2-N-acylaminoalkylindoles: design and quantitative structure-activity relationship studies leading to MT2-selective melatonin antagonists.

Several indole analogues of melatonin (MLT) were obtained by moving the MLT side chain from C(3) to C(2) of the indole ring. Binding and in vitro functional assays were performed on cloned human MT1 and MT2 receptors, stably transfected in NIH3T3 cells. Quantitative structure-activity relationship studies showed that 4-methoxy-2-(N-acylaminomethyl)indoles, with a benzyl group in position 1, were selective MT2 antagonists and, in particular, N-[(1-p-chlorobenzyl-4-methoxy-1H-indol-2-yl)methyl]propanamide (12) behaved as a pure antagonist at MT1 and MT2 receptors, with a 148-fold selectivity for MT2. We present a topographical model that suggests a lipophilic group, located out of the plane of the indole ring of MLT, as the key feature of the MT2 selective antagonists.

Synthesis, pharmacological characterization and QSAR studies on 2-substituted indole melatonin receptor ligands.

A number of 6-methoxy-1-(2-propionylaminoethyl)indoles, carrying properly selected substituents at the C-2 indole position, were prepared and tested as melatonin receptor ligands. Affinities and intrinsic activities for the human cloned mt1 and MT2 receptors were examined and compared with those of some 2-substituted melatonin derivatives recently described by us. A quantitative structure activity relationship (QSAR) study of the sixteen 2-substituted indole compounds, 5a-k, 1, 8-11, using partial least squares (PLS) and multiple regression analysis (MRA) revealed the existence of an optimal range of lipophilicity for the C2 indole substituent. There are also indications that planar, electron-withdrawing substituents contribute to the affinity by establishing additional interactions with the binding pocket. No mt1/MT2 subtype selectivity was observed, with the relevant exception of the 2-phenethyl derivative 5e, which exhibited the highest selectivity for the h-MT2 receptor among all the compounds tested (MT2/mt1 ratio of ca. 50). Conformational analysis and superposition of 5e to other reported selective MT2 ligands revealed structural and conformational similarities that might account for the MT2/mt1 selectivity of 5e.

Structure-property relationships on histamine H3-antagonists: binding of phenyl-substituted alkylthioimidazole derivatives to rat plasma proteins.

The binding of a series of H3-antagonists to rat plasma proteins was investigated by dialysis experiments, with RP-HPLC measurement of the free ligand. The series was composed of 4(5)-phenyl-2-[[2-[4(5)-imidazolyl]ethyl]thio]imidazoles having, on the phenyl ring, meta- and para-substituents, with different physico-chemical characteristics. As high protein binding had been proposed as being one of the features limiting brain access for the reference H3-antagonist thioperamide, the title series was employed to test the possibility of achieving lower protein binding by modulation of lipophilicity, while maintaining good receptor affinity. The compounds tested showed quotas of bound drug ranging from 60 to 97.5%, while for thioperamide a 78% bound drug quota was observed at high total concentrations, with a steep increase in bound percentage at lower concentrations. Two of the tested compounds, having a carboxamide substituent, showed lower protein binding compared to thioperamide over a wide range of total concentration, without a significant loss in affinity with respect to the parent compound. A strict dependence of protein binding on lipophilicity was observed, and a QSPR model was derived which could also account for the protein binding observed for thioperamide, while receptor affinity had been reported to be quite insensitive to phenyl ring substitution. It is therefore possible to modulate protein binding of these H3-antagonists, through lipophilicity adjustment, without losing receptor affinity; this finding could help in the design of new compounds with improved brain access.

Synthesis and biological assays of new H3-antagonists with imidazole and imidazoline polar groups.

New histamine H3-receptor antagonists were synthesised and tested on rat brain membranes and on electrically stimulated guinea-pig ileum. The new compounds have a central polar group represented by a 2-alkylimidazole or a 2-thioimidazoline nucleus. The effect of the polar group basicity on the optimal length of the alkyl chain, connecting this group to a 4(5)-imidazolyl ring, was investigated. The best affinity values, obtained by displacement of [3H]-RAMHA from rat brain, were obtained for the 2-alkylimidazole derivatives (2a-f) with tetramethylene chain (pKi 8.03-8.97), having an intermediate basicity between that of the previously reported 2-thioimidazoles (1a-i) and that of 2-alkylthioimidazolines (3a-h). In contrast, a general lowering of affinity (pKi 5.90-7.63) was observed for compounds of the last series (3a-h), with a complex dependence on the terminal lipophilic group and chain length.

Experimental and theoretical analysis of the interaction of (+/-)-cis-ketoconazole with beta-cyclodextrin in the presence of (+)-L-tartaric acid.

1H NMR spectroscopy was used for determining the optical purity of cis-ketoconazole enantiomers obtained by fractional crystallization. The chiral analysis was carried out using beta-cyclodextrin in the presence of (+)-L-tartaric acid. The mechanism of the chiral discrimination process, the stability of the complexes formed, and their structure in aqueous solution were also investigated by 1H and 13C chemical shift analysis, two-dimensional NOE experiments, relaxation time measurements, and mass spectrometry experiments. Theoretical models of the three-component interaction were built up on the basis of the available NMR data, by performing a conformational analysis on the relevant fragments on ketoconazole and docking studies on the components of the complex. The model derived from a folded conformation of ketoconazole turned out to be fully consistent with the molecular assembly found in aqueous solution, as inferred from NOE experiments. An explanation of the different association constants for the complexes of the two enantiomers is also provided on the basis of the interaction energies.

Melatonin.

Melatonin is the principal hormone secreted by the pineal gland, produced in humans with a circadian rhythm characterized by elevated blood levels during the night. It is involved in the regulation of several rhythmic functions in various vertebrates, and participates in the processing of photoperiodic information. Although its role in human physiologic and pathologic processes is not yet completely understood, MLT exerts a number of actions, in physiological or pharmacological concentrations, which could be of interest for future therapeutic uses. The mechanisms involved in MLT actions include interaction with membrane receptors, recently classified as mt1/MT2/MT3, and with nuclear sites corresponding to orphan members of the nuclear receptor superfamily, RZR/ROR; MLT also acts as a radical scavenger, exerting a protective action against various oxidative injuries. The present review is mainly addressed to the medicinal chemistry of ligands at the MLT membrane receptors, focusing on the models of binding interaction published in the literature. Several different pharmacophore and 3D-QSAR models have been reported so far, and a re-consideration of known active compounds, in the light of the recently developed biological tests on cloned receptors, could help to resolve the incongruities among these models; to this end, additional information is becoming available from new, conformationally constrained ligands, and from antagonist compounds with a selective affinity for receptor subtypes.

Melatonin receptor ligands: synthesis of new melatonin derivatives and comprehensive comparative molecular field analysis (CoMFA) study.

The CoMFA methodology was applied to melatonin receptor ligands in order to establish quantitative structure-affinity relationships. One hundred thirty-three compounds were considered: they were either collected from literature or newly synthesized in order to gain information about the less explored positions. To this end, various melatonin derivatives were prepared and their affinity for quail optic tecta melatonin receptor was tested. Compounds were aligned on the putative active conformation of melatonin proposed by our previously reported pharmacophore search, and their relative affinities were calculated from the displacement of 2-[125I]-iodomelatonin on different tissues expressing aMT receptors. Compounds were grouped into three sets according to their topology. Subset A: melatonin-like compounds; subset B: N-acyl-2-amino-8-methoxytetralins and related compounds; subset C:N-acyl-phenylalkylamines and related compounds. CoMFA models were derived for each set, using the steric, electrostatic, and lipophilic fields as structural descriptors; the PLS analyses were characterized by good statistical parameters, taking into account the heterogeneity of the binding data, obtained with different experimental protocols. From the CoMFA model for the melatonin-like compounds, besides the well-known positive effect of 2-substitution, a low steric tolerance for substituents in 1, 6, and 7, and a negative effect of electron-rich 4-substituents were observed; the information provided by the newly synthesized compounds was essential for these results. Moreover, a comprehensive model for the 133 compounds, accounting for a common alignment and a common mode of interaction at the melatonin receptor, was derived (Q2 = 0.769, R2 = 0.905). This model validates our previously reported pharmacophore search and offers a clear depiction of the structure-affinity relationships for the melatonin receptor ligands.

2-[N-Acylamino(C1-C3)alkyl]indoles as MT1 melatonin receptor partial agonists, antagonists, and putative inverse agonists.

The synthesis of several novel indole melatonin analogues substituted at the 2-position with acylaminomethyl (8-11), acylaminoethyl (5a-k), or acylaminopropyl (13) side chains is reported. On the basis of a novel in vitro functional assay (specific binding of [35S]GTPgammaS), which can discriminate agonist from partial agonist, antagonist, and inverse agonist ligands, 5a,g, h,j and 13 were shown to be partial agonists, 5d,e and 8-11 competitive antagonists, and 5b,c,k putative inverse agonists. Binding and functional assays were performed on cloned human MT1 receptor. Structure-activity relationship considerations indicate that N-[1-aryl-2-(4-methoxy-1H-indol-2-yl)(C1-C2)alkyl]alkanamides represent a lead structure for this type of ligands.

H3-receptor antagonists: synthesis and structure-activity relationships of para- and meta-substituted 4(5)-phenyl-2-[[2-[4(5)-imidazolyl]ethyl]thio]imidazoles.

We report the synthesis, octanol/water partition coefficient (log P), dissociation constants (pKa), H3-receptor affinity (pKi in rat brain membranes, [3H]-N alpha-methylhistamine), and H3-antagonist potency (pA2 in guinea ileum, (R)-alpha-methylhistamine) of novel H3-receptor antagonists obtained by introducing a para or meta substituent on the phenyl ring of the lead compound 4(5)-phenyl-2-[[2-[4(5)-imidazolyl]ethyl]thio]imidazole (3a). The substituents were chosen to obtain broad and uncorrelated variation in their lipophilic, electronic, and steric properties. The log P values of the neutral species cover almost 3 orders of magnitude (from 1.40 to 4.11). The pKa,2 values (protonation of the 2-thioimidazole fragment) vary from 3.13 to 4.34, indicating that this fragment, which incorporates the so-called polar group common to many H3-receptor antagonists, is neutral at physiological pH. The compounds had pKi values in a range too narrow (from 7.28 to 8.03) to derive QSAR equations. In one case (3g), a biphasic displacement curve was observed (pKi,1 = 8.53; pKi,2 = 6.90). The pA2 values ranged 2 orders of magnitude (from 6.83 to 8.87) and yielded a QSAR model (PLS) indicating that antagonist potency depends parabolically on lipophilicity and is decreased by bulky para substituents. The compounds of this series, therefore, maintain a fair-to-good affinity for rat brain H3-receptor and a fair-to-good H3-antagonist potency on guinea pig ileum, although varying markedly in their lipophilicity. The series thus appears as a good candidate for pharmacokinetic optimization leading to brain-penetrating H3-receptor antagonists.

Conformationally restrained melatonin analogues: synthesis, binding affinity for the melatonin receptor, evaluation of the biological activity, and molecular modeling study.

The design, synthesis, and biological profile of several indole melatonin analogues with a conformationally restricted C3 amidoethane side chain are presented. Examination of the accessible conformations of the melatonin side chain led us to explore some of its fully or partially restricted analogues, 2-12, the binding affinity values of which were utilized to gain further insight on the melatonin binding site. Two pharmacophoric models have been devised for melatonin and the active compounds by conformational analysis and superimposition performed using the DISCO program. In these models, the melatonin side chain can adopt a gauche/anti conformation out of the indole plane. Another contribution of this study regards the observation of a possible binding point interaction around the C2 position of the indole, as suggested by the remarkably increased binding affinity observed in the C2-substituted analogues 6 and 9 and especially in the more rigid analogue 5. The biological activity and the efficacy of the new compounds were tested by measuring the inhibition of the forskolin-stimulated cAMP accumulation and the GTP gamma S index. Both analyses demonstrated that all of the compounds were full agonists with the exception of 4 and 9, which showed a slight reduction in efficacy and would seem to be partial agonists.

H3-receptor antagonists: conformational analysis of thioperamide and x-ray crystal structure of the analog N-cyclohexyl-4-methylpiperidine-1-carbothioamide.

Thioperamide (N-cyclohexyl-4-[4(5)-imidazolyl]piperidine-1-carbothioamide) is a potent H3-receptor antagonist, the low conformational flexibility of which could be a favourable feature in the design of new H3-receptor antagonists using its structure as a template. Minimum-energy conformations of thioperamide were studied with the molecular mechanics approach, integrated by X-ray crystallography on an analogue, N-cyclohexyl-4-methylpiperidine-1-carbothioamide (1). Compound 1 was synthesized, and its structure has been solved by X-ray diffraction in order to verify the conformation of the piperidine-1-carbothioamide fragment, and to compare the crystallographic results with those of molecular mechanics. Conformational analysis on the free-rotating bonds of thioperamide was performed with different search methods in order to find the minimum-energy conformations and to estimate rotational barriers. For steric reasons, the rotation around the bond connecting the cyclohexane ring with the carbothioamide nitrogen is more hampered than that around the bond connecting imidazole with piperidine. The rotation around the first bond presents two symmetrical energy minima separated from a third minimum by an energy barrier of 40 KJ/mol. The spatial disposition of compound 1 in the crystal and the common part of thioperamide in one of its minimum-energy conformations are very similar. The minimum-energy conformations of thioperamide calculated by molecular mechanics are therefore reliable and they can be used for structural comparisons with other H3-receptor antagonists.

Plasma concentration and brain penetration of the H3-receptor antagonist thioperamide in rats.

Thioperamide is a potent and selective H3-receptor antagonist, whose in vivo effects have been reported after systemic administration. Some questions have arisen about its ability to cross the blood-brain barrier, since different experimental conditions have given different results in rats, namely a low brain/blood ratio at low doses (10 mg/Kg) and a much higher one at higher doses (60 mg/Kg). In this work we demonstrate the dose-dependence of thioperamide pharmacokinetics, measuring its plasma and cerebral levels after i.p. administration of different doses to rats. Both the plasma half-life and brain penetration of thioperamide resulted as being dose-dependent: when administered to 80 g body weight Wistar rats at 10 mg/Kg i.p., the drug has a short half life (120') and a rather poor brain penetration, but increasing the dose (to 20, 40 and 60 mg/Kg) gives rise to a prolongation of its persistence in the blood (up to 600' at highest dose) and a higher brain penetration. Also, the profile of the plasma concentration curve varies from the dose of 10 to that of 20 mg/Kg, passing from a mono-exponential decrease to a more complex one characterized by an apparent distribution phase. The different distribution processes can be interpreted in the light of thioperamide protein binding and affinity for lipophilic tissues: protein binding can prevent brain penetration (but not distribution to other tissues) at lower doses, while at higher doses the free plasma fraction increases and it can allow passive distribution to lipophilic tissues such as brain tissues. A re-distribution from these tissues and plasma is probably responsible for the strong increase in half-life at high doses.

Ligands of the histamine H3-receptor: new potent antagonists of the 2-thioimidazole type.

An overview of H3-receptor ligands is presented, with particular attention to antagonists. The protein binding of the classical H3-receptor antagonist thioperamide and its effect on in vivo distribution are discussed. A series of H3-receptor antagonists characterised by the presence of an imidazole ring, a spacer (ethylthio-, ethylamino-, propylthio- or propylamino-chain), a second heterocycle nucleus and a lipophilic group is described. Their H3-receptor antagonist potency has been measured on electrically stimulated guinea-pig intestine, and their affinity for central H3-receptor has been determined by competitive inhibition of [3H]N alpha-methylhistamine binding to rat cortex. Biphasic inhibition curves have been observed in some cases. Compounds endowed with interesting activity belong mostly to the class of 2-[[2-[4(5)-imidazolyl]ethyl]thio]imidazole, having a phenyl or a cyclohexyl group.

Rat protein binding and cerebral phospholipid affinity of the H3-receptor antagonist thioperamide.

The binding of thioperamide, a known H3-receptor antagonist, to rat plasma and proteins and its affinity for rat cerebral phospholipids are investigated. Thioperamide is strongly bound to plasma proteins (95-80% at plasma concentrations of 3.5-400 micrograms mL-1), and its binding can be resolved into two components a high-affinity, saturable component and a non-specific component. The drug has a high affinity for cerebral phospholipids, with a partition coefficient of approximately 100 (log K = 2.06 +/- 0.14), which should promote brain penetration and accumulation. Protein binding and cerebral phospholipid affinity can suggest the explanation of some differences reported in the literature on thioperamide distribution data: at low plasma concentrations of the drug, its protein binding (95% at 3.5 micrograms mL-1) can prevent brain accumulation, while at higher concentrations the free plasma fraction suddenly increases (> 10% at 18 micrograms mL-1) and it allows passive distribution to lipophilic tissues such as brain tissue.

Biological studies on 1,2-benzisothiazole derivatives V. Antimicrobial properties of N-alkanoic, N-arylalkanoic and N-aryloxyalkanoic derivatives of 1,2-benzisothiazolin-3-one: QSAR study and genotoxicity evaluation.

N-alkanoic, N-arylalkanoic and N-aryloxyalkanoic acids of 1,2-benzisothiazolin-3-one, esters and amides of N-arylalkanoic and N-aryloxyalkanoic acids and 1,1-dioxide derivatives of N-arylalkanoic acids and esters were investigated in vitro antimicrobial activity. N-arylalkanoic and N-aryloxyalkanoic acids (compounds 4-12) and their esters and amides (compounds 13-26) exhibited a good antimicrobial activity against Gram positive bacteria, with several compounds showing potencies 10-20 times higher than 1,2-benzisothiazolin-3-one. None of the chemicals tested inhibited the growth of E. coli. Yeasts and moulds possess a considerable susceptibility to compounds 12-23. The logP (octanol-water) of esters and amides of N-arylalkanoic and N-aryloxyalkanoic acids were measured by the shake-flask technique and the potencies against Gram positive bacteria of the compounds tested was related to their lipophilicity. QSAR analysis showed a bilinear relation, with a logD0 around 3 for the activity on B. subtilis. The phenoxyacetic and phenoxybutyric acid derivatives are positive outliers, showing a potency higher than that predicted from their lipohilicity. The most active compounds were further tested against different Gram positive bacteria and moulds, including Bacilli, Sarcina lutea, Staphylococcus epidermidis, Candida spp. and dermatophytes. The antibacterial and antifungal activity was specific for 1,2-benzisothiazolin-3-ones, the corresponding 1,1-dioxide derivatives being inactive. The genotoxic properties of the compounds studied were evaluated by the Bacillus subtilis rec-assay and Salmonella-microsome test. None of the compounds showed DNA-damaging or mutagenic activity.

QSAR study on H3-receptor affinity of benzothiazole derivatives of thioperamide.

Starting from the structure of thioperamide, a known H3-antagonist, a new series of compounds with a benzothiazole nucleus instead of the cyclohexylcarbothioamide moiety was synthesized. Various substituents, selected by experimental design, were introduced in position 6 of the benzothiazole nucleus, in order to change its physico-chemical characteristics. The lipophilicity of the synthesized compounds was measured by means of RP-HPLC, and their H3-receptor affinity was evaluated by competitive binding assays on rat cortex synaptosomes, with the labelled ligand N alpha-[3H]methylhistamine. A QSAR analysis was performed on the experimental data, using also substituent constants taken from the literature. The newly synthesized compounds showed lower H3-affinities than thioperamide; quantitative structure-activity relationships, described by models obtained with PLS and MRA techniques, were observed among benzothiazole derivatives. According to these relationships, any attempt to improve the potency of these compounds should involve the substitution of the benzothiazole moiety with less bulky and/or more flexible structures, which should also be less lipophilic and allow better electronic interactions with the binding site. 1-(Benzothiazol-2-yl)-4-[(1H)-imidazol-4-yl]piperidine represents a limit structure for H3-activity, since it seems impossible to improve its affinity by means of substitution in the studied position of the benzothiazole nucleus, as shown by predictions performed by a PLS model.

Synthesis and binding assays of H3-receptor ligands.

The preparation of a representative group of derivatives of the known H3-antagonist thioperamide is described. Binding affinity for histamine H3-receptors of thioperamide and its derivatives, which were obtained by substitution on the imidazole ring, was measured on rat brain cortex synaptosomes. Competitive binding assays were performed with two different labelled ligands, the physiological agonist [3H]histamine ([3H]HA) and the potent H3-agonist N alpha-[3H]methyl-histamine ([3]NAMHA). We observed a remarkable difference in Ki values obtained versus the two labelled ligands, both for thioperamide and its derivatives. In particular, 5-methylthioperamide showed a considerable selectivity for the system recognized by [3H]NAMHA, being about 100 times more potent versus this system than versus the system recognized by [3H]HA. On the basis of these observations, we suggest that it is necessary to consider this difference in evaluating the affinity of new compounds for the H3-receptors.

HPLC detection of thioperamide from biological samples and its determination in rat blood and brain after systemic administration.

Thioperamide is a potent and selective antagonist on histamine H3 receptors. A method for its isolation and quantitation by HPLC from rat plasma and brain samples has been developed. Using this technique, thioperamide concentrations in rat plasma and brain were measured after systemic administration, in order to evaluate its persistence in blood and its ability to cross the blood-brain barrier. We observed that, at a dose of 60 mg/Kg, thioperamide undergoes a slow elimination from plasma, with a half-life of 10 hours, and can readily cross the blood-brain barrier.

4-(1,2-Benzisothiazol-3-yl)alkanoic and phenylalkanoic acids: synthesis and anti-inflammatory, analgesic and antipyretic activities.

Continuing their studies on benzisothiazolyl derivatives, Authors refer to the preparation and pharmacological properties of 4-(1,2-benzisothiazol-3-yl) alkanoic and phenylalkanoic acids. All substances were tested for anti-inflammatory, analgesic and antipyretic properties. As reference compounds, 1,2-benzisothiazolin-3-one and 4-(3-oxo-1,2-benzisothiazolin-3-yl) phenylacetic acid, as prototypes of benzisothiazolinonic derivation. Ibuprofen, as a prototype of substituted arylalkanoic acids, and Phenylbutazone were used. Analysis of the data leaded to the following conclusions. Introduction of the aryl moiety, passing from benzisothiazolylalkanoic to benzisothiazolyl-phenylalkanoic structures, produced a remarkable increase of activity. 2-[4-(1,2-benzisothiazol-3-yl)phenyl] propionic and 2-[4-(1,2-benzisothiazol-3-yl)phenyl]butyiric acids showed anti-inflammatory, analgesic and antipyretic properties comparable to those of Ibuprofen. Substantial differences in variations in activities were observed comparing the properties of benzisothiazolylphenylalcanoic acids with those of the benzisothiazolinonic series, object of preceding studies.