Synthesis of fluorinated 3β-hydroxypregn-5-en-20-one derivatives

Treatment of the unstable 3β-hydroxy-20, 20-dimethoxypregn-5-ene 3-acetate with acetic anhydride at reflux temperature gave a mixture of 3β-hydroxy-20-methoxypregna-5, 17(20)-diene and 3β-hydroxy-20-methoxypregna-5, 20-diene 3-acetates. Fluorination of this mixture with perchloryl fluoride afforded after fractionated crystallization 3β-hydroxy-17-fluoro-20-methoxypregna-5, 20-diene 3-acetate. Acid hydrolysis of the reaction mixture and subsequent Chromatographic separation led to 3β-hydroxy-17-fluoropregn-5-en-20-one 3-acetate and 3β-hydroxy-21-fluoropregn-5-en-20-one 3-acetate. 3β-Hydroxy-17-fluoro-20-methoxypregna-5, 20-diene 3-acetate did not react further with perchloryl fluoride even under forcing conditions. Fluorination of 3β-hydroxy-20-(N-ethyl benzylamino)-pregna-5, 17(20)-diene gave 3β-hydroxy-17, 21-difluoro-pregn-5-en-20-one, exclusively.     

Synthesis of some steroidal oximes, lactams, thiolactams and their antitumor activities

The antiproliferative activity of previously synthesized (Z)-cholest-4-en-6-one oxime (1), (E)-cholest-4-en-6-one oxime (2), 7-aza-B-homocholest-4-en-6-one (3) and 6-aza-B-homocholest-4-en-7-one (4) and newly synthesized 6-thioxo-7-aza-B-homocholest-4-ene (5) and 6-aza-7-thioxo-B-homocholest-4-ene (6) was tested for their possible effects against two human tumor cell lines, cervical carcinoma (HeLa) and chronic myelogenous leukemia (K-562). Compounds 1-6, exerted a dose-dependent antiproliferative effect toward cell lines used in experimental design, showing high selectivity in their action for tumor cells in comparison to normal immunocompetent cells (non-stimulated PBMC and PHA-stimulated PBMC). Compounds 2, 3 and 4 exhibited a very high but selective antitumor activity, by inducing apoptosis in sensitive, for that purpose targeted tumor cell line (HeLa cells). Low toxic effect upon both non-stimulated, and PHA stimulated PBMCs from control, healthy volunteers, has been detected for compounds 1, 2, 3 and 4. The possible reasons for profound differences in the effects of this spectrum of steroidal compounds between tumor cell lines and normal stimulated and non-stimulated PBMCs are discussed. The molecular mechanisms for apoptotic events in HeLa cell line are suggested. The guidelines for further research are underlined.     

Anticonvulsant evaluation and mechanism of action of benzylamino enaminones

The mechanism of anticonvulsant action was evaluated for the benzylamino enaminones. The most potent enaminone in this series was the unsubstituted benzylamine analog (30; methyl 4-benzylamino-6-methyl-2-oxocyclohex-3-en-1-oate) which had an oral effective dose (ED50) in rats of 27 mg/kg against maximal electroshock seizures, and a concentration 10-fold less than this dose depressed excitatory synaptic transmission, and action potential firing in the rat brain in vitro.     

Synthesis and Octopaminergic Agonist Activity of 2-(Substituted benzylamino)-2-thiazolines

2-(Substituted benzylamino)-2-thiazolines (SBAT) were synthesized by a hydrochloric acid-catalyzed cyclization of the corresponding thioureas, using a reaction of 2-methylthio-2-thiazoline with substituted benzylamines or by alkylating 2-amino-2-thiazoline. 2-(Alkylthio)-2-thiazolines were obtained by alkylating 2-mercaptothiazoline. Most of the SBAT compounds activated adenylate cyclase in homogenates of cockroach ventral nerve cords; the effect of introducing substituents at the phenyl of the SBAT compounds on octopaminergic agonist activity was not significant. 2-[β-(Substituted phenyl)ethylamino]-2-thiazolines and 2-(alkylthio)-2-thiazolines were not significant octopaminergic agonists. Washing removed nearly all of the activity of one of the SBAT compounds, suggesting that the SBAT compounds bound reversibly to the octopaminergic receptor.     

Synthesis of (25R)-26-hydroxy-15-ketosterols

(25R)-3beta,26-Dihydroxy-5alpha-cholest-8(14)-en-15-one (1) and (25R)-3beta,26-dihydroxy-5alpha,14beta-cholest-16-en-1 5-one (2) were synthesized from (25R)-3beta,26-dibenzoyloxy-5alpha,14alpha-chole st-16-ene (4). Oxidation of 4 with CrO3-3,5-dimethylpyrazole at -20 degrees C gave (25R)-3beta,26-dibenzoyloxy-5alpha,14alpha-chole st-16-en-15-one (5) along with (25R)-3beta,26-dibenzoyloxy-5alpha-cholest-16alpha+ ++,17alpha-epoxide (6). Oxidation of 5 with selenium dioxide afforded (25R)-3beta,26-dibenzoyloxy-5alpha-cholest-8(14),16-++ +dien-15-one (7) and (25R)-3beta,26-dibenzoyloxy-5alpha,14beta-choles t-16-en-15-one (8). Selective hydrogenation of 7 followed by hydrolysis in alcoholic potassium hydroxide yielded (25R)-3beta,26-dihydroxy-5alpha-cholest-8(14)-en-15-one (1). Hydrolysis of 5 and 8 in alcoholic potassium hydroxide provided (25R)-3beta,26-dihydroxy-5alpha,14beta-cholest-16-en-1 5-one (2).     

Inhibitors of sterol synthesis. Synthesis and spectral properties of 3 beta-hydroxy-24-dimethylamino-5 alpha-chol-8(14)-en-15-one and its effects on HMG-CoA reductase activity in CHO-K1 cells.

A simple, three-step synthesis of the 25-aza analog of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I) is described. Treatment of 3 beta-acetoxy-24-hydroxy-5 alpha-chol-8(14)-en-15-one (III) with 1.75 equivalents of tosyl chloride in pyridine for 24 h at 5 degrees C gave 3 beta-acetoxy-24-tosyloxy-5 alpha-chol-8(14)-en-15-one (IV). In contrast, treatment of III with 3.95 equivalents of tosyl chloride in pyridine for 12 h at 48 degrees C gave 3 beta-acetoxy-24-chloro-5 alpha-chol-8(14)-en-15-one (V). Treatment of IV with dimethylamine in dioxane yielded 3 beta-acetoxy-24-dimethylamino-5 alpha-chol-8(14)-en-15-one (VI). Hydrolysis of VI with LiOH.H2O in methanol gave 3 beta-hydroxy-24-dimethylamino-5 alpha-chol-8(14)-en-15-one (VII). 1H- and 13C-NMR assignments are presented for compounds IV-VII. The 25-aza analog (VII) of the 15-ketosterol (I), at a concentration of 1.0 microM, caused a 47% lowering of the level of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in Chinese hamster ovary cells.     

Synthesis and biological evaluation of 1,3-diphenylprop-2-en-1-ones possessing a methanesulfonamido or an azido pharmacophore as cyclooxygenase-1/-2 inhibitors

A group of (E)-1,3-diphenylprop-2-en-1-one derivatives (chalcones) possessing a MeSO(2)NH, or N(3), COX-2 pharmacophore at the para-position of the C-1 phenyl ring were synthesized using a facile stereoselective Claisen-Schmidt condensation reaction. In vitro COX-1/COX-2 structure-activity relationships were determined by varying the substituents on the C-3 phenyl ring (4-H, 4-Me, 4-F, and 4-OMe). Among the 1,3-diphenylprop-2-en-1-ones possessing a C-1 para-MeSO(2)NH COX-2 pharmacophore, (E)-1-(4-methanesulfonamidophenyl)-3-(4-methylphenyl)prop-2-en-1-one (7b) was identified as a selective COX-2 inhibitor (COX-2 IC(50)=1.0 microM; selectivity index >100) that was less potent than the reference drug rofecoxib (COX-2 IC(50)=0.50 microM; SI>200). The corresponding 1,3-diphenylprop-2-en-1-one analogue possessing a C-1 para-N(3) COX-2 pharmacophore, (E)-1-(4-azidophenyl)-3-(4-methylphenyl)prop-2-en-1-one (7f), exhibited potent and selective COX-2 inhibition (COX-1 IC(50)=22.2 microM; COX-2 IC(50)=0.3 microM; SI=60). A molecular modeling study where 7b and 7f were docked in the binding site of COX-2 showed that the p-MeSO(2)NH and N(3) substituents on the C-1 phenyl ring are oriented in the vicinity of the COX-2 secondary pocket (His90, Arg513, Phe518, and Val523). The structure-activity data acquired indicate that the propenone moiety constitutes a suitable scaffold to design new acyclic 1,3-diphenylprop-2-en-1-ones with selective COX-1 or COX-2 inhibitory activity.     

Synthesis and cell growth inhibitory properties of substituted (E)-1-phenylbut-1-en-3-ones

A series of (E)-1-phenylbut-1-en-3-ones, based on the naturally occurring (E)-1-(4′-hydroxyphenyl)but-1-en-3-one [IC₅₀ (K562) 60 μM], was synthesised and screened for cytotoxic activity against the K562 human leukaemia cell line. (E)-1-(Pentafluorophenyl)but-1-en-3-one [IC₅₀ (K562) 1.8 μM] was found to be over 30-fold more active than 1.     

Synthesis of 6-oxy functionalized campest-4-en-3-ones: efficient hydroperoxidation at C-6 of campest-5-en-3-one with molecular oxygen and silica gel

As a reference compound library for the investigation of biosynthesis of brassinosteroids, focused on a pathway from campesterol (1) to campestanol (2), 6-oxy functionalized campest-4-en-3-ones as well as campest-5-en-3-one (7) and campestane-3,6-dione were prepared from 1. Oxidation of 1 with pyridinium chlorochromate buffered by calcium carbonate gave 5-en-3-one (7) in 76% yield. Treatment of 7 with silica gel under an oxygen atmosphere in ethyl ether at room temperature produced efficient hydroperoxidation at the C-6 position to give 6alpha-hydroperoxycampest-4-en-3-one and 6beta-hydroperoxycampest-4-en-3-one in 34% and 49% yields, respectively. These compounds were converted to 6alpha-hydroxycampest-4-en-3-one and 6beta-hydroxycampest-4-en-3-one by reduction with triethyl phosphite. This provided the first example of the practical use of hydroperoxidation at C-6 of a Delta(5(6))-unsaturated 3-oxo-steroid with molecular oxygen and silica gel. On the other hand, oxidation of 1 with pyridinium chlorochromate in the absence of calcium carbonate gave campest-4-ene-3,6-dione in 64% yield. This compound was then converted in a highly stereoselective manner to campestane-3,6-dione with A/B trans ring junction by reduction with titanium (III) chloride in 85% yield.     

Inhibitors of sterol synthesis. Characterization of beta,gamma-unsaturated analogs of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one and their effects on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells

Treatment of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (1), a potent regulator of cholesterol metabolism, with perchloric acid in methanol resulted in its partial isomerization to the beta,gamma-unsaturated 15-ketosterols, 3 beta-hydroxy-5 alpha,14 beta-cholest-8-en-15-one (2) and 3 beta-hydroxy-5 alpha,14 beta-cholest-7-en-15-one (3), which were easily separated from 1 by chromatography. Isomers 1, 2, and 3 could be distinguished by their chromatographic retention times as well as by their physical and spectral properties. Reduction of 2 with sodium borohydride gave 5 alpha,14 beta-cholest-8-ene-3 beta,15 beta-diol (4), for which the C-15 configuration was established from the lanthanide-induced shifts of its 3 beta-tert-butyldimethylsilyl ether. 1H and 13C NMR chemical shift differences between 2, 3, and 4 indicated the involvement of variable populations of conformers that differ in the flexible C-D ring system and in the side chain. Compounds 2, 3, and 4 lowered the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells.     

Microbial transformation of 17alpha-ethynyl- and 17alpha-ethylsteroids, and tyrosinase inhibitory activity of transformed products

The microbial transformation of the 17alpha-ethynyl-17beta-hydroxyandrost-4-en-3-one (1) (ethisterone) and 17alpha-ethyl-17beta-hydroxyandrost-4-en-3-one (2) by the fungi Cephalosporium aphidicola and Cunninghamella elegans were investigated. Incubation of compound 1 with C. aphidicola afforded oxidized derivative, 17alpha-ethynyl-17beta-hydroxyandrosta-1,4-dien-3-one (3), while with C. elegans afforded a new hydroxy derivative, 17alpha-ethynyl-11alpha,17beta-dihydroxyandrost-4-en-3-one (4). On the other hand, the incubation of compound 2 with the fungus C. aphidicola afforded 17alpha-ethyl-17beta-hydroxyandrosta-1,4-dien-3-one (5). Two new hydroxylated derivatives, 17alpha-ethyl-11alpha,17beta-dihydroxyandrost-4-en-3-one (6) and 17alpha-ethyl-6alpha,17beta-dihydroxy-5alpha-androstan-3-one (7) were obtained from the incubation of compound 2 with C. elegans. Compounds 1-6 exhibited tyrosinase inhibitory activity, with compound 6 being the most potent member (IC(50)=1.72 microM).     

Synthesis and antihepatotoxic activity of 5-(2,3-dihydro-1,4-benzodioxane-6-yl)-3-substituted-phenyl-4,5-dihydro-1H-pyrazole derivatives

In continuance of our search for newer antihepatotoxic agents some novel pyrazoline derivatives containing 1,4-dioxane ring system were synthesized starting from 3-(2,3-dihydro-1,4-benzodioxane-6-yl)-1-substituted-phenylprop-2-en-1-one. Some of the synthesized compounds were evaluated for antihepatotoxic activity against CCl(4)-induced hepatotoxicity in rats. Among them some compounds have shown significant antihepatotoxic activity comparable to standard drug silymarin.     

Synthesis and antimalarial activity of new heterocyclic hybrids based on chloroquine and thiazolidinone scaffolds

A series of new 21 chloroquine heterocyclic hybrids containing either benzylamino fragment or N-(aminoalkyl)thiazolidin-4-one moiety were synthesized and screened for their antimalarial activity against chloroquine (CQ)-sensitive 3D7 and multidrug-resistance Dd2 strains of Plasmodium falciparum. Although no compounds more active than CQ against 3D7 was found; against Dd2 strain, six compounds, four of them with benzylamino fragment, showed an excellent activity, up to 3-fold more active than CQ. Non specific cytotoxicity on J774 macrophages was observed in some compounds whereas only two of them showed liver toxicity on HepG2 cells. In addition, all active compounds inhibited the ferriprotoporphyrin IX biocrystalization process in concentrations around to CQ. In vivo preliminary results have shown that at least two compounds are as active as CQ against Plasmodium berghei ANKA.     

Inhibitors of sterol biosynthesis. Synthesis and activities of ring C oxygenated sterols

The chemical syntheses of a number of C27 ring C oxygenated sterols have been pursued to permit evaluation of their activity in the inhibition of sterol biosynthesis in cultured mammalian cells. Thus, 5 alpha-cholest-7-ene-3 beta, 11 alpha-diol, 3 alpha-hydroxy-5 alpha-cholest-9(11)-en-12-one, and the previously unreported 11 alpha-hydroxy-5 alpha-cholest-7-en-3-one, 5 alpha-cholest-9(11)-ene-3,12-dione, and 3 beta-hydroxy-5 alpha-cholest-9 (11)-en-12-one have been synthesized. The effects of these compounds on the synthesis of digitonin-precipitable sterols from labeled acetate in mouse L cells and on the levels of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in the same cells have been investigated and compared with previously published data on other ring C oxygenated sterols. 5 alpha-Cholest-7-ene-3 beta, 11 alpha-diol was shown to be the most potent inhibitor of sterol synthesis.     

A novel synthesis of acyclonucleosides via allylation of 3-[1-(phenylhydrazono)-L-threo-2,3,4-trihydroxybut-1-yl]quinoxalin-2(1H)one

The allylation of 3-[1-(phenylhydrazono)-L-threo-2,3,4-trihydroxybut-1-yl]quinoxalin-2(1H)one (1) gave, in addition to the anticipated 1-N-allyl derivative (2), a dehydrative cyclized product, 1-N-allyl-3-[5-(hydroxymethyl)-1-phenylpyrazol-3-yl]quinoxalin-2-one (4) and its isomeric O-allyl derivative 3. The O-allyl group in 3 underwent acetolysis under acetylation conditions, in addition to the acetylation of the hydroxyl group, to afford 2-acetoxy-3-[5-(acetoxymethyl)-1-phenylpyrazol-3-yl]quinoxaline (8) instead of the O-acetyl derivative of 3. Allylation of the tri-O-acetyl derivative of 1 caused the elimination of a molecule of acetic acid in addition to N-allylation to give 1-N-allyl-3-[3,4-di-O-acetyl-2-deoxy-1-(phenylhydrazono)but-2-en-1-yl]quinoxalin-2-one (11). Hydroxylation of the allyl group gave a glycerol-1-yl acyclonucleoside which can be alternatively obtained by a displacement reaction of the tosyloxy group in 2,3-O-isopropylidene-1-O-(p-tolylsulfonyl)glycerol (14), followed by deisopropylidenation. 1-N-(2,3-Dibromopropyl)-3-[5-(hydroxymethyl)-1-(4-bromophenyl)pyrazol-3-yl]quinoxalin-2-one (15) underwent azidolysis to give a 2,3-diazido derivative. The assigned structures were based on spectral analysis. The activity of compounds 2, 4, 6, and 15 against hepatitis B virus was studied.     

Active nonaromatic intermediates in the conversion of steroidal estrogens into catechol estrogens

A mechanism is proposed for mixed-function oxidase-catalyzed formation of the catechol estrogens 2-hydroxy- and 4-hydroxyestradiol from estradiol. This mechanism involves nonaromatic epoxyenones as intermediates. The isomeric 1 alpha,2 alpha-epoxy-17 beta-hydroxyestr-4-en-3-one and 1 beta,2 beta-epoxy-17 beta-hydroxyestr-4-en-3-one (the latter as its 17-acetate) were synthesized from 17 beta-hydroxy-5 alpha-estran-3-one. The isomeric 4 alpha,5 alpha-epoxy-17 beta-hydroxyestr-1-en-3-one and 4 beta,5 beta-epoxy-17 beta-hydroxyestr-1-en-3-one were prepared from 19-nortestosterone. From incubations of [6,7-3H]estradiol with microsomes from MCF-7 human breast cancer cells, which principally catalyze the formation of 2-hydroxyestradiol from estradiol, we were able to isolate a 3H-labeled product with the chromatographic properties of 1 beta, 2 beta-epoxy-17 beta-hydroxyestr-4-en-3-one (as its 17-acetate). The soluble protein fraction of homogenates of rat liver, which is devoid of estrogen 2-/4-hydroxylase activity, has been shown to catalyze the formation of 2- and 4-hydroxyestradiol from the 1 alpha,2 alpha-epoxide and from the 4 alpha,5 alpha- and 4 beta,5 beta-epoxides, respectively. We suggest that these results taken together strongly support a role for epoxyenones as intermediates in the formation of catechol estrogens.     

New polyoxygenated steroids from the Antarctic octocoral Dasystenella acanthina

The chemical study of the Antarctic octocoral Dasystenella acanthina has led to the isolation of the new polyoxygenated steroids (24R,22E)-24-hydroxycholest-4,22-dien-3-one (1), 23-acetoxy-24,25-epoxycholest-4-en-3-one (2), 12beta-acetoxycholest-4-en-3,24-dione (3), 12beta-acetoxy-24,25-epoxycholest-4-en-3-one (4), (22E)-25-hydroxy-24-norcholest-4,22-dien-3-one (5), 3alpha-acetoxy-25-hydroxycholest-4-en-6-one (6), and 3alpha,11alpha-diacetoxy-25-hydroxycholest-4-en-6-one (7), whose structures have been established by spectroscopic analysis. The absolute stereochemistry at C-24 in compound 1 has been determined through the 1H NMR study of the corresponding (R)- and (S)-MPA esters. All the new compounds showed significant activities as growth inhibitors of several human tumor cell lines. In addition, cytostatic and cytotoxic effects were also observed on selected tumor cell lines.     

Synthesis and biological evaluation of benzamides and benzamidines: structural requirement of a pyrimidine ring for inhibition of EGFR tyrosine kinase

The benzamides 1 and the benzamidines 2-3 were synthesized as the mimics of 4-anilinoquinazolines, which possess inhibition of epidermal growth factor receptor (EGFR) tyrosine kinase, and tested for cytotoxicity toward A431 and inhibitory activity toward autophosphorylation by the enzyme assay. High cell growth inhibition was observed in a series of the cyclic benzamides 3: the IC(50) values are 0.09-0.32 mM. The benzamidines 3a and 3b exhibited high inhibition of EGFR tyrosine kinase at a 1.0 microM concentration, although the benzamides 1 and the benzamidines 2 did not show significant kinase inhibition at a 10 microM concentration.     

Synthesis and screening of a combinatorial library of naphthalene substituted chalcones: inhibitors of leukotriene B4.

A combinatorial mini library of naphthalene substituted chalcones has been prepared by solution phase chemistry. Screening of these mixtures for leukotriene B4 inhibitory activity using human whole blood assay (HWBL) afforded a lead compound, 1-(6-butoxy-2-naphthyl)-3-(4-nitrophenyl)-prop-2-en-1-one (K4A3) with an IC50 value of 18.5 microM.     

Two new sesquiterpenoids and anti-HIV principles from the root bark of Zanthoxylum ailanthoides

Two new sesquiterpenes, 10beta-methoxymuurolan-4-en-3-one (1) and 10alpha-methoxycadinan-4-en-3-one (2), were isolated from the root bark of Zanthoxylum ailanthoides. The structures of 1 and 2 were elucidated from spectroscopic data. Sixty-seven compounds obtained from the root bark of the same plant were evaluated for inhibition of HIV replication in H9 lymphocyte cells, and 14 compounds demonstrated significant activity. Among them, decarine, gamma-fagarine, and (+)-tembamide were the most potent anti-HIV compounds, with EC50 values of <0.1 microg/mL and TI values of >226, >231, and >215, respectively.