1.9Hz,1H),7.25(dd,J=9.0,2.2Hz,1H),6.56(s,3H),5.83(dd,J=12.0,5.2Hz,1H),4.18( q,J=6.8Hz,1H),3.97(dd,J=18.1,12.2Hz,1H),3.36-3.18(m,3H),3.03-2.66(m,2H),2.33(t,J=7.3Hz ,1H),2.20(t,J=7.3Hz,1H),1.89-1.70(m,1H),1.47(dd,J=14.7,7.3Hz,1H),1.39(t,J=6.9Hz,2H) ,0.87(t,J=7.4Hz,2H). ¹³ C NMR(101MHz,DMSO-d ₆ ,ppm)δ180.76,180.36,170.20,160.33,153.68,148.13,137.54,130.45,129.94,129.31,128.60,122.02 120.25, 106.57, 97.38, 63.66, 62.05, 40.41, 37.61, 35.00, 32.74, 20.73, 18.83, 14.39, 13.66.

Accurately weigh substance 3 (30mg, 0.033mmol) into a 25mL round bottom flask, add DMF to dissolve, stir until clear, add hexanoic anhydride (15.20μL, 0.066mmol) to the solution, under argon protection, 45°C avoid After light stirring for 24h, a light yellow solution was obtained. After the reaction, DMF was removed by rotary evaporation, and purified by thin-layer chromatography to obtain substance 3b as light yellow solid powder, yield: 23.30 mg, yield: 63.96%, purity: 98.68%. ¹ H NMR (400MHz, DMSO-d ₆ , ppm) δ7.95(d, J=9.1Hz, 1H), 7.84(d, J=8.5Hz, 1H), 7.59(d, J=8.5Hz, 1H) ,7.34(d,J=2.3Hz,1H),7.25(dd,J=9.0,2.4Hz,1H),6.88-6.33(m,3H),5.83(dd,J=12.0,5.3Hz,1H), 4.18(q,J=6.9Hz,1H),4.04-3.85(m,1H),3.28(dd,J=18.1,5.2Hz,1H),2.88(tdd,J=23.4,15.6,7.5Hz,1H) ,2.33(t,J=7.4Hz,1H),2.21(t,J=7.5Hz,1H),1.90-1.70(m,1H),1.56-1.43(m,1H),1.39(d,J=7.0 Hz, 1H), 1.26(dd, J=7.1, 3.9Hz, 2H), 0.86(t, J=6.9Hz, 1H). ¹³ C NMR (101MHz, DMSO-d ₆ , ppm) δ180.87, 180.37, 170.32, 170.20,160.23,153.60,148.02,137.54,130.63,129.93,129.21,128.55,121.91,120.55,106.92,97.59,63.66,35.94,35.65,32.85,31.12,24.80,22.00,20.65,14.39,13.74.HR-MS( ESI)m/z calcd for C ₃₁ H ₄₀ Cl ₃ IN ₅ O ₆ Pt[M+H] ⁺ :1006.07156,found:1006.07227.

Accurately weigh substance 3 (30mg, 0.033mmol) into a 25mL round-bottomed flask, add DMF to dissolve, stir until clarification, add caprylic anhydride (19.61μL, 0.066mmol) to the solution, and store under argon protection at 45°C After light stirring for 24h, the solution turned into a light yellow solution. After the reaction was completed, the DMF in the reaction solution was removed by rotary evaporation, and purified by thin-layer chromatography to obtain a light yellow solid powder of substance 3c, yield: 20.5 mg, yield: 61.75%, purity: 97.96%. ¹ H NMR (400MHz, DMSO-d ₆ , ppm) δ7.95 (d, J = 9.0Hz, 1H), 7.84 (d, J = 8.3Hz, 1H), 7.59 (d, J = 8.3Hz, 1H) ,7.34(s,1H),7.26(d,J=8.9Hz,1H),6.56(s,2H),5.95-5.76(m,1H),4.35-4.07(m,1H),3.97(dd,J =18.0,12.1Hz,1H),3.28(dd,J=18.2,5.1Hz,1H),3.07-2.75(m,1H),2.33(t,J=7.1Hz,1H),2.21(t,J= 7.4Hz, 1H), 1.89-1.70(m, 1H), 1.44(d, J=6.9Hz, 1H), 1.39(t, J=6.9Hz, 1H), 1.24(s, 3H), 0.86(t, J＝6.5Hz, 1H). ¹³ C NMR (101MHz, DMSO-d ₆ , ppm) δ180.86, 170.20, 160.33, 153.68, 148.13, 137.54, 130.44, 128.61, 122.02, 106.57, 97.39, 63.65, 40.08, 39.8 ,39.45,39.25,39.04,38.83,35.65,32.74,31.18,28.56,25.42,22.07,20.72,14.39,13.96.HR-MS(ESI)m/z calcd for C ₃₃ H ₄₄ Cl ₃ IN ₅ O ₆ Pt[ M+H] ⁺ :1034.10293,found:1034.10339.

Accurately weigh substance 3 (30mg, 0.033mmol) and place it in a 25mL round bottom flask, add DMF to dissolve, stir until clear, add dodecanoic anhydride (25.25mg, 0.066mmol) to the solution, under argon protection, 45 °C and stirred in the dark for 24 hours to obtain a pale yellow solution. After the reaction, the DMF in the reaction solution was removed by rotary evaporation, and purified by thin-layer chromatography to obtain a light yellow solid powder of substance 3d, yield: 20.5 mg, yield: 63.94%, purity: 96.54%. ¹ H NMR (400MHz, DMSO-d ₆ , ppm) δ7.95 (d, J = 9.0Hz, 1H), 7.84 (d, J = 8.4Hz, 1H), 7.59 (d, J = 8.4Hz, 1H) ,7.34(d,J=2.1Hz,1H),7.25(dd,J=9.0,2.3Hz,1H),6.56(s,3H),5.83(dd,J=12.0,5.2Hz,1H),4.18( q,J=6.8Hz,1H),3.97(dd,J=18.0,12.1Hz,1H),3.28(dd,J=18.1,5.1Hz,1H),3.01-2.74(m,1H),2.33(t ,J=7.3Hz,1H),2.21(t,J=7.4Hz,1H),1.94-1.74(m,1H),1.45(s,1H),1.40(dd,J=13.5,6.6Hz,2H) ,1.23(s,8H),0.85(t,J＝6.7Hz,2H). ¹³ CNMR(101MHz,DMSO-d ₆ ,ppm)δ180.87,180.36,170.19,160.33,153.66,148.13,137.54,130.44,129.92, 129.30,128.59,122.01,120.24,106.56,97.37,63.65,35.67,35.01,32.75,31.28,29.04,29.02,28.97,28.92,28.71,28.61,25.43,22.08,1340.3993,

Accurately weigh substance 3 (30mg, 0.033mmol) into a 25mL round-bottomed flask, add DMF to dissolve, stir until clear, add palmitic anhydride (32.65mg, 0.066mmol) to the solution, and protect it under argon at 45°C in the dark After stirring for 24h, a light yellow solution was obtained. After the reaction, the DMF in the reaction solution was removed by rotary evaporation, and purified by thin-layer chromatography to obtain a light yellow solid powder of substance 3e, yield: 26.0 mg, yield: 68.73%, purity: 98.84%. ¹ H NMR (400MHz, DMSO-d ₆ , ppm) δ7.96 (d, J = 8.8Hz, 1H), 7.84 (d, J = 8.1Hz, 1H), 7.59 (d, J = 8.2Hz, 1H) ,7.34(s,1H),7.26(d,J=9.0Hz,1H),6.63(d,J=61.2Hz,3H),5.83(dd,J=12.0,5.0Hz,1H),4.19(d, J＝6.9Hz, 1H), 3.97(dd, J＝17.7, 12.1Hz, 1H), 3.45(s, 1H), 3.28(dd, J＝18.1, 5.0Hz, 1H), 3.06-2.72(m, 1H ),2.33(t,J=7.0Hz,1H),2.21(t,J=7.3Hz,1H),1.89-1.75(m,1H),1.45(s,1H),1.41(dd,J=16.5, 9.7Hz, 2H), 1.23(s, 11H), 0.85(t, J=6.3Hz, 1H). ¹³ CNMR(101MHz, DMSO-d ₆ , ppm) δ181.37, 180.87, 170.70, 160.83, 154.16, 148.64, 138.04 .

Accurately weigh substance 2 (50.11 mg, 0.15 mmol) into a 25 mL round-bottom flask, add DMSO to dissolve, and stir the reaction solution at 60° C. in the dark for 30 min under the protection of argon, and the solution becomes colorless and clear. Take another accurately weighed substance TDRL-543 (0.15mmol) and TBTU (0.30mmol), add DMSO to dissolve, add 70μL triethylamine to the reaction solution, and react with ultrasonic vibration at room temperature for 30min, add the reaction solution to the solution of substance 2, and The reaction bottle was stirred at 60°C in the dark for 12 hours under the protection of argon to obtain a reddish-brown clear liquid. The reaction solution was washed with water and centrifuged to obtain a pale yellow solid precipitate. Using dichloromethane-methanol (15:1) as the developing solvent, thin-layer chromatography purified to obtain substance 4 as light yellow solid powder. Yield: 38.3 mg, Yield: 29.7%. ¹ H NMR (400MHz, DMSO-d ₆ , ppm) δ7.96 (d, J = 8.5Hz, 1H), 7.75 (d, J = 8.3Hz, 1H), 7.66 (d, J = 8.4Hz, 1H) ,7.33(s,1H),7.25(d,J=8.9Hz,1H),6.21-5.77(m,4H),4.19(dd,J=13.8,6.8Hz,1H),3.98(dd,J=18.0 ,12.1Hz,1H),3.22(dd,J=36.5,4.9Hz,1H),2.98-2.73(m,1H),2.27(t,J=7.2Hz,1H),1.82(dd,J=14.8, 7.5Hz, 1H), 1.39 (t, J=6.9Hz, 2H); ¹³ C NMR (101MHz, DMSO-d ₆ , ppm) δ180.81, 170.58, 160.55, 160.16, 153.59, 148.35, 131.93, 130.45, 129.55, 128.93 , 123.90, 122.24, 120.44, 106.83, 63.87, 48.78, 40.71, 36.05, 33.16, 21.22, 14.59; HR-MS (ESI, positive-ion mode) m/z: calcd for C ₂₅ H ₂₉ BrCl ₃ N ₅ O ₅ Pt[M+H] ⁺ :858.0065; found: 858.0107.

Accurately weigh substance 2 (50.11 mg, 0.15 mmol) into a 25 mL round-bottom flask, add DMSO to dissolve, and stir the reaction solution at 60° C. in the dark for 30 min under the protection of argon, and the solution becomes colorless and clear. Take another accurately weighed substance TDRL-505 (0.15mmol) and TBTU (0.30mmol), add DMSO to dissolve, add 70μL triethylamine to the reaction solution, and react with ultrasonic vibration at room temperature for 30min, add the reaction solution to the solution of substance 2, and The reaction bottle was stirred at 60°C in the dark for 12 hours under the protection of argon to obtain a reddish-brown clear liquid. The reaction solution was washed with water and centrifuged to obtain a pale yellow solid precipitate. Using dichloromethane-methanol (13:1) as developing solvent, thin-layer chromatography purified to obtain substance 5 as light yellow solid powder. Yield: 35.2 mg, Yield: 27.7%. ¹ H NMR (400MHz, DMSO-d ₆ , ppm) δ8.02(s, 1H), 7.91(d, J=8.9Hz, 1H), 7.73(d, J=8.3Hz, 2H), 7.66(d, J＝8.2Hz, 2H), 7.33(s, 1H), 7.25(d, J＝8.9Hz, 1H), 6.27-5.67(m, 7H), 4.18(d, J＝6.9Hz, 2H), 3.99( dd,J=17.8,12.2Hz,1H),3.27(s,1H),3.03(d,J=44.8Hz,2H),2.55(d,J=7.1Hz,1H),1.39(t,J=6.8 Hz,3H); ¹³ C NMR(101MHz,DMSO-d ₆ ,ppm)δ180.12,170.06,160.32,153.47,148.14,134.96,133.29,131.74,130.24,129.87,129.26,128.65,123.77,210.6,1122. _{Found _ _ _ _} ^_ :843.9970.

Accurately weigh substance 2 (50.11 mg, 0.15 mmol) into a 25 mL round-bottom flask, add DMSO to dissolve, and stir the reaction solution at 60° C. in the dark for 30 min under the protection of argon, and the solution becomes colorless and clear. Take another accurately weighed substance TDRL-550 (0.15mmol) and TBTU (0.30mmol), add DMSO to dissolve, add 70 μ L triethylamine to the reaction solution, and react with ultrasonic vibration at room temperature for 30 minutes, add the reaction solution to the substance 2 solution, and The reaction bottle was stirred at 60°C in the dark for 12 hours under the protection of argon to obtain a reddish-brown clear liquid. The reaction solution was washed with water and centrifuged to obtain a pale yellow solid precipitate. Using dichloromethane-methanol (13:1) as developing solvent, thin-layer chromatography purified to obtain substance 6 as light yellow solid powder. Yield: 34.8 mg, Yield: 25.9%. ¹ H NMR (400MHz, DMSO-d ₆ , ppm) δ8.01(s, 1H), 7.91(d, J=9.0Hz, 1H), 7.83(d, J=8.3Hz, 2H), 7.57(d, J＝8.3Hz, 2H), 7.33(s, 1H), 7.26(d, J＝8.9, 2.2Hz, 1H), 6.32-5.70(m, 7H), 4.18(d, J＝6.9Hz, 2H), 3.98(dd,J=17.9,12.1Hz,1H),3.27(dd,J=18.1,5.2Hz,1H),3.15-2.87(m,2H),2.62-2.52(m,1H),1.39(t, J＝6.9Hz, 3H); ¹³ C NMR (101MHz, DMSO-d ₆ , ppm) δ180.34, 170.27, 160.56, 153.97, 148.38, 137.83, 130.71, 130.09, 129.52, 128.80, 122.30, 120.52, 1376.286, 9 , 57.66, 40.74, 36.03, 31.35, 30.08, 14.65; HR-MS (ESI, positive-ionmode) m/z: calcd for C ₂₄ H ₂₇ Cl ₃ IN ₅ O ₅ Pt[M+H] ⁺ :891.9770; found :891.9813.

Embodiment 2—compound (3a-3e) and antitumor activity evaluation of cisplatin

MTT colorimetry is one of the methods used to detect cell growth and survival in experimental research. In the mitochondria of living cells, exogenous MTT can be reduced by intracellular succinate dehydrogenase to formazan, a purple-blue crystal formazan that is insoluble in water. Formazan is deposited in the cytoplasm of cells, but dead cells do not This phenomenon occurs. When formazan encounters DMSO, the formazan deposited in the cells will be dissolved by DMSO. Use a microplate reader to detect the formazan optical density value (OD value) at a wavelength of 570nm to indirectly reflect the number of living cells. The amount of MTT crystals formed is positively correlated with the number of living cells. Use Graphpad Prism 6 software to process data and calculate IC ₅₀ The _IC50 value refers to the half-inhibitory concentration of the measured drug on cell growth. The experimental results show that, as shown in the table below, the novel platinum (IV) RPA small molecule inhibitor prodrug of the present invention has significantly better antitumor activity than classic platinum drugs such as cisplatin in HeLa, A549, NCI-H460 and other cell lines. The novel platinum (IV) RPA small molecule inhibitor prodrug of the present invention still maintains excellent anti-tumor activity in NCI-H460cisR and other cisplatin-resistant cell lines. The _IC50 values of the series of prodrugs of the present invention and cisplatin in different cell lines for 72h ^a

^{a The} IC ₅₀ value ± standard deviation (μM) was obtained by doubling half-dilution method, with three replicate wells in each group, repeated once, and dosed and incubated for 72 hours to fit the dose-effect curve. ^b FI (fold increase) is the ratio of IC ₅₀ (CDDP) to IC ₅₀ (substance 3b). ^c RF (resistance coefficient) ratio of IC ₅₀ (NCI-H460cisR) to IC ₅₀ (NCI-H460). ^b FI (fold increase) is the ratio of IC ₅₀ (CDDP) to IC ₅₀ (substance 3). ^e ND, not determined.

Example 3

We use cell cycle and apoptosis experiments to verify the anti-tumor activity of the novel platinum (IV) RPA small molecule inhibitor prodrug of the present invention. The cell cycle refers to the process of cells from the completion of one mitosis to the end of the next mitosis. It can be divided into three phases: G0/G1 phase, S phase, and G2/M phase. The G0/G1 phase is the preparation period for mitosis, and the synthesis of RNA and related proteins is doubled to make necessary preparations for the synthesis of genetic material DNA; the S phase is the DNA synthesis phase, in which all DNA synthesis is completed; the G2/M phase is During protein synthesis and mitosis, the synthesized chromosomes and other components are evenly distributed to two newly formed daughter cells, and the above process repeats itself to realize cell proliferation. The cell cycle plays a very important role in the regulation of cell proliferation and apoptosis.

As shown in accompanying drawings 31 and 32, the results of cell cycle experiments show that the novel platinum (IV) RPA small molecule inhibitor prodrug of the present invention is distributed in G2 and S phases as the concentration of substance 3b increases (0.1-10 μM). The number of cells showed an upward trend; at the same time, the proportion of cells in G1 phase gradually decreased. This indicates that after the action of the drug, cells gather in the G2 phase and S phase during the cell cycle, and substance 3b can arrest HeLa cells in the G2 phase and S phase. Substance 3b at a concentration of 1 μM in the treatment group is mainly S Phase arrest, which turns into G2 phase arrest at 10 μM concentration. Compared with the control group, the cycle arrest of HeLa cells was more pronounced by 3b. The results of cell apoptosis experiments showed that compared with the control group cisplatin, single substitution (3) and combination drug (551+Pt), the apoptosis rate of substance 3b in the treatment group was significantly increased, and with the increase of administration concentration High, the apoptosis rate of the treatment group increased significantly, and the apoptotic area was mainly concentrated in the late withering area. This shows that the novel platinum (IV) RPA small molecule inhibitor prodrug 3b of the present invention can effectively induce the apoptosis of HeLa cells.

Example 4

We further studied the in vivo anti-tumor activity and anti-drug resistance activity evaluation of the novel platinum (IV) RPA small molecule inhibitor prodrugs of the present invention through in vivo anti-tumor experiments. Using 4-year-old nude mice as experimental animals, a nude mouse subcutaneous xenograft tumor model was constructed with a human tumor cell line (HeLa) with relatively sensitive in vitro activity. After the model was successfully constructed, the tumor-bearing nude mice were randomly divided into groups and administered by tail vein injection. The body weight and tumor volume of the animals were observed and recorded. The experiment lasted for 4 weeks. After the experiment was over, the experimental animals were sacrificed by cervical dislocation, and the tumor tissues and major organs were collected for HE staining and mechanism analysis. The experimental results show that in the six experimental models of PBS, CDDP, 551+Pt, TDRL-551, substance 3 and substance 3b, after a 24-day experimental period, compared with other groups, the substance 3b treatment group has obvious tumor inhibition As a result, the tumor volume of nude mice in the four groups of PBS, TDRL-551, 551+Pt and substance 3 showed an overall upward trend. Although the CDDP administration group also had an obvious tumor-suppressing effect, the weight loss of nude mice in the CDDP group was too obvious. It shows that the CDDP compound has great toxic and side effects on the nude mice themselves.

The adjustment of the process parameters according to the content of the present invention can realize the preparation of the platinum compound of the present invention, and exhibit the anticancer performance basically consistent with the examples. The present invention has been described as an example above, and it should be noted that, without departing from the core of the present invention, any simple deformation, modification or other equivalent replacements that can be made by those skilled in the art without creative labor all fall within the scope of this invention. protection scope of the invention.

Claims (10)

1. The replication protein A targeted platinum compound is characterized by having a structure shown as the following chemical formula:

wherein R is ₁ Is I or Br; r is ₂ Is hydrogen atom, butyryl, hexanoyl, octanoyl, dodecanoyl or hexadecanoyl; n is 2 or 3.

2. The replication protein a-targeted platinum-based compound of claim 1, wherein R is ₁ Is I; r is ₂ Is a hydrogen atom, a hexanoyl group; n is 2 or 3.

3. The preparation method of the platinum compound with the replication protein A targeting is characterized in that a tetravalent cisplatin prodrug is used as a matrix, an RPA small molecular inhibitor and an aliphatic chain are combined successively at an axial position to synthesize a target compound, and the preparation method is carried out according to the following conditions:

substance 3

(1) Preparation of substance 3 is obtained by reacting substance 2 with substance TDRL, wherein substance TDRL is in excess with respect to substance 2 and the molar ratio of substance TDRL to substance 2 is (1.1-1.2): 1, the reaction temperature is 50-60 ℃, the reaction time is 12-36h, the reaction process is carried out under the protection of inert protective gas and in a dark environment, and n is 2 or 3;

substance 2

(2) Preparation of substances 3a-3 e: obtained by reacting substance 3 with a series of corresponding fatty acid anhydrides in a molar ratio of substance 3 to fatty acid anhydrides of 1: (2-2.5), reacting for 4-8h at the reaction temperature of 45-55 ℃ in the dark under the protection of inert protective gas;

4. the method for preparing the replication protein A-targeted platinum compound as claimed in claim 3, wherein in the preparation of the substance 3, the reaction temperature is 55-60 ℃, the reaction time is 18-24 h, and the inert protective gas is argon, nitrogen or helium; magnetic stirring is selected, 200-300 revolutions per minute.

5. The method of claim 3, wherein the organic solvent used in the preparation of substance 3 is dimethylsulfoxide, N' -dimethylformamide, or tetrahydrofuran.

6. The method for preparing replication protein A targeted platinum compound according to claim 3, wherein in the preparation of the substance 3, catalysts O-benzotriazole-N, N, N ', N' -tetramethyluronium tetrafluoroborate and triethylamine are used, and the molar ratio of the substances TDRL and O-benzotriazole-N, N, N ', N' -tetramethyluronium tetrafluoroborate is 1:2, the molar ratio of substances TDRL and triethylamine is 1:4.

7. the method of claim 3, wherein substance 2 is prepared from cisplatin and 30% by volume H ₂ O ₂ The aqueous solution is generated by refluxing and stirring reaction at 70-80 ℃ in the dark for 4-6h.

8. The method for preparing the replication protein A targeted platinum compound as claimed in claim 3, wherein in the preparation of the substances 3a to 3e, the reaction temperature is 50 to 55 ℃, the reaction time is 6 to 8 hours, and the inert protective gas is argon, nitrogen or helium; magnetic stirring is selected, and 200-300 revolutions per minute are carried out; the organic solvent is dimethyl sulfoxide, N' -dimethylformamide or tetrahydrofuran.

9. Use of the replication protein a-targeted platinum compound of claim 1 or 2 in the preparation of an anti-tumor medicament.

10. The use of claim 9, wherein the neoplasm is lung cancer or cervical cancer.

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