The synthesized derivatives were characterized and their chemical structures were established by various physicochemical methods such as IR, Mass, 1H-NMR, 13C-NMR and elemental analysis. The synthesized compounds were tested for their neuropharmacological potential. The compounds exhibited significant antidepressant and anti-anxiety activities against various behavioral in vivo models. Compounds PFC-3 and PFC-12 were found to be the most active derivatives in the series. The 2-pyrazoline analogs, having 2-hydroxyphenyl and anthracen-9-yl substitution at 3rd position while 4-benzyloxyphenyl and 4-methylphenyl substitution at 5th position, were decisive in eliciting good antidepressant and anxiolytic properties, respectively. The docking experiments revealed that the synthesized derivatives were potential inhibitors of MAO-A protein, which plays a central role in managing depression and anxiety disorders. The most potent derivatives were found to be involved in some key interactions with Tyr407, Tyr444, Phe352 and Ala68 amino acid residues at the binding site of MAO-A protein. All the synthesized derivatives successfully passed the pharmacokinetic barriers of absorption, distribution, metabolism and elimination as predicted using in silico techniques without showing any substantial indication of acute and neurotoxicity. This was further confirmed in the laboratory by performing acute toxicity studies as per OECD guidelines.A novel series of 1,3,5-trisubstituted-2-pyrazoline derivatives  The proper functioning of synaptic neurotransmission is due to rapid degradation of biogenic amines, which are critically important for the regulation of emotional behaviors and other brain functions  is a FAD-dependent major neurotransmitter degrading enzyme present in the outer mitochondria of neuronal, glial and other cells, that catalyse the aerobic oxidation of structurally diverse xenobiotic arylalkylamine substrates including neurotransmitters and exogenous amines to the corresponding aldehyde and imines with the generation of hydrogen peroxide . In mamal., 2002). The unst, 3rd and 5th positions by different aromatic and heteroaromatic units inspired the researchers to carry out structural modifications with an array of pharmacological activities such as antimicrobial, anti-mycobacterial, antifungal, anti-amoebic, anti-malarial, anti-inflammatory, anti-depressant, antiepileptic, anticancer, anti-HIV, antitubercular, antibacterial, photoluminescence, polarity probes, insecticidal, antinociceptive, antioxidant, hypotensive, cholesterol inhibitory, cannabinoid (CB1) receptor antagonist, nitric oxide synthase (NOS) inhibitor and MAO inhibitor . Howevebinoid CB receptorAzam 2005; Lange eAzam 2005; Patil aAzam 2005; Prasad Azam 2005; RathishAzam 2005; ShaharyAzam 2005; Yar et Azam 2005; ZampierAzam 2005; Zhang eAzam 2005; ChimentAzam 2005; Gokhan-Azam 2005).st, 3rd and 5th position of the pyrazoline nucleus, was in particular the focus of the study and was further corroborated by the computational studies. In addition to the conventional heating methods, microwave assisted organic synthesis approach was employed to prepare the proposed derivatives. A technique which can be used to rapidly explore “chemical space” and increase the vast interaction of the compounds produced is known as microwave assisted organic synthesis. Finally, to predict the binding affinity and ascertain the interactions of the proposed derivatives with the biological target, molecular docking studies were performed.In order to understand the structural requirements, for both inhibition and selectivity towards the two MAO isoforms (MAO-A/ MAO-B), it was decided to synthesize some novel 1, 3,5 trisubstituted-2-pyrazoline derivatives. The influence on the biological behavior, due to the substitutions of different aromatic/heteroaromatic rings at 1-1. 1H-NMR and 13C-NMR spectra were recorded on Bruker Avance-400, FTNMR spectrometer  at 400 MHz and the chemical shifts are reported in parts per million , taking TMS (δ 0 ppm for 1H NMR) as the internal standard. Mass spectra were recorded on Waters UPLC-TQD Mass Spectrometer instrument  using LC-ESI or APCI-MS Technique. Elemental analysis was performed on Perkin Elmer-2400, Series-II Analyzer .The chemicals and reagents for synthesis were procured from S. D. Fine Chemicals and Sigma Aldrich, Mumbai, India and the pre-coated TLC sheets were obtained from Merck Chemicals, India and were used as such. Reagent grade solvents were used and were purified and dried by standard methods. Raga's Scientific Microwave System  was used for the microwave assisted organic synthesis (MAOS). Melting points were determined by open capillary method and are uncorrected. IR spectra were recorded on Bruker FT-IR, ALPHA-T (Eco-ATR) spectrophotometers,  and the values are expressed in cmSynthesis of the proposed 2-pyrazoline analogs was carried out in three steps using conventional methods (stirring and refluxing) as well as an environmentally benign green synthetic approach under the exposure of microwave irradiations. It has been observed that the entire reactions performed using microwave assisted organic synthesis (MAOS) produced compounds in better yields and at a faster rate.Conventional synthesis: Ethanolic KOH  solution was added with stirring over a period of 15 min-2 hrs to the solution of substituted ketones (0.01 M) and aldehydes (0.01 M). The stirring was continued for 24-48 hrs at a low temperature (0-10 °C). On completion of the reaction, the reaction mixture was poured into ice-cold water, and then neutralized to pH 2 with hydrochloric acid (6N). The yellow colored products obtained were filtered, washed, and re-crystallized from methanol .MAOS: Substituted ketones (0.01 M) and aldehydes (0.01 M) were reacted, in presence of hydro-alcoholic solution of KOH , under microwave irradiation . On completion of the reaction, the reaction mixture was poured into ice-cold water, and then neutralized to pH 2 with hydrochloric acid (6N). The yellow colored products obtained were filtered, washed, and re-crystallized from methanol .Conventional synthesis: The obtained chalcone derivatives were refluxed for 3-6 hrs with excess of hydrazine hydrate in dry ethanol. The hot reaction mixture was poured in ice-cold water to obtain the crude product, which was washed and re-crystallized from appropriate solvent (ethanol/acetone/ethyl acetate) to afford the respective pyrazoline derivative .MAOS: The chalcone derivatives obtained were mixed with hydrazine hydrate (in excess) in dry ethanol under the exposure of microwave irradiation . The reaction mixture was poured in ice-cold water to get the crude product, which was washed and re-crystallized from appropriate solvent (ethanol/acetone/ethyl acetate) to give the respective pyrazoline derivative .Conventional method: 3,5-Disubstituted-2-pyrazolines (0.001M) were reacted with 0.002 M of 4-chlorobenzenesulfonylchloride by stirring, taking tetra hydro furan (10 mL) as the solvent. Stirring was continued for 1-4 hrs. After completion of reaction, the reaction mixture was poured on a petri plate and solvent was evaporated to dryness. The sticky crude product was re-precipitated using acetonitrile or methanol. Recrystallization was done with acetonitrile or methanol to obtain the pure product. MAOS: Appropriately substituted 3,5-disubstituted-2-pyrazolines (0.001 M) were reacted with 0.002 M of 4-chlorobenzenesulfonylchloride under of microwave irradiation , taking tetra hydro furan (10 mL) as the solvent. After completion of reaction, the reaction mixture was poured on a petri plate and solvent was evaporated to dryness. The sticky crude product was re-precipitated using acetonitrile or methanol. Recrystallization was done with acetonitrile or methanol to obtain the pure product.Characterization of the intermediate compounds was done with the help of TLC, melting point and mass spectrometry. However, complete physicochemical Table 1 and spec2--phenol(PFC-1)1): N-H str (3531), C-H Ar (3036), C=N str (1596), C-H deform (1491), S=O. 1H NMR : 2.00-2.06 , 2.54-2.58 , 3.70-3.73 , 5.23-5.27 , 6.50-7.96 . 13C NMR : 39.2 (CH2 pyrazoline), 45.1 (CH pyrazoline), 113.8-146.3 (12CH benzene), 149.6-157.2 (6C benzene), 161.5 (C pyrazoline). MS (m/z): 429 . Anal.Calcd. for C21H17ClN2O4S: C, 59.66; H, 4.32; N, 6.32. Found: C, 59.64; H, 4.36; N, 6.29.IR (cm-2--phenol(PFC-2)1): N-H str (3361), C-H Ar (3049), C=N str (1602), C-H deform (1460), S=O . 1H NMR : 2.30-2.48 , 3.51-3.70 , 3.22-3.50 , 7.23-7.86 , 8.62 . 13C NMR : 40.8 (CH2 pyrazoline), 47.2 (CH pyrazoline), 76.5 , 113.4-117.1 (4CH aromatic), 119.1 (2C benzene), 126.8-131.7 (8CH aromatic), 135.2 (C benzene), 144.7 (C benzene), 158.5 (C pyrazoline), 160.4-160.8 (2C benzene). MS (m/z): 443.2 . Anal.Calcd. for C22H19ClN2O4S: C, 59.66; H, 4.32; N, 6.32. Found: C, 59.70; H, 4.31; N, 6.35.IR (cm-2--phenol(PFC-3)1): N-H str (3258), C-H Ar (3115), C=N str (1605), C-H deform (1448), S=O . 1H NMR : 2.27-2.51 , 3.67-3.94 , 3.15-3.63 , 7.11-7.82 , 8.62 . 13C NMR : 41.5 (CH2 pyrazoline), 49.6 (CH pyrazoline), 79.2 , 112.7-119.8 (4CH aromatic), 118.4 (C benzene), 127.3-130.7 (8CH aromatic), 135.2 (C benzene), 141.6 (C benzene), 157.3 (C pyrazoline), 159.4-160.0 (2C benzene). MS (m/z): 519.6 . Anal. Calcd. for C28H23ClN2O4S: C, 64.80; H, 4.47; N, 5.40. Found: C, 64.83; H, 4.45; N, 5.41.IR (cm-2--phenol (PFC-4)1): N-H str (3456), C-H Ar (3083), C=N str (1582), C-H deform (1414), S=O . 1H NMR : 2.58-2.71 , 3.70-3.96 , 3.11-3.56 , 4.09 , 7.14-7.91 , 9.02 . 13C NMR : 26.5 , 41.7 (CH2 pyrazoline), 48.6 (CH pyrazoline), 112.7-128.7 (12CH aromatic), 134.2-137.6 (3C benzene), 157.3 (C pyrazoline), 160.1-161.9 (2C benzene). MS (m/z): 428.1 . Anal. Calcd. for C22H19ClN2O3S: C, 61.89; H, 4.49; N, 6.56. Found: C, 61.91; H, 4.53; N, 6.57.IR (cm-4--phenol(PFC-5)1): N-H str (3459), C-H Ar (2994), C=N str (1598), C-H deform (1451), S=O . 1H NMR : 2.20-2.48 , 3.56-3.85 , 3.15-3.63 , 4.12 , 7.21-7.79 , 8.70 . 13C NMR : 41.5 (CH2 pyrazoline), 49.6 (CH pyrazoline), 60.3 , 115.5-127.3 (12CH aromatic), 128.2-136.4 (3C benzene), 158.6 (C pyrazoline), 159.4-160.0 (2C benzene). MS (m/z): 443.7 . Anal. Calcd. for C22H19ClN2O4S: C, 59.66; H, 4.32; N, 6.32. Found: C, 59.70; H, 4.30; N, 6.33.IR -1-(4-chloro-benzenesulfonyl)-4,5-dihydro-1H-pyrazole(PFC-9)1): N-H str (3301), C-H Ar (3051), C=N str (1608), C-H deform (1452), S=O. 1H NMR : 2.60-2.72 , 3.69-3.87 , 3.24-3.58 , 5.11 , 7.43-7.96 . 13C NMR : 41.8 (CH2 pyrazoline), 49.6 (CH pyrazoline), 78.3 , 128.7-134.4 (21CH aromatic), 138.4 (5C aromatic), 160.3 (C pyrazoline), 161.4 (C aromatic). MS (m/z):603.1 . Anal. Calcd. For C36H27ClN2O3S: C, 71.69; H, 4.51; N, 4.64. Found: 71.71; H, 4.47; N, 4.65.IR (cm-3-Anthracen-9-yl-1-(4-chloro-benzenesulfonyl)-5-(4-methoxy-phenyl)-4,5-dihydro-1H-pyrazole(PFC-10)1): N-H str (3379), C-H Ar (3105), C=N str (1611), C-H deform (1446), S=O . 1H NMR : 3.02-3.11 , 3.67-3.91 , 327.1-3.56 , 3.73 , 7.11-7.82 . 13C NMR : 42.6 (CH2 pyrazoline), 47.3 (CH pyrazoline), 57.2 (CH3 methyl), 125.7-134.3 (17CH aromatic), 138.4 (7C aromatic), 157.6 (C pyrazoline), 160.0 (C aromatic). MS (m/z): 528.5 . Anal. Calcd. for C30H23ClN2O3S: C, 68.37; H, 4.40; N, 5.32. Found: 68.36; H, 4.43; N, 5.28.IR (cm-4--phenol(PFC-11)1): N-H str (3340), C-H Ar (3050), C=N str (1575), C-H deform (1421), S=O . 1H NMR : 2.20-2.35 , 3.61-3.90 , 3.15-3.64 , 7.23-7.91 , 8.55 . 13C NMR : 39.8 (CH2 pyrazoline), 49.5 (CH pyrazoline), 116.7-127.3 (17CH aromatic), 135.2 (7C aromatic), 157.3 (C pyrazoline), 162.1 (C aromatic). MS (m/z): 528.5 . Anal. Calcd. for C29H21ClN2O3S: C, 67.90; H, 4.13; N, 5.46. Found: C, 67.92; H, 4.16; N, 5.47.IR (cm-3-Anthracen-9-yl-1-(4-chloro-benzenesulfonyl)-5-p-tolyl-4,5-dihydro-1H-pyrazole(PFC-12)1): N-H str (3273), C-H Ar (2925), C=N str (1668), C-H deform (1438), S=O . 1H NMR : 2.40 , 3.23-3.61 , 5.15-5.22 , 7.25-7.58 , 7.77-8.49 . 13C NMR : 21.4 (CH2 pyrazoline), 50.7 (C pyrazoline), 76.8-77.6 (2CH aromatic), 124.5-134.3 (14CH aromatic), 136.2-138.8 (6C aromatic), 159.4 (C pyrazoline). MS (m/z): 512.3 . Anal. Calcd. for C30H23ClN2O2S: C, 70.51; H, 4.54; N, 5.48. Found: C, 70.50; H, 4.51; N, 5.44.IR (cm-5-(4-Benzyloxy-phenyl)-1-(4-chloro-benzenesulfonyl)-3-(1H-pyrrol-2-yl)-4,5-dihydro-1H-pyrazole(PFC-13)1): N-H str (3195), C-H Ar (3024), C=N str (1602), C-H deform (1444), S=O . 1H NMR : 2.26-2.52 , 3.66-3.90 , 3.18-3.60 , 5.47 , 6.14 , 7.12-7.87 . 13C NMR : 39.8 (CH2 pyrazoline), 52.5 (CH pyrazoline), 76.2 , 112.7-129.8 (12CH aromatic), 118.4 (3CH pyrrole), 127.3-130.7 (8CH aromatic), 137.2-139.2 (4C aromatic), 157.8 (C pyrazoline), 159.1 (C aromatic). MS (m/z): 492.3 . Anal. Calcd. for C26H22ClN3O3S: C, 63.47; H, 4.51; N, 8.54. Found: C, 63.52; H, 4.49; N, 8.71. IR (cm-1-(4-Chloro-benzenesulfonyl)-5-(4-methoxy-phenyl)-3-(1H-pyrrol-2-yl)-4,5-dihydro-1H-pyrazole(PFC-14)1): N-H str (3382), C-H Ar (3010), C=N str (1600), C-H deform (1440), S=O . 1H NMR : 2.18-2.44 , 3.81-3.93 , 3.12-3.66 , 3.51 , 5.28 , 6.39 , 7.17-7.85 . 13C NMR : 39.8 (CH2 pyrazoline), 52.5 (CH pyrazoline), 59.8 , 112.7-129.8 (8CH aromatic), 121.1 (3CH pyrrole), 117.3-126.5 (8CH aromatic), 137.2-138.6 (3C aromatic), 157.8 (C pyrazoline), 160.5 (C aromatic). MS (m/z): 416.6 . Anal. Calcd. for C20H18ClN3O3S: C, 57.76; H, 4.36; N, 10.10. Found: C, 57.75; H, 4.39; N, 10.08.IR (cm-4--phenol(PFC-15)1): N-H str (3325), C-H Ar (2997), C=N str (1623), C-H deform (1462), S=O . 1H NMR : 2.61-2.84 , 3.63-3.99 , 3.44-3.71 , 5.28 , 6.39 , 7.24-7.68 , 8.23 . 13C NMR : 40.3 (CH2 pyrazoline), 49.8 (CH pyrazoline), 114.1-128.5 (8CH aromatic), 116.7 (3CH pyrrole), 135.2-139.6 (3C aromatic), 156.2 (C pyrazoline), 162.1 (C aromatic). MS (m/z): 402.8 . Anal. Calcd. for C19H16ClN3O3S: C, 56.79; H, 4.01; N, 10.46. Found:C, 56.82; H, 3.97; N, 10.41. IR (cm-1-(4-Chloro-benzenesulfonyl)-3-(1H-pyrrol-2-yl)-5-p-tolyl-4,5-dihydro-1H-pyrazole(PFC-16)1): N-H str (3378), C-H Ar (3182), C=N str (1605), C-H deform (1438), S=O . 1H NMR : 2.08-2.34 , 3.61-3.95 , 3.18-3.60 , 2.51 , 5.33 , 6.06 , 7.13-7.69 . 13C NMR : 39.5 (CH2 pyrazoline), 54.6 (CH pyrazoline), 33.7 , 118.7-126.8 (8CH aromatic), 118.9 (3CH pyrrole), 137.2-138.6 (3C aromatic), 153.8 (C pyrazoline), 155.6 (C aromatic).MS (m/z): 401.3 . Anal. Calcd. for C20H18ClN3O2S: C, 60.07; H, 4.54; N, 10.51. Found: C, 60.11; H, 4.57; N, 10.49.IR  were obtained from the animal house of Faculty of Pharmacy, BBDNIIT, Lucknow, U.P., India and kept in polymeric cages under standard living conditions of 26±2 ºC temperature and 55±5 percent humidity, with regular light and dark cycles, having free access to standard food and water. Animals were treated humanely as per the Institutional Animal Ethics Committee (IAEC) guidelines. The pharmacological studies were approved by the IAEC with protocol number BBDNIIT/IAEC/008/2014. For the study, animals were divided into 29 groups, each group consisting of 6 animals (n=6). All the mice were treated at respective doses according to body weight. Treatments including standard/vehicle/test compounds were given at 1 mL/100 g body weight per oral (p.o.). Imipramine (10 mg/kg body weight) for antidepressant activity, and Diazepam (2 mg/kg body weight) for anti-anxiety activity was respectively taken as the standard drugs. Carboxy methyl cellulose (0.5 % CMC solution) was taken as the vehicle (control group). The groups and respective doses are given in Table 2PFC1-PFC16). These behavioral in vivo tests have been successfully used to screen and predict the efficacy and potency of various antidepressant treatments including MAO inhibitors  and Tail suspension test (TST) in mice model were used to screen the antidepressant potential of the prepared compounds .Antidepressant potential of the synthesized compounds was tested using Porsolt's behavioral despair test, i.e. FST. Typical immobility behaviors of mice are induced when they are forced to swim in a limited space from where they cannot flee. During the test, mice were plunged into a glass cylinder containing water and it was observed that the animals, after the first 2 min of the initial struggle, became motionless. The duration of stillness was considered when mice started floating in water in a vertical position, with modest activities in order to prevent sinking, and was documented specially for the last 4 min of the 6 min test. This behavior is explained as a state of despair which can be minimized by different therapeutically effective antidepressants, measured by comparing animals treated at numerous doses of test or standard drugs with control  and anxiogenic drugs with opposite effects. The values are represented in percent of control. Open arm exploratory time is increased while motor activity is decreased by benzodiazepines and valproates .The elevated plus-maze is used to determine the anxiety-related behavior, measured by the extent to which the mouse avoids to visit the open arm of the maze. It is a standard test of fear and anxiety where animals are positioned at the middle of an elevated four-arm maze, in which two arms were open (50 X 10 X 40 cm) and 2 arms are closed (50 X 10 X 40 cm), with an open roof situated on the opposite side. The maze was elevated to a height of 50 cm. After, one hour of oral administration of the standard drug (Diazepam at a dose of 2 mg/kg b.w.), test compound  and control (0.5 % aqueous CMC suspension), the mice were put in the middle of the maze, facing the closed arm. The observations were recorded during the 6 min test duration: time spent in closed arm and total number of the arm entries  of a test substance on the CNS. Several behavioral studies  are employed to identify potential neurotoxicity in mice model. Rotarod and actophotometer tests are the two most often used methods to identify the effect of chemical compounds (test compound) on neuromuscular coordination and locomotor activities, respectively .The rotarod test is valuable to find out the consequences of drug on motor coordination, i.e. neurotoxicity. This test was introduced by Dunham and Miya in 1956, where rLocomotor activities of animals, and humans, are affected by majority of the CNS acting drugs and therefore it is utilized as an index of their mental alertness. Some CNS depressant drugs like barbiturates and alcohols have been found to reduce it. However, CNS stimulants like amphetamines and caffeine exhibit significant increase in the motor activity. The locomotor activity was examined for a 10 minute duration, using Medicraft Actophotometer , to rule out such CNS depressant or stimulant type of effects of the tested compounds. During the test, animal moves in a square arena of dimension 30x30x25 cm with mesh wire bottom. Photoelectric cells connected in circuit with a counter are used to operate the apparatus, where light beam striking on the photo cell is cut off by the animal to record a count .50) of two most active final derivatives (PFC-3 and PFC-12) was performed as per guidelines laid by Organization for Economic Co-operation and Development (OECD) guideline No 423 “Acute Oral Toxicity - Acute Toxic Class Method”. The compounds were administered orally at four fixed dose levels , after 4 hours of fasting. After dose administration, food but not water was withheld for 2 hours. The body weight of all the mice were recorded on study days D0 (initiation), D1, D7 and D14. The animals were observed for 4 hours post dose treatment and thereafter for 14 days for mortality, including various signs of toxicity such as, changes in skin and fur, behavior patterns, convulsions, tremors and death , using GLIDE program. The X-ray crystal structure of MAO protein was retrieved from the Protein Data Bank (PDB) and optimized using “protein preparation wizard”. Ligands were prepared by LigPrep module 3.2 version v25111, using OPLS  2005 force field to give the consequent energy minima. Default settings were applied to other parameters. Rigid docking was performed during these calculations. Re-docking experiment was conducted to validate the docking procedure, in which the coordinates of crystal ligand were obtained from the PDB structure of MAO-A protein-ligand complex and docked back into the binding pocket. GLIDE  program perfectly reproduced the tentative pose of the ligand and accurately predicted the binding mode.in silico prediction of ADME properties of the synthesized derivatives.Majority of the drug candidates do not succeed in clinical trials due to poor pharmacokinetic properties, such as absorption, distribution, metabolism, and excretion (ADME). The outlay in developing new drug significantly increases due to these later-stage failures, which can be radically reduced if the problematic candidates are detected in early phases. In this study, the QikProp module (version 4.2) of Schrödinger  software program was successfully employed for in silico toxicity of the compounds. Two of these softwares, used in this study are; LAZAR and OSIRIS Property explorer. LAZAR provides a generic tool for predicting complex toxicological end points, such as carcinogenicity, long-term toxicity, and reproductive toxicity. Virtual Computational Chemistry Laboratory maintains OSIRIS as a fundamental part of Actelion's in house substance registration system. This calculates various drug-related properties of chemical structures including some toxicity parameters and drug likeness. Predicted outcomes are rated and color coded. Properties such as mutagenicity or a poor intestinal absorption having higher risks of undesired effects are shown in red, whereas a green color indicates drug-conform behavior .All the values of the experimental results are expressed as mean±SD and analyzed by one-way ANOVA, followed by Dunnett's test for the possible significance (P<0.05) identification between various groups. Statistical analysis was carried out using Graph Pad Prism 5.0 . (PFC1 to PFC-16) were synthesized using conventional as well as a green chemistry approach of microwave assisted organic synthesis (MAOS) as given in Figure 1-1), N-H stretching (3456-3105 cm-1) and C-H deformation (1428-1357 cm-1) along with a characteristic peak of sulfonyl group absorption bands (1340-1180 cm-1) in the corresponding regions of IR spectra.A series of thirteen 2-pyrazoline derivatives a/Hb) at δ 2.92-3.38 ppm and 3.70-3.93, coupled with each other and in turn with the vicinal methine proton (Hx) at δ 6.68-7.04 were recorded in the 1H-NMR spectra. All the other protons of various aliphatic/aromatic/heteroaromatic groups were anticipated at respective places.The presence of two non-equivalent protons of a methylene group .Detailed SAR studies revealed that 4-chlorobenzenesulfonyl substitution at N1 position was decisive in exhibiting good antidepressant and anti-anxiety activities. It was also observed that 2-hydroxyphenyl substitution at 3PFC-3 and PFC-12) were screened for their acute toxicities, at the tested doses. During these studies; behavioral pattern, changes in skin and fur, convulsions, tremors and death were not observed in any of the animals, in due course of 14 days of acute toxicity studies, as per the OECD guidelines.All the synthesized derivatives were also evaluated for their possible neurotoxicological effects such as neuromuscular coordination (using rotarod test) and locomotor activity (using actophotometer test). It was observed that none of the synthesized compounds showed serious neurotoxicity (disturbances in the motor co-ordinations and locomotor activity) threats at the tested doses Table 2. TherefoPFC-12) displayed additional key interactions of this moiety with side chain and backbone residues of the amino acids at the binding pocket. This included a hydrophobic interaction of benzene ring with Tyr 407, Tyr444 residues, and H-bond interaction with Ala68 and Tyr444 residues , Lucknow, India and Department of Chemistry, Banasthali Vidyapith University, Banasthali, Rajasthan, India for providing the library and sophisticated analytical instrument facilities. Authors are thankful to the All India Council for Technical Education (AICTE), New Delhi, India, for providing grant under the Research Promotion Scheme (Grant No.: 8023/RID/RPS/30 (Pvt.) 2011-12), through which the computational software facility has been made available at the host institute. We also acknowledge the technical support team/application scientists of Schrodinger Inc. for their help during computational studies.The authors declare that they have no conflict of interest.


