Cngb1 locus-encoded β-subunit of rod cGMP-gated cation channel and associated glutamic acid rich proteins (GARPs) are required for phototransduction, disk morphogenesis, and rod structural integrity. To probe individual protein structure/function of the GARPs, we have characterized several transgenic mouse lines selectively restoring GARPs on a Cngb1 knockout (X1−/−) mouse background. Optical coherence tomography (OCT), light and transmission electron microscopy (TEM), and electroretinography (ERG) were used to analyze 6 genotypes including WT at three and ten weeks postnatal. Comparison of aligned histology/OCT images demonstrated that GARP2 accelerates the rate of degeneration. ERG results are consistent with the structural analyses showing the greatest attenuation of function when GARP2 is present. Even 100-fold or more overexpression of GARP1 could not accelerate degeneration as rapidly as GARP2, and when co-expressed GARP1 attenuated the structural and functional deficits elicited by GARP2. These results indicate that the GARPs are not fully interchangeable and thus, likely have separate and distinct functions in the photoreceptor. We also present a uniform murine OCT layer naming nomenclature system that is consistent with human retina layer designations to standardize murine OCT, which will facilitate data evaluation across different laboratories.The  The β-subunit and GARP2 have critical roles in phototransduction, disk morphogenesis, and maintenance of outer segment structural integrity1314Cngb1 locus, these proteins are identical in sequence for 318 amino acids. GARPs are intrinsically disordered proteins161615202215In the rod photoreceptor, the CNG channel is a heterotetramer composed of three α- and one β-subunits which are arranged around a central pore781011−/−), results in loss of function by one month, nearly undetectable levels of CNG α-subunit by two months, and retina degeneration by four months−/−) also results in loss of structure and function, including disruption of disk/plasma membrane interactions−/− mice occurs 4–5 months earlier and the levels of the CNG α-subunit are significantly lower than in X1−/− mice. To further dissect individual protein structure/function of the GARPs in rods, we have characterized several new transgenic mouse models, which selectively restore the absent GARP proteins to the X1−/− mouse retina. Additionally, we present a system for murine optical coherence tomography (OCT) layer designation, that conforms with recently published human OCT guidelinesKnockout of the CNG channel β-subunit, while leaving GARPs intact  and ten weeks (0.042 mm ± 0.01), but no change in FRT. Consistent with progressive retinal degeneration in X26−/− mice, OCT analysis of transgenic mice showed significant reduction in ONL thickness (from 0.052 mm ± 0.01 to 0.033 mm ± 0.01) and FRT (from 0.215 mm ± 0.02 to 0.182 mm ± 0.01) over time. The effects of overexpression of G1 on the X1−/− background were also assessed. X1−/− G1Tg showed reduction in ONL thickness between three (0.052 mm ± 0.01) and ten weeks (0.038 mm ± 0.02), but no change in FRT. X1−/− G2Tg showed a reduction in ONL thickness between three (0.051 mm ± 0.01) and ten weeks (0.019 mm ± 0.01), as well as a reduction in FRT (from 0.205 mm ± 0.02 to 0.163 mm ± 0.02). X1−/− G1Tg G2Tg also showed a reduction in ONL thickness between three (0.050 mm ± 0.01) and ten weeks (0.028 mm ± 0.01), as well as a reduction in FRT (from 0.214 mm ± 0.01 to 0.176 mm ± 0.02). All of the genotypes except WT showed a reduction in ONL thickness over time. However, neither X1−/− or X1−/−G1Tg showed a reduction in FRT, as was observed for all other genotypes.To assess the effects of the transgene alleles on retinal degeneration in X1en weeks  and 4 in−/− (0.212 mm ± 0.01), X1−/− G1Tg (0.207 mm ± 0.02), X26−/− (0.215 mm ± 0.02), X1−/− G2Tg (0.205 mm ± 0.02), and X1−/− G1Tg G2Tg (0.214 mm ± 0.01) all showed reduced FRT compared to WT (0.227 mm ± 0.02). At ten weeks X1−/−  and X26−/−  retinas showed reduced ONL and FRT thickness compared to WT . X26−/− (0.033 mm ± 0.01) retinas showed reduced ONL thickness compared to X1−/− (0.042 mm ± 0.01) and X1−/− G1Tg (0.038 mm ± 0.02). X1−/− G1Tg (0.038 mm ± 0.02) showed reduced ONL thickness compared to WT (0.051 mm ± 0.01) and X1−/− (0.042 mm ± 0.01), as well as reduced FRT (0.205 mm ± 0.02) compared to WT (0.218 mm ± 0.02). X1−/− G2Tg (0.019 mm ± 0.01) showed reduced ONL thickness compared to WT (0.051 mm ± 0.01), X1−/− (0.042 mm ± 0.01), X26−/− (0.033 mm ± 0.01), X1−/− G1Tg (0.038 mm ± 0.02) and X1−/− G1Tg G2Tg (0.028 mm ± 0.01), as well as reduced FRT (0.163 mm ± 0.02) compared to WT (0.218 mm ± 0.02), X26−/− (0.182 mm ± 0.01) and X1−/− G1Tg (0.205 mm ± 0.02). X1−/− G1Tg G2Tg (0.028 mm ± 0.01) showed reduced ONL thickness compared to WT (0.051 mm ± 0.01), X1−/− (0.042 mm ± 0.01), X26−/− (0.033 mm ± 0.01), and X1−/− G1Tg (0.038 mm ± 0.02), as well as reduced FRT (0.176 mm ± 0.02) compared to WT (0.218 mm ± 0.02) and X1−/− G1Tg (0.205 mm ± 0.02). Genotypes expressing GARP2 in the absence of β-subunit  had significant reduction in ONL thickness compared to those that did not express GARP2 (X1−/− and X1−/− G1Tg) or expressed GARP2 and β-subunit (WT).OCT also revealed between group differences in ONL thickness and FRT at each time point . At thre−/−, X26−/− and X1−/− G1Tg than in WT (p < 0.001). ONL thinned more rapidly in X1−/− G2Tg than in WT, X1−/−, X26−/−, X1−/− G1Tg, or X1−/− G1Tg G2Tg. ONL thinned more rapidly in X1−/− G1Tg G2Tg than in WT, X1−/−, or X1−/− G1Tg. Therefore, the greatest rate of ONL thinning is in X1−/− G2Tg. There is ONL thinning in X1−/− G1Tg, but at a much slower rate than in X1−/− G2Tg. Expression of both GARP1 and GARP2 in X1−/− G1Tg G2Tg shows a slower rate of ONL thinning than X1−/− G2Tg, but faster than X1−/− G1Tg. Thus, in order of decreasing rates of thinning, the comparative rate of thinning per genotype is X1−/− G2 > X1−/− G1Tg G2Tg > X26−/− > X1−/− G1Tg > X1−/− > WT. The only difference between X26−/− and X1−/− G1Tg G2Tg, is that the latter expresses more GARP1 than the former. This indicates that co-expression of GARP2 along with increased GARP1 (X1−/− G1Tg G2Tg) results in increased rates of thinning. However, increased expression of GARP1 without GARP2 (X1−/− G1Tg) has a lower rate of thinning than endogenous expression of GARP1 with GARP2 (X26−/−), suggesting that GARP2 expression has the strongest effect on the rate of thinning.The rate of thinning over time was compared between groups, a measurement of the interaction between time and genotype . In gene−/− and X26−/− than in WT or X1−/− G1Tg. The rate of FRT thinning is greater in X1−/− G2Tg than WT or X1−/− G1Tg. Finally, the rate of FRT thinning is greater in X1−/− G1Tg G2Tg than WT or X1−/− G1Tg. Considering all genotypes, the rate of thinning from fastest to slowest is X1−/− G2Tg, X1−/− G1Tg G2Tg, X1−/−, and X26−/− > X1−/− G1Tg and WT. The only genotype that did not have faster FRT thinning than WT was X1−/− G1Tg.The rate of FRT thinning  is great−/−, 0.039 mm ± 0.005 for X26−/−, 0.040 mm ± 0.003 for X1−/− G1Tg, 0.042 mm ± 0.003 for X1−/− G2Tg and 0.037 mm ± 0.003 for X1−/− G1Tg G2Tg. At ten weeks WT ONL is 0.036 mm ± 0.002, compared to 0.032 mm ± 0.003 for X1−/−, 0.026 mm ± 0.003 for X26−/−, 0.029 ± 0.004 for X1−/− G1Tg, 0.021 mm ± 0.002 for X1−/− G1Tg G2Tg, and 0.025 mm ± 0.004 for X1−/− G2. Similarly to OCT results, there was a significant within group difference between 3 and 10 weeks for all genotypes except WT and X1−/−. While OCT results did show a significant thinning for X1−/− between 3 and 10 weeks, this was the smallest difference and the least statistically significant change of all of the genotypes. Additionally, there were between group differences for ONL thickness at 10 weeks. OCT results showed a significant thinning in ONL at 10 weeks in X26−/−, X1−/− G1Tg G2Tg, and X1−/− G2Tg retinas compared to WT. However, histology measurements showed a significant thinning in ONL at 10 weeks for all genotypes compared to WT. At 3 weeks, WT FRT is 0.182 mm ± 0.006, compared to 0.179 mm ± 0.005 for X1−/−, 0.245 mm ± 0.025 for X26−/−, 0.233 mm ± 0.013 for X1−/− G1Tg, 0.220 mm ± 0.012 for X1−/− G2Tg, and 0.200 mm ± 0.012 for X1−/− G1Tg G2Tg. At 10 weeks, WT FRT is 0.205 mm ± 0.010, compared to 0.197 mm ± 0.014 for X1−/−, 0.190 mm ± 0.013 for X26−/−, 0.179 mm ± 0.011 for X1−/− G1Tg, 0.201 mm ± 0.020 for X1−/− G2Tg, and 0.155 mm ± 0.006 for X1−/− G1Tg G2Tg. While OCT results only showed a significant FRT thinning over time in WT, X26−/−, X1−/− G2Tg, and X1−/− G1Tg G2Tg, histology measurements showed significant FRT thinning over time in all genotypes. OCT results at 10 weeks showed a significantly thinner FRT for X26−/−, X1−/− G1Tg, X1−/− G1Tg G2Tg, and X1−/− G2Tg compared to WT. Similar results were observed for histology measurements, except histology did not show a difference in FRT between WT and X26−/− retinas. Overall, histology measurements of ONL and FRT confirmed OCT observations. In addition, histology also showed that photoreceptor outer segments are less uniform and more disorganized in X1−/−, X1−/− G1Tg and X1−/− G1Tg G2Tg. Photoreceptor outer segments are almost completely lost in X1−/− G2Tg at 10 weeks. At 10 weeks, GARP2 accelerates thinning; however GARP1 does not affect the rate of thinning and partially counteracts the effect of GARP2 in the X1−/− G1Tg G2Tg hybrid.To validate OCT results, histologic analysis was performed on each genotype at three and ten weeks . At thre−/−, X1−/− G1Tg, X1−/− G2Tg, and X1−/− G1Tg G2Tg retinas  images revealed structural changes as early as three weeks in X1 retinas . In WT, Cngb1) at 1 month postnatal , without the N-Cngb1 primary antibody showed no signal in any genotype.Because there is an ongoing degeneration it could not be assumed that the transgene expressed GARPs are properly localizing in photoreceptors. Therefore, we determined the localization of GARPs in WT and the transgenic animals. Using an antibody that recognizes the N-terminus of the β-subunit and GARPs . When GARP1 is overexpressed without GARP2 (X1−/−G1Tg) or co-expressed with GARP2 (X1−/−G1TgG2Tg) it was readily apparent. GARP2 was easily detected in WT, X1−/−G2Tg, X1−/−G1TgG2Tg, and X26−/− mice. Protein expression is shown as the percent of GARP2 expression in WT retina  in X1−/−G1Tg mice and 215% (±26%) in X1−/−G1TgG2Tg mice. GARP2 was expressed at 42% (±25%) in X1−/−G2Tg mice, 204% (±72%) in X1−/−G1TgG2Tg mice, and 18% (±12%) in X26−/− mice. Despite a 30% increase in GARP1 expression in X1−/−G1TgG2Tg compared to X1−/−G1Tg mice, this difference was not statistically significant. However, there was significantly lower expression of GARP2 in X1−/−G2Tg and X26−/− mice compared to X1−/−G1TgG2Tg mice. Despite a 20% increase in GARP2 expression in X1−/−G2Tg mice compared to X26−/− mice, this difference was not statistically significant.To compare proteins levels of channel β-subunit, GARP1, and GARP2 in the various genotypes, automated capillary-based Western analysis was performed, using an internal standard for normalization. Similar to previously published resultsT retina , lane WTT retina . While G2 flash were significantly higher than X1−/− , X26−/− , X1−/− G1Tg , X1−/− G1Tg G2Tg  and X1−/− G2Tg . In addition, the X1−/− a-wave was significantly higher than X1−/− G2Tg, and the b-wave was significantly higher than X1−/− G2Tg and X1−/− G1Tg G2Tg. At 10 weeks , X26−/− , X1−/− G1Tg , X1−/− G1Tg G2Tg  and X1−/− G2Tg . These data are consistent with a negative effect of GARP2 on rod function in the absence of the β-subunit. Scotopic responses were also measured at lower intensities (0.0001–10 cd*s/m2) and showed similar results as the saturating 25 cd*s/m2 flash .Using the mouse OCT nomenclature described in −/− G2Tg mice. Co-expression of GARP1 and GARP2 on the X1−/− reduced this attenuation, but it was still greater than the attenuation observed in X1−/− G1Tg. These results provide further evidence that the negative effect of GARP2 is dominant over GARP1, and suggests a possible interaction between GARP1 and GARP2. This also further supports the conclusion that GARP2, in the absence of the β-subunit, drives functional and structural decline in the