L. lucidus was subjected for isolation of new bioactive compounds, which enabled isolation of five new pimarane-type diterpenoids, namely, 3β, 8β, 12β, 18-tetrahydroxy pimar-15-ene (10), 7α, 8β, 12β, 18-tetrahydroxy pimar-15-ene (11), 3β, 8β, 11β, 12α, 18-pentahydroxy pimar-15-ene (12), 12β acetoxy, 8β, 3β, 18-trihydroxy pimar-15-ene (13), and 3β acetoxy, 8β, 12β, 18-trihydroxy pimar-15-ene (14), along with nine known compounds. The structures were elucidated by spectroscopic analysis and comparison with literature data. The isolated new pimarane diterpenoids were examined for antimicrobial activity against Gram-negative and Gram-positive bacteria strains. Among them, the compound 3β, 8β, 12β, 18-tetrahydroxy pimar-15-ene (10) was most effective, exhibiting minimum inhibitory concentration (MIC) values of 15.62 µg/mL against Staphylococcus epidermidis, 31.25 µg/mL against Staphylococcus aureus, 62.5 µg/mL against Pseudomonas aeruginosa, and 125 µg/mL against Escherichia coli.The ethyl acetate fraction obtained from aerial parts of  Lycopus of family Lamiaceae (Labiatae) contains around 16 species with wide distribution in Europe, Asia, and North America -. 1H-NMR  δ: 7.33 , 7.28 , 6.77 . 1³C-NMR  δ: 167.3 , 155.0 (C-4), 144.9 (C-3), 121.9 (C-5), 121.7 (C-1), 116.5 (C2), 115.1 (6).White gray crystalline powder; HR-ESI-MS m/z: 137.0251[M-H]-. 1H-NMR  δ: 9.69 , 7.25 , 7.22 , 6.90 . 1³C-NMR  δ: 190.0 , 152.1 (C-4), 145.8 (C-3), 129.8 (C-5), 124.49 (C-1), 115.5 (C2), 114.3 (6).White gray crystalline powder; HR-ESI-MS m/z: 167.148[M-H]-. 1H-NMR  δ: 7.43 , 7.42 , 6.83 , 3.801 . 1³C-NMR  δ: 167.12 , 151.01 (C-4), 147.18 (C-3), 123.44 (C-1), 121.74 (C-6), 114.98 (C-5), 112.71 (C-2), 55.526 .White gray crystalline powder; HR-ESI-MS m/z: 137.12[M-H]-. 1H-NMR  δ: 7.78 , 6.81 . 1³C-NMR  δ: 167.1 (-COOH), 161.5 (C-4), 131.5 (C-2& C6), 121.4 (C-1), 115.0 (C-3&C5).White crystalline powder; HR-ESI-MS m/z: 179.159[M-H]-. 1H-NMR  δ: 7.55 , 7.07 , 6.95 , 6.81 , 6.224 . 1³C-NMR  δ: 171.2 (C-9), 149.5 (C-4), 147.0 (C-7), 146.9 (C-3), 127.9 (C-1), 122.9 (C-6), 116.6 (C-5), 115.7 (C-8), 115.2 (C-2).White crystalline powder; HR-ESI-MS m/z: 359.29[M-H]-. 1H-NMR  δ: 7.51 , 7.03 , 6.82 , 6.72 , 6.66 , 6.64 , 6.57 , 6.28 , 5.20 , 3.06 , 3.00 . 1³C-NMR  δ: 171.3 (C-9), 166.8 (C-9′), 149.1 (C-4′), 146..6 (C-7′), 146.4 (C-3′), 145.8 (C-3), 144.9 (C-4), 129.1 (C-1), 127.3 (C-1′), 121.5 (C-6′), 117.3 (C-6), 116.6 (C-2), 115.6 (C-5′), 116.4 (C-5), 114.3 (C-2′), 114.2 (C-8′), 73.8 (C-8), 37.0 (C-7).White crystalline powder; HR-ESI-MS m/z: 373.24[M-H]-. 1H-NMR  δ: 7.55 , 7.04 , 6.95 , 6.78 , 6.70 , 6.69 , 6.57 , 6.26 , 5.19 , 3.7 , 3.06 , 3.00 . 1³C-NMR  δ: 172.3 (C-9), 168.4 (C-9′), 149.9 (C-4′), 148.1 (C-7′), 146.9 (C-3′), 146.3 (C-3), 145.5 (C-4), 128.8 (C-1), 127.7 (C-1′), 123.3 (C-6′), 121.9 (C-6), 117.6 (C-2), 116.6 (C-5′), 116.4 (C-5), 115.3 (C-2′), 114.2 (C-8′), 74.8 (C-8), 52.8 (-OCH3), 38.0 (C-7).White crystalline powder; HR-ESI-MS m/z: 463.371[M-H]-. 1H-NMR  δ: 7.71 , 7.58 , 6.87 , 6.39 , 6.20 , 5.25 , 3.37–3.60 . 1³C-NMR  δ: 179.6 (C-4), 166.1 (C-7), 163.2 (C-5), 159.1 (C-2), 158.5 (C-9), 149.9 (C-4′), 146.0 (C-3′), 135.7 (C-3), 123.3 (C-6′), 123.2 (C-1′), 117.6 (C-5′), 116.1 (C-2′), 105.8 (C-10), 104.4 , 100.0 (C-6), 94.8 (C-8), 78.5 , 78.2 , 75.8 , 71.3 , 62.7 .Yellow crystalline powder; HR-ESI-MS m/z: 185.24[M-H]-. 1H-NMR  δ: 12.97 , 7.42 , 7.39 , 6.88 , 6.66 , 6.43 , 6.18 . 1³C-NMR  δ: 181.6 (C-4), 164.1 (C-7), 163.8 (C-2), 161.4 (C-5), 157.2 (C-9), 149.7 (C-4′), 145.7 (C-3′), 121.4 (C-1′), 118.9 (C-6′), 115.9 (C-5′), 113.3 (C-2′), 103.6 (C-10), 102.8 (C-3), 98.82 (C-6), 93.8 (C-8).Yellow amorphous powder; HR-ESI-MS m/z 337.2354 and molecular formula C20H34O4.13C  and 1H-NMR  spectroscopic data are given in Tables White crystalline needles; freely soluble in methanol and ethyl acetate; HR-ESI-MS [M-H]- ion at − ion at m/z 337.2322 and molecular formula C20H34O4.13C  and 1H-NMR  spectroscopic data are given in Tables White crystalline needles, freely soluble in ethyl acetate and methanol; HR-ESI-MS [M-H]m/z 353.2269 with molecular formula C20H34O5. 13C  and 1H-NMR  spectroscopic data are given in Tables White crystalline needles; freely soluble in ethyl acetate and methanol, HR-ESI-MS [M-H]- ion at − ion at m/z 379.2479 with molecular formula C22H36O5. 13C  and 1H-NMR  spectroscopic data are given in Tables White crystalline needles; freely soluble in ethyl acetate and methanol, HR-ESI-MS [M-H]− ion at m/z 379.2473 with molecular formula C22H36O5. 13C  and 1H-NMR  spectroscopic data are given in Tables White crystalline needles; freely soluble in acetone; sparingly soluble in ethyl acetate and methanol, HR-ESI-MS [M-H] S. aureus (ATCC 9144) and S. epidermidis (ATCC 12228) and Gram-negative bacteria (P. aeruginosa (ATCC 27853) and E. coli (ATCC 14948) were purchased from National Path Lab, Butwal, Nepal. All the ATCC strains were subcultured on different culture media and investigated for Gram staining and biochemical test. The detail of microbial analysis of all the strains is given on supplementary data  was transferred into each vial. For the sample solution preparation, 25000 µg/mL of stock solution was prepared in DMSO, subjected to serial dilution, using a 1 : 1 mixture of DMSO and water to prepare sample solutions of 10 different concentrations (25000 µg/mL-48.8298125 mg/mL). After that, 40 µL of sample solution was transferred into a corresponding vial containing 960 µL of MHB, so that the final concentration of sample ranged from 1000 µg/mL to 1.95 µg/mL. Bacteria with an inoculum of about 1 × 105 CFU/mL were loaded into each vial. For the preparation of microorganism inocula, broth culture was incubated for 12 h, and turbidity of the suspension was adjusted to the turbidity of 0.5 McFarland standards. One inoculated vial was used as a negative control, to ensure suitability broth for growth of microorganism growth. Also, 4% DMSO was tested as a blank control. Streptomycin sulfate and Vancomycin were considered as a positive control for Gram-negative and Gram-positive microorganisms, respectively. After the incubation of the sample containing broth media for 24 h at 37 0C, the MIC value was determined. The MIC value was considered as the minimum concentration of compound that prevented the microorganism growth. The bacterial cell viability was determined by using 3-2-5-diphenyl tetrazolium bromide (MTT) by incubating at 37°C for further 2 h and visual inspection of formazan formation - ion at m/z 337.2354 and molecular formula C20H34O4. The 1H-NMR showed three olefin protons, twelve methylene groups, two oxygenated geminal methylene protons, two oxygenated methine protons, and two other normal methine protons, along with three methyl protons. The 13C-NMR exhibited 20 carbon signals consisting of two olefin carbon signals at δC 153.4 (C-15) and 108.9 (C-16), four oxygenated carbon signals at δC 75.6 (C-12), 74.9 (C-8-tertiary carbon), 68.5 (C-3), and 64.5 (C-18). Three methyl signals were detected at δC 33.7 (C-17), 25.0 (C-19), and 22.1 (C-20). Similarly, six other normal methylene carbons at δC 36.7 (C-1), 28.5 (C-2), 17.9 (C-6), 39.2 (C-7), 27.1 (C-11), and 45.9 (C-14) and two normal methine carbon signals at δC 43.9 (C-5) and 40.5 (C-9) were also recorded. Four carbon signals at δC 74.8 (C-8), 45.5 (C-4), 37.7 (C-13), and 33.9 (C-10) were found to be tertiary carbons through HMQC analysis. All protons were assigned to their corresponding carbons using HMQC experiment. Correlation of two oxygenated germinal methylene protons at δH 3.89  and 3.54  to carbon δC 64.5 (C-18) suggests presence of CH2OH group. Among three olefinic protons, two were found to be connected with carbon δC 153.4 (C-15). The presence of an ABX spin system in its 1H-NMR spectra due three vinyl protons of monosubstituted double bond at d 4.9 , d 4.79 , and d 5.78  along with the respective carbon resonances in the 13C-NMR spectra  ensured the compound is pimar-15-en derivative - ion at m/z 337.2322 and molecular formula C20H34O4. It differs from compound 10 by presence of C-7-hydroxyl group instead of C-3. Hydroxylation at C-7 was confirmed by its chemical shift (δC-74.1). Final structure of compound is confirmed as 7α, 8β, 12β, 18-tetrahydroxy pimar-15-ene.Compound 12 was found to be white amorphous powder. Its molecular formula C20H34O5 was deduced by using HR-ESI-MS [M-H]- ion at m/z 353.2269. Spectral data of compound 12 was similar to compound 10 except new addition of hydroxyl group at C-11, which was ensured by its chemical shift value (δC-73.1). ROESY ensured the chemical structure of compound 12 as 3β, 8β, 11β, 12α, 18-pentahydroxy pimar-15-ene.Compound 13 was white in color with crystalline needles. Its molecular formula C22H36O5 was deduced by using HR-ESI-MS [M-H]- ion at m/z 379.2479. Spectral data of compound 13 was different from compound 10 by addition of new methyl proton as an acetyl moiety at δH 2.06  along with one new tertiary carbonyl carbons signal at δC 172.2  and one methyl carbon at δH 22.4 (C-22). Increment of mass of compound 13 by 42 units [-COCH3-H+] also confirmed the addition of one acetyl moiety. Position of acetyl group was ensured accurately by other 2D-NMR analysis. In the HMBC data, carbon–proton correlations (C-21/H-22) illustrated attachment of the methyl group with carbonyl carbon. Correlation (C-12/H-21) verified the position of acetyl group. ROESY analysis illustrated the chemical name of compound 13 as 12β acetoxy, 8β, 3β, 18-trihydroxy pimar-15-ene.Compound 14 also was white crystalline needles having similar molecular formula, mass fragmentation, and spectral data with compound 13. It differed from compound 13 only by position of acetyl moiety. Chemical shift of C-3 (δH 65.9) gives an idea about its oxygenation. From the HMBC data, carbon–proton correlations (C-21/H-22) illustrated that methyl group is attached with carbonyl carbon and another association (C-22/H-18) suggested the location of acetyl group at C-3. Chemical name compound was deduced as 3β acetoxy, 8β, 12β, 18-trihydroxy pimar-15-ene. The 13C and 1H-NMR spectroscopic data of compound 10–14 are given in Tables Compound ations C-1/H-22 ilS. epidermidis (ATCC 12228) and S. aureus (ATCC 9144) and Gram-negative bacteria P. aeruginosa (ATCC 27853) and E. coli (ATCC 14948), using a twofold serial broth dilution technique and MIC was evaluated. As shown in 10, which manifested MIC values of 15.62 and 31.25 µg/mL against S. epidermidis and S. aureus, respectively. Also, this compound was effective against P. aeruginosa and E. coli with MICs of 62.5 µg/mL and 125 µg/mL, respectively. It is to be noted that compounds 13, 14, and 15 were ineffective against both Gram-negative strains at examined concentrations.The isolated new pimarane diterpenoids were examined for their antimicrobial potency in response to Gram-positive bacteria 10 and 11, were slightly different. The structural difference between these compounds is only the position of one hydroxyl group (3rd position OH group in compound 10 is shifted to 7th position in compound 11). Although the exact structure-activity relationship is not known, this may signify that a change in position of the same functional group may also alter the antibacterial effect. On the other hand, the antibacterial activity of compounds 13 and 14 was reported to be reduced, in which -OH group was replaced by an electron-withdrawing acetyl group. In a previous study, 3- and 19-hydroxyl groups of isopimarane compounds were acetylated to investigate the role of substituents on antimicrobial activity. The results indicated that if acetylation occurs in the 3-hydroxyl position or both 19- and 3-hydroxyl groups, the antibacterial activity gets reduced significantly [In the search for natural products as effective antimicrobial agents, numerous investigations have proved the promising bactericidal effect revealed by diterpenoids. However, very limited researches have been reported for the antibacterial potency of pimarane diterpenoids . It has ficantly . Hence, ficantly , 28, 29.L. lucidus and their chemical structures were elucidated completely through instrumental data including 2D-NMR.Five new pimarane diterpenoids along with nine known compounds (polyphenol and flavonoids) were isolated from the ethyl acetate fraction of 6 (rosmarinic acid) was isolated in the largest amount. Compound 2  and compound 4  were isolated for the first time from this plant. Besides, antibacterial activity screening for newly isolated compounds showed that pimarane diterpenoids are more sensitive towards Gram-positive bacteria. Among the isolated new diterpenoids, compounds 10 and 11 were found to be most effective with 15.62 µg/mL and 31.25 µg/mL MICs values, respectively, against S. epidermidis. In addition, this screening showed that small changes in position and/or nature of the oxygenated functional group in diterpenoids can result in a significant variation in antibacterial activity.Among them, compound


Fusarium oxysporum isolated from the plant Aglaonema hookerianum Schott. And to investigate the cytotoxic and other pharmacological properties of the isolated compounds as part of the drug discovery and development process. Different chromatographic techniques were adopted to isolate the bioactive compounds that were identified by spectroscopic techniques. The cytotoxic properties of the compounds were assessed in the Vero cell line via the trypan blue method. Moreover, physicochemical, pharmacokinetic, bioactivity and toxicity profiles of the compounds were also investigated through in silico approaches. After careful spectral analysis, the isolated compounds were identified as 3β,5α-dihydroxy-ergosta-7,22-dien-6-one (1), 3β,5α,9α-trihydroxy-ergosta-7,22-dien-6-one (2), p-hydroxybenzaldehyde (3), 3-(R)-7-butyl-6,8-dihydroxy-3-pent-11-enylisochroman-1-one (4) and beauvericin (5). An in vitro study in the Vero cell line revealed that the presence of the compounds reduced the number of cells, as well as the percentage of viable cells, in most cases. An in silico cytotoxic analysis revealed that compounds 1, 2 and 5 might be explored as cytotoxic agents. Moreover, compounds 3 and 4 were found to be highly mutagenic. The present study suggested that further thorough investigations are necessary to use these molecules as leads for the cytotoxic drug development process.The present study was intended to characterize the secondary metabolites of the endophyte  Fusarium as a repository of bioactive compounds, including anti-fungal agents 1H NMR : δ 9.76 , 7.73 , 6.89 . 13C NMR : δ 191.4 (-CHO), 163.2 (C-4), 132.4 , 128.8 (C-1), 115.8 .White crystal; + m/z 327.1565 . 1H NMR : δ 11.44 , 6.21 , 5.51 , 5.43 , 4.53 , 2.84 , 2.65 , 2.21 , 1.94 , 1.66 , 1.54 , 1.42 , 0.96 . 13C NMR : δ 170.5 (C-1), 162.2 (C-8), 160.0 (C-6), 138.2 (C-4a), 129.5 (C-11), 126.4 (C-12), 114.7 (C-7), 105.9 (C-5), 101.6 (C-8a), 78.5 (C-3), 34.5 (C-9), 32.9 (C-4), 30.9 (C-15), 27.7 (C-10), 22.8 (C-16), 22.3 (C-14), 17.9 (C-13), 13.9 (C-17).White powder; ESI-MS: [M + Na]+ m/z 806.3984 . 1H NMR : δ 7.24 , 5.58 , 4.88 , 3.4 -8b), 3.04 , 2.97 , 1.99 , 0.82 , 0.41 . 13C NMR : δ 169.9 , 136.5 (C-9), 128.8 , 128.6 , 126.8 (C-12), 75.6 (C-1), 57.2 (C-7), 34.7 (C-8), 32.2 (N-CH3), 29.7 (C-2), 18.3 (CH3-3), 17.4 (CH3-4).Needle shaped white crystal; ESI-MS: [M + Na]6 cells and incubated for 24 h. After the incubation period, the treated cells were collected using 0.5% trypsin. Trypan blue (0.4% w/v) was used to make the unviable cells stained and viable cells remained unstained. An automated cell counter was used to count the number of unviable (stained) cells  × 100.http://www.swissadme.ch/index.php) (accessed on 10 November 2021). The Molinspiration online tool version 2018.03 (https://www.molinspiration.com/) (accessed on 11 November 2021) was used to assess the bioactivity score of the compounds against drug targets such as GPCR, kinase enzymes, ion channels, nuclear receptors and other enzymes. The toxicity risks such as the mutagenic, tumorigenic, irritant and reproductive effects of the compounds were determined using the Osiris Property Explorer, and other toxic features were analyzed via the pkCSM online tool (http://biosig.unimelb.edu.au/pkcsm/) (accessed on 7 November 2021). In each case, canonical simplified molecular input line entry system (SMILES) notations of the isolated compounds were fed into the tools to obtain the outcomes.Several online tools were used to assess the physicochemical, bioactivity and ADMET properties of the isolated compounds. The physicochemical, pharmacokinetic and medicinal chemistry aspects of the compounds were screened through the SwissADME tool (http://way2drug.com/Cell-line/) (accessed on 15 November 2021) known as the Cell Line Cytotoxicity Predictor (CLC-Pred), where the cytotoxicity of chemicals is predicted based on a PASS (Prediction of Activity Spectra for Substances) algorithm using a training dataset of 59,882 cytotoxic compounds obtained from the experimental data of ChEMBL (version 23) (https://www.ebi.ac.uk/chembldb/) (accessed on 15 November 2021) to generate and establish the ‘structure-cytotoxicity’ relationship models against 278 cancer cell lines and 27 normal cell lines of humans [The cytotoxic effect of the compounds was screened in both normal (non-tumor) and cancer cell lines by a web service . Data are stated as mean ± SEM (standard error of mean). A student’s t-test or ANOVA followed by Post Hoc Tukey’s test were used for analyzing the cytotoxicity data. To determine the correlation between variables, a linear regression was carried out. Statistical significance was considered when


Overall, 75 compounds are categorized into five major groups: gorgost-5-ene, 5,6-epoxygorgostane, 5,6-dihydroxygorgostane, 9,11-secogorgostane, and 23-demethylgorgostane, in addition to miscellaneous gorgostane. The structural diversity, selectivity for marine organisms, and biological effects of gorgostane steroids have generated considerable interest in the field of drug discovery research.Gorgostane steroids are isolated from marine organisms and consist of 30 carbon atoms with a characteristic cyclopropane moiety. From the pioneering results to the end of 2021, isolation, biosynthesis, and structural elucidation using  Marine natural products are an untapped reservoir for discovering biologically active phytomolecules, with a significant array of bioactivities reported against many aliments ,2. AntibClavularia viridis [Sarcophyton species [Klyxum flaccidum [Gorgosterol was the first sterol reported to contain a cyclopropane ring on the side chain, and since then, several additional sterols with this ring structure and sterols with polyoxygenated functionalities have been isolated . The rep viridis , antibac species , and cytlaccidum .13C-NMR spectroscopic data are summarized. Hopefully, this review will contribute to the elucidation of the structure and identification of this class of compounds.Gorgostane-type steroids have some structural variability in the number of carbon atoms and the degree of oxygenation patterns. According to the number of carbon atoms and structural features, gorgostane-type steroids can be classified into five groups. In this review, 75 gorgostane-type steroids with their Plexaura flexuosa [Pinnigorgia sp. -dien-3β-ol, 5α,8α-epidioxygorgost-6-en-3β-ol, and 5α,8α-epidioxygorgost-6,9(11)-dien-3β-ol, were isolated only from the genus Sinularia. Nine of the ten compounds, which belong to 5, 6-epoxy gorgostane, were isolated from Isis hippuris. Also, the majority of demethylgorgostane compounds were separated from Clavularia viridis.Several classes of sterols were isolated from different marine organisms . Howeverflexuosa . Seventyrgia sp. . The sigPeridinium foliaceum, Crypthecodinium cohnii, and the cultured zooxanthella symbiont of Cassiopea xamachana. They found that a decrease in S-adenosylmethionine concentration concomitantly with an increase in dimethylpropiothetin biosynthesis is linked with the attenuation of the production of gorgosterol in aposymbiotic zooxanthellae [Pseudoplexaura porosa, P. flagellosa, and P. wagenaari and Pseudopterogorgia americana soft corals were used as a source of zooxanthellae to detect the conversion of labelled farnesyl pyrophosphate to squalene. They concluded that zooxanthellae obtained from P. porosa contributed to the part of the pathway from mevalonate to gorgosterol  and gorgosta-5,25-dien-3β-ol (22), with characteristic 13C-NMR signals at 149.0 (C-11), 121.1 (C-9) and 156.9 (C-25), and 106.0 (C-26) [13C-NMR of C-7, as in crassumsterol (2) and klyflaccisteroid E (5), to 65.3 and 74.9 ppm, respectively [α-orientation of the hydroxyl group in crassumsterol and β-orientation in klyflaccisteroid [7), and at position 7 and 11, as in klyflaccisteroid D (8), with a characteristic 13C-NMR signal at 214.1 (C-11) and 211.6 (C-11) in klyflaccisteroid D [β-Acetoxy-1α,11α-dihydroxygorgost-5-en-18-oic acid (18) with a characteristic carboxylic group at position 18 with a signal at 176.9 ppm. The existence of a carboxylic group at position 18 leads to a downfield shift in C-13 by 3 ppm in comparison with gorgost-5-en-3β-ol (1) [Gorgost-5-ene is characterized by the presence of unsaturation between C-5 and C-6 . In 13C-ectively . The difisteroid . Gorgosten-3β-ol , with chteroid D  and 3β-Aβ-ol (1) ,20. 13C-NMR, the two carbons of the epoxide, C-5 and C-6, showed characteristic signals at 62.9 and 63.7 ppm, respectively. The degree of substitution of the A and B rings greatly affects the previous chemical shifts of C-5 and C-6. The substitutions, mainly hydroxyl or acetoxy groups, usually occur at C-1, C-3, C-7, C-11, C-12, and C-15. 5β,6β-Epoxygorgosterol (23) [24) [25) [26) [24) except for the presence of an acetoxy group at C-12. The chemical shift is more downfield from 83.6 to 86.2 ppm. 5β,6β-Epoxygorgostane-3β,7α,11α,12β-tetrol 11-acetate (27) [28) [29) [30) [31) and 5β,6β-epoxygorgostane-3β,7α,11α, 12β,15α-pentol 11,15-diacetate (32) [5,6-Epoxygorgostane is characterized by the presence of an epoxide of β orientation at C-5 and C-6 . In 13C-rol 23)  is the p  is the 24) [25)  has a hy25) [26)  is the s28) [29)  has a di29) [30)  has a hyate (32)  have sim13C-NMR, C-5 and C-6 of xeniasterol C (33) showed characteristic signals at 75.9 and 76.3 ppm, respectively. The chemical shifts of C-5 and C-6 of 5, 6-dihydroxy gorgostane consequently changed according to the oxygenation pattern on A and B rings. The substitutions, mainly hydroxyl or acetoxy groups, usually occur at C-1, C-3, C-7, C-9, C-12, and C-20. Xeniasterol C (33) [34) [35) [36) [37) [38) [35), except the presence of a hydroxyl group at C-20 with an α orientation has more downfield shift (76.3 ppm). Gorgostane-3β,5α,6β,9α,11α-pentol (39) [34), except the presence of a hydroxyl group at C-9 with an α orientation has more downfield shift (80.8 ppm); moreover, the neighboring carbons, C-8 and C-10, shift downfield at 33.2 and 43.5 ppm, respectively. 11α-Acetoxygorgostane-3β,5α,6β,12α-tetrol (40) [41) [42) [41) and 9.9 ppm (42). Gorgostane-1α,3β,5α,6β,11α,12β-hexol 12-acetate (43) [5,6-Dihydroxygorgostane is characterized by the presence of two hydroxyl groups at C-5 with an α orientation and C-6 with a β orientation . In 13C-l C (33)  is the p33) [34)  is the s34) [35)  has an a35) [36)  has an a36) [37)  has an a37) [38)  is the stol (39)  is the s41) [42)  have theate (43)  has the 13C-NMR, C-9 and C-11 of 3β,11-dihydroxy-9,11-secogorgost-5-en-9-one (44) showed characteristic signals at 217.4 and 60.0 ppm, respectively. The chemical shifts of C-9 and C-11 of 9,11-secogorgostane consequently changed according to substitutions. Compounds 3β,11-dihydroxy-9,11-secogorgost-5-en-9-one (44), 3β,11,24-trihydroxy-9,11-secogorgost-5-en-9-one (45), and ameristerol A (46) [47), and 5α,6α-epoxy-3β,11-dihydroxy-9,11-secogorgostan-9-one (48) [49) and 5α,6α-epoxy-1β,3β,11-trihydroxy-9,11-secogorgostan-9-one (50) [51) [52) [53) [9,11-Secogorgostane is characterized by the opening of ring C at C-9 and C-11. It is characterized by the presence of a ketone carbonyl carbon at C-9 and a primary alcohol group at C-11 . In 13C-l A (46) ,34,35 haone (48) ,37 are s51) [52)  is a 5α,52) [53)  is a 9, 13C-NMR, the chemical shift values for the cyclopropane moiety  in 23-demethylgorgostane  are different from those of gorgostane  with a 24-methyl group . This group could be 5, 6-epoxy, 5, 6-dihydroxy, or 5, 8-epidioxy gorgostane groups with different substitutions, mainly hydroxyl, carbonyl, and acetoxy or other groups such as chloride. These substitutions usually occur at C-1, C-2, C-3, C-4, C-7, and C-11. Stoloniferone M (54) [13C-NMR chemical shift in C-2 and C-10. Further, 5, 6-β-epoxy has an upfield chemical shift of up to 1 ppm more than 5, 6-α-epoxy gorgostane. Compounds 5α,6α-epoxy-23-demethylgorgost-8-ene-3β, 7α-diol (55) and 5α,6α-epoxy-23-demethylgorgost-8(14)-ene-3β, 7α-diol (56) [59) [58) [60-66) [23-Demethylgorgostane is characterized by the presence of only 29 carbon atoms and the lack of a methyl group that arises from the cyclopropane moiety at C-23 . In 13C-e M (54)  is a 23-iol (56)  have the56) [59) . In addi59) [58)  has 28 c [60-66) ,44,45,4667) and 5α,8α-epidioxygorgosta-6,9(11)-dien-3β-ol (68) [69) and 1α,3β,5β,11α-tetrahydroxygorgostan-6-one (70) [71) and gorgost-4-en-3-one (75) [71) has 3α, 6β-dihydroxy substitution at C-3 and C-6, in contrast to the latter compound, which has a carbonyl group at C-3. Ameristerenol A and B  [73) has an additional acetoxy group at C-3. Furthermore, the chemical shift of C-3 is more downfield to 71.9 and 74.0 ppm, respectively, similar to the gorgost-5-ene group. Compound klyflaccisteroid L (74) [Miscellaneous Gorgostane has an undefined chemical structure . Compoun-ol (68)  have theone (70) ,49 are rone (75) ,51 have   are 9,11d L (74)  has an u13C-NMR spectroscopic data, which are considered an added value to the structural identification of gorgostane derivatives. Further investigation of this class of biological activity explaining its mechanisms of action in treating different diseases is required. These studies will assist in the discovery and development of new drugs from natural sources.This review provides an exploration of the structural diversity of gorgostane-type steroids isolated from marine sources and


