Background of the invention 1. Field of the invention

This invention relates to 17-substituted steroids and their use in the treatment of androgen-dependent and oestrogen-dependent disorders, especially prostatlc cancer and breast cancer respectively. 2. Description of the related art

The 17α-hydroxylase/C ₁₇ _20 ¹ y ^se enzyme complex ⁽ hereinafter "hydroxyl se/lyase") is known to be essential for the biosynthesis of androgens and oestrogens. In the treatment of androgen-dependent disorders, especially prostatlc cancer, there is a need for strong inhibitors of hydroxylase/lyase. Certain anti-androgenic steroids are well known, for example Cyproterone acetate (17α-acetoxy-6-chloro-lα,2α-methylene-4,6-pregnadiene- 3,20-dione). Many other steroids have been tested as hydroxylase/lyase Inhibitors. See, for example, PCT Specification WO 92/00992 (Schering AG) which describes anti-androgenic steroids having a pyrazole or trlazole ring fused to the A ring at the 2,3- position, or European Specifications EP-A 288053 and EP-A 413270 (Merrell Dow) which propose 17(3-cyclopropyl mino- androst- 5-en-3β-ol or -4-en-3-one and their derivatives. Summary of the invention

It has now surprisingly been found that steroids lacking a C20 side chain and having a 17-(3-pyridyl) ring in its place, together with a 16,17-double bond, are powerful hydroxylase/lyase inhibitors, useful for the above-stated purposes. According to the invention, there are provided compounds of the general formula

wherein X represents the residue of the A, B and C rings of a steroid, R represents a hydrogen atom or an alkyl group of 1 - 4 carbon atoms, R ¹⁴ represents a hydrogen atom, a halogen atom or an alkyl group of 1 to 4 carbon atoms and each of the R ¹ **- substituents independently represents a hydrogen atom or an alkyl or alkoxy group of 1-4 carbon atoms, a hydroxy group or an al ylcarbonyloxy group of 2 to 5 carbon atoms or together represent an oxo or methylene group or R ¹⁴ and one of the R ¹ - ^* - groups together represent a double bond and the other R^ group represents a hydrogen atom or an alkyl group of 1 to 4 carbon atoms, and R ¹ ^ represents a hydrogen atom, halogen atom, or an alkyl group of 1 to 4 carbon atoms, in the form of the free bases or pharmaceutically acceptable acid addition salts.

The term "steroid" herein includes any compound having the steroidal B and C rings, but in which all or part of the A ring is missing e.g. ring not closed (lacking the 2- or 3-position C-atom or both) or takes the form of a cyclopentane ring. It also includes azasteroids having a ring nitrogen atom in place of a ring carbon atom, especially in the A-ring such as in 4-azasteroids.

In general, the compounds of formula (1) are new and such compounds per se are included in tήe invention. However, certain of them have been disclosed as intermediates in the synthesis of certain steroids having a 3-pyridyl or 3-pyridonyl group in the

17β-position, see J. Wicha and M. Masnyk, Bulletin of the Polish Academy of Sciences: Chemistry 3_3_ (1-2), 19-27 and 29-37 (1985). The first of these papers says that a 17β-side chain of the form -C-C-C-0 or -C-C-C-N favours cardiotonic properties and describes the synthesis of 17β- ⁽ 3-pyridyl)-14β-androst-4-ene-3β,14-diol , while the second uses this compound to prepare 17 3-pyr1d-2(1H ⁾ onyl]-l4β-androst-4-ene-3β,14-diol . Those final compounds differ from those of the present invention by having a saturated D-ring and the paper contains no test results. Insofar as certain compounds within formula (1) are known as intermediates in these syntheses, the invention extends to the compounds themselves only for use in therapy.

These are 17- ⁽ 3-pyridyl)androsta-5,14,16-trien-3β-ol and 15α- and 15β-acetoxy-17- ⁽ 3-pyridyl ⁾ androsta-5,16-dien-3β-ol and their 3-acetates. See also 3 Wicha. £fc. aj_., Heterocycles 2Q, 231-234 (1983 ⁾ which 1s a preliminary communication of the first of the above two papers.

J. Wicha £±. _ _. _t Bulletin of the Polish Academy of Sciences, Chemistry 21 <l-2), 75-83 (1984) have also described the preparation of 3β-methoxy-17β-(3-pyridyl)androstane and pyridone analogues thereof via the Intermediate 3β-methoxy-17-<3-pyridyl) androst-16-ene. Accordingly, the invention extends to the latter compound only for use in therapy. A preliminary communication of this paper, by J. Wicha and M. Masynk, appeared in Heterocycles Ifi, 521-524 (1981).

The invention also includes pharmaceutical compositions comprising a compound of formula (1) in association with a pharmaceutically acceptable diluent or carrier. The terminology "pharmaceutical compositions" implies that Injectlble formulations are sterile and pyrogen-free and thereby excludes any compositions comprising the compound of formula (1) and a non-sterile organic solvent, such as may be encountered in the context of the final stages of preparing these above-mentioned compounds of formula (1) which have been described in the literature but without any therapeutic use being mentioned.

Description of the preferred embodiments

In the compounds of the invention the essential structural features comprise all of: a 3-pyridyl ring in the 17-position - a ring double bond in the 16,17-position of the D-ring the 18-positlon methyl group

It is critical that the pyridine nitrogen atom be in the 3-posit1on, not the 2- or 4-position. It is also critical that the pyridine ring be joined directly to the 17-carbon atom. This critlcallty is demonstrated by tests of inhibiting activity against hydroxylase and lyase (Table 1). The concentration of test compound required to achieve 50% inhibition of the enzyme is far greater for the 2-pyridyl, 4-pyridyl and 2-pyridyl ethyl compounds tested than for the 3-pyridyl. The methods of determination were as described in the Examples hereinafter.

TABLE 1 Effect of variations in the 17-substitutent on inhibition of hydroxyl ase and lyase, demonstrating the criticality of the 17-substituent in this invention.

>17 IC ₅₀ (μM) lYEfe Lvase Hvdroylase

2-Pyridyl 0.13 0.32

(for comparison)

3-pyridyl 0.003 0.004 (present invention)

4-pyridyl 2.0 5.0 (for comparison)

XX 2-picolyl >10 >10

^■ H (for comparison)

Note: all the compounds of formula (2) tested were poor inhibitors of aromatase: IC5 _Q >20 μM.

Our modelling of the geometry of the putative transition state of the lyase component of the hydroxyl se-lyase enzyme complex, in the putative mechanism of action of the lyase component, suggests that the 16, 17-double bond is essential to allow the 3-pyridlne ring to adopt the orientation required for co-ordination to the haem group of the hydroxylase-lyase complex.

Elsewhere, the D-r1ng can have any other simple substltuent.

Certain simple substituents are defined 1n connection with the preferred general formula (1), but it will be appreciated that others could be substituted for those of formula (1). In the compounds of formula (1 ⁾ , R ¹⁵ is preferably hydrogen or alkyl of

1 to 3 carbon atoms, R ¹⁶ hydrogen, alkyl of 1 to 3 carbon atoms, fluorine, chlorine, bromine or iodine, and R hydrogen or methyl, in the 5-position of the pyridine ring. The remainder of the molecule, designated "X" In formula (1), can be of any kind conventional in steroid chemistry or have any other feature known In steroids having ant1-androgen1c activity, for example the pyrazole or triazole ring, fused to the A ring at the 2- and 3- positions, disclosed In the above-cited Specification WO 92/00992, or oxazole ring fused in the same positions.

