Vision loss in patients with wet/exudative age-related macular degeneration (AMD) is associated with choroidal neovascularization (CNV), and AMD is the leading cause of irreversible vision impairment in older adults. Interleukin-17A (IL-17A) is a component of the microenvironment associated with some autoimmune diseases. Previous studies have indicated that wet AMD patients have elevated serum IL-17A levels. However, the effect of IL-17A on AMD progression needs to be better understood. We aimed to investigate the role of IL-17A in a laser-induced CNV mouse model.We established a laser-induced CNV mouse model in wild-type (WT) and IL-17A-deficient mice and then evaluated the disease severity of these mice by using fluorescence angiography. We performed enzyme-linked immunosorbent assay (ELISA) and fluorescence-activated cell sorting (FACS) to analyze the levels of IL-17A and to investigate the immune cell populations in the eyes of WT and IL-17A-deficient mice. We used ARPE-19 cells to clarify the effect of IL-17A under oxidative stress.+CD4+RORγt+ T cells, Treg cells, and neutrophils were decreased and the number of macrophages was increased in the eyes of IL-17A-deficient mice compared with WT mice. In WT mice, IL-17A-producing γδ T-cell numbers increased in a time-dependent manner from day 7 to 28 after laser injury. IL-6 levels increased and IL-10, IL-24, IL-17F, and GM-CSF levels decreased in the eyes of IL-17A-deficient mice after laser injury. In vitro, IL-17A inhibited apoptosis and induced the expression of the antioxidant protein HO-1 in ARPE-19 cells under oxidative stress conditions. IL-17A facilitated the repair of oxidative stress-induced barrier dysfunction in ARPE-19 cells.In the laser-induced CNV model, the CNV lesions were larger in IL-17A-deficient mice than in WT mice. The numbers of γδ T cells, CD3Our findings provide new insight into the protective effect of IL-17A in a laser-induced CNV model and reveal a novel regulatory role of IL-17A-producing γδ T cells in the ocular microenvironment in wet AMD.The online version contains supplementary material available at 10.1186/s12974-023-02952-1. Age-related macular degeneration (AMD) is a retinal disease that causes damage to the macula and leads to loss of central vision. AMD is usually frequent in elderly people, and there are two types of macular degeneration associated with age: dry (atrophic) and wet (exudative) AMD . Most AMThe clinical efficacy of intravitreal anti-VEGF drugs has been widely demonstrated in wet AMD . HoweverReactive oxygen species (ROS) formation is a byproduct of the normal metabolism of oxygen and plays important roles in cell signaling and homeostasis . ROS levCytokines are critical mediators of communication for the immune system. TNF-α stimulates the expression of proangiogenic VEGF  and faciIL-17A is a pleiotropic cytokine and is mainly produced by T helper 17 cells (Th17 cells), type three innate lymphoid cells (ILC3s), and gamma delta T cells (γδ T cells). IL-17A binds to a heterodimeric receptor complex, which consists of IL-17RA and IL-17RC, to activate the MAPK and NF-κB signaling pathways and further induce the production of chemokines. These chemokines further recruit immune cells, such as monocytes and neutrophils, to sites of local inflammation. Therefore, IL-17A is critical for host defense against microbes because it promotes the recruitment of neutrophils and induces the production of antimicrobial peptides –22. IL-1There is much evidence indicating that AMD is associated with the immune system, which includes complement, macrophages, and T cells , 25. Th1Previous studies reported that serum IL-17A levels were elevated in patients with AMD , 35. HowWild-type (WT) C57BL/6J mice were purchased from the National Laboratory Animal Center . IL-17A-deficient mice on a C57BL/6J genetic background were maintained in the animal center of National Cheng Kung University. Both IL-17A-deficient and WT mice (6–8 weeks old) were used and kept on a 12-h light–dark cycle at 22 ± 2 °C. The research procedures were approved by the Animal Ethics Committee of National Cheng Kung University . The methods were carried out in accordance with the approved guidelines.Mice were anesthetized by intraperitoneal injection of Zoletil  and Rompun , and the pupils were dilated with one drop of 1% tropicamide ophthalmic solution . After anesthesia and dilation, CNV was induced with a 532-nm laser photocoagulator  with a microscopic delivery system . In all eyes, four spots were applied around the optic disc and between retinal vessels four times. A bubble formed immediately at the site of laser application, indicating successful rupture of Bruch’s membrane. Spots with subretinal hemorrhage following laser photocoagulation were excluded. Fundus fluorescein angiography (FFA) was used to evaluate CNV leakage by performing with the retinal imaging microscopy  7, 14, 21, and 28 days after laser photocoagulation. Mice were anesthetized, and their pupils were dilated and intraperitoneally injected with 2% sodium fluorescein  at 20 μg/gram body weight. The FFA images were taken with a retinal imaging microscope at 5 min after fluorescein injection. The fluorescein leakage area of each lesion was contoured and evaluated quantitatively with ImageJ software .To isolate the immune cells in the retina and choroid, the tip of angled scissors was inserted between the skin and the eyeball, and then the mouse eyes were enucleated. To avoid contamination, the eye was briefly dipped in 70% ethanol and washed in PBS. The anterior segment  was removed. Both eyes were pooled for each animal and enzymatically digested in DMEM-F12 serum-free medium  supplemented with 2 mg/ml dispase II (Sigma‒Aldrich) at 37 °C for 40 min. The posterior segment of the eyes, including the sclera, choroid, and retina, was then disrupted in the digestion medium. The sample was filtered with a 70 μm cell strainer  and then centrifuged at 4 °C (1800 rpm/610 g for 5 min). The supernatant was removed, and the cells were resuspended in medium for further flow cytometry analysis.2 at 37 °C. Cells were stained with surface marker antibodies including APC-Cy7-conjugated anti-CD45 (BD Pharmingen™) and PE-conjugated anti-CD4 (BD Pharmingen™). Then, the cells were fixed and permeabilized for 30 min at 4 °C using BD Cytofix/Cytoperm buffer (BD Biosciences), washed twice with BD Perm/Wash buffer and then stained with Alexa 488-conjugated anti-IL-17A (BD Pharmingen™) in the dark for 30 min at 4 °C. The cell samples were acquired using a BD FACS Canto II Flow Cytometer (BD Biosciences) using BDFACS DIVA software (BD Biosciences) and analyzed with FlowJo software .The cells were suspended in FACS buffer  to produce a single-cell suspension. Myeloid cells were stained with surface marker antibodies, including BV 421-conjugated anti-CD45 , 7-AAD-conjugated anti-CD11b (BD Pharmingen™), Alexa 647-conjugated anti-F4/80 , Alexa 488-conjugated anti-Ly6G (BD Pharmingen™), and Alexa 647-conjugated anti-Ly6C . Lymphoid cells were stained with surface marker antibodies including PE-conjugated anti-CD45 (BD Pharmingen™), BV 421-conjugated anti-TCRγδ (BD Pharmingen™), and Alexa 488-conjugated anti-CD4 (BioLegend), in the dark for 30 min at 4 °C. For transcription factor staining, cells were fixed and permeabilized for 30 min at 4 °C using BD Cytofix/Cytoperm buffer , washed twice with BD Perm/Wash buffer and then stained with transcription factor marker antibodies, including 7-AAD-conjugated RORγt (BD Pharmingen™) and Alexa 647-conjugated Foxp3 (BioLegend), in the dark for 30 min at 4 °C. For intracellular cytokine staining, cells isolated from the retina were activated with 20 ng/ml PMA (Sigma‒Aldrich), 1 μM ionomycin (Sigma‒Aldrich), and BFA  and cultured for 5 h in 5% CO® Gold antifade reagent with DAPI . Images were taken using a scanning confocal laser microscope  to visualize the stained cells.Eyes from mice were harvested and fixed in neutral buffered 4% paraformaldehyde. After paraffin embedding, retinal cross-sections were cut perpendicularly (5 μm). Hematoxylin and eosin (H&E) staining and immunofluorescence staining were performed following standard protocols. For immunofluorescence staining, the primary antibodies used were anti-IL-17A  and anti-ZO-1 . Isotype-matched control antibodies were used as a negative control. The samples were washed with PBS and stained with fluorescence-conjugated secondary antibody at room temperature for 1 h. The slides were mounted with ProLongIL-17A concentrations in serum from mice with laser-induced CNV and healthy mice were measured using a mouse IL-17A ELISA Kit  according to the manufacturer’s instructions. To analyze dynamic changes in cytokine levels in healthy mice and mice with laser-induced CNV on day 7, day 14, day 21, and day 28, serum samples were collected and evaluated using a customized LEGENDplex™ kit according to the manufacturer’s instructions (BioLegend). The bead-based multiplex assay panel allowed simultaneous quantification of IL-17F, IL-17A, IL-22, IL-23, TNF-α, IFN-γ, IL-5, IL-13, and IL-6 using a sandwich immunoassay. Data were acquired on a BD FACSCanto II (BD Biosciences). The data were analyzed using BioLegend’s LEGENDplex™ data analysis software.–ΔΔCt. All sequences of primers used in this study are listed in Additional file Total RNA was extracted from total mouse ocular cells or cultured cells with QIAzol Lysis Reagent  according to the manufacturer’s instructions. RNA samples 1 μg) were then subsequently used for reverse transcription using the PrimeScript™ RT Reagent Kit  , and the cDNA samples were further diluted 10 times in diethylpyrocarbonate (DEPC)-treated water. cDNA was then amplified on a thermocycler  with gene-specific primers. The PCR mixture contained 2 µl of diluted cDNA samples, 5 µl of SYBR Green Master Mix (QIAGEN), 1.6 μl of RNase-free water (QIAGEN), and 0.7 µl of each primer at 10 pmol/µl in a final reaction volume of 10 μl. The amplification conditions were as follows: activation at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 10 sec and extension at 60 °C for 20 sec. mRNA quantification analysis results were normalized to β-actin or GAPDH, which was used as the internal control. Relative fold changes in mRNA expression were determined by calculating 2 μg were 2.The ARPE-19 human retinal pigment epithelial cell line was obtained from the American Type Culture Collection (ATCC). ARPE-19 cells were cultured in a 1:1 mixture of DMEM/Ham’s F-12 medium with L-glutamine and 15 mM HEPES (GeneDireX) and were supplemented with 2.48 g/L sodium bicarbonate , 10% FBS , and 1% antibiotic–antimycotic (GeneDireX). Cells were incubated at 37 °C with 95% air and 5% CO2O2  for 24 h. After treatment, the cells were incubated with 3--2,5-diphenyltetrazolium bromide  at a final concentration of 0.2 mg/ml for 3 h. The MTT solution was removed, and 150 μl of DMSO (Sigma‒Aldrich) was added to