N-imidazolium-borates were prepared, and their deprotonation reactions were investigated. Addition of BH3•THF to N-benzylimidazoles and N-mesitylimidazoles leads to imidazolium-trihydridoborate adducts. Ammonium tetraphenylborate reacts with benzyl- or mesityl-imidazoles with the loss of one of the phenyl groups yielding the corresponding imidazolium-triphenylborates. Their authenticity was confirmed by CHN analysis, 1H-NMR, 13C-NMR, 11B-NMR, FT-IR spectroscopy, and electrospray ionization mass spectrometry (ESI-MS). 3-Benzyl-imidazolium-1-yl)trihydridoborate, (HImBn)BH3, and (3-mesityl-imidazolium-1-yl)trihydridoborate, (HImMes)BH3, were also characterized by X-ray crystallography. The reactivity of these new compounds as carbene precursors in an effort to obtain borate-NHC complexes was investigated and a new carbene-borate adduct (which dimerizes) was obtained via a microwave-assisted procedure.In this study, four new  N-heterocyclic carbenes (NHCs) , Cp2TiCl, VCl3, and ScCl3 yielded the corresponding metal complexes  and [Ca{N(SiMe3)2}2(THF)2] . A. A3− ,673− [5 oworkers  reported2(THF)2] .N-heterocyclic carbene ligands, the use of anionic NHC-borates is still scarce Br2  [2B(HTzBn)2]Br Br , H2C(HTz2)2COO−) . RecentlH2CH3)2) ,81 or th2)3SO3−) .3B(5-(CF3)pz)] and Na[H3B(3-(NO2)pz)] and related copper(I) and silver(I) phosphane complexes + complex and NaBPh4 in acidic conditions + and [(HImMes)BH3 + Na]+, respectively. In addition the ESI(+)MS spectra displayed peaks due to the fragmentation species [HImR + H]+ and to the aggregates [(HImR)2BH2]+ (R = Bn or Mes). Analogously, the ESI(+)-MS spectra of Compounds 3 and 4 were dominated by the peaks at m/z 83 and 159 due to the [HImCH3 + H]+ and [HImBn + H]+, respectively, along with a fragment at m/z 247 ([(HImCH3)BPh2]+, 25%) and an aggregate at 481 ([(ImBn)2BPh2]+, 45%), in 3 and 4 respectively.In the ESI(+)-MS spectra of N-imidazolium-borates and study their reactivity and investigate their reactivity as carbene precursors in an effort to obtain borate-NHCs silver(I) complexes. However, treatment of Compounds 1–4 with nBuLi to yield the imidazol-2-ylidenes always led to decomposition species. Further direct reactions of 1–4 with Ag2O, in different reaction conditions , or with silver acetate (in CH3OH or CH3CN) to give silver carbene complexes were unsuccessful: only mixtures of unreacted or decomposition products were detected. The only partially isolable product of the reaction of 4 and Ag2O in CH3CN was the imidazabole species 5. After these efforts, we found that the direct synthesis of imidazaboles +, 181 (40) [HImBn + Na]+, 195 (90) [(HImBn)BH3 + Na]+, 329 (100) [(HImBn)2BH2]+. Anal. Calcd. for C10H13BN2: C 69.82, H 7.62, N 16.28%. Found: C 69.52, H 7.30, N 15.91%.1-Benzylimidazole  was dissolved in dry THF (50 mL) under N2 atmosphere and BH3•THF complex  was added drop by drop. The reaction mixture was stirred at room temperature for 24 h. Then, the volatiles were removed under reduced pressure to give a brown oil. It was re-crystallized by CHCl3/diethyl ether/n-hexane (1/3/3) solution to obtain a brown precipitate; it was filtered, washed with diethyl ether, and dried under reduced pressure to give 1 in 68% yield (0.680 g). Single crystals of 2 suitable for X-ray analysis were obtained by slow evaporation of a CHCl3/THF solution of 2. Melting point: 109–111 °C. IR (cm−1): 3177w, 3155w, 3132w, 3061w, 3028w (C-H); 2374m, 2338m, 2323m, 2300m, 2259m (B-H); 1526s (C=C/C=N). 1H-NMR : δ 2.03 , 2.3 , 2.37 , 6.90 , 7.02 , 7.31 , 7.75 . 1H-NMR : δ 2.04 , 2.1 , 2.35 , 7.08 , 7.23 , 7.25 , 8.14 . 13C{1H}-NMR : δ 17.33, 21.06 (CH3Mes), 121.25, 128.12, 129.49, 131.71, 134.86, 136.85 (CH), 140.34 (2-CH). 11B-NMR : δ −19.21 . ESI-MS , m/z (%): 187 (15) [HImMes + H]+, 223 (55) [(HImMes)BH3 + Na]+, 385 (100) [(HImMes)2BH2]+. Anal. Calcd. for C12H17BN2: C 72.03, H 8.56, N 14.00%. Found: C 71.81, H 8.25, N 13.60%.1-mesityl-imidazole  was dissolved in dry THF (30 mL) under N3CN, 60 mL). Then, ammonium tetraphenylborate  was added to the solution. A white precipitate was formed, but the solution became limpid after 1 h. The reaction proceeded for 70 h at reflux under magnetic stirring. At the end, the solution was dried at reduced pressure, obtaining a white solid. Et2O was added to the round-bottom flask to purify the residue from the starting materials that did not react. The resulting suspension was filtered, dried under reduced pressure, and furthe purified with CHCl3 to precipitate the excess of NH4BPh4. The mixture was filtered and the mother liquors were dried at reduced pressure to give the white ligand (HImCH3)BPh3 (3) in 76% yield (1.293 g). Melting point: 209–212 °C. IR (cm−1): 3158w, 3133m, 3085w, 3064m, 3054mbr, 3010mbr (C-H); 1546m, 1531m, 1483mbr (C=C/C=N). 1H-NMR : δ 3.79 , 6.90 , 7.03–7.15 , 7.44 , 8.09 . 13C{1H}-NMR : δ 35.13 (NCH3), 122.34, 124.85, 126.42, 127.02, 134.49, 138.58 (CH). 11B-NMR : δ −6.52 . ESI-MS , m/z (%): 83 (100) [HImCH3 + H]+, 247 (25) [(HImCH3)BPh2]+. Anal. Calcd. for C22H21BN2: C 81.50, H 6.53, N 8.64. Found: C 81.14, H 6.56, N 8.38.A large excess of 1-methylimidazole  was dissolved in acetonitrile (CH3CN (60 mL). Then, NH4BPh4  was added to the solution. A white precipitate was formed, but the solution became limpid after 1 h. The reaction proceeded for 70 h at reflux under magnetic stirring. At the end, the solution was dried at reduced pressure, obtaining a white solid. EtOH was added to the round-bottom flask to purify the residue from the starting materials that did not react. The resulting suspension was filtered and dried at reduced pressure to give the white ligand (HImBn)BPh3 (4) in 50% yield (0.570 g). Melting point: 175–178 °C. IR (cm−1): 3163m, 3140m, 3125m, 3064mbr, 3023m (C-H); 1531mbr, 1506m, 1489mbr (C=C/C=N). 1H-NMR : δ 5.39 , 6.91 , 7.04–7.43 , 7.49 , 8.37 . 13C{1H}-NMR : δ 51.24 (CH2Ph), 121.25, 124.90, 127.23, 128.21, 129.33, 129.47, 133.46 (CH), 136.33 (2-CH). 11B-NMR : δ −6.37 . ESI-MS , m/z (%): 91 (80) [C7H7]+, 159 (100) [HImBn + H]+, 242 (50) [BPh3 + H]+, 481 (45) [(ImBn)2BPh2]+. Anal. Calcd. for C28H25BN2: C 84.01, H 6.29, N 7.00. Found: C 83.72, H 6.03, N 7.06.A large excess of 1-benzylimidazole  was dissolved in CH4 , silver oxide , and CH3CN (25 mL) were added. The reaction mixture was heated in the microwave reactor following a pre-set heating ramp, up to 80 °C. Once the temperature was reached, the reaction proceeded for 1 h and then it was cooled following a pre-set cooling ramp, to room temperature. All the steps were performed always under magnetic stirring. At the end, the mixture was filtered and the obtained mother liquors were dried at reduced pressure to give the oily brownish residue (ImBnBPh2)2 (5) in 54% yield (0.157 g). IR (cm−1): 3161m, 3143m, 3113sh, 3087m, 3064m, 3038m, 3024m, 3010m, 2999m, 2972m, 2938wbr (C-H); 1600m, 1587m, 1571m, 1534s, 1509s, 1496sbr (C=C/C=N). 1H-NMR : δ 5.18 , 6.91 , 7.18–7.36 , 7.77 . 1H-NMR : δ 5.13 , 6.92 , 7.11–7.44 , 7.67 . 13C{1H}-NMR : δ 50.04 (CH2Ph), 120.14, 127.98, 128.28, 128.79, 128.94, 129.17, 130.56, 134.47 (CH), 159.18 (2-C). 11B-NMR : δ 1.43 (s). ESI-MS , m/z (%): 91 (95) [C7H7]+, 159 (100) [HImBn + H]+. Elemental analysis for C29H27AgBN2 (%): calculated: H 5.94, C 82.01, N 8.69; found: H 6.04, C 81.27, N 8.89.In a 100-mL PTFE vessel equipped with a magnetic stir bar, Compound N oil was selected and mounted on a Cryo-loop and immediately placed in the low temperature nitrogen stream. X-ray intensity data were measured at 100(2) K on a Bruker SMART APEX II CCD area detector system equipped with an Oxford Cryosystems 700 series cooler, a graphite monochromator, and a Mo Kα fine-focus sealed tube (λ = 0.71073 Å). Intensity data were processed using the Bruker ApexII program suite. Absorption corrections were applied by using SADABS. Initial atomic positions were located by direct methods using XS, and the structures of the compounds were refined by the least-squares method using SHELXL [http://www.ccdc.cam.ac.uk/conts/retrieving.html or from the Cambridge Crystallographic Data Centre (CCDC), 12 Union Road, Cambridge, CB2 1EZ, UK).A suitable crystal covered with a layer of hydrocarbon/Paratone-g SHELXL . All the3•THF to N-benzyl- and N-mesitylimidazoles. In addition, two imidazolium-triphenylborates were obtained by displacement of one phenyl group of ammonium tetraphenylborate reacting with methyl- or benzyl-imidazoles. 3-Benzyl-imidazolium-1-yl)trihydridoborate and (3-mesityl-imidazolium-1-yl)trihydridoborate were also characterized by X-ray crystallography. The reactivity of these new compounds as carbene precursors was investigated and a new dimeric carbene-borate adduct was obtained via a microwave-assisted procedure. The intermolecular rearrangement pathway to the head-to-tail imidazabole dimer prevented the isolation of this type of compounds and the development of their characteristic NHC chemistry.Two imidazolium-trihydridoborate adducts were obtained by addition of BH


