CN112266396A

CN112266396A - Integrated prodrug based on bio-orthogonal chemistry, preparation method and medical application thereof

Info

Publication number: CN112266396A
Application number: CN202011077271.9A
Authority: CN
Inventors: 黄张建; 张奕华; 孙涛; 吴建兵; 朱杰; 朱明超
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2020-10-10
Filing date: 2020-10-10
Publication date: 2021-01-26
Anticipated expiration: 2040-10-10
Also published as: CN112266396B

Abstract

The invention discloses an integrated prodrug based on bioorthogonal chemistry, a preparation method thereof and application of the integrated prodrug as a novel integral bioorthogonal prodrug in antitumor drugs. The compound can be selectively activated in tumor cells, tetravalent platinum in the structure of the compound is reduced into cisplatin, meanwhile, the cisplatin is used as a novel bioorthogonal catalyst to catalyze part of NO donor fragments to release NO, and the NO donor fragments and the cisplatin cooperatively play an anti-tumor activity; the compound does not have the above process in normal cells, so that the compound has better safety.

Description

Integrated prodrug based on bio-orthogonal chemistry, preparation method and medical application thereof

Technical Field

The invention belongs to the technical field of pharmacy, and relates to a preparation method and application of a Nitric Oxide (NO) donor containing a tetravalent platinum complex, in particular to a novel micromolecule which has tumor selectivity and combines the tetravalent platinum complex and an NO donor azonium dialkoxide fragment, wherein the micromolecule and the NO donor azonium dialkoxide fragment are connected through a succinyl group.

Background

At present, the morbidity and mortality of malignant tumors always show a significant trend, and chemotherapy is once the standard therapy for tumor treatment. However, the traditional small molecule drugs lack targeting property, and can kill normal cells by mistake while killing tumor cells to cause serious toxic and side effects, so that the wide application in clinic is limited. Bio-orthogonal chemistry, an emerging and developing discipline, is widely used to label, track, manipulate and activate target molecules by means of bond generation and bond cleavage. In recent years, some bioorthogonal bond cleavage reactions catalyzed by metal catalysts have been successfully applied to tumor therapy. Compared with the traditional cytotoxic drugs or the antitumor drugs which rely on enzyme activation, the prodrug activation strategy which relies on the biological orthogonal bond breakage of the exogenous catalyst can better realize the aim of targeting tumors and reduce side effects and adverse reactions to the maximum extent.

Bioorthogonal metal catalysts

The most widely used bio-orthogonal chemical metal catalysts currently include gold, copper and palladium. The structure of the metal catalyst is difficult to modify, and the metal catalyst and the prodrug are required to be separately administrated, so that the curative effect of the compound is greatly limited, the selectivity and the targeting property in vivo are lacked, and the metal catalyst is easy to accumulate in vivo and difficult to metabolize. Therefore, to solve the above problems, the discovery of a new catalyst is crucial for bio-orthogonal chemistry. As platinum and palladium are in the same main group of the periodic table of elements, cisplatin can be found as a new bioorthogonal catalyst after a series of verification. However, cisplatin has strong cytotoxicity and cannot be directly administered, so that modification of cisplatin is required. The tetravalent platinum prodrug can be selectively reduced into cisplatin under the action of tumor cell reduction mediators, so that the tetravalent platinum prodrug plays a catalytic role. Meanwhile, cisplatin can be crosslinked with DNA in tumor cells to play a role in cytotoxicity, so that a tetravalent prodrug of cisplatin is selected as a precursor form of a catalyst.

Tetravalent platinum prodrugs

Tetravalent platinum prodrugs are a new class of molecules that may improve the pharmacological properties of divalent platinum anti-tumor drugs. The central platinum ion of the tetravalent platinum complex relative to the divalent platinum complex in planar configuration is d⁶The electronic configuration is increased by two axial orbitals, and a stable octahedral coordination configuration can be formed. Chemical ligand modification of the axial position of the tetravalent platinum complex may further influence and adjust the physical and chemical properties of the tetravalent platinum complex. Different ligand modifications to the axial position of tetravalent platinum can affect the fat solubility, reducibility, targeting property and other biological activities of the tetravalent platinum complex. The tetravalent platinum complex is reduced by transfer of two electrons, which is reduced to divalent platinum with antitumor activity, accompanied by release of two axial ligands.

