Conformational considerations in the design of dual antagonists of platelet-activating factor (PAF) and histamine.

Following the discovery of the first dual antagonist of platelet-activating factor (PAF) and histamine, 1-acetyl-4-(8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin- 11-ylidene)piperidine, Sch 37370, 1, a related series of structures, exemplified by (+/-)-1-acetyl-4-(8-chloro-5,6-dihydro-11H-benzo[5,6]-cyclohepta[1,2-b] pyridin-11-yl)piperazine, Sch 40338, 2, were prepared. Interestingly, the compounds exhibited a parallel structure antiallergy activity relationship, suggesting that the two series may adopt a common conformation at the PAF receptor. Conformational analysis led to a proposal for this bioactive conformation accessible to both series. The synthesis of novel conformationally constrained analogues that might mimic the proposed bioactive conformation of these compounds, and the evaluation of their in vitro antiallergy activity form the subject matter of this report.     

1,2,4-Triazino-[5,6b]indole derivatives: effects of the trifluoromethyl group on in vitro antimalarial activity

In an attempt to search for new and alternative antimalarial agents, a series of unsubstituted and 6-trifluoromethyl-1,2,4-triazino[5,6b]indole and 5H-1,2,4-triazolo[1',5',2,3]-1,2,4-triazino[5,6b]indole derivatives were synthesized and their chemical structures confirmed by 1H NMR and 13C NMR, elemental, IR and mass spectrophotometric analyses. The in vitro antimalarial activities of these compounds were evaluated against the chloroquine-sensitive (D10) and the chloroquine-resistant (RSA11) strains of Plasmodium falciparum. The 1,2,4-triazino[5,6b]indole derivatives (4, 6 and 8) with a trifluoromethyl group at position 6 exhibit increased in vitro activity when compared to the unsubstituted analogues, which are all devoid of activity. The presence of the trifluoromethyl group in the 5H-1,2,4-triazolo[1',5',2,3]-1,2,4-triazino[5,6b]indole ring system leads to compounds with diminished antimalarial activity when compared to the corresponding unsubstituted analogues. The compounds associate with ferriprotoporphyrin IX and interact with DNA to more or less the same extent.     

Synthesis of (8Z,11Z,14Z)-eicosatrien-5-ynoic (5,6-dehydroarachidonic) acid and its transformation into [5,6-3H]arachidonic acid and [5,6-3H]prostaglandins E2 and F2 alpha

8Z,11Z,14Z-Eicosatriene-5-ynoic acid and its tritium-labelled analogue, [5,6-3H]arachidonic acid, have been synthesized on the basis of acetylenic compounds. [5,6-3H]Arachidonic acid has been used as substrate for the enzymatic synthesis of [5,6-3H]PGE2 and [5,6-3H]PGF2 alpha.     

Synthesis and preliminary pharmacological evaluation of 5-hydroxy- and 5,6-dihydroxy-1,2,3,7,12,12a-hexahydrobenzo[5,6]cyclohepta[1,2,3-ij]isoquinoline derivatives as dopamine receptor ligands.

A series of 5-hydroxy- and 5,6-dihydroxy-1,2,3,7,12,12a-hexahydrobenzo[5,6]cyclohepta[1,2,3-ij]isoquinoline derivatives (5a--e and 6a--e) were synthesized as conformationally rigid analogues of 1-benzyltetrahydroisoquinoline and evaluated for their affinity at D(1) and D(2) dopamine receptors. All compounds showed lower D(1) and D(2) affinities than dopamine. The 5-hydroxy-1-methyl-2,3,12,12a-hexahydrobenzo[5,6]cyclohepta[1,2,3-ij]isoquinoline 5a and the 5,6-dihydroxy analogue 6a showed D(2) agonist activity. This was proved by their effects on prolactin release from primary cultures of rat anterior pituitary cells. Molecular modeling studies showed that the geometric parameters (namely the distances from meta and para hydroxyl oxygens to the nitrogen and the height of nitrogen from the hydroxylated phenyl ring plane) of the dopaminergic pharmacophore embedded in our compounds have lower values in comparison with those observed in D(1) and D(2) selective ligands.     

Synthesis of some new [1,2,4]triazolo[3,4-b][1,3,4]thiadiazines and [1,2,4]triazolo[3,4-b][1,3,4] thiadiazoles starting from 5-nitro-2-furoic acid and evaluation of their antimicrobial activity

New series of fused 1,2,4-triazoles such as, 6-(aryl)-3-(5-nitrofuran-2-yl)-5,6-dihydro-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles 4-8, 6-(alkyl/aryl amino)-3-(5-nitrofuran-2-yl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles 9-13 and 6-(4-substituted phenyl)-3-(5-nitrofuran-2-yl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines 14-18 have been synthesized via the reaction of 4-amino-5-(5-nitrofuran-2-yl)-4H-1,2,4-triazole-3-thiol 3 with various reagents such as hetero aromatic aldehydes, alkyl/aryl isothiocyanates and 4-substituted phenacyl bromides, respectively. The structures of the newly synthesized compounds have been confirmed on the basis of elemental analysis and spectral studies. The newly synthesized triazolo derivatives have been investigated for their in vitro antibacterial activity. Most of the tested compounds showed interesting antibacterial activity against Staphylococcus aureus. Furthermore, the most potent antibacterial compounds 11-13 were evaluated for their in vitro cytotoxic activity against human cancer cell lines. It was found that compounds 11 and 13 showed higher cytotoxicity against Hep-G2 cell line as compared to standard.     

Synthesis and antiproliferative activity of new cytotoxic tri- and tetraazabenzo[3,2-a]fluorene-5,6-dione derivatives

A new series of substituted tri-/tetraazabenzo[3,2-a]fluorene-5,6-diones and their corresponding oxime derivatives have been synthesized and spectroscopically characterized. The antiproliferative activities of all compounds were evaluated on at least three different cell lines.     

Complexation behaviour of chiral tetradentate polypyridines derived from α-pinene

A series of ligands, where two pinene-bipyridine moieties are either connected directly, or through a p-xylene bridge are investigated with respect to their complexation behaviour in solution. The bridged [5,6]-CHIRAGEN[p-xyl] ligands, which are substituted in 5′ or 6′ positions show self-assembly reactions, which lead to similar supramolecular species as the unsubstituted bis-pinene-bipyridines ligands studied before. The directly connected [5,6]-CHIRAGEN[0] derivatives, which are substituted at positions 5′ or 6′, form mononuclear complexes with helical chirality at the metal centre.     

Synthesis of new 5,6-dihydrobenzo[h]quinazoline 2,4-diamino substituted and antiplatelet/antiphlogistic activities evaluation

In pursuing our research on some 2,4-diamino-benzopyranopyrimidines and 2-amino-5,6-dihydrobenzo[h]quinazolines, previously reported as antiplatelet and analgesic/anti-inflammatory agents respectively, we designed and synthesized a new series of 5,6-dihydrobenzo[h]quinazoline 2,4-diamino substituted. The insertion of amino substituents at positions 2 and 4 of the benzoquinazoline scaffold resulted in compounds endowed with a potent ASA-like antiplatelet activity, combined with an anti-inflammatory activity comparable, in some cases, to that of indomethacin, used as a reference drug.     

Analogues of 1-(3,10-dibromo-8-chloro-6,11-dihydro-5H-benzo-[5,6]-cyclohepta [1,2-b]pyridin-11-yl)piperidine as inhibitors of farnesyl protein transferase.

The synthesis of several 4-pyridylacetyl N-oxide derivatives of 4-(3-bromo-6,11-dihydro-5H-benzo[5,6]-cyclohepta[1,2-b]-pyridin-11-yl)pi perazine/piperidine 3 is described. This study was aimed at identifying fomesyl protein transferase (FPT) inhibitors in these two series of tricycles containing different phenyl ring substituents. The in vitro activity profile of the initial group of compounds 7a-7g led to the synthesis of the 8-methyl-10-methoxy and 8-methyl-10-bromo analogues 7i, 13i, and 13j. The 11R(-) enantiomers of these compounds were found to exhibit potent in vitro FPT inhibition activity.     

The effect of the bay-region 12-methyl group on the stereoselective metabolism at the K-region of 7,12-dimethylbenz[a]anthracene by rat liver microsomes.

The enantiomers of a trans-5,6-dihydrodiol formed in the metabolism of 7,12-dimethylbenz[a]anthracene by rat liver microsomes (microsomal fractions) were resolved by chiral stationary-phase high-performance liquid chromatography. The major 7,12-dimethylbenz[a]anthracene trans-5,6-dihydrodiol enantiomer and its hydrogenation product 5,6,8,9,10,11-hexahydro-trans-5,6-diol were found to have 5S,6S absolute configurations by the exciton chirality c.d. method. The R,R/S,S enantiomer ratios of 7,12-dimethylbenz[a]anthracene trans-5,6-dihydrodiol formed in the metabolism of 7,12-dimethylbenz[a]anthracene by liver microsomes from untreated, 3-methylcholanthrene-treated and phenobarbital-treated male Sprague-Dawley rats were found to be 11:89, 6:94, and 5:95 respectively. These findings and those reported previously on the metabolic formations of trans-5,6-dihydrodiols from 7-methylbenz[a]anthracene and 12-methylbenz[a]anthracene suggest that the 12-methyl group in 7,12-dimethylbenz[a]anthracene plays an important role in determining the stereoselective metabolism at the K-region 5,6-double bond. Furthermore, the finding that formation of 5S,6S-dihydrodiol as the predominant enantiomer was not significantly affected by the isoenzymic composition of cytochrome P-450 present in microsomes prepared from the livers of the rats pretreated with the different inducing agents indicates that the stereoselectivity depends on the substrate metabolized rather than on the precise nature of the metabolizing-enzyme system.     

Synthesis and antibacterial evaluation of new,unsymmetrical triaryl bisamidine compounds

Herein we describe the synthesis and antibacterial evaluation of a new, unsymmetrical triaryl bisamidine compound series, [Am]-[indole]-[linker]-[HetAr/Ar]-[Am], in which [Am] is an amidine or amino group, [linker] is a benzene, thiophene or pyridine ring, and [HetAr/Ar] is a benzimidazole, imidazopyridine, benzofuran, benzothiophene, pyrimidine or benzene ring. When the [HetAr/Ar] unit is a 5,6-bicyclic heterocycle, it is oriented such that the 5-membered ring portion is connected to the [linker] unit and the 6-membered ring portion is connected to the [Am] unit. Among the 34 compounds in this series, compounds with benzofuran as the [HetAr/Ar] unit showed the highest potencies. Introduction of a fluorine atom or a methyl group to the triaryl core led to the more potent analogs. Bisamidines are more active toward bacteria while the monoamidines are more active toward mammalian cells (as indicated by low CC₅₀ values). Importantly, we identified compound P12a (MBX 1887) with a relatively narrow spectrum against bacteria and a very high CC₅₀ value. Compound P12a has been scaled up and is currently undergoing further evaluations for therapeutic applications.     

Epoxy derivatives of aromatic polycyclic hydrocarbons. The preparation and metabolism of epoxides related to 7,12-dimethylbenz[a]-anthracene

The syntheses of 7,12-dimethylbenz[a]anthracene 5,6-oxide, 7-acetoxymethyl-12-methylbenz[a]anthracene 5,6-oxide and a product that appears to be mainly 7-hydroxymethyl-12-methylbenz[a]anthracene 5,6-oxide are described. The compounds readily rearranged to phenols in the presence of mineral acid, and 7,12-dimethylbenz[a]anthracene 5,6-oxide and its 7-hydroxymethyl derivative reacted slowly with water to yield trans-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz[a] anthracene and trans-5,6-dihydro-5,6-dihydroxy-7-hydroxymethyl-12-methylbenz [a]anthracene respectively. Both epoxides were converted enzymically by rat liver microsomal fractions and homogenates into the related trans-dihydrodiols. The epoxides reacted chemically with GSH to form conjugates that were identical with the conjugates formed when the epoxides were incubated with rat liver homogenates. The GSH conjugates were more stable to acid than conjugates derived from other arene oxides. In the alkylation of 4-(p-nitrobenzyl)pyridine, 7,12-dimethyl-benz[a]anthracene 5,6-oxide was more active than the 5,6-oxides of 7-methylbenz[a]-anthracene and benz[a]anthracene.     

Analogs of 4-(3-bromo-8-methyl-10-methoxy-6,11-dihydro-5H-benzo[5,6]-cyclo hepta[1,2-b]pyridin-11-yl)-1-(4-pyridinylacetyl)piperidine N-oxide as inhibitors of farnesyl protein transferase.

A series of 3-substituted analogs 3 of 4-(3-bromo-8-methyl-10-methoxy-6,11-dihydro-5H-benzo[5,6]-cyclohepta[1,2 b]pyridin-11-yl)-1-(4-pyridinylacetyl)piperidine N-oxide 2 was prepared and evaluated as FPT inhibitors. The objective of this study was to identify other substituents at C3 in this series of FPT inhibitors that would have the FPT potency enhancement similar to that found for a C3 bromo substituent. The 3-methyl analog 17b was found to be tenfold less active than 2, and other C3 substituents having more steric bulk were found to cause a further reduction in activity.     

Synthesis and biological evaluation of new donepezil-like Thiaindanones as AChE inhibitors

Pharmacomodulations of previously reported thiaindanones related to donepezil were achieved with the aim to enhance their AChE inhibitory activity. Condensation of the cyclopentane carbonyl group into hydrazone or cyanolefine derivatives, as well as its hydrogenation and the subsequent substitution of the resulting hydroxyl group led to new 2-(4-benzylpiperazin-1-yl)-N-(1,3-dibromo-6-hydroxy-5,6-dihydro-4H-cyclopenta[c]thien-4-yl) acetamides. The in vitro evaluation of this new series, according to the method of Ellman, shows however that it conserved only partially the biological activity. The best compound remains the alcohol 11 (IC(50) = 0.40 microM, against 0.02 microM for donepezil).     

Gender-specific metabolism of benz[a]anthracene in hepatic microsomes from Long-Evans and Hooded Lister rats

The cytochrome P450 isoforms responsible for the regio-selective metabolism of benz[a]anthracene (BA) are poorly defined but as with other polycyclic aromatic hydrocarbons (PAHs) may include members of the CYP2C sub-family. Since the expression of some of these is regulated in a gender-specific manner and may be altered by age, rat strain or by phenobarbital treatment, the effects of these variables on metabolism of BA to diols was investigated. These studies used hepatic, microsomal membranes from immature and adult Long-Evans rats and adult Hooded Lister rats. BA-diols were resolved by normal phase HPLC into three discrete peaks identified as benz[a]anthracene-5,6-diol (BA-5,6-diol), benz[a]anthracene-10, 11-diol (BA-10,11-diol) and a mixture of benz[a]anthracene-3,4- and -8,9-diols (BA-3,4-diol and BA-8,9-diol and termed Peak(3/8)). Significant gender-related differences were found in the rates of diol formation in adults of both the Long-Evans and Hooded Lister rat strains. Formation of BA-10,11-diol and to a lesser extent the components of Peak(3/8) were greater in the male compared to female animals by factors of at least 14 and two, respectively. An age-dependent effect is also observed in the Long-Evans rat since these differences are still apparent in prepubertal animals but to a lesser extent (gender ratio male:female BA-10,11-diol 9X; Peak(3/8) 1.4X). In contrast BA-5,6-diol was formed at similar rates by membranes from female and male rats whether mature (Long-Evans and Hooded Lister) or immature (Long-Evans). Phenobarbital treatment of the adult Long-Evans rats resulted in a moderate increase in the formation of each diol other than at the 10,11-position and the induction was not gender specific. The rate of formation of BA-10, 11-diol was decreased in phenobarbital-treated male rats suggesting modulation of a male specific isoform. Measurement of microsomal epoxide hydrolase revealed no gender or age differences and suggests that this enzyme is not rate limiting in BA-diol formation and thus is not responsible for the differences in BA-diol formation observed. The results suggest that CYP2C11 along with a male-specific isoenzyme not regulated by age are important in the formation of BA-10,11-diol and a component(s) of Peak(3/8) in males. CYPs 2B2 and/or 2C6 appear to be involved in formation of BA-5,6-diol in male and female. Identification of the CYPs involved in the regio-selective metabolism of BA may lead to an explanation of the lower carcinogenic potency of this PAH compared to dimethylbenz[a]anthracene and this study provides novel clues concerning the identities of the CYPs, which are important.     

Collisionally induced dissociation of epoxyeicosatrienoic acids and epoxyeicosatrienoic acid-phospholipid molecular species.

Four isomers of epoxyeicosatrienoic acid (EET) can be formed by cytochrome P-450 oxidation of arachidonic acid: 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid. The collision-induced dissociation of the [M-H]- anion at m/z 319 from each of these isomers, using negative-ion fast atom bombardment ionization and a triple quadrupole mass spectrometer, resulted in a series of common ions as well as ions characteristic of each isomer. The common ions were m/z 301 [M-H2O]- and 257 [M-(H2O + CO2)]-. Unique ions resulted from cleavages alpha to the epoxide moiety to form either conjugated carbanions or aldehydes. Mechanisms involving charge site transfer are suggested for the origin of these ions. A distonic ion series that may involve a charge-remote fragmentation mechanism was also observed. The epoxyeicosatrienoic acids were also incorporated into cellular phospholipids following incubation of the free acid with murine mast cells in culture. Negative fast atom bombardment mass spectrometry of purified glycerophosphoethanolamine-EET species and glycerophosphocholine-EET species yielded abundant [M-H]- and [M-CH3]- ions, respectively. The collision-induced dissociation of these specific high-mass ions revealed fragment ions characteristic of the epoxyeicosatrienoic acids incorporated (m/z 319, 301, and 257) and the same unique ions as those seen with each isomeric epoxyeicosatrienoic acid. With this direct method of analysis, phospholipids containing the four positional isomers of EET, including the highly labile (5,6-EET), could be identified as unique molecular species in mast cells incubated with EET.     

A facile synthesis and anti-avian influenza virus (H5N1) screening of some novel pyrazolopyrimidine nucleoside derivatives

Treatment of 5-amino-1-(9-methyl-5,6-dihydronaphtho[1',2':4,5]thieno[2,3-d]pyrimidin-11-yl)-1H-pyrazole-4-carbonitrile (1) with formic acid afforded pyrazolo[3,4-d]pyrimidin-4-one derivative 2. The sodium salt of the latter compound (generated in situ) was treated with some alkyl halides to afford the corresponding N-substituted compounds 3-7. The siloxy derivative 8 (generated also in situ from 2) was ribosylated and glycosylated to yield compounds 9 and 11, respectively. Deprotection of compounds 9 and 11 in methanolic ammonia produced the free nucleosides 10 and 12, respectively. Moreover, the prepared compounds were tested for antiviral activity against H5N1 virus [A/chicken/Egypt/1/2006] and some of them revealed moderate results compared with the other tested compounds.     

Cantharidin analogues: synthesis and evaluation of growth inhibition in a panel of selected tumour cell lines

Diels-Alder addition of furans (furan, furfuryl alcohol, and 3-bromofuran) to maelic anhydride yields three distinct 5,6-dehydronorcantharidins. Hydrogenation of (4,10-dioxatricyclo[5.2.1.0]decane-3,5-dione) (4a), in dry ethanol affords the monoester (7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic aid monoethyl ester) (6). Subsequent transesterification affords a series of monoesters (7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid monomethyl ester (7)), 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid monopropyl ester (8), (7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid monohexyl ester (9)) and differentially substituted diesters (7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid 2-ethyl ester 3-isopropyl ester) (10), and (7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid 2-ethyl ester 3-phenyl ester) (11). Analogues were firstly screened for their ability to inhibit protein phosphatases 1 (PP1) and 2A (PP2A) as the lead compounds cantharidin (1) and norcantharidin (2) are known PP1 and PP2A inhibitors. Only analogues 4a, 6-8 displayed good PP1 and PP2A inhibition (PP1 IC(50)'s=2.0, 2.96, 4.71, and 4.82 microM, respectively; PP2A IC(50)'s=0.2, 0.45, 0.41, and 0.47 microM, respectively). All analogues were also screened for their anti-cancer potential against a panel of tumour cell lines, HL60, L1210, SW480, WiDr, HT29, HCT116, A2780, ADDP, and 143B, producing GI(50) values ranging from 6 microM to >1000 microM. Analogues possessing good PP1 and/or PP2A inhibition also returned moderate to good anti-cancer activity. Analogues with substituents directly attached to the intact bicyclo[2.2.1]heptane skeleton were poor to moderate anti-cancer agents. This correlates well with their lack of PP1 or PP2A activity. Analogues capable of undergoing a facile ring opening of the anhydride or with a single carboxylate were good PP1 and PP2A inhibitors, largely correlating to the observed anti-cancer activity in all cases, except 11. Analogue 11, whist neither a PP1 nor a PP2A inhibitor shows anti-cancer activity comparable to 1 and 2. We believe that intracellular esterases generate the corresponding dicarboxylate, which is a potent PP1 and PP2A inhibitor, and that it is this species which is responsible for the observed anti-cancer activity.     

The metabolism of 7- and 12-methylbenz[a]-anthracene and their derivatives

1. 7- and 12-Methylbenz[a]anthracene were converted by rat-liver homogenates into the corresponding hydroxymethyl derivatives, products that are probably the 8,9-dihydro-8,9-dihydroxy and the 5,6-dihydro-5,6-dihydroxy derivatives, and a number of phenolic products. 2. Both hydrocarbons were converted into glutathione conjugates; that from 7-methylbenz[a]anthracene was also formed, together with 5,6-dihydro-5,6-dihydroxy- and 5-hydroxy-benz[a]anthracene, from 5,6-epoxy-5,6-dihydro-7-methylbenz[a]anthracene. 3. 7- and 12-Hydroxymethyl-benz[a]anthracene were converted into products that are probably 8,9-dihydro-8,9-dihydroxy derivatives, and into phenols. 4. The preparation of a number of derivatives of the hydrocarbons is described. 5. The oxidation of the hydrocarbons with lead tetra-acetate was investigated.     

One step synthesis of 6-oxo-cholestan-3β,5α-diol

Cholesterol metabolism has been recently linked to cancer, highlighting the importance of the characterization of new metabolic pathways in the sterol series. One of these pathways is centered on cholesterol-5,6-epoxides (5,6-ECs). 5,6-ECs can either generate dendrogenin A, a tumor suppressor present in healthy mammalian tissues, or the carcinogenic cholestane-3β,5α,6β-triol (CT) and its putative metabolite 6-oxo-cholestan-3β,5α-diol (OCDO) in tumor cells. We are currently investigating the identification of the enzyme involved in OCDO biosynthesis, which would be highly facilitated by the use of commercially unavailable [¹⁴C]-cholestane-3β,5α,6β-triol and [¹⁴C]-6-oxo-cholestan-3β,5α-diol. In the present study we report the one-step synthesis of [¹⁴C]-cholestane-3β,5α,6β-triol and [¹⁴C]-6-oxo-cholestan-3β,5α-diol by oxidation of [¹⁴C]-cholesterol with iodide metaperiodate (HIO₄).