Expression of liver-specific member of the 3 beta-hydroxysteroid dehydrogenase family, an isoform possessing an almost exclusive 3-ketosteroid reductase activity.

We have recently characterized two types of rat 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) isoenzymes expressed in adrenals and gonads. In addition, we have cloned a third type of cDNA encoding a predicted type III 3 beta-HSD protein specifically expressed in the male rat liver which shares 80% similarity with the two other isoenzymes. Transient expression in human HeLa cells of the cDNAs reveals that the type III 3 beta-HSD protein does not display oxidative activity for the classical substrates of 3 beta-HSD, in contrast to the type I 3 beta-HSD isoenzyme. However, in the presence of NADH, type III isoenzyme, in common with the type I isoform, converts 5 alpha-androstane-3,17-dione (A-dione) and 5 alpha-dihydrotestosterone (DHT) to the corresponding 3 beta-hydroxysteroids. In fact, the type I and the type III isoenzymes have the same affinity for DHT with Km values of 5.05 and 6.16 microM, respectively. When NADPH is used as cofactor, the affinity for DHT of the type III isoform becomes higher than that of the type I isoform with Km values of 0.12 and 1.18 microM, respectively. The type III isoform is thus a 3-ketoreductase using NADPH as preferred cofactor which is responsible for the conversion of 3-keto-saturated steroids such as DHT and A-dione into less active steroids.     

Synthesis, biochemical evaluation and rationalisation of the inhibitory activity of a range of 4-hydroxyphenyl ketones as potent and specific inhibitors of the type 3 of 17beta-hydroxysteroid dehydrogenase (17beta-HSD3)

We report the synthesis and biochemical evaluation of a number of 4-hydroxyphenyl ketones as potential inhibitors of the enzyme 17beta-hydroxysteroid dehydrogenase (17beta-HSD). In particular, we evaluated compounds against the catalysis of the conversion of androstenedione (AD) to testosterone (T) [17beta-HSD type 3 (17beta-HSD3)], furthermore, in an effort to determine the specificity of our compounds, we evaluated the ability of the compounds to inhibit the catalysis of the conversion of estrone (E1) to estradiol (E2) [17beta-HSD type 1 (17beta-HSD1)] as well as the conversion of dehydroepiandrosterone (DHEA) to AD [by 3beta-hydroxysteroid dehydrogenase (3beta-HSD)]. The results of our study suggest that the synthesised compounds are, in general, able to inhibit 17beta-HSD3 whilst being weak inhibitors of 17beta-HSD1. Against 3beta-HSD, we discovered that all of the synthesised compounds were weak inhibitors (all were found to possess less than 50% inhibition at [I]=500 microM). More specifically, we discovered that 1-(4-hydroxy-phenyl)-nonan-1-one (15) was the most potent against 17beta-HSD3 (IC(50)=2.9 microM) whilst possessing poor inhibitory activity against 17beta-HSD1 ( approximately 36% inhibitory activity against this reaction at [I]=100 microM) and less than 10% inhibition for the conversion of DHEA to AD. We have therefore provided good lead compounds in the design and synthesis of novel non-steroidal inhibitors of 17beta-HSD3.     

Flavonoid inhibition of overexpressed human 3beta-hydroxysteroid dehydrogenase type II

The inhibitory effects of various flavonoids on human 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase type II (3beta-HSD type II), overexpressed in baculovirus, were investigated, and the structure-inhibition relationship was examined. The isoflavone derivatives daidzein, genistein, formononetin and biochanin A inhibited 3beta-HSD type II activity at a concentration of 10 microM and of these, genistein was the most potent inhibitor. 6-Hydroxyflavone (6-HF), a synthetic flavone, also strongly inhibited 3beta-HSD activity but 5-HF, 7-HF and other natural flavones were less potent. Energy minimization structures of the flavonoids, as produced using MOE software, showed that isoflavones and flavones have an almost flat A-C ring structure, and that flavonoids that acted as inhibitors had similar steric structures to DHEA. Genistein, 6-HF and cyanoketone, which is known as a typical 3beta-HSD inhibitor, were found to act as competitive inhibitors with K(i) values of 0.12 microM, 0.19 microM and 0.67 nM, respectively. Furthermore, the LUMO (lowest unoccupied molecular orbital (LUMO)) values, as calculated using WinMOPAC (Fujitsu, Japan), of the inhibitors were correlated with the IC(50) values (r2 = 0.84). From these results, it appears that inhibitory effects of flavonoids are due to the combination of steric structure and electron affinity between the active center of 3beta-HSD type II and the flavonoid molecule.     

Characterization and regulation of the 3beta-hydroxysteroid dehydrogenase isomerase enzyme in the rat sciatic nerve

In the peripheral nervous system, progesterone (PROG) has a stimulatory effect on myelination. It could be derived from local synthesis, as Schwann cells in culture express the 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and convert pregnenolone (PREG) to PROG. Although 3beta-HSD mRNA can be detected by RT-PCR in peripheral nerves, the activity of the enzyme has so far not been demonstrated and characterized in nerve tissue. In this study, we show that homogenates prepared from rat sciatic nerves contain a functional 3beta-HSD enzyme and we have analysed its kinetic properties and its regulation by steroids. The activity of 3beta-HSD in homogenates was evaluated using 3H-labelled PREG as a substrate and NAD+ as a cofactor, the levels of steroids formed were calculated either by extrapolating the relationship between tritiated peaks obtained by TLC to the initial amount of PREG, or by gas chromatography/mass spectrometry determination. A rapid increase in PROG formation was found between 0 and 50 min of incubation and no further significant changes were observed between 1 and 4 h. The calculated Km value (1.06 +/- 0.19 microm) was close to the values described for the 3beta-HSD type-I and type-IV isoforms. Trilostane, a competitive inhibitor of the 3beta-HSD caused a potent inhibition of the rate of conversion of PREG to PROG (IC50 = 4.06 +/- 2.58 microm). When the effects of different steroids were tested, both oestradiol and PROG significantly inhibited the conversion of PREG to PROG.     

3 beta-hydroxysteroid dehydrogenase isomerase (3beta-HSD) activity in the rat sciatic nerve: kinetic analysis and regulation by steroids

We have shown that progesterone (PROG) has a stimulatory effect on myelin formation after sciatic nerve injury. PROG is synthesized from pregnenolone (PREG) by the enzyme 3 beta-hydroxysteroid dehydrogenase isomerase (3beta-HSD). At the occasion of the 15th International Symposium of the Journal of the Steroid Biochemistry and Molecular Biology, we presented some of our recent results demonstrating, expression and activity of the enzyme 3beta-HSD in the rat sciatic nerve. We determined the kinetic properties of 3beta-HSD and its regulation by PROG and estradiol. The expression of 3beta-HSD protein was assessed by Western-blot analysis, and the 3beta-HSD activity was evaluated by incubating homogenates with [3H]-PREG as substrate and NAD(+) as cofactor. Levels of steroids formed were calculated either by extrapolation of the relationship between the tritiated peaks obtained by thin layer chromatography (TLC) and the initial amount of PREG, or by gas chromatography-mass spectrometry (GC-MS) determination. A rapid increase in PROG formation was found between 0 and 50min of incubation and no significant change was observed between 1 and 4h. The calculated K(m) value was close to the values obtained for the 3beta-HSD types I and IV isoforms. Trilostane caused a potent inhibition of the rate of conversion of PREG to PROG. When we tested the effects of progesterone and estradiol on 3beta-HSD activity, a significant inhibition was obtained.     

Localization of delta 5-3 beta- and 17 beta-hydroxysteroid dehydrogenase activity in the efferent ducts, epididymis and vas deferens of the rabbit, hamster and marmoset monkey

The presence and distribution of delta 5-3 beta-hydroxysteroid dehydrogenase (delta 5-3 beta-HSD: EC 1.1.1.51) and 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD: EC 1.1.1.51) were studied histochemically in the excurrent ducts of the rabbit, hamster and marmoset monkey. Dehydroepiandrosterone (DHEA) and testosterone were used as substrates for delta 5-3 beta-HSD and 17 beta-HSD respectively, while phenanthroline monohydrate was used to eliminate non-specific staining due to other tissue dehydrogenases. The rabbit possessed least enzyme activity, which was confined to tubules in the middle segment of the epididymis. Enzyme activity was demonstrable throughout the excurrent ducts of the hamster and marmoset, with maximal staining occurring in the middle segment of the epididymis in both species. The region of maximum activity of hydroxysteroid dehydrogenase is where spermatozoa first develop their fertilizing capacity.     

Effects of flavonoid phytochemicals on cortisol production and on activities of steroidogenic enzymes in human adrenocortical H295R cells

Inhibitory effects of flavonoid phytochemicals, flavones, flavonols and isoflavones on cortisol production were examined in human adrenal H295R cells stimulated with di-buthylyl cAMP. In addition, the inhibitory effects of these chemicals on the activity of P450scc, 3beta-HSD type II (3beta-HSD II), P450c17, P450c21 and P45011beta, steroidogenic enzymes involved in cortisol biosynthesis, were examined in the same cells. Exposure to 12.5 microM of the flavonoids 6-hydroxyflavone, 4'-hydroxyflavone, apigenin, daidzein, genistein and formononetin significantly decreased cortisol production (by 6.3, 69.6, 47.5, 26.6, 13.8 and 11.3%, respectively), and biochanin A significantly decreased cortisol production (by 47.3%) at a concentration of 25 microM without any significant cytotoxic effects or changes in cell number. Daidzin, the 7-glucoside of daidzein, did not alter cortisol production by H295R cells at concentrations over 10 microg/ml (24 microM). Daidzein-induced reduction of cortisol production by H295R cells was not inhibited by the estrogen receptor antagonist ICI 182,780. The flavonoids 6-hydroxyflavone, daidzein, genistein, biochanin A and formononetin strongly and significantly inhibited microsomal 3beta-HSD II activity at concentrations from 1 to 25 microM, and I(50) values were estimated to be 1.3, 2, 1, 0.5 and 2.7 microM, respectively. In addition, these flavonoids significantly inhibited microsomal P450c21 activity at 12.5 and/or 25 microM. In addition, 6-hydroxyflavone inhibited activity of microsomal P450c17 and mitochondrial P45011beta at 12.5 and/or 25 microM. Results of Lineweaver-Burk's plot analysis indicate that daidzein is a competitive inhibitor of the activity of 3beta-HSD II and P450c21. K(m) and V(max) values of 3beta-HSD II for DHEA were estimated to be 6.6 microM and 328pmol/minmg protein, respectively. K(m) and V(max) values of P450c21 for progesterone were estimated to be 2.8 microM and 16pmol/minmg protein, respectively. K(i) values of 3beta-HSD II and P450c21 for daidzein were estimated to be 2.9 and 33.3 microM, respectively.     

Astrocytes and neurosteroids: metabolism of pregnenolone and dehydroepiandrosterone. Regulation by cell density

The rat central nervous system (CNS) has previously been shown to synthesize pregnenolone (PREG) and convert it to progesterone (PROG) and 7 alpha-hydroxy-PREG (7 alpha-OH PREG). Astrocytes, which participate to the regulation of the CNS function, might be involved in the metabolism of neurosteroids. Purified type 1 astrocytes were obtained from fetal rat forebrain with the use of selective culture conditions and were identified by immunostaining with specific antibodies (GFAP+, A2B5-). They were plated at low, intermediate, or high densities (2.5-5 x 10(5), 1-2 x 10(6), or 4-8 x 10(6) cells/dish, respectively) and maintained for 21 d. They were then incubated with 14C-PREG and 14C-DHEA for 24 h and the steroids extracted from cells and media were analyzed. Most radioactive derivatives were released into incubation media. Two metabolic pathways were mainly observed. PREG and DHEA were oxidized to PROG and androstenedione (ADIONE), respectively, [3 beta-hydroxysteroid-dehydrogenase, delta 5-->4 3- ketosteroid-isomerase (3 beta-HSD) activity], and converted to 7 alpha- OH PREG and 7 alpha-OH DHEA, respectively (7 alpha-hydroxylase activity). After low density plating, the formation of PROG and ADIONE was approximately 10% of incubated radioactivity, tenfold larger than that of 7 alpha-hydroxylated metabolites. In contrast, after high density plating, low levels of PROG and ADIONE were formed, whereas the conversion to either 7 alpha-OH PREG or 7 alpha-OH DHEA was > or = 50%. The results expressed per cell indicated that the 3 beta-HSD activity was almost completely inhibited at high cell density, in contrast to the 7 alpha-hydroxylation which was maintained or increased. The pattern of steroid metabolism was related to cell density at the time of measurement and not to an early commitment of cells: when primary cultures were plated at high density (8 x 10(6) cells/dish), then subcultured after several dilutions (3-, 9-, or 27-fold), the 3 beta- HSD activity was recovered only at low density. Furthermore, when 5 x 10(5) cells were centrifuged and the resulting clusters were plated, 3 beta-HSD activity was decreased, whereas steroid 7 alpha-hydroxylation was enhanced. This implies that cell density per se, but neither cell number nor a diffusible factor(s) is involved in the regulation of steroid metabolism. We conclude that astrocytes in culture metabolize PREG and DHEA, and that the metabolic conversions and, therefore, the related enzymatic activities depend on cell-to-cell contacts.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chalcones are potent inhibitors of aromatase and 17beta-hydroxysteroid dehydrogenase activities

Chalcones were tested for estimating anti-aromatase, anti-3beta-hydroxysteroid dehydrogenase delta5/delta4 isomerase (3beta-HSD) and anti-17beta-hydroxysteroid dehydrogenase (17beta-HSD) activities in human placental microsomes. In the present study, we have demonstrated for the first time that chalcones are potent inhibitors of aromatase and 17beta-hydroxysteroid dehydrogenase activities: these enzymes being considered as important targets in the metabolic pathways of human mammary hormone-dependent cells. Our results showed that naringenin chalcone and 4-hydroxychalcone were the most effective aromatase and 17beta-hydroxysteroid dehydrogenase inhibitors with IC50 values of 2.6 and 16 microM respectively. In addition, inhibitory effects of some flavones and flavanones were compared to those of the corresponding chalcones. A structure-activity relationship was established and regions or/and substituents essential for these inhibitory activities were determined.     

Pregnenolone metabolized to 17alpha-hydroxyprogesterone in yeast: biochemical analysis of a metabolic pathway

The cDNA coding for the human 3beta-hydroxy-5-ene steroid dehydrogenase/5-ene-4-ene steroid isomerase (3beta-HSD) has been expressed in yeast. When expressed from identical vectors except for the coding sequence, the specific activity of the type I is lower than that of the type II enzyme. A mutant of the human 3beta-HSD type II lacking the putative membrane spanning domain 1 was generated by site directed mutagenesis: its apparent K(m) for pregnenolone (PREG) is significantly increased and its V reduced to the level of the type I enzyme. The influence of the kinetic properties of 3beta-HSD in the accumulation of 17alpha-hydroxyprogesterone was probed by co-expression of the bovine 17alpha-hydroxylase cytochrome P450 (P45017alpha) cDNA. The metabolism of PREG was followed with time using the membrane fraction. Kinetic properties of the 3beta-HSD were modulated such that its activity was in excess, limiting or balanced with respect to the activity of the P45017alpha and the accumulation of intermediates and products recorded. Conditions for the generation of the by-products resulting from the 17,20-Lyase activity of the P45017alpha were found. The potential applications of the system are discussed.     

Kinetic analysis of human placental, ovarian, and adrenal 3 beta-hydroxysteroid dehydrogenase inhibition by epostane in vitro

The effect of epostane [(2 alpha,4 alpha,5 alpha,17 beta)-4,5-epoxy-17-hydroxy-4,17-dimethyl-3-oxo- androstane-2-carbonitrile] on the conversion of pregnenolone to progesterone and of dehydroepiandrosterone (DHA) to androstenedione was studied in human term placental microsomes and in comparison with human ovarian and adrenal microsomes. Using pregnenolone as substrate, 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity in the three tissues had a similar Km (3-6 microM) but Vmax ranged from 1.3 nmol/mg protein per min in ovary to 10 nmol/mg protein per min in placenta. Epostane inhibited 3 beta-HSD activity in all three tissues with the characteristics of a pure competitive inhibitor: mean Ki values were 1.7 microM for placenta, 0.5 microM for adrenal and 0.1 microM for ovary. Moreover, in placental microsomes epostane inhibited the conversion of DHA to androstenedione with a Ki of 0.6 microM. The mechanism of action of epostane explains its effectiveness in blocking progesterone synthesis during the luteal phase and in pregnancy in women, and its strong anti-steroidogenic effect in other endocrine tissues in vitro.     

The use of fluorescein 5'-isothiocyanate for studies of structural and molecular mechanisms of soybean lipoxygenase

Human estrogenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD1) plays a crucial role in the last step of the synthesis of estrogens. A detailed kinetic study demonstrated that the enzyme shows about 240 fold higher specificity towards estrone reduction than estradiol oxidation at physiological pH using tri-phosphate cofactors. The kcat/Km values are 96 +/- 10 and 0.4 +/- 0.1 s-1 (microM)-1 respectively for the above two reactions. However, it has been shown that this difference is closely linked to the use of NADPH and NADP cofactors. A binding study using equilibrium dialysis indicated similar KD (equilibrium dissociation constant) of 11 +/- 1 and 4.7 +/- 0.9 microM for estrone and estradiol, respectively. The binding affinity of 17beta-HSD1 to estrone was significantly increased with a KD of 1.6 +/- 0.2 microM in the presence of NADP, the latter used as an analogue of the NADPH. The results of binding studies agree with the steady-state kinetics, which showed that the Km of estrone is 12-fold lower when using NADPH as a cofactor than when using NADH. These results strongly suggest that the cofactor plays a crucial role in the stimulation of the specificity for estrogen reduction.     

Inhibitors of human and rat testes microsomal 17beta-hydroxysteroid dehydrogenase (17beta-HSD) as potential agents for prostatic cancer

In a screening programme for inhibitors of human testis 17beta-hydroxysteroid dehydrogenase (17beta-HSD type 3), as potential agents for the treatment of hormone-dependent prostatic cancer, we have used crude human testis microsomal 17beta-hydroxysteroid dehydrogenase as a convenient source of the enzyme. Crude human enzyme was shown to have a similar substrate profile to recombinant Type 3 17beta-HSD from the same source as determined by the low Km/Vmax ratio for the reduction of androstenedione compared to the oxidation of testosterone, and a low level of activity in reduction of oestrone. Screening of a wide range of compounds of different structural types as potential inhibitors of the microsomal enzyme in the reduction step revealed that certain p-benzoquinones and flavones/isoflavones were potent inhibitors of the enzyme, diphenyl-p-benzoquinone (2.7 microM), phenyl-p-benzoquinone (5.7 microM), 7-hydroxyflavone (9.0 microM), baicalein (9.3 microM) and biochanin A (10.8 microM). Some structure-activity relationships within the flavone/isoflavone series are discussed. Studies with rat testis microsomal 17beta-HSD showed that it differed from the human enzyme mainly in its greater ability to accept oestrone as substrate and the pH-optimum for oxidation of testosterone. It was found to be much less sensitive to inhibition by the compounds studied so negating it use as a more readily available tissue for the screening of potential inhibitors.     

Intracrinology and the skin

The skin, the largest organ in the human body, is composed of a series of androgen-sensitive components that all express the steroidogenic enzymes required to transform dehydroepiandrosterone (DHEA) into dihydrotestosterone (DHT). In fact, in post-menopausal women, all sex steroids made in the skin are from adrenal steroid precursors, especially DHEA. Secretion of this precursor steroid by the adrenals decreases progressively from the age of 30 years to less than 50% of its maximal value at the age of 60 years. DHEA applied topically or by the oral route stimulates sebaceous gland activity, the changes observed being completely blocked in the rat by a pure antiandrogen while a pure antiestrogen has no significant effect, thus indicating a predominant or almost exclusive androgenic effect. In human skin, the enzyme that transforms DHEA into androstenedione is type 1 3beta-hydroxysteroid dehydrogenase (type 1 3beta-HSD) as revealed by RNase protection and immunocytochemistry. The conversion of androstenedione into testosterone is then catalyzed in the human skin by type 5 17beta-HSD. All the epidermal cells and cells of the sebaceous glands are labelled by type 5 17beta-HSD. This enzyme is also present at a high level in the hair follicles. Type 1 is the 5alpha-reductase isoform responsible in human skin for the conversion of testosterone into DHT. In the vagina, on the other hand, DHEA exerts mainly an estrogenic effect, this effect having been demonstrated in the rat as well as in post-menopausal women. On the other hand, in experimental animals as well as in post-menopausal women, DHEA, at physiological doses, does not affect the endometrial epithelium, thus indicating the absence of DHEA-converting enzymes in this tissue, and avoiding the need for progestins when DHEA is used as hormone replacement therapy.     

Inter-conversion of 7alpha- and 7beta-hydroxy-dehydroepiandrosterone by the human 11beta-hydroxysteroid dehydrogenase type 1

The dehydroepiandrosterone (DHEA) 7alpha-hydroxylation in humans takes place in the liver, skin, and brain. These organs are targets for the glucocorticoid hormones where 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) activates cortisone through its reduction into cortisol. The putative interference of 7alpha-hydroxy-DHEA with the 11beta-HSD1-catalyzed reduction of cortisone into cortisol has been confirmed in preliminary works with human liver tissue preparations of the enzyme demonstrating the transformation of 7alpha-hydroxy-DHEA into 7-oxo-DHEA and 7beta-hydroxy-DHEA. However, the large production of 7beta-hydroxy-DHEA could not be explained satisfactorily. Therefore our objective was to study the role in the metabolism of oxygenated DHEA by recombinant human 11beta-HSD1 expressed in yeast. The 7alpha- and 7beta-hydroxy-DHEA were each oxidized into 7-oxo-DHEA with quite dissimilar K(M) (70 and 9.5 microM, respectively) but at equivalent V(max). In contrast, the 11beta-HSD1-mediated reduction of 7-oxo-DHEA led to the production of both 7alpha- and 7beta-hydroxy-DHEA with equivalent K(M) (1.1 microM) but with a 7beta-hydroxy-DHEA production characterized by a significantly greater V(max). The 7alpha-hydroxy-DHEA produced by the cytochrome CYP7B1 in tissues may exert anti-glucocorticoid effects through interference with the 11beta-HSD1-mediated cortisone reduction.     

Synthesis, biochemical evaluation and rationalisation of the inhibitory activity of a range of phenyl alkyl imidazole-based compounds as potent inhibitors of the enzyme complex 17alpha-hydroxylase/17,20-lyase (P450(17alpha))

The cytochrome P450 enzyme, 17alpha-hydroxylase/17,20-lyase (P450(17alpha)), is a potential target in hormone-dependent cancers. We report the synthesis, biochemical evaluation and rationalisation of the inhibitory activity of a number of azole-based compounds as inhibitors of the two components of P450(17alpha), i.e., 17alpha-hydroxylase (17alpha-OHase) and 17,20-lyase (lyase). The results suggest that the imidazole-based compounds are highly potent inhibitors of both components, with N-7-phenyl heptyl imidazole (21) (IC(50)=0.32 microM against 17alpha-OHase and IC(50)=0.10 microM against lyase) and N-8-phenyl octyl imidazole (23) (IC(50)=0.25 microM against 17alpha-OHase and IC(50)=0.21 microM against lyase) being the two most potent compounds within the current study, in comparison to ketoconazole (KTZ) (IC(50)=3.76 microM against 17alpha-OHase and IC(50)=1.66 microM against lyase). Furthermore, consideration of the inhibitory activity against the two components show that the compounds tested are less potent towards the 17alpha-OHase component, a desirable property in the development of novel inhibitors of P450(17alpha). Structure-activity relationship determination of the range of compounds synthesised suggests that logP (log of the partition coefficient) is a key physicochemical factor in determining the overall inhibitory activity. In an effort to determine the viability of these compounds becoming potential drug candidates as well as to show specificity of these compounds, we undertook the biochemical evaluation of the synthesised compounds against two isozymes of 17beta-hydroxysteroid dehydrogenase [namely type 1 (17beta-HSD1) and type 3 (17beta-HSD3)] and 3beta-hydroxysteroid dehydrogenase (3beta-HSD). Consideration of the inhibitory activity possessed by the compounds considered within the current study against 3beta-HSD, 17beta-HSD1 and 17beta-HSD3 shows that there is no clear structure-activity relationship and that the compounds appear to possess similar inhibitory activity against both 3beta-HSD and 17beta-HSD3 whilst against 17beta-HSD1, the compounds appear to possess poor inhibitory activity at [I]=100 microM. Indeed, two of the most potent inhibitors of P450(17alpha), (compounds 21 and 23), were found to possess relatively good levels of inhibition against the three enzymes-compound 21 was found to possess approximately 32%, approximately 21% and approximately 37% inhibition whilst compound 23 was found to possess approximately 38%, approximately 30% and approximately 28% inhibition against 3beta-HSD, 17beta-HSD1 and 17beta-HSD3 respectively. We therefore concluded that the azole-based compounds synthesised within the current study are not suitable for further consideration as potential drug candidates due to their lack of specificity.