Self-assembled trimers of 17-O-acetyl-17-β estradiol, their preparation, activity and applications

Abstract

The present invention discloses a self-assembled trimer of 17-O-acetyl-17-β estradiol of the formula [17-O-acetyl-17-β estradiol]3, a preparation method and characterization method of [17-O-acetyl-17-β estradiol]3, and further discloses the therapeutic effect of [17-O-acetyl-17-β estradiol]3 on ovariectomized female mice. Therefore, the present invention discloses the use of [17-O-acetyl-17-β estradiol]3 in the preparation of a drug for treating female menopausal reactions. #imgabs0#

Description

Technical Field

[0001] The present invention relates to a self-assembled trimer of 17-O-acetyl-17-β-estradiol, [17-O-acetyl-17-β-estradiol]3, a preparation method and characterization of [17-O-acetyl-17-β-estradiol]3, and further to the therapeutic effects of [17-O-acetyl-17-β-estradiol]3 on ovariectomized female mice. The present invention thus illustrates the use of [17-O-acetyl-17-β-estradiol]3 in the preparation of a drug for treating female menopausal reactions. The present invention belongs to the field of biomedicine. Background Art

[0002] For decades, it was generally believed that the ovaries of healthy premenopausal women secrete a type of estrogen called 17-β-estradiol (Lieveritz, RW, Amer. J. of Obstetrics and Gynecology, Vol. 156, pp. 1289-1293, 1987). Disease, oophorectomy, traumatic injury, and aging can lead to 17-β-estradiol deficiency. With aging, ovarian ovulation becomes less frequent and less predictable. Consequently, the amount of 17-β-estradiol secreted by the ovaries gradually decreases. This gradual loss of ovarian function occurs naturally between the ages of 45 and 55, ultimately leading to the cessation of menstrual cycles, commonly known as menopause.

[0003] For decades, it was believed that during a normal ovulatory cycle, the ovaries secrete 17-β-estradiol in the range of 60-600 μg / day, resulting in circulating 17-β-estradiol concentrations ranging from 40-400 pg / mL. Circulating 17-β-estradiol concentrations in premenopausal women fluctuate throughout the menstrual cycle. After menopause, women gradually experience irreversible ovarian failure, with ovarian 17-β-estradiol production gradually decreasing from 60-600 μg / day to less than 20 μg / day, and circulating 17-β-estradiol concentrations dropping significantly from 40-400 pg / mL to less than 30 pg / mL (Stumpf, P.G., Obstetrics and Gynaecology, Vol. 75 (suppl.), pp. 95-135, 1990). Such low levels of 17-β estradiol can lead to typical postmenopausal symptoms such as hot flashes, urogenital atrophy, and mineral-losing osteoporosis (Marsh, M.S. et.al., British Medical Bulletin, Vol. 48, pp. 426-457, 1992).

[0004] Over the past few decades, it has been further recognized that the most active naturally occurring human estrogen is unconjugated 17-β-estradiol, leading to the development of estrogen replacement therapy (HRT). The goal of HRT is to restore circulating 17-β-estradiol levels in postmenopausal women to premenopausal physiological levels by supplementing 17-β-estradiol, thereby combating postmenopausal symptoms.

[0005] Decades of HRT supplementation with 17-β-estradiol have revealed that it doesn't produce the expected results. While it can combat postmenopausal osteoporosis, it doesn't improve the urogenital atrophy associated with postmenopausal hot flashes. Even more surprising, it has side effects such as carcinogenicity and the risk of blood clots.

[0006] During their long-term research on chemically modified 17-β-estradiol, the inventors were forced to ponder the reasons for the problematic effects of 17-β-estradiol supplementation. According to clinical evidence, premenopausal women face virtually no risk of cancer, thrombosis, or urogenital atrophy associated with hot flashes. However, postmenopausal women who supplemented with 17-β-estradiol experienced these risks. This reflection gradually led the inventors to recognize a clear logical contradiction. This logical contradiction led them to suspect that the estrogen secreted by the ovaries might not be 17-β-estradiol. Therefore, the inventors removed the ovaries of female mice and homogenized them. The ovarian homogenate was mixed with spectrally grade methanol and repeatedly sonicated. The methanol extract of the ovarian homogenate was dried with nitrogen and then extracted with spectrally grade methanol. The extract was then analyzed by HPLC-FT-ICR-MS. Surprisingly, the HPLC-FT-ICR-MS spectrum of the methanol extract of the female mouse ovarian homogenate showed no peaks for 17-β-estradiol, but rather peaks for 17-O-acetyl-17-β-estradiol. The inventors then prepared and extracted homogenates of female mouse organs and blood. The extracts were then subjected to HPLC-FT-ICR-MS analysis. Similarly, the HPLC-FT-ICR-MS spectrum of the methanol extract of the female mouse organ and blood homogenate showed no 17-β-estradiol peak, but rather a 17-O-acetyl-17-β-estradiol peak. Based on this discovery, the inventors developed the present invention. Summary of the Invention

[0007] The first aspect of the present invention is to provide a self-assembled trimer of 17-O-acetyl-17-β estradiol of the following formula [17-O-acetyl-17-β estradiol] 3.

[0008]

[0009] The second content of the present invention is to provide a method for preparing [17-O-acetyl-17-β estradiol] 3, which comprises the following steps:

[0010] 1) dissolving estradiol in acetic anhydride, and stirring the resulting reaction solution at room temperature for 12 hours;

[0011] 2) slowly diluting the solution stirred at room temperature for 12 hours with distilled water, wherein the volume of distilled water used for dilution is 5-20 times the volume of acetic anhydride, preferably 10 times;

[0012] 3) The above diluted solution is concentrated under reduced pressure to remove as much dilute acetic acid as possible;

[0013] 4) The residue was neutralized with saturated sodium bicarbonate aqueous solution to pH 8 at 0°C, and the generated precipitate was fully precipitated under ultrasound, and filtered to obtain the self-assembled [17-O-acetyl-17-β estradiol] 3;

[0014] 5) Structural characterization of self-assembled [17-O-acetyl-17-β estradiol] 3.

