Effectiveness of vigabatrin against focally evoked pilocarpine-induced seizures and concomitant changes in extracellular hippocampal and cerebellar glutamate, gamma-aminobutyric acid and dopamine levels, a microdialysis-electrocorticography study in freely moving rats

Limbic seizures were evoked in freely moving rats by intrahippocampal administration of the muscarinic agonist pilocarpine via the microdialysis probe (10 mM for 40 min at 2 microl/min). This study monitored changes in extracellular hippocampal gamma-aminobutyric acid (GABA), glutamate and dopamine levels after systemic (30 mg/kg/day) or local (intrahippocampal or intranigral, 5 mM or 600 microM for 180 min at 2 microl/min) vigabatrin administration, and evaluated the effectiveness of this antiepileptic drug against pilocarpine-induced seizure activity. Extracellular GABA and glutamate overflow in the ipsilateral cerebellum was studied simultaneously. Microdialysis was used as an in vivo sampling technique and as a drug-delivery tool. Electrophysiological evidence for the presence or absence of seizures was recorded with electrocorticography. The observed alterations in extracellular hippocampal amino acid levels support the hypothesis that muscarinic receptor stimulation by the intrahippocampal administration of 10 mM pilocarpine is responsible for the seizure onset, and that the amino acids maintain the sustained seizure activity. The focally evoked pilocarpine-induced seizures were completely prevented by intraperitoneal vigabatrin premedication for 7 days or by a single intraperitoneal injection. Effective protection was reflected in a lack of sustained elevations of hippocampal glutamate levels. Rats receiving vigabatrin intrahippocampally or intranigrally still developed seizures, although there appeared to be a partial protective effect. During the intrahippocampal perfusion with 5 mM vigabatrin, extracellular hippocampal GABA levels increased, whereas the extracellular glutamate and dopamine overflow decreased. The lack of a complete neuroprotection after local vigabatrin treatment is discussed.     

2,3'-disubstituted-2-(2'-carboxycyclopropyl)glycines as potent and selective antagonists of metabotropic glutamate receptors

2-(9-Xanthylmethyl)-2-(2'-carboxycyclopropyl) glycine 6e is a novel metabotropic glutamate receptor antagonist. A series of alpha, C-3' disubstituted (carboxycyclopropyl)glycines 6f-n were prepared. Antagonist activity was observed for all these compounds at group 2 and group 3 mGluRs. Although they were slightly less active on group 2 mGluRs than non C-3' substituted 6e, the compounds 6f-n were more selective with lesser or no activity on group 1 mGluR subtypes (IC50 values greater than 100 microns).     

Evaluation of 2-benzylidenecyclohexanones and 2,6-bis(benzylidene)cyclohexanones for antitumor and cytotoxic activity and as inhibitors of mitochondrial function in yeast: metabolism studies of (E)-2-benzylidenecyclohexanone

Some 2-benzylidenecyclohexanones, 2,6-bis(benzylidene)cyclohexanones, and related compounds were evaluated for antitumor and cytotoxic activities; (E)-2-benzylidenecyclohexanone (Ia) was shown to have significant cytotoxic properties and a potent inhibitory effect on yeast mitochondria. After intraperitoneal injection of Ia, unchanged drug and a metabolite, tentatively identified as 2-(p-hydroxybenzyl)cyclohexanol, were found in the urine. No metabolites were found in the feces. Oral administration of Ia afforded three unidentified metabolites in the urine and three unidentified metabolites in the feces.     

7-(2-Aminomethyl-1-azetidinyl)-4-oxoquinoline-3-carboxylic acids as potent antibacterial agents: design, synthesis, and antibacterial activity

2-Aminomethyl-1-azetidinyl, -1-pyrrolidinyl, and -1-piperidinyl groups were designed as novel C-7 substituents for potential antibacterial quinolone agents. Of the three substituents, the 2-aminomethyl-1-azetidinyl group (compound 12a) was found to be the most favorable for enhancing the activity of the 6,8-difluoroquinoline molecule 12. Therefore the 2-aminomethyl-1-azetidinyl group was introduced into a variety of quinolines (giving 24-26a, and 28a) and naphthyridines (giving 31a and 32a). Through optical resolution of 1-benzylazetidine-2-carboxamide (19) and chiral synthesis of its R-isomer, both enantiomers of 2-aminomethyl-1-azetidinyl quinolines 12a and 24-26a were also prepared. The most active of all the compounds was 5-amino-6,8-difluoroquinoline (R)-26a. The activity of (R)-26a was more potent than those of the corresponding 1-piperazinyl derivative (3) and sparfloxacin (1), and was comparable to those of the corresponding 3-amino-1-pyrrolidinyl (4), 3-aminomethyl-1-pyrrolidinyl (5), and 3-amino-1-azetidinyl (6) derivatives.     

Heteroaromatic analogs of 1-[2-(diphenylmethoxy)ethyl]- and 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazines (GBR 12935 and GBR 12909) as high-affinity dopamine reuptake inhibitors

A new series of heteroaromatic GBR 12935 [1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)-piperazine] (I) and GBR 12909 [1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine] (2) analogs was synthesized and evaluated as dopamine transporter (DAT) ligands. Analogs 5-16, in which the benzene ring in the phenylpropyl side chain of the GBR molecule had been replaced with a thiophene, furan, or pyridine ring, exhibited high affinity and selectivity for the DAT vs serotonin transporter (SERT) and stimulated locomotor activity in rats in a manner similar to the parent compound 2. In cocaine and food self-administration studies in rhesus monkeys, both thiophene-containing (6 and 8) and pyridine-containing (14 and 16) derivatives displayed potency comparable to 2 in decreasing the cocaine-maintained responding at the doses tested (0.8, 1.7, and 3 mg/kg). However, these compounds did not produce the degree of separation between food- and cocaine-maintained responding that was seen with 2. Among the bicyclic fused-ring congeners 17-38, the indole-containing analog of 2, 22, showed the greatest affinity for binding to the DAT, with IC50 = 0.7 nM, whereas the corresponding indole-containing derivative of 1, 21, displayed the highest selectivity (over 600-fold) at this site vs the SERT site.     

2-Amino-3-substituted-6-[(E)-1-phenyl-2-(N-methylcarbamoyl)vinyl]imid azo[1,2-a]pyridines as a novel class of inhibitors of human rhinovirus: stereospecific synthesis and antiviral activity

A series of 2-amino-3-substituted-6-[(E)-1-phenyl-2-(N-methylcarbamoyl)vinyl]+ ++imid azo[1,2-a]pyridines 1a-i, structurally related to Enviroxime and its analogous benzimidazoles, was designed and prepared for testing as antirhinovirus agents. The imidazo ring in this class of compounds was constructed starting from the aminopyridine after tosylation and subsequent treatment with the appropriate acetamides. The key steps in the synthesis include the development and use of a new Horner-Emmons reagent for the direct incorporation of methyl vinylcarboxamide. The reaction was stereospecific in the substrates 5a-f leading exclusively to the desired E-isomer and avoiding the use of reverse-phase preparative HPLC for the separation of both possible isomers before antiviral activity evaluation. The isopropylsulfonyl group, known as the best substituent at the 1-position in the benzimidazole SAR in terms of activity, was introduced in this new series of imidazo[1,2-a]pyridines via halogen-metal exchange and subsequent treatment with isopropyl isopropanethiolsulfonate. Compounds 1a-i were evaluated in plaque reduction assay and in a cytopathic effect assay. Compounds 1b-d,h exhibited a strong antirhinovirus activity, and no apparent cellular toxicity was visible. The substitution at the 3-position was required for activity. Surprisingly the isopropylsulfonyl in this family of compounds did not enhance the activity as in the case of benzimidazoles. Instead, compound 1i was 4 times less active than its phenyl and sulfide partners. The chemistry as well as the biological evaluation are discussed.     

Syntheses and conformational analyses of glutamate analogs: 2-(2-carboxy-3-substituted-cyclopropyl)glycines as useful probes for excitatory amino acid receptors

An hypothesis that each subtype of glutamate receptors requires a specific conformation of L-glutamate for its selective activation was examined using the conformationally constrained analogs of L-glutamate, L-2-(2-carboxycyclopropyl)glycines (CCGs), and L-2-[2-carboxy-3-(methoxymethyl)cyclopropyl)glycines (MCGs). All MCG isomers were newly synthesized in a stereoselective manner via the common synthetic intermediate 5a starting with the oxazolidine aldehyde 1. The synthesis of the four MCG isomers was characterized by a stereoselective inversion of alpha-cyclopropyl acyl anion (e.g., from 10 to 11). The spectroscopic studies, in particular, pH vs J correlation experiments of CCGs and MCGs using 1H NMR and their molecular mechanics calculations, revealed that these analogs possessed an antiperiplanar conformation regarding the H-C2-C1'-H bond as a majority among the other possible rotamers in aqueous solution. The fact that each CCG and MCG exhibited potent and selective activities to the distinct types of glutamate receptors allowed us to extract an active conformation of L-glutamate. Thus, the conformational requirement of metabotropic glutamate receptors was speculated to be the anti-anti conformation (aa-A) because the conformations of CCG-1 and cis and trans-MCG-I, selective agonists of the receptors, closely mimicked the rotamer A of L-glutamate. On the other hand, N-methyl-D-aspartate and kainate receptors, representative ionotropic glutamate receptors, would require glutamate g+g+ rotamer E which was deduced from the conformation-activity relationship studies of the selective agonists CCG-IV, cis-MCG-IV, and trans-MCG-IV and the related analogs.     

2-substituted (2SR)-2-amino-2-((1SR,2SR)-2-carboxycycloprop-1-yl)glycines as potent and selective antagonists of group II metabotropic glutamate receptors. 2. Effects of aromatic substitution, pharmacological characterization, and bioavailability

In this paper we describe the synthesis of a series of alpha-substituted analogues of the potent and selective group II metabotropic glutamate receptor (mGluR) agonist (1S,1'S,2'S)-carboxycyclopropylglycine (2, L-CCG 1). Incorporation of a substitutent on the amino acid carbon converted the agonist 2 into an antagonist. All of the compounds were prepared and tested as a series of four isomers, i.e., two racemic diastereomers. On the basis of the improvement in affinity realized for the alpha-phenylethyl analogue 3, in this paper we explored the effects of substitution on the aromatic ring as a strategy to increase the affinity to these compounds for group II mGluRs. Affinity for group II mGluRs was measured using [3H]glutamic acid (Glu) binding in rat forebrain membranes. Antagonist activity was confirmed for these compounds by measuring their ability to antagonize (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid-induced inhibition of forskolin stimulated cyclic-AMP in RGT cells transfected with human mGluR2 and mGluR3. Meta substitution on the aromatic ring of 3 with a variety of substituents, both electron donating (e.g., methyl, hydroxy, amino, methoxy, phenyl, phenoxy) and electron withdrawing (e.g., fluorine, chlorine, bromine, carboxy, trifluoromethyl) gave from 1.5- to 4.5-fold increases in affinity. Substitution with p-fluorine, as in 97 (IC50 = 0.022 +/- 0.002), was the exception. Here, a greater increase in affinity was realized than for either the ortho- or meta-substituted analogues; 97 was the most potent compound resulting from monosubstitution of the aromatic. At best, only modest increases in affinity were realized for certain compounds bearing either two chlorines or two fluorines, and two methoxy groups gave no improvement in affinity (all examined in a variety of substitution patterns). Three amino acids, 4, 5, and 104, were resolved into their four constituent isomers, and affinity and functional activity for group II mGluRs was found to reside solely in the S,S,S-isomers of each, consistent with 1. With an IC50 = 2.9 +/- 0.6 nM, the resolved xanthylmethyl compound 168 was the most potent compound from this SAR. Amino acid 168 demonstrated high plasma levels following intraperitoneal (i.p.) administration and readily penetrated into the brain. This compound, however, had only limited (approximately 5%) oral bioavailability. Systemic administration of 168 protected mice from limbic seizures produced by the mGluR agonist 3,5-dihydroxyphenylglycine, with an ED50 = 31 mg/kg (i.p., 60 min preinjection). Thus, 168 represents a valuable tool to study the role of group II mGluRs in disease.     

The synthesis and reactivity of 3 beta-(2-alkynylsulfonyl)- and 3 beta-(2-alkynylsulfonylmethyl) androst-5-en-17-ones as inhibitors of glucose-6-phosphate dehydrogenase

3 beta-(Hexadec-2-ynylsulfonyl)androst-5-en-17-one, 2c, was designed as an analog of dehydroepiandrosterone sulfatide 1c, a potent, natural inhibitor of glucose-6-phosphate dehydrogenase (G6PDH). Nucleophilic substitution of 1-bromo hexadec-2-yne 11 with 3 beta-mercaptoandrost-5-en-17-one followed by oxidation afforded 2c. The propargylic sulfone 2c may tautomerize to the electrophilic allenic sulfone 3a and thus function as a masked affinity label of the steroidal binding site of G6PDH. Since 2c demonstrated low potency as an inhibitor of G6PDH, a sulfonylmethyl analog 4b was also designed and synthesized. Synthesis of 4b began by methylenation of androst-5-en-3,17-dione 17-ketal 6 with the Tebbe reagent, to yield the 3-methyleneandrost-5-ene 7. Hydroboration, followed by oxidation, gave a mixture of 3 alpha- and 3 beta-hydroxymethyl isomers 8a and 8b, respectively. The 3 beta alcohol 8b was converted to the thiol 10. Alkylation of 10 with 1-bromo-2-hexadecyne 11, followed by selective oxidation, gave the desired acetylenic sulfone 4b. Insertion of the methylene in 4a and 4b significantly increased their G6PDH inhibitory properties over the initial compounds, 2b and 2c.     

Synthesis and cellular uptake of 2'-substituted analogues of (E)-5-(2-[125I]iodovinyl)-2'-deoxyuridine in tumor cells transduced with the herpes simplex type-1 thymidine kinase gene. Evaluation as probes for monitoring gene therapy

A useful synthetic methodology was developed to synthesize and radiolabel a series of (E)-5-(2-[125I]iodovinyl)uracil nucleoside substrates for herpes simplex virus type-1 thymidine kinase (HSV-1 TK). (E)-5-(2-[125I]Iodovinyl)-2'-deoxyuridine ([125I]IVDU, 10), (E)-5-(2-[125I]iodovinyl)-2'-fluoro-2'-deoxyuridine ([125I]IVFRU, 11), (E)-5-(2-[125I]iodovinyl)-2'-fluoro-2'-deoxyarabinouridine ([125I]IVFAU, 12), and (E)-5-(2-[125I]iodovinyl)arabinouridine ([125I]IVAU, 13) were synthesized in 63-83% radiochemical yield by reaction of the unprotected (E)-5-(2-(trimethylsilyl)vinyl) precursors (6-9) with [125I]ICl. Cellular uptake of these labeled compounds (10-13) was evaluated in vitro. All compounds showed minimal uptake in the KBALB cell line. However, increased uptake was observed for all compounds in KBALB-STK cells which are transduced with a replication incompetent Moloney murine leukemia virus vector encoding the HSV-1 TK gene. The results indicate that uptake of these compounds in KBALB-STK cells is variable and highly dependent on the nature of the sugar 2'-substituent. When a fluoro (12) or a hydroxy (13) substituent is present in the arabinofuranosyl (up) configuration at the 2'-position, there is diminished cellular uptake in KBALB-STK cells relative to hydrogen (10) or fluorine (11) in the ribofuranosyl (down) configuration at the 2'-position. Our results indicate that radiolabeled IVFRU (11) is most promising for further in vivo studies.     

Some 1-[(benzofuran-2-yl)methyl]imidazoles as inhibitors of 17 alpha-hydroxylase: 17, 20-lyase (P450 17) and their specificity patterns

Four 1-[(benzofuran-2-yl)methyl]imidazoles (1-4) have been evaluated as in-vitro inhibitors of human testicular and bovine adrenal microsomal 17 alpha-hydroxylase: 17,20-lyase (P450 17) as potential anti-prostatic agents. Their specificity towards other steroidogenic and liver enzymes has been compared with that of ketoconazole. All four compounds were inhibitors of the testicular enzyme (2, IC50 (concentration resulting in 50% inhibition) 0.185 microM; 4, IC50 0.18 microM) but less potent than ketoconazole (IC50 0.03 microM). Towards bovine adrenal enzyme 2 and 4 were 35- and 31-fold more potent than ketoconazole (IC50 = 39.8 microM). Compound 2 is a useful lead compound but although less potent than ketoconazole towards P450SCC and P450 11 beta, but not P450C21, at the enhanced dose required for equivalent effects in-vivo on P450 17 it is likely that cortisol and aldosterone production will be affected to a greater extent than with ketoconazole.     

Thiopyrano[2,3,4-cd]indoles as 5-lipoxygenase inhibitors: synthesis, biological profile, and resolution of 2-[2-[1-(4-chlorobenzyl)-4-methyl-6-[(5-phenylpyridin-2-yl)methoxy]-4,5 -dihydro-1H-thiopyrano[2,3,4-cd]indol-2-yl]ethoxy]butanoic acid

Leukotriene biosynthesis inhibitors have potential as new therapies for asthma and inflammatory diseases. The recently disclosed thiopyrano[2,3,4-cd]indole class of 5-lipoxygenase (5-LO) inhibitors has been investigated with particular emphasis on the side chain bearing the acidic functionality. The SAR studies have shown that the inclusion of a heteroatom (O or S) in conjunction with an alpha-ethyl substituted acid leads to inhibitors of improved potency. The most potent inhibitor prepared contains a 2-ethoxybutanoic acid side chain. This compound, 14d (2-[2-[1-(4-chlorobenzyl)-4-methyl-6-[(5-phenylpyridin-2-yl)methox y]- 4,5-dihydro-1H-thiopyrano[2,3,4-cd]indol-2-yl]ethoxy]-butanoic acid, L-699,333), inhibits 5-HPETE production by human 5-LO and LTB4 biosynthesis by human PMN leukocytes and human whole blood (IC50s of 22 nM, 7 nM and 3.8 microM, respectively). The racemic acid 14d has been shown to be functionally active in a rat pleurisy model (inhibition of LTB4, ED50 = 0.65 mg/kg, 6 h pretreatment) and in the hyperreactive rat model of antigen-induced dyspnea (50% inhibition at 2 and 4 h pretreatment; 0.5 mg/kg po). In addition, 14d shows excellent functional activity against antigen-induced bronchoconstriction in the conscious squirrel monkey [89% inhibition of the increase in RL and 68% inhibition in the decrease in Cdyn (0.1 mg/kg, n = 3)] and in the conscious sheep models of asthma (iv infusion at 2.5 micrograms/kg/min). Acid 14d is highly selective as an inhibitor of 5-LO activity when compared to the inhibition of human 15-LO, porcine 12-LO and ram seminal vesicle cyclooxygenase (IC50 > 5 microM) or competition in a FLAP binding assay (IC50 > 10 microM). Resolution of 14d affords 14g, the most potent diastereomer, which inhibits the 5-HPETE production of human 5-LO and LTB4 biosynthesis of human PMN leukocytes and human whole blood with IC50s of 8 nM, 4 nM, and 1 microM respectively. The in vitro and in vivo profile of 14d is comparable to that of MK-0591, which has showed biochemical efficacy in inhibiting ex vivo LTB4 biosynthesis and urinary LTE4 excretion in clinical trials.