HK1197414B

HK1197414B - Estra-1,3,5(10),16-tetraene-3-carboxamide derivatives, process for preparation thereof, pharmaceutical preparations comprising them, and use thereof for production of medicaments

Info

Publication number: HK1197414B
Application number: HK14110909.3A
Authority: HK
Inventors: U．伯特; N.巴拉克; M．布泽曼; O．M．菲舍尔; A．罗特盖里; I．加邵; I.哈通; T.马夸特
Original assignee: 拜耳知识产权有限责任公司; 拜耳医药股份有限公司
Priority date: 2011-09-29
Filing date: 2012-09-24
Publication date: 2016-08-12

Description

Estra-1, 3,5(10), 16-tetraene-3-carboxamide derivatives, method for the production thereof, pharmaceutical preparations containing the same, and use thereof for producing medicaments

The present invention relates to AKR1C3 inhibitors and to a process for their preparation, to their use for the treatment and/or prevention of diseases and also to their use for the preparation of medicaments for the treatment and/or prevention of diseases, in particular of bleeding disorders and endometriosis.

AKR1C3 is a multifunctional enzyme and catalyzes, inter alia, the reduction of 4-androstene-3, 17-dione (weak androgen) to testosterone (strong androgen) and the reduction of estrone (weak estrogen) to 17 β -estradiol (strong estrogen). In addition, reduction of Prostaglandin (PG) H2 to PGF2 α and reduction of PGD2 to 9 α,11 β -PGF2 are inhibited (T.M. Penning et al, 2006,' Aldo-key reduction (AKR)1C3: Rolein protein diseases and molecular encapsulation of specific inhibition 248(1-2), 182-191).

The local formation of estradiol (E2) plays a major role in the development and progression of breast cancer disorders and endometriosis. The reduction of the tissue concentration of estrogen, in particular estradiol, is achieved by the therapeutic administration of aromatase inhibitors (for inhibiting the formation of estrogen from androgen) and sulfatase inhibitors (for blocking the formation of estrone from estrone sulfate). However, both treatments have the disadvantage of drastically reducing systemic estrogen concentrations (a.osteter et al, j.med.chem.2010,53, 8176-. Recently, experiments have demonstrated that the endometriosis foci are able to locally synthesize estradiol (b.delvoux et al, jclinencrinolometer 2009,94, 876-. For the ovarian endometriosis subtype, overexpression of AKR1C3mRNA has been described (T. Smuc et al, MolCellEndocrinol.2009Mar25;301(1-2): 59-64).

There is a great need to identify new inhibitors of the enzyme aldehyde-ketone reductase 1C3(AKR1C3) (synonyms: 17 β -hydroxysteroid dehydrogenase type 5 or prostaglandin F synthase) because of their potential to treat hormone-dependent disorders such as endometriosis as well as hormone-independent disorders (m.c. byrins, y.jin, t.m. penning, journal biochemical and molecular biology (2010); a.l. loveringet.al, cancer res64(5), 1802-. In addition to endometriosis, this includes Prostate cancer (K.M. Yang et al, EndocrRelatcancer13(1),169- "180"), prostatic hyperplasia (R.O.Roberts et al, Prostate66(4),392- "404"), endometrial cancer (T.L.Rizner et al, MolCellEndocrinol2006248(1-2),126- "135"), polycystic ovarian syndrome (K.Qin et al, JEndocrine Metab2006,91(1),270- "276-"), lung cancer (Q.Lan et al, Carcinogenesia 2004,25(11),2177- "2181"), non-Hodgkin lymphoma (Q.Lan et al, HumGenet2007, 662 (2),161- "168"), alopecia (L.Colomberbbe et al, Exmatrol 2007,16 (762), Integren et al, HumGenetJ 769, obesity Fangy, WaltGenetJ 5, Mortek et al, (Sshy Fa.) (III, ShinJ 5, ShinJ.), (III-13-), (Mullkuneh et al, (Sshy, St. J5, Sshy, No. 2009, No. 13, No. 2009, No. 11), multiple myeloma # 2181, No. 11, No., 147-.

Some inhibitors of AKR1C3 are known (see: JoannaMDay, HelenaJTutll, Atul purohitand Michael Reed, Endocrine-related cancer (2008)15,665-. Steroid substances which have been described are, for example, EM-1404, which are based on an estratriene skeleton having a spirolactone unit in position 17 (f. labrie et al, U.S. patent No. 6,541,463, 2003).

Other steroid substances with lactone units are described in P.Bydal, VanLuu-The, F.Labrie, D.Poirier, European journal of medicinal chemistry2009,44, 632-644. Fluorinated estratriene derivatives have been described in d.deluca, g.moller, a.rosinus, w.elger, a.hillisch, j.adamski, mol.cell.endocrinol.2006,248, 218-224.

The compounds of the invention are based on estra-1, 3,5(10), 16-tetraene skeletons substituted at the 17-position by an aromatic heterocycle and at the 3-position by an aminocarbonyl group. US5604213 by barrie et al describes 17- (3-pyridyl) estra-1, 3,5(10), 16-tetraen-3-ol derivatives as inhibitors of 17 alpha-hydroxylase/C17-20 lyase (Cyp17a 1). Specifically, the substance 17- (3-pyridyl) estra-1, 3,5(10), 16-tetraen-3-ol is reported. However, US5604213 does not describe any 17- (3-pyridyl) estra-1, 3,5(10), 16-tetraene derivative substituted at the 3-position with an aminocarbonyl group. The compounds of the invention claimed herein additionally have a carboxyl group as functional group, which gives further structural differences from the substances described in US 5604213. Surprisingly, it has now been found that the compounds of the invention claimed herein are potent inhibitors of AKR1C3 (see example 31) and at the same time inhibit Cyp17a1 (see example 32) only very weakly, if at all.

The substitution of aminocarbonyl (-CONH) in the 3-position is described in US2005/0203075₂) Radical-substituted estra-1, 3,5(10), 16-tetraene derivatives. However, these derivatives are not substituted by a heterocycle at the 17-position of the estra-1, 3,5(10), 16-tetraene skeleton.

Reviews of 17-pyridylalandrostane derivatives and 17-pyrimidylalandrostane derivatives described as Cyp17a1 inhibitors are found in v.m. moreira et al, currentmedicinialchemistry, 2008vol.15, No. 9.

It is an object of the present invention to provide alternative substances which are useful as AKR1C3 inhibitors. The novel AKR1C3 inhibitors claimed herein additionally show significantly improved solubility in water compared to the known AKR1C3 inhibitor EM-1404 (see example 33). Thus, it is easier to formulate the compounds of the present invention in an aqueous delivery medium.

The present invention provides compounds of formula (I), and salts, solvates and solvates of said salts thereof:

wherein

R1 and R2 independently of one another denote hydrogen, fluorine, chlorine, nitrile, trifluoromethyl, pentafluoroethyl, methoxy, ethoxy, trifluoromethoxy, -OCH₂CF₃、CH₃SO₂-、CH₃CH₂SO₂-、-(C=O)CH₃Carboxyl group, C₁-C₄-alkyl, hydroxy, -CH₂OH、-C(CH₃)₂OH、-CONH₂- (C = O) NH-alkyl, - (C = O) N (CH)₃)₂、-SO₂NH₂、-SO₂NHCH₃、-SO₂N(CH₃)₂Or the C-H group in the pyridine ring is replaced by a nitrogen atom, and

r3 and R4 represent hydrogen, or

R3 represents hydroxy, fluoro, methoxy or ethoxy and R4 represents hydrogen, or

R3 represents hydrogen and R4 represents hydroxy, fluoro, methoxy or ethoxy, and

r5 and R6 represent hydrogen, or

R5 represents fluorine, hydroxy, methoxy or ethoxy and R6 represents hydrogen, or

R5 represents hydrogen and R6 represents fluorine, and

r7 represents hydrogen or C₁-C₄Alkyl radical, C₃-C₆-cycloalkyl, cyclopropylmethyl, trifluoromethyl or 2,2, 2-trifluoroethyl, and

r8 represents-CR^aR^b-COOH, wherein

R^aAnd R^bIndependently of one another, represent hydrogen, methyl, ethyl, or

R^aAnd R^bTogether represent- (CH)₂)_n-, wherein n =2, 3,4 or 5, wherein said CH₂Up to 4 hydrogen atoms of the radical may be replaced by fluorine atoms, or

R^aAnd R^bTogether represent-CH₂-O-CH₂-、-CH₂-NH-CH₂-、-CH₂-N(CH₃)-CH₂-、-CH₂CH₂-O-CH₂CH₂-、-CH₂CH₂-NH-CH₂CH₂-、-CH₂CH₂-N(CH₃)-CH₂CH₂-, or

R^aRepresents hydrogen, methyl or ethyl, and R^bTogether with R7 for- (CH)₂)_n-, where n =1, 2,3, 4, where said CH₂A single hydrogen atom or up to 4 hydrogen atoms of the group may be replaced by fluorine atoms, or

R^aTogether with R7 representing-CH₂-O-CH₂CH₂-、-CH₂-N(CH₃)-CH₂CH₂-, and R^bRepresents hydrogen, methyl or ethyl, or

R8 represents-CR^cR^d-CR^eR^f-COOH, wherein

R^c、R^d、R^e、R^fRepresents hydrogen, or

R^c、R^dIndependently of one another, methyl, ethyl or together- (CH)₂)_n-, where n =2, 3,4,5, or together represent-CH₂CH₂-O-CH₂CH₂-, and R^e、R^fRepresents hydrogen, or

R^c、R^dRepresents hydrogen, and R^e、R^fIndependently of one another, methyl, ethyl or together- (CH)₂)_n-, where n =2, 3,4,5, or together represent-CH₂CH₂-O-CH₂CH₂-、-CH₂CH₂-NH-CH₂CH₂-、-CH₂CH₂-N(CH₃)-CH₂CH₂-or-CH₂-O-CH₂-, or

R^cRepresents methyl, ethyl, trifluoromethyl, and R^d、R^eAnd R^fRepresents hydrogen, or

R^c、R^dAnd R^fRepresents hydrogen, and R^eRepresents methyl, ethyl, trifluoromethyl, hydroxy or methoxy, or

R^cAnd R^eTogether represent- (CH)₂)_n-, where n =1, 2,3 or 4, and R^dAnd R^fRepresents hydrogen, or

R8 represents-CH₂-CH₂-CHR^g-COOH, wherein

R^gRepresents hydrogen, or

R^gAnd R7 together represent-CH₂-or-CH₂CH₂-。

The invention also provides compounds of formula (II) and formula (III), and salts, solvates and solvates of said salts:

wherein

R1 represents hydrogen, fluorine, chlorine, nitrile, methoxy, ethoxy, trifluoromethoxy, methyl, ethyl, trifluoromethyl, - (C = O) CH₃And an

R3 and R4 represent hydrogen, or

R3 represents hydroxy and R4 represents hydrogen, or

R3 represents hydrogen and R4 represents hydroxy, and

r5 represents hydrogen or fluorine, and

r7 represents hydrogen or C₁-C₄-alkyl, and

r8 represents-CR^aR^b-COOH, wherein

R^aAnd R^bIndependently of one another, represent hydrogen, methyl or ethyl, or

R^aAnd R^bTogether represent- (CH)₂)_n-, where n =2, 3,4 or 5, or

R^aRepresents hydrogen, and R^bTogether with R7 for- (CH)₂)_n-, where n =3 or 4, or

R8 represents-CR^cR^d-CR^eR^f-COOH, wherein

R^c、R^d、R^e、R^fRepresents hydrogen, or

R^c、R^dRepresents hydrogen, and R^e、R^fIndependently of one another, methyl, ethyl or together- (CH)₂)_n-, where n =2, 3,4,5, or together represent-CH₂CH₂-O-CH₂CH₂-, or

R^cRepresents methyl or ethyl, and R^d、R^eAnd R^fRepresents hydrogen, or

R8 represents-CH₂-CH₂-CHR^g-COOH, wherein

R^gRepresents hydrogen, or

R^gAnd R7 together represent-CH₂CH₂-。

The invention also provides compounds of formula (II) and formula (III), and salts, solvates and solvates of said salts, wherein

R1 represents hydrogen, fluorine, chlorine, nitrile, methoxy, trifluoromethyl, and

r3 and R4 represent hydrogen, or

R3 represents hydroxy and R4 represents hydrogen, or

R3 represents hydrogen and R4 represents hydroxy, and

r5 represents hydrogen or fluorine, and

r7 represents hydrogen, methyl or ethyl, and

r8 represents-CR^aR^b-COOH, wherein

R^aAnd R^bIndependently of one another, represent hydrogen, methyl or ethyl, or

R8 represents-CR^cR^d-CR^eR^f-COOH, wherein

R^c、R^d、R^e、R^fRepresents hydrogen, or

R^c、R^dRepresents hydrogen, and R^e、R^fIndependently of one another, methyl or ethyl, or together- (CH)₂)_n-, where n =2, 4,5, or together represent-CH₂CH₂-O-CH₂CH₂-, or

R^cRepresents a methyl group, and R^d、R^eAnd R^fRepresents hydrogen, or

R^cAnd R^eTogether represent- (CH)₂)_n-, where n =3 or 4, and R^dAnd R^fRepresents hydrogen, or

R8 represents-CH₂-CH₂-CHR^g-COOH, wherein

R^gRepresents hydrogen, or

R^gAnd R7 together represent-CH₂CH₂-。

Furthermore, the present invention provides compounds of formula (II) and formula (III), and salts, solvates and solvates of said salts, wherein

R1 represents hydrogen, fluorine, methoxy, trifluoromethyl, and

r3 and R4 represent hydrogen, or

R3 represents hydroxy and R4 represents hydrogen, or

R3 represents hydrogen and R4 represents hydroxy, and

r5 represents hydrogen or fluorine, and

r7 represents hydrogen or methyl, and

r8 represents-CR^aR^b-COOH, wherein

R^aAnd R^bIndependently of one another, represents hydrogen or methyl, or

R^aRepresents hydrogen, and R^bTogether with R7 for- (CH)₂)₃-, or

R8 represents-CR^cR^d-CR^eR^f-COOH, wherein

R^c、R^d、R^e、R^fRepresents hydrogen, or

R^cAnd R^dRepresents hydrogen, and R^eAnd R^fRepresents methyl or together represents- (CH)₂)_n-, where n =2 or 4, or together represent-CH₂CH₂-O-CH₂CH₂-, or

R^cRepresents a methyl group, and R^d、R^eAnd R^fRepresents hydrogen, or

R^cAnd R^eTogether represent- (CH)₂)₃-, and R^dAnd R^fRepresents hydrogen, or

R8 represents-CH₂-CH₂-CHR^g-COOH, wherein

R^gRepresents hydrogen, or

R^gAnd R7 represents-CH₂CH₂-。

Furthermore, the present invention provides the following compounds, and salts, solvates and solvates of said salts:

4- [ ({ [17- (5-fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } amino) methyl ] -3,4,5, 6-tetrahydro-2H-pyran-4-carboxylic acid

N- { [17- (5-Fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } -N-methyl-beta-alanine

1- [ ({ [17- (5-fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } amino) methyl ] cyclopropane-1-carboxylic acid

1- [ ({ [17- (5-fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } amino) methyl ] cyclopentane-1-carboxylic acid

3- ({ [17- (5-Fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } amino) -2, 2-dimethylpropanoic acid

1- { [17- (5-fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } piperidine-4-carboxylic acid

N- { [17- (5-Fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } -2-methylalanine

4- ({ [17- (5-Fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } amino) butanoic acid

N- { [17- (5-Fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } -beta-alanine

N- { [17- (5-Fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } glycine

(1R, 2S) -2- ({ [17- (5-fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } amino) cyclopentane-1-carboxylic acid

(S) -3- ({ [17- (5-Fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } amino) butanoic acid

(R) -3- ({ [17- (5-Fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } amino) butanoic acid

3- ({ [17- (5-methoxypyridin-3-yl) estra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } amino) -2, 2-dimethylpropionic acid

N- { [17- (5-methoxypyridin-3-yl) estra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } -beta-alanine

N- { [17- (5-methoxypyridin-3-yl) estra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } -N-methyl-beta-alanine

N- { [17- (pyrimidin-5-yl) estra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } -beta-alanine

4- ({ [17- (pyrimidin-5-yl) estra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } amino) butanoic acid

N-methyl-N- { [17- (pyrimidin-5-yl) estra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } -beta-alanine

2, 2-dimethyl-3- ({ [17- (pyrimidin-5-yl) estra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } amino) propanoic acid

N- ({17- [5- (trifluoromethyl) pyridin-3-yl ] estra-1, 3,5(10), 16-tetraen-3-yl } carbonyl) -beta-alanine

N-methyl-N- ({17- [5- (trifluoromethyl) pyridin-3-yl ] estra-1, 3,5(10), 16-tetraen-3-yl } carbonyl) -beta-alanine

N- { [17- (5-Fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } -L-proline

N- { [17- (5-Fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } -D-proline

4- ({ [11 β -fluoro-17- (5-fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } amino) butanoic acid

N- { [17- (5-Fluoropyridin-3-yl) -15 α -hydroxyestra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } -N-methyl- β -alanine

N- { [17- (5-Fluoropyridin-3-yl) -15 β -hydroxyestra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } -N-methyl- β -alanine

N-methyl-N- { [17- (6-methylpyridazin-4-yl) estra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } -beta-alanine

N-methyl-N- { [17- (3-pyridin-3-yl) estra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } -beta-alanine

4- ({ [17- (5-methoxypyridin-3-yl) estra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } amino) butanoic acid.

Estra-1, 3,5(10), 16-tetraene-3-carbonylamino derivatives provided by the present invention have been found to act as AKR1C3 inhibitors. For most of the claimed structural range, these substances show strong Inhibition (IC) of AKR1C3 in vitro₅₀Values less than 50nM), and even most exhibit IC₅₀Value of<20 nM. Furthermore, these derivatives only show very low inhibition of Cyp17a1 (if any).

The compounds of the present invention are compounds of formula (I) and salts, solvates and solvates of said salts, compounds of the formulae mentioned below and salts, solvates and solvates of said salts encompassed by formula (I), and compounds of the formulae mentioned below and encompassed by formula (I) and salts, solvates and solvates of said salts, mentioned below as examples, with the proviso that the compounds mentioned below and encompassed by formula (I) are not salts, solvates and solvates of said salts.

Depending on their structure, the compounds of the invention may exist in stereoisomeric forms (diastereomers). In the compounds of formula (I), a stereogenic centre may be present in the group R8 (and, if R7 and R8 together form a ring, also in this ring). Accordingly, the present invention includes diastereomers and their corresponding mixtures. From such mixtures of diastereomers, stereoisomerically homogeneous components can be separated in a known manner.

If the compounds of the invention can exist in tautomeric forms, the invention includes all tautomeric forms.

In the context of the present invention, preferred salts are physiologically acceptable salts of the compounds of the invention. However, the present invention also includes salts which are not suitable for pharmaceutical use per se, but which can be used, for example, for isolating or purifying the compounds of the invention.

Physiologically acceptable salts of the compounds of the invention include acid addition salts of inorganic acids, carboxylic acids and sulfonic acids, for example the following: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, acetic acid, formic acid, trifluoroacetic acid, propionic acid, lactic acid, tartaric acid, malic acid, citric acid, fumaric acid, maleic acid, and benzoic acid.

Physiologically acceptable salts of the compounds of the invention also include salts of customary bases, such as, for example and preferably, alkali metal salts (e.g. sodium and potassium salts), alkaline earth metal salts (e.g. calcium and magnesium salts) and ammonium salts derived from ammonia or organic amines having from 1 to 16 carbon atoms, such as, for example and preferably, ethylamine, diethylamine, triethylamine, ethyldiisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanol, procaine, dibenzylamine, N-methylmorpholine, arginine, lysine, ethylenediamine and N-methylpiperidine.

In the context of the present invention, it is,solvatesIs a form of the compound of the invention which is in a solid or liquid state and forms a complex by coordination with solvent molecules. Hydrates are a particular form of solvates in which the coordination is with water. In the context of the present invention, the preferred solvate is a hydrate.

In addition, the present invention also includes prodrugs of the compounds of the present invention. The term "prodrug" includes compounds that may themselves be biologically active or inert, but which convert (e.g., metabolically or hydrolytically) to the compounds of the invention during the time they spend in the body.

In the context of the present invention, unless indicated otherwise, the substituents have the following meanings:

C ₁ -C ₄ -alkyl radicalRepresents a linear or branched alkyl group having 1 to 4 carbon atoms, such as and preferably methyl, ethyl, propyl, butyl, isopropyl, tert-butyl, isobutyl.

C ₃ -C ₆ -cycloalkyl radicalRepresents cycloalkyl having 3 to 6 carbon atoms, wherein the ring may also be partially unsaturated, such as and preferably cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

Furthermore, the present invention provides a process for the preparation of the compounds of formula (I) according to the invention. The preparation of compound (I) of the present invention can be exemplified by the following synthetic route:

some of the compounds of the invention can be prepared starting from 17-oxoestra-1, 3,5(10) -triene-3-carboxylic acid methyl ester, which is known from the literature (Steroids,1995,60,3, 299-propanoic acid 306) (scheme 1):

by conversion to intermediate 1 using trifluoromethanesulfonic anhydride or N, N-bis (trifluoromethanesulfonyl) aniline in the presence of a base such as pyridine, 2, 6-lutidine or 2, 6-di-tert-butylpyridine or in the presence of a tertiary amine such as triethylamine or diisopropylethylamine, or by conversion to intermediate 1 using an alkali metal hexamethylsilazane or Lithium Diisopropylamide (LDA) (J.Med.Chem.,1995,38, 2463-2471, J.Org.Chem.,1985,50, 1990-1992, JACS,2009,131, 9014-9019, Archi v derrmazie (Weinheim, Germany),2001,334,12, 373-374). Preference is given to reacting with trifluoromethanesulfonic anhydride in methylene chloride in the presence of 2, 6-di-tert-butylpyridine.

Intermediate 2 was prepared using a Suzuki reaction known to those skilled in the art. To this end, intermediate 1 is reacted with a nitrogen-containing aromatic boronic acid (boronicacid), a boronic ester such as boronic pinacol ester, MIDA boronic ester (d.m. knapp et al, j.am. chem. soc.2009,131,6961), orReacted with trifluoroborate (g.a. molander et al, j.org.chem.2009,74,973). Suitable catalysts are a number of palladium-containing catalysts, for example tetrakis (triphenylphosphine) palladium (0), bis (triphenylphosphine) palladium (II) dichloride or [1, 3-bis (2, 6-diisopropylphenyl) imidazol-2-ylidene](3-Chloropyridyl) palladium (II) dichloride (CAS 905459-27-0). Alternatively, a palladium-containing source such as palladium (II) acetate, palladium (II) chloride or Pd (dba)₂In combination with phosphorus-containing ligands such as triphenylphosphine, SPhos (d.m.knapp et al, j.am.chem.soc.2009,131,6961) or RuPhos (g.a.molander, j.org.chem.2009,74,973). Preferably, the boronic acid is reacted with tetrakis (triphenylphosphine) palladium (0) or [1, 3-bis (2, 6-diisopropylphenyl) imidazol-2-ylidene](3-chloropyridyl) palladium (II) dichloride.

Intermediate 3 is prepared by hydrolysis of the methyl ester according to methods known to those skilled in the art. For this purpose, intermediate 2 is mixed with aqueous sodium hydroxide solution or aqueous lithium hydroxide solution in a solvent such as Tetrahydrofuran (THF), methanol or dimethyl sulfoxide (DMSO) or in a mixture of methanol and THF. If desired, the mixture is heated. The reaction in THF and methanol at 40 ℃ in the presence of aqueous sodium hydroxide solution or aqueous lithium hydroxide solution is preferred.

Starting from intermediate 3, the preparation of the exemplary compounds proceeds in two steps by amide coupling with an ester of an amino acid and by subsequent conversion of the carboxylic ester to a carboxylic acid. Suitable for the amide coupling (step A) are reagents known to the person skilled in the art, such as N, N '-Dicyclohexylcarbodiimide (DCC), N- [3- (dimethylamino) propyl ] -N' -ethylcarbodiimide hydrochloride (EDC) [ CAS25952-53-8] or HATU (O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate). In addition, reagents such as 1H-benzotriazol-1-ol hydrate (HOBt hydrate [ CAS123333-53-9]) or 4-Dimethylaminopyridine (DMAP) may also be employed as additives. Suitable as base are, for example, pyridine, triethylamine or diisopropylethylamine. Preference is given to the reaction using EDC, HOBt hydrate and triethylamine. For the conversion of the carboxylic ester to the carboxylic acid (step B), if the ester is, for example, a methyl, ethyl or benzyl ester, the hydrolysis method described for the preparation of intermediate 3 can be used. If the ester is a tert-butyl carboxylate, it can be converted into the carboxylic acid by methods known to the person skilled in the art, for example by reaction with trifluoroacetic acid in dichloromethane or chloroform or by reaction with hydrogen chloride in1, 4-dioxane. The reaction with trifluoroacetic acid in dichloromethane is preferred.

A subset of compounds of formula (I) of the present invention wherein R5= F and R6= H can be prepared as described in scheme 2:

3,11 α -dihydroxyestra-1, 3,5(10) -trien-17-one is converted to intermediate 4 using acetic anhydride and pyridine in the presence of 4-Dimethylaminopyridine (DMAP) in dichloromethane. The conversion to intermediate 5 was carried out in methanol using sodium bicarbonate. Reaction of intermediate 5 with 1,1,2,2,3,3,4,4, 4-nonafluorobutane-1-sulfonyl fluoride and potassium carbonate yielded intermediate 6, which was converted to intermediate 7 under a carbon monoxide atmosphere in an autoclave, in methanol and DMSO together with palladium (II) acetate, 1, 3-bis (diphenylphosphino) propane, triethylamine. The conversion to intermediate 8 was carried out using the method described for the preparation of intermediate 1. The conversion of intermediate 8 to intermediate 9 was carried out using the method described for the preparation of intermediate 2. Intermediate 9 was hydrolyzed using potassium carbonate and methanol to yield intermediate 10. The conversion to intermediate 11 was carried out in THF using 1, 8-diazabicyclo [5.4.0] undec-7-ene and 1,1,2,2,3,3,4,4, 4-nonafluorobutane-1-sulfonyl fluoride. Hydrolysis of intermediate 11 was carried out using the conditions described for the preparation of intermediate 3 to give intermediate 12. Preference is given to the reaction in THF and methanol in the presence of aqueous lithium hydroxide solution. The preparation of a subset of exemplary compounds starting from intermediate 12 is performed similarly to the preparation of exemplary compounds starting from intermediate 3 as described in scheme 1.

A subset of compounds of formula (I) of the present invention with substituents defined as R3= OH and R4= H or R3= H and R4= OH can be prepared as exemplified in scheme 3. The reaction is carried out using microorganisms, for example certain suitable fungal strains which allow regioselective or stereoselective hydroxylation. In this way, hydroxyl groups can be introduced, for example, in a regioselective and stereoselective manner into the 15-position of the steroid skeleton. The resulting 15-OH derivatives are exemplary compounds for the purposes of the present invention and may be further modified in subsequent chemical reactions.

In an unforeseeable manner, the compounds of the present invention exhibit a useful spectrum of pharmacological activity and advantageous pharmacokinetic properties. They are therefore suitable as medicaments for the treatment and/or prophylaxis of diseases in humans and animals. For the purposes of the present invention, the term "treatment" includes prophylaxis. The pharmaceutical efficacy of the compounds of the invention can be explained by their role as AKR1C3 inhibitors. Accordingly, the compounds of the present invention are particularly suitable for the treatment and/or prevention of endometriosis, uterine leiomyomas, uterine bleeding disorders, dysmenorrhea, prostate cancer, prostatic hyperplasia, acne, seborrhea, alopecia, premature maturation, polycystic ovary syndrome, breast cancer, lung cancer, endometrial cancer, renal cell carcinoma, bladder cancer, non-hodgkin's lymphoma, Chronic Obstructive Pulmonary Disease (COPD), obesity or inflammatory pain.

Furthermore, the present invention provides the use of a compound of the invention for the preparation of a medicament for the treatment and/or prevention of disorders, in particular of the disorders mentioned above.

Furthermore, the present invention provides methods for the treatment and/or prophylaxis of disorders, in particular of the disorders mentioned above, using an effective amount of a compound of the invention.

Furthermore, the present invention provides the use of the compounds according to the invention for the treatment and/or prophylaxis of disorders, in particular of the disorders mentioned above.

Furthermore, the present invention provides a compound of the invention for use in a method for the treatment and/or prevention of the above-mentioned disorders.

Furthermore, the present invention provides medicaments comprising at least one compound according to the invention and at least one or more further active compounds, in particular for the treatment and/or prophylaxis of the abovementioned conditions. The following active compounds suitable for combination may be mentioned by way of illustration and preference: selective Estrogen Receptor Modulators (SERMs), Estrogen Receptor (ER) antagonists, aromatase inhibitors, 17 β HSD1 inhibitors, steroid sulphatase (STS) inhibitors, GnRH agonists and antagonists, kisspeptin receptor (KISSR) antagonists, Selective Androgen Receptor Modulators (SARMs), androgens, 5 α -reductase inhibitors, Selective Progesterone Receptor Modulators (SPRMs), progestogens, antiprogestagens (antiprogestagens), oral contraceptives, inhibitors of mitogen-activated protein (MAP) kinase and MAP kinase (Mkk3/6, Mek1/2, Erk1/2) inhibitors, protein kinase B (PKB α/β/γ; Akt1/2/3), inhibitors of phosphoinositide 3-kinase (PI3K), inhibitors of cyclin dependent kinases (1/2), inhibitors of CDK dependent kinases (CDK1/2), Inhibitors of hypoxia-induced signal transduction pathways (HIF1 a inhibitors, activators of prolyl hydroxylase), Histone Deacetylase (HDAC) inhibitors, prostaglandin F receptor (FP) (PTGFR) antagonists, and non-steroidal anti-inflammatory drugs (NSAIDs).

The invention also relates to pharmaceutical preparations containing at least one compound of general formula I (or a physiologically acceptable addition salt thereof with an organic or inorganic acid) and to the use of these compounds for producing medicaments, in particular for the above-mentioned indications.

The compounds may be used for the above indications after oral administration and after parenteral administration.

The compounds of the invention may have systemic and/or local effects. For this purpose, they can be administered in a suitable manner, for example orally, parenterally, pulmonarily, nasally, sublingually, buccally, rectally, dermally, transdermally, conjunctivally, otically or as an implant or stent.

For these routes of administration, the compounds of the present invention may be administered in a suitable dosage form.

Suitable for oral administration are dosage forms comprising the compounds of the invention in crystalline and/or amorphous (amorphized) and/or dissolved form which function according to the prior art and rapidly release and/or modulate the release of the compounds of the invention, for example tablets (uncoated or coated tablets, e.g. enteric coatings or coatings with a delayed dissolution or insoluble coatings which control the release of the compounds of the invention), tablets which disintegrate rapidly in the oral cavity or films/wafers (wafers), films/lyophilisates, capsules (e.g. hard or soft gelatine capsules), sugar-coated tablets, granules, pellets, powders, emulsions, suspensions, aerosols or solutions.

Parenteral administration can be carried out, avoiding absorption steps (e.g., intravenous, intra-arterial, intracardiac, intraspinal, or lumbar administration) or involving absorption (e.g., intramuscular, subcutaneous, intradermal, transdermal, or intraperitoneal administration). Formulations suitable for parenteral administration are, in particular, solutions, suspensions, emulsions, lyophilized products or injectable and infusible preparations in the form of sterile powders.

Suitable dosage forms for other routes of administration are, for example, pharmaceutical forms for inhalation (in particular powder inhalers, nebulizers), nasal drops, solutions and sprays; tablets for lingual, sublingual or buccal administration, films/wafers or capsules, suppositories, otic or ocular preparations, vaginal capsules, aqueous suspensions (lotions, shakes), lipophilic suspensions, ointments, creams, transdermal therapeutic systems (e.g. patches), milk preparations (milk), pastes, foams, dusting powders, implants, intrauterine systems, vaginal rings or stents.

The compounds of the invention may be converted into the dosage forms described. This can be done in a manner known in the art by mixing with inert, non-toxic, pharmaceutically suitable adjuvants. These include carriers (e.g. microcrystalline cellulose, lactose, mannitol), solvents (e.g. liquid polyethylene glycols), emulsifiers and dispersing or wetting agents (e.g. sodium lauryl sulphate, polyoxysorbitan oleate), binders (e.g. polyvinylpyrrolidone), synthetic and natural polymers (e.g. albumin), stabilisers (e.g. antioxidants such as ascorbic acid), colourants (e.g. inorganic pigments such as iron oxide) and taste and/or odour correctors, among others.

The invention also relates to pharmaceutical products comprising at least one compound according to the invention, usually together with one or more inert, non-toxic, pharmaceutically suitable adjuvants, and to the use thereof for the above-mentioned purposes.

In the case of oral administration, the daily amount is about 0.01-100mg/kg body weight. The amount of the compound of formula I to be administered varies within a wide range and may cover each effective amount. The amount of compound administered may be from 0.01 to 100mg/kg body weight/day, depending on the condition to be treated and the method of administration.

Optionally, however, deviations from the stated amounts may be required, depending primarily on body weight, route of administration, individual response to the active substance, type of formulation, and the point or interval at which administration is carried out. Thus, in some cases it may be sufficient to use less than the minimum amount mentioned above, while in other cases the upper limit mentioned must be exceeded. In the case of relatively large amounts of administration, it may be advisable to divide them into a plurality of individual doses over the entire day.

Unless otherwise specified, percentages in the following tests and examples are percentages by weight; parts are parts by weight. In each case, the solvent ratio, dilution ratio and information relating to the concentration of the liquid/liquid solution refer to the volume.

List of abbreviations (chemistry)

Abbreviations and acronyms

DMAP	4-dimethylaminopyridine
		DMF	Dimethyl formamide
DMSO	Dimethyl sulfoxide
		h	Hour(s)
HPLC	High pressure high performance liquid chromatography
		LC-MS	Liquid chromatography-mass spectrometry
ES-MS	Electrospray mass spectrometry
		min	Minute (min)
MS	Mass spectrometry
		NMR	Nuclear magnetic resonance spectrum
RT	At room temperature
		TFA	Trifluoroacetic acid
THF	Tetrahydrofuran (THF)

Purification of the Compounds of the invention

In some cases, the compounds of the invention can be purified by preparative HPLC, for example using an automated purifier apparatus from Waters (detecting compounds by UV detection and electrospray ionization) in combination with a commercially available prepackaged HPLC column (e.g. XBridge column (from Waters), C18,5 μm,30 x 100 mm). The solvent system used was acetonitrile/water with addition of formic acid. Other additives known to those skilled in the art may be used, such as ammonia, ammonium acetate or trifluoroacetic acid. Instead of acetonitrile, it is also possible to use, for example, methanol.

In some cases, preparative HPLC separations were performed using the following methods:

lyophilization or vacuum centrifugation was used to remove the HPLC solvent mixture. If the resulting compounds are present as TFA salts or formates, they can be converted to the corresponding free bases by standard laboratory procedures known in the art.

In some cases, the compounds of the invention may be purified by silica gel chromatography. For this purpose, for example, a prepackaged silica gel column (e.g., from Separtis,flashchicagel) with a flashmaster ii chromatograph (Argonaut/Biotage) and a chromatographic solvent or solvent mixture (e.g., hexane, ethyl acetate and dichloromethane and methanol).

Structural analysis of the Compound of the present invention

In some cases, compounds of the invention were analyzed by LC-MS:

in some cases, the following analytical methods were used:

the instrument comprises the following steps: WatersAcquisyteUPLC-MSSQD 3001; column: AcquisytUPLCBEHC181.750X 2.1 mm; mobile phase A: water +0.1 vol% formic acid, mobile phase B: acetonitrile; gradient: 1-99% B at 0-1.6min, 99% B at 1.6-2.0 min; the flow rate is 0.8ml/min; temperature: 60 ℃; sample introduction: 2 mu l of the solution; DAD scan: 210-400nm

The following symbols are used for NMR data of the compounds of the invention:

s	single peak
		d	Doublet peak
t	Triplet peak
		q	Quartet peak
quin	Quintuple peak
		m	Multiple peaks
br	Broad peak
		mc	Central multiplet

Synthesis of Compounds of the invention

Intermediate 1

17- { [ (trifluoromethyl) sulfonyl ] oxy } estra-1, 3,5(10), 16-tetraene-3-carboxylic acid methyl ester

To a mixture of 5.00g (16.0mmol) of methyl 17-oxoestra-1, 3,5(10) -triene-3-carboxylate (Steroids,1995,60,3, 299-propan 306) in 100ml of dichloromethane and 5.3ml of 2, 6-di-tert-butylpyridine, 3.2ml of trifluoromethanesulfonic anhydride were added dropwise and the mixture was stirred at RT for 20 h. The mixture was carefully poured into 250ml of saturated aqueous sodium bicarbonate solution and stirred for 40min, the phases were separated, the aqueous phase was extracted twice with dichloromethane and the combined organic phases were washed with saturated sodium bicarbonate solution and sodium chloride solution, dried over sodium sulfate and concentrated. Trituration with hexane yielded 4.55g of the title compound as a solid.

¹H-NMR (300MHz, chloroform-d) [ ppm ]]=1.01(s,3H),1.37-1.74(m,5H),1.81(td,1H),1.88-2.02(m,2H),2.05-2.19(m,1H),2.27-2.55(m,3H),2.83-3.11(m,2H),3.90(s,3H),5.63(dd,1H),7.32(d,1H),7.68-7.90(m,2H)。

Intermediate 2-a

17- (5-Fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid methyl ester

8.00g (2.25mmol) of 17- { [ (trifluoromethyl) sulfonyl ] oxy } estra-1, 3,5(10), 16-tetraene-3-carboxylic acid methyl ester and 3.55g (1.4 equiv.) of 5-fluoropyridine-3-boronic acid are initially taken in 60ml of toluene and 40ml of ethanol. Then, 1.53g (2.0 equivalents) of lithium chloride, 24ml of 2M aqueous sodium carbonate solution and 1.04g (5mol%) of tetrakis (triphenylphosphine) palladium (0) were added and the mixture was heated at 100 ℃ for 3.5 h. Water was added, the mixture was extracted three times with ethyl acetate, and the extract was washed with saturated aqueous sodium bicarbonate solution and aqueous sodium chloride solution and concentrated. Purification by column chromatography on silica gel (hexane/ethyl acetate) gave 5.5g (78% of theory) of the title compound.

¹H-NMR (400MHz, chloroform-d) [ ppm ]]=1.06(s,3H),1.47-1.63(m,1H),1.63-1.78(m,3H),1.84(td,1H),1.98-2.06(m,1H),2.13-2.26(m,2H),2.35-2.51(m,3H),2.98(dd,2H),3.90(s,3H),6.10(dd,1H),7.32-7.44(m,2H),7.76-7.86(m,2H),8.36(br.s.,1H),8.48(s,1H)。

Intermediate 2-b

17- (5-methoxypyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid methyl ester

In analogy to the preparation of intermediate 2-a, 2.00g (4.50mmol) of intermediate 1 are reacted with 0.96g (1.4 eq) of (5-methoxypyridin-3-yl) boronic acid in the presence of 260mg of tetrakis (triphenylphosphine) palladium (0) overnight at 100 ℃ to yield 1.4g (76% of theory) of the title compound.

¹H-NMR (300MHz, chloroform-d) [ ppm ]]=1.05(s,3H),1.43-1.60(m,1H),1.62-1.89(m,4H),1.95-2.08(m,1H),2.10-2.25(m,2H),2.30-2.53(m,3H),2.98(dd,2H),3.88(s,3H),3.90(s,3H),6.00-6.08(m,1H),7.16-7.22(m,1H),7.35(d,1H),7.75-7.83(m,2H),8.20(d,1H),8.28(d,1H)。

Intermediate 2-c

17- (pyrimidin-5-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid methyl ester

In analogy to the preparation of intermediate 2-a, 3.00g (6.75mmol) of intermediate 1 are reacted with 1.17g (1.4 equivalents) of pyrimidin-5-ylboronic acid in the presence of 390mg of tetrakis (triphenylphosphine) palladium (0) overnight at 100 ℃ to yield 1.70g (64% of theory) of the title compound.

¹H-NMR (400MHz, chloroform-d) [ ppm ]]=1.06(s,3H),1.47-1.59(m,1H),1.65-1.80(m,3H),1.85(td,1H),1.98-2.06(m,1H),2.12-2.25(m,2H),2.36-2.53(m,3H),2.98(dd,2H),3.90(s,3H),6.14(dd,1H),7.35(d,1H),7.76-7.85(m,2H),8.76(s,2H),9.09(s,1H)。

Intermediate 2-d

17- [5- (trifluoromethyl) pyridin-3-yl ] estra-1, 3,5(10), 16-tetraene-3-carboxylic acid methyl ester

In analogy to the preparation of intermediate 2-a, 1.66g (3.75mmol) of intermediate 1 are reacted with 1.00g (1.4 equivalents) of [5- (trifluoromethyl) pyridin-3-yl ] boronic acid in the presence of 216mg of tetrakis (triphenylphosphine) palladium (0) overnight at 100 ℃ to yield 1.20g (73% of theory) of the title compound.

¹H-NMR (300MHz, chloroform-d) [ ppm ]]=1.08(s,3H),1.49-1.89(m,6H),1.97-2.09(m,1H),2.09-2.28(m,2H),2.35-2.54(m,3H),2.98(dd,2H),3.90(s,3H),6.15(dd,1H),7.36(s,1H),7.77-7.85(m,2H),7.88(s,1H),8.83(s,2H)。

Intermediate 2-e

17- (6-Methylpyridazin-4-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid methyl ester

In analogy to the preparation of intermediate 2-a, 180mg (3.74mmol) of intermediate 1 are reacted at 100 ℃ with 125mg (1.4 eq) of 3-methyl-5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyridazine in the presence of 14mg of bis (triphenylphosphine) palladium (II) chloride. Aqueous workup (aquousway-up) as described in the preparation of intermediate 2-a yielded 201mg of crude product, which was used without further purification for the preparation of intermediate 3-e.

Intermediate 2-f

17- (pyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid methyl ester

In analogy to the preparation of intermediate 2-a, 500mg (1.13mmol) of intermediate 1 are reacted with 194mg (1.4 equivalents) of pyridin-3-ylboronic acid in the presence of 39mg of bis (triphenylphosphine) palladium (II) chloride at 100 ℃ for 18 h. Aqueous workup yielded 462mg of crude product, which was used without further purification for the preparation of intermediate 3-f.

Intermediate 3-a

17- (5-fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid

First 372mg (0.95mmol) of intermediate 2-a are added to 50ml of THF and 3ml of methanol. A solution of 120mg of lithium hydroxide in 3ml of water was added and the mixture was stirred at RT for 18 h. An additional 5 equivalents of lithium hydroxide were added and the mixture was stirred at RT for 24h and at 40 ℃ for 18 h. The mixture was diluted with water, acidified to pH4 with 10% strength aqueous citric acid solution, ethyl acetate was added and the solid was filtered off, yielding 153mg (43% of theory) of the title compound after washing the solid with ethyl acetate and water and drying. The organic phase of the filtrate was separated and the aqueous phase was extracted twice with ethyl acetate. The combined organic phases were washed with sodium chloride solution, dried over sodium sulfate and concentrated to give a residue which was triturated with ether. Drying gives a further 143mg (40% of theory) of the title compound.

¹H-NMR(300MHz,DMSO-d₆):[ppm]=0.99(s,3H),1.38-1.78(m,5H),1.83-1.97(m,1H),2.05-2.21(m,2H),2.25-2.43(m,3H),2.89(dd,2H),6.27(dd,1H),7.36(d,1H),7.58-7.72(m,3H),8.43(d,1H),8.49(t,1H)。

Intermediate 3-b

17- (5-methoxypyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid

A solution of 1.4g (3.47mmol) of 17- (5-methoxypyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid methyl ester in 30ml THF, 4ml methanol and 8.7ml aqueous 2M sodium hydroxide solution was stirred at RT overnight and then warmed at 40 ℃ for 8.5 h. The mixture was diluted with water, acidified to pH =4 with a 10% strength citric acid solution and extracted three times with ethyl acetate, and the organic phase was washed with sodium chloride solution and concentrated. The crude product is triturated with diethyl ether to give 1.2g (89% of theory) of the title compound.

¹H-NMR(300MHz,DMSO-d₆):[ppm]=0.98(s,3H),1.34-1.81(m,5H),1.84-1.97(m,1H),2.03-2.19(m,2H),2.21-2.43(m,3H),2.89(dd,2H),3.81(s,3H),6.12–6.20(m,1H),7.20-7.29(m,1H),7.36(d,1H),7.59-7.70(m,2H),8.15(d,1H),8.20(d,1H)。

Intermediate 3-c

17- (pyrimidin-5-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid

A mixture of 1.70g (4.54mmol)17- (pyrimidin-5-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid methyl ester, 40ml THF, 11.3ml2M aqueous sodium hydroxide solution and 5ml methanol was stirred at RT overnight, then at 40 ℃ for 8.5h and then at RT overnight. The mixture was diluted with water and acidified to pH =4 with 10% strength citric acid solution and ethyl acetate was added. The insoluble solid was filtered off and dried. This gives 1.3g (79% of theory) of the title compound.

¹H-NMR(300MHz,DMSO-d₆):[ppm]=0.99(s,3H),1.39-1.79(m,5H),1.84-1.97(m,1H),2.06-2.21(m,2H),2.26-2.44(m,3H),2.89(dd,2H),6.28-6.33(m,1H),7.36(d,1H),7.59-7.69(m,2H),8.83(s,2H),9.04(s,1H),12.7(br.s.,1H)。

Intermediate 3-d

17- [5- (trifluoromethyl) pyridin-3-yl ] estra-1, 3,5(10), 16-tetraene-3-carboxylic acid

1.2g17- [5- (trifluoromethyl) pyridin-3-yl ] estra-1, 3,5(10), 16-tetraene-3-carboxylic acid methyl ester are initially added to 12ml THF, a solution of 0.23g lithium hydroxide in 12ml water is added and the mixture is stirred at 40 ℃ overnight. The mixture was diluted with water, acidified to pH =4 with 10% strength citric acid solution, and extracted three times with ethyl acetate. The extract was washed with sodium chloride solution and concentrated, and the residue was triturated with ether. This gave 850mg of the title compound in solid form.

¹H-NMR(400MHz,DMSO-d₆):[ppm]=1.01(s,3H),1.37-1.50(m,1H),1.50-1.69(m,3H),1.76(td,1H),1.86-1.95(m,1H),2.08-2.19(m,2H),2.27-2.44(m,3H),2.90(dd,2H),6.36(dd,1H),7.36(d,1H),7.61-7.68(m,2H),8.04(s,1H),8.82-8.86(m,1H),8.90(d,1H),12.7(br.s.,1H)。

Intermediate 3-e

17- (6-methylpyridazin-4-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid

201mg of 17- (6-methylpyridazin-4-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid methyl ester (crude product) are initially added to 3ml of THF and 0.5ml of methanol, 1.3ml of 2M aqueous sodium hydroxide solution are added, and the mixture is stirred at 40 ℃ overnight. The mixture was diluted with water, acidified to pH =4 with 10% strength citric acid solution, and extracted three times with ethyl acetate, and the extract was concentrated. The residue was purified by preparative HPLC to yield 42mg of the title compound as crude product.

C₂₄H₂₆N₂O₂(374.5). Measured MS-ES + mass: 374.20.

Intermediate 3-f

17- (pyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid

462mg of 17- (pyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid methyl ester (crude product) was dissolved in 4ml of THF and 1ml of methanol, 3ml of 2M aqueous sodium hydroxide solution was added, and the mixture was stirred at 40 ℃ overnight. The mixture was diluted with water and acidified to pH =4 with 10% strength citric acid solution and ethyl acetate was added. The remaining insoluble solid was filtered, washed with water and ethyl acetate, and dried under reduced pressure. This gives 375mg (84% of theory) of the title compound.

C₂₄H₂₅NO₂(359.47). Actually measured MS-ES + mass: 359.00.

¹H-NMR(300MHz,DMSO-d₆):[ppm]=0.99(s,3H),1.35-1.78(m,6H),1.84-1.96(m,1H),2.03-2.18(m,2H),2.21-2.44(m,4H),2.89(dd,2H),6.10-6.14(m,1H),7.29-7.39(m,2H),7.57-7.68(m,2H),7.77(dt,1H),8.42(dd,1H),8.59(d,1H)。

intermediate 4

17-oxoestra-1, 3,5(10) -triene-3, 11 alpha-diyl diacetate

To a solution of 10.0g (34.9mmol) of 3,11 α -dihydroxyestra-1, 3,5(10) -trien-17-one in 100ml of dichloromethane are added dropwise 13.2ml (4.0 equivalents) of acetic anhydride at RT and the reaction mixture is cooled to 5 ℃. Then 14.1ml pyridine was added dropwise and after 10min the mixture was warmed to RT and stirred for 4 h. A spatula tip of DMAP was added and the mixture was stirred at RT for 72 h. The mixture is poured into 500ml of water, the phases are separated, the aqueous phase is extracted with dichloromethane and the combined organic phases are washed with 1M hydrochloric acid, water and sodium chloride solution, dried over sodium sulfate and concentrated. This gives 12.9g (99% of theory) of a white solid.

¹H-NMR(400MHz,DMSO-d₆):[ppm]=0.81(s,3H),1.29(t,1H),1.43-1.72(m,4H),1.79-2.00(m,2H),2.00-2.06(m,3H),2.06-2.19(m,2H),2.19-2.25(m,3H), 2.42-2.57 (m, overlaid by DMSO signals), 2.76(t,2H),5.26(td,1H),6.82-6.89(m,2H),6.97(d, 1H).

Intermediate 5

3-hydroxy-17-oxoestra-1, 3,5(10) -trien-11 alpha-yl acetate

To 12.9g (34.7mmol) 17-oxoestra-1, 3,5(10) -triene-3, 11 α -diyl diacetate in 100ml methanol was added 14.6g (5 equivalents) sodium bicarbonate and the mixture was stirred at RT overnight. 100ml of water and 1ml of 1M hydrochloric acid were added and the mixture was stirred for 30 min. The mixture was extracted four times with ethyl acetate. The solid precipitated from the organic phase, was filtered off with suction and dried. This gives 3.74g (33% of theory) of the title compound. Furthermore, 6.39g (56% of theory) of the title compound are isolated by washing the organic phase with saturated sodium chloride solution, drying over sodium sulfate, concentrating, triturating the residue with ethyl acetate, suction-filtering and drying under reduced pressure.

¹H-NMR(300MHz,DMSO-d₆):[ppm]=0.79(s,3H),1.25(t,1H),1.36-1.69(m,4H),1.75-1.98(m,2H),1.98-2.18(m,5H),2.34-2.43(m),2.68(t,2H),5.16(td,1H),6.43-6.55(m,2H),6.76(d,1H),9.07(s,1H)。

Intermediate 6

3- { [ (1,1,2,2,3,3,4,4, 4-nonafluorobutyl) sulfonyl ] oxy } -17-oxoestra-1, 3,5(10) -trien-11 α -ylacetate

To a solution of 10.1g (31mmol) of 3-hydroxy-17-oxoestra-1, 3,5(10) -trien-11 α -yl acetate in 20ml of THF were added 12.8g (3 equivalents) of potassium carbonate and 6.5ml (1.2 equivalents) of 1,1,2,2,3,3,4,4, 4-nonafluorobutane-1-sulfonyl fluoride and the mixture was heated at reflux for 4h and stirred at RT for 18 h. Another 1ml of 1,1,2,2,3,3,4,4, 4-nonafluorobutane-1-sulfonyl fluoride was added and the mixture was heated at reflux for 3 h. Water and saturated sodium chloride solution were added, the mixture was stirred for 20min, the phases were separated and the aqueous phase was extracted three times with 50ml of ethyl acetate in each case. The combined organic phases were washed twice with in each case 50ml of water and twice with 50ml of saturated sodium chloride solution, dried over sodium sulfate, filtered and concentrated. Purification by silica gel column chromatography (hexane/ethyl acetate) gave 18.1g (96% of theory) of 3- { [ (1,1,2,2,3,3,4,4, 4-nonafluorobutyl) sulfonyl ] oxy } -17-oxoestra-1, 3,5(10) -trien-11 α -ylacetate.

¹H-NMR(400MHz,DMSO-d₆):[ppm]0.85(s,3H),1.26 to 1.37(m,1H),1.47 to 1.76(m,4H),1.83 to 2.02(m,2H),2.03 to 2.25(m,5H, s contained at 2.06 ppm), 2.41 to 2.47(m),2.59(t,1H),2.77 to 2.95(m,2H),5.29(td,1H),7.15(d,1H),7.23 to 7.29(m, 2H).

Intermediate 7

11 alpha-acetoxy-17-oxoestra-1, 3,5(10) -triene-3-carboxylic acid methyl ester

First, 10.0g (16.4mmol) of 3- { [ (1,1,2,2,3,3,4,4, 4-nonafluorobutyl) sulfonyl ] oxy } -17-oxoestra-1, 3,5(10) -trien-11 α -ylacetate, 230mg (6mol%) of palladium (II) acetate and 440mg (6mol%) of 1, 3-bis (diphenylphosphino) propane were loaded in an autoclave under argon, and 36ml of methanol, 54ml of DMSO and 6ml of triethylamine were added. The reaction mixture was flushed three times with carbon monoxide and stirred at RT for 30min at 7.5 bar carbon monoxide pressure. The autoclave was then vented and evacuated, and the mixture was stirred at 70 ℃ for 3.5h under 6.8 bar carbon monoxide pressure. The mixture was concentrated and the residue was dissolved in water and ethyl acetate. The phases were separated and the aqueous phase was extracted twice with ethyl acetate. The combined organic phases were washed with 1M hydrochloric acid and saturated sodium bicarbonate solution, dried over sodium sulfate and concentrated. Purification by column chromatography on silica gel (hexane/ethyl acetate) gave 5.96g (98% of theory) of the title compound in solid form.

¹H-NMR(300MHz,DMSO-d₆):[ppm]=0.81(s,3H),1.29(t,1H),1.40-1.76(m,4H),1.78-2.00(m,2H),2.00-2.21(m,5H, s contained at 2.03 ppm), 2.37-2.52 (m, shaded by DMSO signal), 2.59(t,1H),2.72-2.93(m,2H),3.79(s,3H),5.29(td,1H),5.23-5.38(m,1H),7.08(d,1H),7.68-7.75(m, 2H).

Intermediate 8

11 α -acetoxy-17- { [ (trifluoromethyl) sulfonyl ] oxy } estra-1, 3,5(10), 16-tetraene-3-carboxylic acid methyl ester

In analogy to the preparation of intermediate 1, 2.96g (7.99mmol) of methyl 11 α -acetoxy-17-oxoestra-1, 3,5(10) -triene-3-carboxylate are converted into 5.13g of the title compound in the form of the crude product (containing residual 2, 6-di-tert-butylpyridine).

¹H-NMR(300MHz,DMSO-d₆):[ppm]=0.93(s,3H),1.41-1.71(m,3H),1.71-1.87(m,1H),1.87-2.16(m,5H, s contained at 2.03 ppm), 2.16-2.40(m,2H),2.67(t,1H),2.74-2.93(m,2H),3.79(s,3H),5.34(td,1H), 5.75-5.82 (m,1H),7.03(d,1H),7.67-7.75(m, 2H).

Intermediate 9

11 alpha-acetoxy-17- (5-fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid methyl ester

In analogy to intermediate 2-a, 2.50g (4.98mmol) of 11 α -acetoxy-17- { [ (trifluoromethyl) sulfonyl group are reacted at reflux temperature]Oxy } estra-1, 3,5(10), 16-tetraene-3-carboxylic acid methyl ester in a molar ratio of 170mg (5mol%) [1, 3-bis (2, 6-diisopropylphenyl) imidazol-2-ylidene](3-Chloropyridyl) Palladium (II) dichloride (PEPSI)^TM-IPr, CAS905459-27-0) with 981mg (1.4 equivalents) of 5-fluoropyridine-3-boronic acid for a period of 5 h. This gave 2.62g of the title compound as crude product.

Intermediate 10

17- (5-Fluoropyridin-3-yl) -11 α -hydroxyestra-1, 3,5(10), 16-tetraene-3-carboxylic acid methyl ester

To 2.62g (5.83mmol)11 α -acetoxy-17- (5-fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid methyl ester in 40ml methanol was added 4.0g (5 equivalents) potassium carbonate and the mixture was stirred at RT for 3 h. The mixture was diluted with water and 1M hydrochloric acid and extracted three times with ethyl acetate. The combined organic phases were washed with water and saturated sodium chloride solution, dried over sodium sulfate and concentrated. Column chromatography on silica gel (hexane/ethyl acetate) gave 1.19g (50% of theory) of the title compound.

¹H-NMR(300MHz,DMSO-d₆The selected signal is [ ppm ]]=0.95(s,3H),1.40-1.61(m,3H),2.78-2.97(m,2H),3.79(s,3H),4.06–4.21(m,1H),4.79–4.92(m,1H),6.26(br.s.,1H),7.59-7.74(m,3H),8.07(d,1H),8.39-8.54(m,2H)。

Intermediate 11

11 beta-fluoro-17- (5-fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid methyl ester

To an ice-cold solution of 531mg (3.49mmol) of 17- (5-fluoropyridin-3-yl) -11 α -hydroxyestra-1, 3,5(10), 16-tetraene-3-carboxylic acid methyl ester in 15ml of THF were added dropwise 0.52ml (1.65 equivalents) of 1, 8-diazabicyclo [5.4.0] undec-7-ene and 0.58ml (1.5 equivalents) of 1,1,2,2,3,3,4,4, 4-nonafluorobutane-1-sulfonyl fluoride, and the mixture was stirred for 3h with cooling in the ice bath. The mixture was concentrated and the product was purified by silica gel column chromatography (hexane/ethyl acetate). This gives 747mg (84% of theory) of the title compound in the form of a crude product.

¹H-NMR(300MHz,DMSO-d₆The selected signal is [ ppm ]]=2.86–2.97(m,2H),5.57–5.83(m,1H),6.26–6.32(m,1H),7.45–7.53(m,1H),7.65-7.78(m,3H),8.39-8.53(m,2H)。

Intermediate 12

11 beta-fluoro-17- (5-fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid

To a mixture of 862mg (2.11mmol) of 11 β -fluoro-17- (5-fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid methyl ester in 10ml THF were added 5ml methanol and 442mg lithium hydroxide monohydrate in 5ml water, and the mixture was stirred at room temperature overnight. Water was added and the reaction mixture was adjusted to pH =4 with 10% strength aqueous citric acid solution. The resulting precipitate was filtered off with suction, washed with ethyl acetate and dried. This gave 498mg (60% of theory) of a white solid.

¹H-NMR(500MHz,DMSO-d₆):[ppm]=1.19(s,3H),1.44-1.59(m,1H),1.80-1.96(m,2H),1.96-2.08(m,2H),2.18-2.29(m,1H),2.32-2.42(m,1H),2.59(td,1H),2.74(dd,1H),2.77(br.s.,1H),2.86-3.00(m,2H),5.66–5.80(m,1H),6.32(dd,1H),7.48(d,1H),7.65-7.78(m,3H),8.47(d,1H),8.54(t,1H)。

Example 1

Step A: firstly, the method100mg (0.26mmol) of 17- (5-fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid are added to 1ml of DMF and 3ml of THF. Then, 119mg (2.0 equivalents) of 4- (aminomethyl) -3,4,5, 6-tetrahydro-2H-pyran-4-carboxylic acid ethyl ester hydrochloride, 41mg (2.0 equivalents) of 1-hydroxy-1H-benzotriazole hydrate, 102mg (2.0 equivalents) of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 0.11ml of triethylamine were added and the mixture was stirred at RT overnight.

And B:then, 0.66ml of 2M aqueous sodium hydroxide solution and 0.50ml of methanol were added and the mixture was stirred at RT overnight. Water was added and the reaction mixture was acidified to pH3-4 with 10% strength aqueous citric acid. The aqueous phase was extracted three times with ethyl acetate, the combined organic phases were concentrated and the residue was purified by preparative HPLC (acetonitrile/water/formic acid). This gives 76mg (55% of theory) of solid.

C₃₁H₃₅FN₂O₄(518.6). Actually measured MS-ES + mass: 518.26.

¹HNMR(300MHz,DMSO-d₆selected signals) ppm0.99(s,3H),1.36-1.99(m,10H),2.05-2.21(m,2H),2.25-2.44(m,3H),2.82-2.95(m,2H),3.39(d,2H), 3.67-3.76 (m,2H), 6.25-6.29(m,1H),7.31(d,1H), 7.49-7.60(m,2H),7.68(dt,1H),8.27(t,1H),8.43(d,1H),8.49(s,1H),12.5 (br.s).

Example 2

Step A: to a mixture of 4.00g (10.6mmol)17- (5-fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid and 3.38g (2 equiv) of tert-butyl N-methyl-. beta. -alanine in 100ml THF and 5ml DMF was added 1.62g (1.0 equiv) 1-hydroxy-1H-benzotriazole hydrate, 4.06g (2.0 equiv) 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 4.4ml triethylamine and the mixture was stirred at RT for 18H. The mixture was diluted with water and extracted three times with ethyl acetate, and the extracts were washed with sodium chloride solution and concentrated. The residue was purified on silica gel (hexane/ethyl acetate) to give 5.1g of N- { [17- (5-fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } -N-methyl-. beta. -alanine tert-butyl ester (93% of theory) in solid form.

And B: 1.00g (1.93mmol) of tert-butyl N- { [17- (5-fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } -N-methyl-. beta. -alanine were initially taken in 15ml of dichloromethane, 1.5ml of trifluoroacetic acid were added and the mixture was stirred at 40 ℃ overnight, poured into ice water, stirred briefly and extracted three times with dichloromethane. The combined organic phases were washed with saturated sodium chloride solution, filtered through a water-repellent filter and concentrated. Diethyl ether was added to the crude product, the mixture was stirred and filtered with suction, and the product was washed with diethyl ether and dried. This gives 0.79g (89% of theory) of N- { [17- (5-fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } -N-methyl-. beta. -alanine.

C₂₈H₃₁FN₂O₃(462.6). Actually measured MS-ES + mass: 462.23.

¹H-NMR(300MHz,DMSO-d6):[ppm]1.00(s,3H), 1.33-1.79 (m,5H),1.82-1.99(m,1H), 2.08-2.21 (m,2H),2.21-2.43(m,3H),2.50(s), 2.74-2.88 (5H, s contained at 2.88 ppm), 3.36-3.71(m),6.27(s, 1H),6.99-7.16(m,2H),7.28(d,1H),7.68(dt,1H),8.43(d,1H),8.49(s,1H),8.48-8.56(m,1H),12.28 (br.s.).

Example 3

In analogy to example 1, 100mg (0.26mmol)17- (5-fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid and 88mg (2.0 equivalents) 1- (aminomethyl) cyclopropane-1-carboxylic acid methyl ester hydrochloride are converted into 72mg (57% of theory) of the title compound.

C₂₉H₃₁FN₂O₃(474.6). Actually measured MS-ES + mass: 474.23.

¹H-NMR(300MHz,DMSO-d6):[ppm]=0.82-0.90(m,2H),0.95-1.06(m,5H),1.33-1.81(m,5H),1.56(d,3H),1.84-1.97(m,1H),1.84-1.97(m,1H),2.05-2.44(m),2.80-2.94(m,2H),3.44-3.54(m,2H),6.27(s.,1H),7.31(d,1H),7.50-7.60(m,2H),7.68(dt,1H),8.18(t,1H),8.43(d,1H),8.49(s,1H),12.3(s,1H)。

example 4

In analogy to example 1 (step B was performed overnight at 50 ℃ and after addition of a further 5 equivalents of 2M aqueous sodium hydroxide solution, by stirring overnight at 60 ℃) 100mg (0.26mmol)17- (5-fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid and 103mg (2.0 equivalents) 1- (aminomethyl) cyclopentane-1-carboxylic acid methyl ester hydrochloride were converted into 63mg (48% of theory) of the title compound.

C₃₁H₃₅FN₂O₃(502.6). Actually measured MS-ES + mass: 502.26.

¹H-NMR(400MHz,DMSO-d6):[ppm]=0.99(s,3H),1.36–1.66(m),1.73(td,1H),1.81-1.95(m,3H),2.07-2.20(m,2H),2.25-2.44(m),2.83-2.95(m,2H),3.45(d,2H),6.25-6.29(m,1H),7.31(d,1H),7.49-7.56(m,2H),7.69(dt,1H),8.14(t,1H),8.43(d,1H),8.49(s,1H),12.2(s)。

example 5

In analogy to example 1 (step B was carried out overnight at 50 ℃ and overnight with stirring at 60 ℃ after addition of a further 5 equivalents of 2M aqueous sodium hydroxide solution), 100mg (0.26mmol) of 17- (5-fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid and 89mg (2.0 equivalents) of methyl 3-amino-2, 2-dimethylpropionate hydrochloride were converted into 63mg (50% of theory) of the title compound.

C₂₉H₃₃FN₂O₃(476.6). Actually measured MS-ES + mass: 476.25.

¹H-NMR(400MHz,DMSO-d6):[ppm]=0.99(s,3H),1.06(s,6H),1.36-1.66(m,4H),1.73(td,1H),1.86-1.95(m,1H),2.07-2.20(m,2H),2.25-2.45(m,3H),2.84-2.92(m,2H),3.37(d, overlapped by a water signal), 6.25-6.29(m,1H),7.31(d,1H),7.50-7.57(m,2H),7.69(dt,1H),8.15(t,1H),8.43(d,1H),8.49(t,1H), 12.2(s).

Example 6

In analogy to example 1 (step B carried out at 50 ℃ C. for a period of 5h), 100mg (0.26mmol) of 17- (5-fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid and 83mg (2.0 equivalents) of ethyl piperidine-4-carboxylate are converted into 65mg (50% of theory) of the title compound.

C₃₀H₃₃FN₂O₃(488.6). Actually measured MS-ES + mass: 488.25.

¹H-NMR(400MHz,DMSO-d₆):[ppm]=1.00(s,3H),1.33-1.96(m,10H),2.03-2.22(m,2H),2.24-2.43(m,3H),2.79-2.94(m,3H),3.02(br.s.,1H),3.57(br.s.,1H),4.26(br.s.,1H),6.25-6.29(m,1H),7.00-7.11(m,2H),7.29(d,1H),7.69(dt,1H),8.43(d,1H),8.47-8.51(m,1H),12.3(s)。

example 7

In analogy to example 1 (step B was performed overnight by heating at 50 ℃ C., and after addition of a further 5 equivalents of 2M aqueous sodium hydroxide solution, stirring overnight at 60 ℃) 100mg (0.26mmol)17- (5-fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid and 81mg (2.0 equivalents) 3-amino-2, 2-dimethylpropionic acid methyl ester hydrochloride were converted into 81mg (66% of theory) of the title compound.

C₂₈H₃₁FN₂O₃(462.6). Actually measured MS-ES + mass: 462.23.

¹H-NMR(300MHz,DMSO-d6):[ppm]=0.99(s,3H),1.39(s,6H),1.4-1.82(m),1.86–1.98(m,1H),2.05-2.43(m,2H),2.20-2.50(m),2.83-2.94(m,2H),6.27(s,1H),7.31(d,1H),7.52-7.61(m,2H),7.65-7.72(m,1H),8.29(s,1H),8.43(d,1H),8.46-8.53(m,1H),12.1(s)。

example 8

In analogy to example 1, 100mg (0.26mmol)17- (5-fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid and 81mg (2.0 equivalents) methyl 4-aminobutyrate hydrochloride are converted into 58mg (48% of theory) of the title compound.

C₂₈H₃₁FN₂O₃(462.6). Actually measured MS-ES + mass: 462.23.

¹H-NMR(300MHz,DMSO-d6):[ppm]=0.99(s,3H),1.39-1.78(m,7H),1.83-1.99(m,1H), 2.05-2.50 (m, superimposed by DMSO signal)Coating), 3.1-3.4 (m, overlapped by water signal), 2.79-2.98(m,2H),6.26(s, 1H),7.31(d,1H),7.52-7.59(m,2H),7.67(dt,1H),8.32(t,1H),8.43(d,1H),8.49(s,1H), 12.0(s).

Example 9

In analogy to example 1, 100mg (0.26mmol)17- (5-fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid and 81mg (2.0 equivalents) beta-alanine ethyl ester hydrochloride are converted into 59mg (50% of theory) of the title compound.

C₂₇H₂₉FN₂O₃(448.5). Actually measured MS-ES + mass: 448.22.

¹H-NMR(300MHz,DMSO-d6):[ppm]=0.99(s,3H),1.38-1.78(m,5H),1.83-1.98(m,1H),2.05-2.21(m,2H), 2.25-2.44(m, overlaid by DMSO signals), 2.80-2.94(m,2H),3.35-3.51(m, overlaid by water signals), 6.26(s, 1H),7.31(d,1H),7.50-7.58(m,2H),7.62-7.74(m,1H),8.36(t,1H),8.43(d,1H),8.49(s,1H), 12.2(s).

Example 10

In analogy to example 1, 100mg (0.26mmol)17- (5-fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid and 67mg (2.0 equivalents) glycine methyl ester hydrochloride are converted into 58mg (50% of theory) of the title compound.

C₂₆H₂₇FN₂O₃(434.5). Actually measured MS-ES + mass: 434.20.

¹H-NMR(400MHz,DMSO-d₆):[ppm]=1.00(s,3H),1.37-1.67(m,4H),1.74(td,1H),1.85-1.97(m,1H),2.07-2.21(m,2H),2.26-2.5(m, overlaid by DMSO signals), 2.84-2.94(m,2H),3.86(d,2H),6.25-6.29(m,1H),7.34(d,1H),7.55-7.61(m,2H),7.64-7.72(m,1H),8.43(d,1H),8.49(t,1H),8.64(t,1H), 12.5(s).

Example 11

In analogy to example 1, 100mg (0.26mmol)17- (5-fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid and 103mg (2.0 equivalents) (1R, 2S) -2-aminocyclopentane-1-carboxylic acid ethyl ester hydrochloride are converted into 63mg (49% of theory) of the title compound.

C₃₀H₃₃FN₂O₃(488.6). Actually measured MS-ES + mass: 488.25.

¹H-NMR(300MHz,DMSO-d₆):[ppm]=1.00(s,3H),1.39-1.66(m,5H),1.68-1.96(m,7H),2.02-2.21(m,2H),2.24-2.41(m),2.78-2.98(m,3H),4.42–4.57(m,1H),6.27(s.,1H),7.30(d,1H),7.46-7.58(m,2H),7.68(dt,1H),8.03(d,1H),8.43(d,1H),8.46-8.52(m,1H),11.9(s)。

example 12

Analogously to example 1 (in step B, a further 5 equivalents of 2M aqueous sodium hydroxide solution are added, the mixture is stirred for 4h, a further 5 equivalents of 2M aqueous sodium hydroxide solution are added, the mixture is stirred for 30min in a microwave oven at 110 ℃/300W, a10 equivalents of 2M aqueous sodium hydroxide solution are added, the mixture is then heated for 60min in a microwave oven at 120 ℃/300W and for 60min in a microwave oven at 130 ℃/300W), 100mg (0.26mmol) of 17- (5-fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid and 84mg of tert-butyl (S) -3-aminobutyrate are converted into 24mg (20% of theory) of the title compound.

C₂₈H₃₁FN₂O₃(462.6). Actually measured MS-ES + mass: 462.23.

¹H-NMR(400MHz,DMSO-d₆):[ppm]=1.02(s,3H),1.16(d,3H),1.38-1.68(m,4H),1.76(td,1H),1.87-2.01(m,1H),2.09-2.24(m,2H),2.27-2.46(m,4H),2.51-2.61(m,1H),2.82-3.00(m,2H),4.31(spt,1H),6.27-6.31(m,1H),7.34(d,1H),7.50-7.61(m,2H),7.70(dt,1H),8.16(d,1H),8.46(d,1H),8.50–8.54(m,1H),12.2(br.s.,1H)。

example 13

In analogy to example 1 (in step B, a further 5 equivalents of 2M aqueous sodium hydroxide solution are added and the mixture is stirred at 50 ℃ for 30h), 100mg (0.26mmol) of 17- (5-fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid and 84mg of tert-butyl (R) -3-aminobutyrate are converted into 34mg (28% of theory) of the title compound.

C₂₈H₃₁FN₂O₃(462.6). Actually measured MS-ES + mass: 462.23.

¹H-NMR(400MHz,DMSO-d₆):[ppm]=1.02(s,3H),1.16(d,3H),1.39-1.69(m,4H),1.76(td,1H),1.88-2.00(m,1H),2.09-2.23(m,2H),2.27-2.47(m,4H),2.51-2.61(m,1H),2.82-2.98(m,2H),4.32(spt,1H),6.24-6.34(m,1H),7.34(d,1H),7.52-7.60(m,2H),7.70(dt,1H),8.15(d,1H),8.46(d,1H),8.50-8.54(m,1H),12.1(br.s.,1H)。

example 14

In analogy to example 1 (step B) was carried out by stirring at 50 ℃ for 7h, 100mg (0.26mmol) of 17- (5-methoxypyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid and 75mg (0.51mmol) of ethyl 3-amino-2, 2-dimethylpropionate were converted into 12mg (10% of theory) of the title compound.

C₃₀H₃₆N₂O₄(488.63). Actually measured MS-ES + mass: 488.27.

¹H-NMR(300MHz,DMSO-d6):[ppm]0.98(s,3H),1.06(s,6H),1.31-1.82(m,5H), 1.84-1.97 (m,1H),2.00-2.19(m,2H),2.20-2.40(m), 2.80-2.95 (m,2H), 3.36-3.38 (m, partially obscured by a water signal), 3.81(s,3H),6.16(s, 1H),7.20-7.37(m,2H),7.46-7.63(m,2H),8.09-8.27(m, 3H).

Example 15

In analogy to example 1 (step B) was carried out by stirring at 50 ℃ for 7h, 100mg (0.26mmol) of 17- (5-methoxypyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid and 79mg (0.51mmol) of beta-alanine ethyl ester hydrochloride were converted into 64mg (54% of theory) of the title compound.

C₂₈H₃₂N₂O₄(460.58). Actually measured MS-ES + mass: 460.24.

¹H-NMR(300MHz,DMSO-d₆):[ppm]=0.98(s,3H),1.38-1.78(m,5H),1.85-1.96(m,1H),2.04-2.18(m,2H),2.21-2.41(m,3H),2.76-2.98(m,2H),3.34-3.50(m,2H),3.81(s,3H),6.14-6.18(m,1H),7.23-7.28(m,1H),7.31(d,1H),7.46-7.63(m,2H),8.16(d,1H),8.20(d,1H),8.38(t,1H),12.2(br.s.,1H)。

example 16

To a mixture of 100mg (0.26mmol)17- (5-methoxypyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid and 82mg (2 equivalents) N-methyl-. beta. -alanine tert-butyl ester in 3ml THF and 1ml DMF were added 39mg (1.0 equivalent) 1-hydroxy-1H-benzotriazole hydrate, 98mg (2.0 equivalents) 1- [3- (dimethylamino) propyl ] -3-ethylcarbodiimide hydrochloride and 0.11ml triethylamine, and the mixture was stirred at RT for 72H. The mixture was diluted with water and extracted three times with ethyl acetate, and the extracts were concentrated. To the residue were added 4ml of dichloromethane and 1ml of trifluoroacetic acid, and the mixture was stirred at room temperature for 17 h. The mixture was concentrated, and after purification of the residue by preparative HPLC, 66mg of the title compound was produced.

C₂₉H₃₄N₂O₄(474.61). Actually measured MS-ES + mass: 474.25.

¹H-NMR(300MHz,DMSO-d₆):[ppm]=0.99(s,3H),1.38-1.78(m,5H),1.82–1.96(m,1H),2.03-2.18(m,2H),2.21-2.43(m,3H),2.79-2.93(m,5H),3.42(br.s.,1H),3.57(br.s.,1H),3.81(s,3H),6.16(s,1H),6.99-7.14(m,2H),7.23-7.31(m,2H),8.13-8.22(m,2H),12.3(br.s.,1H)。

example 17

In analogy to example 1 (step B) was carried out by stirring at 50 ℃ for 18h, 100mg (0.28mmol) of 17- (pyrimidin-5-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid and 85mg (2.0 equivalents) of beta-alanine ethyl ester hydrochloride were converted into 63mg (50% of theory) of the title compound.

C₂₆H₂₉N₃O₃(431.5). Actually measured MS-ES + mass: 431.22.

¹H-NMR(300MHz,DMSO-d₆):[ppm]=0.99(s,3H),1.36-1.79(m,5H),1.84-1.97(m,1H),2.06-2.20(m,2H),2.25-2.41(m,4H),2.82-2.93(m,2H),3.35-3.45(m,2H),6.28-6.33(m,1H),7.31(d,1H),7.50-7.58(m,2H),8.38(t,1H),8.83(s,2H),9.04(s,1H),12.19(br.s.,1H)。

example 18

In analogy to example 1 (step B) was carried out by stirring at 50 ℃ for 18h, 100mg (0.28mmol) of 17- (pyrimidin-5-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid and 85mg (2.0 equivalents) of methyl 4-aminobutyrate hydrochloride were converted into 61mg (47% of theory) of the title compound.

C₂₇H₃₁N₃O₃(445.6). Actually measured MS-ES + mass: 445.24.

¹H-NMR(300MHz,DMSO-d₆):[ppm]=0.99(s,3H),1.39-1.79(m,7H),1.85-1.98(m,1H),2.05-2.26(m,4H),2.26-2.41(m,3H),2.81-2.95(m,2H),3.15-3.25(m,2H),6.27-6.34(m,1H),7.31(d,1H),7.50-7.61(m,2H),8.34(t,1H),8.83(s,2H),9.04(s,1H),12.04(br.s.,1H)。

example 19

To a mixture of 100mg (0.26mmol)17- (pyrimidin-5-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid and 88mg (2 equivalents) of tert-butyl N-methyl-beta-alanine in 3ml THF and 1ml DMF was added 42mg (1 equivalent) of 1-hydroxy-1H-benzotriazole hydrate, 106mg (2.0 equivalents) of 1- [3- (dimethylamino) propyl ] -3-ethylcarbodiimide hydrochloride and 0.12ml triethylamine, and the mixture was stirred at RT for 72H. The mixture was diluted with water and extracted three times with ethyl acetate, and the extracts were concentrated. To the residue were added 3ml of dichloromethane and 1ml of trifluoroacetic acid, and the mixture was stirred at room temperature for 72 h. The mixture was concentrated, and after purification of the residue by preparative HPLC, 56mg of the title compound was produced.

C₂₇H₃₁N₃O₃(445.6). Actually measured MS-ES + mass: 445.24.

¹H-NMR(300MHz,DMSO-d₆):[ppm]=1.00(s,3H),1.36-1.77(m,5H),1.85-1.94(m,1H),2.07-2.20(m,2H),2.26-2.5(m, shaded), 2.80-2.92(m,5H),3.42(br.s.,1H),3.57(br.s.,1H), 6.27-6.33 (m,1H),7.02-7.11(m,2H),7.29(d,1H),8.83(s,2H),9.04(s,1H),12.3(br.s, 1H).

Example 20

In analogy to example 1 (step B was carried out by stirring at 50 ℃ C. for 5h), 100mg (0.28mmol) of 17- (pyrimidin-5-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid and 81mg (0.55mmol) of ethyl 3-amino-2, 2-dimethylpropionate were converted into 10mg (8% of theory) of the title compound.

C₂₈H₃₃N₃O₃(459.6). Actually measured MS-ES + mass: 459.25.

¹H-NMR(600MHz,DMSO-d₆):[ppm]=1.02(s,3H),1.09(s,6H),1.42-1.50(m,1H),1.53–1.68(m,3H),1.77(td,1H),1.91-1.97(m,1H),2.13-2.20(m,2H),2.31-2.39(m,2H),2.41-2.47(m,1H),2.89-2.94(m,2H),3.40(d,2H),6.33(dd,1H),7.34(d,1H),7.53-7.59(m,2H),8.16–8.21(m.,1H),8.85(s,2H),9.07(s,1H),12.25(br.s.,1H)。

example 21

In analogy to example 1 (step B) was carried out by stirring at 50 ℃ for 18h, 100mg (0.23mmol) of 17- (5- (trifluoromethyl) pyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid and 72mg (2.0 equivalents) of beta-alanine ethyl ester hydrochloride were converted into 65mg (56% of theory) of the title compound.

C₂₈H₂₉F₃N₂O₃(498.6). Actually measured MS-ES + mass: 498.21.

¹H-NMR(300MHz,DMSO-d₆):[ppm]=1.01(s,3H),1.35-1.69(m,4H),1.76(td,1H),1.84-2.00(m,1H),2.03-2.21(m,2H),2.24-2.41(m),2.78-2.96(m,2H),3.35-3.53(m,2H),6.33–6.38(m,1H),7.31(d,1H),7.47-7.62(m,2H),8.03(s,1H),8.35(t,1H),8.78-8.86(m,1H),8.86-8.97(m,1H),12.2(br.s.,1H)。

example 22

Step A: to a mixture of 100mg (0.23mmol)17- [5- (trifluoromethyl) pyridin-3-yl ] estra-1, 3,5(10), 16-tetraene-3-carboxylic acid and 74mg (2 equivalents) N-methyl- β -alanine tert-butyl ester in 3ml THF were added 36mg (1 equivalent) 1-hydroxy-1H-benzotriazole hydrate, 90mg (2.0 equivalents) 1- [3- (dimethylamino) propyl ] -3-ethylcarbodiimide hydrochloride and 98. mu.l triethylamine, and the mixture was stirred at RT for 4H. The mixture was diluted with water and extracted three times with ethyl acetate, and the extracts were concentrated.

And B: 2ml dichloromethane and 180. mu.l trifluoroacetic acid were added and the mixture was stirred at a bath temperature of 40 ℃ for 18 h. Another 90 microliters of trifluoroacetic acid was added and the mixture was stirred at 40 ℃ for 5 h. Water was added, the phases were separated and the aqueous phase was extracted twice with dichloromethane. The organic phase was concentrated and the residue was purified by HPLC (acetonitrile/water/formic acid). This gives 83mg (69% of theory) of the title compound.

C₂₉H₃₁F₃N₂O₃(512.58). Actually measured MS-ES + mass: 512.23.

¹H-NMR(300MHz,DMSO-d₆):[ppm]=1.01(s,3H),1.36-1.51(m,1H),1.52-1.81(m,4H),1.84-1.98(m,1H),2.04-2.21(m,2H),2.25-2.40(m),2.79-2.96(m,5H),3.42(br.s.),3.55(br.s.),6.35(s,1H),7.00-7.15(m,2H),7.28(d,1H),8.03(s,1H),8.81-8.87(m,1H),8.87-8.95(m,1H),12.3(br.s.,1H)。

example 23

In analogy to example 22, 100mg (0.26mmol)17- (5-fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid are reacted with 91mg (2.0 equivalents) tert-butyl L-proline. Purification by preparative HPLC gives 65mg (50% of theory) of the title compound.

C₂₉H₃₁FN₂O₃(474.6). Actually measured MS-ES + mass: 474.23.

¹H-NMR(300MHz,DMSO-d₆):[ppm]=1.00(s,3H),1.39-1.94(m),2.05-2.43(m),2.77–2.94(m,2H),3.40-3.59(m,2H),4.27-4.40(m,1H),6.27(s.,1H),7.04-7.34(m,3H),7.64-7.73(m,1H),8.43(d,1H),8.49(s,1H),12.5(br.s.,1H)。

the title compound was analyzed by analytical HPLC:

system for controlling a power supply	Waters:Alliance2695,DAD996
		Column:	Chiralpak AS-RH5μm150×4.6mm
solvent:	H₂o (0.1% by volume formic acid)/acetonitrile 50:50(v/v)
		Flow rate:	1.0ml/min
temperature:	25℃
		solution:	1.0mg/ml ethanol/methanol 2:1
Sample introduction:	5.0μl
		and (3) detection:	DAD254nm
peak(s)	Rt in minutes
		1	8.15

[0436]Example 24

In analogy to example 22, 100mg (0.26mmol)17- (5-fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid are reacted with 91mg (2.0 equivalents) tert-butyl D-proline. Purification by preparative HPLC gives 66mg (52% of theory) of the title compound.

C₂₉H₃₁FN₂O₃(474.6). Actually measured MS-ES + mass: 474.23.

¹H-NMR(300MHz,DMSO-d₆):[ppm]=1.00(s,3H),1.34-1.97(m),2.05-2.43(m),2.75–2.94(m,2H),3.42-3.59(m,2H),4.25-4.40(m,1H),6.27(s.,1H),7.02-7.36(m,3H),7.68(d,1H),8.43(d,1H),8.49(s,1H),12.5(br.s.,1H)。

the title compound was analyzed by analytical HPLC:

system for controlling a power supply	Waters:Alliance2695,DAD996
		Column:	Chiralpak AS-RH5μm150×4.6mm
solvent:	H₂o (0.1% by volume formic acid)/acetonitrile 50:50(v/v)
		Flow rate:	1.0ml/min
temperature:	25℃
		solution:	1.0mg/ml ethanol/methanol 2:1
Sample introduction:	5.0μl
		and (3) detection:	DAD254nm
peak(s)	Rt in minutes
		2	9.50

[0444]Example 25

In analogy to example 1, 100mg of 11 β -fluoro-17- (5-fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid and 78mg (2.0 equivalents) of methyl 4-aminobutyrate hydrochloride are converted into 80mg (66% of theory) of the title compound.

C₂₈H₃₀F₂N₂O₃(480.6). Actually measured MS-ES + mass: 480.22.

¹H-NMR(400MHz,DMSO-d₆):[ppm]=1.14(s,3H),1.40–1.55(m,1H),1.70(quin,2H),1.74-2.03(m,4H),2.13-2.27(m,3H),2.27-2.37(m,1H),2.49-2.60(m,1H),2.60–2.77(m,1H),2.81-2.97(m,2H),3.22(q,2H),5.58-5.80(m,1H),6.21-6.34(m,1H),7.40(d,1H),7.48-7.61(m,2H),7.72(dt,1H),8.36(t,1H),8.45(d,1H),8.50(s,1H),12.0(br.s.,1H)。

example 26

A 0.2ml dmso/ice culture of the calonectriaceae strain (atccno.14767) was inoculated into a 100ml erlenmeyer flask containing 20ml of a 20ml aqueous nutrient solution (adjusted to ph6.2) containing 1% corn steep liquor and 1% soybean meal that had been sterilized in an autoclave at 121 ℃ for 20 minutes and the erlenmeyer flask was shaken at 165 revolutions per minute on a rotary shaker at 21 ℃ for 48 hours. 8ml of this preculture were inoculated into 500ml conical flasks filled with 100ml of sterile medium (final composition identical to that described for the preculture). The flask was shaken on a rotary shaker at 165 revolutions per minute for 48 hours at 21 ℃.50ml of this preculture were each inoculated into two 2l Erlenmeyer flasks each containing 1l of a sterile nutrient solution comprising 3% glucose monohydrate, 1% ammonium chloride, 0.2% sodium nitrate, 0.1% potassium dihydrogen phosphate, 0.2% dipotassium hydrogen phosphate, 0.05% potassium chloride, 0.05% magnesium sulfate heptahydrate and 0.002% iron (II) sulfate heptahydrate. After a growth phase at 27 ℃ for 6 hours at 27 ℃ on a rotary shaker at 165 rpm, a solution of 50mg of N- { [17- (5-fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } -N-methyl-. beta. -alanine in 10ml of DMF was divided into these two flasks. The flask was shaken for a further 43 hours and then worked up. The two culture broths were combined and extracted with 1l of isobutyl methyl ketone at 40 rpm for 19 hours in a 5l glass extraction vessel. The organic phase was dried over sodium sulfate and concentrated to dryness. The residue was washed with methanol to remove silicone oil. This gave 328mg of crude product. The crude product was adsorbed onto celite and chromatographed: the method comprises the following steps: biotage isolera,10g snap column, solvent gradient from 2% to 20% methanol in ethyl acetate (1% glacial acetic acid added). This gave 42mg of the title compound.

HPLCRt=4.8min

HPLC conditions: a is water with 0.05% formic acid, B is acetonitrile with 0.1% formic acid, gradient 0min:60:40A/B, 12min:30:70A/B, flow rate 0.8ml/min, column LunAC18(2)5 μ 125X 4.6, detection wavelength 244nm

¹H-NMR(400MHz,DMSO-d6):[ppm]=1.08(s,3H), 1.50-1.58 (m,2H), 1.61-1.67 (m,2H), 1.76-1.85 (m,1H), 2.08-2.12 (m,1H), 2.30-2.35 (m,2H), 2.40-2.45 (m,1H),2.55(2H is overlaid by a DMSO signal), 2.85-2.89 (m,2H),2.91(s,3H),3.45(br.s,1H),3.62(br.s,1H),4.62(d,1H),4.95(br.s,1H),6.15(s,1H),7.05(s,1H),7.11(d,1H),7.31(d,1H),7.71(d,1H),8.49(d,1H), 1H (1H), 1.51.51 (s, 1H).

Example 27

A0.2 ml DMSO/ice culture of Mucoralomyces (Mucorallus) strain (CBSNo.29563) was inoculated into a 100ml Erlenmeyer flask containing 20ml of a nutrient aqueous solution (adjusted to pH6.0) containing 3% glucose monohydrate, 1% corn steep liquor, 0.2% sodium nitrate, 0.1% potassium dihydrogen phosphate, 0.2% dipotassium hydrogen phosphate, 0.05% potassium chloride, 0.05% magnesium sulfate heptahydrate, and 0.002% ferrous sulfate heptahydrate (II) that had been sterilized in an autoclave at 121 ℃ for 20 minutes, and the Erlenmeyer flask was shaken at 27 ℃ on a rotary shaker at 165 rpm for 65 hours. 8ml of this preculture were inoculated into 500ml conical flasks filled with 100ml of sterile medium (final composition identical to that described for the preculture). The flask was shaken on a rotary shaker at 165 revolutions per minute at 27 ℃ for 72 hours. 50ml of this preculture were each inoculated into two 2l Erlenmeyer flasks each containing 1l of a sterile nutrient solution comprising 3% glucose monohydrate, 1% ammonium chloride, 0.2% sodium nitrate, 0.1% potassium dihydrogen phosphate, 0.2% dipotassium hydrogen phosphate, 0.05% potassium chloride, 0.05% magnesium sulfate heptahydrate and 0.002% iron (II) sulfate heptahydrate. After a growth phase at 27 ℃ for 6 hours at 27 ℃ on a rotary shaker at 165 rpm, a solution of 50mg of N- { [17- (5-fluoropyridin-3-yl) estra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } -N-methyl-. beta. -alanine in 10ml of DMF was divided into two of these flasks. The flask was shaken for a further 43 hours and then worked up. The two culture broths were combined and extracted with 1l of isobutyl methyl ketone at 40 rpm for 19 hours in a 5l glass extraction vessel. The organic phase was dried over sodium sulfate and concentrated to dryness. The residue was washed with methanol to remove silicone oil. This gave 236mg of crude product as a brown oil. The crude product was adsorbed onto celite and chromatographed: the instrument comprises the following steps: biotage isolera,10g snap column, solvent gradient from 2% to 20% methanol in ethyl acetate (1% glacial acetic acid added). This gave 35mg of the title compound. HPLCRT =5.4min

¹H-NMR(400MHz,DMSO-d6):[ppm]=1.28(s,3H);1.38–1.60(m,4H);1.69–1.78(m,1H);2.01–2.08(m,1H);2.20–2.28(m,1H) 2.30-2.40 (m,1H),2.55(2H overlaid by DMSO signal), 2.85-2.90 (m,5H),3.10(s,1H), 3.45(br.s,1H), 3.57(br.s,1H), 4.50(s,1H), 4.69(br.s,1H), 6.30(s,1H), 7.05(s,1H), 7.08(d,1H), 7.28(d,1H), 7.71(d7,1H), 8.47(d,1H), 8.52(s,1H), 12.1(br.s, 1H).

Example 28

42mg of 17- (6-methylpyridazin-4-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid (impure) and 36mg of tert-butyl N-methyl-. beta. -alanine (2 equivalents) were dissolved in 2.5ml of THF and 0.5ml of DMF. 43mg of 1- [3- (dimethylamino) propyl ] -3-ethylcarbodiimide hydrochloride (EDC), 17mg of 1-hydroxy-1H-benzotriazole hydrate and 0.047ml of triethylamine were added, and the mixture was stirred at room temperature for 18H. The reaction mixture was diluted with water and extracted three times with ethyl acetate. The combined organic phases were concentrated, 2ml dichloromethane and 0.5ml trifluoroacetic acid were added to the residue and the mixture was stirred at room temperature for 6 h. The mixture was concentrated and the product was purified by preparative HPLC. This gave 18mg of the title compound.

C₂₈H₃₃N₃O₃(459.59). Actually measured MS-ES + mass: 459.25.

¹H-NMR(400MHz,DMSO-d6):[ppm]=1.02(s,3H),1.35-1.77(m,5H),1.84–1.95(m,1H),2.07-2.42(m,6H),2.58(s,3H),2.78-2.95(m,5H),3.41(br.s),3.57(br.s),6.54–6.59(m,1H),7.02-7.13(m,2H),7.29(d,1H),7.49(d,1H),9.10(d,1H),12.3(br.s,1H)。

example 29

N-methyl-N- { [17- (pyridin-3-yl) estra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } -beta-alanine

100mg of 17- (pyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid and 89mg of N-methyl-. beta. -alanine tert-butyl ester (2 equivalents) were dissolved in 3ml of THF and 0.5ml of DMF. 107mg of 1- [3- (dimethylamino) propyl ] -3-ethylcarbodiimide hydrochloride (EDC), 43mg of 1-hydroxy-1H-benzotriazole hydrate and 0.116ml of triethylamine were added, and the mixture was stirred at room temperature for 18H. The reaction mixture was diluted with water and extracted three times with ethyl acetate. The combined organic phases were concentrated, 3ml dichloromethane and 1ml trifluoroacetic acid were added to the residue and the mixture was stirred at room temperature for 20 h. The mixture was concentrated and the product was purified by preparative HPLC. This gave 78mg of the title compound.

C₂₈H₃₂N₂O₃(444.58). Actually measured MS-ES + mass: 444.24.

¹H-NMR(400MHz,DMSO-d₆):[ppm]=0.99(s,3H),1.37-1.67(m,4H),1.73(td,1H),1.86-1.94(m,1H),2.06-2.17(m,2H),2.24-2.45(m,3H),2.80-2.93(m,5H),3.42(br.s.),3.57(br.s.),6.12(dd,1H),7.02-7.11(m,2H),7.26-7.35(m,2H),7.77(dt,1H),8.42(dd,1H),8.59(d,1H),12-3(br.s.,1H)。

example 30

4- ({ [17- (5-methoxypyridin-3-yl) estra-1, 3,5(10), 16-tetraen-3-yl ] carbonyl } amino) butanoic acid

In analogy to example 1, 100mg (0.26mmol)17- (5-methoxypyridin-3-yl) estra-1, 3,5(10), 16-tetraene-3-carboxylic acid and 79mg 4-aminobutyric acid methyl ester hydrochloride are converted into 64mg (53% of theory) of the title compound.

C₂₉H₃₄N₂O₄(474.61). Actually measured MS-ES + mass: 474.25.

¹H-NMR(300MHz,DMSO-d₆):[ppm]=0.99(s,3H),1.38-1.78(m,7H),1.86-1.96(m,1H),2.04-2.43(m,7H),2.83-2.92(m,2H),3.15-3.26(m,2H),3.81(s,3H),6.16(s,1H),7.23-7.34(m,2H),7.51-7.59(m,2H),8.16(d,1H),8.20(d,1H),8.33(t,1H),12.0(br.s.,1H)。

in vitro pharmacological testing of Compounds of the invention

Example 31(AKR1C3 inhibitory Activity)

The AKR1C3 inhibitory activity of the agents of the invention was measured in the AKR1C3 assay described in the following paragraphs.

Basically, enzyme activity is measured by quantifying Coumberol from coumberon (Halim, m., ye, d.j. and Sames, d., j.am. chem. soc.130, 14123-14128 (2008), and Yee, d.j., Balsanek, v., Bauman, d.r., Penning, t.m. and Sames, d.natl.acad.sci.103, 13304-13309 (2006)). In this assay, the increase in superfluorescent Coumberol due to NADPH- (nicotinamide adenine dinucleotide phosphate) -dependent reduction of the nonfluorescent Coumberone by AKR1C3 can be determined.

The enzyme used was recombinant human AKR1C3 (aldehyde-ketone reductase family 1 member C3) (GenBank accession No. NM — 003739). It is expressed as a GST (glutathione S transferase) fusion protein in e.coli (e.coli) and purified by glutathione sepharose affinity chromatography. GST was removed by digestion with thrombin followed by size exclusion chromatography (Dufort, i., Rheault, p., Huang, XF., Soucy, p., andLuu-The, v., endocrinology140,568-574 (1999)).

For this assay, 50nl of 100-fold concentrated solution of the test substance in DMSO is pipetted into a black small-volume 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), and AK is addedR1C3 in assay buffer [50mM potassium phosphate buffer pH7, 1mM DTT, 0.0022% (w/v) Pluronic F-127, 0.01% BSA (w/v) and protease inhibitor cocktail (complete EDTA-free protease inhibitor cocktail from Roche)]And the mixture was incubated for 15min to pre-bind the substance to the enzyme prior to the enzyme reaction. Then, the enzyme reaction was initiated by adding NADPH (16.7. mu.M → 10. mu.M final concentration in 5. mu.l assay volume) and Coumberone (0.5. mu.M → 0.3. mu.M final concentration in 5. mu.l assay volume) to the solution in assay buffer (3. mu.l), and the resulting mixture was incubated at 22 ℃ for a reaction time of 90 min. The concentration of AKR1C3 was adapted to the respective activity of the enzyme preparation and adjusted so that the assay was performed in the linear range. Typical concentrations are in the region of 1 nM. By addition of the inhibitor EM-1404[ F. Labrie et al, U.S. Pat. No. 6,541,463,2003]The reaction was stopped with 5. mu.l of stop solution consisting of (2. mu.M → 1. mu.M final concentration in 5. mu.l of assay volume). The fluorescence of the Coumberol was then measured at 520nm (excitation at 380 nm) using a suitable measuring instrument (Pherastar from BMGLAbtechnologies). The fluorescence intensity was used as a measure of the amount of coulombles formed and thus of the enzymatic activity of AKR1C 3. Data were normalized (enzyme reaction without inhibitor =0% inhibition; with all other assay components but no enzyme =100% inhibition). Typically, test substances are tested on the same microtiter plate at 11 different concentrations ranging from 20 μ M to 96.8pM (20 μ M, 5.9 μ M, 1.7 μ M, 0.5 μ M, 0.15 μ M, 44nM, 12.9nM, 3.8nM, 1.1nM, 0.3nM and 96.8pM, dilution series prepared by successive 1:3 dilutions with 100% DMSO based on the level of 100-fold concentrated solution prior to assay), each concentration is tested twice, and IC is calculated using a 4-parameter fit₅₀The value is obtained.

As described, the inhibitory activity of the claimed pharmacological substances on AKR1C3 enzyme was examined (see table 1). For most of the claimed structural range, these compounds show strong inhibition of AKR1C3 in vitro (IC)₅₀Value of<50nM), and even most exhibit IC₅₀Value of<20nM。

Table 1: AKR1C3 inhibition of Compounds of the invention

(for most compounds, values determined in two tests are shown)

Example 32 (inhibition of Cyp17A1)

CYP17A1 (synonyms: 17 alpha-hydroxylase/17.20-lyase) is an enzyme that adds a hydroxyl group to the 17-position of the steroid D ring of pregnenolone and progesterone and thereby forms 17 alpha-hydroxyprogesterone and 17 alpha-hydroxyprogesterone. Subsequently, dehydroepiandrosterone and androstenedione are formed. Known CYP17a1 inhibitors abiraterone are for example used in the therapy of metastatic castration-resistant prostate cancer following failure of docetaxel-based chemotherapy (Urologe2010,49, 64-68). Abiraterone blocks androgen synthesis and estrogen synthesis throughout the entire body of an androgen and thereby reduces hormone production in a non-tissue specific manner, which leads to undesirable side effects (see FDA, manuscript at 28.4.2001, U.S. foodand drug administration).

Surprisingly, it has been found that the compounds of the present invention inhibit CYP17a1 (if any) only very weakly, however they have an aromatic nitrogen-containing heterocycle at the 17-position of the steroid skeleton.

Description of the assay:

the test compounds were evaluated for inhibition of CYP17a1 using the recombinant enzyme. Human CYP17A1 is expressed in E.coli (Ehmer, P.B. et al; J.Steroid biochem.mol.biol.,75, 57-63 (2000)). At 37 deg.C, with progesterone (24.95. mu.M) and³a mixture of H-Progesterone (0.05. mu.M, 101.3Ci/mmol), 50. mu.M ADPH regeneration System (in phosphate buffer with 10mM ADP +, 100mM glucose 6-phosphate and 2.5U glucose 6-phosphate dehydrogenase) and the appropriate test substance (in 5. mu.l DMSO) pre-incubate the microsomal fraction and 140. mu.L phosphate buffer (50mM sodium phosphate, 1mM MgCl, respectively)₂、0.1mMEDTA、0.1mM dithiothreitol, pH7.4) for 5 minutes. The reaction was started by addition of enzyme and after incubation at 37 ℃ for 30min, the reaction was stopped by addition of 50. mu.l of 1N hydrochloric acid.

The steroids were extracted with ethyl acetate. After evaporation of the organic phase, the steroid is dissolved in acetonitrile. 16 α -hydroxyprogesterone, 17 α -hydroxyprogesterone and progesterone were separated on a C18 reverse phase chromatography column (Nucleodur C18 gradient, 3 μm, Macherey-Nagel, Duren, Germany) on an HPLC system (Agilent1100Series, Agilent technologies, Waldbronn, Germany) using acetonitrile/water (45:55) as mobile phase. Detection and quantification of steroids is performed using a radioflow detector (bertholttech technologies, BadWildbad, Germany). Inhibition was calculated using the formula:

each value was calculated from at least three independent experiments. Final IC₅₀The value is calculated as 3 or 4 independent ICs₅₀Average of the values.

The compounds of the invention show no or only very weak inhibition of CYP17a1 (table 2) and compared to the known CYP17a1 inhibitor abiraterone (used as the free base), IC₅₀The value is greater than 10. mu.M.

Table 2: inhibition of human CYP17

^a% inhibition at 200. mu.M substance concentration

Example 33 (solubility in aqueous buffer ph 6.5):

determination of thermodynamic solubility in aqueous buffer at pH6.5 (Shake flask method)

The thermodynamic solubility was determined according to the shaking flask method (shake-flash method) [ literature: Edwards H.Kerns and LiDi (2008) SolubiityMethodsin: Drug-like Properties: Concepts, Structure Designationdmethods, p276-286.Burlington, MA, academic Press ].

Here, a saturated solution of the active compound in a buffer of ph6.5 was prepared and stirred for 24h to ensure that an equilibrium was formed between the solid and the material in solution. Then, the solution was centrifuged, and the concentration of the obtained solution was quantified with the aid of a calibration line.

For the samples, 2mg of solid matter was accurately weighed and placed into a 4ml glass bottle. 1ml of phosphate buffer solution at pH6.5 was added. The solution was stirred on a stirrer for 24h at room temperature. The solution was then centrifuged. To prepare a control for calibration, 2mg of solid material was accurately weighed and dissolved in 30ml of acetonitrile. After a short sonication, the solution was diluted to 50ml with water.

HPLC with UV detection was used to quantify samples and controls. Each sample was injected three times per injection volume (5. mu.l and 50. mu.l). For control, three injection volumes (5. mu.l, 10. mu.l and 20. mu.l) were injected.

The following chromatographic conditions were selected:

HPLC column: XterraMSC182.5 μm 4.6X 30mm

Sample volumes of 3X 5. mu.l and 3X 50. mu.l

Controls 5. mu.l, 10. mu.l, 20. mu.l

Flow rate 1.5ml/min

Mobile phase acid gradient:

a water/0.01% trifluoroacetic acid (TFA)

B acetonitrile/0.01% TFA

0min→95%A5%B

0-3min → 35% A65% B, linear gradient

3-5min → 35% A65% B, isocratic

5-6min → 95% A5% B, isocratic

UV detector: wavelength close to absorption maximum (between 200nm and 400 nm)

The area of the sample and control injection was determined using HPLC software (Waters empower2FR) and the calculation of solubility (in mg/l).

For the compound of the invention example 2, a solubility of 354mg/l was measured; the known AKR1C3 inhibitor EM-1404 showed a solubility of 0.1 mg/l.

Example 34 (endometriosis model)

To test the in vivo efficacy of the compound of exemplary example 2, an endometriosis model of tamarin was used. 4-8 years old female tamarin marmoset (body weight 340-460 g) was used. In these animals, endometriosis is induced by piercing the uterus during laparotomy and flushing with sterile media to pass uterine cells through an upper catheter into the abdomen [ Einspanier et al, molhum reprod2006 ]. After 3 months, the procedure was repeated. Before the actual start of the treatment, the animals were subjected to laparotomy and examined for the presence of endometriosis foci on the bladder, uterus and ovary. After 6 weeks, treatment was started. Two treatment groups were used, each group scaled to n =6 animals/group. Group 1 was treated with vehicle only (strawberry/banana juice) and group 2 with the test substance administered in vehicle. 30mg/kg of the test substance is administered orally once a day. The treatment period was 6 weeks. Immediately after the end of the treatment, the 2 nd laparoscopy was performed and the number and size of the lesions on the uterus, ovary and bladder were determined again. Since hardly any lesions were found on the ovaries before and after treatment, no ovarian type lesions were considered during the evaluation.

Claims

1. Compounds of formula (I), and salts thereof,

wherein

R1 and R2 independently of one another denote hydrogen, fluorine, chlorine, trifluoromethyl, pentafluoroethyl, methoxy, ethoxy, trifluoromethoxy, -OCH₂CF₃Or C₁-C₄-an alkyl group,

or the C-H group in the pyridine ring is replaced by a nitrogen atom, and

r3 and R4 represent hydrogen, or

R3 represents hydroxy and R4 represents hydrogen, or

R3 represents hydrogen and R4 represents hydroxy, and

r5 and R6 represent hydrogen, or

R5 represents fluorine and R6 represents hydrogen, or

R5 represents hydrogen and R6 represents fluorine, and

r7 represents hydrogen or C₁-C₄-alkyl, and

r8 represents-CR^aR^b-COOH, wherein

R^aAnd R^bIndependently of one another, represent hydrogen, methyl, ethyl, or

R^aRepresents hydrogen, and R^bTogether with R7 for- (CH)₂)_n-, where n is 1,2, 3,4, or

R8 represents-CR^cR^d-CR^eR^f-COOH, wherein

R^c、R^d、R^e、R^fRepresents hydrogen, or

R^c、R^dRepresents hydrogen, and R^e、R^fIndependently of one another, methyl, ethyl or together- (CH)₂)_n-, where n ═ 2,3, 4,5, or together represent-CH₂CH₂-O-CH₂CH₂-, or

R^cRepresents methyl or ethyl, and R^d、R^eAnd R^fRepresents hydrogen, or

R^cAnd R^eTogether represent- (CH)₂)_n-, where n ═ 1,2, 3 or 4, and R^dAnd R^fRepresents hydrogen, or

R8 represents-CH₂-CH₂-CHR^g-COOH, wherein

R^gRepresents hydrogen, or

R^gAnd R7 together represent-CH₂-or-CH₂CH₂-。

2. The compound of claim 1 of formula (II) and formula (III), and salts thereof,

wherein

R1 represents hydrogen, fluorine, chlorine, methoxy, ethoxy, trifluoromethoxy, methyl, ethyl, trifluoromethyl, and

r3 and R4 represent hydrogen, or

R3 represents hydroxy and R4 represents hydrogen, or

R3 represents hydrogen and R4 represents hydroxy, and

r5 represents hydrogen or fluorine, and

r7 represents hydrogen or C₁-C₄-alkyl, and

r8 represents-CR^aR^b-COOH, wherein

R^aAnd R^bIndependently of one another, represent hydrogen, methyl or ethyl, or

R^aRepresents hydrogen, and R^bTogether with R7 for- (CH)₂)_n-, where n is 3 or 4, or

R8 represents-CR^cR^d-CR^eR^f-COOH, wherein

R^c、R^d、R^e、R^fRepresents hydrogen, or

R^cRepresents methyl or ethyl, and R^d、R^eAnd R^fRepresents hydrogen, or

R8 represents-CH₂-CH₂-CHR^g-COOH, wherein

R^gRepresents hydrogen, or

R^gAnd R7 together represent-CH₂CH₂-。

3. A compound of formula (II) and formula (III) as claimed in claim 1 or 2, and salts thereof, wherein

R1 represents hydrogen, fluorine, chlorine, methoxy, trifluoromethyl, and

r3 and R4 represent hydrogen, or

R3 represents hydroxy and R4 represents hydrogen, or

R3 represents hydrogen and R4 represents hydroxy, and

r5 represents hydrogen or fluorine, and

r7 represents hydrogen, methyl or ethyl, and

r8 represents-CR^aR^b-COOH, wherein

R^aAnd R^bIndependently of one another, represent hydrogen, methyl or ethyl, or

R8 represents-CR^cR^d-CR^eR^f-COOH, wherein

R^c、R^d、R^e、R^fRepresents hydrogen, or

R^c、R^dRepresents hydrogen, and R^e、R^fIndependently of one another, methyl or ethyl, or together- (CH)₂)_n-, where n ═ 2, 4,5, or together represent-CH₂CH₂-O-CH₂CH₂-, or

R^cRepresents a methyl group, and R^d、R^eAnd R^fRepresents hydrogen, or

R^cAnd R^eTogether represent- (CH)₂)_n-, where n is 3 or 4, and R^dAnd R^fRepresents hydrogen, or

R8 represents-CH₂-CH₂-CHR^g-COOH, wherein

R^gRepresents hydrogen, or

R^gAnd R7 together represent-CH₂CH₂-。

4. A compound of formula (II) and formula (III) as claimed in claim 1 or 2, and salts thereof, wherein

R1 represents hydrogen, fluorine, methoxy, trifluoromethyl, and

r3 and R4 represent hydrogen, or

R3 represents hydroxy and R4 represents hydrogen, or

R3 represents hydrogen and R4 represents hydroxy, and

r5 represents hydrogen or fluorine, and

r7 represents hydrogen or methyl, and

r8 represents-CR^aR^b-COOH, wherein

R^aAnd R^bIndependently of one another, represents hydrogen or methyl, or

R^aRepresents hydrogen, and R^bTogether with R7 for- (CH)₂)₃-, or

R8 represents-CR^cR^d-CR^eR^f-COOH, wherein

R^c、R^d、R^e、R^fRepresents hydrogen, or

R^cAnd R^dRepresents hydrogen, and R^eAnd R^fRepresents methyl or together represents- (CH)₂)_n-, where n ═ 2 or 4, or together represent-CH₂CH₂-O-CH₂CH₂-, or

R^cRepresents a methyl group, and R^d、R^eAnd R^fRepresents hydrogen, or

R8 represents-CH₂-CH₂-CHR^g-COOH, wherein

R^gRepresents hydrogen, or

R^gAnd R7 together represent-CH₂CH₂-。

5. A compound, and salts thereof, having the following names

6. A compound according to any one of claims 1 to 5 for use in the treatment and/or prevention of a disease.

7. Use of a compound according to any one of claims 1 to 5 for the preparation of a medicament for the treatment and/or prophylaxis of diseases.

8. A compound as claimed in any one of claims 1 to 5 for use in the treatment and/or prevention of endometriosis, uterine leiomyoma, uterine bleeding disorders, dysmenorrhea, prostate cancer, prostatic hyperplasia, acne, seborrhea, alopecia, premature maturation, polycystic ovary syndrome, breast cancer, lung cancer, endometrial cancer, renal cell carcinoma, bladder cancer, non-Hodgkin's lymphoma, chronic obstructive pulmonary disease, obesity or inflammatory pain.

9. A medicament comprising a compound according to any one of claims 1 to 5 in combination with one or more compounds selected from the group consisting of: selective estrogen receptor modulators, estrogen receptor antagonists, aromatase inhibitors, 17 β HSD1 inhibitors, steroidal sulfatase inhibitors, GnRH agonists and antagonists, kisspeptin receptor antagonists, selective androgen receptor modulators, androgens, 5 α -reductase inhibitors, selective progesterone receptor modulators, progestogens, antiprogestins, oral contraceptives, inhibitors of mitogen-activated protein kinase and inhibitors of mitogen-activated protein kinase, inhibitors of protein kinase B, inhibitors of phosphoinositide 3-kinase, inhibitors of cyclin-dependent kinase, inhibitors of hypoxia-induced signal transduction pathways, histone deacetylase inhibitors, prostaglandin F receptor antagonists and non-steroidal anti-inflammatory drugs.