To study the role of the Rho/ROCK pathway in Ang II and TGF-β1-induced atrial remodeling.A canine atrial fibrillation (AF) model was established by rapid atrial pacing (RAP) of the left atrium. The roles of TGF-β1, the RhoA/ROCK signaling pathway and connective tissue growth factor (CTGF) in atrial remodeling were studied via both in vitro and in vivo experiments. Each of the dogs that received RAP developed persistent AF within 4 weeks. The mRNA expression levels of TGF-β1 (1.32±0.38), Collagen-I(1.33±0.91), CTGF(5.83±3.71), RhoA(1.23±0.57) and ROCK-1 (1.02±0.27) in the left atrium were significantly increased following 4 weeks of RAP. Angiotensin II (Ang II) induced the proliferation of atrial fibroblasts and up-regulated the expression of both CTGF and ROCK-1 in a dose-dependent manner. Simvastatin and Y27632 reversed Ang II-induced CFs proliferation, as well as ROCK-1 and CTGF  expression. The expression mRNA of ROCK-1(1.74±0.13) and CTGF (2.28±0.11) can upregulated by TGF-β1, and down-regulated by Simvastatin (1.22±0.03 vs 2.27±0.11), Y27632 (1.01±0.04 vs 1.64±0.03), Los (1.04±0.11 vs 1.26±0.05), respectively. Losartan and Simvastatin attenuated the effects of TGF-β1, inhibited RhoA activity as opposed to RhoA protein expression. Y27632 had no effect on either the expression or the activity of RhoA.The increased expression of profibrotic factors  played an important role in our RAP-induced AF model. Increased atrial profibrotic factors involve the activation of either the TGF-β1/RhoA/ROCK-1 or the TGF-β1/Smad2/3 signaling pathway. Atrial fibrillation (AF) is the most common cardiac arrhythmia encountered in clinical practice and is common among the elderly, with an annual incidence of approximately 3% among patients 85 years of age and older. UnderlyTGF-β1 activity has been observed during pathologic cardiac remodeling in a variety of animal models. Furthermore, the overexpression of TGF-β1 preferentially results in the fibrosis of atrial cells. TGF-β1 The role of atrial remodeling in AF and the mechanism underlying remain unknown. It is unclear whether the Rho pathway is involved in atrial fibrosis, downstream of the RAS. The relationship between the TGF-β1/RhoA/ROCK-1 pathway and the TGF-β1/Smad2/3 pathway in atrial remodeling must also be elucidated. In the present study, we aimed to determine the regulatory factors involved in the development of AF, as well as to develop a means of either attenuating or preventing AF.Angiotensin II, Simvastatin, and Pentobarbital sodium were each purchased from Sigma . Losartan and Y-27632 were obtained from Selleck . TGF-B1 was purchased from PROSPEC . The ROCK-1, CTGF, Collagen–I (α1), TGF-β1, and Smad 2/3 antibodies were obtained from Santa Cruz Biotechnology . The RhoA pull-down was provided by Cytoskeleton . All DNA probes were synthesized by Invitrogen . The HE and Masson trichrome stains were both purchased from Maxin . The hydroxyproline detection kits were purchased from the Nanjing Jiancheng Bioengineering Institute . Fetal calf serum and Dulbecco's modified Eagle medium (DMEM) were obtained from Gibco BRL . All buffer solutions used in this study were prepared in our lab. Additional commonly used chemicals and materials were purchased from standard commercial sources.All experimental protocols involving the use of animals conformed to the Guide for the Care and Use of Laboratory Animals and complied with the guidelines specified by the Chinese Heart Association policy pertaining to research animal use and the Public Health Service policy pertaining to the use of laboratory animals. The experimental protocols were reviewed and approved by our institutional ethics committee . The beagles were purchased from Kangda Animal Science and Technology . The animals were maintained in the Animal Experiment Center of Sun Yat-sen University in temperature-controlled animal housing with 12 h alternating light and dark cycles and were provided adequate nutrition and water ad libitum.Fifteen beagles of both sexes (12–16 kg) were randomized into a pacing group (n = 9) and a control group (n = 6). Left atrial rapid pacing was performed as described previously, with some modifications. The beaThe pacing group was maintained on high rate left atrial pacing (400 beats/min) for 4 weeks, with a brief break to allow for the measurement of both electrophysiological and mechanical parameters. The control group underwent an identical surgical procedure but did not undergo RAP. Postoperative care included the administration of both antibiotics and analgesics.Electrocardiograms were recorded on days 1, 7, 14, 21 and 28 following surgery. An electrophysiological study was performed at 4 weeks following surgery, as was the case in previous studies, 21.At the end of the experiment, all beagles were sacrificed with euthanized using pentobarbital sodium (30 mg/kg), and their hearts were removed and weighed. The atria were subsequently removed and cut into three  sections. Each section was divided equally into ten pieces. One piece was used for the hydroxyproline content analysis. Three pieces were paraffin-embedded for hematoxylin and eosin staining, Masson's trichrome staining and immunohistochemistry analysis. The remaining pieces were frozen in liquid nitrogen and maintained at -80°C for both mRNA and protein analysis. A hydroxyproline content analysis was performed using a hydroxyproline kit according to the manufacturer’s instructions.Fifty micrograms of total protein from homogenized atrial tissue were solubilized for 5 min at 95°C in loading buffer . The samples were subsequently loaded onto a 10% SDS-PAGE gel for electrophoresis and transferred to PVDF membranes. The membranes were blocked using 5% nonfat dry milk in PBS (containing 0.05% Tween 20) and incubated overnight at 4°C with the primary antibodies . Following washing, the membranes were incubated with either an anti-rabbit or an anti-mouse IgG, HRP-linked antibody  for 2 hours at room temperature. Detection was performed using an ECL Western blotting kit . The band densities were quantified using Image J software .2O2 in methanol for 30 minutes. Following several washes in phosphate buffered saline (PBS), the slides were heated in a microwave oven at 121°C for antigen retrieval. After being cooled at room temperature and washed with PBS, the sections were subsequently incubated with blocking solution for one hour at room temperature. Following PBS washing, the tissues were bordered using a pap-pen before being incubated with mouse monoclonal antibodies against Collagen I (α1), ROCK-1, CTGF, TGF-β1 and anti-SMAD 2/3  for 30 minutes at room temperature in accordance with standard protocols. The histological sections stained with HE and Masson trichrome solution were analyzed via light microscopy. A minimum of 3 sections per atrial site were examined.For the immunohistochemical analyses, the sections were deparaffinized and digested using 0.05% subtilisin. The inactivation of endogenous peroxidase activity was performed via incubation in 3% H15 primers and M-MLV reverse transcriptase  according to the manufacturer’s instructions. All primers were designed using Primer 5 software. The experiments were performed using Power SYBR Green PCR Master Mix . All measurements were performed in duplicate. The relative quantification of expression was performed using the “2-ΔΔCT method”[Gapdh) for normalization.Total RNA was extracted from the atrial tissue in accordance with standard techniques using an RNeasy Fibrous Tissue Mini Kit and Micro Kit . The isolated RNA was quantified via UV spectrophotometry . Quantitative real-time polymerase chain reaction (RT-qPCR) was used to validate the levels of desmoplastic factor expression. As much as 1 μg of RNA per sample was reverse transcribed into cDNA using oligo-dTT method” via the 2 at 37°C, and subsequently passaged via trypsinization. The experiments were performed using early passage cells (passages 2–5).Atrial tissue freshly obtained from Neonatal Sprague Dawley (SD) rat hearts. Neonatal SD rat were killed by cervical dislocation and hearts excised and placed in ice-cold isolation solution for mincing and digesting via incubation with 0.25% trypsin digest solution  for 4 h at 37°C. The atrial cardiac fibroblasts cells were collected via centrifugation, washed with DMEM ) and resuspended in full growth medium , 2 mmol/L L-glutamine, 100 μg/mL penicillin G, 100 μg/mL streptomycin and 0.25 μg/mL amphotericin). The cells were plated into cell culture flasks for 30 min to allow the fibroblasts to adhere, followed by the removal of any non-adherent cells. The atrial CFs were cultured until they reached 80–90% confluence in fresh full growth medium, in a humidified atmosphere of 5% CO3 cells per well) in 150 μL of full growth medium. Following overnight incubation, the cells were growth-arrested in serum-free medium (SFM) for 48 h. The SFM was subsequently replaced with 150 μL of minimal growth medium (medium containing 2.5% FCS) containing the appropriate drugs. Four time points  were designated in this study. Following the culture, viable cells were detected using an MTT assay as described previously[The atrial CFs were seeded into 96-well plates , both with and without Ang II (10-6mol/L). Group 3 was treated with Simvastatin , both with and without Ang II (10-6mol/L). Group 4 was treated with Y27632 , both with and without Ang II (10-6mol/L).The atrial CFs were divided into the four following treatment groups, and each group was divided into several subgroups: group 1 was used to investigate the effect of Ang II on atrial CF proliferation. Group 2 was treated with Losartan (104 cells per well) in 1500 μL of full growth medium. When the cells reached 80–90% confluence, they were growth-arrested in serum-free medium for 24 h, followed by 24 h of drug treatment. The SFM was subsequently replaced with 1500 μL of full growth medium for another 24 h culture. Two time points (4 h and 6 h following the addition of the drugs) were designated in this study.The atrial CFs were seeded into 12-well plates , and group 8 . Following treatment, the cells were collected, and the mRNA expression levels of ROCK-1 and CTGF were measured via RT-PCR. The expression levels of CTGF and phosphorylated-Smad2/3 were analyzed via Western blotting. RhoA activity was detected using a RhoA kit  according to the manufacturer’s instructions.The cells were divided into the following 8 groups: group 1 , group 2 , group 3 , group 4 , group 5 , group 6 , group 7 , group 3 , group 4 , group 5 , and group 6 . Following treatment, the cells were collected, and the mRNA expression levels of both ROCK-1 and CTGF were analyzed via RT-PCR. The expression levels of CTGF and ROCK-1, TGF-β1 were analyzed via Western blotting.The effect of the RhoA/ROCK-1 signal pathway on the Ang II induced expression of CTGF was subsequently studied. The cells were treated as follows: group 1 , group 2 . Comparisons between the quantitative data were made using the AF was induced by S1S2  and burst (600 bpm) stimulation, which was repeated 3 times. Eleven cases of paroxysmal AF were observed 36 times following S1S2 stimulation in the pacing group, whereas only 1 case was observed in the control group. Fifteen events of paroxysmal AF and 12 cases of persistent AF were induced via burst stimulation in the pacing group. However, only 6 cases of paroxysmal AF and no cases persistent AF were induced via burst stimulation in the control group. The incidences of paroxysmal AF in the control group and the pacing group were 33.3% (6/18) and 55.6% (15/27), respectively. The Incidences of permanent AF in the control group and the pacing group were 0% and 44.4% (12/27), respectively. Each of the dogs that received electrical pacing developed persistent AF within 4 weeks.p = 0.71, data not shown). The ERPAs at different pacing cycle lengths  were 134.46±21.30 ms (P<0.05 vs Group C), 126.66±10.00 ms (P<0.01 vs Group C) and 132.44±21.54 ms (P<0.05 vs Group C), respectively. The ERPAV increased from 160.00±0.00 ms to 167.78±4.41 ms (P<0.01vs Group C). CSNRT increased from 103.33±2.58 ms to 115.44±8.24 ms (P<0.01 vs Group C). The AVN-Wen increased from 328.33±24.01 bpm to 294.44±26.03 bpm (P<0.05 vs Group C)  .Significant structural changes were observed in the heart following electrical pacing. The tissue adjacent to the atrium exhibited significant dilatation. Following 4 weeks of pacing, the left atrial anteroposterior diameter (LAap), left atrial upper-bottom diameter (LAud) and left atrial left-right diameter (LAh) were 24.33±3.39 mm (P<0.05), 27.11±4.01 mm (P<0.05), and 28.44±4.69 mm (P<0.05), respectively. The right atrial upper-bottom diameter (RAud) was 22.33±2.60 mm (P>0.05) at week 4, and no changes in the ventricular dimensions were observed .Following 4 weeks of RAP, the expression levels of hydroxyproline at different anatomic locations of the heart were analyzed. The results indicated that the expression of hydroxyproline was elevated following RAP . CompareThe mRNA expression levels of TGF-β1 (1.32±0.38 vs 0.58±0.02), Collagen-I (1.33±0.91 vs 0.35±0.33) and CTGF(5.83±3.71 vs 1.53±0.76) in the left atrium were significantly increased following 4 weeks of RAP . The mRNThe representative histological sections of the atrial myocardium of each group are depicted in −8, 10−7, 10−6, 10−5, 10−4 mol/L was 0.56,0.54,0.59,0.56,0.69 respectively vs 0.06 in control group,(P<0.01)  .−8,10−7,10−6,10−5,10−4 mol/L was 0.35,0.33,0.29,0.29,0.29 respectively vs 0.38 in Ang II(10-6mol/L) group,(P<0.05). Optical density in different concentration of Simvastatin 0.01μM,0.1μM,1μM was 0.29,0.23,0.21 respectively vs 0.34 in Ang II(10-6mol/L) group (P<0.05). Optical density in different concentration of Y27632 10−8,10−7,10−6,10−5,10-4mol/L was 0.30,0.29,0.26,0.28,0.13 respectively vs 0.34 in Ang II(10-6mol/L) group (P<0.05). (The Ang II receptor antagonist Losartan and the RhoA/ROCK-1 inhibitors (Sim and Y27632) significantly inhibited the Ang II induced proliferation of the atrial CFs in a dose dependent manner. Optical density in different concentration of Los 10P<0.05). . However−8, 10−7, 10−6) was 1.22±0.02,1.35±0.06,1.73±0.07 respectively at 21 hours vs 0.95±0.01,1.36±0.03,1.59±0.08 at 6 hours (P<0.01). The expression mRNA of CTGF in different concentration of Ang II was 0.94±0.01,1.10±0.03,1.20±0.02 respectively at 21 hours vs 0.95±0.02,1.08±0.01,1.08±0.01 at 6 hours (P<0.01) (-6mol/L) group. The expression mRNA of CTGF in different inhibitors  was 0.87±0.04,0.91±0.02,1.15±0.01,1.23±0.01 respectively vs 1.20±0.02 in Ang II(10-6mol/L) group. Simvastatin and Losartan downregulated the expression of TGF-β1, and their combined administration exerted synergistic effects. Y27632 had no effect on Ang II induced TGF-β1 expression. The results indicated that TGF-β1 and RhoA/ROCK-1 are both downstream factors of Ang II in atrial CFs  . Each ofrial CFs .P<0.01). The expression mRNA of ROCK-1 in different time induced by TGF-β1was 1.74±0.13,1.20±0.07 respectively. Furthermore, the expression levels of CTGF and ROCK-1 correlated strongly (P<0.05), but the combined administration of the inhibitors did not exert synergistic effects. The expression mRNA of ROCK-1 in different inhibitors was 1.22±0.03,1.01±0.04,1.04±0.11,1.19±0.01 respectively vs 1.74±0.13 in TGF-β1 group. The expression mRNA of CTGF in different inhibitors was 2.27±0.11,1.64±0.03,1.26±0.05,2.09±0.07 respectively vs 2.28±0.11 in TGF-β1 group  (p>0.05)  vs control group (0.83±0.03),(P<0.01) . The exp(p>0.05) . Further(p>0.05) .In this study, we observed that electrical parameters such as AVN-Wen, CSNRT and ERPAV were significantly increased, whereas ERPA was markedly decreased, following 4 weeks of RAP. Meanwhile, the expression of hydroxyproline was increased in the left atrium, and both the left atrial upper-bottom diameter and left atrial left-right diameter were significantly increased. AF is a progressive disease that develops secondary to continuous structural remodeling and electrical remodeling of the atria, often due to AF itself. The development of regional fibrosis results in the electrical uncoupling of adjacent muscle bundles, subsequently resulting in discontinuities in transverse conduction, localized regions of conduction slowing and block, and increased heterogeneity, which ultimately promotes both focal and macro-re-entry and resuDesmoplastic material  plays an important role in atrial fibrosis. Increased levels of CTGF, Collagen-I and TGF-β1 mRNA and protein expression were observed in the pacing group, findings consistent with those of previous studies. ROCK-1 and RhoA mRNA expression and ROCK-1 protein expression were also increased, which paralleled the increases in desmoplastic material expression. However, whether RhoA and ROCK-1 participate in atrial fibrosis has not been determined. The RhoA/ROCK-1 pathway plays a significant role in myocardial fibrosis. FluvastCTGF is a downstream factor of TGF-β1, 13 and TGF-β1 mediates atrial fibrosis, and the Smads are important downstream effectors of this process . In 2011Ang II activates multiple intracellular signaling molecules and pathways, including the RhoA/ROCK-1 pathway. We have demonstrated that AT1 receptor antagonist inhibited the Ang II-induced increase in TGF-β1 expression in isolated adult rabbit cardiac fibroblasts, and downregulated excessive atrial collagen synthesis in animal left atria after pacing. RhoA/ROIn conclusion, Ang II/TGF-β1/RhoA/ROCK-1 signaling plays an important role in atrial remodeling. CTGF is an important mediator of the above signaling pathway. RhoA is an additional downstream effector of TGF-β1 and affects the activity of Smad 2/3 and the RhoA/ROCK-I pathway. These findings may provide clinicians and researchers with new insight regarding the mechanisms underlying AF induced atrial fibrosis, as well as information regarding potential therapeutic targets in the management of AF.


The first one-arm phase II trial aimed to evaluate and predict efficacy and safety of S-1 plus oral leucovorin (S-1/LV) as first-line chemotherapy for patients with advanced gastric cancer (AGC), using S-1 pharmacogenetic pathway approach.A total of 39 patients orally took S-1 at conventional dose and LV simultaneously at a dose of 25 mg twice daily for a week, within a 2-week cycle. The primary endpoint was overall response rate (ORR), while the secondary endpoints were progression-free survival (PFS), time to failure (TTF), overall survival (OS), disease control rate (DCR), and adverse events (AEs). Peripheral blood was sampled prospectively for baseline expression of dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyltransferase (OPRT), thymidine phosphorylase (TP), and thymidylate synthase (TS), CYP2A6 gene polymorphisms, and 5-FU pharmacokinetics.0–24h of 5-FU and metastatic/recurrent sites ≤2 (vs. >3) independently predicted prolonged PFS. Low baseline plasmic DPD independently predicted prolonged OS.The ORR and DCR were 41.0 and 76.9%. The median PFS, TTF, and OS were 4.13, 3.70, and 11.40 months. Grade 3–4 AEs occurred in only 13 patients, and grade 4 AEs occurred in only 1 of them. High OPRT/TS and peritoneal metastasis (vs. liver metastasis) independently predicted responding. High OPRT/DPD independently predicted grade 3–4 AEs. High AUCTwo-week, oral S-1/LV regimen demonstrated promising efficacy and safety as first-line chemotherapy for AGC.NCT02090153The online version of this article (doi:10.1007/s00280-016-3209-1) contains supplementary material, which is available to authorized users. A globally accepted standard regimen for advanced gastric cancer (AGC) has not been established. S-1, a fourth-generation oral fluorouracil (5-FU), has opened new perspectives with simplicity and convenience over the traditional backbone of chemotherapy for AGC, 5-FU.S-1 contains tegafur/gimeracil/oteracil. Gimeracil reduces the degradation of 5-FU by inhibiting dihydropyrimidine dehydrogenase (DPD), and oteracil improves its gastrointestinal tolerability by inhibiting orotate phosphoribosyltransferase (OPRT). S-1 plus cisplatin is recommended as standard treatment by the Japanese Gastric Cancer Association and has been approved in Asian and European countries .One of the potential partners of S-1 is leucovorin (LV), known to enhance the efficacy of 5-FU by forming a ternary complex with 5-FU metabolite and thymidylate synthase (TS) which prolongs the inhibition of TS and blocks DNA synthesis. It was proved UFT, a third-generation oral 5-FU, had favorable activity and tolerability when combined with LV in AGC patients . A phasePreclinical studies have highlighted the importance of TS, the cellular target of 5-FU–folinic acid mechanism of action . Low TS This current phase II study of S-1/LV is deemed necessary to explore its efficacy and safety as first-line chemotherapy for AGC patients. Considering the frequent grade 3 toxicities, dose reduction, and period prolongation in that previous trial of mCRC , the 4-wAs a one-arm, single-center, open phase II clinical trial, it is approved by the independent Institute Research Ethics Committee at the Sun Yat-sen University Cancer Center and conducted in accordance with the Declaration of Helsinki.The eligibility criteria included (1) histologically confirmed metastatic or recurrent gastric cancer; (2) at least one measurable lesion; (3) an age of ≥18; (4) adequate oral intake; (5) no previous antitumor therapy within 5 years ; (6) an Eastern Cooperative Oncology Group performance status <2; and (7) adequate bone marrow, hepatic, and renal function.The exclusion criteria included (1) known hypersensitivity to any of the study drugs, or usage of drugs interacting with S-1; (2) serious concomitant conditions; (3) extensive bone, brain, or meningeal metastasis; (4) another synchronous cancer; (5) surgery within 3 months; (6) participating in other clinical studies; (7) pregnant women; (8) subjects with reproductive potential who were unwilling to use an effective method of contraception.2), 50 mg (1.25 ≤ BSA < 1.50 m2) and 60 mg (BSA ≥ 1.50 m2) b.i.d. in combination with LV given simultaneously at a fixed dose of 25 mg b.i.d. on days 1–7, followed by a 7-day rest.S-1 (20-mg capsules) and LV (25-mg tablets) were provided by DaPeng Co., Ltd, Japan. All patients were orally treated with S-1 in doses of 40 mg (body surface area (BSA) < 1.25 mClinical and laboratory examinations were carried out within 7 days before enrollment and each cycle of chemotherapy afterward. Tumor measurement was conducted on the basis of computed tomographic scans, within 15 days before enrollment and every 3 cycles afterward, according to Response Evaluation Criteria in Solid Tumors guidelines (version 1.1). Patients were considered response-assessable if they had overt clinical or radiological evidence of early PD within the first three cycles. All treatment-related adverse events (AEs) were categorized according to the National Cancer Institute’s Common Terminology Criteria for Adverse Events version 4.0 (NCI-CTCAE v4.0).Peripheral blood was prospectively, anonymously sampled for each patient on the first day of the first cycle at 0 h (pre-dose) and 0.5, 1, 2, 4, 8, 24 h post-S-1/LV morning dosing. The 2 ml plasma at 0 h was separated for measuring baseline protein expression level of DPD, OPRT, TS, and TP. The 4-ml blood cells at 0 h were separated for CYP2A6 gene polymorphism. The 2 ml plasma at 0, 0.5, 1, 2, 4, 8, 24 h was separated for plasma concentrations of 5-FU.The plasmic protein expression level of DPD, OPRT, TS, and TP was determined by an enzyme-linked immunosorbent assay (ELISA), as described by Cui et al. .Polymerase chain reaction (PCR)–restriction fragment length polymorphism were used to determine common variant alleles that affect CYP2A6 activity or expression in Asian population , *13, and the wild-type allele (CYP2A6*1), as previously described .0–24h), maximum concentration (Cmax), time taken to reach maximum concentration (Tmax), half-time (T1/2), area under the first moment curve (AUMC0–24h), mean resistance time (MRT0–24h), and plasma clearance (CL) were derived with non-compartmental methods using WinNonlin version 3.1  was independently predictive of grade 3–4 AEs. The rates of grade 3–4 AEs differed between patients with high OPRT/DPD and low OPRT/DPD . Figure Thirteen patients experienced grade 3–4 AEs and 26 patients did not. By univariate logistic regression analysis, high OPRT (accuracy 79.0%), high OPRT/DPD (80.8%), high OPRT/TP (77.8%), high OPRT/TS (71.0%), high OPRT/DPD + TP + TS (78.7%), high OPRT/DPD + TS (77.8%), high OPRT/TP + TS (77.2%), and high OPRT/DPD + TP (80.1%) were all associated