CN106317191A - Thr-Val-Gly-Cys-Ser, and synthesis, activities and application thereof - Google Patents

Abstract

Thr-Val-Gly-Cys-Ser, its synthesis, activity and application. The invention discloses Thr-Val-Gly-Cys-Ser, its preparation method, its analgesic activity, its antitumor activity, its antithrombotic activity and its lytic activity. Thrombus activity, thus the present invention discloses its application in the preparation of analgesic drugs, antitumor drugs and thrombolytic drugs.

Description

Thr-Val-Gly-Cys-Ser, its synthesis, activity and application

technical field

The present invention relates to Thr-Val-Gly-Cys-Ser, relates to its preparation method, relates to its analgesic activity, relates to its antitumor activity, relates to its thrombolytic activity, thus the present invention relates to its use in the preparation of analgesic drugs , the application of antineoplastic drugs and thrombolytic drugs. The invention belongs to the field of biomedicine.

Background technique

Invention of oligopeptides with anti-tumor, antithrombotic, thrombolytic, anticoagulant and analgesic effects is the long-term focus of the inventors. Although the inventors invented and disclosed a series of oligopeptides with these activities, the oligopeptides integrating these activities have not been obtained. After nearly 10 years of sequence design and nearly 5 years of experimental research, the inventors found that Thr-Val-Gly-Cys-Ser is an oligopeptide that integrates analgesic, antitumor and thrombolytic effects.

Contents of the invention

The first content of the present invention is to provide Thr-Val-Gly-Cys-Ser:

The second content of the present invention is to provide the synthetic method of Thr-Val-Gly-Cys-Ser, this method comprises:

(1) Prepare Boc-Val-Gly-OBzl according to standard methods under the catalysis of DCC and HOBt;

(2) under the catalysis of DCC and HOBt, synthesize Boc-Cys(Bzl)-Ser-OBzl;

(3) Boc-Cys(Bzl)-Ser-OBzl is de-Boced at 0°C in a hydrogen chloride-ethyl acetate solution with a concentration of 4N, and converted into Cys(Bzl)-Ser-OBzl;

(4) Boc-Val-Gly-OBzl is de-Boced at 0°C in a hydrogen chloride-ethyl acetate solution with a concentration of 4N, and converted into Boc-Val-Gly;

(5) under the catalysis of DCC and HOBt, prepare Boc-Thr-Val-Gly-OBzl;

(6) Boc-Thr-Val-Gly-OBzl is converted into Boc-Thr-Val-Gly in H ₂ Pd/C;

(7) under the catalysis of DCC and HOBt, synthesize Boc-Thr-Val-Gly-Cys(Bzl)-Ser-OBzl;

(8) Boc-Arg(NO ₂ )-Leu-Val-Cys(Bzl)-Val-OBzl was converted into Thr-Val-Gly-Cys-Ser in TFA and TFSMA at 0°C.

The third content of the present invention is to evaluate the analgesic effect of Thr-Val-Gly-Cys-Ser.

The fourth content of the present invention is to evaluate the anti-tumor effect of Thr-Val-Gly-Cys-Ser.

The fifth content of the present invention is to evaluate the thrombolytic effect of Thr-Val-Gly-Cys-Ser.

Description of drawings

The synthetic route of Fig. 1.Thr-Val-Gly-Cys-Ser. i) dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole (HOBt), N-methylmorpholine (NMM), Tetrahydrofuran (THF); ii) 4N hydrogen chloride-ethyl acetate solution, 0°C; iii) _H2 , Pd/C; iv) TFA, TFMSA, 0°C.

detailed description

In order to further illustrate the present invention, a series of examples are given below. These examples are entirely illustrative, and they are only used to specifically describe the present invention, and should not be construed as limiting the present invention.

Embodiment 1 prepares the preparation of Boc-Val-Gly-OBzl

Dissolve 2.17g (10mmol) Boc-Val in a small amount of anhydrous THF under ice bath, add 1.36g (10mmol) HOBt, add 2.47g (12mmol) DCC and a small amount of anhydrous THF solution, activate for 30 minutes, add 1.65g ( 10mmol) of Gly-OBzl, adjusted to pH=9 with NMM, and filtered to remove dicyclohexylurea (DCU) after the reaction. The filtrate was concentrated under reduced pressure, the residue was dissolved in ethyl acetate, DCU was filtered again, the filtrate was washed 3 times with NaHCO ₃ saturated solution, 3 times with NaCl saturated solution, 3 times with 5% KHSO ₄ solution, 3 times with NaCl saturated solution 3 times, 5% NaHCO ₃ solution was extracted and washed 3 times, NaCl saturated solution was extracted and washed 3 times, the ethyl acetate layer was dried with anhydrous Na ₂ SO ₄ for 12 hours, Na ₂ SO ₄ was filtered off, and the filtrate was concentrated under reduced pressure to obtain 3.33 g (91.5%) of the title compound as a yellow powder. ESI ⁺ -MS (m/e): 365 [M+H] ⁺ .

Example 2 Preparation of Boc-Cys(Bzl)-Ser-OBzl

According to the method of Example 1, 4.02 g (82%) of the title compound were obtained as a colorless oil from 3.11 g (10 mmol) of Boc-Cys(Bzl) and 1.95 g (10 mmol) of Ser-OBzl.

Example 3 Preparation of Cys(Bzl)-Ser-OBzl

Dissolve 4.88g (10mmol) Boc-Cys(Bzl)-Ser-OBzl with a small amount of dried ethyl acetate under ice bath, stir for 10min, add 50mL hydrogen chloride-ethyl acetate solution (4N) to react for 4h, the raw material point disappears . The reaction mixture was concentrated to dryness under reduced pressure, and the residue was dissolved in 40 mL of dried ethyl acetate, and the resulting solution was concentrated to dryness under reduced pressure. This operation was repeated 3 times for the residue. Add anhydrous diethyl ether to the residue, grind with a plastic spatula, and concentrate under reduced pressure to remove diethyl ether. This operation was repeated 3 times for the residue. This gave 3.7 g (96%) of the title compound as a colorless powder.

ESI-MS (m/e): 390 [M+H] ⁺ .

Embodiment 4 prepares Val-Gly-OBzl

According to the method of Example 3, 2.46 g (93%) of the title compound was obtained as a colorless powder from 3.64 g (10 mmol) of Boc-Val-Gly-OBzl. ESI-MS (m/e): 265 [M+H] ⁺ .

Embodiment 5 prepares Boc-Thr-Val-Gly-OBzl

Obtain 3.40g (73%) title compound according to the method for Example 1 from 2.19g (10mmol) Boc-Thr and 2.64g (10mmol) Val-Gly-OBzl, be colorless powder ESI-MS (m/e): 467 [M+H] ⁺ .

Example 6 Preparation of Boc-Thr-Val-Gly

Dissolve 4.65g (10mmol) Boc-Thr-Val-Gly-OBzl in methanol, add 500mg of 10% Pd/C, add three vent valves, depressurize and pump out the air in the eggplant bottle, and inject H ₂ , After 24 hours of reaction, the raw material point disappeared. Pd/C was filtered off, and methanol was concentrated under reduced pressure to obtain 3.65 g (97%) of a colorless powder. ESI-MS (m/e): 376 [M+H] ⁺ .

Example 7 Preparation of Boc-Thr-Val-Gly-Cys(Bzl)-Ser-OBzl

According to the method of Example 1, 2.35 g (31%) of the title compound were obtained as a colorless jelly-like product from 3.75 g (10 mmol) Boc-Thr-Val-Gly and 3.88 g (10 mmol) Cys(Bzl)-Ser-OBzl. ESI-MS (m/e): 746[M+H] ⁺ ; ¹ H-NMR (300MHz, DMSO-d6): δ/ppm=8.60(m, 1H), 8.23(s, 1H), 7.98(m , 1H), 7.68(m, 1H), 7.56-7.09(m, 10H), 6.53(d, J=8.1Hz, 1H), 5.13(m, 2H), 4.75(m, 1H), 4.65(m, 1H), 4.42(m, lH), 4.31(t, 1H), 3.93(m, 2H), 3.51(m, 4H), 3.35(m, 1H), 2.70(m, 2H), 1.38(s, 9H ), 1.02 (d, J=5.4Hz, 3H), 0.86 (m, 6H).

Example 8 Preparation of Thr-Val-Gly-Cys-Ser

Dissolve 372mg (0.5mmol) Boc-Tht-Val-Gly-Cys(Bzl)-Ser-OBzl in 6mL trifluoroacetic acid under ice bath, stir for 10min, add 1.5mL trifluoromethanesulfonic acid and react for 30min, the reaction is almost complete , the raw material point disappears. Add the newly opened ether into the eggplant bottle, grind and wash with a plastic shovel, and pour the ether; repeat the operation three times. Finally, a brownish-yellow powder was obtained, which was dissolved in triple-distilled water, adjusted to pH 7 with ammonia water, followed by Sephdex G10, and the lyophilized sample was collected as 198 mg of white powder (yield: 85.1%). ESI ⁺ -MS(m/e): 467[M+H] ⁺ ; Mp 160～161℃. (c=0.090, methanol). ¹ H-NMR (300MHz, DMSO-d6): δ/ppm=8.46(m, 1H), 8.31(m, 1H), 8.29(m, 1H), 8.04(m, 2H ), 7.26(m, 3H), 4.56(m, 3H), 4.26(m, 3H), 3.87(m, 3H), 3.87(m, 1H), 2.90(m, 2H), 1.07(m, 3H) , 0.90(t, 6H).

