Preparation method of theophylline-platinum (IV) complex and application of theophylline-platinum (IV) complex in tumor drugs

Abstract

The invention discloses a preparation method of a theophylline-platinum (IV) complex and application of the theophylline-platinum (IV) complex in tumor drugs, one molecule of theophylline is introduced to a single side in the axial direction of a platinum (IV) coordination center or two molecules of theophylline are introduced to two sides of the platinum (IV) coordination center to form a monosubstituted tetravalent platinum complex or a symmetrical disubstituted tetravalent platinum complex; or, one molecule of theophylline is introduced to one axial side of the platinum (IV) coordination center, and one molecule of carbon long-chain group is introduced to the other axial side of the platinum (IV) coordination center, so that the asymmetric disubstituted tetravalent platinum complex is formed, the prepared compound has stronger killing ability on tumor cells, and the anti-proliferative activity in various cell lines is obviously improved. The theophylline-platinum (IV) complex disclosed by the invention is simple in synthesis process and low in cost, greatly improves the curative effect of drug combination, and has the advantages of good curative effect, small side effect and the like compared with traditional bivalent platinum and similar tetravalent platinum drugs.

Description

Technical Field

[0001] This invention belongs to the field of anticancer chemical drug technology, and in particular relates to a method for preparing a theophylline-platinum(IV) complex and its application in tumor drugs. Background Technology

[0002] Cancer, as a significant global public health challenge, seriously threatens human life. Ovarian cancer encompasses various types of malignant tumors. The WHO classifies ovarian tumors into four main categories based on their histology: epithelial tumors, germ cell tumors, metastatic tumors, and sex cord-stromal tumors. Studies have shown that ovarian cancer has a hereditary and familial aggregation tendency, with approximately 20 known genetic susceptibility genes associated with it. Among these, the breast cancer susceptibility gene (BRCA) has the most significant impact. BRCA1 and BRCA2, as tumor suppressor genes, encode proteins that play a crucial role in DNA double-strand repair to maintain genomic stability. When BRCA1 and BRCA2 genes mutate, the synthesis of tumor suppressor proteins is inhibited, and the DNA repair pathway of homologous recombination is blocked, altering intracellular genetic material and thus inducing cancer. Statistics show that individuals carrying BRCA1 and BRCA2 gene mutations have a 54% and 23% risk of developing ovarian cancer, respectively. Therefore, developing new drug candidates is urgently needed to improve the survival time and quality of life for ovarian cancer patients.

[0003] Chemotherapy is an effective anti-tumor treatment. Before the 1960s, all drugs used to treat cancer were purely organic compounds. However, in the late 1960s, a simple coordination compound with anti-cancer properties—cisplatin—was unexpectedly discovered. Cisplatin was initially discovered as a substance that inhibits bacterial growth; later, researchers found that it had a powerful killing effect on cancer cells, opening up new avenues for cancer treatment. Currently, platinum-based drugs occupy an important position in clinical cancer treatment, covering a variety of cancers including head and neck cancer, ovarian cancer, testicular cancer, neuroblastoma, cervical cancer, and non-small cell lung cancer. Of particular note is the successful application of cisplatin in germ cell tumors, specifically its introduction into combination therapy for testicular cancer, which increased the chemotherapy cure rate for testicular cancer from 5% to 80%. The structures of currently approved platinum-based anticancer drugs are as follows: Figure 1 As shown, these include first-generation drugs such as cisplatin, second-generation drugs such as carboplatin and nedaplatin, and third-generation drugs such as heptaplatin, oxaliplatin, and lobaplatin, which are widely used in clinical practice worldwide.

[0004] As an effective antitumor drug, cisplatin's antitumor mechanism of action involves passive diffusion across the plasma membrane and active transport mediated by membrane proteins into tumor cells. Before binding to DNA, cisplatin undergoes a chlorine ligand substitution activation step, where the chlorine atom in its structure is replaced by a water molecule, generating a monohydrate or dihydrate complex, namely cis-[Pt(NH3)2Cl(OH2)]. + cis-[Pt(NH3)2(OH2)2] 2+ Platinum hydrate cations can enter the cell nucleus and covalently bind to the N7 position of guanine and adenine in DNA, forming Pt-DNA chelates. This process disrupts the normal double helix structure of DNA, affects the normal function of DNA in tumor cells, and induces apoptosis in cancer cells, thereby achieving an anti-tumor effect.

[0005] Poly(ADP-ribose) polymerase (PARP) is an important signal transduction enzyme that uses nicotinamide adenine dinucleotide+ (NAD+) as a donor to transfer ADP-ribose units to the substrate, forming a covalent poly(ADP-ribosylation) modification. The PARP family currently contains 17 members, with PARP-1 being one of the most characteristic, responsible for over 90% of the functions of the PARP family. PARP-1 plays a crucial role in DNA damage repair. Specifically, in the DNA damage response, PARP-1 acts as a "detector." When cells suffer DNA damage, PARP-1 is rapidly activated and recruited to the damage site, subsequently forming a PAR chain through its own modification. It then recruits and modifies other response factors, activating downstream pathways. PARP-1 repairs DNA single-stranded breaks (SSBs) through the base excision repair pathway. Simultaneously, PARP-1 is also a major component of many transcription factors involved in tumor development and inflammation-related processes.

[0006] Theophylline (1,3-dimethylxanthine) is an alkaloid extracted from tea and coffee, widely used in the treatment of respiratory diseases, particularly asthma and chronic obstructive pulmonary disease (COPD). One of the mechanisms of action of theophylline is through antagonism of adenosine receptors. As a non-specific adenosine antagonist, its antagonistic effects on A1, A2, and A3 receptors are almost identical. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a method for preparing theophylline-platinum (IV) complexes and their application in tumor drugs.

[0008] The technical solution adopted in this invention is: a theophylline-platinum (IV) complex, wherein theophylline is connected to both sides of the tetravalent platinum coordination center along the axis; or, theophylline is connected to one side of the tetravalent platinum coordination center along the axis, and a long carbon chain group is connected to the other side.

[0009] Preferably, the structural formula is as shown in Formula 1-1, Formula 1-2 or Formula 1-3;

[0010]

[0011]

[0012] in, Selected from cisplatin, oxaliplatin, carboplatin, heptaplatin, nedaplatin, lorplatin, or miplatin.

[0013] Preferably Cisplatin, oxaliplatin, or carboplatin;

[0014] R1 is -CnH2n-, where n is an integer and 1≤n≤6, preferably n=1 or 3;

[0015] R2 is -CmH2m+1, where m is an integer and 1≤m≤20, preferably m≤16, and more preferably 2≤m≤16;

[0016] Preferably, R1 and / or R2 are straight-chain groups.

[0017] Preferably, it is represented by any of the structural formulas in Equation 2-11:

[0018]

[0019]

[0020] A method for preparing theophylline-platinum(IV) complexes involves esterifying compounds of formula 12 and formula 13 in the presence of a first condensing agent and a first acid-binding agent to obtain compounds of formula 1-1 or formula 1-2.

