Sargassum paradoxum, resulted in the isolation and identification of four new  and nine previously reported  bioactive meroditerpenoids. HPLC-NMR and HPLC-MS were central to the identification of a new unstable compound, sargahydroquinal (9), and pivotal in the deconvolution of eight  other meroditerpenoids. In particular, the complete characterization and identification of the two main constituents (1 and 2) in the crude dichloromethane extract was achieved using stop-flow HPLC-NMR and HPLC-MS. This study resulted in the first acquisition of gHMBCAD NMR spectra in the stop-flow HPLC-NMR mode for a system solely equipped with a 60 μL HPLC-NMR flow cell without the use of a cold probe, microcoil, or any pre-concentration.A phytochemical investigation of a southern Australian marine brown alga,  Sargassum genus  number in excess of 300 species −) suggested the previously described meroditerpenoid sargahydroquinoic acid (2)  , did not contain a secondary alcohol functional group; this led to peak D being identified as the previously reported compound 2--6-methyl-1,4-benzenediol (6)  , both ofe moiety . On the diol (6) . This reak C see . The str7) [m/z 425.2694 [M − H]−.Peak E was determined to be the previously described meroditerpenoid fallaquinone  [7], basmax 290 nm), this compound also possessed a hydroquinone moiety [m/z of 409.2745 [M − H]− obtained via HPLC-MS suggested a molecular formula of C27H38O3. The stop-flow WET1D proton NMR spectrum showed a distinct proton signal at δH 10.07, s, confirming the presence of an aldehyde moiety. The MarinLit database indicated that sargaquinal (8), which possesses a p-benzoquinone moiety and an aldehyde functional group, had been previously reported from the Sargassum genus -2,3-dimethyl-1,4-benzoquinone, attributed the trivial name paradoxquinol (10).The UV profile (λe moiety . The HPL2) and was identified as the previously reported sargahydroquinoic acid (11) −) obtained via HPLC-MS, in consultation with a SciFinder search, peak J was identified as previously described meroditerpenoid 2--6-methyl-1,4-benzoquinone (12) -6-methyl-1,4-benzenediol (6) in a ratio of approximately 3:5. While complete 2D NMR characterization was conducted on the mixture, these components were obtained as separate entities via stop-flow HPLC-NMR. By analysis of the data obtained from both chemical profiling and off-line isolation, the compounds could be identified. In the first instance, the previously reported compound 2--6-methyl-1,4-benzenediol (6) could be confirmed -6-methyl-1,4-benzenediol, and attributed the trivial name paradoxhydroquinone (5). Unfortunately, 5 and 6 oxidized to 15 and 12 before any NOESY NMR experiments could be carried out. Therefore a bsNOESY NMR experiment was carried out on 15 and 12, respectively, and the results related back to 5 and 6. On biosynthetic grounds, the configuration of the double bonds was therefore confirmed as 2′E, 6′E, and 10′E for 5, and 2′E, 6′E, and 10′Z for 6.Paradoxhydroquinone (onfirmed . The com (C-20′) . The oxy10), unlike many of the previously isolated meroditerpenoids, which show the presence of an AB aromatic system, only displayed a single aromatic proton NMR signal at δ 6.46, s (H-3), suggesting further substitution of the p-benzoquinone ring. The gHMBCAD NMR spectrum confirmed the structure of 10 as proposed by chemical profiling. The two deshielded methyl groups [δH 2.03  and 2.00 ] showed correlations to carbons at δC 140.6 (C-5), 141.5 (C-6), and 187.7 (C-1), and δ 140.6 (C-5), 141.5 (C-6), and 187.9 (C-4), respectively (10 as the new meroditerpenoid 5--2,3-dimethyl-1,4-benzoquinone, attributed the trivial name paradoxquinol (10). A single irradiation nOe NMR experiment confirmed the configuration of the double bond at position C-10′ as 10′E (irradiation of H-10′ showed a key enhancement to H-12′). Although this represents the first occurrence in a marine alga of a meroditerpenoid containing the position 8 methyl, this biosynthetic feature has been reported in terrestrial natural products -6-methyl-1,4-benzoquinone (12) in a ratio of approximately 3:5. While complete NMR characterization was conducted on the mixture, the latter of these was identified as a separate entity via stop-flow HPLC-NMR. By analysis of the data obtained from both chemical profiling and off-line isolation, the latter of these compounds could be identified as the known meroditerpenoid 2--6-methyl-1,4-benzoquinone (12). This compound had previously been reported as a derivative of a natural product; however, this represents the first report of its natural occurrence  pulsed field gradient flow probe with a 60-μL active volume flow cell coupled to a Varian Prostar 210 solvent delivery system, a Prostar 430 Autosampler, and a Prostar 335 PDA detector. The HPLC-NMR analyses were carried out using CORBA communication and operated with VnmrJ software. The 2H resonance observed from the D2O was used to obtain a field-frequency lock. The resonances from the HOD and the methyl of the acetonitrile were suppressed using the water enhanced through transverse gradients (WET) solvent suppression experiment -6-methyl-1,4-benzoquinone (12) as an inseparable mixture , and sargaquinal (8) . The 40% dichloromethane/ethyl acetate to 60% dichloromethane/ethyl acetate silica gel column fractions were combined and subjected to Sephadex LH-20 size exclusion column chromatography using 100% methanol. The third fraction resulting from this analysis was subjected to reversed phase HPLC (85% CH3CN/H2O) to yield fallaquinone (7)  and paradoxquinol (10) . The remaining half of the dichloromethane extract was purified separately using reversed-phase HPLC (90% CH3CN/H2O) to yield fallahydroquinone (1) , sargahydroquinoic acid (2) , paradoxhydroquinone (5) and 2--6-methyl-1,4-benzenediol (6) as an inseparable mixture , sargaquinoic acid (11) , and 9--6-methyl-2-(4-methyl-3-penten-1-yl)-2,6-nonadienal (14) . The percentage yields are reported on the basis of the dry mass of the alga extracted (refer to the supporting information for details of the bioassay-guided isolation scheme adopted).The frozen marine alga  was extracted with 3:1 methanol/dichloromethane (2 L). The crude extract was then decanted and concentrated under reduced pressure and sequentially solvent partitioned (triturated) into dichloromethane- and methanol-soluble extracts, respectively. Approximately 450 mg of the dichloromethane extract was subjected to flash silica gel column chromatography (20% stepwise elution from petroleum spirits (60–80 °C) to dichloromethane to ethyl acetate and finally to methanol). The 40% petroleum spirits/dichloromethane silica gel column fraction was subjected to reversed phase HPLC (100% CHS-Hydroxy-11-(hydroxymethyl)-3,7,15-trimethyl-2,6,10,14-hexadecatetraen-1-yl]-6-methyl-1,4-benzenediol  (1): HPLC-NMR WET1D NMR  obtained from stop-flow mode .2--6-methyl-1,4-benzenediol (paradoxhydroquinone) (6): HPLC-NMR WET1D NMR  obtained from stop-flow mode δ 7.25 , 7.22 , 6.10 , 6.06 , 5.95 , 4.84 , 4.05 , 2.95–3.04 (m), 2.97 , 2.95 , 2.93 , 2.52 , 2.48 , 2.41  ND  *signals interchangeable; HRESILCMS m/z 411.2899 .2--6-methyl-1,4-benzenediol (S-Hydroxy-11-(hydroxymethyl)-3,7,15-trimethyl-2,6,10,14-hexadecatetraen-1-yl]-6-methyl-1,4-benzoquinone  (7): HPLC-NMR WET1D NMR  obtained from stop-flow mode δ 7.42 , 7.28 , 6.30 , 6.00 , 5.95 , 5.91 , 4.95 , 4.89 , 4.87 , 3.93 , 2.95–3.10 (m), 3.08 , 3.03 , 2.96 , 2.50 , 2.46 , 2.42  ND ; HRESILCMS m/z 425.2694 .5--2,3-dimethyl-1,4-benzoquinone (paradoxquinol) (10): HPLC-NMR WET1D NMR  obtained from stop-flow mode δ 7.27 , 6.29 , 5.99 , 5.92 , 4.95 , 4.88 , 4.87 , 3.92 , 2.95-3.10 (m), 2.49 , 2.45 , 2.41  ND ; HRESILCMS m/z 439.2850 .5--6-methyl-1,4-benzoquinone -2--6-methyl-1,4-benzenediol  (1): unstable yellow oil;c 0.035, CHCl3); CD (EtOH) λmax (Δε) 202 (+6.1), 221 (+1.6), 224 (1.8), 243 (−0.2), 260 (+2.0), 275 (+0.5), 290 (+0.6); UV (EtOH) λmax (log ε) 252 (3.60), 288 (3.58); IR νmax 3391, 2925, 2855, 1652, 1607, 1470, 1381, 1197, 1037 cm−1. All off-line NMR spectroscopic and mass spectrometric data were identical to those reported in the literature; however, some carbon and proton NMR chemical shift reassignments have been made -6-methyl-1,4-benzenediol (paradoxhydroquinone) (5): dark yellow oil; 1H and 13C NMR  .(2′Cl3) see ; ESIMS mE,6′E,10′Z)-2--6-methyl-1,4-benzenediol (6): dark yellow oil. All off-line NMR spectroscopic and mass spectrometric data were identical to those reported in the literature -6-methyl-1,4-benzoquinone  (7): unstable yellow oil which darkened with time;c 0.065, CHCl3); CD (EtOH) λmax (Δε) 206 (+1.8), 220 (+1.0), 238 (+2.1), 247 (+2.3), 262 (+1.6), 270 (+1.9), 286 (+0.1), 294 (+0.3); UV (EtOH) λmax (log ε) 254 (4.10); IR νmax 3392, 2924, 2855, 1652, 1614, 1510, 1436, 1378, 1295, 1195, 1156 cm−1. All off-line NMR spectroscopic and mass spectrometric data were identical to those reported in the literature -2,3-dimethyl-1,4-benzoquinone (paradoxquinol) (10): unstable yellow oil which darkened with time; c 0.0125, CHCl3); CD (EtOH) λmax (Δε) 218 (+6.2), 220 (+6.4), 235 (−4.2), 245 (+2.4), 255 (+1.2); UV (EtOH) λmax (log ε) 232 (4.17), 254 (4.32); IR νmax 3401, 2924, 2853, 1652, 1511, 1456, 1249 cm−1; 1H and 13C NMR  .(2′Cl3) see ; ESIMS mE,6′E,10′Z)-6,10-Dimethyl-12--2-(4-methyl-3-pentenyl)-2,6,10-dodecatrienoic acid (sargaquinoic acid) (11): yellow oil which darkened with time. All off-line NMR spectroscopic and mass spectrometric data were identical to those reported in the literature -6-methyl-1,4-benzoquinone (12): dark yellow oil. All off-line NMR spectroscopic and mass spectrometric data were identical to those reported in the literature -6-methyl-1,4-benzoquinone (paradoxquinone) (15): yellow oil which darkened with time; 1H and 13C NMR  .(2′Cl3) see ; ESIMS mS. paradoxum successfully implemented the use of HPLC-NMR and HPLC-MS, in combination with the use of databases, to rapidly dereplicate, identify, and deduce the structures of nine meroditerpenoids . In particular, chemical profiling was an invaluable tool for the on-line identification of sargahydroquinal (9), which rapidly degraded when an off-line bioassay-guided isolation was employed, as well as for the meroditerpenoids 5, 6, and 12, which could be resolved and identified as individual components via stop-flow HPLC-NMR. This study reports, for the first time, the complete 1D and 2D NMR characterization and identification of two known (1 and 2) natural products via stop-flow HPLC-NMR and HPLC-MS. The bioassay-guided isolation approach confirmed the identity of the compounds responsible for the dichloromethane crude extract antimicrobial activity. Subsequent biological evaluation of the isolated meroditerpenoids indicated that there were no significant differences in antimicrobial activity for 11 of the meroditerpenoids isolated. Off-line bioassay-guided isolation also permitted the first complete NMR characterization of 2--6-methyl-1,4-benzenediol (6) and 2--6-methyl-1,4-benzoquinone (12) to be carried out. It is proposed that the presence of fallahydroquinone (1) and fallaquinone (7) could be used as potential chemotaxonomic markers for both S. paradoxum and S. fallax, which have only been reported from these two species of Sargassum.The study of the marine brown alga


Scutellaria platystegia (family Labiatae). Methanolic (MeOH) extract of aerial parts of S. platystegia and SPE fractions of methanolic extract , growing in East-Azarbaijan province of Iran were found to have radical scavenging activity by DPPH  assay. Dichloromethane (DCM) extract of this plant exhibited animalarial activity by cell free method providing IC50 at 1.1876 mg/mL. Crude extracts did not exhibit any toxicity assessed by brine shrimp lethality assay.This study aimed to determine biological activity and phytochemical study of Phytochemical study of methanolic extract by using reverse phase HPLC method and NMR instrument for isolation and identification of pure compounds respectively, yielded 2-(4- hydroxy phenyl) ethyl-O-β-D- glucopyranoside from 10% and apigenin 7-O-glucoside, verbascoside and martynoside from 40% SPE fraction. Occurance of verbascoside and martynoside as biochemical markers appeared to be widespread in this genus. in-vitro and in-vivo studies and clinical trials to develop new therapeutical alternatives.Antioxidant and antimalarial activity of MeOH and DCM extracts, respectively, as well as no general toxicity of them could provide a basis for further  Scutellaria which belongs to Labiatae family, spread throughout the world, with approximately 350 currently recognized species  were gathered from Yam region of East Azarbaijan province of Iran in June 2009. A voucher specimen (Tbz-Fph-724) for this collection has been deposited in the Herbarium of pharmacy faculty, Tabriz University of Medical Sciences, Tabriz, Iran. 100 g of the air dried and grounded sample was extracted by the aid of Soxhlet apparatus using hexane, dichloromethane (DCM) and methanol (MeOH) respectively (1 L for each solvent). Obtained extracts were individually concentrated under vacuum in a rotary evaporator , yielding 1.67 g, 1.50 g and 24.50 g respectively. Aerial parts of Fractionation of methanolic extractFractionation of dried methanolic extract (2 g) was carried out using SPE (solid phase extraction) cartridge (Sep- pak ODS C18), eluting with methanol and water mixture . All SPE fractions were dried by rotary evaporator at a temperature not exceeding 50 ºC. In order to increase the available SPE fractions, procedure was repeated 3 times yielding 1.421, 0.279, 0.655, 0.220, 0.162 and 0.190 g of each fraction respectively. DPPH assay50 that was extrapolated from dose-response curve. Tests were carried out in duplicate  (sigma). In order to obtain antioxidant activity, different sample solution series were prepared. 5 mL of each concentration of methanolic extract and SPE fractions were added to 5 mL of 0.004% methanolic solution of DPPH. After 30 min incubation of solutions at room temperature and bleaching of DPPH, absorption of samples was monitored at 517 nm against a blank. Inhibition of DPPH was calculated as RCuplicate . Antimalaria assayet al. .This test is proposed as a preliminary and simple assay to study general toxicity of plant extracts. The eggs of Isolation of compoundsi.d, particle size 10 µm, Dr. Maisch, Germany) column. The mobile phase consisted of (A) methanol and (B) water. The following mobile phase program was used over 60 min to isolate glycosylated phenylethanoid , equipped with a Reprosil 100 C18 : Aglycone moiety: δ7.04 , 6.68 , 3.8-4.1 , 2.7 , Glucose moiety: δ 4.29 , 3.2-3.8 ,13CNMR : Aglycone moiety: δ 153.88(C4), 130.42(C1), 130.19 , 115.28, 73.02(C8), 34.22(C7). 2-(4- hydroxy phenyl) ethyl-O-β-D- glucopyranoside ; pale gr1HNMR . Aglycone moiety: δ 7.90 , 6.93 , 6.85 , 6.63 , 6.46 . Glucose moiety: δ 4.65 , 3.2-3.8 .Apigenin 7-O-glucoside ; brown s1HNMR : aglycone moiety: δ 7.6 , 7.06, 6.98, 6.78, 6.69, 6.64, 6.58, 6.25 , 3.8-4.1 , 2.79 , glucose moiety: δ 4.37 , 3.2-3.8 , rhamnose moiety: δ 5.19 , 3.2-3.8 , 1.085 . 13CNMR  Aglycone moiety: δ 166.8(C9′), 148.4(C4′), 146.6(C7′), 145.4(C3′), ), 143.2(C3), 130(C1), 126.2(C1′), 121.8(C6′), 119.8(C6), 115.7(C2), 115.7(C5′), 114.86(C5), 114.7(C4), 113.7 (C2′), 113.2 (C8′), 72.3(C8), 35.1(C7), Glucose moiety: δ 101.6(C1″), 80.2(C3″), 74.8(C2″), 74.6(C5″), 69(C4″), 60.9(C6″), Rhamnose moiety: 102.77(C1′′′), 72.3(C4′′′), 70.9(C2′′′), 70.6(C3′′′), 69.1(C5′′′), 17(C6′′′). Data were in agreement with the published data : aglycone moiety: δ 7.63 , 7.16, 7.03, 6.79, 6.79, 6.69, 6.67, 6.40 , 3.8-4.1 , 3.87 , 3.75 , 2.8 , glucose moiety: δ 4.34 , 3.2-3.8 , Rhamnose moiety: δ 5.18 , 3.2-3.8 , 1.085 . 13C NMR ; Aglycone moiety: δ 166.85 (C9'), 150.81 (C4'), 149.41 (C3'), 148.03 (C7'), 147.98 (C3), 147.37(C4), 132.93 (C1), 127.71 (C1'), 124.46 (C6'), 122.94 (C6), 117.19 (C5), 116.66 (C5'), 115.19 (C8'), 112.95 (C2), 111.91 (C2'), 102.79 (C1'''), 101.58 (C1''), 81.65 (C3''), 76.24 (C2''), 76.04 (C5''), 73.87 (C4'''), 72.42 (C2'''), 72.35 (C8), 72.20 (C3'''), 70.74 (C4''), 70.31 (C5'''), 62.49 (C6''), 53.49 (O-CH3), 53.39 (O-CH3), 35.12 (C7), 17.03 (C6'''). Data were in agreement with the published data  yielded 2-(4- hydroxy phenyl) ethyl-O-β-D- glucopyranoside from 10% ,one flavonoid (Apigenin 7-O-glucoside) and 2 phenyl ethanoid glycosides (verbascoside and martynoside) from 40% SPE fractions. In fact compounds 1-4 have been identified previously from other species of this genus  ethyl-O-β-D- glucopyranoside (from 10% SPE fraction), Apigenin-7- O- glucoside, verbascoside and martynoside (from 40% SPE fraction) were isolated and identified by UV and NMR analysis. All spectroscopic data were in agreement with respective published data. Identified components have been reported from other species of despread . So, it in-vitro and in-vivo studies and clinical trials to develop new therapeutical alternatives. More over the results of present study show that it is worth to do further phytochemical studies on Iranian Scutellaria species and to isolate compounds responsible for antioxidant and antimalarial activities. It can be concluded that antioxidant and antimalarial activity of MeOH and DCM extracts, respectively, as well as no general toxicity of them, could provide a basis for further


Fonsecinone A (2) exhibited the most potent antibacterial activity, with minimum inhibitory concentrations (MICs) of 4.26, 17.04, and 4.26 μg/mL against ESBL-producing E. coli, P. aeruginosa, and E. faecalis, respectively. The inhibitory effects of fonsecinones A (2) and C (3) against E. coli and ESBL-producing E. coli were comparable to those of amikacin. Molecular docking-based target identification of naphtho-γ-pyrones 1–8 revealed bacterial enoyl-acyl carrier protein reductase (FabI) as an antibacterial target, which was further validated by FabI affinity and inhibition assays. Fonsecinones A (2) and C (3) and aurasperones A (6) and E (7) bound FabI specifically and produced concentration-dependent inhibition effects. This work is the first report of anti-drug-resistant bacterial activities of naphtho-γ-pyrones 1–8 and their possible antibacterial mechanism of action and provides an example of the successful application of in silico methods for drug target identification and validation and the identification of new lead antibiotic compounds against drug-resistant pathogens.Four naphtho- Staphylococcus aureus (MRSA), Enterococcus faecalis , extended-spectrum β-lactamase-producing Escherichia coli (ESBL-producing E. coli), Klebsiella pneumoniae, and Pseudomonas aeruginosa express a remarkable array of resistance and virulence factors, which contributes to their prominent roles in hospital-acquired infections34Aspergillomarasmine A, a fungal metabolite discovered 50 years ago, was recently repurposed based on its ability to overcome antibiotic resistance associated with metallo-β-lactamase89The emergence and spread of antibiotic-resistant bacteria have potentially drastic consequences for human health globallyγ-pyrones  that possess potent inhibitory activities against MRSA and ESBL-producing E. coli in vitro. We then analyzed the antimicrobial activities and possible mechanisms of action of relevant analogues (compounds 1–8) isolated by our group in detail. Naphtho-γ-pyrones are an important group of fungal polyketides, some of them have been shown to exert antimicrobial activities against S. aureus, B. cereus, M. tuberculosis, E. coli, plant pathogenic bacteria, and C. albicans111213γ-pyrones against drug-resistant bacteria14Our efforts to identify novel antibiotics from microbial sources yielded four naphtho-γ-pyrones 1–8 against MRSA, ESBL-producing E. coli, K. pneumoniae, P. aeruginosa, and E. faecalis were investigated. We also performed structure-based virtual screening and confirmatory bioassays to identify the possible antibacterial targets of these bioactive naphtho-γ-pyrones.In this study, the antimicrobial effects of naphtho-1–8 were determined by 1H NMR, 13C NMR, MS, and ECD data analysis and by comparison with the literature161718191922R)-configured dimeric naphtho-γ-pyrones exhibit a negative Cotton effect in the long-wavelength region and a positive one at shorter wavelengths. The ECD spectra of compounds 1–8 (see R). Moreover, the calculated ECD spectra of compounds 3, 4, and 7 matched well with their experimental ECD spectra  were used to screen antibacterial activity against antimicrobial-resistant pathogenic bacteriain vitro. Fonsecinones A (2) and C (3) and aurasperones A (6) and E (7) exhibited potent inhibitory activities against MRSA and ESBL-producing E. coli at 100 μg/mL. The polycyclic skeletons of these compounds , fonsecinone B (4), rubrofusarin B (5), and asperpyrone C (8)—were purified by HPLC (≥95%) for further study to confirm this result. Flavasperone (1) and rubrofusarin B (5) are monomers with linear and angular polycyclic skeletonsγ-pyrones were classified as asperpyrone-type naphtho-γ-pyrones with C-10′-C-9 (2 and 3), C-10-C-7′ , or C-6-C-7′ (4) linkages13E. coli (ATCC 35218), and four common nosocomial infection strains: P. aeruginosa (ATCC 27853), K. pneumoniae (ATCC 700603), E. faecalis (ATCC 29212), and E. coli (ATCC 29212). The MICs of the tested compounds against these bacterial are presented in MRSA (ATCC 43300) and ESBL-producing i ATCC 3518 were uompounds  are simiγ-pyrones displayed significant antibacterial activity against gram-negative bacteria. Fonsecinones A (2) and C (3) and aurasperones A (6) and E (7) possessed potential antibacterial activities against E. coli, ESBL-producing E. coli, P. aeruginosa, E. faecalis and MRSA in the micromolar range. Fonsecinone A (2) exhibited the most potent antibacterial activity. The MICs of Fonsecinone A for ESBL-producing E. coli, P. aeruginosa and E. faecalis were 4.26, 17.04, and 4.26 ug/mL, respectively. The inhibitory effects of fonsecinones A (2) and C (3) against E. coli and ESBL-producing E. coli were comparable to those of amikacinμg/mL, respectively. The structure-activity relationships in these compounds indicated that the compounds with C-10′-C-9 linkages, fonsecinones A and C, had the highest antibacterial activities against the targeted pathogenic bacteria, followed by the compounds with C-10′-C-7 linkages, fonsecinone B and aurasperones A and E. Asperpyrone C, which features a C-6-C-7′ linkage, had the lowest antibacterial activities. The removal of electron-donating substituents (OH) at C2/C2′ was associated with higher antibacterial activity for these naphtho-γ-pyrones. Flavasperone, a monomer with an angular skeleton, exhibited higher antibacterial activity than its linear isomer (5).Most of the investigated naphtho-1–8, which exhibited potent antimicrobial activity, were subjected to further investigation to determine their possible antimicrobial mechanisms. A thorough literature survey indicated that several flavones26γ-pyrone with a chaetochromin-skeleton, are inhibitors of cellular fatty acid biosynthesis in bacteria. Bacterial fatty acid biosynthesis enzymes are excellent targets for novel antibacterial agent discovery because of their essential roles in the synthesis of the cell membranein silico target identification by small-scale inverse docking of 14 enzymes involved in fatty acid metabolism291–8. Targets with lower calculated binding energies are considered to have higher binding affinities for a specific compound31γ-pyrones. As the negative control, four unrelated targets3334351–8, the results were shown in Compounds E. coli, S. aureus, E. faecalis and P. aeruginosa, and has no homologue in mammals30293831+ and exhibited a key π-π accumulation effect on the nicotine ring of NAD+ 38FabI is highly conserved among most bacteria, including γ-pyrones could bind FabI. Microscale thermophoresis (MST) was then performed to quantitatively measure bindingE. coli and other pathogens have been reported to be very similarE. coli FabI to quantify the dissociation constant (Kd) for compound binding to the protein. Fonsecinones A (2) and C (3) and aurasperones A (6) and E (7) had Kd values of 215 ± 28.8, 270 ± 9.1, 289 ± 31.1, and 329 ± 29.7 μM, respectively  inhibited E. coli FabI; their IC50 values are presented in 2) exhibited the most potent inhibition, with an IC50 of only 2.78 μM. The order of inhibitory potencies was 2 > 3 > 6 > 7 > 1 > (≈) 4. The antibacterial activities of these compounds generally agreed with the experimentally determined FabI inhibitory activities. Among these compounds, fonsecinones A and C, had moderate antibacterial activities against E. coli and S. aureus, which express FabI as the sole enoyl reductase in the FASII system; however, no obvious inhibition of K. pneumonia, which produces only FabK, was observed29E. coli FabI was comparable to that of cephalochrominE. coli FabI and exert selective inhibitory effects are comparable, as observed in the molecular docking experiment  as potent antibiotics against a panel of drug-resistant microbial pathogens. These compounds exhibit FabI inhibition as one antibacterial mode of action. Notably, fonsecinones A and C (2 and 3) are both potent FabI inhibitors of fungal origin and exhibited promising antibacterial activities against the nosocomial infection pathogens MRSA, P. aeruginosa, E. faecalis, E. coli, and ESBL-producing E. coli, with MIC values in the micromolar range. These bioactive naphtho-γ-pyrones may be suitable as new antibiotics because of their potent antimicrobial properties and low toxicities15γ-pyrones found in this study may provide novel chemical scaffolds for the discovery of antibacterial agents.In conclusion, a combination of 3 (δH 7.26/δC 77.16). Semi-preparative HPLC was conducted on an Agilent 1100 liquid chromatography apparatus with a C18 column  and variable wavelength scanning ultraviolet detector (wavelength range: 190–600 nm). MPLC was conducted on a QuikSep-50 chromatography system . TLC was performed on silica gel GF254 . Column chromatography were performed using silica gel , ODS , and Sephadex LH–20 . The ELISA reader used SOFTmax PRO software .UV spectra were measured on a Shimadzu UV-2401A spectrophotometer. IR spectra were determined on a Bruker Vertex 70 FT-IR spectrophotometer. ECD spectra were obtained with a JASCO J-810 spectrometer. HRESIMS was performed on an APIQSTAR Pulsar spectrometer mass spectrometer. NMR spectra were recorded on a Bruker AM–400 NMR spectrometer, and chemical shifts were referenced to the solvent peaks for CDCl1–8 were isolated from Aspergillus sp. Z120. The strain was obtained from the Marine Culture Collection of China  and was isolated from the Pacific Ocean. The DNA sequence data for the fungus have been deposited in GenBank under the accession number FJ798688. All information and strains collected can be obtained from the website http://www.mccc.org.cn/ and the collection center. The fungus was cultured in three 500-mL Erlenmeyer flasks each containing 100 mL of PDB at 20 °C on a rotary shaker (110 rpm) for 8 d to obtain the seed culture. Then, the seed culture was added to 500-ml Erlenmeyer flasks, each containing solid rice medium (80 g) and H2O (120 mL). The fungal strain was grown under static conditions at 20 °C for 28 days. The mycelium-containing culture medium was divided into small portions, extracted with AcOEt (3 × 5 L), and concentrated under reduced pressure to obtain the crude extract (53.2 g). The crude extract was separated into 7 fractions, Frs. 1–7, on a SiO2 column via step-gradient elution of CH2Cl2/MeOH . Fr. 4 (4.5 g) was separated by MPLC (MeOH–H2O from 20:80 to 100:0) into six subfractions, Frs. 4.1–4.6. Fr. 4.4 (119.3 mg) was further separated by CC  followed by semi-prep HPLC (65% MeOH–H2O) to isolate compounds 1  and 5 . Fr. 5 (6.5 g) was subjected to Sephadex LH-20 in CH2Cl2–MeOH (1:1) to isolate the major component, which was then separated by MPLC (MeOH–H2O from 10:90 to 100:0) to obtain Frs. 5.3.1–5.3.10. Fr. 5.3.6 (96.9 mg) was purified by semi-prep HPLC (75% MeOH–H2O) to yield compound 6 . Compounds 7  and 8  were isolated from Fr. 5.3.8 (67.0 mg) by semi-prep HPLC (50% MeCN–H2O). The separation of Fr. 6 (12 g) followed a procedure similar to that used for Fr. 5 to produce compounds 4, 2, and 3 by semi-prep HPLC .Compounds All investigated compounds were ≥95% pure . NMR data for compounds 3, 4 and 7 were completed for the first time in the present study. The NMR spectra of the compounds are provided in the 1): 1H-NMR : 2.48 , 3.90 , 3.95 , 6.26, 6.39 , 6.57 , 6.85 , 12.81 . HRESIMS: m/z [M+H] calcd for C16H14O5, 287.0875, found 287.0874.Flavasperone (2): 1H-NMR : 2.10 , 2.46 , 3.41 , 3.59 , 3.76 , 4.01, 5.98 , 6.17, 6.31 , 6.40 , 6.95 , 7.03 , 12.81 , 15.23 . HRESIMS: m/z [M+H] calcd for C32H26O10, 570.1560, found 570.1562.Fonsecinone A (3): 1H-NMR : 1.45 , 2.45 , 2.92 , 3.38, 3.62 , 3.81 , 3.99 , 6.12 , 6.31 , 6.36 , 6.96 , 7.01 , 12.73 , 14.49 . 13C-NMR : 197.4 (C-4′), 183.1 (C-4), 166.9 (C-2), 165.3 (C-8′), 162.7 (C-8), 162.1 (C-6′), 160.3 (C-10), 156.8 (C-5), 155.8 (C-5′), 155.2 (C-10b), 151.4 (C-10′a), 142.8 (C-6a), 140.7 (C-9′a), 118.4 (C-9), 110.9 (C-5a), 109.7 (C-10a), 108.1 (C-5′a), 108.1 (C-3), 106.8 (C-10), 106.3 (C-4a), 103.7 (C-4′a), 102.2 (C-6), 100.4 (C-2′), 97.5 (C-7′), 96.7 (C-9′), 61.4 (10-OCH3), 56.4 (6′-OCH3), 56.2 (8-OCH3), 55.4 (8′-OCH3), 46.9 (C-3′), 29.3 (2′-CH3), 20.7 (2-CH3). HRESIMS: m/z [M+H] calcd for C32H28O11, 588.1665, found 588.1667.Fonsecinone C (4): 1H-NMR : 1.78 , 2.09 , 2.99 , 3.39, 3.60 , 3.73 , 3.99 , 5.94 , 6.18, 6.38 , 6.68 , 6.82 , 14.13 , 15.23 . 13C-NMR : 196.7 (C-4′), 184.8 (C-4), 167.8 (C-2), 164.3 (C-8′), 162.9 (C-8), 161.6 (C-6′), 161.2 (C-6), 161.2 (C-5), 159.7 (C-5′), 153.5 (C-10a), 151.0 (C-10′a), 142.9 (C-9a), 140.9 (C-9′a), 117.0 (C-7), 110.9 (C-5a), 108.7 (C-5a′), 107.4 (C-3), 105.4 (C-10′),104.4 (C-4a), 103.7 (C-4′a), 102.9 (C-9), 102.1 (C-10), 100.3 (C-2′), 97.2 (C-7′), 96.6 (C-9′), 62.1 (6-OCH3), 56.4 (6′-OCH3), 56.1 (8-OCH3), 55.4 (8′-OCH3), 47.5 (C-3′), 29.0 (2′-CH3), 20.9 (2-CH3). HRESIMS: m/z [M+H] calcd for C32H28O11, 588.1665, found 588.1667.Fonsecinone B (5): 1H-NMR : 2.35 , 3.91 , 3.99 , 5.98 , 6.38 , 6.56 , 6.98 , 15.0 . HRESIMS: m/z [M+H] calcd for C16H14O5, 287.0875, found 287.0879.Rubrofusarin B (6): 1H-NMR : 2.10 , 2.39 , 3.44 , 3.60 , 3.76 , 4.00, 5.96 , 6.03 , 6.18 , 6.39 , 6.95 , 7.13 , 14.82 , 15.23 . HRESIMS: m/z [M+H] calcd for C32H26O10, 570.1560, found 570.1557.Aurasperone A (7): 1H-NMR : 1.47 , 2.38 , 2.92 , 3.40, 3.61 , 3.79 , 3.97 , 6.01 , 6.09 , 6.33 , 6.94 , 7.09 , 14.52 , 14.77 . 13C-NMR : 197.8 (C-4′), 184.7 (C-4), 168.0 (C-2′), 165.1 (C-8), 162.3 (C-8′), 162.0 (C-6′), 161.9 (C-6), 160.4 (C-5), 153.3 (C-5′), 151.6 (C-10′a), 142.8 (C-10a), 140.4 (C-9a), 118.9 (C-9′a), 111.5 (C-7′), 110.4 (C-5′a), 107.9 (C-5a), 107.5 (C-3′), 106.6 (C-10), 104.9 (C-4′a), 104.0 (C-4a), 101.9 (C-9′), 101.5 (C-10′), 100.4 (C-2), 97.5 (C-7), 96.3 (C-9), 62.0 (6′-OCH3), 56.4 (6-OCH3), 56.1 (8′-OCH3), 55.4 (8-OCH3), 47.2 (C-3), 28.9 (2-CH3), 21.0 (2′-CH3). HRESIMS: m/z [M + H] calcd for C32H28O11, 588.1665, found 588.1667.Aurasperone E (8): 1H-NMR : 2.38 , 2.51 , 3.59 , 3.62 , 3.79 , 3.97 , 6.01 , 6.23, 6.30 , 6.40 , 6.95 , 7.09 , 13.09 , 14.72 ; HRESIMS: m/z [M+H] calcd for C32H26O10, 570.1560, found 570.1558.Asperpyrone C  distance was less than 0.5 Å. Semi-empirical PM3 quantum mechanical geometry optimisations were performed on the conformations using the Gaussian 0913 program. The duplicated conformations after geometry optimisation were then identified and removed. Remaining conformations were further optimised at the B3LYP/6-31G* level of theory in methanol solvent with the IEFPCM314 solvation model using the Gaussian 09 program, and duplicated conformations emerging after these calculations were removed according to the same RMS criteria indicated above. The harmonic vibrational frequencies were performed to confirm the stability of obtained conformers . Oscillator strengths and rotational strengths of the 20 weakest electronic excitations of each conformer were calculated using the TDDFT methodology at the B3LYP/6-311++G** level of theory with methanol as the solvent by the IEFPCM solvation model implemented in the Gaussian 09 program. The ECD spectra for each conformers were then simulated using Gaussian function with a bandwidth σ = 0.45 eV. The calculated spectra for each conformations were combined after Boltzmann weighting according to their population contributions.The conformational analyses were carried out for compounds 5/mL) were inoculated into Mueller–Hinton broth and dispensed at 0.1 mL/well into a 96-well microtitre plate. MICs were determined in triplicate by serial 2-fold dilutions of test compounds. The MIC was defined as the concentration of a test compound that completely inhibited cell growth during a 24h incubation at 30 °C. Bacterial growth was determined by measuring the absorption at 600 nm using a microtitre ELISA reader.Whole-cell antimicrobial activity was determined by the broth microdilutionE. coli str. K-12 substr. MG1655, complete genome) was amplified from E. coli genomic DNA using the following forward and reverse primers: 5′-GGAATTCATATGGGTTTTCTTTCCGGTAAGCGC-3′ and 5′-GGGTGCCTCGAGTTATTTCAGTTCGAGTTCGTT-3′. The gene was cloned into a modified pET21b vector containing a 6 His-tag coding region at the N-terminus of the insert. After verifying the recombinant plasmids by sequencing, the plasmids were used to transform E. coli BL21 (DE3) cells. The transformed cells were grown in LB medium at 37 °C to an OD of 0.8–1.0 and induced with 0.5 mM isopropyl-D-thiogalactopyranoside (IPTG) at 18 °C for 18 h. The cells were harvested by centrifugation at 4,100 rpm for 10 min and re-suspended in lysis buffer containing 20 mM Tris, pH 8.5, 200 mM NaCl, and 10 mM imidazole, followed by disruption on a French press. Cell debris was removed by centrifugation at 21,000 rpm for 30 min. The protein was bound to Ni-agarose affinity resin, washed with buffer containing 20 mM Tris, pH 8.5, 200 mM NaCl, and 10 mM imidazole, and eluted with buffer containing 20 mM Tris, pH 8.8, 250 mM NaCl, and 150 mM imidazole. The protein was further purified by anion-exchange chromatography using a linear gradient of 10 mM to 1 M NaCl and size exclusion chromatography at pH 8.5 in 200 mM NaCl.The full-length FabI gene (E. coli FabI was labeled with the Monolith NT™ Protein Labeling Kit RED (Cat#L001) according to the supplied labeling protocol. Labelled FabI was kept constant at 50 nM, while all samples tested were diluted in a 20 mM HEPES (pH 7.4) and 0.05 (v/v)% Tween-20. After 10 min incubation at room temperature, samples were loaded into MonolithTM standard-treated capillaries and the thermophoresis was measured at 25 °C after 30 min incubation on a Monolith NT.115 instrument . Laser power was set to 20% or 40% using 30 seconds on-time. The LED power was set to 100%. The dissociation constrant Kd values was fitted by using the NTAnalysis software .Recombinant 50 values of the investigated compounds were determined using 1 μg of enzyme, 50 μM crotonoyl-CoA as the substrate, and 100 μM NADH as a cofactor30NADH, NADPH, crotonoyl-CoA, and HEPES reagents were obtained from Sigma. All other chemicals were of analytical grade. The assay was performed in half 96-well microtiter plates. All compounds in this study were dissolved in DMSO to produce 100 mM stocks. The IChttp://www.rcsb.org)3839424344454647484950For the small scale inverse docking31p < 0.05 was considered statistically significant.Statistical analysis of the data was performed using Graph Pad Prism 4.0 software. The data were expressed as the means ± SD. Values were analyzed using SPSS version 12.0 software by one-way analysis of variance (ANOVA), and How to cite this article: He, Y. et al. Fungal naphtho-γ-pyrones: Potent antibiotics for drug-resistant microbial pathogens. Sci. Rep.6, 24291; doi: 10.1038/srep24291 (2016).


