Botrytis cinerea. These substrates were incubated with fungal enzymes, and the reactions were monitored using state-of-the-art analytical methods. Under such conditions, a series of dimers, trimers, and tetramers were generated. The reactions were optimized and scaled up. The resulting mixtures were purified by high-resolution semi-preparative HPLC combined with dry load introduction. This approach generated a series of 23 phenylpropanoid derivatives, 11 of which are described here for the first time. These compounds are divided into 12 dimers, 9 trimers , and 2 tetramers. Elucidation of their structures was performed with classical spectroscopic methods such as NMR and HRESIMS analyses. The resulting compound series were analyzed for anti-Wnt activity in TNBC cells, with several derivatives demonstrating specific inhibition.In this study, a series of complex phenylpropanoid derivatives were obtained by chemoenzymatic biotransformation of ferulic acid, caffeic acid, and a mixture of both acids using the enzymatic secretome of  Throughout history, natural products (NPs) have been a source of inspiration for the development of a large number of therapeutics . New cheThe classical way of obtaining bioactive NPs is based on the bioassay-guided fractionation of an active extract from plant or microbial origin . This stIn this context, chemoenzymatic methods using common NPs as substrates could be an interesting option to generate chemically diverse and well-characterized NP derivative libraries. Biotransformation reactions represent an interesting tool for chemo-diversification of NPs covering a large chemical space from sustainable sources . Rather p-hydroxycinnamyl alcohol monomers and related compounds  is responsible for 627,000 deaths worldwide in 2018, being the most widespread cancer type in women. In triple-negative breast cancer (TNBC), expression of the estrogen and progesterone receptors, as well as human epidermal growth factor receptor 2 (HER2) is missing, making it unsusceptible to the targeted treatment and thus resulting in a drastically poor prognosis and a strong risk of relapse in the first 5 years following surgery .The Wnt signaling pathway is reported in many sources to be a target for TNBC . The sigB. cinerea secretome to obtain a series of more complex structures for evaluation of their Wnt inhibition properties.In this study, the aim was to perform biotransformations of these representative phenylpropanoid units using the B. cinerea secretome was added last. The amount of substrate, acetone, and water remained constant, but that of the secretome varied at 1, 5, and 10% (m/z 300–400), trimer (m/z 450–600), and tetramer (>m/z 680) derivatives  were, therefore, carefully adjusted to optimize the consumption of starting materials while avoiding polymer generation , ferulic acid (1 mg), and a mixture of both (0.5 mg of each) acids as substrates. The compounds were first solubilized in acetone, then diluted in water, and the  and 10% . The subivatives . UV tracivatives   and semi-preparative HPLC scale by geometric gradient transfer . The staFrom the 3 biotransformation reactions, 24 compounds were isolated and fully characterized by HRMS and NMR including 12 dimer, 9 trimer, and 2 tetramer derivatives . In addirel-(2E)-3-[(2S)-2-[(R)-hydroxymethyl]-1,3-benzodioxol-5-yl]-2-pro penoic acid from the biotransformation of caffeic acid with H2O2 and a Momordica charantia peroxidase.Among the 12 dimers produced, 9 were already known and 3 are new derivatives. Compound 6 resulted from an 8-O-4′ oxidative coupling between two caffeic acid units . This cod6, compound 18 fully decarboxylated rapidly (less than 24 h) to give off a new derivative (19) (Three 8-5′-benzofuran dimers formed by 8-5′ oxidative coupling followed by 4′-O-7 cyclization  were ideive (19) .m/z 327.0872 [M – H]– corresponding to a molecular formula of C18H15O6 , confirming the dimerization of caffeic and ferulic acids. The position of the methoxy group was deduced from the HMBC correlation of this methoxyl at δH 3.83  to C-3 at δC 147.8 and from H-5 at δH 6.77  to C-1 (δC 129.2) and C-3. Compound 19 was thus identified as (E)-3--4-hydroxy-3-methoxystyryl)phenyl)acrylic acid.The ESI(-)-HRMS analysis of compound 19 showed a molecular ion at Inonotus sp. , 6.77 , 6.71 , 5.67 , and 4.15 , whereas those of ferulic acid were observed at δH 6.99 , 6.86 , 6.79 , 5.72 , 4.08 , and 3.8  because of loss of symmetry. This compound was named dehydrocaffeicferulic acid dilactone (20).The biotransformation reactions also generated dilactone dimers. For example, the dehydrodicaffeic acid dilactone (5) and dehydrodiferulic acid dilactone (9) resulted from an 8-8′ coupling , 5 was kotus sp. , and 9 wotus sp. . A new dSurprisingly, in each reaction, dilactones were accompanied by a more polar and less intense peak: 5′ for the caffeic acid reaction , 9′ for Furthermore, a degradation process also occurred under our conditions for these dilactones. For example, we observed that one of the lactone rings can open to give off almost quantitatively phellinsin A (23) in the case of dehydrodicaffeic acid dilactone (5) . The samcis isomer of compound 7 was also isolated as 8 and had never been described. On the ROESY spectrum, both 7 and 8 showed correlations between H-8 and H-2′/H-6′, indicating an E configuration of the double bond. For 7 the coupling constant between H-7 and H-8 was 2.9 Hz and an ROE correlation was observed between H-8 and H-2, confirming the trans configuration of H-7 and H-8 protons, whereas 8 cis configuration was indicated by the ROE correlation between H-7 and H-8 and a 3JH7–H8 value of 7.6 Hz.The 7,8-E,2′E)-3,3′-[[2-hydroxy-2-(4-hydroxy-3-methoxyphen yl)ethylidene]bis[oxy]]bis-2-propen oic acid (11) formed through two successive 8-O-4′ couplings and H2O addition (rel-(2E)-3-[4-[[-5-[(1Z)-2-Carboxyethenyl]-2,3-dihyd ro-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-3-benzofuranyl] oxy]-3-methoxyphenyl]-2-propenoic acid (15) that resulted from 8-5′ coupling followed by 8-O-4′′ coupling from compound 24 (rel-(2E) -3-[1]benzopyr an-8-yl]-2-propenoic acid (12) already identified by Momordica charantia peroxidase. It was formed by an 8-5′ coupling of two ferulic acids followed by 8-8′′ coupling with a third ferulic acid -3--1--4-(4-hydroxy-3-methoxyphenyl)-6-methoxy-3-oxo-1,3a,4,9b-tet rahydro-3H-furochromen-8-yl)acrylic acid.Concerning the 9 trimers formed, 3 were already described. Compounds 11 and 15 were isolated by addition . The secpound 24 . The thilic acid . Our ream/z 537.1401 [M – H]–, . The 1H and COSY NMR data indicated the presence of two 1,3,4-trisubstituted aromatic cycles at δH 6.35 , 6.47 , and 6.57  for the first one and δH 6.64 , 6.73 , and 6.82  for the second one. The presence of two methoxy groups at δH 3.71  and 3.73  indicated that the trimer resulted from the coupling of two ferulic acids and one caffeic acid. The ROE correlations between 3OCH3 and H-2 and between 3′′OCH3 and H-2′′ positioned these methoxy groups on the two 1,3,4-trisubstituted aromatic cycles. The HMBC correlations from H-2′′ and H-6′′ to the oxymethine H-7′′ at δH 5.14 , from H-2 and H-6 to the oxymethine H-7 at δH 5.01  and the COSY correlations from H-7 to H-8, H-8 to H-8′′, and H-8′′ to H-7′′ allowed to link these two ferulic entities. The HMBC correlation from the aromatic singlet H-5′ at δH 6.97 to H-8 and the second aromatic singlet H-2′ at δH 6.79 to the ethylenic proton H-7′ at δH 7.19  positioned the caffeic acid group. Finally, a 7-O-9′′ cyclization made it possible to match the structure to the HRMS data. The NMR spectra recorded in DMSO-d6 showed a broad singlet at δH 5.41, which gives ROE correlation with H-7′ confirming the position of the hydroxy group. The trans–trans relative configuration of γ-butyrolactone was given by the ROE correlation from H-2 and H-6 to H-7 and H-8, from H-5′ to H-8, H-7 and H-8′′, and from H-7′′ to H-8 and H-8′′. The relative configuration in C-7′ was defined thanks to the following ROE correlations from H-7′′ to H-8′′ and H-8 and from H2′′/H-6′′ to H-8 and H-7′ (2O addition in C-7′′ (rel-(E)-3--4-((R)-hydroxy(4-hydroxy-3-methoxyphenyl)methyl)-2-(4-hydroxy-3-methoxyphenyl)-5-oxotetrahydrofuran-3-yl)phenyl)acrylic acid.Compound 16 is a trimer, as indicated by its ESI(-)-HRMS ion at and H-7′ . This coin C-7′′ . Compoun– ion at m/z 519.1294 corresponding to a molecular formula of C28H23O10  and thus to a trimer. The 1H and edited HSQC confirmed the presence of two trans olefinic protons at δH 6.15  and 7.62 , as well as three aromatic groups with the same substitution as that of 16, i.e., two 3-methoxy-4-hydroxyphenyl and one caffeic acid substituted in C-6. In addition, the following signals were observed: an oxymethine at δH 5.61  with HMBC correlations to C-5′ and C-6′, a methine at δH 4.38  with HMBC correlations to C-1 and C-2, and a downfield olefinic proton at δH 7.65  with HMBC correlations to C-2′′ and C-6′′. A 3-methylenedihydrofuranone was identified thanks to the HMBC correlations from H-7, H-8, and H-7′′ to the carbonyl C-9′′ at δC 172.3. The ROE correlations from H-6′′ to H-7 defined the double bond as (E) and those from H-7 to H-5′ and from H-8 to H-2 a trans configuration of H-7 and H-8 protons. This compound should be formed by an 8-6′ coupling between ferulic acid and caffeic acid followed by decarboxylation. Then a 7-8′′ coupling with another ferulic acid followed by cyclization between the acid and C-8 should occur (rel-(E)-3--4-((E)-4-hydroxy-3-meth oxybenzylidene)-3-(4-hydroxy-3-methoxyphenyl)-5-oxotetrahy drofuran-2-yl)phenyl)acrylic acid.The ESI(-)-HRMS of compound 21 displayed a [M – H]ld occur . Compoun1H NMR data of 1, 3, and 17 showed that they share the same aromatic profile: four singlet protons, three protons belonging to a 3,4-dihydroxyphenyl group, and two trans olefinic protons. Compound 1 displayed an ESI(-)-HRMS ion at m/z 507.0943 [M - H]–,  indicating a trimer. The 1H and edited-HSQC indicated that in addition to the aromatic signals, the following signals were observed: an oxymethine proton at δH 4.89  with its OH proton at δH 5.69 , an oxymethine at δH 4.88 , and two methine protons at δH 3.12 , and 2.96 . These protons were linked together scalar coupling from 7-OH to H-7, H-8, H-8′, and H-7′. The 3,4-dihydroxyphenyl group at δH 6.34 , 6.48 , and 6.53  were linked to H-7′ thanks to the HMBC correlations of H-2′ and H-6′ to C-7′ at δC 81.4 (C-7′). The HMBC correlations of H-7, H-8, and H-7′ to the carbonyl at δC 173.5 (C-9) allowed to form butyrolactone. The correlations from H-2 and H-5 to the same sp2 carbon at δC 125 (C-6), 132.2 (C-1), 145 (C-4), and 145.1 (C-3) showed that they belonged to the same aromatic group and the correlation from H-2 to C-7 at δC 65.9 (C-7) linked this 3,4-dihydroxyphenyl group substituted in C-6 to C-7. The correlations from both H-2′′ at δH 6.3  and H-5′′δH 6.39  to the hydroxy sp2 carbon C-4′′ at δC 146.3 and the quaternary carbon C-6′′ at δC 47.7 positioned H-2′′ and H-5′′ on the same ring but also indicated a 2-hydroxycyclohexa-2,5-dien-1-one ring, as confirmed by the correlation of H-5′′ to the carbonyl C-3′′ at δC 181.1. The trans olefinic protons were linked to C-1′′ because of the HMBC correlation of H-8′′ at δH 5.55  with C-1′′ at δC 156.9. Finally, the correlation between H-5 and C-6′′ allowed to establish the structure of 1 , which indicated that the compound obtained was the result of decarboxylation and e fusion of three units of caffeic acid. The NMR data of 3 showed close similarities to those of compound 1, except that the oxymethine proton H-7 with its OH proton and the methine proton H-8 of compound 1 was not present in 3. On the contrary, an additional olefinic proton was detected in 3 at δH 7.53 . The HMBC correlations of this proton to C-8′ at δC 48.6, C-2 at δC 118.1, C-6 at δC 127.1, and C9 at δC 167.5 allowed to position this proton and to elucidate 3 as the new biotransformed form of caffeic acid described in The ESI(-)-HRMS of 3 displayed a [M – H]H 3.65  was observed in 17. The methoxy was positioned in C-3′ thanks to its ROE correlation with H-2′ at δH 6.9 . All the NMR and HRMS data agree with the structure depicted in O-methylspirocaffeic acid trimer.The NMR data of 17 showed close similarities to those of 3, except that a methoxy group at δ– ions at m/z 681.2004 and 681.2005, respectively, corresponding to a molecular formula of C38H33O12 and thus to tetramers. The NMR data of these compounds showed protons corresponding to a 3-methoxy,4-hydoxypenyl group, two meta-coupled aromatic protons, two trans olefinic protons, and an oxymethine and methine protons. These data were thus closed to those of 8-5′-benzofuran diferulic acid (10), except that only one carbonyl was observed at δC 171 for 13 and 171.3 for 14. This carbonyl was assigned to the acid linked to the trans olefinic carbons and thus no acid was detected in C-8. This information, combined with the HRMS, data led to the conclusion that 13 and 14 are symmetric dimers. The 3J coupling constant between H-7 and H-8 was 5.6 Hz for 13 and 3.4 Hz for 14, but both compounds showed ROE correlations from H-8 to H2/H-6, suggesting a trans configuration between H-7 and H-8 in both molecules. The difference should thus be the configuration between H-8a and H-8b  and more deshielded aromatic H-2, H-3, and H-6 protons. Compounds 13 and 14 were named meso-8-5′-benzofuran tetra-ferulic acid and threo-8-5′-benzofuran tetra-ferulic acid, respectively.The ESI(-)-HRMS of compounds 13 and 14 displayed [M – H]and H-8b . The mesBotrytis cinerea secretome  activity. Interestingly, we have found that biotransformation resulted in the generation of several low-potency Wnt inhibition-specific scaffolds  and that the original compounds possessed no specific activity.The biotransformation products were analyzed for their activity against the Wnt pathway . To thisNone of these compounds have ever been reported to possess a Wnt inhibitory activity before. Surprisingly, we did not observe any long-term inhibition of TNBC cell proliferation in a 3-day MTT assay by the Wnt-inhibiting compounds. On the other hand, the majority of the compounds (except 17) that showed toxicity in the Renilla assay (24 h) demonstrated comparatively less potency in inhibition of cell proliferation over 3 days. Combined with a similar trend for compounds inhibiting Wnt signaling, this suggests that these derivatives are sufficiently stable under culture conditions for only 24 h and that the cells recover quickly from the initial impact.1H, COSY, ROESY, HSQC, and HMBC) were recorded in DMSO-d6 or CD3OD. Chemical shifts were reported in parts per million (δ) and coupling constants (J) in Hz. The residual DMSO-d6 signal  and CD3OD signal  were used as internal standards for 1H and 13C NMR, respectively. Trans-caffeic acid  was purchased from Sigma-Aldrich , and trans-ferulic acid  was purchased from Roth .The UV and ECD spectra were recorded on a JASCO J-815 spectrometer  in MeOH or MeCN using a 1-cm cell. Scan speed was set at 200 nm/min in continuous mode between 600 and 190 nm. NMR spectroscopic data were recorded on a Bruker Avance Neo 600 MHz NMR spectrometer equipped with a QCI 5-mm cryoprobe and a SampleJet automated sample changer . One-dimensional (1D) and 2D NMR experiments , and maintained by regular transplanting. The fungus was grown in an oatmeal agar medium (Difco), and conidia were sampled by vacuum aspiration and stored dry at −80°C until use. B. cinerea was cultivated in a 1.5-L liquid medium (in 5 L bottles) at 22°C under alternating light and dark (12 h/12 h). The medium was filtrated through a double layer of folded filter paper . The filtrate was brought to 80% saturation  and centrifuged . The supernatant was discarded, and the resulting pellet was solubilized in nanopure water  (the protein crude extract) and submitted to dialysis  against nanopure water overnight at 4°C. The resulting protein extract was concentrated against polyethylene glycol beads  in the dialyzed tube. Protein content was determined by the method of Bradford, using a Bio-Rad protein assay kit with BSA as a standard. The volume of the extract was adjusted to obtain a final protein concentration of 2 μg/μl. The resulting extract (considered as secretome) was aliquoted to 1 ml and stored at −20°C until use.The biotransformation reactions of caffeic acid, ferulic acid, and the mixture of both were performed on an analytical scale in 2-ml Eppendorf tubes. The total volume of the reactions was 1 ml, with a total substrate concentration of 1 mg/ml (0.5 mg/ml of each compound for the mixture) and an acetone volume of 10% (100 μl). The secretome was always added last, and its amount varied from 1 to 10% (10–100 μl). Water volume was adapted accordingly to fit the total volume (890–800 μl). Once prepared, the mixtures were incubated at room temperature in the dark under constant gentle agitation. Aliquots were collected after 15 min and 5 h and dried under vacuum in a centrifugal evaporator. The reactions were stopped after 24 h by removing the solvent under vacuum in a centrifugal evaporator. A total of 1 ml of MeOH was then added to the dry deposit. The sample was centrifugated, and the supernatants were analyzed by UHPLC-PDA-ELSD-MS.m/z range of 150–1,000 Da. Chromatographic separation was performed on an Acquity UPLC BEH C18 column  at 6 ml/min, 40°C with H2O (A) and MeCN (B), both containing.1% formic acid, as solvents. The gradient was carried out as follows: 5–100% B in 7 min, 1 min at 100% B, and a re-equilibration step at 5% B for 2 min. The ELSD temperature was fixed at 45°C, with a gain of 9. The PDA data were acquired from 190 to 500 nm, with a resolution of 1.2 nm. The sampling rate was set at 20 points/s. Data were processed with the MassLynx software .The crude reaction mixtures were analyzed on an ultra-high-performance liquid chromatography system equipped with a photodiode array, an evaporative light-scattering detector, and a single quadrupole detector by heated electrospray ionization (UHPLC-PDA-ELSD-MS) . ESI parameters were the following: capillary voltage 800 V, cone voltage 15 V, source temperature 120°C, and probe temperature 600°C. The acquisition was performed in negative ionization (NI) mode with an 2O (A) and MeCN (B), both containing 1% formic acid, as solvents. The gradient was carried out as follows: 5–100% B in 7 min, 1 min at 100% B, and a re-equilibration step at 5% B in 2 min. The ionization parameters were the same as those used in The pure compounds were analyzed on a Waters Acquity UHPLC system equipped with a Q-Exactive Focus mass spectrometer , using a heated electrospray ionization source (HESI-II). Chromatographic separation was carried out on an Acquity UPLC BEH C18 column  at 6 ml/min, 40°C with HThe biotransformation reactions were performed on a large scale by keeping the most efficient parameters tested on the analytical scale. The biotransformation reaction of caffeic acid was performed in a 100-ml Schott bottle with 100 mg of starting material and a total volume of 100 ml (1 mg/ml substrate concentration) for 24 h. The percentage of acetone was kept at 10% (10 ml), the amount of secretome was 1% (1 ml), and the water volume was 89 ml. The biotransformation reaction of ferulic acid was performed in a 100-mL Schott bottle with 100 mg of starting material and a total volume of 100 ml (1 mg/ml substrate concentration) for 24 h. The percentage of acetone was kept at 10% (10 ml), the amount of secretome was 1% (1 ml), and the water volume was 89 ml. The biotransformation reaction of caffeic acid/ferulic acid mixture was performed in a 100-ml Schott bottle with 100 mg of starting material (50 mg of caffeic acid and 50 mg of ferulic acid) and a total volume of 100 ml (1 mg/ml substrate concentration) for 24 h. The percentage of acetone was kept at 10% (10 ml), the amount of secretome was 1% (1 ml), and the water volume was 89 ml. The reaction mixtures were incubated at room temperature in the dark under constant gentle magnetic stirring. After evaporation of the solvent with a rotatory evaporator, the dry deposit was resuspended in MeOH and filtered through.45-μm filters . These crude reaction mixtures were analyzed by UHPLC-PDA-ELSD-MS for comparison with analytical scale reactions before moving to gradient optimization and semi-preparative HPLC fractionation.2O (A) and MeOH (B), both containing 1% formic acid, as solvents. The optimized gradient conditions for each reaction were geometrically transferred by gradient transfer to the analytical and semi-preparative HPLC scale (2O (A) and MeOH (B), both containing 1% formic acid, as solvents. UV absorbance was measured at 210 and 254 nm, and UV-PDA spectra were recorded between 190 and 600 nm (step 2 nm). The geometrically transferred gradients were used on the semi-preparative scale in a Shimadzu system equipped with an LC-20 A module pump, an SPD-20 A UV/VIS, a 7725I Rheodyne® valve, and an FRC-40 fraction collector . Separation was performed on a Waters XBridge C18 column  equipped with a Waters C18 pre-column cartridge holder  at 17 ml/min, with H2O (A) and MeOH (B), both containing 1% formic acid, as solvents. UV detection was set at 210 and 254 nm. The mixtures were injected on the semi-preparative HPLC column using a dry load methodology developed in our laboratory  at 4 ml/min, 40°C with Hboratory .tR 14 min), 2 , 3 , 4 , 5 , and 6 .Two semi-preparative HPLC injections of about 40 mg each yielded 50 fractions each. The fractions were analyzed by UHPLC-PDA-ELSD-MS and mixed together according to their composition. Six compounds were obtained pure: 1 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , and 15 .Two semi-preparative HPLC injections of about 40 mg each yielded 45 fractions each. The fractions were analyzed by UHPLC-PDA-ELSD-MS and mixed together according to their composition. Nine compounds were obtained pure: 7 , 17 , 19 , 20 , 21 , and 22 . Two compounds were isolated first as 5  and as 10 , but ring opening occurred during the NMR analysis of 5 that led to compound 23, and decarboxylation occurred during the NMR analysis of 10 that led to compound 24.Two semi-preparative HPLC injections of about 45 mg each yielded 55 fractions each. The fractions were analyzed by UHPLC-PDA-ELSD-MS and mixed according to their composition. Six compounds were obtained pure: 16  δ 2.96 , 3.12 , 4.88 , 4.89 , 5.55 , 5.69 , 5.97 , 6.3 , 6.34 , 6.39 , 6.44 , 6.48 , 6.53 , 7.03 , 8.9 , 9.03 , 9.06 , 9.07 ; 13C NMR  δ 44.6 (C-8), 47.7 (C-6′′), 49.6 (C-8′), 65.9 (C-7), 81.4 (C-7′), 112.2 (C-5), 114.5 (C-5′), 114.8 , 118.7 (C-6′), 120.3 (C-5′′), 125 (C-6), 126.3 (C-1′), 127.8 (C-2′′), 132.2 (C-1), 144.7 (C-3′), 145 (C-4), 145.1 (C-3), 145.9 (C-4′), 146.3 (C-4′′), 156.9 (C-1′′), 173.5 (C-9), 181.1 (C-3′′). ESI(-)-HRMS m/z 507.0943 [M – H]–, . MS/MS spectrum: CCMSLIB00006718008.Spiro caffeic acid trimer (1). UV (MeOH) λmax (log ε) 237 (sh) (4.05), 287 (3.91), 330 (3.59) nm. 1H NMR  δ 6.9 , 7.23 , 7.27 , 9.7 ; 13C NMR  δ 113.9 (C-2), 115.2 (C-5), 124.1 (C-6), 128.4 (C-1), 145.5 (C-3), 151.8 (C-4), 190.6 (C-7). ESI(-)-HRMS m/z 137.0245 [M – H]–, .3,4-Dihydroxybenzaldehyde (2) . 1H NMR 1H NMR  δ 3.73 , 4.89 , 5.74 , 6.03 , 6.22 , 6.43 , 6.54 , 6.6 , 6.73 , 6.87 , 7.02 , 7.53 ; 13C NMR  δ 48.6 (C-8′), 48.9 (C-6′′), 81.5 (C-7′), 113.1 (C-5), 114.7 (C-2′), 115 (C-5′), 118.1 (C-5′′), 118.1 (C-2), 118.6 (C-6′), 123 (C-8), 123.7 (C-1), 126.8 (C-8′′), 127.1 (C-6), 128 (C-1′), 130.8 (C-2′′), 131.8 (C-7), 138.2 (C-7′′), 145 (C-3′), 145.3 (C-3), 146 (C-4′), 147.5 (C-4′′), 148.8 (C-4), 155.2 (C-1′′), 166.3 (C-9′′), 167.5 (C-9), 181 (C-3′′). ESI(-)-HRMS m/z 489.0839 [M – H]–, . MS/MS spectrum: CCMSLIB00006717996.7-Hydroxyspirocaffeic acid trimer (3). UV (MeOH) λmax (log ε) 264 (4.13), 318 (3.83), 350 (3.74) nm. 1H NMR  δ 4.18 , 5.84 , 6.2 , 6.65 , 6.71 , 6.74 , 7 , 7.1 , 7.45 ; 13C NMR  δ 55.6 (C-8), 87 (C-7), 113.2 (C-2), 115.5 (C-5), 115.8 (C-8′), 115.9 (C2′), 116.2 (C-6′), 117.2 (C-6), 127.6 (-1′), 131.2 (C-1), 144.3 (C-7′), 145.3 (C3), 145.5 (C-4), 148.7 (C-4′), 167.7 (C-9′), 171.7 (C-9). ESI(-)-HRMS m/z 357.0611 [M – H]–, . MS/MS spectrum: CCMSLIB00006718010.8-5′-Benzofuran-dicaffeic acid (4) . 1H NMR 1H NMR  δ 4.03 , 5.66 , 6.72 , 6.76 , 6.8 , 9.09 , 9.2 ; 13C NMR  δ 48.3 , 81.9 , 113.7 , 115.7 , 117.4 , 129.1 , 145.5 , 146.1 , 175.3 . ESI(-)-HRMS m/z 357.0621 [M – H]–, . MS/MS spectrum: CCMSLIB00006718000.Dehydrodicaffeic acid dilactone (5) . 1H NMR rel-(2E)-3-[(2S)-2-[(R)-hydroxymeth yl]-1,3-benzodioxol-5-yl]-2-propenoic acid (6)  δ 4.64 , 5.79 , 6.26 , 6.35 , 6.68 , 6.83 , 6.85 , 7.08 , 7.25 , 7.47 ; 13C NMR  δ 72.3 (C-7), 105.8 (C-2′), 107.6 (C-5′), 112.8 (C-8), 114.7 (C-2), 115 (C-5), 116.6 (C-8′), 118.2 (C-6), 124.2 (C-6′), 128.1 (C-1′), 129.8 (C-1), 143.9 (C-7′), 144.8 (C-4), 148.5 (C-3′), 149.6 (C-4′), 167.8 (C-9′). ESI(-)-HRMS m/z 329.0671 [M – H]–, . MS/MS spectrum: CCMSLIB00006717997.acid (6) . UV (MeOtrans-8-8-γ-Lactone-diferulic acid (7)  δ 3.82 , 3.88 , 4.16 , 5.71 , 6.77 , 6.8 , 6.85 , 6.89 , 7.14 , 7.29 , 7.65 ; 13C NMR  δ 55.3 (C-8), 56.4 (3O CH3), 56.5 (3′O CH3), 83 (C-7), 110.2 (C-2), 114 (C-2′), 116.5 (C-5), 116.6 (C-5′), 119.3 (C-6), 120.6 (C-8′), 126.8 (1′), 127.2 (C-6′), 132.6 (C-1), 141.8 (C-7′), 148.3 (C-4), 149.3 (C-3), 149.4 (C-3′), 150.9 (C-4′), 174.1 (C-9). ESI(-)-HRMS m/z 385.0932 [M – H]–, . MS/MS spectrum: CCMSLIB00006718011.7,8-acid (7) . 1H NMR cis-8-8-γ-Lactone-diferulic acid (8).1H NMR  δ 3.87 , 3.89 , 4.51 , 5.72 , 6.79 , 6.86 , 6.91 , 7.06 , 7.12 , 7.25 , 7.57 ; 13C NMR  δ 54.2 (C-8), 55.9 (3OCH3), 56.3 (3′OCH3), 81.6 (C-7), 110.8 (C-2), 113.4 (C-2′), 115.5 (C-5), 116.2 (C-5′), 120.3 (C-6), 123.0 (C-8′), 126.8 (C-6′), 127.1 (C-1′), 128.1 (C-1), 139.6 (C-7′), 147.7 (C-4), 148.5 (C-3), 149 (C-3′), 150.4 (C-4′), 173.3 (C-9), 174.2 (C-9′). ESI(-)-HRMS m/z 385.0932 [M – H]–, . MS/MS spectrum: CCMSLIB00006718012.7,8-1H NMR  δ 3.8 , 4.2 , 5.73 , 6.8 , 6.87 , 7 , 9.26 ; 13C NMR  δ 48.1 , 55.8 , 82.1 , 110.6 , 115.4 , 119.2 , 129 , 147.4 , 147.9 , 175.4 . ESI(-)-HRMS m/z 385.0933 [M – H]–, . MS/MS spectrum: CCMSLIB00006717999.Dehydrodiferulic acid dilactone (9) .1H NMR  δ 3.83 , 3.91 , 4.31 , 6.02 , 6.36 , 6.8 , 6.85 , 6.96 , 7.2 , 7.27 , 7.63 ; 13C NMR  δ 56.4 (3OCH3), 56.8 (3′OCH3), 56.9 (C-8), 89.1 (C-7), 110.6 (C-2), 113.9 (C-2′), 116.3 (C-5), 116.9 (C-8′), 119.2 (C-6′), 119.9 (C-6), 128.2 (C-5′), 130 (C-1′), 132.9 (C-1), 146.1 (C-3′), 146.5 (C-7′), 148.1 (C-4), 149.3 (C-3), 151.4 (C-4′), 170.8 (C-9′), 174.0 (C-9). ESI(-)-HRMS m/z 385.0933 [M – H]–, . MS/MS spectrum: CCMSLIB00006717995.8-5′-Benzofuran-diferulic acid (10) . 1H NMR 1H NMR  δ 3.68 , 3.78 , 3.84 , 4.95 , 6 , 6.33 , 6.37 , 6.74 , 6.77 , 6.94 , 6.96 , 7.03 , 7.08 , 7.14 , 7.16 , 7.52 , 7.55 ; 13C NMR  δ 56.4 , 56.5 (3′′OCH3), 75.8 (C-7), 106.3 (C-8), 112.6 , 112.6 (C-2), 115.7 (C-5), 118.5 (C-8′), 118.6 (C-8′′), 120.2 (C-5′), 120.8 (C-5′′), 121.7 (C-6), 122.6 (C-6′), 122.7 (C-6′′), 131.6 (C-1′), 131.8 (C-1′′), 132.2 (C-1), 145.6 , 147.4 (C-4), 148.7 (C-3), 149.1 (C-4′), 149.3 (C-4′′), 152.1 (C-3′), 152.3 (C-3′′), 170.8 . ESI(-)-HRMS m/z 551.1558 [M – H]–, . MS/MS spectrum: CCMSLIB00006717991.-3,3′-[[2-hydroxy-2-(4-hydroxy-3-methoxyphenyl) ethylidene]bis[oxy]]bis-2-Propenoic acid (11) . UV -1,3a,4,9b-tetrahydro-1,4-bis(4-h ydroxy-3-methoxyphenyl)-6-methoxy-3-oxo-3H-furo[1] benzopyran-8-yl]-2-Propenoic acid (12)  δ 3.5 , 3.83 , 3.85 , 3.87 , 3.89 , 5.23 , 5.42 , 6.18 , 6.51 , 6.8 , 6.9 , 6.92 , 7.07 , 7.09 , 7.11 , 7.4 ; 13C NMR  δ 44.7 (C-8), 46.2 (C-8′′), 75.8 (C-7′′), 88.5 (C-7), 110.6 (C-2′), 111.7 (C-2), 112.3 (C-2′′), 116 (C-5′′), 116.4 (C-5), 118 (C-8′), 121.6 (C-6), 121.6 (C-6′′), 122.2 (C-5′), 123.1 (C-6′), 129.1 (C-1′), 129.9 (C-1), 130.1 (C-1′′), 145.7 (C-7′), 147.5 (C-4′), 148.2 (C-4′′), 148.9 (C-4), 149 (C-3′′), 149.6 (C-3), 150.7 (C-3′), 170.8 (C-9′), 176 (C-9′′). ESI(-)-HRMS m/z 533.1455 [M – H]–, . MS/MS spectrum: CCMSLIB00006717994.(2E)-cid (12) . UV (MeCmeso-8-5′-Benzofuran tetra-ferulic acid (13). UV (MeOH) λmax (log ε) 234 (sh) (4.53), 290 (4.39), 324 (4.44) nm. 1H NMR  δ 3.78 , 3.91 , 4.06 , 5.45 , 6.2 , 6.68 , 6.78 , 6.8 , 6.82 , 7.2 , 7.54 ; 13C NMR  δ 54.4 (C-8), 56.4 (3OCH3), 56.8 (3′OCH3), 89.5 (C-7), 110.9 (C-2), 114.1 (C-2′), 116.5 (C-5), 117.2 (C-8′), 118.7 (C-6′), 120.3 (C-6), 130.2 (C-1′), 130.4 (C-5′), 133.3 (C-1), 146.1 (C-3′), 146.3 (C-7′), 148.3 (C-4), 149.3 (C-3), 152.3 (C-4′), 171 (C-9′). ESI(-)-HRMS m/z 681.2004 [M – H]–, . MS/MS spectrum: CCMSLIB00006717998.threo-8-5′-Benzofuran tetra-ferulic acid (14). UV (MeOH) λmax (log ε) 232 (sh) (4.61), 289 (4.48), 323 (4.52) nm. 1H NMR  δ 3.64 , 3.98 , 4.07 , 5.44 , 6.23 , 6.33 , 6.42 , 6.63 , 7.12 , 7.23 , 7.56 ; 13C NMR  δ 56.2 (C-8), 56.3 (3OCH3), 56.9 (3′OCH3), 88.3 (C-7), 109.6 (C-2), 113.8 (C-2′), 116.1 (C-5), 117.9 (C-8′), 118.5 (C-6), 119.4 (C-6′), 130.2 (C-5′), 130.5 (C-1′), 134.3 (C-1), 145.8 (C-7′), 146.2 (C-3′), 147.5 (C-4), 149 (C-3), 152.3 (C-4′), 171.3 (C-9′). ESI(-)-HRMS m/z 681.2005 [M – H]–, . MS/MS spectrum: CCMSLIB00006717993.rel-(2E)-3-oxy]-3-meth oxyphenyl]-2-propenoic acid (15)  δ 3.74 , 3.86 , 3.95 , 5.76 , 5.91 , 6.34 , 6.43 , 6.7 , 6.75 , 6.76 , 7.02 , 7.16 , 7.2 , 7.29 , 7.3 , 7.61 , 7.63 ; 13C NMR  δ 56.4 (3OCH3), 56.5 (3′′OCH3), 56.9 (3′OCH3), 87.9 (C-8), 92 (C-7), 110.4 (C-2), 112.7 (C-2′′), 114.8 (C-2′), 116.5 (C-5), 117.5 (C-8′), 118.6 (C-8′′), 119.6 (C-6), 120.2 (C-5′′), 120.6 (C-6′), 123.0 (C-6′′), 128 (C-5′), 130.3 (C-1′), 131.1 (C-1), 131.6 (C-1′′), 145.7 (C-7′′), 146.1 (C-7′), 146.5 (C-3′), 148.1 (C-4), 149.3 (C-3), 149.6 (C-4′′), 152.6 (C-3′′), 153.2 (C-4′), 170.8 (C-9′′), 170.9 (C-9′). ESI(-)-HRMS m/z 533.1455 [M – H]–, . MS/MS spectrum: CCMSLIB00006717992.cid (15) . UV (MeOrel-(E)-3--4-((R)-hydroxy(4-hy droxy-3-methoxyphenyl)methyl)-2-(4-hydroxy-3-methoxyphen yl)-5-oxotetrahydrofuran-3-yl)phenyl)acrylic acid (16). UV (MeOH) λmax (log ε) 232 (sh) (4.37), 284 (4.15), 328 (3.98) nm. 1H NMR  δ 3.67 , 3.71 , 3.73 , 3.78 , 5.01 , 5.14 , 5.71 , 6.35 , 6.47 , 6.57 , 6.64 , 6.73 , 6.79 , 6.82 , 6.97 , 7.19 ; 13C NMR  δ 48.3 (C-8), 56.1 (3′′OCH3), 56.4 (3OCH3), 56.4 (C-8′′), 74.3 (C-7′′), 88.9 (C-7), 109.9 (C-2), 111.3 (C-2′′), 113.7 (C-2′), 114.8 (C-5′), 115.6 (C-5), 115.8 (C-5′′), 117.8 (C-8′), 120.7 (C-6′′), 121.2 (C-6), 127.2 (C-1′), 129.3 (C-1), 130.9 (C-6′), 133.3 (C-1′′), 143.2 (C-7′), 145.8 (C-3′), 147.2 (C-4′′), 148.3 (C-4), 148.6 (C-3′′), 149.4 (C-4′), 149.4 (C-3), 170.7 (C-9′), 178.4 (C-9′′). ESI(-)-HRMS m/z 537.1401 [M – H]–, . MS/MS spectrum: CCMSLIB00006718002.O-Methylspirocaffeic acid trimer (17). UV (MeOH) λmax (log ε) 237 (sh) (4.72), 267 (4.76), 318 (4.48), 356 (4.35) nm. 1H NMR  δ 3.65 , 3.96 , 4.97 , 5.71 , 6.05 , 6.21 , 6.53 , 6.63 , 6.71 , 6.9 , 6.93 , 7.03 , 7.53 , 9.11 , 9.17 , 9.36 , 9.67 , 12.57 ; 13C NMR  δ 55.2 (3′OCH3), 48.8 (C-6′′), 48.5 (C-8′), 81.6 (C-7′), 111.1 (C-2′), 112.9 (C-5), 114.3 (C-5′), 117.8 (C-2), 118.3 (C-5′′), 120.8 (C-6′), 123.6 (C-1), 125.6 (C-8′′), 126.8 (C-6), 127.4 (C-1′), 130.8 (C-2′′), 131 (C-7), 138.8 (C-7′′), 145 (C-3), 147 (C-4′), 147.2 , 148.6 (C-4), 154.6 (C-1′′), 165.9 (C-9′′), 167.2 (C-9), 180.7 (C-3′′). ESI(-)-HRMS m/z 503.0981 [M – H]–, . MS/MS spectrum: CCMSLIB00006718001.2′-1H NMR  δ 3.75 , 4.17 , 5.92 , 6.18 , 6.76 , 6.79 , 6.96 , 6.97 , 7.11 , 7.44 .5-[(E)-2-carboxyvinyl]-7-hydroxy-2-(4-hydroxy-3-methoxyp henyl)-2,3-dihydro-1-benzofuran-3-carboxylic acid (8-5′-ben zofuran-caffeic-ferulic acid dimer) (18). 1H NMR  δ 3.83 , 6.25 , 6.77 , 6.92 , 6.96 , 7.12 , 7.19 , 7.39 , 7.44 ; 13C NMR  δ 55.5 (3OCH3), 109.7 (C-2), 112.4 (C-2′), 115.6 , 118 (C-6′), 119.8 (C-6), 119.9 (C-8), 124.9 (C-1′), 125 (C-5′), 129 (C-7), 129.2 (C-1), 144.6 (C-7′), 145.7 (C-4′), 146.5 (C-4), 147.8 (C-3), 168 (C-9′). ESI(-)-HRMS m/z 327.0872 [M – H]–, . MS/MS spectrum: CCMSLIB00006718006.(E)-3--4-hydroxy-3-methoxystyryl)phe nyl)acrylic acid (19). UV (MeOH) λmax (log ε) 240 (sh) (4.1), 294 (4.12), 321 (4.17) nm. 1H NMR  δ 3.8 , 4.08 , 4.15 , 5.67 , 5.72 , 6.71 , 6.77 , 6.79 , 6.81 , 6.86 , 6.99 ; 13C NMR  δ 48.1 (C-8′), 48.2 (C-8), 55.8 (3′OCH3), 81.9 (C-7), 82.1 (C-7′), 110.6 (C-2′), 113.7 (C-2), 115.4 (C-5′), 115.7 (C-5), 117.3 (C-6), 119.3 (C-6′), 129 (C-1′), 129.1 (C-1), 145.5 (C-3), 146.1 (C-4), 147.4 (C-4′), 147.9 (C-3′), 175.3 (C-9), 175.4 (C-9′). ESI(-)-HRMS m/z 371.0769 [M – H]–, . MS/MS spectrum: CCMSLIB00006718007.Dehydrocaffeicferulic acid dilactone (20). rel-(E)-3--4-((E)-4-hydroxy-3-met hoxybenzylidene)-3-(4-hydroxy-3-methoxyphenyl)-5-oxotetrah ydrofuran-2-yl)phenyl)acrylic acid (21). UV (MeOH) λmax (log ε) 237 (sh) (4.31), 288 (4.16), 328 (4.12) nm. 1H NMR  δ 3.51 , 3.68 , 4.38 , 5.61 , 6.15 , 6.57 , 6.64 , 6.71 , 6.71 , 6.85 , 7.01 , 7.11 , 7.62 , 7.65 ; 13C NMR  δ 51.7 (C-7), 55.2 (3′′OCH3), 55.4 (3OCH3), 82.3 (C-8), 111.5 (C-2), 112 (C-5′), 113.6 , 115.5 (C-5′′), 116.3 (C-5), 118.1 (C-8′), 118.8 (C-6), 122.9 (C-1′), 124.5 (C-1′′), 126.1 (C-6′′), 131 (C-1), 131.2 (C-6′), 139.2 (C-7′′), 139.9 (C-7′), 145.6 (C-3′), 145.8 (C-4), 147.4 (C-3′′), 147.7 (C-3), 148 (C-4′), 149.2 (C-4′′), 167.4 (C-9′), 172.3 (C-9′′). ESI(-)-HRMS m/z 519.1294 [M – H]–, . MS/MS spectrum: CCMSLIB00006718003.rel-(E)-3--1--4-(4-hyd roxy-3-methoxyphenyl)-6-methoxy-3-oxo-1,3a,4,9b-tetrahydro-3H-furochromen-8-yl)acrylic acid (22). UV (MeOH) λmax (log ε) 236 (sh) (4.29), 288 (4.15), 322 (4.05) nm. 1H NMR  δ 3.56 , 3.75 , 3.78 , 3.81 , 5.29 , 5.44 , 6.26 , 6.45 , 6.76 , 6.78 , 6.84 , 6.85 , 6.91 , 7.04 , 7.24 , 7.28 ; 13C NMR  δ 42.2 (C-8), 43.7 (C-8′′), 55.6 (3′OCH3), 55.7 (3′′OCH3), 73.5 (C-7′′), 85.7 (C-7), 109.2 (C-2′), 111.8 (C-2′′), 114.6 (C-2), 115.1 (C-5′′), 115.6 (C-5), 117.4 (C-8′), 118.7 (C-6), 120.5 (C-6′′), 120.8 (C-5′), 121.7 (C-6′), 127 (C-1′), 128 (C-1), 128.3 (C-1′′), 143.7 (C-7′), 145.6 (C-4′), 146.2 (C-4), 146.9 (C-4′′), 147.4 (C-3′′), 148.8 (C-3′), 167.7 (C-9′), 173.6 (C-9′′). ESI(-)-HRMS m/z 519.1291 [M – H]–, . MS/MS spectrum: CCMSLIB00006718005.rel--2--4-[methylene]tetrahydro-5-oxo-3-furancarboxylic acid = phe llinsin A (23)  δ 4.41 , 5.66 , 6.64 , 6.7 , 6.79 , 6.81 , 6.97 , 7.03 , 7.36 ; 13C NMR  δ 51.5 (C-8), 79 (C-7), 113.5 (C-2), 115.1 (C-5), 115.8 (C-5′), 117.3 (C-6), 117.5 (C-2′), 121.5 (C-8′), 123.2 (C-6′), 125.1 (C-1′), 126.5 (C-1), 137.6 (C-7′), 144.9 (C-3), 145.3 (C-4), 145.6 (C-3′), 148.4 (C-4′), 170.3 (C-9), 171.2 (C-9′). ESI(-)-HRMS m/z 357.0617 [M – H]–, . MS/MS spectrum: CCMSLIB00006718013.n A (23) . 1H NMR 1H NMR  δ 3.83 , 3.87 , 6.47 , 6.75 , 6.97 , 7.12 , 7.21 , 7.52 , 7.53 ; 13C NMR  δ 55.5 (3OCH3), 56.1 (3′OCH3), 109 (C-2′), 109.7 (C-2), 115.7 (C-5), 116.2 (C-8′), 119.4 (C-8), 119.6 (C-6′), 119.9 (C-6), 125.4 (C-1′), 129.4 (C-7), 130.5 (C-1), 144.7 (C-7′), 145.9 (C-4′), 146.5 (C-4), 147.8 (C-3), 148 (C-3′), 167.8 (C-9). ESI(-)-HRMS m/z 341.1029 [M – H]–, . MS/MS spectrum: CCMSLIB00006718004.(2E)-3-[4-Hydroxy-3-[(1E)-2-(4-hydroxy-3-methoxyphenyl) ethenyl]-5-methoxyphenyl]-2-propenoic acid = poacic acid (24) . UV -2-carb oxy-ethenyl]-3-2,3-dihydro-2-]3,4-dihy-droxyphe-nyl]-(E)-2-propenoic acid, the trans -3-[4-[[6-[(E)-2-carboxy-ethenyl]-3--2,3 -dihydro-2-]oxy]-3-hydroxyphenyl](E)-2-pro penoic acid obtained by R,3S)-6-[(1E)-2-Carboxyethenyl]-3--2,3-dihydro-1,4-benzodioxin-2-carboxylic acid described by It is well known in the literature that biotransformation reactions carried out on caffeic acid, ferulic acid, or the mixture of both with purified enzymes can generate very different compounds depending on the experimental conditions used. Indeed, conditions such as nature of the solvent or co-solvent and its proportion, concentration of the starting material, the value of pH , or presMore surprisingly, our reactions allowed us to biosynthesize 11 new phenylpropanoid derivatives, including 4 novel scaffolds. Several hypotheses could explain this like the experimental conditions used  or the use of an enzymatic secretome instead of a purified enzyme. Indeed, this enzymatic pool could explain that certain reactions are not only due to laccases but other enzymes. A more detailed study of this precise point will be carried out.Careful monitoring of the biotransformation reactions by UHPLC coupled to a triple detection system (ELSD-PDA-MS) allowed us to find the best reaction conditions. On the other hand, an innovative compound isolation process  allowed for the isolation of a large number of compounds including the minor ones.From a drug discovery point of view, the advantages of this approach to generate libraries of natural product derivatives seem obvious to us: the reactions can be easily carried out using a mixture of enzymes  as a catalyst, which is non-toxic and environmentally friendly. Furthermore, there is no need to use a cofactor, since laccases catalyze the process using oxygen as an oxidant . FinallySome of the generated compounds have complex structures, and some of the new structural skeletons are described here for the first time. This exemplifies the potential of this approach for the chemo-diversification of common NPs leading to the generation of libraries of unusual derivatives for biological screening. Having reproducible reactions allows a particular compound to be reproduced in case of interesting biological activities and the need for further studies.While the starting phenylpropanoids (caffeic acid and ferulic acid) showed no specific Wnt inhibition activity, compounds 3, 6, 14, 21, 23, and 24 exhibited a moderate Wnt inhibition that has never been reported. However, the lack of long-term inhibition of TNBC cell proliferation in the compounds and the potential relationship of this to their instability suggest that this method can be considered as an excellent first step in the identification of an active pharmacophore, but that the products require further optimization to become stable, selective, and efficient.https://doi.org/10.26037/yareta:gccdworqzfejhffuukqr4moexu. The MS/MS spectrum of each isolated compound has its own accession number from CCMSLIB00006718008 to CCMSLIB00006718013 on the Global Natural Product Social Molecular Networking (GNPS) (accessed via: https://gnps.ucsd.edu/ProteoSAFe/static/gnps-splash.jsp).The raw data supporting the conclusions of this article are available on the UNIGE website Yaretta at doi: EFQ, KG, RH, SS, LM, AK, and VK contributed to the conception and design of the study. RH, LM, EM, and AK organized and performed the experiments. EFQ, KG, SS, LM, RH, and AK performed the data analysis. LM, RH, AK, VK, J-LW, KG, SS, and EFQ wrote the first draft of the article and edited the further versions. All authors read, reviewed, and approved the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Dihydrocarvone-hybrid derivatives have been synthesized under sonication conditions and characterized by FTIR, NMR, and HRMS. Antifungal efficacy against both strains of M. fructicola was determined by half maximal effective concentration (EC50) and toxicity using the brine shrimp lethality test (BSLT). Among the synthesized compounds, 7 and 8 showed the best activity against both strains of M. fructicola with EC50 values of 148.1 and 145.9 µg/mL for strain 1 and 18.1 and 15.7 µg/mL for strain 2, respectively, compared to BC 1000®  but lower than Mystic® 520 SC. However, these compounds showed low toxicity values, 910 and 890 µg/mL, respectively, compared to Mystic® 520 SC, which was highly toxic. Based on the results, these hybrid compounds could be considered for the development of more active, less toxic, and environmentally friendly antifungal agents against phytopathogenic fungi.The aim of this study was to synthesize a series of novel and known dihydrocarvone-hybrid derivatives ( Monilinia fructicola (G. Winter) Honey , is a leading constraint in world stone fruit production D22 = −1.76 ; FTIR (υmax cm−1): 2949, 2360, 1730; 1450; 1290. 1H NMR : δ 7.44 ; 7.28 ; 7.21 ; 6.88 ; 5.00 ; 4.67 ; 3.82 ; 3.55 ; 2.36 ; 2.10–1.79 ; 1.87–1.62 ; 1.82 ; 1.17 . 13C NMR : δ 205.88 (C-1); 158.1 (C-3′); 146.4 (C-7); 130.0 (C-1′); 129.9 (C-5′); 129.4 (C-7′); 124.7 (C-2′); 120.0 (C-6′); 114.9 (C-8); 110.3 (C-4′); 55.4 (OCH3); 46.0 (C-3); 45.4 (C-6); 28.3 (C-5); 27.0 (C-4); 21.5 (C-9); 15.9 (C-10). HRMS-ESI: [M + 1]+ 271.1676 .(3S)-3-isopropenyl-2-(2-methoxybenzylidene)-6-methylcyclohexanone (4): Yellow oil. [α]D22 = −0.92 ; FTIR (υmax cm−1): 2954, 2357, 1712, 1454, 1310. 1H NMR : δ 7.60 ; 7.27 ; 6.96 ; 6.93 ; 6.89 ; 5.01 ; 4.71 ; 3.79 ; 3.66 ; 2.38–1.82 ; 1.86 ; 1.46–1.28 ; 1.25 . 13C NMR : δ 206.4 (C-1); 159.9 (C-4′); 144.9 (C-2); 140.2 (C-7); 137.3 (C-1′); 136.3 (C-2′); 129.5 (C-6′); 122.4 (C-7′); 115.6 (C-8); 114.3 (C-5′); 109.7 (C-3′); 55.2 (OCH3); 45.8 (C-3); 44.9 (C-6); 29.7 (C-5); 29.7 (C-4); 21.5 (C-9); 15.9 (C-10). HRMS-ESI: [M + 1]+ 271.1685 .(3S)-3-isopropenyl-2-(3-methoxybenzylidene)-6-methylcyclohexanone (5): Yellow oil. [α]D22 = −0.86 ; FTIR (υmax cm−1): 2918, 2360, 1734, 1500, 1297. 1H NMR : 1H NMR : δ 7.60 ; 7.26 ; 6.90 ; 5.01 ; 4.71 ; 3.78 ; 3.65 ; 2.03 ; 1.91–1.81 ; 1.87 ; 1.45-1.28 ; 1.25 . 13C NMR : δ 203.2 (C-1); 159.4 (C-5′); 145.8 (C-2); 139.1 (C-7); 136.5 (C-1′); 129.3 (C-3′ and C-7′); 122.6 (C-2′); 115.1 (C-8); 113.9 (C-4′ and C-6′); 55.3 (OCH3); 44.9 (C-3); 31.9 (C-6); 29.7 (C-5); 29.3 (C-4); 21.5 (C-9); 14.1 (C-10). HRMS-ESI: [M + 1]+ 271.1679 .(3S)-3-isopropenyl-2-(4-methoxybenzylidene)-6-methylcyclohexanone (6): Yellow oil. [α]D22 = −1.15 ; FTIR (υmax cm−1): 2943, 2360, 1701, 1502, 1305. 1H NMR : 1H NMR : δ 7.67 ; 7.26 ; 7.00 ; 6.87 ; 5.02 ; 4.75 ; 3.90 ; 3.85 ; 3.66 ; 2.01 ; 1.92–1.28 ; 1.89 ; 1.25 . 13C NMR : δ 203.3 (C-1); 150.1 (C-4′); 148.6 (C-5′); 145.5 (C-2); 140.1 (C-7); 134.7 (C-1′); 128.0 (C-2′); 123.4 (C-7′); 114.9 (C-8); 113.0 (C-6′); 110.9 (C-3′); 55.9 (OCH3); 55.7 (OCH3); 44.9 (C-3); 32.5 (C-6); 29.7 (C-5); 29.3 (C-4); 21.5 (C-9); 14.1 (C-10). HRMS-ESI: [M + 1]+ 301.1806 .(3S)-2--3-isopropenyl-6-methylcyclohexanone (7): Dark yellow oil. [α]D22 = -3.72 ; FTIR (υmax cm−1): 2893, 2359, 1684, 1489. 1H NMR : δ 7.58 ; 6.91 ; 6.85 ; 6.79 ; 5.99 ; 4.98 ; 4.70 ; 3.66 ; 2.03 ; 1.91–1.28 ; 1.89 ; 1.25 . 13C NMR : δ 203.3 (C-1); 148.4 (C-5′); 147.7 (C-4′); 145.4 (C-2); 139.5 (C-7); 136.6 (C-1′); 129.3 (C-2′); 125.0 (C-7′); 115.4 (C-8); 109.7 (C-6′); 108.4 (C-3′); 101.4 (OCH2O); 44.7 (C-3); 32.5 (C-6); 29.7 (C-5); 29.3 (C-4); 21.4 (C-9); 14.1 (C-10). HRMS-ESI: [M + 1]+ 285.1480 .(3S)-2--3-isopropenyl-6-methylcyclohexanone (1) (3.28 mmol) and commercial benzaldehyde G and H  and Yb(OTf)3  was taken in 100 mL round-bottom flask. The mixture was irradiated in the water bath of an ultrasonic cleaner at 55–65 °C for 5 h. After the reaction was complete, the system was cooled to r.t.; then, the reaction was diluted with 10 mL alcohol and 15 mL deionized water, and the contents were stirred for 10 min. Later, the mixture was extracted with EtOAc (3 × 30 mL). The catalyst remaining in the aqueous phase was recovered by removing the water by heating and then drying under reduced pressure at 100 °C for 2 h and the organic layer was dried with Na2CO3, filtered, and separated with column chromatography using a hexane/EtOAc mixture increased polarity, obtaining compounds 8 and 9 in yields between 45.2% and 53.7%.A mixture of dihydrocarvone (8): Orange viscous oil. [α]D22 = −1.42 ; FTIR (υmax cm−1): 3310, 2924, 2362, 1695, 1593. 1H NMR : δ 7.41 ; 7.26 ; 7.21 ; 6.86 ; 6.81 ; 4.99 ; 4.65 ; 3.66 ; 2.35 ; 2.02 ; 1.86–1.77 ; 1.64 ; 1.33-1.25 ; 1.17 . 13C NMR : δ 206.4 (C-1); 155.7 (C-4′); 145.9 (C-2); 141.6 (C-7); 137.0 (C-1′); 134.9 (C-2′); 129.5 (C-6′); 121.7 (C-7′); 116.4 (C-5′); 115.7 (C-8); 115.1 (C-3′); 45.8 (C-3); 45.5 (C-6); 28.1 (C-5); 27.0 (C-4); 21.5 (C-9); 16.0 (C-10). HRMS: [M + 1]+ 257.1534 .(3S)-2-(3-hydroxybenzylidene)-3-isopropenyl-6-methylcyclohexanone (9): Brown viscous oil. Spectroscopic data of compound 9 were consistent with those reported in the literature [(3S)-2-(4-hydroxybenzylidene)-3-isopropenyl-6-methylcyclohexanone (terature .M. fructicola were kindly provided by the phytopathology laboratory of the Servicio Agrícola y Ganadero de Chile (SAG), Santiago, Chile. The isolate S1 was recovered from infected peach from commercial orchards in the O’Higgins region, Chile, and the isolate S2 was recovered from infected nectarines from commercial orchards in the province of Maipo, Metropolitan Region, Chile and identified to species level with a PCR assay developed by the molecular biology laboratory belonging to SAG; these were maintained on potato dextrose agar (PDA) and incubated for 120 h at 24 °C.Both strains (S1 and S2) of 2–9) against both strains of M. fructicola was determined by the radial test previously reported [v/v) and added to the potato dextrose agar medium (PDA) medium in the petri dishes to obtain a final concentration of 10, 25, 50, 150, and 250 µg/mL. The mycelial growth diameters were measured after 120 h of incubation at 24 °C in the dark, and the inhibition percentages were calculated with respect to the negative ethanol control. The commercial fungicides Mystic® 520 SC   and BC-1000® (grapefruit seed and pulp extract (Citrus x paridisi), 50% w/v (500 g/L)) were used as positive controls and were measured under the same conditions as the compounds.The antifungal activity of all dihidrocarvone-hybrid derivatives (reported . The tes50) that reduced mycelial growth by 50%. This value was determined by regressing the values of the percentage inhibition of radial growth against the compound concentration values. The fit analysis was performed using the Origin Pro® V. 8.0 software  [n = 6). Significant differences were determined using a one-way analysis of variance, followed by a pairwise comparison of means  (Microsoft Office Excel® 2016).The results were expressed as the effective concentration  . These eArtemia salina L. eggs were inserted into a box containing seawater; the box was placed under a UV lamp; after 48 h, the eggs hatched into larvae and were ready for the test. All of the compounds were dissolved in methanol at final concentrations of 1000 to 10 µg/mL. After 24 h, the live and dead shrimp were counted. The experiment was conducted in triplicate. The median lethal concentrations (LD50) with 95% confidence intervals were determined using the Probit analysis method. For LC50 values, probit analysis was used with Minitab V. 15 software .The assay was carried out according to the principle and protocol previously described ,48. BrieM. fructicola. It can also be concluded that the choice of acidic or basic medium is key in condensation due to the nature of the aldehyde substituent, which plays a key role since the ultrasound-assisted one-step protocol led to better yield of benzylidene-cycloalkanones. There is a difference in sensitivity to these compounds, with strain 2 being more sensitive than strain 1. Furthermore, compounds 7 and 8 showed the major antifungal effect of hybrid derivatives for both strains. These new compounds could be successful in the control of other fungal phytopathogens closely related to members of the Sclerotiniaceae family such as Botrytis cinerea, B. aclada, Sclerotinia sclerotiorum, S. cepivorum, and Ciborinia camelliae. Future studies could validate dihydrocarvone-hybrid derivatives as useful new fungicides for fruit production industry.The results suggest that studied dihydrocarvone-hybrid derivatives have potential as new antifungal agents against


N-methyl-acridone, xanthone, and thioxanthone was investigated, leading to the acridone- and xanthone-derived products methyl 2-methoxy-2-(10-methylacridin-9 (10H)-ylidene)acetate (7) and methyl 2-methoxy-2-(9H-xanthen-9-ylidene)acetate (10) in low yields with the de-methoxylated product methyl 2-(10-methylacridin-9 (10H)-ylidene)acetate (8) and the reduced compound methyl 2-methoxy-2-(9H-xanthen-9-yl)acetate (11) as the major products from N-methyl acridone and xanthone. From thioxanthone, only the rearrangement and reduction products (14) and (15) resulted. The photophysical properties of compounds (7), (8), and (10) were investigated in the presence and absence of the Brønsted acid TFA by NMR, UV–VIS absorption, and fluorescence spectroscopy. Protonation of the acridone-derived alkenes (7) and (8) led to strong bathochromic and hyperchromic fluorescence shifts and a substantial increase in Stokes shift. The photooxygenation experiments with these substrates showed an unusual reactivity pattern in the singlet oxygen processes: whereas the electron-rich enolether (7) was chemically unreactive, (8) and (10) were oxidatively cleaved, presumably via intermediate 1,2-dioxetanes.A synthetic route to new heterocyclic 1,1-donor–acceptor-substituted alkenes starting with  Subsequr oxygen  and lumir oxygen ,8 to perr oxygen ,10,11, wr oxygen ,13. In or oxygen ,15,16. F opening . Besides opening ,19. El-Sayed’s rule  cycloaddition with singlet oxygen and adamantylidene adamantine  cycloadditions and the oxidative cleavage products, acridone and xanthone, both highly fluorescent ketones. That this substitution pattern allows efficient dioxetane formation has already been shown for non-heterocyclic tetrasubstituted alkenes  cycloaddition with singlet oxygen (The photooxygenation of the xanthenylidene derivative (t oxygen . Compari7) and (8) towards singlet oxygen is therefore an interesting topic. While (7) possesses a geminal acceptor–donor structure, (8) only has an electron-withdrawing ester group that is expected to deactivate the carbon–carbon double bond in its reaction with singlet oxygen.The photooxygenation of other acridinylidene derivatives has already been proven to be a viable option for clearly visible chemiluminescence . Investi7) did not react with singlet oxygen within the standard reaction time of 12 h (1H-monitored photooxygenation of (8) is depicted in 6) with significant signals at 3.92, 7.31, 7.55, 7,74, and 8.58 ppm. The corresponding second cleavage product methyl 2-oxoacetate could not be detected in the NMR . CDCl3  was used as the solvent. The coupling constant J is indicated in Hz. The fine structure is designated using the following abbreviations: s (singlet), d (doublet), t (triplet), q (quartet), quin (quintet), sxt (sextet), br (broad), and m (multiplet). Infrared spectra were measured with a Nicolet 380 FTIR . The wave numbers are categorized from 4000 to 800 cm−1. The signals are listed with the following abbreviations: w (weak), m (medium), s (strong), vs (very strong), and br . The melting points of solid compounds were determined with a Melting Point apparatus B-545 . Absorption spectra were measured with a UV–VIS spectrometer Type Lambda 35 . Every sample was measured in quartz absorption cells with a diameter of 1 cm. Fluorescence spectra were measured with a luminescence spectrometer, LS-55B (PerkinElmer). Quartz fluorescence cells with a diameter of 1 cm were used for the measurements. High-resolution mass spectra were measured with a MAT 900 S and with an LTQ Orbitrap XL  via electrospray ionization (ESI). Crystal structure analyses were performed on a Nonius KappaCCD-Circle diffractometer. The structure was resolved using SHELXS-97 and SHELXL-97. Flash chromatography was performed on silica gel 60 Å, particle size 0.035–0.070 mm .All reagents and solvents were purchased from commercial sources. The degree of purity of the compounds was at least 95%, and they were used without any further treatment. DCM was refluxed over calcium hydride under argon atmosphere and freshly distilled prior to use. 4 (1.25 eq.) was slowly added under argon atmosphere to the ketone compound (1.00 eq.) dissolved in dry CH2Cl2 (15 mL per mmol ketone compound). The mixture was stirred for 15 min at room temperature. Methyl 2-methoxyacetate (2.00 eq.) and triethylamine or DIPEA (13.5 eq.) were added consecutively to the reaction mixture, which was subsequently heated under reflux for 24 h. An aqueous solution of HCl (1M) was added to the cooled-down reaction mixture until a clear solution emerged and extracted three times with CH2Cl2. The combined organic phases were washed with brine, dried over either MgSO4 or Na2SO4, and filtered, and the solvent was removed in vacuo. The residue was purified via column chromatography on SiO2.General procedure for olefination: In a double-heated three-necked flask with a reflux condenser and septum, TiCl3 (1 mL). An amount of 0.1 mg of TPP (c = 1.63 × 10−4 mol/L) was added, and the solution was saturated with oxygen over an inlet under light irradiation with two halogen lamps (150 W each) at 25 °C. The reaction progress was monitored via 1H-NMR after certain periods of time. After the reaction was completed, the product/products were not worked up or purified.General procedure for photooxygenation: In a NMR tube, the olefin (20 mg) was dissolved in CDClMethyl 2-methoxy-2-(10-methylacridin-9(10H)-ylidene)acetate (7) and methyl 2-(10-methylacridin-9(10H)-ylidene)acetate (8)6) , methyl 2-methoxyacetate , triethylamine , TiCl4  in DCM (60 mL). The crude products were purified by flash column chromatography with n-hexane/EtOAc (18:1). Products (7) and (8) were both obtained as a green viscous oil with a yield of 0.04 g  and 0.12 g  respectively.They were prepared according to the general procedure for olefinations, using 10-methylacridin-9(10H)-one . 1H-NMR : δ [ppm] = 3.49 , 3.50 , 3.68 , 6.95 , 7.01 , 7.03 , 7.05 , 7.23 , 7.30 , 8.13 . 13C-NMR : δ [ppm] = 33.6 (CH3), 51.5 (CH3), 57.7 (CH3), 112.0 (CHarom), 112.3 (CHarom), 120.3 (CHarom), 120.4 (CHarom), 121.2 (Cq), 121.4 (Cq), 123.0 (Cq), 127.4 (CHarom), 128.3 (CHarom), 128.6 (CHarom), 128.7 (CHarom), 139.1 (Cq), 141.7 (Cq), 142.1 (Cq), 166.4 (Cq). IR (FTIR): ῦ [cm−1] = 2943 (w), 2833 (w), 2361 (w), 1712 (s), 1592 (m), 1496 (w), 1463 (vs), 1431 (w), 1351 (m), 1333 (w), 1257 (vs), 1217 (m), 1190 (w), 1168 (w), 1152 (w), 1132 (w), 1115 (m), 1063 (w), 1051 (w), 1024 (w), 940 (w), 962 (w), 911 (w), 888 (w), 858 (w), 806 (w). HRMS (ESI): calcd. [M + H]+: 296.12811, found: 296.12873; calcd. [M + Na]+: 318.11006, found: 318.11049.Analytical data for 8: Rf = 0.27 . 1H-NMR : δ [ppm] = 3.59 , 3.73 , 6.00 , 7.08 , 7.10 , 7.13 , 7.16 , 7.42 , 7.44 , 7.75 , 7.95 . 13C-NMR : δ [ppm] = 34.0 (CH3), 51.1 (CH3), 108.6 (CH), 112.9 (CHarom), 113.1 (CHarom), 119.5 (CHarom), 119.9 (Carom), 121.4 (Carom), 124.0 (Cq), 124.2 (CHarom), 129.8 (CHarom), 130.3 (CHarom), 130.6 (CHarom), 140.1 (Cq), 141.4 (Cq), 145.3 (Cq), 167.6 (Cq). IR (FTIR): ῦ [cm−1] = 2946 (w), 1703 (m), 1591 (s), 1487 (w), 1462 (vs), 1431 (w), 1356 (w), 1301 (w), 1269 (w), 1156 (vs), 1130 (vs), 1060 (w), 1049 (w), 971 (w), 938 (w), 888 (w), 856 (w). HRMS (ESI): calcd. [M + H]+: 266.11756, found: 266.11771; calcd. [M + Na]+: 282.11247, found: 282.11268.Analytical data for Methyl 2-methoxy-2-(9H-xanthen-9-ylidene)acetate (10) and methyl 2-methoxy-2-(9H-xanthen-9-yl)acetate (11)9) , methyl 2-methoxyacetate , triethylamine , TiCl4  in DCM (60 mL). The crude products were purified by flash column chromatography with c-hexane/EtOAc (20:1→10:1). Both separated products were then freed of remaining xanthone via Kugelrohr distillation . Product (10) was obtained as a yellow solid and (11) as a yellow viscous oil with yields of 0.08 g  and 0.20 g , respectively.They were prepared according to the general procedure for olefinations, using xanthone . 1H-NMR : δ [ppm] = 3.59 , 3.74 , 7.05 , 7.15 , 7.19 , 7.21 , 7.30 , 8.15 . 13C-NMR : δ [ppm] = 51.9 (CH3), 57.6 (CH3), 116.2 (CHarom), 116.5 (CHarom), 117.1 (Cq), 121.0 (Cq), 121.4 (Cq), 122.8 (CHarom), 123.1 (CHarom), 126.7 (CHarom), 128.7 (CHarom), 128.9 (CHarom), 129.1 (CHarom), 140.9 (Cq), 152.3 (Cq), 152.6 (Cq), 165.9 (Cq). IR (FTIR): ῦ [cm−1] = 2942 (w), 1716 (vs), 1656 (w), 1597 (w), 1581 (w), 1476 (m), 1450 (vs), 1337 (m), 1252 (vs), 1226 (vs), 1208 (s), 1161 (m), 1128 (s), 1109 (s), 1101 (s), 1043 (m), 1022 (s), 962 (w), 940 (w), 914 (w), 898 (w), 881 (w), 864 (w), 816 (w). M.p.: 72–75 °C. Analytical data for +: 283.09648, found: 283.09679; calcd. [M + Na]+: 305.07843, found: 305.07868.HRMS (ESI): calcd. [M + H]11: Rf = 0.07 . 1H-NMR : δ [ppm] = 3.21 , 3.60 , 3.83 , 4.40 , 7.07 , 7.11 , 7.12 , 7.22 , 7.24 , 7.26 , 7.28 . 13C-NMR : δ [ppm] = 43.1 (CH), 51.8 (CH3), 59.0 (CH3), 86.5 (CH), 116.4 (CHarom), 116.5 (CHarom), 120.8 (Cq), 121.0 (Cq), 123.0 (CHarom), 123.2 (CHarom), 128.4 (CHarom), 128.5 (CHarom), 128.8 (CHarom), 129.4 (CHarom), 152.8 (Cq), 153.0 (Cq), 171.2 (Cq). IR (FTIR): ῦ [cm−1] = 2949 (w), 2831 (w), 1748 (m), 1651 (w), 1601 (w), 1577 (w), 1458 (s), 1355 (w), 1301 (w), 1254 (vs), 1201 (m), 1154 (w), 1118 (s), 1027 (w), 1003 (w), 963 (w), 942 (w), 927 (w), 893 (w), 861 (w), 830 (w). HRMS (ESI): calcd. [M + Na]+: 307.09408, found: 307.09427.Analytical data for Methyl 2-methoxy-2-(9-(2-oxoethyl)-9H-thioxanthen-9-yl)acetate (14) and methyl 2-methoxy-2-(9H-thioxanthen-9-yl)acetate (15)12) , methyl 2-methoxyacetate , DIPEA , TiCl4  in DCM (60 mL). The crude products were purified by flash column chromatography with n-hexane/EtOAc (4:1). Product (14) was obtained as a colorless solid with a camphor-like smell and (15) as an orange solid with yields of 0.32 g  and 0.41 g , respectively.They were prepared according to the general procedure for olefinations, using thioxanthone . 1H-NMR : δ [ppm] = 2.99 , 3.27 , 3.71 , 3.97 , 4.48 , 7.19 , 7.22 , 7.26 , 7.29 , 7.30 , 7.41 , 7.48 , 9.61 . 13C-NMR : δ [ppm] = 40.5 (CH2), 48.9 (Cq), 51.6 (CH3), 58.9 (CH3), 78.5 (CH), 126.5 (CHarom), 126.6 (CHarom), 127.1 (CHarom), 127.2 (CHarom), 127.3 (CHarom), 127.4 (CHarom), 127.7 (CHarom), 128.9 (CHarom), 131.9 (Cq), 133.1 (Cq), 133.3 (Cq), 134.4 (Cq), 170.6 (Cq), 202.1 . IR (FTIR): ῦ [cm−1] = 3583 (w), 3564 (w), 3445 (w), 2924 (w), 2852 (w), 2360 (w), 2343 (w), 1738 (vs), 1717 (vs), 1464 (m), 1445 (w), 1425 (w), 1362 (w), 1311 (w), 1301 (w), 1272 (w), 1219 (m), 1197 (s), 1176 (w), 1113 (s), 1064 (w), 1051 (w), 1012 (w), 956 (m), 945 (w), 913 (w), 811 (w). M.p.: 131.4 °C. HRMS (ESI): calcd. [M + H]+: 343.09985, found: 343.09992; calcd. [M + Na]+: 365.08180, found: 365.08170.Analytical data for 15: Rf = 0.36 . 1H-NMR : δ [ppm] = 3.10 , 3.42 , 4.13 , 4.36 , 7.17 , 7.19 , 7.21 , 7.23 , 7.25 , 7.40 , 7.42 . 13C-NMR : δ [ppm] = 51.6 (CH3), 52.6 (CH), 58.6 (CH3), 79.0 (CH), 126.3 (CHarom), 126.4 (CHarom), 126.6 (CHarom), 126.9 (CHarom), 127.2 (CHarom), 127.4 (CHarom), 129.4 (CHarom), 131.3 (CHarom), 132.2 (Cq), 132.8 (Cq), 133.4 (Cq), 134.1 (Cq), 171.8 (Cq). IR (FTIR): ῦ [cm−1] = 3059 (w), 2996 (w), 2934 (w), 2832 (w), 2359 (w), 2343 (w), 1744 (vs), 1585 (w), 1465 (m), 1445 (m), 1430 (w), 1365 (w), 1313 (w), 1295 (w), 1277 (w), 1243 (w), 1227 (w), 1203 (s), 1176 (m), 1151 (w), 1113 (s), 1078 (w), 1065 (w), 1041 (w), 1030 (w), 1007 (m), 982 (w), 961 (w), 947 (w), 928 (w), 887 (w), 873 (w), 860 (w), 830 (w). HRMS (ESI): calcd. [M + Na]+: 323.07123, found: 323.07155.Analytical data for 8).Photooxygenation of methyl 2-(10-methylacridin-9(10H)-ylidene)acetate (8)  and TPP  dissolved in CDCl3 (1 mL). After 12 h, the reaction was completed, and 10-methoxyacridin-9(10H)-one (19) and an unidentifiable compound were obtained.The reaction was performed according to the general procedure for photooxygenation using (10)Photooxygenation of methyl 2-methoxy-2-(9H-xanthen-9-ylidene)acetate (10)  and TPP  dissolved in CDCl3 (1 mL). After 6 h, the reaction was completed, and xanthone (9) and dimethyloxalate were obtained.The reaction was performed according to the general procedure for photooxygenation using (7) and (10) were synthesized via Knoevenagel condensation from 10-methylacridin-9(10H)-one (6) and xanthone (9) with titanium (IV) chloride. An additional acceptor-substituted compound (8) that does not represent an acceptor–donor system was also included in the photooxygenation study. The results of the singlet oxygen reactions showed that (8) and (10) are versatile substrates for singlet oxygen [2 + 2] cycloaddition reactions, whereas (7) surprisingly does not show chemical quenching abilities, possibly an effect of a strong physical quenching of singlet oxygen by the tertiary amine (7). Concerning their photophysics, the heteroaromatic compounds (7) and (8) were only slightly fluorescent and (10) showed no fluorescence at all. After the addition of 500 eq. of TFA, however, a strong increase in fluorescence intensity was detected for (7) and (8), while the xanthenylidene derivative (10) did not change in fluorescence. NMR analyses revealed the formation of the corresponding acridinium cation structures (7H+) and (8H+).Two new heterocyclic donor–acceptor-substituted alkenes (


Aedes aegypti L. is known as the most relevant vector mosquito for viruses such as yellow fever, chikungunya, dengue, and Zika, especially in places with unplanned urbanization, and erratic water supply. Plants used in folk medicine have become a useful source of active compounds with the potential to control the dissemination of Ae. aegypti. Compounds isolated from Malvaceae sensu lato have been previously reported as larvicides, repellents, and insecticides. Recent studies have demonstrated the anti Ae. aegypti activity of sulfated flavonoids, an uncommon type of flavonoid derivatives. This research reports the phytochemical investigation of Sidastrum paniculatum (L.) Fryxell, a Malvaceae species with the potential against Ae. aegypti. Chromatographic procedures resulted in the isolation of the compounds: stearic acid (1), N-trans-feruloyltyramine (2), acacetin (3), apigenin (4), tiliroside (5), along with the sulfated flavonoids: wissadulin (6), 7,4′-di-O-methyl-8-O-sulfate flavone (7), yannin (8), beltraonin (9a), 7-O-sulfate isoscutellarein (paniculatumin) (9b), and condadin (10). This is the first report of compound 7-O-sulfate isoscutellarein (9b). The structures were elucidated by spectroscopic analysis . The sulfated flavonoids identified were submitted to a ligand-based and structure-based virtual screening against two targets: 1YIY (from adult Ae. aegypti) and 1PZ4 (from Ae. aegypti larvae). The results indicated that when the O-sulfate group is bearing the position 7, the structures are potentially active in 1PZ4 protein. On the other hand, flavonoids with the O-sulfate group bearing position 8 were showed to be more likely to bind to the 1YIY protein. Our findings indicated that S. paniculatum is a promising source of sulfated flavonoids with potential against Ae. aegypti. Mosquito-transmitted diseases are a great threat to billions of people in all tropical regions of the globe. Considering the global warming, researches have projected the increasing risk of mosquito-transmitted diseases in a warmer and more populated planet . AccordiAedes aegypti L. is known as the most relevant vector for viruses such as yellow fever, chikungunya, dengue, and Zika  Fryxell is a Malvaceae species known in Brazil as “malva roxa” and “malvavisco”. Previous studies on S. paniculatum showed its antileishmanial activity and led to the isolation of steroids, phaeophytins, terpenoids, and phenolic compounds , using deuterated solvents: Methanol-d4 (CD3OD) or chloroform (CDCl3). The chemical shifts were expressed as parts per million (ppm) and the coupling constants (J) in Hz.Chromatographic glass columns were used for purification of the compounds, packed with Silica gel 60 , Sephadex LH-20 (Sigma-Aldrich®) or Amberlite XAD-2 (Sulpeco®) as stationary phases. Thin layer chromatography (TLC) was carried out using Silica gel plates (Whatman®). The isolated compounds were identified by Infrared (IR), Perkin-Elmer apparatus, FT-IR-1750 and Shimadzu—Prestige 21 models using KBr disc; and 1D and 2D NMR (2O (solvent A) and acetonitrile (solvent B). The flow rate was 300 μl/min. The obtained results were analyzed using Xcalibur 2.2 (Thermo Fisher Scientific)  (LC-HRMS (Accela 600 HPLC system combined with an Exactive (Orbitrap)—Thermo Fisher Scientific  was used to obtain the high-resolution mass spectra in negative or positive mode. The samples were solubilized in methanol (HPLC grade) to obtain a concentration of 1 mg/ml. The injection volume was 20 µl. The column used was a reverse phase ACE C-18  from HiChrom . The mobile phase gradient was a mixture of 0.1% formic acid in HGermany) .S. paniculatum, were collected in Pedra da Boca Park, located in Araruna City, Paraiba/Brazil, in June 2008 (SISBIO Authorization Number 46923-2). The plant was identified by Prof. Dr. Maria de Fátima Agra, and a voucher specimen (JPB-6051) was authenticated and deposited at Lauro Pires Xavier Herbarium (CCEN/UFPB). This research has been registered at National System of Genetic Resource Management and Associated Traditional Knowledge (SisGen—A568B8A).The botanical material  of n-hexane (Hex), chloroform (CHCl3), ethyl acetate (AcOEt) and n-butanol, resulting in their respective phases, and hydroalcoholic phase.The plant material was dried in an oven in a temperature of 40°C for 72 h. After that, it was grounded in a mechanical mill, yielding 3.7 kg of a powder which was submitted to maceration with ethanol for three consecutive days. This process was repeated in order to maximum extraction of chemical constituents. The obtained ethanol extractive solution was concentrated in a rotatory evaporator, resulting 250 g of crude ethanol extract (CEE). The CEE was solvent-extracted with 3 (1:1), resulting in 30 fractions. The obtained fractions were combined after thin layer chromatography (TLC) analyses. Fraction 7–25 (1.03 g) was submitted to chromatographic column (CC) using Silica gel eluted with hexane, AcOEt and MeOH in increasing polarity mixtures. Fraction 17–26 (100 mg) was again submitted to a similar procedure, resulting 24 fractions. The fraction 9 (amorphous solid) was found to be pure, named as compound 1 (5.0 mg) and fraction 16 (green amorphous solid) named as compound 2 (15.0 mg).The precipitate from chloroform phase (2.0 g) was submitted to chromatographic column using Sephadex LH-20 and eluted with MeOH and MeOH:CHCl2O, H2O:MeOH (1:1), Acetone, AcOEt, and Hex. Acetone fraction (300.0 mg) was submitted to Sephadex (LH-20), as the mobile phase, MeOH, and MeOH:CHCl3 (7:3). After TLC, the fractions were combined and selected to new Sephadex CC. The fraction 3–5 (15.0 mg) was submitted to Silica Flash CC and eluted with hexane, AcOEt and MeOH, the fraction 2–3  was pure, named as compound 3 (5.0 mg).N-butanol phase (5.0 g) was submitted to CC using Amberlite XAD-2, eluted with H2O:MeOH (1:1) (2.0 g) was chromatographed in successive Sephadex CC, using as the mobile phase MeOH and MeOH: CHCl3 (1:1). This procedure provided compounds 7 (15.0 mg), 8 (15.0 mg), 9a, and 9b (9.0 mg) as yellow amorphous solids.The fraction H3 (1:1). The fractions 14–20 (50.0 mg) were submitted to Silica gel CC using Hex, AcOEt, and MeOH, which led to isolation of the compounds 4 (6.0 mg) and 5 (10.0 mg) as yellow amorphous solids. The fraction 26–28 (20.0 mg) was submitted the same method previously described, resulting in the isolation of the compound 6 (7.0 mg), a yellow amorphous solid.Methanol fraction (800 mg) was submitted to Sephadex CC eluted with MeOH and MeOH: CHCl2O, H2O:MeOH, acetone, AcOEt, and hexane. Methanol fraction (1.0 g) was submitted to Silica gel CC twice, resulting in 10 fractions. These fractions were analyzed by TLC and combined. The fraction 9 (50.0 mg) was submitted to a similar chromatography. From this procedure, the fraction 10 showed one spot at TLC and it was named as compound 10 (10.0 mg).Hydroalcoholic phase (5.0 g) was chromatographed using Amberlite XAD-2 CC, eluted with HAe. aegypti.From the ChEMBL database, we select a set of chemical structures for the construction of a predictive model. The set was composed by 162 diverse chemical structures that have been studied and inhibited larvae of 50 (mol/L) = pIC50, which led us to assign 85 active (pIC50 ≥ 4.15) and 76 inactive compounds (pIC50 < 4.15). In this case, the IC50 represented the concentration required for 50% inhibition of larvae of Aedes aegypti. Through a bibliographical search, 11 flavonoids  (http://www.chemaxon.com)] to canonize structures, add hydrogens, perform aromatic form conversions, clean the molecular graph in three dimensions, and save compounds in sdf. format. Three dimensions structures (3D) were used as input data in the software Dragon 7.0 to generate descriptors  . We usedxon.com)  - ion, was m/z 379.0120 (C16H12O9S).However, comparisons with 4′-avonoids . The pre9b showed singlets at δH 6.69 attached to C-3 (δC 103.7) and δH 7.16 attached to C-6 (δC 104.4)  and positions C-6 and C-8 more deshielded, corroborating with the indication of the O-sulfate group at C-7. The The compound 9a was identified as 4′-O-methyl-7-O-sulfate isoscutellarein (beltraonin) (9b as 7-O-sulfate isoscutellarein (paniculatumin) (9b is being reported for the first time in the literature.The minor constituent C 104.4) . Comparitraonin)  and complatumin) . The com1): IR  3,459, 1,642, 1,093, 471; 1H NMR  2.23 , 1.64 , 1.29–1.38 , 2.02 , 0.89 ; 13C NMR  175.3 (C-1), 31.9 (C-2), 29.6 (C-3/C-10), 31.8 (C-11), 22.7 (C-16), 14.1 (C-17). The 1H- and 13C-NMR spectral data are consistent with published data (Stearic acid (hed data .trans-feruloyltyramine (2): 1H NMR  7.43 , 7.10 , 7.04 , 7.03 , 6.79 , 6.71 , 6.4 , 3.87 , 3.46 , 2.74 ; 13C NMR  169.15 (C-9), 156.86 (C-4′), 149.87 (C-3), 149.22 (C-4), 142.02 (C-7), 131.26 (C-1′), 130.73 , 128.22 (C-1), 123.21 (C-6), 118.69 (C-8), 116.43 (C-5), 116.24 , 111.45 (C-2), 56.33 (3- OCH3), 42.54 (C-8′), 35.88 (C-7’). The 1H- and 13C-NMR spectral data are consistent with published data (N-hed data .3): 1H-NMR  6.64 , 6.22 , 6.47 , 7.95 , 7.09 , 3.89 ; 13C-NMR  165.8 (C-2), 104.7 (C-3), 183.9 (C-4), 162.4 (C-5), 100.5 (C-6), 163.4 (C-7), 95.4 (C-8), 159.4 (C-9), 105.3 (C-10), 124.5 (C-1′), 129.5 , 115.8 , 164.3 (C-4′), 56.46 (4′-OCH3). The 1H- and 13C-NMR spectral data are consistent with published data (Acacetin (hed data .4): 1H-NMR  6.60 , 6.22 , 6.46 , 7.85 , 6.93 ; 13C-NMR  164.9 (C-2), 103.5 (C-3), 182.4 (C-4), 161.8 (C-5), 99.9 (C-6), 164.8 (C-7), 94.7 (C-8), 158.0 (C-9), 103.9 (C-10), 121.9 (C-1′), 129.5 , 116.6 , 161.3 (C-4’). The 1H- and 13C-NMR spectral data are consistent with published data (Apigenin (hed data .5): 1H-NMR  6.13 , 6.30 , 8.00 , 6.82 , 5.23 , 3.30–3.50 , 4.18–4.30 , 7.33 , 6.80 , 7.40 , 6.10 ; 13C-NMR  159.4 (C-2), 135.1 (C-3), 179.3 (C-4), 162.9 (C-5), 100.4 (C-6), 167.1 (C-7), 95.3 (C-8), 158.7 (C-9), 105.3 (C-10), 122.7 (C-1′), 132.4 , 117.0 , 161.6 (C-4′), 104.2 (C-1″), 75.6 (C-2″), 78.3 (C-3″), 71.7 (C-4″), 75.6 (C-5″), 64.4 (C-6″), 127.1 (C-1‴), 130.9 , 116.3 , 161.3 (C-4‴), 146.6 (C-7‴), 114.8 (C-8‴), 168.8 (C-9‴). The 1H- and 13C-NMR spectral data are consistent with published data (Tiliroside (hed data .6): IR  3,452, 2,925, 1,628, 1,384, 1,253, 1,183, 830; 1H-NMR  6.72 , 7.96 , 7.11 , 3.89 ; 13C-NMR  166.4 (C-2), 104.2 (C-3), 106.4 (C-10), 124.6 (C-8), 129.5 , 115.7 , 164.5 (C-4′), 56.1(4′-OCH3); HRMS (FTMS + pESI) m/z, observed: 363.0165; C16H12O8S [M]- required: 363.0180. The 1H- and 13C-NMR spectral data are consistent with published data (Wissadulin (hed data .7): IR  3,476, 3,092, 1,654, 1,604, 1,246, 1,065, 995; 1H-NMR  6.67 , 6.52 , 8.21 , 7.08 , 3.95 , 3.89 ; 13C-NMR  166.6 (C-2), 103.8 (C-3), 184.3 (C-4), 159.6 (C-5), 96.9 (C-6), 160.9 (C-7), 123.4 (C-8), 151.4 (C-9), 105.5 (C-10), 124.7 (C-1′), 130.2 , 115.5 , 164.5 (C-4′), 56.0 (4′- OCH3), 57.0 (7- OCH3); HRMS (FTMS + pESI) m/z, observed: 393.0262; C17H14O9S [M]- required: 393.0285. The 1H-and 13C-NMR spectral data are consistent with published data (7.4′-di-O-metil-8-O-sulfate flavone (hed data .8): IR  3,445, 1,655, 1,609, 1,371, 1,251, 1,051; 1H-NMR  6.63 , 6.30 , 8.04 , 6.93 ; 13C-NMR  166.4 (C-2), 103.3 (C-3), 183.9 (C-4), 157.7 (C-5), 100.6 (C-6), 159.2 (C-7), 122.1 (C-8), 151.5 (C-9), 105.3 (C-10), 123.1 (C-1′), 129.9 , 116.8 , 162.7 (C-4’). HRMS (FTMS + pESI) m/z, observed: 364.9962; C15H10O9S [M]- required: 364.9972. The 1H- and 13C-NMR spectral data are consistent with published data (Yannin (hed data .9a): Full NMR data are presented in 1H- and 13C-NMR spectral data are consistent with published data (Beltraonin (hed data .O-sulfate isoscutellarein (paniculatumin) (9b): Full NMR data are presented at 7-10):  3,445, 1,655, 1,609, 1,371, 1,251, 1,051; 1H-NMR  6.63 , 6.52 , 7.66 , 7.08 , 7.75 ; 13C-NMR  166.7 (C-2), 103.5 (C-3), 184.0 (C-4), 159.6 (C-5), 96.7 (C-6), 160.8 (C-7), 123.0 (C-8), 151.5 (C-9), 105.1 (C-10), 124.8 (C-1′), 114.2 (C-2′), 147.9 (C-3′), 152.8 (C-4′), 112.6 (C-5′), 120.8 (C-6′), 56.8 (4′- OCH3), 56.2 (7- OCH3). HRMS (FTMS + pESI) m/z, observed: 409.0230 [M-H]-; C17H14O10S [M]- required: 410.0308. The 1H- and 13C-NMR spectral data are consistent with published data  for generation evaluation of the The graphic of ROC curve that analyze the model performance was generated for the external test set and obtained an area under the curve of 0.93, and 0.91 for the internal cross validation . ConsideAe. aegypti model was used in order to triage the sulfates flavonoids isolated from S. paniculatum for the identification of possible bioactive molecules against Ae. aegypti larvae. The six flavonoids tested showed a potential activity of more than 53% (The than 53% .It is possible to observe in S. paniculatum were tested with the two Ae. aegypti target proteins (1YIY and 1PZ4) in order to screen the compounds with the highest potential larvicidal activity, thus a virtual screening based on the receptor structure. Then, calculations were made to obtain the molecules with the best probabilities of active potential against Ae. aegypti larvae. These calculations were carried out using the following formula:MT is the energy of the test molecule, EM is the lowest energy obtained from the molecules tested and EL is the energy of the ligand obtained from the crystallography of the test protein, the fraction is conditioned to the energy of the test molecule being smaller than the energy of the ligand, that is, and only molecules which have obtained binding energy lower than the binding energy of the crystallographic binder will be considered potentially active  were performed. The analysis of the mean square deviation (RMSD) of the sterol protein (PDB ID 1PZ4) showed that the enzyme reached conformations ranging from 1,5 to 2,4 nm in size in 1 ns. While Kinurenine aminotransferase (PDB ID 1YIY) protein showed a RMSD with conformations ranging from 1.5 to 3.5 nm in 1 ns. The results show that the protein Kinurenine aminotransferase is more unstable, requiring greater mobility and interaction with its ligands , 5.The mean square root fluctuations (RMSF) of the individual residues were also calculated. It was verified that residues 1, 23, 25, 105, 106, and 110 of the Sterol -sulfated flavonoid complexes  presented higher fluctuation values, indicating the key residues in the conformational change of the protein structure . Only thThe Coulombic and Lennard-Jones interaction energies of the protein-ligand complexes were calculated and the flavonoid linkers  were shown to have a higher interaction stability with the sterol enzyme when compared to the ligand PDB PLM . As for Although the RMSD analysis of the sterol-PLM complex is better when compared to the flavonoids, we can suggest that through RMSF and interaction energy calculations, flavonoids interact better with the two proteins because they allow greater flexibility and interaction with amino acids, and interaction stability.O-sulfate isoscutellarein (paniculatumin) (9b), along with eleven known compounds, including a fatty acid (1), a tyramine derivative (2), and flavonoids, contributing to the phytochemical knowledge of Malvaceae sensu lato family.This research presented the isolation of the new sulfated flavonoid: 7-1) by S. paniculatum has been mentioned recently by Sida, Sidastrum, and Wissadula  has been previously isolated from the specie Sida acuta , apigenin (4), and tiliroside (5). These compounds have been previously reported from Malvaceae species such as Sidastrum micranthum  is referred to as the most prevailing flavonoid produced by Malvaceae species (5), such as antioxidant, vasorelaxant, modulator of bacterial resistance, and anti-inflammatory and larvicidal activity against Ae. aegypti , 7,4′-di-O-metil-8-O-sulfate isoscutellarein (7), yannin (8), beltraonin (9a), the new compound paniculatumin (9b), and condadine (10).The phytochemical study on the polar fractions of O-sulfated substituents attached to the 2-phenyl-benzyl-γ-pyrone nucleus are considered the most uncommon flavonoids derivates found in natural products (O-sulfate) was published in 1937. After that, studies have pointed few species from the families Asteraceae (Compositae), Bixaceae, Malvaceae, Dilleniaceae, Umbelliferae, and Verbenaceae as producers of sulfated flavonoid , 7,4′-di-O-metil-8-O-sulfate isoscutellarein (7), yannin (8), and beltraonin (9a) have been originally isolated from Wissadula periplocifolia. In fact, phytochemical investigations on W. periplocifolia suggested very similar metabolites production for Wissadula and Sidastrum species  have been previously reported from H. velutina along with other sulfated structures  for the activity against Ae. aegypti  were showed to be more likely to bind to the 1YIY protein. Our findings indicated that S. paniculatum is a promising source of sulfated flavonoids with the potential to be used against Ae. aegypti.In this perspective, we submitted the identified sulfated flavonoids to a ligand-based and structure-based virtual screening techniques that were combined to evaluate the sulfated flavonoids against


Donor-acceptor 3-methyl-2-siloxycyclopropenecarboxamide is rapidly formed from the reactant enoldiazoamide and undergoes catalytic ring opening to give only the Z-γ-substituted metallo-enolcarbene. Elimination from 3-siloxy-2-diazo-3-pentenamide to form the conjugated 3-siloxy-2,4-pentadienamide is competitive but minimized at low temperature.A substituted donor–acceptor cyclobutenecarboxamide is synthesized with modest enantiocontrol through a chiral copper(I) complex catalyzed [3 + 1]-cycloaddition reaction of α-acyl diphenylsulfur ylides with 3-siloxy-2-diazo-3-butenamides. With a methyl substituent on the 4-position of the 3-butenamide, the  The four-membered carbon ring is an important structural framework present in natural products and biologically active compounds, but is less accessible than are other ring structures ,2,3. Fur1 = H) 2-siloxycyclobutenecarboxylates in good yields with uniformly high enantiocontrol. Diastereoselectivity for the formation of 1,2,3,4-tetrasubstituted (R1 = Me) 2-siloxycyclobutenecarboxylates was good (>10:1) to excellent (>20:1) with the trans isomer dominant; however, even in this earlier study there were indications that formation of the cis-diastereomer could be competitive.Previous research from our laboratory established that catalytic [3 + 1]-cycloaddition of silyl group-protected enoldiazoacetate esters with α-acyl dimethylsulfur ylides was effective in forming stable donor−acceptor cyclobutene derivatives b 18]. T. T18]. TPrior research has suggested that diazoamides are more stable and more selective in their catalytic reactions emanating from metal carbene intermediates ,20, and N,N-dimethyl-enoldiazoacetamide 1a with α-benzoyl dimethylsulfur ylide 2a using the same copper(I) catalyst and chiral ligand  and SaBox (sidearmed bis-oxazoline) ligands  brought about an increase in enantioselectivity (34% ee) using Box L2 4 in place of CuOTf·Tol1/2 had no meaningful influence on % yield or enantioselectivity  or trans (E-3b) diastereomer + 416.2459; found: 416.2448.To an oven-dried sealable 2-dram vial equipped with a stir bar were added CuOTf·Tol1/2  and sulfur ylide 2b . After the vial was evacuated and backfilled with N2 three times, dry DCM (2.0 mL) was added via a syringe; and the solution was stirred at room temperature for 5 min before 2-diazo-N,N-dimethyl-3-(triisopropylsiloxy)but-3-enamide  dissolved in dry DCM (2.0 mL) was added dropwise via a syringe for 1 h. The reaction solution was stirred at room temperature for 16 h then filtered through a short pad of Celite and washed with DCM. The filtrate was concentrated, and the residue was purified by flash chromatography on silica gel using 10:3 hexanes:ethyl acetate as the eluent to afford the corresponding racemic [3 + 1]-cycloaddition product 4-benzoyl-N,N-dimethyl-2-(triisopropylsiloxy)cyclobut-1-ene-1-carboxamide (Racemic 3a) as a colorless liquid . Enantiomer composition was determined by HPLC analysis + 312.2102; found 312.2096.To a 100 mL oven-dried round bottom flask containing a magnetic stirring bar, commercially available 2-diazo-1/2  and bisoxazoline ligand L14 . After the vial was evacuated and backfilled with N2 three times, dry DCM (1.0 mL) was added via a syringe and the resulting solution was stirred at room temperature for 1 h before sulfur ylide 2b  dissolved in dry DCM (0.5 mL) was added dropwise via a syringe. The reaction was stirred at room temperature for 5 min and then a solution of cyclopropenecarboxamide 1b  in dry DCM (1.0 mL) was then added dropwise for 1 h. The reaction mixture was stirred at the same temperature for 24 h, filtered through a short pad of silica gel and washed with DCM. The filtrate was further concentrated and directly subjected to analysis by 1H-NMR. After that, the residue was purified by flash chromatography on silica gel using 10:0 to 10:3 hexanes:ethyl acetate as the eluent to afford the expected [3 + 1]-cycloaddition product Z-3b, E-3b and diene (4).To an oven-dried sealable 2-dram vial equipped with a stir bar were added CuOTf·Tol1b (Z:E-1b = 1:3) using Ligand L14, Ylide 2b at rt, 24 h: Scale 0.2 mmol, dr 6:1 (Z-3b:E-3b), 47 mg of Z-3b as colorless liquid, 57% yield, 64% ee; 10% of E-3b, 7% ee and 15 mg (22%) of 4 as colorless liquid; HPLC conditions for determination of enantiomeric excess of Z-3b: Chiralpak OD-H column, 254 nm, hexanes/i-PrOH = 97:3, 1.0 mL/min, tr (minor) = 13.15 min, tr (major) = 17.19 min; HPLC conditions for determination of enantiomeric excess of E-3b: Chiralpak OD-H column, 254 nm, hexanes/i-PrOH = 97:3, 1.0 mL/min, tr (minor) = 11.31 min, tr (major) = 9.63 min.For 1b (Z:E-1b = 25:1) using Ligand L14, Ylide 2b at rt, 24 h: Scale 0.2 mmol, dr 9:1 (Z-3b:E-3b), 56 mg of Z-3b as colorless liquid, 69% yield, 68% ee; 8% of E-3b, 20% ee and 10 mg (15%) of 4 as colorless liquid; HPLC conditions for determination of enantiomeric excess of Z-3b: Chiralpak OD-H column, 254 nm, hexanes/i-PrOH = 97:3, 1.0 mL/min, tr (minor) = 13.61 min, tr (major) = 18.10 min; HPLC conditions for determination of enantiomeric excess of E-3b: Chiralpak OD-H column, 254 nm, hexanes/i-PrOH = 97:3, 1.0 mL/min, tr (minor) = 11.34 min, tr (major) = 9.61 min.For 1/2  and bisoxazoline ligand L14 . After the vial was evacuated and backfilled with N2 three times, dry DCM (1.0 mL) was added via a syringe and the resulting solution was stirred at room temperature for 1 h before sulfur ylide 2b  dissolved in dry DCM (0.5 mL) was added dropwise via a syringe for 1 h. The reaction was stirred at room temperature for 5 min and then a solution of cyclopropenecarboxamide 5  in dry DCM (0.5 mL) was then added dropwise. The reaction mixture was stirred at the same temperature for 24 h, filtered through a short pad of silica gel and washed with hexanes/EtOAc . The filtrate was further concentrated and directly subjected to analysis by 1H-NMR. After that, the residue was purified by flash chromatography on silica gel using 10:0 to 10:3 hexanes:ethyl acetate as the eluent to afford to afford the expected [3 + 1]-cycloaddition product Z-3b, E-3b and diene (4).To an oven-dried sealable 2-dram vial equipped with a stir bar were added CuOTf·Tol5 using Ligand L14, Ylide 2b at rt, 24 h: Scale 0.2 mmol, dr 12:1 (Z-3b:E-3b), 56 mg of Z-3b as colorless liquid, 67% yield, 71% ee; 5.5% of E-3b, 16% ee and 5.5 mg (8%) of 4 as colorless liquid; HPLC conditions for determination of enantiomeric excess of Z-3b: Chiralpak OD-H column, 254 nm, hexanes/i-PrOH = 97:3, 1.0 mL/min, tr (minor) = 13.65 min, tr (major) = 17.76 min; HPLC conditions for determination of enantiomeric excess of E-3b: Chiralpak OD-H column, 254 nm, hexanes/i-PrOH = 97:3, 1.1 mL/min, tr (minor) = 11.50 min, tr (major) = 9.74 min.For -4-Benzoyl-3-methyl-N,N-dimethyl-2-(triisopropylsiloxy)cyclobut-1-ene-1-carboxamide (Z-3b): 1H-NMR  δ 7.94 , 7.54 , 7.44 , 4.64 , 3.39–3.33 , 3.29 , 2.97 , 1.23–1.05 , 0.97 ; 13C-NMR  δ 199.4, 164.3, 150.3, 137.2, 132.9, 128.5, 128.2, 110.6, 44.5, 43.0, 37.9, 34.7, 17.5, 13.0, 12.6; HRMS (ESI) m/z calcd for C24H37NO3Si: [M + H]+ 416.2615; found: 416.2610.-4-Benzoyl-3-methyl-N,N-dimethyl-2-(triisopropylsiloxy)cyclobut-1-ene-1-carboxamide (E-3b): 1H-NMR  δ 8.06 , 7.58–7.54 , 7.48 , 4.01 , 3.21 , 3.01–2.95 , 2.93 , 1.42 , 1.23–1.05 .N,N-Dimethyl-3-triisopropylsiloxy-2,4-pentadienecarboxamide (4): 1H-NMR : δ 6.20 , 5.64 , 5.32 , 5.24 , 3.06 , 2.97 , 1.24–1.18 , 1.16–1.08 ; 13C-NMR  δ 167.3, 153.3, 135.3, 117.0, 37.9, 34.6, 17.9, 13.5; HRMS (ESI) m/z calcd for C16H31NO2Si: [M + H]+ 298.2197; found: 298.2193.N,N-dimethyl-3-oxopentanamide + 170.0924; found 170.0923.To a stirred solution of tanamide   was prepared from 2-diazo-N,N-dimethyl-3-oxopentanamide (8) according to reported + 326.2258; found 326.2253.reported ,32 proceN,N-dimethyl-3-((triisopropylsiloxy)pent-3-enamide (Z:E-1b = 25:1). The title compound includes a small percent (5–10%) of cyclopropenecarboxamide 5 because enoldiazoacetamide undergoes dinitrogen extrusion to form cyclopropenecarboxamide, even when stored at 0 °C. Compound 1b (Z:E = 25:1) in common NMR solvents  is converted (at 0 °C) to the corresponding cyclopropene (5), but was found to be stable at −20 °C and should be stored at −20 °C. At 0 °C and rt, 20% and 70%, formation of 5 was observed after 6 h respectively.The reported  procedurN,N-dimethyl-3-oxopentanamide  was added, and the system was filled with nitrogen. THF (30 mL) was then added, and the reaction solution was cooled to −78 °C (dry ice/acetone bath), LiHMDS  was introduced dropwise over 2 min, followed by the addition of TIPSOTf  at −78 °C. The resulting solution was stirred at −78 °C until the reaction was complete . The reaction was quenched at −78 °C with ice-cold brine, and solvent was removed at 15 °C under reduced pressure, and the residue was directly purified by column chromatography on silica gel which was pre-treated with 5 vol.% triethylamine/hexanes (eluent: pure hexanes then 10% EtOAc in hexanes) to give the desired product 2-diazo-N,N-dimethyl-3-(triisopropylsiloxy)pent-3-enamide, 1b (Z:E-1b = 25:1) as yellow liquid which was immediately stored −20 °C. 1H-NMR of Z1b-  δ 5.00 , 3.01 , 1.70 , 1.32–1.02 ; 13C-NMR of Z1b- including 5  δ 160.7, 142.5, 79.6, 37.1, 34.6, 34.5, 31.5, 25.8, 25.2, 22.6, 20.3, 17.7, 17.4, 17.4, 14.1, 12.3, 12.0, 11.8 ppm; HRMS (ESI) m/z calculated for C16H31N3O2Si: [M + H]+ 326.2258; found 326.2254.To a 100-mL oven-dried vial equipped with a magnetic stirring bar, 2-diazo-N,N-dimethyl-3-(triisopropylsiloxy)pent-3-enamide, 1b (Z:E-1b = 1:3)  at room temperature was added to a 20 mL oven dried vial containing a magnetic stirring bar and 10 mL CHCl3, and then the vial was screwed close. The sealed reaction mixture was heated in oil bath at 50 °C for 2 h, during this time the diazo compound was converted to the corresponding cyclopropene. The color of the solution changed from yellow to colorless. Solvent was removed under reduced pressure to afford 980 mg (>99%) of N,N,3-trimethyl-2-(triisopropylsiloxy)cycloprop-1-ene-1-carboxamide (5) was obtained as colorless liquid and was characterized directly without further purification. Compound 5 was also obtained heating 1b (Z:E-1b = 25:1) in CHCl3 at 50 °C for 1 h. 1H-NMR  δ 3.16 , 2.99 , 2.38 , 1.30 , 1.36–1.24 , 115–1.10 ; 13C-NMR  δ 160.7, 142.5, 79.6, 37.1, 34.5, 25.8, 20.3, 17.9, 17.8, 17.7, 17.7, 17.5, 17.4, 17.4, 12.3, 12.0 ppm; HRMS (ESI) m/z calculated for C16H31NO2Si: [M + H]+ 298.2197; found 298.2193.2-Diazo-1b is surprisingly diverse. Diastereoselectivity favoring the cis isomer ranges from 2:1 with SaBox ligand L1  providing the highest diastereocontrol. The highest enantioselectivity is also found with L14  and low enantioselectivity, which further signaled the inherent complexity of stereocontrol in these reactions.The influence of ligands on stereocontrol with γ-methyl-substituted igand L1 , entry 1 L1  was minimized, we thought that the diene might arise from the ligand, acting as a base, to effect elimination from the intermediate metallo-enol carbene. Indeed, using 2.4 mol% triethylamine in place of the ligand resulted in diene formation  catalyzed asymmetric [3 + 1] cycloaddition of α-benzoyl diphenylsulfur ylide