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).
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.
Inhibitors of thromboxane A2 (TxA2) synthase are regarded as potentially useful agents in the treatment of cardiovascular diseases and in the prevention of tumour cell metastases. We report here a novel in vitro assay for the evaluation of TxA2 synthase inhibitors. For the determination of inhibitory activity, malondialdehyde (MDA) formation by TxA2 synthase in whole blood was utilized. After reaction with thiobarbituric acid MDA was quantified spectrofluorimetrically. The blank value was obtained by incubation with a selective TxA2 synthase inhibitor. For the screening of compounds the simple MDA assay represents an alternative to the rather expensive and time consuming radioimmunoassay, HPLC and TLC methods. Only for compounds which have been shown to be good inhibitors in the MDA assay should a radioimmunoassay for selective inhibition of TxA2 synthase be performed.
Key Words: tetrahydroquinoline, imidazole-substituted; thromboxane A2 synthase (P450 TxA2) inhibitors; aromatase (P450 arom); 17a-hydroxylase-CI 7,204yase (P450 17); antitumor drugsIn recent years our research interest was focused on the development of inhibitors of steroidogenic enzymes, e.g. aromatase (P450 arom, CYP 19) and 17a-hydroxylase-C17,20-lyase (P450 17, CYP 17) [']. Inhibitors of these enzymes are promising candidates for the treatment of estrogen and androgen dependent diseases like breast and prostate cancer. Structurally related tetralins containing carboxylic residues at the benzene nucleus are reported in the literature to be inhibitors of thromboxane A2 synthase (P450 TxA2, CYP 5f2], an enzyme which is also P450 dependentf3]. A fascinating new indication for P450 TxA2 inhibitors could be the prophylaxis of tumor metastases. Several tumor tissues contain high levels of TxA214] and in various cancer cell lines a cell proliferation could be achieved by the addition of TxA2Prostacyclin -the arachidonic acid metabolite which is increased in concentration as a consequence of P450 TxA2 inhibition -and its stable analogs display antimetastatic activity in spontaneously metastasizing tumors[6a1. Moreover, experimental hepatic metastases can be prevented by the application of a P450 TxA2 inhibitor[6b1. Many authors claim that the structural requirements for strong TxA2 synthase inhibition are a heterocycle with an accessible sp2-hybridized nitrogen and a carboxylic group at a distance of about 8 A[71. While the lone electron pair of the nitrogen coordinates with the heme iron of the enzyme[*], the function of the carboxylic group has not been yet elucidated. Some authors assume that it mimics the C1-carboxylic residue of the substrate prostaglandin H2 (Scheme 1). Nevertheless, we tested some of our tetralins -which do not contain a carboxylic group -on in vitro P450 TxA2 inhibition and were surprised to find rather good inhibitors. We therefore looked more thoroughly into this class of compounds and tested about 150 compounds Scheme 1) using a malonaldehyde assay dewas tested on selective inhibition (determination of TxB2 and PGE2 concentration by radioimmunoassay). All of the tested compounds proved to be selective P450 TxA2 inhibitors.The in vitro studies revealed some interesting structure activity relationships: As found with other compounds[2. 71, the heterocyclic nitrogen is a basic requirement for P450 TxA2 inhibition. The inhibitory activity strongly depends on the position and the type of substituent at the benzene nucleus. Hydrophilic and dipolar groups as well as hydrogen bond acceptors increase the inhibitory activity (Scheme 2). Recent insights into the structure of P450 TxA2 -obtained from a molecular modelling study based on the bacterial fatty acid monooxygenase P450 BM-3[l0] -suggest that the C15-hydroxy group of prostaglandin H2 plays the major role in substrate binding to the enzyme (Scheme 1). Inside a hydrophobic canal, the amino acids Arg-410 and Glu-433 are located within a distanc...
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