Trimer of Asn-Gly-Pro, its preparation and application

Abstract

The present invention discloses a trimer of Asn-Gly-Pro, namely [Asn-Gly-Pro]3, discloses its preparation method and its application in the treatment of venous thrombotic diseases. Experiments have proved that [Asn-Gly-Pro]3 of the present invention not only has a good anti-venous thrombosis effect, but also the anti-venous thrombosis effect is significantly stronger than that of Asn-Gly-Pro. Therefore, it is proposed that the present invention provides an effective technical means for anti-venous thrombosis.

Description

Technical Field

[0001] The present invention relates to a trimer of Asn-Gly-Pro, i.e., [Asn-Gly-Pro]3, and discloses its preparation method and application in the treatment of venous thrombotic diseases. Experiments have proved that [Asn-Gly-Pro]3 of the present invention not only has a good anti-venous thrombosis effect, but also has a significantly stronger anti-venous thrombosis effect than that of Asn-Gly-Pro. The present invention belongs to the field of biomedicine. Background Art

[0002] Cynomorium songaricum Rupr. is a perennial fleshy parasitic herb without chlorophyll. The whole plant is reddish-brown, and most of the whole herb is buried in the sand. Cynomorium songaricum Rupr. buds of different sizes parasitize on the roots. The Cynomorium songaricum Rupr. buds gradually mature from the initial spherical shape to 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. grows at the top of the stem, protrudes above the ground, is club-shaped, 5-16 cm long, and 2-6 cm in diameter. There are very dense small flowers 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 light 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 deserts along rivers and lakes. It is distributed in many provinces and regions in China. Cynomorium songaricum Rupr. can tonify the kidney, benefit essence, moisten dryness, and is mainly used for treating impotence and spermatorrhea, soreness and weakness of the waist and knees, and constipation due to intestinal dryness, and has a certain effect on paralysis and improving sexual function weakness. It is worth noting that when the inventor evaluated the anti-venous thrombosis effect of Cynomorium songaricum Rupr., Asn-Gly-Pro was found in venous thrombosis. The inventor knew that aggregates of Asn-Gly-Pro often showed a stronger anti-venous thrombosis effect. So the inventor prepared a trimer of Asn-Gly-Pro, i.e., [Asn-Gly-Pro]3. Further experiments have proved that [Asn-Gly-Pro]3 of the present invention not only has a good anti-venous thrombosis effect, but also has a significantly stronger anti-venous thrombosis effect than that of Asn-Gly-Pro. 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 a trimer of Asn-Gly-Pro, i.e., [Asn-Gly-Pro]3, has a better anti-venous thrombosis effect than Asn-Gly-Pro. To achieve the above object, the present invention adopts the following four technical means.

[0004] The first technical measure is to propose the trimer of Asn-Gly-Pro of the present invention, namely [Asn-Gly-Pro]3, and the [Asn-Gly-Pro]3 is composed of three Asn-Gly-Pro molecules through non-covalent bonds.

[0005] The second technical measure is to propose a method for preparing the trimer of Asn-Gly-Pro, namely [Asn-Gly-Pro]3, and this method includes the following 5 steps:

[0006] 1) Prepare Boc-Gly-Pro-OBzl;

[0007] 2) Prepare Gly-Pro-OBzl;

[0008] 3) Prepare Boc-Asn-Gly-Pro-OBzl;

[0009] 4) Prepare Asn-Gly-Pro;

[0010] 5) Prepare [Asn-Gly-Pro]3.

[0011] The third technical measure is to confirm the role of the trimer of Asn-Gly-Pro, namely [Asn-Gly-Pro]3, in the preparation of drugs for inhibiting venous thrombosis.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0013] The present invention provides a trimer composed of three Asn-Gly-Pro molecules through non-covalent bonds, namely [Asn-Gly-Pro]3. Through experiments, it is proved that the [Asn-Gly-Pro]3 of the present invention not only has a good anti-venous thrombosis effect, but also the anti-venous thrombosis effect is significantly stronger than that of Asn-Gly-Pro. Therefore, it is proposed that the present invention provides an effective technical measure for the field of drugs for inhibiting venous thrombosis. Description of the Drawings

[0014] Figure 1 It is the synthesis route diagram of Asn-Gly-Pro: i) HOBt, DCC, NMM, THF; ii) ethyl acetate solution of hydrogen chloride with a concentration of 4N; iii) Pd / C, hydrogen, CH3OH.

[0015] Figure 2 It is the FT-ICR-MS spectrum of [Asn-Gly-Pro]3 and the qCID spectrum of [Asn-Gly-Pro]3. Detailed Embodiments

[0016] 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.

[0017] Example 1 Preparation of Asn-Gly-Pro

[0018] Example 1.1 Preparation of Boc-Gly-Pro-OBzl

[0019] At 0 °C, Boc-Gly (1.76 g, 9.93 mmol) and N-hydroxybenzotriazole (HOBt, 1.34 g, 9.93 mmol) were dissolved in anhydrous tetrahydrofuran. A solution of dicyclohexylcarbodiimide (DCC, 10.24 g, 9.94 mmol) in anhydrous tetrahydrofuran was added to this solution. The two solutions were stirred well for 20 minutes to obtain activated Boc-Gly / HOBt. At 0 °C, HCl·Pro-OBzl (2.40 g, 9.93 mmol) was added to the activated Boc-Gly / 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 (methylene chloride / methanol = 20 / 1) showed the disappearance of HCl·Pro-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 2.96 g (99%) of the title compound as a pale yellow solid, which was directly used in the next step of the reaction. ESI-MS (m / e): 363 [M+H] + 。

[0020] Example 1.2 Preparation of Gly-Pro-OBzl

[0021] Dissolve Boc-Gly-Pro-OBzl (2.00 g, 2.21 mmol) in 15 mL of anhydrous ethyl acetate. Add 20 mL of an anhydrous ethyl acetate solution of hydrogen chloride with a concentration of 4 N while cooling in an ice-salt bath. The reaction mixture is stirred for 120 minutes while cooling in an ice-salt bath. TLC (dichloromethane / methanol = 20 / 1) shows the disappearance of Boc-Gly-Pro-OBzl. The reaction mixture is concentrated under reduced pressure to remove free hydrogen chloride gas. The residue is redissolved in anhydrous ethyl acetate and concentrated under reduced pressure to remove free hydrogen chloride gas. This operation is repeated three times to completely remove free hydrogen chloride gas. The residue is triturated with petroleum ether three times to obtain 1.93 g (97%) of the title compound as a colorless solid, which is directly used in the next reaction. ESI-MS (m / e): 263 [M+H] + .

[0022] Example 1.3 Preparation of Boc-Asn-Gly-Pro-OBzl

[0023] The crude product obtained from Boc-Asn (1.50 g, 6.41 mmol) and Gly-Pro-OBzl (1.68 g, 6.41 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): 477 [M+H] + ; 1 1H NMR (300 MHz, DMSO-d6): δ / ppm = 7.78 (m, 1H), 7.37 (dd, J1 = 3.9 Hz, J2 = 3.4 Hz, 5H), 7.27 (s, 1H), 6.99 (dd, J1 = 8.3 Hz, J2 = 4.2 Hz, 1H), 6.89 (s, 1H), 5.12 (d, J = 2.4 Hz, 2H), 4.33 (td, J1 = 27.3 Hz, J2 = 8.8 Hz, J3 = 4.3 Hz, 2H), 4.00 (dt, J1 = 17.9 Hz, J2 = 5.2 Hz, 1H), 3.83 (dd, J1 = 17.4 Hz, J2 = 4.7 Hz, 1H), 3.56 (dt, J1 = 9.9 Hz, J2 = 4.3 Hz, 2H), 3.42 (m, 1H), 2.46 (d, J = 4.8 Hz, 1H), 2.36 (dd, J1 = 15.2 Hz, J2 = 8.6 Hz, 1H), 2.16 (m, 1H), 1.89 (m, 3H), 1.38 (s, 9H); 1313C NMR (75 MHz, DMSO-d6): δ / ppm = 172.12, 172.00, 157.33, 136.44, 128.93, 128.48, 128.17, 66.29, 63.12, 59.04, 52.51, 45.71, 29.06, 28.65, 24.84, 8.85, 7.70.

[0024] Example 1.4 Preparation of Asn-Gly-Pro

[0025] Dissolve Boc-Asn-Gly-Pro-OBzl (500 mg, 1.05 mmol) in methanol, add 75 mg of Pd / C, and stir to make it homogeneous. 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-Asn-Gly-Pro-OBzl. The reaction mixture is filtered to remove Pd / C, and the filtrate is concentrated to dryness under reduced pressure. The residue is triturated with ether (30 mL × 3) to obtain Boc-Asn-Gly-Pro as a colorless powder. Dissolve the Boc-Asn-Gly-Pro powder in 2 mL of anhydrous ethyl acetate solution at 0 °C, and mix the resulting ethyl acetate solution with 5 mL of 4N hydrogen chloride in anhydrous ethyl acetate solution. The reaction compound is stirred for 2 hours, and TLC (ethyl acetate / acetic acid / water = 2 / 1 / 1) shows the disappearance of Boc-Asn-Gly-Pro. The reaction mixture is concentrated to dryness under reduced pressure. The residue is dissolved in 5 mL of anhydrous ethyl acetate and concentrated under reduced pressure to remove the free hydrogen chloride gas. This operation is repeated three times to completely remove the free hydrogen chloride gas. The residue is purified by C18 column chromatography to obtain 265 mg (92%) of the title compound. M.p. 131 - 132 °C; ESI-MS (m / e): 287 [M + H] + ; 1 1H-NMR (300 MHz, DMSO-d6): δ / ppm = 8.59 (dt, J1 = 12.1 Hz, J2 = 5.3 Hz, 1H), 8.22 (m, 1H), 7.77 (m, 1H), 7.24 (d, J = 2.2 Hz, 1H), 4.24 (dd, J1 = 8.8 Hz, J2 = 3.4 Hz, 1H), 4.13 (m, 1H), 4.01 (m, 1H), 3.52 (dt, J1 = 10.0 Hz, J2 = 5.0 Hz, 2H), 2.75 (m, 1H), 2.62 (m, 1H), 2.15 (m, 1H), 1.91 (m, 3H); 1313C NMR (75 MHz, DMSO-d6): δ / ppm = 173.64, 171.21, 168.91, 166.62, 59.11, 49.51, 45.97, 41.71, 36.15, 29.14, 24.79.

[0026] Example 2 Preparation of the trimer of Asn-Gly-Pro

[0027] Dissolve 100 mg of Asn-Gly-Pro powder in 5 mL of ultrapure water. The resulting solution was vortexed for 15 minutes and then sonicated in an ultrasonic bath for 4 hours until the solution became completely colorless and transparent. The colorless and transparent solution was concentrated to dryness under reduced pressure to obtain the trimer of Asn-Gly-Pro, i.e., [Asn-Gly-Pro]3. The structure of [Asn-Gly-Pro]3 was confirmed by ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Figure 2 The FT-ICR-MS spectrum gave peaks for [Asn-Gly-Pro]+H at 287.13795 (theoretical value 287.13500), for [Asn-Gly-Pro]2+H at 573.27072 (theoretical value 573.26272), and for [Asn-Gly-Pro]3+H at 859.40722 (theoretical value 859.39044).

[0028] To clarify the relationship between the peaks of [Asn-Gly-Pro]+H, [Asn-Gly-Pro]2+H, and [Asn-Gly-Pro]3+H, the qCID spectrum of [Asn-Gly-Pro]3+H was measured. Figure 2 The qCID spectrum of [Asn-Gly-Pro]3 gave a peak for [Asn-Gly-Pro]+H at 287.13717 (theoretical value 287.13500) and a peak for [Asn-Gly-Pro]2+H at 573.26625 (theoretical value 573.26272). That is, [Asn-Gly-Pro]3 is the only form in which Asn-Gly-Pro exists.

[0029] Example 3 Evaluation of the antithrombotic activity of [Asn-Gly-Pro]3

[0030] The antithrombotic effect of [Asn-Gly-Pro]3 was evaluated in a rat model of inferior vena cava ligation. Male SD rats (250±20 g) were fasted for one day at 25-26 °C and used to establish the rat model of inferior vena cava ligation. During model establishment, the rats were anesthetized by intraperitoneal injection of 20% urethane solution, and the anesthetized rats were fixed in the supine position on the operating board, and the skin was prepared and disinfected. Then, the abdominal cavity was opened along the linea alba of the rat. The abdominal incision was from the coagulating gland to the lower part and to the upper part to expose a corner of the liver. The small intestine and other organs were removed from the abdominal cavity and wrapped with a gauze soaked in normal saline. The connective tissue around the blood vessels was bluntly dissected to expose the inferior vena cava and its branches. The abdominal aorta and the inferior vena cava were dissected below the renal vein, and then the inferior vena cava was ligated at the junction of the inferior vena cava and the left renal vein with a suture soaked in normal saline. The intestine and other organs removed from the abdominal cavity and wrapped with a gauze soaked in normal saline were returned to the abdominal cavity according to the anatomical position, and the abdominal cavity was sutured layer by layer with sutures. Then, the rats were randomly divided into groups of 10 rats each. The rats were orally administered normal saline (dose: 10 mL / kg / day, once a day for 7 consecutive days) or a normal saline solution of warfarin (dose: 0.82 μmol / kg / day, once a day for 7 consecutive days) or a normal saline solution of [Asn-Gly-Pro]3 (dose: 0.1 μmol / kg / day, once a day for 7 consecutive days) or a normal saline solution of Asn-Gly-Pro (dose: 1 μmol / kg / day, once a day for 7 consecutive days). 30 minutes after the last oral administration, the rats were anesthetized by intraperitoneal injection of 20% urethane solution, and the anesthetized rats were fixed in the supine position on the operating board. First, 2 cm of the inferior vena cava was removed from the ligation site at the junction of the inferior vena cava and the left renal vein, and then the thrombus was taken out from the inferior vena cava and weighed.

[0031] The data in Table 1 showed that [Asn-Gly-Pro]3 at an oral dose of 0.1 μmol / kg / day effectively inhibited venous thrombosis (P < 0.01 compared with normal saline). The data in Table 1 also showed that the weight of venous thrombosis in rats orally administered [Asn-Gly-Pro]3 at 0.1 μmol / kg / day was significantly less than that in rats orally administered warfarin at 0.82 μmol / kg / day (P < 0.01 compared with warfarin). [Asn-Gly-Pro]3 had prominent technical effects. The data in Table 1 further showed that the weight of venous thrombosis in rats orally administered [Asn-Gly-Pro]3 at 0.1 μmol / kg / day was significantly less than that in rats orally administered Asn-Gly-Pro at 1 μmol / kg / day (P < 0.01 compared with Asn-Gly-Pro). [Asn-Gly-Pro]3 had unexpected technical effects.

[0032] Table 1 Effects of [Asn-Gly-Pro]3 on Venous Thrombosis in Rats

[0033] Therapeutic agent Dose Venous thrombosis weight, mean ± SD mg Normal saline 10 mL / kg / day 14.55±2.34 Warfarin 0.82 μmol / kg / day 8.71±1.13 <![CDATA[[Asn-Gly-Pro]3]]> 0.1 μmol / kg / day <![CDATA[5.11±0.65 a > Asn-Gly-Pro 1 μmol / kg / day <![CDATA[8.35±1.02 b >

[0034] a) P < 0.01 compared with normal saline, warfarin, and Asn-Gly-Pro; b) P < 0.01 compared with normal saline, P > 0.05 compared with warfarin; n = 10.

Claims

1. A trimer of Asn-Gly-Pro, characterized in that, The trimer is [Asn-Gly-Pro]3, and the [Asn-Gly-Pro]3 is composed of three Asn-Gly-Pro molecules through non-covalent bonds.

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

3. Use of the trimer of Asn-Gly-Pro according to claim 1 in the preparation of an anti-venous thrombosis drug.

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