WO2018201939A1 - Prodrug of benzoindazole derivative, preparation method therefor, and application thereof - Google Patents

Abstract

The present invention relates to a prodrug of a benzoindazole derivative and a pharmaceutically acceptable salt thereof, a preparation method therefor, and an application thereof. The benzoindazole derivative prodrug has a general structural formula of formula I, wherein R is selected from various amino acids, sugars, and other basic groups. The benzoindazole derivative prodrug has the following advantage: the benzoindazole derivative prodrug of the present invention and its salt structure resolve the problem of poor water solubility of compounds in existing patents, and can be used for intravenous injections. The benzoindazole derivative prodrug and the salt thereof of the present invention can inhibit tubulin polymerization, and have antitumor activity for leukemia, non-small cell lung cancer, colon cancer, central nervous system tumor, melanoma, ovarian cancer, renal cancer, prostate cancer, breast cancer, and the like.

Description

Prodrug of benzoxazole derivative, preparation method and application thereof Technical field

The invention relates to the technical field of medicinal chemistry, in particular to a prodrug of a benzoxazole derivative (TCd-1), a preparation method thereof and an application thereof.

Background technique

Tumors, especially malignant tumors, have seriously threatened human health. The Global Cancer Report 2014 published by the World Health Organization (WHO) shows that the incidence of cancer in developing countries in Africa, Asia and Central and South America is the most severe. In 2012, there were 14 million new cancer cases worldwide and 8.2 million deaths. Among them, China added 3.07 million cancer patients and caused about 2.2 million deaths, accounting for 21.9% and 26.8% of the global total, respectively. The report predicts a rapid increase in global cancer cases, from 14 million in 2012 to 19 million in 2025 and 24 million by 2035. Although anti-tumor drugs have contributed greatly to prolonging patients' survival and improving their quality of life, most drugs are cytotoxic drugs, and their lack of selectivity can damage a large number of normal cells and are prone to drug resistance. Therefore, tumor vascular targeting drugs have become a research hotspot.

Microtubules are composed of tubulin and microtubule-binding proteins and are the main components of the cytoskeleton. Microtubules have the kinetic properties of polymerization and depolymerization, and play an important role in maintaining cell morphology, cell division and proliferation, organelle composition and transport, and signal material conduction. The anti-tumor drug targeting microtubules utilizes its kinetic properties, or promotes its depolymerization or inhibits its polymerization, thereby directly affecting the mitosis of cells, affecting many normal physiological functions of cells, and stopping cell division in M phase. . Studies have shown that there are three different drug binding sites in the microtubule: the paclitaxel site, the vincristine site, and the colchicine site. Since the cavity volume of the colchicine site is small and the structure of the corresponding inhibitor is relatively simple, research on its inhibitor has also received much attention in recent years.

The inhibitory colchicine treatment window originally discovered at this site is narrow, which limits its clinical application. The discovery of the stilbene natural inhibitor, combretastatins, has attracted great attention from researchers, representing the compound Combretastatin A-4 (CA-4), which exhibits high inhibition of microtubule polymerization and antitumor activity. Tumor vascular blocking effect. The researchers have carried out a large number of structural modifications to CA-4, and several analogs have entered clinical research, showing good prospects for development. Among them, CA-4P granted its status as an orphan drug for ovarian cancer in July 2013. Currently, CA-4P has carried out clinical research on undifferentiated thyroid cancer, neuroendocrine tumor and ovarian cancer.

Chinese Patent Application No. 201510740269.8, filed on Nov. 4, 2015, the filing date of which is hereby incorporated by reference in its entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all all However, the prodrug of the benzoxazole derivative (TCd-1) of the present invention, its preparation method and application have not been reported yet.

Summary of the invention

The object of the present invention is to provide a prodrug of a benzoxazole derivative (TCd-1) against the deficiencies in the prior art.

Still another object of the present invention is to provide a process for preparing a prodrug of a benzoxazole derivative (TCd-1).

Another object of the present invention is to provide a prodrug of a benzoxazole derivative (TCd-1).

In order to achieve the above object, the technical solution adopted by the present invention is: a compound represented by the formula (I),

Wherein R is selected from the group consisting of amino acids, saccharides, and other groups having a basic group.

The basic group is an amino group.

The structural formula of the compound is:

A pharmaceutically acceptable salt of the compound.

The salt is the hydrochloride salt.

The structural formula of the salt is:

In order to achieve the above second object, the technical solution adopted by the present invention is:

The synthetic route is:

In order to achieve the above third object, the technical solution adopted by the present invention is: application in the preparation of a tubulin polymerization inhibitor.

The use in the preparation of a medicament for treating a tumor disease.

The tumor disease is leukemia, non-small cell lung cancer, colon cancer, central nervous system tumor, melanoma, ovarian cancer, kidney cancer, prostate cancer or breast cancer.

The advantages of the invention are:

1. The benzoxazole derivative prodrug of the present invention and its salt structure solve the problem of poor water solubility of the compound in the prior patent, and can be used for intravenous injection.

2. The prodrug of the benzoxazole derivative of the present invention inhibits tubulin polymerization, for leukemia, non-small cell lung cancer, colon cancer, central nervous system tumor, melanoma, ovarian cancer, renal cancer, prostate cancer, and breast. Cancer and the like have antitumor activity.

detailed description

The specific embodiments provided by the present invention will be described in detail below with reference to the embodiments.

The specific synthetic route of the compounds of the invention is as follows:

Reagents and conditions: (a) BOC-L-serine, propylphosphoric anhydride (T3P), triethylamine, anhydrous dichloromethane, reflux; (b) methanolic hydrochloric acid solution, methanol.

Example 1: (S)-3-Hydroxy-2-((tert-butoxycarbonyl)amino)-N-(2-methoxy-5-(6,7,8-trimethoxy-4,5- Dihydro-2H-benzo[e]carbazole-1-position)phenyl)propanamide (TCd-1-PB)

The starting material 6,7,8-trimethoxy-1-(3-amino-4-methoxyphenyl)-2-phenyl-4,5-dihydro-2H-benzo[e]carbazole TCd-1) (10 mmol, 1.0 equiv) and BOC-L-serine (12 mmol, 1.2 equiv) were dissolved in 100 mL of anhydrous dichloromethane, and then evaporated, and then triethylamine (27 mmol, 2.7 equiv). Then propylphosphoric anhydride (T3P) (17 mmol, 1.7 equiv) was slowly added under ice-cooling conditions and stirred for 10 minutes. The temperature was then raised to reflux for 6 hours and monitored by TLC. After the reaction was completed, it was cooled to room temperature, and 100 mL of water was added. After stirring for 5 minutes, the mixture was separated and evaporated, evaporated, evaporated, evaporated The yield was 39%. _{^{1 H NMR (400MHz, CDCl 3}} ) δ9.08 (s, 1H), 8.56 (s, 1H), 7.34 (dd, J = 8.5,2.0Hz, 1H), 6.82 (d, J = 8.3Hz, 1H) , 6.75 (s, 1H), 6.01 (d, J = 7.6 Hz, 1H), 4.38 (s, 1H), 4.30 (dd, J = 11.5, 2.6 Hz, 1H), 3.86 (s, 3H), 3.85 ( s, 3H), 3.83 (s, 2H), 3.75 (dd, J = 11.9, 4.6 Hz, 1H), 3.52 (s, 3H), 2.95 (t, J = 7.1 Hz, 2H), 2.78 (t, J = 7.3 Hz, 2H), 1.49 (s, 9H). ESI-MS m/z = 569.3 [M + 1] ⁺ .

Example 2: (S)-3-Hydroxy-2-amino-N-(2-methoxy-5-(6,7,8-trimethoxy-4,5-dihydro-2H-benzo[ e] carbazole-1-position) phenyl) propionamide hydrochloride (TCd-1-PH)

Starting material (S)-3-hydroxy-2-((tert-butoxycarbonyl)amino)-N-(2-methoxy-5-(6,7,8-trimethoxy-4,5-dihydro -2H-benzo[e]carbazole-1-position)phenyl)propanamide (TCd-1-PB) (1.7 g, 3 mmol) was dissolved in 30 mL of methanol. M, 40 mL), then stirred at room temperature overnight. After the completion of the reaction, most of the solution was removed under reduced pressure, poured into 50 mL of ice water, and the mixture was adjusted to neutral with a saturated sodium hydrogen carbonate solution, and a white floc was precipitated. The free base was filtered, and the free base was dried and dissolved in methylene chloride (40mL), and 4M hydrochloric acid was slowly added dropwise, and a white solid was precipitated. When the appearance of the appearance of hydrochloric acid was observed by visual observation, the dropping was stopped. The beige product was obtained by filtration, yield 72%. ^{1 H NMR (400MHz, DMSO)} δ9.92 (s, 1H), 8.35 (s, 1H), 8.34 (s, 1H), 8.28 (d, J = 1.6Hz, 1H), 7.36 (d, J = 8.4 Hz, 1H), 7.23 (d, J = 8.5 Hz, 1H), 6.70 (s, 1H), 6.17 (s, 1H), 4.23 (dd, J = 9.7, 5.0 Hz, 1H), 3.91 (s, 3H) ), 3.82 (d, J = 4.6 Hz, 2H), 3.75 (s, 3H), 3.71 (s, 3H), 3.45 (s, 3H), 2.89 (t, J = 7.0 Hz, 2H), 2.74 (t , J = 6.9 Hz, 2H). ESI-MS m/z = 469.3 [M + 1] ⁺ .

Example 3: Determination of Solubility of Original Drug TCd-1 and Prodrug Hydrochloride TCd-1-PH

The HPLC method was used to test the solubility of the sample. The standard curve was obtained by accurately weighing the appropriate amount of the sample, gradually adding physiological saline, shaking it to a sufficient concentration in the shaker, and detecting by HPLC. A sample saturated solution was then configured in a similar manner and tested by HPLC. The results showed that the solubility of the compound TCd-1 in physiological saline was much less than 0.001 g/mL, while the solubility of the prodrug hydrochloride TCd-1-PH was more than 0.945 g/mL, which was more than 945 times higher than that of the original drug.

Example 4: Prodrug hydrochloride TCd-1-PH in vivo activity test

The anti-tumor effect of the sample was evaluated using a human colon cancer colo205 model transplanted into nude mice. The sample was injected intravenously 4 times at a dose of 20 mg/kg once every 4 days. Compared with the control group, the sample has a tumor inhibition rate of >50% on human colon cancer colo205 xenografts in nude mice, and has obvious anti-tumor activity. The body weight of the nude mice in the experimental group was not significantly different from that of the control group, and the sample showed no obvious toxicity.

Example 5: Synthesis method and activity experiment of related compounds

Starting material 6,7,8-trimethoxy-1-(3-amino-4-methoxyphenyl)-2-phenyl-4,5-dihydro-2H-benzo[e]carbazole (TCd) For the synthesis of -1), see the patent "Benzoxazole derivatives and their preparation methods and applications" (Application No. 201510740269.8).

The synthesis of the starting material 6-methoxy-3,4-dihydronaphthalene-2(1H)-one (1) is described in J Med Chem 2012, 55, 5720-5733.

For the synthesis of the starting material 5,6,7-trimethoxy-3,4-dihydronaphthalen-2-one (2), see the patent "Substituted benzylidene tetralone derivatives and preparation methods and applications" (application) No.: 201510064131.0).

6-methoxy-1-(3,4,5-trimethoxyphenyl)-3,4-dihydronaphthalene-2(1H)-one (S-dk)

The raw material 6-methoxy-3,4-dihydronaphthalene-2(1H)-one (1) (5.68 mmol, 1.0 equiv) was dissolved in 20 mL of anhydrous THF, protected with nitrogen, and added to a salt bath. LDA (6.24 mmol, 1.1 equiv) was added. After stirring for 4 hr, a solution of 3,4,5-trimethoxybenzoyl chloride in THF (6.24 mmol, 1.1 equiv) was added. After completion of the reaction, the mixture was quenched by the addition of a saturated aqueous solution of ammonium chloride. The reaction mixture was extracted with EtOAc (EtOAc (EtOAc)EtOAc. The product was a yellow solid with a yield of 61%. _{^{1 H NMR (600MHz, CDCl 3}} ) δ6.77 (s, 2H), 6.72 (d, J = 2.3Hz, 1H), 6.66 (d, J = 8.6Hz, 1H), 6.47 (dd, J = 8.6, 2.5 Hz, 1H), 3.92 (d, J = 6.2 Hz, 1H), 3.88 (s, 3H), 3.75 (s, 3H), 3.69 (s, 6H), 2.93 (t, J = 6.8 Hz, 2H) , 2.68–2.52 (m, 2H). HRMS (ES+) m/z found 371.1486 (M+H ⁺ ); C ₂₁ H ₂₃ O ₆ (M+H ⁺ ) require 371.1489.

7-Methoxy-1-(3,4,5-trimethoxyphenyl)-4,5-dihydro-2H-benzo[e]carbazole (SC1)

Ethanol, intermediate S-dk and an equivalent amount of hydrazine hydrate were successively added to a 100 mL flask, stirred overnight, and concentrated and purified to give a white solid. 172-173 ° C, ¹ H NMR (600 MHz, CDCl ₃ ) δ 7.35 (d, J = 8.5 Hz, 1H), 6.84 (s, 2H), 6.82 (d, J = 2.5 Hz, 1H), 6.64 ( Dd, J = 8.5, 2.7 Hz, 1H), 3.92 (s, 3H), 3.83 (s, 6H), 3.79 (s, 3H), 2.99 (t, J = 7.2 Hz, 2H), 2.83 (t, J = 7.2 Hz, 2H), ¹³ C NMR (150MHz, CDCl ₃ ) δ 157.92, 153.57, 148.74, 140.29, 138.47, 136.92, 126.69, 124.28, 123.15, 114.38, 113.61, 111.66, 105.49, 61.01, 56.28, 55.28, 30.46, 21.81.HRMS(ES+)m/z found 367.1653(M+H ⁺ ); C ₂₁ H ₂₃ N ₂ O ₄ (M+H ⁺ )requires 367.1652.

7-Methoxy-2-methyl-1-(3,4,5-trimethoxyphenyl)-4,5-dihydro-2H-benzo[e]carbazole (SC2)

Referring to the synthesis of SC1, the intermediate S-dk and methylhydrazine were reacted to give SC2 as a white solid. 157-158 ° C, ¹ H NMR (600 MHz, CDCl ₃ ) δ 7.33 (d, J = 8.5 Hz, 1H), 6.89 (s, 2H), 6.81 (d, J = 2.4 Hz, 1H), 6.55 ( Dd, J=8.5, 2.6 Hz, 1H), 3.95 (s, 3H), 3.90 (s, 3H), 3.84 (s, 6H), 3.78 (s, 3H), 3.03 (t, J = 7.5 Hz, 2H) ), 2.89 (t, J = 7.3 Hz, 2H). ¹³ C NMR (151MHz, CDCl ₃ ) δ 157.84, 153.78, 138.74, 136.76, 125.73, 123.66, 114.42, 111.74, 106.95, 61.05, 56.37, 55.26, 36.66, 30.34 , 21.92.HRMS(ES+)m/z found 381.1821(M+H ⁺ ); C ₂₂ H ₂₅ N ₂ O ₄ (M+H ⁺ )requires 381.1809.

7-Methoxy-2-hydroxyethyl-1-(3,4,5-trimethoxyphenyl)-4,5-dihydro-2H-benzo[e]carbazole (SC3)

Referring to the synthesis of SC1, the intermediate S-dk and hydroxyethylhydrazine were reacted to give SC3 as a white solid. Melting point 143-144 ° C, ¹ H NMR (600 MHz, CDCl ₃ ) δ 6.86 (d, J = 8.5 Hz, 1H), 6.78 (d, J = 2.6 Hz, 1H), 6.63 (s, 2H), 6.55 ( Dd, J = 8.5, 2.7 Hz, 1H), 4.07 - 4.04 (m, 2H), 3.97 (d, J = 5.1 Hz, 2H), 3.95 (s, 3H), 3.84 (s, 6H), 3.77 (s , 3H), 3.01 (t, J = 7.2 Hz, 2H), 2.89 (t, J = 7.2 Hz, 2H). ¹³ C NMR (150 MHz, CDCl ₃ ) δ 157.72, 153.73, 149.38, 138.61, 138.08, 136.80, 125.94 ,123.71,123.16,114.81,114.39,111.69,107.20,62.17,61.03,56.34,55.24,50.31,30.46,22.19.HRMS(ES+)m/z found 411.1910(M+H ⁺ );C ₂₃ H ₂₇ N ₂ O ₅ (M+H ⁺ )requires 411.1914.

7-Methoxy-2-phenyl-1-(3,4,5-trimethoxyphenyl)-4,5-dihydro-2H-benzo[e]carbazole (SC4)

Referring to the synthesis of SC1, the intermediate S-dk and phenylhydrazine were reacted to give SC4 as a gray solid. Mp 142-143 ° C, ¹ H NMR (600 MHz, CDCl ₃ ) δ 7.30 - 7.20 (m, 5H), 7.08 (d, J = 8.5 Hz, 1H), 6.83 (d, J = 2.6 Hz, 1H), 6.60 (dd, J=8.6, 2.7 Hz, 1H), 6.52 (s, 2H), 3.92 (s, 3H), 3.79 (s, 3H), 3.66 (s, 6H), 3.06 (t, J = 7.1 Hz) , 2H), 3.00–2.94 (m, 2H). HRMS (ES+) m/z found 443.1969 (M+H ⁺ ); C ₂₇ H ₂₇ N ₂ O ₄ (M+H ⁺ ) require 443.1965.

Ethyl 2-(7-methoxy-1-(3,4,5-trimethoxyphenyl)-4,5-dihydro-2H-benzo[e]indazole-2-)acetate (SC5 )

Referring to the synthesis of SC1, the intermediate S-dk and ethyl 2-mercaptoacetate were reacted to give SC4 as a gray solid. Melting point 118 - 119 ° C, ¹ H NMR (600 MHz, CDCl ₃ ) δ 6.90 (d, J = 8.5 Hz, 1H), 6.79 (d, J = 2.6 Hz, 1H), 6.66 (s, 2H), 6.56 ( Dd, J = 8.5, 2.7 Hz, 1H), 4.70 (s, 2H), 4.20 (q, J = 7.1 Hz, 2H), 3.95 (s, 3H), 3.81 (s, 6H), 3.77 (s, 3H) ), 3.02 (t, J = 7.2 Hz, 2H), 2.91 (t, J = 7.2 Hz, 2H), 1.26 (t, J = 7.1 Hz, 3H). ¹³ C NMR (151 MHz, CDCl ₃ ) δ 168.71, 157.77 , 153.67, 150.06, 138.62, 138.38, 136.94, 125.80, 123.78, 123.19, 115.21, 114.33, 111.72, 106.98, 102.71, 61.68, 61.01, 56.25, 55.24, 50.62, 42.65, 30.44, 25.62, 23.07, 22.21, 14.16, 14.02 .HRMS(ES+)m/z found 453.2023(M+H ⁺ ); C ₂₅ H ₂₉ N ₂ O ₆ (M+H ⁺ )requires 453.2020.

2-(7-Methoxy-1-(3,4,5-trimethoxyphenyl)-4,5-dihydro-2H-benzo[e]indazol-2-)acetic acid (SC6)

Ethyl 2-(7-methoxy-1-(3,4,5-trimethoxyphenyl)-4,5-dihydro-2H-benzo[e]indazole-2-)acetate ( SC5) was dissolved in ethanol, added with 5 times equivalent of aqueous NaOH solution, stirred overnight, neutralized with dilute hydrochloric acid to pH <7, extracted with dichloromethane, and concentrated with organic layer to obtain SC6 as a white solid. _{^{1 H NMR (600MHz, CDCl 3}} ) δ6.90 (d, J = 8.5Hz, 1H), 6.78 (d, J = 2.6Hz, 1H), 6.63 (s, 2H), 6.57 (dd, J = 8.5, 2.7 Hz, 1H), 4.74 (s, 2H), 3.95 (s, 3H), 3.82 (s, 6H), 3.77 (s, 3H), 3.12 (m, 2H), 2.96 (m, 2H). HRMS ( ES+)m/z found 425.1699(M+H+); C23H25N2O6(M+H+)requires 425.1707.

5,6,7-trimethoxy-1-(4-methoxyphenyl)-3,4-dihydronaphthalene-2(1H)-one (T-dk-a)

Referring to the synthesis of S-dk, T-dk-a was prepared by reacting 5,6,7-trimethoxy-3,4-dihydronaphthalen-2-one (2) with 4-methoxybenzoyl chloride. It is a yellow solid. Melting point 86-87 ° C, ¹ H NMR (600 MHz, CDCl ₃ ) δ 7.51 (d, J = 8.8 Hz, 2H), 6.85 (d, J = 8.8 Hz, 2H), 6.06 (s, 1H), 3.88 ( s, 3H), 3.88 (s, 1H), 3.85 (s, 3H), 3.83 (s, 3H), 3.29 (s, 3H), 2.92 (t, J = 6.9 Hz, 2H), 2.61 - 2.53 (m , 2H).HRMS(ES+)m/z found 371.1481(M+H ⁺ ); C ₂₁ H ₂₃ O ₆ (M+H ⁺ )requires 371.1489.

5,6,7-trimethoxy-1-(3-fluoro-4-methoxyphenyl)-3,4-dihydronaphthalene-2(1H)-one (T-dk-b)

Preparation of T by reaction of S-dk with 5,6,7-trimethoxy-3,4-dihydronaphthalen-2-one (2) and 3-fluoro-4-methoxybenzoyl chloride -dk-b is a yellow solid. ¹ H NMR (600 MHz, CDCl ₃ ) δ 7.32 (dd, J = 11.7, 1.9 Hz, 1H), 7.30 (d, J = 9.2 Hz, 1H), 6.89 (t, J = 8.4 Hz, 1H), 6.05 (s, 1H), 3.91 (s, 3H), 3.89 (s, 3H), 3.85 (s, 3H), 3.33 (s, 3H), 2.93 (t, J = 6.9 Hz, 2H), 2.61 - 2.52 ( m,2H). ¹³ C NMR (150MHz, CDCl ₃ ) δ 197.21, 180.59, 152.02, 150.51, 150.38, 149.72, 149.65, 139.64, 128.69, 128.66, 127.86, 125.91, 120.28, 116.75, 116.62, 111.92, 108.89, 107.78, 60.72, 60.52, 55.86, 55.09, 33.81, 19.47.

5,6,7-trimethoxy-1-(3-nitro-4-methoxyphenyl)-3,4-dihydronaphthalene-2(1H)-one (T-dk-c)

According to the synthesis of S-dk, the raw material 5,6,7-trimethoxy-3,4-dihydronaphthalen-2-one (2) and 3-nitro-4-methoxybenzoyl chloride were reacted. T-dk-c is a yellow solid. ¹ H NMR (600MHz, CDCl ₃ ) δ 8.10 (d, J = 2.2 Hz, 1H), 7.71 (dd, J = 8.8, 2.2 Hz, 1H), 7.02 (d, J = 8.8 Hz, 1H), 6.04 (s, 1H), 3.99 (s, 3H), 3.90 (s, 3H), 3.86 (s, 3H), 3.36 (s, 3H), 3.00–2.89 (m, 2H), 2.64–2.54 (m, 2H) ).

6,7,8-Trimethoxy-1-(4-methoxyphenyl)-4,5-dihydro-2H-benzo[e]carbazole (TCa-1)

Referring to the synthesis of SC1, the intermediate T-dk-a and hydrazine hydrate were reacted to give TCa-1 as a brown solid. Melting point 105-106 ° C, ¹ H NMR (600 MHz, CDCl ₃ ) δ 7.55 (d, J = 8.5 Hz, 2H), 6.99 (d, J = 8.5 Hz, 2H), 6.70 (s, 1H), 3.88 ( s, 3H), 3.86 (s, 6H), 3.54 (s, 3H), 3.00 (t, J = 7.2 Hz, 2H), 2.82 (t, J = 7.2 Hz, 2H); ¹³ C NMR (150 MHz, CDCl) ₃ ) δ160.13, 151.63, 151.43, 140.45, 129.88, 126.55, 123.49, 121.13, 114.20, 113.39, 103.08, 60.98, 60.95, 55.75, 55.41, 22.10, 21.73. HRMS(ES+)m/z found 367.1652 (M+H ⁺ ); C ₂₁ H ₂₃ O ₆ (M+H ⁺ )requires 367.1662.

6,7,8-Trimethoxy-1-(4-methoxyphenyl)-2-methyl-4,5-dihydro-2H-benzo[e]carbazole (TCa-2)

Referring to the synthesis of SC1, the intermediate T-dk-a and methylhydrazine were reacted to give TCa-2 as a brown solid. Melting point 126-127 ° C, ¹ H NMR (600 MHz, CDCl ₃ ) δ 7.37 (d, J = 8.5 Hz, 2H), 7.05 (d, J = 8.5 Hz, 2H), 6.30 (s, 1H), 3.88 ( s, 3H), 3.86 (s, 4H), 3.83 (s, 3H), 3.70 (s, 3H), 3.42 (s, 3H), 3.00 (t, J = 7.4 Hz, 2H), 2.85 (t, J = 7.4 Hz, 2H). ¹³ C NMR (151 MHz, CDCl ₃ ) δ 160.18, 151.51, 151.39, 149.28, 140.16, 138.11, 131.43, 130.14, 126.65, 123.03, 120.90, 114.81, 114.37, 113.71, 102.43, 60.94, 60.88, 55.43, 36.47, 29.70, 22.14, 21.74. HRMS(ES+)m/z found 381.1805(M+H ⁺ ); C ₂₁ H ₂₃ O ₆ (M+H ⁺ )requires 381.1809.

6,7,8-Trimethoxy-1-(3-fluoro-4-methoxyphenyl)-4,5-dihydro-2H-benzo[e]carbazole (TCb-1)

Referring to the synthesis of SC1, the intermediate T-dk-b and hydrazine hydrate were reacted to give TCb-1 as a purple solid. Mp 147 - 148 ° C, ¹ H NMR (600 MHz, CDCl ₃ ) δ 7.38 (dd, J = 11.8, 2.0 Hz, 1H), 7.35 - 7.30 (m, 1H), 6.97 (t, J = 8.5 Hz, 1H ), 6.70 (s, 1H), 3.93 (s, 3H), 3.88 (s, 3H), 3.87 (s, 3H), 3.57 (s, 3H), 3.01 - 2.92 (m, 2H), 2.79 - 2.70 ( m,2H); ¹³ C NMR (150MHz, CDCl ₃ ) δ 152.93, 151.77, 151.44, 151.30, 147.98, 147.91, 140.48, 126.34, 124.70, 120.92, 116.47, 116.34, 113.56, 113.28, 103.11, 61.00, 60.98, 56.28, 55.77, 21.98, 21.28. HRMS(ES+)m/z found 385.1564(M+H ⁺ ); C ₂₁ H ₂₃ O ₆ (M+H ⁺ )requires 385.1558.

6,7,8-Trimethoxy-1-(3-fluoro-4-methoxyphenyl)-2-hydroxyethyl-4,5-dihydro-2H-benzo[e]carbazole (TCb -3)

Referring to the synthesis of SC1, the intermediate T-dk-b and hydroxyethylhydrazine were reacted to give TCb-3 as a brown solid. Melting point 150-151 ° C, ¹ H NMR (600 MHz, CDCl ₃ ) δ 7.22 (dd, J = 11.4, 2.0 Hz, 1H), 7.20 - 7.16 (m, 1H), 7.11 (t, J = 8.4 Hz, 1H ), 6.25 (s, 1H), 4.06 - 4.01 (m, 2H), 3.97 (s, 3H), 3.95 (dd, J = 9.1, 4.5 Hz, 2H), 3.86 (s, 3H), 3.84 (s, 3H), 3.45 (s, 3H), 2.99 (t, J = 7.4 Hz, 2H), 2.84 (t, J = 7.4 Hz, 2H). ¹³ C NMR (151 MHz, CDCl ₃ ) δ 153.17, 151.69, 151.49, 149.42 ,148.73,148.66,140.75,137.58,126.76,125.39,122.39,122.34,121.07,118.20,118.08,115.35,113.79,102.68,61.77,60.95,60.90,56.42,55.50,50.35,21.77,21.45.HRMS(ES+)m /z found 429.1830(M+H ⁺ ); C ₂₁ H ₂₃ O ₆ (M+H ⁺ )requires 429.1820.

6,7,8-Trimethoxy-1-(3-fluoro-4-methoxyphenyl)-2-phenyl-4,5-dihydro-2H-benzo[e]carbazole (TCb- 4)

Referring to the synthesis of SC1, the intermediate T-dk-b and phenylhydrazine were reacted to give TCb-4 as a brown solid. 159-160 ° C, ¹ H NMR (600 MHz, CDCl ₃ ) δ 7.32 - 7.28 (m, 2H), 7.26 - 7.22 (m, 3H), 7.13 (dd, J = 11.6, 2.0 Hz, 1H), 7.09 –7.05 (m, 1H), 6.98 (t, J = 8.5 Hz, 1H), 6.40 (s, 1H), 3.92 (s, 3H), 3.89 (s, 3H), 3.87 (s, 3H), 3.48 ( s, 3H), 3.05 (t, J = 7.3 Hz, 2H), 2.93 (t, J = 7.2 Hz, 2H). ¹³ C NMR (151 MHz, CDCl ₃ ) δ 153.44, 151.72, 151.51, 151.39, 151.55, 148.34, 148.27,140.96,138.93,136.30,128.97,127.48,126.88,125.36,125.10,122.98,121.68,120.65,118.21,118.09,116.67,115.32,113.48,103.06,60.98,56.29,55.57,21.84,21.63.HRMS(ES+) m/z found 461.1870(M+H ⁺ ); C ₂₁ H ₂₃ O ₆ (M+H ⁺ )requires 461.1871.

6,7,8-Trimethoxy-1-(3-nitro-4-methoxyphenyl)-2-phenyl-4,5-dihydro-2H-benzo[e]carbazole (TCc -1)

Referring to the synthesis of SC1, the intermediate T-dk-c and hydrazine hydrate were reacted to give TCc-1 as a brown solid. Melting point 108-109 ° C, ¹ H NMR (600 MHz, CDCl ₃ ) δ 8.16 (d, J = 2.2 Hz, 1H), 7.84 (dd, J = 8.6, 2.2 Hz, 1H), 7.16 (d, J = 8.7 Hz, 1H), 6.65 (s, 1H), 4.02 (s, 3H), 3.89 (s, 3H), 3.88 (s, 3H), 3.60 (s, 3H), 3.01 (t, J = 7.4 Hz, 2H) ), 2.87 - 2.80 (m, 2H). ¹³ C NMR (151MHz, CDCl ₃ ) δ 152.68, 151.90, 151.59, 140.81, 139.68, 134.04, 125.87, 125.43, 124.97, 120.86, 114.05, 113.62, 102.98, 61.00, 56.70, 55.89, 21.89, 21.04.

6,7,8-Trimethoxy-1-(3-amino-4-methoxyphenyl)-2-phenyl-4,5-dihydro-2H-benzo[e]carbazole (TCd- 1)

Acetic acid was added to 10 mL of 75% ethanol, adjusted to pH = 3-4, iron powder (90 mg, 1.6 mmol) was added with stirring, and the mixture was heated to 90 ° C and stirred for one hour. Further, compound TCc-1 (66 mg, 0.16 mmol) was added, and stirring was continued for 30 minutes, and the reaction was completed. The reaction mixture was neutralized with EtOAc (EtOAc m. The filtrate was extracted with EtOAc. EtOAc (EtOAc)EtOAc. _{^{1 H NMR (600MHz, CDCl 3}} ) δ6.95 (t, J = 5.4Hz, 2H), 6.84 (s, 1H), 6.83 (d, J = 8.1Hz, 1H), 4.19 (s, 2H), 3.89 (s, 3H), 3.87 (s, 3H), 3.86 (s, 3H), 3.58 (s, 3H), 3.48 (s, 1H), 3.02 - 2.92 (m, 2H), 2.83 - 2.73 (m, 2H) ¹³ C NMR (150 MHz, CDCl ₃ ) δ 151.56, 151.33, 150.26, 147.71, 140.29, 140.10, 136.49, 126.72, 123.68, 121.09, 118.61, 114.63, 113.04, 110.30, 103.27, 60.99, 60.95, 55.79, 55.61, 22.10 , 21.72.

The cytotoxic effect of the target on Colo205 cell line.

First, experimental materials and methods:

1. Experimental materials

(1) Experimental cell strain

In this experiment, human colon cancer cell Colo205 was used as a screening target, and the cancer cell lines were all provided by the pharmacology research laboratory of Shanghai Pharmaceutical Industry Research Institute.

(2) The culture medium is DMEM+10% FBS+ double antibody, and the model of the automatic microplate reader used is: Varioskan Flash, manufacturer: Thermo scientific. Import 96-well culture plates, etc.

2, test methods

The in vitro antitumor activity was determined by the MTT method.

MTT method. 100 μl of a cell suspension having a concentration of 5 × 10 ⁴ /ml was added to each well of a 96-well plate, and placed in a 37 ° C, 5% CO ₂ incubator. After 24 h, the sample solution was added, 10 μl/well, and three replicate wells were set at 37 ° C, 5% CO _{2 for} 72 h. 20 μl of 5 mg/ml MTT solution was added to each well. After 4 hours, the solution was added, 100 μl/well, placed in an incubator, and dissolved, and the OD value of 570 nm was measured by a full-wavelength multi-plate reader.

Second, the experimental results:

Table 1 Inhibition of in vitro proliferation of human colon cancer cell Colo205 by target compound

Note: This compound is compound 1 in patent CN103130632A

Inhibition of tubulin polymerization experiments

First, experimental materials and methods:

1. Experimental materials

(a) tubulin

In this experiment, bovine tubulin was used as a screening target, and the manufacturer: Cytoskeleton.

(2) Buffer

General Tubulin Buffer: 80 mM PIPES pH = 6.9, ₂ mM MgCl ₂ , 0.5 mM EGTA, manufacturer: Cytoskeleton.

Tubulin Glycerol Buffer: 60% glycerol, manufacturer: Cytoskeleton.

GTP: 100 mM, manufacturer: Cytoskeleton

The fully automatic microplate reader model used is: Synergy 4, manufacturer: BioTek. Imported 96-well half-area culture plates, etc.

2, test methods

The literature method (Cell Biochemistry and Biophysics, 2003, 38: 1-21), and the tubulin kit method (CytoDYNAMIX Screen 03) were used.

Sample preparation: After dissolving in DMSO (Merck), buffer was added to prepare a corresponding concentration of the solution or a homogeneous suspension, and then diluted with a buffer.

The experimental steps are as follows:

Add 10 μl of the test compound to each well in a 96-well culture plate and place it in a 37 °C microplate reader. After 2 min, add 3 mg/ml tubulin solution, 100 μl/well, and set double duplicate wells at 37 °C. For 1 h, the 340 nm OD value was measured every minute.

Second, the experimental results

Table 2 The effect of some compounds on inhibition of tubulin polymerization

The experimental results show that the inhibition activity of tubulin of TCd-1 is even better than that of CA-4, which is worthy of further study as an antitumor drug.

The above description is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention. These improvements and additions should also be considered. It is the scope of protection of the present invention.

Claims (10)

a compound of the formula (I),

Wherein R is selected from the group consisting of amino acids, saccharides, and other groups having a basic group. The compound of claim 1 wherein said basic group is an amino group. The compound of claim 1 wherein said compound has the structural formula:

A pharmaceutically acceptable salt of a compound according to any one of claims 1 or 2. The salt according to claim 3, wherein the salt is a hydrochloride. The salt according to claim 4, wherein the salt has the structural formula:

The method of preparing a salt according to claim 6, wherein the synthetic route of the preparation method is:

Use of a compound according to any one of claims 1 or 2, a salt according to any one of claims 3-5, for the preparation of a tubulin polymerization inhibitor. The use of the compound according to any one of claims 1 or 2, the salt according to any one of claims 3-5, for the preparation of a medicament for treating a tumor disease. The use according to claim 8, wherein the tumor disease is leukemia, non-small cell lung cancer, colon cancer, central nervous system tumor, melanoma, ovarian cancer, renal cancer, prostate cancer or breast cancer.