Caenorhabditis elegans. We call this scaffold Nemacol and show that it inhibits the vesicular acetylcholine transporter (VAChT), a target recognized by commercial animal and crop health groups as a viable anthelmintic target. We demonstrate that it is possible to create Nemacol analogs that maintain potent in vivo activity whilst lowering their affinity to the mammalian VAChT 10-fold. We also show that Nemacol enhances the ability of the anthelmintic Ivermectin to paralyze C. elegans and the ruminant nematode parasite Haemonchus contortus. Hence, Nemacol represents a promising new anthelmintic scaffold that acts through a validated anthelmintic target.Nematode parasites of humans and livestock pose a significant burden to human health, economic development, and food security. Anthelmintic drug resistance is widespread among parasites of livestock and many nematode parasites of humans lack effective treatments. Here, we present a nitrophenyl-piperazine scaffold that induces motor defects rapidly in the model nematode  Harrington et al report their discovery of Nemacol, which is a small molecule inhibitor of the vesicular acetylcholine transporter (VAChT). VAChT loads synaptic vesicles with acetylcholine and is a key point of vulnerability in animals. Harrington et al show that Nemacol has nematode selectivity and potential utility against nematode parasites. Ancylostoma duodenale and Necator americanus) and whipworm (Trichuris trichiura) are recognized to have inadequate efficacy1. Furthermore, the top four marketed anthelmintics used in non-human animals, which include macrocyclic lactones (e.g. Ivermectin), tetrahydropyrimidines (e.g. Pyrantel), imidazothiazoles (e.g. Levamisole) and benzimidazoles (e.g. Thiabendazole), have demonstrated pervasive resistance in cattle and small-ruminants2 as well as companion animals7. The dire need for new anthelmintics has been recognized by academic, industry and governmental experts for some time9. Towards identifying novel candidate anthelmintic scaffolds, our group has recently carried out small molecule screens for those that affect the motor activity of the free-living nematode Caenorhabditis elegans10. One scaffold that we focus on here inhibits cholinergic signalling. The neurotransmitter acetylcholine (ACh) is the primary signalling molecule in most animals that triggers muscle contraction11. Without careful regulation of ACh, animals lose muscular control and die14. The essentiality of cholinergic signaling is a major point of vulnerability that has been repeatedly exploited both in nature as a target of toxins and venoms15 and by humans as a pesticide strategy17.Nematodes that parasitize humans and non-human animals including livestock are a significant burden to human health, food security and economic development. Unfortunately, most frontline anthelmintic molecules suffer from inadequacies. For example, anthelmintics used to treat human hookworm (21 and antagonized by Derquantel24. Acetylcholinesterases (AChE), which break down ACh at the neuromuscular junction and are inhibited by organophosphates and carbamates that in turn lead to catastrophically high levels of synaptic ACh. Finally, vesicular acetylcholine transporter (VAChT), which packages ACh into presynaptic vesicles26, is inhibited by Vesamicol and by the relatively novel spiroindoline scaffold27. Spiroindolines have been investigated as nematode and/or insect parasiticides by Zoetis (patent US20160296499A128), Intervet Inc (patent US9096599B2)29 and Syngenta (patent US9174987B2)30, but none have yet brought a spiroindoline to market27.Three protein groups within the cholinergic pathway are targeted by anthelmintic and nematicidal molecules. These include nicotinic ACh receptors (nAChRs), which are agonized by the imidazothiazoles and tetrahydropyrimidines27), we call this scaffold Nemacol. Nemacol rapidly induces C. elegans worms to coil their bodies, which is a phenotype shared by mutants of the CHA-1 choline acetyltransferase enzyme, which makes ACh, and mutants of the VAChT worm ortholog (UNC-17)26. Herein, we describe the kinetics of the Nemacol-induced phenotypes and provide chemical-genetic and biochemical evidence to show that Nemacol inhibits nematode VAChT. We demonstrate that Nemacol also disrupts the motor activity of the commercially important animal parasites Dirofilaria immitis (dog heartworm) and Haemonchus contortus (a nematode parasite of ruminants). We also demonstrate that Nemacol can enhance the macrocyclic lactone Ivermectin to kill C. elegans and H. contortus. Finally, we show that select Nemacol analogs can maintain their low micromolar potency in nematodes but reduce their affinity for the mammalian VAChT receptor tenfold relative to the Nemacol parent, demonstrating the potential for an expanded therapeutic window. We conclude that Nemacol is a novel small molecule scaffold with anthelmintic potential.Here, we present a novel nitrophenyl-piperazine scaffold that inhibits VAChT. Because of its structural similarity to the VAChT inhibitor Vesamicol  and elicit similar uncoordinated motor phenotypes  locomotion’  antagonizes both the metabolism of Nemacol-1 (P < 0.05) and the dissipation of these phenotypes (P < 0.001)  31 should be hypersensitive to the compound, which is what we observed (P < 0.001)  Fig. .Fig. 2NeC. elegans UNC-17 C391Y mutation called md414 had previously been shown to disrupt Vesamicol binding32. We reasoned that if Nemacol inhibits VAChT through a shared binding site with Vesamicol, then unc-17(md414) mutant animals should suppress Nemacol phenotypes, which is what we observed (P < 0.001)  and aldicarb (a carbamate) in a dose-dependent manner   Fig. . The abi15) Fig. .Fig. 3Ne3H]Vesamicol bound to rat VAChT expressed in rat PC12A123.7 cells, which is an assay previously established for measuring small molecule affinity for VAChT34. We found that Nemacol-1 binds mammalian VAChT, but with 37-fold less affinity than Vesamicol (P < 1E-15)  Fig. . TogetheC. elegans worms, but has 37-fold less affinity than Vesamicol for the mammalian VAChT target. This comparison raised the possibility that the Nemacol scaffold could be modified to create nematode-selective VAChT inhibitors. Although co-crystal structures of Vesamicol with VAChT are not available, several residues known to be important for interaction are known36. Inspection of a VAChT multiple sequence alignment shows that residues immediately flanking Vesamicol-interacting residues are divergent in nematodes . We found many analogs to be active against C. elegans and Pristionchus, and two analogs (Nemacol-1 and Nemacol-5) to have low micromolar activity against Dirofilaria  reveals an activity ratio of 0.11. By contrast, Nemacol-63’s equivalent ratio is 1.10, which is an improvement of over tenfold in selectivity. The two analogs with the poorest VAChT affinity (Nemacol-62 and 63) share a cyclopentylmethyl group in the R2 position, suggesting that this feature diminishes affinity with mammalian VAChT. These results suggest that it may be possible to modify the Nemacol scaffold to achieve nematode selectivity.Next, we chose 12 diversely structured analogs that had good activity against ity Fig. b, c. Com− channel agonist39. Nemacol inhibits VAChT and consequently depresses cholinergic signaling by decreasing synaptic vesicle ACh content. We reasoned that Nemacol, like derquantel, might therefore synthetically interact with macrocyclic lactones to disrupt nematode neuromuscular function. Indeed, we found that in 3-day liquid viability assays (see 'Methods'), combinations of Ivermectin and Nemacol-1 could yield effective killing of C. elegans at concentrations that had negligible effect on their own  synergy score of 20.0, which exceeds the ZIP score threshold of synergy (10)42 . Notably, the double drug treatment remains synergistic in the triple mutant  reduction-of-function mutant was more sensitive to the effects of Ivermectin. Indeed, unc-17(e245) demonstrated a sensitivity to Ivermectin (P < 1E-15 relative to wild type’s sensitivity to Ivermectin) that was comparable to 30 μM Nemacol treatment (P < 1E-15 relative to wild type’s sensitivity to Ivermectin)  Fig. . This suDirofilaria immitis is known to be refractory to Ivermectin in in vitro assays50, we asked whether enhancement may be seen with the ruminant nematode parasite Haemonchus contortus51. A small survey indicated that Nemacol-53 has activity against H. contortus, with an EC30 of 26 μM   Fig. . These d52. Hence, the identification of novel and selective nematicidal compounds is a key step in protecting human health and food security. Here, we have identified the Nemacol scaffold that disrupts nematode motor function via the inhibition of VAChT. Select Nemacol analogs maintain nematode activity whilst losing affinity for mammalian VAChT, suggesting that nematode selectivity can be achieved with this scaffold. Nemacol is detoxified in C. elegans over the course of 24 h. However, assays performed here against multiple parasitic nematodes over an equivalent timespan (or longer) .In contrast to Vesamicol, the spiroindoline scaffold has been rigorously pursued as a candidate anthelmintic because it selectively incapacitates nematodes and insects56 highlights that compounds with a Log P greater than 4 have accumulative potential in adipose tissue of animals. The primary spiroindoline lead pursued by Syngenta (SYN876 in ref. 27) has a SwissADME predicted consensus LogP of 5.53 suggesting that this lead may have concerning accumulative potential. SwissADME is a highly cited chemoinformatics tool that is widely used in the field57. In contrast, 92% of Nemacol analogs had a SwissADME predicted consensus Log P ≤ 4.0 with a median of 2.9357  .Relative to the structural complexity of the spiroindoline scaffold (MW 453.53 with a SwissADME synthetic accessibility score of 4.23)see Fig. . The hig30. One reason for this is it may be difficult to mutate VAChT to a state that reduces an inhibitor’s efficacy without compromising the transporter itself. The VAChT mutant residue that confers resistance to Vesamicol disrupts the transporter’s ability to interact with the inhibitor, reduces the transporter’s interaction with acetylcholine and lead to motor defects in C. elegans31. Hence, missense mutations that alter the ability of VAChT inhibitors to interact with the transporter will likely confer a clear selective disadvantage to worms in the absence of Nemacol pressure. Ensuring persistence of drug-sensitive alleles within population is becoming a more common practice of parasite management59. A second reason for the keen interest in the VAChT target may be because of its high conservation across nematodes60  operated in accordance with the local Animal Care and Use Committee and governmental authorities (LANUV#200/A176 and #200/A154). The Zamanian group sources their Dirofilarial nematodes from the NIH FR3 (BEI Resources) resource center. Animal research at the FR3 complies with all relevant ethical regulations and operates under the University of Georgia IACUC (AUP#: A2019 04-010-Y1-A0). The INVENesis group performs animal experimentations in the Infectiology of Farm, Model, and Wildlife Animals Facility . Experimental protocols were designed in compliance with French law  concerning the use of laboratory animals. Care and euthanasia of animals were practiced according to the national ethical guidelines and approved by the local ethics committee for animal experimentation : APAFIS#17560. The authors are committed to the principles of the 3Rs: reduction, refinement, and replacement of experimental animals.C. elegans used in the experiments is hermaphroditic. There are no visible markers of sex differentiation in the Dirofilaria or Haemonchus larvae used herein and there is no evidence of drug effects unevenly distributed across larvae. Given the numbers of Dirofilaria and Haemonchus used in the Kulke, Zamanian and INENesis experiments, both sexes were likely well-represented.The sex of 62. The N2 (wild-type) strain of Caenorhabditis elegans and C. elegans mutant strains were obtained from the C. elegans Genetic Center (University of Minnesota). Synchronization of worm developmental stages was achieved using standard bleach preparation protocols63. Synchronized young adult animals are acquired by incubating synchronized L1s  food for 64–72 h at 20 °C. Worms were maintained on Modified Youngren’s, Only Bacto-peptone (MYOB) media containing 2% agar with a surface lawn of OP50 strain Escherichia coli64. Photomicrographs shown in Fig. All nematode strains were cultured using standard methods at 20 °C unless otherwise indicatedE. coli bacteria culture from a saturated Luria Broth culture was added by pipette onto the surface of the culture media. Plates were allowed to dry on a benchtop proximal to flame for 15 min. Plates were covered and wrapped in tinfoil and were used the following day.Molten Modified Youngren’s, Only Bacto-peptone (MYOB) containing 2% agar was equilibrated to 55 °C in a water bath. Small-molecules solvated in DMSO were spiked into at least 4 mL of molten media mixture in 15 mL conical tubes, inverted five times and vortexed. The final concentration of dimethyl sulfoxide (DMSO) in each of the wells was 1% v/v. One mL of media containing small-molecule was pipetted into wells of a 24-well plate using a 10 mL Sarstedt serological pipette. Plates were dried under sterile air flow in a laminar flow hood for 90 min. After 90 min 25 μL of OP50 strain Escherichia coli on each well. Each compound was added to the MYOB substrate before pouring to achieve the desired final concentrations of 30 µM or 60 µM after diffusion through the media. Synchronized young adult worms are transferred into each well using a platinum wire pick. A Leica MZ75 stereomicroscope was used to visualize the movement of worms on the solid substrate. The specific dominant locomotor phenotype  was scored after the touch on the head with a platinum wire over a ~3–5 s of observation. Animals were scored as having either Punc locomotion if animals exhibit a lack of smooth locomotion with frequent abrupt pauses before and after a touch on the head; as coiler if animals are observed in a coiled position ) seeded with 25 µL of OP50 C. elegans larval development assays were conducted in 96-well clear flatbottom plates. ~20 L1 larvae in 10 μL of M9 buffer were pipetted into each test wells containing 40 μL of Nematode Growth Media (NGM)65 media supplemented with HB101 E. coli with the desired test compound (+0.6% dimethyl sulfoxide (DMSO) as the chemical solvent). Plates were wrapped in three layers of brown paper towels soaked with water. After either 3 days of incubation the number of C. elegans animals of different larval stages were recorded using a Leica MZ75 stereomicroscope.i data are the Ki of nemacol analog as determined from competitive radioligand [3H]Vesamicol binding assays using [3H]Vesamicol and PC12 cells expressing ratVAChT. [3H]Vesamicol was procured from PerkinElmer , Am = 1591 GBq/mmol)). Stably transfected PC12 cells expressing ratVAChT were shared by Dr. Ali Roghani  and radioligand competition experiments were conducted using standard protocols67.Rat VAChT KDirofilaria immitis in two labs. In the first set of experiments, shown as part of Fig. D. immitis microfilariae and larval stage 3 (L3) worms were assayed in the laboratories of Bayer Animal Health GmbH . For microfilariae immobility assays, approximately 250 freshly purified microfilariae were cultured in single wells of a 96-well microtiter plate containing supplemented RPMI 1640 medium69. Compounds were added in the following concentrations: 50 µM, 10 µM, 2 µM, 0.4 µM, 0.08 µM, 0.016 µM and 0.0032 µM. Microfilariae exposed to medium substituted with 1% DMSO were used as negative controls. Motility of microfilariae was evaluated after 72 h of drug exposure using an image-based approach—DiroImager, developed by Bayer Technology Services. As described in detail in refs. 69, the DiroImager is an automated high-throughput platform that allows for high-resolution optical imaging of an entire 96-well microtiter plate. Data are reported as the EC50 (µM) calculated from the tested concentration series.We conducted experiments with the Missouri isolate of Dirofilaria immitis larval development assays, freshly isolated L3s were cultured in wells of a 96-well microtiter plate with 10 L3s per well. All wells contained supplemented RPMI 1640 medium69 and a test compound at one of the following concentrations: 10 µM, 2 µM, 0.4 µM, 0.08 µM, 0.016 µM and 0.0032 µM. L3s exposed to DMSO only (1%) were used as negative controls. All drug concentrations were tested in duplicate and drug effects were evaluated after 72 h of incubation, after which DMSO-only controls are L4s and the shed L3 cuticles are evident in the wells. Compounds that have dramatic acute effects retard or arrest the growth of the nematodes and they remain L3s, while other compounds have less severe effects and the nematodes grow to the L4 stage. Motility was scored for each sample in the following standard manner: The 20 worms total from the two duplicate sample wells are considered. If only one of the 20 worms moves (as detected by pixel displacement with the DiroImager), the score for that sample is considered as 5% motility. Data on a given day were considered as valid only if the DMSO-only controls exhibited at least 90% motility. Data is then reported as the EC50 (µM) calculated from the tested concentration series.For the Dirofilaria immitis experiments in the lab of M. Zamanian (Univeristy of Wisconsin). For this experiment, shown in Supplementary Fig. 70. Blood was drawn and shipped approximately 24 h before use. Upon arrival, blood was warmed to 37 °C then combined with a 0.85% sodium chloride and 0.2% saponin solution in a 1:11 volumetric ratio and incubated in a 37 °C water bath for 15 min. The hemolyzed solution was passed through a 25 mm 5.0 μm pore size syringe filter. The used filter disks were transferred to petri dishes filled with RPMI 1640 culture media with 1% penicillin/streptomycin (0.1 mg/mL) and incubated at 37 °C for 2 h while microfilariae (mf) separated from the disks. Disks were discarded and mf titered to 5 mf/μL.We also conducted 2. Videos of the plates were taken every 24 h for 72 h using an ImageXpress Nano . A full protocol for the imaging process can be found here: 10.21203/rs.3.pex-1916/v2. After acquiring the 72-h timepoint video, viability staining was performed using the CellTox Green kit (Promega); a full protocol for this procedure can also be found at the previous link. Image analysis and the subsequent measurement of optical flow and fluorescence was performed using wrmXpress v1.3.0271.Aliquots of titered mf were incubated on a heating block at 60 °C for 1 h to produce heat killed positive controls. One μL of 100× drug was aliquoted to each well of a 96-well plate; 100 μL of live mf were added to treatment wells and 100 μL of heat killed mf were added to positive control wells . Plates were sealed with a breathable plate cover and maintained at 37 °C and 5% atmospheric CO72, measures the effect of compounds on the third larval (L3) stage of Haemonchus contortus nematodes . Approximately 300 L3s were exposed to the drug or control treatments in each well of a 96-well plate. In all experiments, non exsheated L3s were exposed for 24 h to the compound in a solution of 1.5% of DMSO and 0.00425% of Tween 20. Larvae were then transferred to a migration plate, which is a 96-well plate allowing H. contortus L3s to migrate from a deposit area to a trap area through a corridor. The mobility of the worms in the trap area is monitored over a 21 min time window by an automated data acquisition system equipped with a Basler acA2000-50gm camera with which pixel displacement is measured. The effect of compounds is expressed as a percent reduction of motility compared to negative controls. Nemacol-53 was tested in three replicates at the following concentrations: 120 µM, 60 µM, 30 µM, 15 µM, 7.5 µM, 3.75 µM, 1.875 µM, 0.9375 µM, and 0 µM. Ivermectin was tested in three replicates at the following concentrations: 20 µM, 5 µM, 1.25 µM, 0.3125 µM, 0.078125 µM, 0.0195 µM, 0.0049 µM, 0.0012 µM and 0 µM alone and along with the estimated dose corresponding to the EC30 of Nemacol-53.The INVENesis Migration Trap Assay (MTA), previously described in ref. C. elegans cytochrome P450 oxidoreductase EMB-8, whose loss-of-function phenotype is embryonic lethality, we used the MJ69 strain harbouring the emb-8(hc69) temperature-sensitive allele in combination with RNAi targeting emb-8 transcript degradation as done previously73. Embryonic lethality is avoided by growing MJ69 at 15 oC, obtaining synchronized L1s, placing the L1 animals on emb-8(RNAi)-inducing bacterial food (in the background of the HT115 E. coli strain), then shifting the temperature to 25  oC. Wild-type controls are treated the same way and grown on L4440 mock-RNAi-inducing bacterial food. The RNAi is used to increase the reduction of function of emb-8. The RNAi-by-feeding approach has been previously described74. Briefly, RNAi-inducing plates were prepared by growing liquid bacterial cultures overnight in the presence of 100 μg/mL ampicillin to saturation. The following day, 6 cm MYOB agar plates with final concentrations of 100 μg/mL carbenicilin and 1 mM IPTG were seeded with 250 μL of the bacterial culture and dried at room temperature overnight. Worms were allowed to feed for 2 days before being transferred to experimental ‘drug’ plates. All ‘drug’ plates contained final concentrations of 100 μg/mL carbenicilin and 1 mM IPTG and were seeded with the emb-8(RNAi) and L4440(RNAi) control bacteria. Samples were then analyzed for phenotype as described above or for ‘drug’ metabolites, described in another section.To disrupt the 75. After three washes in gelatin-M9, 500 µL of worm suspension was added to each well of Pall ACropPrep 96-well filter plates . The buffer was drained from the wells by vacuum and the worms were resuspended in 50 µL of M9 buffer and frozen at −80 °C. The samples were later lysed by adding 35 μl of a 2× lysis solution  to each tube at 56 °C for 1 h. Prior to HPLC coupled to a diode array detector (HPLC-DAD) analyses analysis, 70 µL of acetonitrile was added to the lysates. The samples were then mixed by vortexing for 10 s, and centrifuged at 17,949×g for 2 min. After centrifugation, 100 µL of the lysate was injected onto a 4.6 × 150 mm Zorbax SB-38 column (5-μm particle size) and eluted with solvent and flow rate gradients over 8.65 min. UV–Vis absorbance was measured every 2 nm between 190 and 602 nm. Using MATLAB (The MathWorks), absorbance intensity values were converted to three-dimensional heat-mapped chromatograms. A sample of 5 nmol pure Nemacol-1 was processed prior to worm samples to determine the compounds elution time and absorbance spectrum. All HPLC was performed a using an HP 1050 system equipped with an autosampler, vacuum degasser, and a variable wavelength diode-array detector. The solvent and flow rate gradients are indicated in the chart below. Data