with grade 3–4 AEs. OPRT/DPD exhibited the highest accuracy (80.8%). By multivariate logistic regression analysis, high OPRT/DPD [>0.754 vs. ≤0.754, OR 15.566 (1.490–162.605), 0–24h of 5-FU and metastatic/recurrent sites ≤2 (vs. >3) were significant predictors of prolonged PFS (supplementary Table S5). Similarly, multivariate analysis demonstrated high AUC0–24h of 5-FU was borderline significant predictor of prolonged TTF (supplementary Table S6).Multivariate analysis with a Cox proportional hazards model demonstrated that high AUC0–24h of 5-FU  2.931 1.155–7.433), 33, P = 00–24h of 5-FU, low DPD , and other 35 patients .To integrate the above predictors, we classified 39 patients into 4 patients with high OPRT/TS, high AUCTo our knowledge, the current study is the first one-arm and the second phase II trial to evaluate efficacy and toxicity of S-1/LV chemotherapy in AGC patients, which is the first to predict outcomes using S-1 pharmacogenetic pathway approach, on the basis that LV is but a cofactor entering the 5-FU metabolism. The enrollment was between July 2011 and July 2012, which was similar to between October 2011 and December 2012 of that randomized phase II study of S-1/LV versus SOL versus SP for AGC [We reported S-1/LV regimen yielded promising ORR, PFS, TTF, and OS, without combination with platinum, taxane, irinotecan, or trastuzumab as first-line treatment. In previous phase II studies of S-1 monotherapy in AGC and previous S-1 monotherapy arms of phase III JCOG9912 study, SPIRITS study, SC-101 study, GC0301/TOP-002 study, and START study, conventional dose for 4 weeks followed by 2-week rest was usually given –22. The To reduce the duration of effective drug concentration and keeping an appropriate rest period could be effective method of improving safety. So we modified the schedule as 1-week S-1/LV, followed by 1-week rest period, the same as S-1/LV group in that randomized phase II study. The efficacy S-1/LV here was quite comparable with S-1/LV group in that randomized phase II study, in terms of ORR (41.0 vs. 43%), PFS (4.13 vs. 4.2 m), and TTF (3.70 vs. 4.1 m) . Fewer pJapan Gastric Cancer Association guideline states that S-1 alone could be considered for patient who is not suitable for S-1 plus cisplatin therapy . PreviouGenotyping the peripheral blood for CYP2A6 polymorphism, quantifying plasmic protein expression of DPD, TS, TP, and OPRT with ELISA, is more optimal, convenient, quicker than evaluating genes, mRNAs, or proteins in tumor tissue, especially when 5-FU plasmic pharmacokinetics were to be integrated at the same time. Thus, there may be more clinical accessibility and prospects. What is more, the cutoff levels were determined by standard statistic analysis, not the simple median or mean. The plasmic expression of DPD in this study was consistent with previously reported with ELISA; however, no report for plasmic OPRT, TP, or TS was available . The freIn clinical setting, numerous studies have reported a low TS, low TP, or a high OPRT expression contributed to a high sensitivity to UFT, S-1, or S-1-based treatment in gastric cancer patients, with or without influencing the PFS or OS –38. TherIn this current study, low baseline plasmic DPD was not related to response; however, it was related to long OS, compatible with previous study that high intratumoral DPD mRNA was predictor of poor OS, not ORR or PFS . PreviouP = 0.024), TS (P = 0.044) expression significantly inversely, while DPD (P = 0.073), TP (P = 0.080) borderline inversely correlated with AUC0–24h of 5-FU. The number of CYP2A6 gene variants (P = 0.889) did not correlate with AUC0–24h. This was consistent with that CYP2A6 gene correlated with tegafur pharmacokinetics, but not with 5-FU pharmacokinetics [0–24h of 5-FU, not OPRT/TS, was predictive of PFS revealed that other factors may also influence PFS by AUC0–24h of 5-FU. We did not demonstrate the number of CYP2A6 gene variants correlated with efficacy. Although some studies demonstrated patients having fewer CYP2A6 variants had better PFS in AGC patients with S-1 plus cisplatin, or S-1 plus docetaxel [We found plasmic OPRT Mean plasma concentration–time curve of 5-FU for the entire population. 5-FU: fluorouracil, AUCSupplementary material 2 (DOCX 19 kb)Below is the link to the electronic supplementary material.


Tabernaemontana longipes was studied in this work as a potential source of antiparasitic agents. The chloroform extract of T. longipes leaves was separated into several fractions, and tested for antitrypanosomal activity. One of the fractions displayed significant growth inhibitory activity against Trypanosoma brucei. The active principle in the fraction was isolated, purified, and characterized by NMR and mass spectrometry. The antitrypanosomal agent in the CHCl3 extract of T. longipes leaves is the pentacyclic triterpenoid bauerenol acetate. A metabolite profiling assay suggest that the triterpenoid influences cholesterol metabolism. The molecular target(s) of bauerenol and its acetate, like many other antiparasitic pentacyclic triterpenoids is/are unknown, but they present privileged structural scaffolds that can be explored for structure-based activity optimization studies using phenotypic assays.The Latin American plant  Trypanosoma brucei gambiense/rhodesiense is still a public health problem in rural and remote regions of the continent, especially in the Democratic Republic of the Congo. Despite a gradual decrease in the number of reported cases in the past few years, the lack of effective and safe medicines, the unavailability of adequate and rapid diagnostic tools, especially in rural and remote places, and the possibility of continuous transmission of the parasite from animal reservoirs to humans, make the disease a continuous threat to millions of people   at 295 K. The structure was solved by Olex2 using SHELXTL and was refined by full-matrix least-squares procedure  νmax 2935, 1732, 1463, 1374, 1249, 1026 cm−1; 1H- and 13C-NMR, see +], m/z 475.4113 [Calculated for C32H52O2Li 475.4128]. Colorless crystals; melting point 289–291 °C; IR (CHCl3) νmax 3379, 2929, 2866, 1457, 1372, 1032 cm−1; 1H- and 13C-NMR, see +], m/z 427.3922 [Calculated for C30H51O2 427.3940].Colorless solid; melting point 202–203 °C; IR (CHClT. brucei (strain 427) cultured in HMI-9-medium supplemented with 10% FBS, 10% Serum plus (SAFC), 0.05 mM bathocuproine sulfonate, 1.5 mM l-cysteine, 1 mM hypoxanthine, 0.2 mM β-mercaptoethanol, 0.16 mM thymidine, 1 mM pyruvate, and 0.0125% Tween 80 was used. Parasites were dispensed into a sterile 96-well plate at 5 × 103 cells/well and treated with compound 1 and 2 for 24 h. The compounds were prepared in DMSO containing 1.25% Tween 80. Final concentration of Tween 80 in the assay wells was 0.0125%. The compounds were screened in triplicate with a total assay volume of 100 µL. Next, Alamar blue (20 µL) was added and the plate was incubated at 37 °C for 4 h. Immediately following incubation, fluorescence signals were read . Suramin was used as positive control. The growth inhibitory activity of the compounds was evaluated using the Alamar blue assay . Bloodstl-glutamine and sodium bicarbonate, 10% FBS, 1% penicillin/streptomycin, 0.0125% Tween 80) incubated at 37 °C in a 5% CO2 environment. Once 80–90% confluent, the cells were washed with phosphate-buffered saline (PBS), treated with 0.25% (w/v) of trypsin/EDTA, counted and suspended in fresh complete media. Into 96-well plates, 5 × 105 cells/mL were seeded and incubated for 24 h. Cells were treated with the compounds prepared in DMSO containing 1.25% Tween 80 for 72 h. Final concentration of Tween 80 in the assay wells was 0.0125%. Thereafter, DMEM:F12 medium containing MTT (5 mg/mL in PBS) was added to the cells and incubated for 1 h. The MTT-containing medium was then gently removed and replaced with DMSO (200 μL/well), the plate was then mixed gently to allow the formazan crystals to dissolve. Absorbance was measured at 550 nm. All compounds were tested in triplicates. SDS (10%) was used as positive control.Human hepatocarcinoma cell line  was used for cytotoxicity studies. The cells were grown in complete medium  for grinding. The cells were grinded for 30 s at 1500 rpm, and centrifuged at 14,000× g for 5 min. The supernatants were transferred to new tubes. The extraction was repeated twice, and the pooled supernatant was concentrated to 500 µL. The samples were silylated using MSTFA + 1% TMCS at 37 °C, and were used for primary metabolite profiling on a LECO Pegasus IV GC-time of flight mass spectrometer  as described by Fiehn and co-workers [-workers . The GC-The samples (0.5 µL each) were analyzed using the splitless mode on a Rtx-5Sil MS capillary column . The gas flow-rate was 1 mL/min. Initial injection temperature was set at 50 °C and ramped up to 250 °C at a rate of 12 °C/s. Initial oven temperature was set at 50 °C for 1 min, then ramped up to 330 °C at a rate of 20 °C/min, and held constant for 5 min. The TOF mass spectrometer  unit mass resolution was set at 17 spectra/s from 80–500 Da at −70 eV ionization energy and 1800 V detector voltage. The transfer line and the ion source temperatures were set at 230 °C and 250 °C, respectively. The primary metabolites were identified and quantified using the quantifier ions shown in


OT demonstrated the ability to recover these disturbances to the level of wild-type mice for 30 min after injection. LOT-2 and LOT-3 partially recovered the behaviors for a short period. Conversely, LOT-1 restored the behavioral parameters, not for 30 min, but for 24 h. These data suggest that the lipidation of OT has some therapeutic benefits, and LOT-1 would be most useful because of its long-last activity.Oxytocin (OT) is a nonapeptide that plays an important role in social behavior. Nasal administration of OT has been shown to improve trust in healthy humans and social interaction in autistic subjects. As is consistent with the nature of a peptide, OT has some unfavorable characteristics: it has a short half-life in plasma and shows poor permeability across the blood-brain barrier. Analogs with long-lasting effects may overcome these drawbacks. To this end, we have synthesized three analogs: lipo-oxytocin-1 (LOT-1), in which two palmitoyl groups are conjugated to the cysteine and tyrosine residues, lipo-oxytocin-2 (LOT-2) and lipo-oxytocin-3 (LOT-3), which include one palmitoyl group conjugated at the cysteine or tyrosine residue, respectively. The following behavioral deficits were observed in CD38 knockout (CD38 Autism spectrum disorder (ASD) is a set of neurodevelopmental conditions characterized by impairments in social communication, social interactions and the presence of repetitive behaviors and restricted interests . There aA great deal of research has focused on a neuroendocrine hormone, oxytocin (OT) ,7. This 1 residue and the phenolic hydroxyl group of the Tyr2 residue [1 residue or the phenolic hydroxyl group of the Tyr2 residue, respectively [One of the successful methods for extending the half-life of peptides in the blood is lipidation, i.e., the conjugation of a given peptide with long fatty acids . Palmito residue . In addiectively . LOT-1 wectively . A singlectively .