Tetramer of Ala-Gln-Gly and its preparation and application

Abstract

The present invention discloses a tetramer of Ala-Gln-Gly, namely [Ala-Gln-Gly]<subgt;4< / subgt;, and further discloses a preparation method of the tetramer of Ala-Gln-Gly and its application in the treatment of arterial thrombotic diseases. Experiments have proved that the tetramer of Ala-Gln-Gly of the present invention has excellent anti-arterial thrombosis effect, and its anti-arterial thrombosis effect is significantly stronger than that of Ala-Gln-Gly. Therefore, it is proposed that the present invention provides an effective technical means for anti-arterial thrombosis.

Description

Technical Field

[0001] The present invention relates to a tetramer of Ala-Gln-Gly, i.e., [Ala-Gln-Gly]4, to a method for preparing the same, and to its use in the preparation of a medicament for treating arterial thrombotic diseases. The present invention belongs to the field of biomedicine. Background Art

[0002] Cynomorium songaricum Rupr. is a perennial fleshy parasitic herb without chlorophyll, with the whole plant being reddish-brown. Most of the whole herb is buried in the sand, and Cynomorium songaricum Rupr. buds of different sizes are parasitized on the roots. The Cynomorium songaricum Rupr. buds gradually mature from the initial spherical shape into the later oval or long cylindrical shape. Most roots of Cynomorium songaricum Rupr. have fibrous roots and shed scale leaves. The stem of Cynomorium songaricum Rupr. is cylindrical, erect, and brownish. The base of the stem of Cynomorium songaricum Rupr. is slightly thickened or swollen, and there are deciduous scale leaves arranged spirally and becoming sparser upwards on the stem. The scale leaves are ovate-triangular. The spadix of Cynomorium songaricum Rupr. is borne at the top of the stem, protruding above the ground, club-shaped, 5-16 cm long, and 2-6 cm in diameter. Very dense small flowers are borne on the spadix. Male flowers, female flowers, and bisexual flowers are intermixed, with a fragrance. The flowering period is from May to July. Cynomorium songaricum Rupr. produces small nuts, nearly spherical or oval, with a white pericarp and a persistent pale yellow style at the top. The fruiting period is from June to July. The seeds of Cynomorium songaricum Rupr. are nearly spherical, dark red, and the seed coat is hard and thick. Cynomorium songaricum Rupr. grows in desert steppes, desert steppe deserts, and along rivers and lakes in desert areas. It is distributed in many provinces and regions in China. Cynomorium songaricum Rupr. can tonify the kidney, benefit essence, and moisten dryness, and is mainly used to treat impotence and spermatorrhea, soreness and weakness of the waist and knees, and intestinal dryness and constipation, and has a certain effect on paralysis and improving sexual function weakness. It is worth noting that when the inventor evaluated the anti-arterial thrombosis effect of Cynomorium songaricum Rupr., Ala-Gln-Gly was found in arterial thrombi. The inventor conjectured that aggregates of Ala-Gln-Gly might show a stronger anti-arterial thrombosis effect. Therefore, the inventor prepared aggregates of Ala-Gln-Gly based on Ala-Gln-Gly. The inventor found that under suitable conditions, Ala-Gln-Gly forms a tetramer, i.e., [Ala-Gln-Gly]4. Through further experiments, the inventor found that the [Ala-Gln-Gly]4 not only has a good anti-arterial thrombosis effect, but also has a significantly stronger anti-arterial thrombosis effect than that of Ala-Gln-Gly. Based on these findings, the inventor proposed the present invention. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to confirm that the tetramer of Ala-Gln-Gly, i.e., [Ala-Gln-Gly]4, is composed of four Ala-Gln-Gly molecules through non-covalent bonds.

[0004] And further confirm that [Ala-Gln-Gly]4 has a better anti-arterial thrombosis effect than Ala-Gln-Gly. To achieve the above object, the present invention adopts the following four technical means.

[0005] The first technical measure is that four Ala-Gln-Gly molecules form a tetramer of Ala-Gln-Gly of the present invention, namely [Ala-Gln-Gly]4, through non-covalent bonds.

[0006] The second technical measure is to propose a method for preparing the Ala-Gln-Gly tetramer, namely [Ala-Gln-Gly]4. This method includes the following 5 steps:

[0007] 1) Prepare Boc-Gln-Gly-OBzl;

[0008] 2) Prepare Gln-Gly-OBzl;

[0009] 3) Prepare Boc-Ala-Gln-Gly-OBzl;

[0010] 4) Prepare Ala-Gln-Gly;

[0011] 5) Prepare [Ala-Gln-Gly]4.

[0012] The third technical measure is to confirm the effective application of the Ala-Gln-Gly tetramer, namely [Ala-Gln-Gly]4, in the preparation of drugs for inhibiting arterial thrombosis.

[0013] Compared with the prior art, the beneficial effects of the present invention are:

[0014] The present invention is a tetramer formed by four Ala-Gln-Gly molecules through non-covalent bonds, namely [Ala-Gln-Gly]4. Experiments have proved that [Ala-Gln-Gly]4 of the present invention not only has a good anti-arterial thrombosis effect, but also the anti-arterial thrombosis effect is significantly stronger than that of Ala-Gln-Gly. Therefore, it is proposed that the present invention provides an effective technical measure for the field of drugs for inhibiting arterial thrombosis. Description of the Drawings

[0015] Figure 1 It is the synthesis route of Ala-Gln-Gly molecule: i) HOBt, DCC, NMM, THF; ii) ethyl acetate solution of hydrogen chloride with a concentration of 4N; iii) Pd / C, hydrogen, CH3OH.

[0016] Figure 2 It is the FT-ICR-MS spectrum of [Ala-Gln-Gly]4 and the qCID spectrum of [Ala-Gln-Gly]4. Detailed Embodiments

[0017] To further illustrate the present invention, a series of embodiments are given below. These embodiments are purely illustrative and are only used to specifically describe the present invention and should not be construed as a limitation of the present invention.

[0018] Example 1 Preparation of Ala-Gln-Gly

[0019] Example 1.1 Preparation of Boc-Gln-Gly-OBzl

[0020] At 0 °C, Boc-Gln (1.75 g, 7.10 mmol) and N-hydroxybenzotriazole (HOBt, 0.96 g, 7.10 mmol) were dissolved in anhydrous tetrahydrofuran. A solution of dicyclohexylcarbodiimide (DCC, 1.47 g, 7.10 mmol) in anhydrous tetrahydrofuran was added to this solution. The two solutions were stirred well for 20 minutes to obtain activated Boc-Gln / HOBt. At 0 °C, HCl·Gly-OBzl (2.40 g, 7.20 mmol) was added to the activated Boc-Gln / HOBt to obtain a reaction mixture. At 0 °C, the pH value of the reaction mixture was adjusted to 9 with N-methylmorpholine (NMM) and stirred for 30 minutes, and then stirred at room temperature for 12 hours. TLC (dichloromethane / methanol = 20 / 1) showed the disappearance of HCl·Gly-OBzl. The insoluble matter in the reaction mixture was filtered off, and the filtrate was concentrated to dryness under reduced pressure. The residue was dissolved in 300 mL of ethyl acetate. The obtained ethyl acetate solution was washed successively with saturated aqueous NaHCO3 (200 mL × 3), saturated aqueous NaCl (200 mL × 3), 5% aqueous KHSO4 (200 mL × 3), saturated aqueous NaCl (200 mL × 3), saturated aqueous NaHCO3 (200 mL × 3) and saturated aqueous NaCl (200 mL × 3). The separated ethyl acetate solution was dried over anhydrous Na2SO4 for 12 hours, filtered, and the filtrate was concentrated under reduced pressure. The residue was triturated repeatedly with ether to obtain 2.27 g (98%) of the title compound as a pale yellow solid, which was directly used in the next step of the reaction. ESI-MS (m / e): 394 [M+H] + 。

[0021] Example 1.2 Preparation of Gln-Gly-OBzl

[0022] Dissolve Boc-Gln-Gly-OBzl (2.00 g, 5.09 mmol) in 15 mL of anhydrous ethyl acetate, and add 20 mL of a 4 N hydrogen chloride solution in anhydrous ethyl acetate while cooling in an ice-salt bath. The reaction mixture was stirred for 120 minutes while cooling in an ice-salt bath. TLC (dichloromethane / methanol = 20 / 1) showed the disappearance of Boc-Gln-Gly-OBzl. The reaction mixture was concentrated under reduced pressure to remove free hydrogen chloride gas. The residue was redissolved in anhydrous ethyl acetate and concentrated under reduced pressure to remove free hydrogen chloride gas. This operation was repeated three times to completely remove free hydrogen chloride gas. The residue was triturated with petroleum ether three times to obtain 1.45 g (97%) of the title compound as a colorless solid, which was directly used in the next reaction. ESI-MS (m / e): 294 [M+H] + 。

