Preliminary Structure-Activity Relationship (SAR) of a Novel Series of Pyrazole SKF-96365 Analogues as Potential Store-Operated Calcium Entry (SOCE) Inhibitors

From a series of (1R, 1S)-1[β-(phenylalkoxy)-(phenetyl)]-1H-pyrazolium hydrochloride as new analogues of SKF-96365, one has an interesting effect for endoplasmic reticulum (ER) Ca2+ release and store-operated Ca2+ entry (SOCE) (IC50 25 μM) on the PLP-B lymphocyte cell line. A successful resolution of (±) 1-phenyl-2-(1H-pyrazol-1-yl)ethan-1-ol has been developed by using the method of “half-concentration” in the presence of (+)-(1S)- or (−)-(1R)-CSA.


Introduction
Cytoplasmic Ca 2+ is very important for fundamental biological processes; this includes cell proliferation, apoptosis, migration, and gene expression [1]. The increase of cytoplasmic Ca 2+ concentration following many plasma membrane receptors is connected to the release of stored Ca 2+ within the endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) associated to the influx of extra-cellular Ca 2+ across the plasma membrane. Store-operated Ca 2+ entry (SOCE) is the typical mechanism to generate Ca 2+ signals that combines the intracellular and extra-cellular processes and represents one of the most common and ubiquitous Ca 2+ influx routes in non-excitable cells [2][3][4]. SOC entry is mediated by calcium selective channels such as the archetypal calcium-release-activated calcium (CRAC) channels in lymphocytes, which are typically supported by Orai1 proteins and regulated by STIM1 (Stromal Interacting Molecule). STIM1 is a trans-membrane protein mainly located in the ER membrane acting as a calcium-sensing protein, whereas Orai1 is located in the plasmalemma forming the calcium-selective pore of the CRAC channel [5]. STIM and Orai are expressed in all tissues and therefore are of first important in the numerous cellular functions. Abnormal SOC channels activities cause several human diseases, such as breast cancer [6][7][8], inflammatory bowel diseases [9], thrombosis [10], and severe combined immunodeficiency (SCID) disorders [11], which leads to an increasing interest in developing small molecule compounds to regulate aberrant SOC and especially CRAC channels function [12]. The therapeutic potential of inhibiting CRAC currents (named I CRAC ) has been established by the clinical use of calcineurin inhibitors (cyclosporine A and tacrolinus) to prevent rejection of organ transplants. Examination of literature showed that the first identified inhibitor of the CRAC channel was SKF-96365 or 1-{β-[3-(4-methoxy-phenyl)propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride [13,14] in 1990 ( Figure 1) and is still used as a tool for the probing of receptor-mediated Ca 2+ entry processes [15] in non-excitable cells [16], but it is interesting to note that the synthesis of SKF-96365 was not detailed in academic literature and also not patented. Recent studies demonstrated that it strongly inhibits voltage-gated sodium current (I Na ) in rat ventricular myocytes using the whole-cell patch voltage-clamp technique [17]. SKF-96365 SOCE inhibitor exhibited potent anti-neoplastic activity by inducing cell-cycle arrest and apoptosis in colorectal cancer cells (HCT-116 and HT29 cells) [18]. Effect of SKF-96365 was also observed on hERG current in HEK 293 cells in a concentration-dependent manner. These blocking properties were similar to those observed previously for hERG channels by the calmodulin inhibitor W-7 [19]. All together, these results suggest a low specificity of this compound for CRAC channels.
A variety of new small molecules blocking the CRAC channels have been identified and developed, i.e., curcumin and caffeic acid phenethyl ester (CAPE) in ORAI1/STIM-co-expressing HEK 293 cells [20], 4 -[(trifluoromethyl)pyrazol-1-yl]carboxanilides exhibiting high selectivity for the CRAC channel over the voltage-operated Ca 2+ (VOC) channels [21], 2-APB with a concentration-dependent effect [22], GSK-7975A by altering the Orai pore geometry [23], Synta 66 with good selectivity for CRAC channels, and no effect for Ca 2+ pumps and K + channels and no interference with STIM1 aggregation [24]. Int trans-membrane protein mainly located in the ER membrane acting as a calcium-sensing protein, whereas Orai1 is located in the plasmalemma forming the calcium-selective pore of the CRAC channel [5]. STIM and Orai are expressed in all tissues and therefore are of first important in the numerous cellular functions. Abnormal SOC channels activities cause several human diseases, such as breast cancer [6][7][8], inflammatory bowel diseases [9], thrombosis [10], and severe combined immunodeficiency (SCID) disorders [11], which leads to an increasing interest in developing small molecule compounds to regulate aberrant SOC and especially CRAC channels function [12]. The therapeutic potential of inhibiting CRAC currents (named ICRAC) has been established by the clinical use of calcineurin inhibitors (cyclosporine A and tacrolinus) to prevent rejection of organ transplants. Examination of literature showed that the first identified inhibitor of the CRAC channel was SKF-96365 or 1-{β-[3-(4-methoxy-phenyl)propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride [13,14] in 1990 ( Figure 1) and is still used as a tool for the probing of receptor-mediated Ca 2+ entry processes [15] in non-excitable cells [16], but it is interesting to note that the synthesis of SKF-96365 was not detailed in academic literature and also not patented. Recent studies demonstrated that it strongly inhibits voltage-gated sodium current (INa) in rat ventricular myocytes using the whole-cell patch voltage-clamp technique [17]. SKF-96365 SOCE inhibitor exhibited potent anti-neoplastic activity by inducing cell-cycle arrest and apoptosis in colorectal cancer cells (HCT-116 and HT29 cells) [18]. Effect of SKF-96365 was also observed on hERG current in HEK 293 cells in a concentration-dependent manner. These blocking properties were similar to those observed previously for hERG channels by the calmodulin inhibitor W-7 [19]. All together, these results suggest a low specificity of this compound for CRAC channels.
A variety of new small molecules blocking the CRAC channels have been identified and developed, i.e., curcumin and caffeic acid phenethyl ester (CAPE) in ORAI1/STIM-co-expressing HEK 293 cells [20], 4′-[(trifluoromethyl)pyrazol-1-yl]carboxanilides exhibiting high selectivity for the CRAC channel over the voltage-operated Ca 2+ (VOC) channels [21], 2-APB with a concentration-dependent effect [22], GSK-7975A by altering the Orai pore geometry [23], Synta 66 with good selectivity for CRAC channels, and no effect for Ca 2+ pumps and K + channels and no interference with STIM1 aggregation [24]. In this context [25], we decided to examine the synthesis of SKF-96365 analogues bearing substituted (or not) pyrazole platforms and the other parameters for this structure-activity relationship (SAR) study are respectively the length of the chain in position Cβ and the presence (or not) of a methoxy polar group in para-position on the phenylethyl skeleton and on the phenylalkyloxy side chain. The generic phenylethyl skeleton is maintained for this SAR study. Effects of these analogues are also examined for endoplasmic reticulum (ER) Ca 2+ release and SOCE on a B lymphocyte cell line [26]. In this context [25], we decided to examine the synthesis of SKF-96365 analogues bearing substituted (or not) pyrazole platforms and the other parameters for this structure-activity relationship (SAR) study are respectively the length of the chain in position Cβ and the presence (or not) of a methoxy polar group in para-position on the phenylethyl skeleton and on the phenylalkyloxy side chain. The generic phenylethyl skeleton is maintained for this SAR study. Effects of these analogues are also examined for endoplasmic reticulum (ER) Ca 2+ release and SOCE on a B lymphocyte cell line [26].

Chemistry
These pyrazole analogues of SKF-96365 were prepared as shown in Scheme 1 using a modified method described in literature [27]. The first step involved reaction of 2-bromoacetophenone 1a or 2-bromo-1-(4-methoxyphenyl)ethan-1-one 1b with various pyrazoles 2 substituted by one or two trifluoromethyl groups (2a: pyrazole, 2b: 3-trifluoromethylpyrazole, and 2c: 3,5-bis-trifluoromethylpyrazole). The reaction was conducted with potassium carbonate in acetonitrile at room temperature. After work-up, the four desired N-substituted pyrazoles 3a-d were obtained in yields ranging from 53% to 69% ( Table 1). Reduction of the ketone function of 3a-d was realized in methanol solution at 25 • C using sodium tetrahyborohydride during 5-7 h. The desired hydroxyl compounds 4a-b were obtained after elimination of volatile compounds in vacuo, and simple treatment of the crude reaction mixture with deionized water afforded 4a-d in good yields (82-98%) after crystallization. Table 1.

Chemistry
These pyrazole analogues of SKF-96365 were prepared as shown in Scheme 1 using a modified method described in literature [27]. The first step involved reaction of 2-bromoacetophenone 1a or 2-bromo-1-(4-methoxyphenyl)ethan-1-one 1b with various pyrazoles 2 substituted by one or two trifluoromethyl groups (2a: pyrazole, 2b: 3-trifluoromethylpyrazole, and 2c: 3,5-bis-trifluoromethylpyrazole). The reaction was conducted with potassium carbonate in acetonitrile at room temperature. After work-up, the four desired N-substituted pyrazoles 3a-d were obtained in yields ranging from 53% to 69% (Table 1). Reduction of the ketone function of 3a-d was realized in methanol solution at 25 °C using sodium tetrahyborohydride during 5-7 h. The desired hydroxyl compounds 4a-b were obtained after elimination of volatile compounds in vacuo, and simple treatment of the crude reaction mixture with deionized water afforded 4a-d in good yields (82-98%) after crystallization. Table 1. Results for the preparation of 1-phenyl-2-(1H-pyrazol-1-yl)ethan-1-one 3a-d, 1-phenyl-2-(1H-pyrazol-1-yl)ethan-1-ol 4a-d and (±)-(1R, 1S) 1-[β-(phenylalkoxy)-phenethyl]-1H-pyrazolium hydrochloride 7a-e from phenylalkyl halides 5a-d. In the next step, for the introduction of molecular diversity on Cβ hydroxyl group of 4 by O-alkylation, we used a series of alkyl halides 5 carrying various chains (n = 0 or 2) and methoxy group in para-position of the phenyl moiety (5a: 4-methoxybenzyl chloride; 5b: 1-(2-bromoethyl)-4-methoxybenzene; 5c: 1-(3-bromopropyl)-4-methoxybenzene; 5d: 3-phenylpropyl bromide). For optimization of the reaction condition parameters, we developed a set of experiments which are the following: (i) the choice of the solvent (DMF, DMSO, MeCN, etc.); (ii) the choice of appropriate base (DIPEA, Et 3 N, KOH); (iii) the number of equivalents for the base; (iv) the reaction time; and (v) appropriate reaction temperature. We obtained good reproducibility (Table 2 for each product) when we applied a reaction time of 48 or 72 h (monitored by thin layer chromatography on silica with appropriate eluent for evaluation of consumption of the starting reagents), 2-5 equivalents of KOH, heating at 50 • C (during 48 or 72 h) after addition of alkyl halide 5 to produce the intermediate 6 which was not isolated (initial attempts to isolate it by flash chromatography on silica gel failed). After the addition of saturated brine to the crude reaction mixture (to solubilize the N-alkylated pyrazolium by-product), the intermediate 6 was extracted with dry diethyl ether and the collected extracts were directly treated with a commercial solution of 1 M HCl in ether. The desired salt 7 was collected by simple filtration after complete crystallization. It should be noted that this protocol yielded compounds 7a-e in 10-33% (Table 1), on the contrary, attempts to prepare the lipophilic trifluoromethyl derivatives 7f-l failed in spite of long crystallization times (168 and 336 h) at 4 • C and modification of the solvent for reaction (THF, dioxane). In the next step, for the introduction of molecular diversity on Cβ hydroxyl group of 4 by O-alkylation, we used a series of alkyl halides 5 carrying various chains (n = 0 or 2) and methoxy group in para-position of the phenyl moiety (5a: 4-methoxybenzyl chloride; 5b: 1-(2-bromoethyl)-4-methoxybenzene; 5c: 1-(3-bromopropyl)-4-methoxybenzene; 5d: 3-phenylpropyl bromide). For optimization of the reaction condition parameters, we developed a set of experiments which are the following: (i) the choice of the solvent (DMF, DMSO, MeCN, etc.); (ii) the choice of appropriate base (DIPEA, Et3N, KOH); (iii) the number of equivalents for the base; (iv) the reaction time; and (v) appropriate reaction temperature. We obtained good reproducibility (Table 2 for each product) when we applied a reaction time of 48 or 72 h (monitored by thin layer chromatography on silica with appropriate eluent for evaluation of consumption of the starting reagents), 2-5 equivalents of KOH, heating at 50 °C (during 48 or 72 h) after addition of alkyl halide 5 to produce the intermediate 6 which was not isolated (initial attempts to isolate it by flash chromatography on silica gel failed). After the addition of saturated brine to the crude reaction mixture (to solubilize the N-alkylated pyrazolium by-product), the intermediate 6 was extracted with dry diethyl ether and the collected extracts were directly treated with a commercial solution of 1 M HCl in ether. The desired salt 7 was collected by simple filtration after complete crystallization. It should be noted that this protocol yielded compounds 7a-e in 10-33% (Table 1), on the contrary, attempts to prepare the lipophilic trifluoromethyl derivatives 7f-l failed in spite of long crystallization times (168 and 336 h) at 4 °C and modification of the solvent for reaction (THF, dioxane).  In the next step, for the introduction of molecular diversity on Cβ hydroxyl group of 4 by O-alkylation, we used a series of alkyl halides 5 carrying various chains (n = 0 or 2) and methoxy group in para-position of the phenyl moiety (5a: 4-methoxybenzyl chloride; 5b: 1-(2-bromoethyl)-4-methoxybenzene; 5c: 1-(3-bromopropyl)-4-methoxybenzene; 5d: 3-phenylpropyl bromide). For optimization of the reaction condition parameters, we developed a set of experiments which are the following: (i) the choice of the solvent (DMF, DMSO, MeCN, etc.); (ii) the choice of appropriate base (DIPEA, Et3N, KOH); (iii) the number of equivalents for the base; (iv) the reaction time; and (v) appropriate reaction temperature. We obtained good reproducibility ( Table 2 for each product) when we applied a reaction time of 48 or 72 h (monitored by thin layer chromatography on silica with appropriate eluent for evaluation of consumption of the starting reagents), 2-5 equivalents of KOH, heating at 50 °C (during 48 or 72 h) after addition of alkyl halide 5 to produce the intermediate 6 which was not isolated (initial attempts to isolate it by flash chromatography on silica gel failed). After the addition of saturated brine to the crude reaction mixture (to solubilize the N-alkylated pyrazolium by-product), the intermediate 6 was extracted with dry diethyl ether and the collected extracts were directly treated with a commercial solution of 1 M HCl in ether. The desired salt 7 was collected by simple filtration after complete crystallization. It should be noted that this protocol yielded compounds 7a-e in 10-33% (Table 1), on the contrary, attempts to prepare the lipophilic trifluoromethyl derivatives 7f-l failed in spite of long crystallization times (168 and 336 h) at 4 °C and modification of the solvent for reaction (THF, dioxane). In the next step, for the introduction of molecular diversity on Cβ hydroxyl group of 4 by O-alkylation, we used a series of alkyl halides 5 carrying various chains (n = 0 or 2) and methoxy group in para-position of the phenyl moiety (5a: 4-methoxybenzyl chloride; 5b: 1-(2-bromoethyl)-4-methoxybenzene; 5c: 1-(3-bromopropyl)-4-methoxybenzene; 5d: 3-phenylpropyl bromide). For optimization of the reaction condition parameters, we developed a set of experiments which are the following: (i) the choice of the solvent (DMF, DMSO, MeCN, etc.); (ii) the choice of appropriate base (DIPEA, Et3N, KOH); (iii) the number of equivalents for the base; (iv) the reaction time; and (v) appropriate reaction temperature. We obtained good reproducibility ( Table 2 for each product) when we applied a reaction time of 48 or 72 h (monitored by thin layer chromatography on silica with appropriate eluent for evaluation of consumption of the starting reagents), 2-5 equivalents of KOH, heating at 50 °C (during 48 or 72 h) after addition of alkyl halide 5 to produce the intermediate 6 which was not isolated (initial attempts to isolate it by flash chromatography on silica gel failed). After the addition of saturated brine to the crude reaction mixture (to solubilize the N-alkylated pyrazolium by-product), the intermediate 6 was extracted with dry diethyl ether and the collected extracts were directly treated with a commercial solution of 1 M HCl in ether. The desired salt 7 was collected by simple filtration after complete crystallization. It should be noted that this protocol yielded compounds 7a-e in 10-33% (Table 1), on the contrary, attempts to prepare the lipophilic trifluoromethyl derivatives 7f-l failed in spite of long crystallization times (168 and 336 h) at 4 °C and modification of the solvent for reaction (THF, dioxane). In the next step, for the introduction of molecular diversity on Cβ hydroxyl group of 4 by O-alkylation, we used a series of alkyl halides 5 carrying various chains (n = 0 or 2) and methoxy group in para-position of the phenyl moiety (5a: 4-methoxybenzyl chloride; 5b: 1-(2-bromoethyl)-4-methoxybenzene; 5c: 1-(3-bromopropyl)-4-methoxybenzene; 5d: 3-phenylpropyl bromide). For optimization of the reaction condition parameters, we developed a set of experiments which are the following: (i) the choice of the solvent (DMF, DMSO, MeCN, etc.); (ii) the choice of appropriate base (DIPEA, Et3N, KOH); (iii) the number of equivalents for the base; (iv) the reaction time; and (v) appropriate reaction temperature. We obtained good reproducibility ( Table 2 for each product) when we applied a reaction time of 48 or 72 h (monitored by thin layer chromatography on silica with appropriate eluent for evaluation of consumption of the starting reagents), 2-5 equivalents of KOH, heating at 50 °C (during 48 or 72 h) after addition of alkyl halide 5 to produce the intermediate 6 which was not isolated (initial attempts to isolate it by flash chromatography on silica gel failed). After the addition of saturated brine to the crude reaction mixture (to solubilize the N-alkylated pyrazolium by-product), the intermediate 6 was extracted with dry diethyl ether and the collected extracts were directly treated with a commercial solution of 1 M HCl in ether. The desired salt 7 was collected by simple filtration after complete crystallization. It should be noted that this protocol yielded compounds 7a-e in 10-33% (Table 1), on the contrary, attempts to prepare the lipophilic trifluoromethyl derivatives 7f-l failed in spite of long crystallization times (168 and 336 h) at 4 °C and modification of the solvent for reaction (THF, dioxane). In the next step, for the introduction of molecular diversity on Cβ hydroxyl group of 4 by O-alkylation, we used a series of alkyl halides 5 carrying various chains (n = 0 or 2) and methoxy group in para-position of the phenyl moiety (5a: 4-methoxybenzyl chloride; 5b: 1-(2-bromoethyl)-4-methoxybenzene; 5c: 1-(3-bromopropyl)-4-methoxybenzene; 5d: 3-phenylpropyl bromide). For optimization of the reaction condition parameters, we developed a set of experiments which are the following: (i) the choice of the solvent (DMF, DMSO, MeCN, etc.); (ii) the choice of appropriate base (DIPEA, Et3N, KOH); (iii) the number of equivalents for the base; (iv) the reaction time; and (v) appropriate reaction temperature. We obtained good reproducibility ( Table 2 for each product) when we applied a reaction time of 48 or 72 h (monitored by thin layer chromatography on silica with appropriate eluent for evaluation of consumption of the starting reagents), 2-5 equivalents of KOH, heating at 50 °C (during 48 or 72 h) after addition of alkyl halide 5 to produce the intermediate 6 which was not isolated (initial attempts to isolate it by flash chromatography on silica gel failed). After the addition of saturated brine to the crude reaction mixture (to solubilize the N-alkylated pyrazolium by-product), the intermediate 6 was extracted with dry diethyl ether and the collected extracts were directly treated with a commercial solution of 1 M HCl in ether. The desired salt 7 was collected by simple filtration after complete crystallization. It should be noted that this protocol yielded compounds 7a-e in 10-33% (Table 1), on the contrary, attempts to prepare the lipophilic trifluoromethyl derivatives 7f-l failed in spite of long crystallization times (168 and 336 h) at 4 °C and modification of the solvent for reaction (THF, dioxane). In the next step, for the introduction of molecular diversity on Cβ hydroxyl group of 4 by O-alkylation, we used a series of alkyl halides 5 carrying various chains (n = 0 or 2) and methoxy group in para-position of the phenyl moiety (5a: 4-methoxybenzyl chloride; 5b: 1-(2-bromoethyl)-4-methoxybenzene; 5c: 1-(3-bromopropyl)-4-methoxybenzene; 5d: 3-phenylpropyl bromide). For optimization of the reaction condition parameters, we developed a set of experiments which are the following: (i) the choice of the solvent (DMF, DMSO, MeCN, etc.); (ii) the choice of appropriate base (DIPEA, Et3N, KOH); (iii) the number of equivalents for the base; (iv) the reaction time; and (v) appropriate reaction temperature. We obtained good reproducibility ( Table 2 for each product) when we applied a reaction time of 48 or 72 h (monitored by thin layer chromatography on silica with appropriate eluent for evaluation of consumption of the starting reagents), 2-5 equivalents of KOH, heating at 50 °C (during 48 or 72 h) after addition of alkyl halide 5 to produce the intermediate 6 which was not isolated (initial attempts to isolate it by flash chromatography on silica gel failed). After the addition of saturated brine to the crude reaction mixture (to solubilize the N-alkylated pyrazolium by-product), the intermediate 6 was extracted with dry diethyl ether and the collected extracts were directly treated with a commercial solution of 1 M HCl in ether. The desired salt 7 was collected by simple filtration after complete crystallization. It should be noted that this protocol yielded compounds 7a-e in 10-33% (Table 1), on the contrary, attempts to prepare the lipophilic trifluoromethyl derivatives 7f-l failed in spite of long crystallization times (168 and 336 h) at 4 °C and modification of the solvent for reaction (THF, dioxane). In the next step, for the introduction of molecular diversity on Cβ hydroxyl group of 4 by O-alkylation, we used a series of alkyl halides 5 carrying various chains (n = 0 or 2) and methoxy group in para-position of the phenyl moiety (5a: 4-methoxybenzyl chloride; 5b: 1-(2-bromoethyl)-4-methoxybenzene; 5c: 1-(3-bromopropyl)-4-methoxybenzene; 5d: 3-phenylpropyl bromide). For optimization of the reaction condition parameters, we developed a set of experiments which are the following: (i) the choice of the solvent (DMF, DMSO, MeCN, etc.); (ii) the choice of appropriate base (DIPEA, Et3N, KOH); (iii) the number of equivalents for the base; (iv) the reaction time; and (v) appropriate reaction temperature. We obtained good reproducibility ( Table 2 for each product) when we applied a reaction time of 48 or 72 h (monitored by thin layer chromatography on silica with appropriate eluent for evaluation of consumption of the starting reagents), 2-5 equivalents of KOH, heating at 50 °C (during 48 or 72 h) after addition of alkyl halide 5 to produce the intermediate 6 which was not isolated (initial attempts to isolate it by flash chromatography on silica gel failed). After the addition of saturated brine to the crude reaction mixture (to solubilize the N-alkylated pyrazolium by-product), the intermediate 6 was extracted with dry diethyl ether and the collected extracts were directly treated with a commercial solution of 1 M HCl in ether. The desired salt 7 was collected by simple filtration after complete crystallization. It should be noted that this protocol yielded compounds 7a-e in 10-33% (Table 1), on the contrary, attempts to prepare the lipophilic trifluoromethyl derivatives 7f-l failed in spite of long crystallization times (168 and 336 h) at 4 °C and modification of the solvent for reaction (THF, dioxane). In the next step, for the introduction of molecular diversity on Cβ hydroxyl group of 4 by O-alkylation, we used a series of alkyl halides 5 carrying various chains (n = 0 or 2) and methoxy group in para-position of the phenyl moiety (5a: 4-methoxybenzyl chloride; 5b: 1-(2-bromoethyl)-4-methoxybenzene; 5c: 1-(3-bromopropyl)-4-methoxybenzene; 5d: 3-phenylpropyl bromide). For optimization of the reaction condition parameters, we developed a set of experiments which are the following: (i) the choice of the solvent (DMF, DMSO, MeCN, etc.); (ii) the choice of appropriate base (DIPEA, Et3N, KOH); (iii) the number of equivalents for the base; (iv) the reaction time; and (v) appropriate reaction temperature. We obtained good reproducibility ( Table 2 for each product) when we applied a reaction time of 48 or 72 h (monitored by thin layer chromatography on silica with appropriate eluent for evaluation of consumption of the starting reagents), 2-5 equivalents of KOH, heating at 50 °C (during 48 or 72 h) after addition of alkyl halide 5 to produce the intermediate 6 which was not isolated (initial attempts to isolate it by flash chromatography on silica gel failed). After the addition of saturated brine to the crude reaction mixture (to solubilize the N-alkylated pyrazolium by-product), the intermediate 6 was extracted with dry diethyl ether and the collected extracts were directly treated with a commercial solution of 1 M HCl in ether. The desired salt 7 was collected by simple filtration after complete crystallization. It should be noted that this protocol yielded compounds 7a-e in 10-33% (Table 1), on the contrary, attempts to prepare the lipophilic trifluoromethyl derivatives 7f-l failed in spite of long crystallization times (168 and 336 h) at 4 °C and modification of the solvent for reaction (THF, dioxane).

Compound
In the next step, for the introduction of molecular diversity on Cβ hydroxyl group of 4 by O-alkylation, we used a series of alkyl halides 5 carrying various chains (n = 0 or 2) and methoxy group in para-position of the phenyl moiety (5a: 4-methoxybenzyl chloride; 5b: 1-(2-bromoethyl)-4-methoxybenzene; 5c: 1-(3-bromopropyl)-4-methoxybenzene; 5d: 3-phenylpropyl bromide). For optimization of the reaction condition parameters, we developed a set of experiments which are the following: (i) the choice of the solvent (DMF, DMSO, MeCN, etc.); (ii) the choice of appropriate base (DIPEA, Et3N, KOH); (iii) the number of equivalents for the base; (iv) the reaction time; and (v) appropriate reaction temperature. We obtained good reproducibility ( Table 2 for each product) when we applied a reaction time of 48 or 72 h (monitored by thin layer chromatography on silica with appropriate eluent for evaluation of consumption of the starting reagents), 2-5 equivalents of KOH, heating at 50 °C (during 48 or 72 h) after addition of alkyl halide 5 to produce the intermediate 6 which was not isolated (initial attempts to isolate it by flash chromatography on silica gel failed). After the addition of saturated brine to the crude reaction mixture (to solubilize the N-alkylated pyrazolium by-product), the intermediate 6 was extracted with dry diethyl ether and the collected extracts were directly treated with a commercial solution of 1 M HCl in ether. The desired salt 7 was collected by simple filtration after complete crystallization. It should be noted that this protocol yielded compounds 7a-e in 10-33% (Table 1), on the contrary, attempts to prepare the lipophilic trifluoromethyl derivatives 7f-l failed in spite of long crystallization times (168 and 336 h) at 4 °C and modification of the solvent for reaction (THF, dioxane).
In the next step, for the introduction of molecular diversity on Cβ hydroxyl group of 4 by O-alkylation, we used a series of alkyl halides 5 carrying various chains (n = 0 or 2) and methoxy group in para-position of the phenyl moiety (5a: 4-methoxybenzyl chloride; 5b: 1-(2-bromoethyl)-4-methoxybenzene; 5c: 1-(3-bromopropyl)-4-methoxybenzene; 5d: 3-phenylpropyl bromide). For optimization of the reaction condition parameters, we developed a set of experiments which are the following: (i) the choice of the solvent (DMF, DMSO, MeCN, etc.); (ii) the choice of appropriate base (DIPEA, Et3N, KOH); (iii) the number of equivalents for the base; (iv) the reaction time; and (v) appropriate reaction temperature. We obtained good reproducibility ( Table 2 for each product) when we applied a reaction time of 48 or 72 h (monitored by thin layer chromatography on silica with appropriate eluent for evaluation of consumption of the starting reagents), 2-5 equivalents of KOH, heating at 50 °C (during 48 or 72 h) after addition of alkyl halide 5 to produce the intermediate 6 which was not isolated (initial attempts to isolate it by flash chromatography on silica gel failed). After the addition of saturated brine to the crude reaction mixture (to solubilize the N-alkylated pyrazolium by-product), the intermediate 6 was extracted with dry diethyl ether and the collected extracts were directly treated with a commercial solution of 1 M HCl in ether. The desired salt 7 was collected by simple filtration after complete crystallization. It should be noted that this protocol yielded compounds 7a-e in 10-33% (Table 1), on the contrary, attempts to prepare the lipophilic trifluoromethyl derivatives 7f-l failed in spite of long crystallization times (168 and 336 h) at 4 °C and modification of the solvent for reaction (THF, dioxane).
In the next step, for the introduction of molecular diversity on Cβ hydroxyl group of 4 by O-alkylation, we used a series of alkyl halides 5 carrying various chains (n = 0 or 2) and methoxy group in para-position of the phenyl moiety (5a: 4-methoxybenzyl chloride; 5b: 1-(2-bromoethyl)-4-methoxybenzene; 5c: 1-(3-bromopropyl)-4-methoxybenzene; 5d: 3-phenylpropyl bromide). For optimization of the reaction condition parameters, we developed a set of experiments which are the following: (i) the choice of the solvent (DMF, DMSO, MeCN, etc.); (ii) the choice of appropriate base (DIPEA, Et3N, KOH); (iii) the number of equivalents for the base; (iv) the reaction time; and (v) appropriate reaction temperature. We obtained good reproducibility ( Table 2 for each product) when we applied a reaction time of 48 or 72 h (monitored by thin layer chromatography on silica with appropriate eluent for evaluation of consumption of the starting reagents), 2-5 equivalents of KOH, heating at 50 °C (during 48 or 72 h) after addition of alkyl halide 5 to produce the intermediate 6 which was not isolated (initial attempts to isolate it by flash chromatography on silica gel failed). After the addition of saturated brine to the crude reaction mixture (to solubilize the N-alkylated pyrazolium by-product), the intermediate 6 was extracted with dry diethyl ether and the collected extracts were directly treated with a commercial solution of 1 M HCl in ether. The desired salt 7 was collected by simple filtration after complete crystallization. It should be noted that this protocol yielded compounds 7a-e in 10-33% (Table 1), on the contrary, attempts to prepare the lipophilic trifluoromethyl derivatives 7f-l failed in spite of long crystallization times (168 and 336 h) at 4 °C and modification of the solvent for reaction (THF, dioxane). For this structure-activity relationship (SAR) study, we were also interested to evaluate the potential impact of chirality's of the compounds 7 on SOC channels activities because our present protocol for preparation of these pyrazole SKF-96365 analogues produced a (±) racemic mixture. Based on this finding, our attention was attracted by the method of half-quantities [28] for resolution of our (±)-(1R, 1S)-1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b mixture (Scheme 2). To find a simple and cost effective resolution procedure for (1R)-4b and (1S)-4b, we used commercial (+)-(1S)-and (−)-(1R)-10-camphorsulfonic acid (CSA). Treatment of (±)-(1R, In the next step, for the introduction of molecular diversity on Cβ hydroxyl group of 4 by O-alkylation, we used a series of alkyl halides 5 carrying various chains (n = 0 or 2) and methoxy group in para-position of the phenyl moiety (5a: 4-methoxybenzyl chloride; 5b: 1-(2-bromoethyl)-4-methoxybenzene; 5c: 1-(3-bromopropyl)-4-methoxybenzene; 5d: 3-phenylpropyl bromide). For optimization of the reaction condition parameters, we developed a set of experiments which are the following: (i) the choice of the solvent (DMF, DMSO, MeCN, etc.); (ii) the choice of appropriate base (DIPEA, Et3N, KOH); (iii) the number of equivalents for the base; (iv) the reaction time; and (v) appropriate reaction temperature. We obtained good reproducibility ( Table 2 for each product) when we applied a reaction time of 48 or 72 h (monitored by thin layer chromatography on silica with appropriate eluent for evaluation of consumption of the starting reagents), 2-5 equivalents of KOH, heating at 50 °C (during 48 or 72 h) after addition of alkyl halide 5 to produce the intermediate 6 which was not isolated (initial attempts to isolate it by flash chromatography on silica gel failed). After the addition of saturated brine to the crude reaction mixture (to solubilize the N-alkylated pyrazolium by-product), the intermediate 6 was extracted with dry diethyl ether and the collected extracts were directly treated with a commercial solution of 1 M HCl in ether. The desired salt 7 was collected by simple filtration after complete crystallization. It should be noted that this protocol yielded compounds 7a-e in 10-33% (Table 1), on the contrary, attempts to prepare the lipophilic trifluoromethyl derivatives 7f-l failed in spite of long crystallization times (168 and 336 h) at 4 °C and modification of the solvent for reaction (THF, dioxane).
In the next step, for the introduction of molecular diversity on Cβ hydroxyl group of 4 by O-alkylation, we used a series of alkyl halides 5 carrying various chains (n = 0 or 2) and methoxy group in para-position of the phenyl moiety (5a: 4-methoxybenzyl chloride; 5b: 1-(2-bromoethyl)-4-methoxybenzene; 5c: 1-(3-bromopropyl)-4-methoxybenzene; 5d: 3-phenylpropyl bromide). For optimization of the reaction condition parameters, we developed a set of experiments which are the following: (i) the choice of the solvent (DMF, DMSO, MeCN, etc.); (ii) the choice of appropriate base (DIPEA, Et3N, KOH); (iii) the number of equivalents for the base; (iv) the reaction time; and (v) appropriate reaction temperature. We obtained good reproducibility ( Table 2 for each product) when we applied a reaction time of 48 or 72 h (monitored by thin layer chromatography on silica with appropriate eluent for evaluation of consumption of the starting reagents), 2-5 equivalents of KOH, heating at 50 °C (during 48 or 72 h) after addition of alkyl halide 5 to produce the intermediate 6 which was not isolated (initial attempts to isolate it by flash chromatography on silica gel failed). After the addition of saturated brine to the crude reaction mixture (to solubilize the N-alkylated pyrazolium by-product), the intermediate 6 was extracted with dry diethyl ether and the collected extracts were directly treated with a commercial solution of 1 M HCl in ether. The desired salt 7 was collected by simple filtration after complete crystallization. It should be noted that this protocol yielded compounds 7a-e in 10-33% (Table 1), on the contrary, attempts to prepare the lipophilic trifluoromethyl derivatives 7f-l failed in spite of long crystallization times (168 and 336 h) at 4 °C and modification of the solvent for reaction (THF, dioxane). In the next step, for the introduction of molecular diversity on Cβ hydroxyl group of 4 by O-alkylation, we used a series of alkyl halides 5 carrying various chains (n = 0 or 2) and methoxy group in para-position of the phenyl moiety (5a: 4-methoxybenzyl chloride; 5b: 1-(2-bromoethyl)-4-methoxybenzene; 5c: 1-(3-bromopropyl)-4-methoxybenzene; 5d: 3-phenylpropyl bromide). For optimization of the reaction condition parameters, we developed a set of experiments which are the following: (i) the choice of the solvent (DMF, DMSO, MeCN, etc.); (ii) the choice of appropriate base (DIPEA, Et3N, KOH); (iii) the number of equivalents for the base; (iv) the reaction time; and (v) appropriate reaction temperature. We obtained good reproducibility ( Table 2 for each product) when we applied a reaction time of 48 or 72 h (monitored by thin layer chromatography on silica with appropriate eluent for evaluation of consumption of the starting reagents), 2-5 equivalents of KOH, heating at 50 °C (during 48 or 72 h) after addition of alkyl halide 5 to produce the intermediate 6 which was not isolated (initial attempts to isolate it by flash chromatography on silica gel failed). After the addition of saturated brine to the crude reaction mixture (to solubilize the N-alkylated pyrazolium by-product), the intermediate 6 was extracted with dry diethyl ether and the collected extracts were directly treated with a commercial solution of 1 M HCl in ether. The desired salt 7 was collected by simple filtration after complete crystallization. It should be noted that this protocol yielded compounds 7a-e in 10-33% (Table 1), on the contrary, attempts to prepare the lipophilic trifluoromethyl derivatives 7f-l failed in spite of long crystallization times (168 and 336 h) at 4 °C and modification of the solvent for reaction (THF, dioxane). For this structure-activity relationship (SAR) study, we were also interested to evaluate the potential impact of chirality's of the compounds 7 on SOC channels activities because our present protocol for preparation of these pyrazole SKF-96365 analogues produced a (±) racemic mixture. Based on this finding, our attention was attracted by the method of half-quantities [28] for resolution of our (±)-(1R, 1S)-1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b mixture (Scheme 2). To find a simple and cost effective resolution procedure for (1R)-4b and (1S)-4b, we used commercial (+)-(1S)-and (−)-(1R)-10-camphorsulfonic acid (CSA). Treatment of (±)-(1R, In the next step, for the introduction of molecular diversity on Cβ hydroxyl group of 4 by O-alkylation, we used a series of alkyl halides 5 carrying various chains (n = 0 or 2) and methoxy group in para-position of the phenyl moiety (5a: 4-methoxybenzyl chloride; 5b: 1-(2-bromoethyl)-4-methoxybenzene; 5c: 1-(3-bromopropyl)-4-methoxybenzene; 5d: 3-phenylpropyl bromide). For optimization of the reaction condition parameters, we developed a set of experiments which are the following: (i) the choice of the solvent (DMF, DMSO, MeCN, etc.); (ii) the choice of appropriate base (DIPEA, Et3N, KOH); (iii) the number of equivalents for the base; (iv) the reaction time; and (v) appropriate reaction temperature. We obtained good reproducibility (Table 2 for each product) when we applied a reaction time of 48 or 72 h (monitored by thin layer chromatography on silica with appropriate eluent for evaluation of consumption of the starting reagents), 2-5 equivalents of KOH, heating at 50 °C (during 48 or 72 h) after addition of alkyl halide 5 to produce the intermediate 6 which was not isolated (initial attempts to isolate it by flash chromatography on silica gel failed). After the addition of saturated brine to the crude reaction mixture (to solubilize the N-alkylated pyrazolium by-product), the intermediate 6 was extracted with dry diethyl ether and the collected extracts were directly treated with a commercial solution of 1 M HCl in ether. The desired salt 7 was collected by simple filtration after complete crystallization. It should be noted that this protocol yielded compounds 7a-e in 10-33% (Table 1), on the contrary, attempts to prepare the lipophilic trifluoromethyl derivatives 7f-l failed in spite of long crystallization times (168 and 336 h) at 4 °C and modification of the solvent for reaction (THF, dioxane).   (Table 2 for each product) when we applied a reaction time of 48 or 72 h (monitored by thin layer chromatography on silica with appropriate eluent for evaluation of consumption of the starting reagents), 2-5 equivalents of KOH, heating at 50 °C (during 48 or 72 h) after addition of alkyl halide 5 to produce the intermediate 6 which was not isolated (initial attempts to isolate it by flash chromatography on silica gel failed). After the addition of saturated brine to the crude reaction mixture (to solubilize the N-alkylated pyrazolium by-product), the intermediate 6 was extracted with dry diethyl ether and the collected extracts were directly treated with a commercial solution of 1 M HCl in ether. The desired salt 7 was collected by simple filtration after complete crystallization. It should be noted that this protocol yielded compounds 7a-e in 10-33% (Table 1), on the contrary, attempts to prepare the lipophilic trifluoromethyl derivatives 7f-l failed in spite of long crystallization times (168 and 336 h) at 4 °C and modification of the solvent for reaction (THF, dioxane). In the next step, for the introduction of molecular diversity on Cβ hydroxyl group of 4 by O-alkylation, we used a series of alkyl halides 5 carrying various chains (n = 0 or 2) and methoxy group in para-position of the phenyl moiety (5a: 4-methoxybenzyl chloride; 5b: 1-(2-bromoethyl)-4-methoxybenzene; 5c: 1-(3-bromopropyl)-4-methoxybenzene; 5d: 3-phenylpropyl bromide). For optimization of the reaction condition parameters, we developed a set of experiments which are the following: (i) the choice of the solvent (DMF, DMSO, MeCN, etc.); (ii) the choice of appropriate base (DIPEA, Et3N, KOH); (iii) the number of equivalents for the base; (iv) the reaction time; and (v) appropriate reaction temperature. We obtained good reproducibility ( Table 2 for each product) when we applied a reaction time of 48 or 72 h (monitored by thin layer chromatography on silica with appropriate eluent for evaluation of consumption of the starting reagents), 2-5 equivalents of KOH, heating at 50 °C (during 48 or 72 h) after addition of alkyl halide 5 to produce the intermediate 6 which was not isolated (initial attempts to isolate it by flash chromatography on silica gel failed). After the addition of saturated brine to the crude reaction mixture (to solubilize the N-alkylated pyrazolium by-product), the intermediate 6 was extracted with dry diethyl ether and the collected extracts were directly treated with a commercial solution of 1 M HCl in ether. The desired salt 7 was collected by simple filtration after complete crystallization. It should be noted that this protocol yielded compounds 7a-e in 10-33% (Table 1), on the contrary, attempts to prepare the lipophilic trifluoromethyl derivatives 7f-l failed in spite of long crystallization times (168 and 336 h) at 4 °C and modification of the solvent for reaction (THF, dioxane).  For this structure-activity relationship (SAR) study, we were also interested to evaluate the potential impact of chirality's of the compounds 7 on SOC channels activities because our present protocol for preparation of these pyrazole SKF-96365 analogues produced a (±) racemic mixture. Based on this finding, our attention was attracted by the method of half-quantities [28] for resolution of our   For this structure-activity relationship (SAR) study, we were also interested to evaluate the potential impact of chirality's of the compounds 7 on SOC channels activities because our present protocol for preparation of these pyrazole SKF-96365 analogues produced a (±) racemic mixture. Based on this finding, our attention was attracted by the method of half-quantities [28]    For this structure-activity relationship (SAR) study, we were also interested to evaluate the potential impact of chirality's of the compounds 7 on SOC channels activities because our present protocol for preparation of these pyrazole SKF-96365 analogues produced a (±) racemic mixture. Based on this finding, our attention was attracted by the method of half-quantities [28]    For this structure-activity relationship (SAR) study, we were also interested to evaluate the potential impact of chirality's of the compounds 7 on SOC channels activities because our present protocol for preparation of these pyrazole SKF-96365 analogues produced a (±) racemic mixture. Based on this finding, our attention was attracted by the method of half-quantities [28]   For this structure-activity relationship (SAR) study, we were also interested to evaluate the potential impact of chirality's of the compounds 7 on SOC channels activities because our present protocol for preparation of these pyrazole SKF-96365 analogues produced a (±) racemic mixture. Based on this finding, our attention was attracted by the method of half-quantities [28] (Table 3). This protocol was also applied to (+)-(1S)-CSA for resolution of the (±) racemic 4b, and after diastereomeric enrichment, the other diastereomer [(+)-(1R)-4b/(+)-(1S)-CSA, (>99% de)] was obtained in 51% yield with [αD] = +6.0 (c 1.0, MeOH). Crystal structures of the salt diastereomers were determined by X-ray crystal structure analysis, as example the diastereomer (+)-(1R)-4b/(+)-(1S)-CSA is shown in Figure 2.  During the resolution of the (±) racemic mixture 4b by the "half-quantities" protocol with (−)-(1R)-CSA or (+)-(1S)-CSA and neutralization of the two diastereomers, all these compounds were submitted to chiral HPLC analysis with a Chiracel OJ-H column (250 × 4.6 i.d. mm) and a UV detector at 220 nm using hexane/i-PrOH as mobile phase with appropriate composition and flow rate. Ortep diagram of (+)-(1R) 1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b/(+)-(1S)-10-camphorsulfonic acid obtained by X-ray diffraction.
Next, access to the free enantiomer (1S)-4b and (1R)-4b was operated by a simple neutralization of the respective diastereomers (−)-(1S)-4b/(−)-(1R)-CSA or (+)-(1R)-4b/(+)-(1S)-CSA using exactly 1 equivalent of MeONa in dry MeOH. After mixing during 12 h followed by elimination of volatile compounds in vacuo, the crude reaction mixture was treated with deionized water and the resulting insoluble enantiomer (−)-(1S)-4b or (+)-(1R)-4b precipitated and was collected by classical filtration. As can be seen in Table 3 During the resolution of the (±) racemic mixture 4b by the "half-quantities" protocol with (−)-(1R)-CSA or (+)-(1S)-CSA and neutralization of the two diastereomers, all these compounds were submitted to chiral HPLC analysis with a Chiracel OJ-H column (250 × 4.6 i.d. mm) and a UV detector at 220 nm using hexane/i-PrOH as mobile phase with appropriate composition and flow rate.  During the resolution of the (±) racemic mixture 4b by the "half-quantities" protocol with (−)-(1R)-CSA or (+)-(1S)-CSA and neutralization of the two diastereomers, all these compounds were submitted to chiral HPLC analysis with a Chiracel OJ-H column (250 × 4.6 i.d. mm) and a UV detector at 220 nm using hexane/i-PrOH as mobile phase with appropriate composition and flow rate. For the (±) racemic mixture 4b, we obtained only two peaks, the retention time of the first and second eluted enantiomers were measured respectively as 65.9 and 71.4 min (hexane/i-PrOH 96/4 v/v, flow rate = 0.6 mL/min). The quality of the results obtained by the "half-quantities" and neutralization methods were confirmed by the presence of only one peak for the two diastereomers For the (±) racemic mixture 4b, we obtained only two peaks, the retention time of the first and second eluted enantiomers were measured respectively as 65.9 and 71.4 min (hexane/i-PrOH 96/4 v/v, flow rate = 0.6 mL/min). The quality of the results obtained by the "half-quantities" and neutralization methods were confirmed by the presence of only one peak for the two diastereomers Progress of the transformation and consumption of the starting reagents were monitored by 1 H NMR and also by thin layer chromatography on silica plates. After extraction with dry Et 2 O and treatment of the collected extracts with a solution of 1 M HCl, to our surprise, we did not observe precipitation of the desired (−)-(1S)-7d and (+)-(1R)-7d. We tried to change the solvent for extraction (dioxane as example) but this modification was not well suited for crystallization at room temperature even for an additional day at 4 • C.

Biology
The synthesized hydrochloride compounds 7a-c as described above were evaluated for their ability on endoplasmic reticulum (ER) Ca 2+ release and SOCE using the PLP-B lymphocyte cell line. For comparison, commercial SKF-96365 hydrochloride was used as reference. All compounds 7 and SKF-96365 were added 3 min before recording intracellular Ca 2+ level variation in the presence of Fura-2 dye loaded on PLP-B lymphocyte cell. Fluorescence measurements were realized in Flex Station™ 3 microplate reader. Fura-2 loaded PLP-B lymphocyte cells were stimulated with 2 µM Thapsigargin (Tg) for 30 min and SOCE was measured after addition of 1.8 mM Ca 2+ in the extracellular medium. Results are respectively summarized in Table 4 and also in Figure 3A-E. For this structure-activity relationship (SAR) study, we examined mainly the effect of MeO substituent on the para-phenyl group of the phenethyl-1H-pyrazolium skeleton and also on the phenylalkoxy chain of Cβ compared to the SKF-96365 reference. With the two pure enantiomers (−)-(1S)-4b, (+)-(1R)-4b on hand, we were stimulated to prepare potentially the compounds (−)-(1S)-7d and (+)-(1R)-7d with the alkyl halide 1-(3-bromopropyl)-4-methoxybenzene 5c according to Scheme 2. We applied the protocol used initially for the preparation of racemic (1R, 1S)-7d using 5 equivalents of KOH in solution of DMSO at 50 °C during 72 h from (−)-(1S)-4b or (+)-(1R)-4b and 4-(3-bromopropyl)-4-methoxybenzene 5c (Scheme 3). Progress of the transformation and consumption of the starting reagents were monitored by 1 H NMR and also by thin layer chromatography on silica plates. After extraction with dry Et2O and treatment of the collected extracts with a solution of 1 M HCl, to our surprise, we did not observe precipitation of the desired (−)-(1S)-7d and (+)-(1R)-7d. We tried to change the solvent for extraction (dioxane as example) but this modification was not well suited for crystallization at room temperature even for an additional day at 4 °C.

Biology
The synthesized hydrochloride compounds 7a-c as described above were evaluated for their ability on endoplasmic reticulum (ER) Ca 2+ release and SOCE using the PLP-B lymphocyte cell line. For comparison, commercial SKF-96365 hydrochloride was used as reference. All compounds 7 and SKF-96365 were added 3 min before recording intracellular Ca 2+ level variation in the presence of Fura-2 dye loaded on PLP-B lymphocyte cell. Fluorescence measurements were realized in Flex Station™ 3 microplate reader. Fura-2 loaded PLP-B lymphocyte cells were stimulated with 2 μM Thapsigargin (Tg) for 30 min and SOCE was measured after addition of 1.8 mM Ca 2+ in the extracellular medium. Results are respectively summarized in Table 4 and also in Figure 3A-E. For this structure-activity relationship (SAR) study, we examined mainly the effect of MeO substituent on the para-phenyl group of the phenethyl-1H-pyrazolium skeleton and also on the phenylalkoxy chain of Cβ compared to the SKF-96365 reference.
Examination of Table 1 and also dose response curves ( Figure 3E) showed that compound 7d (IC50 25 μM) was a better potent SOCE inhibitor than SKF-96365 (IC50 60 μM). This means that a slight modification of the heterocyclic platform, particularly the position of the second nitrogen atom (N-3 position in imidazole for SKF-96365 and N-2 position for 7d), has a direct impact on SOCE activity. The effect of the length for the Cβ-(4-methoxyphenylalkoxy) side chain appeared to be important in Figure 3C when we compared compounds 7c and 7d: for the same concentration of 7, the curve obtained for compound 7d gave higher effect on SOCE inhibition level (%). Again, the presence or absence of MeO group in para-position for the phenethyl-1H-pyrazolium skeleton of 7 and for the Cβ-phenylpropoxy side chain afforded variation of IC50, i.e., comparison of 7b (IC50 34 μM) and 7d (IC50 25 μM) showed that absence of MeO group in the skeleton of 7b has a lower effect on SOCE IC50. On the other hand, absence of MeO group of 7c (IC50 48 μM) on the side chain led to a more important effect and the IC50 decreased. Similar observations were also made when a more physiological stimulation of SOCE activation was implemented (Figure 4). When B cells are stimulated with an antigen (M Immunoglobulin: IgM), B cell receptor (BCR) activation leads to an increase of Ca 2+ concentration mainly due SOCE. However, inhibition by 7c and 7d compounds is quite identical and less than what was observed with SKF-96365. Examination of Table 1 and also dose response curves ( Figure 3E) showed that compound 7d (IC 50 25 µM) was a better potent SOCE inhibitor than SKF-96365 (IC 50 60 µM). This means that a slight modification of the heterocyclic platform, particularly the position of the second nitrogen atom (N-3 position in imidazole for SKF-96365 and N-2 position for 7d), has a direct impact on SOCE activity. The effect of the length for the Cβ-(4-methoxyphenylalkoxy) side chain appeared to be important in Figure 3C when we compared compounds 7c and 7d: for the same concentration of 7, the curve obtained for compound 7d gave higher effect on SOCE inhibition level (%). Again, the presence or absence of MeO group in para-position for the phenethyl-1H-pyrazolium skeleton of 7 and for the Cβ-phenylpropoxy side chain afforded variation of IC 50 , i.e., comparison of 7b (IC 50 34 µM) and 7d (IC 50 hydrochloride salt 7d). Effects of compounds 7a-d on endoplasmic reticulum (ER) Ca 2+ and SOCE were evaluated on PLP-B lymphocyte cell line, and 7d was identified as a better SOCE inhibitor than SKF-96365. However, the inhibitory effects of 7c and 7d compounds on SOCE are inferior to what was observed for SKF-96365 when evaluated with BCR stimulation. This preliminary SAR study showed that the MeO group in para-position of the phenethyl-1H-pyrazolium skeleton or for the Cβ-phenylpropoxy side chain of 7 influenced the SOCE activity. These results offered possibilities to increase molecular diversity for a complete SAR study, and we are also currently exploring the potential of this synthetic methodology.      Table 4.
Results for effects of (±)-(1R, 1S) 1-[β-(phenylalkoxy)-phenethyl]-1H-pyrazolium hydrochloride 7a-e on store-operated Ca 2+ entry with PLP-B lymphocyte. Store-operated Ca 2+ entry was induced by depletion of Endoplasmic Reticulum Ca 2+ stores (ER Ca 2+ stores) with thapsigargin (Tg) in a Ca 2+ -free medium and measured following the addition of 1.8 mM CaCl 2 in the extracellular medium.  Table 4. Results for effects of (±)-(1R, 1S) 1-[β-(phenylalkoxy)-phenethyl]-1H-pyrazolium hydrochloride 7a-e on store-operated Ca 2+ entry with PLP-B lymphocyte. Store-operated Ca 2+ entry was induced by depletion of Endoplasmic Reticulum Ca 2+ stores (ER Ca 2+ stores) with thapsigargin (Tg) in a Ca 2+ -free medium and measured following the addition of 1.8 mM CaCl2 in the extracellular medium.  of hydrochloride salt 7d). Effects of compounds 7a-d on endoplasmic reticulum (ER) Ca 2+ and SOCE were evaluated on PLP-B lymphocyte cell line, and 7d was identified as a better SOCE inhibitor than SKF-96365. However, the inhibitory effects of 7c and 7d compounds on SOCE are inferior to what was observed for SKF-96365 when evaluated with BCR stimulation. This preliminary SAR study showed that the MeO group in para-position of the phenethyl-1H-pyrazolium skeleton or for the Cβ-phenylpropoxy side chain of 7 influenced the SOCE activity. These results offered possibilities to increase molecular diversity for a complete SAR study, and we are also currently exploring the potential of this synthetic methodology.  Table 4. Results for effects of (±)-(1R, 1S) 1-[β-(phenylalkoxy)-phenethyl]-1H-pyrazolium hydrochloride 7a-e on store-operated Ca 2+ entry with PLP-B lymphocyte. Store-operated Ca 2+ entry was induced by depletion of Endoplasmic Reticulum Ca 2+ stores (ER Ca 2+ stores) with thapsigargin (Tg) in a Ca 2+ -free medium and measured following the addition of 1.8 mM CaCl2 in the extracellular medium.  of hydrochloride salt 7d). Effects of compounds 7a-d on endoplasmic reticulum (ER) Ca 2+ and SOCE were evaluated on PLP-B lymphocyte cell line, and 7d was identified as a better SOCE inhibitor than SKF-96365. However, the inhibitory effects of 7c and 7d compounds on SOCE are inferior to what was observed for SKF-96365 when evaluated with BCR stimulation. This preliminary SAR study showed that the MeO group in para-position of the phenethyl-1H-pyrazolium skeleton or for the Cβ-phenylpropoxy side chain of 7 influenced the SOCE activity. These results offered possibilities to increase molecular diversity for a complete SAR study, and we are also currently exploring the potential of this synthetic methodology.  Table 4. Results for effects of (±)-(1R, 1S) 1-[β-(phenylalkoxy)-phenethyl]-1H-pyrazolium hydrochloride 7a-e on store-operated Ca 2+ entry with PLP-B lymphocyte. Store-operated Ca 2+ entry was induced by depletion of Endoplasmic Reticulum Ca 2+ stores (ER Ca 2+ stores) with thapsigargin (Tg) in a Ca 2+ -free medium and measured following the addition of 1.8 mM CaCl2 in the extracellular medium. This preliminary SAR study showed that the MeO group in para-position of the phenethyl-1H-pyrazolium skeleton or for the Cβ-phenylpropoxy side chain of 7 influenced the SOCE activity. These results offered possibilities to increase molecular diversity for a complete SAR study, and we are also currently exploring the potential of this synthetic methodology.

100
−   This preliminary SAR study showed that the MeO group in para-position of the phenethyl-1H-pyrazolium skeleton or for the Cβ-phenylpropoxy side chain of 7 influenced the SOCE activity. These results offered possibilities to increase molecular diversity for a complete SAR study, and we are also currently exploring the potential of this synthetic methodology.  Table 4. Results for effects of (±)-(1R, 1S) 1-[β-(phenylalkoxy)-phenethyl]-1H-pyrazolium hydrochloride 7a-e on store-operated Ca 2+ entry with PLP-B lymphocyte. Store-operated Ca 2+ entry was induced by depletion of Endoplasmic Reticulum Ca 2+ stores (ER Ca 2+ stores) with thapsigargin (Tg) in a Ca 2+ -free medium and measured following the addition of 1.8 mM CaCl2 in the extracellular medium. This preliminary SAR study showed that the MeO group in para-position of the phenethyl-1H-pyrazolium skeleton or for the Cβ-phenylpropoxy side chain of 7 influenced the SOCE activity. These results offered possibilities to increase molecular diversity for a complete SAR study, and we are also currently exploring the potential of this synthetic methodology.   This preliminary SAR study showed that the MeO group in para-position of the phenethyl-1H-pyrazolium skeleton or for the Cβ-phenylpropoxy side chain of 7 influenced the SOCE activity. These results offered possibilities to increase molecular diversity for a complete SAR study, and we are also currently exploring the potential of this synthetic methodology.

Chemistry Section
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 silica 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 (New Castle, PA, USA). All reagents and solvents were purchased from Acros Fisher (Illkirch, France), Sigma-Aldrich Chimie (St. Quentin Fallavier, France), and Fluka Chimie (Paris, France) and were used without further purification. 1 H NMR spectra were recorded on Bruker AC 300 P (300 MHz) spectrometer and 13 C NMR spectra on Bruker AC 300 P (75 MHz) spectrometer. 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 at the "Centre Régional de Mesures Physiques de l'Ouest" platform (CRMPO platform, ScanMAT UMS 2001 CNRS, Rennes, France). Melting points were determined on a Kofler melting point apparatus and were uncorrected. Optical rotations [α D ] were measured on a Perkin-Elmer 214 polarimeter at room temperature (25 • C) and are recorded in units of deg cm −3 g −1 dm −1 (c in g cm −3 in MeOH) with a 1.0 cm cell. The ee-and de-values were determined by chiral HPLC analysis using Chiracel OJ-H column (250 × 4.60 mm) with UV detector at 220 nm using hexane/i-PrOH as mobile phase with appropriate composition and flow rate.
1-Phenyl-2-(1H-pyrazol-1-yl)ethan-1-one (3a). To a solution of 2-bromoacetophenone 1a (4 g, 20.1 mmol) in 20.8 mL of acetonitrile, pyrazole 2a (1.45 g, 21.3 mmol, 1.06 equiv.) was added in small portions under vigorous magnetic stirring (550 rpm) at room temperature, and mixing was pursued until complete dissolution of the reagents. To this homogeneous solution, K 2 CO 3 (2.92 g, 21.1 mmol, 1.05 equiv.) was poured and the resulting suspension was stirred for 8 h at 25 • C and monitored by thin layer chromatography on 0.2 mm plates of silica gel 60 F-254 (Merck) using cyclohexane/AcOEt (1:1 v/v) as eluent. The reaction mixture was diluted with 20 mL of AcOEt, and the resulting solution was filtered on a Büchner funnel (porosity N • 4) and the residual precipitate was washed with AcOEt (2 × 10 mL). The collected filtrate was transferred into a separating funnel. The organic phase was washed successively with deionized water (3 × 80 mL), brine (3 × 80 mL), and dried over magnesium sulfate. After filtration on a filter paper, the filtrate was concentrated in a rotary evaporator under reduced pressure and the oily residue was submitted to purification by preparative chromatography 1-(4-Methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-one (3b). To a solution of 2-bromo-1-(4-methoxyphenyl)ethan-1-one 1b (4.6 g, 20.1 mmol) in 20.8 mL of acetonitrile, pyrazole 2a (2.74 g, 40.2 mmol, 2 equiv.) was added in small portions under vigorous magnetic stirring (550 rpm) at room temperature, and mixing was pursued until complete dissolution of the reagents. To this homogeneous solution, K 2 CO 3 (5.56 g, 40.2 mmol, 2 equiv.) was poured and the resulting suspension was stirred for 12 h at 25 • C and monitored by thin layer chromatography on 0.2 mm plates of silica gel 60 F-254 (Merck) using cyclohexane/AcOEt (3:7 v/v) as eluent. The reaction mixture was diluted with 20 mL of AcOEt, and the resulting suspension was filtered on a Büchner funnel (porosity N • 4) and the residual precipitate was washed with AcOEt (2 × 10 mL). The collected filtrate was transferred into a separating funnel. The organic phase was washed successively with deionized water (3 × 80 mL), brine (3 × 80 mL), and dried over magnesium sulfate. After filtration on a filter paper, the filtrate was concentrated in a rotary evaporator under reduced pressure and the oily residue was submitted to purification by preparative chromatography (Combi Flash R f 200 psi apparatus with a DAD 200/360 nm detector) on pre-packed column of silica gel 60 F-254 (Merck) using a stepwise gradient of cyclohexane/AcOEt (0-70%) for elution. Pooling for 60 min and elimination of the solvent in vacuo gave 4.35 g (66% yield) of the pure desired compound 3b as yellowish needles.

2-(3-Trifluoromethyl-1H-pyrazol-1-yl)-1-(4-methoxyphenyl)ethan-1-one (3c).
To a solution of 2-bromo-1-(4-methoxyphenyl)ethan-1-one 1b (0.5 g, 2.18 mmol) in 2.25 mL of acetonitrile, 3-trifluoromethylpyrazole 2b (0.89 g, 6.54 mmol, 3 equiv.) was added in small portions under vigorous magnetic stirring (550 rpm) at room temperature, and mixing was pursued until complete dissolution of the reagents. To this homogeneous solution, K 2 CO 3 (0.905 g, 6.54 mmol, 3 equiv.) was poured and the resulting suspension was stirred for 12 h at 25 • C and monitored by thin layer chromatography on 0.2 mm plates of silica gel 60 F-254 (Merck) using cyclohexane/AcOEt (1:1 v/v) as eluent. The reaction mixture was diluted with 5 mL of AcOEt, and the resulting solution was filtered on a Büchner funnel (porosity N • 4) and the residual precipitate was washed with 5 mL of AcOEt. The collected filtrate was transferred into a separating funnel. The organic phase was washed successively with deionized water (3 × 20 mL), brine (3 × 20 mL), and dried over magnesium sulfate. After filtration on a filter paper, the filtrate was concentrated in a rotary evaporator under reduced pressure and gave a solid residue. After addition of 40 mL of hexane and mixing for 4 h, the solid was filtered on a Büchner funnel (porosity N • 4) then dried at 60 • C for 3 h and gave 0.

2-(3,5-Bis
To a solution of 2-bromo-1-(4-methoxyphenyl)ethan-1-one 1b (0.5 g, 2.18 mmol) in 2.25 mL of acetonitrile, 3,5-bis-trifluoromethylpyrazole 2c (1.778 g, 8.72 mmol, 4 equiv.) was added in small portions under vigorous magnetic stirring (550 rpm) at room temperature, and mixing was pursued until complete dissolution of the reagents. To this homogeneous solution, K 2 CO 3 (1.205 g, 8.72 mmol, 4 equiv.) was poured and the resulting suspension was stirred for 24 h at 25 • C and monitored by thin layer chromatography on 0.2 mm plates of silica gel 60 F-254 (Merck) using cyclohexane/AcOEt (1:1 v/v) as eluent. The reaction mixture was diluted with 5 mL of AcOEt, and the resulting solution was filtered on a Büchner funnel (porosity N • 4) and the residual precipitate was washed with 5 mL of AcOEt. The collected filtrate was transferred into a separating funnel. The organic phase was washed successively with deionized water (3 × 20 mL), brine (3 × 20 mL), and dried over magnesium sulfate. After filtration on a filter paper, the filtrate was concentrated in a rotary evaporator under reduced pressure and gave a solid residue which was dried under high vacuum (10 −2 Torr) at 25 •  To a solution of 1-phenyl-2-(1H-pyrazol-1-yl)ethan-1-one 3 (5 mmol) in an appropriate volume of anhydrous methanol (9-40 mL), sodium borohydride NaBH 4 (0.189 g, 5 mmol, 1 equiv.) was added in small portions at 0 • C (ice bath) under magnetic stirring (300 rpm). The resulting mixture was stirred (500 rpm) for an appropriate reaction time (5-7 h) at room temperature, and the reaction solution was monitored by thin layer chromatography (TLC) on 0.2 mm plates of silica gel 60 F-254 (Merck) using an appropriate mixture of solvents as eluent. The reaction mixture was concentrated in a rotary evaporator under reduced pressure and the resulting solid residue was washed with deionized water (3 × 45-3 × 47 mL) on a Büchner funnel. Then, the desired compounds 3a-c were dried at 60 • C for 3 h.
According to the standard procedure, the compound 4b was prepared from 1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-one 3b (1.08 g, 5 mmol) in 14.4 mL of anhydrous methanol after a reaction time of 7 h using CH 2 Cl 2 /MeOH 8:2 v/v as eluent for thin layer chromatography. Washing work-up was realized with 3 × 47 mL of deionized water and gave 1.  To a solution of 2-(3,5-bis-trifluoromethyl-1H-pyrazol-1-yl)-1-(4-methoxyphenyl)ethan-1-one 3d (1.76 g, 5 mmol) in 31 mL of anhydrous methanol, sodium borohydride NaBH 4 (0.378 g, 10 mmol, 2 equiv.) was added in small portions at 0 • C (ice bath) under magnetic stirring (300 rpm). The resulting mixture was stirred (500 rpm) for 7 h at room temperature and the reaction solution was monitored by thin layer chromatography on 0.2 mm plates of silica gel 60 F-254 (Merck) using hexane/AcOEt 1:1 v/v as eluent. After concentration of the reaction mixture in vacuo, 44 mL of deionized water was added in the crude oily residue, and extraction was conducted with AcOEt (3 × 44 mL) in a separating funnel. The combined extracts were washed with 44 mL of brine and dried over magnesium sulfate. After filtration on a filter paper, the filtrate was concentrated in a rotary evaporator under reduced pressure and gave an oily residue which was dried under high vacuum (10 −2 Torr) at 25 • C for 2 h. The desired compound 4d (1.771 g) was obtained as yellowish mobile oil in 98% yield. 1 13 C NMR (DMSO-d 6 ) δ = 55.1 (OCH3), 58.8 (C-2), 70.6 (C-1), 106.8 (C-4 ), 113.7 (C-3", C-5"), 127.2 (C-2", C-6"), 133.5 (C-1"), 158.9 (C-4"). ES + HRMS, m/z = 377.0700 found (calculated for C 14  Pellets of potassium hydroxide KOH (0.112 g, 2 mmol, 2 equiv.) were added to a solution of 1-phenyl-2-(1H-pyrazol-1-yl)ethan-1-ol 4 (1 mmol, 1 equiv.) in 3.76 mL of dry dimethylsulfoxide pa at room temperature. The resulting mixture was stirred vigorously (550 rpm) for 30 min until complete dissolution of KOH, then commercial arylalkyl halide 5 (1 mmol, 1 equiv.) was added in one portion in the reaction mixture. The reaction mixture was heated at 50 • C under magnetic stirring (300 rpm) for 48 h. After cooling down to room temperature, 18.7 mL of saturated brine was added to the reaction mixture and the resulting solution was transferred into a separating funnel. Extraction was conducted with 18.7 mL of diethyl ether Et 2 O, then the organic phase was dried over magnesium sulphate and filtered on a filter paper. The filtrate was concentrated in a rotary evaporator under reduced pressure. The oily crude residue containing1-[β-(phenylalkoxy)-phenethyl]-1H-pyrazole 6 was dissolved in appropriate volume of diethyl ether Et 2 O pa under a stream of argon with magnetic stirring (200 rpm). To this homogeneous solution, a commercial solution of 1 M HCl (1 equiv.) in ether was added dropwise rapidly. During mixing at room temperature, the initial pale yellow oil crystallized progressively on the circumference of the round flask and mixing (from 24 h to 5 days) was pursued until complete crystallization of the desired 1-[β-(phenylalkoxy)-phenethyl]-1H-pyrazolium hydrochloride 7. The resulting precipitate was collected by filtration on a Büchner funnel (porosity N • 4) and washed with dry diethyl ether pa. The desired compound 7 was dried at room temperature for 4 h and stored in a dessicator.
Starting from 200 mg of the pure salt (+)-(1R)-4b/(+)-(1S)-CSA, a mixed suspension was prepared in 2.5 mL of dry methanol, and 1 equiv. of commercial MeONa was added in one portion. The resulting reaction mixture was stirred at room temperature for 12 h and then concentrated in a rotary evaporator under reduced pressure. The crude solid residue was washed thoroughly with 4 × 2 mL of deionized water. The insoluble compound was collected by filtration on a Büchner funnel (porosity N • 4) and dried in vacuum which gave 0.078 g (41% yield) of the desired ( To a stirred solution of (±) 1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b (0.5 g, 2.29 mmol) in 5 mL of dry acetone, a solution of (+)-(1S)-10-camphorsulfonic acid (0.266 g, 1.145 mmol) in 2 mL of dry acetone was added dropwise at room temperature for 5 min. Stirring (300 rpm) was pursued for 12 h at 25 • C. It is interesting to note that during the addition of the solution of (+)-(1S)-CSA, the formation of a white fine suspension of diastereomer appeared in the reaction mixture. This first fraction of diastereomer salt was collected by filtration on a Büchner funnel (porosity N • 4), washed thoroughly with 4 × 0.3 mL of dry acetone, and dried in vacuum to give 0.358 g of the desired the salt (+)-(1R)-4b/(+)-(1S)-CSA (>87% de). This salt was recrystallized in dry acetonitrile and afforded