Compounds 16a,b,d also inhibited the growth of MCF-7/ADR cells with IC50 in the range of 0.52–6.26 μM. Interestingly, the new compounds were less cytotoxic against normal MRC-5 cells (IC50=0.155–17.08 μM). Mechanistic studies revealed the ability of compounds 16a,b,d to inhibit tubulin polymerisation and multiple oncogenic kinases. Moreover, compounds 16a,b,d induced preG1 and G2/M cell cycle arrest and early apoptosis in MCF-7 cells. The molecular docking analyses of compounds 16a,b,d into the active site in tubulin, CDK-2, and EGFR proteins revealed higher binding affinities compared to the co-crystallised ligands. These preliminary results suggested that compounds 16a,b,d could serve as promising lead compounds for the future development of new potent anticancer agents.Two new series of pyrrolizines bearing 3,4,5-trimethoxyphenyl moieties were synthesized.16a,b,d displayed the highest cytotoxicity against the three cancer cell lines.Compounds 16a,b,d.Kinase profiling test revealed inhibition of multiple oncogenic kinases by compounds 16a,b,d exhibited weak to moderate inhibition of tubulin-polymerization.Compounds 16a,b,d induced preG1 and G2/M cell cycle arrest and early apoptosis in MCF-7 cells.Compounds 16a,b towards tubulin and CDK-2.Docking studies revealed high binding affinities for compounds In the present study, two new series of pyrrolizines bearing 3,4,5-trimethoxyphenyl moiety were designed, synthesised, and evaluated for their cytotoxic activity. The benzamide derivatives   A 2D pharmacophore of these TPIs 1–6 is generated in Tubulin polymerisation is one of the promising targets in the development of new anticancer agents2, and 3  are examd 3  group which restricts the free rotation and keep the two aromatic rings in cis-conformation. However, this type of restriction is absent in compounds 4–6, where the two phenyl rings can adopt different orientations relative to each other , and platelet-derived growth factor receptor β (PDGFR-β). Compound 7 also displayed superior in vivo activity in reducing tumour size and vascularity compared to sunitinib, and docetaxelIncorporation of tubulin-binding moieties such as the TMP moiety into anticancer agents was succeeded as a new strategy to produce multi-target anticancer agents . Compoun8, another multi-target anticancer agent designed by combining the pharmacophoric groups of the TBI (colchicine) with those needed to inhibit HDAC8 displayed potent cytotoxic activity (IC50=2–105 nM) against a panel of cancer cell lines mediated by moderate inhibition of HDAC and tubulin polymerisation and activity 9a,b revealed inhibitory activities against COX-2  and/or multiple oncogenic kinases. However, the results of the MTT assay revealed the ability of the two compounds to inhibit the growth of MCF-7 cells at a concentration lower than those required for COX-2 inhibition. In addition, compound 9b exhibited poor selectivity towards A2780 and HT29 cells . Accordingly, we perform the current study to optimise the cytotoxic potential of compounds 9b and investigate other potential targets which could contribute to their cytotoxic activities. However, to keep the ability of the new derivatives targeting oncogenic kinases, only small structural modifications in the scaffold of compound 9a were allowed.Recently, we reported compounds 9a,b  among a 9a,b by trimethoxy-benzylidene/benzoyl moieties .The study of structure–activity relationship (SAR) of scaffold A  was achi9aSupplementary data (Tables S1 and S2). Based on these results, 10 of the designed derivatives which showed good scores were selected for the synthesis.In addition, a preliminary docking study was performed to evaluate the binding affinities of the designed derivatives into the binding site of colchicine in tubulin protein. The derivatives which displayed high binding scores towards tubulin were also evaluated for their binding affinities towards two of the oncogenic kinases . The selection of these kinases was based on the results of the mechanistic study of compound 2.2.1.−1). The proton magnetic spectra were recorded on BRUKER AVANCE III at 500 MHz  in CDCl3/DMSO-d6. The J constant is given in Hz. The 13C NMR spectra of the new compounds in CDCl3/DMSO-d6 were done at 125 MHz. Mass spectra were recorded on Shimadzu GCMS QP5050A spectrometer , at 70 eV (EI) at the regional centre for mycology and biotechnology, Al-Azhar University . Elemental analyses were done in the microanalytical centre, Cairo University . Compounds 1113a–e,14a–e18191H NMR, 13C NMR, 13C NMR, DEPT C135, and mass spectra for each of the new compounds are provided in Supplementary .All the chemical reagents and solvents used were purchased from Sigma-Aldrich . Solvents were dried according to the literature when necessary. The purity of the new compounds was checked with TLC using the benzene–ethanol mixture (9:1). Melting points (m.p.) are uncorrected and were determined by IA 9100MK-Digital melting point apparatus. BRUKER TENSOR 37 spectrophotometer  was used to perform the infra-red (IR) spectra of the new compounds, the spectra were recorded using KBr disc and were expressed in wavenumber  and 3,4,5-trimethoxybenzaldehyde , 0.5 mL glacial acetic acid in absolute ethanol (30 mL) was stirred under reflux for 5 h  with trimethoxy benzoyl chloride  according to the general procedure A. Compound 15a was obtained as a yellow amorphous solid product, m.p. 242–4 °C, yield 71%. IRʋmax/cm−1 3271, 3236, 3183 (NH), 3076, 3024 (aromatic C–H), 2967, 2941  2211 (CN), 1672 (COs), 1609, 1578, 1556 , 1432, 1318, 1232 . 1H NMR : 2.57–2.63 , 3.09 , 3.97 , 3.98 , 4.58 , 7.13 , 7.19 , 7.35 , 7.68 , 9.11 , 10.68 . 13C NMR : 24.59 (pyrrolizine CH2-2), 25.46 (pyrrolizine CH2-1), 50.14 (pyrrolizine CH2-3), 56.35 , 61.09 (4″-OCH3), 84.71 (pyrrolizine C-7), 105.90 , 116.31 (pyrrolizine C-6), 117.71 (CN), 119.79 , 124.12 (Ph CH-4′), 129.04 , 130.75 (Ph C-1″), 138.23 (pyrrolizine C-5), 139.40 (pyrrolizine C-7a), 142.18 (Ph CH-1′), 148.19 (Ph C-4″), 153.82 , 158.48 (PhNHCO) 159.52 (N═CH). DEPT135 : δ 24.59 (pyrrolizine CH2-2), 25.47 (pyrrolizine CH2-1), 50.14 (pyrrolizine CH2-3), 56.35 , 61.09 (4″-OCH3), 105.90 , 119.79 , 124.12 (Ph CH-4′), 129.04 , 159.52 (N═CH). MS (EI): m/z (%) 445 , 444  443 , 367 (4), 352 (36), 322 (5), 308 (6), 294 (4), 278 (12), 277 (45), 249 (13), 222 (7), 196 (13), 195 (100), 194 (7), 186 (10), 174 (13), 168 (15), 154 (5), 139 (11), 119 (5), 122 (2), 107 (10), 92 (6), 77 (10). Anal. Calcd. for C25H24N4O4 (444.48): C, 67.55; H, 5.44; N, 12.60. Found: C, 67.38; H, 4.98; N, 12.69.The title compound was prepared from the reaction of compound 2.1.1.2.14b  with 3,4,5-trimethoxybenzaldehyde  according to the general procedure A. Compound 15b was obtained as a yellow amorphous solid product, m.p. 251–3 °C, yield 78%. IRʋmax/cm−1 3275, 3232, 3179 (NH), 3070 (aromatic C–H), 2962, 2926 , 2211 (CN), 1668 (COs), 1611, 1576, 1552 , 1459, 1337, 1233 . 1H NMR : 2.33 , 2.54–2.60 , 3.06 , 3.94 , 3.95 , 4.54 , 7.12 , 7.16 , 7.54 , 9.08 , 10.58 . 13C NMR : δ 20.88 (CH3), 24.58 (pyrrolizine CH2-2), 25.47 (pyrrolizine CH2-1), 50.13 (pyrrolizine CH2-3), 56.35 , 61.09 (4″-OCH3), 105.87 , 116.36 (pyrrolizine C-7), 117.82 (CN), 119.75 , 129.52 , 130.79 (pyrrolizine C-6), 133.74 (Ph C-1″), 135.67 (pyrrolizine C-5), 139.23 (pyrrolizine C-7a), 142.13 (Ph C-1′), 142.69 (Ph C-4′), 148.08 (Ph C-4″), 153.80 , 158.39 (PhNHCO), 159.39 (N═CH). DEPT135 : δ 20.88 (CH3), 24.58 (pyrrolizine CH2-2), 25.47 (pyrrolizine CH2-1), 50.13 (pyrrolizine CH2-3), 56.36 , 61.09 (4″-OCH3), 105.82 , 119.75 , 129.52 . MS (EI): m/z (%) 460 , 459 , 458 , 457 (7), 429 (4), 353 (21), 352 (100), 336 (4), 322 (7), 308 (7), 292 (11), 291 (54), 280 (2), 278 (3), 263 (8), 229 (4), 196 (3), 195 (21), 194 (2), 186 (9), 168 (9), 154 (3), 106 (2), 91 (2), 77 (4). Anal. Calcd. for C26H26N4O4 (458.51): C, 68.11; H, 5.72; N, 12.22. Found: C, 68.53; H, 5.32; N, 12.67.The title compound was prepared from the reaction of compound 2.1.1.3.14c  with 3,4,5-trimethoxybenzaldehyde  according to the general procedure A. Compound 15c was obtained as a yellow amorphous solid product, m.p. 263–5 °C, yield 74%. IRʋmax/cm−1 3281, 3240, 3185 (NH), 3077, 3000 (aromatic C–H), 2942, 2841  2208 (CN), 1674 (COs), 1605, 1578, 1511 , 1334, 1234 . 1H NMR : δ 2.55–2.61 , 3.06 , 3.82 , 3.95 , 3.99 , 4.52 , 6.88 , 7.28 , 7.61 , 8.96 , 10.70 . 13C NMR : δ 24.62 (pyrrolizine CH2-2), 25.48 (pyrrolizine CH2-1), 50.10 (pyrrolizine CH2-3), 55.54 (4′-OCH3), 56.42 , 61.20 (4″-OCH3), 106.45 , 106.72 (pyrrolizine C-7), 114.16 , 114.77 (CN), 116.08 (pyrrolizine C-6), 118.05 (Ph C-1′), 121.49 , 129.60 (Ph C-1″), 131.29 (pyrrolizine C-7a), 148.00 (pyrrolizine C-5), 153.66 (Ph C-4″), 153.71 , 156.32 (Ph C-4′), 158.06 (CONH), 160.30 (N═CH). DEPT C135 : δ 24.62 (pyrrolizine CH2-2), 25.48 (pyrrolizine CH2-1), 50.10 (pyrrolizine CH2-3), 55.54 (4′-OCH3), 56.41 , 61.20 (4″-OCH3), 106.42 , 114.16 , 121.48 . MS (EI): m/z (%) 474 , 449 (8), 422 (5), 396 (34), 383 (43), 361 (58), 332 (100), 320 (37), 261 (28), 190 (11), 120 (22), 93 (8), 69 (9). Anal. Calcd. for C26H26N4O5 (474.51): C, 65.81; H, 5.52; N, 11.81. Found: C, 65.44; H, 5.21; N, 12.15.The title compound was prepared from the reaction of compound 2.1.1.4.14d  with 3,4,5-trimethoxybenzaldehyde  according to the general procedure A. Compound 15d was obtained as a yellow amorphous solid product, m.p. 271–3 °C, yield 64%. IRʋmax/cm−1 3277, 3234, 3177 (NH), 3065 (aromatic C–H), 2996, 2939  2208 (CN), 1676 (COs), 1611, 1577, 1547 , 1417, 1398, 1291 . 1H NMR : 2.55–2.61 , 3.07 , 3.94 , 3.96 , 4.53 , 7.15 , 7.28 , 7.60 , 9.08 , 10.67 . 13C NMR : δ 24.60 (pyrrolizine CH2-2), 25.45 (pyrrolizine CH2-1), 50.14 (pyrrolizine CH2-3), 56.37 , 61.11 (4″-OCH3), 105.95 , 116.18 (pyrrolizine C-7), 117.42 (CN), 120.90 , 126.45 (pyrrolizine C-6), 128.98 (Ph C-1″), 129.05 , 130.65 (Ph C-4′), 138.87 (pyrrolizine C-5), 139.60 (pyrrolizine C-7a), 142.34 (Ph C-1′), 148.37 (Ph C-4″), 153.85 , 158.46 (PhNHCO), 159.75 (N═CH). DEPT135 : δ 24.61 (pyrrolizine CH2-2), 25.45 (pyrrolizine CH2-1), 50.14 (pyrrolizine CH2-3), 56.37 , 61.12 (4″-OCH3), 105.95 , 120.90 , 129.05 , 159.75 (N═CH). MS (EI): m/z (%) 480 , 479 , 478 , 458 (7), 353 (13), 352 (73), 323 (4), 322 (8), 311 (11), 308 (10), 292 (12), 291 (44), 278 (8), 263 (13), 252 (9), 229 (8), 222 (11), 196 (15), 195 (100), 186 (20), 168 (28), 154 (12), 147 (6), 111 (4), 106 (5), 91 (7), 77 (12). Anal. Calcd. for C25H23ClN4O4 (478.93): C, 62.70; H, 4.84; N, 11.70. Found: C, 63.14; H, 5.07; N, 11.22.The title compound was prepared from the reaction of compound 2.1.1.5.14e  with 3,4,5-trimethoxybenzaldehyde  according to the general procedure A. Compound 15e was obtained as a yellow amorphous solid product, m.p. 277–80 °C, yield 68%. IRʋmax/cm−1 3172 (NH), 3063 (aromatic C–H), 2961, 2939, 2838  2209 (CN), 1679 (COs), 1610, 1578, 1545 , 1418, 1334, 1232 . 1H NMR : δ 2.57–2.62 , 3.08 , 3.96 , 3.99 , 4.52 , 7.21 , 7.44 , 7.59 , 9.01 , 10.77 . 