analysis was done using HP Chemstation software. The area under the curve (AUC) was automatically integrated by the software for quantification of Nemacol-1 absorbance peaks. For each sample, the ratio of parent and individual compound metabolite  AUC to the total AUC for all compound related peaks  were calculated. As an example, for the calculation of the parent compound ratio, the AUC for the parent compound was divided by the total area under the curve for the parent compound and all metabolite related peaks.Young adult worms were prepared on RNAi-inducing plates as described above. Adult worms were then incubated on drug plates for 6 h at 25 °C unless otherwise noted with 2000 worms per plate. After incubation, worms were washed off the agar plates and suspended in M9 buffer containing 0.5% gelatinHPLC solvent and flow rate gradientst tests, or Chi-square tests were conducted between control and treatment groups where appropriate and as indicated in figure legends. In Fig. P values were calculated using the extra sum-of-squares F-tests comparing EC50 curves generated for dose-response data in GraphPad Prism (version 9.3.1). In Fig. P value (P < E-15) was calculated using Fisher’s Method with extra sum-of-squares F test comparing the binding curve of (−)Vesamicol to the binding curve of nemacol-1 conducted in parallel (on the same day with the same cell preparation).Unpaired one- or two-sided Nemacol analogs were procured from several commercial sources as dry compound. Molecules were sourced from Chembridge Inc., Enamine and OTAVA chemicals, Ltd. Vesamicol was purchased from Sigma-Aldrich as (±)-Vesamicol hydrochloride (product ID V100).4) stain. Flash column chromatography was performed on Siliaflash P60 40-63 µm silica gel purchased from Silicycle. NMR characterization data were obtained at 293 K on a Varian Mercury 300 MHz, Varian Mercury 400 MHz, Bruker Advance III 400 MHz, Agilent DD2 500 MHz equipped with a 5 mm Xses cold probe or Agilent DD2 600 MHz. 1H spectra were referenced to the residual solvent signal . 13C{1H} spectra were referenced to the residual solvent signal . Data for 1H NMR are reported as follows: chemical shift (δ ppm), multiplicity , coupling constant (Hz), integration. NMR spectra were recorded at the University of Toronto Department of Chemistry NMR facility (http://www.chem.utoronto.ca/facilities/nmr/nmr.html). Infrared spectra were recorded on a PerkinElmer Spectrum 100 instrument equipped with a single-bounce diamond/ZnSe ATR accessory in the solid state and are reported in wavenumber (cm−1) units. High-resolution mass spectra (HRMS) were recorded at the Advanced Instrumentation for Molecular Structure (AIMS) in the Department of Chemistry at the University of Toronto (https://www.chem.utoronto.ca/chemistry/AIMS.php).Unless otherwise stated, all reactions were carried out under an atmosphere of dry argon, using glassware that was either oven (120 °C) or flame-dried. Work-up and isolation of compounds was performed using standard benchtop techniques. All commercial reagents were purchased from chemical suppliers  and used without further purification. Dry solvents were obtained using standard procedures . Reactions were monitored using thin-layer chromatography (TLC) on EMD Silica Gel 60 F254 plates. Visualization was performed under UV light (254 nm) or using potassium permanganate , potassium carbonate , dimethyl sulfoxide (4.7 mL) then the corresponding fluorobenzene derivative  and the mixture was stirred at 100 °C. Once the reaction was complete as indicated by TLC (approximately 22 h), the reaction mixture was diluted with ethyl acetate, transferred to a separating funnel and washed with water (three times) then saturated aqueous sodium chloride. The organic phase was dried with Na2SO4, filtered, then concentrated on a rotary evaporator and the substituted 1-phenylpiperazine (1a–f) was obtained. The crude solid was used in the next step without further purification.The procedure was modified from literature1 , dry acetonitrile (1.0 mL), triethylamine  then the alkyl halide or toluenesulfonate  and the mixture was stirred at reflux until completion as indicated by TLC (~24 h). The mixture was diluted with ethyl acetate, transferred to a separating funnel and washed with water (three times) then saturated aqueous sodium chloride. The organic phases were combined and dried with Na2SO4, filtered, and concentrated on a rotary evaporator. The residue was purified by column chromatography with the indicated eluent and the alkylated arylpiperazine (2) was obtained.To a round-bottom flask were added 77. To a round-bottom flask were added cyclopentylmethanol  and dry dichloromethane (1.6 mL) and the flask was submerged in an ice-water bath. Triethylamine  was added to the flask followed by 4-(dimethylamino)pyridine  then p-toluenesulfonyl chloride  and the mixture was warmed to room temperature and stirred until completion as indicated by TLC (~3 h). Dichloromethane and water were added, then the mixture was transferred to a separating funnel and the layers were separated. The organic phase was washed with water (two times) followed by saturated aqueous sodium chloride, then dried over Na2SO4, filtered, and concentrated on a rotary evaporator. The residue was purified by column chromatography (19/1 v/v pentanes/ethyl acetate) and cyclopentylmethyl 4-methylbenzenesulfonate  was obtained as a colourless oil. The spectral data were in accordance with literature.The procedure was modified from literature2a was isolated as a bright yellow solid (MP = 61–63 °C). 1H NMR : δ 7.46 , 6.83 , 3.32–3.28 , 2.52–2.48 , 2.15 , 1.78 , 1.74–1.63 , 1.50 , 1.28–1.11 , 0.93–0.83 . 13C NMR : δ 153.6, 133.5, 120.2, 114.1, 99.9, 65.6, 53.3, 47.2, 35.1, 31.9, 26.9, 26.2. IR (neat): 2922, 2849, 2780, 2216, 1599, 1515, 1447, 1247, 1177, 818. HRMS (DART): calc. for C18H26N3 284.2127 [M + H]+, found 284.2132.Synthesized according to general procedures A and B from 4-fluorobenzonitrile and (bromomethyl)cyclohexane. Compound 2b was isolated as a bright yellow solid (MP = 52–54 °C). 1H NMR : δ 8.05–8.00 , 6.98 , 3.04 , 2.61–2.52 , 2.35 , 2.20 , 1.85–1.64 , 1.59–1.47 , 1.32–1.14 , 0.90 . 13C NMR : δ 157.7, 142.3, 132.2, 126.8, 122.8, 118.2, 65.7, 53.8, 51.2, 35.1, 32.0, 26.9, 26.3, 19.0. IR (neat): 2918, 2842, 2810, 1583, 1504, 1447, 1328, 1228, 1011, 893. HRMS (DART): calc. for C18H28N3O2 318.2182 [M + H]+, found 318.2178.Synthesized according to general procedures A and B from 1-fluoro-2-methyl-4-nitrobenzene and (bromomethyl)cyclohexane. Compound 2c was isolated as a bright yellow solid (MP = 75–77 °C). 1H NMR : δ 8.06 , 6.68 , 6.61 , 3.39–3.35 , 2.62 , 2.53–2.49 , 2.16 , 1.82–1.75 , 1.75–1.64 , 1.50 , 1.29–1.12 , 0.94–0.83 . 13C NMR : δ 154.0, 139.0, 137.3, 127.9, 116.2, 111.2, 65.6, 53.3, 47.1, 35.1, 32.0, 26.9, 26.2, 22.8. IR (neat): 2920, 2843, 1638, 1600, 1533, 1249, 1226, 1001, 925, 822. HRMS (DART): calc. for C18H28N3O2 318.2182 [M + H]+, found 318.2189.Synthesized according to general procedures A and B from 4-fluoro-2-methyl-1-nitrobenzene and (bromomethyl)cyclohexane. Compound 2d was isolated as a white solid (MP = 66–70 °C). 1H NMR : δ 8.17 , 6.92 , 3.40 , 2.43 , 2.12 , 1.76–1.70 , 1.69–1.58 , 1.50 , 1.26–1.07 , 0.89–0.77 . 13C NMR : δ 155.2, 146.3, 108.0, 64.6, 52.6, 45.5, 34.2, 31.2, 26.4, 25.5. IR (neat): 2919, 2849, 1638, 1600, 1533, 1249, 1132, 1001, 989, 805. HRMS (DART): calc. for C16H26N3 260.21267 [M + H]+, found 260.2120.Synthesized according to general procedures A and B from 4-chloropyridine hydrochloride and (bromomethyl)cyclohexane. Compound 3a. Compound 2e was isolated as a light orange solid (MP = 49–51 °C). 1H NMR : δ 7.47 , 6.84 , 3.34–3.29 , 2.59–2.52 , 2.30 , 2.08 , 1.80–1.71 , 1.65–1.48 , 1.26–1.16 . 13C NMR : δ 153.6, 133.6, 120.3, 114.1, 100.0, 64.5, 53.1, 47.2, 37.2, 31.5, 25.3. IR (neat): 2947, 2936, 2864, 2816, 2211, 1601, 1513, 1448,1166, 921. HRMS (DART): calc. for C17H24N3 270.19702 [M + H]+, found 270.1968.Synthesized according to general procedures A, B and C from 4-fluorobenzonitrile and 2f was isolated as a dark orange solid. The characterization data are in accordance with literature78. 1H NMR : δ 8.12 , 6.82 , 3.46 , 2.68 , 2.32 , 0.90 , 0.58–0.54 , 0.16–0.12 . 13C NMR : δ 155.0, 138.5, 126.1, 112.7, 63.7, 52.8, 47.1, 8.4, 4.1.Synthesized according to general procedures A and B from 1-fluoro-4-nitrobenzene and (bromomethyl)cyclopropane. Compound Further information on research design is available in the Supplementary Information.Supplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Supplementary Movie 7Description of additional supplementary filesReporting SummaryPeer Review File


The biochar doses and irrigation methods significantly (p < 0.05) increased maize growth and yield characteristics, irrigation water use efficiency (IWUE), and fertilizer N use efficiency (FNUE) and enhanced the soil properties. In the BI and DI method, the NB1, NB2, and NB3 treatments increased yield by 4.96%–6.10%, 8.36%–9.85%, and 9.65%–11.41%, respectively, compared to NB0. In terms of IWUE and FNUE, the non-biochar treatment had lower IWUE and FNUE compared to biochar combined with N fertilizer treatments under both BI and DI methods. In the BI method, the IWUE in NB2 and NB3 ranged from 3.36 to 3.43 kg kg−1, and in DI, it was maximum, ranging from 5.70 to 5.94 kg kg−1. Similarly, these medium and high doses of biochar increased the FNUE of maize. The FNUEs in NB2 and NB3 under BI ranged from 38.72 to 38.95 kg kg−1 and from 38.89 to 39.58 kg kg−1, while FNUEs of these same treatments under DI ranged from 48.26 to 49.58 kg kg−1 and from 48.92 to 50.28 kg kg−1. The effect of biochar was more obvious in DI as compared to the BI method because soil water content (SWC) and soil N concentrations (SNCs) were higher at rhizosphere soil layers under DI. Biochar improved SWC and SNC at 0–20 cm and 20–40 cm soil layers and decreased below 60-cm soil layers. In contrast, despite biochar-controlled SWC and SNCs, still, values of these parameters were higher in deeper soil layers. In the BI method, the SNCs were higher at 60–80 cm and 80–100 cm compared to the top and middle soil layers. Depth-wise results of SNC demonstrated that the biochar’s ability to store SNC was further enhanced in the DI method. Moreover, biochar increased soil organic matter (OM) and soil aggregate stability and maintained pH. The NB0 treatment increased soil OM by 11.11%–14.60%, NB2 by 14.29%–19.42%, and NB3 by 21.98%–23.78% in both irrigation methods. This increased OM resulted in improved average soil aggregates stability by 2.45%–11.71% and 4.52%–14.66% in the BI and DI method, respectively. The results of our study revealed that combined application of N fertilizer with a medium dose of biochar under the DI method would be the best management practice, which will significantly increase crop yield, improve SWC, enrich SNC and OM, improve soil structure, and maintain pH.Long-term application of nitrogen (N) fertilizer adversely degrades soil and decreases crop yield. Biochar amendment with N fertilizer not only can increase yield but also can improve the soil. A 3-year field experiment was conducted to determine the effect of biochar doses with N fertilizer on maize yield and soil N and water dynamics under border irrigation (BI) and drip irrigation (DI) methods. Treatments were 260 kg N ha Maize cNitrogen is a key component of chlorophyll, proteins, and nucleic acids, which are involved in various physiological processes, such as photosynthesis, respiration, and enzyme activity . An adeqBiochar, a multifunctional porous substance with a tiny particle size, a large surface area, a low bulk density, a high adsorption capacity, and an abundant carbon content, has received a great deal of attention because it provides numerous advantageous functions for crop production and soil science . Biochar4+  and high FNUE and crop yield (The combined application of N fertilizer and biochar has received a lot of interest because of its particular advantages in boosting soil N content (SNC) and improving FNUE . Mixed a4+ ; (iii) i4+ ; (iv) it4+ ; and (v)4+ . The ben4+  and higop yield . Also, bop yield . HoweverBorder irrigation (BI) and drip irrigation (DI) are two commonly used irrigation methods in maize production systems. BI comprises flooding the field with water through border ridges, allowing the water to seep into the soil . This meThe combined application of N fertilizer rates with different biochar dosages under BI and DI methods could have interactive effects on maize growth, nutrient uptake, and yield. The interaction between N and biochar may influence nutrient availability and water retention at different depths of the soil, thereby affecting plant growth and productivity. 22.1The experiment site is located in Qiqihar (47°21′15.65″ N latitude and 123°55′5.47″ E longitude), in the west-central part of Heilongjiang Province, northeastern China. The region has a temperate continental monsoon climate at a high altitude, spring is dry and windy, and summer is hot and rainy. Autumn is shorter, and winter is chilly and long, where the 24-h average summer temperature is 21.5°C, and the average annual rainfall is 410–540 mm; approximately 70% of rainfall occurs in the summer season. In the case of weather conditions during the present study, the rainfall was low in 2017 compared to 2018 and 2019. Rainfall mainly occurred in June and July as well as in August. The temperature and rainfall during the maize growing season are shown in 2.2−1 alone and combined with low, medium, and high doses of biochar (B), namely, 15.5 t ha−1, 30.7 t ha−1, and 45.3 t ha−1 , respectively, under BI and DI methods. There were three replications. The maize variety was Nendan 19, and sowing dates were 7–9 May of each year. To achieve the recommended plant population of 67,000 ha−1, 28–30 kg·ha−1 seed was used. Seeds were sown in rows approximately 75 cm apart, and plant-to-plant spacing was 30 cm. The biochar used in this study was provided by Liaoning Biochar Engineering Technology Center and made from corn straw pyrolysis. It contained NPK 9.85 N, 1.63 P, 19 K, 55 Si, 2.9 Mg, 3.7 Ca, 163 O, and 595 OC g·kg−1. The basic pH of biochar was 7.5, and particle composition % was as follows: 14 (<0.1 mm), 61.3 (0.1–2 mm), and 25.1 (>2 mm). In the DI method, the irrigation amount was 220 mm and applied in small doses, with each application being 31.42 mm, while in the BI method, the irrigation amount was 300 mm and applied five times, with each application being 60 mm. In the DI method, the sub-main pipeline system supplied water to the laterals, while the lateral pipes on the plot carried water directly to the root zones of the crops. The inner diameter of the lateral pipes was 1.5 cm at a flow rate of 1.3 L h−1. The treatment plots were separated from adjoining plots by 1-m-wide isolation strips in order to account for the marginal effects of various irrigation techniques, and each plot (144 m2) was 18 m long and 8 m wide. In the experimental plot, the planting density was 7–8 plants/m2. The spacing between plants within a row was 14.4 cm, and the drip tapes were spaced apart by 110 cm. The plants were sowed in alternately wide and narrow rows, measuring 0.8 m and 0.3 m. A water reading meter was used to measure the irrigation water amount. To promote optimal seed germination and vigorous seedling establishment, the total dose of phosphorus (50 kg ha−1) and potassium fertilizers (250 kg ha−1) was incorporated into the soil during seedbed preparation. The source of phosphorus was monoammonium phosphate with an NPK content of 12:61:0, produced by Guizhou Kai Phosphorus Group Co., Ltd. . Potassium sulfate was used as the source of potassium, and its NPK ratio was 12:0:50, which is produced by Luobupo Potassium Salt Co., Ltd. . To control the weed infestation in the experimental field, mechanical plough operation was performed between space of rows at the 3–5 leaf stage, and manual hand hoeing was performed at the 6–7 leaf stage. The cropping scheme is shown in The field experiments were conducted from 2017 to 2019. Treatments were 260 kg N ha2.3−1 KCl  to estimate the soil mineral N concentration (SNC), which was then measured using a continuous flow analyzer . The leaf area index (LAI) was measured on different growth stages using a portable leaf area meter. When maize reached its full physiological maturity and grain moisture dropped below 13%, then it was harvested, and grain yields and dry matter (DM) were obtained. The irrigation water use efficiency (IWUE), fertilizer nitrogen use efficiency (FNUE), and yield changes were calculated as follows:, P is precipitation (mm), ET is evapotranspiration (mm), WUE is water use efficiency, IWUE is irrigation use efficiency, and FNUE is fertilizer N use efficiency. NB0 is 260 kg N ha−1 without biochar amendment; NB1, NB2, and NB3 are combinations of N fertilizer with low, medium, and high doses of biochar, namely, 15.5 t ha−1, 30.7 t ha−1, and 45.3 t ha−1, respectively.where 2.4p < 0.05 using Duncan’s multiple ranges. Simple data processing was done in Excel and graphically visualized using Origin2021 software.The data were statistically analyzed using IBM SPSS Statistics 26. Analysis of variance (ANOVA) was used to determine differences between the control treatment, chemical fertilizer alone, and the interaction of biochar with chemical fertilizer on maize yield. The significant differences were detected at 33.10, the SWC was high at 60–80 cm and 80–100 cm soil depths under the BI method, and it was also higher in the DI method as well. All biochar-added treatments decreased ineffective SWC and maintained SWC at the optimum level. The NB1, NB2, and NB3 treatments under the BI method decreased SWC content at the 80[cm soil depth compared to NB0. The same treatments, NB1, NB2, and NB3, under the DI method, decreased SWC at 60-cm to 100-cm soil depths. The biochar doses were high in NB2 and NB3 treatments; therefore, these treatments mainly enhanced SWC at 20-cm to 40-cm soil depths. Despite the high dose of biochar in NB2 and NB3 treatments restricting water from infiltrating in the deeper soil layers, water still moved into deeper soils under the BI method. However, this treatment showed a clear effect in the DI method. Considering the last season of the experiment, the SWC showed a different pattern of water distribution in the soil profile as compared to the first season. It could be because biochar does not decompose well in the early stage, so biochar particles absorb and store water. In the last season, still, the effect of biochar on different soil depths was positive under the DI method as well as the BI method. Biochar application also decreased ineffective SWC in the soil profile during the last season of the experiment, particularly in the deep soil layer. Maize is a shallow-rooted crop; therefore, it is critical that SWC content must be higher in the topsoil layer. Overall, experimental results related to SWC indicate that biochar application would be effective in BI and DI methods; however, more benefits of biochar can be achieved under the DI method as compared to the BI method.3.23.2.10 and higher in 60–80 cm and 80–100 cm depths. This could be due to the maximum transportation of N along with water from the topsoil layers and accumulation in deeper soil layers. Despite the irrigation amount being high in the BI method, biochar holds N in upper soil layers in all treatments containing biochar sources. Biochar combined treatments, including NB1, NB2, and NB3, resulted in maximum SNC at 0–20 cm, 20–40 cm and 40–60 cm depths and decreased SNC in 60–80 cm and 80–100 cm soil depths. This trend of N accumulation in soil was similar in the 2017, 2018, and 2019 seasons. However, SNCs were relatively higher in the last year of the experimental field. In the BI method, SNC level of NB0 ranged from 2.81 to 3.41 mg·kg−1 at 0–20 cm soil depth, from 5.86 to 5.55 mg·kg−1 at 20–40 cm depth, from 6.45 to 7.91 mg·kg−1 at 40–60 cm depth, from 7.41 to 8.12 mg·kg−1 at 60–80 cm depth, and from 5.06 to 7.50 mg·kg−1 at 80–100 cm soil depth. In NB1, NB2, and NB3 treatments, the SNC ranged from 5.41 to 8.13 mg·kg−1 at 0–20 cm soil depth, from 9.86 to 10.76 mg·kg−1 at 20–40 cm depth, from 7.27 to 9.86 mg·kg−1 at 40–60 cm depth, from 2.88 to 3.32 mg·kg−1 at 60–80 cm depth, and from 2.46 to 3.72 mg·kg−1 at 80–100 cm soil depth.In this irrigation method, the SNC at 0–20 cm, 20–40 cm, and 40–60 cm depths were significantly lower in non-biochar treatment NB3.2.21, NB2, and NB3 treatments. Unlike the BI method, the DI method decreased SNC at 60–80 cm and 80–100 cm deeper soil layer depths. This could be because N did not transport along with water application from the topsoil layers and accumulated in topsoil layers because the amount of water in the DI method was significantly lower than in the BI method. Overall, results indicated that biochar holds maximum N in upper soil layers under the DI method. In all treatments, the SNC at 60–80 cm and 80–100 cm were substitutionally lower than in the BI method. The biochar’s ability to store SNC was further enhanced in the DI method. In the DI method, SNC in NB0 ranged from 7.41 to 7.84 mg·kg−1 at 0–20 cm soil depth, from 9.90 to 11.72 mg·kg−1 at 20–40 cm depth, from 6.79 to 8.72 mg·kg−1 at 40–60 cm depth, from 1.77 to 5.41 mg·kg−1 at 60–80 cm depth, and from 1.17 to 1.50 mg·kg−1 at 80–100 cm soil depth. In NB1, NB2, and NB3 treatments, SNC ranged from 9.41 to 10.27 mg·kg−1 at 0–20 cm soil depth, from 13.16 to 14.01 mg·kg−1 at 20–40 cm depth, from 4.26 to 7.99 mg·kg−1 at 40–60 cm depth, from 1.50 to 2.33 mg·kg−1 at 60–80 cm depth, and from 1.30 to 1.76 mg·kg−1 at 80–100 cm soil depth. Thus, experimental results related to SNC indicate that biochar application would be effective in both BI and DI methods; however, soil N holding efficiency of biochar under the DI method can be high compared to the BI method.In this irrigation method, compared to the BI method, the SNCs at 0–20 cm and 20–40 cm were significantly higher in all treatments, including non-biochar treatment, mainly SNCs in NB3.31, NB2, and NB3 were significantly higher than in NB0. OM content gradually increased from the first season to the third season of the experiment. The OM contents were 13.3–14.3 g·kg−1, 14.8–16.1 g·kg−1, 15.2–17.0 g·kg−1, and 15.5–17.7 g·kg−1 in NB0, NB1, NB2, and NB3 treatments, respectively under BI and DI methods. Compared to non-biochar treatment under the BI method, NB1 increased OM by 11.28%–14.60%, NB2 by 14.29%–18.98%, and NB3 by 21.98%–23.36% from the first to the last season of the experiment. In the DI method, NB1 increased OM by 11.11%–14.39%, NB2 by 14.81%–19.42%, and NB3 by 16.30%–23.78%, compared to the NB0 treatment. This indicated that soil OM highly changes due to biochar application rates, but the irrigation method has some effect. The medium and high doses of biochar contributed largely to soil OM increments. The higher the biochar application rate, the higher the soil OM content, because biochar is a charred organic substance itself. Furthermore, the increase in OM content increased soil aggregate stability. Results showed that compared to non-biochar treatment, the NB1, N1B2, and N1B3 treatments improved the weight diameter of soil aggregates, and it is varied with irrigation methods as well. In the BI method, NB1, NB2, and NB3 increased the average soil aggregates stability by 2.45%, 4.01%, and 11.71%, and in the DI method, it increased by 4.52%, 8.19%, and 14.66%, respectively. In terms of soil pH, results revealed that biochar combination with N fertilizer could regulate soil pH under BI and DI methods. The NB1, NB2, and NB3 treatments significantly decreased soil pH compared to N fertilizer alone. In 3 years, NB1 decreased soil pH on average by 1.93%–4.99% and NB2 and NB2 by 2.44%–6.36%, compared to NB0 treatment under BI and DI methods. The pH, OM, and soil aggregate stability indicators indicated that biochar application would be crucial to improve soil properties.3.4p < 0.05) effect on maize LAI development, and LAI development is also influenced by biochar doses, but the final LAI was not significantly different (p > 0.05). This could be due to the effect of rainfall or the maize plant recovered vegetative growth rate at the maturity stage, but it did not contribute to yield. The LAI was relatively larger during the second and third seasons as compared to the first season. It could be due to biochar increased nutrient availability in soil with time. The final LAI of maize in NB0 under the BI method was 3.80 m2 m−2, 3.90 m2 m−2, and 3.97 m2 m−2, and under the DI method, it was 4.20 m2 m−2, 4.40 m2 m−2, and 4.36 m2 m−2 in 2017, 2018, and 2019, respectively. The LAI of maize in NB1 under the DI method was 3.90 m2 m−2, 4.00 m2 m−2, and 4.07 m2 m−2, and under the DI method, it was 4.30 m2 m−2, 4.40 m2 m−2, and 4.46 m2 m−2 in 2017, 2018, and 2019, respectively. The maximum LAI of maize was in NB2 and NB3 under both BI and DI methods. The LAI of maize in NB2 and NB3 reached up to 4.56 m2 m−2. Compared to a maximum value of LAI in NB0 treatment under the BI method, the LAI of maize in NB2 and NB3 increased by 14%–15.11% under the DI method. Results demonstrated that the application of biochar could promote maize vegetative growth, and it could be clearly measured at different growth stages. In this regard, further research work is required.3.5p < 0.05 and p < 0.01.3.5.11, and NB2 treatments were 27,956 kg, 29,342 kg, and 30,294 kg, respectively, from 2017 to 2019. In t he NB0 non-biochar treatment, the maize yield was 9,270 kg ha–1 in 2017, 9,337 kg ha–1 in 2018, and 9,823 kg ha–1 in 2019. In NB1, the maize yield was 9,719 kg ha–1 in 2017, 9,800 kg ha–1 in 2018, and 12,470 kg ha–1 in 2019. In NB2, the maize yield was 10,067 kg ha–1 in 2017, 10,100 kg ha–1 in 2018, and 10,127 kg ha–1 in 2019. In NB3, the maize yield was 10,112 kg ha–1 in 2017, 10,251 kg ha–1 in 2018, and 10,291 kg ha–1 in 2019. Thus, on an annual average basis, NB0 had 9,319 kg ha–1, NB1 had 9,781 kg ha–1, NB2 had 10,098 kg ha–1, and NB3 had 10,218 kg ha–1. Compared to NB0, the NB1, NB2 and NB3 treatments increased maize yield by 4.96%, 8.36%, and 9.65%, respectively. Regarding IWUE, the non-biochar treatment NB0 had a lower IWUE compared to biochar doses combined with fertilizer. Maize IWUE in NB0 was 3.09 kg kg−1, 3.11 kg kg−1, and 3.12 kg kg−1 in 2017, 2018, and 2019, respectively, whereas maize IWUE in NB1 ranged from 3.24 to 3.27 kg kg−1. The NB2 and NB2 showed maximum IWUE, ranging from 3.36 to 3.43 kg kg−1. Considering the annual average IWUE of maize under different biochar treatments, the IWUE of NB1, NB2, and NB3 treatments increased by 5%–10% compared to non-biochar treatment. Similarly, biochar amendments also improved the FNUEs of maize crops; the maize had higher FNUE in NB1, NB2, and NB3 treatments. FNUE of the NB0 treatment ranged from 35.65 to 35.96 kg kg−1., whereas FNUE of the NB1 treatment ranged from 37.38 to 37.78 kg kg−1, FNUE of the NB2 treatment ranged from 38.72 to 38.95 kg kg−1, and FNUE of NB3 ranged from 38.89 to 39.58 kg kg−1. The order of yield, IWUE, and FNUE was NB3 ≥ NB2 > N1B1 > N1B0.In this irrigation method, the total maize productions of NB0, NB3.5.20, NB1, NB2, and NB3 treatments were 36,863 kg, 36,863 kg, and 38,169 kg, respectively, from 2017 to 2019. In the NB0 non-biochar treatment, the maize yield was 11,400 kg ha–1 in 2017, 11,576 kg ha–1 in 2018, and 11,770 kg ha–1 in 2019. In NB1, the maize yield was 12,100 kg ha–1 in 2017, 12,293 kg ha–1 in 2018, and 12,470 kg ha–1 in 2019. In NB2, the maize yield was 12,547 kg ha–1 in 2017, 12,732 kg ha–1 in 2018, and 12,891 kg ha–1 in 2019. In NB3, the maize yield was 12,720 kg ha–1 in 2017, 12,916 kg ha–1 in 2018, and 13,072 kg ha–1 in 2019. Thus, on an annual average basis, NB0 had 11,582 kg ha–1, NB1 had 12,288 kg ha–1, NB2 had 12,723 kg ha–1, and NB3 had 12,903 kg ha–1. Compared to NB0, the NB1, NB2, and NB3 treatments increased yield by 6.10%, 9.85%, and 11.41%, respectively. In terms of IWUE, the non-biochar treatment NB0 had a lower IWUE compared to biochar combined with fertilizer treatments. Maize IWUE in NB0 was 5.18 kg kg−1, 5.26 kg kg−1, and 5.35 kg kg−1 in 2017, 2018, and 2019, respectively, whereas maize IWUE in NB1 ranged from 5.50 kg kg−1 to 5.67 kg kg−1. The NB2 and NB2 showed higher IWUE, ranging from 5.70 kg kg−1 to 5.94 kg kg−1. Considering the annual average IWUE of maize under different biochar treatments, the IWUE of NB1, NB2, and NB3 treatments increased by 6%–11% compared to non-biochar treatment. Similarly, biochar amendments also improved the FNUEs of maize crops; the maize had higher FNUE in NB1, NB2, and NB3 treatments. FNUE of NB0 treatment ranged from 43.85 to 45.27 kg kg−1, whereas FNUE of the N1B1 treatment ranged from 46.54 to 47.96kg kg−1, FNUE of the NB2 treatment ranged from 48.26 to 49.58 kg kg−1, and FNUE of NB3 ranged from 48.92 to 50.28 kg kg−1. The order of FNUE of maize under different treatments was the same as the BI method. However, in the DI method, the FNUE of maize was much higher than in the BI method. The order of yield, IWUE, and FNUE was NB3 ≥ NB2 > N1B1 > N1B0 in the DI method, whereas considering the irrigation method, the order of yield, IWUE, and FNUE was DI > FI.In this irrigation method, the total maize production of NB44.1p < 0.05. The addition of low, medium, and high biochar doses 15.5 t ha−1, 30.7 t ha−1, and 45.3 t ha−1 with 260 kg N ha−1 increased maize yield by 4.96%, 8.36%, and 9.65% in the BI method, respectively. The benefit of biochar amendment was further enhanced in the DI method. The same combination of chemical fertilizer with biochar in the DI method increased maize yield by 6.10%, 9.85%, and 11.41%, respectively. These results are consistent with previous research that has shown the obvious impact of biochar with N fertilizer on crop yield . Our results are also aligned with ); (ii) it supplies minerals and soil organic carbon for plant growth ; and (iii) it enhances soil nutrient storage, transformation, and absorption .The fertilization and irrigation methods significantly affect maize yield, IWUE, and FNUE. Chemical N fertilizer alone was not efficient enough to increase yield. Biochar is a carbon-rich substance made from the pyrolysis of biomass. Its combined application with N fertilizer stimulates water retention capacity, water, and N uptake . Thus, iop yield . Our ret growth ; and  showed higher IWUE, ranging from 3.24 to 3.43 kg kg–1. These results are consistent with –1 in NB0, while the N fertilizer with biochar doses exhibited higher IWUE, ranging from 5.50 to 5.94 kg kg–1. These findings are in line with previous studies that have highlighted the water-saving potential of biochar in DI systems. Regarding IWUE, results demonstrated that the N fertilizer application with biochar doses has a greater impact on maize IWUE than the non-biochar dose. Incorporating biochar into the soil can increase the IWUE of maize production systems. In the BI method, the IWUE varied from 3.09 to 3.12 kg kg–1 in NB0, while the N fertilizer with biochar doses showed higher FNUE, ranging from 37.38 to 39.58 kg ha–1. These results related to FNUE are in line with previous research that has reported improved FNUE with biochar amendments  highly increased OM content; the increase in soil OM content ranged from 11.11% to 23.78% in both BI and DI methods compared to the non-biochar treatment. These findings are consistent with previous works demonstrating that biochar amendments might increase soil OM accumulation. −1 increased soil OM by 2%–59%; and 2 and NB3 treatments increased the average soil aggregate stability by 4.01%–14.66% in both irrigation methods. Because soil structure is strangely related to OM. About a 3%–26% increase in soil aggregate stability and consistency is also reported by 1, NB2, and NB3 treatments resulted in a significant drop in soil pH compared to the NB0 treatment each year. The reduction in soil pH ranged from 1.93 to 6.36% in BI and DI methods. This finding suggests that biochar application with N fertilizer can help in pH adjustment, which is also confirmed by Biochar has the ability to boost soil OM content by functioning as a long-term carbon sink in the soil as a stable form of organic carbon . Biochar5−1) and high (45.3 t ha−1) doses of biochar could increase maize yield by 8.36%–9.65% and 9.85%–11.41% under BI and DI methods, respectively, compared to N fertilizer alone. Moreover, biochar increased FNUE and IWUE in both irrigation methods. Biochar application improved the SWC in topsoil layers. Results showed that in the BI method, the NB0 treatment decreased SNC at topsoil layers and increased at deep soil layers because N was transported along with a heavy application of water. When the irrigation method changed from BI to DI, the same treatment increased the SNC at the top layers and decreased it at deeper soil depths. The application of biochar doses further influenced the change in SNC. All combinations of N fertilizer with low, medium, and high biochar doses, namely, 15.5 t ha−1, 30.7 t ha−1, and 45.3 t ha−1, respectively, decreased N deposition in deeper soil layers. The SNC deposition in deeper soil layers under the DI method was much lower than in the BI method, indicating that biochar N holding efficiency in the topsoil layer was further enhanced in the DI method. Considering maize yield, FNUE, IWUE, and SNC dynamics, biochar application would be effective in the DI method, and a medium dose of biochar would be enough. Moreover, the effect of biochar was obvious on soil OM, soil aggerate stability, and regulation of pH. The soil OM increased with the application dose of biochar. Thus, it improved soil aggregate stability. The biochar application would be crucial to increase soil OM, improve soil structure, and maintain pH. In future studies, the long-term effect of biochar on soil properties should be studied with different N fertilizer application rates, and the optimal ratio of chemical fertilizer and biochar should be determined.In this study, the effect of different biochar doses on maize grain yields, N, and water dynamics was compared between conventional BI and DI methods. It was concluded that the application of medium (30.7 t haThe original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.LJ: funding acquisition, conceptualization, and supervision. JW: resources and project administration. LW and SL: investigation, writing—original draft, and methodology. LW, SL, NW, and MP: data collection. LW, SL, and JW: formal analysis. LJ, NW, and MP: writing—review and editing. All authors read and approved the final manuscript.


