Pharmacological analysis of immepip and imetit homologues. Further evidence for histamine H(3) receptor heterogeneity?

Following a previous report by our research group on discriminative properties of a series of aliphatic histamine homologues, we now studied immepip, imetit and its lower and higher sidechain homologues as ligands for the histamine H(3) receptor in a [(125)I]-iodophenpropit binding assay using rat cerebral cortex membranes, and two functional H(3) receptor models (inhibition of the neurogenic contraction of the guinea pig jejunum and inhibition of [(3)H]-noradrenaline release in rat cerebral cortex slices). The immepip homologues behaved as competitive H(3)-receptor antagonists in both functional systems. The potencies (pA(2) values) observed at the guinea pig jejunum were 8.4 and 6.2 for the immepip homologues VUF 4929 and VUF 4735, respectively, whereas on the electrically evoked release of [(3)H]-noradrenaline from cortical slices the pA(2) values were 7.1 and 5.5 for VUF 4929 and VUF 4735, respectively. Moreover, immepip, but not the (R)-alpha-methylhistamine, showed almost a tenfold higher agonistic potency in the rat cerebral cortex than in the guinea pig jejunum. For imetit and its homologues important discrepancies in the potencies in the two functional assays were noticed as well. VUF 8328 acts as a potent (pD(2)=8.0) partial agonist in the brain, but as a very active (pA(2)=9.4) competitive antagonist in the guinea pig jejunum. The partial agonistic activity of VUF 8328 in the brain was confirmed by GTP gamma S-sensitive, biphasic displacement of [(125)I]-iodophenpropit binding to rat cerebral cortex membranes. The differences in potencies shown by the various ligands are discussed in relation to H(3) receptor heterogeneity.

Thiazole and thiadiazole analogues as a novel class of adenosine receptor antagonists.

Novel classes of heterocyclic compounds as adenosine antagonists were developed based on a template approach. Structure-affinity relationships revealed insights for extended knowledge of the receptor-ligand interaction. We replaced the bicyclic heterocyclic ring system of earlier described isoquinoline and quinazoline adenosine A(3) receptor ligands by several monocyclic rings and investigated the influence thereof on adenosine receptor affinity. The thiazole or thiadiazole derivatives seemed most promising, so we continued our investigations with these two classes of compounds. The large difference between a pyridine and isoquinoline ring in binding adenosine A(1) and A(3) receptors showed the importance of the second ring of the isoquinoline ligands. We prepared several N-[4-(2-pyridyl)thiazol-2-yl]benzamides, and these compounds showed adenosine affinities in the micromolar range. Most surprising in the series of the N-[4-(2-pyridyl)thiazol-2-yl]amides were the retained adenosine affinities by introduction of a cylopentanamide instead of the benzamide. A second series of compounds, the thiadiazolobenzamide series of compounds, revealed potent and selective adenosine receptor antagonists, especially N-(3-phenyl-1,2,4-thiadiazol-5-yl)-4-hydroxybenzamide (LUF5437, 8h) showing a K(i) value of 7 nM at the adenosine A(1) receptor and N-(3-phenyl-1,2,4-thiadiazol-5-yl)-4-methoxybenzamide (LUF5417, 8e) with a K(i) value of 82 nM at the adenosine A(3) receptor. 4-Hydroxybenzamide 8h is the most potent adenosine A(1) receptor antagonist of this new class of compounds. Structure--affinity relationships showed the existence of a steric restriction at the para-position of the benzamide ring for binding adenosine A(1) and A(3) receptors. The electronic nature of the 4-substituents played an important role in binding the adenosine A(3) receptor. Cis- and trans-4-substituted cyclohexyl derivatives were made next to the 4-substituted benzamide analogues. We used them to study the proposed specific interaction between the adenosine A(1) receptor and the 4-hydroxy group of this class of thiadiazolo compounds, as well as a suggested special role for the 4-methoxy group in binding the A(3) receptor. Both the adenosine A(1) and A(3) receptor slightly preferred the trans-analogues over the cis-analogues, while all compounds showed low affinities at the adenosine A(2A) receptor. Our investigations provided the potent and highly selective adenosine A(1) antagonist N-(3-phenyl-1,2,4-thiadiazol-5-yl)-trans-4-hydroxycyclohexanamide (VUF5472, 8m) showing a K(i) value of 20 nM. A third series of compounds was formed by urea analogues, N-substituted with thiazolo and thiadiazolo heterocycles. The SAR of this class of compounds was not commensurate with the SAR of the previously described quinazoline urea. On the basis of these findings we suggest the existence of a special interaction between adenosine receptors and a region of high electron density positioned between the thia(dia)zole ring and phenyl(pyridyl) ring. Molecular electrostatic potential contour plots showed that for this reason the ligands need either a thiadiazole ring instead of a thiazole or a 2-pyridyl group instead of a phenyl. The derived novel classes of antagonists will be useful for a better understanding of the molecular recognition at the adenosine receptors.

Design, synthesis, and evaluation of Phe-Gly mimetics: heterocyclic building blocks for pseudopeptides.

Enantiopure heterocyclic Boc-protected Phe-Gly dipeptidomimetics containing 1,3,4-oxadiazole, 1,2,4-oxadiazole, and 1,2,4-triazole ring systems have been synthesized as building blocks in the synthesis of pseudopeptides. Three derivatives (1-3) have the carboxylic acid function directly bound to the heterocyclic ring, and three derivatives (4-6) have an extra methylene group between the heterocyclic ring and the acid function to allow for an increased conformational flexibility. The mimetics were used as Phe-Gly replacements in the biologically active peptides dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH(2)) and substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-MetNH(2), SP). The pseudopeptide synthesis was performed using solid-phase methodology on a MBHA-resin using Boc-chemistry. The biological evaluation was performed by testing the micro- and delta-opioid receptor affinities of the dermorphin pseudopeptides and the NK(1) receptor affinities of the SP pseudopeptides. The results showed that all mimetics except 3 were excellent replacements of Phe-Gly in dermorphin since they displayed affinities for the micro-receptor (IC(50) = 12-31 nM) in the same range as dermorphin itself (IC(50) = 6.2 nM). The agonist activity of three pseudopeptides at human micro-receptors was also evaluated. It was shown that the tested compounds retained their agonist activity. The SP pseudopeptides showed considerably lower affinities (IC(50) > 1 microM) for the NK(1) receptor than SP itself (IC(50) = 1.5 nM) indicating that the Phe-Gly replacements prevent the pseudopeptides from adopting bioactive conformations.

N6,5'-Disubstituted adenosine derivatives as partial agonists for the human adenosine A3 receptor.

5'-(Alkylthio)-substituted analogues of N6-benzyl- and N6-(3-iodobenzyl)adenosine were synthesized in 37-61% overall yields. The affinities of these compounds for the adenosine A1, A2A, and A3 receptors were determined using rat brain cortex, rat brain striata, and stably transfected human A3 receptors in HEK 293 cells, respectively. The compounds proved to be selective for the adenosine A3 receptor and displayed affinities in the nanomolar range. Compounds 8, 10, and 11 had the highest affinities for the A3 receptor with Ki values ranging from 8.8 to 27.7 nM. In the N6-benzyl series, compound 4 (LUF 5403), with a 5'-methylthio group, maintained a reasonable affinity and had the highest selectivity for the A3 receptor. Compound 12 (LUF 5411), with an N6-(3-iodobenzyl) group and a 5'-(n-propylthio) substituent, had the highest A3 selectivity of all of the compounds and also displayed high affinity for this receptor (Ki = 44.3 nM). The compounds were also evaluated for their ability to stimulate [35S]GTPgamma[S] binding in cell membranes expressing the human adenosine A3 receptor. It appeared that the N6,5'-disubstituted adenosine derivatives behaved as partial agonists. Compounds 2, 4, 8, and 10 had the highest intrinsic activities. Additionally, when tested in a cAMP assay, these compounds also behaved as partial agonists.

Characterization of the binding site of the histamine H3 receptor. 1. Various approaches to the synthesis of 2-(1H-imidazol-4-yl)cyclopropylamine and histaminergic activity of (1R,2R)- and (1S,2S)-2-(1H-imidazol-4-yl)-cyclopropylamine.

Various approaches to the synthesis of all four stereoisomers of 2-(1H-imidazol-4-yl)cyclopropylamine (cyclopropylhistamine) are described. The rapid and convenient synthesis and resolution of trans-cyclopropylhistamine is reported. The absolute configuration of its enantiomers was determined by single-crystal X-ray crystallographic analysis. The distinct trans-cyclopropylhistamine enantiomers were tested for their activity and affinity on the histamine H3 receptor. (1S,2S)-Cyclopropylhistamine (VUF 5297) acts as an agonist both on the rat cortex (pD2 = 7.1; alpha = 0.75) and on guinea pig jejunum (pD2 = 6.6; alpha = 0.75). Its enantiomer, (1R, 2R)-cyclopropylhistamine (VUF 5296), is about 1 order of magnitude less active. Both enantiomers show weak activity on H1 and H2 receptors. All synthetic attempts to cis-cyclopropylhistamine were unsuccessful. Nevertheless, the results of this study provide an ideal template for molecular modeling studies of histamine H3 receptor ligands.

Histamine homologues discriminating between two functional H3 receptor assays. Evidence for H3 receptor heterogeneity?.

We studied several histamine homologues as potential ligands for the histamine H3 receptor in two binding assays ([125l]iodophenpropit and N alpha-[3H]methylhistamine binding to rat brain cortex membranes) and two functional H3 receptor models (inhibition of the neurogenic contraction in the guinea pig jejunum and of [3H]noradrenaline release in mouse brain cortex slices). The histamine homologues acted all as competitive H3 antagonists at the guinea pig jejunum. The potency in this model and/or the affinity for N alpha-[3H]methylhistamine binding was higher for the butylene (pA2 = 7.7; pKi = 9.4) and pentylene homologue (impentamine, pA2 = 8.4; pKi = 9.1) than for the propylene, hexylene and octylene homologues (pA2 = 5.9-7.8; pKi = 6.1-7.6). In the mouse brain cortex the propylene, butylene and pentylene homologues acted as partial agonists (alpha = 0.3-0.6) and the hexylene and octylene homologues acted as antagonists. [125I]Iodophenpropit binding was displaced monophasically by the propylene, hexylene and octylene homologues and biphasically by the butylene and pentylene homologues. Biphasic displacement curves were converted to monophasic ones by 10 microM guanosine-5'-O-(3-thiotriphosphate. In conclusion, the homologue of histamine with five methylene groups is a more potent H3 receptor antagonist in the guinea pig jejunum than the other homologues tested. Furthermore, the propylene, butylene and pentylene homologues can discriminate between the two functional H3 receptor models in the guinea pig jejunum and mouse brain. These data are discussed in relation to the efficiency of receptor coupling and receptor heterogeneity.

New analogs of burimamide as potent and selective histamine H3 receptor antagonists: the effect of chain length variation of the alkyl spacer and modifications of the N-thiourea substituent.

Burimamide was one of the first compounds reported to antagonize the activation of the histamine H3 receptor by histamine. We have prepared a large series of burimamide analogs by variation of the alkyl spacer length of burimamide from two methylene groups to six methylene groups and also by replacement of the N-methyl group with other alkyl and aryl groups. All analogs are reversible, competitive H3 antagonists as determined on the guinea pig intestine. Elongation of the alkyl chain from an ethylene chain to a hexylene chain results in an increase of the H3 antagonistic activity. The H3 selective pentylene and hexylene analogs of burimamide are about 10 times more potent than burimamide. The N-thiourea substituents, however, have no beneficial influence on the affinity.

Homologs of histamine as histamine H3 receptor antagonists: a new potent and selective H3 antagonist, 4(5)-(5-aminopentyl)-1H-imidazole.

The influence of alkyl chain length variation on the histamine H3 receptor activity of histamine homologs 1 was investigated. A series of 4(5)-(omega-aminoalkyl)-1H-imidazoles 1 was prepared with an alkyl chain length varying from one methylene group to 10 methylene groups. Besides the H3 activity, the affinities of these compounds for the H1 and H2 receptors were determined. The ethylene chain of histamine is optimal for agonistic activity on all three histamine receptor subtypes. For the H3 receptor, elongation of the alkyl chain from three methylene groups on leads to compounds with antagonistic properties. 4(5)-(5-Aminopentyl)-1H-imidazole (impentamine, 1e) is the most potent and selective H3 antagonist from this series of 4(5)-(omega-aminoalkyl)-1H-imidazoles 1, with a pA2 value of 8.4 (on guinea pig jejunum). A specific antagonistic binding site for this compound is proposed.

Characterization of the binding of the first selective radiolabelled histamine H3-receptor antagonist, [125I]-iodophenpropit, to rat brain.

1. The binding of the first selective radiolabelled histamine H3-receptor antagonist [125I]-iodophenpropit to rat cerebral cortex membranes was characterized. 2. [125I]-iodophenpropit, radiolabelled to a high specific activity of 1900 Ci mmol-1, saturably bound to a single class of sites with a KD of 0.57 +/- 0.16 nM (n = 4) and Bmax of 268 +/- 119 fmol mg-1 protein. 3. Specific binding at a concentration below 1 nM represented 50 to 60% of total binding. 4. Binding of [125I]-iodophenpropit to rat cerebral cortex membranes was readily displaced by histamine H3-agonists and antagonists. In contrast, the inhibitory potencies of selective histamine H1- and H2-receptor ligands were very low. 5. [125I]-iodophenpropit was biphasically displaced by the histamine H3-receptor antagonists, burimamide and dimaprit, which may indicate the existence of histamine H3-receptor subtypes. Other histamine H3-receptor antagonists showed a monophasic displacement. 6. Competition binding curves of H3-agonists were biphasic and showed a rightward shift upon the addition of the nonhydrolysable GTP analogue, guanosine 5'-o-(3-thio) triphosphate (GTP gamma S; 100 microM) which implicates the interaction of histamine H3-receptors with G-proteins. The affinities of the H3-receptor antagonists iodophenpropit, thioperamide and burimamide were not altered by GTP gamma S. 7. Histamine competition binding curves were shifted to the right by different nucleotides (100 microM) with a rank order of potency GTP gamma S > Gpp(NH)p, GTP. 8 In vitro autoradiographic studies revealed a heterogeneous distribution of [125I]-iodophenpropitbinding sites in rat brain, with highest densities observed in specific cerebral cortical areas and layers,the caudate-putamen complex, the olfactory tubercles, the hippocampal formation, the amygdala complex, the hypothalamic area and the mammillary bodies.9 It is concluded that the histamine H3-receptor antagonist, [125I]-iodophenpropit, meets the criteria fo ra suitable radioligand for histamine H3-receptor binding studies in rat brain.

A simple and rapid in vitro test system for the screening of histamine H3 ligands.

A simple and rapid functional test system for the screening of histamine H3 ligands is described. It is based on the inhibitory effect of histamine H3 agonists on electrically-evoked contractile response of isolated guinea pig intestine. Whole jejunum segments are continuously stimulated maximally (15 V) by electrical pulses with a frequency of 0.1 Hz and a duration of 0.5 msec. The resulting twitches are recorded isotonically (1.0 g) and can be completely abolished by atropine (0.1 mcM).