CN1077887C - Preparation method of antihypertensive and antitumor compound - Google Patents

Abstract

The present invention relates to compounds of formula A and formula B, their preparation methods and their use in preparing blood pressure lowering and anti-tumor drugs.

Description

Preparation method of antihypertensive and antitumor compound

The invention relates to two compounds with hypotensive and antitumor activities, their preparation methods, and their application in the preparation of medicines.

Since Fleming discovered penicillin from fungi in 1929, fungal metabolites have become a rich source of drugs. Most clinically used antibiotics are derived from fungi and bacteria. Fungal metabolites have other medicinal values. Such as anti-tumor, treatment of cardiovascular diseases, immunomodulators, enzyme inhibitors, etc. Due to the particularity of the marine environment, marine fungi can provide metabolites that terrestrial fungi cannot provide. Some compounds with unique structures have been discovered from marine fungi internationally, which have antibacterial, antiviral, antitumor and neurocardiovascular activities. For example, cephalosporins produced from Acremonium ehrysogenum have been developed into a large class of semi-synthetic antibiotics and are widely used in clinical practice. For other examples, see the article "Marine fungi a profile resource of biologically active natural products?" by Kerstin Liberra. "[Pharmazie 50 (1995) H.9: 583-588], international research in this area has shown a trend of accelerated development since the 1980s.

The purpose of the present invention is to provide a new class of compounds with potential medical value and their extraction and separation method, as well as their use in the preparation of antihypertensive and antitumor drugs.

The fungus Halorosellinia oceanicum is isolated from the subtropical marine ecological environment. Its ascocarps are arranged singly or in groups and lack well-developed ascus. In a petri dish with oat agar as a batch medium, after 4 weeks of inoculation, the colony can grow The surface of the culture medium is full. In the initial stage of growth, the colony is white, covered with scattered hairs, and clings to the culture medium. It has irregular ring patterns with dense and complete edges, and then the color of the ring patterns gradually deepens. The bottom surface of the culture has no color. Ascocarps solitary or several side by side, linear or ring-shaped, leathery on fresh cultures, 8 spores in the ascus, ascospores in single row, or partly double row in the top of the ascus, gray-green or opaque Brown, ascospore wall smooth, slightly thick, without attachments or epispores, ascospore buds can usually be clearly seen on the back side, straight and obvious. There is no report on the metabolites of this genus of fungi.

The inventor extracted and isolated two compounds with novel structures from the fermentation culture of the marine fungus Halorosellinia oceanicum 323 in the South China Sea. The structure is composed of phenethylamine and irregularly substituted diterpenes. This structure is unprecedented.

Compound of the present invention is shown in following formula (A) and formula (B) (hereinafter referred to as compound A and B respectively for short):

The compounds A and B of the present invention can be obtained by extracting and separating from the fermentation broth of the fungus Halorosellinia oceanicum 323.

The fungus Halorosellinia oceanicum 323 used in the present invention has been preserved in the China Center for Type Culture Collection (CCT, China, within the campus of Wuhan University), the preservation number is CCTCC No: 99008, and the preservation date is May 31, 1999.

The method for preparing compounds A and B of the present invention comprises the following steps;

a. Seed culture of the fungus Halorosellinia oceanicum 323 CCTCC No: M99008: medium composition by weight: glucose 0.5-1.5, yeast extract 0.05-0.15, peptone 0.1-0.3, agar 1-1.5, sodium chloride 3-5 , water 100, make a test tube slant, pick the strains and insert them into the slant, and cultivate them at 30-35°C for 5-7 days;

b. Fermentation culture of the fungus Halorosellinia oceanicum 323 CCTCC No: M99008: The composition of the fermentation medium is: glucose 0.5-1.5, yeast extract 0.05-0.15, peptone 0.1-0.3, sodium chloride 3-5, water 100, Pick the cultured strains in the slope into the fermentation medium, and let it stand at room temperature 25-35°C for 1-2 months;

c. filtering the above-mentioned cultivated fermented liquid to remove the thallus;

d. The fermented liquid is heated and concentrated to 1/3-1/5 of the volume of the stock solution, extracted several times with ethyl acetate, concentrated ethyl acetate extract, and chromatographically separated in a silica gel column, using ethyl acetate:petroleum ether= 1%-100% is gradient elution of eluent;

e. Collect 70%-90% ethyl acetate:petroleum ether eluate and concentrate to obtain white needle crystals to obtain a mixture of A and B. Recrystallize multiple times in methanol to separate A and B.

Animal experiments showed that compounds A, B and isoproterenol could all inhibit the automatic rhythmic contraction of the ileum, and the final concentrations of compounds A and B were within the dose range of 5×10 ^-6 -5×10 ^-3 mg/ml, Slowly relax the smooth muscle until it is completely relaxed in 15 minutes, and the relaxation of the smooth muscle can last for 2.5 hours (washing once every 20 minutes), while the relaxation effect of isoproterenol is fast, and it can be recovered in about 20 minutes after washing. The concentration is 5×10 ^-5 mg/ml.

The effect of the compound of the present invention on rabbit vascular smooth muscle is at a final concentration of 5×10 ^-6 -5×10 ^-3 mg/ml, and it can inhibit the contraction of rabbit aortic vascular smooth muscle caused by adrenaline, and the concentration is greater than 5×10 ^-3 At mg/ml, it directly relaxes the smooth muscle of rabbit aorta.

The dose of the compound of the present invention is 0.2 mg/kg, which has obvious antihypertensive effect in the test of anesthetized rats, and has not recovered after 2 hours.

Both compounds A and B of the present invention can inhibit the growth of tumor cell lines. In the MTT reduction method detection anti-tumor activity test using the mouth floor cancer cell line as the target cell, the _IC50 of the compound A is 124 μg/ml, and the compound The _IC50 of B was 129 μg/ml.

The above results show that compounds A and B have good smooth muscle relaxation effects, and the duration is longer than that of the existing drug isoproterenol. Compounds A and B have obvious antihypertensive effects, which have not recovered after 2 hours. At the same time, both compounds A and B can effectively inhibit the growth of tumor cell lines. Therefore, the compound A or/and B of the present invention can be used in the preparation of antihypertensive and antitumor drugs.

The present invention will be further described below in conjunction with embodiment.

Example 1

The separation and preparation method of compound A and B

a. Seed culture of the fungus Halorosellinia oceanicum 323 CCTCC No: M99008: medium composition by weight: glucose 0.5-1.5, yeast extract 0.05-0.15, peptone 0.1-0.3, agar 1-1.5, sodium chloride 3-5 , water 100, make a test tube slope, pick the strains into the slope, and cultivate at 30-35°C for 5-7 days;

b. Fermentation culture of the fungus Halorosellinia oceanicum 323 CCTCC No: M99008: The components of the fermentation medium are: glucose 0.5-1.5, yeast extract 0.05-0.15, peptone 0.1-0.3, sodium chloride 3-5, water 100, Pick the cultured strains in the slope into the fermentation medium, and let it stand at room temperature 25-35°C for 1-2 months;

c. filtering the above-mentioned cultivated fermented liquid to remove the thallus;

d. The fermented liquid is heated and concentrated to 1/3-1/5 of the volume of the stock solution, extracted several times with ethyl acetate, concentrated ethyl acetate extract, and chromatographically separated in a silica gel column, using ethyl acetate:petroleum ether= 1%-100% is gradient elution of eluent;

e. Collect 70%-90% ethyl acetate:petroleum ether eluate and concentrate to obtain white needle crystals to obtain a mixture of A and B. Recrystallize multiple times in methanol to separate A and B.

Both A and B are white needle crystals, the melting point of A is 280°C (decomposition), and the melting point of B is 274°C (decomposition).

Experimental data of compound A: Elemental analysis: C ₃₀ H ₃₇ NO ₆ (A and B are the same) % Hydrocarbon nitrogen A measured value 70.86 7.53 2.25B measured value 71.03 7.58 2.37 calculated value 71.01 7.30 2.76MS (FAB) m/z: 509, 490, 479, 448, 430, 412, 402, 388, 357, 348, 289, 265, 254, 172, 165, 91, 77. IR(KBr)cm ^-1 : 3416, 3233, 3128, 2973, 2974, 2860, 1743, 1693, 1454, 1370, 1271, 1229, 1046, 1011, 962, 906, 779, 744, 702. UV (CHCl ₃ ) λmax nm: 238 (ε12845), 260 (ε11379), 281 (ε7706) ¹ HNMR (CDCl ₃ , TMS) ppm: 7.32 (t, 8, 8Hz, 2H), 7.26 (t, 8, 8Hz , 1H), 7.14(d, 8Hz, 2H)), 6.11(dd, 2, 15.5Hz, 1H), 5.72(bs, 1H), 5.69(dd, 10, 15.5Hz, 1H), 3.24(bs, 1H ), 2.85(t, 10, 10Hz, 1H), 2.81(dd, 5, 13.5Hz, 1H), 2.74(dq, 7, 12.5Hz, 1H), 2.70(d, 3.5, 7Hz, 1H), 2.68( dd, 9.5.13.5Hz, 1H), 2.51(ddd, 10.5, 12.5, 13Hz, 1H), 2.15(dd, 3.5, 5Hz, 1H), 2.27(s, 3H), 1.51(s, 3H), 1.21( d, 6.5Hz, 3H), 0.94(d, 6.5Hz, 3H), 1.92(br, 2H) ¹³ C-NMR (CDCl ₃ ): C 210.24, 173.73, 169.66, 147.5, 137.16, 77.67, 53.28CH 134.04, 132.25, 130.56, 127.52, 127.02, 69.77, 77.10, 53.56, 49.85, 46.91, 42.27, 32.60, 129.07×2, 128.88×2 CH ₂ 114.41, 45.18, 37.68 CH ₃ 26.12, 190.79,

Test data of compound B: NS(FAB) m/z: 508, 490, 479, 60, 448, 43, 412, 48, 20, 89, 79, 19, 165, 136, 120, 107, 9177, 65, 51IR(KBr)cm ^-1 : 3416, 3198, 3121, 2973, 2924, 2853, 1743, 1700, 1461, 1377, 1236, 1046, 1011, 962UV(CHCl ₃ ) λmax nm: 238(ε6457), 281(ε2660 ), 301 (ε1875), 328 (ε1640) ¹ HNMR (CDCl ₃ , TMS) ppm: 7.32 (t, 7, 7Hz, 2H), 7.25 (t, 7, 7Hz, 1H), 7.17 (d, 7Hz, 2H ), 6.01(dd, 2, 15.5, 1H), 5.90(t, 2, 2Hz, 1H), 5.89(dd, 15.5, 10.5Hz, 1H), 5.34(ddd, 15.5, 10.5, 5.5H2, 1H), 5.16(dd, 2, 15.5Hz, 1H), 3.78(d, 10Hz, 1H), 3.34(dd, 7.5, 6.5Hz, 1H), 2.96(dd, 7.5, 14Hz, 2H), 2.74(dq, 7, 12.5Hz, 1H), 2.471(bs, 1H), 2.26(s), 1.70(s, 3H), 1.51(s, 3H), 1.46(s, 3H), 1.20(d, 7Hz, 3H). ^13C -NMR (CDCl ₃ ) ppm: C 210.00, 174.40, 169.60, 137.60, 131.40, 126.30, 77.86, 52.64, CH 134.11, 131.99, 131.0, 128.35, 127.02, 75.02, 68.10, 60.461, 42.9, 50.8 2, 128.92×2.CH ₂ 44.79.37.64CH ₃ 24.32, 2.79, 19.36, 17.17, 13.84

Example 2

Effects of Compounds A and B on Guinea Pig Ileum Smooth Muscle

a. Accurately weigh 15 mg each of compounds A and B, dissolve them with 8 drops of dimethyl sulfoxide, then slowly add 10 ml of normal saline prepared with double distilled water to a concentration of 1.5 mg/ml.

b. The guinea pig was stunned and the ileum was dissected, and the ileum was taken with pre-cooled Tyrode nutrient solution (1000mlH ₂ O, NaCl 8g, KCl 0.2g, MgCl ₂ 0.1g, NaH ₂ PO ₄ .2H ₂ O 0.05g, NaHCO ₃ 1g, CaCl ₂ 0.2g, glucose 1g, pH 7.4) wash the food residue in the intestine, then cut it into a length of 3 cm, clamp both ends with frog heart clips, place in the perfusion bath, and fix the frog heart clip at the bottom of the bath , the upper end of the frog heart clip is connected to the tension transducer of the second physiological recorder (Chengdu Instrument Factory, LMS-2B) with a line, and the automatic rhythmic contraction of the ileum is recorded. Pure oxygen, when the ileum is hung up, apply a pulling force of 1g, and start to balance for 40 minutes (change Tyrode's nutrient solution every 20 minutes). First record a section of normal contraction curve of the ileum, and then add isoproterenol and the positive control drug respectively.

The test results are as follows:

Compounds A, B and isoproterenol can all inhibit the automatic rhythmic contraction of the ileum. Compounds A and B slowly relax smooth muscle in the dose range of 5×10 ^-6 -5×10 ^-3 mg/ml in the final concentration. It relaxes completely at 15 minutes, and the relaxation of smooth muscle can last for 2.5 hours (washing every 20 minutes), while the relaxation effect of isoproterenol is fast, and it can be restored in about 20 minutes after washing. The final concentration of isoproterenol is 5× 10 ^-5 mg/ml.

The results show that compounds A and B have a good smooth muscle relaxation effect, and the duration is longer than that of the existing drug isoproterenol.

Example 3

Effects of Compounds A and B on Rabbit Aortic Strips

One New Zealand white rabbit was decapitated, and the chest was opened. The thoracic aorta was quickly taken out, and put into pre-cooled Lock's solution (1000ml H ₂ O, NaCl 9g, KCl 0.35g, MgSO ₄ 7H ₂ O 0.35g, KH ₂ PO ₄ 0.16g, NaHCO ₃ 1g), quickly wash away the blood, then carefully cut off the hoof tissue outside the blood vessel, and cut it into a 2-3mm wide artery strip at an angle of 45 degrees to the longitudinal diameter of the tube for testing use.

Take an artery strip with a length of about 2 cm, clamp both ends with frog heart clips, place it in a perfusion bath with a constant temperature of 38°C, and inject oxygen into the bath liquid, fix the lower end of the frog heart clip to the bottom of the bath, and connect the upper end of the frog heart clip with a wire Tension transducer, and the contraction force of the blood vessel strips were recorded with a two-channel physiological recorder (Chengdu Instrument Factory, LMS-2B). The arterial strips were placed in the perfusion bath, equilibrated for 80 minutes to start the test, during which the Lock’s solution was changed every 20 minutes, and compound A with a concentration of 5×10 ^-3 mg/ml was added to relax the blood vessels, and the final concentration was 1.6×10 ^-6 mg /ml of adrenaline, record the contraction curve of the blood vessel strip, and wash it away when the contraction height no longer increases, and wash it 3 times in total. After 40 minutes, when the baseline of the blood vessel strip returns, first add 5×10 ^-4 mg/ml Compound A acts for 10 minutes. It can be seen that at this concentration, Compound A has no direct effect on relaxing vascular smooth muscle. Then add epinephrine with a final concentration of 1.6×10 ^-6 mg/ml. It can be seen that the added adrenaline can only relax blood vessels. The shrinkage of the bar was increased to 1/3 of the original, and the results of compound B were similar to A.

The test results show that compound A directly relaxes blood vessels when the concentration is greater than 5×10 ^-3 mg/ml, and does not directly relax vascular smooth muscle when the concentration is lower than this concentration, but can reduce the contractility of epinephrine on blood vessels. The results for compound B were similar to A.

Example 4

Antihypertensive effect of compounds A and B

SD rats were anesthetized with 40 mg/ml pentobarbital sodium, fixed on the test bench in the dorsal position, cut off the neck hair, cut the skin, separated the left common carotid artery, clamped the proximal end with an arterial clip, and The end of the heart is tied with woolen thread, and a small V-shaped opening is cut near the woolen thread with ophthalmological scissors, and an arterial cannula filled with heparin saline is inserted. The arterial cannula is connected to a blood pressure transducer, and the blood pressure transducer is connected to a computer-controlled three-channel Physiological and pharmacological recording system, the right femoral vein of rats was isolated and cannulated for drug injection. After the blood pressure is stabilized (that is, after the blood pressure contraction curve is stable), the test is started. Compound A was injected at a dose of 0.2 mg/kg, and it can be seen that there is a significant antihypertensive effect.

The test results are: time (minutes) 0 5 15 60 mean arterial pressure (Kpa) 13.8 8.7 9.0 9.5 The results of compound 8 are similar to those of A.

The results showed that compounds A and B had obvious and lasting blood pressure lowering effects.

Example 5

Anti-tumor activity test of compounds A and B detected by MTT reduction method

1. Materials:

1.1 Tetrazolium salt (MTT): Dissolve MTT (3-(4,5-dimethylthiazol-z-yl)2,5-diphenytetrazolium bromide, SIGMA) with 0.01mol/L phosphate buffered saline (PBS) at a final concentration of 5mg/L ml, sterilized by filtration, and stored in the dark at 4°C after aliquoting.

1.2 Preparation of MTT lysate: Dissolve 80g of sodium dodecylsulfonate (SDS, Huabing Bioengineering Co., Ltd.) in 200ml of N-N-dimethylformamide (Beijing Chemical Plant), heat in a water bath to aid dissolution, add 200ml Distilled water, mixed with 80% acetic acid and 1N hydrochloric acid (1:1) to adjust the pH to 4.7.

1.3 Preparation of target cells: recovery and culture of KBS cells

a. Take out the cryopreservation tube of KBS cells from the liquid nitrogen tank, quickly place it in a 37°C water bath, shake it constantly to dissolve it quickly, and transfer it into a centrifuge tube aseptically;

b. Add the whole culture medium to 10ml, centrifuge at 1000rpm for 5s, and discard the supernatant;

c. Repeat the above operation once;

d. Pipette the cells with the whole culture solution to mix the cells, then transfer them to a culture bottle, and culture at 5% CO ₂ at 37°C;

e. Observe the growth of the cells, replace the culture medium in time, and divide the bottles.

1.4 KBS cell counting, 96-well plate preparation

a. Select the cells in the logarithmic growth phase, trypsinize, terminate the whole culture, transfer to a centrifuge tube, add whole culture to 10ml;

b. Take one drop and drop it into the groove on one side of the counting plate, count the total number of cells in the four grids under the microscope, divide by 4, and multiply by 10 ⁴ , which is the number of cells contained in each ml of culture medium;

c. Adjust the number of cells to 1×10 ⁵ ml;

d. Put the 96-well plate in the super mirror stage and irradiate it under ultraviolet light for 4 hours, within a distance of 30cm.

1.5 Preparation of Compounds A and B: Add a certain amount of Compounds A and B to the whole culture, adjust the concentration to 500 μg/ml, ultrasonically emulsify, filter and sterilize, and store at 4°C.

1.6 Test method

a. 180 μl of KBS cells (1×10 ⁵ /ml) were added to each well of a 96-well plate, and cultured at 37° C. for 4 hr in 5% CO ₂ .

b. Add 20 pl of each test subject at different concentrations, add 2 pl of whole culture to the control, and continue to cultivate for 48 hours.

c. Remove 100 μl of culture solution from each well, add 10 μl of MTT (5 mg/ml each), and continue culturing for 4 hr.

d. Add 100 μl of MTT lysate to each well, and shake gently for 5-10 minutes to dissolve the particles overnight.

e. Measure the OD value of each well with an enzyme-linked immunosorbent analyzer at 570 nm.

f. Calculate the inhibition rate:

Tumor cell killing rate%=(average OD value determined by the control group-average OD value determined by the drug-dosed group)/average OD value determined by the control group×100%.

g. Plot the logarithm of the inhibitory rate against the drug concentration to obtain the IC ₅₀ value.

Take lgc as the abscissa and the inhibition rate as the ordinate to obtain the IC ₅₀ value.

1.7 Results

It was found that both compounds A and B could inhibit the growth of KBS cell line, and the IC ₅₀ value of A was 124 μg/ml; the IC ₅₀ value of B was 124 μg/ml.

Claims (1)

1. the preparation method of the compound of following structural formula A and B,

It is characterized in that the method comprises the following steps: a. Seed culture of the fungus Halorosellinia oceanicum 323 CCTCC No: M99008: medium composition by weight: glucose 0.5-1.5, yeast extract 0.05-0.15, peptone 0.1-0.3, agar 1-1.5, sodium chloride 3-5 , water 100, make a test tube slant, pick the strains and insert them into the slant, and cultivate them at 30-35°C for 5-7 days; b. Fermentation culture of the fungus Halorosellinia oceanicum 323 CCTCC No: M99008: The composition of the fermentation medium is: glucose 0.5-1.5, yeast extract 0.05-0.15, peptone 0.1-0.3, sodium chloride 3-5, water 100, Pick the cultured strains in the slope into the fermentation medium, and let it stand at room temperature 25-35°C for 1-2 months; c. filtering the above-mentioned cultivated fermented liquid to remove the thallus; d. The fermented liquid is heated and concentrated to 1/3-1/5 of the volume of the stock solution, extracted several times with ethyl acetate, concentrated ethyl acetate extract, and chromatographically separated in a silica gel column, using ethyl acetate:petroleum ether= 1%-100% is gradient elution of eluent; e. Collect 70%-90% ethyl acetate:petroleum ether eluate and concentrate to obtain white needle crystals to obtain a mixture of A and B. Recrystallize multiple times in methanol to separate A and B.