The 16,17-double bond is needed for irreversible inhibition of human cytochrome p45017alpha by abiraterone (17-(3-pyridyl)androsta-5, 16-dien-3beta-ol) and related steroidal inhibitors.

Abiraterone (17-(3-pyridyl)androsta-5,16-dien-3beta-ol, 1) is a potent inhibitor (IC50 4 nM for hydroxylase) of human cytochrome P45017alpha. To assist in studies of the role of the 16,17-double bond in its mechanism of action, the novel 17alpha-(4-pyridyl)androst-5-en-3beta-ol (5) and 17beta-(3-pyridyl)-16,17alpha-epoxy-5alpha-androst-3beta-ol (6) were synthesized. 3beta-Acetoxyetienic acid was converted in three steps into 5 via photolysis of the thiohydroxamic ester 8. Oxidation of an appropriate 16,17-unsaturated precursor (21) with CrO3-pyridine afforded the acetate (23) of 6. Inhibition of the enzyme by 1, the similarly potent 5,6-reduced analogue 19 (IC50 5 nM), and the 4, 16-dien-3-one 26 (IC50 3 nM) and by the less potent (IC50 13 nM) 3,5, 16-triene 25 is slow to occur but is enhanced by preincubation of the inhibitor with the enzyme. Inhibition following preincubation with these compounds is not lessened by dialysis for 24 h, implying irreversible binding to the enzyme. In contrast under these conditions the still potent (IC50 27 nM) 17alpha-(4-pyridyl)androst-5-en-3beta-ol (5) showed partial reversal after 5 h of dialysis and complete reversal of inhibition after 24 h. This behavior was also shown by the less potent 16,17-reduced 3-pyridyl compounds 3 and 24. Further, in contrast to the compounds (1, 19, 25, 26) with the 16,17-double bond, the inhibition of the enzymic reaction was not enhanced by preincubation either with 5 or with the 17beta-pyridyl analogues 3, 4, and 24 which also lack this structural feature. The results show that the 16,17-double bond is necessary for irreversible binding of these pyridyl steroids to cytochrome P45017alpha. However oxidation to an epoxide is probably not involved since epoxide 6 was only a moderately potent inhibitor (IC50 260 nM).

Biochemistry and pharmacokinetics of potent non-steroidal cytochrome P450(17alpha) inhibitors.

Two potent non-steroidal inhibitors (CB7645 and CB7661) of human cytochrome P450(17alpha) were tested for in vivo activity in WHT mice. There were no signs of toxicity, but there was no effect on the androgen-dependent organs. The pharmacokinetics and biochemistry of the compounds in mice were investigated. Following i.p. administration of 0.5 mmol/kg of CB7645 and CB7661, peak plasma levels of 13.4 and 3.4 microM, respectively, occurred after 2-4 h, both compounds were cleared rapidly (terminal half-lives 2.7 and 3.3 h, respectively) and neither was detectable at 24 h. CB7645 produced some decrease in plasma testosterone at 4 h, but this was not sustained. When tested in vitro against the WHT testicular enzyme, the CB7645 and CB7661 were competitive inhibitors with K(i) values of 10 and 13 nM, respectively. However, the K(m) for the substrate progesterone was lower at 4.3 nM. These data indicate that, for effective and continuous inhibition of the murine cytochrome P450(17alpha) enzyme, higher peak levels of the compounds would be required, and these levels would need to be maintained throughout the treatment period.

3- and 4-pyridylalkyl adamantanecarboxylates: inhibitors of human cytochrome P450(17 alpha) (17 alpha-hydroxylase/C17,20-lyase). Potential nonsteroidal agents for the treatment of prostatic cancer.

