Juncus maritimus Lam. was subjected to phytochemical and pharmacological investigations. The isolation process was carried out by using combined extraction and chromatographic methods. The structures of the obtained chemical compounds were elucidated by spectroscopic analysis, including HRESIMS, 1D , and 2D  NMR spectra. Four new [maritins A–D (1–4)] and seven known phenanthrenes (5–11) were isolated from the plant, of which two (4 and 11) are phenanthrene dimers composed of effusol monomers. Maritin C (3) has an unusual 4,5-ethanophenanthrene skeleton most likely produced by biosynthetic incorporation of a vinyl group into a cyclohexadiene ring. Compounds 1–11 were tested for their antiproliferative activity on seven human tumor cell lines  and one normal cell line (MRC-5) using the 3--2,5-diphenyltetrazolium bromide (MTT) assay. The dimeric phenanthrenes showed strong antiproliferative activity against T-47D cells with IC50 values of 9.1 and 6.2 µM, respectively.Juncaceae family represents an abundant source of phenanthrenes. In continuation of our work aiming at the isolation of biologically active compounds from Juncaceae species,  Phenanthrenes, a small group of aromatic secondary metabolites, have recently gained considerable attention due to their structural diversity and promising pharmacological properties. To date, various phenanthrene derivatives have been described from plant species belonging to the Annonaceae, Aristolochiaceae, Cannabaceae, Combretaceae, Euphorbiaceae, Dioscoreaceae, Lauraceae, Malpighiaceae, Orchidaceae, Stemonaceae, and Juncaceae families . PhenantJuncus maritimus Lam. is a perennial halophyte herb native to coastal salt marshes regularly flooded with seawater. In the Algerian, Moroccan, and Tunisian folk medicines, preparations of the plant have long been used as analgesic, antiseptic, and anti-inflammatory remedies to treat various ailments, such as infections of the urinary and reproductive systems, injuries, wounds, and skin diseases  and seven known (5–11) phenanthrenes from the methanol extract of J. maritimus. The antiproliferative activity of the isolated phenanthrenes was investigated on seven human cancer cell lines  and one normal cell line (MRC-5).As part of our ongoing research program, we describe here the isolation and structure determination of four new − . The 1H-NMR spectrum displayed signals of two ortho-coupled aromatic methines , an aromatic proton singlet (δH 6.92), two methylenes , an oxymethylene , a methyl group , and a vinyl moiety   . The 18 1H-1H COSY correlations defined three sequences of correlated protons, namely, –CH2–CH2– (H2-9–H2-10), –CH=CH2 (H-12–H-13a and H-13b), and –CH=CH– (H-3–H-4) fragments (1 was assembled with the aid of an HMBC experiment. Heteronuclear long-range correlations of H-3 and H2-10 with C-4a (δC 127.2), H-4, H-6, and H2-9 with C-5a (δC 128.3), H2-9, H2-10, and H2-14 with C-8a (δC 141.2), as well as of H-4, H2-9, H2-10, and H3-11 with C-1a (δC 140.1) established a 9,10-dihydrophenanthrene skeleton. HMBC correlations from H-3, H-4, and H3-11 to C-2 (δC 155.1), and from H-6 and H2-14 to C-7 (δC 155.3) suggested that compound 1 contains two hydroxy groups at the positions of C-2 and C-7. The location of the H3-11 methyl group at C-1 was dictated by its HMBC correlations with C-1, C-1a, and C-2. The two- and three-bond correlations between H2-14 (δH 4.79), C-7, C-8 (δC 124.4), and C-8a demonstrated that the freely rotating hydroxymethyl substituent is attached to C-8. The location of the vinyl moiety at C-5 (δC 136.6) was confirmed by the H-6/C-12 and H-13/C-5 HMBC correlations. The NOE cross-peaks between H-4/H-12, H-13a/H-6, H2-9/H2-14, and H2-10/H3-11 were consistent with the proposed structure of 1, as shown in The ragments . The str2 (maritin B) was obtained as a white amorphous solid. Its molecular formula was deduced to be C18H18O based on the protonated molecule in the HRESIMS at m/z [M + H]+ 251.1429 . The 1H-NMR spectrum contained signals of two pairs of ortho-coupled aromatic protons , two methylenes , a vinyl substituent , and two methyl groups  (3-11 (δH 2.24) to C-1 (δC 120.9), C-1a (δC 137.7), and C-2 (δC 153.3), and further correlations between H-3 (δH 6.73), H-4 (δH 7.47), and C-2 showed that a methyl and a hydroxy group are situated on the adjacent carbons C-1 and C-2, respectively. The locations of another methyl (δH 2.32) and a vinyl substituent at C-7 and C-8, respectively, were apparent from the HMBC correlations H3-12/C-6, H3-12/C-7, H3-12/C-8, H-6/C-8, H2-9/C-8, and H-14/C-8. Further heteronuclear correlations were detected between H-3, H-5 (δH 7.49), H2-10 (δH 2.74), and C-4a (δC 128.4), H-4, H-6 (δH 7.11), H2-9 (δH 2.88), and C-5a (δC 133.3), and from H-5, H2-9, and H2-10 to C-8a (δC 134.1). The NOE cross-peaks H-6/H3-12, H3-12/H-13, H2-9/H-14b, and H2-10/H3-11 supported the proposed structure of compound 2.Compound each 3H) . The HMB3 (maritin C) as an orange amorphous solid. According to a peak of the deprotonated molecule at m/z 279.1027 [M − H]− in the HRESIMS data, the molecular formula C18H16O3  was assigned to 3. The 1H-NMR spectrum exhibited two aromatic methines coupled with each other , two aromatic singlets (δH 7.17 and 7.02), two methyl groups , and signals of an oxymethine  and a saturated methylene . The 1H-1H COSY spectrum afforded two structural elements, the aforementioned –CH=CH– (δH 7.79 and 7.56) and a –CH(OR)–CH2– fragment . The proton signals at δH 7.02 (H-3) and δH 2.49 (H3-11) gave HMBC correlations with a downfield shifted, nonprotonated carbon displayed at δC 153.4, while the aromatic singlet at δH 7.17 (H-8) and the methyl group at δH 2.50 (H3-14) gave HMBC correlations to a carbon resonating at δC 155.1. Thus, it was deduced that this phenanthrene bears hydroxy groups at C-2 and C-7. The two methyls were placed onto C-1 and C-6 on the basis of the corresponding H3-11/C-1, H3-11/C-1a, H3-11/C-2, H3-14/C-5, H3-14/C-6, and H3-14/C-7 HMBC correlations. Further long-range correlations from H-9 (δH 7.56) to C-1a, C-5a, and C-8, as well as from H-10 (δH 7.79) to C-1, C-4a, and C-8a established a phenanthrene skeleton with an aromatic ring B. Considering the HMBC cross-peaks of H-13a (δH 3.38) with C-3 (δC 117.0), C-4 (δC 130.9), C-4a (δC 122.5), and C-5 (δC 134.6), it was clear that a vinyl group was incorporated into an oxygen-substituted cyclohexadiene ring. From a biosynthetic point of view, compound 3 was likely formed from a dehydrojuncusol precursor through the modification of its vinylic double bond, followed by a ring closure between C-4 and C-13. The depicted structure of maritin C was corroborated by NOE cross-peaks between H-3/H-13a and b, H-12/H3-14, H-8/H-9, and H-10/H3-11. The specific optical rotation of 3 was recorded as zero, therefore, it was isolated as a racemic mixture.Separation of the plant extract yielded compound 4 (maritin D) has the molecular formula C34H30O4 compatible with its protonated molecule at m/z 503.2203 [M + H]+  in the HRESIMS data. The 34 carbon signals displayed in the 13C-JMOD NMR spectrum suggested that compound 4 is a phenanthrene dimer , a –CH=CH–  and two –CH2–CH2– structural portions , and two methyls  in 4 (Compound ne dimer . The 1H-3H) in 4 . meta-coupled aromatic protons  were also identified via weaker 4JH-H (W-type) COSY cross-peaks and three-bond HMBC correlations between the corresponding methine groups. Further analysis of the HMBC correlations unambiguously determined that 4 is comprised of two monomers of a known 9,10-dihydrophenanthrene, effusol, which was also isolated as an individual compound (6) from the plant. Taking into account the HMBC correlations from H-4′ and H3-11′ to C-2′ (δC 144.6), it was concluded that oxygen atoms are connected to both of the vicinal carbons C-2′ and C-3′ (δC 156.6). Although no HMBC correlations were observed between the monomers, NOE cross-peaks H-4′/H3-11 and H-13′b/H-12 indicated the close proximity of these protons, and consequently implied that the monomers must be attached through an ether bond between C-2/C-2′ or C-2/C-3′. In order to determine the exact structure, energy-minimized structures were generated for each of the hypothetical compounds by using the MM2 force field method. A minimum energy conformation , seven known phenanthrenes, namely, juncusol (5) . This separation yielded 14 fractions (A-N). The fractions were combined according to their TLC patterns.The plant material  was air-dried (2.2 kg) at room temperature. Thereafter, it was ground and percolated with 40 L methanol at room temperature. After evaporation, the extract was dissolved in 50% aqueous methanol, and repetitive solvent-–solvent partition was performed with 6 × 0.5 L 2Cl2-–MeOH (1:1) as eluent. Fraction B/2 was separated by normal-phase HPLC under gradient conditions, using cyclohexane–EtOAc  as mobile phase to obtain compounds 5  and 2 . Purification of fractions D/4 and D/5 by preparative TLC afforded compounds 6 (4.3 mg) and 7 (3.4 mg).All major fractions were purified by Sephadex LH-20 gel chromatography using CH2O (from 8:2 to 1:0). Subfraction E/1 was then further purified by reversed-phase HPLC under gradient conditions, using MeOH–H2O  as mobile phase to yield compound 10 . Subfraction E/2 was separated by preparative TLC on silica gel using cyclohexane–EtOAc–EtOH (20:10:1) as solvent system to yield compounds 9 (3.5 mg) and 3 (4.5 mg).Fraction E was chromatographed by reversed-phase MPLC using MeOH–H2O (from 8:2 to 1:0). Subfraction HI/3-4/1 was separated by preparative TLC on silica gel using cyclohexane–EtOAc–EtOH (20:10:1) as eluent to isolate compound 8 (10.4 mg). HI/3-4/1/2 was purified by reversed-phase HPLC under gradient conditions, using MeOH–H2O  as mobile phase, and compound 1  was isolated. Subfraction HI/3-4/7 was further fractionated by normal-phase HPLC under gradient conditions, using cyclohexane–EtOAc  as mobile phase, to afford compound 11 . Subfraction HI/3-4/9 was separated by preparative TLC on silica gel using an isocratic cyclohexane–EtOAc–EtOH (60:30:3) eluent, and then HI/3-4/9/3 was purified by reversed-phase HPLC under gradient conditions, using acetonitrile–H2O  as mobile phase to yield compound 4 .Fractions H/3 and I/4 were combined (HI/3-4) because of their similar chemical composition and were purified by reversed-phase MPLC using a stepwise gradient solvent system composed of MeOH–H1)Maritin A (1H- and 13C-JMOD NMR (in methanol-d4) data, see m/z 281.1183 [M − H]− .Yellow amorphous solid; for 2)Maritin B (1H- and 13C-JMOD NMR (in CDCl3) data, see m/z [M + H]+ 251.1430 .White amorphous solid; for 3)Maritin C ; for 1H-and 13C-JMOD NMR (in methanol-d4) data, see m/z 279.1027 [M − H]− C18H16O3 .Orange amorphous solid; [α]4)Maritin D (1H- and 13C-JMOD NMR (in methanol-d4) data, see m/z [M + H]+ 503.2203 .Yellow amorphous solid; for 10):Jinflexin A : δH 6.67 , 6.87 , 6.86 , 2.87 , 2.37 , 2.88 , 2.38 , 2.22 , 4.85 , 1.57 , 2.19 , 2.92 . 13C NMR : δC 121.8 (C-1), 140.5 (C-1a), 154.9 (C-2), 112.4 (C-3), 127.5 (C-4), 127.4 (C-4a), 129.3 (C-5a), 138.4 (C-5), 111.5 (C-6), 155.1 (C-7), 121.4 (C-8), 139.8 (C-8a), 27.6 (C-9), 26.7 (C-10), 11.8 (C-11), 76.5 (C-12), 23.4 (C-13), 11.8 (C-14), 55.7 (12-OCH3).