Yucca schidigera Roezl (Mojave), a kind of ornamental plant belonging to the Yucca genus (Agavaceae), whose extract exhibits important roles in food, beverage, cosmetic and feed additives owing to its rich spirostanol saponins. To provide a comprehensive chemical profiling of the spirostanol saponins in it, this study was performed by using a multi-phase liquid chromatography method combining a reversed phase chromatography T3 column with a normal phase chromatography silica column for the separation and an ESI-Q-Exactive-Orbitrap MS in positive ion mode as the detector. By comparing the retention time and ion fragments with standards, thirty-one spirostanol saponins were identified. In addition, according to the summary of the chromatographic retention behaviors and the MS/MS cleavage patterns and biosynthetic pathway, another seventy-nine spirostanol saponins were speculatively identified, forty ones of which were potentially new ones. Moreover, ten novel spirostanol saponins (three pairs of (25R/S)-spirostanol saponin isomer mixtures) were targeted for isolation to verify the speculation. Then, the comprehensive chemical profiling of spirostanol saponins from Y. schidigera was reported here firstly. Yucca schidigera Roezl (Mojave), belonging to the genus Yucca, Agavaceae family, is mainly distributed in the southwestern United States and the northern deserts of Mexico + fragment ion, or lose 142 Da to form a [M-C8H14O2 + H]+ fragment ion, while the 25-CH3 spirostanol saponins could lose 114 Da to yield a [M-C6H10O2 + H]+ fragment ion, or 144 Da was lost, forming a [M-C8H16O2 + H]+ fragment ion. The different m/z as well as the different kinds of cleavage fragment ions , it was found that the retention times of this class of compounds were mainly affected by the substitution type of the C-12 and C-25 of the aglycon, the stereochemistry of 25-CH3, as well as the type of substituted glycosyl (By comparing the chromatographic elution order of reference standards (peaks glycosyl .R/S)-spirostanol saponins would be described in detail. What′s more, the speculation would be verified by their subsequent targeted isolation.Using the rules summarized above, another seventy-nine spirostanol saponins have been tentatively identified. In this section, the tentative identification of ten novel 25(10 (m/z 903.4550), characteristic ions at m/z 447.3109, 411.2903, 393.2797, 333.2435, 315.2324, 285.1842 could be observed -5β-spirostan-2β,3β-ol-12-one spirostanol. It was speculated that its glycosyl substituent consisted of two molecules of hexose and one pentose molecule because of its neutral loss of 294 Da and 162 Da. According to the biosynthetic pathway of Y. schidigera, the structure of peak 10 could be tentatively speculated to be either 25(R/S)-5β-spirostan-2β,3β-ol-12-one-3-O-xylopyranosyl(1→3)-[glucopyranosyl(1→2)]-glucopyranoside or 25(R/S)-5β-spirostan-2β,3β-ol-12-one-3-O-xylo- pyranosyl(1→3)-[glucopyranosyl(1→2)]-galactopyranoside. In order to clarify the correctness of above speculation, a targeted separation was conducted, and peak 10 was finally unambiguously identified as a novel compound, named as 25(R)-5β-spirostan-2β,3β-ol-12-one-3-O-xylopyranosyl(1→3)-[glucopyranosyl(1→2)]-galactopyranoside.In the MS/MS spectra of peak observed . The rel14 (m/z 889.4772), 15 (m/z 889.4772), 34 (m/z 727.4261), 36 (m/z 727.4261), 40 (m/z 595.3868), and 41 (m/z 595.3866), characteristic ions at m/z 433.3312, 397.3101, 379.2995, 301.2526, 289.2162, 283.2420, 271.2056, 253.1951 could be observed  and 433.3323(162 Da loss)], peak 15 , peak 34 , peak 36 [m/z 727.4261 and 433.3314 (294 Da loss)], peak 40 [m/z 595.3864 and 433.2562 (162 Da loss)], peak 41 [m/z 595.3864 and 433.2563 (162 Da loss)] were indicated to be substituted by two hexoses and one pentose, two hexoses and one pentose, one hexose and one pentose, one hexoseand one pentose, one hexose and one hexose, respectively. The MS/MS spectra of peaks 14 and 15, 34 and 36, 40 and 41 were almost identical, and their retention times were also very close, therefore, they were conjectured to be three pairs of (25R/S)-isomers with the YS-7 aglycon. Considering the biosynthetic pathway of Y. schidigera as well as the chromatographic elution order, peaks 14, 15, 34, 36, 40 and 41 were speculated to be 25(S)-5β-spirostan-3β,12β-diol 3-O-xylopyranosyl(1→3)-[glucopyranosyl(1→2)]-glucopyranoside (14), 25(R)-5β-spirostan-3β,12β-diol 3-O-xylopyranosyl(1→3)-[glucopyranosyl(1→2)]-glucopyranoside (15), 25(S)-5β-spirostan-3β,12β-diol 3-O-xylopyranosyl(1→3)-glucopyranoside (34), 25(R)-5β-spirostan-3β,12β-diol 3-O-xylopyranosyl(1→3)-glucopyranoside (36), 25(S)-5β-spirostan-3β,12β-diol 3-O-glucopyranoside (40) and 25(R)-5β-spirostan-3β,12β-diol 3-O-glucopyranoside (41), respectively. Furthermore, they were all novel compounds, and targeted for separation to verify the above speculation.In the MS/MS spectrum of peaks observed . Accordi27 (m/z 887.4648), 38 (m/z 887.4665), 51 (m/z 755.4223), characteristic ions at m/z 431.3156, 413.3050, 395.2945, 377.2839, 299.2369, 281.2264, 269.1900 could be observed in their MS/MS spectra , 593.3700 (162 Da loss) and 431.3167 (162 Da loss)], peak 38 , and peak 51  suggested they were substituted by two hexose molecules and one pentose molecule, two hexose molecules and one pentose molecule and two hexose molecules, respectively. By referring to references as well as comparing them to reference standards, peaks 24 and 32 were the isomers of peaks 27 and 38, which have been identified to be 25(R)-5β-spirostan-3β-ol-12-one 3-O-xylopyranosyl(1→3)-[glucopyranosyl(1→2)]-galactopyranoside (24) and 25(R)-5β-spirostan-3β-ol-12-one 3-O-xylopyranosyl(1→3)-[glucopyranosyl(1→2)]-glucopyranoside (32), respectively. Thus peaks 27 and 38 were tentatively speculated to be YS-8 substituted by two hexose molecules and one pentose molecule. During the targeted separation and structure elucidation, they were unambiguously identified as 25(R)-5β-spirostan-3β-ol-12-one 3-O-apiofuranosyl(1→3)-[glucopyranosyl(1→2)]-glucopyranoside (27) and 25(R)-5β-spirostan-3β-ol-12-one 3-O-xylopyranosyl(1→3)-[glucopyranosyl(1→6)]-glucopyranoside (38), respectively. Similarly, peak 51 was unambiguously identified as 25(R)-5β-spirostan-3β-ol-12-one 3-O-glucopyranosyl(1→3)-glucopyranoside (51). Peaks 27, 38 and 51 are all novel compounds.For peaks  spectra , which iSimilar MS/MS cleavage patterns and chromatographic elution orders as well as the comparisons with references  were use10, 14, 15, 27, 34, 36, 38, 40, 41, 51 was carried out. In the light of the MPLC-MS analysis results, peak 10 was found to be enriched in the “Preparation of the YSESs Test Solutions” fraction 9; peaks 14, 15 and 38 were found to be enriched in the “Preparation of the YSESs Test Solutions” fraction 8; peak 27 was found to be enriched in the “Preparation of the YSESs Test Solutions” fraction 7; peaks 34, 36, 40, 41 and 51 were found to be enriched in the “Preparation of the YSESs Test Solutions” fraction 6. Then silica gel, ODS CC and preparative HPLC (pHPLC) were used to isolate these fractions and the spirostanol saponins 10, 14/15, 27, 34/36, 38, 40/41, 51 were thus obtained.For the purpose of verifying the above speculations, targeted separation of peaks D25 –7.7, MeOH). Its molecular formula, C44H70O19, was deduced by positive-ion ESI-Q-Exactive-Orbitrap MS . Acid hydrolysis yielded d-galactose, d-glucose, and d-xylose [1H- and 13C-NMR ], one β-d-glucopyranosyl [δ 5.57 ], together with one β-d-xylopyranosyl [δ 5.23 ]. The 13C-NMR signals attributed to the sugar moieties of 10 were consistent with those of yucca spirostanoside D1 (5β-spirost-25(27)-en-2β,3β-diol-12-one 3-O-β-d-xylopyranosyl(1→3)-[β-d-glucopyranosyl(1→2)]-β-d-galactopyranoside) , three oxygenated methine protons at δH , together with one carboxyl carbon at δC 212.7 (C-12). The 1H 1H COSY spectrum of 10 suggested the presence of the three partial structures indicated by bold lines in 3-18 to C-12–14, C-17; H3-19 to C-1, C-5, C-9, C-10; H3-21 to C-17, C-20, C-22; H2-26 to C-22; H2-27 to C-24–26. Moreover, the connection positions of glycosyl units were determined by the long-range correlations from H-1′ to C-3, H-1” to C-2′, H-1′′′ to C-3′ observed in the HMBC experiment. The 1H- and 13C-NMR data for the protons and carbons in the A–E rings were identical to those of Yucca spirostanoside D1  with those of (25R)-5-spirostan  -β-d-galactopyranoside.A white powder with negative optical rotation ([α] 13C-NMR  spectra  (C-22)] , further1H- and 13C-NMR -5β-spirostan-3β,12β-diol. The difference was that the 2-OH was lacking in the structures of 14/15, 34/36 and 40/41, meanwhile the 12-C=O of 10 has been changed into a 12-OH group. The long-range correlations from H-14, 17 and H3-18 to C-12 observed in their HMBC spectra as well as the correlations between H2-2 and H-3 displayed in their 1H-1H COSY spectra verified the correctness of the above speculation. By comparing the C-22–26 and 27 carbon signals of 10, 14/15, 34/36 and 40/41 they were determined to be 25R and 25S isomer mixtures. As their 1H- and 13C-NMR data were consistent with those of 25(R and S) schidigera-saponin F1 , one β-d-glucopyranosyl [δ 5.65 ], together with one β-d-xylopyranosyl [δ 5.29 ]; 34/36 possessed one β-d-glucopyranosyl [δ 4.90 ], and one β-d-xylopyranosyl [δ 5.27 ]; 40/41 possessed one β-d-glucopyranosyl [δ 4.92 ]. Finally, combining the long-range correlations from H-1′ to C-3, H-1” to C-2′, and H-1′′′ to C-3” for 14/15; H-1′ to C-3, and H-1” to C-3” for 34/36; H-1′ to C-3 for 40/41, their structures were elucidated to be 25(S/R)-5β-spirostan-3β,12β-diol 3-O-β-d-xylopyranosyl(1→3)-[β-d-glucopyranosyl(1→2)]-β-d-glucopyranoside (14/15), 25(S/R)-5β-spirostan-3β,12β-diol 3-O-β-d-xylopyranosyl(1→3)-β-d-glucopyranoside (34/36) and 25(S/R)-5β-spirostan-3β,12β-diol 3-O-β-d-glucopyranoside (40/41), respectively. According to the chromatographic elution order summarized in S)-spirostanol saponin was shorter than that of 25(R)-spirostanol saponin, thus the six peaks were finally identified as 25(S)-5β-spirostan-3β,12β-diol 3-O-β-d-xylopyranosyl(1→3)-[β-d-glucopyranosyl(1→2)]-β-d-glucopyranoside (14), 25(R)-5β-spirostan-3β,12β-diol 3-O-β-d-xylopyranosyl(1→3)-[β-d-glucopyranosyl(1→2)]-β-d-glucopyranoside (15), 25(S)-5β-spirostan-3β,12β-diol 3-O-β-d-xylopyranosyl (1→3)-β-d-glucopyranoside (34), 25(R)-5β-spirostan-3β,12β-diol 3-O-β-d-xylopyranosyl(1→3)-β-d-glucopyranoside (36), 25(S)-5β-spirostan-3β,12β-diol 3-O-β-d-glucopyranoside (40) and 25(R)-5β-spirostan-3β,12β-diol 3-O-β-d-glucopyranoside (41), respectively.The  spectra , 14/15 p27, 38 and 51 were all white powders with negative optical rotations. They were determined to possess the same aglycon by comparing their 1H- and 13C-NMR (5D5N) data with each other, that was identical to the speculation in 1H-, 13C-NMR and 2D-NMR  spectra of the aglycon indicated that it was similar to that of 14/15, the difference being that the 12-OH group was lacking while a 12-C=O appeared in the structures of these three compounds. This speculation was proven by the long-range correlations from H-14, H-17 and H3-18 to C-12 observed in their HMBC spectra , 5.35 ] suggested the presence of two β-d-glucopyranosyls. In its HMBC spectrum, long-range correlations from H-1′ to C-3, H-1” to C-3′ could be observed. As a result, their structures were finally elucidated to be (25R)-5β-spirostan-3β-ol-12-one 3-O-β-d-apiofuranosyl-(1→3)-[β-d-glucopyranosyl(1→2)]-β-d-glucopyranoside (27), (25R)-5β-spirostan-3β-ol-12-one 3-O-β-d-xylopyranosyl(1→3)-[β-d-glucopyranosyl(1→6)]-β-d-glucopyranoside (38) and (25R)-5β- spirostan-3β-ol-12-one 3-O-β-d-glucopyranosyl(1→3)-β-d-glucopyranoside (51), respectively. The appearance of a β-d-apiofuranosyl moiety provided new possibilities for speculation about the structure of other compounds.Compounds  13C-NMR , C5D5N d spectra . In addiuranosyl . The HSQY. schidigera.These results not only confirmed the speculation based on chromatographic retention behaviors and the MS/MS cleavage patterns, but also made supplemented the assignment of the spirostanol saponin glycosyl substituents of Y. schidigera, especially, the targeted isolation experiment made the analysis results more convincing. Moreover, the rules summarized in this paper also provided more accurate references to identify this kind of compounds.In general, based on the phytochemistry researches reported before, this study accomplished a comprehensive chemical profiling of the spirostanol saponins in 1, A2, B1, B2, B3, C1, C2, C3, D1, schidigera-saponin A1, schidigera-saponin A3, 5β-spirost-25(27)-en-3β-ol-12-one 3-O-β-d-glucopyranosyl(1→2)-O-[β-d-glucopyranosyl(1→3)]-β-d-glucopyranoside, schidigera-saponin C2, schidigera-saponin C1  were used for LC/MS analysis.Acetonitrile (ACN), methanol (MeOH), formic acid (FA) of HPLC grade , ultra-pure water prepared with a Milli-Q purification system  and different HPLC columns , nine fractions  were given. YSEM7–YSEM9 were evaporated to dryness under reduced pressure, and then dissolved with MeOH to get three test stock solutions (5 mg/mL) due to their enrichment of spirostanol saponin. YSEM7–YSEM9 stock solutions were stored at 4 °C in darkness and brought to room temperature before use.The section “Preparation of the YSEMs Test Solutions” YSE stock solution (30.0 g) was subjected to normal pressure silica gel  column [CH2Cl2→CH2Cl2-MeOH ], and  were obtained. YSES6–YSES9 stock solutions (5 mg/mL) were prepared by using the similar method as YSEM7–YSEM9 and stored at 4 °C in darkness and brought to room temperature before use.The section “18, HSS C18, C18 and T3), mobile phase , pH (0.1% and 0.01% FA) and column temperature (30 °C and 35 °C) . Samples were separated on a Waters ACQUITY UPLC® T3  using a mobile phase composed of H2O (A) and FA-MeOH-ACN  (B) in the gradient program: 0–15 min, 12–24% B; 15–30 min, 24–32% B; 30–42 min, 32–40% B; 42–47 min, 40–60% B; 47–65 min, 60–95% B. An equilibration of 3 min was used between successive injections. The flow rate was 0.3 mL/min, and column temperature was 30 °C. An aliquot of 3 µL of each sample was injected for analysis.Separation of spirostanol saponins was performed on a Thermo UltiMate 3000 UHPLC instrument equipped with a quaternary pump, an autosampler. After the optimization of stationary phase (BEH C2) and high purity nitrogen (N2) were used as the collision gas and the sheath/auxiliary gas, respectively. The ESI source parameters were 350 °C for capillary temperature, 300 °C for ion source heater temperature, 40 arbitrary units for sheath gas (N2), 10 arbitrary units for auxiliary gas (N2), and the collision energy of the quadrupole ranged between 15 and 45 V were used. The mass range of the Orbitrap analyzer scanner was m/z 150 to 1500. Selected precursors analyzed more than two times were actively excluded from analysis for 60 s. Monitoring time was 0–65 min. Data recording and processing were performed using the Xcalibur 4.0 software . The accuracy error threshold was fixed at 5 ppm. Automatic components extraction was accomplished by a Sieve v2.2 SP2 (Thermo Fisher Scientific) with the time range from 0 to 65 min and the BP minimum count at 10,000. Mass resolution is 70,000 and 17,500 for MS and MS2, respectively.Spirostanol saponins identification was carried out by using a Thermo ESI-Q-Exactive-Orbitrap MS (in the positive ESI mode (capillary voltage: 3.2 kV). Ultra-high purity nitrogen (NPreparation of the YSESs Test Solutions” fraction YSES6 (6.0 g) was separated by normal pressure ODS CC  [MeOH-H2O ] and 14 fractions  were obtained. Fraction 6-11 (298.3 mg) was purified by silica gel CC ) to obtain 25(S/R)-5β-spirostan-3β,12β-diol 3-O-β-D-xylopyranosyl(1→3)-β-D-glucopyranoside . Fraction 6-11-5 (28.7 mg) was subjected to silica gel CC [CH2Cl2-MeOH ], and 25(S/R)-5β-spirostan-3β,12β-diol 3-O-β-d-glucopyranoside  was given. Fraction 6-12 (400.5 mg) was purified by PHPLC  to obtain (25R)-5β-spirostan-3β-ol-12-one 3-O-β-d-glucopyranosyl(1→3)-β-d-glucopyranoside .The section “Preparation of the YSESs Test Solutions” fraction YSES7 (5.0 g) was subjected to PHPLC , and 12 fractions  were obtained. Fraction 7-5 (356.3 mg) was separated by PHPLC  to afford (25R)-5β-spirostan-3β-ol-12-one 3-O-β-d-apiofuranosyl(1→3)-[β-d-glucopyranosyl(1→2)]-β-d-gluco pyranoside .The section “Preparation of the YSESs Test Solutions” fraction YSES8 (5.0 g) was separated by PHPLC  to yield 17 fractions . Fraction 8-5 (92.4 mg) was subjected to PHPLC  and PHPLC  successively to obtain 25(S/R)-5β-spirostan-3β,12β-diol 3-O-β-d-xylopyranosyl(1→3)-[β-d-glucopyranosyl(1→2)]-β-d-glucopyranoside . Fraction 8-13 (22.1 mg) was isolated by PHPLC , and (25R)-5β-spirostan-3β-ol-12-one 3-O-β-d-xylopyranosyl(1→3)-[β-d-glucopyranosyl(1→6)]-β-d-glucopyranoside  was obtained.The section “Preparation of the YSESs Test Solutions” fraction YSES9 (6.0 g) was isolated by PHPLC  to obtain 16 fractions . Fraction 9-7 (110.1 mg) was separated successively by PHPLC  and PHPLC  to obtain 25(R)-5β-spirostan-2β,3β-ol-12-one-3-O-xylopyranosyl(1→3)-[glucopyranosyl (1→2)]-galacto pyranoside .The section “O-β-d-xylopyranosyl(1→3)-[β-d-glucopyranosyl(1→2)]-β-d-galactopyranoside (10): White powder; [α]D25: −7.7° ; IR νmax (KBr) cm−1: 3353, 2928, 2872, 1704, 1455, 1377, 1159, 1074, 1043, 983; 1H-NMR  and 13C-NMR  data δ: see m/z 903.4550 [M + H]+ .(25R)-5β-Spirostan-2β,3β-diol-12-one 3-O-β-d-xylopyranosyl(1→3)-[β-d-glucopyranosyl(1→2)]-β-d-glucopyranoside (14/15): White powder; [α]D25: −8.0° ; IR νmax (KBr) cm−1: 3398, 2930, 2876, 1644, 1453, 1373, 1160, 1077, 1043, 990; 1H-NMR  and 13C-NMR  data δ: see m/z 889.4799 [M + H]+ .25(S/R)-5β-Spirostan-3β,12β-diol 3-O-β-d-apiofuranosyl(1→3)-[β-d-glucopyranosyl(1→2)]-β-d-glucopyranoside (27): White powder; [α]D25: −10.3° ; IR νmax (KBr) cm−1: 3412, 2929, 2874, 1705, 1453, 1161, 1079, 1028, 982; 1H-NMR  and 13C-NMR  data δ: see m/z 887.4658 [M + H]+ .(25R)-5β-Spirostan-3β-ol-12-one 3-O-β-d-xylopyranosyl(1→3)-β-d-glucopyranoside (34/36): White powder; [α]D25: −36.0° ; IR νmax (KBr) cm−1: 3396, 2928, 2869, 1646, 1546, 1453, 1372, 1161, 1045, 986; 1H-NMR  and 13C-NMR  data δ: see m/z 727.4282 [M + H]+ .25(S/R)-5β-Spirostan-3β,12β-diol 3-O-β-d-xylopyranosyl(1→3)-[β-d-glucopyranosyl(1→6)]-β-d-glucopyranoside (38): White powder; [α]D25: −16.2° ; IR νmax (KBr) cm−1: 3423, 2930, 2874, 1705, 1456, 1377, 1164, 1073, 1039, 985; 1H-NMR  and 13C-NMR  data δ: see m/z 887.4632 [M + H]+ .(25R)-5β-Spirostan-3β-ol-12-one 3-O-β-d-glucopyranoside (40/41): White powder; [α]D25: −44.7° ; IR νmax (KBr) cm−1: 3414, 2930, 2871, 1646, 1563, 1452, 1376, 1166, 1061, 1022, 990; 1H-NMR  and 13C-NMR  data δ: see m/z 595.3847 [M + H]+ .25(S/R)-5β-Spirostan-3β,12β-diol 3-O-β-d-glucopyranosyl(1→3)-β-d-glucopyranoside (51): White powder; [α]D25: −0.3° ; IR νmax (KBr) cm−1: 3388, 2928, 2871, 1706, 1453, 1375, 1159, 1077, 1036, 986; 1H-NMR  and 13C-NMR  data δ: see m/z 755.4223 [M + H]+ .(25R)-5β-Spirostan-3β-ol-12-one 3-Y. schidigera, a MPLC-MS/MS analysis technique was used to accomplish the qualitative analysis of the spirostanol saponins from its extract, and a comprehensive characterization method for spirostanol saponins was set up for the first time. This will lay a foundation for the quality evaluation of Y. schidigera. On the other hand, the chromatographic retention behaviors and the MS/MS cleavage patterns of spirostanol saponins were summarized. In conclusion, in order to provide references for the quality assessment standard establishment of tR) and the exact mass-to-charge ratio (m/z), thirty-one compounds were unambiguously identified by comparing them to references. Meanwhile, the MS/MS fragmentation pattern and chromatographic elution order rules have been generalized by using the standard compounds as references, seventy-nine compounds were tentatively identified and forty of them were potential new ones. Among them, ten were targeted for separation to prove the correctness of our speculations. During the verification process, the appearance of a β-d-apiofuranosyl moiety was found for the first time, which provided new possibilities for speculation about the structure of other compounds. As a result, an accurate and comprehensive chemical composition profiling of the aerial part of the Y. schidigera was realized, which lays a foundation for the quality evaluation of the plant.In summary, according to the retention time (