13C NMR : δ 24.64 (pyrrolizine CH2-2), 25.45 (pyrrolizine CH2-1), 50.15 (pyrrolizine CH2-3), 56.42 , 61.20 (4″-OCH3), 106.22 , 106.72 (pyrrolizine C-7), 116.55 (CN), 117.54 (pyrrolizine C-6), 121.28 , 130.46 (Ph C-4′), 131.73 (Ph C-1″), 131.97 , 132.62 (pyrrolizine C-5), 137.34 (pyrrolizine C-7a), 148.41 (Ph C-1′), 153.67 (Ph C-4″), 153.78 , 158.31 (PhNHCO), 160.29 (N═CH). DEPT C135 : δ 24.64 (pyrrolizine CH2-2), 25.44 (pyrrolizine CH2-1), 50.15 (pyrrolizine CH2-3), 56.40 , 61.20 (4″-OCH3), 106.18 , 121.25 , 131.97 . MS (EI): m/z (%) 525 , 524 , 523 , 522 , 368 (28), 352 (100), 313 (22), 236 (13), 195 (17), 109 (16), 97 (26), 69 (35). Anal. Calcd. for C25H23BrN4O4 (523.38): C, 57.37; H, 4.43; N, 10.70. Found: C, 56.93; H, 4.57; N, 11.16.The title compound was prepared from the reaction of compound 2.1.2.14a–e (3 mmol) and 3,4,5-trimethoxybenzoyl chloride  in dry benzene (30 mL) was stirred for 48 h at room temperature  with 3,4,5-trimethoxy benzoyl chloride  according to the general procedure B. Compound 16a was obtained as a white solid product, m.p. 253–5 °C, yield 63%. IRʋmax/cm−1 3190, 3131 (NHs), 3064, 3014 (aromatic C–H), 2968, 2909  2224 (CN), 1656, 1639 (COs), 1584, 1567 , 1465, 1338, 1297 . 1H NMR : 2.47–2.51 , 3.04 , 3.73 , 3.84 , 4.32 , 7.06 , 7.31 , 7.35 , 7.58 , 9.78 , 10.34 . 13C NMR : δ 24.88 (pyrrolizine CH2-2), 25.71 (pyrrolizine CH2-1), 49.64 (pyrrolizine CH2-3), 56.57 , 60.63 (4″-OCH3), 84.98 (pyrrolizine C-7), 105.80 , 115.26 (CN), 119.06 (Ph C-1″), 119.75 , 124.16 (Ph CH-4′), 128.80 (pyrrolizine C-5), 128.84 (pyrrolizine C-7a), 129.34 , 139.00 (pyrrolizine C-6), 141.23 (Ph C-1′), 146.44 (Ph C-4″), 153.21 , 157.92 (PhNHCO), 166.38 (PhCONH). DEPT C135 : δ 24.88 (pyrrolizine CH2-2), 25.71 (pyrrolizine CH2-1), 49.64 (pyrrolizine CH2-3), 56.56 , 60.63 (4″-OCH3), 105.78 , 119.75 , 124.16 (Ph CH-4′), 129.34 . MS (EI): m/z (%) 460 , 373 (2), 368 (2), 367 (6), 344 (2), 266 (1), 169 (10), 195 (100), 173 (11), 167 (3), 147 (9), 145 (22), 122 (5), 117 (20), 106 (5), 105 (17), 93 (6), 91 (40), 78 (2). Anal. Calcd. for C25H24N4O5 (460.48): C, 65.21; H, 5.25; N, 12.17. Found: C, 65.54; H, 5.17; N, 12.64.The title compound was prepared from the reaction of compound 2.1.2.2.14b  with 3,4,5-trimethoxy benzoyl chloride  according to the general procedure B. Compound 16b was obtained as a white solid product, m.p. 265–7 °C, yield 66%. IRʋmax/cm−1 3187, 3112 (NHs), 3038 (aromatic C–H), 2968, 2870  2221 (CN), 1656, 1638 (COs), 1586, 1549 , 1466, 1389, 1263 . 1H NMR : 2.23 , 2.46–2.54 , 3.03 , 3.74 , 3.85 , 4.31 , 7.10 , 7.35 , 7.47 , 9.65 , 10.32 . 13C NMR : δ 20.87 (CH3), 24.88 (pyrrolizine CH2-2), 25.72 (pyrrolizine CH2-1), 49.63 (pyrrolizine CH2-3), 56.63 , 60.64 (4″-OCH3), 85.07 (pyrrolizine C-7), 105.89 , 115.21 (CN), 119.32 (Ph C-1″), 119.77 , 127.09 (pyrrolizine C-5), 128.70 (pyrrolizine C-7a), 129.69 , 133.23 (Ph C-1′), 136.47 (pyrrolizine C-6), 141.36 (Ph C-4′), 146.32 (Ph C-4″), 153.24 , 157.69 (PhNHCO), 166.40 (PhCONH). DEPT C135 : δ 20.87 (CH3), 24.88 (pyrrolizine CH2-2), 25.72 (pyrrolizine CH2-1), 49.63 (pyrrolizine CH2-3), 56.63 , 60.64 (4″-OCH3), 105.89 , 119.77 , 129.69 . MS (EI): m/z (%) 475 , 474 , 368 (34), 367 (37), 352 (6), 341 (4), 284 (6), 280 (1), 200 (11), 196 (11), 195 (100), 174 (2), 167 (6), 146 (2), 145 (1), 122 (4), 117 (2), 107 (30), 106 (5), 92 (3), 78 (2), 77 (7). Anal. Calcd. for C26H26N4O5 (474.51): C, 65.81; H, 5.52; N, 11.81. Found: C, 65.30; H, 5.71; N, 11.99.The title compound was prepared from the reaction of compound 2.1.2.3.14c  with 3,4,5-trimethoxy benzoyl chloride  according to the general procedure B. Compound 16c was obtained as a light buff solid product, m.p. 259–61 °C, yield 69%. IRʋmax/cm−1 3196 (NHs), 3068, 3003 (aromatic C–H), 2963, 2941, 2838  2223 (CN), 1657, 1634 (COs), 1586, 1511 , 1464, 1339, 1234 . 1H NMR : δ 2.39–2.44 , 2.88 , 3.71 , 3.72 , 3.81 , 4.25 , 6.85 , 7.45 , 7.52 , 10.69 . 13C NMR : δ 24.68 (pyrrolizine CH2-2), 25.56 (pyrrolizine CH2-1), 49.47 (pyrrolizine CH2-3), 55.66 (4′-OCH3), 56.39 , 60.58 (4″-OCH3), 84.33 (pyrrolizine C-7), 105.76 , 106.44 (CN), 114.35 , 116.20 (Ph C-1′), 120.89 , 121.46 (Ph C-1″), 132.77 (pyrrolizine C-5), 140.40 (pyrrolizine C-7a), 145.92 (pyrrolizine C-6), 152.32 (Ph C-4″), 152.96 , 155.57 (Ph C-4′), 158.33 (PhNHCO), 166.33 (PhCONH). DEPT C135 : δ 24.68 (pyrrolizine CH2-2), 25.56 (pyrrolizine CH2-1), 49.47 (pyrrolizine CH2-3), 55.66 (4′-OCH3), 56.39 , 60.58 (4″-OCH3), 105.75 , 114.35 , 120.89 . MS (EI): m/z (%) 492 , 491 , 490 , 382 (4), 368 (100), 341 (16), 267 (6), 195 (45), 123 (41), 77 (7). Anal. Calcd. for C26H26N4O6 (490.51): C, 63.66; H, 5.34; N, 11.42. Found: C, 63.78; H, 5.61; N, 11.69.The title compound was prepared from the reaction of compound 2.1.2.4.14d  with 3,4,5-trimethoxy benzoyl chloride  according to the general procedure B. Compound 16d was obtained as a white solid product, m.p. 281–3 °C, yield 61%. IRʋmax/cm−1 3193, 3108 (NHs), 3062, 3006 (aromatic C–H), 2969, 2937  2220 (CN), 1644 (COs), 1586, 1524 , 1440, 1296, 1235 . 1H NMR : 2.47–2.51 , 3.04 , 3.73 , 3.84 , 4.31 , 7.33 , 7.37 , 7.63 , 9.94 , 10.31 . 13C NMR : δ 24.90 (pyrrolizine CH2-2), 25.69 (pyrrolizine CH2-1), 49.62 (pyrrolizine CH2-3), 56.58 , 60.63 (4″-OCH3), 84.98 (pyrrolizine C-7), 105.82 , 115.23 (CN), 118.67 (Ph C-4′), 121.29 (Ph C-1″), 121.41 , 127.70 (pyrrolizine C-5), 128.87 (pyrrolizine C-7a), 129.21 , 138.03 (pyrrolizine C-6), 141.21 (Ph C-1′), 146.58 (Ph C-4″), 153.19 , 158.05 (PhNHCO), 166.24 (PhCONH). DEPT C135 : δ 24.90 (pyrrolizine CH2-2), 25.70 (pyrrolizine CH2-1), 49.61 (pyrrolizine CH2-3), 56.57 , 60.63 (4″-OCH3), 105.80 , 121.40 , 129.21 . MS (EI): m/z (%) 496 , 494 , 480 (1), 450 (6), 436 (4), 400 (7), 396 (5), 387 (7), 372 (6), 352 (20), 340 (14), 339 (11), 329 (17), 328 (7), 327 (9), 313 (13), 299 (12), 298 (15), 280 (4), 279 (11), 273 (21), 269 (15), 268 (48), 254 (20), 240 (26), 228 (30), 210 (40), 196 (23), 195 (31), 174 (100), 169 (31), 147 (29), 145 (17), 123 (31), 117 (29), 107 (44), 92 (36), 77 (14). Anal. Calcd. for C25H23ClN4O5 (494.93): C, 60.67; H, 4.68; N, 11.32. Found: C, 61.13; H, 4.87; N, 11.06.The title compound was prepared from the reaction of compound 2.1.2.5.14e  with 3,4,5-trimethoxy benzoyl chloride  according to the general procedure B. Compound 16e was obtained as a light buff amorphous solid product, m.p. 286–8 °C, yield 64%. IRʋmax/cm−1 3222 (NHs), 3060 (aromatic C–H), 2972, 2938, 2835  2220 (CN), 1644 (COs), 1587, 1524 , 1414, 1388, 1296 . 1H NMR : δ 2.46–2.51 , 3.04 , 3.73 , 3.83 , 4.30 , 7.32 , 7.50 , 7.57 , 9.91 , 10.29 . 13C NMR : δ 24.90 (pyrrolizine CH2-2), 25.69 (pyrrolizine CH2-1), 49.61 (pyrrolizine CH2-3), 56.56 , 60.63 (4″-OCH3), 84.86 (pyrrolizine C-7), 105.77 , 115.23 (CN), 115.73 (Ph C-4′), 118.67 (Ph C-1″), 121.76 , 127.62 (pyrrolizine C-5), 128.81 (pyrrolizine C-7a), 132.13 , 138.43 (pyrrolizine C-6), 141.17 (Ph C-1′), 146.61 (Ph C-4″), 153.19 , 158.04 (PhNHCO), 166.23 (PhCONH). DEPT C135 : δ 24.90 (pyrrolizine CH2-2), 25.69 (pyrrolizine CH2-1), 49.61 (pyrrolizine CH2-3), 56.55 , 60.63 (4″-OCH3), 105.75 , 121.75 , 132.13 . MS (EI): m/z (%) 543 , 541 , 538 , 490 (31), 457 (12), 422 (89), 368 (100), 341 (46), 318 (37), 296 (18), 200 (18), 175 (18), 121 (6), 100 (17), 73 (31). Anal. Calcd. for C25H23BrN4O5 (539.38): C, 55.67; H, 4.30; N, 10.39. Found: C, 55.83; H, 3.91; N, 10.62.The title compound was prepared from the reaction of compound 2.1.3.19  and 3,4,5-trimethoxybenzaldehyde , 0.5 mL glacial acetic acid in absolute ethanol (30 mL) was stirred under reflux for 5 h , 3068 (aromatic C–H), 2938, 2875, 2824  2209 (CN), 1677 (COs), 1609, 1594, 1575 , 1481, 1330, 1230 . 1H NMR : δ 1.91–1.95 , 2.02–2.05 , 2.97 , 3.95 , 3.98 , 4.54 , 7.12 , 7.26 , 7.32 , 7.69 , 8.95 , 10.96 . 13C NMR : δ 18.78 (indolizine CH2-7), 22.94 (indolizine CH2-8), 23.02 (indolizine CH2-6), 46.96 (indolizine CH2-5), 56.42 , 61.18 (4′-OCH3), 82.19 (indolizine C-1), 106.54 , 106.73 (CN), 115.79 (indolizine C-2), 119.63 (Ph C-1′), 120.06 , 124.10 (Ph C-4″), 128.72 (indolizine C-3), 128.97 , 129.42 (indolizine C-8a), 138.35 (Ph C-1″), 142.92 (Ph C-4′), 153.73 , 158.80 (N═CH), 161.09 (PhNHCO). DEPT C135 : δ 18.78 (indolizine CH2-7), 22.94 (indolizine CH2-8), 23.03 (indolizine CH2-6), 46.97 (indolizine CH2-5), 56.41 , 61.19 (4′-OCH3), 106.50 , 120.05 , 124.02 (Ph C-4″), 128.98 . MS (EI): m/z (%) 460 , 458 , 422 (83), 396 (30), 382 (30), 356 (47), 332 (100), 296 (15), 106 (13), 89 (26), 77 (12), 69 (31). Anal. Calcd. for C26H26N4O4 (458.51): C, 68.11; H, 5.72; N, 12.22. Found: C, 67.88; H, 5.46; N, 12.53.A mixture of indolizine-3-carboxamides  for 5 h . The sol2.1.4.19  and 3,4,5-trimethoxybenzoyl chloride  in dry benzene (30 mL) was stirred for 48 h at room temperature , 3061, 3040, 3015 (aromatic C–H), 2960, 2944, 2839 , 2221 (CN), 1648 (COs), 1587, 1566 , 1317, 1235 . 1H NMR : δ 1.72–1.76 , 1.85–1.89 , 2.71 , 3.71 , 3.76 , 4.24 , 6.97 , 7.23 , 7.37 , 7.44 , 7.58 , 10.92 . 13C NMR : δ 19.15 (indolizine CH2-7), 22.75 (indolizine CH2-8), 22.86 (indolizine CH2-6), 45.90 (indolizine CH2-5), 56.21 , 60.56 (4′-OCH3), 88.27 (indolizine C-1), 105.69 , 116.52 (CN), 119.25 , 123.00 (Ph C-1′), 125.71 (indolizine C-3), 128.80 (Ph CH-4″), 129.10 , 129.38 (indolizine C-8a), 139.84 (indolizine C-2), 140.12 (Ph C-1″), 140.20 (Ph C-4′), 152.77 , 159.62 (PhNHCO), 166.29 (PhCONH). DEPT C135 : δ 19.15 (indolizine CH2-7), 22.75 (indolizine CH2-8), 22.86 (indolizine CH2-6), 45.90 (indolizine CH2-5), 56.21 , 60.56 (4′-OCH3), 105.68 , 119.25 , 128.80 (Ph CH-4″), 129.10 . MS (EI): m/z (%) 475 , 474 , 381 (82), 366 (6), 262 (4), 195 (64), 152 (6), 109 (20), 93 (57), 81 (71), 69 (98). Anal. Calcd. for C26H26N4O5 (474.51): C, 65.81; H, 5.52; N, 11.81. Found: C, 66.21; H, 5.78; N, 11.66.A mixture of indolizine-3-carboxamides perature . The rea2.2.2.2.1.2.2.1.1.2, 95% air, and 100% relative humidity, to maintain continuous logarithmic growth. MCF-7, A2780, and HCT116 were maintained in RPMI-1640 media . MRC-5 cells were maintained in EMEM . Doxorubicin  added for RPMI-1640 media containing MCF7 cells was used to develop doxorubicin-resistant MCF7/ADR cells. All cancer and normal cell lines were cultured according to our previous reportMCF-7 (human breast adenocarcinoma), A2780 (human ovarian cancer) and HCT116 , and MRC-5  cell lines were bought from the ATCC and were cultured in flasks which were incubated at 37 °C in an atmosphere of 5% CO2.2.1.2.,3 cells , 1 × 104 cells (HCT116), to a volume of 180 μL/well of culture medium. A Neubauer haemocytometer was used for cell counting. Cells were incubated at 37 °C overnight to allow attachment to the wells. The final concentrations of each compound in wells were: 0–50 μM in 200 μL of media (DMSO 0.1%). Lapatinib  was used as a positive control at the same final concentrations. Medium only (20 μL) was added to each control well, and each concentration was tested in triplicates (n = 3). Following incubation for 72 h, 50 μL MTT was added into each well. Plates were incubated for 3 h, the supernatant was