Tetramer of succinyl-Arg-Gly-Asp-Ser, its synthesis, activity and application

Abstract

The present invention discloses a tetramer of arbutinyl-Arg-Gly-Asp-Ser, and the tetramer has the structure of the following formula. The structure of the following formula means that the tetramer is composed of four arbutinyl-Arg-Gly-Asp-Ser molecules through non-covalent bonds. In addition, the present invention also discloses a preparation method of the tetramer of arbutinyl-Arg-Gly-Asp-Ser, as well as the anti-osteoporosis activity of the tetramer of arbutinyl-Arg-Gly-Asp-Ser and its application in the preparation of anti-osteoporosis drugs.

Description

Technical Field

[0001] The present invention relates to a tetramer of arbutinyl-Arg-Gly-Asp-Ser, and further relates to a preparation method of the tetramer of arbutinyl-Arg-Gly-Asp-Ser, as well as the anti-osteoporosis activity of the tetramer of arbutinyl-Arg-Gly-Asp-Ser and its application in the preparation of anti-osteoporosis drugs. The present invention belongs to the field of biomedicine. Background Art

[0002] The inventor filed a Chinese invention patent application with the application number 202211061697.4 in 2022. Based on virtual screening by molecular docking in this application, it was confirmed that the docking score of arbutinyl-Arg-Gly-Asp-Ser entering the TNF-α active pocket was significantly lower than that of arbutolic acid. The inventor further confirmed based on anti-osteoporosis experimental research in the above application that the anti-osteoporosis activity of arbutinyl-Arg-Gly-Asp-Ser was significantly better than that of arbutolic acid.

[0003] However, at the experimental research level, the lowest oral effective dose of arbutinyl-Arg-Gly-Asp-Ser for anti-osteoporosis was 10 μmol / kg / day, and the oral dose was relatively high. To overcome the above defects, the inventor learned through further research that the molecular aggregates of arbutinyl-Arg-Gly-Asp-Ser were beneficial to reducing the oral effective dose. Therefore, the inventor prepared the molecular aggregates of arbutinyl-Arg-Gly-Asp-Ser. The inventor found that under suitable conditions, arbutinyl-Arg-Gly-Asp-Ser was prepared to form a tetramer. The inventor also found that the anti-osteoporosis activity of the tetramer of arbutinyl-Arg-Gly-Asp-Ser with an oral dose of 1 μmol / kg / day was significantly better than that of arbutinyl-Arg-Gly-Asp-Ser with an oral dose of 10 μmol / kg / day. Based on these findings, the inventor proposed the present invention. Summary of the Invention

[0004] The first object of the present invention is to provide a tetramer of arbutinyl-Arg-Gly-Asp-Ser represented by the following formula structure, and the tetramer is composed of four arbutinyl-Arg-Gly-Asp-Ser molecules through non-covalent bonds.

[0005]

[0006] The second object of the present invention is to provide a preparation method of the tetramer of arbutinyl-Arg-Gly-Asp-Ser with the above structure, and the method includes five steps:

[0007] 1) Prepare Boc-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl;

[0008] 2) Prepare HCl·Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl;

[0009] 3) Using 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate as a condensing agent, condense ursolic acid with HCl·Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl to prepare

[0010] ursolyl-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl;

[0011] 4) Use H2 / Pb to remove the OBzl and NO2 protecting groups of ursolyl-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl to prepare ursolyl-Arg-Gly-Asp-Ser;

[0012] 5) Prepare a tetramer of ursolyl-Arg-Gly-Asp-Ser.

[0013] The third object of the present invention is to confirm the anti-osteoporosis effect of the tetramer of ursolyl-Arg-Gly-Asp-Ser represented by the above structure and its application in the preparation of anti-osteoporosis drugs.

[0014] Compared with the prior art, the beneficial effects of the present invention are:

[0015] The present invention is composed of four ursolyl-Arg-Gly-Asp-Ser molecules through non-covalent bonds to form the ursolyl-Arg-Gly-Asp-Ser. The submission of the present invention overcomes the defect of the high oral dose of the existing ursolyl-Arg-Gly-Asp-Ser molecule for anti-osteoporosis. At the same time, through experiments, it is proved that the tetramer of ursolyl-Arg-Gly-Asp-Ser of the present invention still has significantly better anti-osteoporosis activity than the existing ursolyl-Arg-Gly-Asp-Ser molecule under the condition of a greatly reduced oral dose. Therefore, the present invention provides an effective technical means for its application in the preparation of anti-osteoporosis drugs. Description of the Drawings

[0016] Figure 1 It is the FT-ICR-MS spectrum of the tetramer of ursolyl-Arg-Gly-Asp-Ser.

[0017] Figure 2 It is the qCID spectrum of the tetramer of ursolyl-Arg-Gly-Asp-Ser. Detailed Embodiments

[0018] 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 to the present invention.

[0019] Example 1 Preparation of Boc-Asp(OBzl)-Ser-OBzl

[0020] Take 3.60 g (11 mmol) of Boc-Asp(OBzl), 1.35 g (10 mmol) of N-hydroxybenzotriazole (HOBt), 80 mL of anhydrous tetrahydrofuran, and 2.50 g (12 mmol) of N,N'-dicyclohexylcarbodiimide (DCC), and stir at 0 °C for 0.5 hour to obtain reaction solution A. Add 2.00 g (10 mmol) of HCl·Ser-OBzl to reaction solution A, and adjust the pH value to 9 with N-methylmorpholine (NMM) at 0 °C. Then, stir the reaction mixture at room temperature for 4 h. TLC shows that Tos·Ala-OBzl disappears, and stop stirring. Concentrate the reaction mixture under reduced pressure, and dissolve the residue in 100 mL of ethyl acetate. Filter the obtained solution, and wash the filtrate three times with 30 mL of saturated NaHCO3 solution, three times with 30 mL of saturated NaCl solution, three times with 30 mL of 5% KHSO4 solution, and three times with 30 mL of saturated NaCl solution. Then, dry with anhydrous Na2SO4 for 12 hours. Filter off Na2SO4, and concentrate the filtrate under reduced pressure to obtain a crude product. The crude product is purified by column chromatography (gradient elution with CH2Cl2-MeOH; CH2Cl2 / MeOH, 40:1, UV detection, Rf = 0.35) to obtain 3.75 g (75%) of Boc-Asp(OBzl)-Ser-OBzl as a colorless solid. ESI-MS (m / e): 501 [M+H] + 。

[0021] Example 2 Preparation of HCl·Asp(OBzl)-Ser-OBzl

[0022] Dissolve 4.60 g (10 mmol) of Boc-Asp(OBzl)-Ser-OBzl in 30 mL of ethyl acetate solution of hydrogen chloride (4 M), and stir at 0 °C for 4 h. TLC shows that Boc-Asp(OBzl)-Ser-OBzl disappears, and stop stirring. Concentrate the reaction mixture under reduced pressure, dissolve the residue in 10 mL of anhydrous ethyl acetate and concentrate under reduced pressure. Repeat this operation 3 times. Suspend the residue in 10 mL of petroleum ether, and concentrate the suspension under reduced pressure. Repeat this operation 3 times. The obtained HCl·Asp(OBzl)-Ser-OBzl is a pale yellow solid and is directly used for the subsequent reaction. ESI-MS (m / e): 401 [M+H] +。

[0023] Example 3 Preparation of Boc-Gly-Asp(OBzl)-Ser-OBzl

[0024] Using the method of Example 1, 1.75 g (10 mmol) of Boc-Gly and 4.00 g (10 mmol) of HCl·Asp(OBzl)-Ser-OBzl were used to obtain a pale yellow crude product of Boc-Gly-Asp(OBzl)-Ser-OBzl. This crude product was purified by column chromatography (gradient elution with CH2Cl2-MeOH; CH2Cl2 / MeOH, 40:1, visualized by UV, Rf = 0.35) to obtain 3.07 g (55%) of Boc-Gly-Asp(OBzl)-Ser-OBzl as a colorless solid. ESI-MS (m / e): 558 [M+H] + 。

[0025] Example 4 Preparation of HCl·Gly-Asp(OBzl)-Ser-OBzl

[0026] Using the method of Example 2, HCl·Gly-Asp(OBzl)-Ser-OBzl was prepared from 5.20 g (10 mmol) of Boc-Gly-Asp(OBzl)-Ser-OBzl, which was a pale yellow solid and was directly used in the subsequent reaction. ESI-MS (m / e): 458 [M+H] + 。

[0027] Example 5 Preparation of Boc-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl

[0028] Using the method of Example 1, 3.20 g (10 mmol) of Boc-Arg(NO2) and 4.60 g (10 mmol) of HCl·Gly-Asp(OBzl)-Ser-OBzl were used to obtain a pale yellow crude product of Boc-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl. This crude product was purified by column chromatography (gradient elution with CH2Cl2-MeOH; CH2Cl2 / MeOH, 15:1, visualized by UV, Rf = 0.20) to obtain 3.81 g (50%) of Boc-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl as a colorless solid. ESI-MS (m / e): 762 [M+H] + 。

[0029] Example 6 Preparation of HCl·Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl

[0030] The method of Example 2 was used to prepare HCl·Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl from 5.20 g (10 mmol) of Boc-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl. It was a pale yellow solid and was directly used in the subsequent reaction. ESI-MS (m / e): 758 [M+H] + .

[0031] Example 7 Preparation of Ursolyl-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl

[0032] Weighed 0.55 g (1.2 mmol) of ursolic acid, 0.46 g (1.2 mmol) of 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, and 10 mL of anhydrous N,N-dimethylformamide. They were stirred and dissolved at room temperature for about 4 hours to obtain reaction solution A. Added 0.70 g (1.0 mmol) of HCl·Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl to reaction solution A, and adjusted the pH to 9 with anhydrous N,N-diisopropylethylamine at 0 °C. Then, the reaction mixture was stirred at room temperature for 36 hours. During the stirring, the pH value was continuously monitored, and anhydrous N,N-diisopropylethylamine was added to maintain the pH value at 8 - 9. The stirring was terminated. The reaction solution was poured into 100 mL of saturated NaCl solution at 0 °C, and allowed to stand. The suspension was washed three times with 30 mL of saturated NaHCO3 solution, three times with 30 mL of saturated NaCl solution, three times with 30 mL of 5% KHSO4 solution, and three times with 30 mL of saturated NaCl solution. Then, it was dried with anhydrous Na2SO4 for 12 hours. The Na2SO4 was filtered off, and the filtrate was concentrated under reduced pressure to obtain a crude product of ursolyl-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl as a pale yellow solid. This crude product was purified by column chromatography (gradient elution with CH2Cl2-MeOH; CH2Cl2 / MeOH, 15:1, UV visualization; Rf = 0.20) to obtain 0.56 g of ursolyl-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl (51%), which was a colorless solid. ESI-MS (m / e): 1098 [M+H] + .

[0033] Example 8 Preparation of Ursolyl-Arg-Gly-Asp-Ser

[0034] Dissolve 1.10 g (1.0 mmol) of arbutinyl-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl in 100 mL of methanol. Add 110 mg of palladium-carbon to the resulting solution. After evacuating the air from the obtained suspension, stir at room temperature and hydrogenate for 12 hours by passing hydrogen. TLC shows the disappearance of arbutinyl-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl, and terminate the hydrogenation. Filter off the palladium-carbon from the suspension, and concentrate the filtrate under reduced pressure to obtain the crude product of arbutinyl-Arg-Gly-Asp-Ser. This crude product is purified by column chromatography. The silica gel column is eluted with a gradient of CH2Cl2 / MeOH = 15 / 1 plus 2% HCO2H to obtain 0.55 g (63%) of arbutinyl-Arg-Gly-Asp-Ser as a white solid. TLC (ethyl acetate / distilled water / glacial acetic acid, 5 / 1 / 1; developed with iodine vapor; Rf = 0.30). FT-MS 872.55326[M+H] + (872.54972); Mp: 300 - 301 °C; IR / cm -1 = 3272, 2926, 2865, 1649, 1601, 1516, 1454, 1377, 1188, 1032, 997, 915, 763, 661; 1 1H NMR (300 MHz, DMSO-d6) δ / ppm = 10.08 (s, 1H), 8.63 (d, J = 8.4 Hz, 1H), 8.49 (s, 1H), 7.30 (d, J = 7.2 Hz, 1H), 7.17 (d, J = 7.2 Hz, 1H), 6.99 (s, 2H), 5.21 (s, 1H), 4.40 (m, 1H), 4.28 - 4.22 (m, 2H), 3.99 (m, 1H), 3.78 (dd, J1 = 16.2 Hz, J2 = 5.7 Hz, 1H), 3.67 - 3.54 (m, 3H), 3.17 - 3.13 (m, 1H), 2.98 (s, 2H), 2.59 (dd, J1 = 16.5 Hz, J2 = 5.4 Hz, 1H), 2.38 (dd, J1 = 18.3 Hz, J2 = 4.8 Hz, 1H), 2.12 (d, J = 10.8 Hz, 1H), 1.98 - 1.85 (m, 2H), 1.85 - 1.75 (m, 2H), 1.67 - 1.61 (m, 5H), 1.55 - 1.31 (m, 12H), 1.29 - 1.15 (m, 3H), 1.02 (s, 3H), 0.95 - 0.82 (m, 15H), 0.67 (s, 3H), 0.64 (s, 1H), 0.58 (s, 3H); 1313C NMR (75 MHz, DMSO-d6) δ / ppm = 176.71, 175.15, 173.14, 171.01, 168.88, 164.64, 138.36, 125.80, 77.29, 62.57, 55.65, 55.27, 52.67, 50.29, 49.01, 47.53, 47.18, 42.03, 39.58, 39.27, 38.84, 38.70, 37.80, 37.20, 36.98, 33.16, 30.84, 28.72, 27.84, 27.47, 24.02, 23.79, 23.36, 21.54, 18.46, 17.49, 16.93, 16.52, 15.68。

[0035] Example 9 Preparation of the tetramer of arbutinyl-Arg-Gly-Asp-Ser

[0036] Dissolve 100 mg of arbutinyl-Arg-Gly-Asp-Ser 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 tetramer of arbutinyl-Arg-Gly-Asp-Ser, namely [arbutinyl-Arg-Gly-Asp-Ser]4. The structure of [arbutinyl-Arg-Gly-Asp-Ser]4 was confirmed by ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Figure 1 A peak of [arbutinyl-Arg-Gly-Asp-Ser]-H at 870.52963 (theoretical value 870.53352) was given, a peak of divalent [arbutinyl-Arg-Gly-Asp-Ser]-H at 1306.28275 was given, and according to the mass-to-charge ratio, this divalent ion corresponded to the trimer [arbutinyl-Arg-Gly-Asp-Ser]3-H, and a peak of divalent [arbutinyl-Arg-Gly-Asp-Ser]-H at 1742.04074 was given, and according to the mass-to-charge ratio, this divalent ion corresponded to the tetramer [arbutinyl-Arg-Gly-Asp-Ser]4-H.

[0037] To clarify the relationship between the peak of [arbutinyl-Arg-Gly-Asp-Ser]-H and the peak of [arbutinyl-Arg-Gly-Asp-Ser]3-H and the peak of [arbutinyl-Arg-Gly-Asp-Ser]4-H, the qCID spectrum of [arbutinyl-Arg-Gly-Asp-Ser]4-H was measured. Figure 2The qCID spectrum of the peak of [Arbutinyl-Arg-Gly-Asp-Ser]4-H gave a peak of the divalent ion trimer [Arbutinyl-Arg-Gly-Asp-Ser]3-H with a mass number of 1306.50756, and gave a peak of [Arbutinyl-Arg-Gly-Asp-Ser]-H with a mass number of 870.52540 (the theoretical value is 870.53352). That is to say, both [Arbutinyl-Arg-Gly-Asp-Ser]3-H and [Arbutinyl-Arg-Gly-Asp-Ser]-H are cleavage products of [Arbutinyl-Arg-Gly-Asp-Ser]4, that is, [Arbutinyl-Arg-Gly-Asp-Ser]4 is the only form in which Arbutinyl-Arg-Gly-Asp-Ser exists.

[0038] Example 10 evaluated the anti-osteoporosis effect of the tetramer of Arbutinyl-Arg-Gly-Asp-Ser

[0039] Arbutin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. SPF-grade male ICR mice (25±2 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. An osteoporosis model was induced by bilateral ovariectomy in ICR female mice. The oral doses of Arbutinyl-Arg-Gly-Asp-Ser were 100 μmol / kg / day, 10 μmol / kg / day and 1 μmol / kg / day; the oral doses of [Arbutinyl-Arg-Gly-Asp-Ser]4 were 10 μmol / kg / day, 1 μmol / kg / day and 0.1 μmol / kg / day; the negative control was CMC-Na.

[0040] When making the model, the mice were induced to anesthesia with an anesthesia machine. That is, the mice were placed face up on the mouse board, the mouth and nose were aligned with the ventilation valve, and the mice were continuously anesthetized with isoflurane gas. After that, the lower abdomen of the mice was disinfected with iodophor and alcohol, the skin of the lower abdomen was cut open with surgical scissors, the muscle layer was cut open along the white line of the abdomen to expose the abdominal cavity, the bladder was found under the lower abdominal fat, the uterus in a "Y" shape was found under the bladder, the bilateral fallopian tubes were found along the uterus, the ovaries were found at the blind ends of the bilateral fallopian tubes. The ovaries were in the shape of small cauliflower and were wrapped with fat. The fallopian tubes were ligated away from the uterus, and the bilateral ovaries were removed. A drop of penicillin solution was dropped at each ligation site. After putting it back into the abdominal cavity, a drop of penicillin solution was dropped before and after suturing the muscle layer, and after suturing the skin layer, the skin suture was disinfected with alcohol and iodophor again. The functions of penicillin, alcohol and iodophor were to prevent infection. After removing the anesthesia device, the mice woke up quickly.

[0041] After the mice in the sham operation group (sham operation) were induced to anesthesia with an anesthesia machine, they were placed on their backs on a mouse board, with their mouths and noses aligned with the ventilation valve, and continuously anesthetized with isoflurane gas. Then, the lower abdomen of the mice was disinfected with iodophor and alcohol. The skin of the lower abdomen was cut open with surgical scissors, and the muscle layer was cut open along the linea alba to expose the abdominal cavity. The bladder was found under the lower abdominal fat, and the "Y"-shaped uterus was found under the bladder. The two fallopian tubes were found along the uterus, and the ovaries were found at the blind ends of the two fallopian tubes. The ovaries were in the shape of small cauliflower and were wrapped in fat. The fallopian tubes were not ligated, nor were the ovaries removed. After putting them back into the abdominal cavity, a drop of penicillin solution was dropped before and after suturing the muscle layer. After suturing the skin layer, the skin suture was disinfected with alcohol and iodophor again. The roles of penicillin, alcohol, and iodophor were to prevent infection. After removing the anesthesia device, the mice woke up quickly.

[0042] The mice were allowed to recover for seven days after the operation and then randomly grouped. Among them, the mice in the ovariectomy group and the sham operation group were intragastrically administered 5‰ CMC-Na solution according to their body weight every day for 28 consecutive days, and their body weights were recorded at the same time. The mice in the Arg-Gly-Asp-Ser treatment group were intragastrically administered Arg-Gly-Asp-Ser according to their body weight at doses of 100 μmol / kg / day, 10 μmol / kg / day, and 1 μmol / kg / day for 28 consecutive days, and their body weights were recorded at the same time. The mice in the [Arg-Gly-Asp-Ser]4 treatment group were intragastrically administered [Arg-Gly-Asp-Ser]4 according to their body weight at doses of 10 μmol / kg / day, 1 μmol / kg / day, and 0.1 μmol / kg / day for 28 consecutive days, and their body weights were recorded at the same time.

[0043] The femurs of the mice were scanned by Bruker skyscan 1276 Micro-CT to calculate the trabecular bone density of the mice. The region of interest at the distal end of the femur with the same height was selected to calculate the trabecular parameters and analyze the trabecular structure of the femur. The trabeculae of the ovariectomized mice were quantitatively analyzed according to six indexes, namely trabecular bone density, bone volume fraction, trabecular number, trabecular thickness, trabecular separation, and trabecular pattern factor. The results of each index were expressed as mean ± SD. The SD values were first analyzed by variance using SPSS software to test the homogeneity of variance, and the t-test was used for statistical comparison between groups.

[0044] In terms of indicators such as trabecular bone density of mouse femurs, arbutinyl-Arg-Gly-Asp-Ser can improve osteoporosis in ovariectomized mice by intragastric administration at doses of 100 μmol / kg and 10 μmol / kg for 28 consecutive days. That is, the lowest effective dose of arbutinyl-Arg-Gly-Asp-Ser for treating osteoporosis is 10 μmol / kg / day. In addition, the lowest effective dose of [Arbutinyl-Arg-Gly-Asp-Ser]4 for treating osteoporosis is 1 μmol / kg / day. The specific results are shown in Tables 1 - 6. The data in the tables show that the present invention has unexpected technical effects.

[0045] Table 1 Trabecular bone density of femurs of mice treated with arbutinyl-Arg-Gly-Asp-Ser and the tetramer

[0046]

[0047]

[0048] a) P < 0.01 compared with ovariectomized mice and mice treated with arbutinyl-Arg-Gly-Asp-Ser (1 μmol / kg / day), P < 0.05 compared with mice treated with arbutinyl-Arg-Gly-Asp-Ser (10 μmol / kg / day); b) P < 0.01 compared with ovariectomized mice, P < 0.05 compared with mice treated with arbutinyl-Arg-Gly-Asp-Ser (1 μmol / kg / day); c) P > 0.05 compared with the ovariectomized group; d) P < 0.01 compared with ovariectomized mice and mice treated with arbutinyl-Arg-Gly-Asp-Ser (10 μmol / kg / day), P < 0.05 compared with mice treated with [Arbutinyl-Arg-Gly-Asp-Ser]4 (1 μmol / kg); e) P < 0.01 compared with ovariectomized mice and mice treated with arbutinyl-Arg-Gly-Asp-Ser (1 μmol / kg / day), P < 0.05 compared with mice treated with [Arbutinyl-Arg-Gly-Asp-Ser]4 (0.1 μmol / kg); f) P > 0.05 compared with ovariectomized mice; n = 10.

[0049] Table 2 Bone volume fraction of femurs of mice treated with arbutinyl-Arg-Gly-Asp-Ser and the tetramer

[0050]

[0051] a) P < 0.01 compared with ovariectomized mice and mice treated with Arg-Gly-Asp-Ser (1 μmol / kg / day), P < 0.05 compared with mice treated with Arg-Gly-Asp-Ser (10 μmol / kg / day); b) P < 0.01 compared with ovariectomized mice, P < 0.05 compared with mice treated with Arg-Gly-Asp-Ser (1 μmol / kg / day); c) P > 0.05 compared with the ovariectomized group; d) P < 0.01 compared with ovariectomized mice and mice treated with Arg-Gly-Asp-Ser (10 μmol / kg / day), P < 0.05 compared with mice treated with [Arg-Gly-Asp-Ser]4 (1 μmol / kg); e) P < 0.01 compared with ovariectomized mice and mice treated with Arg-Gly-Asp-Ser (1 μmol / kg / day), P < 0.05 compared with mice treated with [Arg-Gly-Asp-Ser]4 (0.1 μmol / kg); f) P > 0.05 compared with ovariectomized mice; n = 10.

[0052] Table 3 Trabecular bone number of femurs in mice treated with Arg-Gly-Asp-Ser and its tetramer

[0053]

[0054] a) P < 0.01 compared with ovariectomized mice and mice treated with Arg-Gly-Asp-Ser (1 μmol / kg / day), P < 0.05 compared with mice treated with Arg-Gly-Asp-Ser (10 μmol / kg / day); b) P < 0.01 compared with ovariectomized mice, P < 0.05 compared with mice treated with Arg-Gly-Asp-Ser (1 μmol / kg / day); c) P > 0.05 compared with the ovariectomized group; d) P < 0.01 compared with ovariectomized mice and mice treated with Arg-Gly-Asp-Ser (10 μmol / kg / day), P < 0.05 compared with mice treated with [Arg-Gly-Asp-Ser]4 (1 μmol / kg); e) P < 0.01 compared with ovariectomized mice and mice treated with Arg-Gly-Asp-Ser (1 μmol / kg / day), P < 0.05 compared with mice treated with [Arg-Gly-Asp-Ser]4 (0.1 μmol / kg); f) P > 0.05 compared with ovariectomized mice; n = 10.

[0055] Table 4 Trabecular bone thickness of femurs in mice treated with Arg-Gly-Asp-Ser and its tetramer

[0056]

[0057] a) P < 0.01 compared with ovariectomized mice and mice treated with succinyl-Arg-Gly-Asp-Ser (1 μmol / kg / day), P < 0.05 compared with mice treated with succinyl-Arg-Gly-Asp-Ser (10 μmol / kg / day); b) P < 0.01 compared with ovariectomized mice, P < 0.05 compared with mice treated with succinyl-Arg-Gly-Asp-Ser (1 μmol / kg / day); c) P > 0.05 compared with the ovariectomized group; d) P < 0.01 compared with ovariectomized mice and mice treated with succinyl-Arg-Gly-Asp-Ser (10 μmol / kg / day), P < 0.05 compared with mice treated with [succinyl-Arg-Gly-Asp-Ser]4 (1 μmol / kg); e) P < 0.01 compared with ovariectomized mice and mice treated with succinyl-Arg-Gly-Asp-Ser (1 μmol / kg / day), P < 0.05 compared with mice treated with [succinyl-Arg-Gly-Asp-Ser]4 (0.1 μmol / kg); f) P > 0.05 compared with ovariectomized mice; n = 10.

[0058] Table 5 Trabecular bone separation in femurs of mice treated with succinyl-Arg-Gly-Asp-Ser and tetramers

[0059]

[0060] a) P < 0.01 compared with ovariectomized mice and mice treated with succinyl-Arg-Gly-Asp-Ser (1 μmol / kg / day), P < 0.05 compared with mice treated with succinyl-Arg-Gly-Asp-Ser (10 μmol / kg / day); b) P < 0.01 compared with ovariectomized mice, P < 0.05 compared with mice treated with succinyl-Arg-Gly-Asp-Ser (1 μmol / kg / day); c) P > 0.05 compared with the ovariectomized group; d) P < 0.01 compared with ovariectomized mice and mice treated with succinyl-Arg-Gly-Asp-Ser (10 μmol / kg / day), P < 0.05 compared with mice treated with [succinyl-Arg-Gly-Asp-Ser]4 (1 μmol / kg); e) P < 0.01 compared with ovariectomized mice and mice treated with succinyl-Arg-Gly-Asp-Ser (1 μmol / kg / day), P < 0.05 compared with mice treated with [succinyl-Arg-Gly-Asp-Ser]4 (0.1 μmol / kg); f) P > 0.05 compared with ovariectomized mice; n = 10.

[0061] Table 6 Osteocalcin pattern factor in the femur of mice treated with arbutinyl-Arg-Gly-Asp-Ser and its tetramer

[0062]

[0063] a) P < 0.01 compared with ovariectomized mice and mice treated with arbutinyl-Arg-Gly-Asp-Ser (1 μmol / kg / day), P < 0.05 compared with mice treated with arbutinyl-Arg-Gly-Asp-Ser (10 μmol / kg / day); b) P < 0.01 compared with ovariectomized mice, P < 0.05 compared with mice treated with arbutinyl-Arg-Gly-Asp-Ser (1 μmol / kg / day); c) P > 0.05 compared with the ovariectomized group; d) P < 0.01 compared with ovariectomized mice and mice treated with arbutinyl-Arg-Gly-Asp-Ser (10 μmol / kg / day), P < 0.05 compared with mice treated with [arbutinyl-Arg-Gly-Asp-Ser]4 (1 μmol / kg); e) P < 0.01 compared with ovariectomized mice and mice treated with arbutinyl-Arg-Gly-Asp-Ser (1 μmol / kg / day), P < 0.05 compared with mice treated with [arbutinyl-Arg-Gly-Asp-Ser]4 (0.1 μmol / kg); f) P > 0.05 compared with ovariectomized mice; n = 10.

Claims

1. A tetramer of arbutinyl-Arg-Gly-Asp-Ser, characterized in that, The described tetramer is composed of four arbutinyl-Arg-Gly-Asp-Ser molecules through non-covalent bonds, and the structural formula of the tetramer is as follows:

2. The preparation method of the tetramer of arbutinyl-Arg-Gly-Asp-Ser according to claim 1, characterized in that, The method comprises the following steps: 1) Prepare Boc-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl; 2) Prepare HCl·Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl; 3) Prepare arbutinyl-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl; 4) Prepare arbutinyl-Arg-Gly-Asp-Ser; 5) Prepare the tetramer of arbutinyl-Arg-Gly-Asp-Ser.

3. Use of the tetramer of arbutinyl-Arg-Gly-Asp-Ser according to claim 1 in the preparation of anti-osteoporosis drugs.

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