N’-substituted 5-chloro-3-methylisothiazole-4-carboxylic acid hydrazide derivatives with anticancer activity. The structure of new compounds was determined by mass spectrometry (MS), elemental analysis, proton nuclear magnetic resonance spectroscopy (1H-NMR), carbon nuclear magnetic resonance spectroscopy (13C-NMR), 1H-13C NMR correlations and infrared spectroscopy (IR). Moreover, the structures of the compounds were confirmed by crystallographic examination. The antiproliferative MTT tests for 11 prepared compounds was conducted towards human biphenotypic B cell myelomonocytic leukemia MV4-11. SRB test was used to examine their potential anticancer activity towards human colon adenocarcinoma cell lines sensitive LoVo, resistant to doxorubicin LoVo/DX, breast adenocarcinoma MCF-7 and normal non-tumorigenic epithelial cell line derived from mammary gland MCF-10A. The most active compound was 5-chloro-3-methyl-N′-[-(3-phenyloprop-2-en-1-ylidene]isothiazole-4-carbohydrazide, which showed the highest antiproliferative activity against all tested cell lines. Thanks to the progress in oncology, pharmacological treatment of cancer is gaining in importance and in the near future anti-cancer chemotherapeutics are expected to be the main method of treatment for cancer diseases. What is more, the search for new anti-cancer compounds with the desired application properties is constantly underway. As a result of designed syntheses, we obtained some new  Chemotherapy is a method of systemic treatment of malignant tumors using cytostatic drugs, which stop the division and spread of cancer cells . The basMany studies have indicated the antitumor potential of isothiazole derivatives. CP-547.632 39, which belongs to the (3-aryl-4-carboxamido-isothiazol-3-yl)-carbamides, is a promising tyrosine kinase inhibitor with antineoplastic activity  used in Another biological target of various isothiazole derivatives are MEK1 and MEK2 kinases ,12,13,14N’-substituted 5-chloro-3-methylisothiazole-4-carboxylic acid hydrazides with the chlorine substitution of the isothiazole ring in the 5-position. 5-Chloro-3-methyl-isothazole-4-carbohydrazide 2 has already been synthetized by Kuczyński et al. -3-methylisothiazole-4-carbohydrazide 3, but it is less activity than compounds from the 5-substituted isothiazole Schiff base series, Cisplatin and 5-Fluorouracil towards sensitive (LoVo) and multi-drug resistant (LoVo/DX) human colon adenocarcinoma cell lines -3-methylisothiazole-4-carbohydrazide 3, which possesses almost two times lower activity towards the MCF-10A normal cell line than against cancer cells. This indicates the potential selectivity of the compound with regards to cancer cells such as leukemia, breast and colon cancer cell lines. In addition, the most active compounds in both series contain substituents at the meta position of the phenyl ring near the azomethine group. In our opinion, the research on low-molecular weight of isothiazole derivatives with antiproliferative activity is very desirable because of the demand for oncological drugs that break the increasing resistance of tumors to cytostatics currently used in therapy.Based on the results of biological studies of the 5-substituted 5-hydrazine-3-methylisothiazole-4-carboxylic acid derivatives  and comp−1. Proton nuclear magnetic resonance (1H-NMR), carbon nuclear magnetic resonance (13C-NMR) and 2D 1H-13C NMR correlation spectra were recorded in deuterated dimethyl sulfoxide (DMSO-d6) using a Bruker ARX-300 spectrometer . Chemical shifts are reported in in parts per million (ppm) units and signal multiplicities were collected by the abbreviations: s (singlet), d (doublet), t (triplet), m (multiplet). The values of coupling constant are reported as J in Hz. Elemental analysis was obtained on NA 1500 equipment . Mass spectrometry (MS) was performed on a compactTM Electrospray Ionisation-Quadrupole-Time of Flight (ESI-Q-TOF) apparatus . The samples for ESI-MS experiments were dissolved in methanol. Monoisotopic mass was calculated  by Compass Data Analysis 4.2.Commercially available reagents were used without further purification. Progress of the reaction was controlled by thin layer chromatography (TLC) on ALUGRAM SIL G/UV pre-coated TLC sheets  and visualized by ultraviolet (UV) light at 254 nm . Melting points of all new compounds were measured by a LLG uniMELT-2 apparatus (LLG). A Thermo Scientific Nicolet iS50 FT-IR spectrophotometer  was used to record infrared specta (IR). The samples were applied as solids and frequencies are given in cm1. The mixture was heating under reflux for 3 h to obtain a clear solution. After cooling, the solution was distilled in an evaporator to give an oily residue. Thereafter benzene (30 mL) was added twice and distilled off in each case to remove residual thionyl chloride. A 45% solution of thionyl chloride dissolved in benzene (90 mL) was added to 33.8 mmol of 5-chloro-3-methylisothiazole-4-carboxylic acid In an ice-cold water bath cooled to 8 °C with a magnetic stirrer, 5-chloro-3-methylisothiazole-4-carboxylic acid chloride  was dissolved in acetone (240 mL). The reaction mixture was kept at a temperature of up to 8 °C at all times. At the same time, sodium azide  was dissolved in distilled water (18 mL) and then slowly added to the reaction mixture. After addition of the sodium azide solution, stirring was continued for 30 min. The separated salt was filtered off. The solution was distilled from the reaction mixture until the semi-liquid form. The suspension was cooled in an ice-cold water bath. The precipitate was filtered off and washed with 2-propanol.max (cm−1): 1622, 3205, 3288; 1H-NMR  2.39 , 4.65 , 9.69 ; 13C-NMR  δ 18.7 (CH3), 132.2 (isothiazole-C3), 150.1 (isothiazole-C4), 160.4 (isothiazole-C5), 165.8 (C=O); anal. C 31.44, H 3.14, N 21.96%, calcd for C5H6ClN3OS C 31.34, H 3.16, N 21.93%; ESI-MS m/z 189.9847 .To azide , 2-propanol (40 mL) and anhydrous hydrazine  was added. Then, the reaction mixture was heated at a temperature of 83 °C for 45 min under reflux. The course of the reaction was monitored by TLC eluting with ethyl acetate. The solid precipitated in the course of the reaction was then filtered off through a paper filter. After cooling the filtrate, colorless crystals fell out of the mixture, which were washed with cold methanol , mp = 163.0 °C; IR ν2, 439.2 mg, 2.3 mmol) and cinnamaldehyde  in EtOH (2.0 mL) was heated at 78 °C for 4 h. At the end of the reaction (monitored by TLC chloroform–ethyl acetate 7:3), reaction mixture was cooled. The separated product was precipitated and then washed with cold methanol . The product was purified by crystallization from acetonitrile to give yellow crystals, mp = 180–181 °C; IR νmax 1578, 1649, 3164 cm−1; 1H-NMR  δ 2.37 and 2.45′ , 7.0  and 7.06 , 7.10′ , 7.32 , 7.34 , 7.38 , 7.41′ , 7.43′ , 7.60′ , 7.94 , 8.06′ , 11.92 and 12.09′ ; 13C-NMR  δ 18.8 (CH3), 124.7 (isothiazole-C3), 125.1 (CH), 127.2 and 128.9′ (N=CH), 131.9 and 132.2′ (arC), 135.7 (arC), 140.0 (arC), 140.3 (arC), 148.2 (arC), 150.1 and 150.6′ (arC), 151.1 (CH), 157.3 (isothiazole-C4), 165.3 (isothiazole-C5), 166.0 (C=O). anal. C 55.34, H 3.84, N 13.70%, calcd for C14H12ClN3OS C 54.99, H 3.96, N 13.74%; ESI-MS m/z 304.0388 .A stirred mixture of 5-chloro-3-methylisothiazole-4-carboxylic acid hydrazide  and 3-chlorobenzaldehyde  in EtOH (1.5 mL) was heated at 78 °C for 4 h. At the end of the reaction (controlled in a TLC chloroform–ethyl acetate 9:1), the reaction mixture was cooled. The separated product was precipitated and washed with cold methanol . The product was purified by crystallization from acetonitrile to give yellow crystal, mp = 174–175 °C. IR νmax 1557, 1648, 3216 cm−1; 1H-NMR  δ 2.39 and 2.46′ , 7.44  and 7.51′ , 7.70–7.74  and 7.81′ , 8.10 and 8.28′ , 12.18 and 12.34′ ; 13C-NMR  δ 18.5 (CH3), 124.9 (C3-isothiazole), 125.7 and 125.9′ (N=CH), 126.4 (arC), 129.5 and 129.8′ (arC), 130.5 and 131.4′ (arC), 133.3 and 133.4′ (arC), 135.7 and 135.7′ (arC), 143.4 and 146.8′ (arC), 150.1 and 151.1′ (isothiazole-C4), 157.4 and 163.5′ (isothiazole-C5), 165.2 and 165.7′ (C=O); anal. C 46.15, H 2.62, N 13.37%, calcd for C12H9Cl2N3OS C 45.87, H 2.89, N 13.17%. ESI-MS m/z 311.9750 .A stirred mixture of 5-chloro-3-methylisothiazole-4-carboxylic acid hydrazide  and 3-nitrobenzaldehyde  in EtOH (1.5 mL) was heated at 78 °C for 4 h. During the reaction, the resulting suspension thickens. At the end of the reaction , the separated product was filtered and washed with cold methanol . The product was purified by crystallization from 70% ethanol to give yellow crystals, mp = 199–200 °C; IR νmax 1560, 1644, 3271 cm−1; 1H-NMR  δ 2.40 and 2.47′ , 7.68–7.77 , 7.90 , 8.18-8.24′ , 8.29′ , 8.42 and 8.58′ , 12.39 ; 13C-NMR  δ 18.1 and 18.8′ (CH3), 121.1 and 121.4′ (isothiazole-C3), 124.3 and 124.6′ (N=CH), 130.5 and 131.6′ (arC), 132.6 and 133.5′ (arC), 135.6 and 135.7′ (arC), 143.0 (arC), 146.4 and 148.2′ (arC), 150.5 and 151.4′ (arC), 157.8 and 158.5′ (isothiazole-C4), 163.9 (isothiazole-C5), 165.5 and 166.0′ (C=O); anal. C 44.37, H 2.60, N 17.13%, calcd for C12H9ClN4O3S C 44.38, H 2.79, N 17.25%; ESI-MS m/z 322.9810 . A stirred mixture of 5-chloro-3-methylisothiazole-4-carboxylic acid hydrazide  and 4-ethylbenzaldehyde  in EtOH (1.5 mL) was heated at 78 °C for 4 h. During the reaction, the resulting suspension thickens. At the end of the reaction (controlled by TLC chloroform–ethyl acetate 7:3), the separated product was filtered and washed with cold methanol . The product was purified by crystallization from acetonitrile to give colorless crystals, mp = 152–154 °C, IR νmax 1551, 1668, 3195 cm−1; 1H-NMR  δ 1.12 , 1.14 , 1.17 , 1.20 , 1.22′ , 2.39 and 2.46′ , 2.57′ , 2.60′ , 2.64′ , 2.68′ , 7.23 , 7.31′  7.39 , 7.67′ , 8.09 and 8.25′ , 11.99 and 12.17′ ; 13C- NMR  δ 15.2 (CH3), 18.8 (CH3), 28.0 (CH2), 126.8 (isothiazole-C3), 127.4 and 128.3′ (N=CH), 131.3 and 132.0′ (arC), 145.3 (arC), 146.2 and 146.6′ (arC), 148.7 (arC), 150.2 and 151.1′ (arC), 157.4 (isothiazole-C4), 163.5 (isothiazole-C5), 165.5 and 166.0′ (C=O); anal. C 54.40, H 4.31, 13.53%, calcd for C14H14ClN3OS C 54.63, H 4.58, N 13.65%; ESI-MS m/z 306.0346 .A stirred mixture of 5-chloro-3-methylisothiazole-4-carboxylic acid hydrazide  and 3-methoxybenzaldehyde  in EtOH (1.5 mL) was heated at 78 °C for 4 h. At the end of the reaction (controlled by TLC chloroform–ethyl acetate 7:3), the formed clear mixture was poured into water. The separated product fell out, and was washed with cold methanol . The product was purified by crystallization from 70% ethanol to give colorless crystals, mp = 139–140 °C; IR νmax 1580, 1666, 3181 cm−1; 1H-NMR  δ 2.40 and 2.47′ , 3.73 and 3.82′ , 6.96–7.05  and 7.30′ , 7.39′ , 8.08 and 8.27′ , 12.07 and 12.26′ ; 13C-NMR  δ 18.7 (CH3), 55.0 (OCH3), 111.5 (isothiazole-C3), 115.9 (N=CH), 119.3 (arC), 120.3 (arC), 130.0 (arC), 131.8 (arC), 135.2 (arC), 144.8 (arC), 148.6 (isothiazole-C4), 157.5 (isothiazole-C5), 159.4 (C=O); anal. C 50.84, H 3.91, N 13.06%, calcd for C13H12ClN3O2S C 50.40, H 3.90, N 13.56%; ESI-MS m/z 308.0237 .A stirred mixture of 5-chloro-3-methylisothiazole-4-carboxylic acid hydrazide  and benzaldehyde  in EtOH (1.5 mL) was heated at 78 °C for 4 h. At the end of the reaction , the reaction mixture was cooled. The separated product was precipitated and then washed with cold methanol . The product was purified by crystallization from acetonitrile to give colorless crystals, mp = 164–165 °C; IR νmax 1557, 1648, 3192 cm−1; 1H-NMR  δ 2.39 and 2.47′ , 7.38–7.40 , 7.46–7.48 ’ , 7.74 , 7.76  8.12 and 8.29′ , 12.09 and 12.21′ ; 13C-NMR  δ 18.8 (CH3), 126.7 (isothiazole-C3), 127.3 and 128.9′ (arC), 130.2 and 130.5′ (N=CH), 131.8 (arC), 133.7 (arC), 145.2 (arC), 148.7 (arC), 150.3 and 151.2′ (arC), 157.5 (isothiazole-C4), 163.7 (isothiazole-C5), 165.5 and 166.0′ (C=O); anal. C 51.67, H 3.35, N 14.92%, calc. for C12H10ClN3OS C 51.52, H 3.60, N 15.02%; ESI-MS m/z 278.0187 .A stirred mixture of 5-chloro-3-methylisothiazole-4-carboxylic acid hydrazide  and 2,4-dimethylbenzaldehyde  in EtOH (2.0 mL) was heated at 78 °C for 4 h. At the end of the reaction , the reaction mixture was cooled. The separated product was precipitated and then washed with cold methanol . The product was purified by crystallization from acetonitrile to give colorless crystals, mp = 232–234 °C; IR νmax 1556, 1667, 3182 cm−1. 1H-NMR  δ 2.23′ , 2.25 , 2.38′ , 2.46 , 7.00  and 7.10′ , 7.31  and 7.76′ , 8.32 and 8.52′ , 11.96 and 12.09′ ; 13C-NMR  δ 18.7 and 18.8′ (CH3), 18.9 and 19.2′ (CH3), 20.9 (CH3), 126.2 (C3-isothiazole) 126.7 and 126.9′ (N=CH), 127.0 and 128.9′ (arC), 131.6 and 131.7′ (arC), 131.9 and 132.3′ (arC), 136.7 and 137.1′ (arC), 139.5 and 140.0′ (arC), 144.9 and 147.4′ (arC), 149.7 and 151.1′ (isothiazole-C4), 157.3 and 163.6′ (isothiazole-C5), 165.3 and 166.0′ (C=O); anal. C 54.72, H 4.55, N 13.67%, calcd for C14H14ClN3OS C 54.63, H 4.58, N 13.65%; ESI-MS m/z 306.0506 .A stirred mixture of 5-chloro-3-methylisothiazole-4-carboxylic acid hydrazide  and 2-methylbenzaldehyde  in EtOH (5.0 mL) was heated at 78 °C for 4 h. At the end of the reaction , the reaction mixture was cooled. The separated product was precipitated and then washed with cold methanol . The product was purified by crystallization from acetonitrile to give colorless crystals, mp = 226–228 °C; IR νmax 1594, 1643, 3173 cm−1; 1H-NMR  δ 2.27 and 2.38′ , 2.43 and 2.46′ , 7.19 , 7.21 , 7.25′ , 7.27 , 7.31′ , 7.42′ , 7.84′ , 8.36 and 8.56′ , 12.03 and 12.15′ ; 13C-NMR  δ 17.9 (CH3), 18.4 (CH3), 125.4 (isothiazole-C3), 128.9 (arC), 130.1 and 130.8′ (N=CH), 131.4 (arC), 135.9 (arC), 136.4 (arC), 143.9 (arC), 156.6 (arC), 162.9 (isothiazole-C4), 164.4 (isothiazole-C5), 165.2 (C=O); anal. C 53.41, H 4.04, N 14.21%, calcd for C13H12ClN3OS C 53.15, H 4.12, N 14.30%; ESI-MS m/z 292.0337 .A stirred mixture of 5-chloro-3-methylisothiazole-4-carboxylic acid hydrazide  and 2-chlorobenzaldehyde  in EtOH (1.5 mL) was heated at 78 °C for 4 h. During the reaction, the resulting suspension thickens. At the end of the reaction , the separated product was filtered and washed with cold methanol . The product was purified by crystallization from 70% ethanol to give yellow crystals, mp = 232–233 °C; IR νmax 1590, 1644, 3167 cm−1; 1H-NMR  δ 2.39 and 2.47′ , 7.33 , 7.36′ , 7.43–7.49 , 7.52′ , 7.56′ , 8.04′ , 8.51 and 8.70′ , 12.27 and 12.39′ ; 13C-NMR  δ 19.1 (CH3), 126.5 (isothiazole-C3), 127.3 and 128.0 (C=NH), 130.3 and 131.2′ (arC), 131.8 and 132.2′ (arC), 133.4 and 133.7′ (arC), 141.7 (arC), 144.9 (arC), 151.7 (arC), 157.9 (isothiazole-C4), 164.1 (isothiazole-C5), 165.8 and 166.3′ (C=O); anal. C 46.16, H 2.81, N 13.08%, calcd for C12H9Cl2N3OS C 45.87, H 2.89, N 13.37%; ESI-MS m/z 311.9587 .A stirred mixture of 5-chloro-3-methylisothiazole-4-carboxylic acid hydrazide  with a Ruby CCD camera (3 and 4) and a Kuma KM-4 CCD with a Sapphire2 CCD camera (8), with graphite monochromatized Mo-Kα radiation (λ = 0.71073 Å) at 100(2) K, using an Oxford Cryosystems cooler. Data collection, cell refinement, data reduction and analysis were carried out with CrysAlisPro  = 0.091, wR(F2) = 0.214, S = 1.04, largest diff. peak and hole = 1.29 e Å−3/−0.45 e Å−3. The diffraction experiment was performed with a twined crystal , 1673 reflections with I > 2σ(I), 173 parameters, R[F2 > 2σ(F2)] = 0.082, wR(F2) = 0.242, S = 1.06, largest diff. peak and hole = 1.02 e Å−3/−0.59 e Å−3.Compound 8: C12H10ClN3OS, rM = 279.74 g mol−1, colorless needles, size 0.55 × 0.20 × 0.12 mm, monoclinic, space group Cc, a = 13.989 (4) Å, b = 10.846 (2) Å, c = 8.6451 (15) Å, β = 103.85 (3)°, V = 1255.4 (5) Å3, Z = 4, Dcalc = 1.480 g cm−3, F(000) = 576, μ= 0.46 mm−1, 7998 measured reflections, 3417 independent reflections [Rint = 0.040], 3195 reflections with I > 2σ(I), 187 parameters, R[F2 > 2σ(F2)] = 0.042, wR(F2) = 0.106, S = 1.06, largest diff. peak and hole = 0.46 e Å−3/−0.29 e Å−3. The isothiazole ring was found to be disordered over two sites and refined with the occupancy factors of 0.758 (3) and 0.242 (3). Disordered atoms of lower occupancy were refined with a common isotropic displacement parameter -3-methylisothiazole-4-carbohydrazide 3, which is the most active compound of this series, possessed almost two times lower activity  towards the MCF-10A normal cell line than against cancer cells. This compound is characterized by potential selectivity toward cancer cells such as leukemia, breast and colon cancer cell lines and low toxicity against healthy cells. Other tested compounds displayed slightly weaker antiproliferative activity against tumor cells, but in the case of normal MCF-10A cells, their activity was about three times lower. On the basis of the conducted research, it can be concluded that the antiproliferative activity of this group of derivatives corresponds to the hydrazide moiety. The position of the azomethine group -N=CH at the 5-position of the isothiazole in Schiff bases [3–11.In recent decades, there has been great progress in designing and obtaining modern oncology drugs . In thisff bases  is more


Fusarium culmorum AM10, Armillaria mellea AM296, Trametes versicolor AM536). The hydroxylation processes were not only regioselective but also stereoselective. The hydroxylation products of each secondary carbon atom in the cyclohexane ring were obtained by the application of the selected fungal strains. The Fusarium culmorum AM10 introduced the hydroxy function at C-3 and C-4, Armillaria mellea AM296 incorporated the hydroxy function at C-3 and C-5 and Trametes versicolor AM536 transformed the substrate to the mixture of C-3, C-4 and C-5 hydroxylactones. The hydroxylactones obtained were enantiomericaly enriched . The in vitro antiproliferative activities of the functionalization products were also evaluated. Regardless of the hydroxy substituent location all tested lactones exhibited similar, significant activity towards selected cancer cell lines (IC50 in the range 22.8–33.9 µg/mL).Searching for the new anticancer compounds we prepared three new β-cyclocitral-derived hydroxyl-γ-lactones by microbial hydroxylation of tetramethyl-substituted bicyclic γ-lactone. The substrate was transformed by the enzymatic system of filamentous fungi. Three out of fifteen strains were selected as effective biocatalysts ( There appears to be an increasing occurrence of cancer in animals. In livestock farming, the biggest problems are cattle and poultry cancers. In medical-veterinary practice, cancer is considered to be the main cause of mortality in dogs. Hematopoietic disorders account for approximately 30% of all canine cancers. Dogs may be a useful model for the study of new anticancer drugs and therapeutic strategies for humans because of the similarity of some canine and human cancers in etiology, pathogenesis and response to treatment as well as anatomical and physiological similarities of dogs to humans . In our Fusarium culmorum AM10, Armillaria mellea AM296, Trametes versicolor AM536) with the ability for selective hydroxylation of tetramethyl-γ-lactone (1). All selected microorganisms are already known for their hydroxylation potential towards steroid compounds nonan-8-one (2), White crystals, m.p. = 131–138 °C; 3, ee = 92%); 1H-NMR  δ: 0.96 and 1.17 2C<), 1.22 , 1.34 , 1.57–1.67 , 2.12 and 2.61 , 3.67 ; 13C-NMR  δ: 16.87 and 21.83 ((CH3)2C<), 17.88 (CH3-1), 21.35 (CH3-6), 26.27 (C-4), 34.98 (C-5), 41.46 (C-6), 41.87 (C-2), 46.28 (C-7), 73.26 (C-3), 93.51 (C-1), 175.47 (C-8); IR : 3512 (s), 1751 (s), 1271 (m), 1006 (m); HRMS (ESI-TOF) m/z [M + Na]+ calcd for C12H20O3 235.1310; found 235.1311; GC tr = 5.343; CGC tr = 209.29, 213.91.(+)-rel--4-Hydroxy-1,2,2,4-tetramethyl-9-oxabicyclo[4.3.0]nonan-8-one (3), White crystals, m.p. = 102–106 °C; 3, ee = 31%); 1H-NMR  δ: 1.05 and 1.10 2C<), 1.24 , 1.29 , 1.37 , 1.57 , 1.86 ,1.79  2.19 and 2.63 , 3.87 ; 13C-NMR  δ: 17.10 (CH3-1), 22.18 (CH3-6), 25.77 and 26.99 ((CH3)2C<), 38.51 (C-2), 43.59 (C-6), 45.75 (C-5), 45.88 (C-3), 46.63 (C-7), 63.14 (C-4), 90.70 (C-1), 175.31 (C-8); IR : 3415 (s), 1764 (s), 1267 (m), 1039 (m); HRMS (ESI-TOF) m/z [M + Na]+ calcd for C12H20O3 235.1310; found 235.1310; GC tr = 5.361; CGC tr = 215.02, 217.54.(−)-rel--5-Hydroxy-1,2,2,4-tetramethyl-9-oxabicyclo[4.3.0]nonan-8-one (4), White crystals, m.p. = 93–97 °C; 3, ee = 65%); 1H-NMR  δ: 1.02 and 1.06 2C<), 1.15 , 1.32 , 1.35 , 1.55–1.75 , 2.50 and 2.68 , 3.50 ; 13C-NMR  δ: 14.01 (CH3-6), 18.29 (CH3-1), 25.36 and 26.80 ((CH3)2C<), 27.13 (C-4), 35.59 (C-3), 36.40 (C-2), 43.11 (C-7), 47.44 (C-6), 73.77 (C-5), 93.48 (C-1), 176.11 (C-8); IR : 3518 (s), 1758 (s), 1269 (m), 1034 (m); HRMS (ESI-TOF) m/z [M + Na]+ calcd for C12H20O3 235.1310; found 235.1311; GC tr = 5.417; CGC tr = 217.56, 218.61.l-glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin and 10% fetal bovine serum (FBS) . The CLB70 cell line was maintained in Advanced RPMI (Gibco) culture medium supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin and 10% heat-inactivated FBS. Cells were cultured in the incubator with 5% CO2 and 95% humidified atmosphere at 37 °C. The antiproliferative assay was performed according to the procedure described earlier [50 value ± SD (p < 0.05.The GL-1 (canine B-cell leukemia) was obtained from Yasuhito Fujino and Hajime Tsujimoto from the Department of Veterinary Internal Medicine University of Tokyo , the CLB earlier . The teslue ± SD . Statist1. Application of microorganisms made it possible to obtain the hydroxyderivatives in one step, which would be difficult to achieve by traditional chemical synthesis. In all biotransformation processes the unactivated methylene carbon atoms  were hydroxylated to give products with OH function in cis orientation to the γ-lactone moiety. All isolated hydroxylactones  to the best of our knowledge were not described in earlier literature. What is also important is that the hydroxylactones 2 and 3 were obtained almost as the single enantiomers, ee values 99% and 96% respectively. The results of antiproliferative assay indicate the significant activity of obtained products towards tested canine cancer cell lines. The hydroxyl group position did not have any influence on antiproliferative activity. All hydroxylactones were slightly more active towards the GL-1 cell line in comparison to CLB70. The antiproliferative activity of hydroxylactones towards leukemic lymphocytes seems promising, therefore further studies to determine the possible mechanism of action for this group of compounds are required.Three fungal strains were selected to transform tetramethyl-γ-lactone


B1–B15, which were in vitro assessed for their anti-Candida potential. Compound B10 was found to be more potent against Candida spp. when compared with the reference drugs Fluconazole and Ketoconazole. A docking study of the newly synthesized Schiff bases was performed, and results showed good binding affinity in the active site of co-crystallized Itraconazole-lanosterol 14α-demethylase isolated from Saccharomyces cerevisiae. An in silico ADMET  study was done in order to predict some pharmacokinetic and pharmacotoxicological properties. The Schiff bases showed good drug-like properties. The results of in vitro anti-Candida activity, a docking study and ADMET prediction revealed that the newly synthesized compounds have potential anti-Candida activity and evidenced the most active derivative, B10, which can be further optimized as a lead compound.In the context of the dangerous phenomenon of fungal resistance to the available therapies, we present here the chemical synthesis of a new series of thiazolyl-triazole Schiff bases  Candida have become a major cause of morbidity and mortality in immunocompromised patients. In clinic, the number of antifungal agents that are available is limited. These drugs belong to major classes of azoles, allylamines, polyenes, fluropyrimidines, and thiocarbamates. The azole molecules are the most widely used [Over the last years, invasive fungal infections caused by ely used . They acely used .Candida albicans, followed by C. glabrata, C. tropicalis, C. parapsilosis and C. krusei. Some types of Candida became resistant to the first and second line of antifungal treatment . Most of the isolated strains are of C. glabrata. A growing concern worldwide is represented by the multidrug-resistant Candida infections, which no longer respond to the treatment with fluconazole or echinocandins. The number of drugs available is surpassed by the large spread of fungal infections. Furthermore, they present a series of disadvantages, such as reduced bioavailability, marked hepatotoxicity and many drug interactions, which limit their use. Due to the facts presented above, the resistance and the toxicity of available antifungals, and the development of new antifungal molecules, which can act by a different mechanism of action, have a better pharmacokinetic and safety profile and are efficient against the resistant strains, are urgently required.Unfortunately, the extensive use of azole molecules as antifungals has led to the appearance of severe fungal resistance. The majority of candidiasis is caused by Schiff bases, key intermediates in organic synthesis and also as common ligands in the coordination chemistry , have beThe pharmacokinetic and pharmacodynamic behavior of molecules inside the human body is influenced by their molecular properties, molecular size, flexibility and the presence of different pharmacophore features. Almost one half of the drug failures in development phases are due to suboptimal pharmacokinetic properties and unacceptable toxicity . The in Saccharomyces cerevisiae, to facilitate the understanding of the antifungal mechanism of action of the synthesized Schiff bases.Docking studies are useful in drug discovery to predict the docked structure of the ligandreceptor complex and to rank the ligand molecules based on their binding energy. Docking protocols help elucidating the most energetically favorable binding pose of a ligand to its receptor. The new derivatives were docked into the active site of lanosterol-14α-demethylase from H-1,2,4-triazole-3-thiol is illustrated in 1. The 4-methyl-2-phenylthiazole-5-carbohydrazide 2 was obtained by treating compound 1 with hydrazine hydrate, under reflux, in a water bath, for 3 h. Then, compound 2 was treated with CS2 and KOH, at room temperature, to give the potassium 2-(4-methyl-2-phenylthiazole-5-carbonyl)hydrazine-carbodithioate 3. Finally, the 4-amino-5-(4-methyl-2-phenylthiazol-5-yl)-4H-1,2,4-triazole-3-thiol 4 was obtained, in good yields (80%), by treating compound 3 with hydrazine hydrate, under reflux, for 2 h. Schiff bases B1–B15 were synthesized in good yields by the condensation of compound 4 with various aromatic or heteroaromatic aldehydes, in the presence of concentrated H2SO4, used as a catalyst -4catalyst .1H-NMR and 13C-NMR) spectra. The results of the C, H, N, S quantitative elemental analysis of all synthesized compounds were in accordance with the calculated values, within ±0.4% of the theoretical values. The spectral data confirmed the formation of the B1–B15 Schiff bases. Details of the synthetic procedures, the yields and the physical, analytical and spectral data of the synthesized compounds are presented in the Experimental Section. Compounds 1–3 were previously reported in the literature [The structures of thiazolyl-triazole Schiff bases were established by elemental analysis and on the basis of their mass spectra (MS), infrared spectra (IR), nuclear magnetic resonance , as suggested by their molecular formulas. The absence of the NH2 asymmetric and symmetric stretching vibrations at 3281 cm−1 and 3186 cm−1 and also the presence of N=CH stretch absorption bands at 1635–1618 cm−1 in the final compounds’ IR spectra provided strong evidences for the formation of Schiff bases B1–B15.Mass spectra recorded for the final products 1H-NMR spectrum of compound 4, a signal was recorded, characteristic for the amino protons, as a singlet, at 5.73 ppm. The absence of this signal from the 1H-NMR spectra of the new synthesized compounds (B1–B15), and also the presence of a singlet characteristic to the N=CH proton at 9.36–9.52 ppm further confirmed the condensation between the 4-amino-5-(4-methyl-2-phenylthiazol-5-yl)-4H-1,2,4-triazole-3-thiol 4 and the corresponding aromatic aldehydes. The 3-mercapto-1,2,4-triazoles could exist in two tautomeric forms , proving that the compounds were in thione form, in solid state. Furthermore, in the 13.91–14.18 ppm region of the 1H-NMR spectra of compounds 4 and B1–B15, the NH proton resonated as a singlet, indicating that the crystal structures of the compounds correspond to the thione form. The 13C-NMR spectra of the newly synthesized compounds were consistent with the proposed structures and exhibited only the presence of thionic tautomeric form (C=S) through the presence of the exocyclic C=S peaks, at 169.88–170.25 ppm. The spectral investigations performed by us proved that thiazolyl-triazole Schiff bases B1–B15 exist in their thione form, in solid state.In the IR spectra of compounds Candida activity was tested in vitro using the disk diffusion method, by measuring the diameters of the inhibition zones. The synthesized compounds B1–B15 were screened against the fungal strain of Candida albicans ATCC 90028. The concentrations used for evaluating the antimicrobial activity were 100 μg/disk for the synthesized compounds and for the reference substance used, fluconazole. Because the compounds are not soluble in water, the solvent used to prepare the solutions of the synthesized compounds, dimethylsulfoxide (DMSO), exhibited no inhibitory activity on the fungal strain used in the study. The obtained results are presented in The anti-Candida activity, we observed that the compounds substituted on the phenyl bound to the azomethinic group with para-Br (B5), meta-nitro (B10) and para-nitro (B11) showed the biggest zone inhibition diameters (20 mm). The other compounds, with the exception of the 2,4-dichloro substituted compound B1 (16 mm), showed the same diameter (18 mm).Analyzing the results obtained, we can observe that all the tested compounds showed zone inhibition diameters inferior to Fluconazole, used as an antifungal reference drug. Concerning the relationships between the structure of the compounds and the anti-Candida activity, eleven Schiff bases having the same inhibition zone diameters  and a non-albicans Candida strain (Candida krusei) [In the first screening test, we observed that the compounds showed similar anti-iameters . In orde krusei) ,17,18, fCandida albicans ATCC 10231, Candida albicans ATCC 18804 and Candida krusei ATCC 6258). Stock solutions (1 mg/mL) were prepared by dissolving the test compounds and the reference antimicrobials  in sterile DMSO. The results are presented in The broth microdilution method was employed for the MIC test. All the synthesized compounds were tested against three strains of fungi (B10 (meta-nitro) is 4-fold more active than Fluconazole and 2-fold more active than the other reference local antifungal drug, Ketoconazole, respectively, on Candida albicans ATCC 10231. On C. albicans ATCC 18804, compounds B8 (meta-hydroxy), B10 and B14 (substituted with thiophene heterocycle) were 2-fold more active than Fluconazole and equal, as potential, with Ketoconazole. Concerning the non-albicans Candida strain, compounds B5 (para-Br) and B10 were 2-fold more active than Fluconazole and with the same activity as Ketoconazole.From the obtained results, we can observe that all the tested compounds showed MIC values equal or inferior to systemic antifungal reference drug, Fluconazole . The Schmeta-hydroxy), B9 (para-hydroxy) and B10 presented the same activity against C. albicans ATCC 18804. Determination of MFC confirmed the results previously obtained when MIC was investigated. Supplementary, we observed that Schiff bases B8  value between the top ranked predicted conformation and the observed X-ray crystal structure of the Protein Data Bank (PDB) deposited structure, was 0.324 Å. A value being below 2 Å the docking protocol was validated.With the aim of elucidating the mechanism of action of synthesized Schiff bases, molecular docking studies were performed on lanosterol-14α-demethylase from B1–B15 and fluconazole, as a control inhibitor, were docked into the active site of lanosterol 14α-demethylase. The predicted binding affinity of Schiff bases into the active site of the enzyme and the predicted polar contacts between them, are presented in The synthesized compounds B1–B15 did not covalently interact with the heme from the active site of lanosterol 14α-demethylase, like classic antifungal azoles. They interact with the amino acids in the access channel to the active site of the lanosterol 14α-demethylase (meta position (B8) and the corresponding meta- and para-substituted nitro-derivatives  form supplementary bonds with the Arg98 from the access groove  which showed the highest inhibition capacity. Non-interacting amino acids in the foreground were removed for clarity. The thiole group is not supposed to interact with amino acids from the enzyme. Our future studies will be directed towards optimizing the structure, in order to obtain compounds with a superior binding affinity and with better anti-Candida activity. Modulation of thiole by introducing one polar substituent, may improve the inhibition ability, because of the possible supplementary interaction with Tyr72.The data obtained from docking shows that all the Schiff bases have higher binding interaction energy, ranging from −10.45 to −12.92 kcal/mol relative to fluconazole  . Figure High oral bioavailability is an important factor for the development of bioactive compounds as therapeutic agents . The preB1–6, B12, B13 and B15 have tPSA values less than 140 Å2, passing the criteria for gastro-intestinal absorption, after oral administration . On the other hand, all the compounds were predicted to have a low blood–brain barrier penetration (tPSA > 90 Å2), so, the risks of central nervous system (CNS) side effects are reduced or absent. Water solubility of our compounds is poor, for all compounds—logS value being between −6 and −10. The compounds B1–B5 are considered “poorly soluble” and the rest are “moderately soluble”.Topological polar surface area (tPSA), that is the surface belonging to polar atoms, is a descriptor that correlates well with passive molecular transport through membranes, including the blood–brain barrier . Compounstration . The othAll Schiff bases are predicted as noninhibitors of CYP2D6, therefore the side effects  are not expected upon the administration of these compounds. Also, all are predicted to be metabolized by CYP2C9 ,25, due P-glycoprotein (P-gp) is a member of the ATP-binding cassette (ABC) transporter family involved in intestinal absorption, drug metabolism, and brain penetration, and its inhibition can seriously alter a drug’s bioavailability and safety . The druB1–15 show good pharmacokinetic properties, but with some limitations, such as moderate gastrointestinal absorption and no blood–brain barrier penetration. All molecules are accepted as drug-like, passing Lipinski’s “Rule of five”. The predicted parameters are within the range of accepted values. However, further optimization should be performed on tested compounds, in order to achieve better ADME properties. A mandatory improvement for the properties of the newly synthesized derivatives would be to increase their bioavailability. Small chemical modulations on the molecules could improve the ADME properties. Potential chemical modulations, such as alkylation, on the ionisable thiol moiety, would severely change the ADME properties of the resulted molecules, in order to extend the potential oral administration.According to the above ADME and toxicity results, the thiazolyl-triazole Schiff bases 1–3 were previously reported and were synthesized by us according to methodologies described in the literature [All chemicals and reagents were obtained from commercial sources and were used as supplied, without further purification. Compounds terature ,14.254 sheets , heptane–ethyl-acetate 3:7 elution system and ultraviolet UV light (254 nm) for visualization. MS analyses were performed at 70 eV with an Agilent gas chromatograph 6890  equipped with an apolar Macherey Nagel Permabond SE 52 capillary column  and with an LCMS Shimadzu Mass Spectrometer . Elemental analysis was registered with a Vario El CHNS instrument . IR spectra were recorded on a JASCO FT-IR-4100 spectrometer  using the ATR technique . Nuclear magnetic resonance (1H-NMR) spectra were recorded at room temperature on a Bruker Avance NMR spectrometer  operating at 400 and 500 MHz, using tetramethylsilane (TMS) as an internal standard  and were in accordance with the assigned structures. The samples were prepared by dissolving the compounds in DMSO-d6 (δH = 2.51 ppm) as a solvent and spectra were recorded using a single excitation pulse of 12 μs (1H-NMR). Spin multiplets are given as s (singlet), d (doublet), t (triplet) and m (multiplet). 13C-NMR spectra were recorded on a Bruker Avance NMR spectrometer , operating at 125 MHz, in DMSO-d6, using a waltz-16 decoupling scheme.Melting points were determined with an Electrothermal melting point meter in the open glass capillary method and are uncorrected. The reaction progress and purity of the synthesized compounds were monitored by analytical thin layer chromatography (TLC) using Merck precoated Silica Gel 60FGeneral procedure for the synthesis of ethyl 4-methyl-2-phenylthiazole-5-carboxylate (1) [late (1) . An amouGeneral procedure for the synthesis of 4-methyl-2-phenylthiazole-5-carbohydrazide (2) [1) (58.7 mmol in 100 mL absolute ethanol). The resulting reaction mixture was refluxed on a water bath, at 100 °C, for 4 h, and then it was concentrated under reduced pressure. Distilled water was added to the obtained precipitate and then the mixture was subjected to low pressure filtration, washed with distilled water, dried and recrystallized from absolute ethanol.zide (2) . An amouGeneral procedure for the synthesis of potassium 2-(4-methyl-2-phenylthiazole-5-carbonyl)hydrazinecarbodithioate (3) [2  was added a suspension of 30 mmol (1.68 g) of KOH in 60 mL of absolute ethanol and 30 mmol (3.04 g) CS2. The reaction mixture was stirred for 3 h at room temperature to obtain a quantitative precipitation. The reaction mass was filtered, washed with diethyl ether and dried in order to yield a bright yellow precipitate.oate (3) . To the General procedure for the synthesis of 4-amino-5-(4-methyl-2-phenylthiazol-5-yl)-4H-1,2,4-triazole-3-thiol (4). The alcoholic solution of potassium 2-(4-methyl-2-phenylthiazole-5-carbonyl) hydrazinecarbodithioate 3 (10 mmol in 40 mL of absolute ethanol) was treated with 10 mmol of hydrazine hydrate (80%). The reaction mixture was refluxed for 2 h. After cooling, the reaction mass was poured onto water and then acidified with a HCl (10%) solution. The reaction mixture was allowed to stand for 2 h for quantitative precipitation and evolution of H2S. The obtained precipitate was filtered and washed with ethanol. The obtained compound was recrystallized from absolute ethanol. Yield 80.0% (0.23 g); m.p. 165 °C; light yellow powder; Anal. Calcd for C12H11N5S2 (289.38): C, 49.81; H, 3.83; N, 24.20; S, 22.16; Found: C, 49.83; H, 3.85; N, 24.24; S, 22.13; IR : 3281 (ν NH2 asym), 3186 (ν NH2 sym), 3110 (ν NHtriazole), 1268 (ν C=S); 1H-NMR : 14.09 , 7.92 , 7.68 , 7.59 , 5.73 , 2.54 ; 13C-NMR : 170.16 (C=S), 159.34 (C), 153.51 (C), 151.07 (C), 144.12 (C), 143.81 (C), 131.12 (2CH), 129.41 (2CH), 127.85 (CH), 16.05 (CH3); MS  m/z (%): 290.08 (M + 1).General procedure for the synthesis ofSchiff basesB1–B15. An amount of 2 mmol (0.578 g) of 4-amino-5-(4-methyl-2-phenylthiazol-5-yl)-4H-1,2,4-triazole-3-thiol 4 was suspended in 10 mL of absolute ethanol. To the resulting suspension was added an alcoholic solution of 2 mmol of aldehyde (aromatic or heteroaromatic) in 5 mL of absolute ethanol and 2–3 drops of concentrated H2SO4 as a catalyst. The reaction mixture was refluxed for 6 h. The obtained precipitate was filtered hot and washed with absolute ethanol, and then it was dried and recrystallized from DMSO.4--5-(4-methyl-2-phenylthiazol-5-yl)-4H-1,2,4-triazole-3-thiol (B1). Yield 67.4% (0.300 g); m.p. 247–249 °C; light yellow powder; Anal. Calcd for C19H13Cl2N5S2 (446.38): C, 51.12; H, 2.31; N, 15.68; S, 14.33; Found: C, 51.32; H, 2.32; N, 15.61; S, 14.36; IR : 3095 (ν NHtriazole), 1633 (ν -N=CH-), 1260 (ν C=S); 1165 (ν C-Cl), 1098 (ν C-Cl); 1H-NMR : 14.15 , 9.42 , 7.93–8.01 , 7.72 , 7.50 , 7.35–7.42 , 2.33 ; 13C-NMR : 170.25 (C=S), 159.14 (C), 157.73 (CH=N), 154.01 (C), 150.98 (C), 144.11 (C), 132.44 (C), 131.09 (C), 130.89 (2CH), 129.33 (2CH), 129.15 (CH), 129.03 (CH), 128.82 (CH), 128.41 (2C), 126.01 (CH), 15.93 (CH3); MS  m/z (%): 446.3 (M + 1).4--5-(4-methyl-2-phenylthiazol-5-yl)-4H-1,2,4-triazole-3-thiol (B2). Yield 70.5% (0.314 g); m.p. 300 °C; light yellow powder; Anal. Calcd for C19H13Cl2N5S2 (446.38): C, 51.12; H, 2.31; N, 15.68; S, 14.33; Found: C, 50.9; H, 2.32; N, 15.61; S, 14.27; IR : 3092 (ν NHtriazole), 1635 (ν -N=CH-), 1265 (ν C=S); 1161 (ν C-Cl), 1095 (ν C-Cl); 1H-NMR : 14.11 , 9.41 , 7.95–8.03 , 7.54–7.59 , 2.39 ; 13C-NMR : 170.09 (C=S), 159.16 (C), 157.61 (CH=N), 153.97 (C), 151.04 (C), 144.01 (C), 133.72 (2C), 132.19 (C), 130.96 (2CH), 129.37 (2CH), 129.11 (CH), 128.75 (CH), 128.64 (C), 128.32 (2CH), 15.86 (CH3); MS  m/z (%): 446.3 (M + 1).4--5-(4-methyl-2-phenylthiazol-5-yl)-4H-1,2,4-triazole-3-thiol (B3). Yield 77.1% (0.687 g); m.p. 285–290 °C; light yellow powder; Anal. Calcd for C19H13Cl2N5S2 (446.38): C, 51.12; H, 2.31; N, 15.68; S, 14.33; Found: C, 51.32; H, 2.32; N, 15.61; S, 14.27; IR : 3096 (ν NHtriazole), 1632 (ν -N=CH-), 1263 (ν C=S); 1159 (ν C-Cl), 1091 (ν C-Cl); 1H-NMR : 14.06 , 9.48 , 7.89–8.93 , 7.71 , 7.54 , 7.35-7.48 , 2.36 ; 13C-NMR : 170.04 (C=S), 159.11 (C), 157.75 (CH=N), 153.97 (C), 151.04 (C), 144.01 (C), 142.88 (C), 134.65 (C), 132.19 (CH), 130.98 (2CH), 130.44 (C), 129.33 (2CH), 128.88 (CH), 128.61 (C), 128.33 (CH), 125.12 (CH), 15.79 (CH3); MS  m/z (%): 446.3 (M + 1).4-(3-Chlorobenzylideneamino)-5-(4-methyl-2-phenylthiazol-5-yl)-4H-1,2,4-triazole-3-thiol (B4). Yield 82.92% (0.781 g); m.p. 275 °C; light yellow powder; Anal. Calcd for C19H14ClN5S2 (411.9): C, 55.34; H, 3.39; N, 16.99; S, 15.53; Found: C, 55.6; H, 3.37; N, 17.05; S, 15.59; IR : 3091 (ν NHtriazole), 1630 (ν -N=CH-), 1271 (ν C=S); 1132 (ν C-Cl); 1H-NMR : 14.18 , 9.52 , 7.97–8.06 , 7.92 , 7.77 , 7.59 , 7.47–7.54 , 2.41 ; 13C-NMR : 170.12 (C=S), 159.15 (C), 157.66 (CH=N), 153.81 (C), 151.07 (C), 143.96 (C), 135.16 (C), 134.51 (C), 131.21 (CH), 130.93 (2CH), 130.29 (CH), 129.29 (2CH), 128.94 (CH), 128.68 (C), 127.36 (CH), 127.14 (CH), 15.92 (CH3); MS  m/z (%): 412.04 (M + 1).4-(4-Bromobenzylideneamino)-5-(4-methyl-2-phenylthiazol-5-yl)-4H-1,2,4-triazole-3-thiol (B5). Yield 75.3% (0.343 g); m.p. 296–298 °C; yellow powder; Anal. Calcd for C19H14BrN5S2 (456.38): C, 49.89; H, 3.06; N, 15.33; S, 14.02; Found: C, 50.1; H, 3.07; N, 15.33; S, 14.07; IR : 3104 (ν NHtriazole), 1618 (ν -N=CH-), 1274 (ν C=S); 1055 (ν C-Br); 1H-NMR : 14.12 , 9.40 , 7.98–8.04 , 7.81–7.83 , 7.60 , 7.44–7.49 , 2.35 ; 13C-NMR : 170.02 (C=S), 159.16 (C), 157.64 (CH=N), 153.61 (C), 151.17 (C), 143.67 (C), 131.85 (2CH), 131.11 (C), 130.89 (2CH), 129.29 (2CH), 128.93 (CH), 128.71 (C), 128.30 (2CH), 125.59 (C), 15.77 (CH3); MS  m/z (%): 456.3 (M + 1).4-(4-Fluorobenzylideneamino)-5-(4-methyl-2-phenylthiazol-5-yl)-4H-1,2,4-triazole-3-thiol (B6). Yield 67.8% (0.268 g); m.p. 294–296 °C; yellow powder; Anal. Calcd for C19H14FN5S2 (395.48): C, 57.65; H, 3.54; N, 17.7; S, 16.18; Found: C, 57.9; H, 3.55; N, 17.62 S, 16.11; IR : 3107 (ν NHtriazole), 1620 (ν -N=CH-), 1270 (ν C=S); 1215 (ν C-F); 1H-NMR : 13.99 , 9.47 , 7.96–8.01 , 7.79–7.82 , 7.46–7.54 , 7.31 , 2.29 ; 13C-NMR : 169.96 (C=S), 165.22 (C), 159.20 (C), 157.58 (CH=N), 153.65 (C), 151.18 (C), 143.53 (C), 130.98 (2CH), 130.81 (2CH), 129.33 (2CH), 128.95 (CH), 128.68 (C), 127.77 (C), 115.59 (2CH), 15.83 (CH3); MS  m/z (%): 396.3 (M + 1).2-((3-Mercapto-5-(4-methyl-2-phenylthiazol-5-yl)-4H-1,2,4-triazol-4-ylimino) methyl)phenol (B7). Yield 46.8% (0.356 g); m.p. 269 °C; yellow powder; Anal. Calcd for C19H15N5OS2 (393.49): C, 57.94; H, 3.81; N, 17.78; S, 16.26; Found: C, 58.2; H, 3.82; N, 17.85 S, 16.19; IR : 3348 (ν O-H), 3115 (ν NHtriazole), 1626 (ν -N=CH-), 1284 (ν C=S); 1243 (ν C-O); 1H-NMR : 13.97 , 9.82 , 9.43 , 7.99–8.05 , 7.68 , 7.44–7.46 , 7.15 , 7.01 , 2.27 ; 13C-NMR : 169.99 (C=S), 161.31 (C), 159.23 (C), 157.88 (CH=N), 153.57 (C), 151.21 (C), 143.51 (C), 132.53 (CH), 132.15 (CH), 130.85 (2CH), 129.41 (2CH), 128.90 (CH), 128.59 (C), 121.76 (CH), 118.18 (C), 116.99 (CH), 15.88 (CH3); MS  m/z (%): 394.2 (M + 1).3-((3-Mercapto-5-(4-methyl-2-phenylthiazol-5-yl)-4H-1,2,4-triazol-4-ylimino) methyl)phenol (B8). Yield 65.3% (0.496 g); m.p. 295–297 °C; light yellow powder; Anal. Calcd for C19H15N5OS2 (393.49): C, 57.94; H, 3.81; N, 17.78; S, 16.26; Found: C, 58.2; H, 3.82; N, 17.85 S, 16.19IR : 3344 (ν O-H), 3112 (ν NHtriazole), 1622 (ν -N=CH-), 1281 (ν C=S); 1245 (ν C-O); 1H-NMR : 13.92 , 9.78 , 9.39 , 8.01–8.04 , 7.54 , 7.48–7.56 , 7.39 , 7.23 , 6.98 , 2.34 ; 13C-NMR : 169.92 (C=S), 159.09 (C), 158.83 (C), 157.67 (CH=N), 153.46 (C), 151.24 (C), 143.44 (C), 135.33 (C), 130.87 (2CH), 130.27 (CH), 129.30 (2CH), 128.92 (CH), 128.64 (C), 121.74 (CH), 118.26 (CH), 115.01 (CH), 15.93 (CH3); MS  m/z (%): 394.2 (M + 1).4-((3-Mercapto-5-(4-methyl-2-phenylthiazol-5-yl)-4H-1,2,4-triazol-4-ylimino) methyl)phenol (B9). Yield 68.15% (0.518 g); m.p. 285 °C; light yellow powder; Anal. Calcd for C19H15N5OS2 (393.49): C, 57.94; H, 3.81; N, 17.78; S, 16.26; Found: C, 57.7; H, 3.82; N, 17.7 S, 16.19; IR : 3341 (ν O-H), 3118 (ν NHtriazole), 1623 (ν -N=CH-), 1285 (ν C=S); 1249 (ν C-O); 1H-NMR : 14.07 , 9.83 , 9.38 , 7.78–7.95 , 7.41–7.47 , 6.98 , 2.32 ; 13C-NMR : 169.94 (C=S), 160.39 (C), 158.88 (2C), 158.42 (C), 157.97 (CH=N), 143.74 (C), 130.97 (2CH), 130.52 (2CH), 129.36 (2CH), 128.94 (CH), 126.33 (C), 116.11 (2CH), 106.06 (C), 16.24 (CH3); MS  m/z (%): 394.2 (M + 1).5-(4-Methyl-2-phenylthiazol-5-yl)-4-(3-nitrobenzylideneamino)-4H-1,2,4-triazole-3-thiol (B10). Yield 80.8% (0.682 g); m.p. 270 °C; light yellow powder; Anal. Calcd for C19H14N6O2S2 (422.48): C, 53.96; H, 3.31; N, 19.88; S, 15.14; Found: C, 53.7; H, 3.32; N, 19.8; S, 15.2; IR : 3131 (ν NHtriazole), 1521 (ν N-O asy), 1629 (ν -N=CH-), 1324 (ν N-O sy), 1257 (ν C=S); 1H-NMR : 14.04 , 9.34 , 8.52 , 8.01–8.22 , 7.49–7.59 , 2.45 ; 13C-NMR : 169.88 (C=S), 159.03 (C), 157.76 (CH=N), 153.45 (C), 151.19 (C), 148.88 (C), 143.66 (C), 135.44 (CH), 134.58 (C), 130.83 (2CH), 129.71 (CH), 129.38 (2CH), 128.83 (CH), 128.55 (C), 126.36 (CH), 121.65 (CH), 16.12 (CH3); MS  m/z (%): 423.2 (M + 1).5-(4-Methyl-2-phenylthiazol-5-yl)-4-(4-nitrobenzylideneamino)-4H-1,2,4-triazole-3-thiol (B11). Yield 50% (0.412 g); m.p. 275 °C; orange powder; Anal. Calcd for C19H14N6O2S2 (422.48): C, 53.96; H, 3.31; N, 19.88; S, 15.14; Found: C, 53.7; H, 3.29; N, 19.95; S, 15.2; IR : 3129 (ν NHtriazole), 1525 (ν N-O asym), 1625 (ν -N=CH-), 1320 (ν N-O sym), 1259 (ν C=S); 1H-NMR ; 13.93 , 9.36 , 8.02–8.29 , 7.45–7.51 , 2.34 ; 13C-NMR : 169.91 (C=S), 159.09 (C), 157.78 (CH=N), 153.35 (C), 151.16 (C), 150.13 (C), 143.59 (C), 138.54 (C), 130.91 (2CH), 129.25 (2CH), 128.90 (CH), 128.57 (C), 127.89 (2CH), 124.16 (2CH), 16.16 (CH3); MS  m/z (%): 423.2 (M + 1).4-(2-Methoxybenzylideneamino)-5-(4-methyl-2-phenylthiazol-5-yl)-4H-1,2,4-triazole-3-thiol (B12). Yield 71.7% (0.603 g); m.p. 285 °C; yellow powder; Anal. Calcd for C20H17N5OS2 (407.51): C, 58.89; H, 4.17; N, 17.17; S, 15.7; Found: C, 58.7; H, 4.15; N, 17.23; S, 15.63; IR : 3093 (ν NHtriazole), 1628 (ν -N=CH-), 1272 (ν C=S); 1254 (ν C-O), 1038 (ν C-O); 1H-NMR : 13.95 , 9.45 , 7.92–8.05 , 7.71 , 7.47–7.55 , 7.29 , 7.09 , 3.86 , 2.41 ; 13C-NMR : 169.97 (C=S), 159.10 (C), 157.88 (CH=N), 157.43 (C), 153.40 (C), 151.17 (C), 143.43 (C), 132.10 (CH), 131.81 (CH), 130.79 (2CH), 129.21 (2CH), 128.93 (CH), 128.72 (C), 121.39 (CH), 117.02 (C), 111.22 (CH), 55.69 (CH3), 15.91 (CH3); MS  m/z (%): 408.49 (M + 1).4-(3-Methoxybenzylideneamino)-5-(4-methyl-2-phenylthiazol-5-yl)-4H-1,2,4-triazole-3-thiol (B13). Yield 80.6% (0.680 g); m.p. 270 °C; yellow powder; Anal. Calcd for C20H17N5OS2 (407.51): C, 58.89; H, 4.17; N, 17.17; S, 15.7; Found: C, 58.7; H, 4.18; N, 17.23; S, 15.63; IR : 3089 (ν NHtriazole), 1621 (ν -N=CH-), 1283 (ν C=S); 1257 (ν C-O), 1034 (ν C-O); 1H-NMR : 13.91 , 9.44 , 7.99–8.05 , 7.58 , 7.38–7.52 , 7.38 , 7.14 , 3.93 , 2.43 ; 13C-NMR : 169.95 (C=S), 160.75 (C), 159.14 (C), 157.88 (CH=N), 153.37 (C), 151.09 (C), 143.41 (C), 134.80 (C), 130.85 (2CH), 129.86 (CH), 129.25 (2CH), 128.83 (CH), 128.66 (C), 121.62 (CH), 116.66 (CH), 111.28 (CH), 55.75 (CH3), 15.89 (CH3); MS  m/z (%): 408.49 (M + 1).5-(4-Methyl-2-phenylthiazol-5-yl)-4-(thiophen-2-ylmethyleneamino)-4H-1,2,4-triazole- 3-thiol (B14). Yield 79.3% (0.536 g); m.p. 280 °C; yellow powder; Anal. Calcd for C17H13N5S3 (383.51): C, 53.19; H, 3.38; N, 18.25; S, 25.03; Found: C, 53.4; H, 3.38; N, 18.32; S, 24.92; IR : 3088 (ν NHtriazole), 1627 (ν -N=CH-), 1288 (ν C=S); 1H-NMR : 14.10 , 9.46 , 7.94–7.99 , 7.74–7.78 , 7.40–7.51 , 2.42 ; 13C-NMR : 170.08 (C=S), 159.13 (C), 157.95 (CH=N), 153.29 (C), 151.09 (C), 144.96 (C), 143.27 (C), 130.98 (2CH), 130.11 (CH), 129.27 (2CH), 128.91 (CH), 128.77 (C), 128.55 (CH), 127.30 (CH), 16.01 (CH3); MS  m/z (%): 384.4 (M + 1).4-(4-(Dimethylamino)benzylideneamino)-5-(4-methyl-2 phenylthiazol-5-yl)-4H-1,2,4-triazole-3-thiol (B15). Yield 77.3% (0.653 g); m.p. 260 °C; Anal. Calcd for C21H20N6S2 (420.55): C, 59.92; H, 4.75; N, 19.97; S, 15.21; Found: C, 59.7; H, 4.76; N, 19.89; S, 15.27; yellow powder; IR : 3086 (ν NHtriazole), 1619 (ν -N=CH-), 1331 (ν C-N), 1258 (ν C=S); 1H-NMR : 14.01 , 9.51 , 7.96–7.98 , 7.45–7.53 , 6.99–7.34 , 3.31 , 2.47 ; 13C-NMR : 170.03 (C=S), 159.09 (C), 157.63 (CH=N), 153.53 (C), 153.28 (C), 151.06 (C), 138.27 (C), 130.97 (2CH), 129.33 (2CH), 128.81 (CH), 128.37 (2CH), 127.94 (C), 121.86 (C), 111.99 (2CH), 43.31 (2CH3), 16.09 (CH3); MS  m/z (%): 421.12 (M + 1).6 CFU/mL). A sterile swab was soaked in suspension and then the Mueller-Hinton agar plates were inoculated by streaking the entire surface. After drying for 10–15 min, six-millimeter diameter wells were cut from the agar using a sterile cork-borer, and a volume of 20 μL of each compound solution (5 mg/mL in dimethyl sulfoxide-DMSO) was delivered into the wells (100 µg/well).The in vitro antifungal activity was determined using the cup-plate agar diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines . For antFluconazole (100 μg/well) was used as the standard drugs. The controls were performed with only sterile broth, overnight culture and 20 μL of DMSO. The plates were incubated at 35 °C. Zone diameters were measured to the nearest whole millimeter at a point in which there will be no visible growth after 24–48 h. Results were obtained in triplicate. The solvent used for the preparation of each compound stock solution (5 mg/mL), DMSO  exhibited no inhibitory activity against the tested fungal strain.Candida albicans ATCC 10231, Candida albicans ATCC 18804, Candida krusei ATCC 6258. The cultures were stored on potato dextrose agar . Prior to antifungal susceptibility testing, each strain was inoculated on potato dextrose agar plated to ensure optical growth characteristics and purity. Then, yeast cells were suspended in saline and adjusted spectrophotometrically to RPMI  1640 medium to a final concentration of 106 CFU/mL.The microorganisms used for antifungal activity evaluation were obtained from the University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca Romania. The antifungal activity was evaluated against cultures of l-glutamine, to pH 7.0 with 3-(n-morpholino)propanesulfonic acid. The initial density of E. coli and S. aureus was 105 CFU/mL and Candida sp was approximately 2 × 106 colony forming units (CFU)/mL. Inoculums  were prepared in a sterile solution of 0.9% NaCl solution. Then, tested strains were suspended in nutrient broth and RPMI 1640 media to give a final density of 2 × 105 CFU/mL. Solutions of the test compounds and suspensions of bacteria and fungi were inoculated onto 96-well microplates. The growth control, sterility control and control of antibacterial/antifungal compounds were used. Plates were incubated under normal atmospheric conditions at 30 °C for 48 h , and next minimum inhibitory concentration (MIC) values have been determined by adding resazurin  followed by incubation for 2 h. The MIC was defined as the lowest concentration required to arrest the growth of the bacteria/fungi. For determination of minimum fungicidal concentration (MFC), a 0.01 mL aliquot of the medium drawn from the culture tubes showing no macroscopic growth at the end of the 24 h culture was subcultured on nutrient agar/potato dextrose agar plates to determine the number of vital organisms and incubated further at 30 °C for 48 h. The MFC was defined as the lowest concentration of the agent at which no colonies are observed. All MIC and MFC experiments were repeated three times.Stock solutions (1 mg/mL) were prepared by dissolving the test compounds, the reference antifungals (fluconazole and ketoconazole) in sterile DMSO. These solutions were stored at 4 °C. A series of double diluting solutions of the above compounds were prepared in RPMI 1640 medium obtaining final concentrations in the range of 500 µg/mL to 0.015 µg/mL. The broth microdilution method was employed for the minimum inhibitory concentration test. Media were placed into each of the 96 wells of the microplates. Sample solutions at high concentration (100 µg/mL) were added into the first rows of the microplates and two-fold dilutions of the compounds were made by dispensing the solutions into the remaining wells. Culture suspensions of 10 µL were inoculated into all the wells. The sealed microplates were incubated at 37 °C for 18 h. Antifungal activity was tested by using the broth microdilution method according to the Clinical and Laboratory Standard Institute (CLSI) guidelines . The medSaccharomyces cerevisiae and to predict the binding mode of our synthesized compounds. The crystallographic structure of the complex between the three-dimensional (3D) structure of the enzyme, with the intact transmembrane domain, and co-crystallized itraconazole (PDB:5EQB), was used for the docking study. The crystal structure of the enzyme obtained by X-ray diffraction was taken from Protein Data Bank [A molecular docking study was carried out using AutoDock 4.2 , in ordeata Bank .B1–B15 was generated using HyperChem 6, with geometry optimization and minimized energy conformation. Further, all input files were prepared using AutoDock Tools 1.5.6 [A dataset of 3D thiazolyl-triazole Schiff bases ls 1.5.6 . Ligand ls 1.5.6 , was takx = y = z = 28 Å in dimension with x = 19.692, y = 14.168, z = 16.077 center coordinates. Grid maps were generated using AutoGrid 4 with 0.375 grid points size space.Using AutoDock Tools, co-crystallized ligand (itraconazole) and water were removed from the active site of the enzyme, polar hydrogen atoms were added, non-polar hydrogen atoms merged, rotatable bonds were defined, amide bonds were set non-rotatable, carboxylic moieties deprotonated and Gasteiger partial charges were assigned. The grid box was defined as 4 , rate of mutation at 0.02, rate of crossover at 0.8, step size for translations at 2 Å and cluster tolerance at 2 Å.AutoDock searched for the best conformers poses, based on the Lamarkian genetic algorithm. For each compound, AutoDock searched for 10 conformers. The maximum number of evaluation was set at 9 × 10In this present investigation, the new thiazolyl-triazole Schiff bases were subjected to a theoretical in silico ADME and toxicity prediction study, under Lipinski’s “Rule of five” . LipinskB1–B15 followed several steps. The 4-amino-5-(4-methyl-2-phenylthiazol-5-yl)-4H-1,2,4-triazole-3 thiol 4 was obtained by treating potassium 2-(4-methyl-2-phenylthiazole-5-carbonyl)hydrazine-carbodithioate 3, previously obtained, with hydrazine hydrate. The final derivatives were synthesized in good yields by the condensation of compound 4 with various aromatic or heteroaromatic aldehydes.Synthesis of Schiff bases Candida potential. The in vitro methods used were the disk diffusion disk, the determination of MIC and MFC, respectively. The activity of the new derivatives was reported to reference drugs, already used in therapy. Some of the Schiff bases showed MICs inferior to those of the references used. Compound B10 showed to be the most promising anti-Candida candidate, inhibiting the growth of all three Candida strains used and being more potent than fluconazole. The obtained results suggest that the new series bearing thiazole and triazole scaffolds may be considered for further investigation and optimization, in designing anti-Candida drugs.Schiff bases were investigated for their anti-B1–15 and presented a rationale for the activity of these compounds. Performed on CYP51 enzyme, this study showed that the thiazolyl-triazole Schiff bases interact with the amino acids in the access channel to the active site of the lanosterol 14α-demethylase with higher binding affinity energy than fluconazole. Moreover, the compounds do not interact with the heme.A docking study of the thiazolyl-triazole Schiff bases was performed on compounds Candida agents with good pharmacokinetic properties and without severe side effects. Furthermore, a virtual study was performed to predict the ADME and the toxicity of the derivatives. The studied compounds showed an acceptable gastro-intestinal absorption and a low to moderate water solubility. We could predict the absence of toxicity at CNS level, due to the fact that the compounds do not pass the blood–brain barrier. All of the newly synthesized compounds were predicted as noninhibitors of CYP2D6, noninducers of phospholipidosis, and they are not a substrate for P-glycoprotein. All these observations will be helpful in designing newer anti-


Balanophora simaoensis for the first time using n-hexane-ethyl acetate-methanol-water  as the optimum solvent system. Accordingly, 1-O-(E)-caffeoyl-3-O-galloyl-β-d-glucopyranose , 1-O-(E)-caffeoyl-3-O-galloyl-4,6-(S)-hexahydroxydiphenoyl-β-d-glucopyranose , and 1-O-(E)-caffeoyl-4,6-(S)-hexahydroxydiphenoyl-β-d-glucopyranose  with 98.0%, 98.5%, and 98.7% purities, respectively, were purified from 210 mg crude extract of B. simaoensis in a one-step separation. The structures of the glucose esters were identified by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance spectra (NMR). Their antioxidant activities were evaluated by measuring their inhibition activity on liver microsomal lipid peroxidation induced by the Fe2+-Cys system in vitro. Compounds I–III showed significant antioxidant activities with IC50 values ranging from 2.51 to 6.68 μm, respectively.High-speed counter-current chromatography was used to separate and purify galloyl, caffeoyl, and hexahydroxydiphenoyl esters of glucoses from the aerial parts of the parasitic plant  Balanophora simaoensis  indigenous to China is a parasitic plant growing on the roots of various plants and mainly distributed in the Yunnan province, China −. 1H-NMR  δ: 7.65 , 7.08 , 7.01 , 6.92 , 6.74 , 6.31 , 5.72 , 5.24 , 4.48 , 4.28 , 3.72 , 3.69 , 3.56 .13C-NMR  δ: caffeoyl: 126.5 (C-1), 114.3 (C-2), 145.4 (C-3), 148.5 (C-4), 115.6 (C-5), 122.8 (C-6), 147.6 (C-7), 113.1 (C-8), 166.3 (C-9); galloyl: 119.7 (C-1), 109.5 , 144.8 , 138.6 (C-4), 166.5 (C-7); glucose: 94.8 (C-1), 72.5 (C-2), 79.1 (C-3), 69.8 (C-4), 78.2 (C-5), 62.1 (C-6). On comparison with the reported data [I was identified as 1-O-(E)-caffeoyl-3-O-galloyl-β-d-glucopyranose. Compound II: ESI-MS m/z: 795 [M − H]−. 1H-NMR  δ: 7.64 , 7.06 , 7.03 , 7.02 , 6.78 , 6.60, 6.45 , 6.32 , 5.76 , 5.42 , 5.32 , 5.07 , 4.30 , 3.84 , 3.84 .13C-NMR  δ:caffeoyl:126.6 (C-1), 114.4 (C-2), 145.3 (C-3), 148.5 (C-4), 115.4 (C-5), 122.7 (C-6), 147.5 (C-7), 113.2 (C-8), 166.0 (C-9); galloyl: 119.6 (C-1), 109.1 , 145.1 , 138.4 (C-4), 166.3 (C-7); HHDP: 115.3, 115.0 , 125.2, 125.0 , 106.9, 107.1 , 144.4, 144.5 , 136.2, 136.5 , 143.1, 143.4 , 167.8, 168.1 ; glucose: 95.1 (C-1), 71.4 (C-2), 74.8 (C-3), 70.1 (C-4), 75.2 (C-5), 65.3 (C-6).Compared with the reported data −. 1H-NMR  δ: 7.66 , 7.04 , 6.98 , 6.79 , 6.59, 6.48 , 6.30 , 5.69 , 5.30 , 4.86 , 4.13 , 3.80 , 3.79 , 3.56 . 13C-NMR  δ: caffeoyl: 127.7 (C-1), 115.5 (C-2), 146.3 (C-3), 149.5 (C-4), 116.4 (C-5), 123.2 (C-6), 148.5 (C-7), 114.2 (C-8), 166.5 (C-9); HHDP: 115.0, 115.3 , 125.3, 125.5 , 106.4, 107.2 , 144.4, 144.8 , 136.3, 136.5 , 143.7, 143.8 , 167.8, 168.1 ; glucose: 96.1 (C-1), 72.7 (C-2), 74.5 (C-3), 73.1 (C-4), 75.5 (C-5), 64.7 (C-6). According to literature [III was identified as1-O-(E)-caffeoyl-4,6-(S)-hexahydroxydiphenoyl-β-d-glucopyranose.Compound 2+-Cys system in vitro. As shown in I–III showed significant antioxidant activities with IC50 values ranging from 2.51 to 6.68 μm, respectively.The antioxidant activities of three glucose esters were evaluated by measuring their inhibition activity on liver microsomal lipid peroxidation induced by the FeThe HSCCC instrument employed in the study was a model TBE-300C high-speed countercurrent chromatography apparatus  with three multilayer coils connected in series , which was equipped with a model TBP-5002S constant-flow pump , a Model TBD2000 UV detector  operating at 254 nm, and a model HW2000 workstation . The temperature of the HSCCC column was controlled at 25 °C using a Model DC-0506 circulatory temperature regulator .1H- and 13C-NMR experiments wereperformed on a Varian 600 MHz NMR spectrometer .The analytical HPLC system consisted of a Waters Empower system  with a model 600 pump, a model 600 multi-solvent delivery system, a model 996 diode-array detector, and an Empower workstation. The ESI-MS data were obtained by an Agilent 1100/MSG1946 mass spectrometer . The 4 and thiobarbituric acid were obtained from Sigma . The whole plants of B. simaoensis were collected in Yunan, China, and identified by Dr. Jia Li .The solvents used for the HSCCC separation and antioxidant assay were analytical grade, and were purchased from Tianjing Chemical Factory . Acetontrile was HPLC grade and purchased from Siyou Special Reagent Factory . The PBS, liver microsomes, and cysteine were bought from Beijing Solarbio Science & Technology Co., Ltd. . The FeSOB. simaoensis (1.5 kg) were powdered and extracted with MeOH at room temperature. The extract was filtered and concentrated in vacuo to afford the crude extract (210 g). Then, the extract was suspended in water (500 mL) and extracted by n-hexane (3 × 500 mL), ethyl acetate (3 × 500 mL), and n-butanol (3 × 500 mL) in sequence. The ethyl acetate extract were evaporated to dryness, yielding 42 g of ethyl acetate extract for further HSCCC separation.The whole plants of K) of the three target compounds. The K values were determined by HPLC [K value was expressed as the peak area of the compound in the upper phase divided by that in the lower phase.The selection of two-phase solvent system was based on the partition coefficient  was used for the HSCCC separation. The solvent system was thoroughly equilibrated in a separation funnel by repeatedly vigorously shaking at room temperature and separated shortly prior to use. The sample solution was prepared by dissolving the dried extract in the mixture solution of lower phase and upper phase  of the solvent system.In the present study, the two-phase solvent system of The upper phase was firstly pumped to the multiplayer coiled column as the stationary phase. Then, the lower aqueous phase was pumped into the column at a suitable flowrate of 2 mL/min, while the apparatus was rotated at a speed of 850 rpm. After hydrodynamic equilibrium was established, the sample solution was injected into the HSCCC and separated with a head-to-tail mode. The effluent of the HSCCC was continuously monitored by a UV detector at 254 nm. The peaks were collected manually according to the elution profile and then analyzed by HPLC. Finally, the retention of the stationary phase was obtained by measuring the ratio of the organic phase to the whole volume in the multilayer-coiled columns.The peak fractions from the crude sample and HSCCC separation were analyzed by HPLC equipped with a Inertsil-ODS-SP column  at room temperature. The mobile phase of acetonitrile (A) and water (B), with gradient 0 min 15% A; 35 min 70% A, was applied at a flow rate of 1.0 mL/min. Chroma grams were recorded at 254 nm. 1H- and 13C- NMR spectra. ESI-MS spectra were analyzed by an Agilent 1100/MS-G1946  mass spectrometer in the negative ionzation mode. The 1H- and 13C-NMR spectra were obtained on a Varian-600NMR spectrometer .The fractions of the target compounds obtained from the HSCCC were determined by MS, I–III were evaluated by measuring their inhibition activities on liver microsomal lipid peroxidation induced by the Fe2+-Cys system in vitro [4 was added, mixed, and incubated for 15 min at 37 °C again. The reaction was terminated by addition an equal volume of 20% TFA. The mixture was centrifuged at 3000× g rpm for 10 min. The supernatant (1 mL) was reacted with 0.67% (w/v) thiobarbituric acid in a boiling H2O bath for 10 min. After cooling, the absorbance was read at 532 nm, and the inhibitory rate was calculated. The value of IC50 was calculated by Origin 8.0 Version software  from the graph plotting inhibition percentage.The antioxidant activities of compounds in vitro . VitaminB. simaoensis. From 210 mg of the crude sample, three glucose esters with purity all over 98.0% were preparative separated and showed significant antioxidant activities by measuring their inhibition activity on liver microsomal lipid peroxidation induced by the Fe2+-Cys system in vitro. The study is of great reference value for preparative separation of glucose esters with high purity from the Balanophora plants, which also demonstrated that HSCCC is a powerful protocol for quick and efficient separation and purification of active compounds from natural products.In this study, an efficient HSCCC method was developed and successfully applied to the separation and purification of glucose esters from the crude extract of


S)-FcCH(OH)CH3 (Fc = ferrocenyl), with various pyrazoles in acidic conditions at room temperature within several minutes. X-ray structural data for racemic -1N-ethyl ferrocene (1) and its (S)-enantiomer (S)-1 were determined. A series of racemic pyrazolylalkyl ferrocenes was separated into enantiomers by analytical HPLC on β- and γ-cyclodextrins (CD) chiral stationary phases. The quantum chemical calculations of interaction energies of β-CD were carried out for both (R)- and (S)-enantiomers. A high correlation between experimental HPLC data and calculated interaction energies values was obtained.Enantiomeric-enriched ferrocene-modified pyrazoles were synthesized via the reaction of the ferrocene alcohol, ( Enantioselective synthesis is a key process in modern chemistry and is particularly important in the field of medicinal chemistry, as the different enantiomers or diastereomers of a molecule often have different biological activities. The thalidomide tragedy in the early 1960s led to much stricter testing requirements for drugs and pesticides before they can be licensed. It is especially noted into Pharmacopoeias of many countries or regions .The unique properties of organometallic compounds, intermediates between those of classical inorganic and organic materials, provide new opportunities in medicinal chemistry. A broad variety of ferrocene compounds are regarded as potential drug candidates for medical treatments ,5,6,7,8.Efficient synthetic approaches towards ferrocene modifications of organic compounds have been summarized and analyzed in the recent reviews ,23. For In this paper, the development of our ferrocene-devoted works ,34,35,36S)-ferrocene-modified pyrazoles were synthesized via the reaction of the enantioenriched ferrocene alcohol (S)-FcCH(OH)Me (Fc = ferrocenyl) with pyrazoles in acidic conditions according to the approach previously developed by us for racemic ferrocene heterocycles −1 (methanol), 1.0 mmol of the corresponding heterocycle in 1.0 mL of methylene dichloride, and 0.18 mL of 45% aqueous solution of fluoroboric acid was added under vigorous stirring. The agitation was continued for 5 min, and then Et2O (15 mL), the same amount of cold water, and 5–10 mg of ascorbic acid were added to the reaction flask. After vigorous shaking of the mixture, the organic solution was separated, washed with cold water, the solvent was removed, and the residue was dried over CaCl2 in a desiccator.To a mixture of 1.0 mmol, 0.23 g of ((S)--α-ethylferrrocene (S)-1, yield 92%, 0.28 g, ee 93%, orange crystals, m.p. 76 °C, 1H-NMR : 1.83 ; 2.20 ; 2.23 ; 4.13 ; 4.15 ; 4.20 ; 4.22 ; 5.30 ; 5.75 Pz). 13C-NMR : 11.6 (CH3), 13.7 (CH3), 20.2 (CH3), 53.9 (CH), 66.7 (C5H4), 67.3 (C5H4), 67.8 (C5H4), 68.3 (C5H4), 68.8 (C5H5), 89.9 (ipso-C5H4), 105.2 , 137.7 , 146.6 . IR : 3010, 2890, 1560, 1470, 1120, 1009, 840, 778, 539, 472. EI-MS, m/z : 308 [M]+ (92).(S)-(3-Trifluoromethyl-5-methylpyrazolyl)-α-ethylferrrocene (S)-3, yield 88%, 0.32 g, yellow crystals, m.p. 47 °C, 1H-NMR : 1.85 ; 2.25 ; 4.04 ; 4.13 ; 4.21 ; 5.33 ; 6.23 Pz). 13C-NMR : 11.5 (CH3), 20.4 (CH3), 55.0 (CH), 66.5 (C5H4), 67.4 (C5H4), 67.8 (C5H4), 68.4 (C5H4), 68.9 (C5H5), 89.3 (ipso-C5H4), 103.7  = 1.8 Hz, Pz, C-4), 124.4  = 266.7 Hz, CF3), 138.8 , 140.5  = 37.5 Hz, Pz, C-3). IR : 3029, 2898, 1564, 1456, 1265, 1120, 1011, 844, 776, 547, 472. EI-MS, m/z : 362 [M]+ (89).(S)--α-ethylferrrocene (S)-4, yield 87%, 0.36 g, orange oil, 1H-NMR : 1.88 ; 4.14–4.22 ; 5.45 ; 6.56 Pz). 13C-NMR : 20.6 (CH3), 58.1 (CH), 66.1 (C5H4), 67.0 (C5H4), 67.7 (C5H4), 68.3 (C5H4), 68.9 (C5H5), 89.6 (ipso-C5H4), 106.3 , 119.8  = 267 Hz, CF3), 122.4  = 267 Hz, CF3), 138.2  = 37.5 Hz, Pz, C-5), 141.4  = 37.5 Hz, Pz, C-3). IR : 3010, 2964, 1575, 1510, 1462, 1415, 1396, 1163, 1040, 987, 840, 715, 547, 498. EI-MS, m/z : 416 [M]+ (100).(S)-(Pyrazolyl)-α-ethylferrrocene (S)-5, yield 86%, 0.24 g, ee 95%, orange crystals, m.p. 54 °C, 1H-NMR : 1.86 ; 4.17–4.29 ; 5.41 ; 6.21 Pz); 7.30 ; 7.51 Pz). 13C-NMR : 21.4 (CH3), 57.3 (CH), 66.2 (C5H4), 67.9 (C5H4), 68.0 (C5H4), 68.5 (C5H4), 68.9 (C5H5), 89.0 (ipso-C5H4), 105.0 , 126.7 (Pz), 138.4 . IR : 3115, 2965, 1525, 1411, 1293, 1120, 1045, 968, 853, 720, 665, 538, 498. EI-MS, m/z : 280 [M]+ (82).N-ethyl ferrocene (R)- and (S)-enantiomers as well as the interaction energies of β-CD with (R)- and (S)-enantiomers were performed by the restricted Becke−Lee−Young−Parr modification (RB3LYP) of the density functional theory [R- and S-enantiomers.The calculations of β-cyclodextrin (β-CD), 1l theory ,63,64. Td6 compounds 1 and (S)-1. X-ray diffraction measurements were carried out using CAD4 Enraf-Nonius and Bruker Smart APEX II diffractometers. The structures were solved by a direct method and refined by the full-matrix least-squares technique against F2 in an anisotropic approximation. Hydrogen atoms were located from the difference Fourier maps and refined in rigid body model. All calculations were performed using the SHELXTL program package [Single crystals suitable for X-ray diffraction analysis were obtained by recrystallization from acetone- package . Details1 and (S)-1 have been deposited in the Cambridge Crystallographic Data Centre (CCDC Nos. 1491349 and 1491350) and can be obtained free of charge via the webpage: http://www.ccdc.cam.ac.uk/Community/Requestastructure/pages/Requestastructure.aspx.Bond lengths and angles, thermal parameters, and experimental conditions of the structures In summary, we reported an efficient, highly enantiospecific synthesis of chiral pyrazole ferrocenes via direct substitution under acidic conditions in two-phase media from ferrocene alcohols and pyrazoles in good yields with very high enantiomeric purity.


Acanthopanax gracilistylus  resulted in the discovery of a new lupane-triterpene compound, acangraciligenin S (1), and a new lupane-triterpene glycoside, acangraciliside S (2), as well as two known ones, 3α,11α-dihydroxy-lup-20(29)-en-23,28-dioic acid (3) and acankoreoside C (4). Their chemical structures were elucidated by mass, 1D- and 2D-nuclear magnetic resonance (NMR) spectroscopy. The chemical structures of the new compounds 1 and 2 were determined to be 1β,3α-dihydroxy-lup-20(29)-en-23, 28-dioic acid and 1β,3α-dihydroxy-lup-20(29)-en-23,28-dioic acid 28-O-[α-l-rhamnopyranosyl-(1→4)-β-d-glucopyranosyl-(1→6)-β-d-glucopyranosyl] ester, respectively. The anti-neuroinflammatory activity of the selective compounds, 1 and 3, were evaluated with lipopolysaccharide (LPS)-induced BV2 microglia. The tested compounds showed moderate inhibitory effect of nitric oxide (NO) production.The phytochemical study on the leaves of  Acanthopanax gracilistylus W. W. Smith belongs to Araliaceae, which is widely distributed in China, and its dried roots and stem barks are listed officially in the Chinese Pharmacopoeia (2015 edition) as Acanthopanax Cortex (named as Wujiapi), which has been used as medicine for the treatment of paralysis, arthritis, rheumatism, lameness, and liver disease − , indicating eight degrees of unsaturation. The 1H-NMR spectrum of 1 (in acetone-d6) showed the following signals: five tertiary methyl groups at δ 0.97, 0.99, 1.07, 1.17 and 1.71 ; two olefinic protons at δ 4.58  and 4.72 ; two hydroxy-substituted methines at δ 3.82  and 3.84 . The 13C-NMR and distortionless enhancement by polarization transfer (DEPT) spectra revealed 30 carbon signals. The skeleton of 1 was recognized to be lupane triterpenoid by 1H- and 13C-NMR analysis  correlated with carbons C-2 (δ 37.36), C-9 (δ 52.98), C-10 (δ 44.33), and C-25 (δ 13.24); protons H-2 at δ 1.82  and H-25 at δ 0.97  correlated with carbon C-1 (δ 75.46); the H-3 proton signal at δ 3.82  correlated with carbons C-5 (δ 45.25) and C-23 (δ 178.16); protons H-2 at δ 1.82  and H-24 at δ 1.17  correlated with carbon C-3 (δ 73.99); protons H-5 at δ 1.95  and H-24 at δ 1.17  correlated with carbon C-23 (δ 178.16); protons H-16 at δ 1.48 , H-18 at δ 1.64 , and H-22 at δ 1.92  correlated with carbon C-28 (δ 177.84); the correlations from H-29 at δ 4.58  and δ 4.72  to C-19 (δ 48.24), C-20 (δ 151.89), and C-30 (δ 19.72); the correlations from H-30 at δ 1.71  to C-19 (δ 48.24), C-20 (δ 151.89), and C-29 (δ 110.26). These pieces of evidence confirmed that the double bond was at C-20/C-29 and the other hydroxyl group was at C-1  was axial and H-3 was equatorial, which, in turn, suggested that the hydroxyl group at C-3 and carboxyl group at C-4 were α-positioned. Furthermore, cross-peaks between H-19 and H-13 confirmed that the isopropenyl group at C-19 was α-positioned -en-23,28-dioic acid, a new compound named acangraciligenin S.Compound ysis see , with ths at C-1 . The nucsitioned . Based o2 was obtained as a white amorphous powder, m.p. 230.5 °C, and gave positive responses in Liebermann–Burchard and Molish tests. Its molecular formula of C48H76O20 was determined on the basis of ESI-MS at m/z = 972.5 [M]+ and HR-ESI-MS at m/z = 971.4865 [M − H]− (negative) , indicating eleven degrees of unsaturation. The 1H-NMR spectrum of 2 (in methanol-d4) showed the following signals: five tertiary methyl groups at δ 0.95, 0.98, 1.03, 1.09 and 1.70 ; one secondary methyl group at δ 1.25 , assigned to H-6″ of the rhamnose; and two olefinic protons at δ 4.58  and 4.72 ; two hydroxy-substituted methines at δ 3.67  and 3.80 . In addition, three anomeric protons were at δ 4.37 , 4.84 , and 5.45 , suggesting the appearance of three sugar units. The 13C-NMR and DEPT spectra revealed 48 carbon signals, of which, 30 signals were assigned to a triterpenoid sapogenol moiety and 18 signals belong to three monosaccharide moieties. The aglycone of 2 was recognized to be lupane triterpene type by 1H- and 13C-NMR analysis , α-hydroxyl group at C-3 (δ 74.14), and two carboxyl groups at C-23 (δ 182.60) and C-28 (δ 176.40) were performed by comparing its spectral data with compound 1  and literature values  ester, a new compound named acangraciliside S.The NOESY spectrum of sitioned . Alkalinobserved . In addiectively . These rcone δ 17.40, betw species ,27,28. C3 and 4 were identified as 3α,11α-dihydroxy-lup-20(29)-en-23,28-dioic acid -2,5-diphenyltetrazolium bromide (MTT) to 1 mL of a cell suspension (1 × 105 cells/mL in 96-well plates) and incubated for 30 min. The formazan formed was dissolved in acidic 2-propanol, and the optical density was measured at 540 nm.BV2 microglias were maintained at 5 × 10w/v) N-(1-naphathyl)-ethylenediamine and 1% (w/v) sulfanilamide in 5% (v/v) phosphoric acid) for 10 min at room temperature. The absorbance of the final product was measured spectrophotometrically at 540 nm using an enzyme-linked immunosorbent assay (ELISA) plate reader. The nitrite concentration in the samples was determined from a standard curve of sodium nitrite prepared in phenol red-free DMEM.The concentration of nitric oxide (NO) in the conditioned media was determined by a method based on the Griess reaction . An aliqt-test). Statistical analysis was performed using GraphPad Prism software, version 3.03 . Statistical significance was accepted at p < 0.05.All values are expressed as the mean ± S.D. Differences between mean values of normally-distributed data were assessed with one-way analysis of variance (ANOVA)  by using HSCCC-HPLC combinatorial chromatography method resulted in the discovery of four lupane-triterpenoids, including two new compounds, acangraciligenin S (1) and acangraciliside S (2), as well as two known species, 3α,11α-dihydroxy-lup-20(29)-en-23,28-dioic acid (3) and acankoreoside C (4). Two selected compounds were also evaluated for their inhibitory activity against lipopolysaccharide (LPS)-induced nitric oxide (NO) production in murine microglia BV2 cells. Compounds 1 and 3 showed moderate abilities to inhibit NO production and had no influence on cell viability, as determined by the MTT method.In summary, in this work, a phytochemical study on the leaves of


Their structures were elucidated via HRESIMS and 1D and 2D NMR spectroscopic analyses, and further verified via transformation methods. In addition, compounds 1–3 and five semisynthetic congeners (1a–e) were assayed for the activity to induce neurite outgrowth in rat pheochromocytoma (PC12) cells. As a result, nepalactone A derivative 1c and nepalarin (3) significantly enhanced nerve growth factor (NGF)-mediated neurite outgrowth in PC12 cells.Two picrotoxane sesquiterpene lactone glycosides, nepalactones A ( Coriaria nepalensis Wall (Coriaria sinica Maxim) (Coriariaceae) is a Chinese medicine herb mainly distributed in the southwest of China. Traditionally, this herb is used to treat numbness, toothache, traumatic injury, and acute conjunctivitis +). Furthermore, the 1H and 13C-NMR data of 1 were similar to those of the known picrotoxane sesquiterpenoid, corialactone D (1b), which has been previously identified from the same plant –CH3(10)–units, whose connectivity was defined by the key HMBC correlations (Compound tra of 1  exhibitespectrum  between  spectra  and HRESelations .1b) of 1 was defined by a NOESY experiment . Additionally, enzymatic hydrolysis of 1 afforded the known intact aglycone 1b, which was identified from 1H NMR, and its absolute configuration  was previously determined from CD spectrum  at C-5 in 3. The location of the isopentenyl unit at C-5 was supported by the HMBC correlations of H-2′′ to C-5, and H-1′′ to C-6 and C-10. Full analyses of the 1H and 13C-NMR spectral data of 3, supported by the 1H-1H COSY, DEPT, HSQC, and HMBC experiments  coumarin and named nepalin C. To our knowledge, this is a rare coumarin with three isopentenyl units in natural source.Compound eriments , permitt1–3, and 1a–1e) were evaluated for the effect on NGF-induced neurite outgrowth in rat pheochromocytoma PC12 cells according to our previously reported procedures +, 846.62 [2M + Na]+; HRESIMS m/z 435.1991 [M + Na]+ .Nepalactone B (2). White powder; c 0.24, MeOH); IR (KBr) νmax 3390, 1769, 1076, 1028 cm−1; 1H and 13C-NMR data, see m/z 823.15 [2M − H]−; HRESIMS m/z 435.1987 [M + Na]+ .Nepalarin (3). Pale yellow needles; UV(MeOH): λmax (logε) nm: 221 (4.28), 336 (4.22); IR (KBr) νmax 3355, 1731, cm−1; IR (KBr) νmax 3319, 1692, 1583, 1458, 1394, 1306, 1067, 1041, 1015 cm−1; 1H-NMR  δ 7.51 , 5.34 , 5.33 , 5.11 , 3.84 , 3.58 , 3.56 , 3.26 , 1.89 , 1.86 , 1.83 , 1.75, 1.72 , 1.71 ; 13C-NMR  δ 162.3 (C-2), 149.5 (C-7), 149.3 (C-9), 142.0 (C-6), 135.9 (C-4), 135.2 (C-3′), 133.1 (C-3′′′), 132.8 (C-3′′), 128.2 (C-10), 124.6 (C-3), 122.6 (C-2′′), 121.1 (C-2′′′), 119.8 (C-2′), 114.3 (C-8), 111.8 (C-5), 62.2 (6-OCH3), 28.5 (C-1′), 25.9 (C-4′), 25.8 (C-5′′′), 25.6 (C-4′′), 24.7 (C-1′′), 22.4 (C-1′′′), 18.1 (C-5′′), 18.0 (C-4′′′), 17.8 (C-5′); ESI-MS m/z 419.49 [M + Na]+, 815.18 [2M + Na]+, 341.19 [M − C4H7]+; ESI-MS m/z 395.41 [M − H]−; HRESIMS m/z 395.2226 [M − H]− .1  in pyridine (1 mL), acetic anhydride (1 mL) was added, and the mixture stood at RT for 24 h. The reaction mixture was concentrated to dryness, and the residue were purified on silica gel CC using EtOAc, affording tetraacetate 1a (ca. 3 mg) as an off-white gum. c 0.2, MeOH); 1H and 13C-NMR data, see m/z [M + Na]+ 603.2416 .To a solution of 1 (2 mg) was hydrolyzed with 2N HCl-dioxane  at 100 °C for 2 h. The reaction mixture was partitioned between chloroform and H2O three times. The aqueous layer was neutralized with 2 M Ag2CO3 and evaporated in vacuo. The residue was dissolved in pyridine (0.5 mL), to which l-cysteine methyl ester hydrochloride in pyridine  was added. After reacting at 60 °C for 1 h, trimethylsilylimidazole (0.05 mL) was added to the reaction mixture and kept at 4 °C for another 8 h. The mixture was analyzed via GC–MS. By comparison of the retention time of the authentic sample, the monosaccharide of 1 was determined to be d-glucose (tR 11.85 min) (tR 12.15 min of l-glucose).Compound 1 (20 mg) in a mixture of a citrate buffer  and water (1 mL), β-gluosidase  was added. After the reaction mixture was incubated at 30 °C for 5 days, it was extracted with EtOAc, dried over anhydrous sodium sulfate, concentrated, and purified via column chromatography using MeOH/CHCl3 (1:15) to yield aglycon 1b as a white solid , which was identified as a sesquiterpenoid, corialactone D, via comparison with the 1H-NMR spectrum. 1H-NMR  δ 1.73 , 2.46 , 4.68 , 2.02 , 2.36 , 2.36 , 1.30 , 1.83 , 1.02 , 1.00 , 2.37 , 2.10 , 4.86 , 5.33 , 5.18 .To a solution of 1b  in CH2Cl2 (1 mL) at room temperature, NaHCO3  and Dess-Martin periodinane (DMP)  were added. After 40 min of stirring, the reaction mixture was poured into a mixture of a saturated aqueous solution of Na2S2O3 (5 mL), a saturated aqueous solution of NaHCO3 (5 mL), and water (5 mL). The layers were separated, and the aqueous phase was extracted with CH2Cl2. The combined organic extracts were washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified via flash chromatography on silica gel (PE/EtOAc 5/1 to 2/1), affording 1c  as a slightly yellow oil; 1H-NMR  δ 6.06 , 5.25 , 4.66 , 2.65 , 2.46 , 2.38 , 2.36 , 2.34 , 1.99 , 1.78 , 1.77 , 1.15 , 0.96 , 0.94 ; 13C-NMR  δ 203.7 (C-12), 176.7 (C-15), 151.9 (C-13), 117.5 (C-14), 79.0 (C-3), 51.2 (C-4), 45.0 (C-5), 40.1 (C-1), 39.8 (C-11), 35.5 (C-6), 33.5 (C-2), 32.2 (C-7), 25.3 (C-8), 20.6 (C-10), 19.73 (C-9); ESIMS m/z 249.08 [M + H]+, 271.15 [M + Na]+; HRESIMS m/z 271.1307 [M + Na]+ .To a solution of 1 (30 mg) was hydrolyzed with 4 N HCl–dioxane  at 100 °C for 2 h. The reaction mixture was partitioned between chloroform and H2O three times. The organic layer was neutralized with 20 M Ag2CO3 and filtered. The resulting solution was washed with brine (50 mL), dried over Na2SO4, and then filtered and evaporated. The residue was subjected to column chromatography on silica gel using petroleum ether and ethyl acetate as eluent to yield 1d  and 1e  as a yellowish oil.Compound 1d: amorphous powder; IR (KBr)νmax 3402, 1765, 1076, 1025 cm−1; 1H-NMR  δ 5.74 , 4.64 , 4.23 , 4.12 , 1.79 , 1.25 , 0.99 , 0.96 ; 13C-NMR  δ 178.1 (C-15), 144.9 (C-13), 130.2 (C-12), 79.6 (C-3), 50.9 (C-4), 46.4 (C-1), 44.7 (C-5), 43.8 (C-6), 40.8 (C-14), 34.0 (C-11), 30.8 (C-2), 29.1 (C-7), 25.8 (C-8), 20.6 (C-10), 19.8 (C-9); HRESIMS m/z 291.1128 [M + Na]+ .1e: amorphous powder; IR (KBr)νmax 3402, 1765, 1076, 1025 cm−1; 1H-NMR  δ 5.59 , 4.65 , 4.18 , 1.79 , 1.15 , 1.00 , 0.97 ; 13C-NMR  δ 178.3 (C-15), 149.1 (C-13), 126.4 (C-12), 79.9 (C-3), 59.8 (C-14), 51.0 (C-4), 46.1 (C-1), 44.8 (C-5), 42.7 (C-6), 34.2 (C-11), 31.4 (C-2), 28.4 (C-7), 25.2 (C-8), 20.7 (C-10), 19.8 (C-9); HRESIMS m/z 273.1470 [M + Na]+ .p < 0.01). Statistical analyses were performed using Dunnett’s t-test.Neurite outgrowth-promoting activity of these isolated compounds was assessed as described previously by our group ,18. Expe1) and B (2) and one new coumarin nepalarin (3), from the root barks of C.nepalensis. In addition, five congeners 1a–e were synthesized from nepalactone A. We for the first time discovered that these compounds, especially compound 1c and 3, could potentiate NGF-induced neurite outgrowth in PC12 cells.In conclusion, the present study reported the identification of three new compounds, including two new picrotoxane sesquiterpene glycosides nepalactones A (


Unexpectedly Pleurotus ostreatus produced together with the hydroxylactone, 2-hydroxy-4,4-dimethyl-9-oxabicyclo[4.3.0]nonane-8-one (3), its own metabolite --3,9,15-trimethyl-4,10,16-trioxacyclohexa-deca-6,12-diene-1,5,8,11,14-pentaone (4). The method presented here, in which this macrosphelide 4 was obtained by biotransformation, has not been previously described in the literature. To the best of our knowledge, this compound has been prepared only by chemical synthesis to date. This is the first report on the possibility of the biosynthesis of this compound by the Pleurotus ostreatus strain. The conditions and factors, like temperature, salts, organic solvents, affecting the production of this macrosphelide by Pleurotus ostreatus strain were examined. The highest yield of macroshphelide production was noticed for halolactones, as well with iodide, bromide, iron and copper (2+) ions as inductors.The aim of the study was to obtain new compounds during biotransformation of two halocompounds, the δ-bromo and δ-iodo-γ-bicyclolactones  Fungi are remarkable organisms that easily produce a wide range of secondary metabolites with broad spectrum biological activity . Forty-tMicrosphaeropsis sp. nonan-8-one (3): 1H-NMR  δ: 0.95 , 1.00 , 1.03 , 1.38 , 1.43 , 1.66 , 1.99 , 2.27 , 2.60 , 2.77 , 3.95 , 4.58 .--3,9,15-Trimethyl-4,10,16-trioxacyclohexa-deca-6,12-diene-1,5,8,11,14-pentaone (4): 1H-NMR  δ: 1.44 , 1.49 , 1.61 , 2.70 , 2.92 , 5.22 , 5.29 , 5.40 , 6.66 , 6.87 , 7.18 , 7.38 , 13C-NMR , δ: 15.93 (CH3C-15), 17.07 (CH3C-9), 19.52 (CH3C-3), 40.64 (C-2), 69.22 (C-3), 75.56 (C-15), 76.35 (C-9), 132.03 (C-6), 132.30 (C-13), 132.33 (C-12), 134.38 (C-7), 163.11 (C-1), 163.49 (C-8), 170.11 (C-14), 195.61 (C-5), 197.46 (C-11).P. ostreatus were prepared as described in the screening procedure. The culture was grown at three different temperatures: 21 °C–22 °C, 25 °C–26 °C and 28 °C–30 °C. After three, five, seven and nine days of biotransformation, one-fourth the volume of the medium with overgrown mycelium was taken from each flask. Such samples were extracted with chloroform, dried and analysed by means of GC.Six Erlenmeyer flasks with the three-day cultures of 2 was added to a culture of P. ostreatus after three or six days. After three, five, seven and nine days of biotransformation the culture was extracted with chloroform and analysed by means of GC. As a control, in one flask the microorganism was grown without substrate 2.Substrate P. ostreatus were prepared as described in the screening procedure. To each flask was added, respectively KI (5 mg), KBr (5 mg), NaCl (3 mg), NaCl (585 mg), DMSO (1 mL), H2O2, , acetone (1 mL), ethanol (1.2 mL), propanol (0.75 mL), propionic acid (0.38 mL), FeSO4 × 7H2O (5 mg) and EDTA (25 mg), FeCl3 × 6H2O (5 mg) and EDTA (25 mg), CuSO4 × 5H2O (5 mg) and EDTA (25 mg), CuCl (5 mg) and EDTA (25 mg), CoCl2 × 6H2O (5 mg) and EDTA (25 mg), ZnCl2 (5 mg) and EDTA (25 mg), MnCl2 (5 mg) and EDTA (25 mg). The salts were dissolved in deionized water. In addition, a control experiment, without any salt, was also performed. After seven days the cultures were extracted with chloroform and analysed by means of GC.Eighteen Erlenmeyer flasks with three-day cultures of 1, whereas iodolactone 2 was transformed by five strains. A complete conversion of the substrate was observed only in the Pleurotus ostreatus culture. Both substrates were converted into hydroxylactone 3 by hydrolytic dehalogenation. When chloroform was used for the extraction instead of methylene chloride, the presence of a fungal metabolite, macrosphelide 4, was observed in the extract in addition to hydroxylactone 3. Further studies have demonstrated that the biosynthesis of macrosphelide 4 was induced not only by substrates 1 and 2, but also by iodide, bromide, iron, copper, manganese and cobalt ions. The present work is the first to discover the production of macrosphelide 4 in Pleurotus ostreatus culture and to investigate the induction of its biosynthesis.From the nine tested microorganism strains, four were able to biotransform bromolactone


Dolichos lablab L. (DLL-Ex) inhibited high-fat diet (HFD)-induced increases in body weight and body fat mass and ameliorated increases in body weight. In the present work, we studyed the molecular mechanism for the inhibitory effect of DLL-Ex or Chikusetsusaponin IVa (CS-IVa), as isolated from Dolichos lablab L. (DLL) seeds extract, on adipocyte differentiation. We evaluated the effect of DLL-Ex, an anti-obesity agent, and CS-IVa, an active component of DLL-Ex, on 3T3-L1 cell differentiation via Oil red O assay and Q-PCR, along with their effects on CCAAT element binding protein alpha (C/EBPα), peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid synthase (FAS), and fatty acid-binding protein 4 (FABP4) mRNA transcriptions. FAS and FABP4 protein expression levels after exposure to CS-IVa were also tested. The results showed that DLL-Ex and CS-IVa have potent inhibitory activity on adipocyte differentiation. Therefore, DLL and CS-IVa may be developed as a functional food material to treat obesity.Obesity, a condition where excess body fat accumulates to the extent, causes a negative effect on health. Previously, we reported the extract of  Dolichos lablab L. (DLL), also known as hyacinth bean and part of the family Fabaceae, is widely grown in Africa and southern Asia, including in India and China − . 1H-NMR  δ 6.26 , 5.40 , 4.95 , 3.34 , 3.16 , 1.28 , 1.26 , 1.07 , 0.96 , 0.90 , 0.87 , 0.81 ; 13C-NMR  δ 176.82 (C-28), 176.78 (CGlcUA-6), 144.49 (C-13), 123.23 (C-12), 107.39 (CGlcUA-1), 96.09 (CGlc-1), 89.44 (C-3), 79.67 (CGlc-5), 79.18 (CGlc-3), 78.53 (CGlcUA-4), 77.66 (CGlcUA-3), 75.77 (CGlcUA-5), 74.43 (CGlc-2), 73.94 (CGlcUA-2), 71.07 (CGlc-4), 62.51 (CGlc-6), 56.12 (C-5), 48.34 (C-9), 47.34 (C-17), 46.55 (C-19), 42.47 (C-14), 42.07 (C-18), 40.25 (C-8), 39.85 (C-4), 39.00 (C-1), 37.29 (C-10), 34.36 (C-21), 33.54 (C-7), 33.53 (C-29), 32.87 (C-22), 31.15 (C-20), 28.61 (C-23), 26.50 (C-2), 28.58 (C-15), 26.88 (C-27), 24.12 (C-16), 24.01 (C-11), 23.74 (C-30), 18.84 (C-6), 17.81 (C-26), 17.35 (C-24), 15.91 (C-25). 1H-NMR spectrum of CS-IVa exhibited seven methyl signals , and an olefin signal H-12 [5.40 ], and the 13C-NMR spectrum of isolated compound showed one oleanolic acid group . The 1H-NMR spectrum of CS-IVa showed two anomeric proton signals  and the 13C-NMR spectrum of CS-IVa also showed the existence of a glucose and a glucuronic acid group . From these results, the structure of isolated compound was identified as Chikusetsusaponin IVa (CS-IVa), indicating the molecular formula as C42H66O14, through a comparison with reference data [CS-IVa was isolated from water soluble fraction of 70% prethanol A extract . The ext 34.36 C-, 33.54 , is a carrier protein for fatty acid that is primarily expressed in adipocyte. FABP4 is similar to the FABPs family and are thought to enhance the transfer of fatty acids through binding to fatty acid ,33. SpecSaponins are potential biological activity compounds that are insisted in various natural plants. Generally, saponins contains triterpenoid saponin and steroid saponin, that triterpenoids have C30 triterpene skeleton and steroid saponins have C27 steroidal skeleton. Some of saponins, especially triterpenoid saponins, are found as saponin glycosides that attach various sugar molecules with the triterpene skeleton, such as glucose, galactose. It is reported that triterpenoid saponins showed many biological activities, such as anti-microbial, hypolipidemic, immunomodulatory, anti-tumor, anti-diabetic, hypocholesterolemia, anti-coagulant, anti-carcinogenic, hepatoprotective, hypoglycemic, neuroprotective, anti-inflammatory, and anti-oxidant activity ,42,43. SOur recent animal study revealed that DLL-Ex not only repressed hepatic steatosis but also repressed weight gain by HFD . PresentCS-IVa, which is a bioactive component of DLL-Ex (0.54% in DLL-Ex), decreased the expression of genes that are important to the adipogenesis process in 3T3-L1 adipocytes, suggesting that it is the active ingredient that suppresses the adipocyte size, number, and intracellular fat accumulation found after treatment with DLL-Ex. The biological availability concentration of CS-IVa is 16.29 mg/mL in raw seeds, or 1.85 mg/mL in DLL-Ex. These results suggest that CS-IVa and DLL-Ex might be developed into a drug for the treatment of obesity and related diseases.