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.

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.

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.

Protein-binding properties of a designed steroidal lactam compound.

Introducing amide bonds into a steroid nucleus or its side chain may reduce the acute toxicity and enhance the pharmaceutical activity. In this work, a designed steroidal amide compound, named 3ß-hydroxy-17-aza-d-homo-5-androsten-17-one (HAAO), was synthesized and identified. The interactions between HAAO and human serum albumin (HSA) were studied by multiple spectroscopic methods and molecular modeling procedures. It was found that HAAO locates in Sudlow's site I in subdomain IIA of HSA molecules, relying on hydrogen bonds and van der Waals power to form HAAO-HSA complexes at ground state. The number of binding sites, binding constants, enthalpy change (ΔH(θ)), Gibbs free energy change (ΔG(θ)) and entropy change (ΔS(θ)) were calculated at different temperatures based on fluorescence quenching theory and classical thermodynamic equation. The percentages content of the HSA's secondary structures in presence of HAAO were detected by circular dichroism (CD) spectra and compared with those in no presence of HAAO. In addition, the experimental results of both binding site and conformational change were further confirmed by molecular modeling investigation, in which more details of the binding were visually unfolded. The information provided by the study may be useful for designing novel chemotherapeutic drugs and be helpful both in the early stages of drug discovery and in clinical practice.

N-substituted piperazinopyridylsteroid derivatives as abiraterone analogues inhibit growth and induce pro-apoptosis in human hormone-independent prostate cancer cell lines.

Nine new 17-(piperazin-1-yl)pyridin-5-yl)steroids as abiraterone analogues were synthesized. Compounds 5d and 5g showed selective activities against 17α-hydroxylase/C17,20-lyase (CYP17A1) and aromatase (CYP19), respectively. IC50 values of 5d were 5.09 and >50 µm, whereas these values for 5g were >50 µm and 7.40 µm, respectively, for CYP17A1 and CYP19. Molecular modelling highlighted that the inhibitor designed to bind cytochrome P450 haem iron is a necessary condition but not the only rationale to explain inhibitory activity. These abiraterone analogues were then evaluated on hormone-independent prostate cancer cell lines DU-145 and PC-3 and on hormone-dependent breast and prostate cancer cell lines MCF-7 and LNCaP, respectively. Compounds 5e, 5g and 5i have showed potent activities only on hormone-independent prostate cancer cell lines DU-145 and PC-3 with 60-85% inhibition of both cell viability and growth at 10 nm with pro-apoptotic mechanism as illustrated in PC-3 cells by DNA ladder assay and Western blotting of Bax, Casp-3 and its substrate, the poly (ADP-ribose) polymerase. We conclude that hybrid heterocycle steroids could be good lead compounds in the drug design especially against hormone-independent prostate cancer.

[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.

Synthesis, antiproliferative activity, acute toxicity and assessment of the antiandrogenic activities of new androstane derivatives.

A number of 17-oxo-5-androsten-3ß-yl esters (9a-9f) and 3ß-alkoxy-5-androsten-17-ones (11a-11e) were synthesized from commercially available (25R)-5-spirosten-3ß-ol (Diosgenin) (4) as starting material. The synthesized compounds were evaluated for their antiproliferative activity against the prostate-specific cancer cell line DU-145, acute toxicity and effect on serum androgen levels, and compared with finasteride as positive control. Some of the compounds exhibited better cytotoxicity and antiandrogenic activity than the reference control. The detailed synthesis, spectroscopic data and biological activity of the synthesized compounds are reported.

Synthesis, biological evaluation, and molecular modeling of abiraterone analogues: novel CYP17 inhibitors for the treatment of prostate cancer.

Abiraterone, a steroidal cytochrome P450 17alpha-hydroxylase-17,20-lyase inhibitor (CYP17), is currently undergoing phase II clinical trials as a potential drug for the treatment of androgen-dependent prostate cancer. Since steroidal compounds often show side effects attributable to their structure, we have tried to replace the sterane scaffold by nonsteroidal core structures. The design and synthesis of 20 new abiraterone mimetics are described. Their activities have been tested with recombinant human CYP17 expressed in E. coli. Promising compounds were further evaluated for selectivity against CYP11B1, CYP11B2, and the hepatic CYP3A4. Compounds 19 and 20 showed comparable activity to abiraterone (IC50 values of 144 and 64 nM vs 72 nM) and similar or even better selectivity against the other CYP enzymes. Selected compounds were also docked into our homology model, and the same binding modes as for abiraterone were found.

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.

Another designer steroid: discovery, synthesis, and detection of 'madol' in urine.

Madol (17alpha-methyl-5alpha-androst-2-en-17beta-ol) was identified in an oily product received by our laboratory in the context of our investigations of designer steroids. The product allegedly contained an anabolic steroid not screened for in routine sport doping control urine tests. Madol was synthesized by Grignard methylation of 5alpha-androst-2-en-17-one and characterized by mass spectrometry and NMR spectroscopy. We developed a method for rapid screening of urine samples by gas chromatography/mass spectrometry (GC/MS) of trimethylsilylated madol (monitoring m/z 143, 270, and 345). A baboon administration study showed that madol and a metabolite are excreted in urine. In vitro incubation with human liver microsomes yielded the same metabolite. Madol is only the third steroid never commercially marketed to be found in the context of performance-enhancing drugs in sports.

Selective antagonism of 5alpha-reduced neurosteroid effects at GABA(A) receptors.

Although neurosteroids have rapid effects on GABA(A) receptors, study of steroid actions at GABA receptors has been hampered by a lack of pharmacological antagonists. In this study, we report the synthesis and characterization of a steroid analog, (3alpha,5alpha)-17-phenylandrost-16-en-3-ol (17PA), that selectively antagonized neurosteroid potentiation of GABA responses. We examined 17PA using the alpha1beta2gamma2 subunit combination expressed in Xenopus laevis oocytes. 17PA had little or no effect on baseline GABA responses but antagonized both the response augmentation and the direct gating of GABA receptors by 5alpha-reduced potentiating steroids. The effect was selective for 5alpha-reduced potentiating steroids; 5beta-reduced potentiators were only weakly affected. Likewise, 17PA did not affect barbiturate and benzodiazepine potentiation. 17PA acted primarily by shifting the concentration response for steroid potentiation to the right, suggesting the possibility of a competitive component to the antagonism. 17PA also antagonized 5alpha-reduced steroid potentiation and gating in hippocampal neurons and inhibited anesthetic actions in X. laevis tadpoles. Analogous to benzodiazepine site antagonists, the development of neurosteroid antagonists may help clarify the role of GABA-potentiating neurosteroids in health and disease.

Synthesis and in vitro antineoplastic evaluation of certain 16-(4-substituted benzylidene) derivatives of androst-5-ene.

In a systematic effort aimed at identifying new steroidal cytotoxic agents with potent antiproliferative activity against cancer cells, we synthesized certain 16-[4-(NO2, CN, and i-Pr)substituted]benzylidene derivatives of androst-5-ene, 7-25, with pyrrolidino functionality in the 3beta-position of the steroid nucleus, i.e., 13-18 and 25. The selected compounds were examined for their cytotoxicity against a panel of three human cancer cell lines at the National Cancer Institute (NCI), Bethesda, USA. The results presented herein provide experimental evidence that compounds 7, 9, 10, 12, 16, and 19-21 induced apoptosis in human cancer cells.

Nucleosteroids: carbocyclic nucleoside analogs of androst-4-en-17 beta-ol.

Steroidal nucleoside analogs were synthesized starting from testosterone. By reduction of the oxime of 17 beta-hydroxy-androst-4-en-3-one (testosterone), a mixture of the two amino epimers of C-3 were obtained. The 3 alpha-amino-androst-4-en-17 beta-ol was crystallized in 73% yield and coupled with 5-amino-4,6-dichloropyrimidine to give 3 alpha-(5'-amino-4'-chloro-pyrimidin-6'-yl)amino-androst-4-en-17 beta-ol. This compound was treated with triethyl orthoformate in acid media to give the corresponding purinyl steroid adduct 3 alpha-(6'-chloro-purin-9'-yl)-androst-4-en-17 beta-ol in 98% yield. This substance, in turn, was converted with good yield into the 6'-thio, 6'-methylamino, and 6'-diethyl aminopurinyl derivatives through nucleophilic reactions at C-6 of the purine nucleus.

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.

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.

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.