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In search of new leads for selective inhibition of estrogen and androgen biosynthesis, respectively, heterocyclic substituted 2-(arylmethylene)-1-tetralones (1-4, 9-17), 2-(aryl-hydroxymethyl)-1-tetralones (5-8), exo-1a,2,3,7b-tetrahydro-1H-cyclopropa[alpha] naphthalenes (18-24), and 3-alkyl substituted 4,5-dihydronaphtho[1,2-c]pyrazoles (25-27) were synthesized and tested for inhibitory activity toward four steroidogenic enzymes (P450 arom, P450 17, P450 18, and P450 scc, as well as another P450 enzyme, thromboxane A(2) (TXA(2)) synthase. The test compounds inhibited human placental P450 arom, showing a wide range of inhibitory potencies. (Z)-4-Imidazolyl compound 17 was the most potent inhibitor, with a relative potency (rp) of 110 [rp of aminoglutethimide (AG) = 1), rp of fadrozole = 359]. A competitive type of inhibition was shown by the (E)-4-imidazolyl compound 16(rp = 71). On the other hand some of these compounds inhibited rat testicular P450 17. Maximum activity was shown by the 3-pyridyl compound 20 (rp = 10, ro of ketoconazole = 1). 20 was the only compound which exhibited a marked inhibition of TXA(2) synthase (IC(50) = 14.5 microM; IC(50) of dazoxiben = 1.1 microM). Regarding selectivity toward the steroidogenic enzymes, compound 16 was relatively selective toward P450 arom, whereas compound 20 was relatively selective toward P450 17. (P450 arom: K(m) testosterone = 42 nM, K(i)16 = 33 nM, K(i)20 = 3 microM. P450 17: K(m)progesterone = 7 microM, K(i)16 = 9 microM, K(i)20 = 80 nM). 17 and 24 were not selective since they showed strong inhibition of P450 arom (K(i)17 = 26 nM, K(i)24 = 0.12 microM) and P450 17 (K(i) 17 = 0.7 microM, K(i)24 = 0.11 microM).

Compounds capable of inhibiting 17 alpha-hydroxylase/17,20-lyase (P450 17 alpha) are of great interest for the therapy of prostatic cancer since they block androgen biosynthesis. In order to evaluate the inhibitory activity of a series of benzocycloalkenes developed in our group, an in vitro assay was established using rat testicular microsomes as source of the enzyme, non labelled progesterone as substrate and a HPLC procedure for separation of the steroids. The inhibitory activities of 33 test compounds were compared to ketoconazole (IC50 67 microM), a known inhibitor of P450 17 alpha, which recently has been successfully used in prostate cancer patients. Several compounds of the present study were stronger inhibitors of P450 17 alpha than ketoconazole. The most active compounds were compound 12(5-methoxy-2-(4-pyridylmethyl)-1-tetralone: IC50 13 microM) and compound 13(5-methoxy-2-(4-pyridyl)-1-tetralone: IC50 13 microM).

The synthesis and biological evaluation of substituted exo-1-(4-pyridyl)-1a,2,3,7b-tetrahydro-1H-cyclopropa[a]naphthalene s as inhibitors of estrogen biosynthesis is described [H (1); 4-OCH3 (2); 5-OCH3 (3); 6-OCH3 (4); 1-CH3, 6-OCH3 (5); 4-OCH3, 7-Br (6); 6-OCH3, 5-Br (7); 4-OH (8); 5-OH (9); 6-OH (10)]. The synthetic key step--the formation of the cyclopropyl ring--was accomplished using the conditions of a modified Wolff-Kishner reduction (N2H5OH/KOH; delta T) and yielded exclusively the exo-configurated diastereomers. The racemic compounds 1-10 showed an inhibition of human placental aromatase (P450 arom) exhibiting relative potencies (rp) from 3.7 to 303 (compounds 8 and 4, respectively; rp of aminoglutethimide (AG) identical to 1, fadrozole = 359). The enantiomers of 4 and 7 were separated by LPLC on tribenzoyl cellulose and by crystallization of the diastereomeric tartrates (4). (1aS,2S,7bS)-(+)-4 (absolute configuration determined by X-ray crystallographic analysis) is the active P450 arom inhibiting enantiomer of 4 and shows a rp value of 617. Compound 4 is a reversible inhibitor showing a competitive type of inhibition and a type II difference spectrum. In vitro 4 influenced other steroidogenic P450 enzymes either not at all (bovine adrenal P450 scc) or only marginally (rat testicular P450 17, bovine adrenal P450 18). In ACTH-stimulated rat adrenal tissue, 4 was less active, inhibiting corticosterone and aldosterone formation compared to AG and fadrozole, respectively. In vivo 4 was not superior to AG as far as the inhibition of the uterotrophic activity of androstenedione (juvenile SD rats) and the reduction of the plasma estradiol concentration (pregnant mares' serum gonadotropin-primed SD rats) are concerned. Compound 4 shows marked antitumor activity in the dimethylbenzanthracene-induced mammary carcinoma of the SD rat: in the postmenopausal model it is at least as active as AG; in the premenopausal experiment it is clearly superior to AG. No induction of hepatic P450 enzymes was observed in the latter experiment. The rp value of 4 toward rat ovarian P450 arom, i.e., 23 (rp of AG identical to 1), is markedly decreased compared to the human enzyme (rp value of 303). From this fact it must be concluded that 4 should be more active in the human than in the rat.

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In search of potential drugs for the treatment of estrogen- and androgen-dependent cancer as well as the prophylaxis of metastases, tetralones, tetralins, and dihydronaphthalenes bearing a OCH3 substituent at the benzene nucleus and an imidazol-4-yl, imidazol-1-yl, or 1,2,4-triazol-1-yl substituent in 2-position were synthesized with and without C1-spacer between the rings (compounds 2-26). The compounds were tested in vitro for inhibition of the three targets enzymes P450 arom (human placental microsomes), P450 17 (rat testicular microsomes), and P450 TxA2 (citrated human whole blood). To examine selectivity, some compounds were further tested in vitro for inhibition P450 18 (bovine adrenal mitochondria), P450 scc (bovine adrenal mitochondria) and corticoid formation (aldosterone, corticosterone; ACTH stimulated rat adrenal tissue). In vitro, selected compounds were examined in Sprague Dawley rats regarding P450 TxA2 inhibition, reduction of plasma testosterone concentration, antiuterotrophic activity (inhibition of the uterotrophic activity of androstenedione), reduction of plasma estradiol concentration (pregnant mares' serum gonadotropin-primed rats), and mammary tumor inhibiting activity (dimethylbenzanthracene-induced tumor; pre-and postmenopausal model). In the series of imidazol-4-yl compounds, which represent a novelty in the field of azole inhibitors of steroidogenic P450 enzymes, strong inhibitors of P450 arom and/or P450 17 were found; 7-OCH3-2-(imidazol-4-ylmethylene)-1-tetralone (4) and 7-OCH3-2-(imidazol-4-ylmethyl)-tetralin (12) are among the most potent inhibitors of P450 arom in vitro known so far. Compound 4 is a selective inhibitor, whereas 12 shows in addition strong inhibition of P450 17. In contrast to 12, the 6-OCH3 derivative (compound 11) is a selective inhibitor of P450 17, being 50 times more potent than ketoconazole. Some imidazol-1-yl compounds show a marked inhibition of P450 TxA2: 2-(imidazol-1-ylmethyl)-1-tetralone (13) is a selective inhibitor of P450 TxA2, whereas 7-OCH3-2-(imidazol-1-ylmethyl)-tetralin (17) as well 2-(imidazol-1-ylmethyl)-tetralin (16) and 7-OCH3-2-imidazol-1-yl-3, 4-dihydronaphthalene (25) additionally show strong inhibition of P450 arom and P450 17. Regarding the other steroidogenic P450 enzymes as well as corticosterone formation, the compounds show only little inhibitory activity. Aldosterone formation, however, is inhibited at low concentrations. Nevertheless, 4 and 12 are more selective, i.e. inhibit aldosterone synthesis less than the well known inhibitor of P450 arom fadrozole. The compounds show activity in the aforementioned in vivo tests.

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