NO donor type medicine

NO donor type drugs generally refer to prodrugs formed from NO donors and related drugs or certain active compounds through various linking groups. It has now been found that NO donors of various structural types, such as nitrosothiols, nitrates, NO-metal complexes (nitroprusside), furazan N-oxides, azonium dialkoxides, etc.; among them, azonium dialkoxides (diazeniumdiolates) have significant advantages in selective and targeted release of NO. On one hand, azonium dialkoxide is extremely unstable under physiological conditions, can automatically release 1-2 molecules of NO, and has half-life period from several seconds to several hours. On the other hand, O of azonium dialkoxide²The site (O attached to the azonium ion is called O¹O attached to the nitrogen atom of the olefinic bond is O²) After alkylation, can be converted into prodrugs that are stable under physiological conditions; removing O by recognition of some specific enzymes in vivo or under the action of special physiological environment²The protective group is converted into unstable azo onium diol salt anions, thereby achieving the selective and targeted release of NO. To date, a variety of O's have been developed²The new policy is protected.

NO and cancer

NO is an organismThe inner water-soluble gas carrying free radicals has redox properties and plays an important role in physiology and pathology. Research has shown that NO is a potential anti-tumor agent that can modulate tumor-related processes including angiogenesis, apoptosis, invasion and metastasis, cell cycle, etc. The direct action mechanism of NO on tumor cells is not clear, and there are 3 main aspects reported at present: (1) NO reacts with intracellular superoxide anion to form peroxynitrite, which is protonated and decomposed into NO₂And hydroxyl radicals, which can bind to various molecules of tumor cells, thereby causing tumor cell damage, such as lipid peroxidation, protein, amino acid crosslinking reaction; (2) NO is easy to form Fe-NO with Fe-S center-containing protein Fe, so that the degradation of Fe-S prosthetic group and aconitase on mitochondrial respiratory chain is caused, the synthesis of intracellular energy is prevented, and apoptosis is induced; (3) NO can directly act on RNA reductase to influence the replication of tumor cell DNA, and can also nitrify the DNA to inhibit the proliferation of tumor cells. Another major mechanism of NO against tumors is the killing of tumor cells by NO-mediated macrophages, and the specific mechanism is: t cell recognition antigen → T cell secretion of cytokines → cytokine stimulation to produce NO → NO killing of tumor cells. In addition, there are studies demonstrating that NK cytotoxicity is also NO-dependent. Therefore, designing and studying NO prodrugs becomes one of the important strategies for the innovation of antitumor drugs. The National Cancer Institute (NCI), some world-known pharmaceutical companies such as Merk, Pfizer, NicOx, Nitromed, etc. all invest in supporting NO donor-type drug development. In recent years, research and development of NO donor type drugs have also been greatly advanced, and NCI has placed JS-K, an anti-tumor drug, into rapid research and development plans.

Disclosure of Invention

Object of the Invention

The present invention provides an organic compound comprising a tetravalent platinum complex and an azonium dialkoxide fragment having an antitumor therapeutic effect. The compound can selectively release NO and cisplatin in tumor cells, thereby exerting a synergistic antitumor effect.

Technical scheme

An organic compound comprising a tetravalent platinum complex and an azonium dialkoxide fragment, which compound is based on a tetravalent platinum complex and a nitric oxide donor molecule, the nitric oxide donor molecule and the tetravalent platinum complex being connected by a chemical coupling process using a succinate bond as a single entity; the nitric oxide donor molecule is N-methylethanolamine azonium dialkoxide; the tetravalent platinum complex is cis-diamine trichlorohydroxyplatine and trans-diamine trichlorohydroxyplatine.

In a first aspect, the present invention provides a compound having N-methylethanolamine azonium dialkoxide as a skeleton, which is a compound represented by the general formula I:

wherein: r¹Selected from methyl, allyl, propargyl; r²Selected from cis-diamine trichlorohydroxyplatine and trans-diamine trichlorohydroxyplatine.

Further, R₁Selected from propargyl, methyl, R₂Selected from cis-diamine trichlorohydroxyplatine and trans-diamine trichlorohydroxyplatine.

