Prasachandaeng (PSD) remedy has been empirically used in Thai traditional medicine to treat fever in bile duct and liver and cancer patients through Thai folk doctors. However, there have been no scientific reports on the bioactive compounds and bioactivities related to inflammation-associated carcinogenesis or cytotoxicity against cancer cell lines. In this study, we investigated the chemical content of the remedy, and evaluated its cytotoxic activity against two cancer cell lines in comparison with a non-cancerous cell line and determined tumor necrosis factor-alpha (TNF-α) production in a murine macrophage cell line (RAW 264.7) to evaluate anti-inflammatory activity. A novel HPLC method was used for quality control of its chemical content.D. cochinchinensis were isolated using bioassay-guided fractionation and chemical content of the PSD remedy was determined using HPLC. The cytotoxic activity against the hepatocarcinoma cell line (HepG2) and cholangiocarcinoma cell line (KKU-M156), in comparison with non-cancerous cell line (HaCaT), were investigated using antiproliferative assay (SRB). The anti-inflammatory activity measured by TNF-α production in RAW 264.7 was determined using ELISA.Pure compounds from the EtOH extract of ****p < 0.0001) in their cytotoxic activity against HepG2, KKU-M156, and HaCaT. The PSD remedy exhibited cytotoxic activity against HepG2 and KKU-M156 with IC50 values of 10.45 ± 1.98 (SI = 5.3) and 4.53 ± 0.74 (SI = 12.2) µg/mL, respectively. Some constituents from C. sappan, D. cochinchinensis, M. siamensis, and M. fragrans also exhibited cytotoxic activity against HepG2 and KKU-M156, with IC50 values less than 10 µg/mL. The isolated compounds, i.e., Loureirin B (1), 4-Hydroxy-2,4’-dimethoxydihydrochalcone (2), and Eucomol (3) exhibited moderate cytotoxicity against two cancer cell lines. None of the crude extracts and isolated compounds showed cytotoxicity against HaCaT. D. cochinchinensis and PSD remedy exhibited higher anti-inflammatory activity measured as TNF-α production than acetaminophen.All crude extracts and isolated compounds exhibited significant differences from vincristine sulfate (D. Cochinchinensis. The results consistent the use clinical activity and use of PSD remedy as a antipyretic treatment for liver and bile duct cancer patients by Thai traditional practitioners.The findings provide evidence of bioactivity for EtOH extracts of PSD remedy and the isolated compounds of  In Thailand's contemporary society, the incidence of primary hepatocellular carcinoma is very high. The occurrence of cancer of the liver and bile duct ranks the highest among male patients 19.5%) and seventh among females 3.8%) 9.5% and . The per.8% 9.5% The current study involves an anti-cancer proliferative activity that has been used for screening the cytotoxicity of herbal medicine extracts and bioactive compounds. The SRB assay is related to the mechanism of antiproliferation of cancer cells . Tumor nThe National List of Essential Medicine (NLEM) of Thailand lists many herbal combinations for treating several diseases, such as headache, dyspepsia, gastritis, fever, etc. Prasachandaeng (PSD) remedy, is an antipyretic medicine used to treat toxic or chronic fever in both adults and children . PSD rem3), ethyl acetate (EtOAc), methanol (MeOH), dimethylsulfoxide (DMSO), hydrochloric acid (HCl) were purchased from RCI Labscan, Thailand. Dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS), minimum essential medium (MEM), penicillin–streptomycin (P/S), and phosphate-buffered saline (PBS) were purchased from Biochrom, Germany. Sodium bicarbonate (NaHCO3) was purchased from BHD, United Kingdom. Sodium hydroxide (NaOH) was purchased from Univar, Australia. Sulforhodamine B sodium salt, Tris [hydroxymethyl] aminoethane, HEPES buffer solution, nutrient mixture F-12 Ham (HAM’s F12), and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich, USA. Trypan blue stain 0.4% and trypsin–EDTA were purchased from Gibco, USA. Silica Gel 60 (particle size 0.063–0.200 mm) for vacuum liquid chromatography (VLC), Silica Gel 60 (particle size 0.040–0.063 mm) for column chromatography (CC), and thin layer chromatography (TLC) silica gel 60 F254 were purchased from Merck, Germany. Anisaldehyde reagent was purchased from Fluka, Switzerland. NMR spectra were obtained with a Bruker Avance 400 spectrometer at 400 and 500 MHz for 1H NMR and 100 and 125 MHz for 13C NMR. The chemical shifts were recorded in δH,δc (ppm) in CDCl3. Rotary evaporator was purchased from Buchi, Switzerland. UV spectrophotometer was purchased from SHIMADZU, Japan. CO2 incubator was purchased from Shellab, USA. Laminar flow cabinet was purchased from Boss tech, Thailand. Microplate reader was purchased from Bio Tek instrument, USA.Ethanol (EtOH), 95%, was purchased from C.M.J. Anchor company, Thailand. Acetic acid, Sulfuric acid and Trichloroacetic acid (TCA) were purchased from Merck, Germany. Analytical grade reagents, i.e., hexane, chloroform  S.C. Chen was the main herbal ingredient of PSD remedy constituting 50% w/w of all proportions and the EtOH extract of D. cochinchinnesis (DC95) showed the most potent cytotoxic activity against two cancer cell lines (HepG2 and KKU-M156). Therefore, the DC95 was chosen for the isolation of pure compounds by bioassay-guided fractionation. DC95 (70 g) was chromatographed in a vacuum liquid chromatography (VLC) using silica gel 60 (300 g) with gradient elution which provided five fractions as follows: hexane , hexane: CHCl3 , chloroform , CHCl3: MeOH  and MeOH , respectively. The percentage of yield as %w/w of the starting weight of crude extracts are shown in Table 50 values of 43.14 ± 1.50 and 42.26 ± 1.07 µg/mL, respectively. F1-5 fractions showed no cytotoxicity against a human normal cell line (HaCaT) with the IC50 > 50 µg/mL. Fraction 3 (7.0 g) was chromatographed in a column chromatography (CC) on silica gel 60 (150 g) with gradient elution to give six fractions as followed: hexane:EtOAC , hexane:EtOAC , hexane:EtOAC , EtOAC (500 mL), EtOAC:MeOH , and MeOH (500 mL), respectively. The eluent was collected and examined by thin layer chromatography (TLC) with UV 254 detector at 356 nm, and was sprayed with anisaldehyde reagent. The structure of the isolated compounds were identified by 1H-NMR, 13C-NMR, DEPT135, DEPT90, COSY, NOESY, HSQC, and HMBC. Subfraction Fr.11 (416 mg) was also purified by CC on silica gel 60 (50 g) and eluted with hexane: CHCl3 (1:9) to afford a 2  as a white amorphous powder and a 3  as colorless crystal. The subfraction Fr.15 (146 mg) was also purified by recrystallization with hexane: CHCl3 (7:3) to provide a 1  as a white amorphous powder.In this study, a gel 60 00 g withThe HPLC method followed a protocol previously described , 12 with2 > 0.99 within the linear range. All quantitatively determined data from the isolated compounds in PSD remedy was expressed as the mean ± standard error of the means (SEM) of at least three independent experiments.The HPLC method was modified from Pipatrattanaseree et al., 2019 . The EtOThe solvent system consisted of a gradient mobile phase of water (A) and acetonitrile (B) which was programmed as follows: 0–5 min, , 5–55 min, , and 55–60 min, , respectively. The flow rate was set at 1 mL/min and the pressure limit was 400 bars. The samples were injected into the HPLC system and detected with diode array detector at 280 nm wavelength.Hepatocellular carcinoma  was cultured in Minimum Essential Media (MEM) supplemented with 10% heated-inactivated fetal bovine serum (FBS) and 1% penicillin–streptomycin (P/S). Cholangiocarcinoma cell line (KKU-M156) was cultured in HAM's F12 supplemented with 10% FBS, 1% P/S, and 12.5 mM HEPES. In addition, one non-cancerous human keratinocyte cell line  was cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1% P/S .3, 3 × 103, and 8 × 103 cells/well, respectively. The 100 µL/well of these cell suspensions were seeded in 96-well plates and incubated at 37 °C with 5% CO2 at 95% humidity for 24 h. Then, 100 µL of a sample solution was added to each well. The control was the medium mixed with 2% DMSO. The 96-well plates were incubated at 37 °C with 5% CO2 at 95% humidity for 72 h. The mixture in the well was removed and washed with 200 µL fresh medium. The 96-well plate was further incubated for 72 h, then the cell in 96-well plates was fixed with 40%TCA and washed with water five times. Finally, the fixed cell in the 96-well plate was strained with Sulforhodamine B sodium salt and the percentage of inhibition of cell growth was measured colorimetrically using the SRB assay -2H-chromen-4-one, which structure has been described in the literature [50 values of 7.12 ± 0.56 and 25.76 ± 1.56 µg/mL, respectively (Table Eucomol 3): Colorless crystal; ly Table . The NMRterature . Eucomol: Colorle2 value greater than > 0.99. (1) showed the highest content (28.71 ± 1.22 mg/g) followed by (3) (24.81 ± 0.17 mg/g) and (2) (18.67 ± 0.14 mg/g), respectively. The chemical constituent contents and HPLC chromatogram of marker compounds are shown in Fig. The contents of the three isolated compounds, Loureirin B (1), 4-Hydroxy-2,4’-dimethoxydihydrochalcone (2), and Eucomol 3), were simultaneously determined by a HPLC method. The three isolated compounds were the major chemical constituents of PSD95. The contents of each isolated compounds were calculated against its corresponded calibration curve which showed the r, were si****p < 0.0001) in their cytotoxic activity against