[0023] Example 1.3 Preparation of Boc-Ala-Gln-Gly-OBzl

[0024] The crude product obtained from Boc-Ala (0.78 g, 4.10 mmol) and Gln-Gly-OBzl (1.20 g, 4.10 mmol) according to the procedure of Example 1.1 was purified by silica gel column chromatography to obtain 1.52 g (96%) of the title compound as a colorless solid. ESI-MS (m / e): 465 [M+H] + ; 1 1H NMR (300 MHz, DMSO-d6): δ / ppm = 8.37 (t, J = 5.9 Hz, 1H), 7.89 (d, J = 8.1 Hz, 1H), 7.44 - 7.27 (m, 5H), 7.21 (s, 1H), 7.00 (d, J = 7.3 Hz, 1H), 6.77 (m, 1H), 5.76 (s, 1H), 5.12 (s, 2H), 4.27 (m, 1H), 3.92 (m, 3H), 2.10 (t, J = 7.9 Hz, 2H), 1.74 (dq, J1 = 15.7 Hz, J2 = 8.0 Hz, 1H), 1.37 (s, 9H), 1.17 (d, J = 7.1 Hz, 4H); 13 13C NMR (75 MHz, DMSO-d6): δ / ppm = 174.21, 173.06, 172.30, 170.03, 155.66, 136.33, 128.90, 128.54, 128.42, 78.65, 66.34, 52.27, 50.29, 41.16, 31.69, 28.66, 18.47。

[0025] Example 1.4 Preparation of Ala-Gln-Gly

[0026] Dissolve Boc-Ala-Gln-Gly-OBzl (500 mg, 1.82 mmol) in methanol, add 75 mg of Pd / C, and stir to make it uniform. First, evacuate the air in the reaction flask under reduced pressure, and then introduce hydrogen gas into the reaction flask for 48 hours to carry out the debenzylation reaction. TLC (methylene chloride / methanol = 20 / 1) shows the disappearance of Boc-Ala-Gln-Gly-OBzl. Filter the reaction mixture to remove Pd / C, concentrate the filtrate under reduced pressure to dryness, and wash the residue with diethyl ether (30 mL × 3) to obtain Boc-Ala-Gln-Gly as a colorless powder. Dissolve the Boc-Ala-Gln-Gly powder in 2 mL of anhydrous ethyl acetate solution at 0 °C, and mix the obtained ethyl acetate solution with 5 mL of 4N hydrogen chloride in anhydrous ethyl acetate solution. Stir the reaction compound for 2 hours, and TLC (ethyl acetate / acetic acid / water = 2 / 1 / 1) shows the disappearance of Boc-Ala-Gln-Gly. Concentrate the reaction mixture under reduced pressure to dryness. Dissolve the residue in 5 mL of anhydrous ethyl acetate and concentrate under reduced pressure to remove the free hydrogen chloride gas. Repeat this operation three times to completely remove the free hydrogen chloride gas. Purify the residue by C18 column chromatography to obtain 440 mg (88%) of the title compound. M.p. 124 - 125 °C; ESI-MS (m / e): 275 [M + H] + ; 1 1H NMR (300 Hz, DMSO-d6): δ / ppm = 8.70 (t, J = 7.2 Hz, 1H), 8.37 (td, J1 = 14.4 Hz, J2 = 13.5 Hz, J3 = 6.1 Hz, 2H), 7.40 (s, 1H), 4.38 - 4.26 (m, 1H), 3.88 - 3.85 (m, 1H), 3.63 (s, 2H), 2.17 (t, J = 7.9 Hz, 2H), 1.95 (td, J1 = 11.2 Hz, J2 = 8.5 Hz, J3 = 4.4 Hz, 1H), 1.77 (m, 1H), 1.36 (d, J = 6.8 Hz, 3H); 13 13C NMR (75 MHz, DMSO-d6): δ / ppm = 174.16, 171.61, 171.49, 169.81, 63.74, 52.65, 48.50, 31.77, 28.74, 17.56.

[0027] Example 2 Preparation of the tetramer of Ala-Gln-Gly

[0028] Dissolve 100 mg of Ala-Gln-Gly powder in 5 mL of ultrapure water. The resulting solution is vortexed for 15 minutes first, and then sonicated in an ultrasonic bath for 4 hours until the solution becomes completely colorless and transparent. The colorless and transparent solution is concentrated to dryness under reduced pressure to obtain the Ala-Gln-Gly tetramer, namely [Ala-Gln-Gly]4. The structure of [Ala-Gln-Gly]4 is confirmed by ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Figure 2 The FT-ICR-MS spectrum shows peaks of [Ala-Gln-Gly]+H at 275.13833 (theoretical value 275.13500), [Ala-Gln-Gly]2+Na at 571.25428 (theoretical value 571.24466), [Ala-Gln-Gly]3+H at 823.41045 (theoretical value 823.39044), and [Ala-Gln-Gly]4+H at 1097.55225 (theoretical value 1097.51816).

[0029] To clarify the relationship among the peaks of [Ala-Gln-Gly]+H, [Ala-Gln-Gly]2+Na, [Ala-Gln-Gly]3+H, and [Ala-Gln-Gly]4+H, the qCID spectrum of [Ala-Gln-Gly]4+H was measured. Figure 2 The qCID spectrum of [Ala-Gln-Gly]4 shows peaks of [Ala-Gln-Gly]3+H at 823.39615 (theoretical value 823.39044), [Ala-Gln-Gly]2+H at 571.24823 (theoretical value 571.24466), and [Ala-Gln-Gly]+H at 275.13689 (theoretical value 275.13500). That is to say, [Ala-Gln-Gly]4 is the only form in which Ala-Gln-Gly exists.

[0030] Example 3: Evaluate the anti-arterial thrombosis activity of [Ala-Gln-Gly]4

[0031] 1) Pull a polyethylene tube into a thin tube with a beveled end, with a fixed length of 10.0 cm, and insert it into the right jugular vein (with a relatively thick diameter) and the left carotid artery (with a relatively thin diameter) respectively; the middle section of the polyethylene tube has a fixed length of 8.0 cm, and the thrombus thread is pressed in the direction of the carotid artery catheterization. Heparin needs to be filled in the tube before catheterization.

[0032] 2) Male Sprague-Dawley rats weighing 200 ± 20 g were acclimated to the environment and fasted for one day before surgery. They were randomly divided into a normal saline group (blank control, oral dose of 0.3 mL / 100 g, 10 rats), an aspirin group (positive control, oral doses of 167 μmol / kg and 16.7 μmol / kg, 10 rats each), a normal saline solution group of Ala-Gln-Gly (oral dose of 1 μmol / kg, 10 rats), and a normal saline solution group of [Ala-Gln-Gly]4 (oral dose of 0.1 μmol / kg, 10 rats). After 30 minutes of oral administration, the rats were anesthetized by intraperitoneal injection of 20% urethane solution (7 mL / kg), and surgery was started 2 minutes later. During the surgery, the rats were placed supine on a fixing plate, the neck skin was incised, the right common carotid artery and the left jugular vein were separated, a precisely weighed silk thread was placed under the blood vessels, the distal end was ligated, a small incision was made at the distal end of the vein, a cannula was inserted into the venous end, heparin was injected, then the syringe for injecting heparin was removed, fixed with a ligature, and then the proximal end of the artery was clamped with an arterial clamp. A small incision was made at the distal end of the artery, the arterial end was ligated, fixed with a ligature, and then the arterial clamp was loosened to establish an extracorporeal circulation bypass. After 15 minutes of circulation, first cut the vein to observe whether the blood circulation was normal. If the blood circulation was normal, the silk thread with thrombus was taken out from the arterial end, the uncoagulated blood was blotted dry with filter paper, the weight of the silk thread with thrombus was precisely weighed, and the weight of the thrombus was obtained by subtracting the weight of the silk thread from the weight of the silk thread with thrombus. The data were listed in Table 1. The thrombus weights in the table showed that Ala-Gln-Gly effectively inhibited arterial thrombosis in rats at an oral dose of 1 μmol / kg (p < 0.01 compared with normal saline and aspirin at an oral dose of 16.7 μmol / kg). The thrombus weights in the table further showed that the anti-arterial thrombosis activity of [Ala-Gln-Gly]4 at an oral dose of 0.1 μmol / kg was significantly stronger than that of Ala-Gln-Gly (p < 0.01 compared with Ala-Gln-Gly). This was an unexpected technical effect.

[0033] Table 1 Effects of [Ala-Gln-Gly]4 on Arterial Thrombosis in Rats

[0034]

[0035] a) p < 0.01 compared with normal saline and aspirin at an oral dose of 16.7 μmol / kg; b) p > 0.05 compared with normal saline; c) p < 0.01 compared with normal saline and Ala-Gln-Gly at an oral dose of 1 μmol / kg; n = 10.

Claims

1. A tetramer of Ala-Gln-Gly, characterized in that, The tetramer is [Ala-Gln-Gly]4, and the [Ala-Gln-Gly]4 is composed of four Ala-Gln-Gly molecules through non-covalent bonds.

2. A method for preparing the tetramer of Ala-Gln-Gly according to claim 1, characterized in that, The preparation method includes the following steps: 1) Prepare Boc-Gln-Gly-OBzl; 2) Prepare Gln-Gly-OBzl; 3) Prepare Boc-Ala-Gln-Gly-OBzl; 4) Prepare Ala-Gln-Gly; 5) Prepare [Ala-Gln-Gly]4.

3. Use of the tetramer of Ala-Gln-Gly according to claim 1 in the preparation of a drug for inhibiting arterial thrombosis.

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