[0021]

[0022] in, Selected from cisplatin, oxaliplatin, carboplatin, heptaplatin, nedaplatin, lorplatin, or miplatin, preferably Cisplatin, oxaliplatin, or carboplatin;

[0023] R1 is -CnH2n-, where n is an integer and 1≤n≤6. Preferably, -C n H2n - is a straight-chain group;

[0024] Preferably, the first solvent is one or a mixture of acetonitrile, dichloromethane, acetone, dimethyl sulfoxide (DMSO), and N,N-dimethylformamide (DMF); preferably, DMSO is used as the first solvent for preparing compound 1-1, and DMF is used as the first solvent for preparing compound 1-2.

[0025] The first condensing agent is 1-hydroxybenzotriazole (HOBT) or O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU), preferably O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU).

[0026] The first acid-binding agent is triethylamine (TEA);

[0027] The reaction temperature is 25℃-100℃, preferably 25℃;

[0028] When preparing compound 1-1, the feed ratio of compound 12, compound 13, first condensing agent and first acid-binding agent is 1:1-1.5:1-1.5:1-1.5, preferably 1:1:1.2:1.2;

[0029] When preparing compounds of formula 1-2, the feeding ratio of compound 12, compound 13, first condensing agent and first acid-binding agent is 1:2-3:2-3:2-3, preferably 1:2:2:2.

[0030] Preferably, in a second solvent, the compound of formula 1-1 is reacted with the compound of formula 14 to obtain the compound of formula 1-3;

[0031]

[0032] Where R2 is -C m H 2m+1 And m≥1.

[0033] Preferably, the second solvent is one or a mixture of acetonitrile, dichloromethane, acetone, dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF), with DMF being the most preferred;

[0034] The reaction temperature is 25℃-100℃, preferably 60℃;

[0035] The feed ratio of compound 1-1 to compound 14 is 1:2-10, preferably 1:5.

[0036] Preferably, compound 15 and compound 16 are dissolved in a third solvent, reacted under the conditions of a second acid-binding agent, and then subjected to ester hydrolysis to obtain compound 13.

[0037]

[0038] Preferably, the third solvent is one or a mixture of acetonitrile, dichloromethane, acetone, dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF), preferably DMF;

[0039] The second acid-binding agent is K2CO3;

[0040] The feeding ratio of compound 15, compound 16 and the first acid-binding agent is 1:1-3:1-3, preferably 1:1.6:1.3;

[0041] The reaction temperature is 25℃-100℃, preferably 85℃;

[0042] The ester hydrolysis reaction conditions are lithium hydroxide monohydrate;

[0043] The reaction temperature is 25℃.

[0044] Application of theophylline-platinum(IV) complex in the preparation of antitumor drugs.

[0045] The advantages and positive effects of this invention are: by combining theophylline with platinum drugs, a new theophylline-platinum(IV) complex is synthesized. Theophylline has potential anti-tumor effects, and its mechanism of action involves multi-pathway synergistic killing of tumor cells, greatly improving the anti-tumor efficacy and anti-proliferative IC50 of platinum drugs. 50 It has a value hundreds of times better than the parent divalent platinum and shows a broad-spectrum killing effect on a variety of cancer cell lines, especially a significant anti-proliferative effect on ovarian cancer, BRCA1 knockdown ovarian cancer, lung cancer, BRCA1 knockdown lung cancer and breast cancer cells.

[0046] The introduction of carbon fatty chains increases drug stability and enhances the lipid solubility and transmembrane capacity of Pt(IV) molecules, promoting the absorption of platinum drugs. The theophylline-platinum(IV) complex with theophylline and carbon fatty chains double substitution significantly improves drug stability and cellular uptake, enhancing antitumor activity while also having the effects of drug detoxification and sustained release.

[0047] Theophylline, as a traditional respiratory disease drug, reduces the severe toxic side effects of traditional platinum drugs. In addition, the prodrug synthesis process is simple and low-cost, which greatly improves the efficacy of combination therapy. Compared with traditional divalent platinum drugs, it has advantages such as better efficacy and fewer side effects, providing a new approach for the modification of tetravalent platinum. Attached Figure Description

[0048] Figure 1 Intracellular release of compound e in BRCA1-knockdown ovarian cancer cells after 4 hours of treatment;

[0049] Figure 2 Statistics on Pt content of compound e in BRCA1 knockdown ovarian cancer cells;

[0050] Figure 3 The effect of 10 μM compound e on DNA damage in BRCA1 knockdown ovarian cancer cells;

[0051] Figure 4 The effect of 1 μM compound e on BRCA1 knockdown ovarian cancer cells to induce ROS.

[0052] Figure 5 The effect of 2 μM compound e on BRCA1 knockdown ovarian cancer cells to induce MMP;

[0053] Figure 6 The effect of 2 μM compound e on BRCA1 knockdown ovarian cancer cells to induce apoptosis;

[0054] Figure 7 The effect of 2 μM compound e on BRCA1 knockdown ovarian cancer cells to induce cell cycle arrest.

[0055] Figure 8 The effect of 0.5 μM compound e on the inhibition of migration in BRCA1-knockdown ovarian cancer cells;

[0056] Figure 9 The effect of 0.5 μM compound e on the inhibition of invasion in BRCA1-knockdown ovarian cancer cells;

[0057] Figure 10 The effect of 1 μM compound e on the expression of regulatory proteins in BRCA1-knockdown ovarian cancer cells;

[0058] Figure 11 The antitumor activity of compound e in a BRCA1 knockdown ovarian cancer xenograft model. Detailed Implementation

[0059] The embodiments of the present invention will now be described with reference to the accompanying drawings.

[0060] This invention relates to a theophylline-platinum(IV) complex, specifically, a theophylline molecule can be attached to one side of the platinum(IV) coordination center to form a monosubstituted tetravalent platinum complex, as shown in Formula 1-1; or a theophylline molecule can be attached to each side of the platinum(IV) coordination center to form a structure as shown in Formula 1-2; or a theophylline molecule can be attached to one side of the platinum(IV) coordination center to form a disubstituted tetravalent platinum complex, as shown in Formula 1-3.

[0061]

[0062] in, Selected from cisplatin, oxaliplatin, carboplatin, heptaplatin, nedaplatin, lorplatin, or miplatin, preferably For cisplatin, oxaliplatin, or carboplatin; R1 is -C n H 2n - where n is an integer and n≥1, preferably n≤6, more preferably n≤2, -C n H 2n - The effect is better when it is a straight-chain group.

[0063] R1 and R2 are any combination of the same or different atoms, alkyl groups, alkenes, alkynes, aryl groups, heterocycles, or several of the above groups; R1 is -C n H 2n -, n is an integer and 1≤n≤6. In some embodiments of the present invention, n can take the value of 1, 2, 3, 4, 5 or 6. Preferably, n is 1 or 3; R2 is -C m H 2m+1 And 1≤m≤20, preferably m≤16, more preferably 2≤m≤16; in some embodiments of the present invention, m can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16; wherein, R1 and / or R2 are preferably straight-chain groups.

[0064] In some embodiments of the present invention, theophylline is attached to one side of the tetravalent platinum coordination center and a carbon aliphatic chain is attached to the other side, forming compounds as shown in Formulas 1-3; the introduction of the carbon aliphatic chain increases the stability of the drug and has the effects of drug detoxification and sustained release.

[0065] Theophylline platinum (IV) complexes can specifically be represented by any of the structures shown in Formula 2-11:

[0066]

[0067]

[0068] Theophylline and related xanthines possess potential anticancer effects, capable of regulating apoptosis, senescence, and proliferation in various cancer cells. When theophylline is used in combination with cisplatin, it can induce apoptosis in various tumor cells. Theophylline can also effectively inhibit PARP-1 expression; treatment with 100 μM theophylline reduces PARP-1 activity by 43%. Therefore, theophylline, a widely available and low-biotoxic natural drug, holds great potential as a small-molecule scaffold in the synthesis and development of safe and effective antitumor drugs.

[0069] In preparing the above-mentioned theophylline-platinum(IV) complexes, a carboxyl group is introduced into the secondary amine position of theophylline through substitution and ester hydrolysis. The modified theophylline is then introduced into tetravalent platinum through a simple ester condensation reaction to obtain a platinum(IV) complex with one or two theophylline-substituted molecules. When it is necessary to prepare a platinum(IV) complex containing both theophylline and a fatty acid chain, a fatty acid chain is introduced into the opposite side of the monosubstituted theophylline platinum(IV) complex through an addition reaction or ester condensation reaction to obtain a platinum(IV) complex with both theophylline and a fatty acid chain substituted. The specific preparation methods are as follows:

[0070] Step 1: Dissolve compounds of formula 15 and formula 16 in a third solvent, react them under the conditions of a second acid-binding agent, and then undergo ester hydrolysis to obtain compound 13.

[0071]

[0072] Where R1 is -C n H 2n - where n is an integer and n≥1; the third solvent is one or a mixture of acetonitrile, dichloromethane, acetone, dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF), preferably DMF; the second acid-binding agent is K2CO3; the feed ratio of compound 15, compound 16 and the first acid-binding agent is 1:1-3:1-3, preferably 1:1.6:1.3; the reaction temperature is 25℃-100℃, preferably 85℃; the ester hydrolysis reaction condition is lithium hydroxide monohydrate; the reaction temperature is 25℃.

[0073] Step 2: The compound of formula 12 and the compound of formula 13 are subjected to esterification reaction in the presence of the first condensing agent and the first acid-binding agent to obtain the compound of formula 1-1 or formula 1-2.

[0074]

[0075] In Equation 12, The first solvent is cisplatin, oxaliplatin, carboplatin, heptaplasm, nedaplatin, leuplatin, or miplatin; preferably cisplatin or oxaliplatin; the first solvent is one or a mixture of acetonitrile, dichloromethane, acetone, dimethyl sulfoxide (DMSO), and N,N-dimethylformamide (DMF); preferably, DMSO is used as the first solvent for compound 1-1 and DMF is used as the first solvent for compound 1-2; the first condensing agent is 1-hydroxybenzotriazole (HOBT) or O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU), preferably O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU); the first acid-binding agent is triethylamine (TEA); the reaction temperature is 25℃-100℃, preferably 25℃; the reaction is carried out under light-protected, anhydrous, and inert gas-protected conditions;

[0076] When preparing compound 1-1, the feed ratio of compound 12, compound 13, the first condensing agent, and the first acid-binding agent is 1:1-1.5:1-1.5:1-1.5, preferably 1:1:1.2:1.2; when preparing compound 1-2, the feed ratio of compound 12, compound 13, the first condensing agent, and the first acid-binding agent is 1:2-3:2-3:2-3, preferably 1:2:2:2.

[0077] Step 3: In the second solvent, react compound 1-1 with compound 14 to obtain compound 1-3;

[0078]

[0079] Where R2 is -C m H 2m+1 And m≥1; the second solvent is one or a mixture of acetonitrile, dichloromethane, acetone, dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF), preferably DMF; the feed ratio of compound 1-1 to compound 14 is 1:2-10, preferably 1:5; the reaction temperature is 25℃-100℃, preferably 60℃; the reaction is carried out under light-protected, anhydrous, and inert gas protection conditions.

[0080] The introduction of long fatty acid chains increases drug stability and enhances the lipophilicity and transmembrane capacity of Pt(IV) molecules, promoting the absorption of platinum drugs. The theophylline-platinum(IV) complex, with double substitution of theophylline and fatty acid chains, exhibits significantly improved drug stability and cellular uptake, enhancing antitumor activity while also providing reduced toxicity and sustained release. The prodrug synthesis process is simple and low-cost, greatly improving the efficacy of combination therapy. Compared with traditional divalent platinum and similar tetravalent platinum drugs, it has advantages such as better efficacy and fewer side effects.

[0081] The present invention will be described below with reference to the embodiments. Experimental methods that do not specify the operation steps are performed in accordance with the corresponding product instructions. Unless otherwise specified, the instruments, reagents and consumables used in the embodiments can be purchased from commercial companies.

[0082] Example 1:

[0083] The structural formula of the theophylline-platinum(IV) complex a in this embodiment is as follows:

[0084]

[0085] The synthetic route for preparing the theophylline-platinum(IV) complex in this embodiment is as follows:

[0086]

[0087] 1. Preparation of compound a1 cisplatin oxide

[0088] Cisplatin was oxidized in hydrogen peroxide for 6 hours, then refrigerated overnight at 0-4°C. It was then recrystallized with water, ice-cold ethanol, and diethyl ether to obtain a pale yellow precipitate Oxoplatin(c,c,t-[Pt(NH3)2Cl2(OH)2]), a1.

[0089] 2. Preparation of compound a2

[0090] Step 2: Theophylline (THEO) and ethyl bromoacetate were dissolved in anhydrous N,N-dimethylformamide (DMF). The reaction was carried out under argon protection at 85°C for 12 hours. The reaction progress was monitored by thin-layer chromatography. After the reaction was complete, the reaction solution was extracted three times with water and ethyl acetate. The combined organic phases were dried over anhydrous Na₂SO₄, and the organic solvent was removed by rotary evaporation. The crude product was mixed with a small amount of silica gel and loaded onto the sample using a dry chromatography method. Purification was performed by column chromatography with dichloromethane:methanol = 50:1 as the eluent, yielding a grayish-white solid intermediate.

[0091] The reaction intermediate was dissolved in a THF:H₂O (1:1) solution, and LiOH·H₂O was added. The reaction was carried out at room temperature for 2 hours. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, THF was removed by rotary evaporation while controlling the temperature. 1 mol / L HCl was added to the reaction solution, and a white precipitate appeared. The white precipitate was washed three times with water and dried under vacuum to obtain a white solid with a yield of 92%.

[0092] pass 1 The obtained compound was characterized by 1H NMR, and the following data were obtained:

[0093] 1 H NMR (400MHz, DMSO-d6): δ 8.06 (s, H), 4.27 (t, J = 6.4 Hz, 2H), 3.42 (s, 3H), 3.23 (s, 3H), 2.20 (t, J = 7.0 Hz, 2H), 2.02 (p, J = 6.5 Hz, 2H).

[0094] 3. Preparation of compound a

[0095] Compound a2 was mixed with dry N,N-dimethylformamide (DMF), and O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroboric acid (TBTU) was added. After activation for a period of time, triethylamine (TEA) was added, followed by activation for another period of time, and then compound a1 (Oxoplatin) was added. The mixture was reacted at 60°C for 24 h. Ethanol and diethyl ether were added to precipitate the precipitate, which was then dried and further purified by silica gel chromatography using dichloromethane and methanol as eluents. A white precipitate of compound a was obtained. Due to its poor solubility and high polarity, a pure product of this compound was not obtained; the purity was determined to be 71% by HPLC.

[0096] Example 2

[0097] The structural formula of the theophylline-platinum(IV) complex b in this embodiment is as follows:

[0098]

[0099] The synthetic route for preparing the theophylline-platinum(IV) complex in this embodiment is as follows:

[0100]

[0101] 1. Preparation of compound b1 cisplatin oxide

[0102] The preparation method of cisplatin oxide of compound b1 is the same as the preparation method of cisplatin oxide of formula a1 in Example 1.

[0103] 2. Preparation of compound b2

[0104] The preparation method of compound b2 is the same as that of compound a2 in Example 1.

[0105] 3. Preparation method of compound b

[0106] Compound b2 was mixed with dry N,N-dimethylformamide (DMF), and O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroboric acid (TBTU) was added. After activation for a period of time, triethylamine (TEA) was added, and after activation for a period of time, compound b1 (Oxoplatin) was added. The mixture was reacted at room temperature for 24 hours. Ethanol and diethyl ether were added to precipitate the precipitate. After drying, the precipitate was further purified by silica gel chromatography with dichloromethane and methanol as eluents, yielding a white precipitate of compound b.

[0107] pass 1 H NMR, 13 The obtained compound was characterized by C NMR and HRMS (ESI), and the following data were obtained:

[0108] 1H NMR (400MHz, DMSO-d6): δ8.13(s,2H),6.62(s,6H),4.32(t,J=6.4Hz,4H),3.43(s,6H),3.23(s,6H),2.13(t,J=7.0Hz,4H),1.97(p,J=6.5Hz,4H). 13 C NMR(101MHz,DMSO-d6)δ179.50,154.31,150.98,148.52,142.84,105.88,48.55,31.85,29.40,27.56,26.55.HR-MS(ESI)(m / z):calcd for C 22 H 32 Cl2N 10 O8Pt + (M+H) + 829.1429, found 829.1487.

[0109] Example 3

[0110] The structural formula of the theophylline-platinum(IV) complex c in this embodiment is as follows:

[0111]

[0112] The synthetic route for preparing the theophylline-platinum(IV) complex in this embodiment is as follows:

[0113]

[0114] 1. Preparation of compound c1

[0115] The preparation method of compound c1 is the same as that of formula a in Example 1.

[0116] 2. Preparation method of compound c

[0117] C1 was mixed with dry N,N-dimethylformamide (DMF), and hexanoic anhydride was added. The mixture was reacted at 60°C for 24 h. DMF was removed using a rotary evaporator under argon vacuum throughout the reaction. The reaction progress was monitored by thin-layer chromatography. After the reaction was complete, the DMF was evaporated to dryness, and the crude product was dissolved in a mixture of methanol and dichloromethane. The solution was then loaded onto a GF254 silica gel plate for separation, with dichloromethane:methanol = 10:1 as the developing solvent. A pale yellow solid c was obtained, washed 2-3 times with diethyl ether, and dried under vacuum to obtain a pale yellow powder, with a yield of 47%.

[0118] pass 1 H NMR, 13 The obtained compound was characterized by C NMR and HRMS (ESI), and the following data were obtained:

[0119] 1 H NMR (400MHz, DMSO-d6): δ8.02(s,1H),6.59(s,6H),5.07(s,1H),3.44(s,3H),3.23(s,3H),2.21(t,J=7.5Hz,2H),1.23(s,6H),0.85(d,J=6.7Hz,3H). 13 C NMR(101MHz,DMSO-d6)δ180.88,173.85,154.87,150.94,147.80,143.37,106.46, 47.17,35.48,30.76,29.45,27.59,25.06,21.88,13.83.HR-MS(ESI)(m / z):calcd for C 15 H 26 Cl2N6O6Pt + (M+H) + 651.0939, found 651.0999.

[0120] Example 4

[0121] The structural formula of the theophylline-platinum(IV) complex d in this embodiment is as follows:

[0122]

[0123] The synthetic route for preparing the theophylline-platinum(IV) complex in this embodiment is as follows:

[0124]

[0125] 1. Preparation of compound d1

[0126] The preparation method of compound d1 is the same as that of formula a in Example 1.

[0127] 2. Preparation method of compound d

[0128] Mix d1 with dry N,N-dimethylformamide (DMF), add lauric anhydride, and react at 60°C for 24 h. Remove DMF using a rotary evaporator under argon vacuum throughout. Monitor the reaction progress using thin-layer chromatography. After the reaction is complete, evaporate the DMF to dryness, dissolve the crude product in a mixture of methanol and dichloromethane, and load the sample. Separate using a GF254 silica gel plate with dichloromethane:methanol = 10:1 as the developing solvent. A pale yellow solid is obtained, washed 2-3 times with diethyl ether, and dried under vacuum to obtain a pale yellow powder d, with a yield of 52%.

[0129] pass 1H NMR, 13 The obtained compound was characterized by C NMR and HRMS (ESI), and the following data were obtained:

[0130] 1 H NMR (400MHz, DMSO-d6): δ8.01(s,1H),6.49(s,6H),5.07(s,2H),3.44(s,3H ),3.23(s,3H),2.21(t,J=7.2Hz,2H),1.24(s,18H),0.85(d,J=6.7Hz,3H). 13 C NMR(101MHz,DMSO-d6)δ180.87,173.84,154.85,152.64,150.93,143.37,106.44,47.15,35.54,31.25, 29.44,29.00,28.98,28.92,28.85,28.66,28.55,27.57,25.39,22.04,13.90.HR-MS(ESI)(m / z):calcd for C 21 H 38 Cl2N6O6Pt + (M+H) + 735.1878, found 735.1916.

[0131] Example 5

[0132] The theophylline-platinum(IV) complex e in this embodiment has the following structural formula:

[0133]

[0134] The synthetic route for preparing the theophylline-platinum(IV) complex in this embodiment is as follows:

[0135]

[0136] 1. Preparation of compound e1

[0137] The preparation method of compound e1 is the same as that of formula a in Example 1.

[0138] 2. Preparation method of compound e

[0139] e1 was mixed with dry N,N-dimethylformamide (DMF), and hexadecanoic anhydride was added. The mixture was reacted at 60°C for 24 h. DMF was removed using a rotary evaporator under argon vacuum throughout the reaction. The reaction was monitored by thin-layer chromatography. After the reaction was complete, the DMF was evaporated to dryness, and the crude product was dissolved in a mixture of methanol and dichloromethane. The solution was then loaded onto a silica gel plate (GF254) for separation using dichloromethane:methanol (10:1) as the developing solvent. A pale yellow solid was obtained, washed 2-3 times with diethyl ether, and dried under vacuum to obtain product e, a pale yellow powder, with a yield of 46.8%.

[0140] pass 1 H NMR, 13 The obtained compound was characterized by C NMR and HRMS (ESI), and the following data were obtained:

[0141] 1 H NMR (400MHz, DMSO-d6): δ8.00(s,1H),6.46(s,6H),5.06(s,2H),3.44(s,3H ),3.23(s,3H),2.21(t,J=7.4Hz,2H),1.23(s,26H),0.85(t,J=6.7Hz,3H). 13 C NMR(101MHz,DMSO-d6)δ180.87,173.84,154.86,150.93,147.80,143.35,106.46,47.16,43.46,35.53,31.25,30.20,29.44, 29.33,29.01,28.96,28.94,28.86,28.65,28.57,27.76,27.57,25.40,22.58,22.04,18.57,13.89.HR-MS(ESI)(m / z):calcd for C 25 H 46 Cl2N6O6P t +(M+H) + 791.2504, found 791.2575.

[0142] Example 6

[0143] The theophylline-platinum(IV) complex f in this embodiment has the following structural formula:

[0144]

[0145] The synthetic route for preparing the theophylline-platinum(IV) complex in this embodiment is as follows:

[0146]

[0147]

[0148] 1. Preparation of compound f1 cisplatin oxide

[0149] Cisplatin was oxidized in hydrogen peroxide for 6 hours, then refrigerated overnight at 0-4°C. It was then recrystallized with water, ice-cold ethanol, and diethyl ether to obtain a pale yellow precipitate Oxoplatin(c,c,t-[Pt(NH3)2Cl2(OH)2]), a1.

[0150] 2. Preparation of compound f2

[0151] Step 2: Theophylline (THEO) and ethyl bromoacetate were dissolved in anhydrous N,N-dimethylformamide (DMF). The reaction was carried out under argon protection at 85°C for 12 hours. The reaction progress was monitored by thin-layer chromatography. After the reaction was complete, the reaction solution was extracted three times with water and ethyl acetate. The combined organic phases were dried over anhydrous Na₂SO₄, and the organic solvent was removed by rotary evaporation. The crude product was mixed with a small amount of silica gel and loaded onto the sample using a dry chromatography method. Purification was performed by column chromatography with dichloromethane:methanol = 50:1 as the eluent, yielding a grayish-white solid intermediate.

[0152] The reaction intermediate was dissolved in a THF:H₂O (1:1) solution, and LiOH·H₂O was added. The reaction was carried out at room temperature for 2 hours. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, THF was removed by rotary evaporation while controlling the temperature. 1 mol / L HCl was added to the reaction solution, and a white precipitate appeared. The white precipitate was washed three times with water and dried under vacuum to obtain a white solid with a yield of 91%.

[0153] pass 1 The obtained compound was characterized by 1H NMR, and the following data were obtained:

[0154] 1 H NMR (400MHz, DMSO-d6): δ13.27(s,1H),8.04(s,1H),5.07(s,2H),3.44(s,3H),3.21(s,3H).

[0155] 3. Preparation of compound f

[0156] Compound a2 was mixed with dry N,N-dimethylformamide (DMF), and O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroboric acid (TBTU) was added. After activation for a period of time, triethylamine (TEA) was added, followed by activation for another period of time, and then compound a1 (Oxoplatin) was added. The mixture was reacted at 60°C for 24 h. Ethanol and diethyl ether were added to precipitate the compound. After drying, the precipitate was further purified by silica gel chromatography using dichloromethane and methanol as eluents, yielding a white precipitate as compound f. Due to its poor solubility and high polarity, a pure product of this compound was not obtained; the purity was determined to be 64% by HPLC.

[0157] Example 7

[0158] The theophylline-platinum(IV) complex g in this embodiment has the following structural formula:

[0159]

[0160] The synthetic route for preparing the theophylline-platinum(IV) complex in this embodiment is as follows:

[0161]

[0162] 1. Preparation of compound g1 cisplatin oxide

[0163] The preparation method of cisplatin oxide of compound g1 is the same as the preparation method of cisplatin oxide of formula a1 in Example 1.

[0164] 2. Preparation of compound g2

[0165] The preparation method of compound g2 is the same as that of compound f2 in Example 1.

[0166] 3. Preparation method of compound g

[0167] Compound g2 was mixed with dry N,N-dimethylformamide (DMF), and O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroboric acid (TBTU) was added. After activation for a period of time, triethylamine (TEA) was added, and after activation for a period of time, compound g1 (Oxoplatin) was added. The mixture was reacted at room temperature for 24 hours. Ethanol and diethyl ether were added to precipitate the precipitate. After drying, the precipitate was further purified by silica gel chromatography with dichloromethane and methanol as eluents, yielding a white precipitate of compound g.

[0168] pass 1 H NMR, 13 The obtained compound was characterized by C NMR and HRMS (ESI), and the following data were obtained:

[0169] 1H NMR (400MHz, DMSO-d6): δ8.13(s,2H),6.62(s,6H),4.32(t,J=6.4Hz,4H),3.43(s,6H),3.23(s,6H),2.13(t,J=7.0Hz,4H),1.97(p,J=6.5Hz,4H). 13 C NMR(101MHz,DMSO-d6)δ179.50,154.31,150.98,148.52,142.84,105.88,48.55,31.85,29.40,27.56,26.55.HR-MS(ESI)(m / z):calcd for C 22 H 32 Cl2N 10 O8Pt + (M+H) + 829.1429, found 829.1487.

[0170] Example 8

[0171] The theophylline-platinum(IV) complex h in this embodiment has the following structural formula:

[0172]

[0173] The synthetic route for preparing the theophylline-platinum(IV) complex in this embodiment is as follows:

[0174]

[0175] 1. Preparation of compound h1

[0176] The preparation method of compound h1 is the same as that of formula f in Example 6.

[0177] 2. Preparation method of compound h

[0178] h1 was mixed with dry N,N-dimethylformamide (DMF), and hexanoic anhydride was added. The mixture was reacted at 60°C for 24 h. DMF was removed using a rotary evaporator under argon vacuum throughout the reaction. The reaction progress was monitored by thin-layer chromatography. After the reaction was complete, the DMF was evaporated to dryness, and the crude product was dissolved in a mixture of methanol and dichloromethane. The solution was then loaded onto a GF254 silica gel plate for separation, with dichloromethane:methanol = 10:1 as the developing solvent. A pale yellow solid c was obtained, washed 2-3 times with diethyl ether, and dried under vacuum to obtain a pale yellow powder, with a yield of 43.6%.

[0179] pass 1 H NMR, 13 The obtained compound was characterized by C NMR and HRMS (ESI), and the following data were obtained:

[0180] 1 H NMR (400MHz, DMSO-d6): δ8.10(s,1H),6.54(s,6H),4.32(t,J=6.4Hz,2H),3.43(s,3H),3.24(s,3H),2.22(t,J=7.5Hz,2H ),2.12(t,J=7.1Hz,2H),1.97(p,J=6.3Hz,2H),1.48(q,J=7.3Hz,2H),1.26(dd,J=7.1,3.7Hz,5H),0.86(t,J=6.9Hz,3H). 13 C NMR(101MHz,DMSO-d6)δ180.94,179.46,154.32,150.99,148.51,142.82,105.90,54.83,4 5.24,35.63,30.79,29.39,27.55,26.56,25.07,21.89,13.81.HR-MS(ESI)(m / z):calcdfor C 17 H 30 Cl2N6O6Pt + (M+H) + 679.1252, found 679.1299.

[0181] Example 9

[0182] The structural formula of the theophylline-platinum(IV) complex i in this embodiment is as follows:

[0183]

[0184] The synthetic route for preparing the theophylline-platinum(IV) complex in this embodiment is as follows:

[0185]

[0186] 1. Preparation of compound i1

[0187] The preparation method of compound i1 is the same as that of formula f in Example 6.

[0188] 2. Preparation method of compound i

[0189] i1 was mixed with dry N,N-dimethylformamide (DMF), and decanoic anhydride was added. The mixture was reacted at 60°C for 24 h. DMF was removed using a rotary evaporator under argon vacuum throughout the reaction. The reaction progress was monitored by thin-layer chromatography. After the reaction was complete, the DMF was evaporated to dryness, and the crude product was dissolved in a mixture of methanol and dichloromethane. The solution was then loaded onto a GF254 silica gel plate for separation, with dichloromethane:methanol = 10:1 as the developing solvent. A pale yellow solid was obtained, washed 2-3 times with diethyl ether, and dried under vacuum to obtain product i, a pale yellow powder, with a yield of 54.8%.

[0190] pass 1 H NMR, 13 The obtained compound was characterized by C NMR and HRMS (ESI), and the following data were obtained:

[0191] 1 H NMR (400MHz, DMSO-d6): δ8.10(s,1H),6.53(s,6H),4.32(t,J=6.4Hz,2H),3.43(s,3H),3.24(s,3H),2.22(t,J=7 .4Hz,2H),2.12(t,J=7.0Hz,2H),1.97(p,J=6.2Hz,2H),1.49–1.40(m,2H),1.24(s,17H),0.86(t,J=6.7Hz,3H). 13 C NMR(101MHz,DMSO-d6)δ180.94,179.45,154.32,150.98,148.51,142.82,105.90,45.25,38.91,35.69,31.81, 31.26,29.38,28.99,28.94,28.87,28.67,28.60,27.55,26.56,25.42,22.05,13.88.HR-MS(ESI)(m / z):calcd for C 23 H 42 Cl2N6O6Pt + (M+H) + 763.2191, found 763.2234.

[0192] Example 10

[0193] The theophylline-platinum(IV) complex j in this embodiment has the following structural formula:

[0194]

[0195] The synthetic route for preparing the theophylline-platinum(IV) complex in this embodiment is as follows:

[0196]

[0197] 1. Preparation of compound j1

[0198] The preparation method of compound j1 is the same as that of formula f in Example 6.

[0199] 2. Preparation method of compound j

[0200] J1 was mixed with dry N,N-dimethylformamide (DMF), and hexadecanoic anhydride was added. The mixture was reacted at 60°C for 24 h. DMF was removed using a rotary evaporator under argon vacuum throughout the reaction. The reaction progress was monitored by thin-layer chromatography. After the reaction was complete, the DMF was evaporated to dryness, and the crude product was dissolved in a mixture of methanol and dichloromethane. The solution was then loaded onto a silica gel plate (GF254) for separation, with dichloromethane:methanol = 10:1 as the developing solvent. A pale yellow solid was obtained, washed 2-3 times with diethyl ether, and dried under vacuum to obtain a pale yellow powder (e) in 54% yield.

[0201] pass 1 H NMR, 13 The obtained compound was characterized by C NMR and HRMS (ESI), and the following data were obtained:

[0202] 1 H NMR (400MHz, DMSO-d6): δ8.11(s,1H),6.54(s,6H),4.32(t,J=6.5Hz,2H),3.43(s,3H),3.23(s,3H),2.21(t,J=7.5 Hz,2H),2.12(t,J=7.1Hz,2H),1.96(p,J=6.6Hz,2H),1.44(q,J=6.1,5.5Hz,2H),1.24(s,26H),0.88–0.83(m,3H). 13 C NMR (101MHz, DMSO-d6) δ180.95,179.45,154.33,151.00,148.53,142.84,105.90,45.25,40.16,35.70,31.24,29.40 29.01,28.87,28.65,28.59,27.57,26.56,25.42,22.04,13.89.HR-MS(ESI)(m / z):calcd for calcd for C 27 H 50 Cl2N6O6Pt + (M+H) + 819.2817, found 819.2817.

[0203] Example 11: In vitro antitumor activity assay

[0204] The theophylline-platinum(IV) complex prepared in the above examples was subjected to in vitro antitumor activity assay.

[0205] This study investigated the antitumor activity of the synthesized compounds using the MTT assay (3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide). All cell lines used in this study were human-derived, including: ovarian cancer cell line (SKOV3), BRCA1 knockdown ovarian cancer cell line (SKOV3 BRCA1 KD), non-small cell lung cancer cell line (A549), BRCA1 knockdown non-small cell lung cancer cell line (A549BRCA1 KD), triple-negative breast cancer cell line (MDA-MB-231), and normal hepatocyte cell line (LO2). The A549 cell line was purchased from Peking Union Medical College, China; the LO2, SKOV3, and MDA-MB-231 cell lines were purchased from the American Type Culture Collection; and the SKOV3 BRCA1 KD and A549 BRCA1 KD cell lines were constructed by members of our research group according to literature methods and were characterized and validated. SKOV3, SKOV3 BRCA1KD, A549, and A549 BRCA1 KD cell lines were cultured in PRMI 1640 medium containing 10% FBS, while MDA-MB-231 and LO2 cell lines were cultured in DMEM medium containing 10% FBS. All cell lines were cultured in a humidified incubator at 37°C with 5% CO2.

[0206] The specific experimental steps are as follows:

[0207] Collect cells in the logarithmic growth phase, count them, and adjust the cell concentration to 3 × 10⁻⁶. 4 Cells / mL were seeded into 96-well plates at 100 μL per well, with a blank group (pure culture medium) and a control group (no drug added). The plates were incubated overnight. After adhesion, the compound was diluted with culture medium to the desired concentration in the first well, followed by 5 / 2 dilutions for subsequent wells, resulting in 18 concentration gradients. The mixture was gently pipetted to mix. After 72 h of incubation, 10 μL (5 mg / mL) of MTT solution was added to each well. The plates were incubated for another 4 h, the supernatant was removed, and 100 μL / well DMSO was added. After thorough shaking, the OD value was measured using a microplate reader at λ = 570 nm. Three independent replicates were performed to ensure the reliability of the results.

[0208] Table 1. IC50 of the compounds after 72 hours of cell treatment. 50 value

[0209]

[0210]

[0211] SI a :selectivity index,IC 50 (in LO2) / IC 50 (in SKOV3 BRCA1 KD).

[0212] FI b :fold increase, IC 50 (CDDP) / IC 50 (e)

[0213] Table 1 shows the antiproliferative activity of the compounds against SKOV3, SKOV3 BRCA1 KD, A549, A549 BRCA1 KD, MDA-MB-231, and LO2 cells, and the IC50 values ​​of compounds c, d, e, h, i, and j. 50 The value was significantly lower than that of cisplatin, THEO, and combined administration, indicating a better anti-proliferative capacity against cancer cells. Compound g exhibited the best anti-proliferative activity in SKOV3 BRCA1 KD cells, with an IC50 value significantly lower than that of cisplatin, THEO, and combined administration. 50 The cytotoxicity of compound e (0.01 ± 0.001 μM) was 581-fold higher than that of cisplatin (5.81 ± 0.32 μM). Notably, compound e exhibited significantly different cytotoxicity in SKOV3 and SKOV3 BRCA1 KD cells. In SKOV3 BRCA1 KD cells, its IC50 value (0.01 ± 0.001 μM) was significantly higher than that of cisplatin (5.81 ± 0.32 μM). 50 The value (0.01±0.001) was 13-fold higher than that in SKOV3 cells (0.13±0.001). Furthermore, compound e showed a selectivity index (SI) of 12 for LO2 and SKOV3 BRCA1KD cells, 14.8 times that of cisplatin (0.81), demonstrating a certain degree of selectivity in vitro. We hypothesize that this increased cytotoxicity may be due to the synergistic antitumor effect of compound e on cisplatin and theophylline. Cisplatin interferes with the replication and transcription of the SKOV3 BRCA1 KD cell genome, causing DNA damage; theophylline inhibits PARP-1 expression, blocking the DNA single-strand repair pathway and further transforming it into DNA double-strand breaks. Simultaneously, the knockdown of the BRCA1 gene inhibits the precise homologous recombination repair pathway, forcing the selection of the highly error-prone non-homologous recombination pathway for repair, ultimately leading to irreversible tumor cell death and increasing selectivity for homologous recombination-deficient tumor cells. However, the IC50 of compound e in A549 and A549 BRCA1 KD cells was [not specified in the original text]. 50The differences in values ​​were not very significant, which we speculate is due to the fact that BRCA1 gene mutations have a more significant impact on ovarian cancer than on non-small cell lung cancer. In summary, compound e exhibited excellent anti-proliferative activity in all tested tumor cell lines (especially SKOV3 BRCA1KD), while showing relatively low toxicity to normal cells. Therefore, we selected compound e, along with SKOV3 and SKOV3 BRCA1 KD cells, for further investigation into its anti-tumor mechanism.

[0214] Example 12: Intracellular Reduction Experiment

[0215] Exploring the intracellular release capacity of prodrugs is crucial for studying the mechanism of action of tetravalent platinum, which needs to be reduced to its divalent form in vivo to exert its effects. To investigate whether theophylline-platinum(IV) complexes can be reduced intracellularly by reducing agents (glutathione, ascorbic acid, etc.) to release divalent platinum and simultaneously achieve a sustained-release effect, we conducted an intracellular reduction experiment. Taking compound e as an example, the specific experimental steps are as follows:

[0216] 1×10 6 SKOV3 BRCA1 KD cells were seeded in 6-well plates. After cell adhesion, the cells were treated with 100 μM compound e and cultured for 4 h. The culture medium was discarded, and the cells were washed three times with PBS. After centrifugation to remove the PBS, the cells were resuspended in a certain amount of methanol and dichloromethane and then transferred to a homogenizer and homogenized for 10 minutes until the cells were completely lysed. After homogenization, the cells were allowed to stand for a period of time, and the supernatant was collected by centrifugation. The solvent was evaporated at room temperature, and the solid was resuspended in 200 μL of chromatographic methanol. The cells were then analyzed by liquid chromatography. The liquid chromatography analysis conditions were as follows: UV detection wavelength 260 nm, mobile phase methanol and water (containing 0.1% formic acid), Venusil XBP C18 column (50 × 4.6 mm, 5 μm), Shima Seiki (LC-20A) high performance liquid chromatograph, gradient elution, methanol 5%-95% (0-10 min), 95% methanol 25 min, flow rate 1 mL / min.

[0217] HPLC detection results are as follows Figure 1 As shown in the figure, two absorption peaks were observed on the band of the intracellular sample extracted from compound e, with retention times of 16.986 and 9.360 min, respectively, corresponding to the standards of compound e and compound THEO-ES. This indicates that under intracellular conditions, the theophylline carboxylic acid derivative THEO-ES can be released from compound e and exert antitumor activity in combination with cisplatin, validating our previous hypothesis regarding the MTT results. These results provide a foundation for further research on the intracellular activity and mechanism of compound e.

[0218] In summary, the theophylline-platinum (IV) complex synthesized in this patent can be reduced intracellularly and has a sustained-release effect.

[0219] Example 13: Study on the in vitro antitumor activity mechanism of drugs

[0220] To investigate the antitumor mechanism of the theophylline-platinum(IV) complex described in this invention, an in vitro antitumor mechanism study was conducted. Taking compound e as an example, the following experiments were performed, and the experimental procedures required for verification can be carried out using conventional methods.

[0221] The intracellular release of compound e into BRCA1-knockdown ovarian cancer cells was studied 4 hours after treatment. The results are as follows: Figure 1 As shown; the statistical analysis of Pt content in BRCA1 knocked-down ovarian cancer cells by compound e is presented in the following figures. Figure 2 As shown in the figure, the effects of 10 μM compound e on DNA damage in BRCA1-knockdown ovarian cancer cells, 1 μM compound e on ROS induction in BRCA1-knockdown ovarian cancer cells, and 2 μM compound e on MMP induction in BRCA1-knockdown ovarian cancer cells were investigated. The results are as follows: Figure 3-5 As shown.

[0222] The effects of 2 μM compound e on BRCA1 knockdown ovarian cancer cells on apoptosis and cell cycle arrest were investigated. The results are as follows: Figure 6-7 As shown; the effects of 0.5 μM compound e on the inhibition of cell migration and cell invasion in BRCA1 knockdown ovarian cancer cells were investigated, and the results are as follows. Figure 8-9 As shown; the study investigated the effect of 1 μM compound e on the expression of regulatory proteins in BRCA1 knockdown ovarian cancer cells, and the results are as follows. Figure 10 As shown.

[0223] The above results indicate that ICP-MS analysis showed that compound e significantly increased platinum accumulation levels in both SKOV3 and SKOV3BRCA1 KD cells, with no statistically significant difference between the two. This suggests that the difference in antiproliferative activity of compound e in these two cell lines was not related to drug uptake but rather due to BRCA1 gene knockdown. Subsequent experiments further investigated the antitumor mechanism of compound e in SKOV3 and SKOV3BRCA1KD cells. Comet assays and immunofluorescence assays showed that compound e significantly induced DNA damage; cell cycle and apoptosis assays showed that compound e induced S-phase arrest and promoted apoptosis; scratch and invasion assays showed that compound e inhibited cell migration and invasion; furthermore, compound e significantly increased the expression of reactive oxygen species and decreased mitochondrial membrane potential, thereby inducing apoptosis; Western blotting experiments showed that compound e inhibited PARP-1 expression and enhanced γ-H2AX expression. In the aforementioned mechanistic experiments, compound e exhibited stronger effects than SKOV3 in both BRCA1 KD cells and SKOV3 cells. Based on these results, we conclude that in BRCA1 knockdown SKOV3 cells, compound e induces DNA damage while inhibiting DNA repair pathways, thereby selectively killing tumor cells deficient in homologous recombination and exerting a synthetic lethal effect. Simultaneously, compound e also inhibited the expression of EMT and TGF-β pathway-related proteins, demonstrating multi-pathway anti-tumor activity. In conclusion, compound e possesses potential anti-tumor therapeutic value and provides a novel drug development strategy for the clinical treatment of gene-mutant cancers.

[0224] Example 14: Study on the in vivo antitumor activity of the drug

[0225] To investigate the antitumor effect of the theophylline-platinum(IV) complex described in this invention, an in vivo antitumor activity study was conducted, the specific steps of which are as follows:

[0226] A SKOV3 BRCA1 KD xenograft tumor model was established using 4-5 week old female Balb / c nude mice. The tumor volume was increased to 50-100 cm³. 3 Mice were then randomly divided into four groups: PBS, CDDP, Olaparib (PARP inhibitor), and compound e. The drug concentration was set at 2.5 mg / kg Pt, and administration was every two days for a total of six administrations. The PBS, CDDP, and compound e groups were administered via tail vein injection, while the Olaparib group was administered via gavage. Mouse body weight and tumor volume were measured every other day. Mice were euthanized two days after the last administration.

[0227] Experimental results are as follows Figure 11 As shown, the tumor inhibition rates at the treatment endpoint in the CDDP, Olaparib, and compound e groups were 50.48%, 47.63%, and 71.70%, respectively. (This is combined with tumor images at the treatment endpoint.) Figure 11 A. Tumor growth curve Figure 11 B and treatment endpoint: tumor weight Figure 11 C, together with other studies, confirmed that compound e's inhibitory effect on tumor growth was significantly superior to CDDP and Olaparib, indicating that compound e has a more significant anti-tumor advantage compared to first-line platinum-based drugs and PARP inhibitors. Simultaneously, changes in mouse body weight... Figure 11 D and survival curves Figure 11 As shown in Figure E, the body weight of mice in the compound e group remained relatively stable, with no significant difference from the control group, and the final survival rate was 100%. In contrast, the body weight of mice in the CDDP group showed a significant decreasing trend, with a final survival rate of 0% (a decrease in body weight to 20% of initial body weight was considered death). This demonstrates that compound e has significantly reduced systemic toxicity compared to first-line platinum-based drugs in clinical practice. In vivo experiments indicate that compound e possesses synergistic and toxicity-reducing antitumor activity. These results collectively demonstrate that theophylline-platinum(IV) complexes based on a synthetic lethal strategy have potential application value in antitumor therapy, providing new strategies and ideas for the treatment of clinical gene-mutant cancers.

[0228] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. A theophylline-platinum(IV) complex, characterized in that: Theophylline is attached to at least one side of the tetravalent platinum coordination center; or, theophylline is attached to one side of the tetravalent platinum coordination center and a long carbon chain group is attached to the other side.

2. The theophylline-platinum(IV) complex according to claim 1, characterized in that: The structure is shown in any of Equations 1-1, 1-2, and 1-3; in, Selected from cisplatin, oxaliplatin, carboplatin, heptaplatin, nedaplatin, lorplatin, or miplatin. Preferably Cisplatin, oxaliplatin, or carboplatin; R1 is -C n H 2n - a straight-chain or branched group, where n is an integer and 1 ≤ n ≤ 6, preferably n = 1 or 3; R2 is -C m H 2m+1 m is an integer and 1≤m≤20, preferably m≤16, preferably R1 and / or R2 are straight-chain groups.

3. The theophylline-platinum(IV) complex according to claim 1 or 2, characterized in that: As shown by any structure in Equation 2-11:

4. A method for preparing the theophylline-platinum(IV) complex according to any one of claims 3, characterized in that: The compound of formula 12 and the compound of formula 13 are dissolved in a first solvent and reacted under the conditions of a first condensing agent and a first acid-binding agent at a reaction temperature of 25-100°C, preferably 25°C, to obtain the compound of formula 1-1 or formula 1-2. in, The active ingredients are cisplatin, oxaliplatin, carboplatin, heptaplatin, nedaplatin, levoplatin, or miplatin.

5. The method for preparing the theophylline-platinum(IV) complex according to claim 4, characterized in that: The first solvent is one or a mixture of acetonitrile, dichloromethane, acetone, dimethyl sulfoxide (DMSO), and N,N-dimethylformamide (DMF); Preferably, DMSO is used as the first solvent for preparing compound 1-1, and DMF is used as the first solvent for preparing compound 1-2. The first condensing agent is 1-hydroxybenzotriazole (HOBT) or O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU), preferably O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU). The first acid-binding agent is triethylamine (TEA); The reaction temperature is 25℃-100℃, preferably 25℃; When preparing compound 1-1, the feed ratio of compound 12, compound 13, first condensing agent and first acid-binding agent is 1:1-1.5:1-1.5:1-1.5, preferably 1:1:1.2:1.2; When preparing compounds of formula 1-2, the feeding ratio of compound 12, compound 13, first condensing agent and first acid-binding agent is 1:2-3:2-3:2-3, preferably 1:2:2:

2.

6. The theophylline-platinum(IV) complex according to claim 4, characterized in that: In a second solvent, the compound of formula 1-1 is reacted with the compound of formula 14 to obtain the compound of formula 1-3; Where R2 is -C m H 2m+1 And m≥1.

7. A method for preparing the theophylline-platinum(IV) complex according to any one of claims 6, characterized in that: The second solvent is one or a mixture of acetonitrile, dichloromethane, acetone, dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF), preferably DMF; The reaction temperature is 25℃-100℃, preferably 60℃; The feed ratio of compound 1-1 to compound 14 is 1:2-10, preferably 1:

5.

8. The method for preparing the theophylline-platinum(IV) complex according to claim 4, characterized in that: Compounds 15 and 16 were dissolved in a third solvent, reacted under the conditions of a second acid-binding agent, and then subjected to ester hydrolysis to obtain compound 13.

9. The method for preparing the theophylline-platinum(IV) complex according to claim 8, characterized in that: The third solvent is one or a mixture of acetonitrile, dichloromethane, acetone, dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF), preferably DMF; The second acid-binding agent is K2CO3; The feeding ratio of compound 15, compound 16 and the first acid-binding agent is 1:1-3:1-3, preferably 1:1.6:1.3; The reaction temperature is 25℃-100℃, preferably 85℃; The ester hydrolysis reaction conditions are lithium hydroxide monohydrate; The reaction temperature is 25℃.

10. The use of the theophylline-platinum(IV) complex according to any one of claims 1-3 in the preparation of antitumor drugs; preferably, for the preparation of drugs for ovarian cancer, lung cancer, or breast cancer; Preferably, it is used to prepare BRCA1 knockdown drugs for ovarian cancer or BRCA1 knockdown lung cancer.

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