HepG2, KKU-M156, and HaCaT. The EtOH extract of PSD remedy (PSD95) exhibited potent cytotoxicity against hepatocellular carcinoma (HepG2) and cholangiocarcinoma (KKU-M156) with IC50 values of 10.45 ± 1.97 (SI = 5.30) and 4.53 ± 0.74 (SI = 12.25) µg/mL, respectively. In addition, PSD95 exhibited moderate cytotoxicity against HaCaT with IC50 values of 55.45 ± 1.73 µg/mL. Some plant ingredients also exhibited strong cytotoxicity against HepG2, i.e., 95% EtOH extract of C. sappan (CS95), D. cochinchinensis (DC95), K. galanga (KG95), L. chuanxiong (LC95), M. siamensis (MS95), M. ferrea (MF95), and M. fragrans (MYF95) with IC50 values of 6.44 ± 0.54 (SI = 6.7), 7.72 ± 1.87 (SI = 5.2), 7.81 ± 2.39 (SI = ND), 11.87 ± 4.43 (SI = ND), 5.67 ± 0.32 (SI = 9.0), 7.10 ± 0.16 (SI = 7.3), and 5.67 ± 0.32 (SI = 4.6) µg/mL, respectively. The chemotherapeutic drug (vincristine sulfate) exhibited cytotoxicity against HepG2, KKU-M156, and HaCaT with IC50 values of 0.012 ± 0.0005 (SI = 0.00058), 0.0026 ± 0.001 (SI = 0.0026), and 0.000007 ± 0.00 µg/mL, respectively. The summarized results of cytotoxic activity are shown in Table All crude extracts and isolated compounds exhibited significant differences from the anti-cancer drug vincristine sulfate (p > 0.05) in their anti-TNF-α production. The results also showed that DC95 and PSD95 exhibited inhibitory activity on TNF- α production 4, and 2.5-fold, respectively, higher than the standard anti-pyretic / analgesic drug, acetaminophen.The DC95 and PSD95 were investigated for anti-inflammatory activity on TNF-α production in the murine macrophage cell line RAW 264.7). The results are shown in Table . The resC.sappan (CS95), D.cochinchinensis (DC95), M.siamensis (MF95), and M. fragrans (MYF95) also exhibited strong cytotoxic activity against cholangiocarcinoma cell line (KKU-M156) with IC50 values less than 10 µg/mL, respectively. The previous study demonstrated that the 70% ethanolic extract of C.sappan showed cytotoxic activity against hepatocellular carcinoma (HepG2) cell line [Streptomyces sp. HUST012 (SPE-B5.4) was isolated from the heartwood of D.cochinchinensis resulted in potent cytotoxic activity against hepatocellular carcinoma cell line (HepG2) with an IC50 value of 0.23 µg/mL [C.sappan and D.cochinchinensis exhibited cytotoxic activity against hepatocellular carcinoma (HepG2) with the IC50 values less than 20 µg/mL. On the other hand, the extract of M.ferrea showed comparably modest cytotoxic activity using MTT assay against cholangiocarcinoma cell line (CL-60) with IC50 value of 48.23 µg/mL [In this study, the ethnopharmacological wisdom of TTM for treating toxic (chronic) fever starting with the need to reduce high temperature  provided initial guidance). According to the TTM, PSD remedy has bitter and cold flavors that can reduce toxic fever. Furthermore, the ingredients exhibited several flavors, i.e., astringent, fragrant, spicy. These combinations of ingredients and the amelioration of related symptoms of chronic diseases are linked. The flavors of PSD ingredients and their bioactivity are shown in Table ell line . Additio23 µg/mL . The res23 µg/mL .Table 8T50 values of 20.02 ± 0.46 and 21.26 ± 3.17 µg/mL, respectively  against cancer cell lines. This is also the first scientific evidence of its cytotoxic activity against cancer cell lines in comparison with a non-cancerous cell line.Compound 1 exhibited cytotoxicity against HepG2 and KKU-M146 with the ICD. cochinchinensis. In our investigations, we have discovered flavonoids that are an important class of natural products. They belong to a class of plant secondary metabolites having a polyphenolic structure widely found in fruits, vegetables, and herbs. There are several well characterized bioactivities of flavonoids such as antioxidant, anti-inflammatory, and anti-carcinogenic properties [D. cochinchinensis exhibited cytotoxic activity against hepatocellular carcinoma (HepG2) with IC50 values less than < 20 µg/mL [Eucomol (3) has three OH groups that can increase the bioactivity . This isoperties . The res20 µg/mL . This is20 µg/mL . QualityThe scientific evidence detailed in these investigations suggests that the three isolated compounds discovered had anti-cancer proliferative activity. The PSD remedy exhibited potent cytotoxic activity against hepatocellular carcinoma (HepG2) and cholangiocarcinoma (KKU-M156). In fact, PSD remedy exhibited a greater anti-inflammatory activity as measured by inhibition of TNF-α production than acetaminophen. The results of this study support the Thai traditional wisdom that the uses the herbal combination as ingredients in traditional remedies may be effective as medicines for cancer, at least in part through their anti-inflammatory, and antipyretic activities. As a result of this study, the Thai traditional practitioners and folk doctors that use PSD remedy for toxic fever in liver and bile duct cancer patients also have additional scientific evidence underlying the rationale of their continued clinical use.


Three molecules of the title compound are present in the asymmetric unit, exhibiting different conformations relative to the ethoxy group. 17H18O3, crystallizes with three molecules in the asymmetric unit. The molecules differ in the conformation related to the ethoxy group and in the orientation of the two phenyl rings, one of which has the ethoxy group disordered over two positions with refined occupancies of 0.735:0.265 (9). In the crystal packing, the molecules are connected by weak C—H⋯π interactions.The title compound, C For example, 4-hydroxybenzoic acid and its esters are widely used as preservatives in cosmetic and pharmaceutical products known as parabens, for which the physical properties and crystal structures have been widely described , 82.6 (6) and 89.6 (7)°, and in the orientation of the two phenyl rings that form a dihedral angle of 46.4 (1), 70.3 (1), and 62.2 (1)°, respectively. A side view of the molecules displayed in Fig. 4et al., 1987C, the ethoxy group O9/C5/C51 is disordered over two sets of sites -3-methoxybenzoate compound, which is an important organic intermediate for the synthesis of the antineoplastic drug Cediranib  and 4.946 (3) Å, but with unsuitable orientations for efficient π-stacking interactions. On the other hand, the crystal packing indicates a series of C—H⋯π ring interactions in the range 2.65–2.94 Å Table 1, as deri4.et al., 2019et al., 2008et al., 2009et al. 2012The conformations of the three independent molecules present in the crystal structure of the title compound agree with previous structurally characterized species containing the (benzyloxy)phenyl fragment, where the two aromatic rings form dihedral angles of 64.5 (2)° , 1258, 1276 ν (C—Oester), 1106, 1102 ν (C—Oether). 1H NMR : δ = 1.37 , 2.36 ,4.35 , 5.07 , 6.98 , 7.20 , 7.31 , 7.99 , ppm. 13C NMR : 14.4 (C11), 21.3 (C7), 60.7 (C10), 70.1 (C8), 114.4 , 123.15 (C1), 127.73 , 129.1 , 131.6 , 133.31 (C1′),138.13 (C4), 162.5 (C4′), 166.4 (C9), ppm. LC–MS (ESI) m/z: [M + H]+. Calculated for C17H18O3 271.13; found 271.13.A mixture of ethyl-4-hydroxybenzoate  and 4-methylbenzylbromide  in acetone (100 ml) was refluxed for 14 h over anhydrous potassium carbonate (20 g). The solvent was removed 6.2CH3 moiety of molecule C was found to be disordered over two sets of sites with refined occupancies of 0.735 (9):0.265 (9). For modelling the minor disordered part, all atoms were refined with isotropic displacement parameters, and C—C and C—O bond lengths were restrained by using DFIX commands.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989022009380/wm5660sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989022009380/wm5660Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989022009380/wm5660Isup3.cmlSupporting information file. DOI: 2174691CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The entire analogs showed a diverse range of activities having IC50 values between 0.80 ± 0.05 µM to 40.20 ± 0.70 µM (α-amylase) and 1.20 ± 0.10 µM to 43.30 ± 0.80 µM (α-glucosidase) under the positive control of acarbose (IC50 = 10.30 ± 0.20 µM) (IC50 = 9.80 ± 0.20 µM) as the standard drug. Among the synthesized scaffolds, seven scaffolds 12d, 10d, 8d, 9d, 11d, 5d, and 14d showed excellent α-amylase and α-glucosidase inhibitory potentials with IC50 values of 4.30 ± 0.10, 2.10 ± 0.10, 1.80 ± 0.10, 1.50 ± 0.10, 0.80 ± 0.05, 5.30 ± 0.20, and 6.40 ± 0.30 µM (against α-amylase) and 3.30 ± 0.10, 2.40 ± 0.10, 1.20 ± 0.10, 1.90 ± 0.10, 8.80 ± 0.20, 7.30 ± 0.40, and 5.50 ± 0.10 µM (against α-glucosidase), respectively, while the remaining 12 scaffolds 19d, 8d, 17d, 16d, 15d, 7d, 4d, 3d, 1d, 2d, 13d and 6 d showed less α-amylase and α-glucosidase inhibitory potentials than standard acarbose but still found to be active. Structure–activity connection studies also showed that scaffolds with electron-withdrawing groups like -Cl, -NO2, and -F linked to the phenyl ring had higher inhibitory potentials for -amylase and -glucosidase than scaffolds with -OCH3, -Br, and -CH3 moieties. In order to better understand their binding sites, the powerful scaffolds 11d and 9d were also subjected to molecular docking studies. The results showed that these powerful analogs provide a number of important interactions with the active sites of both of these targeted enzymes, including conventional hydrogen bonding, pi–pi stacking, pi–sulfur, pi–anion, pi–pi, pi–sigma, T-shaped, and halogen (fluorine). Furthermore, various techniques (spectroscopic), including 1H, 13C-NMR, and HREI-MS mass, were used to explore the correct structure of newly afforded hybrid scaffolds based on quinoline-bearing triazole ring.The 7-quinolinyl-bearing triazole analogs were synthesized  In the past few decades, DM (diabetes mellitus) was known to be the most familiar epidemic disorder, which adversely affects the metabolic functions performed by the endocrine system. It is growing and spreading rapidly all over the world. Islets of Langerhans are well-known specialized cells that are responsible for producing insulin in the human body when a diabetic patient does not either produce insulin or is incapable of consuming insulin properly. Due to this dysfunction, the glucose (sugar) level of blood increases in the body, resulting in hyperglycemia. DM is categorized into two types, insulin-reliant, which is due to a lack of production of insulin in the human body termed type-1; however, in type-2 diabetes, either insulin is not produced by the human body or is incapable of utilizing insulin properly. After cardiovascular and cancer diseases, which result in mortality, DM is among the most frequent and rapidly growing chronic diseases . HoweverIn addition to the usage of several approaches, the inhibitors of α-glucosidase are used as a therapeutic approach for the treatment of DM . The α-gQuinoline scaffolds find application in numerous pharmaceutically potent drugs and received much attention in the field of medicinal chemistry. Quinoline scaffolds were reported to show a broad range of biological activities such as antifungal , antibacIt is well known that scaffolds harboring the 1,2,4-triazole motif have a variety of biological and pharmacological properties, including anticonvulsant, anti-inflammatory, antiproliferative, and antifungal activities . Further(1d–19d) with the hope of further expanding their inhibitory potentials as dual α-amylase and α-glucosidase inhibitors  based on the quinoline-bearing triazole skeleton, methyl quinoline-7-carboxylate (0.5 mmol) (a) solution stirred in EtOH (10 ml) was reacted and refluxed with hydrazine hydrate  for 5 h. As the reaction was completed, a solid residue (0.5 mmol) was obtained by evaporation of the solvent and then washed with cold water and dried to afford quinoline-7-carbohydrazide (b). In the second step, the mixture of quinoline-7-carbohydrazide (0.5 mmol), the corresponding phenyl isothiocyanates , and Et3N (1 ml) in THF (10 ml) was refluxed for 12 h and then quenched with water (10 ml) to form a precipitate, which was further filtered and using EtOH (5 ml) recrystallized to access an intermediate, which further underwent cyclization with 2% NaOH (10 ml) followed by neutralization with dilute HCl (5 ml) to afford the targeted quinoline-based triazole (1d–19d) scaffolds in the appropriate yield  and 1.20 ± 0.10 µM and 43.30 ± 0.80 µM (α-glucosidase) under the positive control of acarbose [(IC50 = 10.30 ± 0.20 µM) (IC50 = 9.80 ± 0.20 µM)] as the standard drug. Among the synthesized scaffolds, seven scaffolds 12d, 10d, 18d, 9d, 11d, 5d and 14d showed superior α-amylase and α-glucosidase inhibitory potentials than standard acarbose drugs with IC50 values of 4.30 ± 0.10, 2.10 ± 0.10, 1.80 ± 0.10, 1.50 ± 0.10, 0.80 ± 0.05, 5.30 ± 0.20, and 6.40 ± 0.30 µM (against α-amylase) and 3.30 ± 0.10, 2.40 ± 0.10, 1.20 ± 0.10, 1.90 ± 0.10, 8.80 ± 0.20, 7.30 ± 0.40, and 5.50 ± 0.10 µM (against α-glucosidase), respectively, while the remaining 12 scaffolds 19d, 8d, 17d, 16d, 15d, 7d, 4d, 3d, 1d, 2d, 13d and 6d showed less α-amylase and α-glucosidase inhibitory potentials than standard acarbose but still found to be active. SAR studies suggest that the entire part of synthetic scaffolds, including quinoline, triazole ring, and N-phenyl ring, actively participates in the activity, and any change observed in the activity was due to alteration in position, number/s, and nature of substituents around phenyl ring B.In this study, nineteen scaffolds based on quinoline-bearing triazole were afforded and then assessed against α-amylase and α-glucosidase to explore their inhibition profile. The entire analogs showed a diverse range of activities having IC3, -OCH3, and–Br groups bearing scaffolds. This enhanced inhibitory potential of fluoro moiety-bearing analogs was due to greater electron withdrawing affinity of the –F moiety, which makes the ph-ring more effective for interactions with α-amylase active pocket. Moreover, it also seemed that the activity was altered by bringing variation in the position of the –F moiety around aryl ring B. In current newly afforded scaffolds, the analog 11d that holds the –F moiety at 4-position of aryl ring B linked to the N-triazole skeleton was found to show ten-fold enhanced potency than standard acarbose drug. However, analogs 9d with ortho-F and 10d having meta-F substitutions displayed less potency than scaffold 11d, although these analogs resemble in their structure with analog 9d but have different positions around phenyl ring B. This variation in potency of these–F moiety-bearing scaffolds was due to different positions of the -F moiety at the phenyl ring, indicating that alteration in the position of the –F moiety may result in diverse potency.It was noteworthy from SAR studies that quinoline-based triazole scaffolds that hold the fluoro moiety as being the electron-withdrawing entity attached to various positions of the aryl ring show a significant role in the inhibition of the α-amylase enzyme, and hence hybrid scaffolds based on the quinoline-bearing triazole moiety showed better inhibition than -CH14d, 12d, and 13d. By comparing scaffold 12d that holds ortho–Cl moiety with scaffolds 14d bearing para-Cl and 13d having meta-Cl substitutions, the ortho-Cl moiety holding analog 12d showed enhanced potency than its counterparts 14d and 13d, although these scaffolds are structurally similar. This indicates that the activity was diminished by shifting ortho-Cl moiety either to para- or meta-position as in scaffolds 14d and 13d. In addition, compound 14d bearing para-Cl substitution showed 2-folds more potency than scaffold 7d bearing para-NO2 moieties on the aryl ring, although the position of substituents around the aryl ring is the same. The activity was different in the case of both these compounds, which may be owing to the diverse nature of attached substituents (-Cl and–NO2). Moreover, by comparing the ortho-Cl moiety bearing scaffold 12d with compound 5d that hold the ortho-NO2 group on the aryl ring of the triazole skeleton, the scaffold 12d has emerged as a better inhibitor of α-amylase than scaffold 5d. This difference in potency between these analogs was caused by the unique properties of both the -Cl and -NO2 moieties. If we compared compound 14d (IC50 = 6.40 ± 0.30 µM) bearing mono-chloro substitution on 4-position of the attached N-ph-ring with its counterpart 2 bearing di-Cl substitution linked to 3-and 4-position of the phenyl ring, the compound 18d showed better activity than compound 14d due to the attached di-Cl substitutions that offered stronger electron-withdrawing effect. Analog 1d having un-substituted attached ph-ring is recognized as the least potent inhibitor among the newly constructed scaffolds. However, the activity may be enhanced when substituents of either electron-withdrawing/electron-donating properties are attached to it at various positions of the ph-ring. Therefore, compounds 8d (having para–OCH3), 4d (having para–CH3), 14d (having (having para–Cl), and 18d  moieties showed enhanced α–amylase inhibitory potentials when compared to compound 1d with un-substituted ph-ring, indicating that the activity was largely dependent on attached electron-withdrawing/electron-donating groups  showed enhanced α–glucosidase inhibitory potentials when compared to Cl-substituted  and bromo-substituted analogs . Similarly, Cl-substituted scaffolds display better activity than bromo-substituted analogs. This shows that the α–glucosidase activity was enhanced by increasing the electron-withdrawing nature of the attached substituent; therefore, halogen-substituted scaffolds showed activity in order of–F > Cl > Br. Moreover, by comparing the para-F moiety bearing analog 11d with compounds 10d bearing meta-F and 9d having ortho-F group, compound 11d showed superior potentials than its counterparts 10d and 9d, which are similar in structure to compound 11d. This difference in activity found in these fluoro-substituted analogs was due to the different positions of the attached electron-withdrawing fluoro moiety around the ph-ring. If we compare analog 14d and 12d with mono-chloro substitution with compound 18d bearing 3, 4-di Cl chloro groups, the di-Cl moieties bearing scaffold 18d showed superior activity than a mono-Cl moiety containing scaffolds 14d and 12d. This enhanced activity of compound 18d may be due to the stronger electron-withdrawing effect offered by the two chloro groups as compared to compound 14d and 12d bearing mono-chloro substitution. Similarly, by comparing scaffold 5d bearing ortho-nitro substitution with its structurally similar analog 7d that holds the nitro group at the para-position of the phenyl ring, the compound 5d showed enhanced activity than its counterpart 7d. This shows that alteration in the position of substitution around the phenyl ring greatly affects the enzymatic activity , and hence these fluoro-substituted analogs were subjected to the molecular docking study to explore their binding site and results obtained