OUCMDZ-1635. Their structures, including the absolute configurations, were fully elucidated on the basis of spectroscopic analysis, ECD spectra, quantum chemical calculations, and chemical methods. Cladodionen (1) showed cytotoxic activities against MCF-7, HeLa, HCT-116, and HL-60 human cancer cell lines with IC50 values of 18.7, 19.1, 17.9, and 9.1 µM.A new hybrid polyketide, cladodionen ( Cladosporium sp. OUCMDZ-1635, isolated from a sponge (1), and a new analogue of abscisic acid, which we named cladosacid (2). Cladodionen (1) was isolated as an inseparable mixture of two geometric isomers because of dynamic interconversion  was isolated as a brown powder and presented as an inseparable mixture of two E/Z geometric isomers (1a and 1b). Although HPLC analysis of 1 through an ODS column (60% MeOH/H2O) showed that it afforded a baseline separation of the two isomers, attempts to separate the two isomers failed due to spontaneous and rapid isomerization between two forms. The positive ESI peak at m/z 234 [M + H]+ , 4.41 , 2.36/2.56 , 6.93 , and 7.70 . These data combined with 1H-1H COSY correlations extending from H-7 to H3-11 and the key HMBC correlations of H-7 to C-3/C-6 and H-10 to C-6/C-8  and 2.10 , along with the HMBC correlations of H3-13 to C-5/C-12/C-14, and H3-14 to C-5/C-12/C-13 (3-14 (δH 2.10) than H3-13 (δH 1.74) due to the deshielding effect of 4-carbonyl group. The remaining 1H and 13C NMR signals and the HMBC correlations of HN-1 (δH 9.79) to C-3 (δC 103.2), C-4 (δC 183.3) and C-5 (δC 129.3)  , along wd 2.10 H-, s, alon1, a methylation was done by adding NaH and CH3I reagents to the N,N-dimethylformamide (DMF) solution of 1 -hexa-2,4-dienoyl)-1,3-dimethyl-5-(propan-2-ylidene)pyrrolidine-2,4-dione (3) by MS (1 could undergo an N-methylation followed by an intramolecular elimination  method  method , respectboth 10S .2) was obtained as an amorphous powder and its molecular formula was determined to be C15H20O3 on the basis of a HRESIMS peak at m/z 249.1496 [M − H]−  to δCOOH of callilongisin D (170.5 ppm)  data, indicating that the α,β-unsaturated carboxylic acid and α,β-unsaturated ketone chromophores in 2 must interact with each other to generate exciton ECDs + ; ESIMS m/z 234 [M + H]+.Cladosacid (2): amorphous powder; max (log ε) 245 (3.11) nm; ECD  λmax (Δε) 239 (+0.44), 218 (−0.32); IR (KBr) νmax 3478, 2929, 1766, 1384, 1096 cm−1; 1H and 13C NMR data, see m/z 251.1645 [M + H] +  and m/z 249.1496 [M − H]− ; ESIMS m/z 249 [M − H]−.N,N- dimethylformamide (DMF) (1.0 mL) was added a solution of compound 1 (5.0 mg) in DMF (1.0 mL) at −5 °C. After stirring for 0.5 h, 15 μL of CH3I was added dropwise and the mixture was stirred for another 0.5 h. Then the reaction was quenched by the addition of saturated aqueous NH4Cl solution (2.0 mL) and extracted with CH2Cl2 (3 × 10 mL). The organic layers were combined and washed with H2O (2 × 10 mL) and concentrated in vacuum. The residue was then purified by HPLC over an ODS column (70% MeOH/H2O) to afford compound 3 .To a stirred suspension of sodium hydride  in Z,4Z)-Hexa-2,4-dienoyl)-1,3-dimethyl-5-(propan-2-ylidene)pyrrolidine-2,4-dione (3)3-pyrrolidine-2,4-dione nucleus, cladodionen (1), along with a new abscisic acid analogue, cladosacid (2), were isolated and identified from a solid cultural products of the marine-derived Cladosporium sp. OUCMDZ-1635. Cladodionen (1) exhibit cytotoxic activities against MCF-7, HeLa, HCT-116, and HL-60 human cancer cell lines with the IC50 values of 18.7, 19.1, 17.9, and 9.1 µM.A new hybrid polyketide with a 3-(2


All of these compounds were evaluated for antifouling activity against cyprids of the barnacle Balanus  amphitrite. The results showed that compounds 1–3 and 6–13 were active, with EC50 values ranging from 2.95 ± 0.59 to 69.19 ± 9.51 μM, respectively. The inhibitive effect of compounds 1–3 and 7 was reversible. This is the first description of the antifouling activity of thielavins against barnacle cyprids.Eleven new depsides—thielavins W–Z ( Winogradskyella Poriferorum + at m/z 511.1604, corresponding to a molecular formula of C27H26O10 , and the UV spectrum revealed absorption at λmax 218.1, 267.8, and 304.2 nm. The 1H, 13C-NMR , 2.40 , 2.29 , 2.06 , 2.06 , 1.99 , 18 olefinic carbons, including fifteen quaternary carbons at δC 138.7, 165.4, 114.2, 149.5, 114.7, 131.8, 150.7, 116.5, 149.1, 120.8, 121.6, 141.0, 161.4, 160.8, and 107.6, and 3 tertiary carbons at δC 110.3, 100.6, 110.3. Three carbonyl signals were also observed at δC 172.0, 167.1, and 166.7. These data suggested that the compound is similar to thielavin H (13) , 1 methoxy signal [δH 3.83 ], and 1 olefinic proton signal [δH 6.40 ]. The 13C-NMR and HSQC results  and δC 150.7 (C-2), 116.0 (C-3) and 148.5 (C-4), between δH 1.96  and δC 120.1 (C-5), 121.4 (C-6), C-4, and between δH 2.13 (6-Me) to δC 132.1 (C-1), C-5 and C-6, and also revealed long range correlations between δH 3.83 (1-COOMe) and δC 168.7 (1-C=O). NOESY results show the correlation from protons 6-Me to methoxy proton (1-COOMe), indicating the presence of a 2-hydroxyl-4-oxygenated-3,5,6-trimethylbenzoic methyl ester subunit, corresponding to the ion fragment of m/z 179.1. The HMBC correlations (δH 1.97 (3′-Me) to δC 162.6 (C-2′), 108.6 (C-3′) and 161.0 (C-4′), from δH 6.40 (H-5′) to δC 102.7 (C-6′), 24.0 (6′-Me), C-3′ and C-4′, and from δH 2.54 (6′-Me) to δC 139.4 (C-1′), 111.0 (C-5′) and C-6′, suggested the presence of a 2-hydroxyl-4-oxygenated-3,6-dimethylbenzoyl subunit, corresponding to an ion fragment with m/z 211.1  and named thielavin Z7.Compound e 1H NMR  revealed results  indicateelations c betweenelations c from δH/z 211.1 d. These shown in  and name12–15 were identified as thielavin A (12), thielavin H (13), thielavin J (14), and thielavin K (15), by comparison of their spectral data with those reported in the literature + ; ISCID data, see Thielavin W (2): White powder, UV ACN λmax 218.1, 267.8, 306.5 nm; 1H and 13C-NMR data, see m/z 525.1727 [M + H]+ ; ISCID data, see Thielavin X (3): White powder, UV ACN λmax 218.1, 276.1, 304.7 nm; 1H and 13C-NMR data, see m/z 525.1765 [M + H]+ ; ISCID data, see Thielavin Y (4): White powder, UV ACN λmax 218.1, 276.1, 306.5 nm; 1H and 13C-NMR data, see m/z 525.1757 [M + H]+ ; ISCID data, see Thielavin Z (1 (5): White powder, UV ACN λmax 218.1, 276.1, 306.9 nm; 1H and 13C-NMR data, see m/z 525.1747 [M + H]+ ; ISCID data, see Thielavin Z2 (6): White powder, UV ACN λmax 218.1, 267.8, 305.2 nm; 1H and 13C-NMR data, see m/z 539.1916 [M + H]+ ; ISCID data, see Thielavin Z3 (7): White powder, UV ACN λmax 214.6, 276.1, 309.1 nm; 1H and 13C-NMR data, see m/z 525.1758 [M + H]+ ; ISCID data, see Thielavin Z4 (8): White powder, UV ACN λmax 218.1, 267.8, 305.2 nm; 1H and 13C-NMR data, see m/z 511.1564 [M + H]+ ; ISCID data, see Thielavin Z5 (9): White powder, UV ACN λmax 213.4, 267.8, 307.0 nm; 1H and 13C-NMR data, see m/z 525.1778 [M + H]+ ; ISCID data, see Thielavin Z6 (10): White powder, UV ACN λmax 215.8, 276.1, 307.0 nm; 1H and 13C-NMR data, see m/z 587.1719/589.1646 [M + H]+ 3:1 ; ISCID data, see Thielavin Z7 (11): White powder, UV ACN λmax 215.8, 276.1, 308.9 nm; 1H and 13C-NMR data, see m/z 375.1464 [M + H]+ ; ISCID data, see Thielavin ZB. amphitrite, adult brood stocks of B. amphitrite were collected from piling at the Pak Sha Wan Pier, Hong Kong . Barnacle larvae were obtained and reared to cyprid stage as previously described by Harder et al. [−1 in dimethyl sulfoxide (DMSO) and further diluted to varying concentrations  immediately before each bioassay. One milliliter of the test solutions was added to each well of a 24-well plate , and approximately 15 larvae were gently pipetted into each well. For the negative controls, 1.0 mL of FSW together with 1.0 μL of DMSO, instead of the testing solution, was added to each well prior to the addition of larvae. The plates were incubated at 28 °C for 48 h in the dark. At the end of the incubation, the numbers of settled and swimming larvae were counted directly under a microscope, and where appropriate, potential toxic effects were noted. The number of settled larvae was expressed as a percentage of the total number of larvae per well. Three replicates of each extract concentration or purified compound were evaluated. EC50 represents the concentration of a compound that inhibits settlement of 50% of the larval population compared with the negative control, while the LC50 represents the concentration of a compound that is required to kill 50% of the larvae in a tested population. The experiments were performed in triplicate.To prepare the larval culture of the barnacle B. amphitrite. Three replicates of each experimental concentration were assessed.In the recovery assay, cyprids used in the negative controls were placed in new polypropylene containers containing FSW for 24 h, as described in the literature . One milThielavia terricola [Thielavia sp. UST030930-004, including 11 new ones, and 11 of them had antifouling activities. Of all of the compounds, Thielavin W (1) showed excellent antifouling activity, and cyprids treated with this compound had the highest recovery rate. Thus, Thielavin W is a promising natural antifoulant.Thielavins are para-depside-type compounds originally isolated from erricola . To dateerricola ,17,18. Terricola ,16,19,20


Undecenoic acid was methylated to methyl 10-undecenoate which was subjected to a thiol–ene reaction with cysteamine hydrochloride. Further amidation of the amine was carried out with different phenolic acids such as caffeic, ferulic, sinapic, coumaric and cinnamic acid. All synthesized compounds were fully characterized and their structures were conﬁrmed by spectral data. The anti-oxidant activity of the synthesized lipoconjugates of phenolic acids was studied by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay and also by the inhibition of linoleic acid oxidation in micellar medium by differential scanning calorimetry (DSC). The prepared compounds were also screened for their cytotoxic activity against five cell lines. It was observed that the lipoconjugates of caffeic acid, sinapic acid, ferulic acid, and coumaric acid displayed anticancer and anti-oxidant properties. The anticancer properties of these derivatives have been assessed by their IC Phenolic compounds are a class of natural compounds which are found ubiquitously in the plant kingdom. They are reported to possess a wide range of biological properties like anti-oxidant, antimicrobial, anti-inflammatory, anticarcinogenic and antiviral activities . The phe1) in quantitative yield. Next, ester 1 was treated with 1,1′-azobis(cyclohexanecarbonitrile) (ABCN) and 2-mercaptoethylamine hydrochloride in dioxane/ethanol 70:30 (v:v) to obtain methyl 11-(2-aminoethylthio)undecanoate (2) in 89% yield. The structures of compounds 1 and 2 were in agreement with the reported literature data +, 428 [M + Na]+; HRMS (m/z): [M + H]+ calcd for C23H36O3NS, 406.24104; found, 406.24077.ications . BrieflySynthesis of methyl 11-((2-((E)-3-(4-hydroxyphenyl)acrylamido)ethyl)sulfanyl)undecanoate (3b): Similarly, methyl 11-((2-((E)-3-(4-hydroxyphenyl)acrylamido)ethyl)sulfanyl)undecanoate (3b) was prepared from 2  and coumaric acid  in 85% yield (0.78 g) and the product was characterized by 1H and 13C NMR, IR, ESIMS and HRMS spectral studies. Mp 64–65 °C; 1H NMR  δ 7.57 , 7.39 , 6.85 , 6.26 , 3.67 , 3.58 , 2.73 , 2.58 , 2.30 , 1.24–1.62 ; 13C NMR  δ 174.71 (-C(O)-OCH3), 166. 84 (-NH-C(O)-), 158.33, 141.51 (-NH-C(O)-CH=CH-),129.64, 128.99, 117.32 (-NH-C(O)-CH=CH-), 115.98, 51.58 (-C(O)-OCH3-), 38.63 (-CH2-NH-), 34.16 (-S-CH2-), 31.89 (-CH2-S-), 31.84 (-CH2-CH2-S-), 29.69–24.96 (-CH2-CH2-); IR : 3409, 2923, 2853, 1729, 1652, 1595, 1519, 1452, 1373; ESIMS (m/z): 422 [M + H]+, 444 [M + Na]+; HRMS (m/z): [M + H]+ calcd for C26H36O4NS, 422.23596; found, 422.23491.Synthesis of methyl 11-((2-((E)-3-acrylamido)ethyl)sulfanyl)undecanoate (3c): Similarly, methyl 11-((2-((E)-3-acrylamido)ethyl)sulfanyl)undecanoate was prepared from 2  and caffeic acid  in 85% yield (0.81 g) and the product was characterized by 1H and 13C NMR, IR, ESIMS and HRMS spectral studies. 1H NMR  δ 7.55 , 7.12 , 6.95  6.87 , 6.24 , 3.68 , 3.56 , 2.73 , 2.58 , 2.30 , 1.24–1.62 ; 13C NMR  δ 174.74 (-C(O)-OCH3), 167. 38 (-NH-C(O)-), 146.87, 144.53, 142.53 (-NH-C(O)-CH=CH-), 127.07, 121.33, 117.12 (-NH-C(O)-CH=CH-), 115.46, 114.71, 51.60 (-C(O)-OCH3-), 39.05 (-CH2-NH-), 38.88 (-S-CH2-), 38.76 (-CH2-S-), 34.16 (-CH2-CH2-S-), 29.59–24.96 (-CH2-CH2-); IR : 3359, 2953, 2854, 1721, 1654, 1599, 1527, 1441, 1365; ESIMS (m/z): 438 [M + H]+, 460 [M + Na]+; HRMS (m/z): [M + H]+ calcd for C23H36O5NS, 438.23087; found, 438.23023.Synthesis of methyl 11-((2-((E)-3-(4-hydroxy-3-methoxyphenyl)acrylamido)ethyl)sulfanyl)undecanoate (3d): Similarly, methyl 11-((2-((E)-3-(4-hydroxy-3-methoxyphenyl)acrylamido)ethyl)sulfanyl)undecanoate was prepared from 2  and ferulic acid  in 84% yield (0.82 g) and the product was characterized by 1H and 13C NMR, IR, ESIMS and HRMS spectral studies. 1H NMR  δ 7.55 , 7.06 , 7.00 , 6.91 , 6.27 , 3.92 , 3.67 , 3.58 , 2.73 , 2.58 , 2.30 , 1.24–1.62 ; 13C NMR  δ 174.36 (-C(O)-OCH3), 166. 21 (-NH-C(O)-), 147.48, 146.76, 141.26 (-NH-C(O)-CH=CH-), 127.33, 122.22, 118.06 (-NH-C(O)-CH=CH-), 114.76, 109.64, 55.95 (-OCH3-), 51.45 (-C(O)-OCH3-), 38.42 (-CH2-NH-), 34.11 (-S-CH2-), 31.97 (-CH2-S), 31.74 (-CH2-CH2-S-), 29.47–24.95 (-CH2-CH2-); IR : 3375, 2926, 2853, 1730, 1656, 1596, 1516, 1433, 1273; ESIMS (m/z): 452 [M + H]+, 474 [M + Na]+; HRMS (m/z): [M + H]+ calcd for C24H38O5NS, 452.24652; found, 452.24475.Synthesis of methyl-11-((2-((E)-3-acrylamido)ethyl)sulfanyl)undecanoate (3e): Similarly, methyl 11-((2-((E)-3-acrylamido)ethyl)sulfanyl)undecanoate was prepared from 2  and sinapic acid  in 85% yield (0.89 g) and the product was characterized by 1H and 13C NMR, IR, ESIMS and HRMS spectral studies. Mp 69–70 °C; 1H NMR  δ 7.53 , 6.76 , 6.28 , 3.93 , 3.67 , 3.58 , 2.73 , 2.58 , 2.30 , 1.24–1.62 ; 13C NMR  δ 174.37 (-C(O)-OCH3), 166.07 (-NH-C(O)-), 147.22, 141.43 (-NH-C(O)-CH=CH-),136.64, 126.29, 118.44 (-NH-C(O)-CH=CH-), 104.82, 56.34 (-OCH3-), 51.48 (-C(O)-OCH3-), 38.39 (-CH2-NH-), 34.11 (-S-CH2-), 31.96 (-CH2-S-), 31.69 (-CH2-CH2-S-), 29.65–24.95 (-CH2-CH2-); IR : 3371, 2926, 2852, 1730, 1658, 1612, 1514, 1455 1285; ESIMS (m/z): 482 [M + H]+, 504 [M + Na]+; HRMS (m/z): [M + H]+ calcd for C25H40O6NS, 482.25709; found, 482.25532.Ac − As)/Ac] × 100 where Ac is the absorbance of the control and As is the absorbance of the tested sample after 40 min.The anti-oxidant activity was determined by the radical scavenging ability using the stable DPPH radical method as reported . BrieflyThe anti-oxidant activity was also evaluated by differential scanning calorimetry using pure linoleic acid as a lipid model system –24. All ® HTB-26™); SKOV3, ovarian cancer (ATCC® HTB-77™); MCF7, breast cancer (ATCC® HTB-22™); DU 145, prostate cancer (ATCC® HTB-81™); HepG2, liver hepatocellular carcinoma (ATCC® HB-8065™) were obtained from the ATCC  and maintained in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a 5% CO2 incubator. After seeding of cells in 96 well culture plates, they were allowed to attach properly. Test compounds of different concentrations ranging from 1 to 50 µM were added in triplicates and incubated for 24 h. The cells were then incubated with MTT (0.5 mg/mL) for 3 h and, to dissolve the insoluble formazan crystals, 100 µL DMSO was added to each well. Finally the absorbance of the plates was measured using a Synergy H1 multi-mode plate reader (USA). Doxorubicin was used as the positive control for comparison.The cytotoxicity assay (MTT) was evaluated for all test compounds as described in our earlier work . Five diFile 11H NMR, 13C NMR, HRMS and DSC spectra.Copies of


The structures of these compounds were elucidated on the basis of extensive spectroscopic analysis. The cytotoxicity of some compounds against a panel of human cancer cell lines is also reported.Eight new sterols ( Petrosia (Strongylophora) collected from the Similan Island, Thailand. Previously, various studies on the chemical constituents of Petrosia sp. led to the isolation of cyclosterols − , three singlet methyls at δ 0.91 (CH3-19), 1.23 (CH3-18), and 1.35 (CH3-21), and two doublet methyls at δ 1.02  and 1.06 . The 13C NMR , which was also isolated in this study and reported previously from the marine sponge Xestospongia testudinaria − also supported this result. The orientation of 3-OH in 3 was determined by the carbon chemical shifts at C-3 and coupling constants of H-3. The downfield chemical shift of C-3 from δC 65.7 in 1 to 71.1 + . After comparing the 1H and 13C NMR spectral data with that of compound 1 . Therefore, compound 4 was established as 26,27-cyclo-24,27-dimethylcholestan-3,3-dimethoxy-7α,12β,20β-triol .Compound mpound 1 , the pro31H54O5 of compound 5 was deduced from APCI-TOF MS. The 1H and 13C NMR spectral data closely resembled those of 4 except that the secondary hydroxyl group at C-7 and methyl group at C-13 in 4 were replaced by a methylene (δH 0.81 and 1.65) and a hydroxymethyl (δH 3.65 and 4.02/δC 60.8) group, respectively in 5. The HMBC correlations from H-18 to C-12 (δ 80.6) and C-17 (δ 61.2) supported the position of the hydroxymethyl at C-13 of the sterol. Thus, compound 5 was elucidated as the new steroid 26,27-cyclo-24,27-dimethylcholestan-3,3-dimethoxy-12β,18,20β-triol.The molecular formula C11 was determined as C31H54O4 by APCI-TOF MS. The 1H and 13C NMR spectra of 11  and the hydroxy-substituted quaternary carbon at C-20 (δC 74.4) in 4 was replaced by the methine (δH 1.85/δC 32.3) group. The 16α-OH group in 11 was assigned on the basis of its correlations of H-16 with C-20 (δ 32.3) and C-14 (δ 51.3) in the HMBC correlations and the cross peak of H-17 and H-16 in the 1H-1H COSY together with the correlation between H-16 and CH3-18 in the NOESY spectrum. In addition, the relative stereochemistry of C-20–C-29 side chain at C-17 of compound 11 was confirmed by the similarity of the 1H and 13C chemical shifts to those of known petrosterol (19) − . The IR spectrum showed characteristic absorption bands of a hydroxyl group at 3518 cm−1 and a carbonyl group at 1713 cm−1. The 1H and 13C NMR spectra of the rings A–D of 12 (8) D) with literature values.Besides these eight new compounds, the eight known structures, aragusterol I (6) , aragust6) c 0.53, CHCl3); IR (ATR) νmax: 3304, 2927, 2926, 2857, 1449, 1372, 1004 cm−1; 1H and 13C NMR data .Compound data see ; APCI-TO3. White amorphous powder; [α]c 0.53, MeOH); IR (ATR) νmax: 3239, 2926, 2855, 1734, 1455, 1374, 1056, 1015 cm−1; 1H and 13C NMR data .Compound data see ; APCI-TO4. White amorphous powder; [α]c 0.90, CHCl3); IR (ATR) νmax: 3230, 2951, 2918, 2870, 1720, 1452, 1373, 1362, 1243, 1175, 1141, 1116, 1044, 1018, 921, 750 cm−1; 1H and 13C NMR data .Compound data see ; ESI-TOF5. White amorphous powder; [α]c 0.30, CH2Cl2); 1H and 13C NMR data .Compound data see ; APCI-TO11. White amorphous powder; 1H and 13C NMR data .Compound data see ; APCI-TO12. White amorphous powder; [α]c 1.82, MeOH); IR (ATR) νmax: 3518, 3248, 2955, 2871, 1713, 1450, 1384, 1226, 1156, 1063, 1041, 1019, 974, 748 cm−1; 1H and 13C NMR data .Compound data see ; APCI-TO13. White amorphous powder; [α]c 0.80, CHCl3); IR (ATR) νmax: 3274, 2946, 2871, 1716, 1456, 1360, 1178, 1055, 970 cm−1; 1H and 13C NMR data .Compound data see ; APCI-TOThe cytotoxic activity toward a panel of mammalian cancer cell lines  were tested using the 3--2,5-diphenyltetrazolium bromide (MTT) assay , while tPetrosia sp. led to the isolation of eight new (1–5 and 11–13) and eight known (6–10 and 14–16) sterols. Their structures were established by the basis of spectroscopic method. Some compounds  were evaluated for their cytotoxicity using a panel of human cancer cell lines. The most potent, compound 15, was cytotoxic, with the IC50 values of 7.10 and 6.11 μM against HepG-2 and HeLa cell lines, respectively.The chemical investigation of Thai marine sponge


Pteris vittata L. is rich in flavonoids which exhibit different bioactivities. In order to investigate the flavonoids components of P. vittata L., extracts of the plant were isolated by column chromatography on silica gel and Sephadex LH-20.1), quercetin (2), kaempferol-3-O-d-glucopyranoside (3) and rutin (4), respectively, on the basis of NMR spectroscopic analyses.Four flavonoids compounds were obtained, the structures were identified as kaempferol (3 was reported for the first time from P. vittata L.Compound The online version of this article (doi:10.1186/s40064-016-3308-9) contains supplementary material, which is available to authorized users. Pteris vittata L., a common fern known as ‘Chinese Brake Fern’, is native from China and widespread all over the world . The voucher specimen (No. PGZ090239) has been deposited at the CANT Herbarium, South China Agricultural University, Guangzhou, P. R. China.The whole plant of 254 silica gel for thin layer chromatography (TLC) were purchased from Qingdao Marine Chemical Ltd., Qingdao, China. Sephadex LH-20 obtained from Amersham Biosciences, Sweden was used for CC. Other reagents were of analytical grade purchased from Guangzhou reagent Co. Ltd. NMR spectra  were determined on a Bruker AV-600 instrument using TMS as an internal reference.Silica gel (100–200 and 200–300 mesh) for column chromatography (CC) and GFP. vittata L. was extracted with methanol for three times at 25 °C. After the solvent was removed under vacuum, the concentrated extract was further extracted successively with petroleum ether, ethyl acetate and n-butanol. The ethyl acetate extract (80 g) was subjected to silica gel column chromatography using gradient hexane–EtOAc (50:1–1:1) to obtain 6 fractions. Fraction 2 (12 g) was subjected to silica gel column chromatography eluted with gradient hexane–acetone (100:1–10:1) to afford compound 1 (52 mg). Fraction 3 (8 g) was subjected repeatedly to silica gel column chromatography eluted with hexane–acetone (100:5) and was finally purified by column chromatography on Sephadex LH-20 eluted with CHCl3–MeOH (1:1) to give compound 2 (53 mg). Fraction 5 (2 g) was subjected to silica gel column chromatography eluted with CHCl3–MeOH (95:5) to give compound 3 (36 mg). Fraction 6 (5 g) was subjected to silica gel column chromatography eluted with CHCl3–MeOH (10:1) led to the isolation of compound 4 (280 mg).The whole plant (7.0 kg) of 1), quercetin (2), kaempferol-3-O-d-glucopyranoside (3) and rutin (4), respectively, by spectral analysis and comparison with the spectroscopic data reported in previous literatures  δH: 6.19 , 6.44 , 6.92 , 8.04 . 13C-NMR  δC: 93.5 (C-8), 98.2 (C-6), 103.0 (C-10), 115.4 , 121.7 (C-1′), 129.5 (C-2′), 130.5 (C-6′), 135.6 (C-3), 146.8 (C-2), 156.2 (C-5), 159.2 (C-4′), 160.7 (C-9), 163.9 (C-7), 175.9 (C-4). It was determined as kaempferol by comparison with the spectroscopic data reported in the literature  δH: 6.19 , 6.40 , 6.88 , 7.54 , 7.67 . 13C-NMR  δC: 93.3 (C-8), 98.1 (C-6), 102.9 (C-10), 114.9 (C-2′), 115.5 (C-5′), 119.9 (C-6′), 121.8 (C-1′), 135.6 (C-3), 144.9 (C-1′), 146.7 (C-1), 147.6 (C-4′), 156.1 (C-5), 160.7 (C-9), 163.8 (C-7), 175.8 (C-4). It was identified as quercetin by comparison with the spectroscopic data reported in the literature  δH: 3.09 , 3.21 , 3.32 , 3.55 , 4.29 , 4.96 , 5.07 , 5.36 , 5.46, 6.21 , 6.43 , 6.88 , 8.04 . 13C-NMR  δC: 61.1 (C-6″), 70.2 (C-4″), 74.5 (C-2″), 76.8 (C-5″), 77.8 (C-3″), 94.0 (C-8), 99.1 (C-6), 101.2 (C-1″), 104.3 (C-10), 115.4 , 121.2 (C-1′), 131.2 , 133.5 (C-3), 156.6 (C-2), 156.7 (C-9), 160.3 (C-4′), 161.5 (C-5), 164.5 (C-7), 177.8 (C-4). It was determined as kaempferol-3-O-d-glucopyranoside by comparison with the spectroscopic data reported in the literature  δH: 1.00 , 3.08 , 3.70 , 4.39 , 5.10 , 5.28 , 5.35 , 6.20 , 6.39 , 7.54 , 7.56  13C-NMR : δC: 17.6 (C-6′″), 66.9 (C-6″), 68.1 (C-5′″), 69.9 (C-4″), 70.2 (C-2′″), 70.4 (C-3′″), 71.7 (C-4′″), 73.9 (C-2″), 75.8 (C-5″), 76.3 (C-3″), 93.5 (C-8), 98.5 (C-6), 100.6 (C-1′″), 101.0 (C-1″), 103.8 (C-10), 115.1 (C-2′), 116.1 (C-5′), 121.0 (C-6′), 121.5 (C-1′), 133.2 (C-3), 144.6 (C-3′), 148.3 (C-4′), 156.3 (C-9), 156.5 (C-2), 161.1 (C-5), 164.0 (C-7), 177.2 (C-4). It was identified as rutin compared with the spectroscopic data reported in the literature , quercetin (2), kaempferol-3-O-d-glucopyranoside (3) and rutin (4), respectively. Compound 1, 2 and 4 were known components which were mentioned in the previous results (Imperato and Telesca 3 was a derivative of kaempferol with a glucopyranoside. It is reported for the first time from P. vittata L. The current results may provide more information about flavonoids profiles of this plant.Phytochemical screening of


As the inhibitor of AKT1 , the aforementioned compounds may have implication in preventing complications of cancers. In recent years, the chemistry of 2-chloroquinoline-3-carbaldehydes have received considerable attention owing to their synthetic and effective biological importance which exhibits a wide variety of biological activity, N1, N4-bis ((2-chloroquinolin-3-yl) methylene) benzene-1, 4-diamine derivatives (3a-3i)  were synthesized, and theoretically evaluated for their inhibitory as Potential Human AKT1 Inhibitors via docking process. The docking calculation was done in GOLD 5.2.2 software using Genetic algorithm.A group of NCompounds 3b (6-Me) and 3d (6-OEt) showed the best inhibitory potency by GOLD score value of 113.76 and 107.58 respectively. Some of the best models formed strong hydrogen bonds with Asn 49, Lys 220, Ser 157, Arg 225 and Trp 76 via quinoline moiety and nitrogen of quinolone ring . pi-pi interaction between Lys 220, Trp 76, Tyr 224, Arg 225, Ile 80, and Asn 49 quinoline moiety was one of the common factor in enzyme-inhibitor junction.It was found that both hydrogen bonding and hydrophobic interactions are important in function of biological molecules, especially for inhibition in a complex. AKT1/PKBa (protein kinase B) is a serine/threonine kinase that belongs to the AKT family AKT1 , also known as «AKT» or protein kinase B (PKB) is an important molecule in mammalian cellular signaling. In humans, there are three genes in the «AKT family»: AKT1, AKT2, and AKT3. These enzymes are members of the serine/threonine-specific protein kinase family  methylene) benzene-1, 4-diamine.GOLD, the first algorithm to be evaluated on a large data set of complex posse, an empirical free energy scoring function that estimates the free energy of binding permitting inhibition constant for protein ligand complex. It is a package of program for structure visualization and manipulation for docking, the post processing and visualization of the results . The obj1, N4-bis ((2-chloroquinolin-3-yl) methylene) benzene-1, 4-diamine (3a-3i). Therefore, we studied our compound as a potential inhibitor for AKT1 enzyme.Quinoline and its derivatives have always attracted both synthetic and biological chemists because of its diverse chemical and pharmacological properties . For exaMaterials and Methods1H NMR, elemental analyses). The purity determinations of the products were accomplished by TLC on silica gel polygram STL G/UV 254 plates. Melting points were determined with an Electrothermal Type 9100 melting point apparatus. Elemental analyses were made by a Thermo Finning Flash EA1112 CHNO-S analyzer and agreed with the calculated values. The FTIR spectra were recorded on an Avatar 370 FTIR Therma Nicolet spectrometer. The NMR spectra were recorded on a Bruker Avance 100 and 400 MHz instrument in CDCl3 and DMSO. The products were characterized by spectroscopic data  methylene) benzene-1, 4-diamine (3a-i).A mixture of 2-chloroquinoline-3-carbaldehydes  (1), benzene-1,4-diamine  (2), few drops glacial acetic acid and 40 mL EtOH in a 100 mL flask was stirred at reflux for 4 h. After completion of the reaction , the resulting solid was separated by filtration, and recrystallized from ethanol to afford pure product.N1, N4-Bis ((2-chloroquinolin-3-yl) methylene) benzene-1, 4-diamine (3a) -1: 3000-3300 (CH aromatic), 2952 , 1616 (C=N imine), 1557 (C=N quinoline), 1520 (C=C quinoline), 1500 (C=C phenyl), 1057 (C-Cl quinolone ring). 1H NMR  δ: 7.46 , 7.65 , 7.84 , 8.09 , 8.10 , 9.09, 9.10. Anal. Calcd. for C26H16Cl2N4: C, 68.58; H, 3.54; Cl,15.57; N, 12.30; Found: C, 68.54; H, 3.57; N, 12.28. Yield: 72%; mp 195 °C; IR (KBr) υcmN1, N4-Bis ((2-chloro-6-methylquinolin-3-yl) methylene) benzene-1, 4-diamine (3b)-1: 3100 (CH aromatic), 2900-3000 , 1619 (C=N imine), 1580 (C=N quinoline), 1525 (C=C quinoline), 1504 (C=C phenyl), 1059 (C-Cl quinolone ring). 1H NMR  δ: 2.46 , 6.36 , 7.24 , 7.86 , 7.88 , 7.95 , 8.87 . Anal. Calcd. for C28H20Cl2N4: C, 69.57; H, 4.17; Cl,14.67; N, 11.59; Found: C, 69.59; H, 4.20; N, 11.62.Yield: 70%; mp 212-215 °C; IR (KBr) υ cmN1, N4-Bis ((2-chloro-6-methoxyquinolin-3-yl) methylene) benzene-1, 4-diamine (3c)-1: 3100-3200 (CH aromatic), 2900-3000 , 1615 (C=N imine), 1572 (C=N quinoline), 1518 (C=C quinoline), 1498(C=C phenyl), 1234(C-O), 1055(C-Cl quinolone ring). 1H NMR  δ: 3.01 , 6.37 , 6.61 , 7.22 , 7.62 , 7.88 , 8.87 . Anal. Calcd. for C28H20Cl2N4O2: C, 65.25; H, 3.91; Cl,13.76; N, 10.87; Found: C, 65.21; H, 3.90; N, 10.89.Yield: 69%; mp 218-220 °C; IR (KBr) υ cmN1, N4-Bis ((2-chloro-6-ethoxyquinolin-3-yl) methylene) benzene-1, 4-diamine (3d)-1: 3100-3150 (CH aromatic), 2895-2950 , 1600 (C=N imine), 1570 (C=N quinoline), 1520 (C=C quinoline), 1500 (C=C phenyl), 1234 (C-O), 1057 (C-Cl quinolone ring). 1H NMR  δ: 1.38 , 2.74 , 6.39 , 6.61 , 7.21 , 7.45 , 7.60 , 8.88 . Anal. Calcd. for C30H24Cl2N4O2: C, 66.30; H, 4.45; Cl,13.05; N, 10.31; Found: C, 66.29; H, 4.41; N, 10.28.Yield: 74%; mp 196 °C; IR (KBr) υ cmN1, N4-Bis  methylene) benzene-1, 4-diamine (3e)-1: 2900-3300 (CH aromatic), 2862 , 1620 (C=N imine), 1573 (C=N quinoline), 1519 (C=C quinoline), 1500 (C=C phenyl), 1055(C-Cl quinolone ring). 1H NMR  δ: 6.42 , 6.62 , 7.86 , 7.90 , 8.37 , 8.88 . Anal. Calcd. For C26H14Cl4N4: C, 59.57; H, 2.69; Cl, 27.05; N, 10.69; Found: C, 59.60; H, 2.71; N, 10.66.Yield: 74%; mp 196-198 °C; IR (KBr) υ cmN1, N4-Bis ((2-chloro-7-methylquinolin-3-yl) methylene) benzene-1, 4-diamine (3f)-1: 3110-3210 (CH aromatic), 2950 , 1612 (C=N imine), 1585 (C=N quinoline), 1519 (C=C quinoline), 1489 (C=C phenyl), 1057 (C-Cl quinolone ring). 1H NMR  δ: 2.60, 7.42 , 7.59 , 7.44 , 7.84 , 7.88 , 9.02 . Anal. Calcd. for C28H20Cl2N4: C, 69.57; H, 4.17; Cl, 14.67; N, 11.59; Found: C, 69.52; H, 4.12; N, 11.61. Yield: 70%; mp 198 °C; IR (KBr) υ cmN1, N4-Bis ((2-chloro-6-ethylquinolin-3-yl) methylene) benzene-1, 4-diamine (3g)-1: 3000-3100 (CH aromatic), 2900-3000 , 1610 (C=N imine), 1583 (C=N quinoline), 1517 (C=C quinoline), 1489 (C=C phenyl), 1061 (C-Cl quinolone ring). 1H NMR  δ: 1.35 , 2.85 , 7.42 , 7.67 , 7.96, , 7.73 , 8.42 , 9.02 . Anal. Calcd. for C30H24Cl2N4: C, 70.45; H, 4.73; Cl, 13.86; N, 10.59; Found: C, 70.47; H, 4.70; N, 10.94.Yield: 69%; mp 190 °C; IR (KBr) υ cmN1, N4-Bis ((2-chloro-6-isopropylquinolin-3-yl) methylene) benzene-1, 4-diamine (3h)-1: 3100-3200 (CH aromatic), 2800-3000 , 1615 (C=N imine), 1581 (C=N quinoline), 1515 (C=C quinoline), 1500 (C=C phenyl), 1064 (C-Cl quinolone ring). 1H NMR  δ: 1.36 , 3.10 , 7.43 , 7.73 , 7.74 , 7.42 , 7.99, , 9.04 . Anal. Calcd. for C32H28Cl2N4: C, 71.24; H, 5.23; Cl, 13.14; N, 10.38; Found: C, 71.24; H, 5.20; N, 10.36.Yield: 68%; mp 200 °C; IR (KBr) υ cmN1, N4-Bis  methylene) benzene-1, 4-diamine (3i)-1: 3000-3200 (CH aromatic), 2907-3005 , 1610 (C=N imine), 1583 (C=N quinoline), 1519 (C=C quinoline), 1477(C=C phenyl), 1060(C-Cl quinolone ring). 1H NMR  δ: 6.48 , 7.70 , 8.24 , 8.04 , 8.85 , 8.82 . Anal. Calcd. For C26H14Cl4N4: C, 59.57; H, 2.69; Cl, 27.05; N, 10.69; Found: C, 59.55; H, 2.69; N, 10.72.Yield: 75%; mp 195 °C; IR (KBr) υ cmStructure optimization-1) . In the second optimization, output files were minimized under Semi empirical AM1 methods   , 17.Crystal structures of AKT1 (EC.2.7.11.1) were retrieved from RCSB Protein Data Bank (PD B entry: 3O96).Molecular dockingDocking was carried out using GOLD 5.2 (Genetic optimization for Ligand Docking) software based on the Gold Score fitness function, that uses the Genetic algorithm (GA). All water molecules and hetero atoms were omitted from the protein to evaluate the two scoring functions in GOLD. For each of the 25 independent GA runs, a maximum number of 100000 GA functions were established on a set of five groups with a population size of 100 individuals. Mutation, migration and operator weights for crossover were set to 95, 10, and 95, respectively. Default cutoff values of 4.0 A for van der Waals distance and 2.5 A (dH-X) for hydrogen bonds were employed. When the top three solutions achieved RMSD values enough 1.5 A, GA docking was terminated. The RMSD values for the docking computations are based on the RMSD matrix of the ranked solutions and observed that the best ranked solutions were always among the first 50 GA runs, and further analyzing of the conformation of molecules performed on the best fitness score. The docking procedure was validated by redocking of tolrestat to the AKT1 crystal structure 3O96.ChemistryTo formation the products (3a-3i), the reaction took place between 2-chloroquinoline-3- carbaldehydes (1a-1i) and benzene-1, 4-diamine  methylene) benzene-1, 4-diamine derivatives (3a-3i). The reactions proceeded very efficiently in relatively high yields as showed in In order to study the generality of this method, we extended our studies to synthesis of some Na) Isolated yieldDockingThe receptor 3O96 is a complex structure of enzyme. The possible active site was identified using Accelrys DS Visualizer. Eight active site residues as Trp 80, Ile 84, Cer 205, Lys 268, Tyr 272, Ile 290, Asp 292 andCys 296 were found. Therefore it is chosen as a most biologically favorable site for docking. Some of the best models formed strong hydrogen bonds with Asn 49, Lys 220, Ser 157, Arg 225 and Trp 76 via quinoline moiety and nitrogen of quinolone ring .. pi - p1, N4-bis ((2-chloroquinolin-3-yl) methylene) benzene-1, 4-diamine derivatives (3a-3i). The 1H NMR spectrum of 3a consist of three singlet signals at δ 7.46, 9.09, 9.10 due to one aromatic and one imine hydrogen and one hydrogen of quinolone ring that posse nitrogen respectively. The doublet signal at δ 8.09 due to the hydrogen on the aromatic ring of quinolines and three multiple for the three hydrogens  were confirmed to the structure. The mass spectrum shows the molecular ion at 454 m/z. Also the absence of the stretching vibration band at 1689 cm-1 due to the carbonyl group (C=O) in IR spectrum confirmed the structure of 3a. The final proof for this structure received by the results of elemental analysis that was in good agreement calculated data. The complete spectral and elemental analytical data of the products confirmed the formation of new N1, N4-bis ((2-chloroquinolin-3-yl) methylene) benzene-1, 4-diamine with AKT1 was performed by using of GoldScore fitness function. The algorithm exhaustively searches the entire rotational and translational space of the ligand with respect to the receptors. The various solutions valuated by a score, which is equivalent to the absolute value of the total energy of the ligand in the protein environment. For each compound the best docking solutions of GOLD score was considered. GoldScore carry out a force field based scoring function and is made up of four components: 1) Ligand internal van der Waals energy ; 2) Ligand intermolecular hydrogen bond energy ; 3) Protein- ligand hydrogen bond energy ; 4) Protein-ligand van der Waals energy . When the total fitness score is computed the external vdw score is multiplied by a factor of 1.375. This is an empirical correction to persuade protein-ligand hydrophobic interaction. The fitness function has been optimized for the divination of ligand binding positions. GoldScore = S (hb_ext) + S (vdw_ext) + S (hb_int) + S (vdw_int), where S (hb_ext) is the protein- ligand hydrogen bond score, S (vdw_ext) is the protein-ligand Van der Waals score, S (hb_int) is the score from intermolecular hydrogen bond in the ligand and S (vdw_int) is the score from intermolecular strain in the ligand. Redocking of 1- quinoxalin- 6-yl) benzyl) piperidin-4-yl)-1H-benzo[d]imidazol- 2(3H)-one  ∆G = -44.43 and Ki = 1.64E-8, bonding model of the mentioned molecule was similar to which was reported in the crystal structure of 3O96. It was noted that GOLD scores of 3b and 3d are 113.76 and 107.58, respectively, which are greater than the other scores as shown in Docking of N1, N4-bis ((2-chloroquinolin-3-yl) methylene) benzene-1, 4-diamine involving condensation reaction of 2- chloroquinoline-3-carbaldehydes and p-phenylenediamine in acidic condition. Docking was carried out using GOLD 5.2. It was found that both hydrogen bonding and hydrophobic interaction play important roles in the structure and function of biological molecules, peculiarly for inhibition in a complex. It was noted that GOLD scores of 3b and 3d are greater than the other scores. In summary we have described an efficient and convenient synthesis of N


Saccharomonospora sp. CNQ-490 has yielded three new α-pyrones, saccharomonopyrones A–C (1–3). The chemical structures of these compounds were assigned from the interpretation of 1D, 2D NMR and mass spectrometry data. Saccharomonopyrone A (1) is the first α-pyrone microbial natural product bearing the ethyl-butyl ether chain in the molecule, while saccharomonopyrones B and C possess unusual 3-methyl and a 6-alkyl side-chain within a 3,4,5,6-tetrasubstituted α-pyrone moiety. Saccharomonopyrone A exhibited weak antioxidant activity using a cation radical scavenging activity assay with an IC50 value of 140 μM.Intensive study of the organic extract of the marine-derived bacterium  Saccharomonospora, a genus in the actinomycete family Pseudonocardiaceae, was first described in 1971 + at m/z 241.1446  indicated the molecular formula C13H20O4, which implied four degrees of unsaturation. UV absorption at 288 nm and IR absorptions at 3402, 1672, and 1569 cm−1 indicated the presence of an α-pyrone moiety.Saccharomonopyrone A , two quaternary sp2 carbons (δC 97.3 and 107.5), along with two methyl singlets [3-CH3  and 5-CH3 ], one methyl triplet  and five methylene groups [CH2-7 /CH2-8 /CH2-9 /CH2-10 /CH2-11 ]. Interpretation of 1H-1H correlation spectroscopy (COSY) correlations allowed the construction of two spin systems: [H-7 (δH 2.70)/H-8 (δH 3.56)] and [H-9 (δH 3.35)/H-10 (δH 1.43)/H-11 (δH 1.26)/H-12 (δH 0.83)]. Heteronuclear multiple bond correlation (HMBC) correlations from H-8 to C-9 (δC 69.6) and from H-9 to C-8 (δC 67.0), as well as the oxygenated carbon chemical shifts of C-8 and C-9, supported the attachment of two spin systems through an oxygen atom , C-3 (δC 97.3), C-4 (δC 164.3), and from 5-CH3 to C-4, C-5 (δC 107.5), C-6 (δC 155.2), indicated the position of the methyl groups on the α-pyrone ring. Lastly, the HMBC cross-peaks from H-7 to C-6 and C-5 supported the connectivity of the α-pyrone ring to the linear chain, thus completing the structure assignment of saccharomonopyrone A (1).The α-pyrone functionality was constructed as 3,5-dimethyl-4-hydroxypyran-2-one with a side chain substituent at C-6 by analysis of 2) was isolated as an amorphous white powder. Its molecular formula C14H22O3 was determined by the observation of a HR-ESIMS ion peak [M + H]+ at m/z 239.1651, . The infrared (IR) absorption at 1673 cm−1 was assigned to a conjugated ketone functionality. The 1H NMR spectrum of 2 was similar to that of 1 except for the absence of two oxygenated methylene protons and the presence of two methyl doublets. COSY NMR correlations (H-7 (δH 2.47)/H-8 (δH 1.51)/H-9 (δH 1.29)/H-10 (δH 1.17), H-12 (δH 0.85)/H-11 (δH 1.51)/H-13 (δH 0.85)) and HMBC correlations of H-12 and H-13 to C-10 (δC 38.4), and from H-9 (δH 1.29) to C-10, and from H-10 to C-9 (δC 26.6) permitted the construction of the aliphatic chain as a 5-methylhexyl moiety. The linkage of the alkyl moiety to the α-pyrone ring was secured from the HMBC correlation from H-7 to C-6 (δC 158.2), completing the structure assignment of saccharomonopyrone B (2) as 4-hydroxy-3,5-dimethyl-6-(5-methylhexyl)-α-pyrone.Saccharomonopyrone B (3) was also obtained as a white amorphous powder. The formula of 3 was deduced as C14H22O3 by the observation of the [M + H]+ ion peak at m/z 239.1665 . Compound 3 had the same molecular formula as 2. The only difference was that the 1H NMR of 3 displayed a methyl triplet. The analysis of 2D NMR spectroscopic data allowed the establishment of 3 as 4-hydroxy-3,5-dimethyl-6-heptyl-α-pyrone.Saccharomonopyrone C  were tested on various assays such as monoamine oxidase (MAO) inhibitory, acetylcholinesterase (AChE) inhibitory, β-site amyloid precursor protein cleaving enzyme 1 (BACE1), anti-osteoporosis, cytotoxicity, anti-tyrosinase, and antibacterial activities. However, the compounds did not display any significant biological activities in these assays. Interestingly, saccharomonopyrone A (1) showed weak antioxidant activity measuring free radical scavenging activity and cation radical scavenging activity in assays with IC50 values of 911 μM and 140 μM, respectively. Saccharomonopyrones A–C (1) has an unusual ethylbutyl ether moiety attached at C-6. There are no reports of an ether moiety attached at C-6 within the α-pyrone class. Some similar molecules with ethyl-methyl sulfide and propyl-methyl sulfide groups were obtained as bioengineered products + .Saccharomonopyrone A (2): amorphous white solid, UV (MeOH) λmax (log ε) 203 (2.53), 292 (2.32) nm; IR (KBr) νmax 3427, 2940, 1673, 1575 cm−1, 1H and 13C NMR data, m/z 239.1651 [M + H]+ .Saccharomonopyrone B (3): amorphous white solid, UV (MeOH) λmax (log ε) 201 (2.53), 291 (2.20) nm; IR (KBr) νmax 3395, 2930, 1665, 1560 cm−1, 1H and 13C NMR data, m/z 239.1665 [M + H]+ .Saccharomonopyrone C  and three Gram-negative  strains following a previously published method [50 21.02 ± 0.82 µM). Data are presented as the mean values ± standard deviation (SD) of three measurements. The free radical scavenging activity of each solution was then calculated as the percent inhibition according to the following equation:MAO inhibitory assay , AChE ind method . The ant50 13.01 ± 0.21 µM). Data are presented as the mean values ± standard deviation (SD) of the three measurements. The extent of decolorization is calculated as a percent reduction in absorbance. The percentage of cation radical scavenging was computed using the following equation:2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) cation radical scavenging activity of the compounds were tested using the spectroscopic method described by Roberta et al. . The ABTStatistical analyses for DPPH and ABTS cation radical scavenging activities were performed using GraphPad Prism 5 . The nonlinear regression was used to determine the dose-response inhibition. Results are expressed as means ± standard deviation of three independent experiments.1–3), were isolated from the marine sediment-derived bacterium Saccharomonospora sp. strain CNQ-490. Saccharomonopyrones A–C (1–3) are 3,4,5,6-tetra-subtituted α-pyrones. Saccharomonopyrone A is unusual in having an ether moiety attached at C-6, observed for the first time in this class of natural products. Analysis of the tetra-substituted α-pyrone biosynthetic gene cluster from this strain may provide an opportunity to discover diverse α-pyrone analogues.Three new α-pyrones, saccharomonopyrones A–C (


Burkholderia sp. MAK1 (DSM102049), capable of using pyridin-2-ol as the sole carbon and energy source, was isolated from soil. Whole cells of Burkholderia sp. MAK1 were confirmed to possess a good ability to convert different pyridin-2-amines and pyridin-2-ones into their 5-hydroxy derivatives. Moreover, several methylpyridines as well as methylated pyrazines were converted to appropriate N-oxides. In conclusion, regioselective oxyfunctionalization of pyridine derivatives using whole cells of Burkholderia sp. MAK1 is a promising method for the preparation of various pyridin-5-ols and pyridin-N-oxides.Pyridinols and pyridinamines are important intermediates with many applications in chemical industry. The pyridine derivatives are in great demand as synthons for pharmaceutical products. Moreover, pyridines are used either as biologically active substances or as building blocks for polymers with unique physical properties. Application of enzymes or whole cells is an attractive strategy for preparation of hydroxylated pyridines since the methods for chemical synthesis of pyridinols, particularly aminopyridinols, are usually limited or inefficient.  N-heterocyclic rings, pyridin-2-ol has considerable chemical and pharmacological importance. Due to a peptidomimetic functionality of 1H-pyridin-2-one tautomer, it plays an essential role as a scaffold in drug design145910The pyridine ring is found in various man-made compounds, such as dyes, industrial solvents, herbicides, pesticides as well as in many natural metabolites. Among the Mycobacterium tuberculosis glutamine synthetase inhibitorsA novel class of phenolic antioxidants, 6-aminopyridin-3-ols, are more effective than many other phenolic-class compounds reported to date1314Preparation of some 5-hydroxy-2-pyridones is achievable by both organo-chemical and biocatalytic approaches1219N-heterocyclic compounds, especially with regard to the production of metabolic intermediates, has received considerable attention in biotechnology as the starting process for the synthesis of fine and commodity chemicals, e.g., pyridine-2,5-diol (an intermediate for production of 5-aminolevulinic acid), 6-hydroxynicotinic acid and other pyridine derivatives252627N-oxides can be prepared using microbial cells and enzymes; however, this has been accomplished using Methylococcus capsulatus monooxygenaseAgrocybe aegerita peroxygenaseVerticillium sp. GF39 cellsOxyfunctionalization of chemical compounds by using enzymes or whole cells is an attractive strategy to obtain the desired products2223Arthrobacter, Achromobacter, Rhodococcus, and Nocardia are able to grow on pyridin-2-ol3233343235Several bacteria belonging to the genera Burkholderia sp. MAK1 was found to be an efficient biocatalyst for the hydroxylation of various pyridin-2-ols and pyridin-2-amines. Moreover, Burkholderia sp. MAK1 was capable of oxidising several N-heterocyclic ring systems to corresponding N-oxides.In this study, the oxyfunctionalization of the pyridine ring by whole bacterial cells was investigated. The pyridin-2-ol-degrading Burkholderia sordidicola. Based on the results of 16S rRNA gene sequence analysis 3235Burkholderia sp. MAK1 described here, pyridin-2-ol was catabolized without the formation of a blue pigment. Assuming that pyridine-2,5-diol is an intermediate in pyridin-2-ol catabolic pathway, the activity of pyridine-2,5-diol 5,6-dioxygenase detected in the pyridin-2-ol-induced cells of Burkholderia sp. MAK1 suggested that this strain possesses an inducible pyridin-2-ol 5-monooxygenase.In the case of Burkholderia sp. MAK1 consumes pyridine-2-ol via pyridine-2,5-diol by supposedly pyridine-2-ol inducible pyridin-2-ol 5-monooxygenase we wanted to test whether Burkholderia sp. MAK1 is capable of hydroxylating other pyridine derivatives. In this study, more than 100 of pyridine, pyrimidine, and pyrazine derivatives were screened for the hydroxylation using Burkholderia sp. MAK1 as a whole-cell biocatalyst  were not able to trigger the induction. Also no hydroxylation occurred when cells were cultivated with other sole carbon source (glucose or succinate) instead of pyridin-2-ol.It is worth mentioning that induction of Burkholderia sp. MAK1 grew poorly in rich nutrient medium, but the growth was observed in mineral medium (EFA or Koser) with pyridin-2-ol as a sole carbon source. The growth reached its peak after 40 h of incubation in EFA medium (OD600 = 0.4). The optimal temperature for cultivation of Burkholderia sp. MAK1 appeared to be 30 °C. At higher tested temperature (37 °C), Burkholderia sp. MAK1 cells were not able to grow. Although bacterial growth was observed at 25 °C it was rather slow compared to 30 °C. The effect of temperature on Burkholderia sp. MAK1-mediated synthesis of hydroxylated pyridine derivatives was also investigated pyridin-2-ol was slowly converted into a detectable new compound whose molecular mass was 16 Da higher than that of the substrate. Burkholderia sp. MAK1 cells were not able to hydroxylate pyridin-2-ols containing carboxyl or methoxy groups at position 3.Another group of potential sition 3 . HPLC-MSBurkholderia sp. MAK1 with 3-amino-6-methyl-pyridin-2-ol, a new compound with a molecular mass of 278 Da accumulated in the reaction mixture. Since the molecular mass of the expected 3-amino-6-methyl-pyridin-2-ol hydroxylation product is 140 Da, it is likely that the oxidation of the substrate is followed by the spontaneous dimerization. When Burkholderia sp. MAK1 cells were incubated with 3-bromo-6-methyl-pyridin-2-ol, neither hydroxylation, nor any other transformation occurred suggesting that 3-bromo functional group disrupted the proper orientation of the substrate.Pyridin-2-ols carrying substituents at positions 3 and 6 were also examined. The pyridin-2-ol-induced cells were able to metabolize 2-hydroxy-6-methyl-pyridine-3-carbonitrile: substrate concentration decreased over time, and no new products were detectable by HPLC-MS. After incubation of Burkholderia sp. MAK1 cells after 20 hours of incubation. However, the intermediate product accumulating in the reaction mixture was detected by HPLC-MS and its absorption spectra as well as molecular mass  were consistent with those of hydroxylated pyridine-2,4-diol , yet none of these spectra matched that of the product  corresponded to that of 6-amino-5-chloro-pyridin-3-ol. The compound showed four peaks in the 1H NMR spectrum : δ = 5.51 , 7.11 , 7.56 , 9.24 , and five peaks in the 13C NMR spectrum : δ = 113.58, 125.21, 133.73, 146.21, 149.46), identifying the product as 6-amino-5-chloro-pyridin-3-ol. The production yield of 6-amino-5-chloro-pyridin-3-ol was 34%.Pyridin-2-amines with methyl, nitro, chloro, bromo, or fluoro substituent at position 3  were allH-dipyridooxazine-3-one) suggesting that Burkholderia sp. MAK1 catalyzes the oxidative dimerization of 2-aminopyridin-3-ol. Also, although another analytical standard, pyridine-2,3-diamine derivative, is commercially unavailable, our results indicate, that MAK1 catalyzes dimerization of pyridine-2,3-diamine as well. These dimers are potential anticancer and antimicrobial drugsBoth pyridine-2,3-diamine and 2-aminopyridin-3-ol were transformed into colored compounds, with a molecular mass of 213 Da (yellow-brown) and 214 Da (yellow-green), respectively. The retention time, UV-Vis spectra, and ionisation profile of the oxidation product of 2-aminopyridin-3-ol matched those of the analytical standard , and their structures were analysed by 1H NMR and 13C NMR. 6-Amino-4-methyl-pyridin-3-ol : δ = 2.18 , 6.41 , 6.61 , 6.70 , 7.87 , 13C NMR : δ = 20.52, 109.39, 113.72, 136.73, 138.00, 150.61), 6-amino-4-chloro-pyridin-3-ol : δ = 6.66 , 6.84–6.83 , 7.0 , 8.04 , 13C NMR : δ = 108.17, 112.44, 131.29, 138.34, 151.70) and 6-amino-4-fluoro-pyridin-3-ol : δ = 6.54 , 6.62 , 7.11 , 8.07 , 13C NMR : δ = 95.45, 100.74, 139.03, 158.91, 161.39) were formed by whole cells of Burkholderia sp. MAK1 with the yield of 34%, 50% and 68%, respectively  occurred. The compounds with 6-chloro or 6-bromo substituents were converted to the corresponding hydroxylated products. The product of oxidation of 6-chloropyridin-2-amine, 6-amino-2-chloropyridin-3-ol, was purified and identified by 1H NMR : δ = 5.90 , 6.38 , 6.84 , 9.79 . While 6-fluoropyridin-2-amine conversion was very slow, the transformation of 6-methoxypyridin-2-amine did not occur at all. The conversion of 6-aminopyridin-2-ol led to several compounds suggesting that the substrate is hydroxylated at position 3 and/or 5, so that a mixture of several products in varying proportions results.The compounds substituted at position 6  were alsBurkholderia sp. MAK1 may be applied for the regioselective synthesis of 6-substituted 2-aminopyridinols . The retention time, UV spectrum and ionisation profile of the bioconversion product matched those of analytical standard, pyridine-N-oxide, suggesting that Burkholderia sp. MAK1 catalyzes pyridine oxidation at position 1. Induction of cells with pyridin-2-ol was necessary for the oxidation of pyridine as well as for pyridin-2-ol and pyridin-2-amine transformation indicating that the same enzyme of Burkholderia sp. MAK1 is responsible for all these biotransformations.The study on pyridin-2-amine and pyridin-2-ol bioconversion by Burkholderia sp. MAK1. The test revealed that the whole cells of Burkholderia sp. MAK1 catalyzed the transformation of 2-methyl-, 3-methyl-, and 4-methylpyridine into corresponding N-oxides whose structures were confirmed by HPLC-MS using analytical standards : δ = 8.34–8.36 , 8.54–8.57 ; 13C NMR : δ = 134.85, 148.94) and pyrazine-1,4-dioxide : δ = 8.28 ; 13C NMR : δ = 137.21).Based on HPLC-MS analysis, the biotransformation of pyrazine resulted in the formation of two products with molecular masses that were 16 Da and 32 Da higher than that of the parent compound. Burkholderia sp. MAK1 has also the ability to oxidize various methylpyrazines. For the oxidation of methylated pyrazines the single free position adjacent to either one of nitrogen atoms was a sufficient condition, e. g. the cells could oxidize 2,3,5-trimethylpyrazine, but not 2,3,5,6-tetramethylpyrazine.Our research revealed that N-hydroxylation of pyridines have been published. The formation of pyridine N-oxides has been observed in fungi Cunninghamella elegans ATCC 26269Verticillium sp. GF39Methylococcus capsulatusDiaphorobacter sp. J5-51fungus Agrocybe aegerita has been found to be active towards pyridine and its derivativesTo date, only a few reports regarding the microbial Burkholderia sp. MAK1 have high activity towards pyridin-2-amines and pyridin-2-ols, and are applicable for the synthesis of pyridin-5-ols from the corresponding substrates. Moreover, unsubstituted pyridine and pyrazine as well as their methylated derivatives can be converted into the corresponding N-oxides using pyridin-2-ol-induced Burkholderia sp. MAK1 . Pyridin-2-ol, pyrimidin-2-ol, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 2-pyridylmethanol, pyridine-2-thiol, 1-methylpyridin-2-one, 3-bromopyridin-2-ol, 3-methylpyridin-2-ol, 3-methoxypyridin-2-ol, pyridine-2,3-diol, pyridine-2,4-diol, pyridine-2,6-diol, 3-methylpyridin-2-amine, 3-methylpyridine-2-carbonitrile, 2,3-dimethylpyridine, 2,4-dimethylpyridine, 2,5-dimethylpyridine, 2,6-dimethylpyridine, 3,4-dimethylpyridine, 3,5-dimethylpyridine, 2,3,5-trimethylpyridine, pyridine-2,3-diamine, pyridine-2,6-diamine, 4-methyl-3-nitro-pyridin-2-ol, 4-methylpyridin-2-amine, 4,6-dimethylpyridin-2-amine, 3-methylpyridin-6-amine, pyridine, pyran-2-one, 2-hydroxy-6-methyl-pyridine-3-carbonitrile, 1-pyrazin-2-ylethanone, 2,3-dimethylpyrazine, 2,5-dimethylpyrazine, 2,6-dimethylpyrazine, 2,3,5-trimethylpyrazine, pyrazin-2-amine, tetramethylpyrazine, 2-(2-pyridyl)ethanol, 2-aminopyridine-3-carbonitrile, 3-methylpyridazine, 2-methylpyridin-3-ol, 4-methylpyridine-2-carbonitrile, pyridine-2,6-dicarbonitrile, 6,7-dihydro-5H-cyclopenta[b]pyridine, 5-methyl-6,7-dihydro-5H-cyclopenta[b]pyrazine, pyrimidine-2,4-diamine, 2-methylpyridin-5-ol, 2-aminopyridin-6-ol, 4-methyl-3-nitro-pyridin-2-amine were obtained from Sigma-Aldrich . Pyridin-2-ol N-oxide, 2-hydroxypyridine-3-carboxylic acid, 6-hydroxypyridine-2-carboxylic acid, 3-hydroxypyridine-2-carboxylic acid, 6-methylpyridin-2-ol, 2-methylpyridine N-oxide, 2-ethylpyridine, 4-ethylpyridine, pyridine-3-carboxamide, 4-[(4-nitrophenyl)methyl]pyridine, pyridine-3-carbonitrile, 2,4,6-trimethylpyridine and pyridin-4-ol were purchased from Fluka . 5-hydroxypyridine-2-carboxylic acid, 3-fluoropyridin-2-ol, 3-chloropyridin-2-ol, 6-chloropyridin-2-ol, 6-bromopyridin-2-ol, 6-methylpyridin-2-ol, 4-chloropyridin-2-ol, 2-oxo-1H-pyridine-4-carbonitrile, 4,6-dimethylpyridin-2-ol, 3-amino-6-methyl-pyridin-2-ol, 3-(trifluoromethyl)pyridin-2-ol, 4-(trifluoromethyl)pyridin-2-ol, 6-(trifluoromethyl)pyridin-2-ol, 6-(trifluoromethyl)pyridin-2-amine, 3-chloropyridin-2-amine, 4-chloropyridin-2-amine, 6-chloropyridin-2-amine, 3-bromopyridin-2-amine, 4-bromopyridin-2-amine, 6-bromopyridin-2-amine, 3,6-dibromopyridin-2-amine, 3-fluoropyridin-2-amine, 4-fluoropyridin-2-amine, 6-fluoropyridin-2-amine, 2-aminopyridin-4-ol, 3-methoxypyridin-2-amine, 6-methoxypyridin-2-amine, ethyl 2-aminopyridine-3-carboxylate, 2-aminopyrimidin-4-ol, 4-chloropyrimidin-2-amine, 4-bromopyridin-2-ol and 3-bromo-6-methylpyridin-2-ol were the products of Combi Blocks Inc . All reagents used in this study were of analytical grade. Nutrient agar (NA) medium and brain heart infusion (BHI) medium were obtained from Oxoid . The 2-amino-3H-dipyridooxazine-3-one prepared by oxidation-dimerization of 2-amino-3-hydroxypyridine as described for 2-aminophenolPyridin-2-amine, 2-chloropyridine, pyridine-2-carboxylic acid, pyridine-H)-one. Yield 170 mg (69%), colourless oil. MS (ESI+): m/z 124.15 [M + H]+. 1H NMR : δ = 1.37 , 4.00 , 6.21 , 6.61 , 7.29–7.37 ; 13C NMR : δ = 14.69, 44.96, 106.47, 120.96, 137.01, 139.46, 162.51.1-Ethylpyridin-2(1H)-one. Yield 200 mg (73%), colourless oil. MS (ESI+): m/z 138.15 [M + H]+. 1H NMR : δ = 0.95 , 1.56–1.69 , 3.75 , 6.11 , 6.42 , 7.35–7.46 , 7.72 ; 13C NMR : δ = 14.00, 29.23, 46.78, 105.55, 119.82, 138.93, 141.43, 162.15.1-Propylpyridin-2(1H)-one. Yield 180 mg (60%), yellowish oil. MS (ESI+): m/z 152.20 [M + H]+. 1H NMR : δ = 0.90 , 1.18–1.34 , 1.52–1.65 , 3.86 , 6.19 , 6.36 , 7.33–7.42 , 7.66 ; 13C NMR : δ = 14.05, 19.70, 31.25, 48.61, 105.50, 120.02, 139.53, 140.13, 161.82.1-Butylpyridin-22SO4 1.0, 2-hydroxypyridine 2.0, MgSO4 × 7H2O 0.2, salt solution 10 ml/l., pH 7.2; Salt solution (g/l): CaCl2 × 2H2O 2.0, MnSO4 × 4H2O 1.0, FeSO4 × 7H2O 0.5, all components were dissolved in 0.1N HCl and added in to EFA medium before cultivation; Koser mineral medium (g/l): NaCl 5.0, NH4H2PO4 1.0, K2HPO4 1.0, MgSO4 × 7H2O 0.4. The final pH was adjusted to 7.0EFA (g/l): KBacteria were cultivated in liquid media with aeration at 30 °C.Burkholderia sp. MAK1 was grown at 30 °C for 20 hours in 1 l flasks containing 200 ml EFA medium. The cells were harvested by centrifugation and washed twice with 10 mM potassium phosphate buffer, pH 7.2.For substrate specificity and bioconversion experiments Rhodococcus, Streptomyces, Mycobacterium) or fungi. After 3–5 days of aerobical cultivation at 30 °C the best growing colonies were selected for further work.0.5 g of soil was suspended in 20 ml of Koser mineral medium. The aliquots (10–100 μl) were spread on Koser agar plates supplemented with 0.1% 2-hydroxypyridine and clotrimazole (20 μg/ml). Clotrimazole is known as cytochrome P450 inhibitor and was used to suppress growth of actinobacteria . Prior the purification the column was equilibrated with water. A mobile phase that consisted of water and methanol delivered in the gradient 10:0 → 10:5 elution mode. The collected fractions were analyzed by HPLC-MS. The fractions containing pure product were joined, and the solvent was removed under reduced pressure. 1H NMR spectra were recorded in DMSO-d6 or CDCl3 on Bruker Ascend 400, 400 MHz, and 13C NMR were recorded on Bruker Ascend 400, 100 MHz. Chemical shifts are reported in parts per million relative to the solvent resonance signal as an internal standard.~2 g of wet biomass of 2 as nebulizing and drying gas. Mass spectrometry data was acquired in both the positive and negative ionization mode. The data was analyzed using the LabSolutions LCMS software.Before the analysis the cells were separated from the reaction mixture by centrifugation. The resultant supernatant was mixed with an equal part of acetonitrile, centrifuged and analyzed using a high performance liquid chromatography system  equipped with a photo diode array (PDA) detector  and a mass spectrometer  equipped with an ESI source. The chromatographic separation was conducted using a YMC Pack Pro column, 3 × 150 mm  at 40 °C and a mobile phase that consisted of 0.1% formic acid water solution (solvent A), and acetonitrile (solvent B) delivered in the 0 → 60% gradient elution mode. Mass scans were measured from m/z 10 up to m/z 700, at 350 °C interface temperature, 250 °C DL temperature, ±4,500 V interface voltage, neutral DL/Qarray, using NBurkholderia sp. MAK1 was grown at 30 °C for 20 hours in two 150 ml flasks, one containing 25 ml EFA medium (pyridin-2-ol induced cells), other containing 25 ml EFA medium where pyridin-2-ol is substituted for succinate . The cells were harvested by centrifugation, washed twice with 10 mM potassium phosphate buffer (pH 7.2), suspended in 5 ml of the same buffer and sonicated. In 1.5 ml tubes three separate reaction mixtures were combined: internal control , negative control  and sample . 100 μl of each reaction mixture was transferred to a 96 well plate and change in absorbance (λmax 320 nm) per 30 minutes was measured. Overall change in absorbance was evaluated by subtracting noise data  from sample data. We were able to achieve 200–250 mU per 1 l medium, where 1 enzyme unit (U) is an amount of enzyme that catalyzes depletion of 1 μmol pyridine-2,5-diol per minute. The measured molar extinction coefficient of pyridine-2,5-diol in ethanol was 9800 M−1∙cm−1.How to cite this article: Stankevičiūtė, J. et al. Oxyfunctionalization of pyridine derivatives using whole cells of Burkholderia sp. MAK1. Sci. Rep.6, 39129; doi: 10.1038/srep39129 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


This compound is the only one described that does not release o-diphenol after the hydroxylation step. Oxytyrosinase hydroxylates the deoxyarbutin in ortho position of the phenolic hydroxyl group by means of an aromatic electrophilic substitution. As the oxygen orbitals and the copper atoms are not coplanar, but in axial/equatorial position, the concerted oxidation/reduction cannot occur and the release of a copper atom to bind again in coplanar position, enabling the oxidation/reduction or release of the o-diphenol from the active site to the medium. In the case of deoxyarbutin, the o-diphenol formed is repulsed by the water due to its hydrophobicity, and so can bind correctly and be oxidized to a quinone before being released. Deoxyarbutin has been characterized with: -1 and o-diphenol products with tyrosinase show how these ligands bind at the copper centre of tyrosinase. The presence of an energy barrier in the release of the o-diphenol product of deoxyarbutin, which is not present in the case of β-arbutin, together with the differences in polarity and, consequently differences in their interaction with water help understand the differences in the kinetic behaviour of both compounds. Therefore, it is proposed that the release of the o-diphenol product of deoxyarbutin from the active site might be slower than in the case of β-arbutin, contributing to its oxidation to a quinone before being released from the protein into the water phase.Deoxyarbutin, a potent inhibitor of tyrosinase, could act as substrate of the enzyme. Oxytyrosinase is able to hydroxylate deoxyarbutin and finishes the catalytic cycle by oxidizing the formed  The catalytic centre of tyrosinase has two copper atoms each coordinated with three histidine residues, similar to of hemocyanin and catechol oxidase 0 → ∞) –47, we oK12 = k−12/k12, if we consider that K9 = k−9/k9 could have a similar value to K12 , K12, since k10 has a low value. If this constant has a high value, K12, as it happens in the case of true substrates of tyrosinase 0 = 100 nM, [D-Arb]0 = 0.5 mM and [H2O2]0 = 20 mM. The spectrophotometric recordings were made every 60 seconds. Inset. Spectrophotometric recordings of the activity of different concentrations of tyrosinase on D-Arb in the presence of H2O2. The experimental conditions were [E]0 (nM) = (a) 25, (b) 50, (c) 75, (d) 100, (e) 150 and (f) 200; [D-Arb]0 = 0.5 mM and [H2O2]0 = 20 mM.Spectrophotometric recordings of the action of tyrosinase on D-Arb in the presence of H(TIF)Click here for additional data file.S3 FigE]0 = 50 nM, [D-Arb]0 = 0.1 mM and [AH2]0 = 0.2 mM. The spectrophotometric recordings were made every 2 minutes.The experimental conditions were [(TIF)Click here for additional data file.S4 FigInset A. Spectrophotometric recordings ((a) before and (b) after D-Arb passed through the column) at 485 nm of the oxidation of D-Arb by sodium periodate in excess to show that there is no contamination by o-diphenol. The experimental conditions were [D-Arb]0 = 0.2 mM and [NaIO4]0 = 0.5 mM. Inset B. Activity of tyrosinase on the eluted D-Arb. The experimental conditions were [D-Arb]0 = 0.25 mM and [E]0 = 50 nM.Scans of (a) D-Arb (0.2 mM) and (b) an aliquot taken after D-Arb was filtered through a Sephadex G-25 column containing aluminium oxide  eluted with ammonium acetate buffer at pH = 6.1. (TIF)Click here for additional data file.S5 Fig2O2]0 = 5 mM and [E]0 = 200 nM. Inset. Spectrophotometric recordings of the main figure. [D-Arb]0 (mM): (a) 0.05, (b) 0.10, (c) 0.15 and (d) 0.20.Final absorbance at 485 nm of the spectrophotometric recordings of the action of tyrosinase on different concentrations of D-Arb until its consumption. The experimental conditions were: [H(TIF)Click here for additional data file.S6 FigE]0 = 500 nM, [Eox]0 = 0.2 x [E]0, [Em]0 = 0.8 x [E]0; [D-Arb]0 = 0.2 mM and [O2]0 = 0.26 mM. The rate constants were: k8 = 2.3 x 108 M-1 s-1, k-8 = 1.07 x 103 s-1, k9 = 1.6 x 105 M-1 s-1, k-9 = 3.8 s-1, k10 = 1.5 s-1, k11 = 400 s-1, k12 = 1.6 x 105 M-1 s-1, k-12 = 3.8 s-1, k15 = 2 x 106 M-1 s-1, k-15 = 10 s-1, k16 = 6.6 x 10−4 s-1.Representation of initial rate values of tyrosinase on D-Arb calculated from the simulated progress curves obtained through numerical integration of the set of differential equations corresponding to the mechanism shown in (TIF)Click here for additional data file.S7 Fig(TIF)Click here for additional data file.S8 Figk-13 and k19, respectively Click here for additional data file.S9 Fig(TIF)Click here for additional data file.S10 FigEmD-ArbOH with the rate constant k-13 and bond to Em and Eox with the rate constants k13 and k17, respectively Click here for additional data file.S11 FigE]0 = 750 nM, [Eox]0 = 0.2 x [E]0, [Em]0 = 0.8 x [E]0; [H2O2]0 = 1.25 μM, [D-Arb]0 = 0.4 mM and [O2]0 = 0.26 mM. The rate constants were: k8 = 2.3 x 108 M-1 s-1, k-8 = 1.07 x 103 s-1, k9 = 1.6 x 105 M-1 s-1, k-9 = 3.8 s-1, k10 = 1.5 s-1, k11 = 400 s-1, k12 = 1.6 x 105 M-1 s-1, k-12 = 3.8 s-1, k13 = 105 M-1 s-1, k-13 = 3 s-1, k15 = 2 x 106 M-1 s-1, k-15 = 10 s-1.Representation of the accumulation of P due to the action of tyrosinase on D-Arb calculated from the simulated progress curves obtained through numerical integration of the set of differential equations corresponding to the mechanism shown in (TIF)Click here for additional data file.S12 FigSurface representation of the configuration poses of β-Arb (A) and D-Arb (B) corresponding to  View from the water phase to the atoms copper buried in the protein structure.(TIF)Click here for additional data file.S13 FigThe ligand configurations are scaled and placed at the binding pose for. Copper atoms are located at about 4.2 nm z-distance. (A) β-ArbOH, (B) D-ArbOH.(TIF)Click here for additional data file.S14 Figo-phenol group (C2-OH) (solid line) and C1-OH (dashed line) hydroxyl groups of D-ArbOH with water. (C) Hydrogen bonds of C2-OH (solid line) and C1-OH (dashed line) hydroxyl groups of D-ArbOH with N260 residue. The asterisks mark the positions of the conformation poses shown in (A) Hydrogen bonds of β-ArbOH (upper trace) and D-ArbOH (lower trace) with water. (B) Hydrogen bonds of (TIF)Click here for additional data file.S15 Fig(A) β-ArbOH at the copper centre binding site. From (B) to (F) conformational structures of D-ArbOH at the positions marked as asterisks in (TIF)Click here for additional data file.S16 Figo-diphenol (L-dopa), Q = o-dopaquinone, Cr = dopachrome.M = monophenol (L-tyrosine), D = (TIF)Click here for additional data file.S17 Fig(TIF)Click here for additional data file.S18 FigA. Representation of initial rate values of tyrosinase on L-tyrosine in the absence (●) and presence (▲) of D-Arb calculated from the simulated progress curves obtained through numerical integration of the set of differential equations corresponding to the mechanism shown in E]0 = 700 nM, [Eox]0 = 0.2 x [E]0, [Em]0 = 0.8 x [E]0; [D-Arb]0 = 0.2 mM, [O2]0 = 0.26 mM and R = [L-dopa]0 / [L-tyrosine]0 = 0.042. The rate constants were: k1 = 2 x 105 M-1 s-1, k-1 = 10 s-1, k2 = 5 x 105 M-1 s-1, k-2 = 10 s-1, k3 = 900 s-1, k4 = 4.8 x 104 M-1 s-1, k-4 = 0.5 s-1, k5 = 12 s-1, k6 = 2.16 x 105 M-1 s-1, k-6 = 10 s-1, k7 = 108 s-1, k8 = 2.3 x 108 M-1 s-1, k-8 = 1.07 x 103 s-1, k9 = 1.6 x 105 M-1 s-1, k-9 = 3.8 s-1, k10 = 1.5 s-1, k11 = 400 s-1, k12 = 1.6 x 105 M-1 s-1, k-12 = 3.8 s-1, k14 = 10 s-1. Inset. Graphical representation of the Lineweaver–Burk equation showing the simulated inhibition of the monophenolase activity of tyrosinase in the absence (●) and presence (▲) of D-Arb. The experimental conditions were the same as those of the main figure. B. Simulation of the inhibition of the diphenolase activity of tyrosinase by D-Arb. Representation of initial rate values of tyrosinase on L-dopa in the absence (●) and presence (▲) of D-Arb calculated from the simulated progress curves obtained through numerical integration of the set of differential equations corresponding to the mechanism shown in E]0 = 700 nM, [Eox]0 = 0.2 x [E]0, [Em]0 = 0.8 x [E]0; [D-Arb]0 = 0.2 mM and [O2]0 = 0.26 mM. The rate constants were: k2 = 5 x 105 M-1 s-1, k-2 = 10 s-1, k3 = 900 s-1, k4 = 4.8 x 104 M-1 s-1, k-4 = 0.5 s-1, k5 = 12 s-1, k6 = 2.16 x 105 M-1 s-1, k-6 = 10 s-1, k7 = 108 s-1, k8 = 2.3 x 108 M-1 s-1, k-8 = 1.07 x 103 s-1, k9 = 1.6 x 105 M-1 s-1, k-9 = 3.8 s-1, k10 = 1.5 s-1, k11 = 400 s-1, k12 = 1.6 x 105 M-1 s-1, k-12 = 3.8 s-1, k14 = 10 s-1. Inset. Graphical representation of the Lineweaver–Burk equation showing the simulated inhibition of the diphenolase activity of tyrosinase in the absence (●) and presence (▲) of D-Arb. The experimental conditions were the same as in the main figure.(TIF)Click here for additional data file.S19 Fig(TIF)Click here for additional data file.S20 Fig(TIF)Click here for additional data file.S21 Fig(TIF)Click here for additional data file.S22 FigE]0 = 700 nM, [Eox]0 = 0.2 x [E]0, [Em]0 = 0.8 x [E]0; [D-Arb]0 (mM) = (a) 0.05, (b) 0.10, (c) 0.25, (d) 0.40, (e) 0.70 and (f) 1; [H2O2]0 = 1.25 μM and [O2]0 = 0.26 mM. The rate constants were: k8 = 2.3 x 108 M-1 s-1, k-8 = 1.07 x 103 s-1, k9 = 1.6 x 105 M-1 s-1, k-9 = 3.8 s-1, k10 = 1.5 s-1, k11 = 400 s-1, k12 = 1.6 x 105 M-1 s-1, k-12 = 3.8 s-1, k15 = 2 x 106 M-1 s-1, k-15 = 10 s-1.Accumulation of P due to the action (with no lag period) of tyrosinase on different concentrations of D-Arb calculated from the simulated progress curves obtained through numerical integration of the set of differential equations corresponding to the mechanism shown in (TIF)Click here for additional data file.S23 Fig2O2 (μM) during a minute, calculated from the simulated progress curves obtained through numerical integration of the set of differential equations corresponding to the mechanism shown in E]0 = 500 nM, [Eox]0 = 0.2 x [E]0, [Em]0 = 0.8 x [E]0; [H2O2]0 = 1.25 μM and [O2]0 = 0.26 mM. After the pre-incubation, the final values of [Em], [Eox] and [H2O2] were used to simulate the activity of the enzyme on D-Arb (0.2 mM). The rate constants were: k8 = 2.3 x 108 M-1 s-1, k-8 = 1.07 x 103 s-1, k9 = 1.6 x 105 M-1 s-1, k-9 = 3.8 s-1, k10 = 1.5 s-1, k11 = 400 s-1, k12 = 1.6 x 105 M-1 s-1, k-12 = 3.8 s-1, k15 = 2 x 106 M-1 s-1, k-15 = 10 s-1.Representation of initial rate values of tyrosinase on D-Arb after pre-incubation with different concentrations of H(TIF)Click here for additional data file.S24 FigEox]0 = 0.2 x [E]0, [Em]0 = 0.8 x [E]0, being [E]0 (nM) = 0, 50,100, 250, 500 and 750, respectively; [H2O2]0 = 1.25 μM, [D-Arb]0 = 0.2 mM and [O2]0 = 0.26 mM. The rate constants were: k8 = 2.3 x 108 M-1 s-1, k-8 = 1.07 x 103 s-1, k9 = 1.6 x 105 M-1 s-1, k-9 = 3.8 s-1, k10 = 1.5 s-1, k11 = 400 s-1, k12 = 1.6 x 105 M-1 s-1, k-12 = 3.8 s-1, k15 = 2 x 106 M-1 s-1, k-15 = 10 s-1. Inset. Representation of initial rate values of tyrosinase on different concentrations of D-Arb calculated from the simulated progress curves obtained through numerical integration of the set of differential equations corresponding to the mechanism shown in Representation of initial rate values of tyrosinase on D-Arb with different concentrations of enzyme calculated from the simulated progress curves obtained through numerical integration of the set of differential equations corresponding to the mechanism shown in (TIF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.