Synthesis of benzylideneacetophenones and their inhibition of lipid peroxidation.

A series of benzylideneacetophenone derivatives have been synthesized and found to show potent inhibition of the lipid peroxidation (LPO) in rat liver microsomes. All 19 compounds prepared in this series are LPO inhibitors. The highest activity was found in para hydroxy derivatives with two meta tert-butyl substituents.

Isoquinoline and quinazoline urea analogues as antagonists for the human adenosine A(3) receptor.

Isoquinoline and quinazoline urea derivatives were found to bind to human adenosine A(3) receptors. Series of N-phenyl-N'-quinazolin-4-ylurea derivatives and N-phenyl-N'-isoquinolin-1-ylurea derivatives were synthesized and tested in radioligand binding assays on their adenosine receptor affinities. A structure-affinity analysis indicated that on the 2-position of the quinazoline ring or the equivalent 3-position of the isoquinoline ring a phenyl or heteroaryl substituent increased the adenosine A(3) receptor affinity in comparison to unsubstituted or aliphatic derivatives. Furthermore, the structure-affinity relationship of substituted phenylurea analogues was investigated. Substituents such as electron-withdrawing or electron-donating groups were introduced at different positions of the benzene ring to probe electronic and positional effects of substitution. Substitution on the 3- or 4-position of the phenyl ring decreased the adenosine A(3) receptor affinity. Substitution at position 2 with an electron-donating substituent, such as methyl or methoxy, increased human adenosine A(3) receptor affinity, whereas substitution on the 2-position with an electron-withdrawing substituent did not influence affinity. Combination of the optimal substituents in the two series had an additive effect, which led to the potent human adenosine A(3) receptor antagonist N-(2-methoxyphenyl)-N'-(2-(3-pyridyl)quinazolin-4-yl)urea (VUF5574, 10a) showing a K(i) value of 4 nM and being at least 2500-fold selective vs A(1) and A(2A) receptors. Compound 10a competitively antagonized the effect of an agonist in a functional A(3) receptor assay, i.e., inhibition of cAMP production in cells expressing the human adenosine A(3) receptor; a pA(2) value of 8.1 was derived from a Schild plot. In conclusion, compound 10a is a potent and selective human adenosine A(3) receptor antagonist and might be a useful tool in further characterization of the human A(3) receptor.

Selective ligands as tools to study histamine receptors.

In this review the histaminergic ligands for the histamine H(1), H(2) and H(3) receptors, which are currently used as tools in pharmacological studies, are described. To study interactions with the histamine H(1) receptor, the H(1) agonist 2-aminoethylthiazole has long since been used. However, during the last decade, 2-phenylhistamine derivatives emerged with interesting binding features. So far no radiolabelled selective H(1) agonist has been commonly used. As H(1) antagonists mepyramine, triprolidine and chlorpheniramine are described together with radiolabelled H(1) antagonists [3H]mepyramine and [3H]doxepin. Special attention has been paid to the PET ligands [11C]doxepin and [11C]mepyramine and the [125I] labelled antagonists [125I]iodobolpyramine and [125I]iodoazidophenpyramine. Concerning H(2) agonists, especially dimaprit, amthamine and impromidine are discussed. There are several H(2) antagonists; amongst them cimetidine, tiotidine and ranitidine are used most frequently. Many of these antagonists behave as inverse agonists. As radiolabelled H(2) antagonists, [3H]cimetidine, [3H]tiotidine, [125I]iodoaminopotentidine and [125I]iodoazidopotentidine are included. Commonly used histamine H(3) agonists are N(alpha)-methylhistamine, (R)alpha-methylhistamine, imetit and immepip. Both methylhistamines are also available as [3H] labelled ligands. As reference compounds, often used H(3) antagonists are thioperamide, clobenpropit, iodophenpropit and impentamine. Most important radiolabelled H(3) antagonists are S-[3H]methylthioperamide, [3H]thioperamide, [125I]iodophenpropit and [125I]iodoproxyfan. The use of all these compounds as a tool in pharmacology is also discussed.

Effect of clobenpropit, a centrally acting histamine H3-receptor antagonist, on electroshock- and pentylenetetrazol-induced seizures in mice.

The anticonvulsant activity of clobenpropit, an isothiourea derivative of histamine and potent H3-receptor antagonist, was investigated in two representative seizure models in mice. In the maximal electroshock seizure threshold test, clobenpropit dose-dependently raised the electroconvulsive threshold for tonic (hindlimb extension) seizures, but a significant increase of about 15% was determined only at the high dose of 40 mg/kg i.p. The protective action of this drug was reduced by immepip and (R)-alpha-methylhistamine, selective H3-receptor agonists. In co-medication with two standard antiepileptics, clobenpropit (20 and 40 mg/kg) significantly increased the anticonvulsant effectiveness of carbamazepine and tended to increase the effectiveness of valproate. Additional studies indicated that the high dose of clobenpropit also significantly enhanced the plasma carbamazepine concentration. One the other hand, in the s.c. PTZ seizure threshold test clobenpropit revealed no protective effects. In the rotarod ataxia test, impaired motor function was observed at 80 mg/kg clobenpropit. In conclusion, the present findings indicated no pronounced anticonvulsant effects of clobenpropit against generalized tonic as well as clonic seizures.

A novel class of adenosine A3 receptor ligands. 1. 3-(2-Pyridinyl)isoquinoline derivatives.

A series of 3-(2-pyridinyl)isoquinoline derivatives was synthesized as potential antagonists for the human adenosine A3 receptor by substitution of the 1-position. The compounds were obtained by various synthetic routes from 1-amino-3-(2-pyridinyl)isoquinoline. The affinity was determined in radioligand binding assays for rat brain A1 and A2A receptors and for the cloned human A3 receptor. A structure-activity relationship analysis indicated that a phenyl group when coupled by a spacer allowing conjugation on position 1 of the isoquinoline ring increased the adenosine A3 receptor affinity. In contrast, such a phenyl group directly bound to position 1 of the isoquinoline ring decreased affinity. Since the combination of a phenyl group together with a spacer raised adenosine A3 receptor affinity, various spacers were investigated. VUF8501 (N-[3-(2-pyridinyl)isoquinolin-1-yl]benzamidine (15) showed an affinity at the human adenosine A3 receptor of 740 nM. Substituent effects on the phenyl group were investigated by in vitro evaluation of a series of substituted benzamidines. Electron-donating groups at the para position of the benzamidine ring increased adenosine A3 receptor affinity. These investigations led to VUF8505 (4-methoxy-N-[3-(2-pyridinyl)isoquinolin-1-yl]benzamidine(22)), which is a moderately potent and selective ligand for the human adenosine A3 receptor with an affinity of 310 nM in our test system having negligible affinity for rat A1 and A2A receptors.

A novel class of adenosine A3 receptor ligands. 2. Structure affinity profile of a series of isoquinoline and quinazoline compounds.

1-Substituted 3-(2-pyridinyl)isoquinolines have been shown to form a novel class of adenosine A3 receptor ligands. In the present study further investigations of this new lead and the structure affinity relationships of this class of compounds are described. First, the influence of an amide group at position 1 of the isoquinoline ring on the adenosine A3 receptor affinity was determined. A carboxamide proved to be a useful spacer between the isoquinoline and a phenyl ring. N-[2-(2-pyridinyl)isoquinolin-4-yl]benzamide (VUF8507, compound 6) had an affinity of 200 nM at the adenosine A3 receptor. Second, we investigated the effects of substitution of the benzamide ring of 6 with a series of mono- and disubstituted N-[3-(2-pyridinyl)isoquinoline]benzamides. The ratio of the tautomers of the benzamides was determined in the solid state and in solution by spectroscopic techniques (IR and NMR). Affinities were determined in radioligand binding assays at rat brain A1 and A2A receptors and at cloned human A3 receptor. The benzamides showed higher adenosine A3 receptor affinity than aliphatic amides. We propose that the adenosine A3 receptor affinity of the different benzamides is related to their presence in either the iminol or amide form. Ligands present in the iminol form showed relatively high adenosine A3 receptor affinity. Finally, we explored the influence of replacement of C4 of the isoquinoline ring by a nitrogen atom. Comparison of isoquinolines with the corresponding quinazolines revealed that both compounds showed similar adenosine A3 receptor affinity. These investigations led to potent and selective human adenosine A3 receptor ligands with affinities in the nanomolar range. The subtype-selective compound 4-methoxy-N-[2-(2-pyridinyl)quinazolin-4-yl]benzamide (VUF8504, 13) with an affinity of 17.0 nM at the human adenosine A3 receptor might become a useful tool in the pharmacological characterization or the investigation of the physiological function of this receptor.

Development of an ear edema model of contact hypersensitivity to avoid false-positive results due to interactions between hapten and test agents.

Ear edema models are regularly used for topical testing of antiinflammatory compounds. However, test compounds are usually applied simultaneously with proinflammatory agents at the same site which may result in mutual interactions. In order to avoid the occurrence of false antiinflammatory effects, a model of oxazolone-induced contact hypersensitivity has been described in which the hapten and test compound are each applied separately to only one side of the ear. By splitting and weighing the dorsal and ventral cutis of the ears, it was shown that the edemateous response of the control nonhapten side was comparable with the hapten-treated side. Some agents with antiinflammatory properties, as for example, dapsone, cimetidine, cyclosporine A, and budesonide, were tested simultaneously with oxazolone on both sides of the ear or applied separately on the dorsal and ventral ear sides, respectively. When dissolving the compounds in solutions of oxazolone, marked colorations of the test solutions were noted, indicating the occurrence of a chemical interaction. On simultaneous application at the same area, almost complete inhibition of the edemateous response was obtained for all compounds tested. In contrast, when applied separately, only budesonide appeared to exhibit antiinflammatory activity. The results indicate that the proposed model can be used to avoid the occurrence of interactions between oxazolone, and possibly other sensitizers, and substances that are being evaluated for topical antiinflammatory activity. By use of this model spurious antiinflammatory activity can be detected.

The agonistic binding site at the histamine H2 receptor. I. Theoretical investigations of histamine binding to an oligopeptide mimicking a part of the fifth transmembrane alpha-helix.

Mutation studies on the histamine H2 receptor were reported by Gantz et al. [J. Biol. Chem., 267 (1992) 20840], which indicate that both the mutation of the fifth transmembrane Asp186 (to Ala186) alone or in combination with Thr190 (to Ala190) maintained, albeit partially, the cAMP response to histamine. Recently, we have shown that histamine binds to the histamine H2 receptor as a monocation in its proximal tautomeric form, and, moreover, we suggested that a proton is donated from the receptor towards the tele-position of the agonist, thereby triggering the biological effect [Nederkoorn et al., J. Mol. Graph., 12 (1994) 242; Eriks et al., Mol. Pharmacol., 44 (1993) 886]. These findings result in a close resemblance with the catalytic triad (consisting of Ser, His and Asp) found in serine proteases. Thr190 resembles a triad's serine residue closely, and could also act as a proton donor. However, the mutation of Thr190 to Ala190-the latter is unable to function as a proton donor-does not completely abolish the agonistic cAMP response. At the fifth transmembrane alpha-helix of the histamine H2 receptor near the extracellular surface, another amino acid is present, i.e. Tyr182, which could act as a proton donor. Furthermore, Tyr182 lies within the proximity of Asp186, so an alternative couple of amino acids, Tyr182 and Asp186, could constitute the histamine binding site at the fifth alpha-helix instead of the (mutated) couple Asp186 and Thr190. In the first part of our present study, this hypothesis is investigated with the aid of an oligopeptide with an alpha-helical backbone, which represents a part of the fifth transmembrane helix. Both molecular mechanics and ab initio data lead to the conclusion that the Tyr182/Asp186 couple is most likely to act as the binding site for the imidazole ring present in histamine.

Structure-activity relationships for the glutathione conjugation of 2-substituted 1-chloro-4-nitrobenzenes by rat glutathione S-transferase 4-4.

In the present study structure--activity relationships (SAR's) are described for the experimentally determined kinetic parameters (Km, kappa cat, and kappa cat/Km) of the GST 4-4-catalyzed reaction between GSH and 10 2-substituted 1-chloro-4-nitrobenzenes. Steric, lipophilic, and electronic parameters were correlated with the kinetic parameters. Moreover, charge distributions and several energy values were calculated for the substrates and the corresponding Meisenheimer intermediates with MeS- as a model nucleophile for the thiolate anion of GSH and used in the regression analyses. The correlations obtained were compared with the corresponding SAR's for the base-catalyzed GSH conjugation reaction at pH 9.2. A high correlation coefficient was found between the kinetic parameter kappa s for the base-catalyzed reaction and the Hammett substituent constant (sigma p). Much lower correlation coefficients were obtained with kappa cat and sigma p and with kappa cat/Km and sigma p. Moreover, the reaction constant rho was significantly higher for the base-catalyzed than for the enzyme-catalyzed reaction. Also, high correlations were found between the kinetic parameters and the charges on the p-nitro substituent in the substrates. When kappa s was plotted against these charges, a linear relationship was found in which the slope was larger than the slope of a corresponding plot with kappa cat/Km. The Hammett sigma p can be divided into an inductive (F) and a resonance (R) component. With multiple regression between the kinetic parameters and F and R, higher correlation coefficients were obtained than with sigma p alone. Our observations suggest that the transition states for the base-catalyzed and the GST 4-4-catalyzed GSH conjugation reaction are different. Moreover, single classical physiochemical and computer-calculated molecular parameters and combinations of them can be an alternative approach for examining SAR's for spontaneous and GST-catalyzed GSH conjugation reactions.

Inhibition of growth of Histoplasma capsulatum yeast cells in human macrophages by the iron chelator VUF 8514 and comparison of VUF 8514 with deferoxamine.

Histoplasma capsulatum requires intracellular iron to survive and multiply within human and murine macrophages (M phi). Thus, iron chelators may be useful compounds in the treatment of histoplasmosis. In the present study we compared the efficacies of five different iron chelators with deferoxamine (DEF) for their capacity to inhibit the growth of H. capsulatum yeast cells in culture medium and within human M phi. Of the agents tested, only one, VUF 8514, a 2,2'-bipyridyl analog, was found to be effective. VUF 8514 inhibited the growth of yeast cells in tissue culture medium and within M phi in a dose-response fashion. In tissue culture medium, the 50% effective dose (ED50) of VUF 8514 was 30 nM and the ED50 of DEF was 1 mM. In human M phi, the ED50 of VUF 8514 was 520 nM and the ED50 of DEF was 4 mM. Thus, VUF 8514 was effective at a concentration 7.7 x 10(3)-fold lower than DEF in inhibiting the growth of yeast cells in M phi. Inhibition of the intracellular growth of yeast cells by VUF 8514 was reversed by holotransferrin and iron nitriloacetate, an iron compound that is soluble at neutral to alkaline pH. Thus, VUF 8514 inhibits the intracellular growth of yeast cells by acting as an iron chelator rather than through its capacity as a weak base. These data suggest that the hydroxamic acid siderophore of H. capsulatum yeast cells competes successfully for iron against some iron chelators but not others and that VUF 8514 may be a potential therapeutic agent for the treatment of histoplasmosis.

Influence of membrane composition of various parasites on the inhibitory activity of a series of bipyridyl analogues.

In a previous report the dependence of the antimycoplasmal activity of bipyridyl analogues on the presence of Cu+ ions has been shown. The inhibitory activity of these compounds has now been studied against Escherichia coli mycobacteria and Candida albicans in the absence and presence of Cu2+ ions using growth kinetic techniques. It was found that the inhibitory activity against E. coli increases in the presence of Cu2+ ions. In contrast, no additional effect of Cu2+ ions is observed for the inhibitory activity against mycobacteria. Some of the derivatives show very promising activity even against Mycobacterium avium strains. More complicated is the effect of Cu2+ ions on the inhibitory activity of the derivatives against C. albicans. For isoquinolones the observed delay in onset of inhibitors is reduced and no influence on the inhibitory activity is observed. The addition of Cu2+ ions to the phenanthrolines leads in contrast to a decrease in antifungal activity. The possible influence of membrane properties of the studied microorganism on the effect of Cu2+ ions is discussed.

New activation model for the histamine H2 receptor, explaining the activity of the different classes of histamine H2 receptor agonists.

Recently we developed amthamine [2-amino-5-(2-aminoethyl)-4-methylthiazole]. This cyclic analogue of dimaprit proved to be the most potent and selective histamine H2 receptor agonist of a series of substituted 4- or 5-(2-aminoethyl)thiazoles. Quantum chemical studies on histamine (N pi-H tautomer), dimaprit, and amthamine revealed that, based upon geometries of molecular electrostatic potentials, it is likely that these agonists accept a proton from the proton-donating receptor site on their double-bonded (heteroaromatic) nitrogen atoms. In contrast to reported models, this new model is able to accommodate and explain the agonistic activities of all known (including nontautomeric) histamine H2 receptor agonists. Quantitative structure-activity relationship studies with a series of substituted histamine derivatives and heterocyclic analogues support the presented model, in which the monocations in extended conformation interact with the receptor surface; their affinities correlate with the proton association constants of the heteroaromatic nuclei. The negatively charged anchoring site for the ethylammonium side chain of these agonists in this model is a functional group with a pKa value of 4.17.

Histamine H2-receptor agonists. Synthesis, in vitro pharmacology, and qualitative structure-activity relationships of substituted 4- and 5-(2-aminoethyl)thiazoles.

It is well known that both histamine and dimaprit show moderate histamine H2-receptor agonistic activities on the guinea pig right atrium. Quantum chemical calculations on these two compounds showed similarities in electron distributions and molecular electrostatic potentials (MEP's), which could be extended to rigid analogues [2-amino-5-(2-aminoethyl)thiazoles] of the latter structure. On the base of these results a series of substituted 4- and 5-(2-aminoethyl)thiazoles was synthesized applying small alkyl substitution variations as reported for histamine. 2-Amino-5-(2-aminoethyl)-4-methylthiazole (Amthamine) proved to be the most potent full histamine H2-receptor agonist on the guinea pig right atrium, being with a pD2 value of 6.21 slightly more potent than histamine. This compound shows no affinity for H1-receptors and is a full but weak agonist on the histamine H3-receptor with a pD2 value of 4.70, thus showing a marked specificity for histamine H2-receptors. In the 5-(2-aminoethyl)thiazole series the presence of a 2-amino substituent proved to be not essential for stimulation of the histamine H2-receptor, leading to the important conclusion that in contrast to histamine, for this series, acceptance of a proton by the thiazole nucleus of the agonist from the active site of the receptor is sufficient for the stimulation of the histamine H2-receptor.

Synthesis and copper-dependent antimycoplasmal activity of amides and amidines derived from 2-amino-1,10-phenanthroline.

A series of both aliphatic and aromatic amides and aromatic amidines derived from 2-amino-1,10-phenanthroline (3) according to the Topliss scheme were synthesized and subsequently tested for antimycoplasmal potency. Although the compounds themselves showed no activity, in the presence of a nontoxic copper concentration of 40 microM all compounds appeared to be very active against Mycoplasma gallisepticum K154. The most active compounds were found in the amide series and show growth inhibition in the nanomolar range. These compounds are 4 times more active than tylosin, a macrolide antibiotic, which is used therapeutically in veterinary practice. In the presence of copper, amides derived from 3 are more active than corresponding amidines. Increased activity following derivatization of 3 may be due to the presence of a third coordination site for copper in the title compounds. Evaluation of biological data revealed that antimycoplasmal activity of amides derived from 3 is dependent on lipophilicity. For these amides a good linear correlation was found between antimycoplasmal activity and hydrophobic fragmental values for substituents considered. This quantitative structure-activity relationship study indicated that antimycoplasmal activity was increased upon a decrease of these hydrophobic fragmental values.

4- or 5- (omega-aminoalkyl) thiazoles and derivatives; new selective H2-receptor agonists.

It is well known that both histamine and dimaprit show moderate H2-receptor agonistic activity (guinea pig right atrium). Quantum chemical calculations indicated that 2-aminothiazole derivatives that might be regarded as cyclic dimaprit analogues, should possess H2-receptor agonistic activity as well. In the present study a series of 4- or 5-(omega-aminoalkyl) thiazoles has been synthesized, showing a moderate to strong H2-receptor agonistic activity as compared to histamine whereas no activity on H1- and H3-receptors could be detected. In contrast to histamine and derivatives, which are supposed to "trigger" the H2-receptor via a tautomeric shift involving two protons viz. one proton of the active site of the receptor and one proton of the heteroaromatic ring system, the thiazole derivatives seem to stimulate the H2-receptor via a one proton mechanism. In a series of impromidine analogues the 3- [4(5)-imidazolyl]propyl moiety was replaced by the more H2-receptor specific 3-(4- or 5-thiazolyl)propyl fragment resulting in potent and selective full H2-receptor agonists.

New selective histamine H1 agonists. Synthesis and pharmacology.

In this article the synthesis and histaminergic H1 activity of a series of substituted 2-phenylhistamines are described. It appeared that substitution of the phenyl ring of these compounds influences the H1 activity substantially. In general, substitution in para position causes a decrease in H1 activity. However, in the meta-substituted members both increases and decreases of H1 activity have been observed; thus the meta nitro and meta methoxy derivatives are four times as potent as the parent 2-phenylhistamine. As far as investigated, neither H1 nor H3 activity could be established.

Copper complexes of 1,10-phenanthroline and related compounds as superoxide dismutase mimetics.

In a preliminary study we tested CuSO4.5H2O, (Cu(II]2[3,5-diisopropylsalicylate]4.2H2O and a number of copper complexes of substituted 1,10-phenanthrolines for superoxide anion dismutase activity. It appeared that this activity depends on the ligands involved and might be governed by the redox potential of the Cu(I) complex/Cu(II) complex couple. The strong superoxide anion dismutase activity of Cu(II)[DMP]2 complex can be expected considering its high redox potential. Rather surprisingly is the superoxide anion dismutase activity of the Cu(I)[DMP]2 complex since it involves oxidation to Cu(II)[DMP]2 complex. From regression analysis it was established that steric and field effects of the substituents of the investigated phenanthrolines play an important role in SOD activity and therefore it is concluded that complex formation is important for the superoxide dismutase-like activity.

Synthesis and copper-dependent antimycoplasmal activity of 1-amino-3-(2-pyridyl)isoquinoline derivatives. 2. Amidines.

In our search for new compounds with antimycoplasmal activity, a series of aromatic amidines derived from 1-amino-3-(2-pyridyl)isoquinoline (1) was synthesized. In the presence of 40 microM copper the most active compounds show growth inhibition of Mycoplasma gallisepticum in the nanomolar range. These compounds are 3 times as active as tylosin, an antimycoplasmal therapeutic agent that is used in veterinary practice. In the presence of copper, amidines derived from 1 are 2-3 times more active than the corresponding amides. Furthermore it was established that for these compounds too, the presence of a 2,2'-bipyridyl moiety is a necessary prerequisite for antimycoplasmal activity. As for the amides, antimycoplasmal activity of amidines derived from 1 is dependent on the hydrophobic fragmental value of the aromatic nucleus of the amidine moiety. A quantitative structure-activity relationship established the optimal hydrophobic fragmental value of this part of the molecule to be zero.