In some embodiments, the compound of formula I is selected from:

in a second aspect, a process for the preparation of said compounds, the synthetic route for the compounds of formula I is as follows:

NOgas: nitric oxide gas, anhydrousether: anhydrous diethyl ether; CH (CH)₃ONa: sodium methoxide; CH (CH)₃OH: methanol; DMF: n, N-dimethylformamide; THF: tetrahydrofuran;CHP: cis-diamminetrichlorohydroxyplatinum; FHP: trans-diamine trichlorohydroxyplatine; et (Et)₃N: triethylamine; TBTU: 2- (1H-benzotriazol L-1-yl) -1,1,3, 3-tetramethyluronium tetrafluoroborate.

In a third aspect, the present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the compound and a pharmaceutically acceptable carrier.

In a fourth aspect, the compound or the solvate thereof is used for preparing a medicament for preventing and/or treating tumor diseases. The application of the pharmaceutical composition in preparing medicines for preventing and/or treating tumor diseases.

Has the advantages that: the invention integrates two fragments of NO donor azonium dialkoxide and tetravalent platinum complex together, and designs the integrated bioorthogonal chemical catalytic NO donor drug. In the specific reducing environment of tumor cells, the compound reduces tetravalent platinum fragments into cisplatin and releases O²Protected azonium dialkoxide fragments. Cisplatin plays an anti-tumor role on one hand, and on the other hand, cisplatin is used as a bioorthogonal catalyst to catalyze azonium dialkoxide O²The breakage of the protecting group further releases NO, thereby exerting the curative effect of synergy anti-tumor. The integrated prodrug molecule has higher tumor specificity, so that cisplatin and NO are specifically released in tumor cells, the toxic and side effects on normal cells caused by low targeting when the integrated prodrug molecule is used alone are avoided, and the problem that the traditional bioorthogonal reaction catalyst lacks selectivity is solved. Meanwhile, the design concept of the whole molecule also improves the traditional mode of separately feeding the catalyst and the prodrug of the bioorthogonal chemistry, and further improves the drug forming property of the whole bioorthogonal prodrug.

The invention synthesizes a new compound I for the first time_5aThe compound I is verified by cell and zebra fish experiments_5aCan be selectively activated in tumor cells, thereby exerting the anti-tumor curative effect. Compound I compared to Normal cells_5aWill preferentially enter tumor cells, then tetravalent platinum is reduced to cisplatin, and the cisplatin catalytic compound releases NO, thereby exerting the antitumor activity in synergy with cisplatin. Andin comparison to the currently existing bioorthogonal prodrugs, Compound I_5aAs an integral prodrug molecule, the compound avoids separate administration, enhances the targeting property of the compound, reduces the toxicity of a catalyst, and greatly improves the druggability of the integrated bioorthogonal chemical catalytic NO donor molecule.

Drawings

FIG. 1 is Compound I_5aThe design concept of (1).

FIG. 2 is Compound I_5aStability in PBS and in the presence of L- (+) -ascorbic acid.

FIG. 3 is I_5aLevels of NO released in PBS and in the presence of L- (+) -ascorbic acid.

FIG. 4 is Compound I_5aLevels of NO were released in a2780 and IOSE80 cells.

FIG. 5 shows the concentrations of Compound I_5aAnd levels of NO released at different times from cell incubation.

FIG. 6 is the NO scavenger carboxy-PTIO vs. Compound I_5aThe effect of the tumor cell proliferation inhibitory activity of (3).

FIG. 7 shows the comparison of A2780 and IOSE80 cells against Compound I_5aAnd the amount of CDDP taken.

FIG. 8 is Compound I_5aAnd CDDP Pt content on DNA in a2780 cells and IOSE80 cells, respectively.

FIG. 9 is a study of Compound I using a cisplatin Probe_5aIn the case of cisplatin conversion in A2780 cells and IOSE80 cells.

FIG. 10 is a photograph of a further study of Compound I_5aAntitumor proliferation inhibitory activity in zebra fish.

FIG. 11 shows further investigation of Compound I_5aNO release in zebrafish.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

The synthetic route for the compounds of formula I is as follows:

CH₃ONa: sodium methoxide; CH (CH)₃OH: methanol; DMF: n, N-dimethylformamide; THF: tetrahydrofuran; CHP: cis-diamminetrichlorohydroxyplatinum; FHP: trans-diamine trichlorohydroxyplatine; et (Et)₃N: triethylamine; TBTU: 2- (1H-benzotriazol L-1-yl) -1,1,3, 3-tetramethyluronium tetrafluoroborate.

In some embodiments, the intermediate compound I₂To compound I₄The synthesis of (a) is as follows:

compound I₂The synthesis of (2): adding N-methyl-2-hydroxyethylamine (I)₁20mL), and 50.79mL of a methanol solution (5.4mol/L) of sodium methoxide were mixed and added to a polytetrafluoroethylene container, and 200mL of anhydrous ether was added thereto to prepare a reaction system N₂After replacement, introducing Nitric Oxide (NO) gas to make the pressure reach 0.4-0.8MPa, and carrying out closed reaction in a greenhouse for 24 h. Post-treatment, discharging excessive unreacted NO gas to reduce pressure to normal pressure, opening the container, pouring the reaction solution into 1L anhydrous ether to separate out a large amount of white solid, filtering, washing the filter cake with ether for 3 times, drying in a vacuum drying oven at room temperature for 2h, and collecting a white product, namely the compound I₂. The resulting compound I₂The reaction was carried out without further purification.

Compound I_3aThe synthesis of (2): take 1g toCompound I₂And 5mL of DMF was added to a 100mL two-necked glass reaction flask, and the flask was placed in ice bath and subjected to N₂And (3) after protection, slowly adding 757.8mg of bromopropyne into a reaction bottle, continuing to react for 0.5h in an ice bath after dripping, and then moving the reaction solution to room temperature to continue to react for 12 h. After-treatment, DMF is removed by rotary evaporation, the residue is prepared into sand, and column chromatography is carried out to obtain colorless oily substance I_3a。

Compound I_3bThe synthesis of (2): 1g of the compound I are taken₂And 5mL of DMF was added to a 100mL two-necked glass reaction flask, and the flask was placed in ice bath and subjected to N₂And (3) protecting, slowly adding 949.8mg of methyl iodide into the reaction bottle, keeping the temperature in an ice bath for 0.5h after dripping, and then moving the reaction solution to room temperature to continue reacting for 12 h. After-treatment, DMF is firstly removed by rotary evaporation, and the residue is subjected to sand preparation and column chromatography to obtain a light yellow oily substance I_3b。

Compound I_4aThe synthesis of (2): taking 700mg of compound I_3aThe reaction mixture was dissolved in 10mL of anhydrous tetrahydrofuran, 122mg of DMAP was added thereto, the mixture was stirred at room temperature for 15mins, 608mg of succinic anhydride was added thereto, and the reaction mixture was refluxed overnight. Post-treatment, filtering the reaction solution, concentrating the filtrate, adding water, extracting with dichloromethane for 5 times, drying, and concentrating to obtain compound I_4a。

Compound I_4bThe synthesis of (2): taking 602mg of compound I_3bThe reaction mixture was dissolved in 10mL of anhydrous tetrahydrofuran, 122mg of DMAP was added thereto, the mixture was stirred at room temperature for 15mins, 608mg of succinic anhydride was added thereto, and the reaction mixture was refluxed overnight. Post-treatment, filtering the reaction solution, concentrating the filtrate, adding water, extracting with dichloromethane for 5 times, drying, and concentrating to obtain compound I_4b。

Example 1: compound I_5a,I_5bPreparation of

The synthetic route is as follows:

217mg of Compound I₄In a single-neck reaction flask, 86.6mg of triethylamine, 265mg of TBTU and 5mL of DMF were added and dissolved, and the reaction mixture was stirred at room temperature for 30 mins. Then, 200mg of CHP or FHP was added to the reaction flask, and the reaction was continued overnight under exclusion of light. Post-treatment, firstly concentrating to remove DMF, preparing sand from residues, and carrying out column chromatography to obtain a target product I_5aAnd I_5b。

Compound I_5a: pale yellow solid, 97mg, yield 28%.¹H NMR(ppm,300MHz,DMSO-d₆)δ6.28-6.03(m,6H),4.82(d,J＝2.4Hz,2H),4.16(t,J＝5.4Hz,2H),3.58(t,J＝2.4Hz,2H),2.98(s,3H),2.49-2.42(m,5H).¹³C NMR(ppm,75MHz,DMSO-d₆)δ178.90,172.01,78.75,60.54,60.39,51.83,40.52,30.94,29.80.HRMS(ESI)calcd for C₁₀H₂₀Cl₃N₅O₆Pt[M+H]⁺:607.0200；Found:607.0191,ppm error-1.5.

Compound I_5bLight yellow solid, 89mg, yield 18%.¹H NMR(ppm,300MHz,DMSO-d₆)δ6.06(br,6H),4.83(d,J＝2.4Hz,2H),4.17(t,J＝5.1Hz,2H),3.60(t,J＝4.2Hz,2H),3.00(s,3H),2.47-2.45(m,5H).ESI-MS m/z:629.1[M+Na]⁺

Example 2: compound I_5cPreparation of

The synthetic route is as follows:

CH₃ONa: sodium methoxide; CH (CH)₃OH: methanol; CH (CH)₃I: methyl iodide; DMF: n, N-dimethylformamide; THF: tetrahydrofuran; CHP: cis-diamminetrichlorohydroxyplatinum; et (Et)₃N: triethylamine; TBTU: 2- (1H-benzotriazol L-1-yl) -1,1,3, 3-tetramethyluronium tetrafluoroborate.

200mg of compound I are taken₄In a single-necked reaction flask, 86.6mg of triethylamine and 265mg of TBTU were added and dissolved in 5mL of DMF, and the reaction mixture was stirred at room temperature for 30 min. Then, 200mg of CHP was added to the reaction flask, and the reaction solution was allowed to react overnight under exclusion of light. Post-treatment, firstly concentrating to remove DMF, preparing sand from residues, and carrying out column chromatography to obtain a target product I of 134mg_5cThe yield thereof was found to be 37%.¹H NMR(ppm,500MHz,DMSO-d₆)δ6.28-6.03(m,6H),4.15(t,J＝5.0Hz,2H),3.90(s,3H),3.52(t,J＝5.0Hz,2H),2.95(s,3H),2.52-2.44(m,4H).¹³C NMR(ppm,125MHz,DMSO-d₆)δ182.01,175.12,63.69,63.57,55.06,43.86,34.06,32.93.HRMS(ESI)calcd for C₈H₂₀Cl₃N₅O₆Pt[M+H]⁺:583.0200；Found:583.0191，ppm error-1.5.

FIG. 1 shows Compound I_5aTransformation in tumor cells and Normal cells, respectivelyAnd (6) carrying out the process. From the above, Compound I_5aCan be stable in normal cells and substantially free of cytotoxicity, while in tumor cells, Compound I_5aThe tetravalent platinum can be reduced to cisplatin first, and the cisplatin catalytic compound releases NO, so that the cisplatin and the cisplatin can exert the antitumor activity synergistically.

FIG. 2 is Compound I_5aStability in PBS and in the presence of L- (+) -ascorbic acid. As can be seen from FIG. 2, I_5aRelatively stable in PBS at 24 deg.c; however, in the presence of L- (+) -ascorbic acid, the degradation can be faster, e.g., more than 50% in 2h, and substantially complete in 24 h.

FIG. 3 is I_5aThe concentration of NO released in PBS and in the presence of L- (+) -ascorbic acid was measured. As can be seen from FIG. 3, I_5aSubstantially NO production in the absence of L- (+) -ascorbic acid; when the L- (+) -ascorbic acid exists, more NO is generated; description of the invention I_5aFirstly, cis-platinum and I are generated under the action of L- (+) -ascorbic acid_4aCDDP further catalyzes I_4aTo thereby release NO.

Table 1 shows Compound I_5aThe proliferation inhibitory activity of human ovarian cancer cell A2780 and human ovarian normal cell IOSE80 was studied.

TABLE 1

As can be seen from table 1, CHP showed higher tumor cell selectivity compared to cisplatin CDDP. And the compound I_4aIC for tumor cell A2780 and normal cell IOSE80₅₀The larger values indicate that the growth inhibitory activity of the cells of 2 strains was weak. When CHP is pre-incubated with A2780 for 10h, I is added_4aShowing a strong proliferation inhibitory activity (I)_4a,IC₅₀＝89.46±4.87μMvs CHP+I_4a,IC₅₀0.81 ± 0.07 μ M), indicating that CHP can be reduced to cisplatin, which catalyzes I, after entering tumor cells_4aNO is produced. Importantly, I_5aShows stronger proliferation inhibition activity on A2780 than CDDP and CHP (I)_5a,IC₅₀＝0.23±0.01μM；CDDP,IC₅₀＝1.08±0.06μM；CHP,IC₅₀1.69 ± 0.06 μ M). At the same time, I_5a(SF ═ 518) shows better selectivity for tumor cells than CDDP (SF ═ 7.8) and CHP (SF ═ 10.9).

