2-Amino-substituted bendamustine derivatives with HDAC inhibitory effect and preparation method and application thereof

Abstract

The present invention discloses a 2-amino substituted bendamustine derivative having HDAC inhibitory effect, a preparation method and application thereof, wherein the structure of the bendamustine derivative is shown in formula (I): the bendamustine derivative can be prepared into a hydrochloride, a sulfate or a tartrate. The compound has a dual effect of HDAC inhibitory effect and alkylation, and has a good efficacy in inhibiting the growth of blood tumors.

Description

Technical Field

[0001] The invention belongs to the pharmaceutical field, and particularly relates to a 2-amino substituted bendamustine derivative with HDAC inhibitory effect, a preparation method thereof and a pharmaceutical use thereof. Background Art

[0002] Histone deacetylase (HDAC) is one of the most representative epigenetic modifiers, playing an important role in chromatin modification, regulation of gene expression, cell cycle differentiation and apoptosis. Epigenetic dysregulation is one of the mechanisms leading to the occurrence and development of cancer, and HDACs inhibitors have significant clinical efficacy in hematological malignancies. HDAC inhibitors that have been approved for marketing worldwide, such as vorinostat, romidepsin, belinostat, panobinostat and cedabendamide, and abexinostat, which is currently in clinical research, are all used to treat hematological malignancies.

[0003] Bendamustine is a bifunctional alkylating agent with anti-tumor and cytocidal effects. The main mechanism of the anti-tumor and cytocidal effects of this product is the cross-linking of DNA single and double strands through alkylation, which disrupts the function and synthesis of DNA, and also causes cross-linking between DNA and protein, and between protein and protein, thereby exerting an anti-tumor effect. It is clinically used to treat chronic lymphocytic leukemia, non-Hodgkin's lymphoma and multiple myeloma.

[0004] Tinostamustine (EDO-S101) is the first alkylating deacetylase inhibitor obtained by structural modification of bendamustine. It has the dual functions of alkylating and deacetylase inhibitors. It is currently in early clinical development for a series of rare and difficult-to-treat blood cancers and advanced solid tumors, including: multiple myeloma, Hodgkin's lymphoma, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, T-PLL, soft tissue sarcoma, small cell lung cancer, triple-negative breast cancer, ovarian cancer, endometrial cancer and MGMT non-methylated glioblastoma. Preclinical studies have shown that tinostamustine has better potential to reach DNA chains in cancer cells, destroy them, and prevent damage repair. These complementary and simultaneous modes of action have the potential to overcome resistance to some other cancer therapies. Summary of the invention

[0005] The purpose of the present invention is to provide a class of bendamustine derivatives with HDAC inhibitory effect. The compound has the dual effects of HDAC inhibitory effect and alkylation effect and has good efficacy in inhibiting the growth of blood tumors.

[0006] In order to achieve the above object, the present invention adopts the following technical scheme: a 2-amino substituted bendamustine derivative having HDAC inhibitory effect, wherein the structure of the 2-amino substituted bendamustine derivative having HDAC inhibitory effect is shown in formula (I):

[0007]

[0008] The bendamustine derivative can be prepared as a hydrochloride, a sulfate or a tartrate.

[0009] A method for preparing the above-mentioned 2-amino substituted bendamustine derivative having HDAC inhibitory effect comprises the following steps:

[0010] Step 1: Dissolve 2-methylamino-5-nitroaniline and methyl cyanamide in water, add sodium thiosulfate as a catalyst under full stirring, raise the temperature to about 70°C, add concentrated hydrochloric acid dropwise to adjust the pH value to 3-4, react for 2h, and obtain [1-methyl-2-(acetylamino)-5-nitro]-1H-benzimidazole;

[0011] Step 2: Dissolve [1-methyl-2-(acetylamino)-5-nitro]-1H-benzimidazole in methanol, add palladium carbon as a catalyst, and hydrogenate at room temperature and pressure for 12 hours to obtain the reactant [1-methyl-2-(acetylamino)-5-amino]-1H-benzimidazole;

