Tirapazamine derivatives and preparation methods and applications thereof

Abstract

The present invention provides a tirapazamine derivative having a structure of formula (I). The derivative provided by the present invention has a more excellent hypoxia activation ability, and while retaining hypoxia selective toxicity, it also exhibits strong cytotoxicity even in tumor cells with a lower degree of hypoxia, and the toxicity in a normoxic environment is significantly reduced, which is expected to solve the problem of poor efficacy of tirapazamine due to insufficient hypoxia in the patient's tumor.

Description

Technical Field

[0001] The present invention relates to the technical field of drug synthesis, in particular to a tirapazamine derivative and a preparation method and application thereof. Background Art

[0002] Tumor hypoxia is a common feature of human and animal solid tumors, a powerful negative clinical biomarker, and is considered one of the best validated therapeutic targets in cancer treatment. On the one hand, tumor hypoxia is a major factor leading to the failure of many anticancer therapies. On the other hand, tumor hypoxia may be an attractive therapeutic target because it underlies the major differences between tumors and normal tissues. Hypoxia-activated prodrugs have selectively higher toxicity to hypoxic cells and lower toxicity in areas of higher oxygen tension such as normal tissues. Therefore, hypoxia-activated prodrugs have the potential to selectively kill hypoxic cells, thereby transforming tumor hypoxia from a problem to a selective therapeutic advantage.

[0003] Tirapazamine (TPZ) is a representative hypoxia-activated prodrug that is selectively cytotoxic to hypoxic cells in solid tumors and inhibits tumor growth by inducing single-strand and double-strand breaks, base damage, and cell death in DNA. Despite the encouraging results of phase II clinical trials, most phase III clinical trials have failed to show the additional benefits of adding TPZ. However, a further sub-study of phase II clinical trial data showed that TPZ could significantly improve the clinical treatment effect if patients with tumor hypoxia were selected, indicating that the failure of TPZ in clinical trials was mainly due to insufficient hypoxia in the tumor, which was insufficient to effectively activate TPZ and achieve potent killing of tumor cells. Currently, most studies are committed to increasing the degree of hypoxia in the tumor site and the degree of activation of TPZ in the tumor site by combining with other treatments to achieve inhibition of tumor growth. Tumor hypoxia levels are usually enhanced by oxygen consumption or vascular occlusion in the tumor. For example, oxygen consumption is achieved by photodynamic therapy or sonodynamic therapy; hepatic artery ligation and vascular blocking agents are used to induce tumor vascular occlusion to increase tumor hypoxia. Currently, a phase II clinical trial combining TPZ and TAE embolization (trans-arterial embolization) is underway. TAE embolization can only be used to treat liver cancer, and photodynamic therapy, sonodynamic therapy, and TAE embolization can only be used for local area treatment, which cannot effectively improve the therapeutic effect of drugs on patients. Currently, there is no effective vascular blocking agent on the market.

[0004] Therefore, it is very necessary to develop a TPZ derivative with more sensitive hypoxic selectivity. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a tirapazamine derivative, which has a more excellent hypoxia activation ability and exhibits strong cytotoxicity even in tumor cells with a lower degree of hypoxia while retaining hypoxia selective toxicity.

[0006] The present invention provides a tirapazamine derivative having a structure of formula (I),

[0007]

[0008] Among them, n is the -CH in brackets 2 -The number of repeating units, 0≤n≤10;

[0009] The R is H, substituted C1-C6 alkyl, unsubstituted C1-C6 alkyl, substituted C3-C7 cycloalkyl, unsubstituted C3-C7 cycloalkyl, substituted C2-C8 heterocycloalkyl, unsubstituted C2-C8 heterocycloalkyl, substituted C6-C12 aryl, unsubstituted C6-C12 aryl, substituted C2-C9 heteroaryl, unsubstituted C2-C9 heteroaryl, hydroxyl, C1-C5 alkylamine, carboxyl, trifluoromethyl, C2-C4 alkynyl, C2-C4 alkenyl, and amide.

[0010] Preferably, the substituent of the substituted alkyl is F, Cl, Br, or I.

[0011] Preferably, R is hydrogen or a structure of formula (a-1) to formula (a-17):

[0012]

[0013]

[0014] Preferably, the tirapazamine derivative of the formula (I) is specifically the following structure:

[0015]

[0016] The present invention provides a method for preparing a tirapazamine derivative of formula (I) according to any one of the above technical solutions, comprising the following steps:

[0017] A) Tirapazamine reacts with p-nitrophenyl chloroformate in a basic solvent to obtain an intermediate product TPZ-NPC;

[0018] B) TPZ-NPC is reacted with an amino compound represented by formula (II) in a solvent to obtain the product;

[0019]

[0020] The R is H, substituted C1-C6 alkyl, unsubstituted C1-C6 alkyl, substituted C3-C7 cycloalkyl, unsubstituted C3-C7 cycloalkyl, substituted C2-C8 heterocycloalkyl, unsubstituted C2-C8 heterocycloalkyl, substituted C6-C12 aryl, unsubstituted C6-C12 aryl, substituted C2-C9 heteroaryl, unsubstituted C2-C9 heteroaryl, hydroxyl, C1-C5 alkylamine, carboxyl, trifluoromethyl, C2-C4 alkynyl, C2-C4 alkenyl, and amide.

[0021] Preferably, the alkaline substance is triethylamine, pyridine, lutidine, diisopropylethylamine; the solvent is tetrahydrofuran, acetonitrile, chloroform, dichloromethane, carbon tetrachloride, benzene, toluene, dimethyl sulfoxide, dimethylformamide.

[0022] Preferably, the reaction temperature in step A) is 25° C. and the reaction time is 24 h.

[0023] Preferably, the reaction temperature of step B) is 25°C and the reaction time is 24h. The present invention provides a use of a tirapazamine derivative of formula (I) and a pharmaceutically acceptable salt thereof as described in any one of the above technical solutions in the preparation of anticancer drugs.

[0024] Preferably, the cancer includes one or more of nasal and paranasal sinus malignancies, nasopharyngeal cancer, oral cancer, laryngeal cancer, intracranial tumors, thyroid cancer, tongue cancer, lung cancer, esophageal cancer, breast cancer, gastric cancer, colorectal cancer, sigmoid colon and rectal cancer, liver cancer, pancreatic cancer and periampullary cancer, biliary tract cancer, kidney cancer, prostate cancer, bladder cancer, testicular malignancies, penile cancer, cervical cancer, endometrial cancer, ovarian cancer, fibrohistiocytic carcinoma, rhabdomyosarcoma, synovial sarcoma, melanoma, osteosarcoma, Ewing's sarcoma, leukemia, lymphoma and multiple myeloma.

[0025] The present invention provides an anticancer drug, comprising a tirapazamine derivative of the formula (I) structure described in any one of the above technical solutions or a tirapazamine derivative of the formula (I) structure prepared by the preparation method described in any one of the above technical solutions.

[0026] Compared with the prior art, the present invention provides a tirapazamine derivative with a structure of formula (I). The derivative provided by the present invention has a more excellent hypoxia activation ability, and while retaining hypoxia selective toxicity, it also exhibits strong cytotoxicity even in tumor cells with a lower degree of hypoxia, and the toxicity in normal cells is significantly reduced, which is expected to solve the problem of poor efficacy of tirapazamine due to insufficient hypoxia in the patient's tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1The present invention provides a synthetic route and nuclear magnetic hydrogen spectrum characterization of the intermediate compound (TPZ-NPC);

[0028] Figure 2 The H-NMR spectrum characterization of compound TPZ-His;

[0029] Figure 3 The H-NMR spectrum characterization of compound TPZ-BZA;

[0030] Figure 4 The H-NMR spectrum characterization of compound TPZ-Hex;

[0031] Figure 5 The H-NMR spectrum characterization of compound TPZ-Acp; DETAILED DESCRIPTION

[0032] The present invention provides a tirapazamine derivative and a preparation method and application thereof. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they all belong to the scope of protection of the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0033] The present invention provides a tirapazamine derivative having a structure of formula (I),

[0034]

[0035] Among them, n is the -CH in brackets 2 -The number of repeating units, 0≤n≤10; preferably, 2≤n≤8;

[0036] The R is H, substituted C1-C6 alkyl, unsubstituted C1-C6 alkyl, substituted C3-C7 cycloalkyl, unsubstituted C3-C7 cycloalkyl, substituted C2-C8 heterocycloalkyl, unsubstituted C2-C8 heterocycloalkyl, substituted C6-C12 aryl, unsubstituted C6-C12 aryl, substituted C2-C9 heteroaryl, unsubstituted C2-C9 heteroaryl, hydroxyl, C1-C5 alkylamine, carboxyl, trifluoromethyl, C2-C4 alkynyl, C2-C4 alkenyl, amide;

[0037] Preferably, R is H, substituted C2-C6 alkyl, unsubstituted C2-C6 alkyl, substituted C3-C7 cycloalkyl, unsubstituted C3-C7 cycloalkyl, substituted C3-C8 heterocycloalkyl, unsubstituted C3-C8 heterocycloalkyl, substituted C6-C10 aryl, unsubstituted C6-C10 aryl, substituted C2-C9 heteroaryl, unsubstituted C2-C9 heteroaryl, hydroxyl, C1-C5 alkylamine, carboxyl, trifluoromethyl, C2-C3 alkynyl, C2-C3 alkenyl, amide;

[0038] Specifically, the substituent of the substituted alkyl group of the present invention is F, Cl, Br, I;

[0039] The substituent of the substituted aryl group is a nitro group;

[0040] In a preferred embodiment of the present invention, R is hydrogen or a structure of formula (a-1) to formula (a-15):

[0041]

[0042]

[0043] According to the present invention, the tirapazamine derivative of the formula (I) is specifically the following structure:

[0044]

[0045] The present invention provides a method for preparing a tirapazamine derivative of formula (I) according to any one of the above technical solutions, comprising the following steps:

[0046] A) Tirapazamine reacts with p-nitrophenyl chloroformate in a basic solvent to obtain an intermediate product TPZ-NPC;

[0047] B) TPZ-NPC is reacted with an amino compound represented by formula (II) in a solvent to obtain the product;

[0048]

[0049] The R is H, substituted C1-C6 alkyl, unsubstituted C1-C6 alkyl, substituted C3-C7 cycloalkyl, unsubstituted C3-C7 cycloalkyl, substituted C2-C8 heterocycloalkyl, unsubstituted C2-C8 heterocycloalkyl, substituted C6-C12 aryl, unsubstituted C6-C12 aryl, substituted C2-C9 heteroaryl, unsubstituted C2-C9 heteroaryl, hydroxyl, C1-C5 alkylamine, carboxyl, trifluoromethyl, C2-C4 alkynyl, C2-C4 alkenyl, and amide.

[0050] Preferably, R is H, substituted C2-C6 alkyl, unsubstituted C2-C6 alkyl, substituted C3-C7 cycloalkyl, unsubstituted C3-C7 cycloalkyl, substituted C3-C8 heterocycloalkyl, unsubstituted C3-C8 heterocycloalkyl, substituted C6-C10 aryl, unsubstituted C6-C10 aryl, substituted C2-C9 heteroaryl, unsubstituted C2-C9 heteroaryl, hydroxyl, C1-C5 alkylamine, carboxyl, trifluoromethyl, C2-C3 alkynyl, C2-C3 alkenyl, amide;

[0051] Specifically, the substituent of the substituted alkyl group of the present invention is F, Cl, Br, I;

[0052] The substituent of the substituted aryl group is a nitro group;

[0053] The preparation method of the tirapazamine derivative of the structure of formula (I) provided by the present invention comprises the following steps: firstly reacting tirapazamine with p-nitrophenyl chloroformate in an alkaline solvent to obtain an intermediate product TPZ-NPC.

[0054] The preferred method is as follows: dissolving tirapazamine in a solvent and placing it in an ice bath; dissolving p-nitrophenyl chloroformate in a solvent and slowly adding dropwise to react; after the reaction is completed, the solvent is dried by spin drying, and the mixture is washed with a mixture of ethyl acetate / n-hexane until the supernatant is colorless; collecting the precipitate by centrifugation, and drying to obtain the target product TPZ-NPC.

[0055] The reaction formula is:

[0056]

[0057] The alkaline substance of the present invention is triethylamine, pyridine, lutidine, diisopropylethylamine;

[0058] The solvent is tetrahydrofuran, acetonitrile, chloroform, dichloromethane, carbon tetrachloride, benzene, toluene, dimethyl sulfoxide, and dimethylformamide.

[0059] Specifically, the reaction temperature of step A) is 25° C. and the reaction time is 24 h.

[0060] The TPZ-NPC is obtained by reacting the amino compound represented by formula (II) in a solvent.

[0061] After the reaction of the present invention, the solvent is preferably drained, and then washed with a washing solvent for 1 to 3 times, centrifuged to obtain a precipitate and dried to obtain a crude product. After dissolving in dichloromethane, column chromatography is performed, and the eluent is dichloromethane and methanol to collect the target compound. The washing solvent is a mixture of ethyl acetate / n-hexane.

[0062]

[0063] The R is H, substituted C1-C6 alkyl, unsubstituted C1-C6 alkyl, substituted C3-C7 cycloalkyl, unsubstituted C3-C7 cycloalkyl, substituted C2-C8 heterocycloalkyl, unsubstituted C2-C8 heterocycloalkyl, substituted C6-C12 aryl, unsubstituted C6-C12 aryl, substituted C2-C9 heteroaryl, unsubstituted C2-C9 heteroaryl, hydroxyl, C1-C5 alkylamine, carboxyl, trifluoromethyl, C2-C4 alkynyl, C2-C4 alkenyl, and amide.

[0064] The amino compounds of the present invention include but are not limited to histamine, benzylamine, n-hexylamine and cyclopropylmethylamine.

[0065] The reaction temperature of the present invention is 25°C and the reaction time is 24h. The reaction formula is:

[0066]

[0067] The present invention provides use of a tirapazamine derivative of formula (I) and a pharmaceutically acceptable salt thereof according to any one of the above technical solutions in the preparation of anticancer drugs.

[0068] The cancer described in the present invention includes one or more of nasal and paranasal sinus malignancies, nasopharyngeal cancer, oral cancer, laryngeal cancer, intracranial tumors, thyroid cancer, tongue cancer, lung cancer, esophageal cancer, breast cancer, gastric cancer, colorectal cancer, sigmoid colon and rectal cancer, liver cancer, pancreatic cancer and periampullary cancer, biliary tract cancer, kidney cancer, prostate cancer, bladder cancer, testicular malignancies, penile cancer, cervical cancer, endometrial cancer, ovarian cancer, fibrohistiocytic carcinoma, rhabdomyosarcoma, synovial sarcoma, melanoma, osteosarcoma, Ewing's sarcoma, leukemia, lymphoma and multiple myeloma.

[0069] The present invention provides an anticancer drug, comprising a tirapazamine derivative of the formula (I) structure described in any one of the above technical solutions or a tirapazamine derivative of the formula (I) structure prepared by the preparation method described in any one of the above technical solutions.

[0070] The present invention has clearly described the above specific structure and preparation method, which will not be repeated here.

[0071] The urea derivative of tirapazamine designed by the present invention can improve its sensitivity to hypoxia while retaining hypoxia selectivity, and exhibits higher cytotoxicity under hypoxic conditions.

[0072] The present invention proposes for the first time the development of urea derivatives of TPZ, which are more sensitive to the hypoxic tumor microenvironment, can effectively achieve high toxicity killing of tumor cells under conditions of lower hypoxia, while showing lower toxicity at the normoxic cell level, and have more excellent tumor hypoxia sensitivity selectivity, and are expected to achieve killing of tumor cells with lower hypoxia levels, inhibit tumor growth, and broaden the applicability to patients with different hypoxia levels.

[0073] In order to further illustrate the present invention, a tirapazamine derivative provided by the present invention and its preparation method and application are described in detail below in conjunction with examples.

[0074] Embodiment 1:

[0075] Weigh 700.0 mg of tirapazamine (3.93 mmol), add it to a 100 mL eggplant flask, dissolve it with 20 mL of ultra-dry acetonitrile, and place it in an ice bath for 5 minutes. Weigh 1188.3 mg of p-nitrophenyl chloroformate (NPC, 5.90 mmol), dissolve it with 10 mL of ultra-dry solvent, and slowly add it dropwise into the reaction system. React at 30 ° C in the dark for 24 hours. After the reaction is completed, spin dry the solvent, wash it with ethyl acetate / n-hexane mixture until the supernatant is colorless, collect the precipitate by centrifugation, and dry it to obtain the target product TPZ-NPC, 1.2 g, with a yield of 90%. Synthesis route ( Figure 1 , a) and H NMR spectrum characterization ( Figure 1 ,b).

[0076] Embodiment 2:

[0077] Weigh 100.0 mg TPZ-NPC and dissolve it in 20 mL ultra-dry acetonitrile, then weigh 64.8 mg amino compound histamine (0.3 mmol), dissolve it in 20 mL ultra-dry acetonitrile, slowly add it to the reaction system, react at 30 ° C in the dark for 48 hours, drain the solvent, wash it three times with ethyl acetate / n-hexane mixture, centrifuge and dry the precipitate to obtain a crude product. After dissolving in dichloromethane, column chromatography was performed with dichloromethane and methanol as eluents to collect the target compound tirapazamine ureido derivative TPZ-His. Nuclear magnetic hydrogen spectrum characterization ( Figure 2 ).

[0078] Embodiment 3:

[0079] Referring to the method of Example 2, benzylamine was used instead of histamine to prepare the ureido derivative TPZ-Bza of tirapazamine. Figure 3 ).

[0080] Embodiment 4:

[0081] Referring to the method of Example 2, n-hexylamine was used instead of histamine to prepare the ureido derivative TPZ-Hex of tirapazamine. Figure 4 ).

[0082] Embodiment 5:

[0083] Referring to the method of Example 2, cyclopropylmethylamine was used instead of histamine to prepare the ureido derivative TPZ-Acp of tirapazamine. Figure 5 ).

[0084] Embodiment 6:

[0085] 4T1 cells in the logarithmic growth phase were used to prepare 2.6×10 3 / mL cell suspension, 190μL per well was inoculated in a 96-well plate; after 12h of culture, the corresponding concentration of the corresponding drug-containing culture medium was added, 3 replicates were set for each concentration, and the final volume of each well was 200μL. After 24h of culture, CCK-8 20μL / well was added, and after 1h of culture, the absorbance at 450nm was detected by an enzyme marker. Cell survival rate = (absorbance value of drug group / absorbance value of control group) × 100%, and IC was calculated by plotting cell survival rate and drug concentration. 50 Table 1 shows the IC50 values ​​of some compounds against 4T1 cells.

[0086] Compound <![CDATA[Normoxia IC50 (μM)]]> <![CDATA[Hypoxia IC50 (μM)]]> TPZ 22.98 9.72 TPZ-His 1.65 0.58 TPZ-Bza 2.06 0.87 TPZ-Hex 1.57 0.79 TPZ-Acp 1.66 0.60

[0087] Table 1

[0088] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A tirapazamine derivative, The specific structure is as follows: Formula (I-1); Formula (I-4); Formula (I-5).

2. A method for preparing the tirapazamine derivative according to claim 1, characterized in that: The steps include: A) Tirapazamine reacts with p-nitrophenyl chloroformate in a basic solvent to obtain an intermediate product TPZ-NPC; B) TPZ-NPC is obtained by reacting an amino compound in a solvent; the amino compound includes cyclopropylmethylamine, n-hexylamine and histamine; 。 3. The preparation method according to claim 2, characterized in that: The alkaline substance is triethylamine, pyridine, lutidine, and diisopropylethylamine; the solvent is tetrahydrofuran, acetonitrile, chloroform, dichloromethane, carbon tetrachloride, benzene, toluene, dimethyl sulfoxide, and dimethylformamide.

4. The preparation method according to claim 2, characterized in that: The reaction temperature of step A) is 20°C-40°C, and the reaction time is 12 h-48 h. The reaction temperature of step B) is 20°C-40°C, and the reaction time is 12 h-48 h.

5. The preparation method according to claim 4, characterized in that: The reaction temperature of step A) is 25°C and the reaction time is 24h; Step B) The reaction temperature is 25°C and the reaction time is 24h.

6. Use of the tirapazamine derivative and the pharmaceutically acceptable salt thereof according to claim 1 in the preparation of anticancer drugs; the cancer is breast cancer.

7. An anti-breast cancer drug, characterized in that: The invention comprises the tirapazamine derivative according to claim 1 or the tirapazamine derivative prepared by the preparation method according to any one of claims 2 to 5.

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