Rickettsia species (spp.) are strict obligate intracellular bacteria, some of which are pathogenic in their mammalian host, including humans. One critical feature of these stealthy group of pathogens is their ability to manipulate hostile cytosolic environments to their benefits. Although our understanding of Rickettsia cell biology and pathogenesis is evolving, the mechanisms by which pathogenic Rickettsia spp. evade host innate immune detection remain elusive. Here, we show that disease severity in wild-type (WT) C57BL/6J mice infected with Rickettsia typhi (the etiologic agent of murine typhus) and Rickettsia rickettsii (the etiologic agent of Rocky Mountain spotted fever), but not with the nonpathogenic species Rickettsia montanensis, correlated with levels of bacterial burden as detected in the spleens of mice, as well as the serum concentrations of proinflammatory cytokine interleukin-1α (IL-1α) and, to a lesser extent, IL-1β. Antibody-mediated neutralization of IL-1α confirmed a key role in controlling mortality rates and bacterial burdens of rickettsia-infected WT mice. As macrophages are a primary source of both IL-1α and IL-1β cytokines, we determined the mechanism of the antirickettsial activities using bone marrow-derived macrophages. We found that pathogenic R. typhi and R. rickettsii, but not nonpathogenic R. montanensis, eluded pro-IL-1α induction and benefited predominantly from the reduced IL-1α secretion, via a caspase-11–gasdermin D (Gsdmd)-dependent pathway, to facilitate intracytosolic replication. Adoptive transfer experiments identified that IL-1α secretion by macrophages was critical for controlling rickettsiosis in WT mice. In sum, we identified a previously unappreciated pathway by which pathogenic, unlike nonpathogenic, rickettsiae preferentially target the caspase-11–Gsdmd–IL-1α signaling axis in macrophages, thus supporting their replication within the host. Listeria, Shigella, Burkholderia, Francisella, Orientia, and Rickettsia species, have developed strategies to induce their own uptake by phagocytosis and to circumvent host innate immune defenses for their intracellular survival . Activated Mϕ play a crucial role in either terminating an infection at an early stage, which commonly is the fate of nonpathogenic Rickettsia spp., or succumbing to bacterial replication and pathogen colonization as well as host dissemination to distant organs  to establish an intracytosolic replication niche. Recently, we reported that pathogenic Rickettsia spp. secret effectors to promote host colonization by modulating endoplasmic reticulum structures or by hijacking the autophagic defense pathway , but also shows that these pathogens likely employ species-specific strategies to evade host cytosolic defense mechanisms. For instance, Rickettsia australis, a pathogenic TRG member, benefited from ATG5-mediated autophagy induction and suppression of inflammasome-dependent IL-1β production to colonize both bone marrow-derived macrophages (BMDMs) , showed that R. typhi is ubiquitinated upon host entry and escapes autolysosomal fusion to establish an intracytosolic niche in nonphagocytic cells  by which pathogenic, but not nonpathogenic, Rickettsia spp. block immune defense responses to establish a replication niche in phagocytic cells, like Mϕ. Specifically, we sought to test the hypothesis that pathogenic, but not nonpathogenic, Rickettsia spp. reduce inflammasome-mediated IL-1 responses, thereby promoting their intracytosolic replication within host cells.In the case of emerging –25. Spec (BMDMs)  and mice (BMDMs) . In cont SFG see  below, u SFG see , 27. In ic cells . These uRickettsia spp. have been described globally, but less than a dozen is known to cause disease in humans, with some being notoriously pathogenic and associated with high morbidity and mortality, while others exert limited or no pathogenicity  and nonpathogenic (Rickettsia montanensis) strains in vivo. We first established a mouse model of mild  or more severe (∼LD50) rickettsiosis in C57BL/6J wild-type (WT) mice. For both R. rickettsii and R. typhi, LD25 or LD50 were achieved with doses of 105 or 106 PFU, respectively  confirmed successful infection with all three Rickettsia spp. at day 3 postinfection, while R. typhi- and R. rickettsii-infected WT mice displayed a significantly higher bacterial burden in the spleens compared to splenic tissues from R. montanensis-infected mice at day 7   Representative images of spleens (at day 7) from C57BL/6J WT mice injected i.v. with R. typhi, R. rickettsii, R. montanensis, or PBS (dose of 106 PFU). (B) Spleen weight from injected animals was evaluated at day 7 day (n = 5). Error bars in panel B represent means ± SEM from five independent experiments. *, P ≤ 0.05; ***, P ≤ 0.005. Download FIG S1, EPS file, 1.7 MB.Splenic data during infection of pathogenic and nonpathogenic Copyright © 2022 Voss et al.2022Voss et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Rickettsia spp. in vivo, IL-1α or IL-1β function was neutralized via tail vein (intravenous [i.v.]) injection with anti-IL-1α, anti-IL-1β, or anti-IgG-isotype control antibodies (Abs) into C57BL/6J WT mice in our established model of mild rickettsiosis   endotoxin-free rIL-1α and rIL-1β proteins following infection with R. typhi, R. rickettsii, and R. montanensis. Pretreatment of mice with rIL-1α and, to a lesser extent, rIL-1β protected C57BL/6J WT mice from pathogenic Rickettsia-induced lethality (Rickettsia spp. in C57BL/6J WT mice.To characterize further the role of IL-1α and IL-1β cytokines in restricting nonpathogenic and pathogenic 105 PFU)  to D. Neted mice  to C andted mice . The effted mice  and F. Nethality  to C, wiethality . Moreoveethality  and F. C10.1128/mBio.02918-21.2FIG S2Rickettsia species. (A) Representative images of spleens (day 7) from C57BL/6J WT mice injected i.v. with R. typhi, R. rickettsii, or R. montanensis (dose of 105 PFU), followed by administration of anti-IL-1β, anti-IL-1α, or IgG isotype control antibody (Ab) (250 μg Ab/mouse) (24 h post-Ab injection). (B) Spleen weight from injected animals was evaluated at day 7 (n = 5). Error bars in panel B represent means ± SEM from five independent experiments. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.005. Download FIG S2, EPS file, 3.2 MB.Splenic data after IL-1 signaling neutralization during infection of pathogenic and nonpathogenic Copyright © 2022 Voss et al.2022Voss et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02918-21.3FIG S3Rickettsia species. (A) Representative images of spleens (day 7) from C57BL/6J WT mice injected i.v. with rIL-1β or rIL-1α protein, followed by administration of R. typhi, R. rickettsii, R. montanensis, or PBS (dose of 106 PFU) at day 7. (B) Spleen weight from injected animals was evaluated at day 7 day (n = 5). Error bars in panel B represent means ± SEM from five independent experiments. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.005. Download FIG S3, EPS file, 4.0 MB.Splenic data after administration of recombinant IL-1α and IL-1β proteins during infection of pathogenic and nonpathogenic Copyright © 2022 Voss et al.2022Voss et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Rickettsia spp. and are considered to play a crucial role in either terminating the infection early at the skin site or allow initial pathogen colonization and subsequent dissemination within the infected host  compared to uninfected WT BMDMs  and Casp-11 [noncanonical]), respectively, we assessed their potential role in regulating replication of nonpathogenic versus pathogenic Rickettsia in BMDMs. In this effort, BMDMs derived from WT, −/−Casp-1, −/−Casp-11, and −/−Casp-1/11 mice were infected with R. typhi, R. rickettsii, and R. montanensis, and the levels of IL-1β and IL-1α and cell death, as well as bacterial burdens, were evaluated over the course of infection. Our assays revealed that Casp-11 was involved in the secretion of cleaved IL-1α upon infection of BMDMs with nonpathogenic and pathogenic Rickettsia spp. (−/−Casp-1) caused a significant decrease in IL-1β production . As our findings indicate that infections with R. typhi and R. rickettsii resulted in a significant reduction of IL-1α secretion, likely via a Casp-11-dependent mechanism, we assessed the expression and activation status of Casp-1 and Casp-11 via Western blot analyses. R. montanensis infection resulted in a robust activation of Casp-1, as indicative of the detection of the Casp-1–p20 fragment (R. typhi and R. rickettsii spp. resulted in a lower activation of Casp-1 (∼5-fold) (R. montanensis resulted in a robust induction of Casp-11 (∼8-fold) compared to infection data using both pathogenic Rickettsia spp. , as indicative by the detection of the Gsdmd-p30 fragment  . Intrigusia spp. . To testnsis see –32, we afragment . In supptanensis . These f10.1128/mBio.02918-21.4FIG S4Rickettsia species resulted in a reduced IL-1α and IL-1β cytokine release by macrophages. (A and B) BMDMs from WT mice were infected with heat-inactivated or non-heat-treated R. typhi, R. rickettsii, or R. montanensis (MOI = 50) for 24 h. Culture supernatants were analyzed for production of IL-1β (A) and IL-1α (B) using Legendplex kits (BioLegend), followed by flow cytometry. (C) Bacterial burden in Rickettsia-infected BMDMs from WT mice was evaluated at 2 and 24 h postinfection by GltA RT-qPCR. Expression of the housekeeping gene Gapdh was used for normalization. Error bars in panels A to C represent means ± SEM from five independent experiments. NS, nonsignificant; **, P ≤ 0.01; ***, P ≤ 0.005; ****, P ≤ 0.001. Download FIG S4, EPS file, 1.7 MB.Heat inactivation of Copyright © 2022 Voss et al.2022Voss et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02918-21.5FIG S5Rickettsia infection. (A and B) BMDMs from WT and −/−Gsdmd mice were infected with R. typhi, R. rickettsii, or R. montanensis (MOI = 50) for 24 h. Culture supernatants were analyzed for production of IL-1β (A) and IL-1α (B) using Legendplex kits (BioLegend), followed by flow cytometry. (C) Bacterial burden in Rickettsia-infected BMDMs from WT and −/−Gsdmd mice was evaluated at 2 and 24 h postinfection by GltA RT-qPCR. Expression of the housekeeping gene Gapdh was used for normalization. Error bars in panels A to C represent means ± SEM from five independent experiments. NS, nonsignificant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.005. Download FIG S5, EPS file, 1.7 MB.Gsdmd is involved in the release of IL-1α and IL-1β by macrophages upon Copyright © 2022 Voss et al.2022Voss et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Rickettsia spp. in vivo, we first injected (i.v.) WT mice with phosphate-buffered saline (PBS)- or dichloromethylene biphosphate (Cl2MBP)-liposomes to deplete endogenous macrophages as described previously (2MBP-liposome-treated WT mice were injected (i.v.) with BMDMs isolated from WT, −/−Il-1β, or −/−Il-1α mice prior to infection with R. typhi, R. rickettsii, or R. montanensis. Strikingly, adoptive transfer of −/−Il-1α BMDMs, but not −/−Il-1β or WT Mϕ, significantly increased the mortality of Cl2MBP- but not PBS-treated WT mice injected with all three Rickettsia spp., reaching levels similar to the survival percentages observed in IL-1α Ab neutralization studies  PBS-treated C57BL/6J WT mice were injected i.v. with WT, −/−Il-1β, or −/−Il-1α BMDMs (5 × 106 cells/mouse), followed by infection (24 h post-Mϕ transfer) with R. typhi (B), R. rickettsii (C), R. montanensis (D), or PBS (dose