Dimer of Glu-Arg-Ala, its preparation and application

Abstract

The present invention discloses a dimer of Glu-Arg-Ala, namely [Glu-Arg-Ala]2, and discloses its preparation method and application in the treatment of vasoconstrictive diseases. Experiments have proved that [Glu-Arg-Ala]2 of the present invention not only has a good vasodilatory effect, but also the vasodilatory effect is significantly stronger than that of Glu-Arg-Ala. Therefore, it is proposed that the present invention provides an effective technical means for vasodilation.

Description

Technical Field

[0001] The present invention relates to a dimer of Glu-Arg-Ala, i.e., [Glu-Arg-Ala]2, to its preparation method and to its application in the treatment of vasoconstrictive diseases. Experiments have shown that [Glu-Arg-Ala]2 of the present invention not only has a good vasodilatory effect, but also has a significantly stronger vasodilatory effect than that of Glu-Arg-Ala. 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, 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 triangles. The spadix of Cynomorium songaricum Rupr. is born at the top of the stem, extends out of 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 mixed and accompanied, with a fragrance. The flowering period is from May to July. Cynomorium songaricum Rupr. produces small nuts, nearly spherical or oval, the pericarp is white, and there is 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, grassland-desert transition zones 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 for treating impotence and spermatorrhea, weakness of the waist and knees, intestinal dryness and constipation, and has a certain effect on paralysis and improving sexual function weakness. It is worth pointing out that when the inventor evaluated the vasodilatory effect of Cynomorium songaricum Rupr., Glu-Arg-Ala was found in the vascular strip. The inventor knows that aggregates of Glu-Arg-Ala often show a stronger vasodilatory effect. Therefore, the inventor prepared a dimer of Glu-Arg-Ala, that is, [Glu-Arg-Ala]2. Experiments have shown that [Glu-Arg-Ala]2 of the present invention not only has a good vasodilatory effect, but also has a significantly stronger vasodilatory effect than that of Glu-Arg-Ala. 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 identify a dimer of Glu-Arg-Ala, i.e., [Glu-Arg-Ala]2, and further confirm that [Glu-Arg-Ala]2 has a more excellent vasodilatory effect than Glu-Arg-Ala. To achieve the above object, the present invention adopts the following four technical means.

[0004] The first technical means is to propose the Glu-Arg-Ala dimer of the present invention, namely [Glu-Arg-Ala]2, and the [Glu-Arg-Ala]2 is composed of two Glu-Arg-Ala molecules through non-covalent bonds.

[0005] The second technical means is to propose a method for preparing the Glu-Arg-Ala dimer, namely [Glu-Arg-Ala]2. This method includes the following 5 steps:

[0006] 1) Prepare Boc-Arg(NO2)-Ala-OBzl;

[0007] 2) Prepare Arg(NO2)-Ala-OBzl;

[0008] 3) Prepare Boc-Glu(OBzl)-Arg(NO2)-Ala-OBzl;

[0009] 4) Prepare Glu-Arg-Ala;

[0010] 5) Prepare [Glu-Arg-Ala]2.

[0011] The third technical means is to confirm the role of the Glu-Arg-Ala dimer, namely [Glu-Arg-Ala]2, in the preparation of vasodilator drugs.

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

[0013] The dimer of the present invention, namely [Glu-Arg-Ala]2, which is composed of two Glu-Arg-Ala molecules through non-covalent bonds, is proven by experiments. Compared with the prior art, the [Glu-Arg-Ala]2 of the present invention has a good vasodilatory effect, and its vasodilatory effect is significantly stronger than that of Glu-Arg-Ala. Therefore, the present invention provides an effective technical means for the preparation of drugs for dilating blood vessels. Description of the Drawings

[0014] Figure 1 Synthesis route diagram of Glu-Arg-Ala: 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 FT-ICR-MS spectrum of [Glu-Arg-Ala]2 and qCID spectrum of [Glu-Arg-Ala]2. Detailed Embodiments