Various 3- and 4-pyridylalkyl 1-adamantanecarboxylates have been synthesized and tested for inhibitory activity toward the 17 alpha-hydroxylase and C17,20-lyase activities of human testicular cytochrome P450(17 alpha). The 4-pyridylalkyl esters were much more inhibitory than their 3-pyridylalkyl counterparts. The most potent was (S)-1-(4-pyridyl)ethyl 1-adamantanecarboxylate (3b; IC50 for lyase, 1.8 nM), whereas the (R)-enantiomer 3a was much less inhibitory (IC50 74 nM). Nearly as potent as 3b was the dimethylated counterpart, the 2-(4-pyridylpropan-2-yl) ester 5 (IC50 2.7 nM), which was also more resistant to degradation by esterases. In contrast to their 4-pyridyl analogs, the enantiomers of the 1-(3-pyridyl)ethyl ester were similarly inhibitory (IC50 for lyase; (R)-isomer 8a 150 nM, (S)-isomer 8b 230 nM). Amides corresponding to the 4-pyridylmethyl ester 1 and the (S)-1-(4-pyridyl)ethyl ester 3b, respectively 11 and 15b, were much less inhibitory than their ester counterparts. On the basis of a combination of inhibitory potency and resistance to esterases, the ester 5 was the best candidate for further development as a potential nonsteroidal inhibitor of cytochrome P450(17 alpha) for the treatment of prostate cancer.

Esters of 3-pyridylacetic acid that combine potent inhibition of 17 alpha-hydroxylase/C17,20-lyase (cytochrome P45017 alpha) with resistance to esterase hydrolysis.

Esters of 3- and 4-pyridylacetic acid have been prepared and tested for inhibitory activity toward the human testicular 17 alpha-hydroxylase/C17,20-lyase and human placental aromatase enzymes. The structural features required for optimal inhibition of the hydroxylase/lyase enzyme were a 3-pyridine ring, methyl substitution alpha to the carbonyl group, and a bulky alkoxycarbonyl substituent. The compounds with the greatest selectivity were isopinocampheyl 2-methyl-2-(3-pyridyl)propanoate, 9, 1-adamantyl 2-methyl-2-(3-pyridyl)propanoate, 12, and 2-methyl-2-adamantyl 2-methyl-2-(3-pyridyl)propanoate, 14, which, while inhibiting the aromatase activity with IC50 values of 30, 35, and 40 microM, respectively, exhibited IC50 values toward hydroxylase/lyase of between 13 and 90 nM. For comparison, ketoconazole gave an IC50 value of 15 microM against aromatase and values of 65 and 26 nM for inhibition of the hydroxylase and lyase activities, respectively. Some of the structural features required for enzyme inhibition also conferred resistance to esterase hydrolysis, in vitro using rat liver microsomes as a source of the esterase activity. Therefore these esters are lead compounds in the development of inhibitors of androgen biosynthesis for the treatment of hormone-dependent prostatic cancer.

Novel steroidal inhibitors of human cytochrome P45017 alpha (17 alpha-hydroxylase-C17,20-lyase): potential agents for the treatment of prostatic cancer.

Steroidal compounds having a 17-(3-pyridyl) substituent together with a 16,17-double bond have been synthesized, using a palladium-catalyzed cross-coupling reaction of a 17-enol triflate with diethyl(3-pyridyl)borane, which are potent inhibitors of human testicular 17 alpha-hydroxylase-C17,20-lyase. The requirement for these structural features is stringent: compounds having 2-pyridyl (9), 4-pyridyl (10), or 2-pyridylmethyl (11) substituents instead of the 3-pyridyl substituent were either poor inhibitors or noninhibitory. Reduction of the 16,17-double bond to give 17 beta-pyridyl derivatives diminished potency with 3-pyridyl substitution (3-->27; IC50 for lyase, 2.9-->23 nM) but increased it with a 4-pyridyl substituent present (10-->28; IC50 1 microM-->53 nM). In contrast, a variety of substitution patterns in rings A-C of the steroid skeleton afforded inhibitors having potencies similar to those most closely related structurally to the natural substrates pregnenolone and progesterone, respectively 17-(3-pyridyl)androsta-5,16-dien-3 beta-ol (3, Kiapp < 1 nM; IC50 for lyase, 2.9 nM) and 17-(3-pyridyl)androsta-4,16-dien-3-one (15; IC50 for lyase, 2.1 nM). Thus compounds having variously aromatic ring A (18), saturated rings A/B (21, 22), and oxygenated ring C (26) exhibited IC50 values for lyase (1.8-3.0 nM) falling within a 2-fold range. The most potent compounds are candidates for development as drugs for the treatment of hormone-dependent prostatic carcinoma.