Escherichia coli and Klebsiella pneumoniae on the in vitro antibacterial effects of imipenem/relebactam (IMR) and ceftazidime/avibactam (CZA).To evaluate effect of inoculum size of extended-spectrum β-Lactamase (ESBL)-producing-, AmpC-producing-, and KPC-producing We compared the impact of inoculum size on IMR and CZA of sixteen clinical isolates and three standard isolates through antimicrobial susceptibility tests, time-kill assays and in vitro PK/PD studies.5 to 107 CFU/mL, an inoculum effect was observed for 26.3% (5/19) and 52.6% (10/19) of IMR and CZA, respectively; time-kill assays revealed that the concentration of CZA increased from ≥ 4 × MIC to 16 × MIC to reach 99.9% killing rate against K. pneumoniae ATCC-BAA 1705  and 60,700 (SHV-27- and DHA-1-producing). While for IMR, a concentration from 1 × MIC to 4 × MIC killed 99.9% of the four strains. When the inoculum size increased to 109 CFU/mL, neither IMR nor CZA showed a detectable antibacterial effect, even at a high concentration. An in vitro PK/PD study revealed a clear bactericidal effect when IMR administered as 1.25 g q6h when inoculum size increased.When inoculum size increased from 10An inoculum effect on CZA was observed more frequent than that on IMR. Among the β-lactamase-producing strains, the inoculum effect was most common for SHV-producing and KPC-producing strains.The online version contains supplementary material available at 10.1186/s12941-023-00660-5. Enterobacteriaceae producing extended-spectrum-β-lactamases (ESBL) and carbapenemases are the main source of multi-drug resistant, and even pan-drug resistant, bacteria worldwide. While the number of treatments is limited, making effective treatment a clinical challenge.Production of β-lactamases is the main reason underlying the antimicrobial resistance (AMR) of Gram-negative bacteria; in addition, the selection of β-lactams is closely related to the frequency and evolution of AMR . EnterobEnterobacteriaceae, with susceptibility rates of 100%, 96.6% and 95.1%, respectively; IMR also shows good in vitro activity against ESBL-, AmpC-, and KPC-producing strains, with susceptibility rates of 100%, 95.1% and 84.5%, respectively [Imipenem/relebactam (IMR) and ceftazidime/avibactam (CZA) are new β-lactam/β-lactamase inhibitor combinations (BLBLIs) approved by the FDA for the treatment of multi-drug resistant bacterial infections. CZA shows potent in vitro activity against ESBL-, KPC- and AmpC-producing ectively .5 CFU/mL [5–106 CFU/mL, while in intra-abdominal infections (IAIs) the bacterial load can be as high as 108–109 CFU/mL, and that in meningitis can reach 109 CFU/mL [vs. 80%, respectively) [vs. 96.3%, respectively) [However, the clinical efficacy may be poor even though the strains are sensitive in an in vitro antimicrobial susceptibility test. Differences in the bacterial load have an impact on the choice of drugs and the dosing regimens; in particular, some antibiotics are susceptible to an inoculum effect , which has an impact on treatment of infections. The recommended standard bacterial inoculum for the microbroth method is 1–5 × 105 CFU/mL ; however9 CFU/mL , 5. A se9 CFU/mL . In clinE. coli and K. pneumoniae on IMR and CZA, thereby providing a reference for clinical application.The purpose of this study was to use antimicrobial susceptibility tests, time-kill assays, and in vitro PK/PD studies to evaluate the in vitro inoculum effect of ESBL-, KPC-, and AmpC- producing E. coli and four clinical isolates of K. pneumoniae were examined, as well as standard strains E. coli ATCC 25922, K. pneumoniae ATCC-BAA 1705 , and 700,603 . Bacterial genomic DNA was extracted using the QIAamp DNA mini kit and subjected to whole genome sequencing to identify β-lactamase types , 107, and 109 CFU/mL. Ceftazidime , avibactam  and imipenem  were purchased from Dalian Meilun Biotechnology Co., Ltd. Relebactam  was provided by MSD. E. coli ATCC25922 was used as a quality control strain. An inoculum effect was defined as an ≥ eightfold increase in the MIC value upon exposure to a higher inoculum. All tests were carried out in triplicate.In accordance with the Clinical and Laboratory Standards Institute (CLSI) recommendations , antibioK. pneumoniae ATCC-BAA 1705 producing KPC-2, OXA-2 and OKP-B-7, E. coli 56,706 co-producing TEM-1and AmpC, K. pneumoniae 60,700 co-producing SHV-27 and DHA-1 and K. pneumoniae 61,089 co-producing KPC-2 and CTX-M-65, respectively. The studies were performed using antibiotics at concentration of 1× , 4× , 16× , and 32× MIC and an initial inoculum size of 105 (standard inoculum), 107 or 109 CFU/mL, respectively. Samples were removed after 0, 1, 2, 4, 6, 8, 12 and 24 h of incubation at 37 °C overnight and plated using an automatic spiral spreading instrument  for viable colony counts. All tests were carried out in triplicate.Four strains with the most significant inoculum effect was selected for the time-kill assays. They are The simulated human serum concentrations of CZA and IMR obtained after multiple intravenous administrations were based on PK data from previous studies , 1.5 mL of the test strain was collected and clones were counted after 18-24 h incubation. Plated using an automatic spiral spreading instrument for colony counts. The limit of clone detection was 30 CFU/mL. Each experiment was performed in triplicate to assure reproducibility.The study was conducted using the in vitro PK Auto Simulation System 400 . The bacterial suspension was injected into 100 mL of broth medium to achieve a initial inoculum of 10The PD parameters, including Maximum Kill Down , the difference in bacterial counts between 0 and 24 h (∆log N24), and the bacterial growth recovery time  were analysed by PASS 400 Analyse Bactericidal Activity software. The area between the control growth curve and bactericidal curves (IE) was calculated by the trapezoidal rule using GraphPad Prism 9; these data were used as the integral parameters for evaluating antimicrobial effects. Data were analysed using one-way analysis of variance, and P < 0.05 was considered statistically significant.5 to 107 CFU/mL, the MIC values for CZA against all strains increased by 2- to 64-fold (from 0.03–128/4 mg/L to 0.25–256/4 mg/L), and the MIC values for IMR increased by 1–16-fold (0.03–128/4 mg/L to 0.125–256/4 mg/L). An inoculum effect on CZA and IMR was observed for 52.6% (10/19) and 26.3% (5/19) of isolates, respectively. The most common (by genotype) were SHV and KPC strains. When the inoculum size was 109 CFU/mL, the MIC value for CZA and IMR against all strains was ≥ 512/4 mg/L. When the concentration of AVI and REL was increased from 4 to 8 mg/L, the MIC values for CZA decreased by 2 to  > 16-fold (from 0.03/4 to > 512/4 mg/L to 0.015/8–512/8 mg/L) and the MIC values for IMR decreased by 2 to 64-fold (from 0.03/4 to > 512/4 mg/L to 0.007/8–128/8 mg/L).When the inoculum size increased from 105 CFU/mL), IMR at 1 × MIC killed 99.9% of K. pneumoniae ATCC-BAA 1705 and E. coli 56,706 (TEM-1- and AmpC-producing) after 8 h, and 99.9% of K. pneumoniae 61,089 (CTX-M-65-and KPC-2-producing) after 12 h; these effects were maintained for over 24 h. By contrast, CZA at 1 × MIC led to a < 1log10 reduction the CFU/mL of K. pneumoniae 60,700 (SHV-27- and DHA-1-producing), but it was unable to match the 99.9% killing effect of IMR. CZA at 4 × MIC killed 99.9% of K. pneumoniae ATCC-BAA 1705 and 61,089 at 105 CFU/mL after 6 h, and 99.9% of K. pneumoniae 60,700 after 8 h; this effect was maintained for over 24 h. When the inoculum size was increased to 107 CFU/mL, CZA at 16 × MIC killed 99.9% of K. pneumoniae ATCC-BAA 1705 and 60,700 after 24 h and 12 h, respectively; however, it had little effect on E. coli 56,706 and K. pneumoniae 61,089. IMR at 4 × MIC killed 99.9% of the four strains after 6 or 8 h, and maintained this for over 24 h. When the inoculum size rose to 109 CFU/mL, CZA and IMR at high concentrations (32 × MIC) still showed no bactericidal effect against four tested strains  showed potent bactericidal effects. CZA 2.5 g q8h and IMR 1.25 g q6h killed 99.9% of four strains  after 24 h. The killing effects against E. coli 56,706 (TEM-1- and AmpC-producing), K. pneumoniae 61,089 (CTX-M-65- and KPC-2-producing), and K. pneumoniae 60,700 (SHV-27- and DHA-1-producing) were maintained for 24 h; however, bacterial growth resumed after 24 in the presence of CZA 1.25 g q8h. When the inoculum size increased to 107 or 109 CFU/mL, all bacteria recovered after 24 h of exposure to CZA and IMR, although it is noteworthy that regrowth after exposure to CZA was more obvious than that after exposure to IMR ). When CZA was administered as 2.5 g q8h, the IE for K. pneumoniae ATCC-BAA 1705 was 63.26, 50.24, and 49.99 lgCFU/mL∙h ; that for E.coli 56,706 was 75.42, 67.47 and 63.47 lgCFU/mL∙h ; that for K. pneumoniae 61,089 was 62.5, 75.24 and 68.14 lgCFU/mL∙h ; and that for K. pneumoniae 60,700 was 73.83, 67.88 and 61.56 lgCFU/mL∙h, . As the inoculum size increased, CZA showed an obvious inoculum effect, although the antibacterial effect of IMR was much more pronounced than that of CZA  did not kill the bacteria. Data from the in vitro PK/PD studies showed that the conventional recommended doses of CZA 2.5 g q8h and IMR1.25 g q6h allowed bacterial regrowth after 2–14 h. At a high inoculum size, a large amount of β-lactamase was produced, negating the effects of AVI and REL. CAZ and IPM are hydrolysed by β-lactamases, which reduces their antibacterial effects. Non-β-lactamase-producing E. coli ATCC 25922 showed an inoculum effect when the inoculum size increased to 109 CFU/mL, which may suggest that β-lactamase-production is not the only factor involved.The mechanisms underlying the inoculum effect are quite complicated. As the inoculum size increases, the concentration of antibacterial drugs that interact with individual bacterial cells decreases , weakeni4 M−1 s−1. Therefore, the high catalytic efficiency of KPC may be the reason underlying the inoculum effect of KPC-producing strains. A previous study on the inoculum effect of ESBL-producing E. coli on piperacillin/tazobactam found no difference in frequency with respect to TEM- producing, SHV- producing, and CTX-M- producing strains [K. pneumoniae but not E. coli; therefore, the type of bacteria may have an impact on the presence of an inoculum effect.The inoculum effect may also be affected by the type of β-lactamases in β-lactamase-producing strains. In this study, the inoculum effect was greatest against KPC-producing strains and SHV-producing strains. Queenan et al.  found th strains . The preE.coli, respectively, while meropenem did not show an inoculum effect [An inoculum effect on cephalosporins was observed more frequent than that on carbapenems , 19. A pm effect . Anotherm effect . CAZ hasm effect . When thm effect . Then upm effect . The difPseudomonas aeruginosa increased from 5 × 104 CFU/mL to 5 × 105 and 5 × 106 CFU/mL, the MIC of IMR remained almost unchanged [Enterobacteriaceae, whereas we tested β-lactamase-producing E. coli and K. pneumoniae.Many studies have showed that the inoculum effect can impact clinical outcomes , 27. Onenchanged . Here, wnchanged , 10, witnchanged . The clinchanged . This diOur study has some limitations. First, the experimental strains produced a variety of β-lactamases simultaneously; the actions of these β-lactamases may have affected the antibacterial efficacy of the drugs. Second, we used only conventional recommended regimens (IMR 1.25 g q6h and CZA 2.5 g q8h) and low-dose regimens (IMR 625 mg q6h and CZA 1.25 g q8h) in the in vitro PK/PD study. The efficacy of other regimens (such as high-dose and continuous dosing regimens) on severe infections needs further study.IMR and CZA are considered reasonable options for the treatment of multidrug-resistant bacterial infections; however, the presence of an inoculum effect may lead to their failure to treat infections with a high bacterial load ; in such cases, IMR may be a better choice. In addition, the presence/absence of an inoculum effect is somewhat determined by the type of β-lactamase. Therefore, the type of β-lactamase should be taken into consideration when selecting antibacterial drugs.Additional file 1:Table S1. PK parameters for various dosing regimens. Table S2. PD parameters of different regimens against four bacterial strains.


Leishmania amazonensis. The cell alterations were evaluated in vitro on promastigote and amastigote forms. The lethal dose (LD50) at 24, 48, and 72 h on the promastigote's forms using lapachol was 75.60, 72.82, and 58.85 μg/mL and for β-lapachone was 0.65, 1.24, and 0.71 μg/mL, respectively. The naphthoquinones significantly inhibited the survival rate of L. amazonensis amastigotes at 83.11, 57.59, and 34.95% for lapachol , and 78.49, 83.25, and 80.22% for β-lapachone . The compounds on the promastigote's forms led to the loss of mitochondrial membrane potential, induced changes in the integrity of the membrane, caused damage to cells suggestive of the apoptotic process, and showed inhibition of tumor necrosis factor (TNF)-α and interleukin (IL)-6 production. The results showed that these naphthoquinones are promising candidates for research on new drugs with anti-Leishmania activity derived from natural products.Leishmaniasis is a neglected disease that affects millions of people worldwide, and special attention should be given to treatment because the available drugs have limitations, which can lead to low therapeutic adherence and parasitic resistance. This study evaluated the activity of the bioactive naphthoquinones, lapachol and β-lapachone, against  Lutzomyia. Cells of the mononuclear phagocytic system are where the parasite survives and replicates, so parasite elimination by macrophages is critical for host resistance to disease progression  is a polymorphic skin and mucosal disease causing single or multiple ulcerative or nodular wounds. It is considered a significant public health problem in some countries due to the several species of parasite and vectors and geographic differences involved. One of the main species responsible for CL is  failure ,4.Leishmania activity has increased and is essential for the development of new therapeutic strategies with lower toxicity and better accessibility ; penicillin G ; streptomycin (Sigma-Aldrich®); 2,3-bis (2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-carboxanilide (XTT) (Sigma-Aldrich®); phenazine methosulfate (PMS) ; dimethyl sulfoxide (DMSO) (Sigma-Aldrich®); triton X-100 (Sigma-Aldrich®); Rhodamine 123 ; annexin V/propidium iodide (PI) (Sigma-Aldrich®); BD CBA Mouse Th1/Th2/Th17 Cytokine kit ; RPMI 1640 medium ; Medium 199 (Gibco®); fetal bovine serum (FBS) (Gibco®); Fast panoptic ; Entellan ; trypan blue (Sigma-Aldrich®).Drugs and reagents used in this study included the following: lapachol (4-hydroxy-3-(3-methylbut-2-enyl)naphthalene-1,2-dione); β-lapachone ; amphotericin B (AmB) , J.H.G. Oliveira 10323 HUM, [lat: -23.4253005981445 long: -51.9385986328125 err: ±19250 WGS84]). The purity of the β-lapachone was calculated to be >98% by HPLC  were obtained from the central animal house of UEM, Brazil.BALB/c peritoneal macrophages were obtained by washing the peritoneal cavity with 8 mL of RPMI 1640 culture medium  was maintained using animal infections, and the cultures were maintained by periodic subculture at 25°C  at pH 7.2. at 25°C . BrieflyL. amazonensis (MHOM/BR/1977/LTB0016) promastigotes was evaluated using the XTT colorimetric method, which consists in the XTT salt degradation by the mitochondria forming formazan crystals. Promastigote forms (2×107 parasites/mL) from a culture in logarithmic growth phase were incubated with lapachol and β-lapachone diluted in 199 culture medium. The drugs were diluted in series, in a ratio of two, starting from 1/2, on a 96-well cell culture plate . The compounds were tested at concentrations from 204.4 to 0.40 µg/mL (lapachol) and 157.5 to 0.31 µg/mL (β-lapachone). After 24, 48, and 72 h of incubation at 25°C, 100 μL of the XTT solution (500 μg/mL) activated with PMS (50 μg/mL) were added to each well and incubated for 4 h at 37°C. The result was measured by absorbance, determined by spectrophotometry . The lethal dose (LD50) was defined as the concentrations of the compounds that reduced survival of L. amazonensis by 50% compared to untreated parasites in culture medium. AmB (1.96 to 0.25 µg/mL) was used as positive control of leishmanicidal activity. The therapeutic selectivity index (TSI CC50/LD50) was calculated by the ratio of toxicity to macrophage vs toxicity to parasites after 24 h incubation  of formalin solution (2%), and the parasites were counted in a Neubauer chamber. The inhibitory concentration (IC50) of promastigotes was calculated by comparing with untreated promastigotes in culture medium. The results were evaluated by linear regression of the inhibition percentage. The selectivity index (SI CC50/IC50) was calculated by the ratio of toxicity concentration to macrophage vs inhibitory concentration to the parasite growth after 24 h incubation (cubation . All ass5 per well). Cells were seeded onto round glass coverslips within 24-well plates in RPMI 1640 medium and incubated for 2 h at 37°C in 5% CO2. After that, the proportion of 6 parasites (L. amazonensis) per macrophage were added, and cultures were incubated for 4 h at 37°C in 5% CO2. Free parasites were removed by washing with RPMI 1640 medium. The infected macrophages were incubated with lapachol  and β-lapachone  for 24 h at 37°C. After the incubation time, the coverslips were removed, washed, stained with Fast Panoptic, and fixed on glass slides with Entellan. For the determination of cytokines, the supernatant was stored at −80°C. The experiments were done twice in triplicate, and treated uninfected cells were used as the cytotoxicity control and non-treated infected cells were used as the positive control of infection. The number of intracellular amastigotes was determined by counting 200 macrophages by concentration in an optical microscope . The infection index was calculated by multiplying the percentage of infected macrophages by the number of parasites per macrophages , followed by 2 h of incubation (5% CO2 at 37°C). J774A.1 macrophages were cultured in RPMI 1640 medium (Gibco), and during the experiment cell suspension adjusted for 5×105 macrophages/mL was distributed in a 96-well culture plate , followed by 48 h of incubation (5% CO2 at 37°C). In both cases, non-adherent cells were removed by washing with RPMI 1640 culture medium. After that, the compounds were tested with concentrations ranging from 408.75 to 0.03 μg/mL (Lapachol) and 630.0 to 0.04 μg/mL (β-lapachone). Cultures were incubated for 24 h at 37°C in 5% CO2, and non-treated cultures were used as viability control. The results were revealed using XTT colorimetric method . After 4 h of incubation in 5% CO2 at 37°C protected from light, the results were measured as absorbance, determined by spectrophotometry  (50) was defined as the dose of the compound that reduced 50% of the survival of macrophages compared with untreated macrophages  . The cyt7 cells) of L. amazonensis untreated (negative control) or treated with 76.62 µg/mL of lapachol and 1.63 µg/mL of β-lapachone were analyzed to observe changes in cell morphology of the parasites induced by the treatment after 24 h of incubation. A validation of IC50 was made in an optical microscope using a Neubauer chamber, and observations were performed in EVOS cell imaging system using transmitted light ; approximately 200 cells were observed in each condition using 1000 magnification. Subsequently, the parasites were analyzed using a BD FacsCalibur flow cytometer  and CellQuest Pro software , and 10,000 events were acquired. Dot plots were generated, the forward scatter (FSC-H) parameter was analyzed as presenting a correlation with the cell volume.Promastigote forms , submitted to critical point drying, and metalized with gold for visualization on a Shimadzu 55-550 (Japan) microscope.Scanning electron microscopy (SEM) also was performed to analyze morphological changes in cell surface topography. After 24 h of treatment, the parasites were centrifuged at 576 Ψm was determined using on BD FacsCalibur™ flow cytometer and CellQuest Pro software, with a total of 10.000 events acquired, using a mitochondrial-specific green-fluorescent dye Rh123 (L. amazonensis (2×107 parasites/mL) were incubated with lapachol (76.62 µg/mL) and β-lapachone (1.63 µg/mL) for 1, 4, and 24 h at 25°C. After washed with PBS, the parasites were incubated with 5 μg/mL Rh123 for 15 min. After the incubation time, the cells were washed and suspended in PBS for additional incubation of 30 min for analysis. The percentage of fluorescence of Rh123, was determined by the (MT/MC)*100 equation, with MT representing the median fluorescence of the treated parasites and the MC the median fluorescence of negative controls. Alteration in ΔΨm was quantified using an index of variation (IV) obtained by the equation IV = (MT - MC)/MC, where negative IV values correspond to depolarization of the mitochondrial membrane. H2O2 (2 mM) was used as a positive control (The Δye Rh123 . Briefly control .L. amazonensis (2×107 parasites/mL) were incubated with lapachol (76.62 µg/mL) and β-lapachone (1.63 µg/mL) for 24 h at 25°C. After treatment, the cells were centrifuged (901 g for 10 min) at room temperature and washed in PBS. According to the manufacturer's instructions, annexin V-FITC and PI (Sigma-Aldrich) were added. Data were acquired using the BD FacsCalibur™ flow cytometer (FL1vs FL2) and CellQuest Pro software, with a total of 10,000 events acquired for analysis of results.L. amazonensis (1×106 promastigotes/mL) was incubated with the IC50 of compounds for 24 h. After validation of IC50 as mentioned above, pellets were re-suspended in digestion buffer , to which 0.5 mg/mL proteinase K was added. The mixture was incubated for 3 h in an ice bath and again for 1 h at 55°C in the presence of 0.1 mg/mL RNase A. DNA material was extracted by phenol/chloroform (1:1) treatment and was precipitated by adding 3 M sodium acetate and ice-cold ethanol (100%). After an overnight incubation at -20°C, the material was centrifuged (13270 g) for 30 min at 25°C temperature. Then, 2.5 volumes of ethanol (70%) were added to the pellet. The material was centrifuged (13270 g) for 15 min again and the pellet was collected, air dried, and re-suspended in Tris-EDTA buffer . DNA aliquots (10 µg) were electrophoresed on 2% agarose gel that contained ethidium bromide (0.5 µg/mL) using Tris-acetate-EDTA buffer (pH 8.0) for 45 min at 100 V and photographed under ultraviolet light. L. amazonensis untreated promastigotes were used as negative control of fragmentation (C-) and promastigotes treated with H2O2 as positive control (C+). The drag of the formed band was observed.The presence of DNA fragmentation was evaluated by electrophoresis in agarose gel according to a previous study . L. amazL. amazonensis (6 parasites/macrophage); samples treated with lapachol at IC50/2, IC50, and 2× IC50 with L. amazonensis (+) or without (-); the same was repeated for β-lapachone). The supernatant was centrifuged, and a 50 μL aliquot was used to determine cytokine levels (in pg/mL) using the BD Cytometric Bead Array (CBA) Mouse Th1/Th2/Th17 Cytokine kit . The dosage was performed in triplicate and according to the manufacturer's standards. This method is based on a suspension that contains beads, in which encoded fluorescent beads have cytokines that capture antibodies to bind the proteins. The final analysis was performed by flow cytometry using the BD FacsCalibur™ flow cytometer and CellQuest Pro software. The kit allows the measurement of seven distinct populations of Th1, Th2, and Th17 cytokines  in a single sample.For the determination of cytokines, the supernatant of the amastigote assays stored at −80°C was used ; MO + t-test was used. When the data did not follow a normal distribution, the Mann-Whitney test was applied. In the analyses that included more than two groups, variance analysis was used, verifying the assumptions of independence, normality, and homoscedasticity of the residues. ANOVA followed by the Tukey post hoc test or Kruskal-Wallis followed by the Mann-Whitney pairwise test were conducted when the data did not follow the criteria described. The value of P<0.05 was considered statistically significant. Analyses were performed using BioEstat 5.0 software  and Past 3.X software .The results are reported as means±SE or means±SD. For comparison between two groups with normal distribution, Student's L. amazonensis. After the treatment in promastigotes forms, alterations in the morphology, cell and nuclear integrity, mitochondrial membrane potential, and cytokine detection were shown.The naphthoquinones lapachol and β-lapachone  at low cL. amazonensis promastigote death , 1.24±0.19 µg/mL (48 h), and 0.71±0.03 µg/mL (72 h) (50 of AmB was 0.97±0.09 µg/mL (24 h), 1.65±0.02 µg/mL (48 h), and 0.80±0.03 µg/mL (72 h) (50 lower than that of lapachol (P<0.05 by ANOVA and Tukey post hoc tests). Comparing β-lapachone with AmB, there was a significant difference between LD50 at 24 and 48 h (P<0.05 by ANOVA and Tukey post hoc tests), which did not occur between lapachol and AmB. Lapachol and β-lapachone also showed an inhibitory effect on the growth of L. amazonensis promastigotes after 24 h of incubation, with concentrations that inhibited 50% of the promastigotes (IC50), ranging from 76.62±1.21 µg/mL for lapachol to 1.63±0.09 µg/mL for β-lapachone (Lapachol and β-lapachone induced te death . After 2ectively . The LD5L (72 h) . The LD5L (72 h) . For allapachone .L. amazonensis amastigotes in 83.11, 57.59, and 34.95% for lapachol and in 78.49, 83.25, and 80.22% for β-lapachone, respectively, at the concentrations tested (The naphthoquinones significantly inhibited the infection rate of s tested . The res50/24 h) were 41.14±2.17 µg/mL for lapachol and 3.21±2.72 µg/mL for β-lapachone. The index for J774A.1 lineage macrophages ranged from 26.66±8.69 µg/mL for lapachol to 3.18±0.04 µg/mL for β-lapachone . Comparing TSI and SI of the compounds for murine macrophages, it was observed that β-lapachone was 3.65 times more selective for inhibiting the growth of Leishmania culture than lapachol, and 9.15 times more selective for Leishmania death. Similar results were observed for J774A.1 macrophages, in which β-lapachone was 5.6 times more selective for inhibiting the growth of Leishmania culture than lapachol and 14 times more selective for Leishmania death.The concentrations for cytotoxicity to 50% peritoneal macrophages  was observed already at 1 h (44%), which increased at 4 h (69%) and decreased at 24 h (30%). The loss of mitochondrial potential was more pronounced for β-lapachone (1.63 µg/mL), which occurred concurrently with a longer time of exposure to treatment . The value observed at 24 h for β-lapachone was similar to values found in the positive control (H2O2), which induced 97, 97, and 98% of changes in the ΔΨm in parasites after 1, 4, and 24 h of treatment  and β-lapachone (1.63 µg/mL) for 4 h showed annexin labeling  of 8.57 and 9.03%, respectively . After 24 h of incubation , it was not possible to observe the increase of this process, so it should be appropriately tested using longer incubations. The level of apoptosis after treatment was comparable to that triggered by the AmB, which presented the same behavior of the compounds.To determine whether the effect of lapachol and β-lapachone on L. amazonensis treated with the IC50/24 h of lapachol and β-lapachone for 24 h promoted DNA fragmentation , which has a T cell hyposensitivity pole and a marked T helper type 2 immune response. These immune conditions can be related to therapeutic failure and parasite resistance , and for β-lapachone was 1.63 µg/mL (6.73 µM). Guimarães et al. (L. amazonensis (strains MHOM/BR/1989/ BA199), and the IC50 found at 72 h was 102.05 µM for lapachol and 1.39 µM for β-lapachone. Moreno et al.  braziliensis (MHOM/BR/94/H-3227) by 88.8% using 50 µg/mL.When the survival rate of en found ,21,34. Fen found  demonstrL. amazonensis promastigotes after 24 h. Flow cytometry analysis and SEM showed decreased cell volume in lapachol and β-lapachone-treated promastigotes. Similar results were described by Mendonça et al.  or intracellular (intrinsic) sources, such as intracellular stress caused by drug treatment . To betta et al.  when resLeishmania protozoa have a single organelle in their cytoplasm and a mitochondrial dysfunction causes irreversible damage leading to cell death because there is no possibility of compensating for injured mitochondria  and lipophilicity to enhance penetration through the membrane  due to the direct reaction with proteins, lipids, and DNA causes oxidative stress, mitochondrial membrane depolarization, and DNA fragmentation ,19. ThesL. amazonensis promastigotes upon lapachol treatment.As mentioned, mitochondrial dysfunction can culminate in cell death by apoptosis, and our results subtly suggested that the compounds exert leishmanicidal activity probably via apoptosis, since we observed cell damage, phosphatidylserine externalization, and DNA fragmentation in the results of the experiments. Exposure to phosphatidylserine is the hallmark of classic apoptosis, as this molecule is a phospholipid present on the inner surface of the cell membrane and its exteriorization occurs during the process of apoptosis. Annexin V is a molecule with affinity for phosphatidylserine, binding to it at the beginning of the apoptotic process. In contrast, PI binds to the DNA of cells in the final phase of apoptosis, when membrane integrity is already lost. Araújo et al.  also desLeishmania parasites is important when we study a new molecule for treatment. In CL, host immunity is one of the most critical factors in disease diagnosis and cure (1 cells and macrophages that produce IFN-γ, TNF-α, IL-12, and other cytokines that promote the activation of microbicidal mechanisms and the killing of the parasite by macrophages. This study observed that macrophages infected with L. amazonensis and treated with lapachol and β-lapachone showed no significant changes in IL-2, IL-4, IFN-γ, IL-17A, and IL-10. However, differences were found in TNF-α and IL-6, and both cytokines were downregulated.The investigation of immune mechanisms in eliminating and cure . InfectiL amazonensis are the major agents of ADCL, a rare clinical form. Although ADCL is the most severe and incurable form of CL, it occurs in about 1% of CL cases each year. In ADCL, nodular skin lesions are rich in amastigote forms, and the parasite is disseminated throughout the body. This suggests a specific failure of protective immunological mechanisms to control infection. As a result, there is preferential activation of the Th2-type immune response, with the production of cytokines that negatively regulate the immune response and culminate with an inadequate response to traditional forms of treatment (Interestingly, in Brazil, reatment . Our resL. amazonensis, inducing several morphological changes on promastigotes, including alterations in mitochondrial potential, loss of membrane integrity, and probably cell damage by the apoptotic process. These data are an essential contribution to the preclinical studies of lapachol and β-lapachone, demonstrating the promising therapeutic action of these new molecules on leishmaniasis. Further investigation is necessary to characterize which additional mechanisms of action are involved with the effects of lapachol and β-lapachone.The present results strengthen the possibility of using lapachol and β-lapachone, a natural and a synthesized compound, respectively, in the treatment of CL. According to de Castro et al.  and our pdf].Click here to view [