WO1992016507A1

WO1992016507A1 - Heterocyclic compounds, their preparation and pharmaceutical use

Info

Publication number: WO1992016507A1
Application number: PCT/GB1992/000509
Authority: WO
Inventors: Susan Elaine Barrie; Michael Jarman; Raymond Mccague; Gerard Andrew Potter; Martin George Rowlands
Original assignee: British Technology Group Limited
Priority date: 1991-03-22
Filing date: 1992-03-20
Publication date: 1992-10-01
Also published as: GB2253851A; PT100275A; AU1417592A; GB2253851B; GB9206062D0; IE920900A1; EP0594629A1; NZ242054A

Abstract

Compounds having the general formula (3), wherein each of R?1 and R?2 independently represents hydrogen or C1-4? alkyl or together represent the residue of a cycloalkyl group of 3 to 6 carbon atoms; A represents O, NR?4 wherein R?4 is defined as for R?1 or R?2 or CR?5R?6 wherein R?5 and R?6 are as defined for R?1 or R?2 as separate substituents; and R?3 represents a bridged alicyclic group; as free bases or their pharmaceutically acceptable salts are useful in treating androgen-dependent, especially prostatic, cancer.

Description

HETEROCYCLIC COMPOUNDS, THEIR PREPARATION

AND PHARMACEUTICAL USE

Background of the Invention

1. Field of the Invention

This invention relates to derivatives containing the 3-pyridylacetyl function, namely esters and amides of 3-pyridylacetic add as well as 3-pyridylmethyl ketones, together with certain derivatives thereof, their preparation and use in treating prostatlc cancer.

2. Description of related art

R. McCague, M. G. Rowlands, S. E. Barrle and J. Houghton, J . Med. Chem. 33, 3050-3055 (1990), have reported that certain esters of 4-pyridylacetic add, of general formula:

wherein R^a represents a specified alicyclic group (e.g. cyclohexyl or a terpene residue) or

wherein R^b represents a hydrogen atom or a methyl group, inhibit the 17α-hydroxylase/C_17-20 lyase enzyme complex which is essential for biosynthesis of androgens. The inhibition of androgen biosynthesis by virtue of the hydroxylase/lyase inhibition indicates that the compounds of McCague et al., supra, could be useful for the treatment of prostate cancer since many such tumours depend on androgens for growth. The compounds of McCague et al. are also inhibitors of aromatase. Aromatase is an enzyme required in the biosynthesis of estrogens. The ability to inhibit aromatase is considered a desirable property in compounds which are to be used to treat breast cancer. It is undesirable, however, for the treatment of prostatic cancer that a compound should be a strong inhibitor of both aromatase and hydroylase/lyase since the inhibition of aromatase would prevent the removal, by further conversion into oestrogens, of any products of the hydroxylase/lyase enzyme complex which escaped the blockade of hydroxylase/lyase. As a result, a patient could lose some of the benefits of hydroxylase/lyase inhibition.

Accordingly, for the present application of treating prostatic cancer it is desirable to keep the ratio:

IC₅₀ versus lyase

IC₅₀ versus aromatase

as low as possible. (A small numerator indicates that the compound is a powerful inhibitor of lyase. A large denominator indicates that it is a poor inhibitor of aromatase). Their best compound from that viewpoint is cyclohexyl 2-methyl-2-(4-pyridyl) propanoate of formula (2) above wherein R^b is methyl and formula (26) in the paper, although it must be borne in mind that their data are based on human aromatase and rat lyase. The same compound was also degraded more slowly by hog Hver esterases than the monomethylated compound [formula (2), R^b - H] or the unmethylated counterpart [formula (1), R^a = cyclohexyl]. The paper envisages similar dimethyl substitution of compound of formula (1) wherein R^a is an alicyclic group of the terpene residue type, notwithstanding that in such compounds the IC₅₀ lyase/aromatase ratio in unmethylated compounds is bigger than for the cyclohexyl compounds (1).