H-1,2,4-triazole was synthesized. The anticonvulsant effect and neurotoxicity of the compounds were calculated with maximal electroshock (MES) test and rotarod tests with intraperitoneally injected mice. Among the synthesized compounds, compound 3-heptyloxy-4-(4-(hexyloxy) phenyl)-4H-1,2,4-triazole (5f) was the most active one and also had the lowest toxicity. In the anti-MES potency test, it showed median effective dose (ED50) of 37.3 mg/Kg, median toxicity dose (TD50) of 422.5 mg/Kg, and the protective index (PI) of 11.3 which is much greater than the reference drug carbamazepine with PI value of 6.4. As well as demonstrating the anti-MES efficacy of compound 5f, its potency against seizures induced by pentylenetetrazole, 3-mercaptopropionic acid, and bicuculline were also established, with the results suggesting that GABA-mediated mechanisms might be involved in its anticonvulsant activity, such as enhancing of GABAergic neurotransmission or activity, activate GAD or inhibit GABA-T, and GABAA-mediated mechanisms.A series of 3-alkoxy-4-(4-(hexyloxy/heptyloxy) phenyl)-4 Epilepsy, a ubiquitous disease characterized by recurrent seizures. For epilepsy treatment, nearly 95% of clinically available drugs were approved before 1985 and they are only effective in reducing the severity and number of seizures in less than 70% of patients. These drugs, however, also cause notable adverse side effects such as drowsiness, ataxia, gastrointestinal disturbance, hepatotoxicity and megaloblastic anemia . ResearcH) -one  quinolones, 1-substituted-7-benzyloxy-4,5-dihydro-triazoloquinolines and 7-alkoxy-4,5-dihydro-triazoloquinolines  -one  were firinolines  were synH-1,2,4 -triazole (compound III). The hypothesis was that a rotatable triazole ring may have higher affinity for the receptor and enhance their anticonvulsant activity.Analyzing the relationship of anticonvulsant activity and the structure of compound II, it was found that triazole ring C may be the main structure combined with receptor, because the 1,2,4-triazole nucleus has been incorporated into a wide variety of therapeutically important agents, such as antimicrobial , 8, antiChemistryGeneral methods1H-NMR spectra were measured on a AV-300 , and all chemical shifts were given in ppm relative to tetramethy-silane. Mass spectra were measured on an HP1100LC . Elemental analyses were performed on a 204Q CHN . Micro-analyses of C, N, and H were performed using a Heraeus CHN Rapid Analyzer. Critical chemicals were purchased from Aldrich Chemical Corporation. All other chemicals were of analytical grade.Melting points were determined in open capillary tubes and are uncorrected. IR spectra were recorded (in KBr) on a FT-IR1730 . Synthesis of N-(4-(hexyloxy/heptyloxy) phenyl) acetamide (2a/2b)N-(4-hydroxyphenyl)acetamide (10 mmol) and K2CO3 (15 mmol) reacted with appropriate bromohexane/bromoheptane to obtain compound 2a/2b in absolute ethanol with stirring at 80 ℃for 15 hours. Then the solution was evaporated to dryness under reduced pressure, and 20 mL water was poured into the flask, and the mixture was stirred for 0.5 h to eliminate excess K2CO3. The crude product was collected through filtration and dried in vacuo.A solution of Synthesis of 4-(hexyloxy/heptyloxy)benzenamine (3a/3b)A mixture of N-(4-(hexyloxy/heptyloxy) phenyl) acetamide (10 mmol) and about 30 mL of hydrochloric acid (15 % water solutions) was refluxed for 8 h. Adjusting the solution’s pH value to 8-9 using saturated NaOH resulted in precipitation of solid. The crude product was collected through filtration and dried in vacuoSynthesis of 4-(4-(hexyloxy/heptyloxy)phenyl)-2H-1,2,4-triazol-3(4H)-one (4a/4b)4-(hexyloxy/heptyloxy) benzenamine (10 mmol), triethyl orthoformate (15 mmol) and methyl hydrazinocarboxylate (15 mmol) were placed into a round-bottomed flask containing 30 mL ethanol. The mixture was refluxed for 24 hours. Then 12 mmol sodium methoxide was put into the flask and continues to be refluxed for 10 hours. After being cooled to the room temperature and concentrated under reduced pressure, the product 4a/4b was collected through filtration.General procedure for the synthesis of 3-alkoxy-4-(4-(hexyloxy/heptyloxy)phenyl) -4H -1,2,4-triazole (5a-5t)H) -one was placed into a 100 mL round-bottomed flask containing 30 mL 15 % NaOH water solutions, stirring at room temperature for half an hour. Then 25 mL ethanol and appropriate alkyl halide (13 mmol) were added to the flask with refluxing and stirring for 7-15 hours (TLC moni-toring). After removing most of the solvent under reduced pressure, the crude product was collected through filtration and recrystallized in acetone to obtain the pure product.4- (4- (hexyloxy/heptyloxy) phenyl) -2H-1,2,4-triazol- 3phenyl)-4H-1,2,4-triazole 5aoC. 1H-NMR : δ 0.93 , 0.99 , 1.27-1.47 , 1.78-1.86 , 3.83 , 3.97 , 6.98 , 7.43 , 7.62 , IR (KBr) cm-1: 2968 (-N=CH-), 1683 (-R-O-C-); MS-EI m/z 290 (M+1). Yield: 10.3%, mp: 121-123 3-Propoxy-4-(4-(hexyloxy)phenyl)-4H-1,2,4-triazole5boC. 1H-NMR : δ 0.92 , 1.01 , 1.35-1.47 , 1.78-1.90 , 3.82 , 3.96 , 6.97 , 7.42 , 7.62 , IR (KBr) cm-1: 2969 (-N=CH-), 1685 (-R-O-C-); MS-EI m/z 304 (M+1).Yield: 11.7%, mp: 115-117 3-Butoxy-4-(4-(hexyloxy)phenyl)-4H-1,2,4-triazole5coC. 1H-NMR : δ 0.89 , 0.96 , 1.31-1.45 , 1.70-1.93 , 3.84 , 3.96 , 6.95 , 7.37 , 7.62 , IR (KBr) cm-1: 2967 (-N=CH-), 1684 (-R-O-C-); MS-EI m/z 318 (M+1).Yield: 17.6%, mp: 111-113 3-Pentyloxy-4-(4-(hexyloxy)phenyl)-4H-1,2,4-triazole5doC. 1H-NMR : δ 0.90 , 1.01 , 1.27-1.35 , 1.38-1.85 , 3.86 , 3.99 , 6.98 , 7.43 , 7.62 , IR (KBr) cm-1: 2969 (-N=CH-), 1685 (-R-O-C-); MS-EI m/z 332 (M+1).Yield: 18.3%, mp: 129-131 3-Hexyloxy-4-(4-(hexyloxy)phenyl)-4H-1,2,4-triazole5eoC. 1H-NMR : δ 0.84 , 0.92 , 0.97-1.42 , 1.44-1.86 , 3.86 , 3.99 , 6.98 , 7.42 , 7.62 , IR (KBr) cm-1: 2969 (-N=CH-), 1687 (-R-O-C-); MS-EI m/z 346 (M+1). Yield: 20.6%, mp: 136-138 3-Heptyloxy-4-(4-(hexyloxy)phenyl)-4H-1,2,4-triazole5foC. 1H-NMR : δ 0.89 , 0.93 , 1.21-1.48 , 1.76-1.85 , 3.85 , 3.97 , 6.98 , 7.42 , 7.62 , IR (KBr) cm-1: 2965 (-N=CH-), 1686 (-R-O-C-); MS-EI m/z 360 (M+1). Yield: 46.8%, mp: 137-139 3-Octyloxy-4-(4-(hexyloxy)phenyl)-4H-1,2,4-triazole 5goC. 1H-NMR : δ 0.90 , 0.93 , 1.27-1.47 , 1.49-1.83 , 3.85 , 3.97 , 6.98 , 7.42 , 7.62 , IR (KBr) cm-1: 2964 (-N=CH-), 1688 (-R-O-C-); MS-EI m/z 374 (M+1). Yield: 46.8%, mp: 139-141 3-Decyloxy-4-(4-(hexyloxy)phenyl)-4H-1,2,4-triazole 5hoC. 1H-NMR : δ 0.89 , 0.93 , 1.27-1.48 , 1.50-1.85 , 3.85 , 3.99 , 6.98 , 7.42 , 7.62 , IR (KBr) cm-1: 2969 (-N=CH-), 1687 (-R-O-C-); MS-EI m/z 402 (M+1).Yield: 45.4%, mp: 123-125 3-Dodecyloxy-4-(4-(hexyloxy)phenyl)-4H-1,2,4-triazole 5ioC. 1H-NMR : δ 0.89 , 0.92 , 1.19-1.37 , 1.43-1.52 , 3.85 , 3.98 , 6.98 , 7.42 , 7.62 , IR (KBr) cm-1: 2968 (-N=CH-), 1685 (-R-O-C-); MS-EI m/z 430 (M+1). Yield: 48.8%, mp: 115-117 3-Benzyloxy-4-(4-(hexyloxy)phenyl)-4H-1,2,4-triazole 5joC. 1H-NMR : δ 0.93 , 0.93 , 1.35-1.50 , 1.77-1.87 , 3.98 , 5.04 , 6.96-7.04 , 7.32-7.62 , 7.64 , IR (KBr) cm-1: 2964 (-N=CH-), 1689 (-R-O-C-); MS-EI m/z 352 (M+1). Yield: 48.8%, mp: 165-167 3-Ethoxy-4-(4-(heptyloxy)phenyl)-4H-1,2,4-triazole5koC. 1H-NMR : δ 0.87 , 0.93 , 1.31-1.74 , 1.76-1.81 , 3.87 , 3.95 , 6.96 , 7.40 , 7.60 , IR (KBr) cm-1: 2965 (-N=CH-), 1686 (-R-O-C-); MS-EI m/z 304 (M+1). Yield: 10.5%, mp: 121-123 3-Propoxy-4-(4-(heptyloxy)phenyl)-4H-1,2,4-triazole5loC. 1H-NMR : δ 0.89 , 0.93 , 1.21-1.48 , 1.76-1.85 , 3.85 , 3.97 , 6.98 , 7.42 , 7.62 , IR (KBr) cm-1: 2967 (-N=CH-), 1687 (-R-O-C-); MS-EI m/z 318 (M+1). Yield: 16.4%, mp: 127-129 3-Butoxy-4-(4-(heptyloxy)phenyl)-4H-1,2,4-triazole 5moC. 1H-NMR : δ 0.89 , 0.96 , 1.31-1.43 , 1.69-1.80 , 3.84 , 3.96 ,6.95 , 7.39 , 7.62 , IR (KBr) cm-1: 2969 (-N=CH-), 1684 (-R-O-C-); MS-EI m/z 332 (M+1). Yield: 15.3%, mp: 133-135 3-Pentyloxy-4-(4-(heptyloxy)phenyl)-4H-1,2,4-triazole5noC. 1H-NMR : δ 0.90 , 0.93 , 1.07-1.47 , 1.65-1.85 , 3.85 , 3.99 , 6.98 , 7.42 , 7.62 , IR (KBr) cm-1: 2968 (-N=CH-), 1684 (-R-O-C-); MS-EI m/z 346 (M+1). Yield: 16.4%, mp: 145-147 3-Hexyloxy-4-(4-(heptyloxy)phenyl)-4H-1,2,4-triazole 5ooC. 1H-NMR : δ 0.79 , 0.98 , 1.32-1.73 , 1.75-1.80 , 3.83 , 3.96 , 6.94 , 7.40 , 7.60 , IR (KBr) cm-1: 2970 (-N=CH-), 1689 (-R-O-C-); MS-EI m/z 360 (M+1). Yield: 26.7%, mp: 149-151 3-Heptyloxy-4-(4-(heptyloxy)phenyl)-4H-1,2,4-triazole5poC. 1H-NMR : δ 0.85 , 0.90 , 1.03-1.40 , 1.42-1.81 , 3.84 , 3.97 , 6.96 , 7.41 , 7.60 , IR (KBr) cm-1: 2971 (-N=CH-), 1682 (-R-O-C-); MS-EI m/z 374 (M+1). Yield: 36.5%, mp: 156-158 3-Octyloxy-4-(4-(heptyloxy)phenyl)- 4H-1,2,4-triazole5qoC. 1H-NMR : δ 0.85 , 0.92 , 1.27-1.69 , 1.71-1.84 , 3.84 , 3.97 , 6.96 , 7.41 , 7.60 , IR (KBr) cm-1: 2972 (-N=CH-), 1670 (-R-O-C-); MS-EI m/z 388 (M+1). Yield: 31.2%, mp: 167-169 3-Decyloxy-4-(4-(heptyloxy)phenyl)-4H-1,2,4-triazole 5roC. 1H-NMR : δ 0.81 , 0.97 , 1.15-1.53 , 1.72-1.92 , 3.83 , 3.97 , 6.96 , 7.40 , 7.60 , IR (KBr) cm-1: 2971 (-N=CH-), 1671 (-R-O-C-); MS-EI m/z 416 (M+1).Yield: 40.8%, mp: 147-149 3-Dodecyloxy-4-(4-(heptyloxy)phenyl)-4H-1,2,4-triazole 5soC. 1H-NMR : δ 0.85 , 0.91 , 1.10-1.48 , 1.68-1.83 , 3.83 , 3.97 , 6.96 , 7.40 , 7.60 , IR (KBr) cm-1: 2970 (-N=CH-), 1673 (-R-O-C-); MS-EI m/z 444 (M+1). Yield: 39.2%, mp: 152-154 3-Benzyloxy-4-(4-(heptyloxy)phenyl)-4H-1,2,4-triazole5toC. 1H-NMR : δ 0.90 , 0.91 , 1.05-1.46 , 1.44-1.83 , 3.97 , 5.02 , 6.95-7.07 , 7.31-7.42 , 7.60 , IR (KBr) cm-1: 2974 (-N=CH-), 1686 (-R-O-C-); MS-EI m/z 366 (M+1). Yield: 46.4%, mp: 168-169 Pharmacologyi.p.) injection at dosages of 100mg/kg to avoid wasting animals. Electro-convulsions were produced by an electric stimulation generator . All compounds, which were dissolved in dimethylsulfoxide (DMSO), were evaluated for anticonvulsant activities with KunMing mice in the 18-22 g weight range purchased from the Laboratory of Animal Research, College of Pharmacy, Yanbian University. The MES test, Chemicals induce epileptic test and rotarod test were carried out according to the procedures described in Anticonvulsant Screening Program with some modification , 23. In The maximal electroshock (MES) testvia ear-clip electrodes for 0.2s. Protection against the spread of MES-induced seizures was defined as the absence of tonic extension of the hind leg. At 30 min after the administration of the compounds, the activities were evaluated in MES test. In preliminary screening, each compound was administered at the dose levels of 200, 100，and 30 mg/Kg for evaluating the preliminary anticonvulsant activity. For determination of the median effective dose (ED50) and the median toxic dose (TD50), the quantitative evaluation was prepared. Groups of 10 mice were given a range of intraperitoneal doses of the tested compound until at least three points were established in the range of 10–90% seizure protection or neurotoxicity. From these data, the respective ED50, TD50 values, and 95% confidence intervals were calculated by probit analysis.Seizures were elicited with a 60 Hz alternating current of 50 mA intensity in mice. The current was applied Rotarod test od test i.p. injection of the test compounds. Neurotoxicity was indicated by the inability of the animal to maintain equilibrium on the rod for at least 1min in each of the trials.The neurotoxicity of the compounds was measured in mice by the rotarod test. The mice were trained to stay on an accelerating rotarod of diameter an inch that rotates at 6 rpm. Trained animals were given Chemical model seizure testPentylenetetrazol-induced seizure test re test At 30 min after the administration of the test compound, 100 mg/Kg of PTZ dissolved in saline was administered sc. The animals placed in individual cages and observed for 30 min. The number of clonic and tonic seizures as well as the number of deaths was noted.Mercaptopropionic acid-induced seizures eizures At 30 min after the administration of the test compound, 40 mg/Kg of 3-MP dissolved in saline solution was injected sc. The animals placed in individual cages and observed for 30 min. The number of clonic and tonic seizures as well as the number of deaths was noted.Bicuculline-induced seizures test es test At 30 min after the administration of the test compound, 5.4 mg/Kg of bicuculline (within 15-45 min after preparation due to instability) was injected sc. The animals placed in individual cages and observed for 30 min. The number of clonic and tonic seizures as well as the number of deaths was noted.Calculation formula50 = lg-1 [Xm - i(∑P-0.5)]EDX50 =i [(∑P -∑P)2 /n-1]1/2S-1(lgED50 ±1.96SX50)The 95% confidence limits= lgThe meaning of the symbol in the formula:Xm: Maximum dose of logarithm.i: Dose the proportion between the value of common logarithm.P: The positive rate of each group.n: The number of animal in each group.X50: The standard error of lgED50. S50 has the same calculation method as above.TDChemistry 1H-NMR, MS and elemental analysis techniques. Their anticonvulsant activity was evaluated by the maximal electroshock test (MES) and their neurotoxicity was evaluated by the rotarod test. To elucidate the possible mechanism of action, the most active compound was tested in pentylenetetrazole (PTZ), 3-mercaptopropionic acid (3-MP), and bicuculline (BIC) induced seizure tests.The structures of those new compounds were characterized using IR,H-1,2,4-triazole (5a-5t). Target compounds were prepared along the reaction sequence in Based on the previous studies in our laboratory, we designed and synthesized a series of 3-alkoxy-4-(4-(hexyloxy/heptyloxy)phenyl)-4Pharmacological evaluationsi.p.) injection at dosages of 200, 100, and 30 mg/Kg. Their anticonvulsant effects and neurovirulence were assessed at 0.5 h intervals following administration in mice.During the preliminary screening process, compounds were administered by intraperitoneal (The results of preliminary (phase I) screening of 5a-5t are summarized in Structure activity relationships1 was n-hexane chain, as the alkyl chain length increased from 5a to 5f, the anticonvulsant activity gradually increased with compound 5f being the most active compound. From 5f to 5i, although the alkyl chain length increased further, the anticonvulsant activity decreased and ultimately disappeared. When R1 was n-heptane chain, as the alkyl chain length increased from 5k to 5p , the anticonvulsant activity gradually increased with compound 5p being the most active compound. From 5p to 5s, although the alkyl chain length increased further, the anticonvulsant activity decreased and ultimately disappeared. This structure-activity relationship might be associated with the lipid-water partition coefficients of the compounds, which affects drug absorption, hydrophobic drug-receptor interactions, metabolism of molecules, and especially the ability to cross the blood-brain barrier (BBB). The compounds 5j and 5t, which substituted with a benzyloxy group, showed less anticonvulsant activity than the best alkyl chain-substituted 5f and 5p.Following activity analysis of the synthesized compounds, the following structure–activity relationships (SARs) were obtained. For the alkyl chain-substituted derivatives 5a-5i and 5k-5s, the length of the alkyl chain appeared to have a direct impact on the anticonvulsant activity of the derivatives. For example when RQuantitative evaluation of the anticonvulsant activity50) and neurotoxicity (indicated by TD50) in mice. Results of the quantitative testing of selected compounds, together with data of the antiepileptic drug carbamazepine (measured in the same condition), are shown in Based on the considerable anticonvulsant activity demonstrated during the preliminary screening test, compounds 5f and 5p were subjected to quantitative evaluation trials for quantification of their anticonvulsant activity  neurotransmission . GABA is3-MP acid was seen as the competitive inhibitor of GABA synthesis enzyme glutamate decarboxylase (GAD), inhibited the synthesis of GABA to decrease the GABA level in the brain . In the A receptor (A-mediated mechanisms. In the BIC induced seizure model, both carbamazepine and 5f inhibited tonic seizures and death, but did not inhibit clonic seizures. Carbamazepine showed inhibition of clonic and tonic seizures and death at rates of 0%, 100%, and 40%, respectively. Compound 5f inhibited the clonic seizures, tonic seizures and lethality at the rates of 0%, 100%, and 80% induced by BIC, respectively, better than carbamazepine in inhibiting lethality . BIC is receptor . As comp


A single-crystal X-ray structural analysis of [Ru(η6-p-cym)(2Me4Claza)Cl2] (6) revealed a typical piano-stool geometry with an N7-coordination mode of 2-methyl-4-chloro-7-azaindole (2Me4Claza). The complexes have been found to be inactive against human ovarian cancer cell line A2780 up to the highest applied concentration (IC50 > 50.0 μM). An inactivity of the complexes is caused by their instability in water-containing solvents connected with a release of the naza N-donor ligand, as proved by the detailed 1H NMR, mass spectrometry and fluorescence experiments.A series of organometallic half-sandwich dichloridoruthenium(II) complexes of the general formula [Ru( The reported complexes 1–8 , we strived to investigate and explain the reasons of their inactivity by means of 1H NMR, ESI+ mass spectrometry and fluorescence studies.Herein we report the synthesis and characterization of the complexes of the general composition 2 was prepared according to the reported synthetic procedure [The chemicals (RuClrocedure .η6-p-cym)Cl]2  was dissolved in methanol (5 mL) and left to react with two molar equivalents of the corresponding naza. The reaction mixture was stirred at ambient temperature for 20 min (reactions with 5Braza and 5Faza), 2 h  or 3 h , until the yellow , or light (for 8) or dark  orange product was formed. The products ([Ru(μ-Cl)(products  were isoη6-p-cym)(aza)Cl2] (1): Anal. Calcd. for RuCl2C17H20N2: C, 48.12; H, 4.75; N, 6.60; found: C, 47.76; H, 4.74; N, 6.37%. 1H NMR : δ 11.00 , 8.68 , 7.94 , 7.31 , 7.11 , 6.44 , 5.58 , 5.28 , 2.96 , 1.83 , 1.19 . 13C NMR : δ 151.1 (C6), 148.7 (C7a), 130.7 (C4), 126.3 (C2), 122.9 (C3a), 166.7 (C5), 103.4 (C14), 101.9 (C3), 97.7 (C11), 83.6 , 81.7 , 30.6 (C17), 22.3 , 18.1 (C20).[Ru(η6-p-cym)(3Claza)Cl2] (2): Anal. Calcd. for RuCl3C17H19N2: C, 44.51; H, 4.18; N, 6.11; found: C, 44.32; H, 4.19; N, 5.95%. 1H NMR : δ 11.12 , 8.78 , 7.99 , 7.29 , 7.21 , 5.60 , 5.31 , 2.95 , 1.85 , 1.30 . 13C NMR : δ 150.0 (C6), 149.4 (C7a), 128.9 (C4), 123.1 (C2), 120.8 (C3a), 117.1 (C5), 103.4 (C14), 105.6 (C3), 97.8 (C11), 81.6 , 83.7 , 30.6 (C17), 22.3 , 18.1 (C20).[Ru(η6-p-cym)(3Iaza)Cl2] (3): Anal. Calcd. for RuCl2C17H19N2I: C, 37.11; H, 3.48; N, 5.09; found: C, 37.05; H, 3.34; N, 5.47%. 1H NMR : δ 11.29 , 8.76 , 7.79 , 7.37 , 7.21 , 5.61 , 5.32 , 2.95 , 1.84 , 1.30 . 13C NMR : δ 150.5 (C6), 150.0 (C7a), 131.5 (C4), 131.0 (C2), 125.3 (C3a), 117.6 (C5), 103.5 (C14), 98.1 (C11), 83.9 , 81.8 , 30.8 (C17), 22.5 , 18.3 (C20).[Ru(η6-p-cym)(5Braza)Cl2] (4): Anal. Calcd. for RuCl2C17H19N2Br: C, 40.57; H, 3.81; N, 5.57; found: C, 40.80; H, 3.82; N, 5.47%. 1H NMR : δ 11.19 , 8.77 , 8.09 , 7.32 , 6.47 , 5.60 , 5.31 , 2.95 , 1.87 , 1.31 . 13C NMR : δ 148.9 (C7a), 133.0 (C4), 127.9 (C2), 123.9 (C3a), 111.2 (C5), 103.4 (C14), 101.5 (C3), 97.8 (C11), 83.7 , 81.7 , 29.7 (C17), 22.3 , 18.1 (C20).[Ru(η6-p-cym)(5Faza)Cl2] (5): Anal. Calcd. for RuCl2C17H19N2F: C, 46.16; H, 4.33; N, 6.33; found: C, 45.90; H, 4.42; N, 6.10%. 1H NMR : δ 11.08 , 8.67 , 7.70 , 7.39 , 6.50 , 5.60 , 5.31 , 2.97 , 1.87 , 1.31 . 13C NMR : δ 148.6 (C7a), 131.1 (C4), 137.6 (C6), 128.9 (C2), 122.7 (C3a), 117.0 (C5), 103.6 (C14), 102.3 (C3), 98.0 (C11), 83.9 , 81.1 , 30.8 (C17), 22.5 , 18.3 (C20).[Ru(η6-p-cym)(2Me4Claza)Cl2] (6): Anal. Calcd. for RuCl3C18H21N2: C, 45.73; H, 4.48; N, 5.92; found: C, 45.40; H, 4.49; N, 5.60%. 1H NMR : δ 11.00 , 8.45 , 7.07 , 6.26 , 5.54 , 5.27 , 2.96 , 2,47 , 1.90 , 1.30 . 13C NMR : δ 151.3 (C6), 147.2 (C7a), 138.4 (C2), 122.9 (C3a), 119.9 (C5), 98.2 (C3), 103.5 (C14), 97.5 (C11), 83.2 , 81.8 , 30.5 (C18), 22.2 , 18.1 (C17), 14.2 (C8).[Ru(η6-p-cym)(3Cl5Braza)Cl2] (7): Anal. Calcd. for RuCl3C17H18N2Br: C, 37.98; H, 3.37; N, 5.21; found: C, 37.88; H, 3.27; N, 4.99%. 1H NMR : δ 11.25 , 8.84 , 8.12 , 7.32 , 5.60 , 5.32 , 2.93 , 1.89 , 1.31 . 13C NMR : δ 151.2 (C6), 148.3 (C7a), 131.4 (C4), 128.8 (C2), 121.8 (C3a), 111.9 (C5), 105.2 (C3), 103.6 (C14), 98.0 (C11), 83.8 , 81.8 , 30.8 (C17), 22.4 , 18.3 (C20).[Ru(η6-p-cym)(3I5Braza)Cl2] (8): Anal. Calcd. for RuCl2C17H18N2BrI: C, 32.46; H, 2.88; N, 4.45; found: C, 32.24; H, 2.77; N, 4.22%. 1H NMR : δ 11.38 , 8.79 , 7.91 , 7.39 , 5.58 , 5.30 , 2.91 , 1.86 , 1.29 . 13C NMR : δ 149.9 (C6), 149.3 (C7a), 133.8 (C4), 132.2 (C2), 126.1 (C3a), 112.1 (C5), 103.4 (C14), 97.9 (C11), 83.7 , 81.6 , 30.6 (C17), 22.2 , 18.2 (C20). (6) were grown from its saturated MeOH solution by slow evaporation method after 48 h of standing at ambient temperature. X-ray diffraction data of 6 were collected with a Rigaku HighFlux HomeLab™ universal dual wavelength (Mo–Kα and Cu–Kα) single crystal diffractometer at 120(2) K, using the Mo-Kα radiation (λ = 0.71075 Å). The diffractometer was equipped with the Eulerian 3 circle goniometer and the Rigaku Saturn724+ (2 × 2 bin mode) detector. Data reduction and correction of the absorption effect were performed using the XDS software package  containing 7-azaindole or its derivatives (naza) as monodentate N-donor ligands. Many representatives of this type of ruthenium(II) organometallic complexes have been recently reported by several research groups as antitumor active against various human cancer cell lines  complexes (e.g. references [cis-[PtCl2(5Braza)2] exhibited IC50 = 1.8, 2.0 and 0.6 μM against A2780, HOS osteosarcoma, and G-361 malignant melanoma, respectively  [This work aimed to prepare and study the ines see  14–22].–22.in viferences –30) showor CDDP) –29.η6-p-cym)(naza)Cl2] Cl]2 dimer and appropriate naza, as described for analogical [Ru(η6-p-cym)(L)Cl2]  –22. The in CDCl3  and a sip-cymene and 7-azaindole-based ligands were found in 1H  and 13C NMR spectra recorded on CDCl3 solutions of 1–8 (see 1H NMR spectrum of 2 in CDCl3. The spectra did not change in time (recorded after 24 and 48 h), which indicated that 1–8 are stable in chloroform.All the signals of the coordinated  1–8 see  section.1–8. The peaks of the monochlorido [Ru(η6-p-cym)(naza)Cl]+ species were found for 1–6 and 8 (see 8), in the case of 3 the [Ru(η6-p-cym)(3Iaza)Cl]+ peak was detected as overlapped with [Ru(η6-p-cym)(3Iaza)(OCH3)]+ in the 2:1 ratio. Moreover, the peaks of the {[Ru(η6-p-cym)(naza)]–H}+ species were found in the ESI+ mass spectra of 1, 2, 6 and 7. Further, the intensive peaks at 271.0, 507.0, 542.9, 570.9 and 576.9 m/z were observed in the ESI+ mass spectra of 1–8, whose mass and isotopic distribution corresponded to {[Ru(η6-p-cym)Cl]}+, [Ru2(η6-p-cym)2Cl]+, {[Ru2(η6-p-cym)2Cl2]+H}+, [Ru2(η6-p-cym)2Cl2(OCH3)]+ and [Ru2(η6-p-cym)2Cl3]+), i.e. the peaks detected also in the spectra of the starting ruthenium(II) dimer +H}+ corresponding to the free naza species.The ESI+ mass spectra were recorded on MeOH solutions of nd 8 see  for 8, iI) dimer . Finallyη6-p-cym)(2Me4Claza)Cl2] (6) suitable for a single-crystal X-ray analysis were prepared by a diffusion of diethyl ether into the saturated chloroform solution of 6. The molecular and crystal structures are depicted in The crystals of [Ru complexes containing various heterocyclic ligands coordinated through endocyclic sp2 nitrogen heteroatom have been deposited within the Cambridge Structural Database (L)Cl2], where L = N-(aminopropyl)imidazole derivative of lonidamine  [2(μ-ac)4(aza)2]PF6∙CH2Cl2  deposited to date within CSD. Similarly, the Ru–Cl bond lengths of 6 (η6-p-cym)(L)Cl2] complexes. The Ru···Cg distance between the metal centre and centroid of p-cymene aromatic ring (η6-p-cym)(NH3)Cl2] with 1.657 Å [η6-p-cym)(L11)Cl2] with 1.663 Å (L11 = methyl isonicotinate) [p-cymene and 7-azaindole moiety is 29.98(4°.The Ru(II) atom of 4(aza)2]  and [Ru26∙CH2Cl2  containigth of 6  is markeths of 6  fell inttic ring  correlat 1.657 Å  or [Ru(ηotinate) . The dihd(N1···Cl2) = 3.030(2) Å and <(N1–H···Cl2) = 135.36 complexes [η6-p-cym)(L)Cl2] complexes containing lonidamine-modified imidazole ligand (IC50 = 19.3 μM) [50 = 0.2 μM) [H-phenanthridin-6-one (IC50 = 38.8 μM) [N-[3-(imidazol-1-yl)propyl](4-dimethylamino)-1,8-naphthalimide (IC50 = 6.1 μM) }+ and/or {[Ru(η6-p-cym)(3Claza)(H2O)2]}2+, and/or products of their protolysis involving the OH−ligands. To get an evidence for the formation of the mentioned species, ESI mass spectrometry experiments were performed on 2 dissolved in 10% DMF/H2O solution. The obtained spectrum of 2 recorded on the fresh solution in 10% DMF/H2O contained many peaks of ruthenium-containing species, including those of the species detected also in mass spectrum of 2 dissolved in MeOH, namely [Ru2(η6-p-cym)2Cl3]+ (578.9 m/z), [Ru(η6-p-cym)(3Claza)Cl]+ (422.9 m/z), {[Ru(η6-p-cym)(3Claza)]–H}+ (387.0 m/z), [Ru(η6-p-cym)Cl]+ (271.0 m/z) and {(3Claza)+H}+ (153.1 m/z). Apart from those, several new peaks showed in the spectrum of 2 in 10% DMF/H2O as compared with the spectrum of 2 in MeOH. In particular, overlapped peaks of [Ru2(η6-p-cym)2(OH)Cl2]+ and [Ru2(η6-p-cym)2(OH)3Cl]+ , and the peaks of the {[Ru2(η6-p-cym)2Cl]+O}+ (522.1 m/z), [Ru(η6-p-cym)(DMF)Cl]+ (343.9 m/z) and [Ru(η6-p-cym)(H2O)Cl]+ (288.9 m/z) species were observed in the spectra. To sum up, although the above mentioned hydrolytic products, e.g. {[Ru(η6-p-cym)(3Claza)(H2O)Cl]}+ and/or {[Ru(η6-p-cym)(3Claza)(H2O)2]}2+, were not detected, it is evident from the ESI+ mass spectrometry results that the complex 2 is decomposed to various species.The mentioned C13,15–H2O into the DMF-d7 solution of 8 led to the formation of white precipitate which was centrifuged, and the isolated solid was dissolved in DMF-d7 and proved to be free 3I5Braza by 1H NMR experiment. After removing of the precipitate, the 1H NMR spectrum of 8 showed, in total, four pairs of doublets belonging to C13,15–H2/C12,16–H2 detected at 5.51/5.31, 5.64/5.44, 5.95/5.75 and 6.10/5.87 ppm, with the integral intensities being 0.18, 0.15, 1.00, and 1.00, respectively 2Cl3]+, [Ru(η6-p-cym)Cl]+, the overlapped peaks of [Ru2(η6-p-cym)2(OH)Cl2]+ and [Ru2(η6-p-cym)2(OH)3Cl]+, {[Ru2(η6-p-cym)2Cl]+O}+, [Ru(η6-p-cym)(DMF)Cl]+ and [Ru(η6-p-cym)(H2O)Cl]+ (288.9 m/z), indicating analogical behaviour of both the studied complexes 2 and 8 in the used mixture of solvents.Addition of Dectively . No 3I5B3I5Braza . Similard7/D2O (or 10% DMF/H2O) led to the release of naza and the formation of low active or most probably non-potent ruthenium-containing species. Since the used mixture of solvents was similar to that one used for in vitro cytotoxicity testing (0.1% DMF in RPMI-1640 medium), it can be anticipated that similar processes proceeded within the performed biological testing, altogether resulting in inactivity of the studied complexes against the used human cancer cell line.Generally said, dissolving of the studied complexes in 10% DMF-3 , as judged by 1H NMR spectra, we strived to investigate their solution behaviour also in another relevant solvent (i.e. methanol), with a coordination ability of –0.4 +2H}+ and {[Ru2(η6-p-cym)2Cl]+2H}+ , [Ru2(η6-p-cym)2Cl2]+ and [Ru2(η6-p-cym)2(OCH3)Cl]+ , and [Ru2(η6-p-cym)2Cl3]+, [Ru2(η6-p-cym)2(OCH3)Cl2]+ and [Ru2(η6-p-cym)2(OCH3)2Cl]+ , as well as the peaks of [Ru(η6-p-cym)(3Claza)Cl]+ (422. η6-p-cym)(3Claza)]–H}+ (387.0 m/z), [Ru(η6-p-cym)Cl]+ (271.0 m/z) and {(3Claza)+H}+ (153.1 m/z). However, some additional peaks were also detected in the spectra of 2 in 10% MeOH/H2O solution as compared with the spectra detected in the MeOH solution. The additional peaks most probably belong, according to their mass and isotopic patterns, to [Ru2(η6-p-cym)2(OH)]+ (488.1 m/z), overlapped [Ru2(η6-p-cym)2(OCH3)(O)]+ and [Ru2(η6-p-cym)2Cl(O)]+ , and {[Ru2(η6-p-cym)2Cl2(O2)]+2H}+ (577.0 m/z) (2(η6-p-cym)2(OH)]+ (488.1 m/z), [Ru2(η6-p-cym)2(OCH3)]+ (503.0 m/z), [Ru2(η6-p-cym)2(OCH3)(O)]+ (518.9 m/z), [Ru2(η6-p-cym)2(OCH3)3]+ (565.0 m/z) and {(3I5Braza)+H}+ (322.8 m/z) species were detected in the fresh 10% MeOH/H2O solution of 8 with no change after 24 and 48 h.It can be concluded that, as in the case of 10% DMF-7.0 m/z) . No chan2 or 8, several new peaks were found after the addition of water . An integral intensity of the signals of the Ru–SG adduct was not consistent with the above mentioned p-cymene signals, proving that the Ru–SG adduct does not contain neither p-cymene nor 3Claza ligand. Interestingly, a ratio of Cys-α and Cys-β signals of free and coordinated glutathione  changed to ca 1: 1 after 48 h of standing at the ambient temperature, meaning that the formed Ru–SG adduct is unstable in the used mixture of solvents, which is connected with a release of GSH.ure data . With re1H NMR spectra recorded on 10% MeOD-d4/D2O solutions of the mixture of 8 with GSH did not contain any signals in the naza and p-cymene region and only the signals of the used biomolecules were detected. The set of signals, detected at 4.49/2.86 ppm and 4.55/2.77 ppm ppm for 2, were also detected in the spectra of the fresh solution of 8 in 10% MeOD-d4/D2O. However, an integral intensity ratio (9: 1) was found to be inverse as compared with 2, and did not change in time, which showed that equilibrium between free GSH and Ru–SG adduct is reached rapidly. Again, the Ru–SG adduct does not contain neither 3Claza nor p-cymene ligands.1H NMR studies proved that interactions of 2 and 8 with GSH led to the formation of Ru–SG adduct and to a complete release of naza from the structures of 2 and 8. These results are consistent with a detection of various adducts with GSH in the ESI+ mass spectra of the studied complexes dissolved in 10% MeOH/H2O. Concretely, the peaks, whose mass and isotopic pattern correspond to [Ru(η6-p-cym)(GS)]+ (542.1 m/z), {[Ru2(η6-p-cym)2(GS)2]–H}+ (1082.7 m/z) and [Ru2(η6-p-cym)2(GS)3]+ (1390.2 m/z) formed by an interaction of GSH with 2  ,37. On t2, 5 or 8, the concentration-dependent fluorescence quenching of EtBr was observed, showing on the ability of the studied complexes or their decomposition/solvolysis product/s to interact with the used ctDNA (5); note: 5 was used together with the representative complexes 2 and 8, because 5Faza contained in its structure showed markedly higher fluorescence as compared with 3Claza  and 3I5Braza . Except the fluorescence maximum of EtBr/ctDNA adduct detected at 615 nm, another peak showed in the obtained fluorescence spectra of 2 (at 415 nm) and 5  complexes ed ctDNA  for 5; ninset in , which pη6-p-cym)(naza)Cl2] complexes  were prepared and thoroughly characterized by relevant techniques including a crystallographic study of [Ru(η6-p-cym)(2Me4Claza)Cl2] (6) showing a piano-stool arrangement with N-donor ligand coordination through its N7 atom. The complexes were studied for their anticancer activity against the A2780 human cancer cell line, however, no cytotoxicity was found up to the tested concentration of 50 μM. That is why we were looking for the reason of the inactivity. Thus, the complexes were studied for their stability in various solvents . These studies revealed the complexes to be highly unstable, because besides a rapid solvolysis they decompose to the starting ruthenium(II) compound, [Ru(μ-Cl)(η6-p-cym)Cl]2 and/or its solvolysis products, which is connected with a release of naza. With respect to instability of the studied complexes in the solvents used, it may be concluded that the anticancer inactivity of the compounds is associated with this property and formation of inactive species (starting ruthenium(II) dimer, released naza, solvolysis products). It has to be noted that such findings are rather unexpected because the literature data clearly reveal that complexes of a general formula [Ru(η6-p-cym)(L)Cl2] should show anticancer activity, as can be seen from references + and overlapped [Ru2(η6-p-cym)2(OCH3)(O)]+ and [Ru2(η6-p-cym)2Cl(O)]+ (1:1 ratio) species (middle) and their calculated simulations (down).The spectrum (depicted at 400–600 (TIF)Click here for additional data file.S7 Fig2O solution of 2 mixed with one molar equivalent of GSH.The spectrum was acquired on the 10% MeOH/H(TIF)Click here for additional data file.S8 Fig(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 Text(PDF)Click here for additional data file.


Unfortunately the original version of this article  containeThe full author list should read:1,2, Ling-Ling Cui1,2, Lin Han1,2, Can Wang1,2, Zhi-Jian Song3, Jia-Wei Shen3, Zhi-Qiang Li3, Jian-Hua Chen3, Zu-Jia Wen3, Xiao-Min Wang1,2, Yong-Yong Shi3*and Chang-Gui Li1,2*Zhao-Wei Zhou*Corresponding authors: Chang-Gui Li licgqd@163.com and Yong-Yong Shi yongyongshi@gmail.com.


The structured phospholipids were obtained in high yields (59–87%) and evaluated in vitro for their cytotoxic activity against several cancer cell lines of different origin: MV4-11, A-549, MCF-7, LOVO, LOVO/DX, HepG2 and also towards non-cancer cell line BALB/3T3 . The phosphatidylcholines modified with monoterpene acid showed a significantly higher antiproliferative activity than free monoterpene acids. The highest activity was observed for the terpene-phospholipids containing the isoprenoid acids in sn-1 position of phosphatidylcholine and palmitic acid in sn-2.The synthesis of novel phosphatidylcholines with geranic and citronellic acids in  Isoprenoids are one of the most widely occurring group of natural compounds. Many of them exhibit wide range of biological activities. Among documented useful properties of isoprenoids the following activities need to be mentioned: fungicidal, antibacterial, antiviral and anti-inflammatory –4. The rIn recent years there has been also a rapid progress in the field of bioengineering and phospholipid technology ,9. Many Keeping in view of the properties of isoprenoids and phospholipids the production of novel hybrid molecules with the combination of these two biologically important products could result in bioactive analogues—terpene-phospholipids, which could be characterized by enhanced biological activities. It has been reported that phospholipid modifications of drugs is the method of modulation their polarity, to prevent degradation and enhance the biological activity of molecules attached to the PLs –20.cis/trans isomers; 23:67) (1a) and racemic citronellic acid (1b) were purchased from Sigma–Aldrich Chemical Co. Phospholipase A2  was a gift from Novozymes. The enantiomerically pure GPC was purchased from Bachem and converted to the cadmium chloride complex (GPC × CdCl2) using the method described elsewhere. All of the solvents used in liquid chromatography were of HPLC grade (LiChrosolv Reagents) and were purchased from Merck.Geranic acid (mixture of 254 plates (Merck) with mixture of chloroform: methanol: water  as developing system. Products were detected by spraying the plates with a solution of 10 g of Ce(SO4)2 and 20 g of phosphomolybdic acid in 1 L of 10% H2SO4 followed by heating or 0.05% primuline solution  followed by UV (365 nm). Column chromatography was performed on silica gel ). 1H, 13C, and 31P NMR spectra were recorded using a Bruker Advance II 600 MHz spectrometer. Chemical shifts are given in ppm downfield from tetramethylsilane (TMS) as the internal standard. In 31P NMR spectra, chemical shifts were referenced to 85% H3PO4 as a standard. Coupling constant (J) values are given in hertz. Elemental analyses were performed on Waters ESI-Q-TOF Premier XE spectrometer using electron spray ionization (ESI) technique.Analytical TLC was performed on silica gel 60, F2 pressure was 0.24 MPa. The system was controlled and data acquisition was carried out using the Chromeleon 6.80 software (Dionex Corporation). HPLC analysis was carried out using a Betasil DIOL 5-μm column .The purity of synthesized products were monitored by HPLC using an Ultimate 3000 DIONEX chromatograph equipped with a DGP-3600A dual-pump fluid control module, a TCC-3200 thermostatted column compartment, and a WPS-3000 autosampler. A Corona charged aerosol detector (CAD) from ESA Biosciences was used. The acquisition range 100 pA, digital filter set to none, the N-1 with solvent A = 1% HCOOH, 0.1% triethylamine (TEA) in water, B = hexane, and C = propan-2-ol. Gradient time: 3/40/57 (%A/%B/%C), at 4 min 10/40/50, at 9 min 10/40/50, at 9.1 min 3/40/57 and at 19 min 43/40/57. The total time of analysis was 19 min. The injection volume was 15 μL in all of the experiments and the cooling temperature for the samples was 20°C.The column temperature was maintained at 30°C. The gradient had a constant flow rate 1.5 mL minsn-glycero-3-phosphocholine (GPC)  in methanol (65 mL) was added to a solution of cadmium chloride (CdCl2 × 21/2H2O)  in cold water (30 mL). A heavy white precipitate appeared and was stored at 0°C for 4 h. The precipitate (GPC × CdCl2) was next filtered, lyophilized and then dried in an Abderhalden’s drying pistol over P2O5 at the boiling temperature of acetone to give 15.65 g of white powder in 91% yield.The solution of sn-1 and sn-2. Geranic acid (1a) or citronellic acid (1b) (0.92 mmol) was dried by repeated evaporation with an anhydrous CH2Cl2 and then dissolved in anhydrous CH2Cl2 (1 mL). To the 100 mg (0.23 mmol) of GPC × CdCl2 complex added 56 mg (0.46 mmol) of DMAP and the solution of 200 mg of DCC (0.97 mmol) in 6 mL of the anhydrous CH2Cl2. The reaction flask was flushed with dry N2 and next tightly sealed. The mixture was stirred at room temperature under protection from light for 72 h. After this time (TLC) the formed during the reaction course precipitate was removed using a Schott funnel. The solution was mixed with ion-exchange resin  for 30 min to dislodge the cadmium chloride and DMAP. DOWEX was filtered off and the solvent was evaporated under vacuum. The crude PC was purified by silica gel column chromatography ). Fractions containing products (Rf 0.3) were collected and evaporated under vacuum (45°C) to afford 1,2-diisoprenoyl-sn-glycero-3-phosphocholine 3 as a mixture of two stereoisomers.The cadmium complex of GPC has been acetylated with isoprenoid acids in positions 1H NMR ), δ: 1.29 , CH3-9sn-1 (B), CH3-9sn-2 (A), CH3-9sn-2 (B)), 1.36 , CH3-8sn-1 (B), CH3-10sn-2 (A), CH3-10sn-2 (B)), 1.81–1.85 , CH2-4sn-1 (B), CH2-5sn-2 (A), CH2-5sn-2 (B)), 2.91 3 (A), N(CH3)3 (B)), 3.32–3.35 , CH2-β (B)), 3.70–3.77 , CH2-3' (B)), 3.91 , one of CH2-1' (B)), 3.96–4.01 , CH2-α (B), 4.06 , one of CH2-1' (B), 4.80 , H-6sn-2 (B)), 4.84 , H-6sn-2 (A)), 4.94 , H-2' (B)), 5.35 , H-2sn-1 (B), H-2sn-2 (A), H-2sn-2 (B)); 13C NMR ) δ: 15.39, 15.44, 15.46, 15.49, 24.70, 24.74, 24.88 (C-8sn-1 (A), C-8sn-1 (B), C-8sn-2 (A), C-8sn-2 (B), C-10sn-1 (A), C-10sn-1 (B), C-10sn-2 (A), C-10sn-2 (B)), 16.88, 16.89, 16.93, 16.95 (C-9sn-1 (A), C-9sn-1 (B), C-9sn-2 (A), C-9sn-2 (B)), 25.58, 25.59, 26.26, 26.28 (C-5sn-1 (A), C-5sn-1 (B), C-5sn-2 (A), C-5sn-2 (B)), 40.52, 40.54, 40.55, 40.57 (C-4sn-1 (A), C-4sn-1 (B), C-4sn-2 (A), C-4sn-2 (B)), 53.65 3 (A), -N(CH3)3 (B)), 58.59 , C-α (B)), 61.73 (C-1' (A), C-1' (B)), 63.24 , C-3' (B)), 66.10 , C-β (B)), 70.28 , C-2' (B)), 114.49, 114.60, 115.00, 115.10 (C-2sn-1 (A), C-2sn-1 (B), C-2sn-2 (A), C-2sn-2 (B)), 121.85, 121.88, 122.35, 122.37 (C-6sn-1 (A), C-6sn-1 (B), C-6sn-2 (A), C-6sn-2 (B)), 128.11, 128.15 (C-7sn-1 (A), C-7sn-1 (B), C-7sn-2 (A), C-7sn-2 (B)), 131.44, 132.13 (C-3sn-1 (A), C-3sn-1 (B), C-3sn-2 (A), C-3sn-2 (B)), 161.05, 161.19, 165.69, 165.78 (C-1sn-1 (A), C-1sn-1 (B), C-1sn-2 (A), C-1sn-2 (B)); 31P NMR ) δ: -0.63; HRMS (ESI): m/z calcd. for C28H48NO8P [M + H]+ 558.3196; found 558.3217Colourless greasy solid ); 1H NMR ), δ: 0.70, 0.71 , CH3-9sn-1 (B), CH3-9sn-2 (A), CH3-9sn-2 (B)), 0.99 , one of CH2-4sn-1 (B), one of CH2-4sn-2 (A), one of CH2-4sn-2 (B)), 1.12 , one of CH2-4sn-1 (B), one of CH2-4sn-2 (A), one of CH2-4sn-2 (B)), 1.36, 1.44 , CH3-8sn-1 (B), CH3-10sn-1 (A), CH3-10sn-1 (B), CH3-8sn-2 (A), CH3-8sn-2 (B), CH3-10sn-2 (A), CH3-10sn-2 (B)), 1.66–1.81 , CH2-5sn-1 (B), CH2-5sn-2 (A), CH2-5sn-2 (B), H-3sn-1 (A), H-3sn-1 (B), H-3sn-2 (A), H-3sn-2 (B)), 1.86–1.95 , one of CH2-2sn-1 (B), one of CH2-2sn-2 (A), one of CH2-2sn-2 (B)), 2.07–2.14 , one of CH2-2sn-1 (B), one of CH2-2sn-2 (A), one of CH2-2sn-2 (B)), 2.99 3 (A), N(CH3)3 (B)), 3.38–3.39 , CH2-β (B)), 3.77 , CH2-3' (B)), 3.92 , one of CH2-1' (B)), 4.03 , CH2-α (B)), 4.19 , one of CH2-1' (B)), 4.84 , H-6sn-1 (B), H-6sn-2 (A), H-6sn-2 (B)), 5.00 , H-2' (B)); 13C NMR ) δ: 16.91, 24.96 (C-8sn-1 (A), C-8sn-1 (B), C-8sn-2 (A), C-8sn-2 (B), C-10sn-1 (A), C-10sn-1 (B), C-10sn-2 (A), C-10sn-2 (B)), 18.77, 18.78. 18.85 (C-9sn-1 (A), C-9sn-1 (B), C-9sn-2 (A), C-9sn-2 (B)), 24.88 (C-5sn-1 (A), C-5sn-1 (B), C-5sn-2 (A), C-5sn-2 (B)), 29.49, 29.53, 29.56 (C-3sn-1 (A), C-3sn-1 (B), C-3sn-2 (A), C-3sn-2 (B)), 36.20, 36.23 (C-4sn-1 (A), C-4sn-1 (B), C-4sn-2 (A), C-4sn-2 (B)), 40.99, 41.15, 41.16 (C-2sn-1 (A), C-2sn-1 (B), C-2sn-2 (A), C-2sn-2 (B)), 53.58 3 (A), -N(CH3)3 (A)), 58.75 , C-α (B)), 62.21 (C-1' (A), C-1' (B)), 63.27 , C-3' (B)), 65.93 , C-β (B)), 69.89, ), 69.91 ), 123.64 (C-6sn-1 (A), C-6sn-1 (B), C-6sn-2 (A), C-6sn-2 (B)), 131.09 (C-7sn-1 (A), C-7sn-1 (B), C-7sn-2 (A), C-7sn-2 (B)), 172.55, 172.56, 172.97 (C-1sn-1 (A), C-1sn-1 (B), C-1sn-2 (A), C-1sn-2 (B)); 31P NMR ) δ: -0.79; HRMS (ESI): m/z calcd. for C28H52NO8P [M + H]+ 562.3509; found 562.3530Colourless greasy solid ); sn-glycero-3-phosphocholine (5) was carried out according to the method described by Morgado et al. + 646.4448; found 646.4451Colourless greasy solid ); 1H NMR ), δ: 0.64 14C(O) (A), CH3(CH2)14C(O) (B)), 0.72 , CH3-9 (B)), 0.96–1.05 12CH2CH2C(O) (A), CH3(CH2)12CH2CH2C(O) (B), one of CH2-4 (A), one of CH2-4 (B)), 1.12 , one of CH2-4 (B)), 1.31–1.39 13CH2CH2C(O) (A), CH3(CH2)13CH2CH2C(O) (B)), 1.36, 1.44 , CH3-8 (B), CH3-10 (A), CH3-10 (B)), 1.65–1.81 , CH2-5 (B), H-3 (A), H-3 (B)), 1.87–1.93 , one of CH2-2 (B)), 2.07 13CH2C(O) (A), CH3(CH2)13CH2C(O) (B)), 2.09–2.15 , one of CH2-2 (B)), 2.99 3 (A), -N(CH3)3 (B)), 3.39 , CH2-β (B)), 3.77 , CH2-3’ (B), 3.92 , one of CH2-1' (B)), 4.03 , CH2-α (B)), 4.17 , one of CH2-1' (B)), 4.84 , H-6 (B)), 5.00 , H-2' (B)); 13C NMR ) δ: 13.44 (CH3(CH2)14C(O) (A), CH3(CH2)14C(O) (B)), 16.97, 25.02 (C-8 (A), C-8 (B), C-10 (A), C-10 (B)), 18.93 (C-9 (A), C-9 (B)), 22.20 (CH3CH2(CH2)13C(O) (A), CH3CH2(CH2)13C(O) (B)), 24.45 (CH3(CH2)12CH2CH2C(O) (A), (CH3(CH2)12CH2CH2C(O) (B)), 24.94 (C-5 (A), C-5 (B)), 28.67, 28.87, 28.89, 29.06, 29.19, 29.21, 29.23, 31.47 (CH3CH2(CH2)11CH2CH2C(O) (A), CH3CH2(CH2)11CH2CH2C(O) (B)), 29.55, 29.56 (C-3 (A), C-3 (B)), 33.78 (CH3(CH2)13CH2C(O) (A), CH3(CH2)13CH2C(O) (B)), 36.28 (C-4 (A), C-4 (B)), 41.06 (C-2 (A), C-2 (B)), 53.66 3 (A), -N(CH3)3 (B)), 58.79 , C-α (B)), 62.12, 62.14 (C-1' (A), C-1' (B)), 63.39 , C-3' (B)), 65.00 , C-β (B)), 69.95 , C-2' (B)), 123.68 (C-6 (A), C-6 (B)), 131.13 (C-7 (A), C-7 (B)), 172.95 (C-1 (A), C-1 (B)), 173.19 (CH3(CH2)13CH2C(O) (A), CH3(CH2)13CH2C(O) (B); 31P NMR ) δ: -0.83; HRMS (ESI): m/z calcd. for C34H66NO8P [M + H]+ 648.4604; found 648.4615Colourless greasy solid ); sn-glycero-3-phosphocholine  was carried out according to the procedure described by Niezgoda et al. + 408.2151; found 408.2175Colourless greasy solid ); 1H NMR ), δ: 0.72 , CH3-9 (B)), 0.96–1.15 , CH2-4 (B)), 1.37, 1.45 , CH3-8 (B), CH3-10 (A), CH3-10 (B)), 1.70–1.82 , CH2-5 (B), H-3 (A), H-3 (B)), 1.93 , one of CH2-2 (B)), 2.14 , one of CH2-2 (B)), 2.99 3 (A), -N(CH3)3 (B)), 3.37–3.39 , CH2-β (B)), 3.62–3.73 , CH2-3' (B)), 3.74–3.77 , H-2' (B)), 3.87–3.95 , CH2-1' (B)), 4.02–4.04 , CH2-α (B)), 4.85 , H-6 (B)); 13C NMR ) δ: 16.92, 24.98 (C-8 (A), C-8 (B) C-10 (A), C-10 (B)), 18.86 (C-9 (A), C-9 (B)), 24.91 (C-5 (A), C-5 (B)), 29.53 (C-3 (A), C-3 (B)), 36.30 (C-4 (A), C-4 (B)), 41.09 (C-2 (A), C-2 (B)), 53.61 3 (A), -N(CH3)3 (B), 58.62 , C-α (B)), 64.49 (C-1' (A), C-1' (B)), 66.01 , C-β (B)), 66.47 , C-3' (B)), 68.30 , C-2' (B)), 123.72 (C-6 (A), C-6 (B)), 131.08 (C-7 (A), C-7 (B)), 173.39 (C-1 (A), C-1 (B)); 31P NMR ) δ: -0.03; HRMS (ESI): m/z calcd. for C18H36NO7P [M + H]+ 410.23075; found 410.2311Colourless greasy solid ); 10a and 10b were prepared from 1-isoprenoyl-2-hydroxy-sn-glycero-3-phosphocholines  (0.14 mmol) by adding palmitic acid (0.56 mmol) dissolved in 2 mL of anhydrous CH2Cl2, DMAP (0.28 mmol) and finally a solution of DCC (0.58 mmol) in 3 mL of CH2Cl2. The reaction was carried out for 72 h under a nitrogen atmosphere in the dark at 40°C. The product was extracted according to the method described for compounds 3a, 3b and pure 1-isoprenoyl-2-palmitoyl-sn-glycero-3-phosphocholine (10) was obtained as the mixture of two stereoisomers.Compounds 1H NMR ) δ: 0.63 14C(O) (A), CH3(CH2)14C(O) (B)), 1.03–1.04 12CH2CH2C(O)), 1.35 13CH2CH2C(O) (A), CH3(CH2)13CH2CH2C(O) (B)), 1.36 , CH3-9 (B)), 1.43 , 1.86–1.92 , CH2-4 (B), CH2-5 (A), CH2-5 (B)), 2.07 13CH2C(O) (A), CH3(CH2)13CH2C(O) (B)), 2.98 3 (A), -N(CH3)3 (B),), 3.36–3.39 , CH2-β (B)), 3.77 , CH2-3' (B)), 3.94 , one of CH2-1' (B)), 4.00–4.05 , CH2-α (B)), 4.18 , one of CH2-1' (B)), 4.83 . H-6 (B)), 4.96–5.03 , H-2' (B)), 5.41 , H-2 (B)); 13C NMR ) δ: 13.38 (CH3CH2(CH2)11CH2CH2C(O) (A), CH3CH2(CH2)11CH2CH2C(O) (B)), 16.95, 16.97 (C-9 (A), C-9 (B)), 22.16 (CH3CH2(CH2)11CH2CH2C(O) (A), CH3CH2(CH2)11CH2CH2C(O) (B)), 24.38, 24.43 (CH3CH2(CH2)11CH2CH2C(O) (A), CH3CH2(CH2)11CH2CH2C(O) (B)), 24.92 (C-8 (A), C-8 (B), C-10 (A), C-10 (B)), 25.55 (C-5 (A), C-5 (B)), 28.59, 28.63, 28.82, 28.85, 29.01, 29.14, 29.16, 29.18 (CH3CH2(CH2)11CH2CH2C(O) (A), CH3CH2(CH2)11CH2CH2C(O) (B)), 33.70, 33.76 (CH3(CH2)13CH2C(O) (A), CH3(CH2)13CH2C(O) (B)), 40.54, 41.13 (C-4 (A), C-4 (B)), 53.57 3 (A), -N(CH3)3 (A)), 58.69 , C-α (B)), 62.50 (C-1' (A), C-1' (B)), 63.17 ), 63.37 ), 65.94 , C-β (B)), 69.85 ), 70.08 ), 113.31, 114.32 (C-2 (A), C-2 (B)), 122.32, 123.03 (C-6 (A), C-6 (B)), 131.48, 132.09 (C-7 (A), C-7 (B)), 141.46 (C-3 (A), C-3 (B)), 161.13, 166.16 (C-1 (A), C-1 (B)), 173.13, 173.23 (CH3CH2(CH2)11CH2CH2C(O) (A), CH3CH2(CH2)11CH2CH2C(O) (B)); 31P NMR ) δ: 0.83; HRMS (ESI): m/z calcd. for C34H64NO8P [M + H]+ 646.4448; found 646.4474Colourless greasy solid ); 1H NMR ) δ: 0.64 14C(O) (A), CH3(CH2)14C(O) (B)), 0.70 , CH3-9 (B)), 0.90–1.34 12CH2CH2C(O) (A), CH3(CH2)12CH2CH2C(O) (B), one of CH2-4 (A), one of CH2-4 (B)), 1.13 , one of CH2-4 (B)), 1.35–1.40 13CH2CH2C(O) (A), CH3(CH2)13CH2CH2C(O) (B)), 1.36, 1.44 , CH3-8 (B), CH3-10 (A), CH3-10 (B)), 1.68–1.78 , CH2-5 (B), H-3 (A), H-3 (B)), 1.87–1.92 , one of CH2-2 (B)), 2.06–2.12 , one of CH2-2 (B)), 2.09 13CH2C(O) (A), CH3(CH2)13CH2C(O) (B)), 2.99 3 (A), -N(CH3)3 (B)), 3.39–3.40 , CH2-β (B)), 3.78 , CH2-3’ (B), 3.93 , one of CH2-1' (B)), 4.01–4.04 , CH2-α (B)), 4.20 , one of CH2-1' (B)), 4.84 , H-6 (B)), 4.99 , H-2' (B)); 13C NMR ) δ: 13.61 (CH3(CH2)14C(O) (A), CH3(CH2)14C(O) (B)), 17.14, 25.19 (C-8 (A), C-8 (B), C-10 (A), C-10 (B)), 18.94, 18.97 (C-9 (A), C-9 (B)), 22.31 (CH3CH2(CH2)13C(O) (A), CH3CH2(CH2)13C(O) (B)), 24.49 (CH3(CH2)12CH2CH2C(O) (A), (CH3(CH2)12CH2CH2C(O) (B)), 25.03 (C-5 (A), C-5 (B)), 28.78, 28.94, 28.99, 29.14, 29.27, 29.29, 29.30, 29.32, 31.57 (CH3CH2(CH2)11CH2CH2C(O) (A), CH3CH2(CH2)11CH2CH2C(O) (B)), 29.69, 29.71 (C-3 (A), C-3 (B)), 33.72 (CH3(CH2)13CH2C(O) (A), CH3(CH2)13CH2C(O) (B)), 36.34, 36.36 (C-4 (A), C-4 (B)), 41.30 (C-2 (A), C-2 (B)), 53.81 3 (A), -N(CH3)3 (B)), 58.86 , C-α (B)), 62.41, 62.43 (C-1' (A), C-1' (B)), 63.42 , C-3' (B)), 66.09 , C-β (B)), 69.95 ), 69.96 ), 123.76 (C-6 (A), C-6 (B)), 131.27 (C-7 (A), C-7 (B)), 172.66, 172.68 (C-1 (A), C-1 (B)), 173.71 (CH3(CH2)13CH2C(O) (A), CH3(CH2)13CH2C(O) (B)); 31P NMR ) δ: -0.88; HRMS (ESI): m/z calcd. for C34H66NO8P [M + H]+ 648.4604; found 648.4630Colourless greasy solid ); in vitro, human cancer cell lines: MV4-11 (human biphenotypic B myelomonocytic leukaemia), A-549 , MCF-7 (breast cancer), LoVo (colon cancer), LoVo/DX (colon cancer drug resistant), HepG2 (liver cancer) and BALB/3T3  were used.Established All lines were obtained from American Type Culture Collection . The cell line is being maintained in the Institute of Immunology and Experimental Therapy, Wroclaw, Poland.2 humidified atmosphere.The leukaemia cell line was cultured in RPMI 1640 medium  with supplemented with 2 mM L-glutamine adjusted to contain 1.0 mM sodium pyruvate, 10% fetal bovine serum . A549, LoVo and LoVo/DX cells were cultured in the mixture of RPMI 1640+Opti-MEM (1:1) , MCF-7 cells in Eagle medium , HepG2 in DMEM medium , BALB/3T3 in DMEM medium  supplemented with 2 mM L-glutamine and 1.0 mM sodium pyruvate, 10% fetal bovine serum . The MCF-7 cell culture was supplemented with 0.8 mg/L of insulin  and the LoVo/DX cell culture was supplemented with 0.1 μg/mL of doxorubicin (Accord). All culture media were supplemented with 100 units/mL penicillin, and 100 μg/mL streptomycin . All cell lines were grown at 37°C with 5% COPrior to usage, the compounds were dissolved in DMSO or in mixture of 99.8% ethanol and DMSO (1:1) to the concentration of 25 or 50 mM, and subsequently diluted in culture medium to reach the required concentrations (ranging from 5 to 625 μM).4 cells per well. An assay was performed after 72 hours exposure to varying concentrations of the tested agents.24 hours before addition of the tested compounds, the cells were plated in 96-well plates at density of 1×10Cytotoxic test MTT was applied for the cytotoxicity screening against leukaemia cells growing in suspension culture and were carried out according to the method described before . Cytotox50 (inhibitory concentration 50)–the concentration of tested agent, which inhibits proliferation of 50% of the cell population. IC50 values were calculated for each experiment separately and mean values ± SD is presented in the The results were calculated as an IC50 values, the resistance indexes (RI) were calculated by dividing the IC50 values of the compounds tested against the cells of drug resistant cell LOVO/DX line by respective values obtained against the cells of drug sensitive LoVo line. According to Harker et al. [Using the obtained ICr et al.  three caStatistical analysis was performed using STATISTICA version 10 . Mann-Whitney U Test was used in the analysis and the results in sn-glycero-3-phosphocholine (DOPC) with terpene alcohols  (1a) or cytronellic acid (CA) (1b) in the sn-1 and sn-2 positions. In second variant, GA or CA was attached to the sn-2 position whereas in sn-1 position saturated fatty acid  was introduced as it is observed in natural phospholipids. Third group of phospholipids possessed geranic or cytronellic acid residue in sn-1 position and palmitic acid in sn-2 position.One of the most widely studied phospholipids is phosphatidylcholine (PC). Yamamoto proposed the transphosphatidylation of 1,2-dioleoyl-, phytol ,20 as th, phytol ,20. Prom, phytol ,28. Instminicola ,30. Its minicola . The bio1a) or citronellic acid (1b) in the sn-1 and sn-2 positions  was obtained by known Steglich esterification method [sn-glycero-3-phosphocholine (GPC×CdCl2) (2) was acylated with terpene acid (TA) 1a and 1b in the presence of 4-pyridine (DMAP) and a coupling agent N,N’-dicyclohexylcarbodiimide (DCC). All the reagents and solvent (dichloromethane) were freshly dried just before reaction to avoid the hydrolysis of DCC to dicyclohexylurea. In described condition using the molar ratio 1: 4: 2: 4.2 of GPC/TA/DMAP/DCC, 1,2-digeranoyl-sn-glycero-3-phosphocholine (3a) and 1,2-dicitroneloyl-sn-glycero-3-phosphocholine (3b) were obtained in 82% and 86% yields respectively after 72 hours of reaction  (sn-3-glycero-phosphocholine was converted to the 2-lysophospholipid (6) by treatment with phospholipase A2. Subsequent reacetylation of 2-OH group of known 1-palmitoyl LPC by isoprenoid acids yielded the mixed diacyl phosphatidylcholines  [The synthesis of asymmetrically substituted phospholipids containing terpene acids (TA) in the cid (PA) . Then 1, .sn-glycero-3-phosphocholine by isoprenoid acids proceeded very slowly  in the reaction mixture was on the level of 58–84%. We decided to use then the higher molar excess of DCC and DMAP in relation of 1-palmitoyl-2-lysophospholipids and isoprenoid acids and increase temperature of reaction to 40°C which resulted the full conversion of substrate after 72 h of the reaction.The time-course of esterification of 1-palmitoyl-2-hydroxy-y slowly . We prol7a and 7b were established based on their spectral data. The 1-palmitoyl-2-geranoyl-sn-glycero-3-phosphocholine (7a) was synthesized earlier by Biodrowska in 27% yield but in our investigations using higher ratio of DCC and DMAP resulted in significantly higher yield (59%) [sn-glycero-3-phosphocholine (7b) has not been reported in the literature and was obtained in 62% yield.The structures of obtained products ld (59%) . The 1-psn-glycero-3-phosphocholines is a task requiring regioselective incorporation of different acyl donors in two positions of the GPC backbone. The literature presents several useful methods for this type of mixed-chain phosphatidylcholines synthesis [sn-glycero-3-phosphocholine. Initially, we applied chemoenzymatic strategy similar to that applied in the synthesis of compounds 7a and 7b. It was based on the hydrolysis of 1,2-diacyl-sn-glycero-3-phosphocholines  obtained previously (Scheme 1) and subsequent reesterification of 1-acyl LPC with palmitic acid (PA) using DCC and DMAP. Unfortunately the yields of hydrolysis of terpene-PLs carried out with the phospholipase A2 (PLA2) were very low and we decided to use tin-mediated mono-functionalization of GPC described by D’Arrigo [in situ using the procedure described by Mattson et al. [sn-glycero-3-phosphocholines  with 75% and 87% yields respectively. The last step was acylation of 9a and 9b with palmitic acid (PA) in the presence of DCC and DMAP, affording two new products 10a and 10b in 67 and 70% yields respectively. The structures of the final products were confirmed by their spectroscopic data.The synthesis of 1-isoprenoyl-2-palmitoyl-ynthesis ,24,37. ID’Arrigo . As it in et al. . We usedn et al. , for the3a, 3b, 7a, 7b, 9a, 9b, 10a, 10b) were obtained as the mixture of diastereoisomers. In the case of phospholipids containing geranic acid  it was the results of composition of cis/trans (23:67) isomers of starting isoprenoid. In the case of phosphatidylcholines modified with citronellic acid  the formation of two diastereoisomers (50:50) was caused by introduction of racemic citronellic acid into optically pure sn-glycero-3-phosphocholine (GPC).All synthesized products  were tested for their antiproliferative activity towards human cancer cell lines, such as MV4-11 (human leukaemia), A549 (lung cancer), MCF-7 (breast cancer), HepG2 (liver cancer), LoVo (colon cancer) and also against doxorubicin-resistant colon cancer LoVo/DX . The experiments of cytotoxicity were also performed towards BALB/3T3 . The data for the in vitro anticancer activity (50 –concentration of the compound (in μM) that inhibits proliferation of cells by 50% compared to the untreated control cells. The results were compared with the activity of free terpene acids (TA). Cisplatin and doxorubicin hydrochloride (for LoVo and LoVo/Dx cells) were used as a positive control.All terpene–modified PCs  containing terpene acids (TA) in the sn-1 and sn-2 position possessed the antiproliferative activity 2-3-fold higher than free acids. These compounds stronger than corresponding acids inhibit the proliferation of cancer cells. Free terpene acids  did not showed any reduction of cells growth of A549, HepG2 even at 625 μM. The highest activity was observed for mixed-chain PC . Their IC50 values were more than 9-fold lower than those determined for free terpene acids 1a, 1b towards leukaemia, LoVo, LOVO/DX and breast cell lines. For cells of lung cancer and HepG2 lines the activity was exhibited only after introduction of terpene acids into PC. Among eight tested terpene-PCs compound 10a showed the strongest antiproliferative effect towards all tested cell lines. Very high antiproliferative activity was demonstrated against leukaemia and colon cancer LoVo cells . During the experiments we observed also that tested compounds exhibit activity against normal mice fibroblasts (BALB/3T3). However it is worth to notice that cytotoxic activity of compounds 7a, 7b, 10a and 10b against BALB/3T3 cells was in most cases lower in comparison to cytotoxicity exhibited against cancer cell lines, which is very promising in contest of potential application of tested compounds as the anticancer drugs. Generally, activity of terpene-modified PC increased in the following sequence: 1-isoprenoyl-LPC < diisoprenoyl-PC < monoisoprenoyl-PC.The aim of this study was to determine whether a joining the terpenes with PC has impact on their biological activity. Increasing the lipophilicity of active compounds was expected to raise their cytotoxicity. We observed that almost all synthesized compounds were cytotoxic against tested cancer cell lines. The exception is compound 50 values of the compounds tested against the cells of drug resistant cell LoVo/DX by respective values obtained against the drug sensitive LoVo cell line .We calculated also the resistance indexes (RI) by dividing the ICell line . All cometc.) influences on the activity of whole molecule. These studies are in progress. The aim of further work is also to elucidate the mechanism of the cytotoxicity terpene-modified PCs.In conclusion, all novel phospholipids containing the isoprenoid residue of geranic and citronellic acid in the non-polar part of phosphatidylcholine were synthesized in good yields (59–87%). It is the first report on the evaluation of anticancer activity of geranic and citronellic acid in the free form and after incorporation into PC. The results of cytotoxic studies confirmed that phospholipid modifications with geranic and citronellic acids enhanced their biological activity. It was indicated by the lower active doses of terpene-PCs comparison to doses of free terpene acids. The low activity observed for lysoderivatives may be a result of significantly increased polarity of those compounds comparing to 1,2-diacylphosphatidylcholines. We found that there is structure-activity relationship among the tested terpene-modified PCs but it is also important to determine how the structure of isoprenoid residue Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S4 Fig(DOCX)Click here for additional data file.S5 Fig(DOCX)Click here for additional data file.S6 Fig(DOCX)Click here for additional data file.S7 Fig(DOCX)Click here for additional data file.S8 Fig(DOCX)Click here for additional data file.S9 Fig(DOCX)Click here for additional data file.S10 Fig(DOCX)Click here for additional data file.S11 Fig(DOCX)Click here for additional data file.S12 Fig(DOCX)Click here for additional data file.S13 Fig(DOCX)Click here for additional data file.S14 Fig(DOCX)Click here for additional data file.S15 Fig(DOCX)Click here for additional data file.S16 Fig(DOCX)Click here for additional data file.S17 Fig(DOCX)Click here for additional data file.S18 Fig(DOCX)Click here for additional data file.S19 Fig(DOCX)Click here for additional data file.S20 Fig(DOCX)Click here for additional data file.S21 Fig(DOCX)Click here for additional data file.S22 Fig(DOCX)Click here for additional data file.S23 Fig(DOCX)Click here for additional data file.S24 Fig(DOCX)Click here for additional data file.S25 Fig(DOCX)Click here for additional data file.S26 Fig(DOCX)Click here for additional data file.S27 Fig(DOCX)Click here for additional data file.S28 Fig(DOCX)Click here for additional data file.S29 Fig(DOCX)Click here for additional data file.S30 Fig(DOCX)Click here for additional data file.S31 Fig(DOCX)Click here for additional data file.S32 Fig(DOCX)Click here for additional data file.S33 Fig(DOCX)Click here for additional data file.S34 Fig(DOCX)Click here for additional data file.S35 Fig(DOCX)Click here for additional data file.S36 Fig(DOCX)Click here for additional data file.S37 Fig(DOCX)Click here for additional data file.S38 Fig(DOCX)Click here for additional data file.S39 Fig(DOCX)Click here for additional data file.S40 Fig(DOCX)Click here for additional data file.