[0015] The third aspect of the present invention is to determine the activity of self-assembled [17-O-acetyl-17-β estradiol] 3.

[0016] The fourth aspect of the present invention is to investigate the side effects of self-assembled [17-O-acetyl-17-β estradiol] 3. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Synthetic route of self-assembled [17-O-acetyl-17-β-estradiol] 3.

[0018] Figure 2 ESI(+)-FT-ICR-MS characterization of self-assembled [17-O-acetyl-17-β-estradiol]3.

[0019] Figure 3 qCID supports that the self-assembled [17-O-acetyl-17-β-estradiol]3 is the only form of 17-O-acetyl-17-β-estradiol.

[0020] Figure 4 NOESY 2D 1 HNMR revealed that 17-O-acetyl-17-β-estradiol formed a self-assembled [17-O-acetyl-17-β-estradiol]3 pattern.

[0021] Figure 5Photographs of uterine tissue morphology: ac, photographs of uterus tissue morphology of sham-operated mice treated with normal saline for 28 consecutive days; df, photographs of uterus tissue morphology of ovariectomized mice treated with normal saline for 28 consecutive days; gi, photographs of uterus tissue morphology of ovariectomized mice treated with 2.3 μmol / kg / day [17-O-acetyl-17-β estradiol]3 for 28 consecutive days; jl, photographs of uterus tissue morphology of ovariectomized mice treated with 0.23 μmol / kg / day [17-O-acetyl-17-β estradiol]3 for 28 consecutive days.

[0022] Figure 6 Photographs of the oviduct's tissue morphology: ac, photographs of the oviduct's tissue morphology of sham-operated mice treated with normal saline for 28 consecutive days; df, photographs of the oviduct's tissue morphology of oviduct of ovariectomized mice treated with normal saline for 28 consecutive days; gi, photographs of the oviduct's tissue morphology of oviduct of ovariectomized mice treated with 2.3 μmol / kg / day of [17-O-acetyl-17-β estradiol]3 for 28 consecutive days; jl, photographs of the oviduct's tissue morphology of oviduct of ovariectomized mice treated with 0.23 μmol / kg / day of [17-O-acetyl-17-β estradiol]3 for 28 consecutive days.

[0023] Figure 7 Histomorphological photographs of the spleen: ac, histomorphological photographs of the spleen of sham-operated mice treated with normal saline for 28 consecutive days; df, histological photographs of the spleen of ovariectomized mice treated with normal saline for 28 consecutive days; gi, histological photographs of the spleen of ovariectomized mice treated with [17-O-acetyl-17-β estradiol]3 at a dose of 2.3 μmol / kg / day for 28 consecutive days; jl, histological photographs of the spleen of ovariectomized mice treated with [17-O-acetyl-17-β estradiol]3 at a dose of 0.23 μmol / kg / day for 28 consecutive days.

[0024] Figure 8 Photographs of the thymus tissue morphology: ac, photographs of the thymus tissue morphology of sham-operated mice treated with normal saline for 28 consecutive days; df, photographs of the thymus tissue morphology of ovariectomized mice treated with normal saline for 28 consecutive days; gi, photographs of the thymus tissue morphology of ovariectomized mice treated with 2.3 μmol / kg / day of [17-O-acetyl-17-β estradiol]3 for 28 consecutive days; jl, photographs of the thymus tissue morphology of ovariectomized mice treated with 0.23 μmol / kg / day of [17-O-acetyl-17-β estradiol]3 for 28 consecutive days. DETAILED DESCRIPTION

[0025] To further illustrate the present invention, a series of examples are provided below. It should be noted that these examples are purely illustrative. These examples are provided to fully demonstrate the significance and content of the present invention and are not intended to limit the present invention in any way.

[0026] Example 1 Preparation of 17-O-acetyl-17-β estradiol self-assembled trimer [17-O-acetyl-17-β estradiol] 3

[0027] Dissolve 1.00 g (3.7 mmol) of 17-β-estradiol in 5 mL of acetic anhydride and stir at room temperature for 12 hours. TLC (petroleum ether / ethyl acetate, 2 / 1) indicates the disappearance of 17-β-estradiol. Slowly dilute the reaction solution with 50 mL of distilled water, and concentrate the solution under reduced pressure to remove as much of the dilute acetic acid as possible. Neutralize the residue with saturated aqueous sodium bicarbonate to pH 8 at 0°C, allowing the resulting precipitate to fully precipitate. Filtration yields 1.11 g (95%) of a self-assembled trimer of 17-O-acetyl-17-β-estradiol as a colorless solid. ESI(+)-FT-ICR-MS (m / e): 965.56758 [M3+Na] + (Theoretical value: 965.56108); 1 H NMR (800MHz, DMSO-d6): δ / ppm=9.001(s,1H),7.033(d,J=8.0Hz,1H),6.508(d,J=8.0Hz,1H),6.439(s, 1H),4.600(t,J=8.0Hz,1H),2.731(m,J=5.6Hz,2H),2.225(m,J=5.6Hz,1H),2.091(m, J=4.0Hz,2H),2.007(d,J=4.0Hz,3H),1.785(m,J=4.8Hz,2H),1.668(m,J=8.0Hz,1H), 1.493(q,J=1.6Hz,1H), 1.361(m,J=5.6Hz,4H), 1.298(m,J=4.0Hz,2H), 0.772(s,3H). 13 C NMR (201 MHz, DMSO-d6): δ / ppm = 169.74, 154.22, 136.49, 129.56, 125.46, 114.26, 112.06, 81.31, 48.47, 41.91, 39.37, 39.27, 38.95, 35.88, 28.50, 26.58, 26.24, 25.28, 22.19, 11.35. The following examples illustrate that the self-assembled [17-O-acetyl-17-β-estradiol]3 of the present invention is the only form of 17-O-acetyl-17-β-estradiol.

[0028] Example 2 Characterization of Self-Assembled [17-O-Acetyl-17-β Estradiol] 3

[0029] 1 μg of the self-assembled [17-O-acetyl-17-β estradiol] 3 prepared above was dissolved in 1 mL of spectral grade methanol, and 1 μL was injected to measure the ESI(+)-FT-ICR-MS spectrum. Figure 2 . Figure 2 ESI(+)-FT-ICR-MS revealed four peaks at masses 315.19770, 337.17980, 651.36993, and 965.56758. According to the accurate molecular weights, 315.19770 represents the 17-O-acetyl-17-β-estradiol monomer with H (theoretical value: 315.19547), 337.17980 represents the 17-O-acetyl-17-β-estradiol monomer with Na (theoretical value: 337.17741), 651.36993 represents the self-assembled dimer [17-O-acetyl-17-β-estradiol]2 with Na (theoretical value: 651.36561), and 965.56758 represents the self-assembled trimer [17-O-acetyl-17-β-estradiol]3 with Na (theoretical value: 965.56108).

[0030] In order to clarify the relationship between the 17-O-acetyl-17-β-estradiol monomer, the self-assembled dimer [17-O-acetyl-17-β-estradiol]2 and the self-assembled trimer [17-O-acetyl-17-β-estradiol]3, the qCID spectrum was measured for the peak with a mass of 965.56758 [17-O-acetyl-17-β-estradiol]3. Figure 3 . Figure 3The qCID yielded three peaks at masses 315.19567, 337.17748, and 651.36413. Similar to the above, 315.19567 represents the monomeric 17-O-acetyl-17-β-estradiol with H added (theoretical value: 315.19547), 337.17748 represents the monomeric 17-O-acetyl-17-β-estradiol with Na added (theoretical value: 337.17741), and 651.36413 represents the self-assembled dimer [17-O-acetyl-17-β-estradiol]2 with Na added (theoretical value: 651.36561). The results indicate that the peaks of 17-O-acetyl-17-β-estradiol monomers and the self-assembled dimer [17-O-acetyl-17-β-estradiol]2 in the ESI(+)-FT-ICR-MS spectrum are fragmentation products of the self-assembled trimer [17-O-acetyl-17-β-estradiol]3. In other words, the self-assembled trimer [17-O-acetyl-17-β-estradiol]3 is the only form of 17-O-acetyl-17-β-estradiol. Under electron bombardment, the self-assembled trimer fragments into monomers and dimers.

[0031] Example 3 Characterization of the Self-Assembly Pattern of 17-O-Acetyl-17-β Estradiol into [17-O-Acetyl-17-β Estradiol] 3

[0032] In order to reveal the mode by which 17-O-acetyl-17-β estradiol spontaneously assembles into self-assembled trimers, the inventors determined the NOESY 2D 1 HNMR spectrum. Results are shown in Figure 4 . Figure 4 NOESY 2D 1 The significant correlation peak given by the HNMR spectrum is the correlation peak between the 2-H and 9-H positions. This correlation peak comes from the 2-H position on the A ring of one 17-O-acetyl-17-β-estradiol molecule and the 9-H position on the B ring of another 17-O-acetyl-17-β-estradiol molecule. The basic requirement for the appearance of this correlation peak is that the distance between the two H positions is less than To this end, the A ring of one molecule must be close to the B ring of another molecule. According to this requirement, the self-assembly pattern of 17-O-acetyl-17-β estradiol monomers into [17-O-acetyl-17-β estradiol] 3 is that the A ring of one molecular monomer and the B rings of the other two molecular monomers should be close to each other to a distance less than

[0033] Example 4 Evaluation of the Anti-Osteoporotic Activity of the Self-Assembled Trimer of 17-O-Acetyl-17-β-Estradiol [17-O-Acetyl-17-β-Estradiol] 3

[0034] Female ICR mice (40, 25 ± 2 g) were anesthetized by intraperitoneal injection of chloral hydrate in saline (5%, 0.1 mL / 10 g). The lower abdomen was then cleaned with iodine-containing benzylpenicillin (BPA) and the epidermal layer, muscle layer, and abdominal cavity were incised along the linea alba. The uterus was then located beneath the bladder. The fallopian tubes were located along the uterus. The blind ends of the tubes were the ovaries. The ovaries are cauliflower-shaped and surrounded by fat. After ligating the fallopian tubes distal to the uterus, the ovaries were removed. A drop of penicillin solution was applied to the ligation site, and the tubes were returned to the abdominal cavity. The muscle layer was then sutured. A drop of penicillin solution was applied before and after suturing to prevent infection. The epidermal layer was then sutured. BPA and iodine were applied before and after suturing to prevent infection. After surgery, the mice were kept warm in a cage. They recovered after 3–5 hours. These mice were then used for modeling.

[0035] Female ICR mice (40, 25 ± 2 g) were anesthetized by intraperitoneal injection of 5% chloral hydrate in saline (0.1 mL / 10 g). The lower abdomen was then cleaned with iodine-containing benzyl alcohol. The epidermis, muscle layer, and abdominal cavity were incised along the linea alba. The uterus was located beneath the bladder. The oviducts were located along the uterus. The blind ends of the oviducts were the ovaries. The ovaries were cauliflower-shaped and surrounded by fat. The oviducts were returned to the abdominal cavity. The muscle layer was sutured. A drop of penicillin solution was applied before and after suturing to prevent infection. The epidermis was then sutured. Both benzyl alcohol and iodine-containing solutions were applied before and after suturing to prevent infection. After surgery, the mice were kept warm in a cage. They recovered 3–5 hours later. These mice were designated as sham-operated (Sham) mice.

[0036] After seven days of recovery after surgery, on the eighth day, the mice were randomly divided into four groups: a saline group, a high-dose group (0.77 μmol / kg / day), a low-dose group (0.077 μmol / kg / day) of the self-assembled trimer of 17-O-acetyl-17-β-estradiol [17-O-acetyl-17-β-estradiol]3, and a sham-operated group. All mice were treated for four consecutive weeks. Tail bleeding time was measured 30 minutes after the last dose. The next day, the mice were weighed, anesthetized with ether, and blood was collected from the orbital cavity. The mice were then sacrificed, and their organs and femurs were dissected. The organs, including the uterus, were weighed and pathological sections were prepared according to standard procedures. Muscle tissue was removed from the femurs, and their anatomical lengths were measured with a vernier caliper. The femurs were then degreased by soaking them twice in a chloroform / methanol (2 / 1) solution (3 hours each time). The dried weights were then measured by drying them in a 120°C oven for 6 hours. The dried weights were then calcined in a muffle furnace at 800°C for 8 hours, and the ash weights were then measured.

[0037] The femoral dry weights of the treated mice are listed in Table 1. The data in Table 1 demonstrate that at both doses, the self-assembled trimer of 17-O-acetyl-17-β-estradiol [17-O-acetyl-17-β-estradiol] 3 effectively prevents bone loss in ovariectomized mice. In other words, at both doses, the self-assembled trimer of 17-O-acetyl-17-β-estradiol [17-O-acetyl-17-β-estradiol] 3 can prevent bone loss in postmenopausal women.

[0038] Table 1 Femoral dry weight of treated mice

[0039]

[0040] a) P < 0.01 compared with normal saline; b) P < 0.05 compared with normal saline; n = 8

[0041] Femoral bone ashes from treated mice are listed in Table 2. The data in Table 2 demonstrate that at both doses, the self-assembled trimer of 17-O-acetyl-17-β-estradiol [17-O-acetyl-17-β-estradiol] 3 effectively prevents bone mineral loss in ovariectomized mice. In other words, at both doses, the self-assembled trimer of 17-O-acetyl-17-β-estradiol [17-O-acetyl-17-β-estradiol] 3 can prevent bone mineral loss in postmenopausal women.

[0042] Table 2 Femoral ash weight of treated mice

[0043]

[0044] a) P < 0.01 compared with normal saline; b) P < 0.05 compared with normal saline; n = 8

[0045] The bone calcium content of the treated mice's ashes was measured using a colorimetric method, and the data are listed in Table 3. The data in Table 3 demonstrate that the self-assembled trimer of 17-O-acetyl-17-β-estradiol [17-O-acetyl-17-β-estradiol] 3 effectively maintains bone calcium at both doses. In other words, the self-assembled trimer of 17-O-acetyl-17-β-estradiol [17-O-acetyl-17-β-estradiol] 3 can prevent bone calcium loss in postmenopausal women at both doses.

[0046] Table 3 Bone calcium content of treated mice

[0047]

[0048] a) P < 0.01 compared with normal saline; b) P < 0.05 compared with normal saline; n = 8

[0049] The body weights of the treated mice are listed in Table 4. The data in Table 4 demonstrate that at both doses, the self-assembled trimer of 17-O-acetyl-17-β-estradiol [17-O-acetyl-17-β-estradiol] 3 effectively prevented weight gain in ovariectomized mice. In other words, at both doses, the self-assembled trimer of 17-O-acetyl-17-β-estradiol [17-O-acetyl-17-β-estradiol] 3 can prevent obesity in postmenopausal women.

[0050] Table 4 Body weight of treated mice

[0051]

[0052] a) P < 0.05 compared with the sham-operated group; b) P > 0.05 compared with the sham-operated group; n = 8

[0053] The tail bleeding time of treated mice is listed in Table 5. The data in Table 5 show that at both doses, the self-assembled trimer of 17-O-acetyl-17-β-estradiol [17-O-acetyl-17-β-estradiol] 3 did not shorten the tail bleeding time of mice. In other words, at both doses, the self-assembled trimer of 17-O-acetyl-17-β-estradiol [17-O-acetyl-17-β-estradiol] 3 did not pose a coagulation risk in postmenopausal women.

[0054] Table 5 Bleeding time of treated mice tail

[0055]

[0056] a) There is no significant difference between the data of each group; n = 8

[0057] The present invention further discovered that the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol] 3 and the composition of pharmaceutically acceptable excipients have similar effects; the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol] 3 and the composition of other sex hormones have similar effects; the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol] 3 and the composition of other progestogens have similar effects.

[0058] Example 5 Evaluation of the therapeutic effect of the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol] 3 on organ damage caused by menopause

[0059] To investigate the therapeutic effects of a self-assembled trimer of 17-O-acetyl-17-β-estradiol (17-O-acetyl-17-β-estradiol) on menopausal-induced female organ damage, the present invention measured uterine weights in sham-operated mice treated with saline for 28 consecutive days, uterine weights in ovariectomized mice treated with saline for 28 consecutive days, and uterine weights in ovariectomized mice treated with a high dose (0.77 μmol / kg / day) and a low dose (0.077 μmol / kg / day) of the self-assembled trimer of 17-O-acetyl-17-β-estradiol (17-O-acetyl-17-β-estradiol) for 28 consecutive days. The data in Table 6 demonstrate that the self-assembled trimer of 17-O-acetyl-17-β-estradiol (17-O-acetyl-17-β-estradiol) effectively mitigated uterine atrophy in ovariectomized mice at both doses. That is, the self-assembled trimer of 17-O-acetyl-17-β-estradiol [17-O-acetyl-17-β-estradiol]3 prevented uterine atrophy in postmenopausal women at both doses.

[0060] Table 6 Uterine weight of treated mice

[0061]

[0062] a) P < 0.01 compared with normal saline; n = 8

[0063] To investigate the therapeutic effects of a self-assembled trimer of 17-O-acetyl-17-β-estradiol, [17-O-acetyl-17-β-estradiol]3, on menopausal-induced female organ damage, the present invention measured the weight of the oviducts of sham-operated mice treated with saline for 28 consecutive days, the weight of the oviducts of ovariectomized mice treated with saline for 28 consecutive days, and the weight of the oviducts of ovariectomized mice treated with a high dose (0.77 μmol / kg / day) and a low dose (0.077 μmol / kg / day) of 17-O-acetyl-17-β-estradiol, [17-O-acetyl-17-β-estradiol]3, for 28 consecutive days. The data in Table 7 demonstrate that [17-O-acetyl-17-β-estradiol]3 effectively mitigated oviduct atrophy in ovariectomized mice at both doses. That is, the self-assembled trimer of 17-O-acetyl-17-β-estradiol [17-O-acetyl-17-β-estradiol] 3 can prevent fallopian tube atrophy in postmenopausal women at two doses.

[0064] Table 7 Treatment of mice oviduct weight

[0065]

[0066]

[0067] a) P < 0.01 compared with normal saline; n = 8

[0068] Uterine and fallopian tube atrophy are the most prominent menopausal symptoms of the female reproductive system. The data in Tables 6 and 7 consistently demonstrate that the self-assembled trimer of 17-O-acetyl-17-β-estradiol [17-O-acetyl-17-β-estradiol] 3 is effective in treating these symptoms of menopause in women with reproductive problems.

[0069] The present invention further discovered that the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol] 3 and the composition of pharmaceutically acceptable excipients have similar effects; the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol] 3 and the composition of other sex hormones have similar effects; the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol] 3 and the composition of other progestogens have similar effects.

[0070] To investigate the therapeutic effects of a self-assembled trimer of 17-O-acetyl-17-β-estradiol (17-O-acetyl-17-β-estradiol) on menopausal-induced female organ damage, the present invention measured spleen weights in sham-operated mice treated with saline for 28 consecutive days, spleen weights in ovariectomized mice treated with saline for 28 consecutive days, and spleen weights in ovariectomized mice treated with high (0.77 μmol / kg / day) and low (0.077 μmol / kg / day) doses of the self-assembled trimer of 17-O-acetyl-17-β-estradiol (17-O-acetyl-17-β-estradiol) for 28 consecutive days. The data in Table 8 demonstrate that the self-assembled trimer of 17-O-acetyl-17-β-estradiol (17-O-acetyl-17-β-estradiol) effectively prevented spleen weight gain in ovariectomized mice at a dose of 0.77 μmol / kg / day. That is, [17-O-acetyl-17-β estradiol]3 at a dose of 0.77 μmol / kg / day can prevent spleen weight gain in postmenopausal women.

[0071] Table 8 Spleen weight of treated mice

[0072]

[0073] a) P < 0.05 compared with normal saline; n = 8

[0074] To investigate the therapeutic effects of a self-assembled trimer of 17-O-acetyl-17-β-estradiol, [17-O-acetyl-17-β-estradiol]3, on menopausal-induced female organ damage, the present invention measured thymus weights in sham-operated mice treated with saline for 28 consecutive days, thymus weights in ovariectomized mice treated with saline for 28 consecutive days, and thymus weights in ovariectomized mice treated with a high dose (0.77 μmol / kg / day) and a low dose (0.077 μmol / kg / day) of a self-assembled trimer of 17-O-acetyl-17-β-estradiol, [17-O-acetyl-17-β-estradiol]3, for 28 consecutive days. The data in Table 9 demonstrate that [17-O-acetyl-17-β-estradiol]3 effectively mitigated thymic hypertrophy in ovariectomized mice at both doses. That is, the self-assembled trimer of 17-O-acetyl-17-β-estradiol [17-O-acetyl-17-β-estradiol]3 prevented thymic hypertrophy in postmenopausal women at both doses.

[0075] Table 9 Thymus weight of treated mice

[0076]

[0077] a) P < 0.01 compared with normal saline; n = 8

[0078] Splenomegaly and thymic hypertrophy are the most prominent menopausal symptoms in female endocrine organs. The data in Tables 8 and 9 consistently demonstrate that the self-assembled trimer of 17-O-acetyl-17-β-estradiol [17-O-acetyl-17-β-estradiol] 3 can effectively treat menopausal symptoms in the female endocrine system.

[0079] The present invention further discovered that the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol] 3 and the composition of pharmaceutically acceptable excipients have similar effects; the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol] 3 and the composition of other sex hormones have similar effects; the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol] 3 and the composition of other progestogens have similar effects.

