Taraxacum officinale. The structural identification of compounds present in hexane (Hex) and ethyl acetate (AcOEt) extracts was performed by mass spectrometry (GC-MS) spectroscopic techniques and nuclear magnetic resonance (NMR) with the major compounds corresponding to different sesquiterpene lactones , monoterpene ]decane-9,10-diol), phytosterol , terpenes , and coumarin . The results obtained show that the Hex extract was highly active against Staphylococcus aureus showing a MIC of 200 μg/mL and moderately active against Escherichia coli and Klebsiella pneumoniae with MIC values of 400 μg/mL and 800 μg/mL for the other Gram-negative strains tested with Proteus mirabilis as uropathogens in vitro. Therefore, the effective dandelion extracts could be used in the development of future products with industrial application.Currently, the most effective treatment for recurrent urinary tract infections in women is antibiotics. However, the limitation for this treatment is the duration and dosage of antibiotics and the resistance that bacteria develop after a long period of administration. With the aim of identifying mainly novel natural agents with antibacterial activity, the present study was undertaken to investigate the biological and phytochemical properties of extracts from the leaves Taraxacum officinale Weber ex F. H. Wigg. (Asteraceae), commonly known as dandelion, for a systematic study on the chemical composition and potential antibacterial properties.The increasing resistance of uropathogens to antibiotics and the recognition of the generally self-limiting nature of uncomplicated urinary tract infection (UTI) suggest that it is time to reconsider the empirical treatment of UTI by using antibiotics. Limitation for this treatment is the duration and dosage of antibiotics and the resistance that bacteria develop after a long period of administration . TherefoT. officinale is an herbaceous perennial plant of the family Asteraceae, native to Asia; it can be found growing in temperate regions of the world, including Chile in the III-IX region. Dandelion is considered a weedy species [ Taraxacum, only few studies concerning its antimicrobial properties consider chemical identification of the extracts obtained and, most of the time, this identification is qualitative . Authors report the presence of terpenoids, triterpenoids, steroids, coumarins, phenols, saponins, flavonoids, flavones, flavonols, chalcones, phlobatannins, and cardiac glycosides in antimicrobial extracts [ species ; it has  species , 4. Ther species –7. These species , 9. For extracts –18, but  Taraxacum officinale plant and its antibacterial activity against selected uropathogenic bacterial strains.Therefore, based on the above reasoning and observations, the aim of the study was to explore the phytochemical of n-hexane and ethyl acetate extracts of the T. officinale are summarized in Tables Results of the gas chromatography analysis of hexane and ethyl acetate extracts from leaves ofα-Amyrin (4.78%), β-sitosterol (4.55%), and (22Z)-Stigmasta-5,22-dien-3β-ol acetate (2.98%). In addition, the composition of both extracts of leaves from T. officinale in which triterpenoids were the predominant portion found was reported, unlike other studies in which it has been described that most compounds constituting the leaf part are polyphenols and flavonoids glycosides [Forty components were identified in the hexane extract: 72.46% were triterpenoids, 16.56% terpenes, 4.25% phthalate ester, 1.46% fatty acids and derivatives, 1.42% aldehydes and ketones, 1.81% alcohols, and 0.55% unknown compounds. The hexane extract was mainly characterized by lupeol acetate (25.09%), betulin (23.81%), lupeol (23.31%), 3,7,11,15-tetramethyl-2-hexadecen-1-ol (13.08%), diethyl phthalate (4.25%), and phytol (3.48%). On the other hand, eighty components were identified in the ethyl acetate extract: 37.06% were triterpenoids, 17.08% terpenes, 3.04% fatty acids and derivatives, 1.50% ketones and alcohols, and 37.86% unknown compounds. The ethyl acetate extract was mainly characterized by lupeol acetate (19.95%), 3,7,11,15-tetramethyl-2-hexadecen-1-ol (15.57%), betulin (6.17%), α-Amyrin, β-sitosterol, and betulin) from both extracts were characterized and identified by different NMR experiments . In addition, all the spectra of these products were compared with literature data and with authentic samples acquired from Sigma-Aldrich. The structures of these terpenes are shown in Most of the metabolites isolated belonging to the group of terpenes ; 4.56 ; 3.18 ; 2.36 ; 1.67 ; 1.62 ; 1.38 ; 1.35 ; 1.25 ; 1.02 ; 0.96 ; 0.94 ; 0.82 ; 0.78 ; 0.67 .13C-NMR. 151.2 (C-20); 109.5 (C-29); 79.2 (C-3); 55.5 (C-5); 50.6 (C-9); 48.5 (C-18); 48.2 (C-19); 43.2 (C-17); 43.0 (C-14); 41.0 (C-8); 40.2 (C-22); 39.0 (C-4); 38.9 (C-1); 38.2 (C-13); 37.3 (C-10); 35.8 (C-16); 34.4 (C-7); 30.0 (C-21); 28.2 (C-23); 27.2 (C-15); 27.6 (C-2); 25.3 (C-12); 21.1 (C-11); 19.5 (C-30); 18.5 (C-6); 18.1 (C-28); 16.3 (C-26); 16.2 (C-25); 15.6 (C-24); 14.7 (C-27) [; 48.2 C-; 43.2 ; 4.57 ; 4.48 ; 2.37 ; 2.04 ; 1.68 ; 1.03 ; 0.94 ; 0.88 ; 0.88 ; 0.70 .13C-NMR. 171.4 (CO); 151.4 (C-20); 109.7 (C-29); 81.3 (C-3); 55.7 (C-5); 50.7 (C-9); 48.3 (C-18); 48.3 (C-19); 43.2 (C-17); 42.8 (C-14); 41.2 (C-8); 40.3 (C-22); 38.7 (C-1); 38.4 (C-4); 38.1 (C-13); 37.4 (C-10); 34.5 (C-16); 34.5 (C-7); 30.2 (C-21); 28.3 (C-23); 27.8 (C-2); 25.4 (C-15); 25.4 (C-12); 21.3 (C-11); 21.1 (CH3CO); 19.6 (C-30); 18.5 (C-6); 18.3 (C-28); 16.8 (C-24); 16.5 (C-25); 16.3 (C-26); 14.8 (C-27) [; 28.3 C-; 27.8 ; 3.16 ; 1.94 ; 1.85 ; 1.76 ; 1.01 ; 0.94 ; 0.93 ; 0.89 ; 0.85 ; 0.73 ; 0.67 .13C-NMR. 139.5 (C-13); 124.4 (C-12); 79.6 (C-3); 59.0 (C-18); 55.1 (C-5); 47.7 (C-9); 42.0 (C-14); 41.5 (C-22); 40.0 (C-8); 39.6 (C-19); 39.6 (C-20); 38.7 (C-1); 38.7 (C-4); 36.9 (C-10); 33.7 (C-17); 32.2 (C-7); 31.2 (C-21); 28.7 (C-2); 28.1 (C-28); 28.1 (C-23); 27.2 (C-15); 26.6 (C-16); 23.3 (C-11); 23.2 (C-27); 21.4 (C-30); 18.4 (C-6); 17.4 (C-29); 16.8 (C-26); 15.6 (C-25); 15.6 (C-24) [; 39.6 C-; 38.7 ; 3.53 ; 1.01 ; 0.93 ; 0.84 ; 0.83 ; 0.81 ; 0.68 .13C-NMR. 140.9 (C-5); 121.9 (C-6); 72.0 (C-3); 56.9 (C-14); 56.3 (C-17); 50.3 (C-9); 46.1 (C-22); 42.6 (C-13); 42.5 (C-4); 39.9 (C-12); 37.5 (C-1); 36.7 (C-10); 36.3 (C-18); 34.2 (C-20); 32.1 (C-7); 32.1 (C-8); 31.9 (C-2); 29.4 (C-25); 28.5 (C-16); 26.3 (C-15); 26.3 (C-21); 23.3 (C-23); 21.3 (C-11); 20.1 (C-26); 19.6 (C-27); 19.2 (C-19); 19.0 (C-28); 12.2 (C-24); 12.0 (C-29) [1H-NMR. 4.70 ; 4.58 ; 3.79 ; 3.33 ; 3.18 ; 1.67 ; 0.99 ; 0.97 ; 0.96 ; 0.80 ; 0.75 .13C-NMR. 150.6 (C-20); 109.8 (C-29); 79.2 (C-3); 60.6 (C-28); 55.4 (C-5); 50.5 (C-9); 48.8 (C-19); 47.9 (C-17); 47.9 (C-18); 42.8 (C-14); 41.0 (C-8); 38.9 (C-1); 38.8 (C-4); 37.4 (C-10); 37.2 (C-13); 34.3 (C-7); 34.1 (C-22); 29.8 (C-21); 29.2 (C-16); 28.1 (C-23); 27.5 (C-2); 27.1 (C-15); 25.3 (C-12); 20.9 (C-11); 19.2 (C-30); 18.4 (C-6); 16.2 (C-25); 16.1 (C-26); 15.4 (C-24); 14.8 (C-27) [; 19.2 C-; 18.4 (C8 (C-27) .α-Amyrin, and β-sitosterol from both extracts with potential antibacterial activity against pathogens that infect the urinary tract. In several investigations, the toxicity of dandelion was found to be low, due to absence of any significant toxins or alkaloids [Recently, other authors have determined from ethanol extract three novel lupane-, bauerane-, and euphane-type triterpenoids and other known triterpenes present in dandelion roots, using spectroscopic analysis, which have potential anti-inflammatory activity , 33. Danlkaloids . TherefoIt was determined that the total fatty acid content of the dandelion leaves, using the Soxhlet method, is 4.8 ± 0.5% raw extract/dry matter, composed of different fatty acids as shown in  T. officinale; seven fatty acids were identified in the raw extracts, from which the most abundant was palmitic acid (16 : 0) with 41 ± 0.8%. The next most abundant fatty acids were linolenic acid (18 : 3n-3) and linoleic acid (18 : 2n-6), corresponding to 26 ± 1.9% and 17 ± 1.0%, respectively. Small and very small amounts of stearic (18 : 0), oleic (18 : 1n-9), myristic (14 : 0), and arachidic (20 : 0) acids were also detected. Paenibacillus (American foulbrood), Melissococcus pluton (European larvae foulbrood), and other microorganisms that inhabit the brood combs of beehives.Manning 