A cyclovirobuxine D diastereomer, preparation method and application

Abstract

The present invention discloses a cyclovirobuxine D diastereomer, a preparation method and an application thereof. The present invention discloses a cyclovirobuxine D diastereomer represented by general formula I or a pharmaceutically acceptable salt thereof; the LD of the diastereomer of the present invention 50 Its 95% confidence interval is 14.7, which is significantly less toxic than cyclovirobuxine D and has higher pharmacological activity. #imgabs0#

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical chemical industry, and specifically relates to a cyclovirobuxine D diastereoisomer and a pharmaceutically acceptable salt thereof, and a preparation method and application thereof. Background Art

[0002] Cyclovirobuxine D is an alkaloid extracted from Buxus microphylla and its plants of the same genus. Liang Bingwen et al. developed Huangyouning Tablets with it as the main ingredient (Chinese Herbal Medicine, 1981, 12, 43.), which can be used to treat cardiovascular diseases such as chest pain, coronary heart disease, and arrhythmia caused by qi stagnation and blood stasis. It has been included in the "Chinese Pharmacopoeia" (2020 edition).

[0003]

[0004] Cycloviroboxine D is an innovative drug developed independently by my country for the prevention and treatment of chest pain and heart pain. In recent years, Chinese researchers have conducted a series of studies on it, showing that cycloviroboxine D can treat cardiovascular diseases by reducing myocardial oxygen consumption, enhancing myocardial contractility, and increasing coronary blood flow (Chinese Herbal Medicine, 2021, 52, 5437-5444.). At the same time, cycloviroboxine D can also be used to prevent and treat ischemic stroke (Chinese Journal of Traditional Chinese Medicine, 2007, 07, 623-626.). However, cycloviroboxine D has shortcomings and deficiencies such as poor water solubility, slow oral onset, low bioavailability, and cumulative toxicity and hepato-renal toxicity (Chinese Journal of Pharmacology, 1982, 3, 101-104.; Chinese Journal of Traditional Chinese Medicine for Emergency, 2010, 20, 1619-1624.). Summary of the invention

[0005] In view of the deficiencies of the prior art, the object of the present invention is to provide a cyclovirobuxine D diastereomer and a pharmaceutically acceptable salt thereof, a preparation method and application thereof.

[0006] The technical solution adopted by the present invention to solve the technical problem is:

[0007] In the first aspect, the present invention protects a cyclovirobuxine D diastereomer or a pharmaceutically acceptable salt thereof as shown in the general formula I:

[0008]

[0009] Although it is only a change in the hydroxyl configuration, it greatly increases its activity and improves the efficacy of the drug.

[0010] In specific embodiments, the pharmaceutically acceptable salts are produced from different organic or inorganic acids;

[0011] Wherein, the inorganic acid is selected from any one of hydrochloric acid, sulfuric acid, phosphoric acid and hydrobromic acid;

[0012] The organic acid is selected from any one of oxalic acid, formic acid, acetic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, citric acid, lactic acid, ferulic acid, glycine, lysine, and aspartic acid.

[0013] In the second aspect, the present invention protects a method for preparing the above-mentioned cyclovirobuxine D diastereomer, and its synthetic route is as follows:

[0014]

[0015] The preparation method comprises the following steps:

[0016] (1) Formula Ⅰ-1 reacts with an amino protecting group and then undergoes an oxidation reaction to obtain Formula Ⅱ;

[0017] (2) Formula II undergoes reduction reaction and then removes the amino protecting group;

[0018] Among them, R 1 The protecting group is selected from hydrogen, an amino protecting group except acetyl and p-nitrobenzyloxycarbonyl.

[0019] In a specific embodiment, R 1 Common amino protecting groups selected from hydrogen, tert-butoxycarbonyl, benzyloxycarbonyl, methoxycarbonyl, ethoxycarbonyl, allyloxycarbonyl, methoxycarbonyl, benzyl and the like except acetyl and p-nitrobenzyloxycarbonyl, preferably hydrogen, tert-butoxycarbonyl, benzyloxycarbonyl, methoxycarbonyl and ethoxycarbonyl.

[0020] In a specific embodiment, in step (1), the oxidant for the oxidation reaction is selected from any one of Dess-Martin reagent, Jones reagent, PCC reagent, potassium permanganate, manganese dioxide, Collins reagent, and chromium trioxide, preferably Dess-Martin reagent.

[0021] In a specific embodiment, in step (2), the reducing agent used in the reduction reaction is selected from sodium borohydride, lithium aluminum hydride, lithium borohydride, aluminum isopropoxide, H 2 Any of .

[0022] In a more specific embodiment, H 2 When used as a reducing agent, the catalyst used is at least one selected from metal palladium, metal platinum or metal nickel catalysts, preferably sodium borohydride or lithium aluminum hydride.

[0023] In a third aspect, the present invention also provides a compound as shown in formula A1,

[0024]

[0025] In a specific embodiment, the present invention provides a crystalline form of the compound as shown in Formula A1, and its single crystal structure data is as follows:

[0026]

[0027] In a fourth aspect, the present invention also protects the use of the above-mentioned cyclovirobuxine D diastereomer or a pharmaceutically acceptable salt thereof in the preparation of drugs for treating cardiovascular and cerebrovascular diseases.

[0028] Beneficial Effects

[0029] The present invention reverses the hydroxyl configuration on cyclovirobuxine D to obtain diastereomers of cyclovirobuxine D and pharmaceutically acceptable salts thereof. Compared with the prior art, the LD of the diastereomers of cyclovirobuxine D of the present invention is 50 Its 95% confidence interval was 14.7 (14.2-15.3), which was significantly less toxic than that of cyclovirobuxine D and had higher pharmacological activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 MS spectrum of compound Ⅰ.

[0031] Figure 2 Compound Ⅰ 1 H-NMR spectrum.

[0032] Figure 3 Compound Ⅰ 13 C-NMR spectrum.

[0033] Figure 4 HPLC spectrum of cyclovirobuxine D.

[0034] Figure 5 HPLC spectrum of compound Ⅰ.

[0035] Figure 6-7 X-ray diffraction pattern of compound A1.

[0036] Figure 8 Effects of different concentrations of compound Ⅰ on cell viability. DETAILED DESCRIPTION

[0037] The specific steps of the present invention are described below by way of examples, but are not limited to the examples.

[0038] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.

[0039] The present invention is further described in detail below in conjunction with specific examples and with reference to data. It should be understood that these embodiments are only for illustrating the present invention and are not intended to limit the scope of the present invention in any way.

[0040] In the following examples, various processes and methods not described in detail are conventional methods well known in the art.

[0041] The present invention is further described below in conjunction with specific implementation modes.

[0042] Example 1

[0043]

[0044] Take the compound of formula Ⅰ-1 (5.0g, 12.42mmol) in a reaction bottle, add 38mL methanol and 25mL dichloromethane to stir and dissolve, cool to below 0°C, take di-tert-butyl dicarbonate (6.5g, 29.80mmol) and dissolve it in 13mL methanol, mix well and add it to the reaction solution, continue stirring for 20min and then take it out, react at room temperature for 4h. Add 1M citric acid and dichloromethane to the reaction solution to quench the reaction, take the organic layer, wash it with saturated brine, dry it with anhydrous sodium sulfate and spin dry it to obtain 7.40g of compound of formula Ⅰ-2, with a yield of 98.85% and a purity of 96.73%.

[0045] MS: 625.45529 [M+Na] +

[0046] Example 2

[0047]

[0048] Take the compound of formula Ⅰ-2 (3.0g, 4.98mmol) in a reaction bottle, add 150mL of dichloromethane to stir and dissolve, cool to below 0℃, take Dess-Martin reagent (3.17g, 7.46mmol) to the reaction solution, continue stirring for 20min, then take it out and react at room temperature for 3h. Add saturated sodium sulfite to the reaction solution to quench the reaction, take the organic layer, wash it with saturated brine, dry it with anhydrous sodium sulfate, and spin dry it to obtain 2.73g of compound of formula Ⅱ, with a yield of 91.31% and a purity of 95.23%.

[0049] MS: 623.43868[M+Na] +

[0050] Example 3

[0051]

[0052] Under nitrogen atmosphere, take the compound of formula II (1.0 g, 1.66 mmol) in a reaction bottle, add 70 mL of anhydrous methanol to stir and dissolve, cool to below 0°C, take sodium borohydride (0.2 g, 5.29 mmol) and add it to the reaction solution, and continue to react for 24 hours. Add 100 mL of dichloromethane and 100 mL of water to the reaction solution to quench the reaction, extract it three times with dichloromethane, combine the organic phases, wash with saturated brine, dry it with anhydrous sodium sulfate, and spin dry to obtain the compound of formula I-3, which is separated and purified by silica gel column (eluent: n-hexane / ethyl acetate = 50:1 to 3:1) to obtain 0.23 g of the compound of formula I-4, with a yield of 22.92% and a purity of 96.48%.

[0053] MS: 625.45392[M+Na] +

[0054] Example 4

[0055]

[0056] Take the compound of formula Ⅰ-4 (0.1 g, 4.98 mmol) into a reaction bottle, add 2 mL of dichloromethane and stir to dissolve, slowly add hydrochloric acid acetone solution (56 μL hydrochloric acid dissolved in 1 mL acetone), continue stirring for 2 hours, then take out and filter to obtain the filter cake, dissolve it with water, adjust the pH value to strong alkalinity with sodium hydroxide, extract it with ethyl acetate, dry it with anhydrous sodium sulfate, and spin dry to obtain 35 mg of compound of formula Ⅰ, with a yield of 52.40% and a purity of 98.14%.

[0057] MS: 403.37261[M+H] +

[0058] 1 H-NMR (500 MHz, CD 3 OD+D 2 O)δ4.53-4.46(m, 1H), 3.56-3.48(m, 1H), 2.88-2.81(m, 1H), 2.76(m, 3H ), 2.72(m, 3H), 2.24-2.18(m, 1H), 2.14-2.06(m, 3H), 1.74-1.61(m, 6H) ,1.53-1.40(m,4H),1.37-1.33(m,3H),1.25-1.15(m,5H),,1.12(s,3H) , 0.97 (s, 3H), 0.94 (s, 3H), 0.91-0.83 (m, 1H), 0.65 (s, 1H), 0.5 (s, 1H).

[0059] Example 5

[0060]

[0061] Take the compound of formula II (0.2 g, 0.33 mmol) into a reaction bottle, add 4 mL of dichloromethane and stir to dissolve, slowly add hydrochloric acid acetone solution (57 μL hydrochloric acid dissolved in 1 mL acetone), continue stirring for 2 hours, then take out and filter to obtain a filter cake, dissolve it with water, adjust the pH value to strong alkalinity with sodium hydroxide, extract it with ethyl acetate, dry it with anhydrous sodium sulfate, and spin dry to obtain 83 mg of compound of formula I, with a yield of 62.24% and a purity of 95.83%.

[0062] MS: 401.35370 [M+H] +

[0063] Example 6

[0064]

[0065] Take the compound of formula Ⅰ-1 (1.0 g, 2.48 mmol) in a reaction bottle, add 25 mL of dichloromethane and stir to dissolve, then add NaHCO 3 (1.04g, 12.42mmol) was dissolved in 25mL water and added to the reaction solution. The temperature was lowered to below 0℃, benzyl chloroformate (1.06g, 6.21mmol) was added to the reaction solution, and the mixture was stirred for 20min before being taken out and reacted at room temperature for 24h. Water and dichloromethane were added to the reaction solution to quench the reaction, and the organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and then spin-dried to obtain a crude compound of formula Ⅰ-5, which was separated and purified by silica gel column (eluent: n-hexane / ethyl acetate = 50:1 to 3:1) to obtain 1.28g of compound of formula Ⅰ-5, with a yield of 76.82% and a purity of 95.81%.

[0066] MS: 693.42408[M+Na] +

[0067] Example 7

[0068]

[0069] Take the compound of formula I-5 (1.0 g, 1.49 mmol) in a reaction bottle, add 150 mL of dichloromethane to stir and dissolve, cool to below 0°C, take Dess-Martin reagent (DMP) (1.06 g, 2.49 mmol) and add it to the reaction solution, continue stirring for 20 min, then take it out and react at room temperature for 3 h. Add saturated sodium sulfite to the reaction solution to quench the reaction, take the organic layer, wash it with saturated brine, dry it with anhydrous sodium sulfate, and spin dry it to obtain 0.89 g of the compound of formula I-6, with a yield of 89.27% ​​and a purity of 95.62%.

[0070] MS: 691.41041[M+Na] +

[0071] Example 8

[0072]

[0073] Under nitrogen atmosphere, take the compound of formula Ⅰ-6 (0.5g, 0.75mmol) in a reaction bottle, add 35mL of anhydrous methanol to stir and dissolve, cool to below 0℃, take sodium borohydride (0.09g, 2.38mmol) and add it to the reaction solution, and continue to react for 24h. Add 50mL of dichloromethane and 50mL of water to the reaction solution to quench the reaction, extract it with dichloromethane three times, combine the organic phases, wash with saturated brine, dry with anhydrous sodium sulfate, and spin dry to obtain the compound of formula Ⅰ-7, which is separated and purified by silica gel column (eluent: dichloromethane / methanol=100:1~5:1) to obtain 0.11g of compound of formula Ⅰ-8, with a yield of 21.93% and a purity of 96.27%.

[0074] MS: 693.42435[M+Na] +

[0075] Example 9

[0076]

[0077] Under hydrogen atmosphere, take the compound of formula Ⅰ-8 (0.08 g, 0.12 mmol) in a reaction bottle, add 2 mL of methanol and excess 10% Pd(OH) 2 , stirred at room temperature for 24 hours, filtered through diatomaceous earth, dissolved with water, adjusted the pH value to strong alkalinity with sodium hydroxide, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and spin-dried to obtain 43 mg of compound I with a yield of 64.57% and a purity of 96.34%.

[0078] Example 10

[0079]

[0080] Take the compound of formula I (30 mg) into a reaction bottle, add 1 mL of dichloromethane, stir and dissolve at room temperature, slowly add hydrochloric acid acetone solution (12 μL hydrochloric acid dissolved in 0.5 mL acetone), continue stirring for 2 h, filter out the solid, and dry it in vacuo at 40 ° C for 3 h to obtain 25 mg of compound of formula I-9, with a yield of 70.56% and a purity of 98.32%.

[0081] Embodiment 11

[0082]

[0083] Take the compound of formula I (30 mg) into a reaction bottle, add 1 mL of dichloromethane, stir and dissolve at room temperature, slowly add 0.5 mL of sulfuric acid acetone solution (42 μL of sulfuric acid dissolved in 5 mL of acetone), continue stirring for 2 hours, filter out the solid, and dry it in vacuo at 40°C for 3 hours to obtain 31 mg of compound of formula I-10, with a yield of 83.10% and a purity of 96.41%.

[0084] Example 12 Preparation of Single Crystal of Compound A1

[0085]

[0086] Take 30 mg of the target compound I and salt it according to the method provided in patent CN 106632573B to obtain 35 mg of compound A1. Take 15 mg and put it in a vial. Add 0.9 mL of ethanol and 0.3 mL of water to dissolve the solid. After sealing with plastic wrap, poke a few small holes with a needle, place it in a 50 mL beaker, cover it with an evaporating dish of appropriate size, and place it in a quiet and cool place to allow the solvent to evaporate until crystals precipitate.

[0087] The single crystal data of compound A1 are shown below:

[0088]

[0089]

[0090] The single crystal structure of compound A1 is shown in Figure 6-7 shown.

[0091] Example 13 Acute toxicity test of intravenous injection in mice

[0092] Experimental method: According to the results of the preliminary experiment, acute toxicity tests of cyclovirobuxine D and compound I were carried out on mice by intravenous injection. 60 mice were selected for each test. The mice were stratified by body weight and randomly divided into 6 groups, with 10 mice in each group, half of which were male and half were female. The highest dose of cyclovirobuxine D was 20.0 mg / kg, and the group ratio was 0.8. The concentration decreased step by step, and the lowest (Group 6) dose was 6.6 mg / kg. The highest dose of compound I was 25 mg / kg, and the group ratio was 0.8. The concentration decreased step by step, and the lowest (Group 6) dose was 8.2 mg / kg. The dosage volume was 5 mL / kg, and the drug was administered once through the tail vein. The mice were observed for 7 consecutive days after administration. The death of mice was recorded and the Bliss method was used to calculate the LD of the two drugs. 50 value.

[0093] Experimental results: According to the animal mortality, the LD of cyclovirobuxine D was calculated by Bliss method. 50 The LD of compound Ⅰ was 11.6 (10.7-12.6). 50and its 95% confidence interval was 14.7 (14.2-15.3), therefore, compound I was less toxic than cyclovirobuxine D. LD 50 The measurement results are shown in Table 1.

[0094] Table 1 LD50 of acute toxicity test in mice after intravenous injection 50 Measurement results

[0095]

[0096] Example 14 Anti-myocardial injury experiment

[0097] Primary rat cardiomyocytes (PNRCMs) were extracted by trypsin digestion, and the PNRCMs injury model was prepared with ALD (10 μmol / L). After treatment with different doses of compound Ⅰa, the cell viability was detected by the MTT method.

[0098] Culture of neonatal rat cardiomyocytes: Take 1-3 days old SD neonatal rats, disinfect them with 75% alcohol after anesthesia, cut the heart tissue, wash it with pre-cooled phosphate buffer saline (PBS), rinse it with double antibody (penicillin-streptomycin mixture) for 30 seconds, wash the double antibody with PBS, and cut the heart into about 1mm 3 Tissue blocks of different sizes were digested with 0.125% trypsin and incubated at 4°C overnight. The next day, 0.08% trypsin was added and incubated at 37°C, 5% CO 2 Incubate in an incubator for 5 min. Use a rubber-tipped pipette to gently blow the tissue, aspirate the supernatant and add it to an equal volume of DMEM (10% FBS) culture medium to terminate digestion. Repeat the above steps until the tissue becomes white and translucent. Centrifuge the cell suspension (1000 r / min -1 , 5min), discard the supernatant, add DMEM (10% FBS) to resuspend, and inoculate into the culture bottle, differentially adhere to the wall for 90min, purify and separate primary cardiomyocytes. After the adhesion is completed, take the cell suspension, count it with trypan blue staining, and inoculate it into the required well plate at the required density, and add a final concentration of 0.1mmol·L -1 5-Br was co-cultured to inhibit the proliferation of non-cardiac cells. After 72 hours of cell culture, the cardiomyocytes and purity were identified by immunofluorescence detection of cardiomyocyte-specific protein cTnT. Only cells with a purity of more than 90% could be used for subsequent experiments.

[0099] Experimental groups: control group (Control), ALD group, positive control group (10μmol·L -1 ALD+1μmol·L - 1 CVB-D), compound I group (10 μmol·L -1ALD+compound I).

[0100] MTT assay to detect cell viability: 1×10 6 pc·mL -1 The cardiomyocytes were seeded into 96-well plates and cultured for 72 h. -1 ) and co-administered with CVB-D (1 μmol·L -1 ), compound Ⅰ (0.1, 0.5, 1, 10 μmol·L -1 ) for 48 hours, with 6 replicate wells in each group, and the experiment was repeated 3 times. MTT was added and incubated for 4 hours, and then DMSO was added to dissolve it, and the absorbance of each well was measured at a wavelength of 490 nm, and the cell survival rate was calculated using the following formula.

[0101] Cell survival rate / % = OD of drug group / OD of blank group × 100%

[0102] Experimental Results

[0103] MTT results showed that administration of different concentrations of compound Ⅰ could improve ALD-induced cell damage in a dose-dependent manner (*P<0.05, **P<0.01). Figure 8 , and with 1μmol·L -1 Compared with CVB-D, 1μmol·L -1 Compound I improved ALD-induced cell damage to a greater extent, indicating that the pharmacological activity of compound I is higher than that of CVB-D.

[0104] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims

1. A compound having the structural formula shown in general formula I or a pharmaceutically acceptable salt thereof:

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The pharmaceutically acceptable salts are produced from different organic or inorganic acids; Wherein, the inorganic acid is selected from any one of hydrochloric acid, sulfuric acid, phosphoric acid and hydrobromic acid; The organic acid is selected from any one of oxalic acid, formic acid, acetic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, citric acid, lactic acid, ferulic acid, glycine, lysine, and aspartic acid.

3. A method for preparing the compound according to claim 1, characterized in that: Its synthetic route is as follows: The preparation method comprises the following steps: (1) Formula Ⅰ-1 reacts with an amino protecting group and then undergoes an oxidation reaction to obtain Formula Ⅱ; (2) Formula II undergoes a reduction reaction and then removes the amino protecting group; (3) separation and purification by silica gel column to obtain the compound represented by the general formula I according to claim 1; Among them, R 1 The protecting group is selected from hydrogen, an amino protecting group except acetyl and p-nitrobenzyloxycarbonyl.

4. The preparation method according to claim 3, characterized in that: The amino protecting group is selected from any one of tert-butyloxycarbonyl, benzyloxycarbonyl, methoxycarbonyl, ethoxycarbonyl, allyloxycarbonyl, tert-methoxycarbonyl and benzyl.

5. The preparation method according to claim 4, characterized in that: The amino protecting group is selected from any one of tert-butyloxycarbonyl, benzyloxycarbonyl, methoxycarbonyl and ethoxycarbonyl.

6. The preparation method according to claim 3, characterized in that: In the step (1), the oxidant for the oxidation reaction is selected from any one of Dess-Martin reagent, Jones reagent, PCC reagent, potassium permanganate, manganese dioxide, Collins reagent, and chromium trioxide.

7. The preparation method according to claim 6, characterized in that: In the step (1), the oxidant for the oxidation reaction is Dess-Martin reagent.

8. The preparation method according to claim 3, characterized in that: In the step (2), the reducing agent used in the reduction reaction is selected from any one of sodium borohydride, lithium aluminum hydride, lithium borohydride, aluminum isopropoxide, and H2.

9. The preparation method according to claim 8, characterized in that: When H2 is used as a reducing agent, the catalyst used is selected from at least one of a metal palladium, a metal platinum or a metal nickel catalyst.

10. The preparation method according to claim 9, characterized in that: When H2 is used as the reducing agent, the catalyst used is sodium borohydride or lithium aluminum hydride.

11. A compound as shown in formula A1:

12. The compound according to claim 11, characterized in that The single crystal structure data of the compound are as follows:

13. Use of the compound according to any one of claims 1 or 2 or a pharmaceutically acceptable salt thereof or the compound according to claim 11 or 12 in the preparation of a medicament for treating cardiovascular and cerebrovascular diseases.

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