Pueraria lobata (Puerariae Flos) is a reddish-purple to violet-purple flower that blooms between July and September. In our preliminary study, Puerariae Flos extract exhibited significant activity against a human ovarian cancer cell line. This research aims to identify the active compounds in Pueraria Flos. By repeated chromatography, one new tryptophan derivative (1), two new flavanones (4 and 5), and 19 known compounds, including tryptophan derivatives (2 and 3), flavonoids (6–9), isoflavonoids (10–20), a flavonolignan (21), and a phenolic compound (22), were isolated from a methanol extract of Puerariae Flos. The structures of new compounds were elucidated as 13-N-benzoyl-l-tryptophan-1-N-β-d-glucopyranoside (1), 2-hydroxy-5-methoxy-naringenin (4), and 2-hydroxy-5-methoxy-naringenin 7-O-β-d-glucopyranoside (5). Among the isolates, afromosin (17), tectorigenin (11), apigenin (8), glycitein (16), (-)-hydnocarpin (21), irilin D (12), irisolidone 7-O-glucoside (14), and genistein (10) showed cytotoxicity against human ovarian cancer cell line A2780. Apigenin (8) and (-)-hydnocarpin (21) were the most active .The flower of  Pueraria lobata (Willd.) Ohwi (Leguminosae) is one of the most important Chinese traditional medicines native to East Asia, Central and South America, and Europe , two meta coupled proton signals  and 6.49 -8)), a methylene signal at δH 3.66 , and a methoxy signal at δH 3.56   to C-5 (δC 160.6)  . The IR 10 cm−1) . The 1H-  , indicatxy group  20]. Th. Th4 was δC 55.7 . The locC 160.6) . The rel5 was obtained as a brown powder. The molecular formula of 5 was established as C22H24O11 from the HR-ESI-Orbitrap-MS  (−1) and benzene ring (1590 and 1502 cm−1) /5.62 ) and signals at δH 4.10–4.52 indicated the existence of the β-glucopyranosyl group in 5. In the 13C- and DEPT NMR spectra, some signals also appeared as dual peaks (5 showed the correlation between the anomeric proton (δH 5.68/5.62) and C-7 (δC 101.42/101.56), confirming the position of the β-glucopyranosyl group at C-7  . The IR 02 cm−1) . The 1H-des of 4 . The anoal peaks . Each peal peaks . The HMBp at C-7 . On the periment . Therefo2–3 and 6–22 were identified as 13-N-malonyl-l-tryptophan (2) [N-caproyl-l-tryptophan-1-N-β-d-glucopyranoside (3) [O-β-d-glucopyranoside (6) [O-β-d-glucopyranoside (7) [8) [9) [10) [11) [12) [13) [O-β-d-glucopyranoside (14) [O-β-D-xylopyranosyl-(1→6)-O-β-d-glucopyronoside (15) [16) [17) [18) [O-β-d-xylopyranosyl-(1→6)-O-β-d-glucopyronoside (19) [20) [21) [22) [P. lobata and puerarin; daidzein, tectoridin (13), and glycitin (18) were the most predominant constituents [2, 6, 7, 21, and 22 were isolated from P. lobata for the first time in this work.Compounds phan (2) , 13-N-caside (3) , 2-hydroside (7) , apigeni (7) [8) , nicotif [8) [9) , geniste[9) [10) , tectori10) [11) , irilin 11) [12) , tectori12) [13) , irisoliide (14) , tectoriide 15) , glycite15) [16) , afromos16) [17) , glyciti17) [18) , 7,4′-diide (19) , gehuain19) [20) , (-)-hyd20) [21) , and hyd21) [22) , by comp , glycit8), genistein (10), tectorigenin (11), irilin D (12), irisolidone 7-O-glucoside (14), glycitein (16), afromosin (17), and (-)-hydnocarpin (21) showed cytotoxic activity (IC50 <100 μM) against A2780 cells (8) and (-)-hydnocarpin (21) were the most active . In contrast, cisplatin, which is widely used as a first-line therapy in ovarian epithelial cancer, and quercetin, which is a well-known flavonoid to have anticancer activities, showed an IC50 value of 10.73 and 19. 45 μM, respectively. To further evaluate the effect of the compounds on the cell viability of non-malignant ovarian epithelial cells, we explored the cytotoxicity of the eight compounds  against the immortalized human ovarian surface epithelial cell line IOSE80PC. (-)-Hydnocarpin (21) showed a mild cytotoxicity with an IC50 value of 65.84 μM against IOSE80PC cells and other seven compounds exhibited no activity (IC50 >100 μM)  as to A2780 cells . To identify anti-tumor constituents in Pueraria Flos, we examined the effects of the 25 isolates on the cell viability of the human ovarian cancer A2780 cell line using the MTT assay. Among the isolated compounds, apigenin  inhibits the proliferation and migration of ovarian cancer cell line A2780 by suppressing the expression of Id1 and focal adhesion kinase [10) inhibits cell proliferation and downregulates VEGF expression in the ovarian cancer cell line OVCAR3 [12) and afromosin (17) in the breast cancer cell line MCF-7 has been reported [12), irisolidone 7-O-glucoside (14), afromosin (17), and (-)-hydnocarpin (21) on a human ovarian cancer cell line, to the best of our knowledge. Interestingly, (-)-hydnocarpin (21), a flavonolignan, exhibited the greatest effect on A2780 cells. (-)-Hydnocarpin (21) exhibited no cytotoxicity against multiple cancer cell lines  [Previous studies demonstrate that apigenin  , but shod MCF-7) ,45. Pueraria lobata (Willd.) Ohwi (Leguminoseae) were purchased from CK Pharm Co.  in June 2019. The origin of the herbal material was identified by prof. Dae Sik Jang and a voucher specimen (PULO5-2019) has been stored in the Lab. of Natural Product Medicine, College of Pharmacy, Kyung Hee University, Seoul, Korea.The flowers of General experimental procedures are in the P. lobata (1.6 kg) were extracted twice with methanol (16 L) at 80 °C for 2 h and the solvent was removed by rotary evaporator at 45 °C. The methanol extract (278.95 g) was fractionated by column chromatography (CC) using Diaion HP-20 with a gradient system of acetone and H2O (0:100 to 100:0 v/v) to give 17 fractions (F1~F17). Dried flowers of 22 (33.0 mg) was purified from F3-4 using medium pressure liquid chromatography . F3-7 was subjected to silica gel CC  to obtain compounds 19 (342.8 mg) and 5 (44.7 mg). F3-10 was subjected to silica gel CC  to give compounds 18 (24.4 mg), 6 (340.9 mg), and 7 (13.7 mg). F4 was fractionated further using Sephadex LH-20 CC (4.7 × 57.6 cm) with 50% methanol and produced six fractions (F4-1~F4-6). F4-5 yielded compound 6 (125.2 mg) by flash CC with Redi Sep-C18 cartridge . F5 was separated by Sephadex LH-20 CC (5.4 × 60.8 cm) with 50% methanol to produced nine fractions (F5-1~F5-9). F5-7 was subjected to MPLC with methanol and H2O mixture (from 30:70 to 35:65 v/v) to afford compound 6 (58.0 mg). F5-8 was subjected to silica gel CC  to isolate compounds 13 (5.7 mg) and 18 (4.2 mg). F6 was fractionated further using Sephadex LH-20 CC  to give nine fractions (F6-1~F6-9). F6-5 was chromatographed over silica gel  to obtain compounds 18 (2.8 mg) and 15 (205.1 mg). F6-7 yielded compound 2 (21.4 mg) by MPLC . Compounds 4 (38.3 mg) and 9 (10.6 mg) were isolated from F6 to F8 by flash CC (CH2Cl2:methanol:H2O = 100:0:0 to 50:45:5 v/v/v). Compound 13 (3.5373 g) was obtained by recrystallization from F7 in methanol. The mother liquor was fractionated further using Sephadex LH-20 CC (4.7 × 66.5cm) to isolate compounds 4 (25.4 mg), 1 (20.1 mg), 20 (5.7 mg), and 18 (23.2 mg). F8 was fractionated by Sephadex LH-20 CC  to obtain compound 3 (380.7 mg). F10 was subjected to Sephadex LH-20 CC  to obtain ten fractions (F10-1~F10-10). Compounds 14 (42.0 mg) and 12 (10.7 mg) were purified from F10-3 and F10-8, respectively, by MPLC. Compound 16 (16.6 mg) was recrystallized in F10-10 with methanol. F12 was separated by Sephadex LH-20 CC  to afford twelve fractions (F12-1~F12-12) and compounds 17 (16.6 mg) and 11 (1.54 g). F12-11 was chromatographed on silica gel  to isolate compound 10 (122.2 mg). Compounds 8 (13.8 mg), 16 (18.2 mg), and 21 (11.2 mg) were purified from F12-12 by silica gel CC .F3 was separated by Sephadex LH-20 CC (5.6 × 59.0 cm) with 45% methanol to afford twelve fractions (F3-1~F3-12). Compound m/z = 471.1758 ; c 0.1, methanol); UV (methanol) λmax nm (log ε): 204 (4.41), 223 (4.50), 272 (3.86); IR (ATR) νmax 1718, 1630, 1522, 1460, 1322, 1222, 1071, 1015, and 712 cm−1; 1H- and 13C-NMR data, see Pale yellow powder; HR-ESI-Orbitrap-MS (positive mode) m/z = 303.0879 ; UV (methanol) λmax nm (log ε): 215 (3.80), 286 (3.71), 324 (3.47); IR (ATR) νmax 1676, 1588, and 1510 cm−1; 1H- and 13C-NMR data, see Brown powder; HR-Q-TOF-MS (positive mode) m/z = 465.1391 ; UV (methanol) λmax nm (log ε): 211 (4.43), 2.86 (4.26); IR (ATR) νmax 1687 and 1590 cm−1; 1H- and 13C-NMR data, see Brown powder; HR-ESI-Orbitrap-MS (positive mode) 1 and 5 (each 1.0 mg) were hydrolyzed with 2N HCl at 80 °C for 4 h and with 1N HCl at 100 °C for 3 h, respectively. Each reaction was stopped by the addition of sodium thiosulfate. Additional hydrolysis was proceeded for identifying the absolute configuration of tryptophan moiety of 1. Compound 1 (1.0 mg) was hydrolyzed with 6N HCl at 85 °C for 24 h. Reaction was stopped by the addition of sodium thiosulfate.Compounds β-glucoses in 1 and 5, the modified analysis method from a reference was conducted [l-cysteine methyl ester hydrochloride (1.2 mg) was added and heated at 60 °C for 1 h. σ-Tolyl isothiocyanate (100 μL) was added and heated again at 60 °C for 1 h. The reaction product was analyzed by HPLC under a gradient system (A: 0.1 % (v/v) formic acid in water, B: 0.1 % (v/v) formic acid in acetonitrile, 10 to 50% B, 45 min). The glucoses in the reaction mixture of 1 and 5 were detected at 28.3 and 28.4 min each. Authentic l- and d-glucose were, respectively, detected at 27.6 and 28.2 min at the same HPLC conditions. Therefore, the absolute configuration of β-glucose in compounds 1 and 5 was identified as the d configuration.To determine the absolute configuration of onducted . The hydl-configuration  when compared to the optical rotation value of l-tryptophan  and d-tryptophan .The hydrolysate to determine the absolute configuration of tryptophan of 1 was subjected to HPLC by under a gradient system . Tryptophan (0.2 mg) was obtained from the hydrolysate. Analyzing the optical rotation dispersion of the obtained tryptophan revealed that it has 2 and 95% air humidified atmosphere at 37 °C. RPMI 1640, FBS, streptomycin sulfate, and penicillin were procured from Life Technologies Inc. . The cells were seeded at a density of 1.0 × 105 cells/mL in a 96-well plate containing 50 μL of RPMI medium in each well and incubated for 24 h. Various concentrations of compounds dissolved in dimethyl sulfoxide (DMSO) were mixed with RPMI 1640 medium and added into cells in each well. The final concentration of DMSO in the medium did not exceed 0.1%. Following 48 h incubation, 50 μL of MTT  solution was added into each well to achieve a final concentration of 0.5 mg/mL and then incubated for an additional 4 h. The medium was discarded, and the formazan blue that formed in the cells was dissolved in 50 μL of DMSO. The optical density was measured at 540 nm by microplate spectrophotometer . Three independent experiments with at least three replicates have been performed for all the tested compounds except quercetin, which has been tested once. IC50 is defined as the concentration that reduces cell number by 50% compared to control cultures. Results shown in Human ovarian endometrioid adenocarcinoma cell line A2780 and immortalized ovarian surface epithelial cell line IOSE80PC were provided by Dr. Ie-Ming Shih  and Dr. N. Auersperg , respectively. The cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 supplemented with 5% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin sulfate (100 μg/mL) in a 5% CO1–22), including one new tryptophan derivative, 13-N-benzoyl-l-tryptophan-1-N-β-d-glucopyranoside (1), and two new flavanones, 2-hydroxy-5-methoxy-naringenin (4) and 2-hydroxy-5-methoxy-naringenin 7-O-β-d-glucopyranoside (5). Apigenin (8), genistein (10), tectorigenin (11), irilin D (12), irisolidone 7-O-glucoside (14), glycitein (16), afromosin (17), and (-)-hydnocarpin (21) showed significant cytotoxicity against human ovarian cancer cell line A2780 and exhibited little cytotoxicity against human ovarian surface epithelial cell line IOSE80PC. (-)-Hydnocarpin (21), a flavonolignan, showed the most potent cytotoxic activity against A2780 cells.An investigation on compounds with cytotoxic activity in Puerariae Flos led to the isolation of 22 compounds (


H-1-chromene series, including the natural product precocene I, and the di-O-alkyl 2,2-dimethyl-2H-1-chromene series, including the natural 2H-1-chromenes precocenes II and III, was achieved. The synthetic compounds were subjected to spectroscopic analysis, 1HNMR,13CNMR, and mass characterization. The antifungal activity of synthesized precocenes I, II, and III, as well as their synthetic intermediates, was evaluated by the poison food technique. Precocene II (EC50 106.8 µg × mL−1 and 4.94 µg mL−1), and its regioisomers 7a (EC50 97.18 µg × mL−1 and 35.30 µg × mL−1) and 7d (EC50 170.58 × µg mL−1), exhibited strong fungitoxic activity against Aspergillus niger and Rhizoctonia solani. Some of the novel chromenes, 11a and 11b, which had never been evaluated before, yielded stronger fungitoxic effects. Finally, docking simulations for compounds with promising fungitoxic activity were subjected to structure–activity relationship analyses against the polygalactouronases and voltage-dependent anion channels. Conclusively, precocenes and their regioisomers demonstrated promising fungitoxic activity; such compounds can be subjected to minor structural modifications to yield promising and novel fungicides.This study aimed to synthesize, characterize, and explore the eco-friendly and antifungal potential of precocenes and their derivatives. The organic synthesis of the mono-O-alkyl-2,2-dimethyl 2 The problems associated with excessive pesticide use, particularly in developing countries such as Egypt, have prompted scientists to investigate eco-friendly and novel methods of plant protection. Secondary metabolites of higher plants, insects, and animals have been investigated as potential natural fungitoxicants for plant disease management. These natural chemicals are physiologically active and are produced to aid in pathogen defense, interspecies competition, and reproductive process facilitation. The biological activity of natural products against phytophagous insects, such as pesticides and crop protection compounds, has gained significant attention over the last couple of decades. Natural products are now considered as promising alternatives to the current arsenal of synthetic compounds . The hetRhizoctonia solani, and Aspergillus niger, especially the former, have long been recognized as a major threat to agricultural production around the globe, causing massive yield losses and control difficulties. R. solani invades the stem and root of the host plants, including the maize, rice, soybean, potato, sugar beet, and wheat ,  and  inhibited the mycelial growth of Lasiodiplodia theobromae at 100 mg × mL−1 concentration , 193 (100) [M-Me], 152 (81), 137 (15), 123 (21), 113 (19), 106 (21), 95 (26), 83 (26), 77 (30), 68 (30), 51 (54).Next, 100.2 g (1 mole) of 3-methyl-but-2-eneoic acid and 1319.7 g  of phosphorus oxychloride were stirred for 10 min. When the reaction mixture cooled down to the 25 ± 2 °C, the flask was charged with 201.5 g (1.4 moles) of zinc chloride. To this mixture, 126.14 g (1.0 mole) of 1,2,3-trihydroxybenzenes  yield 8 . The rea2–164 °C ), the wh1HNMR (DMSO-d6) spectral data for compound 3a: 2.0 , 1H, J = 9.4 Hz, 5′-H), 6.90 , 7.37 , 8.6 , 10.5 , 13.29 .The 1HNMR (DMSO-d6) spectral data for compound 3b: 2.0 , 2.11 , 6.28 , 6.76 , 7.23 , 8.75 , 10.39 , 12.85 .The 8: Rt = 17.370 min, MS : m/z (%) = 192 (5) [M+], 177 (100) [M+-Me], 137 (31), 108 (7), 81 (11), 69 (11), 51 (16).The GC-MS spectral data for compound 1HNMR (DMSO-d6) spectral data for compound 8: 2.01 , 2.15 , 6.25 , 6.35 , 6.91 , 7.84 , 10.57 , 13.26 .The 1HNMR, 13CNMR, and mass spectra of all prepared compounds in the experimental section are available in the The 3a or 3b compounds or 76.8 g (0.4 moles) of compound 8 was dissolved in 1 L of 5% aqueous NaOH solution (1.25M) and stirred for 1.5–2.0 h at room temperature. After the reaction was completed, the solution was diluted with 500 mL of cold distilled water. Then, the solution was acidified by dropping 36% HCl until pH 1 was obtained. The 4c  was prepared directly from 1,3,5-trihydroxybenzene (1c), and 162.14g (1.0 mole) of 3-methyl-but-2-eneoic acid and POCl3/ZnCl2 were mixed. The reaction was set overnight at 25 °C, as described in 4a and re-crystallization from ethanol–water (9:1) to obtain 4b, 4c, and 8. The reaction yields 95.1%, 87.6%, 51.8%, and 74.2% and MPs were 197–198, 207, 142, and 173 °C for 4a, 4b, 4c, and 8, respectively , Rt = 19.763 min, MS : m/z (%) = 208 (39) [M+], 193 (40.5), 152 (100) [M+-CH2=C(CH3)2], 124 (13.4), 106 (14.1), 113 (19), 106 (14), 95 (8), 68 (11), 53 (13).Afterwards, 100 g (0.48 moles) of ctively, ). The GC1HNMR (DMSO-d6) spectral data for compound 4a: 1.39 , 2.66 , 6.45 , 7.11 , 8.48 , 9.97 .The 4b , Rt = 21.313 min, MS : m/z (%) = 208 (48) [M+], 193 (100) [M+-Me], 153 (27), 124 (41), 106 (14.1), 96 (20), 78 (11), 51 (13).The GC-MS spectral data for compound 1HNMR (DMSO-d6) spectral data for compound 4b: 1.34 , 2.59 , 6.27 , 7.04 , 9.04 , 10.14 .The 4c , Rt = 19.790 min, MS : m/z (%) = 207 (100) [M+], 167 (78), 138 (39), 123 (15), 110 (17), 69 (38), 51 (15).The GC-MS spectral data for compound 1HNMR (DMSO-d6) spectral data for compound 4c: 1.38 , 2.77 , 5.82 , 10.73 , 12.11 .The 9: 7-hydroxy 2, 2-dimethyl chroman-4-one, Rt = 16.521 min, MS : m/z (%) = 192 (55) [M+], 177 (100) [M+-Me], 137 (61), 108 (56), 95 (5), 80 (20), 69 (18), 51 (21).The GC-MS spectral data for compound 1HNMR (CDCl3) of compound 8 had the following attributes; 1.46 , 2.68 , 5.81 , 6.35 , 6.48 , 7.80 .The (5a–f). Under inert gas (argon) conditions, 40 g (0.19 moles) of 4a–c or 19.2 g (0.1 moles) of compound 9 was charged with 100 mL of dry N,N-dimethylformamide for 4a–c or 100 of dry acetone for compound 9, and then 26.6 g (0.19 moles) or 14.0 g (0.1 moles) of anhydrous K2CO3 was added to the solutions and stirred at 25 °C for 1h. The resultant solution was refluxed at 80 °C. Then, 29.81 g  of 99% methyl iodide used to yield 5a–c and 15.61 g  used to prepare 10a, or 32.76 g  of 99% ethyl iodide used to yield 5d–f and 17.16 g  used to yield 10b, was dropped by a programable syringe pump  for over 1.5–6.0 h. The progress of the reaction was monitored by TLC (9:1 hexane: ethylacetate). The reaction times for individual compounds are listed in A regioselective alkylation was used to prepare the monoalkoxy and monohydroxy 2,2-dimethylchroman-4-one series 2Cl2. The organic phase was discarded, and the alkaline aqueous phase was then acidified with conc. HCl (pH 1–2). The filtered-out solid was dried overnight under reduced pressure. The product was subjected to re-crystallization using an ethanol–water (5% water) extraction. The recorded MPs were 78, 108, 54, 141–143, 106–107, and 74 °C for 5a–f, respectively, while the reported MPs were 70, 113–114, 68, 141, 109, and 78 °C, respectively , 167 (100) [M+-CH=C(CH3)2], 207 (90), 192 (8.9), 152 (7.1), 148 (75), 137 (28), 120 (91), 95 (51), 67 (22), 53 (31).The GC-MS spectral data for compound 1HNMR (CDCl3) spectral data for compound 5a: 1.51 , 2.72 , 3.96 , 5.42 , 6.6 , 7.5 .The 5b : Rt = 22.062 min, MS : m/z (%) = 222 (50) [M+], 167 (100) [M+-CH=C(CH3)2], 207 (99), 192 (10), 138 (8), 123 (71), 111 (9), 95 (16), 79 (9), 53 (51).The GC-MS spectral data for compound 1HNMR (CDCl3) spectral data for compound 5b: 1.45 , 2.67 , 3.93 , 5.30 , 6.41 , 7.36 .The 5c : Rt = 19.920 min, MS : m/z (%) = 222 (46) [M+], 207 (100) [M+-Me], 166 (24), 138 (24), 123 (6), 110 (13), 95 (32), 69 (25), 53 (14).The GC-MS spectral data for compound 1HNMR (CDCl3) spectral data for compound 5c: 1.49 , 2.72 , 3.84 , 5.9 , 6.3 , 12.05 .The 5d : Rt = 21.871 min, MS : m/z (%) = 236 (81) [M+], 152 (100) [M+-C4H10O], 221 (81), 193 (26), 181 (76), 162 (52), 134 (44), 123 (42), 106 (26), 95 (42), 79 (25), 68 (27), 53 (25). The 1HNMR (CDCl3) spectral data for compound 5d: 1.52 (triplet (t), 3H, J = 7.3 Hz, OCH2CH3), 1.57 , 2.77 , 4.25 (quartet (q), 2H, J = 7.3 Hz, OCH2CH3), 5.49 , 6.63 , 7.5 .The GC-MS spectral data for compound 5e : Rt = 23.031 min, MS : m/z (%) = 236 (57) [M+], 221 (100) [M+-Me], 193 (47), 180 (56), 153 (55), 124 (14), 107 (10), 95 (12), 82 (35), 53 (48).The GC-MS spectral data for compound 1HNMR (CDCl3) spectral data for compound 5e: 1.45 , 1.49 , 2.66 , 4.13 , 5.3 , 6.38 , 7.36 .The 5f : Rt = 20.623 min, MS : m/z (%) = 236 (58) [M+], 221 (100) [M+-Me], 193 (49), 181 (50), 153 (45), 124 (49), 96 (19), 69 (46), 55 (17).The GC-MS spectral data for compound 1HNMR (CDCl3) spectral data for compound 5f: 1.4 , 1.5 , 2.68 , 4.02 , 5.93 , 5.99 , 12.01 .The 10a  Rt = 15.119 min, MS : m/z (%) = 206 (31) [M+], 191 (100) [M+-Me], 151 (80), 122 (50), 107 (30), 95 (11), 79 (28), 63 (25), 51 (29).The GC-MS spectral data for compound 1HNMR (CDCl3) spectral data for compound 10a: 1.46 , 2.68 , 3.84 , 6.38 , 6.54 , 7.8 .The 10b : Rt = 15.991 min, MS : m/z (%) = 220 (67) [M+], 205 (100) [M+-Me], 165 (49), 136 (69), 108 (49), 80 (17), 69 (18), 51 (20).The GC-MS spectral data for compound 1HNMR (CDCl3) spectral data for compound 10b: 1.42 , 1.45 , 2.66 , 4.05 , 6.35 , 6.52 , 7.78 .The 6a–f, 0.1 moles of respective 5a–f in 100 mL of N,N-dimethylformamide was treated with 15.4g (0.11 moles) of anhydrous K2CO3, and 19.87 g  of CH3I (99%) was added using a programmable syringe pump , as described in 2Cl2. The organic layer was washed two times with NaOH 2% and water, and dried over anhydrous sodium sulfate. After removing the solvent, the product was re-crystallized from ethanol. The GC-MS spectral data for compound 6a : Rt = 20.227 min, MS : m/z (%) = 236 (85) [M+], 221 (100) [M+-Me], 181 (93), 152 (89), 137 (51), 120 (33), 109 (34), 94 (34), 78 (17), 66 (31), 53 (34), 51 (12).To obtain 1HNMR (CDCl3) spectral data for compound 6a: 1.52 , 2.71 , 3.8 , 3.9 , 6.6 , 7.66 .The 6b : Rt = 17.603 min, MS : m/z (%) = 236 (49) [M+], 221 (100) [M+-Me], 205 (5), 181 (79), 165 (32), 137 (33), 109 (10), 109 (10), 53 (37).The GC-MS spectral data for compound 1HNMR (CDCl3) spectral data for compound 6b: 1.47 , 2.68 , 3.89 , 3.89 , 6.41 , 7.27 .The 6c : Rt = 18.097 min, MS : m/z (%) = 236 (54) [M+], 180 (100) [M+-CH2=C(CH3)2], 221 (25), 152 (59), 137 (62), 109 (20), 95 (18), 79 (16), 53 (25).The GC-MS spectral data for compound 1HNMR (CDCl3) spectral data for compound 6c: 1.44 , 2.64 , 3.82 , 3.88 , 6.03 .The 6d : Rt = 19.256 min, MS : m/z (%) = 250 (2.6) [M+], 219 (100) [M+-OCH3], 234 (12), 191 (77), 176 (12), 147 (6), 69 (17), 53 (5).The GC-MS spectral data for compound 1HNMR (CDCl3) spectral data for compound 6d: 1.44 , 1.49 , 2.68 , 3.84 , 4.1 , 6.56 , 7.61 .The 6e : Rt = 22.209 min, MS : m/z (%) = 250 (2.6) [M+], 235 (100) [M+-Me], 195 (76), 179 (8), 167 (72), 137 (20), 123 (20), 111 (6), 95 (16), 69 (78), 53 (5).The GC-MS spectral data for compound 1HNMR (CDCl3) spectral data for compound 6e: 1.46 , 1.55 , 2.66 , 3.87 , 4.13 , 6.39 .The 6f : Rt = 22.962 min, MS : m/z (%) = 250 (51) [M+], 166 (100) [M+-C5H8O], 150 (24), 138 (27), 123 (24), 69 (36).The GC-MS spectral data for compound 1HNMR (CDCl3) spectral data for compound 6f: 1.39 , 1.44 , 2.64 , 3.87 , 4.03 , 6.01 .The H-1-chromene compounds (7a–f) were prepared following the reduction and dehydration of the corresponding 6a–f compounds. In total, 0.05 moles of compounds 6a–f was dissolved in 100 mL of dry methanol (absolute) and then treated with 5 g (0.13 moles) of NaBH4 dissolved in 50 mL of dry methanol (absolute) using a dropping funnel over one hour under a stream of argon gas condition. Similarly, 7-O-methyl-2,2-dimethylchromene  and 7-O-ethyl-2,2-dimethylchromene (11b) were synthesized by reducing 0.07 moles of compounds 10a–b, respectively, with 5.31 g (0.13 moles) of LiAlH4 in 50 mL of dry tetrahydrofuran (THF). After the reduction process, the reaction was stopped by adding 100 mL of water, and the product was extracted from the mixture with CH2Cl2. The reaction was monitored by TLC with hexane–ethyl acetate in a ratio of 1:1. Subsequently, the solvent was removed, and the residue was subjected to dehydration with 100 mL (4 mol × L−1) of HCl in dry THF at 5 °C using a dropping funnel over 1h. The time required to complete the reduction and dehydration is presented in 2SO4. The product was obtained by column chromatography (9:1 hexane: Et2O).The 27a : Rt = 17.348 min, MS : m/z (%) = 220 (16) [M+], 205 (100) [M+-Me], 190 (14), 161 (14), 144 (7), 91 (7), 51 (6).The GC-MS spectral data for compound 1HNMR (CDCl3) spectral data for compound 7a: 1.47 , 3.84 , 3.87 , 5.5 , 6.26 , 6.41 , 6.68 .The 13CNMR (CDCl3) spectral data for compound 7a: 28.1 (2CH3), 60.9 (OCH3-7), 64.6 (OCH3-8), 76.5 (C-2), 105.5 (C-6), 116.3 (C-4a), 120.8 (C-5), 122.3 (C-4), 128.6 (C-3), 138.1 (C-8), 146.7 (C-8a), 152.98 (C-6).The 7b : Rt = 17.781 min, MS : m/z (%) = 220 (16) [M+], 205 (100) [M+-Me], 189 (13), 161 (15), 91 (10), 77 (10), 69 (11), 51 (6).The GC-MS spectral data for compound 1HNMR (CDCl3) spectral data for compound 7b: 1.41 , 3.84 , 3.85 , 5.46 , 6.24 , 6.43 , 6.254 .The 13CNMR (CDCl3) spectral data for compound 7b: 27.9 (2CH3), 56.1 (OCH3-6/7), 56.7 (OCH3-6/7), 76.8 (C-2), 101.2 (C-8), 109.9 (C-5), 113.2 (C-4a), 122.1 (C-4), 128.4 (C-3), 143.2 (C-8a), 147.3 (C-7), 149.8 (C-6).The 7c : Rt = 18.287 min, MS : m/z (%) = 220 (14) [M+], 205 (100) [M+-Me], 190 (18), 161 (11), 147 (8), 77 (8), 69 (8), 51 (4).The GC-MS spectral data for compound 1HNMR (CDCl3) spectral data for compound 7c: 1.41 , 3.76 , 3.78 , 5.40 , 6.01 , 6.57 .The 13CNMR (CDCl3) spectral data for compound 7c: 27.7 (2CH3), 55.2 (OCH3-5/7), 55.4 (OCH3-5/7), 76.1 (C-2), 91.4 (C-6), 94.1 (C-8), 104.2 (C-4a), 116.7 (C-4), 125.7 (C-3), 154.7 (C-8a), 156.1 (C-7), 161.01 (C-5).The 7d : Rt = 18.041 min, MS : m/z (%) = 236 (25) [M+], 219 (100) [M+-Me], 191 (51), 176 (37), 115 (10), 91 (20), 77 (12), 51 (10).The GC-MS spectral data for compound 1HNMR (CDCl3) spectral data for compound 7d: 1.41 , 1.74 , 3.86 , 4.06 , 5.51 , 6.28 , 6.41 , 6.65 .The 13CNMR (CDCl3) spectral data for compound 7d: 15.4 (OCH2CH3), 28.41 (2CH3), 61.23 (OCH3), 64.94 (OCH2CH3), 76.77 (C-2), 105.85 (C-6), 116.65 (C-4a), 121.14 (C-5), 122.56 (C-4), 128.93 (C-3), 138.93 (C-8), 147.3 (C-8a), 153.29 (C-7).The 7e : Rt = 18.826 min, MS : m/z (%) = 236 (25) [M+], 219 (100) [M+-Me], 191 (91), 176 (24), 91 (22), 77 (20), 69 (23), 51 (12).The GC-MS spectral data for compound 1HNMR (CDCl3) spectral data for compound 7e: 1.42 , 1.48 , 3.8 , 4.06 , 5.45 , 6.24 , 6.42 , 6.54 .The 13CNMR (CDCl3) spectral data for compound 7e: 14.9 (OCH2CH3), 27.8 (2CH3), 56.8 (OCH3), 64.4 (OCH2CH3), 76.8 (C-2), 102.3 (C-8), 110.3 (C-4a), 113.3 (C-5), 122.2 (C-4), 128.4 (C-3), 143.1 (C-8a), 147.4 (C-7), 149.5 (C-6).The 7f : Rt = 16.081 min, MS : m/z (%) = 236 (2) [M+], 219 (100) [M+-Me], 234 (20), 191 (66), 176 (24), 147 (19), 91 (17), 69 (22), 51 (10).The GC-MS spectral data for compound 1HNMR (CDCl3) spectral data for compound 7f: 1.38 , 1.41 , 3.79 , 4.0 , 5.41 , 6.02 , 6.58 .The 13CNMR (CDCl3) spectral data for compound 7f: 14.8 (OCH2CH3), 28.1 (2CH3), 55.4 (OCH3), 63.0 (OCH2CH3), 76.59 (C-2), 92.11 (C-6), 94.82 (C-8), 104.53 (C-4a), 116.79 (C-4), 126.17 (C-3), 155.01 (C-8a), 158.65 (C-7), 160.76 (C-5).The 11a : Rt = 12.582 min, MS : m/z (%) = 190 (10) [M+], 175 (100) [M+-Me], 160 (10), 132 (14), 115 (4), 103 (4), 91 (4), 77 (8), 63 (6), 51 (8).The GC-MS spectral data for compound 1HNMR (CDCl3) spectral data for compound 11a: 1.41 , 3.75 , 5.45 , 6.26 , 6.36 , 6.4 , 6.86 .The 13CNMR (CDCl3) spectral data for compound 11a: 28.02 (2CH3), 55.1 (OCH3), 76.7 (C-2), 102.09 (C-8), 106.8 (C-4a), 115.03 (C-5), 122.02 (C-4), 127.3 (C-3), 128.5 (C-6), 154.5 (C-8a), 161.06 (C-7).The 11b : Rt = 13.432 min, MS : m/z (%) = 204 (20) [M+], 161 (100) [M+-C2H4O], 189 (92), 132 (11), 115 (9), 105 (7), 91 (7), 77 (21), 63 (7), 51 (8).The GC-MS spectral data for compound 1HNMR (CDCl3) spectral data for compound 11b: 1.37 , 1.42 , 4.01 , 5.45 , 6.26 , 6.36 , 6.4 , 6.86 .The 13CNMR (CDCl3) spectral data for compound 11b: 15.2 (OCH2CH3), 28.4 (2CH3), 63.8 (OCH2CH3), 76.7 (C-2), 102.9 (C-8), 107.6 (C-4a), 114.9 (C-5), 122.3 (C-4), 127.3 (C-3), 128.1 (C-6), 154.5 (C-8a), 160.4 (C-7).The R. solani and A. niger, using the poisoned food technique [A. niger, while PDA media were used as growth media for R. solani. The fungitoxic activity of 29 compounds (chromanones and chromenes) was tested against A. niger and R. solani in vitro at various concentrations, ranging from 100 to 800 μg × mL−1, while amphotericin-B  was used at 5–25 μg × mL−1. An in vitro assay was performed on PDA and CDA growth media treated with gradient concentrations of all the synthesized compounds (100–800 µg × mL−1 in sterilized DMSO (5%) + Tween-20 as a dispersing agent). Then, 1 mL of a solution containing different compounds was poured into sterilized melted media, homogenized, and plated into Petri dishes (90 × 15 mm). The media-containing compounds were incubated for 48 h at 25 °C. After incubation, all plates were inoculated with agar plugs containing fungi and incubated again at 22 °C for 8 days. To assess mycelial inhibition, fungal growth diameters (mm) were measured daily, and radial inhibition was calculated when negative control plates were fully covered with the fungal mycelial. For all treatments, four replicates were used, and the percentage of mycelial growth inhibition was calculated according to the equation suggested by Pandey et al. [The antifungal activity of all synthesized compounds and the standard antifungal drug amphotericin-B was evaluated in vitro against two phytopathogenic fungi, echnique . Pure fuy et al. .% myceldc = the diameter of the fungal colony in the negative control;dt = the diameter of the fungal colony in treatment.−1. The EC50 and EC90 for all treatments were calculated using a regression equation between the log concentrations and the probit of the percentage growth inhibition of fungi, according to Abd El-Naeem et al. [The positive control (inoculated media with fungus and DMSO + Tween-20) was used to evaluate the toxicity of the solvent and the dispersing agent. In addition, the synthetic antibiotic drug, amphotericin-B , was also used as a standard antifungal drug at concentrations of 5–25 µg × mLm et al. .A. niger- and R. solani-encoded PGUs with different synthetic chromenes and chromanones, two available structures of A. niger-encoded PGUs (PDB ID 1CZF and 1NHC) were retrieved from the Protein Data Bank  (accessed on 8 June 2022). Two additional PGU sequences (accession numbers KP896518 and KP896519) were retrieved from the NCBI Data Bank (https://pubmed.ncbi.nlm.nih.gov/) (downloaded on 7 June 2022) and their 3D structures were inferred using iTASSER (https://zhanggroup.org/I-TASSER/) (accessed on 10–15 June 2022) and Alpha fold2  (accessed on 10–16 June 2022) tools. Fusarium solani-encoded VDAC mRNA sequences (accession # XM0462760) were retrieved from the NCBI Data Bank (https://pubmed.ncbi.nlm.nih.gov/) (accessed on 16 June 2022) and its 3D structure was inferred using the iTASSER (https://zhanggroup.org/I-TASSER/) (accessed on 16 June 2022) webserver. Additionally, a mouse-encoded VDAC (PDB id 3EMN) was retrieved to compare and validate the interactions.To determine the interaction mode of To prepare protein input files, all water molecules, ligands, and ions were removed, and polar hydrogens were added from the PDB file using AutoDock Vina (version 1.10) . Finallyhttps://pubchem.ncbi.nlm.nih.gov/compound) (accessed on 16 June 2022) or drawn using ChemBiochem Drew Ultra (version 12). All the ligands were used in the structure data file (sdf) format.Three-dimensional structures of different chromenes and chromanones were either downloaded from the Webchem webpage . The selective O-alkylation on the benzene ring produced stepwise mono and di-O-alkoxy derivatives of chromanones 5a–f, 6a–f, and 10a–b. The following reduction step by NaBH4 and in situ dehydration produced the corresponding 2H-1-chromene compounds 7a–f and 11a–b. The poison feed protocol was used to evaluate the antifungal activity of two categories of compounds (i) chroman-4-one with mono and di-O-alkoxy analogues and (ii) 2H-1-chromenes against A. niger and R. solani. The tested compounds showed different antifungal attribution to changes in the type and position of the substituent in both categories of compounds. These results were supported by molecular docking. The synthesized chromene and chromanone compounds are eco-friendly compounds because they are not organo-metalic, organophosphoures, or halogenated compounds which could be harmful to the environment. More studies are required to evaluate the effect of chromenes and chromanones in both field experiments with economically important plant pathogenic fungi on the molecular level.In conclusion, the chemical synthesis of naturally occurring precocenes I, II, and III in addition to their analogous, was performed by the reaction of corresponding di and trihydroxybenzenes with α,β-unsaturated carboxylic acid under mild conditions from POCl


N-Heterocyclic carbenes (NHCs) have seen more and more use over the years. The go-to systems that are usually considered are derivatives of benzimidazole or imidazole. Caffeine possesses an imidazole unit and was already utilized as a carbene-type ligand; however, its use within a tridentate bis-NHC system has—to the best of our knowledge—not been reported so far. The synthesis of the ligand is straightforward and metal complexes are readily available via silver-salt metathesis. A platinum(II) and a palladium(II) complex were isolated and a crystal structure of the former was examined. For the Pt(II) complex, luminescence is observed in solid state as well as in solution. N-heterocyclic carbenes (NHC) has gained significant attention in recent years + (M + Na+) requires 486.1609.Synthesis of 7,7′-)bisbis(tetrafluoborate) (4). Compound 3  was suspended in anhydrous CH2Cl2 (40 mL); Me3OBF4  and K2CO3 (100 mg) were added to the reaction mixture and stirred overnight. The solvent was removed under reduced pressure, subsequently MeOH (50 mL) was added and removed under reduced pressure. This sequence was repeated three times. The resulting solid was dissolved in a minimal amount of CH3CN, filtered and then precipitated by the addition of diethyl ether. The colourless solid was dried to yield the product . 1H-NMR : δ = 8.76 , 7.89 , 7.52 , 5.70 , 4.15 , 3.74 , 3.25  ppm. 13C-NMR : δ = 154.6 (1-C), 153.4 (4/5-C), 151.5 (5/4-C), 140.9 (d-C), 140.3 (a-C), 140.2 (3-C), 124.4 (b-C), 108.7 (2-C), 53.4 (c-C), 38.3 (9′-C), 32.3 (3′-C), 29.2 (1′-C) ppm. HRMS : 246.6088, [C23H27N9O4]2+ (M2+) requires 246.6088.Synthesis of the Ag(I) complex 7,7′-)-bis silver tetrafluoroborate (5). Protoligand 4  was dissolved in a mixture of CH3CN (50 mL) and CH2Cl2 (20 mL) as well as Ag2O  were added. The mixture was stirred at 50 °C for 2 h. The reaction progress was monitored by 1H-NMR spectroscopy and heating was stopped when the signal of the imidazolium proton had disappeared. The reaction mixture was filtered to remove the suspended solid and then precipitated into diethyl ether to yield the product as a grey solid . 1H-NMR : δ = 7.73 , 7.10 , 5.50 , 4.25 , 3.76 , 3.24  ppm. 13C-NMR : δ = 189.7 (d-C), 156.4 (1-C), 154.9 (4/5-C), 151.9 (5/4-C), 142.0 (a-C), 140.3 (3-C), 122.9 (b-C), 110.2 (2-C), 54.7 (c-C), 40.5 (9′-C), 32.4 (3′-C), 29.0 (1′-C) ppm. HRMS : 598.1055, [C23H25AgN9O4]+ (M+) requires 598.1075.Synthesis of the Pd(II) complex 7,7′-)-bis palladiumchlorido hexafluorophosphate (6). PdCl2  was dissolved in anhydrous CH3CN (20 mL) at 60 °C and after 1 h the silver salt 5  was added. The resulting yellow-orange suspension was filtered after 18 h and the solvent was removed under reduced pressure. The residue was purified by column chromatography  and the second yellow fraction was collected. NH4PF6 (1.0 g) in H2O (10 mL) was added and the solution was extracted with CH2Cl2 (50 mL). The solvent was removed under reduced pressure to yield the product as a yellow solid . 1H-NMR : δ = 8.07 , 7.73 , 6.43 , 5.70 , 4.28 , 3.73 , 3.32  ppm. 13C-NMR : δ = 174.2 (d-C), 155.6 (1-C), 154.8 (4/5-C), 151.9 (5/4-C), 143.1 (a-C), 141.9 (3-C), 127.1 (b-C), 109.4 (2-C), 53.7 (c-C), 39.4 (9′-C), 32.8 (3′-C), 28.9 (1′-C) ppm. HRMS : 632.0730, [C23H25ClN9O4Pd]+ (M+) requires 632.0730.Synthesis of Pt(II) complex 7,7′-)-bis platninumchlorido hexafluorophosphate (7). The silver salt 5  and K2PtCl4  were dissolved in anhydrous CH3CN (20 mL) and the reaction mixture was stirred overnight at 60 °C. The resulting light-yellow suspension was filtered and the solvent was removed under reduced pressure. The residue was purified by column chromatography  and the second yellow fraction was collected. NH4PF6 (1.0 g) in H2O (10 mL) was added and the solution extracted with CH2Cl2 (50 mL). The solvent was removed under reduced pressure to yield the product as a slightly yellow solid . 1H-NMR : δ = 8.08 , 7.75 , 6.57 , 5.41 , 4.24 , 3.74 , 3.32  ppm. 13C-NMR : δ = 171.2 (d-C), 155.8 (1-C), 155.1 (4/5-C), 152.2 (5/4-C), 142.9 a-C), 142.6 3-C), 127.7 (b-C), 109.4 (2-C), 53.9 (c-C), 33.1 (9′-C), 30.1 (3′-C), 29.2 (1′-C) ppm. HRMS : 721.1334, [C23H25ClN9O4Pt]+ (M+) requires 721.1360.


Zingiber officinale rhizomes. Many scientific studies have reported the diverse biological activities of 6-shogaol. However, the major drawback of 6-shogaol is its instability at room temperature. We synthesised new shogaol thiophene compounds (STCs) by replacing the pentyl group in the sidechain with thiophene derivatives. The STCs were tested for their nuclear factor erythroid 2-related factor 2 (NRF2) activation ability in murine hepatoma cells (Hepa1c1c-7) by determining their NAD(P)H quinone oxidoreductase 1 (NQO1) inducing ability and expression of NRF2-associated antioxidant genes. The anti-inflammatory activity of STCs was determined in Escherichia coli lipopolysaccharide (LPSEc)-stimulated NR2-proficient and -silenced mouse microglial cells (BV-2) by measuring the inflammatory markers, cytokines, and mediators. The modes of action  of NRF2 activation by STCs were determined using commercially available kits. The in vitro metabolic stability of the STCs in liver microsomes  was also investigated. The molecular docking and molecular dynamics studies were conducted to identify the binding poses, stability, and molecular interactions of the STCs in the binding pockets of Kelch and BTB domains of KEAP1 and GSK-3β enzyme. The new STCs were synthesised in good yields of > 85%, with a purity of about 95%, using a novel synthesis method by employing a reusable proline–proline dipeptide catalyst. The STCs are more potent than 6-shogaol in activating NRF2 and reducing inflammation. The nature of substituents on thiophene has a profound influence on the bioactivity of the STCs. Phenylthiophene STC (STC5) is the most potent, while thiophenes containing electron-withdrawing groups showed weaker bioactivity. The bioactivity of 6-shogaol is in the micromolar range, whereas STC5 showed bioactivity in the sub micromolar range. The STCs showed anti-inflammatory effects via NRF2-dependent and NRF2-independent mechanisms. The STCs improved NRF2 activity through multiple (KEAP1-independent and -dependent) mechanisms. The STCs showed decreased reactivity with thiols than 6-shogaol and thus may possess fewer side-effects than 6-shogaol. The STCs were more metabolically stable than 6-shogaol. 6-shogaol is a natural and the most potent bioactive vanilloid in dried  Zingiber officinale (commonly known as ginger). Our group has reported that 6-shogaol is the most potent antioxidant, anti-inflammatory, and anticancer agent among natural vanilloids bi-indolylidene-3,2′-dione-3-oxime (PDB ID: 1Q41) with a resolution of 2.10 A° [+ and Cl− ions) were added to neutralise the charges. The MD was performed in the NPT ensemble (at 300 K and 1.63 bar) for 100 ns. The OPLS-3e force field was used. The root means square deviation (RMSD) of ligand and protein over the 100 ns MD trajectory were recorded.The binding poses of STCs and their molecular interactions with amino acid residues in the binding sites of KEAP1 Kelch domain and GSK-3β were determined using Fast Rigid Exhaustive Docking (FRED) 4.2.0.1  ,48. The  1.97 A°  and (2)  2.10 A°  were use 2.10 A°  with def 2.10 A°  applicat 2.10 A°  was used 2.10 A° . In addi 2.10 A°  and primp < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****)) was calculated using Brown–Forsythe and Welch ANOVA tests, followed by the DunnetteT3 test. The statistical analysis of the results was carried out using GraphPad Prism 9.5.0, and results were presented as mean ± SD. The statistical significance : δ 2.81, 3.71, 5.14, 6.44, 6.59, 6.73 , 7.18 , 7.37, 7.43 13CNMR : δ 29.96(5-CH2), 42.66(4-CH2), 55.90(8″-OCH3), 111.34(2″-CH), 114.54(5″-CH), 120.84(6″-CH), 125.29(4′-HC), 126.10(3′-CH), 127.80(1′-CH), 128.86(2′-C), 133.14(2-CH), 136.29(1″-C), 138.29(C-1), 144.60(4″-C), 146.89(3″-C), 199.98(3-CO).16HO3SMolecular Formula: CElemental Analysis shown: C, 66.64; H, 5.59; O, 16.65; S, 11.12Elemental Analysis found: C, 66.69; H, 5.55; O, 16.61; S, 11.13Percentage yield: 87%1H NMR : δ 2.75 , 3.63 , 5.18, 6.38, 6.53, 6.71, 6.85, 7.23, 7.5113CNMR : δ 29.42(5-CH2), 42.89(4-CH2), 55.90(8″-OCH3), 111.42(2″-CH), 114.58(5″-CH), 120.77(6″-CH), 124.68(3′-CH), 128.61(2′-CH), 129.04(4′-CH), 131.81(1-CH), 132.99(2-CH), 135.28(1″-C), 139.81(1-C’), 144.25(4″-C), 146.87(3″-C), 199.27(3-CO)16H16O3SChemical formula: CElemental analysis shown: C, 66.64; H, 5.59; O, 16.65; S, 11.12Elemental analysis found: C, 66.69; H, 5.55; O, 16.61; S, 11.13Percentage yield: 89%1H NMR : δ 2.27, 2.88, 3.84, 5.93, 6.41, 6.71, 6.68, 6.82, 7.22, 7.7213CNMR : δ 15.52(5′-CH), 30.06(5-CH2), 43.31(4-CH2), 55.90(8″-OCH3), 111.33(2″-CH), 114.55(5″-CH), 120.55(6″-CH), 123.77(1′-C), 127.45(1-CH), 131.42(1″-C), 133.11(2-CH), 133.84(4′-CH), 133.90(3′-CH), 142.42(2′-C), 144.05(4″-C), 146.62(3″-C), 199.18(3-CO)17H18O3SChemical formula: CElemental analysis shown: C, 67.52; H, 6.00; O, 15.87; S, 10.60Elemental analysis found: C, 67.55; H, 6.04; O, 15.84; S, 10.58Percentage yield: 86%1H NMR : δ 2.88, 3.75, 5.62, 6.42, 6.61, 6.63, 6.73, 7.04, 7.16, 7.4213CNMR : δ 29.49 (6″-CH), 43.18(5″-CH), 55.90(8″-OCH3), 109.10(4-CH2), 111.16(2″-CH), 114.48(5″-CH), 120.51(6″-CH), 125.70(2′-CH), 132.91(5-CH2), 133.00(1″-C), 133.58(2″-CH), 140.59(2-CH), 143.96(4″-C), 144.01(3″-C), 146.52(1″-CH), 198.74 (3-CO)16H15BrO3SChemical formula: CElemental analysis shown: C, 52.33; H, 4.12; Br, 21.76; O, 13.07; S, 8.73 Elemental analysis found: C, 52.36; H, 4.15; Br, 21.72; O, 13.07; S, 8.76Percentage yield: 86%1H NMR : δ 2.85, 3.78, 5.49, 6.47, 6.65, 6.76, 7.24, 7.33, 7.40, 7.46, 7.48, 7.61.13CNMR : δ 29.94 (5-CH2), 43.14(4-CH2), 55.91(8″-OCH3), 111.14(2″-CH), 114.38(5″-CH), 120.85(4′-CH), 123.49(6″-CH), 125.05, 126.27(8′-CH), 127.72, 128.97(2′-CH), 130.41(1-CH), 133.09(2-CJ), 134.88(1″-C), 135.04(5′-C), 140.40(1′-C), 143.46(3′-C), 143.96(4″-C), 146.45(3″-C), 198.90(3-CO)22H20O3SChemical formula: CElemental analysis shown: C, 72.50; H, 5.53; O, 13.17; S, 8.80 Elemental analysis found: C, 72.47; H, 5.55; O, 13.14; S, 8.83Percentage yield: 87%1H NMR : δ 2.75, 3.76, 6.32, 6.49, 6.61, 6.65, 6.74, 7.12.13CNMR : δ 29.88 (5-CH2), 43.59(4-CH2), 55.90(8″-OCH3), 109.10(2″-CH), 111.77(5″-CH), 114.44(4′-C), 117.87(6″-CH), 120.74(3′-CH), 120.81(2′-CH), 123.37(1-CH), 127.51(2-CH), 132.98(1″-C), 143.94(1′-C), 146.49(4″-C), 152.92(3″-C), 198.93(3-CO)16H15BrO3SChemical formula: CElemental analysis shown: C, 52.33; H, 4.12; Br, 21.76; O, 13.07; S, 8.73Elemental analysis found: C, 52.36; H, 4.15; Br, 21.72; O, 13.07; S, 8.76Percentage yield: 88%1H NMR : δ 1.31, 2.84, 2.89, 3.85, 5.57, 6.41, 6.71, 6.82, 7.07, 7.5813CNMR : δ 15.69 (5-CH2), 24.01(4-CH2), 30.24(8″-OCH3), 43.01(5′-CH), 55.96(6′-CH), 111.20(2″-CH), 114.42(5″-CH), 120.89(4′-C), 123.65(6″-CH), 125.02(3′-CH), 132.50(2′-CH), 133.28(1-CH), 135.82(2-CH), 137.46(1″-C), 143.97(1′-C), 146.51(4″-C), 152.34(3″-C), 199.17(3-CO)18H20O3SChemical formula: CElemental analysis shown: C, 68.33; H, 6.37; O, 15.17; S, 10.13 Elemental analysis found: C, 68.36; H, 6.34; O, 15.15; S, 10.17Percentage yield: 88%1H NMR : δ 2.78, 3.81, 5.77, 6.61, 6.65, 6.72, 7.11, 7.42, 7.7513CNMR : δ 29.66(5-CH2), 47.44(4-CH2), 55.97(8″-OCH3), 111.20(2″-CH), 114.50(5″-CH), 120.87(6″-CH), 128.62(3′-CH), 129.23(1-CH), 129.63(2-CH), 132.69(1″-C), 132.96(2′-CH), 144.08(1′-C), 145.99(4″-C), 146.58(3″-C), 152.21(4′-C), 198.29(3-CO)16H15NO5SChemical formula: CElemental analysis shown: C, 57.65; H, 4.54; N, 4.20; O, 24.00; S, 9.62 Elemental analysis found: C, 57.59; H, 4.56; N, 4.17; O, 24.05; S, 9.66Percentage yield: 89%The cytotoxic effect of STCs on Hepa-1c1c7 cells is shown in the Ec (200 ng/mL)-challenged BV-2 cells; thus, a safe dose of the STCs (in the presence of LPSEc) on BV-2 cells was determined via their cytotoxicity on BV-2 cells. The results are shown in the Ec showed less than 90% cell viability at concentrations ≥ 5 μM. The combination of STC-8 plus LPSEc showed less than 90% cell viability at concentrations ≥ 2.5 μM. All other STCs (in the presence of LPSEc) showed less than 90% cell viability at 10 μM. The anti-inflammatory activity of STCs was investigated using LPSThe qPCR studies were carried out to determine the effect of 6-Shogoal and STCs (at 1 μM) on the expression of the NRF2 gene and its downstream representative eleven antioxidant genes . The gene expression results are shown in Ec-stimulated NRF2-Proficient and -silenced BV-2 cells (The anti-inflammatory activity of STCs (at 1 μM concentration) was assessed by measuring nitric oxide (NO), proinflammatory cytokines , and inflammatory mediators  production in LPS-2 cells . The abi-2 cells . Ec (200 ng/mL) produced NO in NRF2-proficient BV-2 cells was 10.53 ± 0.22 μM, and in NRF-2 silenced cells, it produced 11.37 ± 0.15 μM NO. All the STCs and 6-shogaol inhibited the NO production. STCs containing 2-thiophene analogues (STC2-7) are more active than 6-shogaol (6.32 ± 0.03 μM in NRF2-proficient BV-2 cells and 8.56 ± 0.36 μM in NRF2-silenced BV-2 cells). STC1 (containing 3-thiophene) and STC8 (containing 5-nitro-2-thiophene) are as active as 6-shogaol. The STC5 is the most active, and NO production was 2.58 ± 0.04 μM in the NRF2-proficient BV-2 cells and 3.0 ± 0.28 μM in NRF2-silenced BV-2 cells. Among the 2-thiophene analogues, STC8 is the least active; NO production was 6.38 ± 0.04 μM in the NRF2-proficient BV-2 cells and 8.86 ± 0.38 μM in NRF2-silenced BV-2 cells. The results (The effect of 6-shogaol and STCs on NO production is shown in  results B have shEc (200 ng/mL) produced IL-1β in NRF2-proficient BV-2 cells 93.53 ± 1.10 pg/mL, and in NRF-2 silenced BV-2 cells, it produced 93.23 ± 0.90 pg/mL. All the STCs and 6-shogaol reversed the elevated levels of IL-1β. All the STCs (except STC1 containing 3-thiophene) are more active than 6-shogaol (43.30 ± 0.62 pg/mL in NRF2-proficient BV-2 cells and 88.57 ± 0.55 pg/mL in NRF2-silenced BV-2 cells). The STC5 is the most active, and IL-6 levels were 26.77 ± 0.85 pg/mL in the NRF2-proficient BV-2 cells and 72.50 ± 1.32 pg/mL in NRF2-silenced BV-2 cells. Among the 2-thiophene analogues, STC8 is the least active; IL-1β levels were 39.93 ± 0.74 pg/mL in the NRF2-proficient BV-2 and 77.33 ± 0.93 pg/mL in NRF2-silenced BV-2 cells. The results (The effect of 6-shogaol and STCs on IL-1β production is shown in  results D have shEc (200 ng/mL) produced IL-6 in NRF2-proficient BV-2 cells, and the amount was 926.33 ± 4.73 pg/mL. In NRF-2 silenced BV-2 cells, it produced 909.67 ± 2.52 pg/mL. All the STCs and 6-shogaol reversed the elevated levels of IL-6. The compounds STC1 and STC8 are equally active as 6-shogaol. All the other STCs (STC 2-7) are more active than 6-shogaol (435.67 ± 7.77 pg/mL in NRF2-proficient BV-2 cells and 852.67 ± 5.69 pg/mL in NRF2-silenced BV-2 cells). The STC5 is the most active, and IL-6 levels were 212.33 ± 6.11 pg/mL in the NRF2-proficient BV-2 cells and 748.67 ± 8.33 pg/mL in NRF2-silenced BV-2 cells. Among the 2-thiophene analogues, STC8 is the least active; IL-6 levels were 431.667 ± 3.06 pg/mL in the NRF2-proficient BV-2 cells and 846.67 ± 11.68 pg/mL in NRF2-silenced BV-2 cells. The results (The effect of 6-shogaol and STCs on IL-6 production is shown in  results F have shEc (200 ng/mL) produced IFN-γ in NRF2-proficient BV-2 cells, and the amount was 40.44 ± 1.69 pg/mL. In NRF-2 silenced BV-2 cells, it produced 41.91 ±1.10 pg/mL. All the STCs and 6-shogaol reversed the elevated levels of IFN-γ. The compounds STC1 and STC8 are equally active as 6-shogaol. All the other STCs (STC 2-7) are more active than 6-shogaol (25.75 ± 0.72 pg/mL in NRF2-proficient BV-2 cells and 37.52 ± 1.01 pg/mL in NRF2-silenced BV-2 cells). The STC5 is the most active, and IFN-γ levels were 14.34 ± 1.10 pg/mL in the NRF-2 proficient BV-2 cells and 31.19 ± 0.85 pg/mL in NRF2-silenced BV-2 cells. Among the 2-thiophene analogues, STC8 is the least active; IFN-γ levels were 24.63 ± 0.64 pg/mL in the NRF2-proficient BV-2 cells and 37.86 ± 1.69 pg/mL in NRF2-silenced BV-2 cells. The results (The effect of 6-shogaol and STCs on IFN-γ production is shown in  results H have shEc (200 ng/mL) produced TNF-α in NRF2-proficient BV-2 cells was 2907.00 ± 51.00 pg/mL, and in NRF-2 silenced BV-2 cells, it produced 3111.00 ± 51.00 pg/mL. All the STCs and 6-shogaol reversed the elevated levels of TNF-α. The compounds (STC1 and STC8) are equally active as 6-shogaol. All the other STCs (STC 2-7) are more active than 6-shogaol (1734.00 ± 51.00 pg/mL in NRF2-proficient BV-2 cells and 3113.00 ± 55.34 pg/mL in NRF2-silenced BV-2 cells). The STC5 is the most active, and TNF-α levels were 850.00 ± 77.90 pg/mL in the NRF2-proficient BV-2 cells and 2581.00 ± 51.00 pg/mL in NRF2-silenced BV-2 cells. Among the 2-thiophene analogues, STC8 is the least active; TNF-α levels were 1751.00 ± 77.90 pg/mL in the NRF2-proficient BV-2 cells and 3130.33 ± 27.47 pg/mL in NRF2-silenced BV-2 cells. The results (The effect of 6-shogaol and STCs on TNF-α production is shown in  results J have shEc (200 ng/mL) produced COX-2 in NRF2-proficient BV-2 cells, and the amount was 6.67 ± 0.15 ng/mL. In NRF-2 silenced BV-2 cells, it produced 6.83 ± 0.15 ng/mL. All the STCs and 6-shogaol reversed the elevated levels of COX-2. The compounds STC1 and STC8 are equally active as 6-shogaol. All the other STCs (STC 2-7) are more active than 6-shogaol (4.30 ± 0.10 ng/mL in NRF2-proficient BV-2 cells and 6.03 ± 0.21 ng/mL in NRF2-silenced BV-2 cells). The STC5 is the most active, and COX-2 levels were 27.00 ± 0.10 ng/mL in the NRF2-proficient BV-2 cells and 3.48 ± 0.09 ng/mL in NRF2-silenced BV-2 cells. Among the 2-thiophene analogues, STC8 is the least active; COX-2 levels were 4.10 ± 0.10 ng/mL in the NRF2-proficient BV-2 cells and 4.47 ± 0.15 ng/mL in NRF2-silenced BV-2 cells. The results (The effect of 6-shogaol and STCs on COX-2 production is shown in  results L have shEc (200 ng/mL) produced NF-κB p65 in NRF2-proficient BV-2 cells, and the amount was 125.96 ± 5.04 μg/mL; in NRF-2 silenced BV-2 cells, it produced 138.56 ± 2.52 μg/mL. All the STCs and 6-shogaol reversed the elevated levels of NF-κB p65. The compounds STC1 and STC8 are equally active as 6-shogaol. All the other STCs (STC 2-7) are more active than 6-shogaol (70.54 ± 5.04 ng/mL in NRF2-proficient BV-2 cells and 132.17 ± 4.52 ng/mL in NRF2-silenced BV-2 cells). The STC5 is the most active, and NF-κB p65 levels were 41.48 ± 0.79 μg/mL in the NRF2-proficient BV-2 cells and 86.28 ± 1.03 μg/mL in NRF2-silenced BV-2 cells. Among the 2-thiophene analogues, STC8 is the least active; NF-κB p65 levels were 75.58 ± 5.04 μg/mL in the NRF2-proficient BV-2 cells and 131.57 ± 0.15 ng/mL in NRF2-silenced BV-2 cells. The results (The effect of 6-shogaol and STCs on NF-κB p65 production is shown in  results N have shThree different mechanisms mainly activate NRF2 by (1) interacting with the Kelch domain of KEAP1, (2) covalent bonding with cysteine sensors (in BTB domain) of KEAP1, and (3) inhibiting GSK-3β activity. The effect of STCs on the different modes of NRF2 activation is shown in 50 values  and STC8  are almost equipotent to 6-shogaol. The most potent compound is STC5, with an IC50 value of 0.04 ± 0.001 μM. The potency of STCs in decreasing order is as follows: STC5 > STC4~STC6 > STC7 > STC3 > STC2 > STC1 > STC8. The dose-dependent effect of 6-shogaol and STCs on NRF2-KEAP1 inhibition is shown in 50 values  is equipotent to 6-shogaol. The compound STC8  is less potent than 6-shogaol. All other STCs (STC2-7) are more potent than 6-shogaol. The most potent compound is STC5, with an IC50 value of 0.05 ± 0.001 μM. The potency of STCs in decreasing order is as follows:The dose-dependent effect of 6-shogaol and STCs on GSK-3β enzyme activity is shown in 0 values B are calSTC5 > STC6 > STC4 > STC7 > STC3 > STC2 > STC1 > STC8. The 6-shogaol and STCs’ ability to interact with thiols in cysteine residues was indirectly estimated by determining the free thiol content. The free thiol content (%) in NRF2-proficient BV-2 cells upon treatment with test compounds is shown in Overall, the results suggest that STCs have the potential to activate NRF2 through (1) interacting with the Kelch domain of KEAP1, (2) interacting with cysteine residues of KEAP1, and (3) inhibiting GSK-3β enzyme activity. The metabolic stability of STCs was compared with 6-shogaol in liver microsomes . The results are shown in 0) of the test compounds in the binding pockets of 4IQK and 1Q41 are shown in The results of in silico studies are shown in Oxidative stress is the leading cause of many chronic diseases. The NRF2 activation is the primary antioxidant defence mechanism to protect from oxidative stress. Thus, NRF2 has emerged as a promising therapeutic target for developing drugs against chronic diseases. Researchers from academia and industry have been extensively investigating to discover new interventions for NRF2 activation. Many NRF2 activating compounds  are reported in the literature. Of all these reported compounds, products of natural origin  have shown potent NRF2 activation . Althoug1H and 13C) and elemental analysis. The polar surface area  is the same as that of 6-shogaol, which is 49.6 A2. The polar surface area is a vital property of the small molecules that determines oral absorption and brain penetration. For passive transportation (by transcellular route) of the drugs, the polar surface area should be < 120 A2 for oral absorption and should be < 60A2 for brain penetration [Eight STCs were synthesised (with yields > 85%) using the reusable catalyst proline–proline dipeptide and characterised using NMR  cells by determining the levels of inflammation maker , proinflammatory cytokines , and inflammatory mediators (COX-2 and NF-κB p65). Nitric oxide production was determined using the Griess assay, and proinflammatory cytokines and inflammatory mediators were determined using ELISA assays. To assess the role of NRF2 activation, the anti-inflammatory activities of the compounds were also assessed in NRF2-silenced BV-2 cells. The modes of action of STCs in NRF2 activation were assessed by measuring their ability to interact with the Kelch domain and cysteine residues of KEAP1 and GSK-3β enzyme activity. The interaction with the Kelch domain and inhibition of GSK-3β enzyme activity was assessed using commercially available kits. The ability of STCs to interact with the cysteine residues was assessed indirectly by determining the free thiol content in NRF2-proficient BV-2 cells upon treatment with STCs. The microsomal stability of the test compounds was assessed in humans, rats, and mice liver microsomes. The binding poses, binding energies, and the key interactions with amino acid residues in the binding pockets of KEAP1’s Kelch domain (4IQK), BTB domain (1Q41), and GSK-3β enzyme were predicted using relevant tools in OpenEye Scientific Software and Schrödinger Drug Discovery Suite. In addition, the stability of the most potent compound (STC5) in the binding pockets of proteins was predicted via molecular dynamics simulation using the Desmond package. In all the experiments, the activities of STCs were compared with those of 6-shogaol. The NRF2 activation of the STCs was elucidated by determining the NQO1 induction (Prochaska microtiter plate assay) and expression of representative antioxidant genes  in mouse hepatoma (Hepa-1c1c7) cells. Then, the anti-inflammatory activity of the STCs was investigated in LPSIn this study, STC1 (containing 3-thiophene) and STC2 (containing 2-thiophene) were initially synthesised and preliminarily evaluated for their NQO1-inducing ability to understand which thiophene gives better activity than 6-shogaol. The results have shown that STC2 is more active than STC1. Thus, six 2-thiophene analogues (STC3-8) containing representative electron-donating , electron-withdrawing , and aromatic (phenyl) substituents on 2-thiophene moiety were observed. All the compounds except STC8 (containing nitro substituent) were less cytotoxic to both cell types (Hepa-1c1c7 and BV-2). In the Prochaska microtiter plate assay, STC8 showed toxicity at > 1 μM concentration and did not double the NQO1 induction at concentrations < 1 μM. All other STCs (STC1-7) showed significant NQO1 induction, and STC5 (containing phenyl ring) is the most active compound. The bromo thiophenes (STC4 and STC6) are the second most active compounds, and the bromo substituent’s position does not significantly influence the activity. Among the alkyl-substituted thiophenes, ethylthiophene (STC7) is more active than methylthiophene (STC3). All the cell-based biological assays (gene expression and anti-inflammatory studies) were carried out with 1μM effective concentration of STCs. In general, in all the assays, the order of activity follows the NQO1 inducing activity order (STC5 > (STC4~STC6) > STC7 > STC3 > STC 2 > (STC1~6-shogaol) > STC8). The STCs’ order of activity is associated with the order of their XlogP values. STC5 has the highest XlogP of 5.0, and STC8 has the lowest XlogP of 1.8  in the cytoplasm and orchestrates NRF2 ubiquitination. NRF2 activators prevent its ubiquitination and translocate it into the nucleus. Then, NRF2 binds with the antioxidant-response element (ARE) in the nucleus to promote the expression of many antioxidant and detoxification genes, such as HMOX1, NQO1, GCLC, GCLM, TXNRD1, PRDX1, GPX2, GSTP1, GSTM2, GSTA1, and G6PDX ,62. TherWe also show that STCs have the potential to activate NRF2 through three independent mechanisms, i.e., by interacting with the Kelch domain of KEAP1, by interacting with cysteine residues of KEAP1, and by inhibiting GSK-3β enzyme activity. However, we addressed these possibilities primarily using in vitro and in silico approaches, and future experiments are needed to establish which of these mechanisms operate in vivo.Ec). In both models, the STC2-7 showed significantly improved anti-inflammatory activity compared to 6-shogaol. It is worth noting that the test compounds also showed anti-inflammatory activity in NRF2-silenced BV-2 cells , suggesting that other mechanisms might be involved in the anti-inflammatory activity. The compounds have reversed the elevated levels of the inflammatory marker (nitric oxide), cytokines  and mediators (COX-2 and NF-κBP65). Many reports confirm that NRF2 activation plays a vital role in the anti-inflammatory process in oxidative-stress-associated neurodegenerative diseases ,64,65. TKEAP1-dependent and KEAP1-independent mechanisms activate NRF2 ,67, and 1/2) than 6-shogaol in all three representative liver microsomes. The increased metabolic stability of STCs could be because of (1) steric hindrance of α,β-unsaturated carbonyl group by thiophene and (2) reduced electron density on the alkene because of thiophene’s electron-withdrawing ability. Metabolic stability refers to the biotransformation of compounds. It plays a vital role in early-phase drug discovery to develop molecules with favourable pharmacokinetic properties . Liver mIn this study, we reported the new 6-shogaol derivatives (shogaol thiophene compounds (STCs)) with improved NRF2 activity through multiple mechanisms (KEAP1-independent and -dependent), decreased reactivity with thiols , and metabolic stability in liver microsomes. The new synthetic method using a reusable catalyst (proline–proline dipeptide) was employed to synthesise STCs in good yields (>85%) and purity (~95%). The drug-like properties and bioactivity of STCs are better than 6-shogaol. The NRF2 activity of STCs significantly depends on the nature of substituents . The STC containing an aromatic ring is the most potent. STCs containing electron-withdrawing show weak activity. STCs containing electron-donating substituents show moderate activity. 6-shogaol activates NRF2 in the micromolar range, whereas the most potent STC  activates NRF2 in the sub-micromolar range. The STCs have shown more significant anti-inflammatory effects than 6-shogaol via NRF2-dependent and NRF2-independent mechanisms (future investigations should be carried out to elucidate this aspect further). The half-life of STC5 in liver microsomes is about 1.5 times longer than that of 6-shogaol. The reactivity of STC5 with thiols is about 70% lower than that of 6-shogaol. The compound STC5 has shown promising in vitro activity, whose potential as a drug-like should be further confirmed in animal and pharmacokinetics studies.


Ganoderma applanatum exhibit significant anti-adipogenesis effects. In order to obtain more structurally diverse lanostane triterpenoids to establish a structure–activity relationship, we continued the study of lanostane triterpenoids from the fruiting bodies of G. applanatum, and forty highly oxygenated lanostane-type triterpenoinds (1–40), including sixteen new compounds (1–16), were isolated. Their structures were elucidated using NMR spectra, X-ray crystallographic analysis, and Mosher’s method. In addition, some of their parts were evaluated to determine their anti-adipogenesis activities in the 3T3-L1 cell model. The results showed that compounds 16, 22, 28, and 32 exhibited stronger anti-adipogenesis effects than the positive control  at the concentration of 20 μM. Compounds 15 and 20 could significantly reduce the lipid accumulation during the differentiation process of 3T3-L1 cells, comparable to the untreated group. Their IC50 values were 6.42 and 5.39 μM, respectively. The combined results of our previous and present studies allow us to establish a structure-activity relationship of lanostane triterpenoids, indicating that the A-seco-23→26 lactone skeleton could play a key role in anti-adipogenesis activity.Our previous research has shown that lanostane triterpenoids from  Tricholoma matsutake, 28D −1.2 ; UV (MeOH); λmax (log ε): 230 (3.36), and 196 (3.33); IR (KBr) vmax 3428, 2953, 2943, 1653, 1636, 1423, 1364, 1212, and 1131 cm−1; 1H NMR and 13C NMR data: see m/z: 561.2696 [M + H]+ .Ganoapplic acid B (2): white powder (MeOH); [α]28D +48.0 ; UV (MeOH); λmax (log ε): 251 (3.75), and 196 (4.08); IR (KBr) vmax 3430, 2953, 2928, 1653, 1636, 1473, 1344, 1211, and 1147 cm−1; 1H NMR and 13C NMR data: see m/z: 527.2653 [M − H]− .Ganoapplic acid C (3): white powder (MeOH); [α]28D +42.0 ; UV (MeOH); λmax (log ε): 251 (3.59), and 196 (3.93); IR (KBr) vmax 3423, 2955, 2930, 1673, 1635, 1428, 1380, 1219, and 1132 cm−1; 1H NMR and 13C NMR data: see m/z: 527.2654 [M − H]− .Methyl ganoapplate C (4): white powder (MeOH); [α]28D +39.5 ; UV (MeOH); λmax (log ε): 251 (3.52), and 196 (3.85); IR (KBr) vmax 3458, 2957, 2928, 1689, 1606, 1473, 1375, 1210, and 1132 cm−1; 1H NMR and 13C NMR data: see m/z: 565.2773 [M + Na]+ .Ganoapplic acid D (5): white powder (MeOH); [α]28D −20.1 ; UV (MeOH); λmax (log ε): 242 (3.71), and 195 (3.79); IR (KBr) vmax 3503, 2967, 2935, 1663, 1635, 1452, 1390, 1200, and 1125 cm−1; 1H NMR and 13C NMR data: see m/z: 511.2707 [M − H]− .Methyl gibbosate M (6): white powder (MeOH); [α]28D −49.64 ; UV (MeOH); λmax (log ε): 307 (3.21), 243 (3.80), and 196 (3.77); IR (KBr) vmax 3438, 2966, 2912, 1688, 1634, 1453, 1364, 1212, and 1132 cm−1; 1H NMR and 13C NMR data: see m/z: 533.2873 [M + Na]+ .Methyl ganoapplate E (7): white powder (MeOH); [α]28D −69.3 ; UV (MeOH); λmax (log ε): 316 (3.13), 243 (3.74), and 196 (3.72); IR (KBr) vmax 3413, 2953, 2916, 1657, 1620, 1454, 1374, 1209, and 1142 cm−1; 1H NMR and 13C NMR data: see m/z: 535.3033 [M + Na]+ .Ganoapplic acid E (8): white powder (MeOH); [α]28D −143.5 ; UV (MeOH); λmax (log ε): 317 (3.57), 244 (4.15), and 196 (4.05); IR (KBr) vmax 3445, 2980, 2906, 1712, 1690, 1458, 1380, 1214, and 1028 cm−1; 1H NMR and 13C NMR data: see m/z: 497.2911 [M − H]− .Methyl gibbosate L (9): white powder (MeOH); [α]28D +20.27 ; UV (MeOH); λmax (log ε): 291 (3.70), 244 (3.94), and 196 (3.85); IR (KBr) vmax 3444, 2976, 2911, 1673, 1658, 1423, 1374, 1219, and 1140 cm−1; 1H NMR and 13C NMR data: see m/z: 533.2876 [M + Na]+ .Ganoapplic acid F (10): white powder (MeOH); [α]28D +20.86 ; UV (MeOH); λmax (log ε): 299 (3.77), and 196 (3.98); IR (KBr) vmax 3437, 2953, 2924, 1678, 1656, 1433, 1374, 1215, and 1132 cm−1; 1H NMR and 13C NMR data: see m/z: 511.2708 [M − H]− .Methyl ganoapplate F (11): white powder (MeOH); [α]28D +5.80 ; UV (MeOH); λmax (log ε): 301 (3.46), and 196 (3.87); IR (KBr) vmax 3439, 2953, 2929, 1723, 1638, 1445, 1334, 1219, and 1138 cm−1; 1H NMR and 13C NMR data: see m/z: 549.2827 [M + Na]+ .Methyl gannosate I (12): white powder (MeOH); [α]28D +95.0; UV (MeOH); λmax (log ε): 249 (3.99), and 195 (3.73); IR (KBr) vmax 3452, 2985, 2921, 1673, 1618, 1425, 1376, 1221, and 1132 cm−1; 1H NMR and 13C NMR data: see m/z: 565.2769 [M + Na]+ .Ganoapplic acid G (13): white powder (MeOH); [α]28D −32.86 ; UV (MeOH); λmax (log ε): 242 (3.99), and 196 (3.86); IR (KBr) vmax 3440, 2978, 2965, 1683, 1628, 1403, 1364, 1200, and 1151 cm−1; 1H NMR and 13C NMR data: see m/z: 535.0000 [M + Na]+ .Methyl ganoapplate G (14): white powder (MeOH); [α]28D +0.80 ; UV (MeOH); λmax (log ε): 243 (3.79), and 196 (3.77); IR (KBr) vmax 3445, 2963, 2931, 1683, 1638, 1453, 1384, 1209, and 1142 cm−1; 1H NMR and 13C NMR data: see m/z: 549.2823 [M + Na]+ .Methyl applate C (15): white powder (MeOH); [α]28D +43.44 ; UV (MeOH); λmax (log ε): 248 (3.99), and 196 (3.95); IR (KBr) vmax 3443, 2956, 2915, 1665, 1624, 1433, 1376, 1205, and 1132 cm−1; 1H NMR and 13C NMR data: see m/z: 547.2672 [M + Na]+ .Methyl gibbosate A (16): white powder (MeOH); [α]28D +18.94 ; UV (MeOH); λmax (log ε): 237 (3.78), and 196 (3.82); IR (KBr) vmax 3430, 2953, 2929, 1703, 1628, 1433, 1354, 1229, and 1147 cm−1; 1H NMR and 13C NMR data: see m/z: 563.2612 [M + Na]+ Crystallographic Data for Ganoapplic acid A (1): C30H40O10·CH4O·H2O, M = 610.68, a = 13.9761(7) Å, b = 6.9089(3) Å, c = 15.8732(7) Å, α = 90°, β = 90.948(2)°, γ = 90°, V = 1532.50(12) Å3, T = 100.(2) K, space group P1211, Z = 2, μ(Cu Kα) = 0.844 mm−1, 26,208 reflections measured, 5973 independent reflections (intR = 0.0593). The final R1 values were 0.0504 (I > 2σ(I)). The final wR(F2) values were 0.1381 (I > 2σ(I)). The final R1 values were 0.0543 . The final wR(F2) values were 0.1436 . The goodness of fit for F2 was 1.042. Flack parameter = 0.07(9).X-ray 4 was performed based on the previous method + , suggesting 11 degrees of unsaturation. Its 13C NMR spectra displayed 30 carbon resonances, of which seven methyls, three ketone carbonyls , an α,β-unsaturated carbonyl , one carboxyl (δC 179.7), two oxygenated methines (δC 80.0 and δC 61.2), and three quaternary carbons containing oxygen  were assigned based on the HSQC and 13C-DEPT NMR spectra , C-22, and the oxygenated quaternary carbon (δC 71.4); of H3-30 with C-15 (δC 80.0); and of H-15  with the oxygenated methine (δC 61.2) and quaternary carbons (δC 71.4). Meanwhile, the proton of the methine containing oxygen  showed the HMBC correlations with C-13, C-14, C-15, and C-20, as well as the 1H-1H COSY correlation with H-15  further confirmed that the absolute configurations of C-15, C-16, C-20, and C-25 were R, S, S, and S -7,12,23-trioxo-8β,20-dihydroxy-16α,17α-epoxy-lanosta-9,11-en-26-oic acid.In the ROESY spectrum B of 1, Hanalysis A of 1  was isolated as a colorless solid. Its molecular formula was determined to be C30H40O8 by the HRESIMS. The 1D NMR and HSQC data of 2 supported the presence of five methyls, an α,β-unsaturated ketone, two carboxyl or ester carbonyls, one oxygenated methylene , three oxygenated methines , one quaternary carbon containing oxygen (δC 67.4), a terminal double bond , and a double bond , which indicated that compound 2 was an A-seco-lanostane triterpenoid and had the similar structure to that of gibbosicolid F. . T. T4) sho5) was isolated as a white powder and its molecular formula was determined to be C30H40O7 based on the HRESIMS at m/z 511.2701 [M − H]− . Its 13C-DEPT spectra (see sp2 and two oxygenated), and eleven quaternary carbons . These data indicated that compound 5 was a lanostane-type triterpenoid and had similar structure to that of gibbosic acid M (22) + . The 1D NMR spectra (see 6 were the same as those of gibbosic acid M (22) + . The 1D NMR spectra of 13 showed the presence of the ketone carbonyl at C-3, 7,8-epoxyl, α,β-unsaturated ketones (C-9/C-11/C-12 and C-20/C-22/C-23), 15-OH, and 27-oic acid, which was further confirmed by the 2D NMR spectra ,20E(22)-dien-26-oic acid. In addition, methyl ganoapplate G (14) was deduced to be the methylation product of 13 on the basis of the HMBC correlation of OMe with C-27.Ganoapplic acid G ( spectra A. The af15) was found to have a similar structure to methyl ganoapplate F (14), except for the presence of a double bond in 15, rather than one methylene and one methine in 14. Furthermore, in the HMBC spectrum of 15, the correlations -conjugated diene was 17,20-Z-. Additionally, the ROESY correlation of H3-30/H-15 demonstrated that 15-OH was β. Finally, the structure of 15 was determined to be methyl 15β-hydroxy-7β,8β-epoxy-3,12,23-trioxolanosta-9(11),16Z,20E(22)-trien-26-oate and named methyl applanate C (15).Methyl applanate C (elations A of H3-316) was considered to be the methylation derivative of gibbosic acid A (29) [β,8β-epoxy was proven by the ROESY correlation of H-17/H3-30. Thus, the structure of 16 was established to be methyl 20-hydroxy-7β,8β-epoxy-3,12,15,23-tetraoxo-lanosta-9,16-dien-26-oate.Methyl gibbosate A (d A (29)  because 17) [18) [19) [20) [21) [22) [23) [24) [25) [26) [27) [28) [29) [30) [31) [32) [33) [34) [35) [36) [37) [38) [39) [40) [In addition, 24 known compounds were identified by comparing their 1D NMR spectra with those reported in the literature, and they were assigned as gibbosic acid G (17) , applano17) [18) , gibbosi18) [19) , gibbosi19) [20) , gibbosi20) [21) , gibbosi21) [22) , gibbosi22) [23) , gibbosi23) [24) , applano24) [25) , gibbosi25) [26) , ganodap27) [28) , ganoapp28) [29) , elfving29) [30) , applano30) [31) , gibbosi31) [32) , gibbosi32) [33) , elfving33) [34) , methyl 34) [35) , applano16, 22, 28, and 32 showed comparable inhibition for lipid accumulation compared to the positive control . Meanwhile, compounds 15 and 20 displayed stronger inhibitory effects than the positive control, even resembling the untreated group , type III (22), type VI (15 and 28), type VII (16 and 32), and type VIII (37). For type II, when the relative configuration of 15-OH was α, the activity was decreased, similar to compound 19, while any changes in the side chain decreased their activities, such as compounds 2–4. For type III, no matter which reactions happened in type III, compounds 5–8 and 23 did not show inhibitory activity. For type VI, compound 27 was the 3-OH analogue of 15 and 28, leading to a decrease in inhibition. In type VII, the carbonyl at C-3 and the carbonyl or hydroxyl at C-15 could be the crucial active functionalities. C24 lanostane triterpenoids possessing a double bond at C-16 and C-17 displayed anti-adipogenesis activity [20 belonging to type II showed the strongest inhibitory activity, suggesting that A-seco-15β-hydroxy-7,8-epoxy-12-oxolanosta-9,11-en-23→27 lactone could play a significant role in the anti-adipogenesis effect ,11(12),20(22)-trien fraction, type IV with a 12-oxo-7(8),9(11)-dien fraction, type V with a 7,11-dioxo-12-hydroxy-8(9)-en fraction, type VI with a 7,8-epoxy-12,23-dioxo-9(11),16(17),20(22)-trien fraction, type VII with a 20-hydroxy-7,8-epoxy-12,23-dioxo-9(11)-en fraction, type VIII with a 12-oxo-7,8-epoxy-9(11)-en-21,22,23,24,25,26,27-norlanostane, and type IX with a 12,23-epoxy-23→27 lactone fraction. The combined results of the previous and present studies showed that bioactive compounds were mainly present in type II  could be of importance for the anti-adipogenic effect. In the next step, we can use type III as a template for further structural modification in order to find the lead compound.Overall, inspired by our previous studies, we investigate the lanostane-type triterpenoids of


Candida. The broth microdilution test was used to determine the Minimum Inhibitory Concentration (MIC) of the compounds and to verify the possible antifungal action mechanisms. The synthetic derivatives were obtained using various reaction methods, and six new compounds were obtained. The structures of the synthesized products were characterized by FTIR spectroscopy: 1H-NMR, 13C-NMR, and HRMS. The coumarin derivative 8 presented the best antifungal profile, suggesting that the pentyloxy substituent at the C-7 position of coumarin ring could potentiate the bioactivity. Compound 8 was then evaluated against the biofilm of C. tropicalis ATCC 13803, which showed a statistically significant reduction in biofilm at concentrations of 0.268 µmol/mL and 0.067 µmol/mL, when compared to the growth control group. For a better understanding of their antifungal activity, compounds 8 and 21 were submitted to a study of the mode of action on the fungal cell wall and plasma membrane. It was observed that neither compound interacted directly with ergosterol present in the fungal plasma membrane or with the fungal cell wall. This suggests that their bioactivity was due to interaction involving other pharmacological targets. Compound 8 was also subjected to a molecular modeling study, which showed that its antifungal action mechanism occurred mainly through interference in the redox balance of the fungal cell, and by compromising the plasma membrane; not by direct interaction, but by interference in ergosterol synthesis. Another important finding was the antifungal capacity of homoisoflavonoids 23 and 24. Derivative 23 presented slightly higher antifungal activity, possibly due to the presence of the methoxyl substituent in the meta position in ring B.A set of twenty-four synthetic derivatives, with coumarin and homoisoflavonoid cores and structural analogs, were submitted for evaluation of antifungal activity against various species of  Candida spp., has been increasing worldwide + = 366.9581, Found = 366.9481 [M + Na]+; calculated value [M + H]+ = 344.9763, Found [M + H]+ = 344.9733. * Interchangeable.2-Oxo-Coumarin-3-carboxylic acid (1.233 mmol), aromatic alcohol (1.0 equiv.) and 4-(dimethylamino)-pyridine  were dissolved in 4.0 mL dichloromethane, the mixture was subjected to constant stirring at room temperature. Then, a solution of dicyclohexylcarbodiimide  in dichloromethane (3.0 mL) was added; the reactions were kept under constant stirring and at room temperature for 24 h. The DCU formed was filtered and the products purified in silica gel 60 column chromatography, using a mixture of hexane and ethyl acetate in different proportions as eluent .9): White solid. Yield: 14.2% . M.P.: 87–88 °C : 3053 (C-H sp2); 1758 (C=O ester), 1698 ; 1619, 1569, 1459 (C=C aromatic); 1215, 1156 (C-O). 1H NMR  δ 8.53 ; 7.65 ; 7.59 ; 7.50–7.46 ; 7.41–7.30 ; 5.39 . 13C NMR  δ 162.91 ; 156.71 (C-2); 155.32 (C-8a); 149.02 (C-7); 135.50 (C-1′); 134.59 (C-4); 129.68 (C-5); 128.77 ; 128.55 (C-4′); 128.45 ; 124.97 (C-6); 118.07 (C-4a); 117.93 (C-3); 116.92 (C-8); 67.58 (C-7′) +. * Interchangeable.N-phenethyl-2H-chromene-3-carboxamide (17): Crystalline solid, Yield: 41.1% (64 mg. 0.2181 mmol). M.P.: 175.2–176.3 °C : 3327 (N-H); 3055 (C-H sp2); 2943 (C-H sp3); 1720 (C=O); 1707 (C=O amide); 1657 ; 1610; 1569; 1525 (C=C aromatic). 1H NMR  δ 8.89 ; 8.87 ; 7.67 ; 7.64 ; 7.39–7.29 ; 7.25–7.20 ; 3.71 ; 2.93 . 13C NMR  δ 161.56 (C=O) *; 161.47 (C-2) *; 154.53 (C-8a); 148.33 (C-7); 138.91 (C-1′); 134.09 (C-4); 129.90 (C-5); 128.89 ; 128.74 ; 126.66 (C-4′); 125.36 (C-6); 118.78 (C-4a); 118.62 (C-3); 116.72 (C-8); 41.47 (C-8′); 35.77 (C-7′) [2-Oxo-8–180 °C . TLC (6:N-butyl-2-oxo-2H-chromene-3-carboxamide (18): Crystalline solid, Yield: 41.5% (107 mg. 0.4365 mmol). M.P.: 90–91 °C (Lit.: 74–76 °C [f = 0.63. IR ʋmax (KBr. cm−1): 3330 (N-H); 3055 (C-H sp2); 2950 (C-H sp3) 1719 (C=O C-2); 1705 (C=O amide); 1661 ; 1609; 1568; 1535 (C=C aromatic). 1H NMR  δ 8.90 ; 8.81 ; 7.68 ; 7.65 ; 7.40–7.34 ; 3.45 ; 1,61 ; 1,41 ; 0.95 . 13C NMR  δ 161.61 (C=O) *; 161.54 (C-2) *; 154.49 (C-8a); 148.34 (C-7); 134.06 (C-4); 129.90 (C-5); 125.37 (C-6); 118.79 (C-4a); 118.65 (C-3); 116.71 (C-8); 39.75 (C-1′); 31.53 (C-2′); 20.30 (C-3′); 13.87 (C-4′)  [74–76 °C ); TLC (6N-isobutyl-2-oxo-2H-chromene-3-carboxamide (19): Crystalline solid, Yield: 47.2 % (122 mg. 0.4974 mmol). M.P.: 117.1–118.1 °C.; TLC (6:4 hexane/EtOAc); Rf = 0.63 (6:4. hexane/EtOAc). IR ʋmax : 3346 (N-H); 3055 (C-H sp2); 2963 (C-H sp3); 1718 (C=O); 1706 (C=O amide); 1655 ; 1609; 1567; 1520 (C=C aromatic). 1H NMR  δ 8.90 ; 8.87 ; 7.68 ; 7.64 ; 7.40–7.34 ; 3.29 ; 1.91 ; 0.98 . 13C NMR  δ 161.65 (C=O) *; 161.58 (C-2) *; 154.48 (C-8a); 148.35 (C-7); 134.05 (C-4); 129.88 (C-5); 125.36 (C-6); 118.78 (C-4a); 118.67 (C-3); 116.70 (C-8); 47.35 (C1′); 28.56 (C-2′); 20.33  [20  [The 2-hydroxyacetophenone (2.203 mmol) and the aldehyde  were dissolved in 2.5 mL of methanol. The mixture was cooled in an ice bath with magnetic stirring to add a chilled solution of NaOH (60%). The reaction was kept in the ice bath for 45 min and at room temperature for 48h. The mixture was poured into ice water and the pH adjusted to 2.0 with a 6N hydrochloric acid (HCl) solution. The yellow precipitate formed was filtered and recrystallized with methanol to obtain the chalcone ; 22.8%) .E)-3-(2-hydroxy-3-methoxyphenyl)-1-(2-hydroxyphenyl)prop-2-en-1-one (20): Yellow solid, Yield: 22.8% . M.P.: 180–181.8 °C : 3346 (O-H); 1631 (C=O); 1607 ; 1584, 1562, 1481 (C=C aromatic). 1H NMR (400 MHz. DMSO-d6) δ 12.47 ; 8.09 ; 8.04 ; 7.81 ; 7.44 ; 7.39 ; 6.94 ; 6.90–6.86 ; 6.74 ; 3.72 . 13C NMR  δ 193.89 (C=O); 161.88 (C-2′); 148.15 (C-3); 146.99 (C-2); 140.17 (C-β); 136.20 (C-4′); 130.63 (C-6′); 121.50 (C-1); 120.92 (C-1′); 120.68 (C-6); 120.05 (C-α); 119.28 (C-5′) *; 119.25 (C-3′) *; 117.82 (C-5); 114.09 (C-4); 56.07 (C-7) [ 130–131 ); TLC  and then concentrated [4-Chromanone (0.684 mmol) and the aldehyde  were dissolved in methanol (2 mL), and pyrrolidine  was added to the solution. The mixture was stirred at room temperature for 24 h. The mixture was poured into chilled water, the precipitate filtered and washed with water, dissolved in dichloromethane, dried with anhydrous sodium sulfate (Naentrated .E)-3-Benzylidenechroman-4-one (21): crystalline white solid, Yield: 72% (0.492 mmol). M.P. 80.8–81.6 °C : 2857 (C-H sp3); 1668 (C=O); 1610 (C=C). 1H NMR : δ 8.03 ; 7.88 ; 7.51–7.40 ; 7.31–7.30 ; 7.09–7.05 ; 6.98–6.95 ; 5.35 . 13C NMR  δ 182.31 (C-4); 161.24 (C-8a); 137.56 (C-9); 135.96 (C-7); 134.50 (C-3); 131.03 (C-1′); 130.08 ; 129.56 (C-4′); 128.83 ; 128.05 (C-5); 122.13 (C-4a); 122.01 (C-6); 118.02 (C-8); 67.71 (C-2) [(: 111 °C ). TLC (971 (C-2) .E)-3-(4-Methoxybenzylidene)chroman-4-one (22): Crystalline yellow solid, Yield: 76% (0.518 mmol). M.P. 133–134.1 °C : 3079 (C-H sp2), 2837 (C-H sp3); 1665 (C=O); 1606 (C=C); 1510; 1478 (C=C aromatic). 1H NMR : δ 8.01 ; 7.83 ; 7.47 ; 7.28–7.26 ; 7.08–7.03 ; 6.98–6.94 ; 5.37 ; 3.85 . 13C NMR  δ 182.26 (C-4); 161.09 (C-8a); 160.85 (C-4′); 137.40 (C-9); 135.76 (C-7); 132.16 ; 129.02 (C-3); 127.99 (C-5); 127.14 (C-1′); 122.24 (C-4a); 121.93 (C-6); 117.93 (C-8); 114.39 ; 67.90 (C-2); 55.51 (OCH3) [(: 118 °C . TLC (9:1 (OCH3) .E)-3-(3-methoxybenzylidene)chroman-4-one (23): Yellow amorphous solid, Yield: 15.5% (0.1049 mmol). M.P. 78.7–80.4 °C : 3071 , 2978 , 1669 (C=O); 1614 (C=C); 1600; 1583 (C=C aromatic). 1H NMR : δ 8.02 ; 7.84 ; 7.49 ; 7.36 ; 7.09–7.05 ; 6.98–6.94 ; 6.89–6.87 ; 6.84 ; 5.35 ; 3.84 . 13C NMR  δ 182.33 (C-4); 161.32 (C-8a); 159.85 (C-3′); 137.50 (C-9); 136.01 (C-7); 135.83 (C-3); 131.30 (C-1′); 129.88 (C-5′); 128.09 (C-5); 122.41 (C-6); 122.16 (C-4a); 122.05 (C-6′); 118.06 (C-8); 115.57 (C-4′); 115.20 (C-2′); 67.79 (C-2); 55.49 (OCH3) [(85–86 °C . TLC (9:9 (OCH3) .E)-3-(2-Methoxybenzylidene)chroman-4-one (24): Crystalline white solid, Yield: 63.3% (0.4277 mmol). M.P. 102.1–103.6 °C. TLC (9:1 hexane/EtOAc); Rf = 0.46. IR ʋmax : 3086 , 2981 , 1677 (C=O); 1611 (C=C); 1578; 1559 (C=C aromatic). 1H NMR  δ 8.06 ; 8.03 ; 7.49 ; 7.41–7.36 ; 7.10–7.07 ; 7.03–7.01 ; 6.97–6.95 ; 5.24 ; 3.85 . 13C NMR  δ 182.54 (C-4); 161.39 (C-8a); 158.30 (C-2′); 135.77 (C-9); 133.97 (C-7); 131.26 (C-4′); 130.93 (C-3); 130.53 (C-6′); 128.02 (C-5); 123.59 (C-4a); 122.29 (C-1′); 121.85 (C-6); 120.37 (C-5′); 117.95 (C-8); 111.03 (3′); 68.18 (C-2); 55.59 (OCH3).: Candida albicans ATCC 90028, Candida albicans ATCC 60193, Candida tropicalis ATCC 13803, Candida krusei ATCC 6258, Candida parapsilosis ATCC 22019 and Candida glabrata ATCC 90030. Nystatin, ketoconazole, DMSO (Dimethyl Sulfoxide), Tween 80% and Ergosterol were obtained from Sigma-Aldrich® Chemical Co. . Sorbitol (anhydrous D-sorbitol) was purchased from INLAB® .Reference strains of 3 CFU/mL, 530 nm, absorbance between 0.08–0.116. Serial dilutions of the compounds placed in 96-well U-bottom microtiter plates containing RPMI, in concentrations ranging from 1000 to 7.81 μg/mL. Nystatin and ketoconazole were used as controls and were tested at concentrations ranging, respectively, from 48 to 0.75 μg/mL and 16 to 0.125 μg/mL. These plates were incubated for 24 h at 35 °C, and the results were read by visually observing cell aggregates at the bottom of the wells. Cell viability controls, sterility of the culture medium, and 5% DMSO solution, were used to prepare the compounds solutions and performed simultaneously with the assay. The MIC was defined as the lowest concentration capable of inhibiting visible growth. The bioactivity of the compounds was determined from the MIC values and classified according to the following categories: (a) very strong bioactivity  strong bioactivity (MIC between 3.515 and 25 µg/mL); (c) moderate bioactivity (MIC between 26–100 µg/mL); (d) weak bioactivity (MIC from 101 to 500 µg/mL); and (e) very weak bioactivity (MIC in the range of 501–2000 µg/mL) [The MIC was determined using the microdilution technique described by the Clinical and Laboratory Standards Institute, 2008 . The yea0 µg/mL) .To determine the MFC, 10 μL aliquots from the wells corresponding to MIC, MICx2, and MICx4 were subcultured on Sabouraud Dextrose Agar . The plates were incubated for 24 h at 35 °C, and reading was performed by visually observing the fungal growth in the solid medium. MFC was defined as the lowest concentration capable of inhibiting visible growth (colonies forming in solid culture medium). The MFC/MIC ratio was calculated to determine whether the substance presented fungistatic  or fungicidal (MFC/MIC less than 4) activity .C. albicans strain ATCC 90028 was used, and the assay was conducted as described for MIC determination. Nystatin was used as a positive control [The MIC in the presence of ergosterol was defined as the lowest concentration of the substance capable of promoting the inhibition of visible microbial growth. The assay was also performed using the microdilution technique, however, in the presence of exogenous ergosterol  at a concentration of 400 µg/mL. The  control .C. albicans ATCC 90028 in the absence and presence of 0.8 μM sorbitol. To conduct this experiment, the procedures described for determining the MIC were performed. After this step, the plates were incubated at 35 °C, and readings were taken 24 h after the incubation period. Caspofungin, at an initial concentration of 4 µg/mL, was used as a positive control. Sorbitol is an osmotic protector of the fungal cell wall and upon addition of this substance. Higher MIC values in the media indicate a possible mode of action on targets that involve cell wall functions [The sorbitol assay was performed using the microdilution technique, aiming to compare MIC values against unctions ,33.C. tropicalis ATCC 13803 inoculum containing about 106 CFU/mL were transferred to a 48-well microdilution plate. Molecule concentrations previously determined by the values of MIC (60 µg/mL–0.258 µmol/mL), MICx2 (30 µg/mL–0.129 µmol/mL), MICx4 (15 µg/mL–0.0645 µmol/mL) were added to the wells, followed by incubation for 48 h at an optimal growth temperature of 35 °C, allowing the yeast to adhere. Likewise, the concentrations of nystatin (positive control) were defined at the following concentrations: 4 µg/mL (0.0043 µmol/mL), 2 µg/mL (0.0021 µmol/mL) and 1 µg/mL (0.0011 µmol/mL) which correspond respectively to the values of MIC, MICx2 and MICx4. Then, the wells were washed with phosphate-buffered saline (PBS) to remove weakly bound cells and fresh medium was added. The plates were incubated for 48 h at 35 °C. For biofilm quantification, the wells were washed twice with PBS, air dried for 45 min and stained with 0.4% crystal violet solution. Absorbance values were read at 600 nm using a plate reader [Aliquots (1000 µL) of the e reader . The unte reader .8 in C. albicans were identified employing the previously reported homology-based target fishing protocol [8 were first predicted with the Similarity Ensemble Approach (SEA) method [C. albicans (tax id: 5476) proteins contained in the Reference proteins (refseq_protein) database. Proteins from the fungus identical in at least 35% to any SEA predicted targets and with their sequences covered in at least 70% by the Blast alignment were considered as potential targets of compound 8 in C. albicans.Potential targets for compound protocol ,72. For ) method . Computa) method  search a8 and partial atomic charges of type am1bcc were added to it with MolCharge (QUACPAC) [C. albicans proteins selected for modeling had no structure deposited in the PDB database, thus homology models were generated for them with the SwissModel web server [OpenEye’s Omega ,76 was uQUACPAC) . Among tQUACPAC)  model ofb server . Severalb server  was seleb server ,82. Brie8, respectively. Topologies and force field modifications for the ligand were generated with antechamber, while for cofactors these were obtained from the Amber parameter database maintained by the Bryce Group at The University of Manchester ). Systems were enclosed in truncated octahedron boxes and solvated with OPC water molecules. Excess charges on the solvated systems were neutralized by adding either Na+ or Cl− counterions. The solvated and neutralized complexes were energy minimized in two stages, the first one of which included constraints for all atoms except the solvent, while during the second one all constraints were removed. The energy minimized systems were then gradually heated from 0 to 300 K for 20 ps before proceeding to the equilibration stage. Equilibration took place in the NTP ensemble maintaining the temperature at 300 K and pressure set to 1 bar. The equilibrated systems were used as input to five different production runs, each one lasting for 4 ns. The atomic velocities were randomly re-initialized before each production run to obtain a better description of the complexes’ conformational space. Free energies of binding were predicted with the MM-PBSA method as implemented in Amber. For this, 20 MD snapshots were evenly extracted from each of the five production runs, totaling 100 MD complex conformations for MM-PBSA calculations. In addition, snapshots for free energy of binding calculations were selected from the 1 ns–4 ns interval. The ionic strength was set to 150 mM and default implicit solvent parameters were used.Amber 20  was used8 and the control ketoconazole were performed with the SwissADME and pkCSM web servers following the procedure described. SwissADME was employed to retrieve the physicochemical parameters and lipophilicity properties. On the other hand, the pkCSM server was used to predict the pharmacokinetics properties and toxicity of the compounds [The ADMET predictions for compound ompounds ,86.Candida, compound 8 exhibited the best antifungal profile, with strong activity against the two tested strains: C. albicans ATCC 90028 and C. tropicalis ATCC 13803, and moderate activity against C. krusei ATCC 6258. Our findings suggest the importance of alkyl chain length to antifungal activity in O-alkylated derivatives at the coumarin C-7 position. Among homoisoflavonoids, the bioactivity of derivatives 23 and 24 stands out; derivative 23 presented slightly higher antifungal capacity, with moderate activity against three strains tested, which may be related to the m-OCH3 substituent of ring B. Compound 8 also showed the ability to reduce C. tropicalis ATCC 13803 biofilm from 73% to 68% at concentrations of 0.268 µmol/mL to 0.067 µmol/mL, respectively. The mode of action studies of compounds 8 and 21, did not evidence direct interaction with plasma membrane ergosterol or the fungal cell wall. Molecular modeling of 8 suggested a mechanism of action involving interaction with several pharmacological targets , involving interference in the redox balance of the C. albicans cell and plasma membrane synthesis, such that membrane impairment does not occur through direct interaction with its components, but through interferences in ergosterol synthesis. The ADMET properties of compound 8 are similar to those represented by the antifungal drug ketoconazole. Therefore, in further studies the development of an antifungal drug candidate may have improved ADMET properties over compound 8. The results of this study may contribute to the development of new antifungal agents.From the series of molecules tested against species of


This study aimed to investigate the cytotoxic effects of C17 polyacetylenes from P. ginseng against ovarian cancer cell lines. Four unreported (1–4) and fifteen known (5–19) C17 polyacetylenes were obtained from the roots of P. ginseng using repeated chromatography . The chemical structures of all the compounds were determined by analyzing their spectroscopic data  and HR-MS. The structures of new polyacetylenes were elucidated as -(-)-heptadeca-9,10-epoxy-4,6-diyne-3,8-diyl diacetate (1), -(−)-heptadeca-1-en-9,10-epoxy-4,6-diyne-3,8-diyl diacetate (2), (−)-haptadeca-9,10-epoxy-8-methoxy-4,6-diyne-3,11-diol (3), and -(+)-3-acetoxy-9,10-dihydroxyheptadeca-1-en-4,6-diyne (4), named ginsenoynes O, P, and Q, and 3-acetyl panaxytriol, respectively. Subsequently, in vitro experiments on A2780 and SKOV3 human epithelial ovarian carcinoma cells were performed to assess the cytotoxic properties of the isolates. Among the isolates, panaquinquecol 4 (15) exhibited the most remarkable cytotoxic effects on both human ovarian cancer cells A2780  and SKOV3 . Therefore, C17 polyacetylenes derived from P. ginseng may warrant further investigation for their therapeutic potential in epithelial ovarian cancer.Although C Ovarian cancer, identified as a group of malignant neoplasms, poses a lethal threat to women worldwide . It is tPanax ginseng Meyer  and its constituents have anti-tumor effects in various cancer cell lines, including ovarian cancer  and two singlet methyl signals , seven methylene , four oxygenated methine , four quatenery carbon , and two carbonyl signals  were observed  . The IR -OCOCH3] . Moreoveobserved . The COSobserved . The HMB and C-8 .1 was determined by considering the 3JHH coupling constants and NOESY NMR data. Consistent correlations between H-9 with H-10 and the coupling constants for 3JH9-H10 (4.0 Hz) agreed well with the cis configuration .,14.1 wasloxyne A . The speeriments . Four prs −12.11 , suggest2 was isolated as yellow oil. The molecular formula of 2 was identified as C21H28O5 by HR-DART-MS  , 5.52 /δc 120.1 (C-1) and olefinic signals [δH 5.83 /δc 131.8 (C-2)]  and the NOESY correlations suggested that 2 had the same relative configuration as 1 -(−)-heptadeca-1-en-9,10-epoxy-4,6-diyne-3,8-diyl diacetate, named ginsenoyne P.Compound 8.22805) . The IR 8 (C-2)] . The aboups in 2 . The J vion as 1 . The absed for 1 . Therefo3 was isolated as yellow oil, and its chemical formula was identified as C18H26O4 by HR-DART-MS , five oxygenated methine protons , exomethylene protons at δH 4.90 (H-17a) and 4.96 (H-17b), a methyl proton at δH 0.99 (H-1), and one methoxy signal at δH 3.41 (8-OC3H) (13C and HSQC NMR spectra presented one olefinic carbon at δC 114.7 (C-16), five oxygenated sp3 carbons , four sp carbons , one methyl signal at δC 9.5 (C-1), and one methoxy signal at δC 57.1 (8-OCH3) . Although the NOESY spectrum exhibited correlations between H-8/H-11 and H-9/H-10 -haptadeca-9,10-epoxy-8-methoxy-4,6-diyne-3,11-diol, named ginsenoyne Q. Compound -DART-MS . Using t(8-OCH3) . The 13C(8-OCH3) . The conOSY data . The posOSY data . The steH-9/H-10 , the rel4 was isolated as yellow oil, and its molecular formula was identified as C19H28O4 by HR-DART-MS -panaxytriol (11), which is a major polyacetylene from ginseng and which was also isolated in this study, except for the presence of an acetyl group in 4  in 4 was the same as that of 11, implicating that they have the same relative configurations at C-9 and C-10. To determine the absolute configuration, deacetylation of 4 was conducted to yield 4a (4a (−24.2) was similar to those of 11 (−19.5) and -panaxytriol in the literature (−18.6) + ; 1H and 13C NMR data. See Yellow oil; [α]D22 −5.96; UV (Acetonitrile) λmax (log ε) 233 nm (2.99), 246 nm (2.69), 260 nm (2.50); IR (neat) νmax 1478, 1676, 2255; DART-MS m/z 378.22787 [M + NH4]+ ; 1H and 13C NMR data. See Yellow oil; [α]D22 −4.90; UV (Acetonitrile) λmax (log ε) 224 nm (3.04), 245 nm (2.83), 258 nm (2.72); IR (neat) νmax 2944, 3003, 3469; DART-MS m/z 324.21625 [M + NH4]+ ; 1H and 13C NMR data. See Yellow oil; [α]D22 33.03; UV (Acetonitrile) λmax (log ε) 222 nm (2.90), 233 nm (2.95), 245 nm (2.82); IR (neat) νmax 1226, 1746, 2307; DART-MS m/z 338.23114 [M + NH4]+ ; 1H and 13C NMR data. See Yellow oil; [α]1 were performed. Using Gaussian 16 software  and modified versions of the reported methods, all geometries and conformers of 1 were optimized using an HF/3-21* level of theory .Acid hydrolysis was performed to confirm the absolute configuration of escribed . CompounA2780 and SKOV3 human ovarian cancer cell lines and RAW264.7 macrophages were obtained from American Type Culture Collection (ATCC). The cell culture and MTT assay were performed using the method described previously ,36.17 polyacetylenes, ginsenoyne O (1), ginsenoyne P (2), ginsenoyne Q (3), and 3-acetyl panaxytriol (4), along with fifteen known ones (5-19), were isolated from the fresh roots of P. ginseng; moreover, the chemical structures of 1-4 were elucidated using 1D- and 2D- NMR spectroscopy and specific rotation calculation. Among the 19 polyacetylenes, acetylpanaxydol (6), -panaxytriol (11), and panaquinquecol 4 (15) exhibited a potent cytotoxic effect against both A2780 and SKOV3 human ovarian cells. This finding warrants further investigation into the anti-tumor activity of these C17 polyacetylenes in human ovarian cancer.Four new C