of 105 PFU) . Survival was monitored for 7 days. Download FIG S6, EPS file, 1.6 MB.Survival data after adoptive transfer of WT, Copyright © 2022 Voss et al.2022Voss et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02918-21.7FIG S7−/−Il-1β, or −/−Il-1α macrophages and infection of pathogenic and nonpathogenic Rickettsia species into dichloromethylene biphosphate (Cl2MBP)-treated C57BL/6J WT mice. (A) Representative images of spleens (day 7) from Cl2MBP-treated C57BL/6J WT mice injected i.v. with WT, −/−Il-1β, or −/−Il-1α BMDMs (5 × 106 cells/mouse), followed by administration (24 h post-Mϕ transfer) of R. typhi, R. rickettsii, R. montanensis, or PBS (dose of 105 PFU). (B) Spleen weight from injected animals was evaluated at day 7 (n = 5). Error bars in panels B represent means ± SEM from five independent experiments. NS, nonsignificant; *, P ≤ 0.05; **, P ≤ 0.01. Download FIG S7, EPS file, 3.8 MB.Splenic data after adoptive transfer of WT, Copyright © 2022 Voss et al.2022Voss et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Rickettsia spp., the roles for both inflammasome and autophagy to restrict their replication in endothelial cells and immune cells, like Mϕ, is only now emerging, although without consistent mechanistical insights  and more severe (∼LD50) disease for two pathogenic spp., R. rickettsii and R. typhi. In fact, data of disease severity correlated with the bacterial burdens detected in the mice spleens. We also observed that infections with pathogenic, but not nonpathogenic, Rickettsia spp. resulted in a reduced serum response of both proinflammatory cytokines, IL-1β and IL-1α, which is likely the result of pathogenic Rickettsia spp. to evade canonical and noncanonical inflammasome-dependent defense sensing, of which the former is in agreement with recent studies using R. australis . However, in the case of strict obligate intracytosolic insights –28, 34. ustralis . Thus, oin vivo. By testing the putative antirickettsial capabilities of these cytokines through employing Ab neutralization and recombinant protein assays, we showed that IL-1α and, to a much lesser extent, IL-1β was able to restrict the replication and colonization of both nonpathogenic and pathogenic Rickettsia spp. in vivo. Our current understanding by which Rickettsia spp. evade host-induced antibacterial activities, and in particular, how cytosolic rickettsiae overcome host immune surveillance in defense cells like Mϕ, is primarily based on reports that are not aligned with one another  of R. typhi and R. rickettsii spp. is likely less immunogenic than that of R. montanensis, which is further supported by our recent reports , and future experiments are under way to address these possibilities  in compliance with the National Institutes of Health guide .Anti-IL-1α (clone ALF-161), anti-IL-1β (clone B122), and an isotype control IgG (Armenian hamster IgG) antibody (Ab) were purchased from BioXCell. Anticaspase (anti-Casp-1) Ab was purchased from Adipogen, while anti-Casp-11 (clone EPR18628) and anti-Gsdmd (clone EPR19828) Abs were obtained from Abcam. Elongation factor Ts (EF-Ts) Ab was obtained from Primm Biotech as previously described , and R. typhi strain Wilmington was obtained from the CDC. All Rickettsia strains were propagated in Vero76 cells grown in DMEM supplemented with 5% FBS at 34°C with 5% CO2. All Rickettsia cells were purified as previously described (Rickettsia spp. were used at a multiplicity of infection (MOI) of 50, to ensure the presence of enough bacteria at early stage of infection, for host response (Rickettsia spp. were heated at 90°C for 20 min (Rickettsia cells (105 to 106 PFU) resuspended in PBS. At days 1, 3, and 7 after administration, blood was collected, and serum cytokine levels were measured by flow cytometry. Splenic tissue specimens were collected at the indicated times and used for bacterial burden analysis by quantitative PCR (qPCR) as described below.Vero76 cells  were maintained in minimal Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 37°C with 5% COresponse . For infr 20 min . Rickett−/−Il-1β, −/−Il-1α, −/−Gsdmd, −/−Casp-1, −/−Casp-11, and −/−Casp-1/11 mice. Femurs from −/−Casp-1, −/−Casp-11, and Casp-1/−/−11 mice were kindly provided by Amal Amer , while bones from −/−Il-1β or −/−Il-1α mice were obtained from Thirumala-Devi Kanneganti . Femurs from −/−Gsdmd were kindly provided by Matthew Welch . Differentiation was induced by culturing bone marrow cells in RPMI 1640 medium supplemented with 10% FBS and 30% L929-conditioned medium (a source of macrophage colony-stimulating factor) with culture for 7 days as described previously  following the manufacturer’s instructions as described previously .6 BMDMs or 100 μL of organ homogenate using the Quick-RNA miniprep kit (ZymoResearch). The iScript reverse transcription supermix kit  was used to synthesize cDNAs from 200 ng of RNA according to the manufacturer's instructions. Quantitative real-time PCR (qRT-PCR) was performed using SYBR green (Thermo Fisher Scientific) and 2 μL cDNA, and for the rickettsial citrate synthase gene (GltA), 1 μM each oligonucleotides 5′-CATAATAGCCATAGGATGAG-3′ (forward [F]) and 5′-ATGATTTATGGGGAACTACC-3′ (reverse [R]) were used and the results analyzed as described previously  as described previously , anti-IL-1β , or an IgG isotype control  Ab 24 h before the induction of mild rickettsiosis using 105 PFU of R. typhi, R. rickettsii, or R. montanensis.For 2MBP)-liposomes 72 and 48 h prior to macrophage transfer as described previously , followed by infection (24 h post-Mϕ transfer) with R. typhi, R. rickettsii, R. montanensis, or PBS (dose of 105 PFU).C57BL/6J WT mice were injected (i.v.) twice with PBS- or dichloromethylene biphosphate  and severe (106 PFU) rickettsiosis were analyzed by using Kaplan-Meir survival curves and the log-rank test . The statistical significance was assessed using analysis of variance (ANOVA) with Tukey’s multiple-comparison posttest (GraphPad). Data are presented as the mean ± standard error of the mean (SEM), unless stated otherwise. The alpha level was set to 0.05.Endpoint studies of mice subjected to mild (10


H)-one (C8H5N3O3), 6-aminoquinazolin-4(3H)-one (C8H7N3O) and 4-aminoquinazoline hemihydrochloride dihydrate (C16H19N6O2) were synthesized and their structures were determined by single-crystal X-ray analysis.6-Nitroquinazolin-4(3 H)-one , 6-aminoquinazolin-4(3H)-one  and 4-aminoquinazolin-1-ium chloride–4-aminoquinazoline–water (1/1/2),  were synthesized and their structures were determined by single-crystal X-ray analysis. In the crystals of I and II, the quinazoline molecules form hydrogen-bonded dimers via N—H⋯O interactions. The dimers are connected by weak intermolecular C—H⋯N and C—H⋯O hydrogen bonds, forming a layered structure in the case of I. In the crystal of II, N—H⋯N and C—H⋯O interactions link the dimers into a three-dimensional network structure. The asymmetric unit of III consists of two quinazoline molecules, one of which is protonated, a chloride ion, and two water molecules. The chloride anion and the water molecules form hydrogen-bonded chains consisting of fused five-membered rings. The protonated and unprotonated quinazolin molecules are linked to the chloride ions and water molecules of the chain by their amino groups.The title compounds, 6-nitroquinazolin-4(3 The hemi-protonated structures may be useful for the preparation of materials important to various branches of science, ranging from biology to nanodevice fabrication and to pharmaceuticals  and unprotonated (B) 4-aminoquinazoline molecules  Å. Other N—H⋯N hydrogen bonds form centrosymmetric via N—H⋯Cl and N—H⋯Ow hydrogen bonds from the lower and upper side –3.9080 (18) Å].The packing analysis of on Fig. 6. The proe Table 6. The chaet al., 2016H)-ones contains a nitro group [refcodes GAPPUK -one and its first metal coordination compound have also been reported [BIHJIO -one  was dissolved in 78 ml of concentrated sulfuric acid at 303 K for 1 h. Then a nitrating mixture (21 ml of nitric acid and 18 ml of concentrated sulfuric acid) was added to the flask under vigorous stirring of the mixture. The reaction mixture was stirred for another hour, maintaining a temperature not higher than 303 K, and then for another hour at room temperature. At room temperature, 45 ml of nitric acid were added dropwise to the reaction mixture over a period of 1 h. The reaction mixture was left at room temperature for 10 h. The contents of the flask were poured into a dish containing ice, the resulting precipitate was filtered off, washed with water and dried and recrystallized from ethanol to obtain 25.7 g of pure compound I as single crystals in 87.4% yield, m.p. 560–562 K.Compound II: In a three-necked flask equipped with a mechanical stirrer and reflux condenser, 12.6 g (56 mmol) of tin (II)2·2H2O) were cooled in an ice bath and 16.98 ml of concentrated (36%) HCl were added, then 3 g (16 mmol) of quinazolin-4-one as a suspension in 20 ml of ethanol and 7 ml of HCl (36%) were added portionwise with stirring of the mixture. The reaction was carried out for 10-15 minutes at ∼273 K, 30 min at room temperature and 2 h in a water bath (∼363 K). The reaction mixture was left overnight at room temperature, diluted with water, and brought to a strongly alkaline medium (pH = 10–11) with 10% of sodium hydroxide, in which the expected 6-amino-3N-quinazoline-4-one was dissolved, so that the chloride was brought to a neutral medium in the presence of acid, and precipitated when converted to an alkaline medium with ammonia. The precipitate was filtered, washed with water until it reached a neutral medium, and dried at room temperature. The precipitate was recrystallized from ethanol and 6.67 g of pure compound II were obtained representing an 88.1% yield, m.p. 589–591 K.Compound III: Crystals of compound III were obtained as a minor additional product in the reaction of 4-chloroquinazoline with ammonia.Uiso(H) = 1.2Ueq(C). Hydrogen atoms of the water molecules and those bonded to nitrogen atoms were located in electron density difference maps and were freely refined.Crystal data, data collection and structure refinement details are summarized in Table 710.1107/S2056989021008823/dj2030sup1.cifCrystal structure: contains datablock(s) I, II, III, GLOBAL. DOI: 10.1107/S2056989021008823/dj2030Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989021008823/dj2030IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989021008823/dj2030IIIsup4.hklStructure factors: contains datablock(s) III. DOI: Click here for additional data file.10.1107/S2056989021008823/dj2030Isup5.