Computational prediction of new CYP17 inhibitors based on pharmacophore modeling, virtual screening and docking approach.

17α-Hydroxylase/C17-20-lyase (P450 17, CYP 17) is an important enzyme in the androgen biosynthesis and inhibitors of this enzyme can be used for the treatment of prostate cancer. With the aim of developing new inhibitors for the target enzyme, we generated a structure-based pharmacophore model to further explain the binding requirements for human CYP17 inhibitors. Seven common features of steroidal CYP17 inhibitors were determined using MOE software. This pharmacophore model was then used to search the Cambridge Structural Database (CSD) with the aim of developing more potent and selective CYP17 inhibitors by identifying new hits. We were able to identify 36 structures as possible active CYP17 inhibitors. Docking studies for the selected compounds from the database were also performed and the best three compounds were chosen as possible hits.

Steering the azido-tetrazole equilibrium of 4-azidopyrimidines via substituent variation - implications for drug design and azide-alkyne cycloadditions.

This paper focuses on an interesting constitutional isomerism called azido-tetrazole equilibrium which is observed in azido-substituted N-heterocycles. We present a systematic investigation of substituent effects on the isomer ratio within a 2-substituted 4-azidopyrimidine model scaffold. NMR- and IR-spectroscopy as well as X-ray crystallography were employed for thorough analysis and characterization of synthesized derivatives. On the basis of this data, we demonstrate the possibility to steer this valence tautomerism towards the isomer of choice by means of substituent variation. We show that the tetrazole form can act as an efficient disguise for the corresponding azido group masking its well known reactivity in azide-alkyne cycloadditions (ACCs). In copper(I)-catalyzed AAC reactions, substituent-stabilized tetrazoles displayed a highly decreased or even abolished reactivity whereas azides and compounds in the equilibrium were directly converted. By use of an acid sensitive derivative, we provide, to our knowledge, the first experimental basis for a possible exploitation of this dynamic isomerism as a pH-dependent azide-protecting motif for selective SPAAC conjugations in aqueous media. Finally, we demonstrate the applicability and efficiency of stabilized tetrazolo[1,5-c]pyrimidines for Fragment-Based Drug Design (FBDD) in the field of quorum sensing inhibitors.

Comparison of envelope 2 CD81 binding regions in PBMC-derived versus serum-derived hepatitis C virus isolates: higher conservation of CD81 region 2 in PBMC isolates.

UNLABELLED: The aim of the present study was to investigate the variability of hepatitis C virus (HCV) CD81 binding regions (CD81-1/2) in peripheral blood mononuclear cells (PBMC)-derived and serum-derived HCV-RNA samples. HCV-RNA was isolated from PBMC (104 cells) and serum samples from 37 patients chronically infected with HCV genotype 1a/1b (n=21/16). The hypervariable regions 1/2 (amino acid 384-410, amino acid 474-482) and regions CD81-1/2 (amino acid 474-494, amino acid 522-551) were analysed. Mutational frequency of amino acid sequences was compared between PBMC-derived and serum-derived HCV variants as well as local accumulation of mutations. Furthermore, CD81 was quantified on PBMC. Mutational frequency was not different between PBMC-derived and serum-derived HCV variants. A trend to lower mutational frequency in genotype 1a PBMC variants compared with serum-derived variants was observed in region CD81-2 (5%vs 10%). Smoothed mutational frequency analysis showed a significantly lower variability within genotype 1a CD81-2 in PBMC-derived compared to serum-derived HCV-RNA (P=0.026). CD81 expression on PBMC was not correlated with the number of mutations within the CD81 binding regions. CONCLUSION: A higher conservation was observed in region CD81-2 in PBMC-derived versus serum-derived HCV-RNA indicating selection of HCV variants on PBMC. The variability in the CD81 binding regions appeared to be independent from CD81 expression.

Etomidate unmasks intraadrenal regulation of steroidogenesis and proliferation in adrenal cortical cell lines.

To characterize intraadrenal adaptations for inhibition of cortisol synthesis, we analyzed the effects of etomidate (ETO) on steroid hormone secretion and expression of key regulators of steroidogenesis and proliferation in human NCI-h295 adrenocortical cancer cells. Etomidate potently blocked 11beta-hydroxylase (CYP11B1), aldosterone synthase (CYP11B2), and side chain cleavage enzyme (CYP11A1). This inhibition of steroidogenesis was associated with increased expression of steroidogenic acute regulatory protein (StAR), and CYP11A1 and 17alpha-hydroxylase/17, 20-lyase (CYP17A1) protein levels, but not of the respective mRNA levels. Promoter activity of CYP11A1 and melanocortin 2 receptor (MC2R) was not increased by etomidate in treated cells compared to controls. The increase in protein levels was partially reversed by cycloheximide suggesting post-transcriptional mechanisms but also protein stabilization as underlying cause. Furthermore, ETO exhibited antiproliferative activity paralleled by a decrease in phosphorylation of MEK and ERK1, 2. In summary, ETO exhibits pleiotropic effects on adrenal function in vitro. Inhibition of steroidogenesis is followed by increased levels of steroidogenic key proteins and reduced proliferation. These changes reflect adaptations to maintain steroidogenesis at the cost of adrenal proliferation.

[Derivatives of exemestane--synthesis and evaluation of aromatase inhibition]. / Exemestan-derivate--Synthese und Prüfung auf Aromatase-Hemmung.

The irreversible aromatase inhibitor exemestane (6) reacts with nitromethane and sodium ethanolate to yield the Michael adduct 9. The aldehyde 10 is obtained by Nef reaction of the nitro compound 9 and affords the 1,4-dihydropyridine (DHP) 11 by Hantzsch reaction using methyl beta-aminocrotonate in acetic acid. The new compounds showed a reduced inhibitory potency towards aromatase (IC50 values: 9, 0.91 microM; 10, 2.5 microM; 11, 10 microM) compared to 6 (IC50 = 0.23 microM). The 1,4-DHP 11 was dehydrogenated with CAN or electrochemically (E1/2 =1.18 V) to yield the corresponding pyridine 12.

In vitro reconstitution of α-pyrone ring formation in myxopyronin biosynthesis.

Myxopyronins are α-pyrone antibiotics produced by the terrestrial bacterium Myxococcus fulvus Mx f50 and possess antibacterial activity against Gram-positive and Gram-negative pathogens. They target the bacterial RNA polymerase (RNAP) "switch region" as non-competitive inhibitors and display no cross-resistance to the established RNAP inhibitor rifampicin. Recent analysis of the myxopyronin biosynthetic pathway led to the hypothesis that this secondary metabolite is produced from two separate polyketide parts, which are condensed by the stand-alone ketosynthase MxnB. Using in vitro assays we show that MxnB catalyzes a unique condensation reaction forming the α-pyrone ring of myxopyronins from two activated acyl chains in form of their ß-keto intermediates. MxnB is able to accept thioester substrates coupled to either N-acetylcysteamine (NAC) or a specific carrier protein (CP). The turnover rate of MxnB for substrates bound to CP was 12-fold higher than for NAC substrates, demonstrating the importance of protein-protein interactions in polyketide synthase (PKS) systems. The crystal structure of MxnB reveals the enzyme to be an unusual member of the ketosynthase group capable of binding and condensing two long alkyl chains bound to carrier proteins. The geometry of the two binding tunnels supports the biochemical data and allows us to propose an order of reaction, which is supported by the identification of novel myxopyronin congeners in the extract of the producer strain. Insights into the mechanism of this unique condensation reaction do not only expand our knowledge regarding the thiolase enzyme family but also opens up opportunities for PKS bioengineering to achieve directed structural modifications.

Influence of alkyl chain ramification on estradiol receptor binding affinity and intrinsic activity of 1,2-dialkylated 1,2-bis(4- or 3-hydroxyphenyl)ethane estrogens and antiestrogens.

The influence of a symmetrical introduction of CH3 substituents in the alpha or beta positions of the 1,2-dialkyl-1,2-bis(4-hydroxyphenyl)ethane estrogens hexestrol (ethyl, HES) and octestrol (n-propyl, OCES) [isopropyl (1), tert-butyl (2), sec-butyl (3), isobutyl (4)] and the 1,2-dialkyl-1,2-bis(3-hydroxyphenyl)ethane antiestrogens metahexestrol (ethyl, MetaHES) and metaoctestrol (n-propyl, MetaOCES) [isopropyl (5), tert-butyl (6), sec-butyl (7), isobutyl (8)] on estradiol receptor (E2R) binding affinity and intrinsic activity is described. The synthesis of compounds 1-8 was accomplished by reductive coupling of (a) the corresponding alpha-alkylbenzyl alcohols with TiCl3/LiAlH4 and separation of the meso diastereoisomers (compounds 2-4, 7, and 8), (b) alpha-tert-butyl-3-methoxybenzyl chloride with CoCl2/EtMgCl and isolation of the meso configurated isomer (6), and (c) the isopropyl phenyl ketones with TiCl4/Zn and subsequent hydrogenation of the corresponding cis-hex-3-enes (compounds 1 and 5). The binding affinity of 1-8 to the calf uterine E2R was measured relative to that of [3H]E2 by a competitive binding assay. Compounds 1 and 5 showed relative binding affinity (RBA) values exceeding those of HES and MetaHES, respectively. All other derivatives showed RBA values smaller than the corresponding parent compounds. The intrinsic activity was monitored in terms of uterotrophic and antiuterotrophic activity. It is striking that the introduction of a CH3 group in the alpha positions of MetaHES and MetaOCES led to compounds with full intrinsic activity (5-7), i.e., estrogens without antiestrogenic properties. No correlation between E2R binding affinity and intrinsic activity was found.

Aromatase inhibitors. Synthesis and evaluation of mammary tumor inhibiting activity of 3-alkylated 3-(4-aminophenyl)piperidine-2,6-diones.

The synthesis and biological evaluation of 3-alkyl-substituted 3-(4-aminophenyl)piperidine-2,6-diones as inhibitors of estrogen biosynthesis are described [H (1), methyl (2), ethyl (3), n-propyl (4), isopropyl (5), n-butyl (6), isobutyl (7), sec-butyl (8), n-pentyl (9), isopentyl (10), 2-methylbutyl (11), sec-pentyl (12), n-hexyl (13), n-heptyl (14)]. In vitro compounds 4-14 showed a stronger inhibition of human placental aromatase compared to aminoglutethimide (AG, compound 3), which recently has become used for the treatment of hormone-dependent breast cancer. The most active derivative, compound 10, showed a 93-fold stronger inhibition than AG. With the exception of 5, 7, and 8, all other compounds exhibited similar or decreased inhibition of bovine adrenal desmolase compared to AG. Compounds 4 and 6-12 showed a stronger inhibition of the plasma estradiol concentration of pregnant mare serum gonadotropin (PMSG) primed Sprague-Dawley (SD) rats compared to the parent compound. Compounds 4, 6-8, 10, and 12 inhibited the testosterone-stimulated tumor growth of ovariectomized 9,10-dimethyl-1,2-benzanthracene (DMBA) tumor-bearing SD rats more strongly than AG. Being stronger and more selective inhibitors of the estrogen biosynthesis than AG, some of the newly developed derivatives of AG might be better candidates for the treatment of the hormone-dependent human breast cancer.

Aromatase inhibitors. Syntheses and structure-activity studies of novel pyridyl-substituted indanones, indans, and tetralins.

The (E)-2-(4-pyridylmethylene)-1-indanones(E)-1-(E)-5[(E)-1,H;(E)-2,4- OCH3; (E)-3,5-OCH3; (E)-4,4-OH;(E)-5,5-OH] were obtained by aldol condensation of the corresponding 1-indanones with 4-pyridinecarboxaldehyde, and in case of the OH compound (E)-4 subsequent ether cleavage of (E)-2. The synthesis of the (Z)-isomers (Z)-1-(Z)-3[(Z)-1,H;(Z)-2,4-OCH3; (Z)-3,5-OCH3] was accomplished by UV irradiation of the corresponding (E)-isomers. Catalytic hydrogenation of (E)-1-(E)-3 gave the 2-(4-pyridylmethyl)-1-indanones 6-8 (6, H; 7,4-OCH3; 8,5-OCH3). The 2-(4-pyridylmethyl)-substituted indans 11-13 (11,H; 12,4-OCH3; 13,5-OCH3) and the tetralins 16-19 (16,H;17,5-OCH3;18,6-OCH3;19,7-OCH3) were obtained by reduction of the corresponding ketones using N2H4/KOH. The 2-(4-pyridylmethyl)-substituted indanones 9 (4-OH) and 10 (5-OH), indans 14 (4-OH) and 15 (5-OH), and tetralins 20-22 (20,5-OH; 21,6-OH; 22,7-OH) were synthesized by ether cleavage of the corresponding OCH3 compounds. All compounds showed inhibition of human placental aromatase exhibiting relative potencies from 0.9 [(E)-4] to 163 [18; aminoglutethimide (AG) potency identical to 1]. Compounds 13 and 18 showed competitive type of inhibition and a type II difference spectrum, indicating the interaction of the pyridyl-N with the central Fe(III) ion of the cytochrome P450 heme component.(ABSTRACT TRUNCATED AT 250 WORDS)

Ring-substituted 1,2-dialkylated 1,2-bis(hydroxyphenyl)ethanes. 1. Synthesis and estrogen receptor binding affinity of 2,2'- and 3,3'-disubstituted hexestrols.

The syntheses of symmetrically 3,3'- and 2,2'-disubstituted meso hexestrol derivatives are described [3,3'-substituents: OH (1), F (2), Cl (3), Br (4), I (5), CH2N(CH3)2 (6), CH3 (7), CH2OCH3 (8), CH2OC2H5 (9), CH2OH (10), NO2 (11), NH2 (12), N(CH3)2 (13), COCH3 (14), and C2H5 (15); 2,2'-substituents: OH (16), F (17), Cl (18), Br (19), CH3 (20), and C2H5 (21)]. The synthesis of 1-3 was accomplished by reductive coupling of the propiophenones with TiCl4/Zn and subsequent hydrogenation of the cis-3,4-diphenylhex-3-enes. Compounds 4-15 were obtained by substitution of hexestrol, while compounds 16-21 were synthesized by coupling the 1-phenyl-1-propanols with TiCl3/LiAlH4 and separation of the meso diastereomers. The binding affinity of these compounds to the calf uterine estrogen receptor was measured relative to that of [3H]estradiol by a competitive binding assay. All test compounds showed relative binding affinity (RBA) values between 32 and less than 0.01% that of estradiol. Only meso-3,4-bis(2,4-dihydroxyphenyl)hexane (16) showed an estrogen receptor binding affinity comparable to that of hexestrol (32 and 27%, respectively). Compounds exhibiting RBA values of greater than 5% were evaluated in the mouse uterine weight test. All of them showed uterotrophic activity. Compounds 2, 7, 16, 17, and 20 were strongly active in very small doses (1 microgram per animal per day), while 1 and 12 produced full uterotrophic effects only in high doses and inhibited the estrone-stimulated uterine growth strongly in small doses (59 and 78% inhibition, respectively).

Ring-substituted 1,2-dialkylated 1,2-bis(hydroxyphenyl)ethanes. 2. Synthesis and estrogen receptor binding affinity of 4,4'-, 5,5'-, and 6,6'-disubstituted metahexestrols.

The syntheses of symmetrically 4,4'-, 5,5'-, and 6,6'-disubstituted derivatives of the mammary tumor inhibiting antiestrogen metahexestrol [meso-3,4-bis(3-hydroxyphenyl)hexane] (1) are described [4,4'-substituents: F, (2), Cl (3), Br (4), I (5), CH2N (CH3)2 (6), CH3 (7), CH2OCH3 (8), CH2OC2H5 (9), CH2OH (10), NO2 (11), NH2 (12), N(CH3)2 (13), COCH3 (14), and C2H5 (15); 5,5'-substituents: OH (16) and Cl (17); 6,6'-substituents: OH (18), F (19), Cl (20), and CH3 (21)]. The synthesis of 1-3, 16, and 19 was accomplished by reductive coupling of the propiophenones with TiCl4 /Zn and subsequent hydrogenation of the cis-3,4- diphenylhex -3- enes . Compounds 17, 18, 20, and 21 were synthesized by coupling the 1-phenyl-1-propanols with TiCl3 /LiAlH4 and separation of the meso diastereomers, while 4-15 were obtained by substitution of metahexestrol . The binding affinity of these compounds to the calf uterine estrogen receptor was measured relative to that of [3H]estradiol by a competitive binding assay. The test compounds showed relative binding affinity (RBA) values between 15 and less than 0.01% that of estradiol. Only compound 21 showed an estrogen receptor binding affinity exceeding that of metahexestrol (15 and 10%, respectively). Compounds exhibiting RBA values of greater than 0.5% were evaluated in the mouse uterine weight test. They showed a similar (2 and 12), slightly increased (19 and 21), or strongly enhanced (7 and 20) estrogenicity compared to that of metahexestrol . Compounds 1, 2, 7, 12, and 21 exhibited antiestrogenic activity inhibiting the estrone-stimulated uterine growth (24 to 60% inhibition).

Influence of alkyl-chain fluorination on the action of mammary tumor inhibiting 2,3-bis(hydroxyphenyl)butanes and 2,3-bis(hydroxyphenyl)but-2-enes.

trans-1,2-Bis(trifluoromethyl)-1,2-bis(4- and 3-hydroxyphenyl)ethenes 2 and 4 were prepared by reductive coupling (TiCl4/Zn/pyridine) of the methoxy-substituted alpha, alpha, alpha-trifluoroacetophenones, separation of the resulting cis- and trans-stilbene derivatives, and ether cleavage with BBr3. The cis-stilbenes were catalytically hydrogenated to give meso-1,1,1,4,4,4-hexafluoro-2,3-bis(4- and 3-hydroxyphenyl)butanes 6 and 8. Compounds 2, 4, 6, and 8 showed 2- to 10-fold increased binding affinities for the estradiol receptor (E2R) and enhanced estrogenicity in the uterine weight test of the immature mouse compared to their unfluorinated analogues. Compound 8 exhibited a 46% inhibition of the estrone-stimulated uterine growth. Antitumor activity was evaluated with use of the transplantable, hormone-dependent MXT mammary tumor of the BD2F1 mouse. All compounds showed tumor growth inhibitory activity corresponding to their RBA values. The most interesting compound 8 led to a significant inhibition of the tumor growth on the DMBA-induced hormone-dependent mammary carcinoma of the Sprague-Dawley rat.

Synthesis and aromatase inhibition of 3-cycloalkyl-substituted 3-(4-aminophenyl)piperidine-2,6-diones.

The synthesis of 3-cycloalkyl-substituted 3-(4-aminophenyl)piperidine-2,6-diones is described [cyclopentyl (1), cyclohexyl (2)]. The enantiomers of 2 were separated either by using HPLC on optically active sorbent or by crystallization of the brucine salt of the phthalamic acid of 2. The absolute configuration of the (+)- and (-)-enantiomers of 2 were assigned as S and R, respectively, by comparing the CD spectra to those of the enantiomers of aminoglutethimide (AG, 3-(4-aminophenyl)-3-ethylpiperidine-2,6-dione). The compounds were tested in vitro for inhibition of human placental aromatase, the cytochrome P450-dependent enzyme which is responsible for the conversion of androgens to estrogens. Compounds 1 and 2 inhibited aromatase by 50% at 1.2 and 0.3 microM, respectively (IC50 AG = 37 microM). According to the findings with AG, the (+)-enantiomer of 2 was responsible for the inhibitory activity, being a 240-fold more potent aromatase inhibitor in vitro than racemic AG. On the other hand, (+)-2 displayed a strongly reduced inhibition of desmolase (cholesterol side-chain cleavage enzyme) compared to AG (relative activity = 0.3). Thus (+)-2 is of interest as a potential drug for the treatment of estrogen-dependent diseases, e.g. mammary tumors.

New aromatase inhibitors. Synthesis and biological activity of pyridyl-substituted tetralone derivatives.

The (E)-2-(4-pyridylmethylene)-1-tetralones 1-7 (1, H; 2, 5-OCH3; 3, 6-OCH3; 4, 7-OCH3; 5, 5-OH; 6, 6-OH; 7, 7-OH) were obtained by aldol condensation of the corresponding 1-tetralones with 4-pyridinecarboxaldehyde, and in the case of the OH compounds 5 and 7 subsequent ether cleavage of the OCH3-substituted 2-(4-pyridylmethylene)-1-tetralones. Catalytic hydrogenation of 1-4 gave the 2-(4-pyridylmethyl)-1-tetralones 8-11 (8, H; 9, 5-OCH3; 10, 6-OCH3; 11, 7-OCH3). Subsequent ether cleavage of 9-11 led to the corresponding OH compounds 12-14 (12, 5-OH; 13, 6-OH; 14, 7-OH). The enantiomers of 11 and 12 were separated semipreparatively by HPLC on triacetylcellulose. All compounds (1-14) showed an inhibition of human placental aromatase exhibiting relative potencies from 2.2 to 213 [compounds 6 and (+)-12, respectively; aromatase inhibitory potency of aminoglutethimide (AG) = 1]. The compounds exhibited no or only a weak inhibition of desmolase [cholesterol side chain cleavage enzyme; maximum activity shown by 12, 23% inhibition (25 microM); AG, 53% inhibition (25 microM)]. In vivo, however, the compounds were not superior to AG as far as the reduction of the plasma estradiol concentration and the mammary carcinoma (MC) inhibiting properties are concerned (PMSG-primed SD rats as well as DMBA-induced MC of the SD rat, pre- and postmenopausal experiments, and the transplantable MXT-MC of the BD2F1 mouse). This is due to a fast decrease of the plasma E2 concentration inhibiting effect as could be shown by a kinetic experiment. In addition, select compounds inhibited rat ovarian aromatase much less than human placental aromatase (12, factor of 10). Estrogenic effects as a cause for the poor in vivo activity of the test compounds could be excluded, since they did not show affinity for the estrogen receptor.

1-imidazolyl(alkyl)-substituted di- and tetrahydroquinolines and analogues: syntheses and evaluation of dual inhibitors of thromboxane A(2) synthase and aromatase.

A series of 1-imidazolyl(alkyl)-substituted quinoline, isoquinoline, naphthalene, benzo[b]furan, and benzo[b]thiophene derivatives was synthesized as dual inhibitors of thromboxane A(2) synthase (P450 TxA(2)) and aromatase (P450 arom). Dual inhibition of these enzymes could be a novel strategy for the treatment of mammary tumors and the prophylaxis of metastases. The most potent dual inhibitors, 5-(2-imidazol-1-ylethyl)-7,8-dihydroquinoline (31) (P450 TxA(2): IC(50) = 0.29 microM; P450 arom: IC(50) = 0.50 microM) and its 5, 6-saturated analogue 30 (P450 TxA(2): IC(50) = 0.68 microM; P450 arom: IC(50) = 0.38 microM), showed a stronger inhibition of both target enzymes than the reference compounds (dazoxiben: IC(50) = 1.1 microM; aminoglutethimide: IC(50) = 18.5 microM). For the determination of the in vivo activity, the influence of selected compounds on serum TxB(2) concentration was examined in rats. Compound 30 (8.5 mg/kg body weight) led to a reduction of the TxB(2) serum level of 78%, 71%, and 51% after 3, 5, and 8 h, respectively (dazoxiben: 60%, 34%, and 36%). Selectivity was studied toward some enzymes of the steroidogenic and eicosanoid pathways. P450 17 was inhibited by selected compounds only at high concentrations. Compound 30 inhibited P450 scc by 13% (25 microM). Compound 31 did not affect cyclooxygenase and lipoxygenase.

Ring-substituted 1,1,2,2-tetraalkylated 1,2-bis(hydroxyphenyl)ethanes. 4. Synthesis, estrogen receptor binding affinity, and evaluation of antiestrogenic and mammary tumor inhibiting activity of symmetrically disubstituted 1,1,2,2-tetramethyl-1,2-bis(hydroxyphenyl)ethanes.

The syntheses of symmetrically 2,2'-disubstituted derivatives of 1,1,2,2-tetramethyl-1,2-bis(4-hydroxyphenyl)ethane (1) and of 5,5'-, and 6,6'-disubstituted derivatives of 1,1,2,2-tetramethyl-1,2-bis(3-hydroxyphenyl)ethane (6) are described (1 and 6 are strong antiestrogens with mammary tumor inhibiting activity exhibiting only slight estrogenic properties): (2,2'-substituents) F (2), Cl (3), OCH3 (4), CH3 (5); (5,5'-substituents) Cl (7); (6,6'-substituents) F (8), Cl (9), OCH3 (10), CH3 (11). The synthesis of 1-11 was accomplished by reductive coupling of the corresponding 2-phenyl-2-propanols with TiCl3 and LiAlH4. The binding affinity of the compounds to the calf uterine estrogen receptor was measured relative to that of [3H]estradiol by a competitive binding assay. With the exception of 7 and 10 all other compounds showed relative binding affinity (RBA) values between 0.5 and 6.4% that of estradiol, 2 (RBA value 6.4), and 8 and 9 (4.0 and 3.5), exceeding those of the corresponding unsubstituted 1 and 6 (3.6 and 3.0). Compounds exhibiting RBA values of greater than 2.5% were evaluated in the mouse uterine weight test. The substituted derivatives showed an increase in uterotrophic and a decrease in antiuterotrophic activity compared to 1 and 6. Compound 2 showed a strong, dose-dependent inhibition on the DMBA-induced hormone-dependent mammary tumor of the SD-rat, exceeding that of the parent compound 1. At a dose of 5 mg/kg per day, 2 reduced total tumor area by 47% and caused a complete remission in 74% of the tumors.

Antiestrogens. Synthesis and evaluation of mammary tumor inhibiting activity of 1,1,2,2-tetraalkyl-1,2 -diphenylethanes.

Among the newly synthesized 1,1,2,2-tetraalkyl-1,2-diphenylethanes, 1,1,2,2-tetramethyl-1,2-bis(4'-hydroxyphenyl)ethane (23) and 1,1,2,2-tetramethyl-1,2-bis(3'-hydroxyphenyl)ethane (26) were the most active compounds regarding estradiol receptor affinity, exhibiting Ka values of 0.73 X 10(8) and 0.67 X 10(8) M-1, respectively. In vivo, 23 and 26 showed only very small uterotrophic activity in the mouse. They strongly inhibited (73%) the estrone-stimulated mouse uterine growth. Tested on the 9,10-dimethyl-1,2-benzanthracene induced hormone-dependent mammary adenocarcinoma of the Sprague-Dawley rat, compounds 23 and 26 exhibited a dose-dependent inhibition of the tumor growth, having a strong effect at a dose of 20 (mg/kg)/day (compound 23).

Potential antiestrogens. Synthesis and evaluation of mammary tumor inhibiting activity of 1,2-dialkyl-1,2-bis(3'-hydroxyphenyl)ethanes.

The syntheses of the meso-1,2-dialkyl-1,2-bis(3'-hydroxyphenyl)ethanes [alkyl substituent: CH3 (19), C2H5 (20), C3H7 (22), C4H9 (23), i-C4H9 (24), and C5H11 (25)] and of d,l-3,4-bis(3'-hydroxyphenyl)hexane (21) are described. In vitro these compounds inhibited the [3H]estradiol receptor interaction competitively, exhibiting Ka values between 0.20 x 10(9) (20) and 0.11 x 10(6) M-1 (24). In vivo the meso compounds reduced the estrone-stimulated mouse uterine growth; the most effective compounds were 20, 22, and 23 (53, 50, and 45% inhibition, respectively). Compounds 20 and 22-24 showed weak estrogenic activity in the mouse uterine weight test and in the vaginal cornification test. Compounds 19 (NSC-297169), 20 (NSC-297170), and 22 (NSC-297171) exhibited a dose-dependent growth inhibition on the MCF-7 human breast tumor cell line (10(-6) to 10(-9) M). These compounds also showed a marked dose-dependent inhibition on the DMBA-induced, hormone-dependent mammary carcinoma of the Sprague-Dawley rat corresponding to their association constants.

Ring-substituted 1,2-dialkylated 1,2-bis(hydroxyphenyl)ethanes. 3. Synthesis, estrogen receptor binding affinity, and evaluation of antiestrogenic and mammary tumor inhibiting activity of 2,2'-disubstituted butestrols and 6,6'-disubstituted metabutestrols.

The synthesis of symmetrically 2,2'-disubstituted butestrols [meso-2,3-bis(4-hydroxyphenyl)butanes] and of 6,6'-disubstituted metabutestrols [meso-2,3-bis(3-hydroxyphenyl)butanes] are described [2,2'-substituents: H (1), OH (2), F (3), Cl (4), Br (5), CH3 (6), and C2H5 (7); 6,6'-substituents: H (8), OH (9), Cl (10), and CH3 (11)]. Compounds 1-11 were obtained by reductive coupling of the corresponding 1-phenylethanols with TiCl3/LiAlH4 and separation of the meso diastereomers. The binding affinity of the test compounds to the calf uterine estrogen receptor was measured relative to that of [3H]estradiol by a competitive binding assay. With the exception of 9, all other compounds showed remarkably high relative binding affinity (RBA) values between 1.0 and 29% that of estradiol. Compounds 3 and 6 (RBA values: 15 and 29), as well as 10 and 11 (1.7 and 5.2), exceeded those of the corresponding unsubstituted compounds 1 and 8 (12 and 1.0). The compounds exhibited strong (3, 4, 6, and 7), moderate (1, 2, and 10), weak (11), or no (8) estrogenic activity in the uterine weight test of the immature mouse. Compounds 1, 2, 8, 10, and 11 showed antiestrogenic activity inhibiting the estrone-stimulated uterine growth (25-35% inhibition). Compound 11 led to a significant inhibition of the tumor growth when tested on the 9,10-dimethyl-1,2-benzanthracene induced, hormone-dependent mammary carcinoma of the Sprague-Dawley rat.

Synthesis and evaluation of 17-aliphatic heterocycle-substituted steroidal inhibitors of 17alpha-hydroxylase/C17-20-lyase (P450 17).

In the search for potent inhibitors of P450 17, the key enzyme in androgen biosynthesis, a series of steroidal inhibitors were synthesized and tested toward rat and human P450 17. Small aliphatic heterocycles (aziridine, oxirane, thiirane, diaziridine, diazirine, azetidine) were introduced into the 17beta-position of anstrost-5-en-3beta-ol. After identifying that aziridine is the most suitable functional group to coordinate with the heme iron, modifications of the steroidal skeleton were performed for further optimization. A wide range of inhibitory potencies toward P450 17 were found for the 21 test compounds. The most potent inhibitors toward the human and rat enzyme were aziridine compounds 3 (IC(50) rat: 0.21 microM, K(i) = 3 nM; IC(50) human: 0.54 microM, K(i) = 8 nM), 5 (IC(50) rat: 0.43 microM, K(i) = 7 nM; IC(50) human: 0.29 microM, K(i) = 4 nM), and 8 (21R:21S = 1:1; IC(50) rat: 0.53 microM, K(i) = 9 nM; IC(50) human: 0.40 microM, K(i) = 6 nM) which were more potent than the reference ketoconazole (IC(50) rat: 67 microM; IC(50) human: 0.74 microM). The inhibitory potency depends markedly on the stereochemistry at C20 of the inhibitors. This effect is more pronounced for the rat enzyme. Tested for selectivity, the highly potent inhibitors show poor inhibitory activity toward P450 arom, P450 scc, P450 TxA(2), and 5alpha-reductase. Tested for in vivo activity, 3 and 8 (0.019 mmol/kg) decreased the plasma testosterone concentration in rats by 81% and 84% after 2 h.