Structure-activity studies on nociceptin analogues: ORL1 receptor binding and biological activity of cyclic disulfide-containing analogues of nociceptin peptides.

Nociceptin/Orphanin FQ is an endogenous peptide ligand for the opioid receptor-like 1 (ORL1) receptor. To investigate the structural and conformational requirements of the nociceptin (NC)-receptor interaction, six cyclic analogues containing Cys disulfide linkages were designed and synthesized. Analogues cyclized at the N-terminal part, cyclo[Cys(0), Cys(7)]NC(1-13)-NH(2) (2) and cyclo[Cys(0), Cys(11)]NC(1-13)-NH(2) (4), and their corresponding linear peptides had very low activities in both the receptor binding and the GTP gamma S functional assays using human ORL1 transfected cell membranes. On the contrary, analogues cyclized at the C-terminal parts by the disulfide linkages at positions 6-10, 7-11, 7-14, and 10-14 sustained relatively high potencies in both assays. Notably, cyclo[Cys(10), Cys(14)]NC(1-14)-NH(2) (12) was found to be a potent NC agonist nearly as active as the parent peptide or NC. The maximum efficacy (Emax) of the C-terminally cyclized analogues and their linear counterparts in the GTP gamma S functional assay showed more than 94% (vs NC as 100%), suggesting that these analogues are full agonists. Analogue 12 is the first conformationally constrained NC analogue with almost full activity, and thus may serve to analyze the bioactive conformations of NC at the receptor site as well as serving as a template for more potent NC agonists.     

Specific inhibition of benzodiazepine receptor binding by some 1,2,3-triazole derivatives

Certain 1,2,3-triazole derivatives were prepared and tested for their ability to displace [3H]diazepam from bovine brain membranes. From these compounds, the quinolytriazole derivatives (14, 15, 16, 17) were clearly the most potent, while the naphthyl- and the naphthyridyl-triazoles were considerably less active. The p-nitrophenyl derivative (15) was the compound that bound with the highest affinity within the quinolyltriazole compounds class. The replacement of the p-nitrophenyl group with other substituents greatly decreased the binding activity. From a Lineweaver-Burk analysis of 11, it appears that the inhibition is competitive.     

Structure-activity relationships of receptor binding of 1,4-dihydropyridine derivatives

The present study was undertaken to investigate binding activity of synthesized 1,4-dihydropyridine (1,4-DHP) derivatives (Compounds 1--124) to 1,4-DHP calcium channel antagonist receptors in rat brain. Sixteen 1,4-DHP derivatives inhibited specific (+)-[3H]PN 200-110 binding in rat brain in a concentration-dependent manner with IC50 value of 0.43 to 3.49 microM. Scatchard analysis revealed that compounds 54, 69, 85, like nifedipine, caused a significant increase in apparent dissociation constant (Kd) for (+)-[3H]PN 200-110, while compounds 68, 69 and 80 caused a significant decrease in maximal number of bindings sites (Bmax). These data suggest that compounds 68, 69 and 80 exert longer-acting antagonistic effects of 1,4-DHP receptors than compounds 54, 69 and 85. The structure-activity relationship study has revealed that 1) ester groups in 3- and 5-positions are the most effective, 2) the aryl group in the 4-position of 1,4-DHP ring is the basic requirement for optimal activity, 3) position and type of electron-withdrawing groups on phenyl group at position 4 would affect the receptor-binding activity. Furthermore, compound 58 exerted alpha1 receptor binding activity, being 1.6 times greater than 1,4-DHP receptors. Compounds 81, 84, 91, 94, 106, 108 and 109 showed significant binding of ATP-sensitive potassium (K ATP) channel, and the binding activities of compounds 81, 84, 108 and 109 were 1.6--3.8 times greater than the binding activity for 1,4-DHP receptors. Compounds 91 and 106 had similar binding activity for K ATP channel and 1,4-DHP receptors. In conclusion, the present study has shown that novel 1,4-DHP derivatives exert relatively high binding affinity to 1,4-DHP receptors and has revealed new aspect of structure-activity relationships of 1,4-DHP derivatives, especially hexahydroquinoline derivatives.     

Studies on the synthesis of condensed pyridazine derivatives. III. Synthesis and benzodiazepine receptor binding studies of condensed triazolopyridazine derivatives

A series of 6H-(1)benzothiopyrano[3,4-e][1,2,4]triazolo[4,3- b]pyridazines and 6,7-dihydro-(1)benzothiepino[4,5- e][1,2,4]triazolo [4,3-b]pyridazines were prepared and tested for their ability to displace [3H] diazepam from rat brain membranes. An approximately planar shape of these molecules was essential for high affinity to the benzodiazepine receptor. Among them, 11-aryl compounds in the latter series were found to have high affinity to the benzodiazepine receptor. 11-Phenyl- and 11-thienyl- 6,7-dihydro-(1)benzothiepino[4,5-e][1,2,4]triazolo[4,3- b]pyridazine (3b-5 and 3b-11 respectively) showed the potent affinity comparable to that of diazepam. The structure-activity relationships are also discussed.     

Structure-activity studies on splitomicin derivatives as sirtuin inhibitors and computational prediction of binding mode

NAD (+)-dependent histone deacetylases (sirtuins) are enzymes that cleave acetyl groups from lysines in histones and other proteins. Potent selective sirtuin inhibitors are interesting tools for the investigation of the biological functions of those enzymes and may be future drugs for the treatment of cancer. Splitomicin was among the first two inhibitors that were discovered for yeast sirtuins but showed rather weak inhibition on human enzymes. We present detailed structure-activity relationships on splitomicin derivatives and their inhibition of recombinant Sirt2. To rationalize our experimental results, ligand docking followed by molecular mechanics Poisson-Boltzmann/surface area (MM-PBSA) calculations were carried out. These analyses suggested a molecular basis for the interaction of the beta-phenylsplitomicins with human Sirt2. Protein-based virtual screening resulted in the identification of a novel Sirt2 inhibitor chemotype. Selected inhibitors showed antiproliferative properties and tubulin hyperacetylation in MCF7 breast cancer cells and are promising candidates for further optimization as potential anticancer drugs.     

Structure-activity relationships in carboxamide derivatives based on the targeted delivery of radionuclides and boron atoms by means of peripheral benzodiazepine receptor ligands

The structure-activity relationship studies on 2-quinolinecarboxamide peripheral benzodiazepine receptor (PBR) ligands have been refined with the aim of using these ligands as carriers of radionuclides and boron atoms. Some new ligands show enhanced affinity and steroidogenic activity with respect to reference compound 1 and are interesting candidates for radiolabeling and PET studies. Moreover, carborane derivative 3q, representing the first example of PBR ligand bearing a carborane cage, can be useful to explore an alternative mechanism in BNCT.     

Theoretical structure-activity studies of benzodiazepine analogues. Requirements for receptor affinity and activity

Conformational and electronic properties of a series of 1,4-benzodiazepine analogues and their specific interaction with a model cationic receptor site have been calculated using both empirical energy and semiempirical molecular orbital methods. The aim of these studies was to identify molecular properties and modes of receptor interaction which are determinants of relative receptor affinities and pharmacological activities for these anxiolytics. Analogues with variations in key positions of the 7-membered (B) ring, at positions C7, C8, and C9 of the fused phenyl (A) ring, and at positions 2' and 4' of the phenyl (C) ring were examined. The results indicate that both active and inactive analogues have similar low-energy conformations, arguing against this property as a modulator of recognition at the receptor. However, calculated molecular electrostatic potentials together with explicit model receptor interactions allowed the deduction that interactions with three cationic receptor sites are required for high-affinity analogues. The specific cationic site interactions are postulated with electron-withdrawing groups at C7, the C2 = O1 group, and the imine nitrogen, N4. Moreover, interactions of N4 with a model cationic receptor site are enhanced by halogen substituents at C2', but only when the phenyl ring is rotated by 30 degrees toward a more planar conformer, corresponding to an induced conformational change. If this enhancement is important, a 2'-Cl substituent on more rigid analogues of the 1,4-benzodiazepines with increased co-planarity of the phenyl C-ring and the C1'--C5 = N4 plane should have an even greater differential effect on receptor affinity.     

Microcalorimetric studies on the binding of some benzodiazepine derivatives to human serum albumin.

The authors have employed a microcalorimetric method to study the nature of the interactions between several benzodiazepin molecules and human serum albumin. The thermodynamic functions of complexation have been determined at different pH, ionic strength and electrolyte support conditions. Both electrostatic and hydrophobic interactions have been demonstrated to exist in proportions which vary according to the drug's molecular structure.     

Different residues in the GABAA receptor benzodiazepine binding pocket mediate benzodiazepine efficacy and binding

Benzodiazepines (BZDs) exert their therapeutic actions by binding to the GABA(A) receptor (GABA(A)R) and allosterically modulating GABA-induced chloride currents (I(GABA)). A variety of ligands with divergent structures bind to the BZD site, and the structural mechanisms that couple their binding to potentiation of I(GABA) are not well understood. In this study, we measured the effects of individually mutating 22 residues throughout the BZD binding pocket on the abilities of eszopiclone, zolpidem, and flurazepam to potentiate I(GABA). Wild-type and mutant α(1)β(2)γ(2) GABA(A)Rs were expressed in Xenopus laevis oocytes and analyzed using a two-electrode voltage clamp. GABA EC(50), BZD EC(50), and BZD maximal potentiation were measured. These data, combined with previous radioligand binding data describing the mutations' effects on BZD apparent binding affinities (J Neurosci 28:3490-3499, 2008; J Med Chem 51:7243-7252, 2008), were used to distinguish residues within the BZD pocket that contribute to BZD efficacy and BZD binding. We identified six residues whose mutation altered BZD maximal potentiation of I(GABA) (BZD efficacy) without altering BZD binding apparent affinity, three residues whose mutation altered binding but had no effect on BZD efficacy, and four residues whose mutation affected both binding and efficacy. Moreover, depending on the BZD ligand, the effects of some mutations were different, indicating that the structural mechanisms underlying the ability of BZD ligands with divergent structures to potentiate I(GABA) are distinct.     

Structure-activity requirements for hypotension and alpha-adrenergic receptor blockade by analogues of atropine

The hypotensive action of various antimuscarinic compounds structurally related to atropine was studied in conscious, unanesthetized rats. The alpha-adrenolytic activity of these agents was assessed both in vivo (blockade of norepinephrine-induced pressor response) and in vitro (displacement of [3H]WB-4101 binding). Benztropine, homatropine and hyoscyamine caused hypotension and produced alpha-adrenergic receptor blockade similar to atropine. Other analogues were either inactive (atroscine, scopolamine, tropic acid and tropine) or evoked nonspecific changes in blood pressure and lacked alpha-adrenolytic activity (benactyzine, eucatropine, methylatropine, methylhomatropine and methylscopolamine). Based on these data, we propose the following structure-activity relationship for hypotension and alpha-adrenolytic activity: (a) the tropine moiety is inactive unless it is attached to another group by an ester linkage, (b) chemical modification of the tropine moiety, including quaternization, decreases potency, (c) the d-stereoisomer appears to be more potent than the corresponding 1-form.     

Structure-activity relationship studies of novel oxygen-incorporated SAHA analogues

Novel oxygen-incorporated SAHA (oxa-SAHA) analogues, in which oxygen was inserted in the alkyl linker connecting the hydroxamic acid moiety and amide group, were synthesized and their inhibitory activities on histone deacetylase were evaluated. The most active oxa-SAHA analogue potently inhibited histone deacetylase, almost as potently as SAHA. Various structural modifications in the amide, but not the hydroxamic acid, significantly affected the inhibitory activities of the derivatives. Based on the inhibitory data, the N-phenyl moiety of the amide turned to be a better modification site for enhancing the inhibitory activity.     

Structure-activity relations for the neurotoxicity of kainic acid derivatives and glutamate analogues

The neurotoxic potency of glutamate analogues and derivatives of kainic acid was examined in the chick neural retina and the rat corpus striatum by measuring the effects of the agents on the activity of neurotransmitier-synthesizing enzymes. In terms of neurotoxic potency, in the retina: $\text{kainate}\text{?}\text{ibotenate}\text{?}\text{quisqualate}\text{> N-}\text{methyl}\text{-}\text{d,l}\text{-}\text{aspartate}\text{?}\text{d,l}\text{-}\text{homocysteate}\text{>}\text{l}\text{-}\text{glutamate}$ whereas in the corpus striatum: $\text{kainate}\text{>}\text{ibotenate}\text{> N-}\text{methyl}\text{-}\text{d,l}\text{-}\text{aspartate}\text{>}\text{d,l}\text{-}\text{homocysteate}\text{?}\text{l}\text{-}\text{glutamate}$. In both the retina and corpus striatum, dihydrokainate, N-acetylkainate and kainic acid dimethylester are at least 45 to 100-fold less toxic than kainate. These studies demonstrate a correlation between the neurotoxic and neuro-excitatory effects of glutamate analogues.     

Rotarod studies in the rat of the GABAA receptor agonist gaboxadol: lack of ethanol potentiation and benzodiazepine cross-tolerance

All benzodiazepines and benzodiazepine site agonists impair motor performance dose-dependently and potentiate the effects of ethanol. In order to evaluate the risk of benzodiazepine and ethanol interaction with the direct acting GABA(A) receptor agonist 4,5,6,7-tetrahydroisoxazolo (5,4-c) pyridin-3-ol (gaboxadol), we studied impairment of motor coordination for combinations of gaboxadol, ethanol and a series of benzodiazepines (flunitrazepam, zolpidem and indiplon) in a rat rotarod model. All compounds produced a dose-dependent motor impairment and, in agreement with earlier data, a supra-additive effect of the benzodiazepine ligands and ethanol 1 g/kg was seen. In contrast, no significant potentiation of the effects of gaboxadol by ethanol was detected, and furthermore, no synergistic interaction between gaboxadol and any of the benzodiazepines was seen. A 30-day tolerance study was conducted with daily injections of gaboxadol (7.9 mg/kg) and zolpidem (1.25 mg/kg), respectively. A time-dependent tolerance developed to the motor impairment produced by both compounds. On day 31, cross-tolerance studies between zolpidem/gaboxadol and gaboxadol/zolpidem were conducted. No cross-tolerance was observed, indicating that the motor coordination effects observed with gaboxadol and zolpidem may arise from interaction with different receptor populations.     

Inhibition of sleep and benzodiazepine receptor binding by a beta-carboline derivative

The effects of systemic injections of beta-carboline-3-carboxylate-t-butyl ester (beta-CCtB) were investigated with regard to normally occurring sleep and several measures of benzodiazepine receptor occupancy in rats. A dose of 30 mg/kg of beta-CCtB was found to have a long time-course of action as measured by an in vivo assay for benzodiazepine binding, with an 84% depletion of [3H]diazepam binding at one hour after the intraperitoneal injection. This dose of beta-CCtB was shown to delay sleep onset, decrease non-REM and total sleep in the first two hours after the injection, and to delay the appearance of REM sleep after the sleep onset. The dose- and time-dependence of the effects on sleep approximated the dose- and time-dependence of inhibitory effects of an IP injection of beta-CCtB on in vitro measures of benzodiazepine receptor affinity and number.     

Specific inhibition of benzodiazepine receptor binding by some N-(indol-3-ylglyoxylyl)amino acid derivatives

Several N-(indol-3-ylglyoxylyl)amino acid derivatives were synthesized and tested for their affinity for the benzodiazepine receptor in bovine cortical membranes. From these compounds, the N-[(5-chloro-, 5-bromo-, or 5-nitroindol-3-yl)glyoxylyl]glycine or -alanine esters were clearly the most potent, while the 5-methoxy analogues were considerably less active. Moreover, esters were more active than the corresponding acids. It is concluded that the affinity of these derivatives for the benzodiazepine receptor is profoundly dependent on amino acid molecular size, as well as the hydrophobic and electronic properties of the compounds.     

Structure-activity studies of cerulenin analogues as protein palmitoylation inhibitors

Activation of ras oncogenes occurs in a high percentage of tumors, making the enzymes involved in the posttranslational processing of their encoded proteins (p21s) attractive targets for the development of new drugs. Although most effort has focused on farnesyl transferase, which catalyzes the first processing step, attachment of palmitate to p21 is required for optimal transformation by H-ras and N-ras. We have demonstrated that the natural product cerulenin ([2R,3S]-2,3-epoxy-4-oxo-7,10-trans,trans-dodecadienamide) inhibits the palmitoylation of H-ras- and N-ras-encoded p21s in parallel with inhibition of cell proliferation. More than 30 analogues of cerulenin, both aromatic and aliphatic, with various chain lengths and amide substitutions, have been synthesized for use in SAR studies. Studies on the inhibition of T24 cell proliferation indicate that the alpha-keto-epoxy moiety is critical for cytotoxicity, while alkyl chain length had only modest effects on potency. Several compounds inhibited the incorporation of [(3)H]palmitate into p21 in intact T24 cells, with the unsubstituted carboxamides being more active than N,N-dimethyl compounds. In contrast to the effects on palmitoylation, the only compounds which inhibited fatty acid synthase contained alkyl side chains of 12 carbons or fewer. Regression analyses indicated that inhibition of palmitoylation is more closely related to inhibition of proliferation than is inhibition of fatty acid synthase. Further characterization of the molecular pharmacology of these and analogous compounds may define a new class of drugs with antitumor activity.     

Stereospecific benzodiazepine receptor binding by the enantiomers of oxazepam sodium hemisuccinate

The ability of the enantiomers of oxazepam sodium hemisuccinate to displace ³H diazepam specifically bound to benzodiazepine receptors has been investigated in crude synaptosomal preparations from rat brain. The relative potencies of the enantiomers to displace ³n diazepam was consistent with their reported differential anticonvulsant activity in animals and anxiolytic activity in man.The benzodiazepines represent a class of drugs with important anxiolytic, hypnotic, muscle relaxant and anticonvulsant properties, (Randall, Schallek, Sternbach &; Ning, 1974). By using ³H diazepam as the ligand in binding studies with crude synaptosomal preparations it has recently been demonstrated that benzodiazepines bind to specific receptors in rat brain. Bound 5n diazepam is not displaced by putative neurotransmitters but the displacement potencies of other benzodiazepines is related to their anxiolytic activities (Squires &; Braestrup, 1977, Möhler &; Okada, 1977).If a drug exhibits stereoisomerism and the enantiomers have differing pharmacological potencies in vivo, then the pharmacological properties most relevant for these effects should also show stereospecificity (Enna, et al., 1976, Crow. &; Johnstone, 1977).The enantiomers of oxazepam sodium hemisuccinate (OXZ) have been reported to exhibit, differing anticonvulsant activity in animal studies (Mussini et al., 1972, de Angelis, Predominate &; Vertua, 1972) and (+) OXZ has recently been shown to be more effective than the racemic mixture in the treatment of anxious patients (Lescovelli, Castellani &; Perbellini, 1976) In view of these findings we have investigated the relative potencies of (+) and (-) OXZ in displacing specific-'H diazepam binding in preparations of rat brain.