Potent and selective neuronal nitric oxide synthase inhibitors with improved cellular permeability

Recently, a series of potent and selective neuronal nitric oxide synthase inhibitors containing two basic nitrogen atoms was reported (Ji, H.; Stanton, B. Z.; Igarashi, J.; Li, H.; Martásek, P.; Roman, L. J.; Poulos, T. L.; Silverman, R. B. J. Am. Chem. Soc. 2008, 130, 3900–3914). In an effort to improve their bioavailability, three compounds (2a–c) were designed with electron-withdrawing groups near one of the basic nitrogen atoms to lower its pK_a. Inhibition studies with these compounds showed that two of them not only retained most of the potency and selectivity of the best analogue of the earlier series, but also showed improved membrane permeability based on data from a cell-based assay.     

Further studies on hepatitis C virus NS5B RNA-dependent RNA polymerase inhibitors toward improved replicon cell activities: benzimidazole and structurally related compounds bearing the 2-morpholinophenyl moiety

Following the discovery of JTK-109 (1) as a potent inhibitor of hepatitis C virus NS5B RNA-dependent RNA polymerase, [(a) Hirashima, S.; Suzuki, T.; Ishida, T.; Noji, S.; Yata, S.; Ando, I.; Komatsu, M.; Ikeda, S.; Hashimoto, H. J. Med. Chem.2006, 49, 4721. (b) Hashimoto, H.; Mizutani, K.; Yoshida, A. Int. Patent Appl. WO 01/47883, 2001.] further studies toward the improvement of the cellular potency have been performed. A greater than 40-fold improvement was achieved through replacing the biphenyl moiety with a 2-morpholinophenyl group and the benzimidazole ring with the tetracyclic scaffold to afford compound 7 with an excellent replicon potency (EC(50)=7.6 nM).     

2-Benzyl-2-methylsuccinic acid as inhibitor for carboxypeptidase A. synthesis and evaluation.

Recently, Asante-Appiah et al. (Asante-Appiah, E.; Seetharaman, J.; Sicheri, F.; Yang, D. S.-C.; Chan, W. W.-C. Biochemistry 1997, 36, 8710 8715) reported that 2-ethyl-2-methylsuccinic acid is a highly potent inhibitor for carboxypeptidase A (CPA), a prototypic zinc protease. The X-ray crystal structure of the complex of the enzyme formed with 2-ethyl-2-methylsuccinic acid revealed that at the active site of CPA there is present a small cavity which accommodates the methyl group of the inhibitor. These investigators postulated that incorporation of a methyl group at the alpha-position to the carboxylate of existing inhibitors of CPA would improve the inhibitory potency. We have synthesized racemic and optically active 2-benzyl-2-methylsuccinic acids and evaluated their inhibitory activities for CPA to find the K(i) values to be 0.28, 0.15, and 17microM for racemic form, (R)-, and (S)-enantiomer, respectively. Contrary to the expectation, the effect on the binding affinity by the incorporation of the methyl group is minimal. The validity of the proposition that the small cavity may be utilized for the improvement of the inhibitory potency appears questionable.     

Design, synthesis, inhibitory activity, and SAR studies of pyrrolidine derivatives as neuraminidase inhibitors

A series of pyrrolidine derivatives were synthesized and evaluated for their ability to inhibit neuraminidase (NA) of influenza A virus (H3N2). All compounds were synthesized in good yields starting from commercially 4-hydroxy-L-proline using a suitable synthetic strategy. These compounds showed potent inhibitory activity against influenza A neuraminidase. Within this series, five compounds, 6e, 9c, 9e, 9f, and 10e, have good potency (IC(50)=1.56-2.71 microM) which are compared to that the NA inhibitor Oseltamivir (IC(50)=1.06 microM), and could be used as lead compoundS in the future.     

3-Aryl-4-hydroxyquinolin-2(1H)-one derivatives as type I fatty acid synthase inhibitors

A series of 3-aryl-4-hydroxyquinolin-2(1H)-ones with fatty acid synthase inhibitory activity was prepared. Starting from a derivative with an IC(50) = 1.4 microM, SAR studies led to compounds with more than 70-fold increase in potency (IC(50) < 20 nM).     