[0012] Step 3: Dissolve [1-methyl-2-(acetylamino)-5-amino]-1H-benzimidazole in an aqueous solution of glacial acetic acid, add ethylene oxide dropwise at low temperature, stir for 5 h, heat and stir for 2 h, and then add K 2 CO 3 The aqueous solution is used to adjust the pH value of the reaction system to 7-8, and the reaction is extracted with chloroform, dried over anhydrous sodium sulfate, and concentrated to obtain [1-methyl-2-(acetylamino)-5-N, N-di(2'-hydroxyethyl)]-1H-benzimidazole; Step 4: adding [1-methyl-2-(acetylamino)-5-N, N-di(2'-hydroxyethyl)]-1H-benzimidazole to concentrated hydrochloric acid, heating and refluxing for 4 hours, and drying the solvent to obtain an oily substance, and pouring the oily substance into water, and cooling under ice-water bath conditions to obtain [1-methyl-2-(amino)-5-N, N-di(2'-chloroethyl)]-1H-benzimidazole hydrochloride;

[0013] Step 5: [1-methyl-2-(amino)-5-N,N-di(2'-chloroethyl)]-1H-benzimidazole hydrochloride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 1-hydroxybenzotriazole were stirred at 30°C for 0.5h; 3-(4-methylformylphenyl)oxypropionic acid, dimethylformamide and triethylamine were added to another reaction bottle and stirred at 30°C for 0.5h; the above two reaction solutions were mixed and stirred for 48h, and then vacuum rotary evaporation was performed to obtain a yellow crude product; silica gel column chromatography was performed with gradient elution, and vacuum rotary evaporation was performed to obtain a 2-amino-substituted bendamustine derivative.

[0014] Furthermore, the molar ratio of 2-methylamino-5-nitroaniline to methyl cyanamide in step 1 is 1:1-2.

[0015] Furthermore, the low temperature in step 3 is 0-5°C, and the temperature is slightly raised to 5-10°C with stirring.

[0016] Furthermore, the reflux temperature in step 4 is 90-95°C.

[0017] Furthermore, in step 5, the molar ratio of [1-methyl-2-(amino)-5-N,N-di(2'-chloroethyl)]-1H-benzimidazole hydrochloride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and 1-hydroxybenzotriazole is 1:(2-4):(1-2); the molar ratio of 3-(4-methylformylphenyl)oxypropionic acid and triethylamine is 1:(1-2).

[0018] Furthermore, the silica gel column chromatography in step 5 is separated by gradient elution using petroleum ether and acetone in a volume ratio of 9 to 1:1.

[0019] A use of the above-mentioned 2-amino substituted bendamustine derivative with HDAC inhibitory effect in the preparation of a drug for treating hematological malignancies, wherein the drug comprises the bendamustine derivative, or its hydrochloride, sulfate or tartrate.

[0020] The bendamustine derivative disclosed in the present invention has the dual effects of HDAC inhibition and alkylation, and has good efficacy in inhibiting the growth of blood tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Graph showing the inhibition rate of the target compound of the example on tumor cell proliferation. DETAILED DESCRIPTION

[0022] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present application.

[0023] It should be noted that the terms "including" and "having" and any variations thereof in the specification and claims of the present application and the above-mentioned drawings are intended to cover non-exclusive inclusions. For example, a process, method, system, product or apparatus comprising a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or apparatuses.

[0024] Synthesis of target compounds

[0025] Synthesis route:

[0026]

[0027] Synthesis operation:

[0028] 1) Synthesis of [1-methyl-2-(acetylamino)-5-nitro]-1H-benzimidazole (3)

[0029] Add 2-methylamino-5-nitroaniline (2, reference Strait Pharmacy, 2011, 23: 227-229 synthesis) 16.7g (0.1mol), methyl cyanamide 15.0g (0.15mol) and 60mL of water to the reaction flask, add 0.65g of sodium thiosulfate under full stirring, raise the temperature to about 70°C, add concentrated hydrochloric acid dropwise to adjust the pH value to 3-4 (5min). Heat to 70°C and react for 2h. After the reaction is completed, wash and filter with 80°C hot water while hot, then stir with 60mL of 5% formaldehyde at 35°C for 3h, then separate, wash with water, and weigh 20.3g after drying. The crude product is directly used for the next step reaction.

[0030] 2) Synthesis of [1-methyl-2-(acetylamino)-5-amino]-1H-benzimidazole (4)

[0031] Compound 3 (9.36 g, 40 mmol) was dissolved in methanol (200 mL), palladium carbon (0.1 g, 5%) was added as a catalyst, and hydrogenated for 12 h at room temperature and pressure. The mixture was filtered and the solvent was dried to obtain a brown solid. MS-ESI (m / z): 235.1 [M+H] + ; 1HNMR (DMSO-d6, 400MHz) δ: 7.16 (d, J = 8.6 Hz, 1H), 6.54 (d, J = 8.6 Hz, 1H), 6.34 (s, 1H), 3.67 (s, 3H), 2.15 (s, 3H).

[0032] 3) Synthesis of [1-methyl-2-(acetylamino)-5-N,N-di(2'-hydroxyethyl)]-1H-benzimidazole (5)

[0033] Compound 4 (4.08 g, 20 mmol) was dissolved in a solution of glacial acetic acid (15 mL) in water (30 mL), and 10 mL of ethylene oxide was added dropwise at 0°C-5°C. The mixture was stirred for 5 h at room temperature and then at 5°C-10°C for 2 h. 2 CO 3 Aqueous solution (0.6 molL -1 ) The pH of the reaction system was adjusted to 7-8, extracted with 100 mL of chloroform, dried over anhydrous sodium sulfate, and concentrated to obtain a yellow colloidal solid 5.

[0034] 4) Synthesis of [1-methyl-2-(amino)-5-N,N-di(2'-chloroethyl)]-1H-benzimidazole hydrochloride (6)

[0035] Compound 5 was added to 10 mL of concentrated hydrochloric acid, heated under reflux at 90-95°C for 4 h, and the solvent was dried to obtain an oily substance. The oily substance was poured into 20 mL of water, cooled in an ice-water bath to obtain 4.2 g of a crude product, which was directly used for the next step reaction.

[0036] 5) Synthesis of target compound (1)

[0037] Compound 6 (3.22 g, 10 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 6.0 g, 30 mmol), 1-hydroxybenzotriazole (HOBt, 0.16 g, 12 mmol) were stirred at 30°C for 0.5 h; 3-(4-methylformylphenyl)oxypropionic acid (2.24 g, 10 mmol), dimethylformamide (DMF, 8 mL), triethylamine (1.0 mL) were added to another reaction bottle and stirred at 30°C for 0.5 h; the reaction solution was mixed and stirred for 48 h, and then vacuum rotary evaporation was performed to obtain a yellow crude product. Silica gel column chromatography (petroleum ether-acetone (9:1-1:1, V:V) gradient elution was performed, and vacuum rotary evaporation was performed to obtain a white solid. EI / MS (m / z): 494.3 [M+H] + ; 1HNMR (DMSO-d6, 400MHz) δ: 7.75 (d, J = 8.7Hz, 2H), 7.61 (d, J = 9.2Hz, 1Hz), 7.09 (d, J = 2.3Hz, 1H), 7.04 (d , J=8.7Hz, 2H), 6.80 (dd, J=9.3, 2.3Hz, 1H), 4.22 (t, J=5.2Hz, 2H), 3.67 (s, 3H), 2.57 (t, J=5.3Hz, 2H).

[0038] In vitro inhibitory activity of target compounds against HDACs

[0039] 2.1 Experimental plan

[0040] 1) Add 50 μL of the test compound solution of different concentrations (the blank group and the 100% control group were replaced by buffer) and 10 μL of enzyme solution (the blank group used buffer instead of enzyme) to a 96-well ELISA plate, and incubate in a 37°C incubator for 5 min;

[0041] 2) Add 40 μL of fluorescent substrate to each well and incubate in a 37°C incubator for 30 min;

[0042] 3) Add 40 μL of stop solution (trypsin) to each well to terminate the reaction, incubate in a 37°C incubator for 20 min, then take out and read the absorbance value at the set wavelength (390 nm / 460 nm) as the fluorescence value of the compound;

[0043] 4) The inhibition rate of the compound can be calculated by the following formula, and then the half inhibition rate IC can be calculated by fitting the S curve (X axis: different concentrations of the compound, Y axis: inhibition rate at different concentrations) 50 value.

[0044] Inhibition rate = (100% fluorescence average - compound fluorescence average) / (100% fluorescence average - blank group fluorescence average) * 100%

[0045] 2.2 Experimental Results

[0046] The target compound has a significant inhibitory effect on HDACs, IC 50 12.8±1.6nmolL -1 .

[0047] Antiproliferative effect of target compounds on tumor cells

[0048] 3.1 Experimental plan

[0049] Human multiple myeloma cell line U266, human chronic myeloid leukemia K562, human promyelocytic leukemia HL60, human acute myeloid leukemia cell line KJ-1, bone marrow stromal cell line HS5 (normal cells). They were stored in a liquid nitrogen environment in the laboratory. After thawing, they were kept at 37°C, 5% CO 2 The cells were cultured in an incubator. The culture medium was RPMI-1640 or DMEM medium supplemented with 10% fetal bovine serum (FBS).

[0050] 1) Preparation of MTT solution: Dissolve 0.25 g of MTT solid in 50 mL of PBS, filter and sterilize, divide into portions, and store in a -20°C refrigerator for later use.

[0051] 2) Cell inoculation: Add 100 μL of culture medium containing cells to a 96-well plate at a number of 5,000 adherent cells / well and 10,000 suspended cells / well. After the cells settle, add 100 μL of culture medium with different concentrations of compounds. The values ​​measured in the wells containing only culture medium solution are taken as the blank group, and a group of wells inoculated with cells without any compound added are taken as the control group. Set H replicates for each group of data and calculate the average value;

[0052] 3) After the cells were cultured in a carbon dioxide incubator at 37°C for 48 hours, 20 μL of 0.5% MTT solution was added to each well and the cells were cultured in the incubator for another 4 hours;

[0053] 4) After centrifugation at 2500 rpm for 12 min, the culture medium was discarded. For adherent cells, the culture medium was directly discarded and DMSO was added at 200 μL / well to dissolve;

[0054] 5) Set the wavelength to 570nm, measure the absorbance value with a microplate reader, and calculate the inhibition rate of the compound using the following formula. Then fit the S curve (X-axis: different concentrations of the compound, Y-axis: inhibition rate at different concentrations) to calculate the half inhibition rate IC 50 value.

[0055] Inhibition rate = (100% absorbance average - compound absorbance average) / (100% absorbance average - blank group absorbance average) * 100%

[0056] 3.2 Experimental Results

[0057] The experimental results are shown in Figure 1 As shown in the figure: the target compound can significantly inhibit the proliferation of human chronic myeloid leukemia K562, human multiple myeloma cell line U266, human promyelocytic leukemia HL60, human acute myeloid leukemia cell line KJ-1 and other tumor cells, and its IC 50The target compound is toxic to the bone marrow stromal cell line HS5 (normal cells), and its IC 50 It is slightly smaller than bendamustine (less than 1 times), indicating that the target compound has good selectivity for tumor cells, and its selectivity is higher than that of bendamustine.

[0058] 3.3 Experimental Conclusion

[0059] The target compound can significantly inhibit the proliferation of human chronic myeloid leukemia K562, human multiple myeloma cell line U266, human promyelocytic leukemia HL60, human acute myeloid leukemia cell line KJ-1 and other tumor cells. 50 The value is significantly lower than that of bendamustine. This indicates that the inhibitory effect of the target compound on the proliferation of the above tumor cells is significantly stronger than that of bendamustine. The toxicity of the target compound to the bone marrow stromal cell line HS5 (normal cells) is significantly lower than that of K562, U266, KJ-1 or HL60. Its IC 50 It is slightly smaller than bendamustine (less than 1 times), indicating that it has good selectivity for tumor cells and is higher than bendamustine.

[0060] It should be understood that the present invention is described by way of example only and can be modified within the scope and spirit of the present invention. The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary skills in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A 2-amino-substituted bendamustine derivative or its hydrochloride, sulfate or tartrate having HDAC inhibitory effect, Features: The structure of the 2-amino-substituted bendamustine derivative having HDAC inhibitory effect is shown in formula (I): 。 2. A method for preparing a 2-amino substituted bendamustine derivative having HDAC inhibitory effect according to claim 1, Features The following steps are involved: Step 1: Dissolve 2-methylamino-5-nitroaniline and methyl cyanamide in water, add sodium thiosulfate while stirring thoroughly, raise the temperature to about 70°C, add concentrated hydrochloric acid dropwise to adjust the pH value to 3-4, and react for 2 h to obtain [1-methyl-2-(acetylamino)-5-nitro]-1H-benzimidazole; Step 2: Dissolve [1-methyl-2-(acetylamino)-5-nitro]-1H-benzimidazole in methanol, add palladium carbon as a catalyst, and hydrogenate at room temperature and pressure for 12 hours to obtain the reactant [1-methyl-2-(acetylamino)-5-amino]-1H-benzimidazole; Step 3: Dissolve [1-methyl-2-(acetylamino)-5-amino]-1H-benzimidazole in an aqueous solution of glacial acetic acid, add ethylene oxide dropwise at low temperature, stir for 5 h, heat and stir for 2 h, and then add K 2 CO 3 The aqueous solution was used to adjust the pH of the reaction system to 7-8, extracted with chloroform, dried over anhydrous sodium sulfate, and concentrated to obtain [1-methyl-2-(acetylamino)-5-N, N-di(2'-hydroxyethyl)]-1H-benzimidazole; Step 4: Add [1-methyl-2-(acetylamino)-5-N,N-di(2'-hydroxyethyl)]-1H-benzimidazole to concentrated hydrochloric acid, heat and reflux for 4 hours, spin dry the solvent to obtain an oily substance, pour the oily substance into water, and cool in an ice-water bath to obtain [1-methyl-2-(amino)-5-N,N-di(2'-chloroethyl)]-1H-benzimidazole hydrochloride; Step 5: [1-methyl-2-(amino)-5-N,N-di(2'-chloroethyl)]-1H-benzimidazole hydrochloride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 1-hydroxybenzotriazole were stirred at 30°C for 0.5h; 3-(4-hydroxyaminoformylphenyl)oxypropionic acid, dimethylformamide and triethylamine were added to another reaction bottle and stirred at 30°C for 0.5h; the above two reaction solutions were mixed and stirred for 48h, and then vacuum rotary evaporation was performed to obtain a yellow crude product; silica gel column chromatography was performed with gradient elution, and vacuum rotary evaporation was performed to obtain a 2-amino-substituted bendamustine derivative.

3. The preparation method according to claim 2, Features: The molar ratio of 2-methylamino-5-nitroaniline to methyl cyanamide in step 1 is 1:1-2.

4. The preparation method according to claim 2, Features: The low temperature in step 3 is 0-5°C, and the temperature is slightly raised to 5-10°C with stirring.

5. The preparation method according to claim 2, Features: The reflux temperature in step 4 is 90-95°C.

6. The preparation method according to claim 2, Features: In the step 5, the molar ratio of [1-methyl-2-(amino)-5-N,N-di(2'-chloroethyl)]-1H-benzimidazole hydrochloride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and 1-hydroxybenzotriazole is 1:(2-4):(1-2); the molar ratio of 3-(4-hydroxyaminoformylphenyl)oxypropionic acid and triethylamine is 1:(1-2).

7. The preparation method according to claim 2, Features: The silica gel column chromatography in step 5 is performed with gradient elution and separation using petroleum ether and acetone in a volume ratio of 9 to 1:

1.

8. Use of the 2-amino substituted bendamustine derivative having HDAC inhibitory effect according to claim 1 in the preparation of a medicament for treating hematological malignancies, Features: The drug comprises the 2-amino substituted bendamustine derivative, or its hydrochloride, sulfate or tartrate.

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