CN101284792A - Bisbenzylisoquinoline compound and its preparation method and application - Google Patents

Abstract

The invention provides a kind of bisbenzylisoquinoline compound in black medicine and its preparation method. The dry tuber root of black medicine is taken as raw material, after being extracted with alcohol solvent, the extract is concentrated under reduced pressure to obtain extract, and the extract is dissolved in water After suspending and acidifying with dilute acid, use organic solvent extraction to remove neutral components, then use weak base to adjust the pH to 10 in the water phase, use organic solvent extraction and solvent recovery to obtain the crude product of total alkaloids, dissolve the crude product in alcohol, and then After adding an appropriate amount of water for precipitation treatment, the refined total alkaloids can be obtained, eluted by chloroform-methanol system, and gradient eluted by water-methanol, the eluted part is collected, concentrated and removed to obtain the final product. The invention is used to carry out inhibitory activity experiments on L1210 and K562 tumor cell lines, and the results show that they all have strong cytotoxic activity and present a dose-effect relationship, and can be used in the preparation of antitumor drugs. The compound of the present invention has the above structural formula.

Description

Bisbenzylisoquinoline compound and its preparation method and application

technical field

The invention belongs to the technical field of medicine, and in particular relates to a method for preparing a new bisbenzylisoquinoline alkaloid from traditional Chinese medicine wuyao and its application in the preparation of antitumor drugs.

Background technique

Wuyao is a commonly used traditional Chinese medicine. It is one of the important medicines for warming the stomach, regulating qi and relieving pain. Modern research has found that the chemical composition of black medicine is complex, rich in furosesquiterpenes and their lactones, flavonoids, volatile oils, isoquinoline alkaloids, etc. Improve central nervous system function, anti-inflammatory and analgesic, prevent and treat diabetic nephropathy, protect liver, regulate blood coagulation function and other pharmacological effects. The alkaloids contained in black medicine are mostly isoquinoline alkaloids, among which laureolistine, boldine and reticuline are the main three isoquinoline alkaloids, It is ubiquitous in Lauraceae plants (Chemical constituents and pharmacological effects of Wuyao. Wang Zhengtao, Xu Luoshan. Chinese Wild Plant Resources, 1999, 18(3), 5-10.). Modern pharmacological studies have shown that the extract of black medicine has obvious inhibitory effect on mouse sarcoma S180. The extract of black medicine can induce the production of cytostatic factors in mice, inhibit tumor growth, prolong the survival period of mice with lung cancer, but does not show any toxicity to normal cells, and the curative effect is positively correlated with the dose (Phytochemical and pharmacological research of black medicine General situation. Wang Junwei, Ruan Bing. Zhejiang Journal of Traditional Chinese Medicine, 2006, 41(11), 675-677.). However, no bisbenzylisoquinoline alkaloids have been found in black medicine so far, and there is no report on the anti-tumor cytotoxic activity of such alkaloids in black medicine.

Contents of the invention

The object of the present invention is to provide a kind of bisbenzyl isoquinoline compound (Linderegatine, I) that extracts in a kind of black medicine, has following structural formula:

The second object of the present invention is to provide the preparation method of bisbenzylisoquinoline compound (I), which is realized by the following steps: the dry tuber root of black medicine is cut into thin slices, and after pulverizing, add 10 times the amount according to the weight/volume ratio of medicinal material/solvent Alcohol, extract at room temperature or reflux for 2-3 times until the color of the extract is lighter. Combine the extracts, concentrate under reduced pressure to obtain the extract, suspend and dissolve the extract with an appropriate amount of water, acidify with an inorganic acid, extract with a medium-polar organic solvent to remove non-alkaloid components, and adjust the pH of the water phase to 10 with a weak base , extracted three times with a medium-polar organic solvent, and the organic solvent layer was backwashed with water to neutrality, and the crude product of total alkaloids was obtained after solvent recovery; the crude product was dissolved with an appropriate amount of alcohol, and water was added to keep the alcohol content ≤ 60% to precipitate Completely, the refined total alkaloids can be obtained after centrifugation or filtration. Take the refined total alkaloids, mix with silica gel, add to the top of the chromatographic column filled with silica gel, wash with chloroform-methanol solvent system from 50:1→25:1 Remove, collect chloroform:methanol 25:1 polar segment elution fraction, concentrate and remove the solvent, go through reversed-phase C-18 silica gel chromatography, use water-methanol solvent system from 80:20→40:60 gradient elution, collect water: The methanol 40:60 eluted fraction was concentrated to remove the solvent to obtain the target compound I.

Another object of the present invention is to provide bisbenzylisoquinoline compounds with strong cytotoxic activity in the preparation of antitumor drugs.

