Studies on the toxicity and efficacy of some ester analogues of dapsone in vitro using rat and human tissues

The toxicity and efficacy of a series of 13 anti-tubercular sulphone esters has been evaluated using human and rat tissues. The toxicity studies involved comparison of the esters' ability to generate rat microsomally mediated NADPH-dependent methaemoglobin with that of dapsone. All the compounds formed significantly less methaemoglobin in the 1 compartment studies compared with dapsone itself. The ethyl, propyl, 3-methyl-butyl cyclopentyl esters and the carboxy parent derivative all yielded less than 5% of the methaemoglobin generated by dapsone. The 3-nitro benzoic acid ethyl and propyl esters generated 30 and 25% of dapsone's methaemoglobin formation. A similar effect was seen in the 2 compartment system, except for the butyl ester, which yielded similar haemoglobin oxidation to dapsone. The low toxicity ethyl and propyl esters, were also low in toxicity using human liver microsomes, producing less than 30% of the dapsone mediated methaemoglobin. All the compounds except the benzoic acid parent were superior to dapsone in terms of suppression of human neutrophil respiratory burst using a lucigenin-based chemiluminescence assay. The most potent derivatives were the phenyl, propyl and 3-nitro benzoic acid ethyl esters, which were between two- and threefold more potent compared with dapsone in arresting the respiratory burst. Overall, the ethyl ester showed the best combination of low toxicity in the rat and human microsomal systems and its IC(50) was approximately 40% lower than that of dapsone in neutrophil respiratory burst inhibition. These compounds indicate some promise for future development in their superior anti-inflammatory capability and lower toxicity compared with the parent sulphone, dapsone.     

Studies on the Toxicity of Analogues of Dapsone In-vitro Using Rat,Human and Heterologously Expressed Metabolizing Systems†

Three metabolizing systems (rat, heterologously expressed CYP3A4 and human liver) were used to evaluate 12 analogues of dapsone (4,4′diaminodiphenylsulphone) in-vitro. Methaemoglobin formation in a two-compartment and cytotoxicity in a single-compartment model were studied using human erythrocytes and neutrophils, respectively, as target cells. In the two-compartment system using rat microsomes as a generating system and methaemoglobin as an end-point, the least potent methaemoglobin formers tested were the 2-methyl-4-propylamino (AXDD14), 2-hydroxy-4-4′amino (ABDD5) derivatives and a sulphone/trimethoprim derivative (K-130). Dapsone itself, a 2-methoxy-4-ethylamino (W10) and a 2-hydroxyl-4-ethylamino compound (ABDD39) were the most toxic. In the single-compartment cytotoxicity test using rat microsomes, AXDD14 was again among the least toxic, as was a 2-methyl 4-cyclopentyl derivative (AXDD17) and surprisingly ABDD39. The most cytotoxic compounds again included dapsone itself as well as two 2-trifluoromethyl derivatives. The only significant methaemoglobin formation and cytotoxicity shown with the heterologously expressed human CYP 3A4 was with AXDD14, which was extensively activated. Interestingly, metabolism of dapsone was low using the expressed CYP 3A4. In the two-compartment system using human liver microsomes, AXDD14, K-130 and ABDD5 were oxidized to a significantly lesser extent compared with dapsone and these preliminary findings indicate that future development of these compounds may be worthwhile.     

The effect of 2,2'-substitution on the metabolism and toxicity of dapsone in vitro and in vivo

The effect of 2,2'-substitution with fluorine, methyl or trifluoromethyl groups on the toxicity, metabolism and pharmacological activity of dapsone has been investigated in vitro and in vivo. There was marked inter-species variation in the bioactivation (N-hydroxylation) of the compounds, as determined by methemoglobin formation. However, the inclusion of fluorine significantly (P<0.01) reduced methemoglobin formation compared with dapsone in all species studied. All three analogs resulted in significantly (P<0.001) less methemoglobinemia than dapsone when given either intraperitoneally or intravenously to the male Wistar rat. Rapid plasma clearance of the analogs through increased lipophilicity and enhanced N-glucuronidation may account for the low toxicity compared with dapsone. Although trifluoromethyl substitution resulted in a loss of activity against respiratory burst in human neutrophils in an in vitro model, all three analogs retained pharmacological activity against Plasmodium berghei malaria in an in vivo mouse model.     