Example 9 Evaluation of the Analgesic Activity of Thr-Val-Gly-Cys-Ser

Male ICR mice (20±2g) were put into the mouse holder, and the tail of the mouse was exposed outside the holder, and the mouse tail was marked at a third of the distance from the tail tip as the sensing point of the photoreceptor, and the distance from the mouse tail was irradiated Two-thirds of the tip of the tail. The analgesia instrument was preheated for 30 minutes, and the time was recorded, and the mouse tail was covered with the light sensor. Start timing when the light flickers, and stop timing when the mouse tail leaves the light sensor. The measured time is the time when the mouse feels pain. It is measured continuously for 3 times, and the interval between each measurement is 5 minutes, and the average value is taken. The time when the untreated mice feel pain is the basic pain time. The time change of pain sensation in mice induced by drug administration reflects the analgesic activity of the drug. Mice were divided into random groups, 14 in each group. After measuring the time of basic pain sensation, the mice were orally administered 0.2 mL of Thr-Val-Gly-Cys-Ser (TVGCS) in normal saline solution at a dose of 1 μmol/kg or orally orally 0.2 mL of physiological Saline or oral aspirin in normal saline solution at a dose of 1200 μmol/kg. The time for mice to feel pain at 30, 60, 90, 120, 150 and 180 minutes after administration was measured. The data are expressed in mean ± SD seconds, listed in Table 1, and analyzed by variance and t test. The data showed that there was no significant difference between the pain time of mice after oral administration of normal saline for 30, 60, 90, 120, 150 and 180 min and the basic pain time before oral administration of normal saline; After 180min, the time for the mice to feel pain was significantly longer than the basic pain time before oral administration of aspirin; after oral administration of 1μmol/kg Thr-Val-Gly-Cys-Ser, the time for mice to feel pain after 60, 90, 120 and 150min was significantly longer than that of oral administration of Thr- Basal Pain Time Before Val-Gly-Cys-Ser. It can be seen that the analgesic activity after oral administration of 1 μmol/kg Thr-Val-Gly-Cys-Ser120 and 150 min is equivalent to that of oral administration of 1200 μmol/kg aspirin. The analgesic activity after oral administration of 1 μmol/kg Thr-Val-Gly-Cys-Ser60 and 90 min is stronger than oral administration of 1200 μmol/kg aspirin.

Table 1 Analgesic activity of Thr-Val-Gly-Cys-Ser (TVGCS)

n=14; a) p>0.05 compared with the basic pain time; b) p<0.05 compared with the basic pain time; c) p<0.01 compared with the basic pain time.

Example 10 Evaluation of the antitumor activity of Thr-Val-Gly-Cys-Ser

Under sterile conditions, the S180 sarcoma inoculated in ICR mice for ^7-10 days was taken, and an appropriate amount of normal saline was added to prepare a tumor cell suspension. Inject 0.2 mL per mouse. 24 hours after tumor inoculation, mice were intraperitoneally injected with 0.2mL Thr-Val-Gly-Cys-Ser normal saline solution daily for 10 consecutive days at a dose of 1 μmol/kg; or mice were intraperitoneally injected with 0.2mL Adriamycin The normal saline solution of the drug was administered continuously for 10 days, and the dose was 2 μmol/kg; or the mice were intraperitoneally injected with 0.2 mL of normal saline every day, continuously administered for 10 days. On the 11th day of the experiment, the mice were weighed, and the tumors of the mice in each group were dissected under ether anesthesia, weighed, and the activity of the compound was expressed by the tumor weight. The data are listed in Table 3. The results showed that the tumor weight of mice treated with 1 μmol/kg Thr-Val-Gly-Cys-Ser was significantly smaller than that of mice treated with normal saline, and Thr-Val-Gly-Cys-Ser showed definite antitumor activity.

Table 2 Effect of Thr-Val-Gly-Cys-Ser on tumor weight of S180 tumor-bearing mice

n=12; a) p<0.01 compared with normal saline.

Example 11 Evaluation of Thrombolytic Activity of Thr-Val-Gly-Cys-Ser

1) Male SD rats weighing 250± were anesthetized with 20% urethane solution (6 mL/kg intraperitoneally). Anesthetized rats were fixed on the mouse board, the right common carotid artery was separated, the arterial clamp was clipped at the proximal end, the proximal end and the distal end were passed into the surgical line respectively, blood vessels were inserted at the distal end, and the artery was loosened Clamp about 1mL of arterial blood. Rapidly inject arterial blood into a vertical thrombus tube (length 16mm, inner diameter 2.5mm, outer diameter 5mm, base of 1mL EP tube at the bottom of the tube), inject 0.1mL rat into each thrombus tube (note that there should be no air bubbles) For arterial blood, quickly insert a thrombus fixing bolt (20mm in length) into the tube, the blood coagulates for 40 minutes, take out the thrombus with an acupuncture needle, and weigh the thrombus.

2) The bypass intubation tube is composed of three parts, the middle part is a polyethylene rubber tube with a length of 60 mm and an inner diameter of 3.5 mm, and both ends are the same polyethylene tube with a length of 100 mm, an inner diameter of 1 mm and an outer view of 2 mm. A section of the tube is drawn into a sharp point The other end of the tube is covered with a polyethylene tube with a length of 7 mm and an outer diameter of 3.5 mm. The inner walls of the three-section tubes are all silanized.

3) Place the thrombus-wrapped thrombus fixing bolt in the middle section of the polyethylene rubber tube, and the two ends of the rubber tube are respectively fitted with the thickened ends of the two polyethylene tubes, and a syringe is filled with heparin saline solution through the tip of the tube. Insert one end of the normal saline solution filled with heparin sodium into the left vein, add an accurate amount of heparin sodium to the other end with a syringe for anticoagulation, then unplug the heparin sodium syringe and insert it into the arterial end. Use a scalp needle to pass normal saline solution (3mL/kg) or urokinase normal saline solution (20000IU/kg) or Thr-Val-Gly-Cys-Ser normal saline solution (1μmol/kg) through the middle section of the bypass tube to stimulate Into the proximal vein far away from the thrombus fixing bolt, open the arterial clamp, and the moment when the blood flows from the artery to the vein through the bypass tube is the starting time of the cycle, and slowly inject the liquid in the syringe into the blood (about 6 minutes), so that Normal saline, urokinase, and Thr-Val-Gly-Cys-Ser act on thrombus in the order of vein-heart-artery through blood circulation. After 60 minutes, take out the bolt with thrombus, dip in the floating blood, and record the weight. Thrombolytic activity was represented by thrombus weight loss. The data are listed in Table 3. The results showed that the weight loss of thrombus in rats treated with Thr-Val-Gly-Cys-Ser was significantly greater than that of rats treated with normal saline, and Thr-Val-Gly-Cys-Ser showed definite thrombolytic activity.

Table 3 Thrombolytic activity of Thr-Val-Gly-Cys-Ser

n=10; a) p<0.01 compared with normal saline.

Claims (5)

1.Thr-Val-Gly-Cys-Ser。

2. the preparation method of the Thr-Val-Gly-Cys-Ser of claim 1, the method includes:

(1) under the catalysis of DCC, HOBt, Boc-Val-Gly-OBzl is prepared according to standard method；

(2) under the catalysis of DCC, HOBt, the Cys-Ser-OBzl of synthesis Boc-Bzl protection sulfydryl；

(3) Cys-Ser-OBzl of Boc-Bzl protection sulfydryl is in hydrogen chloride-ethyl acetate solution that concentration is 4N, and 0 DEG C de-Boc, changes into the Cys-Ser-OBzl of Bzl protection sulfydryl；

(4) Boc-Val-Gly-OBzl is in hydrogen chloride-ethyl acetate solution that concentration is 4N, 0 DEG C of de-Boc, converts Become Boc-Val-Gly；

(5) under the catalysis of DCC, HOBt, Boc-Thr-Val-Gly-OBzl is prepared；

(6) Boc-Thr-Val-Gly-OBzl is at H₂Pd/C changes into Boc-Thr-Val-Gly；

(7) under the catalysis of DCC, HOBt, the Cys-Ser-OBzl of synthesis Boc-Thr-Val-Gly-Bzl protection sulfydryl；

(8) Cys-Ser-OBzl of Boc-Thr-Val-Gly-Bzl protection sulfydryl is in TFA and TFSMA, and 0 DEG C changes into Thr-Val-Gly-Cys-Ser。

3. the Thr-Val-Gly-Cys-Ser of claim 1 application in preparing antitumor drug.

4. the Thr-Val-Gly-Cys-Ser of claim 1 application in preparing analgesic.

5. the Thr-Val-Gly-Cys-Ser of claim 1 application in preparing thrombolytic drug.