CN101260136B

CN101260136B - Oestrogen derivatives as inhibitors of steroid sulphatase

Info

Publication number: CN101260136B
Application number: CN2008100858356A
Authority: CN
Inventors: M·利斯; A·普罗希特; M·J·里德; S·P·纽曼; S·K·钱德; F·茹尔当; B·V·L·波特
Original assignee: Strix Ltd
Current assignee: Strix Ltd; Sterix Ltd
Priority date: 2003-03-24
Filing date: 2004-02-20
Publication date: 2011-09-07
Anticipated expiration: 2024-02-20
Also published as: CN101260136A; CN100384868C; GB0306717D0; CN1791610A

Abstract

The present invention provides a compound comprising a steroidal ring system and an optional group R<1> selected from any one of -OH, a sulphamate group, a phosphonate group, a thiophosphonate group, a sulphonate group or a sulphonamide group; wherein the D ring of the steroidal ring system is substituted by a group R<2> of the formula -L-R<3>, wherein L is an optional linker group and R<3> is selected from groups which are or which comprise one of a nitrite group, an alcohol, an ester, an ether, an amine and an alkene, provided that when R<3> is or comprises an alcohol, L is present; and wherein the A ring of the steroidal ring system is substituted at position 2 or 4 with a group R<4>, wherein R<4> is a hydrocarbyl group.

Description

The oestrogen derivatives of steoid sulfatase inhibitor

The present invention is dividing an application of following application: the applying date: on February 20th, 2004; Application number: 200480013919.9 (PCT/GB2004/000705); Denomination of invention: " oestrogen derivatives of steoid sulfatase inhibitor ".

Invention field

The present invention relates to a kind of compound.

Particularly, the present invention relates to a kind of compound and relate to a kind of medicinal compositions that comprises this compound.The present invention also relates to the purposes that this compound or composition (composition) are applied to treat.

Background of invention

Evidence suggests that oestrogenic hormon relates to promote the main mitogen of tumor growth in internal secretion-dependency tissue (for example mammary gland and uterine endometrium).Though women's blood plasma estrogen concentrations of suffering from mammary cancer or not suffering from mammary cancer is similarly, oestrone in the breast tumor and the estradiol level level in normal galactophore tissue or the blood.The oestrogenic hormon original position is synthetic to be considered to high-caliber estrogenic important factor in the tumour, and therefore, oestrogenic hormon biosynthesis inhibitor, particularly specific inhibitor have potential to treatment internal secretion-dependent tumors and be worth.

In the past twenty years, pay close attention to is that this path changes into oestrone with the androgen precurosor rotex on the inhibitor of exploitation virtueization enzyme path quite a lot ofly.Yet, evidence suggests oestrone sulphatase (E1-STS) path at present, be about to the oestrone sulfuric ester and be hydrolyzed into oestrone (E1S to E1), and estrogenic generation in the soluble breast tumor of virtueization enzyme (being that rotex changes into oestrone).

Fig. 1 and 2 is for showing the sketch that relates to by some enzyme of the synthetic oestrone of oestrone sulfuric ester, estradiol and rotex original position.

In Fig. 2, this figure has schematically shown the source of postmenopausal women's estrogens sterol, " ER " represents estrogen receptor, " DHA-S " represents trans-dehydroandrosterone-sulfuric ester, " Adiol " represents androstenediol, and " E1-STS " represents oestrone sulphatase, " DHA-STS " expression DHA-sulfatase, " Adiol-STS " expression androstenediol sulfatase, and " 17B-HSD " expression estradiol 17B-hydroxysteroid dehydrogenase.

As can be seen, relating to two kinds of main enzymes of the peripheral synthetic of oestrogenic hormon is virtueization enzyme and oestrone sulphatase.

In brief, the virtueization enzyme is converted into oestrone with a large amount of excretory rotex of adrenal cortex.Nearest report shows that some flavones can suppress the activity of virtueization enzyme.

Yet a large amount of oestrone that so form have changed into oestrone sulfuric ester (E1S), and present a considerable amount of evidence shows that the E1S in blood plasma and the tissue works as the bank that the effect by oestrone sulphatase forms oestrone.

In this respect, believe that now it is estrogenic main source in the breast tumor that oestrone sulphatase (E1-STS) path-be oestrone sulfuric ester is hydrolyzed into oestrone (E1S to E1).This theory obtains suffering from mammary cancer and accepts the postmenopausal women's of aromatase inhibitor (for example aminoglutethimide and 4-hydroxyandrostenedione) treatment the appropriate support that reduces of blood plasma estrogen concentrations, and also obtains high relatively this true support of blood plasma E1S concentration maintenance that these accept aromatase inhibitor treatment patient.Compare with the oestrogenic hormon (20 minutes) that yoke not closes, in the blood E1S the long half-lift (10-12 hour) and liver, normally reach in the malignant galactophore tissue high-caliber steoid sulfatase activity and also tend to support this theory.

Therefore, the oestrogenic hormon that forms in malignant galactophore and endometrial tissue by the sulfatase path plays a major role to the high estrogen concentration that exists in these tumours.

The medicinal compositions that PCT/GB92/01587 has described the new steroid sulfatase inhibiting agent and contained them is used for the treatment of especially mammary cancer of oestrone dependent tumors.These steoid sulfatase inhibitor are sulfamate, N for example, N-dimethyl oestrone-3-sulfamate, preferred oestrone-3-sulfamate (being also referred to as " EMATE " in addition).EMATE has following structure:

Known EMATE is effective E1-STS inhibitor, and it is in complete MCF-7 cell, show inhibition E1-STS activity greater than 99% with 0.1nM concentration.EMATE also suppresses the E1-STS enzyme with time and concentration dependent mode, and this shows that it is as the inactivator of active-site directed and work.Suppress though the EMATE initial design is E1-STS, it also suppresses trans-dehydroandrosterone sulfatase (DHA-STS), and this enzyme is considered to play a crucial role in the biosynthesizing of regulating oestrogenic hormon steroide androstenediol.Evidence suggests also that at present androstenediol may rise even more important role as the breast tumor growth stimulant.EMATE also has activity in vivo, when per os or subcutaneous administration, almost completely suppresses rat liver E1-STS (99%) and DHA-STS (99%) activity.In addition, EMATE demonstrates in rat and has memory raising effect.To studies show that of mouse DHA-STS active with the immune response of adjusting part between getting in touch.Can think that this also may take place in the mankind.It is important that the bridge joint O-atom pairs of the thionamic acid ester moiety among the EMATE suppresses activity.Therefore, when the 3-O-atom was replaced by other heteroatomss, as oestrone-3-N-sulfamate and oestrone-3-S-sulfamate, these analogues were more weak non-time-dependent manner inactivator.

Except that oestrone, other have the most of steroides oestrogenic hormon characteristic, that produced by the postmenopausal women is the androstenediol (see figure 2).

Although be male sex hormone, androstenediol can be bonded to estrogen receptor (ER) and can stimulate the growth of the positive breast cancer cell of ER and the growth of the rat breast tumor that carcinogenic substance brings out.Importantly, 90% look through after androstenediol that the women produced derive from by a large amount of excretory male sex hormone of adrenal cortex trans-dehydroandrosterone sulfuric ester (DHA-S).DHA-S is converted into DHA by the DHA sulfatase, and this sulfatase can be identical or different with the oestrone sulphatase of responsible hydrolysis E1S.

Among the 10-15 in the past, carried out considerable research to develop effective aromatase inhibitor, wherein some are gone on the market now.Yet, suffering from breast cancer and accepting in nearest three reports of postmenopausal women of aromatase inhibitor treatment, blood plasma E1S concentration remains between the 400-1000pg/ml.

Therefore the oestrogenic hormon original position is synthetic is considered to high-caliber oestrogenic hormon in the tumour is played an important role, so specificity oestrogenic hormon biosynthesis inhibitor has potential value to treatment internal secretion-dependent tumors.

In addition, even the oestrogenic hormon that forms in malignant galactophore and endometrial tissue by the sulfatase path plays a major role to high estrogen concentration, still have other to synthetic enzymatic path of working in the oestrogenic hormon body.

The summary of the invention aspect

The present invention is based on following accident finds: the D ring have be selected from for or the steroide that comprises one of itrile group, alcohol, ester, ether, amine and alkene group can be used as effective steoid sulfatase (STS) inhibitor; The Cycle Regulation agent; The apoptosis conditioning agent; Cell growth regulator; Glucose uptake prevention and/or inhibitor; Tumor-blood-vessel growth preventive or inhibitor; The microtubule disrupting agent; And/or cell death inducer.

The compounds of this invention can comprise other substituting groups.These other substituting groups for example can further strengthen the active of The compounds of this invention and/or increase stability (external and/or in vivo).

The detailed Description Of The Invention aspect

According to an aspect of the present invention, provide a kind of steroid loop systems and optional radicals R of comprising ¹Compound, optional radicals R ¹Be selected from-OH, any in sulphamate group, phosphonate group, phosphonothionic acid ester group, sulfonate group or the sulfoamido; Wherein the D of steroid loop systems ring is by formula-L-R ³Radicals R ²Replace, wherein L is optional linking group and R ³Be selected from for or comprise the group of one of itrile group, alcohol, ester, ether, amine and alkene, condition is to work as R ³For or when comprising alcohol, L exists; Wherein steroid loop systems A encircles at 2 or 4 by radicals R ⁴Replace, wherein R ⁴Be alkyl (hydrocarbyl).

According to an aspect of the present invention, provide a kind of i of comprising) compound as herein defined; Reach the ii) composition of biological response modifier.

According to an aspect of the present invention, provide a kind of medicinal compositions, said composition comprises (a) (i) compound as herein defined, or (ii) composition as herein defined, and (b) pharmaceutically acceptable carrier, thinner, vehicle or auxiliary agent.

According to an aspect of the present invention, provide (i) that be used for medicine compound as herein defined, or (ii) composition as herein defined.

According to an aspect of the present invention, provide (i) compound as herein defined, or (ii) composition as herein defined, in the preparation prevention and/or suppress purposes in the medicine of tumor growth.

According to an aspect of the present invention, (i) is provided compound as herein defined, or (ii) composition as herein defined, be used for the treatment of purposes in the medicine of illness or disease, described illness or disease and steoid sulfatase (STS) activity in preparation; Cell cycle; Apoptosis; The cell growth; Tumour is to the picked-up of glucose; Tumor-blood-vessel growth; Microtubule forms; And one or more are relevant in the apoptosis.

According to an aspect of the present invention, (i) is provided compound as herein defined, or (ii) composition as herein defined, be used for the treatment of purposes in the medicine of illness or disease, described illness or disease and reverse (adverse) steoid sulfatase (STS) activity in preparation; Cell cycle; Apoptosis; The cell growth; Tumour is to the picked-up of glucose; Tumor-blood-vessel growth; Microtubule forms; And one or more are relevant in the apoptosis.

According to an aspect of the present invention, provide (i) compound as herein defined, or (ii) composition as herein defined, be used for the purposes of the medicine of following one or more situations in preparation: suppress steoid sulfatase (STS) activity; Regulate the cell cycle; Regulate apoptosis; The growth of adjusting cell; Prevention and/or inhibition tumour are to the picked-up of glucose; Prevention and/or inhibition tumor-blood-vessel growth; Destroy microtubule; And cell death inducing.

According to an aspect of the present invention, provide (i) compound as herein defined, or (ii) composition as herein defined, suppressed purposes in steoid sulfatase (STS) active medicine in preparation.

According to an aspect of the present invention, provide (i) compound as herein defined, or (ii) composition as herein defined, regulated purposes in the cell growth medicine in preparation.

According to an aspect of the present invention, provide a kind of methods of treatment, this method comprises patient (i) compound as herein defined that the treatment needs are arranged, or (ii) composition as herein defined.

According to an aspect of the present invention, provide a kind of methods of treatment to be used to suppress steoid sulfatase (STS) activity; Regulate the cell cycle; Regulate apoptosis; The growth of adjusting cell; Prevention and/or inhibition tumour are to the picked-up of glucose; Prevention and/or inhibition tumor-blood-vessel growth; Destroy microtubule; And/or cell death inducing, this method comprises patient (i) compound as herein defined that the treatment needs are arranged, or (ii) composition as herein defined.

According to an aspect of the present invention, provide a kind of method, this method comprises that (a) tests in the following situation one or more with one or more candidate compounds defined herein: steoid sulfatase (STS) suppresses; Cycle Regulation; Apoptosis is regulated; Cell cycle regulation; Prevention and/or inhibition tumour are to the picked-up of glucose; Tumor-blood-vessel growth prevention and/or inhibition; Microtubule destroys; And apoptosis-inducing; (b) determine whether that one or more can realize in the following situation one or more in the described candidate compound: steoid sulfatase (STS) suppresses; Cycle Regulation; Apoptosis is regulated; Cell cycle regulation; Prevention and/or inhibition tumour are to the picked-up of glucose; Tumor-blood-vessel growth prevention and/or inhibition; Microtubule destroys; And apoptosis-inducing; And (c) select one or more can realize in the following situation one or more described candidate compound: steoid sulfatase (STS) suppresses; Cycle Regulation; Apoptosis is regulated; Cell cycle regulation; Prevention and/or inhibition tumour are to the picked-up of glucose; Tumor-blood-vessel growth prevention and/or inhibition; Microtubule destroys; And apoptosis-inducing.

In any the inventive method, can there be one or more other steps.For example, method also can comprise in step (for example by chemistry and/or zymotechnic) of modifying determined candidate compound and the following situation of the testing this modified compound one or more optional other step: steoid sulfatase (STS) suppresses; Cycle Regulation; Apoptosis is regulated; Cell cycle regulation; Prevention and/or inhibition tumour are to the picked-up of glucose; Tumor-blood-vessel growth prevention and/or inhibition; Microtubule destroys; And apoptosis-inducing.By further example, this method also can comprise the step (for example by using crystallization technique) of determining the candidate compound structure of identifying and carry out The study of computer simulation-for example be further its activity of increase then.Therefore, the present invention also comprises the computer (for example crystallography adjusting) that described evaluation candidate compound is had data set.The present invention comprises that also being provided for computer screening analyzes-for example enzyme and/or protein bound institute identify the candidate compound.

According to an aspect of the present invention, provide by the inventive method compounds identified.

The present invention also comprises new compound of the present invention (for example those that are proposed) herein, with and preparation method thereof (for example method that is proposed herein) and be used for the new intermediate (for example those that are proposed) of those methods herein.

The generalized aspect

According to the present invention broad aspect, provide compound preparation be used for the treatment of with following situation in purposes in the medicine of one or more relevant illnesss or disease: the cell cycle; Apoptosis; The cell growth; Tumour is to the picked-up of glucose; Tumor-blood-vessel growth; Microtubule forms; And apoptosis; Wherein this compound comprise the steroid loop systems and be selected from-OH, sulphamate group, phosphonate group, phosphonothionic acid ester group, sulfonate group or sulfoamido in any optional radicals R ¹Wherein the D of steroid loop systems ring is by formula-L-R ³Radicals R ²Replace, wherein L is optional linking group and R ³Be selected from for or comprise the group of one of itrile group, alcohol, ester, ether, amine and alkene, condition is to work as R ³For or when comprising alcohol, L exists.

According to the present invention broad aspect, provide composition preparation be used for the treatment of with following situation in purposes in the medicine of one or more relevant illnesss or disease: the cell cycle; Apoptosis; The cell growth; Tumour is to the picked-up of glucose; Tumor-blood-vessel growth; Microtubule forms; And apoptosis; Wherein said composition comprises i) comprise the steroid loop systems and be selected from-OH, any optional radicals R in sulphamate group, phosphonate group, phosphonothionic acid ester group, sulfonate group or the sulfoamido ¹Compound; Wherein the D of steroid loop systems ring is by formula-L-R ³Radicals R ²Replace, wherein L is optional linking group and R ³Be selected from for or comprise the group of one of itrile group, alcohol, ester, ether, amine and alkene, condition is to work as R ³For or when comprising alcohol, L exists; Reach ii) biological response modifier.

According to the present invention another broad aspect provides compound to be used for the treatment of purposes in the medicine of illness or disease in preparation, and one or more are relevant in described illness or disease and the reverse following situation: the cell cycle; Apoptosis; The cell growth; Tumour is to the picked-up of glucose; Tumor-blood-vessel growth; Microtubule forms; And apoptosis; Wherein this compound comprise the steroid loop systems and be selected from-OH, sulphamate group, phosphonate group, phosphonothionic acid ester group, sulfonate group or sulfoamido in any optional radicals R ¹Wherein the D of steroid loop systems ring is by formula-L-R ³Radicals R ²Replace, wherein L is optional linking group and R ³Be selected from for or comprise the group of one of itrile group, alcohol, ester, ether, amine and alkene, condition is to work as R ³For or when comprising alcohol, L exists.

According to the present invention another broad aspect provides composition to be used for the treatment of purposes in the medicine of illness or disease in preparation, and one or more are relevant in described illness or disease and the reverse following situation: the cell cycle; Apoptosis; The cell growth; Tumour is to the picked-up of glucose; Tumor-blood-vessel growth; Microtubule forms; And apoptosis; Wherein said composition comprises i) comprise the steroid loop systems and be selected from-OH, any optional radicals R in sulphamate group, phosphonate group, phosphonothionic acid ester group, sulfonate group or the sulfoamido ¹Compound; Wherein the D of steroid loop systems ring is by formula-L-R ³Radicals R ²Replace, wherein L is optional linking group and R ³Be selected from for or comprise the group of one of itrile group, alcohol, ester, ether, amine and alkene, condition is to work as R ³For or when comprising alcohol, L exists; Reach ii) biological response modifier.

According to the present invention another broad aspect provides compound to be used for purposes in one or more the medicine of following situation in preparation: to regulate the cell cycle; Regulate apoptosis; The growth of adjusting cell; Prevention and/or inhibition tumour are to the picked-up of glucose; Prevention and/or inhibition tumor-blood-vessel growth; Destroy microtubule; And cell death inducing; Wherein this compound comprise the steroid loop systems and be selected from-OH, sulphamate group, phosphonate group, phosphonothionic acid ester group, sulfonate group or sulfoamido in any optional radicals R ¹Wherein the D of steroid loop systems ring is by formula-L-R ³Radicals R ²Replace, wherein L is optional linking group and R ³Be selected from for or comprise the group of one of itrile group, alcohol, ester, ether, amine and alkene, condition is to work as R ³For or when comprising alcohol, L exists.

According to the present invention another broad aspect provides composition to be used for purposes in one or more the medicine of following situation in preparation: to regulate the cell cycle; Regulate apoptosis; The growth of adjusting cell; Prevention and/or inhibition tumour are to the picked-up of glucose; Prevention and/or inhibition tumor-blood-vessel growth; Destroy microtubule; And cell death inducing; Wherein said composition comprises i) comprise the steroid loop systems and be selected from-OH, any optional radicals R in sulphamate group, phosphonate group, phosphonothionic acid ester group, sulfonate group or the sulfoamido ¹Compound; Wherein the D of steroid loop systems ring is by formula-L-R ³Radicals R ²Replace, wherein L is optional linking group and R ³Be selected from for or comprise the group of one of itrile group, alcohol, ester, ether, amine and alkene, condition is to work as R ³For or when comprising alcohol, L exists; Reach ii) biological response modifier.

Broad aspect according to the present invention, the purposes that provides compound to be used for regulating cell growth medicine in preparation; Wherein this compound comprise the steroid loop systems and be selected from-OH, sulphamate group, phosphonate group, phosphonothionic acid ester group, sulfonate group or sulfoamido in any optional radicals R ¹Wherein the D of steroid loop systems ring is by formula-L-R ³Radicals R ²Replace, wherein L is optional linking group and R ³Be selected from for or comprise the group of one of itrile group, alcohol, ester, ether, amine and alkene, condition is to work as R ³For or when comprising alcohol, L exists.

Broad aspect according to the present invention, the purposes that provides composition to be used for regulating cell growth medicine in preparation; Wherein said composition comprises i) comprise the steroid loop systems and be selected from-OH, any optional radicals R in sulphamate group, phosphonate group, phosphonothionic acid ester group, sulfonate group or the sulfoamido ¹Compound; Wherein the D of steroid loop systems ring is by formula-L-R ³Radicals R ²Replace, wherein L is optional linking group and R ³Be selected from for or comprise the group of one of itrile group, alcohol, ester, ether, amine and alkene, condition is to work as R ³For or when comprising alcohol, L exists; Reach ii) biological response modifier.

Broad aspect according to the present invention provides a kind of methods of treatment to be used to regulate the cell cycle; Regulate apoptosis; The growth of adjusting cell; Prevention and/or inhibition tumour are to the picked-up of glucose; Prevention and/or inhibition tumor-blood-vessel growth; Destroy microtubule; And/or cell death inducing; This method comprises the patient of treatment needs compound, this compound comprises the steroid loop systems and be selected from-OH, sulphamate group, phosphonate group, phosphonothionic acid ester group, sulfonate group or sulfoamido in any optional radicals R ¹Wherein the D of steroid loop systems ring is by formula-L-R ³Radicals R ²Replace, wherein L is optional linking group and R ³Be selected from for or comprise the group of one of itrile group, alcohol, ester, ether, amine and alkene, condition is to work as R ³For or when comprising alcohol, L exists.

Broad aspect according to the present invention provides a kind of methods of treatment to be used to regulate the cell cycle; Regulate apoptosis; The growth of adjusting cell; Prevention and/or inhibition tumour are to the picked-up of glucose; Prevention and/or inhibition tumor-blood-vessel growth; Destroy microtubule; And/or cell death inducing; This method comprises the patient of treatment needs composition, and said composition comprises i) comprise the steroid loop systems and be selected from-OH, any optional radicals R in sulphamate group, phosphonate group, phosphonothionic acid ester group, sulfonate group or the sulfoamido ¹Compound; Wherein the D of steroid loop systems ring is by formula-L-R ³Radicals R ²Replace, wherein L is optional linking group and R ³Be selected from for or comprise the group of one of itrile group, alcohol, ester, ether, amine and alkene, condition is to work as R ³For or when comprising alcohol, L exists; Reach ii) biological response modifier.

According to the present invention broad aspect provides compound to be used for the treatment of purposes in the medicine of illness relevant with carbonic anhydrase or disease in preparation; Wherein this compound comprise the steroid loop systems and be selected from-OH, sulphamate group, phosphonate group, phosphonothionic acid ester group, sulfonate group or sulfoamido in any optional radicals R ¹Wherein the D of steroid loop systems ring is by formula-L-R ³Radicals R ²Replace, wherein L is optional linking group and R ³Be selected from for or comprise the group of one of itrile group, alcohol, ester, ether, amine and alkene, condition is to work as R ³For or when comprising alcohol, L exists.

According to the present invention broad aspect provides composition to be used for the treatment of purposes in the medicine of illness relevant with carbonic anhydrase or disease in preparation; Wherein said composition comprises i) comprise the steroid loop systems and be selected from-OH, any optional radicals R in sulphamate group, phosphonate group, phosphonothionic acid ester group, sulfonate group or the sulfoamido ¹Compound; Wherein the D of steroid loop systems ring is by formula-L-R ³Radicals R ²Replace, wherein L is optional linking group and R ³Be selected from for or comprise the group of one of itrile group, alcohol, ester, ether, amine and alkene, condition is to work as R ³For or when comprising alcohol, L exists; Reach ii) biological response modifier.

According to the present invention another broad aspect provides compound to be used for the treatment of purposes in the medicine with active relevant illness of reverse carbonic anhydrase or disease in preparation; Wherein this compound comprise the steroid loop systems and be selected from-OH, sulphamate group, phosphonate group, phosphonothionic acid ester group, sulfonate group or sulfoamido in any optional radicals R ¹Wherein the D of steroid loop systems ring is by formula-L-R ³Radicals R ²Replace, wherein L is optional linking group and R ³Be selected from for or comprise the group of one of itrile group, alcohol, ester, ether, amine and alkene, condition is to work as R ³For or when comprising alcohol, L exists.

According to the present invention another broad aspect provides composition to be used for the treatment of purposes in the medicine with active relevant illness of reverse carbonic anhydrase or disease in preparation; Wherein said composition comprises i) comprise the steroid loop systems and be selected from-OH, any optional radicals R in sulphamate group, phosphonate group, phosphonothionic acid ester group, sulfonate group or the sulfoamido ¹Compound; Wherein the D of steroid loop systems ring is by formula-L-R ³Radicals R ²Replace, wherein L is optional linking group and R ³Be selected from for or comprise the group of one of itrile group, alcohol, ester, ether, amine and alkene, condition is to work as R ³For or when comprising alcohol, L exists; Reach ii) biological response modifier.

Another broad aspect according to the present invention, the purposes that provides compound to be used for regulating the carbonic anhydrase active medicine in preparation; Wherein this compound comprise the steroid loop systems and be selected from-OH, sulphamate group, phosphonate group, phosphonothionic acid ester group, sulfonate group or sulfoamido in any optional radicals R ¹Wherein the D of steroid loop systems ring is by formula-L-R ³Radicals R ²Replace, wherein L is optional linking group and R ³Be selected from for or comprise the group of one of itrile group, alcohol, ester, ether, amine and alkene, condition is to work as R ³For or when comprising alcohol, L exists.

Another broad aspect according to the present invention, the purposes that provides composition to be used for regulating the carbonic anhydrase active medicine in preparation; Wherein said composition comprises i) comprise the steroid loop systems and be selected from-OH, any optional radicals R in sulphamate group, phosphonate group, phosphonothionic acid ester group, sulfonate group or the sulfoamido ¹Compound; Wherein the D of steroid loop systems ring is by formula-L-R ³Radicals R ²Replace, wherein L is optional linking group and R ³Be selected from for or comprise the group of one of itrile group, alcohol, ester, ether, amine and alkene, condition is to work as R ³For or when comprising alcohol, L exists; Reach ii) biological response modifier.

Broad aspect according to the present invention provides a kind of active methods of treatment of carbonic anhydrase that is used to regulate, and this method comprises the patient of treatment needs compound; Wherein this compound comprise the steroid loop systems and be selected from-OH, sulphamate group, phosphonate group, phosphonothionic acid ester group, sulfonate group or sulfoamido in any optional radicals R ¹Wherein the D of steroid loop systems ring is by formula-L-R ³Radicals R ²Replace, wherein L is optional linking group and R ³Be selected from for or comprise the group of one of itrile group, alcohol, ester, ether, amine and alkene, condition is to work as R ³For or when comprising alcohol, L exists.

Broad aspect according to the present invention provides a kind of active methods of treatment of carbonic anhydrase that is used to regulate, and this method comprises the patient of treatment needs composition; Wherein said composition comprises i) comprise the steroid loop systems and be selected from-OH, any optional radicals R in sulphamate group, phosphonate group, phosphonothionic acid ester group, sulfonate group or the sulfoamido ¹Compound; Wherein the D of steroid loop systems ring is by formula-L-R ³Radicals R ²Replace, wherein L is optional linking group and R ³Be selected from for or comprise the group of one of itrile group, alcohol, ester, ether, amine and alkene, condition is to work as R ³For or when comprising alcohol, L exists; Reach ii) biological response modifier.

In these broad aspect, preferred R ¹, R ², R ³And L as defined herein.

For ease of reference, now of the present invention these are reached many-sided suitable sub-section titles discussion of pressing.Yet the content in each trifle needn't be defined in each specific trifle.

Some advantages

A key advantages of the present invention is that The compounds of this invention can suppress steoid sulfatase (STS) activity.

A key advantages of the present invention is that The compounds of this invention can be regulated the cell cycle.

A key advantages of the present invention is that The compounds of this invention can be regulated apoptosis.

A key advantages of the present invention is that The compounds of this invention can be regulated the cell growth.

A key advantages of the present invention is that The compounds of this invention can prevent and/or suppress the picked-up of tumour to glucose.

A key advantages of the present invention is that The compounds of this invention can prevent and/or suppress tumor-blood-vessel growth.

A key advantages of the present invention is that The compounds of this invention can destroy microtubule.

In this respect, microtubule is with the part of the cytoskeleton system of microfilament and intermediate filament body formation cell.Microtubule is responsible for many motion-examples of cell, comprises beating and transport membrane vesicle in tenuigenin of cilium and flagellum.All these motions are produced by the polymerization of microtubule and the effect of depolymerization or microtubule motor protein therbligs and kinesin.Some other cell movements, for example during reduction division and mitotic division chromosomal arrangement with separate, produce by two kinds of mechanism.Microtubule also guides cell movement, but in some cases, microtubule has structure function purely.

Microtubule is made up of alpha-tubulin and the monomeric heterodimer of 'beta '-tubulin subunit.Two kinds of microtubule colonies are arranged: microtubule stable, that the life-span is long and microtubule dynamic, that the life-span is short.When needing assembling rapidly and dismounting, micro-tubular structure finds dynamic microtubule.For example, during mitotic division, the kytoplasm microtubule network characterization of interface cell disappears and wherein tubulin often forms the fusiformis device of equally karyomit(e) being distributed to daughter cell.Finish as silk division, this shuttle shape thing decomposes and forms interface microtubule network again.

Suppress mitotic medicine and be provided at the useful means of handling microtubule in the cell.Three kinds of medicines: colchicine, vinealeucoblastine(VLB) and safe plain-by plant purifying-turned out to be the force probe that has of microtubule function, part because they only are incorporated into tubulin or microtubule debond in other albumen, and also because can easily control their concentration in cell.

Because they are to mitotic effect, the microtubule inhibitor has been widely used for treating disease and has been used as cancer therapy drug recently, because the formation of blocking-up shuttle shape thing will preferentially suppress the rapid splitted cell as cancer cells.Extremely effectively ovarian cancer resistance medicament is safe plain.In ovarian cancer cell, carry out cell fission rapidly, mitotic division is blocked by safe extract for treating and other functions of being carried out by complete microtubule are unaffected.Visible " Molecular Cell Biology " (the 1995 WH Freeman and Co.New York pp 1051-1122 such as Ed:Lodish) of the comprehensive review of microtubule.

But a key advantages of the present invention is the The compounds of this invention cell death inducing.

Apoptosis is induced by the MT-targeted drug, and this process can relate to the proteic phosphorylation of apoptosis conditioning agent bcl-2 (and inactivation) (Halder, Cancer Res.57:229,1997).

The present invention is based on compound this unexpected discovery of effective treatment of cancer is provided.

Another advantage of The compounds of this invention is that they in vivo may be effectively.

Some The compounds of this invention can be non-estrogen compound.Herein, term " non-oestrogenic hormon " refers to not show or does not show substantially estrogen activity.Herein, term " non-oestrogenic hormon " refers to not show or does not show substantially the general estrogen activity, for example by scheme 4 determined activity.

Concerning some were used, this compounds had estrogen effect.

Another advantage may not be metabolised to the compound that shows or induce hormonal activity for some compounds.

Concerning some were used, preferably this compounds had reversible action.

Concerning some were used, preferably this compounds had irreversible effect.

The advantage of some The compounds of this invention is that also they may be oral effective.

Some The compounds of this invention can be used for preventing and/or treating cancer for example mammary cancer and (perhaps) non-malignant disorders, for example prevent and/or treat any or multiple relevant illness in inflammatory conditions-for example and the following illness: autoimmune disorder for example comprises rheumatoid arthritis, I type and type ii diabetes, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, thyroiditis, vasculitis, ulcerative colitis and Crohn disease, tetter for example acne, psoriatic and contact dermatitis; Graft versus host disease (GVH disease); Eczema; Asthma and transplanting back organ rejection.The compounds of this invention is particularly useful when medicine may need to begin to take from the less age.

In one embodiment, The compounds of this invention is used for the treatment of mammary cancer.

Therefore, except treatment internal secretion dependence cancer, for example treat autoimmune disorder, some The compounds of this invention also think to have therepic use.

For ease of reference, now of the present invention these are reached many-sided suitable sub-section titles discussion of pressing.Yet the content under each trifle is not necessarily limited to each specific trifle.

Preferred aspect

Compound

As mentioned above, the invention provides a kind ofly comprise the steroid loop systems and be selected from-OH, sulphamate group, phosphonate group, phosphonothionic acid ester group, sulfonate group or sulfoamido in any optional radicals R ¹Compound; Wherein the D of steroid loop systems ring is by formula-L-R ³Radicals R ²Replace, wherein L is optional linking group and R ³Be selected from for or comprise the group of one of itrile group, alcohol, ester, ether, amine and alkene, condition is to work as R ³For or when comprising alcohol, L exists; Wherein steroid loop systems A encircles at 2 or 4 by radicals R ⁴Replace, wherein R ⁴Be alkyl.

One preferred aspect in, compound can be realized in the following situation one or more: suppress steoid sulfatase (STS) activity; Regulate the cell cycle; Regulate apoptosis; The growth of adjusting cell; Prevention and/or inhibition tumour are to the picked-up of glucose; Prevention and/or inhibition tumor-blood-vessel growth; Destroy microtubule; And cell death inducing.

The steroid loop systems

The compounds of this invention has cyclopentanoperhydro-phenanthrene integral part-that is to say have cyclopenta (cyclopentano) luxuriant and rich with fragrance skeleton or its bioisostere.

As well-known in the art, typical steroid ring structure has following general formula:

In the said structure formula, will encircle mark and numbering in a usual manner.

On the one hand, this steroid ring structure can contain any one or a plurality of C, H, O, N, P, halogen (comprising Cl, Br and I), S and P.

At least a cyclic group can be heterocyclic group (heterocycle) or non-heterocyclic group in this steroid ring structure.

At least a cyclic group can be saturated rings structure or unsaturated ring structure (for example aryl) in this steroid ring structure.

At least a cyclic group is an aromatic ring in preferred this steroid ring structure.

The example of bioisostere for when among ring A, B, C and the D any one or a plurality of for heterocyclic ring and/or when among ring A, B, C and the D any one or a plurality of be substituted and/or in encircling A, B, C and D any one or a plurality of during by modification; But wherein bioisostere has the steroide characteristic.

In this respect, the structure of preferred steroid ring structure can be expressed as:

Wherein encircle independent separately heterocyclic ring or the non-heterocyclic ring represented of A ', B ', C ' and D ', this class ring can independently be replacement or unsubstituted saturated or undersaturated.

As an example, any one or a plurality of can be independently the replacement by suitable group among ring A ', B ', C ' and the D ',, suitable group has for example alkyl, allyl group, hydroxyl, halogen, alkyl, oxygen base alkyl etc.

Term used herein " alkyl " refers to comprise at least the group of C and H and can choose wantonly and comprises one or more other suitable substituents.The substituent example of this class can comprise halogen-, alkoxyl group-, nitro-, hydrocarbon polymer (hydrocarbon) group, N-acyl group, cyclic group etc.May be except that substituting group for the cyclic group, substituent combination can form cyclic group.If alkyl comprises more than a C, then those carbon needn't interconnect.For example, at least two carbon can connect by suitable element or group.Therefore, alkyl can contain heteroatoms.Suitable heteroatoms is conspicuous to those skilled in the art, and comprises for example sulphur, nitrogen and oxygen.

In a preferred embodiment of the invention, alkyl is a hydrocarbon group.

Term " hydrocarbon polymer " refers to any alkyl, thiazolinyl, alkynyl, acyl group herein, and this class group can be straight chain, side chain or ring-type, or refers to aryl.The term hydrocarbon polymer also comprises wherein their optional substituted those groups.If hydrocarbon polymer is for wherein having substituent branched structure, then this substituting group can be on the main-chain hydrocarbon or on side chain; Perhaps these substituting groups can be on the main chain of hydrocarbon polymer and on side chain.

In a preferred embodiment of the invention, alkyl is an oxygen base alkyl.

Term used herein " oxygen base alkyl " refers to comprise at least the group of C, H and O and can choose wantonly and comprises one or more other suitable substituents.The substituent example of this class can comprise halogen-, alkoxyl group-, nitro-, alkyl, cyclic group etc.May be except that substituting group for the cyclic group, substituent combination can form cyclic group.If oxygen base alkyl comprises more than a C, then those carbon needn't interconnect.For example, at least two carbon can connect by suitable element or group.Therefore, oxygen base alkyl can contain heteroatoms.Suitable heteroatoms is conspicuous to those skilled in the art, and comprises for example sulphur and nitrogen.

In a preferred embodiment of the invention, oxygen base alkyl is an oxygen base hydrocarbon group.

Term " oxygen base hydrocarbon polymer " refers to any alkoxyl group, oxygen base thiazolinyl, oxygen base alkynyl herein, and this class group can be straight chain, side chain or ring-type, or refers to oxygen Ji Fangji.Term oxygen base hydrocarbon polymer also comprises wherein their optional substituted those groups.If oxygen base hydrocarbon polymer is for wherein having substituent branched structure, then this substituting group can be on the main-chain hydrocarbon or on side chain; Perhaps these substituting groups can be on the main chain of hydrocarbon polymer and on side chain.

Preferred oxygen base alkyl is an alkoxyl group.Preferred oxygen base alkyl has formula C _1-6O (C for example _1-3O).

The example of D ' is for having at least one substituent five yuan or hexa-atomic non-heterocyclic ring.

In a preferred embodiment, ring D ' is replaced by ethynyl.