each well. The optical densities at 570 nm were read on a microplate spectrophotometer.ARPE-19 cells were cultured in 96-well plates and incubated in DMEM/F12 containing IL-17A  or 300 μM H4 cells/well and then treated with 300 μM H2O2 (Honeywell Fluka™) and IL-17A  in DMEM/F12 with 2% FBS for 24 h. Cells were harvested and stained with a FITC Annexin V apoptosis detection kit  according to the manufacturer’s instructions. The cells were subjected to apoptosis analysis using a BD FACSCalibur (BD Biosciences).ARPE-19 cells were grown overnight in a 12-well plate at a density of 8 × 104 cells/well overnight. Cells were cultured in DMEM/F12 with 1% FBS for 7 days until they reached the appropriate cell confluence. The cells were then treated with 300 μM H2O2 and IL-17A (100 ng/ml) for 16 h. After treatment, 10 kDa FITC-dextran  was added to the upper chamber of the Transwell, and then collected 50 μl aliquots from the lower chamber at 10–16 h to detect and measure the fluorescence signal at 485/538 nm excitation/emission wavelengths using a luminometer.ARPE-19 cells were seeded on 24-mm diameter, 0.4-μm pore size, polyester Transwell filters  at a density of 1 × 10Mouse ocular tissues or ARPE-19 cells were lysed with commercial Cell Lysis Buffer . Proteins were separated by SDS‒PAGE and transferred electrophoretically to 0.45 μm PVDF membranes . The membranes were blocked with 5% (w/v) nonfat milk in TBST for 1 h at room temperature and then incubated overnight at 4 °C with primary antibodies: anti-NQO-1 , anti-HO-1 , anti-Nrf2 , anti-GAPDH  and anti-tubulin . After binding of primary antibodies, the membranes were washed four times with TBST and incubated for 1 h at room temperature with species-specific horseradish peroxidase-labeled secondary antibodies. After binding of secondary antibodies, the membranes were washed five times with TBST. The binding of secondary antibodies was detected with SuperSignal West Pico Chemiluminescent Substrate (Millipore), and the chemiluminescence signals were visualized and imaged on a luminescence imaging system following exposure and development of Hyperfilm ECL molecules (Invitrogen).Prism 9.0  was used for the statistical analysis. GraphPad Prism software was employed to process the initial data and plot graphs. Normality tests were performed before statistical tests. In this study, the data were normally distributed, and variances were homogeneous in all groups; parametric analysis (t test for the comparison of two groups or one-way ANOVA with Tukey's multiple comparisons test for the comparison of three or more groups) was used, and data are presented as the mean ± SEM. Two-way ANOVA with Sidak's multiple comparisons test was used to analyze multiple groups. The specific test that was used is indicated in the figure legends. P < 0.05 was considered to indicate statistical significance.A previous study  reported+CD4+RORγt+ T cells, Treg cells (CD3+CD4+Foxp3+), and γδ T cells (CD3+TCRγδ+). FACS analysis showed no significant differences in these immune cell populations in the cervical lymph nodes of WT and IL-17A-deficient mice after laser injury , MDSCs (CD11b+Ly6G−Ly6Chi), and neutrophils (CD11b+Ly6G+Ly6C+) in WT and IL-17A-deficient mice after laser injury. FACS analysis showed that IL-17A deficiency reduced neutrophil recruitment but increased macrophage infiltration into the eyes at 28 days after laser injury  and γδ T cells (CD3+TCRγδ+IL-17A+) were the two major cellular sources of IL-17A in the eyes of mice with laser-induced CNV , anti-inflammatory cytokines (IL-10 and TGF-β), and Th17-associated cytokines . There was no significant change in TNF-α, IL-1β, and TGF-β expression between IL-17A-deficient and WT mice Fig. C–E. IL-12O2 to test the protective effect of IL-17A on H2O2-induced cytotoxicity, the viability of ARPE-19 cells was examined after H2O2 (0–2 mM) exposure for 24 h. We found that the IC50 value of H2O2 after stimulation for 24 h was 300 μM (data not shown). Therefore, we selected this experimental condition for the following experiments. Next, ARPE-19 cells were cotreated with IL-17A and H2O2, and then cell viability was analyzed. MTT assays showed that IL-17A did not affect ARPE-19 cell viability during exposure to H2O2-induced oxidative stress  formed by the tight junctions between RPE cells is critical for maintaining retinal homeostasis. The integrity of the outer BRB prevents choroidal neovascularization from invading the retina , 44. Theoup Fig. A. We obsyer Fig. A. Additioup Fig. B. To furoup Fig. C–G. Thes+CD4+RORγt+ T cells, γδ T cells, macrophages, and neutrophils and affected the expression of ocular proinflammatory molecules. IL-17A inhibited cell apoptosis and induced the expression of the antioxidant protein HO-1 in ARPE-19 cells under oxidative stress. IL-17A has the ability to repair oxidative stress-induced barrier dysfunction in ARPE-19 cells. These findings suggest a protective effect of IL-17A in the laser-induced CNV model.Oxidative stress is a critical inducer of RPE cell dysregulation in wet AMD; this process causes RPE barrier dysfunction and might result from disruption of intercellular tight junctions. We demonstrated that IL-17A deficiency exacerbated disease severity in a laser-induced CNV mouse model. IL-17A deficiency influenced ocular Treg cells, CD3To confirm the elevated serum IL-17A levels in AMD patients, we established a laser-induced CNV mouse model and found that IL-17A was expressed mainly in CNV lesions. We did not observe a significant change in serum IL-17A levels in mice after laser injury. However, we found that the number of IL-17A-expressing cells in the eyes increased after laser injury. IL-17A deficiency influenced Treg, Th17, and γδ T-cell populations in the eyes but not in the cervical lymph nodes. In addition, IL-17A deficiency decreased neutrophil infiltration and increased macrophage infiltration into the retina. Therefore, the laser-induced CNV mouse model might involve a local inflammatory response and disruption of the ocular microenvironment but not the systemic microenvironment 28 days after laser injury. In addition, we analyzed the dynamic changes in the proportions of IL-17A-expressing immune cells and observed that the number of γδ T cells significantly increased from day 7 to 28 after laser injury; this change occurred in a time-dependent manner. These data suggested that IL-17A-secreting γδ T cells might play a critical role in maintaining ocular homeostasis at later time points after laser-induced injury.Previous studies –49 reporClinical trials targeting IL-17A in rheumatoid arthritis  and uveiPrevious studies , 56, 57 A previous study  reportedIL-10 was upregulated in ocular macrophages of aged mice after laser injury. In addition, IL-10 regulates macrophage function and promotes angiogenesis . IL-6 waTh17 cells, ILC3s, and γδ T cells mainly produce IL-17A. Neutrophils and macrophages also secrete IL-17A. In addition, a previous study  reportedIL-17A acts as a pathogenic cytokine in a variety of inflammatory diseases, including multiple sclerosis , 27, infPrevious studies reported that IL-17A promoted VEGF production in corneal neovascularization , osteoarIntravitreal injections of anti-VEGF agents have become a first-line treatment for wet AMD. However, some patients still have persistent fluid or recurrent exudation, which is called refractory neovascular AMD . A previIn summary, our findings demonstrate a possible effect of IL-17A-producing γδ T cells on the progression of laser-induced CNV in mice. IL-17A can exert a protective effect against laser-induced CNV injury. The underlying mechanism is that IL-17A induces the expression of the antioxidant protein HO-1, which might inhibit RPE cell apoptosis and prevent redundant macrophage infiltration into the retina and the subsequent generation of an excessive immune response. IL-17A repaired oxidative stress-induced barrier dysfunction. Our findings provide insight into the protective mechanisms of IL-17A in the ocular microenvironment, which may provide a new direction for understanding wet AMD.Additional file 1: Figure S1. Expression of VEGFA in the laser-induced CNV mouse model. A Immunofluorescence staining for VEGFA expression in ocular tissue in WT and IL-17A−/− mice 28 days after laser injury (N = 4). Magnification: 200X. B, C The ocular tissues from WT and IL-17A−/− mice 28 days after laser injury (N = 3) were collected and analyzed using immunoblotting with specific antibodies against VEGFA. GAPDH was used as an internal control. D The mRNA expression of VEGFA in eyes isolated from WT and IL-17A−/− mice (N = 6) 28 days after laser injury was analyzed using RT‒qPCR with specific primers. Statistical differences were determined by two-way ANOVA and Sidak’s multiple comparisons test. Data are presented as the mean ± SEM . Figure S2. Analysis of CD3+CD4+RORγt+ T cells, γδ T cells, and Treg cells in cervical lymph nodes of mice in a laser-induced CNV mouse model. A–C Representative flow cytometry data of CD3+CD4+RORγt+ T cells, γδ T cells, and Treg cells in cervical lymph nodes of WT and IL-17A−/− mice at 28 days after laser injury. N = 5 mice per group. Data are presented as the mean ± SEM. The data shown are representative of three independent experiments with similar results. Figure S3. Gating strategy for flow cytometry analysis for Fig. + population. These were then further gated for the subsets of interest, namely, CD3+CD4+ T cells, CD3+CD4+RORγt+ T cells, Treg cells (CD3+CD4+Foxp3+), γδ T cells (CD3+TCRγδ+), macrophages (CD11b+F4/80+), MDSCs (CD11b+Ly6G−Ly6Chi), and neutrophils (CD11b+Ly6G+Ly6C+). Data were analyzed using FlowJo software, and population frequencies were expressed as percentages of the CD45+ parent population. Figure S4. Gating strategy for flow cytometry analysis for Fig. + population. These were then further gated for the subsets of interest, namely, IL-17A+ immune cells (IL-17A+CD45+), IL-17A+ nonimmune cells (IL-17A+CD45−), CD3+IL-17A+ T cells, CD3+CD4+IL-17A+ T cells, CD3+CD8+IL-17A+ T cells, γδ T cells (CD3+TCRγδ+IL-17A+), Th17 cells (CD3+CD4+RORγt+IL-17A+), and Treg cells (CD3+CD4+Foxp3+IL-17A+). Data were analyzed using FlowJo software, and population frequencies were expressed as percentages of the IL-17A+ parent population. Figure S5. Dynamic changes in systemic cytokine levels in a laser-induced CNV mouse model. Serum samples were collected from healthy mice and WT and IL-17A−/− mice with laser-induced CNV (N = 5) at 7 days, 14 days, 21 days, and 28 days after laser injury. A–I The cytokine levels of IL-17F, IL-17A, IL-22, IL-23, TNF-α, IFN-γ, IL-5, IL-13, and IL-6 were measured by LEGENDplex™. Statistical differences were determined by two-way ANOVA and Sidak's multiple comparisons test. Data are presented as the mean ± SEM . The data shown are representative of two independent experiments with similar results. Table S1. List of primers sequences used in this study.