Posidonia oceanica) was studied in order to identify its antimicrobial constituents and further characterize the composition of its metabolome. It was identified as Fusarium petroliphilum by in-depth phylogenetic analyses. The ethyl acetate extract of that strain exhibited antimicrobial activities and an ability to inhibit quorum sensing of Staphylococcus aureus. To perform this study with a few tens of mg of extract, an innovative one-step generic strategy was devised. On one side, the extract was analyzed by UHPLC-HRMS/MS molecular networking for dereplication. On the other side, semi-preparative HPLC using a similar gradient profile was used for a single-step high-resolution fractionation. All fractions were systematically profiled by 1H-NMR. The data were assembled into a 2D contour map, which we call “pseudo-LC-NMR,” and combined with those of UHPLC-HRMS/MS. This further highlighted the connection within structurally related compounds, facilitated data interpretation, and provided an unbiased quantitative profiling of the main extract constituents. This innovative strategy led to an unambiguous characterization of all major specialized metabolites of that extract and to the localization of its bioactive compounds. Altogether, this approach identified 22 compounds, 13 of them being new natural products and six being inhibitors of the quorum sensing mechanism of S. aureus and Pseudomonas aeruginosa. Minor analogues were also identified by annotation propagation through the corresponding HRMS/MS molecular network, which enabled a consistent annotation of 27 additional metabolites. This approach was designed to be generic and applicable to natural extracts of the same polarity range.An endophytic fungal strain isolated from a seagrass endemic to the Mediterranean Sea ( The rapid and efficient identification of novel natural products (NPs) in complex biological systems is a priority for the search for new lead compounds . In this1H-NMR hyphenations have permitted us to partly characterize metabolites in crude extracts  and nuclear magnetic resonance (NMR) data to obtain a definitive identification . To speeextracts . Howeverextracts . One wayextracts . Severalextracts . A limitStaphylococcus aureus (MRSA) and Pseudomonas aeruginosa (PA). The gram-positive bacterium S. aureus causes superficial and potentially fatal infections, such as sepsis and pneumonia  was chosen in this work. P. oceanica is an underwater seagrass endemic to the Mediterranean Sea; it forms dense meadows from the surface down to a depth of 40 m. This marine vascular plant is known for its longevity and being, potentially, the host for a diverse microbial community including endophytes and epiphytes  A bioactivity screen was performed on a set of Posidonia fungal endophytes. 2) The most active strain was selected for the study. 3) An aliquot from the extract was subjected to UHPLC-HRMS and automated data-dependent acquisition MS/MS for metabolite profiling, followed by dereplication of known compounds through molecular network (MN) analysis. 4) Since the annotated compounds were not reported to act as QS inhibitors, their unambiguous characterization had to be performed. 5) In order to localize and identify the bioactive compounds, the fungal extract was thoroughly analyzed by pseudo-LC-NMR. The pseudo-LC-NMR process was developed for the purpose of this study but was intended to be generic in order to rapidly provide complementary NMR information to the LC-MS metabolite profiling of crude extracts available in limited amounts.In order to identify a fungal strain that produces bioactive compounds and to rapidly identify the metabolites responsible for this activity, the following procedure was applied in this study. 1) an optimized geometrical transfer was applied from the scale of UHPLC to analytical HPLC and then to semi-preparative HPLC (2) The crude extract was injected by LC on the semi-preparative scale with automated fraction collection every 30 s. 3) All fractions were submitted to 1H-NMR analyses, followed by LC-HRMS/MS. 4) 1H-NMR spectra were stacked and plotted in a 2D map sequenced according to the retention time generating the pseudo-LC-NMR plot (5) The concomitant processing of the pseudo-LC-NMR with the LC-MS profile allowed us to increase the level of annotation and to conduct specific 2D-NMR experiments when needed.The key steps for pseudo-LC-NMR were as follows: ive HPLC . 2) The NMR plot . 5) The In order to localize the bioactivity, the fractionation strategy had to be adapted to the sensitivity of the assays. In this specific study, the measurement of antibacterial and anti-QS activity required larger quantities than those obtained from the 30 s/fraction on the semi-preparative scale described above. Thus, a pooling of fractions was designed to highlight the area of chromatographic activity . This fiF. petroliphilum (Q.T. Chen and X.H. Fu)  (2013) (GB accession number: LC512834), F. macroceras (Wollenweber and Reinking 1925) (MH854821), and F. solani (Mart. 1842) (MH855493). These species, based on ITS BLAST results, belong to the F. solani species complex in which many species are still not formally described  were available in GB. After a similarity search of the 28S (MH866321) of F. macroceras in GB, that sequence appeared to be 100% similar to several sequences for F. solani  with 100% sequence coverage but also to four sequences for F. petroliphilum  but with 90% sequence coverage. Consequently, these two taxa are likely to be identical but without sequences for other more variable loci  for F. macroceras, and without type sequences for these species, it is not possible to clarify the situation. Combined analyses for the Fusarium 5 locus-64 taxa  and a strain of Pseudomonas aeruginosa (ATCC 27853) (F. petroliphilum (FEP 16) presented an antibacterial activity against MRSA with an MIC at 32 μg/ml but no activity against the Gram-negative P. aeruginosa strain.The minimum inhibitory concentration (MIC) of the extracts of all strains was tested against a methicillin-resistant strain of C 27853) . The cruP. aeruginosa, we performed a reporter assay based on percentage of fluorescence to evaluate the potential of the crude extract on QS (pqsA and lasB) normally induced by QS in P. aeruginosa  inhibition for ct on QS . Interesruginosa . The pqse enzyme . These p6, which yielded 1900 features in the PI mode and 2270 in the NI mode for building the corresponding MNs (in silico MS/MS fragmentation database (ISDB-DNP)  and negative (NI) ionization modes; MS/MS spectra of all detected features were recorded by data-dependent analysis (DDA). Feature-based molecular networks  for bothding MNs . The preding MNs  and predSDB-DNP) .Fusarium, and 28 features were reported in the closely related species F. solani. In the NI mode, 705 features were annotated out of 2720, 200 of them belonged to Nectriaceae, 135 of them were reported in the same genus, and 19 of them have been found in F. solani. The annotated metabolites corresponding to the most intense MS peaks detected in both PI (threshold > 5 × 107) and NI (threshold > 107) are presented in https://doi.org/10.6084/m9.figshare.14706198.v1). The same MN will be used later on in this study to apply annotation propagation through the location of isolated molecules in the MN.For each node of the MN, possible structure candidates were listed according to MS/MS similarity  up to a maximum of the top 50 compounds reported to occur in fungi. To increase the level of confidence in annotation, a reweighting step based on taxonomy was performed. This step takes into consideration the matching between the biological source reported in the DNP at the level of species > genus > family, resulting in a maximum of the top 5 candidates  for each annotated node . As a reFusarium species . The optimum HPLC conditions were determined with UV monitoring at 254 nm. This latter linear gradient method was then geometrically transferred to the semi-preparative scale (To effectively link the expected fractionation of this extract with the metabolite profiling data (see above), a chromatographic gradient transfer method was used to find the correct separation parameters, ideally for a single separation at the semi-preparative level. In practice, an intermediate step at the analytical HPLC level  was neceve scale .In order to obtain an efficient high-resolution separation of this complex mixture and to avoid any loss of chromatographic resolution, the crude extract (40 mg) was introduced into a dry loading cell according to our previously published protocol . Using td6 and submitted to 1H-NMR analysis. DMSO-d6 was selected since it is known to have good solubility properties and for its compatibility with bioassays. The 1H-NMR spectra of all fractions were obtained by automated acquisition . In order to visualize all 1H-NMR signals from the 135 fractions (F1-F135), a 2D plot was generated. For this, all individual spectra were binned and combined into a single matrix (ppm vs. Rt or fraction Nº). This plot simulates the actual output of a classic on-flow LC-NMR analysis (https://oolonek.github.io/pseudo_lcnmr_plotter/2dNMR.html).With the idea to obtain a comprehensive NMR profile of all fractions and be able to align all spectra over the extended polarity range of the various metabolites that were separated, each fraction was diluted in 600 µl of DMSO-analysis . The chranalysis . An inte1H-NMR signals are perfectly aligned, since all spectra are recorded with the same solvent across the whole separation (DMSO-d6). By comparing our approach to on-flow LC-NMR, no solvent suppression is necessary. In addition, compared to at-line LC-NMR detection methods, such as LC-SPE-NMR , a main constituent of the fungal membrane, could be identified in F132, yielding an intense signal in the histogram (1H-NMR spectrum was in good agreement with reported data (20) could be confirmed in F116 by comparing its 1H-NMR spectrum with those in the literature , which were easily identified at the beginning of the chromatogram (F1-F27). In this part of the semi-preparative HPLC chromatogram, the LC-UV profile exhibited main peaks that were not fully separated  (4) (5)   was iden (1) (4)  in fract (4) (5)  in fract (4) (5)  (Table 2Fusarium were dereplicated by HRMS/MS and confirmed by 1H-NMR as bostrycoidin (11) (7) and 3-O-methylfusarubin (8) . Another class of molecules (cyclohexanones) could be highlighted in fractions F64–F65 and F103–F104, which were characterized by their signals between δH 5.2 and 5.3 (orange). The analysis of NMR spectra of these fractions, for which no confident HR-MS/MS annotations were obtained, revealed three new compounds , as shown in In the intermediate region of the chromatogram (fractions F28–F109), three known compounds which are common to din (11)  in F60 aubin (8)  in F43 aavanicin  and solaavanicin  were deravanicin . In thisd6, the low field region (δH 11.0–14.0) of the pseudo-LC-NMR 2D plot highlighted characteristic deshielded mobile protons. Among them, the acidic protons of fatty acids were clearly seen (δH 12.0) in F113–F119, and