Dimer of arbutinoyl-Asp-Gly-Glu-Ala, its synthesis and application

Abstract

The present invention discloses an ursolic acid-Asp-Gly-Glu-Ala dimer, represented by the following structure. This structure indicates that the dimer is composed of two ursolic acid-Asp-Gly-Glu-Ala molecules non-covalently bonded. Furthermore, the present invention discloses a method for preparing the ursolic acid-Asp-Gly-Glu-Ala dimer, as well as the anti-osteoporosis activity of the ursolic acid-Asp-Gly-Glu-Ala dimer and its use in the preparation of an anti-osteoporosis drug.

Description

Technical Field

[0001] The present invention relates to an ursolic-Asp-Gly-Glu-Ala dimer composed of two ursolic-Asp-Gly-Glu-Ala molecules formed by a non-covalent bond. It further relates to a method for preparing the ursolic-Asp-Gly-Glu-Ala dimer, as well as the anti-osteoporosis activity of the ursolic-Asp-Gly-Glu-Ala dimer and its use in the preparation of anti-osteoporosis drugs. The present invention belongs to the field of biomedicine. Background Art

[0002] In the Chinese invention patent application numbered 202211065344.1 filed by the inventors in 2022, virtual screening based on molecular docking confirmed that the docking score of ursolic acid-Asp-Gly-Glu-Ala into the TNF-α active pocket was significantly lower than that of ursolic acid. In application number 202211065344.1, the inventors further confirmed, based on experimental anti-osteoporosis studies, that the anti-osteoporosis activity of ursolic acid-Asp-Gly-Glu-Ala was significantly superior to that of ursolic acid.

[0003] However, experimentally, the effective oral dose of ursolic acid-Asp-Gly-Glu-Ala for osteoporosis prevention was 10 μmol / kg / day. This relatively high effective dose prompted the inventors to further investigate this molecule. Further research led the inventors to understand that molecular aggregates of ursolic acid-Asp-Gly-Glu-Ala could help lower the effective oral dose. Therefore, the inventors prepared molecular aggregates of ursolic acid-Asp-Gly-Glu-Ala. Further experiments revealed that under appropriate conditions, ursolic acid-Asp-Gly-Glu-Ala could form dimers. The inventors also discovered that the anti-osteoporotic activity of ursolic acid-Asp-Gly-Glu-Ala dimers at an oral dose of 1 μmol / kg / day was significantly superior to that of ursolic acid-Asp-Gly-Glu-Ala at an oral dose of 10 μmol / kg / day. Based on these findings, the inventors proposed the present invention. Summary of the Invention

[0004] The first object of the present invention is to provide a dimer of ursolic acid-Asp-Gly-Glu-Ala represented by the following structural formula, wherein the dimer is composed of two ursolic acid-Asp-Gly-Glu-Ala molecules through a non-covalent bond.

[0005]

[0006] The second object of the present invention is to provide a method for preparing the dimer of ursolic acid-Asp-Gly-Glu-Ala represented by the structural formula, which method comprises five steps:

[0007] 1) Preparation of Boc-Asp(OBzl)-Gly-Glu(OBzl)-Ala-OBzl;

[0008] 2) Preparation of HCl·Asp(OBzl)-Gly-Glu(OBzl)-Ala-OBzl;

[0009] 3) Ursolic acid was condensed with HCl·Asp(OBzl)-Gly-Glu(OBzl)-Ala-OBzl using 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate as a condensation agent to prepare ursolic acid-Asp(OBzl)-Gly-Glu(OBzl)-Ala-OBzl;

[0010] 4) using H2 / Pb to remove the OBzl protecting group of ursolic-Asp(OBzl)-Gly-Glu(OBzl)-Ala-OBzl to prepare ursolic-Asp-Gly-Glu-Ala;

[0011] 5) Preparation of a dimer of ursolic acid-Asp-Gly-Glu-Ala.