retina. Finally, coupled with the structural data, this also suggests GARP1 and GARP2 have distinct functions in the rods.As expected, photoreceptor (a-wave) and bipolar cell function (b-wave), measured via ERG, were reduced in all genotypes compared to WT. In addition, photoreceptor and bipolar cell function (attenuated a- and b-waves in ERG) were consistent with OCT findings, with the greatest attenuation in X1−/− background which increased the GARP1 levels to approximately endogenous levels of GARP2 expression. A GARP2 transgene (at endogenous levels of protein when expressed on a WT background) was also crossed onto the X1−/− background. In addition, the two GARP expressing animals were then crossed to enable examination of expression of the two transgenes in the absence of the CNG β-subunit. Finally, endogenous GARP1 and GARP2 were both expressed in the X26−/− mouse which only lacks the CNG β-subunit. If the two proteins have identical function one would expect identical phenotypes when expressed in various genetic backgrounds where phenotypes are discernible. Furthermore, one would expect the co-expression of both GARPs to further accelerate the thinning. The ONL in X26−/− mice thins more slowly than in X1−/− G2Tg.In WT mice, GARP1 is 20-fold less abundant than GARP2 and four-fold less than the CNG β-subunit−/−, X26−/−, and a G2 overexpression mouse131419−/−, and the G2 overexpression mouse, GARP/β-subunit expression is confined to the OS with variable OPL signal. Unsurprisingly, GARP/β-subunit expression is absent in the X1−/− mouse. GARP expression in the X1−/− G1Tg, X1−/− G2Tg, and X1−/− G1Tg G2Tg mice was confined primarily to the OS, with minimal expression in the IS and variable signal in OPL. This shows that the transgene, even when overexpressed, primarily localizes to the OS.Previous studies have examined GARP/β-subunit expression in WT, X1−/− mice is ~18% of the endogenous GARP2 expression, while GARP2 expression in X1−/− G2Tg mice is ~42%. This higher level of GARP2 expression could explain why the ONL in X26−/− mice thins more slowly than in X1−/− G2Tg. Crossing the X1−/− G1Tg (185% of WT GARP2 expression) and X1−/− G2Tg (42% of WT GARP2 expression) mice results in offspring that express GARP2 at ~200% of WT GARP2 levels. X1−/− G1Tg mice do not have significantly faster ONL thinning than X26−/−, but the X1−/− G1Tg G2Tg mice (with the 200% increases in GARP2 expression) do have significantly faster rates of thinning than X26−/− mice. However, the increased GARP2 expression in X1−/− G1Tg G2Tg is not enough to increase the rate of thinning compared to X1−/− G2Tg mice, which have lower levels of GARP2. Thus, the presence of GARP1 must be slowing the rate of thinning. This also indicates differing roles for GARP1 and GARP2 and the possibility that increased expression of GARP1 is somehow involved in increasing expression of GARP2. Taken together, our results lead us to conclude that GARP1 and GARP2 are not interchangeable as they do not produce identical phenotypes, nor are they additive in effect, and thus these proteins may have distinct and separate roles in the rod photoreceptor.Total GARP2 protein expression in X26−/−−/−One possible mechanism for the acceleration of degeneration in our GARP transgenic mice could be inhibition of α-subunit transport (in the absence of the β-subunit) from IS to OS which is consistent with the significantly lower α-subunit levels found in X26−/−)−/−)Tg, 100-fold overexpressed) using a 4.4 kb opsin promoter, mouse protamine poly-A construct generated by replacing the PDE6 γ-subunit insertTg, construct shown in −/− G1Tg G2Tg mice carrying both alleles were generated by crossing G1Tg and G2Tg transgenic mice on a homozygous X1−/− background and identifying the presence of both alleles by PCR genotyping (see below). All animal lines were made congenic on a C57BL/6J background. All animal use protocols were approved by the University of Alabama at Birmingham (UAB) Institutional Animal Care and Use Committee and are consistent with the Association for Research in Vision and Ophthalmology guidelines for the use of animals in research. All animals were maintained on a standard 12/12-hour light/dark cycle, fed standard rodent chow, and housed with standard rodent bedding.An exon 1, 2 and predicted promoter Cngb1 knockout null allele  in digestion buffer consisting of 5 mM EDTA, 200 mM NaCl, 100 mM Tris-HCl, and 0.2% SDS. Tails were digested overnight at 55 °C. 100% ethanol was added to the lysate, which was then centrifuged at 16,000 × g for thirty minutes, washed with 70% ethanol, centrifuged at 16,000 × g for twenty minutes, and then the DNA pellet was dissolved in TE buffer, pH 8.0. PCR was performed using the following cycling parameters: 94 °C denaturation, five minutes, for one cycle; thirty five cycles of 94 °C, thirty seconds; for GARP1, 58 °C, thirty seconds; for GARP2, 62 °C, forty five seconds; 72 °C, fifty seconds; and one cycle of 72 °C for seven minutes. Primers sequences for X26TM 1.4 software  rectangular volume scan of 1.4 mm diameter with one thousand A-scans/B-scan, one hundred B-scans/volume, and one frame/B-scan or (scan parameter two) rectangular volume scan of 1.6 mm in diameter with one thousand A-scans/B-scan, three B-scans/volume, and forty eight frames/B-scan. The one thousand A-scan frames collected in parameter two were averaged to reduce noise and increase signal. These averaged images were then converted to bmp files. To determine the ONL thickness, the difference in distance between the outer plexiform layer (OPL) and the external limiting membrane (ELM) was calculated. To determine the FRT, the difference in distance between the inner limiting membrane (ILM) and retinal pigment epithelium (RPE) was calculated. Measurements of ONL thickness and FRT were performed with Bioptigen Diver 2.4 software on retinal OCT images, captured using scan parameter one, of three week and ten week old mice at eight equidistant eccentricities  was performed to visualize the posterior segment in living mice. The Bioptigen OCT instrument has a transverse resolution of 2.5 μm and an axial resolution of 1.6 μm. Animals were anesthetized with intraperitoneal injection of ketamine/xylazine  at 100 mg/kg and 10 mg/kg body weight, respectively. Pupils were dilated using 1% tropicamide  and 0.5% proparacaine , and artificial tears  were applied frequently, with saline wash throughout to maintain corneal clarity. Scans were captured with the beam centered on the optic nerve. Both horizontal and vertical linear scans were obtained with Bioptigen InVivoVuericities , in one ricities . ONL thiricities  were takLinear regression mixed models were used to assess ONL and FRT in five groups representing each transgenic line and WT mice. Linear regression mixed models were used to account for the within-animal correlation that occurs when multiple observations are taken from the same subject. First, we examined whether the mean ONL and FRT across the entire retina differed within each transgenic line group over time (three weeks vs. ten weeks). T-tests were also used to examine this association at each eccentricity . Next, we examined whether there were any between-group differences at each time point. Finally, a statistical interaction term was added to the model to assess if the changes over time differed by transgenic group. Separate linear mixed models were used to assess mean ONL and mean FRT. For five of the animals, data was collected from the same animal at both time points. Therefore, two separate analyses were employed to account for the paired nature of these measurements. First, data collected at ten weeks was treated as independent observations from those collected at time three. Second, the analyses were re-run excluding data collected at ten weeks for just those five animals. In both approaches, the pattern of results was similar. The results from the first analysis are presented; however, differences in results are noted where applicable. Adjustment of p-values for multiple comparisons was not performed. P-values < 0.05 were considered statistically significant.Cngb1; QEPPEPKDPPKPPGC) (1:50)Morphologic analysis was performed as previously describedCngb1; QEPPEPKDPPKPPGC) was used (1:500) to detect expression of the β-subunit and GARP2. N-Cngb1 was generated in rabbit and affinity purified . Data analysis was performed using the Compass Software (ProteinSimple) and quantitation was determined by normalizing the area under the curve of the β-subunit and GARP2 peaks by the area under the curve of the ProteinSimple internal standard peak. Representative computer generated electerophoretic images were generated by the Compass Software. Images shown contain information from capillaries run over multiple trials. The computer generated electrophoretic images displayed larger product size than is normally observed for these proteins. The very low pI of the proteins is likely the basis for the altered migration, which is even more pronounced in the capillary than is observed in an acrylamide gelWestern analysis on blots for GARP1 were performed as previously described2 and 25 cd*s/m2 were delivered through a Ganzfeld dome on an Ocuscience ERG  instrument manufactured for functional analysis of animal eyes. ERG data, including time/response and waveforms, were viewed using proprietary ERGView 4.860 A software and analyzed after extraction into Microsoft Excel 2013 (ver. 15.0.4763.1003). Standard t-tests were used to analyze differences in the maximum saturating a- and b-wave response (μV) between genotypes at each light intensity.Scotopic ERG responses were measured at 6 different intensities of light in each genotype at 3 weeks and 10 weeks of age. Mice to be used for full-field ERG analysis were dark adapted overnight, sedated with 3% isoflurane in a closed chamber, and anesthesia was maintained at 2% via a certified isoflurane vaporizer . Eyes were anesthetized with proparacaine drops  and pupils dilated with topical phenylephrine HCL and tropicamide . Only eyes receiving light stimuli were dilated. Methylcellulose 2.5%  was applied to the corneal surface as well as to the contact lens placed on top of the silver-embedded recording electrode. The reference and ground electrodes were stainless steel subdermal needles placed in the cheek and lower back, respectively. During recordings the equipment and animal were enclosed in a Faraday cage and body temperature maintained at 37 °C by a digital heating pad placed under the animal. Defined light pulses between 0.1 cd*s/mHow to cite this article: DeRamus, M. L. et al. GARP2 accelerates retinal degeneration in rod cGMP-gated cation channel β-subunit knockout mice. Sci. Rep.7, 42545; doi: 10.1038/srep42545 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The dihydropyrimidine ring adopts a twist-boat conformation while the quinone ring is slightly non-planar. In the crystal, molecules are linked by weak C—H⋯O and C—H⋯S hydrogen bonds and C—H⋯π interactions. In addition, a short intermolecular S⋯N contact occurs. 24H18N2O4S, crystallizes in the orthorhombic P212121 space group, indicating the existence of only one enantiomer with an S configuration of the chiral center in the crystal phase. The dihydropyrimidine ring adopts a twist-boat conformation while the quinone ring is slightly non-planar. In the crystal, molecules are linked by weak C—H⋯O and C—H⋯S hydrogen bonds and C—H⋯π interactions. In addition, a short intermolecular S⋯N contact of 3.250 (3) Å indicates an interaction between the S atom and the π-system of the thiazole ring.The title compound, C It was expected that the reaction would proceed with substitution of one or both chlorine atoms and the formation of a thiazole ring in the product 3  compounds that can be modified easily depending on the starting materials used during the reaction  and 0.458 (2) Å, respectively. Such a conformation is common for 1,6-dihydroaromatic heterocycles  and −160.5 (3)°, respectively].The dihydropyrimidine ring adopts a twist-boat conformation with puckering parameters  contact of 3.250 (3) Å  indicates an interaction between the S atom and the π-system of the thiazole ring  was added to a solution of 2,3-dichloronaphthalene-1,4-dione  in DMF (20 mL) and the mixture was kept under reflux for 3 h. After that, the reaction mixture was cooled, and the precipitated solid product was filtered off and purified via recrystallization from MeOH:DMF:H2O (2:2:1) to give product 3 in the form of dark-red crystals in 78% yield (0.35 g), m.p. 520.3– 522.0 K.Ethyl 6-methyl-4-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate Uiso(H) = nUeq(C) .Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989018000117/zq2240sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989018000117/zq2240Isup2.hklStructure factors: contains datablock(s) I. DOI: 1814348CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


EXE) is an endocrine therapy commonly used by postmenopausal women with hormone‐responsive breast cancer due to its potency in inhibiting aromatase‐catalyzed estrogen synthesis. Preliminary in vitro studies sought to identify phase I EXE metabolites and hepatic cytochrome P450s (CYP450s) that participate in EXE biotransformation. Phase I metabolites were identified by incubating EXE with HEK293‐overexpressed CYP450s. CYP450s 1A2, 2C8, 2C9, 2C19, 2D6, 3A4, and 3A5 produce 17β‐dihydroexemestane (17β‐DHE), an active major metabolite, as well as two inactive metabolites. 17β‐DHE formation in pooled human liver microsomes subjected to isoform‐specific CYP450 inhibition was also monitored using tandem mass spectrometry. 17β‐DHE production in human liver microsomes was unaffected by isoform‐specific inhibition of CYP450s 2A6, 2B6, and 2E1 but decreased 12–39% following inhibition of drug‐metabolizing enzymes from CYP450 subfamilies 1A, 2C, 2D, and 3A. These results suggest that redundancy exists in the EXE metabolic pathway with multiple hepatic CYP450s catalyzing 17β‐DHE formation in vitro. To further expand the knowledge of phase I EXE metabolism, the impact of CYP450 genetic variation on 17β‐DHE formation was assessed via enzyme kinetic parameters. Affinity for EXE substrate and enzyme catalytic velocity were calculated for hepatic wild‐type CYP450s and their common nonsynonymous variants by monitoring the reduction of EXE to 17β‐DHE. Several functional polymorphisms in xenobiotic‐metabolizing CYP450s 1A2, 2C8, 2C9, and 2D6 resulted in deviant enzymatic activity relative to wild‐type enzyme. Thus, it is possible that functional polymorphisms in EXE‐metabolizing CYP450s contribute to inter‐individual variability in patient outcomes by mediating overall exposure to the drug and its active metabolite, 17β‐DHE.Exemestane ( Overall, glucuronidation (Vmax/KM) of 17β‐DHE in the HLM panel was 36‐fold lower in UGT2B17‐null homozygotes (*2/*2) compared to wild‐type homozygotes (*1/*1) , such as the CYP450s, can lead to genotype‐dependent variability in clinical responses for certain drugs , Tokyo Chemical Industry Co.  or Sigma‐Aldrich . Thin‐layer chromatography plates and silica columns used to purify the synthesized steroids were purchased from Bonna‐Agela Technologies Inc.  and Yamazen Corp. , respectively. Variant CYP450 overexpression vectors were made using a QuikChange II Site‐Directed Mutagenesis Kit from Agilent , as well as oligonucleotides manufactured by Integrated DNA Technologies . Negative control baculosomes, SuperScript II First‐Strand Synthesis System for RT‐PCR, cell culture medium, fetal bovine serum, penicillin/streptomycin, and G418 were acquired from Invitrogen . Choice‐Taq polymerase was purchased from Denville Scientific . LC/MS grade organic solvents, tris base, glycine, tetramethylethylenediamine (TEMED), ammonium persulfate (APS), sodium dodecyl sulfate (SDS), SuperSignal West Femto Maximum Sensitivity Substrate, Lipofectamine 2000, oligo(dT) primer, Pierce BCA protein assay kit, PVDF membranes, and monoclonal HRP‐conjugated V5 epitope antibody  were procured from Thermo Fisher Scientific . Nonfat dry milk used as a blocking agent for Western blotting was prepared by BioRad . The nicotinamide adenine dinucleotide phosphate (NADPH) regeneration system included in kinetic assays was purchased from Corning . Ampicillin, dithiothreitol (DTT), Tween 20, and acrylamide/bis‐acrylamide solution were acquired from Sigma‐Aldrich . Pooled mixed gender liver microsomes from 50 human donors were obtained from XenoTech . The ethnic composition of the pooled hepatic microsomes was 84% Caucasian, 8% Hispanic, 6% African American, and 2% Asian. (‐)‐N‐3‐benzylphenobarbital (NBP) is a product of Cypex Ltd , whereas all other compounds used for CYP450 isoform‐specific inhibition were obtained from Sigma‐Aldrich.EXE and its phase I metabolites were synthesized at Washington State University using previously published protocols  Variation Viewer . Search 20 was used to prime reverse transcription of pooled hepatic total RNA from five human donors (Penn State Cancer Institute Biorepository). The resultant first‐strand cDNA was further amplified using CYP450 isoform‐specific primers and Taq polymerase. cDNA encoding wild‐type CYP450s 1A2, 2C8, 2C9, 2C19, 2D6, 3A4, or 3A5 was introduced into the pcDNA3.1/V5‐His‐TOPO expression vector for stable overexpression in mammalian cells. CYP2D6*2 and CYP2C19*1B cDNA were also amplified from pooled human liver RNA. Additional constitutive overexpression vectors encoding nonsynonymous variants with MAF > 0.01 were produced for CYP450s 1A2, 2C8, 2C9, 2C19, and 3A4 via site‐directed mutagenesis (SDM) using wild‐type plasmid as template. Two common CYP2D6 haplotypes were likewise derived from the CYP2D6*2 overexpression vector through SDM. Oligonucleotide pairs used to amplify wild‐type CYP450 cDNA or prime mutagenesis are detailed in Data Oligo(dT)μg/mL G418 selective pressure in DMEM supplemented with 4.5 g/L glucose, l‐glutamine, 110 mg/L sodium pyruvate, 10% FBS, and penicillin/streptomycin. Data Each expression vector was transformed into chemically competent BL21. Transformants were then grown overnight on ampicillin selection plates at 37°C. Sanger sequencing was used for sequence confirmation prior to transfecting HEK293 with overexpression plasmids using Lipofectamine 2000 per the manufacturer instructions. Transfected HEK293 were grown for at least 3 weeks under 700 μL reaction contained 2.5 mmol/L EXE substrate, 3 μL NADPH regeneration system, and 20 μg of microsomal protein from a wild‐type CYP450‐overexpressing HEK293 cell line in phosphate‐buffered saline (PBS), pH 7.4. Similar incubations were conducted to assess background activity of endogenous metabolizing enzymes in non‐transfected HEK293, as well as commercially available negative control baculosomes. These incubations included 100 μM EXE, 50 μg of microsomes, or 50 μg of baculosomes in addition to PBS, pH7.4, and an NADPH regeneration system. Enzymatic incubations were terminated with 50 μL of ice‐cold acetonitrile before centrifugation at 13,200g for 15 min at 4°C. The resulting supernatant was analyzed by ultra‐pressure liquid chromatography paired with tandem mass spectrometry (UPLC/MS/MS) on a Waters ACQUITY platform configured with a 0.2 μm inline filter preceding a 1.7 μm ACQUITY UPLC BEH C18 column . The UPLC gradient used to separate EXE and its phase I metabolites has been described previously , 17α‐DHE (m/z 299.14 → 135.07), 17β‐DHE (m/z 299.20 → 135.13), and 6‐HME (m/z 312.89 → 158.98). 0.01 sec dwell time was optimal for all four compounds, whereas 25 V of collision energy was used for EXE and 6‐HME. 20 V of collision energy was used for 17α‐ and 17β‐DHE. An additional non‐targeted screening for phase I EXE metabolites was performed using UPLC/MS to detect positively charged molecular ions with m/z ranging from 100 to 450. The identity of all metabolites observed was verified by comparing the observed retention times and m/z with those of purified standards.CYP450‐derived phase I EXE metabolites were identified in 45‐min incubations performed at 37°C. Each 50‐β‐DHE production was measured in 45‐min incubations at 37°C in PBS, pH 7.4 with varying concentrations of EXE (25–2500 μmol/L). Each reaction included 20 μg of microsomal protein from CYP450‐overexpressing HEK293, as well as an NADPH regeneration system. The chosen incubation length and protein concentration fell within the linear range of EXE reduction velocity curves for the seven wild‐type CYP450s assayed (data not shown). Cold acetonitrile (50 μl) was used to terminate each reaction. After centrifugation for 15 min at 4°C at 13,200g, 17β‐DHE formation was monitored in the supernatant according to a previously established UPLC/MS/MS method and quantitated against a standard curve constructed of known 17β‐DHE concentrations . Furafylline (1 μmol/L) was used to inhibit CYP1A2, whereas 0.5 μmol/L tranylcypromine (TCP) and 10 μmol/L thioTEPA inhibited CYPs 2A6 and 2B6, respectively. Other compounds used to systematically inhibit hepatic CYP450s included 0.5 μmol/L montelukast (CYP2C8 inhibitor), 1 μmol/L sulfaphenazole (CYP2C9 inhibitor), 0.5 μmol/L NBP (CYP2C19), 1 μmol/L quinidine (CYP2D6 inhibitor), 5 μmol/L clomethiazole (CYP2E1 inhibitor), and 1 μmol/L ketoconazole (CYP3A inhibitor). Initial dosages were selected from the literature and then further titrated to determine the lowest dose producing maximum isoform‐specific inhibition  and analyzed by UPLC/MS/MS using the aforementioned method. A negative control reaction was run in parallel and received ethanol vehicle rather than inhibitor. Organic solvent constituted less than 1% of each incubation.Reduction of EXE to 17KM and relative Vmax values were calculated in GraphPad Prism 6 according to the Michaelis–Menten equation . Vmax is expressed as pmol min−1 mg−1 and was normalized to total protein content to account for differences in CYP450 expression between cell lines as determined by Western blot analysis. Two‐sided unpaired t‐tests were used to compare the wild‐type catalytic activity of CYP450s 1A2 and 3A4 to their respective variants. Wild‐type CYP450s 2C8, 2C9, 2C19, and 2D6 were compared to their nonsynonymous variants using one‐way ANOVA supplemented with Dunnett's multiple comparison test. In all instances, the threshold for statistical significance was set at a two‐tailed P < 0.05. The percent change in 17β‐DHE formation associated with each inhibitor in pooled HLM was likewise calculated in GraphPad Prism 6. An uninhibited reaction receiving vehicle was considered maximum catalytic activity for the purposes of comparison. All experimental results represent triplicate assays performed independently.