aspirated, and 100 μL of DMSO was added to each well. The optical density (OD) of the purple formazan is proportional to the number of viable cells. When the amount of formazan produced by treated cells is compared with the amount of formazan produced by untreated control cells, the strength of the drug in causing growth inhibition can be determined, through plotting growth curves of absorbance against drug concentration, thus formulation concentration causing 50% inhibition (IC50) compared to control cell growth (100%) were determined. GraphPad Prism version 5.00 for Windows was used for analysis . Cytotoxicity of the new compounds was calculated after treatment of cancer/normal cell lines for 72 h. The absorbance of the reduced MTT was determined using a microplate reader A570 nm.The MTT assay was used to measure the cytotoxicity and growth inhibitory activities and cytotoxicity of the new compounds according to our previous report2.2.2.16a,b,d to induce apoptotic changes in MCF-7 cells was determined by Annexin V-FITC/PI staining according to the previous report,,The ability of compounds 2.2.3.2.2.3.1.16a,b,d and imatinib were evaluated for their inhibitory activities against 20 kinases at a single concentration (10 μM). The test was performed using the radiolabeled ATP determination method . The test was performed following the previous reports,Compounds 2.2.3.2.16a,b,d to inhibit the activity of CDK-2 was determined using ADP-Glo assay . The assay was done according to the manufacturer’s instructions and as described in the previous reportThe ability of compounds 2.2.4.16a,b,d at 0.1 µM concentration for 24 h was determined using FC500 flow cytometer . The cell cycle analysis was performed according to our previous reportCell cycle perturbations of MCF-7 cells treated with compounds 2.2.5.2.2.5.1.16a,b,d on tubulin polymerisation was measured in vitro using tubulin polymerisation fluorescence-based assay . The assay was performed following the previous report2, 0.5 mM EGTA pH 6.9, 10 µM fluorescent reporter, 1 mM GTP, 15% glycerol) to a final concentration of 10 mg/mL. Then tubulin solution was transferred to a pre-warmed 96-well plate that contained the test compounds 16a,b,d (5 μM), paclitaxel (3 μM), CaCl2 (0.5 μM), and control buffer. The polymerisation of tubulin was monitored as fluorescence at 37 °C for 60 min. Relative fluorescence units (RFU) were determined at 360 nm for excitation and at 460 nm for emission using the Varioskan Flash spectral scanning multimode reader  at a reading speed of 1 cycle/min.The effect of compounds 2.2.5.2.5/well) were treated with the indicated concentration of control, colchicine (3 μM), paclitaxel (3 μM), and the test compound 16b (5 μM) for 24 h as previously reported® 488 secondary antibodies , after being washed with PBS for 1 h in a darkroom. Cellular microtubules were observed with the Nikon Eclipse Ti microscope .The MCF-7 cancer cells plated on coverslips on six-well plate (1 × 102.3.http://www.rcsb.org/pdb). Ligand and protein files were prepared according to the previous report,,The molecular docking simulation of the new compounds was carried out by AutoDock 4.23.3.1.11 was prepared from the reaction of pyrrolidin-2-one 10 and malononitrile as previously reported13a–e were obtained from the reaction of (un)substituted anilines 13a–e with chloroacetyl chloride according to the reported procedures14a–e were prepared from the reaction of compounds 11 and 13a–e in acetoneIn this work, compound 15a–e were obtained on refluxing compounds 14a–e with 3,4,5-trimethoxybenzaldehyde in absolute ethanol in the presence of glacial acetic acid. On the other hand, the benzamide derivatives 16a–e were prepared from the reaction of compounds 14a–e with 3,4,5-trimethoxybenzoyl chloride in dry benzene .The Schiff bases  benzene . Structu18 (Scheme 2) was obtained on the treatment of piperidin-2-one 17 with dimethyl sulphate and malononitrile according to the reported procedures19 was synthesised using the same reaction conditions adopted for the synthesis of compound 14a with the replacement of 2-(pyrrolidin-2-ylidene)malononitrile 11 by 2-(piperidin-2-ylidene)malononitrile 18.In addition, compound 20 was also obtained from the condensation of 3,4,5-trimethoxybenzaldehyde and 2-aminoindolizine-3-carboxamide 19 in ethanol. On the other hand, acylation of compound 19 with 3,4,5-trimethoxybenzoyl chloride yielded the benzamide derivative 21. Spectral  and elemental analyses were performed to confirm the chemical structure of compounds 20 and 21. The spectra of the two compounds are also provided in Supplementary data .The Schiff base 3.2.3.2.1.3.2.1.1.15a–e, 16a–e, 20, and 21) was evaluated in vitro using MTT assay9a,b,,The cytotoxic activity of the new compounds  compared to their corresponding benzylidene analogues (15a–e and 20).The results of the MTT assay  revealed16b was the most potent in inhibiting the growth of MCF-7 cells, while compounds 16c,d exhibited the highest cytotoxic activity against HCT116 and A2780 cells, respectively. On the other hand, lapatinib exhibited cytotoxic activity against the three cancer cell lines with IC50 values in the range of 6.690–10.043 μM  were also selected to evaluate their cytotoxicity against doxorubicin-resistant MCF-7/ADR cells. The evaluation was performed using MTT assay50 in the range of 0.52–6.26 μM. Among the three derivatives, compound 16d with the electron-withdrawing chloro atom exhibited the highest cytotoxic activity against MCF-7/ADR cells, while the methyl analogue 16b was the least active. These results suggested that electron-withdrawing groups on the phenyl ring of scaffold A could be used to enhance cytotoxic activity against MCF-7/ADR cells.Encouraged by the high cytotoxic activity of the new compounds against MCF-7 cells , three o3.2.1.3.15a–e, 16a–e, 20, and 21), their growth inhibitory activities against normal human foetal lung fibroblast MRC-5 cells were evaluated using the MTT assay. The results expressed as IC50 values are presented in 50=0.155–17.080 μM), compared to 25.810 and 9.00 μM for compounds 9a,b, respectively15c,e, and 16a,b,d were more potent and/or more selective than compound 9a. The selectivity of the new compounds towards each of the three cancer cell lines was calculated by dividing their IC50 values against normal MRC-5 cells by their IC50 values against the corresponding cancer cell line (Selectivity towards cancerous cells is one of the important issues which must be considered during the discovery and development of new anticancer agents. To assess the toxicity and selectivity of the new compounds (ell line .15a–e exhibited lower selectivity (SI = 0.05–118.45) towards the three cancer cell lines than their benzamide analogues 16a–e (SI = 0.45–270). Among the new compounds, compound 16d was the most selective towards MCF-7 and A2780 cells, while compound 15e showed the highest selectivity to the HCT116 cancer cell line  and very weak cytotoxicity against A2780 and MCF-7 cancer cell lines with IC50 values of 25.76 and 36.88 μM, respectively. On the other hand, replacement of the 3,4,5-trimethoxybenzylidene)amino in compound 15a with 3,4,5-trimethoxybenzamide moiety resulted in a significant increase in the cytotoxic activities against the three cancer cell lines.The study of SAR of the new compounds is represented in 15a–e, substitution on para-position of the phenyl ring of compound 15a with electron-donating (OCH3) or electron-withdrawing (Cl/Br) atoms resulted in a significant increase (1.7–628.3-fold) in cytotoxic activity against MCF-7 and A2780 cells, while substitution with CH3 group decreased cytotoxicity against both HCT116 and A2780 cancer cell lines (The substituents on the phenyl ring showed variable effects on cytotoxicity of the new compounds. In Schiff base derivatives ll lines .16a–e showed that substitution on the phenyl ring of compound 16a with the electron-donating (OCH3) or electron-withdrawing (Br) groups resulted in a significant decrease (3.3–20.8-fold) in the cytotoxicity against MCF-7 and A2780, while substitution with CH3/Cl groups resulted in a very slight increase in cytotoxic activity against both MCF-7 and HCT116 cell lines  resulted in 1.4–27.5-fold increase in cytotoxicity against the three cancer cell lines  in cytotoxic activities against the three cell lines. Expansion of the pyrrolidine ring in compound 16a to piperidine ring resulted in an increase in cytotoxic activity against the three cancer cell lines.Replacement of the 3,4,5-trimethoxybenzylidene-amino moiety in compound In conclusion, the 3,4,5-trimethoxybenzamido moiety and the electron-withdrawing atoms on the phenyl ring at C-5 on the pyrrolizine scaffold are favoured for high cytotoxic outcomes.3.2.2.9a was mediated by the induction of apoptosis in MCF-7 cells,16a,b,d, the most active in the MTT assay (16a increased apoptosis (early and late events) in MCF-7 cells to 19% compared to 1% in the control. Moreover, compound 16b caused a significant increase of MCF-7 apoptotic cells which was similar to the effect of compound 16a. However, compound 16d was more potent in inducing apoptosis in MCF-7 cells (26%) compared to compounds 16a and 16b.The cytotoxic activity of our previously reported pyrrolizine-5-carboxamide TT assay  were sel6a,d caused slight increases  in the necrotic events compared to by the control (1.90%). On the other hand, compound 16b caused a 7.11% increase in necrotic events.The test compounds also caused variable degrees of necrosis in MCF-7 cells. Compounds 116a,b,d induced higher apoptotic events in MCF-7 cells at lower concentration compared to compound 9a  (Compound %=3–25%) .16a,b,d, the most active in MTT assay were also selected for the kinase profiling test to investigate their potential mechanism of action. The three compounds were tested at the same concentration (10 μM) against the same 20 kinases used in the previous report,In the current work, compounds 16a,b,d with inhibition% in the range of 1–38%. The activities of seven of these kinases were inhibited by the three compounds (2–28%)  .16a,b,d resulted in 19–28% inhibition in the activity of the kinase compared to only 2% and 4% inhibition by imatinib and compound 9a, respectively. Compounds 16a,b also showed inhibitory activity against EGFR with inhibition% of 30 and 11%, respectively. Moreover, compound 16d exhibited 14% inhibition in the activity of Aurora A kinase compared to 38% inhibition for imatinib.Treatment of CDK-2/cyclin A1 with compounds 16a,b,d. In addition, compounds 16a,b displayed inhibitory activity against EPHA1, MSK1, p38 Alpha, and SGK1 with inhibition% in the range of 5–22% , BLK (2–11%), CK1 Alpha 1 (7–15%), and DYRK3 (4–17%) was also observed following the treatment with compounds of 5–22% . Althoug16a,b,d compared to compound 9a. This improvement was observed in the number of oncogenic kinases (8–13 kinases) inhibited by the new compared to only five kinases inhibited by