A Convenient Four-Step Synthesis of 1-{ β-[ 3-( 4-Methoxy-phenyl ) Propoxy ]-4-Methoxyphenethyl }-1 H-Imidazole Hydrochloride as a Probing Tool for SOCE Assays

1-{β-[3-(4-Methoxy-phenyl)propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride 7, or SKF-96365, was synthesized in four steps with an overall yield of 9%. The structure of 7 was confirmed by 1H-, 13C-NMR, HRMS, and elemental analysis. The intermediates 3 and 4 were also isolated and characterized by 1H-, 13C-NMR, and HRMS.


Introduction
Variations in cytoplasmic Ca 2+ concentration ([Ca 2+ ] i ) play an important role in mediating fundamental biological activities for cell proliferation, differentiation, apoptotosis, and gene expression.Calcium influx through store-operated Ca 2+ entry (SOCE) triggered by the depletion of endoplasmic reticulum (ER) Ca 2+ stores largely contributes to changes in ([Ca 2+ ] i ) and regulates a large variety of cellular functions [1][2][3].The first identified inhibitor of the CRAC (Ca 2+ release-activated Ca 2+ channel) channel, the archetypal SOCE channel, was the 1-{β-[3-(4-methoxy-phenyl)propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride, also known as SKF-96365 [4], and this imidazole derivative inhibited thapsigargin-induced SOCE in Jurkat cells with an IC 50 of 12 µM in a dose-dependant manner [5].Although this inhibitor, in a mouse model of breast cancer, prevented the development of tumor metathesis [6].In the imidazole group, econazole and miconazole have been also identified as inhibitors of CRAC channels, but some studies mentioned that these imidazole coumpounds showed non-specific effects by inhibiting the cytochrome P450 activity [7] or other Ca 2+ channels [8].However, SKF-96365 is still used as a tool for the probing of receptor-mediated Ca 2+ entry processes, particularly in non-excitable cells [9][10][11].To our surprise, the synthesis of SKF-96365 was not patented or not detailed in academic literature, but it is interesting to note that the enantiomers has been subjected to a chiral high pressure liquid chromatography (HPLC) study [12].In this context and due to the recent interest of SKF-96365 for their anticancer effect in a mouse xenograph model [13], we developed efforts on a complete and detailed preparation of 1-{β-[3-(4-methoxy-phenyl)propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride.

Results
The synthetic route for the preparation of the title compound 7 was achieved in four steps, as presented in Scheme 1.In the first step, reaction of commercial imidazole 1 with 2-bromo-4'-methoxyacetophenone 2 produced mainly the corresponding 2-(1H-imidazol-1-yl)-1-[4methoxyphenyl)ethanone 3 in a 51% yield.This N-alkylation was conducted in DMF at 5 • C and after a reaction time of two hours; the crude reaction mixture was treated with deionized water in order to eliminate the undesired N-protoned derivative 3. detailed in academic literature, but it is interesting to note that the enantiomers has been subjected to a chiral high pressure liquid chromatography (HPLC) study [12].In this context and due to the recent interest of SKF-96365 for their anticancer effect in a mouse xenograph model [13], we developed efforts on a complete and detailed preparation of 1-{β-[3-(4-methoxy-phenyl)propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride.

Results
The synthetic route for the preparation of the title compound 7 was achieved in four steps, as presented in Scheme 1.In the first step, reaction of commercial imidazole 1 with 2-bromo-4'-methoxyacetophenone 2 produced mainly the corresponding 2-(1H-imidazol-1-yl)-1-[4-methoxyphenyl)ethanone 3 in a 51% yield.This N-alkylation was conducted in DMF at 5 °C and after a reaction time of two hours; the crude reaction mixture was treated with deionized water in order to eliminate the undesired N-protoned derivative 3.For the second step, the acetophenone derivative 3 in a dry methanol solution was converted into its corresponding alcohol 4 [14] in a 63% yield by the addition of sodium borohydride (2 equiv).In the third step, treatment of alcohol 3 with 4-methoxyphenylpropylbromide 5 in a basic (KOH, 5 equiv) solution of dimethylsulfoxide led to the desired O-alkyl compound 6 together with the N-alkyl derivative of 6 as a by-product.Compound 6 was purified by preparative chromatography on silica gel using a step-wise gradient of CH2Cl2/MeOH (0-1%).Finally, from a solution of 1M HCl in diethyl ether, pure O-alkyl imidazole intermediate 6 was transformed into imidazolium chloride 7. It is interesting to note that the crystallization was achieved after one day at 25 °C in a 29% yield.Compound 7 was prepared with an overall yield of 9%.The structure of 7 was confirmed by 1 H-NMR, and the data were in agreement with those previously described in literature [12]. 13C-NMR, HRMS, and elemental analysis of 7 completed the data and are in accordance with this structure.

General Information
Preparative chromatography was realized on a Combi Flash Rf 200 psi UV ref. 208K20284 (Serlabo Technologies, Entraigues-sur-la-Sorgue, France) using pre-packed column of alumina gel 60 F 254 Merck equipped with a DAD UV/Vis 200-360-nm detector .Thin-layer chromatography (TLC) was accomplished on 0.2-mm precoated plates of neutral alumina gel 60 F-254 (Merck) with appropriate eluent.Visualization was made with ultraviolet light (254 and 365 nm) or with a fluorescence indicator.Solvents were evaporated with a BUCHI rotary evaporator.All reagents and solvents were purchased from Acros Fisher, Sigma-Aldrich Chimie, and Fluka France and were used without further purification. 1H-NMR spectra were recorded on a BRUKER AC 300 P (300 MHz) spectrometer, and 13 C-NMR spectra on BRUKER AC 300 P (75 MHz) spectrometer (Bruker France Scientifique, Voisins-le-Bretonneux, France).Chemical shifts are expressed in parts per million downfield.Data are given in the following order: δ value, multiplicity (s: singlet; d: doublet; For the second step, the acetophenone derivative 3 in a dry methanol solution was converted into its corresponding alcohol 4 [14] in a 63% yield by the addition of sodium borohydride (2 equiv.).In the third step, treatment of alcohol 3 with 4-methoxyphenylpropylbromide 5 in a basic (KOH, 5 equiv.)solution of dimethylsulfoxide led to the desired O-alkyl compound 6 together with the N-alkyl derivative of 6 as a by-product.Compound 6 was purified by preparative chromatography on silica gel using a step-wise gradient of CH 2 Cl 2 /MeOH (0-1%).Finally, from a solution of 1M HCl in diethyl ether, pure O-alkyl imidazole intermediate 6 was transformed into imidazolium chloride 7. It is interesting to note that the crystallization was achieved after one day at 25 • C in a 29% yield.Compound 7 was prepared with an overall yield of 9%.The structure of 7 was confirmed by 1 H-NMR, and the data were in agreement with those previously described in literature [12]. 13C-NMR, HRMS, and elemental analysis of 7 completed the data and are in accordance with this structure.

General Information
Preparative chromatography was realized on a Combi Flash R f 200 psi UV ref. 208K20284 (Serlabo Technologies, Entraigues-sur-la-Sorgue, France) using pre-packed column of alumina gel 60 F 254 Merck equipped with a DAD UV/Vis 200-360-nm detector .Thin-layer chromatography (TLC) was accomplished on 0.2-mm precoated plates of neutral alumina gel 60 F-254 (Merck) with appropriate eluent.Visualization was made with ultraviolet light (254 and 365 nm) or with a fluorescence indicator.Solvents were evaporated with a BUCHI rotary evaporator.All reagents and solvents were purchased from Acros Fisher, Sigma-Aldrich Chimie, and Fluka France and were used without further purification. 1H-NMR spectra were recorded on a BRUKER AC 300 P (300 MHz) spectrometer, and 13 C-NMR spectra on BRUKER AC 300 P (75 MHz) spectrometer (Bruker France Scientifique, Voisins-le-Bretonneux, France).Chemical shifts are expressed in parts per million downfield.Data are given in the following order: δ value, multiplicity (s: singlet; d: doublet; t: triplet; q: quartet; quint: quintuplet: m: multiplet; br: broad), number of protons, coupling constants J is given in Hertz.The high-resolution mass spectra (HRMS) were recorded in positive mode using direct electrospray infusion, respectively on Waters Q-Tof 2 or on Thermo Fisher Scientific Q-Exactive spectrometers, and the purity of the final compound 1-{β-[3-(4-methoxy-phenyl)propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride 7 was performed on a microanalyzer Flash EA1112 CHNS/O Thermo Electron (Thermo Electron, Villebon-sur-Yvette, France).The obtained results were within ±0.4% of the theorical values at the "Centre Régional de Mesures Physiques de l'Ouest" SFS ScanMAT (CRMPO SFS ScanMAT, Rennes, France).Melting points were determined on a Kofler melting point apparatus and were uncorrected.

Supplementary Materials:
Copies of the 1 H-, 13 C-NMR for compounds 3, 4, and 7, and MS spectra of 7, as well as the molfile for 7, can be found at http://www.mdpi.com/1422-8599/2016/4/M909/s1.Acknowledgments: One of us (C.D.D.) wishes to thank the "Fondation Benianh International" and the "Ministère de l'Enseignement Supérieur et de la Recherche Scientifique de Côte d'Ivoire" for grants.Financial support of this program carried out under the French National Cancer Institute "Cancéropôle Grand Ouest" by contract "Ion Channel-Network CGO 2012", is gratefully acknowledged.The authors are grateful to the assistance