H)-ones as lead for the development of potent adenosine triphosphate (ATP) competitive protein kinase inhibitors with implications as anti-cancer drugs. Initially, we identified the pyrazin-2(1H)-one scaffold from hamacanthins  by Molecular Modeling studies as core binding motif in the ATP pocket of receptor tyrosine kinases (RTK), which are validated drug targets for the treatment of various neoplastic diseases. Structure-based design studies on a human RTK member PDGFR (platelet-derived growth factor receptor) suggested a straight forward lead optimization strategy. Accordingly, we focused on a Medicinal Chemistry project to develop pyrazin-2(1H)-ones as optimized PDGFR binders. In order to reveal Structure-Activity-Relationships (SAR), we established a flexible synthetic route via microwave mediated ring closure to asymmetric 3,5-substituted pyrazin-2(1H)-ones and produced a set of novel compounds. Herein, we identified highly potent PDGFR binders with IC50 values in an enzymatic assay below µM range, and possessing significant activity against PDGFR dependent cancer cells. Thus, marine hamacanthin-derived pyrazin-2(1H)-ones showing interesting properties as lead for their further development towards potent PDGFR-inhibitors.In this study, we report on pyrazin-2(1 H)-one scaffold, respectively  inhibitor (MRSA-PK inhibitor) with an IC50 value of 0.016 µM and significant selectivity over human protein kinase isoforms . In. InH)-on5 inhibited VEGF-R2/3 in the low µM range, and most potently inhibited PDGFRβ with an IC50 of 0.5 μM. Moreover, compound 5 was shown to exhibit promising selectivity over the other PK enzymes tested as part of the panel .5 in the active site of PDGFRβ, we subsequently aimed to optimize this compound by performing a virtual screen against a focused set of compounds, with particular focus upon varying the indole at the pyrazin-2(1H)-one 5′ position, as the pyrazin-2(1H)-one 3′- moiety had already been shown to be required in order to inhibit PDGFRβ -one 5′-position was systematically varied by synthetically feasible decoration patterns yielding a set of virtual compounds. The subsequent ligand preparation, docking and scoring campaign using Schrödinger Glide SP  produced a list with compound 8 as the one that was predicted to inhibit PDGFRβ most potently (8 is comparable to the binding pose of compound 5 with the pyrazin-2(1H)-one core addressing H-bonds to Glu101 and Cys103  and 13C (75 MHz) NMR were recorded on a Bruker Avance III 300 spectrometer  at 300 K with a multinuclear probe head using the manufacturer’s pulse programs. The data are reported as follows: chemical shift in ppm from Me4Si (TMS) as external standard, multiplicity and coupling constant (Hz). NMR spectra were obtained on a 1H (300 MHz) and 13C spectra (75 MHz) were referenced either to TMS or to internal DMSO-d5 (1H NMR δ 2.50) and internal DMSO-d6 (13C NMR δ 39.5) or internal CHCl3 (1H NMR δ 7.26) and internal CDCl3 (13C NMR δ 77.0). All coupling constants  are quoted in Hz. The following NMR abbreviations are used: br (broad), s (singlet), d (doublet), t (triplet), m (unresolved multiplet). The labelling scheme of structures to correlate NMR signals can be found in Supporting Information.Mass spectra of the compounds were recorded after chromatographic separation. Mixtures were separated with an Agilent 1100 HPLC system  consisting of a thermostated autosampler, diode array detection and an Agilent Zorbax Eclipse XDB-C8 column . Elution was achieved with a solvent gradient system of water and acetonitrile, with 0.1% of acetic acid and a flow rate of 1 mL/min. The eluent flow was splitted to the mass spectrometer.Mass spectra with nominal resolution were recorded with an Esquire ~LC mass spectrometer , with electrospray ionization operating in the positive ion mode, with the following parameters: drying gas nitrogen 8 L/min, nebulizer 35 psi, dry gas heating 350 °C, HV capillary 4000 V, HV EndPlate offset −500 V. GC/MS was performed on a HP6890 Series System. EI-Mass spectra were recorded on a Varian MAT 311A (70 eV). HRMS spectra were recorded on a MAT-95 (Finnigan).Melting points/decomposition temperatures were determined on a Büchi apparatus according to Dr. Tottoli and are uncorrected.ca. 60 nm). Column chromatography for test compounds was performed using a La-Flash-System (VWR) with Merck silica gel 60 (0.015–0.040 mm) or RP8 columns. The progress of the reactions was monitored by thin-layer chromatography (TLC) performed with Merck silica gel 60 F-245 plates. Where necessary, reactions were carried out in a nitrogen atmosphere using 4Å molecular sieves. All reagents and solvents were obtained from commercial sources and used as received (THF was used after distillation over K/benzophenone). Reagents were purchased from Sigma-Aldrich Chemie, Steinheim, Germany; Lancaster Synthesis, Mühlheim, Germany or Acros, Nidderau, Germany.Where appropriate, column chromatography was performed for crude precursors with Merck silica gel 60 (0.063–0.200 mm) or Acros organics silica gel . All key compounds were proven by this method to show ≥98% purity. HPLC analysis was performed on a Hewlett-Packard HP 1090 Series II using a Thermo Betasil C8  column  in N-methylpyrrolidone and reacted at room temperature for 1 h. Then, tryptamine (1 equiv) was added and the mixture was stirred overnight. Then, water was added to quench the reaction and the mixture was extracted three times with EtOAc. The combined organic phase was evaporated under reduced pressure and the residue purified by flash silica gel chromatography to afford N-[2-(1H-indol-3-yl)ethyl]-2-oxo-2-acetamide (3). Yield: ; mp 119 °C; 1H NMR  δ: 2.98 , 3.57 , 3.77 , 6,98 , 7.07 , 7.20 , 7.28 , 7.34 , 7.57 , 8.99 , 10.82 ; 13C NMR  δ: 24.8 (CH2-2′″), 39.1 (CH2-1′″), 56.0 , 60.3 (4′-OMe), 107.3 , 111.3 (CH-7″), 111.4 (Cq-3″), 118.2 , 120.9 (CH-6″), 122.7 (CH-2″), 127.1 (Cq-3a″), 128.0 (Cq-1′), 136.2 (Cq-7a″), 143.1 (Cq-4′), 152.8 , 164.6 (CO-1), 188.9 (CO-2); LC-MS m/z 383 [M + H]+.CDI  was added to a solution of 1 equivalent 2-oxo-2-acetic acid (3 in THF/H2O (9:1) at 0 °C, DDQ (1.5 equiv. dissolved in THF) was added dropwise and stirred for 1 h. Then the solvent was evaporated to dryness. To the residual mixture, methanol was added. The precipitate was filtered off and washed with H2O and methanol to afford N-[2-(1H-indol-3-yl)-2-oxo-ethyl]-2-oxo-2-acetamide (4). Yield: ; mp 201 °C; 1H NMR  δ: 3.80 , 3.93 , 4.69 , 7.23 , 7.51 , 7.57 , 8.16 , 8.51 , 9.21 , 12.08 ; 13C NMR  δ: 45.4 (CH2-1′″), 56.1 , 60.3(4′-OMe), 107.5 , 112.2 (CH-7″), 113.8 (Cq-3″), 121.0 (CH-4″), 122.0, 122.9 , 125.4 (Cq-3a″), 128.2 (Cq-1′), 133.9 (CH-2″), 136.4 (Cq-7a″), 143.1 (Cq-4′), 152.9 , 166.3 (CO-1), 189.1 (CO-2), 189.7 (CO-2′″); LC-MS m/z 397 [M + H]+.To a solution of 5, 6 and 8a) +. HRMS: m/z calculated for [M]+ C21H19N3O4: 377.1375; found 377.1363.By using the general procedure for pyrazinone ring closure we obtained 5-(1H-indol-3-yl)-1H-pyrazin-2-one (6) was synthesized from N-[2-(1H-indole-3-yl)-2-oxoethyl]-2--2-oxoacetamide +. HRMS: m/z calculated for [M]+ C20H17N3O3: 347.1270; found: 347.1254.By using general procedure for pyrazinone ring closure, 3--5-(1cetamide . Yield: H-indol-3-yl)-1H-pyrazin-2-one (7) was synthesized from N-[2-(1H-indole-3-yl)-2-oxoethyl]-2-(4-methoxyphenyl)-2-oxoacetamide +. HRMS: m/z calculated for [M]+ C19H15N3O2: 317.1164; found: 317.1175.By using general procedure for pyrazinone ring closure, 3-(4-methoxyphenyl)-5-(1cetamide . Yield: H-indol-3-yl)-3--1H-pyrazin-2-one, compound 8a was synthesized from N-[2-(5-benzoxy-1H-indol-3-yl)-2-oxo-ethyl]-2-oxo-2-acetamide +. HRMS: m/z calculated for [M]+ C28H25N3O5: 483.1794; found: 483.1776.By using general procedure for pyrazinone ring closure 5-(5-benzyloxy-1cetamide . Yield: 8a (100 mg), cyclohexene (90 mg), Pd/C 10% (20 mg) and methanol (1.5 mL). The vial was sealed and stirred 10 min at 100 °C in a microwave synthesizer. The solution was filtered and cooled to room temperature to form a precipitate which was filtered and washed with methanol and Et2O to afford 5-(5-hydroxy-1H-indole-3-yl)-3-pyrazin-2(1H)-one, compound 8. Yield: ; mp 340 °C; 1H NMR  δ: 3.76 , 3.88 , 6.68 , 7.23 , 7.64 , 7.79 , 7.80 , 8.00 , 8.67 , 11.05 , 12.47 ; 13C NMR  δ: 55.7 (2 OMe), 60.1 (OMe), 104.7 (CH-4″), 106.0 , 111.3 (Cq-3″), 111.8, 111.9 , 123.5 , 125.7 (Cq-4′), 131.9 , 132.8 (Cq-3a″), 138.8 (Cq-4′), 151.2 (COH-5″), 152.3 , 154.0 (CO-2); LC-MS m/z 394 [M + H]+. A microwave vial (5 mL) was equipped with compound All modeling was performed on a DELL 8 core system. For visualization and building the structures Maestro (version 9.3) from Schrödinger  was used . The ilAll inhibitor solutions were prepared freshly in DMSO prior to each experiment and used immediately.http://www.proqinase.com/pages/science -ATP (approx. 5 × 105 cpm per well), recombinant protein kinase (50–400 ng). Depending upon the kinase being assayed, appropriate substrates were used and were as follows (substrates shown in parentheses): AKT1 (GSK3/14-27), ARK5 (autophosphorylation), Aurora-A, Aurora-B (Tetra(LRRWSLG)), B-Raf-VE (MEK1 KM), CDK2/CycA (histone H1), CDK4/CycD1 (Rb-CTF), CK2-A1 (Casein), EGF-R, EPHB4, ERBB2, FAK, IGF1-R, SRC, VEGF-R2, VEGF-R3  4:1), FLT3, INS-R, MET, PDGFRβ  6:2:5:1), PLK1 (Casein), SAK (autophosphorylation), TIE2  4:1), COT (autophosphorylation). The IC50 values were measured by testing 10 concentrations of compounds by single sampling. The final DMSO concentration in the assay was 1% (v/v). The data were fitted using the 4-parameter logistic fit option of GraphPad Prism 5.Protein kinase Assay. A proprietary protein kinase assay . In addition to the test plates, one plate was prepared for the reference measurement at day zero. All plates were incubated for 24 h at 37 °C in a humidified atmosphere with 5% CO2. Compounds 5 and 8 that were dissolved in 100% DMSO (v/v) were added to test plates using the Echo 550® Liquid Handler . The final DMSO concentration in the assay was 0.5% v/v. The viability of the cells in the day zero control plates were determined on the same day without adding any compounds. The CellTiter-Glo® Viability Assay was used to determine the viability of cells using the standard protocol for this assay . The luminescence signal was measured at the EnSpire® Multimode Plate Reader . Test plates were incubated for further 48 h and the cell viability was defined as just described. Measured raw data were converted into percent of cell growth by using the high control (0.5% DMSO v/v without compound) and the day zero control. For dose-response studies, 11 different concentrations of compounds were tested in quadruplicates. The IC50 values were calculated using the 4-parameter logistic fit option of GraphPad Prism 5.The cells were grown in RPMI 1640 Glutamax with 10% FCS, 100 µg/mL streptomycin and 100 U/mL Penicillin G and incubated in a 5% COH)-ones as potent and PDGFRβ inhibitors based on marine derived hamacanthins. Modeling studies showed the core moiety of hamacanthins to bind in the ATP binding pocket of RTK and suggested a straightforward strategy towards potent PDGFRβ binders. For subsequent optimization of hamacanthin derivatives as PDGFRβ inhibitors, a flexible synthetic route via microwave-mediated ring closure to asymmetric 3,5-substituted pyrazin-2(1H)-ones was established and a set of novel compounds was produced. Herein, we identified highly potent PDGFRβ binders with IC50 values in an enzymatic assay below µM range with compound 5 possessing significant activity against PDGFR dependent cancer cells. Thus, marine hamacanthin-derived pyrazin-2(1H)-ones showed interesting properties as lead for the further development of highly potent and selective PDGFRβ-inhibitors.In this study, we developed pyrazin-2(1


Their structures were determined on the bases of extensive spectroscopic analyses, including 1D and 2D NMR data. Among them, pseudozoanthoxanthins III and IV (1–2), 8-hydroxy-6β-methoxy-14- oxooplop-6,12-olide (6) and 3β-methoxyguaian-10(14)-en-2β-ol (7) were new, 1 and 3 showed mild anti-HSV-1 activity, and 7 showed significant antilarval activity towards Balanus amphitrite larvae.Five zoanthoxanthin alkaloids ( Echinogorgia pseudossapo belongs to the genus Echinogorgia that is known to produce sesquiterpenes and sterols +}. The weak HMBC correlations of H-1′ with C-2 /C-3a  and comparison of the 13C NMR data of C-3a in 1 and 4 (2–CH2–CH2–CH2–CH2–COOH unit should be attached on the nitrogen atom N(3) instead of the another nitrogen atom attached at C(2). So, the structure of 1 was determined as shown and the compound was named pseudozoanthoxanthin III.Compound anthin A , zoanthoin A −) and NMR spectra. Comparison of 1H and 13C NMR spectral data , 5.50 , δC 130.8, 131.8]. Extensive 2D NMR analyses, including HSQC, HMBC and 1H–1H COSY spectra proved that 1 and 2 had the same skeleton. Moreover, the HMBC spectrum showed correlations of H-1′ (δH 4.14) with C-2′ (δC 130.8)/C-3′ (δC 131.8), H-2′ (δH 5.59) with C-1′ (δC 58.6)/C-3′/C-4′ (δC 27.7), H-3′ (δH 5.50) with C-1′ (δC 58.6)/C-2′/C-4′/C-5′ (δC 25.9), H-4′ (δH 2.14) with C-3′/C-5′/C-6′ (δC 34.3), H-5′ (δH 1.69) with C-2′/C-3′/C-4′/C-6′, and H-6′ (δH 2.31) with C-4′/C-5′/C-7′ (δC 177.6), which suggested the presence of an –N–CH2–CH=CH– CH2–CH2–CH2–COOH unit.Compound ral data  revealed1H–1H COSY spectrum  indicated that geometric configuration of double bond H-2′/H-3′ was E. The weak HMBC correlations of H-1′ with C-2 /C-3a  and comparison of the 13C NMR data of C(3a) in 2 and 4 (2–CH=CH–CH2–CH2–CH2–COOH unit should be attached to the nitrogen N(3). Thus, the structure of 2 was determined as shown and named pseudozoanthoxanthin IV.This suggestion was supported by the spectrum  showing  2 and 4  suggeste6 had the molecular formula of C16H22O5 as deduced from EIMS and NMR spectra. Its 1H NMR spectrum displayed four methyls at δH 1.80 , 1.36 , 2.26 , 3.14 . The 13C and DEPT-135 NMR spectra showed 17 carbons consisting of four methyls , three methylenes , three methines , two oxygenated quaternary carbons , one double bond , one lactone group (δC 171.9), and one keto group (δC 208.2). The 1H and 13C NMR spectral data of 6 showed similarity to those of 7β-hydroxyoplop-11-enone  with H-9/H-2 , and H-3  with H-2/H-4, suggesting the presence of A-ring unit. The suggestion was supported by HMBC correlations of H-1 with C-4/C-8/C-9, H-2 with C-1 (δC 23.4)/C-3 (δC 52.1)/C-4/C-9, and H-3 with C-2 (δC 27.0)/C-4. Furthermore, HMBC correlations of H-15  with C-3/C-14 (δC 208.2), and H-3 with C-14 indicated that an acetyl group was attached on C(3).Compound spectrum , correlaspectrum  showed c6 was deduced from the NOESY spectrum , 0.95 , 1.15 , 3.37  and two oxymethines at δH 3.67 , 4.15 . The 13C NMR spectrum showed 16 carbons including four methyls , three methylenes , five high-filed sp3 methines , two oxymethines , and one double bond . The 13C and 1H NMR data of 7 were similar to those of guaia-1(10),11-diene and guaia-9,11-diene  with C-1 (δC 53.3)/C-9 (δC 31.5)/C-10 suggested one double bond between C-10 and C-14. HMBC correlations of H-12  and H-13  with C-7 (δC 43.7)/C-11 (δC 29.1), and H-11  with C-7/C-12 (δC 15.8)/C-13 (δC 22.0) indicated that an isopropyl unit attached on C-7 of the seven membered ring substructure. Meanwhile, HMBC correlations of H-16  with C-3 (δC 90.6), and H-15  with C-4 (δC 44.5), indicated that one methoxy group and one methyl were connected with C-3 and C-4, respectively.The suggestion was supported by  spectra . The pre7 was determined by a NOESY experiment -en-2β-ol.The relative configuration of periment  and compIn vitro antiviral activity of 1–4 against HSV-1 was evaluated using plaque reduction assay. First, the completely non-toxic concentration (CC0) of 1–4 and positive control ACV on Vero cells were tested to be 270.3, 523.6, 185.2, 195.3, >7500 μM by MTT assays, respectively, then for further antivirus studies, the concentrations of tested compounds were kept below their CC0 values. The antiviral assays displayed that 1–4 exhibited anti-HSV-1 activity with EC50 (50% effective concentration required to inhibit virus-induced cytopathicity 50%) values of 108.1, 471.2, 70.4, 117.2, 6.08 μM, respectively. The results suggested that the side chain at the nitrogen N(3) in 1–4 could affect their antiviral activity. Although 1 and 3 showed mild anti-HSV-1 activity, their activities were far lower than that of the positive control ACV.7 was evaluated for its antilarval activity against B. amphitrite and B. neritina larvae. The results showed that 7 had significant antilarval activity towards B. amphitrite larvae with EC50 value of 17.2 μg/mL (68.2 μM), and showed 50% inhibition towards the settlement of B. neritina larvae at concentration of 25 μg/mL. The EC50 value of 7 is lower than the standard requirement of an EC50 of 25 μg/mL established by the US Navy program as an efficacy level for natural antifoulants, indicating that 7 is a potential natural antifouling agent.Compound 1H, 13C NMR and 2D NMR spectra were recorded on a Bruker AV-500 MHz NMR spectrometer with TMS as internal standard. MS spectral data were obtained on an LCQDECA XP HPLC/MSn spectrometer for ESIMS.Optical rotations were measured with a Horiba SEAP-300 spectropolarimeter. UV spectra were measured with a Shimadzu double-beam 210A spectrophotometer in MeOH solution. E. pseudossapo  was collected in Sanya, Hainan Province, China in October 2007 and identified by Research Assistant Xiubao Li, the South China Sea Institute of Oceanology, Academia Sinica (SCSIO). A voucher specimen (No. 2007-SCSIO-3) was deposited in SCSIO, Guangzhou, China.The South China Sea gorgonian coral E. pseudossapo were exhaustively extracted with EtOH/CH2Cl2 (2:1) three times at room temperature, and the solvent was evaporated in vacuo. The residue was partitioned in H2O and extracted with EtOAc and n-BuOH in turn three times, respectively. The n-BuOH extract was concentrated in vacuo to afford 10.2 g of residue, and then the n-BuOH portion was subjected to column chromatography on silica, using CHCl3/MeOH (from 10:0 to 0:10) as eluent. By combining the fractions with TLC (GF254) monitoring, 8 fractions were obtained. Fraction 2 was chromatographed over Sephadex LH-20 eluting with CHCl3/MeOH (1:1) to obtain three sub-fractions (A–C). Sub-fraction B was purified over semi-preparative HPLC with MeOH/water (50:50) to yield 1 (10 mg) and 3 (4.0 mg). Sub-fraction C were purified over semi-preparative HPLC eluted with MeOH/H2O (60:40) to yield 2 (10.1 mg), 4 (13.0 mg), and 5 (2.3 mg). The EtOAc extracts were concentrated in vacuo to afford 33.5 g of residue. The EtOAc portion was subjected to column chromatography (CC) on silica, using petroleum ether-EtOAc (from 10:1 to 0:10) as eluent. By combining the fractions with TLC (GF254) monitoring, 16 fractions were obtained. Fraction 7 was purified by silica gel column, eluted with petroleum ether-EtOAc (2:1) to yield 8 (17.0 mg). Fraction 8 was subjected to CC on silica gel, eluted with CHCl3-Me2CO (from 100:5 to 0:10), and then purified with semi-preparative HPLC, using MeOH-water as eluent to afford 6 (10.0 mg) and 9 (6.4 mg). Fraction 10 was chromatographed over Sephadex LH-20 eluting with CHCl3/MeOH(1:1), then repeatedly subjected to CC on Si gel, eluted with CHCl3/MeOH (from 10:0 to 6:4) to yield 7 (10.3 mg).The frozen specimens of Pseudozoanthoxanthin III (1): Yellow oil; UV (MeOH) λmax 221, 257, 304, 362 nm; IR (KBr) νmax 3400, 3300, 1750, 1690, 1620 cm−1; 1H NMR and 13C NMR spectral data, see m/z 371 [M + H]+; HRESIMS m/z 371.2159 [M + H]+ .Pseudozoanthoxanthin IV (2): Yellow oil; UV (MeOH) λmax 221, 257, 304, 362 nm; IR (KBr) νmax 3407, 3313, 1752, 1694, 1623 cm−1; 1H NMR and 13C NMR spectral data see m/z 381 [M − H] −; HRESIMS m/z 381.2075 [M − H] − .8a-hydroxy-6β-methoxy-14-oxooplop-6,12-olide (6): Colorless oil; [α]25 D +0.3 ; UV (MeOH): 225 nm; IR (KBr) νmax 3276, 1723, 1625 cm−1; 1H NMR δH: 1.94, 1.63 , 2.28, 1.77 , 3.31 , 2.65 , 2.53, 1.77 , 1.84 , 1.36 , 1.80 , 2.26 , 3.14 ; 13C NMR  δC: 23.4 (C-1), 27.0 (C-2), 52.1 (C-3), 42.1 (C-4), 157.5 (C-5), 106.9 (C-6), 51.2 (C-7), 71.5 (C-8), 56.7 (C-9), 21.9 (C-10), 121.4 (C-11), 171.9 (C-12), 8.7 (C-13), 208.2 (C-14), 28.7 (C-15), 50.0 (C-16); HR-EI-MS m/z 294.1472 [M]+ .3β-methoxyguaian-10(14)-en-2β-ol (7): Colorless oil; [α]25 D +0.8 ; IR (KBr) νmax 3446, 1648, 1456 cm−1; 1H NMR δH: 2.97 , 4.15 , 3.67 , 1.73 , 2.14 , 1.97, 1.53 , 1.28 , 1.67 , 2.04, 2.24 , 1.73 , 0.78 , 0.95 , 4.64, 4.62 , 1.15 , 3.37 ; 13C NMR  δC: 53.3 (C-1), 78.3 (C-2), 90.6 (C-3), 44.5 (C-4), 45.3 (C-5), 25.9 (C-6), 43.7 (C-7), 24.8 (C-8), 31.5 (C-9), 147.8 (C-10), 29.1 (C-11), 15.8 (C-12), 22.0 (C-13), 109.2 (C-14), 30.3 (C-15), 57.1 (C-16); HR-EI-MS m/z 252.2082 [M]+ .HSV-1 (15577) strain and Vero cells were obtained from American Type Culture Collection. Cytotoxicity assay and cytopathic effect reduction assay were undertaken with the reported methods . ACV wasBalanus amphitrite and Bugula neritina larvae. The procedures were the same as previously reported [Antilarval activity of the compounds was evaluated in settlement inhibition assays with laboratory-reared reported .E. pseudossapo led to the obtainment of five zoanthoxanthin alkaloids (1–5) and four sesquiterpenes (6–9). Among these compounds, 1, 2, 6 and 7 were new, 1 and 3 showed moderate anti-HSV-1 and anti-RSV activity, and 7 showed significant antilarval activity towards B. amphitrite larvae. The results elucidate the basis of medicinal substances of E. pseudossapo, and suggest that 7 is a potential natural antifouling agent.In conclusion, our investigation on the chemical constituents of gorgonian


Spectroscopic and X-ray single-crystal diffraction analyses determined the structures of the four β-diketiminates. While thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) showed two distinct endothermic peaks for each β-diketiminate at temperatures of 92.55°C and 221.50°C (4a), 93.51°C and 238.82°C (4b), 109.60°C and 329.22°C (4c), 115.43°C and 243.25°C (4d), respectively, corresponding to their melting and boiling points.Four novel unsymmetrical β-diketiminates 2-amino-4-(phenyl)imino-2-pentene ( In addition, the solid state structures of the compounds 4a-d have been analyzed by single crystal X-ray diffraction.The availability of straightforward, multigram syntheses for many classes of β-diketiminates has generated widespread popularity of the ligand for coordination and organometallic chemistry. Prototypical symmetrical β-diketimines with N-aryl substituents can be synthesized in one step from commercially available anilines and diones through simple condensation reactions (McGeachin 4a-d were synthesized in two steps: (1) condensation of acetylacetone with one equiv. of primary aromatic amine 2,6-diisopropylaniline in methanol, with formic acid as catalyst, afforded the (Z)-4-pent-3-en-2-one intermediate 3; (2) another equiv. of the other aromatic amine  was pre-treated with para-toluenesulfonic acid in a 1 : 1 ratio for 3 h to afford para-toluenesulfonate, which was then reacted with the (Z)-4-pent-3-en-2-one intermediate 3 to give unsymmetrical β-diketimines 4a-d in good yields. In the second step, the one-pot reaction of (Z)-4-pent-3-en-2-one 3, aromatic amine and para-toluenesulfonic acid failed to give any target product; the reaction of (Z)-4-pent-3-en-2-one with the para-toluenesulfonic acid took place instead, preventing further reaction with the aromatic amine.The synthetic pathway for unsymmetrical β-diketiminates described in this work is outlined in Scheme 1H NMR spectra of all the unsymmetrical β-diketimines show a characteristic downfield shift in the range δ = 12.87-13.14 ppm for the NH proton and high field shift in the range δ = 4.79-4.84 ppm for the methyne proton attributable to the formation of unsymmetrical β-diketimines from (Z)-4-pent-3-en-2-one and amines. Two sharp singlets observed at the range 1.69-1.83 and 1.56-1.65 ppm are assigned to the protons of the two methyl groups (CH3C=NAr and CH3CNHAr) of the unsymmetrical β-diketiminates. The resonance due to the four CH3 protons (CH(CH3)2) were observed as a two doublets at the range 1.15-1.19 and 1.09-1.14 ppm while that of CH appeared as a septet at the range 3.12-3.18 ppm.The 13C NMR is in good agreement with the proposed unsymmetrical β-diketiminate structures as well. The 13C-NMR spectra of unsymmetrical β-diketiminates showed a peak at the range 161.8-164.1 ppm which is assigned to carbon of the C=N group. The methyne carbon appears at the range 95.9-96.6 ppm. Four peaks at the range 24.3-28.7, 20.3-20.6, 21–22.8 and 20.7-21.2 ppm are due to carbons of the six methyl groups 2) respectively. In addition, their identities have also been confirmed by a molecular ion peak [M+] from GC-MS mass spectra.The 4a-d are recorded in the solid state using the KBr disc technique at the region from 400 to 4000 cm-1. The (C=N) bands are observed at 1645–1557 (ν) cm-1; the position of these bands varies with the molecular structure, though no regularity can be pointed out. The bands at the range 3055–3072 (ν) cm-1 is typical of the NH group. Weak to medium absorptions around 3100–3000 cm-1 observed corresponding to the =C–H stretch of aromatic ring.The FT-IR spectra for the compounds 4a-d were grown by the slow evaporation method using methanol as the solvent at room temperature. The solid state structures of 4a-d with an atom-numbering scheme are shown in Figures 4a-d are displaced in Figures 4a-d is shown in Table 4a-d shows that they are coplanar. Compounds 4a-d have two aromatic rings and a central linkage. Compounds 4a-d crystallized in the triclinic space group P4a-d is planar; the bond lengths  259.39, found: 259.21. Anal. Calcd for C17H25NO: C, 78.72; H, 9.71; N, 5.40; Found: C, 78.61; H, 9.59; N, 5.36. IR  ν: 3418 (m), 2972 (w), 16015 (s), 1573 (m), 1515 (m), 1467 (w), 1411 (m), 1397 (m), 1344 (m), 1299 (w), 1203 (w), 1137 (m), 1076 (w), 983 (m), 842 (w), 722 (w), 601 (m), 513 (m), 484 (w).2-amino-4-(phenyl)imino-2-pentene (4a). Aniline , and para-toluenesulfonic acid monohydrate  in 100 mL of toluene were stirred for approximately 3 h at room temperature. To the obtained yellow suspension, 4-amino-3-penten-2-one  was added. A Dean-Stark apparatus was attached and the mixture was heated at reflux for 24 h to remove the water. The reaction mixture was cooled to room temperature and all the volatiles were removed under reduced pressure to give a yellow solid. The solid was treated with diethyl ether (100 mL), water (100 mL) and sodium carbonate , and the obtained mixture was kept stirring. After complete dissolution, the aqueous phase was separated and extracted with diethyl ether. The combined organic phase was dried over MgSO4 and rotary evaporated to dryness under reduced pressure to afford a brownish yellow solid. Yellow crystals  were obtained after recrystallization from methanol. 1H NMR  δ (ppm): 13.14 , 7.16-7.14 , 7.10-7.09 , 6.97-6.94 , 6.88-6.85 , 4.84 N), 3.18 2), 1.82 , 1.63 , 1.19 2), 1.14 2). 13C NMR  δ (ppm): 163.1 (NCCH3), 157.3 (NCCH3), 144.9 (Ar-C), 142.8 (Ar-C), 141.1 (Ar-C), 129.2 (Ar-C), 125.2 (Ar-C), 123.4 (Ar-C), 123.3 (Ar-C), 122.7 (Ar-C), 96.6 (CH), 28.7 (CH3C=NAr), 24.3 (CH(CH3)2), 22.7 (CH(CH3)2), 21.0 (CH(CH3)2), 20.6 (CH3CNHAr). Melting point: 93.0°C. Mass data (TOF MS EI+): Calcd for C23H30N2 [M+] 334.50, found: 334.49. Anal. Calcd for C23H30N2: C, 82.59; H, 9.04; N, 8.37; Found: C, 82.55; H, 9.01; N, 8.41. IR  ν: 3055 (w), 2960 (w), 2920 (w), 2875 (w), 1925 (w), 1873 (w), 1800 (w),1740 (w), 1630 (s), 1547 (s), 1489 (w), 1360 (m), 1275 (m), 1260 (m), 1155 (s), 1100 (w), 1030 (w), 800 (s), 798 (s), 750 (s), 699 (m), 595 (w), 501 (w), 425 (m).2-amino-4-(4-methylphenyl)imino-2-pentene (4b). β-Diketiminate 4b was synthesized by the same procedure as 4a. Yield . 1H NMR  δ (ppm): 13.11 , 7.13-7.11 , 6.89-6.85 , 4.83 N), 3.16 2), 2.05 ,1.83 , 1.63 , 1.17 2), 1.13 2). 13C NMR  δ (ppm): 163.8 (NCCH3), 156.6 (NCCH3), 143.7 (Ar-C), 141.6 (Ar-C), 140.5 (Ar-C), 132.8 (Ar-C), 129.8 (Ar-C), 124.8 (Ar-C), 123.4 (Ar-C), 123.1 (Ar-C), 96.4 (CH), 28.7 (CH3Ar), 24.3 (CH3C=NAr), 22.7 (CH(CH3)2), 21.1 (CH(CH3)2), 20.7 (CH(CH3)2), 20.5 (CH3CNHAr). Melting point: 94.0°C Mass data (TOF MS EI+): Calcd for C24H32N2 [M+] 348.52, found: 348.47. Anal. Calcd for C24H32N2: C, 82.71; H, 9.25; N, 8.04; Found: C, 82.69; H, 9.17; N, 8.03. IR  ν: 3149 (w), 3072 (w), 3015 (w), 2960 (m), 1635 (w), 1598 (s), 1542 (m), 1468 (m), 1439 (m), 1388 (w), 1255 (m), 1236 (s), 1180 (w), 1120 (m), 1105 (m), 1037 (m), 760 (m), 741(m), 664 (w), 615 (w), 602 (w), 557 (w).2-amino-4-(4-methoxyphenyl)imino-2-pentene (4c). β-Diketiminate 4c was synthesized by the same procedure as 4a. Yield . 1H NMR  δ (ppm): 13.04 , 7.14-7.11 , 6.87-6.84 , 6.65-6.63 , 4.84 N), 3.26 , 3.17 2), 1.81 , 1.65 , 1.19 2), 1.14 2). 13C NMR  δ (ppm): 164.1 (NCCH3), 156.8 (NCCH3), 156.6 (Ar-C), 144.2 (Ar-C), 140.2 (Ar-C), 136.6 (Ar-C), 125.0 (Ar-C), 124.6 (Ar-C), 123.4 (Ar-C), 114.5 (Ar-C), 95.9 (CH), 54.9 (OCH3Ar), 28.7 (CH3C=NAr), 24.2 (CH(CH3)2), 22.8 (CH(CH3)2), 21.2 (CH(CH3)2), 20.3 (CH3CNHAr). Melting point: 110.0°C. Mass data (TOF MS EI+): Calcd for C24H32N2O [M+] 364.52, found: 364.36. Anal. Calcd for C24H32N2O: C, 79.08; H, 8.85; N, 7.68; Found: C, 79.10; H, 8.72; N, 7.57. IR  ν: 3057 (m), 2971 (m), 2925 (w), 2878 (m), 2832 (w), 1631 (m), 1548 (m), 1520 (s), 1472 (s), 1400 (s), 1320 (m), 1289 (m), 1260 (w), 1251 (m), 1233 (w), 1169 (m), 1115 (m), 930 (w), 795 (m), 759 (m), 753 (w), 740 (m), 679 (w), 641 (w), 597 (m), 578 (w), 415 (m).2-amino-4-(4-methoxyphenyl)imino-2-pentene (4c). β-Diketiminate 4d was synthesized by the same procedure as 4a. Yield . 1H NMR  δ (ppm): 12.87 , 7.14-7.08 , 7.00-6.98 , 6.63-6.60 , 4.79 N), 3.12 2), 1.69 , 1.56 , 1.15 2), 1.09 2). 13C NMR  δ (ppm): 161.8 (NCCH3), 159.2 (NCCH3), 144.9 (Ar-C), 142.2 (Ar-C), 141.0 (Ar-C), 129.2 (Ar-C), 128.3 (Ar-C), 125.9 (Ar-C), 123.6 (Ar-C), 123.5 (Ar-C), 96.6 (CH), 28.7 (CH3C=NAr), 24.4 (CH(CH3)2), 22.7 (CH(CH3)2), 20.7 (CH(CH3)2), 20.6 (CH3CNHAr). Melting point: 116.0°C. Mass data (TOF MS EI+): Calcd for C23H29ClN2 [M+] 368.94, found: 368.92. Anal. Calcd for C23H29ClN2: C, 74.88; H, 7.92; Cl, 9.61; N, 7.59; Found: C, 74.89; H, 7.90; Cl, 9.58; N, 7.55. IR  ν: 3062 (s), 2960 (m), 1670 (s), 1558 (m), 1530 (w), 1495 (m), 1450 (m), 1417 (s), 1325 (m), 1271 (w), 1239 (m), 1215 (w), 1182 (m), 1120 (m), 1051 (m), 1030 (m), 930 (w), 864 (m), 837 (w), 800 (w), 745 (m), 677 (w), 635 (w).


Their antiproliferative potency was evaluated in three human cancer cell lines representing ovarian (CH1), lung (A549), and colon carcinoma (SW480). In cisplatin-sensitive CH1 cancer cells, cytotoxicity was found in the low micromolar range, whereas, in inherently cisplatin-resistant A549 and SW480 cells, the activity was very low or negligible. Astonishingly, raise in lipophilicity of the complexes, as found in the case of cisplatin analogs, did not result in a significant enhancement of the cytotoxic effect.A series of eight novel diamminetetrakis(carboxylato)platinum(IV) complexes was synthesized and characterized by multinuclear  Introduction. - Cisplatin, carboplatin, and oxaliplatin , Pt-based treatment still builds the basis for combination cancer therapy.IV complexes seems to be most promising . Con. ConIV ci) Therefore, they can be administered orally via absorption through the gastrointestinal tract, if lipophilic enough. ii) Platinum(IV) complexes act as prodrugs which can be reduced to the PtII species featuring a higher reactivity/activity (activation by reduction) in the hypoxic (oxygen deficiency) milieu of many solid tumors, accompanied by release of the axial ligands . It was found that, with increasing lipophilicity, the cellular accumulation and the antiproliferative potency were enhanced as well. In case that this behavior is a general characteristic, it should then be possible to improve the cytotoxicity of kinetically more inert diaminetetrakis(carboxylato)plati-num(IV) complexes just by increasing their lipophilicity. To validate this hypothesis, eight novel (OC-6–33)-diamminebis(carboxylato)malonatoplatinum(IV) complexes were synthesized, characterized, and their cytotoxicity was evaluated in three human cancer cell lines.II precursor, (SP-4–2)-diammineplati-num(II) (1), was prepared starting from the diamminediiodido complex via reaction with AgNO3 and subsequent coordination of malonate. Oxidation with 30% H2O2 was performed in aqueous solution at ambient temperature resulting in the octahedrally configured dihydroxido compound 2 . The latter was carboxylated with succinic anhydride in DMF as published recently -4-oxobutanoato}platinum(IV) (4e). The synthesis was carried out as described for 4a: CDI  in abs. DMF (8 ml), 3  in abs. DMF (8 ml), iPrONa (15 mg Na in 10 ml of iPrOH). The crude product was purified by CC (AcOEt/MeOH 3 :1) to yield a white solid, which was dried in vacuo. Yield: 66 mg (11%). 1H-NMR: 6.78 ; 4.92 ); 3.62 ); 2.60 ); 2.48 ); 1.20 ). 13C-NMR: 179.4 (C(3)); 172.4 (C(2)); 171.9 (C(6)); 67.4 (C(7)); 46.9 (C(1)); 30.3 (C(4)); 30.2 (C(5)); 21.3 (C(8)). 15N-NMR: -54.4.195Pt-NMR: 3543. Anal. calc. for C17H30N2O12Pt · H2O (667.52): C 30.59, H 4.83, N 4.20; found C 30.82, H 4.93, N 4.11.(OC-6–33)-Diamminebis{[4-(2-methoxyethoxy)]-4-oxobutanoato}platinum(IV) (4f). The synthesis was carried out as described for 4a. CDI  in abs. DMF (8 ml), 3  in abs. DMF (8 ml), of 2-methoxyethanolate (15 mg Na in 10 ml 2-methoxyetha-nol). The crude product was purified by CC  to yield a white solid, which was dried in vacuo. Yield: 130 mg (21%). 1H-NMR: 6.76 ; 4.19 ); 3.62 ); 3.58 ); 3.32 ); 2.61 ); 2.54 ). 13C-NMR: 179.2 (C(3)); 172.5 (C(6)); 172.4 (C(2)); 70.2 (C(8)); 63.3 (C(7)); 58.0 (C(9)); 46.9 (C(1)); 30.3 (C(4)); 29.8 (C(5)). 15N-NMR: -54.3. 195Pt-NMR: 3542. Anal. calc. for C17H30N2O14Pt· H2O (699.52): C 29.19, H 4.61, N 4.00; found C 29.10, H 4.33, N 4.00.(OC-6–33)-Diamminebis{(4-propylamino)-4-oxobutanoato}platinum(IV) (4g). A soln. of CDI  in abs. DMF (8 ml) was added to a soln. of 3  in abs. DMF (8 ml). The mixture was stirred at 608 for 15 min, then cooled down to r.t., and flushed with Ar to remove the formed CO2. PrNH2  was added, and the soln. was stirred for 48 h at r.t. DMF was removed under reduced pressure, and the crude product was purified by CC (AcOEt/MeOH 3 :1) to yield a white solid, which was dried in vacuo. Yield: 246 mg (41%). 1H-NMR: 7.80 ; 6.77 ; 3.62 ); 3.09 ); 2.55 ); 2.40 ); 1.46 ); 0.87 ). 13C-NMR: 180.2 (C(3)); 172.4 (C(2)); 171.5 (C(6)); 46.9 (C(1)); 40.8 (C(7)); 31.6 (C(4)); 31.3 (C(5)); 22.7 (C(8)); 11.1 (C(9)). 15N-NMR: 93.6 (CONH); -54.9 (NH3). 195Pt NMR: 3541. Anal. calc. for C17H32N4O10Pt · 2 H2O (683.57): C 29.87, H 5.31, N 8.20; found C 29.93, H 4.96, N 8.13.(OC-6–33)-Diamminebis{(4-cyclopentylamino)-4-oxobutanoato}malonatoplatinum(IV) (4h). The synthesis was carried out as described for 4g: CDI  in abs. DMF (8 ml), 3  in abs. DMF (8 ml), cyclopentylamine . The crude product was purified by CC (AcOEt/MeOH 3:1) to yield a white solid, which was dried in vacuo. Yield: 382 mg (60%). 1H-NMR: 7.79 ; 6.77 ; 4.08 ); 3.62 ); 2.53 ); 2.37 ); 1.83 ); 1.65 ); 1.52 ); 1.43 ). 13C-NMR: 180.3 (C(3)); 172.4 (C(2)); 171.1 (C(6)); 50.8 (C(7)); 46.9 (C(1)); 32.5 (C(8)); 31.6 (C(4)); 31.3 (C(5)); 23.6 (C(9)). 15N-NMR: 106.7 (CONH); - 54.4 (NH3). 195Pt-NMR: 3541. Anal. calc. for C21H36N4O10Pt · H2O (717.62): C 35.15, H 5.34, N 7.81; found C 35.12, H 5.23, N 7.73.Cell Lines and Culture Conditions. CH1  cells were a generous gift from Lloyd R. Kelland . A549  and SW480  cells were kindly provided by Brigitte Marian . All cell lines were grown in 75-cm2 culture flasks in Minimal Essential Medium (MEM) containing 10% heat-inactivated fetal bovine serum, 1 mm sodium pyruvate, 2 mm l-glutamine and 1% (v/v) non-essential amino acids  at 378 in a humidified atmosphere of 95% air and 5% CO2.Cytotoxicity Tests in Cancer Cell Lines. The cells were harvested from culture flasks by using 0.25% trypsin. Then, 100 ml of cell suspensions in MEM were seeded in densities of 1.0 x 103 (CH1), 4.0 x 103 (A549), and 2.5 x 103 (SW480) cells per well into 96-well microculture plates and incubated for 24 h to restore adherent growth. After dissolving and serially diluting the test compounds in MEM, 100 ml of these solns. were added to each well, and plates were incubated for 96 h at 378. Thereafter, the MEM containing the test compounds was replaced with 100 ml of RPMI1640 medium and 20 ml 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) in phosphate-buffered saline . After 24 h incubation at 378, the mixture was aspirated, and the purple formazan product was dissolved in 150 ml of DMSO per well. The absorbance of the resulting colored solns. was quantified spectrophoto-metrically at a wavelength of 550 nm and a reference wavelength of 690 nm with a microplate reader (Tecan Spectra Classic). Results were averaged from at least three independent experiments.


N-arylidene substituents, was synthesized for evaluation as anti-inflammatory agents. The starting material 2-(2-Phenoxyphenyl) acetohydrazide was synthesized from 2-phenoxybenzoic acid in several steps according to the previous published method. Various substituted arylidene-2-phenoxynicotinic acid hydrazide derivatives were synthesized by the reaction of hydrazide 17 with selected aldehydes and screened for their potential anti-inflammatory activity. The structure of synthesized compounds was confirmed by different nuclear magnetic resonance technique, Fourier transform infrared spectroscopy (FTIR) and Mass-spectrometry data format. Qualitative structure-activity relationship data, acquired using the carrageenan-induced rat paw edema assay, showed that this group of arylidene-2-phenoxybenzoic acid hydrazides exhibit anti-inflammatory activity with significant reduction of rat paw edema  in comparison with control group and a moderate to good activity range in comparison with diclofenac as the reference drug. Compounds 9a, 9d and 9e exhibited the most prominent and consistent anti-inflammatory activity. The compound, N-(4-Chlorobenzylidene)-2-(2-phenoxyphenyl) acetohydrazide (9d), exhibited the most in-vivo activity (32-58% reduction in inflammation) compared to the reference drug diclofenac (35-74% reduction in inflammation) in a carrageenan induced rat paw-edema assay.Diclofenac sodium has been used for its anti-inflammatory actions for about 28 years, but since all the non-steroidal anti-inflammatory drugs (NSAIDs) suffer from the lethal gastro intestinal (GI) toxicities, diclofenac sodium is not an exception. The free –COOH group is thought to be responsible for the GI toxicity associated with all traditional NSAIDs. In the present research, the main motto was to develop new chemical entities as potential anti-inflammatory agents with no GI toxicities. A new type of 2-(2-phenoxyphenyl) acetohydrazide possessing  Non-steroidal anti-inflammatory drugs (NSAIDs) are the most prescribed drugs in the treatment of pain and inflammation, particularly for different types of arthritis -3. The pDiclofenac, Mefenamic acid and Indomethacin are three traditional NSAIDs belonging to the class of fenamates . N-phenyl (or heteroaryl) anthranilic acid that are employed for their analgesic, anti-inflammatory and anti-pyretic properties. Unlike most of the NSAIDs, the fenamates also appear to compete with prostaglandins for binding at the prostaglandin receptor site and, thus, potentially antagonizing the physiopathological effects of prostaglandins that have already been formed. Fenamates arestill endowed with most of the adverse effects induced by NSAIDs, particularly gastrointestinal (GI) bleeding, ulceration, and perforation. Several studies have described the derivatization of the carboxylate function of representative NSAIDs with amide or N-acylarylhydrazone having less acidic amide hydrogen resulted in an increased anti-inflammatory activity with reduced ulcerogenicity (N-Acylhydrazones (NAH) have been widely described as potent anti-inflammatory, anti-nociceptive, and anti-platelet compounds, due to their ability to mimic the bis-allylic moiety of unsaturated fatty acids and amides, for example, arachidonic acid (AA), precursor of the eicosanoid biosynthesis, involved in the endocannabinoid system. This can be rationalized through the relative acidity in amide hydrogen of NAH group as well as its capacity of stabilizing the free radicals  spectra were measured through a Brucker FT-500 MHz spectrometer  in CDCl3 or DMSO-d6 with tetramethylsilane (TMS) as the internal standard, where j-values (coupling constant) are estimated in Hertz. Spin multiples are given as s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), m (multiplet), and b (broad). Mass spectra were obtained, usinga Finnigan Mat triple-stage quadrupole (TSQ 70) spectrometer . All solvents and reagents were purchased from the Fluka , or Merck Chemical Company . Male Wistar rats used in the anti-inflammatory screens were purchased from Pasteur Institute  and experiments were carried out using protocols approved by the ethics committee of Tehran University of Medical Sciences. Compounds 10-16 have been synthesized according to the published methods in literatures.Melting points were determined with a Reichert-Jung hot-stage microscope  and are uncorrected. Infrared spectra were recorded on a Nicolet Magna 550-FT spectrometer . 2-(2-Phenoxyphenyl) acetohydrazide (17)°C;1H-NMR : δ 7.3-7.36 , 7.21-7.24 , 7.1 , 6.94 , 6.87 , 3.76 , 3.57 ; IR (KBr): Cm-1 3308 (N-H), 3037 , 1654 (C=O), 1236 (C-O), 1096 (C-N); MS: m/z (%) 242 , 210 (68), 183 (100), 165 (32), 155 (54), 77 (45), 51 (36).A solution of compound 16  and hydrazine hydrate  in 20 mL methanol was stirred at room temperature for 5 h. The reaction mixture poured immediately into a mixture of crushed ice water, gave a crude precipitate collected by filtration and recrystallized in ethanol/water to give 2.6 g (94%) of hydrazide derivative 17, as a light yellow amorphous solid, m.p.: 62-64General procedure for preparing N-arylidene-2-(2-phenoxyphenyl) acetohydrazides (9a-g)The synthesis of final hydrazones 9a-g was carried out by the reaction of hydrazide 17 and the corresponding aldehydes in ethanol. A mixture of hydrazide 17 (1.5 mmol) and the corresponding aldehyde (1.5 mmol) in ethanol (10 mL) was stirred at room temperature for 1 to 24 h in the presence of hydrochloric acid (2 drops) as the catalyst. The completion of the reaction was monitored by thin layer chromatography (TLC). The reaction mixture was concentrated under reduced pressure and was neutralized with a 10% aqueous solution of sodium bicarbonate. The resulting precipitate was filtered, washed with water, and crystallized using a suitable solvent to give 9.N-Benzylidene-2-(2-phenoxyphenyl) acetohydrazide (9a)°C (Ethanol/Water); as a mixture of diastereomers in ratio of 2.59:1; 1H-NMR : δ 9.01 , 8.77 , 7.80 ,7.72-7.63 , 7.62-7.55 , 7.49 , 7.45-7.32 , 7.27-7.18 , 7.17-7.08 , 7.05-6.88 , 4.17 , 3.74 ; IR (KBr): Cm-1 3249 (N-H), 3056 , 1677 (C=O), 1235 (C-O); MS: m/z (%) 330 , 240 (10), 227 (5), 210 (8), 183 (89), 165 (28), 155 (38), 149 (12), 134 (100), 119 (23), 104 (10), 89 (23), 77 (41), 68 (32), 55 (22).M.p.: 169-171N-(4-Methyl-benzylidene)-2-(2-phenoxyphenyl) acetohydrazide (9b)°C (Ethanol/Water); as a mixture of diastereomers in ratio of 2.03:1; 1H-NMR : δ 9.40 , 8.73 , 7.73 , 7.64 , 7.55 , 7.49 , 7.36 , 7.27-7.20 , 7.20-7.8 , 7.07-6.97 , 6.97-6.87 , 4.17 , 3.72 , 2.38 , 2.36 ; IR (KBr): Cm-1 3262 (N-H), 3083 , 1685 (C=O), 1242 (C-O); MS: m/z (%) 344 , 279 (10), 237 (3), 228 (3), 196 (3), 181 (50), 165 (20), 149 (70), 134 (65), 127 (12), 118 (20), 104 (30), 91 (32), 77 (60), 69 (62), 57 (97), 41 (100).M.p.: 196-198N-(4-Methoxybenzylidene)-2-(2-phenoxyphenyl) acetohydrazide (9c)°C (Ethanol/Water); as a mixture of diastereomers in ratio of 2.03:1; 1H-NMR : δ 9.43 , 8.74 , 7.71 , 7.63 , 7.60 , 7.53 , 7.48 , 7.40 , 7.35 , 7.28-7.19 , 7.17-7.07 , 7.05-6.85 , 4.16 , 3.84 , 3.82 , 3.72 ; IR (KBr): Cm-1 258 (N-H), 3075 , 1677 (C=O), 1308 (C-O); MS: m/z (%) 360 , 279 (6), 257 (5), 243 (5), 218 (8), 211 (4), 194 (6) , 183 (55), 165 (21), 149 (84), 134 (100), 121 (14) , 105 (16), 97 (30), 81 (41), 69 (89), 57 (84), 43 (88).M.p.: 200-202N-(4-Chlorobenzylidene)-2-(2-phenoxyphenyl) acetohydrazide (9d)°C (Ethanol/Water); as a mixture of diastereomers in ratio of 2.33:1; 1H-NMR : δ 9.34 , 8.82 , 7.79 , 7.63 , 7.59 , 7.51 , 7.48 , 7.39 , 7.38-7.30 , 7.29-7.20 , 7.19-7.07 , 7.05-6.96 , 6.95-6.85 , 4.15 , 3.73 ; IR (KBr): Cm-1 3246 (N-H), 3083 , 1681 (C=O), 1246 (C-O); MS: m/z (%) 366 , 330 (3), 282 (20), 257 (5),236 (12), 227 (10), 210 (8) , 196 (35), 183 (100), 195 (30), 134 (100), 165 (30) , 155 (56), 134 (89), 127 (15), 99 (7), 77 (30), 57 (28).M.p.: 215-218N-(4-Fluorobenzylidene)-2-(2-phenoxyphenyl) acetohydrazide (9e)°C (Ethanol/Water); as a mixture of diastereomers in ratio of 2.46:1; 1H-NMR : δ 9.24 , 8.80 , 7.80 , 7.70-7.60 , 7.57 , 7.48 , 7.40 , 7.36 , 7.26-7.18 , 7.15-7.09 , 7.08-6.97 , 6.96-6.85 , 4.16 , 3.73 ; IR (KBr): Cm-1 3458 (N-H), 3222 , 1677 (C=O), 1237 (C-O), 1096 (C-N); MS: m/z (%) 348 , 256 (7), 227 (5), 210 (5), 183 (100), 165 (31), 155 (42), 149 (70), 134 (65), 134 (80), 122 (10), 107 (14), 95 (17), 77 (28), 69 (40).M.p.: 210-215N--2-(2-phenoxyphenyl) acetohydrazide (9f)°C (Ethanol/Water); as a mixture of diastereomers in ratio of 1.4:1; 1H-NMR : δ 8.00, 7.99 , 7.75, 7.73 , 7.46 , 7.39 , 7.33 , 7.27-7.18 , 7.16-7.07 , 7.05-6.95 , 6.92-6.87 , 6.60-6.52 , 6.48 , 6.46-6.44 , 6.42 , 4.14 , 3.87, 3.88, 3.84, 3.83, 3.82, 3.81 ; IR (KBr): Cm-1 1670 (C=O), 1207 (C-O), 1031 (C-N); MS: m/z (%) 390 , 359 (7), 227 (5), 210 (5), 183 (100), 164 (31), 150 (70), 137 (65), 122 (10), 106 (14), 95 (17),75 (28), 69 (40).M.p.: 250-252N--2-(2-phenoxyphenyl) acetohydrazide (9g)°C (Ethanol/Water); as a mixture of diastereomers in ratio of 1.69:1; 1H-NMR : δ 9.55 , 8.75 , 7.76 , 7.61 , 7.47 , 7.40 , 7.36 , 7.29-7.20 , 7.15-7.08 , 7.04-6.95 , 6.95-6.87 , 6.88 , 6.82 , 4.15 , 3.87, 3.86 , 3.85 , 3.72 .; IR (KBr): Cm-1 3243 (N-H), 3222 , 1667 (C=O),1237 (C-O), 1065 (C-N); MS: m/z (%) 420 , 389 (10), 226 (5), 212(5), 184 (100), 170 (31), 152 (42), 149 (70), 136 (65), 121 (10), 107 (14), 90 (17), 77 (28), 69 (40).M.p.: 258-260Anti-inflammatory activityin-vivo using the carrageenan-induced rat paw edema test. Edema was induced in the right hind paw of all rats by subcutaneous injection of 0.1 mL of 1% (w/v) carrageenan  in saline into their footpads 0.5 h after the intra-peritoneal (IP) administration of compounds (18). The paw thickness was measured from the ventral to the dorsal surfaces using a dial caliper immediately before and then two and four h after the carrageenan injection. The edema was calculated as the thickness variation between thickness of paw before and after carrageenan injection. The anti-inflammatory activity was expressed similar to the inhibition percentage of edema when compared with the control group and was calculated using the following formula:The anti-inflammatory activity was determined Inhibition% = (1 - Tt / Tc) × 100t and Tc are defined as the thickness variation of test group and control group, respectively.In this formula, TStatisticsThe results are expressed as mean ± SEM of n animals per group. The data were statistically analyzed by one-way analysis of variance (ANOVA) followed by Tukey multi comparison test.Differences with p < 0.05 among the experimental groups were considered statistically significant.ChemistryThe synthetic reactions leading to the substituted arylidene-2-(2-phenoxyphenyl)- aceto- hydrazides 9a-g are outlined in 1H-NMR and Mass spectral data for synthesized compounds are reported in experimental protocols.2-(2-Phenoxyphenyl) acetohydrazide  is the k1H-NMR spectral data, all protons were seen according to the expected chemical shift and integral values. The aromatic protons appeared as multiplet peaks within the range of 6.9-7.7 ppm and the singlet signals derived from hydrazide 17 (–NH–NH2) structure appeared at 3.76 ppm. Methylene protons resonated as singlet at 4.1-4.2 ppm. The 1H-NMR spectra of compounds 9a-g displayed singlet due to –NH– groups around 8.1, 9.01 and 9.4 ppm (probably due to their ability to get exchanged with D2O) while each signal showed integration for one proton. For the compounds 9a-g, the signals which belonged to benzylidene group were observed at aromatic region, while the signals of –NHNH2 disappeared indicating the functionalization of hydrazide to hydrazone with substituted aromatic aldehydes.The FTIR spectra of Schiff bases exhibited very similar features and showed the expected bands for the characteristic groups which were present in the compounds such as C–H and the C=N stretching vibrations and another specific band for Ar–C–N vibrations. Compounds of 9a–g have C=O stretching bands in the range of 1677-1685. In the proton 1H-NMR for 9a-g,enabled us to detect the presence oftwo singlet signals related to the imine hydrogen, which was attributed to respective (E) and (Z)-diastereomers. The existent ratio between the two diastereomers could be defined from the relative integration of imine-attached hydrogen in the corresponding 1H- NMR spectra (The next step of this work was to determine the relative configuration of the imine double bond of arylidene benzoic acid derivatives 9a-g, in order to assure the stereoisomeric ratio essential for the complete understanding of the biological effect.The careful analysis of the  spectra .Anti-inflammatory activityIn-vivo pharmacological evaluation of 9a-g was carried out to assess their potential anti-inflammatory activity. Qualitative structure-activity relationship data acquired using the carrageenan-induced rat paw edema assay (ma assay , showed ma assay . As shown in Various substituted arylidene-2-phenoxynicotinic acid hydrazide derivatives were synthesized and screened for their potential anti-inflammatory activity. Most compounds exhibited moderate to good anti-inflammatory activities in comparison with diclofenac. Compounds 9a, 9d and 9e exhibited the most prominent and consistent anti-inflammatory activity.


The synthesized compounds were screened for their in vitro growth inhibiting activity against different strains of bacteria viz., Klebsilla penumoniae, Escherichia coli, Bassilus subtilis and Streptococcus aureus and antifungal activity against Aspergillus niger and Crysosporium pannical and results have been compared with the standard antibacterial streptomycin and antifungal griseofulvin. Compounds exhibits significant antibacterial activity and antifungal activity. Compounds 4a and g exhibited equal while 4c, d, i and j slightly less antibacterial activity than standard streptomycin. Compounds 4a and g exhibited equal while 4b, c, d, f and i displayed slightly less antifungal activity than standard griseofulvins.An efficient electrochemical method for the preparation of 2-amino-5-substituted-1,3,4-oxadiazoles (4a-k) at platinum anode through the electrooxidation of semicarbazone (3a-k) at controlled potential electrolysis has been reported in the present study. The electrolysis was carried out in the acetic acid solvent and lithium perchlorate was used as supporting electrolyte. The products were characterized by IR, All In the context of green chemistry, some 5-substituted-2-amino-1,3,4-oxadiazoles (4) have been synthesized by electrooxidative cyclization of semicarbazone (3) as a new general environmentally benign synthetic method. The development of ecofriendly synthetic methods are the need of the hour. In this respect, organic synthesis involving multi-component reactions under reagent-free conditions is a basic protocol because multistep conventional synthesis produces considerable amounts of environmentally unfavorable wastes, mainly due to a series of complex isolation procedures often involving expensive, toxic and hazardous solvents and reagents after each step. The application of electricity as a non conventional energy source for activation of reactions in suitable solvents has now gained popularity over the usual homogeneous and heterogeneous reactions, as it provides chemical processes with special attributes, such as enhanced reaction rates, better selectivity, higher yield of pure products and several eco-friendly advantages. These reactions do not require oxidizing reagents and can be performed at ordinary room temperature.-1.1H NMR and13C NMR spectra were measured on Bruker DRX 300 MHz FT spectrometer instruments using DMSO-d6 as solvent with TMS and CDCl3 as internal standards . Carbon multiplities were assigned by DEPT techniques. The structures of the newly synthesized compounds were assigned on the basis of elemental analysis and were recorded on a Elementar Vario EL III. Carbon, hydrogen and nitrogen analyses were within ±0.4% of the theoretical values. Mass spectra were taken out on a Jeol SX 102/DA-6600 mass spectrometer using Argon/Xenon  as the FAB gas. All the chemicals used were of synthetic and AR grade and was procured from Agros-Organics, USA, S. D. Fine Chem. Ltd., Mumbai and Merck, Mumbai, India.Column chromatography was carried out by using Merck silica gel 60. The purity of the synthesized compounds were ascertained by TLC on pre-coated silica gel plates in various solvent systems using iodine vapors and UV light as detecting agent. The melting points were recorded on an electrothermal apparatus and were uncorrected. Infra red spectra were recorded on a Shimadzu 8201 PC IR spectrophotometer in KBr pellets and reported in cm4 were dissolved in (100 ml) acetic acid.Semicarbazide hydrochloride  (2) and NaOAc (12.2 mmol) was dissolved in (10 ml) water and then an aldehyde  (1) was added with continuous stirring. The mixture was left overnight, which evolved a solid product semicarbazone (3) which was used as initial compound for the electrolysis. Semicarbazone  (3) and LiClO-1of electricity was passed for the electrolysis which is very small in comparison to energy used in other conventional methods. The physical and analytical data of the synthesized compounds (4a-k) has been depicted in the 1H NMR,13C NMR and MS) and analytical data are in good agreement with their structures.Preparative scale controlled potential electrolysis–30 were -1): 3360 (NH), 3045 (C-H aromatic), 1613 (C=N-N=C), 1470 (C=C aromatic), 1265, 1072 (C-O-C), 980, 890, 750, 595 (substituted benzene);1H NMR : 7.75 , 6.94-7.14 ;13C NMR : 158.9 (C), 147.7 (C), 140.6 (C), 132.9 (CH), 131.9 (CH), 125.1 (C), 115.4 (C), 106.4 (CH); MS m/z: 240 (M+), 241  for C8H6N3OBr.Brownish crystals; mp: 68-69°; IR : 3362 (NH), 3042 (C-H aromatic), 1620 (C=N-N=C), 1473 (C=C aromatic), 1265, 1072 (C-O-C), 980, 890, 7505, 595 (substituted benzene);1H NMR : 7.75 , 6.94-7.24 ;13C NMR : 158.9 (C), 147.7 (C), 140.6 (C), 132.9 (CH), 131.9 (CH), 125.1 (C), 115.4 (C), 106.4 (CH); MS m/z: 240 (M+), 241  for C8H6N3OBr.Brownish crystals; mp: 75-76°; IR : 3360 (NH), 3045 (C-H aromatic), 1615 (C=N-N=C), 1475 (C=C aromatic), 1275, 1075 (C-O-C), 985, 890, 755, 597 (substituted benzene);1H NMR : 7.75 , 7.71 , 7.64 ;13C NMR : 158.9 (C), 147.7 (C), 140.6 (C), 132.9 (CH), 131.9 (CH), 125.1 (C), 115.4 (C), 106.4 (CH); MS m/z: 240 (M+), 241  for C8H6N3OBr.Brownish crystals; mp: 69-70°; IR : 3341 (NH), 3035 (C-H aromatic), 1607 (C=N-N=C), 1550 (N=O), 1465 (C=C aromatic),1275, 1070 (C-O-C), 985, 865, 810, 730 (substituted benzene);1H NMR : 7.75 , 7.25-7.69 ;13C NMR : 171.3 (C), 147.1 (C), 146.8 (C), 140.8 (C), 136.8 (C), 134.7 (CH), 130.4 (CH), 121.2 (CH); MS m/z: 252 (M+), 253  for C8H6N5O5.Dark yellowish needles; mp: 71-73°; IR : 3350 (NH), 3037 (PyC-H), 1628 (C=N-N=C), 1472 (C=C aromatic), 1430-1600 (C=C and C=N str), 1070 (C-O-C);1H NMR : 7.18-8.56 , 7.75 ;13C NMR : 171.2(C), 167 (C), 149.8 (CH), 135.7 (CH), 129 (C), 123.6 (CH), 122.1 (CH); MS m/z: 162 (M+), 163  for C7H6N4O.Dark yellowish crystals; mp: 67-68°; IR : 3360 (NH), 3062 (C-H), 1618 (C=N-N=C), 1280, 1066 (C-O-C), 680 (C-Cl);1H NMR : 7.75 , 3.8 ;13C NMR : 170.6 (C), 167.6 (C), 24.9 (CH2); MS m/z: 133.5 (M+), 134.5  for C3H4N3OCl.Brownish crystals; mp: 61-62°; IR : 3360 (NH), 3065 (C-H), 1609 (C=N-N=C), 1280, 1066 (C-O-C), 690 (C-Cl);1H NMR : 7.75 , 3.9 ;1H NMR : 172.1 (C), 163.2 (C), 51.2 (CH); MS m/z: 167 (M+), 168  for C3H3N3OCl2.Brownish crystals; mp: 64-65°; IR : 3270 (NH), 3045 (C-H aromatic), 3010 (C-H), 2927, 1602 (C=N-N=C), 1473 (C=C aromatic), 1265, 1069 (C-O-C), 960, 765 (substituted benzene); H1NMR  7.75 , 7.69 , 7.62 , 1.12 ; C13NMR  178.2(C), 149.9 (C), 141.3(CH), 138.6 (CH), 136.5 (CH), 129 (CH), 127.8 (CH), 126.5 (CH), 20.6 (CH3); MS m/z: 175 (M+), 176  for C9H9N3O.Light brown needles; mp: 74-75°; IR : 3261 (NH), 3045 (C-H aromatic), 2815 (OCH3), 1609 (C=N-N=C), 1470 (C=C aromatic), 1270, 1069 (C-O-C), 915, 870, 790 (substituted benzene);1H NMR : 7.75 , 6.46-7.70 , 3.11 ;13C NMR : 172.4 (C), 167.5 (C), 146.7 (C), 146.3 (C), 134.9 (C), 129.5 (C), 105.7 (CH), 106.5 (CH), 54.3 and 44.6 (CH3); MS m/z: 251 (M+), 252  for C11H13N3O4.Dark brownish needles; mp: 84-85°; IR : 3330 (NH), 3045 (C-H aromatic), 1612 (C=N-N=C), 1475 (C=C aromatic), 1055 (C-O-C), 775 (substituted aromatics);1H NMR : 7.75 , 7.25-7.69 ;13C NMR : 171.5 (C), 149.5 (C), 133.7 (C), 128 (CH), 126 (C); MS m/z: 227 (M+), 228  for C12H9N3O2.Dark brownish needles; mp: 94-95°; IR : 3335 (NH), 3045 (C-H aromatic), 1622 (C=N-N=C), 1468 (C=C aromatic), 1045 (C-O-C), 775 (substituted aromatics);1H NMR : 7.75 , 7.25-7.69 ;13C NMR : 171.6 (C), 149.5 (C), 133.7 (C), 128 (CH), 126 (C); MS m/z: 227 (M+), 228  for C12H9N3O2.Dark brownish needles; mp: 96-97°; IR . The standard groups received phenylbutazone 50 mg/kg body weight po, suspended in 1% w/v of carboxymethylcellulose (CMC) in distilled water. The test group received synthesized compounds (4a-k) . The control group received corresponding amount of vehicle (1% w/v of CMC). All the test compounds and standard drug were administered 30 min prior to carrageenan injection.Antiinflammatory activity was determined by carrageenan-induced rat paw method of Winter. Male WiAcute edema was induced in the right hind paw of rats by injecting 0.1 ml of freshly prepared 1% w/v of aqueous solution of carrageenan  in the subplanter region of right hind paw. After the carrageenan injection the paw volume was measured before and after 1, 2 and 3 h by plethysmometer . The difference between the left and right paw was taken as a measure of oedema. Any significant reduction in the volume of the paw compared to the control group was considered as anti-inflammatory response. Percent inhibition of inflammation after 3 h was calculated by applying Newbould formula. % inhibition = 100×1–(a – x)/(b – y), where, x= mean paw volume of rats before the administration of carrageenan injection in the test and the standard groups, y = mean paw volume of rats before the administration of carrageenan injection in the control group, a = mean paw volume of rats after the administration of carrageenan and test compound or standard compound, b = mean paw volume of rats after the administration of carrageenan injection in control group. The results are presented in 4) as an electrolyte that can be handled very easily without major precautions.It is obvious that an environmentally benign synthetic method for the synthesis of 2-amino-5-substituted-1,3,4-oxadiazoles would be highly attractive. Keeping this objective in the mind, we have synthesized 2-amino-5-substituted-1,3,4-oxadiazole 4 derivatives through the electrooxidation of semicarbazone 3 at a platinum electrode in controlled potential electrolysis in acetic acid . This elThe first step of the mechanism  represenKlebsilla penumoniae, Escherichia coli, Basillus subtilis, Streptococus aureus organisms at the two concentrations (25 and 50 ppm) taking Streptomycin as the standard. The majority of the compounds exhibited significant antibacterial activity against E. coli, K. pneumonia, B. subtilis and S. aureus as compared to standard streptomycin. The screening results of antibacterial activity revealed that compound 4a and g exhibited approximately similar activity to the standard Streptomycin. Compounds 4c, d, i and j exhibited slightly less antibacterial activity. Compound 4b exhibited weak while other compounds have zero or negligible antibacterial activity against all bacterial strains used for our evaluation. The screening results showed that compounds 4b, c d, f and i displayed better antifungal activity against Aspergillus niger and C. pannical along with the standard fungicide Griseofulvins. The screening results revealed that compounds 4a and g showed equal antifungal activity when compared with the griseofulvins.The antimicrobial activity indicated that compounds 4a, b, c, d, f, g and i were found to be active against The antimicrobial activity of the compounds varied upon the type and position of the substituents at 5-substituted-2-amino-1,3,4-oxadiazole moiety. It can be concluded from the antimicrobial screening results that when 5-substituted-2-amino-1,3,4-oxadiazoles were substituted with aryl halide the antimicrobial activity was altered to an appreciable extent.The results of antiinflammatory activity  shows th


Staphylococcus aureus (MRSA) strains exemplifies the urgent need for new antibiotics. The marine natural product, marinopyrrole A, was previously shown to have potent antibiotic activity against Gram-positive pathogens, including MRSA. However, its minimum inhibitory concentration (MIC) against MRSA was increased by >500 fold in the presence of 20% human serum, thus greatly limiting therapeutic potential. Here we report our discovery of a novel derivative of marinopyrrole A, designated 1a, featuring a 2–4 fold improved MIC against MRSA and significantly less susceptibility to serum inhibition. Importantly, compound 1a displayed rapid and concentration-dependent killing of MRSA. Compared to the natural product counterpart, compound 1a provides an important natural product based scaffold for further Structure Activity Relationship (SAR) and optimization.Infections caused by drug-resistant pathogens are on the rise. The ongoing spread of methicillin-resistant  Staphylococcus aureus (MRSA) several years ago . In . In 1 an2 was obtained using a published procedure -2-yl)methanone (10a). To a solution of compound 9a  in DCM (5 mL) at 0 °C, SO2Cl2  was added dropwise, and the solution was allowed to stir at 0 °C for 1 h. Saturated aqueous NaHCO3 solution (2 mL) was added and the resulting mixture was extracted with DCM (3 × 4 mL). The combined organic layers were dried with anhydrous MgSO4, filtered, and concentrated. The residue was purified by flash column chromatography  to afford 10a  as an off-white solid. 1H NMR  δ 10.38 , 7.25–7.21 , 7.13 , 6.96–6.94 , 6.76 , 6.68 , 6.31 , 3.80 , 3.72 ; HRMS (ESI-TOF) [M + H]+ calcd for C24H16Cl5N2O4 570.9547, found 570.9537; HPLC purity, 95.2%.(4-Chloro-2-methoxyphenyl)-1′H-1,3′-bipyrrol-2-yl)methanone (10b). The same procedure described for 10a was used to synthesize 10b . 1H NMR  δ 10.28 , 7.36 , 7.31–7.27 , 7.19 , 7.07 , 6.88 , 6.79 , 6.73 , 6.33 , 3.78 , 3.73 .(5-Chloro-2-methoxyphenyl)-1′H-1,3′-bipyrrol-2-yl)methanone (10c). The same procedure described for 10a was used to synthesize 10c . 1H NMR  δ 10.30 , 7.57 , 7.37–7.27 , 7.11 , 6.85 , 6.76 , 6.67 , 6.57 , 3.83 , 3.73 .(3-Chloro-2-methoxyphenyl)-1′H-1,3′-bipyrrol-2-yl)methanone (1a). To a solution of 10a  in anhydrous DCM (1 mL) was slowly added 1.0 M solution of BBr3 in DCM  via a syringe under N2 at −78 °C. After being stirred for 0.5 h, the mixture was quenched by addition of MeOH (0.5 mL) and extracted with DCM (3 × 10 mL). The combined organic layers were dried over anhydrous MgSO4, filtered and concentrated in vacuum. The residue was purified by column chromatography  to give 1a  as a yellow solid. 1H NMR  δ 11.34 , 10.39 , 9.77 , 7.47 , 7.43 , 7.36 , 7.04 , 6.95–6.90 , 6.87 , 6.68 , 6.53 ; HRMS (ESI-TOF) [M + H]+ calcd for C22H12Cl5N2O4 542.9234, found 542.9237; HPLC purity 96.6%.(4-Chloro-2-hydroxyphenyl)-1′H-1,3′-bipyrrol-2-yl)methanone (1b). The same procedure as 1a was followed to obtain 1b  from 10b . 1H NMR  δ 11.10 , 10.33 , 9.89 , 8.06 , 7.95 , 7.83 , 7.71–7.64 , 7.49–7.41 , 6.97 , 6.87 ; HRMS (ESI-TOF) [M + Na]+ calcd for C22H11Cl5N2O4Na 564.9054, found 564.9055; HPLC purity, 95.0%.(5-Chloro-2-hydroxyphenyl)-1′H-1,3′-bipyrrol-2-yl)methanone (1c). The same procedure as 1a was followed to obtain 1c  from 10c . 1H NMR  δ 11.74 , 10.48 , 9.93 , 8.06 , 7.94 , 7.82 , 7.67 , 7.61 , 7.45 , 6.96 , 6.83 ; HRMS (ESI-TOF) [M + Na]+ calcd for C22H11Cl5N2O4Na 564.9054, found 564.9058; HPLC purity, 97.4%.(3-Chloro-2-hydroxyphenyl)-1′TCH1516, a USA300 strain of community-associated MRSA, was obtained from the American Type Culture Collection and used for biological assays. MICs were determined by broth microdilution according to Clinical and Laboratory Standards Institute guidelines except that Todd-Hewitt broth (THB) was used in place of Mueller-Hinton broth. Assays were done in 96-well tissue-culture treated plates. Vancomycin served as a control antibiotic. MIC assays in 20% human serum were assessed by bacterial metabolic activity in resazurin as described [1a against the CA-MRSA isolate TCH1516 were assessed by time-kill analysis essentially as described previously [5 cfu/mL) were added to duplicate 5 mL polystyrene round-bottom tubes  containing 20×, 10×, 1×, none of the MIC of derivative 1a (0.39 μM) or an equivalent amount of DMSO vehicle control (10 cfu/mL).The bactericidal activity of derivative eviously ,20,21. B control , none. T1a (MIC = 0.19–0.39 μM), exhibits significantly greater activity than linezolid and vancomycin against MRSA. Moreover, in vitro time-kill kinetics of 1a demonstrated potent concentration-dependent killing. These qualities were similar to those displayed by the parent natural product in time-kill studies, but 1a was 2–4-fold more potent at lower compound concentrations. One of the main drawbacks of the natural product has been its significant inhibition in the presence of human serum. For example, the antibiotic activity of (−)-1 was inactivated (MIC increased 256–522×) in the presence of serum [1a again exhibits an advantage, with only 32–64 fold increase in MIC in 20% human serum (In summary, we designed and synthesized a novel series of asymmetrical derivatives of marine natural product, marinopyrrole A, and discovered that a novel derivative, of serum . Here, 1an serum . Further


Alternaria alternata strain D2006, isolated from the marine soft coral Denderonephthya hemprichi, which was selected as its profound antimicrobial activities. The compounds were assigned as pyrophen (1), rubrofusarin B (2), fonsecin (3), and fonsecin B (5) beside to the four dimeric naphtho-γ-pyrones; aurasperone A (6), aurasperone B (7), aurasperone C (8), and aurasperone F (9). Structures of the isolated compounds were identified on the basis of 1D and 2D NMR spectroscopy and mass  data, and by comparison with the literature. Configuration of the four dimeric naphtho-γ-pyrones 6-9 was analyzed by CD spectra, exhibiting an identical stereochemistry.Eight bioactive pyrone derivatives were identified from the culture of  Mycobacterium tuberculosis, among them more than 80% of TB patients living in sub-Africa and Asia +. The 1H NMR spectra (Table δ 14.96), two m-coupled doublets  and two singlets (δ 6.94 and 5.98), along with two methoxy signals  and an sp2 linked methyl (δ 2.35). The 13C/HMQC spectra (Table sp2 methines (δ 107.3-97.2), 3 sp2-oxy carbons (δ 162.6-160.6), 1 carbonyl of γ -lactone (δ 184.2) 284-285 +359274-22843.4, then one trough between [θ]270-267 -151670-339938 and the last elliptical peak was shown at [θ]227-219 +107899-5629. As the dimer 6 has no further chiral elements, the chiral axis is dominating the absolute configuration. Based on the revealed features from the CD spectroscopic data, the four dimeric compounds (6-9) have identical (S)-configurations around their corresponding axis between C-10' and C-7 . The absolute configurations of dimeric naphtho-terature , (S)-con1-8 was examined against 11 microbial test organisms using the agar diffusion method (40 μg/disc) (Table 1) and rubrofusarin B (2) displayed high (28 mm) and moderate (12 mm) activity against C. albicans, respectively, while aurosperone A (6) was active (13 mm) against the plant pathogenic fungi, Rhizoctonia solani. In the brine shrimp assay (10 μg/mL), all studied compounds here showed weak cytotoxicity (approx. 4-11%).The antibiotic activity of compounds c) Table . Accordin method 0 μg/discRf values were measured on Polygram SIL G/UV254 . Size exclusion chromatography was done on Sephadex LH-20 .The NMR spectra were measured on a Bruker AMX 300 (300.135 MHz), a Varian Unity 300 (300.145 MHz) and Varian Inova 500 (499.876 MHz) spectrometers. EI mass spectra were recorded on a Finnigan MAT 95 spectrometer (70 eV). ESI MS was recorded on a Finnigan LCQ with quaternary pump Rheos 4000 (Flux Instrument). HRMS were recorded by ESI MS on an Apex IV 7 Tesla Fourier-Transform Ion Cyclotron Resonance Mass Spectrometer . Optical rotation was measured on a Perkin-Elmer Polarimeter, model 343. Flash chromatography was carried out on silica gel (230-400 mesh). D. hemprichi was collected from the Red Sea; approx. 30 km offshore from Safaga (east Egypt) at a depth of approx. 30 m. Pieces of the coral were rinsed three times with sterile seawater and then aseptically cut into smaller pieces and shaken for 2 h. The aqueous supernatant was serially diluted, and each 200 μL were inoculated onto 15-cm Petri dishes, each containing 50 mL of yeast extract/starch agar  +, 36), 288 ; (-)-ESI MS m/z 286 [M+H]-; (+)-HRESI MS m/z 288.12301 ; 310.10490 .Colourless semisolid, UV-absorbing, no colour reaction on spraying with anisaldehyde/sulphuric acid; Rf = 0.78 (CH2Cl2/5% MeOH); 1H NMR  and 13C NMR  see Table EI MS m/z (%) = 286.2 , 268.1 , 257.2 , 240.2 (8) 213.2 (5), 43.1 (7); (+)-ESI MS m/z (%) = 594.9 , 287 ; (+)-HRESI MS m/z 287.09137 .Yellow solid, UV-green fluorescence (365 nm), orange with anisaldehyde/sulphuric acid; Rf = 0.38 (CH2Cl2/5% MeOH); 1H NMR  and 13C NMR  see Table EI MS m/z (%) = 290.2 , 272.2 , 243.2 (8), 232.1 (21), 189.1 (7), 175.1 (16), 101.1 (15), 85.1 (22), 59.1 (36), 43.1 (100); (+)-ESI MS m/z (%) = 291 ([M+H]+); (-)-ESI MS m/z (%) = 289 ([M-H]-); (+)-HRESI MS m/z 291.08631 .Yellow solid, UV-green fluorescence (365 nm), turned dark red with anisaldehyde/sulphuric acid; Rf = 0.44 (CH2Cl2/5% MeOH); 1H NMR  δ = 14.09 , 7.00 , 6.68 , 6.55 , 6.38 , 3.84 , 3.14 , 2.72 , 1.61 ; EI MS m/z (%) = 304.3 , 286.3 , 262.3 (8), 247.2 (28), 246.2 (60), 220.2 (20), 218.2 (10), 149.2 (20), 145.2 (34), 127.2 (12), 116.2 (64), 101.2 (48), 84.1 (36), 66.1 (24), 59.1 (63), 43.1 (100).Yellow solid, UV-green fluorescence (365 nm), turned dark red with anisaldehyde/sulphuric acid; Rf = 0.82 (CH2Cl2/5% MeOH); [α]D20 = -18.9 ; CD  [θ]400 0 [θ]284 +22843, [θ]270 -36396, [θ]219 +5629; 1H NMR  δ = 7.35 , 7.25 , 6.51 , 6.23 , 6.15 , 6.08 , 3.95 , 3.79 , 3.59 , 3.46 , 2.42 , 2.13 ; EI MS m/z (%) = 570.5 , 539.5 , 513.4 (5), 286 (7), 167.2 (7), 145.2 (44), 116.2 (100), 85.1 (39), 55.1 (22), 43.1 (24).Yellow solid, UV-green fluorescence (365 nm), turned orange with anisaldehyde/sulphuric acid; Rf = 0.48 (CH2Cl2/5% MeOH); [α]D20 = -18.3 ; CD  [θ]400 0, [θ]284 +143232, [θ]267 -151670, [θ]227 +46610; 1H NMR  δ = 14.51 , 14.08 , 6.84 , 6.72 , 6.37 , 6.14 , 3.99 , 3.78 , 3.63 , 3.39 , 3.02, , 2.89 , 1.79 , 1.46 ; EI MS m/z (%) = 570.3 , 539.4 ([M-(2H2O+OCH3)]+, 74), 524.3 (5), 299.2 (12), 272.2 (13), 269.7 (24), 230.2 (18), 193.1 (12), 154.2 (14), 149.1 (19), 130.1 (48), 91.1 (54), 57.1 (30), 43.1 (57); (+)-HRESI MS m/z 607.18100 , m/z 629.16294 .Yellow solid, UV-green fluorescence (365 nm), turned orange with anisaldehyde/sulphuric acid; Rf = 0.26 (CH2Cl2/5% MeOH); [α]D20 = -33.5 ; CD  [θ]400 0, [θ]285 +359273, [θ]268 -339938, [θ]226 +107899.87; 1H NMR : δ = 6.84 , 6.57 , 6.38 , 6.20 , 3.93 , 3.60 , 3.50 , 3.30-3.29 , 1.69 , 1.49 ; EI MS m/z (%) = 574.3 , 556.3 , 525.3 ([M-(2H2O+OCH3)]+, 32), 264.2 (7) 58.2 (28), 43.1 (100); (+)-HRESI MS m/z 615.14779 , m/z 593.16570 .Yellow solid, UV-green fluorescence (365 nm), turned orange with anisaldehyde/sulphuric acid; Rf = 0.55 (CH2Cl2/5% MeOH); 1H NMR  δ = 6.87 , 6.55 , 6.51 , 6.36 , 6.08 , 3.95 , 3.63 , 3.43 , 3.35-3.25, , 2.16 , 1.65 ; EI MS m/z (%) = 556.5 , 286.3 ([rubrofusarin B (2)]+, 8), 84.1 (12), 57.2 (10), 44.1 (100); (+)-ESI MS m/z (%) = 1172 , 575 ; (-)-ESI MS m/z (%) = 1721 , 1147 , 573 .Yellow solid, UV-green fluorescence (365 nm), turned orange with anisaldehyde/sulphuric acid; 1-8 were dissolved in CH2Cl2/10% MeOH at a concentration of 1 mg/mL. Aliquots of 40 μL were soaked on filter paper discs  and dried for 1 h at room temperature under sterilized conditions. The paper discs were placed on inoculated agar plats and incubated for 24 h at 38°C for bacterial and 48 h (30°C) for the fungal isolates, while the algal test strains were incubated at room temperature in day light.Compounds P.aeruginosa, S. aureus, C. albicans and A. niger were used. Both bacterial and yeast strains were grown on nutrient agar medium (g/L): Beef extract 3; peptone, 10; and agar, 20. The pH was adjusted to 7.2. The fungal strain was grown on Czapek-Dox medium (g/l): Sucrose, 30; NaNO3, 3; MgSO4.7H2O, 0.5l; KCl, 0.5; FeSO4, 0.01; K2HPO4, 1; and agar, 20. The pH was maintained at 6.0. The disc diffusion test has been done according to Collins and Lyne [g from the culture extract, and located on the surface of the agar plates (150 mm diameter containing 50 mL of solidified media). The paper discs were placed on inoculated agar plats and incubated for 24 h at 38°C (bacteria and yeast) and 48 h at 30°C (fungi).For the fungal extract examination, representative test microbes; and Lyne . Filter The cytotoxic assay was performed according to Takahashi et al.  and SajiA. alternata strain D2006, isolated from the marine soft coral D. hemprichi. Selection of the strain was based on its profound antibiotic and antimicrobial activities. Structures of the isolated compounds were identified on the basis of 1D and 2D NMR spectroscopy and mass  data, and by comparison with the literature. Configuration of the four dimeric naphtha-γ-pyrones 6-9 was analyzed by CD spectra, exhibiting an identical stereochemistry. The biological activity  of the fungal extract and its corresponding isolated compounds were comparatively studied. This is as a trial to find out new leading drugs to overcome some of the recently discovered diseases.In this research article, eight bioactive pyrone derivatives were identified from the culture of The authors declare that they have no competing interests.Spectral data of Pyrophen (1). Ten charts (chart 1-10) containing the mass  and NMR  spectral data of Pyrophen (1).Click here for fileSpectral data of Rubrofusarin B (2). Thirteen charts (chart 11-23) containing the mass  and NMR  spectral data of Rubrofusarin B (2).Click here for fileSpectral data of Fonsecin (3). Nine charts (chart 24-32) containing the mass  and NMR  spectral data of Fonsecin (3).Click here for fileSpectral data of Fonsecin B (5). Two charts (chart 33-34) containing the mass (EI MS) and NMR (1HNMR) spectral data of Fonsecin B (5).Click here for fileSpectral data of Aurasperone A (6). Two charts (chart 35-36) containing the mass (EI MS) and NMR (1HNMR) spectral data of Aurasperone A (6)Click here for fileSpectral data of Aurasperone B (7). Three charts (chart 37-39) containing the mass  and NMR (1HNMR) spectral data of Aurasperone B (7)Click here for fileSpectral data of Aurasperone C (8). Four charts (chart 40-43) containing the mass  and NMR (1HNMR) spectral data of Aurasperone C (8)Click here for fileSpectral data of Aurasperone F (9). Three charts (chart 44-46) containing the mass  and NMR (1HNMR) spectral data of Aurasperone F (9)Click here for fileCD Spectra of Aurasperones A-C (6-8). Three charts (chart 47-49) containing the CD spectral data of Aurasperones A-C (6-8).Click here for file


Anaplasma phagocytophilum is an intracellular organism in the Order Rickettsiales that infects diverse animal species and is causing an emerging disease in humans, dogs and horses. Different strains have very different cell tropisms and virulence. For example, in the U.S., strains have been described that infect ruminants but not dogs or rodents. An intriguing question is how the strains of A. phagocytophilum differ and what different genome loci are involved in cell tropisms and/or virulence. Type IV secretion systems (T4SS) are responsible for translocation of substrates across the cell membrane by mechanisms that require contact with the recipient cell. They are especially important in organisms such as the Rickettsiales which require T4SS to aid colonization and survival within both mammalian and tick vector cells. We determined the structure of the T4SS in 7 strains from the U.S. and Europe and revised the sequence of the repetitive virB6 locus of the human HZ strain.vir genes, there is great diversity within these loci among strains. This is particularly evident in the virB2 and virB6 which are postulated to encode the secretion channel and proteins exposed on the bacterial surface. VirB6-4 has an unusual highly repetitive structure and can have a molecular weight greater than 500,000. For many of the virs, phylogenetic trees position A. phagocytophilum strains infecting ruminants in the U.S. and Europe distant from strains infecting humans and dogs in the U.S.Although in all strains the T4SS conforms to the previously described split loci for virB2 is in both the total number of copies, which varied from 8 to 15 in the herein characterized strains, and in the sequence of each copy. The diversity in virB6 is in the sequence of each of the 4 copies in the single locus and the presence of varying numbers of repetitive units in virB6-3 and virB6-4. These data suggest that the T4SS should be investigated further for a potential role in strain virulence of A. phagocytophilum.Our study reveals evidence of gene duplication and considerable diversity of T4SS components in strains infecting different animals. The diversity in  Anaplasma phagocytophilum is a tick-borne pathogen in the Order Rickettsiales that is increasingly recognized as a cause of disease in humans and animals world-wide . Plugs were made immediately on completion of the isolation procedure. Briefly, following the final centrifugation of the purified organisms, the pellet was resuspended in cell suspension buffer using 40–50 μl for every 108 host cells used at >95% infectivity. The sample was passed 2x through a 31 G needle  to ensure homogeneity of the thick suspension, and an equal volume of 1% low melting point SeaPlaque GTG agarose [(Lonza #50111) dissolved in DEPC-treated water  and held at 55°C] was immediately added. Following mixing, 100 μl aliquots were dispensed into plug molds  and allowed to set for 1 hr at 4°C prior to transfer into a 50 ml tube containing 5–10 ml, 50°C NDSK solution . The tube was incubated upright at 50°C with mild shaking (40 rpm) for 8–24 hrs, until the plugs turned clear and colorless. Plugs were gently washed 3x in 5 ml 0.5 M EDTA-Na2 (pH 9.5), then transferred to a fresh tube and stored in EDTA at 4°C. Optical Mapping data generated from the BamHI-digested ApDog1 chromosome was analyzed using the OpGen MapSolver software.ApHZ, ApDog1 and ApMRK. Genome coverage range was 31.3x to 72.1x. For each strain, the SFF format flow files, returned by ICBR for bioinformatics analysis, were first combined and converted to .fasta and .qual files (or the two combined in .fastq format) using Roche/454 Genome Sequencer FLX System software. Genome drafts were assembled using the CLC Genomics Workbench software suite (version 4.0-4.9) by mapping reads initially against the fully annotated, Sanger sequenced ApHZ genome (GenBank CP000235), then against the completed ApDog1 genome. Default parameters were used: length fraction, 0.5; similarity, 0.8; and for paired end reads, minimum distance, 1500/maximum distance, 4500. To obtain the vir loci, the resulting consensus sequence and underlying aligned reads were inspected for conflicts and mismatched paired ends suggesting the presence of insertions and/or deletions not mirrored in the consensus. These were manually corrected. Gaps were also manually closed where possible. Briefly, overlapping reads covering at least 2 kb of sequence on both sides of a gap and extending into it were individually extracted from the alignment. A new consensus for each side was obtained by assembling the reads against each other, and 250 N’s were added to its ends. These were individually used as the reference sequence against which all the 454 reads were re-mapped to pull out novel reads extending into the unknown region. The process was repeated multiple times, allowing for the incremental filling of the gap. PCR, followed by sequencing was performed when sequences extrapolated in this fashion spanned complex tandem repeat regions such as repeat regions 1 and 3  core facilities, University of Florida for library construction and pyrosequencing on the Roche/454 Genome Sequencer according to standard manufacturer protocols. Regular read libraries were generated for all strains. Additionally, 3 kb paired end libraries were made for http://www.phylosoft.org/archaeopteryx). Hydrophobicity analyses were conducted using the method of Hopp and Woods [http://www.ch.embnet.org/software/TMPRED_form.html.Amino acid sequences were aligned with MAFFT  and dispnd Woods ,54 at wevirB6-4 gene repeat regions were performed in the presence of 1.5-1.7 M Betaine (Sigma). The 8.36 kb PCR product spanning R3 and R4 in the ApHZ strain , which is more permissive to repeat-laden foreign DNA. Recombinants containing the correct size insert were end sequenced to verify their identity.Due to difficulties in amplifying tandem repeat-containing DNA, all PCR reactions spanning the ApHZ, equimolar amounts of the TA clone were cut with either HindIII or EcoRV. Following pooling and EtOH precipitation, the linearized DNA mix was submitted to ICBR/UF for SMRTbell library construction and sequencing. Libraries were constructed using a commercial strobe library preparation kit  following standard manufacturer protocols. To further increase the likelihood of full coverage, the strobe-sequencing run was performed using two different conditions: I) 45 min light period (continuous collection time); and II) , followed by . The ApJM and ApDog1 constructs were double-digested with HindIII/XbaI to excise the ~10.3 kb inserts. Following separation on 0.5% agarose gels, the inserts were recovered from agarose slices by electroelution and further purified and concentrated by passage over QIAquick spin columns following the PCR Purification kit protocol (Qiagen). SMRTbell libraries were made as above then sequenced using a single 75 min movie time run.In preparation for sequencing with the long-read length Pacific Biosciences (PacBio) next-generation sequencing RS instrument, constructs were linearized with restriction enzymes which cut the vector only, but on opposite sides of the insert within the Multiple Cloning Site. For de novo failed to yield a sequence of the expected size. Therefore, for each construct, reads >3 kb were selected from the multi-fasta files using the Galaxy suite [virB64 R4 repeat signature-sequences  or not determined for the other strains except ApHz. We provide a revised sequence of virB64 for the previously sequenced [ApHZ strain.The sequences of equenced ApHZ straApDog1:JX415845 - JX415868B2-1 B2-2, B2-3, B2-4, B2-5, B2-6, B2-7, B2-8, B2-9, B3, B4-1, B4-t1, B4-2, B6-1, B6-2, B6-3, B6-4, B8-1, B8-2, B9-1, B9-2, B10, B11, D4ApJM:JX415869 - JX415892B2-1, B2-2, B2-3, B2-4, B2-5, B2-6, B2-7, B2-8, B2-9, B3, B4-1, B4-t1, B4-2, B6-1, B6-2, B6-3, B6-4, B8-1, B8-2, B9-1, B9-2, B10, B11, D4ApDog2:JX415893 - JX415915 (virB6-4 submitted separately as gapped)B2-1, B2-2, B2-3, B2-4, B2-5, B2-6, B2-7, B2-8, B2-9, B3, B4-1, B4-t1, B4-2, B6-1, B6-2, B6-3, B8-1, B8-2, B9-1, B9-2, B10, B11, D4ApNorLambV2:JX415916 - JX415938B2-1, B2-2, B2-3, B2-4, B2-5, B2-6, B2-7, B2-8, B2-9, B3, B4-1, B4-t1, B4-2, B6-1, B6-2, B6-3, B8-1, B8-2, B9-1, B9-2, B10, B11, D4ApNorLambV1:JX415939 - JX415966B2-1, B2-2, B2-3, B2-4, B2-5, B2-6, B2-7, B2-8, B2-novel1, B2- novel2, B2-novel3, B2-novel4, B2-novel5, B2-novel6, B3, B4-1, B4- t1, B4-2, B6-1, B6-2, B6-3, B8-1, B8-2, B9-1, B9-2, B10, B11, D4ApHZvirB6-4:JX415967ApVar1:JX415968 - JX415996B2-1, B2-2, B2-3, B2-4, B2-5, B2-6, B2-7, B2-8, B2-novel1, B2- novel2, B2-novel3, B2-novel4, B2-novel5, B2-novel6, B2-novel7, B3, B4-1, B4-t1, B4-2, B6-1, B6-2, B6-3, B8-1, B8-2, B9-1, B9-2, B10, B11, D4ApMRK:JX415997 - JX416019B2-1, B2-2, B2-3, B2-4, B2-5, B2-6, B2-7, B2-8, B2-9, B3, B4-1, B4-t1, B4-2, B6-1, B6-2, B6-3, B8-1, B8-2, B9-1, B9-2, B10, B11, D4virB6-4Gapped:JX416020.ApDog2The authors declare that they have no competing interests.in vitro the JM, MRK, Dog2 and Ap variant 1 strains, and prepared Dog2 and Ap variant 1 strain gDNA. ARA and SMM established the Dog1 strain. All authors read and approved the final manuscript.BAK and AFB conceived the study, performed bioinformatics analyses and drafted the manuscript. BAK grew infected HL-60 cell cultures, purified organisms, isolated gDNA, designed and supervised PCR and submitted sequences to GenBank. AML performed PCR analyses and cloning and supervised data transfer between units. SS and EGG isolated the European sheep strains, infected and monitored sheep, and prepared organisms at maximal parasitemia. UGM and CMN isolated and cultured Figure S1. Multiple sequence alignment of VirB6-3 amino acid sequences from different strains of A. phagocytophilum. Arrows indicate the locations of C-terminal 41-mer repeats.Click here for file Figure S2. Structure of the virB6-4 repeat regions R3 and R4 in four US A.phagocytophilum strains.A. Comparative maps of AB1393/AB1466 PCR products detailing the repeat unit content of R3 and R4 in the human, rodent and dog strains. ApJM and ApDog1 have identical virB6-4 genes and are, therefore, represented by one map. Moderate variability in the number and sequence of the R3 405 bp repeat units (light blue arrows) is apparent. The small bar at the end of R3 corresponds to the 3′-most partial repeat unit present in all strains. The colored arrows within R4 represent the five repeat types T1a (yellow), T1b (green), T2a, (red), T2b (dark blue) and T2c (grey). The repeat pattern in ApHZ shows no relationship to that of ApJM/ApDog1, which is also 2.87 kb larger, totaling 9.76 kb. This region was not fully characterized in ApDog2 as indicated by a broken line, but the repeat pattern of the 5′- and 3′-most repeats is clearly different from that of the other strains. The small bar downstream of the second repeat unit represents a partially characterized type 2 repeat unit. Lines above and below the ApHZ and ApJM/ApDog1 maps delineate segments of sequence identity within the respective R4 regions. Their sizes are specified. B. Alignment of the nucleic acid sequence of all virB6-4 R4 repeat unit types identified to date. Type 1 repeats are shown in black, type 2 in blue. Differences between sub-types are highlighted. A single BamHI site present in all type 2 repeats is underlined. With the exception of only a few nucleotides at each end, type 1 and type 2 repeat units do not share any sequences. C. Alignment of the amino acid sequences of the repeat units shown in B. The single nucleotide differences between sub-types do not lead to changes in amino acid sequence.Click here for file


Rhodococcus ruber strain BKS 20-38, isolated from the palm tree rhizosphere soil of Bhitarkanika National Park, Odhisha, India. The draft genome sequence of strain BKS 20-38 consists of 6,126,900 bp, with a G+C content of 69.72%, 5,716 protein-coding genes, and 49 RNAs.We report the 6.1-Mb genome sequence of  Rhodococcus was first proposed by Zopf (1891) and emended by Tsukamura (1974) (Rhodococcus ruber was first proposed by Kruse (1896) and later on emended by Goodfellow and Alderson (1977)  of 101 bp. We used NGS QC Toolkit v2.3  comprising 6,126,900 nucleotides (nt) was annotated with the help of the RAST (Rapid Annotation using Subsystem Technology) system (The genus name  by Zopf 891 and ea 1974) . Rhodocon (1977) . We have . RhodocKruse 189 and lateRhodococcus jostii strain RHA1 , Rhodococcus erythropolis strain PR4 , and Rhodococcus opacus strain B4  are the closest neighbors of the strain BKS 20-38. Genome annotation available at RAST indicates that strain BKS 20-38 contains genes for glycolysis and gluconeogenesis, the tricarboxylic acid (TCA) cycle, and the pentose phosphate pathway. In the RAST annotation, we also found genes for fatty acid metabolic clusters, 3-ketoacyl coenzyme A (3-ketoacyl-CoA) thiolase (EC 2.3.1.16), 3-oxoacyl-[acyl carrier protein] reductase (EC 1.1.1.100), and acyl dehydratase. Also present in the annotation were genes for branched-chain amino acid biosynthesis, 3-isopropylmalate dehydratase small and large subunits (EC 4.2.1.33), 3-isopropylmalate dehydrogenase (EC 1.1.1.85), acetolactate synthase small and large subunits (EC 2.2.1.6), branched-chain amino acid aminotransferase (EC 2.6.1.42), dihydroxy-acid dehydratase (EC 4.2.1.9), ketol-acid reductoisomerase (EC 1.1.1.86), leucine dehydrogenase (EC 1.4.1.9), and threonine dehydratase (EC 4.3.1.19). Genes involved in isoleucine degradation, i.e., those for 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35), 3-ketoacyl-CoA thiolase (EC 2.3.1.16), branched-chain acyl-CoA dehydrogenase (EC 1.3.99.12), branched-chain alpha-keto acid dehydrogenase, E1 component, alpha and beta subunits (EC 1.2.4.4), branched-chain amino acid aminotransferase (EC 2.6.1.42), butyryl-CoA dehydrogenase (EC 1.3.99.2), and enoyl-CoA hydratase (EC 4.2.1.17), were also found in the annotation. Valine degradation genes, i.e., those encoding 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31), 3-hydroxyisobutyryl-CoA hydrolase (EC 3.1.2.4), methylmalonate-semialdehyde dehydrogenase (EC 1.2.1.27), acyl-CoA dehydrogenase, short-chain specific (EC 1.3.99.2), and butyryl-CoA dehydrogenase (EC 1.3.99.2), are also present in the genome of strain BKS 20-38.RAST annotation indicates that AOEX00000000. The version described in this paper is the first version, number AOEX01000000. Genome assembly and annotation data can be downloaded from our genomics web portal at http://crdd.osdd.net/raghava/genomesrs/.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number


All the synthesis compounds have been characterized by 1HNMR, IR and Mass spectral data. The compounds were purified by column chromatography. All synthesized derivatives were determined by the carrageenan-induced rat-paw-oedema model for anti-inflammatory activity. The entire compound gives good response for the anti-inflammatory activity:  oxadiazole-2yl] benzamide (C4), and  oxadiazole-2yl] benzamide (C7). For this activity, indometacin was used as a standard drug and compared to new synthesized drugs. Some new synthesized drugs have shown better activities for the anti-inflammation. A series of five-membered heterocyclic rings were synthesized by the reaction between benzoyl chloride and various chlolro-nitro-benzoyl chlorides and semi carbazide to form (C The number of so many synthetic compounds with oxadiazole nucleus used for antibacterial , antifun254 (4:1) for preparation of TLC plates and also used the solvent system 5% ethyl acetate in pet. Ether and spots were seen under iodine vapours and UV light chamber. IR spectra were obtained on a Perkin-Elmer 1720 FT-IR-Spectrometer (KBr-soln/pellets). 1HNMR spectra were noted in Brucker Ac-400 MHz spectrometer using TMS as internal standard in DMSO/CDCl3 and mass spectra (m/z %) recorded by VG ZAB-HS (FAB) instrument.All chemicals were supplied by . Melting point (m.p) was determined by open capillary tube method. Purification of compounds was checked by column chromatography and silica gel-G (60-120 mess) and silica gel GFMaterial and methods1 to C7 ):- (1:1 molar ratio) Aromatic, phosphorus pentachloride and benzene were taken in RBF, fitted with air condenser and calcium chloride guard tube. The mixture was heated gently to melt with vigorous shaking at around 50°C. After 30 min excess POCl3 was distilled out. The residue was dried well and used for the next reaction. Then, semicarbazide was added to the respected acid chloride and reflux for 5 h. These programs of the reaction were monitored by checking the TLC. The excess benzene was distilled out, neutralizing with aq. NaHCO3 and the compound was extracted with chloroform. The crude was obtained through distillation of chloroform under reduce pressureGeneral procedure for synthesis of compounds  were divided into 3 groups as control, test and standard . Overnight fasted animals were used and during that period only tap water was given. Generally, indomethacin was used as standard drug. Both test and standard drugs were suspended in 1% carboxymethyl cellulose (CMC) and administered orally through gastric gavage needle. One percent of CMC was administered in control group. After 1 h of administrating the compound, we induced the carrageenan (1%) by the sub planner surface of the right hind paws of animals. The initial paw volume and also the paw volume after 3 and 6 h of administrating carrageenan were measured. Percent paw oedema inhibition was calculated.1H NMR and MASS spectra. Then, the pharmacological activity was done. The entire compound had a good response for Anti-inflammatory activity :  oxadiazole-2yl]benzamide (C4),and  oxadiazole-2yl]benzamide (C7). Substitution of 2-chloro-benzoic acid, at 2,5- position anti-inflammatory activity greater (C2) than 3-chloro substituted compound (C3)and 4-cloro-benzoic acid compound (C4) substituted at 2,5 position anti-inflammatory activity greater than 2-chloro-substituted compound (C2). While substitution of 4-nitro-compounds (C7) at 2,5-position greater than other 2-nitro and 3-nitro substituted compounds (C5 and C6).At the end of the experiment, it has been concluded that the compounds synthesized in the project have good yield value. The synthesized oxadiazole compounds were identified and characterized by IR, Compound 1: [N--benzamide]1HNMR, 8.72  , 7.72 , 7.82 , 7.62 :m/z (%), 266 (23), 262 (14), 260 (100), 249 (17), 248 (100). analytical calculated for C15H11N3O2 c = 67.90, H = 4.23, N = 15.86, O = 12.12, found = C = 67.92, H = 4.15, N = 15.84, O = 12.07.IR : 3214( NH) ,1664(C=O) , 1070( N-N) ,1232(C-O-C) ; Compound 2: oxadiazol-2-yl]benzamide]15H9N3O2Cl2; C = 49.60, H = 2.96, N = 23.48. found C = 49.62, H = 2’86, N = 23.44.IR: 3270(NH),1670( C=O) ,1072( N-N)1240( C-O-C),776( C-Cl) ;1HNMR  ,7.96  ,7.78  ,7.72 , 7-7.8 ; MASS (CSM), m/z (%), analytical calculated for CCompound 3:  oxadiazol-2-yl]-benzamide] IR  P: 3272 ( NH) , 1668 (C=O), 1076 (N-N), 1242 (C-O-C), 778 (C-Cl); 1HNMR , 7.76 , 7.68 , 7.73 ,7.78 ; MASS (C-SI), m/z (%) analytical calculated for C15H9N3O2Cl2, C = 48.98, H = 2.83, N = 23.48, found, C = 47.96, H = 2.81, H = 23.32. Compound 4:  oxadiazole-2yl]benzamide IR : 3268 (NH), 1668 (C=O), 1072 (N-N), 1242 (C-O-C), 725 (C-Cl); 1HNMR  ,7.72-7.75 , 7.78 . MASS (CSM); M\Z%-Anal calculator for C15H9N3O2Cl2, C = 48.41, H = 2.79, N = 23.25, Found C = 47.98, H = 2.76; N = 23.16. Compound 5:  oxadiazole-2yl] benzamide IR (KBr cm-1) :- 3272 (NH), 1670 (C=O), 1078 (N-N), 1260 (C-O-C), 780 (C-Cl) H1NMR (DMS+ds400M H2):- 7.70 , 7.78 , 7.78 , 7.25  Mass (CSM) (M/Z (%)-Anal calculator for C14H9N5O6, C = 48.97, H = 2-93, N = 30.48, formed C = 48.27, N = 2.90, N = 30.42. Compound 6:  oxadiazole-2yl]benzamide IR (KBr Cm-1):- 3271(NH), 1668(C=O), 1070(N-H), 1265(C-O-C), 786(C-Cl) H1NMR (DMS+ds400M H2):- 7.71, 7.77, 7.73 , 7.22 , Mass,(CSM) M/Z (%)-Anal calculator for C14H9N5O6 C = 48.92, H = 2.44, N = 30.47, found C = 48.46, H = 2.90, N = 30.45.Compound 7:  oxadiazole-2yl] benzamideIR(KBr Cm-1) :- 3272(NH), 1665 (C=O), 1078 (N-N), 1260 (C-O-C), 783(C-Cl) H1NMR (DMS+ds400M H2):- 7.70, 7.2 , 7.74 , 7.26 , Mass, (CSM) M/Z(%)-Anal calculator for C14H9N5O6 C = 48.98, H = 292, H = 30.46, formed C = 48.92, H = 2.48, N = 30.36.