Preliminary evaluation of the toxicity and efficacy of novel 2,4-diamino-5-benzylpyrimidine-sulphone derivatives using rat and human tissues in vitro

Four novel combined dapsone and trimethoprim analogues, K-120, K-150, K-138 and DRS-506, have been compared with dapsone in their methaemoglobin forming abilities as well as their anti-inflammatory properties using rat and human tissues in vitro. All four compounds formed consistently less methaemoglobin compared with dapsone in both the rat and human microsomes. Using human microsomes from five livers, K-120 was significantly less toxic than the other analogues in three of the five livers (P < 0.01). DRS-506 and K-138 both inhibited the human neutrophil respiratory burst to a significantly greater degree compared with dapsone at 0.5 mM (P < 0.01), while K-120 and K-150 showed no significant effect at 0.5 mM. At 1 mM, DRS-506, K-120 and K-138 were more potent than dapsone (P < 0.01), although K-150 appeared to increase the neutrophil activation. All four analogues caused a significant reduction in neutrophil adhesion to human umbilical vein cells at 0.1 mM. In view of its efficacy and low toxicity, K-120 shows considerable promise for future clinical evaluation.     

The inhibitory potency and selectivity of arginine substrate site nitric-oxide synthase inhibitors is solely determined by their affinity toward the different isoenzymes

We have investigated various nitric oxide (NO) synthase inhibitors for their affinity and selectivity toward the three human isoenzymes in radioligand binding experiments. Therefore, we developed the new radioligand [(3)H]2-amino-4-picoline to measure binding of these compounds to the three human NO synthase (NOS) isoenzymes. Aminopicoline is a potent and nonselective inhibitor of all three isoforms. [(3)H]2-amino-4-picoline bound saturably and with high affinity to human NOSs. Affinity constants (K(D) values) of 59, 111, and 136 nM were obtained for the inducible, neuronal, and endothelial NOS isoforms (iNOS, nNOS, eNOS). Binding of [(3)H]2-amino-4-picoline was competitive with the substrate arginine. From all the inhibitors tested, AMT (2-amino-5, 6-dihydro-6-methyl-4H-1,3-thiazine hydrochloride) showed the highest affinity and no selectivity. L-NIL [L-N(6)-(1-Iminoethyl)lysine hydrochloride] and aminoguanidine were moderately iNOS-selective while L-NA (N(G)-nitro-L-arginine) and L-NAME (N(G)-nitro-L-arginine methyl ester hydrochloride) showed selectivity toward the constitutive isoforms. High iNOS versus eNOS selectivity was found for 1400W, whereas several isothiourea derivatives and 1400W displayed moderate n- versus eNOS selectivity. To relate the affinity of these compounds to their inhibitory potency, we measured the inhibitory potency under almost identical conditions using a new microtiter plate assay. The inhibitory potency of selective and nonselective NOS inhibitors was almost exactly mirrored by their affinity toward the different isoenzymes. Highly significant correlations were obtained between the potency of enzyme inhibition and the inhibition of [(3)H]2-amino-4-picoline binding for all three isoenzymes. These data show that the potency and selectivity of NOS inhibitors are solely determined by their affinity toward the different isoforms. Furthermore, these data identify the new radioligand [(3)H]2-amino-4-picoline as a very useful radiolabel for the investigation of the substrate binding site of all three isoforms.     

Cyclic AMP-dependent inhibition of human neutrophil oxidative activity by substituted 2-propynylcyclohexyl adenosine A(2A) receptor agonists