Pulmonary hypertension (PH) is a lethal vascular disease with limited therapeutic options. The mechanistic connections between alveolar hypoxia and PH are not well understood. The aim of this study was to investigate the role of mitotic regulator Polo-like kinase 1 (PLK1) in PH development.Plk1 heterozygous knockout mice and PLK1 inhibitors (BI 2536 and BI 6727)-treated mice were checked for the significance of PLK1 in the development of PH.Mouse lungs along with human pulmonary arterial smooth muscle cells and endothelial cells were used to investigate the effects of hypoxia on PLK1. Hypoxia- or Sugen5416/hypoxia was applied to induce PH in mice. Plk1 were partially resistant to hypoxia-induced PH. PLK1 inhibitors ameliorated PH in mice.Hypoxia stimulated PLK1 expression through induction of HIF1α and RELA. Mice with heterozygous deletion of Augmented PLK1 is essential for the development of PH and is a druggable target for PH. Pulmonary hypertension (PH) is a severe disorder of pulmonary vasculature. Excessive proliferation of vascular cells, increased extracellular matrix deposition, and accumulation of inflammatory cells collectively cause high pulmonary pressure and eventually leads to right heart failure even death , 2. Hypo2-sensitive α-subunit (mainly HIF1α and HIF2α) and a constitutively expressed β-subunit , incubated on ice for 15 min. The samples were resuspended by vortexing and incubated at 30 °C for 15 min. Cells were then lysed step by step in Buffer B , Buffer C , and Buffer D  for 5 min each on ice. Cells were then sonicated to shear DNA under conditions established to ensure that the DNA fragments were between 500 and 1500 bp. After removal of insoluble material by centrifugation, supernatant was diluted in ChIP dilution buffer . The samples were precleared with 20 μL of blocked Protein A/G Agarose 50% slurry  for 1 h at 4 °C. 200 μL (20%) from supernatant was added with 6 μL 5 M NaCl  and incubated overnight at 65 °C to determine DNA concentration and fragment size. The rest of supernatant was immunoprecipitated with polyclonal anti-HIF1α antibody, or polyclonal p65 antibody, or rabbit control IgG overnight at 4 °C on a rotating wheel. The immunocomplexes were recovered by addition of 20 μL of Protein A/G Agarose 50% slurry for 1 h at 4 °C. The samples were then sequentially washed for 5 min twice in ChIP low-salt wash buffer , ChIP high-salt wash buffer , ChIP LiCl buffer  and TE buffer [10 mM Tris (pH 8.0) and 1 mM EDTA]. The immunocomplexes were extracted twice with the elution buffer containing 1% SDS and 0.1 M NaHCOSYBR Green PCR analysis with iQ SYBR Green Supermix was processed on an iCycler (Bio-Rad). For all primer sets, identical thermocycler conditions were used: stage 1, 3 min at 95 °C; stage 2, 40 cycles, with 1 cycle consisting of 15 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C. Each PCR was performed in duplicate. Because PCR amplification was being monitored by SYBR Green, the absence of nonspecific amplification was determined by analyzing the melting curve of the PCR amplification products. Data collected were analyzed using Microsoft Excel and plotted using GraphPad Prism software. Ten percent of the amount of chromatin was used for the input in our assay, the input dilution factor was therefore 10 times. We used delta-delta-CT method to analyze the fold enrichment following RT-PCR. Briefly the calculation was (1) DeltaCt = Ct ChIP − (Ct input – Log2(Input Dilution Factor)), (2) delta-deltaCt = deltaCt – Ct IgG, (3) old Enrichment = 2^ (− delta-deltaCt) .Primers were designed within the two predicted binding regions (PBR of HIF1a and PBR of RELA) and at 1–2 kb downstream of the transcription start site as no-binding control (NBR) using Primer Premier 5. The primers indicated are:RELA-PBR-F: 5′-GGGCGTCCGTGTCAATCAGGTT-3′, RELA-PBR-R: 5′-GCGGGCGGGTTTGGATTTTAA-3′, HIF1α-PBR-F: 5′-GGCGGGAGGATTGCTTGAACC-3′, HIF1α-PBR-R: 5′-CCCCCCACGCCTTTTTTTTGT-3′, RELA-NBR-F: 5′-GGGCCCCAGAGTTAGAGTGAGTGTC-3′, RELA-NBR-R: 5′-GCACCAGTCTCCCAGCCACAAC-3′, HIF1α-NBR-F: 5′-GCCCTCTCCTTCCCACCCACAGT-3′, HIF1α-NBR-R: 5′-GCCCAGCTTGAGGTCTCGATGAATA-3′.All animal experiments were approved by the Animal Research Committee, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College. Experiments were designed and performed in accordance with the relevant guidelines and regulations , 37. At 2 and 90% N2) and were opened twice a week for cleaning and replenishment of food and water. To assess the influence of hypoxia on PLK1 expression, 8-week-old male C57BL/6 mice  were randomly assigned to be kept in either 10% oxygen or in room air (21% oxygen) [Plk1/−+ mice and Plk1/++ male littermates (provided by Xiaochun Yu and Junjie Chen [Normobaric hypoxic chambers were maintained with approximately 6 L/min flow of hypoxic air (10% O oxygen) . At 0, 4jie Chen ) were kejie Chen , or 0.9%Mice were anesthetized with Ketamin (100 mg/kg) and fentanyl (0.05 mg/kg) intraperitoneally as described . A Milla2 were subcutaneously injected with Sugen5416  for 3 weeks and then continually kept in hypoxia chamber  for another 3 weeks. During the last 3 weeks, these mice were treated randomly with either 0.5% hydroxyethyl cellulose (vehicle) (n = 10) or BI 6727 (15 mg/kg body weight biweekly) (n = 10) by gavage. Twenty control mice were subcutaneously injected with DMSO once per week for 3 weeks under normoxia. These mice were then treated randomly with either vehicle (n = 10) or BI 6727 (15 mg/kg body weight biweekly) (n = 10) by gavage for 3 weeks [Sugen5416 is a potent, selective VEGFR (Flk-1/KDR) inhibitor with an IC50 of 1.23 μM  and a ha 3 weeks .Firstly, hemodynamic measurements were performed with echocardiography. Echocardiography was evaluated with the high-resolution Micro-Ultrasound system . Mice were anesthetized with 1.5% isoflurane. Right ventricle wall thickness during diastole (RVWTD) were obtained from parasternal short axis view at papillary muscle level using M-mode. Pulmonary arterial (PA) peak flow velocity and PA acceleration time/ejection time (AT/ET) were obtained from parasternal short axis view at aortic valve level using pulsed Doppler mode –51. The After hemodynamic measurements and exsanguination, the left lungs were fixed for histology in 10% neutral buffered formalin, and the right lungs were snap-frozen in liquid nitrogen. H&E and immunofluorescence (IF) staining experiments were conducted as previously described . Nuclei Mouse lungs were fixed, embedded and sectioned into 5 μm thick slices. TdT-mediated dUTP Nick-End Labeling (TUNEL) assay was performed using TUNEL Apoptosis Assay Kit  according to the manufacturer’s instruction. Nuclei were labeled by DAPI staining. Images were captured with a Nikon microscope digital camera system.Plk1 with Actb as internal control. The primers used were as follows:Total RNA was extracted from cells and reversely transcribed, cDNA was then used as template in a quantitative PCR reaction. Oligonucleotide primers were synthesized to detect Hif1α Forward: 5′-GGCGGCGAGAACGAGAAG-3′, mouse Hif1α Reverse: 5′-GACCACCGGCATCCAGAAG-3′; mouse Plk1 forward-1: 5′-CTTCGCCAAATGCTTCGAGAT-3′, mouse Plk1 reverse-1: 5′-TAGGCTGCGGTGAATTGAGAT-3′; mouse Plk1 forward-2: 5′-CTAGCACACCAACACGTCGTA-3′, mouse Plk1 reverse-2: 5′-ACCTCCAGATCCTCGTTCAGG-3′; mouse Actb forward: 5′-AGAGGGAAATCGTGCGTGAC-3′, mouse Actb reverse: 5′-CAATAGTGATGACCTGGCCGT-3′.mouse Primers were synthesized by Sangon Biological Engineering Technology & Services .Total RNA was extracted from lung and heart using Trizol method . The first strand cDNA was synthesized from 1 μg of total RNA with HIScript-II Q RT SuperMix for qPCR . Quantitative RT-PCR was performed on an iCycler IQ system (Bio-Rad) utilizing SYBR Green Master Mix (Transgen) according to the manufacturer’s instructions. The thermal cycle conditions were operated as 94 ℃ for 30 s, 94 ℃ for 5 s, 60 ℃for 1 min and a total of 40 cycles. Target mRNA was determined using GAPDH as the comparative cycle threshold method. All samples were run in duplicate. The primer sequences were listed below:Gapdh: 5′-GGTTGTCTCCTGCGACTTCA-3′; 5′-GGTGGTCCAGGGTTTCTTACTC-3′; ANF: 5′-CACAGATCTGATGGATTTCAAGA-3′; 5′-CCTCATCTTCTACCGGCATC-3′; BNP: 5′-GAAGGTGCTGTCCCAGATGA-3′; 5′-CCAGCAGCTGCATCTTGAAT-3′; Collagen I: 5′-GAGTACTGGATCGACCCTAACCA-3′; 5′-ACGGCTGAGTAGGGAACACA-3′; Collagen III: 5′-TCCCCTGGAATCTGTGAATC-3′; 5′-TGAGTCGAATTGGGGAGAAT-3′; Il-1β: 5′-CTTCCCCAGGGCATGTTAAG-3′; 5′-ACCCTGAGCGACCTGTCTTG-3′; Il-6: 5′-GCTACCAAACTGGATATAATCAGGA-3′; 5′-CCAGGTAGCTATGGTACTCCAGAA-3′.Immunoblotting was conducted as described previously –54. Tisst-test between two groups. One-way of variance (ANOVA), followed by Dunnett’s post hoc test, was used between multiple groups. Quantitative data of cell experiments were reported as mean ± SD, Excel software was used to analyze cell proliferation and quantitative real-time RT–PCR data. A p value of < 0.05 was considered significant.All data were analyzed using GraphPad Prism 7.0 . Quantitative data of mouse experiments were reported as median and interquartile ranges (IQR). Normally distributed data were assessed by two-tailed, unpaired Student’s To investigate the role of PLK1 in PH, we checked the expression of PLK1 in mice with pulmonary hypertension. The averaged value of RVSP (ranging from 16 to 26 mmHg) is 21 mmHg in the 8-week-old male C57BL/6 mice . RVSP, tHif1α compromised Plk1 in hPASMCs  induced time-dependent HIF1α and PLK1 protein elevation in hPASMCs and hPAECs Fig. A, B. BotMCs Fig. E. We theMCs Fig. F. Real-tMCs Fig. G.Fig. 2HRELA reduced PLK1 expression in hPASMCs  to 21.1 mmHg   and ejection fraction (EF) compared with Plk1/++ mice under hypoxia condition (Table We assessed the development of hypoxia-induced PH in out mice . H&E staxia Fig. A, B. Plkons Fig. C. The luxia Fig. C. WT lit reduced . This ca reduced . AngiogrWe used BI 2536, an ATP-competitive PLK1 inhibitor , to testBI 6727 is also an ATP-competitive inhibitor of PLK1 and has better pharmacokinetic profile than that of BI 2536 –60. WhilPlk1 knockdown, markedly induced apoptosis of hPASMCs, as determined by Annexin V-FITC analysis and cell morphology (Fig. Proliferation of smooth muscle cells causes progressive obliteration of PA which in return increases resistance to blood flow . We examogy Fig. B, C. BI ogy Fig. D. The thPlk1 heterozygous mice are partially resistant to the development of hypoxia-mediated PH. Pharmaceutical inhibition of PLK1 attenuates PH. Therefore, PLK1 is essential for PH progression.PH is a devastating pulmonary arterial disease with high morbidity and mortality. In this study, we demonstrate that hypoxia stimulates PLK1 expression through induction of HIF1α and RELA. The elevation of PLK1 correlates with the rise of pulmonary arterial pressure in either hypoxia-induced or SuHx-induced PH mice. Hypoxia is a strong stimulus for PH. Alveolar hypoxia induces excessive proliferation of vSMCs that remodels pulmonary vascular wall –54, 58. Plk1 heterozygous knockout mice as homozygous Plk1 deficient mice were embryonic lethal. Plk1 heterozygous knockout mice were resistant to hypoxic induction of PH. PH is a lethal respiratory disease without effective treatment [To test the potential role of augmented PLK1 in the pathogenesis of hypoxia-induced PH, we checked the development of PH in reatment . As PLK1Enhanced vSMC proliferation and attenuated apoptosis are the major features of pulmonary vascular remodeling during the development of PH. Excessive proliferation and/or too little apoptosis may contribute to sustained pulmonary vasoconstriction and excessive pulmonary medial hypertrophy in PH. As PLK1 inhibitors perturbed mitotic progression, rapidly dividing cells are more sensitive to PLK1 inhibition. Here, PLK1 inhibitor BI 2536 reduced the viability and induced apoptosis of PASMCs. BI 6727 increased apoptosis of lungs in SxHu-treated mice. These findings are consistent with what observed in PH-PASMCs exposed to siPLK1 or PLK1 inhibitor BI 6727 . InductiIn summary, hypoxia stimulates PLK1 overexpression through activation of HIF1α and RELA. Augmented PLK1 plays essential roles in the development of hypoxia/SuHx-induced PH. As inhibition of PLK1alleviates mouse PH, targeting PLK1 is therefore feasible for the treatment of PH. Due to the nature of largely animal study, these findings could not be directly extrapolated to PH patients. All these laboratory findings thus warrant validation from clinical trials of PLK1 inhibitors on PH.


A divalent copper one-dimensional ribbon copper(II) coordination polymer, with 1,3-bis(pyridin-3-yl)urea and succinate ligands, was structurally characterized by single-crystal X-ray diffraction. 2(C4H4O4)2(C11H10N4O)]n, mono-periodic coordination polymer ribbons are held into the crystal structure by means of N—H⋯O hydrogen bonding and crystal packing forces.In the title compound, [Cu Previously, our group had isolated a series of cadmium succinate coordination polymers featuring isomeric dipyridylamide coligands. Structural topologies were highly dependent on the specific dipyridylamide ligand used 4} paddlewheel dimers with a Cu⋯Cu separation of 2.657 (1) Å. The full span of the gauche-conformation succ ligands connect the dimeric clusters into [Cu2(succ)2]n coordination polymer chains oriented parallel to the b crystal direction 2]n chain motifs, affording [Cu2(succ)2(3-dpu)]n coordination polymer ribbons oriented parallel to the b crystal direction 2(3-dpu)]n motifs aggregate into supramolecular layers parallel to the bc crystal planes by means of N—H⋯O hydrogen bonding between the urea groups of 3-dpu ligands in one ribbon, and succ carboxylate O atoms in the next ribbon 2·2.5H2O , succinic acid , 3,3′-dipyridylurea , and 0.75 ml of a 1.0 M NaOH solution were placed into 10 ml distilled water in a Teflon-lined acid digestion bomb. The bomb was sealed and heated in an oven at 393 K for 48 h, and then cooled slowly to 273 K. Green crystals of the title complex were obtained in 43% yield.Cu I, global. DOI: 10.1107/S2414314623007472/bh4078Isup2.hklStructure factors: contains datablock(s) I. DOI: 2290886CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


II complex was synthesized by reacting 5-methyl-3-pyrazolamine and copper(II) acetate in water under ambient conditions. Diethanolamine was added to facilitate carbon dioxide adsorption, creating an alkaline environment. Structural analysis revealed that the complex crystallizes in the P21/c space group of the monoclinic crystal system, with the central copper(II) atom in a square-planar coordination environment N2O2. Co-crystallized water molecules are present, forming O—H⋯O hydrogen bonds with the CuII mononuclear complex. Hirshfeld surface analysis highlighted the importance of various interactions, including H⋯O/O⋯H, H⋯C/C⋯H, and H⋯N/N⋯H, in providing crystal structure packing.A mononuclear square-planar Cu II complex of (5-methyl-1H-pyrazol-3-yl)carbamate, [Cu(C5H6N3O2)2]·4H2O, was synthesized using a one-pot reaction from 5-methyl-3-pyrazolamine and copper(II) acetate in water under ambient conditions. The adsorption of carbon dioxide from air was facilitated by the addition of diethanolamine to the reaction mixture. While diethanolamine is not a component of the final product, it plays a pivotal role in the reaction by creating an alkaline environment, thereby enabling the adsorption of atmospheric carbon dioxide. The central copper(II) atom is in an (N2O2) square-planar coordination environment formed by two N atoms and two O atoms of two equivalent (5-methyl-1H-pyrazol-3-yl)carbamate ligands. Additionally, there are co-crystallized water molecules within the crystal structure of this compound. These co-crystallized water molecules are linked to the CuII mononuclear complex by O—H⋯O hydrogen bonds. According to Hirshfeld surface analysis, the most frequently observed weak intermolecular interactions are H⋯O/O⋯H (33.6%), H⋯C/C⋯H (11.3%) and H⋯N/N⋯H (9.0%) contacts.A mononuclear square-planar Cu Co-crysII ion displays a square-planar coordination environment (N2O2) formed by two nitrogen atoms of pyrazole rings and two oxygen atoms of carboxylate group of (5-methyl-1H-pyrazol-3-yl)carbamate ligands. The Cu1—N1 distances are 1.931 (2) Å while the Cu1–O1 distances are shorter and account to 1.9140 (17) Å. The O1–Cu1–O1i and N1—Cu1—N1i bond angles are 180°, which is typical for a square-planar arrangement -to-plane distance of 0.182 (3) Å.The Cunt Fig. 1. At the 2] units form layers with Cu1 centres lying in the ab plane. The plane-normal-to-plane-normal angle between the horizontal N1—O1—N1i—O1i planes of two adjacent layers is 74.762 (2)°.In the crystal structure, monomeric  molecular complexes. Geometric parameters for intermolecular hydrogen bonds are given in Table 2All the components of the structure are associated s Figs. 2, 3 ▸. π–2] neutral complexes. The plane-to-plane distance for these π–π contacts is 3.324 (3) Å with the plane-to-plane shift being 1.498 (5) Å. It is also worth noting very weak C—H⋯π contacts between two contiguous [Cu(5-MeHpzCarb)2] units with a carbon-atom-to-plane distance of 3.586 (4) Å.Interestingly, four water molecules and the carboxyl group form a five-membered supramolecular ring Fig. 3. In addi4.Crystal Explorer 21.5 software  to 1.1041 (blue) a.u. There are eight red spots on the dnorm surface . Visualizations were performed using a red–white–blue colour scheme, where red highlights shorter contacts, white is used for contacts around vdW separation, and blue depicts longer contacts. The red spots on the 3D dnorm Hirshfeld surfaces indicate the direction and strength of the intermolecular E—H⋯O hydrogen bonds , as well as weak C—H⋯O and C—H⋯π contacts. The overall two-dimensional fingerprint plots for the selected interactions are shown in Fig. 4b.The Hirshfeld surface analysis was performed and the associated two-dimensional fingerprint plots were generated using ce Fig. 4a. VisuaThe most significant contributions to the overall crystal packing are from H⋯H (32.2%), H⋯O/O⋯H (33.6%), H⋯C/C⋯H (11.3%), H⋯N/N⋯H (9.0%) and C⋯N/N⋯C (4.1%) interactions. The H⋯O/O⋯H contacts form a pair of spikes on the sides of the corresponding two-dimensional plot, which are indicative of strong intermolecular interactions between atoms. At the same time, the H⋯N/N⋯H and H⋯C/C⋯H contacts form less pronounced spikes, indicating that these interactions are weaker.5.et al., 20162(CO2)2 moiety [four-coordinated copper atom with an N2O2 coordination environment] were found. Most similar to the title compound, complexes forming a four-coordinated N2O2 coordination environment, are trans-bisbiscopper(II) copper(II) biscopper(II) dicopper(II) , copper(II) acetate  and diethanolamine  were mixed together, and dissolved in water. After 3 days, clear, light-violet crystals were collected by filtration, dried for less than a minute, and then placed under crystallographic oil for further measurements.5-Methyl-3-pyrazolamine —H, Cp—H and Ow—H groups  were positioned geometrically and refined as riding atoms, with C—H = 0.95 Å and Uiso(H) = 1.2Ueq(C) for Cp—H groups, N—H = 0.88 Å and Uiso(H) = 1.2Ueq(N) for N—H groups and O—H = 0.87 Å and Uiso(H) = 1.5Ueq(O) for Ow—H groups. Methyl H atoms were positioned geometrically and were allowed to ride on C atoms and rotate around the C—C bond, with C—H = 0.98 Å and Uiso(H) = 1.5Ueq(C) for the CH3 groups.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989023008575/tx2076sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989023008575/tx2076Isup2.hklStructure factors: contains datablock(s) I. DOI: 2298123CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Schistosoma mansoni's cathepsin B1 enzyme (SmCB1), which plays a crucial role in hemoglobin degradation within the worm's gut and its nutrition processes. Consequently, SmCB1 has emerged as a promising target for novel schistosomiasis therapies. Moreover, chloroquinoline exhibits characteristics in its aromatic structure that hold promise for developing SmCB1 inhibitors, along with its interaction with hemoglobin's heme group, potentially synergizing against the parasite's gut. In this context, we report the synthesis of 22 hybrid analogs combining hydrazones and quinolines, evaluated against S. mansoni. Five of these hybrids demonstrated schistosomicidal activity in vitro, with GPQF-8Q10 being the most effective, causing worm mortality within 24 h at a concentration of 25 µM. GPQF-8Q8 proved to be the most promising in vivo, significantly reducing egg presence in feces (by 52.8%) and immature eggs in intestines (by 45.8%). These compounds exhibited low cytotoxicity in Vero cells and an in in vivo animal model (Caenorhabditis elegans), indicating a favorable selectivity index. This suggests their potential for the development of new schistosomiasis therapies. Further studies are needed to uncover specific target mechanisms, but these findings offer a promising starting point.The chemical classes of semicarbazones, thiosemicarbazones, and hydrazones are present in various compounds, each demonstrating diverse biological activities. Extensive studies have revealed their potential as schistosomicidal agents. Thiosemicarbazones, in particular, have shown inhibitory effects on  Semicarbazones, thiosemicarbazones, and hydrazones are chemical classes of compounds with a broad range of biological activities. Due to their resemblance to the peptide bonds in proteins, they are regarded as privileged chemical groups, since they mislead macromolecules that initially identify peptide connections. Moreover, they present additional points for intermolecular interaction, which allows them to have greater affinity for the target Fig. A.Figure 1 conducted in vitro studies with ten thiosemicarbazones and hydrazones. They selected an analog, the LpQM-45, which significantly reduced worm motility, male–female pairing, and oviposition after 144 h, with an IC50 value of 32.09 μg/mL. However, the macromolecular target of these compounds, was not determined in this study. In 2015, Fonseca et al.2 synthesized and tested 29 thiosemicarbazones with a direct action on cathepsin B1 from Schistosoma mansoni (SmCB1), revealing a set of five compounds with IC50 values under 10 μM. However, all the other analogs, showed only moderate inhibitory activity hydrazin‐1‐yl]quinoline were synthesized through the coupling of 7‐chloro‐4‐hydrazinylquinoline with various aldehydes or ketones, as described in Methods Session and illustrated in Fig. Table The ease of synthesis, along with good yields, the use of relatively inexpensive reagents, and the production of solid compounds, all represent desirable characteristics for drug candidates targeting neglected diseases like schistosomiasis.S. mansoni, only chemical compounds or natural extracts that demonstrated any activity at this concentration were considered significant for further in vitro and in vivo testing. Accordingly, five out of the 22 final compounds met these criteria and were selected to follow with the experimental investigations.All compounds were subjected to an initial trial at 50 µM. In the context of testing compounds against GPQF-8Q9/GPQF-8Q17, and GPQF-8Q10/GPQF-8Q18, exhibited contrasting biological responses at 50 µM, with only the aldehydes derivatives displaying activity. This strongly suggests the presence of a macromolecular target with specific stereochemical and/or volume requirements. The ketone derivatives, which possess a three-dimensional structure distinct from the typically flat structure of the azomethines derived from aldehydes, were inactive. These initial results point towards a stereo-specific binding site, where only the flat derivatives could exert significant inhibitory activity.This first trial also uncovered some intriguing insights into the relationships between the chemical properties of the compounds and their biological behavior. Notably, all ketone derivatives proved to be inactive against the adult worms, at least within the established concentrations limits of the test. Even compounds with similar overall structures but derived from equivalent aldehydes or ketones, such as the pairs The five active analogs were subsequently subjected to in vitro assays at lower concentrations. The results, which include reductions in worm motor activity categorized as normal, slight, significant, or absent and are separated by sex, are presented in Fig. All compounds exhibited significant interference with worm motility at concentrations of 50 µM or lower. Distinctions in their biological behavior became apparent at 25 µM, where some compounds demonstrated effective interference with motility and lethality against the worms Fig. .Figure 4GPQF-8Q10 proved to be the most effective analog in vitro, killing the worms within the first 24 h at 25 µM. A noticeable reduction in motility followed a similar pattern. It appears that there may be differences in sensitivity between males and females, although these results do not provide a detailed understanding of how each gender responds to the treatment. Notably, females appeared to be affected by all the active analogs to varying degrees and at different time intervals, with the exception of GPQF-8Q2.With regard to lethality, SmCB1 suggests that they induced death by interfering with nutrition, a process that naturally takes some time to occur.Another noteworthy observation is the compounds require some time to exert their lethal effects, as none of them killed the worms immediately. This time-dependent activity aligns with the design concept of these compounds, as their potential inhibition of 17. The cytotoxicity concentration that led to a 50% cell death rate (CC50) was determined. The results, including the effective concentration that caused 50% inhibition (EC50) and the selectivity index, are summarized in Table The active compounds were subjected to testing against Vero cells (African green monkey kidney cells), a commonly employed cell line in toxicity studies, especially in the realm of antischistosomal drug discovery50 values confirm the initial in vitro observations. The Vero cells assay indicated low toxicity of these compounds against these cells and the calculated selectivity indexes suggests that good selectivity could be achieved.The measured ECThe next step in this investigation was the in vivo tests Fig. .Figure 5GPQF-8Q10, did not exhibit the same level of effectiveness in vivo. It might be tempting to associate this lack of in vivo activity with its high partition coefficient (CLogP 5.06). However, this does not appear to be the case, as the analog GPQF-8Q2, which had the highest CLogP value (5.17), showed the best reduction in oviposition. The most significant reductions in worm burden were achieved by compounds GPQF-8Q8 and GPQF-8Q9, with reductions of 50.8 and 58.7%, respectively. GPQF-8Q8 displayed the most comprehensive in vivo biological activity, as it was also the most effective compound in interfering with egg production. It led to reductions in both eggs found in feces (52.8%) and immature eggs found in the intestines (45.8%). The presence of the thiophene ring in this compound also contributed positively to lipophilicity, which could suggest that this factor plays a role in the action on oviposition.Remarkably, the best-performing compound in in vitro tests, GPQF-8Q9 and GPQF-8Q11 (both having a CLogP of 4.26 as they are position isomers), exhibited intriguing in vivo biological behaviors. Interestingly, only GPQF-8Q9 showed significant in vivo activity, suggesting the presence of an important steric factor related to theses structures. Ortho substituents are well-known to influence the conformation of rings, forcing them out of the plane of the molecule, in this case, the plane of the 7-chloroquinoline ring. This could explain why GPQF-8Q11 did not exhibit any in vivo activity at all. Since the only difference between these two compounds is the steric positioning of hydroxy group, the significantly different biological responses to both compounds strongly indicate the presence of an