Dimer of arbutinoyl-Tyr-Gly-Phe-Gly-Gly, its synthesis and application

Abstract

The present invention discloses a dimer of arbutinyl-Tyr-Gly-Phe-Gly-Gly, and the dimer has the structure of the following formula. The structure of the following formula means that the dimer is composed of two arbutinyl-Tyr-Gly-Phe-Gly-Gly molecules through non-covalent bonds. Experiments have proved that the dimer of arbutinyl-Tyr-Gly-Phe-Gly-Gly of the present invention has excellent anti-osteoporosis activity. Its anti-osteoporosis effect is significantly stronger than the existing anti-osteoporosis activity of arbutinyl-Tyr-Gly-Phe-Gly-Gly. Therefore, it is proposed that the present invention provides an effective technical means for the application in the preparation of anti-osteoporosis drugs.

Description

Technical Field

[0001] The present invention relates to a dimer of arbutin-Tyr-Gly-Phe-Gly-Gly, and further relates to a preparation method of the dimer of arbutin-Tyr-Gly-Phe-Gly-Gly, as well as the anti-osteoporosis activity of the dimer of arbutin-Tyr-Gly-Phe-Gly-Gly and its application in the preparation of anti-osteoporosis drugs. The present invention belongs to the field of biomedicine. Background Art

[0002] The inventor submitted a Chinese patent application with the application number 202211069067.1 in 2022. Based on virtual screening by molecular docking, it was confirmed that the docking score of arbutin-Tyr-Gly-Phe-Gly-Gly entering the TNF-α active pocket was significantly lower than that of ursolic acid. The inventor further confirmed based on anti-osteoporosis experimental research in the application of 202211069067.1 that the anti-osteoporosis activity of arbutin-Tyr-Gly-Phe-Gly-Gly was significantly better than that of ursolic acid.

[0003] However, at the experimental research level, the oral effective dose of arbutin-Tyr-Gly-Phe-Gly-Gly for anti-osteoporosis was 10 μmol / kg / day, with a relatively high dose. The inventor conjectured that the molecular aggregate of arbutin-Tyr-Gly-Phe-Gly-Gly might be beneficial for reducing the oral effective dose. Therefore, the inventor prepared the molecular aggregate of arbutin-Tyr-Gly-Phe-Gly-Gly. Through further research and experimental analysis, the inventor found that under suitable conditions, arbutin-Tyr-Gly-Phe-Gly-Gly was prepared to form a dimer. The inventor also found that the anti-osteoporosis activity of the dimer of arbutin-Tyr-Gly-Phe-Gly-Gly at an oral dose of 1 μmol / kg / day was significantly better than that of arbutin-Tyr-Gly-Phe-Gly-Gly at 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 dimer of arbutin-Tyr-Gly-Phe-Gly-Gly composed of two arbutin-Tyr-Gly-Phe-Gly-Gly molecules through non-covalent bonds, and the dimer has the following structure:

[0005]

[0006] The second object of the present invention is to provide a method for preparing a dimer of arbutinoyl-Tyr-Gly-Phe-Gly-Gly of the said structure, and this method comprises five steps:

[0007] 1) Prepare Boc-Tyr-Gly-Phe-Gly-Gly-OBzl;

[0008] 2) Prepare HCl·Tyr-Gly-Phe-Gly-Gly-OBzl;

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

[0010] 4) Remove the OBzl protecting group of arbutinoyl-Tyr-Gly-Phe-Gly-Gly-OBzl by H2 / Pb to prepare arbutinoyl-Tyr-Gly-Phe-Gly-Gly;

[0011] 5) Prepare a dimer of arbutinoyl-Tyr-Gly-Phe-Gly-Gly.

[0012] The third object of the present invention is to evaluate the anti-osteoporosis activity of the dimer of arbutinoyl-Tyr-Gly-Phe-Gly-Gly represented by the said structure and its application in the preparation of anti-osteoporosis drugs.

[0013] Compared with the prior art, the beneficial effects of the present invention are:

[0014] On the basis of arbutinoyl-Tyr-Gly-Phe-Gly-Gly, the present invention conducts molecular aggregation, and a dimer of arbutinoyl-Tyr-Gly-Phe-Gly-Gly is formed by two arbutinoyl-Tyr-Gly-Phe-Gly-Gly molecules through non-covalent bonds. Experiments have found that such a dimer of arbutinoyl-Tyr-Gly-Phe-Gly-Gly is beneficial to reducing the oral effective dose and overcomes the defect that the oral dose of arbutinoyl-Tyr-Gly-Phe-Gly-Gly for anti-osteoporosis in the prior art is too high. At the same time, the molecular dimer aggregate of the present invention has more excellent anti-osteoporosis activity compared with the existing arbutinoyl-Tyr-Gly-Phe-Gly-Gly. Therefore, the present invention provides a more effective technical means for its application in the preparation of anti-osteoporosis drugs. Description of the Drawings

[0015] Figure 1FT-ICR-MS spectrum of the dimer of arbutinoyl-Tyr-Gly-Phe-Gly-Gly

[0016] Figure 2 qCID spectrum of the dimer of arbutinoyl-Tyr-Gly-Phe-Gly-Gly Detailed implementation mode

[0017] To further illustrate the present invention, a series of examples are given below. These examples are entirely illustrative and are only used to specifically describe the present invention and should not be construed as a limitation to the present invention.

[0018] Example 1 Preparation of Boc-Gly-Gly-OBzl

[0019] Take 1.75 g (10 mmol) of Boc-Gly, 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), stir at 0 °C for 0.5 hour to obtain reaction solution A. Add 3.40 g (10 mmol) of Tos·Gly-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·Gly-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, concentrate the filtrate under reduced pressure to obtain 3.15 g (98%) of Boc-Gly-Gly-OBzl, which is a pale yellow solid. TLC (petroleum ether / ethyl acetate, 4 / 1; UV visualization; Rf = 0.25), ESI-MS (m / e): 323 [M+H] + 。

[0020] Example 2 Preparation of HCl·Gly-Gly-OBzl

[0021] 3.00 g (9.3 mmol) of Boc-Gly-Gly-OBzl was dissolved in 30 mL of ethyl acetate solution of hydrogen chloride (4 M), and stirred at 0 °C for 4 hours. TLC showed the disappearance of Boc-Gly-Gly-OBzl, and the stirring was terminated. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in 10 mL of anhydrous ethyl acetate and concentrated under reduced pressure. This operation was repeated 3 times. The residue was suspended in 10 mL of petroleum ether, and the suspension was concentrated under reduced pressure. This operation was repeated 3 times. The obtained HCl·Gly-Gly-OBzl was a pale yellow solid and was directly used for the subsequent reaction. ESI-MS (m / e): 223 [M+H] + 。

[0022] Example 3 Preparation of Boc-Gly-Phe-OBzl

[0023] Using the method of Example 1, 4.08 g (99%) of Boc-Gly-Phe-OBzl was obtained from 1.75 g (10 mmol) of Boc-Gly and 2.90 g (10 mmol) of HCl·Phe-OBzl as a colorless solid. TLC (CH2Cl2 / MeOH, 40 / 1; UV visualization; Rf = 0.35), ESI-MS (m / e): 413 [M+H] + 。

[0024] Example 4 Preparation of Boc-Gly-Phe

[0025] 4.00 g (9.7 mmol) of Boc-Gly-Phe-OBzl was dissolved in 100 mL of methanol. 400 mg of palladium on carbon was added to the obtained solution. The obtained suspension was evacuated and stirred at room temperature and hydrogenolyzed with hydrogen for 12 hours. TLC showed the disappearance of Boc-Gly-Phe-OBzl, and the hydrogenolysis was terminated. The palladium on carbon was removed from the suspension, and the filtrate was concentrated under reduced pressure to obtain 3.10 g (99%) of Boc-Gly-Phe as a colorless solid. ESI-MS (m / e): 323 [M+H] + 。