from molecular docking show that the most potent analogs 11d and 9d not only display better potency (in vitro) but also furnish numerous important interactions (in silico) with the active site of the amino acid.To explore the active part in newly constructed quinoline-based triazole scaffolds against α-amylase and α-glucosidase, molecular docking was carried out. Among newly afforded scaffolds being tested, the analogs that hold fluoro moiety showed enhanced inhibitory potentials , GLN-63 (H-B), LEU-165 (π–sigma), and TRY-62 (H-B) as shown in It was shown by the PLI profile of potent analog ractions  such as 11d having para-F moiety on the extended aryl ring interacts with α-glucosidase via several interactions, including MET-470 (π–S), ALA-234 (π–R), ASP-568 (π–anion), TRP-329 (π–π T-shaped), TRP-432 (π-sigma), ARG-552 (H-B), and ASP-232 (π–anion) as shown in 9d exhibited the following interactions against alpha glucosidase such as TRP-432 (π–π T-shaped), ASP-232 (H-F), MET-470 (π–S), ALA-234 (π–R), ASP-568 (H-F), TRP-329 (π–π T-Stacked), and ARG-552 (H-B) along with van der Waals interaction , π–π T-shaped, π–sulfur, π–alkyl, π–sigma, π–π stacking, and π–anion interactions with the active part of the amino acid of α-amylase and α-glucosidase than standard acarbose drug which furnish only conventional HB van der Waals and carbon HB interactions. Therefore, scaffolds 11d and 9d were found to be more potent against α-amylase and α-glucosidase than acarbose standard drugs.It was observed by comparison of the protein–ligand interaction profile of most active scaffolds 11d and 9d have different potency (in vitro) for both enzymes being targeted and hence established a diverse range of interactions (molecular docking) with active sites of these enzymes (in silico) possessed by these scaffolds toward enzymes being targeted was due to different positions of the attached fluoro moiety around the aryl part of the quinoline-triazole skeleton. It was shown (11d and 9d (possess the–F at a different position) furnish a better binding mode of interactions with the active part of enzymes being targeted (α-amylase and α-glucosidase) than standard acarbose drug which established only conventional HB, van der Waals, and carbon HB interactions with the active part of targeted enzymes (α-amylase and α-glucosidase).The most potent scaffolds at  enzymes –4. This as shown  from doc(1d–19d) based on a quinoline-bearing triazole moiety in their core structure. The entire constructed analogs were also additionally assessed for α-amylase and α-glucosidase (in vitro) inhibitory activities. All scaffolds being synthesized were found to display a diverse range of inhibitory potentials between 0.80 ± 0.05µM and 40.20 ± 0.70 µM (against α-amylase) and 1.20 ± 0.10 µM and 43.30 ± 0.80 µM (against α-glucosidase). Compound 11d (IC50 = 0.80 ± 0.05 µM) (IC50 = 1.20 ± 0.10 µM) holding para-fluoro substitution on the phenyl ring emerged as the most active analog among the synthesized series. Moreover, compound 9d with fluoro substitution at the ortho-position of the phenyl ring was identified as the second most active analog against α-amylase and α-glucosidase enzymes. Therefore, it was concluded based on SAR studies that scaffolds bearing electron-withdrawing groups such as–NO2, -Cl, and –F attached to the phenyl ring, resulting in enhanced inhibitory potentials. When these active scaffolds 11d and 9d underwent molecular docking analysis, they created a number of significant contacts with the active sites of the two targeted enzymes. The protein–ligand contact profile for compound 11d against α-amylase shows that it established numerous important interactions such as His-305 (donor interactions), Tyr-62 (π–π stacked), His-299 (donor interactions), Asp-3002 (H-B), Glu-233 (H-B), Leu-162 (π–R), and Trp-59 (π–π T-shaped), while against α-glucosidase it also furnishes numerous important interactions, including Met-470 (π-S), Ala-234 (π–R), Asp-568 (π–anion), Trp-329 (π–π T-shaped), Trp-432 (π–sigma), Arg-552 (H-B), and Asp-232 (π–anion). Furthermore, the structures of all the newly synthesized quinoline-based triazole analogs were confirmed by employing different techniques, including 1H NMR, HREI-MS, and 13C-NMRspectroscopy.In conclusion, the present study synthesized novel hybrid analogs


Among the agonists against three peroxisome proliferator-activated receptor (PPAR) subtypes, those against PPARα (fibrates) and PPARγ (glitazones) are currently used to treat dyslipidemia and type 2 diabetes, respectively, whereas PPARδ agonists are expected to be the next-generation metabolic disease drug. In addition, some dual/pan PPAR agonists are currently being investigated via clinical trials as one of the first curative drugs against nonalcoholic fatty liver disease (NAFLD). Because PPARα/δ/γ share considerable amino acid identity and three-dimensional structures, especially in ligand-binding domains (LBDs), clinically approved fibrates, such as bezafibrate, fenofibric acid, and pemafibrate, could also act on PPARδ/γ when used as anti-NAFLD drugs. Therefore, this study examined their PPARα/δ/γ selectivity using three independent assays—a dual luciferase-based GAL4 transactivation assay for COS-7 cells, time-resolved fluorescence resonance energy transfer-based coactivator recruitment assay, and circular dichroism spectroscopy-based thermostability assay. Although the efficacy and efficiency highly varied between agonists, assay types, and PPAR subtypes, the three fibrates, except fenofibric acid that did not affect PPARδ-mediated transactivation and coactivator recruitment, activated all PPAR subtypes in those assays. Furthermore, we aimed to obtain cocrystal structures of PPARδ/γ-LBD and the three fibrates via X-ray diffraction and versatile crystallization methods, which we recently used to obtain 34 structures of PPARα-LBD cocrystallized with 17 ligands, including the fibrates. We herein reveal five novel high-resolution structures of PPARδ/γ–bezafibrate, PPARγ–fenofibric acid, and PPARδ/γ–pemafibrate, thereby providing the molecular basis for their application beyond dyslipidemia treatment. The obtained crystals were briefly soaked in a cryoprotection buffer ; afterward, these were flash cooled in a stream of liquid nitrogen until X-ray crystallography was conducted.Cocrystallization of PPARδ-LBD was performed in hanging-drop mixtures of 0.5 µL of PPARδ-LBD (10 mg/mL in buffer A’), 0.1 µL of 10 mM ligand, 0.3 µL of buffer A’, 0.1 µL of 5% Rmerge < 0.5, Rpim < 0.3, and completeness > 0.9) were set by the highest resolution shell. All structures were determined using molecular replacement in PHASER [http://www.pymol.org; accessed on 21 April 2020). All collection data and refinement statistics are summarized in Datasets were collected by BL-5A or BL-17A beamline at the Photon Factory  using a synchrotron radiation of 1.0 Å. Diffraction data were collected at 0.1° oscillation per frame, and a total of 1800 frames (180°) were recorded for 1.0 Å X-ray crystallography. Data processing and scaling were carried out using XDS X-ray detector software and AIMLESS, respectively . Resolutn PHASER  with PDBn PHASER . The str


Microbial-based biosynthesis, as an ecofriendly method, has been used for GA and pyrogallol production. In GA and pyrogallol biosynthetic pathways, the low hydroxylation activity of kcat/Km of 0.059 μM−1 s−1 towards 3,4-DHBA, which was 4.92-fold higher than the reported mutant (Y385F/T294A PobA). Molecular docking simulation provided the possible catalytic mechanism explanation of the high activity mutant. Expression of this mutant in E. coli BW25113 (Fʹ) can generate 840 ± 23 mg/L GA from 1000 mg/L 3,4-DHBA. After that, this mutant was assembled into a de novo GA biosynthetic pathway. Subsequently, this pathway was introduced into a 3,4-DHBA-producing strain (E. coli BW25113 (Fʹ)ΔaroE) to achieve 301 ± 15 mg/L GA production from simple carbon sources. Similarly, assembling this mutant into a de novo pyrogallol biosynthetic pathway enabled 129 ± 15 mg/L pyrogallol production.This work reported a high activity PobA mutant (Y385F/T294A/V349A PobA) towards 3,4-DHBA. This mutant was screened out from a PobA random mutagenesis library through a novel naked eye visual screening method. In vitro enzyme assay showed this mutant has a This work established an efficient screening method and generated a high activity PobA mutant. Assembling this mutant into de novo GA and pyrogallol biosynthetic pathways achieved the production of these two compounds from glucose. Besides, this mutant has great potential for the production of GA or pyrogallol derivatives. The screening method could be used for other GA biosynthesis-related enzymes.The online version contains supplementary material available at 10.1186/s13068-022-02142-w. Gallic acid (GA), a natural phenolic acid of plants, has diverse biological activities such as antioxidant, antibacterial and anti-inflammatory activities –7. PyrogTraditional method of GA production mainly relies on the hydrolysis of tannins through acids or bases . In addiEscherichia coli (E. coli) endogenous compound generated from the shikimate pathway [p-hydroxybenzoate hydroxylase (PobA). For pyrogallol biosynthesis, PDC was generally introduced into GA-producing strain to convert GA into pyrogallol [Pseudomonas fluorescens (P. fluorescens) displays high hydroxylase activity towards 4-HBA, but very weak or negligible activity towards 3,4-DHBA [In recent years, microbial-based biosynthesis has been attempted to produce many value-added compounds, including GA and pyrogallol , 23, fro pathway . 4-HBA c3,4-DHBA . To avoi3,4-DHBA . To addr3,4-DHBA , 32. Bas3,4-DHBA . Further3,4-DHBA . After t3,4-DHBA . Maxel e3,4-DHBA .E. coli to produce GA or pyrogallol. Assembling Y385F/T294A/V349A PobA into de novo GA or pyrogallol biosynthetic pathway achieved the efficient biosynthesis of the corresponding products. This work constructed an efficient screening method and then used this method to screen out a high activity PobA mutant.Though PobA mutants with the ability of hydroxylating 3,4-DHBA have been obtained, the hydroxylation activity still cannot satisfy the demand of high-level GA and pyrogallol production. Specifically, the low activity of PobA towards 3,4-DHBA can lead carbon source to flow into the byproduct catechol biosynthetic pathway. Therefore, PobA mutant with higher activity urgently needs to be investigated. Rational design of PobA mutants generally required to deeply understand the catalytic mechanism of PobA. In some cases, the mutants generated from rational design did not have the expected high activity . Random 3. After 2 h, we found the mixture with a pH of 9.3 displayed green color visible to naked eyes  was screened out from PobA mutagenesis library. Subsequently, this mutant was expressed and purified. SDS-PAGE in Additional file Km of Y385F/T294A/V349A PobA was 30.3 ± 10.4 μM towards 3,4-DHBA  was used as template for simulating Y385F/T294A PobA and Y385F/T294A/V349A PobA. After that, molecular docking of the mutants with substrate 3,4-DHBA and cofactor FAD were conducted to generate the corresponding complexes. As shown in Fig. 2), the SASA of the binding pocket in Y385F/T294A/V349A PobA was increased (1764.048 Å2). These suggested the binding pocket of Y385F/T294A/V349A PobA became bigger than that of Y385F/T294A PobA, which would provide more additional space for substrate 3,4-DHBA entering and then improve the binding of 3,4-DHBA to FAD and active amino acids of Y385F/T294A/V349A PobA, finally resulting in the high activity.Besides, amino acid residues within the distance of 5 Å around 3,4-DHBA in Y385F/T294A PobA , generating strains CTT1 and CTT2, respectively. 1000 mg/L 3,4-DHBA was added to the culture at 5.5 h. As shown in Fig. E. coli BW25113 (Fʹ) with Y385F/T294A/V349A PobA expression exhibits higher in vivo conversion ability towards 3,4-DHBA than E. coli BW25113 (Fʹ) with Y385F/T294A PobA expression.To test the in vivo conversion ability of PobA mutants towards 3,4-DHBA, Y385F/T294A PobA and Y385F/T294A/V349A PobA were individually expressed in E. coli BW25113 (Fʹ). Plasmids pZE-PobA** and pZE-PobA*** were individually introduced into E. coli BW25113 (Fʹ) harboring pCS-PDC, resulting in strains CTT3 and CTT4, respectively. 1000 mg/L 3,4-DHBA was fed to CTT3 or CTT4 at 5.5 h. As shown in Fig. 600 value of 6.62 ± 0.05 at 12 h. Within 12 h, 9.17 ± 5.20 mg/L GA, 171 ± 26 mg/L pyrogallol and 503 ± 3 mg/L byproduct catechol accumulated in the culture. During the next 36 h, the titer of pyrogallol increased to 222 ± 16 mg/L at 48 h, while the titer of catechol decreased to 465 ± 9 mg/L. Though CTT3 can convert 3,4-DHBA into pyrogallol, large accumulation of byproduct catechol was accompanied.Further, in vivo conversion of 3,4-DHBA into pyrogallol were achieved by expressing PobA mutants and decarboxylase PDC in 600 value of 6.66 ± 0.11 at 12 h  could achieve the in vivo conversion of 3,4-DHBA into pyrogallol. Moreover, Y385F/T294A/V349A PobA coupling with PDC represents higher in vivo ability of converting 3,4-DHBA into pyrogallol when compared with Y385F/T294A PobA coupling with PDC.As a comparison, CTT4 grew rapidly in the first 12 h and has an OD2 h Fig. D. MeanwhE. coli [E. coli BW25113 (Fʹ) was engineered to produce 3,4-DHBA from 3-dehydro-shikimate (DHS)  was blocked through knockout of gene aroE (strain CTT5) or knockout of genes aroE and ydiB (strain CTT6). AroE and YdiB are isoenzymes that can catalyze DHS to produce shikimate. Additional file 600 values of CTT5 and CTT6 increased with the extension of culture time. At 15 h, CTT5 and CTT6 reached maximum OD600 values, 4.13 ± 0.10 and 4.15 ± 0.13, respectively. After this time point, the growth of CTT5 and CTT6 stopped. As a comparison, the original strain E. coli BW25113 (Fʹ) has a maximum OD600 value of 4.61 ± 0.13 at 16 h, which was close to that of CTT5 and CTT6 at 15 h. After 16 h, E. coli BW25113 (Fʹ) stopped growing. These results suggest knockout of aroE or knockout of aroE and ydiB in E. coli BW25113 (Fʹ) did not significantly affect the cell growth.Construction of an efficient 3,4-DHBA biosynthetic pathway was significant for achieving the de novo production of GA and pyrogallol. In previous study, through expression of heterogenous PobA 3,4-DHBA can be produced from 4-HBA in E. coli . For GA E. coli BW25113 (Fʹ), CTT5 and CTT6 to generate strains CTT7, CTT8 and CTT9, respectively. Figure 600 values of CTT8 and CTT9 raised rapidly in first 12 h and have no significant improvement during the next 36 h. At 48 h, CTT8 produced 752 ± 17 mg/L 3,4-DHBA. Meanwhile, the OD600 value was 2.03 ± 0.06. For CTT9, 420 ± 26 mg/L 3,4-DHBA accumulated in the culture at 48 h, which was 1.79-fold lower than that of CTT8. These indicate the ability of strain CTT8 to produce 3,4-DHBA was higher than that of CTT9. As a comparison, CTT7 has negligible 3,4-DHBA accumulation throughout the 48-h fermentation, suggesting without knockout of aroE or ydiBE. coli BW25113 (Fʹ) could not synthesize 3,4-DHBA in large amount. These results suggest the engineered E. coli BW25113 (Fʹ) (CTT8 or CTT9) has ability to de novo produce 3,4-DHBA and can be used as host for de novo GA and pyrogallol production.To achieve the de novo production of 3,4-DHBA, 3-dehydroshikimate dehydratase (AroZ) which can catalyze 3-dehydro-shikimate to produce 3,4-DHBA, was individually introduced into ** was individually introduced into E. coli BW25113 (Fʹ), CTT5 and CTT6, generating strains CTT10, CTT11 and CTT12, respectively. The fermentation results are displayed in Fig. aroE significantly increased the ability of E. coli BW25113 (Fʹ) to de novo produce GA. For strain CTT12, GA titer and OD600 value continued to increase during the 48-h fermentation ΔaroEΔydiB has lower ability to de novo synthesize GA when compared with E. coli BW25113 (Fʹ)ΔaroE.To achieve the de novo production of GA, plasmid pZE-AroZ-PobA*** was individually introduced into E. coli BW25113 (Fʹ), CTT5 and CTT6, generating strains CTT13, CTT14 and CTT15, respectively. As shown in Fig. 600 value were similar to that of CTT11 ΔaroE demonstrates stronger ability for de novo producing GA when compared with E. coli BW25113 (Fʹ) or E. coli BW25113 (Fʹ)ΔaroEΔydiB  harboring pZE-AroZ-PobA** and pCS-PDC (CTT16), CTT5 harboring pZE-AroZ-PobA** and pCS-PDC (CTT17) and CTT6 harboring pZE-AroZ-PobA** and pCS-PDC (CTT18) were constructed to de novo produce pyrogallol. The fermentation results of CTT16 showed trace amount of 3,4-DHBA, GA, pyrogallol and byproduct catechol accumulated in the culture throughout the 48-h fermentation ΔaroE performed better than E. coli BW25113 (Fʹ)ΔaroEΔydiB for de novo production of pyrogallol.-PDC CTT1 and CTT6-PDC CTT1 and CTT6-PDC CTT1 and CTT6*** and pCS-PDC were co-transferred into E. coli BW25113 (Fʹ), CTT5 and CTT6, resulting in strains CTT19, CTT20 and CTT21, respectively. As shown Fig. E. coli containing the designed artificial pathway could achieve the de novo biosynthesis of pyrogallol. Among the engineered strains, E. coli BW25113 (Fʹ)ΔaroEΔydiB with overexpression of Y385F/T294A/V349A PobA and PDC demonstrates strongest ability for de novo production of pyrogallol  with high activity towards 3,4-DHBA was screen out from a PobA random mutagenesis library. Y385F/T294A/V349A PobA possesses higher catalytic efficiency towards 4-HBA or 3,4-DHBA when compared with the reported mutant (Y385F/T294A PobA). Moreover, Y385F/T294A/V349A PobA represents higher in vivo ability of converting 3,4-DHBA into GA or pyrogallol when compared with Y385F/T294A PobA. Assembling Y385F/T294A/V349A PobA into the de novo GA or pyrogallol biosynthetic pathway achieved GA or pyrogallol production from simple carbon sources. In all, this work constructed an efficient method for screening high activity hydroxylase PobA, and this method could be applied for screening other GA biosynthesis-related enzymes. The high activity PobA which was obtained in this study has great potential for the production of GA or pyrogallol derivatives.4, 0.05 mM CaCl2, 2 g/L MOPS and 5 g/L yeast extract, was used for feeding experiments and de novo production of GA and pyrogallol. Terrific Broth (TB) medium which contains 12 g/L tryptone, 24 g/L yeast extract, 4 g/L glycerol, 12.5 g/L K2HPO4 and 2.31 g/L KH2PO4, was used for protein expression. If needed, 100 μg/mL ampicillin or 50 μg/mL kanamycin was added to the culture. E. coli XL10-Gold and E. coli BL21(DE3) were used for plasmid construction and protein expression, respectively, while E. coli BW25113 (Fʹ) was used for feeding experiments and de novo biosynthesis of GA and pyrogallol. Plasmids pZE12-luc and pCS27 were used for pathway construction. Plasmid pETDuet-1 was used for protein expression. Plasmids pKD46 and pCP20 were used for knockout of genes. Strains and plasmids used in this study are depicted in Table Luria–Bertani (LB) medium containing 10 g NaCl, 10 g tryptone and 5 g yeast extract per liter, was used for cell inoculation and propagation. For solid medium, 20 g/L agar was added. Modified M9 (M9Y) medium which contains 11.28 g/L 5 × M9 Minimal Salt, 10 g/L glycerol, 2.5 g/L glucose, 1 mM MgSOY385F/T294A pobA [Y385F/T294A/V349A pobA were individually cloned into pZE-luc by Gibson assembly, generating plasmids pZE-PobA** and pZE-PobA***. Genes Y385F/T294A pobA and Y385F/T294A/V349A pobA were individually cloned into pETDuet-1, generating plasmids pETDuet-PobA** and pETDuet-PobA***. To realize the efficient biosynthesis of 3,4-DHBA, gene aroZ (GenBank: VCW80955.1) was synthesized by OE-PCR, and was then inserted into pZE-luc by Gibson assembly, generating pZE-AroZ. In order to achieve GA production from simple carbon sources, PLlacO1-AroZ amplified from pZE-AroZ was individually inserted into pZE-PobA** and pZE-PobA*** by Gibson assembly, resulting in plasmids pZE-AroZ-PobA** and pZE-AroZ-PobA***. Plasmid pCS-PDC [Genes 94A pobA  and Y385 pCS-PDC  was usedpobA mutagenesis library, the in vivo activity and in vivo conversion ability of the reported PobA mutants  and 73.5 μL ddH2O. The amplification program was as follows: 94 °C initial denaturation for 3 min, and then 30 cycles of 94 °C denaturation for 1 min, 58 °C annealing for 1 min and 72 °C extension for 1.5 min. After that, the PCR product was confirmed and purified via agarose gel electrophoresis. The purified product was digested by DpnI at 37 °C for 1.5 h and then cloned into pZE-luc, generating a PobA mutagenesis library. This mutagenesis library contained 500 colonies.To construct a 94A PobA , Y385F/L94A PobA , L199R/T94A PobA  and V47I94A PobA ) were cod-thiogalactopyranoside (IPTG) and 1 g/L 3,4-DHBA in 96-deep-well plates. The cultures were left at 30 °C for incubation. At the same time, the remaining seed cultures were pre-saved for DNA sequencing. After 12 h, samples were taken. The samples were firstly centrifuged at 12,000 rpm for 10 min to remove the cells and sediments in medium. After that, 50 μL supernatant was taken into 96-well plates which contained 0.1 M NaHCO3. After reaction for 2 h, the samples which with deepest green color among all the samples were screened out. Then, microplate reader (BioTek Cytation 3) was used to detect the optical densities of the screened samples at 640 nm. The mutations in the screened pobAs were confirmed through DNA sequencing.The single colonies of PobA mutagenesis library were pre-inoculated into 96-deep-well plates which contained 1 mL LB and 100 μg/mL ampicillin, and then aerobically cultured at 37 °C for 12 h to generate seed cultures. After that, 10 μL seed cultures were transferred into 990 μL M9Y medium which was supplemented with 100 μg/mL ampicillin, 0.5 mM isopropyl-β-E. coli BL21 (DE3) containing pETDuet-PobA** or pETDuet-PobA*** was pre-inoculated in 5 mL LB medium which contained 100 μg/mL ampicillin, and was then cultured overnight at 37 °C. Then, 1 mL of pre-inoculum was transferred into 100 mL of fresh TB containing 100 μg/mL ampicillin and cultured at 37 °C until OD600 reached around 0.6. After that, 0.5 mM IPTG was added to the culture to induce protein expression at 16 °C. After 12 h, the cells were harvested via centrifugation (4000 rpm for 30 min at 4 °C) and then resuspended in 20 mL lysis buffer . The cells were disrupted by ultrasonic processor. The generated mixture was then centrifuged . The supernatant was collected. The proteins were purified by nickel column. Quick Start™ Bradford Protein Assay (BIO-RED) was used for measuring protein concentrations.For HPLC detection, a 1000-μL reaction system which contained 100 mM Tris–HCl (pH 8.0), 10 μM FAD, 1000 μM NADPH, 500 nM purified enzyme and 0–1000 μM 4-HBA or 3,4-DHBA, was prepared. For NADPH detection, a 500-μL reaction system which contained 100 mM Tris–HCl (pH 8.0), 10 μM FAD, 1000 μM NADPH, 500 nM purified enzyme and 0–1000 μM 4-HBA or 3,4-DHBA, was prepared. The reactions were conducted at 30 °C for 2 min. The kinetic parameters were estimated through non-linear regression of the Michaelis–Menten equation in OriginPro8.5.Structural models of Y385F/T294A PobA and Y385F/T294A/V349A PobA were built with the SWISS-MODEL server using Y385F PobA (PDB ID: 6JU1) as template . The comE. coli BW25113 (Fʹ) containing pZE-PobA** (CTT1), E. coli BW25113 (Fʹ) containing pZE-PobA*** (CTT2), E. coli BW25113 (Fʹ) containing pZE-PobA** and pCS-PDC (CTT3), and E. coli BW25113 (Fʹ) containing pZE-PobA*** and pCS-PDC(CTT4) were used. The single colonies were pre-inoculated into 5 mL LB with 100 μg/mL ampicillin and then cultured at 37 °C overnight. 200 μL overnight cultures were inoculated into 20 mL M9Y medium containing 100 μg/mL ampicillin. The cultures were cultivated at 37 °C for 2.5 h, and then induced with 0.5 mM IPTG at 30 °C. After induction for 3 h, 1000 mg/L 3,4-DHBA was fed to the culture. For GA production, samples were taken at 5.5, 6.5, 9, 12, 24, 36 and 48 h. For pyrogallol production, samples were taken at 12, 24, 36 and 48 h. Cell growth was confirmed through measuring OD600. Products and intermediates were analyzed by UHPLC.To testify the in vivo conversion efficiency of PobA mutants towards 3,4-DHBA, feeding experiments were conducted. aroE or ydiB of E. coli BW25113 (Fʹ) was knocked out. First, donor fragments for pre-knockout genes needed to be constructed. For knockout of gene aroE, 530 bp at the 5ʹ-terminus of aroE, FRT from pRecA-FRT [kan, FRT and 340 bp at the 3ʹ-terminus of aroE were sequentially assembled through OE-PCR, generating aroE-donor. For knockout of gene ydiB, ydiB-donor was constructed like aroE-donor. To construct E. coli BW25113 (Fʹ)ΔaroE, the aroE-donor fragment was transferred into E. coli BW25113 (Fʹ) containing pKD46, and then cultured at 30 °C overnight. To eliminate pKD46, the overnight cultures were then spread on LB solid medium with 50 μg/mL kanamycin and then cultured at 37 °C overnight. After that, plasmid pCP20 was transferred into the generated strain and cultured at 30 °C overnight. To induce FLP recombinase and eliminate plasmid pCP20, the single colonies were picked and individually incubated on LB solid medium with 100 μg/mL ampicillin, LB solid medium with 50 μg/mL kanamycin and LB solid medium overnight at 43 °C. The colonies which can grow on LB solid medium and cannot grow on LB solid medium with ampicillin or kanamycin, were the colonies of strain E. coli BW25113 (Fʹ)ΔaroE (CTT5). The construction of E. coli BW25113 (Fʹ)ΔaroEΔydiB (CTT6) was similar to that of CTT5.To acquire 3,4-DHBA-producing strains, gene RecA-FRT , kan, FRE. coli BW25113 (Fʹ) containing pZE-AroZ (CTT7), CTT5 containing pZE-AroZ (CTT8) and CTT6 containing pZE-AroZ (CTT9), were used for de novo biosynthesis of 3,4-DHBA. E. coli BW25113 (Fʹ) containing pZE-AroZ-PobA** (CTT10), CTT5 containing pZE-AroZ-PobA** (CTT11), CTT6 containing pZE-AroZ-PobA** (CTT12), E. coli BW25113 (Fʹ) containing pZE-AroZ-PobA*** (CTT13), CTT5 containing pZE-AroZ-PobA*** (CTT14) and CTT6 containing pZE-AroZ-PobA*** (CTT15), were used for de novo biosynthesis of GA. E. coli BW25113 (Fʹ) containing pZE-AroZ-PobA** and pCS-PDC (CTT16), CTT5 containing pZE-AroZ-PobA** and pCS-PDC (CTT17), CTT6 containing pZE-AroZ-PobA** and pCS-PDC (CTT18), E. coli BW25113 (Fʹ) containing pZE-AroZ-PobA*** and pCS-PDC (CTT19), CTT5 containing pZE-AroZ-PobA*** and pCS-PDC (CTT20) and CTT6 containing pZE-AroZ-PobA*** and pCS-PDC (CTT21), were used for de novo biosynthesis of pyrogallol. Transformants were pre-inoculated into 5 mL LB medium with appropriated antibiotics and cultured overnight at 37 °C. Then, 200 μL seed cultures were transferred into 20 mL M9Y medium containing appropriated antibiotics and 0.5 mM IPTG. The cultures were cultivated at 30 °C for 48 h. Samples were collected at 12, 24, 36 and 48 h. OD600 values were measured. UHPLC was used for analyzing the products and intermediates.The standards  were purchased from J&K Chemicals. UHPLC (Agilent Technologies 1290 Infinity II), equipped with a reverse phase column , was used for analyzing and quantifying standards and samples. Firstly, the samples were centrifuged at 12,000 rpm for 10 min to remove the cells and sediments in medium. Then, the supernatants were filtered by 0.22 μm membrane and loaded. The column temperature was set at 30 °C. Flowing phase containing solvent A (water with 0.1% formic acid) and solvent B (100% methanol) were used, with a flow rate of 1 mL/min. The gradients were used as follows: 5% to 40% solvent B for 20 min, 100% solvent B for 2 min, 100% to 5% solvent B for 2 min and 5% solvent B for an additional 6 min. 3,4-DHBA, GA, catechol and pyrogallol were quantified based on their peak areas at 268 nm.Additional file 1: Figure S1. Analyzing the mixture of GA and NaHCO3 by UHPLC. Figure S2. MS analysis of the reaction mixture and speculation of compound in the reaction mixture. Figure S3. SDS-PAGE of Y385F/T294A PobA and Y385F/T294A/V349A PobA. Figure S4. The non-linear regression curves of PobA mutants towards 4-HBA and 3,4-DHBA through the Michaelis–Menten equation. Figure S5. Close view of the catalytic pocket of Y385F PobA with FAD and 3,4-DHBA complex. The hydrogen bonds were shown as dashed line. Figure S6. Verification of E. coli BW25113 (Fʹ), E. coli BW25113 (Fʹ)ΔaroE (CTT5) and E. coli BW25113 (Fʹ)ΔaroEΔydiB (CTT6). Figure S7. In vivo conversion of 3,4-DHBA into GA. Table S1. Plasmids and strains used in this study. Table S2. Kinetic parameters of PobA mutants towards 4-HBA and 3,4-DHBA.


We also investigated downstream kinases associated with β-arrestin-dependent signal transduction. We discovered that δOR agonist-induced seizure activity strongly and positively correlates with β-arrestin 2 efficacy for the agonist, but that indirect inhibition of ERK activation using the MEK inhibitor SL327 did not inhibit seizure potency and duration. Inhibition of the PI3K/AKT/mTOR signaling with honokiol but not PQR530, attenuated SNC80 seizure duration in β-arrestin 1 knockout, but honokiol did not reduce SNC80-induced seizures in wild-type mice. Ultimately, our results indicate that β-arrestin 2 is correlated with δOR agonist-induced seizure intensity, but that global β-arrestin 1 knockout mice are a poor model system to investigate their mechanism of action.The δ-opioid receptor (δOR) has been considered as a therapeutic target in multiple neurological and neuropsychiatric disorders particularly as δOR agonists are deemed safer alternatives relative to the more abuse-liable µ-opioid receptor drugs. Clinical development of δOR agonists, however, has been challenging in part due to the seizure-inducing effects of certain δOR agonists. Especially agonists that resemble the δOR-selective agonist SNC80 have well-established convulsive activity. Close inspection suggests that many of those seizurogenic δOR agonists efficaciously recruit β-arrestin, yet surprisingly, SNC80 displays enhanced seizure activity in β-arrestin 1 knockout mice. This finding led us to hypothesize that perhaps β-arrestin 1 is protective against, whereas β-arrestin 2 is detrimental for δOR-agonist-induced seizures. To investigate our hypothesis, we characterized three different δOR agonists  in cellular assays and  ADL5859 is a δOR agonist that was deemed safe enough to enter phase II clinical trials, whereas ARM390 is considered a low-internalizing δOR agonist (The δ-opioid receptor (δOR) is a G-protein coupled receptor (GPCR) within the opioid family that has been considered as therapeutic target for the treatment of migraine, chronic pain, anxiety, and major depressive disorder . δOR ago agonist . We also agonist , attenuaad libitum. Adult male (19–25 g) and female (16–23 g) mice between 8–16 weeks old were used for our experiments. All purchased mice were acclimated to the animal facility for at least 1 week prior to experimentation.Animal studies were carried out in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals. All animal protocols (#1305000864) describing the care and experimental use of animals were preapproved by the Purdue University Institutional Animal Care and Use Committee. Wild-type C57BL/6 male and female mice were purchased from Envigo  and β-arrestin 1 or 2 global knockout mice were bred in our facility  were dissolved in distilled water, with ADL5859 administered intraperitoneally and ARM390 administered orally via gavage (OG); SNC80  was diluted in slightly acidic saline (pH 5–6) and administered intraperitoneally. Doses for each δOR agonist were equipotent for their ability to impact behavior through δOR . SL327 . Honokion = 6 was used comprising of 3 male and 3 female mice being tested for each drug . One exception is that 5 male and 5 female β-arrestin 1 knockout were tested with SNC80; this was because these tests were used as a positive control. For experiments testing SL327 and SNC80, 3 male and 3 female wild-type and β-arrestin 1 knockout mice were tested with SL327 + SNC80, and only SL327 . For experiments testing 40 mg/kg Honokiol and SNC80, 3 male and 3 female β-arrestin 1 knockout mice were tested with Honokiol + SNC80, and only Honokiol . For experiments testing 80 mg/kg Honokiol and SNC80, 3 female and 2 male β-arrestin 1 knockout mice were tested . 3 male and 3 female β-arrestin 1 KO mice were tested with PQR530 and SNC80 . 6 female wild-type mice were tested with Honokiol + SNC80 and 6 female wild-type mice were tested with only SNC80 . After last injection, mice were immediately placed in a clear, plexiglass cage and recorded for 60 or 180 min. Videos footage was analyzed using the Modified Racine score  . For stune score , where en = 3 per sex) or saline . Tissue was collected as previously described , mouse anti-p44/42 MAPK (Erk1/2) (L34F12) , mouse anti-GAPDH (1D4)  and Li-Cor secondary antibodies: anti-mouse IRdye 680LT  and anti-rabbit IRDye 800CW . Prepared samples were scanned using the Li-Cor Odyssey® CLx Scanner (Li-Cor). For statistical analysis, all ERK bands were normalized to GAPDH.Samples (20 µL containing 10 µg protein) were loaded per well of a NuPage 4–12% Bis-Tris gradient gels , and the SDS-Page gel was subsequently transferred to nitrocellulose membranes  and Western blot was carried out using the following primary rabbit anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP2. For β-arrestin recruitment assays, U2OS-δOR-β-arrestin 1 cells or CHO-δOR-β-arrestin 2 cells were plated in low -volume 384 well plates at a concentration of 2,500 cells per well (10 µL) a day prior to running the assay. The next day, cells were stimulated with 2.5 µL of drug solution for 90 min at 37 °C/5% CO2 as previously described  was used as reference molecule.U2OS-δOR-β-arrestin 1 cells  and Chinese hamster ovarian CHO-δOR-β-arrestin 2 cells  were cultured as recommended by the manufacturer and maintained at 37 °C/5% COescribed . Followip < 0.05 was identified, and an appropriate post-hoc analysis was performed as recommended by Prism . For in-vitro assays, nonlinear regression was used to determine pEC50 (β-arrestin recruitment assays). Each data point for β-arrestin recruitment assays was performed as a technical duplicate in at least three biological triplicates. For each experiment, a positive control or reference compound was used to normalize the data and determine the log bias value.All data is presented as means ± standard error of the means (SEM). Data analysis was performed using GraphPad Prism 9 software . Seizure assays were analyzed for statistical significance using student t-test, one-way or two-way Analysis of Variance (ANOVA) depending on the number of treatment groups and independent variables. If a significant deviation of the mean p < 0.0001 and a time × agonist interaction effect F  = 2.39, p < 0.0001]. We noticed that SNC80 is the only agonist of the three to reach Racine scores higher than 2 throughout the 60 min.While it has been well-established that the δOR agonist SNC80 induces seizures , other δp < 0.0001 and a time × agonist interaction effect F  = 3.73, p < 0.0001]. Tukey’s multiple comparison found that the SNC80-induced seizure effect was significantly different from ARM390 at each time point, and different from ADL5859 starting at 9 min; ADL5859-induced seizure effect differed from ARM390 for the first 30 min post administration.SNC80 has also previously been shown to induce enhanced seizure activity in β-arrestin 1 knockout male and female mice . Given tp = 0.002, F  = 20.24, with Sidak’s multiple comparison indicating no difference between male and female over the first 60-min period post administration but a significant effect when measuring seizure activity across the 180-min period post administration  = 3.25  = 93.87. β-arrestin 1 knockout mice displayed significantly higher Racine scores compared to wild-type and β-arrestin 2 knockout mice in response to SNC80 and ADL5859; while there is no significant difference among the genotypes for ARM390, β-arrestin 1 knockout mice continue to show slightly higher scores compared to wild-type and β-arrestin 2 knockout mice. There is no significant difference between wild-type and β-arrestin 2 knockout mice in seizure response to any of the drugs tested. Altogether, these results demonstrate that β-arrestin 1 knockout mice specifically have higher propensity towards convulsive behavior in response to δOR agonists, compared to wild-type or β-arrestin 2 KO mice.Based on the confirmed finding that δOR agonists exhibit enhanced seizure activity in β-arrestin 1 knockout mice, we hypothesized that β-arrestin 1 may be protective against seizures. Under this hypothesis, we predicted that β-arrestin 2 knockout mice, that lack β-arrestin 1, would have milder seizures than wild-type mice. When we analyzed β-arrestin 2 knockout mice seizure responses to SNC80, ADL5859 and ARM390, seizure activities are similar to wild-type mice where SNC80 induced highest level of Racine scores but statistically significantly different from ADL5859 and ARM390 at the 15-min timepoint  = 8.11, ) = 3.25 . To comp) = 3.25 . We founmax (max) and the intensity of seizures they induced at the equi-analgesic doses of 10 mg/kg for SNC80 and ARM390 and 30 mg/kg ADL5859. We found that there exists a weak to moderate correlation between β-arrestin 1 recruitment Emax and Racine score achieved by the mice over the 60 min (AUC) post dosing    . Alternae: 0.99) .p < 0.0001, F  = 108.2), without a sex difference  = 3.85). We note the caveat that the male and female samples were not run on the same blot and thus no firm conclusion can be drawn for sex differences. To test our hypothesis that δOR-agonist induced activation of ERK in the hippocampus contributes to its seizure activity, we utilized the MEK inhibitor, SL327, which indirectly but effectively inhibits ERK  = 29.05, with Tukey’s multiple comparison displaying no significant effect between ‘SL327 + SNC80’ to SNC80 at any time points)  = 29.05, with Tukey’s multiple comparison “SL327 + SNC80” versus SNC80 p = 0.52)  = 143.1, with Tukey’s multiple comparison displaying a significant effect between “SL327 + SNC80” to SNC80 starting at the 36 min time point)  = 143.1, with Tukey’s multiple comparison “SL327 + SNC80” versus SNC80 p < 0.01) , we next aimed to investigate potential mechanisms of action for δOR-agonist induced seizures as it relates to β-arrestin 2 signaling. Several kinases are known to scaffold with β-arrestin 2 including extracellular signal-related kinase (ERK). ERK signaling is involved with various cellular processes and has been shown to be stimulated downstream of δOR activation . Thus, wbits ERK  in our mbits ERK . Against points) . When an = 0.52) . Though e point) . Analysi < 0.01) .p < 0.0001, F  = 68.8 and treatment x time effect p < 0.0001, F  = 2.81, with Tukey’s multiple comparison finding a significant difference between SNC80 and 40 mg/kg Honokiol + SNC80 at 18, 57 and 60 min, and between SNC80 and 80 mg/kg Honokiol + SNC80 to SNC80 at 24, 36 and 48–60 min) (p = 0.23), there the larger number of occurrences for significant difference with 10 mg/kg SNC80 does suggest that 80 mg/kg was more potent at inhibiting SNC80-induced seizures p = 0.16, F  = 2.27 and a small treatment x time effect p < 0.05, F  = 1.67, p = 0.16, We next hypothesized that δOR-agonist activation of the “mTOR pathway”, through a mechanism involving β-arrestin 2 scaffolding with AKT is responsible for their seizure activity. To determine this, we utilized the pI3K/AKT/mTOR pathway inhibitor honokiol, which has previously been used in mice and, importantly, can cross the blood brain barrier . We expo–60 min) . While tmax for 10 mg/kg SNC80 in max for 32 mg/kg SNC80 in p = 0.048 - post-hoc analysis did not find significant differences at any time-point; time × agonist interaction effect F  = 0,072, p = 0.90]  = 1.20, p = 0.26] (In the absence of reproducible antibodies in the PI3K pathway, we opted to confirm the role of the PI3K/AKT/mTOR pathway in β-arrestin 1 knockout mice using a different inhibitor. We chose to administer β-arrestin 1 knockout mice with 25 mg/kg PQR530, based on reports that this inhibitor of PI3K/mTOR is brain penetrable  and was  = 0.26] .We had hypothesized that β-arrestins were involved in the induction of seizures by certain δOR agonists as those δOR agonists producing the strongest seizures are known to efficaciously recruit β-arrestin  and becaWe initially hypothesized that β-arrestin 1 was protective against δOR-induced seizures given that β-arrestin 1 knockout mice exhibit increased seizure sensitivity compared to wild type mice in response to SNC80 . HoweverThus, we explored the hypothesis that β-arrestin 2 contributes to severe seizure activity and to investigate if kinases known to scaffold with β-arrestin 2 could be linked to δOR agonist-induced seizure. We decided to use both the wild-type and β-arrestin 1 knockout mice, the latter as a model system with reduced complexity of the cellular environment by ensuring cells only express β-arrestin 2. For this part of the study, we narrowed our approach by only testing SNC80 and by selecting the hippocampus to investigate kinase activity. The rationale, was that microinjection of various δOR agonists, including SNC80, deltorphin, and DSTLE, into the dorsal and ventral hippocampal areas produced convulsive behavior and epileptic-like EEG recordings  suggestiWe were particularly interested in studying ERK and PI3K/AKT signaling, as these are known pathways to be modulated by δOR selective agonists in the central nervous system . We firsUnder most conditions SNC80 induces seizures, however in rats 30 mg/kg SNC80 reportedly has anti-convulsant activity in reducing pilocarpine-induced seizures , highligLastly, it is also necessary to call attention to the potential sex differences seen with these seizures. Our 3-h study found that male β-arrestin 1 knockout mice had stronger seizures compared to female mice in response to SNC80; but no difference was seen within the first hour, which was why our later studies only analyzed the first hour post drug exposure. Prior studies have indicated sex differences for δOR signaling in hippocampal areas . PreviouIn conclusion, β-arrestin 1 knockout mice show a sensitized response in seizure effects . A highe


N-oxide cation and a methanesulfonate anion.The title compound is a pyridinium salt that consists of a sulfonated pyridine  6H8NO3S+·CH3O3S−, consists of a cationic sulfonated pyridine N-oxide moiety and a methanesulfonate anion. An N—O bond length of 1.4004 (15) Å is observed in the cation. In the crystal, weak C—H⋯O interactions link the components into a three-dimensional network.The title molecular salt, C N-oxide and trifluoromethanesulfonic anhydride. The reactivity of O-sulfonyl pyridinium salts toward nucleophiles and their substitution of the 2-position as reaction products were described by Umemoto et al.  Å is similar to that observed in 1-{[(trifluoromethyl)sulfonyl]oxy}pyridin-1-ium trifluoromethanesulfonate pyridin-1-ium methanesulfonate, Cre Fig. 1 consistsIn the crystal, the components are linked by C—H⋯O interactions into a three-dimensional network Table 1; the C5—et al.  in DCM (100 ml) was treated dropwise with a solution of methanesulfonic anhydride  in DCM at −30°C. After complete addition, the reaction mixture was stirred for 2 h and allowed to warm to room temperature. The white precipitate was filtered and washed with fresh DCM (30 ml). Additional drying in vacuo yields the title compound . Colourless needles suitable for X-ray crystal structure analysis were obtained by cooling a warm saturated acetonitrile solution to −30°C (Caution: heating to > 50°C leads to decomposition of the title compound.). 1H NMR  δ 8.7 , 8.1 , 7.9 , 3.5 , 2.6 . 13C NMR  δ 140.62, 129.10, 41.78, 39.65. IR : 3108 (w), 3013 (w), 2986 (w), 2943 (w), 1606 (w), 1479 (w), 1428 (w), 1381 (m), 1330 (w), 1315 (w), 1289 (w), 1182 (s), 1163 (s), 1144 (m), 1040 (s), 1002 (m), 984 (s), 818 (m), 789 (s), 762 (s), 672 (m), 655 (s), 602 (w), 554 (s), 520 (s), 507 (s), 489 (m), 456 (m), 421 (m). Analysis (%) calculated for C7H11NO6S2: C, 31.22; H, 4.12; N, 5.20; S, 23.81. Found: C, 31.02; H, 4.61; N, 4.93; S, 23.62.Following a modified literature procedure of Rössler  al. 2019, a stirrCrystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314621010269/hb4392sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2414314621010269/hb4392Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314621010269/hb4392Isup3.cmlSupporting information file. DOI: 2113673CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


As a result of the presence of two bromomethyl substituents in 3,5-bis(bromomethyl)phenyl acetate, molecular dimers formed by crystallographically non-equivalent molecules are connected to structurally different two-dimensional aggregates in which the bromine atoms participate in Br⋯Br bonds of type I and type II. In the case of 5-hydroxybenzene-1,3-dicarbaldehyde,which possesses three donor/acceptor substituents, the molecular association in the crystal creates a close three-dimensional network comprising C 10H12O2; 1), 3,5-bis(bromomethyl)phenyl acetate  and 5-hydroxybenzene-1,3-dicarbaldehyde  were determined by single-crystal X-ray analysis. The crystals of 1 are composed of strands of C—H⋯O=C bonded molecules, which are further arranged into layers. As a result of the presence of two bromomethyl substituents in compound 2, molecular dimers formed by crystallographically non-equivalent molecules are connected to structurally different two-dimensional aggregates in which the bromine atoms participate in Br⋯Br bonds of type I and type II. In the case of compound 3, which possesses three donor/acceptor substituents, the molecular association in the crystal creates a close three-dimensional network comprising Caryl—H⋯Ohydroxy, Cformyl—H⋯Oformyl and O—H⋯Oformyl bonds.The crystal structures of the title compounds, methyl 3,5-dimethylbenzoate (C The binding properties of these compounds depend on the nature of their building blocks, among others, the type of bridging units that connect two aromatic platforms , whereas compound 2 crystallizes in the triclinic space group PA and B) in the asymmetric unit of the cell. In compound 1   Fig. 3, the mol3.1, the molecules are arranged into layers extending parallel to the crystallographic [101] plane  2.57 Å; Table 1et al., 2009Cg distance of 2.77 Å. Fig. 5In the crystal structure of 2 shown in Fig. 6A are connected via Br⋯Br bonds  = 3.562 (1) Å; θ1 = 150.2°, θ2 = 158.5°] and of type II  as well as C—H⋯Br hydrogen bonds to form two-dimensional aggregates extending parallel to crystallographic [011] plane, in which the bromine atoms contribute to the formation of a cyclic four-membered synthon (Br4) and an eight-membered bonding motif . The structure of the domains created by molecule B is fundamentally different from those formed by molecule A. In them, the dimers are linked in a strand-like fashion via type I Br⋯Br interactions  , which are part of an eight-membered ring motif. In the direction of the crystallographic a-axis, the connection of the dimers occurs through π–·π (face-to-face) interactions  = 1.2–1.5Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989022005643/ex2057sup1.cifCrystal structure: contains datablock(s) 1, 2, 3, global. DOI: 10.1107/S2056989022005643/ex20571sup4.hklStructure factors: contains datablock(s) 1. DOI: Click here for additional data file.10.1107/S2056989022005643/ex20571sup5.cmlSupporting information file. DOI: 10.1107/S2056989022005643/ex20572sup2.hklStructure factors: contains datablock(s) 2. DOI: Click here for additional data file.10.1107/S2056989022005643/ex20572sup6.cmlSupporting information file. DOI: 10.1107/S2056989022005643/ex20573sup3.hklStructure factors: contains datablock(s) 3. DOI: Click here for additional data file.10.1107/S2056989022005643/ex20573sup7.cmlSupporting information file. DOI: 2174617, 2174616, 2174615CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The structure of compound 1 was established by the analysis of NMR and MS data while its chirality was established by comparison of optical rotation values with synthetically prepared authentics. Compound 1 inhibited the growth of Bacillus subtilis and Staphylococcus aureus at MIC of 50 μg/mL and 100 μg/mL, respectively.A new antibacterial 3-monoacyl- Nostoc commune, an unusual antibacterial n−1 fatty acid from N. verrucosum + observed by high-resolution ESITOFMS . Three degrees of unsaturation, calculated from the molecular formula, were accounted for by a carboxyl group (δC 175.3) and two double bonds  observed in the 13C NMR spectrum , a bisallylic methylene , a methylene adjacent to a carboxyl group , two allylic methylenes , and an aliphatic methyl group . Along with these resonances, several oxygenated (δH ca. 4.13–3.54) and aliphatic signals (δH 1.62 and ca. 1.39–1.30) were observed, implying that compound 1 is a derivative of a fatty acid. Indeed, all oxygenated protons constituted a spin system (CH21'–CH2'–CH23') in the COSY spectrum , a C8 internal hydrocarbon chain with two degrees of unsaturation (C12–C11=C10–C9–C8=C7–C6–C5), three consecutive methylenes (C4–C3–C2) with a carboxy-termination, and a glyceryl moiety (Z-geometry was deduced for both double bonds (Δ7 and Δ10) from shielded chemical shift values of the allylic carbons (C6: 27.9 ppm and C12: 28.0 ppm) D22.4 −5.9 ; UV (MeOH) λmax, nm (log ε): 200 (1.7); HRMS–ESIMS (m/z): [M + Na]+ calcd for C19H34NaO4+, 349.2349; found, 349.2348; IR (ATR) νmax: 3350, 2921, 2852, 1601, 1457, 1195, 1103, 1015, 875, 696 cm−1.Nostochopcerol  exceeded 0.1. The liquid culture was diluted to adjust the turbidity to ABS600 0.09–0.1 (0.5 McFarland), which corresponds to a cell density of 1.5 × 108 cfu/mL. This was further diluted by 75 times to prepare a cell suspension of 2.0 × 106 cfu/mL, of which 100 μL were dispensed to the wells to give microcultures with the final cell density of 1.0 × 106 cfu/mL. The plates were incubated at 37 °C for 48 h and the concentration at which the growth of microbes was completely inhibited was defined as the minimum inhibitory concentration (MIC).To each well of a sterile 96-well microtiter plate was dispensed 100 μL of tryptic soy broth. Additionally, 98 μL of the same medium and 2 μL of the solutions of test compounds in MeOH or a reference antibiotic, kanamycin monosulfate, in H1), copies of NMR spectra for 1, 3-linoleoyl-1,2-O-isopropylidene-sn-glycerol (2b), and 1-linoleoyl-sn-glycerol (3a).Supporting information features procedures for synthesis of chiral α-linoleoyl glycerols, physicochemical properties of synthetic compounds, HRESITOF mass spectrometric analysis of nostochopcerol (File 1Experimental details, characterization data and copies of spectra.