Pyrrolo[1',2':1,2]imidazo[4,5-b]pyridines, pyrrolo- [2',1':2,3]imidazo[4,5-c]pyridines and pyrrolo[2,1-f]purines as potential benzodiazepine ligands

The synthesis of some 7,8,8a,9-tetrahydro-6H-pyrrolo[1',2':1,2]imidazo[4,5-b]pyridin-6-ones, 5,5a,6,7-tetrahydro-8H-pyrrolo[2',1':2,3]imidazo[4,5-c]pyridin-8-ones and 7,8,8a,9-tetrahydro-6H-pyrrolo[2,1-f]purin-6-ones is reported. The structure of the obtained compounds has been assigned by means of 1H-NMR spectra assisted by NOESY measurements. In addition, the ability to displace [3H]-flunitrazepam binding from rat brain membranes is determined. Only the pyrrolopurine derivative 5d binds to the benzodiazepine receptor (BZR) with appreciable potency.     

Studies on quinazolines and 1,2,4-benzothiadiazine 1,1-dioxides. 8.1, 2 synthesis and pharmacological evaluation of tricyclic fused quinazolines and 1,2,4-benzothiadiazine 1,1-dioxides as potential alpha1-adrenoceptor antagonists

A series of 2-substituted methyl 2,3-dihydroimidazo[1, 2-c]quinazolin-5(6H)-ones (4), 3-substituted methyl 2, 3-dihydroimidazo[1,2-c]quinazolin-5(6H)-ones (5), 3-substituted methyl 2,3-dihydro-5H-thiazolo[2,3-b]quinazolin-5-ones (15a,b), 3-substituted methyl 2,3-dihydroimidazo[2,1-b]quinazolin-5(1H)-ones (16a,b), 3-substituted methyl 2,3-dihydro-1H-imidazo[1,2-b][1,2, 4]benzothiadiazine 5,5-dioxides (33a,b), 2-substituted methyl imidazo[1,2-c]quinazolin-5(6H)-ones (42-45a,b), 3-substituted methyl imidazo[1,2-c]quinazolin-5(6H)-ones (50-53a,b), 3-substituted methyl 5H-thiazolo[2,3-b]quinazolin-5-ones (55-56a,b), and 3-substituted methyl 5-(methylthio)-2,3-dihydroimidazo[1,2-c]quinazoline (57) were synthesized as compound 1conformational rigid congeners for pharmacological evaluation as potential alpha1-adrenoceptor antagonists. Compounds 4, 5, 33a,b, 44a,b, 45a,b, 52a,b, 53a,b, and 57 were found to possess high affinity for the alpha1-adrenoceptor. Compounds 5 and 57 were the most highly selective and potent alpha1 antagonists with Ki = 0.21 +/- 0.02 and 0.90 +/- 0.08 nM, respectively. The S-enantiomers of these two compounds (Ki = 0.13 +/- 0.01 nM for (S)-(-)-5; Ki = 1.0 +/- 0.2 nM for (S)-(+)-57) were 144-200-fold more potent than the R-enantiomers (Ki = 26 +/- 8 nM for (R)-(+)-5; Ki = 144 +/- 23 nM for (R)-(-)-57). Compound 4 showed 8-fold higher affinity to alpha1A-AR better than alpha1B-AR. These compounds possessed weak to no activity against the 5-HT1A receptor.     

Antitumor agents. 178. Synthesis and biological evaluation of substituted 2-aryl-1,8-naphthyridin-4(1H)-ones as antitumor agents that inhibit tubulin polymerization

As part of our continuing search for potential anticancer drug candidates in the 2-aryl-1,8-naphthyridin-4(1H)-one series, we have synthesized two series of 3'-substituted 2-phenyl-1,8-naphthyridin-4(1H)-ones and 2-naphthyl-1,8-naphthyridin-4(1H)-ones. All compounds showed significant cytotoxic effects (log GI50 < -4.0; log molar drug concentration required to cause 50% growth inhibition) against a variety of human tumor cell lines of the National Cancer Institute's in vitro screen, including cells derived from solid tumors such as non-small cell lung, colon, central nervous system, melanoma, ovarian, prostate, and breast cancers. All 3'-substituted compounds demonstrated strong cytotoxic effects in almost all tumor cell lines. Introduction of an aromatic ring at the 2'- and 3'-positions also generated compounds with potent antitumor activity. Incorporation of an aromatic ring at the 3'- and 4'-positions produced compounds with reduced activity. Interestingly, introduction of a halogen at the 3'-position yielded compounds with different selectivity for the tumor cell lines tested. All 3'-halogenated compounds (29-36) and compounds 38 and 42-44 were potent inhibitors of tubulin polymerization with activities nearly comparable to those of the potent antimitotic natural products colchicine, podophyllotoxin, and combretastatin A-4. Active agents also inhibited the binding of [3H]colchicine to tubulin.     

Synthesis and pharmacological activity of some N,N-disubstituted 1-amino-2-phenyl-3H,12H-naphtho[1,2-b]pyrano[2,3-d]pyran-3-ones

The synthesis of some N,N-disubstituted 1-amino-2-phenyl-3H,12H-naphtho[1,2-b]pyrano[2,3-d]pyran-3-ones 4, by reaction of phenylchloroketene with a series of N,N-disubstituted 3-aminomethylene-2,3-dihydro-4H-naphtho[1,2-b]pyran-4-ones, followed by dehydrochlorination in situ of the primary adducts with DBN, is described. Some compounds 4 showed antiarrhythmic and analgesic activities.     

GYKI 52466 and related 2,3-benzodiazepines as anticonvulsant agents in DBA/2 mice

The behavioural and anticonvulsant effects of several 1-aryl-3,5-dihydro-4H-2,3-benzodiazepin-4-ones (2,3-BZs) and of 11b-aryl-7,11-dihydro-3-phenyl[1,2,4]oxadiazolo[5,4-a][2,3]benz odiazepin-6-ones (2,3-OBZs) were studied after intraperitoneal (i.p.) administration in DBA/2 mice, a strain genetically susceptible to sound-induced seizures. The seizures were evoked by means of auditory stimulation (109 dB, 12-16 kHz) in animals placed singly under a hemispheric Perspex dome. The 2,3-benzodiazepines studied after 30 min pretreatment were generally less potent than the related derivative 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466) except 3,5-dihydro-7,8-dimethoxy-1-phenyl-4H-2,3-benzodiazepin-4-one (2,3-BZ-2) and 2,3-BZ-2M (3-methyl derivative of 2,3-BZ-2) which showed comparable activity. Thirty minutes after i.p. administration of 2,3-benzodiazepines, the rank order of potency for anticonvulsant activity against clonus was 2,3-BZ-2 > GYKI 52466 > 2,3-BZ-2M > 2,3-BZ-1 > 2,3-BZ-3, > 2,3-OBZ-1, > 2,3-OBZ-2 2,3-OBZ-3. The intracerebroventricular (i.c.v.) injection of aniracetam on it own (12.5 - 100 nmol/mouse) had no convulsant activity, but it reversed the anticonvulsant effects of some 2,3-benzodiazepines. In particular, the pharmacological actions of GYKI 52466, 2,3-BZ-2 and 2,3-BZ-2M, which proved to be the most potent 2,3-benzodiazepine derivatives as anticonvulsants, were significantly reduced by an i.c.v. pretreatment with aniracetam (50 nmol/mouse). Concomitant treatment with aniracetam (50 nmol/mouse) shifted to the right the dose-response curves and significantly increased the ED50 values for GYKI 52466, 2,3-BZ-2 and 2,3-BZ-2M. After 30 min pretreatment 2,3-BZ-2 showed a similar potency to GYKI 52466 in antagonizing seizures induced by i.c.v. administration of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), thus suggesting a clear involvement of AMPA receptors in the anticonvulsant activity of these compounds. In addition, 2,3-BZ-2 and 2,3-BZ-2M showed anticonvulsant properties longer lasting than GYKI 52466.     

Stability of alpha-tocopherol: pre-analytical conditions in its determination in blood samples

Some amides, 1-substituted methylenediamines and 2-substituted thiazolidin-4-ones, all containing the aminopyrazinyl moiety, were synthesized and tested in the experimental models of acute and chronic inflammations and as potential analgesic agents. The first series of compounds are inactive, whereas the N,N'-di-(2-pyrazinyl)-methylene-diamines and the 3-(2'-pyrazinyl)-thiazolidinones 6, 7, 11, 12 and even more 16 and 17 were found to be endowed with antiinflammatory and analgesic properties and low acute toxicity, the two latter being the most interesting.     

A case of primary splenic large cell lymphoma with a t(9;14)(p13;q32)

Appropriately substituted 2,3-dihydro-7H-thiazolo[3,2-a]pyrimidin-7-ones 9-12 and 18 were considered as annulated analogues of HEPT (1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)-thymine), and some of these compounds were also found active against HIV-1, the most active one being 2,3-dihydro-5-[(3,5-dimethylphenyl)methyl]-3-ethoxy-6-ethyl-7H- thiazolo[3,2-a]pyrimidin-7-one (10b). S-Alkylation of 5-alkyl-6-(arylmethyl)-2-thiouracils 1-4 was performed with 2-bromoacetaldehyde acetals to furnish the S-[bis(alkoxy)ethyl] derivatives 5-8 and with allyl bromide to furnish S-allyl derivatives 17. The target compounds 9-12 were obtained by an N1 regioselective intramolecular cyclization reaction of silylated 5-8 using trimethylsilyl trifluoromethanesulfonate (TMS triflate) as the catalyst. Treatment of the S-allyl derivatives 17 with bromine in dry methylene chloride afforded the 3-(bromomethyl) derivatives 18.     

High-performance liquid chromatographic determination of the enantiomers of the benzoquinolinone LY191704, a human type 1 5 alpha-reductase inhibitor, in plasma

A stereoselective reversed-phase liquid chromatographic method for the determination of compounds LY300502 and LY300503 (enantiomers of LY191704) in rat and dog plasma was developed. The assay involved extraction of the compounds using a strong cation-exchange solid-phase extraction column, from which the compounds are eluted with 1% of 1 M HCI in methanol. The enantiomers were separated on a Daicel Chiralcel OD-R column. The mobile phase consisted of water-acetonitrile-methanol (50:40:10, v/v) at a flow-rate of 0.3 ml/min. UV detection was achieved at 220 nm. The disposition of the enantiomers of LY191704 in rats and dogs was found to be stereoselective and species specific.     

A comparison of the bronchodilator activity of (+/-) 11-deoxy prostaglandin E1 with its 15-methyl analogue, (doxaprost)

The bronchodilator activity of (+/-) 11-deoxy prostaglandin E1 was compared to the activity of its (+/-) 15-methyl analogue, (doxaprost). Both compounds inhibited histamine-induced bronchoconstriction in the anesthetized guinea pig where changes in tracheal pressure were measured. Doxaprost was 73 and 32 times more potent that (+/-) 11-deoxy PGE1 by the aerosol and i.v. routes, respectively. Doxaprost also demonstrated a longer duration of effect. Both compounds decreased pulmonary resistance in the 5HT tonal cat. There was no difference in the potency of the two compounds. However, doxaprost had a longer duration of effect. Both compounds caused a fall in mean arterial blood pressure after i.v. administration in the guinea pig but not after aerosol administration in the guinea pig and cat. Both compounds caused relaxation of the isolated guinea pig tracheal strip when tone was induced with carbachol. There was no difference in the potency of the two compounds. The increased activity in vivo but not in vitro of the 15-methyl analogue doxaprost is consistent with a lack of enzyme inactivation.     

Comparison of skin-prick test and specific serum IgE determination for the diagnosis of latex allergy

The synthesis and anticonvulsant activity of novel 7,8-methylenedioxy-4H-2,3-benzodiazepin-4-ones 3a-e, structurally-related to GYKI 52466 1, a well-known noncompetitive AMPA-receptor antagonist, are reported. The new compounds possess marked anticonvulsant properties and, in analogy to 1, antagonize seizures induced by AMPA. In addition, when compared to the model compound 1, compounds 3 show a longer-lasting anticonvulsant activity and a lower toxicity.     

Benign intracranial cysts and certain congenital neoplasms

Carzelesin (U80,244, I) is a cyclopropylpyrroloindole prodrug analog. The compound exerts its cytotoxic activity after conversion, via U-76,073 (II) to U-76,074 (III) by binding to the DNA minor groove in a sequence selective fashion. In pre-clinical investigations the drug displayed a broad spectrum activity against human xenografts in mice. To enable pharmacokinetic monitoring during the phase I clinical trials, we have developed a selective and sensitive assay for the parent compound and its two metabolites. Sample pre-treatment has been automated by using the ASPEC system and involves solid-phase extraction of the diluted plasma sample (1:3 in 20% v/v acetic acid) on an SPE-C18 precolumn followed by two consecutive washings with water and acetonitrile. The compounds are eluted with 600 microliters of dimethylacetamide and 500 microliters is injected on a Spherisorb-CN column. The sample is chromatographed using a linear gradient from 24% to 60% (v/v) acetonitrile in 20 mM phosphate buffer (pH 6.5). The eluate fraction containing the three compounds is heart-cutted in a 2-ml sample loop, switched onto a Spherisorb-ODS column and separation is accomplished using a mobile phase of acetonitrile-20 mM phosphate buffer pH 6.5 (64:36, v/v). UV detection is used with absorbance monitored at 360 nm. This highly selective method offers a lower limit of detection of less than 1 ng/ml for each of the compounds using 1000 microliters of plasma and enables the quantification of the analytes with an acceptable precision and accuracy over a range of 1 to 50 ng/ml. The assay has been implemented in a phase I clinical trial.     

Loss of heterozygosity on chromosome 11p15 during histological progression in microdissected ductal carcinoma of the breast

Two enone reductases catalyzing irreversibly the enantioselective reduction of alpha,beta-unsaturated carbonyls have been purified 165-fold and 257-fold, respectively, from the cytosolic fraction of Saccharomyces cerevisiae by means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, blue Sepharose CL-6B chromatography and Superdex 200 preparation-grade filtration. One enzyme (E I) was NADPH-dependent, showed a molecular mass of 75 kDa and decomposed under denaturing electrophoretic conditions into two subunits of 34 kDa and 37 kDa. The other enzyme (E II) was NADH linked and the molecular mass estimated by means of Superdex 200 preparation-grade filtration, was 130 kDa. The enzyme decomposed into subunits of 56 kDa and 64 kDa on SDS/PAGE. Both enzymes were most active at pH 4.8 and accepted 1-octen-3-one, 1-hexen-3-one, 3-alken-2-ones, 4-alken-3-ones, 2-cyclohexen-1-ones, 2-alkenals, 2,4-alkadienals, 2-phenyl-2-alkenals, and 2-alkyl-2-alkenals as substrates. Both enzymes showed their highest activities with 1-hexen-3-one and 1-octen-3-one, respectively. The Km values for 1-octen-3-one were estimated as 0.54 mM (E I) and 0.20 mM (E II), respectively. Both enzymes catalyzed the enantioselective reduction of cis- and trans-2-phenyl-2-butenal into (R)-2-phenylbutanal.     

Synthesis and inhibitory action on HMG-CoA synthase of racemic and optically active oxetan-2-ones (beta-lactones)

A homologous series of both C3-unsubstituted and C3-methyl substituted oxetan-2-ones (beta-lactones) was investigated as potential inhibitors of yeast 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase. Several reported methods for racemic beta-lactone synthesis were studied for preparation of the target series. In addition, a novel aluminum-based Lewis acid obtained by combination of Et2AlCl with (1R,2R)-2-[(diphenyl)hydroxymethyl] cyclohexan-1-ol was studied for the asymmetric [2 + 2] cycloaddition of aldehydes and trimethylsilylketene. This Lewis acid exhibited good reactivity but variable enantioselectivity (22-85% ee). In in vitro assays using both native and recombinant HMG-CoA synthase from Saccharomyces cerevisiae, oxetan-2-ones mono-substituted at C4 with linear alkyl chains gave IC50s that decreased monotonically with chain length up to 10 carbons and then rose rapidly for longer chains. The trans isomers of 3-methyl-4-alkyl-oxetan-2-ones showed a similar trend but had 1.3- to 5.6-fold lower IC50s. The results imply a substantial hydrophobic pocket in this enzyme that interacts with both C-3 and C-4 substituents of oxetan-2-one inhibitors.     

6-aminoalkyloxazolo[4,5-b]pyridin-2(3H)-ones: synthesis and evaluation of antinoceptive activity

Series of 6-aminoalkyloxazolo[4,5-b]pyridin-2(3H)-ones incorporating structural modifications both in the alkyl chain and basic amino moiety were tested for their analgesic efficacy and safety in mice and rats. Two of the synthesised compounds, 4a (3-methyl-6-[(4-phenyl-1-piperazinyl)methyl]oxazolo[4,5-b]pyridin-2(3H)-one) and 12a (3-methyl-6?1-[2-(4-phenyl-1-piperazinyl)ethan-1-ol]?oxazolo[4,5-b]pyridin- 2(3H)-one) were found to be more potent than aspirin with ED50 values of 26 (16.1-42.4) and 15.5 (11.4-21.2) mg/kg po (mouse, phenylquinone writhing test) respectively and 6 (3.1-9.8) and 5.5 (3.5-8.8) mg/kg po (rat, acetic acid writhing test). Compounds 4a and 12a proved to be potent nonopioid nonantiinflammatory analgesics but unfortunately have sedative properties at relatively low doses (respectively 64 and 16 mg/kg po, mice).     

Enhanced mitosis in cultured protoplasts: beneficial effects of oxygenated perfluorocarbon and haemoglobin

Cell suspension-derived protoplasts of Petunia hybrida cv. Comanche were cultured for up to 10 d in (1) aqueous medium, (2) aqueous medium overlaying an oxygenated (10 mbar, 15 min) perfluorochemical liquid (perfluorodecalin; PFC), (3) aqueous medium containing 1:50 (v:v) of the commercial haemoglobin (Hb) solution (Erythrogen), (4) aqueous medium supplemented with 1:50 (v:v) of Erythrogen overlaying oxygenated PFC. Treatments 2, 3, and 4 enhanced mean mitotic division by 111%, 80% and 139% respectively, compared to controls. Both fresh-Hb and stored-Hb significantly (P < 0.05) enhanced mitotic division, although this was 45% greater with fresh-Hb compared to stored-Hb. Both oxygenated PFC and Erythrogen provide approaches for enhancing cellular oxygen supply to eukaryotic cells. Commercially, the recoverability and recycleability of PFCs make these compounds a more attractive option in comparison to Erythrogen, despite a high initial investment cost.     

Biochemical characterization of human osteoclast integrins. Osteoclasts express alpha v beta 3, alpha 2 beta 1, and alpha v beta 1 integrins

The study of osteoclast integrins has been previously hampered by the lack of a source of large numbers of purified osteoclasts. Osteoclastoma, a human giant cell tumor of bone, supplied a rich source of osteoclasts within a tissue containing many diverse cell types. Osteoclastoma integrin immunostaining confirmed the presence of the integrin alpha v beta 3 complex and the alpha 2 and beta 1 integrin subunits on osteoclasts. However, weak integrin expression, for example with alpha v beta 5, was difficult to interpret. Purification with magnetic beads coated with vitronectin receptor monoclonal antibody (13C2) enabled osteoclast membranes to be isolated with high purity and yield (57%) from osteoclastoma tissue. Positively (osteoclast-enriched) selected membranes were biochemically assessed for integrin expression by immunoprecipitation and visualization by non-radioactive enhanced chemiluminescence. alpha 1, alpha 4, alpha 6, alpha 8, alpha M, alpha X, gpIIb, beta 4, beta 6, and beta 8 integrin chains were undetectable at a sensitivity of 1 ng. alpha 3, alpha 5, alpha L, beta 2, and alpha v beta 5 were found in the negatively selected osteoclastoma tissue but not in the positively purified osteoclast membranes. The presence of alpha v beta 1 and alpha 2 beta 1 dimers was demonstrated biochemically on the immunoisolated osteoclast membranes. Osteoclast alpha v beta 3 isolation by Arg-Gly-Asp (RGD) affinity chromatography for NH2-terminal amino acid sequencing confirmed that the osteoclast vitronectin receptor was identical to that previously characterized on other cell types. In situ hybridization using human alpha v riboprobes in osteoclasts from human and rodent bone further demonstrated the high level and specificity of expression of alpha v vitronectin receptor in osteoclasts.     

A biomechanical comparison of intramedullary nail and crossed lag screw fixation for tibiotalocalcaneal arthrodesis

A series of Pyrazolo[1,5-a]pyrido[3,4-e]pyrimidin-6-ones (4a-p) was prepared by a simple synthetic procedure based on the reaction of hydroxylamine or methoxyamine with 2,3-substituted ethyl 7-dimethylaminovinyl pyrazolo[1,5-a]pyrimidin-6-carboxylates (3a-p). The antimicrobial activity of the obtained compounds was evaluated on a series of standard strains of Gram positive, Gram negative bacteria and fungi. None of the tested compounds showed significant activity.     

Biological studies on 1,2-benzisothiazole derivatives VI Antimicrobial activity of 1,2-benzisothiazole and 1,2-benzisothiazolin-3-one derivatives and of some corresponding 1,2-benzisoxazoles

Numerous 1,2-benzisothiazole and 1,2-benzisothiazolin-3-one derivatives, variously substituted in the different positions of the molecule, were tested for their in vitro antimicrobial activity. Some corresponding 1,2-benzisoxazoles and 1,2-benzisoxazolin-3-ones were also considered. Several compounds possess a potent and broad antibacterial and antifungal activity, particularly against Gram positive microorganisms, yeasts and dermatophytes. 1,2-Benzisothiazolin-3-ones were found to be the most active substances. On the contrary, the benzisoxazoles and the benzisoxazolin-3-ones considered were devoid of activity. The results obtained are discussed on the basis of structure-activity relationships.     

Enantioselectivity of some 1-(benzofuran-2-yl)-1-(1-H-imidazol-1-yl) alkanes as inhibitors of P450Arom

The low stereospecificity of the enantiomers of 1-[(benzofuran-2-yl)-4-chlorophenylmethyl]imidazole (6, R=H, R'=4'-Cl) and the corresponding 4-fluoro compound as inhibitors of aromatase (P450Arom) has been explored using 1-(5,7-dichlorobenzofuran-2-yl)-1-(1H-imidaz-1-yl)ethane (7, R1=R2=Cl, R=CH3), -propane (7, R1=R2=Cl, R=C2H5), and the corresponding 5,7-dibromo compounds resolved as their dibenzoyl-D (or -L) tartrates. Low enantioselectivity ratios of 4.8 (5,7-diCl) and 12.6 (5,7-diBr) were shown for the ethanes. The values for the corresponding propanes were 8.3 and 5.2, respectively, and for these compounds the stereoselectivity was reversed.