[0012] The third purpose of the present invention is to evaluate the anti-osteoporosis activity of the dimer of ursolic acid-Asp-Gly-Glu-Ala having the structure and the application of the dimer in the preparation of anti-osteoporosis drugs.

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

[0014] The present invention discloses an ursolic-Asp-Gly-Glu-Ala dimer composed of two ursolic-Asp-Gly-Glu-Ala molecules formed by a non-covalent bond. Experiments have shown that the ursolic-Asp-Gly-Glu-Ala dimer of the present invention overcomes the disadvantage of the prior art ursolic-Asp-Gly-Glu-Ala, which requires a relatively high oral dosage for osteoporosis treatment. Furthermore, the molecular dimer of the present invention exhibits superior anti-osteoporosis activity compared to the prior art ursolic-Asp-Gly-Glu-Ala. Therefore, the present invention provides an effective technical means for use in the preparation of anti-osteoporosis drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the FT-ICR-MS spectrum of the dimer of ursolic acid-Asp-Gly-Glu-Ala.

[0016] Figure 2 This is the qCID spectrum of the dimer of ursolic acid-Asp-Gly-Glu-Ala. DETAILED DESCRIPTION

[0017] In order to further illustrate the present invention, a series of examples are given below. These examples are purely illustrative and are only used to specifically describe the present invention and should not be understood as limiting the present invention.

[0018] Example 1 Preparation of Boc-Glu(OBzl)-Ala-OBzl

[0019] 4.00 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) were stirred at 0°C for 0.5 hours to obtain reaction solution A. 2.10 g (10 mmol) of Tos·Ala-OBzl was added to reaction solution A, and the pH was adjusted to 9 with N-methylmorpholine (NMM) at 0°C. The reaction mixture was then stirred at room temperature for 4 hours. Stirring was terminated when TLC indicated the disappearance of Tos·Ala-OBzl. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in 100 mL of ethyl acetate. The resulting solution was filtered, and the filtrate was washed sequentially with 30 mL of saturated NaHCO3 solution (three times), 30 mL of saturated NaCl solution (three times), 30 mL of 5% KHSO4 solution (three times), and 30 mL of saturated NaCl solution (three times). The mixture was then dried over anhydrous Na2SO4 for 12 hours. The Na2SO4 was filtered off, and the filtrate was concentrated under reduced pressure to give 4.49 g (90%) of Boc-Glu(OBzl)-Ala-OBzl as a colorless solid. TLC (CH2Cl2 / MeOH, 50 / 1; UV; Rf = 0.35), ESI-MS (m / e): 499 [M+H] + .

[0020] Example 2 Preparation of HCl·Glu(OBzl)-Ala-OBzl

[0021] Dissolve 5.00 g (10 mmol) of Boc-Glu(OBzl)-Ala-OBzl in 30 mL of 4 M hydrogen chloride in ethyl acetate and stir at 0°C for 4 hours. TLC indicates the disappearance of Boc-Glu(OBzl)-Ala-OBzl, and discontinue stirring. The reaction mixture is concentrated under reduced pressure, and the residue is dissolved in 10 mL of anhydrous ethyl acetate and concentrated under reduced pressure. Repeat this procedure three times. The residue is suspended in 10 mL of petroleum ether, and the suspension is concentrated under reduced pressure. Repeat this procedure three times. The resulting HCl·Glu(OBzl)-Ala-OBzl is a light yellow solid, which is used directly in the subsequent reaction. ESI-MS (m / e): 398 [M+H] + .

[0022] Example 3 Preparation of Boc-Gly-Glu(OBzl)-Ala-OBzl

[0023] Using the method of Example 1, 5.28 g (95%) of Boc-Gly-Glu(OBzl)-Ala-OBzl was obtained from 2.10 g (12 mmol) of Boc-Gly and 4.30 g (10 mmol) of HCl·Glu(OBzl)-Ala-OBzl as a colorless solid. TLC (CH2Cl2 / MeOH, 40 / 1; UV illumination; Rf = 0.35), ESI-MS (m / e): 556 [M+H] + .

[0024] Example 4 Preparation of HCl·Gly-Glu(OBzl)-Ala-OBzl

[0025] HCl·Gly-Glu(OBzl)-Ala-OBzl was prepared from 5.50 g (10 mmol) of Boc-Gly-Glu(OBzl)-Ala-OBzl using the method of Example 2. The resulting solid, a pale yellow solid, was used directly in the subsequent reaction. ESI-MS (m / e): 456 [M+H] + .

[0026] Example 5 Preparation of Boc-Asp(OBzl)-Gly-Glu(OBzl)-Ala-OBzl

[0027] Using the method of Example 1, a pale yellow crude product of Boc-Asp(OBzl)-Gly-Glu(OBzl)-Ala-OBzl was obtained from 3.90 g (12 mmol) of Boc-Asp(OBzl) and 4.90 g (10 mmol) of HCl·Gly-Glu(OBzl)-Ala-OBzl. The crude product was purified by column chromatography (gradient elution with CH2Cl2-MeOH; CH2Cl2 / MeOH, 40:1, UV visualization, Rf = 0.35) to obtain 6.86 g (90%) of Boc-Asp(OBzl)-Gly-Glu(OBzl)-Ala-OBzl as a colorless solid. ESI-MS (m / e): 762 [M+H] + .

[0028] Example 6 Preparation of HCl·Asp(OBzl)-Gly-Glu(OBzl)-Ala-OBzl

[0029] Using the method of Example 2, HCl·Asp(OBzl)-Gly-Glu(OBzl)-Ala-OBzl was prepared from 7.60 g (10 mmol) of Boc-Asp(OBzl)-Gly-Glu(OBzl)-Ala-OBzl as a pale yellow solid and was directly used in the subsequent reaction. ESI-MS (m / e): 661 [M+H] + .

[0030] Example 7 Preparation of Arbutinyl-Asp(OBzl)-Gly-Glu(OBzl)-Ala-OBzl

[0031] 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 0.70 g (1.0 mmol) of HCl·Asp(OBzl)-Gly-Glu(OBzl)-Ala-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 with anhydrous Na2SO4 for 12 hours. Filter out Na2SO4, and concentrate the filtrate under reduced pressure to obtain a crude product of ursolyl-Asp(OBzl)-Gly-Glu(OBzl)-Ala-OBzl as a pale yellow solid. This crude product is purified by column chromatography (gradient elution with CH2Cl2-MeOH; CH2Cl2 / MeOH, 40:1, UV visualization; Rf = 0.20) to obtain 0.44 g of ursolyl-Asp(OBzl)-Gly-Glu(OBzl)-Ala-OBzl (40%) as a colorless solid. ESI-MS (m / e): 1099 [M-H] - .

[0032] Example 8 Preparation of Ursolyl-Asp-Gly-Glu-Ala

[0033] Dissolve 1.10 g (1.0 mmol) of ursolyl-Asp(OBzl)-Gly-Glu(OBzl)-Ala-OBzl in 100 mL of methanol. Add 110 mg of palladium carbon to the resulting solution. After evacuating the air from the resulting suspension, stir at room temperature and hydrogenate with hydrogen for 12 hours. TLC shows the disappearance of ursolyl-Asp(OBzl)-Gly-Glu(OBzl)-Ala-OBzl, and stop the hydrogenation. Filter out the palladium carbon from the suspension, and concentrate the filtrate under reduced pressure to obtain a crude product of ursolyl-Asp-Gly-Glu-Ala. 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 0.36 g (44%) of ursolyl-Asp-Gly-Glu-Ala as a white solid. TLC (ethyl acetate / distilled water / glacial acetic acid, 10 / 1 / 1; iodine vapor visualization; Rf = 0.30). FT-MS 851.48113 [M+Na] +(851.47823);Mp:175-177℃; IR / cm -1 =3303,2926,1715,1648,1517,1453,1389,1186,1029,996,920,830,804,660; 1 H NMR (300MHz, DMSO-d6) δ / ppm=12.38(s,3H),8.15(d,J=7.2Hz,1H),8.13(s,1H),7.91(d,J=8.1Hz,1H),7.67(t,J=5.4Hz,1H),7.50(d,J=7.2Hz, 1H),5.17(t,J=3.6Hz,1H),4.47(q,J=6.6Hz,1H),4.30(td,J1=8.1Hz,J2=5.4Hz,1H),4.17(m,1H),3.70(d,J=5.7Hz,2H),2.99(dd,J1=9.9Hz,J 2=6.0Hz,1H),2.67(dd,J1=16.5Hz,J2=5.7Hz,1H),2.50(dd,J1=17.1Hz,J2=7.5Hz,1H),2.25(t,J=8.1Hz,2H),2.14(d,J=10.8Hz,1H),1.88~1. 98(m,2H),1.82~1.70(m,4H),1.64~1.54(m,3H),1.50~1.32(m,9H),1.2 7(d,J=7.2Hz,6H),1.02(s,3H),0.91~0.80(m,15H),0.67~0.63(m,7H); 13 C NMR(75MHz,DMSO-d6)δ / ppm=177.10,174.36,174.31,172.47,171.45,171.13,168.68,1 63.41,138.55,125.37,77.36,55.30,52.54,51.92,50.22,49.05,47.95,47.57,47.14,4 2.64,42.11,39.60,38.98,38.84,38.77,36.97,36.24,33.20,30.85,30.47,28.73,28.00,27.69,27.48,24.00,23.68,23.34,21.56,18.45,17.52,17.39,17.20,16.52,15.69.

[0034] Example 9 Preparation of Ursolic Acid-Asp-Gly-Glu-Ala Dimer

[0035] Dissolve 100 mg of arbutinyl-Asp-Gly-Glu-Ala powder in 5 mL of ultrapure water. The resulting solution is vortexed for 15 minutes first, and then sonicated in an ultrasonic bath for 4 hours until the solution becomes completely colorless and transparent. The colorless and transparent solution is concentrated to dryness under reduced pressure to obtain the dimer of arbutinyl-Asp-Gly-Glu-Ala, i.e., [arbutinyl-Asp-Gly-Glu-Ala]2. The structure of [arbutinyl-Asp-Gly-Glu-Ala]2 is confirmed by ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Figure 1 Peaks of [arbutinyl-Asp-Gly-Glu-Ala]+H at 829.51958 (theoretical value: 829.49574) and [arbutinyl-Asp-Gly-Glu-Ala]2+Na at 1680.08492 (theoretical value: 1679.96614) are given.

[0036] To clarify the relationship between the peak of [arbutinyl-Asp-Gly-Glu-Ala]+H and the peak of [arbutinyl-Asp-Gly-Glu-Ala]2+Na, the qCID spectrum of [arbutinyl-Asp-Gly-Glu-Ala]2+Na is measured. Figure 2 The qCID spectrum of [arbutinyl-Asp-Gly-Glu-Ala]2+Na gives a peak of [arbutinyl-Asp-Gly-Glu-Ala]+H at 829.50756 (theoretical value: 829.49574). That is to say, the peak of [arbutinyl-Asp-Gly-Glu-Ala]+H comes from the cleavage of [arbutinyl-Asp-Gly-Glu-Ala]2+Na, i.e., [arbutinyl-Asp-Gly-Glu-Ala]2 is the only form in which arbutinyl-Asp-Gly-Glu-Ala exists.

[0037] Example 10 Evaluating the anti-osteoporosis effect of the dimer of arbutinyl-Asp-Gly-Glu-Ala

[0038] Ursolic acid is purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. SPF-grade male ICR mice (25±2 g) are purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. An osteoporosis model is established by bilateral ovariectomy of ICR female mice. The oral doses of arbutinyl-Asp-Gly-Glu-Ala are 100 μmol / kg / day, 10 μmol / kg / day, and 1 μmol / kg / day; the oral doses of [arbutinyl-Asp-Gly-Glu-Ala]2 are 10 μmol / kg / day, 1 μmol / kg / day, and 0.1 μmol / kg / day; the negative control is CMC-Na.

[0039] When making the model, 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. 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, and the muscle layer was cut along the white line of the abdomen 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 ligated away from the uterus, and both 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 was disinfected with alcohol and iodophor again. The role of penicillin, alcohol and iodophor is to prevent infection. After removing the anesthesia device, the mice woke up quickly.

[0040] In the sham operation group (sham operation), after the 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. 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, and the muscle layer was cut along the white line of the abdomen 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 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 was disinfected with alcohol and iodophor again. The role of penicillin, alcohol and iodophor is to prevent infection. After removing the anesthesia device, the mice woke up quickly.

[0041] The mice recovered for seven days after the operation and were randomly grouped. Among them, the mice in the ovariectomy group and the sham operation group were intragastrically administered 5‰ CMC-Na solution according to body weight every day for 28 consecutive days, and the body weight was recorded at the same time. The mice in the Arbutinyl-Asp-Gly-Glu-Ala treatment group were intragastrically administered Arbutinyl-Asp-Gly-Glu-Ala according to body weight at doses of 100 μmol / kg / day, 10 μmol / kg / day and 1 μmol / kg / day for 28 consecutive days, and the body weight was recorded at the same time. The mice in the dimer treatment group of Arbutinyl-Asp-Gly-Glu-Ala were intragastrically administered the dimer of Arbutinyl-Asp-Gly-Glu-Ala at doses of 10 μmol / kg / day, 1 μmol / kg / day and 0.1 μmol / kg / day for 28 consecutive days, and the body weight was recorded at the same time.

[0042] Mouse femurs were scanned using a Bruker Skyscan 1276 Micro-CT scanner to calculate trabecular bone density. Regions of interest (ROIs) at the distal femur were selected at the same height to calculate trabecular bone parameters and analyze femoral trabecular structure. Trabecular bone in ovariectomized mice was quantitatively analyzed using six parameters: trabecular BMD, bone volume fraction, trabecular number, trabecular thickness, trabecular separation, and trabecular pattern factor. Results are presented as mean ± SD. SD values were analyzed for homogeneity of variance using SPSS software, and t-tests were used for statistical comparisons between groups.

[0043] Ursolic acid-Asp-Gly-Glu-Ala improved osteoporosis in ovariectomized mice at doses of 100 μmol / kg and 10 μmol / kg administered orally for 28 consecutive days, demonstrating that the minimum effective dose of ursolic acid-Asp-Gly-Glu-Ala for osteoporosis treatment is 10 μmol / kg / day. Furthermore, the minimum effective dose of ursolic acid-Asp-Gly-Glu-Ala dimer is 1 μmol / kg / day. Specific results are shown in Tables 1-6. The data in these tables demonstrate the unexpected technical benefits of the present invention.

[0044] Table 1 Femoral trabecular bone density of mice treated with ursolic acid-Asp-Gly-Glu-Ala and its dimer

[0045]

[0046] a) Compared with the ovariectomized mice and the mice treated with ursolic acid-Asp-Gly-Glu-Ala (1 μmol / kg / day), P<0.01; compared with the mice treated with ursolic acid-Asp-Gly-Glu-Ala (10 μmol / kg / day), P<0.05; b) Compared with the ovariectomized mice, P<0.01; compared with the mice treated with ursolic acid-Asp-Gly-Glu-Ala (1 μmol / kg / day), P<0.05; c) Compared with the ovariectomized group, P>0.05; d) Compared with the ovariectomized mice and the mice treated with ursolic acid-Asp-Gly-Glu-Ala (10 μmol / kg / day), P<0.05 Compared with mice treated with lu-Ala (10 μmol / kg / day), P < 0.01, and compared with mice treated with [ursolic-Asp-Gly-Glu-Ala]2 (1 μmol / kg), P < 0.05; e) compared with ovariectomized mice and mice treated with ursolic-Asp-Gly-Glu-Ala (1 μmol / kg / day), P < 0.01, and compared with mice treated with [ursolic-Asp-Gly-Glu-Ala]2 (0.1 μmol / kg), P < 0.05; f) compared with ovariectomized mice, P > 0.05; n = 10.

[0047] Table 2 Femoral bone volume fraction of mice treated with arbutinyl-Asp-Gly-Glu-Ala and its dimer

[0048]

[0049] a) P < 0.01 compared with ovariectomized mice and mice treated with arbutinyl-Asp-Gly-Glu-Ala (1 μmol / kg / day), P < 0.05 compared with mice treated with arbutinyl-Asp-Gly-Glu-Ala (10 μmol / kg / day); b) P < 0.01 compared with ovariectomized mice, P < 0.05 compared with mice treated with arbutinyl-Asp-Gly-Glu-Ala (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-Asp-Gly-Glu-Ala (10 μmol / kg / day), P < 0.05 compared with mice treated with [arbutinyl-Asp-Gly-Glu-Ala]2 (1 μmol / kg); e) P < 0.01 compared with ovariectomized mice and mice treated with arbutinyl-Asp-Gly-Glu-Ala (1 μmol / kg / day), P < 0.05 compared with mice treated with [arbutinyl-Asp-Gly-Glu-Ala]2 (0.1 μmol / kg); f) P > 0.05 compared with ovariectomized mice; n = 10.

[0050] Table 3 Number of trabecular bone in the femur of mice treated with arbutinyl-Asp-Gly-Glu-Ala and its dimer

[0051]

[0052] a) Compared with the ovariectomized mice and the mice treated with ursolic acid-Asp-Gly-Glu-Ala (1 μmol / kg / day), P<0.01; compared with the mice treated with ursolic acid-Asp-Gly-Glu-Ala (10 μmol / kg / day), P<0.05; b) Compared with the ovariectomized mice, P<0.01; compared with the mice treated with ursolic acid-Asp-Gly-Glu-Ala (1 μmol / kg / day), P<0.05; c) Compared with the ovariectomized group, P>0.05; d) Compared with the ovariectomized mice and the mice treated with ursolic acid-Asp-Gly-Glu-Ala (10 μmol / kg / day), P<0.05 Compared with mice treated with lu-Ala (10 μmol / kg / day), P < 0.01, and compared with mice treated with [ursolic-Asp-Gly-Glu-Ala]2 (1 μmol / kg), P < 0.05; e) compared with ovariectomized mice and mice treated with ursolic-Asp-Gly-Glu-Ala (1 μmol / kg / day), P < 0.01, and compared with mice treated with [ursolic-Asp-Gly-Glu-Ala]2 (0.1 μmol / kg), P < 0.05; f) compared with ovariectomized mice, P > 0.05; n = 10.

[0053] Table 4 Femoral trabecular thickness of mice treated with ursolic acid-Asp-Gly-Glu-Ala and its dimer

[0054]

[0055]

[0056] a) Compared with the ovariectomized mice and the mice treated with ursolic acid-Asp-Gly-Glu-Ala (1 μmol / kg / day), P<0.01; compared with the mice treated with ursolic acid-Asp-Gly-Glu-Ala (10 μmol / kg / day), P<0.05; b) Compared with the ovariectomized mice, P<0.01; compared with the mice treated with ursolic acid-Asp-Gly-Glu-Ala (1 μmol / kg / day), P<0.05; c) Compared with the ovariectomized group, P>0.05; d) Compared with the ovariectomized mice and the mice treated with ursolic acid-Asp-Gly-Glu-Ala (10 μmol / kg / day), P<0.05 Compared with mice treated with lu-Ala (10 μmol / kg / day), P < 0.01, and compared with mice treated with [ursolic-Asp-Gly-Glu-Ala]2 (1 μmol / kg), P < 0.05; e) compared with ovariectomized mice and mice treated with ursolic-Asp-Gly-Glu-Ala (1 μmol / kg / day), P < 0.01, and compared with mice treated with [ursolic-Asp-Gly-Glu-Ala]2 (0.1 μmol / kg), P < 0.05; f) compared with ovariectomized mice, P > 0.05; n = 10.

[0057] Table 5 Separation of femoral trabecular bone in mice treated with ursolic acid-Asp-Gly-Glu-Ala and its dimer

[0058]

[0059] a) Compared with the ovariectomized mice and the mice treated with ursolic acid-Asp-Gly-Glu-Ala (1 μmol / kg / day), P<0.01; compared with the mice treated with ursolic acid-Asp-Gly-Glu-Ala (10 μmol / kg / day), P<0.05; b) Compared with the ovariectomized mice, P<0.01; compared with the mice treated with ursolic acid-Asp-Gly-Glu-Ala (1 μmol / kg / day), P<0.05; c) Compared with the ovariectomized group, P>0.05; d) Compared with the ovariectomized mice and the mice treated with ursolic acid-Asp-Gly-Glu-Ala (10 μmol / kg / day), P<0.05 Compared with mice treated with lu-Ala (10 μmol / kg / day), P < 0.01, and compared with mice treated with [ursolic-Asp-Gly-Glu-Ala]2 (1 μmol / kg), P < 0.05; e) compared with ovariectomized mice and mice treated with ursolic-Asp-Gly-Glu-Ala (1 μmol / kg / day), P < 0.01, and compared with mice treated with [ursolic-Asp-Gly-Glu-Ala]2 (0.1 μmol / kg), P < 0.05; f) compared with ovariectomized mice, P > 0.05; n = 10.

[0060] Table 6 Femoral trabecular bone pattern factors of mice treated with ursolic acid-Asp-Gly-Glu-Ala and its dimer

[0061]

[0062] a) Compared with the ovariectomized mice and the mice treated with ursolic acid-Asp-Gly-Glu-Ala (1 μmol / kg / day), P<0.01; compared with the mice treated with ursolic acid-Asp-Gly-Glu-Ala (10 μmol / kg / day), P<0.05; b) Compared with the ovariectomized mice, P<0.01; compared with the mice treated with ursolic acid-Asp-Gly-Glu-Ala (1 μmol / kg / day), P<0.05; c) Compared with the ovariectomized group, P>0.05; d) Compared with the ovariectomized mice and the mice treated with ursolic acid-Asp-Gly-Glu-Ala (10 μmol / kg / day), P<0.05 Compared with mice treated with lu-Ala (10 μmol / kg / day), P < 0.01, and compared with mice treated with [ursolic-Asp-Gly-Glu-Ala]2 (1 μmol / kg), P < 0.05; e) compared with ovariectomized mice and mice treated with ursolic-Asp-Gly-Glu-Ala (1 μmol / kg / day), P < 0.01, and compared with mice treated with [ursolic-Asp-Gly-Glu-Ala]2 (0.1 μmol / kg), P < 0.05; f) compared with ovariectomized mice, P > 0.05; n = 10.

Claims

1. A dimer of arbutinyl-Asp-Gly-Glu-Ala, characterized in that, The dimer is composed of two ursolic acid-Asp-Gly-Glu-Ala molecules through non-covalent bonds, and the structural formula of the dimer is as follows:

2. The preparation method of the dimer of arbutinyl-Asp-Gly-Glu-Ala according to claim 1, characterized in that, The method comprises the following steps: 1) Preparation of Boc-Asp(OBzl)-Gly-Glu(OBzl)-Ala-OBzl; 2) Preparation of HCl·Asp(OBzl)-Gly-Glu(OBzl)-Ala-OBzl; 3) Preparation of ursolic acid-Asp(OBzl)-Gly-Glu(OBzl)-Ala-OBzl; 4) preparing ursolic acid-Asp-Gly-Glu-Ala; 5) Preparation of a dimer of ursolic acid-Asp-Gly-Glu-Ala.

3. Use of the dimer of ursolic acid-Asp-Gly-Glu-Ala according to claim 1 in the preparation of anti-osteoporosis drugs.

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