An Immediately following companion paper by C. A. Laughton and S. Neidle, J. Med. Chem. 33, 3055-3060 (1990) attempts to explain the data of the McCague et il paper in terms of mimicry of the natural steroid substrates for aromatase and hydroxylase/ lyase by the 4-pyridylacetates and propanoates with reference to cyclohexyl 4-pyridyl acetate [formula (1); R^a = cyclohexyl] and Its α-methyl derivative, cyclohexyl 2-(4-pyridyl)propanoate [formula (2); R^b = H], since the α-methyl substitution lowers the IC₅₀ lyase/aromatase ratio from 67 to 1.0. The conclusions of the paper are rather hard to discern. As regards aromatase inhibition, the paper suggests that the carbon atom adjacent to the ester carbonyl function occupies a spatial position mimidng that of the C(2)-atom of the natural steroid substrate (testosterone), and that the conformation of the overall molecular "fit" is favourable, but that the α-methyl substituent would then mimic a substituent on C(2) of the steroid. Since other evidence has suggested that sterlc bulk in the C(2) region is unfavourable for aromatase inhibition, the poorer inhibition of the α-methyl substituted ester could be rationalised in this manner. However, as regards hydroxylase/lyase inhibition, there are no such literature precedents, and the authors make many assumptions in suggesting that the α-methyl group of the cyclohexyl 2-(4-pyridyl)propanoate lies in a spatial position mimidng the C(16) or C(20)-atom of the natural steroid substrate pregnenolone, and conclude that the inhibitory activity stems from hydrophobic interactions of the methyl group with the active site of the enzyme. This view does not, however, explain why the hydroxylase/lyase activity of compounds lacking the α-methyl group, e.g. 4-ethyl cyclohexyl 4-pyridylacetate, Is just as good as the α-methyl compound used for modelling and thereby suggests that the structural requirements for hydroxylase/lyase activity are unpredictable.

Summary of the Invention

It has now surprisingly been found that 3-pyridylacetyl compounds of formula (3) below have useful hydroxylase/lyase inhibitory activity with low IC₅₀ lyase/aromatase ratios, and are therefore of potential value in treating androgen-induced cancers such as prostatic cancer. These compounds have the general formula:

wherein each of R¹ and R² independently represents hydrogen or lower alkyl or together represent the residue of a cycloalkyl group of 3 to 6 carbon atoms;

A represents 0, NR⁴ where R⁴ is defined as for R¹ and R², or CR⁵R⁶ where R⁵ and R⁶ are defined as for R¹ or R² as separate substituents; and

R³ represents a bridged all cyclic group;

as free bases or their pharmaceutically acceptable salts, especially acid addition salts. The term "lower" herein signifies that the group has 1 to 4 carbon atoms. The invention includes each of the optical Isomers and mixtures thereof, especially racemic mixtures.

Description of the preferred embodiments

The R³ group in the preferred compounds of the invention can be defined in various ways, all reflecting the fact that R³ is hydrocarbyl, cyclic and non-aromatic and has at least one bridge across a ring. Because R³ is defined as bridged, it contains at least two alicyclic rings. It can contain more than two alicyclic rings, either by having more than one bridge or by having one or more other fused rings (not resulting from a bridge). In this invention, a bridge is regarded as joining two non-adjacent carbon atoms of the ring by means of at least one intermediate carbon atom. A fused ring is produced when two non-adjacent carbon atoms are joined directly by a bond.

In the preferred compounds it is possible to regard the R³ group as a substituted cyclohexyl group in which the substituents comprise bridging members. Examples of preferred R³ groups fitting this definition are shown below, along with the adjacent oxygen atom:-

isopinocampheyl (cyclohexyl substituted by a 3,5-1 sopropylidene bridge and additionally having a 2-methyl substituent)

borneyl (cyclohexyl substituted by a 2,5-1 sopropylidene bridge and additionally having a 2-methyl substituent)

isoborneyl

endo-norborneyl (cyclohexyl substituted by a 2,5-methylene bridge)

exo-norborneyl

adamantyl (cyclohexyl substituted by a first 1,3-(1,3-propylene), bridge and further substituted by a second bridge between its 5-carbon atom and the middle carbon atom of the first bridge)

methyladamantyl (cyclohexyl substituted by a first 2,4-(1,3- propylene) bridge, further substituted by a second bridge between its 6-carbon atom and the middle carbon of the first bridge, and additionally having a 1-methyl substituent on the cyclohexane ring)

cedryl (cyclohexyl substituted by a 2,4-isobutylene bridge, having a methyl-substituted cyclopentane ring fused to the 4 carbon atom of the cyclohexane ring and the 1-carbon atom of the bridge and additionally having a 1-methyl substituent on the cyclohexane ring).