Pharmacology of novel steroidal inhibitors of cytochrome P450(17) alpha (17 alpha-hydroxylase/C17-20 lyase).

Medical or surgical castration for the treatment of prostatic cancers prevents androgen production by the testes, but not by the adrenals. Inhibition of the key enzyme for androgen biosynthesis, cytochrome P450(17) alpha, could prevent androgen production from both sources. The in vivo effects of 17-(3-pyridyl)androsta-5,16-dien-3 beta-ol (CB7598) and 17-(3-pyridyl)androsta-5,16-dien-3-one (CB7627), novel potent steroidal inhibitors of this enzyme, on WHT mice were compared with those of castration and two clinically active compounds, ketoconazole and flutamide. Flutamide and surgical castration caused significant reductions in the weights of the ventral prostate and seminal vesicles. CB7598, in its 3 beta-O-acetate form (CB7630), and CB7627 caused significant reductions in the weights of the ventral prostate, seminal vesicles, kidneys and testes when administered once daily for 2 weeks. Ketoconazole, given on the same schedule, caused no reductions. Plasma testosterone was reduced to < or = 0.1 nM by CB7630, despite a 3- to 4-fold increase in the plasma level of luteinizing hormone. Adrenal weights were unchanged following treatment with CB7630 or CB7627 but were markedly increased following ketoconazole, indicating no inhibition of corticosterone production by these steroidal compounds. These results indicate that CB7598, CB7630 or CB7627 may be useful in the treatment of hormone-dependent prostatic cancers.

Evaluation of some 4-fluoro- and 4-cyano derivatives of delta 4,3-ketosteroids as inhibitors of testosterone 5 alpha-reductase.

Some 4-fluorinated analogues of 3-oxo-delta 4 steroids and 4-cyano derivatives of progesterone and androstenedione were evaluated as inhibitors of steroid 5 alpha-reductase activity. Inhibitors of this enzyme may be useful in treating prostatic cancer. 4-Fluoroandrostenedione was a modest inhibitor of the rat enzyme (IC50 = 4.08 microM), while 4-cyanoprogesterone was a potent inhibitor of both the rat and human enzymes (IC50 values = 0.045 microM and 0.050 microM respectively). These two steroids were tested in vivo for activity against androgen sensitive organs in WHT mice. 4-Fluoroandrostenedione caused increases in organ weights, suggesting it is an androgen agonist, while the 4-cyano compound displayed modest androgen ablation. Therefore substitutions at the 4-position may produce compounds of therapeutic use in treating prostate cancer.

Inhibition of enzymes of estrogen and androgen biosynthesis by esters of 4-pyridylacetic acid.

A variety of esters of 4-pyridylacetic acid have been prepared by base mediated exchange from the methyl ester. Several of the esters of alcohols that contained a cyclohexyl ring were potent inhibitors of human placental aromatase and of the rat testicular 17 alpha-hydroxylase/C17-20lyase complex. The most potent agents found against both enzyme complexes were the borneyl, isopinocampheyl, and 1-adamantyl esters. These were over 100 times more potent than aminoglutethimide against aromatase and of greater potency than ketoconazole against hydroxylase/lyase. Potency against either enzyme complex was reduced if the ester function was borne on the cyclohexyl ring in an axial rather than an equatorial position. Some differential selectivity could be introduced since whereas methyl substitution adjacent to the carbonyl group reduced the inhibition of aromatase, it increased that against hydroxylase/lyase.

Hydroxyperfluoroazobenzenes: novel inhibitors of enzymes of androgen biosynthesis.