If any one is a heterocyclic ring among ring A ', B ', C ' and the D ', then preferred this heterocyclic ring comprises the combination of C atom and at least one N atom and/or at least one O atom.Other heterocyclic atoms can exist in ring.

The compounds of this invention example suitable, preferred steroidal nuclear ring A '-D ' comprises the ring A-D of oestrone and trans-dehydroandrosterone.

The preferred steroidal nuclear ring of The compounds of this invention A '-D ' comprises the ring A-D of following compounds:

Oestrone and replacement oestrone, that is:

Oestrone

2-OH-oestrone

2-alkoxyl group-oestrone (C for example _1-6Alkoxyl group-oestrone, for example 2-methoxyl group-oestrone)

4-OH-oestrone

6 α-OH-oestrone

7 α-OH-oestrone

16 α-OH-oestrone

16 β-OH-oestrone

Estradiol and replacement estradiol, that is:

The 2-OH-17 beta estradiol

2-alkoxyl group-17 beta estradiol (C for example _1-6Alkoxyl group-17 beta estradiol, for example 2-methoxyl group-17 beta estradiol)

The 4-OH-17 beta estradiol

6 α-OH-17 beta estradiol

7 α-OH-17 beta estradiol

The 2-OH-17 alpha-estradiol

2-alkoxyl group-17 alpha-estradiol (C for example _1-6Alkoxyl group-17 alpha-estradiol, for example 2-methoxyl group-17 alpha-estradiol)

The 4-OH-17 alpha-estradiol

6 α-OH-17 alpha-estradiol

7 α-OH-17 alpha-estradiol

16 α-OH-17 alpha-estradiol

16 α-OH-17 beta estradiol

16 β-OH-17 alpha-estradiol

16 β-OH-17 beta estradiol

17 alpha-estradiols

17 beta estradiols

17 α-ethynyl-17 beta estradiol

17 β-ethynyl-17 alpha-estradiol

Trihydroxy-oestrin and replacement trihydroxy-oestrin, that is:

Trihydroxy-oestrin

The 2-OH-trihydroxy-oestrin

2-alkoxyl group-trihydroxy-oestrin (C for example _1-6Alkoxyl group-trihydroxy-oestrin, for example 2-methoxyl group-trihydroxy-oestrin)

The 4-OH-trihydroxy-oestrin

6 α-OH-trihydroxy-oestrin

7 α-OH-trihydroxy-oestrin

Trans-dehydroandrosterone and replacement trans-dehydroandrosterone, promptly:

Trans-dehydroandrosterone

6 α-OH-trans-dehydroandrosterone

7 α-OH-trans-dehydroandrosterone

16 α-OH-trans-dehydroandrosterone

16 β-OH-trans-dehydroandrosterone

Loop systems A ' B ' C ' D ' can contain various noiseless substituting groups in generic term.Particularly, loop systems A ' B ' C ' D ' can contain the especially rudimentary (C of one or more hydroxyls, alkyl ₁-C ₆) alkyl, for example methyl, ethyl, n-propyl, sec.-propyl, normal-butyl, sec-butyl, the tertiary butyl, n-pentyl and other amyl group isomer, and n-hexyl and other hexyl isomer, the especially rudimentary (C of alkoxyl group ₁-C ₆) alkoxyl group, for example methoxyl group, oxyethyl group, propoxy-etc., alkynyl is ethynyl for example, or halogen fluoro substituents for example.

Substitute with in the embodiment at one, polynuclear compound can not contain or examine based on steroidal.In this respect, polynuclear compound can contain or based on on-steroidal loop systems-for example diethylstilbestrol, stilboestrol, tonka bean camphor and other loop systems.Other are suitable is used for or is found in US-A-5567831 as the non-steroids of the present composition.

R ¹And R ²

One preferred aspect in, compound is a formula I compound

Formula I

R wherein ¹For be selected from-OH, sulphamate group, phosphonate group, phosphonothionic acid ester group, sulfonate group or sulfoamido in any optional group.

One preferred aspect in, compound is a formula II compound

Formula II

One preferred aspect in, compound is the formula III compound

Formula III

One preferred aspect in, compound is a formula IV compound

Formula IV

To easily understand the A ring of steroid loop systems among the formula I-IV in addition by

radicals R

⁴2 or 4 replacements.

R ¹

It will be appreciated by those skilled in the art that R ¹For existing or can non-existent optional group.One preferred aspect in, R ¹Exist.In aspect this, R ¹For be selected from-OH, sulphamate group, phosphonate group, phosphonothionic acid ester group, sulfonate group or sulfoamido in any group.

Sulphamate group

On the one hand, R ¹Be optional sulphamate group.

Term " sulfamate " comprises ester or the ester of thionamic acid N-substitutive derivative or their salt of thionamic acid.

On the one hand, R ¹Be sulphamate group.In this respect, The compounds of this invention can be described as the sulfamate compounds thing.

Preferred R ¹Sulphamate group be the sulphamate group of following formula

R wherein ⁵And R ⁶Independently be selected from H or alkyl.

Preferred R ⁵And R ⁶Independently be selected from H, alkyl, cycloalkyl, thiazolinyl, acyl group and aryl, or its combination, or represent alkylidene group together, wherein be somebody's turn to do or optional one or more heteroatomss or the group of containing of each alkyl, cycloalkyl, thiazolinyl or aryl.

When being substituted, N-substitution compound of the present invention can contain one or two N-alkyl, N-thiazolinyl, N-cycloalkyl, N-acyl group or N-aryl substituent, preferably contains or each self-contained maximum 10 carbon atom.Work as R ⁵And/or R ⁶During for alkyl, preferred value is R wherein ⁵And R ⁶Independently be selected from those values of the low alkyl group that contains 1-5 carbon atom separately, i.e. methyl, ethyl, propyl group etc.Preferred R ⁵And R ⁶Be methyl.Work as R ⁵And/or R ⁶During for aryl, representative value is phenyl and tolyl (PhCH ₃Adjacent-,-or right-).Work as R ⁵And R ⁶During representation ring alkyl, representative value is cyclopropyl, cyclopentyl, cyclohexyl etc.R when combining ⁵And R ⁶Typically representative provides the alkylidene group of 4-6 carbon atom chain, optional insert one or more heteroatomss or group for example-O-or-NH-to be to provide 5-, 6-or 7-first heterocycle, for example morpholino, tetramethyleneimine-1-base or piperidines-1-base.

In alkyl, cycloalkyl, thiazolinyl, acyl group and aryl value, we comprise the group of replacement, and the group that this class replaces contains just like not disturbing the compound sulfatase that comes into question to suppress the substituting group of active one or more groups herein.Exemplary noiseless substituting group comprises hydroxyl, amino, halogen, alkoxyl group, alkyl and aryl.The non-limiting example of alkyl is an acyl group.

In some embodiments, sulphamate group can by with the steroid loop systems in or one or more atoms on the steroid loop systems condense (or connection) and form ring structure.

In some embodiments, can have more than a sulphamate group.As an example, two sulphamate groups (being the bis-amino sulfonate compound) can be arranged.

In some preferred embodiments, R ⁵And R ⁶In at least one is H.

In some preferred embodiments, R ⁵And R ⁶H respectively does for oneself.

In some preferred embodiments, R ¹For sulphamate group and this compound are suitable as oestrone sulphatase (E.C.3.1.6.2) inhibitor.

In some preferred embodiments, form sulfate compound if the sulphamate group on the sulfamate compounds thing is substituted by sulfate group, then this sulfate compound will be by steoid sulfatase (E.C.3.1.6.2) hydrolysis.

In some preferred embodiments, if the sulphamate group on the sulfamate compounds thing is substituted by sulfate group and forms sulfate compound, and arise from pH 7.4 and 37 ℃ of incubations with steoid sulfatase (EC.3.1.6.2), with the K that provides less than 50mM _mValue.

In some preferred embodiments, if the sulphamate group on the sulfamate compounds thing is substituted by sulfate group and forms sulfate compound, and arise from pH 7.4 and 37 ℃ of incubations with steoid sulfatase (EC.3.1.6.2), with the K that provides less than 50 μ M _mValue.

Phosphonate group

If The compounds of this invention comprises phosphonate group, then this The compounds of this invention is called phosphonate compound.

Typically, phosphonate group has formula:

(R ¹⁸)-P(O)(OH)-O-

Wherein preferred R ¹⁸Be H, alkyl, cycloalkyl, thiazolinyl, acyl group or aryl or its combination, wherein be somebody's turn to do or optional one or more heteroatomss or the group of containing of each alkyl, cycloalkyl, thiazolinyl or aryl.

Work as R ¹⁸During for alkyl, R ¹⁸Can be the low alkyl group that contains 1-6 carbon atom, i.e. methyl, ethyl, propyl group etc.As an example, R ¹⁸Can be methyl.Work as R ¹⁸During for aryl, representative value is phenyl and tolyl (PhCH ₃Adjacent-,-, right-).Work as R ¹⁸During representation ring alkyl, representative value is cyclopropyl, cyclopentyl, cyclohexyl etc.R ¹⁸Even can comprise the alkylidene group that 4-6 carbon atom chain is provided, and optional one or more heteroatomss or the group of inserting, for example so that 5 yuan of heterocycles to be provided, for example morpholino, tetramethyleneimine-1-base or piperidines-1-base.

The group that comprises replacement in alkyl, cycloalkyl, thiazolinyl, acyl group and aryl value, the group that this class replaces contains just like not disturbing the compound sulfatase that comes into question to suppress the substituting group of active one or more groups herein.Exemplary noiseless substituting group comprises hydroxyl, amino, halogen, alkoxyl group, alkyl and aryl.

In some embodiments, phosphonate group can with the steroid loop systems in or one or more atoms on the steroid loop systems condense (or connection) and form ring structure.

In some embodiments, can have more than a phosphonate group.As an example, two phosphonate groups (i.e. two-phosphonate compound) can be arranged.If these compound-bases are examined in steroidal, second preferably (or other at least one) phosphonate group is positioned at 17 of steroidal nuclear.These groups needn't be identical.

The phosphonothionic acid ester group

If The compounds of this invention comprises the phosphonothionic acid ester group, then this The compounds of this invention is called the phosphonothionic acid ester cpds.

Typically, the phosphonothionic acid ester group has formula:

(R ¹⁹)-P(S)(OH)-O-

Wherein preferred R ¹⁹Be H, alkyl, cycloalkyl, thiazolinyl, acyl group or aryl or its combination, wherein be somebody's turn to do or optional one or more heteroatomss or the group of containing of each alkyl, cycloalkyl, thiazolinyl or aryl.

Work as R ¹⁹During for alkyl, R ¹⁹Can be the low alkyl group that contains 1-6 carbon atom, i.e. methyl, ethyl, propyl group etc.As an example, R ¹⁹Can be methyl.Work as R ¹⁹During for aryl, representative value is phenyl and tolyl (PhCH ₃Adjacent-,-, right-).Work as R ¹⁹During representation ring alkyl, representative value is cyclopropyl, cyclopentyl, cyclohexyl etc.R ¹⁹Even can comprise the alkylidene group that 4-6 carbon atom chain is provided, and optional one or more heteroatomss or the group of inserting, for example so that 5 yuan of heterocycles to be provided, for example morpholino, tetramethyleneimine-1-base or piperidines-1-base.

In some embodiments, the phosphonothionic acid ester group can with the steroid loop systems in or one or more atoms on the steroid loop systems condense (or connection) and form ring structure.

In some embodiments, can have more than a phosphonothionic acid ester group.As an example, two phosphonothionic acid ester groups (i.e. two-phosphonothionic acid ester cpds) can be arranged.If these compound-bases are examined in steroidal, second preferably (or other at least a) phosphonothionic acid ester group is positioned at 17 of steroidal nuclear.These groups needn't be identical.

Sulfonate group

If The compounds of this invention comprises sulfonate group, then this The compounds of this invention is called sulfonate compound.

Typically, sulfonate group has formula:

(R ²⁰)-S(O)(O)-O-

Wherein preferred R ²⁰Be H, alkyl, cycloalkyl, thiazolinyl, acyl group or aryl or its combination, wherein be somebody's turn to do or optional one or more heteroatomss or the group of containing of each alkyl, cycloalkyl, thiazolinyl or aryl.

Work as R ²⁰During for alkyl, R ²⁰Can be the low alkyl group that contains 1-6 carbon atom, i.e. methyl, ethyl, propyl group etc.As an example, R ²⁰Can be methyl.Work as R ²⁰During for aryl, representative value is phenyl and tolyl (PhCH ₃Adjacent-,-, right-).Work as R ²⁰During representation ring alkyl, representative value is cyclopropyl, cyclopentyl, cyclohexyl etc.R ²⁰Even can comprise the alkylidene group that 4-6 carbon atom chain is provided, and optional one or more heteroatomss or the group of inserting, for example so that 5 yuan of heterocycles to be provided, for example morpholino, tetramethyleneimine-1-base or piperidines-1-base.

In some embodiments, sulfonate group can with the steroid loop systems in or one or more atoms on the steroid loop systems condense (or connection) and form ring structure.

In some embodiments, can have more than a sulfonate group.As an example, two sulfonate groups (i.e. two-sulfonate compound) can be arranged.If these compound-bases are examined in steroidal, second preferably (or other at least one) sulfonate group is positioned at 17 of steroidal nuclear.These groups needn't be identical.

Other substituting groups

The compounds of this invention can contain the substituting group except those substituting groups of formula I.As an example, these other substituting groups can be one or more following substituting groups: the oxygen base alkyl of one or more sulphamate groups, one or more phosphonate group, one or more phosphonothionic acid ester group, one or more sulfonate group, one or more sulfoamido, one or more halogen, one or more O group, one or more hydroxyl, one or more amino, one or more sulfur-containing group, one or more alkyl-for example.

R ²

The D ring of The compounds of this invention steroid loop systems is by formula-L-R ³Radicals R ²Replace, wherein L is optional linking group and R ³Be selected from for or comprise the group of one of itrile group, alcohol, ester, ether, amine and alkene.

In some preferred embodiments, R ²Be formula-R ³, in other words, no group L exists.

In aspect some are preferred, radicals R ²Be α conformation (conformation).Preferred group R ²On 17 of D ring is the α conformation.

L is an alkyl in some embodiments.L is hydrocarbon group, for example alkylidene group in some embodiments.

L can typically be C _1-10Alkylidene group, C _1-5Alkylidene group, C ₁Or C ₂Alkylidene group.

R ³

As discussed above, R ³Be selected from for or comprise the group of one of itrile group, alcohol, ester, ether, amine and alkene.In aspect some are preferred, R ³Be selected from nitrile, alcohol, ester, ether, amine and thiazolinyl.Preferred R ³For or comprise itrile group.More preferably R ³Be itrile group.

R ³Can be cyclic group or non-annularity group.

Work as R ³During for cyclic group, can form condense with steroide D ring or not with steroide D ring condensed ring.Work as R ³When the D of formation and steroide encircles the condensed cyclic group, preferred R ³Form the ring that connects D ring neighbouring part, more preferably R ³Form the ring that connects 16 and 17 on D ring.

In aspect some are preferred, R ³Be selected from formula=CH ₂,=CH-CH ₃,=C (CN) ₂,=C (CH ₃) (CN) reach-(R ⁷) _n(CR ¹⁴R ¹⁵) _pR ⁸, wherein n is 0 or 1, p is an integer, R ⁷Be selected from=CH-,-O-and NR ¹³R ⁸Be selected from-SO ₂-R ⁹,-C (O) OR ¹⁷,-OR ¹⁰, (CH ₂) _q-X-R ¹⁶,-C ≡ N ,-NR ¹¹R ¹²-C ≡ CH reaches-CH=CH ₂R ⁹Be selected from H and alkyl, R ¹⁰Be selected from H and alkyl; R ¹¹And R ¹²Independently be selected from H and alkyl separately; R ¹³Be selected from H and alkyl, R ¹⁴And R ¹⁵Independently be selected from H and alkyl separately, q is an integer, and X is O or S, R ¹⁶Be selected from H and alkyl, R ¹⁷Be selected from H and alkyl.

In aspect some are preferred, R ³Be formula-(R ⁷) _n(CR ¹⁴R ¹⁵) _pR ⁸Group, wherein n is 0 or 1, p is an integer, R ⁷Be selected from=CH-,-O-and NR ¹³R ⁸Be selected from-SO ₂-R ⁹,-C (O) OR ¹⁷,-OR ¹⁰, (CH ₂) _q-X-R ¹⁶,-C ≡ N ,-NR ¹¹R ¹²-C ≡ CH reaches-CH=CH ₂R ⁹Be selected from H and alkyl, R ¹⁰Be selected from H and alkyl; R ¹¹And R ¹²Independently be selected from H and alkyl separately; R ¹³Be selected from H and alkyl, R ¹⁴And R ¹⁵Independently be selected from H and alkyl separately, q is an integer, and X is O or S, R ¹⁶Be selected from H and alkyl, R ¹⁷Be selected from H and alkyl.

In aspect some are preferred, R ³Be formula-(CR ¹⁴R ¹⁵) _pR ⁸Group, p is an integer, R ⁸Be selected from-SO ₂-R ⁹,-C (O) OR ¹⁷,-OR ¹⁰, (CH ₂) _q-X-R ¹⁶,-C ≡ N ,-NR ¹¹R ¹²-C ≡ CH reaches-CH=CH ₂R ⁹Be selected from H and alkyl, R ¹⁰Be selected from H and alkyl; R ¹¹And R ¹²Independently be selected from H and alkyl separately; R ¹⁴And R ¹⁵Independently be selected from H and alkyl separately, q is an integer, and X is O or S, R ¹⁶Be selected from H and alkyl, R ¹⁷Be selected from H and alkyl.

In aspect some are preferred, R ³Be formula-(CH ₂) _pR ⁸Group, p is an integer; R ⁸Be selected from-SO ₂-R ⁹,-C (O) OR ¹⁷,-OR ¹⁰, (CH ₂) _q-X-R ¹⁶,-C ≡ N ,-NR ¹¹R ¹²-C ≡ CH reaches-CH=CH ₂R ⁹Be selected from H and alkyl, R ¹⁰Be selected from H and alkyl; R ¹¹And R ¹²Independently be selected from H and alkyl separately, q is an integer, and X is O or S, R ¹⁶Be selected from H and alkyl, R ¹⁷Be selected from H and alkyl.

In aspect some are preferred, R ³Be formula-(R ⁷) _nR ⁸Group, wherein n is 0 or 1, R ⁷Be selected from=CH-,-O-and NR ¹³R ⁸Be selected from-SO ₂-R ⁹,-C (O) OR ¹⁷,-OR ¹⁰, (CH ₂) _q-X-R ¹⁶,-C ≡ N ,-NR ¹¹R ¹²-C ≡ CH reaches-CH=CH ₂R ⁹Be selected from H and alkyl, R ¹⁰Be selected from H and alkyl; R ¹¹And R ¹²Independently be selected from H and alkyl separately; R ¹³Be selected from H and alkyl, q is an integer, and X is O or S, R ¹⁶Be selected from H and alkyl, R ¹⁷Be selected from H and alkyl.

P can be any integer.P can be 0-20.P can be 0-10.Typically p is 0-5.P is 0,1 or 2 on the one hand.

Q can be any integer.Q can be 0-20.Q can be 0-10.Typically q is 0-5.Q is 0,1 or 2 on the one hand.

R ⁸Be selected from-SO ₂-R ⁹,-C (O) OR ¹⁷,-OR ¹⁰, (CH ₂) _q-X-R ¹⁶,-C ≡ N ,-NR ¹¹R ¹²-C ≡ CH reaches-CH=CH ₂One preferred aspect in, R ⁸For-SO ₂-R ⁹One preferred aspect in, R ⁸For-SO ₂-R ⁹, R wherein ⁹Be alkyl.Preferably in aspect this, R ⁷For-O-, n is 1, p is 0, so that R ³For-O-SO ₂R ⁹

R ⁹Be selected from H and alkyl.One side R ⁹Be alkyl.In a preferred embodiment of the invention, R ⁹Be selected from H, C ₁-C ₂₀Alkyl, C ₁-C ₁₀Alkyl, C ₁-C ₅Alkyl, C ₁-C ₃Alkyl, hydrocarbon group, C ₁-C ₂₀Hydrocarbon polymer, C ₁-C ₁₀Hydrocarbon polymer, C ₁-C ₅Hydrocarbon polymer, C ₁-C ₃Hydrocarbon polymer, alkyl, C ₁-C ₂₀Alkyl, C ₁-C ₁₀Alkyl, C ₁-C ₅Alkyl and C ₁-C ₃One of alkyl.

One side R ⁹Be selected from H and C _1-10Alkyl.One side R ⁹Be C _1-10Alkyl.One side R ⁹Be selected from H and C _1-5Alkyl.One side R ⁹Be C _1-5Alkyl.One side R ⁹Be selected from H and C _1-3Alkyl.One side R ⁹Be C _1-3Alkyl.Preferred R ⁹For-CH ₂CH ₃

One side R ⁹For replacing or unsubstituted amine.When being substituted, N-substitution compound of the present invention can contain one or two N-alkyl, N-thiazolinyl, N-cycloalkyl, N-acyl group or N-aryl substituent, preferably contains or contain separately maximum 10 carbon atoms.One preferred aspect in, R ⁹Be unsubstituted amine, i.e. R ⁹Be NH ₂

R ¹⁰Be selected from H and alkyl.In a preferred embodiment of the invention, R ¹⁰Be selected from H, C ₁-C ₂₀Alkyl, C ₁-C ₁₀Alkyl, C ₁-C ₅Alkyl, C ₁-C ₃Alkyl, hydrocarbon group, C ₁-C ₂₀Hydrocarbon polymer, C ₁-C ₁₀Hydrocarbon polymer, C ₁-C ₅Hydrocarbon polymer, C ₁-C ₃Hydrocarbon polymer, alkyl, C ₁-C ₂₀Alkyl, C ₁-C ₁₀Alkyl, C ₁-C ₅Alkyl and C ₁-C ₃One of alkyl.

One side R ¹⁰Be selected from H and C _1-10Alkyl.One side R ¹⁰Be selected from H and C _1-5Alkyl.One side R ¹⁰Be selected from H and C _1-3Alkyl.One side R ¹⁰Be C _1-3Alkyl.Preferred R ¹⁰For-H or-CH ₃

As previously mentioned, NR ¹¹R ¹²R ¹¹And R ¹²Independently be selected from H and alkyl separately.When being substituted, N-substitution compound of the present invention can contain one or two N-alkyl, N-thiazolinyl, N-cycloalkyl, N-acyl group or N-aryl substituent, preferably contains or contain separately maximum 10 carbon atoms.Work as R ¹¹And/or R ¹²During for alkyl, preferred value is R wherein ¹¹And R ¹²Independently be selected from those values of the low alkyl group that contains 1-5 carbon atom separately, i.e. methyl, ethyl, propyl group etc.Preferred R ¹¹And R ¹²Both are methyl.Work as R ¹¹And/or R ¹²During for aryl, representative value is phenyl and tolyl (PhCH ₃Adjacent-,-or right-).Work as R ¹¹And R ¹²During representation ring alkyl, representative value is cyclopropyl, cyclopentyl, cyclohexyl etc.R when combining ¹¹And R ¹²Typically representative provides the alkylidene group of 4-6 carbon atom chain, optional one or more heteroatomss or the group of inserting, for example-O-or-NH-, so that 5-, 6-or 7-to be provided first heterocycle, for example morpholino, tetramethyleneimine-1-base or piperidines-1-base.

One side R ¹¹And R ¹²Independent separately is alkyl.In a preferred embodiment of the invention, R ¹¹And R ¹²Independently be selected from H, C separately ₁-C ₂₀Alkyl, C ₁-C ₁₀Alkyl, C ₁-C ₅Alkyl, C ₁-C ₃Alkyl, hydrocarbon group, C ₁-C ₂₀Hydrocarbon polymer, C ₁-C ₁₀Hydrocarbon polymer, C ₁-C ₅Hydrocarbon polymer, C ₁-C ₃Hydrocarbon polymer, alkyl, C ₁-C ₂₀Alkyl, C ₁-C ₁₀Alkyl, C ₁-C ₅Alkyl and C ₁-C ₃One of alkyl.

One side R ¹¹And R ¹²Independently be selected from H and C separately _1-10Alkyl.One side R ¹¹And R ¹²Independent separately is C _1-10Alkyl.One side R ¹¹And R ¹²Independently be selected from H and C separately _1-5Alkyl.One side R ¹¹And R ¹²Independent separately is C _1-5Alkyl.One side R ¹¹And R ¹²Independently be selected from H and C separately _1-3Alkyl.One side R ¹¹And R ¹²Independent separately is C _1-3Alkyl.Preferred R ¹¹And R ¹²Independently be selected from-H or-CH ₃

R ¹³Be selected from H and alkyl.In a preferred embodiment of the invention, R ¹³Be selected from H, C ₁-C ₂₀Alkyl, C ₁-C ₁₀Alkyl, C ₁-C ₅Alkyl, C ₁-C ₃Alkyl, hydrocarbon group, C ₁-C ₂₀Hydrocarbon polymer, C ₁-C ₁₀Hydrocarbon polymer, C ₁-C ₅Hydrocarbon polymer, C ₁-C ₃Hydrocarbon polymer, alkyl, C ₁-C ₂₀Alkyl, C ₁-C ₁₀Alkyl, C ₁-C ₅Alkyl and C ₁-C ₃One of alkyl.

One side R ¹³Be selected from H and C _1-10Alkyl.One side R ¹³Be selected from H and C _1-5Alkyl.One side R ¹³Be selected from H and C _1-3Alkyl.One side R ¹³Be C _1-3Alkyl.Preferred R ¹³For-H.

R ¹⁴And R ¹⁵Independently be selected from H and alkyl separately.One side R ¹⁴And R ¹⁵Independent separately is alkyl.In a preferred embodiment of the invention, R ¹⁴And R ¹⁵Independently be selected from H, C separately ₁-C ₂₀Alkyl, C ₁-C ₁₀Alkyl, C ₁-C ₅Alkyl, C ₁-C ₃Alkyl, hydrocarbon group, C ₁-C ₂₀Hydrocarbon polymer, C ₁-C ₁₀Hydrocarbon polymer, C ₁-C ₅Hydrocarbon polymer, C ₁-C ₃Hydrocarbon polymer, alkyl, C ₁-C ₂₀Alkyl, C ₁-C ₁₀Alkyl, C ₁-C ₅Alkyl and C ₁-C ₃One of alkyl.

One side R ¹⁴And R ¹⁵Independently be selected from H and C separately _1-10Alkyl.One side R ¹⁴And R ¹⁵Independent separately is C _1-10Alkyl.One side R ¹⁴And R ¹⁵Independently be selected from H and C separately _1-5Alkyl.One side R ¹⁴And R ¹⁵Independent separately is C _1-5Alkyl.One side R ¹⁴And R ¹⁵Independently be selected from H and C separately _1-3Alkyl.One side R ¹⁴And R ¹⁵Independent separately is C _1-3Alkyl.Preferred R ¹⁴And R ¹⁵Independently be selected from-H and-CH ₃

X is selected from O or S.X is S on the one hand.X is O on the one hand.

R ¹⁶Be selected from H and alkyl.In a preferred embodiment of the invention, R ¹⁶Be selected from H, C ₁-C ₂₀Alkyl, C ₁-C ₁₀Alkyl, C ₁-C ₅Alkyl, C ₁-C ₃Alkyl, hydrocarbon group, C ₁-C ₂₀Hydrocarbon polymer, C ₁-C ₁₀Hydrocarbon polymer, C ₁-C ₅Hydrocarbon polymer, C ₁-C ₃Hydrocarbon polymer, alkyl, C ₁-C ₂₀Alkyl, C ₁-C ₁₀Alkyl, C ₁-C ₅Alkyl and C ₁-C ₃One of alkyl.

One side R ¹⁶Be selected from H and C _1-10Alkyl.One side R ¹⁶Be selected from H and C _1-5Alkyl.One side R ¹⁶Be selected from H and C _1-3Alkyl.One side R ¹⁶Be C _1-3Alkyl.Preferred R ¹⁶For-H.

One very preferably aspect in, R ³For be selected from=CHC (O) OEt ,-CH ₂C (O) OEt ,=CHCH ₂OH ,-CH ₂CH ₂OH ,-CH ₂C ≡ N ,=CHC ≡ N ,-NHCH ₂CH ₂N (Me) ₂,-OCH ₂CH ₂The group of-OMe.

Particularly preferred R ³Group is selected from following given D ring and replaces

One side R ³D shown in below can being selected from replaces, and wherein each Q independently is selected from O, S, NH and CH ₂, and y is the integer of 3-8, is preferably 5,6,7 or 8.

As previously mentioned, the A of steroid loop systems ring is by R ⁴Group replaces, wherein R ⁴Be alkyl.

One preferred aspect in, R ⁴Be oxygen base alkyl.

As discussed above, used herein, about R ⁴Term " oxygen base alkyl " refer to comprise at least the group of C, H and O, and can choose wantonly and comprise one or more other suitable substituents.The substituent example of this class can comprise halogen-, alkoxyl group-, nitro-, alkyl, cyclic group etc.May be except that substituting group for the cyclic group, substituent combination can form cyclic group.If oxygen base alkyl comprises more than a C, then those carbon needn't interconnect.For example, at least two carbon can connect by suitable element or group.Therefore, oxygen base alkyl can contain heteroatoms.

Suitable heteroatoms is conspicuous to those skilled in the art, and comprises for example sulphur and nitrogen.

In a preferred embodiment of the invention, R ⁴Be oxygen base hydrocarbon group.

Term " oxygen base hydrocarbon polymer " refers to or R herein ⁴For any in alkoxyl group, oxygen base thiazolinyl, the oxygen base alkynyl, these groups can be straight chain, side chain or ring-type, or are oxygen Ji Fangji.Term oxygen base hydrocarbon polymer also comprises wherein their optional substituted those groups.If oxygen base hydrocarbon polymer is for wherein having substituent branched structure, then this substituting group can be on the main-chain hydrocarbon or on side chain; Perhaps these substituting groups can be on the main chain of hydrocarbon polymer and on side chain.

Preferred oxygen base alkyl R ⁴Be alkoxyl group.Preferred oxygen base alkyl R ⁴Be formula C _1-6O (C for example _1-3O) group.Preferred oxygen base alkyl R ⁴Be formula-O (CH ₂) _1-10CH ₃,-O (CH ₂) _1-5CH ₃,-O (CH ₂) _1-2CH ₃Group.One very preferably aspect in, R ⁴Be methoxyl group.

Preferred oxygen base alkyl R ⁴Be ether.Preferred oxygen base alkyl R ⁴Be formula C _1-6OC _1-6(C for example _1-3OC _1-3) group.Preferred oxygen base alkyl R ⁴Be formula-(CH ₂) _1-10O (CH ₂) _1-10CH ₃,-(CH ₂) _1-5O (CH ₂) _1-5CH ₃,-(CH ₂) _1-2O (CH ₂) _1-2CH ₃Group.In aspect very preferably, R ⁴For-CH ₂OCH ₃

In a preferred embodiment of the invention, R ⁴Be hydrocarbon group.Preferred R ⁴Be alkyl.Preferred this alkyl is C _1-6Alkyl (C for example _1-3Alkyl).Preferred alkyl R ⁴Be formula-(CH ₂) _1-10CH ₃,-(CH ₂) _1-5CH ₃,-(CH ₂) _1-2CH ₃Group.In aspect very preferably, R ⁴Be ethyl.

In a preferred embodiment of the invention, R ⁴Be selected from C ₁-C ₁₀Alkyl, C ₁-C ₅Alkyl, C ₁-C ₃Alkyl, hydrocarbon group, C ₁-C ₁₀Hydrocarbon polymer, C ₁-C ₅Hydrocarbon polymer, C ₁-C ₃Hydrocarbon polymer, alkyl, C ₁-C ₁₀Alkyl, C ₁-C ₅Alkyl and C ₁-C ₃One of alkyl.

In a preferred embodiment of the invention, R ⁴Be the alkyl sulfenyl.

Term " alkyl sulfenyl " refers to comprise at least the group of alkyl (as defined herein) and sulphur.This sulfenyl can be chosen wantonly oxidized.Preferred alkyl sulfenyl is formula-S-alkyl, and wherein alkyl as described herein.

Used herein, about R ⁴Term " alkyl sulfenyl " refer to comprise at least the group of C, H and S, and can choose wantonly and comprise one or more other suitable substituents.The substituent example of this class can comprise halogen-, alkoxyl group-, nitro-, alkyl, cyclic group etc.May be except that substituting group for the cyclic group, substituent combination can form cyclic group.If the alkyl sulfenyl comprises more than a C, then those carbon needn't interconnect.For example, at least two carbon can connect by suitable element or group.Therefore, the alkyl sulfenyl also can contain heteroatoms.Suitable heteroatoms is conspicuous to those skilled in the art, and comprises for example nitrogen.

In a preferred embodiment of the invention, R ⁴Be the hydrocarbon polymer sulfenyl.Used herein, about R ⁴Term " hydrocarbon polymer sulfenyl " refer to the group formed by C, H and S.Preferred hydrocarbon polymer sulfenyl is formula-S-hydrocarbon polymer, and wherein hydrocarbon polymer as described herein.

Preferred hydrocarbon polymer sulfenyl R ⁴Be formula C _1-6S (C for example _1-3S).Preferred oxygen base alkyl R ⁴Be formula-S (CH ₂) _1-10CH ₃,-S (CH ₂) _1-5CH ₃,-S (CH ₂) _1-2CH ₃One very preferably aspect in R ⁴For-S-Me.

As previously mentioned, R ⁴Be positioned at 2 or 4 that A encircles.Therefore compound can have formula

Preferred R ⁴Be positioned at 2 that A encircles.

One further preferred aspect in, when the A ring by R ¹And R ⁴During replacement, R ⁴With respect to R ¹Be the ortho position.

In a preferred embodiment, The compounds of this invention has following formula:

R wherein ⁴Be alkyl, R ¹⁴And R ¹⁵Independently be selected from H and alkyl separately, and p is an integer.

R wherein ⁴Be oxygen base hydrocarbon group or hydrocarbon group, R ¹⁴And R ¹⁵Independently be selected from H and C separately _1-10Alkyl, and p is the integer of 0-5.

In this respect, preferred R ⁴Be alkoxyl group C for example _1-6O group, or alkyl C for example _1-6Alkyl.Preferred R ⁴Be methoxyl group or ethyl.

In this respect, preferred R ¹⁴And R ¹⁵Independently be selected from H and CH separately ₃, more preferably R ¹⁴And R ¹⁵Be H.

In this respect, preferred p is 0,1 or 2.More preferably p is 1.

In a highly preferred embodiment, The compounds of this invention has following formula:

In this respect, preferred p is 0,1 or 2.More preferably p is 1.

The present invention's compound exhibits very preferably is following and can be selected from:

Composition

As mentioned above, according to an aspect of the present invention, provide a kind of medicinal compositions, said composition comprises (a) (i) compound as herein defined, or (ii) composition as herein defined, and (b) pharmaceutically acceptable carrier, thinner, vehicle or auxiliary agent.

According to the present invention, the present composition can comprise more than a kind of biological response modifier.

Term biological response modifier (" BRM ") comprising: cytokine, immunomodulator, somatomedin, the hemopoietic regulatory factor, G CFS, chemotaxis, haemolysis and the thrombolysis factor, cell surface receptor, part, leukocyte adhesion molecule, monoclonal antibody, preventative and therapeutic vaccine, hormone, extracellular matrix components, fibronectin etc.

BRMs works in immunity and the inflammatory reaction in regulating disease.The example of BRMs comprises: tumour necrosis factor (TNF), granulocyte colony-stimulating factor, erythropoietin, rhIGF-1 (IGF), Urogastron (EGF), transforming growth factor (TGF), platelet-derived somatomedin (PDGF), Interferon, rabbit (IFNs), interleukin, tissue plasminogen activator, P-, E-or L-select albumen, ICAM-1, VCAM, selection albumen, addressin etc.

Preferred biological response modifier is a cytokine.

Cytokine (often being soluble protein) is the molecule that immunocyte is linked up each other.These molecules are by bringing into play its biological function at the special receptor of target cell surface expression.Discharging of acceptor in conjunction with causing the biochemical signals cascade, and then affect the behavior (Poole, S 1995 TibTech 13:81-82) of the cell that has acceptor dearly.Identified many cytokines and acceptor (Paul and Sedar 1994, Cell 76:241-251) thereof at molecular level, and prepared suitable therapeutic value molecule and treatment target with itself.

The more detailed data of cytokine can find in Molecular Biology andBiotechnology (Pub.VCH, Ed.Meyers, 1995, the 202,203,394,390,475,790 pages).

The example of cytokine comprises: interleukin (IL)-for example IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-19; Tumour necrosis factor (TNF)-for example TNF-α; Interferon alpha, β and γ; TGF-β.

For the purpose of the present invention, the preferred cell factor is tumour necrosis factor (TNF).

More preferably cytokine is TNF-α.

TNF is by the scavenger cell of transmitting inflammation and immunopathology reaction and the cytokine that lymphocyte produces.TNF relates to advancing of disease, and these diseases include but not limited to that (neovascularization), metastases, apoptosis, Sepsis and endotoxemia take place for immunomodulatory imbalance, infection, cell proliferation, blood vessel.

The necrosis effect of TNF relates generally to the capillary vessel damage in the body.TNF not only causes necrosis in tumor tissues but also in granulation tissue.It is causing morphologic variation and cytotoxicity (1987 Ciba Found Symp131:140-153 such as Haranka) aspect the cultured vascular endothelial growth-inhibiting.

For the preferred aspect of the present invention, the TNF-that TNF can be any kind is TNF-α, TNF-β for example, comprises its derivative or mixture.

The content of TNF aspect can find in WO-A-98/08870 of technology-for example and WO-A-98/13348.

TNF can maybe can extract from the source with the chemical process preparation.Preferred TNF is by using the recombinant DNA technology preparation.

With regard to of the present invention this on the one hand with regard to, the present composition is more effective than independent compound or independent TNF in vivo.And in some respects, the combination of compound and TNF is stronger than the usefulness of the independent compound of expection, promptly is conspiracy relation between them.

In addition, the present invention expects that the present composition also comprises the interior inductor of body of the inductor of biological response modifier-biological example reaction control agent.

According to the present invention, the composition of composition can add mixture simultaneously or sequentially.In addition, according to the present invention, can be formed up to small part composition (for example in vivo) by the expression original position of inducing or increase one of composition.For example, can induce or increase for example expression of TNF of biological response modifier.As an example, can induce or increase the expression of TNF by adding bacteria lipopolysaccharide (LPS) and Muramyl dipeptide (MDP).In this respect, bacterium LPS and MDP combination can and stimulate tumour to dwindle Biull EkspBiol Med 1987 104:497-499 such as () Fuks in the generation of stimulated in vitro murine splenocyte TNF in vivo.

In methods of treatment, patient's preferred mammal, more preferably human.For some application, the preferred mankind are the women.

The present invention also comprises the new intermediate that is used to prepare The compounds of this invention.For example, the new pure precursor of inclusion compound of the present invention.As further example, the precursor of the two protections of inclusion compound of the present invention.The example of each is provided in these precursors herein.The present invention also comprises each of a kind of these precursors that comprise synthetic The compounds of this invention or both methods.

Steoid sulfatase

Steoid sulfatase-be sometimes referred to as steoid sulfatase or steryl sulfatase or several sulphating steroid of abbreviation " STS "-hydrolysis, for example oestrone sulfuric ester, trans-dehydroandrosterone sulfuric ester and cholesterol sulfate.The enzyme number of STS is EC 3.1.6.2.

STS is cloned and is expressed.For example can be referring to (Cell 49:443-454 (1987)) such as (J.Biol.Chem.264:13865-13872 (1989)) such as Stein and Yen.

STS relates to the enzyme of numerous disease situation.

As an example, the researchist finds that lacking STS fully can produce ichthyosis.According to some researchists, the STS deficiency is quite general in Japan.Same researchist (Sakura etc., JInherit Metab Dis 1997 Nov; 20 (6): 807-10) also reported allergic disease-for example bronchial asthma, allergic rhinitis or atopic dermatitis-may be relevant with deficiency of steryl-sulfatase.

, the STS activity causes the disease that STS increased activity level also may cause disease condition except lacking fully.As an example, and as above indication, there is very strong evidence to support STS in growth of breast cancers with the effect in shifting.

STS also relates to the other diseases situation.As an example, (Behav Genet1999 March such as Le Roy; 29 (2): 131-6) determined between the initiation of steoid sulfatase concentration and mouse aggressive behaviour, may have genetic correlation.This author reaches a conclusion: the sulphating of steroide may be the main prime mover of complex network, comprises that demonstration relates to the gene that causes attack by sudden change.

STS suppresses

Think that with the active relevant some diseases situation of STS be because the sulphating oestrone of non-activity changes into the oestrone of activated non-sulfuric acid esterification.In the disease condition relevant, be to suppress the STS activity ideally with the STS activity.

Herein, term " inhibition " comprises the deleterious effect that reduces and/or eliminate and/or cover and/or prevent STS.

The STS inhibitor

According to the present invention, The compounds of this invention can work as the STS inhibitor.

Herein, used herein, refer to suppress STS activity-for example reduce and/or eliminate and/or cover and/or the prevent compound of the deleterious effect of STS about the term " inhibitor " of The compounds of this invention.The STS inhibitor can be used as antagonist and works.

Compound suppresses the active ability of oestrone sulphatase and can estimate with complete JEG3 choriocarcinoma cell or placenta microsome.In addition, can use animal model.The details of suitable testing scheme is provided in following trifle.Should note: other experiments also can suppress in order to determine STS activity and STS.For example, also can be with reference to the content of WO-A-99/50453.

On the one hand, for some application, if compound characteristic also be feature promptly sulphamate group replaced by sulfate group and form sulfate derivative, then this sulfate derivative can be by having the active enzymic hydrolysis of steoid sulfatase (E.C.3.1.6.2)-promptly when with steoid sulfatase EC 3.1.6.2 during in pH7.4 and 37 ℃ of incubations.

In a preferred embodiment, if the sulphamate group of compound is replaced forming sulfate compound by sulfate group, then when with steoid sulfatase EC 3.1.6.2 during in pH7.4 and 37 ℃ of incubations, this sulfate compound can be had the active enzymic hydrolysis of steoid sulfatase (E.C.3.1.6.2) and can be produced Km value less than 200 mmoles, preferably less than 150 mmoles, preferably less than 100 mmoles, preferably less than 75 mmoles, preferably less than 50 mmoles.

In a preferred embodiment, The compounds of this invention is not had the active enzymic hydrolysis of steoid sulfatase (E.C.3.1.6.2).

For some application, preferred The compounds of this invention has the selectivity at least about 100 times to target (for example STS and/or virtueization enzyme), preferably target had selectivity at least about 150 times, preferably target had selectivity at least about 200 times, preferably target had selectivity at least about 250 times, preferably target is had selectivity at least about 300 times, preferably target is had selectivity at least about 350 times.

Should note: The compounds of this invention may also have other useful characteristics except having the ability that suppresses STS and/or virtueization enzymic activity, perhaps possess both one of.

Determine the active test of STS with cancer cells

(scheme 1)

The active inhibition of sulfatase in the JEG3 cell

Measure the steoid sulfatase activity with complete JEG3 choriocarcinoma cells in vitro.This clone can be used for studying the control of mankind mastopathy cell's growth.It has significant steoid sulfatase activity (Boivin etc., J.Med.Chem., 2000,43:4465-4478) and by American Type Culture Collection (ATCC) sell.

Cell is kept at (MEM) (FlowLaboratories of minimum medium (Minimal Essential Medium), Irvine, Scotland) in, this substratum contains 20mM HEPES, 5% foetal calf serum, 2mM glutamine, nonessential amino acid and 0.075% sodium bicarbonate.Use above-mentioned substratum, with about 1 * 10 ⁵Cell/flask inoculation can reach the 25cm of 30 replicate(determination)s at most ²Tissue culture flasks.Make cell grow into 80% degrees of fusion (confluency) and change substratum every three days.

(EBSS, from ICN Flow, High Wycombe U.K.) washs in triplicate 25cm with the EarleShi balanced salt solution ²Complete individual layer JEG3 cell in the tissue culture flasks, and in 37 ℃, with 5pmol (7 * 10 in serum-free MEM (2.5ml) ⁵Dpm) [6,7-3H] (specific activity 60Ci/mmol is from New England Nuclear, Boston for oestrone-3-sulfuric ester, Mass., U.S.A.) and oestrone-3-sulfamate (5 concentration: 1nM, 10nM, 100nM, 1000nM and 10 μ M) incubation 3-4 hour together.Behind the incubation, cool off each flask and substratum (1ml) moved into respectively with suction pipe and contain [14C] oestrone (7 * 103dpm) (specific activity 97Ci/mmol, from Amersham Intemational Radiochemical Centre, Amersham is in test tube U.K.).This mixture was with toluene (5ml) shake well 30 seconds.Test shows: through this processing,＞90%[14C] oestrone and＜0.1%[3H] oestrone-3-sulfuric ester removes from water.Separate part (2ml) organic phase, evaporation and with 3H and 14C content in the flicker optical spectroscopy mensuration residue.The oestrone of hydrolysis-3-sulfuric ester quality according to the 3H of gained counting (proofreaies and correct used substratum and organic phase volume, and proofread and correct add the rate of recovery of [14C] oestrone) and the specific activity calculating of substrate.Every batch of test comprises from the microsome of sulfatase positive human placenta (positive control) preparation and the incubation of acellular flask (to estimate tangible substrate non-enzymatic hydrolysis).After handling cell monolayer with Zaponin, determine nucleus quantity in every flask with the Coulter counter.Flask of every batch of usefulness is estimated the cytolemma situation and is used trypan blue exclusion method evaluated for viability (Phillips, H.J. (1973) In:Tissue culture and applications, [eds:Kruse, D.F.﹠amp; Patterson, M.K.]; The 406-408 page or leaf; Academic press, New York).

The active result of steoid sulfatase represents with mean value ± 1 S.D. of the gross product (oestrone+estradiol) that between incubation period (3-4 hour) forms, calculate with 106 cells, with respect to its minimizing of incubation (inhibition) percentages show statistical significance that does not contain oestrone-3-sulfamate.With not matching the statistical significance of Student t-verification test result.

Measure the active test of STS with the placenta microsome

(scheme 2)

The active inhibition of steoid sulfatase in the placenta microsome

The sulfatase positive human placenta of normal epoch pregnancy is thoroughly shredded and (pH 7.4, and 50mM) washing once and then is suspended in the cold phosphate buffered saline buffer (5ml/g tissue) with cold phosphate buffered saline buffer with scissors.Finish homogenize with the Ultra-Turrax homogenizer, per three times 10 seconds pulse is stirred the back and was cooled off 2 minutes in ice.To examine and cell debris centrifugal (4 ℃) was removed in 30 minutes with 2000g, and will part (2ml) supernatant liquor in 20 ℃ of storages.With the protein concn (Anal.Biochem., 72,248-254 (1976)) in the Bradford method mensuration supernatant liquor.

With protein concn be 100mg/ml, concentration of substrate be 20mM [6,7-3H] oestrone-3-sulfuric ester (specific activity 60Ci/mmol, from New England Nuclear, Boston, Mass. U.S.A.) carries out incubation (1ml) and in 37 ℃ of incubations 20 minutes.If necessary, adopt six kinds of concentration: 0.1nM, 1.0nM, 10nM, 100nM, 1000nM and the 10 μ M of compound.Behind the incubation, move into respectively with suction pipe with the cooling of each sample and with substratum (1ml) and to contain [14C] oestrone (7 * 103dpm) (specific activity 97Ci/mmol, from Amersham InternationalRadiochemical Centre, Amersham is in test tube U.K).This mixture was with the thorough jolting of toluene (5ml) 30 seconds.Test shows: through this processing,＞90%[14C] oestrone and＜0.1%[3H] oestrone-3-sulfuric ester removes from water.Separate part (2ml) organic phase, evaporation and with 3H and 14C content in the flicker optical spectroscopy mensuration residue.The oestrone of hydrolysis-3-sulfuric ester quality is calculated according to the 3H counting (proofread and correct used substratum and organic phase volume, and proofread and correct [14C] oestrone rate of recovery that is added) and the specific activity of this substrate of gained.

Determine the active animal test model of STS

(scheme 3)

The active inhibition of oestrone sulphatase in the body

Available Research of Animal Model for Study The compounds of this invention is particularly used ovariectomized rat.In this model, estrogen compound stimulates uterine growth.

Per os gives rat compound (0.1mg/Kg/ days, totally 5 days), and another treated animal is only accepted solvent (propylene glycol).When research finishes, obtain the hepatic tissue sample and measure the oestrone sulphatase activity as substrate, (see PCT/GB95/02638) as previously mentioned with 3H oestrone sulfuric ester.

Determine the animal test model of estrogen activity

(scheme 4)

Per os gives rat compound (0.1mg/Kg/ days, totally 5 days), and another treated animal is only accepted solvent (propylene glycol).When research finishes, obtain the uterus and weigh, with uterus weight/whole body weight * 100 ecbatics.

The compound that uterine growth is not had remarkable effect is not an estrogen compound.

Determine the active biotechnology test of STS

(scheme 5)

Compound suppresses the active ability of oestrone sulphatase, and also nucleotide sequence or its active fragments, derivative, homologue or the variant of available amino end acid sequence or coding STS carry out for example high flux screening evaluation.

Can use any or multiple suitable target-for example aminoacid sequence and/or nucleotide sequence, determine with any drug screening technology whether medicine can regulate STS.The target that adopts in this type of test can be free in the solution, is fixed in solid carrier, is connected in cell surface or is positioned at cell.Can measure active removal of target or target and the formation that is subjected to combine between the reagent thing mixture.

Test of the present invention can be the screening method of the many medicines of test.On the one hand, test method of the present invention is a high-throughput screening method.

Drug screening technology can be based on the method for Geysen description in the european patent application of announcing on September 13rd, 1,984 84/03564.In a word, for example a large amount of different little peptide test-compounds have been synthesized on plastics pin or some other surfaces at solid substrate.With peptide test-compound and suitable target or reaction of its fragment and washing.For example detect the bonded entity then by appropriate method well-known in the art.Also the target that can directly purifying be crossed is applied on the plate, and this plate is used for drug screening technology.Perhaps, available not neutralizing antibody is caught this peptide and is fixed on the solid carrier.

The present invention also expects and uses the competitive drug screening assay test, wherein can specificity competes with test-compound in conjunction with the neutralizing antibody of target to combine with target.

Another triage techniques offers the medicament high flux screening (HTS) that material is had appropriate combination avidity, and based on the method for describing in detail among the WO 84/03564.

Expect that test method of the present invention will not only be suitable on a small scale but also be suitable for large-scale test-compound screening, and be suitable for quantitative test.

One preferred aspect in, the present invention relates to a kind of selectivity of identifying and regulate the method for the medicine of STS, this compounds has formula (I).

Reporter gene

A large amount of reporter genes can be used for test method of the present invention (and screening), and preferred reporter gene provides conventional detectable signal (for example passing through spectrography).As an example, reporter gene can change catalysis the enzyme coding of the reaction of optical absorption characteristics.

Other schemes comprise enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activation cell divide (FACS).Even can use 2 points, based on monoclonal immunoassay, utilize the monoclonal antibody that two kinds of noiseless epitopes are responded.In other position, these and other test has been described in (1983, J Exp Med 15 8:121 1) such as (1990, Serological Methods, A Laboratory Manual, APS press, St Paul MN) such as Hampton R and Maddox DE.

The example of reporter molecule includes but not limited to (beta-galactosidase enzymes, saccharase, green fluorescent protein matter, luciferase, paraxin, Transacetylase, (glucuronidase, outer-dextranase and glucoamylase.Perhaps, the Nucleotide of radio-labeled or fluorescent mark substance markers can be attached in the first delayed early transcription, when being incorporated into oligonucleotide probes, it be identified subsequently.

As further example, many companies for example Pharmacia Biotech (Piscataway, NJ), Promega (Madison, WI) and US Biochemical Corp (Cleveland OH) provides commercially available reagent box and measure the program scheme.Suitable reporter molecule or mark comprise those radionuclides, enzyme, fluorescence, chemoluminescence or developer and substrate, cofactor, inhibitor, magnetic particle etc.Instruction uses the patent of this type of mark to comprise US-A-3817837, US-A-3850752, US-A-3939350, US-A-3996345, US-A-4277437, US-A-4275149 and US-A-4366241.

Host cell

The term that the present invention relates to " host cell " comprises any cell that comprises the target of medicine of the present invention.

Therefore, another embodiment of the present invention provides by target of the present invention or expresses the polynucleotide conversion of target of the present invention or the host cell of transfection.Preferably described polynucleotide being loaded into will become target and maybe will express in the carrier that duplicates and express of polynucleotide of target.Cell will be selected the cell compatible with described carrier, and for example can be protokaryon (as bacterium), fungi, yeast or vegetable cell.

Gram negative bacterium intestinal bacteria (E.coli) are widely used as the host that heterogenous gene is expressed.Yet a large amount of foreign protein matter are tending towards in the cell inner accumulated.The purifying desired protein may be difficult sometimes from a large amount of intestinal bacteria (E.coli) intracellular protein subsequently.

(E.coli) compares with intestinal bacteria, because they advance the ability of substratum with protein secreting, bacterium is well suited for as the xenogenesis host Bacillus (Bacillus).Other bacteriums that are suitable as the host are those bacteriums from streptomyces (Streptomyces) and Rhodopseudomonas (Pseudomonas).

According to the character of the polynucleotide of code book invention polypeptide and/or to the needs of proteinic further processing to be expressed, eukaryote host for example yeast or other fungies can be preferably.Generally speaking, yeast cell is better than the fungal cell, because their easy handlings.Yet some protein were both seldom secreted from yeast cell, can not suitably be handled (for example in the glycosylation of yeast camber) again in some cases.In these cases, should select different fungal host organisms.

The example of suitable expressive host within the scope of the present invention has fungi for example Aspergillus (Aspergillus) (for example those that describe) and Trichoderma (Trichoderma) in EP-A-0184438 and EP-A-0284603; Bacterium is Bacillus (Bacillus) (for example those that describe among EP-A-0134048 and the EP-A-0253455), streptomyces (Streptomyces) and Rhodopseudomonas (Pseudomonas) for example; Reach yeast for example genus kluyveromyces (Kluyveromyces) (for example those that describe among EP-A-0096430 and the EP-A-0301670) and yeast belong (Saccharomyces).As an example, typically expressive host can be selected from aspergillus niger (Aspergillus niger), Aspergillus niger var.tubigenis, Aspergillus niger var.awamori, Aspergillus aculearis, Aspergillus nidulans (Aspergillus nidulans), Aspergillus orvzae, Trichoderma reesei, subtilis (Bacillus subtilis), Bacillus licheniformis (Bacillus licheniformis), bacillus amyloliquefaciens (Bacillusamyloliquefaciens), Kluyveromyces lactis (Kluyveromyces lactis) and yeast saccharomyces cerevisiae (Saccharomyces cerevlsiae).

Use proper host cell-for example yeast, fungi and plant host cell-posttranslational modification (for example myristoylization, glycosylation, cut-out, lapidation (lapidation) and tyrosine, Serine or Threonine phosphorylation) can be provided, and this may be that to give recombination expression product of the present invention with the biological activity of the best required.

Organism

The term that the present invention relates to " organism " comprises the organism of any product that may comprise target of the present invention and/or therefrom obtain.The example of organism can comprise fungi, yeast or plant.

The term that the present invention relates to " transgenic organism " comprises any organism that comprises target of the present invention and/or products therefrom.

The conversion of host cell/host organisms

As early pointing out, host organisms can be prokaryotic organism body or most eukaryotes.Suitable prokaryotic hosts example comprises intestinal bacteria (E.coli) and Bacillus subtilus (Bacillussubtilis).The data that prokaryotic hosts transforms is a lot of in this area, for example can be referring to (Molecular Cloning:A Laboratory Manual such as Sambrook, second edition, 1989, Cold Spring Harbor Laboratory press) and Ausubel etc., Current Protocols inMolecular Biology (1995), John Wiley ﹠amp; Sons, Inc.

If the use prokaryotic hosts, then nucleotides sequence may need suitably to modify before being listed in and transforming-for example remove intron.

Transgenic organism can be yeast in another embodiment: in this respect, yeast also is widely used as the vehicle that heterogenous gene is expressed.Yeast saccharomyces cerevisiae (Saccharomycescerevisiae) has the permanent history of industrial use, comprises that it is used for heterogenous gene and expresses.Goodey etc. (1987, Yeast Biotechnology, editors such as D R Berry, pp 401-429, Allen and Unwin, London) and King etc. (1989, Molecular and Cell Biology ofYeasts, E F Walton and G T Yarronton edit pp 107-133, Blackie, Glasgow) expression in yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) is summarized to heterogenous gene.

Owing to some reasons, yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) is expressed heterogenous gene and is well suited for.At first, it is nonpathogenic to the mankind and it can not produce some intracellular toxin.Secondly, after the various purpose business developments that reach several centuries, it has the history that long-term safety is used.This has caused public's acceptance widely.The 3rd, this organism wide range of commercial is used and studied, cause producing much knowledge about yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) genetics and physiology and large scale fermentation characteristic aspect.

E Hinchcliffe E Kenny to heterogenous gene in yeast saccharomyces cerevisiae (Saccharomycescerevisiae) expression and the secretion principle of gene product carried out summarizing (1993, " Yeast asa vehicle for the expression of heterologous genes (yeast is used for heterogeneic expression as vehicle) ", Yeasts, Vol 5, Anthony H Rose and J Stuart Harrison edit, second edition, Academic Press Ltd.).

Can obtain the yeast vector of several types, comprise needing the integrative vector of recombinant host genome to keep, and the autonomously replicating plasmid carrier.

In order to prepare transgenic yeast, by nucleotide sequence being inserted in the designed structure of in yeast expression with the preparation expression constructs.Several constructions that are used for heterogenous expression have been developed.These constructions contain promoter activity in the yeast of fusion nucleus nucleotide sequence, be generally the promotor in yeast source, for example use the GAL1 promotor.Usually use the signal sequence in yeast source, the sequence of the SUC2 signal peptide of for example encoding.Active this expression system that stops of zymic terminator.

For yeast conversion, several conversion schemes have been developed.For example, transgenic yeast of the present invention can be by (1978, Proceedings of the National Academy ofSciences of the USA 75,1929) such as Hinnen; Beggs, J D (1978, Nature, London, 275,104) and Ito, H etc. (1983, J Bacteriology 153,163-168) described method preparation.

Transformed yeast cells is selected with various selected markers.At the mark that is used for transforming for example for example aminoglycosides antibiotic marker such as G418 of LEU2, HIS4 and TRP1 and dominance antibiotics resistant mark of many nutrient defect type marks arranged.

Another kind of host organisms is a plant.The ultimate principle that makes up genetically modified plant is that genetic information is inserted in the Plant Genome, is inserted the stable maintenance of genetic material to obtain.The technology that has several insertion genetic information, two kinds of cardinal principles are directly to import genetic information and import genetic information by the use carrier system.Can in the article of Potrykus (Annu Rev Plant PhysiolPlant Mol Biol[1991] 42:205-225) and Christou (Agro-Food-IndustryHi-Tech, in March, 1994/April 17-27) etc., find the summary of current techique.The content of Plant Transformation also can find in EP-A-0449375.

Therefore, the present invention also provides a kind of method with the nucleotide sequence transformed host cell, and this nucleotides sequence is classified target as or expressed target.Can under the condition that is suitable for the proteins encoded expression, cultivate by the nucleotide sequence transformed host cells.The protein that reconstitution cell produces can be presented on this cell surface.Understand if desired and by those skilled in the art, contain the expression vector available signal sequences Design of encoding sequence, this signal sequence is by specific prokaryotic cell prokaryocyte film or eukaryotic cell membrane direct secretion encoding sequence.Other recombination to construct things can be attached to this encoding sequence in the nucleotide sequence in coded polypeptide territory, and it will help the purifying ((1993) DNA Cell Biol 12:441-53 such as Kroll DJ) of soluble protein.

Variant/homologue/derivative

Remove concrete aminoacid sequence as herein described and nucleotide sequence, the present invention also comprises the purposes of their variant, homologue and derivative.Herein, term " homology " can be equal to " consistence ".

In this article, homologous sequence comprises the aminoacid sequence of at least 75%, 85% or 90% unanimity, preferably at least 95% or 98% unanimity.Although homology also can be regarded as similarity (amino-acid residue that promptly has similar chemical property/function), in the context of the present invention preferably with the consistent homology of expressing of sequence.

The homology comparison can be undertaken by eyes, or more commonly carries out by means of the sequence comparison program that obtains easily.These commercially available computer programs can calculate the homology percentage ratio between two or more sequences.

Homology percentage ratio can calculate on continuous sequence, i.e. a sequence and another sequence alignment, with each amino acid in the sequence directly and the corresponding amino acid in another sequence compare next residue.This is called " no room " comparison.Usually, this class does not have room comparison and only carries out on few relatively residue number.

Although this is very simple and consistent method, but it reckons without for example to insert or delete a residue the sequence centering of alternate manner unanimity and will cause the amino-acid residue departure ratio of back right, therefore may cause homology percentage ratio to reduce significantly when totally comparing.So most sequence comparative approach are designed to generate best comparison, this comparison is considered possible insertion and deletion and is excessively reduced total homology and keep the score.This realizes with maximization local homology by insert " room " in sequence alignment.

Yet, these more complicated methods are given " gap penalty " in each room that occurs in the comparison, thereby to the consistent amino acid of similar number, the sequence alignment (reflect between two sequences that compare and have higher dependency) with the least possible room will obtain higher keeping the score than the sequence alignment with many rooms." affinity room cost " is generally used for the existence in room is required high relatively cost and each the follow-up residue in the room is applied less point penalty.This is the most frequently used room points-scoring system.The high vacancy point penalty will generate the best comparison with less room in the nature of things.Most comparison programs allow to revise gap penalty.Yet, preferably using this class software to carry out adopting when sequence compares default value.For example when using GCGWisconsin Bestfit bag (referring to following), the acquiescence gap penalty of aminoacid sequence is: the room is-12, and each is extended for-4.

Therefore the calculating of maximum homology percentage ratio at first requires to generate the best comparison of considering gap penalty.The suitable computer program that carries out this class comparison is GCG Wisconsin Bestfit bag (University of Wisconsin, U.S.A.; Devereux etc., 1984, Nucleic AcidsResearch 12:387).Other software instances that can carry out the sequence comparison include but not limited to BLAST bag (referring to Ausubel etc., 1999 ibid-18 chapters), FASTA (Atschul etc., 1990, J.Mol.Biol., 403-410) and GENEWORKS compare tool group.BLAST and FASTA can carry out off line and online query (referring to Ausubel etc., 1999 ibid, 7-58 to 7-60 page or leaf).Yet preferably use GCG Bestfit program.

Other useful reference is found in FEMS Microbiol Lett on May 15th, 1999; 174 (2): (errata of publication is seen FEMS Microbiol Lett on August 1st, 1999 to 247-50; 177 (1): 187-8).

Although final homology percentage ratio can be measured with consistence, comparison method itself is not usually based on all or none paired comparisons.The opposite classification similarity matrix of keeping the score that uses usually will be kept the score and give each paired comparison based on chemical similarity or evolutionary distance.Normally used this matroid example is the BLOSUM62 matrix, i.e. the default matrix of blast program group.Public default value or self-defined symbol comparison sheet (if provide, relevant further details are referring to user manual) are provided the GCGWisconsin program usually.The GCG bag is preferred to use public default value, or uses default matrix (for example BLOSUM62) in other software conditions.

In case software generates best comparison, just might calculate homology percentage ratio, preferred sequence consistence percentage ratio.Software is carried out this usually as the part of sequence comparison, and generates numeric results.

Sequence also can have disappearance, insertion or the displacement of amino-acid residue, and this produces silent mutation and produces functional equivalent.Can carry out amino-acid substitution intentionally based on polarity, electric charge, solubleness, hydrophobicity, wetting ability and/or residue amphoteric similarity, as long as the secondary of reservation material is in conjunction with activity.For example electronegative amino acid comprises aspartic acid and L-glutamic acid; Positively charged amino acid comprises Methionin and arginine; Neutral polar head group, the amino acid with similar hydrophilicity value comprise leucine, Isoleucine, Xie Ansuan, glycine, L-Ala, l-asparagine, glutamine, Serine, Threonine, phenylalanine and tyrosine.

For example can carry out conservative substitution according to following table.Amino acid in amino acid in the second hurdle same sector, preferred third column are gone together mutually can be replaced mutually:

Expression vector

The nucleotide sequence of expressing target as target or be used for can be integrated into the reorganization replicable vector.Carrier is used in and/or duplicates and express nucleotide sequence from compatible host cell.Expression can be controlled by using regulating and controlling sequence, and described regulating and controlling sequence comprises promotor/enhanser and other express conditioning signal.Can use prokaryotic promoter and the promotor of function is arranged in eukaryotic cell.Can using-system specificity or stimulation specificity promoter.Also can use the chimeric promoters that comprises the sequential element that derives from two or more above-mentioned different promoters.

Can be secreted or be included in the cell by the protein that the expression nucleotide sequence generates by host's reconstitution cell, this depends on employed sequence and/or carrier.Encoding sequence can design and have signal sequence, and this signal sequence instructs the secretion of material encoding sequence by concrete protokaryon or eukaryotic cell membrane.

Fused protein

Target amino acid sequence can be used as fused protein and generates, and for example helps to extract and purifying.The accrete example of fused protein comprises glutathione-S-transferase (GST), 6xHis, GAL4 (DNA combination and/or transcription activating territory) and-tilactase.Can comprise between fused protein annexation and target protein sequence easily that also the proteolysis position is to remove the fused protein sequence.The activity that preferred fused protein does not hinder target.

Fused protein can comprise antigen or the antigenic determinant that is fused to material of the present invention.In this embodiment, fused protein can be the fused protein that non-natural exists, and this protein comprises the material that can be used as adjuvant generally to stimulate so that immunity system to be provided.Antigen or antigenic determinant can be attached to the amino or the carboxyl terminal of material.

In another embodiment of the invention, aminoacid sequence can be connected to heterologous sequence and come encoding fusion protein matter.For example for influencing the active medicine of material from the screening of peptide storehouse, the chimeric material of coding expressing heterologous epi-position is useful, and this epi-position is by commercially available antibody recognition.

Treatment

Compound of the present invention can be used as medicine, promptly is used for the treatment of in the application.

Term " treatment " comprises curative effect, improves effect and preventive effect.

Treatment can be used for the human or animal, preferred jenny.

Medicinal compositions

In one aspect, the invention provides a kind of medicinal compositions, this medicinal compositions comprises compound of the present invention and optional pharmaceutically acceptable carrier, thinner or vehicle (comprising their combination).

Medicinal compositions can be used for human or animal's purposes in people and veterinary science, and comprises any or multiple pharmaceutically acceptable thinner, carrier or vehicle usually.The acceptable carrier or the thinner that are used for the treatment of purposes are known by pharmaceutical field, and see and be set forth in for example Remington ' s Pharmaceutical Sciences (Remington's Pharmaceutical Science), Mack PublishingCo. (A.R.Gennaro edit.1985).Can select pharmaceutical carrier, vehicle or thinner according to predetermined route of administration and standard pharmacy practice.Medicinal compositions can comprise as (or except that carrier, vehicle or thinner) any suitable binder, lubricant, suspension agent, Drug coating, solubilizing agent.

Can provide sanitas, stablizer, dyestuff and even seasonings in the medicinal compositions.The example of sanitas comprises Sodium Benzoate, Sorbic Acid and p-Hydroxybenzoate.Can also use antioxidant and suspension agent.

Has different compositions/preparation requirement according to different transfer systems.As an example, medicinal compositions of the present invention can be formulated as and use the pony pump transmission or by mucosal route transmission (for example be formulated as nasal spray or inhalation aerosol and maybe can take solution) or parenteral transmission (wherein composition is formulated as injectable forms, by for example intravenously, intramuscular or subcutaneous route transmission).Perhaps, preparation can be designed to by two kinds of approach transmission.

When medicine will carry out the mucous membrane transmission by gastrointestinal mucosa, it should be able to keep stable by gi tract the time; For example it should tolerate proteolytic degradation, and is stable and tolerate biliary soil-removing action under acid pH.

If it is suitable, medicinal compositions can pass through inhalation, form administration with suppository or vaginal suppository, form topical with lotion, solution, ointment, ointment or epipasxtic, by using the skin patch administration, with the tablet form that comprises vehicle (for example starch or lactose) or with capsule or pill (separately or and mixed with excipients) form or with the form oral administration of the elixir, solution or the suspension agent that comprise seasonings or tinting material, or they can carry out parenteral injection, for example intravenously, intramuscular or subcutaneous injection.To administered parenterally, composition can be preferably uses with the aseptic aqueous solution form, and this aqueous solution can comprise other materials, for example enough salt or monose so that solution and blood etc. ooze.To through cheek or sublingual administration, composition can be with the tablet of ordinary method preparation or the form administration of lozenge.

Combination medicine

Compound of the present invention can be united use with one or more other active medicines (for example one or more other medical active medicines).

As an example, compound of the present invention can for example aromatase inhibitor (for example 4-hydroxy-androstane alkene diketone (4-OHA)) and/or steroid (organic compound of for example naturally occurring neurosteroid trans-dehydroandrosterone sulfuric ester (DHEAS) and Pregnenolone sulfate (PS) and/or other structural similitudies) be united use with other STS inhibitor and/or other inhibitor.The example of other STS inhibitor can be found in the above-mentioned reference.As an example, STS inhibitor used in the present invention comprises and one of compound that this paper provides 5 similar 2-ethyls and 2-methoxyl group 17-deoxy compound or both.

In addition or, compound of the present invention can be united use with biological response modifier.

Term biological response modifier (" BRM ") comprises cytokine, immunomodulator, somatomedin, hematopoiesis regulatory factor, G CFS, chemokine, hemolytic factor and the thrombolysis factor, cell surface receptor, part, leucocyte adherence molecule, monoclonal antibody, preventative and therapeutic vaccine, hormone, extracellular matrix components, Fibronectin etc.To some application, preferred biological response modifier is a cytokine.The example of cytokine comprises: interleukin (IL) (for example IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-19); Tumour necrosis factor (TNF) (for example TNF-α); Interferon alpha, β and γ; TGF-β.To some application, the preferred cell factor is tumour necrosis factor (TNF).Some is used, and TNF can be the TNF (for example TNF-α, TNF-β comprise their derivative or mixture) of any kind.More preferably cytokine is TNF-α.The instruction of relevant TNF can be found in technology, for example among WO-A-98/08870 and the WO-A-98/13348.

Administration

In general, the attending doctor determines the only actual dose of individual patient, and this dosage is according to age, body weight and concrete reaction and different.Following dosage is the example of average situation.Adopt higher or lower dosage range yes individual cases.

Composition of the present invention can pass through direct injection.Composition can be formulated as parenteral, mucous membrane, intramuscular, intravenously, subcutaneous, intraocular or transdermal administration.As required, medicine can be by the dosed administration of 0.01-30 mg/kg body weight, for example 0.1-10 mg/kg body weight, more preferably 0.1-1 mg/kg body weight.

As further example, medicine of the present invention can be according to 1-4 time scheme administration every day, preferred every day 1 time or 2 times.Concrete dosage level can be different to any concrete patient with dose frequency, and depend on various factors, comprise the metabolic stability of activity, this compound of the particular compound that is adopted and action time length, age, body weight, general health situation, sex, diet, administering mode and time, excretion rate, medication combined, the severity of concrete illness and the main body that stands to treat.

Except that above-mentioned typical transfer mode, term " administration " also comprises by for example lipid mediation transfection, liposome, immunoliposome, lipofection (lipofectin), cationic surface amphiphile (CFA) and their technology administrations such as combination.The approach of this class transport mechanism includes but not limited to mucous membrane, nose, mouth, parenteral, stomach and intestine, part or hypogloeeis approach.

Term " administration " includes but not limited to by mucosal route transmission (for example as nasal spray or inhalation aerosol or as taking solution); By parenteral route transmission (by the injectable forms transmission, for example intravenously, intramuscular or subcutaneous route).

Therefore to drug administration, STS inhibitor of the present invention can be in any suitable manner, adopt conventional medicine compounding process and pharmaceutical carrier, auxiliary agent, vehicle, thinner etc. are prepared, and are generally used for administered parenterally.Approximate effective dose rate can be the 1-1000 mg/day, for example the 10-900 mg/day or even the 100-800 mg/day, depend on the independent activity of the compound of discussing and patient's mean body weight (70 kilograms).Preferably the more common dose rate with more effective compound is the 200-800 mg/day, more preferably 200-500 mg/day, most preferably 200-250 mg/day.They can continue medication a couple of days by single dose scheme, divided dose scheme and/or multiple doses scheme.To oral administration, they can be formulated as tablet, capsule, solution or the suspension agent that per unit dosage comprises 100-500 milligram compound.Perhaps preferred compound is formulated in the suitable administered parenterally carrier through administered parenterally, and the odd-numbered day dose rate of 200-800 milligram is provided, preferred 200-500 milligram, more preferably 200-250 milligram.Yet the effective per daily dose of this class changes according to the intrinsic activity and the weight in patients of effective constituent, and this class changes the technology that belongs to the attending doctor and judges in the category.

Cell cycle

Compound of the present invention can be used in the cell cycle treatment of diseases method.

As " Molecular Cell Biology (molecular cytobiology) " the 3rd edition, Lodish etc., the 177-181 page or leaf is described, and different eukaryotic cells can significantly different speed growth and division.For example yeast cell can divide once in per 120 minutes, and the division first of zygote only needs 1530 minutes in the embryonic cell of sea urchin and insect, because division again takes place a big cell that is pre-existing in.Yet most growing plants and zooblast need quantity was increased doubly in 10-20 hour, and some duplicates with slower speed.Many cells (for example neurocyte and line shape muscle cell) of adult do not divide; Other are grown on demand as the inoblast that helps healing wound, but are tranquillization in other cases.

Yet each splitted eukaryotic cell must be given identical genetic material two daughter cells.DNA in the eukaryotic cell is synthetic not to be taken place in cell division cycle, but is limited to the part before the cell fission.

Synthetic and the fissional relation of eukaryotic DNA can grow and the culture of splitted mammalian cell in obtain analysing in depth.Compare with bacterium, finding only to spend portion of time by eukaryotic cell, to be used for DNA synthetic, and just finish in cell fission (mitotic division) preceding a few hours.So time of occurrence gap after DNA is synthetic and before the cell fission; After division and next round DNA another time slot appears before synthesizing.This analysis reach a conclusion into, the eukaryotic cell cycle is by M (mitotic division) phase, G ₁Phase (first gap), S (DNA is synthetic) phase, G ₂Phase (second gap) is formed, and returns M.Each phase (G between the mitotic division ₁, S and G ₂) be called the interkinesis jointly.

Before many Unseparated Cells in the tissue (for example all tranquillization inoblasts) will terminate in after the mitotic division in the cycle and DNA synthesizes; This class " dormancy " cell is considered to break away from the cell cycle and is in G ₀Attitude.

When cell is in one of three stages interkinesis of cell cycle, can come recognizing cells by the relative dna content of measuring them with fluorescence-activated cell sorting device (FACS): be in G ₁The cell of (before DNA is synthetic) has the DNA that set amount is x; (dna replication dna) is x to 2x during the S phase; When being in G ₂When (or M), it has the DNA of 2x.

The mitotic division in the zooblast and the stage of division of cytoplasm are as follows

(a) interkinesis.Intermitotic G ₂Stage begins prior to mitotic division.Chromosomal DNA duplicates and is attached on the protein during S mutually, but karyomit(e) is not counted as obvious structure yet.Nucleus is the unique nuclear structure of visible under opticmicroscope.Two kinds of form karyomit(e)s that have every type in the diploid cell before dna replication dna, cell is called as 2n.At G ₂Behind the middle dna replication dna, cell is 4n.Each chromosomal DNA has 4 copies.Because sister chromosome is also not separated from one another, they are called as sister chromatid.

(b) preprophase.Each centriole begins cytotropic antipole with the daughter centriole of new formation and moves; Karyomit(e) is visible as long line.It is folliculus that nuclear membrane begins depolymerization.

(c) in and late prophase.The karyomit(e) polycondensation is finished; Each visible chromosome structure is combined in their kinetochore by two chromatids and forms.Each chromatid comprises one of two daughter DNA molecules that newly duplicate.The microtubule spindle body begins to carry out radiation from contiguous Centriolar zone, and centriole moves the utmost point near them.Some spindle fiber reaches another utmost point from a utmost point; The overwhelming majority reaches chromatid and is attached on the kinetochore.

(d) mid-term.Move in the cytotropic equator of karyomit(e), and they line up in the plane under the line at this paper.Sister chromatid does not also separate.

(e) later stage.Two sister Chromosome Separation Correlative are karyomit(e) independently.Each comprises the kinetochore that is connected to a utmost point by spindle fiber, and karyomit(e) is moving to Ghandler motion.Therefore each chromosomal copy is endowed each daughter cell.Cell stretches simultaneously, as extremely utmost point spindle body being done.When beginning to form, a minute dehiscence furrow begins division of cytoplasm.

(f) latter stage.Around daughter nucleus, form new film; Karyomit(e) launches and becomes not too obvious, and as seen nucleus becomes again, forms nuclear membrane around each daughter nucleus.Division of cytoplasm is almost finished, and spindle body disappears with the depolymerization of microtubule and other fibers.In the whole process of mitotic division, " son " centriole of each utmost point grows to and reaches total length.In latter stage, duplicating of each archicenter grain finished, and new daughter centriole generated during the next interkinesis.

(g) interkinesis.Division of cytoplasm is once finishing, and cell enters the G of cell cycle ₁Again carry out mutually and along the cycle.

Be understandable that the cell cycle is very important cell processes.Departing from the normal cell cycle can cause many medical conditions.Enhancing and/or unconfined cell cycle can cause cancer.The cell cycle that reduces can cause the sex change illness.Use compound of the present invention that the method for this class disease of treatment and illness can be provided.

Therefore, compound of the present invention is applicable to treatment cell cycle disease (for example cancer comprises hormonal dependent and hormonal independent cancer).

In addition, compound of the present invention is applicable to treatment cancer, for example mammary cancer, ovarian cancer, carcinoma of endometrium, sarcoma, melanoma, prostate cancer, carcinoma of the pancreas etc. and other solid tumors.

To some application, the cell cycle is suppressed and/or prevents and/or stagnates, and preferably wherein the cell cycle is prevented and/or stagnates.In one aspect, the cell cycle can be suppressed and/or prevent and/or stagnate in G ₂/ M phase.In one aspect, the cell cycle can irreversibly be prevented and/or be suppressed and/or be stagnated, and preferably wherein the cell cycle is irreversibly prevented and/or stagnates.

Term " irreversibly prevent and/or suppress and/or stagnate " is meant after using compound of the present invention, removes the effect that compound still can be observed compound, promptly prevents and/or suppresses and/or stagnate the cell cycle.More particularly, term " irreversibly prevent and/or suppress and/or stagnate " is meant that the cell relative comparison cell of handling with target compound demonstrates growth more weak after the scheme I stage 2 when measuring according to the The cell cycle scheme that this paper provided.The details of this scheme is as follows.

Therefore, the invention provides such compound: by prevention and/or suppress and/or stagnate the cell cycle and the growth in vitro of estrogen receptor positive (ER+) and ER feminine gender (ER-) breast cancer cell is produced inhibition; And/or cause nitroso-group in the intact animal (promptly not spay)-methyl urea (NMU) inductive mammary tumor to be dwindled, and/or prevention and/or cell cycle of suppressing and/or stagnating cancer cells; And/or by prevention and/or suppress and/or stagnate the cell cycle and effect and/or as the cell cycle agonist in the body.

The cell cycle

(scheme 7)

Method

Stage 1

The density of MCF-7 breast cancer cell with 105 cells/well is inoculated in the porous culture plate.Allow cytoadherence and growth until about 30% fusion, do following processing to them this moment:

Any processing of control group-do not have

Target compound (COI) 20 μ M

Cell was grown 6 days in comprising the growth medium of COI, per 3 days replacing substratum/COI.When finish this period, use Coulter cell counter counting cells quantity.

Stage 2

Handling cell after 6 days with COI, with cell with 10 ⁴The density of cells/well is inoculated once more.Do not add any further processing.Allow cell continued growth 6 days again in the presence of growth medium.When finish this period, counting cells quantity once more.

Cancer

As described, compound of the present invention can be used for treating the cell cycle disease.Concrete cell cycle disease is a cancer.

Cancer remains the major cause of mortality ratio in most of western countries.The development of cancer therapy has up to now comprised blocking-up functions of hormones or the growth of synthesizing to come the inhibitory hormone dependent tumors.Yet application at present has more aggressive chemotherapy and treats the hormonal independent tumour.

Therefore, the development of the anticancer therapy medicine of hormonal dependent and/or hormonal independent tumour, and lack some or all chemotherapy dependency side effect, the treatment progress that representative is main.

Known that oestrogenic hormon is in its synthetic back many hydroxylations of experience and conjugation reaction.It is believed that as of late the reaction of this class is the part of metabolic process and finally give oestrogenic hormon with water-soluble and promote them to eliminate from health.(for example the oestrone sulfuric ester is E1S) significant to determining oestrogenic hormon some complex reaction in vivo for verified now some hydroxy metabolite thing (for example 2-hydroxyl and 16 Alpha-hydroxies) and conjugate.

Researchists after deliberation 2-and the estrogenic formation of 16-hydroxylation and the relation that changes mammary cancer risk.Proved now that improving the active factor of 2-hydroxylase relates to the risk of cancer of reduction, and the factor that improves 16 'alpha '-hydroxylations can increase the risk of mammary cancer.Further interest to estrogen metabolism thing biological action is excited by ever-increasing evidence, and promptly the 2-methoxyestradiol is the endogenous metabolism thing with antimitotic character.2-MeOE2 is formed by catechol estrogen methyltransgerase (a kind of enzyme that extensively distributes in vivo) by 2-hydroxyl estradiol (2-OHE2).

Researchists have shown that 2-MeOE2 suppresses growth of tumor in vivo, and described tumour generates by subcutaneous injection Meth A sarcoma, B16 melanoma or MDA-MB-435 estrogen receptor negative (ER-) breast cancer cell.It is gone back inhibition of endothelial cell proliferation and migration and extracorporeal blood vessel and generates.The ability that people propose 2-MeOE2 inhibition tumor growth in vivo may suppress the ability of tumor inducing vasculogenesis owing to it, rather than directly suppresses the propagation of tumour cell.

The mechanism that 2-MeOE2 brings into play its effective antimitotic and angiogenesis inhibitor effect is still waiting to illustrate.Evidence suggests that when high density it can suppress microtubule polymerization and be attached to the weak inhibitor of tubulin as colchicine.Yet under the mitotic concentration of blocking-up, intracellular tubulin silk is not found depolymerization but is had and finding same modality after safe plain the processing recently.Therefore, might be as safe plain (medicine that is used for the treatment of mammary gland and ovary mammary cancer), 2-MeOE2 works by stabilize microtubules kinetics.

Though confirming 2-MeOE2 is the new important progress of novel remedies for cancer representative, peroral administration estrogenic bioavailability is very poor.In addition, they experience metabolism widely during first by liver.As the part of development research plan of the steoid sulfatase inhibitor of treatment mammary cancer, oestrone-3-O-sulfamate (EMATE) is confirmed to be effective active fixed point inhibitor.Unexpectedly, the EMATE proof has effective oestrogenic hormon characteristic, and its oral uterus nutritional activities in rat is higher 100 times than estradiol.The oestrogenic hormon of its raising is considered to derive from it and is absorbed by red corpuscle (rbcs), and this protects it to avoid inactivation by liver the time and as storer, is used for it and slowly discharges in long time period.Synthetic and tested many A rings and modified analogues, comprise 2-methoxyestrone-3-O-sulfamate.Though this compound is when the steoid sulfatase inhibitor and the EMATE equivalence, it does not have oestrogenic hormon.

We believe that compound of the present invention provides the particularly methods of treatment of mammary cancer of cancer.

In addition or or, compound of the present invention can be used for blocking the growth of cancer, described cancer comprises leukemia and solid tumor, for example mammary gland, uterine endometrium, prostate gland, ovary and pancreatic neoplasm.

About estrogenic treatment

We believe that some compound of the present invention can be used in the control volume, the intravital estrogen level of women particularly.Therefore, some compound can be used for providing the method for control fertility, for example oral contraception tablet, pill, solution or lozenge.Perhaps The compounds of this invention can be the form of implant or patch.

Therefore, compound of the present invention can be used for treating estrogen related hormone illness.

In addition or or, compound of the present invention can be used for treating the hormone illness except that those estrogen related illnesss.Therefore, compound of the present invention can also influence hormonal activity and can influence immunne response.

Neurodegenerative disease

We believe that some The compounds of this invention can be used for treating neurodegenerative disease and similar conditions.

As an example, think that the memory function that the STS inhibitor can be used for improving the patient or seeks the individuality that memory improves, described patient suffer from for example dementia etc. after dementia, senile dementia, vascular dementia and the apoplexy after amnesia, head injury, alzheimer's disease, epileptic dementia, presenile dementia, the wound of disease.

TH1

We believe that some The compounds of this invention can be used for the TH1 purposes.

As an example, think that providing the existence of STS inhibitor in the antigenic cell can cause reducing responsive T cell at scavenger cell or other improves the ability that TH1 (high IL-2, IFN γ hang down IL-4) reacts.Therefore other steroides for example the normal regulating influence of glucocorticosteroid will preponderate.

Inflammatory conditions

We believe that some The compounds of this invention can be used for treating the illness of inflammatory conditions-for example and following any or multiple disease-related: autoimmune disorder for example comprises rheumatoid arthritis, I type and type ii diabetes, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, thyroiditis, vasculitis, ulcerative colitis and Crohn disease, tetter for example psoriatic and contact dermatitis; Graft versus host disease (GVH disease); Eczema; Organ rejection response after asthma and the transplanting.

As an example, think that the STS inhibitor can prevent DHEA or the relevant steroide normal physiological effect to immunity and/or inflammatory reaction.

The compounds of this invention can be used for preparing the medicine that shows the effect of endogenous glucocorticosteroid sample.

Other treatment

Also can understand The compounds of this invention/composition and can have other important medical purposes.

For example, The compounds of this invention or composition can be used for treating listed disease among the WO-A-99/52890, that is:

In addition, perhaps The compounds of this invention or composition can be used for treating listed disease among the WO-A-98/05635.The part of listed disease now is provided for ease of reference: cancer, inflammation or inflammatory diseases, tetter, heating, cardiovascular effect, hemorrhage, condense and acute phase reaction, emaciation, apositia, acute infection, HIV infection, shock state, graft-vs-host reaction, autoimmune disorder, reperfusion injury, meningitis, migraine and acetylsalicylic acid dependency anti-thrombi; Tumor growth, invasion and attack and diffusion, vasculogenesis, transfer, malignant tumour, ascites and malignant pleural chamber hydrops; Cerebral ischemia, ischemic heart disease, osteoarthritis, rheumatoid arthritis, osteoporosis, asthma, multiple sclerosis, neurodegeneration, alzheimer's disease, arteriosclerosis, shock, vasculitis, Crohn disease and ulcerative colitis; Periodontitis, gingivitis; Psoriatic, atopic dermatitis, chronic ulcer, epidermolysis bullosa; Keratohelcosis, retinopathy and operation wound heal; Rhinitis, anaphylaxis conjunctivitis, eczema, anaphylaxis; Restenosis, congestive heart failure, endometriosis, arteriosclerosis or internal cause sclerosis (endosclerosis).

In addition, perhaps The compounds of this invention or composition can be used for treating listed disease among the WO-A-98/07859.For ease of reference, now provide the part of listed disease: cytokine and cell proliferation/differentiation activity; Immunosuppressor or immunostimulant activity (for example are used for the treatment of immune deficiency, comprise the infected person immunodeficiency virus; Regulate lymphocyte growth; Treatment cancer and many autoimmune disorders, and prevention transplant rejection or induced tumor immunity); Regulate hemoposieis, for example treat marrow or lymph disease; Promote the growth of bone, cartilage, tendon, ligament and nervous tissue, for example healing wound, burn, ulcer and periodontal disease and neurodegenerative treatment; The inhibition or the activation of follicle stimulating hormone (adjusting fertility); Chemotaxis/chemical kinetics activity (for example mobilizing the special cells type) to damage or infection site; Hemostasis and thrombus activity (for example treating hemophilia and apoplexy); Anti-inflammatory activity (being used for the treatment of for example septic shock or Crohn disease); As biocide; For example metabolism or behavior regulator; As pain killer; Treat concrete defective disease; Treat for example psoriatic with human or veterinary drug.

In addition, perhaps the present composition can be used for treating listed disease among the WO-A-98/09985.The part of listed disease now is provided for ease of reference: scavenger cell suppresses and/or T cell inhibitory activity and anti-inflammatory activity; Anti-immunocompetence, promptly the restraining effect of pair cell and/or humoral immune reaction comprises not relevant with inflammation reaction; Suppress scavenger cell and T cell attachment in the ability of extracellular matrix components and fibronectin, and the expression of fas acceptor in the T cell of raising; Suppress unnecessary immune response and comprise the inflammation of sacroiliitis, comprise rheumatoid arthritis, the inflammation relevant with allergy, atopic reaction, asthma, systemic lupus erythematosus, collagenosis and other autoimmune disorders, the inflammation relevant with atherosclerosis, arteriosclerosis, atherosclerotic heart disease, reperfusion injury, asystolia, myocardial infarction, the vasculitic disease, respiratory distress syndrome or other heart and lung diseases, the inflammation relevant with peptide ulceration, ulcerative colitis and other gastrointestinal tract disease, hepatic fibrosis, liver cirrhosis or other hepatopathys, thyroiditis or other gland diseases, glomerulonephritis or other kidneys and urinary tract disorder, otitis or other otorhinolaryngology diseases, dermatitis or other tetter, periodontal disease or other odontopathies, testitis or epididymitis, infertility, testis wound or other immune-related testis disease, the placenta dysfunction, placenta insufficiency, habitual abortion, faint from fear, preeclampsia and other immunity and/or inflammation dependency gynaecopathia, back uveitis, middle uveitis, anterior uveitis, conjunctivitis, choroidoretinitis, the uvea retinitis (uveoretinitis), optic neuritis, intraocular inflammation is the retinitis or cystoid macular edema for example, the sympathetic nerve ophthalmia, scleritis, retinitis pigmentosa, the immunity and the inflammatory component of (fondus) disease dissolved in sex change, the inflammatory component of eye wound, the eye inflammation that infection causes, the proliferative vitreoretinopathy, acute ischemic ophthalmic nerve disease, excessively cicatrization for example forms scar after glaucoma filtration surgery, ocular implant immunity and/or inflammatory reaction and other immunity and inflammation dependency ophthalmic diseases, with autoimmune disorder or inflammation or the relevant inflammation of obstacle, wherein in central nervous system (CNS) or in any other organ, it will be useful that immunity and/or inflammation suppress, Parkinson's disease, complication and/or side effect that the Parkinson's disease treatment causes, the dementia that AIDS is relevant merges the relevant encephalopathic of HIV, the DevicShi disease, the Sydenham tarantism, alzheimer's disease and other degenerative diseases, CNS illness or obstacle, the stokes inflammatory component, post poliomyelitis syndrome, the immunity of psychiatric disorders and inflammatory component, myelitis, encephalitis, subacute sclerosing panencephalitis, encephalomyelitis, acute neuropathy, subacute neuropathy, chronic neuropathic, Guillaim-Barre syndrome, Sydenham chora, myasthenia gravis, pseudotumor cerebri, the Down Cotard, Huntington Chorea, amyotrophic lateral sclerosis, the inflammatory component that CNS compressing or CNS wound or CNS infect, amyotrophy and underfed inflammatory component and immunity and inflammation are diseases related, maincenter and external application neurological conditions or obstacle, inflammation after the wound, septic shock, transmissible disease, inflammatory complication or side effect that operation causes, bone marrow transplantation or other complication of transplant and/or side effect, inflammatory and/or immunologic complication and side effect that gene therapy causes, the for example infection that causes by the virus carrier, or the inflammation relevant with AIDS, suppress body fluid and/or cell immune response, treat or improve for example leukemia of monocyte or white corpuscle proliferative disease by reducing monocyte or lymphocytic amount, transplanting natural or artificial cell, tissue and organ be cornea for example, marrow, organ, lens, pacemaker, prevent and/or treat transplant rejection under the situation of natural or artificial skin tissue.

Compound

The compounds of this invention can pass through suitable alcohol and suitable muriate prepared in reaction.As an example, sulfamate compounds thing of the present invention can be by with suitable alcohol and suitable formula R ⁵R ⁶NSO ₂The sulphonamide chlorine prepared in reaction of Cl.

The representative condition that carries out this reaction is as follows.

In the anhydrous dimethyl formamide solution of 0 ℃ of alcohol that sodium hydride and sulphonamide chlorine is joined stirring.Subsequently, make this reaction be warming up to room temperature, and continue again to stir 24 hours.This reaction mixture is poured in the cold saturated sodium bicarbonate solution, and with dichloromethane extraction gained water.With the anhydrous MgSO of organic extract that merges ₄Dry.Filter, vacuum evaporating solvent then, and use the toluene coevaporation, and obtaining thick residue, it further passes through the flash chromatography purifying.

Preferably before reaction, use sulphonamide chlorine suitably with pure derivatize.In case of necessity, this blocking group or those blocking groups can and be removed with the currently known methods protection by Chun functional group when reaction finishes.

Preferably according to (1990 Tetrahedron 46 such as Page; Method 2059-2068) prepares the sulfamate compounds thing.

Can be suitably in conjunction with (1990 Tetrahedron 46 such as Page; 2059-2068) and the method for PCT/GB92/01586 prepare phosphonate compound.

Can suitably adopt (1990 Tetrahedron 46 such as Page; 2059-2068) and the method for PCT/GB92/01586 prepare sulfonate compound.

Can suitably adopt (1990 Tetrahedron 46 such as Page; 2059-2068) and the method for PCT/GB91/00270 prepare the phosphonothionic acid ester cpds.

Preferred manufacturing procedure also provides hereinafter.

Embodiment

The present invention will describe in further detail by embodiment and with reference to accompanying drawing now, wherein:

Fig. 1 shows concise and to the point flow process;

Fig. 2 shows concise and to the point flow process;

Fig. 3 shows figure; With

Fig. 4 shows figure;

Fig. 5 display panel;

Fig. 6 display panel;

Fig. 7 shows figure;

Fig. 8 shows figure;

Fig. 9 shows figure;

Figure 10 display panel;

Figure 11 shows figure;

Figure 12 shows figure;

Figure 13 shows figure;

Figure 14 shows fluorescence photo;

Figure 15 shows figure;

Figure 16 shows figure;

Figure 17 shows figure;

The present invention will only describe by embodiment now. Yet, it is to be understood that these embodiment also represent the preferred compound of the present invention, and prepare the preferred routes of these compounds and for the preparation of the useful intermediates of these compounds.

Synthetic

According to following flow process

The 17-alkenyl amino sulphonate of Synthetic 2-replacement

The functional conversion of 17-alkenyl esters

The EMATES's that nitrile is functionalized is synthetic

17-alkyl-EMATEs

The amino oestrogenic hormon of 17-

Wherein P is a blocking group.

Method A

With sulphonamide chlorine (680 μ l, 0.745M toluene solution vacuum concentration 0.507mmol) (bath temperature is below 30 ℃), and in ice bath, cool off before adding DMA (1.5ml).Add suitable phenol (0.253mmol) then and make this reaction reach ambient temperature overnight.Also (3 * 10ml) extract this mixture with ethyl acetate to add entry.Merge organic constituent, and water (5 * 10ml) and salt solution (10ml) washing, dry (Na ₂SO ₄) and vacuum concentration.

Oestrone 3-O-t-butyldimethylsilyl ether 1

Method B

With oestrone (10g, 37mmol), imidazoles (6.4g, 94mmol) and t butyldimethylsilyl chloride (6.7g 44.4mmol) is dissolved in the dimethyl formamide (130ml), and under nitrogen stirred overnight at room temperature.Add entry and use methylene dichloride (3 * 100ml) extraction mixtures.The organic constituent water that merges (2 * 100ml) and salt solution (10ml) washing, dry (Na ₂SO ₄) and vacuum concentration.

Ethyl alcohol recrystallization, the oestrone white, needle-shaped crystals that is protected (11.34g, 29.6mmol, 80%).Mp.172-173 ℃ (literature value m.p.170-172 ℃).

δ _H(CDCl ₃) 0.19[6H, s, Si (CH ₃) ₂)], 0.91 (3H, s CH ₃), 0.98[9H, s, C (CH ₃) ₃], 1.36-1.68 (5H, m, alkyl H), 1.90-2.55 (7H, m, alkyl H), 2.82-2.88 (2H, m, alkyl H), 6.57 (1H, d, J=2.3, ArH-4), 6.62 (1H, dd, J=8.6,2.3, ArH-2), 7.12 (1H, d, J=8.6, ArH-1).

J.Org.Chem.52:1987 such as m.p.:Fevig, 247-251.

[3-(tertiary butyl-dimethyl-silanyloxy base)-13-methyl-6,7,8,9,11,12,13,14,15,16-decahydro-cyclopenta [a] phenanthrene-17-subunit]-ethyl acetate 2

Method C

Under room temperature, nitrogen, (520mg 17.4mmol) dissolves up to all solids to stir sodium hydride among the anhydrous THF (15ml).(7.14mmol) and adding TBS oestrone 1 (2g 5.2mmol) stirs 10 minutes before for 3.20g, 2.84ml to add the phosphonic acids triethyl acetate.Gained mixture reflux is spent the night.Add entry and use ethyl acetate (3 * 50ml) extraction mixtures.The organic constituent water that merges (2 * 50ml) and salt solution (10ml) washing, dry (Na ₂SO ₄) and vacuum concentration.

Rapid column chromatography (SiO ₂Hexane: ethyl acetate 9: 1) obtain two kinds of mixture of isomers, be white crystalline solid (1.73g, 3.77mmol, 73%).Determine that by NMR the ratio of isomer is 5: 1.m/z(EI ⁺)454(M ⁺，50％)，397(90％)，82.9(100％)。HRMS (FAB ⁺) calculated value C ₂₈H ₄₂O ₃Si454.2903, measured value 454.2910.

Method: Tetrahedron such as Ewers, 54:1998,4277-4282.

(3-hydroxyl-13-methyl-6,7,8,9,11,12,13,14,15,16-decahydro-cyclopenta [a] phenanthrene-17-subunit)-ethyl acetate 3

Method D

Under room temperature, nitrogen, stir TBS ether 2 (100mg, 0.220mmol), the THF solution of 1M tertiary butyl Neutral ammonium fluoride (300 μ l, 0.300mmol) and THF (5ml) 2 hours.Add entry and use ethyl acetate (3 * 5ml) extraction mixtures.Merge organic constituent, and water (2 * 5ml) and salt solution (5ml) washing, dry (Na ₂SO ₄) and vacuum concentration.

Rapid column chromatography (SiO ₂Hexane: ethyl acetate 9: 1) obtain two kinds of mixture of isomers, be white crystalline solid (74mg, 0.218mmol, 99%).m.p.118-120℃。Determine that by NMR the ratio of isomer is 5: 1.

δ _H(CDCl ₃) 0.86 (3H, s, CH ₃), 1.30 (3H, t, J=7.0, CH ₃), 1.41-2.91 (15H, m, alkyl H), 4.17 (2H, q, J=7.0), 5.20 (1H, s, OH), 5.59 (1H, t, J=2.3, alkene H), 6.58 (1H, d, J=2.7, ArH-4), 6.64 (1H, dd, J=8.2,2.7, ArH-2), 7.15 (1H, d, J=8.2, ArH-1);

M/z (FAB ⁺) 341.1[(MH) ⁺, 100%]; HRMS (FAB ⁺) calculated value C ₂₂H ₂₈O ₃+ H 341.2116, measured value 341.2111.

2-[3-(tertiary butyl-dimethyl-silanyloxy base)-13-methyl-6,7,8,9,11,12,13,14,15,16-decahydro-cyclopenta [a] phenanthrene-17-subunit]-ethanol 4

Method E

Under-78 ℃, nitrogen, to TBS ether 2 (300mg, add in THF 0.66mmol) (5ml) solution 1.5M diisobutyl hydrogenation ammonium toluene solution (1ml, 1.5mmol).This solution is warming up to 0 ℃ and stirred 1.5 hours.Add the first alcohol and water and make this solution be warming up to room temperature in 0 ℃, stirred 30 minutes.With ethyl acetate (3 * 10ml) extract this turbid solution, and water (2 * 10ml) and the organic constituent that merges of salt solution (10ml) washing, dry (Na ₂SO ₄) and vacuum concentration.

Gained white solid (179mg, 0.434mmol, 66%) need not to be further purified and can use.M.p.128-130 ℃ (literature value m.p.128-130 ℃). δ _H(CDCl ₃) 0.18[6H, s, Si (CH ₃) ₂)], 0.81 (3H, s CH ₃), 0.98[9H, s, C (CH ₃) ₃], 1.03-1.61 (7H, m, alkyl H), 1.74-1.97 (3H, m, alkyl H), 2.16-2.42 (3H, m, alkyl H), 2.75-2.86 (2H, m, alkyl H), 4.07-4.23 (2H, m, CH ₂OH), 5.26-5.31 (1H, m, alkene H), 6.55 (1H, d, J=2.3, ArH-4), 6.61 (1H, dd, J=8.6,2.3, ArH-2), 7.13 (1H, d, J=8.6, ArH-1).Determine that by NMR the ratio of isomer is 8: 1.

M/z (FAB ⁺) 412.1 (M ⁺, 7%), 73.0 (100%), 147 (35%); HRMS (FAB ⁺) calculated value C ₂₆H ₄₀O ₂Si 412.2798, measured value 412.2778.

Method: the patent No.s such as Tanabe: US 6,281,205 B1.2001.Estrogen antagonist steroidal compounds and relevant medicinal compositions and using method.

Tetrahedron such as m.p.:Ewers, 54:1998,4277-4282.

17-(2-hydroxyl-ethylidene)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-3-alcohol, 5

(179mg 434mmol) carries out this reaction by the description of method D with allyl alcohol 4.Rapid column chromatography (SiO ₂Hexane: ethyl acetate 4: 1) obtain two kinds of mixture of isomers, be white powder (74mg, 0.218mmol, 99%).m.p.200-202℃。δ _H(CDCl ₃) 0.81 (3H, s, CH ₃), 1.13-1.56 (7H, m, alkyl H), 1.82-1.98 (3H, m, alkyl H), 2.16-2.43 (3H, m, alkyl H), 2.82-2.87 (2H, m, alkyl H), 5.27-5.32 (2H, m, CH ₂OH), 4.53 (1H, s, OH), 5.27-5.32 (1H, m, alkene H), 6.56 (1H, d, J=2.3, ArH-4), 6.63 (1H, dd, J=8.6,2.3, ArH-2), 7.17 (1H, d, J=8.6, ArH-1); M/z (FAB ⁺) 298.1 (M ⁺, 75%), 281.1 (100%); HRMS (FAB ⁺) calculated value C ₂₀H ₂₆O ₂298.1933, measured value 298.1935.

[3-(tertiary butyl-dimethyl-silanyloxy base)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-17-yl]-ethyl acetate 6

Method F

Under hydrogen atmosphere, (300mg, 0.66mmol) ethanol (5ml) solution with 5% palladium/lime carbonate (17mg) spends the night to stir ester 2.This reaction mixture is filtered and uses washing with alcohol by diatomite (Celite) bed.The filtrate vacuum concentration.

Rapid column chromatography (SiO ₂Hexane: ethyl acetate 4: 1) obtain the white crystalline solid (233mg, 0.51mmol, 77%) of reduzate 6.m.p.64-66℃.

δ _H(CDCl ₃) 0.18[6H, s, Sl (CH ₃) ₂)], 0.64 (3H, s CH ₃), 0.97[9H, s, C (CH ₃) ₃], 1.27 (3H, t, J=7.4, CH ₃), 1.30-1.56 (5H, m, alkyl H), 1.73-2.42 (9H, m, alkyl H), 2.79-2.82 (2H, m, alkyl H), 4.13 (2H, q, J=7.4, CH ₂), 6.54 (1H, d, J=2.7, ArH-4), 6.60 (1H, dd, J=8.6,2.7, ArH-2), 7.11 (1H, d, J=8.6, ArH-1);

M/z (FAB ⁺) 456.1 (M ⁺, 100%); HRMS (FAB ⁺) calculated value C ₂₈H ₄₄O ₃Si456.3060, measured value 456.3041.

(3-hydroxyl-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-17-yl)-ethyl acetate 7

(100mg 0.211mmol) carries out this reaction by the description of method D with ester 6.Rapid column chromatography (SiO ₂Hexane: ethyl acetate 4: 1) obtain the grey spicule (56mg, 0.164mmol, 78%) of phenol 7.m.p.126-128℃。

δ _H(CDCl ₃) 0.63 (3H, s CH ₃), 1.27 (3H, t, J=7.0, CH ₃), 1.30-2.43 (14H, m, alkyl H), 2.78-2.84 (2H, m, alkyl H), 4.13 (2H, q, J=7.0, CH ₂), 4.60 (1H, s, OH), 6.56 (1H, d, J=2.7, ArH-4), 6.62 (1H, dd, J=8.2,2.7, ArH-2), 7.14 (1H, d, J=8.2, ArH-1).

M/z (FAB ⁺) 342.1 (M ⁺, 100%); HRMS (FAB ⁺) calculated value C ₂₂H ₃₀O ₃342.2195, measured value 342.2201.

2-(3-hydroxyl-13-methyl-6,7,8,9,11,12,13,14,15,16-decahydro-cyclopenta [a] phenanthrene-17-subunit)-ethyl propionate 8

With TBS oestrone 1 (1g, 2.6mmol) and 2-phosphono propionic acid triethyl ester (1.7g, 1.53ml 7.14mmol) carry out this reaction by the description of method C.Rapid column chromatography (SiO ₂Hexane: ethyl acetate 9: 1) obtain protecting the individual isomer of product 8, be white powder (170mg, 0.48mmol, 18%).m.p.113-115℃。δ _H(CDCl ₃) 0.91 (3H, s CH ₃), 1.40 (3H, t, J=6.6, CH ₃), 1.43-1.68 (7H, m, alkyl H), 1.91-2.28 (6H, m, alkyl H), 2.37-2.42 (2H, m, alkyl H), 2.87-2.91 (2H, m, CH ₂), 4.00 (2H, q, J=7.0, CH ₂), 6.64 (1H, d, J=2.7, ArH-4), 6.71 (1H, dd, J=8.6,2.7, ArH-2), 7.19 (1H, d, J=8.6, ArH-1).

[3-(tertiary butyl-dimethyl-silanyloxy base)-13-methyl-6,7,8,9,11,12,13,14,15,16-decahydro-cyclopenta [a] phenanthrene-17-subunit]-acetonitrile 9

With TBS oestrone 1 (5g, 13.0mmol) and diethyl (cyano methyl) phosphonic acid ester (4.02g, 4.4ml 22.7mmol) carry out this reaction by the description of method C.Aftertreatment crude product mixture subsequently, its TLC reaches ¹H NMR shows that starting raw material no longer exists, and this product is that ratio is the mixture of two kinds of isomer 9 of 6: 1.This mixture need not to be further purified and can use.

(3-hydroxyl-13-methyl-6,7,8,9,11,12,13,14,15,16-decahydro-cyclopenta [a] phenanthrene-17-subunit)-acetonitrile 10

(100g 0.246mmol) carries out this reaction by the description of method D with silyl ether 9.Rapid column chromatography (SiO ₂Hexane: ethyl acetate 4: 1) obtain the white powder (53mg, 0.18mmol, 73%) of phenol 10 individual isomer.m.p.264-266℃。δ _H(DMSO) 0.84 (3H, s CH ₃), 1.24-1.50 (8H, m, alkyl H), 1.82-1.87 (2H, m, alkyl H), (1.93-1.97 1H, m, alkyl H), 2.09-2.16 (1H, m, alkyl H), 2.18-2.34 (1H, m, alkyl H), 2.66-2.77 (2H, m, alkyl H), 5.37 (1H, m, alkene H), 6.44 (1H, d, J=2.64, ArH-4), 6.51 (1H, dd, J=8.2,2.6, ArH-2), 7.06 (1H, d, J=8.2, ArH-1), 9.02 (1H, s, OH); M/z (FAB ⁺) 293.1 (M ⁺, 100%); HRMS (FAB ⁺) calculated value C ₂₀H ₂₃ON 293.1780, measured value 293.1783.

13-methyl-17-methylene radical-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-3-alcohol 11

(220mg 1.96mmol) joins among the anhydrous THF (5ml) and stirred 10 minutes, and this salt is dissolved fully with potassium tert.-butoxide.With methyltriphenylphospbromide bromide (700mg, 1.96mmol) gradation joins in this salts solution.(500mg 1.85mmol) is dissolved in THF (5ml) and be added drop-wise in this pale yellow solution with syringe with oestrone.After the stirring at room 2 hours, should react reflux and spend the night.Should react cooling, also (3 * 10ml) extract this mixture with ethyl acetate to add entry (10ml).Merge organic constituent, and water (2 * 10ml) and salt solution (10ml) washing, drying (Na ₂SO ₄) and vacuum concentration.Rapid column chromatography (SiO ₂Hexane: ethyl acetate 9: 1) also use the toluene/hexane recrystallization, obtain the white crystalline solid (53mg, 0.198mmol, 11%) of alkene 11.Mp.130-132 ℃ (literature value m.p.134-137 ℃).δ _H(CDCl ₃) 0.82 (3H, s CH ₃), 1.20-1.62 (6H, m, alkyl H), 1.78-1.85 (1H, m, alkyl H), (1.90-1.99 2H, m, alkyl H), 2.16-2.38 (3H, m, alkyl H), 2.50-2.59 (1H, m, alkyl H), 2.78-2.92 (2H, m, alkyl H), 4.50 (1H, s, OH), 4.67 (2H, t, J=2.0, alkene H), 6.57 (1H, d, J=2.7, ArH-4), 6.63 (1H, dd, J=8.6,2.7, ArH-2), 7.17 (1H, d, J=8.6, ArH-1).

Method: Williams, Preparation of Alkenes (preparation of alkene), OxfordUniversity press, 1996,32 pages.

J.Org.Chem.46:1981 such as m.p.:Forcellese, 3326-3328.

3-(tertiary butyl-dimethyl-silanyloxy base)-2-ethyl-13-methyl-6,7,8,9,11,12,13,14,15,16-decahydro-cyclopenta [a] phenanthrene-17-ketone 12

(4g 13.4mmol) carries out this reaction by the description of method B with 2-ethyl oestrone.Rapid column chromatography (SiO ₂Hexane: ethyl acetate 9: 1) obtain the white crystalline solid (5.02g, 12.2mmol, 91%) of silyl ether 12.δ _H(CDCl ₃) 0.24[6H, s, Si (CH ₃) ₂)], 0.98 (3H, s, CH ₃), 1.01[9H, s, C (CH ₃) ₃], 1.17 (3H, t, J=7.8, CH ₃), 1.40-1.66 (6H, m, alkyl H), 1.94-2.52 (7H, m, alkyl H), 2.57 (2H, q, J=7.8, CH ₂), 2.82-2.86 (2H, m, alkyl H), 6.50 (1H, s, ArH), 7.06 (1H, s, ArH).

[3-(tertiary butyl-dimethyl-silanyloxy base)-2-ethyl-13-methyl-6,7,8,9,11,12,13,14,15,16-decahydro-cyclopenta [a] phenanthrene-17-subunit]-ethyl acetate 13

With silyl ether 12 (412mg, 1mmol) and the phosphonic acids triethyl acetate (271mg, 240 μ l 1.2mmol) carry out this reaction by the description of method C.Rapid column chromatography (SiO ₂Hexane: ethyl acetate 19: 1) obtain the colorless oil (290mg, 0.602mmol, 60%) of two kinds of isomer 13.By ¹H NMR determines that the ratio of isomer is 6: 1.δ _H(CDCl ₃) 0.22[6H, s, Si (CH ₃) ₂)], 0.97 (3H, s CH ₃), 1.00[9H, s, C (CH ₃) ₃], 1.17 (3H, t, J=7.8, CH ₃), 1.29 (3H, t, J=7.0, CH ₃), 1.59-2.44 (11H, m, alkyl H), 2.56 (2H, q, J=7.8, CH ₂), 2.76-2.92 (4H, m, alkyl H), 4.15 (2H, q, J=7.0, CH ₂), 5.62 (1H, t, J=2.0, alkene H), 6.48 (1H, s, ArH), 7.06 (1H, s, ArH); M/z (FAB ⁺) 482.1 (M ⁺, 90%), 73.0 (100%); HRMS (FAB ⁺) calculated value C ₃₀H ₄₆O ₃Si482.3216, measured value?

Z-and E-(2-ethyl-3-hydroxyl-13-methyl-6,7,8,9,11,12,13,14,15,16-decahydro-cyclopenta [a] phenanthrene-17-subunit)-ethyl acetate 14a and 14b

(290mg 0.639mmol) carries out this reaction by the description of method D with ester 13.Rapid column chromatography (SiO ₂Hexane: ethyl acetate 19: 1) obtain .m.p.157-159 ℃ of the white powder (28mg, 0.0757mmol, 12%) of Z-isomer 14a.δ _H(CDCl ₃) 1.04 (3H, s CH ₃), 1.22 (3H, t, J=7.4, CH ₃), 1.29 (3H, t, J=7.0, CH ₃), 1.35-2.48 (13H, m, alkyl H), 2.60 (2H, q, J=7.4, CH ₂), 2.78-2.84 (2H, m, alkyl H), 4.09-4.12 (2H, m, CH ₂), 4.46 (1H, s, OH), 5.68 (1H, t, J=2.0, alkene H), 6.50 (1H, s, ArH), 7.04 (1H, s, ArH); M/z (FAB ⁺) 368.1 (M ⁺, 100%); HRMS (FAB ⁺) calculated value C ₂₄H ₃₂O ₃368.2351, measured value 368.2364.

Further wash-out obtains the light yellow oil (184mg, 0.497mmol, 78%) of E isomer 14b.δ _H(CDCl ₃) 0.86 (3H, s CH ₃), 1.23 (3H, t, J=7.4, CH ₃), 1.29 (3H, t, J=7.0, CH ₃), 1.33-2.45 (11H, m, alkyl H), 2.60 (2H, q, J=7.4, CH ₂), 2.74-2.91 (4H, m, alkyl H), 4.15 (2H, q, J=7.0, CH ₂), 4.52 (1H, s, OH), 5.59 (1H, t, J=2.3, alkene H), 6.51 (1H, s, ArH), 7.06 (1H, s, ArH).

(2-ethyl-13-methyl-3-sulfamoyloxy-6,7,8,9,11,12,13,14,15,16-decahydro-cyclopenta [a] phenanthrene-17-subunit)-ethyl acetate 15

(132mg 0.357mmol) carries out this reaction by the description of method A with ester 14b.Use preparation HPLC purifying sulfamate 15, obtain white solid (28mg, 0.0757mmol, 21%).δ _H(d ₆-acetone) 0.91 (3H, s CH ₃), 1.18 (3H, t, J=7.7, CH ₃), 1.23 (3H, t, J=7.2, CH ₃), 1.28-1.65 (6H, m, alkyl H), 1.86-2.08 (5H, m, alkyl H), 2.24-2.34 (1H, m, alkyl H), 2.47-2.54 (1H, m, alkyl H), (2H, q, J=7.7, CH ₂), 2.81-2.89 (2H, m, alkyl H), 4.10 (2H, q, J=7.2, CH ₂), 5.56 (1H, t, J=2.5, alkene H), 7.09 (1H, s, ArH), 7.26 (1H, s, ArH).

2-[3-(tertiary butyl-dimethyl-silanyloxy base)-2-ethyl-13-methyl-6,7,8,9,11,12,13,14,15,16-decahydro-cyclopenta [a] phenanthrene-17-subunit]-ethanol 16

(412mg 1mmol) carries out this reaction by the description of method E with ester 14b.TLC and 1HNMR show that all starting raw material no longer exists, and product vinyl alcohol 16 need not to be further purified and can use.

Z-and E-2-ethyl-17-(2-hydroxyl-ethylidene)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-3-alcohol 17a and 17b

(520mg 1.18mmol) carries out this reaction by the description of method D with vinyl alcohol 16.Rapid column chromatography (SiO ₂Hexane: ethyl acetate 19: 1) obtain the white powder (27mg, 0.0831mmol, 7%) of Z-phenol 17a.m.p.205-207℃。δ _H(CDCl ₃) 0.93 (3H, s CH ₃), 1.23 (3H, t, J=7.4, CH ₃), 1.26-1.61 (5H, m, alkyl H), 1.72-1.79 (2H, m, alkyl H), 1.89-1.92 (1H, m, alkane n base H), 2.18-2.39 (4H, m, alkyl H), 2.47-2.53 (1H, m, alkyl H), 2.60 (2H, q, J=7.4, CH ₂), 2.74-2.87 (2H, m, alkyl H), 4.21 (1H, dd, J=7.0,12.1, CHOH), 4.35 (1H, dd, J=12.1,7.0, CHOH), 4.62 (1H, s, OH), 5.33-5.37 (1H, m, alkene H), 6.50 (1H, s, ArH), 7.04 (1H, s, ArH); M/z (FAB ⁺) 326.1 (M ⁺, 100%); HRMS (FAB ⁺) calculated value C ₂₂H ₃₀O ₂326.2246, measured value 326.2259.

Further wash-out obtains the white powder (245mg, 0.754 mmol, 64%) of E-phenol 17b.m.p.169-171℃。δ _H(CDCl ₃) 0.79 (3H, s CH ₃), 1.21 (3H, t, J=7.4, CH ₃), 1.27-1.61 (5H, m, alkyl H), 1.74-1.97 (3H, m, alkyl H), 2.20-2.48 (5H, m, alkyl H), 2.59 (2H, q, J=7.4, CH ₂), 2.77-2.83 (2H, m, alkyl H), 4.11-4.60 (2H, m, CH ₂OH), 4.50 (1H, s, OH), 5.26-5.37 (1H, m, alkene H), 6.48 (1H, s, ArH), 7.06 (1H, s, ArH); M/z (FAB ⁺) 326.1 (M ⁺, 50%), 73.0 (100%); HRMS (FAB ⁺) calculated value C ₂₂H ₃₀O ₂326.2246, measured value 326.2256.

Thionamic acid 2-ethyl-13-methyl-17-vinyl-7,8,9,11,12,13,14,15-octahydro-6H-cyclopenta [a] phenanthrene-3-base ester 18

(65mg 0.200mmol) carries out this reaction by the description of method A with 17b.Rapid column chromatography (SiO ₂CHCl ₃) obtain the colorless oil (36mg, 0.093mmol, 47%) of sulfamate 18.δ _H(CDCl ₃) 0.91 (3H, s CH ₃), 1.21 (3H, t, J=7.4, CH ₃), 1.27-1.55 (2H, m, alkyl H), 1.61-1.73 (4H, m, alkyl H), 1.85-2.07 (2H, m, alkyl H), 2.18-2.39 (4H, m, alkyl H), 2.69 (2H, q, J=7.4, CH ₂), 2.83-2.92 (2H, m, alkyl H), 4.88-4.97 (1H, m, alkene H-16), 4.99 (2H, sNH ₂), 5.35 (1H, d, J=18.0, alkene H-22), 5.73 (1H, br s, alkene H-22), 6.32 (1H, dd, J=18.0,11.4, alkene H-21), 7.10 (1H, s, ArH), 7.18 (1H, s, ArH).

2-ethyl-17-(2-hydroxyl-ethyl)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-3-alcohol 19

(76mg 0.233mmol) carries out this reaction by the description of method F with vinyl alcohol 16.Rapid column chromatography (SiO ₂Hexane: ethyl acetate 5: 1) obtain white powder glycol 19 (51mg, 0.155mmol, 67%).m.p.162-164℃。δ _H(CDCl ₃) 0.63 (3H, s CH ₃), 1.22 (3H, t, J=7.4, CH ₃), 1.24-1.50 (10H, m, alkyl H), 1.72-1.88 (4H, m, alkyl H), 2.15-2.32 (2H, m, alkyl H), 2.59 (2H, q, J=7.4, CH ₂), 2.76-2.81 (2H, m, alkyl H), 3.61-3.75 (2H, m, CH ₂OH), 5.0 (1H, s, OH), 6.49 (1H, s, ArH), 7.05 (1H, s, ArH); δ _C(CDCl ₃) 12.6 (CH ₃), 14.4 (CH ₃), 23.0 (CH ₂), 24.4 (CH ₂), 26.5 (CH ₂), 27.9 (CH ₂), 28.4 (CH ₂), 29.3 (CH ₂), 33.7 (CH ₂), 37.8 (CH ₂), 38.9 (CH), 42.5 (C), 44.2 (CH), 47.2 (CH), 54.7 (CH), 62.7 (CH ₂), 115.2 (CH), 126.3 (CH), 127.1 (C), 132.8 (C), 135.5 (C), 151.1 (C) .m/z (FAB ⁺) 328.1 (M ⁺, 100%); HRMS (FAB ⁺) calculated value C ₂₂H ₃₂O ₂328.2402, measured value 328.2407.

Thionamic acid 2-ethyl-13-methyl-17-(2-sulfamoyloxy-ethyl)-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-3-base ester 20

(35mg 0.107mmol) carries out this reaction by the description of method A with glycol 19.With preparation HPLC purifying sulfamate 20, obtain white solid (28mg, 0.0576mmol, 54%).δ _H(d ₆-acetone) 0.69 (3H, s CH ₃), 1.18 (3H, t, J=7.4, CH ₃), 1.29-1.62 (10H, m, alkyl H), 1.76-1.42 (6H, m, alkyl H), 1.70 (2H, q, J=7.4, CH ₂), 2.80-2.84 (2H, m, alkyl H), 4.16-4.18 (2H, m, CH ₂), 6.63 (2H, s, NH ₂), 7.09 (1H, s, ArH), 7.13 (2H, s, NH ₂), 7.24 (1H, s, ArH); M/z (FAB-) 485.1[(M-H) ⁺, 100%]; HRMS (FAB ⁺) calculated value C ₂₂H ₃₄O ₆N ₂S ₂486.1858, measured value 486.1854.

[3-(tertiary butyl-dimethyl-silanyloxy base)-2-ethyl-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-17-yl]-ethyl acetate 21

(100mg 0.220mmol) carries out this reaction by the description of method F with ester 13. ¹H NMR shows that starting raw material no longer exists, and product ester 21 need not to be further purified and can use.

(2-ethyl-3-hydroxyl-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-17-yl)-ethyl acetate 22

(66mg 0.145mmol) carries out this reaction by the description of method D with ester 21.Rapid column chromatography (SiO ₂Hexane: ethyl acetate 19: 1) obtain colorless oil saturated ester 22 (38mg, 0.103mmol, 71%).δ _H(CDCl ₃) 0.63 (3H, s CH ₃), 1.22 (3H, t, J=7.4, CH ₃), 1.27 (3H, t, J=7.4, CH ₃), 1.24-2.00 (11H, m, alkyl H), 2.12-2.22 (3H, m, alkyl H), 2.27-2.33 (1H, m, alkyl H), 2.41 (1H, dd, J=14.5,5.1, alkyl H), 2.59 (2 H, q, J=7.4, CH ₂), 2.73-2.81 (2H, m, alkyl H), 4.13 (2H, q, J=7.4, CH ₂), 4.62 (1H, s, OH), 6.49 (1H, s, ArH), 7.06 (1H, s, ArH); M/z (FAB) 370.2 (M ⁺, 100%); HRMS (FAB ⁺) calculated value C ₂₄H ₃₄O ₃370.2508, measured value 370.2537.

(2-ethyl-13-methyl-3-sulfamoyloxy-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-17-yl)-ethyl acetate 23

(100mg 0.270mmol) carries out this reaction by the description of method A with ester 22.Rapid column chromatography (SiO ₂Hexane: ethyl acetate 9: 1) obtain white powder sulfamate 23 (83mg, 0.185mmol, 68%).m.p.120-122℃。δ _H(CDCl ₃) 0.63 (3H, s CH ₃), 1.21 (3H, t, J=7.4, CH ₃), 1.27 (3H, t, J=7.0, CH ₃), 1.24-1.50 (7H, m, alkyl H), 1.74-1.98 (5H, m, alkyl H), 2.11-2.42 (4H, m, alkyl H), 2.68 (2H, q, J=7.4, CH ₂), 2.82-2.84 (2H, m, alkyl H), 4.12 (2H, q, J=7.0, CH ₂), 5.08 (2H, s, NH ₂), 7.05 (1H, s, ArH), 7.16 (1H, s, ArH).δ _C(CDCl ₃) 12.5,14.2,14.6,23.0,24.2,26.2,27.5,28.2,29.2,35.4,37.3,38.4,42.3,44.2,46.9,54.3,60.2,121.4,126.9,133.6,136.0,139.6,146.1,174.0; M/z (FAB) 449.1 (M ⁺, 10%), 135.0 (100%); HRMS (FAB ⁺) calculated value C ₂₄H ₃₅O ₅NS 449.2236, measured value 449.2237.

[3-(tertiary butyl-dimethyl-silanyloxy base)-2-ethyl-13-methyl-6,7,8,9,11,12,13,14,15,16-decahydro-cyclopenta [a] phenanthrene-17-subunit]-acetonitrile 24

With silyl ether 12 (1g, 2.6mmol) and diethyl (cyano methyl) phosphonic acid ester (691mg, 631 μ l 3.9mmol) carry out this reaction by the description of method C.Rapid column chromatography (SiO ₂Hexane: ethyl acetate 20: 1) obtain two kinds of isomer 24 of colorless oil (641mg, 1.47mmol, 57%).M/z (FAB) 435.3 (M ⁺, 100%); HRMS (FAB ⁺) calculated value C ₂₈H ₄₁ONSi435.2957, measured value 435.2961.

(2-ethyl-3-hydroxyl-13-methyl-6,7,8,9,11,12,13,14,15,16-decahydro-cyclopenta [a] phenanthrene-17-subunit)-acetonitrile 25

(540mg 1.24mmol) carries out this reaction by the description of method D with silyl ether 24.Rapid column chromatography (SiO ₂Hexane: ethyl acetate 4: 1) obtain light yellow oily phenol 25 (284mg, 0.88mmol, 71%).δ _H(CDCl ₃) 0.88 (3H, s CH ₃), 1.22 (3H, t, J=7.4, CH ₃), 1.25-1.62 (6H, m, alkyl H), 1.90-1.97 (3H, m, alkyl H), 2.18-2.24 (1H, m, alkyl H), 2.40-2.45 (1H, m, alkyl H), 2.59 (2H, q, J=7.4, CH ₂), 2.63-2.84 (4H, m, alkyl H), 4.53 (1H, s, OH), 5.04 (1H, t, J=2.3, alkene H), 6.49 (1H, s, ArH), 7.02 (1H, s, ArH); δ _H(CDCl ₃) 14.4 (CH ₃), 18.0 (CH ₃), 23.0 (CH ₃), 23.5 (CH ₂), 26.3 (CH ₂), 27.4 (CH ₂), 29.1 (CH ₂), 30.3 (CH ₂), 34.7 (CH ₂), 38.5 (CH), 43.7 (CH), 46.4 (C), 52.8 (CH), 87.7 (CH), 115.2 (CH), 117.5 (C), 126.2 (CH), 127.4 (C), 131.7 (C), 135.2 (C), 151.4 (C), 181.1 (C); M/z (FAB) 321.3 (M ⁺, 100%); HRMS (FAB ⁺) calculated value C ₂₂H ₂₇ON 321.2093, measured value 321.2088.

[3-(tertiary butyl-dimethyl-silanyloxy base)-2-ethyl-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-17-yl]-acetonitrile 26

(100mg 0.230mmol) carries out this reaction by the description of method F with silyl ether 24.Rapid column chromatography (SiO ₂Hexane: ethyl acetate 19: 1) obtain colorless oil saturated nitriles 26 (81mg, 0.185mmol, 81%).δ _H(CDCl ₃) 0.22[6H, s, Si (CH ₃) ₂)], 0.67 (3H, sCH ₃), 1.00[9H, s, C (CH ₃) ₃], 1.16 (3H, t, J=7.4, CH ₃), 1.26-1.53 (7H, m, alkyl H), 1.78-2.17 (5H, m, alkyl H), 2.20-2.29 (2H, m, alkyl H), 2.31-2.40 (2H, m, CH ₂CN), 2.56 (2H, q, J=7.4, CH2), 2.74-2.81 (2H, m, alkyl H), 6.47 (1H, s, ArH), 7.04 (1H, s, ArH); M/z (FAB) 437.4 (M ⁺, 100%); HRMS (FAB ⁺) calculated value C ₂₈H ₄₃OSiN 437.3114, measured value 437.3121.

(2-ethyl-3-hydroxyl-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-17-yl)-acetonitrile 27

(73mg 0.167mmol) carries out this reaction by the description of method D with nitrile 26.Rapid column chromatography (SiO ₂Hexane: ethyl acetate 9: 1) obtain .m.p.195-197 ℃ of white solid phenol 27 (38mg, 0.118mmol, 70%).δ _H(CDCl ₃) 0.67 (3H, s CH ₃), 1.22 (3H, t, J=7.4, CH ₃), 1.27-1.53 (7H, m, alkyl H), 1.74-1.89 (3H, m, alkyl H), 1.99 (1H, dt, J=12.1,3.1, alkyl H), 2.03-2.12 (1H, m, alkyl H), 2.14-2.42 (4H, m, alkyl H), 2.59 (2H, q, J=7.4, CH ₂), 2.75-2.86 (2H, m, alkyl H), 4.67 (1H, s OH), 6.49 (1H, s, ArH), 7.04 (1H, s, ArH); M/z (FAB) 323.2 (M ⁺, 100%); HRMS (FAB ⁺) calculated value C ₂₂H ₂₉ON 323.2249, measured value 323.2252.

Thionamic acid 17-cyano methyl-2-ethyl-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-3-base ester 28 (STX 564)

(182mg 0.591mmol) carries out this reaction by the description of method A with phenol 27.Rapid column chromatography (CHCl ₃) obtain white needles sulfamate 28 (120mg, 0.299mmol, 51%).m.p.175-177℃。δ _H(d ₆-acetone) 0.73 (3H, s CH ₃), 1.17 (3H, t, J=7.4, CH ₃), 1.26-1.56 (7H, m, alkyl H), 1.77-2.13 (5H, m, alkyl H), 2.26-2.58 (3H, m, alkyl H), 2.69 (2H, q, J=7.4, CH ₂), 2.80-2.83 (2H, m, alkyl H), 7.08 (1H, s, ArH), 7.12 (2H, s, NH ₂), 7.23 (1H, s, ArH); M/z (FAB) 402.0 (M ⁺, 100%); HRMS (FAB ⁺) calculated value C ₂₂H ₃₀O ₃N ₂S 402.1977, measured value 402.1975.

[3-(tertiary butyl-dimethyl-silanyloxy base)-2-methoxyl group-13-methyl-6,7,8,9,11,12,13,14,15,16-decahydro-cyclopenta [a] phenanthrene-17-ylidenylmethyl]-methylene radical-amine 29

With 2-methoxyl group-3-tertiary butyl dimethyl silanyl oxygen base oestrone (1g, 2.42mmol) and diethyl (cyano methyl) phosphonic acid ester (712g, 650 μ l 4.02mmol) carry out this reaction by the description of method C.Rapid column chromatography (SiO ₂Hexane: ethyl acetate 19: 1) obtain two kinds of isomer (481mg, 1.14mmol, 27%) of colorless oil alkene 29.Determine that by NMR the ratio of two kinds of isomer is 1: 1.M/z (FAB ⁺) 438.1[(MH) ⁺, 50%], 380.1 (100%), 73.0 (80%); HRMS (FAB ⁺) calculated value C ₂₇H ₃₉O ₂NSi 437.2750, measured value 437.2731.

Z-and E-(3-hydroxyl-2-methoxyl group-13-methyl-6,7,8,9,11,12,13,14,15,16-decahydro-cyclopenta [a] phenanthrene-17-subunit)-acetonitrile 30

(400mg 0.95mmol) carries out this reaction by the description of method D with silyl ether 29.Rapid column chromatography (SiO ₂Hexane: ethyl acetate 9: 1) obtain two kinds of mixture of isomers, be white powder (91mg, 0.281mmol, 30%).

Further wash-out obtains white solid E isomer 30 (115mg, 0.356mmol, 37%).δ _H(CDCl ₃) 0.89 (3H, s CH ₃), 1.25-1.64 (6H, m, alkyl H), 1.90-1.99 (3H, m, alkyl H), 2.22-2.27 (1H, m, alkyl H), 2.35-2.40 (1H, m, alkyl H), 2.60-2.69 (1H, m, alkyl H), 2.74-2.82 (3H, m alkyl H), 3.86 (3H, sOCH ₃), 5.05 (1H, t, J=2.7, alkene H), 5.43 (1H, s, OH), 6.65 (1H, s, ArH), 6.77 (1H, s, ArH); M/z (FAB ⁺) 323.1 (M ⁺, 100%); HRMS (FAB ⁺) calculated value C ₂₁H ₂₅O ₂N 323.1885, measured value 323.1885.

Thionamic acid 2-methoxyl group-13-methyl-17-methene amido methylene radical-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-3-base ester 31 (STX639)

(40mg 0.124mmol) carries out this reaction by the description of method A with phenol 30.Rapid column chromatography (SiO ₂Hexane: ethyl acetate 4: 1) obtain white needles sulfamate 31 (36mg, 0.0896mmol, 72%).m.p.202-204℃。δ _H(d ₆-acetone) 0.95 (3H, s CH ₃), 1.25-1.65 (7H, m, alkyl H), 1.94-2.08 (2H, m, alkyl H), 2.26-2.35 (1H, m, alkyl H), 2.46-2.66 (2H, m, alkyl H), 2.74-2.84 (3H, m, alkyl H), 3.84 (3H, s OCH ₃), 5.27 (1H, t, J=2.3, alkene H), 6.90 (2H, s, NH ₂), 7.02 (1H, s, ArH), 7.04 (1H, s, ArH); M/z (FAB ⁺) 402.0 (M ⁺, 100%); HRMS (FAB ⁺) calculated value C ₂₁H ₂₆O ₄N ₂S 402.1613, measured value 402.1611.

(3-hydroxyl-2-methoxyl group-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-17-yl)-acetonitrile 32

(100mg 0.310mmol) carries out this reaction by the description of method F with alkene 30.Rapid column chromatography (SiO ₂Hexane: ethyl acetate 4: 1) obtain white solid saturated nitriles 32 (81mg, 0.249mmol, 80%).m.p.172-174℃。δ _H(d ₆-acetone) 0.71 (3H, s CH ₃), 1.22-1.52 (7H, m, alkyl H), 1.73-1.90 (3H, m, alkyl H), 1.98-2.08 (2H, m, alkyl H), 2.14-2.22 (1H, m, alkyl H), 2.30-2.39 (2H, m, alkyl H), (2.45-2.54 1H, m, alkyl H), 2.65-2.80 (2H, m, alkyl H), 3.80 (3H, s OCH ₃), 6.52 (1H, s, ArH), 6.84 (1H, s, ArH), 8.00 (1H, s, OH) .m/z[FAB+] 325 (100%, M ⁺); HRMS[FAB+] measured value 325.20418, C ₂₁H ₂₇NO ₂Theoretical value 325.20418; Calculated value C, 77.5%; H, 8.36%; N, 4.30%; Measured value C, 77.2%; H, 8.41%; N, 4.01%.

Thionamic acid 17-cyano methyl-2-methoxyl group-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-3-base ester 33 (STX641)

(60mg 0.185mmol) carries out this reaction by the description of method A with nitrile 32.Rapid column chromatography (SiO ₂Hexane: ethyl acetate 4: 1) also use the acetone/hexane recrystallization, obtain white crystalline solid sulfamate 33 (20.3mg, 0.0547mmol, 30%).m.p.183-185℃。

δ _H(CDCl ₃) 0.69 (3H, s CH ₃), 1.20-1.55 (7H, m, alkyl H), 1.70-1.94 (3H, m, alkyl H), 1.98-2.50 (6H, m, alkyl H), 2.74-2.84 (2H, m, alkyl H), 3.87 (3H, s OCH ₃), 5.06 (2H, s, NH ₂), 6.92 (1H, s, ArH), 7.04 (1H, s, ArH); δ _C(CDCl ₃) 12.2 (CH ₃), 17.6 (CH ₂), 23.9 (CH ₂), 26.3 (CH ₂), 27.4 (CH ₂), 28.3 (CH ₂), 28.6 (CH ₂), 37.3 (CH ₂), 38.3 (CH), 42.5 (C), 44.3 (CH), 46.7 (CH), 54.4 (CH), 56.4 (CH ₃), 110.4 (CH), 119.5 (C), 124.2 (CH), 130.2 (C), 138.2 (C), 140.4 (C), 149.0 (C); M/z (FAB ⁺) 404.1 (M ⁺, 100%);

HRMS (FAB ⁺) calculated value C ₂₁H ₂₈O ₄N ₂S 404.1770, measured value 404.1767; C ₂₁H ₂₈O ₄N ₂S theoretical value C, 62.35%; H, 6.98%; N, 6.93%, measured value C, 62.50%; H, 6.96%; N, 6.85%.

3-O-(tertiary butyl-dimethyl-silyl)-17-(2-dimethylamino-ethylamino)-oestrone 34

To 3-OTBS oestrone (200mg, add in THF 0.52mmol) (20mL) solution 2-methoxyethyl amine (226 μ L, 4eq), then, after 10 minutes, add sodium triacetoxy borohydride (449mg, 4.5eq) and acetate (150 μ L).Should react with sodium hydroxide (4mL, the 1M aqueous solution) quencher after 4 days in stirring at room, and dilute with ethyl acetate (50mL).The standard water aftertreatment obtains required amine 34, is white solid, its demonstration

δ _H(400MHz, CDCl ₃, with reference to TMS=0) 7.09 (1H, d, J8.4, ArH), 6.58 (1H, dd, J8.4 and 2.5, ArH), 6.52 (J 2.5 for 1H, d, ArH), and 3.34-3.51 (2H, m, OCH ₂), 3.34 (3H, s, OMe), 2.57-2.92 (5H, m, 6-CH ₂, CH ₂N and CHN), 1.20-2.30 (14H, m), 0.96 (9H, s, Bu), 0.74 (3H, s, 18-CH ₃) and 0.17 (6H, s, SiMe ₂); δ ₀153.6,137.8,133.2,126.1,119.9,117.1,72.4,69.3,58.7,52.4,48.5,44.0,43.2,38.7,38.3,29.7,29.6,27.5,26.4,25.7,23.5,1801,11.8 and-4.4.FAB+444.3.

17-(2-dimethylamino-ethylamino)-oestrone 35

Handle THF (10mL) solution of the estratriene 34 (160mg) of TBS protection with TBAF (0.4mL, 1M THF solution).The stirring laggard column criterion water aftertreatment of spending the night obtains required product 35, its demonstration

δ _H(400MHz, CDCl ₃, with reference to TMS=0) 7.10 (1H, d, J8.6, ArH), 6.58 (1H, dd, J8.6 and 2.7, ArH), 6.52 (1H, d, J2.7, ArH), 3.48-3.52 (2H, m, OCH ₂), 3.34 (3H, s, OMe), 2.62-2.94 (5H, m, 6-CH ₂, CH ₂N and CHN), (15H is m) with 0.75 (3H, s, 18-CH for 1.24-2.52 ₃)

17-(2-dimethylamino-ethylamino)-2-methoxyl group-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-3-alcohol 36

In the THF of 2-MeO-3-OTBS oestrone (415mg) (10mL) solution, add N, the N-dimethyl-ethylenediamine (439 μ L, 4mmol), then, after 10 minutes, add sodium triacetoxy borohydride (954mg, 4.5mmol) and acetate (300 μ L).Should react with sodium hydroxide (4mL, the 1M aqueous solution) quencher after 3 days in stirring at room, and dilute with ethyl acetate (50mL).The standard water aftertreatment obtains the required amine of light yellow oily, its demonstration

δ _H(400MHz, CDCl ₃, with reference to TMS=0) 6.76 (1H, s, ArH), 6.53 (1H, s, ArH), 3.76 (3H, s, OMe), 2.65-2.82 (4H, m, 6-CH ₂And CH ₂N), 2.62 (1H, dd, J8.6 and 8.6, CHN), 2.36-2.48 (2H, m, CH ₂N), (20H, m comprise 2.23 (6H, s, NMe to 1.20-2.30 ₂)), 0.99 (9H, s, Bu), 0.76 (3H, s, 18-CH ₃) and 0.15 (6H, s, SiMe ₂).

Amino steroid with the TBS protection is dissolved in THF (20mL) and uses TBAF (1.5mL, 1M THF solution) and KF (10mg) processing then, stirs then 8 hours.Add ethyl acetate (50mL) then, (2 * 50mL) wash gained solution, dry (Na with sodium hydrogen carbonate solution (50mL, saturated), water (50mL) and salt solution ₂SO ₄) and evaporation.Gained light yellow oil column chromatography purification (CHCl ₃/ MeOH 99: 1-1: 1), obtain the clear, colorless oily matter of required amine 36, grind with ether/hexane then and obtain white powder, and m.p.108 ℃, its demonstration

δ _H(400MHz，CDCl ₃，TMS＝0)6.77(1H，s，ArH)，6.61(1H，s，ArH)，3.85(3H，s，OMe)，2.68-2.84(4H，m，6-CH ₂?CH ₂N)，2.63(1H，dd，J8.68.6，CHN)，2.38-2.46(2H，m，CH ₂N)，1.20-2.30(21H，m?2.24(6H，s，NMe ₂))0.76(3H，s，18-CH ₃).δ _C?144.6，143.4，131.5，129.3，114.7，108.1，69.4，59.3，56.0，52.4，46.3，45.5，44.3，43.2，38.8，38.3，29.7，29.1，27.6，26.9，23.5?11.9.

2-methoxyl group-17-(2-morpholine-4-base-ethylamino)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-3-alcohol 37

To 2-MeO-3-OTBS oestrone (315mg, add in THF 0.76mmol) (10mL) solution 4-(2-amino-ethyl) morpholine (395mg, 4eq), then, after 10 minutes, add sodium triacetoxy borohydride (723mg, 4.5eq) and acetate (240 μ L).Should react with sodium hydroxide (4mL, the 1M aqueous solution) quencher after 4 days in stirring at room, and dilute with ethyl acetate (50mL).The standard water aftertreatment obtains required amine, is white powder, its demonstration

δ _H(400MHz, CDCl ₃, with reference to TMS=0) 6.78 (1H, s, ArH), 6.54 (1H, s, ArH), 3.78 (3H, s, OMe), 3.66-3.76 (4H, m, 2 * OCH ₂), 1.20-2.88 (25H, m) 0.99 (9H, s, ' Bu), 0.76 (3H, s, 18-CH ₃) and 0.16 (6H, s, SlMe ₂)

Amino steroid with the TBS protection is dissolved in THF (20mL) then, and handles with TBAF (1.5mL, 1M THF solution) and KF (10mg), stirs then 8 hours.Add ethyl acetate (50mL) then, (2 * 50mL) wash gained solution, dry (Na with sodium hydrogen carbonate solution (50mL, saturated), water (50mL) and salt solution ₂SO ₄) and evaporation.Gained light yellow oil column chromatography purification (CHCl ₃/ MeOH 99: 1-1: 1), obtain the clear, colorless oily matter of required amine 37, grind with ether/hexane then and obtain white powder, m.p.144-146 ℃, it shows δ _H(400MHz, CDCl ₃, with reference to TMS=0) 6.75 (1H, s, ArH), 6.61 (1H, s, ArH), 3.84 (3H, s, OMe), 3.67-3.71 (4H, m, 2 * OCH ₂) 2.72-2.86 (4H, m, 6-CH ₂And CH ₂N), 2.65 (1H, dd, J9.0 and 8.2, CHN), 2.52 (2H, dd, J6.3 and 6.3, CH ₂N) 2.46-2.50 (4H, m, 2 * CH ₂N), (15H is m) with 0.76 (3H, s, 18-CH for 1.20-2.26 ₃).δ _C144.5,143.4,131.3,129.3 (all C), 114.6,108.0 (two CH), 69.2 (CH), 67.0,57.9 (two CH ₂), 56.0 (CH ₃), 53.7 (CH ₂), 52.3 (CH), 45.0 (CH ₂), 44.3 (CH), 43.2 (C), 38.8 (CH), 38.2,29.4,29.1,27.6,26.9,23.5 (all CH ₂) and 12.0 CH ₃).

2-ethyl-17-[(furans-2-ylmethyl)-amino]-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-3-alcohol 38

To 2-Et-3-O-TBS oestrone (414mg, add in THF 1mmol) (10mL) solution chaff amine (354 μ L, 4mmol), then, after 10 minutes, add sodium triacetoxy borohydride (954mg, 4.5mmol) and acetate (300 μ L).Should react and dilute with sodium hydroxide (4mL, the 1M aqueous solution) quencher after 3 days in stirring at room with ethyl acetate (50mL).The standard water aftertreatment obtains required amine, is light yellow oil.In the THF solution of the steroide of protecting, add TBAF (1.5mL) and Potassium monofluoride (10mg).Add ether (50mL) and water (50mL) after 14 hours in stirring at room.Separate organic layer then, with sodium hydrogen carbonate solution (25mL), water (25mL) and salt solution (25mL) washing, dry then and evaporation obtains light yellow oil (270mg).Then, required product 38 uses column chromatography (0-3% methyl alcohol/chloroform), obtains clear, colorless oily matter, its demonstration

δ _H(CDCl ₃, TMS=0) 7.37 (1H, dd, J1.8 ﹠amp; 0.8), 7.03 (1H, s, ArH), 6.49 (1H, s, ArH), 6.32 (1H, dd, J3.2 ﹠amp; 1.8), 6.24 (1H, dd, J3.2 ﹠amp; 0.8), 3.85-3.95 (2H, m, CH2N), 2.76-2.84 (2H, m, 6-CH ₂), 2.67 (1H, dd, 8.8 ﹠amp; 8.5 CHN), 2.60 (J 7.6, CH for 2H, q ₂Me), (1H, m), (1H, m), (13H, m), 1.23 (J 7.6, CH for 3H, t for 1.26-2.07 for 2.14-2.22 for 2.27-2.35 ₂Me) and 0.81 (3H, s, 18-CH ₃).

(2-ethyl-3-methoxymethoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-17-yl)-pyridine-2-ylmethyl-amine dihydrochloride 39

Stir 2-Et-3-O-MOM oestrone (342mg, 1mmol) and 2-(amino methyl) pyridine ((848mg 4mmol) handles to use acetate (300mg) and sodium triacetoxy borohydride then for 515 μ l, THF 5mmol) (10mL) solution 1 hour.Stir and add sodium hydroxide (5mL, the 2M aqueous solution) after 3 days, add ethyl acetate (30mL) subsequently.Separate organic layer then, water and salt water washing, dry and evaporation.The gained yellow oil is dissolved in the diethyl ether solution of also using HCl among the THF, and (1mL 2M) handles, and solvent removed in vacuo is ground the gained jelly with ether then, obtains the grey powder of product 39 dihydrochlorides, its demonstration

δ _H(CD ₃OD, TMS=0) 8.86 (J 5.1 for 1H, app d), 8.38 (J 7.8 for 1H, app t), 8.06 (J 7.8 for 1H, app d), 7.87 (1H, dd, J 7.8 ﹠amp; 5.1), 7.02 (1H, s, ArCH), 6.73 (1H, s ArH), 5.14 (2H, s, OCH ₂), 4.59 (2H, br, CH ₂N), 3.43 (3H, s, OMe), 2.78-2.86 (2H, m, 6-CH ₂), 2.59 (J 7.4, CH for 2H, q ₂Me), and 1.30-2.41 (12H, m), 1.17 (3H, t, J7.4, CH ₂Me) and 1.01 (3H, s, 18-CH ₃).

2-ethyl-13-methyl-17-[(pyridine-2-ylmethyl)-amino]-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-3-alcohol 40

, join then among the oestrone MPL03139 of MOM protection in 0 ℃ of prepared in reaction HCl methanol solution (Methanolic HCl) by Acetyl Chloride 98Min. (957 μ L) and methyl alcohol (2.5mL), be evaporated to this reactant dried after ultrasonic 2 minutes.This HCl salt is separated out in the methyl alcohol ether but too easily moisture absorption and can't collecting, and the substitute is this compound is dissolved in ethanol, uses NaHCO ₃Ethyl acetate extraction is used in (saturated) alkalization then.The organic layer water, use the salt water washing then, dry and evaporation obtains the product 40 of colorless oil, its demonstration

δ _H(CDCl3, TMS=0) 8.54-8.57 (1H, m), 7.65 (1H, ddd, J 7.87.8 ﹠amp; 1.9), 7.39 (J 7.8 for 1H, app d), 7.16 (1H, m), 7.03 (1H, s, ArH), 6.45 (1H, s, ArH), 3.97 (2H, s, NCH2), 2.73-2.78 (2H, m, 6-CH ₂), 2.67 (1H, dd, 9.0﹠amp; 8.6, CHN), 2.60 (2H, q, J 7.4CH ₂Me), (13H, m), 1.22 (J 7.4, CH for 3H, t for 1.25-2.32 ₂Me) and 0.77 (3H, s, 18-CH ₃).

(3-benzyloxy-2-methoxyl group-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-17-yl)-(2-morpholine-4-base-ethyl)-amine 41

Stir 2-MeO-3-O-benzyl oestrone (390mg, 1mmol) and 4-(2-ethylamino) morpholine ((954mg 4.5mmol) handles to use acetate (300mg) and sodium triacetoxy borohydride then for 651mg, THF 5mmol) (15mL) solution 1 hour.Stir and add sodium hydroxide (5mL, the 2M aqueous solution) after 5 days, add ethyl acetate (30mL) subsequently.Separate organic layer then, water and salt water washing, dry and evaporation.The required product 41 of yellow oily shows

δ _H(CDCl ₃, TMS=0) 7.25-7.46 (5H, m), 6.84 (1H, s, ArH), 6.62 (1H, s, ArH), 5.10 (2H, s, PhCH ₂O), 3.86 (3H, s, OMe), 3.68-3.74 (4H, m, 2 * CH ₂O), (25H is m) with 0.75 (3H, s, 18-CH for 1.20-2.80 ₃).

(3-benzyloxy-2-methoxyl group-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-17-yl)-pyridine-2-ylmethyl-amine 42

Stir 2-MeO-3-O-benzyl oestrone (390mg, 1mmol) and 2-(amino methyl) pyridine ((954mg 4.5mmol) handles to use acetate (300mg) and sodium triacetoxy borohydride then for 515 μ L, THF 5mmol) (20mL) solution 1 hour.Stir and add sodium hydroxide (5mL, the 2M aqueous solution) after 6 days, add ethyl acetate (30mL) subsequently.Separate organic layer then, water and salt water washing, dry and evaporation.Yellow required amine 42 shows

δ _H(CDCl ₃, TMS=0) 8.52 (1H, m), 7.62 (1H, ddd, J 7.8,7.8 ﹠amp; 1.9) 7.24-7.46 (7H, m), 7.12-7.17 (1H, m), 6.84 (1H, s, ArH), 6.61 (1H, s, ArH), 5.10 (2H, s, PhCH ₂O), 3.96 (2H, s, CH ₂Ar) 3.86 (3H, s, OMe), 2.63-2.82 (3H, m, 6-CH ₂And CHN) (13H is m) with 0.75 (3H, s, 18-CH for 1.20-2.32 ₃).

(3-benzyloxy-2-ethyl-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-17-yl)-(2-methoxyl group-ethyl)-amine 43

Stir 2-Et-3-O-benzyl oestrone (220mg, 0.57mmol) and the 2-methoxyethyl amine ((540mg 2.55mmol) handles to use acetate (150mg) and sodium triacetoxy borohydride then for 197 μ L, THF 2.26mmol) (10mL) solution 1 hour.Stir and add sodium hydroxide (5mL, the 2M aqueous solution) after 3 days, add ethyl acetate (30mL) subsequently.Separate organic layer then, water and salt water washing, dry and evaporation.Yellow required amine 43 shows

δ _H(CDCl ₃, TMS=0) 7.27-7.44 (5H, m), 7.08 (1H, s, ArH), 6.61 (1H, s, ArH), 5.02 (2H, s, PhCH ₂O), 3.47-3.51 (2H, m, OCH ₂), 2.74-2.92 (4H, m, 6-CH ₂﹠amp; CH ₂N), 2.58-2.70 (3H, m, CHN ﹠amp; ArCH ₂Me), (14H, m), 1.21 (J 7.6, CH for 3H, t for 1.24-2.34 ₂Me) and 0.75 (3H, s, 18-CH ₃).

N '-(2-ethyl-3-methoxymethoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-17-yl)-N, N-dimethyl-second-1,2-diamines 44

(342mg, 1mmol) and N, ((848mg 4mmol) handles the N-dimethyl-ethylenediamine to use acetate (300mg) and sodium triacetoxy borohydride then for 549 μ L, THF 5mmol) (10mL) solution 1 hour to stir 2-Et-3-O-MOM oestrone.Stir and add sodium hydroxide (5mL, the 2M aqueous solution) after 3 days, add ethyl acetate (30mL) subsequently.Separate organic layer then, water and salt water washing, dry and evaporation.Gained yellow oil column chromatography purification (10%MeOH/CHCl ₃) obtain required product 44, its demonstration

δ _H(CDCl ₃, TMS=0) 7.08 (1H, s, ArCH), 6.78 (1H, sArH), 5.17 (2H, s, OCH ₂), 3.48 (3H, s, OMe), 2.24-2.86 (9H, m, 6-CH ₂, ArCH ₂With 2 * NCH ₂And NCH), 2.23 (6H, s, NMe ₂), 1.20 (J 7.6, CH for 3H, t ₂Me) and 0.75 (3H, s, 18-CH ₃).

N '-(3-benzyloxy-2-methoxyl group-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-17-yl)-N, N-dimethyl-second-1,2-diamines 45

(390mg, 1mmol) and N, ((954mg 4.5mmol) handles the N-dimethyl-ethylenediamine to use acetate (300mg) and sodium triacetoxy borohydride then for 439 μ L, THF 4mmol) (10mL) solution 1 hour to stir 2-MeO-3-O-benzyl oestrone.Stir and add sodium hydroxide (5mL, the 2M aqueous solution) after 3 days, add ethyl acetate (30mL) subsequently.Separate organic layer then, water and salt water washing, dry and evaporation.Yellow oil product 45 shows

δ _H(CDCl ₃, TMS=0) 7.28-7.46 (5H, m), 6.84 (1H, s, ArH), 6.61 (1H, s, ArH), 5.10 (2H, s, PhCH ₂O), 3.86 (3H, s, OMe), 2.37-2.83 (7H, m, 6-CH ₂, 2 * NCH ₂And NCH), 2.22 (6H, s, NMe ₂), (14H is m) with 0.75 (3H, s, 18-CH for 1.24-2.14 ₃).

[2-(3-benzyloxy-2-methoxyl group-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-17-base oxygen base)-the oxyethyl group]-tertiary butyl-dimethyl-silane 46

Handle 3-O-Bn-2-MeO-estradiol (340mg with sodium hydride (2eq), 0.86mmol) toluene (8mL) solution, being heated to 130 ℃ then in sealed tube kept 0.25 hour, be cooled to room temperature, use 2-(t-butyldimethylsilyl oxygen base) monobromoethane (429 μ L then, 2mmol) handle, heated 16 hours in sealed tube in 130 ℃ then.After the standard water aftertreatment, thick material obtains the white solid (208mg) of required ether 46, its demonstration through column chromatography purification

δ _H(400MHz, CDCl ₃, with reference to TMS=0) 7.26-7.46 (5H, m, ArH), 6.85 (1H, s, ArH), 6.62 (1H, s, ArH), 5.11 (2H, s, OCH ₂Ph), 3.87 (3H, s, OMe), 3.71-3.77 (2H, m, OCH2), 3.39-3.69 (3H, m, OCH ₂And HCO), 2.72-2.82 (2H, m, 6-CH2), 1.10-2.40 (13H, m), 0.91 (9H, s, tBu), 0.80 (3H, s, 18-CH ₃) and 0.10 (6H, s, SiMe ₂).

17-[2-(tertiary butyl-dimethyl-silicon alkoxyl group)-oxyethyl group]-2-methoxyl group-13-methyl 7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-3-alcohol 47

(200mg, (10mL) solution of THF 0.36mmol) also place down in hydrogen atmosphere and spend the night to handle ether 46 under the stirring, the degassing with 10%Pd/C (20mg).Then reactant is also evaporated through diatomite filtration, obtains the required phenol 47 of clear, colorless oily,

δ _H6.80 (1H, s, ArH), 6.65 (1H, s, ArH), 5.55 (1H, s, OH), 3.87 (3H, s, OMe), 3.52-3.82 (4H, m, 2 * OCH ₂) .3.42-3.48 (1H, m, 17-CH), 2.72-2.84 (2H, m, 6-CH ₂), 1.20-2.36 (13H, m), 0.94 (9H, s, ^tBu), 0.89 (3H, s, 18-Me) and 0.12 (6H, s, SiMe ₂),

17-[2-(hydroxyl)-oxyethyl group]-2-methoxyl group-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-3-alcohol 48

(250mL 0.25mmol) handles alcohol 47 (110mg, THF 0.24mmol) (5mL) solution that TBS protects with TBAF.After spending the night, stirring uses ethyl acetate (25mL) extractive reaction thing, organic layer water and salt water washing, dry and evaporation.The gained light yellow oil obtains the required glycol 48 of clear, colorless oily (65mg), its demonstration through column chromatography purification (2% methyl alcohol/chloroform)

δ _H6.78 (1H, s, ArH), 6.64 (1H, s, ArH), 5.51 (1H, s, OH), 3.86 (3H, s, OMe), 3.40-3.68 (5H, m, 2 * OCH ₂And CHOR), 2.72-2.82 (2H, m, 6-CH ₂), 1.20-2.30 (14H, m) and 0.81 (3H, s, 18-Me); δ _C144.6,143.5,131.7,129.5,114.6,109.1,89.4,70.9,62.1,56.1,50.2,44.2,38.6,38.0,28.9,28.1,27.2,26.6,23.0 and 11.7.

Thionamic acid 2-methoxyl group-13-methyl-17-(2-sulfamoyloxy-oxyethyl group)-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-3-base ester 49

(1ml, toluene solution 0.69mmol) is evaporated to dried, is cooled to 0 ℃, uses N,N-DIMETHYLACETAMIDE (1.5mL) and glycol 48 (45mg) to handle then successively with sulphonamide chlorine.After 3 hours, reactant dilutes with ethyl acetate (20mL) in stirring at room, and water (10ml) is handled then, and also (5 * 10mL) extract the separation organic layer, and dry also the evaporation obtains yellow oil with salt solution.With the required bis-amino sulphonate 49 of column chromatography (gradient 5-10% methyl alcohol/chloroform) purifying, obtain clear, colorless oily matter (21mg), its demonstration

δ _H(CDCl ₃, TMS=0) 7.03 (1H, s, ArH), 6.90 (1H, s, ArH), 5.02 (4H, br, 2 * NH ₂), 4.37-4.42 (2H, m, CH ₂OS), 3.87 (3H, s, OMe), 3.75-3.82 (2H, m, OCH ₂), 3.48 (1H, dd, J 8.6 ﹠amp; 7.9, OCH), 2.74-2.85 (2H, m, 6-CH ₂), (H is m) with 0.79 (3H, s, 18-CH for 1.25-2.50 ₃); M/z 504.6.

The tertiary butyl-[2-methoxyl group-17-(2-methoxyl group-oxyethyl group)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-3-base oxygen base]-dimethyl-silane 50

With sodium hydride (98mg, 2.45mmol) handle the 2-MeO-3-O-TBS estradiol (340mg, toluene 0.81mmol) (20mL) solution make it reflux then 1 hour, add then 2-(methoxyl group) monobromoethane (153 μ L, 1.62mmol).Reaction refluxed 14 hours, then, use again aliquot sodium hydride (80mg, 2mmol) and 2-(methoxyl group) monobromoethane (141 μ l 1.5mmol) handle, and reflux 8 hours, and then the alkali and the alkyl bromide of adding aliquot again.After refluxing again 14 hours, this reaction is cooled to room temperature,, uses saturated ammonium chloride (20mL) to handle then with ethyl acetate (30mL) dilution.Separate organic layer, water (2 * 20mL), the salt water washing, dry and evaporation.Required then ether 50 obtains the white needles thing through column chromatography for separation (eluent hexane/ethyl acetate 11: 1), mp 98-100 ℃ (276mg, %), its demonstration

δ _H(400MHz, CDCl ₃, with reference to TMS=0) 6.76 (1H, s, ArH), 6.54 (1H, s, Ar, H), 3.77 (3H, s, OMe), 3.41-3.73 (7H, m), 3.40 (3H, s, OMe), 2.68-2.80 (2H, m, 6-CH ₂), 1.16-2.27 (11H, m), 0.99 (9H, s, ' Bu), 0.82 (3H, s, 18-CH ₃) and 0.15 (6H, s, SiMe ₂) δ _C148.6,142.9,133.2,129.0,121.0,110.0,89.6,72.3,69.5,59.2,50.4,43.4,38.5,38.2,29.7,28.8,28.2,27.4,26.6,25.8,23.0,18.5,11.7 and 4.6.m/z FAB+473.2,

2-methoxyl group-17-(2-methoxyl group-oxyethyl group)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-3-alcohol 51

Handle THF (10mL) solution of the ether 50 (232mg) of TBS protection with TBAF (0.5mL, 1M THF solution).The stirring laggard column criterion water aftertreatment of spending the night, (4: 1Hex/EA) purifying obtains white solid to required phenol 51, and it shows δ through column chromatography _H(400MHz, CDCl ₃, with reference to TMS=0) 6.77 (1H, s, ArH), 6.63 (1H, s, ArH), 5.50 (1H, s, OH), 3.85 (3H, s, ArOMe), 3.41-3.72 (5H, m, 2 * OCH ₂And CHOR), 3.40 (3H, s, OMe), 2.69-2.83 (6-CH ₂), 116-2.26 (13H, m) and 0.81 (3H, s, 18-Me); δ _C144.5,143.4,131.7,129.5 (all C), 114.6,108.1,89.6 (all CH), 72.3,69.5 (two CH ₂), 59.1 (CH), 56.0 (CH ₃), 50.3,44.2 (two CH), 43.3 (C), 38.6 (CH), 38.1,28.9,28.1,27.2,26.7,23.0 (all CH ₂) and 11.6 (CH ₃).

2-ethyl-3-methoxymethoxy-13-methyl-6,7,8,9,11,12,13,14,15,16-decahydro-cyclopenta [a] phenanthrene-17-ketone 52

Use the tosic acid pyridine

(333mg) handle 2-Et-3-O-MOM-17-(the ethylidene dioxy base ketal) acetone (200mL) of oestrone (15g) and the solution of water (10mL), it was refluxed 14 hours.Rotary evaporation removes and desolvates then, obtains white solid, and this white solid is used ethanol water recrystallization then, obtains the white solid (5.6g) of required product 52, its demonstration

δ _H(400MHz, CDCl ₃, with reference to TMS=0) 7.09 (1H, s, ArH), 6.81 (1H, s, ArH), 5.18 (2H, s, OCH ₂), 3.49 (3H, s, OMe), 2.84-2.92 (2H, m, 6-CH ₂), 2.61 (2H, q, J7.4, CH ₂Me), and 1.38-2.56 (13H, m), 1.19 (3H, t, J7.4, CH ₂Me) and 0.91 (3H, s, 18-CH ₃); δ _C220.1,152.8,134.8,132.6,130.6,126.2,114.0,94.3,56.0,50.4,48.1,44.1,38.4,36.0,31.7,29.6,26.7,26.0,23.6,21.7,14.9 and 14.0.

2-ethyl-3-methoxymethoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-17-alcohol 53

To stir, 2-Et-3-O-MOM oestrone 52 under the room temperature (743mg, add in THF 2.18mmol) (10mL)/methyl alcohol (40mL) solution sodium borohydride (76mg, 2mmol).Add this reaction of ammonium chloride (2mL saturated solution) quencher after one hour, with ethyl acetate (50mL) and water (25mL) dilution, separate organic layer and water, salt water washing, dry and evaporation.Column chromatography (hexane/ethyl acetate gradient) obtains required pure 53 (650mg) of clear, colorless oily, its demonstration

δ _H(400MHz, CDCl ₃, with reference to TMS=0) 7.09 (1H, s, ArH), 6.78 (1H, s, ArH), 5.17 (2H, s, OCH ₂), 3.69-3.78 (1H, m, CHOH) 3.48 (3H, s, OMe), 2.78-2.86 (2H, m, 6-CH ₂), 2.61 (J 7.4, CH for 2H, q ₂Me), and 1.24-2.38 (13H, m), 1.19 (3H, t, J7.4, CH ₂Me) and 0.77 (3H, s, 18-CH ₃).

2-ethyl-17-(2-methoxyl group-oxyethyl group)-3-methoxymethoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] luxuriant and rich with fragrance 54

With sodium hydride (80mg, 2mmol) handle 2-Et-3-O-MOM estradiol 53 (170mg, toluene 0.5mmol) (10mL) solution make it reflux then 0.5 hour, add then 2-(methoxyl group) monobromoethane (141 μ L, 1.5mmol).This reaction refluxed 14 hours, and then with the sodium hydride of aliquot (80mg, 2mmol) and 2-(methoxyl group) monobromoethane (141 μ l, 1.5mmol) processing, and then refluxed 8 hours do not have remaining starting raw material existence thereafter.This reaction is cooled to room temperature,, uses saturated ammonium chloride (20mL) to handle then with ethyl acetate (30mL) dilution.Separate organic layer, water (2 * 20mL), the salt water washing, dry and evaporation.Required ether 54 is through column chromatography for separation (gradient 0-20% ethyl acetate/hexane), obtain clear, colorless oily matter (128mg, %), its demonstration

δ _H(400MHz, CDCl ₃, with reference to TMS=0) 7.08 (1H, s, ArH), 6.78 (1H, s, Ar, H), 5.17 (2H, s, OCH ₂), 3.67-3.74 (1H, m), 3.52-3.56 (2H, m), 3.48 (3H, s, OMe), 3.43 (1H, dd, J 8.6 and 8.2, CHO), 3.40 (3H, s, OMe), 2.78-2.86 (2H, m, 6-CH ₂), 2.63 (J 7.4, CH for 2H, q ₂Me), and .2.37-2.44 (1H, m), 2.14-2.22 (1H, m), 2.01-2.11 (2H, m), 1.83-1.90 (1H, m), 1.23-1.72 (8H, m), 1.19 (3H, t, J7.4, CH ₂Me) and 0.81 (3H, s, 18-CH ₃) δ _C152.6,135.0,133.2,130.3,126.1,114.0,94.3,89.6,72.3,69.6,59.2,56.0,50.4,44.1,43.5,38.7,38.3,29.7,28.3,27.4,26.6,23.6,23.2,15.0 and 11.7.

According to following flow process reaction.

(E-and the Z-)-2-ethyl-3-O-tertiary butyl-dimethyl-silyl-17-methyl sulfenyl methylene radical oestrone A and

(E-and Z-)-2-ethyl-17-methyl sulfenyl methylene radical oestrone B

(120mg, 3mmol), nitrogen is dry down, uses THF (5mL) to handle then with the hexane (each 1mL) of three equal parts washing sodium hydride.(526 μ L 3mmol) are incorporated in the suspension, acutely emit gas before the formation clear colorless solution with diethyl (methylthiomethyl) phosphonic acid ester then.(412mg, this reaction mixture of THF 1mmol) (5mL) solution-treated refluxed 48 hours then to use 2-Et-3-OTBS oestrone then.With ethyl acetate (30mL) dilution refrigerative reactant, in the impouring water (20mL) and separate water layer.Then the organic layer water (3 * 35mL), salt solution (50mL) washing, dry then and evaporation.Gained oil is through column chromatography (5% ethyl acetate/hexane) purifying, and first component obtains the isomer mixture of the alkene A product of required TBS protection, is colorless oil (196mg; 43%); second component is a colorless oil, is the alkene B of desilylationization (150mg, 44%) through conclusive evidence.

F1 δ _H(CDCl ₃) 7.05 (1H, s, ArH), 6.54,6.47 (1H, s, ArH, two kinds of isomer), 5.48-5.55 (1H, m: CHSMe, two kinds of isomer), 2.75 (2H, m, 6-CH ₂), 2.56 (J 7.4, CH for 2H, q ₂Me), 2.26,2.21 (3H, s, the SMe of two kinds of isomer), 1.19 (J 7.4, CH for 3H, t ₂Me), 1.00 (9H, s, tBu), 0.92,0.81 (3H, s, 18-CH ₃), and 0.21 (6H, s, SiMe ₂).M/z[FAB+] measured value 456.28822, C ₂₈H ₄₄SOSi theoretical value 456.28821.

F2 δ _H(CDCl ₃) 7.04 (1H, s, ArH), 6.48 (1H, s, ArH), 5.51 (t, 1.9: CHSMe), 5.49 (t, 2.3: CHSMe), and 4.43-4.46 (1H, m, OH), 2.72-2.88 (2H, m, 6-CH ₂), 2.59 (J 7.4, CH for 2H, q ₂Me), 2.27 and 2.22 (3H, 2 * s, the SMe of two kinds of isomer), 1.22 (J 7.4, CH for 3H, t ₂Me), 0.93 and 0.81 (3H, s, the 18-CH of two kinds of isomer ₃); M/z[FAB+] measured value 342.20174, C ₂₂H ₃₀SO theoretical value 342.20173.

2-ethyl-17-methyl sulfenyl methyl oestrone C

(E-and Z-)-2-ethyl-17-methyl sulfenyl methylene radical oestrone B (150mg) is dissolved in THF (1mL) and the ethanol (10mL), uses Pd/C (25mg, 5%) to handle then, the degassing is also stirred 16h under hydrogen atmosphere.Reactant filters and evaporation by Celite pad then.Required reduzate C obtains through column chromatography for separation, is colorless oil (85mg), its demonstration

δ _H(CDCl ₃) 7.04 (1H, s, ArH), 6.48 (1H, s, ArH), 4.48 (1H, s, OH), 2.74-2.84 (2H, m, 6-CH ₂), 2.58 (J 7.4, CH for 2H, q ₂Me); 2.11 (SMe), 1.21 (J 7.4, CH for 3H, t for 3H, s ₂Me) and 0.65 (3H, s, 18-CH ₃),

2-ethyl-3-O-sulfamyl-17-methyl sulfenyl methyl oestrone E

Sulphonamide chlorine (1.5mL, 0.56M solution) is evaporated to dried, is dissolved in DMA (1.5mL) in 0 ℃, join then among 2-ethyl-13-methyl-17-methyl sulfenyl methyl oestrone C (80mg).Be reflected in 14 hours under stirring and reach room temperature.Add then ethyl acetate (10mL) and water (3 * 10mL) and salt solution (10mL) wash this solution, dry (Na ₂SO ₄), be evaporated to dried then.Crude product obtains the required sulfamate E of white foam shape (65mg), its demonstration through column chromatography (0-7.5% ethyl acetate/chloroform) purifying

δ _H(CDCl ₃) 7.18 (1H, s, ArH), 7.06 (1H, s, ArH), 4.90 (2H, br, NH ₂), 2.78-2.88 (2H, m, 6-CH ₂), 2.68 (J 7.4, CH for 2H, q ₂Me), 2.11 (SMe), 1.21 (J 7.4, CH for 3H, t for 3H, s ₂Me) and 0.66 (3H, s, 18-CH ₃).

2-hydroxymethyl-3-O-methoxymethyl-17-ethylidene dioxy base oestrone

(189mg 5mmol) handles 2-formyl radical-3-O-methoxymethyl-17-ethylidene dioxy base oestrone (2.00g, THF 5.18mmol) (10mL) and methyl alcohol (20mL) solution with sodium borohydride.Do not have the residue starting raw material after 10 minutes, add ammonium chloride and destroy excessive hydroborate.Reactant is extracted to ethyl acetate (50mL), water (2 * 25mL), salt solution (25mL) washing, dry and evaporation obtains white foam shape 2-hydroxymethyl-3-O-methoxymethyl-17-ethylidene dioxy base oestrone (1.98g),

δ _H(CDCl ₃) 7.23 (1H, s, ArH), 6.82 (1H, s, ArH), 5.20 (2H, s, OCH ₂), 4.64-4.70 (2H, m, ArCH ₂O), 3.88-4.00 (4H, m, OCH ₂CH ₂O), 3.49 (3H, s, OCH ₃), 2.82-2.88 (2H, m, 6-CH ₂), (14H is m) with 0.88 (3H, s, 18-CH for 1.30-2.40 ₃); δ _C152.9,137.7,133.9,127.2,126.2,119.3,114.5,94.6,65.2,64.5,61.9,56.1,49.3,46.1,43.6,38.9,34.2,30.6,29.7,26.9,26.0,22.3 and 14.3; M/z[ES+] 411.2 (100%, M ⁺+ Na).

2-methoxymethyl-3-O-methoxymethyl-17-ethylidene dioxy base oestrone

(1.98g adds sodium hydride (306mg) in THF 5.10mmol) (25mL) solution to 2-hydroxymethyl-3-O-methoxymethyl-17-ethylidene dioxy base oestrone under room temperature, stirring, adds methyl-iodide after 0.5 hour.Reaction stirred is spent the night, and carries out the standard water aftertreatment then.The starting raw material (600mg) that chromatography (hexane/ethyl acetate) obtains the required product of colorless oil (1g) and reclaims.Product 2-methoxymethyl-3-O-methoxymethyl-17-ethylidene dioxy base oestrone shows

δ _H(CDCl ₃) 7.26 (1H, s, ArH), 6.80 (1H, s, ArH), 5.16 (2H, s, OCH ₂), 4.43-4.50 (2H, m, ArCH ₂O), 3.86-3.98 (4H, m, OCH ₂CH ₂O), 3.46 (3H, s, OCH ₃), 3.39 (3H, s, OMe), 2.80-2.86 (2H, m, 6-CH ₂), (13H is m) with 0.86 (3H, s, 18-CH for 1.26-2.40 ₃); δ _C152.6,137.4,133.6,126.4,124.4,119.3,114.3,94.4,69.5,65.2,64.5,58.1,55.9,49.3,46.1,43.6,38.9,34.2,30.6,29.6,26.9,26.1,22.3 and 14.3; M/z[ES+] 425.3 (100%, M ⁺+ Na) .HRMS[FAB+] 402.24063.

2-methoxymethyl 3-O-TBS oestrone

With HCl methanol solution (10mL, 4M) THF (10mL) solution of processing 2-methoxymethyl-3-O-methoxymethyl-17-ethylidene dioxy base oestrone (1g) is 0.25 hour, after the standard water aftertreatment product is dissolved in DMF (10mL), and handles with imidazoles (425mg) and TBSCl (453mg).After 14 hours this reactant is extracted to ethyl acetate (25mL), then water (5 * 20mL) and salt solution (20mL) washing, dry and evaporation.Column chromatography (0-15% ethyl acetate/hexane) obtains the white solid (360mg) of required product, its demonstration

δ _H(CDCl ₃) 7.25 (ArH), 6.51 (ArH) 4.43 (J 11.7, ArCH for 1H, d for 1H, s for 1H, s _a), 4.40 (J 11.7, ArCH for 1H, d _b), 3.39 (3H, s, OCH ₃), 2.82-2.88 (2H, m, 6-CH ₂), 2.42-2.54 (2H, m), 1.92-2.29 (5H, m), 1.36-1.68 (6H, m), 1.01 (9H, s, t-Bu), 0.91 (3H, s, 18-CH ₃) and 0.22 (6H, s, SiMe ₂); δ _C221.1,151.3,136.7,132.3,126.4,126.1,118.6,69.8,58.2,50.4,48.0,44.0,38.3,35.9,31.5,29.4,26.6,25.9,25.7,21.6,18.2,13.8 and-4.3.

2-methoxymethyl-3-O-TBS-17-cyano group methylene radical oestrone

(506 μ L, THF 3mmol) (10mL) solution is after gas is emitted and stopped, with THF (5mL) solution-treated of 2-methoxymethyl 3-O-TBS oestrone (320mg) to handle diethyl (cyano methyl) phosphonic acid ester with sodium hydride (124mg).Reaction is heated to 70 ℃ kept 1.5 hours, carry out aftertreatment then.Product obtains 1: 1 isomer mixture (240mg) through chromatography purification (7: 1 hexane/ethyl acetate), and this clear, colorless oily matter shows δ _H(CDCl ₃) 7.24 ﹠amp; 7.25 (1H, 2 * s, ArH), 6.51 ﹠amp; 6.50 (1H, 2 * s, ArH), 5.13 (dd, J 2.3 and 2.0:CHCN) ﹠amp; (5.05 dd, J 2.5 and 2.3) (1H, 2 * dd: two kinds of isomer of CHCN), 4.43 (1H, d, J12.8, OCH _a), 4.40 (J 12.8, OCH for 1H, d _b), 3.39 ﹠amp; 3.39 (3H, 2 * s, OCH ₃), 0.99 and 0.88 (3H, 2 * s, 18-CH ₃, two kinds of isomer).

2-methoxymethyl-3-O-TBS-17-beta-cyano methyl oestrone

With the hydrogenation 14 hours under the existence of Pd/C (200mg, 5%) of the THF (1mL) of 2-methoxymethyl-3-O-TBS-17-cyano group methylene radical oestrone (220mg) and ethanol (5mL) solution.Reactant is by diatomite filtration then, and evaporation obtains the required product 2-methoxymethyl of clear, colorless oily-3-O-TBS-17-beta-cyano methyl oestrone (200mg), its demonstration

δ _H(CDCl ₃) 7.23 (ArH), 6.50 (ArH), 4.42 (J 11.8, ArCH for 1H, d for 1H, s for 1H, s _a), 4.41 (1H, d, J11.8, ArCH _b), 3.39 (3H, s, OCH ₃), 2.72-2.86 (2H, m, 6-CH ₂), 1.15-2.46 (16H, m), 1.01 (9H, s, t-Bu), 0.67 (3H, s, 18-CH ₃) and 0.21 (6H, s, SlMe ₂); δ _C151.20,136.9,132.7,126.4,125.9,119.8,118.6,69.8,58.2,54.4,46.8,44.0,42.7,38.8,37.3,28.6,28.4,27.6,26.3,25.7,24.0,18.2,17.6,12.3 and-4.1.

2-methoxymethyl-17-beta-cyano methyl oestrone

Handle THF (5mL) solution of 2-methoxymethyl-3-O-TBS-17-beta-cyano methyl oestrone (200mg) with TBAF (1mL, 1M THF solution).0.5 after hour with the reactant aftertreatment.Chromatography (hexane/ether) obtains the crystalline solid (120mg) of required product 2-methoxymethyl-17-beta-cyano methyl oestrone, its demonstration

δ _H(CDCl ₃) 7.22 (ArH), 6.91 (ArH), 6.62 (OH), 4.64 (J 12.5, ArCH for 1H, d for 1H, s for 1H, s for 1H, s _a), 4.59 (1H, d, J12.5, ArCH _b), 3.43 (3H, s, OCH ₃), 2.78-2.86 (2H, m, 6-CH ₂), (15H is m) with 0.67 (3H, s, 18-CH for 1.24-2.42 ₃); δ _C153.8,138.0,131.5,125.0,119.8,119.4,116.3,74.1,58.1,54.3,46.7,43.6,42.5,38.7,37.3,29.3,28.2,27.6,26.2,25.6,23.9,17.6 and 12.2.m/z[APCI-] 338.3 (100%, M ⁺-H).

2-methoxymethyl-3-O-sulfamyl-17-beta-cyano methyl oestrone

(1.7mmol) is cooled to 0 ℃ with sulphonamide chlorine, is dissolved in N,N-DIMETHYLACETAMIDE (1.5mL), handles with 2-methoxymethyl-17-beta-cyano methyl oestrone (60mg) after 5 minutes.Remove exterior cooling after 15 minutes and make this react on envrionment temperature and stirred 3 hours.Then reactant is diluted with ethyl acetate (15mL), impouring salt solution (15mL) also separates organic layer.The organic extraction water (3 * 15mL) and salt solution (15mL) washing, dry and evaporation.Chromatography (0-5%MeOH/DCM) obtains colorless oil product 2-methoxymethyl-3-O-sulfamyl-17-beta-cyano methyl oestrone (65mg).Chloroform/hexane crystallization obtains white crystalline solid, its demonstration

δ _H(CDCl ₃) 7.29 (1H, s, ArH), 7.17 (1H, s, ArH), 5.55 (2H, s.NH ₂), 4.48 (2H, s, OCH ₂), 3.44 (3H, s, OCH ₃), 2.86-2.92 (2H, m, 6-CH ₂), (15H is m) with 0.69 (3H, s, 18-CH for 1.26-2.42 ₃); δ _C147.3,139.4,139.3,128.5,126.8,122.7,119.6,70.6,58.3,54.3,46.6,43.9,42.6,38.2,37.3,29.2,28.2,27.2,26.1,23.9,17.6 and 12.2.m/z[APCl-] 417.3 (100%, M ⁺-H).

2-ethyl-17-methylene radical oestrone

Handle the methyl triphenyl iodate with sodium hydride (400mg)

DMSO (10mmol) (15mL) solution.0.5 after hour in the gained yellow

DMSO (15mL) solution that adds 2-ethyl oestrone (600mg) in the salt makes to be reflected at 60 ℃ of maintenances 16 hours.Reaction is cooled to room temperature, and impouring frozen water (50mL) is with ether (3 * 50mL) extractions, the organic extraction water of merging (3 * 50mL) washings, dry and evaporation.Chromatography (10% ether/hexane) obtains the required product 2-ethyl of white foam shape-17-methylene radical oestrone (390mg), its demonstration

δ _H(CDCl ₃) 7.05 (1H, s, ArH), 6.47 (1H, s, ArH), 4.65-4.68 (2H, m: CH ₂), 4.53 (1H, s, OH), 2.72-2.87 (2H, m, 6-CH ₂), 2.59 (J 7.4, CH for 2H, q ₂Me), (13H, m), 1.22 (J 7.4, CH for 3H, t for 1.25-2.56 ₂Me) and 0.81 (3H, s, 18-CH ₃); δ _C161.6,150.9,135.4,132.6,127.0,126.2,115.1,100.7,53.5,44.4,44.1,38.9,35.8,29.5,29.4,27.7,26.8,24.0,23.1,18.7 and 14.6; HRMS[FAB+] 296.21402.

2-ethyl-17-ethylidene oestrone

Handle ethyltriphenylphosphiodide iodide with sodium hydride (400mg) DMSO (10mmol) (15mL) solution.0.5 after hour in the gained yellow

Add DMSO (15mL) solution of 2-ethyl oestrone (600mg) in the salt, and make and react on 60 ℃ and kept 16 hours.Reaction is cooled to room temperature, and impouring frozen water (50mL) is with ether (3 * 50mL) extractions, the organic extraction water of merging (3 * 50mL) washings, dry and evaporation.Chromatography (10% ether/hexane) obtains the required product of white foam shape suitable-2-ethyl-17-ethylidene oestrone (460mg), its demonstration

δ _H(CDCl ₃) 7.03 (1H, s, ArH), 6.47 (1H, s, ArH), 5.14 (J 7.0,2.0,2.0 for 1H, qdd: CHMe), 4.45 (1H, s, OH), 2.72-2.88 (2H, m, 6-CH ₂), 2.59 (J 7.4, CH for 2H, q ₂Me), and 2.14-2.46 (5H, m), 1.87-1.95 (1H, m), 1.70-1.78 (1H, m), 1.69 (J 7.0,2.0,2.0, MeHC for 3H, ddd :), (6H, m), 1.22 (J 7.4, CH for 3H, t for 1.25-1.62 ₂Me) and 0.91 (3H, s, 18-CH ₃); δ _C150.8,150.1,135.4,132.7,126.9,126.1,115.1,113.3,55.2,44.6,43.9,38.5,37.3,31.5,29.4,27.7,27.1,24.3,23.1,17.1,14.7 and 13.3; HRMS[FAB+] 310.22967.

2-ethyl-3-O-sulfamyl 17-methylene radical oestrone

In methylene dichloride (5mL) solution of 2-ethyl-17-methylene radical oestrone (231mg), add 2,6-di-t-butyl-4-picoline (320mg) and sulphonamide chlorine (1.56mmol).After stirring at room 14 with reactant with ethyl acetate (20mL) dilution, then water (3 * 20mL) and salt solution (20mL) washing, dry and evaporate.Column chromatography obtains the required product 2-ethyl of clear, colorless oily-3-O-sulfamyl 17-methylene radical oestrone (200mg), and it is placed after fixing and shows δ _H(CDCl ₃) 7.19 (1H, s, ArH), 7.09 (1H, s, ArH), 5.06-5.14 (2H, br, NH ₂), 4.56-4.60 (2H, m: CH ₂), 2.82-2.87 (2H, m, 6-CH ₂), 2.69 (J 7.4, CH for 2H, q ₂Me), (13H, m), 1.22 (J 7.4, CH for 3H, t for 1.32-2.60 ₂Me) and 0.82 (3H, s, 18-CH ₃); δ _C161.3,145.9,139.5,135.8,126.8 (all C), 126.8,121.3 (two CH), 100.9 (: CH ₂), 53.5 (CH), 44.3 (C), 44.3,38.4 (two CH), 35.7,29.5,29.3,27.5,26.5,24.0,23.1 (all CH ₂), 18.6 and 14.7 (two CH ₃); HRMS[FAB+] 375.18681.

2-ethyl-3-O-sulfamyl-17-ethylidene oestrone

In methylene dichloride (5mL) solution of 2-ethyl-17-ethylidene oestrone (329mg), add 2,6-di-t-butyl-4-picoline (435mg) and sulfamic acid chloride (2.12mmol).After stirring at room 14 with reactant with ethyl acetate (20mL) dilution, then water (3 * 20mL) and salt solution (20mL) washing, dry and evaporate.Column chromatography obtains the required product of clear, colorless oily suitable-2-ethyl-3-O-sulfamyl 17-ethylidene oestrone, it is placed after fixing and also shows δ _H(CDCl ₃) 7.17 (1H, s, ArH), 7.04 (1H, s, ArH), 5.15 (J 7.0,2.0,2.0 for 1H, qdd: CHMe), 5.09 (2H, s, NH ₂), 2.80-2.86 (2H, m, 6-CH ₂), 2.69 (2H, q, J7.4, CH ₂Me), and 2.18-2.46 (5H, m), 1.89-1.96 (1H, m), 1.70-1.78 (1H, m), 1.69 (J 7.0,2.0,2.0, MeHC for 3H, ddd :), 1.23-1.63 (6H, m), 1.21 (3H, t, J7.4, CH ₂Me) and 0.90 (3H, s, 18-CH ₃); δ _C149.8,145.9,139.5,135.8,133.4 (all C), 126.8,121.2,113.4,55.4 (all CH), 44.5 (C), 44.1,38.0 (two CH), 37.2,31.5,29.3,27.4,26.9,24.2,23.1 (all CH ₂), 17.0,14.7 and 13.3; HRMS[FAB+] 389.20246.

2-methoxyl group-3-benzyloxy-17-O-methoxymethyl estradiol

DMF (10mL) solution with sodium hydride (120mg) processing 2-methoxyl group-3-benzyloxy estradiol (800mg) adds chloromethyl methyl ether (266 μ L) after 0.25 hour.In stirring at room reaction 14 hours, carry out the standard water aftertreatment then.Chromatography (6: 1 hexane/ethyl acetate) obtains the required product of clear, colorless oily (590mg), and it places after fixing, m.p.87-88 ℃ and demonstration

δ _H(CDCl ₃) 7.24-7.45 (5H, m), 6.85 (1H, s, ArH), 6.62 (1H, s, ArH), 5.10 (2H, s, OCH ₂), 4.66 (2H, s, OCH ₂), 3.86 (3H, s, OMe), 3.62 (J 8.4,8.2 for 1H, dd, 17-α H), 3.37 (3H, s, OCH ₃) 2.70-2.80 (2H, m, 6-CH ₂), 1.20-2.32 (13H, m) and 0.82 (3H, s, 18-CH ₃) .m/z[FAB+] 436.1 (100%, M ⁺+ H) .HRMS [FAB+] 436.26136.

2-methoxyl group-17-O-methoxymethyl estradiol

With the hydrogenation (1atm) 16 hours under the existence of Pd/C (50mg, 5%) of the THF (2mL) of 2-methoxyl group-3-benzyloxy-17-O-methoxymethyl estradiol (500mg) and ethanol (10mL) solution.Reactant is carried out aftertreatment by diatomite filtration, and evaporation obtains the white solid (500mg) of required product.The ether/hexane recrystallization obtains white crystals,

δ _H(CDCl ₃) 6.78 (1H, s, ArH), 6.63 (1H, s, ArH), 4.65 (2H, s, OCH ₂), 3.86 (3H, s, OMe), 3.56-64 (1H, m, 17-α H), 3.37 (3H, s, OCH ₃) 2.72-2.80 (2H, m, 6-CH ₂), 1.15-2.30 (13H, m) and 0.82 (3H, s, 18-CH ₃).

2-methoxyl group-3-O-sulfamyl-17-O-methoxymethyl estradiol

Handle DMA (2mL) solution of 0 ℃ sulphonamide chlorine (1.7mmol) with 2-methoxyl group-17-O-methoxymethyl estradiol (170mg).After 3 hours with reactant by carry out aftertreatment with ethyl acetate (15mL) dilution, then water (4 * 15mL) and salt solution (15mL) washing, dry and evaporate.Chromatography (acetone/chloroform gradient) obtains the white solid (160mg) of required product 2-methoxyl group-3-O-sulfamyl-17-O-methoxymethyl estradiol, uses the ether/hexane crystallization then,

δ _H(CDCl ₃) 7.02 (1H, s, ArH), 6.91 (1H, s, ArH), 4.99 (2H, s, NH ₂), 4.65 (1H, d, 9.0, CH _aH _bO), 4.64 (1H, d, 9.0, CH _aH _bO), 3.86 (3H, s, OMe), 3.60 (J 8.6,8.2 for 1H, dd, 17-α H), 3.37 (3H, s, OCH ₃) 2.76-2.82 (2H, m, 6-CH ₂), 1.15-2.30 (13H, m) and 0.82 (3H, s, 18-CH ₃); δ _C148.7,140.4,136.6,130.1,124.0,110.4,96.0,86.5,56.4,55.2,50.0,44.5,43.0,38.2,37.3,28.7,27.1,26.5,23.2 and 11.9.

2-ethyl-3-benzyloxy-17-O-methylsulfonyl estradiol 58

Under nitrogen, stir and be cooled to add in 0 ℃ the 5ml anhydrous pyridine solution of 2-ethyl-3-O-benzyl estradiol (1mmol) Methanesulfonyl chloride (0.09ml, 1.2mmol).Stir this solution 2 hours in 0 ℃, add entry (20ml) then.(2 * 60ml) extract also water and salt water washing organic layer successively to organism, use MgSO then with ethyl acetate ₄Dry.Obtain the white solid 0.36g (77%) of 2-ethyl-3-benzyloxy-17-O-methylsulfonyl estradiol behind solvent removed in vacuo and the column chromatography (hexane/ethyl acetate 5: 1), mp=133 ℃

¹H NMR (CDCl ₃, 270MHz): 0.87 (s, 3H, CH ₃), 1.22 (t, J _H-H=7.4Hz, 3H, CH ₃), 1.25-1.60 (m, 6H), 1.70-1.95 (m, 3H), 2.05 (m, 1H), 2.15-2.45 (m, 3H), 2.68 (q, J _H-H=7.4Hz, 2H, CH ₂), 2.85 (m, 2H, H6), 3.02 (s, 3H, C H ₃SO ₂), 4.57 (m, 1H, H17), 5.05 (s, 2H, C H ₂Ph), 6.64 (s, 1H, ArH, 7.10 (s, 1H, ArH), 7.36-7.44 (m, 5H, Ph). ¹³C NMR (CDCl3): 11.7 (CH ₃), 14.6 (CH ₃), 23.0,23.4,26.0,27.1,27.9,29.536.4,38.2,38.6,43.3,43.7,49.0,69.8 ( CH ₂Ph), 89.5 (C17), 111.8,126.2,127.0,127.6,128.4130.3,131.7,134.7,137.6 and 154.5

2-ethyl-17-O-methylsulfonyl estradiol 59

In the 10ml THF of 2-ethyl-3-benzyloxy-17-O-methylsulfonyl estradiol (0.5mmol) and 40ml ethanolic soln, add 30mg 10%Pd/C.Stirred this mixture 14 hours down in room temperature, hydrogen.With the suspension diatomite filtration, with THF washing and vacuum concentration organic layer.(hexane/ethyl acetate 1: 0-2: 1), separate the white solid 145mg (77%) that obtains 2-ethyl-17-O-methylsulfonyl estradiol, mp=195 ℃ behind the column chromatography

¹H NMR (CDCl ₃, 270MHz): 0.86 (s, 3H, CH ₃), 1.21 (t, J _H-H=7.7Hz, 3H, CH ₃), 1.25-1.60 (m, 6H), 1.71-1.91 (m, 3H), 2.03 (m, 1H), 2.13-2.38 (m, 3H), 2.58 (q, J _H-H=7.7Hz, 2H, CH ₂), 2.79 (m, 2H, H6), 3.01 (s, 3H, C H ₃SO ₂), 4.53 (s, 1H, OH), 4.56 (dd, 1J _H-H=9.1 and 7.9Hz, 1H, H17), 6.49 (s, 1H, ArH, 7.03 (s, 1H, ArH). ¹³C NMR (CDCl ₃): 11.7 (CH ₃), 14.6 (CH ₃), 23.0,23.4,26.0,27.1,27.9,29.536.4,38.2,38.6,43.3,43.7,49.0,89.5 (C17), 115.2,126.3,127.3,132.1,135.2 and 151.2 MS m/z:350.16 (M ⁺) HPLC100%.

Trace analysis: C:66.30 (theoretical value 66.63); H:7.80 (theoretical value 7.99)

2-ethyl-3-O-sulfamyl-17-O-methylsulfonyl estradiol 60

Be dissolved in 1ml DMA with the toluene solution vacuum concentration of 1ml 0.559M sulphonamide chlorine and with sulphonamide chlorine.This solution is cooled to 0 ℃, and under nitrogen, is added in 2-ethyl-17-O-methylsulfonyl estradiol (0.2mmol).In this solution of stirring at room 15 hours.After adding 5ml water, (2 * 50ml) extract organism with ethyl acetate.Organic layer is water and salt water washing successively, uses MgSO ₄Dry also vacuum concentration.(hexane/ethyl acetate 5: 2) obtains white solid 2-ethyl-3-O-sulfamyl-17-O-methylsulfonyl estradiol 60mg (66%), mp=179 ℃ behind the column chromatography.

¹H NMR (CDCl ₃, 270MHz): 0.85 (s, 3H, CH ₃), 1.20 (t, J _H-H=7.4Hz, 3H, CH ₃), 1.30-1.55 (m, 6H), 1.73-187 (m, 3H), 2.04 (m, 1H), 2.16-2.36 (m, 3H), 2.68 (q, J _H-H=7.4Hz, 2H, CH ₂), 2.82 (m, 2H, H6), 3.01 (s, 3H, C H ₃SO ₂), 4.57 (dd, 1J _H-H=8.7 and 8.1Hz, 1H, H17), 5.08 (s, 2H, NH ₂), 6.49 (s, 1H, ArH, 7.03 (s, 1H, ArH), ¹³C NMR (CDCl ₃): 14.1 (CH ₃), 17.0 (CH ₃), 25.4,23.4,28.2,29.2,30.3,31.438.6,40.5,40.6,45.6,46.3,51.4,91.5 (C17), 123.6,129.2,135.9,137.9,141.1 and 148.3.LRMSm/z:457.32 (M ⁺); HPLC 100%;

Trace analysis: C:53.40 (theoretical value 55.12); H:6.38 (theoretical value 6.34); N:3.09 (theoretical value 3.06).

2-ethyl-3-O-TBS-17-dicyano methylene radical oestrone

The 150ml toluene and the 30ml acetic acid solution of 2-ethyl-3-O-TBS oestrone (2mmol), propane dinitrile (6mmol) and Beta-alanine were refluxed 3 days.And, the propane dinitrile of adding 2mmol aliquot after 24 hours and 48 hours.After this solution is cooled to room temperature, stir with 50ml water, with ethyl acetate (2 * 100ml) extractions with solvent evaporation and with residual solids.MgSO is used in organic layer water, salt water washing ₄Dry also solvent removed in vacuo.Obtain white powder 2-ethyl-3-O-TBS-17-dicyano methylene radical oestrone 840mg (91%) behind the column chromatography, mp=168 ℃ of 1H NMR (CDCl ₃, 270MHz): 0.21 (s, 6H, CH ₃Si) .1.00 (s, 9H, (CH ₃) ₃Si), 1.06 (s, 3H, CH ₃), 1.15 (t, J=7.4Hz, 3H, CH ₃), 1.40-1.80 (m, 6H), 1.85-2.05 (m, 2H), 2.26 (m, 1H), 2.45-3.05 (m, 7H), 6.4 (s, 1H, ArH), 7.02 (s, 1H, ArH). ¹³C NMR (CDCl ₃) :-4.2 and-4.1 (CH ₃Sl), 14.6 (CH ₃), 16.6,18.2,23.2,23.6,25.7 (( CH ₃) ₃CSl), 26.3,27.5,29.1,34.0,34.7,38.3,43.4,49.5,54.2,79.6 (C17), 111.1,112.4,118.3,126.1,131.2,132.2,134.3,151.5 and 196.2,

2-ethyl-17-dicyano methylene radical oestrone

With THF (50ml) solution of 2-ethyl-3-O-TBS-17-dicyano methylene radical oestrone (.5mmol) be cooled to 0 ℃ and be added dropwise to TBAF/THF 1M (0.6mmol, 0.6ml).Stir this solution 2 hours in 0 ℃, be warming up to room temperature then.Add entry (10ml) and ethyl acetate (80ml), organic layer is water and salt water washing successively, uses MgSO ₄Dry also vacuum evaporating solvent.Residual solids is through column chromatography (hexane/ethyl acetate 5: 1-2: 1) obtain white solid 2-ethyl-17-dicyano methylene radical oestrone behind the purifying.125mg(72％)；mp＝270℃；

1H NMR (CDCl ₃, 270MHz): 1.06 (s, 3H, CH ₃), 1.21 (t, J=7.5Hz, 3H, CH ₃), 1.39-1.79 (m, 6H), 1.89-2.05 (m, 2H) .2.25 (m, 1H), 2.45-2.52 (m, 1H), 2.61 (q, J=7.5Hz, 2H, CH ₂), 2.63-3.02 (m, 5H), 4.52 (s, 1H, OH), 6.50 (s, 1H, ArH), 7.02 (s, 1H, ArH). ¹³C NMR (CDCl ₃): 14.3,16.6 (CH ₃), 23.0,23.2,26.4,27.4,28.9,33.9,34.7,38.3,43.3,49.4,54.1,79.6 (C17), 111.2,112.5,115.2,126.2,127.5,131.3,135.0,151.4 and 196.1.HPLC:100%

2-ethyl-3-O-sulfamyl-17-dicyano methylene radical oestrone

With the NH that is cooled to 0 ℃ ₂SO ₂DMA (2ml) solution of Cl (0.6mmol) joins in 2-ethyl-17-dicyano methylene radical oestrone (0.2mmol), and stirs this mixture 14 hours down in room temperature, nitrogen.Add entry (10mL) then and use ethyl acetate (2 * 50ml) extraction organism.Organic layer is water and salt water washing successively, uses MgSO ₄Drying, solvent removed in vacuo then.Residual solids obtains 2-ethyl-3-O-sulfamyl-17-dicyano methylene radical oestrone 62mg (73%), mp=228 ℃ through column chromatography (hexane/ethyl acetate 5/1-3/1) purifying;

1HNMR (CDCl ₃, 270MHz): 1.06 (s, 3H, CH ₃), 1.20 (t, J=7,4Hz, 3H, CH ₃), 1.44-1.75 (m, 6H), 1.92-2.03 (m, 2H), 2.29 (m, 1H), 2.44-2.55 (m, 1H), 2.69 (q, J=7.4Hz, 2H, CH ₂), 2.75-3.04 (m, 5H), 5.03 (s, 2H, NH ₂), 7.09 (s, 1H, ArH), 7.16 (s, 1H, ArH). ¹³C NMR (CDCl ₃): 14.6,16.6 (CH ₃), 23.0,23.2,26.2,27.2,28.9,33.8,34.7,37.8,43.4,49.3,54.1,79.7 (C17), 111.0,112.3,121.5,126.9,134.0,135.4,138.0,146.4 and 195.8.HPLC 100%;

Trace analysis: C:64.90 (theoretical value 64.92); H:6.45 (theoretical value 6.40); N:9.68 (theoretical value 9.87).

2-ethyl-3-O-TBS-17 β-dicyano methyl 17-deoxidation oestrone

The methyl alcohol (50ml) and THF (5ml) solution of 2-ethyl-3-O-TBS-17-dicyano methylene radical oestrone (1mmol) are cooled to-10 ℃, and gradation adds NaBH ₄(76mg, 2mmol).Stirred this mixture 4 hours in-10 ℃.Add entry (50ml) and use NH ₄This solution of Cl acidifying.(MgSO is used in 2 * 60ml) extractions, and water and salt water washing organic layer successively to organism with ethyl acetate ₄Dry also vacuum evaporating solvent.Residual solids obtains 2-ethyl-3-O-TBS-17 β-dicyano methyl 17-deoxidation oestrone 380mg (83%), mp=162 ℃ through column chromatography purification

1H NMR (CDCl ₃): 0.22 (s, 6H, CH ₃Si) .0.80 (s, 3H, CH ₃), 1.00 (s, 9H, (CH ₃) ₃CSi), 1.16 (t, J=7.5Hz, 3H, CH ₃), 1.33-1.66 (m, 6H), 1.85 (m, 2H), 2.10-2.40 (m, 6H), 2.55 (q, J=7.5Hz, 2H, CH ₂), 2.78 (m, 2H, H6), 3.56 (d, J=9.9Hz, 1H, CH (CN) ₂), 6.47 (s, 1H, ArH), 7.03 (s, 1H, ArH), ¹³C NMR (CDCl ₃) :-4.2 and-4,1 (CH ₃Sl), 12.4 (CH ₃), 14.7,18.2,23.4,25.7 (( CH ₃) ₃CSi), 26.2,27.6,27.7,29.2,37.5,38.8,43.2,43.6,50.3,54.6,112.7,118.2,126.1,131.9,132.1,134.5, and 151.3.

2-ethyl-17 β-dicyano methyl 17-deoxidation oestrone

THF (50ml) solution of 2-ethyl-3-O-TBS-17 β-dicyano methyl 17-deoxidation oestrone (.5mmol) is cooled to 0 ℃, be added dropwise to TBAF/THF 1M (0.6mmol, 0.6ml).Stir this solution 2 hours in 0 ℃, be warming up to room temperature then.Add entry (10ml) and ethyl acetate (80ml), organic layer is water and salt water washing successively, uses MgSO ₄Dry also vacuum evaporating solvent.(hexane/ethyl acetate 5: 1-2: 1) purifying obtains white solid 2-ethyl-17 β-dicyano methyl 17-deoxidation oestrone to residual solids through column chromatography.124mg(71％)mp＝248℃；

1H NMR (CDCl ₃, 270MHz): 0.80 (s, 3H, CH ₃), 1.21 (t, J=7.4Hz, 3H, CH ₃), 1.30-1.61 (m, 7H), 1.85 (m, 2H), 2.10-2.41 (m, 5H), 2.59 (q, J=7.4Hz, 2H, CH ₂), 2.81 (m, 2H, H6), 3.56 (d, J=9.9Hz, 1H, CH (CN) ₂), 4.51 (br, 1H, OH), 6.49 (s, 1H, ArH), 7.02 (s, 1H, ArH). ¹³C NMR (CDCl ₃): 12.4 (CH ₃), 14.4 (CH ₃), 22.9,23.4,26.3,27.6,27.7,29.0,37.5,38.8,43.2,43.6,50.3,54.5,112.7,115.2,126.3,127.3,131.9,135.2, and 151.3.HPLC:100%;

Trace analysis: C:78.96 (theoretical value 79.27); H:8.03 (theoretical value 8.10); N:7.79 (theoretical value 8.04).

2-ethyl-3-O-sulfamyl-17 β-dicyano methyl 17-deoxidation oestrone

With the NH that is cooled to 0 ℃ ₂SO ₂DMA (2ml) solution of Cl (0.6mmol) joins in 2-ethyl-17 β-dicyano methyl 17-deoxidation oestrone (0.2mmol), and stirs this mixture 14 hours down in room temperature, nitrogen.Add entry (10mL) then and use ethyl acetate (2 * 50ml) extraction organism.Organic layer is water and salt water washing successively, uses MgSO ₄Drying, solvent removed in vacuo then.Residual solids obtains 2-ethyl-3-O-sulfamyl-17 β-dicyano methyl 17-deoxidation oestrone white powder 170mg (80%), mp=169 ℃ through column chromatography (hexane/ethyl acetate 5/1-3/1) purifying;

1H NMR (CDCl3,270MHz): 0.80 (s, 3H, CH ₃), 1.21 (t, J=7.4Hz, 3H, CH ₃), 1.32-1.65 (m, 7H), 1.87 (m, 2H), 2.10-2.38 (m, 5H), 2.68 (q, J=7.4Hz, 2H, CH ₂), 2.83 (m, 2H, H6), 3.57 (d, J=9.9Hz, 1H, CH (CN) ₂), 4.91 (br, 2H, NH ₂), 7.08 (s, 1H, ArH), 7.16 (s, 1H, ArH). ¹³C NMR (CDCl3,270MHz): 12.4 (CH ₃), 14.4 (CH ₃), 22.9,23.4,26.3,27.6,27.7,29.0,37.5,38.8,43.2,43.6,50.3,54.5,112.7,115.2,126.3,127.3,131.9,135.2, and 151.3.HPLC:100%;

Trace analysis: C:64.64 (theoretical value 64.61); H 7.80 (theoretical value 6.84); N:9.62 (theoretical value 9.83).

2-ethyl-3-O-TBS-17 β-(1-cyano group) ethyl 17-deoxidation oestrone

In-78 ℃, following THF (50ml) solution that stirs 2-ethyl-3-O-TBS-17 beta-cyano methyl 17-deoxidation oestrone (0.5mmol) of nitrogen, then with dropping mode, usefulness 1M LDA (1.2mmol, 1.2ml) processing.Add CH after 30 minutes ₃(1.5mmol 212mg), stirred these mixtures 4 hours in-78 ℃ to I, then in stirring at room 12 hours.Add ethyl acetate (100ml) and water (30ml) then, MgSO is used in organic layer water and salt water washing ₄Dry also vacuum evaporating solvent.The gained yellow oil obtains colorless oil 2-ethyl-3-O-TBS-17 β-(1-cyano group) ethyl 17-deoxidation oestrone 176mg (78%) through column chromatography (hexane/ethyl acetate 20: 1) purifying, is (1: 1) non-enantiomer mixture,

¹H NMR (CDCl3,270MHz): 0.23 (s, 6H, CH ₃Sl), 0.76and 0.78 (s, 3H, CH ₃), 1.01 (s, 9H, (CH ₃) ₃CSi), 1.17 (t, J=7.4Hz, 2H, CH ₃), 1.25-1.96 (m, 13H), 2.05-2.50 (m, 3H), 2.57 (q t, J=7.3Hz, 2H, CH ₂), 2.66-2.88 (m, 4H, CH ₂), 6.48 and 6.50 (m, 1H, ArH), 7.03-7.06 (m, 1H, ArH).

2-ethyl-17-deoxidation 17 β-(1-cyano group) ethyl oestrone

THF (50ml) solution of 2-ethyl-3-O-TBS-17 β-(1-cyano group) ethyl 17-deoxidation oestrone (1mmol) is cooled to 0 ℃, and be added dropwise to 1M TBAF/THF (1.2mmol, 1.2ml).Stir this solution 2 hours in 0 ℃, be warming up to room temperature then.Add entry (10ml) and ethyl acetate (80ml), organic layer is water and salt water washing successively, uses MgSO ₄Dry also vacuum evaporating solvent.(hexane/ethyl acetate 5: 1-2: 1) purifying, 6: 1 recrystallizations of hexane/ethyl acetate obtain 220mg (71%) white powder 2-ethyl-17-deoxidation 17 β-(1-cyano group) ethyl oestrone to residual yellow solid (400mg) then through column chromatography.mp：226-228℃

¹H?NMR(CDCl ₃，270MHz)：0.75(s，3H，CH ₃)，1.20-1.65(m，9H)，1.85-1.92(m，5H)，2.10-2.58(m，4H)，2.5(m，2H，H6)，6.55-6.62(m，2H，ArH)，7.07(m，tH，ArH).HPLC：100％

Trace analysis: C:81.60 (theoretical value 81.51); H:8.79 (theoretical value 8.79); N:4.40 (theoretical value 4.53).

2-ethyl-3-O-sulfamyl-17-deoxidation 17 β-(1-cyano group) ethyl oestrone

With the NH that is cooled to 0 ℃ ₂SO ₂DMA (2ml) solution of Cl (0.6mmol) joins in 2-ethyl-17-deoxidation 17 β-(1-cyano group) ethyl oestrone (0.2mmol), and stirs this mixture 14 hours down in room temperature, nitrogen.Add 10mL water then and use ethyl acetate (2 * 50ml) extraction organism.Organic layer is water and salt water washing successively, uses MgSO ₄Drying, solvent removed in vacuo then.Residual solids is through column chromatography (hexane/ethyl acetate 5/1-2/1) purifying, and recrystallization (hexane/ethyl acetate 5: 1) obtains 42mg (54%) 2-ethyl-3-O-sulfamyl-17-deoxidation 17 β-(1-cyano group) ethyl oestrone, mp:157-159 ℃ then;

¹H NMR (CDCl3,270MHz): 0.79 (s, 3H, CH ₃), 1.25-1.64 (m, 11H), 1.73-1.81 (m, 1H), 1.85-2.03 (m, 2H), 2.23-2.37 (m, 2H), 2.40-2.53 (m, 2H), 2.89 (m, 2H, H6), 7.03 (d, J=2.7Hz, 1H, ArH), 7.07 (dd, J=9.0 and 2.7Hz, 1H, ArH), 7.31 (d, J=9.0Hz, 1H, ArH) .HPLC:100%

Trace analysis: C:64.90 (theoretical value 64.92); H:7.52 (theoretical value 7.26); N:7.23 (theoretical value 7.21).

2-ethyl-3-O-benzyl-17 α-(methyl) cyano methyl estradiol

In-78 ℃, following anhydrous THF (50ml) solution that stirs propionitrile (0.05mol) of nitrogen, handle with dropping mode, usefulness LDA (0.05mol) then.Stir this mixture 1 hour, and added the 20ml THF solution (in 3 hours, dripping) of 2-ethyl-3-O-benzyl oestrone (8mmol) then.This mixture of restir 3 hours is used NH then ₄The quencher of the Cl aqueous solution is also used ethyl acetate extraction.Organic layer is water and salt water washing successively, uses MgSO ₄Dry also vacuum evaporating solvent.Thick oily matter obtains white powder 2-ethyl-3-O-benzyl-17 α-(methyl) cyano methyl estradiol 2.70g (76%), mp=75-77 ℃ through column chromatography (hexane/ethyl acetate 10/1-4/1) purifying

¹H?NMR(CDCl ₃，270MHz)：0.97(s，3H，CH ₃)，1.20(t，J＝7.6Hz，3H，CH ₃)，1.25-1.90(m，12H)，1.97-2.07(m，2H)2.15-2.23(m，1H)，2.41-2.49(m，1H)，2.66(q，J＝7.6Hz，2H，CH ₂)，2.77-2.91(m，3H)，5.04(s，2H，C H ₂Ph)，)，6.62(s，1H，ArH)，7.09(s，1H，ArH)，7.28-7.46(m，5H，Ph).

2-ethyl-3-O-benzyl 17-(1-cyano group) ethylidene oestrone

2-ethyl-3-O-benzyl estradiol-17 α-propionitrile (0.05mol) is joined among the TEA (2ml) and DCM (25ml) solution that is cooled to 0 ℃, and adding CH ₃SO ₂Cl (0.055mol, 0.44ml) after, stirred this solution 8 hours in 0 ℃.This solution of water quencher also extracts organism with DCM.Organic layer is water and salt water washing successively, uses MgSO ₄Dry also solvent removed in vacuo.Thick oily matter obtains 2-ethyl-3-O-benzyl 17-cyano group ethylidene oestrone through column chromatography (hexane/ethyl acetate 10/1) purifying.Productive rate 85%, mp=63-64 ℃

¹H?NMR(CDCl ₃，270MHz)：0.95(s，3H，CH ₃)，1.21(t，J＝7.3Hz，3H，CH ₃)，1.30-1.71(m，6H)，1.84(s，3H，CH ₃)，1.85-1.96(m，2H)，2.21-2.51(m，4H)，2.64(q，J＝7.3Hz，2H，CH ₂)，2.75-2.90(m，3H)，5.04(s，2H，C H ₂Ph)，6.63(s，1H，ArH)，7.10(s，1H，ArH)，7.28-7.48(m，5H，Ph).

2-ethyl-17-deoxidation 17 β-(1-cyano group) ethyl oestrone

In the 5mLTHF/45mL methanol solution of 2-ethyl-3-O-benzyl 17-(1-cyano group) ethylidene oestrone (1mmol), add 50mg 5%Pd/C, and under hydrogen, stirred this mixture 16 hours.This suspension is filtered through diatomite/sand, with THF washing and vacuum concentration organic layer.Column chromatography (hexane/ethyl acetate 10: 1-1: 1) and behind the recrystallization (hexane/ethyl acetate 5: 1), separate obtaining white solid 2-ethyl-17-deoxidation 17 β-(1-cyano group) ethyl oestrone, white powder 225mg (67%), mp=235-236 ℃;

¹H?NMR(CDCl ₃，270MHz)：0.76(s，3H，CH ₃)，1.20(t，J＝7.4Hz，3H，CH ₃)，1.24-1.69(m，13H)，1.71-2.00(m，2H)，2.10-2.48(m，2H)，2.57(q，J＝7.3Hz，2H，CH ₂)，2.71(m，1H，C H(CH ₃)CN)，2.78(m，2H，H6)，4.47(br，1H，OH)，6.49(s，1H，ArH)，7.03(s，1H，ArH).

Trace analysis: C:81.50 (theoretical value 81.85); H:9.18 (theoretical value 9.26); N:3.99 (theoretical value 4.15).

2-ethyl-3-O-sulfamyl-17-deoxidation 17 β-(1-cyano group) ethyl oestrone

With the NH that is cooled to 0 ℃ ₂SO ₂DMA (2ml) solution of Cl (1mmol) joins among the 21a (0.3mmol), and stirs this mixture 14 hours down in room temperature, nitrogen.Add entry (10mL) then and use ethyl acetate (2 * 50ml) extraction organism.Organic layer is water and salt water washing successively, uses MgSO ₄Drying, solvent removed in vacuo then.Residual solids is through column chromatography (hexane/ethyl acetate 5/1-2/1) purifying; recrystallization (hexane/ethyl acetate 5: 1) then; obtain white powder 2-ethyl-3-O-sulfamyl-17-deoxidation 17 β-(1-cyano group) ethyl oestrone 86mg (69%), mp=171-172 ℃

¹H?NMR(CDCl3，270MHz)：0.76(s，3H，CH ₃)，1.20(t，J＝7.4Hz，3H，CH ₃)，1.22-1.64(m，11H)，1.75-2.35(m，6H)，2.66(q，J＝7.3Hz，2H，CH ₂)，2.68(m，1H，C H(CH ₃)CN)，2.84(m，2H，H6)，4.92(br，2H，NH ₂)，7.07(s，1H，ArH)，7.16(s，1H，ArH). ¹³C?NMR(CDCl3，270MHz)：12.4(CH ₃)，14.6(CH ₃)，18.6(CH ₃)，23.0，23.8，26.2，26.8，27.0，27.4，29.1，38.2，38.6，42.9，44.0，53.3，54.8，121.4，123.0(CN)，126.9，133.6，135.9，139.3，146.2.

Trace analysis: C:66.40 (theoretical value 66.31); H:7.73 (theoretical value 7.74); N:6.52 (theoretical value 6.72).

2-ethyl-3-O-benzyl-17 β-(2-hydroxyethyl) 17-deoxidation oestrone

(1.84g, 30ml anhydrous THF solution 4mmol) are cooled to 0 ℃ to nitrogen, and gradation adds LiAlH then with (3-benzyloxy-2-ethyl-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-17-yl)-ethyl acetate down ₄(0.30g, 8mmol).In 0 ℃ after 3 hours, this mixture NH ₄The quencher of the Cl aqueous solution is also used the ethyl acetate extraction organism.MgSO is used in organic layer water, salt water washing ₄Dry also vacuum evaporating solvent.(hexane/ethyl acetate 15/1-8: 1) purifying obtains 1.47g (88%) white powder 2-ethyl-3-O-benzyl-17 β-(2-hydroxyethyl) 17-deoxidation oestrone, mp=98-99 ℃ to thick oily matter through chromatography

¹H?NMR(CDCl ₃，270MHz)：0.63(s，3H，CH ₃)，1.20(t，J＝7.4Hz，3H，CH ₃)，1.22-1.58(m，9H)，1.70-1.92(m，5H)，2.16-2.34(m，2H)，2.66(q，J＝7.4Hz，2H，CH ₂)，2.82(m，2H，H6)，3.68(m，2H，C H ₂OH)，5.03(s，2H，CH ₂Ph)，6.62(s，1H，ArH)，7.10(s，1H，ArH)，7.28-7.45(m，5H，Ph).

2-ethyl-3-O-benzyl-17 β-(2-methoxy ethyl) 17-deoxidation oestrone

Under the nitrogen with 2-ethyl-3-O-benzyl-17 β-(1.26g, 30ml anhydrous THF solution 3mmol) is cooled to 0 ℃ to (2-hydroxyethyl) 17-deoxidation oestrone.Gradation adds NaH (0.16g, the dispersion liquid of 4mmolNaH 60% mineral oil), stirs this solution 30 minutes, is added dropwise to CH then ₃I (0.37ml, 6mmol).Make this solution be warming up to room temperature and stirred 18 hours.After adding entry (20ml), the organism ethyl acetate extraction.MgSO is used in organic layer water and salt water washing ₄Dry also vacuum evaporating solvent.Thick oily matter obtains 1.25g (96%) white powder 2-ethyl-3-O-benzyl-17 β-(2-methoxy ethyl) 17-deoxidation oestrone, mp=70-71 ℃ through chromatography (hexane/ethyl acetate 20/1) purifying

¹H?NMR(CDCl ₃，270MHz)：0.66(s，3H，CH ₃)，1.25(t，J＝7.4Hz，3H，CH ₃)，1.26-1.60(m，9H)，1.76-1.96(m，5H)，2.20-2.42(m，2H)，2.70(q，J＝7.4Hz，2H，CH ₂)，2.86(m，2H，H6)，3.38(s，3H，CH ₃O)，3.44(m，2H，CH ₂O)，5.07(s，2H，CH ₂Ph)，6.66(s，1H，ArH)，7，15(s，1H，ArH)，7.31-7.50(m，5H，Ph).

2-ethyl-17 β-(2-methoxy ethyl) 17-deoxidation oestrone

2-ethyl with 35-3-O-benzyl-17 β-(2-methoxy ethyl) 17-deoxidation oestrone 0.87g (2mmol) is dissolved in THF (5mL) and the methyl alcohol (50mL), adds 50mg 5%Pd/C and stirred this mixture 16 hours under hydrogen.This suspension is filtered through diatomite/sand, with THF washing and vacuum evaporating solvent.The little yellow oil of thick shape obtains white powder 2-ethyl-17 β-(2-methoxy ethyl) 17-deoxidation oestrone 0.66g (97%), mp=58-59 ℃ through column chromatography (hexane/ethyl acetate 15/1-10/1) purifying;

¹H NMR (CDCl ₃, 270MHz): 0.62 (s, 3H, CH ₃), 1.19-1.56 (m, 12H), 1.72-1.91 (m, 5H), 2.13-2.34 (m, 2H), 2.60 (q, J=7.4Hz, 2H, CH ₂), 2.77 (m, 2H, H6), 3.37 (s, 3H, CH ₃O), 3.45 (m, 2H, CH ₂O), 5.26 (s, 1H, OH), 6.48 (s, 1H, ArH), 7.06 (s, 1H, ArH). ¹³C NMR (CDCl ₃): 12.4 (CH ₃), 12.5 (CH ₃), 23.0,24.4,26.5,27.9,28.3,29.3,30.2,37.7,38.9,42.4,44.2,47.3,54.6,58.4,72.4,115.1,126.2,127.2,132.6,135.4, and 151.3.HPLC:100%;

HRMS (FAB+): measured value 342.255493; Calculated value C ₂₃H ₃₄O ₂342.2558812-ethyl-3-O-sulfamyl-17 β-(2-methoxy ethyl) 17-deoxidation oestrone

With the NH that is cooled to 0 ℃ ₂SO ₂DMA (2ml) solution of Cl (3mmol) join 2-ethyl-17 β-(2-methoxy ethyl) 17-deoxidation oestrone (342mg, 1mmol) in, and stirred this mixture 24 hours down in room temperature, nitrogen.After adding entry (10mL), (2 * 50ml) extract organism with ethyl acetate.Organic layer is water and salt water washing successively, and uses MgSO ₄Dry.Solvent removed in vacuo, residual solids is through column chromatography (hexane/ethyl acetate 10/1-4/1) purifying, recrystallization (hexane/ethyl acetate 5: 1) then, obtain white powder 2-ethyl-3-O-sulfamyl-17 β-(2-methoxy ethyl) 17-deoxidation oestrone 0.35g (84%), mp=168-169 ℃;

¹H NMR (CDCl ₃, 270MHz): 0.61 (s, 3H, CH ₃), 1.20 (t, J=7.4Hz, 3H, CH ₃), 1.22-1.54 (m, 9H), 1.70-1.95 (m, 5H), 2.15-2.33 (m, 2H), 2.68 (q, J=7.4Hz, 2H, CH ₂), 2.82 (m, 2H, H6), 3.33 (s, 3H, CH ₃O), 3.35 (m, 2H, CH ₂O), 4.95 (s, 2H, NH ₂), 7.06 (s, 1H, ArH), 7.18 (s, 1H, ArH). ¹³C NMR (CDCl ₃, 270MHz): 12.4 (CH ₃), 14.6 (CH ₃), 23.0,24.4,26.3,27.7,28.3,29.3,30.4,37.6,38.5,42.4,44.4,47.4,54.9,58.7,72.6,121.4,127.0,133.5,136.1,139.9, and 146.1.; HPLC:100%;

Trace analysis: C:65.40 (theoretical value 65.52); H:8.29 (theoretical value 8.37); N:3.14 (theoretical value 3.32).

2-ethyl-3-O-benzyl-17 β-(2-(OTBDPS) ethyl) 17-deoxidation oestrone

Under nitrogen, room temperature, stir 2-ethyl-3-O-benzyl-17 β-(2-hydroxyethyl) 17-deoxidation oestrone (0.419g, 1mmol), imidazoles (0.136g, 2mmol) and the 10ml anhydrous DMF solution of TPDPSC1 (1.1mmol) 24 hours.After adding entry (10mL), (2 * 50ml) extract organism with ethyl acetate.Organic layer is water and salt water washing successively, and uses MgSO ₄Dry.Solvent removed in vacuo, residual oily matter obtain white powder [2-(3-benzyloxy-2-ethyl-13-methyl-7 through column chromatography (hexane/ethyl acetate 25/1) purifying, 8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-17-yl)-oxyethyl group]-tertiary butyl-phenylbenzene-silane 590mg (90%), mp=48-50 ℃;

¹H?NMR(CDCl ₃，270MHz)：0.63(s，3H，CH ₃)，1.12(s，9H，(CH ₃) ₃C)1.15-1.60(m，11H)，1.71-2.01(m，5H)2.20-2.44(m，2H)，2.69(q，J＝7.4Hz，2H，CH ₂)，2.88(m，2H，H6)，3.65-3.81(m，2H，CH ₂O)，5.09(s，2H，CH ₂Ph)，6，68(s，1H，ArH)，7.20(s，1H，ArH)，7.32-7.51(m，11H，Ph)，7.72-7.77(m，4H，Ph).

2-ethyl-17 β-(2-(OTBDPS) ethyl) 17-deoxidation oestrone

In the THF (2mL) of 2-ethyl-3-O-benzyl-17 β-(2-(OTBDPS) ethyl) 17-deoxidation oestrone (1mmol) and ethanol (20mL) solution, add 50mg 5%Pd/C, and under hydrogen, stir this mixture.Reaction is monitored with TLC.After reaction is finished, suspension is filtered and vacuum evaporating solvent through diatomite/sand.Residual oily matter obtains white solid 17-[2-(tertiary butyl-phenylbenzene-silicon alkoxyl group)-ethyl through column chromatography (hexane/ethyl acetate 20/1 was to 3: 1) purifying]-2-ethyl-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-3-alcohol, mp=134-135 ℃, 0.49g (87%);

¹H?NMR(CDCl ₃，270MHz)：0.69(s，3H，CH ₃)，1.08(s，9H，(CH ₃) ₃C)，1.18-1.56(m，12H)，1.68-1.89(m，5H)，2.10-2.33(m，2H)，2.61(q，J＝7.4Hz，2H，CH ₂)，2.78(m，2H，H6)，3.61-3.78(m，2H，CH ₂O)，4.71(s，1H，OH)，6.49(s，1H，ArH)，7.07(s，1H，ArH)，7.38(m，6H，Ph)，7.70(m，4H，Ph).

2-ethyl-3-O-sulfamyl-17 β-(2-(OTBDPS) ethyl) 17-deoxidation oestrone

With the NH that is cooled to 0 ℃ ₂SO ₂DMA (2ml) solution of Cl (3mmol) join 39c (566mg, 1mmol) in, and stirred this mixture 24 hours down in room temperature, nitrogen.After adding entry (10mL), (2 * 50ml) extract organism with ethyl acetate.Organic layer is water and salt water washing successively, and uses MgSO ₄Dry.Solvent removed in vacuo, (hexane/ethyl acetate 15/1-8/1 purifying obtains white solid 2-ethyl-3-O-sulfamyl-17 β-(2-(OTBDPS) ethyl) 17-deoxidation oestrone 470mg (72%), mp=185-186 ℃ to residual solids through column chromatography;

¹HNMR (CDCl ₃, 270MHz): 0.57 (s, 3H, CH ₃), 1.05 (s, 9H, (CH ₃) ₃C), and 1.18-1.55 (m, 13H), 1.68-1.89 (m, 4H), 2.15-2.31 (m, 2H), 2.68 (q, J=7.4Hz, 2H, CH ₂), 2.82 (m, 2H, H6), 3.57-3.75 (m, 2H, CH ₂O), 4.91 (br, 2H, NH ₂), 7.06 (s, 1H, ArH), 7.19 (s, 1H, ArH), 7.34-7.45 (m, 6H, Ph), 7.67 (m, 4H, Ph). ¹³C NMR (CDCl ₃): 12.9 (CH ₃), 15.1 (CH ₃), 20.0,23.5,24.9,26.8,27.3,28.1,28.8,29.7,33.9,38.1,38.8,42.7,44.8,47.6,55.0,63.9,121.5,127.2,127.7,129.7,133.7,134.3,135.7,136.3,140.1, and 146.2.

HRMS (FAB+): measured value 645.326294, calculated value C ₂₃H ₃₄O ₂645.330810

2-ethyl-3-O-sulfamyl-17 β-(2-hydroxyethyl) 17-deoxidation oestrone

With 2-ethyl-3-O-sulfamyl-17 β-(323mg, 20ml THF solution 0.5mmol) is cooled to 0 ℃ to (2-(OTBDPS) ethyl) 17-deoxidation oestrone, is added dropwise to 0.6ml 1MTBAF/THF then.Stirred this solution 6 hours in 0 ℃, add entry (10ml) then, organism is with ethyl acetate (2 * 50ml) extractions.Organic layer is water and salt water washing successively, uses MgSO ₄Dry.Solvent removed in vacuo, residual solids is through column chromatography (hexane/ethyl acetate 10/1-2/1) purifying, 4: 1 recrystallizations of hexane/ethyl acetate obtain white powder 2-ethyl-3-O-sulfamyl-17 β-(2-hydroxyethyl) 17-deoxidation oestrone 145mg (72%), mp=157-158 ℃ then;

¹H NMR ((CD ₃) ₂CO) 0.62 (s, 3H, CH ₃), 1.17-1.55 (m, 12H), 1.70-1.94 (m, 5H), 2.16-2.33 (m, 2H), 2.68 (q, J=7.4Hz, 2H, CH ₂), 2.83 (m, 2H, H6), 3.60-3.76 (m, 2H, CH ₂O), 4.87 (br, 2H, NH ₂), 7.06 (s, 1H, ArH), 7.19 (s, 1H, ArH); ¹³C NMR (CD ₃COCD ₃, 400MHz): 11.8 (CH ₃), 13.9 (CH ₃), 22.3,23.7,25.7,27.1,27.7,28.5,33.0,37.1,38.0,41.7,43.8,46.7,54.1,61.2,121.1,126.0,133.1,134.9,138.4 and 145.8;

Trace analysis: C:64.78 (theoretical value 64.83); H:8.12 (theoretical value 8.16); N:3.41 (theoretical value 3.44).

2-(3-benzyloxy-2-ethyl-13-methyl-6,7,8,9,11,12,13,14,15,16-decahydro-cyclopenta [a] phenanthrene-17-subunit)-ethanol

Nitrogen is down with (3-benzyloxy-2-ethyl-13-methyl-6,7,8,9,11,12,13,14,15,16-decahydro-cyclopenta [a] phenanthrene-17-subunit)-(0.92g, 20ml anhydrous THF solution 2mmol) is cooled to 0 ℃ to ethyl acetate, adds LiAlH in gradation adding mode then ₄(0.15g, 4mmol).In 0 ℃ after 6 hours, use NH ₄This mixture of Cl aqueous solution quencher, the organism ethyl acetate extraction.MgSO is used in organic layer water and salt water washing ₄Dry also vacuum evaporating solvent.(hexane/ethyl acetate 15/1-6: 1) purifying obtains 2-(3-benzyloxy-2-ethyl-13-methyl-6,7,8,9 to thick oily matter through chromatography, 11,12,13,14,15,16-decahydro-cyclopenta [a] phenanthrene-17-subunit)-ethanol 0.75g (89%), be white powder, mp=61-62 ℃;

¹H?NMR(CDCl ₃，270MHz)：0.84(s，3H，CH ₃)，1.20-1.66(m，9H)，1.82-2.02(m，3H)，2.21-2.51(m，3H)，2.71(q，J＝7.4Hz，2H，CH ₂)，2.87(m，2H，H6)，4.16(m，2H，CH ₂OH)，5.07(s，2H，CH ₂Ph)，5.31(m，1H，C HCH ₂OH)，6.66(s，1H，ArH)，7.15(s，1H，ArH)，7.30-7.48(m，5H，Ph).

3-benzyloxy-2-ethyl-17-(2-methoxyl group-ethylidene)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene

(3-benzyloxy-2-ethyl-13-methyl-6,7,8,9,11,12,13,14,15,16-decahydro-cyclopenta [a] phenanthrene-17-subunit)-(0.62g, 10ml anhydrous THF solution 1.5mmol) is cooled to 0 ℃ to ethanol with 2-under the nitrogen.Gradation adds NaH (0.08g, the mineral oil dispersion liquid of 2mmol NaH 60%), stirs this solution 30 minutes, is added dropwise to CH then ₃I (0.19ml, 3mmol).Make this solution be warming up to room temperature and stirred 18 hours.After adding entry (20ml), the organism ethyl acetate extraction.MgSO is used in organic layer water and salt water washing ₄Dry also vacuum evaporating solvent.Thick oily matter obtains 3-benzyloxy-2-ethyl-17-(2-methoxyl group-ethylidene)-13-methyl-7,8,9 through chromatography (hexane/ethyl acetate 20/1) purifying, 11,12,13,14,15,16, the 17-decahydro-luxuriant and rich with fragrance 0.59g of 6H-cyclopenta [a] (91%) is colorless oil;

¹H?NMR(CDCl ₃，270MHz)：0.84(s，3H，CH ₃)，1.24(t，J＝7.4Hz，3H，CH ₃)，1.26-1.66(m，6H)，1.82-2.02(m，3H)，2.20-2.51(m，3H)，2.70(q，J＝7.4Hz，2H，CH ₂)，2.87(m，2H，H6)，3.36(s，3H，CH ₃O)，3.94(m，2H，CH ₂OMe)，5.06(s，2H，CH ₂Ph)，5.27(m，1H，C HCH ₂OH)，6.66(s，1H，ArH)，7.15(s，1H，ArH)，7.31-7.48(m，5H，Ph).

2-ethyl-17-(2-methoxyl group-ethylidene)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-3-alcohol

With 3-benzyloxy-2-ethyl-17-(2-methoxyl group-ethylidene)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] luxuriant and rich with fragrance (0.43g, 50ml t-butanol solution 1mmol) refluxes, and in 6 hours with aliquot add Na (0.46g, 20mmol).This mixture refluxed 18 hours, was cooled to room temperature and added the 2-propyl alcohol to destroy excessive sodium.With solvent evaporation and with in the residual solids impouring water (20ml).The organism ethyl acetate extraction, MgSO is used in organic layer water and salt water washing ₄Dry.Vacuum evaporating solvent.(hexane/ethyl acetate 15/1-8: 1) purifying obtains white powder 2-ethyl-17-(2-methoxyl group-ethylidene)-13-methyl-7,8,9,11 to thick oily matter through chromatography, 12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-3-alcohol, 230mg (68%), mp=133-134 ℃;

¹H NMR (CDCl ₃, 270MHz): 0.79 (s, 3H, CH ₃), 1.22 (t, J=7.4Hz, 3H, CH ₃), 1.28-1.60 (m, 6H), 1.75-1.96 (m, 3H), 2.18 (m, 1H), 2.39 (m, 3H), 2.59 (q, J=7.4Hz, 2H, CH ₂), 2.79 (m, 2H, H6), 3.35 (s, 3H, CH ₃O), 3.94 (m, 2H, CH ₂OMe), 5.11 (s, 1H, OH), 5.24 (m, 1H, C HCH ₂OH), 6.48 (s, 1H, ArH), 7.05 (s, 1H, ArH). ¹³C NMR (CDCl ₃, 400MHz): 14.9 (CH ₃), 19.2,23.5,24.4,26.7,27.1,28.1,29.7,36.4,39.1,44.4,44.8,53.5,58.2,70.2,112.8,115.7,126.4,127.5,132.6,135.5,151.5, and 157.1.HPLC:98%;

HRMS (FAB ⁺): measured value 340.24023, calculated value C ₂₃H ₃₂O ₂340.24023.

2-ethyl-3-O-sulfamyl 17-(2-methoxyl group-ethylidene) oestrone

With the NH that is cooled to 0 ℃ ₂SO ₂DMA (2ml) solution of Cl (2mmol) joins 2-ethyl-17-(2-methoxyl group-ethylidene)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta [a] phenanthrene-3-alcohol (170mg, 0.5mmol) in, and stirred this mixture 24 hours down in room temperature, nitrogen.After adding entry (10mL), (2 * 50ml) extract organism with ethyl acetate.Organic layer is water and salt water washing successively, and uses MgSO ₄Dry.Solvent removed in vacuo, residual solids obtain white solid 2-ethyl-3-O-sulfamyl 17-(2-methoxyl group-ethylidene) oestrone through column chromatography (hexane/ethyl acetate 10/1-5/1) purifying, 142mg (68%),

¹H NMR (CDCl ₃, 270MHz): 0.79 (s, 3H, CH ₃), 1.20 (t, J=7.4Hz, 3H, CH ₃), 1.22-1.67 (m, 6H), 1.78-1.98 (m, 3H), 2.17-2.47 (m, 4H), 2.68 (q, J=7.4Hz, 2H, CH ₂), 2.85 (m, 2H, H6), 3.32 (s, 3H, CH ₃O), 3.90 (m, 2H, CH ₂OMe), 5.14 (s, 2H, NH ₂), 5.22 (m, 1H, C HCH ₂OH), 7.06 (s, 1H, ArH), 7.19 (s, 1H, ArH). ¹³C NMR (CDCl ₃, 400MHz): 14.6 (CH ₃), 18.8,23.1,24.0,26.4,26.5,27.4,29.2,35.8,38.2,44.3,44.4,53.2,57.8,69.8,112.9,121.4,127.0,133.7,136.0,139.6,146.2 and 156.4.HPLC:99.7%;

HRMS (FAB+): measured value 419.21303, calculated value C ₂₃H ₃₃O ₄NS 419.21303

2-ethyl-3-O-ethanoyl-17-cyano group-17-hydroxyl-oestrone

In stirring at room 2-ethyl estrone acetate (5mmol) and KCN (3.26g, 20ml methyl alcohol 50mmol) and 5ml acetic acid solution 3-5 days.To ice and water joins in this mixture, wash with the gained solid filtering and with massive laundering.White solid is dissolved in the ethyl acetate (100ml), and organic layer is water and salt water washing successively, and uses MgSO ₄Drying, solvent removed in vacuo obtains white solid 2-ethyl-3-O-ethanoyl-17-cyano group-17-hydroxyl-oestrone, 1.6g (87%), its demonstration

¹H?NMR(CDCl ₃，270MHz)：0.85(s，3H，CH3)，1.16(t，J＝7.4Hz，3H，CH ₃)，1.33-1.70(m，6H)，1.80-2.06(m，5H)，2.30(s，3H，CH ₃CO)，2.25-2.35(m，1H)，2.38-2.58(m，3H)，2.70(q，J＝7.4Hz，2H，CH ₂)，2.82(m，2H，H6)，6.71(s，1H，ArH)，7.15(s，1H，ArH).

Acetate 17-cyano group-13-methyl-7,8,9,11,12,13,14,15-octahydro-6H-cyclopenta [a] phenanthrene-3-base ester

2-ethyl-3-O-ethanoyl-17-cyano group-17-hydroxyl-oestrone (4mmol) is dissolved in the 10ml anhydrous pyridine, and is added dropwise to SOCl ₂(1.46ml, 20mmol).This solution refluxed 1 hour under the nitrogen, was cooled to 0 ℃ and also used the 5M hydrochloric acid hydrolysis to pH 1.Organism ethyl acetate extraction, and organic layer water and salt water washing successively, and use MgSO ₄Dry.Vacuum evaporating solvent, (hexane/ethyl acetate 8: 1-6: 1) purifying obtains white powder acetate 17-cyano group-2-ethyl-13-methyl-7,8 to gained dark oil thing through chromatography, 9,11,12,13,14,15-octahydro-6H-cyclopenta [a] phenanthrene-3-base ester, 0.50g (36%), mp176-177 ℃;

¹HNMR (CDCl ₃, 270MHz): 0.94 (s, 3H, CH ₃), 1.16 (t, J=7.4Hz, 3H, CH ₃), 1.36-1.52 (m, 1H), 1.60-1.73 (m, 4H), 1.84-1.94 (m, 1H), 2.05-2.26 (m, 3H), 2.30 (s, 3H, CH ₃CO), 2.39-2.52 (m, 4H), 2.86 (m, 2H, H6), 6.65 (dd, J=3.5 and 2.0Hz, 1H, H16), 6.72 (s, 1H, ArH), 7.14 (s.1H, ArH).

2-ethyl-3-O-ethanoyl-17-cyano group-17-deoxidation oestrone

To acetate 17-cyano group-13-methyl-7,8,9,11,12,13,14, add 5%Pd/C (30mg) in the THF (5mL) of 15-octahydro-6H-cyclopenta [a] phenanthrene-3-base ester (1mmol) and methyl alcohol (30mL) solution, and stirred this suspension 24 hours down in room temperature, hydrogen.Suspension is filtered and vacuum evaporating solvent through diatomite/sand.(hexane/ethyl acetate 8: 1-6: 1) purifying obtains white powder 2-ethyl-3-O-ethanoyl-17-cyano group-17-deoxidation oestrone, 330mg (94%), mp=195-196 ℃ to residual solids through chromatography;

¹H?NMR(CDCl ₃，270MHz)：0.99(s，3H，CH ₃)，1.21(t，J＝7.4Hz，3H，CH ₃)，1.22-1.32(m，1H)，1.37-1.53(m，4H)，1.56-1.67(m，1H)，1.86-1.95(m，2H)，1.99-2.08(m，1H)，2.12-2.17(m，1H)，2.19-2.32(m，2H)，2.35(s，3H，CH ₃CO)，2.38-2.47(m，2H)，2.53(q，J＝7.4Hz，2H，CH ₂)，2.88(m，2H，H6)，6.76(s，1H，ArH)，7.19(s，1H，ArH).

2-ethyl-17-cyano group-17-deoxidation oestrone

2-ethyl-3-O-ethanoyl-17-cyano group-17-deoxidation oestrone (0.8mmol) is dissolved in acetone (10ml) and the methyl alcohol (10ml), and in this solution, adds K ₂CO ₃(0.55g, 4mmol).In this mixture of stirring at room 24 hours.With the salt elimination and with solvent evaporation.Gained solid NH ₄The Cl aqueous solution is handled (to pH 1), and uses the ethyl acetate extraction organism.Organic layer is water and salt water washing successively, and uses MgSO ₄Dry.Vacuum evaporating solvent, (hexane/ethyl acetate 8: 1-6: 1) purifying, and hexane/ethyl acetate (4: 1) recrystallization obtain white powder 2-ethyl-17-cyano group-17-deoxidation oestrone, 210mg (85%), mp=284-285 ℃ to the gained solid through chromatography;

¹H NMR (CDCl ₃, 270MHz): 0.95 (s, 3H, CH ₃), 1.21 (t, J=7.4Hz, 3H, CH ₃), 1.30-1.60 (m, 6H), 1.79-1.91 (m, 2H), 1.94-2.07 (m, 2H), 2.09-2.24 (m, 2H), 2.33-2.42 (m, 2H), 2.58 (q, J=7.4Hz, 2H, CH ₂), 2.78 (m, 2H, H6), 4.50 (s, 1H, OH), 6.49 (s, 1H, ArH), 7.03 (s, 1H, ArH); ¹³C NMR (CDCl ₃, 400MHz): 14.4 (CH ₃), 14.5,23.1,24.3,26.4,26.7,27.8,29.1,37.1,39.2,40.4,43.7,44.7,53.5,115.2,121.3,126.4,127.4,131.9,135.2 and 151.3;

Trace analysis: C:81.15 (theoretical value 81.51); H:8.72 (theoretical value 8.79); N:4.33 (theoretical value 4.53).

2-ethyl-3-O-sulfamyl-17-cyano group-17-deoxidation oestrone

With the NH that is cooled to 0 ℃ ₂SO ₂DMA (2ml) solution of Cl (0.8mmol) joins in 2-ethyl-17-cyano group-17-deoxidation oestrone (0.3mmol), and stirs this mixture 24 hours down in room temperature, nitrogen.After adding entry (10mL), (2 * 50ml) extract organism with ethyl acetate.Organic layer is water and salt water washing successively, and uses MgSO ₄Dry.Solvent removed in vacuo, residual solids is through column chromatography (hexane/ethyl acetate 5/1) purifying and hexane/ethyl acetate (5/1) recrystallization, obtain white powder 2-ethyl-3-O-sulfamyl-17-cyano group-17-deoxidation oestrone, 100mg (86%), mp=193-194 ℃;

¹H NMR (CDCl ₃, 270MHz): 0.95 (s, 3H, CH ₃), 1.20 (t, J=7.4Hz, 3H, CH ₃), 1.28-1.62 (m, 6H), 1.86 (m, 2H), 1.96-2.28 (m, 4H), 2.37 (m, 2H), 2.68 (q, J=7.4Hz, 2H, CH ₂), 2.83 (m, 2H, H6), 4.94 (s, 2H, NH ₂), 7.08 (s, 1H, ArH), 7.18 (s, 1H, ArH); ¹³C NMR (CDCl _s, 400MHz): 14.4 (CH ₃), 14.7,23.1,24.3,26.1,26.6,27.5,29.1,37.0,38.8,40.3,43.8,44.6,53.5,121.2,121.5,127.0,133.8,135.7,138.8 and 146.3.

Trace analysis: C:64.95 (theoretical value 64.92); H:7.29 (theoretical value 7.26); N:7.11 (theoretical value 7.21).

Biological data

Compound mentioned herein can be determined or be determined by the STX code name by employed compound number in the above-mentioned composite part.Its corresponding STX code name of compound number is following listed.

Compound number	The STX code name
		14a	441
14b	442
		15	563
17a	590
		17b	504
19	536
		20	637
22	506
		23	589

27	505
		28	564
30	626
		31	639
32	640
		33	641
36	621
		51	591
59	941
		60	940

In addition, following compounds refers to:

The inhibition of steoid sulfatase

Compound number	The IC that suppresses placenta microsome steoid sulfatase ₅₀Value (nM)
		28	945
31	9000
		33	109
60	＞10,000

Cell proliferation

With microtiter plate test (Cell Titer 96proliferation assay, Promega, Hampshire, UK) effect of mensuration medicine on cell proliferation.

Compound number	The cytostatic IC of MCF-7 ₅₀(μM)
		14a	30.6
14b	31.1
		15	1.85
17a	0.67
		17b	0.77
19	2.52
		20	5.64
22	26.4
		23	4.19
27	3.24
		28	0.06
30	3.22
		31	0.03
32	0.30
		33	0.15

36	3.25
		51	6.02
59	5.58
		60	0.09

Compound S TX 140 in following test with comparing.

The human umbilical vein cell; 96 orifice plate MTS proliferation tests, on average suppress three holes+/-Sds

Concentration	STX?140	Compound 33	Compound 28
				0.05μM	7％+/-4	53％+/-13	0％+/-6
0.10μM	18％+/-1 ^*	60％+/-8	5％+/-5
				1.00μM	59％+/-3	48％+/-6	54％+/-10
5.00μM	49％+/-5	42％+/-3	49％+/-7

^*Only duplicate reading.

Human skin fibroblast; 96 orifice plate MTS proliferation tests, on average suppress three holes+/-Sds

Concentration	STW?140	Compound 33	Compound 28
				0.05μM	0％+/-6	2％+/-2 ^*	2％+/-1
0.10μM	2％+/-6	10％+/-4	0％+/-2
				1.00μM	9％+/-9	8％+/-4	1％+/-4
5.00μM	18％+/-12	0％+/-7	6％+/-5

^*Only duplicate reading

The A2780 ovarian cancer cell; Three days research, cell number

STX 641

And STX 140

The reversibility of STX 641 and STX 140 drug-induced effects has been studied in expression respectively.For these research, removed medicine at the 3rd day, use the PBS washed cell, add substratum then.These studies show that: in most of the cases the effect of medicine is irreversible.

These data show in Fig. 8.

The PC3 prostate cancer cell; Three days research, cell number

STX 641

And STX 140

These data show in Fig. 9.

Clone in the A2780 ovarian cancer cell is taken place

These data show in Figure 10.

Suppress safe plain inductive microtubule polymerization

Medicine suppresses the ability of safe plain inductive tubulin polymerization by the turbidity measurement of 350nm place.Ultimate density be 1mg/ml tubulin (Cryoskeleton Inc., Denver, CO) in 32 ℃, at G-PEM damping fluid [80mM piperazine-N, N '-two (2-ethanesulfonic acid) sesquialter sodium (sequisodium) salt, 1mM MgCl ₂, 1mM EGTA and 1mM GTP (pH 6.8)] in, in the presence of safe plain, having or do not having an incubation under the situation of female ketone derivatives [20 μ M].

Fig. 3 shows the inhibition to the plain inductive tubulin polymerization of Thailand of STX641 and related compound.STX641 has been considered to anti-microtubule drug effect, promptly destroys the microtubule function.In order to show this effect, tubulin is incubation in the presence of safe plain (being called the compound that promotes tubulin polymerization).Safe plain external evoked effective microtubule assembling increases with the turbidity of 350nm place mensuration and to reflect.Except STX641, STX 505, STX564 or STX640 can block the tubulin assembling effectively to tubulin and safe plain reaction mixture, and the turbidity that detects seldom increases.These results show that STX641 and related compound suppress tubulin polymerization and this compounds may disturb microtubule kinetics (and therefore interrupting tumor growth) in vivo external.

Suppress glucose uptake

In order measuring glucose to be taken in cell inhibiting, cell to be placed 12 hole porous plates and grows to fusion.Cell is contained 1 μ Ci 2-deoxidation-D-[1-with phosphate buffered saline (PBS) (PBS) washing and in every hole ³H] glucose (NEN-Dupont, in incubation buffering liquid UK), potential inhibitor (0.1-10 μ M) lack or in the presence of incubation 15 minutes.By stop picked-up with cold (4 ℃) PBS washed cell.Be dissolved in cell among the Triton-X in 0.01M sodium hydroxide and carry out liquid scintillation counting(LSC) (Smgh etc., Molecular and Cellular Endocrinology, 160:61-66,2000)

Fig. 4 shows the inhibition of glucose uptake.The increase of glucose (as energy derive) picked-up is the characteristic feature of most of cancer cells.Glucose be conveyed into cell by many glucose transporter and in the malignant galactophore tissue a kind of this translocator (GLUT 1) by overexpression.Fig. 4 show STX641 (and related compound) suppress MCF-7 (estrogen receptor positive, ER+) and the glucose uptake of MDA-MB-231 (ER-) breast cancer cell.The inhibition of glucose uptake should suppress tumor growth in the body, and this can be STX641 and related compound is used to suppress the important mechanism that patient tumors is grown.

Effect to tubule formation

((Bucks UK) estimates medicine forms (with its angiogenesis inhibitor potentiality marker determination) to tubule effect to TCS-Cellworks Ltd with the vasculogenesis test kit.For this reason, Human umbilical vein endothelial cells (HUVECs) is cultivated with 24 orifice plates in human skin diploid fibroblast matrix.In entire test, cultured cells is at 37 ℃, 5%CO altogether ₂Incubation in the humidified incubator.At the 1st day, remove substratum and also replace with containing the substratum of studying Chinese traditional medicine.At the 4th, 7 and 9 day, replace substratum with containing the fresh culture of studying Chinese traditional medicine.At the 11st day, cell washed 30 minutes with fixed cell with the PBS and 70% ethanol (1ml) that join in each hole.After fixing, cell is with blocking-up damping fluid (1ml PBS+1% bovine serum albumin, Sigma, UK) washing and dye with the von WillebrandShi factor or CD31.The degree that tubule forms is by manual counting or quantitative by Computer Analysis.With Kodak DC120 digital camera capture image.In addition, the change details of drug-induced tubule formation also scans record by the high definition plate with some Photoshop processing graphical representation scanning.

If grow blood vessel network, the size of most of solid tumors only can increase 1-2mm, so they can obtain to support the basic nutrition (process that is called vasculogenesis) of its growth.Therefore the medicine of blocking this angiogenesis should be able to suppress the growth of solid tumor widely.

In this test, the common cultivation with HUVECs and skin flbroblast detects STX641 (and related compound) as the ability of angiogenesis inhibitor effect.In this system, endotheliocyte forms island at first in inoblast matrix.They are bred subsequently and enter migration phase, and they move by matrix and form the little tubular construction of wire in this stage.These also give birth to the network that forms closed tubule.Fig. 5 and 6 is for showing the representative diagram of STX641 to tubule formation effect.In Fig. 5, for the common cultivation of untreated (contrast) HUVECs and skin flbroblast, the wire character of widely little managed network is obvious visible.On the contrary, for the co-cultured cell of handling with STX641 (0.1 μ M), tubule is destroyed fully, shows that this medicine has effective angiogenesis inhibitor characteristic.In Fig. 6, plate is also also handled by the high-definition scanning record and is provided pixelation (pixalated) image.In addition, for the coculture of be untreated (contrast), the wire tubule obviously as seen, but the STX641 of 0.05 μ M (and STX140) has eliminated tubule formation fully.

The inhibition degree that tubule forms can be by Computer Analysis quantitative (Fig. 7).As shown in, STX641, the STX564 of 0.05 μ M and 0.1 μ M and STX639 suppress tubule fully and form, and have determined the angiogenesis inhibitor potentiality of these compounds.

Suppressing capillary blood vessel forms

In each situation, being calibrated to 1.0 pixels and image area is 3211216.VEGF is a vascular endothelial growth factor.

Show these data among Figure 11.

Cell cycle arrest and apoptosis

The MCF-7 breast cancer cell

40%MCF-7 and 500nM compound fusion colony are carried out fluidic cell metering cell cycle analysis.

Compare with contrast (16%-27%), significantly induce G2/M to stagnate at 8 hours STX 140, STX 640 and STX 641.

Compare with contrast (28%), STX 140 (28%) and STX 640 (18%), be exposed to STX64124 hour after, 68% cell is stuck in G2/M.The increase of this G2/M cell colony is accompanied by the minimizing (be respectively 37%-15%, reach 30%-9%) of G1-and S-stage colony.In the cell that STX 140 handles, observe the remarkable increase of Asia-G1 colony, this is indicator cells apoptosis (36%, compare with 2% in the control cells) normally.

After 48 hours, the cell that STX 641-handles still is stuck in G2/M, and apoptotic Asia-G1 colony does not have remarkable increase.Continue to show the increase of Asia-G1 cell colony with the cell of STX 140 processing.

The A2780 ovarian cancer cell

Figure 12 display density is the effect of 641 pairs of A2780 ovarian cancer cells of STX of 1 μ M, show 24 hours after STX 641 induce G2/M to stagnate (left hand edge graph).Right hand edge graph shows the FACs analytical results with annexin V antibody staining cell.For contrast, but lack the indicated cell surface expression in peak in the M2 zone.On the contrary, the cell that STX 641 handles shows that the dyeing of this apoptosis mark significantly increases, and as shown in the increase in M2 zone, shows this compound cell death inducing.

Xenotransplantation nude mouse model

MCF-7 mammary cancer nude mouse model

Give female ICRF naked (nu/nu) injected in mice 10 * 10 of growing up ⁶MCF-7 cell/0.1mlMatrigel s.c, and monitor tumor growth weekly.When gross tumor volume reaches 100-150mm ³, mouse is divided into following group:

A: contrast solvent 0.1ml one week of p.o 5 times, totally 3 weeks

B:STX one week of 140 20mg/kg p.o 5 times, totally 3 weeks

C:STX one week of 641 20mg/kg p.o 5 times, totally 3 weeks

Before putting to death 20 minutes, give mouse mainline 0.1ml 25mg/ml FITC-dextran solution in case tumor-blood-vessel growth show one's color and quantize.

The result of this research shows in following figure.

Figure 13 shows the effect that STX 140 and STX 641 (20mg/kg p.o) grow to MCF-7 breast cancer cell knurl in the ICRF nude mouse.

STX 140 causes 59% tumour to be dwindled, and dwindles and STX 641 produces 25% tumour during 3 all administrations.

Figure 14 represents fluorescence photo, shows STX 140 and STX 641 (20mg/kg p.o) effect to the tumor vascular system treatment.

Tumour FITC imaging shows control having the vascular system of abundant clear and definite structure, but in the animal that STX 140 and STX 641 handle, tumor vascular system is destroyed and clear and definite hardly.

Figure 15 shows STX-140 and STX641 (20mg/kg) effect to MCF-7 breast tumor vasculogenesis.

The FITC of tumor-blood-vessel growth shows that quantitatively STX 140 and STX 641 all produce significant the inhibition, and the inhibition of tumor-blood-vessel growth is respectively 40% and 60%.

Figure 16 shows that STX 140 and 641 (20mg/kg op) is to liver in the nude mouse and the active effect of tumour sulfatase.

Liver sulfatase activity is almost completely suppressed by STX 140 and STX 641.

Figure 17 shows the influence of STX 140 and 641 (20mg/kg op) 3 weeks of treatment to the nude mouse body weight.

During administration, in any treated animal, all do not observe and lose weight.

Carbonic anhydrase

Compound	CA2?IC ₅₀s
		Acetazolamide	25nM
STX?140	400nM
		STX?641	1μM
The 2-methoxyestradiol	Do not detect inhibition

The inhibition activity of CA2 and CA9 be associated (Vullo 2003, BioOrg.Med.Chem.).

All publications and the patent mentioned in the above-mentioned specification sheets are attached to herein by reference.

Various modifications and variations of the present invention will be conspicuous to those skilled in the art, not deviate from aim of the present invention and scope.Though the present invention is described in conjunction with concrete preferred embodiment, should understands claimed the present invention and should not be defined in these specific embodiments inadequately.Even, be conspicuous for the various modifications of described realization mode of the present invention to chemistry, biology or various equivalent modifications, and will fall in the scope of claims.

Claims

1. the compound that has following formula:

2. pharmaceutical composition, it comprises

A) the defined compound of claim 1 and

B) pharmaceutically acceptable carrier.

3. the defined compound of (i) claim 1 that is used for medicine, or the (ii) defined composition of claim 2.

4. the defined compound of (i) claim 1, or (ii) the defined composition of claim 2 in the preparation prevention and/or suppress purposes in the tumor growth medicine.

5. the defined compound of (i) claim 1, or (ii) the defined composition of claim 2 is used for the treatment of purposes in the medicine of illness relevant with one or more following situations or disease in preparation: the steoid sulfatase activity; Cell cycle; The cell growth; Tumour is to the picked-up of glucose; Tumor-blood-vessel growth; Microtubule forms; And apoptosis.

6. the defined compound of (i) claim 1, or (ii) the defined composition of claim 2 is used for suppressing the purposes of the active medicine of steoid sulfatase in preparation.

7. the defined compound of (i) claim 1, or (ii) the defined composition of claim 2 is used for regulating the purposes of the medicine of cell growth in preparation.