Alternatively, the preferred R³ groups can be defined by reference to the largest carbocyclic ring which is bridged, which in cedryl is a cycloheptane ring and in adamantyl is a cyclooctane ring. According to this definition, R³ represents a bridged alicyclic group having from 6 to 8 ring atoms (excluding any bridge atoms) and optionally having one or more alicyclic groups fused to the bridged ring, e.g. a substituted cyclopentane or cyclohexane ring. In this definition the bridges are normally of 1 or 2 carbon atoms in linear length (counting only those carbon atoms lying within the bridge, between the ring atoms, not counting as within the bridge the ring atoms with which the bridge starts or finishes and not counting as within the linear length any carbon atoms pendant from a bridge atom, e.g. in the two methyls of an isopropyl1dene bridging group).

The all cyclic groups of R³ which are bridged are cycloalkane rings, which can contain unsaturation, but are not aromatic, and can be substituted by one or more simple hydrocarbyl substituents such as alkyl of 1-4 carbon atoms, especially methyl. The bridges need not be wholly linear and thus may have pendant C_1-4 alkyl, especially methyl groups, for example. Likewise the said cycloalkane rings or bridges or both can have cycloalkane, especially cyclopentane or cyclohexane, rings fused thereto. The fused rings may themselves be simply substituted, as mentioned above for the bridged rings, especially by alkyl of 1 to 4 carbon atoms.

The invention includes optically active forms of the compounds of formula (3), particularly with reference to borneyl, isoborneyl, cedryl and isopinocampheyl.

The A group in formula (3) is preferably -O-, but when It Is -CH₂- potentially hydrolysable ester and amide bonds are not present, which is also advantageous.

All "lower alkyl" groups herein are preferably methyl or ethyl.

R¹ and R² are preferably both methyl except when R³ is an extremely bulky group, such as cedryl, in which substituents pendant from a bridge extend into the vicinity of the ester oxygen atom. In such an event preferably no more than one of R¹ and R² is a lower alkyl group and most preferably they are both hydrogen. Alternatively R¹ and R² together with the carbon atom to which they are attached can complete a ring of 3, 4, 5 or 6 carbon atoms, cyclopentane being preferred.

In the divalent amino group NR⁴, R⁴ is preferably hydrogen, but can have any of the other meanings for the individual R¹ and R² substituents, of which lower alkyl, especially methyl, is preferred.

In the ketones, R⁵ and R⁶ are preferably hydrogen, one is methyl and the other hydrogen or both are methyl. R⁵ and R⁶ do not together represent a cycloalkyl or alkylene group.

The compounds of the invention can be prepared in various ways, conveniently starting from 3-pyrldylacetic add or an ester thereof. Preferably, the starting ester Is the methyl or ethyl ester. The starting compounds (A = -O-) have the general formula (4):

wherein X represents -OH or a reactive substltutent and R¹ and R² are as defined for formula (3). Simple ester1f1cat1on or trans-esteriflcation with an alcohol of formula R³-OH where R³ is as defined for formula (3) leads to the esters of formula (3).

The reactive substituent X is any reactive for the purpose of forming an ester or amide of formula (3). For preparation of amides (A = -NH-) the compounds of formula (4) can be reacted with primary amines in the usual way.

To prepare the ketones of formula (3) in which A = -CH₂-, a suitable procedure would involve the reaction between an alkali metal salt of 3-picoline, e.g. 3-picolyllithium (C. G. Screttas, T. F. Estham, C. W. Kamienskl, Chimia, 24, 109-111 , 1970) and an appropriate methyl ester R³ACO₂Me where A is another CR⁵R⁶ group, according to the method used by 3. L. Bond, D. L. Krottlnger, R. M. Schumacher, E. H. Sund and T. 3. Weaver, Journal of Chemical and Engineering Data, 18, 349-350, (1973) to make alkyl 4-pyridylmethylketones from 4-picolyl sodium and RCO₂Me.

Where the compounds of formula (3) being prepared are those in which R¹ or R² is other than hydrogen, it may be convenient to use as the starting compound of formula (4) an unsubstituted pyridyl acetic add compound wherein R¹ and R² are hydrogen, prepare the corresponding compound of formula (3) and subsequently introduce the desired R¹ or R² substituent by the action of an alkali metal hydride followed by a lower alkyl or cycloalkyl bromide or Iodide. Methyl ene (CH₂=) or ethylidene (CH₃CH₂=) derivatives may be prepared from corresponding methyl and ethyl derivatives by thermal decomposition of phenylsulphoxide B. M. Trost el al., J . Amer. Chem. Soc, 52, 4887-4902 (1976).

The compounds may be prepared as salts, e.g. the hydrochloride 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, Upsomes or micro-reservoirs, especially compositions in orally ingest1ble or sterile injectable form. The preferred form of composition contemplated is 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, carboxymethyl cellulose 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-dependent 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 dally during the course of treatment. This works out (for humans) at 20-800 mg/patient 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 following Examples illustrate the invention. Temperatures are in °C.

Example 1

(1S,2S,3S,5R)-Isopinocampheyl 3-pyridylacetate.

A stirred solution of (+)-isopinocampheol (3.086 g, 20 mmol) in dry tetrahydrofuran (20 ml) under N₂ was cooled with an Ice- salt bath. A solution of n-butyllithium (1.6 M, 12.5 ml, 20 mmol) in hexane was added followed, after 5 min, by a solution of ethyl 3-pyridylacetate (2.746 g, 16.7 mmol) in tetrahydrofuran (5 ml) and the clear yellow solution allowed to attain room temperature. After 4 h, the mixture was partitioned between dlethyl ether and water and the ether layers were concentrated. Chromatography of the residue gave on elutlon with 50:50:1 light petroleum (bp 60-80°)-diethyl ether-triethylamine the title compound (3.79 g, 76%) as an oil. By passing hydrogen chloride gas through a solution of the product in diethyl ether, the hydrochloride was obtained. This was recrystallised from dioxan-ether 1:1, mp 158-160°C. Anal. Calcd: C, 65.90: H, 7.81; N, 4.52. Pound: C, 65.36; H, 7.62; N, 4.65%.

Example 2

(1S,2S,3S,5R)-Isopinocampheyl 2-(3-pyridyl)propanoate.

A solution of the free base product of Example 1 (912 mg,

3.34 mmol) in dry tetrahydrofuran (3 ml) was added to a stirred suspension of potassium hydride (35% by weight dispersion in on, 383 mg, 3.34 mmol) in tetrahydrofuran (10 ml) under nitrogen at 0°C. After 10 min, methyl iodide (0.17 ml, 380 mg, 2.68 mmol) was added, and after 1 h at 20°, worked up as above and column chromatographed with 5:4 d1ethyl ether-light petroleum to give the title compound (345 mg, 36%) as an oil. 1H-NMR (CDCl₃) inter alia S 0.97, 1.20 (2s, 6H, Me₂C), 1.53 (d, 3H, J = 6.8 Hz, COCHCH₃), 3.75 (q, 1H, COCHCH₃), 5.10 (m, 1H,

OCH), 7.30 (dd, 1H, J_ortho = 4.8, 7-95 Hz, H-5), 7.80 (m, 1H, H-4), 8.60 (m, 2 H, H-2 and H-6). Anal. Calcd: C, 75.23; H, 8.77; N, 4.87. Found: C, 75.31; H, 8.86; N, 4.69%.

Example 3

(1S,2S,3S,5R)-Isopinocampheyl 2-methyl-2-(3-pyridyl)propanoate.

A solution of (1S,2S,3S,5R)-isopinocampheyl 3-pyridylacetate (706 mg, 2.58 mmol) in dry tetrahydrofuran (8 ml) was added to a stirred suspension of potassium hydride (35% by weight dispersion in oil, 650 mg, 5.68 mmol) in tetrahydrofuran (6 ml) under argon at 0°C. After 10 min, methyl iodide (733 mg, 5.16 mmol) was added in two equal portions, each in tetrahydrofuran (2 ml). On addition of 1 equivalent, the mixture became cloudy and hydrogen evolved. On adding the second equivalent the yellow solution turned colourless. After 20 min, the reaction was quenched by addition of Isopropanol (0.5 ml). Work-up as in Example 1 with chromatography in the same solvent mixture gave the title compound (539 mg, 69%) as a colourless oil which similarly gave a crystalline hvdrochloride. mp 144-146°C. Anal. Calcd: C, 67.57; H, 8.35; N, 4.15; Cl 10.49. Found: C, 67.56; H, 8.30; N, 4.11; Cl 10.61%.

Example 4

(1S,2R)-Borneyl 3-pyridylacetate.

By essentially the procedure of Example 1, using (-)-borneol (2.232 g, 14.47 mmol) in dry tetrahydrofuran (15 ml), n-butyllithium (5.79 ml, 14.47 mmol) and ethyl 3-pyridylacetate (1.91 g, 11.57 mol) in tetrahydrofuran (5 ml) afforded the title compound, isolated after elution with 1:1 diethyl ether-light petroleum as a colourless oil (2.87 g, 73%). ¹H-NMR inter alia δ 0.77 (s, 3H, CCH₃), 0.84, 0.87 (2S, 6H, C(CH₃)₂), 3.64 (S, 2H, COCH₂), 4.90 (m, 1H, CHOCO), 7.26 (m, 1H, H-5), 7.64 (m, 1H, H-4), 8.50 (m, 2H, H-2 and H-6). The hvdrochloride had mp 151-153°. Anal. Calcd: C, 65.90; H, 7.81; N, 4.52; Cl, 11.44. Found: C, 65.61; H, 7.69; N, 4.46; Cl , 11.49%.

Example 5

(1R,2S)-Borneyl 3-pyridylacetate

The procedure followed that of Example 4 but using

(+)-borneol and afforded the title compound as a colourless oil (2.95g, 75%). ¹H-NMR data was the same as given in Example 4.

Anal. Calcd (free base): C, 74.69; H, 8.48; N, 5.13. Found:

C, 74.71; H, 8.62; N, 4.87%.

Example 6

1-Adamantyl 3-pyridyl acetate.

The method essentially followed that described in Example 1, using 1-adamantanol (3.35g, 22mmol) in dry THF (20ml), n-butyllith1um (1.6M, 12.5ml, 20mmo1) in hexane, and methyl 3-pyrldylacetate (3.02g, 20mmol) in THF (8ml). After allowing the reaction mixture to attain room temperature it was heated under reflux for 18h. The product obtained following work-up and chromatography, as described in Example 1, contained unreacted 1-adamantanol. This was further purified by conversion to the hydrochloride, which was washed with dry ether, and the free base reliberated to afford the title compound (1.30g, 24%) which crystallised from hexane (mp 71-72ºC). ¹H-NMR (CDCI₃) 61.70 and 2.08 (2S, 12H, adamantyl CH₂), 2.14 (s, 3H, adamantyl CH), 3.54 (s, 2H, COCH₂), 7.27 (m, 1H, H-5), 7.66 (m, 1H, H-4), 8.50 (m, 2H, H-2 and H-6). Anal. Calcd: C, 75.25; H, 7.80; N, 5.16. Found: C, 75.12; H, 7.89; N, 5.03%.

Example 7

1-Adamantyl 2-(3-pyridyl)propanoate.

The method essentially followed that described in Example 2, using 1-adamantyl 3-pyridylacetate (542mg, 2.0mmol) in dry THF (2ml), potassium hydride (35% w/w dispersion in oil, 229mg, 2.0mmol) in THF (6ml) and methyl iodide (0.10ml, 1.6mmol). Chromatography, upon elutlon with 50:50:1 light petroleum (bp 60-80°)-diethyl ether-triethyl ami ne, gave the title compound (143mg, 25%), as an oil. ¹H-NMR (CDCl₃) 61.47 (d, 3H, J = 7.2 Hz, CHMe), 1.64 and 2.04 (2s, 12H, adamantyl CH₂), 2.14 (s, 3H, adamantyl CH), 3.63 (q, 1H, J = 7.2 Hz, CHMe), 7.26 (m, 1H, H-5), 7.66 (m, 1H, H-4), 8.52 (m, 2H, H-2 and H-6). Anal. Calcd: C, 75.76; H, 8.12; N, 4.81. Found: C, 75.76; H, 8.28, N, 4.54%.

Example 8

1-Adamantyl 2-methyl-2-(3-pyridyl)propanoate.

The method essentially followed that described in Example 3, using 1-adamantyl 3-pyridylacetate (542mg, 2.0mmol) in dry THF (2ml), potassium hydride (35% w/w dispersion in oil, 504mg, 4.4mmol) in THF (6ml), and methyl Iodide (0.25ml, 4.0mmol). Chromatography upon elutlon with 50:50:1 light petroleum (bp 60-80ºC)-diethyl ether-triethylamine afforded the title compound (262mg, 39%) as an oil. ¹H-NMR (CDCI₃) 61.56 (s, 6H, CMe₂), 1.63 and 2.03 (2s, 12H, adamantyl CH₂), 2.13 (s, 3H, adamantyl CM), 7.26 (m, 1H, H-5), 7.65 (m, 1H, H-4), 8.47 (m, 1H, H-2 or 6), 8.62 (m, 1H, H-2 or 6). Anal. Calcd: C, 67.95; H, 7.80; N, 4.17. Found: C, 68.00; H, 7.86; N, 4.17%.

Example 9

(1S,2R,5S,7R,8R)-Cedryl 3-pyridylacetate.

The method essentially followed that described in Example 1, using (+)-cedrol (2.45g, 11mmol) in dry THF (15ml), n-butyllithium (2.5M, 4.4ml, 11mmol) in hexane, and methyl 3-pyridylacetate (1.51g, 10 mmol) in THF (5ml). After allowing the reaction mixture to attain room temperature, stirring was maintained for an additional 24h. Following work-up and chromatography, eluting with 250:50:1 light petroleum (bp 60-80ºC)-diethyl ether-triethylamine, the product obtained contained some unreacted cedrol. This was further purified by forming the hydrochloride, which was washed with dry diethyl ether, and the free base reliberated to afford the title compound (1.30g, 38%) as an oil. ¹H-NMR (CDCI3) inter alia 60.83 (d, 3H, J = 7.2Hz, cedryl CHMe), 0.96 and 1.08 (2s, 6H, cedryl CMe₂), 1.52 (s, 3H, cedryl OCMe), 3.53 (s, 2H, COCH₂), 7.26 (m, 1H, H-5), 7.63 (m, 1H, H-4), 8.51 (m, 2H, H-2 and H-6). Anal. Calcd: C, 77.37; H, 9.15; N, 4.10. Found: C, 77.56; H, 9.19; N, 3.99%.

Example 10

2-Methy1-2-adamantyl 3-pyridylacetate

The method essentially followed that described in Example 1, but using 2-methyl-2-adamantanol (3.66g, 22 mmol) in dry THF (30ml), n-butyllithium (2.5M, 8.8ml, 22mmol) in hexane, and methyl 3-pyridylacetate (3.02g, 20mmol) in THF (10ml). After allowing the reaction mixture to attain room temperature, stirring was maintained for an additional 96h. Following work-up and chromatography, eluting with 250:50:1 light petroleum-diethyl ether-triethylamine, the product obtained contained some unreacted 2-methyl-2-adamantanol. This was further purified by forming the hydrochloride, which was washed with diethyl ether, and the free base reliberated to afford the title compound (1.77g, 31%) as an oil, ¹H-NMROBCl₃) inter alia 6 1.59 (s, 3H, adamantyl OCMe), 3.60(s, 2H, COCH₂), 7.26 (m, 1H, H-5), 7.65 (m, 1H, H-4), 8.52 (m, 2H, H-2 and H-6). FAB-MS m/z 286 (M+1). Anal, Calcd: C, 75.75; H, 8.12; N, 4.91. Found: C, 75.19; H, 8.16; N, 4.83%.

Example 11

N-(1-Adamantyl)-3-pyridylacetamide

To a solution of 3-pyridylacetic add hydrochloride (2.60g, 15mmol) in dry HMPA (30ml) and dry THF (15ml) was added 1,1'-carbonyldiimidazole (2.43g, 15mmol). After stirring for 30 min 1-adamantanamine (2.50g, 16.5 mmol) was added and stirring continued for 12h. The mixture was poured into water (50ml), basified with aqueous sodium hydroxide (1M) and extracted with diethyl ether (3 × 50ml). The ether extracts were combined, dried (Na₂CO₃), and concentrated. Chromatography, on elution with 15:5:1 ethyl acetate-dichloromethane-triethylamine, afforded the title compound (2.63g, 65%) as white crystals, mp 176-177ºC, IR v_max 1649 cm^-1; ¹ H-NMR (CDCI₃) 6 1.66 and 1.96 (2m, 12H, adamantyl CH₂), 2.05(s, 3H, adamantyl CH), 3.46 (s, 2H, COCH₂), 5.10 (s, 1H, NH), 7.29 (m, 1H, H-5), 7.68 (m, 1H, H-4), 8.51 (m, 2H, H-2 and H-6). FAB-MS m/z 271 (M+1).

Example 12

(1S,2R,5S,7R,8R)-Cedryl 1-(3-pyridyl)cyclopentanecarboxylate

A solution of (1S,2R,5S,7R,8R)-cedryl 3-pyridylacetate (341mg, 1.0 mmol) in dry THF (3 ml) was added to a stirred suspension of potassium hydride (35% w/w dlsperson in oil, 252mg, 2.2 mmol) in THF (2 ml) under argon. After 20 min, 1,4-diicodobutane (132μl, 1.0 mmol) was added, and after 30 min. the mixture was partitioned between dlethyl ether and water, the ether extracts dried (Na₂CO₃) and concentrated. Chromatography, on elutlon with light petroleum-diethyl ether (3:1), afford the title compound (96mg, 24%) which crystallised from the light petroleum at -20ºC, m.p. 81-82ºC. ¹H-NMR (CDCl₃) inter alia δ 0.73 and 0.87 (2s,6H, cedryl CMe2), 0.80 (d,3H,J=7.1Hz, cedryl CHMe), 1.34 (s,3H, cedryl OCMe), 7.22 (m,1H,5-M), 7.66 (m,1H,4-H), 8.45 (m,1H,6-H), 8.62 (m,1H,2-H). Anal. Calcd: C, 78.94; H, 9.43, N, 3.54. Found: C, 79.01; H, 9.61; N, 3.48%.

Test results

Assay of the rat 17α-hydroxylase/C17-C20 lyase.

The assay was carried out as described by S. E. Barrle et al., J. Steroid Blochem. 6, 1191-5, (1989) except that recently the radioactivity in the peaks of interest has been monitored on-line by mixing the HPLC effluent with Ecosdnt A (National Diagnostics) scintillation fluid, 1:1, and passing the mixture through a Bertold LB506C radiochemical monitor.

Assay of the human 17α-hydroxylase/C₁₇-C₂₀ lyase.

Human testes were obtained from previously untreated patients undergoing orchldectomy 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. Barrle et al., supra. The material was then thawed, finely chopped, and homogenised in 0.25M sucrose (5ml/gm 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 1hr. The pellet was washed by being resuspended in 0.25M sucrose and repel leted. 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.

The enzyme activities were measured separately.

For the 17α-hydroxylase: The basic assay mixture was similar to that used for the rat enzyme except that the EDTA concentration was 0.2mM, and the substrate, ³H-progesterone, concentration was 3μM. The human enzyme was more sensitive to ethanol than the rat one, and so the compound under test were dissolved in 50% DMSO and the final concentrations of ethanol and DMSO were 1% each. For the compound of Example 2 and Its 4-pyridyl analogue, reaction was carried out for 15min. For all other compounds, the reaction time was extended to 1 hour. It was terminated by the addition of 2 vols. of methanol/ acetonitrile (2/1 v/v) containing approx. 100μM unlabelled progesterone, 17α-hydroxyprogesterone, androstenedione, testosterone, and 16α-hydroxyprogesterone. The last steroid was added as it appeared that the human enzyme was capable of !6α-hydroxylat1on as well as 17α-hydroxylat1on.

For the compound of Example 2 and Its 4-pyrldyl analogue, the separation of the steroids by HPLC was by the same method as described for the rat assay in S.E. Barrle et al., supra. For all other compounds, the separation was carried out on a 10cm. "Nucleosil" 5μ C18 column with a "Nucleosil" pre-column. Elutlon was with 60% methanol at 1ml /min. The effluent was mixed on-line 1:1 with Ecoscint A containing 25% acetonitrile and then passed through a Berthold LB506C radlochemical detector. In all cases, the hydroxylase activity was measured as the production of 17α-hydroxyprogesterone, androstenedione and testosterone.

For the C₁₇-C₂₀ lyase: The mixture was the same as described above for the 17α-hydroxylase except that the substrate was ³H-17α-hydroxyprogesterone. The reaction was carried out for Ihr. and was stopped by the addition of 2 vols. of methanol /acetonitrlle (2/1 v/v) containing approx. 100μM 17α-hydroxyprogesterone, androstenedione and testosterone.

The HPLC separation used for the lyase involved a 10cm 5μ Apex C18 column with a 5cm PELL ODS C18 precolumn. The eluant was 38:12:50 methanol:acetonitrile:water flowing at 1ml /min. The effluent was mixed 1:1 with Ecosdnt A containing 10% methanol and the radioactivity was measured directly by a Berthold LB506C radlochemical detector. The lyase activity was measured as the production of androstenedione and testosterone. Calculation of IC₅₀.

The enzyme activity was measured in the presence of at least 4 concentrations of each compound, and the data were fitted by linear regression to the Dixon equation (Dixon, M., Webb, E. C. Enzymes, 2nd ed., Academic Press, New York, 1964). The IC₅₀0nd its 95% confidence limits were calculated from the slope and Its 95% confidence limits. Where the full determination of the IC₅₀ has not been carried out the values given are approximate and no confidence limits are shown.

Results are shown in Table 1 below.

Assay of aromatase activity

Aromatase activity was determined by the method of A. B. Foster et al., J. Med. Chem. 22, 50-54 (1983), using human placenta! mlcrosomes. For the microsomes used, the Michael is constant K_m for [1β - ³H] androstenedione was 0.039μM. K_i values were obtained from Dixon plots of reciprocal velocity of reaction versus concentration of inhibitor at two concentrations of the androstenedione substrate. K_i values are given in parenthesis after IC₅₀ values. The comparative results are shown in Table 2.

Table 2 : Aromatase activities, IC₅₀ μM with K_i values in parenthesis in μM, human enzyme

3-PYRIDYL SERIES 4 -PYRIDYL SERIES

Compound

Ex . 1 6 ( 1 .01 ± 0.07) 0 .12 (0.015 ± 0.005)

Ex. 2 8.9 (0. 81 + 0.09) 1 . 32 (0. 12 ± 0.03)

Ex . 3 30 (5.2 ± 1 .0) 3 .85 (0.35 ± 0.06)

Ex . 4 3.0 (0. 207 ± 0.03) 0.097 (0.009 ± 0.0003)

Ex. 5 2.9 not tested

Ex . 6 8.9 "

Ex. 7 9.1 "

Ex . 8 35 "

Ex. 9 1 .7 "

The poorer inhibition of aromatase demonstrated by the compounds of the 3-pyridyl series would benefit the IC₅₀ lyase/aromatase ratio.

Claims

1. Compounds having the general formula:

wherein each of R¹ and R² independently represents hydrogen or C_1-4 alkyl or together represent the residue of a cycloalkyl group of 3 to 6 carbon atoms;

A represents O, NR⁴ wherein R⁴ is defined as for R¹ or R² or CR⁵R⁶ wherein R⁵ and R⁶ are as defined for R¹ or R² as separate substituents; and

R³ represents a bridged allcyclic group;

as free bases or their pharmaceutically acceptable salts.

2. Compounds according to claim 1 wherein A represents, 0, NH or CH₂.

3. Compounds according to claim 1 or 2 wherein R³ represents a bridged allcyclic group having from 6 to 8 ring atoms (excluding bridge atoms), which is unsubstltuted or substituted by alkyl of 1 to 4 carbon atoms or by a fused alicyclic group.

4. Compounds according to claim 3 wherein the fused alicyclic group Is a cyclopentane or cyclohexane ring which Itself is unsubstltuted or substituted by alkyl of 1 to 4 carbon atoms.

5. Compounds according to claim 1, 2, 3 or 4 wherein R³ has one or two bridges, each of 1 or 2 carbon atoms in linear length, unsubstltuted or substituted by alkyl of 1 to 4 carbon atoms.

6. Compounds according to claim 1, 2, 3, 4 or 5 wherein any alkyl group of 1 to 4 carbon atoms is a methyl group.

7. Compounds according to claim 1, 2, 3, 4, 5 or 6 wherein R³ represents a borneyl, Isoborneyl, cedryl or Isopinocampheyl group, in each of their optically active forms, or an adamantyl group.

8. Compounds according to claim 1, 2, 3, 4, 5 or 6 wherein R³ represents a methyladamantyl group.

9. A pharmaceutical composition comprising a compound claimed in claim 1, 2, 3, 4, 5, 6, 7 or 8 in association with a therapeutically acceptable carrier or diluent.

10. A compound claimed in claim 1, 2, 3, 4, 5, 6, 7 or 8 for use in treating prostatic cancer.