compound 9a. In addition, compounds 16a,b,d exhibited higher inhibitory activities (1–38%) than those of compound 9a (3–25%)  . These r3.2.3.2.16a,b,d. Among these kinases, CDK-2 was inhibited by the three compounds with inhibition% in the range 19–28%. To confirm the activity against CDK-2, the three compounds were also evaluated for their CDK-2 inhibitory activity using the ADP-Glo kinase assay kit  according to the previous reports50 values of the three compounds are presented in The results of the kinase profiling test  revealed16a,b,d compared to compound 9a, which was matched with the results of the kinase profiling test (16a with the unsubstituted phenyl ring was the most potent as a CDK-2 inhibitor (IC50=0.086 μM). Moreover, compound 16d also exhibited potent inhibitory activity against CDK-2 (IC50=0.113 μM), while 16b with the electron-donating methyl group was the least active among the three benzamide derivatives (3) group.The results revealed higher inhibition in activity CDK-2 by compounds ing test . Among tivatives . These r3.2.4.16a,b,d were selected to investigate their effect on cell cycle phases of MCF-7 cells. The cancer cells were treated with the test compounds at 0.1 μM and vehicle (control) for 24 h according to the previous report1 (11–14-fold) and G2/M (5–7-fold) cell cycle phases at the expense of 5–10-fold decrease of G1 events, all compared to control. On the other hand, the S phase was the least affected with less than a twofold decrease. Compound 16d showed the strongest preG1 (27.56%) and G2/M (45.62%) cell cycle effects in MCF-7 cells  revealed blockade of MCF-7 cells at the S phase with more than a threefold increase compared to the control (17.3–55.6%), at the expense of decreases in other cell cycle phases16a,b,d (0.1 µM) showed completely different effects on cell cycle phases of MCF-7 cells at a much lower concentration compared to compound 9a. The three compounds 16a,b,d increased the preG1 and G2/M cell cycle phases in MCF-7 cells at the same time point (24 h)  .3.2.5.3.2.5.1.16a,b,d, the most active in MTT assay were selected to investigate their effects on the kinetics of the three phases of tubulin polymerisation . In this assay, light scattering by microtubules which is directly proportional to the concentration of the microtubule polymer was used as a measure of tubulin polymerisation. The study was performed using a fluorescence-based tubulin polymerisation assay kit  following the manufacturer’s instructions (https://www.cytoskeleton.com/bk011p) and according to the previous reportIn the current study, the new compounds were designed bearing the trimethoxy phenyl moiety aiming to obtain new cytotoxic agents that can interfere with tubulin polymerisation in cancer cells. Herein, compounds 2, 0.5 μM) was used as a positive control (inhibitor) as per the manufacturer’s instructions (https://www.cytoskeleton.com/pdf-storage/datasheets/bk011p.pdf). The vehicle was also used to evaluate the polymerisation when tubulin is incubated in the absence of the test compound. The effect of the selected compounds (5 μM) on tubulin polymerisation was evaluated and compared with those of the positive control. The results are represented in Paclitaxel (3 μM) was used as a tubulin polymerisation enhancer , while calcium chloride  indicates the total light scattered and corresponds to the total mass of the polymerised tubulin  for 24 h. The nuclei of the cancer cell stained blue with 4′,6-diamidino-2-phenylindole (DAPI), while microtubules were stained (green) with anti-β-tubulin mouse monoclonal antibody and Alexa Fluor® 488 secondary antibodies .To assess the effect of the new compounds on microtubule cytoskeleton in living cells, compound on assay  was sele16b on the microtubule cytoskeleton. The results are represented in 16b showed shrank and partially disorganised microtubules which split partially into fragments. These results indicated the ability of compound 16b to induce a disruption in the formation of microtubules.The immunofluorescence images of the untreated/treated cancer cells were taken to visualise the effect of the test compounds 3.3.16a,b,d. The binding affinities, modes, orientations, and interactions of the new compounds into the relevant protein were investigated using AutoDock 4.2In the current work, the molecular docking studies were performed into protein kinases (PKs) and tubulin to understand the mechanism of action of compounds 3.3.1.16a,b,d compared to the lead compound 9a, a molecular docking study of the new compounds into the active sites of CDK-2, EGFR, and Aurora A were performed. The crystal structure of CDK-2 (pdb code: 3TNW)https://www.rcsb.org/). AutoDock (v.4.2) was used to perform the docking studyTo rationalise the improvement of kinase inhibitory activity of the new compounds 16a,b,d into the selected kinases revealed nice fitting into the active site of CDK-2 and EGFR. These results were confirmed by the absence of any steric clashes or unfavourable interactions with the amino acids in the active sites of the two kinases. Moreover, compounds 16a,b,d exhibited higher binding affinities towards CDK-2 and EGFR compared to CAN508, and erlotinib, respectively  towards CDK-2 compared to −6.47 kcal/mol for CAN508 (IC50=3.5 μM against CDK-2)16a showed the highest affinity towards CDK-2 followed by the chloro analogue 16d. This order of binding affinities was in concordance with the inhibitory activities of the three compounds against CDK-2  in the ATP binding site of CDK-2. These binding interactions were similar to those reported for several CDK-2 inhibitors16b,d displayed only three conventional hydrogen bonds with amino acids in CDK-2. The fewer number of conventional hydrogen bonds could also account for the lower inhibitory activities of compounds 16b,d compared to compound 16a  although it did not show in vitro inhibitory activity against EGFR  and the amino acids in EGFR were investigated. The results revealed that compound 16a exhibited three conventional hydrogen bonds with THR830 and ASP831 .However, compound nst EGFR . To expld ASP831 . Compounh CYC773 . These r16a,b,d, only compound 16d showed weak inhibitory activity against aurora A (14% inhibition) in the kinase profiling test. The docking study of compounds into the active site of aurora A (pdb code: 3E5A) revealed the highest affinity for compound 16d, Supplementary data (Table S3).In addition, among the three derivatives 3.3.2.https://www.rcsb.org/structure/5lyj). The protein and ligand files were prepared following the previous report,In this part, another molecular docking study was performed into the binding site of CA-4/colchicine in tubulin protein to investigate the binding mode/interactions of the new compounds correlate the obtained results with the inhibitory effects of these compounds on tubulin polymerisation  and 9. T1 was re-docked into the binding site of the co-crystallised ligand (7BA) in tubulin protein. The results revealed the superposition of the re-docked CA-4 with the co-crystallised ligand with RMSD of 0.9 Å. Moreover, the re-docked CA-4 showed similar binding mode, orientation, and exhibited the same binding interactions as those of the co-crystallised ligand  d ligand .Investigation of the binding interactions of CA-4 with the amino acids in tubulin protein revealed one conventional hydrogen bond with THR179, one pi-sulphur interaction with MET259, and three carbon hydrogen bonds with VAL238, VAL315, and ASN350. In addition, several hydrophobic interactions with CYS241, ALA250, LEU255, ALA316, and LYS352 were observed .16a–e and 21) displayed higher binding affinity  compared to −8.72 kcal/mol for CA-4, Supplementary data (Table S4). Moreover, the benzamide derivatives (16a–e and 21) also displayed higher binding free energies than their corresponding Schiff bases (15a–e and 20). These results were also matched with the results of the cytotoxic activities of the two series  exhibited binding orientations similar to that of CA-4. The two phenyl rings in each of the six benzamide derivatives adopted orientations superposing on the two phenyl rings of CA-4 with the trimethoxyphenyl moieties extended towards the α/β interface  adopted completely different orientations from CA-4. The difference in the binding orientations of the two series could account even partially for the difference in their binding affinities towards tubulin and their cytotoxic activities.On the other hand, the Schiff base derivatives . The results of this study are provided in the supplementary data (Table S4).The docking study into the binding site of CA-4 in tubulin protein was also performed for the remaining compounds (4.15a–e and 16a–e) bearing 3,4,5-trimethoxyphenyl moiety were designed, synthesised, and evaluated for their cytotoxic activities. In addition, two new indolizines (20 and 21) were also synthesised and for their cytotoxic activities. Structural confirmation of these compounds was carried out by spectral  and quantitative elemental analyses. The results of the MTT assay revealed weak to potent cytotoxic activity for the new compounds against three  cancer cell lines with IC50 in the range of 0.03–36.88 μM. Among the new derivatives, compounds 16a,b,d exhibited the highest cytotoxicity against MCF-7 cells (IC50=0.068–0.082 μM), while compounds 16c,d were the most active against HCT116 and A2780 cells, respectively. On the other hand, the new compounds showed weak cytotoxicity against the normal MRC-5 cells (IC50=0.155–17.08 μM) compared to 13.66 μM for lapatinib. Compounds 16a,b,d exhibited also cytotoxic activities against doxorubicin-resistance MCF-7/ADR cells (IC50=0.52–6.26 μM). The study of SAR revealed higher cytotoxic activity and selectivity for the benzamide derivatives (16a–e and 21) compared to their corresponding Schiff bases (15a–e and 20). The kinase profiling test of compounds 16a,b,d revealed improvement in their inhibitory activities against several kinases (inhibition%=1–38%) compared to compound 9a. Moreover, the three compounds exhibited higher inhibitory activity against CDK-2 than compound 9a. Mechanistic studies of compounds 16a,b,d also revealed weak to moderate inhibitory activity against tubulin polymerisation. Cell cycle analyses of MCF-7 cells treated with compounds 16a,b,d revealed dramatic increases in the preG1 (11–14-fold) and G2/M (5–7-fold) cell cycle phases at the expense of 5–10-fold decrease of G1 events, all compared to the control. Compound 16d showed the strongest preG1 (27.56%) and G2/M (45.62%) cell cycle effects. The results of the docking study into tubulin/CDK-2/EGFR showed higher binding affinities for compounds 16a,b,d compared to the co-crystallised ligands (CAN508 and erlotinib). Taken together, compounds 16a,b,d could serve as promising lead compounds for the future development of new potent anticancer agents.In the present study, two new series of pyrrolizine derivatives (Click here for additional data file.


Syringa oblata L. (S. oblata) is a traditional Chinese medicine that exhibited a significant therapeutic effect on various inflammatory diseases. Its commercial drug Yan Li Xiao (YLX) capsule is used as a cure for the treatment of inflammatory diseases, such as bacillary dysentery, tonsillitis, bronchitis, and acute gastroenteritis. However, studies about S. oblata relieving acute pharyngitis are rare. In this study, high-performance liquid chromatography (HPLC) was used to analyze the chemical profile of S. oblata, and the bioactive phytoconstituents were isolated and identified by nuclear magnetic resonance (NMR) and mass spectrometry. An ammonia-induced acute pharyngitis rat model was established to estimate the protective effect of S. oblata in vivo for the first time. The severity of pharyngitis was observed by appearance index and HE staining. The cytokines expression was performed by ELISA. Crucial proteins expression associated with TLR4/NF-κB/MAPK and NLRP3 inflammasome signaling pathways were analyzed by western blotting and immunohistochemistry. Furthermore, the anti-inflammatory effect of isolated compounds was evaluated by suppressing lipopolysaccharide- (LPS-) induced NO generation and regulating the cytokines levels in RAW 264.7 cells. The results showed that S. oblata exhibited a protective effect in the ammonia-induced acute pharyngitis rat model, and the compounds exert potential anti-inflammatory properties against LPS-activated RAW 254.7 cells.Acute pharyngitis is an inflammation of the pharyngeal mucous membrane and submucous lymphoid tissues. Unsatisfactory treatment and repeated occurrences might cause chronic pharyngitis and other complications.  Acute pharyngitis is the most common seasonal nonspecific inflammation among upper respiratory tract diseases –3. PatieκB) and mitogen-activated protein kinases (MAPKs) can be activated via interaction of dimerized TLR4 with its downstream effector MyD88 to regulate the release of inflammatory cytokines for controlling inflammation, including interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and other proinflammatory cytokines － peaks at m/z  δ: 7.52 , 7.06 , 6.72, 5.48 , 4.69 , 4.47 , 4.28, 3.92 , 3.64 , 3.62 , 3.38 , 3.33 , 3.23 , 3.02 , 2.85 , 2.82 , 2.44 , 2.10 , 1.50 . 13C  δ: 174.75 (C-7), 167.84 (C-11), 157.13 (C-4′), 154.43 (C-3), 130.97 (C-2′), 130.94 (C-6′), 116.32 (C-3′), 116.26 (C-5′), 109.61 (C-4), 100.68 (C-1″), 96.37 (C-1), 77.93 (C-5″), 75.76 (C-8), 74.72 (C-2″), 71.71 (C-4″), 66.51 (C-8′), 62.92 (C-6″), 41.86 (C-9), 35.30 (C-7′), 34.65 (C-6), 28.26 (C-5), 21.81 (C-10). HR-ESI-MS m/z 510.1584 [M-H]− .Syringalactone A (1): amorphous powder; 1H  δ: 7.44 , 7.04 , 6.72 , 5.61 , 4.69 , 4.25 , 3.71 , 3.64 , 3.39 , 3.22 , 3.21 , 3.20 , 3.19 , 2.83 , 2.54 , 2.45 , 2.33 , 2.11 , 1.13 . 13C  δ: 219.81 (C-7), 168.38 (C-11), 157.06 (C-4′), 153.20 (C-3), 130.93 , 130.12 (C-1′), 116.31, 111.22 (C-4), 100.22 (C-1′), 95.44 (C-1), 78.40 (C-3″), 77.97 (C-5″), 74.65 (C-2″), 71.56 (C-4″), 66.31 (C-8′), 62.74 (C-6″), 46.50 (C-9), 44.65 (C-8), 35.30 (C-7′), 28.26 (C-5), 13.70 (C-10). HR-ESI-MS m/z 494.1692 [M-H]− .Syringopicroside (2): amorphous powder; 1H  δ: 7.51 , 5.90 , 4.81 , 4.22 , 4.21 , 4.08 , 3.95 , 3.88 , 3.71  3.67 , 3.42 , 3.39 , 3.34 , 3.32 , 2.82 , 2.71 , 2.44 , 1.64 . 13C  δ: 173.36 (C-2), 168.67 (C-11), 155.15 (C-3), 145.87 (C-3′), 142.41 (C-4′), 131.02 (C-1′), 130.06 (C-9), 124.88 (C-8), 121.08 (C-6′), 116.32 (C-2′), 116.32 (C-5′), 109.43 (C-4), 100.88 (C-1″), 95.18 (C-1), 78.49 (C-5″), 77.99 (C-3″), 74.81 (C-2″), 71.53 (C-4″), 66.91 (C-8′), 62.80(C-6″), 51.90 (11-OMe), 41.26 (C-6), 35.19 (C-7′), 31.85 (C-5), 13.56 (C-10). HR-ESI-MS m/z 538.1490 [M-H]− .Oleuropein (3): amorphous powder; 1H  δ: 7.51 , 7.06 , 6,72 , 6.07 , 5.92 , 4.80 , 4.21 , 4.10 , 3.95 , 3.88 , 3.71 , 3.67  3.41 , 3.40 , 3.33 , 3.31 , 2.83 , 2.71 , 2.44 , 1.64 . 13C  δ: 173.21 (C-7), 168.67 (C-11), 157.11 (C-4′), 155.15 (C-3), 131.02 , 130.53 (C-9), 130.06 (C-1′), 124.88 (C-8), 116.32 , 109.44 (C-4), 100.88 (C-1″), 95.18 (C-1), 78.49 (C-5″), 77.99 (C-3″), 74.81 (C-2″), 71.53 (C-4″), 66.91 (-8′), 62.80 (C-6″), 51.90 (11-OMe), 41.31 (C-6), 35.20 (C-7′), 31.86 (C-5), 13.55 (C-10). HR-ESI-MS m/z 524.1702 [M-H]− .Ligstroside (4): amorphous powder; The severity of lesions on the pharyngeal was evaluated according to score criteria. As shown in β, TNF-α, IFN-γ, IL-4) and inflammation mediators  in tissue were assessed using ELISA kits. The results showed that the levels of IL-6, IL-1β, TNF-α, IFN-γ, PGE2, and COX-2 were significantly increased in ammonia-induced acute pharyngitis rats compared to the control group, while the levels of IL-4 and EGF were decreased. In contrast, the balance between cytokines was regulated after treatment with S. oblata and YLX. The production of IL-6, IL-1β, TNF-α, IFN-γ, PGE2, and COX-2 was dramatically inhibited in treatment groups, whereas the release of IL-4 and EGF was promoted   was used to evaluate the histopathological changes of pharyngeal tissue in ammonia-induced acute pharyngitis. As shown in 5 mg/kg)  were lowκB/MAPK signal pathway in macrophages [κB/MAPK signal pathway could promote and amplify the inflammatory response during cell and tissue damage and pathogen invasion. Thus, the expression of key proteins was examined by western blot and immunohistochemistry, respectively. As shown in Figures κB, JNK1/2/3, p-JNK1/2/3, ERK1/2, p-ERK1/2, p38, and p-p38 compared to the control group while the protein expression in treatment groups significantly reduced compared to the model group. Moreover, immunohistochemical staining further confirmed this result , syringopicroside (2), oleuropein (3), and ligstroside (4) on cell viability in RAW 264.7 cells. The four iridoids at the concentration ranging from 5.5 to 176.0 .7 cells . TherefoμM. Furthermore, all compounds showed an evidently dose-dependent manner except for syringopicroside (2). Hence, the 44.0, 88.0, and 176.0 μM were used for the analysis of other inflammatory cytokines.As shown in α levels while decreasing the IL-4 level. However, pretreatment with test compounds  significantly reversed this situation, suggesting that the four compounds were the inhibitors of inflammation at the tested concentrations , an adaptor (ASC), and an effector (caspase-1). It is a major intracellular inflammatory pathway, mainly driving autoimmune and inflammatory disease processes by regulating the productions of proinflammatory cytokines . Assemblectively . Severaltivation . TherefoNO is a crucial mediator contributing to the pathogenicity of inflammation. Therefore, regulating the release of NO played an important role in the investigation of the anti-inflammatory effect of test compounds. The anti-inflammatory activity in vitro of four iridoids was determined by inhibiting NO production and regulating inflammatory cytokines levels. Interestingly, test compounds exhibited a significant effect in a dose-dependent manner except for syringopicroside. This phenomenon may be caused by the contradictory effect of syringopicroside on macrophages. The result of syringopicroside in vitro was consistent with the previous study . The pheS. oblata. S. oblata significantly regulated the balance between proinflammatory and anti-inflammatory cytokines. The results indicated that S. oblata could alleviate ammonia-induced acute pharyngitis by inhibiting the activation of TLR4/NF-κB/MAPK and NLRP3 inflammasome signaling pathways. Furthermore, four iridoids were isolated from the leaves of S. oblata and identified by NMR methods. Results indicated that all of them exerted anti-inflammatory activity in LPS-stimulated RAW 264.7 cells in different degrees. This study provided a potential basis for future clinical treatment and research in inflammation-related diseases.In this study, the ammonia-induced acute pharyngitis model was used for evaluating the therapeutic effect of


Reaction of 3 with two equivalents of α-halocarbonyl compounds 6–8, 10, and 12a–d afforded the corresponding bis-thiazolidines 9, 11, and 13a–d, respectively. Condensation of bis-thiazolidin-4-one 9 with different aromatic aldehydes furnished bis-thiazolidin-4-ones 14a–d. Compounds 5a–g, 9, and 13a,c,d were screened in vitro for their cytotoxic activities in a panel of cancer cell lines. Compounds 5a–c, 5f–g, and 9 exhibited remarkable cytotoxic activities, especially compound 5c with potent IC50 value 0.6 nM  and compound 5f with high IC50 value 6 nM . Compound 5f–induced appreciated apoptotic cell death was measured as 82.76% associated with cell cycle arrest at the G1 phase. The apoptotic pathways activated in KF-28 cells treated with 5a, 5b, and 5f were further investigated. The upregulation of some pro-apoptotic genes, bax and puma, and the downregulation of some anti-apoptotic genes including the Bcl-2 gene were observed, indicating activation of the mitochondrial-dependent apoptosis. Together with the molecular docking studies of compounds 5a and 5b, our data revealed potential Pim-1 kinase inhibition through their high binding affinities indicated by inhibition of phosphorylated C-myc as a downstream target for Pim-1 kinase. Our study introduces a set of bis-thiazoles with potent anti-cancer activities, in vitro.A series of  For example, thiazoles demonstrated antimicrobial, antiviral, anti-diabetic, diuretic, anticonvulsant, antioxidant, anti-HIV, analgesic, anti-inflammatory, neuroprotective, and antitumor activities. These wide and potent activities were extensively published in several review articles . The antpioglitazone and epalrestat (anti-diabetic drugs), etozoline (diuretic drug), and darbufelone (anti-inflammatory drug)  .Multi-component reaction (MCR) is attracting the attention of chemists for the construction of diverse heterocyclic scaffolds. MCR is a powerful synthetic methodology in which three or more reactive substrates are mixed in one reaction flask to form a new product employing all substrates with minimized by-products and good yields .bis-thiazole structures for examining their potency against several of human cancer cell lines as well as investigating their effects as an inducer of apoptosis on the tested cancer cells.Our research interest is focused on the synthesis and biological evaluation of thiazole-based heterocycles . Therefobis-thiosemicarbazone 3 was reported once in the literature in 1982 as an analytical reagent for spectrophotometric estimation of copper, but no more applications of this substrate in organic synthesis were published so far  as well as elemental analyses confirmed the assigned bis-thiazole structures 5a–g, −1, and their mass spectra revealed, in each case, a peak due to their molecular ions (M+). The 1H NMR spectrum of compound 5b, for example, revealed three singlet signals at δ 2.25, 2.51, and 10.41 (D2O-exchangeable) due to the thiazole-CH3, p-tolyl, and thiazole-NH protons, respectively, and two multiplets in the regions 2.73–2.74 and 2.89–2.90 assigned to cyclohexane protons plus two doublet peaks at 7.12 and 7.23 (J = 8.4 Hz) corresponding to the para-substituted aromatic protons. The 13C NMR spectrum of 5b exhibited 14 peaks corresponding to six aliphatic carbon atoms and eight aromatic carbon atoms.1,4-Cyclohexanedione-d so far . Thus, wInterestingly, a multi-component one-pot procedure was investigated for the construction of the products 5a–c as outlined in bis-thiosemicarbazone 3 with ethyl chloroacetate (6) in 1:2 M ratios in refluxing ethanol using triethylamine as a base led to production of a compound that was established as the bis-4-thiazolidinone derivative (9) in 73% yield  appeared in its mass spectrum. The 1H NMR spectrum of compound 9 revealed two multiplet peaks in the regions δ 2.53–2.57 and 2.68–2.73 assignable to cyclohexane protons and a singlet peak at δ 3.81 for thiazolidinone-CH2 protons besides a broad peak at δ 11.72 (D2O-exchangeable) due to thiazolidinone-NH protons. 13C NMR of compound 9 exhibited five peaks at δ 25.35, 32.23, 32.72, 166, and 173.93. Two alternative methods for synthesis of the bis-thiazolidinone derivative 9 were conducted as shown in bis-thiosemicarbazone 3 with chloroacetic acid (7) in ethanol solvent at reflux temperature employing sodium acetate as a base or with ethyl bromoacetate (8) in dimethylformamide and triethylamine as a base yielded the same product in 62 and 68% yields, respectively, which was identical in all aspects to compound 9 obtained above. Similarly, treatment of compound 3 with two equivalents of α-chloroacetylacetone (10), under a typical reaction procedure above, furnished the 1,4-diylidene-bis-thiazolidine derivative 11 in 71% yield.In the same way, treatment of 3% yield  based onbis((4-arylthiazol-2-ylidene)hydrazono)cyclohexane derivative 13. Thus, when bis-thiosemicarbazone 3 was allowed to react with 2-(bromoacetyl)benzothiazole (12a) in a 1:2 M ratio in ethanol at reflux conditions using triethylamine as a base, it furnished a single product that was established as 1,4-bis((4-(2-benzothiazolyl)thiazol-2-ylidene)hydrazono)-cyclohexane (13a) in 72% yield , thiosemicarbazide (2), and 2-(bromoacetyl)benzothiazole (12a) in 1:2:2 M ratios in one flask, dissolved in ethanol and then heated at reflux temperature and catalyzed by triethylamine. The 1H-NMR spectrum of compound 13a exhibited three multiplet peaks in the regions δ 2.58–2.71, 7.43–7.53, and 8.01–8.09 corresponding to the cyclohexane and benzothiazole protons, respectively, in addition to two singlets at δ 7.73 and 11.03 assignable to thiazole-5-CH and thiazole-3-NH protons, respectively. 13C NMR revealed four aliphatic carbon atoms at δ 23.94, 26.18, 29.29, and 31.50 and nine aromatic carbon atoms at 109.32, 122.34, 122.54, 125.15, 126.52, 134.54, 144.65, 153.59, and 154.11 in addition to 162.79 (C=N) and 170.37 (C=O). Similarly, reaction of bis-thiosemicarbazone 3 with three extra examples of α-bromoketones, namely, 2-(bromoacetyl)benzofuran (12b), 3-(bromoacetyl)coumarin-2-one (12c), and 4-chlorophenacyl bromide (12d), under a typical reaction procedure described above, gave the corresponding 1,4-bis((4-arylthiazol-2-ylidene)hydrazono)cyclohexane derivatives 13b–d in high yields as shown in The scope of our strategy was extended to encompass synthesis of the new 1,4-2% yield  on the bbis(thiazolidin-4-one) derivatives 14a–d was also carried out. Thus, condensation reaction of the bis-thiazolidin-4-one derivative 9 with a number of aromatic aldehydes in ethanol heated at reflux conditions, using piperidine as a base, afforded the 1,4-bis((5-arylidene)-4-oxo-thiazolidin-2-ylidene)hydrazono-cyclohexane derivatives 14a–d -5-arylidene-thiazolidin-4-one derivatives , while compound 5a displayed a notable cytotoxic effect with an IC50 value of 1.51 µM against the MDA-MB-231 cells (treated for 72 h). Finally, the results from cervical cancer, Hela, cells showed a pronounced response when treated with compound 5c with the most significant inhibition potency having IC50 value 0.00065 µM (72 h treatment). Therefore, compounds 5a, 5b, and 5f were thought worthwhile to further investigate their effects as either cytotoxic or antiproliferative effects and whether any of them is an inducer of apoptotic cell death in the representative cancer cell lines KF-28 and A2780.The synthesized derivatives were screened for their cytotoxic activities against five cancer cell lines . This study was performed using serial concentrations of 25, 50, 100, 150, and 200 µM of each compound using the MTT assay. Percentages of cell viability of the tested compounds with serial concentration against the tested panel of cell lines are depicted in bis-thiazole derivatives against various cancer cell lines with promising IC50 values. However, the MTT technique used just indicates the viability under different treatments; therefore, this encouraged us to further investigate the cellular and molecular mechanisms behind such cytotoxicity.Our results agree with those of several previous studies , which iStudying the cell cycle phases by measuring DNA content is a crucial test that helps in evaluating the impact of a given drug(s) on dividing cells and may reflect the mechanism through which this drug(s) affects those cells. Hence, we used the ovarian cancer cells (KF-28) as a representative cellular model to study the effect of the synthesized compounds on the cell cycle. Compounds 5a, 5b, 5c, 5f, 5g, and 9 were added to similar numbers of cells at 200 µM for 48 h, and then, the different cell cycle phases were assessed by flow cytometric analysis.DNA content analysis of KF-28 cells treated with compounds 5a, 5b, 5c, 5f, 5g, and 9 showed that compound 5a significantly accumulated the cells at the S phase , while other compounds such as 5b and 9 showed appreciated reduction in the S phase population of the cells tested measured as 4.5 and 6.5%, respectively , caspases 3, 8 9, anti-apoptotic gene (Bcl-2), and Pim-1 kinase genes in KF-28 cells.5a resulted in a remarkable activation of p53 mRNA relative expression (≈4.07-fold) with a concomitant activation of the puma and bax mRNA levels with a maximum increase of ≈5.08-fold and 5.2-fold, respectively. Compound 5a was able to significantly increase the mRNA levels of caspase 3, 8, 9 genes with a maximum increase of ≈6.2-fold, 2.9-fold, and 5.6-fold, respectively, while it exceptionally inhibited the anti-apoptotic Bcl-2 expression (maximum decrease of ≈0.27-fold) and the Pim-1 kinase expression (maximum decrease of ≈0.31-fold). Additionally, treatment of KF-28 cells with compound 5b significantly activated the level of p53 expression (≈3.41-fold) with consequent activation of the puma and bax expression levels with a maximum increase of ≈4.14-fold and 3.33-fold, respectively, as shown in bis-thiazole derivative 5a was also able to significantly increase the mRNA levels of caspase 3, 8, 9 genes with a maximum increase of ≈4.2-fold, 2.9-fold, and 4.7-fold, respectively. At the same time, it significantly inhibited the anti-apoptotic Bcl-2 expression (maximum decrease of ≈0.5-fold) and the Pim-1 kinase expression (maximum decrease of ≈0.48-fold). Finally, in the same As shown in bis-thiazole derivatives used here  were able to induce apoptosis in the representative cancer cell lines. This apoptosis is mainly a mitochondrial-dependent one as indicated by the gene expression experiment. These compounds were able to enhance the pro-apoptotic genes and inhibit the anti-apoptotic ones, transcriptionally.Based on the fact that cancer is known as one of the scenarios where too little apoptosis occurs , testingPim-1 kinase has been observed to be highly expressed in some solid tumors . Therefobis-thiazole derivatives toward Pim-1 kinase (PDB = 2OBJ) was investigated at the gene expression level using the RT-PCR in the treated FK-28 cells with compounds 5a, 5b, and 5f as mentioned above , as shown in The three active compounds 5a, 5b, and 5f against KF-28 with ICPim-1 has been classified as a weak oncogene. However, a very strong synergism with regard to tumorigenicity occurs between Pim-1 and C-myc. This synergism is observed with bitransgenic mice overexpressing C-myc and Pim-1 in lymphoid tissue . To furt1H NMR), and chemical shifts are given in ppm from TMS. 13C NMR spectra were recorded with a Varian Mercury 300 spectrometer . The 1H NMR samples were measured in 5 mm NMR tubes with a concentration of about 20 mg of the product in 0.6 ml of solvent (DMSO-d6) and about 75–100 mg of the product in about 0.6 ml of solvent for 13C NMR. Mass spectra were recorded on DI Analysis Shimadzu QP-2010 Plus. Elemental analyses were carried out at the Microanalytical Center, Cairo University. 1,4-Cyclohexanedione, ethyl chloroacetate, chloroacetic acid, ethyl bromoacetate, α-chloroacetylacetone, and thiosemicarbazide were used as purchased from Aldrich. The starting materials 4a–g were prepared as reported in the literature  were recorded on a Fourier transform infrared spectrophotometer (IRAffinity-1S from Shimadzu Corporation). NMR spectra were measured with a Varian Gemini 300 spectrometer (300 MHz terature .2), 1,597 (C=N) cm−1; 1H NMR (DMSO-d6) δ 2.42–2.50 , 2.54–2.74 , 7.59 , 8.05 , 9.9–10.11 .To a solution of 1,4-cyclohexanedione (1)  in absolute ethanol (20 ml), thiosemicarbazide (2)  was added, and then the reaction mixture was heated under reflux for 10 min with stirring. The reaction mixture was then left to cool to room temperature, and the solid product was collected by filtration, washed with ethanol, dried, and finally recrystallized from ethanol/DMF to give compound 3 as colorless crystals, 0.18 g (65%), mp. 213–214°C [Lit. mp. 213–214°C ]; IR (KBbis-thiosemicarbazone (3)  and the appropriate hydrazonoyl chlorides 4a–g (2 mmol) in absolute ethanol (10 ml), triethylamine (0.1 ml) was added, and the resulting mixture was refluxed for 6–8 h and then left to cool to room temperature. The solid product formed was collected by filtration, washed with ethanol, dried, and finally recrystallized from the ethanol/DMF mixture to give the corresponding bis-thiazole derivatives 5a–g.To a mixture of 1,4-cyclohexane-bis-thiazole derivatives 5a, 5b, and 5c in 77, 76, and 60% yields, respectively.To a mixture of 1,4-cyclohexadione (1) , thiosemicarbazide (2) , and the appropriate hydrazonoyl chlorides 4a–c (2 mmol) in absolute ethanol (10 ml), triethylamine (0.1 ml) was added, and then the reaction mixture was heated under reflux for 8 h. The reaction mixture was then left to cool to room temperature for complete precipitation. The solid product was collected by filtration, washed with ethanol, dried, and finally recrystallized from the ethanol/DMF mixture to afford the 1,4-Bis((5-phenylazo-4-methylthiazol-2-ylidene)hydrazono)cyclohexane (5a): Brown crystals, yield , mp. 211–212 °C; IR (KBr) ν 3,229 (NH), 1,600 (C=N) cm−1; MS m/z (%): 77 (100%), 93 (71.2%), 108 (31%), 218 (40%), 542 ; 1H NMR (DMSO-d6) δ 2.56 , 2.73–2.75 , 2.89–2.91 , 6.9–7.33 , 10.44 . Anal. calcd. for C26H26N10S2 (542.68): C, 57.54; H, 4.83; N, 25.81%. Found: C, 57.24; H, 4.64; N, 25.59%.1,4-Bis((5-(4-tolylazo)-4-methylthiazol-2-ylidene)hydrazono)cyclohexane (5b): Brown crystals, yield , mp. 221–222 °C; IR (KBr) ν 3,297 (NH), 1,615 (C=N) cm−1; MS m/z (%): 91 (100%), 106 (53%), 232 (28%), 570 ; 1H NMR (DMSO-d6) δ 2.25 , 2.51 , 2.73–2.74 , 2.89–2.90 , 7.12 , 7.23 , 10.41 ; 13C NMR (DMSO-d6) δ 16.53, 20.49, 25.96, 30.88, 32.25, 35.89, 114.25, 120.70, 129.83, 131.19, 141.25, 162.42, 171.01, 177.69. Anal. calcd. for C28H30N10S2 (570.74): C, 58.93; H, 5.30; N, 24.54%. Found: C, 58.72; H, 5.10; N, 24.34%.1,4-Bis((5-(4-chlorophenylazo)-4-methylthiazol-2-ylidene)hydrazono)cyclohexane (5c): Brown crystals, yield , mp. 269–270 °C; IR (KBr) ν 3,250 (NH), 1,620 (C=N) cm−1; MS m/z (%): 92 (82%), 111 (100%), 252 (42%), 611 ; 1H NMR (DMSO-d6) δ 2.5 , 2.73–2.75 , 2.9 , 7.30–7.37 , 10.50 . Anal. calcd. for C26H24Cl2N10S2 (611.57): C, 51.06; H, 3.96; N, 22.90%. Found: C, 50.89; H, 3.76; N, 22.65%.1,4-Bis((5-(4-nitrophenylazo)-4-methylthiazol-2-ylidene)hydrazono)cyclohexane (5d): Green crystals, yield , mp. >300 °C; IR (KBr) ν 3,271 (NH), 1,627 (C=N) cm−1; MS m/z (%): 55 (100%), 71 (64%), 263 (19.4%), 632 ; 1H NMR (DMSO-d6) δ 2.59 , 2.77 , 2.92 , 7.44 , 8.19 , 10.98 . Anal. calcd. for C26H24N12O4S2 (632.68): C, 49.36; H, 3.82; N, 26.57%. Found: C, 49.06; H, 3.62; N, 26.36%.1,4-Bis((5-(2-tolylazo)-4-methylthiazol-2-ylidene)hydrazono)cyclohexane (5e): Brown crystals, yield , mp. 224–225 °C; IR (KBr) ν 3,200 (NH), 1,624 (C=N) cm−1; MS m/z (%): 65 (33%), 77 (19.4%), 91 (100%), 106 (42%), 232 (4%), 339 (4%), 452 (2%), 542 (3%), 570 ; 1H NMR (DMSO-d6) δ 2.34 , 2.55 , 2.75 , 2.9 , 6.94–7.43 , 9.39 . Anal. calcd. for C28H30N10S2 (570.74): C, 58.93; H, 5.30; N, 24.54%. Found: C, 58.63; H, 5.27; N, 24.34%.1,4-Bis((5-(2-chlorophenylazo)-4-methylthiazol-2-ylidene)hydrazono)cyclohexane (5f): Brown crystals, yield , mp. >300 °C; IR (KBr) ν 3,270 (NH), 1,620 (C=N) cm−1; MS m/z (%): 92 (54%), 127 (100%), 217 (12%), 359 (1.3%), 611 ; 1H NMR (DMSO-d6) δ 2.58 , 2.73–2.77 , 2.91 , 7.06 , 7.37 , 7.45 , 7.59 , 9.33 . Anal. calcd. for C26H24Cl2N10S2 (611.57): C, 51.06; H, 3.96; N, 22.90%. Found: C, 50.98; H, 3.76; N, 22.70%.1,4-Bis((5-(2-nitrophenylazo)-4-methylthiazol-2-ylidene)hydrazono)cyclohexane (5g): Brown crystals, yield , mp. 220–221 °C; IR (KBr) ν 3,379 (NH), 1,651 (C=N) (cm−1); MS m/z (%): 71 (100%), 92 (57%), 230 (19.4%), 263 (49%), 372 (3%), 539 (5%), 630 ; 1H NMR (DMSO-d6) δ 2.58 , 2.73–2.76 , 2.88–2.91 , 7.55–8.05 , 10.73 . Anal. calcd. for C26H24N12O4S2 (632.68): C, 49.36; H, 3.82; N, 26.57%. Found: C, 49.16; H, 3.63; N, 26.38%.bis-thiosemicarbazone (3)  and 2 mmol of the appropriate α-halocarbonyl derivatives  in a 1:1 DMF/ethanol mixture (10 ml), triethylamine (0.1 ml) was added, and the resulting mixture was heated at reflux temperature for 8 h and then left to cool to room temperature. The solid product obtained was collected by filtration, washed with ethanol, dried, and finally recrystallized from the ethanol/DMF mixture to give compound 9. Yields of compound 9 obtained from α-chlorocarbonyls 6, 7, and 8 were , , and , respectively.(A) To a mixture of 1,4-cyclohexane-(B) Reaction of 1,4-cyclohexane-bis-thiosemicarbazone (3)  and α-chloroacetylacetone (10) (2 mmol) was carried out under typical reaction conditions as in procedure (A) to give compound 11 in 71% yield (0.15 g).1,4-Bis((4-oxo-thiazolidin-2-ylidene)hydrazono)cyclohexane (9): Yellow crystals, yield , mp. 279–280 °C; IR (KBr) ν 3,320 (NH), 1712 (C=O), 1,638 (C=N) cm−1; MS m/z (%): 87 (40%), 107 (100%), 223 (11%), 338 ; 1H NMR (DMSO-d6) δ 2.53–2.57 , 2.68–2.73 , 3.81 , 11.72 . 13C NMR (DMSO-d6) δ 25.35, 32.23, 32.72, 166, 173.93. Anal. calcd. for C12H14N6O2S2 (338.40): C, 42.59; H, 4.17; N, 24.83%. Found: C, 42.30; H, 4.42; N, 24.63%.1,4-Bis((5-acetyl-4-methylthiazol-2-ylidene)hydrazono)cyclohexane (11): Yellow crystals, yield , mp. 280–281 °C; IR (KBr) ν 3,194 (NH), 1,695 (C=O), 1,597 (C=N) cm−1; MS m/z (%): 107 (83%), 113 (77%), 141 (100%), 156 (49%), 233 (24%), 263 (90%), 418 ; 1H NMR (DMSO-d6) δ 3.36 , 4.47 , 2.55–2.65 , 11.4 . Anal. calcd. for C18H22N6O2S2 (418.53): C, 51.66; H, 5.30; N, 20.8%. Found: C, 51.89; H, 5.22; N, 21.03%.bis-thiosemicarbazone (3)  and the appropriate α-bromoketone derivatives 12a–d (2 mmol) in absolute ethanol (10 ml), triethylamine (0.1 ml) was added, and the resulting mixture was refluxed for 8 h and then left to cool to room temperature. The solid product was collected by filtration, washed with ethanol, dried, and finally recrystallized from the ethanol/DMF mixture to give the corresponding bis-thiazoles 13a–d.To a mixture of cyclohexane-1,4-1,4-Bis((4-(2-benzothiazolyl)thiazol-2-ylidene)hydrazono)cyclohexane (13a): Green solid, yield , mp. >300 °C; IR (KBr) ν 3,250 (NH), 1,635 (C=N) cm−1; 1H NMR (DMSO-d6) δ 2.58–2.71 , 7.43 , 7.53 , 7.73 , 8.01 , 8.09 , 11.03  ppm. 13C NMR (DMSO-d6) δ 23.94, 26.18, 29.29, 31.50, 109.32, 122.34, 122.54, 125.15, 126.52, 134.54, 144.65, 153.59, 154.11, 162.79, 170.37 ppm. Anal. calcd. for C26H20N8S4 (572.07): C, 54.52; H, 3.52; N, 19.56%. Found: C, 54.42; H, 3.46; N, 19.46%.1,4-Bis((4-(2-benzofuryl)thiazol-2-ylidene)hydrazono)cyclohexane (13b): Brown powder, yield , mp. 269–270 °C; IR (KBr) ν 3,325 (NH), 1,612 (C=N) cm−1; MS m/z (%): 102 (48%), 145 (25.6%), 216 (100%), 305 (14%), 419 (2%), 461 (4.3%), 538 ; 1H NMR (DMSO-d6) δ 2.55–2.72 , 7.03 , 7.23–7.32 , 7.55 , 7.64 , 10.79 . Anal. calcd. for C28H22N6O4S2 (538.64): C, 62.44; H, 4.12; N, 15.60%. Found: C, 62.50; H, 4.25; N, 15.33%.1,4-Bis((4-(3-coumarinyl)thiazol-2-ylidene)hydrazono)cyclohexane (13c): Yellow solid, yield , mp. 241–242 °C; IR (KBr) ν 3,250 (NH), 1,697 (C=O), 1,620 (C=N) cm−1; MS m/z (%): 102 (47%), 244 (100%), 452 (0.7%), 487 (3%), 523 (3%), 593 ; 1H NMR (DMSO-d6) δ 2.57–2.73 , 7.35–7.45 , 7.62 , 7.71 , 7.81 , 8.54 , 10.75 . Anal. calcd. for C30H22N6O4S2 (594.66): C, 60.59; H, 3.73; N, 14.13%. Found: C, 60.87; H, 3.63; N, 14.08%.1,4-Bis((4-(4-chlorophenyl)thiazol-2-ylidene)hydrazono)cyclohexane (13d): Green solid, yield , mp. 230–231 °C; IR (KBr) ν 3,327 (NH), 1,609 (C=N) cm−1; MS m/z (%): 89 (48%), 168 (52%), 210 (100%), 408 (2%), 433 (3%), 482 (2%), 527 ; 1H NMR (DMSO-d6) δ 2.73 , 2.88 , 7.29 , 7.44 , 7.86 , 10.65 . Anal. calcd. for C24H20Cl2N6S2 (527.49): C, 54.65; H, 3.82; N, 15.93%. Found: C, 54.55; H, 3.75; N, 15.89%.bis-thiazolidinone derivative 9  and the appropriate aromatic aldehyde derivatives (2 mmol) in DMF/EtOH , sodium hydroxide (0.08 g) was added, and the resulting mixture was refluxed for 8 h and then left to cool to room temperature. The solid product obtained was collected by filtration, washed with ethanol, dried, and finally recrystallized from the ethanol/DMF mixture to give 14a–d.To a mixture of the 1,4-Bis((5-benzylidene-4-oxo-thiazolidin-2-ylidene)hydrazono)cyclohexane (14a): Yellowish-red crystals, yield , mp. >300 °C; IR (KBr) ν 3,124 (NH), 1,697 (C=O), 1,597 (C=N) cm−1; 1H NMR (DMSO-d6) δ 2.66–2.73 , 2.79–2.88 , 7.07–7.66 , 12.2 . Anal. calcd. for C26H22N6O2S2 (514.62): C, 60.68; H, 4.31; N, 16.33%. Found: C, 60.59; H, 4.52; N, 16.30%.1,4-Bis((5-(4-methylbenzylidene)-4-oxo-thiazolidin-2-ylidene)hydrazono)cyclohexane (14b): Yellow crystals, yield , mp. 269–270 °C; IR (KBr) ν 3,232 (NH), 1,690 (C=O), 1,630 (C=N) cm−1; MS m/z (%): 107 (44.6%), 148 (100%), 218 (13%), 325 (19%), 514 (2%), 542 ; 1H NMR (DMSO-d6) δ 2.36 , 2.25–2.63 , 2.65–2.81 , 7.35 , 7.57–7.76 , 12.45 . Anal. calcd. for C28H26N6O2S2 (542.68): C, 61.97; H, 4.83; N, 15.49%. Found: C, 61.91; H, 4.75; N, 15.39%.1,4-Bis((5-(4-chlorobenzylidene)-4-oxo-thiazolidin-2-ylidene)hydrazono)cyclohexane (14c): Orange-red crystals, yield , mp. > 300°C; IR (KBr) ν 3,124 (NH), 1,697 (C=O), 1,635 (C=N) cm−1; MS m/z (%): 55 (100%), 71 (44%), 139 (32%), 278 (13.4%), 435 (1.5%), 581 , 587 ; 1H NMR (DMSO-d6) δ 2.66 , 2.82 , 7.56–7.66 , 12.5 . Anal. calcd. for C26H20Cl2N6O2S2 (583.51): C, 53.52; H, 3.45; N, 14.4%. Found: C, 53.45; H, 3.41; N, 14.31%.1,4-Bis((5-(4-bromobenzylidene)-4-oxo-thiazolidin-2-ylidene)hydrazono)cyclohexane (14d): Orange-red crystals, yield , mp. 290–291°C; IR (KBr) ν 3,210 (NH), 1,697 (C=O), 1,643 (C=N) cm−1; MS m/z (%): 89 (100%), 133 (39%), 214 (8%), 284 (4%), 481 (0.5%), 566 (6%), 673 ;1H NMR (DMSO-d6) δ 2.50–2.72 , 2.79–2.87 , 7.47–7.81 , 12.55 . Anal. calcd. for C26H20Br2N6O2S2 (672.41): C, 46.44; H, 3.00; N, 12.50%. Found: C, 46.30; H, 3.25; N, 12.25%..All biological assays were carried out at Faculty of Science, Port Said University, Egypt, as well as at Department of Chemistry, Faculty of Science, Suez Canal University, Ismailia, Egypt2 atmosphere (NuAire). All cancer cell lines were cultured according to the routine tissue culture work as discussed by the Freshney group , and A2780 cell line was purchased from the ECACC (ECACC 93112519). The cells were maintained in RPMI-1640 . The cervical cancer cell line, Hela cells, breast cancer cell line, MCF-7, and MDA-MB-231 were purchased from the ATCC and cultured in DMEM. Both types of media were supplemented with 2 mM L-glutamine , 10% FBS , and 1% penicillin/streptomycin . All cells were incubated at 37°C in 5% COey group . The celey group . 20 μL ong Excel .5 cells/well) and incubated overnight at 37°C, under 5% CO2. The cells were then treated with indicated compounds for 48 h. Next, media supernatants and the cells were collected and washed with ice-cold PBS. Next, the cells were stained with PI (10 mg/ml) for 30 min and analyzed for DNA contents using flow cytometry analysis using an FL2 (λex/em 535/617 nm) signal detector . For each sample, 12,000 events are acquired. Cell cycle distribution is calculated using a CytoFLex machine and analyzed by CytExpert software. Flow cytometric methodologies  are carried out as previously described , and then total RNA was purified with the phenol/chloroform extraction method. Concentration and purity of RNA samples were measured using NanoDrop. The RNA purity was assessed as OD260/OD230 (≥1.5) using NanoDrop. gDNA was eliminated by DNase. The RNA from three biological samples was then pooled for mRNA analysis, cDNA was synthesized using the iScript kit  according to the manufacturer’s recommendation, and RT-PCRs consisted of 8.34 µL Fluocycle® II SYBR® , 0.5 µL of both 10 µM forward and reverse primers, 1 µL cDNA, and 6.34 µL of H2O. All reactions were performed for 35 cycles using the following temperature profile: 95°C for 5 min , 95°C for 15 min (denaturation), 55°C for 30 min , and 72°C for 30 min (extension) (The cells were grown in 10 cmtension) . Then, tCell lysates were prepared by lysing cells in RIPA buffer (10 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% Na deoxycholate), supplemented with protease inhibitor cocktail (Sigma). Protein concentration of whole cell lysates was determined by the BSA assay using the BSA kit (Pierce), and then equal protein amounts were heated to 95 °C for 5 min with sodium dodecyl sulfate (SDS) sample buffer  and run on 15% SDS polyacrylamide gel electrophoresis (SDS-PAGE). Protein samples were then blotted onto PVDF membranes . The membranes were incubated in blocking solution (5% non-fat milk in PBS) for 1 h and then in primary antibody  or GAPDH mAb ) overnight. After 3 × 10 min washes in TBS (0.1% Tween-20 in PBS), the membrane was incubated for 1 h at room temperature with horseradish-peroxidase–linked (HRP) secondary anti-rabbit or anti-mouse Ab . Signals on the membrane were developed using ECL reagent  and then were imaged with the Chemidoc system .https://www.rcsb.org/structure/2OBJ) was also optimized following the routine work as discussed by Poli et al.  exhibited pronounced cytotoxic activities with high IC50 values 0.6 and 6 nM against the Hela and KF-28 cell lines, respectively. Taken together, the tested compounds exhibited remarkable cytotoxic activities against the tested cancer cell lines with apoptosis induction through inhibition of Pim-1 kinase. Therefore, compounds 5c and 5f are worthy to be further tested in vivo for developing chemotherapeutic anti-cancer agents.In this work, we successfully prepared 1,4-cyclohexanedione-


These results indicated that t9,c12,c15-C18:3 was an intermediate of the α-linolenic acid shifted rumen biohydrogenation pathway. This study hypothesized a pathway for α-linolenic acid biohydrogenation in rumen.Few studies have investigated a relationship between t9,c12,c15-C18:3 as an isomer of α-linolenic acid , has been recently detected in milk, but has not been found in the rumen. This study hypothesized that it may be a biohydrogenation product of ALA in rumen and aimed to explore whether it was present in the rumen and help to understand the rumen biohydrogenation mechanisms of ALA. The in vitro experiment included two treatments, a control check (CK group) with 50 µL ethanol added, and ALA group with 50 µL ethanol and 2.6 mg ALA ; each sample of fermentation fluid had the composition of C18 fatty acids analyzed at 0, 0.5, 1, 2, 3, 4, 5, and 6 h. The results showed that no t9,c12,c15-C18:3 was detected in the CK group, but ALA addition increased the concentration of t9,c12,c15-C18:3 in fermentation fluid. The content of t9,c12,c15-C18:3 peaked 1 h after fermentation, then declined gradually. At 1 h, no t9c12c15-C18:3 was detected in the fermentation fluid of the CK treatment. The results suggested that ALA converted to the isomer t9,c12,c15-C18:3 through biohydrogenation in the rumen. The addition of ALA can also increase the concentration of t9,c12-C18:2, c9,t11-C18:2, c12-C18:1, t11-C18:1, t9-C18:1, and c6-C18:1 in fermentation fluid. It was concluded using an in vitro experiment that t9,c12,c15-C18:3 was a product of rumen biohydrogenation of ALA.The  The in situ isomerization of ALA has been reported in the processing of food. A previous study reported that ALA could isomerize to t9,c12,c15-C18:3 through frying [t9,c12,c15-C18:3 was present in milk [t9,c12,c15-C18:3 through biohydrogenation in rumen. The purpose of this experiment was to explore whether there was t9,c12,c15-C18:3 in rumen fluid using an in vitro fermentation test and to investigate the relationship between t9,c12,c15-C18:3 and ALA. The t9,c12,c15-C18:3 identified in this study may provide a theoretical basis for the exploration of rumen biohydrogenation.The supplementation of α-linolenic acid  and diluted with ethanol. Rumen fluid was collected from three ruminal cannulated Holstein cows fed a diet that met the feeding standards of dairy cattle in China according to the Ministry of Agriculture of China Feeding Standard of Dairy Cattle . Rumen contents were withdrawn before the first meal in the morning, pooled in equal proportions into a container with CO2, then filled with 200 mg diet (Eighty fermentation bottles were separated into two groups as a control (CK group), with 50 µL ethanol added and ALA group with 50 µL ethanol and 2.6 mg ALA . The bottles were prewarmed to 39 °C and flushed by CO mg diet  and 30 mv/v = 3/2) solution was used to extract the fat. The methyl esterification of the fat was then performed using 10% acetyl chlorocarbinol followed by 2% methanolic NaOH. The FAMES were analyzed using an Agilent 7890A gas chromatograph  fitted to a 5975C MS mass spectrometer detector . A sample of 2 μL FAME mixed with hexane was injected through the split injection port in a 10:1 ratio into a capillary column CP-Sil 88 fused silica 100 m × 0.25 mm × 0.20 μm . The injector temperature was set at 250 °C, and oven temperature was initially 120 °C. After holding for 2 min, the temperature was increased by 3 °C per min to 180 °C and then increased by 1.5 °C per min to 200 °C. This temperature was maintained for three min, then increased by 2 °C per min to 220 °C and held for 20 min. The transfer line and MS ion source temperatures were maintained at 250 and 280 °C, respectively, and the ionizing energy was 70 eV.The extraction of fatty acids and fatty acid methyl esters (FAMEs) from ruminal fluid was according to a previous protocol . A 2 mL t9,t12-C18:2, c9,t12-C18:2, t9,c12-C18:2 and c9,c12-C18:2), α-linolenic acid methyl ester mix CRM 47792 including , CLA methyl ester mix Nu-Check prep including c9,t11-C18:2, t10,c12-C18:2 , c6,c9,c12-C18:3, t6-C18:1, t9-C18:1, t11-C18:1, c6-C18:1, c9-C18:1, c11-C18:1, and C18:0 FAME standards individually from Nu-Check Prep , c8-C18:1 and c12-C18:1 FAME individual standards from Cayman , c9-C18:1, c9,c12-C18:2 and c9,c12,c15-C18:3 triacylglycerols (TAGs) individual standards from Nu-Check Prep , and c6,c9,c12-C18:3 TAGs from BePure . The method of Chen et al. [The FAME peaks were identified using known FAME standards, including linoleic acid methyl ester mix CRM 47791 . The following statistical model was used:t9,t12,c15-C18:3, t9,c2,t15-C18:3, c6,c9,c12-C18:3, c9,c12,t15-C18:3 and c9,t12,c15-C18:3 were undetected in this experiment, and t9,t12,t15-C18:3, c9t12t15-C18:3, and ALA were detected in the CK and ALA groups. The t9,c12,c15-C18:3 was found in the ALA group after fermentation. Previous studies reported that t9,c12,c15-C18:3 has been found in the processing of food [t9,c12,c15-C18:3 in milk [The composition of C18 fatty acids detected in fermentation in vitro of the two groups is shown in p < 0.01). With the extension of fermentation time, the content of ALA was decreased gradually, and the decline was the fastest in the first 0.5 h of fermentation. The concentration of t9,c12,c15-C18:3 decreased gradually with the fermentation time and the content of t9,c12,c15-C18:3 reached a peak 1 h after fermentation, then declined gradually. The addition of ALA increased the concentration of t9,c12-C18:2, c9,t11-C18:2, c12-C18:1, t11-C18:1, t9-C18:1, and c6-C18:1 in the fermentation fluid (p < 0.01). The change of t9c12-C18:2 showed the same trend, peaking 1 h after fermentation, then declining gradually. In this study, the change in c12-C18:1, t11-C18:1 showed a similar trend, the highest at 3 h after fermentation, then declined gradually and the concentration of c6-C18:1 increased at 3 h after fermentation. Through the biohydrogenation of ALA, the content of ALA decreased and converted to other C18 fatty acids [c9,t11-C18:2 was one of the ALA biohydrogenation products present in the rumen. In the in vivo experiment, enhancing the content of ALA in the diet of cows also increased c9,t11-C18:2 in the rumen [t10,c12-C18:2 increased with ALA increase in rumen [t9,c12-C18:2. A study reported that ALA supplementation could increase the content of t9,c12-C18:2 in milk fat [t9,c12-C18:2 might originate from the rumen. A previous study reported that c12-C18:1, t11-C18:1, and t9-C18:1 were products of ALA [t11-C18:1 was the main product of ALA biohydrogenation in the rumen [c12-C18:1, t11-C18:1, and t9-C18:1 in the fermentation fluid when ALA was added to the fluid, where t11-C18:1 increased from 1.804 ng/mL to 3.118 ng/mL at 3 h, c12-C18:1 from 0.322 ng/mL to 0.609 ng/mL at 3 h, and t9-C18:1 increased from 0.256 ng/mL to 0.404 ng/mL at 5 h.In this experiment, ALA influenced the content of C18 fatty acids in fermentation, as showed in he rumen , the sams of ALA , and t11he rumen . This stt9,c12,c15-C18:3 increased in the rumen fermentation fluid, so it was speculated that ALA could convert to t9,c12,c15-C18:3. The concentration of t9,c12,c15-C18:3 was less than 0.048 ng/mL, so the conversion of ALA to t9,c12,c15-C18:3 might not be a major biohydrogenation pathway. A previous study reported that t10,c12,c15-C18:3 could convert to t10,c15-C18:2 in the rumen [cis double bond. This study showed that ALA supplementation can increase the content of t9,c12,c15-C18:3 and t9,c12-C18:2 in fermentation fluid, so it is likely that t9,c12,c15-C18:3 could convert to t9,c12-C18:2, but t9,c12,c15-C18:3 was not the main source of t9,c12-C18:2, due to the low concentration of t9,c12,c15-C18:3 in fermentation fluid. To the best of our knowledge, no article has reported the next transformation pathway of t9,c12-C18:2. However, t11,c15-C18:2 was one of the isomers of t9,c12-C18:2 reported that t11,c15-C18:2 could covert to c15-C18:1, t11-C18:1, and t16-C18:1 via t11,c15-C18:2 to c15-C18:1, t11,c15-C18:2 to c15-C18:1 and t11,c15-C18:2to t11-C18:1) [c12-C18:1, t11-C18:1, and t9-C18:1 might also be products of t9c12-C18:2 biohydrogenation, and a higher content of c12-C18:1, t11-C18:1, and t9-C18:1 were also observed in ALA group fermentation fluid. The c12-C18:1, t11-C18:1, and t9-C18:1 were further hydrogenated to C18:0 [t9,c12,c15-C18:3 was the biohydrogenation product of ALA in the rumen and the biohydrogenation pathways may exist in the rumen as t9,c12,c15-C18:3 to t9,c12-C18:2 to c12-C18:1/t11-C18:1/t9-C18:1 to C18:0 as shown by the red line in t9,t11,c15-C18:3 contains two trans configurations, and was previously reported as a biohydrogenation product of ALA, and t9,t11,c15-C18:3 was converted from c9,t11,c15-C18:3 [t9,c12,c15-C18:3 might also isomerize to t9,t11,c15-C18:3 in the rumen, but due to the lesser standard of t9,t11,c15 C18:3, this study did not detect it. Further analysis could explore whether t9,c12,c15-C18:3 could convert to t9,t11,c15-C18:3.As shown in he rumen  and the 15-C18:3 . The t9,t9,c12,c15-C18:3 was the product of ALA biohydrogenation in the rumen, which may provide a new biohydrogenation pathway in the rumen. The cis-trans isomerization of ALA from c9,c12,c15 C18:3 isomerizes to t9,c12,c15-C18:3 could happen in situ and the ortho-position c9,c12,c15-C18:3 isomerized to t10,c12,c15-C18:3. The enzymes and microorganisms involved in isomerization may be different, and any further study should focus on the manipulation of enzymes and microorganisms involved in the biohydrogenation pathway.Overall, this study found that