β‐DHE. Low levels of 6‐HME and 17α‐DHE formation were observed for all CYPs studied. In all cases, formation of the 17α‐dihydroexemestane (17α‐DHE) and 6‐hydroxymethylandrosta‐1,4,6‐triene‐3,17‐dione (6‐HME) metabolites exceeded the minimum threshold of detection. No EXE metabolite formation was observed in incubations using microsomes from the parent HEK293 cell line  followed by CYP2C8 (KM = 0.66 ± 0.10 mmol/L), CYP3A5 (KM = 0.69 ± 0.18 mmol/L), CYP1A2 (KM = 0.74 ± 0.5 mmol/L), and CYP3A4 (KM = 0.83 ± 0.16 mmol/L) . CYP1A2 catalyzed 17β‐DHE production at a rate of 61 ± 17 pmol min−1 mg −1 followed by CYP2C19 (51 ± 8 pmol min−1 mg −1) and CYP2D6 (49 ± 3 pmol min−1 mg −1). Catalytic velocities for CYP450s 3A4 and 3A5 were not significantly different (39 ± 7 and 36 ± 4 pmol min−1 mg −1). Intrinsic clearance (Vmax/KM) was calculated for each wild‐type enzyme as an indication of its overall catalytic activity against EXE. CYP2C8 exhibited the highest intrinsic clearance value (194 nL min−1 mg−1) followed by CYP2D6 (86 nL min−1 mg−1), CYP1A2 (82 nL min−1 mg−1), CYP2C19 (55 nL min−1 mg−1), CYP3A5 (52 nL min−1 mg−1), and CYP3A4 (47 nL min−1 mg−1). In addition to having the slowest rate of EXE reduction, CYP2C9 also exhibited the lowest overall catalytic activity against EXE substrate (27 nL min−1 mg−1).Representative kinetic curves of 17In all, 23 nonsynonymous polymorphisms with MAF > 0.01 were detected using the NCBI Variation Viewer to search for common variants in hepatic CYP450s involved in xenobiotic metabolism. Four polymorphisms of interest were identified for CYP2C9. Two variants were gleaned for CYP2C19, whereas a single variant was reported for both CYP450s 1A2 and 3A4. No variants matching the search criteria were listed for CYP3A5. Variation Viewer search filters returned four common single nucleotide polymorphisms (SNPs) in CYP2C8. In the present study, two of the CYP2C8 polymorphisms were investigated as a single haplotype (CYP2C8*3). In all, 11 nonsynonymous polymorphisms occur in CYP2D6 at > 1%. Although CYP2D6 is notoriously polymorphic, only a handful of common haplotypes are associated with clinically relevant alterations in drug metabolism Zhou . For thiVmax was comparable between wild‐type CYP1A2 and CYP1A2Ser298Arg, the variant enzyme exhibited significantly increased affinity for EXE with KM values of 0.74 ± 0.5 and 0.59 ± 0.2 mmol/L, respectively. No notable differences were observed in affinity between wild‐type CYP450s 2C8, 2C9, 2C19, or 3A4 and their respective variants (Table KM = 0.95 ±0.10 mmol/L) and CYP2D6*10 (KM = 1.04 ± 0.01 mmol/L) were both associated with decreased affinity relative to wild‐type CYP2D6 (KM = 0.57 ± 0.03 mmol/L). The CYP2C8*2 and CYP2C8*4 polymorphisms were associated with 2.2 and 1.7‐fold increases in velocity of EXE reduction compared to wild‐type. CYP2C9*8 and CYP2C9*9 variants were likewise associated with increased rates of 17β‐DHE formation (Vmax = 73 ± 13 and 116 ± 13 versus 26 ± 0.6 pmol min−1 mg−1 for wild‐type). The Vmax for EXE reduction by CYP2D6*2 (23 ± 0.6 pmol min−1 mg −1) is 53% lower than that of wild‐type CYP2D6 (49 ± 3 pmol min−1 mg −1). Vmax values were comparable between CYP2C19 and its variants CYP2C19Ile331Val and CYP2C19Glu92Asp. No significant difference in Vmax was observed between wild‐type CYP3A4 and CYP3A4Arg162Gln .Although β‐DHE production . Similar results were obtained when either thioTEPA or clomethiazole were included to chemically inhibit CYP2B6 (112.5 ± 8.3% control activity) or CYP2E1 (97.3 ± 5.3% control activity), respectively. Inhibition of CYP2D6 decreased EXE reduction by 27%, whereas furafylline‐induced CYP1A2 inhibition reduced formation of the active 17β‐DHE metabolite by 19%.In incubations of EXE with pooled mixed gender HLMs, inhibition of CYP3A isoforms with ketoconazole resulted in a 39% decrease in 17ion Fig. . Isoformβ‐DHE represents a major metabolic pathway for this commonly prescribed aromatase inhibitor . CYPs 3A4 and 3A5 exhibited intermediate intrinsic clearance values in the present study , but the literature indicates that CYP3A isoforms are well expressed in the liver comprising 30% of total CYP450 content and accounting for nearly 55% of xenobiotic metabolism (Chang and Kam −1 mg−1) was second only to that of CYP2C8 (194 nL min−1 mg−1) in kinetic assays. CYP2D6 is estimated to account for 30% of drug metabolism despite constituting only 2% of total hepatic CYP450 content (Chang and Kam INT = 82 nL min−1 mg−1), accounts for only 2% of overall CYP450‐mediated xenobiotic metabolism in human liver  in the presence of the isoform‐specific inhibitor ketoconazole suggests that CYP3As are the major hepatic CYP450s responsible for EXE reduction in human liver microsomes  in the present study. CYP1A1 expression, in turn, is low in human liver diminishing the likelihood that it is a key participant in phase I EXE metabolism . Despite its high incidence in African populations (MAF = 0.0893), a three‐dimensional structure examining conformational changes in CYP1A2 arising from arginine substitution is currently unavailable , CYP2C8Arg139Lys,Lys399Arg (*3), and CYP2C8Ile264Met (*4) are comparable to wild‐type CYP2C8 in affinity for EXE substrate in vitro. Kaspera et al.  and 6‐hydroxyl‐ (M‐23) cerivastatin metabolites. Although E. coli‐expressed CYP2C8*2 yielded individual Vmax values similar to wild‐type CYP2C8 for M‐1 and M‐23 formation, a 53% increase in the sum of cerivastatin metabolite clearance was noted . Kaspera et al.  and CYP2C9Ile359Leu (*3) were similar to those of wild‐type CYP2C9. The CYP2C9*2 and *3 polymorphisms are relatively common (MAF > 0.01) in South Asian, European, and Hispanic populations (see Data Arg150His (*8) and CYP2C9His251Arg (*9) polymorphic protein reduced EXE to 17β‐DHE 2.8‐ and 4.5‐fold faster than wild‐type CYP2C9, respectively. The CYP2C9*8 allele also increased clearance of the antidiabetic tolbutamide in vitro  relative to wild‐type CYP2D6 due to significant decreases in affinity and 17β‐DHE formation rate. Sakuyama et al.  increased the KM value for EXE substrate by 82% causing a 56% decrease in clearance compared to CYP2D6*1. CYP2D6*10 is highly prevalent in Asians and has previously been associated with decreased catalytic activity in vitro  is common in individuals of African heritage and is likewise considered a reduced function allele  and production of the major active metabolite 17β‐DHE (Vmax) indicate that EXE metabolism by common allelic variants of CYP2C19 and CYP3A4 is comparable to that of their respective wild‐type CYP450s. The neutral effect of the CYP2C19Ile331Val (*1B) polymorphism on EXE reduction confirms prior observations of equivalent catalytic activity for the CYP2C19*1A and *1B alleles  did not directly impact 17β‐DHE formation, it cosegregates with approximately 20% of CYP2C19*2 poor metabolizing alleles in Caucasians  polymorphism does not appreciably impact EXE metabolism. However, kinetic studies of the variant enzyme with additional substrates are needed to confirm its overall effect on CYP3A4 activity.Kinetic parameters measuring substrate affinity (α‐DHE, as well as the active metabolite 17β‐DHE. Earlier studies suggested that 17β‐DHE may partially determine overall drug exposure by acting as an androgen agonist, contributing to estrogen blockade through aromatase inhibition, and by serving as a gateway to phase II conjugation and excretion (Ariazi et al. β‐DHE formation or clearance may contribute to inter‐individual variation in the overall therapeutic efficacy of EXE by altering a major metabolic pathway. This possibility is bolstered by the observation that three of the variant CYP450s included in this study had altered affinity for EXE substrate leading to differential 17β‐DHE production while five variants had deviant catalytic rates of EXE reduction. To our knowledge, this is the first study to report the impact of common nonsynonymous polymorphisms in CYP450s on EXE reduction to its 17β‐DHE metabolite. A previous in vitro study demonstrated the capacity of genetic variation to alter EXE metabolism by the cytosolic ketosteroid reductases CBR1 and AKR1C1‐4 (Platt et al. The present in vitro study augments existing pharmaceutical knowledge by examining the role of hepatic CYP450s in the metabolism of EXE, a widely used endocrine therapy for hormone‐responsive breast cancer. Qualitative enzymatic incubations with EXE confirm that multiple hepatic monoxygenases from CYP450 families 1, 2, and 3 catalyze the production of 6‐HME, 17There are no conflicts of interest.Data S1. Digital Content 1.doc.Click here for additional data file.Data S2. Digital Content 2.doc.Click here for additional data file.Data S3. Digital Content 3.doc.Click here for additional data file.Data S4. Digital Content 4.doc.Click here for additional data file.


The title crystal is a co-crystal with the 1,8-naphthyridine derivative crystallizing with one molecule of succinimide per formula unit. In the crystal, the two molecules are mutually linked by N—H⋯O and N—H⋯N hydrogen bonds. The packing is consolidated by C—H⋯ hydrogen bonds and π–π stacking interactions. 17H13Br2N3O·C4H5NO2, is a co-crystal of N-benzamide and pyrrolidine-2,5-dione (succinimide). The benzamide molecule exhibits pseudo-mirror symmetry, with an r.m.s. deviation of the non-H atoms of 0.09 Å (except for the two Br atoms). The angle between the least-squares planes of the two molecules is 26.2 (2)°. In the crystal, the two molecules are mutually linked by N—H⋯O and N—H⋯N hydrogen bonds. The packing is consolidated by C—H⋯ hydrogen bonds and π–π stacking interactions.The title compound, C The naphthyridine ring system makes a dihedral angle of 2.2 (2)° with the benzene ring and is oriented at an angle of 26.2 (2)° relative to the succinimide. The conformation of the C=O and the N—H bonds of the amide segment are anti to one another, similar to that reported for benzamide moiety in N-{4-[(6-chloropyridin-3-yl)-methoxy]phenyl}-2,6-difluorobenzamide  Å , forming a three-dimensional supramolecular network N—H⋯N bond bifurcated Table 1. In the rk Fig. 3.et al., 2016et al., 2013In the Cambridge Structural Database benzamide  were added to an dry acetonitrile (30 ml) solution under nitrogen atmosphere. The mixture was refluxed at room temperature in the presence of light with a 250 W infrared lamp for 4 h. Excess solvent was removed and the crude product was purified by column chromatography using dichloromethane/methanol (120:1) as the mobile phase to give a light-yellow powder . Crystals suitable for X-ray analysis were obtained by slow diffusion of a dichloromethane solution at ambient temperature. Several cycles of purification by chromatography were used to reduce the amount of succinimide.Uiso(H) = 1.5Ueq(C) for methyl H atoms and Uiso(H) = 1.2Ueq(C) for all other H atoms, and with N—H = 0.86 Å, Uiso(H) = 1.2Ueq(N).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016019034/wm5334sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989016019034/wm5334Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016019034/wm5334Isup3.cmlSupporting information file. DOI: 1519551CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


II atoms within N2S2 donor sets because of the presence of chelating thiosemicarbazone anions. Supramolecular chains are found in the crystal owing to the formation of thioamide-N—H⋯S(thione) hydrogen bonds and eight-membered thioamide {⋯HNCS}2 synthons.The title thiosemicarbazone compound features tetrahedrally coordinated Zn II complex, [Zn(C19H20N3OS)2] {systematic name: bis[(N-ethyl-N′-{(Z)-[(2E)-3-(4-methoxyphenyl)-1-phenylprop-2-en-1-ylidene]amino}carbamimidoyl)sulfanido]zinc(II)}, features a tetrahedrally coordinated ZnII ion within an N2S2 donor set provided by two N,S-chelating thiosemicarbazone anions. The resulting five-membered Zn,C,N2,S chelate rings adopt different conformations, i.e. almost planar and an envelope with the Zn atom being the flap atom. The configuration about the imine bond within the chelate ring is Z but those about the exocyclic imine and ethylene bonds are E. In the crystal, supramolecular [100] chains mediated by thioamide-N—H⋯S(thione) hydrogen bonds and eight-membered thioamide {⋯HNCS}2 synthons are observed. A range of interactions, including C—H⋯O, C—H⋯π, C—H⋯π(chelate ring) and π(methoxybenzene)—π(chelate ring) consolidate the packing. The Hirshfeld surface analysis performed on the title complex also indicates the influence of the interactions involving the chelate rings upon the packing along with the more conventional contacts.The title Zn R1R2C=N—N(R3)—C(=S)NR4R5 for R1–5 = H/alkyl/aryl, are numerous and multi-functional. Their preparation is often facile, being formed from the condensation reaction between an aldehyde (or a ketone) with the amine group of a thiosemicarbazide precursor. In the same way, the diversity in ligand construction ensures a rich coordination chemistry ° for the S1—Zn—N3 chelate angle to 127.92 (4)° for S1—Zn—S2. The assignment of four-coordinate geometries can be quantified by comparing the calculated value of τ4, in this case 0.74, with the ideal values for an ideal tetrahedron, i.e. 1.00, and perfect square-planar geometry, i.e. 0.00 2 chelate rings, and these adopt different conformations. Whereas, the  ring is almost planar (r.m.s. deviation = 0.0325 Å), the  chelate ring is best described as an envelope with the Zn atom lying 0.205 (5) Å out of the plane of the remaining four atoms (r.m.s. deviation = 0.0011 Å). The dihedral angle between the mean planes through the chelate rings is 79.68 (8)°. To a first approximation, the thiosemicarbazone ligands comprise two planar regions. Thus, the non-hydrogen, non-phenyl atoms of the atoms of the S1-ligand define one plane (r.m.s. deviation = 0.1910 Å), which forms a dihedral angle of 54.53 (8)° with the (C14–C19) ring, consistent with a near perpendicular relationship. The comparable values for the S2-ligand are 0.2800 Å and 75.09 (11)°, respectively.The mode of the coordination of the thiosemicarbazone ligands leads to the formation of five-membered ZnSCNc-axis direction sustained by eight-membered thioamide {⋯HNCS}2 synthons, Fig. 2a and Table 2b, it is evident that two rows of molecules, each with a right-angle topology, are connected by N—H⋯S(thione) hydrogen bonds. Centrosymmetrically related right angles are connected into a supramolecular tube, Fig. 2c, via imine-phenyl-C—H⋯O(methoxy), imine-phenyl-C—H⋯π(imine-phenyl) and imine-phenyl-C—H⋯π(methoxybenzene) interactions, Table 2et al., 2014et al., 2016v with a ring centroid–ring centroid separation of 3.778 (2) Å and angle of inclination = 15.04 (17)° for symmetry operation (v): 2 − x, 1 − y, 1 − z. A review has appeared very recently on the topic of π(chelate ring)–π(arene) and π(chelate ring)–π(chelate ring) interactions where it was suggested that interactions of the former type provide comparable energies of stabilization to molecular packing as do weak conventional hydrogen bonds  ring and between ethylene-C24—H and the  ring, Table 2a. The donors and acceptors of the intermolecular N—H⋯S and C—H⋯O interactions are also viewed as blue and red regions about respective atoms in the images of Fig. 4a and b, respectively.The Hirshfeld surfaces calculated for (I)a, and those delineated into H⋯H, C⋯H/H⋯C, S⋯H/H⋯S and O⋯H/H⋯O contacts de + di ∼ 2.1 Å in Fig. 6a is due to a short interatomic H⋯H contact  are also reflected from the small but important contribution from C⋯N/N⋯C and C⋯S/S⋯C contacts, Table 4The overall two dimensional fingerprint plot for (I)t Table 3 and the N′-{(E)--amino}-N-ethylcarbamimidothioato-κ2N′,S)zinc(II) molecule, which differs from (I)et al., 20172S2 coordination geometries are found with values of τ4 of 0.70 and 0.74 for the two independent molecules comprising the asymmetric unit. Indeed, in the publication describing this structure =NN=CR′R′′]2 and all structures adopt the same basic structural motif as described herein for (I)The most relevant structure available for comparison is that of the recently described bis was dissolved in hot absolute ethanol (30 ml), which was added to a solution of Zn(CH3COO)2·2H2O  in hot absolute ethanol (40 ml). The mixture was heated and stirred for about 10 min, followed by stirring for 1 h at room temperature. The yellow precipitate obtained was filtered, washed with cold ethanol and dried in vacuo. Single crystals were grown at room temperature from the slow evaporation of the title compound in a mixed solvent system containing dimethylformamide and acetonitrile . IR (cm−1): 3351 ν(N—H), 1597 ν(C=N), 1009 ν(N—N), 420 ν(M—N), 362 ν(M—S).Analytical grade reagents were used as procured and without further purification. 4-Ethyl-3-thiosemicarbazide  and 4-methoxychalcone  were dissolved separately in hot absolute ethanol (30 ml) and mixed while stirring. About five drops of concentrated hydrochloric acid were added to the mixture to catalyse the reaction. The reaction mixture was heated and stirred for about 20 min, and stirring was continued for another 30 min at room temperature. The resulting yellow precipitate, 4-methoxychalcone-4-ethyl-3-thiosemicarbazone, was filtered off, washed with cold absolute ethanol and dried Uiso(H) set to 1.2–1.5Ueq(C). The nitrogen-bound H atoms were located in a difference-Fourier map but were refined with a distance restraint of N—H = 0.88±0.01 Å, and with Uiso(H) set to 1.2Ueq(N). The maximum and minimum residual electron density peaks of 1.10 and 0.59 e Å−3, respectively, are located 1.04 and 0.71 Å from the Zn atom.Crystal data, data collection and structure refinement details are summarized in Table 510.1107/S2056989018000282/hb7725sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989018000282/hb7725Isup2.hklStructure factors: contains datablock(s) I. DOI: 1814817CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


II ion is coordinated by two pyridine N atoms from two symmetry-related unsymmetrical dipyridyl ligands and two chloride anions in a highly distorted tetrahedral geometry. Each unsymmetrical dipyridyl ligand links two HgII ions into polymeric zigzag chains. In the crystal, the chains are linked into a three-dimensional supramolecular network by intermolecular N/C—H⋯Cl hydrogen bonds and weak C—H⋯π interactions. Weak C—Cl⋯π interactions and Cl⋯Cl contacts between the network and the solvent chloroform molecules are also observed.In the title compound, each Hg LCl2]·0.5CHCl3}n (L = N-(pyridin-4-ylmethyl)pyridin-3-amine, C11H11N3), contains one HgII ion, one bridging L ligand, two chloride ligands and a chloroform solvent molecule with half-occupancy that is disordered about a crystallographic twofold rotation axis. Each HgII ion is coordinated by two pyridine N atoms from two symmetry-related L ligands and two chloride anions in a highly distorted tetrahedral geometry with bond angles falling in the range 99.05 (17)–142.96 (7)°. Each L ligand bridges two HgII ions, forming polymeric zigzag chains propagating in [010]. In the crystal, the chains are linked by intermolecular N/C—H⋯Cl hydrogen bonds together with weak C—H⋯π interactions, resulting in the formation of a three-dimensional supramolecular network, which is further stabilized by C—Cl⋯π interactions between the solvent chloroform molecules and the pyridine rings of L [chloride-to-centroid distances = 3.442 (11) and 3.626 (13) Å]. In addition, weak Cl⋯Cl contacts [3.320 (5) Å] between the chloroform solvent molecules and the coordinating chloride anions are also observed.The asymmetric unit of the title compound, {[Hg The solvent molecule is disordered over two orientations of equal occupancy about the crystallographic twofold rotation axis. As shown in Fig. 1II ion is highly distorted tetrahedral with two coordination sites being occupied by two pyridine N atoms from two symmetry-related L ligands. The geometry of the HgII ion is completed by the coordination of two chloride ions. The tetrahedral angles around the HgII ion fall in the range of 99.05 (17)–142.96 (7)°  Å. In the L ligand, the Cpy—N—C—Cpy torsion angle is −70.9 (7)° while the dihedral angle between two terminal pyridine ring planes is 85.0 (2)°. The conformation of the L ligand, along with the the Npy—Hg—Npy coordination angle [99.05 (17)°], may induce the zigzag topology of the chain.Each is Fig. 2. The sepbc plane  Å, C12—Cl4⋯Cg2 = 170.7 (8)°, Cl5⋯Cg2iv = 3.626 (13) Å and C12—Cl5⋯Cg2iv 144.1 (8)°  are observed.In the crystal, adjacent zigzag chains are linked by intermolecular N—H⋯Cl hydrogen bonds and weak intermolecular C—H⋯π interactions Table 2, forminge Figs. 2 and 3 ▸.L ligand was synthesized according to a literature method  = 0.93 Å for Csp2—H, 0.97 Å for methylene C—H, 0.98 Å for methine C—H, and 0.86 Å for amine N—H, and were refined as riding with Uiso(H) = 1.2Ueq.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989016015310/sj5509sup1.cifCrystal structure: contains datablock(s) I, New_Global_Publ_Block. DOI: 10.1107/S2056989016015310/sj5509Isup2.hklStructure factors: contains datablock(s) I. DOI: 1507232CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


These findings provide additional understanding of the NBC series and will aid in the development of these NLRP3 inhibitors as tool compounds or therapeutic candidates for sterile inflammatory diseases.The NLRP3 inflammasome is an important regulator of the sterile inflammatory response, and its activation by host‐derived sterile molecules leads to the intracellular activation of caspase‐1, processing of the pro‐inflammatory cytokines interleukin‐1β (IL‐1β)/IL‐18, and pyroptotic cell death. Inappropriate activation of NLRP3 drives a chronic inflammatory response and is implicated in several non‐communicable diseases, including gout, atherosclerosis, type II diabetes and Alzheimer's disease. In this study, we report the design, synthesis and biological evaluation of novel boron compounds (NBCs) as NLRP3 inflammasome inhibitors. Structure–activity relationships (SAR) show that 4‐fluoro substituents on the phenyl rings retain NLRP3 inhibitory activity, whereas more steric and lipophilic substituents diminish activity. Loss of inhibitory activity is also observed if the CCl Sterile inflammation is a host‐driven immune response to injury in the absence of infection.NLRP3 gene causes spontaneous IL‐1β release in patients with cryopyrin‐associated periodic syndrome (CAPS) diseases that are characterised by fever, rashes and extensive joint pain.IL‐1β and NLRP3 activation are well characterised in a number of non‐communicable diseases involving sterile inflammation including gout,Given the critical role of NLRP3 and IL‐1β in human disease,A number of small molecule inhibitors of the NLRP3 inflammasome have been previously described.1), BC23 (2) and NBC6  group. However, the impact of phenyl ring substitutions on IL‐1β release was not assessed. Additionally, the presence of the CCl3 group significantly contributes to the high lipophilicity of these oxazaborine inhibitors, limiting their drug‐likeness.We recently reported on the discovery of new boron‐based small molecules as potent NLRP3 inhibitors.2) and NBC6 (3), in addition to seeking alternative bioisosteres of the CCl3 group in the search for new NLRP3 inhibitors with improved potency and drug‐like properties.Here we explored structure–activity relationships (SAR) of phenyl ring substitutions based on the known NLRP3 inhibitors BC23  with magnesium turnings or isopropylmagnesium chloride (iPrMgCl), followed by treatment with one molar equivalent of trimethyl borate (B(OMe)3) to afford symmetrical borinic acids (5).4 (when X=MgBr), such as p‐tolylmagnesium bromide, with a stoichiometric quantity of phenylboronic acid pinacol ester (7) to give the asymmetric borinic acid (6).Using the Topliss scheme for aromatic substituents,5 and 6 were then reacted with either (Z)‐2‐acetyl‐3‐amino‐4,4,4‐trichlorobut‐2‐enamide or (Z)‐4‐amino‐5,5,5‐trichloropent‐3‐en‐2‐oneThe synthesised borinic acids m,AN0128, a known borinic acid picolinate ester prepared by Anacor Pharmaceuticals, was also synthesised in a two‐step method according to the reported procedure  and NBC6 (3) were then designed where the CCl3 group was replaced with the bioisosteric trifluoromethyl (CF3) group  to give analogues NBC41 and NBC42  to prime the cells and induce expression of pro‐IL‐1β. The primed cells were then incubated with vehicle (0.5 % DMSO) or inhibitor (10 μm) for 15 minutes before the NLRP3 inflammasome activator nigericin  was added to cells. Supernatants were removed and IL‐1β levels were analysed by ELISA. Nigericin induced release of IL‐1β and this was inhibited by our parent molecule BC23 as expected  had minimal effect on inhibitory activity  and 4‐CH3 (NBC38) derivatives all inhibited IL‐1β release to a similar extent. This observation is further supported by the significantly decreased activities of 3,4‐Cl2 (NBC39) and 3‐Cl,4‐Me (NBC40) derivatives which possess additional substitutions around the phenyl rings. In contrast, there was an observed correlation between increasing lipophilicity and decreasing inhibitory activity across the BC23 series (NBC35 < NBC36 ≈NBC37 ≈NBC38 < NBC39). These observations were also in agreement across the NBC6 series, where the disubstituted‐p‐tolyl analogue NBC32 had slightly lower IL‐1β inhibitory activity than the monosubstituted‐p‐tolyl analogue NBC33 and parent compound NBC6 in THP‐1 cells . These results suggest that aryl ring substitution, particularly with more steric and lipophilic substituents, is unlikely to enhance the activity of the NBCs.We initially tested the effect of modifying the phenyl rings of NBCs on their ability to inhibit NLRP3 inflammasome‐dependent release of IL‐1β from macrophages. Immortalised bone marrow derived macrophages (iBMDMs) were treated with LPS (1 μg mLIt was surprising to note the lack of inhibitory activity of AN0128 in the IL‐1β release assay given its known anti‐inflammatory effects against IL‐1β and TNF‐α.3 group had been replaced with the bioisosteric trifluoromethyl (CF3) group  and NBC42  were ineffective under these conditions . Therefore these initial studies suggest that the CCl3 group is not labile under these reaction conditions. It was noted that during these experiments, nucleophiles were found to attack the boron atom and undergo decomplexation. For example, BC23 cleanly hydrolyses into diphenylborinic acid and (Z)‐4‐amino‐5,5,5‐trichloropent‐3‐en‐2‐one in 9:1 [D6]DMSO/D2O solvent at 37 °C with a half‐life of ≈24 h, as monitored by 1H NMR spectroscopy .Given that the CClThe NLRP3 inflammasome is a multi‐molecular protein complex that is critical for inflammatory responses. Its formation leads to activation of caspase‐1, which cleaves and activates IL‐1β.3 group.3 group is key to inhibitory activity. These discoveries provide new insight into the activity of the NBC series and will aid future development of the NBC molecules as inflammasome inhibitors.We recently published the NBC series of NLRP3 inflammasome inhibitors reporting that key features required for bioactivity were the oxazaborine ring and CClGeneral: (Z)‐4‐Amino‐5,5,5‐trichloropent‐3‐en‐2‐one and (Z)‐2‐acetyl‐3‐amino‐4,4,4‐trichlorobut‐2‐enamide intermediates were prepared as previously described.1H, 13C, 11B and 19F NMR spectra were recorded on a Bruker Avance 400 or 300 MHz spectrometer. Chemical shifts (δ) are defined in parts per million (ppm). 1H NMR spectra were referenced to tetramethylsilane  or residual undeuterated solvent . 13C NMR spectra were referenced to residual undeuterated solvent  as an internal reference. 11B NMR chemical shifts were referenced to external reference BF3⋅OEt2 (δ=0.0 ppm). 19F NMR chemical shifts were referenced using the deuterium lock signal of the solvent. ESI and APCI mass spectrometry was carried out on a Waters Acquity UPLC system connected to a Waters SQD2 mass spectrometer. Accurate mass determination was carried out on a Thermo Exactive Plus EMR Orbitrap LC–MS system. Molecular ion peaks are defined as mass/charge (m/z) ratios. Infrared spectroscopy was recorded on a JASCO FT/IR‐4100 spectrophotometer using the Spectra Manager II (JASCO) software package. Melting points were measuring using a Stuart SMP10 melting point apparatus. Lyophilisation was carried out using a Christ alpha1‐4 plus freeze dryer equipped with an Edwards vacuum pump. Microwave irradiation was carried out on a Biotage Initiator Classic microwave using 2–5 mL Biotage glass vials. Analytical thin‐layer chromatography (TLC) was performed using silica gel 60 on aluminium sheets coated with F254 indicator. All spots were visualised with KMnO4 or ultraviolet light using a MV Mineralight lamp (254/365) UVGL‐58. Flash column chromatography was performed using silica gel with particle size 40–63 μm. Evaporation of solvents was conducted on a Büchi Rotavapor R‐200.p‐tolylborinic acid (5 a)Di‐: To an oven‐dried Schlenk flask under N2 was added p‐tolylmagnesium bromide  in anhydrous THF (5 mL). B(OMe)3  was added dropwise to the reaction mixture and stirred at room temperature for 3 h. HCl  was then added and stirred for 30 min to quench the reaction. The reaction mixture was extracted with EtOAc (3×10 mL), washed with brine (1×10 mL), dried over MgSO4, filtered and evaporated in vacuo. The crude product was then purified by flash column chromatography  to give 5 a as a colourless oil . 1H NMR : δ=7.63 ), 7.18 ), 5.65 , 2.33 ppm ; 13C NMR : δ=141.2 (B‐Ar(p)), 134.8 (B‐Ar(o)), 128.7 (B‐Ar(m)), 21.7 ppm (CH3), B‐Ar(i) quaternary signal not observed.p‐tolyl)‐4‐(trichloromethyl)‐2,3‐dihydro‐1,3,2‐oxazaborinin‐1‐ium‐2‐uide (NBC32)5‐Acetyl‐6‐amino‐2,2‐bis(: (Z)‐2‐Acetyl‐3‐amino‐4,4,4‐trichlorobut‐2‐enamide  was added to a solution of 5 a  in anhydrous THF (5 mL). The mixture was stirred at 50 °C under N2 for 16 h. The mixture was concentrated in vacuo and purified by flash column chromatography . The collected fractions were combined, evaporated in vacuo and stirred in cold n‐hexane (15 mL) for 30 min. The precipitate was then filtered and dried under vacuum to give NBC32 as a yellow solid ; mp: 145–148 °C (dec); 1H NMR : δ=9.23 , 7.58 C=C), 7.29 ), 7.12 ), 5.92 , 2.34 , 2.32 ppm ; 13C NMR : δ=198.1 (CH3CO), 169.2 (CONH2), 165.5 (Cl3C(NH)C=C), 156.1 (B‐Ar(i)), 136.2 (B‐Ar(p)), 132.0 (B‐Ar(o)), 128.2 (B‐Ar(m)), 97.9 (Cl3C(NH)C=C), 94.5 (CCl3), 34.0 (CH3CO), 21.3 ppm (CH3); IR (neat): ν˜=3388 (N−H), 3313 (N−H), 1644 (C=O), 1608 , 1561 cm−1 (C=C‐NH); MS(ES−) (m/z): 434.2 −, 435.2 −, 436.2 −, 437.2 −, 438.2 −, 439.2 −; MS(ES+) (m/z): 344.1 +, 345.0 +, 346.0 +, 347.0 +, 349.1 +, 439.1 +; HRMS(ES+) (m/z): [M+H]+ calcd for C20H2111B35Cl3N2O2, 437.0756; found, 437.0738, error: 4.1 ppm.p‐tolyl)borinic acid (6 a)(Phenyl) in anhydrous THF (5 mL). Phenylboronic acid pinacol ester  in anhydrous THF (5 mL) was added dropwise to the reaction mixture and stirred at room temperature for 3 h. HCl  was then added and stirred for 30 min to quench the reaction. The reaction mixture was extracted with EtOAc (3×10 mL), washed with brine (1×10 mL), dried over MgSO4, filtered and evaporated in vacuo. The crude product was then purified by flash column chromatography  to give 6 a as a colourless oil . 1H NMR : δ=7.72 ), 7.64 ), 7.32–7.47 ), 7.19 ), 5.74 , 2.33 ppm ; 13C NMR : δ=141.4 (B‐Ar(p)), 135.0 (B‐Ph(o)), 134.6 (B‐Ar(o)), 130.9 (B‐Ph(p)), 128.8 (B‐Ar(m)), 127.9 (B‐Ph(m)), 21.7 ppm (CH3), B‐Ar(i) and B‐Ph(i) quaternary signals not observed; MS(ES−) (m/z): 195.1 −; HRMS(ES−) (m/z): [M−H]− calcd for C13H1211BO, 195.0987; found, 195.0973, error: 7.2 ppm.p‐tolyl)‐4‐(trichloromethyl)‐2,3‐dihydro‐1,3,2‐oxazaborinin‐1‐ium‐2‐uide (NBC33)5‐Acetyl‐6‐amino‐2‐(phenyl)‐2‐(: (Z)‐2‐Acetyl‐3‐amino‐4,4,4‐trichlorobut‐2‐enamide  was added to a solution of 6 a  in anhydrous THF (5 mL). The mixture was stirred at 50 °C under N2 for 16 h. The mixture was concentrated in vacuo and purified by flash column chromatography . The collected fractions were combined, evaporated in vacuo and stirred in cold n‐hexane (15 mL) for 30 min. The precipitate was then filtered and dried under vacuum to give NBC33 as a yellow solid ; mp: 141–143 °C; 1H NMR : δ=9.25 , 7.59 C=C), 7.40 ), 7.23–7.34  & B‐Ar(o)), 7.12 ), 5.94 , 2.34 , 2.31 ppm ; 13C NMR : δ=192.8 (CH3CO), 163.9 (Cl3C(NH)C=C), 160.3 (CONH2), 131.1 (B‐Ar(p)), 126.8 (B‐Ph(o)), 126.6 (B‐Ar(o)), 123.0 (B‐Ar(m)), 122.1 (B‐Ph(m)), 121.5 (B‐Ph(p)), 92.7 (Cl3C(NH)C=C), 89.2 (CCl3), 28.7 (CH3CO), 16.0 ppm (CH3), B‐Ar(i) and B‐Ph(i) quaternary signals not observed; MS(ES−) (m/z): 420.2 −, 421.1 −, 422.2 −, 423.2 −, 424.1 −, 425.1 −; HRMS(ES+) (m/z): [M+H]+ calcd for C19H1911B35Cl3N2O2, 423.0600; found, 423.0605, error: 1.2 ppm.General procedure for synthesis of 2,2‐bisaryl‐1,3,2‐oxazaborines: Using an adapted procedure,2 was added magnesium turnings (2.2 equiv), anhydrous THF (5 mL) and a small crystal of I2. The reaction was stirred at 40 °C for 30 min until complete decolourisation. A solution of aryl bromide (2 equiv) and B(OMe)3 (1 equiv) in anhydrous THF (5 mL) was then added dropwise to the reaction mixture and then stirred for an additional 3 h at 40 °C. After cooling to room temperature, 1 m HCl (10 mL) was added and stirred for 30 min to quench the reaction. The reaction mixture was extracted with EtOAc (3×10 mL), washed with brine (1×10 mL), dried over MgSO4, filtered and evaporated in vacuo to give the corresponding crude bisarylborinic acid, typically as a solid. (Z)‐4‐Amino‐5,5,5‐trichloropent‐3‐en‐2‐one (1.5 equiv) was then added to crude bisarylborinic acid (1 equiv) in anhydrous THF (5 mL). The reaction was stirred at 50 °C for 16 h under N2. The reaction mixture was concentrated and purified by flash column chromatography using the indicated solvent system. Collected fractions were evaporated in vacuo and stirred in the minimum amount of cold n‐hexane for 30 min to induce precipitation. The precipitate was then filtered and dried under vacuum to give the corresponding oxazaborine product. Percentage yields are reported over two steps.2,2‐Bis(4‐fluorophenyl)‐6‐methyl‐4‐(trichloromethyl)‐2,3‐dihydro‐1,3,2‐oxazaborinin‐1‐ium‐2‐uide (NBC35): EtOAc/n‐hexane, 1:9. Reaction scale: B(OMe)3 , 1‐bromo‐4‐fluorobenzene  and magnesium turnings  gives bis(4‐fluorophenyl)borinic acid . Bis(4‐fluorophenyl)borinic acid  and (Z)‐4‐amino‐5,5,5‐trichloropent‐3‐en‐2‐one  gives NBC35 as a yellow solid ; mp: 130–131 °C; 1H NMR : δ=7.31 ), 7.00 ), 5.83 C=CH), 2.25 ppm , NH signal is overlapping with triplet at 7.31 ppm; 13C NMR : δ=186.5 (CH3CO), 166.1 (Cl3C(NH)C=CH), 162.6 ), 133.4 ), 114.4 ), 92.8 (CCl3), 92.0 (Cl3C(NH)C=CH), 24.9 ppm (CH3CO), B‐Ar(i) quaternary signal not observed; 11B{1H} NMR : δ=4.11 ppm; 19F{1H} NMR : δ=−116.1 ppm; MS(ES−) (m/z): 399.1 −, 400.1 −, 401.1 −, 402.1 −, 403.1 −, 404.1 −; HRMS(ES−) (m/z): [M−H]− calcd for C17H1211B35Cl319F2NO, 400.0051; found, 400.0048, error: 0.7 ppm.2,2‐Bis(4‐chlorophenyl)‐6‐methyl‐4‐(trichloromethyl)‐2,3‐dihydro‐1,3,2‐oxazaborinin‐1‐ium‐2‐uide (NBC36): EtOAc/n‐hexane, 1:9. Reaction scale: B(OMe)3 , 1‐bromo‐4‐chlorobenzene  and magnesium turnings  gives bis(4‐chlorophenyl)borinic acid . Bis(4‐chlorophenyl)borinic acid  and (Z)‐4‐amino‐5,5,5‐trichloropent‐3‐en‐2‐one  gives NBC36 as a yellow solid ; mp: 106–107 °C; 1H NMR : δ=7.25 , 7.19 ), 5.76 C=CH), 2.18 ppm ; 13C NMR : δ=186.7 (CH3CO), 166.3 (Cl3C(NH)C=CH), 133.2 (B‐Ar(o)), 133.1 (B‐Ar(p)), 127.8 (B‐Ar(m)), 92.7 (CCl3), 92.2 (Cl3C(NH)C=CH), 24.8 ppm (CH3CO), B‐Ar(i) quaternary signal not observed; 11B{1H} NMR : δ=3.82 ppm; MS(ES−) (m/z): 431.0 −, 432.0 −, 433.0 −, 434.0 −, 435.0 −, 436.0 −, 437.0 −, 438.0 −; HRMS(ES−) (m/z): [M−H]− calcd for C17H1211B35Cl5NO, 431.9460; found, 431.9460, error: 0.0 ppm.6‐Methyl‐4‐(trichloromethyl)‐2,2‐bis(4‐(trifluoromethyl)phenyl)‐2,3‐dihydro‐1,3,2‐oxazaborinin‐1‐ium‐2‐uide (NBC37): EtOAc/n‐hexane, 1:19. Reaction scale: B(OMe)3 , 4‐bromobenzotrifluoride  and magnesium turnings  gives bis(4‐trifluorophenyl)borinic acid . Bis(4‐trifluorophenyl)borinic acid  and (Z)‐4‐amino‐5,5,5‐trichloropent‐3‐en‐2‐one  gives NBC37 as a yellow solid ; mp: 103–105 °C; 1H NMR : δ=7.56 ), 7.46 ), 7.41 , 5.89 C=CH), 2.30 ppm ; 13C NMR : δ=187.1 (CH3CO), 166.7 (Cl3C(NH)C=CH), 131.8 (B‐Ar(o)), 129.2 ), 124.7 , 124.4 ), 92.7 (Cl3C(NH)C=CH), 24.8 ppm (CH3CO), B‐Ar(i) and CCl3 quaternary signals not observed; 11B{1H} NMR : δ=3.56 ppm; 19F{1H} NMR : δ=−62.4 ppm; MS(ES−) (m/z): 499.1 −, 500.1 −, 501.1 −, 502.1 −, 503.1 −, 504.1 −; HRMS(ES−) (m/z): [M−H]− calcd for C19H1211B35Cl319F6NO, 499.9987; found, 499.9990, error: 0.6 ppm.2,2‐Bis(4‐methylphenyl)‐6‐methyl‐4‐(trichloromethyl)‐2,3‐dihydro‐1,3,2‐oxazaborinin‐1‐ium‐2‐uide (NBC38): EtOAc/n‐hexane, 1:19. Reaction scale: B(OMe)3 , 4‐bromotoluene  and magnesium turnings  gives bis(4‐methylphenyl)borinic acid . Bis(4‐methylphenyl)borinic acid  and (Z)‐4‐amino‐5,5,5‐trichloropent‐3‐en‐2‐one  gives NBC38 as a yellow solid ; mp: 105–107 °C; 1H NMR : δ=7.41 , 7.28 ), 7.13 ), 5.77 C=CH), 2.34 , 2.23 ppm ; 13C NMR : δ=186.2 (CH3CO), 136.3 (B‐Ar(p)), 131.9 (B‐Ar(o)), 128.4 (B‐Ar(m)), 91.5 (Cl3C(NH)C=CH), 24.9 (CH3CO), 21.5 ppm (CH3), B‐Ar(i) and Cl3C(NH)C=CH quaternary signals not observed; 11B{1H} NMR : δ=4.61 ppm; MS(ES−) (m/z): 392.1 −, 393.1 −, 394.1 −, 395.1 −, 396.1 −; MS(ES+) (m/z): 301.2 +, 302.1 +, 303.1 +, 304.1 +, 305.1 +, 306.0 +; HRMS(ES−) (m/z): [M−H]− calcd for C19H1811B35Cl3NO, 392.0553; found, 392.0555, error: 0.6 ppm.2,2‐Bis‐6‐methyl‐4‐(trichloromethyl)‐2,3‐dihydro‐1,3,2‐oxazaborinin‐1‐ium‐2‐uide (NBC39): EtOAc/n‐hexane, 1:19. Reaction scale: B(OMe)3 , 4‐bromo‐1,2‐dichlorobenzene  and magnesium turnings  gives bisborinic acid . Bisborinic acid  and (Z)‐4‐amino‐5,5,5‐trichloropent‐3‐en‐2‐one  gives NBC39 as a cream solid ; mp: 109–110 °C; 1H NMR : δ=7.37 , 7.36 , 7.10 , 5.88 C=CH), 2.28 ppm , NH signal is observed but overlapping with CHCl3 at 7.26 ppm; 13C NMR : δ=187.1 (CH3CO), 166.7 (Cl3C(NH)C=CH), 133.5 (B‐Ar‐C6), 132.1 (B‐Ar‐C3), 131.1 (B‐Ar‐C4), 130.9 (B‐Ar‐C2), 129.9 (B‐Ar‐C5), 92.8 (Cl3C(NH)C=CH), 92.5 (CCl3), 24.9 ppm (CH3CO), B‐Ar‐C1 quaternary signal not observed; 11B{1H} NMR : δ=3.10 ppm; MS(ES−) (m/z): 499.9 −, 500.9 −, 501.9 −, 502.9 −, 503.9 −, 504.9 −, 505.9 −, 506.9 −, 507.9 −; HRMS(ES−) (m/z): [M−H]− calcd for C17H1011B35Cl7NO, 499.8681; found, 499.8681, error: 0.0 ppm.5‐Acetyl‐6‐amino‐2,2‐bis(3‐chloro‐4‐methylphenyl)‐4‐(trichloromethyl)‐2,3‐dihydro‐1,3,2‐oxazaborinin‐1‐ium‐2‐uide (NBC40): Using an adapted procedure,iPrMgCl  was added dropwise to a solution of 2‐chloro‐4‐iodotoluene  in anhydrous THF (5 mL) in an oven‐dried Schlenk flask under N2. The reaction was stirred at 0 °C for 5 h. B(OMe)3  was then added and the reaction mixture was stirred overnight allowing to warm to room temperature. HCl  was added and the reaction mixture was extracted with EtOAc (3×10 mL), washed with brine (1×10 mL), dried over MgSO4, filtered and evaporated in vacuo to give crude bis(3‐chloro‐4‐methylphenyl)borinic acid as a cream solid in quantitative yield. To a portion of this intermediate  in anhydrous THF (5 mL) was added (Z)‐2‐acetyl‐3‐amino‐4,4,4‐trichlorobut‐2‐enamide . The reaction was stirred at 50 °C for 16 h under N2. The reaction mixture was concentrated and purified by flash column chromatography . Collected fractions were evaporated in vacuo and stirred in the minimum amount of cold n‐hexane for 30 min to induce precipitation. The precipitate was then filtered and dried under vacuum to give NBC40 as a white solid ; mp: 147–148 °C; 1H NMR : δ=11.22 C=C), 7.38 , 7.18 , 7.10 , 6.16 , 5.89 , 2.32 , 2.26 ppm ; MS(ES−) (m/z): 502.1 −, 503.1 −, 504.1 −, 505.1 −, 506.1 −, 507.1 −, 508.1 −, 509.1 −; HRMS(ES−) (m/z): [M−H]− calcd for C20H1711B35Cl5N2O2, 502.9831; found, 502.9833, error: 0.4 ppm.3‐Hydroxypyridine‐2‐carbonyloxy‐bis(3‐chloro‐4‐methylphenyl) borane (AN0128): Prepared according to a previously published method.iPrMgCl  was added dropwise to a solution of 2‐chloro‐4‐iodotoluene  in anhydrous THF (5 mL) in an oven‐dried Schlenk flask under N2. The reaction was stirred at 0 °C for 5 h. B(OMe)3  was then added and the reaction mixture was stirred overnight allowing to warm to room temperature. HCl  was then added and the reaction mixture was extracted with EtOAc (3×10 mL), washed with brine (1×10 mL), dried over MgSO4, filtered and evaporated in vacuo to give crude bis(3‐chloro‐4‐methylphenyl)borinic acid as a cream solid in quantitative yield. A portion of this intermediate  was dissolved in EtOH (5 mL) and heated at reflux. 3‐Hydroxypicolinic acid  was added in portions to the hot solution and after the last addition, the reaction mixture was stirred at reflux for 15 min. The reaction was then cooled, resulting in the precipitation of product from the solution. The reaction mixture was partially concentrated and the precipitate was re‐crystallised in EtOH to give AN0128 as a white solid ; mp: 166–167 °C (lit.: 165.0–166.5 °C)1H NMR : δ=12.17 , 8.58 , 7.93 , 7.22 , 7.20 , 7.11 , 2.27 ppm ; 13C NMR : δ=162.2 (COO), 156.2 (Py‐C9), 134.2 (Py‐C8), 134.0 (Py‐C12), 133.0 (B‐Ar‐C4), 132.1 (B‐Ar‐C3), 131.9 (B‐Ar‐C6), 131.5 (Py‐C11), 130.7 (B‐Ar‐C5), 130.5 (B‐Ar‐C2), 127.5 (Py‐C10), 19.4 ppm (CH3), B‐Ar‐C1 quaternary signal not observed; 11B{1H} NMR : δ=6.76 ppm; MS(ES−) (m/z): 397.1 −, 398.1 −, 399.1 −, 400.1 −, 401.1 −; HRMS(ES−) (m/z): [M−H]− calcd for C20H1511B35Cl2NO3, 398.0528; found, 398.0526, error: 0.5 ppm. All data were in agreement with literature values.3‐pentane‐2,4‐dione (8): Using a previously reported procedure,3CN was slowly generated by dropwise addition of a solution of trifluoroacetic anhydride  in anhydrous pyridine (40 mL) to a solution of trifluoroacetamide  in anhydrous pyridine (20 mL) under N2 in a three‐neck round‐bottom flask equipped with a N2 gas inlet and gas outlet. The gas outlet was connected to a two‐neck round‐bottom flask containing a solution of acetylacetone  and Zn(acac)2  in anhydrous CH2Cl2 (10 mL) that was equipped with a dry ice condenser connected to a bubbler outlet. CF3CN was bubbled into the stirring solution at room temperature for several hours until complete consumption of the trifluoroacetic anhydride solution. The reaction was further stirred at room temperature for 16 h. The reaction mixture was extracted with CH2Cl2 (3×10 mL), washed with brine (1×10 mL), dried over MgSO4, filtered and evaporated in vacuo to give 8 as a white solid . 1H NMR : δ=2.46 , 2.18 ppm ; 13C NMR : δ=202.1 (CH3CO), 196.4 (CH3CO), 145.7 C=C), 120.2 , 112.8 (F3C(NH2)C=C), 32.2 , 29.2 ppm (CH3CO); 19F{1H} NMR : δ=−66.4 ppm. All data were in agreement with literature values.Z)‐4‐Amino‐5,5,5‐trifluoropent‐3‐en‐2‐one (10) was dissolved in EtOH (10 mL) and a saturated solution of K2CO3 (20 mL) was added. The reaction was stirred at 50 °C for 24 h. The reaction mixture was then extracted with CHCl3 (3×10 mL), washed with brine (1×10 mL), dried over MgSO4, filtered and evaporated in vacuo. The crude mixture was purified by flash column chromatography  to give 10 as an orange solid . 1H NMR : δ=5.52 C=CH), 2.18 ppm ; 13C NMR : δ=199.6 (CH3CO), 147.1 C=CH), 120.4 , 94.1 C=CH), 30.5 ppm (CH3CO); 19F{1H} NMR : δ=−71.8 ppm. All data were in agreement with literature values.6‐Methyl‐2,2‐diphenyl‐4‐(trifluoromethyl)‐2,3‐dihydro‐1,3,2‐oxazaborinin‐1‐ium‐2‐uide (NBC41): Using an adapted procedure,10  was added to a solution of DPBA  in anhydrous THF (5 mL). The reaction mixture was stirred at 50 °C under Ar for 16 h. The reaction mixture was then concentrated and purified by flash column chromatography  to give NBC41 as a yellow solid ; mp: 96–97 °C (lit.: 99–100 °C);1H NMR : δ=7.14–7.31 , 6.95 , 5.44 C=CH), 2.17 ppm ; 13C NMR : δ=188.3 (CH3CO), 156.7 C=CH), 131.8 (B‐Ph(o)), 127.6 (B‐Ph(m)), 127.1 (B‐Ph(p)), 118.8 , 91.6 (F3C(NH)C=CH), 24.9 ppm (CH3CO); 11B{1H} NMR : δ=4.37 ppm; 19F{1H} NMR : δ=−72.9 ppm; MS(ES−) (m/z): 316.11 −; MS(ES+) (m/z): 340.11 +; HRMS(ES−) (m/z): [M+Na]− calcd for C17H1511BF3NO, 340.1091; found, 340.1085, error: 1.8 ppm. All data were in agreement with literature values.Z)‐2‐Acetyl‐3‐amino‐4,4,4‐trifluorobut‐2‐enamide (9) in anhydrous pyridine (40 mL) to a solution of trifluoroacetamide  in anhydrous pyridine (20 mL) under N2 in a three‐neck round‐bottom flask equipped with a N2 gas inlet and gas outlet. The gas outlet was connected to a two‐neck round‐bottom flask containing a solution of acetoacetamide  and Zn(acac)2  in anhydrous CH2Cl2 (10 mL) that was equipped with a dry ice condenser connected to a bubbler outlet. CF3CN was bubbled into the stirring solution at room temperature for several hours until complete consumption of the trifluoroacetic anhydride solution. The reaction was further stirred at room temperature for 16 h. The reaction mixture was extracted with EtOAc (5×50 mL), washed with brine (3×50 mL), dried over MgSO4, filtered and evaporated in vacuo. Et2O (10 mL) was added to induce precipitation and the precipitate was filtered, washed with additional Et2O and dried to give 9 as a white solid ; mp: 192–193 °C; 1H NMR : δ=9.06 , 7.75 , 7.40 , 2.18 ppm ; 13C NMR : δ=196.5 (CH3CO), 168.3 (CONH2), 144.9 C=C), 120.3 , 107.0 (F3C(NH2)C=C), 28.0 ppm (CH3CO); 19F{1H} NMR : δ=−65.4 ppm; MS(ES−) (m/z): 195.04 −; MS(ES+) (m/z): 219.03 +; HRMS(ES−) (m/z): [M+Na]− calcd for C6H7F3N2O2, 219.0352; found, 219.0346, error: 2.7 ppm.5‐Acetyl‐6‐amino‐2,2‐diphenyl‐4‐(trifluoromethyl)‐2,3‐dihydro‐1,3,2‐oxazaborinin‐1‐ium‐2‐uide (NBC42): 9  was added to a solution of DPBA  in anhydrous THF (5 mL). The reaction mixture was stirred at 50 °C under Ar for 16 h. The reaction mixture was then concentrated and purified by flash column chromatography . The collected fractions were combined, evaporated in vacuo and precipitated in n‐hexane (10 mL) to give NBC42 as a white solid ; mp: 118–120 °C; 1H NMR : δ=10.05 , 7.22–7.36 , 6.17 , 2.26 ppm ; 13C NMR : δ=196.3 (CH3CO), 169.8 (CONH2), 156.7 C=C), 131.8 (B‐Ar(o)), 127.5 (B‐Ar(m)), 127.0 (B‐Ar(p)), 119.4 , 97.2 (F3C(NH)C=C), 30.5 ppm , B‐Ar(i) quaternary signal not observed; 11B{1H} NMR : δ=2.55 ppm; 19F{1H} NMR : δ=−65.1 ppm; MS(ES−) (m/z): 227.0 −, 359.1 −, 523.3 −; MS(ES+) (m/z): 219.0 +, 399.1 +, 219.0 +, 563.2 +; HRMS(ES+) (m/z): [M+H]+ calcd for C18H1711BF3N2O2, 361.1330; found, 361.1338, error: 2.3 ppm.Cell culture: Immortalised murine bone marrow‐derived macrophages (iBMDMs) were cultured in DMEM, 10 % fetal bovine serum (FBS), 100 U mL−1 penicillin and 100 μg mL−1 streptomycin (PenStrep). Cells were seeded overnight at 0.75×106 cells per mL and then stimulated with LPS , and then incubated with vehicle (0.5 % DMSO) or drug as indicated (10 μm) for 15 min before activation of NLRP3 using nigericin . IL‐1β release was measured by a specific ELISA (R&D systems).Data presentation and statistical analysis: Data are presented as mean values ± standard error of the mean (SEM) of at least three separate experiments. Statistical analyses performed were one‐way analysis of variance (ANOVA) with Dunnett's multiple comparisons test post hoc. Accepted levels of significance were *p<0.05, ***p<0.001. Statistical analyses were carried out using GraphPad Prism.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Hydrogen bonding through the water molecules gives two-dimensional sheets lying parallel to (100).The present paper describes the synthesis and crystal structure of a cobalt(II) isobutyrate dihydrate, based on a slightly distorted CoO 3)2CO2}2(H2O)]·H2O}n, the Co2+ ion is hexacoordinated in a slightly distorted octahedral coordination environment defined by two O atoms from two bridging water molecules and four O atoms from four bridging carboxylate ligands. The carboxylates adopt two different coordination modes, μ-(κ2O:O′) and μ-(κ2O:O), forming a one-dimensional polymeric chain extending along [010]. The intra-chain cobalt⋯cobalt separation is 3.2029 (2) Å. The polymeric chains are linked by hydrogen bonds involving the water molecules of solvation, giving a two-dimensional network structure lying parallel to (100).In the title cobalt(II) coordination polymer with isobutyrate ligands, {[Co{CH(CH Oligo- and polynuclear cobalt carboxylates in turn have attracted great attention because of their utilization in homogeneous oxidation catalysis 2CO2}2(H2O)]·H2O}n, (I)As a part of our ongoing studies on these compounds, we describe here synthesis and crystal structure of the title compound, {[Co{CH(CH2+ cation coordinated by four O atoms from four bridging isobutyrate ligands and two O atoms from two bridging water molecules (O1W) in a distorted octahedral coordination. A water molecule of solvation (O2W) is also present –110.31 (2)°. This data correlates with the angles and the distances in cobalt(II) acetate dihydrate which has a similar structure nt Fig. 1. The Co—nt Fig. 1 and the ∞[Co(H2O)((CH3)2CHCOO)2] composition, extending along [010]  and μ-(κ2O:O). The C—O bond lengths of the first group (involving O1A and O2A) have close values [1.2755 (10) and 1.2533 (10) Å], whereas those of the second group (involving O1B and O2B) have a more striking difference [1.2878 (9) and 1.2510 (11) Å]. The carboxylate O2B atom of the second group forms an inter-unit hydrogen bond with the bridging water molecule [O1W—H⋯O2Bi = 2.6206 (9) Å] 0] Fig. 2. The Co⋯Å] Fig. 2.W) by a system of hydrogen bonds, forming a sheet structure arranged parallel to (100)  Table 2. Only weet al., 2016∞[Co(RCOO)2(H2O)]: acetate  was added. The reaction mixture was periodically stirred in an ultrasonic bath at room temperature until the liberation of carbon dioxide ceased. The unreacted CoCO3·6H2O was removed by filtration, and the filtrate was allowed to stand at room temperature for slow evaporation. Red single crystals of (I)The title compound was synthesized using a similar procedure as for the synthesis of the analogous carboxylates cobalt(II) propionate dihydrate (Fischer Uiso(H) set to 1.5Ueq(O). Other hydrogen atoms were placed in calculated positions and refined using a riding model with d(C—H) = 0.98 Å, Uiso(H) = 1.2Ueq(C) for the tertiary carbon atoms and d(C—H) = 0.96 Å, Uiso(H) = 1.5Ueq(C) for the methyl groups.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989017001360/zs2371sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 1529830CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


C=O⋯π and π–π stacking interactions also occur. Hirshfeld surface analysis was used to confirm and quantify the supramolecular interactions.The structure of a coumarin ester is reported and compared with the results of a quantum chemical calculation. In the crystal, intermolecular C—H⋯O contacts generate an infinite C(6) chain along the  20H18O4, the benzoate ring is oriented at an acute angle of 33.10 (12)° with respect to the planar (r.m.s deviation = 0.016 Å) coumarin ring system. An intramolecular C—H⋯O hydrogen bond closes an S(6) ring motif. In the crystal, C—H⋯O contacts generate infinite C(6) chains along the b-axis direction. Also present are π–π stacking interactions between neighbouring pyrone and benzene rings [centroid–centroid distance = 3.7034 (18) Å] and C=O⋯π interactions [O⋯centroid = 3.760 (3) Å]. The data obtained from quantum chemical calculations performed on the title compound are in good agreement with the observed structure, although the calculated C—O—C—C torsion angle between the coumarin ring system and the benzoate ring (129.1°) is somewhat lower than the observed value [141.3 (3)°]. Hirshfeld surface analysis has been used to confirm and quantify the supramolecular interactions.In the title compound, C Coumarin and its derivatives have been reported to serve as anti-bacterial  ring motif arises from an intramolecular C6—H6⋯O4 hydrogen bond, and generates a pseudo-tricyclic ring system em Fig. 1. The couc-axis direction, as shown in Fig. 3B = 2.38 Å] is found at a distance shorter than the sum of the van der Waals radii. An unusual C10=O4⋯π interaction , is also present. The resulting supramolecular aggregation is completed by the presence of π–π stacking  = 3.7035 (18) and Cg3⋯Cg1  = 3.7034 (18) Å, where Cg1 and Cg3 are the centroids of the pyrone and the C11–C16 benzene rings, respectively] that are less than 3.8 Å, the maximum regarded as suitable for an effective π–π interaction  and 3.6143 (13) Å, respectively, and the distances between Cg1 and a perpendicular projection of Cg3 on ring 1 (slippage) are 0.726 and 0.807Å, respectively.In the crystal, two types of intermolecular hydrogen-bonding interactions are present Table 1. The C8—ng Fig. 4 between et al., 2016et al.,1982et al., 1985et al., 2011et al., 2014meta-substituted coumarin esters dnorm surfaces mapped over a fixed colour scale of −0.39 (red) to 1.4 Å (blue) with the program CrystalExplorer 3.1 dnorm highlights six red spots showing distances shorter than the sum of the van der Waals radii. These dominant interactions correspond to intermolecular C—H⋯O hydrogen bonds, O⋯π and π–π stacking interactions between the surface and the neighbouring environment. The mapping also shows white spots with distances equal to the sum of the van der Waals radii and blue regions with distances longer than the sum of the van der Waals radii. The surfaces are transparent to allow visualization of the molecule , as shown by the X-ray study. The most significant contribution to the Hirshfeld surface (46.8%) is from H⋯H contacts, which appear in the central region of the FP . H⋯O/O⋯H interactions with a 24.1% contribution appear as blue spikes in Fig. 6c and show the presence of O⋯H contacts, whereas the C⋯H/H⋯C plot (17.3%) gives information about intermolecular hydrogen bonds . Other visible spots in the Hirshfeld surfaces show C⋯O/O⋯C and O⋯O contacts, which contribute only 4.0 and 1.0%, respectively .Molecular Hirshfeld surfaces of 2-oxo-2le Fig. 5. Furtherns Fig. 6a, as shFP Fig. 6b. H⋯O/Ods Fig. 6d. Otherly Fig. 6e and 6f++G basis set. The crystal structure in the solid state was used as the starting structure for the calculations. The DFT calculations are performed with the GAUSSIAN09 program package tert-butylbenzoyl chloride  in dry tetrahydrofuran (30 to 40 ml), was added dry trimethylamine  and 7-hydroxycoumarin  in small portions over 30 min. The mixture was then refluxed for four h and poured into 40 ml of chloroform. The solution was acidified with diluted hydrochloric acid until the pH was 2–3. The organic layer was extracted, washed with water to neutrality, dried over MgSO4 and the solvent removed. The resulting precipitate (crude product) was filtered off with suction, washed with petroleum ether and recrystallized from chloroform. Colourless crystals of the title compound were obtained in a good yield: 90%; m.p. 406–408 K.To a solution of 4-Uiso(H) constrained to 1.2 (aromatic) or 1.5 (methyl) times Ueq(C) of the respective parent atom.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989018004188/sj5549sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989018004188/sj5549Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018004188/sj5549Isup3.cmlSupporting information file. DOI: 1828991CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


ASBTapical sodium‐dependent bile acid transporterBAbile acidBSEPbile salt export pumpCAcancer antigenCDCAchenodeoxycholic acidCholcholesterolCol1α, collagen 1α; COX2cyclooxygenase 2eNOSendothelial NO synthaseET1endothelin 1FAfatty acidFFAfree fatty acidFGFfibroblast growth factorFGFRFGF receptorFXRfarnesoid X receptorHCChepatocellular carcinomaHDLhigh‐density lipoproteinIFNinterferonIGF1Rinsulin‐like growth factor 1 receptorILinterleukiniNOSinducible NO synthaseKCKupffer cellLDLlow‐density lipoproteinLDLrLDL receptorLp(a), lipoprotein a; LSECliver sinusoidal endothelial cellMCP1monocyte chemoattractant protein 1MDRmultidrug resistanceMMPmatrix metalloproteinaseNAFLDnonalcoholic fatty liver diseaseNASHnonalcoholic steatohepatitisNFκBnuclear factor kappa BNOnitric oxideNTCPsodium/taurocholate cotransporting polypeptidePLphospholipidsSR‐BIscavenger receptor class B type 1p‐STAT3phosphorylated signal transducer and activator of transcription 3TGtriglycerideTGFtransforming growth factorTIMPtissue inhibitor of metalloproteinaseTJtight junctionTNFtumor necrosis factorVCAMvascular cell adhesion moleculeVLDLvery low‐density lipoproteinZO‐1zona occludens 1.


Offset π–π interactions are also present.The title hydrated salt, C 10H12N3O3S+·Cl−·H2O, the benzimidazolium ring system is almost planar (r.m.s. deviation = 0.006 Å) and the nitro group is inclined at an angle of 4.86 (9)° to this plane. In the crystal, C—H⋯O hydrogen bonds form centrosymmetric R22(20) dimers and these are further aggregated through N—H⋯O and O—H⋯Cl hydrogen bonds involving the water molecules and chloride anions. Aromatic π–π stacking interactions are also found between two parallel benzene rings or the benzene and imidazolium rings, with centroid–centroid distances of 3.5246 (9) and 3.7756 (9) Å, respectively. Analysis of the bond lengths and comparison with related compounds show that the nitro substituent is not involved in conjugation with the adjacent π-system and hence has no effect on the charge distribution of the heterocyclic ring.In the cation of the title hydrated molecular salt, C The centroid–centroid separations are less than 3.8 Å, the maximum regarded as suitable for an effective π–π interaction  in dimethyl sulfoxide (DMSO) (10 ml). The reaction mixture was agitated for 5 h at room temperature. 50 ml of water was then added to the reaction mixture, and the products were extracted with dichloromethane (3 × 50 ml). The combined organic extracts were washed with ammonium chloride solution (10 g of ammonium chloride in 100 ml of water), dried over Na2SO4, filtered and evaporated under reduced pressure. The residue was purified by column chromatography on silica gel . The resulting powder was dissolved in dichloromethane and after three days, yellow crystals suitable for single-crystal X-ray diffraction analysis were obtained in 72% yield with a melting point of 425 K.2-Chloroethanol  and potassium carbonate  were added to 2-methylthio-5-nitro-11H NMR  δ(p.p.m.): 2.7 ; 3 ; 3.7 ; 4.3 ; 5 ; 7.5–8.5 .13C  δ (p.p.m.): 114.28 (CH3); 47 (CH2O); 59 (CH2N); 106.56; 110.03; 112.87; 117.13; 136.38; 147.37; 155.52 ; 162.23 (C=N).Uiso(H) were refined with O—H distances restrained to be 0.82 Å with a standard deviation of 0.02 Å. Other H atoms were placed in calculated positions  and refined using a riding-model approximation with Uiso(H) constrained to 1.2  or 1.5  times Ueq of the respective parent atom.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016013657/sj5502sup1.cifCrystal structure: contains datablock(s) I, New_Global_Publ_Block. DOI: 10.1107/S2056989016013657/sj5502Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016013657/sj5502Isup3.cmlSupporting information file. DOI: 1500918CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Molecules (I) and (II) are nearly planar, while molecule (III) is not planar. In compounds (I) and (II), molecules are linked into chain by C—H⋯π interactions. In compound (III), molecules are linked by a pair of C—H⋯O hydrogen bonds, forming inversion dimers. Weak C—Br⋯π interactions are present in (III). viz. (E)-1-[4-(4-bromobutoxy)phenyl]-3-phenylprop-2-en-1-one, C19H19BrO2, (I), (E)-1-[4-(4-bromobutoxy)phenyl]-3-(4-methoxyphenyl)prop-2-en-1-one, C20H21BrO3, (II), and (E)-1-[4-(4-bromobutoxy)phenyl]-3-prop-2-en-1-one, C21H23BrO4, (III), are reported. In all molecules, the conformation of the keto group with respect to the olefinic bond is s-cis. Molecules of (I) and (II) are nearly planar, while molecule (III) is not planar. In the crystal of compounds (I) and (II), molecules are linked into chains parallel to the c axis by C—H⋯π interactions. In the crystal of compound (III), molecules are linked by a pairs of C—H⋯O hydrogen bonds, forming inversion dimers. Weak C—Br⋯π interactions are also observed in (III).The crystal structures of three chalcones with a bromo-substituted butoxy side chain,  D—π⋯A type molecule. When the electron-rich groups are located on the 4 and/or 4′ positions, the electron flow follows a Λ-shaped path, and therefore the molecule is called a Λ-shaped molecule  anti-tumor activity  connected by an enone moiety with a bromobutoxy side chain attached at the 4-position of one of the phenyl rings. In molecule (I)B. All of them crystallize in the monoclinic space group P21/c with Z = 4. All three molecules adopt an s-cis conformation about the central olefinic C12=C13 bond with O2—C11—C12—C13 torsion angles of −3.2 (4), −1.6 (5) and −21.5 (4)°, respectively, and the hydrogen atoms of the central enone groups are trans-arranged with respect to the C12=C13 double bond. Molecules (I)A and B are 3.10 (13), 5.34 (11)° in compound (I)et al., 2007B with torsion angles of −3.3 (4)° (C20—O3—C17—C16) in molecule (II)The molecular structures of the title compounds (I)A with a C6—C5—O1—C4 torsion angle of 179.7 (3)°.In compounds (I)et al., 2003In compounds (I)et al., 1995et al., 2013et al., 2005via atom H2A of ring A and the centroid of ring B of a symmetry-related molecule (Table 1c axis. In compound (II)c axis by two C—H⋯π interactions involving the C2 and C3 carbon atoms via atoms H2B and H3A of ring A and the centroid of ring B of two symmetry-related molecules (Table 2The packing for molecules (I)e Table 1, formings Table 2.A via atom H10 and the O3 oxygen atom  Å; Cg is the centroid of ring B; symmetry code: (ii) 1 − x, −y, z] having a ‘face-on’ geometry m Table 3. In addiet al., 2016A search of the Cambridge Structural Database  with benzaldehyde (1 equiv.) for compound (I)2Cl2 (3 × 100 ml). The organic layers were separated, washed with brine (1 × 150 ml), dried over anhydrous Na2SO4 and evaporated to give the crude bromo compounds, which were purified by column chromatography (SiO2) using a mixture of hexane/CHCl3 (9:2 v/v) as eluent to afford yellow solids. The compounds were recrystallized by slow evaporation of chloroform solutions.Mixtures of chalcone (1 equiv.), 1,4-dibromobutane (1.2 equiv.) and anhydrous potassium carbonate (2 equiv.) in dry acetone (40 mL) were then stirred at 333 K for 12 h. After completion of reactions, the solvents were evaporated under reduced pressure and the residues extracted with CHUiso(H) values were set to 1.2Ueq(C) or 1.5Ueq(C) for methyl H atoms.Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989017010052/rz5217sup1.cifCrystal structure: contains datablock(s) I, II, III, global. DOI: 10.1107/S2056989017010052/rz5217Isup5.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989017010052/rz5217IIsup6.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989017010052/rz5217IIIsup7.hklStructure factors: contains datablock(s) III. DOI: Click here for additional data file.10.1107/S2056989017010052/rz5217Isup5.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989017010052/rz5217IIsup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989017010052/rz5217IIIsup7.cmlSupporting information file. DOI: 1545201, 1545200, 1545196CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: