Divalent platinum complexes and their derivatives, preparation methods, pharmaceutical compositions and uses

Abstract

The present invention discloses a class of divalent platinum complexes containing non-steroidal anti-inflammatory drug structural units, their derivatives, preparation methods, pharmaceutical compositions and applications. The structure of this class of divalent platinum complexes is as follows: The derivatives of this class of divalent platinum complexes refer to isomers of the said divalent platinum complexes or mixtures thereof. When this class of divalent platinum complexes and their derivatives act on cancer cells, they can cause apoptosis by inducing DNA damage and the production of reactive oxygen species, and can also effectively inhibit the growth of cancer cells by down-regulating the expression of cyclooxygenase 2 to reduce inflammation. At the same time, this class of divalent platinum complexes with C=N double bonds can also target tumors using the acidic microenvironment of tumors, reduce tissue toxicity, show the characteristics of high efficiency and low toxicity in anti-tumor, and can overcome cisplatin resistance to a certain extent, and can be prepared into anti-tumor drugs. In addition, the synthesis method of this class of divalent platinum complexes is simple and easy to operate.

Description

Technical Field

[0001] The present invention relates to a class of divalent platinum complexes containing non-steroidal anti-inflammatory drug structural units, their derivatives, preparation methods, pharmaceutical compositions and applications, and in particular to a divalent platinum complex and its derivatives, preparation methods, pharmaceutical compositions and applications that can be prepared into an anti-tumor drug capable of overcoming cisplatin resistance to a certain extent, reducing tumor inflammation, and having high efficiency and low toxicity. Background Art

[0002] A large number of clinical and epidemiological studies have shown that 15% - 20% of malignant tumors are caused by infections and non-controllable inflammations. For example, inflammatory bowel disease is associated with colon cancer, chronic hepatitis B virus infection can lead to liver cancer, Helicobacter pylori infection is significantly associated with gastric cancer, human herpesvirus infection can cause nasopharyngeal carcinoma, and human papillomavirus infection can cause cervical cancer or Burkitt lymphoma, etc. Chronic inflammation is involved in all pathological processes such as the occurrence, development, invasion, and metastasis of malignant tumors. Therefore, inflammation is also known as the eighth major feature of malignant tumors.

[0003] Non-steroidal anti-inflammatory drugs (NSAIDs) are a class of anti-inflammatory drugs that do not contain a steroid structure. NSAIDs were found to be beneficial for the treatment of head and neck squamous cell carcinoma in 1981. This was the first discovery of the effect of NSAIDs on cancer. In subsequent studies, the effects of NSAIDs on different cancers have been continuously reported. However, as cyclooxygenase-2 (COX-2) inhibitors, when used alone, NSAIDs only have moderate killing activity against cancer cells.

[0004] Platinum-based drugs are one of the most important anti-cancer drugs in clinical practice, and they play a crucial role in tumor treatment. However, divalent platinum-based drugs represented by cisplatin have serious toxicity, and their application is also limited by drug resistance caused by reduced platinum uptake, increased efflux, drug inactivation, and DNA damage repair.

[0005] Clinical studies have shown that the combined administration of NSAIDs with various anti-tumor drugs (such as cisplatin, paclitaxel, or doxorubicin) has certain synergistic effects. However, the combined administration of multiple drugs often leads to complex metabolites, resulting in some toxic side effects. In addition, the efficacy of some combined administrations is not ideal. In recent years, the literature has reported some anti-tumor prodrugs formed by binding NSAIDs at the axial position of tetravalent platinum. Although they can also down-regulate the expression of COX-2 while anti-tumor, these tetravalent platinum complex prodrugs usually have certain defects in aspects such as preparation, drug metabolism, and quality control, making it difficult to be used as anti-tumor drugs. Summary of the Invention

[0006] Objective of the Invention: Aiming at the deficiencies of existing non-steroidal anti-inflammatory drugs, some anti-tumor drugs used alone or in combination, and anti-tumor prodrugs formed by the combination of non-steroidal anti-inflammatory drugs and tetravalent platinum complexes, the present invention aims to provide a class of divalent platinum complexes containing non-steroidal anti-inflammatory drug structural units, their derivatives, preparation methods, pharmaceutical compositions and applications that can, to a certain extent, overcome cisplatin resistance, reduce tumor inflammation, and have high efficiency and low toxicity.

[0007] Technical Solution: As the first aspect of the present invention, the divalent platinum complexes and their derivatives of the present invention have the following structures:

[0008]

[0009] The derivatives are isomers of the divalent platinum complexes or mixtures thereof;

[0010] Wherein:

[0011] Q is a structural fragment without a carboxyl group in the chemical structures of etodolac, ibuprofen, naproxen, diclofenac, indomethacin, fenoprofen, flurbiprofen, ketoprofen, ketorolac, mefenamic acid, oxaprozin, salsalate, sulindac or tolmetin.

[0012] In the present invention, some non-steroidal anti-inflammatory drugs containing carboxylic acid groups are bonded to known divalent platinum complexes. Specifically, non-steroidal anti-inflammatory drugs containing a carboxylic acid group are bonded to anti-tumor platinum compounds containing a ketone carbonyl group through a linking group hydrazine, and a class of single molecular entities that can both inhibit tumor growth and reduce tumor inflammation are synthesized, which is very beneficial for the discovery of new anti-tumor drugs.

[0013] Preferably, in the structures of the divalent platinum complexes and their derivatives:

[0014] Q is a structural fragment without a carboxyl group in the chemical structures of etodolac (Eto), ibuprofen (Ibu), naproxen (Nap) or diclofenac (Dic).

[0015] Preferably, the divalent platinum complexes and their derivatives are any of the following compounds:

[0016]

[0017]

[0018] Preferably, the divalent platinum complexes and their derivatives have the following structures:

[0019]

[0020] Wherein:

[0021] Q is a structural fragment without a carboxyl group in the chemical structures of etodolac, ibuprofen, naproxen or diclofenac.

[0022] As the second aspect of the present invention, the preparation method of the divalent platinum complex and its derivatives of the present invention is as follows:

[0023]

[0024] The divalent platinum complex is obtained by acylating and condensing Compound 1;

[0025] Among them, the definition of Q is as described above, and the structures of DN603 and DN604 are as follows:

[0026]

[0027] As the third aspect of the present invention, the pharmaceutical composition of the present invention contains the divalent platinum complex and / or its derivatives and a pharmaceutically acceptable carrier.

[0028] The divalent platinum complex and its derivatives can be made into common pharmaceutical preparations by adding pharmaceutically acceptable carriers, such as tablets, capsules, syrups, suspensions or injections, and the preparations can be added with common pharmaceutical excipients such as flavors, sweeteners, liquid / solid fillers, diluents, etc.

[0029] As the fourth aspect of the present invention, the divalent platinum complex and its derivatives, and the pharmaceutical composition of the present invention are applied to the preparation of anti-tumor drugs, specifically for treating breast cancer, liver cancer or ovarian cancer, and are particularly suitable for cisplatin-resistant tumors.

[0030] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages:

[0031] (1) This type of divalent platinum complex can cause apoptosis of cancer cells by inducing DNA damage and the production of reactive oxygen species, and can also down-regulate the expression of cyclooxygenase 2 to reduce inflammation and effectively inhibit the growth of cancer cells; the representative compounds have better anti-tumor activity against cisplatin-sensitive and cisplatin-resistant cancer cells than cisplatin, and have less toxicity to normal liver cells than cisplatin;

[0032] (2) This type of divalent platinum complex can not only enhance the toxicity of the original platinum compound to cancer cells, but also improve the sensitivity of the original platinum compound to cisplatin-resistant cells to a certain extent;

[0033] (3) This type of divalent platinum complex with a C=N double bond can target tumors using the acidic microenvironment of tumors and reduce tissue toxicity;

[0034] (4) The preparation method is simple and easy to operate. Description of the Drawings

[0035] Figure 1Hydrolysis results of Eto-DN604 (5 μM, PBS) in an acidic system (pH 6.0) within 24 h;

[0036] Figure 2 Hydrolysis results of Eto-DN604 (5 μM, PBS) in a neutral system (pH 7.4) within 24 h;

[0037] Figure 3 Results of cancer cell apoptosis, where (A) shows the results after treating A2780 cells with cisplatin (5 μM) and Eto-DN604 (5 μM) for 24 h respectively; (B) shows the results after treating A2780 / CDDP cells with cisplatin (5 μM) and Eto-DN604 (5 μM) for 24 h respectively;

[0038] Figure 4 Results of DNA damage in cancer cells, where (A) shows the DNA damage results after treating A2780 and A2780 / CDDP cells with cisplatin (5 μM) and Eto-DN604 (5 μM) for 12 h respectively; (B) shows the DNA quantitative analysis results; (C) shows the expression levels of γ-H2AX after treating A2780 and A2780 / CDDP cells with cisplatin (5 μM) and Eto-DN604 (5 μM) for 12 h respectively;

[0039] Figure 5 Results of ROS production after treating A2780 and A2780 / CDDP cells with cisplatin (5 μM) and Eto-DN604 (5 μM) for 12 h respectively;

[0040] Figure 6 Results of COX-2 expression, where (A) shows the regulatory results of COX-2 expression after treating A2780 cells with cisplatin (5 μM), Eto (5 μM), Eto + DN604 (5 μM), and Eto-DN604 (5 μM) for 24 h respectively; (B) shows the COX-2 expression quantitative analysis results. Detailed implementation manners

[0041] The technical solutions of the present invention will be further described below in conjunction with embodiments.

[0042] Example 1: Preparation of 1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-acetylhydrazide (Compound 2a)

[0043]

[0044] Weigh 574 mg of etodolac (1a) into a flask, add 10 mL of methanol, continue to add 2 drops of concentrated sulfuric acid under stirring at room temperature, and continue stirring for 4 h at room temperature. Monitor the reaction using TLC. After the reaction is completed, concentrate under reduced pressure, extract with dichloromethane and saturated brine for 1 - 2 times, and rotary evaporate the dichloromethane layer. Then dissolve it with 10 mL of methanol, add 1 g of hydrazine hydrate, heat it in an oil bath to 80 °C and reflux. Monitor the reaction by TLC after 2 h. After the reaction is completed, rotary evaporate, extract with dichloromethane and saturated brine for 1 - 2 times, rotary evaporate, and perform column chromatography to obtain 500 mg of white solid with a yield of 83.1%.

[0045] 1 H NMR(600MHz,DMSO-d6)δ10.54(s,1H),8.93(s,1H),7.23(d,J=7.6Hz,1H),6.92–6.87(m,2H),4.27(s,2H),3.97–3.89(m,2H),2.85–2.81(m,2H),2.75(d,J=14.1Hz,1H),2.70–2.66(m,1H),2.64(d,J=14.1Hz,1H),2.62–2.58(m,1H),2.07–2.00(m,2H),1.26(t,J=7.5Hz,3H),0.63(t,J=7.3Hz,3H)ppm.

[0046] Example 2: Preparation of (S)-2-(4-isobutylphenyl)propionyl hydrazide (Compound 2b)

[0047]

[0048] The synthesis method is the same as that in Example 1, white solid, yield 79.5%.

[0049] 1 H NMR(600MHz,DMSO-d6)δ9.13(s,1H),7.22(d,J=8.0Hz,2H),7.06(d,J=8.1Hz,2H),4.17(s,2H),3.48(q,J=7.0Hz,1H),2.39(d,J=7.1Hz,2H),1.81–1.76(m,1H),1.31(d,J=7.1Hz,3H),0.85(d,J=6.6Hz,6H)ppm.

[0050] Example 3: Preparation of (S)-2-(6-methoxynaphthalen-2-yl)propionyl hydrazide (Compound 2c)

[0051]

[0052] The synthesis method is the same as that in Example 1, white solid, yield 79.5%.

[0053] 1 1H NMR (600 MHz, DMSO-d6) δ 9.22 (s, 1H), 7.77 (d, J = 9.0 Hz, 1H), 7.74 (d, J = 8.5 Hz, 1H), 7.72 (s, 1H), 7.45 (dd, J = 8.5, 1.7 Hz, 1H), 7.26 (d, J = 2.4 Hz, 1H), 7.14 (dd, J = 8.9, 2.5 Hz, 1H), 4.21 (s, 2H), 3.85 (s, 3H), 3.66 (q, J = 7.0 Hz, 1H), 1.42 (d, J = 7.1 Hz, 3H) ppm.

[0054] Example 4: Preparation of 2-(2-((2,6-dichlorophenyl)amino)phenyl)acetohydrazide (Compound 2d)

[0055]

[0056] The synthesis method was the same as that in Example 1. It was a white solid with a yield of 79.5%.

[0057] 1 1H NMR (600 MHz, DMSO-d6) δ 9.50 (s, 1H), 8.54 (s, 1H), 7.51 (d, J = 8.1 Hz, 2H), 7.18–7.13 (m, 2H), 7.04 (t, J = 7.7 Hz, 1H), 6.85 (t, J = 7.4 Hz, 1H), 6.30 (d, J = 8.0 Hz, 1H), 4.36 (s, 2H), 3.52 (s, 2H) ppm.

[0058] Example 5: Preparation of Compound Eto-DN604

[0059]

[0060] 150 mg of Compound 2a and 192 mg of DN604 were separately taken in a flask, 50 mL of methanol was added, and the mixture was heated to 60 °C in an oil bath. 2 drops of acetic acid were added dropwise, and the reaction was stirred for 24 h, and then monitored by TLC. After the reaction was completed, the product was filtered and washed with methanol and distilled water respectively to obtain 200 mg of a white solid with a yield of 60.0%.

[0061] 11H NMR (600 MHz, DMSO-d6) δ 10.48 (s, 1H), 10.03 (s, 1H), 7.23 (d, J = 6.6 Hz, 1H), 6.90 (d, J = 6.4 Hz, 1H), 6.88 (s, 1H), 4.21 (s, 6H), 4.00–3.89 (m, 2H), 3.67–3.64 (m, 2H), 3.51–3.43 (m, 2H), 2.98 (d, J = 14.2 Hz, 1H), 2.84–2.75 (m, 3H), 2.68–2.60 (m, 2H), 2.10–2.03 (m, 2H), 1.25 (t, J = 7.0 Hz, 3H), 0.66–0.59 (m, 3H) ppm. 13 13C NMR (150 MHz, DMSO-d6) δ 175.82, 165.24, 154.20, 136.45, 134.45, 126.52, 126.07, 119.67, 118.74, 115.42, 106.90, 75.65, 59.96, 48.95, 43.50, 42.53, 41.20, 30.70, 23.75, 21.89, 14.43, 7.87 ppm. HRMS (m / z) (ESI): calcd for C 23 H 32 N5O6Pt [M+H] + : 669.1995; found: 669.1973.

[0062] Example 6: Preparation of Compound Ibu-DN604

[0063]

[0064] The synthesis method was the same as that in Example 5, white solid, yield 88.7%.

[0065] 1 1H NMR (600 MHz, DMSO-d6) δ 10.42 (s, 1H), 7.25 (d, J = 7.8 Hz, 2H), 7.08 (d, J = 7.9 Hz, 2H), 4.20 (s, 6H), 3.78–3.72 (m, 1H), 3.68–3.58 (m, 2H), 3.54–3.41 (m, 2H), 2.40–2.36 (m, 2H), 1.82–1.77 (m, 1H), 1.34–1.31 (m, 3H), 0.85 (d, J = 6.5 Hz, 6H) ppm. 1313C NMR(150MHz,DMSO-d6)δ175.86,175.84,169.62,154.59,139.32,139.15,128.77,127.01,48.89,44.24,43.42,42.50,41.96,29.60,22.19,22.17,18.58ppm.HRMS(m / z)(ESI):calcd for C 19 H 29 N4O5Pt[M + H] + :588.1780; found:588.1743.

[0066] Example 7: Preparation of Compound Nap-DN604

[0067]

[0068] The synthesis method was the same as that in Example 5. It was a white solid with a yield of 85.9%.

[0069] 1 1H NMR(600MHz,DMSO-d6)δ10.51(s,1H),7.79–7.69(m,3H),7.48(dd,J = 8.4,1.3Hz,1H),7.27(d,J = 2.2Hz,1H),7.14(dd,J = 8.8,2.4Hz,1H),4.19(s,6H),3.96–3.90(m,1H),3.86(s,3H),3.69–3.60(m,2H),3.56–3.44(m,2H),1.43(d,J = 7.0Hz,3H)ppm. 13 13C NMR(150MHz,DMSO-d6)δ175.85,175.81,169.56,157.01,154.75,137.05,133.16,129.10,128.34,126.62,126.45,125.32,118.59,105.69,55.13,48.90,43.39,42.85,42.01,18.64ppm.HRMS(m / z)(ESI):calcd for C 20 H 25 N4O6Pt[M + H] + :612.1416; found:612.1420.

[0070] Example 8: Preparation of Compound Dic-DN604

[0071]

[0072] The synthesis method was the same as that in Example 5, white solid, yield 82.2%.

[0073] 1 1H NMR (600 MHz, DMSO-d6) δ 10.76 (s, 1H), 8.28 (s, 1H), 7.52 (d, J = 8.1 Hz, 2H), 7.27–7.15 (m, 2H), 7.06–7.04 (m, 1H), 6.87–6.83 (m, 1H), 6.31–6.28 (m, 1H), 4.22 (s, 6H), 3.96 (s, 1H), 3.70–3.66 (m, 3H), 3.58–3.49 (m, 2H) ppm. 13 13C NMR (150 MHz, DMSO-d6) δ 175.79, 167.66, 155.74, 142.98, 137.09, 130.48, 129.48, 129.20, 127.33, 125.13, 125.05, 120.71, 115.93, 48.88, 43.36, 41.98, 37.51 ppm. HRMS (m / z) (ESI): calcd for C 20 H 22 Cl2N5O5Pt [M+H] + : 677.0640; found: 678.0598.

[0074] Example 9: Preparation of Compound Eto-DN603

[0075]

[0076] 129 mg of compound 2a and 200 mg of DN603 were separately taken in a flask, 50 mL of methanol was added, heated to 60 °C in an oil bath, 2 drops of acetic acid were added dropwise, and after stirring for 24 h, the reaction was monitored by TLC. After the reaction was completed, it was concentrated, dissolved in dichloromethane, and ether was continuously added dropwise, filtered, and the insoluble matter was rinsed with distilled water to obtain 270 mg of a grayish-white solid, yield 84.1%.

[0077] 11H NMR (600 MHz, DMSO-d6) δ 10.53–10.42 (s, 1H), 10.08 (s, 1H), 7.23 (d, J = 7.2 Hz, 1H), 6.92–6.87 (m, 2H), 6.02–5.91 (m, 2H), 5.32–5.20 (m, 2H), 3.99–3.90 (m, 3H), 3.74–3.71 (m, 1H), 3.50–3.40 (m, 2H), 3.02–2.98 (m, 1H), 2.85–2.79 (m, 3H), 2.68–2.60 (m, 2H), 2.05–1.97 (m, 4H), 1.88–1.81 (m, 2H), 1.56–1.46 (m, 2H), 1.32–1.24 (m, 5H), 1.10–1.01 (m, 2H), 0.66–0.58 (m, 3H) ppm. 13 13C NMR (150 MHz, DMSO-d6) δ 175.70, 165.24, 150.40, 136.45, 134.47, 126.55, 126.07, 119.65, 118.73, 115.40, 106.88, 75.67, 62.12, 59.96, 55.89, 48.85, 46.39, 43.72, 42.54, 40.88, 31.52, 30.73, 24.05, 23.75, 21.91, 14.44, 7.87 ppm. HRMS (m / z) (ESI): calcd for C 29 H 40 N5O6Pt [M+H] + : 749.2621; found: 749.2609.

[0078] Example 10: Preparation of Compound Ibu-DN603

[0079]

[0080] The synthesis method was the same as that in Example 9, and a white solid was obtained with a yield of 81.9%.

[0081] 11H NMR (600 MHz, DMSO-d6) δ 10.44–10.30 (m, 1H), 7.26–7.23 (m, 1H), 7.20–7.16 (m, 1H), 7.09–7.05 (m, 2H), 6.06–5.95 (m, 2H), 5.32–5.20 (m, 2H), 3.87–3.62 (m, 3H), 3.51–3.37 (m, 2H), 3.17–3.05 (m, 1H), 2.40–2.34 (m, 2H), 2.10–2.05 (m, 2H), 1.83–1.77 (m, 2H), 1.45–1.40 (m, 2H), 1.38–1.31 (m, 3H), 1.21 (s, 2H), 1.01 (s, 2H), 0.85 (d, J = 6.5 Hz, 6H) ppm. 13 13C NMR (150 MHz, DMSO-d6) δ 175.73, 175.69, 169.64, 154.81, 139.32, 139.14, 128.76, 127.03, 62.10, 55.88, 48.79, 44.25, 42.51, 41.60, 31.47, 29.61, 24.04, 22.17, 18.56 ppm. HRMS (m / z) (ESI): calcd for C 25 H 37 N4O5Pt [M+H]+ + : 668.2406; found: 668.2406.

[0082] Example 11: Preparation of Compound Nap-DN603

[0083]

[0084] The synthesis method was the same as that in Example 9. It was a white solid with a yield of 79.1%.

[0085] 11H NMR(600MHz,DMSO-d6)δ10.57–10.39(m,1H),7.80–7.69(m,3H),7.49–7.43(m,1H),7.26(s,1H),7.13(d,J=8.8Hz,1H),6.08–6.01(m,2H),5.32–5.26(m,2H),4.05–3.98(m,1H),3.85(s,3H),3.65–3.56(m,2H),3.45–3.40(m,1H),3.21–3.12(m,1H),2.05(s,2H),1.87–1.81(m,2H),1.50–1.41(m,5H),1.22(s,2H),1.00(s,2H)ppm. 13 13C NMR(150MHz,DMSO-d6)δ175.91,175.76,169.68,157.04,154.95,137.79,133.16,129.11,128.41,126.74,126.53,125.27,118.61,105.69,62.14,59.97,55.17,51.61,48.83,47.69,43.51,42.86,31.54,24.07,20.06,18.64ppm.HRMS(m / z)(ESI):calcd for C 26 H 33 N4O6Pt[M+H] + :692.2042;found:692.2192.

[0086] Example 12: Preparation of Compound Dic-DN603

[0087]

[0088] The synthesis method was the same as that in Example 9, white solid, yield 80.0%.

[0089] 11H NMR(600MHz,DMSO-d6)δ10.82–10.70(m,1H),8.28(s,1H),7.52–7.50(m,2H),7.26–7.19(m,2H),7.06–7.02(m,1H),6.87–6.83(m,1H),6.31–6.28(m,1H),6.03–5.87(m,2H),5.31–5.22(m,2H),3.97–3.85(m,1H),3.77–3.64(m,3H),3.55–3.48(m,2H),2.07(s,2H),1.86–1.81(m,2H),1.46(s,2H),1.23(s,2H),1.01(s,2H)ppm. 13 13C NMR(150MHz,DMSO-d6)δ175.68,175.62,167.69,155.86,142.97,137.10,130.97,130.48,129.45,129.19,127.30,125.10,120.72,115.94,64.89,62.09,48.74,41.63,37.49,31.52,24.03,15.15ppm.HRMS(m / z)(ESI):calcd for C 26 H 30 Cl2N5O5Pt[M+H] + :757.1266;found:758.1234.

[0090] Example 13: Cytotoxic Activity of Platinum Complex

[0091] Experimental Method: The MTT method was used to evaluate the in vitro cytotoxicity of the synthesized target compounds. Cells in the logarithmic phase with good condition were digested with trypsin and then seeded into 96-well plates, about 1×10 4Cells were added to the culture medium and placed in an incubator overnight at a constant temperature. After the cells adhered to the wall, a drug administration test was carried out. Solutions of platinum complexes with different concentrations (solvent: DMSO) and cisplatin (solvent: H2O) were prepared and added to 96-well plates respectively. Three replicates were set for each concentration. In the blank group, an equal volume of the prepared solution was added. After adding, it was placed in the incubator and incubated for another 72 h. After the incubation ended, 10 μL of MTT solution with a concentration of 5 mg / mL was added respectively, and then incubated in the incubator for another 4 h. Then, the supernatant was removed, and 150 μL of DMSO was added and shaken well to dissolve the formazan completely. Finally, the absorbance value (OD value) of each well was measured at 490 nm using a microplate reader, and the cell growth inhibition rate was calculated. Combining with SPSS (Statistical Product and Service Solutions) software, the IC 50 value was calculated. The test results were the average values of three experiments. The results are shown in Table 1.

[0092] Table 1 Cytotoxic activities of platinum complexes

[0093]

[0094] *. RF (resistance factor) = IC 50 (A2780 / CDDP) / IC 50 (A2780).

[0095] The MTT method was used to analyze the cytotoxicity of the synthesized platinum complexes against human breast cancer (MCF-7), human liver cancer (HepG2), human ovarian cancer (A2780), cisplatin-resistant human ovarian cancer (A2780 / CDDP) cells and human normal liver cells LO2. Cisplatin, DN604 and DN603 were used as positive controls. As shown in Table 1, among all the synthesized platinum complexes, Eto-DN604 showed the strongest cytotoxicity against the tested cancer cell lines, and the IC 50 value ranged from 1.10 to 3.82 μM. In addition, Eto-DN604 showed the highest anti-proliferative activity against A2780, and the IC 50 value was significantly better than that of DN604, etodolac and the mixture of DN604 (molar ratio 1:1). More importantly, compared with cisplatin and DN604, Eto-DN604 still retained strong cytotoxicity against cisplatin-resistant A2780 / CDDP cells, and the IC 50The value is 3.82 μM, approximately 16 times and 2.7 times that of its parent compound DN604 (62.19 μM) and cisplatin (9.68 μM), respectively. In addition, the toxicity of Eto-DN604 to human normal liver cells LO2 is lower than that of cisplatin, indicating that Eto-DN604 has the characteristics of high efficiency and low toxicity. Among this series of compounds, for A2780 and A2780 / CDDP cell lines, the resistance factors of some compounds are less than 2.0, showing a certain degree of ability to overcome cisplatin resistance. The above results indicate that the single molecular entity formed by combining the non-steroidal anti-inflammatory drug structural unit into the platinum complex can not only enhance the toxicity of the original platinum compound to cancer cells, but also increase the sensitivity of the original platinum compound to cisplatin-resistant cancer cells to a certain extent.

[0096] Example 14: Platinum uptake and DNA platination levels of Eto-DN604 in cancer cells

[0097] Experimental method: Transfer the well-grown A2780 and A2780 / CDDP cells into 6-well plates respectively, and then place them in an incubator at 37 °C with 5% carbon dioxide until the cells adhere and grow to about 80% confluence. Subsequently, change the medium to a new one, add the prepared test compound solution, and continue to incubate in the incubator for 4 h. Then, collect the cells and the medium together and transfer them into a centrifuge tube. After centrifugation to remove the supernatant, collect the cells, wash the cells three times with ice-cold PBS, resuspend the cells with PBS, centrifuge to remove PBS after counting, add 200 μL of 65% nitric acid, digest at 65 °C for 4 h, and finally detect the platinum content in the cells by ICP-MS. To detect the amount of DNA platination, use the DNA Extraction Kit to extract DNA. PCR is used to determine the DNA content, and ICP-MS is used to analyze the platinum content in DNA. Each experiment is carried out three times, and the results are the average of the three times. The results are shown in Table 2.

[0098] Table 2 Platinum uptake and DNA platination amounts of Eto-DN604 in cisplatin-sensitive and -resistant ovarian cancer cells

[0099]

[0100] The platinum uptake in A2780 and A2780 / CDDP cells and the platinum content in DNA were determined by inductively coupled plasma mass spectrometry (ICP-MS). As shown in Table 2, the platinum uptake in A2780 cells after Eto-DN604 treatment was significantly increased, and the platinum level in DNA in the cells was also significantly higher than that in the cisplatin control group. Due to cisplatin resistance, the platinum content and the level of DNA platination in cisplatin-treated A2780 / CDDP cells were decreased to a certain extent compared with A2780 cells. However, A2780 / CDDP cells treated with Eto-DN604 still had a high level of platinum accumulation and DNA platination, which was consistent with the results of cytotoxic activity.

[0101] Example 15: Stability of Eto-DN604 in solution

[0102] Experimental method: Weighed a certain amount of the compound and dissolved it in a little DMSO, then added PBS solution with pH 6.0 to dilute to a concentration of 5.0 μM. After filtering through an organic filter head, HPLC injection detection was carried out at 0, 4, 12, and 24 h respectively. The mobile phase was acetonitrile and water containing 0.3% trifluoroacetic acid (acetonitrile: water = 60:40), the flow rate was 1.0 mL / min, and the detection wavelength was 254 nm. The results are shown in Figure 1 .

[0103] Due to the acidic microenvironment characteristics of tumor tissues, we selected the platinum complex Eto-DN604 with higher in vitro antitumor activity to investigate its hydrolysis behavior in an acidic environment (pH 6.0). As Figure 1 shown, Eto-DN604 was gradually hydrolyzed in the acidic environment to release DN604 with the increase of time. However, Eto-DN604 did not change within 24 h in the neutral environment ( Figure 2 ). These results indicate that this type of platinum complex with C=N double bond can target tumors using the acidic microenvironment of tumors and reduce tissue toxicity.

[0104] Example 16: Induction of apoptosis by Eto-DN604

[0105] Experimental method: Transfer A2780 and A2780 / CDDP cells in the logarithmic phase to a 6-well plate and culture them in a constant-temperature cell incubator. When the cells adhere and grow to about 80%, add the prepared test compound with a final concentration of 5 μM, and then place it in the constant-temperature cell incubator for continued incubation for 24 h. After the incubation, collect all the culture medium and cells (including adherent cells digested with trypsin) in a centrifuge tube, centrifuge (2000 rpm) to remove the supernatant, and collect the precipitated cells. Wash the cells once with ice-cold PBS, then add 500 μL of the pre-prepared Binding Buffer solution to suspend the cells. After sufficient suspension, add 5 μL of Annexin V, shake well and incubate in the dark for 5 min, then immediately add 5 μL of PI and continue to incubate for 15 min. Finally, perform apoptosis detection of the cells by flow cytometry (the excitation wavelength and emission wavelength are Ex = 488 nm and Em = 530 nm, respectively). The results are shown in Figure 3 。

[0106] The apoptosis-inducing ability of Eto-DN604-treated A2780 and A2780 / CDDP cells was evaluated by Annexin V-FITC / PI staining. As Figure 3 shown, cisplatin effectively induced apoptosis in A2780 cells with an apoptosis rate of 33.73%. Compared with cisplatin, Eto-DN604 showed a more significant apoptosis rate (41.6%) in A2780 cells. In addition, in A2780 / CDDP cells, the apoptosis induced by Eto-DN604 (39.59%) was also significantly higher than that of cisplatin (27.5%). These results indicate that compared with cisplatin, Eto-DN604 can more effectively induce apoptosis in cancer cells and increase the sensitivity of cancer cells to platinum compounds.

[0107] Example 17: Comet assay

[0108] Experimental method: After co-incubating A2780 and A2780 / CDDP cells with the test compound for 12 h, wash and collect the cells. Drop normal melting point agarose on a frosted glass slide and solidify it at 4 °C for 10 min. Take 10 μL of the cell suspension (containing about 10 4 cells) and mix it with 75 μL of 0.7% low melting point agarose, and immediately transfer it to the first layer of gel. Place the slide at 4 °C and solidify it for 10 min. Then soak it in cold lysis buffer and lyse it at 4 °C for 1 h. Wash the slide with PBS and soak it in alkaline electrophoresis buffer for 30 min, then perform electrophoresis at 25 V for 30 min. After electrophoresis, neutralize it 3 times with 0.4 mM Tris-HCl (pH 7.5), and then stain it with PI in the dark for 10 min, and image it with a confocal microscope. The results are shown in Figure 4 A.

[0109] We evaluated the ability of Eto-DN604 to induce DNA damage using the comet assay. As Figure 4 shown in A, obvious fluorescence tailing was found in A2780 and A2780 / CDDP cells treated with Eto-DN604 or cisplatin, respectively. Quantitative analysis showed ( Figure 4 B) that the tail DNA of A2780 and A2780 / CDDP cells after treatment with Eto-DN604 was significantly higher than that of the cisplatin treatment group, indicating that Eto-DN604 could effectively induce DNA damage and had better anti-tumor effects.

[0110] Example 18: Expression level of γ-H2AX in cancer cells

[0111] Experimental method: Cells were cultured and collected in the same way as in Example 17 experiment. The cells were resuspended in RIPA lysis buffer, lysed on ice for 1 h, and then centrifuged at 13,000 r / min for 30 min in a 4 °C centrifuge. The OD value of the protein was measured using a BCA kit. Then, the proteins separated by 10% SDS polyacrylamide gel electrophoresis were transferred to a nitrocellulose filter membrane. The membrane was soaked in 5% skim milk at 35 °C for 1 h and then soaked in the corresponding primary antibody overnight. Subsequently, the membrane was incubated with an HRP-conjugated antibody for 1 h and detected by a Tanon automatic chemiluminescence imaging analysis system. The results are shown in Figure 4 C.

[0112] γ-H2AX, as a member of the histone H2A family, is considered a typical marker of DNA double-strand breaks. We evaluated the expression of γ-H2AX by Western blot (WB) experiment to detect whether Eto-DN604 induced a higher level of DNA damage than cisplatin. As Figure 4 shown in C, after treatment with Eto-DN604 or cisplatin, an increase in the protein expression level of γ-H2AX was found in A2780 and A2780 / CDDP cells. Compared with the cisplatin group, the Eto-DN604 group showed a higher expression level of γ-H2AX, indicating that Eto-DN604 had a stronger ability to induce DNA damage than cisplatin.

[0113] Example 19: ROS production in cancer cells

[0114] Experimental method: A2780 and A2780 / CDDP cells were cultured in a 6-well plate overnight, and then the prepared test compound solution was added and the cells were incubated in an incubator. After 24 h, the supernatant was removed and the cells were washed 3 times with PBS. Then, the ROS probe DCFH-DA was added and incubated at room temperature for 30 min. Confocal microscopy imaging was used. The results are shown in Figure 5 .

[0115] ROS-mediated reactions are associated with various pathogenic DNA damage processes. We investigated the generation of intracellular ROS induced by Eto-DN604. As Figure 5 shown, significant ROS generation was observed in both A2780 and A2780 / CDDP cells after treatment with Eto-DN604 or cisplatin, and the ROS levels in A2780 and A2780 / CDDP cells treated with Eto-DN604 were higher than those treated with cisplatin. These results indicate that Eto-DN604 can induce the generation of ROS, causing a higher level of DNA damage than cisplatin.

[0116] Example 20: Expression of COX-2 in cancer cells

[0117] Experimental method: Seed A2780 cells in a 6-well plate. When the cells are completely adherent, remove the supernatant, add fresh DMEM medium, and then add 5 μM of the complex Eto-DN604 to be tested and the positive control, and incubate for 24 h. Collect the cells, centrifuge, wash once with 1 mL of PBS, centrifuge again, pour out the supernatant completely, and add an appropriate amount of lysis buffer (PAPI lysis buffer:PMSF = 100:1), lyse on ice for 1 h, then centrifuge at 13300 rpm at 4 °C for 30 min, collect the supernatant, take 2 μL from it for protein content determination, and add the remaining supernatant to the protein loading buffer, heat in a 100 °C water bath for 10 min, and then store in a -20 °C refrigerator for later use. Add 18 μL of pure water, 200 μL of the prepared BCA reagent, and 2 μL of the protein sample to a 96-well plate, incubate at 37 °C for 0.5 h, measure the OD value at 570 nm, and calculate the loading amount according to the OD value. Next, perform a western blot experiment. First, perform SDS-PAGE electrophoresis to separate proteins with different molecular weights extracted. Then perform membrane transfer. Transfer the bands on the gel to the NC membrane through the formation of a membrane transfer structure of "positive electrode - three layers of filter paper - membrane - gel - three layers of filter paper - negative electrode". Under the action of current, proteins move out of the polyacrylamide gel and adsorb on the membrane surface. Incubate with 5% milk at 37 °C for 1 h to remove non-specific bands. Place the membrane in the prepared antibody (primary antibody) of the target protein and incubate overnight. The next day, wash away the unbound antibody with PBST so that only the primary antibody bound to the target protein remains on the membrane. Then perform enzyme immuno-localization with the labeled secondary antibody. Develop and analyze with ECL developer. Finally, analyze the results. In the experiment, cisplatin, etodolac, and a mixture of etodolac and DN604 (1:1) were used as positive controls. The results are shown in Figure 6 。

[0118] COX-2 is a biochemical target of non-steroidal anti-inflammatory drugs and is overexpressed in human ovarian cancer. We measured the effect of Eto-DN604 on the expression of COX-2 in human ovarian cancer cells. As Figure 6As shown, in A2780 cells, Eto-DN604 significantly downregulated the expression of COX-2 in cells, while cisplatin or etodolac had no significant effect on the expression of COX-2. When etodolac was combined with DN604, the expression of COX-2 in A2780 cells was slightly downregulated. The results indicate that Eto-DN604 can effectively downregulate the expression of COX-2 in cancer cells and reduce tumor inflammation.

Claims

1. A divalent platinum complex, characterized in that, It has the following structure: The divalent platinum complex is any one of the following compounds:

2. A method for preparing a divalent platinum complex as described in claim 1, characterized in that, The preparation method is as follows: The divalent platinum complex is obtained by acylating and condensing compound 1; Among them, the structures of DN603 and DN604 are as follows:

3. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the divalent platinum complex as described in claim 1 and a pharmaceutically acceptable carrier.

4. The pharmaceutical composition according to claim 3, wherein Its dosage form is selected from tablets, capsules, syrups, suspensions or injections.

5. Use of a divalent platinum complex as described in claim 1 in the preparation of a drug for treating breast cancer, liver cancer or ovarian cancer.

6. Use of a divalent platinum complex as described in claim 1 in the preparation of a drug for treating cisplatin-resistant tumors, wherein the cisplatin-resistant tumor is ovarian cancer.

7. Use of a pharmaceutical composition as described in claim 3 in the preparation of a drug for treating breast cancer, liver cancer or ovarian cancer.

8. Use of a pharmaceutical composition as described in claim 3 in the preparation of a drug for treating cisplatin-resistant tumors, wherein the cisplatin-resistant tumor is ovarian cancer.

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