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989021008823/dj2030IIsup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989021008823/dj2030IIIsup7.cmlSupporting information file. DOI: 2104939, 2104938, 2104937CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The initial step of the reaction involved the preparation of the intermediate 9,10-dihydro-9,10-ethanoanthracene-11,12-dicarboxylic anhydride (3) through [4 + 2]-cycloaddition between anthracene and maleic anhydride in refluxing xylene which was then condensed with para-aminophenol to give compound 5. Compounds 5 and 8 were characterized by their physical, elemental, and spectroscopic data. 2D-NMR  techniques were used to confirm the structure of compound 5. Compounds 5 (MIC = 32–128 μg/mL) and 8 (MIC = 16–256 μg/mL) along with the precursor 3 (MIC = 64–128 μg/mL) displayed moderate to low antimicrobial activities against selected bacterial and fungal species when compared with those of nystatin (MIC = 0.50–2 μg/mL) and ciprofloxacin (MIC = 0.50–16 μg/mL) used as reference drugs.A new diazo derivative of a pyrrolidine-2,5-dione (The online version contains supplementary material available at 10.1186/s13065-022-00801-5. Pyrrolidine derivatives known as succinimid are cyclic imides with five vertices . In addicrobial –14.Staphylococcus aureus, vancomycin resistant Enterococcus; which sets the limits of the therapeutic treatments currently used + from which the molar mass of the compound was deduced to be m/z: 367 corresponding to the raw formula C24H17NO3.Its mass spectrum shows two peaks of pseudo molecular ions, one at 390 (100%) corresponding to [M + Na]1H NMR spectrum shows, despite the symmetry, that the aromatic protons in dibenzobarrelene moiety are not equivalent due to the molecular arrangements + from which the molar mass of the compound was deduced to be m/z: 471 corresponding to the gross formula C30H21N3O3  [M+-OH], 394 (24%) [M+ -C6H5], 377 (18%) [M+-OH -C6H5], 311 (11%) [M+-OH-C6H5-CO2-C-N2 + H2O], 278 (15%) [M+-OH-C14H10].On its mass spectrum in ESI3 Scheme . The mas1H NMR spectrum of compound 8, the protons of the aniline moiety H-2′′, H-3′′, H-4′′, H-5′′ and H-6′′ were exhibited at 7.45  overlapping with the protons of the dibenzobarrelene nucleus. It’s also exhibited as in compound 5 the aromatic protons of the dibenzobarrelene moiety respectively at 7.40 , 7.32 , 7.19 . The benzylic protons appeared at 4.75  and 3.25 . The phenolic moiety presents three types of protons, two doublets and a singlet. The singlet at 6.98  is attributable to the proton H-6′ whereas, the doublets at 6.68  and 6.18  are respectively attributed to the protons H-2′ and H-3′. The 13C NMR of this compound showed more than sixteen carbons instead of thirteen as in compound 5; confirming therefore the above suggested substitution pattern. In addition to all the carbons present in compound 5, there are new carbon signals at 160.7 attributable to the depleted carbon C-1′′ of aniline carrying the azo group; that at 130.8 attributable to carbon C-5′ of the phenolic fragment bearing the azo group. Furthermore, one can notice an overlapping of the signals due to carbons C-2′′, C-3′′, C-4′′, C-5′′, C-6′′ at 129.1.On the 5 and 8 are different from each other  μg/mL, (32–128) μg/mL and (16–64) μg/mL on bacteria and 128 μg/mL, (64–128) μg/mL, (64–256) μg/mL on yeasts. It is noted that the introduction of the phenol fragment into compound 3 induced an increase in the activity of compound 5 in particular on Staphylococcus aureus, Candida tropicalis PK233, Cryptococcus neoformans H99 and remains unchanged on the other microorganisms. Furthermore, the introduction of the azo function in compound 5 resulted into an increase of the activity of compound 8, in particular on Staphylococcus aureus, Vibrio cholera SG24, Vibrio cholera CO6. The yeasts have shown a low sensitivity with respect to the azo compound, thus showing that this compound has much better antibacterial than antifungal activity on these types of microorganisms.The antimicrobial activities were evaluated on seven species of microorganisms including bacteria and yeasts and the data are summarized in Table S. aureus, V. cholera NB2 and V. cholera SG24 with compounds 8, 5 and 3 while fungicide effect (MFC/CMI ≤ 4) was recorded on C. neoformans H99 with compounds 8 and 3 and on C. tropicalis PK233 with compound 3. Compounds 8, 5 and 3 showed bacteriostatic effect (MBC/CMI > 4) against V. cholera CO6 and fungistatic effect on Candida albicans ATCC10231. Compounds 8 and 5 displayed fungistatic effect (MFC/CMI > 4) on C. tropicalis PK233 while fungistatic effect was recorded on C. neoformans H99 with compound 3.All the compounds tested showed weak biological activities compared to the reference drugs. Bactericidal effects (MBC/CMI ≤ 4) were observed on The variations in the susceptibilities observed between the microorganisms and the compounds tested would be due to the differences in genetic constitutions that exist between the different microbial strains tested .The cytotoxic activity of azo compounds against red blood cells (RBCs) was investigated using Triton X-100 as a positive control. Interestingly, none of the tested compounds showed cytotoxic activity against RBCs at concentrations up to 256 μg/mL (results not shown). This finding supports the selective toxicity of the tested compounds towards the tested bacteria and fungi.3, 5, 8 possess antimicrobial activities. Although being less active than the compound taken as a reference, the azo compound has better antibacterial activity than the other two compounds especially on Staphylococcus aureus, Vibrio. Cholera SG24 and Vibrio cholera CO6 strains. The antimicrobial screenings revealed that all the tested compounds 8, 5 and 3 have moderate to low antibacterial and antifungal activities. These results show that the azo function (N = N) is indeed a pharmacophore and would be responsible for the biological activity in the azo molecules.The results of biological tests showed that compounds α1 radiation  in transmission geometry with a DECTRIS® MYTHEN 1 K detector . HR-ESI–MS spectra were performed with a spectrometer  equipped with a HR-ESI source. The spectrometer operates in positive ion mode  with automatic gain control to provide high-accuracy mass measurements within 0.40 ppm deviation using Na formate as calibrant. The following parameters were used for experiments: spray voltage of 4.5 kV, capillary temperature of 200 °C. Nitrogen was used as sheath gas (10 l/ min). The spectrometer was attached to an Ultimate 3000  UHPLC system consisting of LC-pump, Diode Array Detector (DAD) (λ: 190–600 nm), auto-sampler (injection volume 10 μl) and column oven (40 °C). The separations were performed using a Synergi MAX-RP 100 A  with a H2O (+ 0.1% HCOOH) (A)/acetonitrile (+ 0.1% HCOOH) (B) gradient  with fluorescent indicator. A mixture of hexane and ethyl acetate (1:2) was used as eluent and iodine was used as revelator for the chromatograms. The IR spectra were measured with a Fourier Transform Infrared spectrometer Brucker Alpha. The UV spectra were recorded with a JENWAY 6715 UV–Vis Spectrophotometer. Combustion analyses were carried out with a C, H, N, and S Euro EA from Hekatech Company, their results were found to be in good agreement (± 0.3%) with the calculated values. XRD data was collected on a STOE Stadi-p X-ray powder diffractometer  with Cu K1H NMR spectra and 13C NMR spectra were recorded in deuterated chloroform on a Bruker SF spectrometer operating respectively at 400 and 100 MHz; TMS was used as internal reference.500 μL/min, injection volume 5 μl). 4.92 g (27.76 mmol) of anthracene and 2.43 g (23.80 mmol) of maleic anhydride are refluxed for 40 min in 50 ml of xylene. The solution obtained is filtered hot and left to stand for about a day for gentle and gradual crystallization of the product. The latter is then filtered, dried and crystallized from xylene to give 6.30 g (83%) of white crystals; mp: 315 °C  previously dissolved in 30 ml of acetic acid is added and the mixture is heated under reflux for 3 h and then cooled to room temperature. The solution is filtered and washed with aqueous ethanol (50%) and dried to give 4.84 g of a gray-colored product 5; mp: 337 °C (Lit [max (DMSO): 250, 355, 395 nm. IR (potassium bromide): 3363 cm−1 (OH), 2973 (CH), 1696 (C = O), 1600–1562 (CAr = CAr), 1273 (C-N), 1202 (C-O), 764 (= CArH) cm−1. 1H NMR (CDCl3) δ 7.32 , 7. 23 , 7.11 . 4.75 , 3.25 , 6.62 , 6.17 . 13C (1H)-NMR (CDCl3) δ177.9 , 157.4 (C-4′), 141.2 , 138.8 , 122.7 (C-1’), 127.6 , 127.1 , 126.8 , 125.1 , 124.3 , 115.9 , 46.9  and 45.8 . Anal. Calcd. for C24H17NO3 (367.12): C, 78.56; H, 4.66; N, 3.8; found: C, 78.35; H, 4.83; N, 3.91.4 g (0.0145 mol) of  °C (Lit : 334–335 °C (Lit : 57%); E6) was dissolved in DMSO (5 mL) and cooled to 0–5 °C. The nitrosyl sulphuric acid solution was added to the amine solutionand the temperature was maintained to 0–5 °C.Dry sodium nitrite  was slowly added over a period of 30 min to concentrated sulphuric acid (5 mL) with occasional stirring. The solution was cooled to 0–5 °C. 1 g (1.07 mmol) of aniline  was dissolved in DMSO (5 mL) and then cooled in an ice-bath at 0–5 °C. The diazonium solution 7 previously prepared was added drop wise over 1 h before neutralizing the sulfuric acid present with a 10 ml sodium acetate (10%) solution. 50 ml of ice-cold water was then added and the solution was filtered off after 30 min and rinsed with iced water. After crystallization from ethanol After crystallization from ethanol (98%), 315 mg of compound 8 was obtained as brown powder; mp: 271 °C, Yield 67%; ESI–MS: 494.22 . UV–Vis: λmax (DMSO) = 385 nm; IR (potassium bromide): 3367 cm−1 (OH), 3060 (CH), 1696 (C = O), 1598 (CAr = CAr), 1465 (-N = N-), 1274 (C-N), 1202 (C-O), 764 (= CArH) cm−1; 1H NMR(CDCl3) δ ppm: 7.45 . 7.40 , 7.32 , 7.19 , 6.98 , 6.68 , 6.18 , 4.75 , 3.25 , 13C NMR (CDCl3) δ ppm: 176.5, 160.7, 152.8, 141.4, 138.8, 130.8, 129.1, 127.8, 127.2, 126.9, 125.2, 124, 122.9, 116.0, 47.0 and 46.1. Anal. Calcd. for C30H21N3O3 (471.16): C, 76.42; H, 4.49; N, 8.91; found: C, 76.12; H, 4.68; N, 8.68.Compound Staphylococcus aureus ATCC25923, three Gram-negative Vibrio cholera NB2, V. cholera SG24 and V. cholera CO6 and three yeast strains Candida albicans ATCC10231, Candida tropicalis PK233 and Cryptococcus neoformans H99. These microorganisms were taken from our laboratory collection. The fungal and bacterial strains were grown at 37 °C and maintained on Sabouraud Dextrose Agar  and nutrient agar  slants respectively.The antimicrobial activity was performed against four bacterial and three fungal species. The selected microorganisms were one Gram-positive 5 cells/mL for yeasts and 106 CFU/mL for bacteria. Dilutions of nystatin  and ciprofloxacin  were used as positive controls for yeasts and bacteria respectively. Broth with 10 µL of DMSO was used as negative control. MICs were assessed visually and were taken as the lowest sample concentration at which there was no growth or virtually no growth. The lowest concentration that yielded no growth after the sub-culturing was considered as the MBCs or MFCs. All the tests were performed in triplicate [The antibacterial and antifungal activity was evaluated by determining the MICs and MMCs as previously described . MICs ofiplicate .Whole blood (10 mL) from albino rats was collected by cardiac puncture in EDTA tubes. The study was conducted according to the institutional guidelines and approved by the Cameroon National Ethical Committee (Reg. No. FWA-IRB00001954) and in compliance with the ARRIVE guidelines. Erythrocytes were harvested by centrifugation at room temperature for 10 min at 1000 xg and were washed three times in PBS buffer .The cytoAdditional file 1: Figure S1. UV Spectrum of compound 5. Figure S2. IR Spectrum of compound 5. Figure S3. Mass Spectrum of compound 5. Figure S4.1H NMR of compound 5. Figure S5. COSY of compound 5. Figure S6.13C NMR of compound 5. Figure S7. HSQCSpectrum of compound 5. Figure S8. HMBC Spectrum of compound 5. Figure S9. UV Spectrum of compound 8. Figure S10. IR Spectrum of compound 8. Figure S11. HRMS ESI-Positive mode of compound 8. Figure S12. Mass Spectrumfragmentation of compound 8. Figure S13.1H NMR of compound 8. Figure S14.13C NMR of compound 8. Table S1. ARRIVE Essential 10. Table S2. ARRIVE Recommended Set.