−/−) male mice displayed a significant effect from 3 to 24 h after injection, during which OT completely lost its effect, while there was no immediate effect at 30 min after injection [Recently, we also showed that the administration of LOT-1 to CD38 knockout  and CD38−/− mice were born in our laboratory colony. The pups were weaned at 21–28 days of age and housed in same-sex groups of five animals until pairing. A male and female of each genotype were paired and housed in a nursing cage in our laboratory under standard conditions  with food and water provided ad libitum. All animal experiments were performed in accordance with the Fundamental Guidelines for the Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science, and Technology of Japan and were approved by the Committee on Animal Experimentation of Kanazawa University .Male and female Slc:ICR mice  were obtained from Japan SLC, Inc.  through a local distributor . The procedure to produce the CD38eviously . The offThe design of the experiments for parental retrieval behavior was described previously ,44. VirgParental retrieval behavior was scored by observing the parent behavior for 10 min following the reunion. Each sire or dam received 2 points for each pup returned completely to the nest, 1 point if the parent had contact with the pup or moved the pup to another place, and 0 if the pup was intact. After finishing of one session, the next sessions were repeated after a 10-min reset period between each session . In the The protocol for the social acquisition and recognition test was described previously ; howeverThe tail suspension test method was previously described ,47. AfteThis test was based on a method that was described previously ,49. The ad libitum. The bottle positions remained constant. Fresh sucrose solution was prepared each day. Cumulative water and sucrose intakes during 24 h were calculated by weighing. Food was provided ad libitum, but food intake was not recorded in this experiment.Experimentally naïve, young adult, male and female mice (6–8 weeks old) were given a two-bottle choice between distilled water and sucrose solution at a 1% concentration based on our previous results , which wSeven-day-old male pups were isolated from their parents in their home cage. Each pup was placed into the recording chamber (22 cm × 22 cm) for 3 min, and the number of grid crossings (4.5 cm × 4.5 cm) was counted .t-test was used for single comparisons between two groups. The rest of the data were analyzed by one-way or two-way analyses of variance (ANOVA) for two or three components, respectively. Post hoc comparisons were performed only when the main effect showed statistical significance. The p-values of the multiple comparisons were adjusted using Bonferroni’s correction. All data are shown as the mean ± standard error of the mean. In all analyses, p < 0.05 indicated statistical significance. All analyses were performed using GraphPad Prism 6 software .The two-tailed Student’s +/+) and CD38 KO (CD38−/−) strains, essentially as described previously [+/+ primiparous dams displayed nearly complete maternal  behavior, with scores of 9.5 ± 1.1 in the first trial and 10.0 in 2nd and 3rd trials (n = 10)  , respectively, which were not significantly lower than those of the wild-type dams  behavior by sires and dams for 5 pups of wild-type CD38+/+ and Ceviously . CD38+/+e of 10) A. The maype dams A.+/+ first-time sires displayed retrieval behavior, with scores of 6.1 ± 0.9, 6.6 ± 1.0 and 7.1 ± 1.2 (n = 10) in the 1st to 3rd trials . Two-tailed Student t-tests indicated a significant difference between CD38+/+ and CD38−/− sires in each trial (p < 0.01).Sires of the ICR strain have been reported to display parental behavior after separation with only their mate dams in a novel cage for 10 min . CD38+/+tely 6–7 ; Figure +/+ sires showed retrieval behavior with an average parental score of 8.2 ± 0.75 (n = 13), while that in CD38−/− sires was 1.0 ± 0.53 (n = 13)  per 100 g of body weight of OT, LOT-1, LOT-2, or LOT-3 were 5.4 ± 1.3 (n = 8), 4.1 ± 1.6 (n = 8), 8.9 ± 0.48 (n = 8) and 8.4 ± 0.56 (n = 8), respectively , LOT-2 (p = 0.001), and LOT-3 (p = 0.001) from CD38−/− sires treated with PBS; however, no significance was observed in those treated with LOT-1 (p = 0.247).Based on the retrieval behavior of dams and sires shown in (n = 13) A. The paectively A. A one-−/− sires, the parental scores for OT, LOT-1, LOT-2, and LOT-3 were 1.6 ± 1.2 (n = 8), 6.5 ± 1.5 (n = 8), 4.3 ± 1.6 (n = 8) and 3.0 ± 1.5 (n = 8), respectively. A one-way ANOVA revealed significant differences: F4,40 = 3.42, p = 0.017. Bonferroni’s post hoc tests revealed a significant difference between the saline- and LOT-1-treated sires (p = 0.015), whereas no significant differences were revealed for the other treatment groups: OT (p = 0.999), LOT-2 (p = 0.515), and LOT-3 (p = 0.999). These data clearly show that CD38−/− sires had impaired paternal retrieval behavior and that their impaired behavior was restored by OT, LOT-2 and LOT-3, but not LOT-1, just after their injection. Conversely, the long-lasting effect was obtained by LOT-1, but not by the others. These results are in accord with previous results using latency in retrieval as the basis for the behavioral scales [The long-term effects of OT and the LOTs were examined using the same paradigm but were observed 24 h after the injections. As shown in l scales ,50.+/+ mice displayed a significant decrease at the 4th meeting with the same (familiar) target mouse from the first meeting (38 ± 1.7 s (n = 10) from 49 ± 1.6, p < 0.001 and 38 ± 1.7 s (n = 10) from 49 ± 1.2 s, p < 0.001, respectively). They also exhibited a significant increase in social contact to a novel (unfamiliar) male at the 5th meeting: 50 ± 1.0 s (n = 10) for CD38+/+ mice with PBS (p < 0.001) and 48 ± 1.2 s (n = 10) (p < 0.001) for CD38+/+ mice with OT.In the experiments on social acquisition and recognition, we examined the investigation time of resident  mice to intruder virgin males for 1 min, as reported previously . At 30 m−/− male mice treated with PBS demonstrated a slight decrease during repeated meetings with familiar males . Surprisingly, at the 5th meeting with a new male, no increase in the investigation time was recorded: 32 ± 1.9 (n = 15) from 33 ± 1.8 s (n = 15) at the 4th meeting. When CD38−/− male mice were treated with OT, however, they displayed a significant decrease at the 4th meeting  compared with the first meeting and a significantly longer investigation time at the 5th meeting   A.−/− males displayed a decrease in interaction time with the repeated meetings: from 36 ± 3.7 s to 27 ± 1.4 s (n = 6) for LOT-1; from 39 ± 3.3 s to 32 ± 3.2 s (n = 5) for LOT-2; and from 44 ± 2.5 s to 28 ± 1.9 s (n = 5) for LOT-3. Significant increases in investigation time were observed at the 5th meeting with a new male from the 4th meeting with a familiar male: from 27 ± 1.4 to 36 ± 3.3 s (n = 6) for LOT-1; from 32 ± 3.2 to 36 ± 4.0 s (n = 5) for LOT-2; and from 28 ± 1.9 to 42 ± 6.0 s (n = 5) for LOT-3.The effects of the LOTs on the investigation time were examined. As shown in −/− males treated with LOT-3. A one-way ANOVA resulted showed a significant difference: F6,49 = 2.779, p = 0.0209. Bonferroni’s post hoc tests revealed a significant difference between CD38−/− mice treated with PBS  and with LOT-3 .The average decrease following the repeated meetings from the first to the 4th meeting is illustrated in −/− males treated with PBS did not exhibit an increase in response to the new male , in wild-type mice treated by OT , and in CD38−/− treated by OT , LOT-2 , and LOT-3  compared to CD38−/− males treated with PBS. It is worth noting that no significance was observed in CD38−/− males treated with LOT-1 , suggesting no immediate effect by LOT-1, as shown in Quantitative data on the percent changes between the 4th and 5th session demonstrated that only CD38n = 15) or OT  treatments in CD38+/+ male mice. In contrast, the time of immobility was significantly lower in CD38−/− male mice treated with PBS  and 145 ± 27 s (n = 6), respectively. A one-way ANOVA revealed and significant difference: F6,54 = 5.922, p = 0.0001. Post hoc comparisons by the Bonferroni’s tests revealed significance for CD38+/+ with PBS (p = 0.0008) or OT (p = 0.009) and for CD38−/− mice treated with OT (p = 0.021) and LOT-2 (p = 0.033), when compared with CD38−/− mice treated with PBS. Statistically, no significance was observed for CD38−/− mice treated with LOT-1  or LOT-3 .The effects of the LOTs were examined on depression-like behavior by the tail suspension test 30 min after the injection. There was no difference in time immobility between PBS . Bonferroni’s post hoc tests revealed significance in CD38+/+ female mice treated by PBS  or OT  and CD38−/− female mice treated with OT  or LOT-2 , when compared to CD38−/− mice . Similar to male mice, treatment with LOT-1  or LOT-3  did not increase immobility time. These data suggest that CD38 knockout adult male and female mice are less depressive, and this phenotype was recovered by OT or LOT-2 in a short time after the injection.Similar findings were observed in the female mice B. A one-+/+ and CD38−/− male mice treated with PBS demonstrated a similar time spent in the open arms (n = 10), respectively, for the wild-type; 27.5 ± 9.1 s and 0.66 ± 0.25 m (n = 5) for CD38−/− mice, respectively. OT slightly increased the time spent and distance travelled in the open arms in both genotypes without statistical significance. A one-way ANOVA did not indicate significance in time spent in open arms  nor distance travelled in the open arms . These data indicate that CD38−/− mice display less anxiety-like behavior, as has been shown previously [In the elevated plus maze task, both CD38pen arms A and dispen arms B: 42.8 ±+/+ and CD38−/− male mice during free-choice between water and sucrose solutions [+/+ and CD38−/− female mice treated with PBS or OT . In sharp contrast, as previously reported [−/− male mice showed significantly lower sucrose consumption  compared with CD38+/+ mice under the PBS injection  or OT administration . A one-way ANOVA revealed significance . The phenotype in CD38−/− was significantly recovered by OT administration , while LOT-1  and LOT-3  were ineffective. A one-way ANOVA indicated an effect for treatment .At 30 min after the administration of the LOTs C, only Ln = 5, p = 0.04), with a tendency towards a long-lasting effect (n = 5), LOT-2  or LOT-3  demonstrated the same level of sucrose preference as treated by PBS. A one-way ANOVA indicated a difference for treatment conditions .Twenty-four hours after the injection, LOT-1 demonstrated an elevation in sucrose consumption  in the grid crossing test, when compared with CD38+/+ pups of the same age treated with PBS  (n = 10 for every cases). A one-way ANOVA indicated significance . Bonferroni’ post hoc tests revealed significance for OT (p = 0.03), LOT-2 (p = 0.0009) or LOT-3 (p = 0.0002), but no significance for LOT-1 (p = 0.99). These data indicate that LOT-2 and LOT-3 restored the knockout phenotype better than OT, while LOT-1 had no effect.Increased pup locomotor activity was also a phenotype that was affected by CD38 gene deletion . The rec 0.0001) . The loc−/− mice displayed impairments in parental retrieval behavior  mouse was observed. In this test, we were clearly able to discriminate between the CD38+/+ and CD38−/− character. Through this, we could show the differential effectiveness between LOT-1 and others, such as OT, LOT-2 or LOT-3. This may lead to a potential usage of LOT-2 and/or LOT-3 individually or in combination with LOT-1 in future.Social recognition was tested by repeated encounters with the familiar mouse of the same sex, which differed from the previous report, in which sires investigated female virgin intruders ,51. The −/− mice than in CD38+/+ mice. This may not be surprising because CD38−/− mice have been shown to be hyperactive [The tail suspension test is commonly used for observation of depression-like behavior. Interestingly, we discovered a shorter duration of immobility in both female and male CD38eractive ,52. Reco−/− mice showed less preference, and this gender-specific phenotype was clearly recovered by OT. At 30 min after the injection, only LOT-2 could replicate the OT effect. At 24 h later, OT, LOT-2 and LOT-3 were ineffective, as expected, while we observed effectiveness with LOT-1.The sucrose preference test is usually used for investigating of anhedonia and reward processes . In our The mechanism how LOT-2 can show overall OT-like effects for short-time period. One of possible mechanisms may be high permeability through BBB, because of lipidation , althoug−/− mice, which had not been examined previously. We confirmed that the lipidation strategy of adding palmitoyl acid improves the drug-like properties of OT, a peptide drug with long-lasting activity. Although the precise mechanism is unknown, these prodrug compounds slowly release side chains and metabolize to OT in the blood stream or in the brain. The next step is to investigate the pharmacodynamics of LOTs. Finally, based on these results together with previous ones, we conclude that LOTs- are beneficial over the native OT and potentially useful candidates for psychiatric diseases, including autism spectrum disorder [Here, we tested the ability of OT or LOTs to elicit the recovery of known deficits in social behaviors in CD38disorder .


Background:Protium species (Burseraceae) have been used in the treatment of various diseases and conditions such as ulcers and wounds. Methods: The essential oil from the oleoresin of Protium amazonicum was obtained by hydrodistillation and analyzed by GC-MS, GC-FID, and chiral GC-MS. P. amazonicum oleoresin oil was screened for antifungal activity against Candida albicans, Aspergillus niger, and Cryptococcus neoformans. Results: A total of 54 components representing 99.6% of the composition were identified in the oil. The essential oil was dominated by δ-3-carene (47.9%) with lesser quantities of other monoterpenoids α-pinene (4.0%), p-cymene (4.1%), limonene (5.1%), α-terpineol (5.5%) and p-cymen-8-ol (4.8%). Chiral GC-MS revealed most of the monoterpenoids to have a majority of levo enantiomers present with the exceptions of limonene and α-terpineol, which showed a dextro majority. P. amazonicum oleoresin oil showed promising activity against Cryptococcus neoformans, with MIC = 156 μg/mL. Conclusions: This account is the first reporting of both the chemical composition and enantiomeric distribution of the oleoresin essential oil of P. amazonicum from Ecuador. The oil was dominated by (−)-δ-3-carene, and this compound, along with other monoterpenoids, likely accounts for the observed antifungal activity of the oil. Protium amazonicum (Cuatrec.) Daly belongs to the Burseraceae, which is comprised of 640 species representing 18 genera throughout the world, mainly distributed in the Neotropics and North Africa [Protium spp. have been used in the treatment of various diseases and conditions such as ulcers and wounds, to treat headaches, toothaches, and rheumatism [P. fimbriatum to treat respiratory infections [Protium oleoresins have been characterized in terms of color, age, odor, as well as volatile and non-volatile chemical characteristics . The tree was identified by Rafael Parducci, and a voucher specimen has been deposited in Saintoil S.A. The essential oil was obtained by hydrodistillation using a Clevenger apparatus as previously described [The oleoresin  of escribed  to give P. amazonicum was analyzed by GC-MS using a Shimadzu GC-MS-QP2010 Ultra  operated in the electron impact (EI) mode (electron energy = 70 eV), with a scan range of 40–400 atomic mass units (amu), a scan rate of 3.0 scans/s, and the GC-MS Solution software . The GC column was ZB-5MS fused silica capillary column  (30 mL × 0.25 mm ID) with a (5% phenyl)-polymethylsiloxane stationary phase with a film thickness of 0.25 μm. The carrier gas was helium with a column head pressure of 551.6 kPa and flow rate of 1.37 mL/min. The injector temperature was 250 °C, and the ion source temperature was 200 °C. The GC oven temperature program was programmed for 50 °C initial temperature, the temperature increased at a rate of 2 °C/min to 260 °C. A 5% w/v solution of the sample in CH2Cl2 was prepared and 0.1 µL was injected with a splitting mode (30:1). Identification of the oil components was based on their retention indices determined by reference to a homologous series of n-alkanes, and by comparison of their mass spectral fragmentation patterns with those reported in the literature [The oleoresin essential oil of v/v with CH2Cl2). The percent composition of the oleoresin essential oil was calculated from raw peak areas without standardization.The gas chromatograph was a Shimadzu GC 2010  equipped with a flame ionization detector, a split/splitless injector, and autosampler AOC-20i . The capillary column was a ZB-5MS  with a film thickness of 0.25 μm. The column temperature was programmed, 50–250 °C at 2 °C/min, the injector temperature was 250 °C, the detector temperature was 280 °C, the carrier gas was nitrogen, and the flow rate was maintained at 1.0 mL/min. Injection mode split with a split ratio of 1:100. The injected volume was 0.3 µL of diluted oil  operated in the EI mode (electron energy = 70 eV), scan range = 40–400 amu, scan rate = 3.0 scans/s. GC equipped with a Restek B-Dex 325 capillary column (30 m × 0.25 mm ID × 0.25 μm film) . Oven temperature was started at 50 °C, and then gradually raised to 120 °C at 1.5 °C/min. The oven was then raised to 200 °C at 2 °C/min and held for 5 min. Helium was the carrier gas and the flow rate was maintained at 1.8 mL/min. The sample was diluted 3% w/v with CH2Cl2 and then a 0.1 µL sample was injected in a split mode with a split ratio of 1:45. The enantiomers of each monoterpene were identified by comparison of retention times to authentic samples obtained from Sigma-Aldrich .Chiral analysis of the Candida albicans (ATCC 18804) and Cryptococcus neoformans var. neoformans (ATCC 24067) were initially grown on potato dextrose agar (PDA) plates for 72 h at 37 °C. A single colony was used to inoculate approximately 5 mL of potato dextrose broth (PDB) which was subsequently grown for an additional 24 h at 37 °C. Aspergillus niger (ATCC 16888) cultures were grown on PDA plates for 5 days at room temperature . A. niger conidia were collected, placed in PDB, and filtered through sterile cheesecloth into fresh PDB. The absorbance of the fresh solution was read at 625 nm and adjusted accordingly with PDB to an absorbance of 0.15. Minimum inhibitory concentrations (MICs) were determined in triplicate using 96-well plates. C. albicans and C. neoformans were diluted in 3-(N-morpholino)propanesulfonic acid (MOPS) buffered Roswell Park Memorial Institute (RPMI) medium to 2000 cells/mL whereas A. niger was diluted with PDB to an OD625 of 0.15. Initially, 50 μL of MOPS buffered RPMI was added to each well of the plate. In the first row, 50 μL of essential oil was added and mixed well, then 50 μL of this mixture was removed and then added to the medium in the next row. This serial dilution process was repeated for each row of the plate, with the removed volume from the last row being discarded. To each well was added 50 μL of cells to achieve a final volume of 100 μL. C. albicans and C. neoformans were incubated at 37 °C for 48 h. The A. niger plates were incubated at RT for 6 days. The MIC was determined from turbidity or growth on the plates in comparison to positive and negative controls. In order to verify the results, MIC determinations were carried out in nine replicates. A combination of Cyprodinil and Fludioxonil served as the positive control with MOPS buffered RPMI serving as the negative control.The broth microdilution method was performed to determine antifungal activity as previously reported ,32. BrieP. amazonicum was obtained in 0.3% yield and analyzed by GC-MS and GC-FID. From a total of 56 peaks, 99.6% of the compounds were identified in the oil (p-cymene (4.1%), limonene (5.1%), α-terpineol (5.5%) and p-cymen-8-ol (4.8%)  and α-pinene (27.0%) as the major components [p-cymene (36.0%), α-terpinene (18.0%), and γ-terpinene (12.0%) [The clear pale yellow oleoresin essential oil from  the oil . The maj.8%) see . δ-3-Caraphyllum ; howeverrved see . Protiumhe resin . The agemponents ; the fre (12.0%) ; and the (12.0%) . The oleP. amazonicum essential oil (see Angelica archangelica showed predominantly (+)-δ-3-carene, but the (−)-enantiomer was detected [Pinus sylvestris (Pinaceae) essential oils, while (−)-limonene predominated [Citrus (Rutaceae) essential oils [Micromeria fruticosa (Lamiaceae) essential oil showed exclusively (+)-α-terpineol while (−)-α-terpineol was found in Laurus nobilis (Lauraceae) essential oil [Pistacia vera showed a predominance of (+)-α-pinene, (+)-limonene, (+)-β-pinene, and exclusively (−)-α-terpineol [Boswellia carterii (Burseraceae) from Ethiopia was composed of (+)-α-thujene, (−)-α-pinene, and (−)-limonene, but the enantiomeric distribution of δ-3-carene was not determined [B. carterii resin oil from Somalia showed (−)-α-thujene, (−)-α-pinene, and (−)-limonene predominating, while B. sacra resin oil from Oman had (+)-α-thujene, (+)-α-pinene, (+)-β-pinene, and (−)-limonene predominating [Chiral GC-MS analysis was performed to evaluate the enantiomeric distribution of the monoterpenes present in  oil see . The levdetected . Only thominated . The (+)ial oils ,40,41,42tial oil . Analysierpineol . Althougtermined . In contminating .P. amazonicum demonstrated antifungal activity against C. albicans, C. neoformans, and A. niger. C. neoformans was most potently inhibited with a promising MIC of 156 μg/mL. Inhibition of C. albicans (MIC = 313 μg/mL) was also rather promising whereas inhibition of A. niger was relatively weak (MIC = 1250 μg/mL). The major component in P. amazonicum oil, δ-3-carene, has shown antifungal activity against several fungi, including C. albicans [The oleoresin essential oil of albicans . In addialbicans , limonenalbicans , and α-t MIC = 12 μg/mL. THeterobasidion parviporum than (−)-α-pinene [Candida albicans [P. amazonicum resin oil has promising potential for further antifungal consideration, in particular against C. neoformans and potentially other yeast-like fungi.The antifungal mechanisms of activity of monoterpenoids are poorly understood. It has been suggested that these hydrophobic compounds disrupt the cytoplasmic membranes or membrane proteins of fungal cells, leading to cytoplasmic leakage, cell lysis, and death . Chiraliα-pinene . Likewisalbicans , and Omralbicans . (+)-δ-3albicans , but theProtium amazonicum. The P. amazonicum resin oil collected in Ecuador was dominated by (−)-δ-3-carene and is therefore, an excellent source of this enantiomer. The abundance of this compound, along with other monoterpenoids, likely account for the observed antifungal activity of the oil. The activity against Cryptococcus neoformans and Candida albicans indicates promise against these opportunistic fungal pathogens. Additional research into this tree species and other Protium species, their chemistry and their biological activities, is needed.This is the first reported chemical analysis of the oleoresin essential oil of


The synthesis of up to pentablock copolymers from various combinations of l-lactide and trimethylene carbonate was accomplished using a dinuclear zinc complex, and the physical, thermal, and mechanical properties of the resulting copolymers evaluated. l-lactide) (PLA) is a bioderived and biodegradable polymer that has limited applications due to its hard and brittle nature. Incorporation of 1,3-trimethylene carbonate into PLA, in a block copolymer fashion, improves the mechanical properties, while retaining the biodegradability of the polymer, and broadens its range of applications. However, the preparation of 1,3-trimethylene carbonate (TMC)/l-lactide (LA) copolymers beyond diblock and triblock structures has not been reported, with explanations focusing mostly on thermodynamic reasons that impede the copolymerization of TMC after lactide. We discuss the preparation of multiblock copolymers via the ring opening polymerization (ROP) of LA and TMC, in a step-wise addition, by a ferrocene-chelating heteroscorpionate zinc complex, {[fc(PPh2)2pz]2)]Zn(μ-OCH2Ph)}2 Zn(μ-OCH2Ph)]2, fc = 1,1′-ferrocenediyl, pz = pyrazole). The synthesis of up to pentablock copolymers, from various combinations of LA and TMC, was accomplished and the physical, thermal, and mechanical properties of the resulting copolymers evaluated.Poly( The resulting thermoplastic elastomers,b-PTMC and PLA-b-PTMC-b-PLA compositions,via an initial TMC polymerization followed by the sequential addition of LA, in the presence of various organic or metal-based catalysts;2)2pz]2)]Zn(μ-OCH2Ph)}2 Zn(μ-OCH2Ph)]2)l-lactide and 1,3-trimethylene carbonate to examine their physical, thermal, and mechanical properties, and we discuss our results herein.Growing concerns over the environmental damage caused by petroleum-based plastic wasteP,B)Zn(μ-OCH2Ph)] toward the ROP of LA and TMC, i.e., its ability to polymerize TMC after LA, we began by studying the solid state molecular structure and the solution behavior of the metal complex. The isolation of [Zn(μ-OCH2Ph)]2 as yellow crystals in a 68.5% yield (eqn (1)) was achieved via the addition of ZnCl·(C7H8)in situ generated KOCH2Ph in THF.Because of the unique behavior of [Zn(μ-OCH2Ph)]2 was determined using single-crystal X-ray diffraction (τ = 0.75).2 fashion via the pyrazole nitrogens, while the phosphine moieties are not coordinated and the benzoxide groups are in a bridging position between the two metal centers.The solid state molecular structure of [Zn complexes.31P{1H} NMR spectrum of [Zn(μ-OCH2Ph)]2 shows a singlet at δ = –15.5 ppm. Similar chemical shifts of δ = –16.4 and –15.5 ppm were observed for a coordinated phosphine in ZnCl and a free phosphine in fc(PPh2)[B(OMe3)2], respectively.31P{1H} NMR spectra between free and zinc(ii)-coordinated phosphines are commonly observed and are attributed to weak interactions between the soft phosphine ligands and the oxophilic zinc(ii) centers.P,B)ZnCl and [Zn(μ-OCH2Ph)]2 Zn(μ-OCH2Ph)]2 to ZnCl is 1.63. This value is somewhat below the expected value of 2 for the dimer, as derived from the comparison of volumes from the solid state structures. However, 1H Nuclear Overhauser Effect Spectroscopy (NOESY) studies of [Zn(μ-OCH2Ph)]2 show a binding motif similar to that observed in the solid state structure. Interactions between the protons of the pyrazole methyl groups and the benzoxide ligand are observed in the 2D plot, while the interactions between the phosphine phenyl groups and the benzoxide are not observed Zn(μ-OCH2Ph)]2 show no significant changes in the range of 298–352 K Zn(μ-OCH2Ph)]2 in C6D6 yields a simple mixture of the two compounds at ambient temperature Zn(μ-OCH2Ph)]2 was evaluated both in the presence and absence of a substrate. In the absence of a monomer, [Zn(μ-OCH2Ph)]2 slowly decomposes in benzene at ambient temperature, reaching 7.0% decomposition after 24 h Zn(μ-OCH2Ph)]2 resulted in the formation of a single major species Zn(PLA)36(OCH2Ph)]2 yielded a diffusion rate of 1.04 × 10–6 m s–2 Zn(μ-OH)]2. The free polymers, PhCH2O(PLA)36H, displayed a diffusion rate of 2.00 × 10–6 m s–2  values  chemistry;The differential scanning calorimetry (DSC) curves for the newly synthesized block copolymers display PLA only . Even at Fig. S63, likely  to PTMC . In genevia dynamic mechanical analysis Zn(μ-OCH2Ph)]2 was lower by 3.3 kcal mol–1 than that of the corresponding monomer, Zn(OCH2Ph), in agreement with the experimental observations.To gain a better understanding of the mechanism, we turned to density functional theory.–1) for the alkoxide nucleophilic attack (TSI–II), the energy for the ring opening step (TSII–III) and the overall activation barrier are lower for the dimeric species than for the monomer by 4.2 and 4.4 kcal mol–1, respectively; furthermore, the two zinc centers participate in the process synergistically when the reaction occurs with the dimer. Similarly, for the initiation of TMC, both activation barriers were lower for the dimer . These results are again in agreement with the experimental observations discussed above that the dimeric zinc complex facilitates the polymerization.Since the energy difference between the dimeric and the monomeric species was small, the free energy surfaces for the reaction with LA and TMC were thus computed for both the monomer and the dimer Zn(μ-OCH2Ph)]2. The solution state behavior of [Zn(μ-OCH2Ph)]2 in the presence and absence of LA and TMC was thoroughly investigated in order to understand why this catalyst does not have the limitations of previously reported compounds that cannot polymerize TMC after LA. Utilizing diffusion ordered NMR spectroscopy, as well as other spectroscopic techniques, the retention of the dimeric state of the zinc complex in solution was confirmed. It was also found that the zinc complex reacts as a dimer when catalyzing the ROP of l-lactide and 1,3-trimethylene carbonate. A combination of molar mass versus conversion, end group analysis, chain extension, and kinetics experiments, as well as great control over the polymer molar masses, suggests that these polymerization processes proceed via a living mechanism.We report the ring opening copolymerization of LA and TMC to obtain pentablock copolymers, by the multiple step-wise addition of either monomer to the other, without the need for synthesizing tailored initiators or using any other additives. These reactions were possible with a dimeric zinc catalyst, 2 was performed on an Exeter Analytical, Inc. CE-440 Elemental Analyzer. Molar masses of the polymers were determined by SEC MALS using a Shimazu Prominence-i LC 2030C 3D equipped with an autosampler, two MZ Analysentechnik MZ-Gel SDplus LS 5 μm, 300 × 8 mm linear columns, a Wyatt DAWN HELEOS-II, and a Wyatt Optilab T-rEX. The column temperature was set at 40 °C. A flow rate of 0.70 mL min–1 was used, and samples were dissolved in chloroform. The number average molar mass and dispersity were found using the known concentration of the sample in chloroform with the assumption of 100% mass recovery to calculate dn/dc from the RI signal. DSC was obtained using a PerkinElmer DSC model 8000 heat flow system with Intracooler II. The method used was to increase the temperature from –40 to 220 °C at 10 °C min–1, held 220 °C for 2 min, then decreased back to –40 °C at 10 °C min–1 for three cycles. Mechanical properties were measured on a TA Instruments RSA III dynamic mechanical analyzer (DMA). Modulus tests were conducted at 20 °C and a frequency of 1 Hz with samples of 6.0 mm wide and ∼40 μm thick loaded onto the DMA with a 3 mm gap between the thin film grips. The stress–strain curves of the films were obtained at 20 °C at a stretching rate of 1 mm s–1. The tested samples used were 6.0 mm wide and ∼40 μm thick with a 3 mm gap between the thin film grips of the DMA. A minimum of three samples was tested per polymer.All reactions were performed using standard Schlenk techniques or in an MBraun drybox  in 5 mL of THF at –78 °C was added HOCH2Ph  drop-wise via syringe until the solution became colorless. A THF solution of ZnCl·(C7H8)  was then added drop-wise and the reaction mixture stirred for 1 h at –78 °C. The reaction vessel was brought to ambient temperature and volatile substances were removed under reduced pressure. The desired product was extracted with 5 mL of toluene and filtered through Celite. Toluene was removed under reduced pressure and the remaining oily orange solids were dissolved in 5 mL of diethyl ether. After several minutes, a copious amount of yellow solids precipitated from diethyl ether. The solids were collected and washed with diethyl ether until the washings became pale yellow. The final product was isolated as yellow crystals in two crops from a THF/diethyl ether (1 : 2) mixture at –35 °C . Crystals of [Zn(μ-OCH2Ph)]2 always contain two molecules of solvent per molecule of compound as a mixture of THF and diethyl ether as supported by NMR spectroscopic data. X-ray quality crystals were obtained from a THF/diethyl ether layering at –35 °C. 1H NMR : δ (ppm) 2.00 , 2.47 , 3.74 , 3.91 , 4.03 , 4.13 , 4.32 , 4.88 , 5.76 , 6.69 , 6.81 , 6.87 , 7.04 , 7.53 . 13C NMR : δ (ppm) 13.5 , 14.0 , 69.9 , 70.5 , 72.7 , 74.1 , 74.3 , 75.7 , 106.0 , 127.3 , 129.3 , 134.4 , 141.0 , 144.3 , 147.7 , 150.2 . 31P{1H} NMR : δ (ppm) –15.5 (s). 11B NMR : δ (ppm) –7.2 (br s). Anal. calcd: [Zn(μ-OCH2Ph)]2·(THF)2 (C86H96B2Fe2N8O2P2Zn2) C, 63.30; H, 5.93; N, 6.87. Found: C, 63.76; H, 5.87; N, 7.01.To KCHP,B)Zn(μ-OCH2Ph)]2 , outside of the glove-box, was added 5 mL of wet diethyl ether and the mixture was stirred for 30 min at ambient temperature. The solution volume was reduced to 2 mL and yellow solids were collected on a frit and washed with 2 × 1 mL of cold diethyl ether. After drying under a reduced pressure for several hours, the final product was isolated as a yellow powder . 1H NMR : δ (ppm) –0.76 , 2.24 , 2.37 , 3.40 , 3.48 , 3.99 , 4.21 , 4.69 , 5.76 , 7.04 , 7.45 . 13C NMR : δ (ppm) 13.4 , 13.7 , 69.9 , 72.6 , 74.5 , 74.6 , 76.0 , 105.1 , 134.3 , 140.1 , 146.6 , 148.6 . 31P{1H} NMR : δ (ppm) –15.6 (s). 11B NMR : δ (ppm) –6.6 (br s). Anal. calcd: [Zn(μ-OH)]2 (C64H68B2Fe2N8O2P2Zn2) C, 58.80; H, 5.24; N, 8.57. Found: C, 59.28; H, 5.26; N, 8.59.To [Zn(μ-OCH2Ph)]2 (5 μmol), the appropriate amount of monomer, and 0.5 mL of C6D6 were added. The contents of the vial were stirred and the homogeneous solution was transferred to a J. Young NMR tube equipped with a Teflon valve. The NMR tube was sealed, taken out of the box and placed in an oil bath. Monomer consumption was monitored by 1H NMR spectroscopy until the desired product was formed.To a small vial, [Zn(μ-OCH2Ph)]2 (2.5 μmol), an external standard, hexamethylbenzene (25 μmol), the appropriate amount of monomer, and up to 1.5 mL of C6H6 in total were added. The Schlenk flask was taken out of the glovebox and placed in an oil bath at 70 °C. Upon completion of each block, the reaction was cooled to room temperature and brought back into the glovebox for the addition of monomer comprising the next block. Typical reaction times for the complete conversion of 100 equivalents of l-lactide are 3–9 h in 0.5–1.5 mL of C6H6; 50 equivalents of TMC, after lactide, are polymerized over a period of 12–24 h in 0.5–1.5 mL of C6H6. The l-lactide and 1,3-trimethylene carbonate used in each polymerization experiment were distributed evenly across each block. Upon completion of the final block, the contents of the Schlenk flask were diluted with 1 mL of dichloromethane and poured into 15 mL of methanol to yield white solids. The product was collected on a glass frit, washed with additional 10 mL of methanol and kept under reduced pressure at 70 °C until it reached a consistent weight.To a Schlenk flask, sealed with a Teflon screw cap, [Zn(μ-OCH2Ph)]2 ratio.Melt polymerizations were carried out in a Schlenk tube equipped with a Teflon screw cap and a stir bar, at 140 °C, with a 600 : 1 monomer to [Zn(μ-OCH2Ph)]2 and l-lactide were added with 1.6 mL as the final volume of C6H6. The flask was then taken out of the glove box and placed in an oil bath at 70 °C. At the appropriate time intervals the flask was removed from the bath and cooled under a flowing stream of cold water prior to being brought back into the glove box. Inside the box, aliquots were poured into hexanes, dried to a constant weight under reduced pressure, and analyzed by 1H NMR spectroscopy.In a glove box a Schlenk flask, sealed with a Teflon screw cap, equipped with a stir bar an appropriate amount of [. Tables with atomic coordinates and equivalent isotropic displacement parameters, with all the distances and angles, and with anisotropic displacement parameters are listed in the cif .X-ray quality crystals were obtained from various concentrated solutions placed in a –40 °C freezer in the glove box unless otherwise specified. Inside the glove box, the crystals were coated with oil (STP Oil Treatment) on a microscope slide, which was brought outside the glove box. The X-ray data collections were carried out on a Bruker SMART 1000 single crystal X-ray diffractometer using MoK2 were replaced by methyl groups to simplify the calculations. Geometry optimizations were performed with B3LYP.All calculations were carried out with the GAUSSIAN 09.There are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file.


Complexes with copper(ii) include a single chelate and chloride ion(s)/water to satisfy a trigonal bipyramidal coordination sphere. The nickel(ii) and zinc(ii) complexes are composed of two chelating groups that adopt an octahedral geometry around the metal ion. Irreversible redox activity was observed with the copper(ii) complex but no redox activity was observed with the ligand alone or zinc(ii) and nickel(ii) complexes. Use of the coumarin carboxylic acid assay shows that the ligand motif is capable of preventing redox cycling of copper in biological conditions and could thus serve as an antioxidant preventative agent. Cellular toxicity studies show that the new triazine molecule could have therapeutic applications in the μM concentration range based on the measured EC50 = 1.183 ± 0.002 mM. Altogether this work shows that by merging triazine chemistry into inorganic compounds, there is potential to explore a range applications thanks to the new architecture.Interest in inorganic applications of triazines is growing. In this report, metal complexes of copper( ii), nickel(ii), and zinc(ii) is explored.A new triazine bearing a pyrazolone moiety and metalation with first row transition metals, copper( The syntheses of purple complex 7 [Ni-12]2+ and white complex 8 [Zn-12]+ were achieved under similar conditions using 1/2 equivalent of nickel chloride hexahydrate (NiCl2·6H2O) and zinc chloride (ZnCl2), respectively.The arrangement of N- and O-atoms in molecule 1 is reminiscent of metal binding molecules derived from triazine cores reported by Melman.omplex 6 , a mixtuO bond lengths (∼1.3 Å) in complexes 6–8 are slightly longer than a traditional CO bond and the observation is attributed to the interaction of the O-atom with the metal center as well as resonance between the imine and enamine forms shown in Samples of complexes 6–8 suitable for analysis by single crystal X-ray diffraction were obtained by slow evaporation of aqueous solution made from previously isolated powders. The data resulted in the solid states structures shown in . Throughii) complexes (H2O)] and 6b, [Cu-1(Cl)2]) with an overall neutral charge in addition to four lattice H2O molecules (τ) for Cu1 (0.06) and Cu2 (0.08).25 The two copper complexes interact with one another in the unit cell through N–H⋯Cl hydrogen bonds between the hydrazine hydrogen atoms and chloride ligands  pyrazole moiety. This strong interaction reduces the H3B–O3–H3A angle to 103.077° from the typical value of 104.5° expected for a perfect tetrahedron. The O1 of morpholine ring forms a strong hydrogen bond with a H2O molecule, which works as a bridge between O1 and O2 of neighboring  pyrazole moiety. This bifurcated strong hydrogen bonding of O6 plays a bridging role in the molecular assembly by holding the three adjacent molecules together through a network of O–H⋯O hydrogen bonds and is a major contributor of crystal packing. Other important hydrogen bonds in 6 are listed in As shown in 2]2+ complex, two Cl− ions, and three H2O molecules (ii) metal center of 7 is bound by two neutral molecule 1 units and exhibits a distorted octahedral geometry  pyrazole moiety, whereas the pyrazole nitrogen (N16) of the remaining 1 interacts through hydrogen bonding with the Cl− counter ion. The N8 of the hydrazine ring is hydrogen bonded to the solvent H2O molecule, which is further connected to the Cl− counter ion. This hydrogen bonding interaction plays a major role in crystal packing. Furthermore, N10 of hydrazine donates H10 to O1 of neighboring  morpholine at a distance of 1.94 Å and forms a strong hydrogen bonding interaction. The N5 nitrogen of each pyrazole moiety in 7 is protonated, thus yielding a neutral ligand. Two chloride counterions serve as charge balance for complex 7. Like 6 the pyrazole, triazine, and hydrazine rings of each ligand are nearly coplanar: the largest deviation from the Ni–N6–N8 plane is 0.219 Å by C5. In addition, there are various hydrogen bonding interactions present in the crystal structure of 7. The pyrazole nitrogen atom (N16) of one molecule 1 has a strong hydrogen bonding interaction with O2 of the neighboring  metal center of 8 exhibits a distorted octahedral geometry  ion is bound to one neutral molecule 1 and one anionic molecule 1, resulting in an overall +1 charge for the complex. The octahedral geometry of 8 compared to 7 is more distorted. For example, the right angle of the N3–Zn–N16 bond angle of 8 is increased to 103.11° compared to 97.099° in 7 and 90° in a perfect octahedron. The angle formed by the two coordinated triazine nitrogen atoms and the metal center (N12–Zn–N3) is 177.511°. The bite angle formed between the coordinated oxygen, metal center, and nitrogen of hydrazine (O3–Zn–N6) is non-linear at 165.00°, which is larger than the same angle (O2–Zn–N16) in the other ligand by 2.1°. This reduction can be attributed to the involvement of the latter ligand in strong hydrogen bonding with H2O molecules, which is absent in the former. The bond length of the apical hydrazine nitrogen atoms (N8 and N9) with the metal center is larger than the other metal nitrogen bonds. Pertinent bond lengths and bond angles involving the metal center are listed in One ·2H2O. The bands at 204, 228, 267, and 298 nm have been assigned as π → π* transitions and a d → d band was observed at 763 nm; tptz = 2,4,6-tris(2-pyridyl)-1,3,5-triazine.26 The absorbance values of the nickel(ii) complex, 7, are in agreement with Abdi et al.,27 which show absorbance bands at, 204, 257, 293 nm (π → π* transitions) and at 669, 778, 923 nm (d → d transition) for the complex [Ni(tptz)(CH3OH)Cl2]. Likewise, ligand based π → π* transitions derived from complex 8 are observed at 231 (74 819 cm−1 M−1), 239 (59 468 cm−1 M−1), and 256(sh) nm (22 543 cm−1 M−1). The absorbance values of the zinc(ii) complex 8 are comparable with Xian Sun et al.28 whom reported absorbance values of the [Zn2(Bpz*eaT)2(HBTC)2](CH3OH)3 complex at 220 nm, corresponding to the π → π* transition, and two LMCT bands at 260 and 280 nm; Bpz*eaT = 2,4-bis-6-diethylamino-1,3,5-triazine.The UV-visible spectra of molecule 1 and complexes 6, 7, and 8 were obtained in Hshown in  and the ii) complex (6) is shown in  complexes are known to be redox active.29 The lack of redox response observed for complex 8 is consistent with the expected redox inert behaviour of diamagnetic zinc(ii) systems. Altogether, this data shows that molecule 1 readily accommodates the +2 oxidation state of all metal ions evaluated, and that the reduced copper(i) species can only be accessed at potentials more negative than approximately −700 mV. The cyclic voltammetry results demonstrate that molecule 1 inhibits the copper(ii) reduction process known to generate toxic reactive oxygen species in vivo.The redox behavior of complexes 6–8 were studied using cyclic voltammetry in aqueous solution. For comparison, cyclic voltammetry of molecule 1 was also carried out and no redox activity was observed in the window studied (1000 to −1000 mV). Therefore, any redox signals observed in the metal complexes is attributed to the metal-ion within the complex. The cyclic voltammogram of the copper calculations help to interpret the ligand protonation states observed in molecules 1, 6, 7, and 8. †2O. The computed enamine and imine C–O bond lengths are slightly longer than the experimental value for molecule 1, 1.27 Å (based on XRD of 6–8). The experimental values are mid-way between typical CO and C–O bonds and are consistent with decreased CO bonding due to ligand–metal interactions. The third alcohol structure is predicted to be relatively unstable, but is most consistent with the deprotonated ligand seen in 6. Overall, the simulations suggest that all three structures could be experimentally relevant, and that coordination to metal could stabilize entry 3.The results in 20 When unregulated metal ions facilitate this process in vivo, the increased levels of oxidative stress have been implicated in a range of diseases.16,18,21 Therefore, there is an increasing interest in finding ways to mitigate such detrimental pathways.21,30,31 The ability of molecule 1 to bind biologically relevant metal ions prompted further exploration of the function to halt the redox cycling of copper ions. The conversion of oxygen into hydroxyl radicals can be simulated in situ. As shown in ), radical production can be quantified with coumarin carboxylic acid (CCA). This species stoichiometrically converts to the fluorescent hydroxy-CCA species in the presence of the hydroxyl radical generated by copper in the presence of ascorbate and oxygen. The process is halted by the addition of molecule 1, presumably by chelation of the copper. As shown in ], 1/2 equivalent of molecule 1 (vs. copper(ii)) results in a marked decrease in fluorescence. A full equivalent of molecule 1 (]), completely inhibits the conversion of CCA to the fluorescent species. The copper(ii): molecule 1 interaction shifts the CuII/I potential outside the accessible range of reduction by ascorbate.20,32 This result is consistent with the cyclic voltammetry data showing very negative CuII/I redox potentials and the characterization of molecule 1 to bind copper(ii) ions. Altogether this assay shows that the metal binding reactivity of molecule 1 has potential as a therapeutic for metal ion misregulation.In aerobic conditions and in the presence of ascorbate, copper can serve as a catalyst to produce hydroxyl radical ions.18,21,33–41 The MTT assay  was employed to evaluate the toxicity of molecule 1 in HEK-293 cells. 50 = 1.183 ± 0.002 mM. Therapeutic windows are typically expected to be an order of 102–103 lower than EC50 toxicities. Therefore, future potential therapeutic applications of molecule 1 would focus on activities achievable within the μM range. Such studies are further encouraged by the capacity for molecule 1 to shift the redox couple of copper(ii) to more negative potential values, thus potentially avoiding redox chemistry known to produce toxic reactive oxygen species in vivo. Future studies would focus on the capacity of molecule 1 to prevent oxidative stress in biological conditions through the molecule's ability to bind to redox active transition metal ions.A preliminary assessment of toxicity was carried out as there are an ever growing number of diseases known to arise from metal ion mis-regulation and molecule 1 shows the capacity to bind biologically relevant transition metal ions.ii), nickel(ii), and zinc(ii). The solid-state structure of 6 shows that 1 serves as a tridentate ligand to a square pyramidal copper(ii) center and the remainder of the coordination sphere is completed by counter ions or solvent molecules. Unlike the copper complex, the coordination spheres of 7 and 8 are complete with two molecules of 1 in an octahedral geometry. Previous studies have shown that incorporating a pyrazole moiety into a triazine based ligand stabilizes the formation of a 5,5,5 ring system in which metal coordination is achieved via a nitrogen atom on the pyrazole. Herein, the ring size has been tuned by functionalizing the pyrazole with a keto group; the asymmetric molecule 1 engenders the formation of a very stable 6,5,5 ring system in which the newly added keto group binds the metal ion. Cyclic voltammetry studies show that the ligand and complexes 7 and 8 are redox inert, but studies of 6 show an irreversible CuII/I event. Cell toxicity data suggests that future work with these types of molecules could include investigating their potential as therapeutics for metal ion mis-regulation as the production of oxidative stress and disease. This represents the first report of this new type of ligand and its coordination chemistry. The results can be used to further explore novel triazine based ligands and their potential applications.In this work, we have successfully prepared a triazine bearing a pyrazolone moiety and explored metalation with first row transition metals, copper, D2O (4.80 ppm), and DMSO-d6 (2.55 ppm). 13C NMR chemical shifts were referenced to CDCl3 (77.23 ppm), and DMSO-d6 (39.52 ppm). The following abbreviations were used to describe multiplets: s (singlet), t (triplet), m (multiplet), and br (broad). The following format was used to report peaks: chemical shift in ppm . Infrared (IR) spectra were recorded on a Jasco FT/IR-4600 and Bruker Alpha FT/IR in the 4000–400 cm−1 range. High-resolution mass spectra (HR-MS) were recorded on an Agilent Technologies 6224 TOF LC/MS system. UV-vis spectra were recorded in a 3 mL quartz cuvette with a path length of 1.0 cm on a 8453 Agilent spectrophotometer between 190 and 1100 nm. The crystalline materials used for XRD analysis were also used for elemental analysis and spectroscopic characterizations.All reagents and solvents were obtained from commercial sources and used as received, unless noted otherwise. Elemental analyses on were performed by Canadian Microanalytical Services Ltd. NMR spectra were recorded on a Bruker Avance III HD 400 MHz spectrometer. 16 To a stirred solution of cyanuric chloride  in acetone (100 mL), a solution of morpholine  and triethylamine  in acetone (100 mL) was added dropwise at 0 °C for 1 h, then quenched with H2O, stirred for 5 minutes, filtered, washed with a cold 1 : 1 mixture of H2O/MeOH and dried under vacuum to yield 2 as a white powder . 1H NMR : δ 3.83 , 3.69 . 13C NMR : δ 170.3, 164.0, 66.3, 44.4. HR-MS (ESI) m/z calcd for C7H9N4Cl2 [M + H]+ 235.0153; found 235.0161.The procedure follows a known synthesis.tert-butyl carbazate  and N,N-diisopropylethylamine  was added. The reaction was stirred for 12 h at room temperature. The solvent was removed by rotary evaporation under reduced pressure and extracted with H2O (100 mL) and DCM (100 mL × 2). The organic layer was collected. Following concentration, the crude product was purified by flash column chromatography (hexanes: EtOAc 2 : 1) to afford compound 3  as white solid. 1H NMR : δ 3.85–3.74 , 3.70–3.63 , 1.44 . 13C NMR : δ 169.3, 167.2, 164.4, 155.4, 81.6, 66.6, 44.0, 28.2. HR-MS (ESI) m/z calcd for C12H20N4O3Cl [M + H]+ 331.1285; found 331.1293.To a stirred solution of 2  in THF (100 mL) 2O. Azeotropy with toluene (50 mL × 2) under reduced pressure by rotary evaporation afforded compound 4  as white solid. 1H NMR : δ 4.57 , 3.75–3.63 , 3.61–3.54 , 1.39 . 13C NMR : δ 168.2, 167.8, 164.9, 156.4, 79.1, 66.5, 43.6, 28.6. HR-MS (ESI) m/z calcd for C12H23N8O3 [M + H]+ 327.1893; found 327.1971.To a stirred solution of 3  in 1,4-dioxane (15 mL) was added hydrazine . The reaction was stirred in a CEM 300 W microwave under dynamic mode at 90 °C for 30 min. The solvent was removed by rotary evaporation under reduced pressure and filtered with HN-methylacetoacetamide . The reaction was stirred at 90 °C for 4 h. The solvent was removed by rotary evaporation under reduced pressure and extracted with H2O (50 mL) and DCM (50 mL × 2). The organic layer was collected and upon concentration, the crude product was purified by flash column chromatography (DCM: MeOH 9 : 1) to afford compound 5  as white solid. 1H NMR : δ 5.54 , 3.80–3.59 , 2.24 . 13C NMR : δ 165.8, 163.4, 162.0, 157.7, 156.1, 153.7, 88.7, 81.5, 66.4, 43.7, 28.2, 14.8. HR-MS (ESI) m/z calcd for C16H25N8O4 [M + H]+ 393.1999; found 393.2760.In a sealed tube, 4  was dissolved in 95/5 THF/pyridine (10 mL) followed by the addition of Lawesson's reagent  and −1): 1638 (CO), 1562, 1574, 1431, 1380, 1301, 1269, 794. 1H NMR : δ 5.54 , 3.80–3.56 , 2.24 . 13C NMR : δ 164.2, 152.5, 90.6, 66.4, 44.4. 1H NMR : δ 3.85–3.45 , 2.16 . 13C NMR : δ 164.3, 163.3, 163.0, 162.1, 154.2, 90.6, 66.1, 44.2, 43.9, 11.6. HR-MS (ESI) m/z calcd for C11H17N8O2 [M + H]+ 293.1474; found 293.1957.To a stirred solution of 5  in MeOH (10 mL) excess conc. HCl (5 mL) was added. The reaction was stirred for 12 h at room temperature. The solvent was removed under reduced pressure and triturated with DCM. Following tituration with DCM compound 1 was isolated in quantitative yields as white solid. Selected IR bands  dissolved in EtOH (3 mL). The reaction was stirred for 12 h at room temperature, filtered, and washed with EtOH (20 mL) to afford compound 6  as a green powder. Single crystals suitable for XRD analysis were grown by the slow evaporation of H2O. Selected IR bands (cm−1): 3211, 3129, 1655 (CO), 1638 (CO), 1613, 1583, 1550, 1496, 1448, 1422, 1348, 1306, 1289, 1174, 1014, 784, 763, 620. HR-MS (ESI) m/z calcd for C11H16ClCuN8O2 [M + H]+ 390.0381; found 390.0937. HR-MS (ESI) m/z calcd for C11H15CuN8O2 [M + H]+ 354.0614; found 354.1092. Anal. calcd for C22H31Cl3Cu2N16O5·4H2O: C, 29.13; H, 4.56; N, 24.71. Found C, 29.20; H, 4.01; N, 24.34.To a stirred solution of 1  in EtOH (5 mL) was added CuCl2 (H2O)6  dissolved in EtOH (3 mL). The reaction was stirred for 12 h at room temperature, filtered, and washed with EtOH (20 mL) to afford compound 7  as purple powder. Single crystals suitable for XRD analysis were grown by the slow evaporation of H2O. Selected IR bands (cm−1): 3130, 1636 (CO), 1576, 1507, 1425, 1284, 1179, 1113, 1061, 1016, 856, 793, 762, 578. HR-MS (ESI) m/z calcd for C22H31N16NiO4 [M + H]+ 641.2068; found 641.3206. Anal. calcd. for C22H32Cl2NiN16O4·7HCl·5H2O: C, 24.94; H, 4.66; N, 21.15. Found C, 24.80; H, 4.73; N, 20.98.To a stirred solution of 1  in EtOH (5 mL) was added NiCl2  dissolved in EtOH (3 mL). The reaction was stirred for 12 h at room temperature, filtered, and washed with a 3 : 1 mixture of acetone: EtOH (20 mL) to afford compound 8 as a white powder . Single crystals suitable for XRD analysis were grown by the slow evaporation of H2O. δ 9.95 , 5.31 , 3.80–3.59 , 2.27 . Selected IR bands (cm−1): 3180, 3132, 1645 (CO), 1612, 1581, 1556, 1513, 1440, 1430, 1406, 1345, 1300, 1287, 1179, 1098, 1061, 1012, 791, 625.1H NMR : δ 5.32 , 3.80–3.60 , 2.27 . HR-MS (ESI) m/z calcd for C22H31N16ZnO4 [M + H]+ 647.2006; found 647.3107, calcd for C11H16ClN8ZnO2 [M + H]+ 391.0376; found 391.0919. Anal. calcd for C26H31Cl ZnN16O4·4HCl·5EtOH: C, 36.24; H, 6.18; N, 21.13. Found C, 36.40; H, 5.79; N, 21.43.To a stirred solution of 1 in EtOH (5 mL) was added ZnCl42 X-ray intensity data were collected on a Bruker D8 Quest diffractometer equipped with a Photon 100 CMOS detector and generator operating at 50 kV and 30 A. For each crystal, an exposure time of 20 seconds was used with a scan angle of 0.5° per frame. The indexing of Bragg intensities was carried out with APEX3 package.43 Data reduction and absorption corrections were performed with the SAINT44 and SADABS45 software packages, respectively. Structures were solved by the direct method using the SHELXL-97  of both parts were constrained to be equal. Due to limited quantity of diffraction data, the positions of two hydrogen atoms bond to O10 were not ascertained. The disorder of the morpholine ring in 7, was observed as residual electron density oriented in approximately a mirror to the major occupancy components. All disordered atoms in 7 including one Cl− counter ion and one water molecule were refined using aforementioned ADP constraint except O6A, O6B, C19A, C19B, C21A and C21B which were refined isotropically. The occupancies of the two components were refined, yielding an approximately 0.538 : 0.462 ratio. The similar procedure was adopted to model the disordered water molecules in 8. The water molecule associated with O5 in 8 is working as a bridge between Cl− counter ion and N16 of the hydrazine ring through hydrogen bonding. Owning to the importance of this long range interaction in crystal packing, O5–H5B distance and H5A-O5-H5B angle were restrained to 0.982 Å and 104.414°, respectively. The disordered water molecules were refined using equal ADPs constraints and the occupancy ratio of the disordered parts was refined to 0.496 : 0.503. The details of data collection, and statistics of structure refinement are summarized Table S1.†The single crystals of 6, 7 and 8 were grown from aqueous solution by slow evaporation at room temperature. The single crystal of each compound, from available crystals, was mounted on the goniometer using Paratone-N oil (cryoprotectant) on the tip of MiTeGen MicroLoops LD™, 45 mm away from the detector and was cooled to a temperature of 100(1) K under the flow of liquid nitrogen using Oxford Cryosystem.HELXL-97  software48,49 The origin of these densities can be attributed to various factors,49 but in the present structures, it can be attributed to the additional disorders especially in 8. Initially, two occupancies were refined freely to identify possible site pairings, but the modelling of multiple occupancies of these disorders was not completely possible with available data. These disorders and residual densities are associated with outer-sphere, non-coordinating chloride counter ions and water molecules only, which ensures the structural accuracy of main coordination moiety.For each modelled structure, larger than desired residual electron density peaks remain after refinement of the data, which is typical for this class of compounds.2 at room temperature was used to obtain all voltammograms. All experiments were carried out in aqueous solution with 0.1 M KCl as the supporting electrolyte and were referenced to Ag/AgCl.A Basi C3 cell stand with a glassy carbon working electrode, Ag/AgCl reference electrode, and platinum wire auxiliary electrode were used to conduct the electrochemical analysis of all compounds. A standard three electrode cell under a blanket of N50 and the correlation functional of Lee, Yang, and Parr (LYP)37)51 (B3LYP) as implemented in Gaussian 09.52 For each calculation, all atoms were optimized via the use of the 6-311g basis set with polarization. A frequency calculation was performed alongside each geometry optimization to ensure the stability of the ground state as ascertained by the absence of imaginary frequencies. Coordinates for each structure were derived from XRD data in complexes 6–8. Optimized structure figures were generated by use of the GaussView program.53DFT calculations were performed using a hybrid functional 2PO4/NaCl [1.5 mM] buffer containing desferryl [1 μM].20,32 except Cu(NO3)2, which was dissolved and diluted in Milli-Q water. Final sample volume = 3 mL. Each experiment was performed in triplicate. Hydroxyl radical production was followed measuring the conversion of CCA into 7-hydroxy-CCA  nm. General order of addition: CCA [500μM], molecule 1 , or copper [40 μM], then ascorbate [300μM].All solutions were prepared in KH−1)/streptomycin (0.1 mg mL−1) (Sigma), 2 mM glutamine (Sigma) and MEM non-essential amino acids (1×) (Sigma), at 37C, 5% CO2, 95% air.Human Embryonic Kidney cells (293HEK) cells (ATCC) were grown in Dulbecco's Modified Eagle's Medium-high glucose (DMEM) supplemented in 10% fetal bovine serum (Sigma) and penicillin . Results are presented as the average of 8 replicates per concentration of drug. GraphPad Prism was used to calculate EC50 values.Cytotoxicity studies were carried out using the MTT assay. Briefly, cells were plated at a density of 5000 cells per well in a 96 well tray. Following an overnight incubation, the cells were treated with the indicated concentrations of drug and further incubated for 16 hours under normal growing conditions. Following this, the medium and drug were removed and 100 μL per well of MTT  was added at a concentration of 1 mg mLThe manuscript was written through contributions of all authors. AY performed the synthesis and coordinated data collection. AM performed the crystallographic analysis. SB performed the titrations for metalation. GA performed cellular assays. KG and MB performed CCA assays. The work was supervised by EES and KNG. All authors have given approval to the final version of the manuscript.There are no conflicts of interest to declare.RA-008-C7RA09459K-s001RA-008-C7RA09459K-s002RA-008-C7RA09459K-s003RA-008-C7RA09459K-s004RA-008-C7RA09459K-s005