A preparation method of HO-1i-Pt(IV) complex and self-assembled nanoparticles and their application in tumor drugs

Abstract

The invention discloses a preparation method of a HO-1i-Pt(IV) complex and a self-assembled nanoparticle and its application in tumor treatment, wherein one molecule of HO-1i is connected to one axial side of a platinum(IV) coordination center, and the other side is OH or Cl to form a monosubstituted tetravalent platinum complex; or one molecule of HO-1i is connected to one axial side of a platinum(IV) coordination center, and the other side is connected to a molecule of a straight or branched fatty chain group to form a disubstituted tetravalent platinum complex; the disubstituted HO-1i-Pt(IV) complex with a specific carbon chain group can be prepared into self-assembled nanoparticles by an anti-solvent method. The prepared complex and nanoparticles have synergistic and broad-spectrum lethality to a variety of tumors, simple synthesis process, low cost, greatly improve the efficacy of combined medication, and compared with traditional divalent platinum and similar tetravalent platinum drugs, have the advantages of good efficacy and small side effects; HO-1i is introduced to exert synergistic anti-tumor efficacy, while activating immunity and reversing cisplatin resistance, significantly increasing the efficacy and reducing the serious toxic side effects of traditional platinum drugs.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical technology, and in particular relates to a preparation method of a HO-1i-Pt (IV) complex and self-assembled nanoparticles and applications thereof in tumor drugs. Background Art

[0002] Cancer, also known as malignant tumor, is one of the most common and frequent diseases that threaten human life and health. The 2022 World Health Statistics Report shows that in 2020, there were 19.29 million new cancer cases and 9.96 million deaths worldwide. Among them, China had 4.57 million new cancer cases, accounting for 23.7% of new cancer cases worldwide; 3 million cancer deaths, accounting for 30% of cancer deaths worldwide. Whether in developing or developed countries, cancer is gradually replacing cardiovascular and cerebrovascular diseases and becoming the number one killer threatening human life and health. Seeking effective cancer treatment methods is an urgent task. Chemotherapy is one of the important means of cancer treatment. It can effectively control or eliminate lesions, and therefore plays an important role in the treatment of advanced malignant and refractory cancer. Since the anti-cancer effects of nitrogen mustard and folic acid antagonists were discovered in the 1940s, the U.S. Food and Drug Administration (FDA) has approved more than 700 drugs for cancer treatment. According to the source and mechanism of action of the drug, it is usually divided into: kinase drugs, cytotoxic drugs, biological response modifiers, monoclonal antibodies, etc. The anti-tumor activity of cisplatin was first reported by Rosenberg et al., a professor at Michigan State University in the United States, in 1969, and was approved by the FDA in 1978 for the treatment of ovarian cancer and testicular cancer, opening up a new field of research on platinum-based anti-tumor drugs. Cisplatin-based platinum-based anti-tumor drugs have been used as the first choice for chemotherapy of various malignant tumors to this day, and are also one of the most common components of clinical combination drug treatments for various malignant tumors. Although it has a significant effect in the clinical treatment of tumors, due to the lack of tumor cell selectivity, cisplatin not only kills tumor cells, but also damages some rapidly dividing normal cells, such as bone marrow cells, gastrointestinal cells, scalp hair follicle cells, etc., causing serious toxic side effects, such as kidney damage, neurotoxicity and ototoxicity. In addition, long-term or high-dose use of cisplatin can also cause severe anemia. In addition to the great toxic side effects, the development of drug resistance is also one of the important factors limiting the clinical application of cisplatin, which significantly reduces the anti-tumor effect of the drug. A comprehensive and in-depth understanding of the mechanism of action of platinum drugs in the body, comparison of the clinical effects of marketed drugs, in-depth research on their drug resistance mechanisms, and the use of novel methods to synthesize new and practical platinum anti-tumor drugs are the keys to achieving further breakthroughs in tumor treatment.

[0003] The emergence of tetravalent platinum prodrugs provides a new idea for overcoming the inherent defects of classic divalent platinum anti-tumor drugs. The central ion of the platinum (IV) complex belongs to d 6The electronic configuration has two more axial positions compared to platinum (II) complexes, which can form a very stable octahedral coordination configuration. Studies have shown that platinum (IV) complexes are relatively stable in the blood and can reduce reactions with nucleophiles in the body, thereby reducing drug losses and reducing toxic side effects. In addition, the axial ligand provides modification space for the construction of various platinum (IV) complexes. On the one hand, targeting molecules, such as folic acid, monoclonal antibodies, peptides and other active molecules, can be introduced to increase the selectivity of compounds for cancer cells and reduce their toxic side effects. On the other hand, biologically active molecules can also be introduced axially, and active molecules can be released by reduction at the lesion site to achieve combined administration and synergistic anti-tumor effects. Therefore, targeted and multi-mechanism attacks on tumor cells can overcome the drug resistance of platinum (II) drugs caused by single treatment and enhance the therapeutic effect. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a method for preparing a HO-1i-Pt(IV) complex and self-assembled nanoparticles and their application in tumor drugs.

[0005] The technical solution adopted by the present invention is: a HO-1i-Pt (IV) complex, in which HO-1i is connected to one axial side of the tetravalent platinum coordination center.

[0006] Preferably, the tetravalent platinum coordination center axis is connected to -OH or -Cl on the other side, or is connected to a straight or branched fatty chain group on the other side.

[0007] Preferably, the structural formula is as shown in Formula 1 or Formula 2;

[0008]

[0009] in, Cisplatin, oxaliplatin, carboplatin, heptaplatin, nedaplatin, levoplatin or miplatin; preferably cisplatin or oxaliplatin;

[0010] R1 is -OH or -Cl;

[0011] R2 is -C n H 2n -a straight or branched chain group, n is an integer and 1≤n≤6, preferably, n=1 or 2;

[0012] R3 is -C m H 2m+1 or -NH-C m H 2m+1 A straight chain or branched group, m is an integer and 1≤m≤20, preferably m≤11.

[0013] Preferably, it is represented by any structure in Formula 3-10:

[0014]

[0015] A method for preparing a HO-1i-Pt(IV) complex, wherein when preparing a compound of formula 1, a compound of formula 11 and a compound of formula 12 are dissolved in a first solvent, and reacted under the conditions of a first condensing agent and a first acid binding agent, and the reaction temperature is 25-100° C., preferably 25° C.;

[0016]

[0017] in, Cisplatin, oxaliplatin, carboplatin, heptaplatin, nedaplatin, levoplatin or miplatin; preferably cisplatin or oxaliplatin;

[0018] R1 is -OH or -Cl;

[0019] R2 is -C n H 2n -a straight or branched chain group, n is an integer and 1≤n≤6, preferably, n=1 or 2;

[0020] The first solvent is a mixture of one or more of acetonitrile, dichloromethane, acetone, dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF); preferably, the first solvent is DMSO;

[0021] The first condensing agent is 1-hydroxybenzotriazole (HOBT) or O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), preferably O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU);

[0022] The first acid binding agent is triethylamine (TEA);

[0023] The feed ratio of the compound of formula 11, the compound of formula 12, 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.

[0024] Preferably, when preparing the compound of formula 2, the compound of formula 1 is reacted with the compound of formula 13 or the compound of formula 14 in a second solvent at a reaction temperature of 25-100° C., preferably 60° C.;

[0025]

[0026] Where R4 is -C m H 2m+1 A straight or branched group, m is an integer and 1≤m≤20, preferably m≤11;

[0027] The second solvent is a mixture of one or more of acetonitrile, dichloromethane, acetone, dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF), preferably DMF;

[0028] The feed ratio of the compound of Formula 5 or Formula 6 to the compound of Formula 13 or Formula 14 is 1:2-10, preferably 1:2.

[0029] Preferably, the compound of formula 15 and the compound of formula 16 are dissolved in a third solvent, and reacted in the presence of a third condensing agent and a third acid binding agent to obtain a compound of formula 12, and the reaction temperature is 25-100° C., preferably 50° C.;

[0030]

[0031] Where R2 is -C n H 2n -a straight or branched chain group, n is an integer and 1≤n≤6, preferably, n=1 or 2;

[0032] The third solvent is a mixture of one or more of acetonitrile, dichloromethane, acetone, dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF), preferably dichloromethane;

[0033] The third condensing agent is 4-dimethylaminopyridine (DMAP), and the third acid binding agent is triethylamine (TEA);

[0034] The feed ratio of the compound of formula 15, the compound of formula 16, the first condensing agent and the first acid binding agent is 1:1-2:0.1-2:1-2, preferably 1:2:0.1:2.

[0035] A HO-1i-Pt(IV) self-assembled nanoparticle, a nanoparticle suspension prepared using a compound of formula 9 or 10, preferably a nanoparticle suspension prepared based on the compound of formula 10.

[0036] A method for preparing HO-1i-Pt(IV) self-assembled nanoparticles, comprising dissolving the HO-1i-Pt(IV) complex in a fourth solvent, dropping the mixture into a fifth solvent, and evaporating the fourth solvent to obtain a self-assembled nanoparticle suspension;

[0037] The fourth solvent is a mixture of one or more of acetonitrile, dichloromethane, acetone, dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF), preferably acetone;

[0038] The fifth solvent is a mixture of one or more of water, methanol and ethanol, preferably water.

[0039] Application of HO-1i-Pt(IV) complex or HO-1i-Pt(IV) self-assembled nanoparticles in the preparation of anti-tumor drugs.

[0040] The advantages and positive effects of the present invention are as follows: the prepared complex and nanoparticles have synergistic and broad-spectrum lethality to various tumors, the synthesis process is simple, the cost is low, the efficacy of combined medication is greatly improved, and compared with traditional divalent platinum and similar quadrivalent platinum drugs, the invention has the advantages of good efficacy and small side effects; the introduction of HO-1i exerts synergistic anti-tumor efficacy, activates immunity and reverses cisplatin resistance, significantly increases the efficacy and reduces the serious toxic side effects of traditional platinum drugs; after the introduction of the fat chain, on the one hand, self-assembled nanoparticles can be prepared through the hydrophilic-lipophilic interaction between the fat chain and HO-1i, and the tumor cell selectivity is increased through the EPR effect, and on the other hand, the drug stability and uptake are increased, and the effects of toxicity reduction and sustained release can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Particle size distribution of self-assembled nanoparticles i;

[0042] Figure 2 Electron microscopy characterization of self-assembled nanoparticles i;

[0043] Figure 3 Cellular uptake of compound h and nanoparticle i in HepG2 and LO2;

[0044] Figure 4 In vitro release profile of self-assembled nanoparticles i;

[0045] Figure 5 Intracellular release of 100 μM nanoparticles i in HepG2 liver cancer cells after 4 hours of action;

[0046] Figure 6 Tumor volume change curves of mice in the PBS, CDDP, Mixture (HO-1i:CDDP=1:1), compound h, and nanoparticle i administration groups;

[0047] Figure 7 Body weight change curves of mice in the PBS, CDDP, Mixture (HO-1i:CDDP=1:1), compound h, and nanoparticle i administration groups;

[0048] Figure 8 Survival curves of mice in the PBS, CDDP, Mixture (HO-1i:CDDP=1:1), compound h, and nanoparticle i administration groups;

[0049] Fig. 9 Statistics of Pt content in main organs and tumors of mice in the PBS, CDDP, Mixture (HO-1i:CDDP=1:1), compound h, and nanoparticle i administration groups detected by ICP-MS;

[0050] Fig.10Final mouse tumor weights in the PBS, CDDP, Mixture (HO-1i:CDDP=1:1), compound h, and nanoparticle i administration groups;

[0051] Fig.11 Final tumor morphology of mice in the PBS, CDDP, Mixture (HO-1i:CDDP=1:1), compound h, and nanoparticle i administration groups;

[0052] Fig.12 Flow cytometric analysis of immune-related surface markers in tumors and spleens of mice in the PBS, CDDP, Mixture (HO-1i:CDDP=1:1), compound h, and nanoparticle i administration groups. DETAILED DESCRIPTION

[0053] The present invention relates to a preparation method of a HO-1i-Pt(IV) complex and self-assembled nanoparticles and their application in tumor treatment. Heme oxygenase (HO-1) is a key enzyme in heme catabolism, which can catalyze the degradation of heme and release biliverdin, CO and Fe 2+ , is also a widely existing antioxidant defense enzyme and endogenous anti-apoptotic protein, and is closely related to many clinical diseases (such as tumors, atherosclerosis, hypertension, etc.). Studies have shown that heme oxygenase inhibitors (HO-1i) can play an anti-tumor role through multiple pathways, such as promoting tumor cell apoptosis, inhibiting tumor metastasis and proliferation, activating immune cells, and inducing macrophage M2 depolarization. HO-1i is combined with a variety of classic chemotherapy drugs including cisplatin to enhance drug sensitivity and synergistically exert a strong anti-tumor effect. At the same time, HO-1i regulates the expression of multiple resistance-related proteins and reverses cisplatin resistance through multiple pathways, such as reducing drug efflux and reducing tumor self-repair ability. HO-1i is combined with platinum drugs to synthesize a new HO-1i-Pt(IV) complex.

[0054] One molecule of HO-1i is connected to the axial side of the platinum (IV) coordination center, and the other side is -OH or -Cl to form a monosubstituted tetravalent platinum complex, the structure of which is shown in Formula 1; or one molecule of HO-1i is connected to the axial side of the platinum (IV) coordination center, and the other side is connected to a molecule of a straight or branched fatty chain group to form a disubstituted tetravalent platinum complex, the structure of which is shown in Formula 2.

[0055]

[0056] in, Cisplatin, oxaliplatin, carboplatin, heptaplatin, nedaplatin, levoplatin or miplatin; preferably cisplatin or oxaliplatin;

[0057] R1 is -OH or -Cl; R2 and R3 are the same or different atoms, alkyl, alkene, alkyne, aryl and heterocycle or any combination of the above groups; R2 is -C n H 2n -a straight chain or branched chain group, n is an integer and 1≤n≤6, preferably, n=1 or 2; R3 is -C m H 2m+1 or -NH-C m H 2m+1 A straight chain or branched group, m is an integer and 1≤m≤20, preferably m≤11.

[0058] In certain embodiments of the present invention, the HO-1i-Pt(IV) complex may be specifically represented by any structure in Formula 3-10:

[0059]

[0060]

[0061] When preparing the above-mentioned HO-1i-Pt(IV) complex, a carboxyl group is introduced into the hydroxyl position of HO-1i through an esterification reaction, and the modified HO-1i is introduced into hydroxyplatinum or chloroplatinum through a simple ester condensation reaction to obtain a single molecule or HO-1i-substituted platinum(IV) complex; when it is necessary to prepare a platinum(IV) complex containing both HO-1i and a linear or branched fatty chain, a linear or branched fatty chain is introduced into the opposite side of a molecule of HO-1i-substituted platinum(IV) complex through an addition reaction or an ester condensation reaction to obtain a platinum(IV) complex double-substituted by HO-1i and a fatty chain; the specific preparation method is as follows:

[0062] Step 1: dissolving the compound of formula 15 and the compound of formula 16 in a third solvent, and subjecting them to addition reaction under the condition of a second acid binding agent to obtain a compound of formula 12;

[0063]

[0064]

[0065] Where R2 is -C n H 2n-straight or branched chain group, n is an integer and 1≤n≤6, preferably, n=1 or 2; the third solvent is a mixture of one or more of acetonitrile, dichloromethane, acetone, dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF), preferably dichloromethane; the third condensing agent is 4-dimethylaminopyridine (DMAP), and the third acid binding agent is triethylamine (TEA); the feed ratio of the compound of formula 15 and the compound of formula 16 to the first condensing agent and the first acid binding agent is 1:1-2:0.1-2:1-2, preferably 1:2:0.1:2; the reaction temperature is 25-100°C, preferably 50°C. The reaction is carried out under light-proof, anhydrous, and inert gas protection conditions.

[0066] Step 2: dissolving the compound of formula 11 and the compound of formula 12 obtained in step 1 in a first solvent, and subjecting them to an esterification reaction in the presence of a first condensing agent and a first acid binding agent to obtain a compound of formula 1;

[0067]

[0068] Among them, in formula 11, is cisplatin, oxaliplatin, carboplatin, heptaplatin, nedaplatin, levoplatin or miplatin; preferably cisplatin or oxaliplatin; R1 is -OH or -Cl; when preparing the compound of formula 1, the first solvent is a mixture of one or more of acetonitrile, dichloromethane, acetone, dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF); preferably, the first solvent is DMSO; the first condensing agent is 1-hydroxybenzotriazole (HOBT) or O-benzotriazole-N,N, N',N'-tetramethyluronium tetrafluoroborate (TBTU), preferably O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU); the first acid binding agent is triethylamine (TEA); the reaction temperature is 25-100°C, preferably 25°C; the feed ratio of the compound of formula 11, the compound of formula 12, 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.

[0069] Step 3: reacting the compound of formula 5 or formula 6 with the compound of formula 13 or formula 14 in a second solvent to obtain a compound of formula 2;

[0070]

[0071] Where R4 is -C m H 2m+1The straight chain or branched chain group, m is an integer and 1≤m≤20, preferably m≤11; the second solvent is a mixture of one or more of acetonitrile, dichloromethane, acetone, dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF), preferably DMF; the reaction temperature is 25-100°C, preferably 60°C; the feed ratio of the compound of formula 5 or formula 6 to the compound of formula 13 or formula 14 is 1:2-10, preferably 1:2. The reaction is carried out under light-proof, anhydrous and inert gas protection conditions.

[0072] HO-1i is combined with platinum drugs to synthesize a new HO-1i-Pt(IV) complex. HO-1i can exert anti-tumor effects through multiple pathways, such as promoting tumor cell apoptosis, inhibiting tumor metastasis and proliferation, activating immune cells, and inducing macrophage M2 depolarization. In particular, HO-1i is combined with a variety of classic chemotherapy drugs including cisplatin to enhance drug sensitivity and synergistically exert a strong anti-tumor effect. At the same time, HO-1i regulates the expression of multiple resistance-related proteins, and reverses cisplatin resistance and chemotherapy drug resistance through multiple pathways such as reducing drug efflux and reducing tumor self-repair ability. Through the above pathways, the HO-1i-Pt(IV) complex greatly improves the anti-tumor efficacy of platinum drugs, and the anti-proliferation IC 50 The value is dozens of times better than that of the parent divalent platinum.

[0073] Furthermore, a lipophilic fatty chain is introduced through structural modification on the basis of the synthesized HO-1i-Pt(IV) complex, as shown in the structure of Formula 2, and self-assembled nanoparticles are synthesized under hydrophilic-lipophilic interactions. The preparation method of the self-assembled carrier-free nano drug delivery system is simple and flexible, with high drug loading capacity and delivery efficiency, long blood circulation half-life, and avoidance of carrier-related toxicity and immunogenic side effects. The synthesized self-assembled nanoparticles increase the stability of the drug and reduce pre-target inactivation on the one hand, and promote the selective distribution of the drug in tumor tissue through the EPR effect to further enhance the anti-tumor effect and reduce the toxicity of normal tissues and organs.

[0074] In certain embodiments of the present invention, the compound of formula 9 or formula 10 is dissolved in a fourth solvent, slowly added dropwise to a fifth solvent, and the fourth solvent is evaporated to obtain a self-assembled nanoparticle suspension. The fourth solvent is a mixture of one or more of acetonitrile, dichloromethane, acetone, dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF), preferably acetone; the fifth solvent is a mixture of one or more of water, methanol, and ethanol, preferably water.

[0075] The HO-1i-Pt(IV) complex and self-assembled nanoparticles synthesized by the above method, the group containing HO-1i can enhance chemotherapy sensitivity and improve the anti-tumor activity of platinum drugs through multifunctional synergistic effects; HO-1i activates immune cells, induces macrophage depolarization, etc., enhances the killing of immune cells to tumor cells and reduces the systemic toxicity caused by platinum drugs; the HO-1i-Pt(IV) complex has a different mode of action from the classic divalent platinum drugs, and HO-1i can regulate the expression of multiple resistance-related proteins, and reverse cisplatin resistance through multiple pathways such as reducing drug efflux and reducing tumor self-repair ability; the HO-1i-Pt(IV) complex is prepared into nanoparticles, and the self-assembled nanoparticles, based on the above advantages, on the one hand, increase the stability of the drug and reduce pre-target inactivation, and on the other hand, promote the selective distribution of the drug in tumor tissue through the EPR effect, further enhance the anti-tumor effect and reduce the toxicity of normal tissues and organs.

[0076] The scheme of the present invention is described below in conjunction with the accompanying drawings, wherein the experimental methods without specific operating steps are all carried out in accordance with the corresponding product instructions, and the instruments, reagents, and consumables used in the examples can all be purchased from commercial companies unless otherwise specified.

[0077] Embodiment 1:

[0078] The structural formula of the HO-1i-Pt(IV) complex a in this embodiment is as follows:

[0079]

[0080] The preparation method and synthesis route of the HO-1i-Pt(IV) complex of this embodiment are as follows:

[0081]

[0082] 1. Preparation of compound a1 chloroplatinum

[0083] Cisplatin and NCS (N-chlorosuccinimide) were oxidized in water for 3 hours. After oxidation, the solvent was removed by rotary evaporator and then recrystallized with ethanol and ether to obtain pale white precipitate Olochloroplatin, a1.

[0084] 2. Preparation of compound a2

[0085] HO-1i and succinic anhydride were dissolved in anhydrous dichloromethane (DCM). Argon was used as protection during the reaction. The reaction temperature was 50°C and refluxed for 10 hours. The reaction solution was concentrated into an oil and separated by a silica gel chromatography column. The eluents were dichloromethane and methanol to obtain a white precipitate a2.

[0086] pass 1H NMR, 13 The obtained compound was characterized by C NMR and HRMS (ESI), and the following data were obtained:

[0087] 1 H NMR (400MHz, DMSO-d6) δ12.33(s,1H),7.60–7.46(m,3H),7.32(d,J=5.5Hz,2H),7.11(s, 1H),6.84(s,1H),5.97(t,J=5.6Hz,1H),4.37(d,J=5.7Hz,2H),2.57(q,2H),2.46(q,2H). 13 C NMR (101MHz, DMSO-d6) δ172.62,171.10,139.85,137.81,131.07,130.57,129. 05,127.21,125.17,121.66,119.99,73.59,50.14,28.80,28.54.HRMS(ESI)m / z calcdfor C 15 H 16 BrN2O4 + (M+H) + 367.0295, found 367.0285.

[0088] 3. Preparation of Compound A

[0089] Compound a2 was mixed with dry dimethyl sulfoxide (DMSO), O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU) was added, triethylamine (TEA) was added after activation for a period of time, compound a1 (Olochloroplatin) was added after activation for a period of time, the mixture was reacted at 60°C for 24h, chloroform was added to precipitate, and after drying, it was further purified by silica gel chromatography with dichloromethane and methanol as eluents to obtain a white precipitate as compound a.

[0090] pass 1 H NMR, 13 The obtained compound was characterized by C NMR and HRMS (ESI), and the following data were obtained:

[0091] 1 H NMR (400MHz, DMSO-d6) δ7.63–7.27(m,5H),7.13(s,1H),6.85(s,1H),6.47–5.85(m,6H),4.39(s,2H),2.54(m,4H). 13C NMR (101MHz, DMSO-d6) δ178.85,171.10,139.94,137.89,131.04,130.64,129. 09,128.08,125.24,121.66,120.01,73.50,50.14,30.86,29.96.HRMS(ESI)m / z calcd for C 15 H 21 BrCl3N4O4Pt + (M+H) + 699.9461, found 699.9455.

[0092] Example 2

[0093] The structural formula of the HO-1i-Pt(IV) complex b in this embodiment is as follows:

[0094]

[0095] The preparation method and synthesis route of the HO-1i-Pt(IV) complex of this embodiment are as follows:

[0096]

[0097] 1. Preparation of Compound b1 Cisplatin Oxide

[0098] The preparation method of the cisplatin oxide of compound b1 is the same as the preparation method of the cisplatin oxide of formula a1 in Example 1.

[0099] 2. Preparation of compound b2

[0100] HO-1i and glutaric anhydride were dissolved in anhydrous dichloromethane (DCM). Argon was used as protection during the reaction. The reaction temperature was 50°C and refluxed for 10 hours. The reaction solution was concentrated into an oil and separated by a silica gel chromatography column. The eluents were dichloromethane and methanol to obtain a white precipitate b2.

[0101] pass 1 H NMR, 13 The obtained compound was characterized by C NMR and the following data were obtained:

[0102] 1H NMR(400MHz,DMSO-d6)δ7.59(s,1H),7.57–7.50(m,2H),7.34(m,2H),7.15(s,1H),6.88(s,1H),6.00 (t,J=5.9Hz,1H),4.49–4.33(m,2H),2.40(t,J=7.1Hz,2H),2.19(t,J=7.2Hz,2H),1.78–1.61(m,2H). 13 C NMR (101MHz, DMSO-d6) δ171.44,139.93,137.82,131.17,130.65,129.13,127. 94,125.22,121.72,120.00,73.34,50.14,32.58,21.05,19.75.HRMS(ESI)m / z calcd for C 16 H 18 BrN2O4 + (M+H) + 381.0452, found 381.0440.

[0103] 3. Preparation method of compound B

[0104] Compound b2 was mixed with dry dimethyl sulfoxide (DMSO), O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU) was added, triethylamine (TEA) was added after activation for a period of time, compound b1 (Olochloroplatin) was added after activation for a period of time, the mixture was reacted at 60°C for 24h, chloroform was added to precipitate, and after drying, it was further purified by silica gel chromatography with dichloromethane and methanol as eluents to obtain a white precipitate as compound b.

[0105] pass 1 H NMR, 13 The obtained compound was characterized by C NMR and HRMS (ESI), and the following data were obtained:

[0106] 1 H NMR(400MHz,DMSO-d6)δ7.73–7.46(m,3H),7.36(q,2H),7.18(s,1H),6.89(s,1H), 6.58–5.80(m,7H),4.58–4.20(m,2H),2.39(m,2H),2.11(m,2H),1.74–1.56(m,2H). 13CNMR(101MHz,DMSO-d6)δ179.47,171.72,140.02,137.88,131.16,130.74,129 .16,125.26,121.70,119.78,73.18,50.14,32.74,20.68,15.14.HRMS(ESI)m / z calcd forC 16 H 23 BrCl3N4O4Pt + (M+H) + 713.9618, found 713.9608.

[0107] Example 3

[0108] The structural formula of the HO-1i-Pt(IV) complex c in this embodiment is as follows:

[0109]

[0110] The preparation method and synthesis route of the HO-1i-Pt(IV) complex of this embodiment are as follows:

[0111]

[0112] 1. Preparation of compound c1 cisplatin oxide

[0113] Cisplatin was oxidized in hydrogen peroxide for 6 hours, refrigerated at 0-4°C overnight, and then recrystallized with water, ice ethanol, and ether to obtain a light yellow precipitate Oxoplatin (c,c,t-[Pt(NH3)2Cl2(OH)2]), c1.

[0114] 2. Preparation of compound c2

[0115] The preparation method of compound c2 is the same as the preparation method of compound a2 in Example 1.

[0116] 3. Preparation of Compound C

[0117] Compound c2 was mixed with dry dimethyl sulfoxide (DMSO), O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU) was added, triethylamine (TEA) was added after activation for a period of time, compound c1 (Oxoplatin) was added after activation for a period of time, the reaction was carried out at room temperature for 24 hours, chloroform was added to precipitate, and after drying, it was further purified by silica gel chromatography column, the eluents were dichloromethane and methanol, and a white precipitate was obtained as compound c.

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

[0119] 1 H NMR (400MHz, DMSO-d6) δ7.58 (s, 1H), 7.55–7.49 (m, 2H), 7.34 (d, J = 6.7Hz, 2H), 7. 13(s,1H),6.85(s,1H),6.34–5.72(m,7H),4.51–4.18(m,2H),2.52–2.32(m,4H). 13 C NMR(101MHz,DMSO-d6)δ179.24,171.30,140.01,137.88,131.01,130.63,129. 09,128.05,125.24,121.64,120.02,73.44,50.15,31.01,30.15.HRMS(ESI)m / z calcd forC 15 H 22 BrCl2N4O5Pt + (M+H) + 681.9800, found 681.9791. Example 4

[0120] The structural formula of the HO-1i-Pt(IV) complex d in this embodiment is as follows:

[0121]

[0122] The preparation method and synthesis route of the HO-1i-Pt(IV) complex of this embodiment are as follows:

[0123]

[0124] 1. Preparation of Compound D1 Cisplatin Oxide

[0125] The preparation method of compound d1 is the same as the preparation method of compound c1 in Example 3.

[0126] 2. Preparation of compound d2

[0127] The preparation method of compound d2 is the same as the preparation method of compound b2 in Example 2.

[0128] 3. Preparation method of compound d

[0129] Compound d2 was mixed with dry dimethyl sulfoxide (DMSO), O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU) was added, triethylamine (TEA) was added after activation for a period of time, compound d1 (Oxoplatin) was added after activation for a period of time, the mixture was reacted at room temperature for 24 hours, chloroform was added to precipitate, and after drying, it was further purified by silica gel chromatography with dichloromethane and methanol as eluents to obtain a white precipitate as compound d.

[0130] pass 1 H NMR, 13 The obtained compound was characterized by C NMR and HRMS (ESI), and the following data were obtained:

[0131] 1 H NMR(400MHz,DMSO-d6)δ7.78(s,1H),7.60(s,1H),7.58–7.50(m,1H),7.36(d,2H),7.25(s ,1H),7.00(s,1H),6.41–5.59(m,7H),4.43(m,2H),2.39(m,2H),2.08(m,2H),1.63(m,2H). 13 C NMR(101MHz,DMSO-d6)δ179.96,171.79,140.03,137.85,131.15,130.74,129.14,128 .21,125.24,121.70,119.77,73.17,50.16,34.92,32.85,20.85.HRMS(ESI)m / zcalcd for C 16 H 24 BrCl2N4O5Pt + (M+H) + 695.9957, found 695.9946.

[0132] Example 5

[0133] The structural formula of the HO-1i-Pt(IV) complex e in this embodiment is as follows:

[0134]

[0135] The preparation method and synthesis route of the HO-1i-Pt(IV) complex of this embodiment are as follows:

[0136]

[0137]

[0138] 1. Preparation of compound e1

[0139] The preparation method of compound e1 is the same as the preparation method of formula c in Example 3.

[0140] 2. Preparation of compound e2

[0141] Valproic acid was mixed with 1-ethyl-3(3-dimethylpropylamine)carbodiimide (EDCI), dissolved in DCM, reacted at room temperature for 24 h, washed with 3% citric acid, 3% NaHCO3 and 3% NaCl solutions respectively until neutral, and then dried with anhydrous Na2SO4 overnight. DCM was removed by rotary evaporator to obtain a white precipitate as compound e2.

[0142] 2. Preparation method of compound e

[0143] Mix e1 and e2, dissolve in DMF, react at 60°C for 24h, remove DMF with a rotary evaporator, add chloroform to precipitate, dry and further purify by silica gel chromatography with dichloromethane and methanol as eluents to obtain a white precipitate as compound e.

[0144] pass 1 H NMR, 13 The obtained compound was characterized by C NMR and HRMS (ESI), and the following data were obtained:

[0145] 1 H NMR(400MHz,DMSO-d6)δ7.70–7.43(m,4H),7.34(d,2H),7.12(d,1H),6.96–6.21(m,6H),5. 95(t,1H),4.40(t,2H),2.55(s,1H),2.25(m,4H),1.58–1.01(m,8H),0.83(t,J=6.9Hz,6H). 13 C NMR(101MHz,DMSO-d6)δ183.96,179.11,171.04,139.93,137.89,131.05,130.65,129.11,128.07 ,125.27,121.65,120.05,73.51,50.17,46.36,34.70,33.77,29.84,20.00,14.15.HRMS(ESI)m / z calcd for C 23 H 36 BrCl2N4O6Pt + (M+H) + 808.0845, found808.0832.

[0146] Example 6

[0147] The structural formula of the HO-1i-Pt(IV) complex f in this embodiment is as follows:

[0148]

[0149] The preparation method and synthesis route of the HO-1i-Pt(IV) complex of this embodiment are as follows:

[0150]

[0151] 1. Preparation of compound f1

[0152] The preparation method of compound f1 is the same as the preparation method of formula d in Example 4.

[0153] 2. Preparation of compound f2

[0154] The preparation method of compound f2 is the same as the preparation method of formula e2 in Example 5

[0155] 2. Preparation method of compound f

[0156] Mix f1 and f2, dissolve in DMF, react at 60°C for 24h, remove DMF with a rotary evaporator, add chloroform to precipitate, dry and further purify by silica gel chromatography with dichloromethane and methanol as eluents to obtain a white precipitate as compound f.

[0157] pass 1 H NMR, 13 The obtained compound was characterized by C NMR and HRMS (ESI), and the following data were obtained:

[0158] 1 H NMR (400MHz, DMSO-d6) δ7.73–7.50(m,4H),7.43–7.30(m,2H),7.16(d,J=14.5Hz,1H),6.78(m,6 H),5.99(m,1H),4.38(dd,2H),2.54(s,1H),2.48–1.93(m,6H),1.44–1.14(m,8H),0.83(t,6H). 13 C NMR(101MHz,DMSO-d6)δ184.08,179.71,171.68,140.02,137.87,131.16,130.74,129.17,128.27,125.2 7,121.71,119.70,73.19,50.14,46.46,40.11,34.70,34.08,32.70,20.63,20.01,14.16.HRMS(ESI)m / z calcd forC24 H 38 BrCl2N4O6Pt + (M+H) + 822.1001, found 822.0989. Example 7

[0159] The structural formula of the HO-1i-Pt(IV) complex g in this embodiment is as follows:

[0160]

[0161] The preparation method and synthesis route of the HO-1i-Pt(IV) complex of this embodiment are as follows:

[0162]

[0163] 1. Preparation of compound g1

[0164] The preparation method of compound g1 is the same as the preparation method of formula c in Example 3.

[0165] 2. Preparation method of compound g

[0166] Mix g1 with dry N,N-dimethylformamide (DMF), add dodecyl isocyanate, react at 60°C for 24 hours, remove DMF with a rotary evaporator, add chloroform to precipitate, and after drying, further purify by silica gel chromatography with dichloromethane and methanol as eluents to obtain a white precipitate as compound g.

[0167] pass 1 H NMR, 13 The obtained compound was characterized by C NMR and HRMS (ESI), and the following data were obtained:

[0168] 1 H NMR(400MHz,DMSO-d6)δ7.70–7.46(m,3H),7.34(d,2H),7.17(s,1H),6.91(s,1H),6.5 9(m,7H),5.95(s,1H),4.41(s,2H),2.54(s,4H),1.24(s,20H),0.85(t,J=6.4Hz,3H). 13C NMR(101MHz,DMSO-d6)δ179.09,171.04,163.84,139.87,137.75,131.07,130.66,129.10,127.47,125.26,121.66,12 0.22,73.44,50.30,31.27,29.86,29.82,29.05,29.02,29.00,28.88,28.69,26.44,22.07,13.93.HRMS(ESI)m / zcalcd for C 28 H 47 BrCl2N5O6Pt + (M+H) + 893.1736, found 893.1727.

[0169] Example 8

[0170] The structural formula of the HO-1i-Pt(IV) complex h in this embodiment is as follows:

[0171]

[0172] The preparation method and synthesis route of the HO-1i-Pt(IV) complex of this embodiment are as follows:

[0173]

[0174] 1. Preparation of compound h1

[0175] The preparation method of compound h1 is the same as the preparation method of formula d in Example 4.

[0176] 2. Preparation of Compound H

[0177] Mix h1 with dry N,N-dimethylformamide (DMF), add dodecyl isocyanate, react at 60°C for 24 hours, remove DMF with a rotary evaporator, add chloroform to precipitate, and after drying, further purify by silica gel chromatography with dichloromethane and methanol as eluents to obtain a white precipitate as compound h.

[0178] pass 1 H NMR, 13 The obtained compound was characterized by C NMR and HRMS (ESI), and the following data were obtained:

[0179] 1H NMR(400MHz,DMSO-d6)δ7.64–7.50(m,3H),7.36(d,2H),7.17(s,1H),6.97–6.44(m,8H),5.99(dd,1H),4.51– 4.24(m,2H),2.47–2.28(m,2H),2.11(m,2H),1.63(m,2H),1.34(s,2H),1.24(s,18H),0.85(t,J=6.8Hz,3H). 13 C NMR(101MHz,DMSO-d6)δ179.73,171.67,163.98,140.01,137.46,131.15,130.73,129.16,128.25,125.26,121.70,119.72 ,73.18,50.16,34.08,32.69,31.28,29.83,29.06,29.03,29.01,28.89,28.70,26.45,22.07,20.65,13.94.HRMS(ESI)m / z calcd for C 29 H 49 BrCl2N5O6Pt + (M+H) + 907.1893, found 907.1886.

[0180] Example 9

[0181] This embodiment is a method for preparing HO-1i-Pt(IV) self-assembled nanoparticles:

[0182] Compound h was dissolved in acetone, and an appropriate amount was slowly added dropwise into water. The acetone was removed by a rotary evaporator to obtain a nanoparticle suspension i.

[0183] The nanoparticle size analyzer and transmission electron microscope were used to characterize the nanoparticles. Figure 1 and Figure 2 , it can be seen that the particle size of the self-assembled nanoparticles in the nanoparticle suspension is uniform.

[0184] Example 10: In vitro anti-tumor activity assay

[0185] In order to better understand the essence of the present invention, the in vitro antitumor activity of the eight HO-1i-Pt(IV) complexes and one HO-1i-Pt(IV) self-assembled nanoparticle synthesized in Examples 1-9, compounds a–i, and their parent controls were determined.

[0186] In this experiment, the anti-tumor activity of compounds a-i, a total of 9 synthetic compounds, and the ligand HO-1i, cisplatin, and the combination of HO-1i and cisplatin (1:1) was studied by the MTT (3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide) method. In this experiment, 5 cancer cells HepG2 (human liver cancer cells), 9464D (mouse neuroblastoma cells), (human non-small cell lung cancer cisplatin-resistant cells), HCT-116 (human colorectal cancer cells), BGC-823 (human gastric cancer cells), A549 (human non-small cell lung cancer cells), A549cisR (human non-small cell lung cancer cisplatin-resistant cells) and a normal human cell LO2 (human normal liver cell) were used for cytotoxicity experiments. All cell lines were cultured in an incubator with saturated humidity at 37°C under a concentration of 5% CO2. The experimental steps are as follows:

[0187] Collect cells in the logarithmic growth phase, count them, and adjust the cell concentration to 3×10 4 cells / mL were inoculated into 96-well plates, 100μL per well, and blank group (Blank, pure culture medium) and control group (Control, no drug addition group) were set up. After the cell culture plate was cultured overnight in a saturated humidity incubator at 37°C and adhered to the wall, the compound was diluted with culture medium to the required concentration of the first row of dosing wells, and then a total of 18 concentration gradients were administered by half-dilution method, and the mixture was gently blown and mixed. After incubation for 72h, 10μL (5mg / mL) of MTT solution was added to each well. After another 4h of incubation in the incubator, the supernatant was removed, 100μL / well DMSO was added, and the OD value was measured with an enzyme marker at λ=570nm after sufficient shaking. Three independent repeated experiments were performed to ensure the reliability of the experimental results.

[0188] Table 1 IC of compounds after 72 hours of cell exposure 50 Value (μM)

[0189]

[0190] a IC 50 (CDDP) / IC 50 (Compound d). b IC 50 (CDDP) / IC 50 (Compound h). c IC 50 (CDDP) / IC 50 (Nanoparticle i). d IC 50 (LO2) / IC 50 (HepG2). e IC 50 (A549cisR) / IC50 (A549).

[0191] As shown in Table 1, the experimental results show that the eight complexes and one self-assembled nanoparticle in the HO-1i-Pt(IV) system have certain anti-tumor activity against the tested tumor cells. Among the HO-1i-substituted HO-1i-Pt(IV) complexes, the compound d substituted with glutaric anhydride as the linker showed a better anti-proliferative effect on tumor cells than cisplatin or the combination of HO-1i and cisplatin. In particular, the IC of compound d in the HepG2 cell line was 50 The selectivity index of compound d for tumor cells HepG2 and normal cell line LO2 reached 12.67, while the selectivity index of cisplatin was only 0.48, which proved that candidate compound d reduced the serious toxic side effects of classic divalent platinum while improving the anti-tumor efficacy.

[0192] In the HO-1i-Pt(IV) complexes with further introduction of linear or branched aliphatic chains, compounds e–h all showed antiproliferative effects on tumor cells that were superior to cisplatin or the combination of HO-1i and cisplatin. In particular, compound h had an IC 50 The selectivity index of compound h for tumor cells HepG2 and normal cell line LO2 was 3.85, which was better than cisplatin. This may be because the introduction of a fat chain of appropriate length can not only improve the antitumor activity of HO-1i-Pt(IV) complex by regulating lipid solubility and promoting drug uptake, but also the fat chain can stabilize the drug, reduce drug pre-target inactivation, enhance the efficacy and reduce the toxicity of platinum drugs.

[0193] Subsequently, the selected compound h with the best anti-tumor activity was prepared into self-assembled nanoparticles i. The IC 50 The value of the compound h was 0.07±0.01, and its antitumor activity was slightly better than that of the free compound h. This may be because the compound self-assembled into nanoparticles and was taken into cells by endocytosis, and its cellular uptake was improved compared with the free compound that entered the cells by passive diffusion, thereby increasing the efficacy (see Cellular uptake Figure 3). More noteworthy is that the selectivity index of nanoparticle compound i for tumor cells HepG2 and normal cell line LO2 is 33.14, which is 8.61 times higher than that of free drugs, proving that HO-1i-Pt(IV) self-assembled nanoparticles significantly reduce the toxicity of platinum drugs. This may be due to the fact that tumor cells have specific acidic and excessive reducing substances (glutathione, etc.) characteristics compared with normal cells. Self-assembled nanoparticles can only depolymerize and release cytotoxic platinum drugs in the tumor environment, and will not be reduced in normal cells, which significantly reduces the toxicity of nanoparticle i (see Nano Release). Figure 4 ).

[0194] In addition to serious toxic side effects, the development of platinum drug resistance is also one of the reasons that limit their clinical application. The common A549cisR (cisplatin-resistant human non-small cell lung cancer cells) was selected to evaluate the effect of HO-1i-Pt(IV) complexes and self-assembled nanoparticles in overcoming drug resistance. The drug resistance index of the preferred compound d, compound h and self-assembled nanoparticle i for A549 / cisR was 1.23, 1.39 and 1.41, respectively. The IC values ​​of the above three compounds in drug-resistant cells were 50 There was no significant difference between the two groups and the non-resistant cells, which proved that the HO-1i-Pt(IV) system significantly overcame cisplatin resistance because of its different cell behavior from cisplatin-like divalent platinum drugs.

[0195] Example 11: Intracellular reduction experiment

[0196] It is well known that tetravalent platinum can only exert its cytotoxic effect when it is reduced to its parent divalent platinum in vivo. Therefore, exploring the intracellular release ability of prodrugs is crucial for studying the mechanism of action of tetravalent platinum. In order to explore whether Wu HO-1i-Pt(IV) self-assembled nanoparticles can be reduced by reducing substances (glutathione, ascorbic acid, etc.) in cells, release divalent platinum and achieve drug sustained release, an intracellular reduction experiment was conducted. The reduction behavior of nanoparticles i in HepG2 cells was detected, and the specific experimental steps are as follows:

[0197] 1×10 6HepG2 cells were seeded in a 6-well plate. After the cells adhered to the wall, 100 μM nanoparticles i acted on the cells and continued to culture for 4 hours. The culture medium was discarded, the cells were washed three times with PBS, and the PBS was removed by centrifugation. The cells were resuspended with a certain amount of methanol and dichloromethane, transferred to a grinder and ground for ten minutes until the cells were completely lysed. After grinding, they were allowed to stand for a period of time, and the supernatant was collected by centrifugation. The solvent was evaporated at room temperature, and then the solid was resuspended with 200 μL of chromatographic methanol. The liquid phase was detected. The liquid phase analysis conditions were: ultraviolet (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 analyzer, gradient elution, methanol 5%-95% (0-10 min), 95% methanol 25 min, flow rate 1 mL / min.

[0198] HPLC test results are as follows Figure 5 As shown in the figure, the strips from bottom to top represent the intracellular extraction sample of nanoparticle i, HO-1i standard, HO-1i ligand intermediate b2 standard and nanoparticle i standard. Since the ultraviolet absorption of cisplatin is weak, the detection of HO-1i ligand is used to characterize the reduction of nanoparticle i. Three main peaks can be observed from the intracellular extraction sample strip of compound i, corresponding to HO-1i, b2 and nanoparticle i from front to back, proving that nanoparticle i can release platinum drugs in vivo. Due to the similarity of the bonding mode of compounds a-h, we can infer that compounds a-h can be detected as original drugs or reduced release products in HPLC under the same conditions, and they also have a sustained release effect.

[0199] Example 12: Study on the anti-tumor activity of drugs in vivo

[0200] In order to explore the anti-tumor effect of the HO-1i-Pt(IV) complex and self-assembled nanoparticles of the present invention, we conducted a study on the anti-tumor activity of the drug in vivo, and the specific steps are as follows:

[0201] First, a Hepa 1-6 tumor model was established using 4-5 week old C57BL / 6 mice. When the tumor volume reached 50-100 cm 3Afterwards, the mice were randomly divided into 6 groups, namely PBS, CDDP (2.5 mg / kg Pt), HO-1i and CDDP in equal amounts (2.5 mg / kg Pt), compound h (2.5 mg / kg Pt), low-dose nanoparticle i (2.5 mg / kg Pt) and high-dose nanoparticle i (5 mg / kg Pt). The drug was given once every 3 days, and the weight and tumor volume of the mice were measured every two days, for a total of 6 doses. Three days after the last dose, the mice were killed, and the organs (heart, liver, spleen, lung, kidney) and tumors were extracted. The organs and tumors were used for ICP-MS platinum content determination. Tumor and spleen lymphocytes were separated and CD3 + T cell content and CD4 + and CD8 + The proportion of T cells was analyzed.

[0202] The experimental results are as follows Figure 6 First, the final tumor inhibition rates of CDDP (2.5 mg / kg Pt), HO-1i and CDDP (2.5 mg / kg Pt), compound h (2.5 mg / kg Pt), low-dose nanoparticle i (2.5 mg / kg Pt) and high-dose nanoparticle i (5 mg / kg Pt) were 43.21%, 50.85%, 58.23%, 65.88%, and 80.35%, respectively. Figure 6 Final tumor weight Fig.10 and eventually tumor Fig.11 All of them proved that the tumor inhibition effect of free drug compound h and self-assembled nanoparticle compound i was significantly better than that of cisplatin and HO-1i combined with cisplatin in equal amounts. Among them, similar to the results of in vitro activity screening, the tumor inhibition effect of nanoparticles was better than that of free drugs, and the tumor inhibition effect of nanoparticles was concentration-dependent. The high-dose nanoparticle administration group showed excellent in vivo antitumor activity. The above results jointly proved that HO-1i-Pt(IV) complexes and self-assembled nanoparticles have excellent in vivo antitumor activity.

[0203] At the same time, the weight changes of mice Figure 7 and survival rate curve Figure 8It showed that under the condition of equal dose, the body weight of the mice in the nanoparticle i group remained basically stable, with no significant difference in body weight compared with the blank group, and the final survival rate was 100%; the body weight of the free drug compound h decreased slightly, and the final survival rate was 100%; the body weight of mice in the cisplatin and HO-1i and cisplatin equal dose combination groups continued to decrease, and the final survival rates were 50% and 66.7%, respectively. However, the body weight of the high-dose group continued to decrease in the early stage of treatment, and the body weight recovered steadily after the fourth dose. The final body weight was even higher than that of the two positive control groups with low doses, and the final survival rate was also 100%. The above results collectively prove that the HO-1i-Pt(IV) complex and self-assembled nanoparticles have significant attenuation effects. Furthermore, the ICP-MS results Fig. 9 It can also be observed that the platinum accumulation of the free drug compound h and the self-assembled nanoparticles i in the kidney is much lower than that of cisplatin, and the damage to the kidney is significantly reduced, which further proves that the compound HO-1i-Pt(IV) complex and the self-assembled nanoparticles significantly reduced the renal damage caused by cisplatin and achieved a detoxification effect.

[0204] The attenuation effect of HO-1i-Pt(IV) complexes and self-assembled nanoparticles is related to the immune activation effect of the introduced HO-1i ligand. Fig.12 The results of flow cytometry analysis of immune-related markers in tumors and spleens after all treatment cycles were completed were shown. First, CD3 is one of the best markers for mature T cells. + T cells represent the sum of all T cells. The compound h and nanoparticle i administration groups showed CD3 + The expression of T cells increased, and the CD3 + T increased in a dose-dependent manner, proving that both the HO-1i-Pt(IV) complex and the self-assembled nanoparticles increased the expression of T cells in vivo, playing an immune activation role, which may be one of the causes of their attenuation effect. + CD4 in T cells + T and CD8 + The results showed that after the action of compound h and nanoparticle i, the CD8 + The proportion of T cells increased, and this increase was also dose-dependent. + T cells, as cytotoxic T lymphocytes, are the main components of T cells attacking tumor cells. + The increase in T ratio demonstrated that HO-1i-Pt(IV) complexes and self-assembled nanoparticles activated T cell-related tumor immune killing effects.

[0205] The HO-1i-Pt(IV) complex and the self-assembled nanoparticles of the present invention have structural correlations. The in vitro anti-tumor test of the eight compounds and one self-assembled nanoparticle in Example 10 showed that they had significant anti-proliferative effects on five tumor cell lines and one drug-resistant cell line, and their effects were improved by tens to hundreds of times compared with the traditional anti-tumor drug cisplatin. The HO-1i-Pt(IV) complex and the self-assembled nanoparticles of the present invention are a new type of highly effective anti-tumor drug candidate, with a broad spectrum of anti-cancer effects, good efficacy, and few side effects, and the preparation method is simple, low cost, and easy to industrialize.

[0206] The embodiments of the present invention are described in detail above, but the contents described are only preferred embodiments of the present invention and cannot be considered to limit the scope of implementation of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A HO-1i-Pt(IV) complex, characterized in that: The structural formula is shown in Formula 1 or Formula 2; Wherein, R1 is -OH or -Cl; R2 is -C n H 2n -a straight or branched chain group, n is an integer and 1≤n≤6; R3 is -C m H 2m+1 or -NH-C m H 2m+1 A straight chain or branched group, m is an integer and 1≤m≤20.

2. The HO-1i-Pt(IV) complex according to claim 1, characterized in that: R2 is -C n H 2n -a straight or branched chain group, n = 1 or 2; R3 is -C m H 2m+1 or -NH-C m H 2m+1 A straight chain or branched group, 1≤m≤11.

3. The HO-1i-Pt(IV) complex according to claim 1, characterized in that: As shown in any structure of Formula 3-10:

4. A method for preparing the HO-1i-Pt(IV) complex according to claim 1, characterized in that: When preparing the compound of formula 1, the compound of formula 11 and the compound of formula 12 are dissolved in a first solvent, and reacted under the conditions of a first condensing agent and a first acid binding agent, and the reaction temperature is 25-100° C.; in, for cisplatin; R1 is -OH or -Cl; R2 is -C n H 2n -a straight or branched chain group, n is an integer and 1≤n≤6; The first solvent is a mixture of one or more of acetonitrile, dichloromethane, acetone, dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF); The first condensing agent is 1-hydroxybenzotriazole (HOBT) or O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU); The first acid binding agent is triethylamine (TEA); The feed ratio of the compound of formula 11, the compound of formula 12, the first condensing agent and the first acid binding agent is 1:1-1.5:1-1.5:1-1.

5.

5. The method for preparing the HO-1i-Pt(IV) complex according to claim 4, characterized in that: The reaction temperature is 25° C., the first solvent is DMSO, the first condensing agent is O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), and the feed ratio of the compound of formula 11, the compound of formula 12, the first condensing agent and the first acid binding agent is 1:1:1.2:1.

2.

6. A method for preparing the HO-1i-Pt(IV) complex according to claim 1, characterized in that: When preparing the compound of formula 2, the compound of formula 1 is reacted with the compound of formula 13 or the compound of formula 14 in a second solvent at a reaction temperature of 25-100° C.; Wherein, R1 is -OH; R2 is -C n H 2n -a straight or branched chain group, n is an integer and 1≤n≤6; Where R3 is -C m H 2m+1 A straight chain or branched chain group, m is an integer and 1≤m≤20; The second solvent is a mixture of one or more of acetonitrile, dichloromethane, acetone, dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF); The feed ratio of the compound of formula 1 to the compound of formula 13 or the compound of formula 14 is 1:2-10.

7. The method for preparing the HO-1i-Pt(IV) complex according to claim 6, characterized in that: The reaction temperature is 60° C., the second solvent is DMF, and the feed ratio of the compound of Formula 1 to the compound of Formula 13 or the compound of Formula 14 is 1:

2.

8. The method for preparing the HO-1i-Pt(IV) complex according to claim 4, characterized in that: The compound of formula 15 and the compound of formula 16 are dissolved in a third solvent, and reacted in the presence of a third condensing agent and a third acid binding agent to obtain a compound of formula 12, and the reaction temperature is 25-100° C.; Where R2 is -C n H 2n -a straight or branched chain group, n is an integer and 1≤n≤6; The third solvent is a mixture of one or more of acetonitrile, dichloromethane, acetone, dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF); The third condensing agent is 4-dimethylaminopyridine (DMAP), and the third acid binding agent is triethylamine (TEA); The feed ratio of the compound of formula 15, the compound of formula 16, the first condensing agent and the first acid binding agent is 1:1-2:0.1-2:1-2.

9. The method for preparing the HO-1i-Pt(IV) complex according to claim 8, characterized in that: The reaction temperature is 50° C., the third solvent is dichloromethane, and the feed ratio of the compound of formula 15, the compound of formula 16, the first condensing agent and the first acid binding agent is 1:2:0.1:

2.

10. A HO-1i-Pt(IV) self-assembled nanoparticle, characterized in that: A nanoparticle suspension prepared from the compound of formula 9 or formula 10 in the HO-1i-Pt(IV) complex according to claim 3.

11. A method for preparing the HO-1i-Pt(IV) self-assembled nanoparticles according to claim 10, characterized in that: Dissolving the HO-1i-Pt(IV) complex in a fourth solvent, adding the mixture dropwise into a fifth solvent, and evaporating the fourth solvent to obtain a self-assembled nanoparticle suspension; The fourth solvent is a mixture of one or more of acetonitrile, dichloromethane, acetone, dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF); The fifth solvent is a mixture of one or more of water, methanol and ethanol.

12. The method for self-assembling HO-1i-Pt(IV) nanoparticles according to claim 11, characterized in that: The fourth solvent is acetone, and the fifth solvent is water.

13. Use of the HO-1i-Pt(IV) complex according to any one of claims 1 to 3 or the HO-1i-Pt(IV) self-assembled nanoparticles according to claim 10 in the preparation of anti-tumor drugs.

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