Their structures were determined as 10β,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-one (2), 6β,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-one (3), 6β,10β,17β-trihydroxy-7α,17α-dimethylestr-4-en-3-one (4), 11β,17β-dihydroxy-(20-hydroxymethyl)-7α,17α-dimethylestr-4-en-3-one (5), 1α,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-one (6), 1α,11β,17β-trihydroxy-7α,17α-dimethylestr-4-en-3-one (7), and 11β,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-one (8), on the basis of spectroscopic studies. All metabolites, except 8, were identified as new compounds. This study indicates that C. blakesleeana, and C. echinulata are able to catalyze hydroxylation at allylic positions, while M. phaseolina can catalyze hydroxylation of CH2 and CH3 groups of substrate 1. Mibolerone (1) was found to be a moderate inhibitor of β-glucuronidase enzyme (IC50 = 42.98 ± 1.24 μM) during random biological screening, while its metabolites 2–4, and 8 were found to be inactive. Mibolerone (1) was also found to be significantly active against Leishmania major promastigotes (IC50 = 29.64 ± 0.88 μM). Its transformed products 3 (IC50 = 79.09 ± 0.06 μM), and 8 (IC50 = 70.09 ± 0.05 μM) showed a weak leishmanicidal activity, while 2 and 4 were found to be inactive. In addition, substrate 1 (IC50 = 35.7 ± 4.46 μM), and its metabolite 8 (IC50 = 34.16 ± 5.3 μM) exhibited potent cytotoxicity against HeLa cancer cell line . Metabolite 2 (IC50 = 46.5 ± 5.4 μM) also showed a significant cytotoxicity, while 3 (IC50 = 107.8 ± 4.0 μM) and 4 (IC50 = 152.5 ± 2.15 μM) showed weak cytotoxicity against HeLa cancer cell line. Compound 1 (IC50 = 46.3 ± 11.7 μM), and its transformed products 2 (IC50 = 43.3 ± 7.7 μM), 3 (IC50 = 65.6 ± 2.5 μM), and 4 (IC50 = 89.4 ± 2.7 μM) were also found to be moderately toxic to 3T3 cell line (mouse fibroblast). Interestingly, metabolite 8 showed no cytotoxicity against 3T3 cell line. Compounds 1–4, and 8 were also evaluated for inhibition of tyrosinase, carbonic anhydrase, and α-glucosidase enzymes, and all were found to be inactive.Seven metabolites were obtained from the microbial transformation of anabolic-androgenic steroid mibolerone ( It is stable to metabolic conversion in the rat ventral prostate. Because of its stability and high affinity binding, it has been used as a ligand for the characterization and quantitation of androgen in prostate, liver, and cultured cells. In addition, mibolerone (1) is more receptor-selective for androgenic receptor than methyltrienolone. The binding interaction of compound 1 with testosterone-estradiol binding globulin of human serum is weaker than 5α-dihydrotestosterone. Mibolerone (1) also acts through the progesterone receptor (PR) as it eliminates progesterone receptor expression at lower doses (1 nM), in contrast to 5α-dehydrotestosterone (10–100 nM), which reduces PR to basal levels. In breast cancer cells, mibolerone (1) has shown a dual action, i.e., androgenic and progestagenic . Reflection intensities were integrated using SAINT software. Absorption correction was done on M-multi-scan. Structures were solved on SHELXTL  R1 = 0.0661, wR2 = 0.2004, R indices R1 = 0.0695, wR2 = 0.2074 for all data, largest diff. peak and hole 0.599 and -0.620 e.A-3.Single-crystal X-ray data: empirical formula Cmax (log ε): 235 (4.38); c 0.004, CH3OH); IR (CHCl3) υmax; 3360 (O-H), 1654 ; EI-MS m/z : 318.2 [M+] (59), 300.0 (63), 290.2 (23), 230.2 (47), 229.1 (64), 43.9 (100); HREI-MS m/z: 318.2192 [M+] ; 1H-NMR : 13C-NMR : 20H30O3; formula weight 318.44; crystal system orthorhombic space group P212121, unit cell, a = 8.338(2) Å, b = 9.733(2) Å, c = 20.949(5) Å, α = β = γ = 90°, volume 1700.1(7) A3, Z = 4, calculated density 1.244 mg/m3, F(000) 696, crystal size 0.31 x 0.11 x 0.07 mm, Ɵ range for data collection 4.22 to 43.50°, reflections collected 3,259, unique reflections 770 (Rint = 0.0465), goodness-of-fit on F2 1.119, final R indices [I>2δ(I)] R1 = 0.0418, wR2 = 0.1008, R indices R1 = 0.0458, wR2 = 0.1035 for all data, largest diff. peak and hole 0.294 and -0.200 e.A-3.White crystalline; yield (percentage yield): 40 mg (4%); melting point 200 ‒ 201°C; UV λmax (log ε): 230 (4.27); c 0.0014, CH3OH); IR (CHCl3) υmax; 3431 (O-H), 1659 ; EI-MS m/z : 318.3 [M+] (16), 300.3 (100), 242.2 (39), 229.2 (98), 174.1 (41), 147.0 (27), 136.0 (26), 95.0 (21), 42.9 (54); HRESI-MS m/z: 318.2196 [M+] ; 1H-NMR : 13C-NMR : White solid; yield (percentage yield): 20 mg (2%); melting point 219‒221°C; UV λmax (log ε): 230 (5.20); c 0.0041, CH3OH); IR (CHCl3) υmax; 3412 (O-H), 1664 ; EI-MS m/z : 334.2 [M+] (100), 298.2 (24), 240.1 (11), 227.1 (64), 172.1 (19), 43.0 (16); HREI-MS m/z: 334.2159 [M+] ; 1H-NMR : 13C-NMR : 20H32O5; formula weight 352.46; crystal system orthorhombic, space group P212121, unit cell, a = 7.6451(10) Å, b = 10.734(2) Å, c = 23.051(3) Å, α = β = γ = 90°, Volume 1891.6(5) A3, Z = 4, calculated density 1.238 mg/m3, F(000) 768, crystal size 0.30 x 0.25 x 0.20 mm, Ɵ range for data collection 7.11 to 70.13°, reflections collected 15,121, unique reflections 2040 (Rint = 0.0240), goodness-of-fit on F2 1.079, final R indices [I>2δ(I)] R1 = 0.0300, wR2 = 0.0799, R indices R1 = 0.0302, wR2 = 0.0802 for all data, largest diff. peak and hole 0.243 and -0.175 e.A-3.White crystalline; yield (percentage yield): 300 mg (30%); melting point 228‒229°C; UV λ1 with M. phaseolina afforded 2.3 g crude extract. Silica gel column chromatography of the crude extract yielded four main fractions (4–7), which were further purified on reverse phase recycling HPLC. Metabolite 5  was obtained from fraction 4 through purification on reverse phase HPLC . Metabolite 6  from fraction 5, compound 7  from fraction 6, and compound 8  from fraction 7 were obtained, on purification with reverse phase recycling HPLC .Incubation of substrate max (log ε): 248 (3.82); c 0.0019, CH3OH); IR (CHCl3) υmax; 3412 (O-H), 1643 ; EI-MS m/z : 334.2 [M+] (25), 317.2 (31), 286.1 (32), 285.1 (80), 267.1 (51), 242.1 (50), 229.1 (100) 133.1 (18), 55.0 (11); HREI-MS m/z: 334.2146 [M+] ; 1H-NMR : 13C-NMR : White solid; yield (percentage yield): 2.9 mg (0.4%); melting point 228 ‒ 230°C; UV λmax (log ε): 247 (2.99); c 0.0049, CH3OH); (CHCl3); υmax 3433 (O-H), 1651 ; EI-MS m/z : 318.9 [M+] (28), 301.9 (35), 300.9 (100), 299.9 (96), 261.9 (61), 228.8 (67), 226.9 (49) 173.9 (53), 135.0 (33); HREI-MS m/z: 318.2193 [M+] ; 1H-NMR : 13C-NMR : White solid; yield (percentage yield): 2.4 mg (0.24%); melting point 170‒174°; UV λmax (log ε): 247 (4.12); c 0.0019, CH3OH); IR (CHCl3) υmax; 3418 (O-H), 1661 ; EI-MS m/z : 334.2 [M+] (2.6), 318.1 (41), 317.1 (90), 259.1 (68), 241.1 (49), 228.1 (100), 214.1 (81) 201.1 (47), 43.0 (36); HREI-MS m/z 334.2140 [M+] ; 1H-NMR : 13C-NMR : White solid; yield (percentage yield): 2.4 mg (0.34%); melting point 280 ‒ 282°C; UV λmax (log ε): 229 (3.88); c 0.0021, IR CH3OH); (CHCl3) υmax; 3410 (O-H), 1651 ; EI-MS m/z : 334.2 [M+] (2.6), 318.1 (41), 317.1 (90), 259.1 (68), 241.1 (49), 228.1 (100), 214.1 (81) 201.1 (47), 43.0 (36); HREI-MS m/z: 318.2211 [M+] ; 1H-NMR : 13C-NMR : 20H30O3; formula weight 318.44; crystal system monoclinic, space group P21, unit cell, a = 5.9750(11) Å, b = 11.550(3) Å, c = 12.642(2) Å, β = 99.728 A3, Z = 2, calculated density 1.230 mg/m3, F(000) 348, crystal size 0.32 x 0.28 x 0.18 mm, Ɵ range for data collection 7.52 to 72.00°, reflections collected 13,571, unique reflections 3,334 (Rint = 0.0236), goodness-of-fit on F2 1.037, final R indices [I>2δ(I)] R1 = 0.0270, wR2 = 0.0734, R indices R1 = 0.0272, wR2 = 0.0736 for all data, largest diff. peak and hole 0.219 and -0.160 e. A-3.White crystalline; yield (percentage yield): 21.1 mg (3%); melting point 249‒252°C; UV λβ-glucuronidase enzyme  by test compounds was determined on a spectrophotometer by measuring the absorbance of p-nitrophenol at 405 nm, produced from the substrate. The reaction mixture comprised 185 μL of 0.1 M acetate buffer, and 5 μL of test compound solution, and 10 μL of (1U) enzyme solution in a 96-well plate. The mixture was incubated at 37°C for 30 min. The test compounds were solubilized in DMSO (100%), and 5 μL volume was added in each well . Similar conditions were used for the standard . The plates were read on a multiplate reader (SpectraMax plus 384) at 405 nm and 37°C, after addition of 50 μL of 0.4 mM p-nitrophenyl-β-D-glucuronide. All assays were performed in triplicate. IC50 values were calculated through EZ-Fit software .The inhibition of Leishmania major  was cultured in a mixture of NNN-biphasic medium, and normal physiological saline solution. L. major promastigotes were grown on RPMI 1640 medium, supplemented with fetal bovine serum (FBS) (10% heat inactivated). Parasites were centrifuged (at log phase) for 10 min at 2,000 rpm and washed thrice with saline. The final density of fresh culture (106 cells/mL) was acquired through dilution of parasites. Sample solution of 1 mg of test compounds was prepared in a mixture 50 μL of DMSO and 950 μL of RPMI media. Fully dissolved compounds were transferred to 96-well plate. The first row of 96-well plate received 180 μL of the medium, while the remaining wells received 100 μL medium. Test compounds (20 μL each) were added into the medium containing wells, followed by serial dilution. The parasite culture (100 μL) was then transferred to each well. Negative controls comprised of only the growth medium, while standard leishmanicidal drugs (amphotericin or pentamidine) were used as positive controls. The plate was then incubated for 72 h at 21–22°C. The inhibitory effect of test compounds on the culture was analyzed microscopically on an improved Neubauer counting chamber. IC50 values were calculated through Ezfit 5.03 Perella Scientific software (USA).® CRL-1658, purchased from American Type Culture Collection, ATCC, Virginia, USA) cell lines. The cells (1 × 105 /well) were plated in 0.2 mL of DMEM high glucose medium/well in 96-well plates. The cells were treated for 24 hours with test compounds in the range of 25, 50, 75, 100, and 200 μM concentrations, respectively. For the MTT assay, the medium from the wells was removed carefully after 24 hours treatment. Each well was washed with 1X PBS for 2–3 times, and 200 μL of MTT (5 mg/mL) was added in to media containing wells (1:10). The plates were incubated for 4 hours at 37°C, in a 5% CO2 incubator. After incubation, MTT was removed and 0.1 mL of DMSO was added to each well and mixed by keeping on a stirrer. The presence of viable cells was visualized by the development of purple color due to formation of the formazan crystals. The plates were observed under spectrophotometer (Spectramax) and absorbance was taken at 545 and 570 nm for cancer and normal cell lines, respectively. Measurements were performed and the concentration required for a 50% inhibition of viability (IC50) was determined graphically.MTT -2,5-diphenyl tetrazolium bromide) assay was employed to study the cytotoxic activity against HeLa , and mouse fibroblast 3T3 , B (C5-C10), C (C8-C9/C11-C14), and D (C13-C17). Ring A exists in a half chair conformation and contains α-β unsaturated carbonyl moiety, whereas trans-fused rings B, and C were in chair conformations. Five membered ring D attains an envelope conformation. A β-oriented hydroxyl group at C-17 is in a pseudo equatorial orientation .Compound chniques  1–4]. S–4. S1 isentation . Single-2 was isolated as white crystals. It displayed the [M+] in HREI-MS at m/z 318.2192 , which suggested the addition of an oxygen in substrate 1 (m/z 302.45). The IR absorbances at 1654 and 3360 cm-1 were due to the presence of α,β-unsaturated ketone, and hydroxyl functionalities, respectively. The 1H-NMR spectrum was distinctly similar to substrate 1 (δ 70.8) was observed in the 13C-NMR spectrum of compound 2, which suggested the hydroxylation of substrate 1  and H2-1  with C-10 (δ 70.8) (δ 4.93) (DMSO-d6) at C-10 was deduced to be β-oriented based on its NOE correlation with the β-oriented H-8  , B (C5-C10), C (C8-C9/C11-C14), and D (C13-C17). Six-membered ring A containing α,β-unsaturated carbonyl moiety, exists in a half-chair conformation, whereas trans-fused rings B, and C were found to be in a chair conformation. Five membered ring D attains an envelope conformation. The two β-oriented hydroxyl groups at C-10 and C-17 are in axial and pseudo equatorial orientations, respectively . The structure of the new compound was thus deduced as 10β,17β-dihydroxy-7α,17α-dimethylestr4-en-3-one 2.Metabolite strate 1 . An addistrate 1 . This OH(δ 70.8) . The OH 1.99, m) . Single-ectively . Single-3 was obtained as a white solid. It displayed the [M+] in HREI-MS at m/z 318.2196 , which suggested the addition of an oxygen in substrate 1 (m/z 302.45). The presence of hydroxyl (3431 cm-1) and α,β-unsaturated ketone (1659 cm-1) were inferred from the IR spectrum. A downfield methine proton signal  in the 1H-NMR spectrum suggested hydroxylation in substrate 1 , suggesting an additional OH group  with the newly appearing methine carbon at δ 78.7 (C-6). The HMBC correlations of H-6  with C-5 (δ 167.3), C-10 (δ 39.8), and C-7 (δ 38.4) further supported an OH at C-6  with H-4 , and vicinal H-7  further confirmed the position of OH at C-6  with H-4  and H-19   . The str4 was obtained as white crystals. The HREI-MS of metabolite 4 showed the [M+] in HREI-MS at m/z 334.2159 , suggesting the addition of two oxygen atoms in substrate 1 (m/z 302.45). The absorbances for the hydroxyl (3412 cm-1) and α,β-unsaturated ketone groups (1664 cm-1) were spotted in the IR spectrum. A downfield methine proton signal  in the 1H-NMR spectrum further indicated hydroxylation in substrate 1  with the newly appearing methine carbon at δ 80.1 (C-6), whereas the second OH group was placed at C-10 on the basis of HMBC correlations of H-4  and H-2  with the downfield quaternary carbon at δ 72.4 (C-10). The HMBC correlations of H-6  with C-5 (δ 160.6), C-10 (δ 72.4), and C-7 (δ 38.6) further supported the positions of OH group at C-6, and C-10  with H-7   with H-4  and H-19 . The OH (δ 4.29) (acetone-d6) at C-10 was deduced to be β-oriented based on its NOE correlations with β-oriented OH-6 (δ 5.26), and β-oriented H-8  , and B (C5-C10), C (C8-C9/ C11-C14), existing in half chair and chair conformations, respectively, while a five-membered ring D (C13-C17) exists in an envelope conformation . The structure of the compound was thus deduced as 6β,10β,17β-trihydroxy-7α,17α-dimethylestr-4-en-3-one 4.Compound strate 1 . The 13Cstrate 1 . One of and C-10 . This wa2.01, m) . The OH 2.30, m) . Single-ormation . Single-5 was obtained as a white solid. HREI-MS of metabolite 5 showed the [M+] at m/z 334.2146 . Increase in mass of 32 amu suggested dihydroxylation in substrate 1 (m/z 302.45). The IR absorbances at 3412 and 1643 cm-1 indicated the presence of OH and α,β-unsaturated ketone functionalities, respectively. The 1H-NMR spectrum showed signals for a new downfield methine  and methylene protons , which suggested dihydroxylation in substrate 1 , and methylene carbons (δ 67.7), confirming dihydroxylation in substrate 1  with the newly formed methine carbon at δ 67.4 (C-11), whereas second OH group was placed at C-20 based on the HMBC correlations of H2-20  with C-13 (δ 46.4), C-16 (δ 33.3), and C-17 (δ 84.5)  with H-9 , and H2-12 , and H2-20  with OH-17 (δ 4.32) (DMSO-d6)  showed NOE correlations with α-oriented H-9 . Therefore, OH at C-11 was deduced to be β-oriented -7α,17α-dimethylestr-4-en-3-one 5.Compound strate 1 . The 13Cstrate 1 . One of (δ 84.5) . The posDMSO-d6) . H-11  which suggested monohydroxylation of substrate 1 302.45). The IR absorbances at 3433 and 1651 cm-1 were due to OH and α, β-unsaturated ketone moieties, respectively. The 1H-NMR spectrum showed signals for a new downfield methine proton , indicating addition of an OH group in substrate 1 (δ 66.3) in the 13C-NMR spectrum supported the above inference  observed with C-3 (δ 200.8), and C-5 (δ 164.9)  with H-10 , and H2-2  further confirmed the structure  showed NOE correlation with β-oriented H-10 . Therefore, OH at C-1 was deduced to be α-oriented  . The COStructure . Geminaloriented . Thus, t7 was obtained as a white solid. The HREI-MS of compound 7 showed the [M+] at m/z 334.2140 . This indicated that dihydroxylation had occurred on substrate 1 (m/z 302.45). The IR absorbances at 3418 and 1661 cm-1 indicated the presence of OH and α,β-unsaturated ketone groups, respectively. The 1H-NMR spectrum showed signals for two new downfield methine protons at δ 4.25, d, and 4.38, t, suggesting dihydroxylation in substrate 1  in 13C-NMR spectrum further supported dihydroxylation  and C-1 (δ 66.3), and the COSY correlations of H-1  with H-10 , and H2-2 . The second OH was placed at C-11 on the basis of HMBC correlations of H-9  and H2-12  with C-11 (δ 67.2), and the COSY correlations of H-11  with H-9 , and H2-12  (δ 4.38) showed NOE correlations with the β-oriented H-10 , while H-11  showed NOE correlations with α-oriented H-9 . Therefore, OH at C-1 as α-oriented, while OH at C-11 was deduced to be β-oriented  . H-1 , indicating monohydroxylation in substrate 1 (m/z 302.45). Single-crystal diffraction showed that compound 8 has the same structural features those of metabolites 2 and 4, and consists of three six-membered rings A, B, and C, possessing half chair, chair, and chair conformations, respectively. The five-membered ring D possesses an envelope conformation. Two OH substituents were attached at C-11, and C-17 in axial, and pseudo equatorial orientations, respectively. All bond angles and lengths were within the normal range, and found to similar to metabolites 2, and 4 . The compound was thus identified as 11β,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-one 8, which has been previously synthesized through biotransformation [Metabolite 2, and 4 . The sinormation .1) and its transformed products 2–4, and 8 were evaluated for their β-glucuronidase inhibitory activity. Compound 1 was identified as a potent inhibitor of β-glucuronidase with IC50 = 42.98 ± 1.24 μM, as compared to the standard drug, D-saccharic acid 1,4-lactone (IC50 = 45.75 ± 2.16 μM) (Mibolerone (2.16 μM) . Interes2.16 μM) .1) and its metabolites 2–4, and 8 were assessed for leishmanicidal activity. Substrate 1 showed a significant leishmanicidal activity against the promastigotes of Leishmania major with IC50 value of 29.64 ± 0.88 μM as compared to the standard drugs, pentamidine (IC50 = 5.09 ± 0.09 μM), and amphotericin B (IC50 = 0.29 ± 0.05 μM) (3 (IC50 = 79.09 ± 0.06 μM) and 8 (IC50 = 70.09 ± 0.05 μM) showed only weak activity against the promastigotes of Leishmania major, while compounds 4 and 5 were found to be inactive (Mibolerone (0.05 μM) . Howeverinactive .1) and its metabolites 2–4, and 8 were evaluated for their cytotoxic activity against HeLa cancer and 3T3 cell lines. Compounds 1, and 8 were found to be strongly active against HeLa cancer cell line  with IC50 values of 35.7 ± 4.46, and 34.1 ± 5.3 μM, respectively, as compared to the standard drug, cisplatin (IC50 = 38.5 ± 1.8 μM)  cell line with IC50 values of 46.3 ± 11.7, 43.3 ± 7.7, 65.6 ± 2.5, and 89.4 ± 2.7 μM, while compound 8 showed no cytotoxicity against 3T3 cell line (Mibolerone ( 1.8 μM) . Compounell line .1) with C. blakesleeana, C. echinulata, and M. phaseolina yielded six new metabolites 2–7, along with a known metabolite 8. During these transformations, hydroxylations at C-1, C-6, C-10, C-11, and C-20 were observed. C-6, C-10, and C-11 were the sites for β-hydroxylation, whereas α-hydroxylation occurred at C-1. Substrate 1 was found to be significantly active against β-glucuronidase enzyme, leishmaniasis, and HeLa cancer, and 3T3 normal cell lines in vitro. Metabolites 2, and 8 were found to be potently active against HeLa cancer cell line, while metabolites 3, and 4 were weakly active. Metabolites 2–4 were toxic to 3T3 cell line, whereas metabolite 8 showed no cytotoxicity against 3T3 cell line. In addition, metabolites 3 and 8 also showed weak leishmanicidal activity in vitro against the promastigotes of Leishmania major. The presented study indicated that compound 8 deserves to be further studied for its therapeutic potential.In conclusion, microbial transformations of mibolerone (S1 Data(PDF)Click here for additional data file.S2 Data(PDF)Click here for additional data file.S3 Data(PDF)Click here for additional data file.S4 Data(PDF)Click here for additional data file.S5 Data(PDF)Click here for additional data file.S6 Data(PDF)Click here for additional data file.S7 Data(PDF)Click here for additional data file.


Salvia willeana (Holmboe) Hedge, growing wild in Cyprus. These compounds comprise one new and one known acetophenone, one megastigmane glucoside, five phenolic derivatives, two caffeic acid oligomers, three flavonoids, two lignans, two triterpene acids, one monoterpene glucoside, and two fatty acids. The structures of the isolated compounds were established by means of NMR  and MS spectral analyses. This is the first report of the natural occurrence of 4-hydroxy-acetophenone 4-O--β-d-glucopyranoside. A chemical review on the non-volatile secondary metabolites has been carried out. Based on the literature data, the analysis revealed that the chemical profile of S. willeana is close to that of S. officinalis L.Twenty compounds were isolated from the aerial parts of  Salvia L. is the largest genus within this family due to the presence of approximately 900 species. Salvia L. spreads in the warm and temperate regions of both the northern and southern hemispheres, and some species of this genus have been cultivated worldwide for use in folk medicine, in perfumery and cosmetics industries, as well as for culinary purposes, like flavoring and aromatic agents [Salvia, is derived from the Latin word “salvare”, which means “to save”, in reference to the curative properties of the plant [The Lamiaceae family consists of more than 250 genera; c agents ,2. Some c agents , antimicc agents ,4, cytotc agents ,4, anti-c agents , and antc agents . It is nhe plant .Salvia willeana  is a low-growing, strongly aromatic suffruticose herb, sometimes carpeting the ground [S. willeana is locally used in aqueous preparations, the purpose of our study was the investigation of secondary metabolites obtained from the polar extract of its aerial parts. Our previous study of its lipophilic extract, revealed the presence of camphor, lupeol, and oleanolic acid, and demonstrated their anti-inflammatory effect [S. willeana, which revealed the presence of the triterpenoids ursolic and oleanolic acids, the diterpenoids carnosic acid and isorosmanol, and the flavonoid salvigenin [e ground . This spe ground . Its exte ground . As S. wy effect . It is olvigenin .2O 5:1) of S. willeana was fractionated by MPLC (medium pressure liquid chromatography), CC (column chromatography), followed by semi-preparative HPLC, preparative TLC, and yielded two triterpenoids, namely ursolic acid (1) and maslinic acid (2), one monoterpene glucoside, -1,8-epoxy-p-menthan-2-yl-O-β-d-glucopyranoside (3), one megastigmane glucoside, -3-oxo-α-ionol β-d-glucopyranoside (4), five phenolic compounds, i.e., hydroxy-tyrosol (5), p-anisic acid (6), eleutheroside B (syringin) (7), 1-O-p-hydroxybenzoyl-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside (8) and eugenylglucoside (9), two acetophenones, 4-O-β-d-glucopyranosyl acetophenone (picein) (10) and 4-hydroxy-acetophenone 4-O--β-d-glucopyranoside (11), two caffeic acid oligomers, rosmarinic acid (12) and salvianolic acid K (13), three flavonoids, luteolin-7-O-β-d-glucoside (cynaroside) (14), 6-hydroxyluteolin 7-O-β-d-glucoside (15) and hesperidin (16), two lignans, syringaresinol-4-O-β-d-glucopyranoside (17), pinoresinol-4-O-β-d-glucopyranoside (18), and two fatty acids: linoleic acid (19) and methyl α-linolenate (20) (The polar extract (MeOH:Hate (20) .11 was obtained as a white amorphous powder. 3OH) λmax: 272 nm. The HREIMS of 11 established its molecular formula as C23H26O11 . The 1H-NMR spectrum  and 7.05 , which were indicative of a 1,4-bisubsituted phenyl group. Thus, these proton signals were recognized belonging to the aromatic ring of the acetophenone moiety [1H-NMR spectrum, a singlet at δH 2.47  was ascribed to the methyl group (CH3-8) attached on the carbonyl group of the acetophenone [δH 7.33 with an integration of two aromatic protons, indicative of a pair of equivalent methine protons, revealed the occurrence of the syringic acid ester structure in the molecule [1H-NMR spectrum, the presence of a β-d-glucopyranose unit was evident based on a characteristic doublet signal with a coupling constant of 7.9 Hz at δH 5.04, assignable to the anomeric proton of the sugar moiety. Moreover, in the 1H-1H-COSY spectrum the correlation peaks between the vicinal protons of the sugar ring were observed. Furthermore, from the 13C-NMR data (δC 101.7 (C-1′), 77.5 (C-3′), 75.5 (C-5′), 74.5 (C-2′), 71.3 (C-4′), and 64.8 (C-6′), matching the reported data of 1,6-disubstituted-β-d-glucose [δC 130.1 (C-2/C-6), 116.2 (C-3/C-5), and 26.5 (C-8), indicative of a 4-hydroxy-acetophenone moiety [δC 108.3 (C-2″/C-6″), 56.0  [δC 199.1 (C-7) with the equivalent protons H-2/H-6 (δH 7.69), as well as an interaction between the carbonyl group (δC 199.1) and the methyl group CH3-8 (δH 2.47). In addition, the linkage of the syringyl moiety with the glucosyl moiety was substantiated by the observation of an HMBC correlation between the carbonyl group at δC 167.3 and the 6′-methylene protons at δH 4.46 . Furthermore, the position of the attachment of the carboxyl group to the quaternary carbon C-1″ of the syringic ester was determined by a diagnostic HMBC cross peak between the equivalent methine protons H-2″/H-6″ at δH 7.33 and the carbonyl carbon C-7″ at δC 167.3, while a long-range coupling between the methyl protons of the methoxy groups at δH 3.83 and the benzylic carbons C-3″/C-5″ at δC 149.0 was also observed. Moreover, the position of the attachment of glucose to the 4-hydroxy-acetophenone moiety was revealed by a ROESY experiment, which displayed correlations between the anomeric proton H-1′ (δH 5.04) and the equivalent protons H-3/H-5 (δH 7.05) of the acetophenone -β-d-glucopyranoside, which is a new natural product, to the best of our knowledge.Compound spectrum  showed se moiety . In the ophenone . MoreoveNMR data  the carb-glucose . The strophenone . On the O-β-d-glucoside (14) [O-β-d-glucoside (15) [16) [19) [α-linolenate (20) [1H-NMR chemical shifts for compounds 1 [2 [3 [4 [7 [9 [10 [12 [17 [18 [The identification of the known flavonoids luteolin-7-lucoside  [14,15,1ide (14) , 6-hydroide (15) , and hes15) [16) ,22,23,2416) [19) ,26 and mate (20) , has beepounds 1 ,29,30, 2nds 1 [2 ,32,33, 3 1 [2 [3 ,35, 4 [3[2 [3 [4 ,37, 7 [3[3 [4 [7 ,40,41,42[4 [7 [9 , 10 [10,7 [9 [10 , 12 [35, [10 [12 ,46,47,48 [12 [17 ,50, and  [17 [18 ,52,53,545, 6, 8, and 13 are not fully recorded in the literature, therefore, they are presented here below.However, the NMR data of compounds Salvia L. is characterized by the presence of several different secondary metabolites, mainly phenolic derivatives and terpenoids [The genus rpenoids .S. willeana polar extracts, i.e., two triterpenoids, namely ursolic acid (1) and maslinic acid (2), one monoterpene glucoside, -1,8-epoxy-p-menthan-2-yl-O-β-d-glucopyranoside (3), one megastigmane glucoside, -3-oxo-α-ionol β-d-glucopyranoside (4), five simple phenolic compounds, i.e., hydroxy-tyrosol (5), p-anisic acid (6), eleutheroside B (syringin) (7), 1-O-p-hydroxybenzoyl-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside (8) and eugenylglucoside (9), two acetophenones, 4-O-β-d-glucopyranosyl acetophenone (picein) (10) and 4-hydroxy-acetophenone 4-O--β-d-glucopyranoside (11), two caffeic acid oligomers, rosmarinic acid (12) and salvianolic acid K (13), three flavonoids, luteolin-7-O-β-d-glucoside (cynaroside) (14), 6-hydroxyluteolin 7-O-β-d-glucoside (15) and hesperidin (16), two lignans, syringaresinol-4-O-β-d-glucopyranoside (17), pinoresinol-4-O-β-d-glucopyranoside (18) and two fatty acids: linoleic acid (19) and methyl α-linolenate (20). In the present study, overall, 20 compounds were isolated from 7, 16, 17, and 18 had not been previously detected in Salvia L. Syringin (7) is reported, here, as a component of the Lamiaceae family for the first time. Moreover, compounds 3, 5, 6, and 8–10 had been previously mentioned only once in the genus, as follows: 3, 9, 10 [6 [8 [14, 15 [S. officinalis L., 5 in S. digitaloides Diels [4 twice in S. nemorosa and S. officinalis L. [1 and 2, these have previously been isolated from the acetone extract of the aerial parts of S. willeana [Salvia species of the over 1000 species suggested have been investigated [S. willeana showed many similarities to S. officinalis L., since most of the isolated simple phenols , as well as the flavonoids 14 and 15, are found only in these two species  Hedge were collected on Troodos Mountain in Cyprus in April 2004 [Aerial parts of ril 2004 . A vouch1H, 13C, and 2D-NMR spectra were recorded in CDCl3 and CD3OD on Bruker DRX 400 (399.95 MHz for 1H-NMR) and Bruker AC 200 (200.13 MHz for 1H-NMR and 50.3 MHz for 13C-ΝMR) instruments at 295 K . Chemical shifts are given in ppm (δ) and were referenced to the solvent signals at 7.24/3.31 and 77.0/49.5 ppm for 1H and 13C-NMR, respectively. COSY, HSQC, HMBC, NOESY, and ROESY (mixing time 950 ms) were performed using standard Bruker microprograms. High-resolution mass spectra were measured on a Q-TOF 6540 UHD . The solvents used were of spectroscopic grade . UV spectra were recorded on a Shimadzu UV-160A spectrophotometer , according to Mabry et al. [18 25 cm × 10 mm Kromasil column on a HPLC system  equipped with an RI detector Shimadzu 10A ; flow rate: 1.0 mL/min; concentration of the samples: 3.5–7.0 mg/mL. All solvents used were of HPLC grade (Merck). MPLC (Medium Pressure Liquid Chromatography) was performed using Büchi C-615 and Büchi 688 chromatographic pump; columns: Büchi Borosilikat 3.3, (41.0 cm × 4.0 cm), flow rate: 10 mL/min; (15.0 cm × 1.5 cm) flow rate: 3 mL/min; vacuum liquid chromatography (VLC): silica gel 60H  [y et al. . Opticalt. 7736) . Column t. 7736)  and Neu’S. willeana (0.43 kg) were successively extracted at room temperature with cyclohexane, dichloromethane, MeOH, and MeOH:H2O (5:1)  [18-MPLC (41.0 × 4.0 cm) using a H2O: MeOH gradient system  to yield twenty three fractions (A–V) of 500 mL each. Fraction D  was similarly purified by RP18-MPLC (15 cm × 1.5 cm) to obtain three sub-fractions (1–3). The first two sub-fractions were combined together , subjected to a Sephadex LH-20 column and eluted with 100% methanol to afford 61 fractions combined in 13 groups (DAA–DAM). Group DAJ (4.2 mg) was identified as salvianolic acid K (13). Group DAC (12.9 mg) was subjected to RP18-HPLC  and afforded 1-O-p-hydroxybenzoyl-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside (8)  and 4-O-β-d-glucopyranosyl-acetophenone (picein) (10) . Group DAD (9.3 mg) was purified by prep. TLC on silica gel using CHCl3:MeOH:AcOH (7:1.5:1.5) as the eluent and yielded hydroxytyrosol (6.3 mg) (5). Fraction H (718.3 mg) was fractionated by CC on a Sephadex LH-20 (25.0 cm × 3.2 cm) using H2O:MeOH (20:80 to 0:100) for gradient elution to afford luteolin 7-O-β-d-glucoside (0.7 mg) (14). Groups HC to HG were combined  and purified by CC (12.2 cm × 2.2 cm) over silica gel with cyclohexane: DM:EtOAc:MeOH mixtures of increasing polarity to yield nine groups (HC′A–HC′I). Group HC′C  was purified by RP18-HPLC  to obtain syringin  (7), -1,8-epoxy-p-menthan-2-yl-O-β-d-glucopyranoside  (3), -3-oxo-α-ionol-β-d-glucopyranoside  (4), eugenyl-glucoside  (9). Combined groups HJ to HL  were subjected to CC over silica gel using cyclohexane: DM:EtOAc:MeOH mixtures of increasing polarity (60 fractions). Purification of fraction HJ′E  was carried out by prep. TLC on silica gel, using CHCl3:MeOH:AcOH (9.0:1.0:0.1) and afforded syringaresinol-4-O-β-d-glucopyranoside (0.9 mg) (17) and pinoresinol-4-O-β-d-glucopyranoside (1.2 mg) (18). Combined groups HM to HP  were fractionated by RP18-HPLC  and yielded rosmarinic acid  (12) and p-anisic acid  (6), and the sub-fraction HM′14b , which was further purified by prep. TLC on silica gel with EtOAc:AcOH:H2O (65:15:20) and led to the isolation of hesperidin (3.0 mg) (16) and of 4-hydroxyacetophenone 4-Ο--β-d-glucopyranoside (2.8 mg) (11). Combined groups HT to HV , subjected to CC on cellulose (11.0 cm × 3.2 cm) using as eluent AcOH:H2O (30:70) afforded 79 fractions. Fraction HT′H (4.8 mg) was purified by prep. TLC on silica gel with EtOAc:AcOH:H2O (65:15:20) to obtain 6-hydroxyluteolin 7-O-β-d-glucoside (0.9 mg) (15). The purification of fraction N (248.5 mg) was performed on silica gel CC (15.2 cm × 2.0 cm) using mixtures of cyclohexane: DM:EtOAc:MeOH of increasing polarity and afforded methyl α-linolenate (C18:3) (1.4 mg) (20) and ursolic acid (4.7 mg) (1). Groups NB′ (7.4 mg) and NF (19.6 mg) were subjected to prep. TLC on silica gel with CHCl3:MeOH:AcOH (9.5:0.5:0.05) and yielded linoleic acid (C18:2) (0.5 mg) (19) and maslinic acid (18.3 mg) (2), respectively. All obtained extracts, fractions, and isolated compounds were evaporated to dryness in vacuum under low temperature and then were put in activated desiccators with P2O5 until their weights had stabilized.The air-dried powdered aerial parts of Compound5: Yellow amorphous powder; 1H-NMR : 2.66 , 3.66 , 6.52 , 6.65 , 6.67 .Compound6: White amorphous powder; 1H-NMR : 3.91 , 6.85 , 7.89 .Compound8: White amorphous powder; 1H-NMR : δ 3.38 , 3.47 , 3.56 , 3.61 , 3.73 , 3.90 , 3.96 , 3.99 , 4.96 , 5.64 , 6.84 , 7.96 .Compound13: Yellow amorphous powder; 1H-NMR : δ 2.90 , 3.07 , 4.24 , 4.88 , 5.02 , 6.31 , 6.38 , 6.62 , 6.66 , 6.74 , 6.76 , 6.82 , 6.84 , 6.98 , 7.01 , 7.47 .


R)-5,28-stigmastadiene-3β,24-diol-7-one (1), (24S)-5,28-stigmastadiene-3β,24-diol-7-one (2), and 24R and 24S-vinylcholesta-3β,5α,6β,24-tetraol (3), together with three known sterols (4–6) were isolated from the green alga Ulva australis. The structures of the new compounds (1–3) were elucidated through 1D and 2D nuclear magnetic resonance spectroscopy as well as mass spectrometry. Compounds 4–6 were identified as isofucoterol (4), 24R,28S and 24S,28R-epoxy-24-ethylcholesterol (5), and (24S)-stigmastadiene-3β,24-diol (6) on the basis of spectroscopic data analyses and comparison with those reported in the literature. Compounds 4–6 were isolated from U. australis for the first time. These compounds, together with the previously isolated secondary metabolites of this alga, were investigated for their inhibitory effects on human recombinant aldose reductase in vitro. Of the compounds, 24R,28S and 24S,28R-epoxy-24-ethylcholesterol (5), 1-O-palmitoyl-3-O-(6′-sulfo-α-d-quinovopyranosyl) glycerol, (2S)-1-O-palmitoyl-3-O-[α-d-galactopyranosyl(1→2)β-d-galactopyranosyl] glycerol, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, and 8-hydroxy-(6E)-octenoic acid weakly inhibited the enzyme, while the three new sterols, 1–3, were almost inactive.Three new sterols, (24 Ulva australis Areschoug is a green alga belonging to the family Ulvaceae and is widely distributed along the coasts of the Yellow Sea and the Bo Sea of China. As an edible seaweed, it contains high nutritional value minerals, vitamins, and noncaloric dietary fibers +, 399 [M − C3H7]+, 381 [M − C3H7 − H2O]+, 329 [M − C7H13O]+, and 285 [M − C10H19O − 2H]+  were close to those of H-26 at δ 0.871  in the 1H NMR spectrum of compound 1. The chemical shift difference between H-27 and H-26 of compound 1 was 0.019 ppm, whereas the chemical shift difference between H-27 and H-21 of compound 1 was 0.041 ppm, which was in accordance with those of 24R-saringosterol. The chemical shift difference between H-27 and H-26 of 24R-saringosterol was 0.020 or 0.020 ppm, whereas the chemical shift difference between H-27 and H-21 24R-saringosterol was 0.032 or 0.033 ppm in the previous reports [1 was elucidated with the structure as shown in Compound  13C NMR  and distO − 2H]+  suggested 18.9 C-), nine s, 29.0 C-, 29.6 , one spd 18.9 C-), nine s, 29.0 C-, 29.6 . Comparison of the 1H and 13C NMR data of compound 2 with those of compound 1 showed that both compounds shared the same sterol skeleton nucleus and side chain, the only difference being that the signal of H-27 in compound 2 appeared at δH 0.91 , whereas the signal of H-27 in compound 1 displayed at δH 0.89 . Stereochemistry at C-24 of compound 2 was established to be S, since the signal attributed to H-27 at δH 0.909  was very close to that of H-21 at δH 0.931  in the 1H NMR spectrum of compound 2. The chemical shift difference between H-27 and H-21 of compound 2 was 0.022 ppm, whereas the chemical shift difference between H-27 and H-26 of compound 2 was 0.038 ppm, which was in accordance with those of 24S-saringosterol. The chemical shift difference between H-27 and H-21 of 24S-saringosterol was 0.019 or 0.020 ppm, whereas the chemical shift difference between H-27 and H-26 24S-saringosterol was 0.027 or 0.027 ppm in the previous reports −  in the negative HR-FAB-MS spectrum. The presence of four hydroxyl groups in compound 3 was indicated by the EI-MS spectrum, absence of the molecular ion peak, which exhibited mass ions for stepwise H2O loss at m/z 444 [M − H2O]+, 426 [M − 2H2O]+, 408 [M − 3H2O]+, and 390 [M − 4H2O]+. Additional prominent fragment peaks were present at m/z 401 [M − C3H7 − H2O]+, 383 [M − C3H7 − 2H2O]+, 365 [M − C3H7 − 3H2O]+, 347 [M − C3H7 − 4H2O]+, 305 [M − C10H19O − 2H]+, 289 [M − C10H19O − H2O]+, 271 [M − C10H19O − 2H2O]+, and 253 [M − C10H19O − 3H2O]+ , 16.4 (C-26), 16.8 (C-19), 17.5 (C-27), and 18.7 (C-21)), a sp2 methylene carbon (δC 112.7/112.6 (C-29)), two oxygenated sp3 methine carbons  and 75.9 ), a sp2 methine carbon (δC 142.2/142.1 (C-28)), two oxygenated sp3 quaternary carbons (δC 75.9 (C-5) and 77.5 (C-24)). The 1H NMR spectrum of compound 3 showed tertiary methyl groups at δ 0.67  and 1.18 , and three secondary methyl groups at δ 0.87 , 0. 90 /0.89 , and 0.914 /0.908 . The 3β, 5α, 6β-trihydroxyl sterol nature was characterized by a multiplet at δ 4.10 , a double doublet at δ 2.08 , a multiplet at δ 1.60 , and a broad singlet at δ 3.49 , while the angular methyl groups at C-19 and C-18 resonated at δ 1.18 (s) and 0.67 (s), respectively + . The 13C signals , which i4–6 were identified by comparing the 1H- and 13C-NMR, as well as the MS spectra with those reported in the literature. They were identified as isofucoterol (4)  glycerol, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, and 8-hydroxy-(6E)-octenoic acid showed weakly inhibitory activities, with inhibition values of 31.28 ± 1.04%, 27.41 ± 1.11%, 33. 89 ± 1.03%, 27.80 ± 0.79%, 33.05 ± 1.32%, and 28.92 ± 0.53%, respectively, which compared with the positive control (71.66 ± 0.52%). In addition, three new sterols 1–3 were almost inactive.Aldose reductase, an enzyme of the aldoketo reductase super-family that catalyzes the conversion of glucose to sorbitol in the polyol pathway of glucose metabolism, has been proved as the molecular target for major complications of diabetes, such as cataract, neuropathy, retinopathy, and nephropathy . In thisnhibitor ,32,33, w3 . Optical rotations were measured by using a JASCO P-1020 automatic digital polarimeter . HPLC was performed on an NPL-500 pump  and a RI-102 detector  using a COSMOSIL Silica 5SL-II Waters column  and a Senshu Pak DOCOSIL column . Open column chromatography was performed with silica gel 60 N , RP-18 reverse-phase silica gel , and Sephadex LH-20 . TLC was carried out on pre-coated TLC plates with silica gel 60 F254 and silica gel RP-18 60 F254 . Detection was achieved by spraying with 10% H2SO4 in MeOH and heating at 110 °C. Aldose reductase recombinant from human muscle cell purchased from Wako Pure Chemical Industries, Ltd. . β-Nicotinamide adenine dinucleotide phosphate tetrasodium salt (NADPH), dl-glyceraldehyde, sodium dihydrogenphosphate dihydrate, disodium hydrogenphosphate 12-water, and dimethyl sulfoxide (DMSO) were bought from Nacalai Tesque, Inc. . Compounds isophitol [O-palmitoyl-3-O-(6′-sulfo-α-d-quinovopyranosyl) glycerol [S)-1-O-palmitoyl-3-O-[α-d-galactopyranosyl(1→2)β-d-galactopyranosyl] glycerol D25 −21.8 , HR-EI-MS m/z 442.3445 ; EI-MS m/z 442 [M]+ (32), 424 (11), 399 (92), 381 (100), 344 (70), 329 (21), 285 (92); 1H NMR  δ 5.81 , 5.69 , 5.20 , 5.14 , 3.67 , 1.20 , 0.931 , 0.890 , 0.871 , 0.68 ; 13C NMR  spectra data, see Compound 2: white amorphous solid; [α]D25 −42.6 , HR-EI-MS m/z 442.3439 ; EI-MS m/z 442 [M]+ (35), 424 (16), 399 (20), 381 (25), 344 (18), 329 (9), 285 (100); 1H NMR  δ 5.81 , 5.69 , 5.19 , 5.14 , 3.67 , 1.20 , 0.931 , 0.909 , 0.871 , 0.68 ; 13C NMR  spectra data, see Compound 3 (a mixture of 24R and 24S): white amorphous solid; negative HR-FAB-MS m/z 461.3625 ; EI-MS m/z 444 (11), 426 (26), 408 (8), 401 (73), 390 (2), 383 (100), 365 (68), 347 (23), 305 (83), 289 (52), 271 (70), 253 (27); 1H NMR  δ 5.81 /5.79 , 5.19 /5.18 , 5.14 /5.13 , 4.10 , 3.49 , 2.08 , 1.60 , 1.18 , 0.914 /0.908 , 0.90 /0.89 , 0.87 , 0.67 ; 13C NMR  spectra data, see Compound dl-glyceraldehyde solution (100 μL), and 3 × 10−2 unit/mL human recombinant aldose reductase solution (100 μL), in the total volume of 1.0 mL. The effects of each sample on the enzyme activity were determined by adding 3 μL of test sample solution  to the reaction mixture. An appropriate blank control mixture and positive control mixture were prepared. The reaction was initiated by the addition of dl-glyceraldehyde solution and the rate of NADPH oxidation was followed by recording the decrease in the absorbance at 340 nm on a UV spectrophotometer . Inhibition percentage (%) was calculated as [1 − (∆As − ∆Ab)/(∆Ac − ∆Ab)] × 100, where As is the decreased absorbance of the sample, Ac and Ab are the decreased absorbances without a sample as a positive control and without a sample and enzyme as a blank control, respectively. An overview about the effects of these substances on human recombinant aldose reductase inhibitory activity is given in The human recombinant aldose reductase inhibition assays were performed according to the method modified by our group . The assU. australis led to the isolation of three new sterols and three known sterols. These compounds, together with the previously isolated secondary metabolites of this alga, were investigated for their inhibitory effects on aldose reductase in vitro. Of the compounds, 24R,28S and 24S,28R-epoxy-24-ethylcholesterol (5), 1-O-palmitoyl-3-O-(6′-sulfo-α-d-quinovopyranosyl) glycerol, (2S)-1-O-palmitoyl-3-O-[α-d-galactopyranosyl(1→2)β-d-galactopyranosyl] glycerol, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, and 8-hydroxy-(6E)-octenoic acid weakly inhibited the enzyme. The obtained results are beneficial to subsequent research on this alga.Phytochemistry investigation of the green alga


Enzymes such as reductase, oxidase, dehydrogenase, and hydrolase from Cephalosporium aphidicola, and Fusarium lini were likely involved in the biotransformation of 1 into new metabolites at pH 7.0 and 26°C, yielding five new metabolites, 2α-methyl-3α,14α,17β-trihydroxy-5α-androstane (2), 2α-methyl-7α-hydroxy-5α-androstan-3,17-dione (3), 2-methylandrosta-11α-hydroxy-1, 4-diene-3,17-dione (6), 2-methylandrosta-14α-hydroxy-1,4-diene-3,17-dione (7), and 2-methyl-5α-androsta-7α-hydroxy-1-ene-3,17-dione (8), along with three known metabolites, 2α-methyl-3α,17β-dihydroxy-5α-androstane (4), 2-methylandrosta-1, 4-diene-3,17-dione (5), and 2α-methyl-5α-androsta-17β-hydroxy-3-one (9), on the basis of NMR, and HREI-MS data, and single-crystal X-ray diffraction techniques. Interestingly, C. aphidicola and F. lini were able to catalyze hydroxylation only at alpha positions of 1. Compounds 1–9 showed a varying degree of cytotoxicity against HeLa , PC3 (human prostate carcinoma), H460 (human lung cancer), and HCT116 (human colon cancer) cancer cell lines. Interestingly, metabolites 4 (IC50 = 49.5 ± 2.2 μM), 5 (IC50 = 39.8 ± 1.5 μM), 6 (IC50 = 40.7 ± 0.9 μM), 7 (IC50 = 43.9 ± 2.4 μM), 8 (IC50 = 19.6 ± 1.4 μM), and 9 (IC50 = 25.1 ± 1.6 μM) were found to be more active against HeLa cancer cell line than the substrate 1 (IC50 = 54.7 ± 1.6 μM). Similarly, metabolites 2 (IC50 = 84.6 ± 6.4 μM), 3 (IC50 = 68.1 ± 1.2 μM), 4 (IC50 = 60.4 ± 0.9 μM), 5 (IC50 = 84.0 ± 3.1 μM), 6 (IC50 = 58.4 ± 1.6 μM), 7 (IC50 = 59.1 ± 2.6 μM), 8 (IC50 = 51.8 ± 3.4 μM), and 9 (IC50 = 57.8 ± 3.2 μM) were identified as more active against PC-3 cancer cell line than the substrate 1 (IC50 = 96.2 ± 3.0 μM). Metabolite 9 (IC50 = 2.8 ± 0.2 μM) also showed potent anticancer activity against HCT116 cancer cell line than the substrate 1 (IC50 = 3.1 ± 3.2 μM). In addition, compounds 1–7 showed no cytotoxicity against 3T3 normal cell line, while compounds 8 (IC50 = 74.6 ± 3.7 μM), and 9 (IC50 = 62.1 ± 1.2 μM) were found to be weakly cytotoxic.In search of selective and effective anti-cancer agents, eight metabolites of anti-cancer steroid, drostanolone enanthate ( In several instances, microbial transformation has been used as an important tool for the regio-, chemo-, and stereo-selective conversions of organic compounds which are difficult to achieve by conventional methods  are anabolic-androgenic steroids (AASs) used by athletes to strengthen their muscles without gaining fat. In addition, drostanolone, and its esters derivatives have the ability to inhibit the production of estrogen. Propionate ester of drostanolone is also used for the treatment of breast cancer, under the brand name of Masteron , the prevalence of cancer is exceeding 6 million cases per year. Cancer cells have high proliferation rate. They spread rapidly in the living system, and can survive against strong chemotherapeutics and DNA damaging agents. Similarly, cytotoxic drugs have many adverse effects on normal cells, and thus their use in cancer chemotherapy is a therapeutic challenge. Due to this, development of safe, effective and selective chemotherapeutic agents is urgently needed against various cancers  through its transformation with Cephalosporium aphidicola, and Fusarium lini. Metabolites 2–9, as well as substrate 1, were evaluated against HeLa , PC-3 (prostate carcinoma), H460 (lung cancer), HCT116 (colon cancer), and 3T3  cell lines, using high-throughput cell-based assay, the most efficient and convenient laboratory method, the MTT assay to predict the response of test compounds in malignancies where they showed specificity against the cancer cells. This study has thus identified anti-cancer metabolites of drostanolone enanthate (1) for further studies.In continuation of our studies on the fungal transformation of bioactive steroids  was used for the analysis of degree of transformation and purity. Silica gel column chromatography  was used for the initial purification of metabolites. In addition, preparative reverse phase recycling , equipped with YMC L-80 , was used for the final purification of metabolites. Structures of metabolites were elucidated with the help of 1H- , and 13C-NMR  spectra, which were recorded on Bruker Avance-NMR spectrometers (France) in CD3OD, CD3COCD3 or DMSO-d6. HREI-MS and IR spectra were performed on JEOL JMS-600H (Japan)  mass spectrometer , and Bruker Vector 22 FT-IR spectrophotometer, respectively. Optical rotations of metabolites were recorded on JASCO P-2000 polarimeter (Japan). Evolution 300 UV-visible spectrophotometer was used to record the UV spectra. The melting points of the transformed products were measured by using Buchi M-560 (Switzerland) instrument. Single-crystal X-ray diffraction data was collected on Bruker APEXII D8 Venture diffractometer, fitted with PHOTON 100 detector (CMOS Technology) and fine-focus sealed tube having X-ray source (Cu Kα radiations α = 1.54178 Å). Reflection intensities were integrated using SAINT software. Absorption correction was done on Multi-scan, and structure was solved by SHELXTL program   and Northern Regional Research Laboratories (NRRL), respectively. Cultures were grown on Sabouraud dextrose agar (SDA) slant, and maintained at 4°C.Microbial culture of C. aphidicola ATCC-28300 was prepared by mixing 200 g of glucose, 4 g of KH2PO4, 8 g of glycine, 4 g of KCl, 8 g of MgSO4.7H2O, and 8 mL of trace elements in 4 L of distilled water. Similarly, four liter of media was prepared for F. lini NRRL 2204 by mixing 40 g of glucose, 20 g of yeast extract, 20 g of peptone, 20 g of KH2PO4, 20 g of NaCl, and 40 mL of glycerol in 4 L of distilled water.Four liter of media for the growth of C. aphidicola and F. lini was prepared by mixing above mentioned ingredients in 4 L of distilled water for each fungus, which was then dispensed equally into 40 Erlenmeyer flasks of 250 mL (100 mL in each flask). All these flasks were autoclaved at 121°C, and were inoculated with SDA slants of C. aphidicola ATCC 28300 and F. lini NRRL 2204 under sterilized conditions. These fungal culture containing flasks were left on the rotary shaker (121 rpm) at 26 ± 2°C for 3–4 days to obtain maximum growth of C. aphidicola and F. lini. Compound 1 (1 g) was dissolved in 20 mL of acetone for each fungus, and was dispensed 0.5 mL in each flask, and again placed on rotary shaker for 12 days. A negative control , as well as positive control (medium + substrate) were also prepared analogously to evaluate the fungal metabolites and degradation of compound 1, respectively. After incubation of 12 days, all the flasks were filtered and extracted with 20 L of dichloromethane (DCM), organic phase was separated, and then was evaporated under reduced pressure on rotary evaporator. This yielded 1.5 g of pale yellowish solid crude. This crude material was fractionated over silica gel column chromatography by elution with gradient hexanes and acetone solvent systems. Column chromatography yielded four main fractions (1–4), which were then purified by recycling HPLC. Metabolites 2 , 3 , 4 , 5 , 6 , 7 , and 9  were purified from fractions 1–7, respectively, through reverse phase recycling HPLC using methanol- water as solvent system . Whereas, metabolite 8 was obtained from fraction 8 through normal phase recycling HPLC .Medium for the growth of c 0.0046, CH3OH); IR (CHCl3): υmax (cm−1), 3531 (OH), 3423 (OH); HREI-MS: m/z 322.2517 [M+], ; EI-MS: m/z (%); 322.2 [M+] (2), 304.2 (74), 271.2 (36), 264.2 (82), 110.0 (99); 1H-NMR : Table 13C-NMR : Table White solid; m. p.: 274–375°C; c 0.0017, CH3OH); IR (CHCl3): υmax (cm−1), 3436 (O-H), 1711 (C = O); HREI-MS: m/z 318.2192 [M+] ; EI-MS m/z (%): 318.1 [M+] (7), 300.1 (36), 147.1 (16.2), 136.1 (100); 1H-NMR : Table 13C-NMR : Table White solid; m. p.: 230–234°C; c 0.0077, CH3OH), IR (CHCl3): υmax (cm−1), 3315 (OH); HREI-MS: m/z 306.2553 [M+] ; EI-MS m/z (%): 306.3 [M+] (75), 291.2 (61), 229.2 (98), 179.1 (93), 121.1 (100); 1H-NMR : Table 13C-NMR : Table White solid; m. p.: 242–245°C; max: 229 nm ; c 0.018, CH3OH); IR (CHCl3): υmax (cm−1), 1737 (C = O stretching), 1665, 1625 ; HREI-MS: m/z 298.1944 [M+] ; EI-MS m/z (%): 298.0 [M+] (55), 280.0 (8), 197.9 (28), 169.9 (23), 152.9 (99), 136.0 (100); 1H-NMR : Table 13C-NMR : Table White solid; m. p.: 198–202°C; UV λmax: 248 nm ; c 0.0012, CH3OH); IR (CHCl3): υmax (cm−1), 3436 (OH),1736 (C = O), 1661, 1621 ; HREI-MS: m/z 314.1897 [M+] ; EI-MS m/z (%): 314.2 [M+] (45), 296.2 (15), 148.1 (11), 136.1 (100), 135.1 (50), 121.1 (16); 1H-NMR : Table 13C-NMR : Table White solid; m. p.: 230–234°C; UV λmax: 248 nm ; c 0.0014, CH3OH); IR (CHCl3): υmax (cm−1), 3468 (OH), 1727 (C = O), 1666, 1628 ; HREI-MS: m/z 314.1861 [M+] ; EI-MS m/z (%): 314.2 [M+] (57), 286.1 (8), 136.1 (62), 135.1 (100), 105.0 (23). 1H-NMR : Table 13C-NMR : Table White solid; m. p.: 224–228°C; UV λmax: 230 nm  c 0.0077, CH3OH), IR (CHCl3): υmax (cm−1), 3417 (OH); 1736 (C = O); HREI-MS: m/z 316.2042 [M+] ; EI-MS m/z (%): 316.2 [M+] (88), 270.2 (50), 159.1 (26.6), 136.1 (59.1), 123.1 (65.4); 1H-NMR : Table 13C-NMR : Table White solid; m. p.: 250–256°C; UV λc 0.00065, CH3OH), IR (CHCl3): υmax (cm−1), 3437 (OH); HREI-MS: m/z 304.2403 [M+] ; EI-MS m/z (%): 304.3 [M+] (63.7), 245.2 (84), 138.1 (18), 95.1 (24), 91.1 (100); 1H-NMR : Table 13C-NMR : Table White solid; m. p.: 151–153°C; 2 atmosphere at 37°C in an incubator.The cytotoxicity of samples was measured against HeLa , PC-3 (human prostate cancer ATCC CRL-1435), NCl-H460 (human lung carcinoma ATCC HTB-177), HCT116 (human colon cancer ATCC CCl-247), and 3T3  cell lines by using standard MTT assay. Cell lines were grown in DMEM F12 media, supplemented with 10% FBS under a 5% COmax at 540 nm in DMSO treated 3T3 fibroblast cells (that are more confluent in morphology) whereas in case of cancer cells, 570 nm gives λmax -2, 5-diphenyl tetrazolium bromide] was used for the evaluation of cell metabolic activity. In this assay, the yellow MTT is reduced to purple formazon in the mitochondria of the living cells  (C27H44O3 [M+] at m/z 416.4), , suggesting the addition of three hydrogen, and an oxygen atom, reduction of C-3 ketonic carbonyl, and hydrolytic cleavage of heptanoate side chain in substrate 1. The IR absorbance at 3,531, and 3,423 cm−1 were due to presence of hydroxyl groups. The 1H-NMR spectrum showed an additional methine proton signal at δ 3.69  with C-3 (δ 71.4)  with C-14 (δ 84.8). The third OH was at C-17, resulted from hydrolytic loss of heptanoate moiety. The O-H group at C-3 was deduced to be α-oriented based on the NOESY correlations of H-3  with β-oriented H-2 , and α-oriented H3-20  . Single-crystal X-ray diffraction analysis further supported the structure of metabolite 2, comprised of three rings in chair conformation , and one in envelop conformation (D). Three OH groups at C-3, C-14, and C-17 were assigned, α-, α-, and β-orientation . The structure of metabolite 2 was thus deduced as 2α-methyl-3α,14α,17β-trihydroxy-5α-androstane.Metabolite 3 was obtained as a white solid. The HREI-MS displayed the [M+] at m/z 318.2192 . This represented the loss of heptanoate side chain, and addition of an oxygen atom in substrate 1. IR spectrum showed absorbances for OH  and ketonic carbonyls . In the 1H-NMR spectrum, signals of heptanoate chain and C-17 methine proton were found missing, whereas a new downfield methine proton signal was observed at δ 3.92  showed HMBC correlations with C-5, and C-8, suggesting an OH at C-7  showed NOESY correlations with axially oriented H-8 , which suggested an α-orientation of OH at C-7 , due to the loss of heptanoate side chain, addition of an oxygen atom, and loss of five hydrogen atoms in substrate 1. IR spectrum showed absorbances for OH , ketone , and enone carbonyl . The 1H-NMR signals for heptanoate protons were found missing, whereas new olefinic  and an oxymethine  protons were appeared , while another C = C was placed between C-4/C-5, based on the HMBC correlations of H3-19 with another newly formed olefinic carbon at δ 171.7 (C-5) and H-1 with olefinic carbon at δ 124.3 (C-4) . NOESY correlations of H-11  with axially oriented H-8 , H3-18 , and H3-19  suggested an OH group at alpha , due to the loss of heptanoate side chain, addition of an oxygen atom, and loss of five hydrogen atoms in substrate 1. IR spectrum showed absorbances for OH , ketone , and enone carbonyl . The 1H-NMR signals for heptanoate protons were found missing, whereas new olefinic protons  appeared , while another C = C was placed between C-4/C-5, based on the HMBC correlations of H3-19 and H2-6 with another newly formed olefinic carbon at δ 171.9 (C-5) and H2-6 with olefinic carbon at δ 123.7 (C-4) . Newly formed ketonic carbonyl was placed at C-17, based on the HMBC correlations of H3-18 and H2-16 with newly formed carbon at δ 223.0 (C-17). The OH-14 was deduced to be α-oriented, based on the NOESY correlations of H-9  with OH-14  (acetone-d6) , due to the loss of heptanoate side chain, addition of an oxygen atom, and loss of three hydrogen atoms in substrate 1. IR spectrum showed absorbances for OH , enone , and carbonyl . The 1H-NMR signals for heptanoate protons were found missing, whereas new olefinic  and an oxymethine protons  appeared in the spectrum of metabolite 8 (Table 13C-NMR spectrum (Table 3-19 with newly formed olefinic carbon at δ 155.6 (C-1), and H3-20 with olefinic carbon at δ 134.3 (C-2) . OH at C-7 was further supported by COSY correlations of H-7 with H2-6 and H-8. Newly formed ketonic carbonyl carbon was deduced to be at C-17, as inferred from the HMBC correlations of H3-18 and H2-16 with newly formed carbon at δ 223.4 (C-17). NOESY correlations of H-7  with axially oriented H-8   (Data sheet 5) (Data sheet 9)  was also obtained through the biotransformation of compound 1. Drostanolone was used as a starting material for the synthesis of drostanolone propionate, and drostanolone enanthate (1), and other derivatives.Metabolites 1, and 5. Compound 1 was comprised of four rings i.e., A, B, C, and D with chair, chair, chair, and envelope conformations. A methyl was present at C-2 with equatorial orientation. Heptanoate ester chain was present at C-17 of ring D . Single-crystal X-ray diffraction analysis data of compounds 1, 2, and 5 is presented in Table Single-crystal X-ray diffraction analyses were also carried out on compounds D Figure . Whereasvia microbial biotransformation due to presence of a range of enzymes in them. Based on reported anti-cancer activity of drostanolone (9), drostanolone propionate, and drostanolone heptanoate (1), compounds 1–9 were evaluated for cytotoxicity against HeLa, PC-3, H460, and HCT116 cancer cell lines. Interestingly, all compounds showed a moderate to potent anti-cancer activity. The anti-cancer activity of compounds 1–9 were inferred from the IC50 values against HeLa , PC-3 (human prostate cancer), H460 (human lung cancer), and HCT116 (human colon cancer) (Table 8 (IC50 = 74.6 ± 3.7 μM), and 9 (IC50 = 62.1 ± 1.2 μM), were found to be non-cytotoxic.Natural products, e.g., flavonoids, steroids, terpenes, etc. can serve as an excellent source for the production of many drugs, as they are reported for many biological activities such as anti-cancer, anti-inflammatory, anti-leishmanial, anti-bacterial activities, etc. Therefore, it is necessary to derivatize them, to study structure-activity relationship which might be helpful for the synthesis of safe and effective drugs. At present, many pharmaceutical products are efficiently synthesized 2 with the absence of ester moiety, and two αO-H groups at C-3, and C-14, and a βO-H group at C-17 showed a lower cytotoxicity against HeLa, H460, and HCT116 cancer cell lines, but its anti-cancer activity was higher against PC-3 cancer cell line, as compared to compound 1. Metabolite 3 with the loss of ester group and two keto groups at C-3, and C-17, and αO-H group at C-7 also showed a lower activity against HeLa, H460, and HCT116 cancer cell lines, but increased activity against PC-3 cancer cell line in comparison to substrate 1. Reduction of C-3 carbonyl to O-H and hydrolytic cleavage of ester moiety in compound 4 increased its activity against both HeLa and PC-3 cancer cell lines to some extent, but its anti-cancer activity against H460, and HCT116 cancer cell lines decreased as compared to substrate 1. Presence of C = C between C-1/C-2 and C-4/C-5, along with C = O at C-3, and C-17 made metabolite 5 a potent anti-cancer agent against HeLa cancer cell line, as compared to substrate 1 and the standard drug (cisplatin). Moreover, its anti-cancer activity against PC-3 cancer cell line was also increased as compared to substrate 1. Interestingly, its anti-cancer activity against H460, and HCT116 cancer cell lines was lower in comparison to compound 1. Presence of C = C between C-1/C-2 and C-4/C-5, C = O at C-3, and C-17, along with αO-H group at C-11 in metabolite 6 increased its anti-cancer activity against HeLa, and PC-3 cancer cell lines in comparison to substrate 1 and showed similar anti-cancer potential against HeLa cancer cell line as standard drug (cisplatin). However, its anti-cancer activity against H460, and HCT116 cancer cell lines decreased in comparison to compound 1. Presence of C = C between C-1/C-2 and C-4/C-5, C = O at C-3, and C-17, along with αO-H group at C-14 in metabolite 7 again increased its anti-cancer activity against HeLa, and PC-3 cancer cell lines, and decreased its anti-cancer activity against H460, and HCT116 cell lines in comparison to substrate 1. Presence of C = C at C-1/C-2, C = O at C-3, and C-17, and αOH at C-7 made metabolite 8 the most potent anti-cancer against HeLa cancer cell line in comparison to substrate 1 and the standard drug (cisplatin). It also showed a good activity against PC-3 cancer cell line but decreased anti-cancer activity against H460, and HCT116 cancer cell lines as compared to substrate 1. Metabolite 9 with only hydrolysis at C-17 showed increased anti-cancer activity against HeLa, PC-3, and HCT116 cancer cell lines but decreased cytotoxicity against H460 cancer cell line in comparison to substrate 1 and the standard drug (cisplatin). On the basis of above discussion, it can be concluded that increased anti-cancer activity of compounds 5–8 against HeLa, and PC-3 cancer cell lines was probably due to the presence of C = C at C-1/C-2, and carbonyl ketone at C-3, and C-17 in comparison to substrate 1.Variations in the structures of metabolites effected their anti-cancer activity. Metabolite 1) with C. aphidicola and F. lini led to the synthesis of eight metabolites, including five new metabolites 2, 3, 6, 7, and 8. Hydroxylation, oxidative cleavage of ester moiety, reduction, and dehydrogenation were the main reactions observed during the transformation. Compounds 1–9 exhibited anti-cancer potential against HeLa , PC-3 (human prostate carcinoma), H460 (human lung cancer), and HCT116 (human colon cancer) cancer cell lines between moderate to potent range. Interestingly, substrate 1 (IC50 = 3.1 ± 3.2 μM) and metabolite 9 (IC50 = 2.8 ± 0.2 μM) were found to be more active against HCT116 cancer cell line than the standard drug, cisplatin (IC50 = 11.2 ± 3.0 μM). Compounds 1 (IC50 = 5.0 ± 1.2 μM), 3 (IC50 = 16.7 ± 2.6 μM), and 4 (IC50 = 12.4 ± 2.3 μM) were found to be more active against H460 cancer cell line than the standard drug, cisplatin (IC50 = 22.2 ± 2.1 μM). Metabolites 5 (IC50 = 39.8 ± 1.5 μM), 8 (IC50 = 19.6 ± 1.4 μM), and 9 (IC50 = 25.1 ± 1.6 μM) were also found more active against HeLa cancer cell line than the standard drug, cisplatin (IC50 = 40.1 ± 2.0 μM). Metabolites 3 (IC50 = 68.0 ± 1.2 μM), 4 (IC50 = 60.4 ± 0.9 μM), 6 (IC50 = 58.4 ± 1.6 μM), 7 (IC50 = 59.1 ± 2.6 μM), 8 (IC50 = 51.8 ± 3.4 μM), and 9 (IC50 = 57.8 ± 3.2 μM) were also found more active against PC-3 cancer cell line than the standard drug, cisplatin (IC50 = 76.5 ± 1.2 μM). Except compounds 8 and 9, all compounds were found to be non-cytotoxic to normal 3T3 cell line. These results indicated specific cytotoxicity of this class of compounds against cancer cell lines, as compared to normal cell line. Thus, the results of presented study will be helpful towards the drug discovery against cervical, prostate, lung, and colon cancers.In conclusion, the microbial transformation of anabolic-androgenic steroid drostanolone heptanoate (The concept of presented research was developed and designed by MIC. MIC was also the project supervisor. All the data were analyzed and interpreted by MIC, A-t-W, and HC. MS, SY, MA, and NF performed the experiments. MIC, A-t-W, and MS wrote the manuscript. All the authors revised the manuscript. The final version of manuscript was also approved by MIC before submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In the recent years, the role of LOX enzymes in the origin of neoplastic diseases such as colorectal, skin, pancreatic and renal cancers has been confirmed. A new series of 1,3,4-thiadiazole derivatives bearing 2-pyridyl moiety was synthesized and the cytotoxicity of the members of this series was assessed using MTT protocol. Enzyme inhibitory activity of the prepared compounds was also tested against 15-lipoxygenase-1 as a novel target for the discovery of anticancer drugs. PC3, HT29 and SKNMC cell lines were utilized and the obtained results were compared with doxorubicin. Overall, nitro containing derivatives exerted a higher cytotoxic activity against PC3 cell line and methoxylated derivatives showed an acceptable activity against SKNMC cell line. Methoxylated derivatives were also the most potent enzyme inhibitors especially at position  Arachidonic acid (AA) and its metabolites have recently generated a heightened interest due to the growing evidence of their significant role in cancer biology . LOXs ar.Several Chemistry254 plates were applied for analytical TLC. Column chromatography was performed on Merck silica gel (70-230 mesh) for purification of intermediate and final compounds. 1H-NMR spectra were recorded using a Varian 400 spectrometer, and chemical shifts are expressed as δ (ppm) with tetramethylsilane (TMS) as internal standard. The IR spectra were obtained on a Shimadzu 470 spectrophotometer (potassium bromide disks). Melting points were determined using elemental analyzer apparatus and are uncorrected. The mass spectra were run on a Finigan TSQ-70 spectrometer  at 70 eV. All cell lines were purchased from Pasteur Institute of Iran. All intermediate and final compounds were prepared according to the All chemical substances such as starting materials, reagents and solvents were purchased from commercial suppliers like Merck and Sigma-Aldrich companies. The purity of the prepared compounds was proved by thin layer chromatography (TLC) using various solvents of different polarities. Merck silica gel 60 GFSynthesis of 2-(pyridin-2-ylmethylene)hydrazinecarbothioamide (2)5 g  of pyridine-2-carbaldehyde were treated with 4.25 g (0.07 mmol) thiosemicarbazide in ethanol (100 mL) solvent. The reaction mixture was refluxed with addition of 1 mL of hydrochloric acid for 5 h. Thin layer chromatography (TLC) was utilized for monitoring the reaction progress. The formed precipitate was filtered and washed by cold water . 1H NMR  δ (ppm): 7.42 , 7.93 , 8.02 , 8.29 , 8.41 , 8.76 , 11.73 . IR  ῡ: 3433 , 3259, 3159 , 3055 , 1608 , 1527 , 1462 , 1296 . MS : 180 , 120 (90), 102 (15), 105 (20), 92 (75), 78 (40), 65 (85). Synthesis of 5-(pyridin-2-yl)-1,3,4-thiadiazol-2-amine (3)4Fe(SO4)2.12 H2O] in water (100 mL) as solvent. Then, 10 g of ammonium ferric sulfate [(NH4Fe(SO4)2.12 H2O] was dissolved in 30 mL of water and added to the reaction medium. The reflux condition was continued for 30 h. Thin layer chromatography (TLC) was applied for monitoring the reaction. The reaction mixture was poured in separating funnel and ethyl acetate was added. Organic phase was washed three times by sodium bicarbonate 2% and brine. Dryness was carried out over anhydrous sodium sulfate and then filtered. The ethyl acetate was evaporated under reduced pressure and obtained powder was washed by n-hexane and diethyl ether (5 g 0.027 mmol) of 2-(pyridin-2-ylmethylene)hydrazinecarbothioamide  δ (ppm): 7.40 , 7.51 , 7.89 , 8.03 , 8.56 . IR  ῡ: 3271 , 3093 , 1620 , 1500 , 1435 , 1130 . MS : 178 , 136 (15), 120 (20), 105 (40), 92 (20), 78 (60). General procedure for synthesis of compounds 4a-4l:N-ethyl-N-dimethylaminopropyl carbodiimide (EDC) in acetonitrile (20 mL). The reaction mixture was stirred at room temperature for 30 min. Then, 200 mg (1.1 mmol) of compound (n-hexane and diethyl ether (Et2O)  hydroxybenzotriazole (HOBt) and r (Et2O) -19, 21. 2-Chloro-N-(5-(pyridin-2-yl)-1,3,4-thiadiazol-2-yl)benzamide (4a)1H NMR  δ (ppm): 7.58 , 7.70 , 8.00 , 8.23 , 8.69 , 13.33 . IR  ῡ: 3429 , 3116 , 1678 , 1585 , 1535 , 1435 , 1311 . MS : 318 , 316 , 281 (85), 253 (20), 141 (55), 139 (100), 122 (20), 111 (60), 78 (40), 51 (20).3-Chloro-N-(5-(pyridin-2-yl)-1,3,4-thiadiazol-2-yl)benzamide (4b)1H NMR  δ (ppm): 7.53 , 7.61 , 7.72 , 7.99 , 8.08 , 8.20 , 8.23 , 8.69 , 12.50 . IR  ῡ: 3147 , 3005 , 1674 , 1535 , 1315 . MS : 318 , 316 , 315 (15), 288 (20), 211 (20), 141 (25), 139 (100), 122 (25), 111 (75), 95 (10), 78 (50), 75 (25), 51 (20).4-Chloro-N-(5-(pyridin-2-yl)-1,3,4-thiadiazol-2-yl)benzamide (4c)1H NMR  δ (ppm): 7.38 , 7.47 , 7.89 , 8.16 , 8.60 . IR  ῡ: 3159 , 3089, 3062, 3008 , 1670 , 1589 , 1523 , 1485 , 1311 . MS : 318 , 316 (M+), 315 (30), 288 (60), 149 (60), 139 (100), 111 (40), 78 (20), 57 (20). 2-Fluoro-N-(5-(pyridin-2-yl)-1,3,4-thiadiazol-2-yl)benzamide (4d)1H NMR  δ (ppm): 7.37 , 7.54 , 7.63 , 7.79 , 8.00 , 13.17 . IR  ῡ: 3120 , 3070 , 1685 , 1616 , 1535 , 1496 , 1319 . MS : 300 , 281 (75), 272 (40), 253 (15), 123 (100), 95 (75), 78 (30), 76 (25), 51 (15).3-Fluoro-N-(5-(pyridin-2-yl)-1,3,4-thiadiazol-2-yl)benzamide (4e)1H NMR  δ (ppm): 7.56 , 8.00 , 8.24 , 8.69 , 13.31 . IR  ῡ: 3132 , 3070 , 1678  1589 , 1531 , 1489 , 1269 . MS : 300 , 299 (20), 272 (55), 123 (100), 95 (90), 78 (40), 51 (20).4-Fluoro-N-(5-(pyridin-2-yl)-1,3,4-thiadiazol-2-yl)benzamide (4f)1H NMR  δ (ppm): 7.39 , 7.53 , 7.99 , 8.23 , 8.68 , 13.35 . IR  ῡ: 3151 , 3066 , 1678 , 1604 , 1539 , 1234 . MS : 300 , 299 (30), 272 (40), 123 (100), 95 (40), 78 (20). 2-Methoxy-N-(5-(pyridin-2-yl)-1,3,4-thiadiazol-2-yl)benzamide (4g)1H NMR  δ (ppm): 3.91 , 7.09 , 7.22 , 7.56 , 7.70 , 7.99 , 8.23 , 8.69 , 12.44 . IR  ῡ: 3294 , 3070 , 1666 , 1600 , 1523 , 1485 , 1238 . MS : 312 , 282 (30), 281 (100), 136 (20), 135 (100), 92 (35), 78 (35), 77 (75), 51 (15).3-Methoxy-N-(5-(pyridin-2-yl)-1,3,4-thiadiazol-2-yl)benzamide (4h)1H NMR  δ (ppm): 3.85 , 7.22 , 7.43 , 7.53 , 7.71 , 7.73 , 7.99 , 8.23 , 8.69 , 13.20 . IR  ῡ: 3109 , 3051 , 2920, 2846 , 1670 , 1585 , 1531 , 1492 , 1276 . MS : 312 , 284 (25), 135 (100), 122 (15), 107 (5), 92 (35), 77 (20). 4-Methoxy-N-(5-(pyridin-2-yl)-1,3,4-thiadiazol-2-yl)benzamide (4i)1H NMR  δ (ppm): 3.81 , 6.99 , 7.36 , 7.51 , 7.69 , 8.05 , 8.56 , 13.25 . IR  ῡ: 3275 , 3097, 3066 , 2931 , 1662 , 1604 , 1261 . m/z, %): 312 , 284 (10), 135 (100), 122 (10), 107 (15), 92 (15), 77 (20). MS (2-Nitro-N-(5-(pyridin-2-yl)-1,3,4-thiadiazol-2-yl)benzamide (4j)1H NMR  δ (ppm): 7.55 , 7.82-7.92 , 8.02 , 8.23 , 8.69 , 13.45 . IR  ῡ: 3097 , 2997, 2897 , 1678 , 1531 , 1311 : 327 , 299 (20), 281 (30), 253 (25), 205 (15), 150 (75), 122 (100), 104 (20), 78 (75), 76 (40), 51 (45).Symmetric). MS (3-Nitro-N-(5-(pyridin-2-yl)-1,3,4-thiadiazol-2-yl)benzamide (4k)1H NMR  δ (ppm): 7.54 , 7.86 , 8.02 , 8.23 , 8.47 , 8.53 , 8.69 , 8.98 , 13.50 . IR  ῡ: 3093 , 2997, 2920 , 1674 , 1531 , 1323 . MS : 327 , 326 (45), 299 (50), 240 (15), 205 (40), 150 (100), 122 (35), 104 (50), 78 (45), 76 (50), 50 (15).4-Nitro-N-(5-(pyridin-2-yl)-1,3,4-thiadiazol-2-yl)benzamide (4l)1H NMR  δ (ppm): 7.51 , 7.68 , 7.98 , 8.21 , 8.67 . IR  ῡ: 3136 , 3070 , 2924 , 1685 , 1527 , 1319 . MS : 327 , 299 (30), 240 (80), 205 (20), 178 (60), 167 (50), 149 (90), 136 (25), 123 (60), 109 (40), 97 (60), 83 (60), 69 (75), 57 (100). MTT assayoC were done, and then the medium was discarded. Dimethyl sulfoxide (60 μL) was added to each well, and the solution was vigorously mixed to dissolve the purple tetrazolium crystals. The absorbance of each well was measured by plate reader  at a test wavelength of 550 nm against a standard reference solution at 690 nm. The amount of produced purple formazan is proportional to the number of viable cells Synthesized derivatives of 1,3,4-thiadiazole (compounds 4a-4l) were tested for cytotoxic activity at 0.1-250 mcg/mL concentration in three human cancer cell lines of PC3 cell (prostate cancer), HT-29 (Colon cancer) and SKNMC (Neuroblastoma). Cells from different cell lines were seeded in 96-well plates at the density of 8000–10,000 viable cells per well and incubated for 48 h to allow cell attachment. The cells were then incubated for another 48-96 h (depends to cell cycle of each cell line) with various concentrations of compounds 4a-4l. Cells were then washed in PBS, and 20 μL of MTT -2,5-diphenyl tetrazolium bromide solution (5 mg/mL) were added to each well. An additional 4 h of incubation at 37 -19.Enzymatic assayThe basis of this method is oxidative coupling of 3-methyl-2-benzothiazolinone hydrazone (MBTH) with 3-(dimethylamino) benzoic acid (DMAB) in a hemoglobin catalyzed reaction. This reaction is initiated in the presence of lipoxygenase reaction product, linoleic acid hydroperoxide and results in a blue color formation which has a peak absorbtion at 590 nm . QuercetCytotoxicity assayin-vitro. Tested compounds rendered a high anticancer activity towards HT29 cell line compared to other cell lines. Nitro containing derivatives  demonstrated a remarkable inhibitory activity against HT29 cell line. Meta positioning of the nitro moiety caused a better cytotoxic potency than other positions. It is likely that the capability of the nitro group for generating free radicals is responsible for its higher anticancer potency in comparison with other moieties. Generally, investigation of the various substituents with electron withdrawing  as well as electron donating (-OCH3) activity were carried out. Chlorinated derivatives showed superior activity against SKNMC cell line than PC3 and HT29. Probably, enhancement of the lipophilic property of these compounds that caused by chlorine substituent is a favorable parameter for improving the anticancer activity. Para positioning of the chlorine atom led to the significant cytotoxic activity towards SKNMC cell line (IC50 = 6.71). A significant potency was observed against PC3 cell line while methoxylated derivatives were investigated. The methoxy moiety showed a superior activity when introduced on the ortho and para positions of the phenyl residue. It is clear that electron donating feature of this group is a beneficial factor for increasing the activity. Movement of the methoxy to the meta position of the phenyl residue end to an outstanding reduction in potency. According to the Enzymatic assayo-methoxy) and 4k (m-methoxy) as methoxylated derivatives displayed the best inhibitory activity in this series. Nitro containing derivatives showed the lowest enzyme inhibitory activity. Besides, fluorinated and chlorinated derivatives were also tested to explore the role of electron withdrawing effect of the moiety. Position para was the best position for fluorine atom as well as chlorine atom to inhibit the lipoxygenase enzyme. Fluorine demonstrated a higher inhibitory potency at position para than chlorine moiety. It is probable increasing the electron withdrawing effect of the moiety and also reducing the lipophilicity are the responsible factors for this evidence.Inhibitory activity of synthesized derivatives 4a-4l was examined against 15-LOX-1 enzyme and obtained results were compared to quercetin as reference compound. Compounds 4j (The authors have been declared no conflict of interest.