quinonic–phenolic groups of fusarubin (7) and 3-O-methylfusarubin (8) were observed . Among them, 13 are original NPs described here for the first time  and presented in Fusarium species: adenosine (1) (4) (5) (7) and 3-O-methylfusarubin (8) (11) (20) (21) (22) (Careful interpretation of the HRMS/MS and sine (1) , gibepyr (1) (4) , aloesol (4) (5) , fusarububin (8) , bostryc(8) (11) , cerevis11) (20) , 6-dehyd20) (21) , and erg21) (22) .6 (F35) was isolated as a pale rose amorphous solid. The HRMS spectrum showed a molecular ion at m/z 288.0846 [M + H]+ . No valid annotations based on MS/MS could be obtained for this compound among all described Nectriaceae metabolites. The 1H and edited-HSQC NMR spectra showed three exchangeable protons at δH 5.06 , 11.39 , and 12.88 , two aromatic protons at δH 6.23  and 6.44 , an oxymethylene at δH 4.75 , a methoxy group at δH 3.89 , and a methyl at δH 2.44 . A methoxy naphthoquinone moiety such as that found in fusarubin (7) was identified, thanks to the HMBC correlations from H-3 to the carbonyl C-4 (δC 191.0) and C-1 (δC 179.5) to the quaternary carbons C-2 (δC 160.5) and C-10 (δC 107.4) and from the methoxy group to C-2 (6 (F35) and 15 (F69) as well as the two furarubins, 7 and 8 (F43-F44). This was also clearly visible for the common H-3 aromatic signal of these molecules (C 152.1), and C-6 (δC 126.8) and from the oxymethylene H2-15 to C-5, C-6, and C-7 allowed us to position these two groups. In addition, a 2-methyl-pyrrole group was placed in C7–C8 according to the HMBC correlations from the aromatic proton H-11 to C-6, C-7, and C-8 (δC 130.5), from the methyl protons H3-14 to C-11 (δC 99.8) and C-12 (δC 156.7), and from NH-13 to C-7, C-8, C-11, and C-12. The ROESY correlations from H-3 to the methoxy H3-16 and from H-11 to H2-15 and H3-14 confirmed the structure. Compound 6 was thus identified as 5-hydroxy-4-(hydroxymethyl)-8-methoxy-2-methyl-1H-benzo[g]indole-6,9-dione.Compound p to C-2 . The metolecules . The HMB15 (F69) showed a molecular ion at m/z 272.0923 [M+H]+ and a molecular formula of C15H13NO4, which actually correspond to one oxygen less than the one of 6. The NMR data of 15 also showed close similarities to those of 6  displayed a molecular ion at m/z 295.1542 [M + H]+, corresponding to the molecular formula C16H22O5. A 4-methoxy-6-substituted-α-pyrone was identified based on the HMBC correlations from the aromatic proton H-5 to C-3, C-6, and C-7, from H-3 to C-5, C-4, and C-2, and from the methoxy signal H3-17 to C-4  found in F55, F91, and F93, suggesting a common pyrone moiety (9 was identified as (6E)-7-(4-methoxy-6-oxo-6H-pyran-2-yl)-3,5-dimethyloct-6-enoic acid.The HRMS spectrum of 7 to C-4 . An alkye moiety . Compoun10 (F55) indeed exhibited close similarities to those of 9 . An additional oxymethylene was observed at δH 3.25  and 3.30  in addition to a hydroxyl at δH 4.34 , whereas the carbonyl of the acid group was no longer observed. The 2D-NMR experiments were in good agreement with the structure presented in m/z 281.1744 [M + H]+ confirmed the structure as 6-((E)-6-ethyl-7-hydroxy-4-methylhept-2-en-2-yl)-4-methoxy-2H-pyran-2-one.The NMR data of ose of 9 ; they sh16, which has a molecular formula of C16H24O3 and ionized at m/z 265.1816 [M + H]+ in F91, shares the same skeleton as 9 and 10. The carboxyl group presented at the end of the side chain of 9 was replaced in this case by a methyl group at δH 0.82  (16 was thus identified as 4-methoxy-6-((E)-4,6-dimethyloct-2-en-2-yl)-2H-pyran-2-one.Compound , H3-13) . Compoun17 (C17H26O3) which was ionized at m/z 279.1956 [M + H]+ in F93 also belongs to the same family. The side chain was the same as that of 16, but an additional methyl group was observed in C-3 at δH 1.69 . The HMBC correlations from the methyl H3-18 to the ester carbon C-2 (δC 179.7), the olefinic carbon C-3 (δC 98.9), and the oxygenated olefinic carbon C-4 (δC 162.0) confirmed this structure as 4-methoxy-3-methyl-6-((E)-4,6-dimethyloct-2-en-2-yl)-2H-pyran-2-one -5,6-dimethylhept-3-en-2-yl)-5-oxo-1H-inden-4-yl)acetic acid.Compound and C-25 . This co13 and 14, both possessing an MF of C21H32O4 and protonated ions at m/z 349.2349, showed great similarities to those of 18 in F103 (C21H32O3), except for the terminal part of the side chain. Compound 13 contains an additional hydroxy group in C-25 as evidenced by the HMBC correlations from the methyls H3-26 and H3-27  to the oxygenated quaternary carbon C-25 (δC 70.5) and the methine C-24 (δC 47.1). In compound 14, the terminal methyl H3-27 was hydroxylated and replaced by an oxygenated methylene at δH/δC 3.17/64.4.The NMR signals of the isomeric 12 in F62 was found to possess an MF of C28H40O6 with 8 degrees of unsaturation, as evidenced by HRMS at m/z 473.2877 [M+H]+ . The 1H-NMR data of 12 showed typical signals of an oxygenated steroid: 2 methyl singlets at δH 0.77  and 0.89 , three methyl doublets at δH 0.85 , 0.90 , and 1.73 , four oxygenated methines at δH 3.27 , 3.59 , 4.18 , and 5.53 , an oxygenated methylene at δH 3.31  and 3.93 , an ethylenic proton at δH 5.22 , and a series of methines and methylenes between δH 1.20 and 2.63. The HMBC also indicated the presence of two oxygenated quaternary carbons at δC 68.1 (C-5) and 74.8 (C-9), two quaternary sp2 carbons at δC 134.1 (C-20) and 142.8 (C-14), and a carbonyl at δC 206.7 (C-15). HMBC correlations from H3-19 to C-1 (δC 25.6), C-5, C-9, and C-10 (δC 37.0), from H3-18 to C-12 (δC 32.1), C-13 (δC 43.1), C-14, and C-17 (δC 51.5), and from the methylene H2-16 at δH 2.16  and 2.63  to C-15 allowed us to identify the four member rings of sterol in compound 12, which was identical to the 5β,6β-epoxy-3β,7α,9α-trihydroxy-ergosta-8(14),22-dien-15-one previously isolated by Polyporus ellisii. The chain attached to C-17 was found to be new; its linkage was established by the HMBC correlations from the methyl H3-21 to C-17 (δC 51.5), C-20, and C-22 (δC 128.1) and, thus, the presence of a double bond between C-20 and C-22. The HMBC correlations from H3-28 to C-23 (δC 79.7), C-24 (δC 42.9), and C-25 (δC 35.9), from H3-26 to C-24, C-25, and C-27 (δC 73.7), and from H2-27 to C-23 indicated the formation of a furan ring. The ROESY correlations from H3-18 to H3-21 and H-11β, from H3-19 to H-11β and H-2β, from H-4α to H-3 and H-6, from OH-7 to H-6, from H-1α to OH-9, from H-23 to H3-21 and H3-28, and from H-22 to H-17 and H-24 allowed us to determine the relative configuration of this new sterol (12) was characterized as 5β,6β-23,26-diepoxy-3β,7α,9α-trihydroxy- ergosta-8(14),20-dien-15-one.Compound w sterol . Compoun19 in F107 showed a sodium adduct ion at m/z 599.4380 [M+Na]+ which correlated with C35H60O6. The NMR spectra of 19 indicated the presence of stigmast-8-en-3-ol with typical signals like two methyl singlets at δH 0.65  and 0.96 , three methyl doublets at δH 0.79 , 0.82 , and 0.90 , one methyl triplet at δH 0.82 , one oximethine at δH 3.46 , and one olefinic carbon detected on the HMBC spectrum from the correlations of methyl H-18 with C-9 (δC 140.4). Additional signals corresponding to a glucose unit were detected at δH 2.89 , 3.01 , 3.06 , 3.12 , 3.40 , 3.64 , and 4.22 . Due to the very small amount of isolated compound and the presence of an overlapping fatty acid in the fraction, it was not possible to obtain a complete assignment of the molecule. However, the ROESY correlation between the H-1' proton of glucose and the H-3 proton of the stigmasterol skeleton allowed the positioning of glucose in C-3 and the identification of 19 as 3-O-β-D-glucopyranoside-stigmast-8-en-3-ol.Compound 1H-NMR spectrum of possibly two to three metabolites. The HRMS data confirmed the presence of two ions at m/z 199.0965 and 155.0703 [M + H]+ which are typical for C10H15O4 and C8H11O3, respectively.In addition to the compounds described in the polar part of the chromatogram (F1-27), F10 exhibited a 4, as indicated by the aromatic H-4 and H-5 at δH/δC 7.35–7.36/140.4–140.5 and 6.26–6.33/100.4–101.4, respectively, and the methyl at δH/δC 1.95–1.96/16.0–16.2. Compounds 2a and 2b (C10H15O4) were diastereoisomers with a 2,3-dihydroxybutan-2-yl side chain characterized by a methyl doublet  at δH 0.97 and 1.03, an oxygenated methine  at δH 3.73 and 3.74, and a methyl singlet (H3-10) at δH 1.37 and 1.24 for 2a and 2b, respectively. The hydroxylation in C-7 and the linkage of the 2,3-dihydroxybutyl chain in C-6 were confirmed by the HMBC correlations from the methyl H3-10 to C-6 , C-7 , and C-8 , and from the aromatic protons H-4 and H-5 to C-6. On the other hand, the 3-methyl-pyran-2-one of 3 (C8H11O3) was substituted by a hydroxyethyl group in C-6 as indicated by the methyl doublet at δH 1.30 , the methine at δH 4.41 , and the HMBC correlation from the methyl to C-6 (δC 165.6) and C-7 (δC 64.9).A detailed 2D-NMR analysis of the fraction revealed the presence of three compounds which share a 3-methyl-pyran-2-one moiety similar to the one of Overall, the combination of the LC-HRMS/MS data and the pseudo-LC-NMR plot together with in-depth 2D-NMR analysis of selected peaks provided a good overview of all the main constituents of fractions F1–F135 in a single semi-preparative HPLC separation.Our workflow permitted us to identify all main compounds from the crude extract in one step. However, as many of the fractions were in very small quantities (in the sub-mg range), we had to find a strategy to collect enough amounts of compounds for the bioassays. This was indeed a limitation since the bioassays could only be conducted with at least 500 µg of pure compound.Staphylococcus aureus (MRSA), the 14 zones were first subjected to MIC tests against this strain. As a result, only zone 9 (Z9) showed significant inhibitory effect at 32 μg/ml, which suggested the presence of an antibacterial compound in fractions F81–F91. In parallel, the 14 zones were evaluated for anti-QS of PA in the same way as for the crude extract and on the same reporter genes (pqsA and lasB).To ensure the accumulation of enough material for the bioassays, the 135 collected fractions were pooled into 14 chromatographic zones (Z1–Z14), where each zone represents a 5-min window of elution time . SubmittpqsA to 31% and that of lasB to 43%  and enabled the bioactivity assessment of compounds 1–14. Since Z9 was also a bioactive target, the apolar hexane fraction was purified similarly. Interestingly, by comparing the dry weights of collected fractions from enriched extract with the equivalent ones from crude extract, all compounds were collected in amounts higher than 500 µg, which was the threshold for the bioassay we selected. This also granted supplementary amounts to perform further bioassay experiments such as quantitative PCR.Taking into account the enrichment factor (2.3 folds), a single semi-preparative HPLC fractionation was carried out on 30 mg of the hydroalcoholic fraction under the same conditions as for the crude extract . This yiAs shown, in our strategy, the precise assignment of the bioactive LC peaks was dependent on the amounts of extract injected. While working with fungal extracts, the pseudo-LC-NMR analysis performed directly at the crude extract level identified all main metabolites and highlighted a high content of fatty acids. Such a profiling based on NMR detection was of interest to provide an unbiased view of the metabolome. However, our fractionation process did not allow a direct biological assessment of all the LC peaks collected, due to lack of assay sensitivity, and necessitated the pooling of fractions. The enrichment procedure followed by the targeted isolation using the same semi-preparative fractionation finally provided a sufficient amount for bioactivity assessments.4–16) that belong to Z3–Z9 were submitted to the same biological tests as described before.In order to assign compounds which are responsible for the antibacterial and anti-QS activity, purified compounds  and 3-O-methylfusarubin (8), which previously located in the active Z5, presented an enhanced and significant QS inhibition (12 and 15%) in pqs and las systems, respectively, at 128 μg/ml. To further confirm these results, we performed quantitative RT-PCR analyses on the QS-regulated genes pqsA, lasB, and rhlA involved in rhamnolipid production (pqsA in the presence of 7 and 8 was two times less expressed than the reference (lasB and rhlA did not show significant effects (data not shown) unlike results obtained by gfp transcriptional fusions  were cleaned under stream water and then dipped