Parkinson’s disease (PD) is characterized by degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc), accompanied by accumulation of α-synuclein, chronic neuroinflammation and autophagy dysfunction. Previous studies suggested that misfolded α-synuclein induces the inflammatory response and autophagy dysfunction in microglial cells. The NLRP3 inflammasome signaling pathway plays a crucial role in the neuroinflammatory process in the central nervous system. However, the relationship between autophagy deficiency and NLRP3 activation induced by α-synuclein accumulation is not well understood.Through immunoblotting, immunocytochemistry, immunofluorescence, flow cytometry, ELISA and behavioral tests, we investigated the role of p38-TFEB-NLRP3 signaling pathways on neuroinflammation in the α-synuclein A53T PD models.Our results showed that increased protein levels of NLRP3, ASC, and caspase-1 in the α-synuclein A53T PD models. P38 is activated by overexpression of α-synuclein A53T mutant, which inhibited the master transcriptional activator of autophagy TFEB. And we found that NLRP3 was degraded by chaperone-mediated autophagy (CMA) in microglial cells. Furthermore, p38-TFEB pathways inhibited CMA-mediated NLRP3 degradation in Parkinson's disease. Inhibition of p38 had a protective effect on Parkinson's disease model via suppressing the activation of NLRP3 inflammasome pathway. Moreover, both p38 inhibitor SB203580 and NLRP3 inhibitor MCC950 not only prevented neurodegeneration in vivo, but also alleviated movement impairment in α-synuclein A53T-tg mice model of Parkinson’s disease.Our research reveals p38-TFEB pathways promote microglia activation through inhibiting CMA-mediated NLRP3 degradation in Parkinson's disease, which could be a potential therapeutic strategy for PD.p38-TFEB pathways promote microglia activation through inhibiting CMA-mediated NLRP3 degradation in Parkinson's disease. In this model, p38 activates NLRP3 inflammasome via inhibiting TFEB in microglia. TFEB signaling negatively regulates NLRP3 inflammasome through increasing LAMP2A expression, which binds to NLRP3 and promotes its degradation via chaperone-mediated autophagy (CMA). NLRP3-mediated microglial activation promotes the death of dopaminergic neurons.The online version contains supplementary material available at 10.1186/s12974-021-02349-y. Parkinson’s disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra, resulting in neuromuscular dysfunction such as bradykinesia, muscular rigidity and rest tremors. Previous studies have shown that neuroinflammation may be a primary cause of PD-related pathogenesis and the degeneration and loss of dopaminergic neurons. Proinflammatory cytokines such as tumor necrosis factor α (TNF-α) and IL-1β are upregulated in the cerebrospinal fluid and blood of PD patients , 3. ReceMitogen-activated protein kinases (MAPKs) regulate various cellular processes. The p38 mitogen-activated protein kinase (p38MAPK), a prominent member of the MAPKs, is not only involved in cell cycle, cell death, development, differentiation, senescence and tumorigenesis, but also functions as a specific class of serine/threonine kinase regulating the inflammation responses. The important role of p38-mediated inflammatory responses was originally confirmed as the target of the pyridinyl imidazole that inhibited inflammatory cytokines release in lipopolysaccharides (LPS)-treated monocytes . The MAPCMA is a pathway for selective degradation of single proteins that is mediated by binding to HSC70 (HSPA8), which recognizes a pentapeptide (KFERQ) in the substrate protein sequence. Then the substrate/chaperon complex binds the lysosome membrane-bound LAMP2A, which triggers the LAMP2A multimerization and followed lysosomal internalization of substrate proteins for degradation. The reduced neuronal CMA activity is observed in the aging brain and neurodegenerative diseases. This decline in CMA activity underpins the pathogenesis of neurodegeneration, including PD .Here, we demonstrate that the p38–TFEB pathway regulates NLRP3 inflammasome degradation via CMA. We not only just identified that NLRP3 is a novel substrate for CMA, but also found that p38 inhibits TFEB activity via phosphorylation, which blocks NLRP3 degradation. Besides, our results reveal that both p38 inhibitor SB203580 and NLRP3 inhibitor MCC950 reduced microglial activation and dopaminergic neuronal loss induced by α-synuclein. All the results suggest the p38–TFEB pathway could be a potential treatment target for NLRP3 inflammasome-driven neurodegenerative diseases.The α-synuclein A53T-tg mice expressing mutant A53T α-synuclein (the 140 amino acid isoform) under the direction of the mouse prion protein promoter have been described previously . The α-sMice were placed on a narrow beam (2*3*60 cm) suspended 20 cm above soft bedding, and their movement from one end towards the other end was recorded. The number of missteps  was scored during the trip.This test was performed based on the method described by Ogawa  used as All animal procedures were approved by the ethics committee of the Southern Medical University. 20 mg/kg MCC950 was used with daily oral administration . Adult CPlasmids: EGFP-α-synuclein A53T (40823), PHM6-α-synuclein-A53T (40825), pAAV α-synuclein WT (36055), pCDNA3 Flag p38α (20352), and pMT3-p38 (12658) were obtained from Addgene. The following antibodies were used: anti-Parkin , anti-LC3A/B , anti-beta Tubulin , anti-p38 , anti-p-p38tyr182 , anti-α-synuclein , anti-synapsin-1 , anti-NLRP3 , anti-IL-1β , anti-TFEB , anti-TH , anti-ACTB , anti-LaminB , anti-SQSTM1/P62 , anti-GAPDH , anti-CASP1 , anti-HSPA8 , anti-ASC , anti-LAMP2A , anti-IBA-1 , anti-GFP , anti-14-3-3 , anti-FLAG . ELISA KITs were purchased from Jiangsu Meimian industrial Co., Ltd including CASP1  and IL-1β . SB203580  and MCC950  were purchased from Selleckchem . AR7  and QX77  were purchased from MCE. The siRNA for p38 was: 5′-GGUCUGUUGGAUGUGUUCAdTdT-3′. PCR primers of α-synucleinA53T-tg were: F 5′-TGTAGGCTCCAAAACCAAGG-3′, R 5′-TGTCAGGATCCACAGGCATA-3′.The paraffin sections of brain tissue were collected for routine immunohistochemistry staining for p-p38 (1:50), TH (1:100), IBA-1 (1:100), IL-1β (1:100), NLRP3 (1:100), LAMP2A (1:100) and TFEB (1:100). The area of the midbrain dopaminergic neurons from brain sections according to the anatomical position and the location of TH-positive neurons has been described previously .4 in sodium cacodylate buffer on ice for 1 h and dehydrated with acetone, and then embedded in resin and polymerized at 60 °C for 48 h. Ultrathin sections were obtained on an ultramicrotome and stained with uranyl acetate and lead citrate before observation under Hitachi H-7500 TEM.SN4741 cells were washed once with PBS, collected with a cell scraper. The sections of midbrain tissue were collected and washed once with cold PBS. Cell and midbrain tissue pellets were re-suspended in the fixative containing 2.5% glutaraldehyde in PBS at 4 °C. Cell and midbrain tissue pellets were fixed for 3 h. After rinsing with sodium cacodylate buffer, they were further fixed in 1% OsOl-glutamine and supplemented with 10% fetal bovine serum at 33 °C. BV2 cells were cultured in DMEM medium containing 10% fetal bovine serum  at 37 °C. Cells were transfected with Lipofectamine 3000 reagent  following the manufacturer's instructions.SN4741 cells were cultured in DMEM medium containing 1% glucose, 100 U/ml penicillin–streptomycin, 1% g for 10 min at 4 °C, and then the supernatant was transferred into a new tube. And then 5 μg of the corresponding first antibody was added to the samples and incubated overnight at 4 °C. The next day, 50 μl agarose beads were added to the samples and incubated 2 h at 4 °C. The immunocomplex was collected with centrifugation at 1500×g for 1 min at 4 °C. And washed 3 times with RIPA buffer. Proteins were eluted from beads with 2 × SDS loading buffer and subjected to immunoblot analysis. The immunoreactive bands were detected by Odyssey Infrared Imaging System. The band intensity was analyzed with Image J analysis software. Antibodies used in Western blotting are diluted by 1000, except TH (1:100) and p-p38(1:100).Cytosolic and nuclear fractions were collected using an isolation kit  following the manufacturer’s instructions. For Western blotting, cells were collected in RIPA buffer containing 20 mM Tris–HCl, pH 7.4, 150 mM NaCl and 1% Triton X-100 with protease inhibitor cocktail . The extracts were centrifuged for 10 min at 13,000 rpm at 4 °C, and the supernatants were used for immunoblot analysis. For co-immunoprecipitation, cells were centrifuged at 13,000×Cell apoptosis of SN4741 cells was measured using a Cell Apoptosis Kit  according to the manufacturer’s instructions. The percent of cell apoptosis were determined by staining with 2 μM Annexin V and 2 μM propidium iodide (PI). The number of cell apoptosis was counted by flow cytometry and images of apoptosis was taken by laser scanning confocal microscopy. Cell death of primary neuron was detected by staining with PI (1 μg/ml) (KeyGEN) according to the manufacturer’s instructions. Images were captured by fluorescence microscope and analyzed by Image J analysis software.The mitochondrial membrane potential in cells was assessed using tetramethylrhodamine methyl ester . Cells were washed with 1 × PBS and then incubated with TMRE (0.1 μM) in the medium for 20 min in the dark. The intensity of fluorescence was monitored using a fluorescence microscope.Tissue specimen: after cutting the specimen, weigh 0.1 g of tissue, add 9 ml of PBS, and homogenize the specimen. Centrifuge for about 20 min  and carefully collect the supernatant that used for ELISA detection following the manufacturer's instructions. The cell culture medium: the cell culture medium was centrifuged and used for ELISA detection following the manufacturer's instructions.Cells were grown on a confocal dish for 2 days, and then cells were washed with PBS three times, then fixed with 4% paraformaldehyde for 15 min at room temperature. After that, cells were permeabilized with frozen methanol for 10 min at − 20 °C and blocked in 5% BSA for 30 min. And then samples were incubated with primary antibody (1:100–1:200) in 5% BSA overnight at 4 °C, and then incubated with secondary antibody (1:100) in 5% BSA for 1 h at room temperature. An Olympus FV1000 confocal microscope with a × 100 objective was used for image capture. The co-localization signal was analyzed with Image Pro Plus software.All data of experiments were analyzed with GraphPad Prism 7 software . Data from the Western blot and immunohistochemistry analysis were performed with the t-test for two groups or ANOVA with Tukey’s post-test (GraphPad Software) for multiple groups. Data from the balance beam test, pole test, transmission electron microscopy, immunofluorescence, were analyzed by two-way ANOVA followed by Tukey’s post-test. All values are expressed as the mean ± SEM, and p values < 0.05 were considered statistically significant. All experiments were repeated at least 3 times.In order to investigate the effects of α-synuclein accumulation and related toxicity, we used α-synuclein A53T transgenic mice (α-synucleinAA53T-tg mice). The accumulation of α-synuclein (SNCA) was identified in both the SNpc and the cortex at 9 months of age . Inspection of the amino acid sequence of mouse NLRP3, four KFERQ-like motifs , were revealed  or QX77 , the latter two chemicals are well-known CMA agonists. The results showed that both serum deprivation and CMA agonists increased the levels of LAMP2A and reduced NLRP3 . The results showed that p38 inhibitor SB203580 significantly elevates TFEB-mediated autophagy, as evidenced by the increased LAMP1, MAP1LC3/LC3, and the decrease of SQSTM1  by mTORC1 has been proved to promote TFEB binding to 14-3-3, we detected the level of TFEB phosphorylation with a phospho-Ser211-specific antibody. It showed that SB203580 reduced TFEB phosphorylation at serine 211  Immunohistochemistry (IHC) demonstrating increased α-synuclein in the cortex and SNpc of 9 months α-synuclein A53T-tg or wild-type mice. Statistical analysis of the average score of α-synuclein staining between α-synuclein A53T-tg and wild-type mice. *p<0.05 (Student’s t-test). (B) Immunohistochemistry (IHC) demonstrating increased IBA-1 in the cortex and SNpc of 9 months α-synuclein A53T-tg or wild-type mice. (C) The co-staining between Iba1 and NLRP3 was detected by IF, which the dynamic changes of NLRP3 inflammasome activation in microglia from 3 months to 9 months of age.  Levels of IL-1β and IL-18 in serum were assessed by ELISA assay in 10 patients suffering from PD and 10 control. Data were performed using the Student’s unpaired t-test.Additional file 2: Fig S2 Lysates from the cortex and SNpc of mice were immunoblotted using the indicated antibodies. The protein levels of p-p65 were statistically analyzed in B. Mean ± SEM, n = 6, *p < 0.05 (Student’s t-test).  Cell lysates from BV2 cells were immunoblotted demonstrating SB203580 decreased the levels of NLRP3, ASC, cleaved CASP1 and shown in D. Mean ± SEM, n = 6, *p < 0.05.  Levels of caspase-1 and IL-1β in tissue homogenates of wild type, α-synuclein A53T-tg and α-synuclein A53T-tg treated with SB203580 were assessed by ELISA assay. (G) The level of mRNA was detected by qPCR. (H) The mouse NLRP3 contains two noncanonical KFERQ-like pentapeptide . (I-K) Cell lysates from primary microglia were immunoblotted to detect the LAMP2A and NLRP3 after starvation and analyzed in J and K. Mean ± SEM, n = 3, *p < 0.05.  Cell lysates from primary microglia were immunoblotted to detect the levels of LAMP2A and NLRP3 after treatment with AR7 and QX77 and statistically analyzed in M. Mean ± SEM, n = 3, *p < 0.05.Additional file 3: Fig S3.  Lysates from SNpc of 9 months α-synuclein A53T-tg or wild-type mice were subjected to subcellular fractionation, the nuclear and cytosolic fractions were immunoblotted using the indicated antibodies to determine the levels of TFEB. Data are shown in C. Mean ± SEM, n = 3. *p < 0.05.  BV2 cells were labeled with Lysosome tracker and visualized lysosome biogenesis under a microscope demonstrating SB203580 increased the levels of lysosome biogenesis and shown in E. Mean ± SEM, n = 10, *p < 0.05. (E) EM images of the lysosomal morphology were shown after SB203580 treatment. (F) The autophagy-related proteins LC3 and P62 were detected in SNpc of brain tissue. (G)Levels of IL-1β and IL-18 were assessed by ELISA. Data were performed using the Student’s unpaired t-test.  Cell lysates from BV2 cells were immunoblotted to detect the levels of pSer211TFEB and statistically analyzed in M. Mean ± SEM, n = 3.Additional file 4: Fig S4.  BV2 cells were labeled with Lysosome tracker and visualized lysosome biogenesis under a microscope demonstrating si-p38 increased the levels of lysosome biogenesis and shown in B. Mean ± SEM, n = 10, *p < 0.05.  Cell lysates from BV2 cells were immunoblotted demonstrating si-p38 decreased the levels of NLRP3, cleaved CASP1 and shown in D. Mean ± SEM, n = 6, *p < 0.05.  Cell lysates from BV2 cells were immunoblotted demonstrating TFEB decreased the levels of NLRP3, cleaved CASP1 and shown in F. Mean ± SEM, n = 6, *p < 0.05.  Cell lysates from BV2 cells were immunoblotted and shown in H. Mean ± SEM, n = 6, *p < 0.05.  Levels of IL-1β and IL-18 in conditional mediate of BV2were assessed by ELISA assay. Data were performed in F.  Cell lysates from BV2 cells were immunoblotted demonstrating autophagy inhibitor CQ eliminate the effect of decreased NLRP3 from SB20350 and shown in L. Mean ± SEM, n = 6, *p < 0.05.Additional file 5: Fig S5.  Cell apoptosis of SN 4741 cells was detected by flow cytometry dyeing with Annexin V-FITC/PI, followed by the treatment with conditioned medium from BV2. Mean ± SEM, n = 3, *p < 0.05. (C) Balance beam foot slips were quantified after PBS or MCC950 injection demonstrating the protective effect of MCC950. (D) Results of the pole test were quantified after PBS or MCC950 injection demonstrating the protective effect of MCC950.  Immunofluorescence (IF) staining of synapsin-1 (SYN-1) in the SNpc of mice. Mean ± SEM, n = 6, *p < 0.05.


O-β-d-glucopyranosyl-(25R)-furost-5-ene-3β,22α,26-triol 3-O-(1−4)-β-d-glucopyranosyl-α-l-rhamnopyranosyl-(1−2)-[α-l-rhamnopyranosyl-(1−4)]-β-d-glucopyranoside [asparacochioside A (1)] was isolated from a hot water extract of the roots of Asparagus cochinchinensis, together with the known steroidal saponins protodioscin (2), methyl protodioscin (3), aspacochioside A (4), aspacochioside C (5), 15−hydroxypseudoprotodioscin (6), and chamaedroside E (7). The structure of the new compound 1 was determined by interpretation of its spectroscopic data (1D- and 2D-NMR and HR−Q−TOF−MS) and sugar analysis. The isolated compounds 1−7 were tested for their in vitro cytotoxicity against human ovarian cancer cell lines (A2780 and SKOV3). Asparacochioside A (1) exhibited a significant cytotoxicity against both A2780 and SKOV3 cells with IC50 values of 5.25 ± 2.2 and 46.82 ± 9.43 μM, respectively. Furthermore, asparacochioside A (1) significantly increased the percentage of Annexin V-positive cells (apoptotic cells), suggesting that asparacochioside A induces ovarian cancer cell death via apoptosis.A new steroidal saponin, 26- Asparagus (Liliaceae) comprises over three hundred species distributed around the world and used in salads and as herbs and vegetables. Asparagus cochinchinensis (Loureio) Merrill is a perennial herb distributed in Eastern Asia, including many provinces of China, Japan, and Korea . Based on the 1H-1H correlation spectroscopy (COSY) and 1H-13C heteronuclear multiple bond correlation (HMBC) data  . The 1H-   10]. Th. Th1 wasnd 140.7 . AccordiBC) data , it was 1 has three glucoses and two rhamnoses [J = 7.5 Hz)] with C−3 (δ 78.0), H−Rha−1′’ [δ 6.42 ] with C−Glc−2′ (δ 78.0), H−Rha−1′’’ [δ 5.87 ] with C−Glc−4′ (δ 78.4), and H−Glc−1′’’’’ [δ 5.24 ] with C−26 (δ 78.5)  analysis. The 1H- and 13C-NMR spectroscopic data of compound 1 were very similar to those of protodioscin (2), except for the presence of an additional β-glucose unit at Rha-4′’’ in 1  -β-d-glucopyranoside and named asparacochioside A.Through analysis of the HMBC and nuclear Overhauser effect correlation spectroscopy (NOESY) spectra , it was hamnoses . The pos(δ 78.5) . The glu′’’ in 1  10]. Th. Th1 hasRha−3′’’ . The abs = 0.32) . Thus, t2) -2,5-diphenyl tetrazolium bromide (MTT) assay.To evaluate the potential anti-tumor activity of the isolates from the hot water extract of 1), protodioscin (2), and methyl protodioscin (3) exhibited a significant cytotoxicity in A2780 cells with IC50 values of 5.25, 10.14, and 21.78 μM, respectively, in a dose dependent manner. In SKOV3 cells, only the new compound asparacochioside A (1) showed a significant cytotoxicity with observed IC50 value of 46.82 μM, while compounds 4−7 were not active against either A2780 or SKOV3 human ovarian cancer cells (>100 μM). Overall, among the seven compounds isolated from A. cochinchinensis, only the novel compound 1 showed a potent cytotoxicity against both A2780 and SKOV3, suggesting its potential anti−cancer activity in human ovarian cancer.As shown in 2) and methyl protodioscin (3) have been reported to show various biological activities such as anti-tumor and anti-inflammatory effects  to afford seven subfractions (C6−1~C6−7). Subfraction C6−4 (1.75 g) was subjected to silica gel CC  with EtOAc−acetone−H2O mixture (from 4:5:1 to 0:8:2 v/v/v), yielding protodioscin . Subfraction C6−5 (7.16 g) was subjected to silica gel CC  eluting with EtOAc−acetone−H2O [4:5:1 to 3.5:5:1.5 v/v/v] and afforded methylprotodioscin . Aspacochioside A  was separated from subfraction C6−2 (392.0 mg) by silica gel CC  with EtOAc−acetone−H2O mixture (4:5:1 v/v/v). Subfraction C6−6 (2.11 g) was fractionated by silica gel CC , eluting with EtOAc−acetone−H2O gradient system [from 4:5:1 to 3.5:5:1.5 v/v/v] to yield compound 1 (724.2 mg).Dried roots of 2O mixture (4:5:1 v/v/v) to generate nine subfractions (C7−1~C7−9). 15−Hydroxy−pseudoprotodioscin  was purified by flash chromatography using a Redi Sep−C18 cartridge  from C7−6 (1.6 g). Protodioscin  by flash chromatography using a Redi Sep−C18 cartridge .Fraction C7 (16.3 g) was subjected to silica gel CC  with EtOAc−acetone−H2O, from 10:90 to 40:60 v/v) to afford aspacochioside C .Fraction C9 (406.6 mg) was fractionated further by flash chromatography using a Redi Sep−C18 cartridge ; IR (ATR) νmax 3410, 1745, 1712 cm−1; HR−Q−TOF−MS (negative mode) m/z = 1209.5892 [M − H]−, ; 1H- and 13C-NMR data, see Pale brown powder; m.p.: 188.2 °C; 1 (10.0 mg) was hydrolyzed with 1N HCl at 80 °C for 3 h. Sodium bicarbonate was added to stop the reaction.Compound β-glucose and α-rhamnose in 1 was determined using the method reported by Tanaka et al. [l-cysteine methyl ester hydrochloride (1.2 mg), and σ-tolyl isothiocyanate (100 μL) and analyzed using HPLC. The glucose and rhamnose in the reaction mixture of 1 was detected at 30.7 and 35.5 min, respectively. The retention times of authentic d-glucoses and l-rhamnose were 30.8 and 35.4 min, respectively, under the same HPLC conditions. Therefore, the absolute configuration of β-glucose in 1 was confirmed as the d configuration, whereas α-rhamnose was confirmed as the l configuration.The absolute configurations of a et al. . The hydA2780 and SKOV3 human ovarian cancer cell lines were obtained from American Type Culture Collection (ATCC). The cells were maintained in Roswell Park Memorial Institute (RPMI) 1640  medium with penicillin (100 U/mL), 5% fetal bovine serum (FBS), and streptomycin sulfate . Cells with medium were kept in humidified atmosphere of 5% CO and 37 °C temperatures.5/mL per well. After 24 h, cells were treated with different concentrations of extract or compounds for 48h. Following incubation, 50 μL of MTT  was added and incubated for 4 h in the 37 °C incubator. The medium was discarded and the formazan crystals were dissolved in DMSO. The absorbance was measured using a microplate spectrophotometer  at 540 nm.The cell growth was assessed using the MTT assay. Briefly, Cells were seeded in a 96-well plate at a density of 1 × 101) for indicated time periods or concentrations. After incubation, both floating and adherent cells from each well collected in tubes and washed with PBS. The cells were then re-suspended in 500 μL of binding buffer and stained with 2.75 μL of FITC-conjugated Annexin V for 15min in darkness. Finally the stained cells were washed with binding buffer and stained with 5 μL of PI (50 mg/mL) prior to analysis using a Guava® easyCyte flow cytometer .Apoptotic cells were detected by the binding of fluorescent Annexin V (Annexin V-FITC). Cells were treated with asparacochioside A . Data are presented as mean ± SD and evaluated by unpaired Student 1‒7, including a new furostane-type steroidal saponin named asparacochioside A (1) were isolated from the roots of A. cochinchinensis in the present study. The chemical structure of the new compound was determined using their spectroscopic data  measurement and by acidic hydrolysis. Isolation of chamaedroside E (7) from the roots of A. cochinchinensis was reported for the first time in this paper. Among the isolates, asparacochioside A (1), protodioscin (2), and methyl protodioscin (3) exhibited a significant cytotoxicity against A2780 cells. In SKOV3 cells, only the new compound asparacochioside A (1) showed a significant cytotoxicity. Compound 1 induced apoptotic cell death in human ovarian cancer cells and significantly increased the percentage of annexin V positive cells (apoptotic cells) in a dose- and time-dependent manner. Thus, asparacochioside A (1) is worthy of additional experiments for its potential as an anti-cancer agent.Seven compounds


The enthalpy changes associated with these sequential processes and with the overall dissociation reaction A:B2(cr) → A(cr) + 2B(cr) were determined by solution calorimetry. It was found that they are all positive, indicating that there is a lattice enthalpy gain when MA:Phe2 is formed, either from the individual precursors or by adding Phe to MA:Phe. Single-crystal X-ray diffraction (SCXRD) analysis showed that MA:Phe2 is best described as a protic salt containing a maleate anion (MA−) and two non-equivalent L-phenylalanine units, both linked to MA− by NH···O hydrogen bonds (H-bond): one of these units is protonated (HPhe+) and the other zwitterionic (Phe±). Only MA− and HPhe+ molecules are present in the MA:Phe lattice. In this case, however, NH···O and OH···O H-bonds are formed between each MA− unit and two HPhe+ molecules. Despite these structural differences, the enthalpy cost for the removal of the zwitterionic Phe± unit from the MA:Phe2 lattice to yield MA:Phe is only 0.9 ± 0.4 kJ mol−1 higher than that for the dissociation of MA:Phe, which requires a proton transfer from HPhe+ to MA− and the rearrangement of L-phenylalanine to the zwitterionic, Phe±, form. Finally, a comparison of the dissociation energetics and structures of MA:Phe and of the previously reported glycine maleate (MA:Gly) analogue indicated that parameters, such as the packing coefficient, density, hydrogen bonds formed, or fusion temperature, are not necessarily good descriptors of dissociation enthalpy or lattice enthalpy trends when bi-component crystals with different molecular composition are being compared, even if the stoichiometry is the same.The energetics of the stepwise dissociation of a A:B In toquinone ,16,17,18oquinone ,22. But oquinone .a priori guiding the synthesis of multi-component crystals, or the inhibition of their formation, is a reasonable understanding of the structure–energetics relationships that determine their thermodynamic stability towards dissociation into the precursors. These relationships are, however, still poorly understood at molecular level  [A central issue for t clear) ,25, and Thermodynamic stability is most appropriately assessed in terms of the Gibbs energy change, eactions , stabiliulations  and the ulations , suggestulations . The genExperimental data are, of course, essential for the understanding of structure–energetics relationships in multi-component crystals, and for the development and validation of theoretical approaches such as those mentioned above. However, in contrast with the large body of available structural information , studiesp-aminobenzoic acid system, there is no correlation between the dissolution rate and the stoichiometry  of multicomponent crystals [One aspect that has been little explored from an experimental perspective is the influence of stoichiometry on the energetics of multi-component crystals. There have been reports showing that in the case of the carbamazepine:crystals , demonstcrystals , or suggcrystals . We are cr” indicates that the dissociation refers to the crystalline state. By the same token, if reaction:If bi-component crystals are regarded as a supermolecules composed of A and B species, then the standard molar enthalpies of the reactions:olecules  and deno2) stoichiometries were synthesized using mechanochemistry. Solvent free grinding was used and the precursor samples