By way of example, X can represent the residue of androstan-3α- or 3β-ol, androst-5-en-3α- or 3β-ol, androst-4-en-3-one, androst-2-ene, androst-4-ene, androst-5-ene, androsta-5,7-d1en-3α or 3β-ol , androsta-l,4-dien-3-one, androsta-3,5-diene, estra-1 ,3,5C10]-triene, estra-1 ,3,5[103-trien-3-ol ,

5α-androstan-3-one,

androst-4-ene-3,11-dione,

6-fluoroandrost-4-ene-3-one or androstan-4-ene-3,6-dione each of which, where structurally permissible, can be further derivatised in one or more of the following ways: to form 3-esters, especially 3-alkanoates and -benzoates, to have one or more carbon to carbon ring double bonds 1n any of the 5,6-, 6,7- 7,8-, 9,11- and 11 ,12-positions as 3-oximes - as 3-methylenes as 3-carboxylates as 3-nitriles as 3-nitros as 3-desoxy derivatives - to have one or more hydroxy, halo, C* _j _4-alkyl, trifluoromethyl, C- _j _4-alkoxy, C- _j ^-alkanoyloxy, benzoyloxy, oxo, methylene or alkenyl substituents in the A, B or C-r1ng to be 19-nor.

Preferred C- _| _4-alkyl and alkoxy groups are methyl and ethoxy. Preferred C- _j ^-alkanoyloxy groups are acetoxy and propanoyloxy.

Preferred halo groups are fluoro, bromo and chloro and the preferred substitution position is the 6-posit1on.

The substituents include, for instance, 2-fluoro, 4-fluoro, 6-fluoro, 9-fluoro, 3-tr1fluoromethyl , 6-methyl , 7-meth l , 6-oxo, 7-oxo, 11-oxo, 6-methylene, 11-methylene, 4-hydroxy, 7-hydroxy, 11-hydroxy or 12-hydroxy, each in any appropriate epimeric form, and, subject to structural compatibility (well known in general steroid chemistry), in any combination of two or more such groups.

Compounds which are likely to be unstable are considered excluded from consideration. Such compounds will be evident to steroid chemists. Compounds having esoteric substituents likely to interfere with the stereochemical alignment of the steroid

molecule with the enzymes to be inhibited, by virtue of steric or electronic distribution effects, are to be avoided. For example, a 2,3,5,6-tetrafluoro-4-trifluoromethylphenoxy substltuent in the 3-posit1on is not recommended. Androst-5-en-3β-ol having such an ether substituent in place of the 3β-hydroxy group has been shown to be a very poor inhibitor for lyase and hydroxyl se.

The currently preferred compounds of formula (1) include those which are saturated and unsubstituted at the 11- and 12-positions and which therefore are of the general formula (3):

wherein Q represents the residue of A, B and C rings of a steroid, and R is a hydrogen atom or an al yl group of 1-4 carbon atoms.

However, 11- and/or 12-substituted compounds are also active. Particularly preferred are 11-oxo and llβ-hydroxy derivatives of compounds of formula (3).

Specifically preferred compounds of the invention comprise 17-(3-pyridyl)androsta-5,l6-dien-3β-ol, 17-(3-pyr1d 1)androsta-3,5,16-tr1ene, 17-(3-pyrid 1)androsta-4,16-dien-3-one, 17-(3-pyridyl)estra-l,3,5C10],16-tetraen-3-ol, 17-(3-pyridyl)-5α-androst-l6-en-3α-ol and their acid addition salts and 3-esters.

Other notable compounds of the invention comprise 17-(3-pyrid l)-5α-androst-l6-en-3-one, 17-(3-pyridyl>-androsta-4,16-diene-3,11-dione, 17-(3-pyridyl)-androsta-3, 5,I6-trien-3-ol,

6α-and 6β-fluoro-17-(3-pyridyl)androsta-4,l6-dien-3-one

17-(3-pyridyl)androsta-4,16-dien-3,6-dione,

17-[3-<5-methyl pyr1dyl)]androsta-5,!6 dien-3β-ol

3α-trif1uorometh 1-17-(3-pyrid 1)androsta-l6-en-3β-ol and their acid addition salts and 3-esters.

Insofar as certain compounds within formula (1) are known j-gr £ and these are compounds which are less easy to prepare than many of the others, a preferred class of compounds of formula (1) is those which do not have a 3β-alkoxy group, a 14, 15-double bond or a 15-ester group.

The compounds of formula (1) can be prepared by a method which is in itself novel and inventive. Starting from a 17-oxo compound of general formula (4):

wherein X, R ¹⁴ , R ¹⁵ and R ¹⁶ are as defined above and any other oxo groups and hydroxy groups in the molecule are first appropriately protected, the method comprises replacing the 17-hydroxy group of compound (4) in its enol form by a leaving group (L) which is capable of being replaced by a 3-pyridyl group 1n a palladium complex-catalysed cross-coupling reaction with a pyridyl ring-substituted boron compound of formula (5):

wherein Z ¹ and Z ² independently represent hydroxy or alkoxy or alkyl of 1-4 carbon atoms each, preferably ethyl or methoxy, and R is as defined above and carrying out said cross-coupling reaction.

The palladium complex-catalysed cross-coupling reaction of the 17-substituted steroid with the boron compound 1s believed to involve the steps indicated in the following illustrative reaction scheme (Py = 3-pyridyl). The pyridyl anionic species is provided by the boron compound.

The replacement of the 17-enol group can be, for example, to form a 16,17-ene trifluoromethanesulphonate of formula (6):

or a 17-1odo or bromo-16, [17]-ene of formula (7) :

(Hal = I or Br)

Compounds of formula (6) can be prepared by reacting the 17-oxo compound of formula (4) with an enol ester-forming trlfluoromethanesulphonic acid derivative such as the anhydride, see S. Cacchi, E. Morera and G. Ortar, Tetrahedron Letters, 21, 4821 (1984). The 17-oxo compound can be considered notionally to exist 1n the enol form, the reaction being one of esterification of the enol.

Compounds of formula (7) can be prepared by first hydrazinatlng the 17-oxo compounds of formula (4) by a standard method to form the 17-hydrazone, which 1s then reacted with bromine or Iodine in the presence of an amine or guanldine base, see D. Barton, G. Bashiardes and 3. Fourrey, Tetrahedron Letters, 24, 1605 (1983).

For the preparation of the 17-position derivatives of formula (6) or (7) any necessary protection of other groups in the molecule is first carried out. For example hydroxyl groups are conveniently protected as their acetates, whilst the 3-oxo group of steroids can be selectively protected as their perfluorotolyl enol ethers, see M. Jarman and R. McCague, J.Chem.Soc. Perkin Trans.1, 1129 (1987).

The 17-positlon derivative is then reacted with the boron compound of formula (5) using as catalyst a palladium(O) phosphine complex, for example tetrakis(tr1phenylphosph1ne)palladium(0), or a palladium (II) phosphine complex which is reducible in situ to a palladlum(O) phosphine species, especially bis ⁽ tr1phenylphosphine>pallad1um (II) chloride.

Further compounds of the invention can be prepared by standard steroid to steroid inter-conversion chemistry, so long as the D-rlng chemical structure 1s not affected thereby. If the D-ring structure is likely to be affected, 1t would usually be necessary to prepare the desired compound d£ novo. i.e. by choosing the appropriate starting compound of formula (4), protected if necessary, and carrying out the reactions of 17-substitution of the enol and cross-coupling with the boron compound as described above.

By way of example, the 3-esters of a steroid 3-ol with an alkanoic acid of 1 to 6 carbon atoms, or a cycloalkanolc acid or aralkanoic add such as phenylacetic or phenylpropionic acid, an aroic acid such as benzole acid, or other simple organic add such as methanesulphonic add, can be converted Into the 3-ol or the 3-ol to the 3-ester. Other examples of simple conversions which would not affect the D-ring structure are

1) Oppenauer oxidation using cyclohexanone and aluminium isopropoxide to convert 3-hydroxy to 3-oxo steroids and notably Δ ⁵ » ⁶ -3-hydroxy to Δ ⁴ » ⁵ -3-oxo steroids; ii) W1tt1g oleflnatlon to convert oxo groups to methylene groups

[D. D. Evans fit al-. J. Chem. Soc, 4312-4317, (1963)]; iϋ) Oxidation of Δ ⁵ -3β-hydroxy to Δ ⁴ -3,6-dione steroids using ϋ-meth lmorpholine ϋ-oxide and tetra-n-propylammonium perruthenate catalyst CM. Moreno et aj.. , Tetrahedron Letters, 21. 3201-3204, (1991)]; iv) 6-Methylenation of Δ ⁴ -3-oxo steroids using formaldehyde dimethylacetal DC. Annen ≤t al., Synthesis, 34-40 (1982)];

v) Conversion of Δ ⁴ -3-oxo to 4,4-dimethyl-Δ ⁵ -3-oxo, Δ^-S-oxo, Δ ¹ . ⁴ - ⁶ -3-oxo, 7α-methyl-Δ -3-oxo, Δ » ⁶ -3-oxo, 6-chloro-Δ- ^4»6 -3-oxo, Δ ² » ⁴ -2,3-isoxazole, 6α-meth l-Δ ⁴ -3-oxo and Δ -3-desoxy; Δ ⁵ -3β-ol to 5α-fluoro-6-oxo, 5α,6,6-trifluoro, 6,6-d1fluoro and 6α-fluoro-Δ ⁴ -3-oxo; and 11-oxo to 11-hydroxy and Δ ⁹ * ¹¹ steroids CD. Lednicer and L. A. Mltscher, The Organic Chemistry of Drug Synthesis, Is. 2 and 3, Wiley (1980 and 1984)] or v ⁾ Electroph111c fluorinatlon of steroids using N-fluoropyridinlurn reagents [T. Umenoto fit al. , Organic Synthesis £2, 129 - 143 (1990)].

The compounds of formula (1) may be prepared as salts, e.g. the hydrochlorlde and converted to the free base form and thereafter to such other conventional pharmaceutically acceptable salts as acetates, citrates and lactates, as may seem appropriate.

The present invention also provides a pharmaceutical composition which comprises a therapeutically effective amount of a compound of the invention, in association with a therapeutically acceptable carrier or diluent. The composition of the invention can, for example, be in a form suitable for parenteral (e.g. Intravenous, Intramuscular or Intracavital), oral, topical or rectal administration. Particular forms of the composition may be, for example, solutions, suspensions, emulsions, creams, tablets, capsules, lipso es or micro-reservoirs, especially compositions in orally Ingestlble or sterile injectable form. The preferred form of composition contemplated 1s the dry solid form, which includes capsules, granules, tablets, pills, boluses and powders. The solid carrier may comprise one or more excipients, e.g. lactose, fillers, disintegrating agents, binders, e.g. cellulose, carboxymethylcellulose or starch or anti-stick agents, e.g. magnesium stearate, to prevent tablets from adhering to tabletting equipment. Tablets, pills and boluses may be formed so as to disintegrate rapidly or to provide slow release of the active ingredient.

Where national patent law permits, the present invention also includes a method of treating androgen- and oestrogen-dependent disorders, especially tumours, and most especially prostatlc tumours, in the mammalian body, which comprises administering a compound of the invention to a mammalian patient in a therapeutically effective dose, e.g. in the range 0.001-0.04 mmole/kg body weight, preferably 0.001-0.01 mmole/kg, administered dally or twice daily during the course of treatment. This works out ⁽ for humans) at 20-800 mg/pat1ent per day. Alternatively the invention includes the compounds of the invention for use in said treatment and their use in the manufacture of medicaments for that purpose. The preferred use is in treating prostatlc cancer. Another use is in treating breast cancer. The following Examples Illustrate the invention. Example 1

(a) 3B-Acetoxvandrosta-5.16-d1en-17-vl trif1uoromethanesul honate

To a stirred solution of dehydroepiandrosterone-3-acetate

(24.8g, 75 mmol) 1n dry dlchloromethane (500 ml) containing 2,6-di-i-butyl-4-methylpyr1d1ne (18.5g, 90 mmol) was added trifluoromethanesulphonic anhydride (12.6 ml, 75 mmol). After 12h the mixture was filtered and washed with water (50 ml), dried ( gSU ), and the solvent evaporated. Chromatography, on elution with light petroleum-dlchloro ethane (6:1), gave firstly androsta-3,5,16-tr1en-17-yl trifluoromethanesulphonate (3.02g, 10%) as an oil. ^-NMRCCDC^) Inter alia δ 0.99 (3H,s,18-CH ₃ ), 1.02(3H,s,19-qH ₃ ), 5.39(lH,m,6-H), 5.59(lH,m,16-P, 5.62 ⁽ lH,m,3- H), 5.93(1H,dm,J 9.4HZ.4-H); MS m/z 402(M ⁺ ). Further elution with light petroleum-dichloromethane (3:1) afforded the title compound (20.Ig, 58%) which crystallised from hexane, m.p. 75-76 ^β C. ¹ H-NMR(CDC1 ₃ ) inter alia δ 1.00(3H,s,18-CH ₃ ), 1.06(3H, s,19-CH ₃ ), 2.04(3H,s,CH ₃ C0 ₂ ), 4.59 ⁽ lH,m,3α-Ji), 5.39(lH,dm,J 4.9 Hz,6-Jl), 5.58(lH,m,16-H). Anal. Calcd: C,57.13; H,6.32; s,6.93. Found: C.57.29; H,6.31; S,6.96%.

(b) 3B-Acetoxy-17- ⁽ 3-Pyridyl ⁾ androsta-5.16-di ene

Diethyl(3-pyridyl)borane (3.38g, 23 mmol) from Aldrich Chemical Co. Ltd. was added to a stirred solution of 3β-acetoxyandrosta-5,16-dien-17-yl trif1uoromethanesulphonate (6.94g, 15 mmol) in THF (75 ml) containing bis(tr1phenylphosphine)pallad1um(II) chloride (0.105g, 0.15 mmol). An aqueous solution of sodium carbonate (2M, 30 ml) was then added and the mixture heated, with stirring, by an oil bath at 80 ^e C for Ih, and allowed to cool. The mixture was partitioned between diethyl ether and water, the ether phase was dried (Na ₂ C0 ₃ ), filtered through a short plug of silica, and concentrated. Chromatography, on elution with light petroleum- diethyl ether (2:1), afforded the title compound (4.95g, 84%) which crystallised from hexane, m.p. 144-145°C, ¹ H-NMR(CDC1 ₃ ) inter alia δ 1.05(3H,s,19-CH ₃ ), 1.08(3H,s,18-CH ₃ ), 2.04(3H,s,CH ₃ C0 ₂ ), 4.60(lH,m,3α-H), 5.42(lH,dm, 3 4.7Hz,6-H), 5.99(lH,m,16-H), 7.23(lH,m,Py 5-H) 7.65(lH,m,Py 4-H), 8.46(lH,m,Py 6-H), 8.62(lH,m,Py 2-H). Anal. Calcd: C, 79.75; H, 8.50; N, 3.58. Found: C, 79.78; H, 8.52; N, 3.54%. Example 2

17-(3-Pyr1dyl)androsta-5.16-dien-3B-ol

To a solution of 3β-acetoxy-l7-(3-pyr1dyl)androsta-5,l6-d1ene (4.90g, 12.5 mmol) in methanol (50 ml) was added an aqueous solution of sodium hydroxide (10% w/v, 10 ml) and the mixture heated, with stirring, on an oil bath at 80 ^e C for 5 min., then allowed to cool. The mixture was poured Into water, neutralised with hydrochloric add (IM), rebasified with saturated sodium bicarbonate solution, and extracted with hot toluene (3 x 100 ml). The toluene extracts were combined, dried (Na2C0 ₃ , and concentrated. Chromatograph , on elution with toluene-diethyl ether (2:1) afforded the title compound (3.45g, 79%) which crystallised from toluene, p 228-229°C; -H-NMR (CDC1 ₃ inter alia δ 1.05(3H,S,19-CH ₃ ), 1.07(3H,s,18-CH ₃ ), 3.54(lH,m,3α-H), 5.40(lH,dm, 3 5.0 Hz, 6-H), 5.99(lH,m,16-H), 7.22(lH,m,Py5-H), 7.65(lH,m,Py 4-H), 8.46(lH,m,Py 6-H), 8.62(lH,m,Py 2-H). Anal. Calcd: C, 82.47; H, 8.94; N, 4.01. Found: C, 82.40; H, 8.91; N, 3.97%.

Example 3 17-(3-Pyridyl )androsta-3.5.16-t ene

The method followed that described in Example 1, using in step (b) diethyl(3-pyridyl)borane (0.88g, 6.0 mmol), androsta- 3,5,16-trien-17-yl trifluoromethanesulphonate (2.01g, 5.0 mmol), prepared in step (a), THF (25 ml), bls(triphenylphosphine)- palladium(II) chloride (35 mg, 0.05 mmol), and aqueous sodium carbonate (2M, 10 ml). Chromatograph , on elution with dichloromethane, afforded the title compound (1.39g, 84%) which crystallised from hexane, m.p. 110-112 ^β C. ^] H-NMR (CDC1 ₃ ) inter alia δ 1.02(3H,s,19-CH ₃ ), 1.07(3H,s,18-CI ₃ ), 5.44(lH,m,6-H), 5.61(lH,m,3-H), 5.95(lH,dm, 39.8Hz, 4-H), 6.01(lH,m,16-H), 7.23(lH,m,Py 5-H), 7.66(lH,m,Py 4-H), 8.46(lH,m,Py 6-H), 8.63(lH,m,Py 2-H); MS m/ ^~ .331 (M ⁺ ). Example 4

S__ 3-r2.3.5.6-tetraf1uoro-4-(trifluoromethvl)phenoxv]androsta-

3.5.16-trien-17-vl trifluoromethanesulphonate

The method followed that described in Example 1(a) but using 3-C2,3,5,6-tetraf1uoro-4-(tr1fluoromethy1)phenox ]androsta-3,5- dien-17-one (5.03g, 10 mmol), prepared as described in M. Jarman and R. McCague, 3. Chem. Soc, Perkin Trans. 1, 1129 (1987), dichloromethane (80 ml), 2.6-d1-i-butyl-4-methylpyr1d1ne (2.87g, 14 mmol), and trlfluoromethanesulphonic anhydride (1.85 ml, 11 mmol). Chromatography, on elution with light petroleum- dichloromethane (10:1), afforded the title compound (1.93α. 30%) which crystallised from ethanol, m.p. 106-107'C. ^- MR (CDC1 ₃ ) inter alia δ 1.02(6H,s,18 and 19-CH-), 5.16(lH,s,4-H), 5.28(lH,m,6-M), 5.59(lH,m,16-H); MS m/z 634 (M ⁺ ). (b) 3-[2.3.5.6-tetrafluoro-4-(trifluoromethv1)phenoxy3-17- (3-Pyrldyl)androsta-3.5.16-triene

The method essentially followed that of Example Kb) but using diethyl(3-pyridyl)borane (0.44g, 3.0 mmol), 3-L2,3,5,6- tetraf1uoro-4-(trif1uoromethy1)phenoxy]androsta-3,5,16-trien - 17-yl trifluoromethanesulphonate (l-27g, 2.0 mmol), THF (10 ml),

bis<triphenylphosphine)palladium(II) chloride (70mg, 0.1 mmol), and aqueous sodium carbonate (2M, 5 ml). Chromatography, on elution with light petroleum-diethyl ether (3:1), afforded the title compound (0.82g, 73%) which crystallised from hexane, m.p. 166.0-166.5°C. ^-NMR <CDC1 ₃ > inter alia δ 1.05(3H,s,19-CH ₃ ), 1.07(3H,s,18-CH ₃ ), 5.18(lH,s,4-H), 5.32(lH,m,6-H), 6.01(lH,m,16-H), 7.23(lH,m,Py 5-H), 7.66(lH,m,Py 4-H), 8.47(lH,m,Py 6-H), 8.63(lH,m,Py 2-H). Anal. Calcd: C, 66.07; H, 5.01; N, 2.49; F, 23.60. Found: C, 65.97; H, 5.02; N, 2.47; F, 23.41%.

(c) 17-(3-Pyridyl)androsta-4.16-d1en-3-one

To a solution of 3-[2,3,5,6-tetrafluoro-4-(trifluoromethyl)- phenoxy]-l7- ⁽ 3-pyr1dyl ⁾ androsta-3,5,l6-triene (0.423g, 0.75 mmol) in THF (5 ml) was added ethanol (5 ml) followed by aqueous hydrochloric acid (IM, 5 ml) and the mixture heated, with stirring, by an oil bath at 65 ^β C for 48h and allowed to cool. The mixture was poured into water (20 ml), neutralised with aqueous sodium hydroxide (IM), and extracted with diethyl ether (3 x 30 ml). The ether extracts were combined, dried (Na C0 ₃ ), and concentrated. Chromatography, on elution with diethyl ether, afforded the title compound (185mg, 71%) which crystallised from diethyl ether, m.p. 148-150 ^β C. IR v ax 1674 cm ^-1 ; ¹ H-NMR(CDC1 ₃ ) inter alia δ 1.07(3H,s,18-CH ₃ ), 1.24(3H,s,19-CH ₃ ), 5.76(lH,s,4-H), 5.99(lH,m,16-H), 7.23(lH,m,Py 5-H), 7.64(lH,m,Py 4-H), 8.47(lH,m,Py 6-H), 8.62(lH,m,Py 2-H); MS m/ ^~ 347 (M ⁺ ).

Example 5

(a) 3-Acetoxyestra-1.3.5π03.16-tetraen-17-yl trifluoromethanesulphonate The method followed that described in Example 1(a), but using oestrone-3-acetate (4.69g, 15 mmol), dichloromethane (120 ml), 2,6-di-t-butyl-4-methylpyridine (4..00g, 19.5 mmol), and trifluoromethanesulphonic anhydride (2.8 ml, 16.5 mmol). Chromatography, on elution with lig t petroleum-dichloromethane (3:1), afforded the title compound (5.21g, 78%). -H-NMR(CDC1 ₃ )

inter alia δ 1.00(3H,s,18-CH ₃ ), 2.29(3H,s,CH ₃ C0 ₂ ), 5.62(lH,m,16-li), 6.81(lH,m,Arϋ), 6.85(lH,m,ArH), 7.26(lH,m,ArH). Anal. Calcd. for C2* _j H2 ₃ 0 ₅ F ₃ S ₁ .%H ₂ 0: C, 55.62; H, 5.34. Found: C, 55.58: H, 5.14%. (b) 3-Acetoxv-17-(3-pvridyl)estra-1.3.5ClQ].16-tetraene

The method followed that described in Example Kb), but using diethyK3-pyridyl)borane (1.65g, 11.2 mmol), 3-acetoxyestra-1,3,- 5C10],16-tetraen-17-yl trifluoromethanesulphonate (3.56g, 8.0 mmol), THF (40 ml), bis(triphenylphosphine)palladium(II) chloride (56mg, 0.08 mmol), and aqueous sodium carbonate (2M, 15 ml).

Chromatography, on elution with light petroleum-diethyl-ether (2:1) afforded the title compound (2.40g, 80%). ^T H-NMR(CDC1 ₃ ) inter,alla δ 1.04(3H, S,Ϊ8-CM), 2.29(3H, s, CJd ₃ C0 ₂ ), 6.03(lH,m,16-Ji), 6.82(lH,m,ArH), 6.85(lH,m,ArH),

7.24(lH,m,Py 5-M), 7.29 ⁽ lH,m,Aril), 7.69(lH,m,Py 4-M), 8.48(lH,m,Py 6-H), 8.65(lH,m,Py 2-H); MS m/z 373. (M ⁺ ). Example 6 l7-(3-Pyr1dyl)estra-1.3.5ClQ].16-tetraen-3-o1 The method followed that described in Example 2, but using 3-acetoxy-17-(3-pyridyl)estra-l,3,5C10],16-tetraene (2.36g, 6.3 mmol), methanol (40 ml), aqueous sodium hydroxide (10% w/v, 5 ml), and the mixture was heated at 80°C for 10 min. Chromatography, on elution with toluene-methanol (8:1), afforded the title compound (1.40g, 67%) which crystallised from toluene, m.p. 256-258 ^# C: ¹ H-NMR(DMS0) Inter alia δ 1.01(3H,s,18-CM ₃ ), 6.15(lH,m,16-D, 6.47(lH,m,Arl), 6.52(lH,m,Arϋ), 7.04(lH,m,ArH ⁾ , 7.35(lH,m,Py 5-H), 7.79(lH,m,Py 4-H), 8.45(lH,m,Py 6-H), 8.62(lH,m,Py 2-H). Anal. Calcd: C, 83.34; H, 7.60; N, 4.23. Found: C, 83.39; H, 7.78; N, 4.06%. Example 7 3o.-Acetoxv-17-(3-pvridvl)-5 -androst-16-ene

The method followed that described in Example 1, using in

step (b) diethyl(3-pyridyl)borane (1.41g, 9.6 mmol), 3α-acetoxy- 5α-androst-16-en-17-yl trifluoromethanesulphonate (3.44g, 7.4 mmol), prepared from the 3α-acetoxy-5α-androstan-17-one by the method of step (a), THF (40 ml), bis(triphenylphosphine)- palladium(II) chloride (52 mg, 0.07 mmol), and aqueous sodium carbonate (2M, 15 mmol). Chromatography, on elution with light petroleum-diethyl ether (2:1), afforded the title compound (2.39g, 82%), ^- MR (CDC1 ₃ ) inter alia δ 0.85(3H,s,19-CH ₃ ), 1.0K3H,S,18-CH ₃ ), 2.06(3H,s,CH ₃ C0 ₂ ), 5.02(1H,m,3β-H), 6.00(lH,m,16-H), 7.24(lH,m,Py 5-H), 7.68(lH,m,Py 4-H), 8.47(lH,m,Py 6-H), 8.63(lH,m,Py 2-H); MS m/z 393 (M ⁺ ). Example 8 17-(3-Pyr1dyl)-5α-androst-l6-en-3--ol

The method followed that described in Example 2, but using 3α-acetoxy-17-(3-pyridyl)-5α-androst-16-ene (2.33g, 5.9 mmol), methanol (40 ml), aqueous sodium hydroxide (10% w/v, 8 ml), and the mixture was heated at 80 ^e C for 20 min. Chromatography, on elution with toluene-methanol (40:1), afforded the title compound (1.62g, 78%) which crystallised from toluene, m.p. 198-199°C; ¹ H-NMR(CDC1 ₃ ) jjitfir alia, δ 0.84(3H,s,19-CH ₃ ), 1.00(3H,s,18-CH ₃ ), 4.06(lH,m,3β-H>, 5.97(lH,m,16-H), 7.2KlH,m,Py 5-H), 7.64(lH,m,Py 4-M), 8.45(lH,m,Py 6-H), 8.61(lH,m,Py 2-H). Anal. Calcd: C, 82.00; H.9.46; N.3.99. Found: C81.78; H.9.47; N.3.96%. Example 9

17-(3-Pyridv1)-5α-androst-16-en-3-one

From a solution of l7-(3-Pyr1dyl)-5α-androst-l6-en-3α-ol (1.05g, 3.0 mmol) in dry toluene (60ml) and cyclohexanone (10ml) was distilled off part of the solvent (20ml) to eliminate moisture. After allowing to cool to 90°C, aluminium isopropoxide (1.02g, 5.0 mmol) was added and the mixture heated under reflux for 90 min. then allowed to cool. The mixture was diluted with diethyl ether (250 ml), washed with aqueous trisodium citrate (15% w/v; 2 x 30ml), dried ( a2C0 ₃ ), and concentrated. Chromatography, on

elution with toluene-methanol (40:1), afforded the title compound (0.90g, 86%) which crystallised from toluene, m.p. 190-192°C. IR vmax 1713 cm" ¹ ; ^] H-NMR (CDC1 ₃ ) inter alia δ 1.04 (3H,s,19-£H_ ₃ ), 1.07 (3H,s,18-Cif ₃ ), 5.98 (1H,M,16-H), 7.22 (lH.m.Py 5-H), 7.64 (lH.m.Py 4-H), 8.46 (IH.m.Py 6-H), 8.61 (!H,m,Py 2-D; MS m/z 349 (M+). Anal. Calcd: C82.47; H.8.94; N,4.01. Found: C82.00; H,8.94; N,3.84% Example 10 a) 3-(tert-Butyldimethylsnoxy)androsta-3.5-diene-ll.l7-d1one To a solution of adrenosterone (6.0g, 20 mmol) 1n dry dichloromethane (120ml) was added triethylamine (8.4ml, 60 mmol) followed by tert-butvldimethyls11vl trifluoromethanesulfonate (5.0ml, 22 mmol) and the mixture stirred at room temperature for 3h. The dichloromethane was evaporated and the residue redissolved In diethyl ether (100ml), then allowed to stand for 30 min, after which time an oil separated. The ether phase was decanted off the oil and the solvent evaporated to give the title compound which was used directly in step (b). IR v ax 1705, 1747 cm" ¹ ; ¹ H-NMR(CDC1 ₃ ) Inter alia δθ.12 (6H,s,Mfi ₂ Si), 0.85 (3H,s,18-£U-), 0.92 1.17(3H,s,19-C ₃ >, 4.73 (IH.dm, J 6.9Hz, 6-JD, 5.36 (lH,m,4-H). hi 13-(tert-Butvldimethylsi1oxv)-l1-oxo-androsta-3.5.16-tr1en-

17-vl trifluoromethanesulfonate

To a solution of the product from step (a) in dry THF (100ml), cooled to -78°C, was added a freshly prepared solution of lithium dϋsopropylamide [prepared by adding n-butyllithium (1.6M; 13.8ml, 22 mmol) in hexane to a solution of dϋsopropyla ine (3.1ml, 22 mmol) in dry THF (25ml) at -18°C] and the resultant yellow solution stirred at -78°C for 30 min. A solution of N-phenyltrifluoromethanesulfonimide (7.15g, 20 mmol) in dry THF (20ml) was then added and after an additional lh. at -78°C was allowed to reach ambient temperature- The reaction mixture was poured into water (200 ml) and extracted with diethyl ether (2 x 200ml), the combined ether extracts were washed with water

(20ml), dried Na ₂ C0 ₃ ), and concentrated to give the title compound which was used directly in step (c). IR vmax 1710 cm ^-1 , H-NMR (CDC1 ₃ ) inter alia 60.13 (6H,S,Mfi ₂ Si), 0.92 (gH.s.tfili Si ⁾ , 1.35 (6H,2s,18-£H ₃ and 19-£U ₃ ), 4.75 (lH,m,6-H) 5.38 (lH,s,4-H ⁾ , 5.68 (lH,m,16-H). c) 3-(tert-Butv1dimethylsiloxy)-17-(3-Pyridyl)androsta-3.5.16 -trien-11-one

The method essentially followed that described in Example 1 (b), but using the 13-(tert-butyldimethylsiloxy)-ll-oxo-androsta- 3,5,16-trien-17-yl trifluoromethanesulfonate from step (b), diethyl (3-pyridyl)borane (3.53g, 24 mmol), THF (100ml), bis(tr1phenylphosph1ne)palladium (II) chloride (280mg, 0.4 mmol), and aqueous sodium carbonate (2M;50ml). Following work-up as described in Example 1 (b) the title compound was obtained, which was used directly in step (d). IR vmax 1705 cπr ¹ , ^" Η-NMR (CDC1 ₃ ) inter alia δθ.13 (3H,s, fi ₂ Si), 0.93 OH.s^fiuSD, 0.99 ⁽ 3H,s,18-C ₃ ), 1.18 (3H,s,19-Cl ₃ ), 4.75 (lH,m,6-H) 5.37 ⁽ lH,m,4-M ⁾ , 6.09 ⁽ lH,m,16-H ⁾ , 7.26 ⁽ !H,m,Py 5-M ⁾ , 7.62 (IH.m.Py 4-M), 8.50 (lH.m.Py 6-H), 8.60 (lH,m,Py Z- ) . MS m/z 475 (M+). d) l7-(3-Pyr1dyl)androsta-4.l6-diene-3.11-dione

To a solution of the product from step (c) in wet THF (60ml ⁾ was added a solution of tetrabutylammonium fluoride (1.0M; 10ml, 10 mmol) in THF, and the mixture stirred at room temperature for 12 h. The mixture was partitioned between diethyl ether and water baslfled with saturated aqueuous sodium bicarbonate, the ether phase isolated, dried (Na ₂ C0 ₃ ), and concentrated. Chromatography, on elution with diethyl ether, afforded the title compound (4.30g, 60% overall yield from adrenosterone) which crystallised from diethyl ether, m.p. 181-183°C.

IR vmax 1669, 1703 cm ^{-1 ,} 'H-NMR(CDC1 ₃ ) inter alia δl.02 (3H,s, 18-£H ₃ ), 45 (3H,s,19-C ₃ ), 5.76 (lH.ClH.s.Py 4-H) , 6.08

(lH,m,16-H) 7.24 (lH,m,Py 5-H), 7.59 (lH,m,Py 4-H),8.50 (lH,m,Py 6-1), 8.59 ⁽ lH,m,Py 2-H). MS m/z 361 (M+). Anal Calcd: C, 79.74; H.7.53: N.3.88. Found: C.79.58; H.7.57; N,3.89%. Example 11 3-Acetoxv-l7-(3-nvridvl)androsta-3.5.16-triene

17-(3-pyridyl)androsta-4,16-dien-3-one (174 mg, 0.50 mmol ⁾ was dissolved in isopropenyl acetate (2 ml). p-Toluenesulfonic add (130 mg, 0.68 rnrnol) was then added and the mixture heated at 80°C for 12h. After allowing to cool the mixture was poured Into diethyl ether, washed with saturated aqueous sodium bicarbonate, dried (Na ₂ C0 ₃ ) and concentrated. Chromatography on elution with light petroleum - diethyl ether (1:1), afforded the title compound (86 mg, 44%), IR vmax 1755 cm" ¹ , ^-NMR (CDC1 ₃ ) inter alia δl.05 (6H,s,18-£H ₃ and 19-CH ₃ ), 2.15 (3H,s,COC ₃ ⁾ 5.44 (lH,m,6-JD, 5.72(lH,m,4-H), 6.00 (lH,m,16-H), 7.25 (lH,m,Py 5-H), 7.66 (lH.m.Py 4-D, 8.47 (lH.M.Py 6-H), 8.63 (lH.m.Py 2-1). MS ffl/Z 389 ⁽ M+ ⁾ .

Example 12 6S-F1uoro-17-(3-pyridvl)androsta-4.16-dien-3-one and

Example 13

6--F1uoro-17-(3-pvridvl)androsta-4.16-dien-3-one To a solution of 3-acetoxy-17-(3-pyridyl)androsta-3,5,16-triene (80mg, 0.21 mmol) in dry dichloromethane (2ml) was added

N-fluoropyridinium trifluoromethanesulfonate (180mg, 0.73 mmol) and the mixture heated under reflux for 12h. The mixture was diluted with diethyl ether (30ml), washed with dilute aqueous sodium hydroxide (0.5M; 2 x 5ml), dried Na ₂ C0 ₃ ), and concentrated. ^H and ¹⁹ F-NMR at this stage showed the

6-fluorinated products were formed as a 3:2 mixture of the β and α-epimers. Chromatography, on elution with light petroleum-diethyl ether (1:2), gave firstly:- i) the title 6B-epimer (13mg), 17%) as white crystals,

m.p. 167-169°C. IR vmax 1684 cm ^-1 ; ^-NMR (CDC1 ₃ ) inter alia δl.ll (3H,s,18-£!i ₃ ), 1.37 (3H,s,19-CJi ₃ ), 5.06 (lH,dd, J _H - _H 2.4 Hz, J _H - _F 49Hz, 6α-H), 5.92 (lH,m,4-H), 6.01 (lH,m,16-H), 7.24 (lH,m,Py 5-H), 7.65 (lH,m,Py 4-H), 8.48 (lH,m,Py 6-H), 8.63 (IH. .Py 2-H). ¹⁹ F-NMR δ -165.9 (dt, J _H _ _F 49 Hz, 6β-F). MS m/z 365 (M+).

Further elution afforded :-

11) The ^* title 6α-epimer (8mg, 11%) as white crystals, m.p. 167-169°C, IR vmax 1681 cm ^"1 ; ^] H-NMR (CDC1 ₃ ) Inter alia 61.07 (3H,s,18-£H ₃ ), 1.24 (3H,s,19-£H ₃ ), 5.18 (lH.dm, J _H - _F 48Hz, 6β-H), 5.98 (ZH,m,4-H and 16-H), 7.26 (IH.m.Py 5-H), 7.64 (lH,m,Py 4-H), 8.40 (lH,m,Py6-H), 8.63 (lH,m,Py 2-H). ¹⁹ F-NMR (CDC1 ₃ ) δ -183.9 (d, J _H _ _F 48 Hz, 6α-F). MS m/z 365 (M+).

Example 14 l7-(3-pvr1dvl)androsta-4.16-d1en-3-one (via Oppenauer Oxidation) This Example illustrates a better method of preparing the compound already prepared In Example 4. The method followed that described 1n Example 9, but using l7-(3-pyr1dyl)androsta-5, 16-dien-3β-ol (1.05g, 3.0 mmol). Chromatography, on elution with toluene-methanol (20:1), afforded the title compound (0.85g, 82%), which crystallised from diethyl ether, m.p. 148-150°C. Spectroscopic data was identical to that given in Example 4(c). Anal. Calcd: C.82.95; H,8.41; N.4.03 Found: C.83.00; H.8.50; N,3.99%

Example 15

17-(3-pyridyl)androsta-4.16-dien-3-one oxime

To a suspension of 17- ⁽ 3-pyridyl)androsta-4,16-dien-3-one (125 mg, 0.36 mmol) in ethanol (2 ml ⁾ was added hydroxylamine hydrochloride (50mg, 0.72 mmol), followed by pyridine (0.2ml), and the mixture heated under reflux for lh. then allowed to cool .

The solvent was evaporated and the crystalline product triturated under water, collected on a sinter, washed with cold water, and dried in vacuo to give the title oxime as a 1:1 mixture of _s_y_n and anti geometric isomers. ^- MR (CDC1 ₃ ) inter alia δl.06 (3H,s,18-£H ₃ ⁾ . 1.13 ⁽ 3H,s,19-£H ₃ ), 5.75 and 5.80 (lH,2m, isomeric 4-H), 6.01 (lH,m,16-H), 7.26 (lH,m,Py 5H), 7.68 and 7.88 (1H, 2m, isomeric Py 4-ϋ ⁾ , 8.48 and 8.53 (1H, 2m, isomeric Py 6-H), 8.63 (lH,m,Py 2-£). MS m/z 362 (M+).

Example 16 17-(3-Pvridvl)aπdrosta-4.16-diene-3.6-dione

To a solution of 17-(3-pyridyl)androsta-5,16-dien-3β-ol (350mg, 1.0 mmol) in dry dichloromethane (10 ml) was added H-methylmorphine N-oxide (351mg, 3.0 mmol) followed by 400mg of freshly dried and powdered 4A molecular sieves and the mixture stirred for 10 min. Tetrapropylammonium perruthenate catalyst (35mg ⁾ , 0.1 mmol) was then added, the reaction flask placed in an ultrasonic bath, and the mixture Irradiated whilst maintaining the temperature between 20-30°C for 2 h. The mixture was then filtered, diluted with diethyl ether, washed with water, dried (Na ₂ C0 ₃ ), and concentrated. Chromatography, on elution with diethyl ether - ethyl acetate (5:1), afforded the title compound (26 mg, 7%) as white crystals m.p. 210-212°C. IR vmax 1680 cm" ¹ ; ^-N R (CDC1 ₃ ) inter alia δl.10 (3H,s,18-£ii ₃ ), 1.44 (3H,s,19-CJi ₃ ⁾ , 4.42 (lH.m.enolic 2-D, 5.84 (lH,s,4-H ⁾ , 6.01 (lH,m,16-Jl), 7.24 (lH.m.Py 5-H), 7.65 (lH,m,Py 4-H), 8.45 (IH.m.Py 4-H), 8.45 (lH,m,Py 6-H), 8.60 (lH.m.Py 2-H). FAB-MS MS ffi/Z 362 (M+l). Example 17 3-.-(Tr1fluoromethvl)-l7-(3-Pyr1dyl)androst-l6-en-3B-ol To a solution of 17-(3-pyridyl)androst-16-en-3-one (100 mg, 0.29 mmol) in THF (2ml) cooled to 0°C was added trifluoromethyltrimethylsilane (200μl , 1.3mmol) followed by tetrabutylammonium fluoride trihydrate (10 mg, 0.03 mmol). After

30 min., dilute aqueous hydrochloric acid (IM; 1ml) was added and the mixture stirred at room temperature for 12h. The mixture was then basified with saturated aqueous sodium bicarbonate and extracted with diethyl ether. The three extracts were combined, dried (Na ₂ C0 ₃ ), and concentrated. Chromatography, on elution with light petroleum - diethyl ether (1:1), afforded the title compound (87mg, 73%) which crystallised from toluene, m.p. 192-193°C -H-NMR (CDC1 ₃ ) Inter alia δθ.92 (3H,s,19-CJ ₃ ) , 1.01 ⁽ 3H,s,18-CJ ₃ ⁾ , 5.98 ⁽ lH,m, 16-HJ , 7.22 ⁽ lH.m.Py 5-H ⁾ , 7.64 (lH. .Py 4-H),8.45 (IH.m.Py 6-H), 8.60 (IH.m.Py 2-H); ¹⁹ F-NMR (CDC1 ₃ ) δ -79.1 (s,3α-CF ₃ ). MS m/z 419 (M+). Anal. Calcd: C.71.57; H.7.69; N.3.34; F,13.59 Found: C.71.67; H.7.71; N,3.25; F,13.30% Example 18 (a) D1ethv1C3-(5-methylpyridyl)] borane

3-Bromo-5-methylpτid1ne, which can be prepared as described in the literature, e.g. L. van der Does and H. 3. van Hertog, Rec. Trav. Chem. Pays Bas £4, 957-960 (1985) or R. A. Abramovitch and M. Saha, Can. 3. Chem. 44, 1765-1771 (1966), 1s reacted with n-butylllthium, according to the method of M. Terashima fit al., Chem. Pharm. Bull. 21, 4573-4577, (1983). The product 1s treated with trlethylborane and then iodine. (b) l7-r3-(5-MethylPyr1dyl)landrosta-5.l6-dien-3B-ol Diethyl C3-(5-methylpyridyl)] borane is reacted with 3β-acetoxyandrosta-5,16-dien-17-yl trifluoromethane sulphonate analogously to Example 1 (b) and the resulting 3β-acetate Is hydrolysed with sodium hydroxide, analogously to Example 2, to yield the title compound. Test results (a) Preparation of testicular material

Human testes were obtained from previously untreated patients undergoing orchidectomy for prostatic cancer. The testes were decapsulated and stored in liquid nitrogen until use. A

microsomal preparation was prepared essentially as described by S. E. Barrie fit al., 3. Steroid Biochem. £, 1191-5, (1989). The material was then thawed, .finely chopped, and homogenised in 0.25M sucrose (5ml/g wet weight) using a Potter homogeniser. The homogenate was centrifuged at 12000g for 30 min,. and then the microsomes were pelleted by spinning the supernatant at 100,000g for lhr. The pellet was washed by being resuspended in 0.25M sucrose ^* and repelleted. The microsomal pellet was then resuspended in 50mM sodium phosphate pH 7.4/glycerol (3/1 v/v) and stored in aliquots in liquid nitrogen. (b) Determination of 17o.-hvdroxylase

The basic assay mixture was EDTA (0.2mM), dithiothreitol (DTT; O.lmM), NADPH (0.25mM), glucose 6-phosphate dehydrogenase (G6PDH; 6.25 μg/ml), MgCl ₂ (lmM), glucose 6-phosphate (G6P; lOmM) and the substrate, ³ H-progesterone (3μM) in sodium phosphate (50mm), pH 7.4. The compounds under test were dissolved in 50% DMSO and the final concentrations of ethanol and DMSO were 1% each. The assay reaction was carried out for 1 hour and was terminated by the addition of 2 vols. of methanol-acetonitrile (2:1) containing approx. 100μM unlabelled progesterone, 17α-hydroxyprogesterone, androstenedione, testosterone, and 16α-hydroxyprogesterone. The last-mentioned steroid was added as it appeared that the human enzyme was capable of 16α-hydroxylation as well as 17α-hydroxylation. The separation of the steroids by HPLC was carried out using an "Uptight" guard column packed with 40-63μm NucleosH C18 and a 10cm main column packed with 5μm Nucleosil C18 and 60% methanol as eluant. The radioactivity in the peaks of interest was monitored on-line by mixing the HPLC effluent 1:1 with Ecosdnt A (National Diagnostics) scintillation fluid, containing 25% acetonitrile, and passing the mixture through a Berthold LB506C radiochemical monitor. The hydroxylase activity was measured as the production of 17α-hydroxyprogesterone, androstenedione and testosterone.

(c) Determination of C- _| --C ₂ ø lyase

The mixture was the same as described above for the

17α-hydroxylase except that the substrate was ³ H-17α-hydroxy- progesterone. The reaction was carried out for l-2h. and was stopped by the addition of 2 vols. of methanol/acetonitrile (2/1 containing approx. lOOμM 17α-hydroxyprogesterone, androstenedione and testosterone.

The HPLC separation used for the lyase involved a mini-re-column "Uptight Guard Column" packed with PELL-ODS

(pellicular c-ctadecyl lilica) and a 10cm. main column "Apex C18" column packed with 5μ APEX-CAT silica.

The eluant was 38:12:50 methanol :acetonitrile:water flowing at lml/min. The effluent was mixed 1:1 with Ecoscint A containing 5% methanol and 5% acetonitrile and the radioactivity was measured directly by a Berthold LB506C radiochemical detector.

The lyase activity was measured as the production of androstenedione and testosterone.

(d) Calculation of IC50.

The enzyme activity was measured In the presence of at least 4 concentrations of each compound. The data were for the 4-pyridyl and 2-picolyl compounds of Table 1 fitted by linear regression to the Dixon equation (M. Dixon, E.C. Webb, Enzymes, 2nd ed., Academic Press, New York, 1964). Data for all the other compounds were fitted by non-linear regression to the median effect equation of Chou, 3. Theoret. Blol. 21, 253-276 (1976 ⁾ . The correlation coefficients were greater than 0.95 except for the compound of Example 1, where it was 0.91. All the assays were carried out with approx. 4nM enzyme (as calculated from kinetic measurements) except those for Ketoconazole and the 2- and 4-pyri yl and 2-picolyl compounds of Table 1, in which 25nM lyase and lOnM hydroxylase were used. The IC ₅₀ values are dependent on enzyme concentration when the inhibitor binds tightly (all the compounds tested except the 4-pyridyl and 2-picolyl). Results are shown in Table 2 below.

TABLE 2 (a) Confirmation that variations in the A and B rings of compounds of the invention have little effect on inhibition of hydroxyl se and lyase.

Compounds tested are of formula (3) wherein R = H:

IC ₅₀ (μM) Lyasg Hvdroxyl ase

TABLE 2 (CONTINUED)

IC ₅₀ (μM) Lvase Hydroxylase

(b ⁾ Confirmation that variation in the C ring of compounds of the invention has little effect on the inhibition of hydroxylase and lyase.

Com ^p ound Tested IC ₅₀ (μM)

Lvase Hydroxylase

(Ex. 10)

The comparative IC50 figures for etoconazole are 0.026 against lyase and 0.065 against hydroxylase.

Assay of aromatase activity Aromatase activity was determined by the method of A. B. Foster fit al. , 3. Med. Chem. 2£, 50-54 (1983), using human placental microsomes. For the microsomes used, the Michaelis constant K-,, for LJβ - ³ H] androstenedione was 0.039μM.

The compounds having a pregnenolone-like skeleton in the A and B rings, i.e. 3β-acetoxy-17-(3-pyridyl)androsta-5,16-diene and its 3-alcohol of Examples 1 and 2, had IC5Q > 20 μM. The compound having a progesterone-li e skeleton i.n the A and B rings, i.e. 17-(3-pyridyl ⁾ -androsta-4,16-dien-3-one of Example 4 exhibited also aromatase inhibitory activity with IC50 = IμM.

In vivo orσan weiσht and endocrine test in mice

Male HWT mice, 12 weeks old, were treated daily for 2 weeks, with 5 animals per treatment group. The test compounds were the compound of Examples 1 and 4 (as representative of compounds of the invention having the pregenolone-like and progesterone-like skeletons respectively). Ketoconazole was also tested at three different doses. The test compounds were made up in 5% benzyl alcohol, 95% safflower oil, and were given i.p.. In addition to an untreated control group of animals, there was also a solvent control group which received the same volume of liquid as the test group (5ml/kg) but no test compound. All animals were sacrificed 24 hours after the last injection. Blood was collected by cardiac puncture into heparinized tubes, and the plasma used for RIA (radio immunoassay) of testosterone and luteinising hormone. The following organs were removed and weighed: adrenals, prostate, seminal vesicles, testes, kidneys. There was no significant body weight loss in any group of mice during the experiments.

Post mortem examination of the mice revealed oil/white deposits i.p. in those treated with compound of Ex. 1 and white deposits throughout the abdomen in those treated with compound of Ex. 4. In all these mice, all organs looked normal. In Ketoconazole- treated animals, adhesions were found in 2/5,2/5,4/5 of the low/middle/top dose groups. The gut and peritoneal wall seemed to be stuck to the seminal vesicles. The livers were brown in the middle/top dose groups.

The weights of organs found in the animals post mortem are shown in Table 3 below. The reductions 1n weight of all of the prostate, seminal vesicles, testes and kidneys were much greater for the test compounds of the invention than for Ketoconazole. Ketoconazole caused an increase in adrenal weight at the two highest doses, whereas the compounds of the invention had no significant effect, suggesting that they did not inhibit corticosterone biosynthesis.

TABLE 3 Compound of Ex 1. Mean weight (mg.) ± standard error.

Dose Adrenals Prostate Seminal Testes Kidneys

Vesicles

controls 4.5 ± 0.1 10.1 ± 0.7 189 ± 9 146 ± 3 709 ± 17 solvent 4.5 ± 0.4 10.2 ± 1.3 171 ± 6 122 ± 7 615 ± 28 controls

0.02mmol/ 4.3 ± 0.2 8.0 ± 0.6 136 ± 4 134 ± 4 604 + 24

/kg/day

0.1 mmol 4.0 ± 0.2 5.3 ± 0.3 51 ± 6 95 ± 3 500 ± 8 /kg/day

0.5 mmol 4.7 ± 0.2 5.6 ± 0.6 25 ± 2 56 ± 2 449 ± 12

/kg/day

Compound of Ex 4.

Dose Adrenal s Prostate Seminal Testes Kidneys

Vesicles

controls 4.3 ± 0.4 8.4 ± 0.2 165 ± 18 142 ± 8 652 ± 45 solvent controls 4.4 ± 0.0 9.2 ± 0.9 152 ± 9 122 ± 8 589 ± 24

0.02mmol/ /kg/day 4.7 ± 0.2 5.9 ± 0.8 108 ± 4 117 ± 9 599 ± 29

0.1 mmol

/kg/day 4.6 ± 0.4 6.4 ± 0.5 61 ± 9 105 ± 5 549 ± 28

0.5 mmol

/kg/day 4.9 ± 0.1 4.1 ± 0.5 25 ± 1 59 ± 2 468 ± 15

Ketoconazole

Dose Adrenals Prostate Seminal Testes Kidneys

Vesicles

controls 4.2 ± 0.2 8.9 ± 0.8 193 ± 8 145 + 4 670 ± 12 solvent controls 4.7 ± 0.4 9.3 ± 1.2 198 ± 18 146 ± 3 615 ± 25

O.Olmmol/ /kg/day 4.8 ± 0.2 9.1 ± 0.8 235 ± 18 141 ± 5 637 ± 22

0.225 mmol

/kg/day 6.1 ± 0.3 10.8 ± 1.4 171 ± 5 127 ± 7 574 ± 23

0.5 mmol

/kg/day 6.9 ± 0.3 9.3 ± 0.9 179 ± 20 133 ± 6 710 ± 30

The results indicate the inhibition by the components of the invention of androgen and particularly testosterone synthesis.

They are confirmed by endocrinological results shown in Table 4.

Although the solvent itself produced marked depression of testosterone levels, probably due .to stress on the animals, the further decrease resulting from the administration of test compounds was much more marked for the compounds of the invention than for ketoconazole. The rise in LH levels is ascribed to a feedback mechanism associated with depletion of testosterone.

TABLE 4

Endocrinological Results (Mean ± standard error)

Testosterone LH nM ng/ml

Compound of Ex. 1

Control 9.8 ± 5.6 0.63 + 0.16 Solvent Control 2.5 ± 1.2 0.80 ± 0.09

0.02Mmol/Kg/Day 2.7 ± 0.5 3.4 ± 0.5 0.1Mmol/Kg/Day 0.2 ± 0.1 2.55 ± 0.45 0.5Mmol/Kg/Day 0.1 ± 0.0 2.25 ± 0.67

Compound of Ex. 4

Control 27.8 + 11.4 Not solvent Control 11.0 ± 5.6 determined

0.02Mmol/Kg/Day 4.5 ± 0.3 0.1Mmol/Kg/Day 3.5 ± 1.0 0.5Mmol/Kg/Day 0.4 + 0.1

Ketoconazole

Control 17.3 ± 7.1 0.66 ± 0.05

Solvent

Control 1.3 ± 0.4 0.25 ± 0.13

0.1Mmol/Kg/Day 0.9 ± 0.2 0.39 ± 0.14

0.225Mmol/Kg/Day 0.7 ± 0.1 0.75 ± 0.02

0.5Mmol/Kg/Day 0.4 + 0.1 0.76 + 0.03