endogenous receptor with selective steric specificities.Even the more “hydrophilic” compounds, GPQF-8Q8 and GPQF-8Q9, were also the only two compounds that predominantly exhibited in vitro activity against females. In a study by Liu et al.5 in 2014, it was demonstrated that S. japonicum expresses a network of proteases in its gut, with Cathepsin B1 being the predominant enzyme. Importantly, they observed that this enzyme was upregulated in female gut. Therefore, the preference of GPQF-8Q8 and GPQF-8Q9 for acting on females could be a strong indication that SmCB1 is indeed the target of these compounds.Another noteworthy observation is that the two best-performing in vivo, SmCB1 could not be the only possible macromolecular target since the in vitro tests do not guarantee the specificity for only one target. Only enzymatic inhibitory assays would prove that this enzyme would be a real target for these compounds, and they have already been performed by our group. But even in this case the selectivity for just one target could not be assured.It is important to highlight, however, that the Caenorhabditis elegans is a nematode commonly employed as an in vivo toxicity model 7. Its utility extends to research aiming to discover new anthelmintic agents 9. In order to delve deeper into the potential toxicity of analogs of 7‐chloro‐4‐[2‐(phenylmethylidene)hydrazin‐1‐yl]quinoline, we assessed the survival of C. elegans exposed to various compound concentrations. As shown in Table GPQF-8Q10 emerging as the most potent compound at 25 µM, leading to the death of all worms within 72 h. Notably, this compound displayed values of 13.3 and 15.6 µM against male and female worms, respectively.Considering the results reported herein, the 7‐chloro‐4‐[2‐(phenylmethylidene)hydrazin‐1‐yl]quinoline scaffold appears to be a promising starting point for the development of new antischistosomal drugs. Among the 22 tested compounds, five demonstrated schistosomicidal activity in the in vitro assay, with GPQF-8Q8 displayed the most comprehensive biological activity, reducing both the number of eggs found in feces (52.8%) and immature eggs found in the intestines (45.8%). The compounds also demonstrated low cytotoxicity against Vero cells and a favorable selectivity index. Furthermore, none of the compounds exhibited any toxic effect in the in vivo toxicity assay using the C. elegans model.In the in vivo assay, Although further studies are needed to determine the target, the collective findings strongly suggest the potential of the hydrazone quinoline scaffold in the quest for new antischistosomal leads.254 Silica gel on TLC aluminum foils, hexane/ethyl acetated 1:2 was employed as eluent and the plates were visualized by ultraviolet (UV) irradiation (254 nm). Melting point analyses were conducted in triplicate using a Marte científica PDF III apparatus. Infrared (IR) analyses were carried out using an IR Affinity-1 Shimadzu Fourier Transform Infrared Spectrophotometer. Samples were prepared with anhydrous KBr, and the analyses covered the wave number region between 4000 and 400 cm−1. 1H-NMR analyses were performed on a Bruker-300 Ultrashield operating at 300 MHz, with solvents DMSO-d6 or CDCl3. The chemical shift values (δ) were expressed in parts per million (ppm), employing tetramethylsilane (TMS) as the internal reference. The following abbreviations were used to describe the multiplicities: s = singlet; d = doublet; dd = double doublet; t = triplet; m = multiplet. Coupling constants (J) were described in Hertz.Reagents and solvents were obtained from commercial suppliers without the need for further purification. Chemical reactions were monitored by thin layer chromatography (TLC) using Merck 60 FAll the structural data collected are provided in the Supplementary Information Fig. .12. A total of 5 mmol of 4,7-Dichloroquinoline was dissolved in 10 mL of ethanol and placed in a round-bottom flask equipped with a reflux system. After 5 min of stirring, 25 mmol of hydrazine hydrate 80% (w/v) was added to the reaction. The solution was then heated under reflux for 15 h at 80–90 °C. After this period, the solution was refrigerated for approximately 8 h. The resulting dark yellow precipitate was washed with cold distilled water and filtered under reduced pressure. The crude compound was subsequently recrystallized from ethanol and a yellow crystalline solid was obtained.The synthesis of the intermediate 7-chloro-4-hydrazinylquinoline was conducted following a previously described methodology in the literatureYield 80%. M.P. 220–224 °C (lit.11 225–226°C). 1H-NMR-(DMSO-d6)—δ = ppm: 4.45 ; 6.86 ; 7.38 ; 7.75 ; 8.16 ; 8.40  8.59 ;. IR—νNH2 and νNH 3282 and 3253 cm-1; νCH 3053 cm−1; νC = C 1612 cm−1; νC = N 1570 cm−1.Prepared in accordance with the synthetic methodology previously described. Yellow crystalline solid. 14. First, (1 mmol) of corresponding aldehyde or ketone was introduced into a round-bottom flask, and in most cases*, approximately 8 mL of a ethanol/acetic acid 25% solution was added to the round-bottom flask. The solution was kept under agitation until the complete solubilization of the corresponding reagents. Then, (1 mmol) of 7-chloro-4-hydrazinylquinoline was added, and the reaction mixture was heated under reflux** (about 80 °C) for about 3–17 h. After the reflux complete, the resulting mixture was concentrated under reduced pressure, and the crude compounds were washed with cold water and cold ethyl ether, then filtered under reduced pressure to obtain the desired products.The quinoline derivatives were obtained following the synthetic methodology reported in the literature(GPQF-8Q1) or a solution of MeOH/HAOc 25% (GPQF-8Q6).*In some cases, the solvent employed was ethanol GPQF-8Q1, 8Q22, 8Q24, and 8Q25, the reaction was kept at room temperature for 24 h.**In the case of Yield 52%. M.P. 220–224 °C (lit.13 223–225°C). 1H-NMR-(DMSO-d6)—δ = ppm: 7.37 ; 7.42–7.51 ; 7.57 ; 7.81 ; 7.86 ; 8.42  8.44 ; 8.47  . IR—νNH 3446 cm−1; νCH 3188 and 2891 cm−1; νC = C 1618 cm−1; νC = N 1577 cm−1.Prepared following the general synthetic procedure of quinoline derivatives, which involved using 7-chloro-4-hydrazinylquinoline (1mmol) and benzaldehyde (1 mmol) as reactants, ethanol was employed as the solvent, and the reaction was maintained at room temperature for 24 h. Yellow solid. Yield 75%. M.P. 320–324 °C. 1H-NMR-(DMSO-d6)—δ = ppm: 7.60 , 7.65 , 7.88 , 7.94 , 8.07 , 8.69 , 8.75–8.78 . IR—νNH 3446 cm−1; νCH 2875 and 2698 cm−1; νC = C 1627 cm−1; νC = N 1612 cm−1.Prepared in accordance with the general synthetic procedure for quinoline derivatives, which involved using 7-chloro-4-hydrazinylquinoline (1mmol) and 4-chlorobenzaldehyde (1 mmol), a solution of ethanol and acetic acid 25% was employed as the solvent. The reaction was conducted under reflux at 80 °C during 3 h. Orange solid. Yield 59%. M.P. 348–350 °C. 1H-NMR-(DMSO-d6)—δ = ppm: 7.74 , 7.93 , 8.08 , 8.19 , 8.36 , 8.73–8.81 . IR—νNH 3446 cm−1; νCH 2646 cm−1; νC = C 1614 cm−1.7-Chloro-4-hydrazinylquinoline (1mmol) and 4-nitrobenzaldehyde (1 mmol) were coupled in the presence of ethanol and acetic acid 25% solution. The reaction mixture was refluxed at 80 °C for 18 h, following the general synthetic procedure. Pale-yellow solid. Yield 63%. M.P. 284–288 °C (lit.10 272–275 °C). 1H-NMR-(DMSO-d6)—δ = ppm: 3.89 ; 6.94 ; 7.25 ; 7.45 ; 7.61 ; 7.84 ; 8.10 ; 8.61 ; 8.77 ; 8.91 ; 9.96 ; 12.98 . IR—νOH and νNH 3421 cm−1; νCH 3209 cm−1; νC = C 1608 cm−1; νC = N 1589 cm−1.7-Chloro-4-hydrazinylquinoline (1 mmol) and 4-hydroxy-3-methoxybenzaldehyde (1 mmol) were coupled following the general synthetic procedure, in the presence of ethanol/acetic acid 25% solution and the reaction was maintained under reflux at 80 °C for 3 h. Yellow solid. Yield 58%. M.P. 284–286 °C (lit.15 238–240 °C). 1H-NMR-(DMSO-d6)—δ = ppm: 7.46 , 7.52 , 7.83 , 7.86 , 8.07 , 8.71 , 8.76  8.82 . IR—νNH 3446 cm−1; νCH 3082 and 2646 cm−1; νC = C 1614 cm−1; νC = N 1589 cm−1.Prepared following the general synthetic procedure for quinoline derivatives, which involved using 7-chloro-4-hydrazinylquinoline (1mmol) and 5-nitrofuran-2-carbaldehyde (1 mmol), a solution of methanol and acetic acid 25% was used as the solvent. The reaction was maintained under reflux at 80 °C for 17 h. Mustard yellowish solid. Yield 61%. M.P. 224–226 °C (lit.15 231–232 °C). 1H-NMR-(DMSO-d6)—δ = ppm: 7.15–7.19 , 7.49 , 7,57 , 7.66 , 7.82 , 8.37–8.43 , 8.66 . IR—νNH 3444 cm−1; νCH 3074cm−1; νC = C 1610 cm−1; νC = N 1550 cm−1.7-Chloro-4-hydrazinylquinoline (1mmol) and thiophene-2-carbaldehyde (1 mmol) were coupled following the general synthetic procedure, in the presence of a solution of ethanol and acetic acid 25%. The reaction mixture was maintained under reflux 80 °C for 16 h. Yellowish bright solid. Yield 77%. M.P. 280–284 °C (lit.10 233–235 °C). 1H-NMR-(DMSO-d6)—δ = ppm: 6.85 , 7.26 , 7.51 , 7.62 , 7.82 , 8.31 , 8.35 ; 8.47  9.97 , 11,03 . ). IR—νO-H and νNH 3425 cm−1; νCH 3030cm−1; νC = C 1608 cm−1; νC = N 1552 cm−1.7-Chloro-4-hydrazinylquinoline (1mmol) and 4-hydroxybenzaldehyde (1 mmol) were coupled following the general synthetic procedure, in the presence of a solution of ethanol and acetic acid 25% and maintained under reflux at 80 °C for 16 h. Orange solid. Yield 96%. M.P. 190–192 °C (lit.12 240–244 °C). 