Trimer of Cys-Thr-Glu and its preparation and application

Abstract

The present invention discloses a trimer of Cys-Thr-Glu, namely [Cys-Thr-Glu]<subgt;3< / subgt;, and discloses its preparation method and therapeutic effect on testes damaged by dexamethasone. Experiments prove that [Cys-Thr-Glu]<subgt;3< / subgt; of the present invention can not only effectively treat testes damaged by dexamethasone, but also has a significantly stronger therapeutic effect on testes damaged by dexamethasone than the therapeutic effect of Cys-Thr-Glu on testes damaged by dexamethasone. Therefore, it is proposed that the present invention provides an effective technical means for treating testes damaged by dexamethasone.

Description

Technical Field

[0001] The present invention relates to a trimer of Cys-Thr-Glu, i.e., [Cys-Thr-Glu]3, to its preparation method and to its therapeutic effect on testes damaged by dexamethasone. Experiments have shown that the [Cys-Thr-Glu]3 of the present invention can not only effectively treat testes damaged by dexamethasone, but also has a significantly stronger therapeutic effect on testes damaged by dexamethasone than the therapeutic effect of Cys-Thr-Glu on testes damaged by dexamethasone. 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 in a spiral pattern 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, 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 together, with a fragrance. The flowering period is from May to July. Cynomorium songaricum Rupr. produces small nuts, which are 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 to treat 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 Cys-Thr-Glu when evaluating the testes of mice damaged by dexamethasone treated with Cynomorium songaricum Rupr. The inventor knows that aggregates of Cys-Thr-Glu often show stronger therapeutic effects. So the inventor prepared a trimer of Cys-Thr-Glu, i.e., [Cys-Thr-Glu]3. Experiments have shown that the [Cys-Thr-Glu]3 of the present invention can not only effectively treat testes damaged by dexamethasone, but also has a significantly stronger therapeutic effect on testes damaged by dexamethasone than the therapeutic effect of Cys-Thr-Glu on testes damaged by dexamethasone. 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 trimer of Cys-Thr-Glu, namely [Cys-Thr-Glu]3. The efficacy of the said [Cys-Thr-Glu]3 in treating dexamethasone-induced testicular injury is significantly stronger than that of Cys-Thr-Glu in treating dexamethasone-induced testicular injury. To achieve the above object, the present invention adopts the following technical means.

[0004] The first technical means is to propose the trimer of Cys-Thr-Glu of the present invention, namely [Cys-Thr-Glu]3. The said [Cys-Thr-Glu]3 is composed of three Cys-Thr-Glu molecules through non-covalent bonds.

[0005] The second technical means is to propose a method for preparing the trimer of Cys-Thr-Glu, namely [Cys-Thr-Glu]3, which method comprises the following 5 steps:

[0006] 1) Prepare Boc-Thr-Glu(OBzl)-OBzl;

[0007] 2) Prepare Thr-Glu(OBzl)-OBzl;

[0008] 3) Prepare Boc-Cys-Thr-Glu(OBzl)-OBzl;

[0009] 4) Prepare Cys-Thr-Glu;

[0010] 5) Prepare [Cys-Thr-Glu]3.

[0011] The third technical means is to confirm that the trimer of Cys-Thr-Glu, namely [Cys-Thr-Glu]3, has excellent efficacy in the treatment of dexamethasone-induced testicular injury.

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

[0013] The trimer of Cys-Thr-Glu of the present invention, namely [Cys-Thr-Glu]3, which is composed of three Cys-Thr-Glu molecules through non-covalent bonds, can not only effectively treat dexamethasone-induced testicular injury, but also the efficacy of treating dexamethasone-induced testicular injury is significantly stronger than that of Cys-Thr-Glu in treating dexamethasone-induced testicular injury. Therefore, the present invention provides an effective technical means for treating dexamethasone-induced testicular injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1Synthetic route diagram for Cys-Thr-Glu: 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 [Cys-Thr-Glu]3 and qCID spectrum of [Cys-Thr-Glu]3. Detailed implementation manners

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

[0017] Example 1 Preparation of Cys-Thr-Glu

[0018] Example 1.1 Preparation of Boc-Thr-Glu(OBzl)-OBzl

[0019] At 0 °C, Boc-Thr (2.90 g, 13.20 mmol) and N-hydroxybenzotriazole (HOBt, 1.78 g, 13.20 mmol) were dissolved in anhydrous tetrahydrofuran, and a solution of dicyclohexylcarbodiimide (DCC, 2.72 g, 13.20 mmol) in anhydrous tetrahydrofuran was added to this solution. The two solutions were stirred well for 20 minutes to obtain activated Boc-Thr / HOBt. At 0 °C, HCl·Glu(OBzl)-OBzl (4.80 g, 13.20 mmol) was added to the activated Boc-Thr / 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 that HCl·Glu(OBzl)-OBzl disappeared. 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 6.88 g (98%) of the title compound, which was a pale yellow solid and was directly used in the next step of the reaction. ESI-MS (m / e): 529 [M+H] + 。

[0020] Example 1.2 Preparation of Thr-Glu(OBzl)-OBzl

[0021] Dissolve Boc-Thr-Glu(OBzl)-OBzl (4.00 g, 7.57 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-Thr-Glu(OBzl)-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 3.04 g (94%) of the title compound as a colorless solid, which is directly used in the next reaction. ESI-MS (m / e): 429 [M+H] + 。

[0022] Example 1.3 Preparation of Boc-Cys-Thr-Glu(OBzl)-OBzl

[0023] The crude product obtained from Boc-Cys (2.64 g, 5.88 mmol) and Thr-Glu(OBzl)-OBzl (2.52 g, 5.88 mmol) according to the procedure of Example 1.1 is purified by silica gel column chromatography to obtain 3.40 g (92%) of the title compound as a colorless solid. ESI-MS (m / e): 1261 [M+H] + ; 1 1H NMR (300 MHz, DMSO-d6): δ / ppm = 8.21 (d, J = 7.6 Hz, 2H), 7.65 (d, J = 8.2 Hz, 2H), 7.35 (m, 20H), 7.26 (m, 2H), 5.10 (s, 4H), 5.07 (s, 4H), 4.85 (d, J = 4.9 Hz, 2H), 4.38 (m, 2H), 4.22 (s, 2H), 4.19 (dd, J1 = 8.2 Hz, J2 = 4.3 Hz, 2H), 3.94 (m, 2H), 3.11 (dd, J1 = 13.5 Hz, J2 = 4.3 Hz, 2H), 2.85 (dd, J1 = 13.4 Hz, J2 = 10.0 Hz, 2H), 2.43 (t, J = 8.3 Hz, 4H), 2.05 (m, 2H), 1.91 (m, 2H), 1.37 (s, 18H), 1.02 (d, J = 6.2 Hz, 6H); 1313C NMR (75 MHz, DMSO-d6): δ / ppm = 172.50, 171.65, 170.82, 170.47, 155.88, 136.57, 136.28, 128.89, 128.86, 128.51, 128.46, 128.37, 128.34, 79.02, 67.02, 66.57, 66.00, 58.56, 54.21, 51.69, 30.17, 28.60, 26.60, 20.16。

[0024] Example 1.4 Preparation of Cys-Thr-Glu

[0025] Dissolve Boc-Cys-Thr-Glu(OBzl)-OBzl (500 mg, 1.12 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-Cys-Thr-Glu(OBzl)-OBzl. Filter the reaction mixture to remove Pd / C, and concentrate the filtrate under reduced pressure to dryness. Wash the residue with diethyl ether (30 mL × 3) to obtain Boc-Cys-Thr-Glu as a colorless powder. Dissolve the Boc-Cys-Thr-Glu 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. 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 it 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 322 mg (83%) of the title compound. M.p. 144 - 145 °C; ESI-MS (m / e): 352 [M + H] + ; 11H-NMR (300 MHz, DMSO-d6): δ / ppm = 8.21 (d, J = 7.6 Hz, 2H), 7.65 (d, J = 8.2 Hz, 2H), 7.35 (m, 20H), 7.26 (m, 2H), 5.10 (s, 4H), 5.07 (s, 4H), 4.85 (d, J = 4.9 Hz, 2H), 4.38 (m, 2H), 4.22 (s, 2H), 4.19 (dd, J1 = 8.2 Hz, J2 = 4.3 Hz, 2H), 3.94 (m, 2H), 3.11 (dd, J1 = 13.5 Hz, J2 = 4.3 Hz, 2H), 2.85 (dd, J1 = 13.4 Hz, J2 = 10.0 Hz, 2H), 2.43 (t, J = 8.3 Hz, 4H), 2.05 (m, 2H), 1.91 (m, 2H), 1.37 (s, 18H), 1.02 (d, J = 6.2 Hz, 6H); 13 13C NMR (75 MHz, DMSO-d6): δ / ppm = 174.29, 173.54, 160.91, 167.59, 67.16, 63.81, 59.92, 52.85, 51.79, 30.49, 26.90, 20.21.

[0026] Example 2 Preparation of the trimer of Cys-Thr-Glu

[0027] Dissolve 100 mg of Cys-Thr-Glu powder in 5 mL of ultrapure water. The resulting solution was vortexed for 15 minutes first 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 Cys-Thr-Glu, namely [Cys-Thr-Glu]3. The structure of [Cys-Thr-Glu]3 was confirmed by ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Figure 2 The FT-ICR-MS spectrum gave peaks of [Cys-Thr-Glu]+H at 352.12098 (theoretical value 352.11730), peaks of [Cys-Thr-Glu]2+H at 703.23869 (theoretical value 703.22732), and peaks of [Cys-Thr-Glu]3+H at 1054.36511 (theoretical value 1054.33734).

[0028] To clarify the relationship between the peaks of [Cys-Thr-Glu]+H, [Cys-Thr-Glu]2+H and [Cys-Thr-Glu]3+H, the qCID spectrum of [Cys-Thr-Glu]3+H was measured. Figure 2The qCID spectrum of [Met-Pro-Ser]3 gave a peak of [Cys-Thr-Glu]2+H at 703.23154 (theoretical value 703.22732), and a peak of [Cys-Thr-Glu]+H at 352.11930 (theoretical value 352.11730). That is to say, [Cys-Thr-Glu]3 is the only form in which Cys-Thr-Glu exists.

[0029] Example 3 evaluated the efficacy of [Cys-Thr-Glu]3 on dexamethasone-damaged testes

[0030] The efficacy of [Cys-Thr-Glu]3 in reversing dexamethasone-induced testicular damage was evaluated in a mouse model. ICR male mice at 6 weeks of age (12 g ± 2 g) were rested for 1 day. Then the mice were orally administered dexamethasone (dose 7 mg / kg / day, once a day for 16 days) to damage the testes, and randomly divided into groups of 16 mice each. The mice were orally administered 5‰ CMC-Na (dose 10 mL / kg / day, once a day for 10 days) or a suspension of [Cys-Thr-Glu]3 and 5‰ CMC-Na (dose 0.3 μmol / kg / day, once a day for 10 days) or a suspension of Cys-Thr-Glu and 5‰ CMC-Na (dose 3 μmol / kg / day, once a day for 10 days). In addition, 16 ICR male mice at 6 weeks of age (12 g ± 2 g) were orally administered 5‰ CMC-Na once a day for 26 days as a control for normally developing testes. On the 27th day, the mice were anesthetized with ether, sacrificed by cervical dislocation, and the testes were removed. The testes were fixed in 4% tissue fixative for 24 hours, then dehydrated, paraffin-embedded, sectioned, and 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 reduced the number of spermatogonia in the early stage of spermatogenesis, significantly reduced the number of spermatogonia in the middle stage of spermatogenesis, and significantly reduced 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 the reversing effect of [Cys-Thr-Glu]3 on dexamethasone-induced testicular dysfunction in mice was manifested by a significant increase in the number of spermatogonia in the early stage of spermatogenesis, a significant increase in the number of spermatogonia in the middle stage of spermatogenesis, and a significant increase in the number of spermatogonia in the late stage of spermatogenesis (p > 0.05 compared with healthy mice). [Cys-Thr-Glu]3 has outstanding technical effects.

[0032] The data in Table 1 further show that the count of early spermatogonia during spermatogenesis in dexamethasone - damaged mice treated with [Cys - Thr - Glu]3 is significantly greater than that in dexamethasone - damaged mice treated with Cys - Thr - Glu, the count of mid - stage spermatogonia during spermatogenesis in dexamethasone - damaged mice treated with [Cys - Thr - Glu]3 is significantly greater than that in dexamethasone - damaged mice treated with Cys - Thr - Glu, and the count of late - stage spermatogonia during spermatogenesis in dexamethasone - damaged mice treated with [Cys - Thr - Glu]3 is significantly greater than that in dexamethasone - damaged mice treated with Cys - Thr - Glu (p < 0.01 compared with Cys - Thr - Glu). [Cys - Thr - Glu]3 has unexpected technical effects.

[0033] Table 1 Effects of [Cys - Thr - Glu]3 on the count of spermatogonia 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 Cys - Thr - Glu; c) p < 0.01 compared with mice treated with 5‰ CMC - Na; n = 16.

[0036] The counts of early, mid - stage, and late - stage spermatocytes during spermatogenesis were calculated based on the morphology of spermatogonia and the pathological sections of seminiferous tubules. The data in Table 2 show that dexamethasone significantly reduces the count of early - stage spermatocytes during spermatogenesis, significantly reduces the count of mid - stage spermatocytes during spermatogenesis, and significantly reduces the count of late - stage spermatocytes during spermatogenesis (p < 0.01 compared with healthy mice). The data in Table 2 also show that the reversing effect of [Cys - Thr - Glu]3 on the testicular function of dexamethasone - damaged mice is manifested in significantly increasing the count of early - stage spermatocytes during spermatogenesis, significantly increasing the count of mid - stage spermatocytes during spermatogenesis, and significantly increasing the count of late - stage spermatocytes during spermatogenesis (p > 0.05 compared with healthy mice). [Cys - Thr - Glu]3 has prominent technical effects.

[0037] The data in Table 2 further show that the count of early spermatocyte in the spermatogenic phase of dexamethasone-injured mice treated with [Cys-Thr-Glu]3 is significantly greater than that of dexamethasone-injured mice treated with Cys-Thr-Glu, the count of mid-spermatocyte in the spermatogenic phase of dexamethasone-injured mice treated with [Cys-Thr-Glu]3 is significantly greater than that of dexamethasone-injured mice treated with Cys-Thr-Glu, and the count of late spermatocyte in the spermatogenic phase of dexamethasone-injured mice treated with [Cys-Thr-Glu]3 is significantly greater than that of dexamethasone-injured mice treated with Cys-Thr-Glu (p < 0.01 compared with Cys-Thr-Glu). [Cys-Thr-Glu]3 has unexpected technical effects.

[0038] Table 2 Effects of [Cys-Thr-Glu]3 on the count of spermatocytes 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 Cys-Thr-Glu; c) p < 0.01 compared with mice treated with 5‰ CMC-Na; n = 16.

[0041] The number of spermatozoa in the early, mid and late spermatogenic phases was calculated based on the morphology of spermatogonia and the pathological sections of seminiferous tubules. The data in Table 3 show that dexamethasone significantly reduces the number of spermatozoa in the early spermatogenic phase, significantly reduces the number of spermatozoa in the mid spermatogenic phase and significantly reduces the number of spermatozoa in the late spermatogenic phase (p < 0.01 compared with healthy mice). The data in Table 3 also show that the reversal effect of [Cys-Thr-Glu]3 on the testicular function of dexamethasone-injured mice is manifested in significantly increasing the number of spermatozoa in the early spermatogenic phase, significantly increasing the number of spermatozoa in the mid spermatogenic phase and significantly increasing the number of spermatozoa in the late spermatogenic phase (p > 0.05 compared with healthy mice). [Cys-Thr-Glu]3 has outstanding technical effects.

[0042] The data in Table 3 further showed that the number of spermatozoa in the early spermatogenic phase of dexamethasone-induced injured mice treated with [Cys-Thr-Glu]3 was significantly greater than that of dexamethasone-induced injured mice treated with Cys-Thr-Glu, the number of spermatozoa in the mid-spermatogenic phase of dexamethasone-induced injured mice treated with [Cys-Thr-Glu]3 was significantly greater than that of dexamethasone-induced injured mice treated with Cys-Thr-Glu, and the number of spermatozoa in the late spermatogenic phase of dexamethasone-induced injured mice treated with [Cys-Thr-Glu]3 was significantly greater than that of dexamethasone-induced injured mice treated with Cys-Thr-Glu (p < 0.01 compared with Cys-Thr-Glu). [Cys-Thr-Glu]3 had unexpected technical effects.

[0043] Table 3 Effects of [Cys-Thr-Glu]3 on the number of spermatozoa at 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 Cys-Thr-Glu; c) p < 0.01 compared with mice treated with 5‰ CMC-Na; n = 16.

Claims

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

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

3. Use of the trimer of Cys-Thr-Glu according to claim 1 in the preparation of a therapeutic agent for treating testes damaged by dexamethasone.

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