FIG. 4 is Compound I_5aLevels of NO were released in a2780 and IOSE 80. As can be seen in FIG. 4, I_4aWhen incubated with cells alone, substantially NO was produced. When CHP and A2780 cells are added for pre-incubation for 10 hours, I is added_4aAfter 8h incubation, more NO was produced, while essentially NO was produced in normal cells IOSE80, indicating that cisplatin-catalyzed O-propynyl cleavage can be selectively performed in tumor cells. I is_5aThe strongest MFI value (25.33) was produced in tumor cells A2780, higher than I_4a+ CHP (MFI 15.16) and JS-K (MFI 7.75).

FIG. 5 shows the concentrations of Compound I_5aAnd levels of NO released at different times from cell incubation. As can be seen from FIG. 5, Compound I_5aThere is essentially NO production in normal cells, while the level of NO released in tumor cells is dose and time dependent, i.e. as the concentration increases or the incubation time increases, the amount of NO released gradually increases.

FIG. 6 is the NO scavenger carboxy-PTIO vs. Compound I_5aThe effect of the tumor cell proliferation inhibitory activity of (3). As can be seen from FIG. 6, when carboxy-PTIO was added, Compound I was reduced_5aInhibitory Activity on tumor cells A2780 and with increasing Carboxy-PTIO concentrations on Compound I_5aThe more significant the decrease in proliferation inhibitory activity of (a). The results show that the compounds I_5aNO produced in tumor cells contributes to the activity of tumor cells.

FIG. 7 shows the comparison of A2780 and IOSE80 cells against Compound I_5aAnd the amount of CDDP taken. From FIG. 7, it can be seen that both the whole cell and the nucleus of A2780 are of pair I_5a(Whole cell, 763.53. + -. 14.81pg Pt/10⁶cells; in the nucleus, 84.4. + -. 1.77pg Pt/10⁶cells) are all significantly higher thanCDDP (Whole cell, 98.34. + -. 23.0pg Pt/10⁶cells; nucleus, 24.78. + -. 1.55pg Pt/10⁶cells)。

FIG. 8 is Compound I_5aAnd CDDP Pt content on DNA in a2780 cells and IOSE80 cells, respectively. As can be seen from FIG. 8, in A2780 cells, Compound I_5a(22.56pg Pt/. mu.gDNA) platinized DNA levels were significantly higher than cisplatin (5.18pg Pt/. mu.gDNA). And compounds I_5aThe level of platinized DNA in A2780 tumor cells (22.56pg Pt/. mu.gDNA) was higher than its level in ovarian normal cells IOSE80 (5.67pg Pt/. mu.gDNA).

FIG. 9 is a study of Compound I using a cisplatin Probe_5aIn the case of cisplatin conversion in A2780 cells and IOSE80 cells. As can be seen from the left panel of FIG. 9, at 0h, the fluorescence in A2780 cells and IOSE80 cells was weak, indicating that there was almost no cisplatin in the cells, but after 3h, the fluorescence in A2780 cells was strong, indicating more cisplatin content, while the fluorescence in IOSE80 cells was still weak, indicating less cisplatin content in the cells. The right graph is a data statistics graph of fluorescence intensity in confocal microscope pictures, and it can be seen that after 3h, the fluorescence intensity in a2780 cells is significantly higher than 0h, and the fluorescence intensity in a2780 cells is also significantly higher than that in IOSE80 cells. From the above, Compound I_5aCisplatin production was significantly higher in A2780 tumor cells than in IOSE80 normal cells.

Example 3: investigation of Compound I_5aAntitumor proliferation inhibitory activity in zebra fish.

