Bariatric surgery is a widely used procedure for the treatment of obesity. Our aim is to describe the main immunological changes in patients who undergo bariatric surgery.A prospective study was conducted within a cohort of patients undergoing bariatric surgery and without previous evidence of systemic or organ-specific autoimmune diseases in whom 3 blood samples were collected – one day before surgery (Time 0), and 5 (Time 1) and 10 months (Time 2) after surgery.p = 0.0002. The CD4/CD8 ratio increased from 2.2(IQR: 1.7–2.7) to 2.4(1.8–2.8), p = 0.0001. As for humoral variables, the C3 fraction of complement decreased from 164 ± 40.6 mg/dL to 112.4 ± 31.4 mg/dL(p < 0.001) and C4 decreased from 29.3 ± 10.1 mg/dL to 22.5 ± 7.1(p = 0.0009) at Time 2. Four patients with negative ANAs at baseline, showed positive ANAs at Time 2.One patient developed anti-citrullinated peptide antibodies >200 IU/mL at Time 2.Thirty four obese patients underwent surgery (Time 0):30(88.24%) were women, mean age 38.3 years. When comparing Time 0 and Time 2, there were statistically significant changes in CD4+T cell count, with an increase from 1074/mL(IQR:860–1316) to 1217.5/mL(IQR:838–1510),Patients undergoing bariatric surgery show immunological changes which might eventually lead to develop an autoimmune disease. •Bariatric surgery is beneficial in treatment of obesity, although little is known about impact of significant weight loss in the immune system.•Bariatric surgery may induce antinuclear antibodies positivity.•Bariatric surgery may induce changes in T lymphocytes population. In a previous article by our group, we described the development of autoimmune diseases following weight loss associated with bariatric surgery . In addiIn this study, we prospectively evaluated 34 obese patients with no clinical or laboratory evidence of autoimmune diseases in whom bariatric surgery was performed. We conducted thorough biochemical and hematological tests, autoantibodies panel and peripheral blood distribution of T and B cells at baseline and in a two-step follow-up after the procedure (5 and 10 months on average) to evaluate any possible changes in the immunological field and to establish any possible mechanisms related to the onset of systemic autoimmune diseases triggered by weight loss.2Between September 2014 and December 2015, a prospective cohort study of patients undergoing bariatric surgery for treating obesity and without previous clinical or laboratory evidence of systemic or organ-specific autoimmune diseases was performed. In these patients, 3 serial blood samples were collected at baseline and 5 and 10 months after the procedure. Inclusion criteria were obese patients over 18 years, candidates for surgical management (sleeve or gastric bypass), who signed an informed consent. Patients with autoimmune rheumatologic diseases, malignancies and immunosuppression were excluded.2.1Prospective descriptive study, where patients were evaluated by a rheumatologist before surgery (Time 0) for a complete medical history and physical examination with anthropometric assessment, and peripheral blood samples were collected for determination of T and B cell peripheral distribution by flow cytometry (processed within the first 6 h of collection) and serum storage at −80 °C for processing ANAs, C3, C4, IgG and IgM anti-cardiolipins, anti-citrullinated peptide antibodies (ACPA), rheumatoid factor (RF), leptin and adiponectin. These parameters were evaluated at two additional timepoints [on average 5 (Time 2) and 10 months (Time 3) after surgery]. All parameters were titrated to determine any possible immunological changes at follow-up. The study protocol was approved by the Ethics Committee, and all patients signed an informed consent.2.2+ markers for T cell separation, CD3+ and CD4+ for helper T cells, CD3+ and CD8+ for cytotoxic lymphocytes, CD19 + for B cells using the Beckman Coulter® tetrachrome CD45-FITC/CD4-RD1/CD8-ECD/CD3-PC5 CYTO-STAT kit . Adipokines (leptin and adiponectin) were measured by solid-phase enzyme-amplified sensitivity immunoassay on microplates using the Inova® DIAsource leptin-EASIA kit  and the Inova® DIAsource adiponectin ELISA kit , respectively.C3 and C4 levels were assessed by immunoturbidimetry using the Roche Cobas® e501 analyzer  with normal values between 90 and 140 mg/dL and 10–40 mg/dL, respectively. The presence of anti-nuclear antibodies (ANAs) was determined by indirect immunofluorescence (IFI) technique using the Inova Kit Hep2 . Anti-cardiolipin IgM, IgG and ACPA titers were determined by ELISA using the Orgentec® kit . Rheumatoid factor was determined by immunoturbidimetry using the Roche® Cobas analyzer . Peripheral blood B and T cell phenotyping was performed by flow cytometry using CD32.3t-test or Wilcoxon signed-rank test, depending on the achievement of normality assumptions. For data analysis, the STATA 12.1 program was used. As for comparisons, p values < 0.05 were considered significant. The Pearson’s correlation coefficient was used to determine correlation between continuous variables.A descriptive exploratory analysis was performed. Continuous variables were expressed as means ± SD, median and interquartile ranges, and were analyzed using a paired 3A total of 34 patients who met the inclusion criteria were included in the study , of whicJoint symptoms: Presence of joint symptoms were evaluated prior to surgery. Eleven patients (32.35%) had no joint symptoms, 9 patients had pain in 1 joint (26.47%), 8 patients had pain in 2 joints (23.53%) and 6 patients had pain in more than 2 joints (17.65%). In all patients, the pain showed mechanical characteristics with no evidence of inflammation or morning stiffness. During the Time 1, 23 patients had no joint symptoms (67.65%), 8 patients had pain in 1 joint (23.53%), and 1 patient had pain in 2 joints (2.94%). At Time 2, 10 months after surgery, 18 patients were asymptomatic for joint involvement. Inflammatory joint involvement was observed in no patients.Dryness symptoms: Prior to surgery, no patients reported either subjective or objective dryness symptoms (dry eye or dry mouth). At time 1, 5 patients (14.71%) had dryness symptoms . At Time 2, this number decreased to 2 patients who had dry mouth only (5.88%).Other symptoms: As for other symptoms, all patients were asymptomatic prior to surgery. During the first follow-up period, 13 patients (38.24%) reported increased hair loss, and one patient (2.94%) had neurological manifestations compatible with Guillain-Barre syndrome.Cellular immunological parameters: B- and T-cell phenotype was assessed in peripheral blood samples at all three different times. + T cell count increased from 1074/mL (IQR: 860–1316) to 1217.5/mL (IQR: 838–1510), p = 0002; while the percentage of CD4+ T cells increased from 44.6 ± 8.3 to 45.8 ± 7.4 with a p value of 0.0179. Although an increased CD8+ lymphocytes count from 494/mL (IQR: 355–634) to 502.5/mL (IQR: 378–632) was evidenced, this result was not significant (p = 0.38). The CD4/CD8 ratio increased from 2.2 (IQR: 1.7–2.7) to 2.4 (1.8–2.8), p = 0.0001. The remaining lymphocytic populations (CD19 and CD20) showed no changes at Time 1 and Time 2.None of the evaluated parameters showed statistically significant changes when comparing Time 0 versus Time 1 data. When comparing Time 0 versus Time 2, several phenotypic changes were evidenced. For instance, CD4Serum immunological parameters: Several serum values also showed statistically significant changes. Complement C3 decreased from 164 ± 40.6 mg/dL to 112.4 ± 31.4 mg/dL  and C4 decreased from 29.3 ± 10.1 mg/dL to 22.5 ± 7.1 mg/dL (p = 0.0009 at Time 2); however, Time 0 versus Time 1 comparisons were not statistically significant.vs. Time 1, leptin decreased from 45.7 ± 9.14 ng/mL to 23.5 ± 12.6 ng/mL  and, when comparing Time 0 versus Time 2, leptin decreased from 44.12 ± 9.75 ng/mL to 22.49 ± 9.7 ng/mL . As for adiponectin, when comparing baseline values versus Time 1, a significant increase from 21.3 (17.8–34.1) ng/mL to 33.5 (23.85–50.05) ng/mL (p < 0.0001) was evidenced. When comparing Time 0 versus Time 2, changes were also statistically significant, with 29.2 ± 20.17 ng/mL for Time 0 and 40.8 ± 23.4 for Time 2 (p = 0.042).When comparing Time 0 Autoantibodies: At Time 0, all patients were negative for ANAs. At Time 2, 4 patients (11.76%) increased ANA concentrations: 3 cases with homogeneous pattern (1:80 titer) and one patient with centriole pattern (1:160 titer).p = 0.8271), or Time 2: 7.8 (6.6–9.7) (p = 0.0916). Despite the lack of changes in ACPA titers, one patient developed positive anti-citrullinated peptide antibodies (ACPA) at the upper limit of 20 IU/mL  at Time 0, with a 10-fold titer increase (200 IU/mL) at Time 2.ACPA antibodies at Time 0 were on average 8 ± 3.2 IU/mL ; no changes were observed at Time 1, with values of 8 ± 4.2  to 3 (2.6–5.2) (p < 0.001).IgG anticardiolipin antibodies showed a statistically significant change when comparing Time 0 versus Time 1, decreasing from 7.4 (6.1–10.4) GPL to 6.4 (5.1–9.4) with a p value of 0.0283. This decrease was also statistically significant when comparing Time 0 versus Time 2, with a decrease of 5.5 ± 1.7 at Time 0 and values of 3.6 ± 1.7 at Time 2, with a p-value of <0.01.IgM anticardiolipin antibodies showed no statistically significant changes when comparing all times. When evaluating RF, there were statistically significant differences, with values of 5.7 ± 1.7 IU/mL at Time 0 and a significant decrease to 5.1 ± 1.8 at Time 1, with a p-value: 0.962), CD8 lymphocyte count , complement C3 , complement C4 , adiponectin (r: 0.16442) and leptin .Then, we evaluated the correlation between percentage loss weight and biological variables at 10 months using the Pearson’s coefficient test. None of the evaluated variables showed a correlation: CD4 lymphocyte count .In terms of positive autoantibody tests, 4 patients (11.76%) were found to develop positive ANAs, which may suggest that, in certain cases, weight loss is associated with immune disorders resulting in the development of antibodies. As for ACPAs, although no significant average changes were observed, one patient had a 10-fold increase in its levels, which could be explained by a genetic predisposition that is necessary for changes to occur in addition to an environmental component.In this study, the role of autoantibodies, complement, cytokines and cellular changes for the future development of autoimmune diseases is unclear and should be assessed during the prospective follow-up of our patients. However, based on the article by Arbuckle et al.  that shoThis study provides information that has been scarcely described in the literature, about the possible effects on the immune system of bariatric surgery and rapid weight loss. On the other hand, some publications have shown the benefit of weight loss on clinical and laboratory outcomes in patients with established autoimmune or inflammatory diseases undergoing bariatric surgery. For example, a retrospective study published by Egeberg et al.  showed tIn the context of patients with established autoimmune diseases and concomitant obesity, the effects of obesity on the course of inflammatory diseases have been demonstrated. For example, a study showed that disease remission in obese patients with RA was less likely than in patients with normal weight .A study by Corcelles et al.  in patiep value of 0.001. In addition, at surgery, 26% of patients were on remission; at 12 months, this percentage had a statistically significant increase to 74%. Likewise, C-reactive protein, erythrocyte sedimentation rate and use of DMARDs showed a significant decrease.A retrospective study conducted in the United States by Sparks et al.  showed fIn conclusion, our study shows that obese patients undergoing bariatric surgery have subsequent immunological changes. Although the development or lack of development of autoimmune diseases is not predictable, awareness about this possibility is mandatory. Further studies are necessary to define any predisposing factors for the development of clinically significant immunological changes.10.13039/501100002411Universidad Icesi and 10.13039/501100003402Fundación Valle del Lili.The study was funded by The authors declare they have no conflicts of interest.


The title compounds were prepared from 1,3-diphenylcyclopentadiene, pyrrolidine, and the corresponding aldehydes in an ethanolic solution. Each compound crystallizes with one molecule per asymmetric unit and exhibits the alternating short and long bond lengths typical of fulvenes. A network of C—H⋯C ring interactions as well as C—H⋯O interactions is observed, resulting in the compact packing found in each structure. 21H18, , 1, and 6--1,3-diphenylfulvene, C30H24O2, {systematic name: 5-[methylidene]-1,3-diphenylcyclopenta-1,3-diene}, 2, were prepared from 1,3-diphenylcyclopentadiene, pyrrolidine, and the corresponding aldehydes in an ethanolic solution. Each structure crystallizes with one molecule per asymmetric unit and exhibits the alternating short and long bond lengths typical of fulvenes. A network of C—H⋯C ring interactions as well as C—H⋯O interactions is observed, resulting in the compact packing found in each structure.The title compounds, 6-cyclopropyl-1,3-diphenylfulvene, C Surprisingly, the rotation of the 6-substituent from the cyclopentadienyl core is greater for fulvene 1 [87.20 (6)°], than for the larger 2,3-dimethoxynaphthalene substituent in fulvene 2, [55.63 (5)°].Compounds 1 Fig. 1 and 2 . Other C—H⋯C contacts  lead to the formation of a network that results in sets of zigzag chains running perpendicular to the a-axis direction and that extend in the direction parallel to the bc plane -1,3-diphenylfulvene, 1. Orange crystals suitable for single crystal X-ray diffraction were obtained from petroleum ether by slow evaporation.6--1,3-diphenylfulvene, 2. Red crystals suitable for single crystal X-ray diffraction were obtained from slow diffusion of petroleum ether into a DCM solution.Uiso(H) = 1.2Ueq(C) or 1.5Ueq(C-methyl).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989020006441/yk2129sup1.cifCrystal structure: contains datablock(s) 1, 2. DOI: 10.1107/S2056989020006441/yk21291sup2.hklStructure factors: contains datablock(s) 1. DOI: Click here for additional data file.10.1107/S2056989020006441/yk21291sup4.cdxSupporting information file. DOI: 10.1107/S2056989020006441/yk21292sup3.hklStructure factors: contains datablock(s) 2. DOI: Click here for additional data file.10.1107/S2056989020006441/yk21291sup5.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020006441/yk21292sup6.cmlSupporting information file. DOI: 2003795, 2003794CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Three new platinum(II) complexes bearing an isopropyl eugenoxyacetate and pyridine derivatives have been synthesized and further characterized by single-crystal X-ray diffraction. 2-2-allyl-4-methoxy-5-{[(propan-2-yloxy)carbonyl]methoxy}phenyl-κC1)chlorido(pyridine-κN)platinum(II), [Pt(C15H19O4)Cl(C5H5N)], (I), carbonyl]methoxy}phenyl-κC1)chlorido(4-methylpyridine-κN)platinum(II), [Pt(C15H19O4)Cl(C6H7N)], (II), and carbonyl]methoxy}phenyl-κC1)chlorido(pyridine-4-carboxylic acid-κN)platinum(II), [Pt(C15H19O4)Cl(C6H5NO2)], (III), have been synthesized and further characterized by single-crystal X-ray diffraction. The PtII atoms exhibit the usual distorted square-planar coordination and are surrounded by one Cl atom, one N atom, and a C atom and C=C double bond of the eugenol ligand. The donor N atom of the pyridine ligand occupies a cis position with respect to the double bond. Complexes (I) and (II) crystallize isomorphously in space group PII) disturbs the π–π interactions. The crystal packing of (III) is characterized by O—H⋯O hydrogen bonding, resulting in the formation of chains of molecules connected in a head-to-tail fashion and running in the [101] direction. The IC50 values for the HepG2 and KB cell lines are 150.9, 122.3 µM for (I) and 138.9, 93.2 µM for (II), respectively.Three new platinum(II) complexes bearing a eugenol and a pyridine derivative, namely (η Complexes (I), (II), (III) are obtained from the reactions of the dinuclear chelate ring complex [Pt(μ-Cl)(iPrEug)]2  with pyridine (Py), 4-methylpyridine (MePy) and pyridine-4-carboxylic acid (PyCOOH), respectively. The synthesis of the three complexes is summarized in Fig. 1In this paper, the crystal structures of three mononuclear platinacyclic complexes namely, (η1—Cl2 (or Pt2—Cl1) bond in complex 1 to form complexes (I), (II), (III). This is due to the weaker Pt1—Cl2 or Pt2—Cl1 bond (2.4773 Å) as compared to the Pt1—Cl1 or Pt2—Cl2 bond (2.3527 Å) (arylolefin-1H)]2 (arylolefin: safrole or eugenol derivatives) analogous to 1 react with different amines  and (II) crystallize isomorphously in the triclinic space group PII atom displays a distorted square-planar coordination with the Cl atom, the N atom of the pyridine or 4-methylpyridine ligand, and completed with a C atom and C=C double bond of the eugenol ligand . The C=C group and N atom are in a cis position with respect to each other. The dihedral angle between the best planes through the pyridine and phenyl rings is 74.90 (15)° for complex (I) and 75.00 (11)° for complex (II). The dihedral angle between the planes through the allyl atoms  and the pyridine ring is 16.0 (2)° for complex (I) and 20.08 (12)° for complex (II). The almost identical conformation is further evidenced by a fit of both structures, excluding H atoms and the methyl substituent in (II), which gives an r.m.s. deviation of 0.1867 Å  also crystallizes in space group PI) and (II) . Although the square-planar coordination of the central PtII atom is identical, the dihedral angle of 21.6 (2)° illustrates that the mutual orientation of the eugenol and pyridine parts is different. The plane through the allyl group makes an angle of 40.9 (3)° with the pyridine plane. An overlay of the identical parts in (I) and (III) gives an r.m.s. deviation of 0.5782 Å, while 0.5507 Å for (II) and (III)  Fig. 2c. AlthoI) Fig. 3.II atom of the three complexes shows that the largest differences occur for the Pt—N distance: 2.139 (2) Å for (I) within experimental error the same as 2.1418 (18) Å for (II), and 2.164 (3) Å for (III).Comparing the bond distances in the coordination sphere of the central PtI) is characterized by C—H⋯O hydrogen bonding, C—H⋯π and π–π interactions  Å; Cg4 is the centroid of ring N22/C23–C27; symmetry code: (v) 2 − x, 2 − y, 1 − z]. The phenyl ring C2–C7 participates in two C—H⋯π interactions.The crystal packing of complex  displays a very similar crystal packing , the π–π stacking is absent  and is in fact replaced by two C—H⋯π interactions between the methyl group and the pyridine ring. This slippage of the pyridine ring also results in an additional C8—H8B⋯Cl21 interaction between the allyl CH2 group and a neighboring Cl atom.Complex  is involved in head-to-tail fashion O—H⋯O interactions resulting in the formation of chains running in the [101] direction  Å; Cg4 and Cg5 are the centroids of rings N22/C23–C27 and C2–C7, respectively; symmetry code: (vi) −x, 1 − y, −z] and C—H⋯O hydrogen-bonding interactions  and (II), but for complex (III) a small void of 37 Å3 is present around .No voids are observed in the crystal packing of complexes  of the C=C group between 0.018 Å  increased significantly. The average C=C bond distance for the complexes in Table 4I)–(III), respectively].A search of the Cambridge Structural Database  and (II) was tested according to the method described in Skehan et al.  and 138.9, 93.2 µM for (II), respectively. This result shows that the presence of the extra methyl group on the pyridine ring in the para position in (II) does not have a notable effect on its anti-cancer activities as compared to those of (I). However, a comparison of complexes that differ solely in the olefin ligand reveals a significant influence. Specifically, complex (I) exhibits much better cytotoxicity against HepG2 and KB cell lines than [PtCl(eugenol-1H)(Py)] (iPrEug)]2 (1) was synthesized according to the synthetic protocol of Thong & Chi (pyridine)] (I). A solution of pyridine  in 10 mL ethanol was slowly added with stirring to a suspension of [Pt(μ-Cl)(iPrEug)]2  in 10 mL acetone. The reaction mixture was stirred at ambient temperature (AT) and filtered off after 30 minutes to remove the insoluble part. Subsequently, slow evaporation of the solvent of the obtained solution at AT gave within 10 h transparent crystals, which were suitable for X-ray diffraction and other analyses. The yield was 515 mg (90%). %Pt : 34.15/34.06. ESI MS , −MS: 1021, 100%, [2M – 2Py + Cl]−; +MS: 1067, 100%, [2M – Py + H]+; 988, 30%, [2M – 2Py + H]+. IR : 3089, 2970 and 2839 (CH); 1748 (C=O); 1597 and 1477 . 1H NMR : 8.79 , 8.04 , 7.65 , 7.04 , 6.66 , 5.07 , 4.83 , 4.54 , 3.81 , 3.78–3.74 , 3.73 , 2.66  = 16.5 Hz, 3JPtH = 110 Hz, 1H, CH2—CH), 1.27 .[PtCl(iPrEug)(4-methylpyridine)] (II). This complex was prepared starting from [Pt(μ-Cl)(iPrEug)]2  and 4-methylpyridine  according to the procedure for the synthesis of I. The yield was 539 mg (92%), transparent crystals were suitable for X-ray diffraction and other analyses. %Pt : 32.34/32.25. ESI MS , −MS: 1021, 100%, [2M – 2MePy + Cl]−; +MS: 1079, 70%, [2M – MePy + H]+; 986, 25%, [2M – 2MePy + H]+;. IR : 2970, 2920 and 2839 (CH); 1748 (C=O); 1616 and 1477 . 1H NMR : 8.60 , 7.46 , 7.04 , 6.65 , 5.07 , 4.79 , 4.54 , 3.78 , 3.76–3.74 , 3.73 , 2.64 , 2.46 , 1.27 .[PtCl(iPrEug)(pyridine-4-carboxylic acid)] (III). A mixture of pyridine-4-carboxylic acid  and [Pt(μ-Cl)(iPrEug)]2  in 10 mL acetone was stirred at AT for 8 h. The resulting precipitate was filtered off and washed consecutively with ethanol (2 × 5 mL) and cold chloroform (2 × 5 mL), then crystallized in chloroform to give a light-yellow powder. The yield was 493 mg (80%). Single crystals suitable for X-ray diffraction were obtained by slow evaporation within 8 h from a concentrated chloroform/ethanol solution at AT. %Pt : 31.58/31.63. ESI MS , -MS: 1021, 100%, [2M – 2PyCOOH + Cl]−; +MS: 1110, 8%, [2M – PyCOOH + H]+; 989, 10%, [2M – 2PyCOOH + H]+. IR : 3267 (OH), 3093, 2974 and 2839 (CH); 1728 (C=O); 1586 and 1477 . 1H NMR : 13.80 , 8.79 , 7.83 , 6.75–6.74 , 5.08 , 4.97 , 4.58/4.51 , 4.33 , 3.93 , 3.79–3.70 , 2.77 , 1.23 .Crystal data, data collection and structure refinement details are summarized in Table 5Uiso(H) values of 1.2Ueq or 1.5Ueq of the parent atoms, with C—H distances of 0.95 (aromatic), 1.00 (CH), 0.99 (CH2) and 0.98 Å (CH3). The carboxylic acid H atom in (III) was refined as rotating group with a O—H distance of 0.84 Å. The displacement parameters of the bonded atoms in the carboxylic acid and isopropyl groups in (III) were restrained to be similar along the bond.The H atoms were placed in idealized positions and included as riding contributions with 10.1107/S2056989020006957/mw2159sup1.cifCrystal structure: contains datablock(s) I, II, III. DOI: 10.1107/S2056989020006957/mw2159Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989020006957/mw2159IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989020006957/mw2159IIIsup4.hklStructure factors: contains datablock(s) III. DOI: 2005230, 2005229, 2005228CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The solid-state structure of the ‘magic mushroom’ natural product norpsilocin is reported, as well as the synthesis and structure of its fumarate salt. N-methyltryptamine (4-HO-NMT); systematic name: 3-[2-(methylamino)ethyl]-1H-indol-4-ol}, C11H14N2O, and its fumarate salt ethyl]methylazanium} but-2-enedioate), C11H15N2O+·0.5C4H2O42−, are reported. The freebase of 4-HO-NMT has a single molecule in the asymmetric unit joined together by N—H⋯O and O—H⋯O hydrogen bonds in a two-dimensional network parallel to the (100) plane. The ethylamine arm of the tryptamine is modeled as a two-component disorder with a 0.895 (3) to 0.105 (3) occupancy ratio. The fumarate salt of 4-HO-NMT crystallizes with a tryptammonium cation and one half of a fumarate dianion in the asymmetric unit. The ions are joined together by N—H⋯O and O—H⋯O hydrogen bonds to form a three-dimensional framework, as well as π–π stacking between the six-membered rings of inversion-related indoles .The solid-state structures of the naturally occurring psychoactive tryptamine norpsilocin {4-hydroxy- Psychoactive tryptamines, particularly psilocybin and psilocin, have recently garnered a great deal of interest because of their potential to treat disorders including anxiety, addiction, and depression , aka psilocin, and the prodrug baeocystin hydrolyses to the active 4-hydroxy-N-methyltryptamine (4-HO-NMT), aka norpsilocin. Norpsilocin was first identified as a natural product of ‘magic mushrooms’ in 2017, and isolated as an amorphous, colorless solid Despite rapidly growing evidence supporting psilocin/psilocybin’s potential for treating mood disorders, very little work has been done to investigate the properties of other structurally similar compounds found in magic mushrooms, N-methyltryptamine (C11H14N2O) molecule. The ethylamine arm (C9–C10–N2–C11) of the tryptamine is modeled as a two-component disorder with a 0.895 (3) to 0.105 (3) occupancy ratio. The rest of the discussion is restricted to the major component. The indole ring system of the tryptamine is near planar with an r.m.s. deviation from planarity of 0.015 Å. The ethylamine arm of the tryptamine is slightly turned, with a C7—C8—C9—C10 torsion angle of 29.3 (3)°. The C10—N2—C11 angle about the amine nitrogen is 113.51 (15)°.The molecular structure of the freebase of norpsilocin, 4-HO-NMT, is shown in Fig. 1N-methyltryptammonium (C11H15N2O+) cation and one half of a fumarate (C4H2O42–) dianion, with the other half generated by inversion. The indole ring system of the tryptamine is near planar with an r.m.s. deviation from planarity of 0.009 Å. Unlike the freebase, the ethyl ammonium arm resides in the same plane as the indole. The planarity of all of the non-hydrogen atoms of the tryptamine is demonstrated with an r.m.s. deviation from planarity of only 0.043 Å. The C10—N2—C11 angle about the ammonium nitrogen is 114.20 (14)°. The fumarate itself is also near planar, with an r.m.s. deviation from planarity of 0.050 Å. The carboxylate unit of the fumarate is delocalized, with C—O distances of 1.2488 (18) and 1.2553 (18) Å.The molecular structure of the fumarate salt of norpsilocin is shown in Fig. 2x + 1, −y + 1, −z + 1) to form a dimer. The indole N—H shows an intermolecular hydrogen bond with the phenol oxygen of another tryptamine molecule  plane through a series of N—H⋯O and O—H⋯N hydrogen bonds Table 1. The pheb.The tryptammonium cations and the fumarate dianions of the fumarate salt of norpsilocin are held together in an infinite three-dimensional framework through a series of N—H⋯O and O—H⋯O hydrogen bonds Table 2. The indet al., 2016N,N-dimethyltryptamine .Single crystals suitable for X-ray analysis were obtained from the slow evaporation of an acetone solution of a commercial sample of 4-hydroxy-N-methyltryptamine, which was dissolved in 10 mL of methanol. 62 mg of fumaric acid was added to the solution and it was stirred overnight under reflux. Solvent was removed in vacuo to yield a dark-blue powder. The powder was triturated with diethyl ether and then recrystallized in acetone to yield colorless crystals suitable for X-ray analysis. 1H NMR : δ 7.12 , 7.10–7.07 , 6.66 , 6.56 , 3.41 , 3.26 , 2.70 ; 13C NMR : δ 171.0 (COOH), 149.7 (ArC), 138.5 (ArC), 134.2 (CH), 123.0 (ArC), 122.8 (ArC), 115.6 (ArC), 108.4 (ArC), 104.2 (ArC), 103.4 (ArC), 50.3 (CH2), 32.4 (CH2), 22.7 (CH3).The fumarate salt was synthesized starting with 101 mg of 4-hydroxy-A, and H2 were found from a difference-Fourier map and were refined isotropically, using DFIX restraints with N—H distances of 0.87 (1) Å and an O—H distance of 0.88 (1) Å. Isotropic displacement parameters were set to 1.2Ueq of the parent nitrogen atom and 1.5Ueq of the parent oxygen atom. All other hydrogen atoms were placed in calculated positions (C—H = 0.93–0.97 Å). Isotropic displacement parameters were set to 1.2Ueq(C) or 1.5Ueq(C-methyl).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989020004077/pk2623sup1.cifCrystal structure: contains datablock(s) I, II, global. DOI: 10.1107/S2056989020004077/pk2623Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989020004077/pk2623IIsup3.hklStructure factors: contains datablock(s) II. DOI: 1992279, 1992278CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


HS66 which colonized in the stems of Isodon xerophilus. Their structures were determined by extensive spectroscopic methods. Furthermore, the structure of 1 was unambiguously confirmed by X-ray diffraction, while those of 2 and 3 were verified through quantum chemical calculation of NMR data and ECD spectra.Arthrinins E–G (The online version of this article (doi:10.1007/s13659-020-00248-y) contains supplementary material, which is available to authorized users. Fungal metabolites have received considerable attention nowadays because their diverse chemical structures and bioactivities greatly facilitate the progress of drug discovery . BotryanIsodon species. As a result, isopenicin A, a potent inhibitor of Wnt signaling  unambiguously verified the aforementioned deduction and assigned the absolute configuration of 1 as 2R,4S,5S, and 8S , indicating three degrees of unsaturation. Comparison of the 1H NMR spectrum of 2 with that of 1 disclosed that 2 was structurally analogous to compound 1 , from H2-10 to C-9. On the other hand, the HMBC correlations from H-5 to C-8 (δC 136.9) and C-13 (δC 31.6), from H2-7 to C-5 (δC 60.9), from H3-12 to C-6 (δC 39.7) and C-7 (δC 51.7), from H3-13 to C-12 (δC 25.6), from H2-14 to C-7 and C-8 defined the ring B fragment and planar structure of 2 ultimately  of 2, randomly assigning H-5 as β-oriented, the observed correlations of H2-10/H3-11/H-5 in the ROESY spectrum, together with the coupling constant (J = 4.4 Hz) between H-1 and H-2 implied that both CH2OH-10 and CH3-11 adopted β-orientation, while the analysis of coupling constant (J = 10.8 Hz) between H-4 and H-5 suggested that H-4 was α-oriented -2 was subjected to quantum chemical calculation of NMR chemical shifts and spin–spin coupling constants at mPW1PW91-SCRF/6–31 + G//B3LYP-D3BJ-SCRF/6-31G(d) and B972-SCRF/pcJ-1//B3LYP-D3BJ-SCRF/6-31G(d) level of theory (both with methanol as solvent and SMD solvent model), respectively, and the calculated results matched their experimental counterparts very well -2 was run at CAM-B3LYP-SCRF/def2-SVP//B3LYP-D3BJ-SCRF/6-31G(d) level of theory in MeOH with SMD solvent model, and the obtained curve, which is entirely consistent with its experimental counterpart, supported the absolute configuration of 2 to be 1R,2R,4S, and 5S  existing in the positive HRESIMS spectrum revealed the molecular formula of 3 to be C15H18O4, which implied seven indices of hydrogen deficiency. Its IR spectrum indicated the presence of hydroxyl (3419 cm–1), methyl (2932 and 2869 cm–1), phenyl (1607 and 1467 cm–1) and ester (1743 and 1040 cm–1) groups. The 1H NMR spectrum gave two characteristic aromatic protons at δH 7.43 , 7.72  and two singlet methyl groups  , C-5 (δC 157.8) and C-11 (δC 173.9), from H-4 to C-2 (δC 126.0) and C-9 (δC 144.7), from H2-10 to C-1, C-2 and C-11 established the structure of the butyrolactone motif (ring C), and its connection to a tetra-substituted benzene ring (ring A). Moreover, the HMBC cross peaks from H-4 to C-6 (δC 50.1), from H2-7 to C-13 (δC 27.2) and C-15 (δC 70.8), from H2-12 to C-5 and C-6, from H3-13 to C-12 (δC 71.3), from H3-14 to C-8 (δC 50.2) and C-9, from H2-15 to C-14 (δC 24.9) determined the existence of a cyclopentane fragment (ring B) and its linkage with ring A. As a result, the planar structure of compound 3 was elucidated as shown in Fig. The 1 δC 145., C-5 -3 were calculated at mPW1PW91-SCRF/6–31 + G//B3LYP-D3BJ-SCRF/6-31G(d) level of theory with methanol as solvent and SMD solvent model, the predicted chemical shifts matched their experimental counterparts very well -3 was obtained at CAM-B3LYP-SCRF/def2-SVP//B3LYP-D3BJ-SCRF/6-31G(d) level of theory in MeOH with SMD solvent model, and the calculated curve matched the experimental one very well and thus supported the absolute configuration of 3 to be 6R,8S. , with cis-platin and paclitaxel as positive controls, however, no compounds showed activity against the tested cell lines .Additionally, compounds δ) are expressed in parts per million (ppm) with reference to the solvent signals. HRESIMS was performed on an API QSTAR spectrometer. Semipreparative HPLC was performed on an Agilent 1200 liquid chromatograph with a Zorbax SB-C18 (9.4 mm × 250 mm) column. LC–MS/MS was performed on Agilent 6530 Accurate-Mass Q-TOF spectrometer coupled to an Agilent 1290 LC system with Zorbax SB-C18 (9.4 mm × 250 mm) column. Column chromatography was performed with silica gel . Fractions were monitored by TLC, and spots were visualized by heating silica gel plates sprayed with 10% H2SO4 in EtOH.Optical rotations were measured with a JASCO P-1020 polarimeter. UV spectra were obtained using a Shimadzu UV-2401 PC spectrophotometer. A Tensor 27 spectrophotometer was used for scanning IR spectroscopy with KBr pellets. 1D and 2D NMR spectra were recorded on Bruker DRX-600 and 500 spectrometers with TMS as internal standard. Chemical shifts  analysis of the ITS region of the rDNA. The fungal strain was cultured on slants of potato dextrose agar at 28 °C for 7 days. Agar plugs were cut into small pieces (about 0.5 × 0.5 × 0.5 cm3) under aseptic conditions, and 15 pieces were used to inoculate three Erlenmeyer flasks (500 mL), each containing 200 mL of media ; the final pH of the media was adjusted to 7.0, and the flasks were sterilized by autoclave. Three flasks of the inoculated media were incubated at 28 °C on a rotary shaker at 170 rpm for 7 days to prepare the seed culture. The detailed lager fermentation procedure was as following: Fermentation was carried out on solid rice medium in 125 Fernbach flasks . Each flask was inoculated with 5.0 mL of the spore inoculum and incubated for 30 days at 28 °C in a static incubator.The fungal strain of 2 in a humidified atmosphere. Cytotoxicity of compounds was determined by MTS method. Briefly, 5 × 103 cells were plated in 96-well plates 12 h before treatment and continuously exposed to test compounds for 48 h. Then MTS (Promega) was added to each well. The samples were incubated at 37 ℃ for 1–4 h and the optical density (OD) was measured at 490 nm using a microplate reader (Bio-Rad Laboratories). The IC50 values were calculated by Reed and Muench’s method D22.2: + 66.6 ; ECD (MeOH) λmax (Δε): 220 (0.02) nm; UV (MeOH) λmax (log ε): 204 (3.03) nm; IR (νmax): 3374, 2927, 2869, 1630, 1607, 1460, 1384, 1366, 1093, 1057, 1026, 1010, 986, 971 cm–1. HRESIMS at m/z 277.1775 . 1H and 13C NMR data, see Tables Arthrinin E : initial2): obtained as a colorless oil; [α]D22.4: + 107.00 ; ECD (MeOH) λmax (Δε): 205 (0.68) nm; UV (MeOH) λmax (log ε): 201 (3.14) nm; IR (νmax): 3383, 2955, 2927, 2890, 2871, 1631, 1609, 1465, 1454, 1384, 1361, 1082, 1026, 994, 978 cm–1. HRESIMS at m/z 263.1618 . 1H and 13C NMR data, see Tables Arthrinin F ( ε): 201 .14 nm; I3): isolated as a colorless oil; [α]D22.8: + 12.55 ; ECD (MeOH) λmax (Δε): 204 (0.21), 240 (– 0.06), 276 (0.01) nm; UV (MeOH) λmax (log ε): 208 (3.22), 241 (2.74), 283 (1.98) nm; IR (νmax): 3419, 2957, 2932, 2869, 1743, 1607, 1467, 1384, 1367, 1040, 1017, 744 cm–1. HRESIMS at m/z 285.1100 . 1H and 13C NMR data, see Tables Arthrinin G : isolate1, deposition number CCDC 1997674) has been deposited in the Cambridge Crystallographic Data Centre database. Copies of the data can be obtained free of charge from the CCDC at www.ccdc.cam.ac.uk.Crystallographic data for the structures of arthrinin E  Å, b = 8.8861(2) Å, c = 31.7909(6) Å, α = 90°, β = 90°, γ = 120°, V = 2173.98(11) Å3, T = 100.(2) K, space group P3221, Z = 6, μ(Cu Kα) = 0.629 mm–1, 29,180 reflections measured, 2838 independent reflections (Rint = 0.0291). The final R1 values were 0.0351 (I > 2σ(I)). The final wR(F2) values were 0.0969 (I > 2σ(I)). The final R1 values were 0.0353 . The final wR(F2) values were 0.0970 . The goodness of fit on F2 was 1.142. Flack parameter = 0.07(3).R*,2R*,4S*,5S*)-2 and -3 were undertaken with the CREST code (version 2.8) using the default iMTD-GC procedure [R*,2R*,4S*,5S*)-2 and -3 were subjected to DFT geometry optimization at B3LYP-D3BJ-SCRF/6-31G(d) level of theory (with MeOH as solvent and SMD solvent model). Frequency analyses of all optimized conformers were undertaken at the same level of theory to ensure that no imaginary frequency exists. Then, thermal correction to Gibbs free energies obtained by frequency analyses were added to the electronic energies obtained at B3LYP-D3BJ-SCRF/6–311 + G level of theory (with MeOH as solvent and SMD solvent model) to get the Gibbs free energies of each conformer. Subsequently, Room-temperature (298.15 K) equilibrium populations were calculated according to Boltzmann distribution law:Pi is the population of the ith conformer; ni the number of molecules in ith conformer; ΔG is the relative Gibbs free energy ; T is room temperature (298.15 K) here; R is the ideal gas constant (0.0019858995). Those conformers with a population of over 2% were subjected to subsequent NMR and ECD calculations.Conformational searching of , where the σTMS was the shielding constant of TMS calculated at the same level of theory. The parameters a and b of the linear regression δcal = aδexp + b; the correlation coefficient, R2; the mean absolute error (MAE) defined as Σn|δcal – δexp|/n; the corrected mean absolute error (CMAE) defined as Σn|δcorr – δexp|/n, where δcorr = /a were calculated [NMR shielding constants were calculated with the GIAO method at mPW1PW91-SCRF/6–31 + G level (with MeOH and SMD solvent model). The obtained shielding constants were converted into chemical shifts by referencing to TMS at 0 ppm  . For eacThe geometry optimization, single-point energy calculation, NMR shielding constant calculation, coupling constant calculation, and TDDFT ECD calculation were all completed in Gaussian 09 program .Electronic supplementary material 1 (PDF 3960 kb)Below is the link to the electronic supplementary material.