2001  mentioneThe total fatty acid content in leaves, relative to dry plant weight, is 3,9 mg per gram and palmitic acid 1.5 mg per gram as the prevailing fatty acid in the sample. The next two most abundant fatty acids were 18 : 2n-6 and 18 : 3n-3 . These v T. officinale were evaluated against four uropathogenic bacteria , Chile, in May 2014 and authenticated by agronomist Lorena Jorquera, Ph.D., from “Pontificia Universidad Católica de Valparaíso”, V region, Chile. The leaves were transported to the laboratory, washed 3 times with water and once with sterile distilled water (WDS), then air-dried on kraft paper, and placed in hot air oven at a temperature of 45.0°C for a period of 4 days until the weight became constant. The dried plant material was converted to a powdered form with the help of clean grinder.The leaves of T. officinale  were obtained from dried plant material (100 g) suspended first in 400 mL of n-hexane and then the residues were further extracted with ethyl acetate (EtOAc) separately; that is, an extraction was performed sequentially and serially. The extraction was carried out by using an orbital shaker (150 rpm) at 25°C for 48 h at room temperature; then the extract was filtered through Whatman N° 1 filter paper , concentrated under reduced pressure with a rotatory evaporator, and finally kept in the refrigerator (−4°C) until the analyses were made.The extracts of1H- and 13C-NMR, respectively, on a Bruker Avance 400 Digital NMR spectrometer operating at 400.1 MHz for 1H and 100.6 MHz for 13C, and comparisons with data reported in the literature were made.The n-hexane and EtOAc extracts were subjected to chromatography over a silica gel column (400 g) and eluted with mixtures of increasing polarity n-hexane and EtOAc. Fractions were combined based on TLC monitoring and purified by repeated CC on silica gel columns. Compounds were identified by chromatographic analysis and spectroscopic data, including n is the number of carbon atoms in the smaller n-alkane, N is the number of carbon atoms in the larger n-alkane, and Tr is the retention time. Components relative concentrations were obtained by peak area normalization.The n-hexane and ethyl acetate extract was diluted with acetone, and analysis by gas chromatography  was carried out according to the method detailed elsewhere . The opeThe gas chromatography analysis was performed on an Agilent Technologies 7890B gas chromatograph using a 30 m capillary column, Supelco Omega-Wax, 0.25 mm (Agilent Corp. CA). Hydrogen was used as a carrier gas at a flow rate of 1 mL/min. Detection was with flame-ionization detection and areal quantitation was made with an auxiliary automatic integrator. A temperature of 170°C was used at the beginning, which was maintained for two minutes, rising from 2,5°C/min to a final temperature of 240°C, which was maintained for 3 min. Injector and detector remained at 250°C and 270°C, respectively. Methyl esters were prepared according to our previously described procedure  by treat Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Proteus mirabilis. The identification of all isolates was confirmed through genomic DNA analysis by sequencing . Strains were cultured in Mueller-Hinton Broth (MHB) and Mueller-Hinton agar at 37°C.The uropathogenic bacteria were clinical isolates belonging to the Chemistry Department, Biological Tests Laboratory (Universidad Técnica Federico Santa María) collection. They comprisedμg/mL to 1600 μg/mL was tested. Later, 1.5 μL of microbial suspension (1 × 106 CFU/mL) of each strain was inoculated into sterile 96-well microplates with equal volume of MHB broth and plant extracts. Then, the 96-well microplates were incubated aerobically at 37°C for 24 h in a shaker at 120 rpm. MIC was determined according to the OD600 obtained in an Accu Reader M965 spectrophotometer. After the cultures were incubated at 37°C for 24 h, the minimum inhibitory concentration (MIC) was confirmed as the lowest concentration of the test extract that demonstrated no visible growth. The plates were incubated for 24 h at 37°C. Chloramphenicol and streptomycin were used as positive control tested at the same concentrations for bacterial strains and 1% ET was used as negative control with inoculum. Also, a negative control was used with 1% ET without inoculum to subtract the OD600 obtained. Each concentration of the extracts was tested in triplicate. Each value represents the mean ± SD of three experiments performed in triplicate, for which the mean and standard deviation for each value were calculated.The minimum inhibitory concentration (MIC) of the plant extract was determined using the broth serial dilution method with modifications, following Andrews 2001 . BrieflyCAbs is the absorbance of the control treatment and TAbs is the absorbance of samples treated with different extracts.Finally, the percentage of bacterial growth inhibition (GI) was calculated using the following formula: T. officinale (Compositae) leaves, mainly known as triterpenoids, and other unknown compounds to a lesser extent. Their presence and structures were elucidated by spectroscopic analyses. In addition, the fatty acids content was determined showing a higher content of palmitic and linolenic acids. Both extracts present antibacterial activity against uropathogenic clinical bacteria, being more active against Gram-positive bacteria . These results suggest that both extracts have potential as antibacterial uropathogenic disease agents.Different secondary metabolites of the n-hexane and ethyl acetate extracts were isolated from


R, 1S)-1[β--(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.From a series of (1 Store-operated Ca2+ entry (SOCE) is the typical mechanism to generate Ca2+ signals that combines the intracellular and extra-cellular processes and represents one of the most common and ubiquitous Ca2+ influx routes in non-excitable cells [CRACI) 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  in dry acetone was submitted to diasteromeric enrichment by addition of 0.02 equivalent of (±) racemic 4b. After work-up the pure (−)-(1S)-4b/(−)-(1R)-CSA was obtained in 48% yield (>99% de) as white powder with [αD] = −6.0  (S)-CSA for resolution of the (±) racemic 4b, and after diastereomeric enrichment, the other diastereomer  was obtained in 51% yield with [αD] = +6.0 . 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 For this structure-activity relationship (SAR) study, we were also interested to evaluate the potential impact of chirality’s of the compounds antities  for reso mixture . To find0, MeOH) . This prS)-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 S)-4b and (+)-(1R)-4b were obtained respectively in isolated yields of 38 and 41% with [αD] = −10.0  for (−)-(1S)-4b (>99% ee) and [αD] = +10.0  for (+)-(1R)-4b (>99% ee).Next, access to the free enantiomer -(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, 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 . 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 (−)-(1S)-4b/(−)-(1R)-CSA, (+)-(1R)-4b/(+)-(1S)-CSA and the two enantiomers (−)-(1S)-4b and (+)-(1R)-4b. These data were summarized in For the (±) racemic mixture S)-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 R, 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 -(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.With the two pure enantiomers (−)- Ca2+ 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 Ca2+ 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 Ca2+ in the extracellular medium. Results are respectively summarized in para-phenyl group of the phenethyl-1H-pyrazolium skeleton and also on the phenylalkoxy chain of Cβ compared to the SKF-96365 reference.The synthesized hydrochloride compounds 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- side chain appeared to be important in 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  on the pyrazole platform in moderate to good yields. For the (±) hydroxyl intermediate 4, we have developed a successful approach for separation of enantiomers using the method of “half-concentration” with commercial (+)-(1S)- and (−)-(1R)-10-camphorsulfonic acid (CSA) followed by neutralization of diastereomers with MeONa in dry MeOH solution. With the pure enantiomers (−)-(1S)-4b and (+)-(1R)-4b, initial attempts to obtain the crystallized (−)-(1S)-7d and (+)-(1R)-7d after treatment of intermediate 6d with a solution of 1 M HCl . Effects of compounds 7a–d on endoplasmic reticulum (ER) Ca2+ 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.Here, we have reported the racemic synthesis of pyrazole SKF-96365 analogues in four steps without substituent  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 . All reagents and solvents were purchased from Acros Fisher , Sigma-Aldrich Chimie , and Fluka Chimie  and were used without further purification. 1H NMR spectra were recorded on Bruker AC 300 P (300 MHz) spectrometer and 13C 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 , 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 . Melting points were determined on a Kofler melting point apparatus and were uncorrected. Optical rotations [αD] 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.General Information. Preparative chromatography was realized on a Combi Flash 1-Phenyl-2-(1H-pyrazol-1-yl)ethan-1-one (3a). To a solution of 2-bromoacetophenone 1a  in 20.8 mL of acetonitrile, pyrazole 2a  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, K2CO3  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 (Combi Flash fR 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 (0–50%) for elution. Pooling for 60 min and elimination of the solvent in vacuo gave 3.74 g (53% yield) of the pure desired compound 3a as yellowish powder. Mp = 96–97 °C. 1H NMR (DMSO-d6) δ = 5.84 , 6.31 , 7.48 , 7.52–7.63 , 7.66–7.77 , 7.97–8.09 . 13C NMR (DMSO-d6) δ = 57.7 (C-2), 105.6 (C-4′), 128.1 , 129.0 , 131.6 (C-5′), 134.0 (C-4″), 134.6 (C-1″), 139.0 (C-3′), 193.7 (C=O). ES+ HRMS, m/z = 209.0689 found .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  in 20.8 mL of acetonitrile, pyrazole 2a  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, K2CO3  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 fR 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. Mp = 102–103 °C. 1H NMR (DMSO-d6) δ = 3.86 , 5.77 , 6.30 , 7.09 , 7.47 , 7.72 , 8.01 . 13C NMR (DMSO-d6) δ = 55.7 (OCH3), 57.3 (C-2), 105.5 (C-4′), 114.2 , 127.4 (C-5′), 130.4 , 131.6 (C-1″), 138.8 (C-3′), 163.6 (C-4″), 191.9 (C=O). ES+ HRMS, m/z = 239.0798 found .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  in 2.25 mL of acetonitrile, 3-trifluoromethylpyrazole 2b  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, K2CO3  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 0.62 g (68% yield) of the desired compound 3c as white powder. Mp = 146–147 °C. 1H NMR (DMSO-d6) δ = 3.87 , 5.94 , 6.78 , 7.12 , 7.95 , 8.02 . 13C NMR (DMSO-d6) δ = 55.8 (OCH3), 58.1 (C-2), 104.4 (C-4′), 104.4 (C-4′), 114.2 , 114.3 , 127.1 (C-1″), 130.4 , 130.5 , 134.2 (C-5′), 134.3 (C-5′), 163.8 (C-4″), 191.0 (C-1). ES+ HRMS, m/z = 307.0671 found ; 285.0862 found .2--1-(4-methoxyphenyl)ethan-1-one (3d). To a solution of 2-bromo-1-(4-methoxyphenyl)ethan-1-one 1b  in 2.25 mL of acetonitrile, 3,5-bis-trifluoromethylpyrazole 2c  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, K2CO3  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 °C for 2 h. The desired compound 3d was obtained in 69% yield as white powder. Mp = 114–115 °C. 1H NMR (DMSO-d6) δ = 3.88 , 6.19 , 7.12 , 7.69 , 8.05 . 13C NMR (DMSO-d6) δ = 55.8 (OCH3), 58.5 (C-2), 107.5 (C-4′), 114.3 , 126.3 (C-1″), 130.8 , 164.2 (C-4″), 189.9 (C=O). ES+ HRMS, m/z = 375.0544 found ; 353.0734 found .H-pyrazol-1-yl)ethan-1-one 3 (5 mmol) in an appropriate volume of anhydrous methanol (9–40 mL), sodium borohydride NaBH4  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.To a solution of 1-phenyl-2-(11-Phenyl-2-(1H-pyrazol-1-yl)ethan-1-ol (4a). According to the standard procedure, the compound 4a was prepared from 1-phenyl-2-(1H-pyrazol-1-yl)ethan-1-one 3a  in 9.3 mL of anhydrous methanol after a reaction time of 5 h using CH2Cl2/MeOH 9:1 v/v as eluent for thin layer chromatography. Washing work-up was realized with 3 × 45 mL of deionized water and gave 0.94 g (82% yield) of the desired compound 4a as white powder. Mp = 132–134 °C. 1H NMR (DMSO-d6) δ = 4.14–4.30 , 4.83–5.01 , 5.65 , 6.18 , 7.19–7.39 , 7.43 , 7.58 . 13C NMR (DMSO-d6) δ = 58.7 (C-2), 71.8 (C-1), 104.7 (C-4′), 126.1 , 127.4 (C-4″), 128.2 , 130.7 (C-5′), 138.5 (C-1″), 142.8 (C-3′). ES+ HRMS, m/z = 211.0848 found .1-(4-Methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol (4b). According to the standard procedure, the compound 4b was prepared from 1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-one 3b  in 14.4 mL of anhydrous methanol after a reaction time of 7 h using CH2Cl2/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 1.091 g (86% yield) of the desired compound 4b as white crystals with de = 50% , retention times Rt(1) = 65.92 and Rt(2) = 71.38 min. [αD] = 0.0 . Mp = 116–118 °C. 1H NMR (DMSO-d6) δ = 3.73 , 4.10–4.27 , 4.87 , 5.54 , 6.17 , 6.88 , 7.15–7.29 , 7.42 , 7.57 . 13C NMR (DMSO-d6) δ = 55.0 (OCH3), 58.7 (C-2), 71.4 (C-1), 104.6 (C-4′), 113.5 , 127.2 , 130.5 (C-1″), 134.8 (C-5′), 138.4 (C-3′), 158.5 (C-4″). ES+ HRMS, m/z = 241.0954 found .1-(4-Methoxyphenyl)-2-(3-trifluoromethyl-1H-pyrazol-1-yl)ethan-1-ol (4c). According to the standard procedure, the compound 4c was prepared from 1-(4-methoxyphenyl)-2-(3-trifluoromethyl-1H-pyrazol-1-yl)ethan-1-one 3c  in 39 mL of anhydrous methanol after a reaction time of 7 h using hexane/AcOEt 1:1 v/v as eluent for thin layer chromatography. Washing work-up was realized with 3 × 47 mL of deionized water and gave 1.431 g (94% yield) of the desired compound 4c as white powder. Mp = 110–112 °C. 1H NMR (DMSO-d6) δ = 3.74 , 4.27 , 4.90 , 5.63 , 6.66 , 6.90 , 7.28 , 7.85 . 13C NMR (DMSO-d6) δ = 55.0 (OCH3), 55.1 (OCH3), 59.3 (C-2), 71.0 (C-1), 103.7, (C-4′), 103.7 (C-4′), 113.6 , 113.6 , 127.2 , 127.3 , 133.1 (C-5′), 133.2 (C-5′), 134.2 (C-1″), 140.13 , 158.7 (C-4″). ES+ HRMS, m/z = 309.0824 found ; 269.0902 found .2--1-(4-methoxyphenyl)ethan-1-ol (4d). To a solution of 2--1-(4-methoxyphenyl)ethan-1-one 3d  in 31 mL of anhydrous methanol, sodium borohydride NaBH4  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. 1H NMR (DMSO-d6) δ = 3.74 , 4.29 , 4.40 , 5.01 , 5.73 , 6.92 , 7.28 , 7.50–7.54 . 13C NMR (DMSO-d6) δ = 55.1 (OCH3), 58.8 (C-2), 70.6 (C-1), 106.8 (C-4′), 113.7 , 127.2 , 133.5 (C-1″), 158.9 (C-4″). ES+ HRMS, m/z = 377.0700 found ; 337.0763 found .H-pyrazol-1-yl)ethan-1-ol 4  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  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 Et2O, 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--phenethyl]-1H-pyrazole 6 was dissolved in appropriate volume of diethyl ether Et2O 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--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.Pellets of potassium hydroxide KOH  were added to a solution of 1-phenyl-2- 1-[β-((4-Methoxybenzyl)oxy)-phenethyl]-1H-pyrazolium hydrochloride (7a). Using the standard procedure from 1-phenyl-2-(1H-pyrazol-1-yl)ethan-1-ol 4a  and 4-methoxybenzyl chloride 5a , the 1-[β-((4-methoxybenzyl)oxy)-phenethyl]-1H-pyrazole 6a  was dissolved in 4.6 mL of dry diethyl ether pa under a stream of argon. The addition of 0.65 mL of 1M HCl  produced a viscous gum on the circumference of the round flask after mixing at room temperature for 5 days. Then, this reaction mixture was stored in a refrigerator at 4 °C for 2 weeks and produced a compact gum. This gum was then manually and carefully triturated in 4 mL of dry hexane and progressively produced a powder. The resulting suspension was stirred (200 rpm) at room temperature for 5 days, and produced a completely divided powder. The desired salt 7a was collected by filtration on a Büchner funnel (porosity N°4) and washed with 0.5 mL of dry diethyl ether pa. After drying at 25 °C for 4 h, the 1-[β-((4-methoxybenzyl)oxy)-phenethyl]-1H-pyrazolium hydrochloride 7a  was obtained as white powder. Mp = 62–65 °C. 1H NMR (DMSO-d6) δ = 3.72 , 4.09 , 4.19–4.32 , 4.40 , 4.78 , 6.22 , 6.82 , 6.99 , 7.27–7.44 , 7.46 , 7.62 . 13C NMR (DMSO-d6) δ = 55.1 (OCH3), 57.0 (C-1′), 69.7 (C-1″), 79.4 (C-2′), 104.9 (C-4), 113.6 , 126.8 , 128.1 (C-4a), 128.6 , 128.8 , 129.9 (C-1b), 130.9 (C-5), 138.6 (C-3), 139.0 (C-1a), 158.6 (C-4b). ES+ HRMS, m/z = 331.1418 found . 