Structure-activity relationships of pentacycloundecylamines at the N-methyl-d-aspartate receptor

Prompted by our interest in neuroprotective agents with multiple mechanisms of action, we assessed the structure-activity relationship of a series of pentacycloundecylamine derivatives previously shown to have both L-type calcium channel blocking activity and N-methyl-d-aspartate receptor (NMDAR) antagonistic activity. We utilized a functional assay to measure NMDAR channel block using (45)Ca(2+) influx into synaptoneurosomes. The cage amine 8-benzylamino-8,11-oxapentacyclo[5.4.0.0(2,6). 0(3,10).0(5,9)]undecane (NPG1-01) proved to be the most potent experimental compound with an IC(50) of 2.98microM, while 8-amino-pentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecane had the next most potent IC(50) of 4.06microM. Increasing the polycyclic cage size of NGP1-01 from a pentacycloundecane to a tridecane cage structure, but retaining the N-benzyl moiety decreased potency 10-fold, indicating a limitation on the volume of the cage that can be accommodated in the channel binding site. In the presence of NGP1-01, NMDA/glycine-induced maximal (45)Ca(2+) influx was attenuated by 34% with an insignificant effect on agonist potency. These results are consistent with uncompetitive antagonism for this group of compounds. Radioligand binding studies with [(3)H]MK-801 or [(3)H]TCP showed little or no displacement of these ligands by pentacycloundecylamines, suggesting that the latter compounds bind to a unique site in the NMDAR channel. The pentacycloundecylamines tested represent a novel group of NMDAR antagonists that have potential as therapeutic agents for neurodegenerative diseases including Parkinson's and Alzheimer's disease.     

Design, synthesis, inhibitory activity, and SAR studies of hydrophobic p-aminosalicylic acid derivatives as neuraminidase inhibitors

A series of hydrophobic p-aminosalicylic acid derivatives containing a lipophilic side chain at C-2 and an amino or guanidine at C-5 were synthesized and evaluated for their ability to inhibit neuraminidase (NA) of influenza A virus (H3N2). All compounds were synthesized in good yields starting from commercially available p-aminosalicylic acid (PAS) using a suitable synthetic strategy. These compounds showed potent inhibitory activity against influenza A NA. Within this series, six compounds, 11, 12, 13e, 16e, 17c, and 18e, have the good potency (IC(50)=0.032-0.049 microM), which are compared to Oseltamivir (IC(50)=0.021 microM) and could be used as lead compounds in the future.     

Bicyclic furo pyrimidine nucleosides with aryloxyphenyl and halophenyl substituted side chains as potent and selective varicella-zoster virus inhibitors

The discovery of potent and selective inhibitors of VZV based on unusual bicyclic alkyl furo pyrimidine nucleosides has been recently reported. Modifications to the side-chain by addition of a phenyl group were found to further enhance the antiviral potency of these compounds. A series of alkoxyphenyl compounds (5a-5g) and two halophenyl derivatives (5h and 5i) were successfully synthesised and displayed anti-VZV activity at low microM concentrations.     

Tyrosinase inhibitory lignans from the methanol extract of the roots of Vitex negundo Linn. and their structure-activity relationship.

Phytochemical investigation of the methanol extract of Vitex negundo afforded eight lignans; negundin A 1, negundin B 2, 6-hydroxy-4-(4-hydroxy-3-methoxy)-3-hydroxymethyl-7-methoxy-3,4-dihydro-2-naphthaledehyde 3, vitrofolal E 4, (+)-lyoniresinol 5, (+)-lyoniresinol-3alpha-O-beta-d-glucoside 6, (+)-(-)-pinoresinol 7, and (+)-diasyringaresinol 8. The structures of these compounds were elucidated unambiguously by spectroscopic methods including 1D and 2D NMR analysis and also by comparing experimental data with literature data. The tyrosinase inhibitory potency of these compounds has been evaluated and attempts to justify their structure-activity relationships have been made in the present work. The compound 5 was found to be the most potent (IC(50)=3.21 microM) while other compounds demonstrated moderate to potent inhibitions. It was found that the substitution of functional group(s) at C-2 and C-3 positions and the presence of the -CH(2)OH group plays a vital role in the potency of the compounds. The compound 5 can act as a potential lead molecule to develop new drugs for the treatment of hyperpigmentation associated with the high production of melanocytes.     

5-Substituted 2-aminothiophenes as A1 adenosine receptor allosteric enhancers

Two series of 5-substituted 2-amino-4-(3-trifluoromethylphenyl)thiophenes were prepared and evaluated as allosteric enhancers at the A(1) adenosine receptor (A(1)AR). In the 3-benzoyl series, a 5-phenyl group was found to confer the greatest potency (9a: ED(50)=2.1 microM, AE score=18%). However, the analogue with no 5-substituent (6b: ED(50)=15.8 microM, AE score=77%) proved to be the most efficacious. In the 3-ethoxycarbonyl series, the 5-(4-chlorophenyl) analogue was clearly the most potent and efficacious (9l: ED(50)=6.6 microM, AE score=57%). The antagonist activity of all compounds was measured using a [(3)H]CPX competitive binding assay.     

Identification of new Cdc25 dual specificity phosphatase inhibitors in a targeted small molecule array

Dual specificity protein phosphatases (DSPases) are key regulators of signal transduction, oncogenesis and the cell cycle. Few potent or specific inhibitors of DSPases, however, are readily available for these pharmacological targets. We have used a combinatorial/parallel synthetic approach to rigidify the variable core region and modify the side chains of 4-(benzyl-(2-[2,5-diphenyl-oxazole-4-carbonyl)-amino]-ethyl)-carbamoyl)- 2-decanoylamino butyric acid (or SC-alphaalphadelta9), which is the most active element in a previously described library of phosphatase inhibitors (Rice, R. L.; Rusnak, J. M.; Yokokawa, F.; Yokokawa, S.; Messner, D. J.; Boynton, A. L.; Wipf, P.; Lazo, J. S. Biochemistry 1997, 36, 15965). Several analogues were identified as effective inhibitors of the protein tyrosine phosphatase (PTPase) PTP1B and the DSPases VHR and Cdc25B2. Two compounds, FY3-alphaalpha09 and FY21-alphaalpha09, were partial competitive inhibitors of Cdc25B2 with Ki values of 7.6+/-0.5 and 1.6+/-0.2 microM, respectively. FY21-alphaalpha09 possessed only moderate activity against PTP1B. Consistent with its in vitro anti-phosphatase activity, FY21-alphaalpha09 inhibited growth in MDA-MB-231 and MCF-7 human breast cancer cell lines. FY21-alphaalpha09 also inhibited the G2/M transition in tsFT210 cells, consistent with Cdc25B inhibition. Several architectural requirements for DSPase inhibition were revealed through modification of the side chain moieties or variable core region of the pharmacophore, which resulted in decreased compound potency. The structure of FY21-alphaalpha09 provides a useful platform from which additional potent and more highly selective phosphatase inhibitors might be generated.     

Cytotoxicity activity of L-proline analogues of anthraquinone-2-carboxylic acid in breast cancer MCF-7 cells

Although prolidase [E.C.3.4.13.9] is found in normal cells, substantially increased levels are found in some neoplastic tissues. Prolidase evokes the ability to hydrolyse the imido-bond of various low molecular weight compounds coupled to L-proline. The synthesis of three proline analogues of anthraquinone-2-carboxylic acid (1-3) has been performed. Treatment of these prodrugs with prolidase generated L-proline and the free drug, demonstrating their substrate susceptibility prolidase. The concentrations of 1, 2 and 3 needed to inhibit [1H]thymidine incorporation into DNA by 50% (IC50) in breast cancer MCF-7 cells were found to be 185 +/- 5 microM, 107 +/- 6 microM and 87 +/- 6 microM, respectively, suggesting a lower cytotoxic potency of these compounds compared to Hoechst 33228 (IC50 = 55 +/- 6 microM).     

Effect of BI-L-239, A-64077 and MK-886 on leukotriene B4 synthesis by chopped guinea pig lung and on antigen-induced tracheal contraction in vitro.

The 5-lipoxygenase (5-LO) inhibitors BI-L-239 and A-64077 were compared with the 5-LO translocation inhibitor MK-886 for the ability to inhibit leukotriene B4 (LTB4) biosynthesis by chopped (1 mm3) guinea pig lung. LTB4 synthesis by ovalbumin-sensitized chopped lung tissue was determined after stimulation with either calcium ionophore (A23187) or antigen. With A23187 stimulation, MK-886 was more potent (IC50 = 0.39 +/- 0.23 microM, mean +/- SEM, p < 0.01) than BI-L-239 (IC50 = 2.48 +/- 0.46 microM) or A-64077 (IC50 = 4.68 +/- 0.70 microM) and BI-L-239 was more potent than A64077 (p < 0.02). Thus, the order of potency was MK-886 > BI-L-239 > A-64077 for inhibition of calcium ionophore-induced LTB4 generation. There was no significant differences in potency of the compounds in chopped lung stimulated with antigen: IC50 for LTB4 synthesis by A-64077 = 3.31 +/- 1.70 microM, for BI-L-239 = 9.06 +/- 4.94 microM, and for MK-886 = 13.33 +/- 7.91 microM. The ability of these compounds to inhibit contraction of tracheal tissue from actively sensitized guinea pigs in response to antigen was also determined in the presence of indomethacin (15 micrograms/ml), mepyramine, and atropine (5 micrograms each/ml). Both 5-LO inhibitors inhibited antigen-induced contraction, with IC50 values for BI-L-239 and A-64077 of 1.58 and 4.35 microM respectively. MK-886 was ineffective at inhibiting antigen-induced tracheal contraction in vitro at concentrations up to 30 microM. In summary, these compounds inhibit antigen-induced and A23187-induced leukotriene biosynthesis in guinea pig tissue. These 5-LO inhibitors were similarly effective at inhibiting antigen-induced tracheal contraction where MK-886 was ineffective.     

Specific interaction of 5-(N-methyl-N-isobutyl)amiloride with the organic cation-proton antiporter in human placental brush-border membrane vesicles. Transport and binding.

The interaction of 5-(N-methyl-N-isobutyl)amiloride (MIBA) with brush-border membrane vesicles isolated from normal human term placentas was investigated using two parameters: binding and transport. The binding of MIBA to placental membranes was specific and temperature- and pH-dependent, and the apparent dissociation constant (Kd) for the process was 58 +/- 2 microM. The binding was inhibited by other amiloride analogs and also by clonidine and cimetidine with a rank order potency: MIBA > benzamil > dimethylamiloride > amiloride > clonidine > cimetidine. These compounds also inhibited Na(+)-H+ exchanger activity in these membrane vesicles, but with a different order of potency: dimethylamiloride > MIBA > amiloride > benzamil > cimetidine > clonidine. The membrane vesicles were also able to transport MIBA into the intravesicular space, and the transport was stimulated many-fold by the presence of an outwardly directed H+ gradient across the membrane. The H+ gradient was the driving force for uphill accumulation of MIBA inside the vesicles. The transport process was electrically silent. The transport of MIBA was inhibited by other amiloride analogs and by clonidine and cimetidine, and the order of potency was the same as the order with which these compounds inhibited the binding of MIBA. The Michaelis-Menten constant (Kt) for the transport process was 46 +/- 2 microM. The binding as well as the transport were also inhibited by Na+ and Li+. Interestingly, tetraethylammonium and N1-methylnicotinamide, two of the commonly used substrates in organic cation transport studies, failed to inhibit the binding and transport of MIBA. Furthermore, although the outwardly directed H+ gradient-dependent uphill transport of tetraethylammonium could be demonstrated in renal brush-border membrane vesicles, there was no evidence for the presence of a transport system for this prototypical organic cation in placental brush-border membrane vesicles. It is concluded that the human placental brush-border membranes possess an organic cation-proton antiporter which accepts MIBA as a substrate, the low affinity binding site for MIBA observed in these membranes represents this antiporter, and that the placental organic cation-proton antiporter is distinct from the widely studied renal organic cation-proton antiporter.     

Discovery of substituted 4-anilino-2-(2-pyridyl)pyrimidines as a new series of apoptosis inducers using a cell- and caspase-based high throughput screening assay. Part 1: structure-activity relationships of the 4-anilino group

A series of 4-anilino-2-(2-pyridyl)pyrimidines has been discovered as a new class of potent inducers of apoptosis using a cell-based HTS assay. Compound 5a was found to arrest T47D cells in G2/M and induced apoptosis. SAR studies showed that a small and electron-donating group at the meta-position of the anilino ring is important for activity. A 20-fold increase in potency, from hit compound 4-(3-methoxyanilino)-2-(2-pyridinyl)-6-(trifluoromethyl)pyrimidine (5a) to lead compound 4-(2,5-dimethoxyanilino)-2-(2-pyridinyl)-6-(trifluoromethyl)pyrimidine (5l), was obtained through the SAR studies. Compound 5l is highly active with an EC50 value of 18 nM in the caspase activation assay in T47D breast cells. Interestingly, 5a and other meta-mono-substituted compounds were active against T47D cells but were not active against H1299 and HT29 cells, while 5l and other 2,5-disubstituted compounds were active against all the three cells. In a tubulin polymerization assay, compound 5l inhibited tubulin polymerization with an IC50 value of < 0.5 microM, while 5a was not active up to 50 microM.     

Differentiation of the drug-binding sites of calmodulin

Calmodulin contains several binding sites for hydrophobic compounds. The apparent specificity of various 'calmodulin antagonists' for these sites was investigated. The Ki values for the inhibition of calmodulin-activated cyclic-nucleotide phosphodiesterase and myosin light-chain kinase was determined. In addition, the Kd values of the same compounds for binding to calmodulin were measured. The compounds could be separated into four groups. Group I and II compounds inhibited competitively the activation of the phosphodiesterase and myosin light-chain kinase by calmodulin. Group I compounds inhibited the activation of the phosphodiesterase and myosin light-chain kinase at identical concentrations. In contrast, group II compounds inhibited the activation of the phosphodiesterase at 5-10-fold lower concentrations than that of myosin light-chain kinase. Group III compounds inhibited the activation of these enzymes by an uncompetitive mechanism. Group IV compounds inhibited the activation of the phosphodiesterase with Ki values above 10 microM and did not affect the activation of myosin light-chain kinase. Binding of [3H]bepridil to calmodulin under equilibrium conditions yielded one high-affinity site (apparent Kd 0.4 microM) and four low affinity sites (apparent Kd 44 microM). Group I compounds interfered with the binding of bepridil to the high and low-affinity sites in a competitive manner. Group II compounds interfered in a non-competitive manner with the high-affinity site and apparently competed only with one of the low-affinity sites. Group III compounds did not compete with any of the bepridil-binding sites. Nimodipine, a group III compound, bound to one site on calmodulin with a Kd value of 1.1 microM. Other dihydropyridines competed with [3H]nimodipine for this site. The group I and II compounds, trifluoperazine and prenylamine, did not affect the binding of [3H]nimodipine. These data show that 'calmodulin antagonists' can be differentiated into at least three distinct groups. Kinetic and binding data suggest that the three groups bind to at least three different sites on calmodulin. Selective occupation of these sites may inhibit specifically the activation of distinct enzymes.     

Synthesis and biological evaluation of methanesulfonamide analogues of rofecoxib: Replacement of methanesulfonyl by methanesulfonamido decreases cyclooxygenase-2 selectivity

A new group of 3-(4-substituted-phenyl)-4-(4-methylsulfonamidophenyl)-2(5H)furanones in which the methylsulfonyl (MeSO(2)) COX-2 pharmacophore present in rofecoxib was replaced by a methanesulfonamido (MeSO(2)NH) moiety, and where the substituent at the para-position of the C-3 phenyl ring was simultaneously varied (H, F, Cl, Br, Me, OMe), were evaluated to determine the combined effects of steric and electronic substituent properties upon COX-1 and COX-2 inhibitory potency and COX isozyme selectivity. Structure-activity relationship (SAR) studies showed that compounds having a neutral (H), or electronegative halogen (F, Cl, Br), substituent at the para-position of the C-3 phenyl ring inhibited both COX-1 and COX-2 with COX-2 selectivity indexes in the 3.1-39.4 range. In contrast, compounds having an electron-donating Me or OMe substituent were selective inhibitors of COX-2 (COX-1 IC(50)>100 microM). These SAR data indicate the 3-aryl-4-(4-methylsulfonamidophenyl)-2(5H)furanone scaffold provides a suitable template to design COX inhibitors with variable COX-2 selectivity indexes.     

Metronidazole acid acyl sulfonamide: a novel class of anticancer agents and potential EGFR tyrosine kinase inhibitors

A series of novel metronidazole derivatives were recently reported as potent anticancer agents targeting EGFR and HER-2 by our group [Qian, Y.; Zhang, H. J.; Zhang, H.; Xu, C.; Zhao, J.; Zhu, H. L. Bioorg. Med. Chem.2010, 18, 4991]. Based on the previous results, we designed and synthesized a new series of metronidazole acid acyl sulfonamide derivatives and a new series of phenylacetyl benzenesulfonamide derivatives and their anticancer activities were evaluated as potential EGFR and HER-2 kinase inhibitors. Among all the compounds, compound 12 displayed the most potent inhibitory activity EGFR and HER-2 (IC(50)=0.39 μM for EGFR and IC(50)=1.53 μM for HER-2) and it also showed the most potent growth inhibitory activity against A549 and B16-F10 cancer cell line in vitro, with an IC(50) value of 1.26 μg/mL for A549 and 0.35 μg/mL for B16-F10. Docking simulation was further performed to position compound 12 into the EGFR active site to determine the probable binding model.     

Characterization df a [3H]Glycine Recognition Site as a Modulatory Site of the N-Methyl-D-Aspartate Receptor Complex

A [3H]glycine recognition site in rat brain synaptic plasma membranes (SPM) has been identified, having characteristics expected of a modulatory component of the N-methyl-D-aspartate receptor complex. Incubation of SPM with [3H]glycine for 10 min at 2 degrees C results in saturable, reversible binding with a KD of 0.234 microM and a Bmax of 9.18 pmol/mg. A pharmacological analysis of this binding site indicates that D-serine (Ki = 0.27 microM), D-alanine (Ki = 1.02 microM), and D-cycloserine (Ki = 2.33 microM) are potent inhibitors of binding, whereas the corresponding L isomers have significantly less activity (Ki = 25.4 microM, 15.9 microM, and greater than 100 microM, respectively). Inactive at concentrations of up to 100 microM were strychnine, L-valine, N,N-dimethylglycine, aminomethylphosphonate, and aminomethylsulfonate. The active compounds were analyzed further for their ability to stimulate [3H]1-[1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) binding to Triton X-100-washed SPM. Results indicate that the affinity of the compounds for the [3H]glycine recognition site correlates with the ability of these analogues to stimulate [3H]TCP binding.     

5-[4-(3,3-Dimethylbutoxycarbonyl)phenyl]-4-pentynoic acid and its derivatives inhibit ionotropic gamma-aminobutyric acid receptors by binding to the 4'-ethynyl-4-n-propylbicycloorthobenzoate site

Acyclic noncompetitive antagonists of ionotropic gamma-aminobutyric acid (GABA) receptors, bearing an ester or ether linkage, were designed, synthesized, and assayed for their inhibition of the specific binding of [3H]4'-ethynyl-4-n-propylbicycloorthobenzoate (EBOB), a radiolabeled noncompetitive antagonist, to rat brain and housefly head membranes. 5-[4-(3,3-Dimethylbutoxycarbonyl)phenyl]-4-pentynoic acid (DBCPP), a butyl benzoate analogue, was found to competitively inhibit the binding of [3H]EBOB in rat brain membranes, with an IC50 of 88 nM. The potency conferred by the p-substituent decreased in the order C(triple bond)C(CH2)2COOH > C(triple bond)C(CH2)2COOCH3 > C(triple bond) CH > Br. Pentyl phenyl ethers were equally potent compared with butyl benzoates, while phenyl pentanoates and benzyl butyl ethers were less pont. These compounds were generally less active in housefly head membranes than in rat brain membranes. The introduction of an isopropyl group into the 1-position of the 3,3-dimethylbutyl group of a butyl benzoate and two benzyl butyl ethers caused an increase in potency in housefly GABA receptors, whereas this modification at the corresponding position of other compounds led to an unchanged or decreased potency. In the case of rat receptors, this modification resulted in a decrease in potency except for a phenyl pentanoate. To confirm that DBCPP interferes with GABA receptor function, we performed whole-cell patch clamp experiments with rat dorsal root ganglion neurons in the primary culture. Repeated co-applications of GABA and DBCPP suppressed GABA-induced whole-cell currents with an IC50 of 0.54 microM and a Hill coefficient of 0.7. These findings indicate that DBCPP and its derivatives inhibit ionotropic GABA receptors by binding to the EBOB site and that there might be structural difference in the noncompetitive antagonist-binding site between rat and housefly GABA receptors.