Firstly, a certain amount of quilt CellTracker is implanted into the yolk part of the young zebra fish in a displacement way^TMA2780 cells marked by CM-Dil fluorescent dye are grown for 24 hours, and strong yellow fluorescence can be seen from the yellow parts of the zebra fish egg under a confocal microscope, so that a zebra fish experimental model of successful tumor cells is constructed. Then the zebra fish which are implanted with tumor cells are respectively mixed with the zebra fish containing I_5aFeeding zebra fish culture solution containing 1 mu M of cisplatin, 1 mu M of cisplatin and no other compounds, observing yellow fluorescence distribution in zebra fish body under confocal microscope after 72h, and counting corresponding fluorescence intensity values, thereby obtaining fluorescent valueEvaluation of Compound I_5aAnti-tumor proliferation inhibiting effect. FIG. 10 is a photograph of a further study of Compound I_5aAntitumor proliferation inhibitory activity in zebra fish. As can be seen from FIG. 10, when zebrafish were compared to the control group with Compound I_5aAfter incubation for 72h, the fluorescence distribution in the zebra fish bodies is obviously reduced, and the compound I is proved_5aEffectively inhibit the growth of tumor cells in zebra fish bodies, and the effect is better than that of cisplatin. The right-hand fluorescence intensity histogram also validates this conclusion.

Example 4: investigation of Compound I_5aNO release in zebrafish.

Firstly, a certain amount of quilt CellTracker is implanted into the yolk part of the young zebra fish in a displacement way^TMA2780 cells marked by CM-Dil fluorescent dye are grown for 24 hours, and strong yellow fluorescence can be seen from the yellow parts of the zebra fish egg under a confocal microscope, so that a zebra fish experimental model of successful tumor cells is constructed. Zebrafish were incubated with the DAF-FM DA probe for 40 minutes at 37 ℃ and then photographed under a confocal microscope to determine their own NO levels in the zebrafish. Then, the zebra fish is raised in a pure zebra fish culture solution for 24 hours, so that the zebra fish metabolizes the NO probe in the body completely. Then mixing zebra fish with the mixture containing I_5a(10. mu.M) and a zebrafish culture broth without addition of other compounds, and after 24 hours, the zebrafish were incubated with a DAF-FM DA (5. mu.M) probe at 37 ℃ for 40 minutes, and then photographed under a confocal microscope, and the corresponding fluorescence intensity values were counted, thereby verifying the compound I_5aWhether NO can be selectively released at the tumor cell part of the zebra fish. FIG. 11 shows further investigation of Compound I_5aNO release in zebrafish. As can be seen from FIG. 11, when zebrafish were mixed with Compound I_5aAfter incubation, the fluorescence intensity at the tumor site was significantly higher than that of the control group, thereby verifying that Compound I_5aThe fluorescent protein can selectively release NO at a tumor site in zebra fish bodies, and the fluorescence statistics on the right side also verify the conclusion.

The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.

Claims

1. A compound of formula I or an optical isomer, enantiomer, diastereomer, racemate or racemic mixture thereof, or a pharmaceutically acceptable salt thereof:

wherein: r₁Selected from methyl, allyl, propargyl; r₂Selected from cis-diamine trichlorohydroxyplatine and trans-diamine trichlorohydroxyplatine.

2. A compound of claim 1, wherein R is₁Selected from methyl and propargyl.

3. The compound of claim 1, wherein the compound of formula I is selected from the group consisting of:

4. a process for the preparation of a compound according to any one of claims 1 to 3, characterized in that the compound of formula I is synthesized as follows:

CH₃ONa: sodium methoxide; CH (CH)₃OH: methanol; DMF: n, N-dimethylformamide; THF: tetrahydrofuran; CHP: cis-diamminetrichlorohydroxyplatinum; FHP: trans-diamine trichlorohydroxyplatine; et (Et)₃N: triethylamine;TBTU: 2- (1H-benzotriazol L-1-yl) -1,1,3, 3-tetramethyluronium tetrafluoroborate.

5. A pharmaceutical composition comprising a therapeutically effective amount of a compound of any one of claims 1-3 and a pharmaceutically acceptable carrier.

6. Use of a compound according to any one of claims 1 to 3 or a solvate thereof for the manufacture of a medicament for the prophylaxis and/or treatment of a neoplastic disease.

7. Use of the pharmaceutical composition of claim 5 for the preparation of a medicament for the prevention and/or treatment of a tumor disease.