1-[β-((3-(4-Methoxyphenyl)propyl)oxy)-phenethyl]-1H-pyrazolium hydrochloride (7b). Using the standard procedure from 1-phenyl-2-(1H-pyrazol-1-yl)ethan-1-ol 4a  and 1-(3-bromopropyl)-4-methoxybenzene 5c , the 1-[β-((3-phenylpropyl)oxy)-phenethyl]-1H-pyrazole 6b  was dissolved in 3 mL of dry diethyl ether pa under a stream of argon. The addition of 0.45 mL of 1M HCl  produced a pale yellow viscous oil that crystallized progressively on the circumference of the round flask, and mixing  was pursued until complete crystallization of the oily gum. The desired salt 7b was collected by filtration on a Büchner funnel (porosity N°4) and washed with 3 × 0.5 mL of dry diethyl ether pa. After drying at 25 °C for 4 h, the 1-[β-((3-(4-methoxyphenyl)propyl)oxy)-phenethyl]-1H-pyrazolium hydrochloride 7b  was obtained as white powder. Mp = 76–80 °C. 1H NMR (DMSO-d6) δ = 1.62 , 2.36 , 3.05 , 3.22 , 3.69 , 4.23 , 4.37 , 4.69 , 6.23 , 6.77 , 6.94 , 7.27–7.44 , 7.46–7.50 , 7.68 . 13C NMR (DMSO-d6) δ = 30.4 (C-3″), 31.1 (C-2″), 54.9 (OCH3), 57.2 (C-1′), 67.4 (C-1″), 80.1 (C-2′), 104.9 (C-4), 113.6 , 126.7 , 128.0 (C-4a), 128.5 , 129.2 , 131.0 (C-5), 133.4 (C-1b), 138.4 (C-3), 139.3 (C-1a), 157.3 (C-4b). ES+ HRMS, m/z = 359.1733 found ; 337.1906 found . 1-[β-((4-Methoxybenzyl)oxy)-(4-methoxyphenethyl]-1H-pyrazolium hydrochloride (7c). Using the standard procedure from 1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b  and 4-methoxybenzyl chloride 5a , the 1--1H-pyrazolium hydrochloride (7d). Using the standard procedure from 1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b  and 1-(3-bromopropyl)-4-methoxybenzene 5c , the 1-[β-((4-methoxyphenyl)-(3-(4-methoxyphenyl)propoxyethyl)]-1H-pyrazole 6d  was dissolved in 2.84 mL of dry diethyl ether pa under a stream of argon. The addition of 0.42 mL of 1M HCl  produced a pale pink viscous oil that crystallized (12 h) on the circumference of the round flask, and mixing  was pursued until complete crystallization of the viscous gum. The desired salt 7d was collected by filtration on a Büchner funnel (porosity N°4) and washed with 3 × 0.5 mL of dry diethyl ether pa. After drying at 25 °C for 4 h, the 1-[β-((4-methoxyphenyl)-(3-(4-methoxyphenyl)propoxyethyl)]-1H-pyrazolium hydrochloride 7d  was obtained as white powder. Mp = 92–94 °C. 1H NMR (DMSO-d6) δ = 1.47–1.70 , 2.36 , 3.03 , 3.19 , 3.69 , 3.75 , 4.18 , 4.35 , 4.62 , 6.22 , 6.77 , 6.86–7.02 , 7.26 , 7.47 , 7.67 . 13C NMR (DMSO-d6) δ = 30.4 (C-3″), 31.1 (C-2″), 54.9 (OCH3), 55.1 (OCH3), 57.2 (C-1′), 67.0 (C-1″), 79.7 (C-2′), 104.9 (C-4), 113.6 , 113.9 , 128.0 , 129.2 , 130.8 (C-3), 131.1 (C-1a), 133.4 (C-1b), 138.5 (C-5), 157.3 (C-4b), 159.0 (C-4a). ES+ HRMS, m/z = 389.1838 found . 1-[β-((4-Methoxyphenyl)-(3-phenylpropoxyethyl)]-1H-pyrazolium hydrochloride (7e). Using the standard procedure from 1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b  and 1-bromo-3-phenylpropane 5d , the 1-[β-((4-methoxyphenyl)-(3-phenylpropoxyethyl)]-1H-pyrazole 6e  was dissolved in 2.5 mL of dry diethyl ether pa under a stream of argon. The addition of 0.37 mL of 1 M HCl  produced a translucent colourless viscous oil that crystallized progressively (24 h) on the circumference of the round flask, and mixing  was pursued until complete crystallization in divided powder. The desired salt 7e was collected by filtration on a Büchner funnel (porosity N°4) and washed with 3 × 0.5 mL of dry diethyl ether pa. After drying at 25 °C for 4 h, the 1-[β-((4-methoxyphenyl)-(3-phenylpropoxyethyl)]-1H-pyrazolium hydrochloride 7d  was obtained as white powder. Mp = 87–89 °C. 1H NMR (DMSO-d6) δ = 1.54–1.76 , 2.37–2.46 , 3.04 , 3.21 , 3.75 , 4.18 , 4.35 , 4.63 , 6.21 , 6.92 , 6.99–7.06 , 7.08–7.17 , 7.17–7.31 , 7.46 , 7.66 . 13C NMR (DMSO-d6) δ = 30.9 (C-2″), 31.4 (C-3″), 55.1 (OCH3), 57.2 (C-1′), 67.1 (C-1″), 79.7 (C-2′), 104.9 (C-4), 113.9 , 125.7 (C-4b), 128.1 , 128.2 , 128.4 , 130.8 (C-5), 131.2 (C-1a), 138.6 (C-3), 141.7 (C-1b), 159.1 (C-4a). ES+ HRMS, m/z = 359.1736 found .(−)-(1S) 1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol (4b): To a stirred solution of (±) 1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b  in 5 mL of dry acetone, a solution of (−)-(1R)-10-camphorsulfonic acid  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 (−)-(1R)-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 0.363 g of the desired the salt (−)-(1S)-4b/(−)-(1R)-CSA (>85% de). This salt was recrystallized in dry acetone and afforded 0.271 g (52% isolated yield) of salt (−)-(1S)-4b/(−)-(1R)-CSA (>97% de). This salt was dissolved in 0.6 mL of dry acetone under magnetic stirring (300 rpm), and to this suspension, 0.02 equiv. of racemic (±) 1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b was added for diastereomeric enrichment. This suspension was stirred for 18 h at room temperature. The enriched salt was successively filtered on a Büchner funnel, washed with 0.5 mL of dry acetone, dried in vacuum, and recrystallized in dry acetone that produced 0.251 g (48% yield) of (−)-(1S)-4b/(−)-(1R)-CSA (>99% de) as white powder; [αD] = −6,0 .S)-4b/(−)-(1R)-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 produced 0.096 g (38% yield) of the desired (−)-(1S) 1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b (99% ee); [αD] = −10,0  and Mp = 113–114 °C.Starting from 200 mg of the pure salt (−)-(1Recovery of the (+)-(1R) 1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol4b: The filtrate of the first fraction was submitted to concentration in a rotary evaporator under reduced pressure and afforded 0.345 g of a crude residue. To this solid residue, 14.5 mL of dry toluene was added and the resulting suspension was submitted to magnetic stirring for 4 h. This suspension was filtered through a Büchner funnel (porosity N°4) and the filtrate was concentrated in a rotary evaporator under reduced pressure and produced 0.190 g (0.87 mmol) of a white powder of (+)-(1R)-4b (65% ee). This white solid was dissolved in 3.8 mL of dry acetone under magnetic stirring and to the resulting solution, a solution of (+)-(1S)-CSA  in 1.52 mL of dry acetone was added dropwise. During the addition of the (+)-(1S)-CSA solution, a fine suspension appeared in the mixture, which was stirred at room temperature for 12 h. The new diastereomer salt was collected by filtration then washed with 4 × 0.25 mL of dry acetone, dried in vacuum, which gave 0.249 g (63% yield) of the (+)-(1R)-4b/(+)-(1S)-CSA salt (96% de) as white powder. From this salt, diastereomeric enrichment was conducted with 0.04 equiv. of racemic (±) 1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b. The enriched suspension was stirred for 18 h at room temperature and the resulting enriched salt was successively submitted to filtration on a Büchner funnel, washing with 0.5 mL of dry acetone, drying in vacuum, and finally recrystallisation in dry acetone which gave 0.202 mg (51% yield) of (+)-(1R)-4b/(+)-(1S)-CSA (>99% de) as white needles; [αD] = +6,0 .R)-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 (+)-(1R) 1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b (99% ee); [αD] = +10,0  and Mp = 110–111 °C.Starting from 200 mg of the pure salt (+)-(1(+)-(1R) 1-(4-Methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol (4b): To a stirred solution of (±) 1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b  in 5 mL of dry acetone, a solution of (+)-(1S)-10-camphorsulfonic acid  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 0.280 g (54% isolated yield) of salt (+)-(1R)-4b/(+)-(1S)-CSA (>96% de). This salt was dissolved in 0.6 mL of dry acetone under magnetic stirring (300 rpm) and to this suspension was added 0.02 equiv. of racemic (±) 1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b for diastereomeric enrichment. This suspension was stirred for 18 h at room temperature. The enriched salt was successively filtered on a Büchner funnel, washed with 0.5 mL of dry acetone, dried in vacuum, and recrystallized in dry acetone that gave 0.229 g (44% yield) of (+)-(1R)-4b/(+)-(1S)-CSA (>99% de) as white powder; [αD] = +6,0 .R)-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 0.089 g (35% yield) of the desired (+)-(1R) 1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b (99% ee); [αD] = +10,0  and Mp = 110–111 °C.Starting from 200 mg of the pure salt (+)-(1Recovery of the (−)-(1S) 1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol4b: The filtrate of the first fraction was submitted to concentration in a rotary evaporator under reduced pressure and afforded 0.366 g of a crude residue. To this solid residue, 14.5 mL of dry toluene was added and the resulting suspension was submitted to magnetic stirring for 4 h. This suspension was filtered through a Büchner funnel (porosity N°4) and the filtrate was concentrated in a rotary evaporator under reduced pressure and produced 0.207 g (0.95 mmol) of a white powder of (−)-(1S)-4b (74% ee). This white solid was dissolved in 4.2 mL of dry acetone under magnetic stirring and to the resulting solution, a solution of (−)-(1R)-CSA  in 1.66 mL of dry acetone was added dropwise. During the addition of the (−)-(1R)-CSA solution, a fine suspension appeared in the mixture, which was stirred at room temperature for 12 h. The new diastereomer salt was collected by filtration then washed with 4 × 0.25 mL of dry acetone and dried in vacuum, which gave 0.285 g (66% yield) of the (−)-(1S)-4b/(−)-(1R)-CSA salt (98% de) as white powder. From this salt, diastereomeric enrichment was conducted with 0.01 equiv. of racemic (±) 1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b. The enriched suspension was stirred for 18 h at room temperature and the resulting enriched salt was successively submitted to filtration on a Büchner funnel, washing with 0.5 mL of dry acetone, drying in vacuum, and finally recrystallisation in dry acetone which gave 0.258 mg (60% yield) of (−)-(1S)-4b/(−)-(1R)-CSA (>99% de) as white needles; [αD] = −6,0 .S)-4b/(−)-(1R)-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 0.090 g (43% yield) of the desired (−)-(1S) 1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b (>99% ee); [αD] = −10,0  and Mp = 113-114 °C.Starting from 200 mg of the pure salt (−)-(1(−)-(1S)-4b/(−)-(1R)-CSA salt: Yield = 48%. White powder, Mp = 168–170 °C. [αD] = −6.0  and >99% de . 1H NMR (DMSO-d6) δ = 0.75 , 1.04 , 1.19–1.40 , 1.81 , 1.80–1.92 , 1.95 , 2.16–2.34 , 2.46 , 2.55–2.76 , 2.93 , 3.73 , 4.10–4.32 , 4.87 , 6.20 , 6.87 , 7.22 , 7.47 , 7.61 . 13C NMR (DMSO-d6) δ = 19.5 (C-7″CH3), 20.0 (C-7″CH3), 24.2 (C-2″), 26.4 (C-5″), 42.1 (C-3″), 42.2 (C-4″), 46.8 (CH2SO3H), 47.1 (C-7″), 55.0 (OCH3), 58.1 (C-6″), 58.6 (C-2), 71.3 (C-1), 104.8 (C-4′), 113.5 , 127.2 , 130.9 (C-5′), 134.7 (C-1a), 138.1 (C-3′), 158.5 (C-4a), 216.0 (C=O). ES+ HRMS, m/z = 241.0946 found .(+)-(1R)-4b/(+)-(1S)-CSA salt: Yield = 51%. White powder, Mp = 169-171 °C. [αD] = +6.0  and >99% de . 1H NMR (DMSO-d6) δ = 0.75 , 1.04 , 1.17–1.42 , 1.81 , 1.78–1.93 , 1.95 , 2.25 , 2.45 , 2.54-2.76 , 2.93 , 3.73 , 4.08–4.32 , 4.87 , 6.20 , 6.87 , 7.22 , 7.43-7.41 , 7.60 . 13C NMR (DMSO-d6) δ = 19.5 (C-7″CH3), 20.0 (C-7″CH3), 24.2 (C-2″), 26.4 (C-5″), 42.1 (C-3″), 42.2 (C-4″), 46.8 (CH2SO3H), 47.1 (C-7″), 55.0 (OCH3), 58.1 (C-6″), 58.6 (C-2), 71.3 (C-1), 104.8 (C-4′), 113.5 , 127.2 , 130.9 (C-5′), 134.7 (C-1a), 138.1 (C-3′), 158.5 (C-4a), 216.0 (C=O). ES+ HRMS, m/z = 241.0946 found .R)-4b/(+)-(1S)-CSA salt: ; M = 450.54. APEXII, Bruker-AXS diffractometer, Mo-Kα radiation (λ = 0.71073 Å), T = 150(2) K; orthorhombic P 21 21 21 (I.T.#19), a = 7.2781(5), b = 9.5344(5), c = 32.8134(19) Å, V = 2277.0(2) Å3. Z = 4, d = 1.314 g·cm−3, μ = 0.182 mm−1. The structure was solved by direct methods using the SIR97 program  = −10.0  and >99% de . 1H NMR (DMSO-d6) δ = 3.73 , 4.06–4.30 , 4.87 , 5.50 , 6.16 , 6.87 , 7.21 , 7.41 , 7.56 . 13C NMR (DMSO-d6) δ = 55.0 (OCH3), 58.7 (C-2), 71.4 (C-1), 104.6 (C-4′), 113.5 , 127.2 , 130.5 C-5′), 134.8 (C-1a), 138.4 (C-3′), 158.5 (C-4a). ES+ HRMS, m/z = 241.0950 found .(+)-(1R) 1-(4-Methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol (4b): Yield = 41%. White powder, Mp = 110–111 °C. [αD] = +10.0  and >99% de . 1H NMR (DMSO-d6) δ = 3.73 , 4.09–4.29 , 4.87 , 5.50 , 6.16 , 6.87 , 7.22 , 7.41 , 7.56 . 13C NMR (DMSO-d6) δ = 55.0 (OCH3), 58.7 (C-2), 71.4 (C-1), 104.6 (C-4′), 113.5 , 127.2 , 130.5 C-5′), 134.8 (C-1a), 138.4 (C-3′), 158.5 (C-4a). ES+ HRMS, m/z = 241.0950 found .® Cell-Tak™ was purchased from Beckton Dickinson  (Ref: 354241). Fura-2 QBT™ Calcium Kit was purchased from Molecular Devices  (Ref: R8198).Dimethyl Sulfoxyde (DMSO-Ref D2438) and Thapsigargin (Tg-Ref: T9033) was purchased from Sigma-Aldrich. CorningThe B-cell leukemia cell line PLP , which p2+ experiments, cells were seeded overnight into four wells plate (NuncTM multidisches 4 wells flat-bottom-Ref: 176740) at 7 × 105 cells/mL in 1 mL culture medium. Cell suspension was then transfered into 1.5 mL Microtube, pelleted, and loaded with Fura-2 acetoxymethyl ester (Fura-2 QBT™) fluorochrome according to the manufacturer’s protocol. Cell suspension was dispensed onto Corning® Cell-Tak™ pre-coated 96 wells black clear bottom plate  and incubated 1 h at 37 °C, 5% CO2. The Fura-2 QBT™ was aspirated and replaced by an equal volume of free Ca2+ Hepes-buffered solution containing (in mM): 135 NaCl, 5 KCl, 1 MgCl2, 1 EGTA, 10 Hepes, 10 glucose, pH adjusted at 7.45 with NaOH. The tested compounds or DMSO control solvent solutions were added in wells (4 µL) and incubated for 5 min at 37 °C until evaluating calcium level on FlexStation 3™ Instrument.For Ca2+ concentration in a 96-well format with the dual-wavelength fluorescent calcium-sensitive dye Fura-2AM. Dual excitation wavelength capability permits ratiometric measurements of Fura-2AM peak emissions (510 nm) after excitations at 340  and 380 nm (unbound to Ca2+). Modifications in the 340/380 ratio reflect changes in intracellular-free Ca2+ concentrations. The FlexStation 3™ temperature was setting at 37 °C during data acquisition. Thapsigargin (2 µM) solution and Hepes-buffered solution with Ca2+ containing (in mM): 135 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 Hepes, 10 glucose, pH adjusted at 7.45 with NaOH were added from a 96-well reservoir plate during Calcium Mobilization Assay running at 100 and 750 s, respectively. Experimental setup parameters were optimized  to minimize disturbance of the cells while ensuring rapid mixing. The data were stored for later analysis by using SoftmaxPro (Molecular Devices), Excel and Graph Pad Prism software .Intracellular calcium levels were monitored by using the FlexStation 3™ , a fluorescence plate reader measuring time–resolved intracellular Ca2+ stores were depleted with 2 μM Thapsigargin an inhibitor of ER SERCA pumps under Ca2+-free conditions to determine the magnitude of intracellular Ca2+ release. Next, cells were returned to Ca2+-containing Hepes-buffered solution to measure SOCE. The magnitude of SOCE was estimated as the maximal values of normalized F340/F340 ratio following Ca2+ re-addition.Intracellular Ca2+ concentration variations are estimated using the ratio of peak RFU at 340 and 380 nm (F340/F380) and for each measurement F340/F380 ratio values are normalized to the initial basal ratio before TG addition. Results are expressed in terms of percentage of response or percentage of inhibition compared to the maximum response on control condition. Inhibition is estimated as follow: % Inhibition = (100 − % response). The EC50 values were estimated from the dose/response curves (log(inhibitor) vs. amplitude of the Ca2+ response) using a regression model (Y = Min + (Max − Min)/(1 + 10(LogIC50−X)×HillSlope) with variable slope (Prism5 GraphPad software). Min and Max are the minimal and maximal inhibition obtained. EC50 is defined as the concentration of drug required to obtain 50% of inhibitory effect.Results are expressed as mean ± SEM for each group. The number of experiments for each group is represented as n. Data analysis for functional assays was performed using Softmax Pro and Graph Pad Prism. (Graph Pad): Ca


Ki value of 16.3 μM, and it exhibited mechanism-based inhibition of CYP2C19-catalyzed [S]-mephenytoin 4′-hydroxylation , CYP2C8-catalyzed amodiaquine N-deethylation , and CYP3A4-catalyzed midazolam 1′-hydroxylation  in human liver microsomes. Fargesin negligibly inhibited CYP1A2-catalyzed phenacetin O-deethylation, CYP2A6-catalyzed coumarin 7-hydroxylation, CYP2B6-catalyzed bupropion hydroxylation, and CYP2D6-catalyzed bufuralol 1′-hydroxylation at 100 μM in human liver microsomes. Dimethyllirioresinol weakly inhibited CYP2C19 and CYP2C8 with IC50 values of 55.1 and 85.0 μM, respectively, without inhibition of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, and CYP3A4 activities at 100 μM. Epimagnolin A, eudesmin, and magnolin showed no the reversible and time-dependent inhibition of eight major CYP activities at 100 μM in human liver microsomes. These in vitro results suggest that it is necessary to investigate the potentials of in vivo fargesin-drug interaction with CYP2C8, CYP2C9, CYP2C19, and CYP3A4 substrates.Magnolin, epimagnolin A, dimethyllirioresinol, eudesmin, and fargesin are pharmacologically active tetrahydrofurofuranoid lignans found in Flos Magnoliae. The inhibitory potentials of dimethyllirioresinol, epimagnolin A, eudesmin, fargesin, and magnolin on eight major human cytochrome P450 (CYP) enzyme activities in human liver microsomes were evaluated using liquid chromatography-tandem mass spectrometry to determine the inhibition mechanisms and inhibition potency. Fargesin inhibited CYP2C9-catalyzed diclofenac 4′-hydroxylation with a  Aristolochia elegans rhizomes, and Zanthoxylum armatum DC. -mephenytoin 4′-hydroxylation and CYP2C8-catalyzed amodiaquine N-deethylation with IC50 values of 55.1 and 85.0 μM, respectively, without inhibition of CYP1A2-catalyzed phenacetin O-deethylation, CYP2A6-catalyzed coumarin 7-hydroxylation, CYP2B6-catalyzed bupropion hydroxylation, CYP2C9-catalyzed diclofenac 4′-hydroxylation, CYP2D6-catalyzed bufuralol 1′-hydroxylation, and CYP3A4-catalyzed midazolam 1′-hydroxylation activities at 100 μM in human liver microsomes   did not cause the IC enzymes , indicat CYP3A4) , Table 1Ki value) and the inhibition mode define the interaction of an inhibitor with a particular enzyme. Fargesin exhibited competitive inhibition of CYP2C9-catalyzed diclofenac 4-hydroxylation with a Ki value of 16.3 μM  values of fargesin were 10.7 μM and 0.082 min−1 for CYP2C8-catalyzed amodiaquine N-deethylation, 3.7 μM and 0.102 min−1 for CYP2C19-catalyzed [S]-mephenytoin 4′-hydroxylation, and 23.0 μM and 0.050 min−1 for CYP3A4-catalyzed midazolam 1′-hydroxylation, respectively, in human liver microsomes  and the potent time-dependent inhibition of CYP2C19, CYP2C8, and CYP3A4 activities  in human liver microsomes. Aschantin, a chemical derivative of fargesin, with a methylenedioxyphenyl moiety also exhibited the reversible and time-dependent inhibition of CYP2C8, CYP2C9, CYP2C19, and CYP3A4 activities in human liver microsomes -mephenytoin 4′-hydroxylation, and CYP3A4-catalyzed midazolam 1′-hydroxylation in pooled human liver microsomes (kinact/Ki ratio) of fargesin against CYP2C8 (7.66 min−1 nM−1) was comparable to that of aschantin (kinact/Ki = 5.49 min−1 nM−1) -mephenytoin, and pooled human liver microsomes  were purchased from Corning Life Sciences . Amodiaquine hydrochloride, N-desethylamodiaquine dihydrochloride, acetaminophen, coumarin, 7-hydroxycoumarin, diclofenac sodium, midazolam, phenacetin, and NADPH were obtained from Sigma-Aldrich . 13C2, 15N-acetaminophen was obtained from Toronto Research Chemicals . Methanol, acetonitrile, and water (liquid chromatography-mass spectrometry [LC-MS] grade) were purchased from Fischer Scientific . All other chemicals were of the highest quality available.Epimagnolin A, eudesmin, fargesin, and magnolin were obtained from PhytoLab GmbH & Co. . Dimethyllirioresinol was a gift from Natural Medicine Research Center in Korea Research Institute of Biology and Biotechnology . Bufuralol hydrochloride, 1′-hydroxybufuralol maleate, d50 values) of dimethyllirioresinol, epimagnolin A, eudesmin, fargesin, and magnolin toward CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 activities in pooled human liver microsomes were evaluated following our previous method using CYP cocktail substrates and liquid chromatography-tandem mass spectrometry (LC-MS/MS) -mephenytoin for CYP2C19, or 2 μM midazolam for CYP3A4), 10 mM MgCl2, 1 mM NADPH, and 50 mM potassium phosphate buffer (pH 7.4) in 90 μL reaction mixtures. The incubation was proceeded for 10 min and stopped by adding 100 μL of ice-cold d9-1′-hydroxybufuralol in methanol. The mixtures were centrifuged at 13,000× g for 4 min at 4 °C, and 50 μL of each supernatant was diluted with 50 μL of water. Aliquots (5 μL) were analyzed by LC-MS/MS, as described above.The mechanism-based inhibition potency of fargesin against human liver microsomal CYP2C8, CYP2C19, and CYP3A4 activities was evaluated. Human liver microsomes (1 mg/mL) were pre-incubated with various concentrations of fargesin and NADPH in 50 mM potassium phosphate buffer (pH 7.4). Aliquots (10 μL) of the pre-incubation mixtures were collected at 5, 10, 15, and 20 min after the pre-incubation and transferred to new tubes containing CYP substrates . Ki, kinact, and the inhibition mode were determined using Enzyme Kinetics ver. 1.1 .The ICKi value of 16.3 μM and exhibited the mechanism-based inhibition of CYP2C19-catalyzed [S]-mephenytoin 4′-hydroxylation, CYP2C8-catalyzed amodiaquine N-deethylation, and CYP3A4-catalyzed midazolam 1′-hydroxylation with Ki values of 3.7, 10.7, and 23.0 μM, respectively, in human liver microsomes. Fargesin negligibly inhibited CYP1A2, CYP2A6, CYP2B6, and CYP2D6 activities at 100 μM. Dimethyllirioresinol weakly inhibited CYP2C19 and CYP2C8 with IC50 values of 55.1 and 85.0 μM, respectively, without inhibition of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, and CYP3A4 activities at 100 μM in human liver microsomes. Epimagnolin A, eudesmin, and magnolin showed no reversible or time-dependent inhibition of 8 major CYP activities at 100 μM in human liver microsomes. These in vitro results suggest that it is necessary to investigate fargesin-induced in vivo drug interaction studies via the inhibition of CYP2C8, CYP2C9, CYP2C19, and CYP3A4 activities.Fargesin competitively inhibited CYP2C9-catalyzed diclofenac 4′-hydroxylation with


For exon 16 in the second to last row, 14-15-16-19-5-6-10-13-14 (19-5) has been corrected to 16-19-5-6-10-13-14-15-16 (19-5), and in the last row, 16-5-6-7-13-14-15-16 (16-5) has been modified to 16-4-5-6-7-13-14-15-16 (16-4).The authors wish to make the following corrections to this paper : Some erAccordingly, These corrections induced a few minor changes in the text of the Results and Discussion sections.circANRIL Isoforms” of Results:In the section “2.3. Characterisation of circANRIL species (4-5-6-7-4 (7-4), 6-4-5-6 (6-4), 6-14-5-6 (14-5), and 14-4-5-6-7-14 (14-4) circANRIL species (4-5-6-7-4 (7-4), 6-4-5-6 (6-4), 6-14-5-6 (14-5), 14-4-5-6-7-14 (14-4), and 6-7-9-10-5-6 (10-5) On page 6, the sixth sentence of paragraph one of the original publication  incorrecANRIL” of Results:In the Section “2.4. Non-Canonical Back-Splicing of ANRIL . Inspection showed that several intron pairs with reverse complementary Alu repeat sequences were found for introns 14-1, 12-1, 11-1, 11-6, 11-7, 6-1, 6-7, 7-5 and 5-1, which could potentially lead to back-splicing events between exons 14-2, 12-2, 11-2, 11-7, 11-8, 6-2, 7, 7-6 and 5-2, respectively . The validated back-spliced junctions found in circANRIL species in the NZM cell lines (On page 8, there are some mistakes in the third sentence and the fourth sentence in the first paragraph. This paragraph should be corrected to, “Analysis was also done to predict possible back-splicing events which may be attributed to the presence of inverted Alu elements present in introns of ll lines  confirmeIn the Discussion section:circANRIL. As an example, exon 10-4 and exon 6-14 junctions do not fit the above category, and alternative mechanisms need to be investigated”. Instead this sentence should read, “Back-splicing due to the presence of inverted repeat-containing introns could therefore be suggested only in the case of one of the isoforms identified in this study, i.e., exon 2-6 (circANRIL. As an example, the exon 10-4 junction does not fit the above category, and alternative mechanisms need to be investigated”.On page 11, Section 3.3, the third-eighth lines in paragraph three of the original publication [These changes have no material impact on the conclusions of our paper. The authors would like to apologize for any inconvenience caused to the readers by these changes.


In this investigation, the synthesis of 2-substituted pyrimidines by the reaction of benzofuran chalcones (3a-d) with urea, thiourea and guanidine hydrochloride was reported. The structures of title compounds (4a-d), (5a-d) and (6a-d) were established on the basis of analytical and spectral data. The synthesized compounds were screened for antimicrobial activity and molecular docking studies. Some of the compounds displayed excellent antimicrobial activity. The molecular docking analysis revealed that compounds 5a and 5c with the lowest binding energy in comparison to others suggesting its potential as best inhibitor of GluN-6-P. Consequently, it is confirmed from the above analysis that the compounds 5a and 5c might serve as a useful backbone scaffold for rational design, adaptation and investigation of more active analogs as potential broad spectrum antimicrobial agents.  N-acetyl glucosamine from which other amino-sugar containing molecules were derived. One of these products, N-acetyl glucosamine, is an important constituent of the peptidoglycan layer of bacterial cell walls and fungal cell wall, chitin. Accordingly, GlcN-6-P serves as a promising target for antibacterial and antifungal drug discovery. In continuation of the research on benzofuran chalcone based pyrimidine derivatives  as internal standard respectively. LC-MS were obtained using C-18 column on Shimadzu, LCMS 2010A, Japan.All reactions were performed at room temperature. High speed stirring was carried out with magnetic force. All chemicals were purchased from Aldrich Chemical Company and solvents were used without further purification. Analytical thin-layer chromatography was performed with E. Merck silica gel GF254 glass plates. Visualization of the developed chromatogram was performed by UV light (254 nm). Column chromatography was performed on silica gel 90, 200–300 mesh. Melting points were determined with Shimadzu DS-50 thermal analyzer. The FT-IR spectra were obtained using KBr pellets on Shimadzu spectrometer. The General procedure for Synthesis of compounds (4a-d) and (5a-d)3. The precipitated product was filtered, dried and recrystallized using ethanol. Equimolar quantity of benzofuran chalcones (1 mmol) (3a-d) were condensed with urea/thiourea in alcoholic KOH in a round bottom flask and the reaction mixture was continuously stirred for about 5-6 h at room temperature. The progress of the reaction was monitored by thin layer chromatography and spots were observed by iodine vapor and/or UV light. After completion of reaction, the reaction mixture was cooled, poured into crushed ice with constant stirring and neutralized using 10% NaHCO4-(1-Benzofuran-2-yl)-6-(thiophen-2-yl) pyrimidin-2-ol (4a)-1): 3464 (OH stretching), 1612 (C=N). 1HNMR (δ ppm): 7.32-7.30 ; 7.53-7.75 ; 8.55 ; 8.78 ; 10.28 .13CNMR (δ ppm): 100.12; 112.50; 112.86; 113.11; 116.96; 118.78; 123.24; 123.91; 127.33; 128.20; 130.38 (C-S), 145.29 (C=C); 151.84 (C-O); 153.78 (C-N); 155.84 (C=N); 159.43 (C-OH).MS, m/z: 295.30[M+1].Yield: 79%. Brown solid (EtOH), mp 160-162 °C; IR -6-(5-methylthiophen-2-yl)pyrimidin-2-ol (4b)-1): 3430 (OH stretching), 1610 (C=N). 1HNMR (δ ppm): 2.42 ; 7.54-7.76 ; 8.54 ; 8.75 ; 10.35 . 13CNMR (δ ppm): 24.23 (CH3); 101.20; 108.20; 110.50; 111.20; 113.84; 115.60; 123.10; 124.76; 128.30; 129.80; 130.14 (C-S); 146.30 (C=C); 152.76 (C-O); 154.72 (C-N); 155.40 (C=N); 159.10 (C-OH). MS, m/z: 309.30[M+1].Yield: 80%. Light brick red (EtOH), mp 154-156 °C. IR -6-(thiophen-2-yl)pyrimidin-2-ol (4c)-1): 3430 (OH stretching), 1615 (C=N), 747(C-Br). 1HNMR (δ ppm): 7.30-7.28 ; 7.50-7.72 ; 8.51 ; 8.76 ; 10.22. 13CNMR (δ ppm): 102.24; 110.30; 111.10; 111.80; 114.92; 116.80; 124.20; 125.86; 129.32; 130.10; 132.34 (C-S); 147.32 (C=C); 153.78 (C-O); 155.80 (C-N); 156.60 (C=N); 160.02 (C-OH). MS, m/z: 372[M+] and 374[M+2].Yield: 86%. Brown solid (EtOH), mp 171-173 °C. IR -6-(5-methylthiophen-2-yl)pyrimidin-2-ol (4d)-1): 3442 (OH stretching), 1614 (C=N), 677 (C-Br). 1HNMR (δ ppm): 2.43 ; 7.53-7.76 ; 8.66 ; 8.74 ; 10.35. 13CNMR (δ ppm): 23.20 (CH3); 100.00; 110.10; 111.20; 112.20; 114.80; 115.90; 123.25; 124.80; 129.22; 130.12; 132.30 (C-S); 147.40 (C=C); 153.74 (C-O); 154.89 (C-N); 156.50 (C=N); 160.12 (C-OH). MS, m/z: 386[M+] and 388[M+2]. Yield: 83%. Brick red (Et-OH), mp 170-172 °C. IR -6-(thiophen-2-yl) pyrimidine-2-thiol (5a)-1): 2564 (SH), 1655 (C=N). 1HNMR (δ ppm): 7.33-7.35 ; 7.53-8.35 ; 8.54 ; 10.35 . 13CNMR (δ ppm):100.50; 112.50; 112.86; 113.11; 116.96; 118.78; 123.24; 124.00; 127.33; 128.20; 130.38 (C-S); 145.29 (C=C); 151.54 (C-O); 153.78 (C-N); 155.84 (C=N); 179.43 (C-SH). MS, m/z: 311.30[M+1].Yield: 85%. Brown (EtOH), mp 158-160 °C. IR -6-(5-methylthiophen-2-yl)pyrimidine-2-thiol (5b)-1): 2560 (SH), 1598 (C=N). 1HNMR (δ ppm): 2.33 ;7.51-8.34 ; 8.45 ; 10.11 .13CNMR (δ ppm): 21.86 (CH3); 101.30; 111.10; 111.84; 114.10; 117.90; 119.75; 124.34; 125.11; 128.44; 129.26; 131.40(C-S); 146.30 (C=C); 152.64 (C-O); 154.73 (C-N); 156.82 (C=N); 180.20 (C-SH). MS, m/z: 325.40[M+1].Yield: 76%. Brown (EtOH), mp 154-156 °C. IR -6-(thiophen-2-yl)pyrimidine-2-thiol (5c)-1): 2558 (SH), 1600 (C=N), 746 (Ar-Br). 1HNMR (δ ppm): 7.30- 7.28 ; 7.52-8.33 ; 8.48 ; 10.24 . 13CNMR (δ ppm):100.20; 112.15; 113.88; 116.18; 118.80; 120.70; 123.30; 123.18; 127.46; 128.36; 130.50 (C-S); 145.40 (C=C); 151.74 (C-O); 154.83 (C-N); 156.92 (C=N); 179.50 (C-SH). MS, m/z: 388[M+] and 390[M+2].Yield: 85%. Brownish yellow (EtOH), mp 157-159 °C. IR -6-(5-methylthiophen-2-yl)pyrimidine-2-thiol (5d)-1): 2556 (SH), 1602 (C=N), 782 (Ar-Br).1HNMR (δ ppm): 2.42 ; 7.54-8.36 ; 8.42 ; 10.28 . 13CNMR (δ ppm): 24.42 (CH3); 102.35; 112.15; 112.88; 115.18; 118.80; 120.70; 121.50; 125.30; 126.18; 129.46; 133.10 (C-S); 148.42 (C=C); 154.14 (C-O); 155.00 (C-N); 156.60 (C=N); 179.46 (C-SH). MS, m/z: 402[M+] and 404[M+2].Yield: 75%. Light Brown (EtOH), mp 162-163 °C. IR -6-(5-substituted thiophen-2-yl)pyrimidin-2-amine (6a-d)3. The precipitated product was filtered dried and recrystallized using ethanol. Equimolar quantity of benzofuran chalcones (1 mmol) (3a-d) was condensed with guanidine hydrochloride (1 mmol) in alcoholic KOH in a round bottom flask and reaction mixture was continuously stirred for about 5-6 h at room temperature. The progress of the reaction was monitored by thin layer chromatography and spots were observed by iodine vapor and/or UV light. After completion of reaction, the reaction mixture was cooled, poured into crushed ice with constant stirring and neutralized using 10% NaHCO4-(1-Benzofuran-2-yl)-6-(thiophen-2-yl)pyrimidin-2-amine (6a)-1): 3318 (NH2), 1625 (C=N). 1HNMR (δ ppm): 7.34-7.30 ; 7.560-7.543 ; 7.60 ; 7.66 ; 8.58 . 13CNMR (δ ppm): 101.20; 108.50; 110.37; 119.22; 120.06; 123.70; 126.09; 126.98; 129.17; 131.93; 134.98 (C-S), 145.82 (C=C);151.60 (C-O); 153.80 (C-N); 155.80 (C=N); 164.24 (C-NH2). MS, m/z: 294.30[M+1].Yield: 83%. Dark Brown (EtOH), mp 168-170 °C. IR -6-(5-methylthiophen-2-yl)pyrimidin-2-amine (6b)-1): 3315 (NH2), 1620 (C=N). 1HNMR (δ ppm): 2.40 ; 7.33-7.29 ; 7.59 ; 7.64 ; 8.54 . 13CNMR (δ ppm): 22.20 (CH3); 100.10; 107.51; 111.17; 118.11; 121.16; 124.60; 127.19; 127.96; 130.18; 131.94; 135.88 (C-S), 146.00 (C=C); 151.66 (C-O); 154.82 (C-N); 156.68 (C=N); 165.00 (C-NH2). MS, m/z: 308.30[M+1].Yield: 79%. Brown (EtOH), mp: 163-165 °C. IR -6-(thiophen-2-yl)pyrimidin-2-amine (6c)-1): 3320 (NH2), 1612 (C=N), 666 (Ar-Br). 1HNMR (δ ppm):7.35-7.31 ; 7.561-7.543 ; 7.51 ; 7.62 ; 9.12 . 13CNMR (δ ppm): 102.30; 109.01; 112.17; 119.10; 122.10; 125.00; 128.29; 128.96; 131.12; 132.84; 136.78 (C-S), 145.10 (C=C); 151.23 (C-O); 155.22 (C-N); 157.60 (C=N); 165.12 (C-NH2). MS, m/z: 371[M+] and 373[M+2].Yield: 84%. Light brown (EtOH), mp 160-162 °C. IR -6-(5-methylthiophen-2-yl)pyrimidin-2-amine (6d)-1): 3318 (NH2), 1623 (C=N), 688 (Ar-Br). 1HNMR (δ ppm): 2.42 ; 7.31-7.28 ; 7.56 ; 7.60 ; 8.94 . 13CNMR (δ ppm): 23.21 (CH3); 101.10; 106.01; 112.07; 119.10; 122.26; 123.68; 128.19; 128.86; 131.08; 132.04; 136.18 (C-S), 145.10 (C=C); 150.96 (C-O); 155.12 (C-N); 156.68 (C=N); 165.00 (C-NH2). MS, m/z: 385[M+] and 387[M+2].Yield: 77%. Brown (EtOH), mp 167-169 °C, IR , gram negative bacteria Escherichia coli (MTCC 1559) and Pseudomonas aeruginosa (MTCC 1034). The antifungal activity against Alternaria alternate (MTCC 3793), Aspergillus niger (MTCC 4325) and Candida albicans (MTCC 1637). Agar well diffusion technique was used for the determination of preliminary antibacterial and antifungal activities  and known GluN-6-P inhibitors (Streptomycin and Fluconazole) were drawn using ChemDraw (Version 10). All the ligands were 3D optimized using PRODRG server . All theProtein preparationThe X-ray crystal structure of GluN-6-P (PDB ID: 1XFF) with a resolution of 1.81 Å was obtained from the Protein Data Bank (http://www.rcsb.org/pdb). All the heteroatoms and water molecules were removed. Polar hydrogens were removed and Gasteiger charges were assigned after merging of non-polar hydrogen atoms. After such preprocessing procedure of the crystal structure, it was saved in PDBQT file format using ADT.Molecular docking 6 energy evaluations, a mutation rate of 0.02, and a crossover rate of 0.80. The grid map was centered at the active site binding pocket of the protein by AutoGrid 4 involving Cys 1, Trp 74, Thr 76, His 77, Gly 99, and Asp 123 as previously reported by other groups  were clustered together and represented by the result with the most favorable free energy of binding. All torsions were allowed to rotate during docking.Automated docking was performed with the Auto Dock 4  using an empirical free energy function and Larmarckian Genetic Algorithm, with an initial population of 250 randomly placed individuals, a maximum number of 10r groups -41. One-Validation of dockingTo ensure that the binding possess obtained from the docking studies are likely to represent authentic binding interactions, the removal and re-docking of the potential ligands/reference ligands (streptomycin/fluconazole) against GluN-6-Pwere performed more than 10 runs. The RMSD of the overlapping structures and reoccurrence of the similar binding pose was ascertained to confirm the docking procedure effectiveness.Absorption-Distribution-Metabolism-Excretion and toxicity (ADME/Tox)The ADME properties of all the ligands were calculated by using the QikProp program (Version 3.4). Qikprop predicts physically significant descriptors and pharmaceutically relevant properties of chemical molecules. Ligprep minimized ligands were given as a source in Qikprop. More than 40 chemical and biological descriptors with relevance to drug-likeliness were analyzed for all the compounds considered in this study .Experimental analysisSynthesis of the target compounds were achieved according to the reactions illustrated in the Chalcone intermediates were obtained by Aldol condensation of corresponding 2-acetyl benzofuran (1) with different thiophene carbaldehydes (2) according to the reported procedure . The con-1 which corresponds to hydroxyl group. Another absorption band in the region of 1612 cm-1 corresponds to C=N stretching vibration. The 1HNMR spectrum of compound 4a exhibited a broad singlet at δ 11.25 ppm due to hydroxyl proton and multiplet between δ 7.30-8.78 ppm for aromatic protons. Further, 13CNMR spectrum of compounds 4a displayed the signals at required positions. The mass spectrum of the compound 4a showed molecular ion peak at m/z 295.30[M+1] which corresponds to molecular weight of the compound. The physical and analytical data of the newly synthesized compounds (4a-d), (5a-d) and (6a-d) are tabulated in The structure of desired pyrimidine analogs (4a-d), (5a-d) and (6a-d) were confirmed using IR, NMR and Mass spectral data. The IR spectrum of compound 4a exhibited a broad absorption band at 3464 cmBiological activityBacillus subtilis. The results of antibacterial and antifungal activity at two different concentrations (50 mg and 100 mg) have been depicted in Though, there are several synthetic drugs in the market, the bacterial mutations assemble their resistance. To overcome this challenge, there is a crucial need to discover the new and specific synthetic molecules against microbes. In view of this, all the synthesized compounds in the present investigation (4a-d), (5a-d) and (6a-d) were evaluated for their antimicrobial activity by a primary screening analysis. All the synthesized compounds exhibited significant antimicrobial activity against bacterial strains Further, the compounds which showed good antimicrobial activity in primary screening were assessed to determine minimum inhibitory concentration by broth dilution method and results were illustrated in Bacillus subtilis with MIC value 18.45 ± 0.23 and 17.32 ± 0.24 µg/mL. The compound 5b exhibit good antibacterial activity against all the tested bacterial strains with MIC value in the range of 16.24 ± 0.26 to 11.47 ± 0.28 µg/mL. Compounds 4a, 5d and 4b showed moderate effect against tested pathogenswith MIC value in the range of 15.32 ± 0.28 to 10.11 ± 0.63 µg/mL. The compounds 4c and 6a showed considerable activity against all the tested bacterial strains with MIC value in the range of 11.22 ± 0.27 to 6.64 ± 0.27 µg/mL. Nevertheless, the remaining compounds showed negligible antibacterial activity against all the tested strains.Recent literature suggests that the structural and electronic parameters of synthesized compounds may have better impact on changing the efficacy of antimicrobial activity . A prima. Henceforth only these potential antimicrobial derivatives  and known GluN-6-P inhibitors (Streptomycin and Fluconazole) were further considered for computational analysis. The antifungal action of newly synthesized compounds also indicate that compounds 4a, 4b, 4c, 5a, 5c, 5d and 6a exhibited excellent antifungal activity against all tested pathogens with MIC value in the range of 15.15 ± 0.48 to 3.21 ± 0.45 µg/mL. The least MIC values were found in the remaining compounds as tabulated in  Computational analysisin-silico analysis was undertaken to identify whether the molecular docking of benzofuran containing pyrimidine derivatives  with GluN-6-P provides any correlation with their in-vitro antibacterial activity. Computationally the molecular docking investigations revealed the similar binding energy values of benzofuran derivatives in comparison to reference compounds like streptomycin and fluconazole . This analysis also revealed that, compound 5a and 5c was nontoxic and has the best human intestinal absorption in comparison to all other ligands .in-vitro observations. Nevertheless, streptomycin had poor HIA in comparison to benzofuran derivatives. All of the above findings demonstrated that computational analysis was in good agreement with  In present work, biologically active benzofuran compounds containing pyrimidine ring were synthesized from benzofuran chalcones having high chemical reactivity and diverse synthetic applications. From antimicrobial activity results, it was found that the presence of hydroxyl, thiol, and amino groups in the pyrimidine ring displayed promising antimicrobial activity. Additionally, the molecular docking analysis revealed that, compounds 5a and 5c with the lowest binding energy were suggestive of the highest binding affinity in comparison to other compounds. Furthermore, computational analysis also revealed favorable ADME/Tox features of compounds 5a and 5c. Among the compounds studied, only 5a and 5c showed significant human intestinal absorption. From all the experimental and computational analysis it may be concluded that benzofuran compounds fused with pyrimidine ring showed significant broad spectrum of antimicrobial activity and had high affinity against GluN-6-P. Henceforth it can serve as new building blocks for synthesis and design of broad spectrum antimicrobial compounds.