®® HTB-22) and the drug-resistant subline of the human breast cancer MCF-7  were purchased from LGC Promochem . Both cell lines were cultured in Eagle’s Minimal Essential Medium  supplemented with a non-essential amino acid mixture, a selection of vitamins, and 10% heat-inactivated fetal bovine serum. In every third passage, 0.56 µg/mL doxorubicin was added to the medium in order to maintain the ABCB1 (P-glycoprotein) expression in KCR cells. A2780 human ovarian cancer cell line  and the cisplatin-resistant human ovarian cancer cell line A2780cis  were purchased from Merck KGaA . The human ovarian cancer cell lines were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum. The RPMI 1640 medium of the cisplatin-resistant cell line A2780 was supplemented with 1 µM cisplatin. HeLa (ATCC® CCL-2™) human cervix carcinoma, HTB-26 breast adenocarcinoma, T-47D (ATCC® HTB-133™) ductal carcinoma, and MRC-5 human embryonal lung fibroblast cell lines (ATCC® CCL-171) were purchased from LGC Promochem . The cells were cultured in Eagle’s Minimal Essential Medium  supplemented with a non-essential amino acid mixture, a selection of vitamins, and 10% heat-inactivated fetal bovine serum. HTB-26 cell line was cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum. T-47D cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 1 mM Na-pyruvate, and 100 mM Hepes. All of the cells were incubated at 37 °C, in a 5% CO2, 95% air atmosphere.Breast cancer cell line MCF-7  was added to the cells. The effects of increasing concentrations of compounds on cell proliferation were tested in 96-well flat-bottomed microtiter plates. The compounds were diluted in the appropriate medium, the dilutions of compounds were performed in separate plates and then added to the cells. The starting concentration of the compounds was 100 μM, and two-fold serial dilution was performed (concentration range: 100-0.19 μM). The culture plates were incubated at 37 °C for 72 h; at the end of the incubation period, 20 μL of MTT  solution (from a stock solution of 5 mg/mL) was added to each well. After incubation at 37 °C for 4 h, 100 μL of sodium dodecyl sulfate (SDS) (Sigma) solution (10% in 0.01 M HCI) were added to each well and the plates were further incubated at 37 °C overnight. Cell growth was determined by measuring the optical density (OD) at 540/630 nm with Multiscan EX ELISA reader . Mean IC50 values were obtained by best fitting the dose-dependent inhibition curves in GraphPadPrism5 program  from four parallel experiments for each cell line. Results are expressed in terms of IC50, defined as the inhibitory dose that reduces the proliferation of the cells exposed to the tested compounds by 50% [The antiproliferative effect of the compounds was determined on the human breast , cervical (HeLa), and ovarian  cancer cells, and on MRC-5 (human embryonic lung fibroblast) cell line. The adherent cells were cultured in 96-well flat-bottomed microtiter plates, using EMEM supplemented with 10% heat-inactivated fetal bovine serum or RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, respectively. The density of the cells was adjusted to 6 × 10s by 50% .1–4), were isolated from the methanolic extract of J. maritimus. All compounds but effusol (7) were reported for the first time from the plant. Some of the new phenanthrenes possess interesting structural features. The obtained compounds are substituted with hydroxy, methyl, hydroxymethyl, and vinyl groups. In cases of three compounds, the C-5 vinyl groups of biosynthetic intermediates were either incorporated into a cyclohexadiene ring  of a rare 4,5-ethanophenanthrene scaffold or further modified into a formyl (7) or a methoxyethyl (10) substituent. The new phenanthrene maritin D (4) contains two effusol monomers attached to each other through a C-2–C-3′ ether bond, which resulted in the formation of a unique diaryl ether skeleton. The two isolated dimers (4 and 11) displayed substantial antiproliferative activity against all investigated cell lines. For both compounds, the highest activities (comparable to the positive control cisplatin) were detected on T-47D ductal carcinoma cells. In general, T-47D cells were the most sensitive to the phenanthrenes, but some of the isolated compounds  on MCF-7 cells; maritin B (2), juncusol (5), and effusol (6) on HeLa cells) exerted outstanding inhibitory potential against other malignant cell lines, too. An assessment of the results of our pharmacological evaluation allowed us to gain a deeper insight into structure-antiproliferative activity relationships of naturally occurring phenanthrenes.Eleven phenanthrenes, including four new ones (


Koch, commonly known as lovage and well known in traditional medicine for its spasmolytic and diuretic effects. The structure of the new compound was established by HRMS and 1D & 2D NMR  spectroscopic analysis. All compounds are reported for the first time from L. officinale. Compounds 1-3 were tested against two Gram negative  and two Gram positive (Staphylococcus aureus and vancomycin-resistant Enterococcus [VRE] faecium) bacteria strains. Compound 3 was active against S. aureus, E. coli and vancomycin-resistant E. faecium with MIC values of 16, 64, and 128 μg/mL, respectively.A new phthalide, namely 7-methoxy-3-propylidenephthalide ( Apiaceae family -ligustilide, (Z)-3-butylidenephthalide, (E)-3-butylidenephthalide, 3-butylphthalide, and levistolide A  mas spectrometer and equipped with an electrospray source with lockspray interface for accurate mass measurements. Silica gel (70-230 mesh) was used for column chromatography, and precoated silica gel F254 (20 × 20 cm) plates for TLC, both supplied by the Merck. C18-reversed phase silica gel used for column chromatography, was purchased from Sigma.NMR spectra were recorded on a Bruker AVANCE III spectrometer operating at 500 MHz for Plant materialL. officinale were collected in July 2015 from the Hezar Mountain of Kerman Province, Iran. The plant material was identified by Prof. Farideh Attar from Tehran University. A voucher specimen (46553-TUH) has been deposited in the herbarium of the Science Faculty of Tehran University, Iran.The roots of Extraction and isolationL. officinale (3 kg) were extracted with n-hexane (3 x 9 L) and then ethyl acetate (3 x 9 L) by maceration at room temperature. The ethyl acetate extract was concentrated by rotary evaporation, to afford 100 g of dried extract. This extract was subjected to silica gel column chromatography , with a gradient of n-hexane–EtOAc and then EtOAc–MeOH as the eluent to give 16 fractions. Fraction 6 (1.8 g) was separated on a silica gel column (230-400 mesh) into five subfractions (6a-6e). Subfraction 6a (50 mg) was applied to a reverse phase silica gel column (8 g) and eluted with methanol-water as eluent, to afford compound 1 (2 mg). Purification of subfraction 6b (40 mg) on a reverse phase silica gel (7 g) column with a gradient of methanol-water as eluent led to the isolation of compound 2 (3 mg). Subfraction 6c (70 mg) was purified on a reverse phase silica (12 g) column to give compound 3 (3.5 mg). Air-dried, powdered roots of 7-methoxy-3-propylidenephthalide (1)1H NMR  δ 7.62 , 7.21 , 6.93 , 5.63 , 4.01 , 2.48 , 1.14 , 13C NMR : δ 158.7 (C-7), 147.0 (C-3), 142.3 (C-3a), 111.3 (C-4), 137.2 (C-5), 111.9 (C-6), 110.6 , 56.1 (-OCH3), 19.3 (C-9), 13.9 (C-10); HRESIMS  for C12H12O3.  Brown oil; 5-hydroxybutylidene phthalide (2)1H NMR  δ 7.76 , 7.02 , 6.97 , 5.57 , 2.43 , 1.54 , 0.98 , 13C NMR : δ 167.4 (C-1), 161.8 (C-5), 145.1 (C-3), 141.8 (C-3a), 127.3 (C-7), 118.2 (C-6), 117.0 (C-7a), 109.6 (C-8), 105.3 (C-4), 27.7 (C-9), 22.5 (C-10), 13.8 (C-11). Brown oil; 7-hydroxybutylidene phthalide (3)1H NMR  δ 7.60 , 7.30 , 6.96 , 5.84  2.39 , 1.65 , 0.1.01 , 13C NMR : δ 158.3 (C-7), 147.7 (C-3), 142.6 (C-3a), 137.0 (C-5), 116.5 (C-6), 111.4 (C-4), 110.6 (C-7a), 109.7 (C-8), 28.0 (C-9), 22.8 (C-10), 13.7 (C-11). Brown oil; Antibacterial activityBacterial strainsIn-vitro antibacterial activity of compounds was assessed against Staphylococcus aureus ATCC 25923, Enterococcus faecium  as Gram-positive bacteria and Escherichia coli ATCC 25922 and Pseudomonas aeruginosa PTCC1430 as Gram-negative bacteria. Some strains (ATCC strains and VRE) were kindly provided by Professor M.M. Feizabadi, Tehran University of Medical sciences. PTCC strain was purchased from Iranian Research Organization for Science and Technology.Determination of MICDetermination of the minimum inhibitory concentration (MIC) was carried out by the broth micro-dilution method as recommended by CLSI  with minor modifications . The serL. officinale led to isolation and identification of a new phthalide (1), together with two knowns compounds namely, 5-hydroxybutylidene phthalide (2) and 7-hydroxybutylidene phthalide (3)  .1 was obtained as a brown oil and its molecular formula, C12H12O3 was established by HR-ESI-MS  with 6-degrees of unsaturation. On the basis of APT and HSQC experiments, 12 resonances in the 13C NMR spectrum  was identified using 1H-NMR and 13C-NMR, as well as by comparison the data with those reported in literature , and S. aureus (1 mg/mL). Antibacterial activity was found for butylidenephthalide against E. coli and S. aureus with MIC values of 4 mg/mL, while (Z)‐ligustilide exhibited moderate antibacterial activity against S. aureus with MIC value of 1 mg/mL (Simple phthalide derivatives usually have low to moderate antibacterial activity with MIC values in the range of 4 – 0.128 mg/mL against  E. coli . For exa 1 mg/mL . L. officinale, including new one. Their structures were elucidated by means of extensive 1D, 2D NMR spectroscopy. Also, the antibacterial activity of the compounds was studied against two Gram negative and two Gram positive bacteria strains. Among them, compound 3 hold a good potential for use in future studies due to their antibacterial properties. To our knowledge, this is the first report on the isolation and structure elucidation of the chemical constituents of L. officinale in Iran. All compounds are reported for the first time from L. officinale. Simple phthalides are not widely reported as showing any significant antibacterial activity. Therefore, the antibacterial activity of compound 3 is notable. In the present study, three phthalide derivatives were isolated from the roots of 1 can be found as Supporting Information.1D and 2D NMR spectra of compound


Numerous substances from different chemical sectors, from the pharmaceutical industry to the many consumer products available for everyday usage, can find their way into water intended for human consumption and wastewater, and can have adverse effects on the environment and human health. Thus, the disinfection process is an essential stage in water and wastewater treatment plants to destroy pathogenic microorganisms but it can form degradation byproducts. Sodium hypochlorite is the most common disinfectant, but the most important drawback associated with this kind of compound is the generation of toxic disinfection byproducts. Many studies have been carried out to identify alternative disinfectants, and in the last few years, peracetic acid has been highlighted as a feasible solution, particularly in wastewater treatment. This study compares the transformations of five emerging pollutants  treated with peracetic acid, to evaluate their degradation and the possible formation of byproducts with those obtained with sodium hypochlorite. Although peracetic acid has many advantages, including a wide field of use against microorganisms and a low toxicity towards animal and plant organisms, it is not as effective in the degradation of the considered pollutants. These ones are recovered substantially and are unchanged quantitatively, producing a very low number of byproducts. Social, productive, and recreational activities require and use a large quantity of water , with thDue to the advantages and future prospects that can be offered by recycling wastewater, new technologies have been created that seek to obtain efficient processes to guarantee a purified water supply at low cost. It is important to note that, due to the scarcity of water resources, many countries allocate purified wastewater for drinking purposes. This practice in countries with very low availability of technology and widespread electricity can be particularly dangerous for human health. Not all wastewater has the same nature and characteristics; therefore, to identify the most suitable depurative treatment system, it is essential to know both the origin of the polluted water and its final destination. Wastewater from human metabolism, and therefore domestic and/or civil waste, has a clear prevalence of organic