1H-NMR-(DMSO-d6)—δ = ppm: 2.36 , 7.29 , 7.34 , 7.56 , 7.69 , 7.84 , 8.38–8.41 , 8.45 . IR—νNH 3446 cm−1; νCH 3022cm−1; νC = C 1618 cm−1; νC = N 1579 cm−1.Prepared following the general synthetic procedure, which involved using 7-chloro-4-hydrazinylquinoline (1mmol) and 4-methylbenzaldehyde (1 mmol), in the presence of a solution of ethanol and acetic acid 25%. The reaction was maintained under reflux at 80 °C for 7 h. Sharp range crystals. Yield 96%. M.P. 222–226 °C (lit.13 150–151 °C). 1H-NMR-(DMSO-d6)—δ = ppm: 6.90–6.95 , 7.27 , 7.48 , 7.69–7.70 , 8.17 , 8.38 , 8.73 . IR—νO-H and νNH 3427 cm−1; νCH 2553cm−1; νC = C 1622 cm−1; νC = N 1554 cm−1.Prepared in accordance with general synthetic procedure. 7-chloro-4-hydrazinylquinoline (1mmol) and 2-hydroxylbenzaldehyde (1 mmol) were coupled using a solution of ethanol and acetic acid 25%. The reaction was conducted under reflux at 80 °C for 17 h. Golden yellow solid. Yield 86%. M.P. 236–241 °C (lit.13 225–226 °C). 1H-NMR-(DMSO-d6)—δ = ppm: 7.28–7.35 , 7.56 , 7.84–7.89 , 8.37–8.42 . IR—νNH 3421 cm−1; νCH 3209cm−1; νC = C 1608 cm−1; νC = N 1571 cm−1.Prepared in accordance with general synthetic procedure. 7-chloro-4-hydrazinylquinoline (1mmol) and 4-fluorobenzaldehyde (1 mmol) were coupled, using a solution of ethanol and acetic acid 25%. The reaction was conducted under reflux at 80 °C for 17 h. Mustard yellow solid. Yield 43%. M.P. 320 °C (decomposition). 1H-NMR-(DMSO-d6)—δ = ppm: 7.70 ; 7.79  7.84–7.92 , 8.03  8.19  8.63–8.69 . IR—νNH 3414 cm−1; νCH 2879cm−1; νC = C 1616 cm−1; νC = N 1589 cm−1.7-Chloro-4-hydrazinylquinoline (1mmol) and 3,4-dichlorobenzaldehyde (1 mmol) were coupled, using a solution of ethanol and acetic acid 25% employed as the solvent, and kept under reflux at 80 °C during 16 h, following the general synthetic procedure. Light yellow solid. Yield 55%. M.P. 340 °C (decomposition). 1H-NMR-(DMSO-d6)—δ 6.40 , 6.45 , 7.46 , 7.75 , 7.82 , 8.08 , 8,57 , 8.81  9.01 , 10.13 , 10.36 . IR—νO-H and νNH 3427 cm−1; νCH 2897 cm−1; νC = C 1631 cm−1; νC = N 1606 cm−1.7-chloro-4-hydrazinylquinoline (1mmol) and 3,4-dihydroxybenzaldehyde (1 mmol) were coupled using a solution of ethanol and acetic acid 25% as the solvent. The reaction was conducted under reflux at 80 °C for 16 h, following the general synthetic procedure. Yellow solid. Yield 52%. M.P. 302–306 °C. 1H-NMR-(DMSO-d6)—δ = ppm: 2.65 , 7.49–7.55 , 7.84 , 8.00 , 8.18 , 8.64 , 8.82 . IR—νNH 3408 cm−1; νCH 2767cm−1; νC = C 1633 cm−1; νC = N 1606 cm−1.Prepared following the general synthetic procedure, which involved using 7-chloro-4-hydrazinylquinoline (1mmol) and 1-phenylethanone (1 mmol) as reactants, and a solution of ethanol and acetic acid 25% as the solvent. The reaction was maintained under reflux at 80 °C for 16 h. Light yellow solid. Yield 54%. M.P. 320 °C (decomposition). 1H-NMR-(DMSO-d6)—δ = ppm: 2.57 , 6.90 , 7.45 , 7.81 , 7.87 , 8.13 , 8.60 , 8.87 , 10.12 . IR—νO-H and νNH 3209 cm−1; νCH 2787cm−1; νC = C 1608 cm−1; νC = N 1585 cm−1.7-Chloro-4-hydrazinylquinoline (1mmol) and 1-(4-hydroxyphenyl)ethanone (1 mmol) were prepared following the general synthetic procedure, using a solution of ethanol and acetic acid 25% as the solvent. The reaction was conducted under reflux at 80 °C for 16 h. Yellow solid. Yield 41%. M.P. 310 °C (decomposition). 1H-NMR-(DMSO-d6)—δ = ppm: 2.38 , 2.60 , 7.32 , 7.51 , 7.83 , 7.90 , 8.08 , 8.82 . IR—νNH 3466 cm−1; νCH 2555 cm−1; νC = C 1610 cm−1; νC = N 1585 cm−1.Prepared in accordance with general synthetic procedure, which involved using 7-chloro-4-hydrazinylquinoline (1mmol) and 1-(4-methylphenyl)ethanone (1 mmol) as reactants, and a solution of ethanol and acetic acid 25% as the solvent. The reaction as maintained under reflux at 80 °C for 16 h. Yellow solid. Yield 49%. M.P. 300–304 °C. 1H-NMR-(DMSO-d6)—δ = ppm: 1.22 ; 2.62 ; 2.68 ; 7.35 ; 7,52 ; 7,85 ; 7,92 ; 8,13 ; 8,65 ; 8,86 . IR—νNH 3473 e 3415 cm−1; νCH 2536 cm−1; νC = C 1608 cm−1; νC = N 1583 cm−1.7-Chloro-4-hydrazinylquinoline (1mmol) and 1-(4-ethylphenyl)ethanone (1 mmol) were coupled in accordance with general synthetic procedure. Ethanol and acetic acid 25% were employed as the solvent, and the reaction was maintained under reflux at 80 °C for 16 h. Sharp yellow crystals. Yield 45%. M.P. 294–298 °C. 1H-NMR-(DMSO-d6)—δ = ppm 2.62 ; 7.57 ; 7.78 ; 7.91 ; 7.99 ; 8.11 ; 8.21 . IR—νNH 3473 and 3417 cm−1; νCH 2640cm−1; νC = C 1606 cm−1; νC = N 1587 cm−1.Prepared following the general synthetic procedure, which involved using 7-chloro-4-hydrazinylquinoline (1mmol) and 1-ethanone (1 mmol) as reactants, and a solution of ethanol/acetic acid 25%. The reaction was conducted under reflux at 80 °C for 16 h. Light orange solid. Yield 71%. M.P. 161–166 °C. 1H-NMR-(DMSO-d6)—δ = ppm: 2.21 ; 2.78 ; 2.98 ; 7.13 ; 7.19–7.23 ; 7.30–7.52 ; 7.77 ; 7.95 ; 8.53 ; 8.64 . 13C-NMR-—δ = ppm: 17.67 (C23); 31.03 (C15); 31.48 (C14); 100.18 (C3); 111.24 (C9); 118.14 (C5); 119.76 (C16); 125.97 (C19); 126.15 (C21); 126.52 (C17); 128.35 (C7); 128.39 (C6); 137.75 (C8); 139.78 (C13); 141.07 (C4); 143.54 (C10); 152.19 (C2). IR—νNH 3421cm−1; νCH 3055cm−1; νC = C 1606 cm−1; νC = N 1554 cm−1.Prepared in accordance with general synthetic procedure, which involved using 7-chloro-4-hydrazinylquinoline (1mmol) and 4-phenylbutan-2-one (1 mmol) as reactants, and a solution of ethanol/acetic acid 25% as the solvent. The reaction was maintained under reflux at 80 °C for 16 h. Beige solid. Yield 77%. M.P. 71–74 °C. 1H-NMR-(CDCl3)—δ = ppm: 1.14 ; 2.09 ; 2.33–2.41 ; 7.22 ; 7.51 ; 7.86 ; 8.37; 8.53  9.38 . 13C-NMR-—δ = ppm: 10.72 (C15); 14.56 (C17); 32.31 (C14); 102.12 (C3); 115.69 (C5); 120.39 (C9); 125.64 (C6 and C7); 134.90 (C8 and C13); 146.92 (C4); 161.88 (C10); 177.40 (C2). IR—νNH 3402 cm−1; νCH 2966 cm−1; νC = C 1610 cm−1; νC = N 1577 cm−1.Prepared using 7-chloro-4-hydrazinylquinoline (1mmol) and propan-2-one (1 mmol), in the presence of a solution of ethanol/acetic acid 25%, and left at room temperature for 24 h. Light yellow solid. Yield 51%. M.P. 250 °C (decomposition). 1H-NMR-(DMSO-d6)—δ = ppm: 0.96 ; 1.63–1.40 ; 2.21 ; 2.43 ; 7.24 ; 7.81 ; 8.12 ; 8.58 ; 8.81 . IR—νNH 3419 cm−1, νCH 2956–2648 cm−1; νC = C 1610 cm−1; νC = N 1587 cm−1.Prepared using 7-chloro-4-hydrazinylquinoline (1mmol) and butan-2-one (1 mmol), in the presence of a solution of ethanol/acetic acid 25%. The reaction was mainteined at room temperature for 24 h. Yellow solid. Yield 59%. M.P. 228–232 °C. 1H-NMR-(DMSO-d6)—δ = ppm: 1.23 ; 2.23 ; 7.27 ; 7.79 ; 8,17 , 8.58 ; 8.83 . 13C-NMR-—δ = ppm: 13.78 (C17); 27.47 ; 39.35 (C14); 100.18 (C3); 114.07 (C9); 119.10 (C5); 126.56 (C7); 126.69 (C6); 138.00 (C8); 139.15 (C13); 142.96 (C4); 152.80 (C10). IR—νNH 3419 cm−1; νCH 2970–2648 cm−1; νC = C 1608 cm−1; νC = N 1585 cm−1.Prepared using 7-chloro-4-hydrazinylquinoline (1mmol) and 3,3-dimethybutan-2-one (1 mmol), with a solution of ethanol/acetic acid 25% as the solvent. The reaction was maintained at room temperature for 24 h. Light yellow solid. Yield 67%. M.P. 290–292 °C. 1H-NMR-(DMSO-d6)—δ = ppm: 2.62 ; 7.58 ; 7.86 ; 8.03 ; 8.08 ; 8.66 ; 8.82 . IR—νNH 3481 and 3414cm−1; νCH 2607cm−1; νC = C 1622 cm−1; νC = N 1587 cm−1.7-Chloro-4-hydrazinylquinoline (1mmol) and 1-(4-chlorophenyl)ethanone (1 mmol) were coupled following the general synthetic procedure. A solution of ethanol/acetic acid 25% was used as the solvent, and the reaction was maintained under reflux at 80 °C for 16 h. Orange solid. S. mansoni (Belo Horizonte strain) is maintained through routine passage through Biomphalaria glabrata snails and Swiss mice at the Research Center on Neglected Diseases . Both rodents and snails were kept under environmentally controlled conditions , with free access to food and water16. Vero cells were obtained from the American Type Culture Collection . Cells were routinely cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 5% heat-inactivated fetal bovine serum and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) at 37 °C in a humidified atmosphere containing 5% CO217.The life cycle of Caenorhabditis elegans wild-type (N2 strain), kindly provided by Dr. Carlos E. Winter (University of São Paulo), was routinely cultured at 22 °C on nematode growth medium (NGM) plates seeded with Escherichia coli (strain OP50) as a food source according to standard protocols 18.20. Briefly, adult parasites, obtained from infected mice at day 42 post-infection, were incubated in flat bottom 24-well plates  in RPMI 1640 culture medium  supplemented with 5% inactivated fetal calf serum, antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin), and buffered with HEPES 25 mM . Compounds were dissolved in dimethyl sulfoxide  and tested at 50 μM using PZQ 2 μM and DMSO 0.5% as positive and negative controls, respectively. Parasites were kept for 72 h  and their viability was assessed using a Motic AE2000 inverted microscope 21. Each experiment was performed in triplicate and repeated at least three times. The negative control  and positive control (praziquantel 2 µM) were included.The antischistosomal assay was performed according to the methodology previously described50), compounds were tested using 1:2 serial dilutions from 50 to 3.12 µM as previously described23. Each concentration was tested in five replicates, and experiments were repeated once.For the determination of an effective concentration of 50%  and incubated with compounds (started at 200 µM and followed a 1:2 dilution series) at 37 °C and 5% CO2 (Panasonic). After 72 h, MTT solution was added to each well and the absorbance at 595 nm was read on a spectrophotometer . The selectivity indices (SI) were calculated by dividing the 50% cytotoxic concentration (CC50) obtained on cells with 50% effective concentration (EC50) values determined on S. mansoni25.The cytotoxicity of the compounds on the Vero cells was determined by thiazolyl blue tetrazolium bromide (MTT) assay (Sigma-Aldrich) as previously describedS. mansoni-infected mice were performed according to standard protocols26. Rodents (3 weeks old) were infected subcutaneously with 80 S. mansoni cercariae each and animals were randomly divided into experimental groups (five mice per group). Compounds were dissolved in 2% ethanol in water (v/v) and tested at a single oral dose (400 mg/kg) administered 42 days post-infection27. For comparison, praziquantel (400 mg/kg) and a corresponding amount of vehicle were administered to groups of five schistosomes-infected animals in the same period29. Two weeks after treatment, animals were placed in a chamber and euthanized by introducing 100% medical grade CO2 and therapeutic efficacy was based on the following aspects: (i) the number of schistosomes collected by portal perfusion (worm burden); (ii) quantitative fecal examination (egg burden determined by Kato-Katz technique), and (iii) eggs in tissue (egg burden in intestine determined by oogram method). as previously described31. The percentage of worm and egg reduction was calculated by means of the following equation: % reduction = [/value of untreated control group] × 100%32. The nonparametric Kruskal–Wallis test was used to compare the medians of the responses between the treatment and control groups using the GraphPad Prism software . Differences were considered statistically significant at P < 0.059.In vivo studies in 18. When at the L4 stage, approximately 25 nematodes were transferred to each well of a 96-wells NGM plates containing the drug of interest at the desired concentration  along with a positive control (levamisole 10 μM) and a negative control (DMSO 0.5%). Nematodes were kept at 22 °C for 24 h, and their viability was verified microscopically using a Motic AE2000 inverted microscope  equipped with an ultra-high definition (UHD) camera and with a 48-inch 4K-UHD monitor system 33. The worm viability was calculated by counting mobile worms and those considered dead which showed no movement on physical stimuli with a fine needle34.For the drug assessment, the age-synchronized L1 stage was cultivated on standard NGM platesAnimal studies are reported in compliance with the ARRIVE guidelines. The protocol for experimental design was reviewed and approved by the Committee for the Ethical Use of Animals in Experimentation at Guarulhos University,  in accordance with the Guidelines for Care and Use of Laboratory Animals as stipulated by Brazilian laws.Supplementary Figures.


The heterocyclic fused ring geometry of the title compound coincides with the geometries of related molecules but without directed intermolecular contacts determining the crystal packing. c]furan-5,7-dione), C6H4O3S2, the observed geometry agrees well with those of its phthalamide, thieno and hydroxy analogs, and with a calculated geometry obtained by density functional theory (DFT) calculations. Specific structural features are an S—C—C—S torsion angle of −70.39 (17)° and S—C bonds to sp2-hybridized C atoms approximately 0.1 Å shorter than those to sp3-hybridized C atoms. Unlike the extended structures of the analogs, there are no directed intermolecular interactions and the head-to-tail rows of molecules that are a prominent structural motif of the packing can be rationalized in terms of optimized dipole–dipole interactions.In the title compound (systematic name: 2,3-dihydro-1,4-dithiino[2,3- We report here the three-dimensional structure of 5,6-dihydro-1,4-dithiine-2,3-dicarboxylic anhydride, which crystallizes in the triclinic space group PThe unit-cell parameters for the title compound have been reported previously . A DFT geometry optimization in vacuo [B3LYP functional, cc-pTVZ basis set; GAMESS  was purchased from Fisher Scientific and recrystalized by slow evaporation at room temperature from tetrahydrofuran solution to yield yellow block-like crystals.OLEX2 system via Hirshfeld atom refinement with nonspherical atomic form factors using NoSpherA2  I, global. DOI: 10.1107/S2414314623006478/hb4438Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314623006478/hb4438Isup3.cmlSupporting information file. DOI: 2284875CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


According to the Hirshfeld surface analysis, non-conventional C—H⋯Cl—Mn hydrogen bonding is predominant in the crystal packing.The 0-D hybrid salt (C a]pyridin-2-ium) tetrachloridomanganate(II), (C8H9N2)2[MnCl4] or [L]2[MnCl4], consists of discrete L+ cations and tetrachloridomanganate(II) anions. The fused heterocyclic rings in the two crystallographically non-equivalent monovalent organic cations are almost coplanar; the bond lengths are as expected. The tetrahedral MnCl42– dianion is slightly distorted with the Mn—Cl bond lengths varying from 2.3577 (7) to 2.3777 (7) Å and the Cl—Mn—Cl angles falling in the range 105.81 (3)–115.23 (3)°. In the crystal, the compound demonstrates a pseudo-layered arrangement of separate organic and inorganic sheets alternating parallel to the bc plane. In the organic layer, pairs of centrosymmetrically related trans-oriented L+ cations are π-stacked. Neighboring MnCl42– dianions in the inorganic sheet show no connectivity, with the minimal Mn⋯Mn distance exceeding 7 Å. The Hirshfeld surface analysis revealed the prevalence of the non-conventional C—H⋯Cl—Mn hydrogen bonding in the crystal packing.The 0-D hybrid salt bis(2-methylimidazo[1,5- In the fused cores, the imidazolium rings show C—N/C bond lengths in the range 1.332 (3)–1.405 (3) Å; the pyridinium rings have normal bond distances; the nitrogen atoms are planar, with a total sum of three angles of 360°. The five- and six-membered rings in the cations are almost coplanar, showing dihedral angles between them of less than 2° [0.61  and 1.46° ].The organic–inorganic hybrid salt ns Fig. 1. In the 42– anion with Mn—Cl distances varying from 2.3577 (7) to 2.3777 (7) Å and the bond angles falling in the range 105.81 (3)–115.23 (3)°  Å] corresponds to the distance between consecutive inorganic planes. Pairs of inversion-related cations in the organic layer are stacked with varying levels of offset, showing the ring-centroid distances of 3.453 (1) , 3.552 (2) and 4.002 (1) Å  with the corresponding interplanar distances being 3.263 (1), 3.526 (1) and 2.401 (1) Å, respectively. In the inorganic layer, the tetrachloridomanganate(II) anions are loosely packed with the shortest Mn⋯Mn separations being about 7.098 Å. The closest Cl⋯Cl distance of 4.649 Å is significantly larger than the double value of the Shannon ionic radii of chloride anion [2r(Cl−) = 2 × 1.81 = 3.62 Å], making magnetic interactions between the metal ions barely possible. Additional structure consolidation is provided by numerous C—H⋯Cl—Mn hydrogen bonds between organic and inorganic sublattices  cover less than 6%  contributed by our research team. Another large group comprises organic salts with substituted L+ cations and inorganic anions such as perchlorate or hexafluorophosphate. NAKNET °] around the Hg center .The ubiquitous tetrachloride anion is found in more than 200 structures stored in the CSD. The average Mn—Cl distance of 2.37 Å in 6.L]2[MnCl4], (I). Solid CH3NH2·HCl  was added to the warm formaldehyde solution prepared by dissolving paraform  in boiling deionized water (10 ml) in a 50 ml conical flask. The solution was stirred vigorously for 1 h at room temperature (r.t.), filtered and left open overnight. On the next day, 2-pyridinecarbaldehyde  was added to the flask, followed by Mn(OAc)2·4H2O  dissolved in 5 ml of water, and the solution was magnetically stirred at r.t. for 30 min, then filtered and allowed to evaporate. Very light-brown needles of I suitable for X-ray crystallography formed within two days in the brown solution. The crystals were filtered off, washed with diethyl ether and dried in air. Yield: 67% (based on Mn). FT–IR : 3430br, 3122s, 3094s, 3050s, 3014, 2954, 2914, 2826, 1654, 1566, 1544, 1454, 1374, 1352, 1328, 1258, 1222, 1148s, 1130, 1038, 920, 800s, 764, 742, 624s, 498, 468, 434. Elemental analysis calculated for C16H18N4MnCl4 (463.08): C 41.50; H 3.92; N 12.10%. Found: C 41.62; H 3.93; N 12.04%.Synthesis of [I in KBr measured in the 4000–400 cm−1 range , the medium intensity bands at 1654 and 1544 cm−1 are associated with heterocyclic ring vibrations, an intense absorption at 1148 cm−1 is ascribed to ν(N–CCH3) vibration and there is a prominent set of three peaks in the out-of-plane C—H-bending region .The FT–IR spectrum of ge Fig. 5 has a di7.Uiso(H) = 1.2Ueq(C) for CH, C—H = 0.98 Å, Uiso(H) = 1.5Ueq(C) for CH3). Anisotropic displacement parameters were employed for the non-hydrogen atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989023002761/jq2027sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989023002761/jq2027Isup2.hklStructure factors: contains datablock(s) I. DOI: 1959314CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: