Simple heteroaryl modifications in the 4,5-diarylisoxazol-3-carboxylic acid scaffold favorably modulates the activity as dual mPGES-1/5-LO inhibitors with in vivo efficacy.

Microsomal prostaglandin E2 synthase-1 (mPGES-1), 5-lipoxygenase (5-LO) and 5- lipoxygenase-activating protein (FLAP) are key for biosynthesis of proinflammatory lipid mediators and pharmacologically relevant drug targets. In the present study, we made an attempt to explore the role of small heteroaromatic fragments on the 4,5-diarylisoxazol-3-carboxylic acid scaffold, which are selected to interact with focused regions in the active sites of mPGES-1, 5-LO and FLAP. We report that the simple structural variations on the benzyloxyaryl side-arm of the scaffold significantly influence the selectivity against mPGES-1, 5-LO and FLAP, enabling to produce multi-target inhibitors of these protein targets, exemplified by compound 18 (IC50 mPGES-1 = 0.16 µM; IC50 5-LO = 0.39 µM) with in vivo efficacy in animal model of inflammation. The computationally modeled binding structures of these new inhibitors for three targets provide clues for rational design of modified structures as multi-target inhibitors. In conclusion, the simple synthetic procedure, and the possibility of enhancing the potency of this class of inhibitors through structural modifications pave the way for further development of new multi-target inhibitors against mPGES-1, 5-LO and FLAP, with potential application as anti-inflammatory agents.

Pd-mediated cross-coupling of C-17 lithiated androst-16-en-3-ol - access to functionalized arylated steroid derivatives.

Herein, we report on Pd-mediated cross-coupling of vinyllithium steroids and aryl bromides to introduce various substituted aryls at C-17 of steroidal frameworks based on the structure of epi-androsterone. Compared to other C-C cross-couplings, this method turned out to be an easy and competitive access to biologically interesting C-17 modified steroids.

Synthesis and in-vivo hypolipidemic activity of some novel substituted phenyl isoxazol phenoxy acetic acid derivatives.

The present study was undertaken to evaluate in-vivo hypolipidemic activity of a novel series of 2-methyl-2-(substituted phenyl isoxazol)phenoxyacetic acid derivatives by triton induced hyperlipidemia in rats. The newly synthesized compounds 5a, 5d and 5g showed significant decrease in the serum TCH, TG, LDL and VLDL along with an increase in serum HDL levels as compared to standard drug Fenofibrate. The treated groups also showed significant decrease in the atherogenic index and increase in % protective activity compared to control group.

[Advances in the study of steroidal inhibitors of cytochrome P45017alpha].

The steroidal enzyme cytochrome P45017alpha catalyzes the conversion of progesterone and pregnenolone into androgens, androstenedione and dehydroepiandrosterone, respectively, the direct precursors of estrogens and testosterone. Dihydrotestosterone is the principal active androgen in the prostate, testosterone is also an active stimulant of the growth of prostatic cancer tissue. Inhibition of this enzyme as a mechanism for inhibiting androgen biosynthesis could be a worthwhile therapeutic strategy for the treatment of PCA. In this paper, four categories of steroidal inhibitors of cytochrome P45017alpha will be reviewed, a diverse range of steroidal inhibitors had been synthesized and shown to be potent inhibitors of P45017alpha.

Discovery of 2-isoxazol-3-yl-acetamide analogues as heat shock protein 90 (HSP90) inhibitors with significant anti-HIV activity.

The recent burst of explorations on heat shock protein 90 (HSP90) in virus research supports its emergence as a promising target to overcome the drawbacks of current antiviral therapeutic regimen. In continuation of our efforts towards the discovery of novel anti-retroviral molecules, we designed, synthesized fifteen novels 2-isoxazol-3-yl-acetamide based compounds (2a-o) followed by analysis of their anti-HIV activity and cytotoxicity studies. 2a-b, 2e, 2j, and 2l-m were found to be active with inhibitory potentials >80% at their highest non-cytotoxic concentration (HNC). Further characterization of anti-HIV activity of these molecules suggests that 2l has ∼3.5 fold better therapeutic index than AUY922, the second generation HSP90 inhibitor. The anti-HIV activity of 2l is a cell type, virus isolate and viral load independent phenomena. Interestingly, 2l does not significantly modulate viral enzymes like Reverse Transcriptase (RT), Integrase (IN) and Protease (PR) as compared to their known inhibitors in a cell free in vitro assay system at its HNC. Further, 2l mediated inhibition of HSP90 attenuates HIV-1 LTR driven gene expression. Taken together, structural rationale, modeling studies and characterization of biological activities suggest that this novel scaffold can attenuate HIV-1 replication significantly within the host and thus opens a new horizon to develop novel anti-HIV therapeutic candidates.

Synthesis of novel 17-triazolyl-androst-5-en-3-ol epimers via Cu(I)-catalyzed azide-alkyne cycloaddition and their inhibitory effect on 17α-hydroxylase/C17,20-lyase.

The regioselective Cu(I)-catalyzed 1,3-dipolar cycloaddition of 17α- and 17ß-azidoandrost-5-en-3ß-ol epimers (3b and 5b) with different terminal alkynes afforded novel 1,4-substituted triazolyl derivatives (8a-k and 9a-k). For the preparation of 5'-iodo-1',2',3'-triazoles (8m-n and 9m-n), an improved method was developed, directly from steroidal azides and terminal alkynes, in reaction mediated by CuI and ICl as iodinating agents. Acetolysis and subsequent hydrolysis of 8n and 9n yielded 5'-hydroxy-1',2',3'-triazoles 8o and 9o. The inhibitory effect of 8a-o, 9a-o, 3, and 5 on rat testicular C17,20-lyase was investigated by means of an in vitro radioincubation technique. The results revealed that the C-17 epimers of steroidal triazoles influence the C17,20-lyase effect. Inhibitors were found only in the 17α-triazolyl series (8a-o), whereas in the C-17 azide pair the 17ß compound (5b) was more potent.

Design, synthesis, biological evaluation and docking studies of new 3-(4,5-dihydro-1H-pyrazol/isoxazol-5-yl)-2-phenyl-1H-indole derivatives as potent antioxidants and 15-lipoxygenase inhibitors.

New candidates of 3-(4,5-dihydro-1H-pyrazol/isoxazol-5-yl)-2-phenyl-1H-indole derivatives (4-7) were designed combining the pyrazoline/isoxazoline heterocycles and 2-phenylindole to explore its potential as 15-lipoxygenase (15-LOX) inhibitors. The design of the new derivatives was based on utilizing the antioxidant properties of pyrazoline, 2-phenylindole and the good 15-LOX inhibition properties of indolylpyrazoline. The derivatives were synthesized adopting simple and laboratory friendly reaction conditions to give the target compounds in quantitative yields. The resulting indolylpyrazolines/isoxazolines were evaluated as antioxidants against 2,2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide (NO) and superoxide dismutase (SOD); indolylpyrazoline (4b) was the most potent antioxidant against SOD assay (IC50 = 1.78 µM) to be superior to ascorbic by 2 folds. Consistently, (4b) was the most potent inhibitor when tested against Soybean 15-LOX (IC50 = 3.84 µM) excelling quercetin as standard inhibitor by 1.8 folds. Some of the new derivatives were docked into the active binding site of human 15-LOX (PDB entry 4NRE) emphasizing the most potent derivative (4b) and the least potent one (4c). Docking solutions of compounds (4b), (4c), (5b) and (6c) revealed that (4b) was the only compound that got stabilized into the catalytic pocket of enzyme by π-cation interaction with the catalytic Fe+ and formation of one hydrogen bond with Ile 676 amino acid. Other derivatives including the least potent one variably got stabilized into the active binding pocket by π-cation interaction with the catalytic Fe+ but failed to form hydrogen bond with Ile 676. For the future optimization of the generated inhibitors, (i) antioxidant activity against SOD, (ii) the inhibitor stabilization by π-cation interaction with the catalytic Fe+3 and (iii) formation of hydrogen bond with Ile 676 should be regarded.

Synthesis of the bis-potassium salts of 5-hydroxy-3-oxopent-4-enoic acids and their use for the efficient preparation of 4-hydroxy-2H-pyran-2-ones and other heterocycles.

5-Hydroxy-3-oxopent-4-enoic acid esters can be efficiently transformed into the stable bis-potassium salts of the corresponding 5-hydroxy-3-oxopent-4-enoic acids, from which the sensitive acids are released in situ, the latter being converted into substituted 4-hydroxy-2H-pyran-2-ones, pyrazoles and isoxazoles under mild conditions; the efficiency of this method is demonstrated by the first synthesis of two naturally occurring pyrones.

Facile synthesis of active antitubercular, cytotoxic and antibacterial agents: a Michael addition approach.

Spiro derivatives of oxindole and isoxazole-5-one were synthesized by using Michael addition reaction, highlighting the regioselective approach towards the synthesis of Michael diadduct followed by condensation of Michael diadduct. The spiro compound 4 showed antitubercular activity against Mycobacterium tuberculosis H37Rv whereas spiro compound 9 possesses pronounced anticancer and antibacterial profile.

Synthesis and biochemical studies of 16- or 19-substituted androst-4-enes as aromatase inhibitors.

Androst-4-en-17-one derivatives [19-acetoxide 4, 16-bromides 14 and 15, 19,19-difluoride 18, and (19R,S)-19-acetylenic alcohol 25] and androst-4-en-17 beta-ol derivatives 3, 5, 10, 12, and 19 were synthesized and tested for their ability to inhibit aromatase in human placental microsomes. All the 17-oxo steroids, except compound 25 and 17,19-diol 3 of this series, were effective competitive inhibitors with apparent Ki's ranging from 170 to 455 nM. 19,19-Difluoro steroid 18 and 19-acetylenic alcohol 25, a weak competitive inhibitor (Ki = 7.75 microM), caused a time-dependent, pseudo-first-order inactivation of aromatase activity with kinact's of 0.0213 and 0.1053 min-1 for compounds 18 and 25, respectively. NADPH and oxygen were required for the time-dependent inactivation, and the substrate, androst-4-ene-3,17-dione, prevented it, but a nucleophile, L-cysteine, did not in each case. The results strongly suggest that aromatase would attack the 19-carbon of steroids 18 and 25.

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).

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.

Design and synthesis of 4,5-diphenyl-4-isoxazolines: novel inhibitors of cyclooxygenase-2 with analgesic and antiinflammatory activity.

4,5-Diphenyl-4-isoxazolines (13a-k) possessing a variety of substituents (H, F, MeS, MeSO2) at the para position of one of the phenyl rings were synthesized for evaluation as analgesic and selective cyclooxygenase-2 (COX-2) inhibitory antiinflammatory (AI) agents. Although the 4,5-phenyl-4-isoxazolines (13a-d,f), which do not have a C-3 Me substituent, exhibited potent analgesic and AI activities, those compounds evaluated (13a, 13b, 13h, and 13k) were not selective inhibitors of COX-2. In contrast, 2,3-dimethyl-5-(4-methylsulfonylphenyl)-4-phenyl-4-isoxazoline (13j) exhibited excellent analgesic and AI activities, and it was a potent and selective COX-2 inhibitor (COX-1, IC(50) = 258 microM; COX-2, IC(50) = 0.004 microM). A related compound 13k having a F substituent at the para position of the 4-phenyl ring was also a selective (SI = 3162) but less potent (IC(50) = 0.0316 microM) inhibitor of COX-2 than 13j. A molecular modeling (docking study) for 13j showed that the S atom of the MeSO2 substituent is positioned about 6.46 A inside the entrance to the COX-2 secondary pocket (Val(523)) and that a C-3 Me (13j, 13k) central isoxazoline ring substituent is crucial to selective inhibition of COX-2 for this class of compounds.

New synthesis of 2 beta-hydroxy-19-oxoandrost-4-ene-3,17-dione and its 2 beta-18O analog.

Treatment of 19-[oxygenated]-androst-4-ene-3,17-dione with Mn(AcO)3 and ClCH2COOH in benzene gave epimeric mixtures of the corresponding 2 zeta-chloroacetates and 2 zeta-acetates. The products were processed to give the title compound. For the synthesis of the 2-18O analog, ClCH2C18OOH was used, which was prepared from ClCH2COCl.

Synthesis of 3 beta,16 beta,19-trihydroxyandrost-5-en-17-one and 16 beta,19-dihydroxyandrost-4-ene-3,17-dione and their 19-oxo derivatives.

3 beta,16 beta,19-Trihydroxyandrost-5-en-17-one (12) was synthesized from 5 alpha-bromo-3 beta-acetoxy-6 beta,19-epoxyandrostan-17-one (2) through acetoxylation at C-16 beta of the enol acetate 4 with lead tetraacetate and reductive cleavage of the epoxide ring with zinc dust yielding the 3 beta,16 beta-diacetoxy-19-hydroxy steroid 11, followed by hydrolysis of the acetoxy groups with sulfuric acid. Jones oxidation of compound 11 followed by the acid hydrolysis gave the 19-oxo steroid 15. 5 alpha-Bromo-3 beta-hydroxy-16 beta-acetoxy-6 beta,19-epoxyandrostan-17-one (8), obtained by selective hydrolysis of the 3-formate 5 with ammonium hydroxide, was oxidized with Jones reagent to afford the 3-oxo steroid 16, which was converted into the 19-hydroxy derivative 17 by treatment with zinc dust. 16 beta,19-Dihydroxyandrost-4-ene-3,17-dione (18) and its 19-oxo derivative 21 were obtained from compound 17 through a similar reaction sequence.

Synthesis of 16 alpha,19-dihydroxy-4-androstene-3,17-dione and 3 beta,16 alpha,19-trihydroxy-5-androsten-17-one and their 17 beta-hydroxy-16-keto isomers.

3 beta,16 alpha,19-Trihydroxy-5-androsten-17-one and 16 alpha,17-dihydroxy-4-androstene-3,17-dione were synthesized from the 5 alpha-bromo-6 beta,19-epoxy-17-ketone derivative 1, using the bromination at C-16 alpha of the 17-ketone 1 and the controlled alkaline hydrolysis of the 16 alpha-bromo-17-ketones 2 and 11 as key reactions. Zinc dust reductive cleavage of the 6 beta,19-epoxy-16 alpha-hydroxy-17-ketones 4 and 12, produced by controlled hydrolysis, gave the corresponding 19-alcohol derivatives 6 and 14, which were rearranged to the 17 beta-hydroxy-16-ketones 7 and 15 when treated with sodium hydroxide. The 3 beta,16 alpha,17 beta,19-tetrol 8 was obtained from the 16 alpha-ketol 6 by reaction with sodium borohydride.

Preparation of androsta - 2,4-dien-17beta -ol.

A simple preparation of androsta-2,4-dien-17beta-ol from testosteroneis described...

Novel silylated steroids as aromatase inhibitors.

Androst-4-en-3-one analogs incorporating a trimethylsilyl or a trimethylsilylmethyl group at C-1, C-2 or C-19 were prepared and evaluated as inhibitors of aromatase. Only 10-[1-hydroxy-2-(trimethylsilyl)ethyl]estr-4-ene-3,17-dione inhibited human placental aromatase. Enzyme kinetic analysis revealed competitive inhibition [apparent dissociation constant (Ki) of 562 +/- 12 nM] associated with marginal time-dependent inhibition.

Synthesis of (19E)-3 beta,7 alpha-dihydroxy-17-oxoandrost-5-en-19-al 19-(O-carboxymethyl)oxime, a new hapten for 7 alpha-hydroxydehydroepiandrosterone (3 beta,7 alpha-dihydroxyandrost-5-en-17-one).

The title compound was prepared in 11 steps from 17,17-ethylenedioxy-19-hydroxyandrost-5-en-3 beta-yl acetate. After tert-butyldimethylsilyl protection of the 19-hydroxyl group, a 7-oxo group was introduced by oxidation with 3,5-dimethylpyrazole-chromium trioxide complex, and then selectively reduced with L-Selectride to give a 7 alpha-hydroxy derivative. This partially protected triol was acetylated and desilylated to 3,7-diacetate. Subsequent oxidation with pyridine-chromium trioxide complex gave 19-aldehyde, which was transformed into the corresponding protected 19-(O-carboxymethyl)oxime. Successive ketal cleavage, deacetylation, and methyl ester splitting gave the final (19E)-3 beta,7 alpha-dihydroxy-17-oxoandrost-5-en-19-al 19-(O-carboxymethyl)oxime, designed as a hapten for 7 alpha-hydroxydehydroepiandrosterone immunoassays.

Reaction of androst-5-en-17-one with hypobromous acid and its use for synthesis of 19-oxygenated 5-ene and 4-en-6-one steroids.

Reaction of androst-5-en-17-one (1) with hypobromous acid using a short reaction time (30 min) along with a careful isolation procedure gave, for the first time, the addition product, 5 alpha-bromo-6 beta-hydroxyandrostan-17-one (3), in 43% yield. This bromohydrin was much more reactive than 5 alpha-bromo-3 beta-acetoxy-6 beta-hydroxyandrostan-17-one (4) towards KHCO3 and HClO4. The high reactivity of compound 3 was found to be a principal reason for the difficulty in isolating this compound by the addition reaction so far. 19-Hydroxyandrost-5-en-17-one (16) and androst-5-ene-17,19-dione (18), as well as 19-hydroxyandrost-4-ene-6,17-dione (28) and androst-4-ene-6,17,19-trione (29), were synthesized through hypoiodite reaction of the bromohydrin 3 as a key reaction.