ANP PHARMACEUTICAL USE Background of the Invention i, Field of the Invention

This Invention relates to derivatives containing the 3-pyr1dylacetyl function, namely esters and amides of 3-pyr1dylacetic add as well as 3-pyrIdylmethyl ketones, together with certain derivatives thereof, their preparation and use 1n treating prostatlc cancer. 2. Description of related art

R. McCague, M. G. Rowlands, S. E. Barrle and J. Houghton, 3. Med. Chem. 22, 3050-3055 (1990), have reported that certain esters of 4-pyr1dylacetic add, of general formula:

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

wherein represents a hydrogen atom or a methyl group, Inhibit the 17α-hydroxylase/Ci7_2o lyase enzyme complex which 1s essential for biosynthesis of androgens. The Inhibition of androgen biosynthesis by virtue of the hydroxylase/lyase Inhibition Indicates that the compounds of McCague e_± aj... supra, could be useful for the treatment of prostate cancer since many such tumours depend on androgens for growth.

The compounds of McCague si, &1- are also Inhibitors of aromatase. Aro atase 1s an enzyme required 1n the biosynthesis of estrogens. The ability to Inhibit aromatase 1s 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 1t 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 1t 1s a poor Inhibitor of aromatase). Their best compound from that viewpoint 1s cyclohexyl 2-methyl-2-(4-pyr1dyl) propanoate of formula (2) above wherein R ^b 1s methyl and formula (26) in the paper, although 1t 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 mono ethy1ated 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 1s an alicyclic group of the terpene residue type, notwithstanding that 1n such compounds the IC ₅₀ 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. 21, 3055-3060 ⁽ 1990 ⁾ attempts to explain the data of the McCague ≤± il paper 1n terms of mimicry of the natural steroid substrates for aromatase and hydroxylase/

lyase by the 4-pyridylacetates and propanoates with reference to cyclohexyl 4-pyridy acetate [formula (1); R ^a - cyclohexyl] and Its α-methyl derivative, cyclohexyl 2-(4-pyr1dyl)propanoate [formula (2); R ^b * H], since the α-methyl substitution lowers the IC ₅₀ 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" 1s favourable, but that the α-methyl substltuent would then mimic a substltuent on C(2) of the steroid. Since other evidence has suggested that sterlc bulk in the C(2) region 1s unfavourable for aromatase Inhibition, the poorer Inhibition of the α-methyl substituted ester could be rationalised 1n 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-pyr1dyl)propanoate lies 1n 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 hydrophoblc 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-ethylcyclohexyl 4-pyr1dyl cetate, 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 1n treating androgen-induced cancers such as prostatic cancer. These compounds have the general formula:

wherein each of R ¹ and R ² independently represents hydrogen or lower alk l or together represent the residue of a cycloalkyl group of 3 to 6 carbon atoms;

A represents 0, NR ⁴ where R ⁴ 1s defined as for R ¹ and R ² , or CR ⁵ R ⁶ where R ⁵ and R ⁶ are defined as for R ¹ or R ² as separate substituents; and

R ³ represents a bridged allcyclic 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 R ³ group in the preferred compounds of the invention can be defined 1n various ways, all reflecting the fact that R ³ 1s hydrocarbyl, cyclic and non-aromatic and has at least one bridge across a ring. Because R ³ 1s defined as bridged, 1t 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 1s 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 1t is possible to regard the R ³ group as a substituted cyclohexyl group 1n which the substituents comprise bridging members. Examples of preferred R ³ groups fitting this definition are shown below, along with the adjacent oxygen atom:-

isopinocampheyl (cyclohexyl substituted by a 3,5-1soprop 11dene bridge and additionally having a 2-methyl substltuent)

borneyl (cyclohexyl substituted by a 2,5-1soprop 11dene bridge and additionally having a 2-methyl substituent)

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

exo-norborneyl

adamantyl ⁽ cyclohexyl substituted by a first 1 ,3-(l ,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-(l,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 substltuent on the cyclohexane ring)

cedryl (cyclohexyl substituted by a 2,4-1sobutylene 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 R ³ 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, R ³ 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 rinr, e.g. a substituted

cyclopentane or cyclohexane ring. In this definition the bridges are normall.' of 1 or 2 carbon atoms 1n 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 1sopropyl1dene bridging group).

The allcyclic groups of R ³ 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- _| _ 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) 1s preferably -0-, but when It Is -CH ₂ - potentially hydrolysable ester and amide bonds are not present, which 1s also advantageous.

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

R ¹ and R ² are preferably both methyl except when R ³ 1s an extremely bulky group, such as cedryl, 1n 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 R ¹ and R ² is a lower alkyl group and most preferably they are both hydrogen. Alternatively R ¹ and R ² 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 NR ⁴ , R ⁴ 1s preferably hydrogen, but can have any of the other meanings for the Individual R ¹

and R ² substituents, of which lower alkyl, especially methyl, is preferred.

In the ketones, R ⁵ and R ^δ are preferably hydrogen, one is methyl and the other hydrogen or both are methyl. R ⁵ and R ⁶ 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 «= -0-) have the general formula (4):

wherein X represents -OH or a reactive substltutent and R ¹ and R ² are as defined for formula (3). Simple ester1f1cat1on or trans-esteriflcation with an alcohol of formula R ³ -0H where R ³ is as defined for formula (3) leads to the esters of formula (3).

The reactive substltuent X 1s 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 1n the usual way.

To prepare the ketones of formula (3) in which A - -CH ₂ -, a suitable procedure would involve the reaction between an alkali metal salt of 3-picoline, e.g. 3-p1colyllithium (C. G. Screttas, T. F. Estham, C. W. Kamienskl, Ch1m1a, 24, 109-111, 1970) and an appropriate methyl ester R ³ AC02 e where A 1s another CR ⁵ R ⁶ 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, IS, 349-350, (1973) to make alkyl 4-pyridylmethylketones from 4-picolylsodium and RC0 ₂ Me.

Where the compounds of formula (3) being prepared are those In which R ¹ or R ² is other than hydrogen, it may be convenient

to use as the starting compound of formula (4) an unsubstituted pyridylacetic add compound wherein R ¹ and R ² are hydrogen, prepare the corresponding compound of formula (3) and subsequently introduce the desired R ¹ or R ² substltuent by the action of an alkali metal hydride followed by a lower alkyl or cycloalkyl bromide or Iodide. Methylene (CH ₂ «) or ethyl1dene ⁽ CH ₃ CH ₂ » ⁾ derivatives may be prepared from corresponding methyl and ethyl derivatives by thermal decomposition of phenylsulphoxlde B. M. Trost e aj.. , 3. 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, 1n 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 intracavltal), 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 1n orally 1ngest1ble 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 exdpients, 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-dependent tumours in the mammalian body, which comprises administering a compound of the invention to a mammalian patient 1n 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 (12,22,32,5fi)-Isop1nocampheyl 3-pyr1dylacetate.

A stirred solution of (+)-1sop1nocampheol (3.086 g, 20 mmol) in dry tetrahydrofuran (20 ml) under N ₂ was cooled with an Ice- salt bath. A solution of n-butyll1th1um (1.6 M, 12.5 ml, 20 mmol) in hexane was added followed, after 5 m1n, by a solution of ethyl 3-pyridylacetate (2.746 g, 16.7 mmol) 1n tetrahydro¬ furan (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°)-d1ethyl ether-tr1ethylamine the title compound (3.79 g, 76 ) as an oil. By passing hydrogen chloride gas through a solution of the product 1n d1ethyl ether, the hydrochloride was obtained. This was recrystalllsed from dioxan-ether 1:1, p 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 (12,22,32,5R)-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

- li ¬ on, 383 mg, 3.34 mmol) 1n tetrahydrofuran (10 ml) under nitrogen at 0°C. Af _^ er 10 m1n, 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. ^-NMR ⁽ CDC1 ₃ ⁾ inter ≤JJLa S 0.97, 1.20 (2s, 6H, Me_ ₂ C), 1.53 (d, 3H, J - 6.8 Hz, COCHCH3 ⁾ , 3.75 (q, 1H, COCHCH3), 5.10 (m, 1H,

OCH ⁾ , 7.30 ⁽ dd, 1H, J _ort ho " ⁴ - ⁸ « ⁷ - ^{95 Hz} » ϋ" ⁵⁾ - ⁷ - ^{80 <m} « ^1H ' H-4 ⁾ , 8.60 (m, 2 H, fl-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 ⁽ l2,22,32,5β ⁾ -Isop1nocampheyl 2-methyl-2-(3-pyr1dyl ⁾ propanoate.

A solution of ⁽ 12,22,32,5β)-isopinocampheyl 3-pyr1dyl- acetate ⁽ 706 mg, 2.58 mmol) in dry tetrahydrofuran (8 ml) was added to a stirred suspension of potassium hydride (35% by weight dispersion 1n oil, 650 mg, 5.68 mmol) 1n tetrahydrofuran (6 ml ⁾ under argon at 0°C. After 10 min, methyl iodide (733 mg, 5.16 mmol ⁾ was added 1n two equal portions, each 1n 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 m1n, the reaction was quenched by addition of Isopropanol (0.5 ml). Work-up as 1n 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

(12,2β)-Borneyl 3-pyrldylacetate. By essentially the procedure of Example 1, using (-)-borneol (2.232 g, 14.47 mmol) 1n dry tetrahydrofuran (15 ml), n-butyll1th1um (5.79 ml, 14.47 mmol) and ethyl 3-pyr1dylacetate (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%). ^] H-NMR inter alia

δ 0.77 ⁽ s, 3H, C£H ₃ ⁾ , 0.84, 0.87 ⁽ 2S, 6H, C ⁽ £U3 ⁾ ₂ >, ³ _' ^{64 (S} ' ^2H ' C0£H ₂ ⁾ , 4.90 ⁽ m, 1H, CE0C0), 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 (l£,22 ⁾ -Borneyl 3-pyr1dyl cetate

The procedure followed that of Example 4 but using

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

Exam le 6

1-Adamantyl 3-pyridylacetate. The method essentially followed that described 1n Example 1, using 1-adamantanol (3.35g, 22mmol) 1n dry THF (20ml), n-butyll1th1um ⁽ 1.6M, 12.5ml, 20mmo1) in hexane, and methyl 3-pyrldyl cetate (3.02g, 20mmo1) in THF (8ml). After allowing the reaction mixture to attain room temperature 1t was heated under reflux for 18h. The product obtained following work-up and chromatograph , as described 1n Example 1, contained unreacted 1-adamantanol . This was further purified by conversion to the hydrochlorlde, which was washed with dry ether, and the free base re11berated to afford the title compound (1.30g, 24%) which crystallised from hexane (mp 71-72'C). ^-N R (CDCI3) 61.70 and 2.08 (2S, 12H, adamantyl CU ₂ ), 2.14 (s, 3H, adamantyl CM), 3.54 ⁽ s, 2H, C0CH ₂ ⁾ , 7.27 ⁽ m, 1H, H-5), 7.66 (m, 1H, M-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-pyr1dyl)propanoate.

The method essentially followed that described 1n 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-tr1ethylamine, gave the title compound (143mg, 25%), as an oil. ^] H-NMR (CDC1 ₃ ) 61.47 (d, 3H, 3 « 7.2 Hz, CHMfi ⁾ , 1.64 and 2.04 ⁽ 2s, 12H, adamantyl CM2>, ² -l ⁴ <s, 3H, adamantyl CM ⁾ , 3.63 (q, IH, 3 - 7.2 Hz, CHMfi), 7.26 (m, IH, H-5), 7.66 (m, IH, M-4), 8.52 (m, 2H, H-2 and M-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-pyr1dyl)propanoate.

The method essentially followed that described 1n Example 3, using 1-adamantyl 3-pyr1dylacetate (542mg, 2.0mmol) in dry THF (2ml), potassium hydride (35% w/w dispersion 1n oil, 504mg, 4.4mmol ⁾ 1n THF ⁽ 6ml), and methyl Iodide (0.25ml, 4.0mmol). Chro atography upon elutlon with 50:50:1 light petroleum (bp 60-80'C)-d1ethyl ether-tr1ethylamine afforded the title compound ⁽ 262mg, 39%) as an oil. ¹ H-NMR (CDCI3) 61.56 (s, 6H, C £ ₂ ), 1.63 and 2.03 ⁽ 2s, 12H, adamantyl U2>. 2.13 (s, 3H, adamantyl CM), 7.26 (m, IH, M-5), 7.65 (m, IH, M-4), 8.47 (m, IH, M-2 or 6), 8.62 (m, IH, M-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

(12,2β,52,7β,8β)-Cedryl 3-pyr1dylacetate. The method essentially followed that described 1n Example 1, using (+)-cedrol (2.45g, llmmol) 1n dry THF (15ml), n-butyl- Hthlu (2.5M, 4.4ml, llmmol) 1n hexane, and methyl 3-pyr1dy1- acetate (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 60-80*C)-d1ethyl ether-tr1ethylamine, the product obtained contained some unreacted cedrol. This was further purified by forming the hydrochlorlde, which was washed with dry d1ethyl ether, and the free base re11berated to afford the title compound (1.30g, 38%) as an oil. ^] H-NMR (CDCI3) inter alia

60.83 (d, 3H, 3 - 7.2Hz, cedryl CHMe_), 0.96 and 1.08 (2s, 6H, cedryl CMe. ₂ ), 1.52 (s, 3H, cedryl OCMe.), 3.53 (s, 2H, C0CM ₂ >, 7.26 (m, IH, H-5), 7.63 (m, IH, H-4), 8.51 ( , 2H, H-2 and M-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-Meth 1-2-adamanty1 3-pyr1d 1acetate

The method essentially followed that described In Example 1, but using 2-methyl-2-adamantanol (3.66g, 22 mmol) 1n dry THF (30ml), n-butyll1th1um (2.5M, 8.8ml, 22mmol) In hexane, and methyl 3-pyridylacetate (3.02g, 20mmol) 1n 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-tr1ethylamine, the product obtained contained some unreacted 2-methyl-2-adamantanol . This was further purified by forming the hydrochlorlde, which was washed with dlethyl ether, and the free base re11berated to afford the title compound (1.77g, 31%) as an oil, ^-NMROBC^) inter ali _. 6 1.59 ⁽ s, 3H, adamantyl OC fi ⁾ , 3.60(s, 2H, C0CM ₂ >, 7.26 (m, IH, M-5), 7.65 ( , IH, M-4), 8.52 (m, 2H, M-2 and M-6). FAB-MS m/z 286 (M+l). Anal, Calcd: C, 75.75; H, 8.12; N, 4.91. Found: C, 75.19; H, 8.16; N, 4.83%.

Example 11 fl-(l-Adamantyl)-3-pyr1dylacetam1de

To a solution of 3-pyr1dylacetic add hydrochlorlde (2.60g, 15mmol) 1n dry HMPA (30ml) and dry THF (15ml) was added l,T-carbonyld11m1dazole (2.43g, lδmmol). After stirring for 30 m1n -adamantanamlne (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 dlethyl ether (3 x 50ml). The ether extracts were combined, dried (Na2C03), and concentrated. Chromatography, on elutlon with 15:5:1 ethyl acetate-dichloromethane-triethylamine, afforded the title compound (2.63g, 65%) as white crystals,

mp 176-177 ^β C, IR _m , _χ 1649 cm" ¹ ; ¹ H-NMR (CDCI3) 6 1.66 and 1.96 ⁽ 2m, 12H, adamantyl CM ₂ ), 2.05(s, 3H, adamantyl CM), 3.46 (s, 2H, COCM ₂ >. 5.10 ⁽ s, IH, NM), 7.29 ( , IH, M-5), 7.68 ( , IH, M-4), 8.51 (m, 2H, H-2 and M-6). FAB-MS ®/z 271 (M+l). Example 12

⁽ 12,2β,52,7β,8β)-Cedryl l-(3-ρyr1dyl)cyclopentanecarboxylate

A solution of (12,2β,52,7β,8β)-cedryl 3-pyr1dylacetate ⁽ 341mg, 1.0 mmol) 1n dry THF (3 ml) was added to a stirred suspension of potassium hydride (35% w/w dlsperson 1n oil, 252mg, 2.2 mmol) 1n THF (2 ml) under argon. After 20 min, l,4-d11codobutane (132μl, 1.0 mmol) was added, and after 30 m1n. the mixture was partitioned between dlethyl ether and water, the ether extracts dried (Na2C0 ₃ ) 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 -20"C, m.p. 81-82'C. ^] H-NMR (CDC1 ₃ ) inter alia δ 0.73 and 0.87 ⁽ 2s,6H, cedryl CMe ₂ ), 0.80 (d,3H «7.1Hz, cedryl Otøe ⁾ , 1.34 ⁽ s,3H, cedryl OCMe), 7.22 (m,lH,5-M), 7.66 ⁽ m,lH,4-M ⁾ , 8.45 ⁽ m,lH,6-M ⁾ , 8.62 (m,lH,2-M). 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-hydroxyl se/Ci7-C2o lyase.

The assay was carried out as described by S. E. Barrle £i ... J. Steroid Blochem. £, 1191-5, (1989) except that recently the radioactivity In the peaks of Interest has been monitored on-Hne by mixing the HPLC effluent with Ecosdnt A (National Diagnostics) scintillation fluid, 1:1, and passing the mixture through a Bertold LB506C radloche ical monitor.

Assay of the human 17α-hydroxylase/Ci7-C2o lyase.

Human testes were obtained from previously untreated patients undergoing orchldectomy for prostatic cancer. The testes were decapsulated and stored 1n liquid nitrogen until

use. A microsomal preparation was prepared essentially as described by S. E. Barrle et j.. , supra. The material was then thawed, finely chopped, and homogenised 1n 0.25M sucrose (5ml/gm wet weight) using a Potter homogeniser. The homogenate was centrlfuged at 12000g for 30 min, and then the mlcroso es 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 aϋquots 1n 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, ³ H-progesterone, concentration was 3μM. The human enzyme was more sensitive to ethanol than the rat one, and so the compound under test were dissolved 1n 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 15m1n. For all other compounds, the reaction time was extended to 1 hour. It was terminated by the addition of 2 vols. of methanol/ acetonitrlle (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 1n S.E. Barrle e_i aj.. , supra. For all other compounds, the separation was carried out on a 10cm. "Nucleosll" 5μ C18 column with a "Nucleosll" pre-column. Elutlon was with 60% methanol at 1ml/min. The effluent was mixed on-line 1:1 with Ecosdnt A containing 25% acetonitrlle and then passed through a Berthold LB506C radlochemlcal detector. In all cases, the hydroxylase activity was measured

as the production of 17α-hydroxyprogesterone, androstenedione and testosterone.

For the C _j 7-C ₂ o lyase: The mixture was the same as described above for the 17α-hydroxylase except that the substrate was ³ H-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:aceton1trile: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 1n 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 IC5 and its 95% confidence limits were calculated from the slope and Its 95% confidence limits. Where the full determination of the IC ₅₀ has not been carried out the values given are approximate and no confidence limits are shown.

Results are shown in Table 1 below.

Table 1 : 17α-hydroxylase/Ci7-C2o lyase activities IC50 (μM) wi th 95% confidence l imi ts i n parenthesi s , measured with the rat enzymes , (R) or human enzymes (H)

3-PYRIDYL SERIES 4-PYRIDYL SERIES

compound Hydroxylase Lyase Hydroxylase Lyase Ex. 1,

R ¹ - R ² - H

R ³ - Isopino- H 0.50 0.10 0.041 0.0066 campheyl (0.44-0.58) (0.09-0.12) (0.038-0.045) (0.006-0.007) R 0.46 0.52 0.26 0.28 (0.42-0.50) (0.48-0.57) (0.24-0.27) (0.24-0.34)

Ex. 2, H 0.1-0.3 0.04 0.02 0.005 R ¹ - CH ₃ , R ² - H R not tested

R ³ - isopino¬ campheyl

Ex. 3, M 0.043 0.016 0.05-0.1 0.01 R ¹ - CH ₃ , R ² - CH ₃ , β 0.047 0.052 not tested (0.042-0.052)(0.045-0.061) R ³ « isopino¬ campheyl

3-PYRIDY ^' . SERIES 4-PYRIDYL SERIES

Compound Hvdroxvl ase Lvase Hvdroxylase Lvase

Ex . 4 , M not tested

R ¹ - R ² - H

R ³ - β 2.0 2.3 1.5 2.2

(-) borneyl ⁽ 1.9-2.1X2.0-2.6 ⁾ (1.4-1.6) (2.0-2.5)

Ex. M 1.5 0.4 not tested Rl R ² - H R ³ (+)borneyl

Ex. 6 M 1.0 0.6 not tested R ¹ - R ² - H R ³ - adamantyl

Ex. 7 H 0.3 0.1 not tested

Rl - CH ₃

R ² - H

R ³ - adamantyl

Ex. 8 M 0.06 0.04 not tested

R ¹ - CH ₃

R ² - CH ₃

R ³ - adamantyl

Ex. 9 M 0.16 0.12 not tested Rl - R ² - H

R ³ - (+)cedryl

Assay of aromatase activity

Aromatase activity was determined by the method of A. B. Foster £i aj.., J. Med. Chem. 22, 50-54 (1983), using human placenta! mlcrosomes. For the microsomes used, the Michaelis constant K _m for [lβ - ³ H] androstenedione was 0.039μM. K values were obtained from Dixon plots of reciprocal velocity of

reaction versus concentration of inhibitor at two concentrations of the androstenedione substrate. K- _j values are given 1n parenthesis after IC50 values. The comparative results are shown in Table 2.

Table 2 : Aromatase activities, IC ₅₀ μM with K- _j values 1n parenthesis in μM, human enzyme

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