CN106902123A - Application of the benzodiazepine compounds of 5 hydrogen 1,4 in liver cancer is treated - Google Patents

Abstract

The invention discloses application of the 5 hydrogen Isosorbide-5-Nitrae benzodiazepine compounds in liver cancer is treated, 5 described hydrogen Isosorbide-5-Nitrae benzodiazepine compounds have good inhibitory action to HepG2 human hepatoma cell strains and Huh7 human hepatoma cell strains；In the seven kind of 5 hydrogen Isosorbide-5-Nitrae benzodiazepine compounds tested, compound 7 when concentration is 30 μM, to the inhibiting rate of HepG2 human hepatoma cell strains up to 70%, to the inhibiting rate of Huh7 human hepatoma cell strains up to 64%.It is present invention firstly discloses purposes of such compound in treatment liver-cancer medicine is prepared and strong for the inhibitory activity of HCC, therefore 5 hydrogen Isosorbide-5-Nitrae benzodiazepine compounds are used to prepare medicines resistant to liver cancer, with good development prospect.

Description

Application of 5-hydrogen-1,4-benzodiazepines in the treatment of liver cancer

technical field

The invention relates to the medical use of 5-hydrogen-1,4-benzodiazepine compounds, in particular to the application of 5-hydrogen-1,4-benzodiazepine compounds in treating liver cancer.

Background technique

Benzodiazepine structural units exist in a series of natural products and drug molecules with physiological activities. For example, 1,5-benzodiazepine compounds are often used as anxiolytics, antidepressants, anticonvulsants, Drugs for sedation and analgesia; and 1,4-benzodiazepines also show good biological activity, such as drugs commonly used as anti-anxiety and antidepressants.

As an important class of 1,4-benzodiazepines, less research has been done on 5-hydrogen-1,4-benzodiazepines; among them, 5-hydrogen-1,4- The biological activity of benzodiazepines has not been reported yet. Therefore, it is of great significance to study the application of such compounds.

Contents of the invention

The present invention provides for the first time the application of 5-hydrogen-1,4-benzodiazepines in the treatment of liver cancer. The 5-hydrogen-1,4-benzodiazepines described in the present invention have HepG2 human liver cancer cell line and Huh7 human liver cancer cell line have good inhibitory effect.

Technical scheme of the present invention is as follows:

The use of 5-hydrogen-1,4-benzodiazepines or pharmaceutically acceptable salts thereof with general structural formula (I) in the preparation of medicines for treating liver cancer,

In the general structural formula (I), R ¹ is selected from hydrogen atom, Me, or F; R ² and R ³ are respectively selected from phenyl and its derivatives, naphthyl or thiophene.

The synthetic method of above-mentioned 5-hydrogen-1,4-benzodiazepines comprises the following steps:

(1) In the reaction device, add 1 to 50 mol% iodine element or iodide catalyst, 1,2-disubstituted acetylene compound and organic solvent respectively, and react under heating at 50 to 140°C;

(2) Afterwards, add 2-aminobenzylamine compound and acidic compound again, continue heating reaction;

(3) After the reaction is complete, the 5-hydrogen-1,4-benzodiazepines are obtained through post-treatment.

The iodide is one of potassium iodide, sodium iodide or iodobenzene diacetate; the catalyst is more preferably simple iodine;

The 1,2-disubstituted acetylene compounds are 1,2-bis(4-methylphenyl)acetylene, 1,2-bis(4-ethylphenyl)acetylene, 1,2-bis(4 - tert-butylphenyl) acetylene, 1,2-bis(4-methoxyphenyl)acetylene, 1,2-bis(3-methoxyphenyl)acetylene, 1,2-bis(4-fluoro phenyl)acetylene, 1,2-bis(1-naphthyl)acetylene, 1,2-bis(2-thienyl)acetylene, 1-phenyl-2-(4-methylphenyl)acetylene, 1 -(4-methoxyphenyl)-2-phenylacetylene, 1-(4-fluorophenyl)-2-phenylacetylene, 1-(4-methoxyphenyl)-2-phenylacetylene , 1-(1-naphthyl)-2-phenylacetylene, 1-(4-methoxyphenyl)-2-(4-methylphenyl)acetylene, 1-(4-fluorophenyl)- One of 2-(4-methoxyphenyl)acetylene or 1-(4-methoxyphenyl)-2-(1-naphthyl)acetylene.

Described organic solvent is ethyl acetate, n-hexane, cyclohexane, tetrahydrofuran, methylene chloride, 1,2-dichloroethane, acetonitrile, toluene, benzene, xylene, 1,4-dioxane, One of methanol, N,N-dimethylformamide, dimethyl sulfoxide or methyl tert-butyl ether; more preferably dimethyl sulfoxide.

The 2-aminobenzylamine compounds are 2-aminobenzylamine, 2-aminomethyl-4-fluoroaniline, 2-aminomethyl-4-methylaniline, 2-aminomethyl-5-methyl One of aniline or 2-aminomethyl-6-methylaniline.

The acidic compound is one of formic acid, acetic acid, benzoic acid, hydrochloric acid, sulfuric acid or phosphoric acid, more preferably acetic acid.

The molar ratio of the catalyst, the alkyne and the 2-aminobenzylamine compound is 1: (1-100): (1-500); more preferably 1: (1-50) :(1-200), particularly preferably 1:(1-20):(1-80).

Compared with the prior art, the beneficial effects of the present invention are as follows:

1. The 5-hydrogen-1,4-benzodiazepine compound of the present invention has a good inhibitory effect on HepG2 human liver cancer cell lines and Huh7 human liver cancer cell lines, and is a potential anticancer active substance; Among the seven tested 5-hydrogen-1,4-benzodiazepine compounds, when the concentration of compound 7 was 30 μM, the inhibitory rate on HepG2 human liver cancer cell line could reach 70%, and on Huh7 human liver cancer The inhibition rate of the cell line can reach 64%. Specifically, the IC ₅₀ value of compound 7 on the HepG2 human liver cancer cell line can reach 11.5 μM, and the IC ₅₀ value on the Huh7 human liver cancer cell line can reach 19.7 μM, as shown in Tables 1 and 2 shown;

2. The present invention also provides the application of a class of 5-hydrogen-1,4-benzodiazepine compounds in liver cancer for the first time, and has strong inhibitory activity on liver cancer cells, providing a basis for future research on this type of compounds Therefore, the 5-hydrogen-1,4-benzodiazepines are used in the preparation of anti-liver cancer drugs and have good development and application prospects.

Of course, implementing any product application of the present invention does not necessarily need to achieve all the above-mentioned advantages at the same time.

Description of drawings

Fig. 1 is a kind of 5-hydrogen-1 of the present invention, the general structural formula of 4-benzodiazepines compound;

Fig. 2 is a synthetic route diagram of a kind of 5-hydrogen-1,4-benzodiazepines of the present invention;

Fig. 3 is the H NMR spectrum of the 2,3-bis(4-methoxyphenyl)-5-hydrogen-1,4-benzodiazepine compound of Example 5 of the present invention;

Fig. 4 is the C-NMR spectrum of the 2,3-bis(4-methoxyphenyl)-5-hydrogen-1,4-benzodiazepine compound of Example 5 of the present invention.

detailed description

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention, not to limit the protection scope of the present invention. Improvements and adjustments made by those skilled in the art according to the present invention in practical applications still belong to the protection scope of the present invention.

As shown in Figure 1, a kind of 5-hydrogen-1 of the present invention, the 4-benzodiazepine compound, its structural general formula (I) is:

Wherein, R ¹ is selected from hydrogen atom, methyl or fluorine; R ² and R ³ are selected from phenyl derivatives, naphthyl or thiophene, respectively.

A kind of 5-hydrogen-1 of the present invention, the synthetic route of 4-benzodiazepine compound is as shown in Figure 2; Its concrete synthetic method comprises the following steps:

(1) In the reaction device, sequentially add 1 to 50 mol% iodine elemental catalyst, 1,2-disubstituted acetylene compound and organic solvent, and react under heating at 50 to 140°C;

(2) After reacting for a period of time, add 2-aminobenzylamine compound and acidic compound successively, continue heating reaction;

(3) After the reaction is complete, the 5-hydrogen-1,4-benzodiazepines are obtained through conventional post-treatment.

In step (1), the reaction temperature can be selected to be 80-140°C, and generally 130°C is more selected during the reaction process; in step (2), the reaction temperature can be selected to be 80-120°C, generally during the reaction The process would be more preferably 100°C.

The present invention utilizes the above synthetic method to prepare compounds 1-7 described in Examples 1-7, and use compounds 1-7 for cell tests on HepG2 human liver cancer cell line and Huh7 human liver cancer cell line to show that the 5- Hydrogen-1,4-benzodiazepines have good inhibitory effect on HepG2 human liver cancer cell line and Huh7 human liver cancer cell line.

1. Synthesis of compounds 1-7:

Example 1

The synthesis of 2,3-diphenyl-5-hydrogen-1,4-benzodiazepine (compound 1), its structural formula is:

Into the reaction tube, add 10.1 mg of iodine, 35.6 mg of 1,2-toluene, and 2 mL of dimethyl sulfoxide in sequence, react in an oil bath at 130°C for 24 hours, cool the reaction solution to room temperature, and then add 97.7 mg of 2-aminobenzylamine and 1 mL of acetic acid were reacted in an oil bath at 100°C for 20 hours, and finally 51.6 mg of compound 1 was obtained by conventional post-treatment, with a yield of 87%.

Compound 1 has a melting point of 131-132°C.

The NMR characterization results of compound 1 are: ¹ H NMR (400MHz, CDCl ₃ ): δ 7.88-7.77(m, 2H), 7.57-7.47(m, 3H), 7.42-7.30(m, 5H), 7.21(t, J=7.0Hz, 4H), 4.86(d, J=10.6Hz, 1H), 3.97(d, J=10.6Hz, 1H); ¹³ C NMR (100MHz, CDCl ₃ ) δ 54.8, 125.7, 126.8, 128.0, 128.3, 128.4, 128.5, 128.6, 129.9, 130.7, 130.8, 137.4, 137.8, 147.5, 160.5, 160.6.

Example 2

The synthesis of 2,3-bis(4-methylphenyl)-5-hydrogen-1,4-benzodiazepine (compound 2), its structural formula is:

Into the reaction tube, add 10.1 mg of iodine, 41.2 mg of 1,2-di(4-methylphenyl)acetylene, and 2 mL of dimethyl sulfoxide in sequence, react in an oil bath at 130°C for 24 hours, and cool the reaction solution to At room temperature, 97.7mg of 2-aminobenzylamine and 1mL of acetic acid were added in sequence, and placed in an oil bath at 100°C for 20 hours. Finally, 48.0mg of compound 2 was obtained by conventional post-treatment, with a yield of 74%.

Compound 2 has a melting point of 150-152°C.

The NMR characterization results of compound 2 are: ¹ H NMR (400MHz, CDCl ₃ ): δ 7.72(d, J=8.0Hz, 2H), 7.51(d, J=7.7Hz, 1H), 7.45-7.30(m, 4H ), 7.21-7.09(m, 3H), 7.00(d, J=7.8Hz, 2H), 4.81(d, J=10.6Hz, 1H), 3.95(d, J=10.6Hz, 1H), 2.31(s , 3H), 2.22(s, 3H); ¹³ C NMR (100MHz, CDCl ₃ ) δ 21.4, 21.5, 54.7, 125.6, 126.5, 127.9, 128.3, 128.5, 129.1, 129.3, 131.0, 134.9, 135.2, 140.0, 141.0 , 147.7, 160.4, 164.7.

The HRMS (ESI) calculation of compound 2 is: C ₂₃ H ₂₁ N ₂ (M+H) ⁺ , 325.1705, found: 325.1694.

Example 3

The synthesis of 2,3-bis(4-ethylphenyl)-5-hydrogen-1,4-benzodiazepine (compound 3), its structural formula is:

Into the reaction tube, add 10.1 mg iodine element, 46.8 mg 1,2-di(4-ethylphenyl) acetylene, and 2 mL dimethyl sulfoxide in sequence, react in an oil bath at 130°C for 24 hours, and cool the reaction solution to At room temperature, 97.7 mg of 2-aminobenzylamine and 1 mL of acetic acid were added sequentially, and placed in an oil bath at 100°C for 20 hours. Finally, 55.0 mg of compound 3 was obtained by conventional post-treatment, with a yield of 78%.

Compound 3 has a melting point of 81-82°C.

The NMR characterization results of compound 3 are: ¹ H NMR (400MHz, CDCl ₃ ): δ 7.76(d, J=8.2Hz, 2H), 7.50(d, J=7.8Hz, 1H), 7.45(d, J=8.2 Hz, 2H), 7.36(t, J=8.0Hz, 1H), 7.21-7.15(m, 3H), 7.05(d, J=8.1Hz, 2H), 4.81(d, J=10.6Hz, 1H), 3.95(d, J=10.6Hz, 1H), 2.64(q, J=7.6Hz, 2H), 2.55(q, J=7.6Hz, 2H), 1.21(t, J=7.6Hz, 3H), 1.13( t, J=7.6Hz, 3H); ¹³ C NMR (100MHz, CDCl ₃ ) δ 15.2, 15.3, 28.7, 28.9, 54.7, 125.6, 126.5, 127.9, 128.0, 128.2, 128.4, 128.5, 128.6, 131.0, 135.1, 135.4, 146.4, 147.3, 147.7, 160.4, 164.8.

The HRMS (ESI) calculation of compound 3 is: C ₂₃ H ₂₅ N ₂ (M+H) ⁺ , 353.2018, found: 353.2015.

Example 4

The synthesis of 2,3-bis(4-tert-butylphenyl)-5-hydrogen-1,4-benzodiazepine (compound 4), whose structural formula is:

Into the reaction tube, add 10.1 mg iodine element, 58.1 mg 1,2-di(4-tert-butylphenyl)acetylene, 2 mL dimethyl sulfoxide in sequence, react in an oil bath at 130°C for 24 hours, and cool the reaction liquid After reaching room temperature, 97.7mg of 2-aminobenzylamine and 1mL of acetic acid were added in sequence, and placed in an oil bath at 100°C for 20 hours. Finally, 69.5mg of compound 4 was obtained by conventional post-treatment, with a yield of 85%.

Compound 4 has a melting point of 136-138°C.

The NMR characterization results of compound 4 are: ¹ H NMR (400MHz, CDCl ₃ ): δ 7.79(d, J=8.5Hz, 2H), 7.49(d, J=8.4Hz, 3H), 7.35(d, J=7.8 Hz, 3H), 7.34(t, J=7.4Hz, 1H), 7.24(d, J=7.8Hz, 2H), 7.16(t, J=7.4Hz, 1H), 4.80(d, J=10.6Hz, 1H), 3.94(d, J=10.6Hz, 1H), 1.30(s, 9H), 1.22(s, 9H); ¹³ C NMR (100MHz, CDCl ₃ ) δ 31.2, 31.3, 34.8, 35.0, 54.7, 125.4 , 125.5, 125.7, 127.9, 128.1, 128.3, 128.5, 131.0, 134.8, 135.1, 147.8, 153.2, 154.2, 160.2, 164.7.

The HRMS (ESI) calculation of compound 4 is: C ₂₉ H ₃₃ N ₂ (M+H) ⁺ , 409.2644, found: 409.2643.

Example 5

The synthesis of 2,3-bis(4-methoxyphenyl)-5-hydrogen-1,4-benzodiazepine (compound 5), whose structural formula is:

Add 10.1 mg iodine, 47.6 mg 1,2-bis(4-methoxyphenyl)acetylene, and 2 mL dimethyl sulfoxide to the reaction tube in sequence, react in an oil bath at 130°C for 24 hours, and cool the reaction liquid After reaching room temperature, 97.7 mg of 2-aminobenzylamine and 1 mL of acetic acid were added in sequence, and placed in an oil bath at 100° C. for 20 hours. Finally, 63.4 mg of compound 5 was obtained by conventional post-treatment, with a yield of 89%.

Compound 5 has a melting point of 148-150°C.

The NMR characterization results of compound 5 are: ¹ H NMR (400MHz, CDCl ₃ ): δ 7.78 (d, J=8.9Hz, 2H), 7.52-7.43 (m, 3H), 7.40-7.36 (m, 2H), 7.17 (td, J=7.5, 1.1Hz, 1H), 6.85(d, J=8.9Hz, 2H), 6.72(d, J=8.9Hz, 2H), 4.78(d, J=10.6Hz, 1H), 3.94 (d, J=10.6Hz, 1H), 3.77(s, 3H), 3.70(s, 3H); ¹³ C NMR (100MHz, CDCl ₃ ) δ 54.6, 55.3, 55.4, 113.8, 114.0, 125.5, 126.3, 127.9 , 128.5, 130.0, 130.3, 130.4, 130.6, 131.0, 147.8, 159.7, 160.9, 161.6, 164.1.

The proton NMR spectrum of compound 5 is shown in Figure 3, and the carbon NMR spectrum is shown in Figure 4.

The HRMS (ESI) calculation of compound 5 is: C ₂₃ H ₂₁ N ₂ O ₂ (M+H) ⁺ , 357.1603, found: 357.1601.

Example 6

The synthesis of 2,3-bis(3-methoxyphenyl)-5-hydrogen-1,4-benzodiazepine (compound 6), whose structural formula is:

Add 10.1 mg iodine, 47.6 mg 1,2-bis(3-methoxyphenyl)acetylene, and 2 mL dimethyl sulfoxide to the reaction tube in sequence, react in an oil bath at 130°C for 24 hours, and cool the reaction liquid After reaching room temperature, 97.7 mg of 2-aminobenzylamine and 1 mL of acetic acid were added sequentially, and placed in an oil bath at 100° C. for 20 hours. Finally, 52.8 mg of compound 6 was obtained by conventional post-treatment, with a yield of 74%.

Compound 6 has a melting point of 145-146°C.

The NMR characterization results of compound 6 are: ¹ H NMR (400MHz, CDCl ₃ ): δ 7.53(d, J=7.7Hz, 1H), 7.49(s, 1H), 7.43-7.34(m, 2H), 7.29(d , J=7.4Hz, 1H), 7.25-7.14(m, 3H), 7.08(t, J=7.7Hz, 1H), 6.99(d, J=7.4Hz, 1H), 6.90(d, J=7.2Hz , 1H), 6.79(d, J=7.2Hz, 1H), 4.85(d, J=10.6Hz, 1H), 3.96(d, J=10.6Hz, 1H), 3.79(s, 3H), 3.71(s , 3H); ¹³ C NMR (100MHz, CDCl ₃ ) δ 54.8, 55.3, 55.4, 112.4, 112.6, 116.3, 117.1, 121.3, 121.7, 125.7, 126.9, 128.0, 128.6, 129.4, 129.6, 130.7, 1398.8, 1 147.5, 159.6, 159.8, 160.4, 164.5.

The HRMS (ESI) calculation of compound 6 is: C ₂₃ H ₂₁ N ₂ O ₂ (M+H) ⁺ , 357.1603, found: 357.1591.

Example 7

The synthesis of 2,3-bis(4-fluorophenyl)-5-hydrogen-1,4-benzodiazepine (compound 7), whose structural formula is:

Add 10.1 mg of iodine, 42.8 mg of 1,2-bis(4-fluorophenyl)acetylene, and 2 mL of dimethyl sulfoxide to the reaction tube, and react in an oil bath at 130°C for 24 hours, and cool the reaction solution to room temperature , and then sequentially added 97.7mg of 2-aminobenzylamine and 1mL of acetic acid, and placed in a 100°C oil bath to react for 20 hours, and finally obtained 51.2mg of compound 7 through conventional post-treatment, with a yield of 77%.

Compound 7 has a melting point of 121-123°C.

The NMR characterization results of compound 7 are: ¹ H NMR (400MHz, CDCl ₃ ): δ 7.80(dd, J=8.7, 5.4Hz, 2H), 7.56-7.44(m, 3H), 7.39(t, J=7.5Hz , 2H), 7.23(t, J=7.5Hz, 1H), 7.04(t, J=8.6Hz, 2H), 6.90(d, J=8.6Hz, 2H), 4.85(d, J=10.6Hz, 1H ), 3.94(d, J=10.6Hz, 1H); ¹³ C NMR (100MHz, CDCl ₃ ) δ 54.8, 115.6(d, J=22.0Hz), 115.9(d, J=22.0Hz), 126.9(d, J=224.0Hz), 127.9(d, J=166.0Hz), 130.5(d, J=8.0Hz), 130.6, 130.7(d, J=9.0Hz), 133.5(d, J=3.1Hz), 133.9( d, J=3.1Hz), 147.4, 159.1, 162.5, 163.0, 165.0, 165.5.

The HRMS (ESI) calculation of compound 7 is: C ₂₁ H ₁₅ N ₂ F ₂ (M+H) ⁺ , 333.1203, found: 333.1205.

2. Antiproliferative activity test of compounds 1-7 on human liver cancer cell lines

(1) Cell lines and cell culture

Two human cell lines, human hepatoma cell line (Huh7) and human hepatoma cell line (HepG2), were selected. The cell lines were all cultured in DMEM culture solution containing 10wt% calf serum, 1U/mL penicillin, and 1U/mL streptomycin, and cultured in an incubator at 37°C containing 5% CO ₂ by volume.

(2) Preparation of test compounds

Dissolve the seven tested 5-hydrogen-1,4-benzodiazepine compounds (compounds 1 to 7) in DMSO solution, and configure the concentration of each compound in 8 wells to be 100 μM , 30 μM, 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM. The inhibitory degree of the above compound on the growth of two kinds of liver cancer cells at this concentration was tested.

(3) Cell growth inhibition test (MTT method)

1) Take the tumor cells, digest them with trypsin, prepare a cell suspension with a concentration of 5000 cells/mL with a culture solution containing 10% calf serum, inoculate 190 μL per well in a 96-well culture plate, and make the cells to be tested Cell density to 1000-10000 wells;

2) Incubate in 5% CO ₂ at 37°C for 12 hours until the cell monolayer covers the bottom of the well, add 10 μL of the compound to be tested in a certain concentration gradient to each well, and set 4 replicate wells for each concentration gradient;

3) 5% CO ₂ , cultured at 37°C for 72 hours, observed under an inverted microscope;

4) Add 10 μL of MTT solution (5 mg/mL PBS, ie 0.5% MTT) to each well and continue to incubate for 4 hours;

5) Terminate the culture, carefully suck off the culture medium in the wells, add 100 μL of DMSO to each well to fully dissolve, mix well with a shaker, and measure the absorbance of each well with a microplate reader;

6) Three experiments were carried out in parallel for each concentration, and the cell inhibition rate was calculated respectively;

7) According to the measured absorbance, the inhibition rate was calculated according to the following formula: survival rate=(absorbance of the experimental group/absorbance of the control group)×100%;

8) According to the obtained tumor cell growth inhibition rate, the IC50 values of the tested compounds against human liver cancer cell lines were respectively calculated by Bliss method, and the statistical results are shown in Tables 1-2.

Inhibitory effect of compounds 1 to 7 in table 1 on the growth of human liver cancer cell line (Huh7)

The inhibitory effect of table 2 compound 1～7 on the cell growth of human liver cancer cell line (HepG2)

The preferred embodiments of the invention disclosed above are only to help illustrate the invention. The preferred embodiments are not exhaustive in all detail, nor are the inventions limited to specific embodiments described. Obviously, many modifications and variations can be made based on the contents of this specification. This description selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can well understand and utilize the present invention. The invention is to be limited only by the claims, along with their full scope and equivalents.

Claims (1)

1. 5- hydrogen-Isosorbide-5-Nitrae-benzodiazepine compounds or its pharmaceutically acceptable salt with general structure (I) are in system The application in the medicine for the treatment of liver cancer is ready for use on,

Wherein, R¹Selected from hydrogen atom, Me or F；R²、R³It is respectively selected from phenyl and its derivative, naphthyl or thiophene.