Tetramer of Met-Pro-Ser and its preparation and application

Abstract

The present invention discloses a tetramer of Met-Pro-Ser, namely [Met-Pro-Ser]<subgt;4< / subgt;, discloses its preparation method and the therapeutic effect on testes damaged by hydrocortisone. Experiments prove that the [Met-Pro-Ser]<subgt;4< / subgt; of the present invention can not only effectively treat testes damaged by hydrocortisone, but also the curative effect of treating testes damaged by hydrocortisone is significantly stronger than that of Met-Pro-Ser in treating testes damaged by hydrocortisone. Therefore, it is proposed that the present invention provides an effective technical means for treating testes damaged by hydrocortisone.

Description

Technical Field

[0001] The present invention relates to the tetramer of Met-Pro-Ser, namely [Met-Pro-Ser]4, to its preparation method and the therapeutic effect on testes damaged by hydrocortisone. Experiments have proved that [Met-Pro-Ser]4 of the present invention can not only effectively treat testes damaged by hydrocortisone, but also has a significantly stronger therapeutic effect on testes damaged by hydrocortisone than that of Met-Pro-Ser in treating testes damaged by hydrocortisone. The present invention belongs to the field of biomedicine. Background Art

[0002] Cynomorium songaricum Rupr. is a perennial fleshy parasitic herb without chlorophyll, and the whole plant is reddish-brown. Most of the whole herb is buried in the sand, and 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. is born at the top of the stem, protruding from the ground, 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 together, 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 of China. Cynomorium songaricum Rupr. can tonify the kidney, benefit essence, 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 the inventor found Met-Pro-Ser when evaluating the testes of mice damaged by hydrocortisone treated with Cynomorium songaricum Rupr. The inventor knows that aggregates of Met-Pro-Ser often show stronger activity. So the inventor prepared the tetramer of Met-Pro-Ser, namely [Met-Pro-Ser]4. Experiments have proved that [Met-Pro-Ser]4 of the present invention can not only effectively treat testes damaged by hydrocortisone, but also has a significantly stronger therapeutic effect on testes damaged by hydrocortisone than that of Met-Pro-Ser in treating testes damaged by hydrocortisone. 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 tetramer of Met-Pro-Ser, namely [Met-Pro-Ser]4, and further confirm that the efficacy of [Met-Pro-Ser]4 in treating testes damaged by hydrocortisone is significantly stronger than that of Met-Pro-Ser in treating testes damaged by hydrocortisone. To achieve the above object, the present invention adopts the following three technical means.

[0004] The first technical means is to propose a tetramer of Met-Pro-Ser of the present invention, namely [Met-Pro-Ser]4. The tetramer is composed of four Met-Pro-Ser molecules through non-covalent bonds.

[0005] The second technical means is to propose a method for preparing a tetramer of Met-Pro-Ser, namely [Met-Pro-Ser]4, which method comprises the following 5 steps:

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

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

[0008] 3) Prepare Boc-Met-Pro-Ser-OBzl;

[0009] 4) Prepare Met-Pro-Ser;

[0010] 5) Prepare [Met-Pro-Ser]4.

[0011] The third technical means is to confirm that the tetramer of Met-Pro-Ser, namely [Met-Pro-Ser]4, has excellent efficacy in drugs for treating testes damaged by hydrocortisone.

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

[0013] The present invention provides a tetramer composed of four Met-Pro-Ser molecules through non-covalent bonds, namely [Met-Pro-Ser]4. Experiments have proved that [Met-Pro-Ser]4 of the present invention can not only effectively treat testes damaged by hydrocortisone, but also the efficacy of treating testes damaged by hydrocortisone is significantly stronger than that of Met-Pro-Ser in treating testes damaged by hydrocortisone. Therefore, the present invention provides an effective technical means for the field of drugs for treating testes damaged by hydrocortisone. Description of the Drawings

[0014] Figure 1Synthetic route diagram for Met-Pro-Ser: 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 [Met-Pro-Ser]4 and qCID spectrum of [Met-Pro-Ser]4. Detailed implementation mode

[0016] To further illustrate the present invention, a series of embodiments are given below. These embodiments are completely illustrative, and they are only used to specifically describe the present invention and should not be construed as a limitation to the present invention.