I showed low micromolar antiproliferative activity, as well as antimigratory activity. Moreover, examination in a model of triple negative breast cancer (TNBC), revealed that, despite not effecting FAK phosphorylation, compound I significantly impairs proliferation whilst impairing focal adhesion growth and turnover leading to reduced migration. Further optimisation and synthesis of analogues of the lead compound I using a four-step synthetic procedure was performed, and analogues were assessed for their antiproliferative activity against three breast cancer  cell lines and one pancreatic cancer (MIAPaCa2) cell line. Compound 5f was identified as a promising lead compound with IC50 values in the range of 4.59–5.28 μM in MDA-MB-231, T47D, BT474, and MIAPaCa2. Molecular modelling and pharmacokinetic studies provided more insight into the therapeutic features of this new series.Focal adhesion kinase (FAK) is a tyrosine kinase that is overexpressed and activated in several advanced-stage solid cancers. In cancer cells, FAK promotes the progression and metastasis of tumours. In this study, we used structure-based virtual screening to filter a library of more than 210K compounds against the focal adhesion targeting FAK-focal adhesion targeting (FAT) domain to identify 25 virtual hit compounds which were screened in the invasive breast cancer line (MDA-MB-231). Most notably, compound  Focal adhesion kinase (FAK) was first described more than 20 years ago and represents a promising target for the treatment of several advanced-stage solid cancers, as well as mesothelioma and haematological malignancies. FAK is a multifunctional non-receptor tyrosine kinase whose overexpression and activation has been linked to tumour progression, survival, migration, invasion, epithelial-to-mesenchymal transition (EMT), and drug resistance through effects on the cancer cells as well as stromal cells of the tumour microenvironment ,2. FAK aFAK exerts its effects on cancer cells, as well as stromal cells of the tumour microenvironment, through both kinase-dependent and kinase-independent effects. In humans, FAK consists of the amino N-terminal region containing the FERM (4.1 protein-ezrin-radixin-moesin) domain, the central kinase domain, and the C-terminal focal adhesion targeting (FAT) domain . FAK posThe first strategy has been to develop ATP-competitive kinase inhibitors which are designed to bind residues surrounding the ATP-binding pocket of FAK around the tyrosine 397 (Y397) autophosphorylation site. As this pocket is similar in many different kinases, there is the potential for off-target effects stemming from a lack of kinase selectivity. For example, compound TAE-226  which isA second strategy towards targeting FAK is based on the inhibition of the key FAK kinase-independent (scaffolding) functions which are not blocked by FAK kinase inhibitors and are possibly enhanced by it. The scaffolding function effects help to explain why FAK kinase inhibition can lead to unpredictable therapeutic outcomes .Moreover, several reports suggest that although FAK kinase activity is necessary for cell motility, it may be not essential for cell proliferation and survival. Additionally, to avoid selectivity problems typically found with competitive inhibitors of the ATP binding site, inhibiting the scaffolding function of FAK might be a useful alternative approach . TargetiIncreased FAK mRNA levels are found in several human malignancies. Large databases such as the Cancer Genome Atlas show that FAK mRNA levels are increased in invasive breast cancers (26%) , and thaMoreover, the mRNA expression levels of the FAK encoding gene (PTK2), FAK phosphorylation as well as the focal adhesion points (FAs) are higher in triple negative breast cancer (TNBC), an aggressive BC type characterised by high metastatic potential. FAK inhibition prevented the oestrogen induced migration of invasive MDA-MB 231 cells . FurtherC4 (chloropyramine hydrochloride) (C4 displays its anticancer activity at a remarkably high micromolar concentration (>100 μM). Our previous lead optimisation work on the chemical structure of chloropyramine (C4) led to the identification of a more active analogue against three human breast cancer lines  with an average antiproliferative activity (IC50) of approximately 23 μM .  in sdf format and prepared using the conformational Search tool in MOE. The virtual screening simulations were performed using the Glide SP within Maestro software using the default settings . The pocket used for the high throughout virtual screening was focused on the area surrounding the three amino acids: Asp 1036, Ser 910, and Lys 1032 which also encompasses the Tyr 925 residue within a 12A° radius. The virtual screening output database was saved as a mol2 file, and the visual inspection of the docking modes was performed in MOE. The crystal structure of the focal adhesion targeting (FAT) domain was downloaded from the Protein Data Bank (PDB code 1K05)  and prep5 cells/mL in 24-well plates. Following 24 h, the media were replaced with fresh RPMI + 5% FCS, in the presence or absence of treatments and a selection of wells counted (day 0 counts). Cells were subsequently counted each day up to 7 days to assess proliferation. Briefly, this entailed the removal of old media and the addition of trypsin/EDTA in order to lift cells before passing gently through a 25 G needle to achieve a single-cell suspension. The resulting solution was then added to isoton in a counting cup and the cell number was determined using a Coulter Multisizer III  Each well was counted twice with all conditions performed in triplicate.To assess proliferation, cells were seeded at 4.2 × 102 for 18 h. After this migratory period was completed, cells were fixed to the underside of the inserts in 3.7% PFA in PBS and stained with 0.5% crystal violet solution. Resulting inserts were then imaged using a standard light microscope.Migration was assessed utilising 24-well, transmembrane permeable support plates  with 6.5 mm microporous membrane (8 μm pore size) inserts, each coated with 10 μg/mL fibronectin in sterile PBS. Inserts were placed into wells containing RPMI + 5% FCS (± treatments), before cells in serum-free RPMI were seeded into the top portion of each insert at a density of 50,000 cells/mL. Cells were then incubated at 37 °C and 5% COY861 and vinculin antibodies were diluted to working concentration  in 1% BSA in PBS. Specific fluorophore-conjugated secondary antibodies were then applied (Alexafluor-488 and Alexfluor-594 diluted 1:1000 in 1% BSA in PBS), and resulting coverslips were briefly washed in PBS and mounted to glass slides using hard-set Vectashield mounting media plus DAPI . Slides were viewed using a 63X oil immersion lens on a Leica DM IRE2 microscope .MDA-MB-231 TNBC cells were seeded at 700,000 cells/mL onto fibronectin coated coverslips and allowed to proliferate until 50% confluent, at which point they were treated with serum-free RPMI for 24 h. Following this, cells were incubated with RPMI+ 5% FCS for 1 h, before being washed briefly in sterile PBS, and fixed in 3.7% PFA for 15 min. The resulting fixed cells were subsequently permeabilised with 0.2% Triton-X100 in PBS for 8 min. This was proceeded by a 40 min block in 10% normal goat serum (in 1% BSA in PBS) prior to 30 min primary antibody incubation. Both FAK3a–i) were prepared by the dropwise addition of the corresponding substituted phenyl magnesium bromide  0.5M solution in THF (5.7mmol) to a solution of the respective aldehyde (5.7mmol) (2a–d) in THF (20 mL) at 0 °C. The reaction mixture was stirred for 24 h from 0 °C to room temperature. The mixture was concentrated under vacuum then quenched by adding saturated aqueous NH4Cl (30 mL) and extracted with ethyl acetate (3 × 30 mL). The combined organic layers were washed with brine (20 mL) and water (30 mL), dried over anhydrous magnesium sulphate, filtered, and concentrated under vacuum. The crude residue was purified by column chromatography eluting with hexane-ethyl acetate gradually increasing from 100:0 to 90:10 v/v. Pure products were obtained in good yields (47%–77%).The diaryl methanol derivatives ((4-tert-butylphenyl)(4-(trifluoromethoxy)phenyl)methanol (3a), 1H NMR (CDCl3) δ 7.45 , 7.41 , 7.31 , 7.21 , 5.86 , 2.57 , 1.34 . 19F (CDCl3) δ −57.84. 13C NMR (CDCl3) δ 150.99 (ArC), 148.40 (ArC), 142.44 (ArC), 140.45 (ArC), 127.86 (ArCH), 126.32 (ArCH), 125.65 (ArCH), 120.90 (ArCH), 120.47 , 75.40 (CH), 34.58 (C(CH3)3), 31.33 (C(CH3)3), MS : calcd for C18H19F3O2 [M + H-OH] 308.1388; found 308.1384.(4-tert-butylphenyl)(4-((trifluoromethyl)thio)phenyl)methanol (3b), 1H NMR (CDCl3) δ 7.67 , 7.48 , 7.44 , 7.30 , 5.79 , 2.95 , 1.39 3). 19F (CDCl3) δ −42.65, 13C NMR (CDCl3) δ 151.09 (ArC), 146.88 (ArC), 140.20 (ArC), 136.35 (ArCH), 129.67 , 127.52 (ArCH), 126.49 (ArCH), 125.68 (ArCH), 75.49 (CH), 31.59 (C(CH3)3), 31.34 (CH3)3. MS : calcd C18H19F3OS [M − H] 339.1031; found 339.1033.(4-tert-Butylphenyl)(4-fluorophenyl) methanol (3c), 1H NMR (CDCl3) δ 7.44–7.36 , 7.34–7.28 , 7.05 , 5.82 , 2.50–2.25 , 1.33 3. 19F (CDCl3) δ −115.24, 13C NMR (CDCl3) δ 162.15 , 150.75 (ArC), 140.78 (ArC), 139.67 (ArC), 128.19 , 126.28 (ArCH), 125.53 (ArCH), 115.22 , 70.87 (CH), 31.50 C(CH3)3, 25.10 (CH3)3. MS : calcd C17H19FO [M − H] 257.1342; found 257.1338 and [M−H2O + H] 241.1393; found 241.1390.(4-tert-butylphenyl)(2-fluorophenyl)methanol (3d), 1H NMR (CDCl3) δ 7.57 , 7.39–7.35 , 7.29–7.26 , 7.19 , 6.16 , 2.26 , 1.33 3). 19F (CDCl3) δ −118.50, 13C NMR (CDCl3) δ 159.91 , 150.71 (ArC), 139.79 (ArC), 131.04 , 129.00 , 127.64 , 126.14 (ArC), 125.46 (ArC), 124.28 , 70.01 (CH), 34.53 (C(CH3)3), 31.33 (CH3)3. MS : calcd C17H19FO [M − H] 257.1342; found 257.1341 and [M−H2O + H] 241.1; found 241.1.(4-tert-Butylphenyl)(3-fluorophenyl) methanol (3e), 1H NMR (CDCl3) δ 7.40 , 7.35–7.29 , 7.21–7.14 , 7.97 , 5.84 , 2.21 , 1.34 3). 19F (CDCl3) δ −112.92, 13C NMR (CDCl3) δ 162.94 , 150.95 (ArC), 146.45 , 140.40 (ArC), 129.87 , 126.35 (ArCH),), 125.60 (ArCH), 122.00 , 114.23 , 113.36 , 75.52 , 34.56 (C(CH3)3), 31.32 (CH3)3. MS : calcd C17H19FO [M − H] 257.1342; found 257.1341 and [M−H2O + H] 241.1; found 241.1.Bis(4-tert-butylphenyl)methanol (3f), 1H NMR (CDCl3) δ 7.40–7.38 , 7.36–7.33 , 5.84 , 2.16 , 1.34 3). 13C NMR (CDCl3) δ 150.40 (ArC), 141.00 (ArC), 126.25 (ArCH), 125.39 (ArCH), 79.95 (CH), 34.52 C(CH3)3, 31.36 (CH3)3. MS : calcd for C21H28O [M − H] 295.2062; found 295.2061 and [M−H2O + H] 279.2113; found 279.2115.(4-Chlorophenyl)(4-trifluoromethoxyphenyl)methanol (3g), 1H NMR (CDCl3) δ 7.38 , 7.35 , 7.30 , 7.21 , 5.80 , 2.57 . 19F (CDCl3) δ −57.85, 13C NMR (CDCl3) δ 148.69 (ArC), 141.99 (ArC), 141.77 (ArC), 133.69 (ArC), 128.81 (ArCH), 128.81 (ArCH), 127.91 (ArCH), 127.86 (ArCH), 121.07 (ArCH), 120.44 , 74.90 (CH). MS : calcd for C14H10ClF3O2 [M + H-H2O] 285.0294; found 285.0298.Bis(4-trifluoromethoxyphenyl)methanol (3h), 1H NMR (CDCl3) δ 7.41 , 7.23 , 5.86 , 2.50 . 19F (CDCl3) δ −57.91, 13C NMR (CDCl3) δ 148.70 (ArC), 141.87 (ArC), 127.93 (ArCH), 121.11 (ArCH), 120.45 , 74.82 (CH). MS : calcd C15H10F6O3 [M − H] 351.0456; found 351.0456.3a–i, 7.20 mmol) in dichloromethane (2 mL) was stirred at room temperature for 2 h. The reaction was concentrated under reduced pressure to give the diaryl chloride. To a solution of the diaryl chloride (7.20 mmol) in dichloromethane (33.12 mL), trimethylsilyl cyanide (7.20 mmol) and titanium tetrachloride  were added. After stirring under argon at room temperature for 2 h, the reaction was quenched with methanol (13.90 mL) and water (41.62 mL) and diluted with dichloromethane (104 mL). The organic layer was washed with saturated, aqueous sodium bicarbonate (68.25 mL) and water (68.25 mL), dried over MgSO4, filtered, and concentrated under reduced pressure to give the crude product.A solution of thionyl chloride (10.80 mmol) and diaryl alcohol (2-(4-tert-butylphenyl)-2-(4-trifluoromethoxyphenyl)acetonitrile (4a), 1H NMR (CDCl3) δ 7.48–7.41 , 7.31 , 7.26 , 5.18 , 1.36 . 