corresponded to the form I polymorphs of MA (CSD Refcode: MALIAC11) [2 was prepared for the first time. Analysis of the jar contents at different grinding times, by X-ray powder diffraction, suggested that MA:Phe is an intermediate of the MA:Phe2 formation , transparent parallelepipedal crystals were obtained.Two bi-component crystals composed by maleic acid and L-phenylalanine with 1:1 (MA:Phe) and 1:2 (MA:PheALIAC11) ,37 and PALIAC11) ,38. IndeALIAC11) ,39) and ALIAC11) ,40. MA:Ption see . Crystal2 crystals carried out at 168 ± 2 K and 293 ± 2 K showed no significant changes in the supramolecular structure with temperature. Therefore, only the 293 ± 2 K results are discussed here, since they correspond to a temperature which is closer to the reference temperature of the thermodynamic data (298 K).Single-crystal X-ray diffraction analysis of MA:Phe2 is compared in 2 involves a proton transfer from maleic acid to one of the L-phenylalanine molecules, with the second L-phenylalanine remaining zwitterionic as in the pure reactant form (−), a L-phenylalanine cation (HPhe+), and a zwitterionic L-phenylalanine (Phe±). The proton transfer from maleic acid to phenylalanine is also present in the case of MA:Phe  Å and 2.41(3) Å) and single (dNH∙∙∙O = 2.02(4) Å) H-bonds between the ammonium group of HPhe+ and the carboxylate group of MA−. The second one  Å and 2.55(3)) and single (dNH∙∙∙O = 2.26(3) Å) H-bonds of N−H∙∙∙O type. Both chains propagate along the b-axis, and they link to each other through a bifurcated O−H∙∙∙O hydrogen bond (dOH∙∙∙O = 1.28(3) Å and 2.54(4) Å) involving the carboxylic and carboxylate groups of adjacent HPhe+ and Phe± molecules, establishing a 2D sheet  Å) and the other between the carboxylic group of a cationic L-phenylalanine and the ammonium group of a zwitterionic L-phenylalanine (dNH∙∙∙O = 2.08(4) Å).From a packing point of view, two 1D chain motifs, both of the irst one a is compcond one b is form2D sheet c. The fi2D sheet d is comp2. The molecules are also organized in +−MA−−HPhe+− one-dimensional patterns that propagate along the b-axis (NH∙∙∙O = 2.20 Å and 2.29 Å) and single (dNH∙∙∙O = 2.10 Å) N−H∙∙∙O hydrogen bonds between the ammonium group of HPhe+ and the COO···OOC framework of MA−  bifurcated hydrogen bonds belonging to the OH∙∙∙O = 1.74 Å) involving the carboxylic group of HPhe+ and an carboxylate oxygen of MA−. The two chains with a common hydrogen bond define the 2D pattern as shown in a-axis by means of an extra N−H∙∙∙O bifurcated hydrogen bond (dNH∙∙∙O = 2.25 Å and 2.45 Å) between MA− and two HPhe+ units . In thibe found b. This ce+ units d.2 (dOH∙∙∙O = 1.69 Å) defining a −.The molecular structure differences observed in the unit cells of pure maleic acid and L-phenylalanine compared to those of MA:Phe and MA:Phe2  are alsodNH∙∙∙O = 1.74 Å and 1.75 Å; bottom: dNH∙∙∙O = 1.98 Å and 2.10 Å).Phenylalanine, two  present b. Each o2, and their precursors are summarized in k) are in the 0.65–0.77 range, which is typical of the close packing range found for spherical or ellipsoid objects [2 are within those of MA (high limit) and Phe (low limit) and become closer to the low limit as the number of Phe units in the crystal lattice increases.The volumetric features of MA:Ph, MA:Phe objects ; (ii) th2 were investigated by differential scanning calorimetry (DSC) and thermogravimetry (TG) at a heating rate of 5 K⋅min−1. For comparison purposes, DSC experiments were also carried out on the MA(cr I) and Phe(cr I) precursors. As shown in Ton) and maximum (Tmax) temperatures of the fusion peaks are summarized in 2, and Phe(cr I).The thermal behaviors of MA:Phe and MA:PheTon = 415.8 ± 0.6 K) and MA:Phe2 (Ton = 444.6 ± 0.6 K) fall within those of pure MA (Ton = 410.7 ± 1.4 K) and Phe (Ton = 529.4 K), and (ii) as the Phe content per formula unit increases the fusion temperature becomes closer to that of pure L-phenylalanine . The first observation is very common for bi-component crystals [Ton = 417.6 ± 1.3 K [Tfus = 411.3 ± 0.4 K) [α-glycine (Tfus = 525.0 ± 0.6 K) [2 from those of pure L-phenylalanine parallels the trend noted for density in Two major conclusions can be drawn from  ± 1.3 K  in the r± 0.4 K)  and α-gl± 0.6 K) . The cloTon = 410.7 ± 1.4 K, −1) were also in close agreement with the results of previous determinations for the same polymorph, namely, Tfus = 411.9 K [Tfus = 411.3 ± 0.4 K [−1 .The temperature and enthalpy of fusion of MA(cr I) in  411.9 K , Tfus =  ± 0.4 K , and ΔfuTon ~ 518 K and is accompanied by thermal decomposition. This is reflected by a complex peak with maxima at 527 and 537 K. As mentioned above, thermal decomposition was also signaled by the presence of a brownish material inside the crucible at the end of the experiment. The same complex melting/decomposition behavior was observed by Lu et al. [Tmax may be due, at least in part, to the higher heating rate used by these authors (10 K∙min−1). The present observations are also consistent with previous TG-FTIR studies indicating that Phe starts to undergo pyrolysis at 515 K with the formation of CO2, NH3, H2O, and benzeneethanamine [The DSC curve obtained for Phe(cr I) a evidencu et al. , who rephanamine . It may 2, reactions (2) and (3) can be written in a compact form as:b = 1 for reaction (2) and b = 2 for reaction (3). The standard molar enthalpy of reaction (4), 2 was obtained from [In the case of MA:Phe and MA:Phened from ,31:(5)Δrn is the amount of substance of water used in the experiments per 1 mol of dissolved solid compound. The obtained results are summarized in In the previous equations, 2, b is the stoichiometric coefficient of the Phe unit (b = 1 for MA:Phe and b = 2 for MA:Phe2). Based on −1 for MA:Phe, −1 for MA:Phe2  . Scheme  MA:Phe2 , ΔsubHmokJ∙mol−1 , and ΔsukJ∙mol−1 , ΔLatHmo−1 is significantly larger than that previously found for the analogous glycine maleate, −1 [k = 0.695, ρ = 1.407 g cm−3, and Ton = 415.8 ± 0.6 K for MA:Phe) than for the latter . This shows that a larger lattice enthalpy does not necessarily imply a larger packing efficiency or fusion and thermal decomposition temperatures when different materials are compared.The value − anion is linked to four protonated amino acid molecules through one O−H···O and three N−H···O hydrogen bonds which, on average, are slightly shorter for MA:Gly (2.02 Å) than for MA:Phe (2.12 Å). Thus, the 20.7 kJ∙mol−1 larger lattice enthalpy of MA:Phe relative to that of MA:Gly should, most likely, reflect the increase in importance of van der Walls interactions and the presence of electrostatic interactions between aromatic rings, associated with the replacement of a hydrogen atom in glycine by a phenyl ring in L-phenylalanine. It is interesting to note that a similar difference (24.2 kJ∙mol−1) exists between the sublimation enthalpies of Phe  and Glyol−1 [−1 ). This ekJ∙mol−1 .2(cr) obtained from the (i)2, MA:Phe, and MA:Gly bi-component crystals are stable towards dissociation into pure components, since positive The MA:Phe(ii)2 should be more stable than MA:Phe, since MA:Phe ± 0.6 K , this is(iii)2, −1, is only slightly larger than the second, −1. This indicates that the enthalpic cost of removing the zwitterionic Phe± unit from MA:Phe2 to produce MA:Phe is not much different from that associated with the disruption of the MA:Phe lattice to yield neutral MA(cr I) and Phe(cr I), which implies the back neutralization of MA− by proton transfer from HPhe+ and the concomitant rearrangement of the latter to a zwitterionic tautomer;The first dissociation enthalpy of MA:Phe(iv)−1) is smaller than that of MA:Gly (9.6 ± 1.0 kJ mol−1), despite the fact that the opposite is observed in terms of lattice enthalpy  and MA:PheElemental C, H, and N analyses (EA) were performed on a Fisons EA1108 apparatus, with an uncertainty of ±0.3% for carbon and nitrogen and ±0.1% for hydrogen.® column , thermostated at 308.15 K. The mobile phase consisted of 0.1% formic acid in ultra-pure water (A) and acetonitrile (B). The elution gradient was as follows: 0 min, 5% B; 20 min, 70% B; 22 min, 100% B; 22–30 min 100% B; 30–45 min, 5% B. The injected sample volume was 10 μL, and the mobile phase flow was 300 μL min−1. Mass spectra obtained in positive ESI mode were acquired using the following optimized parameters: spray voltage = +4.5 kV, transfer capillary voltage = −18 V, ion transfer lens offset = +63 V, nebulizing gas (N2) pressure = 80 arbitrary units, drying gas (N2) pressure = 20 arbitrary units, transfer capillary temperature = 543 K. Data acquisition and processing were performed using Xcalibur 2.2 software.HPLC-ESI/MS analyses were carried out on an Ultimate RS system with a diode array detector coupled to an LCQ Fleet mass spectrometer  equipped with an ESI ionization source. Chromatographic separation was achieved with a Phenomenex 100 Å C18 Luna1 (λ = 1.5406 Å) tube, operated at 40 kV and 40 mA. The data collection was performed in the 2θ range 5–35° with a step size of 0.02°. Glass sample holders were used. The indexation of the powder patterns was performed with CellRef [Powder X-ray diffraction measurements were carried out at 296 ± 2 K on a D8 Advance Bruker diffractometer  e quipped with a LinxEye detector. The radiation was produced with a Cu-Kα CellRef .4H4O4: expected C 41.39%, H 3.47%; found C 41.31 ± 0.13%, H 3.37 ± 0.11% . The purity given by HPLC-ESI/MS analysis was >99.9%. The powder X-ray diffraction pattern was indexed as monoclinic, space group 1/cP2, a = 7.481 ± 0.033 Å, b = 10.010 ± 0.015 Å, c = 7.620 ± 0.032 Å, β = 123.66 ± 0.28o (see Supporting Information). This indicated that the sample corresponded to form I maleic acid, previously characterized by single-crystal X-ray diffraction  was used as received. Elemental analysis for C9H11O2N: expected C 65.44%, H 6.71%, N 8.48%; found C 65.60 ± 0.52%, H 6.64 ± 0.18%, N 8.49 ± 0.08% (average of two determinations). The purity given by HPLC-ESI/MS analysis was >99.9%. The powder X-ray diffraction pattern was indexed as monoclinic space group 1P2, a = 8.800 ± 0.012 Å, b = 6.042 ± 0.006 Å, c = 31.488 ± 0.035 Å, β = 96.58 ± 0.34o (see Supporting Information). These results correspond to form I L-phenylalanine  was used as received. Elemental analysis for Curements ,38).2 samples were prepared by mechanochemistry. Milling was performed with a Retsch MM400 apparatus and a 25 cm3 cylindrical stainless-steel jar containing two 5 mm diameter stainless-steel spheres. The frequency was set to 29 Hz and the grinding time was 15 min. The precursor materials were weighted with a precision of ±0.01 mg in a Mettler Toledo XS205 balance. In the case of MA:Phe, 0.08266 g (0.71 mmol) of maleic acid and 0.11770 g (0.71 mmol) of L-phenylalanine were used. Elemental analysis for C13H15NO6: expected C 55.51%, H 5.38%, N 4.98%; found C 55.51 ± 0.09%, H 5.34 ± 0.08%, N 4.96 ± 0.02% (average of two determinations). The powder pattern of the obtained product was indexed as monoclinic, space group 1P2, a = 11.067 ± 0.010 Å, b = 5.342 ± 0.003 Å, c = 11.481 ± 0.010 Å, β = 101.10 ± 0.13°  of maleic acid with 0.14827 g (0.90 mmol) of L-phenylalanine. Elemental analysis for C22H26N2O8: expected C 59.19%, H 5.87%, N 6.27%; found C 58.75 ± 0.12%, H 5.95 ± 0.09%, N 6.23 ± 0.02% (average of two determinations). The powder pattern was indexed as monoclinic, space group 1P2, a = 13.974 ± 0.020 Å, b = 5.425 ± 0.003 Å, c = 15.415 ± 0.022 Å, β = 108.78 ± 0.16° (see Supporting Information). These results are in excellent agreement with the corresponding data obtained in this work from single-crystal X-ray diffraction experiments . MA:F2 using SHELXL-2014/7 [The selected single crystals were coated with Paratone-N oil and mounted in a Kapton loop. The single-crystal X-ray diffraction data were collected at 168 ± 2 K and 293 ± 2 K, using a BRUKER D8 QUEST  diffractometer, with a PHOTON II detector or a Bruker AXS-KAPPA APEX II diffractometer. The radiation was produced by applying a potential difference and current of 50 kV and 30 mA, respectively, to a graphite-monochromated Mo-Kα source (λ = 0.71073 Å). The X-ray data collection was monitored by the Bruker APEX3 software . An empiL-2014/7 , both prL-2014/7 . Atoms wL-2014/7 , also inL-2014/7 . A summa−1 in the temperature range 298–618 K. The sample and balance chambers were purged with nitrogen gas , with flows of 23 cm3∙min−1 and 38 cm3∙min−1, respectively. The mass scale of the apparatus was calibrated using a 100 mg standard mass. The calibration of the temperature scale was based on the Curie temperatures (TC) of nickel  and alumel .Thermogravimetry analyses were performed on a Perkin Elmer  TGA-7 apparatus. The samples with 2–15 mg mass were contained in a platinum crucible and studied at a heating rate of 5 K min3 min−1 flow of nitrogen , in the temperature range 298–570 K, and at a heating rate of 5 K min−1. The temperature scale of the instrument was calibrated at the same heating rate by measuring the onset temperatures (Ton) for the fusion of indium  and zinc . The calibration of the energy scale was based on the enthalpy of fusion of indium (∆fush = 28.45 J∙g−1).The DSC experiments were made on a Perkin Elmer  DSC-7. The samples with 1–7 mg mass were sealed inside aluminum crucibles and weighted with a precision of ±0.1 µg on a Mettler XP2U ultra-micro balance. The studies were carried out under a 30 cmThe DSC and TG apparatus were both controlled by the Perkin Elmer  Pyris V. 7.0.0.0110 software, which was also used for data acquisition and analysis.3 cylindrical vessel closed by a lid that supports the sample drop system, the stirrer, and the calibration resistance. In a typical experiment, a pellet of sample with 10–15 mg mass, was weighted with a precision of ±0.1 µg on a Mettler XP2U ultra-micro balance and placed in the drop system. Approximately 13 cm3 of solvent were added to the cell body and weighted with a precision of ±0.01 mg using a Mettler XS 205 balance. The calorimetric cell was assembled and transferred to the calorimeter. Stirring (80 rpm) was started and, after an appropriate base line was acquired, the sample was dropped into the solvent. The molar enthalpy of the dissolution process, m and M are the mass and molar mass of sample, respectively, A is the area of the observed calorimetric curve, ε is the calibration constant. The value of Ab = −(0.978 ± 0.072) mV·s was determined from a set of five experiments, where the drop mechanism was activated without a sample. The constant, ε, was obtained from a series of calibration experiments where a potential difference, V, was applied to 20 Ω resistance placed inside the calorimetric cell, causing a current of intensity, I, to flow through the resistance during a pre-programed time, t. This resulted in the dissipation of an amount of heat, Q = VIt, by Joule effect, which was reflected by a curve of area, Acal. The value of ε was calculated from:iV and iI are the voltage and current intensity at a given time it, respectively, and ∆it~1 s is the time difference between two consecutive readings.Enthalpies of solution were determined at 298.15 K on a LKB 2277 Thermal Activity Monitor, using an in-house designed calorimetric cell made of stainless steel. The cell consists of a 20 cmThe control of the experiment and data acquisition were performed with the CBCAL 3.0 program . EasyGra2, were synthesized from maleic acid (form I) and L-phenylalanine (form I), using mechanochemistry and characterized from structural and energetics points of view. Single-crystal X-ray diffraction (SCXRD) analysis showed that they are best described as organic salts: MA:Phe2 includes a maleate anion (MA−) and two L-phenylalanine units, one protonated (HPhe+) and another zwitterionic (Phe±); MA:Phe contains only MA− and HPhe+ ions. In the first case, the main H-bond motif between the molecular units is of the N−H···O type and in the second N−H···O and O−H···O hydrogen bonds are present.Two bi-component crystals with different stoichiometries, MA:Phe and MA:Phe2 are both stable relative to dissociation into their precursors; (ii) the enthalpic cost of removing Phe± from MA:Phe2 to yield MA:Phe (the first dissociation energy of MA:Phe2) is only 0.9 ± 0.4 kJ mol−1 larger than the cost of removing HPhe+ from MA:Phe ; (iii) the latticed enthalpy difference between the two bi-component crystals, −1, is comparable with the sublimation enthalpy of L-phenylalanine, −1, indicating that, at least in this case, Calorimetric determinations showed that on enthalpic grounds: (i) MA:Phe and MA:PheFinally, the comparison of MA:Phe with the previously reported glycine maleate (MA:Gly), in terms of structure and energetics, suggested that structural/volumetric descriptors, such as packing coefficient, density, and hydrogen bond patterns, may not appropriately reflect dissociation enthalpy or lattice enthalpy trends in families of bi-component crystals differing in one of the co-formers, even if the stoichiometry is the same.


N-phenylcarboxamido groups.The asymmetric unit of the title molecule consists of four molecules that differ primarily in the orientations of the styryl and the  25H21N3O2S, comprises four molecules. Their conformations differ primarily in the orientations of the styryl and the N-phenylcarboxamido groups. In the crystal, intermolecular N—H⋯N, C—H⋯O and C—H⋯S hydrogen-bonding contacts as well a C—H⋯π(ring) interactions lead to the formation of a layer structure parallel to (010). Hirshfeld surface analysis revealed that H⋯H interactions represent the main contributions to the crystal packing.The asymmetric unit of the title compound, C Weaker C33—H33C⋯O8iii and C75—H75⋯S4v interactions between a methyl group and an a carbonyl O atom, and between a phenyl CH group and a thiophene S atom, respectively, consolidate the packing. Together with three sets of C—H⋯π(ring) interactions, supramolecular layers parallel to the ac plane with a width corresponding to b/2 are formed  mapped over dnorm in a fixed colour scale of −0.3297 (red) to + 1.5167 (blue) a.u. for molecule I, −0.3246 (red) to +1.4683 (blue) a.u. for molecule II, −0.3890 (red) to +2.0338 (blue) a.u. for molecule III, and −0.3870 (red) to +1.8555 (blue) a.u. for molecule IV. The red spots on the Hirshfeld surface are indicative of contacts shorter than van der Waals separations and represent N—H⋯N, N—H⋯O, C—H⋯O and C—H⋯S contacts. Fig. 7For the four molecules I, II, III and IV, intermolecular interactions Table 4 were quThe fact that the same interactions result in different contributions to the Hirshfeld surface for molecules I, II, III and IV can be attributed to the different environments of each molecule in the crystalline state.et al., 2016b]pyridine moiety yielded ten structures closely related to the title compound: ethyl 3-amino-6-methyl-2-[(4-methylphenyl)carbamoyl]-4-[(E)-2-phenylethenyl]thienopyridine-5-carboxylate hydrate -2-phenylethenyl]thienopyridine-2,5-dicarboxylate diphosphonate) °], while pairwise intermolecular N—H⋯O hydrogen bonding forms dimers across centres of symmetry. In the crystal of XEBPIF, an intramolecular N—H⋯Ocarbonyl hydrogen bond closes an S(6) ring. Supramolecular chains along [01b]pyridine unit. In the crystal of AWETIH, molecules are linked by pairs of N—H⋯O hydrogen bonds, forming inversion dimers with an a-axis direction, there is a weak π–π interaction involving inversion-related thiophene rings. In the crystal of ULAROQ, the conformation of the title molecule is partially determined by an intramolecular N—H⋯O hydrogen bond, forming an S(6) loop, and an N—H⋯π interaction involving the centroid of the 4-chlorophenyl ring. In the crystal, molecules are linked by pairs of N—H⋯O hydrogen bonds, forming inversion dimers with an b-axis direction by intermolecular N—H⋯O and N—H⋯N hydrogen bonds. WUVZES crystallizes with two molecules in the asymmetric unit. The crystal structure is stabilized by intermolecular and intramolecular C—H⋯O hydrogen bonds. The asymmetric unit of NEQSUA likewise comprises two molecules, which differ primarily in the orientations of the acetyl and p-anisyl substituents. In the crystal, N—H⋯O hydrogen bonds form chains extending parallel to (110). The asymmetric unit of the molecular salt RUTRUV comprises two cations and two anions. Each cation features an intramolecular N—H⋯O hydrogen bond, which closes an S(6) ring; in each case the hydropyridine ring adopts a half-chair conformation. In the crystal, O—H⋯O and N—H⋯O hydrogen bonds link the components into [100] chains. Numerous C—H⋯O interactions cross-link the chains into a three-dimensional network.In the crystal of TACXED, mutual N—H⋯O hydrogen bonds form dimers, which are then associated into chains parallel to the N-phenyl-2-chloroacetamide  in an ethanol solution (60 ml) was added, together with sodium ethoxide . The resulting mixture was refluxed for 10 minutes. The solid that precipitated after cooling was collected and recrystallized from ethanol to give the title compound in the form of yellow crystals, yield 92%; m.p. 481–483 K. IR (cm−1): 3452, 3292, 3220 , 3027 , 1701  and 1633 . 1H NMR: δ 9.59 , 7.85–7.88 , 7.07–7.69 , 6.79 , 6.71–6.74 , 2.52 , δ 2.42 . 13C NMR: δ 205.61, 164.34, 158.93, 154.69, 148.61, 140.86, 139.61 (CH of CH=CH), 139.18, 136.00, 133.67, 129.59 (CH), 129.30 (CH), 128.89 (CH), 127.85 (CH), 124.12 (CH), 122.21 (CH of CH=CH), 122.02 (CH), 121.84 (CH), 121.85, 121.25, 98.87, 32.87 (CH3 of acetyl group), 23.27 (CH3 attached to pyridine ring). MS: m/z 427.14 . Analysis calculated for C25H21N3O2S (427.13): C 70.24, H 4.95, N 9.84%. Found: C 70.51, H 4.85, N, 9.90%.To a suspension of 5-acetyl-3-cyano-1,2-dihydro-6-methyl-4-styryl-2-thioxopyridine , Crystal data, data collection and structure refinement details are summarized in Table 610.1107/S2056989022000743/wm5633sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989022000743/wm5633Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989022000743/wm5633Isup3.cmlSupporting information file. DOI: 2143707CCDC reference:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information: