Synthesis and evaluation of N-alkyl-S-[3-(piperidin-1-yl)propyl]isothioureas: high affinity and human/rat species-selective histamine H(3) receptor antagonists.

S-Alkyl-N-alkylisothiourea compounds containing various cyclic amines were synthesized in the search for novel nonimidazole histamine H3 receptor (H3R) antagonists. Among them, four N-alkyl S-[3-(piperidin-1-yl)propyl]isothioureas 18, 19, 22, and 23 were found to exhibit potent and selective H3R antagonistic activities against in vitro human H3R, but were inactive against in vitro human H4R. Furthermore, three alkyl homologs 18-20 showed inactivity for histamine release in in vivo rat brain microdialysis, suggesting differences in antagonist affinities between species. In addition, in silico docking studies of N-[4-(4-chlorophenyl)butyl]-S-[3-piperidin-1-yl)propyl]isothiourea 19 and a shorter homolog 17 with human/rat H3Rs revealed that structural differences between the antagonist-docking cavities of rat and human H3Rs were likely caused by the Ala122/Val122 mutation.

[Synthetic studies of imidazole C-nucleosides toward biofunctional molecules].

Synthetic studies of C4-linked imidazole C-nucleosides toward biofunctional molecules are described, in which the following items are covered. 1) Stereoselective synthesis of imidazole C- and pyrazole C-nucleosides via diazafulvene intermediates. 2) Synthesis of tetrahydrofuranylimidazoles using a PhSe group efficiently and its application to the new human histamine H(3) receptor (hH(3)R) agonist, imifuramine, and the first selective human histamine H(4) receptor (hH(4)R) agonist, OUP-16. 3) Synthesis of imidazole ribonucleoside phosphoramidite (Imz-PA) with pivaloyloxymethyl (POM) group for probing the catalytic mechanism of ribozymes. 4) Synthesis of a two-carbon-elongated homologue (Imz-C(2)-PA) with a combination of POM and 2-cyanoethyl groups. 5) Incorporation of C(2)-imidazole nucleoside into position 638 of VS ribozyme using Imz-C(2)-PA and catalytic activities of the thereby generated modified VS ribozyme (G638C(2)Imz).

Synthesis of novel C4-linked C2-imidazole ribonucleoside phosphoramidite and its application to probing the catalytic mechanism of a ribozyme.

The synthesis of a novel C4-linked C2-imidazole ribonucleoside phosphoramidite (ICN-C2-PA 1) with a two-carbon linker between imidazole and ribose moieties is described. In the phosphoramidite, POM and 2-cyanoethyl groups were selected to protect the endocyclic amine function of imidazole and the 2'-hydroxyl function of D-ribose, respectively. The C2-imidazole nucleoside, a flexible structural mimic of a purine nucleobase, was successfully incorporated using ICN-C2-PA 1 into position 638 of the VS ribozyme through 2'-TBDMS chemistry to study the role of G638 in general acid-base catalysis. The modified VS ribozyme (G638C2Imz) exhibited significantly greater catalytic activity than observed with the C0-imidazole that has no carbon atoms linking the ribose and the C4-imidazole. Imidazole nucleoside analogues with variable spacer lengths could provide a valuable general methodology for exploring the catalytic mechanisms of ribozymes.

Efficient approaches to S-alkyl-N-alkylisothioureas: syntheses of histamine H3 antagonist clobenpropit and its analogues.

S-Alkyl-N-alkylisothioureas were efficiently synthesized via synthetic approach (A) using 3-phenylpropionyl isothiocyanate (PPI). The utility of the approach was proved by the syntheses of clobenpropit, a potent histamine H(3) antagonist, and its analogues. Alternatively, clobenpropit could be prepared via intramolecular amide cleavage (B) with use of 2-nitrophenylacetyl isothiocyanate (NPAI).

Synthesis of novel 4(5)-(5-aminotetrahydropyran-2-yl)imidazole derivatives and their in vivo release of neuronal histamine measured by brain microdialysis.

The (2R,5S)-trans- and (2S,5S)-cis-stereoisomers 1a and 1b of 4(5)-(5-aminotetrahydropyran-2-yl)imidazole, which have two chiral centers and adopt a stable chair conformation, were synthesized via cyclization of diol intermediates 7 using L-glutamine as the starting material. Their enantiomers, (2S,5R)-trans-1c and (2R,5R)-cis-1d, were synthesized by the same methodology from D-glutamine. Stereo isomers 1a-d were converted into cyanoguanidines 11a-d, and into N-isopropyl and N-3,3-dimethylbutyl derivatives 12a-d and 13a-d, respectively. The results of in vivo brain microdialysis of the derivatives apparently indicated that only (2S,5R)-isomers increased the release of neuronal histamine. Among the many (2S,5R)-N-alkyl derivatives, 13c (OUP-133) and 18 (OUP-153) increased histamine release to 180-190% and 180-200% of basal levels, respectively, and were found to be novel histamine H(3) antagonists.

RNA folding and the origins of catalytic activity in the hairpin ribozyme.

The nucleolytic ribozymes catalyse site-specific phosphodiester cleavage and ligation transesterification reactions in RNA. The hairpin ribozyme folds to generate an intimate loop-loop interaction to create the local environment in which catalysis can proceed. We have studied the ion-induced folding using single-molecule FRET experiments, showing that the four-way helical junction accelerates the folding 500-fold by introducing a discrete intermediate that juxtaposes the loops. Using FRET we can observe individual hairpin ribozyme molecules as they undergo multiple cycles of cleavage and ligation, and measure the rates of the internal reactions, free of uncertainties in the contributions of docking and substrate dissociation processes. On average, the cleaved ribozyme undergoes several docking-undocking events before a ligation reaction occurs. On the basis of these experiments, we have explored the role of the nucleobases G8 and A38 in the catalysis. Both cleavage and ligation reactions are pH dependent, corresponding to the titration of a group with pKa=6.2. We have used a novel ribonucleoside in which these bases are replaced by imidazole to investigate the role of acid-base catalysis in this ribozyme. We observe significant rates of cleavage and ligation, and a bell-shaped pH dependence for both.

Nucleobase catalysis in the hairpin ribozyme.

RNA catalysis is important in the processing and translation of RNA molecules, yet the mechanisms of catalysis are still unclear in most cases. We have studied the role of nucleobase catalysis in the hairpin ribozyme, where the scissile phosphate is juxtaposed between guanine and adenine bases. We show that a modified ribozyme in which guanine 8 has been substituted by an imidazole base is active in both cleavage and ligation, with ligation rates 10-fold faster than cleavage. The rates of both reactions exhibit bell-shaped dependence on pH, with pK(a) values of 5.7 +/- 0.1 and 7.7 +/- 0.1 for cleavage and 6.1 +/- 0.3 and 6.9 +/- 0.3 for ligation. The data provide good evidence for general acid-base catalysis by the nucleobases.

Nucleobase participation in ribozyme catalysis.

We constructed a modified form of the VS ribozyme containing an imidazole ring in place of adenine at position 756. The novel ribozyme is active in both cleavage and ligation reactions. The reaction is efficient, although relatively slow. The results are consistent with a role for nucleobase catalysis in the catalytic mechanism of this ribozyme.

Efficient synthesis of trans- or cis-4(5)-(5-aminomethyltetrahydrofuran-2-yl)imidazoles via diazafulvene intermediates: synthetic approach toward human histamine H4)-ligands.

(+)-4(5)-[(2R,5R)-5-aminomethyltetrahydrofuran-2-yl]imidazole [(+)-1, imifuramine] and its 2R,5S-stereoisomer (+)-2 were expected as base compounds to develop selective human histamine H4-receptor ligands. The improved synthesis of (+)-1 was done via cyclization of a diazafulvene intermediate generated by Bu3P/N,N,N',N'-tetramethylazodicarboxamide (TMAD) treatment of a diol 17ab bearing an unsubstituted imidazole moiety in good yields. This methodology also afforded an alternative synthetic route to trans- and cis-ethyl 4(5)-(5-hydroxymethyltetrahydrofuran-2-yl)imidazole carboxylates (5 and 6), reported previously. Also, 4(5)-[(2R,5S)-5-aminomethyltetrahydrofuran-2-yl]imidazole (+)-2 was synthesized from ethyl 4(5)-(2-deoxy-beta-D-ribofuranosyl)imidazole-1-carboxylate (35) via the four steps involving deoxygenation.

A selective human H(4)-receptor agonist: (-)-2-cyano-1-methyl-3-[(2R,5R)-5- [1H-imidazol-4(5)-yl]tetrahydrofuran-2-y] methylguanidine.

A series of 16 compounds related to chiral 4(5)-(5-aminomethyltetrahydrofuran-2-yl)imidazoles (1) have been designed, synthesized, and examined in vitro by radioligand displacement studies and functional assays for both the human H(3)- and H(4)-receptors expressed in SK-N-MC cells. Among them, the (2S,5S)-isomer 1d of amino compounds showed approximately 300-fold higher selectivity at the H(3)-receptor than the H(4)-receptor. On the other hand, (2R,5S)- and (2R,5R)-cyanoguanidines 3b and 3c, in which the amino group of the compounds 1b and 1c was substituted by the cyanoguanidino moiety, bound to the H(4)-receptor with a pEC(50) value of 6.65 and 7.11, respectively, and had >40-fold selectivities over the H(3)-receptor. As such, 3b and 3c are the first selective H(4) receptor agonists.

Synthesis of (+/-)-trans- or cis-(5-aminomethyltetrahydrofuranyl)imidazole by Mitsunobu cyclization: synthetic studies toward novel histamine H3 or H4-ligands.

The (+/-)-trans- or cis-4(5)-(5-aminomethyltetrahydrofuranyl)imidazole [1 and 2] were synthesized by the Mitsunobu cyclization, starting from L-glutamic acid.