[0017] Example 1 Preparation of Met-Pro-Ser

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

[0019] At 0 °C, Boc-Pro (2.40 g, 10.36 mmol) and N-hydroxybenzotriazole (HOBt, 1.40 g, 10.36 mmol) were dissolved in anhydrous tetrahydrofuran. A solution of dicyclohexylcarbodiimide (DCC, 2.20 g, 10.36 mmol) in anhydrous tetrahydrofuran was added to this solution. The two solutions were stirred well for 20 minutes to obtain activated Boc-Pro / HOBt. At 0 °C, HCl·Ser-OBzl (2.40 g, 10.36 mmol) was added to the activated Boc-Pro / HOBt to obtain a reaction mixture. At 0 °C, the pH value of this 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·Ser-OBzl. The insoluble substances in the reaction mixture were 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 ground repeatedly with ether to obtain 3.72 g (91%) of the title compound, which was a colorless solid and was directly used in the next step of the reaction. ESI-MS (m / e): 393 [M+H] + 。

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

[0021] Dissolve Boc-Pro-Ser-OBzl (2.00 g, 5.12 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 under cooling in an ice-salt bath. The reaction mixture is stirred for 120 minutes under cooling in an ice-salt bath. TLC (dichloromethane / methanol = 20 / 1) shows the disappearance of Boc-Pro-Ser-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 three times with petroleum ether to obtain 1.46 g (98%) of the title compound as a colorless solid, which is directly used in the next reaction. ESI-MS (m / e): 293 [M+H] + 。

[0022] Example 1.3 Preparation of Boc-Met-Pro-Ser-OBzl

[0023] The crude product obtained from Boc-Met (1.23 g, 4.93 mmol) and Pro-Ser-OBzl (1.44 g, 4.10 mmol) according to the operation of Example 1.1 is purified by silica gel column chromatography to obtain 2.00 g (91%) of the title compound as a colorless solid. ESI-MS (m / e): 448 [M+H] + ; 1 1H NMR (300 MHz, DMSO-d6): δ / ppm = 8.18 (d, J = 7.7 Hz, 1H), 7.04 (d, J = 7.7 Hz, 1H), 4.45 (dd, J1 = 8.5 Hz, J2 = 3.5 Hz, 1H), 4.30 (m, 2H), 3.63 (m, 7H), 2.08 (m, 5H), 1.87 (m, 3H), 1.75 (m, 3H), 1.36 (s, 9H); 13 13C NMR (75 MHz, DMSO-d6): δ / ppm = 172.36, 170.97, 170.67, 155.87, 136.39, 128.83, 128.46, 128.23, 78.47, 66.41, 61.68, 59.29, 55.21, 51.42, 47.18, 31.22, 29.94, 29.44, 28.68, 24.82, 15.16。

[0024] Example 1.4 Preparation of Met-Pro-Ser

[0025] Dissolve Boc-Met-Pro-Ser-OBzl (500 mg, 1.12 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-Met-Pro-Ser-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-Met-Pro-Ser as a colorless powder. Dissolve the Boc-Met-Pro-Ser powder in 2 mL of anhydrous ethyl acetate solution at 0 °C. The obtained ethyl acetate solution is mixed with 5 mL of 4N hydrogen chloride in anhydrous ethyl acetate solution. Stir the reaction compound for 2 hours. TLC (ethyl acetate / acetic acid / water = 2 / 1 / 1) shows the disappearance of Boc-Met-Pro-Ser. 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 free hydrogen chloride gas. This operation is repeated three times to completely remove free hydrogen chloride gas. Purify the residue by C18 column chromatography to obtain 329 mg (88%) of the title compound. M.p. 137 - 138 °C; ESI-MS (m / e): 344 [M + H] + ; 1 1H-NMR (300 MHz, DMSO-d6): δ / ppm = 8.35 (s, 2H), 8.28 (d, J = 7.7 Hz, 1H), 4.57 (m, 1H), 4.25 (m, 2H), 3.75 (m, 1H), 3.70 (d, J = 4.9 Hz, 1H), 3.62 (m, 2H), 3.50 (m, 1H), 2.66 (m, 1H), 2.57 (m, 1H), 2.07 (d, J = 2.1 Hz, 4H), 2.00 (m, 1H), 1.86 (m, 3H); 13 13C NMR (75 MHz, DMSO-d6): δ / ppm = 172.33, 171.58, 167.03, 61.75, 59.69, 55.16, 50.44, 47.59, 30.41, 29.61, 28.21, 24.97, 14.89.

[0026] Example 2 Preparation of the tetramer of Met-Pro-Ser

[0027] Dissolve 100 mg of Met-Pro-Ser 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 tetramer of Met-Pro-Ser, namely [Met-Pro-Ser]4. The structure of [Met-Pro-Ser]4 is confirmed by ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Figure 2 The FT-ICR-MS spectrum gives the peak of [Met-Pro-Ser]+H at 334.14631 (theoretical value is 334.14312), the peak of [Met-Pro-Ser]2+H at 667.28758 (theoretical value is 667.27896), the peak of [Met-Pro-Ser]3+H at 1000.43605 (theoretical value is 1000.41480), and the peak of [Met-Pro-Ser]4+H at 1333.59464 (theoretical value is 1333.55064).

[0028] To clarify the relationship between the peaks of [Met-Pro-Ser]+H, [Met-Pro-Ser]2+H, [Met-Pro-Ser]3+H and [Met-Pro-Ser]4+H, the qCID spectrum of [Met-Pro-Ser]4+H was measured. Figure 2 The qCID spectrum of [Met-Pro-Ser]4 gives the peak of [Met-Pro-Ser]3+H at 1000.42585 (theoretical value is 1000.41480), the peak of [Met-Pro-Ser]2+H at 667.28298 (theoretical value is 667.27896), and the peak of [Met-Pro-Ser]+H at 334.14519 (theoretical value is 334.14312). That is to say, [Met-Pro-Ser]4 is the only form in which Met-Pro-Ser exists.

[0029] Example 3 Evaluating the Efficacy of [Met-Pro-Ser]4 on Testes Damaged by Hydrocortisone

[0030] [Met-Pro-Ser]4's efficacy in reversing hydrocortisone-induced testicular injury was evaluated in a mouse model. Six-week-old ICR male mice (12 g ± 2 g) were rested for 1 day. Then, the mice were orally administered hydrocortisone (at a dose of 69 μmol / kg / day, once a day for 16 days) to cause testicular injury and were randomly divided into groups of 16 mice each. The mice were either orally administered 5‰ CMC-Na (at a dose of 10 mL / kg / day, once a day for 10 days) or an aqueous suspension of [Met-Pro-Ser]4 and 5‰ CMC-Na (at a dose of 0.3 μmol / kg / day, once a day for 10 days) or an aqueous suspension of Met-Pro-Ser and 5‰ CMC-Na (at a dose of 3 μmol / kg / day, once a day for 10 days). In addition, 16 six-week-old ICR male mice (12 g ± 2 g) were orally administered 5‰ CMC-Na once a day for 26 days as a control for testes with normal development. On the 27th day, the mice were anesthetized with ether, sacrificed by cervical dislocation, and their testes were removed. The testes were fixed in 4% tissue fixative for 24 hours, then dehydrated, embedded in paraffin, sectioned, stained with HE, and finally pathological sections of the testes were obtained.

[0031] Based on the morphology of spermatogonia and the pathological sections of seminiferous tubules, the numbers of spermatogonia in the early, middle, and late stages of spermatogenesis were calculated. The data in Table 1 showed that hydrocortisone significantly decreased the number of spermatogonia in the early stage of spermatogenesis, significantly decreased the number of spermatogonia in the middle stage of spermatogenesis, and significantly decreased the number of spermatogonia in the late stage of spermatogenesis (p < 0.01 compared with healthy mice). The data in Table 1 also showed that [Met-Pro-Ser]4's reversing effect on the testicular function damaged by hydrocortisone was manifested in significantly increasing the number of spermatogonia in the early stage of spermatogenesis, significantly increasing the number of spermatogonia in the middle stage of spermatogenesis, and significantly increasing the number of spermatogonia in the late stage of spermatogenesis (p > 0.05 compared with healthy mice). [Met-Pro-Ser]4 has prominent technical effects.

[0032] The data in Table 1 further showed that the number of spermatogonia in the early stage of spermatogenesis in hydrocortisone-damaged mice treated with [Met-Pro-Ser]4 was significantly greater than that in hydrocortisone-damaged mice treated with Met-Pro-Ser, the number of spermatogonia in the middle stage of spermatogenesis in hydrocortisone-damaged mice treated with [Met-Pro-Ser]4 was significantly greater than that in hydrocortisone-damaged mice treated with Met-Pro-Ser, and the number of spermatogonia in the late stage of spermatogenesis in hydrocortisone-damaged mice treated with [Met-Pro-Ser]4 was significantly greater than that in hydrocortisone-damaged mice treated with Met-Pro-Ser (p < 0.01 compared with Met-Pro-Ser). [Met-Pro-Ser]4 has unexpected technical effects.

[0033] Table 1. Effects of [Met-Pro-Ser]4 on spermatogonial cell counts at different spermatogenic phases

[0034]

[0035] a) p < 0.01 compared with healthy mice; b) p > 0.05 compared with healthy mice, p < 0.01 compared with mice treated with 5‰ CMC-Na and Met-Pro-Ser; c) p < 0.01 compared with mice treated with 5‰ CMC-Na; n = 16.

[0036] Based on the morphology of spermatogonial cells and pathological sections of seminiferous tubules, the numbers of spermatocytes in the early, middle, and late spermatogenic phases were calculated. The data in Table 2 showed that hydrocortisone significantly decreased the number of spermatocytes in the early spermatogenic phase, significantly decreased the number of spermatocytes in the middle spermatogenic phase, and significantly decreased the number of spermatocytes in the late spermatogenic phase (p < 0.01 compared with healthy mice). The data in Table 2 also showed that the reversing effect of [Met-Pro-Ser]4 on the testicular function damaged by hydrocortisone was manifested in significantly increasing the number of spermatocytes in the early spermatogenic phase, significantly increasing the number of spermatocytes in the middle spermatogenic phase, and significantly increasing the number of spermatocytes in the late spermatogenic phase (p > 0.05 compared with healthy mice). [Met-Pro-Ser]4 has prominent technical effects.

[0037] The data in Table 2 further showed that the number of spermatocytes in the early spermatogenic phase of hydrocortisone-damaged mice treated with [Met-Pro-Ser]4 was significantly greater than that of hydrocortisone-damaged mice treated with Met-Pro-Ser, the number of spermatocytes in the middle spermatogenic phase of hydrocortisone-damaged mice treated with [Met-Pro-Ser]4 was significantly greater than that of hydrocortisone-damaged mice treated with Met-Pro-Ser, and the number of spermatocytes in the late spermatogenic phase of hydrocortisone-damaged mice treated with [Met-Pro-Ser]4 was significantly greater than that of hydrocortisone-damaged mice treated with Met-Pro-Ser (p < 0.01 compared with Met-Pro-Ser). [Met-Pro-Ser]4 has unexpected technical effects.

[0038] Table 2. Effects of [Met-Pro-Ser]4 on spermatocyte counts at different spermatogenic phases

[0039]

[0040] a) p < 0.01 compared with healthy mice; b) p > 0.05 compared with healthy mice, p < 0.01 compared with mice treated with 5‰ CMC-Na and Ala-Ala-Thr; c) p < 0.01 compared with mice treated with 5‰ CMC-Na; n = 16.

[0041] The number of spermatozoa in the early, middle, and late spermatogenic phases was calculated based on the morphology of spermatogonia and the pathological sections of seminiferous tubules. The data in Table 3 showed that hydrocortisone significantly decreased the number of spermatozoa in the early spermatogenic phase, significantly decreased the number of spermatozoa in the middle spermatogenic phase, and significantly decreased the number of spermatozoa in the late spermatogenic phase (p < 0.01 compared with healthy mice). The data in Table 3 also showed that the reversal effect of [Met-Pro-Ser]4 on the testicular function damaged by hydrocortisone was manifested in significantly increasing the number of spermatozoa in the early spermatogenic phase, significantly increasing the number of spermatozoa in the middle spermatogenic phase, and significantly increasing the number of spermatozoa in the late spermatogenic phase (p > 0.05 compared with healthy mice). [Met-Pro-Ser]4 has outstanding technical effects.

[0042] The data in Table 3 further showed that the number of spermatozoa in the early spermatogenic phase of hydrocortisone-damaged mice treated with [Met-Pro-Ser]4 was significantly greater than that of hydrocortisone-damaged mice treated with Met-Pro-Ser, the number of spermatozoa in the middle spermatogenic phase of hydrocortisone-damaged mice treated with [Met-Pro-Ser]4 was significantly greater than that of hydrocortisone-damaged mice treated with Met-Pro-Ser, and the number of spermatozoa in the late spermatogenic phase of hydrocortisone-damaged mice treated with [Met-Pro-Ser]4 was significantly greater than that of hydrocortisone-damaged mice treated with Met-Pro-Ser (p < 0.01 compared with Met-Pro-Ser). [Met-Pro-Ser]4 has unexpected technical effects.

[0043] Table 3 Effects of [Met-Pro-Ser]4 on the number of spermatozoa in different spermatogenic phases

[0044]

[0045] a) p < 0.01 compared with healthy mice; b) p > 0.05 compared with healthy mice, p < 0.01 compared with mice treated with 5‰ CMC-Na and Met-Pro-Ser; c) p < 0.01 compared with mice treated with 5‰ CMC-Na; n = 16.

Claims

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

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

3. Use of the tetramer of Met-Pro-Ser according to claim 1 in the preparation of a therapeutic agent for testes damaged by hydrocortisone.

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