into 70% ethanol for 3 min. Samples of all plant parts were cut into 1-cm2 pieces and placed in a culture plate containing potato dextrose agar (PDA). Fungal tips were transferred to a new PDA culture plate as soon as they appeared and were left to grow for 30 days.http://bio2mar.obs-banyuls.fr), who performed the amplification and sequencing of the internal transcribed spacers plus the 5.8S (ITS). After the removal of small and large subunit ITS flanking regions, a first identification of this fungal isolate was performed, searching for similarity of that ITS sequence to those deposited in GenBank  (https://blast.ncbi.nlm.nih.gov/Blast.cgi) used the “blastn” (Megablast) option excluding “uncultured/environmental sample sequences.”Samples of the fungal cultures were sent to Bio2Mar, France  . SequencFusarium strain FEP 16 more precisely, DNA was extracted from a sample of the fungal culture placed in 500 µl of cetyl-trimethyl-ammonium bromide buffer (CTAB 1x). DNA extraction was performed according to TEF1-α) using primers EF1-1F and EF1-1R . The agar was suspended in artificial sea water , followed by 20 min of ultrasound sonication, and filtered through Whatman™ No. 1. This process was repeated three times with fresh solvent. The filtrates were gathered, and the solvent was evaporated to dryness using a rotary evaporator  to yield 300 mg of crude extract.For the whole study, the same strain of ea water  and thenea water . This seThe NMR spectroscopic data were recorded at 298 K on a Bruker Avance III HD 600 MHz. An NMR spectrometer equipped with a QCI 5 mm Cryoprobe and a SampleJet automated sample changer  was used. Chemical shifts are measured in parts per million (δ) using the residual DMSO-d6  as the internal standard for 1H and 13C, respectively, and coupling constants (J) are reported in Hz. Complete assignments were performed based on 2D-NMR experiments (correlation spectroscopy (COSY), rotational nuclear Overhauser effect spectroscopy (ROESY), heteronuclear single quantum correlation (HSQC), and heteronuclear multiple bond coherence (HMBC)). Optical rotations were measured using a Jasco P-2000 polarimeter . UV absorbance was measured using a JASCO FT/IR-4100 spectrometer (JASCO Corporation) equipped with a PIKE MIRacle™ (JASCO Corporation).® v4.2 (Waters), and the ESI and MS acquisition conditions were set according to the work of (2O (A) and MeOH (B), both containing 0.1% formic acid (FA). The separation conditions were optimized by decreasing the slope of the LC linear gradient to ensure the best distribution of the metabolites across the chromatographic window. The gradient was set as follows: 34–100% of B in 16.57 min, followed by 3 min of washing at 100% B. The flow rate was 0.3 ml/min, and the separation was conducted at 40°C.UHPLC-DAD-MS-ELSD analyses were conducted on an Acquity UHPLC system , equipped with DAD and MS single quadrupole (Acquity QDa) as detectors supplemented with an electrospray ionization source (ESI). DAD detection was set between 190 and 500 nm. The system was controlled using MassLynx work of . For the2O (A) and MeCN (B), both containing 0.1% FA. The gradient mode was as follows: 5–100% of B in 18 min, followed by 4 min of 100% B. The flow rate was 0.6 ml/min, and the separation was conducted at 25°C. Metabolite profiling of all fractions was conducted on the same system and the same ESI settings; 2 µl was injected through an Acquity BEH C18 column . The mobile phase was H2O (A) and MeCN (B), both containing 0.1% FA. The gradient mode was 5–100% of B in 4 min, followed by 2 min of washing and reconditioning.UHPLC-HRMS/MS analyses was performed on a Thermo Dionex Ultimate 3000 UHPLC system interfaced with a Q Exactive Plus MS  supplemented with a heated electrospray ionization source (HESI-II). Thermo Scientific Xcalibur 3.1 software was used for instrument control. The HESI-II parameters were set according to the work of . For the6) and MS level 2 (noise level at 0). The ADAP chromatogram builder was used with the threshold set to 4 × 105. Chromatogram deconvolution  was set within its default parameters, except for the Rt wavelet range, which was set as 0–0.1. The most intense isotopes were kept through the isotope peak grouper. Peak alignment was performed using the join aligner method where absolute Rt tolerance is at 0.03 min. Adduct search was performed for Na+, K+, NH4+, and ACN+ in the positive mode and [M-2H+Na]− and [M-2H+K]− in the negative mode  and MeOH (B), both containing 0.1% FA. The flow rate was 1 ml/min, the injection volume was 10 μl, the separation temperature was 25°C, and the sample concentration was 10 mg/ml dissolved in MeOH. The gradient conditions were set following a gradient transfer from UHPLC to HPLC according to the work of  using an X-Bridge C18 column . The HPLC conditions were as follows: mobile phase H work of . The sep1H-NMR spectra of all 135 fractions were divided into equally sized bins (0.01 ppm) in a range from −1 to 15 ppm and then exported as an Excel file (.csv) using MNova v14 . Sample names (number of fractions) were added manually to the Excel file. The file was loaded into an R script using Rstudio V1.2.5042, which was written to create an interactive 2D plot that combines all binned spectra in a matrix (sample vs. ppm). The following R packages were used: “plotly,” “stringr,” “reshape2,” “dplyr,” and “readr.” The R script is freely available here: (https://github.com/oolonek/pseudo_lcnmr_plotter/blob/main/src/plotter/NMR_data_plotter.Rmd). To improve the visualization, retention time (Rt) of the semi-preparative separation was manually added to the created plot . The hydroalcoholic fraction yielded 70 mg, and the hexane fraction yielded 90 mg.® SPD-20A, Kyoto, Japan) through an X-Bridge C18 column . The gradient transfer from analytical to semi-preparative HPLC was calculated according to the work of .The original crude extract and enriched extracts were purified using semi-preparative HPLC-UV equipment (Shimadzu work of . The flo work of . Collecthttps://doi.org/10.26037/yareta:tmpbgvbqsvfrvltepotmjuunua).The spectral data for all new NPs are summarized below; those recorded for previously isolated compounds can be found in the supplementary materials. The 1D- and 2D-NMR spectra of all described compounds below can be found in the supplementary materials . All NMR6--3-methyl-2H-pyran-2-one (2): white amorphous solid; 2a: 1H-NMR  δ 0.97 , 1.31 , 1.95 , 3.73 , 4.67 , 5.19 , 6.32 , 7.35 ; 13C-NMR  δ 16.0 (C-11), 17.4 (C-9), 21.8 (C-10), 70.2 (C-8), 74.5 (C-7), 101.4 (C-5), 121.3 (C-3), 140.4 (C-4), 162.7 (C-2), 167.0 (C-6); 2b: 1H-NMR  δ 1.03 , 1.24 , 1.95 , 3.74 , 4.59 , 5.09 , 6.33 , 7.35 ; 13C-NMR  δ 16.0 (C-11), 17.1 (C-9), 22.6 (C-10), 70.4 (C-8), 75.0 (C-7), 101.4 (C-5), 120.9 (C-3), 140.4 (C-4), 162.7 (C-2), 167.2 (C-6). HRMS m/z 199.0962 [M + H]+ .6-(1-hydroxyethyl)-3-methyl-2H-pyran-2-one (3): white amorphous solid; 1H-NMR  δ 1.30 , 1.96 , 4.41 , 5.56 , 6.26 , 7.36 ; 13C-NMR  δ 16.2 (C-9), 21.5 (C-8), 64.9 (C-7), 100.4 (C-5), 122.0 (C-3), 140.5 (C-4), 162.7 (C-2), 165.6 (C-6). HRMS m/z 155.0703 [M + H]+ . 2a, 2b and 3 occurred as a mixture in a fraction 10 (0.6 mg)g]indole-6,9-dione5-hydroxy-4-(hydroxymethyl)-8-methoxy-2-methyl-1H-benzo+ .E)-7-(4-methoxy-6-oxo-6H-pyran-2-yl)-3,5-dimethyloct-6-enoic acid(6 (9): Red amorphous solid (0.4 mg); [α]20D +22.3 ;UV (DAD) λmax 218, 269; 1H-NMR  δ 0.89 , 0.96 , 1.21 , 1.34 , 1.79 , 1.86 , 1.96 , 2.25 , 2.67 , 3.88 , 5.52 , 6.11 , 6.15 , 11.99 ; 13C-NMR  δ 12.3 (C-16), 19.8 (C-14), 19.9 (C-15), 27.6 (C-11), 29.9 (C-9), 40.9 (C-12), 43.3 (C-10), 56.4 (C-17), 88.7 (C-3), 108.7 (C-5), 124.3 (C-7), 140.0 (C-8), 159.6 (C-6), 167.2 (C-4), 173.7 (C-13), 180.2 (C-2). HRMS m/z 295.1542 [M + H]+ .E)-6-ethyl-7-hydroxy-4-methylhept-2-en-2-yl)-4-methoxy-2H-pyran-2-one6-(( (10): Red amorphous solid (0.7 mg); [α]20D +82.7 ; UV (DAD) λmax 286, 422; 1H-NMR  δ 0.81 , 0.98 , 1.22 , 1.23 , 1.27 , 1.35 , 1.86 , 2.69 , 3.25 , 3.30 , 3.88 , 4.34 , 5.52 , 6.10 , 6.14 ; 13C-NMR  δ 10.5 (C-13), 12.1 (C-16), 20.4 (C-15), 22.6 (C-12), 29.9 (C-9), 37.8 (C-10), 39.1 (C-11), 56.3 (C-17), 62.9 (C-14), 88.6 (C-3), 108.6 (C-5), 124.1 (C-7), 140.4 (C-8), 159.6 (C-6), 167.2 (C-4), 180.2 (C-2). HRMS m/z 281.1745 [M + H]+, calculated for C16H25O4, 281.1747.5β,6β-23,26-diepoxy-3β,7α,9α-trihydroxy-ergosta-8(14),20-dien-15-one (12): pale yellow amorphous solid (0.4 mg); [α]20D +18.9 ; UV λmax 248, 229, 229; 1H-NMR  δ 0.77 , 0.85 , 0.89 , 0.90 , 1.20 , 1.45 , 1.66 , 1.69 , 1.73 , 1.85 , 2.00 , 2.06 , 2.16 , 2.26 , 2.35 , 2.63 , 3.27 , 3.59 , 3.93 , 4.18 , 4.77 , 4.77 , 4.91 , 5.22 , 5.53 ; 13C-NMR  δ 11.2 (C-28), 13.5 (C-26), 17.6 (C-18), 18.8 (C-21), 19.8 (C-19), 25.6 (C-1), 26.7 (C-11), 30.2 (C-2), 32.1 (C-12), 35.9 (C-25), 37.0 (C-10), 39.5 (C-16), 39.9 (C-4), 42.9 (C-24), 43.1 (C-13), 51.5 (C-17), 59.7 (C-7), 60.4 (C-6), 66.5 (C-3), 68.1 (C-5), 73.7 (C-27), 74.8 (C-9), 79.7 (C-23), 128.1 (C-22), 134.1 (C-20), 142.8 (C-14), 206.7 (C-15). HRMS m/z 473.2877 [M + H]+ .E)-6-hydroxy-5,6-dimethylhept-3-en-2-yl)-7a-methyl-5-oxo-1H-inden-4-yl)acetic acid2-: pale yellow amorphous solid (0.4 mg); [α]20D +21.5 ; UV λmax 247, 207; 1H-NMR  δ 0.92 , 0.99 , 1.03 , 1.03 , 1.06 , 1.42 , 1.50 , 1.78 , 1.79 , 2.01 , 2.16 , 2.17 , 2.19 , 2.22 , 2.99 , 3.02 , 5.26 , 5.41 ; 13C-NMR  δ 14.9 (C-28), 16.2 (C-18), 20.8 (C-21), 26.0 (C-27), 27.0 (C-16), 28.2 (C-26), 30.8 (C-7), 32.7 (C-11), 35.8 (C-12), 38.1 (C-20), 44.7 (C-13), 47.1 (C-24), 55.5 (C-17), 70.5 (C-25), 131.1 (C-23), 135.0 (C-22), 171.7 (C-6), 175.2 (C-14). HRMS m/z 349.2383 [M + H]+ .E)-7-hydroxy-5,6-dimethylhept-3-en-2-yl)-7a-methyl-5-oxo-1H-inden-4-yl)acetic acid2-: pale yellow amorphous solid (0.3 mg); [α]20D +19.8 ; UV λmax 245, 212; 1H-NMR  δ 0.75 , 0.94 , 1.03 , 1.06 , 1.43 , 1.51 , 1.78 , 2.17 , 2.22 , 2.53 , 2.98 , 3.02 , 3.16 , 3.17 , 5.27 ; 13C-NMR  δ 13.1 (C-26), 16.3 (C-18), 18.3 (C-28), 21.0 (C-21), 27.3 (C-16), 30.9 (C-7), 32.7 (C-11), 35.9 (C-12), 37.0 (C-24), 38.2 (C-20), 40.6 (C-25), 44.6 (C-13), 55.5 (C-17), 64.4 (C-27), 130.9 (C-23), 135.0 (C-22), 171.8 (C-6), 175.2 (C-14). HRMS m/z 349.2349 [M + H]+ .H-benzo[g]indole-6,9-dione5-hydroxy-8-methoxy-2,4-dimethyl-1 (15): pale yellow amorphous (0.1 mg) solid; UV λmax 280, 239; 1H NMR  δ 2.36 , 2.44 , 3.89 , 6.21 , 6.28 , 11.39 , 12.84 ; 13C-NMR  δ 12.0 (C-15), 13.8 (C-14), 56.6 (C-16), 98.9 (C-11), 107.3 (C-10), 108.8 (C-3), 123.9 (C-6), 129.8 (C-8), 137.6 (C-7), 146.2 (C-12), 152.2 (C-5), 160.5 (C-2), 179.1 (C-1). HRMS m/z 258.0771 [M + H]+, calculated for C14H12NO4, 258.0766.E)-4,6-dimethyloct-2-en-2-yl)-2H-pyran-2-one4-methoxy-6-(( (16): light brown amorphous solid (0.1 mg); [α]20D +17.5 ; UV λmax 220,274; 1H-NMR  δ 0.82 , 0.84 , 0.96 , 1.08 , 1.14 , 1.32 , 1.86 , 2.66 , 3.88 , 5.52 , 6.10 , 6.16 ; 13C-NMR  δ 11.0 (C-13), 12.3 (C-16), 19.3 (C-14), 20.1 (C-15), 28.6 (C-12), 30.1 (C-9), 31.5 (C-11), 43.5 (C-10), 56.3 (C-17), 88.8 (C-3), 108.9 (C-5), 124.2 (C-7), 140.5 (C-8), 159.7 (C-6), 167.4 (C-4). HRMS m/z 265.1816 [M + H]+, calculated for C16H25O3, 265.1803.E)-4,6-dimethyloct-2-en-2-yl)-2H-pyran-2-one4-methoxy-3-methyl-6-(( (17): light brown amorphous solid (0.1 mg); [α]20D +19.7 ; UV λmax 218, 252; 1H-NMR  δ 0.83 , 0.85 , 0.98 , 1.24 , 1.34 , 1.69 , 1.88 , 2.66 , 4.02 , 6.17 , 6.19 ; 13C-NMR  δ 6.5 (C-18), 11.1 (C-13), 12.3 (C-16), 19.3 (C-14), 20.1 (C-15), 28.7 (C-12), 30.1 (C-9), 31.4 (C-11), 43.5 (C-10), 56.0 (C-17), 98.9 (C-3), 108.3 (C-5), 124.4 (C-7), 140.1 (C-8), 158.3 (C-6), 162.0 (C-4), 179.7 (C-2). HRMS m/z 279.1967 [M + H]+ .E)-5,6-dimethylhept-3-en-2-yl)-5-oxo-1H-inden-4-yl)acetic acid2-: pale yellow amorphous solid (0.6 mg); [α]20D +14.9 ; UV λmax 246, 239; 1H-NMR  δ 0.81 , 0.83 , 0.91 , 1.03 , 1.06 , 1.42 , 1.48 , 1.51 , 1.78 , 1.79 , 1.88 , 2.15 , 2.18 , 2.22 , 2.34 , 2.46 , 2.56 , 2.98 , 3.02 , 5.25 , 5.29 ; 13C-NMR  δ 16.3 (C-18), 17.3 (C-28), 19.4 (C-27), 19.7 (C-26), 20.9 (C-21), 27.2 (C-16), 27.3 (C-15), 30.9 (C-7), 32.4 (C-25), 32.7 (C-11), 35.8 (C-12), 38.1 (C-20), 42.0 (C-24), 44.6 (C-13), 55.4 (C-17), 125.0 (C-8), 132.0 (C-23), 134.7 (C-22), 171.7 (C-6), 175.2 (C-14), 196.3 (C-9). HRMS m/z 333.2426 [M + H]+ .O-β-D-glucopyranoside-Stigmast-8-en-3-ol3- (19): pale yellow amorphous solid (0.1 mg); [α]20D +6.2 ; UV λmax 234, 207; 1H-NMR  δ 0.65 , 0.79 , 0.82 , 0.82 , 0.88 , 0.90 , 0.91 , 0.96 , 0.97 , 1.01 , 1.09 , 1.20 , 1.25 , 1.30 , 1.33 , 1.47 , 1.63 , 1.79 , 1.80 , 2.12 , 1.81 , 2.12 , 2.37 , 2.89 , 3.01 , 3.06 , 3.12 , 3.40 , 3.46 , 3.64 , 4.22 , 4.42 , 4.85 , 4.86 , 4.88 ; 13C-NMR  δ 11.6 (C-18), 11.8 (C-29), 18.6 (C-21), 19.0 (C-27), 19.1 (C-19), 19.6 (C-26), 22.5 (C-28), 28.7 (C-25), 29.3 (C-2), 33.3 (C-22), 35.4 (C-20), 36.3 (C-10), 36.7 (C-1), 38.3 (C-4), 39.1 (C-12), 41.8 (C-13), 45.1 (C-24), 49.6 (C-5), 55.4 (C-17), 56.1 (C-14), 61.0 (C-6'), 70.1 (C-4'), 73.4 (C-2'), 76.7 (C-5'), 76.8 , 100.7 (C-1'), 140.4 (C-9). HRMS m/z 599.4280 [M + Na]+, calculated for C35H59O6Na, 599.4287.Staphylococcus aureus  and Pseudomonas aeruginosa (ATCC 27853) strains were used for the antibacterial assay. The minimum inhibitory concentration (MIC) of the extract and the isolated compounds were determined in triplicate according to P. aeruginosa and chloramphenicol for S. aureus were used as controls.Methicillin-resistant pqsA-gfp was constructed using the following primers, GCTCTAGATCGAGCAAGGGTTGTAACGGTTTTTG and GCTGCTGCATGCGACAGAACGTTCCCTCTTCAGCGA, to amplify the pqsA gene promoter and cloned using usual molecular methods into XbaI-SphI sites of the lasB-gfp plasmid  supplied with gentamicin 50 μg/ml and each sample at 128 μg/ml. Azithromycin (2 μg/ml) was used as the positive control. Plates were incubated at 37°C and 160 rpm. After 15 h, OD600 and fluorescence at 480/520 nm were measured using a microplate reader (SynergyHT BioTek). Results are represented in percentage of fluorescence compared to the solvent control (DMSO) fixed at 100%.The assay was performed according to the protocol proposed by  plasmid , in placrnaIII-lacZ (Staphylococcus caprae auto-inducing peptide (AIP) (2CO3 (0.4M), and fluorescence was read using a microplate reader (SynergyHT BioTek) at 360/460 nm. Results are represented in percentage of fluorescence compared to the solvent control (DMSO) fixed at 100%.The assay was performed according to the protocol proposed by III-lacZ  was growde (AIP)  at a conS. aureus PR01 and incubated for 10 min at 37°C, or 100 μl TE (pH 8) and lysozyme (1 mg/ml) and incubated for 5 min at RT for P. aeruginosa PAO1. RNA was extracted using an RNeasy kit (Qiagen) according to the manufacturer’s protocol. RNA was eluted in 40 µl of RNAse-free water, quantified using a Qubit 2.0 fluorometer (Invitrogen), and DNase treated with RQ1 RNase-free DNase (Promega) according to the manufacturer’s instructions. Then, 500 ng of RNA were reverse-transcribed into cDNA using random primers (Promega) and Improm-II reverse transcriptase (Promega) according to the manufacturer. qPCR was performed using SYBR select master mix (Thermo Fisher). Primers for the amplification of target genes are listed in HU and oprF genes were used for normalization for S. aureus and P. aeruginosa, respectively.Cultures were grown in triplicate for 4 h in the presence of the compound of interest at 128 μg/ml. Then, 1 ml of bacteria was treated with RNA protect Bacteria Reagent (Qiagen) before centrifugation and storage at −20°C. Pellets were resuspended in 100 μl TE (pH 8) and 2.5 µl of lysostaphin (10 mg/ml) for


Ambroxol (ABX) is a mucolytic agent used for the treatment of respiratory diseases. Bioactivity has been demonstrated as an enhancement effect on lysosomal acid β-glucosidase (β-Glu) activity in Gaucher disease (GD). The positive effects observed have been attributed to a mechanism of action similar to pharmacological chaperones (PCs), but an exact mechanistic description is still pending. The current study uses cell culture and in vitro assays to study the effects of ABX on β-Glu activity, processing, and stability upon ligand binding. Structural analogues bromohexine, 4-hydroxybromohexine, and norbromohexine were screened for chaperone efficacy, and in silico docking was performed. The sugar mimetic isofagomine (IFG) strongly inhibits β-Glu, while ABX exerts its inhibitory effect in the micromolar range. In GD patient fibroblasts, IFG and ABX increase mutant β-Glu activity to identical levels. However, the characteristics of the banding patterns of Endoglycosidase-H (Endo-H)-digested enzyme and a substantially lower half-life of ABX-treated β-Glu suggest different intracellular processing. In line with this observation, IFG efficiently stabilizes recombinant β-Glu against thermal denaturation in vitro, whereas ABX exerts no significant effect. Additional β-Glu enzyme activity testing using Bromohexine (BHX) and two related structures unexpectedly revealed that ABX alone can refunctionalize β-Glu in cellula. Taken together, our data indicate that ABX has little in vitro ability to act as PC, so the mode of action requires further clarification. The pharmacological chaperones (PCs) represent a novel form of therapy for diseases involving protein misfolding, such as lysosomal storage disorders (LSDs). PCs specifically bind to folded or partially folded proteins to provide enhanced stabilization and enable trafficking of mutant but intrinsically functional protein to the target organelle ,2. Most GBA1 gene (MIM# 606463) encoding for the lysosomal enzyme acid β-glucosidase . GD has an estimated incidence of 1/40,000 in the general population and an incidence of 1/1000 in the Ashkenazi Jewish population +, 59 (56), 57 (100), 43 (15), 41 (45), 39 (12). HRMS : calcd. for C5H11NO3 [M] 133.0733; found 133.0739. IR : 3356(m), 3282(w), 2979(w), 2940(w), 1679(s), 1514(m), 1459(m), 1393(m), 1367(s), 1282(s), 1252(s), 1161(s), 1119(s), 1048(m), 1014(m), 861(m), 774(m), 752(m), 517(s), 479(s).Following the literature procedure , a suspetert-Butyl-2-oxa-3-azabicyclo[2.2.2]oct-5-ene-3-carboxylate was synthesized following a modified literature procedure +, 111 (13), 80 (39), 79 (66), 78 (12), 77 (12), 57 (100), 41 (30), 39 (12). HRMS : calcd. for C11H17NO3 [M] 211.1203; found 211.1208. IR : 2979(m), 2937(w), 1673(s), 1615(w), 1455(m), 1395(s), 1360(s), 1293(m), 1253(s), 1162(s), 1125(m), 1110(s), 1076(s), 1049(s), 993(m), 957(m), 918(m), 879(s), 849(m), 818(m), 797(m), 745(s), 713(s), 680(m), 666(m), 614(m), 492(m), 429(m).rocedure . CuCl  in tetrahydrofuran (50 mL) was added dropwise to a stirring suspension of LiAlH4  in tetrahydrofuran (50 mL) at 0 °C. The ice-bath was removed, and the reaction mixture was stirred at ambient temperature for 1 h. Afterwards, the reaction mixture was cooled to 0 °C and quenched carefully with methanol and aqueous K2CO3 (pH = 10). The insoluble material was filtered off, and volatiles were removed under reduced pressure. The crude product was dissolved in dichloromethane and the aqueous phase was extracted with dichloromethane (5 × 50 mL). The combined organic layers were dried over Na2SO4, and the crude product was purified by column chromatography , affording 3-N-methyl-2-oxa-3-azabicyclo[2.2.2]oct-5-ene  as a brown oil. Rf = 0.60 . 1H NMR  δ 6.66-6.56 , 6.33-6.22 , 4.39-4.28 , 3.57-3.46 , 2.37 , 2.11-1.93 , 1.48-1.18 . 13C NMR  δ 132.8, 129.4, 68.0, 56.0, 44.8, 23.2, 22.4. GCMS : 125 (10) [M]+, 124 (92), 111 (10), 97 (17), 96 (17), 95 (20), 83 (10), 81 (13), 80 (91), 79 (100), 78 (24), 77 (49), 69 (12), 68 (16), 67 (37), 57 (13), 55 (15), 54 (13), 46 (70), 43 (11), 41 (23), 39 (17). HRMS : calcd. for C7H11NO [M] 125.0841; found 125.0849. IR : 3051(w), 2953(m), 2877(m), 1452(m), 1431(m), 1368(m), 1310(w), 1220(w), 1179(m), 1164(m), 1087(w), 1058(m), 998(m), 986(m), 955(s), 931(s), 853(m), 816(m), 801(m), 718(s), 704(s), 654(s), 569(m), 478(m), 438(s).3-rocedure . A solutR,4S)-3-methyl-2-oxa-3-azabicyclo[2.2.2]oct-5-ene 22  in dry methanol (40 mL) was hydrogenated in the presence of 5% Pd/C  at RT under atmospheric pressure of H2  for 24 h. The catalyst was removed by filtration through celite, and the crude product was purified by column chromatography  and sublimation  to afford (syn)-4-(methylamino)cyclohexan-1-ol  as a colorless solid. Rf = 0.06 . m.p. 84–85 °C. 1H NMR  δ 3.87-3.73 , 2.59 , 2.49-2.35 , 2.37 , 1.78-1.43 . 13C NMR  δ 67.3, 57.1, 33.7, 31.6, 27.5. GCMS : 129 (12) [M]+, 71 (17), 70 (100), 57 (38), 42 (7), 30 (4). HRMS : calcd. for C7H15NO [M] 129.1154; found 129.1162. IR : 3261(m), 3087(m), 2974(w), 2936(s), 2849(s), 2785(s), 2676(w), 1563(w), 1499(w), 1430(m), 1368(s), 1353(m), 1330(m), 1309(w), 1284(m), 1261(s), 1218(m), 1137(m), 1119(s), 1090(m), 1060(m), 1031(m), 974(s), 902(s), 824(m), 763(m), 686(m), 651(m), 507(m), 494(m), 464(w), 416(m).Following a modified literature procedure , a solut4 (2.0 eq) was added dropwise to the stirring reaction mixture. After the reaction was continued at 40 °C for 24 h, NaBH4 (2.5 eq) was added to the reaction solution in portions and stirred at 40 °C for an additional 24 h. After completion, H2O was added to the solution to quench the reaction and precipitate a large amount of solid, which was removed by suction filtration. The solvent was evaporated, the aqueous phase adjusted to pH = 10, extracted with EtOAc (3 × 20 mL), dried, and purified by column chromatography.2-amino-3,5-dibromobenzaldehyde (1.0 eq) and cyclohexylamine-derivative (1.5 eq) were dissolved in dry iPrOH (0.1 M) in a preheated flask under Ar and at 40 °C. Then, Ti(OiPr)cis-4-(methylamino)cyclohexan-1-ol  were first treated with Ti(OiPr)4  and then with NaBH4 . Purification by column chromatography  yielded the title compound D1  as a yellow viscous oil. Rf = 0.25 . 1H NMR  δ 7.46 , 7.04 , 5.45 , 3.99 , 3.60 , 2.50-2.34 , 2.14 , 1.93-1.69 , 1.66-1.41 . 13C NMR  δ 144.1, 133.2, 131.8, 110.2, 108.2, 65.8, 61.2, 58.0, 36.6, 32.2, 22.1. GCMS : 394 (7) [M]+, 392 (13) [M]+, 390 (7) [M]+, 335 (10), 333 (20), 331 (10), 307 (10), 305 (21), 303 (11), 295 (32), 291 (34), 266 (28), 335 (10), 264 (57), 262 (30), 142 (15), 128 (29), 104 (16), 77 (10), 70 (100), 57 (10), 44 (14), 42 (15). HRMS : calcd. for C14H20ON279Br2 [M] 389.9937; found 389.9928; calcd. for C14H20ON279Br81Br [M] 391.9916; found 391.9925. IR : 3429(w), 3258(w), 2926(m), 2850(m), 2792(w), 1604(m), 1575(w), 1550(m), 1459(s), 1379(m), 1356(m), 1291(m), 1256(m), 1232(m), 1196(m), 1123(m), 1100(m), 1072(m), 1031(s), 987(m), 958(s), 929(m), 905(m), 885(m), 856(s), 816(m), 736(m), 679(s), 646(s), 551(s), 444(s).Following the general procedure GP1, 2-amino-3,5-dibromobenzaldehyde  and 4  and then with NaBH4 . Purification by column chromatography  yielded the title compound D2  as a light yellow oil. Rf = 0.13 . 1H NMR  δ 7.46 , 7.09 , 5.41 , 3.79 , 2.54-2.34 , 1.98-1.83 , 1.79-1.66 , 1.65-1.55 , 1.39-0.97 . 13C NMR  δ 144.1, 133.2, 131.4, 127.1, 110.5, 108.3, 56.2, 50.3, 33.6, 26.2, 25.0. GCMS : 364 (8) [M]+, 362 (15) [M]+, 360 (8) [M]+, 293 (13), 291 (27), 289 (15), 281 (49), 280 (10), 279 (100), 277 (52), 266 (42), 264 (87), 262 (45), 183 (10), 112 (12), 104 (16), 293 (13), 98 (67), 56 (18), 55 (12), 41 (11). HRMS : calcd. for C13H18N279Br81Br [M] 361.9811; found 361.9806; calcd. for C13H18N281Br2 [M] 363.9790; found 375.9799. IR : 3435(w), 3265(w), 2922(s), 2849(m), 1606(m), 1577(w), 1552(m), 1459(s), 1408(m), 1371(m), 1346(m), 1284(w), 1242(m), 1195(m), 1144(w), 1111(m), 1077(s), 988(w), 956(m), 889(m), 858(s), 818(m), 799(m), 758(m), 682(s), 621(s), 551(s), 509(m), 448(s).Following the general procedure GP1, 2-amino-3,5-dibromobenzaldehyde 18  and cyclohexylamine  were first treated with Ti(OiPr)All statistical analyses were calculated using GraphPad Prism, versions 5.0 and 9.1.0 (for Windows).Pharmacological chaperones merit attention as candidate drugs for the treatment of Gaucher disease, specifically because they promise hope for the treatment of Type III GD. The present report juxtaposes the specific properties of the effects of ambroxol with those of the known PC isofagomine using molecular and biochemical examinations. ABX increases β-Glu activity measured in lysates of GD patient fibroblasts, quantitatively indistinguishable from IFG. Nevertheless, the ability of ABX to stabilize recombinant β-Glu in vitro is limited compared with IFG, and cellular stabilization of mutant β-Glu is also less effective, as verified by evidence of incomplete cellular processing.Closely related structural ABX analogues show no chaperone effect on β-Glu, and in silico docking reveals a possible position reversal in the binding pocket of compounds with an aminomethyl group, suggesting that ABX and derivatives might enter the active site in reverse position, with similar binding enthalpies but different stabilizing efficiency. Thus, the efficacy of ABX as a GCase binder and stabilizer in GD (and likely as a protein aggregation inhibitor for Parkinson’s disease) must be questioned. Our data emphasize the possibility that ABX function may be due to an as-yet-unexplained mode of action in the cellular context.


Polygala species are frequently used worldwide in the treatment of various diseases, such as inflammatory and autoimmune disorders as well as metabolic and neurodegenerative diseases, due to the large number of secondary metabolites they contain. The present study was performed on Polygala inexpectata, which is a narrow endemic species for the flora of Turkey, and resulted in the isolation of nine known compounds, 6,3′-disinapoyl-sucrose (1), 6-O-sinapoyl,3′-O-trimethoxy-cinnamoyl-sucrose (tenuifoliside C) (2), 3′-O-(O-methyl-feruloyl)-sucrose (3), 3′-O-(sinapoyl)-sucrose (4), 3′-O-trimethoxy-cinnamoyl-sucrose (glomeratose) (5), 3′-O-feruloyl-sucrose (sibiricose A5) (6), sinapyl alcohol 4-O-glucoside (syringin or eleutheroside B) (7), liriodendrin (8), and 7,4′-di-O-methylquercetin-3-O-β-rutinoside (ombuin 3-O-rutinoside or ombuoside) (9). The structures of the compounds were determined by the spectroscopic methods including 1D-NMR , 2D-NMR , and HRMS. The isolated compounds were shown in an in silico setting to be accommodated well within the inhibitor-binding pockets of myeloperoxidase and inducible nitric oxide synthase and anchored mainly through hydrogen-bonding interactions and π-effects. It is therefore plausible to suggest that the previously established anti-inflammatory properties of some Polygala-derived phytochemicals may be due, in part, to the modulation of pro-inflammatory enzyme activities. Polygala L. is the largest genus of the family Polygalaceae with more than 700 recognized species and is distributed in all continental areas except the Arctic and New Zealand -fructofuranosyl-α-d-[6-O-(4-methoxybenzoyl)]-glucopyranoside, were shown to attenuate the release of pro-inflammatory cytokines in LPS-stimulated BV2 microglial cells +; Negative ion HR-MS: m/z 753.2246 [M–H]−.6,3′-disinapoyl-sucrose  (2): Chemical formula C35H44O19; Mol. Wt. 768.2477; 1H-NMR : 13C-NMR : m/z 791.2362 [M + Na]+; Negative ion HR-MS: m/z 767.2393 [M–H]−.6-O-(O-methyl-feruloyl)-sucrose (3): Chemical formula C23H32O14; Mol. Wt. 532.4950; 1H-NMR : 13C-NMR : m/z 555.1677 [M + Na]+; Negative ion HR-MS: m/z 531.1709 [M–H]−.3′-O-(sinapoyl)-sucrose (4): Chemical formula C23H32O15; Mol. Wt. 548.4890: 1H-NMR : 13C-NMR : m/z 571.1624 [M + Na]+; Negative ion HR-MS: m/z 547.1727 [M–H]−.3′-O-trimethoxy-cinnamoyl-sucrose (glomeratose) (5): Chemical formula C23H35O15; Mol. Wt. 562.5210; 1H-NMR : 13C-NMR : m/z 585.1781 [M + Na]+.3′-O-feruloyl-sucrose (sibiricose A5) (6): Chemical formula C22H30O14; Mol. Wt. 518.4680; 1H-NMR : 13C-NMR : m/z 541.1523 [M + Na]+; Negative ion HR-MS: m/z 517.1561 [M–H]−.3′-O-glucoside (syringin or eleutheroside B) (7): Chemical formula C17H24O9; Mol. Wt. 372.1420; 1H-NMR : Sinapyl alcohol moiety: 6.58 ; 6.50 ; 5.82 ; 4.35 ; 3.87 ; glucose moiety: 4.91 ; 3.49 ; 3.50 ; 3.45 ; 3.45 ; 3.80 ; 3.70 .13C-NMR : Sinapyl alcohol moiety: 134.73 (C-1); 107.96 (C-2/C-6); 153.97 (C-3/C-5); 134.44 (C-4); 132.50 (C-7); 131.46 (C-8); 59.78 (C-9); 57.06 (OCH3 × 2); glucose moiety: 105.22 (C-1′); 75.66 (C-2′); 77.56 (C-3′); 71.24 (C-4′); 78.29 (C-5′); 62.47 (C-6′). Positive ion HR-MS: m/z 395.1306 [M + Na]+.Sinapyl alcohol 4-8): Chemical formula C34H46O14; Mol. Wt. 742.7240; 1H-NMR : Pinoresinol moiety: 6.67 ; 4.66 ; 3.09 ; 4.21 ; 3.84 ; 3.77 . Glucose moieties: 4.88 ; 3.21 ; 3.15 ; 3.05 ; 4.21 ; 3.42 . 13C-NMR : Pinoresinol moiety: 137.60 (C-1/C-1′); 104.61 (C-2/C-2′ and C-6/C-6′); 153.08 (C-3/C-3′ and C-5/C-5′); 134.09 (C-4/C-4′); 85.53 (C-7/C-7′); 54.07 (C-8/C-8′); 71.83 (C-9/C-9′); 56.88 (OCH3 × 4). Glucose moieties: 103.11 (C-1″/C-1‴); 74.61 (C-2″/C-2‴); 76.94 (C-3″/C-3‴); 70.34 (C-4″/C-4‴); 77.63 (C-5″/C-5‴); 61.33 (C-6″/C-6‴). Positive ion HR-MS: m/z 765.2576 [M + Na]+.Liriodendrin (O-rutinoside (ombuoside)  (9): Chemical formula C29H34O16; Mol. Wt. 638.5750; 1H-NMR : Flavonol moiety: 6.36 ; 6.67 , 7.55 ; 7.04 ; 7.73 ; 3.85 ; 3.86 ; 12.56 ; glucose moiety: 5.39 ; 3.26 ; 3.27 ; 3.51 ; 3.30 ; 3.72 ; 3.35 ; rhamnose moiety: 4.40 ; 3.41 ; 3.10 ; 3.08 ; 3.29 ; 0.97 ; 13C-NMR : Flavonol moiety: 157.21 (C-2); 134.27 (C-3); 178.13 (C-4); 161.43 (C-5); 98.56 (C-6); 165.75 (C-7); 92.84 (C-8); 157.02 (C-9); 105.61 (C-10); 122.13 (C-1′); 116.38 (C-2′); 146.46 (C-3′); 150.63 (C-4′); 111.92 (C-5′); 123.00 (C-6′); 56.67 (7-OCH3); 56.21 (4′-OCH3); glucose moiety: 101.76 (C-1″); 74.63 (C-2″); 76.97 (C-3″); 70.42 (C-4″); 76.40 (C-5″); 67.49 (C-6″). rhamnose moiety: 101.38 (C-1‴); 70.94 (C-2‴); 71.17 (C-3‴); 72.39 (C-4‴); 68.84 (C-5‴); 18.32 (C-6‴); Positive ion HR-MS: m/z 639.1909 [M + H]+, m/z 661.1726 [M + Na]+; Negative ion HR-MS: m/z 637.1761 [M–H]−.Ombuin 3-P. inexpectata secondary metabolites were generated based on the corresponding SMILES strings using myPresto programs available at https://demo1.biomodeling.co.jp/, accessed on 11 June 2021. The X-ray crystallographic structures of (i) human myeloperoxidase (MPO) in complex with a potent triazolopyridine compound [https://www.rcsb.org/, accessed on 23 July 2021. These enzymes not only engage in inflammatory processes but also in the generation of free radicals and the oxidation of nucleic acids, lipids, and proteins  hucompound , and (iicompound  were retcompound  availablng JAMDA ,102,103,P. inexpectata from Turkey. The plant appears to be comparable to other Polygala species with medicinal uses in that it has a rich content of sucrose esters. Liriodendrin and ombuoside are the isolated lignin and flavone glycosides, respectively, and are normally rare in this genus. The findings of the molecular docking calculations highlight the obtained compounds’ potential to inhibit the pro-inflammatory enzymes iNOS and MPO. The very encouraging in silico results indicate that these phytochemicals may have a future role in anti-inflammatory drug development research. Further in vitro and/or in vivo studies, however, are required to demonstrate their efficacy. These data could prove valuable in extending the knowledge of the phytochemistry and medicinal properties of the Polygala taxa.Overall, the present study attempted to explore the phytochemical composition of the endemic taxon


Based on previously identified dicarboximides with significant anticancer and immunomodulatory activities, a series of 26 new derivatives were designed and synthesized by the Diels–Alder reaction between appropriate diene and maleimide or hydroxymaleimide moieties. The resulting imides were functionalized with alkanolamine or alkylamine side chains and subsequently converted to their hydrochlorides. The structures of the obtained compounds were confirmed by 1H and 13C NMR and by ESI MS spectral analysis. Their cytotoxicity was evaluated in human leukemia , cervical cancer (HeLa), and normal endothelial cells (HUVEC). The majority of derivatives exhibited high to moderate cytotoxicity and induced apoptosis in K562 cells. Microarray gene profiling demonstrated upregulation of proapoptotic genes involved in receptor-mediated and mitochondrial cell death pathways as well as antiapoptotic genes involved in NF-kB signaling. Selected dicarboximides activated JNK and p38 kinases in leukemia cells, suggesting that MAPKs may be involved in the regulation of apoptosis. The tested dicarboximides bind to DNA as assessed by a plasmid DNA cleavage protection assay. The selected dicarboximides offer new scaffolds for further development as anticancer drugs. Cancer diseases are an important problem in the clinical medicine and pharmacology of the 21st century. Annually, more than eight million people die from cancer worldwide. In Europe alone, three million new cases are diagnosed and 1.7 million people die from cancer. In Poland, cancer kills nearly 110,000 people, of which about 100,000 die from malignant tumors. It is estimated that the ongoing increase in the incidence of cancer will make it the main cause of death in Poland and worldwide in the coming decades  380.1257; found 380.1259.M.W. = 357.4018; CA mixture of 2,5-diethyl-3,4-diphenylcyclopenta-2,4-dien-1-one (0.01 mol) and maleimide (0.012 mol) in benzene (15 mL) was refluxed for 14 h. When the reaction was completed, the solvent was evaporated under reduced pressure, and the solid residue was crystallized from benzene.25H23NO3; Yield: 92%; white powder, m.p. 206.8–209 °C (from benzen); 1HNMR : 0.87 ; 2.05 , 3.58 ; 6.97 ; 7.18 ; 11.70 ; Anal. Calc for C25H23NO3: 77.93% C; 5.98% H, 3.63% N; found 77.94% C; 5.97% H; 3.62% N; 13CNMR: δ 9.1, 19.2, 43.9, 55.3, 60.4, 128.0, 129.4, 133.1, 142.4, 171.6, 197.8; HRMS (m/z): calculated value for [M + Na+] 408.1570; found 408.1570.M.W. = 385.455; CA mixture of 1,2,3,4-tetraphenylcyclopenta-1,3-diene (0.02 mol) and maleimide (0.022 mol) was heated for 6 h in 20 mL of benzene. When the reaction was completed, the solvent was evaporated under reduced pressure, and the solid residue was crystallized from benzene.33H25NO2; Yield: 81%, white powder, m.p. 246.5–246.9 °C (from benzen); 1HNMR : 2.23 , 3.23 , 4.20 ; 6.60 ; 6.88 ; 7.24 ; 7.74 ; 11.44 ; Anal. Calc for C33H25NO2: 84.79% C; 5.35% H, 3.00% N; found 84.85% C; 5.39% H; 3.08% N; 13CNMR: δ 52.9, 62.9, 65.1, 126.4, 126.6, 127.1, 127.7, 129.1, 129.6, 134.5, 139.8, 145.0, 178.0; HRMS (m/z): calculated value for [M + Na+] 490.1778; found 490.1777.M.W. = 467.5571; C4- 2,5-dimethyl-3,4-diphenylcyclopenta-2,4-dien-1-one (0.02 mol) for compound 5 and N-hydroxymaleimide (0.022 mol) was heated for 6 h in 20 mL of benzene. When the reaction was completed, the solvent was evaporated under reduced pressure, and the solid residue was crystallized from benzene.- 1,2,3,4-tetraphenylcyclopenta-1,3-diene (0.02 mol) for compound A mixture of an appropriate diene:2,6]dec-8-ene-3,5,10-trione (4)4-(2-hydroxyethyl)-1,7-dimethyl-8,9-diphenyl-4-azatricyclo 424.1519; found 424.1519.M.W. = 401.4544; C5)4-(2-hydroxyethyl)-1,7,8,9-tetraphenyl-4-azatricyclodec-8-ene-3,5-dione ; 1HNMR : 2.12 , 2.32 , 3.17 , 3.74 ; 4.17 , 6.55 ; 6.89 ; 7.32 ; 7.70 ; 13CNMR: δ 42.2, 52.2, 60.5, 63.6, 65.9, 126.8, 127.1, 127.4, 128.1, 129.0, 129.8, 133.8, 139.2, 145.0, 177.1; HRMS (m/z): calculated value for [M + Na+] 543.5988; found 534.2045.M.W. = 511.6096; Ca (0.01 mol) in acetonitrile (30 mL), 1,2-dichloroethane (0.02 mol) was added. The reaction was heated for 72 h. When the reaction was completed, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography on silica gel (eluent: chloroform).To the solution of imide 2,6]dec-8-ene-3,5,10-trione (1h)4-(2-chloroethyl)-1,7-dimethyl-8,9-diphenyl-4-azatricyclo 442.8893; found 442.1175.M.W. = 419.9000; C2CO3 (0.01 mol) and an appropriate amine (0.01 mol) were added. The reaction was carried out in reflux, respectively, for 24–48 h in the boiling temperature of the solvent. The reaction was monitored by TLC. When the reaction was completed, the solvent was evaporated, and the residue was purified by column chromatography on silica gel (eluent: chloroform or chloroform/methanol 50:0.2 v/v).The 4-(2-chloroethyl)-1,7-dimethyl-8,9-diphenyl-4-azatricyclodec-8-ene-3,5,10-trione was dissolved in acetonitrile (30 mL), and then anhydrous K2,6]dec-8-ene-3,5,10-trione (1j)4-[2-(morpholin-4-yl)ethy]-1,7-dimethyl-8,9-diphenyl-4-azatricyclo 471.2278; found 471.2276.M.W. = 470.5595 (free amine); C2,6]dec-8-ene-3,5,10-trione (1i)4-[2-(piperidin-1-yl)ethy]-1,7-dimethyl-8,9-diphenyl-4-azatricyclo 469.2486; found 469.2485.M.W. = 468.5866 (free amine); CI: the mixture of an appropriate imide:1: 1,7-dimethyl-8,9-diphenyl-4-azatricyclodec-8-ene-3,5,10-trione2: 1,7-diethyl-8,9-diphenyl-4-azatricyclodec-8-ene-3,5,10-trione3: 1,7,8,9-tetraphenyl-4-azatricyclodec-8-ene-3,5-dioneThe new alkanolamine derivatives were obtained in two-step reactions according to the method described previously . Step I:2CO3 (0.01 mol) was stirred on a magnetic stirrer under reflux with a tube with CaCl2. The reaction was carried out. at room temperature for 15 h. When the reaction was completed (TLC control), the inorganic precipitate was filtered off and the filtrate was concentrated. The obtained oily product was purified by column chromatography on silica gel (eluent: chloroform and chloroform/methanol 50:0.2). Step II: to the solution of an appropriate 4-(oxirane-2-ylmethyl)-4-imide  (0.001 mol) in a mixture of methanol/water (10:1 v/v), the appropriate amine (0.001 mol) was added. Reactions were carried out at room temperature on a magnetic stirrer under reflux for 15–20 h. When the reaction was completed (TLC control), the excess of the solvent was evaporated and the crude product was purified by column chromatography on silica gel ).