E responded in opposite manner 5 min post‐exercise compared to E + C (p = 0.013) and C (p = 0.032) in routine respiration, and to E + C in free routine activity (p = 0.013). E + C > C > E was observed for increased lactate levels and decreased isometric force that correlated with routine respiration . Lactate and pH change did not affect bioenergetics of PBMCs. Acute resistance exercise affected cellular respiration of PBMCs, with training volume and the amount of metabolic stress appear influential. Results suggest that acute inflammation response does not contribute to changes seen in cellular respiration, but the level of peripheral muscle fatigue and metabolic stress could be explaining factors.Little is known how acute exercise‐induced inflammation and metabolic stress affect immune cell bioenergetics and the portion of its components. Therefore, we investigated acute effects of eccentric‐only (E), concentric‐only (C) and combined eccentric‐concentric resistance exercise (E + C) bouts on cellular respiration of peripheral blood mononuclear cells (PBMCs). Twelve strength‐trained young men performed bench press resistance exercises in randomized order. Venous blood samples were drawn at pre‐, 5 min post‐ and 24 h post‐exercise. Several PBMC respiration states were measured using high‐resolution respirometry. Levels of leukocytes, interleukin 6 (IL‐6), C‐reactive protein (CRP), creatine kinase (CK), blood lactate and maximum voluntary isometric force were measured from the same time points. Effects of blood lactate and pH change on bioenergetics of PBMCs were investigated ex vivo. PBMC routine respiration ( Resistance exercise with different workloads have distinct effect on cellular respiration of PBMCs. Training volume and amount of metabolic stress are influential. However, an exercise model comparing responses to eccentric and concentric exercise could reveal differences in bioenergetics of PBMCs accompanying the expected differences in metabolic cost and inflammation response. Consequently, our study investigated the acute effects of three resistance exercise bouts, eccentric‐only (E), concentric‐only (C) and combined eccentric and concentric contractions (E + C), on cellular respiration of PBMCs. It was hypothesized that metabolic cost and inflammation response to C versus E are different and would lead to different respiration responses of PBMCs. Combined eccentric and concentric contractions would reveal whether high metabolic cost and high inflammation response has an additive effect on cellular respiration of PBMCs. Finally, we studied ex vivo if lactate concentration and change in pH specifically affects cellular respiration of PBMCs.22.1Twelve healthy male subjects were recruited to the study through local advertisement . Inclusion criteria were experience in resistance training (>100 kg bench press or ≥1.25 times bodyweight). We hypothesized that inflammation response in untrained test subjects might vary largely, therefore only trained test subjects were chosen to obtain more homogenous responses. In addition, inclusion criteria included no use of anabolic agents such as steroids or medication known to affect the inflammatory status.n with calculated effect sizes for study design by using G*Power program  with 80% power.We estimated the appropriate n = 11.All subjects gave their written informed consent prior to participation. All participants filled a PAR‐Q+ questionnaire  . All exercise protocols were completed with this isokinetic bench press device. The machine operated with a speed of 0.1 m/s in all three exercises, independent of the force produced against it. Ground reaction forces were recorded with a 2‐D strain gauge force plate  positioned under the bench. During the concentric and eccentric loadings, the bar's movement paused for 500 ms at the top and bottom position, to aid pacing the contractions. In E, subjects were asked to start to maximally contract their muscles, once the bar stopped at the top position and continue pushing as hard as possible until the bar stopped at the bottom position . In C, subjects were asked to maximally contract their muscle once the bar stopped at the bottom position and continue pushing until the bar stopped at the up position. During the unloaded traveling phase of the machine, subjects were instructed to rest their arms while grasping the bar to avoid activation of the targeted muscles. During E + C, only 1 ms ‘2.4Maximal isometric force was recorded at 90° of elbow flexion, measured with a custom‐made isometric bench press  were drawn from the antecubital vein from each subject while seated pre‐, 5 min post‐ and 24 h post‐exercise to Vacutainer VACUETTE K3EDTA (Greiner Bio‐One) tubes containing anticoagulant (EDTA). Whole blood count and the number of lymphocytes and monocytes were assessed by Sysmex XP‐300 (Sysmex Corporation) to obtain the number of leukocytes and platelets. 18 ml of blood was used for isolation of PBMCs and 12 ml for serum isolation.g). Serum was stored in −20°C for later analysis, which was performed using IMMULITE 2000 XPi Immunoassay System  according to manufacturers´ instructions. Lactate was measured from fingertip blood samples, which were collected into capillary tubes (20 μl) at all three time points. Samples were then placed in a 1 ml hemolyzing solution and analyzed automatically with Biosen C‐line lactate analyzer .For the detection of IL‐6, CK and CRP, serum was isolated from blood samples by centrifugation . Standing time did not have a statistical effect on cellular respiration.g . The layer of PBMCs and platelets was collected and washed twice with RPMI‐1640  in order to reduce PLT/PBMC ratio below 7  with 15 ml of Ficoll‐Paque TM PLUS  . Substrate‐uncoupler‐inhibitor titration 3 (SUIT3) protocol designed for intact cells was chosen for measurements  from five resistance trained men and isolated PBMCs and measured the cellular respiration by using the same methods as described above.To test whether lactate had an impact on the cellular respiration of PBMCs, 20 mmol of sodium L‐lactate (Sigma Aldrich) was added to the HRR measurement chamber after measurements of routine respiration of PBMCs and SUIT3 protocol was completed ‐lactic acid (Sigma Aldrich) to the HRR measurement chamber after routine respiration of PBMCs and SUIT3 protocol was completed. According to studies, a vigorous exercise can drop the pH of blood near 7.10 and could be under 7.0 in skeletal muscle .Statistical analyses were carried out using the Statistical Package for Social Sciences . Values are reported as mean ± standard deviation (SD), unless otherwise stated. All values were verified for outliers, which were excluded from the analysis (>3× interquartile range [IQR]). Significance level was set at 0.05 in all analyses. ANOVA with repeated measures was used to examine whether there were differences in the study variables between the groups over time . Mauchly's test was used to test the assumption of sphericity and Greenhouse–Geisser corrections were applied if the assumption was violated. To study the effect of time separately within resistance exercises and group × time interaction between adjacent time points, within‐subject contrasts were used as post hoc comparison. Hedges' 33.1Cellular respiration of PBMCs was analyzed through several respiration states Table , which i1) = 8.2, p = 0.017, g = −0.83), free routine activity (F(1) = 6.8, p = 0.025, g = −0.73) and ET‐ capacity (F(1) = 5.7, p = 0.038, g = −0.69) decreased significantly immediately after E + C  = 10.8, p = 0.008, g = 0.95)  = 6.5, p = 0.032, g = 0.73) and E + C (F(1) = 9.6, p = 0.013, g = 0.94) in routine respiration  = 9.5, p = 0.013, g = 0.87)  in respiration states immediately after resistance exercise (from pre‐exercise to 5 min post‐exercise response) were compared between resistance exercises Figure . Post hon Figure . Free ro) Figure .1) = 7.4, p = 0.026, g = 1.17) .Main effects of group and time, and their interaction did not show statistical significances on CRP, CK or IL‐6, hence there were no major changes between time points or between resistance exercises. Mean values and SDs of the data are represented in supplementary data  = 89.3, p < 0.001, g = −2.74; E: −22 ± 6%, F(1) = 131.2, p < 0.001, g = −3.32; E + C: −41 ± 10%, F(1) = 160.6, p < 0.001, g = −3.84) and this was reversed at 24 h post‐exercise  = 46.1, p < 0.001, g = −2.14) and C (F(1) = 6.4, p = 0.032, g = −0.79) showed a statistically greater decrease than E  = 13.8, p = 0.005, g = 1.19)  in isometric force (from pre‐ to 5 min post‐exercise response) were compared between resistance exercises Figure . E + C  Figure .3.2.31) = 103.1, p < 0.001, g = 3.08; E: F(1) = 64.0, p < 0.001, g = 2.32; E + C: F(1) = 171.6, p < 0.001, g = 3.80)  Figure  and this1) = 14.8, p = 0.004, g = 1.27) and C (F(1) = 5.3, p = 0.047, g = 0.70) showed a statistically greater increase than E. Also, the increase from E + C was greater than the increase from C (F(1) = 5.2, p = 0.048, g = 0.69)  in lactate levels (from pre‐ to 5 min post‐exercise response) were compared between resistance exercises Figure . E + C  Figure .3.3Pearson correlation coefficients for 5 min post‐exercise response in routine respiration, free routine activity and ET‐capacity in relation to 5 min post‐exercise response in lactate, isometric force and inflammation and muscle injury markers are presented in Table 3.43) = −3.583, p = 0.035)  Figure .3.5WBCs, as well as lymphocytes and monocytes, which are the main components of PBMCs, were counted at each time point and analyzed between timepoints and resistance exercises. The effect of plasma volume change was examined (according to Dill & Costill 1974] equation)  = 37.4, p < 0.001, g = 1.70; E: F(1) = 9.9, p = 0.010, g = 0.91; E + C: F(1) = 18.7, p = 0.001, g = 1.20) (Table 1) = 5.7, p = 0.039, g = −0.72) and E + C (F(1) = 9.0, p = 0.012, g = −0.84). In C, the difference between pre‐ and 24 h post‐exercise WBC counts were also significantly different (F(1) = 14.0, p = 0.003, g = 1.04)  Table . The dif1) = 18.6, p = 0.001, g = 1.16; E: F(1) = 8.8, p = 0.014, g = 0.83; E + C: F(1) = 20.6, p = 0.001, g = 1.22) (Table 1) = 11.7, p = 0.006, g = −0.92). The difference between pre‐ and 24 h post‐exercise lymphocyte count was significantly different in C (F(1) = 11.1, p = 0.007, g = 0.89). The number of monocytes was elevated after C (F(1) = 6.5, p = 0.027, g = 0.68) and E (F(1) = 5.3, p = 0.045, g = 0.64) and pre‐ and 24 h post‐exercise count reached statistically significant difference in E (F(1) = 7.7, p = 0.019, g = 0.77).Each resistance exercise elevated the number of lymphocytes (C: F(2) Table . The dif1) = 11.7, p = 0.007, g = 0.66) and E (F(1) = 5.0, p = 0.048, g = 0.55). The number of lymphocytes was significantly higher after E + C when compared to C (F(1) = 10.1, p = 0.010, g = 0.66). There were no significant differences in the response between exercises among monocytes.Change (∆) in WBC, lymphocyte and monocyte counts (5 min post‐ exercise response) were compared between resistance exercises  contractions caused an acute decrease in cellular respiration of PBMCs as assessed by routine respiration, free routine capacity and ET‐capacity Figure . ResultsChanges seen in routine control ratio were comparable with respiration results normalized to cell concentration Figure . E + C c4.1PBMCs have a central role in muscle repair and exercise‐related anti‐inflammatory response by producing anti‐inflammatory cytokines  were somewhat lower than a previous study (10–12 mmoL/L)  correlated negatively with the change in routine respiration Table , indicat4.3Peripheral muscle fatigue is a result of several mechanisms including metabolic acidosis and reduction of ATP  axis eventually leading to elevated cortisol levels  exercise‐induced inflammation nor (2) anaerobic metabolites were contributing factors to the acute respiration responses.Experimental design: M.L., S.W., E.I.L., M.K. and I.K. Investigation and data collection: E.I.L, M.K., I.K., E.L. and A.K. Writing and manuscript preparation: E.I.L, M.K., A.K., H.K., S.W. and M.L. Review and editing of the manuscript: S.W., M.L., E.I.L. and H.K. Funding acquisition: H.K. All authors have read and agreed the final version of the manuscript.No conflicts of interest are declared by the authors.


The title compound arose from an unexpected alcoholysis of a prodrug by the methanol solvent. 14H13ClN2O2, was obtained during an attempt to grow single crystals of 4-acetylphenyl 2-[(3-chloro-2-methylphenyl)amino]nicotinate in methanol, and was probably generated by alcoholysis. Two intramolecular hydrogen bonds are formed, one between the N—H group and the carbonyl O atom of the ester and the other between the ortho sp2CH group of the benzene ring and the pyridine N atom. Aromatic π–π stacking [shortest centroid–centroid separation = 3.598 (2) Å] is observed in the extended structure.The title compound, C I) was first synthesized when preparing esters of anthranilic acid as possible analgesic and anti-inflammatory agents ° between the C1–C6 benzene and N2/C8–C12 pyridine rings  Å]: both of these close S(6) rings. The cohesion of the crystal structure is ensured by aromatic π–π stacking between the benzene and pyridine rings [shortest centroid–centroid separation = 3.598 (2) Å] and hydrophobic interactions amino]nicotinate, synthesized by a condensation reaction between clonixin and paracetamol  global, I. DOI: 10.1107/S2414314621005393/hb4383Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314621005393/hb4383Isup3.cmlSupporting information file. DOI: 2085247CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The crystal structure of the title compound is consolidated by N—H⋯Cl and C—H⋯Cl hydrogen bonding interactions. 2H8N2)3][ZnCl4], exists as discrete ions. The [Zn(C2H8N2)3]2+ cation exhibits a distorted octahedral shape. In the [ZnCl4]2− anion, the ZnII atom is in an almost regular tetrahedral environment. The crystal packing is consolidated by N—H⋯Cl and C—H⋯Cl hydrogen bonds.The title complex, [Zn(C X hydrogen bonds. Metal complexes containing an ethylenediamine (–NCH2CH2N) backbone have attracted significant interest as potential anticancer agents because of their rich redox chemistry and relative ease of manipulation  is a common chelating ligand that is widely used in transition-metal complexes. It cannot only chelate metal cations by two nitrogen atoms, but also offers hydrogen atoms to form N—H⋯Metals such as zinc act as a key structural component in many proteins and enzymes, including transcription factors, cellular signalling proteins and DNA repair enzymes 3][ZnCl4], which comprises an ZnCl42− anion and a [Zn(en)3]2+complex cation. The ZnII atom of the tetrachloridozincate(II) anion is in an almost regular Cl4 tetrahedral environment, with Zn—Cl bond lengths in the range 2.255 (1)-2.272 (9) Å. The zinc cation displays a distorted octahedral coordination geometry defined by six N atoms from three ethylenediamine ligands, with Zn—N distances in the range of 2.173 (3)–2.219 (3) Å. The N—Zn—N angles of the en ligands are about 80°. They are noticeably smaller than the ideal octahedral angle of 90°. The five-membered chelate rings are non-planar, with N—C—C—N torsion angles of −57.5 (4), −55.4 (4) and −55.9 (5)°. All of the three en ligands assume a synclinal conformation about the C—C bond.Fig. 1via intermolecular hydrogen bonds. The N—H⋯Cl hydrogen-bonding interactions between the N atoms of the ethylenediamine ligands and Cl atoms of the tetrachloridozincate anion connect the molecules, together with the weak C—H⋯Cl intramolecular interactions, generating a three-dimensional network  mixture. To this solution, ethylenediamine  in 25 ml of an HCl/EtOH (2:3 v/v) mixture was added dropwise. The mixture was stirred and heated to 338 K for 2 h and allowed to stand at room temperature until colourless crystals separated (3–4 weeks). Crystals suitable for single-crystal XRD were collected after recrystallization using acidified water.Zinc chloride  was dissolved in 25 ml of EtOH/HCrystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314620006185/bt4091sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2414314620006185/bt4091Isup2.hklStructure factors: contains datablock(s) I. DOI: 2001547CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In Table 1, the fraction of p‐c‐Jun+neurons in NeuroTrace+ neurons for AR group (Estimate and 95%CI) is shown as “10.3% (10.1–10.5%).” This should be “13.0% (12.8–13.3%).” The corrected Table 1 is shown below:<New Table 1 here>In Table S3, the fraction of p‐c‐Jun+neuronal population for AR group (Estimate and 95%CI) is shown as “10.3% (10.1–10.5%).” This should be “13.0% (12.8–13.3%).” Corrected version of Table S3 is corrected online.In the article by Ogino et al.,


The structure of hexaaquanickel(II) bis(3-carboxy-4-hydroxybenzenesulfonate) dihydrate consists of alternating layers of inorganic cations and organic anions linked by O—H⋯O hydrogen bonds that also include non-coordinated water molecules of crystallization. The structure of hexaaquacobalt(II) bis(3-carboxybenzenesulfonate) dihydrate is also built of alternating layers of complex cations and organic anions without direct coordination to the metal by the protonated carboxylate or unprotonated sulfonate groups. A robust O—H⋯O hydrogen-bonding network involving primarily the coordinated and non-coordinated water molecules and sulfonate groups directs the packing. 2O)6][C6H3(CO2H)(OH)SO3]2·2H2O, (I), crystallizes in the triclinic space group P2O)6][C6H4(CO2H)SO3]2·2H2O, (II), also crystallizes in triclinic PHexaaquanickel(II) bis(3-carboxy-4-hydroxybenzenesulfonate) dihydrate, [Ni(H As a result of the symmetry, the nickel ion has a very regular octahedral coordination of six water molecules  −x + 1, −y + 2, −z], and two in which the water hydrogen atoms H41 and H42 are donors to sulfonate oxygen atoms O1 and O2, respectively.The product of the reaction of nickel nitrate hexahydrate and 5-sulfosalicylic acid (3-carboxy-4-hydroxybenzenesulfonic acid) is [Ni6](C6H4(CO2H)SO3)2·2H2O, (II)Pet al., 1993vs C7—O5 = 1.213 (2) Å] and rotated slightly out of the plane of the ring [torsion angle C2—C3—C7—O4 = 4.6 (2)°]. The sulfonate group is rotated about 19° from its position in (I)vs −25.29 (12)° in (I)]. Presumably this difference is driven by the hydrogen-bonding patterns. The non-coordinated water molecule has a different hydrogen-bonding environment , respectively. These interactions are somewhat longer and less linear than those seen in (I)The reaction of cobalt nitrate hexahydrate and sodium 3-sulfobenzoate (3-carboxybenzenesulfonate) produced crystals that have been identified as (C6H3(CO2H)(OH)SO3)2·4H2O structure has also been reported for cobalt 6](C6H3(CO2H)(OH)SO3)2·2H2O have been reported for manganese  salt of 3-carboxy-4-hydroxybenzenesulfonate has been reported previously ns Fig. 4. Two watns Fig. 4. The nonns Fig. 4, and as ns Fig. 4. Other O4.et al., 2016et al., 2005aet al., 2004et al., 2003det al., 2005et al., 2003bet al., 2003aet al., 2003eet al., 2003cet al., 2011et al., 2019A search of the Cambridge Structural Database 2.6H2O (Aldrich) in 50 ml of water. The resulting clear green solution was stirred for about 30 minutes and transferred to a porcelain evaporating dish that was set out to evaporate in a fume hood. After several days, the water had completely evaporated leaving behind large elongated (>1 cm) green slab-shaped crystals, 2.57 g of which were collected by hand from the dish. These were identified as (I)3)2.6H2O (Aldrich) in 50 ml of water. The resulting red solution was stirred for 30 minutes, transferred to a porcelain dish, and set out to evaporate. The final red product was primarily polycrystalline but some small red–pink plates were found to be suitable for single-crystal X-ray analysis, leading to their identification as (II)A 2.54 g (10.0 mmol) sample of 5-sulfosalicylic acid (3-carboxy-4-hydroxybenzenesulfonic acid)  was dissolved in 100 ml of water. To this colorless solution was added a green solution of 2.91 g (10.0 mmol) of Ni(NO6.Uiso constrained to be 1.2 times the Ueq of the bonding atom. Oxygen-bound hydrogen atoms were located in difference electron-density maps and refined with isotropic displacement parameters while the O—H distances were restrained to 0.84 (1) Å.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989022008295/mw2190sup1.cifCrystal structure: contains datablock(s) I, II, global. DOI: 10.1107/S2056989022008295/mw2190Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989022008295/mw2190IIsup3.hklStructure factors: contains datablock(s) II. DOI: 2202460, 2202459CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the crystal structure of the title compound, the dimesitylboron group acts to reduce the delocalization of the nitrogen atom’s lone pair into the pyrrole ring, with increases in the two N—C bond lengths compared to pyrrole itself. The N—B bond is 1.44125 (15) Å long. 22H26BN, the B atom acts to reduce the delocalization of the nitrogen lone-pair electron density into the pyrrole ring, so that the two N—C bonds increase in length to 1.4005 (14) and 1.3981 (14) Å. The N—B bond length is 1.4425 (15) Å, which is longer than a typical N—B bond because the nitrogen lone pair is not fully available to participate in the bond.In the crystal structure of the title compound, C This molecule has been investigated previously for its fluorescence, and shows a large Stokes shift, which involves twisted intramolecular charge transfer (TICT) of the excited state, by rotation about the N—B bond  and 3.736 (3) Å], and is supported by the widening of the C—B—C bond angle [125.18 (9)°], compared to the two N—B—C bond angles [118.30 (10) and 116.42 (9)°]. The mesityl groups’ planes lie at 77.14 (4)° to each other, and at 69.18 (3) and 67.06 (4)° to the pyrrole ring’s best plane. The hydrogen atoms of three methyl groups  were modelled in two orientations. The positions and displacement parameters of hydrogen atoms on the pyrrole ring were refined, and for those attached to the Cα atoms, the H—Cα—Cβ angle showed widening to 131–132°, similar to that in pyrrole (pyrrol-1-yl)borane ca 0.04 Å longer than in similar compounds where the nitrogen atom is attached to two sp3 carbon atoms  and where the nitrogen lone pair is fully available for donation to boron. Compared to the molecular geometry of pyrrole itself, as determined by X-ray crystallography at 103 K  and 1.3514 (17) Å and the Cβ—Cβ bond length is 1.4290 (17) Å. Thus, in contrast to pyrrole, the N—Cα and Cα—Cβ bonds are no longer similar in length, due to a reduction in the contribution of the nitrogen atom’s lone pair to the electronic π system of the pyrrole ring. When the mesityl groups are replaced by pentafluorophenyl groups in derivative 2, the N—B bond is considerably shorter than in 1 [1.4094 (9) cf. 1.4425 (15) Å] due to greater lone-pair donation from nitrogen towards the more electron-deficient boron. Consequently, compared to 1, the pyrrole ring shows slightly longer N—C bonds [1.4033 (6) Å] and a longer Cβ—Cβ bond [1.4418 (9) Å], though similar lengths for the Cα—Cβ bonds [1.3553 (6) Å] (Table 11 is similar to that when the pyrrole nitrogen atom is substituted with a carbonyl group to form an amide (Table 1The N—B bond is 1.4425 (15) Å long. This is ] Table 1. For come Table 1.3.ab plane . Within a layer, the molecules are related by centres of symmetry and translations along a and b. Adjacent layers are related by the twofold screw and n-glide planes. The four shortest intermolecular C⋯H distances are in the range 2.81–2.83 Å. Two of these involve the meta-C atom, C18, with a methyl hydrogen atom and a pyrrole ring’s hydrogen atom, which are directed to opposite sides of the phenyl ring [C18⋯H12A and C18⋯H3]. The two others involve both a meta and a para-C atom of the second phenyl ring and the pyrrole hydrogen atom H2 [C8⋯H2 and C9⋯H2].The molecules are packed in layers in the ne Fig. 2. There a4.1 bearing two 2′-thienyl groups in the pyrrole’s 2- and 5-positions –1.457 (3) Å] with a correlation between the increasing angle between bonding planes and longer N—B bonds  and 1.440 (3) Å, similar to those in 1  using hexane:dichloromethane (8:1) as eluent to give 1  as a slightly oily white solid, from which crystals were grown using ethyl acetate, m.p. 411 K. 1H NMR  [ppm]: δ = 6.84 , 6.81 , 6.37 , 2.32 , 2.11 ; 13C NMR  [ppm]: δ = 141.7 , 139.0 (2 × 4′-C), 136.5 br (2 × 1′-C), 128.3 , 126.5 , 114.6 , 22.8 , 21.5 (2 × 4′-CH3); IR (ATR): 2920, 2853, 1606, 1472, 1451, 1421, 1399, 1378, 1329, 1310, 1287, 1252, 1156, 1122, 1080, 1074, 1043, 1030, 850, 817, 763, 733, 717, 677, 656, 619, 560, 516 cm−1.A solution of pyrrole  in THF (10 mL) under nitrogen was treated with sodium hydride  and the mixture stirred at 293 K for 2 h. Dimesitylboron fluoride (CARE: gives HF with moisture)  in dry THF (10 mL) was added at room temperature. The mixture was left to stir overnight. The bright orange–yellow mixture was quenched with water (20 mL), extracted with ether (2 × 30 mL) and the combined organic phase was dried over MgSO6.Ueq of the parent atom for aromatic groups and at 1.5Ueq for methyl groups.Crystal data and details of data collection and structure refinement are summarized in Table 210.1107/S2056989022011768/zv2023sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989022011768/zv2023Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989022011768/zv2023sup3.docxThis is NMR data requested by the referee. DOI: Click here for additional data file.10.1107/S2056989022011768/zv2023Isup4.cmlSupporting information file. DOI: 2166138CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Due to a production error, there were inaccuracies in the section numbering. The corrected subsection numbering should read as follows:1. Introduction2. Immunotheraputic Strategies Targeting the TME 2.1 Therapeutic Strategies Based on TME Inhibitory Signaling  2.1.1 Targeting TME Physical Barriers  2.1.2 Targeting Immune Checkpoints  2.1.3 Targeting Immunosuppresive Cells  2.1.4 Targeting Inhibitory Cytokines  2.1.5 Targeting Metabolic Inhibition Signaling 2.2 Therapeutic Strategies Based on TME Stimulatory Signals  2.2.1 Targeting Stimulatory Checkpoints  2.2.2 Application of Stimulating Cytokines  2.2.3 Enhancing Antigen Presentation  2.2.4 Application of Immune Effector cells3. Challenges Faced by Time Research and Solutions 3.1 Complexity of TIME 3.2 Spatiotemporal Heterogenity of TIME 3.3 Systemic Immunity Affects TME Immune Response4. Summary and ProspectThe publisher apologizes for this mistake.The original version of this article has been updated.


MAPK can underlie the pro-differentiation effect of CP-673451 on GBM cells. Overall, the present study identifies a potential novel therapeutic option that could benefit GBM patients in the future, through differentiation of residual GSCs post-surgery, with the aim to limit recurrence and improve quality of life.Glioblastoma (GBM) is the most common and fatal primary brain tumour in adults. Considering that resistance to current therapies leads to limited response in patients, new therapeutic options are urgently needed. In recent years, differentiation therapy has been proposed as an alternative for GBM treatment, with the aim of bringing cancer cells into a post-mitotic/differentiated state, ultimately limiting tumour growth. As an integral component of cancer development and regulation of differentiation processes, kinases are potential targets of differentiation therapies. The present study describes how the screening of a panel of kinase inhibitors (KIs) identified PDGF-Rα/β inhibitor CP-673451 as a potential differentiation agent in GBM. We show that targeting PDGF-Rα/β with CP-673451 in vitro triggers outgrowth of neurite-like processes in GBM cell lines and GBM stem cells (GSCs), suggesting differentiation into neural-like cells, while reducing proliferation and invasion in 3D hyaluronic acid hydrogels. In addition, we report that treatment with CP-673451 improves the anti-tumour effects of temozolomide in vivo using a subcutaneous xenograft mouse model. RNA sequencing and follow-up proteomic analysis revealed that upregulation of phosphatase DUSP1 and consecutive downregulation of phosphorylated-p38 Glioblastoma (GBM) is the most common and fatal primary brain tumour in adults , 2 with While numerous therapeutic approaches aim for cancer cell death, a few studies have instead started exploring treatments that induce cancer cell differentiation into non-neoplastic cells as a novel strategy . Indeed,GBM treatment could highly benefit from differentiation therapies. High resistance to classic cytotoxic treatments and the presence of pluripotent cancer stem cells (CSCs) in residual tumour tissues due to incomplete surgical excision make GBM a good candidate for differentiation therapies –16. SeveIn accordance with these reports, the present study describes the screening of a panel of KIs that led to the identification of a receptor tyrosine kinase (RTK) inhibitor, namely CP-673451, which targets the platelet-derived growth factor receptor α/β (PDGF-Rα/β) as a potential differentiation agent in GBM. Herein, we propose that CP-673451 treatment can induce the upregulation of phosphatase dual-specificity phosphatase 1 (DUSP1) and consecutive downregulation of phosphorylated p38 mitogen-activated protein kinase (MAPK), leading to the onset of differentiation of GBM cells, thus limiting proliferation and invasion in vitro, while improving the anti-tumour effects of temozolomide in vivo.50, no previously identified undesirable effects  at the required concentrations and with the ability to cross the blood-brain barrier (BBB) [p-value) can be found in Table Forty-seven KIs were selected from the KI library (SelleckChem) on the basis of high potency with low ICer (BBB) –27. A fun = 47) for 24 h before nine images were taken per well. All images were analysed for total neurite-like process length and normalised by cell number to obtain an average neurite-like process length per cell , pink, blue and red triangles indicate KIs with significant effects compared to the control . Inhibitors showing effects with p ≤ 0.001 were selected for further experiments, namely: LY2835219, a potent and selective inhibitor of CDK4/6 [Neurite-like process outgrowth was quantified following treatment to select KIs with potential differentiating effects. A schematic diagram illustrates the experimental design Fig.  with U87ell Fig. . The gref CDK4/6 , CP-6734f CDK4/6  and pacrf CDK4/6  Fig. 1Bn = 47 fop ≤ 0.001), 30.5 ± 5.1 μm (p ≤ 0.01), 52.8 ± 6.9 μm (p ≤ 0.001) and 32.1 ± 3.6 μm (p ≤ 0.01), respectively. In AS, a significant increase of the average neurite-like process length per cell was observed following treatment with CP-673451 (72.1 ± 24.63 μm (p ≤ 0.05)) as compared to the control (28.2 ± 5.3 μm). CP-673451 similarly caused the average neurite-like process length per cell to increase significantly in LN229 cells (25.3 ± 7.7 μm (p ≤ 0.01)) as compared to the control (0.9 ± 0.5 μm). Finally, LY2835219, pelitinib and CP-673451 treatments caused a significant increase in the average neurite-like process length (15.3 ± 5.2 μm (p ≤ 0.05), 15.9 ± 6.4 μm (p ≤ 0.05) and 56.0 ± 9.6 (p ≤ 0.001) respectively) in the U138 cells compared to the control (3.9 ± 0.3 μm) , as a normal brain cell type control . Neuriteμm) Fig. .Fig. 2Sep ≤ 0.01) compared to the control, while a slight decrease in viability was observed in U138 and LN229 GBM cells following treatment with pacritinib (0.9 ± 0.05 RAU (p ≤ 0.05) and 0.8 ± 0.06 RAU (p ≤ 0.05), respectively). Treatments with LY2835219, pelitinib and pacritinib were also observed to significantly affect LN229 cell proliferation compared to the control (0.8 ± 0.09 RAU (p ≤ 0.01), 0.8 ± 0.1 RAU (p ≤ 0.05) 0.8 ± 0.1 RAU (p ≤ 0.05), respectively). Altogether, treatment with 1 μM of the selected KIs appeared to stimulate neurite-like process outgrowth in most studied GBM cell lines and astrocytes, with limited impact on cell viability and proliferation.To examine how various treatments affected cell viability and growth, metabolic activity and proliferation were measured following treatment with the inhibitors of interest using WST-1 ) in G166 cells as compared to control (1.2 ± 0.6 μm). In addition, 1 µM LY2835219, CP-673451 and pacritinib led to significant increases in the average neurite-like process length (35.9 ± 7.0 μm (p ≤ 0.01), 21.6 ± 2.7 μm (p ≤ 0.01) and 20.3 ± 5.1 μm (p ≤ 0.05), respectively) in GS090 GSCs compared to the control (13.8 ± 2.5 μm). Finally, 1 µM LY2835219, pelitinib and CP-673451 significantly increased the average neurite-like process length per cell (26.7 ± 6.0 μm (p ≤ 0.05) 25.2 ± 3.2 μm (p ≤ 0.05) and 28.1 ± 4.6 μm (p ≤ 0.05), respectively) in G179 GSCs, compared to the control (12.3 ± 4.4 μm). Representative light microscopic images are shown, with neurite-like processes indicated by red arrows.As one of the drivers of GBM recurrence with multipotent abilities, GSCs are the putative main targets of differentiation therapy. For these reasons, the selected KIs were used to treat different GSC lines, including G166, GS090 and G179. Their morphology was observed using light microscopy following treatment and the average neurite-like process length per cell was calculated as before Fig. . Treatmep ≤ 0.05) and 0.9 ± 0.07 RAU (p ≤ 0.05), respectively). On the other hand, cell viability increased significantly following treatment with CP-673451 (1.04 ± 0.02 RAU (p ≤ 0.05)) as compared to the control. Further, proliferation assays indicated that G166 cell proliferation decreased after pelitinib treatment (0.9 ± 0.04 RAU (p ≤ 0.01)) but increased upon treatment with CP-673451 (1.3 ± 0.14 RAU (p ≤ 0.05)) as compared to the control. Regarding GS090 cells, CP-673451 treatment decreased proliferation (0.99 ± 0.01 RAU (p ≤ 0.01)) as compared to the control. None of the KI treatments caused significant changes in G179 proliferation.GSC viability (WST-1) -PDGF-Rα/β (tyr849/tyr857) was measured as well as PDGF-Rβ upon treatment with CP-673451. U87 GBM cells were treated with 1 µM CP-673451 for 0, 15 min, 1, 4, 24 and 48 h ), PROM-1 (1.64 ± 0.18 fc (p ≤ 0.01)) and VEGFA (1.29 ± 0.05 fc (p ≤ 0.001)). Treatment also indicated significant decreases in GFAP (0.54 ± 0.07 fc (p ≤ 0.001)), CSPG4 (0.57 ± 0.04 fc (p ≤ 0.0001)) and CD44 (0.7 ± 0.03 fc (p ≤ 0.001)). Upon treatment with 1 μM CP-673451, G179 GSCs also showed a significant increase in the expression of NEFL (3.47 ± 0.51 fc (p ≤ 0.0001) and significant decreases in the expression of nestin and PROM1 (0.69 ± 0.09 fc (p ≤ 0.001) and 0.18 ± 0.37 fc (p ≤ 0.01), respectively), CSPG4 (0.65 ± 0.06 fc (p ≤ 0.001) and VEGFA (0.66 ± 0.03 fc (p ≤ 0.001).Gene expression of specific differentiation markers was quantified using qRT-PCR in order to determine which specific normal brain cell lineage  the GBM cells were differentiating into upon CP-673451 treatment. The selected differentiation markers were as follows; Astrocytic (GFAP and ALDH1L1), Neuronal (β3-tubulin and NEFL), stem cell (nestin and PROM-1 (CD133), oligodendrocyte (OLIG2 and CSPG4) and mesenchymal (VEGFA and CD44). Upon treatment of U87 GBM cells with 1 µM CP-673451 for 96 h Fig. , cells sAltogether our data showed that treatment with CP-673451 could trigger a neuron-like differentiation in GBM cells, as supported by the formation of neurite-like processes and expression of the neuronal NEFL marker in GBM cells, with concomitant decrease of expression of markers for stem cells and other brain cell types.p ≤ 0.05)). Conversely, a significant decrease in cell proliferation was observed in GS090 cells upon treatment with 5 µM CP-673451 (0.7 ± 0.03 RAU (p ≤ 0.01)), as compared to the control. All three cell lines showed a significant decrease in proliferation when treated with 10 µM CP-673451: U87 (0.7 ± 0.04 RAU (p ≤ 0.001)), GS090 (0.6 ± 0.07 RAU (p ≤ 0.001)) and G179 (0.7 ± 0.06 RAU (p ≤ 0.01)), as compared to the control.To further characterise the effect of CP-673451 on proliferation, additional experiments with increasing concentrations of the inhibitor were performed. The U87 GBM cell line and GS090 and G179 GSCs were treated with increasing concentrations of CP-673451  for 48 h, while a proliferation assay  was also performed Fig. . A signip ≤ 0.001) and G179: 0.6 ± 0.07 (p ≤ 0.001). Following CP-673451 treatment, significant decreases in cell colony size were observed at 1, 5 and 10 µM in U138 (0.7 ± 0.17 (p ≤ 0.05), 0.6 ± 0.11 (p ≤ 0.01), 0.5 ± 0.05 (p ≤ 0.0001)) and GS090 (0.8 ± 0.09 (p ≤ 0.05) 0.7 ± 0.08 (p ≤ 0.01) and 0.5 ± 0.1 (p ≤ 0.01)). No significant alterations of the number of colonies were observed in the tested cell lines upon CP-673451 treatment. Yet, a significant decrease in U87 cell metabolic activity  and 0.8 ± 0.06 (p ≤ 0.01), respectively). Similarly, U138 showed a significant decrease in metabolic activity upon treatment with 1, 5 and 10 μM CP-673451 (0.7 ± 0.05 (p ≤ 0.001), 0.7 ± 0.07 (p ≤ 0.01), 0.7 ± 0.06 (p ≤ 0.001)), while both GS090 and G179 GSCs showed a significant decrease in metabolic activity upon CP-673451 treatment at 10 µM (0.6 ± 0.11 (p ≤ 0.01)) and (0.9 ± 0.04 (p ≤ 0.01)), respectively. Altogether, it appears that treatment with CP-673451 limits the proliferation and invasiveness of GBM cells while stimulating their neuron-like differentiation in vitro.A HA based hydrogel assay was implemented to assess the invasiveness and colony forming ability of the U87 and U138 GBM cell lines and GS090 and G179 GSCs in a 3D model when treated with increasing concentrations of CP-673451 . Cell metabolic activity was also measured in parallel (using a CellTiter-Glo luminescent cell viability assay). Both the colony areas Fig.  and coloity Fig.  was measIn the interest of ensuring the effects from pharmacological inhibition in U87 GBM cells upon CP-673451 treatment were due to specific inhibition of its targets PDGF-Rα and PDGF-Rβ, gene silencing using siRNA was performed.p ≤ 0.0001) and 95% 0.05 ± 0.03 (p ≤ 0.0001), respectively. In addition, PDGF-Rβ expression was also reduced at both 50 nM and 100 nM by 47% 0.53 ± 0.18 (p ≤ 0.01) and 46% 0.53 ± 0.23 (p ≤ 0.05), respectively. KD of PDGF-Rα and β was also validated in G179 cells ) and PDGF-Rβ 48% (0.52 ± 0.2 (p ≤ 0.01)) compared to the control. At 100 nM siRNA, the gene expression of PDGF-Rα was reduced by 57% (0.43 ± 0.1 (p ≤ 0.0001)) while gene expression of PDGF-Rβ was reduced by 48% (0.52 ± 0.02 (p ≤ 0.001)). Further validating the siRNA transfection, the protein expression of PDGF-Rα and PDGF-Rβ was decreased in U87 GBM cells and G179 GSCs. PDGF-Rβ protein expression was reduced by 72% in U87 GBM cells at both 50 nM and 100 nM concentrations of siRNA compared to control (0.28 ± 0.1 (p ≤ 0.0001))  and PDGF-Rβ by 33% 0.67 ± 0.2 (p ≤ 0.05). 100 nM siRNA reduced PDGF-Rα protein expression by 26% 0.74 ± 0.1 (p ≤ 0.05) and PDGF-Rβ by 40% 0.60 ± 0.2 (p ≤ 0.05) ). Treatment of U87 GBM cells with 100 nM PDGF-Rβ siRNA compared to the control (18.9 ± 1.7 µm) displayed a significant increase in neurite-like process length average per cell (29.8 ± 2.9 µm (p ≤ 0.001)), as did the combination of PDGF-Rα and β siRNA (29.1 ± 6.8 µm (p ≤ 0.05)). Similarly, KD PDGF-Rα in G179 cells with siRNA at 50 and 100 nM significantly increased the average length of neurite-like process per cell (10.7 µm ± 2.1 (p ≤ 0.05)) compared to the control (7.9 ± 1.5 µm) and (12.4 ± 2.9 µm (p < 0.05)) compared to the control (8.8 ± 1.3 µm), respectively. KD of both PDGF-Rα and β also increased neurite-like process outgrowth compared to the controls (10.7 ± 1.2 µm (p ≤ 0.05)) ) Fig. .Fig. 4GeAs specific gene silencing of PDGF-Rα and PDGF-Rβ enhanced neurite-like process growth in U87 GBM cells and G179 GSCs, these data suggest that the observed impact of CP-673451 treatment on GBM cells should be, at least partly, due to specific PDGF-Rα/β inhibition.3): TMZ: 16.75 ± 5.4 mm3 (p ≤ 0.01), CP-673451: 35.9 ± 16.9 mm3 (p ≤ 0.01) and TMZ + CP-673451: 6.06 ± 2.3 mm3 (p ≤ 0.01). Tumour volumes from the combination treatment group were significantly reduced compared to both singular treatments.The in vivo anti-tumour activity of CP-673451 was evaluated using a U87 xenograft GBM mouse model Fig. . Treatmep ≤ 0.01), CP-673451: 352.4 ± 115.6 (p ≤ 0.05) and TMZ + CP-673451: 36.2 ± 12.1 (p ≤ 0.001). There were significantly fewer ki67-positive cells in the TMZ + CP-673451 group compared to CP-673451 treatment alone. Altogether it appeared that CP-673451 treatment can improve the inhibitory effect of TMZ on tumour growth in vivo.Tumour samples were stained for proliferation marker Ki67 Fig. . The numTo decipher the signalling mechanism underlying the effect of CP-673451 on GBM cell differentiation, RNA-seq was performed on U87 GBM cells treated with CP-673451 for 48 h (compared to DMSO control). Whole transcriptome correlation matrix showed a high similarity between the replicate samples Fig. . DESEQ2 p-value = 0.003) and Hallmark NF-κB signalling  in comparison to U87 GBM cells treated with DMSO  in CP-673451 treated U87 GBM cells compared to control [p ≤ 0.0001), 2.5 ± 1.0 (ns), respectively) ) and 45% (0.55 ± 0.14 fc (p ≤ 0.001)) compared to the control siRNA (siCTRL)  simultaneously at 50 nM for 24 h. G179 GSCs were treated by reverse transfection with 100 nM DUSP1 siRNA (SMPs 3 and 4) simultaneously for 48 h. DUSP1 KD was validated by qRT-PCR in U87 GBM cells and G179 GSCs 62% (0.38 ± 0.04 fc (vs 53.8 ± 8.27 μm (p ≤ 0.01)) and G179 GSCs (30% (13.0 ± 1.1 µm vs 18.5 ± 2.2 µm (p ≤ 0.01)) ) and 21% (14.2 ± 0.8 µm vs 18.0 ± 2.3 μm (p ≤ 0.05)), respectively ) Fig. . In addiely Fig. .Fig. 7DUp ≤ 0.05)) and G179 GSCs (3.7 ± 1.4 fold increase (p ≤ 0.05)) compared to control  fold in U87 GBM cells and 30.8 ± 13.1 fold (p ≤ 0.05) in G179 GSCs compared to control . In G179 GSCs, transfection with the DUSP1 plasmid increased the average neurite-like process length by 47% compared to the control 7.9 ± 0.3 µm vs 5.4 ± 0.6 μm (p ≤ 0.01)  and 0.72 ± 0.16 (p ≤ 0.05), respectively). Co-treatment with DUSP1 inhibitor BCI abrogated this decrease in p38MAPK phosphorylation  is essential to the growing neurites of the developing neurons , 35, 36.MAPK, one of DUSP1 down-stream targets, also including p44/p42MAPK (ERK1/2) and SAPK/JNK [via p38MAPK inhibition due to DUSP1 activity. Interestingly, a separate study reported that high DUSP1 levels correlate with increased GBM patient survival. Authors revealed that overexpression of DUSP1 in GSCs impedes self-renewal and induces differentiation via deactivation of p38MAPK in vitro, reducing tumourigenicity and increasing sensitivity to TMZ therapy [Further supporting this observation, our RNA-seq analysis identified DUSP1 expression to be significantly upregulated in GBM cells upon treatment with CP-673451. DUSP1 has been reported to be essential in the early hours of neuronal differentiation during embryogenesis. Its activity is required for both limiting cell proliferation and ensuring proper neurite outgrowth. DUSP1 is also directly implicated in the maintenance of neuronal integrity . PreviouSAPK/JNK , 38, 41.Taken together, these results thus suggest that CP-673451 treatment could hold great promise as part of a novel therapeutic strategy against GBM. CP-673451 is a low molecular weight kinase inhibitor (molecular weight = 417.52)  with thevia DUSP1 is being reported. Yet, our present investigation presents some limitations that would need to be addressed in follow-up studies. Among these, the upstream mechanism between PDGF-R inhibition and upregulation of DUSP1 is yet to be elucidated. However, our RNA-seq data demonstrated a significant enrichment of genes linked to the NF-κB pathway upon treatment of GBM cells with CP-673451. Accordingly, the promoter region of the DUSP1 gene contains binding sites for NF-κB [To our knowledge, this is the first time such an effect of PDGF-R inhibition on GBM cell differentiation or NF-κB . In addior NF-κB , 45. Lasor NF-κB , 51.Altogether, this study has identified a KI, CP-673451, able to induce differentiation in GBM cells, with the potential to target GSCs, which are known to be directly implicated in GBM therapeutic resistance and inevitable recurrence. CP-673451 treatment could thus refine therapeutic strategies against GBM, through reducing side effects and enhancing response to current therapies, consequently improving patients’ quality of life.2 atmosphere. Astrocytes and established human GBM cell lines were purchased from the American Type Culture Collection (ATCC). Astrocytes (AS) were seeded onto flasks and plates pre-treated with 2 μg/cm2 poly-L-lysine (Sigma-Aldrich) and required Astrocyte growth medium (Sigma-Aldrich) 10% foetal bovine serum (FBS) (Sigma-Aldrich) and 100 units ml−1 penicillin, 100 μg ml−1 streptomycin and L-glutamine (1% PSG) (Sigma-Aldrich). U87 and U138 cells required minimum essential medium (MEM) 10% FBS and 1% PSG. LN229 cells required Dulbecco’s modified eagle medium (DMEM) 10% FBS and 1% PSG.Cells were maintained at 37 °C in a humidified 5% COPatient-derived GSCs  were a kind gift from Dr. Angela Bentivegna, University of Milan-Bicocca, Italy and Dr. David Nathanson, University of California, Los Angeles, US. GSCs were isolated from GBM tumour samples following local ethical board approval. GSCs were maintained as neurospheres in DMEM/F12 Ham (Sigma-Aldrich), 1% B27 without vitamin A (Gibco), 1% Glutamax (Gibco) and 1% PSG with a mix of growth factors . Medium was changed twice a week. GBM cell lines and astrocytes were detached at confluence using trypsin/EDTA (Sigma-Aldrich). GSCs were disassociated using TrypleE express enzyme (Gibco) and separated into single cells through a 70 μm cell strainer (Starstedt). All cells were tested for mycoplasma at the beginning of the study.3 cells per well) into a 96-well plate and treated with 1 μM of 47 KIs from a library of 378 (SelleckChem) for 24 h in 10% FBS. The automated liquid handler Biomek 4000 was used for performing this screening (Beckman Coulter). Dimethyl sulfoxide (DMSO) was used as a vehicle control. GSCs were treated with 10% FBS to enhance cell attachment. Following 24 h (GBM cell lines) and 48 h (GSCs) treatment with KIs, images of the cells were taken, and length of neurite-like process was measured as described below.Cells were counted using the automated cell counter Countess (Invitrogen), seeded (2 × 10Light microscopic images were taken at 20x magnification (mag) using the Olympus IX71 microscope with Micromanager software. The length of long thin extensions termed ‘neurite-like processes’ were analysed. Three technical repeats were performed with nine images taken per well and all neurite-like processes analysed. The semi-automatic Fiji (ImageJ) plugin ‘simple neurite tracer’ was used to measure the lengths of the neurite-like processes. The total lengths of neurite-like processes per well were quantified and normalised by the total number of cells per well to calculate the average length of neurite-like process per cell. Graphs display the mean ± SD of at least three independent experiments, representative images shown, red arrows indicating neurite-like processes.3 or 5 × 103 cells per well) into a 96-well plate with 10% FBS 1% PSG for 24 h before the treatments were applied. Cells were then treated with 0, 1, 5 or 10 µM of the stated KIs. Following 24 h (GBM cell lines) or 48 h (GSCs) treatment, cells were fixed with 4% paraformaldehyde (PFA) in 1x phosphate buffered saline (PBS) (Sigma-Aldrich) and incubated at room temperature (temp) for 30 min. PFA was removed and plates left to dry at room temp for 10 min. They were then washed once with PBS and stained with 0.1% crystal violet solution at room temp for 30 min. The solution was then aspirated and washed in 1x PBS twice and water once and left open overnight to dry. When ready to read, 50 μl of 10% acetic acid was added to each well, plates were agitated on a plate rocker for 20 min and absorbance was recorded at 600 nm using a GloMax Explorer reader (Promega). Graphs show the mean ± SD of at least three independent experiments.Cells were seeded (2 × 103 cells per well) into a 96-well plate with 10% FBS 1% PSG for 24 h before the treatments were applied. Cells were then treated with DMSO or 1 µM of the stated KIs. Following 24 h (GBM cell lines) or 48 h (GSCs) treatment, 10 μl of water-soluble tetrazolium salts-1 (WST-1) reagent was added to each well and incubated at 37 °C, 5% CO2 for 2 h. The plate was then agitated on a plate rocker at room temp for 1 min and the absorbance recorded at 450 nm using a GloMax Explorer reader (Promega). Graphs show the mean ± SD of at least three independent experiments.Cells were seeded (2 × 10U87 GBM cells were treated with DMSO or CP-673451 (CP) for 48 h and RNA was purified following PureLink RNA Mini Kit (Life technologies) protocol, as described before . All conhttp://hannonlab.cshl.edu/fastx_toolkit/license.html). The quality of reads were confirmed using the fastqc tool kit (v 0.11.5) (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and only high-quality clean read were used for down-stream analyses. The high-quality reads were mapped to the ENSEMBL built GRCH37 using the STAR aligner (v2.5.3a) [p value (Padj ≤ 0.05) and Log2 fold change ≥ |1| were used as the threshold to screen significance of differentially expressed genes (DEGs). Gene set enrichment function from the enrichplot package (https://github.com/YuLab-SMU/enrichplot) was used to perform gene set enrichment analysis for Hallmark gene sets and CAHOY CNS neural cell type gene sets deposited in the MSIGDB (https://www.gsea-msigdb.org/gsea/msigdb/index.jsp).High-quality clean reads were obtained by trimming the adaptor sequences and removing reads that contained poly-N or were of low-quality from the raw data using the fastX tool kit (v 0.0.14) (v2.5.3a)  with thev2.5.3a) . Differe5 cells per T75 cm2 or 1 × 105 per well into a 6-well plate) and were treated with drug/control or target/control siRNA plasmid/control for the concentration and time stated. Cells were lysed, RNA was then purified following PureLink RNA Mini kit (Life technologies) protocol. Reverse transcription was carried out using a cDNA synthesis kit (Applied Biosystems). Taqman (Applied Biosystems) or SYBR Green (Applied Biosystems) gene expression master mix and synthesised cDNA was mixed with primers (Applied Biosystems) (Tables Cells were seeded (5 × 10) Tables  and run 5 cells were seeded per T75cm2 flask and incubated for 24 h before treatment with: 1 µM CP-673451 for 0, 15 min, 1, 4, 24 and 48 h vs DMSO (control) in 0% FCS medium; 1 μM CP-673451 and 1 μM DUSP1 inhibitor BCI ((E)−2-benzylidene-3-(cyclohexylamino)−2,3-dihydro-1H-inden-1-one) (Merck Millipore) vs 1 μM BCI and DMSO vs 1 μM CP-673451 vs DMSO (control) for 24 h in 10% FCS medium. Cells were collected and protein extracted using RIPA buffer (Sigma) including fresh protease and phosphatase inhibitors (Roche). Standard western blotting protocol was performed [MAPK , anti-p38MAPK , anti-phos-SAPK/JNK , anti-SAPK/JNK , anti-phos-p44/p42MAPK , anti-p44/p42MAPK  and anti-β-actin (Genscript #A00702 1:1000 dilution)) were prepared in the same solution used for blocking and incubated on a rocker at 4 °C overnight (Table 5 × 10erformed  with a 15 cells per well) into a 96-well HA hydrogel assay plate. As per instructions, 40 µl of cell suspension was added as a drop on top of the gel, incubated for 30 min then 100 µl media was added, as previously described [Cells were seeded . Cells were incubated at room temp for 30 min before 100 µl CellTiter-Glo reagent was added to each well, agitated on a plate rocker for 2 min and incubated at room temp for 10 min. Luminescence was recorded with an integration time of 0.3 s using a GloMax Explorer reader (Promega). Graphs show the mean ± SD of at least three independent experiments.3 (U87 GBM cells) or 5 × 103 (G179 GSCs) cells per well) into a 96-well plate and incubated at 37 °C, 5% CO2 for 24 h. Transfection was performed according to the Lipofectamine 3000 (Invitrogen) user guide. Lipofectamine and siRNA complexes were diluted separately in Opti-MEM (Gibco). These solutions were mixed and incubated at room temp for 15 min. Media was then aspirated, fresh media  was added to the cells and the mixed siRNA/Lipofectamine/Opti-MEM solution was added dropwise. Cells were transfected for 48 h with 50 nM and 100 nM siRNA (control (CTRL), PDGF-Rα, PDGF-Rβ or a combination of PDGF-Rα and β) (Table 5 cells per well) into a 6-well plate, transfected as described with 50 nM and 100 nM CTRL siRNA or a combination of PDGF-Rα and PDGF-Rβ siRNA. At 48 h cells were lysed, RNA purified, cDNA synthesised and mixed with SYBR Green master mix (Applied Biosystems) plus primers (Table Cells were seeded (2 × 10Plasmid transfection was performed with 1 ng/µl pCMV6 empty vector or DUSP1 plasmid (Origene RC205220) for 48 h using transfection reagent HiPerFect. Graphs display the mean ± SD of at least three independent experiments, representative images shown.6 U87 GBM cells subcutaneously into 6-week-old immunocompromised athymic nude mice . Once tumours were palpable (tumour diameter ~ 3 mm3), mice were randomly divided into the separate groups: DMSO control (n = 6), TMZ (25 mg/kg/day) (n = 9), CP-673451 (40 mg/kg/day) (n = 9) or combination of TMZ (25 mg/kg/day) and CP-673451 (40 mg/kg/day) (n = 9). Treatments were administered by oral gavage 5 days per week for 3 weeks after which the mice were sacrificed, tumours extracted and tumour volumes measured using the formula: (volume (mm3) = (length x height2)/2). Paraffin-embedded tumours were sectioned at a thickness of 4 mm with five tissue samples obtained per tumour. Samples were placed in a pressure cooker for 15–20 min in 0.01 M citrate buffer (pH 6.0) to remove aldehyde links formed during initial fixation of tissues. Specimens were incubated with antibodies specific for Ki-67 (1:100) overnight at 4 °C and immunodetection performed the following day using 3,3′-diaminobenzidine (DAB) (Dako) according to manufacturer’s instructions. Images were obtained using Olympus BX63 microscope (200x mag) and the number of cells per field quantified.Following local authority ethical approval by Sun Yat-sen University Animal Care and Use Committee, xenograft mouse models were developed by injecting 2 × 10T-tests were used to compare control group with each treatment group. Differences were considered statistically significant at p ≤ 0.05. .Sample size was set to a minimum of three independent experiments  and experimental findings were reliably reproducible. GraphPad Prism was used for data analysis and graphs. All data are presented as mean ± SD. Supplementary Figures 1–5


The results obtained from crystal packing and DFT analysis suggest that the molecules are held together by forces such as hydrogen bonding and π–π interactions.Three  o-nitrosulfonamides and N-cycloamino-o-sulfanilamides were synthesized and characterized using techniques including 1H NMR, 13C NMR and FT–IR spectroscopy, and single-crystal X-ray diffraction (SC-XRD). The calculated density functional theory (DFT)-optimized geometry of the molecules showed similar conformations to those obtained by SC-XRD. Molecular docking of N-piperidinyl-o-sulfanilamide and N-indolinyl-o-sulfanilamide supports the notion that o-sulfanilamides are able to bind to human carbonic anhydrase II and IX inhibitors . Hirshfeld surface analyses and DFT studies of three o-nitrosulfonamides {1-[(2-nitrophenyl)sulfonyl]pyrrolidine, C10H12N2O4S, 1, 1-[(2-nitrophenyl)sulfonyl]piperidine, C11H14N2O4S, 2, and 1-[(2-nitrophenyl)sulfonyl]-2,3-dihydro-1H-indole, C14H12N2O4S, 3} and three N-cycloamino-o-sulfanilamides  suggested that forces such as hydrogen bonding and π–π interactions hold molecules together and further showed that charge transfer could promote bioactivity and the ability to form biological interactions at the piperidinyl and phenyl moieties.In the search for new ‘sulfa drugs’ with therapeutic properties,  Sulfonamides E are derivatives of sulfanilamide and remain an important class of drugs, with antibacterial and non-antibacterial potencies, such as diuretic, antimicrobial, anti-epileptic, antileprotic, antimalarial, hypoglycemic, antiretroviral, antithyroid and anti-inflammatory activities sulfonyl]pyrrolidine, 1, 1-[(2-nitrophenyl)sulfonyl]piperidine, 2, and 1-[(2-nitrophenyl)sulfonyl]-2,3-dihydro-1H-indole, 3, and the N-cycloamino-o-sulfanilamides 2-(pyrrolidine-1-sulfonyl)aniline, 4, 2-(piperidine-1-sulfonyl)aniline, 5, and 2-aniline, 6. The crystal structures, density functional theory (DFT) studies, Hirshfeld surface analysis, molecular electrostatic potential and electronic properties of the title sulfonamides and sulfanilamides (1–6) have been discussed. Molecular docking experiments with carbonic anhydrase II (PDB entry 4iwz) and IX (5fl4) active sites were conducted in order to predict their binding interactions with 1–6  solvent system visualized under a UV lamp (254 nm). Column chromatography was performed with silica gel (70–230 mesh ASTM) and mobile phases were as indicated. Sample crystallization was achieved by the slow evaporation of the indicated solvent systems at ambient temperature. IR spectra were obtained using a Bruker Tensor 27 platinum ATR–FT–IR spectrometer. The ATR–FT–IR spectra were acquired in a single mode with a resolution of 4 cm−1 over 32 scans, in the region 4000–650 cm−1. 1H and 13C NMR spectra were recorded, in CDCl3, on a Bruker 400 MHz spectrometer. Chemical shift (δ) values were measured in parts per million (ppm) downfield from tetramethylsilane (TMS) and coupling constants (J) are reported in hertz (Hz). Theoretical studies were performed for the compounds and, in each case, their SC-XRD structures were used for optimization and global reactivity descriptor (GRD) calculations.All reagents were purchased from Millipore Sigma (Germany and South Africa) and were used without purification. The melting points were determined on an Electrothermal digital melting-point apparatus and are uncorrected. Reactions were monitored by thin-layer chromatography (TLC) on Merck silica gel 60 Fo-Nitrobenzenesulfonyl chloride (1.00 mmol) was added slowly to a stirring dried toluene solution (30 ml) of the cycloamine (2.20 mmol) at ambient temperature and stirred for 12 h, monitored by TLC. The reaction mixture was then diluted with dichloromethane (30 ml) and washed with distilled water (3 × 10 ml). The organic layer was separated, dried over anhydrous sodium sulfate, filtered and concentrated to an oil, which was purified by column chromatography on silica gel . Crystals were obtained by the slow solvent evaporation of the requisite eluates at ambient temperature, except for 5, which was recrystallized from dichloromethane, slowly evaporated and filtered to give single crystals.1. o2.2.1.1. N-Pyrrolidinyl-o-nitrobenzenesulfonamide, -Nitrobenzenesulfonyl chloride  and pyrrolidine . Yellow crystals ; RF = 0.44 ; m.p. 81.7–81.9 °C. IR : 3080 (aryl C—H str.), 2968 (sp3-C—H str.), 1597 (aryl C=C str.), 1543 (asym C—NO2 str.), 1344 (sym C—NO2 str.), 1342 (asym SO2—N str.), 1163 (sym SO2—N str.), 1078 (C—N str.). 1H NMR : 7.94 , 7.62 , 7.54 , 3.37–3.35 , 1.85 . 13C NMR : 148.4, 133.5, 132.1, 131.5, 130.6, 123.9 (ArH), 48.2 (–CH2NCH2–), 25.9 (–CH2CH2–).2. o2.2.1.2. N-Piperidinyl-o-nitrobenzenesulfonamide, -Nitrobenzenesulfonyl chloride  and piperidine . Yellow crystals ; RF = 0.56 ; m.p. 91.6–91.8 °C. IR : 3076 (aryl C—H str.), 2947 (sp3-C—H str.), 1552 (aryl C=C str.), 1550 (asym C—NO2 str.), 1354 (sym C—NO2 str.), 1350 (asym SO2—N str.), 1166 (sym SO2—N str.), 1056 (C—N str.). 1H NMR : 7.96 , 7.70 , 7.59 , 3.26–3.24 , 1.64–1.63 , 1.55–1.54 . 13C NMR : 148.5, 133.6, 131.6, 131.5, 130.8, 123.5 (ArH), 47.0 (–CH2NCH2–), 25.4 (–CH2CH2CH2–), 23.5 (–CH2CH2CH2–).3. o2.2.1.3. N-Indolinyl-o-nitrobenzenesulfonamide, -Nitrobenzenesulfonyl chloride  and indoline . Yellow crystals ; RF = 0.79 ; m.p. 106.5–106.8 °C. IR : 3077 (aryl C—H str.), 2976 (sp3-C—H str.), 1594 (aryl C=C str.), 1536 (asym C—NO2 str.), 1356 (sym C—NO2 str.), 1355 (asym SO2—N str.), 1161 (sym SO2—N str.), 1051 (C—N str.). 1H NMR : 7.95 , 7.71 , 7.62 , 7.48 , 7.21 , 7.05 , 4.17 , 3.10 . 13C NMR : 148.4, 141.1, 134.1, 131.8, 131.7, 131.6, 130.2, 127.8, 125.5, 124.3, 124.2, 114.5 (ArH), 50.5 (–NCH2–), 28.0 (–NCH2CH2–).N-cycloamino-o-nitrobenzenesulfonamides 1–3 (15.63 mmol) dissolved in ethanol (30 ml), at ambient temperature, and 10% palladium-on-charcoal catalyst (3.35 mol%) was added, with stirring. Hydrogen gas was then introduced via a balloon and stirring continued at ambient temperature for 12 h. The reaction mixture was filtered and the solvent was evaporated in vacuo. The resulting residue was extracted into dichloromethane (50 ml), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to afford an oil, which was purified on a silica-gel column using dichloromethane and n-hexane (2:1 v/v). Crystals were obtained via slow solvent evaporation of the eluates at ambient temperature.An evacuated nitrogen-gas-filled round-bottomed flask was charged with 4. N2.2.2.1. N-Pyrrolidinyl-o-sulfanilamide, -Pyrrolidinyl-o-nitrobenzenesulfonamide 1  with 10% palladium-on-charcoal catalyst . Off-white crystals ; RF = 0.40 ; m.p. 75.2–75.4 °C. IR : 3464, 3363 (N—H str.), 3003 (aryl C—H str.), 2947 (sp3-C—H str.), 1620 (aryl C=C str.), 1323 (asym SO2—N str.), 1132 (sym SO2—N str.), 1307 (C—N str.). 1H NMR : 7.63 , 7.28 , 6.74 , 5.13 , 3.31 , 1.80 . 13C NMR : 146.4, 134.0, 130.2, 119.1, 117.6, 117.1 (ArH), 47.8 (–CH2NCH2–), 25.2 (–CH2CH2–).5. N2.2.2.2. N-Piperidinyl-o-sulfanilamide, -Piperidinyl-o-nitrobenzenesulfonamide 2  with 10% palladium-on-charcoal catalyst . Off-white crystals ; RF = 0.57 ; m.p. 76.6–76.8 °C. IR : 3487; 3383 (N—H str.), 3072 (aryl C—H str.), 2947 (sp3-C—H str.), 1606 (aryl C=C str.), 1309 (asym SO2—N str.), 1136 (sym SO2—N str.), 1336 (C—N str.). 1H NMR : 7.48 , 7.21 , 6.67 , 6.64 , 4.99 , 3.03–3.00 , 1.56–1.53 , 1.39–1.37 . 13C NMR : 146.3, 134.0, 130.1, 118.0, 117.6, 117.0 (ArH), 46.8 (–CH2NCH2–), 25.2 (–CH2CH2CH2–), 23.6 (–CH2CH2CH2–).6. N2.2.2.3. N-Indolinyl-o-sulfanilamide, -Indolinyl-o-nitrobenzenesulfonamide 3  with 10% palladium-on-charcoal catalyst . Off-white crystals ; RF = 0.80 ; m.p.: 111.9–112 °C. IR : 3448, 3367 (N—H str.), 3070 (aryl C—H str.), 2924 (sp3-C—H str.), 1597 (aryl C=C str.), 1327 (asym SO2—N str.), 1138 (sym SO2—N str.), 1330 (C—N str.). 1H NMR : 7.48 , 7.16 , 7.08 , 7.03 , 6.90 , 6.58 , 6.55 , 5.00 , 3.96 , 2.86 . 13C NMR : 146.4, 142.3, 134.4, 132.1, 129.8, 127.6, 125.1, 123.7, 119.4, 117.7, 117.3, 115.1 (ArH), 50.0 (–NCH2–), 28.1 (–NCH2CH2). Reaction synthesis of nitrosulfonamides 1–3 and aminosulfonamides 4–6 are presented in Scheme S1 in the supporting information. The FT–IR, MS and 1H/13C NMR spectra of compounds 1–6 are also presented in the supporting information.1–6 (CIF files) were imported directly into the Schrödinger Suite -2-(thiophen-2-yl)acetamide (A) and 5-thiophene-2-sulfonamide (B) were based on chemical structures downloaded from the PubChem (https://pubchem.ncbi.nlm.nih.gov/) website in SDF format. A and B were used as reference compounds because they are natural ligands in the crystalline state of 5fl4 and 4iwz. The Ligprep module of the molecular modelling platform of the Schrödinger Suite  Protein Data Bank (PDB). Retrieved crystal coordinates were prepared in the ‘Protein Preparation Wizard’ of the Schrödinger Suite  and XII (4iw7) isoforms will provide a selectivity profile that may be interesting for the development of novel anticancer agents with limited side effects. The hCA II (PDB entry 5fl4) and XII (4iw7) carbonic anhydrase isoforms have recently emerged as excellent targets for the design of novel therapeutic strategies for cancer, due to their involvement in the survival of tumour cells, as well as in the insurgence of resistance to classical anticancer protocols  mode of the 1–6 whereupon the SC-XRD structures of the compounds were used for optimization and global reactivity descriptor (GRD) calculations. Computational studies and molecular electrostatic potential (MEP) for 1–6 were carried out using the GAUSSIAN16 software package .Theoretical studies were performed for compounds Crystal data, data collection and structure refinement details are summarized in Table 1N-cycloamino-o-sulfanilamides 4–6 were prepared via a two-step reaction, starting from the condensation reaction of o-nitrobenzenesulfonyl chloride with alicyclic amines in toluene, at ambient temperature, to afford N-cycloamino-o-nitrobenzenesulfonamide adducts 1–3 (Scheme S1 in the supporting information). The use of toluene as a nonpolar reaction medium was, amongst other reasons, to drive the forward reaction. In the second step, adducts 1–3 were hydrogenated with hydrogen gas, in ethanol at ambient temperature, in the presence of 10% palladium-on-activated charcoal catalyst to give the target N-cycloamino-o-sulfanilamides 4–6 in 72–86% yield. The reactions were monitored by TLC.The 1H/13C NMR and MS spectra. In the IR spectra of o-nitrosulfonamide adducts 1–3, the strong absorption bands observed at 1355–1342 and 1171–1161 cm−1 were ascribed to the asymmetric and symmetric stretching frequencies, respectively, of the SO2—N moiety, thereby alluding to the formation of the sulfonamide bond. The disappearances of the SO2—Cl (1420 and 1220 cm−1) and N—H (3286–3265 cm−1) stretching bands in the IR spectra of o-nitrobenzenesulfonyl chloride and cycloamines, respectively, were good indicators of a successful condensation reaction. This was corroborated by the shift of the sulfonyl (–SO2–) absorption bands from 1420 and 1220 (in o-nitrobenzenesulfonyl chloride) to 1355–1342 and 1171–1161 cm−1 (in 1–3). It is noteworthy that the lower wavenumbers observed in the IR spectra of o-nitrosulfonamides 1–3 for –SO2– were not unusual as the Cl atom bonded to it had been replaced by a less electronegative N atom. In the IR spectra of o-sulfanilamides 4–6, the appearance of two N—H stretching bands in the higher frequency region around 3467 ± 20 and 3383 ± 10 cm−1, and the disappearance of the nitro (NO2) absorption bands (observed at 1550–1536 and 1369–1342 cm−1) in the spectra of 1–3 were attributed to the successful catalytic reduction of the nitro group to the amino group.All the compounds synthesized were characterized by their melting points and IR, 1H NMR spectra of o-nitrosulfonamides 1–3 were additive of the individual spectra of the starting materials (i.e. o-nitrobenzenesulfonyl chloride and cycloamines), with the disappearance of the nitrogen proton peaks of cycloamines. The aromatic protons of o-sulfanilamides 4–6 resonated upfield in comparison to the same aromatic protons in precursors 1–3. This general shift towards tetramethylsilane (TMS) was credited to the newly formed amino groups whose lone-pair electrons are suspected of having caused the increased mesomeric shielding of the aromatic protons. D2O-exchangeable singlets were also observed in the 1H NMR spectra of 4–6 between 5.13 and 4.99 ppm for the newly-formed amino protons. The success of the catalytic hydrogenation of nitro adducts 1–3 was corroborated by the 13C NMR spectra of 4–6, where the requisite C atoms (C—NO2 → C—NH2) resonated upfield in the range 133.9–130.1 ppm. The spectroscopic data analyses of the synthesized compounds were consistent with the assigned structures of the compounds.The 1–3 and 4–6 crystallized in the monoclinic space group P21/n or P21/c (No. 14), except for 5, which crystallized in the orthorhombic space group Pbca (No. 61). In addition, they all had one molecule in the asymmetric unit, with the exception of 4, with two independent molecules per asymmetric unit cell. The two molecules per unit cell of compound 4 were identical but for the conformation of the pyrrolidine group . It is noteworthy that the pyrrolidine ring in 1 is disordered. The molecular structures of 1–3 and 4–6 are shown in Fig. 2o-nitrosulfonamides 1–3 and N-cycloamino-o-sulfanilamides 4–6 are presented in Table 1para-sulfanilamide and ortho-sulfanilamide, which crystallize in the orthorhombic Pbca (No. 61) and monoclinic P21/c (No. 14) space groups, respectively .The molecules of 2) group in N-cycloamino-o-sulfanilamides 4–6 contributed significantly to their hydrogen-bond interactions (cf. Table 2S(6) graph-set descriptors  descriptors. Interestingly, no infinite chain interaction was observed in 4; instead, four molecules were linked into a ring structure with an p-sulfanilamide  strength  Å were observed between the centrosymmetric indoline moieties. An N=O⋯π ring interaction of 3.657 (2) Å was also evident in 3, whereas intermolecular C—H⋯(π ring) interactions of 2.97 Å and S=O⋯(π ring) interactions of 3.5773 (15) Å were present in the structure of its hydrogenated analogue 6. The packing diagrams of the crystal structures of compounds 1–6 are shown in Fig. S2 in the supporting information.The only π–π stacking interaction of note occurred in et al., 20171–6 showed intermolecular interactions such as O—H⋯O, O—H⋯N and C—H⋯π. Two sharp O—H spikes typical of an O—H⋯O interaction from 1 contributed the highest O⋯H interaction of 42.3%. The fingerprint plots showed that C⋯H contacts were highest for 6 (30.4%), and this is closely related to C—H⋯π interactions  visible on the dnorm surfaces typically indicate that the molecule has a donor site(s) (e.g. amine and/or sulfone) or interactions with proteins.The molecular Hirshfeld surfaces, mapped as o-nitrosulfonamides 1–3 and N-cycloamino-o-sulfanilamides 4–6 in full and resolved into C⋯H, O⋯H and N⋯H are presented in Fig. S4 (supporting information). The intermolecular O⋯H and N⋯H interactions appear as two distinct spikes of almost equal length in the 2D  fingerprint plots in the region 1.2 < (de + di) < 2.9 Å as light-sky-blue patterns in full fingerprint 2D plots and characterized to be 2.56 ± 0.21 Å corresponds to O⋯H contacts which contributes the majority of the surface area. 2D fingerprint plots reveal the contributions of these interactions in the crystal structure quantitatively and are presented in Table 4dnorm, di and de provided). Complementary regions are also visible in the fingerprint plots , where one molecule acts as a donor (de > di) and the other acts as an acceptor (de < di). This finding was validated by the calculated molecular electrostatic potential of 1–6 . The negative potential (acceptor) is indicated as a red surface around the O atoms attached to sulfur (–SO2) and the N atoms attached to oxygen (–NO2). The blue/purple surface area indicates that the positive potential (donor) is mapped in the proximity of the H atoms .Fingerprint plots of 1–6 presented bond lengths similar to those obtained from the crystal data. A comparison of selected torsion angles of the crystal structures of 1–6 and the DFT-optimized molecules showed that conformation of the molecules did not change significantly in the DFT-optimized state . Generally, the observed, almost flat, O—S—N—C torsion angle of the DFT-optimized molecules suggest that the lone pairs on sulfur may have contributed to the π-electron delocalization that is observed in the DFT molecules.The full geometry of optimized molecules 3 and 5. The LUMO is mainly spread over the phenyl moieties. This indicates that there is a transfer of charge between the indolinyl/pyrrolidinyl rings and the phenyl moieties within the molecule.The highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) electrons are distributed around various moieties within the various molecules Fig. 4. Generalet al., 2020N-Indolinyl-o-nitrobenzenesulfonamide 3 displayed the smallest energy gap (3.24 eV), indicating that it was the softest molecule with good polarizability and reactivity, whereas N-piperidinyl-o-sulfanilamide 5 presented the largest energy gap of 4.924 eV, thereby corroborating its high chemical hardness of 2.462 eV (cf. Table 53 (ELUMO = −3.175 eV), indicating that it is the best electron acceptor of the molecules analyzed, whereas 6 was the best electron donor in the series, with the highest HOMO energy (EHOMO) of −6.142 eV , electron affinity (A), chemical potential (μ), electronegativity (χ), global hardness (η), global softness (S) and global electrophilicity (ω) values were calculated using the HOMO and LUMO energy values and are collated in Table 5I value of 6.142 eV originated from sulfanilamide 6, whereas sulfonamide 3 gave the largest A value of 3.175 eV. Amongst the compounds studied, 2 gave the highest χ value of 5.1795 eV. Interestingly, sulfanilamide 5 displayed the highest η value of 2.462 eV and the lowest chemical softness (S) of 0.406 eV, thus alluding to its having the most reactive nature of all the molecules investigated. The highest global electrophilicity of 29.597 eV was also recorded for sulfonamide 2, indicating that it is a strong electrophile. In general, the chemical reactivities of compounds 1–6 have been shown to vary with the groups attached to the compounds   -2-(thiophen-2-yl)acetamide (A) and 5-thiophene-2-sulfonamide (B) were also docked with respective proteins 4iwz and 5fl4, and taken as reference or standard drugs. Docking poses for the synthesized compounds are displayed in Figs. S7–S18, while those for the reference drugs are shown in Figs. 5Docking studies of synthesized 4iwz and A (reference drug) displayed a docking score of −2.252 kcal mol−1, which is higher than for all synthesized compounds 1–6  and HIE64 (2.24 Å) via hydrogen-bonding interactions and with amino acid residue HIS94 (4.75 Å) via π–π stacking interactions  and TRP5 (2.11 Å), and through π–π stacking with THR199 (1.81 Å) .Also,  A Fig. 5. Compoun5fl4, and the results obtained were compared with the docked results of the reference drug B. We observed that the reference drug interacts with amino acid residues ASP13 (1.59–2.73 Å) and VAL130 (2.53 Å) via hydrogen bonding, and with HID94 (5.49 Å) via π–cation interactions , displayed significantly good docking scores; however, they were lower than the reference drug .To determine the mode of interaction of the synthesized compounds with human carbonic anhydrase IX inhibitor (hCA IX), the synthesized compounds were docked into the active site of 31 Fig. 6. Benzene2 with 4iwz and 5fl4 are close to those obtained for A with 4iwz and B with 5fl4. Docking scores of molecules with ring structures 1 and 3–6  also correlated with the electronegativity and electrophilicity values presented in Table 5et al., 2018We observed that the docking scores of o-Nitrosulfonamides 1–3 and N-cycloamino-o-sulfanilamides 4–6 have been successfully synthesized, characterized and the intermolecular interactions analysed, as well as being tested in silico for carbonic anhydrase II (4iwz) and IX (5fl4) inhibitory activities. The results obtained from crystal packing and DFT analysis suggests that the molecules are held together by forces such as hydrogen bonding and π–π interactions. The results of the DFT study of compounds 1–6 were correlated with the molecular docking data and indicate that electronegativity and electrophilicity of the title compounds play an important role in their interaction with carbonic anhydrase II (4iwz) and IX (5fl4).O-Nitrosulfonamide 2 displayed a good docking score against 4iwz (lower than the reference drug) and the best against 5fl4 (higher than the reference drug). These results provided a valuable synthesis approach and structural and docking information for compounds 1–6 that may be used for the development of potent antibacterial drugs.10.1107/S2053229622010130/oj3005sup1.cifCrystal structure: contains datablock(s) ka097, ja198, ja250, ja192, ka115, ja189, global. DOI: 10.1107/S2053229622010130/oj3005ka097sup2.hklStructure factors: contains datablock(s) ka097. DOI: 10.1107/S2053229622010130/oj3005ja198sup3.hklStructure factors: contains datablock(s) ja198. DOI: 10.1107/S2053229622010130/oj3005ja250sup4.hklStructure factors: contains datablock(s) ja250. DOI: 10.1107/S2053229622010130/oj3005ja192sup5.hklStructure factors: contains datablock(s) ja192. DOI: 10.1107/S2053229622010130/oj3005ka115sup6.hklStructure factors: contains datablock(s) ka115. DOI: 10.1107/S2053229622010130/oj3005ja189sup7.hklStructure factors: contains datablock(s) ja189. DOI: Click here for additional data file.10.1107/S2053229622010130/oj3005ka097sup8.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2053229622010130/oj3005ja198sup9.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2053229622010130/oj3005ja250sup10.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2053229622010130/oj3005ja192sup11.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2053229622010130/oj3005ka115sup12.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2053229622010130/oj3005ja189sup13.cmlSupporting information file. DOI: 10.1107/S2053229622010130/oj3005sup14.pdfAdditional figures, tables and spectra. DOI: 2014232, 2039638, 2014231, 2014230, 2014229, 2039639CCDC references: