3-(1-(2, 4-dichlorobenzyl)-1H-indazole-3-yl) benzoyl hydrazine as well as preparation method and application thereof

Abstract

The invention discloses 3-(1-(2, 4-dichlorobenzyl)-1H-indazole-3-yl) benzoyl hydrazine with a chemical formula of C21H16Cl2N4O, and also discloses a preparation method of the 3-(1-(2, 4-dichlorobenzyl)-1H-indazole-3-yl) benzoyl hydrazine and an application of the 3-(1-(2, 4-dichlorobenzyl)-1H-indazole-3-yl) benzoyl hydrazine in synthesis of male contraceptive drugs. The 3-(1-(2, 4-dichlorobenzyl)-1H-indazole-3-yl) benzoyl hydrazine compound disclosed by the invention can cause the reduction of sperm concentration of male SD (Sprague Dawley) rats and ICR (Induced Cyclic Resonance) mice and the disorder of arrangement of testicular spermatogenic cells, and has a spermatogenic inhibition effect.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to 3-(1-(2,4-dichlorobenzyl)-1H-indazole-3-yl)benzoylhydrazide and a preparation method and application thereof. Background Art

[0002] As the world's overall economic level improves, people are facing increasing pressure to have children, and most couples have a need for contraception. Every year, there are as many as 90 million unintended pregnancies due to lack of contraception or contraceptive failure. Unintended pregnancies and the subsequent question of whether to have an abortion cause serious damage to women's physical and mental health. The development of new contraceptive methods can expand the range of contraceptive options for men and women and reduce the number of unintended pregnancies. Appropriate contraceptive technology can not only avoid unintended pregnancies, but also be beneficial to the health of women and families.

[0003] Among the many contraceptive methods, the most commonly used methods for women are oral long-acting contraceptives or short-acting contraceptives. Some women cannot take contraceptives due to their health conditions or the side effects of contraceptives. The most commonly used contraceptive methods for men are condoms and surgical vasectomy, but these two mainstream contraceptive methods are not suitable for all men. Some men are allergic to the latex material of condoms or are worried about irreversible damage caused by surgical operations and cannot effectively prevent pregnancy. The development of male contraceptives has progressed with the development of the basics of reproductive medicine. The occurrence and maturation of sperm are affected by many factors. Spermatogenesis requires spermatogonial stem cells to undergo mitosis and meiosis to form mature sperm. In the process of spermatogenesis, the normal development process is destroyed, spermatogenesis is impaired, and the number of mature sperm produced is reduced or the formed sperm is deformed and loses vitality, which can achieve the effect of male contraception.

[0004] In the study of lonidamine as a male contraceptive, it was found that the types of spermatogenic cells in the testicles changed, mainly the proportion of elongated spermatids decreased, the concentration of sperm in the epididymal head decreased, and the morphological changes of supporting cells. However, taking this drug is generally accompanied by many side effects such as myalgia, testicular pain, nausea and vomiting, and drowsiness. Since the contraceptive rate of lonidamine cannot reach 100% and the accompanying side effects, the research on its contraceptive effect has gradually shifted to the derivatives of lonidamine. By modifying the structure of lonidamine, the contraceptive efficiency can be improved and the side effects can be reduced. AF2785 (1-(2,4-dichlorobenzyl)-indazole-3-acrylic acid) targets rat sperm mitochondria and induces sperm mitochondria to reduce ATP production. AF1312 / TS can reduce the number of supporting cells and germ cells after oral administration. H2-gamendazole works by damaging the specialized function of the apical ectoplasm, but the recovery rate is only 57%. Adjudin (1-(2,4-dichlorobenzyl)-indazole-3-carboxylic acid hydrazide) acts locally in the apical compartment of the seminiferous epithelium behind the blood-testis barrier, destroying the adhesion of germ cells, especially the adhesion of sperm to supporting cells, causing immature sperm to be released from the epithelium, leading to infertility. In the research of these derivatives, Adjudin has entered the clinical trial stage, but due to the side effects of Adjudin such as liver muscle toxicity, the research on it has gradually stopped in recent years. These lonidamine derivatives mostly show good contraceptive effects on rats. At present, few studies on lonidamine derivatives are conducted on mice, so it is unknown whether these derivatives have contraceptive effects on mice, and there is little hope for their application to other mammals or even humans. Considering the good spermatogenesis inhibitory effect of lonidamine and its derivatives, the present invention transforms the structure of lonidamine to synthesize a new reversible male oral contraceptive compound.

[0005] The chemical structure modification of drugs usually retains the core functional groups, and then changes the structures of other functional groups, thereby changing the pharmacokinetic properties of the drugs, improving the efficacy of the drugs, reducing their toxic side effects, etc. In the modification ideas of lonidamine, considering the bioisosteric replacement effect of the benzene ring in the drug-target interaction, the bioisostere can be changed, the adverse effects related to the bioisosteric elements can be eliminated, or the bioavailability of the drug can be increased. Fluorine atoms are small in size and have high electronegativity. After the introduction of fluorine atoms, the electronic configuration of the entire molecule can be changed, intermolecular hydrogen bonds can be constructed, and the conformation of the molecule can be adjusted. Changing the electronic configuration of the molecule can reduce the alkalinity of the molecule, improve the affinity of the molecule with the receptor, thereby increasing the bioavailability of the drug, reducing the hydrolysis rate and enhancing the stability of the molecule. At the same time, it can also protect the CH bond, making the molecule easier to bind to the biological receptor. Amides and hydrazides are stable to mild alkaline hydrolysis conditions for releasing esters. Hydrazides are often used in the synthesis of antibiotics and peptides. In the synthesis of peptides, they can be converted into azides by nitrous acid, making condensation reactions easy to occur. Therefore, it is considered to introduce a hydrazide structure into lonidamine. Summary of the invention

[0006] Based on the above ideas, the purpose of the present invention is to provide a new lonidamine derivative, as well as a method for preparing the substance and its application in the preparation of male contraceptive drugs.

[0007] The present invention adopts such a technical solution: 3-(1-(2,4-dichlorobenzyl)-1H-indazol-3-yl)benzoylhydrazide has the following structural formula: .

[0008] In order to prepare the above-mentioned substance, the following technical scheme is also adopted: a method for preparing 3-(1-(2,4-dichlorobenzyl)-1H-indazol-3-yl)benzoylhydrazide, comprising the following steps: 1 equivalent of indazole, 2 equivalents of potassium hydroxide and 2 equivalents of iodine were mixed, 15 mL of N,N-dimethylformamide was added, and the mixture was stirred at room temperature for 2 hours. Sodium thiosulfate and sodium chloride solution were newly added until the color became transparent. After the solution became transparent, a precipitate appeared, which was extracted, washed and dried. The product was then dissolved in acetone, 1.1 equivalents of 2,4-dichloro-1-(chloromethyl)benzene and 4.4 equivalents of potassium carbonate were mixed, and the mixture was refluxed at 70° C. in an oil bath overnight. After the reaction was completed, the mixture was cooled to room temperature, filtered, and the residue was washed with acetone. The filtrate was concentrated by a rotary evaporator to obtain product A, which was then washed with dichloromethane. 1 equivalent of compound A, 1.5 equivalents of p-methoxycarbonylphenylboronic acid, 0.1 mmol of palladium acetate, 4 mL of saturated sodium carbonate aqueous solution, 1 mL of ethanol and 10 mL of toluene were mixed and stirred at 80°C for 12 hours. The reaction was completed after monitoring by TLC (thin layer chromatography), and the reaction mixture was extracted; washed, dried, concentrated in vacuo, and purified by petroleum ether / ethyl acetate silica column chromatography to obtain a white solid compound B; Dissolve 1 equivalent of compound B in ethanol, add 50 equivalents of hydrazine hydrate, mix, reflux at 80°C for 4 hours, remove volatiles under reduced pressure, wash and dry, and reduce pressure again to obtain the target crude product. The crude product is purified by column chromatography to obtain a white solid 3-(1-(2,4-dichlorobenzyl)-1H-indazol-3-yl)benzoylhydrazide.

[0009] The compound obtained above has the chemical formula C 21 H 16 C l2 N 4 O, physical and chemical properties: white solid, melting point 168-170℃, molecular weight 411.0774.

[0010] The compound obtained above can be used for preparing male contraceptive drugs.

[0011] Animal experiments have shown that the 3-(1-(2,4-dichlorobenzyl)-1H-indazol-3-yl)benzoylhydrazide compound of the present invention can cause a decrease in sperm concentration in male SD rats and ICR mice, disordered arrangement of testicular spermatogenic cells, and has a spermatogenic inhibitory effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The synthetic route map of the compound of the present invention; Figure 2 is the hydrogen nuclear magnetic resonance spectrum of 3-(1-(2,4-dichlorobenzyl)-1H-indazol-3-yl)benzohydrazide of the present invention; Figure 3 is the carbon nuclear magnetic resonance spectrum of 3-(1-(2,4-dichlorobenzyl)-1H-indazol-3-yl)benzoylhydrazide of the present invention; Figure 4 HE staining of the testicles of mice in each group after intragastric administration of different doses of the compounds of the present invention for 2 weeks; Figure 5 HE staining of epididymis of mice in each group after intragastric administration of different doses of the compounds of the present invention for 2 weeks; Figure 6 The figure is a line graph of the weight growth of mice in each group after intragastric administration of different doses of the compound of the present invention for 2 weeks, and an organ index graph of the heart, liver, lung, kidney and spleen; Figure 7 HE staining of the liver of mice in each group after intragastric administration of different doses of the compounds of the present invention for 2 weeks; Figure 8 HE staining of kidneys of mice in each group after intragastric administration of different doses of the compounds of the present invention for 2 weeks; Fig. 9 HE staining of spleen of mice in each group after intragastric administration of different doses of the compounds of the present invention for 2 weeks; Fig.10 HE staining images of the testicles of mice in the high- and low-dose groups after 6 and 12 weeks of drug administration in the present invention; Fig.11 HE staining images of epididymis of mice in high and low dose groups after 6 and 12 weeks of drug administration in the present invention; Fig.12 The fertility statistics of mice in the high and low dose groups after 6 and 12 weeks of drug administration in the present invention; Fig.13 The HE staining images of rat testicles after the rats were given the drug for 2 weeks and 6 weeks according to the present invention; Fig.14 This is a statistical chart of rat fertility after the drug of the present invention was administered to rats for 2 weeks and 6 weeks. DETAILED DESCRIPTION

[0013] Example 1 Preparation of Compound 3-(1-(2,4-dichlorobenzyl)-1H-indazol-3-yl)benzohydrazide See also Figure 1 . Mix 1 equivalent of indazole, 2 equivalents of potassium hydroxide and 2 equivalents of iodine, add 15 mL of N,N-dimethylformamide, stir at room temperature for 2 hours, add new sodium thiosulfate and sodium chloride solution until the color becomes transparent, and a precipitate will appear after the solution becomes transparent, extract, wash and dry. The product is then dissolved in acetone, 1.1 equivalents of 2,4-dichloro-1-(chloromethyl)benzene, 4.4 equivalents of potassium carbonate are mixed, and refluxed at 70°C in an oil bath overnight. After the reaction is completed, cool to room temperature, filter, and wash the residue with acetone. The filtrate is concentrated by rotary evaporator, and the concentrated product A is washed with dichloromethane. The yield of this step is 81%.

[0014] 1 equivalent of compound A, 1.5 equivalents of p-methoxycarbonylphenylboronic acid, 0.1mmol of palladium acetate, 4mL of saturated sodium carbonate aqueous solution, 1mL of ethanol and 10mL of toluene were mixed and stirred at 80°C for 12 hours. The reaction was completed by TLC monitoring and the reaction mixture was extracted. The product obtained after washing, drying and vacuum concentration was purified by petroleum ether / ethyl acetate silica column chromatography to make compound B a white solid. 1 equivalent of compound B was dissolved in ethanol, 50 equivalents of hydrazine hydrate were added, and the mixture was refluxed at 80°C for 4 hours. The volatiles were removed under reduced pressure, washed and dried, and the target crude product was obtained after reduced pressure again. The crude product was purified by column chromatography to obtain a white solid 3-(1-(2,4-dichlorobenzyl)-1H-indazol-3-yl)benzoylhydrazide.

[0015] The obtained compounds were tested for molecular biological activity using nuclear magnetic resonance (NMR), and the following Figure 2 , and the spectra shown in 3. Figure 2 The hydrogen spectrum of the NMR (400 MHz, solvent: CDCl 3 ): Chemical shift (δ) 8.36 (triplet, coupling constant 1.6 Hz, 1H); Chemical shift (δ) 8.15 (double triplet, coupling constant 7.6, 1.2 Hz, 1H); Chemical shift (δ) 8.06 (doublet, coupling constant 8.0 Hz, 1H); Chemical shift (δ) 7.79 (double triplet, coupling constant 8.0, 1.2 Hz, 1H); Chemical shift (δ) 7.60 (triplet, coupling constant 7.6 Hz, 1H); Chemical shift (δ) 7.50 (singlet, 1H); Chemical shift (δ) 7.44 (doublet, coupling constant 2.4 Hz, 1H); chemical shift (δ) 7.43-7.37 (multiplet, 2H); chemical shift (δ) 7.31-7.27 (multiplet, 1H); chemical shift (δ) 7.09 (doublet of doublets, coupling constant 8.4, 2.4 Hz, 1H); chemical shift (δ) 6.72 (doublet, coupling constant 8.4 Hz, 1H); chemical shift (δ) 5.73 (singlet, 2H); chemical shift (δ) 4.15 (singlet, 2H).

[0016] Figure 3 From the carbon spectrum, it can be seen that the carbon NMR spectrum (100 MHz, solvent: DMSO), the chemical shift (δ) are 165.9, 142.9, 141.4, 134.2, 133.9, 133.3, 133.21, 133.16, 130.7, 129.6,129.2, 129.1, 127.8, 127.0, 126.6, 125.5, 121.9, 121.2, 120.9, 110.3, 49.3 respectively.

[0017] Example 2 Animal Experiment (1) Experiment on the effect of drugs on spermatogenesis in ICR mice 1) Grouping: Divide the mice into 5 groups, with 10 ICR male mice in each group; 2) Oral gavage, at doses of 50 mg / kg, 100 mg / kg, 200 mg / kg and 500 mg / kg; 3) After intragastric administration, the mice were weighed and recorded every day; 4) Two weeks after oral administration, the mice were euthanized by cervical dislocation, and the liver, heart, spleen, lung, kidney, testis, and epididymis were removed and weighed and recorded. The liver, spleen, kidney, testis, and epididymis were taken for paraffin section HE staining; 5) Organ index calculation formula: organ weight (mg) / mouse body weight (g) × 100%; (2) Mouse fertility restoration experiment 1) Divide the mice into 3 groups, with 30 mice in each group; 2) Group A was the normal control group, Group B was the low-dose group (50 mg / kg), and Group C was the high-dose group (500 mg / kg); 3) Gavage administration, 2 weeks later, 10 mice were selected from each group, 5 of which had their testicles taken for paraffin section HE staining, and the other 5 were placed in a cage with two 8-week-old ICR female mice for one week. After 19-24 days, the fertility of the female mice was observed and the number of pups was counted; 4) After 6 weeks, 10 mice were selected from each group, and testicles of 5 mice were taken for paraffin section HE staining. The other 5 mice were placed in a cage with two 8-week-old ICR female mice for one week. After 19-24 days, the fertility of the female mice was observed and the number of pups was counted. 5) After 12 weeks, 10 mice were selected from each group, and testicles of 5 mice were taken for paraffin section HE staining. The other 5 mice were placed in a cage with two 8-week-old ICR female mice for one week. After 19-24 days, the fertility of the female mice was observed and the number of pups was counted. 6) Calculation method of fertility index: number of pregnant female mice in each group / number of female mice in each group cage × 100%.

[0018] (3) Experiment on the effect of drugs on spermatogenesis in SD rats 1) Rats were divided into 2 groups, with 20 rats in each group; 2) Group A was the normal control group, and Group B was the compound administration group, which was intragastrically administered at a dose of 100 mg / kg; 3) Two weeks after oral administration, 5 rats were taken from each group for carbon dioxide euthanasia, and the testicles and epididymis were removed and placed in MDF fixative for HE staining; another 5 rats were taken and placed in cages with 2 8-week-old female rats for one week. After 19-24 days, the birth of the female rats was observed and the number of pups was counted; 4) After 6 weeks of administration, 5 rats were taken from each group and euthanized with carbon dioxide. The testicles and epididymis were removed and placed in MDF fixative for HE staining. Another 5 rats were placed in cages with 2 8-week-old female rats for one week. After 19-24 days, the birth of the female rats was observed and the number of pups was counted.

[0019] The experimental results can be found in Figures 4 to 14 .

[0020] Figure 4 The results showed that HE staining of the testes of mice after oral administration of different doses of compounds for 2 weeks showed that the degree of morphological damage of the testes of mice in each group gradually increased with the increase of drug dose. In the 50mg / kg group, part of the germ cell layer was retained in the testes, and the diameter of the seminiferous tubule lumen did not change significantly; in the 100mg / kg group, more testicular germ cells fell off, the germ cells were arranged in disorder, and the diameter of the seminiferous tubule lumen became smaller; in the 200mg / kg group, the testicular germ cells fell off significantly, and only one layer of the remaining germ cells remained, and the diameter of the seminiferous tubule was deformed and reduced; in the 500mg / kg group, almost all the testicular germ cells fell off, and the diameter of the seminiferous tubule was vacuolated, and only a small number of residual germ cells were observed, the diameter of the tubule became smaller, and the number of interstitial cells increased.

[0021] Figure 5 The results showed that HE staining of the mouse epididymis 2 weeks after oral administration of different doses of the compound showed that in the 50 mg / kg group, most of the epididymal lumens were free of sperm, but a small number of them were found; in the 100 mg / kg group, no sperm was found in the epididymal lumens, and there was no obvious change in the diameter of the lumens; in the 200 mg / kg group, no sperm was found in the epididymal lumens, and vacuoles were present; in the 500 mg / kg group, no sperm was found in the epididymal lumens, the cells were disordered, and a large number of vacuoles were present.

[0022] Figure 6 The line graphs of the weight growth of mice in each group during the two-week oral administration of different doses of compounds are shown, and the organ indexes of the heart, liver, lung, kidney and spleen of mice in each group after two weeks of oral administration. The weight of mice at 50mg / kg and 100mg / kg showed a slow growth trend every day, and the weight decreased slightly on the 14th day; the weight of mice at 200mg / kg showed a slow growth trend every day, and the weight decreased slightly on the 10th day, and then continued to increase, and the weight decreased slightly on the 14th day; the weight increase of mice at 500mg / kg was the smallest, and the weight decreased slightly on the 10th day, and then continued to increase, and the weight decreased slightly on the 14th day. There was no statistical difference in the weight changes of mice after oral administration of different doses. The heart index of mice gavaged with 100mg / kg, 200mg / kg and 500mg / kg drugs decreased slightly, but the decrease in heart index in each group was not statistically different from that in the control group; there was no significant difference in the liver, lung, kidney and spleen indexes among the groups.

[0023] Figure 7 HE staining of mouse livers 2 weeks after oral administration of different doses of compounds is shown. The liver sections of mice in different dose groups all show clear demarcation of the hepatic lobule, a central vein in the center of the hepatic lobule, and hepatocytes arranged radially around the central vein. The nuclei are large and round, centered, with little heterochromatin and light coloring, and the nucleoli can be clearly seen. The hepatocyte morphology is intact, the nucleus is not enlarged, and the hepatic lobule morphology is intact without inflammatory cell infiltration.

[0024] Figure 8HE staining of the mouse kidneys showed that after oral administration of different doses of compounds for 2 weeks, the cells in the kidneys of the mice in each group did not enlarge, no germinal centers or transformed cells appeared, and no abnormal changes such as inflammation were found.

[0025] Fig. 9 HE staining of the spleen of mice 2 weeks after oral administration of different doses of compounds showed that the spleen trabeculae in the spleen of mice in each group had intact structure, the red pulp contained a large number of red blood cells, lymphocytes, macrophages, plasma cells, etc., and the arrangement of splenic bodies was not disordered.

[0026] Fig.10 HE staining of the testicles of mice at 6 and 12 weeks after administration showed that 6 weeks after administration, most of the seminiferous tubules in the testicles of the 50 mg / kg group mice returned to normal morphology, and a small number of vacuoles were present in some seminiferous tubules in the testicles of the 500 mg / kg group mice; 12 weeks after administration, the testicles of both groups had basically returned to normal morphology.

[0027] Fig.11 The results showed that HE staining of the epididymis of mice 6 and 12 weeks after administration showed that 6 weeks after administration, a large number of sperm were seen in the epididymis of mice in the 50 mg / kg group, while fewer sperm were seen in the epididymis of mice in the 500 mg / kg group. 12 weeks after administration, a large number of sperm were seen in the epididymis of both groups.

[0028] Fig.12 The fertility statistics of mice were 2 weeks, 6 weeks and 12 weeks after administration. The pregnancy rates of female mice paired with male mice in both groups were 0 2 weeks after administration. The pregnancy rates of female mice paired with male mice in the 50 mg / kg group were about 60% and 500 mg / kg group respectively 6 weeks after administration. The pregnancy rates of female mice paired with male mice in the 50 mg / kg group and 500 mg / kg group respectively 12 weeks after administration reached 100% and 90%.

[0029] Fig.13 HE staining of the testicles and epididymis of rats 2 and 6 weeks after administration showed that 2 weeks after administration, the structure in the testis was obviously destroyed, the diameter of the seminiferous tubules became smaller, and a large number of germ cells fell off; after 6 weeks of administration, almost all the germ cells fell off; no sperm was visible in the epididymis.

[0030] Fig.14 The fertility statistics of rats were 2 weeks and 6 weeks after drug administration. The pregnancy rate of female rats paired with rats before drug administration was 100%, the pregnancy rate of female rats paired with male rats 2 weeks after drug administration was 0%, and the pregnancy rate of female rats paired with male rats 6 weeks after drug administration was 15%.

[0031] The above animal experiments show that the fertility of the test subjects in the examples is significantly suppressed in the short term, but can be restored after a period of time. From the data, the present invention can achieve the purpose of the invention.

Claims

1. 3-(1-(2,4-dichlorobenzyl)-1H-indazol-3-yl)benzoylhydrazide, characterized in that: It has the following structural formula: 。 2. The method for preparing 3-(1-(2,4-dichlorobenzyl)-1H-indazol-3-yl)benzoylhydrazide according to claim 1, comprising the following steps: 1 equivalent of indazole, 2 equivalents of potassium hydroxide and 2 equivalents of iodine were mixed, 15 mL of N,N-dimethylformamide was added, and the mixture was stirred at room temperature for 2 hours. Sodium thiosulfate and sodium chloride solution were newly added until the color became transparent. After the solution became transparent, a precipitate appeared, which was extracted, washed and dried. The product was then dissolved in acetone, 1.1 equivalents of 2,4-dichloro-1-(chloromethyl)benzene and 4.4 equivalents of potassium carbonate were mixed, and the mixture was refluxed at 70° C. in an oil bath overnight. After the reaction was completed, the mixture was cooled to room temperature, filtered, and the residue was washed with acetone. The filtrate was concentrated by a rotary evaporator to obtain product A, which was then washed with dichloromethane. 1 equivalent of compound A, 1.5 equivalents of p-methoxycarbonylphenylboronic acid, 0.1 mmol of palladium acetate, 4 mL of saturated sodium carbonate aqueous solution, 1 mL of ethanol and 10 mL of toluene were mixed and stirred at 80°C for 12 hours. The reaction was completed after monitoring by TLC (thin layer chromatography), and the reaction mixture was extracted; washed, dried, concentrated in vacuo, and purified by petroleum ether / ethyl acetate silica column chromatography to obtain a white solid compound B; Dissolve 1 equivalent of compound B in ethanol, add 50 equivalents of hydrazine hydrate, mix, reflux at 80°C for 4 hours, remove volatiles under reduced pressure, wash and dry, and reduce pressure again to obtain the target crude product. The crude product is purified by column chromatography to obtain a white solid 3-(1-(2,4-dichlorobenzyl)-1H-indazol-3-yl)benzoylhydrazide.

3. The 3-(1-(2,4-dichlorobenzyl)-1H-indazol-3-yl)benzohydrazide of claim 1 is used in the preparation of male contraceptive drugs.

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