When using the H9C2 myocardial cell line hypoxia-reoxygenation model to screen drugs with cardioprotective effects, it was found that total alkaloids at lower concentrations of 10 and 30 μg/ml can increase the survival rate of cells and reduce the activity of LDH in cells, but in At a higher concentration of 90μg/ml, it has certain toxicity to cardiomyocytes. Therefore, it is speculated that the trace components in the total alkaloids may have strong cytotoxic activity. Subsequent research focuses on the acquisition of trace amounts of active ingredients. Under the guidance of the activity experiment, compound I was obtained through multi-stage chromatographic separation (including silica gel, alumina, reversed-phase silica gel and gel chromatography), and was tested by various spectral techniques, including ultraviolet spectroscopy (UV), infrared spectroscopy (IR) , mass spectrometry (MS), nuclear magnetic resonance (NMR), and after detailed analysis of the spectral information, the structure was confirmed to be a bisbenzylisoquinoline alkaloid containing a highly unsaturated conjugated system. Compound I was formulated into three concentrations of 90 μM/L, 30 μM/L and 10 μM/L to carry out inhibitory activity experiments on L1210 and K562 tumor cell lines. The results showed that the three concentrations had strong cytotoxic activity, and showed a dose effect relation.

The benefits of the present invention are:

(1) For the first time, a bisbenzylisoquinoline alkaloid with a highly unsaturated conjugated system was discovered from wuyao, and the preparation method and spectral data of compound I were provided, which provided a basis for the discovery of this type of novel structure from wuyao in the future. Compounds lay the groundwork;

(2) For the first time, it was discovered that compound I has a strong inhibitory effect on tumor cells. After conducting structure-activity relationship research on compound I, it will be possible to obtain more active anti-tumor lead compounds or drugs through chemical synthesis after computer-aided rational design;

(3) Compound I has almost no toxicity to normal cells at a concentration that produces a strong inhibitory effect on tumor cells, suggesting that this compound has a good prospect of being developed into a new anti-tumor drug.

Description of drawings

Figure 1 is a high-performance liquid chromatogram of total alkaloids of black medicine.

Detailed ways

The present invention is further described in conjunction with specific embodiments and accompanying drawings.

The physicochemical and UV, IR, MS spectral data of embodiment 1 compound I are as follows:

Compound I is light yellow solid, [α] _D ²⁰ -16 ° (c 1.0, MeOH); Electrospray mass spectrometry (ESIMS) (positive ion positive) m/z 595[M+H] ⁺ (100); (negative ion type negative) m/z 593 (100); UV (MeOH) λ _max nm (log ε): 207 (5.30), 288 (4.87); IR (KBr) v _max cm ^-1 3405, 2937, 2842, 1664, 1588 , 1510, 1450, 1371, 1276, 1228, 1135, 1028, 756.

The ¹ H NMR of compound I, ¹³ C NMR data and assignment see Table 1, the assignment of each hydrogen, carbon signal through testing two-dimensional NMR spectrum (carbon-hydrogen correlation spectrum, carbon-hydrogen long-distance correlation spectrum, nuclear Overhauser effect II Dimensional correlation spectrum) obtained.

Table 1. ¹ H (400MHz) and ¹³ C (100MHz) NMR data and signal assignment of compound I (CD ₃ OD solvent)

no δ _H (multi, J in Hz) _δC no δ _H (multi, J in Hz) _δC 1 167.8 1' 4.45 (dd, 6.1, 8.0) 57.3 3 3.79(2H, m) 47.8 3′ a 3.37(1H, m)b 3.15(1H, m) 40.6 4 2.83(2H, m) 26.0 4′ 2.89(2H, m) 26.9 4a 131.4 4'a 124.6 5 6.90(1H,s) 111.9 5' 6.68(1H, s) 112.6 6 152.6 6′ 148.8 7 146.4 7' 146.2 8 6.67(1H, s) 114.4 8' 6.48(1H, s) 114.4 8a 120.3 8a′ 126.4 α 194.4 α' a 3.25(1H,dd,14.0,6.1)b 3.03(1H,dd,14.0,8.0) 40.7 9 130.3 9' 130.8 10, 14 7.91 (2H, d, 8.8) 133.6 10' 6.93(1H, s) 124.8 11, 13 6.91 (2H, d, overlap) 117.0 11′ 144.0 12 165.0 12′ 152.3 6-OCH ₃ 3.90(3H,s) 56.5 13′ 7.13 (1H, d, 8.4) 114.6

14′ 7.20 (1H, d, 8.3) 128.7 6′-OCH ₃ 3.78(3H, s) 56.3 12′-OCH ₃ 3.74(3H, s) 56.3

Example 2

Cut 5kg of dried root tubers of black medicine into thin slices, add 50L ethanol after crushing, and extract twice by percolation. The two extracts were combined and then concentrated under reduced pressure to obtain an extract, which was suspended and dissolved in 10 L of water, acidified with 2N HNO ₃ and extracted three times with an equal volume of ethyl acetate, and the ethyl acetate layer was discarded after recovering the solvent. The aqueous phase was then adjusted to pH 10 with 5% _Na2CO3 , extracted twice with an equal volume of ethyl acetate, combined with the ethyl acetate extracts, washed with distilled water until neutral, and 32.3 _g was obtained after recovering ethyl acetate under reduced pressure Crude total alkaloids. Dissolve the crude product in 3L of ethanol, add water to make the ethanol content 60%, let it stand for complete precipitation, filter and dry to obtain 12.8g of refined total alkaloids.

Take the above-mentioned refined total alkaloids of black medicine, mix with 15g of silica gel, add to the top of the chromatographic column equipped with 600g of silica gel, elute with chloroform-methanol solvent system from 50:1→25:1, collect chloroform:methanol 25:1 The fraction eluted in the neutral section was concentrated to remove the solvent, and then subjected to reverse-phase C-18 silica gel chromatography, and the water-methanol solvent system was used for gradient elution from 80:20→40:60. The fraction eluted with water:methanol 40:60 was collected, concentrated and removed. After solvent, 316 mg of compound I were obtained.

Example 3

Cut 3kg of dried root tubers of black medicine into thin slices, add 30L methanol after crushing, and reflux extraction twice, the first time is 2 hours, after the extract is filtered, add 24L methanol to continue reflux extraction for 1 hour. After combining the two extracts, concentrate under reduced pressure to obtain the extract, suspend and dissolve the extract with 6L of water, acidify with 3N HCl, extract with equal volume of chloroform three times, and discard the chloroform layer after recovering the solvent. The pH of the aqueous phase was adjusted to 10 with ammonia water, extracted twice with an equal volume of chloroform, the combined chloroform extracts were washed with distilled water until neutral, and 21.7 g of crude total alkaloids were obtained after recovering the chloroform under reduced pressure. Dissolve the crude product in 3L of methanol, add water to make the methanol content 50%, let it stand for complete precipitation, filter and dry to obtain 8.6g of refined total alkaloids.

Take the above-mentioned refined total alkaloids of black medicine, mix with 10g alumina, add to the top of the chromatographic column equipped with 260g alumina, elute with chloroform-methanol solvent system from 50:1→30:1, collect chloroform:methanol 30: 1. The elution fraction of the polar segment was concentrated to remove the solvent and then subjected to gel Sephadex LH-20 column chromatography with methanol as the eluent. The fraction containing Compound I was obtained by TLC detection. After concentration and removal of the solvent, 198 mg of Compound I was obtained.

The total alkaloids obtained by the method of Examples 1-3 are relatively high in purity, and mainly contain three components, and the content sum of the three compounds (compounds a, b, c) is measured by the high performance liquid chromatography HPLC normalization method More than 85%, in addition, it also contains a trace component compound I, see accompanying drawing 1, wherein 1 is compound a (norboldine); 2: compound b (boldine); 3: compound c (reticuline); 4: compound I (linderegatine) , the structural formula is as follows:

Compound a Compound b Compound c

The pilot scale preparation of embodiment 4 compound I

Cut 100kg of dried root tubers of black medicine into thin slices, add 1000L ethanol after crushing, reflux extraction twice, the first time is 2 hours, add 800L ethanol to continue reflux extraction for 1 hour after filtering the extract. Combine the two extracts and concentrate under reduced pressure to obtain the extract, suspend and dissolve the extract with 200L of water, acidify with 2N HCl and extract three times with an equal volume of ethyl acetate, and discard the ethyl acetate layer after recovering the solvent. The aqueous phase was adjusted to pH 10 with aqueous ammonia, extracted twice with an equal volume of ethyl acetate, the ethyl acetate extracts were combined, washed with distilled water until neutral, and 766 g of crude total alkaloids were obtained after recovery of ethyl acetate under reduced pressure. Dissolve the crude product in 80L of ethanol, add water to make the ethanol content 40%, let it stand for complete precipitation, filter and dry to obtain 362g of refined total alkaloids. Take the above-mentioned refined total alkaloids of black medicinal herbs, mix them with 400g of alumina, add to the top of the chromatographic column equipped with 8kg of alumina, elute with chloroform-methanol solvent system from 50:1→30:1, collect chloroform:methanol 30: 1 The fraction eluted in the polar segment was concentrated to remove the solvent, and then subjected to reverse-phase C-18 silica gel chromatography, eluted with a water-methanol solvent system from 80:20→40:60 gradient, and the fraction eluted with water:methanol 40:60 was collected. The fractions containing Compound I were obtained by TLC detection, and 7.9 g of Compound I were obtained after being combined and concentrated to remove the solvent.

Example 5 The Pharmacological Activity Research of Total Alkaloids and Compound I

5.1. Experimental materials

L1210, K562 tumor cell line, PAA serum, H-DMEM

5.2. Experimental method

5.2.1. Drug preparation: the total alkaloids (LAA) of black medicine were formulated into three concentrations of 90 μg/ml, 30 μg/ml and 10 μg/ml, and the compound I (Linderegatine) was formulated into 90 μM/L, 30 μM/L and 10 μM/L L three concentrations.

5.2.2. Inoculate H9C2, L1210, and K562 cell lines into 96-well plates at 10,000 cells/well, add various concentrations of drugs, culture for 48 hours, and incubate with MTT for 4 hours to detect cell survival rate.

5.2.3. Statistical method: All data were statistically analyzed with t-text.

5.3. See Table 2 for the results.

Table 2. Cytotoxic activity test data of total alkaloids and Linderegatine

Through the cytotoxicity experiment, it can be seen that the total alkaloids of agaricus aeginae have a certain toxicity to cardiomyocytes at a higher concentration of 90 μg/ml, and compound I has a significant effect on two tumor cell lines L1210 and K562 at a lower concentration of 10 and 30 μM. Strong cytotoxic activity, but almost no toxicity to normal cardiomyocytes at a concentration of 10 μM, suggesting that this compound has potential anti-tumor value, and it is expected to be developed into a new anti-tumor drug or as a lead compound for research and development.

Embodiment 6. Preparation of compound I (Linderegatine) preparation

6.1. Making Tablets

Mix 3.0 g of compound I (Linderegatine) with 10 g of starch, add 3 g of 10% starch slurry to make a soft material, add 0.3 g of magnesium stearate, mix with 2 g of dry starch, and press to make 100 tablets. Each tablet contains 30mg of total alkaloids.

6.2. Made into injection

Take 1.0g of compound I (Linderegatine), add 10g of mannitol, add water for injection to 1000ml, heat and dissolve, filter through a 0.22μm microporous membrane, and put 2ml of each bottle into 5ml vials, each bottle contains 2.0mg of Linderegatine, Put it into a vacuum cold dryer to freeze-dry, take it out and pack it with a cap.

Claims (5)

1. a preparation method of bisbenzylisoquinoline compound is characterized in that it has the following structural formula:

2. A preparation method of bisbenzylisoquinoline compound, characterized in that it is realized through the following steps: the dry tuber root of black yaoyao is cut into thin slices, after pulverizing, 10 times of alcohol is added according to the weight/volume ratio of medicinal material/solvent, and extracted at room temperature Or reflux extraction 2-3 times until the color of the extract is lighter. Combine the extracts, concentrate under reduced pressure to obtain the extract, suspend and dissolve the extract with an appropriate amount of water, acidify with an inorganic acid, extract with a medium-polar organic solvent to remove non-alkaloid components, and adjust the pH of the water phase to 10 with a weak base , extracted three times with a medium-polar organic solvent, and the organic solvent layer was backwashed with water to neutrality, and the crude product of total alkaloids was obtained after solvent recovery; the crude product was dissolved with an appropriate amount of alcohol, and water was added to keep the alcohol content ≤ 60% to precipitate Completely, after centrifugation or filtration, the refined total alkaloids can be obtained. Take the refined total alkaloids, mix the sample with silica gel, add to the top of the chromatographic column filled with silica gel, wash with chloroform-methanol solvent system from 50:1→25:1 Remove, collect chloroform:methanol 25:1 polar segment elution fraction, concentrate and remove the solvent, go through reversed-phase C-18 silica gel chromatography, use water-methanol solvent system for gradient elution from 80:20→40:60, collect water: The methanol 40:60 eluted part was concentrated to remove the solvent to obtain the target compound I. 3. the application of the bisbenzylisoquinoline compound described in claim 1 in the preparation of antineoplastic drugs. 4. The application of the bisbenzylisoquinoline compound according to claim 2, characterized in that: the bisbenzylisoquinoline compound and pharmaceutical adjuvants allowed by the formulation are prepared into medicine. 5. the application of bisbenzylisoquinoline compound according to claim 3 is characterized in that: the preparation form of described medicine is injection or tablet.

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