Correction to: J Foot Ankle Res 13, 31 (2020)https://doi.org/10.1186/s13047-020-00403-1Following publication of the original article , we haveOn Page 1, Abstract, ‘Methods’ section, line 6: ‘intra-class coefficients’ should read ‘intra-class correlation coefficients’. This error is also present on page 8 in the ‘Abbreviations’ section.In Table 1 on page 5, within the final sentence of the notes section at the bottom of the table: the cut-off values used for the Arch Index ‘Normal: 0.21 to 0.28, high: < 0.21 and low: > 0.28’, should read ‘Normal: 0.21 to 0.26, high: < 0.21 and low: > 0.26 [23]’


N,N’‐diarylated dihydroazaacene radical cations were prepared by oxidation of neutral N,N’‐diarylated dihydroazaacenes synthesized via palladium‐catalyzed Buchwald‐Hartwig aminations of aryl iodides with N,N’‐dihydroazaacenes. Both neutral as well as oxidized species were investigated via UV‐vis spectroscopy, single crystal analysis, and DFT calculations. All the radical cations are surprisingly stable—their absorption spectra in dichloromethane remain unchanged in ambient conditions for at least 24 hours.Three stable  Oxidation ofN,N’‐diarylated dihydroazaacenes furnishes three stable dihydroazaacene radical cations characterized by means of UV‐vis spectroscopy, single crystal analysis, and DFT calculations. Acene‐based radicals and radical ionsTAP) is an n‐type semiconductor with electron mobilities reaching up to 10 cm2 V−1 s−1 in organic field‐effect transistors (OFETs).−.TAP . Later, our group reported that the radical anion 4TAP−.Br  with around 20 % absorption intensity loss after 9 h in ambient surroundings, whereas its oxidation product, +.Quino is stable; its absorption spectrum remains unchanged for at least 24 h under the same conditions. In this contribution, we exploit acene‐based radical ions, N,N’‐dihydroazaacene‐based radical cations.6,13‐Bis(triisopropylsilylethynyl)‐5,7,12,14‐tetraazapentacene ), although their dications were observed via cyclic voltammetry .DDAs+. in DCM. +.1 and +.3 display intense doublets with g‐values of 2.0022 and 2.0021. +.2 exhibits a defined triplet spectrum with a g‐value of 2.0019.Figure DDAs and their radical cations in DCM were measured, and TD‐DFT calculations of the vertical excited states give insight into the electronic transitions. Compound 1 displays the most hypsochromic absorption maximum (λmax) at 369 nm . The simulated absorption spectra of +.1, +.2, and +.3 display their λmax at 673, 1166, and 1839 nm, respectively, which are mainly contributed by the HOMO (β)→LUMO (β) transitions.UV‐vis absorption spectra of In addition, Figure S3 (see SI) shows the evolution of absorption intensity of the radical cations as a function of time in DCM. All of the three radical cations are stable—their absorption spectra remain unchanged under ambient conditions for at least 24 h.1 and 3 monooxidize readily . 2 is the least easily oxidized . Driving force is the generation of an aromatic acene backbone. We do not observe any reduction events of the neutral specimens, due to the electron‐rich and anti‐aromatic 4n π‐electron N,N’‐dihydroazacene nuclei. The CVs of the radical cation species display both a reversible oxidation and a reversible reduction wave. From +.1 to +.3, the oxidation potentials increase from 0.14 V to 0.26 V, while the reduction potentials decrease from 0.63 V to 0.34 V. The radical cations become more difficult to oxidize and easier to reduce as a consequence of extension of the π‐conjugated systems.All dihydroacenes exhibit two reversible oxidation waves in their cyclic voltammograms , attributed to the formation of their radical cations and dications. zz calculationsN,N’‐dihydropyrazine .N,N’‐dihydropyrazine rings increases dramatically, and both the N,N’‐dihydropyrazine rings of +.2 and +.3 now are aromatic with the NICS‐values decreasing to −2.02 and −4.55 respectively.NICS(1)DDAs and DDAs+. are depicted in Figure S6 in the SI. For the neutral species, their highest occupied molecular orbitals (HOMOs) are distributed over the whole backbone, while the lowest occupied molecular orbitals (LUMOs) are mainly located on the electron‐withdrawing para‐trifluoroaryl side groups, differing from aryl‐substituted acenes such as azarubrenes,DDAs+., their singly occupied molecular orbitals (SOMOs) are mostly distributed on the dihydrodiazaacene cores, as expected.The frontier molecular orbital distributions of 2 and 3 for XRD analysis by slow evaporation of saturated solutions of DCM under ambient conditions. The single crystal structure of 1 was already reported.+.1, +.2, and +.3 were grown successfully by slow evaporation of acetone solutions under air. For all of the six compounds, their N,N’‐(dihydro)diazaacene backbones are planar zz calculations.We cultivated single crystals of the neutral N,N’‐dihydropyrazine units. N,N’‐Diarylated dihydroazaacenes, 1, 2, and 3, were synthesized through Buchwald‐Hartwig amination of aryl iodides with N,N’‐dihydroazaacenes. Upon monooxidation of the neutral compounds, the radical cations +.1, +.2, and +.3 formed, stable in DCM under ambient conditions for at least 24 h. In comparison with the neutral species, the radical cation species display bathochromically‐shifted absorption spectra with a λmax of 769, 1291, and 1986 nm, respectively. The alternation of bond lengths between the neutral compounds and their radical cations in single crystal structures as well as the NICS(1)zz calculations indicate the N,N’‐dihydropyrazine rings of the radical cations to be less antiaromatic. In the future, we will prepare N,N’‐dihydrodiazaacene radical cations with longer backbones, N,N’‐dihydrodiazahexacene and N,N’‐dihydrodiazapentacene radical cations. Further attempts to electrochemically oxidize the N,N’‐dihydroazaacenes to their fully aromatic dications are under way.In conclusion, we present a practical strategy to exploit stable radical cations based on 2. 5,10‐Di(4‐trifluoromethylphenyl)‐5,10‐dihydrophenazine (1),b]‐phenazine,b,i]phenazineThe radical cations were synthesized in a glove box under NGeneral procedure (GP1): To a stirring solution of N,N’‐disubstituted dihydrophenazine derivative ) in 15 mL DCM was added dropwise NO+SbF6− (1.05 equiv.) in 1.5 mL CH3CN and the reaction mixture was stirred for 12 h at room temperature (r.t.). After that, the mixture was filtered through filter paper and the solvent was removed under reduced pressure to give the corresponding radical cations.General procedure for Buchwald‐Hartwig amination (GP2): The dihydrophenazine derivative (1.00 equiv.), 1‐iodo‐4‐(trifluoromethyl)benzene (4.00 equiv.), RuPhos Pd G1 (0.05 equiv.), tBuOK (6.00 equiv.), and RuPhos (0.10 equiv.) were added into a flask under N2. After exposing the flask to a flow of N2 for 30 min to obtain an inert atmosphere, toluene was added. The mixture was allowed to heat to 100 °C and stirred for 36 h. Thereafter, the solution was filtered through a silica pad with ethyl acetate (EE), and then concentrated under reduced pressure before further purification steps were undertaken.+⋅1: 1  and NO+SbF6−  were used in combination with +.GP1. 1 was obtained as a deep green solid. Yield: 69.1 mg, 97.8 μmol, 92 %. Single crystals suitable for X‐ray crystallography were grown from saturated acetone solution. IR: ṽ=3086, 2920, 2851, 1604, 1559, 1466, 1319, 1060, 649 cm−1. MS (MALDI) m/z: [M]+: calcd for C26H16N2F6+⋅: 470.121; found 470.172; correct isotope distribution; elemental analysis calcd for C30H18N2F6+⋅: C: 44.22 %, H: 2.28 %, N: 3.97 %, found: C: 44.16 %, H: 2.50 %, N: 4.22 %.b]phenazine (2)5,12‐Di(4‐trifluoromethylphenyl)‐5,12‐dihydrobenzophenazine , 1‐iodo‐4‐(trifluoromethyl)benzene , RuPhos Pd G1 , tBuOK , and RuPhos  were used in combination with GP2 in toluene (20 mL). After purification via column chromatography  and evaporation of the solvent in vacuo, the crude product was further washed with petroleum (PE) and a faint yellow solid was obtained. Yield: 1.08 g, 2.07 mmol, 96 %. Melting point (mp): 305 °C. 1H NMR : δ=8.05–7.92 , 7.66–7.60 , 7.09–7.03 , 7.00–6.95 , 6.42–6.33 , 5.85–5.80 , 5.75–5.67  ppm. 13C{1H} NMR : δ=143.7, 136.6, 134.7, 132.5, 131.0, 129.3, 126.0, 125.5, 124.7, 123.7, 121.6, 113.4, 108.0 ppm. IR: ṽ=3059, 1475, 1332, 1120, 1060, 843, 732 cm−1. MS (MALDI) m/z: [M]+: calcd for C30H18N2F6: 520.478; found 520.098; correct isotope distribution.+⋅2: 2  and NO+SbF6−  were used in combination with +.GP1. 2 was obtained as a dark red solid. Yield: 69.0 mg, 91.3 μmol, 95 %. Single crystals suitable for X‐ray crystallography were grown from saturated acetone solution. IR: ṽ=3063, 2980, 2915, 1600, 1435, 1323, 1065, 645 cm−1. MS (MALDI) m/z: [M]+: calcd for C30H18N2F6+⋅: 520.137; found 520.206; correct isotope distribution; elemental analysis calcd for C30H18N2F6+⋅: C: 47.65 %, H: 2.40 %, N: 3.70 %, found: C: 47.79 %, H: 2.83 %, N: 3.68 %.b,i]phenazine (3)6,13‐Di(4‐trifluoromethylphenyl)‐6,13‐dihydrodibenzophenazine , 1‐iodo‐4‐(trifluoromethyl)benzene , RuPhos Pd G1 , tBuOK , and RuPhos  were used in combination with GP2 in toluene (20 mL). After purification by column chromatography  and evaporation of the solvent in vacuo, the residue was washed by PE to give a yellow solid. Yield: 226 mg, 395 μmol, 56 %. Mp: >400 °C (decomposition). 1H NMR : δ=8.10–8.01 , 7.74–7.67 , 7.18–7.12 , 7.06–6.99 , 6.02–5.97  ppm. 13C{1H} NMR : δ=143.7, 136.6, 134.7, 132.5, 131.0, 129.3, 126.0, 125.5, 124.7, 123.7, 121.6, 113.4, 108.0 ppm. IR: ṽ=3059, 1614, 1457, 1305, 1129, 1060, 843, 746 cm−1. MS (MALDI) m/z: [M]+: calcd for C34H20N2F6: 570.538; found 570.373; correct isotope distribution; elemental analysis calcd for C34H20N2F6: C: 71.58 %, H: 3.53 %, N: 4.91 %, found: C: 71.43 %, H: 3.81 %, N: 4.94 %.+⋅3: 3  and NO+SbF6−  were used in combination with +.GP1. 3 was obtained as a brown solid. Yield: 65.0 mg, 80.6 μmol, 92 %. Single crystals suitable for X‐ray crystallography were grown from saturated acetone solution. IR: ṽ=3054, 2925, 2856, 2754, 1591, 1314, 1055, 645 cm−1. MS (MALDI) m/z: [M]+: calcd for C34H20N2F6+⋅: 570.153; found 570.243; correct isotope distribution.+.1), 2018607 (2), 2018608 (+.2), 2018609 (3), and 2018610 (+.3)Deposition number(s) 2018606  should be addressed to the authors.SupplementaryClick here for additional data file.


The molecule has twofold crystallographic symmetry and is isomorphous to its Mn, Co, Ni, Cu and Zn counterparts.In the title complex molecule, [Cd(C The largest deviation from planarity is only 0.145 (3) Å, observed for atom C12 of the pyrazine ring. The Cd1 atom is nearly coplanar with each of the two ligands (deviation = 0.316 Å). The dihedral angle between the two ligands is 78.705 (16)°. The oxygen atom O1 of the phenolic group remains protonated, and forms an intramolecular hydrogen bond O1—H1⋯N1 .meta or para position {3-hydroxy-N′-[1-(pyrazin-2-yl)ethylidene]benzohydrazide (L2) or 4-hydroxy-N′-[1-(pyrazin-2-yl)ethylidene]benzohydrazide (L3)}, where no intramolecular hydrogen bond can be formed, crystallization is substantially delayed and a much longer time is required for the complexes to crystallize.The intramolecular hydrogen bond stabilizes the planar geometry of the ligand. The presence of the intramolecular hydrogen bond does also appear to affect the propensity of the metal complex towards crystallization. We found that when the hydroxyl group is in the M(L)2 complexes, the ligands are also close to planar (the mean deviation from the average plane ranges from 0.0608 to 0.0754 Å). In dimethylformamide (DMF)-solvated Ni and Cu complexes of similar ligands L2 {3-hydroxy-N′-[1-(pyrazin-2-yl)ethylidene]benzohydrazide} and L3 {4-hydroxy-N′-[1-(pyrazin-2-yl)ethylidene]benzohydrazide} [M(L2)2]·2(DMF)  and [Cu(L3)2]·2(DMF) (M = Ni and Cu), the planarity of the ligands is reduced, with a mean deviation from the average plane between 0.2164 to 0.2290 Å.In the isomorphous Mn, Co, Ni, Cu and Zn 2+ complexes closely related to the title compound, such as bis{N′-[1-(pyridin-2-yl)ethylidene]benzohydrazidato}cadmium(II) picolinohydrazonamide]cadmium(II) methylene]benzohydrazidato}cadmium(II) , 2.273 (3) and 2.277 (4) Å, respectively, which are close to typical for Cdtrans angles range from 139.07 (10) to 170.63 (17)°, while the cis angles vary between 69.83 (11) and 117.27 (11)°.The coordination environment of the Cd ion is highly distorted octahedral, caused by the rigidity of the ligand and its small N—N and N—O bite angles of only 69.86 (11) (N3—Cd1—N2) and 69.83 (11)° (N2—Cd1—O2). As a result, the N—Cd—O, N–Cd—N and O—Cd—O angles in the title compound deviate substantially from the values of 180 and 90° expected for an idealized octahedral complex. The d complex of the series. The trend of the N—M—O angle (within the same ligand) is opposite to that of the metal ion radius, and first increases and then decreases, with the maximum value appearing for the Ni complex —M—N(mid) angles, which range from 172.30 to 174.46° for the 3d complexes, while the value for the 4d Cd complex is 170.63 (17)°.Bond distances and angles within the isomorphous series of the Mn, Co, Ni, Cu, Zn, and Cd complexes follow a trend consistent with the metal ion radius Table 1. Bond leii and C12—H12⋯O1i, symmetry code given in Table 2a) form a sheet parallel to the crystallographic bc plane. Adjacent sheets of the complex are connected to each other via a weak C4—H4⋯N4iii interaction, forming a three-dimensional network (Table 2b). Intermolecular π–π stacking is observed between the pyrazine rings and benzene rings of ligands in neighbouring complexes  Fig. 3.et al., 2016N′-[1-(pyrazin-2-yl)ethylidene]benzohydrazide ligand resulted in seven related metal complexes with exactly the same ligand. These are the already discussed isomorphous Mn, Co, Ni, Cu and Zn [MII(L)2] complexes  ethylidene]benzohydrazide  ethylidene]benzohydrazide  with M = Cu2+, Ni2+, Zn2+ and Fe2+ ·0.25(CH3OH) Br (NO3) Cl2]·H2O 2Cl2 2Cl2. A Zn complex, Zn(L1)2·H2O 2]·2(DMF)  and [Cu(3L)2]·2(DMF)  2] core are distorted octahedral, and all metal centres have a mer geometry. All ligands L, L1, L2, L3, L4 and L5 are tridentate chelating.There are also complexes of ligands et al., 2020et al., 20193)2·4H2O (0.01 mmol) in water (2 mL). After two weeks of static volatilization in a test tube at room temperature, clear light-yellow block-shaped crystals of Cd(L)2 were obtained  , m.p. > 543 K. IR (KBr): ν (cm−1) = 1594 s, 1534 s, 1518 s, 1489 s, 1458 s, 1401 w, 1349 s, 1299 s, 1248 m, 1225 m, 1198 m, 1162 m, 1147 s, 1106 w, 1072 s, 1042 m, 1029 m, 910 w, 850 w, 833 w, 786 w, 764 m, 701 m, 662 w, 565 w, 541 w, 492 w, 419 w, 406 w.The title complex and ligand were synthesized according to literature procedures  and were included in the refinement in a riding-model approximation, with Uiso(H) set to 1.2Ueq(Csp2) and 1.5Ueq(Csp3). The O-bound H atom was located based on a difference-Fourier map and its position was freely refined. It was assigned Uiso(H) = 1.5Ueq(O).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989021000657/zl5004sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989021000657/zl5004Isup2.hklStructure factors: contains datablock(s) I. DOI: 2051612CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Hypoxia-inducible factor 1α (HIF-1α) and HIF-2α are master transcription factors that regulate cellular responses to hypoxia, but the exact function in regulatory T (Treg) cells is controversial. Here, we show that Treg cell development is normal in mice with Foxp3-specific knockout (KO) of HIF-1α or HIF-2α. However, HIF-2α-KO (but not HIF-1α-KO) Treg cells are functionally defective in suppressing effector T cell-induced colitis and inhibiting airway hypersensitivity. HIF-2α-KO Treg cells have enhanced reprogramming into IL-17-secreting cells. We show crosstalk between HIF-2α and HIF-1α, and that HIF-2α represses HIF-1α expression. HIF-1α is upregulated in HIF-2α-KO Treg cells and further deletion of HIF-1α restores the inhibitory function of HIF-2α-KO Treg cells. Mice with Foxp3-conditional KO of HIF-2α are resistant to growth of MC38 colon adenocarcinoma and metastases of B16F10 melanoma. Together, these results indicate that targeting HIF-2α to destabilize Treg cells might be an approach for regulating the functional activity of Treg cells. HIF-1α is known to repress regulatory T (Treg) cell function, but less is known about the effect of HIF-2α on these cells. Here, the authors use three different mouse models of inflammatory diseases to show that HIF-2α limits HIF-1α expression and is critical for Treg cell function. Treg cells are classified into thymus-derived CD4+CD25+Foxp3+ regulatory T (tTreg) cells, peripheral regulatory T (pTreg) cells and induced regulatory T (iTreg) cells1. Treg cells are characterized by their expression of the master transcription factor forkhead box P3 (Foxp3), which dictates their development and function4. Persistent expression of Foxp3 is required throughout the lifetime of Treg cells to maintain their suppressive activities6. Significant efforts have been dedicated to establishing how Foxp3 gene expression is regulated7. Notably, the extent of methylation in the Treg-specific demethylation region (TSDR) of the Foxp3 gene determines Foxp3 stability9. Direct dendritic cell stimulation or IL-2 treatment promotes Treg stability through TSDR demethylation of Foxp3 (refs. 11). Foxp3 protein stability is further regulated by several post-translational modifications, including ubiquitination, acetylation and phosphorylation14. Some Treg cells are converted into inflammatory effector cells following Foxp3 destabilization, which may lead to loss of their suppressive activity and expression of inflammatory cytokines such as IFN-γ or IL-17 (refs. 17). There are reports that Treg cells can be reprogrammed into inflammatory cells in certain autoimmune diseases22. Recent studies have demonstrated that ex vivo expansion of Treg cells can be used to treat autoimmune diseases and transplantation rejection25. Maintenance of stable suppressive activity is essential for successful therapeutic applications of Treg cells. However, Treg cells contribute to immunosuppressive environments and are a major element inhibiting intratumor anticancer immunity28. Treg-selective deletion of essential Treg factors such as Neuropilin-1 (Nrp-1), suppressor of cytokine signaling 1 (SOCS1), enhancer of zeste homolog 2 (EZH2), Eos (IKzf4), or CARMA1 leads to Foxp3 downregulation and inflammatory cytokine secretion, conferring resistance to tumor growth in the host33.CD437. In the presence of O2, HIF-1α and HIF-2α are hydroxylated at key proline residues by prolyl hydroxylase domain protein 2 (PHD2)/PHD3, leading to recognition by the von Hippel-Lindau (VHL)-containing E3 complex that ubiquitinates HIF-1α and HIF-2α for their proteasomal degradation. Inactivation of PHD2/PHD3 under hypoxic conditions stabilizes HIF-1α and HIF-2α proteins38. HIF-1α and HIF-2α are then heterodimerized with HIF-1β subunits to activate expression of target genes involved in hypoxic responses37. In addition, HIF-1α can be upregulated in T cells by continuous T cell receptor (TCR) stimulation under normoxic conditions40.Hypoxia-inducible factor 1α (HIF-1α) and HIF-2α are master transcription factors that regulate physiological hypoxic responses41. HIF-1α regulates T cell metabolism and participates in glycolysis, thereby suppressing Treg cell development42. Foxp3 protein stability is increased by an insertional mutation that blocks binding of HIF-1α43. Consistent with those findings, VHL-knockout (KO) Treg cells lose their suppressive function and produce excess IFN-γ, whereas additional HIF-1α-KO restores Treg cells activity18. Similarly, persistent expression of HIF-1α by deleting PHD1, PHD2, and PHD3 in T cells leads to a significant increase in the ratio of IFN-γ+ effector T cells to Treg cells30. Previously, we also report that Treg cells become highly unstable in vivo in the absence of the E3 ligase deltex1 that downregulates HIF-1α44, supporting the inhibitory role of HIF-1α on Treg cells. In addition, iTreg differentiation is inhibited by hypoxia, which can be reversed by HIF-1α deficiency45, further confirming the suppressive activity of HIF-1α in Treg cell differentiation. By contrast, a putative hypoxia-responsive element is found on the promoter of Foxp3, and HIF-1α-KO CD4+CD25+ Treg cells fail to protect mice from effector T-cell-induced colitis46. Thus, the exact role of HIF-1α in Treg cell differentiation and action remains uncertain.The role of HIF-1α in Treg cells has been explored in many studies. HIF-1α has been shown to bind Foxp3 and to promote Foxp3 degradation, thereby inhibiting Treg cell differentiation47). Lack of HIF-2α in the myeloid lineage results in decreased tumor-associated macrophage infiltration and alleviates tumor progression48. Loss of HIF-2α from myeloid cells also increases neutrophil apoptosis and reduces neutrophilic inflammation49. However, the function of HIF-2α in the differentiation and function of T cells and Treg cells is unclear50.Most studies of the function of HIF-2α in immune responses have used myeloid cells. HIF-2α is induced by T helper 2 (Th2) cytokines during M2 macrophage polarization and specifically regulates expression of arginase-1  and Treg-specific knockout of HIF-2α (Foxp3CreHif2af/f). Deletion of HIF-2α was confirmed by RT-PCR . The proportions of CD4+Foxp3+ T cells from lymph nodes, thymus and spleen were comparable between wild-type , Foxp3CreHif1af/f and Foxp3CreHif2af/f mice  iTreg cells, which was reversed by HIF-1α knockout . Similar to Hif2af/f iTreg cells, Cd4CreHif2af/f iTreg differentiation was suppressed by hypoxia  iTreg cells differentiated at a low concentration (0.5 ng ml−1) of TGF-β under hypoxic conditions 46. Overall, the development of HIF-2α-KO tTreg and iTreg cells is comparable with that of WT tTreg and iTreg cells, supporting that deficiency of HIF-2α does not interfere with Treg cell development. Moreover, our experiments reveal that HIF-1α may fine-tune the differentiation of iTreg cells in a more complex way than previously recognized.For comparison with Treg cell development in ice Fig. . Upon dills Fig. . Expresslls Fig. . Howeverxia Fig. . The inhlls Fig. . These rCd4CreHif2af/f tTreg cells proved as effective as WT (Hif2af/f) tTreg cells in inhibiting proliferation, as well as IL-2 and IFN-γ production, of effector T cells. By contrast, HIF-1α-KO tTreg cells were ineffective in inhibiting effector T cell proliferation and secretion of IL-2  tTreg cells protected RAG-1-KO mice from CD45.1+ effector T cell-induced body weight loss, colitis, diarrhea, tissue damage and inflammatory infiltration  were as effective as Hif2af/f tTreg cells in suppressing colitis and associated pathologies in RAG-1-KO mice receiving effector T cells  from RAG-1-KO mice after colitis induction and determined the expression of IL-17 and IFN-γ. We found that the fraction of IL-17+ or IFN-γ+ cells among the transferred HIF-2α-KO tTreg cells (CD45.2+) had increased relative to WT tTreg cells  transferred into RAG-1-KO mice revealed increased IL-17 production , but comparable IFN-γ-production, was also found among activated Cd4CreHif2af/f iTreg cells destabilized with Th0-, Th1- or Th17-priming cytokines  iTreg cells, but not by Cd4CreHif2af/f iTreg cells . HIF-2α-KO iTreg cells were less effective than OVA + WT iTreg cells in suppressing ovalbumin re-challenge-triggered allergic responses . Recovery of the transferred iTreg cells (CD45.2+) from allergic mice (CD45.1+) revealed that Foxp3 expression, measured by flow cytometry, was also comparable between HIF-2α-KO and WT iTreg cells  exhibited a similar effect on HIF-1α protein as HIF-2α knockout. Treatment of activating WT CD4+ T cells with PT2385 led to dose-dependent upregulation of HIF-1α  were significantly higher in Cd4CreHif2af/f tTreg cells  mice , and also tunes up glycolysis to suppress Treg development42. Here, we find that HIF-1α knockout does not affect the development or expression of Foxp3 in tTreg cells  in iTreg cells are higher in HIF-1α-KO iTreg cells relative to control when differentiated under normoxic conditions  iTreg cells at sub-optimal conditions (0.5 ng ml−1 TGF-β) under hypoxia was greater than for Cd4CreHif1af/f and Cd4CreHif2af/f iTreg cells, but not at higher TGF-β concentrations , which led to the suggestion that hypoxia activates Foxp3 expression through HIF-1α and the HIF-responsive element on the Foxp3 promoter46. Notably, enhanced hypoxia-mediated WT iTreg differentiation at low TGF-β was not observed in iTreg cells differentiated from Foxp3Cre T cells, relative to Foxp3CreHif1af/f and Foxp3CreHif2af/f iTreg cells . As discussed above, Foxp3 expression is not affected by HIF-1α deficiency in vivo, but may be HIF-1α-dependent in vitro in particular contexts. It is possible that the difference between the suppressive activity of HIF-1α-KO Treg cells in vitro and in vivo is linked to HIF-1α-dependent in vitro Foxp3 expression, since persistent Foxp3 expression is required for Treg inhibitory activity6. It is also possible that during an in vitro suppressive analysis comprising only Treg cells, presenting cells and effector T cells, the inhibitory effect is quantitated from the close proximity between Treg cells and the target cells, but such bystander suppression would not be measured in an in vivo suppression assay that involves specific cognation58. Further studies are needed to examine these possibilities.Recent studies have revealed that the in vivo inhibitory activity of many different Treg cells is distinct from their in vitro suppressive activities , reduced Treg stability (Ubc13), or destabilization of Foxp3-driven gene expression (Ezh2). Moreover, in vitro co-culture of Treg cells may not fully recapitulate the in vivo inflammatory microenvironments in which Treg cells exert their action. We speculate that the in vivo instability of HIF-2α-KO Treg cells likely contributes to the loss of their suppressive function in vivo but not in vitro. It has also been suggested that proliferating memory-type Treg cells are the main Treg populations responsible for suppression in vivo, but their functions have not been determined in an in vitro suppression assay52. Accordingly, HIF-2α is likely involved in the functions of memory-type Treg cells but not in other subsets of Treg cells. Further characterization will help establish the validity of that scenario.How HIF-2α regulates Treg differentiation and function had been unclear. We found that the in vitro suppressive function of tTreg cells and iTreg cells was not affected by HIF-2α deficiency, but HIF-2α-KO tTreg cells were functionally defective in suppressing effector T cell-induced colitis and in inhibiting airway inflammation. HIF-2α-KO tTreg cells and HIF-2α-KO iTreg cells that were stimulated in vitro secreted higher levels of IL-17, but not IFN-γ, relative to WT tTreg cells Fig. . By cont41) and IFN-γ30. Here, we have further demonstrated that elevated HIF-1α in HIF-2α-KO Treg cells contributes to the instability of HIF-2α-KO Treg cells in vivo, as further knockout of HIF-1α restored the function of HIF-2α-KO tTreg cells and HIF-2α-KO iTreg cells in suppressing effector T cells-induced colitis and allergen-triggered airway inflammation, respectively , mouse IL-1α (400-ML), mouse IL-1β (401-ML), and anti-mouse/human HIF-1α-Alexa Fluor 700  were purchased from R&D systems . Antibodies against GAPDH  was obtained from Santa Cruz Biotechnology . Antibodies against HIF-1α  were purchased from Cayman Chemical . Antibodies against actin  were purchased from Merck Millipore . Recombinant mouse IL-4 (PMC0045), mouse IL-6 (RMIL6I), mouse IL-2 (RP-8608), anti-CD4-FITC , anti-CD44-PE-Cy7 , anti-CTLA-4-PE , anti-FR4-PE , anti-Foxp3-Pacific blue or APC , anti-LAG-3-PE , anti-CD45.1-PE , and anti-CD45.2-PE-Cy7  were purchased from Thermo Fisher . Recombinant mouse IL-12 (577002), anti-IL-2 (JES6-1A12), anti-IL-4 (11B11), anti-IL-12 (C17.8), anti-IFN-γ (R4-6A2), anti-IL-4-PE , anti-CD8-APC , anti-CD62L-APC , anti-CD25-FITC , anti-CD25-PE , anti-GITR-PE , anti-IFN-γ-APC , anti-IL-17-FITC , anti-mouse/human Helios-PE , and anti-mouse CD304-Brilliant Violet 421  were purchased from BioLegend . Anti-CD3 (2C11) and anti-CD28 (37.51) were purchased from BioXCell , and anti-mouse CD4 (RL172.4) was purified in our laboratory. ELISA kits for mouse IFN-γ (88-7314-88), mouse IL-17A (88-7371-88), mouse IL-10 (88-7105-88), and mouse TGF-β (88-8350-88) were purchased from Thermo Fisher. Ovalbumin (A2512), 12-O-Tetradecanoylphorbol 13-acetate  and calcium ionophore A23187 (C7522) were obtained from Sigma-Aldrich .B16F10 melanoma cells were obtained from ATCC . MC38 colon adenocarcinoma cells were purchased from Kerafast . MC38 and B16F10 cells were cultured in DMEM  supplemented with 10% FCS (Life Technologies), 1% Penicillin-Streptomycin, 1% L-Glutamine, and 1% HEPES.Cd4Cre mice were obtained from Taconic Farms. Rag1/−− (RAG-1-KO) mice, Hif1atm3Rsjo/J (Hif1af/f) mice, Epas1tm1Mcs/J (Hif2af/f) mice, and NOD/ShiLt-Tg(Foxp3-EGFP/cre)1cJbs/J mice  were obtained from Jackson Laboratories. NOD.Foxp3Cre mice were back-crossed to C57BL/6 mice for 12 generations (Foxp3Cre)44. Cd4Cre mice were bred with Hif1af/f or Hif2af/f for T cell-specific deletion of Hif1a or Hif2a, respectively. Foxp3Cre mice were crossed with Hif1af/f or Hif2af/f to produce mice with Treg-specific deletion of Hif1a or Hif2a. Mice were housed in SPF animal facility of Institute of Molecular Biology (Academia Sinica), with ambient temperature at 21 °C, humidity of 55%, dark/light cycle of 10 h/14 h, and air exchange rate of 12–15 times per hour. In vivo experiments were conducted on sex-matched male and female mice. Both male and female mice were used for in vitro and in vivo experiments. All mice used in our study were 8–10 weeks old. All mouse experimental protocols were approved by the Institutional Animal Care and Utilization Committee of Academia Sinica.+ T cells were stimulated with plate-bound anti-CD3 and anti-CD28 for 24 h to detect IL-2 production and for 48 h to measure proliferation and IFN-γ levels (Thermo Fisher). T cell proliferation was measured by thymidine incorporation using a direct beta-counter .Single-cell suspensions were prepared from lymph nodes or spleen, and immunoglobulin panning was used to isolate total T cells. Specific T cell populations were sorted on a FACSAria II SORP system , and purified T cells were cultured in complete RPMI 1640 medium. Purified CD4+CD25+ thymus-derived regulatory T (tTreg) cells were purified from peripheral lymph nodes and spleens by sorting on a FACSAria II SORP system. Intracellular staining of Helios and Foxp3 was performed on paraformaldehyde-fixed splenocytes pre-labelled for CD4 and CD25 using the Foxp3/Transcription Factor Staining Buffer Set . Neuropilin-1 (Nrp-1) staining was conducted with anti-Nrp-1. Naïve IL-10 and TGF-β cytokine secretion from tTreg cells was estimated by ELISA as per the manufacturer’s instructions (Thermo Fisher) upon stimulating tTreg cells with immobilized anti-CD3 (4 µg ml−1) and anti-CD28 (2 µg ml−1) for 48–96 h. Gating and sorting strategies for tTreg cells purification used in in vitro and in vivo functional analyses are described in Supplementary Fig. CD4−1) and anti-CD28 (1 µg ml−1) in the presence of TGF-β (5 ng ml−1) and IL-2 (20 µg ml−1) for 5 days. CD4+CD25+ cells were collected after 5 days of differentiation and used for in vitro and in vivo suppression analysis. Respective gating and sorting strategies are described in Supplementary Fig. For induced Treg (iTreg) differentiation, FACS-sorted naïve T cells were stimulated with immobilized anti-CD3 (2 µg ml+CD25−effector T (Teff) cells (1 × 105) were co-cultured with tTreg cells or iTreg cells in the presence of antigen-presenting cells  and 1 μg ml−1 soluble anti-CD3 in 96-well U-bottom plates. The ratios of Treg cells to Teff cells were 0:1, 1:1, 1:2, 1:4, or 1:8. After 72 h, CD4+CD25− Teff cell proliferation was measured by thymidine incorporation, and IL-2 and IFN-γ levels were detected by ELISA in supernatants after 40 h of culture.CD4+CD25−Teff cells (4 × 105) from CD45.1+ mice were administered with or without 1 × 105 CD4+CD25+ tTreg cells from 8 to 10 week-old CD45.2+ mice by intraperitoneal injection into sex matched 8-10 week-old CD45.1+ RAG-1-KO mice. We scored body wasting as follows: 0, no wasting; 1 for up to 10 % initial body weight loss; 2 for 10-20 % initial body weight loss; and 3 for >20% initial body weight loss. Rectal prolapse was scored as 0 for absence and 1 for presence. Diarrhea was scored as 0 for absence (solid stool), 1 for soft stool, and 2 for watery/bloody stool. Body weight and score was assessed every 3 days. Five weeks after T-cell transfer, mouse colons were removed, cleaned using PBS, fixed in 4% paraformaldehyde, embedded in paraffin, and then sectioned and stained with H&E. CD45.2+ T cells (representing transferred Treg cells) from lymph nodes and spleen were isolated from CD45.1+ RAG-1-KO mice, and reactivated by TPA/A23187 for 6 h and stained intracellularly for expression of Foxp3, IFN-γ, and IL-17A. Respective gating strategies are described in Supplementary Fig. CD46 iTreg cells or PBS on day 0, and then sensitized by intraperitoneal injections of 50 μg ovalbumin (OVA) emulsified in 2 mg of aluminum hydroxide  on day 1 and day 14. Three weeks later, mice were challenged by inhalation (30 min daily for four consecutive days) of aerosolized 1% OVA in saline (0.9% NaCl) generated by a Nebulizer 646 in an aerosol therapy system (DeVilbliss). To evaluate lung function, an allergy-induced airway hyperreactivity (AHR) assay was performed. Briefly, mice were anesthetized with pentobarbital  at 100 mg/kg body weight after the last OVA challenge. Anesthetized mice were tracheotomized, intubated, and mechanically ventilated at a tidal volume of 0.20 mL and a frequency of 150 breaths/min. Resistive index (RI) values were recorded at baseline and following a 10 s exposure to an increasing concentration of methacholine   through the FinePointe RC System . Immediately after AHR measurement, bronchoalveolar lavage fluid (BALF) was collected by three instillations of 1 mL cold saline, and amounts of IL-4, IL-5, IL-6, IL-13 and IFN-γ in BALF from the first collection were measured by cytometric bead assay . The relative number of different types of leukocytes in BALF was determined from differentials based on 200 cells by Differential Quik III Staining . Results are given as cells ml−1 in BALF.WT mice were intravenously injected with 2.5 × 10−1) plus anti-CD28 (2 μg ml−1) and in the presence of IL-2 (20 ng ml−1) with or without IL-12 (50 ng ml−1), or IL-6 (50 ng ml−1) plus IL-1 . These cells were harvested after 5 days and Foxp3 expression was determined using an LSRII-18P flow cytometry system (BD Biosciences). IFN-γ and IL-17A levels were measured by ELISA in supernatants collected from Treg cells reactivated using TPA (20 ng ml−1) and A23187 (200 ng ml−1) for 16 h.tTreg and iTreg cells were stimulated by plate-bound anti-CD3 (4 μg ml+CD25−CD44loCD62Lhi naïve T cells (1 × 106 ml−1) were stimulated with plate-bound anti-CD3 (2 µg ml−1) and anti-CD28 (1 µg ml−1) in the presence of 20 ng ml−1 mouse IL-12 and 10 µg ml−1 anti-mouse IL-4 antibody for Th1 differentiation. For Th2 differentiation, mouse IL-4 (20 ng ml−1), anti-IL-12 (10 µg ml−1) and anti-IFN-γ antibody (10 µg ml−1) were added. Cultures were also supplemented with 20 ng/mL IL-2 during Th1 and Th2 differentiation. For Th17 differentiation, sorted naïve T cells (2 × 106 ml−1) were stimulated with immobilized anti-CD3 (4 µg ml−1) and anti-CD28 (2 µg ml−1) supplemented with 30 ng ml−1 mouse IL-6, 5 ng ml−1 TGF-β, and 10 µg ml−1 each of anti-mouse IL-2, anti-mouse IL-4, anti-mouse IL-12, and anti-IFN-γ antibodies. Cells were collected after 5 days of differentiation and stimulated for 5 h with PMA (50 ng ml−1) and ionomycin (500 ng ml−1) in the presence of monensin (2 nM). Expression of IFN-γ, IL-4, and IL-17 was measured by intracellular staining. The priming of Treg cells into Th0-, Th1- and Th17-like cells is described in Fig. Sorted CD4+ T or tTreg cells was collected using a GENEzol triRNA Pure Kit . The eluted RNA was reverse-transcribed using SuperScript III Reverse Transcriptase (Thermo Fisher). cDNA was synthesized from 1 μg of RNA from each sample and analyzed for expression of HIF-1α, HIF-2α or HIF-1α target genes on a LightCycler 480 Real-Time PCR System using software v1.5.1.621.5.1.62 . The primers used are listed in Supplementary Table Actin expression.Total RNA from CD42, 0.2 mM EDTA, 0.1 mM Na3VO4, 50 mM NaF, 0.5 mM dithiothreitol and 10% glycerol) on ice for 30 min. Debris was removed by centrifuging on an Eppendoff microfuge . Protein concentrations in the supernatants were quantitated by Bio-Rad protein assay. Protein samples were resolved by SDS polyacrylamide gel electrophoresis, before being transferred to PVDF membrane in transfer buffer  at room temperature for 1.5 h at 400 mA. The membrane was blocked with blocking buffer  at room temperature for 1 h. The treated membrane was incubated with primary antibodies overnight at 4 °C, washed, and incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1.5 h. The membrane was developed using an ECL Western blotting detection kit. Luminescence was detected by X-ray film. Western blot images have been cropped for presentation. Full size images are presented in Supplementary Fig. To prepare whole-cell lysates, cells were lysed by whole-cell extract buffer  or Foxp3CreHif2af/f mice. Tumor size was determined by measuring tumor length and width, using volume as readout. Volumes (V) were calculated using the equation: V = L × W2/2, where L is the long diameter and W is the short diameter. To evaluate the effect of HIF-2α-suppressed iTreg cells, C57BL/6 mice were lightly irradiated (2 Gy), followed by subcutaneous transplantation with 5 × 105 MC38 cells. Sorted CD4+CD25+ iTreg cells were untreated or treated with 20 μM of the HIF-2α-specific inhibitor PT2385  for 16 h and then adoptively transferred into mice on days 3, 6 and 10 after tumor implantation.B16F10 melanoma cells were obtained from ATCC . MC38 colon adenocarcinoma cells were purchased from Kerafast . MC38 and B16F10 cells were cultured in DMEM  supplemented with 10% FCS (Life Technologies), 1% Penicillin-Streptomycin, 1% L-Glutamine, and 1% HEPES. A total of 1 × 10Foxp3CreHif2af/f mice by injecting 2 × 105 B16F10 melanoma cells in 100 μL PBS via the tail vein. On day 12, the mice were euthanized and then perfused with PBS. Numbers of tumor nodules were counted. Lungs were then fixed in 4% paraformaldehyde followed by 70% alcohol for histologic evaluation. Lung sections were stained with hematoxylin and eosin (H&E) to visualize lung architecture and malignant nodules.Lung metastases were established in WT or t-tests and paired two-tailed Student t-tests were used to compare results between two groups. Data are presented as mean with standard deviation (SD) or standard error of the mean (SEM), as indicated in the figure legends. Weight loss and clinical score in CD4+ T cell–mediated colitis in mice were analyzed by repeat measures two-way ANOVA with Tukey’s multiple comparisons test, with the Greenhouse-Geisser correction. Changes in lung resistance (RL) in response to increasing doses of methacholine and cell composition of BALF were analyzed by two-way ANOVA with Tukey’s multiple comparisons test. P values < 0.05 were considered significant.Lung inflammation experiments and colitis histology were blinded. Other data in this study were collected randomly but not blind, several experiments were confirmed independently by a second person. No data were excluded from this study. Our data generally met the assumptions of the statistical tests applied . GraphPad Prism 5 and Microsoft Office Excel 2016 were used for data analysis. Flow cytometry data were collected using FACSdiva software version 8.0.2 (BD Biosciences), and were analyzed on FlowJo version 10 (BD Biosciences). Unpaired two-tailed Student Further information on research design is available in the Supplementary informationPeer Review FileReporting Summary


N,N-dimethyltryptamine (DMT) derivatives, the synthetic psychedelics N-ethyl-N-propyltryptamine (EPT) and N-allyl-N-methyltryptamine (MALT), are reported.The structures of the hydrofumarate salts of two  N,N-dialkyltryptamines, namely N-ethyl-N-propyltryptammonium (EPT) hydrofumarate {systematic name: [2-(1H-indol-3-yl)ethyl](methyl)propylazanium 3-carboxyprop-2-enoate}, C15H23N2+·C4H3O4−, and N-allyl-N-methyltryptammonium (MALT) hydrofumarate {systematic name: [2-(1H-indol-3-yl)ethyl](methyl)(prop-2-en-1-yl)azanium 3-carboxyprop-2-enoate}, C14H19N2+·C4H3O4−, are reported. Both compounds possess a protonated tryptammonium cation, and a hydrofumarate anion in the asymmetric unit. The ethyl group of the EPT cation is modeled as a two-component disorder with 50% occupancy for each component. In the extended structure, N—H⋯O and O—H⋯O hydrogen bonds generate infinite two-dimensional networks parallel to the (001) plane for both compounds.The solid-state structures of the hydrofumarate salts of two  Psychotria viridis plant contain about 0.3% of N,N-dimethyltryptamine (DMT) by mass, which is the primary psychoactive in ayahuasca. The bark of the Banisteriopsis caapi vine contains many different β-carbolines; these β-carbolines function as monoamine oxidase (MAO) inhibitors, which prevent the degradation of DMT in the human gut. Without the inhibition of monoamine oxidase, DMT is not orally active  is orally active in the absence of MAO inhibitors, indicating that the 4-hydroxy substitution makes the compound more resistant to deamination by MAO  and N-methyl-N-allyltryptamine (MALT), both of which have very limited reports in literature N-ethyl-N-propylamine is disordered over two orientations, with a 0.50:0.50 ratio for C14, C15 and C14A, C15A. The hydrofumarate anion deviates slightly from planarity with an r.m.s. deviation of 0.135 Å, and a carboxylate to carboxylate plane twist angle of 16.63 (14)°.The asymmetric unit of N-methyl-N-allyltryptammonium hydrofumarate, (II)The asymmetric unit of x, y, 1 − z). The hydroxy group of the hydrofumarate interacts with a carboxylate oxygen of another hydrofumarate anion through an O3—H3A⋯O2 hydrogen bond . The hydrofumarate anions are linked together in chains along [100], which are linked together by the tryptammonium cations along [010], joining the ions into infinite two-dimensional networks parallel to the (001) plane . One carbonyl oxygen, O2, of the hydrofumarate, the indole nitrogen, N1, of another tryptammonium cation , and a carbonyl oxygen, O4, of a different hydrofumarate anion  combine to form a three-centred (bifurcated) N—H⋯ hydrogen bond. The hydrofumarate anions are linked together in chains along [100], and the tryptammonium cations link these chains together along [010]. The net result in an infinite two-dimensional networks parallel to the (001) plane, similar to what was observed for the EPT .Single crystals of N-methyl-N-allyltryptammonium hydrofumarate, 134 mg of a commercial sample of N-methyl-N-allyltryptamine (The Indole Shop), which is a waxy solid that does not crystallize well, were dissolved in 10 mL of methanol, and 68 mg of fumaric acid were added. The mixture was refluxed for 12 h and solvent was removed in vacuo to obtain a waxy, yellow product. The material was recrystallized from ethanol to yield colorless single crystals suitable for X-ray diffraction. The product was also characterized by nuclear magnetic resonance. 1H NMR : δ 7.69 , 7.54 , 7.34 , 7.29 , 7.21 , 6.66 , 5.92–5.82 , 5.60–5.56 , 3.88–3.83 , 3.77–3.72 , 3.68–3.57 , 3.44–3.37 , 3.34–3.21 , 2.90 . 13C NMR : δ 172.2 (COOH), 137.0 (CH), 135.5 (ArC), 127.3 (ArC), 126.9 (ArC), 126.2 (ArC), 124.8 (ArC), 122.9 (ArC), 120.1 (ArC), 118.9 (ArC), 112.7 (sp2C), 109.0 (sp2C), 58.7 (AkC), 55.6 (AkC), 40.1 (AkC), 20.6 (AkC).To prepare A =1.50 ± (10), C14—C15 = C14A—C15A = 1.54 ± (1), N1—H1 = N2—H2 = 0.87± (1) Å for (I)A 0.88 ± (1) Å for (II)Uiso(H) = 1.2Ueq(C) or 1.5Ueq(C-methyl). The ethyl group of the EPT cation was modeled as a two-component disorder with 50% occupancy for each component for both compounds.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989020008683/dx2028sup1.cifCrystal structure: contains datablock(s) I, II. DOI: 10.1107/S2056989020008683/dx2028Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989020008683/dx2028IIsup3.hklStructure factors: contains datablock(s) II. DOI: 2012495, 2012494CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Chronic myelogenous leukemia (CML) is a clonal myeloproliferative neoplasm resulting from BCR–ABL-transformed hematopoietic stem cells. Previous research has implicated multifunctional proinflammatory cytokines in CML development. It has been reported that Sirtuin 1 (SIRT1) as well as ADP-ribosyltransferase and deacetylase may influence CML cell viability and inflammation.This study was directed toward exploring the SIRT1-involved in the mechanism of lipopolysaccharide (LPS)-triggered inflammation in CML k562 cells.In our study, the LPS-induced inflammation in k562 cells was reflected by increases in levels of diverse inflammatory cytokines, including interleukin (IL)-10, IL-1β, IL-6, interferon-γ, tumor necrosis factor (TNF)-α and TNF-β. LPS also decreased SIRT1 expression and nuclear location in k562 cells. Furthermore, SIRT1 overexpression inhibited the release of the above mentioned cytokines in LPS-treated cells. We also determined that LPS stimulation could activate Toll-like receptor 4 (TLR4), the nuclear factor κ B (NFκB) subunit, and p65 and produce reactive oxygen species (ROS) in k562 cells. Nevertheless, SIRT1 overexpression decreased TLR4 expression, thereby repressing the phosphorylation of the NFκB subunit and p65 and decreasing ROS production.These findings suggest that SIRT1 is a latent therapeutic target for mitigating LPS-induced inflammation via the TLR4–NFκB–ROS signaling axis. Chronic myelogenous leukemia (CML) is a clonal hematological malignancy resulting from BCR–ABL-transformed hematopoietic stem cells (HSCs) . There aSirtuins (SIRTs) are nicotinamide adenine dinucleotide (NAD)-dependent protein deacetylases that are highly conserved from yeast to mammalian cells. Seven SIRTs (SIRT1–SIRT7) in mammalian cells exhibit functional significance on aging, diabetes, cardiovascular diseases, and cancers . SIRT1, Cytokines create a proinflammatory environment during CML development, and provide a proliferative advantage to leukemic cells , 11. Sev2), followed by exposure to 10 μg/mL of LPS for 4 h.CML k562 cells  were cultured in Roswell Park Memorial Institute 1640 medium containing new-born calf serum (10%), streptomycin (100 μg/mL), and penicillin (100 U/mL) at 37 °C in a damp condition (5% CO6 cells were transfected with pCDNA (4 μg), pCDNA-SIRT1 (4 μg), siRNA-NC (8 μg), or siRNA-SIRT1 (8 μg) using the Lipofectamine2000 transfection agent (Thermo Fisher). These cells were used for experiments 36 h after the transfection.About 4 × 10Cells were subjected to radioimmunoprecipitation assay buffer to prepare lysate. Bicinchoninic acid assay was then used to determine the protein concentration. After resolving via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, proteins were transferred to polyvinylidene difluoride membranes (0.45 µm), which were then blocked with 5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS)-Tween20 (PBS-T) at 25 °C for 60 min and incubated at 4 °C for 12 h with the following immunoglobulins: rabbit anti-IL-1β , anti-IL-6 , anti-TNF-α , mouse anti-SIRT1 , anti-TLR4 , anti-p-NFκB , anti-NFκB , and anti-GAPDH . Subsequently, the resulting membranes were incubated with horseradish peroxidase-connected secondary antibodies at 4 °C for another 60 min. Blots were observed through an chemiluminescence reaction via a blot scanner.Total RNA was isolated with Trizol as per the manufacturer’s instructions, and messenger RNA (mRNA) levels were measured through qPCR with GAPDH as the internal standard. qPCR was performed using SYBR Green master mix via a real-time PCR system. The PCR reaction was performed at 95 °C for 10 min and cycled 40 times for 15 s and at 60 °C and 72 °C for 30 s each. The 2-ΔΔCT method was used for determining the copy number of objective genes. The primer sequences were as follows: IL-1β F: 5′-CCA CAG ACC TTC CAG GAG AAT G-3′, IL-1β R: 5′-GTG CAG TTC AGT GAT CGT ACA GG-3′; IL-6 F: 5′-AGA CAG CCA CTC ACC TCT TCA G-3′, IL-6 R: 5′-TTC TGC CAG TGC CTC TTT GCT G-3′; TNF-α F: 5′-GAT CTC AAA GAC AAC CAA CAT GTG-3′, TNF-α R: 5′-CTC CAG CTG GAA GAC TCC TCC CAG-3′; IL-10 F: 5′-CAC AAA GCA GCC TTG CAG AA-3′, IL-10 R: 5′-AGA GCA GGC AGC ATA GCA GTG-3′; IFN-γ F: 5′-ATC CCG TGG AGA CTC CTC AA-3′, IFN-γ R: 5′-CCA AAC ACG TAG ACT GGG TAT CC-3′; TNF-β F: 5′-CCC ATG GCA TCC TGA AAC-3′, TNF-β R: 5′-GGA GGC CTG GAA TCC AAT-3′; SIRT1 F: 5′-TGC TGG CCT AAT AGA GTG GCA-3′, SIRT1 R: 5′-CTC AGC GCC ATG GAA AAT GT-3′; TLR4 F: 5′-CCC TGA GGC ATT TAG GCA GCT A-3′, TLR4 R: 5′-AGG TAG AGA GGT GGC TTA GGC T-3′; p65 F: 5′-GGT CCA CGG CGG ACC GGT-3′, TLR4 R: 5′-GAC CCC GAG AAC GTG GTG CGC-3′; GAPDH F: 5′-CAT GGC CTT CCG TGT TCC TA-3′, GAPDH R: 5′-TGT CAT CAT ACT TGG CAG GTT TCT-3′.IL-1β (BMS224), IL-6 (BMS213), IL-10 (BMS215-2), IFN-γ (KHC4021), TNF-α (BMS223), and TNF-β (BMS202) levels were detected using an ELISA kit from Thermo Fisher in accordance with the manufacturer’s protocol. Absorbance was recorded at OD 543 using a microplate reader POLARstar Omega (BMG Labtech).6) were plated on 6 cm dishes and grown for 36 h. Then, the cells were harvested via scraping into a 500 μL cell lysis buffer containing 10 mM HEPES (pH 7.4), 10 mM NaCl, 1 mM KH2PO4, 5 mM NaHCO3, 1 mM CaCl2, 0.5 mM MgCl2, and 5 mM EDTA with complete protease inhibitor cocktail. Cells were allowed to swell for 5 min, followed by Dounce homogenization for 50-time strokes. Then, the cells were centrifuged at 7500 rpm for 5 min, generating a pellet containing nuclei and debris and a supernatant of cytosol and plasma. Pellets were resuspended in a 1 mL buffer containing 10 mM Tris (pH 7.5), 300 mM sucrose, 1 mM EDTA, and 0.1% NP40 with complete protease inhibitor cocktail, and then pelleted, resuspended, and washed twice. The final pellets were pure nuclei.Cells . Briefly, the assays were performed by incubating the whole cell lysate and Fluoro-Substrate Peptide (0.2 mM), Developer, and NAD (2 mM) at 37 °C for 30 min. Fluorescent intensity was measured on a microplate reader POLARstar Omega (BMG Labtech) with excitation set at an Ex/Em = of 350/460 nm.ROS production in k562 cells was determined using dichloro-dihydro-fluorescein diacetate (DCFH-DA), a fluorescence probe. Following the LPS treatment for 4 h, cells were incubated with DCFH-DA (10 μM) away from light at 37 °C for 30 min. A fluorescence microscope was used to determine fluorescence intensity (Ex 488/Em 525 nm) using a microplate reader POLARstar Omega (BMG Labtech). For NAC treatment, 5 μM NAC was used to treat k562 cells for 4 h.IFAs were performed using the Leica SP8 confocal laser scanning microscope. Coverslips carrying k562 cells were washed with PBST (PBS plus 1% Triton X-100) and then fixed with 4% paraformaldehyde. Cells were then blocked with 4% BSA dissolved in PBST and stained with anti-SIRT1 antibody. The secondary antibodies were tetramethyl rhodamine isocyanate-conjugated anti-rabbit IgG.Data were expressed as mean ± standard deviation. An analysis of variance or t-test was applied to analyze significant differences in variables among the groups. A P-value of < 0.05 indicated significance.First, we examined the proinflammatory role of LPS in k562 cells. These cells were subjected to 10 μg/mL of LPS for 4 h. Subsequently, ELISA and qPCR were used to assess the levels of diverse proinflammatory cytokines. LPS treatment markedly increased in the mRNA levels of inflammatory cytokines in k562 cells (Fig. Recently, different roles of SIRT1 in several hematologic malignancy subtypes have been reported . In the To determine the influence of SIRT1 on inflammatory factor production in k562 cells, these cells were first transfected with SIRT1-expressing plasmid or empty plasmid and then treated with either LPS or the control formulations. SIRT1 expression was detected via WB. Following SIRT1 overexpression, SIRT1 mRNA and protein levels were dramatically increased, even in the LPS group Fig. a, d. Sub–NFκB–ROS signaling axis has been highly associated with the onset of inflammation [The TLR4ammation , 17; theWe hypothesized that reducing ROS production could result in the SIRT1-mediated inactivation of the TLR4–NFκB–ROS axis and the alleviation of inflammation. To explore this, k562 cells were cotreated with 5 μM of NAC, a ROS scavenger , and LPSThe present study results showed that LPS-triggered inflammation accompanied reduced SIRT1 expression in k562 cells. SIRT1 overexpression also could decrease LPS-induced inflammatory cytokine production and obviously reduce inflammation-relevant TLR4 sensors and the activation of downstream NFκB p65. In addition, ROS generation via TLR4–NFκB p65 activation was also reduced by SIRT1 overexpression. The administration of the ROS inhibitor NAC to LPS-treated k562 cells also led to amelioration of inflammation. The study demonstrated that SIRT1 inhibited LPS-induced inflammatory reactions in k562 cells through the abrogation of the TLR4–NFκB–ROS axis.CML is a clonal hematological malignancy that arises resulting from BCR–ABL-transformed HSCs . SIRT1 eTLRs play an essential roles in nonspecific immunity, identify numerous exogenetic PAMPs and pathogens and initiate endogenic immune reactions , 26. TLRIncreased levels of ROS have been reported to be deleterious to cells due to the oxidative stress, resulting from the interactions of ROS with nucleic acids, lipids, and proteins ; ROS alsOur data suggest that SIRT1 exhibits a protective effect in CML k562 cells against LPS-triggered inflammation. SIRT1 ameliorates the inflammation elicited by LPS in k562 and KU812 cells, probably by inactivating the TLR4–NFκB–ROS axis. These results may help find a new treatment method for inflammation in CML cells.