Synthesis of new 5-phenyl[1,2,4]triazole derivatives as ligands for the 5-HT 1A serotonin receptor

A series of 4-amino-3-[[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]thio]-5-(substituted phenyl)[1,2,4]triazoles (5a-f) and the isomeric 4-amino-2-[2-[4-(2-methoxyphenyl)-1- piperazinyl]ethyl]-5-(substitutedphenyl)-2,4-dihydro-3H[1,2,4]triazole-3-thiones ( 6a-f ) and a series of 3-[[2-[4-(2-methoxy or 2-nitrophenyl)1-piperazinyl]ethyl]thio]-5-(substituted phenyl)[1,2,4]triazoles ( 8a-i ) were synthesized with the aim of obtaining new selective 5-HT 1A ligands, with reduced affinity for the α 1 -adrenoceptor subtypes. New compounds were tested in radioligand binding experiments where many of them showed a preferential affinity for the 5-HT 1A receptor.


Introduction
5-HT 1A serotonin receptors belong to the seven-transmembrane-domain (7-TM) receptor superfamily 1 and mediate many physiological effects of the serotonin.6][7] To date several potent 5-HT 1A receptor ligands are known, however their potential use as drugs or pharmacological tools is often limited by their undesired affinity for other receptor types such as an example the dopaminergic D 2 receptor and the α 1 -adrenoceptor (α 1 -AR).][7] In a previous paper we have described the synthesis of new 5-phenyl [1,2,4]triazole derivatives (1-2, n=2, Figure 1) as 5-HT 1A receptor ligands. 8These molecules present a phenylpiperazinyl (PP) residue that forms an essential part of the molecule for 5-HT 1A affinity and a 5-aryl [1,2,4]triazole moiety connected by a propyl chain.Some of them had shown high affinity and selectivity for 5-HT 1A receptor respect to the α 1 -AR.We now report a further development of the structure-activity relationships (SAR) on these classes of compounds.In particular, in this paper we synthesized and tested for affinity and selectivity for 5HT 1A over α 1 -AR, a new series of 5-aryl [1,2,4]triazoles in which the main modification is the length of the chain connecting PP and triazole moieties, that here is constituted by two methylene groups instead of three (1,2, n = 1, Figure 1).In an effort to optimize the 5-position of the triazole ring, a phenyl group substituted with a number of electronically dissimilar residue was chosen.
Attempt to conduct the reaction described in scheme 1 in the same experimental conditions reported in a previous paper, (ethanol in the presence of potassium hydroxide and a catalytic amount of potassium iodide), 8 failed.In fact, in these experimental conditions, 1- 9 thus reducing the yield in the desired compounds.A similar reactivity had been shown by other chloroethylamines in ethanol in presence of potassium hydroxide. 10In order to improve yields of both S-and N-alkylated isomers, we carried out the reaction using refluxing acetone and potassium carbonate, experimental conditions often used when 1-(chloroalkyl)-4-(2-substitutedphenyl)piperazines where employed as alkylating agents. 11,12In these experimental conditions, using 3a-f as starting materials, both the S-alkylated and the N-alkylated isomers were obtained (scheme 1).Flash chromatography of the crude material allowed the recovery of both isomers with an average yield of 45-50% for the Salkylated isomers and 12-18% for the N-alkylated (ratio 3:1).As reported for triazoles previously described, 8 analysis of the 1 H NMR spectra of the two isomeric series is diagnostic for the assignment of structure.Both spectra present in the range from 5.7 to 6.2 δ, a broad singlet signal which integrates for two hydrogens and disappears in the presence of D 2 O; it is analogous to the signal seen in the 1 H NMR spectra of 4-amino-5-aryl-2,4-dihydro-3H [1,2,4]triazole-3-thiones 3a-f and it has to be attributed to the NH 2 group.This implies that alkylation of compounds 3a-f does not take place at the NH 2 substituent.Furthermore, in both spectra, a triplet for the methylene group of the ethyl chain that connects the PP moiety and the triazole part of the molecule, is observed.[10][11][12][13] These results were confirmed by 13 C-NMR spectra where a signal at 29.66 d was observed for the S-alkylated derivative 5d, whereas the same signal is shifted to 46.37 δ for the N-alkylated derivative 6d.

Scheme 1
The preparation of derivatives 8a-i (scheme 2), which lack the amino substituent in the 4position of the triazole, was accomplished using similar reaction conditions as for the synthesis of compounds 5a-f and 6a-f.Derivatives (7a-g) and the appropriate 1-(2-chloroethyl)-4-(2substitutedphenyl)piperazine (4a-b) were reacted in alkaline medium.In this case, only the Salkylated isomers 8a-i were isolated from the reaction mixture.

Scheme 2
][16][17] All the synthesized compounds were tested in binding experiments to evaluate their affinity and selectivity for the 5-HT 1A receptor over the α 1 -AR.The results are presented in tables 1 and 2 and are expressed as K i (nM).
Previously we described a new class of 5-aryl [1,2,4]triazole derivatives as ligands for the 5-HT 1A serotonin receptor selective over the α 1 AR. 8In this paper we analyzed three series of molecules: compounds 5a-f and 8a-i, characterized by a thioethyl chain between the 5aryl [1,2,4]triazole and the PP portions, and compounds 6a-f, in which an ethyl chain is present.As a general trend, most compounds 5a-f and 8a-i showed good and preferential affinity for the 5-HT 1A receptor over the α 1 -AR.On the other hand most of compounds 6a-f, displayed lower affinity and selectivity.Such results indicated that the point of the attachment of the phenylpiperazinethyl moiety (3 or 2-position of the phenyl triazole ring) is critical for affinity and selectivity.Moreover, a comparison of binding data of new synthesized compounds with those of previously reported analogues, 8 shows that the shortening of connecting alkyl chain generally led to a slight decrease of both affinity and selectivity.Substituents on the phenyl ring in the 5-position of the triazole seem to have little effect on 5-HT 1A receptor affinity with the exception of bulkier substituents that reduce the affinity.The presence of an amino group in the 4-position of the triazole ring generally reduced the affinity for both class of receptors, but the α 1 -AR are more sensitive.As described in the previous paper, 8 the 2-methoxyphenyl-piperazinyl derivatives showed higher affinity at both receptors than their respective 2-nitrophenyl analogues (compare 8a and 8h; 8e and 8i).Among tested molecules, compound 5a resulted the most interesting derivative of this series showing good affinity and selectivity for the 5-HT 1A receptors.c selectivity for 5-HT 1A over α 1 receptors is expressed as the ratio K i α 1 /K i 5-HT 1A .

Table 1. Structure and binding properties of compounds 5a-f and 6a-f
In conclusion, a new series of 4-amino [1,2,4]triazoles (8a-i) were synthesized and tested in radioligand binding assays to evaluate their affinity and selectivity for the 5-HT 1A over α 1 -adrenergic receptor.Generally this compounds showed K i values in the nanomolar range and selectivity for the 5-HT 1A receptor.

Experimental Section
General Procedures.Melting points were determined using a Buchi 510 apparatus and are uncorrected.Infrared spectra were recorded on a Perkin-Elmer FTIR 1600 spectrometer in KBr disk. 1 H NMR spectra were recorded on a Varian 200 MHz instrument (200 MHz for 1 H NMR and 50 MHz for 13 C NMR) in DMSO-d 6 solution.Chemical shifts are given in ppm (δ) relative to tetramethylsilane as internal standard; coupling costants (J) are given in Hz.Signals were characterized as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) and br (broad signals).Elemental analyses were performed on a Carlo Erba Elemental Analyzer Mod.1108 apparatus and the data of C, H, N are within ± 0.4% of calculated values.All the compounds synthesized were tested for purity on TLC (aluminum sheets coated with silica gel 60 F 254 , Merck) and visualized by UV (λ =254 and 366 nm).Preparative chromatographic separations were conducted by means of flash chromatography using Merck Silica gel 60 0.040-0.063mm.[16][17]
).The desired compound was obtained as a white solid.
).The desired compound was obtained as a white solid.