Novel 2-propynylcyclohexyl-5'-N:-ehtylcarboxamidoadenosines, trans-substituted in the 4-position of the cyclohexyl ring, were evaluated in binding assays to the four subtypes of adenosine receptors (ARs). Two esters, 4-(3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl)-cyclohexanecarboxylic acid methyl ester (ATL146e) and acetic acid 4-(3-[6-amino-9-(5-ethylcarbamoyl-3, 4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl] -prop-2-ynyl)-cyclohexylmethyl ester (ATL193) were >50 x more potent than 2-[4-(2-carboxyethyl)phenethylamino]-5'-N:-ethylcarboxamidoadenosine (CGS21680) for human A(2A) AR binding. Human A(2A) AR affinity for substituted cyclohexyl-propynyladenosine analogues was inversely correlated with the polarity of the cyclohexyl side chain. There was a comparable order of potency for A(2A) AR agonist stimulation of human neutrophil [cyclic AMP](i), and inhibition of the neutrophil oxidative burst. ATL146e and CGS21680 were approximately equipotent agonists of human A(3) ARs. We measured the effects of selective AR antagonists on agonist stimulated neutrophil [cyclic AMP](i) and the effect of PKA inhibition on A(2A) AR agonist activity. ATL193-stimulated neutrophil [cyclic AMP](i) was blocked by antagonists with the potency order: ZM241385 (A(2A)-selective)>MRS1220 (A(3)-selective)>N-(4-Cyano-phenyl)-2-[4-(2,6-dioxo-1,3-dipropyl-2,3,4,5,6,7-hexahydro-1H-purin-8-yl)-phenoxy]-acetamide (MRS1754; A(2B)-selective) approximately 8-(N-methylisopropyl)amino-N(6)-(5'-endohydroxy-endonorbornyl)-9-methyladenine (WRC0571; A(1)-selective). The type IV phosphodiesterase inhibitor, rolipram (100 nM) potentiated ATL193 inhibition of the oxidative burst, and inhibition by ATL193 was counteracted by the PKA inhibitor H-89. The data indicate that activation of A(2A)ARs inhibits neutrophil oxidative activity by activating [cyclic AMP](i)/PKA.     

The effect of putative protein kinase C inhibitors, K252a and staurosporine, on the human neutrophil respiratory burst activated by both receptor stimulation and post-receptor mechanisms.

1. Two compounds, reported to be potent inhibitors of protein kinase C (PKC), K252a and staurosporine, have been examined in order to gain further information as to the possible role played by PKC in the signal transduction sequence of the neutrophil respiratory burst as determined by superoxide (O2-) production. 2. A number of stimuli were used in the study, some acting at receptors i.e. fMet-Leu-Phe (fMLP), opsonized zymosan and heat-aggregated IgG (HAGG), one acting on a G-protein, fluoride, and two direct PKC activators, dioctanoylglycerol (diC8) and phorbol myristate acetate (PMA). 3. K252a and staurosporine inhibited the respiratory burst with all the stimuli but the order of agonist sensitivity was very different with the two inhibitors. 4. For K252a-induced inhibition of O2- release, the order of potency was fluoride greater than fMLP, HAGG greater than opsonized zymosan greater than PMA, DiC8. For staurosporine-induced inhibition of O2- release, the order of potency changed to fluoride greater than DiC8, PMA greater than HAGG, fMLP greater than opsonized zymosan. The significance of this unexpected difference in relative rank order of potency is discussed with reference to the reported mechanism of action of the two inhibitors and the events involved in the oxidative burst. 5. Staurosporine at low concentrations increased the fMLP-stimulated O2- response by 100%, the maximum effect occurring at 35 nM. 6. To the extent that the compounds used are specific inhibitors of PKC, these findings support a role for the enzyme PKC in stimulus-activation coupling in O2- generation with all the stimuli used in this study.     

Preliminary pharmacological investigation on 38 aminophosphonic acids and their derivatives

Central pharmacological properties of 38 aminophosphonic acids and their derivatives, mostly newly synthesized, were investigated on mice and rats. Acute toxicity, neurotoxic activity, influence on spontaneous locomotor activity, body temperature, electrogenic and pentetrazol convulsions, on cerebral GABA level were tested. The most active compounds were (in a decreasing order of activity): 2-amino-7-phosphonoheptanoic, 2-amino-5-phosphonovaleric, 2-amino-8-phosphonooctanoic, 2-amino-2-methyl-3-methylphosphonopropionic, and 3-amino-3-hydroxy-5-phosphonovaleric acid.     

Structural determinants for AMPA agonist activity of aryl or heteroaryl substituted AMPA analogues. Synthesis and pharmacology

We have previously reported the synthesis and pharmacological characterization of analogues of 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA, 1a), in which the methyl group was replaced by a phenyl group (APPA, 1b) or heteroaryl groups. While 2b and its 3-pyridyl analogue 2-amino-3-[3-hydroxy-5-(3-pyridyl)-4-isoxazolyl]propionic acid (3-Py-AMPA, 3) show very low affinity for AMPA receptors, introduction of heteroaryl substituents containing heteroatom in the 2-position provides potent AMPA receptor agonists. We here report the synthesis and pharmacology of 2-amino-3-(3-hydroxy-5-pyrazinyl-4-isoxazolyl)propionic acid (7) (IC50 = 1.2 microM), which is weaker as an AMPA agonist than AMPA (IC50 = 0.040 microM; EC50 = 3.5 microM) but comparable in potency with 2-Py-AMPA (4) (IC50 = 0.57 microM; EC50 = 7.4 microM), as determined in radioligand binding and electrophysiological experiments, respectively. The AMPA analogues 8a-c, containing 2-, 3-, or 4-methoxyphenyl substituents, respectively, and the corresponding hydroxyphenyl analogues, 9a-c, were also synthesized and evaluated pharmacologically. With the exception of 2-amino-3-[3-hydroxy-5-(2-hydroxyphenyl)-4-isoxazolyl]propionic acid (9a), which is a very weak AMPA agonist (IC50 = 45 microM; EC50 = 324 microM), none of these compounds showed detectable effect at AMPA receptors.     

3-Amino-5-phenoxythiophenes: syntheses and structure-function studies of a novel class of inhibitors of cellular L-triiodothyronine uptake.

A series of substituted 3-amino-5-phenoxythiophenes was synthesized starting from malodinitrile and carbon disulfide. The resulting dicyanoketenedithiolate reacts via Thorpe-Dieckmann cyclization with halogen methanes bearing electron-withdrawing groups to give thiophene-2-thiolates, which can be transformed into 3-amino-5-(methylsulfonyl)thiophene-4-carbonitriles. Replacement of the methylsulfonyl groups by substituted phenolates provides the substituted 3-amino-5-phenoxythiophenes. Some of the derivatives show a considerable inhibitory potency for the L-T3 uptake in inhibition studies on human HepG2 hepatoma cells with maximum values of about 60% at a dose of 10(-5) M for the most potent 2-benzoyl derivatives. The structure of the phenoxythiophenes fits well into a general concept derived for other classes of L-T3 uptake inhibitors, which postulates an angular and perpendicular orientation of the ring systems in these compounds as a prerequisite for an inhibitory potency. Docking studies for the phenoxythiophenes with transthyretin as a receptor model show their preferred attack at the L-T4/L-T3 binding channel.     

Synthesis of pyranochromene and pyranopyrimidine derivatives from substituted natural coumarin isolated from Ammi majus L. and their biological evaluation

A series of novel coumarin derivatives were synthesized from 6-hydroxy-7-methoxy-4-methyl coumarin which was isolated from the aerial parts of the Egyptian medicinal plant Ammi majus L. (Apiaceae). The key intermediate 3-amino-5-methoxy-1-(4-methoxyphenyl)-10-methyl-8-oxo-1,8-dihydropyrano[3,2-f]chromene-2-carbonitrile (3c) was obtained in one-pot synthesis by treating α-cyanocinnamonitrile (1-c) with the natural compound: 6-hydroxy-7-methoxy-4-methyl coumarin (2). Chemical, elemental and spectroscopic evidences confirmed the structures of the synthesized compounds. Some of the newly synthesized compounds exhibited better anti-inflammatory activities at low concentrations compared with indomethacin as positive control.     

Enkephalinase inhibitors. 1. 2,4-Dibenzylglutaric acid derivatives

The synthesis of two new series of dicarboxylic acid dipeptides and two sulfhydryl-containing inhibitors are described. The in vitro enkephalinase inhibition data and some in vivo analgesic data are presented for these compounds. For the dibenzylglutaric acid series structure-activity relationships and in vivo analgesic activity are discussed. The reverse amides, i.e., 4-amino-2,4-dibenzylbutyric acid derivatives, are also discussed. Two sulfhydryl-containing inhibitors showed good in vivo potency in the mouse jump-latency hot-plate test after peripheral administration at moderate low doses.     

Novel potent AMPA/kainate receptor antagonists: synthesis and anticonvulsant activity of a series of 2-[(4-alkylsemicarbazono)-(4-amino-phenyl)methyl]-4,5-methylenedioxyphenylacetic acid alkyl esters

In this paper we describe the synthesis of a series of novel 2-[(4-alkylsemicarbazono)-(4-aminophenyl)-methyl]-4,5-methylenedioxyphenylacetic acid alkyl esters (10-19) carrying an alkylsemicarbazono moiety at a benzylic site. The influence of this group on the biological activity was evaluated by testing the corresponding derivatives 20-22 in which the 4-alkylsemicarbazono moiety was removed (compound 20) or its alkylureido portion shifted at position 1 (compounds 21-22). Furthermore, the involvement of the 4-aminobenzyl moiety in the anticonvulsant activity was evaluated by testing derivative 23. The anticonvulsant activity of all compounds was assayed against audiogenic seizures induced in DBA/2 mice. Within this series of derivatives, 2-[(4-aminophenyl)-(4-methylsemicarbazono)-methyl]-4,5-methylenedioxyphenylacetic acid methyl ester (10) proved to be the most active compound. It displayed a potency 5-fold higher than that shown by 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (1, GYKI 52466), a well-known noncompetitive 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptor antagonist. Compound 10 was also effective in suppressing seizures induced in Swiss mice by maximal electroshock (MES) or pentylenetetrazole (PTZ). Furthermore, it antagonized in vivo seizures induced by icv administration of AMPA or kainate (KA). Using the patch-clamp technique in primary cultures of granule neurons we tested compounds 10 and 21 for their ability to modulate currents evoked by KA and 2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propionic acid (ATPA). These two derivatives reduced KA and ATPA currents to a larger extent than that shown by reference compound 1. Compounds 10 and 21 were also able to reduce neuronal cell death induced by the application of KA (100 microM).     

Some derivatives of the 2-[4-pyrazolinyl]-1,3,4-thiadiazole system

The synthesis of some 2-[5-amino-1-methyl-3-oxo-4-pyrazolynyl]-1,3,4-thiadiazole derivatives is accomplished by reacting 5-amino-1-methyl-3-oxopryrazolynyl-4-dithiocarbohydrazide with carboxylic acid derivatives. The structure of the compounds obtained is verified by means of 1H and 13C-N.M.R. spectra. Antimicrobial and antifungal activity of some of the described compounds was tested in a preliminary screening.     

Structure-activity relationships in the haemolytic activity and nephrotoxicity of derivatives of 1,2- and 1,4-naphthoquinone

Naphthoquinone derivatives are under investigation as potential therapeutic agents. Some such compounds are known, however, to be toxic to both animals and humans. Many naphthoquinone derivatives are haemolytic agents, while others cause necrosis of tubular epithelial cells. In the present study, the short-term toxicity of 16 derivatives of 1,2- and 1,4-naphthoquinone has been examined in rats in order to give information on structure-activity relationships. All the naphthoquinones except one caused haemolytic anaemia, but only hydroxy and amino derivatives were nephrotoxic. Among derivatives of 2-amino-1,4-naphthoquinone, substitution in the 3-position decreased haemolytic activity and abolished nephrotoxicity. Methylation of the hydroxyl group of 2-hydroxy-1,4-naphthoquinone had a similar effect. In contrast, methylation of the amino group of 2-amino-1,4-naphthoquinone increased the severity of both haemolysis and renal damage. Among the 1,2-naphthoquinones tested, the 4-methoxy and 4-amino derivatives were more toxic than the corresponding 1,4-isomers, although 4-methyl-1,2-naphthoquinone was less toxic than 2-methyl-1,4-naphthoquinone. At present, the toxicity of naphthoquinone derivatives cannot accurately be predicted on the basis of their chemical structure. In developing naphthoquinone derivatives for use in humans, toxicological studies in animals should be conducted at an early stage, bearing in mind that clinical studies have shown that humans appear to be particularly vulnerable to the nephrotoxic action of these compounds, and that certain individuals are unusually susceptible to their haemolytic action.     

Mechanism of xanthine-induced relaxation of guinea-pig isolated trachealis muscle.

1. Four 3-alkylxanthines (3-methylxanthine, 3-n-propylxanthine (enprofylline), 3-n-butylxanthine and 3-iso-butylxanthine) and four 1-methyl-3-alkylxanthines (1-methyl-3-methylxanthine (theophylline), 1-methyl-3-n-propylxanthine, 1-methyl-3-n-butylxanthine and 1-methyl-3-iso-butylxanthine (IBMX], were compared in terms of cyclic AMP phosphodiesterase (PDE) inhibition and trachealis muscle relaxation. The relationship between xanthine structure and cyclic AMP PDE inhibition was also studied. 2. Xanthine induced relaxation of guinea-pig isolated trachealis muscle was measured against spontaneous tone. 3. The four 1-methyl-3-alkylxanthines were each significantly more potent than the corresponding 3-alkylxanthines in relaxing the isolated trachealis muscle. The 1-methyl-3-alkylxanthines were similarly more potent than the corresponding 3-alkyl derivatives in inhibiting low Km cyclic AMP PDE. There was a strong positive correlation between low Km cyclic AMP PDE inhibition and the tracheal smooth muscle relaxation evoked by the xanthine derivatives. 4. Since methylation of the 1-position of each 3-alkylxanthine increased the potency of the derivative in inhibiting low Km cyclic AMP PDE and in relaxing trachealis muscle and since a strong positive correlation was observed between the relaxant EC50 and the Ki value of each xanthine derivative, it is suggested that low Km cyclic AMP PDE inhibition by xanthines plays an important role in their tracheal relaxant effect.     

A new class of isoxazole derivatives: the M 1-9 series of compounds with immunotropic activity

The isoxazole derivatives are interesting objects for synthesis in the search for various sorts of biological activity. Looking for more active immunomodulators we synthesized a series of 5-amino-3-methyl-4-isoxazolecarboxylic acid semicarbazides and thiosemicarbazides in the reaction of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide with isocyanates and isothiocyanates. The biological effect of these compounds on the proliferative response of human mononuclear peripheral blood cells to phytohemagglutinin A (PHA) was described.     

Characterization of a series of 3-amino-2-phenylpropene derivatives as novel bovine chromaffin vesicular monoamine transporter inhibitors

A series of 3-amino-2-phenylpropene (APP) derivatives have been synthesized and characterized as novel competitive inhibitors, with K(i) values in the microM range, for the bovine chromaffin granule membrane monoamine transporter(s) (bVMAT). Although, these inhibitors are structurally similar to the bVMAT substrate tyramine, none of them were measurably transported into the granule. Structure-activity studies have revealed that, while the 3'- or 4'-OH groups on the aromatic ring enhance the inhibition potency, Me or OMe groups in these positions reduce the inhibition potency. Halogen substitution on the 4'-position of the aromatic ring causes gradual increase of the inhibition potency parallel to the electron donor ability of the halogen. Substituents on the NH(2) as well as on the 3-position of the alkyl chain reduce the inhibition potency. Comparative structure-activity analyses of APP derivatives with tyramine and the neurotoxin 1-methyl-4-phenylpyridinium suggest that the flexibility of the side chain and the relative orientation of the NH(2) group may be critical for the efficient transport of the substrate through the bVMAT. Comparable bVMAT affinities of these inhibitors to that of DA and other pharmacologically active amines suggest that they are suitable for the structure-activity and mechanistic studies of monoamine transporters and may also be useful in modeling the mechanism of action of amphetamine-related derivatives.     

[H]ATPA: a high affinity ligand for GluR5 kainate receptors

The pharmacological properties of [³H]ATPA ((RS)-2-amino-3(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid) are described. ATPA is a tert-butyl analogue of AMPA (-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid) that has been shown to possess high affinity for the GluR5 subunit of kainate receptors. [³H]ATPA exhibits saturable, high affinity binding to membranes expressing human GluR5 (GluR5) kainate receptors (K_d13 nM). No specific binding was observed in membranes expressing GluR2 and GluR6 receptors. Several compounds known to interact with the GluR5 kainate receptor inhibited [³H]ATPA binding with potencies similar to those obtained for competition of [³H]kainate binding to GluR5. Saturable, high affinity [³H]ATPA binding (K_d4 nM) was also observed in DRG neuron (DRG) membranes isolated from neonatal rats. The rank order potency of compounds to inhibit [³H]ATPA binding in rat DRG and GluR5 membranes were in agreement. These finding demonstrate that [³H]ATPA can be used as a radioligand to examine the pharmacological properties of GluR5 containing kainate receptors.     

Synthesis and biological activity of substituted 2,4,6-s-triazines

Reaction of 7-hydroxy-4-methyl coumarin with amido/ imido alcohols in ethanol containing concentrated hydrochloric acid afforded 8-aralkyl amido/imido-alkyl-7-hydroxy-4-methyl-coumarins (1a-f). Interaction of 1a-f with hydrazine hydrate in pyridine resulted in 1-amino-8-aralkyl amido/imido-alkyl-7-hydroxy-4-methyl-2-oxo-quinolines (2a-f). Treatment of 2 with formaldehyde in ethanol resulted in 1,3,5-tris-(8-aralkyl amido/imido-alkyl-7-hydroxy-4-methyl-2-oxo-quinolinyl)-2,4,6-hexahydro-s-triazines (3a-c). Antiviral activity of compounds 2a-d and 3a, 3b upon Japanese encephalitis virus (JEV) and Herpes simplex virus-1 (HSV-1) was evaluated on vero cells in vitro. 3a-c were also screened for their antihypertensive activity.