[0016] To further illustrate the present invention, a series of embodiments are given below. These embodiments are entirely 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 Glu-Arg-Ala

[0018] Example 1.1 Preparation of Boc-Arg(NO2)-Ala-OBzl

[0019] At 0 °C, Boc-Arg(NO2) (3.55 g, 11.13 mmol) and N-hydroxybenzotriazole (HOBt, 1.50 g, 11.13 mmol) were dissolved in anhydrous tetrahydrofuran. To this solution was added a solution of dicyclohexylcarbodiimide (DCC, 2.30 g, 11.13 mmol) in anhydrous tetrahydrofuran. The two solutions were stirred well for 20 minutes to obtain activated Boc-Arg(NO2) / HOBt. At 0 °C, HCl·Ala-OBzl (2.40 g, 11.13 mmol) was added to the activated Boc-Arg(NO2) / 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, then stirred at room temperature for 12 hours. TLC (dichloromethane / methanol = 20 / 1) showed the disappearance of HCl·Ala-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 solution (200 mL × 3), saturated aqueous NaCl solution (200 mL × 3), 5% aqueous KHSO4 solution (200 mL × 3), saturated aqueous NaCl solution (200 mL × 3), saturated aqueous NaHCO3 solution (200 mL × 3) and saturated aqueous NaCl solution (200 mL × 3). The separated ethyl acetate solution was dried with anhydrous Na2SO4 for 12 hours, filtered, and the filtrate was concentrated under reduced pressure. The residue was triturated repeatedly with ether to obtain 4.21 g (95%) of the title compound as a pale yellow solid, which was directly used in the next step of the reaction. ESI-MS (m / e): 481 [M+H] + 。

[0020] Example 1.2 Preparation of Arg(NO2)-Ala-OBzl

[0021] Dissolve Boc-Arg(NO2)-Ala-OBzl (2.00 g, 4.16 mmol) in 15 mL of anhydrous ethyl acetate, and 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-Arg(NO2)-Ala-OBzl. The reaction mixture is concentrated under reduced pressure to remove free hydrogen chloride gas. The residue is dissolved again 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 three times with petroleum ether to obtain 1.91 g (96%) of the title compound as a colorless solid, which is directly used in the next reaction. ESI-MS (m / e): 381 [M+H] + 。

[0022] Example 1.3 Preparation of Boc-Glu(OBzl)-Arg(NO2)-Ala-OBzl

[0023] The crude product obtained from Boc-Glu(OBzl) (1.50 g, 4.42 mmol) and Arg(NO2)-Ala-OBzl (1.44 g, 4.42 mmol) according to the operation of Example 1.1 is purified by silica gel column chromatography to obtain 2.31 g (89%) of the title compound as a colorless solid. ESI-MS (m / e): 700 [M+H] + ; 1 1H NMR (300 MHz, DMSO-d6); δ / ppm = 8.44 (d, J = 6.8 Hz, 1H), 7.86 (d, J = 8.0 Hz, 1H), 7.34 (m, 10H), 6.99 (d, J = 8.1 Hz, 1H), 5.11 (m, 4H), 4.31 (m, 2H), 4.01 (m, 1H), 3.37 (s, 2H), 2.40 (t, J = 7.8 Hz, 2H), 1.84 (m, 2H), 1.67 (d, J = 15.6 Hz, 1H), 1.51 (d, J = 7.6 Hz, 2H), 1.26 (m, 12H); 13 13C NMR (75 MHz, DMSO-d6): δ / ppm = 172.74, 172.68, 171.90, 171.75, 155.79, 136.65, 136.37, 128.88, 128.52, 128.43, 128.30, 128.27, 78.76, 66.40, 65.90, 53.91, 52.05, 48.15, 30.60, 30.04, 28.58, 27.60, 17.16。

[0024] Example 1.4 Preparation of Glu-Arg-Ala

[0025] Dissolve Boc-Glu(OBzl)-Arg(NO2)-Ala-OBzl (500 mg, 0.72 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-Glu(OBzl)-Arg(NO2)-Ala-OBzl. Filter the reaction mixture to remove Pd / C, and concentrate the filtrate under reduced pressure to dryness. The residue is triturated with ether (30 mL × 3) to obtain Boc-Glu-Arg-Ala as a colorless powder. Dissolve the Boc-Glu-Arg-Ala 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-Glu-Arg-Ala. Concentrate the reaction mixture under reduced pressure to dryness. 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 234 mg (86%) of the title compound. M.p. 151 - 153 °C; ESI-MS (m / e): 375 [M + H] + ; 1 1H-NMR (300 MHz, DMSO-d6); δ / ppm = 8.80 (d, J = 7.6 Hz, 1H), 8.19 (d, J = 7.2 Hz, 1H), 8.09 (s, 1H), 7.25 (s, 3H), 4.29 (s, 1H), 4.09 (m, J = 7.2 Hz, 1H), 3.84 (t, J = 6.3 Hz, 1H), 3.61 (d, J = 5.1 Hz, 1H), 3.11 (m, J = 6.2 Hz, 2H), 2.36 (m, 2H), 1.92 (dt, J1 = 11.6 Hz, J2 = 7.2 Hz, 2H), 1.74 (s, 2H), 1.57 (s, 2H), 1.25 (d, J = 7.2 Hz, 3H); 13 13C NMR (75 MHz, DMSO-d6): δ / ppm = 174.73, 174.44, 170.82, 169.50, 157.56, 52.70, 52.30, 48.69, 40.90, 30.60, 29.46, 27.64, 25.16, 17.99。

[0026] Example 2 Preparation of Glu-Arg-Ala Dimer

[0027] Dissolve 100 mg of Glu-Arg-Ala 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 Glu-Arg-Ala dimer, i.e., [Glu-Arg-Ala]2. The structure of [Glu-Arg-Ala]2 is confirmed by ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). The FT-ICR-MS spectrum gives a peak of [Glu-Arg-Ala]+H at 375.20243 (theoretical value: 375.19866) and a peak of [Glu-Arg-Ala]2+H at 749.40200 (theoretical value: 749.39004).

[0028] To clarify the relationship between the peak of [Glu-Arg-Ala]+H and the peak of [Glu-Arg-Ala]2+H, the qCID spectrum of [Glu-Arg-Ala]2+H is measured. The qCID spectrum of [Glu-Arg-Ala]2+H gives a peak of [Glu-Arg-Ala]+H at 375.20077 (theoretical value: 375.19866). That is to say, [Glu-Arg-Ala]2 is the only form in which Glu-Arg-Ala exists.

[0029] Example 3 Evaluating the vasodilatory activity of [Glu-Arg-Ala]2

[0030] Vasoconstrictive diseases, such as hypertension, cardiac arrest caused by anaphylactic shock, bronchial asthma, and migraine, have become global health problems. Adrenaline, ephedrine, and dopamine, which are clinically used to treat vasoconstrictive diseases, have various problems. Developing drugs for treating vasoconstrictive diseases is a clinical need. In response to this need, the inventors evaluated the vasodilatory activity of [Glu-Arg-Ala]2. During the evaluation, SD rats were first anesthetized with ether and then sacrificed by cervical dislocation. Then, the thoracic aorta of the rats was removed by opening the chest cavity. Finally, the thoracic aorta of the rats was cut into thoracic aortic rings 2 mm in length. The prepared rat thoracic aortic rings should be placed in oxygenated K-H buffer to maintain their viability.

[0031] Norepinephrine is recognized as a vasoconstrictor, and acetylcholine is recognized as a vasodilator. When evaluating the vasodilatory activity of [Glu-Arg-Ala]2 (final concentration 0.1 μg / mL), norepinephrine (NE, final concentration 20 μg / mL) was used to induce the contraction of rat thoracic aortic rings, and acetylcholine (final concentration 2 μg / mL) was used as a positive control. When evaluating the vasodilatory activity of Glu-Arg-Ala (final concentration 1 μg / mL), norepinephrine (final concentration 20 μg / mL) was used to induce the contraction of rat thoracic aortic rings, and acetylcholine (final concentration 2 μg / mL) was used as a positive control.

[0032] During the evaluation, turn on the physiological two-channel instrument (the operating software is M3000), turn on the circulating water in the incubation tank, fill the incubation tank with K-H buffer solution and oxygenate it. Set the tension transducer and calibrate it. Hang the rat thoracic aortic ring on the tension transducer, and slowly lower the tension transducer. The rat thoracic aortic ring follows the descending tension transducer and slowly immerses in the K-H buffer solution in the incubation tank. After the tension of the rat thoracic aortic ring stabilizes at the baseline, add 20 μL of norepinephrine aqueous solution to the incubation tank to cause the contraction of the rat thoracic aortic ring. After the tension of the rat thoracic aortic ring stabilizes at a high level, slowly raise the tension transducer, gently wash away the norepinephrine attached to the rat thoracic aortic ring with K-H buffer solution, replace the K-H buffer solution in the incubation tank, and slowly lower the tension transducer. The rat thoracic aortic ring follows the descending tension transducer and slowly immerses in the K-H buffer solution in the incubation tank. Add 20 μL of acetylcholine aqueous solution, or 20 μL of [Glu-Arg-Ala]2 aqueous solution, or 20 μL of Glu-Arg-Ala aqueous solution to the incubation tank. After the tension stabilizes, calculate the decrease value of the tension of the rat thoracic aortic ring, that is, calculate the antagonistic effect of acetylcholine or [Glu-Arg-Ala]2 or Glu-Arg-Ala on the contraction of rat thoracic aortic rings caused by norepinephrine. According to the above operation, each determination was repeated 6 times.

[0033] The data in Table 1 show that the vasodilatory activity of [Glu-Arg-Ala]2 at a final concentration of 0.1 μg / mL is significantly stronger than that of acetylcholine at a final concentration of 2 μg / mL (P < 0.01 compared with acetylcholine). [Glu-Arg-Ala]2 has outstanding technical effects. The data in Table 1 further show that the vasodilatory activity of [Glu-Arg-Ala]2 at a final concentration of 0.1 μg / mL is significantly stronger than that of Glu-Arg-Ala at a final concentration of 1 μg / mL (P < 0.01 compared with Glu-Arg-Ala). [Glu-Arg-Ala]2 has unexpected technical effects.

[0034] Table 1 Vasodilatory activity of [Glu-Arg-Ala]2

[0035] Therapeutic agent Final concentration Relaxation rate of rat thoracic aortic rings contracted by NE, mean ± SD% Acetylcholine 2 μg / mL 33.46±2.13 <![CDATA[[Glu-Arg-Ala]2]]> 0.1 μg / mL <![CDATA[38.89±2.05 a > Glu-Arg-Ala 1 μg / mL <![CDATA[32.15±1.32 b >

[0036] a) P < 0.01 compared with acetylcholine and Glu-Arg-Ala; b) P > 0.05 compared with acetylcholine; n = 6.

Claims

1. A dimer of Glu-Arg-Ala, characterized in that, The dimer is [Glu-Arg-Ala]2, and the [Glu-Arg-Ala]2 is composed of two Glu-Arg-Ala molecules through non-covalent bonds.

2. A method for preparing the dimer of Glu-Arg-Ala according to claim 1, characterized in that, The preparation method includes the following steps: 1) Prepare Boc-Arg(NO2)-Ala-OBzl; 2) Prepare Arg(NO2)-Ala-OBzl; 3) Prepare Boc-Glu(OBzl)-Arg(NO2)-Ala-OBzl; 4) Prepare Glu-Arg-Ala; 5) Prepare [Glu-Arg-Ala]2.

3. Use of the dimer of Glu-Arg-Ala according to claim 1 in the preparation of antihypertensive drugs.

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