In a search for inhibitors of 17 alpha-hydroxylase-C17,20-lyase and testosterone-5 alpha-reductase, target enzymes in the development of drugs to treat hormone-dependent prostatic cancer, we have identified certain compounds chemically derived by the hydrolysis of decafluoroazobenzene (4) as novel inhibitors for these two enzymes. Hydrolysis of 4 gave the known 4-hydroxynonafluoroazobenzene (1) and the novel 2-hydroxynonafluoroazobenzene (2). By AlI3 demethylation of 4,4'-dimethoxyoctafluoroazobenzene (5) or by hydrolysis of 4 under phase-transfer conditions 4,4'-dihydroxyoctafluoroazobenzene (3) was obtained. Compounds 1 and 2 were inhibitors of the hydroxylase (IC50 values, respectively, 30 and 63 microM) and of the lyase (IC50 values 33 and 16 microM) steps on the pathway of androgen biosynthesis. The 2-hydroxy compound 2 underwent spontaneous conversion into octafluorodibenz[b,f][1,4,5]oxadiazepine (6) which had IC50 values, respectively, of 50 and 15 microM for the hydroxylase and lyase steps and which contributed to the observed activity of 2. Effective inhibitors of the 5 alpha-reductase were 1 (Ki 10 microM) and 3 (Ki4 microM): the activities of 1 and 3 were markedly pH dependent, with respective IC50 values of 14 and 5 microM at pH 7.4 and of 2 and 0.8 microM at pH 6.6.

Inhibition of 17 alpha-hydroxylase/C17-C20 lyase by bifluranol and its analogues.

A simple assay for the measurement of the activities of both 17 alpha-hydroxylase and C17-C20 lyase is described. No extraction procedures are required. The separation of substrate and products is achieved using HPLC which allows the collection of the components of interest and the monitoring of the recovery of various steroids. Using this assay, bifluranol (known to show anti-prostatic activity in vivo) and some analogues were tested for inhibitory activity towards these enzyme activities. Each compound was active, although less potent than ketoconazole, and this activity may contribute towards the in vivo action.

Selective inhibition of cholesterol side-chain cleavage by potential pro-drug forms of aminoglutethimide.

Potential pro-drugs for aminoglutethimide (1) an agent used for the treatment of hormone-dependent breast cancer have been synthesized, namely the azo-(2), azoxy-(3) and hydrazo-(4) derivatives. These compounds have been tested for inhibitory action towards the two main target enzymes for 1, aromatase and the cholesterol side-chain cleavage enzyme complex, P-450scc. None inhibited aromatase but 3 and 4 inhibited P-450scc, the respective IC50 values being about twice and one-half that for 1. Compounds 1 and 3 were also tested as inhibitors of the 17 alpha-hydroxylase-C17,20 lyase complex in comparison with ketoconazole which acts against prostatic cancer by this mechanism. The azoxy-derivative 3 was a weak inhibitor but 1 was inactive.

Dinucleotide analogues as inhibitors of thymidine kinase, thymidylate kinase, and ribonucleotide reductase.

P1-(Adenosine-5')-Pn-(thymidine-5') tri-, tetra-, penta-, and hexaphosphates (ApnT) plus the analogues with a methylene group alpha, beta to the thymidine residue (ApncpT) were synthesized by coupling the appropriate two nucleotides, having activated one by morpholine. These were tested as potential dinucleotide inhibitors of thymidine kinase, thymidylate kinase, and ribonucleotide reductase. All three enzymes bind ATP and thymidine or its nucleotides and therefore might be inhibited by dinucleotides containing adenosine and thymidine. Ap5T and Ap6T strongly inhibited all three enzymes (IC50 = 2.4-20 microM). Ap4cpT and Ap5cpT also strongly inhibited the two kinases (IC50 = 4-20 microM) but were much weaker inhibitors of the reductase (IC50 = 130 and 230 microM).

5-Ethyl-2'-deoxyuridine: an explanation for its lack of cytotoxic action in vivo.

The aim of this study was to explain why 5-ethyldeoxyuridine (EUdR) showed cytotoxic activity against Ehrlich ascites tumour (EAT) cells in vitro but not in vivo. In vitro studies showed that EUdR was phosphorylated to nucleotides which inhibit thymidylate synthetase and DNA polymerase. Toxicity in tissue culture appeared to be related to the inhibition of one or both of these enzymes; and could be prevented/reversed by thymidine (TdR). In vivo EAT cells also formed active EUdR nucleotides at levels which in vitro would have been associated with cytotoxicity but these levels were not maintained. EUdR has been shown to compete with TdR for catabolism by pyrimidine nucleoside phosphorylases from mouse liver and gut. In the ascitic fluid it was found that the level of EUdR fell rapidly while that of TdR and 5-ethyl-uracil increased. It is proposed that competition for catabolism in vivo resulted in the rise in TdR which then compromised the antitumour effect of EUdR.

A reappraisal of the effect upon thymidine kinase of thymidine derivatives carrying large groups at the 5'-position.

Several thymidine derivatives with hydrophobic 5'-substituents, linked by chemically stable amide and ether links, were synthesized as potential thymidine kinase inhibitors. None of these was active nor were several derivatives of thymidine 5'-acetate, which were previously reported to be inhibitors. It was shown that the apparent inhibition by the latter compounds was due to their facile hydrolysis in aqueous solution with release of thymidine. These results must cast doubt on any conclusions drawn from biological studies with 5'-esters of thymidine.

Myocardial enzyme activities in congestive cardiomyopathy.

A number of enzyme activities concerned with various catabolic pathways have been measured in biopsy specimens of the left ventricle in patients with congestive cardiomyopathy and compared with those in patients undergoing surgery for valvar or congenital heart disease. The myopathic patients showed a diminution in glycolytic capacity, in hexokinase activity and in the capacity to transfer reducing equivalents across the mitochondrial membrane.

Myocardial enzyme activities in guinea pigs during development.

The activities of hexokinase, glucose-6-phosphate dehydrogenase, and glycolytic enzymes were higher in the fetal myocardium of the guinea pig than at birth and fell progressively during the 1st mo of life. The alphaHBDH/LDH ratio of H to M subunits of lactate dehydrogenase, was low in the fetus and continued to rise during the 1st mo after birth. The distinction between the left and right ventricular activities of lactate dehydrogenase, which is clear in adult guinea pigs, was absent in the fetus and appeared during postnatal development. Glycogen phosphorylase activity was low in the fetus and at birth. The activities of beta-hydroxyacylcoenzyme A dehydrogenase, succinate dehydrogenase, malate dehydrogenase, and aspartate aminotransferase were low in the fetus, but had reached, or even temporarily exceeded, normal adult levels at birth. Palmitylcarnitine transferase activity was also low in the fetal heart compared with the newborn but continued to increase substantially during the first 2 wk after birth.

Effects of chronic hypoxia and dietary restriction on myocardial enzyme activities.

Enzyme activities were measured in homogenates of left and right ventricles of guinea pigs after 14 and 28 days' exposure to 400 mmHg barometric pressure. All animals developed anorexia and right ventricular hypertrophy. Two control groups of animals were used, one free fed and the other restricted to the amount of food chosen by the hypobaric group. The factorial design of the experiment allowed some distinction between the effects of anorexia, hypertrophy, and hypoxia. Dietary restriction was associated with a decrease in glycogen phosphorylase, hexokinase, and succinate dehydrogenase activity and an increase in the M-subunits of lactate dehydrogenase. Myocardial hypertrophy was associated with an increase in the activity of the enzymes of the glycolytic pathway down as far as phosphoglycerate kinase and an increase in the M-subunits of lactate dehydrogenase. Chronic hypoxia seemed specifically to be associated with an increase in the H-subunits of lactate dehydrogenase and possibly a slight transient increase in succinate dehydrogenase activity. Mixing studies indicated that changes in enzyme activities were likely to be due to changes in enzyme concentrations.