19F (CDCl3) δ −57.87. 13C NMR (CDCl3) δ 151.68 (ArC), 148.98 (ArC), 134.79 (ArC), 132.26 (ArC), 129.28 (ArCH), 127.39 (ArCH), 126.34 (ArCH), 121.58 (ArCH), 120.41 , 119.46 (CN), 41.58 (CH), 34.63 (C(CH3)3), 31.24 (C(CH3)3). MS : calcd for C19H18F3NO [M + Na], 356.1233; found, 356.1236.2-(4-tert-butylphenyl)-2-(4-(trifluoromethyl)thiophenyl)acetonitrile (4b), 1H NMR (CDCl3) δ 7.69 , 7.47–7.42 , 7.30–7.27 , 5.17 , 1.34 3). 19F (CDCl3) δ -42.45, 13C NMR (CDCl3) δ 151.81 (ArC), 139.04 (ArC), 136.89 (ArCH), 131.88 (ArC), 129.40 , 128.83 (ArCH), 127.43 (ArCH), 126.37 (ArCH), 124.61 , 119.13 (CN), 41.93 (CH), 34.64 (C(CH3)3), 31.23 (CH3)3. MS : calcd C19H18F3NS [M + H] 350.1190; found 350.1185, [M + H − HCN] 323.1081; found 323.1082.2-(4-tert-butylphenyl)-2-(4-fluorophenyl)acetonitrile (4c), 1H NMR (CDCl3) δ 7.46 , 7.39 , 7.36 , 7.10 , 5.17 , 1.38–1.37 3). 19F (CDCl3) δ -113.86. 13C NMR (CDCl3) δ 162.45 , 151.492 (ArC), 132.82 (ArC), 132.14 (ArC), 129.55 , 127.40 (ArCH), 126.29 (ArCH), 116.06 (CN), 116.15 , 41.46 (CH), 34.63 (C(CH3)3), 31.31 (CH3)3. MS : calcd C18H18FN [M + NH4] 285.1767; found 285.1766.2-(4-tert-Butylphenyl)-2-(2-fluorophenyl)acetonitrile (4d), 1H NMR (CDCl3) δ 7.38 , 7.30 , 7.26–7.22 , 7.10 , 7.01 , 5.34 , 1.22 3). 19F (CDCl3) δ -117.28. 13C NMR (CDCl3) δ 159.70 , 151.43 (ArC), 131.65 (ArC), 130.26 , 129.31 , 127.17 (ArCH), 126.15 (ArCH), 124.92 , 123.66 , 119.05 (CN), 116.25 , 35.66 , 34.59 C(CH3)3, 31.25 (CH3)3. MS : calcd C18H18FN [M + NH4] 285.1767; found 285.1765.2-(4-tert-butylphenyl)-2-(3-fluorophenyl)acetonitrile (4e), 1H NMR (CDCl3) δ 7.43 , 7.41–7.35 , 7.29 , 7.20 , 7.11- 7.02 , 5.13 , 1.34 3). 19F (CDCl3) δ -111.36. 13C NMR (CDCl3) δ 162.99 , 151.65 (ArC), 138.37 , 132.15 (ArC), 130.73 , 127.38 (ArCH), 126.28 (ArCH), 123.41 , 119.31 (CN), 115.28 , 114.99 , 41.89 , 34.62 C(CH3)3, 31.24 (CH3)3. MS : calcd C18H18FN [M + NH4] 268.1501; found 268.1494.2,2-Bis(4-tert-butylphenyl)acetonitrile (4f), 1H NMR (CDCl3) δ 7.42–7.40 , 7.31–7.29 , 5.11 , 1.33 3). 13C NMR (CDCl3) δ 151.20 (ArC), 133.03 (ArC), 127.39 (ArCH), 126.08 (ArCH), 120.04 (CN), 41.82 (CH), 34.57 C(CH3)3, 31.27 (CH3)3. MS : calcd C22H27N [M + NH4] 323.2487; found 323.2487.2-(4-Chlorophenyl)-2-(4-trifluoromethoxyphenyl)acetonitrile (4g), 1H NMR (CDCl3) δ 7.42–7.37 , 7.31 , 7.26 , 5.16 . 19F (CDCl3) δ -57.90.13C NMR (CDCl3) δ 149.19 (ArC), 134.73 (ArC), 134.04 (ArC), 133.78 (ArC), 129.59 (ArCH), 129.23 (ArCH), 129.06 (ArCH), 121.75 (ArCH), 120.35 , 118.83 (CN), 41.37 (CH). MS : calcd for C15H9ClF3NO [M] 311.0325; found 311.0327.2,2-Bis(4-trifluoromethoxyphenyl)acetonitrile (4h), 1H NMR (CDCl3) δ 7.41 , 7.28 , 5.21 . 19F (CDCl3) δ -57.97, 13C NMR (CDCl3) δ 149.26 (ArC), 133.93 (ArC), 129.27 (ArCH), 121.76 (ArCH), 120.36 , 118.83 (CN), 41.30 (CH). MS : calcd C16H9F6NO2 [M] 361.0537; found 361.0531, [M + H] 362.0616; found 362.0595, [M + H-HCN] 335.0507; found 335.0530.2,2-Bis(4-methoxyphenyl)acetonitrile (4i), 1H NMR (CDCl3) δ 7.27 , 6.91 , 5.08 , 3.83 . 13C NMR (CDCl3) δ 159.40 (ArC), 128.82 (ArCH), 128.30 (ArC), 120.13 (CN), 114.50 (ArCH), 55.36 (2xCH3), 41.07 (CH). MS : calcd for C16H15NO2 [M − H] 252.1019; found 252.1021.4a–i, 0.78 mmol) in diethylether (10 mL) was added dropwise. After stirring under nitrogen for 24 h, the reaction was quenched with water (10 mL). The reaction mixture was extracted with ethyl acetate (3 × 50mL). The combined organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure to give the crude product. The crude mixture was treated with a 2 M anhydrous HCl solution in diethyl ether (5–10 mL), and the resulting amine hydrochloride salts were washed with diethyl ether. If the amine hydrochloride salts did not form a precipitate, the diethyl ether solution was concentrated under reduced pressure and rinsed with diethyl ether to give the hydrochloride salts.To a suspension of lithium aluminum hydride (2.35 mmol) in anhydrous diethylether (20 mL) at 0 °C, a solution of nitrile (2-(4-tert-butylphenyl)-2-(4-trifluoromethoxyphenyl)ethan-1-aminium hydrochloride (5a), 1H NMR (CDCl3) δ 8.12 , 7.35 , 7.31 , 7.17 , 7.14 , 4.44 , 3.57 -3.42 , 1.29 . 19F (CDCl3) δ −57.81. 13C NMR (CDCl3) δ 150.89 (ArC), 148.50 (ArC), 138.30 (ArC), 135.90 (ArC), 129.53 (ArCH), 127.40 (ArCH), 126.19 (ArCH), 121.49 (ArCH), 120.39 , 47.57 (CH), 43.78 (CH2), 34.49 (C(CH3)3), 31.23 (C(CH3)3). MS : calcd for C19H23ClF3NO [M + H] 338.1727; found 338.1726.2-(4-tert-butylphenyl)-2-(4-(trifluoromethyl)thiophenyl)ethan-1-aminium chloride (5b), 19F (CDCl3) δ −42.45, 1H NMR (CDCl3) δ 8.05 , 7.43–7.32 , 7.27–7.10 , 4.49 , 3.60–3.30 , 1.31 3). 13C NMR (CDCl3) δ 151.49 (ArC), 139.63 (ArC), 136.98 (ArCH), 130.78 (ArC), 128.24 , 128.01 (ArCH), 127.41 (ArCH), 126.38 (ArCH), 42.15 (CH), 34.43 C(CH3)3, 31.30 C(CH3)3. MS : calcd C19H23ClF3NS [M + H]+354.1498; found 354.1501.2-(4-tert-butylphenyl)-2-(4-fluorophenyl)ethan-1-aminium chloride (5c), 19F (CDCl3) δ −114.15, 1H NMR (CDCl3) δ 8.07 , 7.36–7.31 , 7.03–6.94 , 4.42 , 3.51–3.50 , 1.29 3). 13C NMR (CDCl3) δ 161.99 , 150.48 (ArC), 136.73 (ArC), 135.58 (ArC), 129.78 , 127.40 (ArCH), 126.03 (ArCH), 125.57 , 34.45 (CH), 34.46 (CH2), 31.25 C(CH3)3, 29.71 (CH3). MS : calcd C18H23ClFN [M-Cl]+ 272.1809; found 272.1808.2-(4-tert-butylphenyl)-2-(2-fluorophenyl)ethan-1-aminium chloride (5d), 1H NMR (CDCl3) δ 8.10 , 7.35–7.29 , 7.22–7.19 , 3.94 , 3.62–3.57 , 3.52–3.48 , 1.29 3). 19F (CDCl3) δ -115.13. 13C NMR (CDCl3) δ 149.70 , 143.94 (ArC), 135.51 (ArC), 130.18 , 129.28 , 127.54 (ArCH), 126.00 (ArCH), 122.95 , 118.30 , 116.32 , 42.82 (CH2), 31.25 , 30.56 (C(CH3)), 30.03 (CH3). C18H23ClFN [M-Cl]+, 272.1809; found 272.1806.2-(4-tert-butylphenyl)-2-(3-fluorophenyl)ethan-1-aminium chloride (5e), 1H NMR (CDCl3) δ 8.15–7.81 , 7.38–7.32 , 7.26–7.20 , 7.19–7.16 , 4.48–4.40 , 3.60–3.40 , 1.31–1.27 3). 19F (CDCl3) δ -111.70. 13C NMR (CDCl3) δ 163.06 , 143.94 (ArC), 135.51 (ArC), 130.18 , 129.28 , 127.54 (ArCH), 126.00 (ArCH), 122.95 , 118.30 , 116.32 , 43.71 (CH2), 47.79 (CH), 34.49 (C(CH3)3), 31.24 ((CH3)3). C18H23ClFN [M-Cl]+, 272.1809; found 272.1808.2,2-Bis(4-tert-butylphenyl)ethan-1-aminium chloride (5f), 1H NMR (CDCl3) δ 8.08 , 7.33 , 7.21 , 3.94 , 3.52 , 1.29 3). 13C NMR (CDCl3) δ 150.41 (ArC), 136.55 (ArC), 127.57 (ArCH), 126.05 (ArCH), 47.65 (CH), 44.08 (CH2), 34.46 C(CH3)3, 31.30 (CH3)3. MS : calcd C22H32ClN [M + H] 310.2529; found 310.2529.2-(4-chlorophenyl)-2-(4-trifluoromethoxyphenyl)ethan-1-aminium chloride (5g), 1H NMR (CDCl3) δ 8.13 , 7.32 , 7.27 , 7.23–7.16 , 4.43 , 3.44 . 19F (CDCl3) δ -57.85, 13C NMR (CDCl3) δ 148.69 (ArC), 137.72 (ArC), 137.15 (ArC), 133.88 (ArC), 129.44 (ArCH), 129.40 (ArCH), 129.31 (ArCH), 121.64 (ArCH), 120.37 , 47.48 (CH), 43.31 (CH2). MS : calcd for C15H14Cl2F3NO [M + H] 316.0711; found 316.0712.2,2-Bis(4-trifluoromethoxyphenyl)ethan-1-aminium chloride (5h), 19F (CDCl3) δ −57.84, 1H NMR (CDCl3) δ 8.15 , 7.42–7.33 , 7.12–7.08 , 4.26–4.20 , 3.35–3.28 . 13C NMR (CDCl3) δ 148.27 (ArC), 142.90 (ArC), 128.88 (ArCH), 121.34 , 120.96 (ArCH), 52.25 (CH), 50.71 (CH2). MS : calcd C16H14ClF6NO2 [M + H]+ 366.0923; found 366.0924.2,2-Bis(4-methoxyphenyl)ethan-1-aminium chloride (5i), 1H NMR (CDCl3) δ 8.04 , 7.17 , 6.85 , 4.38 , 3.76 , 3.48 . 13C NMR (CDCl3) δ 158.59 (ArC), 133.15 (ArC), 128.95 (ArCH), 114.31 (ArCH), 55.25 (2xCH3), 53.27 (CH), 45.23 (CH2). MS : calcd for C16H20ClNO2 [M + H] + 258.1489; found 258.1486.All in vitro biological evaluations were performed by Cyprotex, according to their internal procedures. The detailed protocols procedure can be found online.The test compound was diluted in buffer to give a range of concentrations  and incubated at 37 °C for 2 hr. Absorbance was measured at a wavelength of 620 nm and the solubility was estimated from the concentration of the test compound that produces an increase in absorbance above the vehicle control . The test compound (10 mM in DMSO) was serially diluted to give solutions of 0.1, 0.3, 1, and 3 mM in DMSO. Each test compound concentration was then further diluted 1 to 100 in buffer  so that the final DMSO concentration was 1%, and the final test compound concentrations were 1, 3, 10, 30, and 100 μM. The experiment was performed at 37 °C and each concentration was incubated in 7 replicate wells. The plates were incubated for 2 h at 37 °C before the absorbance was measured at 620 nm. Nicardipine and pyrene were included as control compounds. The solubility of nicardipine is pH dependent whereas the solubility of pyrene is pH independent.The solubility was estimated from the concentration of the test compound that produced an increase in absorbance above the vehicle control .An estimated precipitation range (lower and upper bound) and a calculated mid-range value were returned, along with any relevant comments, in the form of an Excel spreadsheet.int) with standard error and t1⁄2 values were measured.The test compound (3 μM) was incubated with pooled liver microsomes at 5 time points over the course of a 45 min experiment, and the test compound was analysed by LC-MS/MS. Intrinsic clearance values , 0.1 M phosphate buffer pH 7.4, and the test compound  were pre-incubated at 37 °C prior to the addition of NADPH  to initiate the reaction. The final incubation volume was 50 μL. A minus cofactor control incubation was included for each compound tested where 0.1 M phosphate buffer pH 7.4 was added instead of NADPH (minus NADPH). Two control compounds were included with each species. All incubations were performed singularly for each test compound.Each compound was incubated for 0, 5, 15, 30, and 45 min. The control (minus NADPH) was incubated for 45 min only. The reactions were stopped by transferring 20 μL of incubate to 60 μL methanol at the appropriate time points. The termination plates were centrifuged at 2500 rpm for 20 min at 4 °C to precipitate the protein.Following protein precipitation, the sample supernatants were combined in cassettes of up to 4 compounds, the internal standard was added, and samples were analysed using Cyprotex generic LC-MS/MS conditions.From a plot of ln peak area ratio  against time, the gradient of the line was determined. Subsequently, half-life and intrinsic clearance were calculated using the equations below:50 value (test compound concentration which produces 50% inhibition). IC50 is delivered, standard error (SE IC50), and n  for the test compound. The experiments were performed on an Ion Works TM automated patch clamp instrument ), which simultaneously performs electrophysiology measurements for 48 single cells in a specialised 384-well plate ). All cell suspensions, buffers, and test compound solutions were at room temperature during the experiment.Mammalian cells expressing the hERG potassium channel were dispensed into 384-well planar arrays and hERG tail-currents measured by whole-cell voltage-clamping. A range of concentrations of the test compound was then added to the cells and a second recording of the hERG current was made. The percent change in hERG current was calculated and used to calculate an ICThe cells used were Chinese hamster ovary (CHO) cells stably transfected with hERG . A single-cell suspension was prepared in extracellular solution  and aliquots added automatically to each well of a Patch Plate TM. The cells were then positioned over a small hole at the bottom of each well by applying a vacuum beneath the plate to form an electrical seal. The vacuum was applied through a single compartment common to all wells which was filled with intracellular solution (buffered to pH 7.2 with HEPES). The resistance of each seal was measured via a common ground-electrode in the intracellular compartment and individual electrodes placed into each of the upper wells.Electrical access to the cell was then achieved by circulating a perforating agent, amphotericin B, underneath the PatchPlateTM. The pre-compound hERG current was then measured. An electrode was positioned in the extracellular compartment and a holding potential of −80 mV applied for 15 s. The hERG channels were then activated by applying a depolarising step to +40 mV for 5 s and then clamped at −50 mV for 4 s to elicit the hERG tail current, before returning to −80 mV for 0.3 s.Compound dilutions were prepared by diluting a DMSO solution (default 10 mM) of the test compound using a factor 5 dilution scheme into DMSO, followed by dilution into extracellular buffer such that the final concentrations tested were typically 0.008, 0.04, 0.2, 1, 5, and 25 μM . The Ion Works TM instrument automatically adds test compound dilutions to the upper wells of the PatchPlateTM. The test compound was left in contact with the cells for 300 s before recording currents using the same voltage-step protocol as in the pre-compound scan. Quinidine, an established hERG inhibitor, was included as a positive control and vehicle control (0.25% DMSO) as negative control.Each concentration was tested in 4 replicate wells on the PatchPlateTM (maximum of 24 data points). Filters were applied to ensure only acceptable cells were used to assess hERG inhibition. The cell must maintain a seal resistance of greater than 50 MOhm and a pre-compound current of at least 0.1 nA and ensure cell stability between pre-and post-compound measurements.For each replicate, the hERG response was calculated using the following equation:50 value calculated:max = mean vehicle control response; x = concentration; IC50 = concentration required to inhibit current by 50%; s = Hill slope.The % hERG response was plotted against concentration for the test compound and, where concentration-dependent inhibition was observed, the data were fitted to the following equation and an ICI. Subsequently, a series of compound I analogues was synthesised and screened for antiproliferative activity in three breast cancer  cell lines and one pancreatic cancer (MIAPaCa2) cell line. Compounds I and 5f displayed low micromolar antiproliferative activity across the four cell lines. Moreover, compounds I and 5f have promising metabolic stability in the liver microsome, and compound 5f lacks cardiotoxicity in the hERG inhibition assay. With no FAK inhibitors currently on the market, compound 5f represents a promising drug-like small molecule that justifies further development as a lead compound for breast and pancreatic cancer therapy.So far, the main approach for FAK inhibition has been focused on FAK catalytic function, which mainly targets the Y397 tyrosine phosphorylation site by interfering with the binding of ATP. This has resulted in the development of several FAK kinase inhibitors such as defactinib in the clinical stage. However, this approach lacks specificity with varying degrees of cross-reactivity with other tyrosine kinases and associated unwanted side effects. Alternatively, the inhibition of the FAK scaffolding function and the downstream signalling is believed to be a favourable strategy, and likely offers more specific FAK inhibitors rather than focusing solely on the kinase function. In this study, we used a structure based virtual screening approach to identify a small molecule hit compound


In the present work, we consider the transmission properties of a Gaussian wavepacket when transmits through few double and multi-slit systems in a fractional medium. For this purpose, we have solved the two-dimensional fractional Schrodinger equation utilizing a split-step Fourier method. Then, we have investigated the effects of different parameters such as the number of slits, slit width, barrier width, layer width, layer heights, fractional order, and wavepacket width on the transmission coefficient, and wavepacket evolution. The quantum mechanical description of the double-slit experiment can change one's ideas about different classical concepts such as waves, particles, movement, location, etc. In a double-slit experiment on 1909 by Taylor, a very dim light emits just a single photon at a time, led to Paul Dirac’s famous claim: each photon interferes only with itself2.Young double-slit experiment . However, this fact is consistent with our common physics insight. In this figure, increasing the layer height to 50 can decreases the transmission coefficient by 50%. Using a small decrease of the fractional parameter α = 1.3, the transmission coefficient at first decreases and then increases if layer height increases. This fact is not consistent with our common physical insight. Panel (C) of this figure shows the variation of the transmission coefficient T as a function of the layer width for standard non-fractional Schrodinger equation with α = 1.9, 1.6, and 1.3. In these two panels, we see that by increasing the layer width the transmission coefficient decreases. Also, by increasing the fractional parameter α = 1.3, the layer width is a less critical parameter in the transmission coefficient because in all of the studied layer widths, the transmission coefficient is approximately 100%.In the panel (A) of the Fig. In the Fig. entclass1pt{minimaentclass1pt{minimaα = 1.6 and 1.3. Besides, in the panel (C), we also observe that increasing the barrier width the transmission coefficient decreases. Panel (C) of this figure, shows the variation of the transmission coefficient T as a function of the slit width for Schrodinger equations with α = 1.9, 1.6 and 1.3. We assumed the layer width = L/30. Finally, the panel (F) illustrates the variation of the transmission coefficient T as a function of the number of slits for Schrodinger equations with Now, in the panel (A) of the Fig. Figure }α) = L/40, 2, (L/}α) = L/40, 2,  is also that the diagram of the transmission coefficient as a function of the slit width in a nonlinear one. Besides, for large values of the slit width that are greater than 3, all three diagrams saturate, and further increasing the slit width does not change the transmission coefficient any more. Also, the Fig. We present the variation of the transmission coefficient T as a function of the second slit width for standard non-fractional Schrodinger equation with α = 1.9, 1.6, 1.3 and 1.1. In this figure, we assumed system length L = 45, number of slits = 2, slit width = L/50, barrier width = L/70, layer width = L/30, layer height = 100, Laplacian coefficient β = 0.5, and nonlinearity strength γ = 0. Comparing the panels (A) and (B) shows that the behavior of the wavepacket in the standard Schrodinger equation is completely different from propagation characteristics in the fractional system. In the panel (A), there are two characteristic wavepacket widths for them that the diagram of the transmission coefficient has a local minimum and a local maximum. The interesting region is the interval between these two characteristic wavepacket widths. In this region, by increasing the wavepacket widths, the transmission coefficient increases that is not consistent with our common physics insight. In the panel (B), there is a similar behavior for the system with fractional orders α = 1.9 for wavepacket width smaller than 0.25. Another interesting fact is that for wavepacket width larger than 0.25, the transmission coefficient is 100% for the systems with the fractional orders α = 1.1. Figure In the panel (A) of the Fig. α = 1.9 and 1.6. As we mentioned in the introduction section, the fractional Laplacian means a non-parabolic dispersion, i.e., the dispersion of the system directly changes. In the standard Schrodinger equation on the panel (A), the transmission coefficient varies monotonically in an increasing manner, when the Laplacian coefficient increases. However, in the studied diagrams in the panel (B), the transmission diagrams have a maximum value. This means there is a critical Laplacian coefficient that leads to the maximum transmission coefficient. Figure γ for standard non-fractional Schrodinger equation with α = 1.9, 1.6, 1.3 and 1.1. In this figure, we assumed system length L = 45, number of slits = 2, slit width = L/100, barrier width = L/60, layer width = L/30, and layer height = 100. When the nonlinearity strength γ increases, the transmission coefficient decreases. However, the fractional order α = 1.9 is an exception, and in this case, we have an increasing diagram. Finally, Fig. γ.Other parameters are the same as the panels (A) of the Fig. Finally, in panel (A) of the Fig. α the transmission coefficient increased. For systems with small slit, the transmission coefficient was quantized when the slit width changed. By increasing the barrier width the transmission coefficient decreased. In some slit number intervals, the transmission coefficient was quantized concerning the number of slits variations and this was true for different values of the Gaussian wave parameter ‘a’ and fractional parameter α. In strongly fractional systems, the wavepacket could transmit through small and extensive slit width systems with approximately the same probability. We observed the wavepacket reconstruction after transmission through the multi-slit systems and this reconstruction was more perfect in the systems with a larger number of slits. For the incident angels greater that π/4, the transmission coefficients were almost perfect for many studied fractional parameters. For some values of the wavepacket widths, by increasing the wavepacket widths, the transmission coefficient increased. By decreasing the fractional parameter α, the wavepacket was localized within a smaller region. Finally, the double slit interference pattern exists for fractional cases, but by increasing the degree of fractionality , the interference effect gradually vanishes.In the current work, we studied the transmission through double and multi-slits systems as well as wavepacket evolution in the fractional Schrodinger framework. We showed that, by increasing the layer height, the transmission coefficient decreases in the standard Schrodinger equation, but in strongly fractional Schrodinger equation, the transmission coefficient at first decreases and then increases if layer height increases. By decreasing the fractional parameter Supplementary video legends.Supplementary video 1-1-1.Supplementary video 1-1-2.Supplementary video 1-1-3.Supplementary video 1-1-4.Supplementary video 1-1-5.Supplementary video 1-1-6.Supplementary video 1-1-7.Supplementary video 1-1-8.Supplementary video 1-1-9.Supplementary video 1-1-10.Supplementary video 1-1-11.Supplementary video 1-1-12.Supplementary video 1-2-1.Supplementary video 1-2-2.Supplementary video 1-2-3.Supplementary video 1-2-4.Supplementary video 1-2-5.Supplementary video 1-2-6.Supplementary video 1-2-7.Supplementary video 1-2-8.Supplementary video 1-2-9.Supplementary video 1-2-10.Supplementary video 1-2-11.Supplementary video 1-2-12.Supplementary video 2-1-1.Supplementary video 2-1-2.Supplementary video 2-1-3.Supplementary video 2-1-4.Supplementary video 2-1-5.Supplementary video 2-1-6.Supplementary video 2-1-7.Supplementary video 2-1-8.Supplementary video 2-1-9.Supplementary video 2-1-10.Supplementary video 2-1-11.Supplementary video 2-1-12.Supplementary video 2-2-1.Supplementary video 2-2-2.Supplementary video 2-2-3.Supplementary video 2-2-4.Supplementary video 2-2-5.Supplementary video 2-2-6.Supplementary video 2-2-7.Supplementary video 2-2-8.Supplementary video 2-2-9.Supplementary video 2-2-10.Supplementary video 2-2-11.Supplementary video 2-2-12.Supplementary video 3-1.Supplementary video 3-2.Supplementary video 3-3.Supplementary video 3-4.Supplementary video 3-5.Supplementary video 3-6.Supplementary video 3-7.Supplementary video 3-8.Supplementary video 3-9.Supplementary video 3-10.Supplementary video 3-11.Supplementary video 3-12.Supplementary video 4-1.Supplementary video 4-2.Supplementary video 4-3.Supplementary video 4-4.Supplementary video 4-5.Supplementary video 4-6.Supplementary video 4-7.Supplementary video 4-8.Supplementary video 4-9.Supplementary video 5-1.Supplementary video 5-2.Supplementary video 5-3.Supplementary video 5-4.Supplementary video 5-5.Supplementary video 5-6.Supplementary video 5-7.Supplementary video 5-8.Supplementary video 5-9.Supplementary video 5-10.Supplementary video 5-11.Supplementary video 5-12.Supplementary video 6-1-1.Supplementary video 6-1-2.Supplementary video 6-1-3.Supplementary video 6-1-4.Supplementary video 6-1-5.Supplementary video 6-1-6.Supplementary video 6-1-7.Supplementary video 6-1-8.Supplementary video 6-1-9.Supplementary video 6-2-1.Supplementary video 6-2-2.Supplementary video 6-2-3.Supplementary video 6-2-4.Supplementary video 6-2-5.Supplementary video 6-2-6.Supplementary video 6-2-7.Supplementary video 6-2-8.Supplementary video 6-2-9.


R,5S,6R,7S,9S,10S)-1,6,9-trihydroxy-eudesm-3-ene-1,6-di-O-β-d-glucopyranoside (1), -1,6,9,11-tetrahydroxy-eudesm-3-ene-1,6-di-O-β-d-glucopyranoside (3), -9-O-(Z-p-coumaroyl)-1,6,9-trihydroxy-eudesm-3-ene-6-O-β-d-glucopyranoside (6), and -9-O-(E-feruloyl)-1,6,9-trihydroxy-eudesm-3-ene-6-O-β-d-glucopyranoside (7), were isolated from a 95% EtOH extract of the leaves of Aster koraiensis by repeated chromatography. Moreover, three sesquiterpenoids  and two caffeoylquinic acids (8 and 9) having previously known chemical structures were isolated during the isolation procedure. The four new compounds  were elucidated by spectroscopic data  interpretation and hydrolysis. Moreover, the absolute configurations of 2, 4, and 5 were determined for the first time in this study. The compounds isolated were tested for their viability on nitric oxide (NO) and prostaglandin E2 (PGE2) production on LPS-stimulated RAW 264.7 cells. Among them, only 7 presented weak inhibitory effects on both NO and PGE2 production.Four new eudesmane-type sesquiterpenoids, (1 Aster koraiensis Nakai (syn. Gymnaster koraiensis (Nakai) Kitamura; Compositae) is an endemic species to Korea that is distributed from Jeju Island to the southern parts of Gangwon-do . The. The4 wa6 was obtained as a pale yellow amorphous powder and the molecular formula was established as C30H42O10 by HR–ESI–MS  , 1.26 , 1.02 , and 0.93 ; one anomeric proton at δH 4.38  and 5.80  , six glucosyl signals including an anomeric carbon , three oxymethine signals , and cis-para-coumaroyl group , indicating that 6 is a p-coumaroyl derivative of 2 -9-O-(Z-p-coumaroyl)-1,6,9-trihydroxy-eudesm-3-en-6-O-β-d-glucopyranoside. A literature survey revealed that 6 is the geometric isomer of 5, 9β-O-(E-p-hydroxycinnamoyl)-1β,6β-dihydroxy-trans-eudesm-3-ene-6-O-β-d-glucopyranoside, which was isolated previously from the aerial parts of A. koraiensis and reported only its relative configuration +, ; NMR data: White powder; m.p.: 167.2 °C; m/z = 397.2237 [M–H]− ; c 0.03, MeOH); UV (MeOH) λmax nm (log ε): 240 (3.37), 296 (3.34); IR (ATR) νmax 1937, 1587, 1348, 1013 cm−1; 1H-NMR  δH 6.05 , 4.48 , 3.94 , 3.88 , 3.83 , 3.65 , 3.36 , 3.29 , 3.27 , 3.17 , 2.31 , 2.19 , 2.08 , 1.93 , 1.69 , 1.07 , 1.06 , 1.01 ; 13C–NMR  δC 141.9 (C-7), 133.3 (C-5), 129.1 (C-4), 118.5 (C-6), 102.4 (Glc-1), 84.9 (C-1), 78.6 (Glc-5), 78.3 (Glc-3), 78.1 (C-9), 75.1 (Glc-2), 71.7 (Glc-4), 63.0 (Glc-6), 43.1 (C-10), 36.4 (C-11), 33.0 (C-8), 32.5 (C-3), 24.6 (C-2), 22.2 (C-12), 21.7 (C-13), 19.2 (C-15), 12.5 (C-14).Pale yellow amorphous powder; HR-Q-TOF-MS c 0.03, MeOH); UV (MeOH) λmax nm (log ε): 239 (3.27), 290 (3.22); IR (ATR) νmax 1569, 1418, 1363, 1016 cm−1; 1H–NMR  δH 6.07 , 3.77 , 3.68 , 2.30 , 2.22 , 2.14 , 1.80 , 1.68 , 1.07 , 1.05 , 0.93 ; 13C–NMR  δC 141.9 (C-7), 133.3 (C-5), 129.1 (C-4), 118.5 (C-6), 78.6 (C-1), 78.3 (C-9), 43.1 (C-10), 36.4 (C-11), 33.0 (C-8), 32.5 (C-3), 24.6 (C-2), 22.2 (C-12), 21.7 (C-13), 19.2 (C-15), 12.5 (C-14).Pale yellow amorphous powder; c 0.1, MeOH); UV (acetonitrile) λmax (log ε): 205 (3.85), 322 (4.02), 359 (4.08), 373 (4.43) nm; IR (ATR) νmax 3314, 2876, 1360, 1011 cm−1; HR-ESI-MS (positive mode) m/z = 617.2791 [M + Na]+ ; NMR data: White powder; m.p.: 230 °C; m/z = 561.2701 [M–H]− ; c 0.1, MeOH); UV (MeOH) λmax nm (log ε): 313 (4.48); IR (ATR) νmax 1705, 1603 1513, 1165, 1074, 989 cm−1; NMR data: Pale yellow amorphous powder; HR-ESI-MS m/z = 575.2845 [M–H2O–H]− ; c 0.1, MeOH); UV (MeOH) λmax nm (log ε): 237 (3.47), 327 (3.22); IR (ATR) νmax 1705, 1596, 1517, 1268, 1159, 1074 cm−1; NMR data: Pale yellow amorphous powder; HR-ESI-MS 1 (10.0 mg) was incubated together with β-glucosidase (25.0 mg), toluene (2 drops), and H2O (15.0 mL) in a CO2 incubator at 35 °C for 3 days. EtOH was added to the reaction mixture to stop the reaction and β-glucosidase was removed by filtration. Compound 2 (2.0 mg) was isolated from the hydrolysate by flash CC with Redi Sep-C18 cartridge . Meanwhile, compound 1 (93.8 mg) was hydrolyzed with 2N HCl at 80 °C for one hour. The reaction was stopped by the addition of sodium bicarbonate and 1a (8.0 mg) was isolated from the hydrolysate by flash CC with a Redi Sep-C18 cartridge . An enzymatic hydrolysis of 1a (8.0 mg) was performed using the same method as 1 to give 1b (1.5 mg).Compound β-glucose in 1 was determined by the previously reported method [l-cysteine methyl ester hydrochloride (1.2 mg) were added in the hydrolysate and the mixture was heated at 60 °C for 1 h. σ-Tolyl isothiocyanate (100 μL) was added in the mixture and heated again at 60 °C for 1 hour. The reaction was analyzed directly by HPLC with a gradient system  formic acid in water, B: 0.1% (v/v) formic acid in acetonitrile). The reaction mixture of 1 was detected at 27.4 min. At the same HPLC conditions, authentic l- and d-glucoses were detected at 26.8 and 27.4 min, respectively. Therefore, the absolute configuration of β-glucose in 1 was established as the d configuration.The absolute configuration of d method . Pyridin1b and 5 were built by using Chem3D modeling. Conformational structure analysis, optimizations, and ECD calculations were performed as described previously [The 3D models of compounds eviously ,23.Cell viability and nitrite levels were measured using MTT and Griess reaction assays, respectively .2 levels in cell culture mediums were determined using EIA kits  as reported in the previous paper [PGEus paper .1, 3, 6, and 7) were obtained from a 95% EtOH extract of the leaves of Aster koraiensis by repeated chromatography, along with five known compounds . The chemical structures of the four new compounds and absolute configurations of the known compounds 2, 4, and 5 were established by their spectroscopic data  measurement and by acidic and enzymatic hydrolysis. Among the isolates, the new compound 7 exhibited weak inhibitory activities on both NO and PGE2 production. The compounds found in this study do not appear to contribute to the anti-inflammatory activity of the extract from which they were isolated. Thus, compounds with higher activity in the leaves of A. koraiensis needs to be identified through further studies.Four new eudesmene-type sesquiterpenoids (


