CN104940188A - Compound for treating and/or preventing estrogen associated diseases - Google Patents

Abstract

The embodiment of the present invention provides the use of the compound of the general formula (I) in the preparation of drugs for the treatment and/or prevention of estrogen-related diseases; the embodiment of the present invention also provides the compound of the general formula (I) and Epimedium extract of the compound of general formula (I).

Description

Compounds for the treatment and/or prevention of estrogen-related diseases

technical field

The present invention relates to the field of phytochemistry, in particular to compounds for treating and/or preventing estrogen-related diseases.

Background technique

A class of active ingredients in plants with estrogen-like or anti-estrogenic effects is called phytoestrogens (PE). It is known in the prior art that in the human body, phytoestrogens have a two-way regulating effect. On the one hand, when the estrogen level in the body is lower than the normal level, it can exert an estrogen-like effect, which can prevent and treat climacteric syndrome in women , prostate cancer, osteoporosis, and cardiovascular disease. On the other hand, when the level of estrogen in the body is higher than the normal level (such as mammary gland hyperplasia, uterine fibroids and other diseases), it can produce estrogen antagonism, effectively weakening the response of target cells to estrogen. Therefore, some people call it "selective estrogen receptor modulator". It is still expected by those skilled in the art to find phytoestrogens with estrogenic activity and no estrogenic side effects, so as to provide new treatment options for the clinic.

Contents of the invention

Epimedium, also known as Xianlingpi, is the dry aerial part of various plants of the genus Epimedium in the family Berberdiaceae. It is a traditional Chinese herbal medicine with the functions of tonifying the kidney and strengthening yang, and expelling wind and dampness. Epimedium has a long history of medicinal use. Li Shizhen said in "Compendium of Materia Medica" that it has the effect of "benefiting essence, strengthening muscles and bones, strengthening waist and knees, and strengthening heart". Modern pharmacological experiments have shown that Epimedium can enhance cardiovascular and cerebrovascular blood flow, promote hematopoietic function, improve immune function, regulate bone metabolism, and also have anti-aging and anti-tumor effects.

The inventors isolated a series of compounds from Epimedium, and surprisingly found that the isolated series of compounds have estrogen-like or anti-estrogenic activity, and can be used as phytoestrogens for the treatment of estrogen related diseases. And based on this, the present invention has been accomplished.

The first aspect of the present invention provides the use of the compound of general formula (I) in the preparation of drugs for the treatment and/or prevention of estrogen-related diseases;

Wherein, R ₁ , R ₂ , R ₃ , R ₄ , and R ₅ each independently represent -H, -OH, -OCH ₃ ,

R ₆ and R ₇ independently represent -H, -OH, -OCH ₃ , Or R ₆ , R ₇ and the carbon atoms connected to R ₆ and R ₇ respectively form an oxa five-membered ring.

In some preferred embodiments of the _first aspect of the present invention, R represents -H,

R ₂ and R ₃ independently represent -OH;

R ₄ represents -H, -OH or

R ₅ represents -H,

R ₆ represents -H, -OH or R ₇ represents -OH or -OCH ₃ , or R ₆ , R ₇ and the carbon atoms connected to R ₆ and R ₇ respectively form 2-(1-hydroxyl-1-methyl-ethyl)-2,3- Dihydrofuran ring.

In some preferred embodiments of the first aspect of the present invention, estrogen-related diseases may refer to diseases caused by lower estrogen levels than normal levels.

Wherein, the diseases caused by estrogen levels lower than normal levels may include: female climacteric syndrome, prostate cancer, osteoporosis and cardiovascular diseases.

In other preferred embodiments of the first aspect of the present invention, estrogen-related diseases may refer to diseases caused by estrogen levels higher than normal.

Among them, diseases caused by estrogen levels higher than normal levels may include: breast hyperplasia, uterine fibroids.

The second aspect of the present invention provides the compound of general formula (I),

Wherein, R represents _- H, R ₂ and R ₃ independently represent -OH; R ₄ represents -H, -OH or R ₅ represents -H, R ₆ represents -H, -OH or R ₇ represents -OH or -OCH ₃ .

In some preferred embodiments of the second aspect of the invention,

R ₂ is the same as R ₃ and represents -OH;

where R1 _stands for _In case R4 represents -H or R ₅ stands for -H or R ₆ represents -H or -OH; R ₇ represents -OH or -OCH ₃ ;

Where R ₁ represents -H, R ₄ represents -OH; R ₅ represents R ₆ stands for R ₇ represents -OH;

where R1 _stands for In the case of , R ₄ represents -H; R ₅ represents R ₆ represents -OH; R ₇ represents -OH.

The compound of the above general formula (I) can be selected from the following compounds:

It should be noted that the dotted lines of the groups represented by R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , and R ₇ indicate that the group forms a bond with the connection of the general formula (I).

The third aspect of the present invention provides the Epimedium extract capable of isolating the compound of general formula (I). The compound of the general formula (I) can be obtained through the epimedium extract, and further, the compound of the general formula (I) can be used to treat estrogen-related diseases.

Detailed ways

The technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments. Apparently, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

Embodiment 1: preparation epimedium extract

Take 20kg of the whole herb of Epimedium, extract it with 95% (v/v) ethanol under heat reflux, the ratio of extraction material to liquid is 1:1.5, and the extraction time is 2 hours each time, for a total of 3 extractions. Distilled under reduced pressure at 50°C, concentrated to no alcohol, the volume is about 6L, and the estimated mass of the extract is about 4kg. Dissolve 3 kg of crude extract in 10 L of water, extract with an equal volume of ethyl acetate, extract 3 times in total, distill the ethyl acetate layer under reduced pressure, and concentrate to dryness to obtain about 1 kg of extract. The water layer was distilled under reduced pressure at 50°C to a thick extract, and the mass of the extract was about 2kg.

Embodiment 2: preparation compound 1-11

The ethyl acetate layer extract and the water layer extract were separated by silica gel column chromatography, macroporous resin main chromatography, Sephadex LH-20 gel column chromatography, ODS column chromatography and preparative liquid chromatography. Compounds 1-11 were obtained, as shown in Table 1.

specific:

Compound 1 was isolated as follows:

Get ethyl acetate extract (1kg), mix sample with 1.5kg silica gel, carry out silica gel column chromatography separation, with dichloromethane-acetone (1:0→10:1→5:1→2:1→1:1 →0:1) for gradient elution, TLC detection and merge the same fractions to obtain 43 fractions (Fr.1～43). Fr.28 (30g) was chromatographed on an open C18 column, gradient eluted with methanol-water (10:90→100:0), detected by HPLC and combined to obtain 11 fractions (Fr.28-1～11) . Compound 1 (22 mg) was obtained from Fr.28-8 (1.4 g) by reverse-phase high-performance liquid chromatography.

Compound 2 was isolated as follows:

Get ethyl acetate extract (1kg), mix sample with 1.5kg silica gel, carry out silica gel column chromatography separation, with dichloromethane-acetone (1:0→10:1→5:1→2:1→1:1 →0:1) for gradient elution, TLC detection and merge the same fractions to obtain 43 fractions (Fr.1～43). Fr.28 (30g) was chromatographed on an open C18 column, gradient eluted with methanol-water (10:90→100:0), detected by HPLC and combined to obtain 11 fractions (Fr.28-1～11) . Compound 2 (22 mg) was obtained from Fr.28-8 (1.4 g) by reverse-phase high-performance liquid chromatography.

Compound 3 was isolated as follows:

Get ethyl acetate extract (1kg), mix sample with 1.5kg silica gel, carry out silica gel column chromatography separation, with dichloromethane-acetone (1:0→10:1→5:1→2:1→1:1 →0:1) for gradient elution, TLC detection and merge the same fractions to obtain 43 fractions (Fr.1～43). Fr.28 (30g) was chromatographed on an open C18 column, gradient eluted with methanol-water (10:90→100:0), detected by HPLC and combined to obtain 11 fractions (Fr.28-1～11) . Compound 3 (40 mg) was obtained from Fr.28-9 (1.3 g) by reverse-phase high-performance liquid chromatography.

Compound 4 was isolated as follows:

Get ethyl acetate extract (1kg), mix sample with 1.5kg silica gel, carry out silica gel column chromatography separation, with dichloromethane-acetone (1:0→10:1→5:1→2:1→1:1 →0:1) for gradient elution, TLC detection and merge the same fractions to obtain 43 fractions (Fr.1～43). Fr.28 (30g) was chromatographed on an open C18 column, gradient eluted with methanol-water (10:90→100:0), detected by HPLC and combined to obtain 11 fractions (Fr.28-1～11) . Compound 4 (1000 mg) was obtained from Fr.28-11 (2.2 g) by reverse-phase high-performance liquid chromatography.

Compound 5 was isolated as follows:

Get ethyl acetate extract (1kg), mix sample with 1.5kg silica gel, carry out silica gel column chromatography separation, with dichloromethane-acetone (1:0→10:1→5:1→2:1→1:1 →0:1) for gradient elution, TLC detection and merge the same fractions to obtain 43 fractions (Fr.1～43). Fr.43 (100g) was chromatographed on an open C18 column, gradient eluted with methanol-water (10:90→100:0), detected by HPLC and combined to obtain 15 fractions (Fr.43-1～15) . Compound 5 (20 mg) was obtained from Fr.43-8 (20.2 g) by reverse-phase high-performance liquid chromatography.

Compound 6 was isolated as follows:

Get ethyl acetate extract (1kg), mix sample with 1.5kg silica gel, carry out silica gel column chromatography separation, with dichloromethane-acetone (1:0→10:1→5:1→2:1→1:1 →0:1) for gradient elution, TLC detection and merge the same fractions to obtain 43 fractions (Fr.1～43). Fr.43 (100g) was chromatographed on an open C18 column, gradient eluted with methanol-water (10:90→100:0), detected by HPLC and combined to obtain 15 fractions (Fr.43-1～15) . Fr.43-8 (20.2 g) was recrystallized from methanol to obtain Compound 6 (1500 mg).

Compound 7 was isolated as follows:

Get ethyl acetate extract (1kg), mix sample with 1.5kg silica gel, carry out silica gel column chromatography separation, with dichloromethane-acetone (1:0→10:1→5:1→2:1→1:1 →0:1) for gradient elution, TLC detection and merge the same fractions to obtain 43 fractions (Fr.1～43). Fr.8 (100g) was chromatographed on an open C18 column, gradient eluted with methanol-water (10:90→100:0), detected by HPLC and combined to obtain 8 fractions (Fr.8-1～8) . Compound 7 (90 mg) was obtained from Fr.8-7 (15.4 g) by reverse-phase high-performance liquid chromatography.

Compound 8 was isolated as follows:

Get ethyl acetate extract (1kg), mix sample with 1.5kg silica gel, carry out silica gel column chromatography separation, with dichloromethane-acetone (1:0→10:1→5:1→2:1→1:1 →0:1) for gradient elution, TLC detection and merge the same fractions to obtain 43 fractions (Fr.1～43). Fr.28 (30g) was chromatographed on an open C18 column, gradient eluted with methanol-water (10:90→100:0), detected by HPLC and combined to obtain 11 fractions (Fr.28-1～11) . Compound 8 (54 mg) was obtained from Fr.28-5 (1.9 g) by reverse-phase high-performance liquid chromatography.

Compound 9 was isolated as follows:

Get ethyl acetate extract (1kg), mix sample with 1.5kg silica gel, carry out silica gel column chromatography separation, with dichloromethane-acetone (1:0→10:1→5:1→2:1→1:1 →0:1) for gradient elution, TLC detection and merge the same fractions to obtain 43 fractions (Fr.1～43). Fr.43 (100g) was chromatographed on an open C18 column, gradient eluted with methanol-water (10:90→100:0), detected by HPLC and combined to obtain 15 fractions (Fr.43-1～15) . Compound 9 (30 mg) was obtained from Fr.43-8 (20.2 g) by reverse-phase high-performance liquid chromatography.

Compound 10 was isolated as follows:

Get ethyl acetate extract (1kg), mix sample with 1.5kg silica gel, carry out silica gel column chromatography separation, with dichloromethane-acetone (1:0→10:1→5:1→2:1→1:1 →0:1) for gradient elution, TLC detection and merge the same fractions to obtain 43 fractions (Fr.1～43). Fr.25 (30g) was chromatographed on an open C18 column, gradient eluted with methanol-water (10:90→100:0), detected by HPLC and combined to obtain 6 fractions (Fr.25-1～6) . Compound 10 (28 mg) was obtained by separating Fr.25-3 (4.2 g) by reverse-phase high performance liquid chromatography.

Compound 11 was isolated as follows:

Get ethyl acetate extract (1kg), mix sample with 1.5kg silica gel, carry out silica gel column chromatography separation, with dichloromethane-acetone (1:0→10:1→5:1→2:1→1:1 →0:1) for gradient elution, TLC detection and merge the same fractions to obtain 43 fractions (Fr.1～43). Fr.38 (100g) was chromatographed on an open C18 column, gradient eluted with methanol-water (10:90→100:0), detected by HPLC and combined to obtain 6 fractions (Fr.38-1～6) . Compound 11 (54 mg) was obtained by separating Fr.38-3 (3.3 g) by reverse-phase high performance liquid chromatography.

Table 1 Compounds 1-11

Embodiment 3: Determination of the structure of compound 1-11

The structure of compound 1-11 was determined by ¹ H, ¹³ C-NMR;

The model of the NMR instrument is: BRUKER AVANCE Ⅲ 500 superconducting nuclear magnetic resonance spectrometer (TMS is the internal standard, Swiss Bruker company);

specific:

Compound 1 was characterized as follows:

¹ H-NMR (500MHz, DMSO-d ₆ ): δ1.64 (3H, s, H-4″), 1.74 (3H, s, H-5″′), 1.77 (3H, s, H-5″ ),2.71(1H,dd,J=7.5,14.0Hz,H-1″’a),2.71(1H,dd,J=5.0,14.0Hz,H-1″’b),3.45(2H,d, J=7.0Hz, H-1″), 4.25 (1H, dd, J=5.0, 7.5Hz, H-2″′), 4.71 (1H, br s, H-4″′a), 4.81 (1H, br s, H-4″′b), 5.24 (1H, t, J=7.0Hz, H-2″), 6.29 (1H, s, H-6), 6.62 (1H, s, H-3), 7.31 (1H, d, J=2.0Hz, H-6'), 7.34 (1H,d, J=2.0Hz, H-2').

¹³ C-NMR (125MHz, DMSO-d ₆ ): δ17.6 (C-5″′), 17.8 (C-5″), 21.3 (C-1″), 25.4 (C-4″), 36.6 ( C-1"'), 73.7(C-2"'), 98.2(C-6), 102.5(C-3), 103.5(C-10), 106.0(C-8), 110.0(C-4" ′), 111.1(C-6′), 120.7(C-2′), 120.8(C-1′), 122.3(C-2″), 126.8(C-3′), 131.0(C-3″) ,145.3(C-5′),147.8(C-3″′),147.9(C-4′),154.3(C-9),159.0(C-5),161.5(C-7),164.0(C -2), 181.8 (C-4).

Compound 2 was characterized as follows:

¹ H-NMR (500MHz, DMSO-d ₆ ): δ1.70 (3H, s, H-4″), 1.71 (3H, s, H-5″), 3.29 (2H, d, J=7.0Hz, H-1″),5.32(1H,t,J=7.0Hz,H-2″),6.20(1H,d,J=2.0Hz,H-6),6.42(1H,d,J=2.0Hz, H-8), 6.62 (1H, s, H-3), 7.28 (1H, d, J=2.0Hz, H-6′), 7.30 (1H, d, J=2.0Hz, H-2′).

¹³ C-NMR (125MHz, DMSO-d ₆ ): δ17.6 (C-5″), 25.4 (C-4″), 28.2 (C-1″), 93.7 (C-8), 98.7 (C- 6), 102.7(C-3), 103.6(C-10), 110.6(C-6′), 119.2(C-2′), 120.6(C-1′), 122.4(C-2″), 128.9 (C-3′), 131.4(C-3″), 145.1(C-5′), 147.4(C-4′), 157.2(C-9), 161.4(C-5), 164.0(C-7 ), 164.0 (C-2), 181.5 (C-4).

Compound 3 is characterized as follows:

¹ H-NMR (500MHz, MeOD): δ1.76 (6H, s, H-4″′, 5″′), 1.79 (3H, s, H-5″), 3.08 (1H, dd, J=7.0 ,14.0Hz,H-1″a),3.15(1H,dd,J=7.0,14.0Hz,H-1″b),3.36(2H,d,J=7.0Hz,H-1″′),4.45 (1H,t,J=7.0Hz,H-2″),4.70(1H,br s,H-4″a),4.76(1H,brs,H-4″b),5.35(1H,t,J =7.0Hz, H-2"'), 6.24(1H, s, H-6), 6.46(1H, s, H-3), 7.26(1H, d, J=2.0Hz, H-6'), 7.29 (1H, d, J = 2.0 Hz, H - 2').

¹³ C-NMR (125MHz, MeOD): δ17.9 (C-5″), 18.0 (C-5″′), 26.0 (C-4″′), 29.4 (C-1″′), 30.3 (C -1″), 76.4 (C-2″), 99.9 (C-6), 103.4 (C-3), 105.3 (C-10), 105.5 (C-8), 111.7 (C-6′), 111.7 (C-4″), 120.7 (C-2′), 123.0 (C-1′), 123.5 (C-2″′), 130.4 (C-3″′), 133.6 (C-3′), 146.5 (C-5′), 148.5(C-3″), 149.0(C-4′), 157.0(C-9), 161.3(C-5), 164.3(C-7), 166.6(C-2) , 184.3 (C-4).

Compound 4 is characterized as follows:

¹ H-NMR (500MHz, MeOD): δ1.66 (3H, s, H-4″), 1.73 (3H, s, H-5″′), 1.75 (3H, s, H-4″′), 1.79(3H,s,H-5″),3.35(2H,d,J=7.5Hz,H-1″′),3.48(2H,d,J=7.0Hz,H-1″),5.27(1H ,t,J=7.0Hz,H-2″’),5.33(1H,t,J=7.0Hz,H-2″),6.23(1H,s,H-6),6.44(1H,s,H -3), 7.22 (1H, d, J=2.0Hz, H-2'), 7.30 (1H, d, J=2.0Hz, H-6').

¹³ C-NMR (125MHz, MeOD): δ17.9 (C-5″′), 18.2 (C-5″), 22.7 (C-1″), 25.9 (C-4″′), 26.0 (C- 4″), 29.1 (C-1″′), 99.6 (C-6), 103.2 (C-3), 105.2 (C-10), 108.3 (C-8), 111.6 (C-2′), 120.4 (C-6′), 123.0(C-1′), 123.2(C-2″), 123.6(C-2″′), 130.2(C-5′), 132.7(C-3″), 134.0( C-3"'), 146.5(C-3'), 149.0(C-4'), 156.4(C-9), 160.8(C-5), 163.4(C-7), 166.5(C-2) , 184.2 (C-4).

Compound 5 is characterized as follows:

¹ H-NMR (500MHz, MeOD): δ0.90 (3H, d, J=6.0Hz, H-6″′), 1.66 (3H, s, H-4″), 1.71 (3H, s, H- 5″),3.28(1H,m,H-3″′),3.32(1H,m,H-4″′),3.43(2H,m,H-1″),3.71(1H,dd,J= 3.0, 9.0Hz, H-5″′), 3.89 (3H, s, OMe-4′), 4.22 (1H, m, H-2″′), 5.19 (1H, t, J=7.0Hz, H- 2″), 5.39 (1H, s, H-1″′), 6.26 (1H, s, H-6), 7.08 (1H, d, J=8.5Hz, H-3′, 5′), 7.86 ( 1H, d, J = 8.5 Hz, H - 2', 6').

¹³ C-NMR (125MHz, MeOD): δ17.7 (C-6″′), 18.2 (C-5″), 22.5 (C-1″), 25.9 (C-4″), 56.0 (OCH ₃ - 4'), 72.0(C-2"'), 72.1(C-3"'), 72.2(C-5"'), 73.2(C-4"'), 99.4(C-6), 103.5(C -1″′), 106.0(C-8), 108.0(C-10), 115.2(C-3′,5′), 123.7(C-2″), 124.2(C-1′), 131.8(C -2′,6′),132.5(C-3″),136.3(C-3),155.8(C-9),158.8(C-4′),160.9(C-2),163.3(C-5 ), 163.4(C-7), 179.9(C-4).

Compound 6 is characterized as follows:

¹ H-NMR (500MHz, MeOD): δ0.90 (3H, d, J = 6.0Hz, H-6″′), 1.64 (3H, s, H-4″), 1.72 (3H, s, H- 5″), 3.26～3.74 (9H, H-2″′, 3″′, 5″′, 2″″, 3″″, 4″″, 5″″, 6″″), 3.89 (3H, s ,OMe-4′),4.22(1H,m,H-4″′),5.07(1H,d,J=8.0Hz,H-1″″),5.20(1H,t,J=7.0Hz,H -2″), 5.41 (1H, d, J=1.5Hz, H-1″′), 6.65 (1H, s, H-6), 7.10 (1H, d, J=8.5Hz, H-3′, 5'), 7.89 (1H, d, J=8.5Hz, H-2', 6').

¹³ C-NMR (125MHz, MeOD): δ17.7 (C-6″′), 18.3 (C-5″), 22.8 (C-1″), 25.9 (C-4″), 56.1 (OCH ₃ - 4′), 62.4(C-6″″), 71.2(C-2″′), 72.0(C-3″′), 72.1(C-4″″), 72.1(C-5″′), 73.2 (C-4″′), 75.0 (C-2″″), 78.3 (C-3″″), 78.4 (C-5″″), 99.4 (C-6), 102.0 (C-1″″) ,103.5(C-1″′),107.6(C-8),110.6(C-10),115.3(C-3′,5′),123.6(C-2″),124.0(C-1′) ,131.9(C-2′,6′),132.7(C-3″),136.5(C-3),155.1(C-9),159.4(C-4′),161.1(C-2),162.1 (C-5), 163.6 (C-7), 180.2 (C-4).

Compound 7 is characterized as follows:

¹ H-NMR (500MHz, MeOD): δ3.77(3H,s,CH ₃ O-3),3.87(3H,s,CH ₃ O-4′),3.90(3H,s,CH ₃ O-5 ),3.91(3H,s,CH ₃ O-7),6.45(1H,d,J=2.0Hz,H-6),6.66(1H,d,J=2.0Hz,H-8),7.05(2H , d, J=9.0Hz, H-3', 5'), 8.05 (2H, d, J=9.0Hz, H-2', 6').

¹³ C-NMR (125MHz, MeOD): δ56.0(CH ₃ O-4′), 56.6(CH ₃ O-5), 56.6(CH ₃ O-7), 60.3(CH ₃ O-3), 93.8 (C-8),97.1(C-6),109.8(C-10),115.1(C-3′,5′),123.9(C-1′),131.1(C-2′,6′), 141.9 (C-3), 155.3 (C-2), 160.3 (C-9), 162.1 (C-5), 163.2 (C-4′), 166.3 (C-7), 176.0 (C-4).

Compound 8 is characterized as follows:

¹ H-NMR (500MHz, MeOD): δ6.18 (1H, br s, H-6), 6.38 (1H, br s, H-8), 6.88 (1H, d, J=8.5Hz, H-5 '), 7.62 (1H, d, J=8.5Hz, H-6'), 7.73 (1H, s, H-2').

¹³ C-NMR (125MHz, MeOD): δ94.5(C-8), 99.3(C-6), 104.6(C-10), 116.0(C-2′), 116.3(C-5′), 121.7 (C-6′), 124.2(C-1′), 137.3(C-3), 146.3(C-3′), 148.0(C-2), 148.8(C-4′), 158.3(C-9 ), 162.5 (C-5), 165.6 (C-7), 177.4 (C-4).

Compound 9 is characterized as follows:

¹ H-NMR (500MHz, DMSO-d ₆ ): δ3.30 (1H, m, H-6″a), 3.32 (1H, m, H-5″), 3.35 (1H, m, H-3″ ),3.46(1H,m,H-6″b),3.57(1H,m,H-2″),3.65(1H,m,H-4″),5.38(1H,d,J＝7.5Hz, H-1″),6.21(1H,d,J=2.0Hz,H-6),6.41(1H,d,J=2.0Hz,H-8),6.82(1H,d,J=8.0Hz,H -5'), 7.53 (1H, d, J=2.0Hz, H-2'), 7.67 (1H, dd, J=2.0, 8.0Hz, H-6').

¹³ C-NMR (125MHz, DMSO-d ₆ ): δ60.0 (C-6″), 67.8 (C-4″), 71.1 (C-2″), 73.1 (C-3″), 75.7 (C -5″),93.4(C-8),98.6(C-6),101.7(C-1″),103.8(C-10),115.1(C-2′),115.8(C-5′), 121.0(C-1′), 121.9(C-6′), 133.4(C-3), 144.7(C-3′), 148.4(C-4′), 156.1(C-9), 156.2(C- 2), 161.1 (C-5), 164.0 (C-7), 177.4 (C-4).

Compound 10 is characterized as follows:

¹ H-NMR (500MHz, MeOD): δ1.68 (3H, s, H-4″′), 1.81 (3H, s, H-5″′), 1.82 (3H, s, H-5″), 2.92(2H,m,H-1″),3.53(2H,d,J=7.0Hz,H-1″′),4.41(1H,t,J=7.0Hz,H-2″),4.79(1H ,br s,H-4″a),4.90(1H,br s,H-4″b),5.27(1H,t,J=7.0Hz,H-2″′),6.26(1H,s,H -6), 6.52 (1H, s, H-8), 7.29 (1H, d, J=2.0Hz, H-6'), 7.31 (1H, d, J=2.0Hz, H-2').

¹³ C-NMR (125MHz, MeOD): δ18.1 (C-5″), 18.3 (C-5″′), 22.7 (C-4″′), 26.0 (C-1″′), 38.4 (C -1″),76.8(C-2″),99.6(C-6),103.7(C-3),105.3(C-10),108.3(C-8),111.6(C-4″),112.5 (C-6′),122.3(C-2′),123.3(C-1′),123.7(C-2″′),128.0(C-3″′),132.7(C-3′),147.1 (C-5′), 148.4(C-3″), 149.5(C-4′), 156.5(C-9), 160.9(C-5), 163.4(C-7), 166.6(C-2) , 184.3 (C-4).

Compound 11 was characterized as follows:

¹ H-NMR (500MHz, MeOD): δ1.30 (6H, s, H-4″′, 5″′), 1.68 (3H, s, H-4″), 1.83 (3H, s, H-5 ″), 3.54 (2H, d, J=7.0Hz, H-1″), 4.38 (1H, d, J=5.0Hz, H-2″′), 5.24 (1H, t, J=7.0Hz, H -2″), 5.39 (1H, d, J=5.0Hz, H-1″′), 6.27 (1H, s, H-6), 6.57 (1H, s, H-3), 7.39 (1H, d , J=2.0Hz, H-2′), 7.56 (1H, d, J=2.0Hz, H-6′).

¹³ C-NMR (125MHz, MeOD): δ18.3 (C-5″), 22.7 (C-1″), 25.4 (C-4″′), 25.5 (C-5″′), 25.9 (C- 4″), 72.0 (C-1″′), 74.0 (C-3″′), 99.5 (C-6), 99.7 (C-2″′), 104.1 (C-3), 105.4 (C-8 ),108.3(C-10),116.1(C-2′),116.5(C-6′),123.7(C-1′),126.2(C-2″),132.8(C-5′),133.0 (C-3″),143.4(C-3′),153.0(C-4′),156.6(C-9),160.9(C-5),163.5(C-7),184.3(C-4) .

Embodiment 4: The estrogen activity experiment of compound 1-11

Reagent: DMEM/F-12, HEPES, no phenol red is Produced by the company; the article number is 11039-021 and the specification is 500ml/bottle;

Fetal bovine serum FBS adsorbed by activated carbon is produced by BioInd Company, the article number is 04-201-1A, and the specification is 500ml/bottle;

penicillin streptomycin solution is produced by hyclone company, the article number is SV30010 and the specification is 100ml/bottle;

FBS produces for Hyclone Company. Item No.: SH30084.03E Specification 500ml;

ERαAntibody (F-10) is produced by Santa Cruz Company. Product number: sc-8002 Specification 200μg/ml;

Donkey Anti-Mouse IgG H&L (Alexa 488) for abcam company production number: ab150105 500ug;

Hochest33342 is produced by SIGMA company. Item number: 14533;

Tamoxifen is produced for Sigma-Aldrich. Item No.: T5648-1G

PBS is prepared from KH2PO4, Na2HPO4, NaCl, KCl powder with ultrapure water, and the powders are all produced by Sigma;

DMSO is produced by Suolaibao Company, cell culture grade, article number: D8371, specification 50ml/bottle;

4% paraformaldehyde is produced by Suleibao Company, article number: P1110, specification 500ml/bottle;

TritonX-100 is produced by Suleibao Company, the product number is T8200, the specification is 100ml/bottle;

Tween-20 is produced by Suleibao Company. Item No.: T8220 Specification 100ml;

Instrument: High Screening Assay (Operetta) was purchased from Perkin Elmer;

The CO2 incubator IL-161HI is produced by STIK;

Adjustable speed and timing vortex mixer (SI-T256)

The electric heating constant temperature water bath is produced by Tianjin Uno Instrument Company;

Pipette gun is produced by Eppendorf;

Nikon ECLIPSE Ti-U Inverted Biological Microscope

1.6R desktop multi-purpose centrifuge is produced by Thermo company;

The water generator is the Milli-Q Century ultrapure water system of the United States, and the models are Milli-Q Academic, Milli-Q Grandient, Milli-Q Biocel, Milli-Q Synthesis;

4℃ Haier refrigerator;

-20℃ Haier refrigerator;

Sample preparation: Divide the weighed sample mass m by the relative molecular mass M of the sample to obtain the substance amount n of the sample, and then add 10*n volumes of DMSO to obtain an initial concentration of 10 ^-1 mol/L. Take 10ul volume of 10 ^-1 mol/L drug, dilute it to 100ul with DMSO to obtain a drug concentration of 10 ^-2 mol/L, and so on, dilute to 10 ^-4 mol/L, and divide into 10ul each tube, stored at -20°C.

Compounds that selectively activate estrogen receptor nuclear import were screened by indirect immunofluorescence.

1. Cells and their culture

MCF-7 cells ( Number: HTB-22 ^TM ) was purchased from ATCC and subcultured by our laboratory; MCF-7 cells were cultured at 37°C in a 5% CO2 cell culture incubator with 10% FBS/DMEM (4500mg/L D-glucose, L-glucose aminoacyl and 110mg/L sodium pyruvate, 100U/ml penicillin, 100μg/ml streptomycin).

2. Nuclear migration experiment steps

MCF-7 cells were cultured in phenol red-free DMEM/F12 medium containing 10% activated carbon-adsorbed fetal bovine serum and 1% double antibody in a cell culture incubator at 37°C and 5% CO ₂ . After 72 hours, the cell density was about 80%. Spread the cells in a 96-well blackboard at a density of 7000/well. The middle 60 wells were experimental wells. Add 1% activated carbon-adsorbed serum and 1% double-antibody-free serum to each well. 100ul of phenol red DMEM/F12 culture solution, fill the surrounding wells with 100ml of PBS, culture overnight, until the cells are completely adhered to the wall. Dilute the drug with phenol red-free DMEM/F12 medium (the drug is diluted to 10 ^-4 M with DMSO, and stored in a -20°C refrigerator) to 10 ^-7 M. 100ul drug solution with a concentration of 10 ^-7 M per well in a 96-well plate, the positive drug is tamoxifen, and the blank control is added with phenol red-free DMEM/F12 medium containing 1‰DMSO, and each drug is set in three replicates. hole. After administration, incubate for half an hour at 37° C. under the condition of 5% CO ₂ , and fix the cells with 4% paraformaldehyde for half an hour (at room temperature). Wash the cells three times with PBS, 200ul per well, for one minute each time. Add 0.2% TritonX-100 to drill the wells for half an hour (at room temperature), wash the cells three times with PBS, 200ul per well, one minute each time. Add 5% fetal bovine serum (Hyclone calf serum, diluted with PBS) to block for 2 hours (room temperature), add primary antibody (0.01% Tween-20+0.5% Hyclone fetal bovine serum+1% primary antibody+PBS, primary antibody For ERα), incubate overnight at 4°C, wash the cells three times with PBST (PBS+0.05% Tween-20), 200ul per well, 5 minutes each time, and then wash three times with PBS, 200ul per well, 1 minute each time. Add secondary antibody (0.01% Tween-20 + 0.5% Hyclone fetal bovine serum + 1/10,000 Hochest + 1/200 secondary antibody + PBS, the secondary antibody is Alex488) in the dark, and incubate at room temperature for two hours. Wash the cells three times with PBST (PBS+0.05% Tween-20), 200ul per well, 5 minutes each time, then wash three times with PBS, 200ul per well, 1 minute each time, and finally add 100ulPBS to the well for high content. After the high-content photographing is completed, the data is automatically processed with high-content, that is, the fluorescence value in the nucleus is compared with the fluorescence value in the cytoplasm, and the average value of three blanks is taken. Use Graphpad to draw graphs and SPSS to process data to determine whether the comparison between the drug group and the blank group is statistically significant. The experimental results are shown in Table 2:

Table 2 Activation results of compounds 1-11 on estrogen receptor α

*p<0.05, **p<0.01, ***p<0.001

From the above activity experiments, it can be seen that compound 1-11 isolated from Epimedium can bind to estrogen receptor ERα and increase its transcriptional activity, confirming that compound 1-11 has an estrogen-like effect. Therefore, it can be used to prevent and treat menopausal syndrome, postmenopausal osteoporosis, breast cancer, uterine cancer, prostate cancer and colon cancer, and its pharmacological effects have been expanded in the fields of cardiovascular system, reproductive system and immune regulation.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present invention are included in the protection scope of the present invention.

Claims (10)

1. the compound of logical formula I is for the preparation of the purposes treated and/or prevented in the medicine of estrogen-related condition;

Wherein, R ₁, R ₂, R ₃, R ₄, R ₅independently represent-H ,-OH ,-OCH separately ₃,

R ₆, R ₇independently represent-H ,-OH ,-OCH separately ₃, or R ₆, R ₇and with respectively with R ₆, R ₇the carbon atom be connected forms oxa-five-membered ring.

2. lead to the purposes of the compound of formula I as claimed in claim 1, wherein,

R ₁representative-H,

R ₂, R ₃respective independently representative-OH;

R ₄representative-H ,-OH or

R ₅representative-H,

R ₆representative-H ,-OH or r ₇representative-OH or-OCH ₃, or R ₆, R ₇and with respectively with R ₆, R ₇the carbon atom be connected forms 2-(1-hydroxyl-1-methyl-ethyl)-DHF ring.

3. lead to the purposes of the compound of formula I as claimed in claim 1 or 2, wherein, described estrogen-related condition is: the disease caused lower than normal level by estrogen level.

4. lead to the purposes of the compound of formula I as claimed in claim 3, wherein, the described disease caused lower than normal level by estrogen level comprises: female climacteric syndrome, carcinoma of prostate, osteoporosis and cardiovascular disease.

5. lead to the purposes of the compound of formula I as claimed in claim 1 or 2, wherein, described estrogen-related condition is: the disease caused higher than normal level by estrogen level.

6. lead to the purposes of the compound of formula I as claimed in claim 5, wherein, the described disease caused higher than normal level by estrogen level, comprising: cyclomastopathy, hysteromyoma.

7. the compound of logical formula I,

Wherein, R ₁representative-H, r ₂, R ₃respective independently representative-OH; R ₄representative-H ,-OH or r ₅representative-H, r ₆representative-H ,-OH or r ₇representative-OH or-OCH ₃.

8. compound as claimed in claim 7, wherein,

R ₂with R ₃identical, representative-OH;

At R ₁representative when, R ₄representative-H or r ₅representative-H or r ₆representative-H or-OH; R ₇representative-OH or-OCH ₃;

At R ₁when representative-H, R ₄representative-OH; R ₅representative r ₆representative r ₇representative-OH;

At R ₁representative when, R ₄representative-H; R ₅representative r ₆representative-OH; R ₇representative-OH.

9. compound as claimed in claim 7 or 8, it is selected from following each compound:

10. one kind comprises the Herba Epimedii extract of the compound of the logical formula I as described in claim 7-9.