(0.01 mol) with 1-chloro-2,3-epoxypropane (50–60 mL), in the presence of an anhydrous K2,6]dec-8-ene-3,5,10-trione (1b)4-[3-(ethylamino)-2-hydroxypropyl]-1,7-dimethyl-8,9-diphenyl-4-azatricyclo  459.2284; found 459.2284.M.W. = 458.5488 (free amine); C2,6]dec-8-ene-3,5,10-trione (1c)4-[3-(tert-butylamino)-2-hydroxypropyl]-1,7-dimethyl-8,9-diphenyl-4-azatricyclo  487.2598; found 487.2597.M.W. = 486.6019 (free amine); C2,6]dec-8-ene-3,5,10-trione (1d)4-[3-(diethylamino)-2-hydroxypropyl]-1,7-dimethyl-8,9-diphenyl-4-azatricyclo 487.2598; found 487.2597.M.W. = 486.6019 (free amine); C2,6]dec-8-ene-3,5,10-trione (1e)4-[3-(dibutylamino)-2-hydroxypropyl]-1,7-dimethyl-8,9-diphenyl-4-azatricyclo  543.3224; found 543.3223.M.W. = 542.7082 (free amine); C2,6]dec-8-ene-3,5,10-trione (1f)4-[2-hydroxy-3-(piperidin-1-yl)propyl]-1,7-dimethyl-8,9-diphenyl-4-azatricyclo 499.2598; found 499.2597.M.W. = 498.6126 (free amine); C2,6]dec-8-ene-3,5,10-trione (1g)4-[-2-hydroxy-3-(morpholin-4-yl)propyl]-1,7-dimethyl-8,9-diphenyl-4-azatricyclo  501.2384; found 501.2379.M.W. = 500.5854 (free amine); C2,6]dec-8-ene-3,5,10-trione (2b)4-[3-(ethylamino)-2-hydroxypropyl]-1,7-diethyl-8,9-diphenyl-4-azatricyclo  487.6093; found 487.2591.M.W. = 486.6019 (free amine); C2,6]dec-8-ene-3,5,10-trione (2c)4-[3-(tert-butylamino)-2-hydroxypropyl]-1,7-diethyl-8,9-diphenyl-4-azatricyclo  515.6625; found 515.2904.M.W. = 514.6551 (free amine); C2,6]dec-8-ene-3,5,10-trione (2d)4-[3-(diethylamino)-2-hydroxypropyl]-1,7-diethyl-8,9-diphenyl-4-azatricyclo  515.6625; found 515.2904.M.W. = 514.6551 (free amine); C2,6]dec-8-ene-3,5,10-trione (2e)4-[3-(dibutylamino)-2-hydroxypropyl]-1,7-diethyl-8,9-diphenyl-4-azatricyclo 571.7688; found 571.3530.M.W. = 570.7614 (free amine); C2,6]dec-8-ene-3,5,10-trione (2f)4-[2-hydroxy-3-(piperidin-1-yl)propyl]-1,7-diethyl-8,9-diphenyl-4-azatricyclo 527.6732; found 527.2904.M.W. = 526.6658 (free amine); C2,6]dec-8-ene-3,5,10-trione (2g)4-[-2-hydroxy-3-(morpholin-4-yl)propyl]-1,7-diethyl-8,9-diphenyl-4-azatricyclo 529.6460; found 529.2697.M.W. = 528.6386 (free amine); C2,6]dec-8-ene-3,5-dione (3b)4-[3-(ethylamino)-2-hydroxypropyl]-1,7,8,9-tetraphenyl-4-azatricyclo 569.7119; found 569.2798.M.W. = 568.7040 (free amine); C2,6]dec-8-ene-3,5-dione (3c)4-[3-(tert-butylamino)-2-hydroxypropyl]-1,7,8,9-tetraphenyl-4-azatricyclo 597.7651; found 597.3117.M.W. = 596.7575 (free amine); C2,6]dec-8-ene-3,5-dione (3d)4-[3-(diethylamino)-2-hydroxypropyl]-1,7,8,9-tetraphenyl-4-azatricyclo 597.7651; found 597.3117.M.W. = 596.7575 (free amine); C2,6]dec-8-ene-3,5-dione (3e)4-[3-(dibutylamino)-2-hydroxypropyl]-1,7,8,9-tetraphenyl-4-azatricyclo 653.8714; found 653.3743.M.W. = 652.8635 (free amine); C2,6]dec-8-ene-3,5-dione (3f)4-[2-hydroxy-3-(piperidin-1-yl)propyl]-1,7,8,9-tetraphenyl-4-azatricyclo 609.7758; found 609.3117.M.W. = 608.7679 (free amine); C2,6]dec-8-ene-3,5-dione (3g)4-[-2-hydroxy-3-(morpholin-4-yl)propyl]-1,7,8,9-tetraphenyl-4-azatricyclo 611.7486; found 611.2904.M.W. = 610.7407 (free amine); CAlkylamine derivatives were obtained according to the methods published previously ,10. Next2,6]-dec-8-ene-3,5,10-trione (Ic*CH3COOH)Acetate of 4-[2-(piperidin-1-yl)ethyl]-1,7-diethyl-8,9-diphenyl-4-azatricyclo 497.2799; found 498.2799.M.W. = 496.2720 (free amine); C2,6]dec-8-ene-3,5,10-trione (Ie * CH3COOH)Acetate of 4-[2-hydroxy-3-(propan-2-ylamino)propyl]-1,7-diethyl-8,9-diphenyl-4-azatricyclo  501.2748; found 501.2748.M.W. = 500.2669 (free amine); C2,6]dec-8-ene-3,5-dione (II*CH3COOH)Acetate of 4-[2-(dimethylamino)ethyl]-1,7,8,9-tetraphenyl-4-azatricyclo 539.2693; found 539.2693.M.W. = 538.2614 (free amine); Cv/v) formic acid and was degassed by the use of the built-in membrane degasser. The methanol gradient grade for HPLC was purchased from Merck  and formic acid from Polish Reagents ; double-distilled water was used. Then, 5 μL of methanolic solutions  of selected samples were applied to the chromatographic column by the use of an autosampler Hitachi L-2200 . The analysis was performed with the flow rate of 0.7 mL min−1 in isocratic mode using various concentrations of organic modifier (methanol) in binary polar mobile phases; percentages of methanol in water were 45–70%  and changed by 5% per step. Chromatograms were detected at 254 nm and the temperature of the column was 25 °C. All experiments were repeated in triplicate, and the final results were taken to be the arithmetic means. Dead time was measured by the use of uracil . Statistical and regression analyses were performed using Statistica (ver. 13.3 for Windows). The chromatographic lipophilicity parameters (The reversed-phase high-performance liquid chromatography (RP-HPLC) analysis was performed on a Merck–Hitachi LaChrom Elite System  with diode array detector L-2455, thermostat L-2300, pump L-2130, and autosampler L-2200 and with the Waters XTerra MS RP-18  chromatographic column  as the stationary phase. The mobile phase consisted of methanol, water, and 0.1% . The HeLa (human cervix carcinoma), K562, and MOLT-4 (leukemia) cells were cultured in RPMI 1640 medium supplemented with antibiotics and 10% fetal calf serum in a 5% CO2–95% air atmosphere; 7 × 103 cells were seeded on each well on a 96-well plate (Nunc). Twenty-four hours later, cells were exposed to the test compounds for additional 48 hours. Stock solutions of test compounds were freshly prepared in DMSO. The final concentrations of the compounds tested in the cell cultures were 2 × 10−1, 1 × 10−1, 5 × 10−2, 1 × 10−2, 1 × 10−3, and 1 × 10−4 mM. The concentration of DMSO in the cell culture medium was 1%. The values of IC50  were calculated from dose–response curves and used as a measure of cellular sensitivity to a given treatment. The cytotoxicity of all compounds was determined by the MTT  assay. Briefly, after 24 or 48 h of incubation with drugs, the cells were treated with the MTT reagent, and incubation was continued for 2 h. MTT-formazan crystals were dissolved in 20% SDS and 50% DMF at pH 4.7, and absorbance was read at 570 and 650 nm on a microplate reader FLUOstar Omega . As a control (100% viability), we used cells grown in the presence of the vehicle (1% DMSO) only.Human umbilical vein endothelial cells  were cultured (according to the manufacturer’s instructions) in Medium 200 containing low serum growth supplement; 1 × 103 K562 cells were seeded on each well of a 96-well plate in RPMI 1640 medium supplemented with 10% fetal calf serum and antibiotics. Cells were grown for 24 h at 37 °C and 5% CO2. Test dicarboximides were dissolved in DMSO and added to cell culture to the final concentration of 5 × IC50. Cells treated with 1% DMSO served as a negative control, while cells incubated with staurosporine  were used as a positive control. Cells were exposed to test compounds for 18 h at 37 °C and 5% CO2. Subsequently, the activity of caspase 3 and 7 was measured by Apo-ONE® Homogeneous Caspase-3/7 Assay  according to the manufacturer’s instructions. Briefly, the cells were lysed and incubated for 1.5 h with a profluorescent substrate for caspase 3 and 7. Next, the fluorescence was read at an excitation wavelength of 485 nm and emission of 520 nm with a FLUOStar Omega microplate reader .First, 20 × 10®human apoptosis array, Life Technologies, Applied Biosystems, Foster City, CA, USA). The RT-PCR experiments were executed twice, each on three separate TLDA plates. K562 tumor cells were seeded on a six-well plate (Nunc) in an amount of 3 × 106 cells/well and were exposed to the test compounds at a concentration of 5 × IC50 for another 18 h. The control cells were exposed to 1% DMSO or 1 μM staurosporine. The total RNA pool was isolated from the cell lysates using TriPure Isolation Reagent  according to the manufacturer’s instruction. The RNA quality was checked with a NanoDrop ND-1000 spectrophotometer . Two micrograms of total RNA were reverse transcribed using a High Capacity cDNA Reverse Transcription kit . The 384-well TLDA (the qRT-PCR based TaqMan low-density array) cards were configured into four identical 96-gene sets consisting of 93 human genes and three endogenous controls: 18S, ACTB, and GAPDH. The primers  were selected to produce amplicons not longer than 100 bp (a mean of 74 bp). Those 93 genes were categorized into multiple classes including intrinsic, extrinsic, regulatory, and execution apoptosis traits. A reaction mixture with a cDNA template (100 ng) and an equal volume of TaqMan® universal master mix (Applied Biosystems) was immediately loaded into each line of the TLDA microfluidic card. Each card was spun twice (each time for 1 min) at 1200 rpm, then was sealed and loaded into an ABI 7900HT fast real-time PCR system , operating for 2 min at 50 °C, 10 min at 94.5 °C, 30 s at 97 °C, and 1 min at 59.7 °C (40 cycles). The results were statistically analyzed using the Student’s t-test, assuming a level of significance value of p < 0.05. The MetaCoreTM software  was used to perform the pathway analysis of the differentially expressed genes.The expression of apoptotic genes was studied using predesigned 384-well microfluidic cards (TLDA TaqMan6 K562 cells per well were seeded on a six-well plate in the complete medium (RPMI 1640 + 10% FBS). Then, 20 µM JNK kinase inhibitor SP600125 or 10 µM p38 inhibitor SB203580  was added for 24 h. Alternatively, K562 cells were exposed to 1% DMSO (control sample), test compound Ic (6 µM), II (3 µM), or staurosporine (1 µM) for 18 h. Cells exposed to staurosporine served as a positive control in ERK1/2 experiments. K562 cells treated with 20 µM anisomycin  for 30 min served as a positive control in JNK and p38 experiments. After incubation, cells were centrifuged , washed once with PBS, and lysed in the buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, and 1% Triton X-100, containing protease  and phosphatase inhibitors . Cells were lysed for 10 min on ice and centrifuged , and supernatants were collected. Total protein concentration in cell lysates was measured with DC Protein Assay . Cell lysates containing 25 µg of total protein were mixed with Laemli buffer supplemented with SDS and β-Mercaptoethanol, denatured , and subjected to SDS-PAGE electrophoresis (4% stacking and 10% resolving gels). Resolved proteins were electrotransferred  to nitrocellulose membranes . Membranes were blocked for 2 h in TBST buffer  containing 5% BSA (BioShop) and probed with primary antibodies (overnight at 4 °C) diluted in TBST buffer with 0.5% BSA. Tubulin served as a loading control for IB experiments. Anti-phospho ERK1/2  and anti-ERK1/2  were from Cell Signaling Technology. Anti-phospho JNK  and anti-JNK  were from Santa Cruz Biotechnology . Anti-phospho p38  and anti-p38  were from BD Transduction Laboratories . Anti-α-tubulin  was from Sigma. After extensive washing with the TBST buffer, membranes were probed with secondary goat anti-rabbit (1:5000 in TBST buffer) or goat anti-mouse HRP-conjugated IgG  for 45 min at room temperature. Membranes were washed in the TBST buffer, and the chemiluminescent signal was developed with ECL Plus Western Blotting Substrate  and visualized in a Syngene G:Box detection system.First, 2 × 10BamHI restriction site was dissolved in a 1× BamHI reaction buffer and incubated overnight at 37 °C with the test compounds or daunorubicin. Daunorubicin, a strong intercalating agent, was used as a positive control. The concentration of the test compounds and daunorubicin samples was 100 µM. In the next step, the reaction mixtures were digested with BamHI restriction endonuclease (2 U/μL) for 3 h at 37 °C. The total reaction volume was 10 μL. Products of the reaction were subjected to the 1% agarose gel electrophoresis in TBE buffer. The gel was stained with ethidium bromide, and DNA fragments were visualized under a UV lamp .First, 0.5 μg of plasmid DNA  containing a unique