The structures were determined on the basis of the 1D and 2D NMR, MS, UV and IR data. The absolute configurations of compounds 3, 6 and 13 were determined by ECD calculations. The NMR data of compounds deoxyhydroxyaspergillic acid (3) and 2-hydroxy-6-(1-hydroxy-1-methylpropyl)-3-sec-buthylpyrazine (4) were first reported. All of the isolated compounds were evaluated for their cytotoxic activities against human nasopharyngeal carcinoma cell lines CNE1, CNE2, SUNE1 and HONE1 and hepatocellular carcinoma cell lines QGY7701 and HepG2. Monarubin B (6) displayed potent cytotoxicities against the cancer cell lines HepG2 and QGY7701 with IC50 values of 1.72 and 0.71 μΜ, respectively; lunatinin (7) showed moderate cytotoxic activities against the cancer cell lines HepG2, QGY7701 and SUNE1 with the IC50 values of 9.60, 7.12 and 28.12 μΜ, respectively.Three new compounds, monarubins A–C ( Since 1990, marine aquatic production in China has ranked first in the world, among them, edible marine shellfishes are exceeding 75% of the total output ,3,4,5. MMonascus ruber (collection number BB5) was isolated from the inner tissue of marine shellfish Meretrix meretrix collected from Hailing island, Yangjiang, China. The genera Monascus mainly consists of four species: M. ruber, M. purpureus, M. anka and M. pilosus +  (−1). UV maxima at 201, 239 and 329 nm indicated the presence of a conjugated system. The 13C NMR and DEPT spectra showed three methyls, three methylenes, two methines and four quaternary carbons (1H NMR and HMQC spectra displayed one amide proton [δH 10.83 (brs)], one aromatic proton , one terminal olefinic double bond , two methylenes  and three methyls  , C-11 (δC 140.8), and H2-12/C-6, C-11 and C-14 (δC 115.0), H3-13/C-11, H-14a (δH 5.35)/C-6, H-14b (δH 5.66)/C-6 and C-11 indicated the connection of the −CH2CH3 fragment with the C-11, which in turn was linked to C-6. The key HMBC correlations of H2-7/C-2 (δC 156.9), C-3 (δC 159.5), H-8/C-3 displayed the connection of the isobutyl to C-3 /H2-12, and H-14a/H3-13 suggested that the H-5, H2-12 and H3-13 were oriented on the same side of the molecule, H-14a being close to the ethyl and away from the pyrazinone ring  , implyin carbons . The 1H δC 22.7] . The corectively . The keyl to C-3 . The NOEone ring . Therefo6 was obtained as a white solid. The HR-(+)ESI-MS spectrum displayed a strong quasi-molecular ion peak at m/z 265.10703 [M + H]+, corresponding to the molecular formula C14H16O5 ], two double-bond protons , one oxygen-bearing methine multiplet , one methoxy group , one methylene group , a methyl group on the benzene ring  and the other methyl group  (2CH(CH3)– moiety was based on the 1H–1H COSY correlations of H3-11/H-10/H2-9 (δH 11.15 (OH-8) to C-7 (δC 112.6), C-8 (δC 159.9) and C-8a (δC 99.9), from the methyl group at δH 2.12 to C-6 (δC 164.6), C-7 and C-8, from the methoxy group H3-13 (δH 3.90) to C-5 (δC 97.2) and C-6, from H-5 to C-4 (δC 106.3), C-6, C-7 and C-8a, from H-4 to C-3 (δC 153.8), C-5 and C-8a revealed the presence of 6-methyoxy-7-methy-8-hydroxy isochromone skeleton (3-13 (δC 43.0) and C-4 was established by HMBC correlations from the methylenic protons H2-9 (δH 2.59 and 2.66) to carbons C-3 and C-4, and from the olefinic proton H-4 (δH 6.30) to carbons C-3 and C-9. Compound C14H16O5 , requiri carbons . The 1H δC 23.2] . The pre-10/H2-9 . The HMBskeleton . Methoxyon  curves -8-hydroxy-3-(2-hydroxypropyl)-6-methoxy-7-methyl-1H-isochromen-1-one, and it was trivially named as monarubin B.The absolute configuration of ) curves . The cal13 was isolated as a colorless oil. The molecular formula was established as C21H30O5 by HR-(+) ESI-MS at m/z 363.21664 [M + H]+   and 5.52 ], three methylenes  and two oxygen-bearing methine multiplets   and C-20 (δC 31.5), H-17 to C-18 (δC 35.0) and C-19 (δC 25.8) and OH-17 to C-18. The 1H−1H COSY cross-peaks of H-17/H-11/H-6 as well as the HMBC correlations from H-11 to C-17 (δC 69.4), C-5 (δC 34.0), C-6 (δC 41.3) and C-12 (δC 174.7) revealed that the 1-hydroxyhexyl side chain was connected with a γ-lactone (ring C). The HMBC correlations of H-1b to C-3 (δC 73.4), C-9 (δC 151.6) and C-10 (δC 129.6), H-3 to C-1 and C-10, H-4a to C-9 and C-10, H-4b to C-3, C-9 and C-10, H-5 to C-6 (δC 41.3), C-7 (δC 82.9), C-9 and C-10, H-6 to C-5 (δC 34.0), C-7 and C-8 (δC 192.4), H3-16 to C-7 and C-8 displayed the connection of ring A with ring B. Ring C was combined with ring B by the HMBC the correlations from H-6 to C-5, C-7, C-8 and C-11 (δC 174.7), H-11 to C-5, C-6 and C-12, H3-16 to C-7 and C-8 and was supported by the 1H−1H COSY correlations of H-11/H-6/H2-5 (13 was established and given the name monarubin C.Compound 3.21664) , which hen atoms . The 1H δC 73.4] . The 1H−CH=CHCH3 . The 1-hH-6/H2-5 . Therefo13 was identified by the NOESY data. The large coupling constant (13.2 Hz) of the proton H-6 [δH 3.04 ] with the proton H-11 [δH 2.75 ] indicated that they were in opposite position on the ring. The NOESY cross peaks of H3-16 (δH 1.42) and H-11 (δH 2.75), H3-16 and H-1b (δH 4.44), H-1b and H-13 (δH 5.52) suggested that these protons were cofacial and assigned as β-orientation. The correlation between H-3 and H-1a (δH 4.37) suggested both of them having the α-orientation. The proton H-6 was determined as having an α-orientation because no NOESY correlation between H3-16 and H-6 was observed  was obtained as white needles. The molecular formula was established as C12H20N2O2 by HR-(+) ESI-MS molecular ion peak at m/z 225.16019 [M + H]+   , implyin5.15975)  and absoD curves . sec-buthylpyrazine (4) was afforded as white needles. The molecular formula was determined as C12H20N2O2 by HR-(+) ESI-MS at m/z 225.15967 [M + H]+  -3-5.15975) , which hfraction . Althougfraction  and abso2, 5 and 7–12 were elucidated as 3,6-diisobutyl-2(1H)-pyrazinone (2)   , 6,8-dim, 7] + .Monarubin A (3). ECD  λmax (Δε) 211 (+0.51), 228 (+4.24), 309 (−2.63) nm. 1H and 13C NMR data, see m/z 225.16019 [M + H]+ .Deoxyhydroxy aspergillic acid (4). 1H and 13C NMR data, see m/z 225.15967 [M + H]+ .2-Hydroxy-6-(1-hydroxy-1-methylpropyl)-3-sec-buthylpyrazine (6). [α]25D + 19 , UV (MeOH) λmax (logε) 331 (3.62), 280 (3.71), 241 (4.49). ECD  λmax (Δε) 289 (+3.79) nm. IR υmax 3380, 2927, 1673, 1634, 1566, 1520, 1423, 1348, 1252, 1124, 939, 796 cm−1. 1H and 13C NMR data, see m/z 265.10703 [M + H]+ .Monarubin B (13). [α]25D + 43 , UV (MeOH) λmax (logε) 243 (3.89). ECD  λmax (Δε) 246 (20.99) nm. IR υmax 3422, 2934, 1781, 1695, 1631, 1392, 1261, 1209, 1094, 1051, 965, 804 cm−1. 1H and 13C NMR data, see m/z 363.21664 [M + H]+ . Monarubin C . The conformers with a Boltzmann distribution over 2% were chosen for ECD calculations by TD-DFT method at the B3LYP/6-311+G  level in methanol (The absolute configurations of compounds methanol . The ECD4 were obtained from MeOH solution. X-ray diffraction data were collected on a Bruker SMART APEX CCD diffractometer with Cu Kα radiation (λ = 1.54184 Å). The structure was solved by direct methods using SHELXS-97, and refined through the program and full-matrix least-squares calculations. Hydrogen atoms were refined at calculated positions, and all non-hydrogen atoms were fixed anisotropically. Crystallographic data of 4 have been deposited at the Cambridge Crystallographic Data Centre (CCDC number: 1958991).Crystals of 4: monoclinic, C12H20N2O2, a = 11.77370(10) Å, b = 6.91360(10) Å, c = 16.5776(2) Å, α = γ = 90°, β = 91.3700(10)°, V = 1349.01(3) Å3, space group P21, Z = 2, Dc = 1.104 g/cm3, μ = 0.607 mm−1, and F(000) = 488.0. Independent reflections: 5242 (Rint = 0.0525). The final R1 value was 0.0570, wR2 = 0.1619 (I > 2σ(I)). The goodness of fit on F2 was 1.039. Flack parameter = −0.04(7)  .®, Suzhou, China) with 10% fetal bovine serum (FBS) (Gibco®). All of the cells were authenticated using short tandem repeat profiling, tested for Mycoplasma contamination, and cultured for less than 2 months.Human nasopharyngeal carcinoma cell lines CNE1, CNE2, SUNE1 and HONE1 and hepatocellular carcinoma cell lines QGY-7701 and HepG2 were generously provided by Professor Mu-Sheng Zeng  and conserved in the State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-sen University. The culture medium was DMEM -2,5-diphenyl-2H-tetrazolium bromide (MTT) assays and the assays were performed in triplicate. The cells were maintained at 37 °C with 5% CO2 and were seeded in 96-well plates. The volume and number of cells seeded was 0.2 mL and 3 × 103 per well. Compounds 1–13 were added to the cultures at different concentrations (1.5625–100 μM); moreover, compound 6 was tested at additional different concentrations (0.037–27 μM). Solvent DMSO was added as the control. After 72 h, 0.5 mg/mL MTT was added to the culture medium, and the plates were incubated for 4 h at 37 °C. The supernatant was removed. The formazan crystals were dissolved in DMSO (150 µL), the absorbance was measured at 570 nm by using a microplate reader , and the data were analyzed with the CalcuSystem software package. Hirsutanol A, a potent anticancer agent isolated from marine fungal metabolites, was used as a positive control, and its cytotoxic activities against the tested cancer cell lines were shown in The in vitro cytotoxic activities of Monascus ruber BB5. Monarubin B (6) showed potent cytotoxic activities against the human hepatocellular cancer cell lines HepG2 and QGY7701 with IC50 values of 1.72 and 0.71 μΜ, and lunatinin (7) exhibited moderate inhibitory effects against the human hepatocellular cancer cell lines HepG2, QGY7701, and nasopharyngeal carcinoma cancer cell line SUNE1 with IC50 values of 9.60, 7.12 and 28.12 μΜ, respectively. Compound 8 exhibited weak cytotoxic activities against the cancer cell lines HepG2, QGY7701 and SUNE1 with IC50 values in the range of 30–50 μΜ. Compounds 6–8 have the same isocoumarin skeleton. By comparing their structures and activities, we can preliminarily conclude that 8-OH is an essential active functional group. Our finding once again demonstrates that Monascus are genius strains with the ability to produce chemodiverse bioactive compounds.Three new metabolites, monarubins A–C, together with ten known compounds containing alkaloids, isocoumarins and polyketides were isolated from the fungal strain