pollutants, natural but also synthesized as drugs or biologically active substances ,11,12,13Disinfection is a fundamental phase in water-purification processes. Today, chlorine and chlorine derivatives dominate the wastewater treatment market, used at more than two-thirds of the plants, and they are able to not only effectively neutralize all species of micro-organisms, but also to degrade organic pollutants, in some cases after a double treatment. However, the various chlorine-based chemicals used in water treatment create degradation byproducts (DPs) that can harm the environment and human health ,11,12,13Several studies have shown that PAA produces almost no toxic or mutagenic degradation byproducts from the reaction with organic material present in the treated wastewater , if not The aim of the present study was to analyze the possible use of peracetic acid in wastewater treatment plants, instead of sodium hypochlorite. In particular, caffeine, tramadol, irbesartan, diclofenac and trazodone were considered, chosen because found as emerging pollutants of surface waters and for the first three of which the results obtained in the treatment with sodium hypochlorite are already known ,12,13, w−5 M), comparable to those detected in surface water or in water entering civil wastewater treatment plants, with a molar ratio of 1:1 contaminant:oxidizing agent + in the EI mass spectrum and 18 carbon signals in the 13C-NMR spectrum. Nuclear magnetic resonance spectra were very similar to the correspondents to tramadol but with few significant differences. The 1H-NMR spectrum showed the three aromatic protons H-6’, H-2’ and H-5’, at δ 6.95, 7.21 and 7.36 and correlated, in the heteronuclear single quantum coherence (HSQC) spectrum, to the carbons at δ 117.34, 109.21 and 130.22, respectively. Moreover, the 1H-NMR spectrum, in addition to the methoxyl signal at δ 3.97, showed the signal of a singlet integrable for three protons at δ 2.37, correlated in a HSQC spectrum to the signal at δ 30.85, identified in a distortionless enhancement by polarization transfer (DEPT) experiment as a methyl, and the 13C-NMR showed the presence of a carbonyl at δ 202.26. The HMBC experiments allowed the assignment of these signals to an aromatic ring 1,2,4-trisubstituted, with a methyl carbonyl bound to C-4’ carbon as well as a methoxyl and a cyclohexane ring bound to the carbons C-3’ and C-1’, respectively.DP4: It has the molecular formula C18H27NO3 according to the presence of the molecular ion at m/z 305.21 [M]+ in the EI mass spectrum and 18 carbon signals in the 13C-NMR spectrum. Nuclear magnetic resonance spectra were very similar to the correspondents to DP3. In fact, the 1H-NMR spectrum showed the three aromatic protons at δ 6.77, 7.26 and 7.57, correlated in the HSQC spectrum to the carbons at δ 113.98, 132.93 and 114.69, respectively, and identified as the protons H-4’, H-5’ and H-2’. Moreover, the 1H-NMR spectrum, in addition to the methoxyl signal at δ 3.84, showed the signal of a singlet integrable for three protons at δ 2.45, correlated in a HSQC spectrum to the signal at δ 30.92, identified in a DEPT experiment as a methyl, and the 13C-NMR showed the presence of a carbonyl at δ 206.96. The heteronuclear multiple bond correlation (HMBC) experiments allowed us to assign the structure of this compound as an isomer of the previous one precisely with the methyl carbonyl function linked to carbon C-6’, instead of carbon C-4’, and the methoxyl and the cyclohexane ring linked to the carbons C-3’ and C-1’, respectively.DP5: The EI mass spectrum analysis showed a molecular ion peak at m/z 447.26 [M + H]+ corresponding to the molecular formula C25H30N6O2. The 1H-NMR spectrum showed the protons of the methylene CH2–11 at δ 4.31. In the HMBC spectrum, these protons were correlated with the signals at δ 142.16 and 131.42, which were identified as the carbons C-12 and C-13/C-17, and with the signals at δ 178.59 and 180.55, which were identified as the carbonyl carbons C-7 and C-9, respectively. The first carbonyl carbon was correlated with the signals at δ 2.41 and 1.50, which were identified as the protons H-29 and H-30 of the alkyl side chain, respectively, whereas the second one was correlated with the signals at δ 2.05 and 2.20, which were identified as the protons H-3 and H-5 of the cyclopentane group, respectively. These correlations support the hydrolysis of the bound C-7/N-6. DP6: The EI mass spectrum analysis showed a molecular ion peak at m/z 336.36 [M + H]+ corresponding to the molecular formula C19H21N5O. The 1H-NMR spectrum showed the protons of the methylene CH2–11 at δ 4.14, which, in the HMBC spectrum, were correlated with the signals at δ 133.34 and 127.48, which were identified as the carbons C-12 and C-13/C-17, and with the signal at δ 171.19, which was identified as the carbonyl carbon C-7. The last one was correlated with the signals at δ 2.06 and 1.53, identified as the protons H-29 and H-30 of the alkyl side chain, respectively. The 1H-NMR spectra do not show the signals of the protons of the cyclopentane group, whose carbon was missing in the 13C-NMR spectrum, as well as the carbonyl carbon C-9. These correlations support the hydrolysis of the bounds C-7/N-6 and C-9/H-8.DP7: It has the molecular formula C19H22ClN5O2 according to the presence of the molecular ion at m/z 387.18 [M]+ in the EI mass spectrum and 19 carbon signals in the 13C-NMR spectrum. Nuclear magnetic resonance spectra were very similar to the correspondents to trazodone. The 1H-NMR spectrum showed the four aromatic signals of the pyridine ring at δ 6.51, 7.08, 7.10 and 7.75, correlated in the HSQC spectrum to the carbons at δ 110.80, 115.37, 130.21 and 123.71 and identified as the protons H-3, H-5, H-4 and H-2, respectively, as well as the four aromatic signals of the benzene ring 1,3-disubstituted at δ 6.79, 6.87, 6.89 and 7.18, correlated in the HSQC spectrum to the carbons at δ 114.49, 120.57, 116.58 and 130.30 and identified as the protons H-25, H-23, H-21 and H-24, respectively. The 1H-NMR spectra also showed the protons at δ 2.57, 3.44 and 4.19, correlated in the HSQC spectrum to the carbons at δ 22.03, 68.57 and 43.01, respectively, and identified as the protons H-12, H-13 and H-14, as well as the protons H-15/H-19 and H-16/H-18 at δ 3.38 and 3.43, correlated in the HSQC spectrum to the carbons at δ 63.66 and 43.74, respectively.After treatment with PAA (pollutant:PAA 1:1) and a reaction times of 1 to 4 h, all five considered pollutants were recovered with percentages greater than 90%, and 75% in the case of trazodone only. The percentages were even higher for short reaction times (less than 1 h), and in any case significant if we compare them to the treatment with sodium hypochlorite which was no more than 37% in the case of caffeine, and only 15% in the case of irbesartan. Unfortunately, there are no data in the literature on the degradation of trazodone shown in Of course, the mineralization percentage, appraised as percentage by weight, was very low and varied between 10% for caffeine and diclofenac, and 3% for tramadol and irbesartan, with a peak of 23% in the case of trazodone. The corresponding percentage in the case of treatment with hypochlorite was much higher, between 40% and 75%, as shown in DP1 and DP2 had already been isolated in the treatment with sodium hypochlorite, whereas DP3 and DP4 were isolated for the first time, and it is easy to hypothesize that they derive from an electrophilic substitution on the aromatic ring, to the ortho and para positions of the methoxy group. In particular, five other degradation byproducts were obtained with sodium hypochlorite and were oxidized on the side chain or on the aliphatic ring -1-(3-methoxyphenyl)cyclohexyl hypochlorite (DP1). White powder. NMR spectra conform to those recorded for the available standard +; found 280.21 [M + H]+, 261.11 [M − H2O]+.1--2-((dimethylamino)methyl)-1-hydroxycyclohexyl)-2-methoxyphenyl)ethanone (DP3). White powder. 1H-NMR : δ 1.25 , 1.40 , 1.63 , 1.68 , 1.90 , 1.96 , 2.00 , 2.13 , 2.15 , 2.29 , 2.37 , 3.97 , 6.95 , 7.21 , 7.36 . 13C-NMR : δ 21.37 (C-5), 22.62 (C-3), 24.85 (C-4), 30.85 (C-8’), 40.91 (C-6), 41.34 (C-2), 42.23 (C-9/C-10), 56.32 (OCH3), 60.50 (C-7), 75.01 (C-1), 109.21 (C-2’), 117.34 (C-6’), 130.22 (C-4’), 130.22 (C-5’), 146.88 (C-1’), 155.24 (C-3’), 202.26 (C-7’). ESI-MS (positive ions): m/z calculated for C18H27NO3m/z 305.20 [M]+; found 305.23 [M]+ and 262.23 [M − CH3CO]+.1--2-((dimethylamino)methyl)-1-hydroxycyclohexyl)-4-methoxyphenyl)ethanone (DP4). White powder. 1H-NMR : δ 1.40 , 1.41 , 1.60 , 1.63 , 1.70 , 1.92 , 2.05 , 2.11 , 2.19 , 2.21 , 2.45 , 3.84 , 6.77 , 7.26 , 7.57 . 13C-NMR : δ 21.04 (C-5), 21.28 (C-3), 27.09 (C-4), 30.92 (C-8’), 41.35 (C-6), 41.35 (C-9/C-10), 46.02 (C-2), 53.41 (C-7), 55.60 (OCH3), 75.90 (C-1), 113.98 (C-4’), 114.69 (C-2’), 120.80 (C-6’), 132.93 (C-5’), 143.39 (C-1’), 158.85 (C-3’), 206.96 (C-7’). ESI-MS (positive ions): m/z calculated for C18H27NO3m/z 305.20 [M]+; found 305.21 [M]+ and 262.25 [M − CH3CO]+.N-((2’-(1H-tetrazol-5-yl)--4-yl)methyl)-1-amino-N-pentanoylcyclopentanecarboxamide (DP5). White powder. 1H-NMR : δ 0.84 , 1.29 , 1.50 , 1.65 , 2.05 , 2.20 , 2.41 , 4.31 , 6.96 , 7.15 , 7.43 , 7.50 , 7.58 , 7.85 . 13C-NMR : δ 17.56 (C-32), 26.31 (C-31), 28.34 (C-1/C-2), 30.40 (C-30), 37.38 (C-29), 39.32 (C-3/C-5), 47.06 (C-11), 79.31 (C-4), 126.65 (C-23), 131.42 (C-13/C-17), 131.76 (C-19), 132.99 (C-14/C-16), 134.52 (C-22), 134.62 (C-21), 135.19 (C-20), 141.88 (C-18), 142.16 (C-12), 145.44 (C-15), 159.06 (C-24), 178.59 (C-9), 180.55 (C-7). MS-TOF (positive ions): m/z calculated for C25H30N6O2m/z 446.24 [M]+; found 447.26 [M + H]+ (68%). N-((2’-(1H-tetrazol-5-yl)--4-yl)methyl)pentanamide (DP6). White powder. 1H-NMR : δ 0.88 , 1.34 , 1.53 , 2.06 , 4.54 , 7.09 , 7.15 , 7.45 , 7.53 , 7.60 , 7.88 . 13C-NMR : δ 14.21 (C-32), 21.06 (C-31), 26.42 (C-30), 35.74 (C-29), 67.13 (C-11), 124.05 (C-23), 127.48 (C-13/C-17), 128.28 (C-21), 130.18 (C-19), 130.30 (C-14/C-16), 130.76 (C-22), 131.03 (C-20), 133.34 (C-12), 140.41 (C-18), 140.68 (C-15), 156.72 (C-24), 171.19 (C-7). MS-TOF (positive ions): m/z calculated for C19H21N5O m/z 335.40 [M]+; found 336.36 [M + H]+ (83%).4-(3-chlorophenyl)-1--yl)propyl)piperazine 1-oxide (DP7). White powder. 1H-NMR : δ 2.57 , 3.38 , 3.43 , 3.44 , 4.19 , 6.51 , 6.79 , 6.87 , 6.89 , 7.08 , 7.10 , 7.18 , 7.75 . 13C-NMR : δ 22.03 (C-12), 43.01 (C-11), 43.74 (C-16/18), 63.66 (C-15/19), 68.57 (C-13), 110.80 (C-3), 114.49 (C-25), 115.37 (C-5), 116.58 (C-21), 120.57 (C-23), 123.71 (C-2), 130.21 (C-4), 130.30 (C-24), 135.12 (C-22), 148.65 (C-9), 150.90 (C-20). MS-TOF (positive ions): m/z calculated for C19H22ClN5O2m/z 387.15 [M]+; found 388.16 [M + H]+ (100%).This paper investigated the fate of five emerging pollutants, that is, caffeine, tramadol, irbesartan, diclofenac and trazodone, after treatment with peracetic acid. For all five pollutants, high percentages of the unchanged product were recovered (from 75% to over 90%), even after very long reaction times (from 2 to 4 h), with consequently very low percentages of mineralization (less than 10%) and few degradation byproducts. The best results were obtained with reaction times of 2 h and with a concentration of peracetic acid between 10 and 100 times higher than those of the pollutant to be degraded. A comparison with similar studies on the same drugs, such as the treatment with sodium hypochlorite, indicates that a far higher number of degradation byproducts was obtained, but also higher mineralization percentages (minimum of 40% but with peaks up to more 70%) and consequently minimum recoveries of the products treated (from 10% to 30%). It is therefore necessary to consider that the replacement of sodium hypochlorite  with peracetic acid may not be beneficial for obtaining the oxidation of the organic material present in wastewater. Additionally, peracetic acid costs almost four times as much as sodium hypochlorite.