[0026] Example 5 Preparation of Boc-Gly-Phe-Gly-Gly-OBzl

[0027] Using the method of Example 1, a crude product of Boc-Gly-Phe-Gly-Gly-OBzl, pale yellow in color, was obtained from 3.00 g (9.3 mmol) of Boc-Gly-Phe and 2.50 g (9.7 mmol) of Tos·Gly-Gly-OBzl. This crude product was purified by column chromatography (gradient elution with CH2Cl2-MeOH; CH2Cl2 / MeOH, 40:1, visualized by UV, Rf = 0.25) to obtain 4.39 g (90%) of Boc-Gly-Phe-Gly-Gly-OBzl as a colorless solid. ESI-MS (m / e): 562 [M+Cl] - 。

[0028] Example 6 Preparation of HCl·Gly-Phe-Gly-Gly-OBzl

[0029] Using the method of Example 2, HCl·Gly-Phe-Gly-Gly-OBzl was prepared from 4.90 g (9.3 mmol) of Boc-Gly-Phe-Gly-Gly-OBzl as a pale yellow solid and was directly used in the subsequent reaction. ESI-MS (m / e): 427 [M+H] + 。

[0030] Example 7 Preparation of Boc-Tyr-Gly-Phe-Gly-Gly-OBzl

[0031] Using the method of Example 1, 4.74 g (74%) of Boc-Tyr-Gly-Phe-Gly-Gly-OBzl as a colorless solid was obtained from 2.60 g (9.3 mmol) of Boc-Tyr and 4.30 g (9.3 mmol) of HCl·Gly-Phe-Gly-Gly-OBzl. TLC (CH2Cl2 / MeOH, 20 / 1; visualized by UV; Rf = 0.20), ESI-MS (m / e): 725 [M+Cl] - 。

[0032] Example 8 Preparation of HCl·Tyr-Gly-Phe-Gly-Gly-OBzl

[0033] Using the method of Example 2, HCl·Tyr-Gly-Phe-Gly-Gly-OBzl was prepared from 4.00 g (5.8 mmol) of Boc-Tyr-Gly-Phe-Gly-Gly-OBzl as a pale yellow solid and was directly used in the subsequent reaction. ESI-MS (m / e): 591 [M+H] + 。

[0034] Example 9 Preparation of Arbutinyl-Tyr-Gly-Phe-Gly-Gly-OBzl

[0035] Weigh 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. Stir and dissolve at room temperature for about 4 hours to obtain reaction solution A. Add 0.63 g (1.0 mmol) of HCl·Tyr-Gly-Phe-Gly-Gly-OBzl to reaction solution A, and adjust the pH to 9 with anhydrous N,N-diisopropylethylamine at 0 °C. Then, stir the reaction mixture at room temperature for 36 hours. During the stirring, continuously monitor the pH value and add anhydrous N,N-diisopropylethylamine to maintain the pH value between 8 and 9. Stop stirring. Pour the reaction solution into 100 mL of saturated NaCl solution at 0 °C, let it stand, and wash the suspension 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 it with anhydrous Na₂SO₄ for 12 hours. Filter out Na₂SO₄, and concentrate the filtrate under reduced pressure to obtain a crude product of ursolyl-Tyr-Gly-Phe-Gly-Gly as a pale yellow solid. This crude product is purified by column chromatography (gradient elution with CH₂Cl₂-MeOH; CH₂Cl₂ / MeOH, 20:1, UV visualization; Rf = 0.20) to obtain 0.38 g (35%) of ursolyl-Tyr-Gly-Phe-Gly-Gly (35%) as a colorless solid. ESI-MS (m / e): 1098 [M+H] + 。

[0036] Example 10 Preparation of Ursolyl-Tyr-Gly-Phe-Gly-Gly

[0037] Using the method of Example 4, a crude product of ursolyl-Tyr-Gly-Phe-Gly-Gly is obtained from 1.00 g (1.0 mmol) of ursolyl-Tyr-Gly-Phe-Gly-Gly-OBzl as a colorless solid. This solid is purified by column chromatography. The silica gel column is eluted with a gradient of CH₂Cl₂ / MeOH = 15 / 1 plus 2% HCO₂H to obtain 0.66 g (71%) of ursolyl-Tyr-Gly-Phe-Gly-Gly as a colorless solid. TLC (ethyl acetate / distilled water / glacial acetic acid, 10 / 1 / 1; iodine vapor visualization; Rf = 0.30). FT-MS 960.55008 [M+Na] + (960.54624); Mp: 185 - 186 °C; IR / cm -1= 3303, 2925, 2868, 1649, 1514, 1453, 1377, 1226, 1105, 1028, 996, 830, 744, 699, 662; 1 HNMR(300 MHz, DMSO-d6) δ / ppm = 12.45 (s, 1H), 9.08 (s, 1H), 8.27 (s, 1H), 8.34 (t, J = 5.7 Hz, 1H), 8.10 (d, J = 8.1 Hz, 1H), 8.03 (t, J = 6.0 Hz, 1H), 7.92 (t, J = 5.4 Hz, 1H), 7.27 - 7.20 (m, 5H), 7.17 (d, J = 8.1 Hz, 1H), 7.03 (d, J = 8.1 Hz, 2H), 6.60 (d, J = 8.1 Hz, 2H), 5.00 (s, 1H), 4.53 (m, 1H), 4.27 (s, 1H), 4.17 (m, 1H), 3.74 (m, 6H), 3.60 (dd, J1 = 17.1 Hz, J2 = 5.1 Hz, 1H), 3.16 (s, 1H), 3.06 (dd, J1 = 13.8 Hz, J2 = 4.8 Hz, 1H), 2.97 (t, J = 7.2 Hz, 1H), 2.85 - 2.67 (m, 4H), 2.01 (d, J = 10.8 Hz, 1H), 1.85 (m, 1H), 1.73 - 1.57 (m, 4H), 1.49 - 1.23 (m, 13H), 1.05 (d, J = 8.7 Hz, 1H), 0.92 - 0.58 (m, 11H), 0.78 - 0.74 (m, 7H), 0.68 (s, 4H), 0.59 (d, J = 9.3 Hz, 1H), 0.19 (s, 3H); 13 C NMR(125 MHz, DMSO-d6) δ / ppm = 176.81, 172.46, 171.64, 171.48, 169.42, 168.97, 156.42, 138.62, 138.21, 130.59, 129.59, 128.57, 128.50, 126.71, 125.03, 115.27, 77.38, 55.79, 55.34, 54.87, 54.52, 52.48, 49.07, 47.56, 46.88, 42.33, 41.95, 41.14, 41.11, 39.33, 39.02, 38.84, 38.74, 38.10, 36.93, 36.63, 36.58, 32.77, 30.81, 28.76, 27.67, 27.49, 23.96, 23.71, 23.28, 21.55, 18.37, 17.55, 16.55, 16.44, 15.66, 0.57.

[0038] Example 11 Preparation of the dimer of arbutinyl-Tyr-Gly-Phe-Gly-Gly

[0039] Dissolve 100 mg of arbutinyl-Tyr-Gly-Phe-Gly-Gly powder in 5 mL of ultrapure water. The resulting solution is vortexed for 15 minutes first and then sonicated in an ultrasonic cleaner 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 dimer of arbutinyl-Tyr-Gly-Phe-Gly-Gly, i.e., [arbutinyl-Tyr-Gly-Phe-Gly-Gly]2. The structure of [arbutinyl-Tyr-Gly-Phe-Gly-Gly]2 is confirmed by ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Figure 1 The FT-ICR-MS spectrum of [arbutinyl-Tyr-Gly-Phe-Gly-Gly]2 gives a peak of [arbutinyl-Tyr-Gly-Phe-Gly-Gly]+H at 938.59011 (theoretical value: 938.56376) and a peak of [arbutinyl-Tyr-Gly-Phe-Gly-Gly]2+NH4 at 1893.26649 (theoretical value: 1893.14678).

[0040] To clarify the relationship between the peak of [arbutinyl-Tyr-Gly-Phe-Gly-Gly]+H and the peak of [arbutinyl-Tyr-Gly-Phe-Gly-Gly]2+NH4, the qCID spectrum of the peak of [arbutinyl-Tyr-Gly-Phe-Gly-Gly]2+NH4 was measured. Figure 2 The qCID spectrum of [arbutinyl-Tyr-Gly-Phe-Gly-Gly]2+NH4 gives a peak of [arbutinyl-Tyr-Gly-Phe-Gly-Gly]+H at 938.57907 (theoretical value: 938.56376). That is to say, the peak of [arbutinyl-Tyr-Gly-Phe-Gly-Gly]+H comes from the cleavage of [arbutinyl-Tyr-Gly-Phe-Gly-Gly]2+NH4, i.e., [arbutinyl-Tyr-Gly-Phe-Gly-Gly]2 is the only form in which arbutinyl-Tyr-Gly-Phe-Gly-Gly exists.

[0041] Example 12 Evaluation of the anti-osteoporosis effect of arbutinyl-Tyr-Gly-Phe-Gly-Gly

[0042] Ursolic acid 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 established by bilateral ovariectomy in ICR female mice. The oral doses of Ursolyl-Tyr-Gly-Phe-Gly-Gly were 100 μmol / kg / day, 10 μmol / kg / day, and 1 μmol / kg / day; the oral doses of [Ursolyl-Tyr-Gly-Phe-Gly-Gly]2 were 10 μmol / kg / day, 1 μmol / kg / day, and 0.1 μmol / kg / day; the negative control was CMC-Na.

[0043] During model establishment, the mice were induced to anesthesia with an anesthesia machine. That is, the mice were placed supine on the 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, the muscle layer was cut open along the linea alba, the abdominal cavity was exposed, the bladder was found under the lower abdominal fat, the "Y"-shaped uterus 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 cauliflower-shaped and wrapped in 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. After suturing the skin layer, the skin suture site 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.

[0044] After the sham operation group (sham operation) mice were induced to anesthesia with an anesthesia machine, they were placed supine on the 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, the muscle layer was cut open along the linea alba, the abdominal cavity was exposed, the bladder was found under the lower abdominal fat, the "Y"-shaped uterus 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 cauliflower-shaped and wrapped in fat. The fallopian tubes were not ligated, and the ovaries were not removed. After putting it 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 site 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.

[0045] The mice recovered for seven days after surgery and were randomly grouped. Among them, the mice in the ovariectomy group and the sham operation group were intragastrically administered 0.3 mL / 25 g of 5‰ CMC-Na solution every day according to their body weight for 28 consecutive days, and their body weights were recorded simultaneously. The mice in the arbutinoyl-Tyr-Gly-Phe-Gly-Gly treatment group were intragastrically administered arbutinoyl-Tyr-Gly-Phe-Gly-Gly at doses of 100 μmol / kg / day, 10 μmol / kg / day, and 1 μmol / kg / day according to their body weight for 28 consecutive days, and their body weights were recorded simultaneously. The mice in the [arbutinoyl-Tyr-Gly-Phe-Gly-Gly]2 treatment group were intragastrically administered [arbutinoyl-Tyr-Gly-Phe-Gly-Gly]2 at doses of 10 μmol / kg / day, 1 μmol / kg / day, and 0.1 μmol / kg / day according to their body weight for 28 consecutive days, and their body weights were recorded simultaneously.

[0046] The femurs of the mice were scanned by Bruker skyscan 1276 Micro-CT, and the trabecular bone density of the mice was calculated. 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 bone structure of the femur. The trabecular bone of the ovariectomized mice was quantitatively analyzed according to six indexes including 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.

[0047] In terms of indexes such as the trabecular bone density of the mouse femur, arbutinoyl-Tyr-Gly-Phe-Gly-Gly could improve osteoporosis in ovariectomized mice by continuous intragastric administration at doses of 100 μmol / kg and 10 μmol / kg for 28 days. That is, the lowest effective dose of arbutinoyl-Tyr-Gly-Phe-Gly-Gly for treating osteoporosis was 10 μmol / kg / day. In addition, the lowest effective dose of [arbutinoyl-Tyr-Gly-Phe-Gly-Gly]2 was 1 μmol / kg / day. It can be seen that the present invention has unexpected technical effects. The specific results are shown in Table 1 - Table 6.

[0048] Table 1 Trabecular bone density of the femur in mice treated with arbutinoyl-Tyr-Gly-Phe-Gly-Gly and its dimer

[0049]

[0050] a) P < 0.01 compared with ovariectomized mice and mice treated with arbutinyl-Tyr-Gly-Phe-Gly-Gly (1 μmol / kg / day), P < 0.05 compared with mice treated with arbutinyl-Tyr-Gly-Phe-Gly-Gly (10 μmol / kg / day); b) P < 0.01 compared with ovariectomized mice, P < 0.05 compared with mice treated with arbutinyl-Tyr-Gly-Phe-Gly-Gly (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-Tyr-Gly-Phe-Gly-Gly (10 μmol / kg / day), P < 0.05 compared with mice treated with [arbutinyl-Tyr-Gly-Phe-Gly-Gly]2 (1 μmol / kg); e) P < 0.01 compared with ovariectomized mice and mice treated with arbutinyl-Tyr-Gly-Phe-Gly-Gly (1 μmol / kg / day), P < 0.05 compared with mice treated with [arbutinyl-Tyr-Gly-Phe-Gly-Gly]2 (0.1 μmol / kg); f) P > 0.05 compared with ovariectomized mice; n = 10.

[0051] Table 2 Femoral bone volume fraction of mice treated with arbutinyl-Tyr-Gly-Phe-Gly-Gly and dimer

[0052]

[0053] a) P < 0.01 compared with ovariectomized mice and mice treated with arbutinyl-Tyr-Gly-Phe-Gly-Gly (1 μmol / kg / day), P < 0.05 compared with mice treated with arbutinyl-Tyr-Gly-Phe-Gly-Gly (10 μmol / kg / day); b) P < 0.01 compared with ovariectomized mice, P < 0.05 compared with mice treated with arbutinyl-Tyr-Gly-Phe-Gly-Gly (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-Tyr-Gly-Phe-Gly-Gly (10 μmol / kg / day), P < 0.05 compared with mice treated with [arbutinyl-Tyr-Gly-Phe-Gly-Gly]2 (1 μmol / kg); e) P < 0.01 compared with ovariectomized mice and mice treated with arbutinyl-Tyr-Gly-Phe-Gly-Gly (1 μmol / kg / day), P < 0.05 compared with mice treated with [arbutinyl-Tyr-Gly-Phe-Gly-Gly]2 (0.1 μmol / kg); f) P > 0.05 compared with ovariectomized mice; n = 10.

[0054] Table 3 Number of femoral trabeculae in mice treated with arbutinyl-Tyr-Gly-Phe-Gly-Gly and its dimer

[0055]

[0056]

[0057] a) P < 0.01 compared with ovariectomized mice and mice treated with arbutinyl-Tyr-Gly-Phe-Gly-Gly (1 μmol / kg / day), P < 0.05 compared with mice treated with arbutinyl-Tyr-Gly-Phe-Gly-Gly (10 μmol / kg / day); b) P < 0.01 compared with ovariectomized mice, P < 0.05 compared with mice treated with arbutinyl-Tyr-Gly-Phe-Gly-Gly (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-Tyr-Gly-Phe-Gly-Gly (10 μmol / kg / day), P < 0.05 compared with mice treated with [arbutinyl-Tyr-Gly-Phe-Gly-Gly]2 (1 μmol / kg); e) P < 0.01 compared with ovariectomized mice and mice treated with arbutinyl-Tyr-Gly-Phe-Gly-Gly (1 μmol / kg / day), P < 0.05 compared with mice treated with [arbutinyl-Tyr-Gly-Phe-Gly-Gly]2 (0.1 μmol / kg); f) P > 0.05 compared with ovariectomized mice; n = 10.

[0058] Table 4 Thickness of femoral trabeculae in mice treated with arbutinyl-Tyr-Gly-Phe-Gly-Gly and its dimer

[0059]

[0060] a) P < 0.01 compared with ovariectomized mice and mice treated with arbutinyl-Tyr-Gly-Phe-Gly-Gly (1 μmol / kg / day), P < 0.05 compared with mice treated with arbutinyl-Tyr-Gly-Phe-Gly-Gly (10 μmol / kg / day); b) P < 0.01 compared with ovariectomized mice, P < 0.05 compared with mice treated with arbutinyl-Tyr-Gly-Phe-Gly-Gly (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-Tyr-Gly-Phe-Gly-Gly (10 μmol / kg / day), P < 0.05 compared with mice treated with [arbutinyl-Tyr-Gly-Phe-Gly-Gly]2 (1 μmol / kg); e) P < 0.01 compared with ovariectomized mice and mice treated with arbutinyl-Tyr-Gly-Phe-Gly-Gly (1 μmol / kg / day), P < 0.05 compared with mice treated with [arbutinyl-Tyr-Gly-Phe-Gly-Gly]2 (0.1 μmol / kg); f) P > 0.05 compared with ovariectomized mice; n = 10.

[0061] Table 5 Trabecular bone separation in femurs of mice treated with arbutinyl-Tyr-Gly-Phe-Gly-Gly and its dimer

[0062]

[0063] a) P < 0.01 compared with ovariectomized mice and mice treated with arbutinyl-Tyr-Gly-Phe-Gly-Gly (1 μmol / kg / day), P < 0.05 compared with mice treated with arbutinyl-Tyr-Gly-Phe-Gly-Gly (10 μmol / kg / day); b) P < 0.01 compared with ovariectomized mice, P < 0.05 compared with mice treated with arbutinyl-Tyr-Gly-Phe-Gly-Gly (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-Tyr-Gly-Phe-Gly-Gly (10 μmol / kg / day), P < 0.05 compared with mice treated with [arbutinyl-Tyr-Gly-Phe-Gly-Gly]2 (1 μmol / kg); e) P < 0.01 compared with ovariectomized mice and mice treated with arbutinyl-Tyr-Gly-Phe-Gly-Gly (1 μmol / kg / day), P < 0.05 compared with mice treated with [arbutinyl-Tyr-Gly-Phe-Gly-Gly]2 (0.1 μmol / kg); f) P > 0.05 compared with ovariectomized mice; n = 10.

[0064] Table 6 Osteocalcin pattern factor of femur trabecula in mice treated with arbutinyl-Tyr-Gly-Phe-Gly-Gly and its dimer

[0065]

[0066] a) P < 0.01 compared with ovariectomized mice and mice treated with arbutinyl-Tyr-Gly-Phe-Gly-Gly (1 μmol / kg / day), P < 0.05 compared with mice treated with arbutinyl-Tyr-Gly-Phe-Gly-Gly (10 μmol / kg / day); b) P < 0.01 compared with ovariectomized mice, P < 0.05 compared with mice treated with arbutinyl-Tyr-Gly-Phe-Gly-Gly (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-Tyr-Gly-Phe-Gly-Gly (10 μmol / kg / day), P < 0.05 compared with mice treated with [arbutinyl-Tyr-Gly-Phe-Gly-Gly]2 (1 μmol / kg); e) P < 0.01 compared with ovariectomized mice and mice treated with arbutinyl-Tyr-Gly-Phe-Gly-Gly (1 μmol / kg / day), P < 0.05 compared with mice treated with [arbutinyl-Tyr-Gly-Phe-Gly-Gly]2 (0.1 μmol / kg); f) P > 0.05 compared with ovariectomized mice; n = 10.

Claims

1. A dimer of arbutinoyl-Tyr-Gly-Phe-Gly-Gly, characterized in that, The dimer is composed of two arbutinoyl-Tyr-Gly-Phe-Gly-Gly molecules through non-covalent bonds, and the structural formula of the dimer is as follows:

2. A method for preparing the dimer of arbutinyl-Tyr-Gly-Phe-Gly-Gly according to claim 1, characterized in that, The method includes the following steps: 1) Prepare Boc-Tyr-Gly-Phe-Gly-Gly-OBzl; 2) Prepare HCl·Tyr-Gly-Phe-Gly-Gly-OBzl; 3) Prepare arbutinoyl-Tyr-Gly-Phe-Gly-Gly-OBzl; 4) Prepare arbutinoyl-Tyr-Gly-Phe-Gly-Gly; 5) Prepare the dimer of arbutinoyl-Tyr-Gly-Phe-Gly-Gly.

3. Use of the dimer of arbutinoyl-Tyr-Gly-Phe-Gly-Gly according to claim 1 in the preparation of anti-osteoporosis drugs.

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