[0080] Example 6 Evaluation of the therapeutic effect of the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol] 3 on tissue damage caused by menopause

[0081] To investigate the therapeutic effects of a self-assembled trimer of 17-O-acetyl-17-β-estradiol (17-O-acetyl-17-β-estradiol) on female tissue damage caused by menopause, the present invention prepared pathological sections of the uterus, oviduct, spleen, and thymus of sham-operated mice treated with saline for 28 consecutive days; pathological sections of the uterus, oviduct, spleen, and thymus of ovariectomized mice treated with saline for 28 consecutive days; and pathological sections of the uterus, oviduct, spleen, and thymus of ovariectomized mice treated with 0.77 μmol / kg / day and 0.077 μmol / kg / day of the self-assembled trimer of 17-O-acetyl-17-β-estradiol (17-O-acetyl-17-β-estradiol) ... The formalin-fixed uterus, fallopian tubes, spleen, and thymus were then dehydrated by immersion in 70%, 80%, 90%, 95%, and 100% ethanol for 2.5 hours. The dehydrated uterus, fallopian tubes, spleen, and thymus were then embedded in a 1:1 xylene paraffin solution for 1 hour. The paraffin-embedded uterus, fallopian tubes, spleen, and thymus were sectioned to a thickness of 5 μm. The sections were heated to dryness at 50°C, stained with hematoxylin and eosin, and then transferred to neutral resin for histomorphological photographs under a light microscope.

[0082] Figure 5 The following are photos of the histological morphology of the uterus of sham-operated mice treated with saline for 28 consecutive days, photos of the histological morphology of the uterus of ovariectomized mice treated with saline for 28 consecutive days, and photos of the histological morphology of the uterus of ovariectomized mice treated with 0.77 μmol / kg / day and 0.077 μmol / kg / day of 17-O-acetyl-17-β-estradiol self-assembled trimer [17-O-acetyl-17-β-estradiol]3 for 28 consecutive days. Figure 5 It can be seen that the epithelial cells of the endometrium of the sham-operated mice treated with normal saline for 28 consecutive days have dense villi, healthy round glands are distributed in the fixed layer of the endometrium, and the circular and longitudinal muscles of the myometrium are clearly and orderly arranged. Figure 5 It can also be seen that in ovariectomized mice treated with saline for 28 consecutive days, no villi can be seen on the epithelial cells of the endometrium, the glands in the fixed layer of the endometrium are atrophied, and the dividing line between the circular and longitudinal muscles of the myometrium disappears and becomes disordered. Figure 5Further analysis revealed that the uterine histology of ovariectomized mice treated with 17-O-acetyl-17-β-estradiol self-assembled trimers (17-O-acetyl-17-β-estradiol) 3 at doses of 0.77 μmol / kg / day and 0.077 μmol / kg / day for 28 days was similar to that of sham-operated mice treated with saline for 28 days. This suggests that at both doses, 17-O-acetyl-17-β-estradiol self-assembled trimers (17-O-acetyl-17-β-estradiol) 3 effectively alleviated uterine damage caused by ovariectomy. This suggests that 17-O-acetyl-17-β-estradiol self-assembled trimers (17-O-acetyl-17-β-estradiol) 3 can treat uterine tissue damage caused by menopause in women.

[0083] Figure 6 The following are photos of the histological morphology of the oviduct of sham-operated mice treated with normal saline for 28 consecutive days, photos of the histological morphology of the oviduct of ovariectomized mice treated with normal saline for 28 consecutive days, and photos of the histological morphology of the oviduct of ovariectomized mice treated with 0.77 μmol / kg / day and 0.077 μmol / kg / day of 17-O-acetyl-17-β-estradiol self-assembled trimer [17-O-acetyl-17-β-estradiol]3 for 28 consecutive days. Figure 6 It can be seen that the epithelial cells of the fallopian tube endometrium of the sham-operated mice treated with normal saline for 28 consecutive days have dense villi, healthy round glands are distributed in the fixed layer of the fallopian tube endometrium, and the circular muscle and longitudinal muscle of the fallopian tube muscularis are clearly and orderly arranged. Figure 6 It can also be seen that in ovariectomized mice treated with saline for 28 consecutive days, villi are no longer visible on the epithelial cells of the endometrium, the glands in the fixed layer of the endometrium are atrophied, and the demarcation lines between the circular and longitudinal muscles of the muscularis obliterans are disorganized. Figure 6 further shows that the histological morphology of the oviducts of ovariectomized mice treated with 0.77 μmol / kg / day and 0.077 μmol / kg / day of the self-assembled trimer [17-O-acetyl-17-β-estradiol]3 for 28 consecutive days is similar to that of sham-operated mice treated with saline for 28 consecutive days. This indicates that at both doses, the self-assembled trimer [17-O-acetyl-17-β-estradiol]3 effectively alleviates the tissue damage caused by ovariectomy. That is to say, the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol] 3 can treat tissue damage of the fallopian tube caused by menopause in women.

[0084] Uterine tissue damage and fallopian tube tissue damage are the most concerned menopausal reactions of female reproductive organs caused by menopause. Figure 5 and Figure 6 The results showed that the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol] 3 can effectively treat tissue damage of the reproductive system of women caused by menopause, which is an unexpected technical effect.

[0085] The present invention further discovered that the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol] 3 and the composition of pharmaceutically acceptable excipients have similar effects; the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol] 3 and the composition of other sex hormones have similar effects; the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol] 3 and the composition of other progestogens have similar effects.

[0086] Figure 7 The following are photos of the spleen tissue morphology of sham-operated mice treated with normal saline for 28 consecutive days, photos of the spleen tissue morphology of ovariectomized mice treated with normal saline for 28 consecutive days, and photos of the spleen tissue morphology of ovariectomized mice treated with 0.77 μmol / kg / day and 0.077 μmol / kg / day 17-O-acetyl-17-β estradiol self-assembled trimer [17-O-acetyl-17-β estradiol]3 for 28 consecutive days. Figure 7 It can be seen that the lymphocytes in the spleen of the sham-operated mice treated with normal saline for 28 consecutive days were densely arranged, the white pulp volume was normal, and the red pulp had sufficient cellular components. Figure 7 It can also be seen that the lymphocytes in the spleen of ovariectomized mice treated with normal saline for 28 consecutive days were arranged loosely, the spleen medullary was congested, the volume of the white pulp was significantly reduced, and the cellular components in the red pulp were significantly reduced. Figure 7 Further analysis revealed that the spleen histology of ovariectomized mice treated with 0.77 μmol / kg / day and 0.077 μmol / kg / day of [17-O-acetyl-17-β-estradiol]3 for 28 consecutive days was similar to that of sham-operated mice treated with saline for 28 consecutive days. This suggests that at both doses, [17-O-acetyl-17-β-estradiol]3 effectively alleviated menopausal-induced tissue damage to the spleen.

[0087] Figure 8The following are photos of the thymus tissue morphology of sham-operated mice treated with saline for 28 consecutive days, photos of the thymus tissue morphology of ovariectomized mice treated with saline for 28 consecutive days, and photos of the thymus tissue morphology of ovariectomized mice treated with 0.77 μmol / kg / day and 0.077 μmol / kg / day 17-O-acetyl-17-β estradiol self-assembled trimer [17-O-acetyl-17-β estradiol]3 for 28 consecutive days. Figure 8 It can be seen that the lymphocytes in the thymus of sham-operated mice treated with normal saline for 28 consecutive days are closely arranged, and the ratio of cortex to medulla is normal. Figure 7 It can also be seen that the arrangement of lymphocytes in the thymus of ovariectomized mice treated with saline for 28 consecutive days was loose, the proportion of cortical cells increased, and the proportion of medullary cells decreased. Figure 8 Further analysis revealed that the thymic histology of ovariectomized mice treated with 0.77 μmol / kg / day and 0.077 μmol / kg / day of [17-O-acetyl-17-β-estradiol]3 for 28 consecutive days was similar to the spleen histology of sham-operated mice treated with saline for 28 consecutive days. This suggests that at both doses, [17-O-acetyl-17-β-estradiol]3 effectively mitigated the thymic tissue damage caused by ovariectomy.

[0088] Splenic tissue damage and thymic tissue damage are the most concerned menopausal reactions of female endocrine organs caused by menopause. Figure 8 The results showed that the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol] 3 can effectively treat tissue damage of the endocrine system in women caused by menopause, which is an unexpected technical effect.

[0089] The present invention further discovered that the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol] 3 and the composition of pharmaceutically acceptable excipients have similar effects; the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol] 3 and the composition of other sex hormones have similar effects; the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol] 3 and the composition of other progestogens have similar effects.

[0090] Example 7 Evaluation of the Anti-Venous Thrombotic Activity of the Self-Assembled Trimer of 17-O-Acetyl-17-β-Estradiol [17-O-Acetyl-17-β-Estradiol] 3

[0091] Male SD rats (250 ± 20 g) were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. and used to establish the rat inferior vena cava ligation model. Before the experiment, the rats were acclimated to a 25-28°C environment and fasted for one day. They were then randomly divided into three groups: a positive control group (warfarin), which received an oral dose of 0.82 μmol / kg; a negative control group (CMC-Na), which received an oral dose of 10 mL / kg; and a group (17-O-acetyl-17-β-estradiol), a self-assembled trimer of 17-O-acetyl-17-β-estradiol, which received an oral dose of 5.3 μmol / kg.

[0092] Thirty minutes after administration, the rats were anesthetized with an intraperitoneal injection of 20% urethane solution. The rats' limbs were secured to the board with rubber bands at the joints. The skin was then prepared. A cotton ball moistened with an appropriate amount of soapy water was wiped across the midsection of the rat's abdomen to moisten the surface fur. Carefully scraped with a scraper, avoiding scratching the skin. After skin preparation, the superficial skin was incised along the midsection of the rat's abdomen to expose the muscle layer and reveal the linea alba. A perforated gauze was moistened with saline and placed around the surgical incision. The muscle layer was incised along the linea alba to expose the abdominal cavity and locate the inferior vena cava. The tissues and vessels surrounding the inferior vena cava were bluntly dissected. The left renal vein was ligated at the junction with the inferior vena cava. Immediately after ligation, a timer was placed, beginning a four-hour deep venous thrombosis procedure. The removed organs were returned to the abdominal cavity, and the skin and muscle layer were sutured. The rat was allowed to rest and blood circulation was restored after four hours of deep venous thrombosis. The abdominal wall was incised, and all branches of the inferior vena cava were ligated before the deep venous thrombosis was removed. The branch vessels were then severed, and a 2 cm section of the inferior vena cava was removed. The thrombus was removed from the inferior vena cava, and uncoagulated blood on the clot was blotted with absorbent paper. The wet weight of the clot was measured to indicate antithrombotic activity, and the data are listed in Table 10. The data in Table 10 demonstrate that the self-assembled trimer of 17-O-acetyl-17-β-estradiol [17-O-acetyl-17-β-estradiol]3 exhibits excellent anti-venous thrombotic activity. This is an unexpected technical result.

[0093] Venous thrombosis is one of the most common menopausal symptoms in women. The discovery that the self-assembled trimer of 17-O-acetyl-17-β-estradiol [17-O-acetyl-17-β-estradiol]3 can effectively treat venous thrombosis in women after menopause is an unexpected result.

[0094] The present invention further discovered that the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol] 3 and the composition of pharmaceutically acceptable excipients have similar effects; the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol] 3 and the composition of other sex hormones have similar effects; the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol] 3 and the composition of other progestogens have similar effects.

[0095] Table 10 Anti-venous thrombotic activity of [17-O-acetyl-17-β estradiol] 3

[0096] therapeutic agents dose Thrombus wet weight (mean ± SD mg) CMC-Na 10 mL / kg 21.1±4.0 Warfarin 0.82 μmol / kg 6.8±2.9 <![CDATA[[17-O-acetyl-17-β estradiol]3]]> 5.3 μmol / kg <![CDATA[13.5±4.6 a ]]>

[0097] a) P < 0.01 compared with the CMC-Na group; n = 9

[0098] Example 8 Evaluation of the Anti-Arterial Thrombotic Activity of Self-Assembled Trimers of 17-O-Acetyl-17-β-Estradiol [17-O-Acetyl-17-β-Estradiol] 3

[0099] Polyethylene tubing was stretched into a thin tube with an oblique end, 10.0 cm in length, for cannulation of the right jugular vein (larger diameter) and the left carotid artery (thinner diameter). The middle section of the polyethylene tubing was 8.0 cm in length. Accurately weighed silk thread for thrombus attachment was placed in the direction of the carotid artery cannulation. The tubes were filled with heparin before cannulation. Male rats weighing 200 ± 20 g were acclimated and fasted for one day before surgery. The rats were randomly divided into three groups: a negative control (CMC-Na) group, which received an oral dose of 10 mL / kg; a positive control (aspirin) group, which received an oral dose of 167 μmol / kg; and a self-assembled trimer of 17-O-acetyl-17-β-estradiol (17-O-acetyl-17-β-estradiol) group, which received an oral dose of 5.3 μmol / kg. Thirty minutes after drug administration, the rats were anesthetized with an intraperitoneal injection of 20% urethane solution (7 mL / kg). Surgery began 2 minutes later. During surgery, the rat was placed supine on a restraining board. The skin of the neck was incised, and the left jugular vein from the right common carotid artery was isolated. A suture was placed under the vascular cord, and the distal end was ligated. A small incision was made near the distal end of the vein, and a cannula was inserted into the vein. Heparin was injected, and the heparin syringe was removed. The suture was then tied to secure the artery. The proximal end of the artery was clamped with an arterial clamp. A small incision was made near the distal end, and the arterial end was ligated. After securing the arterial clamp, the arterial clamp was released to establish extracorporeal bypass. After 15 minutes of circulation, the venous end was cut and blood circulation was observed for normality. If normal, the suture with thrombus was removed from the arterial end. Uncoagulated blood on the suture with thrombus was blotted with absorbent paper and accurately weighed. The weight gain of the suture was calculated to indicate antithrombotic activity, and the data are shown in Table 11. The data in Table 11 show that the self-assembled trimer of 17-O-acetyl-17-β-estradiol [17-O-acetyl-17-β-estradiol] 3 has excellent anti-arterial thrombotic activity, which is an unexpected technical effect.

[0100] Arterial thrombosis is one of the most common menopausal symptoms in women. The discovery that the self-assembled trimer of 17-O-acetyl-17-β-estradiol [17-O-acetyl-17-β-estradiol]3 can effectively treat arterial thrombosis in women after menopause is an unexpected result.

[0101] The present invention further discovered that the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol] 3 and the composition of pharmaceutically acceptable excipients have similar effects; the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol] 3 and the composition of other sex hormones have similar effects; the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol] 3 and the composition of other progestogens have similar effects.

[0102] Table 11 Anti-arterial thrombotic activity of [17-O-acetyl-17-β estradiol] 3

[0103] therapeutic agents dose Thrombus wet weight (mean ± SD mg) CMC-Na 10 mL / kg 34.2±7.4 aspirin 167 μmol / kg 18.9±4.7 <![CDATA[[17-O-acetyl-17-β estradiol]3]]> 5.3 μmol / kg <![CDATA[23.5±7.8 a ]]>

[0104] a) Compared with the CMC-Na group, P<0.01; n=9.

Claims

1. A self-assembled trimer of 17-O-acetyl-17-β estradiol having the following structural formula [17-O-acetyl-17-β estradiol] 3, 2. A method for preparing the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol] 3 according to claim 1, the method comprising the following steps: 1) Dissolve estradiol in acetic anhydride, and stir the resulting solution at room temperature for 12 hours; 2) slowly diluting the reaction solution stirred at room temperature for 12 hours with distilled water, wherein the volume of distilled water used for dilution is 5-20 times the volume of acetic anhydride; 3) Concentrate the above diluted solution under reduced pressure to remove the dilute acetic acid; 4) The residue was neutralized with saturated sodium bicarbonate aqueous solution to pH 8 at 0°C to allow the generated precipitate to fully precipitate, and filtered to obtain the self-assembled trimer of 17-O-acetyl-17-β-estradiol [17-O-acetyl-17-β-estradiol]3; 5) Structural characterization of the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol] 3.

3. Use of the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol] 3 according to claim 1 in the preparation of a drug for preventing osteoporosis in postmenopausal women.

4. Use of the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol] 3 according to claim 1 in the preparation of an anti-venous thrombotic drug.

5. Use of the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol]3 according to claim 1 in the preparation of an anti-arterial thrombotic drug.

6. Use of the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol] 3 according to claim 1 in the preparation of a drug for preventing reproductive organ damage caused by menopause in women.

7. Use of the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol] 3 according to claim 1 in the preparation of a drug for preventing spleen damage caused by menopause in women.

8. Use of the self-assembled trimer of 17-O-acetyl-17-β estradiol [17-O-acetyl-17-β estradiol]3 according to claim 1 in the preparation of a drug for preventing thymus damage caused by menopause in women.

9. The preparation method according to claim 2, characterized in that The volume of distilled water used for dilution in the described method is 10 times the volume of acetic anhydride.

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