Their structures were elucidated by extensive spectroscopic analysis. Their absolute configurations were determined using the modified Mosher’s method and through comparisons of experimental and calculated electronic circular dichroism data. In addition, muyocopronone B (2) was found to exhibit a weak antibacterial activity against some Gram-positive bacteria.Two new azaphilones, namely muyocopronones A ( Monascus, Penicillium, and Aspergillus spp.) and Sordariomycetes   and five sp2 methine groups . The 13C NMR and DEPT135 spectra  and three conjugated carbonyl carbons at δC 193.0, 193.3 , and 194.4 (C-15). The presence of a 2,4-dimethyl-3-hydroxyhexanoate moiety was indicated by the DQF-COSY sequences of H-3′/H-2′/H3-7′ and H-5′/H3-6′, and the HMBC correlations of H3-6′/C-4′, C-5′, H3-7′/C-1′, C-2′, C-3′, and H3-8′/C-3′, C-4′, C-5′ , a singlet corresponding to the methyl group (H3-9), and six non-protonated carbon atoms  including two conjugated carbonyl carbon atoms (C-6 and C-8), closely resembled the chemical shifts of the azaphilone skeleton observed in S-15183a and S-15183b . Af. AfS andeoisomer . Thus, t2) was also obtained as yellow amorphous solid, and its ESIMS was consistent with the molecular formula C25H32O7, thereby suggesting the addition of two hydrogen atoms relative to 1. A comparison of the 13C NMR and DEPT135 data of compound 2 with that of 1 indicated that two methylene groups  and 41.0 (C-14)) were present in 2 instead of the vicinal-coupled olefinic protons observed in the case of 1  configuration, which was confirmed using the modified Mosher’s method, whereby similar results were obtained as for compound 1 -stereoisomer was in good agreement with the experimental spectrum of 1  stereoisomer.Muyocopronone B (ase of 1 . Thereforum of 1 . Therefo1) and B (2) were screened for their antibacterial activity against five strains of Gram-positive bacteria, namely Staphylococcus aureus (PAGU 273T), methicillin-resistant S. aureus , S. epidermidis (PAGU 283T), Enterococcus faecalis (PAGU 102T), and vancomycin-resistant E. faecalis (PAGU 100). Although muyocopronone B (2) exhibited an antibacterial activity against both antibiotic-resistant and antibiotic-susceptible strains, with minimum inhibitory concentration (MIC) values of ≈128 μg/mL  were reported as intercellular adhesion molecule-1 (ICAM1) expression inhibitors in a patent literature + calcd for C25H31O7, 443.2064; found, 443.2073.Yellow amorphous solid; [α]D22 +73 ; UV (MeOH) λmax (log ε) 218 (4.14), 331 (4.28) nm; ECD  λext (Δε) 219 (−2.80), 253 (+2.10), 275 (−1.99), 351 (+4.19) nm; IR (KBr) νmax: 3480, 2963, 2936, 2876, 1717, 1669, 1634, 1551, 1456, 1373, 1332, 1267, 1231, 1184, 1123, 1088, 970, 914, 878, 735 cm−1; 1H and 13C NMR data, see m/z): [M + H]+ calcd for C25H33O7, 445.2221; found, 445.2228.Yellow amorphous solid; [α]1 , DMAP , and triethylamine  in CH2Cl2 (1.2 mL) was added (R)-MTPACl . After allowing to stand in the dark at 25 °C for 6 h, the resulting solution was directly subjected to silica gel CC with n-hexane/ethyl acetate (1:1) to obtain (S)-MTPA diester 1a (10.5 mg). Similarly, a mixture of 1 , DMAP , and triethylamine  in CH2Cl2 (1.0 mL) was treated with (S)-MTPACl  in the same manner to obtain (R)-MTPA diester 1b (3.5 mg).To a solution of 1H NMR δH 0.92 , 0.95 , 1.16 , 1.21 , 1.25 , 1.45 , 1.49 , 1.87 , 2.26 , 2.66 , 2.85 , 3.08 , 3.49 and 3.58 , 5.28 , 5.41 , 5.85 , 6.16 , 6.34 , 7.27 , 7.31–7.44 .Yellow gum; 1H NMR δH 0.76 , 0.89 , 1.07 , 1.16 , 1.28 , 1.36 , 1.49 , 1.81 , 2.26 , 2.68 , 2.85 , 3.08 , 3.59 and 3.62 , 5.28 , 5.40 , 5.85 , 6.16 , 6.31 , 7.28 , 7.33–7.62 .Yellow gum; Calculation of the ECD spectra were performed using CONFLEX 8, Gaussian 16, and SpecDis software as described previously –14. GeomStaphylococcus aureus (PAGU 273T), methicillin-resistant S. aureus , Staphylococcus epidermidis (PAGU 283T), Enterococcus faecalis (PAGU 102T), and vancomycin-resistant E. faecalis . Piperacillin, vancomycin, and amikacin were used as positive controls. The isolated substances and the antibiotics were serially diluted (over a concentration range of 1–1024 μg/mL) into the wells of a 96-well plate. Following addition of the bacterial inoculum, the microtiter plate was incubated at 37 °C for 18 h. The microorganisms were cultured in CAMHB (cation-adjusted Mueller-Hinton broth) medium at a concentration of 1 × 106 CFU/mL. The recorded MIC was the lowest concentration at which no growth was observed.To evaluate the antibacterial activities of the isolated substances, five strains of Gram-positive bacteria were used, namely File 12a and 2b, phylograms of ECN-279 and related species, and copies of 1D and 2D NMR data for 1 and 2.Preparations of MTPA diesters


Increasing concentration (10−6 M) reduces the biological activities of analogs as compared to brassinolide.The metabolism of brassinosteroid leads to structural modifications in the ring skeleton or the side alkyl chain. The esterification and glycosylation at C-3 are the most common metabolic pathways, and it has been suggested that conjugate brassinosteroids are less active or inactive. In this way, plants regulate the content of active brassinosteroids. In this work, the synthesis of brassinosteroid 24-norcholane type analogs conjugated at C-3 with benzoate groups, carrying electron donor and electron attractant substituents on the aromatic ring, is described. Additionally, their growth-promoting activities were evaluated using the Rice Lamina Inclination Test (RLIT) and compared with that exhibited by brassinolide (used as positive control) and non-conjugated analogs. The results indicate that at the lowest tested concentrations (10 Brassinosteroids (BRs) are an important group of polyhydroxylated sterol plant growth regulators in multiple developmental processes, at nanomolar to micromolar concentration, including cell division, cell elongation, vascular differentiation, reproductive development, and modulation of gene expression . BRs als1) (Since the discovery of brassinolide (1)  6], 70 , 70 1)  .,46.27 wa28 were carried out according to the methodology reported for other steroidal nuclei   were added; then, the mixture was homogenized by magnetic stirring for 10 min. Later, 100 μL of OsO4 solution  were added, and the mixture reaction was stirred at room temperature for 5 h. The end of the reaction was verified by TLC; then, H2O (10 mL) and a saturated solution of Na2S2O3.5H2O (2 mL) were added. The mixture was stirred for another 20 min. Later, it was extracted with EtOAc (2 × 35 mL) and washed with water (2 × 35 mL), and both organic phases were combined, dried over MgSO4, and filtered. The solvent was evaporated under reduced pressure. The crude was re-dissolved in CH2Cl2 (1.0 mL) and chromatographed on silica gel with EtOAc/cyclohexane (16:4) mixture. A mixture of 18a/18b = 1.0/1.0 was obtained . Separation by HPLC of an analytical sample allowed the separation and obtaining of the pure compounds 18a and 18b.To a mixture of 18a was obtained as a colorless solid, m.p. = 74.1 ± 4.6 °C. 1H-NMR  ; 7.24 ; 5.36–5.34 ; 3.80 ; 3.66–3.61 ; 3.52 ; 2.66 ; 2.41 ; 2.32 ; 0.921 ; 0.783 ; 0.681 . 13C-NMR  (2-Ar); 143.65 (C4′-Ar); 129.61 ; 129.20 ; 128.15 (C1′-Ar); 74.14 (C-22); 69.36 (C-3); 66.16 (C-23); 56.71 (C-14); 53.96 (C-17); 53.00 (C-9); 52.47 (C-5); 46.79 (C-7); 42.94 (C-13); 41.46 (C-10); 39.53 (C-20); 38.09 (C-12); 37.97 (C-8); 32.78 (C-1); 27.72 (C-16); 25.55 (C-2); 25.24 (C-4); 23.95 (C-15); 21.78 (CH3-Ar); 21.22 (C-11); 12.73 (C-21); 12.55 (C-19); 12.00 (C-18). HRMS (API+) (31H45O5 ([M + H]+) 497.3267, found 497.3262.Compound , CDCl3) : δ (ppm), CDCl3) : δ (ppm)S (API+) : calcula18b was obtained as a colorless solid, m.p. = 83.0 ± 5.0 °C. 1H-NMR  ; 7.23 ; 5.36–5.34 ; 3.83–3.76 ; 3.69–3.57 ; 3.51 ; 2.65 ; 2.41 ; 2.32 ; 0.953 ; 0.778 ; 0.679 . 13C-NMR  (2-Ar); 143.65 (C4′-Ar); 129.60 (C2′-Ar and C6′-Ar); 129.18 (C3′-Ar and C5′-Ar); 128.16 (C1′-Ar); 73.92 (C-22); 69.36 (C-3); 62.54 (C-23); 56.42 (C-14); 54.06 (C-17); 53.02 (C-9); 52.92 (C-5); 46.79 (C-7); 43.48 (C-13); 41.43 (C-10); 40.15 (C-20); 39.47 (C-12); 38.04 (C-8); 32.81 (C-1); 27.47 (C-16); 25.55 (C-2); 25.23 (C-4); 24.12 (C-15); 21.78 (CH3-Ar); 21.21 (C-11); 13.15 (C-21); 12.55 (C-19); 11.85 (C-18). HRMS (API+) (31H45O5 ([M + H]+) 497.3267, found 497.3265.Compound , CDCl3) : δ (ppm), CDCl3) : δ (ppm)S (API+) : calculat-butanol/water , alkene 29 , DHQD-CLB , CH3SO2NH2 , K2CO3 , and K3[Fe(CN)6]  were added; then, the mixture was homogenized by magnetic stirring for 10 min. Later, 100 μL of OsO4 solution  were added, and the mixture reaction was stirred at room temperature for 5 h. The end of the reaction was verified by TLC; then, H2O (10 mL) and a saturated solution of Na2S2O3.5H2O (2 mL) were added. The mixture was stirred for another 20 min. Later, it was extracted with EtOAc (2 × 35 mL) and washed with water (2 × 35 mL), and both organic phases were combined, dried over MgSO4, and filtered. The solvent was evaporated under reduced pressure. The crude was re-dissolved in CH2Cl2 (1.0 mL) and chromatographed on silica gel with an EtOAc/cyclohexane (16:4) mixture. A mixture of 19a/19b = 1.0/1.0 was obtained (65 mg: 80.8% yield). The separation by HPLC of an analytical sample allowed the separation and obtaining of the pure compounds 19a and 19b.To a mixture of 19a was obtained as a colorless solid, m.p. = 56.9 ± 4.6 °C. 1H-NMR  ; 7.56–7.48 ; 7.21 ; 7.13 ; 5.40–5.39 ; 3.87–3.76 ; 3.75–3.39 ; 2.71 ; 2.31 ; 0.917 ; 0.776 ; 0.676 . 13C-NMR  ; 161.93 ; 134.49 ; 132.31 ; 124.13 ; 117.17 ; 116.99 ; 75.56 (C-22); 70.33 (C-3); 61.15 (C-23); 56.67 (C-14); 53.85 (C-17); 52.79 (C-9); 52.45 (C-5); 46.78 (C-7); 43.47 (C-13); 42.93 (C-10); 41.41 (C-20); 39.51 (C-12); 38.09 (C-8); 32.55 (C-1); 27.72 (C-16); 25.49 (C-2); 25.20 (C-4); 23.94 (C-15); 21.21 (C-11); 12.73 (C-21); 12.61 (C-19); 12.00 (C-18). 19F-NMR  δ (ppm) = −108,91 . HRMS (API+) (30H42O5F ([M + H]+) 501.3016, found 501.3015.Compound , CDCl3) : δ (ppm), CDCl3) : δ (ppm)S (API+) : calcula19b was obtained as a colorless solid, m.p. = 75.4 ± 1.5 °C. 1H-NMR  ; 7.54–7.49 ; 7.21 ; 7.13 ; 5.40–5.39 ; 3.84–3.76 ; 3.71–3.69 ; 3.51 ; 2.70 ; 2.31 ; 0.954 ; 0.777 ; 0.681 . 13C-NMR  ; 160.87 ; 134.48 ; 132.34 ; 124.13 ; 118.37 ; 117.04 ; 73.90 (C-22); 70.32 (C-3); 62.52 (C-23); 56.37 (C-14); 53.95 (C-17); 52.90 (C-9); 52.79 (C-5); 46.78 (C-7); 43.48 (C-13); 41.38 (C-10); 40.15 (C-20); 39.46 (C-12); 38.04 (C-8); 32.57 (C-1); 27.47 (C-16); 25.48 (C-2); 25.20 (C-4); 24.11 (C-15); 21.19 (C-11); 13.13 (C-21); 12.62 (C-19); 11.85 (C-18). 19F-NMR  δ (ppm) = -108,90 . HRMS (API+) (30H42O5F ([M + H]+) 501.3016, found 501.3014.Compound , CDCl3) : δ (ppm), CDCl3) : δ (ppm)S (API+) : calculaOryza sativa) provided by the Institute of Agricultural Research (INIA-Quilamapu-Chile) as previous studies.The biological activity of the growth of the compounds was evaluated by the rice lamina inclination test ,56, acco18a, 18b, 19a, 19b, 20a, and 20b and positive control (1)) needed to reach final concentrations equal to 1 × 10−8 M; 1 × 10−7 M; and 1 × 10−6 M. The negative control only contained sterile distilled water. All treatments were incubated by 48 h at 25 °C in darkness, and the angles developed between the blade and the sheath were measured. Each experiment was performed by duplicate.The seeds were sown and cultivated until the seedlings presenting the second internode of the rice blade were selected for cutting. Six segments per treatment were incubated in Petri dishes containing 60 mL of distilled water, and the amount of test compound  using twelve angle measurements. Statistical analysis was done using a statistical package Excel by applying mean values using one-way ANOVA with the post-hoc least square differences (LSD) test to determine if there was a significant difference between the positive control and the treatments. A S and R on the C-22 carbon of the side chain have been synthesized and characterized. The synthesis uses hyodeoxycholic acid as the starting material, and epimers with different configuration at C-22 are obtained. These epimers have been separated, and their growth-promoting activity was measured using RLIT. The results show that the esterification of BRs analog at C-3 has no effect on the biological activity of synthetic analogs. This suggest that reducing activity by esterification at C-3 requires a long chain carboxylic acid. In addition, the presence of a hydroxyl group at C-3 is not an essential structural feature for activity. This result confirms previous SAR where it has been proposed that activity is not determined by the presence or absence of specific groups in the BR structure.Brassinosteroid 24-norcholane type analogs conjugated at C-3 and configurations


Their structures were elucidated by extensive spectroscopic analyses and time dependent density functional theory (TDDFT), calculation of electronic circular dichroism (ECD) and optical rotation (OR) data. Compound 2 displayed weak inhibitory activity against Escherichia coli GIM1.201 (minimum inhibitory concentration (MIC) value of 0.35 mM). Compounds 4 and 5 displayed significant cytotoxicity against human cancer cell line HepG2 , more potent than the positive control 5-fluorouracil .Two new polyketides, cytospyrone ( Alternaria, Aspergillus, Cladosporium, Colletotrichum, Fusarium, Paecilomyces, Penicillium, Pestalotiopsis, Phoma, Phomopsis, Phyllosticta, and Trichoderma are considered as the predominant producer of the most notable bioactive secondary metabolites ..Cytospor1) was isolated as a white amorphous powder, and has the molecular formula C20H30O5 established by high resolution electrospray ionization mass spectroscopy (HR-ESI-MS) at m/z 373.1988 [M + Na]+ , indicating six degrees of unsaturation. The 1H and 13C nuclear magnetic resonance (NMR) data of 1 in association with distortionless enhancement by polarization transfer (DEPT) and heteronuclear single quantum coherence (HSQC) spectrum suggested the presence of six upfield methyls , a methoxy group , three olefinic signals , four methines , and six quaternary carbons . Two substructures, an α-pyrone moiety and a linear aliphatic chain, were assigned by analyses of detailed interpretation of 1H-1H correlation spectroscopy (COSY) and heteronuclear multiple bond correlation (HMBC) correlations  with C-4 (δC 169.1) and nuclear overhauser effect spectroscopy (NOESY) correlations between 3-CH3/4-OCH3 and H-5/4-OCH3. A linear aliphatic chain with a thirteen carbon unit was determined by the interpretation of 1H–1H COSY and HMBC spectroscopic data. The COSY cross-peaks  and HMBC correlations from 2′-CH3 to carbons C-1′, C-3′, from 3′-CH3 to carbons C-2′, C-3′ and C-4′, from 7′-CH3 to carbons C-6′, C-7′and C-8′, and from H-9′ to C-7′ permitted the construction of a linear aliphatic chain. The above two structural fragments were finally connected to construct the planar structure of compound 1 from the key HMBC correlations from H-1′ and H-2′ to C-6, from H-5 to C-2′. The relative configuration of 1 was deduced by analysis of the NOESY data. The NOESY correlations -calculated electronic circular dichroism (ECD) spectrum at the B3LYP/6-31+g  level was compared with the experimental circular dichroism (CD) spectrum -1,8-dihydroxy-2,3,5,7-tetramethylnona-3,6-dienyl)-4-methoxy-3-methyl-2H-pyran-2-one.Cytospyrone A (elations . The α-pelations  between spectrum . Therefo2), a yellow amorphous solid, was found to have the molecular formula C13H14O6, established by HR-ESI-MS , implying seven degrees of unsaturation. The 1H and 13C NMR data of 2 indicated that six of the seven units of unsaturation could be due to an aromatic ring, a double bond, and one carbonyl group. Thus, the remaining unit of unsaturation was attributed to a ring formation. The ultraviolet (UV) absorption maxima at 247 and 318 nm suggested 2 is an isocoumarin derivative. The 1H and 13C NMR data of 2 and the DEPT and HSQC spectra revealed the presence of two methyl groups, including a methoxy group  and a terminal alkyl methyl , a methylene  and seven quaternary carbons, including a carbonyl . The 1H and 13C NMR and HSQC spectra of compound 2 closely resembled those of an isocoumarin compound peniisocoumarin G 20D = +89.6°(MeOH)) of 2 using TDDFT (2 was determined as (S)-6,8-dihydroxy-3-(2-hydroxypropyl)-5-methoxy-1H-isochromen-1-one and named as cytospomarin.Cytospomarin , Pseudomonas aeruginosa GIM1.200, Candida albicans GIM2.169, together with three plant pathogens including Bacillus subtilis, Colletotrichum gloeosporioides and Magnaporthe oryze, and the results are summarized in 1−5 were also tested in vitro against the human cancer cell line HepG2 with 5-fluorouracil as the positive control. Among them, compound 2 exhibited weak inhibitory activity against E. coli GIM1.201 (minimum inhibitory concentration (MIC) = 0.35 mM) and M. oryzae (MIC = 1.41 mM). Compounds 4 and 5 displayed significant cytotoxicity against human cancer cell line HepG2 with IC50 values of 5.98 ±  0.12 μM and 9.97 ± 0.06 μM, respectively, more potent than the positive control 5-fluorouracil (IC50 = 43.50 ± 3.69 μM) (Compounds 3.69 μM) .1), cytospomarin (2), together with three known metabolites (3–5) were isolated from the rice culture of Cytospora sp. from Chinese mangrove Ceriops tagal. Structurally, 1 and pestalotiopyrones A–H 20D = −23.1° ; UV(MeOH) λmax 212 nm; 1H NMR δH 6.47 , 5.32 , 5.29 , 4.10 , 4.08 , 3.94 , 3.33–3.42 , 2.79 , 1.85 , 1.67 , 1.65 , 1.18 , 1.07 , 1.03 ; 13C-NMR  δC 169.1 (C-4), 168.5 (C-2), 167.8 (C-6), 138.1 (C-7′), 135.3 (C-4′), 134.2 (C-3′), 130.1 (C-6′), 101.5 (C-3), 97.5 (C-5), 80.7 (C-1′), 73.9 (C-8′), 57.3 (4-OCH3), 44.1 (C-2′), 32.3 (C-5′), 21.9 (C-9′), 21.7 (5′-CH3), 16.2 (3′-CH3), 11.8 (7′-CH3), 10.8 (3′-CH3), 8.4 (3-CH3); HR-ESI-MS m/z 373.1991 [M + Na]+  (Cytospyrone (73.1991) .2): a yellow amorphous solid (MeOH); [α]20D = +11.7° ; UV(MeOH)λmax 318 nm, 247 nm; 1H NMR  δH 10.8 , 6.53 , 6.33 , 3.92–4.00  3.68 , 2.50–2.57 , 1.12 ; 13C-NMR  δC 165.6 (C-1), 161.1 (C-8), 158.6 (C-6), 154.7 (C-3), 134.3 (C-5), 130.3 (C-5a), 102.6 (C-7), 99.9 (C-4), 95.5 (C-8a), 64.0 (C-10), 60.4 (5-OCH3), 42.8 (C-9), 23.3 (C-11); HRESIMS m/z 289.0674 [M + Na]+  (Cytospomarin (89.0610) .3): white amorphous powder (MeOH); [α]20D = +123° , UV(MeOH)λmax 216 nm, 1H NMR δH 6.55 , 5.40 , 4.05 , 3.92 , 2.14 , 1.59 ; 13C-NMR  δC 168.3 (C-1), 164.2 (C-5), 157.6 (C-7), 152.9 (C-3a), 120.5 (C-6), 109.7 (C-7a), 97.9 (C-4), 76.4 (C-3), 62.1 (7-OCH3), 56.1 (5-OCH3), 29.7 (C-9), 20.7 (C-8); HRESIMS m/z 233.0969 [M + H]+ . The data of 1H NMR and 13C NMR are basically consistent with those reported in literature 20D = +97.0° ; UV (MeOH) max 280,259,218 nm; 1H NMR 6.23 , 6.19 , 6.14 , 6.10 , 5.28 , 4.82 , 2.81–2.85 , 2.45 , 2.03 , 1.66–1.76 , 1.54–1.57 , 1.40–1.42 , 1.31–1.35 , 0.99 , 0.94 ; 13C-NMR  δ 173.1 (C-1′), 172.7 (C-1′′),165.9 (C-3′), 163.5 (C-5′), 159.2, 149.0 (C-7′), 146.3 (C-6), 111.9 (CH-6′), 107.9 , 105.8 (C-2′), 101.9 (CH-4′), 101.0 (CH-3), 76.7 (CH-14), 75.5 (CH-15′), 37.7 a (CH2-8′), 37.4 a (CH2-15), 37.4 a (CH2-16′), 36.9 (CH2-7), 35.2 (CH2-13), 35.2 (CH2-14′), 33.6 (CH2-9′), 32.3 (CH2-8), 31.0 a (CH2-10′), 30.7 a (CH2-11′), 30.5 a (CH2-12′), 30.5 a (CH2-11), 30.4 a (CH2-10), 30.2 (CH2-9), 26.6 (CH2-13′), 26.4 (CH2-12), 21.2 (CH3-2′′), 19.9 (CH2-16), 19.6 (CH2-17′), 14.3 (CH3-18′), 14.2 (CH3-17), chemical shifts could be interchanged; ESI-MS [M + H]+ m/z 629.4 (C37H57O8). The data of 1H NMR and 13C NMR are basically consistent with those reported in literature 20D = +37.8° ; UV (MeOH) λmax 300, 264, 230 nm; 1H NMR δ 11.98 , 7.61 , 6.21 , 6.19 , 6.14 , 6.10 , 5.20 , 3.48–3.52 , 2.80 , 2.40 , 1.63–1.67 , 1.59–1.63 , 1.48–1.55 , 1.36–1.39 , 1.34–1.36 , 1.28–1.30 , 1.23–1.28 , 0.91 , 0.88 ; 13C-NMR  δ 172.3 (C-1′), 165.5 (C-3′), 162.8 (C-5′), 158.4, 149.1 (C-7′), 146.1 (C-6), 111.8 (CH-6′), 107.8 , 105.1(C-2′), 101.6 (CH-4′), 100.6 (CH-3), 76.3 (CH-14), 71.9 (CH-15′), 40.0 (CH2-16′), 37.9 (CH2-8′), 37.6 (CH2-15), 37.2 (CH2-7), 36.6 (CH2-13), 35.0 (CH2-14′), 33.1 (CH2-9′), 31.9 (CH2-8), 30.7 a (CH2-10′), 30.5 a (CH2-11′), 30.4 a (CH2-12′), 30.1 a (CH2-11), 30.0 a (CH2-10), 29.9 (CH2-9), 26.4 (CH2-13′), 26.2 (CH2-12), 19.5 (CH2-16), 19.4 (CH2-17′), 14.4 (CH3-18′), 14.3 (CH3-17), a chemical shifts could be interchanged; ESIMS [M + H]+ m/z 587.4 (C35H55O7). The data of 1H NMR and 13C NMR are basically consistent with those reported in literature [Integracin B  and GraphPad Prism 5  from dipole-length rotational strengths by applying Gaussian band shapes with sigma = 0.3 eV.Monte Carlo conformational searches were carried out by means of Spartan 14 software using Merck molecular force field (MMFF). The conformers with Boltzmann population of over 1% were chosen for ECD calculations, and then the conformers were initially optimized at B3LYP/6-31g level in gas. The theoretical calculation of ECD was conducted in MeOH using time dependent density functional theory (TDDFT) at the B3LYP/6-31+g  level for all conformers of compounds 2.Monte Carlo conformational searches were carried out by means of Spartan 10 software using Merck molecular force field (MMFF). The conformers with Boltzmann population of over 1% were chosen for OR calculations, and then the conformers were initially optimized at B3LYP/6-31+g  level in MeOH using the conductor-like polarizable continuum model (CPCM) polarizable conductor calculation model. The theoretical calculation of OR was conducted in MeOH using time dependent density functional theory (TDDFT) at the B3LYP/6-31+g  level for all conformers of compound Escherichia coli GIM1.201, methicillin-resistant Staphylococcus aureus GIM1.771 (MRSA), Pseudomonas aeruginosa GIM1.200, Candida albicans GIM2.169 and plant pathogenic fungi Bacillus subtilis, Colletotrichum gloeosporioides and Magnaporthe oryze . The bacteria was cultured in Luria-Bertani (LB) medium  at 37 °C (160 rpm) for 24 h, and the fungi was cultured in potato dextrose broth (PDB) medium  at 28 °C (160 rpm) for 48 h. All isolated compounds were subjected to antimicrobial assays using a serial dilution technique, as previously described [The antimicrobial activity was determined against human pathogens escribed . The minThe human hepatoma cell (HepG2) cells  were grown in RPMI-1640 culture medium. Cytotoxicity against HepG2 cells was evaluated using the 3--2,5-diphenyltetrazolium bromide (MTT)  method, as described previously  and 5-Fl1), cytospomarin (2) and three known metabolites (3−5) were obtained from the ethyl acetate extract of the fermented cultures of the fungus Cytospora sp., which was isolated from the Chinese mangrove Ceriops tagal. Compound 2 exhibited weak inhibitory activity against Escherichiacoli GIM1.201 and compounds 4 and 5 displayed potent cytotoxicity against human cancer cell line HepG2.In the present work, two new polyketides cytospyrone (


Interestingly, at 70 °C, 10 bar of CO2 pressure and 1 mol % of loading, the dinuclear thioaryloxide [Zn(bpzaepe)2{Zn(SAr)2}] (4) behaves as an efficient and selective one-component initiator for the synthesis of poly(cyclohexene carbonate) via ring-opening copolymerization of cyclohexene oxide (CHO) and CO2, affording polycarbonate materials with narrow dispersity values.New mono- and dinuclear chiral alkoxide/thioalkoxide NNO-scorpinate zinc complexes were easily synthesized in very high yields, and characterized by spectroscopic methods. X-ray diffraction analysis unambiguously confirmed the different nuclearity of the new complexes as well as the variety of coordination modes of the scorpionate ligands. Scorpionate zinc complexes  Yield: . Anal. Calcd. For C60H78N10O4Zn2: C, 63.54; H, 6.93; N, 12.35. Found: C, 63.59; H, 6.98; N, 12.43. 1H NMR , δ 7.17 , 7.04 , 6.75 , 6.59 , 5.51 , 5.43 , 5.42, 5.18 , 2.95 , 2.63, 2.30 2-OAr), 2.16 , 1.97 , 1.72 , 1.12 , 0.89 . 13C {1H} NMR , δ 158.1 (ipso-C-OAr), 156.5-153.9 , 132.0 (N-Pho), 128.4 (o-C-OAr), 127.9 (m-C-OAr), 124.2 (p-C-OAr), 111.5 (N-Phm), 102.3 (C4′), 101.1 (C4), 76.9 (Ca), 71.4 (Cb), 39.9 (Me2-OAr), 39.1 (N-CH2CH3), 13.4, 13.3 , 12.1 (N-CH2CH3), 10.2, 9.8 .The synthesis of 2{Zn2}] 3.Synthesis of   and 2,6-dimethylthiophenol  to give 3 as a pale yellow solid. Yield: . Anal. Calcd. For C56H70N10O2S2Zn2: C, 60.59; H, 6.36; N, 12.62. Found: C, 60.63; H, 6.40; N, 12.55. 1H NMR : δ 7.78 , 6.95 , 6.81 , 6.81 , 6.21 , 6.10  5.78, 5.26 , 2.62 2), 2.58 2-SAr), 2.33 , 1.81 , 1.63 , 1.49 . 13C {1H} NMR , δ 158.3 (ipso-C-SAr), 142.5-138.0 , 128.9 (N-Pho), 128.6 (o-C-SAr), 128.3 (m-C-SAr), 127.5 (p-C-SAr), 111.8 (N-Phm), 106.6 (C4′), 106.5 (C4), 77.6 (Ca), 70.0 (Cb), 24.5 (Me2-SAr), 19.8 (N-CH3), 13.8, 13.7 , 10.2, 10.1 .The synthesis of 2{Zn2}] 4.Synthesis of   and 2,6-dimethylthiophenol , to give 4 as a pale yellow solid. This complex was crystallized in 20 mL of THF and crystals sustainable for X ray diffraction analysis were obtained. Yield: . Anal. Calcd. For C60H78N10O2S2Zn2: C, 61.79; H, 6.74; N, 12.01. Found: C, 61.89; H, 6.63; N, 12.00. 1H NMR : δ 7.77 , 6.98 , 6.82 , 6.81 , 6.21 , 6.11  5.79, 5.25 , 3.10 , 2.58 2-SAr), 2.15, 1.82 , 1.63 , 1.50 , 1.00 . 13C {1H} NMR , δ 159.1 (ipso-C-SAr), 142.2-138.1 , 129.3 (N-Pho), 128.6 (o-C-SAr), 128.3 (m-C-SAr), 126.3 (p-C-SAr), 111.9 (N-Phm), 107.3 (C4′), 107.2 (C4), 78.0 (Ca), 70.1 (Cb), 44.0 (N-CH2CH3), 24.6 (Me2-SAr), 13.7, 13.6 , 12.5 (N-CH2CH3), 10.2, 10.1 .The synthesis of 2C6H3S)2(Hbpzampe)] 5.Synthesis of  6.Synthesis of  ethoxide} or bpzaepe = {2,2-bis-1-[4-(diethylamino)phenyl]ethoxide}], with several aromatic alcohols or thioalcohols, and new complexes that contain aryloxide or thioaryloxide ligands were isolated after methane elimination. Thus, the alcoholysis or thioalcoholysis reaction of these monoalkyl complexes with ArEH , in a 1:1 molar ratio, yields the chiral dinuclear zinc complexes [Zn(OAr)(κ2-NNμ-O)]21–2  and [Zn(κ2-NN-μ-O)2{Zn(SAr)2}] 3–4 , whereas when this reaction was carried out in a 1:2 molar ratio of thioalcohol, the mononuclear compounds [Zn(SAr)2(κ2-NN-OH)] 5–6  were obtained (κ3-NNO)]  2 -1-para-tolylethoxide], previously reported  for Zn(1)–N(1) and Zn(1)–N(3) in complex 5, are in good agreement with other values determined for zinc scorpionate complexes such as [Zn(CH3)(bpzbe)] [3)(pbptamd)] [5 are similar to those found for complex 4.Complexes (bpzbe)]  or [Zn(Cbptamd)]  prepared2 and 4, and the mononuclear 6 were initially assessed for the conversion of cyclohexene oxide (CHO) into poly at 80 °C and 40 bar of carbon dioxide pressure in the absence of a co-catalyst under solvent-free conditions for 16 h, using 1 mol% of complexes 2, 4 and 6  or polyether–polycarbonate 9  7, in conjunction with trans-cyclohexene carbonate 8, as a result of backbiting reactions , the catalyst loading was finally optimized to 1 mol %   , entry 17, but led to an important decrease in selectivity , which led to the maintenance of selectivity at a PCHC/CHC ratio of 92/8. Conversely, the increasing of the reaction temperature up to 100 °C did not significantly increase the conversion of CHO into 4 was observed when decreasing the reaction pressure from 40 to 1 bar , with the TOF 4.27 h−1. Not expectedly, increasing of pressure at 30 bar did not produce a significant growth on the conversion value, while a lack of PCHC/CHC selectivity was observed (83/17). It was also noteworthy that the carbonate linkage (>99%) remained essentially constant in the range of 1 to 40 bar , the conversion decreased from 72% to 17% (Đ) values ranging from 1.33–1.05  and syndiotactic (153.7–153.0 ppm) diads were identified  in all polymer chains (see ♦), another series bearing one thioaryloxide group and one hydroxyl end-group (●) and a third series containing one cyclohexenyl group and one hydroxyl end-group (♦), which suggest that chain transfer reactions take place due to the existence of either adventitious water and/or cyclohexenediol (produced by the hydrolysis of the CHO) through the polymerization event. This behaviour has also been previously observed  (4) that behaves as an effective and selective initiator for poly(cyclohexene carbonate) production under milder conditions. Thus, catalyst 4 shows high catalytic activity (72% conversion and TOF up to 4.3 h−1), carbonate linkage (>99%) and polycarbonate selectivity (95%), at 70 °C, 10 bar of CO2 pressure after 16 h, under solvent-free conditions, which constitutes a further step forward in the development of inexpensive, more efficient and non-toxic metal-based catalysts for the CO2 fixation into the selective production of poly(cyclohexene carbonate) with narrow dispersities [These complexes can act as efficient catalysts for the synthesis of poly(cyclohexene carbonate) via ring-opening copolymerization of cyclohexene oxide and carbon dioxide in the absence of a co-catalyst. More interestingly, catalytic efficiency is highly dependent on catalyst nuclearity and substituents. Thus, this new approach has led to the development of an interesting dinuclear thioalkoxide zinc scorpionate [Zn(bpzaepe)ersities ,24,25,51


Trichopodus pectoralis isolated from Malaysia (introduced), Viet Nam (native) and Thailand (native). The mitogenome length range from 16,397 to 16,420 bp. The final partitioned nucleotide alignment consists of 14,002 bp and supports the monophyly of the genus Trichopodus (95% ultrafast bootstrap support) with T. trichopterus forming a sister group with the members of T. pectoralis.We sequenced and assembled three whole mitogenome sequences of the commercially important snakeskin gourami  Trichopodus pectoralis commercially used as ornamental and food fish, and it is the largest and most significant species among the trichogastrids. It is native to the central plain of Thailand, extending from Chao Phraya Basin to Cambodia and Vietnam, Laos . Additional nucleotide similarity search to verify the taxonomy of Trichopodus microlepis (NC_027238) was performed using BlastN  were partitioned and aligned based on gene and codon position using TranslatorX : Partition 1 = 12S and 16S rRNA; Partition2 = ATP6-1,NAD3-1,NAD4-1,COB-1,NAD4L-1,NAD1-1,NAD2-1,ATP8-1, NAD5-1,COX1-1,COX2-1,COX3-1,NAD6-1; Partition3 = ATP6-2, NAD4-2,NAD6-2,NAD2-2,ATP8-2,NAD5-2,COB-2,NAD4L-2,NAD1-2, NAD3-2,COX2-2,COX1-2,COX3-2; Partition 4 = ATP6-3,NAD1-3,NAD3-3,NAD5-3,NAD4-3,COX3-3,NAD4L-3,COB-3,ATP8-3,COX2-3, NAD2-3,COX1-3 and Partition 5 = NAD6-3.Trichopodus (95% ultrafast bootstrap support) with Trichopodus trichopterus forming a sister group with members of Trichopodus pectoralis reported in this study , suggesting potential misidentification and the need to sequence the whole mitogenome of a correctly identified Trichopodus microlepis specimen.The final partitioned nucleotide alignment consists of 14,002 bp length and supports the monophyly of the genus is study . It is a


The present study was designed to synthesize different aromatic hydrazones assembled by various combinations of aromatic hydrazides and aromatic benzaldehydes containing different substituents such as hydroxyl and polyhydroxyl groups as key structural features. Thus, incorporating such moieties and simultaneously creating highly-conjugated systems was expected to create novel species to mimic as much as possible natural phenolics, chalcones and stilbenes. Compounds of aromatic hydrazones synthesized in the present study were tested in vitro for their direct and indirect antioxidant activities using different methods such as DPPH, ABTS and FTC. The antioxidant activities of the new compounds ranged from very weak to very high activity. In addition, the inhibition of tyrosinase and cholinesterase by these compounds was tested. The new compounds containing two or three hydroxyl groups attached to aldehyde rings exhibited significantly greater inhibition effects on tyrosinase or cholinesterase activities in comparison to other compounds of the same series containing only one hydroxyl group.There has been substantial interest over the past many years in the design of novel chemical compounds containing the azomethine group (-NH-N=CH) and exhibiting various medicinal properties such as antibacterial, antiviral, antifungal, and anti-inflammatory activities. Herein, hydrazones were synthesized via the chemical reaction of substituted aromatic hydrazides with various aromatic aldehydes. The obtained products were confirmed using different physical and spectroscopic techniques, such as m.p., IR,  Organic Chemistry, Aromatic hydrazones, Antioxidant activity, Cholinesterase inhibition, Tyrosinase inhibition, DPPH, ABTS, FTC. Consequently, the chemotherapeutic potential of this class of organic compounds provided the impetus that attracted many researchers involved in drug discovery and development to explore the synthesis and testing of their biological activity in the hope of finding some hydrazones with potent bioactivities. Aromatic hydrazones are compounds derived from phenolic acid hydrazides and aromatic aldehydes. Generally, these compounds contain the azomethine group (-NH-N=CH-) and are prepared by refluxing stoichiometric amounts of the aromatic hydrazide and aldehyde or ketone dissolved in a suitable solvent. Therefore, hydrazides and hydrazones are nowadays of considerable technical and commercial importance due to their wide utilization as drugs in medicine and as versatile ligands in coordination chemistry . It is nin vitro for their cytotoxicity and anticancer activity. These new reagents have been tested against different human tumor cell lines and have shown to be promising anti-cancer agents (Many organic compounds containing the azomethine group (-NH-N=CH-) have been synthesized and tested for their biological activities, proving very effective as antimicrobial  and antir agents .N-arylidene hydrazones has also been investigated in vitro as well as in inhibiting the activities of the proteolytic enzymes cathepsin Е and human neutrophil elastase which play important role in the development of autoimmune diseases , cardiovascular diseases, inflammation, among others. For instance, Reis recently reported novel hydrazone hybrid derivatives that exhibited antioxidant and photoprotective activities and proved useful for the prevention of skin cancer and use in sunscreen formulations . These pdiseases .Therefore, different types of organic compounds containing the azomethine moiety have been previously synthesized and tested for their ability to remove free radicals . The antDue to the considerable attention that has been given in recent years to the chemistry of hydrazones and polyphenols and the diverse biological and pharmaceutical applications encompassed by such a class, we were encouraged to delve further into this area of research. Therefore, the present study was designed to construct structurally-different hydrazones derived from the coupling of benzoic and phenolic acids hydrazides and aromatic aldehydes containing different substituents, most notably the hydroxyl groups. These compounds feature highly-conjugated systems enhanced by the presence of hydroxyl groups. Thus, it was expected that the new compounds would closely mimic natural chalcones and stilbenes, which are well known natural antioxidants. There is continuous need for new chemical entities, such as antioxidants, that can have desired biological attributes with minimal or no side effects, and compounds to treat and prevent diseases related to oxidative stress, such as, skin cancer, AD and others.22.12.1.1Benzoic acid, phenolic acids, aromatic aldehydes, hydrazine solution (78–82%), DPPH, ABTS, L-DOPA, and acetylthiocholine iodide were obtained from Sigma and used without any further purification. The cholinesterase enzyme was purchased from Sigma, while tyrosinase was prepared in our laboratory from mushroom. Methyl esters of benzoic and phenolic acids and also benzoic and phenolic acids hydrazides were prepared in our laboratory according to published procedures.2.1.21H-NMR, 13C-NMR, and 13C-DEPT 90 NMR, Bruker Avance II 400 MHz), and UV/VIS spectrophotometer . The obtained data was consistent with those reported in the literature for all known compounds.The chemical structures of the phenolic acids hydrazones were confirmed using various instruments and techniques, such as, melting point instrument , IR spectrophotometer , NMR spectrophotometer  was dissolved in ethanol and was treated with a solution of benzoic acid hydrazide (10 mmol) or phenolic acid hydrazide (10 mmol) in ethanol (25 mL), followed by three drops of glacial acetic acid. The mixture was stirred vigorously and refluxed using a water bath for three hours. The reaction mixture was then cooled down to ambient temperature and the resulting precipitate was filtered off by gravity filtration, dried and recrystallized from ethanol to give products .Image 1S2.2.21–21) are presented below and the corresponding spectra are shown in the supporting information section:The yields, m.p. and spectral data of compounds ((E)-N'-(2-hydroxybenzylidene)benzohydrazide (1) (Yield (%): 52%; m.p. °C: 175–177; IR: 3267 (N-H & OH), 3061 (=C-H), 1666 (C=O), 1645 (C=N), 1543-1483 (C=C Ar ring), 1360, 1263 (C-O); 1H-NMR : δ 8.50 , 7.90 , 7.54–7.48 , 7.47–7.40 , 7.27–7.18 , 6.90 , 7.84 ; 13C-NMR : δ 163.1 (C=O), 157.9 (C-O), 149.5 (N=CH), 132.6 (ArC-C=O), 131.4 (CH), 130.8 (CH), 130.1 (2xCH), 127.9 (2xCH), 127.3 (CH), 118.6 (CH), 117.6 (ArCCH=N), 116.3 (CH) ppm.zide (1) :- Yield (E)-N'-(3-hydroxybenzylidene)benzohydrazide (2) (Yield (%): 57%; m.p. °C: 209–211; IR: 3250 (N-H & OH), 3066 (=C-H), 1638 (C=O), 1610 (C=N), 1577-1533 (C=C Ar ring), 1281 (C-O); 1H-NMR : δ 11.50 , 9.09 , 8.30 , 7.86 , 7.50–7.42 , 7.41–7.45 , 7.26–7.17 , 7.16–7.05 , 6.79 ; 13C-NMR : δ 163.8 (C=O), 157.2 (C-O), 148.2 (N=CH), 135.1 (ArC-C=O), 133.3 (ArCCH=N), 131.1 (CH), 129.1 (CH), 127.9 (2xCH), 127.3 (2xCH), 118.6 (CH), 117.2 (CH), 113.2 (CH) ppm.zide (2) :- Yield (E)-N'-(4-hydroxybenzylidene)benzohydrazide (3) (Yield (%): 53%; m.p. °C: 242–244; IR: 3262 (N-H & OH), 3073 (=C-H), 1647 (C=O), 1600 (C=N), 1499 (C=C Ar ring), 1280 (C-O); 1H-NMR : δ 11.33 , 9.39 , 8.27 , 7.84 , 7.52 , 7.49–7.42 , 7.41–7.35 , 6.76 ; 13C-NMR : δ 163.6 (C=O), 159.1 (C-O), 148.4 (N=CH), 133.5 (ArC-C=O), 130.9 (CH), 128.7 (2xCH), 127.8 (2xCH), 127.2 (2xCH), 124.9 (ArCCH=N), 115.2 (2xCH) ppm.zide (3) :- Yield (E)-N'-benzohydrazide (4) (Yield (%): 62%; m.p. °C: 220–222; IR: 3262 (N-H & OH), 3058 (=C-H), 1646 (C=O), 1600 (C=N), 1506 (C=C Ar ring), 1280 (C-O); 1H-NMR : δ 11.28 , 8.63 , 8.19 , 7.83 , 7.54–7.34 , 7.30 , 6.93 , 6.74 ; 13C-NMR : δ 163.6 (C=O), 148.5 (N=CH), 147.3 (C-O), 144.9 (C-O), 133.5 (ArC-C=O), 130.9 (CH), 127.8 (2xCH), 127.2 (2xCH), 125.7 (ArCCH=N), 120.4 (CH), 114.9 (CH), 113.1 (CH) ppm.zide (4) :- Yield (E)-N'-benzohydrazide (5) (Yield (%): 51%; m.p. °C: 268–270; IR: 3246 (N-H & OH), 3065 (=C-H), 1647 (C=O), 1600 (C=N), 1506 (C=C Ar ring), 1271 (C-O); 1H-NMR : δ 11.58 , 9.47 , 8.37 , 7.85 , 7.51–7.33 , 6.99 , 6.32 ; 13C-NMR : δ 163.0 (C=O), 160.5 (C-O), 159.9 (C-O), 150.2 (N=CH), 132.8 (ArC-C=O), 131.7 (CH), 131.3 (CH), 127.9 (2xCH), 127.3 (2xCH), 110.0 (ArCCH=N), 107.3 (CH), 102.8 (CH) ppm.zide (5) :- Yield (E)-N'-benzohydrazide (6) (Yield (%): 55%; m.p. °C: 226–228; IR: 3410-3175 (N-H & OH), 3058 (=C-H), 1640 (C=O), 1616 (C=N), 1498 (C=C Ar ring), 1232 (C-O); 1H-NMR : δ 11.68 , 8.77 , 8.36 , 7.86 , 7.53–7.35 , 6.60 , 6.37 ; 13C-NMR : δ 162.8 (C=O), 150.3 (N=CH), 148.1 (C-O), 147.2 (C-O), 132.8 (ArC-C=O), 132.3 (C-O), 131.3 (CH), 127.9 (2xCH), 127.3 (2xCH), 121.4 (CH), 110.5 (ArCCH=N), 107.3 (CH) ppm.(6) zide (6) :- Yield (E)-N'-(4-hydroxy-3-methoxybenzylidene)benzohydrazide (7) (Yield (%): 74%; m.p. °C: 195–197; IR: 3490-3255 (N-H & OH), 3060 (=C-H), 1647 (C=O), 1602 (C=N), 1499 (C=C Ar ring), 1280 (C-O), 1200 (C-O); 1H-NMR : δ 11.47 , 8.71 , 8.31 , 7.88 , 7.72–7.27 , 6.95 , 6.79 , 3.78 ; 13C-NMR : δ 164.0 (C=O), 149.0 (N=CH), 147.6 (C-O), 133.5 (ArC-C=O), 131.1 (CH), 127.9 (2xCH), 127.4 (2xCH), 125.6 (ArCCH=N), 122.7 (CH), 114.6 (CH), 108.3 (CH), 55.5 (OCH3) ppm.zide (7) :- Yield (E)-N'-(3-methoxybenzylidene)benzohydrazide (8) (Yield (%): 86%; m.p. °C: 194–196; IR: 3222 (N-H), 3056 (=C-H), 1647 (C=O), 1600 (C=N), 1545-1451 (C=C Ar ring), 1248 (C-O); 1H-NMR : δ 11.11 , 8.37 , 7.57–7.43 , 7.14–6.78 , 6.59–6.37 , 3.34 ; 13C-NMR : δ 163.8 (C=O), 157.5 (C-O), 144.0 (N=CH), 133.2 (ArC-C=O), 131.1 (CH), 130.9 (CH), 127.8 (2xCH), 127.4 (2xCH), 126.2 (CH), 122.2 (ArCCH=N), 120.2 (CH), 110.5 (CH), 55.0 (OCH3) ppm.zide (8) :- Yield (E)-2-hydroxy-N'-(2-hydroxybenzylidene)benzohydrazide (9) (Yield (%): 82%; m.p. °C: 284–286; IR: 3230 (N-H & OH), 3058 (=C-H), 1634 (C=O), 1608 (C=N), 1554-1490 (C=C Ar ring); 1H-NMR : δ 11.89 , 8.51 , 7.85 , 7.44–7.17 , 7.00–6.78 ; 13C-NMR : δ 165.0 (C=O), 160.0 (C-O), 157.8 (C-O), 149.9 (N=CH), 133.8 (CH), 131.0 (CH), 130.0 (CH), 127.6 (CH), 118.8 (CH), 118.4 (CH), 117.7 (ArCCH=N), 117.3 (CH), 116.3 (CH), 113.9 (ArC-C=O) ppm.zide (9) :- Yield (E)-2-hydroxy-N'-(3-hydroxybenzylidene)benzohydrazide (10) (Yield (%): 50%; m.p. °C: 253–255; IR: 3270 (N-H), 3070 (C-H), 1647 (C=O), 1585 (C=N),1545-1451 (C=C Ar ring), 1303 (N-N); IR: 3230 (N-H & OH), 3058 (=C-H), 1634 (C=O), 1608 (C=N), 1554-1490 (C=C Ar ring); 1H-NMR : δ 12.03 , 11.61 , 9.11 , 8.31 , 7.87 , 7.39–7.29 , 7.25–7.18 , 7.17–7.06 , 6.95–6.76 ; 13C-NMR : δ 165.6 (C=O), 160.3 (C-O), 157.2 (C-O), 149.1 (N=CH), 134.7 (ArCCH=N), 133.6 (CH), 129.1 (CH), 127.4 (CH), 118.6 (CH), 118.2 (CH), 117.4 (CH), 117.3 (CH), 114.0 (ArC-C=O), 113.3 (CH) ppm.ide (10) :- Yield 2-Hydroxy(4-hydroxy-benzylidene)benzohydrazide (11) (Yield (%): 51%; m.p. °C: 239–241; IR: 3262 (N-H), 1640 (C=O), 1595 (C=N), 1506 (C=C Ar ring), 1220 (C-O); IR: 3262 (N-H & OH), 1640 (C=O), 1595 (C=N), 1506 (C=C Ar ring), 1220 (C-O); 1H-NMR : δ 12.11 , 11.51 , 9.44 , 8.29 , 7.87 , 7.54 , 7.34 , 6.89 , 6.83 , 6.78 ; 13C-NMR : δ 165.5 (C=O), 160.3 (C-O), 155.5 (C-O), 149.5 (N=CH), 133.5 (CH), 128.9 (2xCH), 127.3 (CH), 124.6 (ArCCH=N), 118.2 (CH), 117.3 (CH), 115.3 (2xCH), 114.0 (ArC-C=O) ppm.ide (11) :- Yield (E)-N'--2-hydroxybenzohydrazide (12) (- Yield (%): 85%; IR: 3273 (N-H & OH), 1639 (C=O), 1590 (C=N), 1487 (C=C Ar ring), 1280 (C-O), 1232 (N-N); 1H-NMR : δ 12.13 , 11.45 , 8.77 , 8.21 , 7.84 , 7.39–7.25 , 6.96 , 6.89 , 6.81 , 6.75 ; 13C-NMR : δ 165.4 (C=O), 160.3 (C-O), 149.5 (N=CH), 147.6 (C-O), 145.0 (C-O), 133.4 (CH), 127.2 (CH), 125.3 (ArCCH=N), 120.5 (CH), 118.1 (CH), 117.3 (CH), 115.0 (CH), 114.0 (ArC-C=O), 113.3 (CH) ppm.ide (12) :- Yield (E)-N'--2-hydroxybenzohydrazide (13) (Yield (%): 55%; m.p. °C: 260–262; IR: 3426-3247 (N-H & OH), 3066 (C-H), 1632 (C=O), 1600 (C=N), 1499 (C=C Ar ring), 1256 (C-O); 1H-NMR : δ 11.98 , 11.69 , 9.51 , 8.38 , 7.82 , 7.38–7.26 , 7.07–6.98 , 6.93–6.76 , 6.41 ; 13C-NMR : δ 164.9 (C=O), 160.7 (C-O), 160.3 (C-O), 160.0 (C-O), 150.8 (N=CH), 133.6 (CH), 131.7 (CH), 127.2 (CH), 118.2 (CH), 117.3 (CH), 113.7 (ArC-C=O), 109.8 (ArCCH=N), 107.4 (CH), 102.8 (CH) ppm.ide (13) :- Yield (E)-2-hydroxy-N'-benzohydrazide (14) (Yield (%): 55%; m.p. °C: 288-282; IR: 3300 (N-H), 1632 (C=O), 1593 (C=N),1506 (C=C Ar ring), 1303 (C-O), 1249 (C-N); IR: 3426-3247 (N-H & OH), 3066 (C=C), 1632 (C=O), 1600 (C=N), 1499 (C=C Ar ring), 1256 (C-O); 1H-NMR : δ 12.03 , 11.72 , 8.37 , 7.82 , 7.40–7.28 , 6.98–6.78 , 6.61 , 6.46–6.34 ; 13C-NMR : δ 165.0 (C=O), 160.3 (C-O), 151.1 (N=CH), 148.2 (C-O), 147.2 (C-O), 133.6 (CH), 132.2 (C-O), 127.2 (CH), 121. 6(CH), 118.2 (CH), 117.3 (CH), 113.6 (ArC-C=O), 110.4 (ArCCH=N), 107.3 (CH) ppm.ide (14) :- Yield (E)-2-hydroxy-N'-(4-hydroxy-3-methoxybenzylidene)benzohydrazide (15) (Yield (%): 65%; m.p. °C: 118–120; IR: 2410 (OH), 3254 (N-H), 2925 (C-H), 1647 (C=O), 1600 (C=N), 1506 (C=C Ar ring), 1303 (C-O), 1232 (C-N); 1H-NMR : δ 12.08 , 11.63 , 9.09 , 8.31 , 7.91 , 7.40–7.30 , 7.02 , 6.90 , 6.83 , 3.84 ; 13C-NMR : δ 165.4 (C=O), 160.1 (C-O), 149.8 (N=CH), 149.1 (C-O), 147.7 (C-O), 133.5 (CH), 127.5 (CH), 125.2 (ArCCH=N), 122.7 (CH), 118.3 (CH), 117.3 (CH), 114.9 (CH), 114.2 (ArC-C=O), 108.5 (CH), 55.4 (OCH3) ppm.ide (15) :- Yield (E)-2,4-dihydroxy-N'-(3-hydroxybenzylidene)benzohydrazide (16) (Yield (%): 40%; m.p. °C: 252–254; IR: 3520 (OH), 3246 (N-H), 1640 (C=O), 1616 (C=N), 1577-1545 (C=C Ar ring), 1274 (C-O); 1H-NMR : δ 12.35 , 11.39 , 9.74 , 9.13 , 8.26 , 7.70 , 7.18 , 7.16–7.04 , 6.78 , 6.38–6.21 ; 13C-NMR : δ 166.0 (C=O), 162.9 (C-O), 162.5 (C-O), 157.3 (C-O), 148.1 (N=CH), 135.0 (ArCCH=N), 129.1 (CH), 128.7 (CH), 118.5 (CH), 117.2 (CH), 113.2 (CH), 107.0 (CH), 105.4 (ArC-C=O), 102.9 (CH) ppm.ide (16) :- Yield (E)-2,4-dihydroxy-N'-(4-hydroxybenzylidene)benzohydrazide (17) (Yield (%): 52%; m.p. °C: 288-282; IR: 3325 (OH), 3270 (N-H), 1632 (C=O), 1608 (C=N), 1506-1436 (C=C Ar ring), 1248 (C-O); 1H-NMR : δ 12.42 , 11.39 , 9.86 , 9.61 , 8.28 , 7.73 , 7.51 , 6.77 , 6.29 , 6.27 ; 13C-NMR : δ 165.7 (C=O), 162.7 (C-O), 162.5 (C-O), 159.4 (C-O), 148.4 (N=CH), 128.8 (CH), 128.7 (2xCH), 124.9 (ArCCH=N), 115.4 (2xCH), 107.0 (CH), 105.4 (ArC-C=O), 102.8 (CH) ppm.ide (17) :- Yield (E)-N'--2,4-dihydroxybenzohydrazide (18):- Yield (%): 45%; m.p. °C: 237–239; IR: 3300 (OH & N-H), 1640 (C=O),1600 (C=N), 1514 (C=C Ar ring), 1280 (C-O); 1H-NMR : δ 12.45 , 11.22 , 9.68 , 8.18 , 7.63 , 7.28 , 6.95 , 6.82–6.72 , 6.37–6.23 ; 13C-NMR : δ 165.8 (C=O), 162.3 (C-O), 160.3 (C-O), 148.6 (N=CH), 147.4 (C-O), 145.0 (C-O), 128.5 (CH), 125.6 (ArCCH=N), 121.4 (CH), 120.3 (CH), 115.0 (CH), 106.9 (CH), 105.5 (ArC-C=O), 102.9 (CH) ppm.(E)-N'--2,4-dihydroxybenzohydrazide (19):- Yield (%): 47%; m.p. °C: 257–259; IR: 3365 (OH), 3255 (N-H), 1625 (C=O), 1570 (C=N), 1506 (C=C Ar ring), 1256 C-O); 1H-NMR : δ 11.57 , 8.5–8.0 , 8.47 , 7.40–7.38 , 7.06–7.04 , 6.47–6.46 , 6.45–6.44 ; 13C-NMR : δ 168.4 (C=O), 162.4 (C-O), 149.3 (C-O), 148.0 (N=CH), 144.5 (C-O), 137.1 (C-O), 134.0 (CH), 131.8 (CH), 123.3 (CH), 120.2 (CH), 111.3 (CH), 110.2 (ArCCH=N), 108.8 (CH), 107.8 (ArC-C=O) ppm.(E)-3,4,5-trihydroxy-N'-(3-hydroxybenzylidene)benzohydrazide (20) (Yield (%): 54%; m.p. °C: 145–147; IR: 3465 (OH), 3340 (N-H), 1695 (C=O), 1616 (C=N),1460 (C=C Ar ring), 1318 (C-O), 1248 (N-N); 1H-NMR : δ 9.45–8.26 , 7.35–6.80 ; 13C-NMR : δ 166.7 (C=O), 161.4 (N=CH), 157.4 (C-O), 145.0 (C-O), 137.7 (C-O), 134.9 (C-O), 129.5 (CH), 120.1 (ArCCH=N), 119.8 (CH), 118.5 (CH), 114.4 (ArC-C=O), 109.0 (2xCH) ppm.ide (20) :- Yield -3,4,5-trihydroxybenzohydrazide (21) (Yield (%): 49%; m.p. °C: 288-282; IR: 3457 (OH), 3333 (N-H), 1702 (C=O), 1632 (C=N), 1522-1460 (C=C Ar ring), 1311 (C-O), 1209 (N-N); δ 165.7 (C=O), 162.7 (C-O), 162.5 (C-O), 159.4 (C-O), 148.4 (N=CH), 128.8 (CH), 128.7 (2xCH), 124.9 (ArCCH = N), 115.4 (2xCH), 107.0 (CH), 105.4 (ArC-C=O), 102.8 (CH) ppm; 13C-NMR : δ 165.0 (C=O), 158.4 (C-O), 151.0 (N=CH), 148.8 (C-O), 147.9 (C-O), 133.6 (CH), 133.2 (C-O), 127.2 (CH), 125.4 (ArCCH=N), 121. 6(CH), 118.2 (CH), 117.3 (CH), 113.6 (ArC-C=O), 107.0 (CH) ppm.ide (21) :- Yield 2.2.32.2.3.1a)2,2-Diphenyl-1-picrylhydrazyl (DDPH) methodc represents the absorbance of 2,2-diphenyl-1-picrylhydrazyl in the absence of the test sample and As stands for the absorbance of 2,2-diphenyl-1-picrylhydrazyl in the presence of the test sample or alternatively, the positive control.b)2,2′-Azino-bis(3-Ethylbenzothiazoline-6-Sulfonic Acid (ABTS) Enzymatic AssayThe antioxidant activity assay was conducted following the method of (7.4 mM) solution and potassium persulfate solution (2.6 mM). The mixture was then reacted for fourteen to sixteen hours in the dark at room temperature to produce ABTS.+. The obtained solution was subsequently diluted by mixing ABTS.+ solution (1 mL) with pH = 7.4 phosphate buffer solution (60 mL) to reach an absorbance of 1.10 ± 0.02 units at a wavelength of 734 nm using a UV/VIS spectrophotometer. In order to prepare the final ABTS.+ solution for each assay, the following procedure was carried out: Add 100 μL of hydrazone solution (prepared as 1 mg/mL in DMSO) to 3.0 mL of ABTS.+ final solution. Incubate for two hours in the dark and measure the absorbance at a wavelength of 734 nm. Trolox (1 mg/mL in DMSO) was used as a positive control. The percent inhibitory rate was worked out according to the following formula:c is the absorbance of control (without samples) and As is the absorbance in the presence of the samples.c)Inhibition of lipid peroxidation by ferric thiocyanate protocolThis enzymatic assay was conducted according to the method described by 2O (3.9 mL). This mixture was placed in an oven maintained at 40 °C in the dark and was designated as a sample solution. Then:This protocol was employed, with some modifications, to investigate the inhibition of lipid peroxidation as reported by μL of a sample solution into a 2 mL screw cap vial, then add 9.7 mL of ethanol (75%), 100 μL μL of NH4SCN (30%) and μL of FeCl2 (20 mM in 3.5% HCl). After three minutes has passed, the absorbance of the solution (red color) was measured at a wavelength of 500 nm. These measurements were repeated every twenty four hours until one day after the absorbance of the control solution (without sample) has reached its maximum. Ascorbic acid (vitamin C) (1 mg/mL) was used as the positive control in this method. The percent Inhibition (%) of lipid peroxidation was found according to the following equation:s represents the absorbance of the sample or positive control on the day when the absorbance of the control was maximum, and Ac represents the absorbance of the control without any sample on the day when it reached its maximum absorbance.Transfer 100 2.2.3.2a)The inhibition of tyrosinaseμL sample. After one minute, 100 μL of the aqueous solution of tyrosinase was added to the mixture and the absorbance was immediately measured at a wavelength of 475 nm for seven minutes. All experimental measurements were repeated thrice (performed in triplicate). The percent inhibitory rate was calculated according to the formula:c represents the absorbance of control devoid of samples and As represents the absorbance in the presence of the samples.b)The inhibition of acetylcholinesteraseThe activity of tyrosinase assay was carried out as previously reported by the group of 2HPO3, 50 mM, pH = 7.7) were taken into a test tube and 100 μL of hydrazone sample dissolved in DMSO (1 mg/mL) was added. Then, 300 μL of enzyme (0.005 U/mL) solution was added to the solution. The solution was incubated at 37 °C for 10 min. Then, 300 μL of acetylthiocholine iodide  and 300 μL of Ellman's reagent  (0.5 mM) were added. After 30 min of incubation at 37 °C, absorbance was measured at a wavelength of 412 nm. Each sample was measured in triplicate. The enzyme percent inhibitory rate was calculated according to the following formula:c represents the absorbance of control devoid of any samples and As represents the absorbance in the presence of the hydrazone sample.Inhibition of acetylcholinesterase (AChE) was measured according to previously described method of 3in vitro whether they have direct or indirect (or both) antioxidant activities using different methods, such as, DPPH, ABTS and ferric thiocyanate (FTC).The aromatic hydrazones obtained in the present investigation were tested 14, 18, 20 and 21). Some of these compounds showed comparable activity to that obtained from the positive control (Vit. C). On the other hand, the antioxidant activities of the present compounds as measured by the ABTS method, which is also a direct method, were in the range of 68.74–93.51 (8). The third indirect method to measure the antioxidant activities of the present hydrazones  to each other give the highest antioxidant activities according to all used methods  and even more active than those having three hydroxyl groups (R1 = R2 = R3 = OH).The direct antioxidant activities of hydrazones as measured by the DPPH method  ranged f74–93.51 , and mosdrazones  was empl2 = R3 = OH) or three hydroxyl groups (R1 = R2 = R3 = OH). Thus, it seems that, the presence of two neighboring hydroxyl groups in this series of aromatic hydrazones (R2 = R3 = OH) is enough to produce optimum antioxidant activities. The obtained aromatic hydrazones containing greater number of hydroxyl groups are expected to have richer conjugated systems in comparison to those that have a lower number hydroxyl groups. Of course, having multiple hydroxyl substituents attached to both aromatic rings created rich conjugated systems and this factor renders the hydrazones as strong antioxidant agents, because they can easily scavenge free radicals in their solutions by undergoing facile hydrogen radical abstraction (H.) and becoming themselves free radicals, although more stable than the former free radicals with which they react. The present results are in agreement with other results obtained by other researchers . As a result, the explanation of the antioxidant activities of the new synthesized series of aromatic hydrazones is based on that of natural antioxidants such as phenolic compounds. Evidence supporting the strong impact exerted by the hydroxyl groups in delocalizing the aromatic ring electrons within and beyond the aromatic rings is shown in 35.0 ppm .Figure 11–20 ranged from δ168.4–162.8 ppm, the imine group was impacted to a greater extent (δ161.4–148.0 ppm).In general, the efficiency of the antioxidant activity is not only related to delocalizing the free radical within aromatic rings, but also outside the rings. Many natural antioxidants exhibit extended conjugation as key features. Thus, in order to examine whether the hydroxyl substituents impact groups outside the rings, the chemical shifts of the carbonyl (C=O) and imine (CH = N) moieties were collected and are summarized in 22). Although a detailed mechanistic study warrants further investigation, it is conceivable that extended conjugation throughout the entire aromatic hydrazone framework may be feasible considering the possible tautomeric structure 23 shown in 5 or from an intermolecular hydrogen abstraction by any of the hydroxyl groups on the other aromatic ring.These above data support extended conjugation from both aromatic ring as shown in It is well known that the generation of reactive oxygen species (ROS) due to metabolic processes leads to various serious diseases such as Alzheimer's disease (AD) and cause skin ageing. There is enough evidence in the literature to relate oxidative stress caused by ROS activity to age-related neurodegenerative ailments and AD. The use of antioxidants has been investigated in an effort to slow down the progression of such diseases . This moGenerally, enzymes can be inhibited by a substance known as enzyme inhibitor which normally reduces the activity of the enzyme by any one of the known mechanisms. Therefore, each one of the present hydrazones may exert different inhibition effect on either tyrosinase or cholinesterase activity. As the nature and the active sites of enzymes are usually different from each other, so the inhibitors of tyrosinase and cholinesterase are expected to be different.5, almost all other compounds affected both enzymes differently and these are considered to be normal as expected. The new data have shown that tyrosinase is moderately inhibited (50.54–62.90) by compounds 5, 6, and 18 . In addition, some of the prepared compounds proved as effective inhibitors of tyrosinase and cholinesterase enzymes.In the present study, a series of 21 aromatic hydrazones were prepared and their chemical structures confirmed by infrared, Ziad Moussa: Analyzed and interpreted the data; Wrote the paper.Mohammed Al-Mamary: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper.Sultan Al-Juhani, Saleh A Ahmed: Performed the experiments.10.13039/501100006013United Arab Emirates University (Grant no. G00003291/Fund no.31S401/Project #852).Ziad Moussa was supported by The authors declare no conflict of interest.No additional information is available for this paper.