Aurora-A kinase is associated with the Aurora kinase family which has been considered a striking anticancer target for the treatment of human cancers.H-pyrazol-4-yl)-3-phenylthiazolidin-4-one derivatives as Aurora-A Kinase inhibitors.To design, synthesize, anticancer evaluation, and docking studies of novel 2-(1-isonicotinoyl-3-phenyl-1P (1–21) were synthesized by using the Vilsmeier Haack reagent which was characterized by FT-IR, 1H NMR, 13C NMR, and Mass spectroscopy. The synthesized derivatives were evaluated for their potential in vitro anticancer activity by MTT assay and Aurora-A kinase inhibition assay.A total of 21 Pyrazole derivatives P-6 exhibited potent cytotoxicity (IC50 = 0.37–0.44 μM) against two cancer (HCT 116 and MCF-7) cell lines, which were comparable to the standard compound, VX-680. Compound P-6 also showed inhibition of Aurora-A kinase with an IC50 value of 0.11 ± 0.03 µM. A Docking study was done to compound P-6 and P-20 into the active site of Aurora A kinase, in order to get the probable binding model for further study.The cytotoxicity assay (MTT assay) showed that compound P(1–21) were designed, synthesized, in vitro anticancer evaluation, and docking studies for Aurora A kinase inhibition. The results established that P-6 is a prospective aspirant for the development of anticancer agents targeting Aurora-A kinase.A series of 21 novel pyrazole derivatives  Cancer is a stage of disease in which a group of cells has an irritating and uncontrollable growth , 2. StraVarious therapeutic agents have been successfully tested and used to eradicate different types of cancer, but side effects have always been shown to be major problems. Furthermore, patient compliance is also compromised . UndoubtTargeted therapies work by influencing the processes that control the growth, division, and spread of cancer cells, and the symptoms that cause natural death in cancer cells, such as monitoring normal cells when they are damaged or aged.The development of new molecules that bind to targets of biological interest is a breakthrough in cancer research.The knowledge of cell cycle mechanism and pathways of cancer pathogenesis has led to novel strategies and targets like Aurora Kinases, Topoisomerases, Check Point Kinases (CPK’s), Cyclin-Dependent Kinases (CDK’s), Oncogenic Human Papillomavirus (HPV), ABC transporters, etc. –11.Aurora kinase is an essential regulator of cell cycle progression in cancer cells which suggests it is an attractive target for the development of anticancer drugs. Aurora kinases are a highly conserved family of serine/threonine protein kinases that play a key role in regulating many fundamental mitotic processes –16.In mammals, three Aurora kinases are found: Aurora-A, Aurora-B, and Aurora-C. Auroras A and B are best known for their distinct roles in controlling mitosis, but the role of auroras C is still unclear. Aurora-A is concerned with the maturation and separation of centrosomes, the integration of bipolar spindles, and mitotic penetration, while Aurora B is essential for accurate chromosomal isolation and cytokinesis –21.Therefore, besieged inhibition of Aurora-A kinase has developed into a striking therapeutic approach in the treatment of cancer. Aurora-A was first described in human neoplastic cell lines but was later discovered to be more uttered in an extremely wide range of human tumors, including primary colorectal carcinoma, breast, ovary, etc. .The scaffolds of the renowned Aurora-A kinase inhibitors could also be categorized into four major groups labeled A-D, as given in Fig. Encouraged by the various biological properties of thiazolidine-4-one and pyrazoles, we have attempted to synthesize the designated compounds using a hybridization approach in the expectation that the resulting novel compounds could react strongly with the Aurora-A kinase domain residues through hydrophobic interactions , 23–27.Most of the anticancer drugs used in the clinic come from heterocyclic. Pyrazole and thiazolidin-4-one nucleus have a scaffold in synthetic chemistry that has been successfully fueling researchers' interest in compounding various derivatives using a hybridization method for multiple chemotherapeutic activities involving cytotoxicity to cause apoptotic cell death in different cancer cell lines as shown in Fig. H-pyrazol-4-)yl)-3-phenylthiazolidin-4-one derivatives as a new class of Aurora-A kinase inhibitors are thought to have a synergistic effect on anticancer activity by inhibiting anti-Aurora A kinase inhibitors. Given the suggestions, we have directed our research to evaluate their anticancer activity in cell lines HCT-116 and MCF-7 to further investigate the initial mechanism of their role in the summary and further expand our research on antitumor compounds with inhibitory activity of Aurora-A kinase, which is considered useful to form a series of 2-(1-isonicotinoyl-3-phenyl-1H-pyrazol-4-yl)-3-phenylthiazolidin-4-one derivatives as a new class of Aurora- Kinases inhibitors as it may show a synergistic effect on anticancer activity by inhibition against Aurora-A kinase.In summary, as a continuation of our study of antitumor compounds with the inhibitory activity of Aurora-A kinase, it is believed to be help form a series of 2- by conventional (refluxing and stirring) as well as by green approach (microwave irradiation) [P (1–21) were synthesized by condensation of Isoniazid with different acetophenone followed by Vilsmeier Haack reaction to form pyrazole-4-carbaldehyde 2(A-C) which was then treated with different anilines to get Schiff bases S (1–21). Schiff bases were further treated with thioglycolic acid in the presence of DMF and Zinc Chloride to form final compounds  , 29. TheP –21 by coP (1–21) displayed absorption bands for C=N, C–H str (aromatic), CH-S str., C=C aromatic and C=O stretching at 1640–1650 cm−1, 3410–3120 cm−1, 2970–2920 cm−1, 1612–1563 cm−1, 2361–2349 cm−1 and 1720–1640 cm−1 respectively. IR spectra of compounds showed a strong and sharp band between 1660–1674 cm−1 and 1725–1714 cm−1 displayed carbonyl stretching of ═N–CO group present at pyrazole nucleus and carbonyl stretching of thiazolidinone nucleus, respectively. The presence of band at 1500 cm−1 and 1325 cm−1 displayed NO2 asymmetric and symmetric stretching respectively supported the presence of the nitro group in the structure of Comp. P-1, P-2, P-3, P-4, P-5, P-6, P-7, P-9, P-10, P-16 & P-17. The IR spectra of Comp. P-15, P-16, P-17, P-18, P-19, P-20 & P-21 showed stretching around 630 cm−1 depicting the presence of Bromo substituted phenyl ring in their structure. The presence of the band at 1222–1160 cm−1 displayed asymmetric C–O–C stretching vibrations whereas the band at 1099–1090 cm−1 displayed symmetric C–O–C stretching supported the presence of methoxy group in the structure of Comp. P-6, P-7, P-8, P-9, P-10, P-11, P-12, P-13, P-14, P-20 & P-21. The IR spectra of Comp. P-4, P-5, P-11, P-12, P-18, and P-19 showed a characteristic strong band at 720–664 cm−1 which was assigned to C–Cl stretching vibrations.The structures of compounds were elucidated by spectral data. The Infrared (IR) spectra of final compounds 1H-NMR spectra  of the compounds had a doublet around 8.06–7.96 ppm (J = 6–8 Hz) and 4.6–3.43 ppm (J = 12–16 Hz) for isonicotine hydrazide and thiazolidinone protons respectively. The chemical shift for the proton = CH– of the pyrazole ring and –CH– of the thiazolidinone ring was observed as a singlet between 8.38 and 8.55 ppm and 5.82–5.89 ppm respectively [13C-NMR spectra  of the compounds displayed peaks for pyridine, pyrazole, and thiazolidin-4-one at 149.8–122.8, 151.8–71.1, 47.8–34 respectively [The ectively , 29. Theectively .P-6 and P-20 exhibited the highest cytotoxicity activity (IC50 = 0.39–0.56 µM). Particularly, it was noticed that compound P-6 is slightly more active than compound P-20 having IC50 = 0.37 ± 0.15 µM for HCT116 and IC50 = 0.44 ± 0.06 µM for MCF-7, comparable to the positive control VX-680 having IC50 = 0.32 ± 0.05 µM for HCT116 and IC50 = 0.40 ± 0.03 µM for MCF-7 cells respectively.MTT assay was engaged to evaluate the anticancer activity of synthesized compounds P1–21) against various human cancer cell lines  –34. As i1 againstResults illustrated in Table para-substituted A ring with electron- withdrawing substituents are of better antitumor activity compared to those with electron-donating substituents, the potency is ordered as NO2 > Br > Cl > H > Me > MeO whereas substitution of B ring at para position with the electron-donating group have improved antiproliferative activity, whereas electron-withdrawing groups  had minor effects. Among all the compounds, P-6 with the p-NO2 group on the A-ring and p-OCH3 on the B-ring respectively led to a noteworthy best activity.By investigating the variation in the selectivity of the tested compounds over two cell lines, it was revealed that different substituents on the B-ring led to different antitumor activities. Among the compounds, the compounds with P (1–21) against the Aurora-A kinase via an enzyme-coupled continuous spectrophotometric assay, and the results were summarized in Table 3, compounds P-6 and P-20 showed the most compelling inhibitory activity with IC50 of 0.18 ± 0.05 µM and 0.22 ± 0.08 µM (positive control VX-680 with an IC50 of 0.11 ± 0.03 µM for Aurora-A kinase) respectively [To examine whether the compounds inhibit Aurora-A kinase, we screened compounds P 1–1 againstectively , 36.The effects of Aurora-A kinase inhibitory activity of the tested compounds are consistent with the structure–activity relationships (SAR) of their antitumor activity. This has shown that the potent antitumor activity of synthetic compounds may be associated with their Aurora-A kinase inhibitory activity.P (1–21) are incorporated into the active site of Aurora kinase A. Both have a positive binding to the binding site as determined by the free binding force (ΔG) and the preferred docking binding mode  , 37 usinde Table . The comP-6 and P-20, VX-680 (positive control) with the receptor in terms of docking score (binding energy) was depicted in Table The interaction of ligands P-6 is bonded to the ATP binding site of Aurora A kinase using hydrophobic bonding and binding is stabilized by hydrogen bond. The oxygen atom of the OCH3 group formed a single hydrogen bond with the amino hydrogen of Asp-404 . An enzyme surface model was shown in Fig. P-6 may be a potential inhibitor of Aurora-A kinase.The binding bond of compound P-6 and Aurora-A kinase are shown in Fig. H-pyrazol-4-yl)-3-phenylthiazolidin-4-one are synthesized and evaluated for their in vitro antitumor activity by MTT assay against HTC-116, MCF-7 cells lines and aurora kinase inhibitory activity. Amongst all compounds, P-6 showed the most potent inhibitory activity which inhibited the growth of HTC-116 and MCF-7 cell lines  and inhibited the Aurora-A kinase with IC50 of 0.18 ± 0.05 µM, which was comparable to the positive control VX-680 (Tozasertib). Molecular docking was further performed to study the inhibitory Aurora-A kinase interactions. The data of this work might be helpful for the design and synthesis of a leading compound P-6 toward the development of a new therapeutic agent to fight against cancer. The vast amount of current research on pyrazole has proven to be an effective aid in the development of new anticancer agents.In conclusion, a series of 2-(1-isonicotinoyl-3-phenyl-113C NMR) are recorded on the Bruker Avance II 500 MHz NMR spectrometer. Mass spectra were performed on the JEOL GC mass spectrometer. The results in the spectrum head show that the molecular mass of the compounds produced was closer to the molecular mass of the expected compounds [All chemicals were procured from SD Fine Chem Co. Ltd and used in experiments. The melting point has been identified in the Bouchi oil melting point apparatus and has not been corrected. The microwave-assisted reactions were carried out in an HPL 2300 ET domestic microwave oven: power: 300 W; frequency: 2.45 GHz; temperature range: 60–250 °C . The purThe comparative procedure  of pyrazole derivatives by the synthetic route as shown in Scheme N'-(1-phenylethylidene) isonicotinohydrazide derivatives 1(A-C).Step 1: Synthesis of 2, 4-OCH3, 4-BrWhereas R = 4-NO(Z)-N′-(1-(4-Nitrophenyl) ethylidene) isonicotinohydrazide (1A) Yellow solid (71.6%); m.pt. 226–228 °C; IR : 3280 , 2900 (C-H str.), 1625 (C=C str.), 1590 (C=N str.), 1135 (C–N str.), 1657 ; 1H NMR  δ (ppm) 9.06 , 7.96 , 7.36 , 8.24, 7.00 .(Z)-N'-(1-(4-Methoxyphenyl) ethylidene) isonicotinohydrazide (1B) Yellow solid (72%); m.pt. 193–195 °C; IR : 3396 , 2922 (C–H str.), 1625 (C=C str.), 1585 (C=N str.), 1150 (C–N str.), 1670 ; 1H NMR  δ (ppm) 9.06 , 7.96 , 7.00 , 7.5 , 6.8 , 3.73 .(Z)-N'-(1-(4-Bromophenyl) ethylidene) isonicotinohydrazide (1C) Brown solid (89%); m.pt. 216–218 °C; IR : 3380 , 2918 (C-H str.), 1620 (C=C str.), 1590 (C=N str.), 1150 (C–N str.), 1690 ; 1H NMR  δ (ppm) 9.06 , 7.96 , 7.00 , 7.45 , 6.8 .Step 2: Synthesis of 1-isonicotinoyl-3-phenyl-4,5-dihydro-1 H-pyrazole-4-carbaldehyde derivatives 2 (A–C)3-(4-Nitrophenyl)-1-isonicotinoyl-1H-pyrazole-4-carbaldehyde (2A) Yellow solid (70%); m.pt. 230–232 °C; IR : 2900 (C–H str.), 1625 (C=C str.), 1590 (C=N str.), 1135 (C–N str.), 1657 , 1558 (NO2 assym. str.), 1298 (NO2 symm. str.); 1H NMR  δ (ppm) 9.06 , 7.96 , δ 7.5 , 7.36 , 8.24, 9.61 .3-(4-Methoxyphenyl)-1-isonicotinoyl-1H-pyrazole-4-carbaldehyde (2B) Yellow solid (72%); mp 198–200 °C; IR : 2980 (C–H str.), 1638 (C=C str.), 1586 (C=N str.), 1136 (C–N str.), 1670 , 1236 (C–O–C asym. str.), 1095 (C–O–C sym. str.); 1H NMR  δ (ppm) 9.06 , 7.96 , δ 3.29 , 7.5 , 7.37 , 6.83 , 9.61 .3-(4-Bromophenyl)-1-isonicotinoyl-1H-pyrazole-4-carbaldehyde (2C) Brown solid (85%); mp 222–224 °C; IR :2996 (C-H str.), 1648 (C=C str.), 1580 (C=N str.), 1136 (C–N str.), 1672 , 570 (C–Br str.); 1H NMR  δ (ppm) 9.06 , 7.96 , 7.37 , 7.49 , 9.61 .Step 3: synthesis of (3-phenyl-4-((phenylimino) methyl)-4,5-dihydropyrazol-1-yl pyridin-4-yl)methanone derivatives/Schiff bases S (1–21).1 = H, 4-NO2, 2-NO2, 4-Cl, 2-Cl, 4-OCH3, 2-OCH3, 4-Br.RStep 4: Synthesis of 2--3-phenyl thiazolidin-4-one derivatives P (1–21).2-(1-isonicotinoyl-3-(4-nitrophenyl)-1H-pyrazol-4-yl)-3-phenylthiazolidin-4-one (P-1) Yellow solid (89.2%); m.pt- 244–246 °C; IIR : 3200 (C-H str. Ar), 2450 (CH-S str.), 1680 (C = O str.pyrazole), 1730 , 1361 (C = N str.), 1224 (Ar-N str.), 1157 (R–C-O str.), 1530 (NO2 asymm.str.), 1380 (NO2 symm.str.), 1200 (Ar-N str.), 1350 (C–C str.), 690–710 .2-(1-isonicotinoyl-3-(4-nitrophenyl)-1H-pyrazol-4-yl)-3-(4-nitrophenyl)thiazolidin-4-one (P-2) Yellow solid (87.4%); m.pt.- 266–268 °C; IR : 3220 (C-H str. Ar), 2990 (CH-S str.), 1720 ,1733 , 1310 (C=N str.), 1230 (Ar-N str.), 1157 (R–C-O str.), 1510 (NO2 asymm. str.), 1360 (NO2 symm. str.), 1270 (Ar-N str.), 1351 (C–C str.), 690–710 .2-(1-isonicotinoyl-3-(4-nitrophenyl)-1H-pyrazol-4-yl)-3-(2-nitrophenyl)thiazolidin-4-one (P-3) Yellow solid (83.1%); m.pt. 282–284 °C; IR : 3220 (C–H str. Ar), 2990 (CH-S str.), 1720 ,1733 , 1310 (C=N str.), 1230 (Ar-N str.), 1157 (R–C–O str.), 1510 (NO2 asymm. str.), 1360 (NO2 symm. str.), 1270 (Ar-N str.), 1351 (C–C str.), 690–710 ; 1H NMR  δ 9.06 , 7.96 , 3.43 , 5.92 , 7.3 , 7.36 , 7.70 , 7.50 , 8.24 .3-(4-chlorophenyl)-2-(1-isonicotinoyl-3-(4-nitrophenyl)-1H-pyrazol-4-yl)thiazolidin-4-one (P-4) Creamy yellow (80%); m.pt.: 286–288 °C; IR : 3201 (C-H str. Ar), 2928 (CH-S str.), 1680 , 1736 , 1361 (C=N str.), 1224 (Ar–N str.), 1157 (R–C–O str.), 1550 (NO2 asymm. str.), 1386 (NO2 symm. str.), 1298 (Ar–N str.), 1350 (C–C str.), 692 (C–Cl str.); 1H NMR  δ 9.06 , 7.96 , 3.43 , 5.92 , 7.3 , 4.6 , 7.5 , 6.8 , 7.38 , 8.22 -2-(1-isonicotinoyl-3-(4-nitrophenyl)-1H-pyrazol-4-yl)thiazolidin-4-one (P-5) Creamy yellow (75.8%); m.pt. 274–276 °C; IR : 3201 (C–H str. Ar), 2928 (CH–S str.), 1680 , 1736 , 1361 (C=N str.), 1224 (Ar–N str.), 1157 (R–C–O str.), 1550 (NO2 asymm. str.), 1386 (NO2 symm. str.), 1298 (Ar- N str.), 1350 (C–C str.), 692 (C–Cl str.).2-(1-isonicotinoyl-3-(4-nitrophenyl)-1H-pyrazol-4-yl)-3-(4-methoxyphenyl)thiazolidin-4-one (P-6) Yellowish brown solid (72.6%); m.pt: 262–264 °C C; IR : 3198 (C–H str. Ar), 2928 (CH–S str.), 1691 ,1726 , 1361 (C=N str.), 1224 (Ar-N str.), 1157 (R–C–O str.), 1514 (NO2 asymm.str.), 1384 (NO2 symm.str.), 1296 (Ar-N str.), 1261 (C–O–C asymm. str.), 1076 (C–O–C symm. str.); 1H NMR  δ 9.06 , 7.96 , 3.43 , 5.92 , 7.3 , 7.9 , 8.2 , 6.64 , 6.70 , 7.22 , 6.60 ; 13C-NMR : 149.8 (4-pyridine C3 & C5), 122.8 (4-pyridine C2 & C6), 167.0 (C=O), 151.8 (pyrazole C3), 46.1 (pyrazole C4), 71.1 (pyrazole C5), 140.0 (4-nitrophenyl C1), 130.0 (4-nitrophenyl C2 & C6), 121.0 (4-nitrophenyl C3 & C5), 60.7 (4-nitrophenyl C4), 47.8 (thiazolidin-4-one C2), 34 (thiazolidin-4-one C5), 170.9 (thiazolidin-4-one C4 (C=O)), 134 (4-methoxyphenyl C1), 122.6 (4-methoxyphenyl C2 & C6), 114 (4-methoxyphenyl C3 & C5), 156.0 (4-methoxyphenyl C4), 55.9 (O-CH3); m/z 503 (M+); Anal.: Calcd. for C25H21N5O5S: C, 59.638; H, 4.28; N, 13.918; S, 6.378; O, 15.898. Found: C, 59.276; H, 4.24; N, 13.886; S, 6.338; O, 15.890.2-(1-isonicotinoyl-3-(4-nitrophenyl)-1H-pyrazol-4-yl)-3-(2-methoxyphenyl)thiazolidin-4-one (P-7) Yellowish brown solid (93.4%); m.pt.: 250–252 °C; IR : 3198 (C–H str. Ar), 2928 (CH–S str.), 1691 ,1726 , 1361 (C=N str.), 1224 (Ar-N str.), 1157 (R–C-O str.), 1514 (NO2 asymm.str.), 1384 (NO2 symm.str.), 1296 (Ar-N str.), 1261 (C–O–C asymm. str.), 1076 (C–O–C symm. str.)2-(1-isonicotinoyl-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)-3-phenylthiazolidin-4-one (P-8) Yellow solid (82.5%); m.pt. 300–302 °C; IR : 3252 (C–H str. Ar), 2932 (CH–S str.), 1688 ,1740 , 1548 (C=N str.), 1226 (Ar–N str.), 1160 (R–C=O str.), 1338 (C=N str.), 1330 (C–O–C asymm. str.), 1128 (C–O–C symm. str.).2-(1-isonicotinoyl-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)-3-(4-nitrophenyl)thiazolidin-4-one (P-9) Yellow solid (76.3%); m.pt. 308–310 °C; IIR : 3152 (C–H str. Ar), 2928 (CH-S str.), 1681 ,1733 , 1649 (C=N str.), 1226 (Ar-N str.), 1157 (R–C=O str.), 1338 (C=N str.), 1514 (NO2 asymm.str.), 1384 (NO2 symm.str.), 1298 (C–O–C asymm. str.), 1108 (C–O–C symm. str.).2-(1-isonicotinoyl-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)-3-(2-nitrophenyl)thiazolidin-4-one (P-10) Yellow white solid (64.7%); m.pt. 322–324 °C; IR : 3137 (C–H str. Ar), 2962 (CH–S str.), 1670 , 1735 , 1617 (C=N str.), 1284 (Ar-N str.), 1175 (R–C–O str.), 1319, 1411 (C=N str.), 1520 (NO2 asymm. str.), 1350 (NO2 symm. str.), 1310 (C–O–C asymm. str.), 1198 (C–O–C symm. str.); 1H NMR  δ 9.06 , 7.96 , 33.43 , 5.92 , 7.3 , 7.9 , 8.2 , 8.24 , 7.50 , 7.70 , 7.36 .3-(4-chlorophenyl)-2-(1-isonicotinoyl-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)thiazolidin-4-one (P-11) Yellowish white (68.8%); m.pt. 318–320 °C; IR : 3137 (C–H str. Ar), 2959 (CH–S str.), 1681 , 1740 , 1588 (C=N str.), 1221 (Ar-N str.), 1157 (R–C–O str.), 1317, 1415 (C=N str.), 696 (C–Cl str.), 1330 (C–O–C asymm. str.), 1180 (C–O–C symm. str.); 1H NMR  δ 9.06 , 7.96 , 3.43 , 5.92 , 7.3 , 6.64 , 6.70 , 7.22 , 6.60 ,7.04 , 7.32 .3-(3-chlorophenyl)-2-(1-isonicotinoyl-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)thiazolidin-4-one (P-12) Yellowish white solid (72.4%); m.pt. 338–340 °C; IR : 3189 , 2919 (CH–S str.), 1669 , 1733 , 1556 (C=N str.), 1222 (Ar-N str.), 1140 (R–C–O str.), 678 (C–Cl str.), 1310 (C–O–C asymm. str.), 1198 (C–O–C symm. str.); 1H NMR  δ 9.06 , 7.96 , 3.43 , 5.92 , 7.3 , 6.61 , 6.75 , 7.20 , 6.66 , 3.73 , 7.11 , 6.98 ; 13C-NMR : 149.8 (4-pyridine C3 & C5), 122.8 (4-pyridine C2 & C6), 167.0 (C=O), 151.8 (pyrazole C3), 46.1 (pyrazole C4), 71.1 (pyrazole C6), 126.0 (4-methoxyphenyl C1), 130.0 (4-methoxyphenyl C2 & C6), 114.0 (4-methoxyphenyl C3 & C5), 163 (4-methoxyphenyl C4), 55.9 (O-CH3), 47.8 (thiazolidin-4-one C2), 34 (thiazolidin-4-one C5), 170.9 (thiazolidin-4-one C4 (C=O)), 143 (3-chlorophenyl C1), 122.6 (3-chlorophenyl C2 & C6), 134.5 (3-chlorophenyl C3), 124.0 (3-chlorophenyl C4), 130.4 (3-chlorophenyl C5); LC–MS: m/z 490 (M+); Anal.: Calcd. for C25H19ClN4O3S Anal. Calcd.: C, 61.168; H, 3.98; N, 11.418; S, 6.538; Cl, 7.228; O, 9.788. Found: C, 61.068; H, 3.68; N, 11.218; S, 6.508; Cl, 7.218; O, 9.748.2-(1-isonicotinoyl-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)-3-(4-methoxyphenyl) thiazolidin-4-one (P-13) Yellow solid (76.6%); m.pt. 320–322 °C; IR : 3354 (C–H str. Ar), 2964 (CH–S str.), 1690 ,1722 , 1593 (C=N str.), 1259 (Ar-N str.), 1174 (R–C–O str.), 1338, 1417 (C=N str.);1292 (C–O–C asymm. str.), 1126 (C–O–C symm. str.); 1H NMR  δ 9.06 , 7.96 , 3.43 , 5.92 , 7.3 , 6.66 , 6.70 , 7.24 , 6.69 , 7.20 ; 13C-NMR : 149.8 (4-pyridine C3 & C5), 122.8 ( 4-pyridine C2 & C6), 167.0 (C=O), 151.8 (pyrazole C3), 46.1 (pyrazole C4), 71.1 (pyrazole C6), 126.0 (4-methoxyphenyl C1), 130.0 (4-methoxyphenyl C2 & C6), 114.0 (4-methoxyphenyl C3 & C5), 163.0 (4-methoxyphenyl C4), 55.9 (O–CH3), 47.8 (thiazolidin-4-one C2), 34 (thiazolidin-4-one C5), 170.9 (thiazolidin-4-one C4 (C=O)), 134 (4-methoxyphenyl C1), 122.6 (4-methoxyphenyl C2 & C6), 114 (4-methoxyphenyl C3 & C5), 156.0 (4-methoxyphenyl C4), 55.9 (O-CH3).; LC–MS: m/z 486 (M+); Anal.: Calcd. for C26H22N4O4S C, 64.188; H, 4.568; N, 11.528; S, 6.598; O, 13.158; Found: C, 63.752; H, 4.576; N, 11.273; S, 6.320; O, 13.614..2-(1-isonicotinoyl-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-4-yl)-3-(2-methoxyphenyl)thiazolidin-4-one (P-14) Yellow solid (84.3%); m.pt. 336–338 °C; IR : 3217 (C–H str. Ar), 2968 (CH–S str.), 1681 , 1735 , 1645 (C=N str.), 1257 (Ar–N str.), 1174 (R–C-O str.), 1338, 1415 (C=N str.), 1310 (C–O–C asymm. str.), 1120 (C–O–C symm. str.); 1H NMR  δ 9.06 , 7.96 , 33.43 , 5.92 , 7.3 , 6.82 , 7.13 , 7.87 , 6.99 , 3.73 .2-(3-(4-bromophenyl)-1-isonicotinoyl-1H-pyrazol-4-yl)-3-phenylthiazolidin-4-one (P-15) Brown solid (84.7%); m.pt. 256–258 °C; IR : 3398 (C–H str. Ar), 2848 (CH–S str.), 1680 , 1730 , 1649 (C=N str.), 1226 (Ar-N str.), 1198 (R–C-O str.), 1420 (C=N str.), 560 (C–Br str.); 1H NMR  δ 9.06 , 7.96 , 33.43 , 5.92 , 7.3 ,7.10 , 7.31 , 7.24 .2-(3-(4-bromophenyl)-1-isonicotinoyl-1H-pyrazol-4-yl)-3-(4-nitrophenyl)thiazolidin-4-one (P-16) Yellowish brown solid (68.9%); m.pt. 268–270 °C; IR : 3398(C-H str. Ar), 2900 (CH-S str.), 1690 ,1740 , 1649 (C=N str.), 1229 (Ar–N str.), 1154 (R–C–O str.), 1418 (C=N str.); 1520 (NO2 asymm. str.), 1310 (NO2 symm. str.), 1150 (Ar–N str.), 570 (C–Br str.).2-(3-(4-bromophenyl)-1-isonicotinoyl-4,5-dihydro-1H-pyrazol-4-yl)-3-(2-nitrophenyl) thiazolidin-4-one (P-17) Brown solid (72.0%); m.pt. 252–254 °C; IR : 3200 (C–H str. Ar), 2850 (CH–S str.), 1690 , 1735 , 1649 (C=N str.), 1226 (Ar-N str.), 1157 (R–C–O str.), 1418 (C=N str.); 1518 (NO2 asymm. str.), 1350 (NO2 symm. str.), 1198 (Ar-N str.), 580 (C–Br str.).2-(3-(4-bromophenyl)-1-isonicotinoyl-1H-pyrazol-4-yl)-3-(4-chlorophenyl)thiazolidin-4-one (P-18) Brown solid (79.8%); m.pt. 244–246 °C; IIR : 3215 (C–H str. Ar), 2936 (CH–S str.), 1680 , 1735 , 1560 (C=N str.), 1228 (Ar–N str.), 1157 (R–C–O str.), 1414 (C=N str.); 690 (C–Cl str.), 578 (C–Br str.); 1H NMR  δ 9.06 , 7.96 , 33.43 , 5.92 , 7.3 , 7.5 ,7.04 , 7.32 .2-(3-(4-bromophenyl)-1-isonicotinoyl-4,5-dihydro-1H-pyrazol-4-yl)-3-(3-chlorophenyl)thiazolidin-4-one (P-19) Brown solid (72.3%); m.pt. 248–250 °C; IR : 3125 (C–H str. Ar), 2910 (CH–S str.), 1670 , 1735 , 1589 (C=N str.), 1224 (Ar–N str.), 1150 (R–C-O str.), 1410 (C=N str.); 694 (C–Cl str.), 570 (C–Br str.).2-(3-(4-bromophenyl)-1-isonicotinoyl-1H-pyrazol-4-yl)-3-(4-methoxyphenyl) thiazolidin-4-one (P-20) Brown solid (83.2%); m.pt. 252–254 °C; IR : 3120 (C-H str. Ar), 2924 (CH-S str.), 1669 , 1740 , 1589 (C=N str.), 1226 (Ar–N str.), 1159 (R–C–O str.), 1340, 1415 (C=N str.); 570 (C–Br str.), 1317 (asymm. C–O–C str.), 1090 (symm. C–O–C str.); 1H NMR : δ 9.06 , 7.96 , 3.43 , 5.92 , 7.3 , 7.5 , 6.61 , 6.75 , 7.20 , 6.66 , 3.73 ; 13C-NMR : 149.8 (4-pyridine C3 & C5), 122.8 (4-pyridine C2 & C6), 167.0 (C=O), 151.8 (pyrazole C3), 46.1 (pyrazole C4), 71.1 (pyrazole C6), 133.0.0 (4-bromophenyl C1), 131.0 (4-bromophenyl C2 & C6), 131.0 (4-bromophenyl C3 & C5), 125.4 (4-bromophenyl C4), 47.8 (thiazolidin-4-one C2), 34 (thiazolidin-4-one C5), 170.9 (thiazolidin-4-one C4 (C=O)), 134 (4-methoxyphenyl C1), 122.6 (4-methoxyphenyl C2 & C6), 114 (4-methoxyphenyl C3 & C5), 156.0 (4-methoxyphenyl C4), 55.9 (O-CH3); LC–MS: m/z 535 (M+); Anal.: Calcd. for C25H19BrN4O3S: C, 56.088; H, 3.588; Br, 14.928; N, 10.468; S, 5.998; O, 8.968. Found: C, 56.078; H, 3.548; Br, 14.878; N, 10.438; S, 5.978; O, 8.938.2-(3-(4-bromophenyl)-1-isonicotinoyl-4,5-dihydro-1H-pyrazol-4-yl)-3-(2-methoxy phenyl) thiazolidin-4-one (P-21) Yellowish brown solid (69.1%); m.pt. 260–262 °C; IR : 3337 , 2924 (CH–S str.), 1670 , 1710 , 1549 (C=N str.), 1225 (Ar–N str.), 1157 (R–C-O str.), 1338, 1415 (C=N str.), 590 (C–Br str.), 1311 (asymm. C–O–C str.), 1095 (symm. C–O–C str.); 1H NMR  δ 9.06 , 7.96 , 3.43 , 5.92 , 7.3 , 7.5 , 5.59 , 6.25 , 7.18 , 6.66 , 3.73 ; 13C-NMR : 149.8 (4-pyridine C3 & C5), 122.8 ( 4-pyridine C2 & C6), 167.0 (C=O), 151.8 (pyrazole C3), 46.1 (pyrazole C4), 71.1 (pyrazole C-6), 133.0.0 (4-bromophenyl C1), 131.0 (4-bromophenyl C2 & C6), 131.0 (4-bromophenyl C3 & C5), 125.4 (4-bromophenyl C4), 47.8 (thiazolidin-4-one C2), 34 (thiazolidin-4-one C5), 170.9 (thiazolidin-4-one C4 (C=O)), 134 (4-methoxyphenyl C1), 120.4 (2-methoxyphenyl C2 & C6), 114 (4-methoxyphenyl C3 & C5), 152.0 (2-methoxyphenyl C4), 55.9 (O-CH3); LC–MS: m/z 535 (M+); Anal.: Calcd. for C25H19BrN4O3S: C, 56.088; H, 3.588; Br, 14.928; N, 10.468; S, 5.998; O, 8.968. Found: C, 56.088; H, 3.548; Br, 14.878; N, 10.438; S, 5.978; O, 8.938.H-tetrazolium (MTT) colorimetric test [When investigating a new drug, whether natural or synthesized, we should examine its safety for the host cell or the cytotoxic effect on the cancer cell. This is illustrious as the cell viability test. There are several methods to determine cell viability, among which the MTT test is a frequently used method. The MTT test was carried out using the 3--2,5-diphenyl-2ric test , 34. MTTric test . HealthyIn the present study, systematic experimental steps as shown in Fig. P (1–21) against HCT116 and MCF-7 cell lines. All the experiments were repeated at least thrice independently, the 50% inhibitory concentration (IC50) of new hybrids was determined (as the anticancer drug concentration causing a 50% reduction in cell viability) and calculated using a trendline equation. IC50 values of the new hybrids in μM against various cell lines are given in Table To test the anticancer activities of the synthesized compounds (selected based on docking studies), we evaluated the antiproliferative activity of compounds P 1–1 againstThe assay was carried out by the following method:2, PEP, LDH, PK, and histones H3), ATP, and inhibitors (selected compounds as per in silico studies) in kinase buffer (HEPES) and this to 96 well plates and incubate for 60 min. Once the ATP substrate was added, the reaction began. With the reaction, NADH was continuously transformed into NAD+ The activity of Aurora-A can be assayed by measuring the consumption of nicotinamide adenine dinucleotide plus hydrogen (NADH) at 340 nm. Absorbance at 340 nm was continuously recorded for 10 min. Inhibition of recombinant human Aurora-A by inhibitors was initially screened via an enzyme-coupled continuous spectrophotometric assay . The aurora kinase inhibitor, VX-680 (Tozasertib)  was used as the positive control [At 30 °C, dilute enzyme (aurora A), substrates  were examined for their capability to inhibit the activity of Aurora A kinase  installed on Windows-7 .Following protocol was followed for the docking studies:Ligand preparation The 2D structures of the designed pyrazole derivatives were drawn by using Chem Sketch (ACDLABS 12.0) and stored in a database in SDF structure format [e format . All theProtein preparation The X-ray co-crystal structure of Aurora-A Kinase protein was obtained from the RCSB protein data bank (http://www.rcsb.org/pdb). After evaluating the numbers of entries, the best protein (PDB entry 2bmc) [ry 2bmc) , 37 was Validation of docking protocol in glide The most suitable method of evaluating the accuracy of the docking procedure is to determine how closely the lowest energy pose predicted by the scoring function resembles an experimental binding mode as determined by X-ray crystallography.Generation of grid and ligand docking Docking studies on designed derivatives prepared through Ligprep were carried out in the active site of the protein. The calculations of grid parameters were accomplished by the Grid tool in ADT. The grid parameter files possessing all the information about the size of grid, protein, ligand, and geometry of search space were prepared and saved as ‘Conf.txt’. The optimized ligand molecules in PDBQT format were docked in the active site of Aurora Kinase A with Auto Dock Vina. Docking runs were launched from the command line, followed by the generation and scoring of best poses, for every ligand using the scoring function. At the end of the docking, ligands with the most favorable free energy of binding were selected. The protein–ligand interactions were further analyzed for the docked ligands by using PyMOL and the best poses in the binding site were drawn [re drawn , 39.


This structure was determined to be 3-hydroxy-1′,16′-didehydro-1′,2′-dihydro-2′,6′-cyclo-β,ψ-carotene-4,4′-dione by UV–VIS, MS, and NMR spectral data. The formation mechanism of roretziaxanthin in the sea squirt was discussed.A novel carotenoid with a unique 2,6-cyclo-ψ-end group, named roretziaxanthin ( S,3′S)-astaxanthin, alloxanthin, mytiloxanthin, mytiloxanthinone, halocynthiaxanthin, amarouciaxanthin A, and amarouciaxanthin B  at room temperature with a Jasco J-500C spectropolarimeter . Preparative high-performance liquid chromatography (HPLC) was performed on a Shimadzu LC-6AD with a Shimadzu SPD-6AV spectrophotometer  set at 450 nm. The column used was a 250mmX10mm i.d. 10 μm Cosmosil 5SL-II and 5C18 II .The UV–visible (UV–VIS) spectra were recorded with a Hitachi U-2001  in diethyl ether  at room temperature. The Me2CO extract was partitioned with hexane/Et2O (1:1) and water. The hexane/Et2O layer was evaporated to dryness and chromatographed on silica gel using increasing Et2O in hexane. The fraction eluted with hexane/Et2O (2:8) was subjected to HPLC on silica gel with Me2CO/hexane (3:7), with a retention time (RT) of 14.1 min. Further purification to remove lipid impurities from 1 was performed by HPLC on ODS with methanol. Finally, 1.5 mg  of roretziaxanthin was obtained as a crimson-colored powder.The tunic (4 kg) of the sea squirt H. roretzi along with roretziaxanthin. The fraction eluted with hexane/Et2O (2:8) was subjected to HPLC on silica gel with Me2CO/hexane (3:7), with an RT of 12.2 min to yield -astaxanthin (3.5 mg), and an RT of 15.0 min for mytiloxanthinone (6.5 mg) (2O/Me2CO (1:1) was subjected to HPLC on silica gel with Me2CO/hexane (3:7): RT of 15.0 min, zeaxanthin (3 mg); RT of 16.2 min, diatoxanthin (9 mg); RT of 18.0 min, alloxanthin (33 mg). The fraction eluted with Me2CO was subjected to HPLC on silica gel with Me2CO/hexane (3:7): RT of 19.0 min, diadinochrome (2.5 mg); RT of 21.2 min, mytiloxanthin (3.5 mg) (The following carotenoids were isolated from the sea squirt (6.5 mg) . The fra3.5 mg) ; RT of 2(3.5 mg) ; and RT .5 mg ; R2O): 463 (shoulder), 482, and 505 nm. HR-FAB MS: m/z 578.3756 (M+) C40H50O3 calcd for 578.3760. Product ions of FAB MS/MS (precursor ion M+): m/z 560 (M-H2O), m/z 486 (M-92), m/z 425 (M-153). HR-ESI MS m/z 601.3641 (M + Na+) C40H50O3Na calcd for 601.3658, m/z 579.3820 (M + H+) C40H51O3 calcd for 579.3838. Product ions of FAB MS/MS (precursor ion M + H+): m/z 561 (M + H-H2O), m/z 486 (M + H-92), m/z 473 (M + H-106), m/z 425 (M–153). CD (in EPA at room temperature): nm (∆ε) 225 (−4), 265 (−12), 300 (0), 325 (−1), 350 (0). 1H-NMR (500 MHz) and 13C-NMR (125 MHz) in CDCl3 are compiled in UV–VIS (Et1), isolated from the sea squirt Halocynthia roretzi, was determined to be 3-hydroxy-1′,16′-didehydro-1′,2′-dihydro-2′,6′-cyclo-β,ψ-carotene-4,4′-dione by UV–VIS, MS, and NMR spectral data. Roretziaxanthin (1) has a unique 2,6-cyclo-ψ-end group in its molecule. Carotenoids with a 2,6-cyclo-ψ-end group have not been found in marine animals or algae. Roretziaxanthin (1) might be formed from rubixanthin through an epoxidation, cyclization, dehydration, or oxidation process, as shown in The structure of the new carotenoid roretziaxanthin (


SCSIO 07395 and the heterologous expression of this BGC in Streptomyces albus. This approach led to the discovery of five new benzoxazole alkaloids microechmycin A–E (1–5), and a previously synthesized compound 6. Their structures were elucidated by HRESIMS and 1D and 2D NMR data. Microechmycin A (1) showed moderate antibacterial activity against Micrococcus luteus SCSIO ML01 with the minimal inhibitory concentration (MIC) value of 8 μg mL−1.Benzoxazole alkaloids exhibit a diverse array of structures and interesting biological activities. Herein we report the identification of a benzoxazole alkaloid-encoding biosynthetic gene cluster ( Heterocyclic compounds play an important role in drug discovery and development . BenzoxaStreptomyces pactum SCSIO 02999 and Streptomyces sp. SCSIO 40010 −, calcd for C15H11N2O4 283.0724, found 283.0725, indicating 11 degrees of unsaturation  NMR data of 1 . The further inspection of the COSY spectrum of 1 revealed two ABC three-spin systems at δH 7.86 (H-3)/7.44 (H-4)/7.93 (H-5) and δH 7.02 (H-12)/6.69 (H-13)/7.57 (H-14) (1 with the intermediate 2-(2-amino-3-hydroxyphenyl)benzoxazole-4-carboxylic acid in the biosynthetic pathway of A33853 +, calcd for C18H18N2O6 359.1239, found 359.1238 /H-17 (δH 3.88) and H-17/H2-18 (δH 3.53). The glycerol moiety was linked to the carboxy group at C-1 in 1 to form an ester bond by HMBC correlations from H2-16 to C-1   .3) was isolated as yellow powder. The molecular formula of 3 was assigned to be C23H20N2O4 by HRESIMS  , and H-19(H-23) to C-16. Based on the HMBC correlation from H2-16 to C-1 (3 was elucidated (Microechmycin C (89.1496) . Analysient in 3 . The pre6 to C-1 , the pheucidated .4) was isolated as yellow oil. The molecular formula of 4 was determined by HRESIMS m/z: [M + H]+, calcd for C33H29N4O9 625.1929, found 625.1931, indicating 22 degrees of unsaturation /H-17 and HMBC correlations from H2-16(H-18) to C-1(C-1’)  . Therefoablished .5) was isolated as yellow oil. The molecular formula of 5 was determined by HRESIMS m/z: [M + H]+, calcd for C33H29N4O9 625.1929, found 625.1923  spectrum   .6) was isolated as yellow oil. The molecular formula of 6 was determined by HRESIMS m/z: [M + H]+, calcd for C16H15N2O4 299.1026, found 299.1030 (6 and 1 were highly similar (δC/δH 52.4/4.03) in 6, which was linked to the carboxy group at C-1 to form an ester bond. This assignment was supported by the HMBC correlations from H3-16 to C-1  of 8 μg mL−1. However, compounds 2–6 did not exhibit any antibacterial activity for the tested strains.Compounds n method . CompounStreptomyces albus Del14 − .Microechmycin A (2): C18H18N2O6; yellow oil; 3CN) λmax (log ε): 202.60 (3.96), 234.60 (3.71), 271.80 (3.36), 284.80 (3.25), 320 (3.41), 378.20 (3.26) nm; Data of 1H  and 13C NMR , see m/z 359.1238 [M + H]+ .Microechmycin B (3): C23H20N2O4; yellow powder; UV (CH3CN) λmax (log ε): 204 (4.12), 271.80 (3.34), 284 (3.25), 310 (3.33), 371.20 (3.18); Data of 1H  and 13C NMR , see m/z 389.1495 [M + H]+ .Microechmycin C (4): C33H29N4O9; yellow oil; UV (CH3CN) λmax (log ε): 202.40 (4.00), 234.60 (4.05), 272 (4.00), 284.80 (4.03), 320.40 (4.19), 378.20(4.28) nm; Data of 1H  and 13C NMR , see m/z 625.1931 [M + H]+ .Microechmycin D (5): C33H29N4O9; yellow oil; [α]25 + 7.50 ; UV (CH3CN) λmax (log ε): 221.60 (3.06), 234.80 (3.19), 271 (3.16), 286 (3.01), 311.80 (3.10), 320.80 (3.48) nm, 375.60 (3.44); Data of 1H  and 13C NMR , see m/z 625.1923 [M + H]+ .Microechmycin E (6): C16H15N2O4; yellow oil; UV (CH3CN) λmax (log ε): 235.40 (2.87), 270.20 (2.99), 284.80 (2.94), 309 (2.77), 319.40 (2.86), 375 (3.22) nm; Data of 1H  and 13C NMR , see m/z 299.1030 [M + H]+ .Microechmycin F  with UV detection at 265 nm under the following program: solvent system ; 38% B (0–35 min), flow rate at 1 mL min−1. Chiral HPLC analysis of compound 5 was conducted by using a chiral column  with UV detection at 265 nm under the following program: solvent system ; 5% B to 100% B (0–20 min), 100% B (21−25 min), 100% B to 5%B (25−26 min), 5% B (26–30 min), flow rate at 1 mL min−1.Chiral HPLC analysis of compound 1–6 was measured against two Gram positive bacteria S. aureus ATCC 29213, M. luteus SCSIO ML01 and four Gram negative bacteria E. coli ATCC 25922, V. alginolyticus 13214, A. baumannii ATCC 19606 and K. pneumoniae ATCC 13883 by the broth micro dilution method [Antibacterial activity of compounds n method . Ciproflmich gene cluster from Micromonospora sp. SCSIO 07395 in the heterologous S. albus host and identified the benzoxazole-containing microechmycins (1–6) as the products of the pathway. Microechmycin D (4) and E (5) can be synthesized through the actions of esterification between glycerol and two molecules of 1. This glycerol esterification has been observed in acautalides C discovery from Acaulium sp H-JQSF [Micromonospora sp. SCSIO 07395, and highlights the potential of searching for novel natural products from marine rare actinomycetes by heterologous expression.In summary, we have expressed the p H-JQSF . The dis


Ligustrum robustum, which have been used as Ku-Ding-Cha, led to the isolation and identification of three new phenylethanoid glycosides and three new phenylmethanoid glycosides, named ligurobustosides R1 (1b), R2–3 (2), R4 (3), S1 (4b), S2 (5), and S3 (6), and five reported phenylethanoid glycosides (7–11). In the bioactivity test, (Z)-osmanthuside B6 (11) displayed strong fatty acid synthase (FAS) inhibitory activity (IC50: 4.55 ± 0.35 μM) as the positive control orlistat (IC50: 4.46 ± 0.13 μM), while ligurobustosides R4 (3) and S2 (5), ligupurpuroside B (7), cis-ligupurpuroside B (8), ligurobustoside N (9), osmanthuside D (10), and (Z)-osmanthuside B6 (11) showed stronger ABTS radical scavenging activity (IC50: 2.68 ± 0.05~4.86 ± 0.06 μM) than the positive control L-(+)-ascorbic acid (IC50: 10.06 ± 0.19 μM). This research provided a theoretical basis for the leaves of L. robustum as a tea with function in treating obesity and diabetes.The phytochemical study on the leaves of  Ligustrum robustum (Roxb.) Blume (Oleaceae), classified as a food by the Chinese Ministry of Health since 2011, has been used as Ku-Ding-Cha in Southwest China 20D −43.9 ; UV (MeOH) λmax: (log ε) 212 (4.1), 227 (4.2), 317 (4.4) nm; IR (film) νmax: 3368, 2930, 1690, 1604, 1515, 1445, 1261, 1039, 981, 829 cm−1; 1H NMR  data, see 13C NMR  data, see m/z 761.2634 [M + Na]+ .Compound 2: white amorphous powder. [α]23D −62.1 ; UV (MeOH) λmax (log ε): 213 (4.1), 226 (4.2), 318 (4.4) nm; IR (film) νmax: 3356, 2931, 1693, 1630, 1603, 1515, 1448, 1263, 1040, 982, 834, 803 cm−1; 1H NMR  data, see 13C NMR  data, see m/z 793.2536 [M + Na]+ .Compound 3: yellow amorphous powder. [α]23D −46.0 ; UV (MeOH) λmax (log ε): 213 (4.1), 227 (4.2), 316 (4.4) nm; IR (film) νmax: 3402, 1652, 1604, 1048, 1029, 823, 761 cm−1; 1H NMR  data, see 13C NMR  data, see m/z 791.2371 [M + Na]+ .Compound 4: white amorphous powder. [α]20D −122.4 ; UV (MeOH) λmax (log ε): 210 (3.9), 230 (3.9), 315 (4.4) nm; IR (film) νmax: 3360, 2929, 1695, 1603, 1449, 1330, 1259, 1157, 1021, 912, 833, 741, 699 cm−1; 1H NMR  data, see 13C NMR  data, see m/z 585.1943 [M + Na]+ .Compound 5: yellowish amorphous powder. [α]20D −18.5 ; UV (MeOH) λmax (log ε): 210 (3.9), 230 (3.9), 315 (4.4) nm; IR (film) νmax: 3369, 2925, 2854, 1706, 1605, 1512, 1452, 1164, 1038, 836, 700 cm−1; 1H NMR  data, see 13C NMR  data, see m/z 585.1947 [M + Na]+ .Compound 6: white amorphous powder. [α]20D −18.5 ; UV (MeOH) λmax (log ε): 210 (3.9), 230 (3.9), 316 (4.4) nm; IR (film) νmax: 3369, 2925, 2854, 1706, 1605, 1512, 1452, 1164, 1038, 836, 700 cm−1; 1H NMR  data, see 13C NMR  data, see m/z 585.1949 [M + Na]+ .Compound 1–6 (2 mg), dissolved in 0.1 mL MeOH, were injected into 2 mL H2SO4 aqueous solution (1 M) and hydrolyzed at 95 °C for 6 h, respectively. Then, 2 mL Ba(OH)2 solution (1 M) was added. The hydrolyzed solution was filtered and condensed. The monosaccharides in the condensed solution were affirmed by TLC  with authentic samples 27D +4.8  -6-O-(trans-p-coumaroyl)-O-β-D-mannopyranoside (ligurobustoside R) [1b ] in 1a was replaced by the cis-p-coumaroyl  in 1b. The acid hydrolysis experiment of 1 gave D-mannose, and L-rhamnose was affirmed by TLC. The HMBC experiment of 1b (δH 4.28 (H-1’ of mannosyl) and δC 72.3 (C-8 of aglycone), between δH 5.17 (H-1’’ of inner rhamnosyl) and δC 83.6 (C-3’ of mannosyl), between δH 5.19 (H-1’’’ of outer rhamnosyl) and δC 81.1 (C-4’’ of inner rhamnosyl), and between δH 4.29 (H-6’a of mannosyl), 4.46 (H-6’b of mannosyl) and δC 168.1 (carbonyl of coumaroyl). The 1H and 13C NMR signals of 1b were assigned by the HMBC experiment (1b was identified as 2-(4-hydroxyphenyl)ethyl 3-O-[α-L-rhamnopyranosyl-(1→4)-α-L-rhamnopyranosyl]-6-O-(cis-p-coumaroyl)-O-β-d-mannopyranoside. It is a novel phenylethanoid glycoside named ligurobustoside R1. In conclusion, compound 1 is a mixture of ligurobustosides R and R1.Compound ta of 1a . was in oside R) . The NMRe R) -4-O-(trans-caffeoyl)-O-β-d-glucopyranoside (ligurobustoside P) -4-O-(trans-p-coumaroyl)-O-β-d-glucopyranoside.Compound um of 2a  revealedum of 2a  showed ant of 2a  displayeeriments . Thus, t2b ] in 2b. The HMBC experiment of 2b (δH 4.43 (H-1’ of glucosyl) and δC 76.7 (C-8 of aglycone), between δH 5.21 (H-1’’ of inner rhamnosyl) and δC 81.1 (C-3’ of glucosyl), between δH 5.06 (H-1’’’ of outer rhamnosyl) and δC 81.5 (C-4’’ of inner rhamnosyl), and between δH 4.90 (H-4’ of glucosyl) and δC 166.8 (carbonyl of coumaroyl). Therefore, the plane structure of 2b was identified as 2-hydroxy-2-ethyl 3-O-[α-L-rhamnopyranosyl-(1→4)-α-L-rhamnopyranosyl]-4-O-(cis-p-coumaroyl)-O-β-d-glucopyranoside.The NMR data of 2b  were simnt of 2b  displaye2 gave (R)-(-)-l-ethane-l,2-diol and (S)-(+)-l-ethane-l,2-diol (9:11), meaning that R/S (9:11) was not equal to 2a/2b (10:3). Based on the above evidence, compound 2 was characterized as a mixture of (2R/S)-2-hydroxy-2-ethyl 3-O-[α-L-rhamnopyranosyl-(1→4)-α-L-rhamnopyranosyl]-4-O-(trans-p-coumaroyl)-O-β-d-glucopyranoside and (2R/S)-2-hydroxy-2-ethyl 3-O-[α-L-rhamnopyranosyl-(1→4)-α-L-rhamnopyranosyl]-4-O-(cis-p-coumaroyl)-O-β-d-glucopyranoside. It is a novel phenylethanoid glycoside named ligurobustoside R2–3.In addition, the enzymatic hydrolysis experiment of 3 was determined as C35H44O19 by HRESIMS . The 1H NMR spectrum of 3 ; (2) a 3,4-disubstituted phenyl at δH 6.89 , 7.46  and 7.47 ; (3) a trans double bond at δH 7.68 and 6.37 ; (4) three anomeric protons at δH 4.54 , 5.06  and 5.22 ; (5) a methylene at δH 4.98 and 5.26 , and two methyl groups at δH 1.06  and 1.10 . The 13C NMR spectrum of 3  linking with hydroxy in 2a was replaced by the carbonyl in 3. The acid hydrolysis experiment of 3 afforded D-glucose and L-rhamnose affirmed by TLC. Additionally, the HMBC experiment of 3 (δH 7.46 (H-2), 7.47 (H-6), 4.98 (H-8a), 5.26 (H-8b) and δC 196.4 (C-7), between δH 4.54 (H-1’ of glucosyl) and δC 72.2 (C-8 of aglycone), between δH 5.22 (H-1’’ of inner rhamnosyl) and δC 81.1 (C-3’ of glucosyl), between δH 5.06 (H-1’’’ of outer rhamnosyl) and δC 81.2 (C-4’’ of inner rhamnosyl), and between δH 4.96 (H-4’ of glucosyl) and δC 167.6 (carbonyl of coumaroyl). The 1H and 13C NMR signals of 3 were assigned by 1H-1H COSY, HSQC, and HMBC experiments -2-oxoethyl 3-O-[α-l-rhamnopyranosyl-(1→4)-α-L-rhamnopyranosyl]-4-O-(trans-p-coumaroyl)-O-β-d-glucopyranoside. It is a novel phenylethanoid glycoside named ligurobustoside R4.Compound rum of 3  showed trum of 3  revealedent of 3  displayeeriments . Therefo4 was determined as C28H34O12 by HRESIMS . The NMR spectra of 4 exhibited 2 stereoisomers 4a and 4b (2:1). The 1H and 13C NMR data of 4a (O-(α-L-rhamnopyranosyl)-4-O-(trans-p-coumaroyl)-O-β-d-mannopyranoside (ligurobustoside S) [4b ] in 4a was replaced by the cis-p-coumaroyl  in 4b. The acid hydrolysis experiment of 4 offered D-mannose and L-rhamnose confirmed by TLC. The HMBC experiment of 4b (δH 4.42 (H-1’ of mannosyl) and δC 72.0 (C-7 of aglycone), between δH 5.16 (H-1’’ of rhamnosyl) and δC 81.6 (C-3’ of mannosyl), and between δH 4.90 (H-4’ of mannosyl) and δC 166.9 (carbonyl of coumaroyl). The 1H and 13C NMR signals of 4 were assigned by the HMBC experiment (4b was identified as benzyl 3-O-(α-L-rhamnopyranosyl)-4-O-(cis-p-coumaroyl)-O-β-d-mannopyranoside. It is a new phenylmethanoid glycoside named ligurobustoside S1. In sum, compound 4 is a mixture of ligurobustosides S and S1.Compound ta of 4a . was in e S) -3-O-(α-L-rhamnopyranosyl)-O-β-d-glucopyranoside   in 5 was replaced by the cis-p-coumaroyl  in 6. The acid hydrolysis experiment of 6 yielded D-glucose and L-rhamnose affirmed by TLC. The HMBC experiment of 6 (δH 4.33 (H-1’ of glucosyl) and δC 72.0 (C-7 of aglycone), between δH 5.15 (H-1’’ of rhamnosyl) and δC 84.1 (C-3’ of glucosyl), and between δH 4.30 (H-6’a of glucosyl), 4.50 (H-6’b of glucosyl) and δC 168.2 (carbonyl of coumaroyl). The 1H and 13C NMR signals of 6 were assigned by 1H-1H COSY, HSQC, and HMBC experiments (6 was identified as benzyl 3-O-(α-L-rhamnopyranosyl)-6-O-(cis-p-coumaroyl)-O-β-d-glucopyranoside. It is a new phenylmethanoid glycoside named ligurobustoside S3.Compound ata of 6  were relent of 6  showed teriments . Thus, c7–11  were identified as reported ligupurpuroside B (7) [cis-ligupurpuroside B (8) [9) [10) [Z)-osmanthuside B6 (11) [1H-1H COSY, HSQC, and HMBC). Compounds 8, 10, and 11 were isolated from this plant for the first time.Compounds de B (7) , cis-ligde B (8) ,22, ligu[9) [10) , and (Z) B6 (11) ,24, by c1–11 from the leaves of L. robustum were measured for the inhibitory effects on FAS, α-glucosidase, α-amylase, and antioxidant activities. The results of the bioactivity assays are shown in 11 (IC50: 4.55 ± 0.35 μM) was as strong as the positive control orlistat (IC50: 4.46 ± 0.13 μM), while the FAS inhibitory effects of compounds 4 (IC50: 6.49 ± 0.27 μM) and 9 (IC50: 5.61 ± 0.44 μM) were weaker than orlistat; the α-glucosidase inhibitory effects of compounds 3 and 5 were moderate and weaker than the positive control acarbose; the α-amylase inhibitory effects of compounds 10 and 11 were moderate and weaker than the positive control acarbose; the DPPH radical scavenging activities of compounds 2, 3, and 9 (IC50: 23.83 ± 0.89~43.17 ± 1.06 μM) were weaker than the positive control L-(+)-ascorbic acid (IC50: 13.66 ± 0.13 μM); the ABTS radical scavenging activities of compounds 3, 5, and 7–11 (IC50: 2.68 ± 0.05~4.86 ± 0.06 μM) were stronger than the positive control L-(+)-ascorbic acid (IC50: 10.06 ± 0.19 μM).Compounds α-glucosidase and α-amylase were two important targets to prevent diabetes and obesity [3–5, 9, and 10, with some FAS, α-glucosidase, and α-amylase inhibitory activities [L. robustum to prevent diabetes and obesity.The previous study revealed that FAS was a potential therapeutic target for anti-obesity drugs ,18; α-gl obesity ,25; and  obesity ,26. Constivities , might bL. robustum resulted in the isolation of eight phenylethanoid glycosides  and three phenylmethanoid glycosides (4–6), including six novel compounds  identified with spectroscopic method , and physical and chemical methods. The biological assays showed that the FAS inhibitory effect of compound 11 (IC50: 4.55 ± 0.35 μM) was as strong as the positive control orlistat (IC50: 4.46 ± 0.13 μM); the α-glucosidase inhibitory effects of compounds 3 and 5, and the α-amylase inhibitory effects of compounds 10 and 11 were moderate; the DPPH radical scavenging activities of compounds 2, 3, and 9 (IC50: 23.83 ± 0.89~43.17 ± 1.06 μM) were weaker than the positive control l-(+)-ascorbic acid (IC50: 13.66 ± 0.13 μM); the ABTS radical scavenging activities of compounds 3, 5, and 7–11 (IC50: 2.68 ± 0.05~4.86 ± 0.06 μM) were stronger than the positive control l-(+)-ascorbic acid (IC50: 10.06 ± 0.19 μM). Together this work and previous studies [L. robustum. This research offered a theoretical basis for the leaves of L. robustum as a functional tea to prevent obesity and diabetes.In summary, the phytochemical investigation on the leaves of  studies ,13, phen


N- and N,O-containing macrocyclic ligands differing by the number of donor sites and cavity size has been obtained. These compounds possess well-pronounced fluorescent properties with emission maxima in a blue region in aprotic solvents and high quantum yields of fluorescence, while in proton media, fluorescence shifts towards the green region of the spectrum. Using macrocycles 5c and 5e as examples, we have shown that such compounds can serve as dual-channel (colorimetric and fluorimetric) pH indicators in water media, with pH transition point and response being dependent on the macrocycle structure due to different sequences of protonation steps.Synthetic approach to fluorescent polyaza- and polyoxadiazamacrocycles comprising a structural fragment of 6,7-diamino-2,3-diphenylquinoxaline has been elaborated using Pd-catalyzed amination providing target compounds in yields up to 77%. A series of nine novel  Elaboration of efficient synthetic approaches to novel macrocyclic compounds of various types  is of great interest due to their unique properties. They find wide applications in supramolecular chemistry ,3,4,5,6,MC) were reported (MC1) ethyldi-tert-butylphosphine) and ligands of this type were noted for its good performance in the Pd-catalyzed amination of halogen heteroarenes 2+ complexes of medium stability, the most stable complex (logβ = 7.57(7)) was observed for a non-cyclic ligand 3, possibly due to conformational lability of its receptor part. Oxygen atoms and nitrogen atoms of the heteroaromatic moiety probably participate in the formation of such complexes.Using the HypSpec program, spectral data for spectrophotometric titrations were subjected to the factor analysis, which provided the composition and binding constants of the complexes, which are collected in 5a and 5b both form the complexes [CuL]2+ and [Cu4(L)3]8+, their stabilities being similar, independent of the fact that these macrocycles differ by the cavity size and the number of oxygen atoms. As macrocycles with enough short linkers cannot form polynuclear complexes, the formation of [Cu4(L)3]8+ could be explained by the association of three [CuL]2+ complexes around one copper(II) center via coordination employing heterocyclic nitrogen atoms. As macrocycles 5c and 5d possess larger cavities with longer polymethylene chains between atoms, they can promote the formation of the complexes of another composition. Indeed, with these two macrocyclic ligands, besides [CuL]2+, complexes featuring two macrocycles and four Cu(II) cations [Cu4(L)2]8+ were observed. Thus, the size and flexibility of the macrocyclic cavity influence stability and composition of the complexes. The studied N,O-ligands can form mono- and polynuclear copper complexes.Macrocycles 5c and tetraazamacrocycle 5e. Quinoxaline derivatives were described as convenient platforms for the development of pH sensors +2 structure, charged protonated aliphatic nitrogen atoms are positioned inevitably closer to the heterocyclic core due to the macrocyclic structure, which leads to lower pK3 5e values compared to 7. Upon further addition of acid new changes are observed in the fluorescence spectrum, which allows us to estimate the value of pK4 1.0 (5c) is observed only in strongly acidic media (pH < 0), which can be explained by a notable difference in the basicity of the [5eH3]3+ particle in the ground and excited states.Macrocycle rocycles ,74,75. Utonation . An atteappeared . This ob 8 and 9 . In the t differ . In macr pK4 1.0 . However2Cl2 and CH3CN were distilled over CaH2, chloroform was distilled over P2O5, MeOH was used freshly distilled. 4,5-Dibromobenzene-1,2-diamine was obtained according to the described 3-step procedure + calcd for C20H12N2Br2: 438.944; found: 438.946. oxaline,  4,5-DibrPalladium-Catalyzed Amination of 6,7-dibromo-2,3-diphenilquinoxaline—General Procedure. A two-neck flask equipped with a condenser and a magnetic stirrer, flushed with dry argon, was charged with corresponding 6,7-dibromo-2,3-diphenilquinoxaline , Pd(dba)2 , phosphine ligand (9 mol%), and absolute dioxane (3–15 mL). The mixture was stirred for 2–3 min, then corresponding amine (0.45–1.2 mmol) and tBuONa  were added, and the reaction mixture was refluxed for 24–48 h. After cooling it down to ambient temperature, the reaction mixture was diluted with CH2Cl2, the solution filtered and evaporated in vacuo, and the residue was chromatographed on silica gel using a sequence of eluents: CH2Cl2, CH2Cl2/MeOH 200:1–3:1, CH2Cl2/MeOH/NH3aq 100:20:1–100:30:5. In the cases of the compounds 5a and 5b, the residue before column chromatography was dissolved in CH2Cl2 (20 mL) and washed with deionized water (2 × 20 mL) to eliminate sodium salts. The organic phase was dried over 3 Å sieves and concentrated in vacuo, and only then subjected to column chromatography according to the general procedure.N,N`-bis(2-methoxyethyl)-2,3-diphenylquinoxaline-6,7-diamine (3) obtained according to General Procedure from 6,7-dibromo-2,3-diphenilquinoxaline (132 mg), amine 2 (90 mg) in the presence of Pd(dba)2 (14 mg), BINAP (17 mg) and tBuONa (87 mg) in dioxane (3 mL) in 24 h. Eluent CH2Cl2/MeOH 100:1. Yield 76 mg (59%), yellowish oil. 1H-NMR  δH 3.39 , 3.44 ), 3.71 ), 4.39 , 7.15 ), 7.27–7.29 ), 7.46–7.49 ). 13C-NMR  δC 43.5 , 58.7 , 70.2 , 104.9 ), 127.7 ), 128.0 ), 129.7 ), 138.4 ), 140.0 ), 141.8 ), 149.0 ). IR (neat): 612 (vw), 624 (vw), 641 (vw), 668 (w), 698 (s), 750 (Vs), 759 (s), 765 (vs), 806 (vw), 832 (w), 853 (w), 917 (vw), 978 (w), 1001 (w), 1024 (m), 1065 (m), 1079 (m), 1117 (m), 1157 (w), 1199 (s), 1240 (m), 1261 (m), 1267 (m), 1274 (m), 1313 (vw), 1354 (m), 1391 (vw), 1447 (m), 1482 (m), 1495 (m), 1518 (m), 1573 (w), 1599 (w), 1615 (w), 1719 (vw), 2816 (w), 2829 (w), 2876 (w), 2885 (w), 2926 (w), 2980 (w), 3054 (vw), 3084 (vw), 3371 (w), 3382 (w) cm−1. HRMS (MALDI-TOF): m/z [M + H]+ calcd for C26H28N4O2+: 429.2285; found: 429.2266.2,3-Diphenyl-6,7,8,10,11,13,14,15-octahydro-dioxadiazacyclododecinoquinoxaline (5a) obtained according to General Procedure from 6,7-dibromo-2,3-diphenilquinoxaline (132 mg), amine 4a (67 mg) in the presence of Pd(dba)2 (14 mg), BINAP (17 mg) and tBuONa (87 mg) in dioxane (15 mL) in 48 h. Eluent CH2Cl2/MeOH 100:1. Yield 59 mg (46%), yellowish oil. 1H-NMR  δH 3.41 ), 3.70 ), 3.74 ) 4.96 , 7.27-7.30 , H3, H4, H5 (Ph)), 7.45-7.47 ). 13C-NMR  δC 45.7 , 69.1 , 69.8 , 110.8 ), 127.9 ), 128.0 ), 129.7 ), 138.8 ), 139.9 ), 143.6 ), 149.7 ). IR (neat): 612 (w), 614 (w), 624 (w), 668 (w), 675 (m), 694 (s), 698 (vs), 712 (w), 749 (s), 759 (s), 763 (s), 790 (w), 805 (w), 830 (m), 847 (w), 857 (w), 868 (w), 887 (w), 928 (w), 945 (w),977 (m), 1001 (w), 1015 (m), 1023 (m), 1033 (m), 1054 (m), 1061 (m), 1071 (m), 1079 (m), 1106 (m), 1116 (m), 1128 (m), 1149 (w), 1158 (w), 1196 (m), 1226 (m), 1264 (m), 1292 (w), 1309 (w), 1339 (m), 1348 (w), 1369 (w), 1407 (w), 1455 (m), 1474 (m), 1493 (w), 1520 (m), 1565 (w), 1598 (w), 1618 (w), 2851 (w), 2880 (w), 2914 (w), 2952 (w), 3051 (vw), 3317 (w) cm−1. HRMS (MALDI-TOF): m/z [M + K–CH2CH2]+ calcd for C24H22N4O2K+: 437.1374; found: 437.1405.2,3,18,19-Tetraphenyl-6,7,8,10,11,13,14,15,22,23,24,26,27,29,30,31-hexadecahydro-tetraoxatetraazacyclotetracosinodiquinoxaline (6a) obtained in mixture with as a by-product in the synthesis of 5a. Eluent CH2Cl2/MeOH 50:1. Yield 4 mg (3%), yellow-brown oil. 1H-NMR  δH 3.47–3.49 ), 3.72 ), 3.90 ) 7.12 ), 7.27–7.31 ), 7.45–7.47 ). NH-protons were not unambiguously assigned due to line broadening. HRMS (MALDI-TOF): m/z [M + Na]+ calcd for C52H52N8O4Na+: 875.4004; found: 875.3983.2,3-Diphenyl-6,7,8,10,11,13,14,16,17,18-decahydro-trioxadiazacyclopentadecino quinoxaline (5b) obtained according to General Procedure from 6,7-dibromo-2,3-diphenilquinoxaline (132 mg), amine 4b (87 mg) in the presence of Pd(dba)2 (14 mg), BINAP (17 mg) and tBuONa (87 mg) in dioxane (15 mL) in 24 h. Eluent CH2Cl2/MeOH 100:1. Yield 43 mg (30%), yellowish oil. 1H-NMR  δH 3.42–3.46 ), 3.66–3.67 ), 3.76–3.78 ), 3.89 ), 4.82 , 7.09 ), 7.27–7.29 ), 7.46–7.49 ). 13C-NMR  δC 42.9 , 68.1  70.0 , 70.2 , 103.7 ), 127.7 ), 128.0 ), 129.9 ), 138.5 ), 140.0 ), 141.5 ), 148.4 ). IR (neat): 609 (w), 668 (vw), 699 (s), 733 (m), 768 (m), 792 (vw), 805 (vw), 844 (m), 882 (vw), 919 (w), 943 (w), 979 (w), 1001 (w), 1025 (m), 1074 (s), 1096 (s), 1110 (s), 1130 (vs), 1202 (s), 1226 (s), 1246 (s), 1265 (m), 1354 (s), 1402 (w), 1417 (w), 1455 (s), 1495 (s),1528 (s), 1577 (w), 1618 (m), 1725 (w), 2867 (m), 2906 (m), 3058 (w), 3077 (w), 3365 (m) cm−1. HRMS (MALDI-TOF): m/z [M + K–CH2CH2]+ calcd for C26H26N4O3K+: 481.1636; found: 481.1605.2,3-Diphenyl-6,7,8,9,11,12,14,15,17,18,19,20-dodecahydro-trioxadiazacyclohepta decinoquinoxaline (5c) obtained according to General Procedure from 6,7-dibromo-2,3-diphenilquinoxaline (132 mg), amine 4c (99 mg) in the presence of Pd(dba)2 (14 mg), BINAP (17 mg) and tBuONa (87 mg) in dioxane (15 mL) in 48 h. Eluent CH2Cl2/MeOH 200:1. Yield 72 mg (48%), yellowish oil. 1H-NMR  δH 2.06 , 3.50 ), 3.68–3.69 ), 3.71–3.74 ), 4.86 , 7.07 ), 7.27–7.28 ), 7.45–7.48 ). 13C-NMR  δC 27.9 , 42.9 , 70.7 , 70.8 , 70.9 , 103.3 ), 127.5 ), 127.9 ), 129.8 ), 138.5 ), 140.4 ), 141.8 ), 148.4 ). IR: 638 (vw), 654 (w), 668 (m), 675 (w), 700 (vs), 734 (s), 768 (m), 804 (m), 832 (m), 871 (w), 896 (w), 916 (w), 937 (w), 979 (m), 1001 (m), 1025 (s), 1066 (vs), 1079 (vs), 1089 (Vs), 1092 (vs), 1112 (vs), 1200 (vs), 1240 (vs), 1264 (s), 1353 (vs), 1437 (m), 1462 (vs), 1482 (s), 1496 (s), 1522 (vs), 1559 (w), 1599 (w), 1616 (m), 2865 (m), 2918 (m), 2942 (w), 3051 (w), 3366 (w) cm−1. HRMS (MALDI-TOF): m/z [M + H]+ calcd for C30H35N4O3+: 499.2704; found: 499.2665.2,3-Diphenyl-6,7,8,9,11,12,13,14,16,17,18,19-dodecahydro-dioxadiazacyclohexa decinoquinoxaline (5d) obtained according to General Procedure from 6,7-dibromo-2,3-diphenilquinoxaline (132 mg), amine 4d (92 mg) in the presence of Pd(dba)2 (14 mg), BINAP (17 mg) and tBuONa (87 mg) in dioxane (15 mL) in 48 h. Eluent CH2Cl2/MeOH 200:1. Yield 69 mg (48%), yellowish oil. 1H-NMR  δH 1.77 , 2.06 , 3.48 ), 3.58 , 3.67 ), 5.15 , 7.05 , 7.27–7.29 ), 7.46–7.48 ). 13C-NMR  δC 26.3 , 28.3 , 44.0 , 70.6 , 70.8 , 102.8 ), 127.5 ), 128.0 ), 129.9 ), 138.5 ), 140.3 ), 141.8 ), 148.1 ). IR (neat): 669 (m), 699 (vs), 732 (m), 804 (m), 830 (s), 979 (m), 1001 (m), 1024 (s), 1065 (s), 1078 (s), 1097 (s), 1109 (s), 1199 (vs), 1240 (s), 1265 (m), 1352 (vs), 1459 (vs), 1487 (s), 1496 (s), 1522 (vs), 1560 (m), 1599 (m), 1617 (m), 2799 (m), 2854 (s), 2918 (s), 3057 (m), 3344 (m), 3388 (m) cm−1. HRMS (MALDI-TOF): m/z [M + H]+ calcd for C30H35N4O2+: 483.2755; found: 483.2725.2,3,22,23-Tetraphenyl-6,7,8,9,11,12,13,14,16,17,18,19,26,27,28,29,31,32,33,34,36,37,38,39-tetracosahydro-tetraoxatetraazacyclodotriacontinodiquinoxaline (6d) obtained as a by-product in the synthesis of 5a. Eluent CH2Cl2/MeOH 50:1. Yield 10 mg (7%), yellow-brown oil. 1H-NMR  δH 1.77 , 2.07 , 3.40–3.43 ), 3.51 ), 3.67 ), 4.92 , 7.09 , 7.27–7.29 ), 7.45–7.47 ). 13C-NMR  δC 26.7 , 28.5 , 43.8 , 71.0 , 71.3 , 103.7 ), 127.6 ), 128.0 ), 129.8 ), 138.5 ), 140.2 ), 142.3 ), 148.5 ). HRMS (MALDI-TOF): m/z [M + K]+ calcd for C60H68N8O4K+: 1003.4995; found: 1003.4983.2,3-Diphenyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-tetraazacyclotetradecinoquinoxaline (5e) obtained according to General Procedure from 6,7-dibromo-2,3-diphenilquinoxaline (132 mg), amine 4e (78 mg) in the presence of Pd(dba)2 (14 mg), JosiPhos (15 mg) and tBuONa (87 mg) in dioxane (15 mL) in 48 h. Eluent CH2Cl2/MeOH 10:1. Yield 105 mg (77%), brown glassy compound. 1H-NMR  δH 2.06 , 2.97-3.07 ), 3.45 ), 7.01 ), 7.26 ), 7.40-7.42 ). 4 NH protons were not unambiguously assigned because of exchange with MeOD. 13C-NMR  δC 24.8 , 44.3 , 46.3 , 49.7 , 103.9 ), 127.9 ), 129,6 ), 138.3 ), 139.3 ), 142.1 ), 148.7 ). IR (neat): 668 (w), 698 (s), 750 (s), 759 (s), 765 (vs), 806 (w), 834 (w), 851 (w), 917 (w), 979 (w), 1001 (w), 1025 (m), 1066 (m), 1078 (m), 1126 (m), 1157 (w), 1200 (s), 1226 (m), 1261 (m), 1267 (m), 1274 (m), 1355 (s), 1459 (s), 1494 (s), 1526 (s), 1563 (m), 1599 (m), 1619 (m), 2846 (m), 2933 (m), 2952 (m), 3054 (m), 3243 (m), 3350 (m) cm−1. HRMS (MALDI-TOF): m/z [M + Na]+ calcd for C28H32N6Na+: 475.2581; found: 475.2589.2,3-Diphenyl-7,8,9,10,11,12,13,14,15,16-decahydro-6H-tetraazacyclotridecinoquinoxaline (5f) obtained according to General Procedure from 6,7-dibromo-2,3-diphenilquinoxaline (132 mg), amine 4f (72 mg) in the presence of Pd(dba)2 (14 mg), JosiPhos (15 mg) and tBuONa (87 mg) in dioxane (15 mL) in 48 h. Eluent CH2Cl2/MeOH 10:1. Yield 73 mg (56%), yellow-brown glassy compound. 1H-NMR  δH 1.92 , 2.86–2.95 ), 3.87 ), 5.70 , 7.27–7.30  H3, H4, H5 (Ph)), 7.42 ). 2 NH-protons were not unambiguously assigned because of exchange with MeOD. 13C-NMR  δC 24.5 , 41.4 , 45.2 , 49.5 , 108.5 ), 127.8 ), 127.9 ), 129,4 ), 137.8 ), 139.1 ), 141.0 ), 149.6 ). IR (neat): 612 (w), 624 (vw), 693 (vs), 699 (vs), 731 (m), 751 (m), 759 (m), 764 (s), 789 (vw), 810 (w), 852 (w), 904 (vw), 917 (w), 955 (vw), 979 (m), 1001 (w), 1024 (m), 1063 (s), 1077 (m), 1113 (m), 1155 (m), 1195 (s), 1217 (s), 1231 (s), 1262 (m), 1350 (s), 1377 (w), 1470 (s), 1494 (m), 1511 (m), 1531 (m), 1577 (w), 1597 (m), 1618 (m), 2846 (m), 2923 (m), 2952 (m), 3056 (m), 3334 (m) cm−1. HRMS (MALDI-TOF): m/z [M + H]+ calcd for C27H31N6+: 439.2605; found: 439.2629.2,3-Diphenyl-7,8,9,10,11,12,13,14,15,16,17,18-dodecahydro-6H-tetraazacyclopenta decinoquinoxaline (5g) obtained according to General Procedure from 6,7-dibromo-2,3-diphenilquinoxaline (132 mg), amine 4g (85 mg) in the presence of Pd(dba)2 (14 mg), JosiPhos (15 mg) and tBuONa (87 mg) in dioxane (15 mL) in 48 h. Eluent CH2Cl2/MeOH 10:1. Yield 71 mg (50%), yellow-brown glassy compound. 1H-NMR  δH 2.00–2.06 , 3.01 ), 3.04–3.08 ), 3.37–3.39 ), 6.85 ), 7.04–7.07 ), 7.12–7.15 ). 4NH-protons were not unambiguously assigned because of exchange with MeOD and overlapping. 13C-NMR  δC 23.3 , 24.0 , 42.3 , 46.7 , 49.7 , 104.2 ), 127.8 ), 127.9 ), 129,6 ), 138.2 ), 139.4 ), 140.9 ), 148.9 ). IR (neat): 649 (w), 659 (w), 663 (w), 668 (s), 674 (w), 679 (w), 698 (m), 721 (vw), 734 (w), 766 (w), 792 (vw), 816 (w), 834 (w), 845 (vw), 924 (vw), 985 (w), 1001 (w), 1025 (w), 1082 (m), 1097 (m), 1118 (m), 1179 (s), 1217 (s), 1255 (m), 1268 (m), 1288 (m), 1332 (m), 1354 (w), 1363 (w), 1395 (m), 1405 (m), 1419 (m), 1430 (m), 1437 (m), 1448 (m), 1457 (m), 1465 (m), 1472 (m), 1491 (m), 1496 (m), 1507 (m),1521 (w), 1540 (m), 1559 (w), 1576 (m), 1606 (m), 1617 (m), 1653 (m), 1684 (w), 1700 (m), 1718 (m), 1730 (s), 1734 (s), 1740 (s), 1743 (s), 2926 (m), 2951 (m), 3054 (m), 3296 (m) cm−1. HRMS (MALDI-TOF): m/z [M + H]+ calcd for C29H35N6+: 467.2918; found: 467.2947.2,3-Diphenyl-7,8,9,10,11,12,13,14,15,16,17,18-dodecahydro-6H-pentaazacyclo pentadecinoquinoxaline (5h) obtained according to General Procedure from 6,7-dibromo-2,3-diphenilquinoxaline (132 mg), amine 4h (85 mg) in the presence of Pd(dba)2 (14 mg), JosiPhos (15 mg) and tBuONa (87 mg) in dioxane (15 mL) in 48 h. Eluent CH2Cl2/MeOH 10:1. Yield 30 mg (21%), yellow-brown glassy compound. 1H-NMR  δH 2.78 ), 3.05 ), 3.23 ), 3.63 ), 5.57 , 6.84 ), 7.11–7.19 ). 3 NH-protons were not unambiguously assigned because of exchange with MeOD and overlapping. 13C-NMR  δC 38.9 , 44.6 , 45.1 , 46.7 , 103.7 , 127.8 ), 129,5 ), 138.2 ), 139.3 ), 139.8 ), 149.1 ). IR (neat): 611 (m), 618 (m), 651 (m), 669 (m), 698 (vs), 732 (s), 777 (m), 807 (m), 835 (m), 919 (w), 978 (m), 1024 (m), 1065 (s), 1131 (m), 1157 (m), 1199 (s), 1242 (s), 1264 (m), 1288 (m), 1355 (s), 1456 (s), 1473 (s), 1481 (s), 1496 (s), 1507 (m), 1521 (m), 1535 (m), 1539 (s), 1559 (m), 1576 (m), 1599 (m), 1617 (m), 2750 (m), 2853 (m), 2928 (m), 2958 (m), 3054 (m), 3313 (m), 3329 (m) cm−1. HRMS (MALDI-TOF): m/z [M + Na]+ calcd for C28H33N7Na+: 490.2690; found: 490.2689.NMR spectra of the compounds are given in the 5c and 5e were performed at room temperature. The solutions were prepared with double-deionized high-purity water (18.2 MΩ cm) obtained from a «Millipore Simplicity» apparatus . Solution concentrations and other experiment conditions are given in the title of the corresponding figures and tables. Protonation studies were conducted in a glass cell equipped with a magnetic stirrer and pH-electrode, adding HClO4 (4 M or 0.01 M) or NaOH (5 M or 0.01 M) to the solutions of ligands . The pKa, spectra of the species and speciation diagrams were calculated using nonlinear least-squares analysis by means of the HypSpec program [Protonation studies of compounds  program  after fa program . The goo3 and 5a–d were performed at room temperature in acetonitrile. Metal-binding experiments were conducted by manual addition of the aliquots of metal salt solutions in acetonitrile by a Hamilton syringe to a solution of ligand placed in a quartz cuvette. All metal salts used were perchlorates of the general M(ClO4)n·xH2O formula. Caution! Although no problems were experienced, perchlorate salts are potentially explosive when combined with organic ligands and should be manipulated with care and used only in very small quantities. 18 metal ions  were tested. Solutions of metal perchlorates were prepared with concentrations of about 1000-fold that of the ligands in order to decrease the influence of the medium changes on the spectra of the studied solutions. Stability constants, spectra of the species and speciation diagrams were calculated using nonlinear least-squares analysis by means of the HypSpec program [Complexation studies of compounds  program  after fa program . The goo5a–h comprising 6,7-diamino-2,3-diphenylquinoxaline moiety in yields up to 77% using Pd-catalyzed amination reaction. Compounds with oxadiamino chains can be obtained using a classical Pd(dba)2/BINAP catalytic system, whereas macrocycles with tetraamino chains demand the application of an advanced Pd(dba)2/JosiPhos system. Compounds thus obtained possess strong luminescence in various solvents, which can be detected by a naked eye. Macrocycles 5a–d were tested for metal cations detection in acetonitrile but did not demonstrate selectivity. Thus, one needs to further modify the receptor unit to achieve this goal. Protonation studies of the macrocycles 5c and 5e were carried out using spectrophotometric and spectrofluorimetric titrations, and their protonation constants were calculated. Significant changes in their absorption and emission spectra at different pH, including a possibility of a naked-eye inspection, make them attractive dual channel indicators working in acidic media. The fabrication of thin films employing the compounds obtained in this work and their derivatives will be the next important step in the application of the sensing abilities of the quinoxaline-based detectors.To sum up, we elaborated an approach to polyoxadiaza- and polyazamacrocycles


Psychotria succulenta Hiern. (Rubiaceae), a Cameroonian medicinal plant traditionally used to cure microbial infections. The ethanol extract was prepared by maceration and extracted with ethyl acetate and n-butanol. The EtOAc (m = 168 g) and n-BuOH (m = 20 g) extracts were further fractionated by silica gel column chromatography to isolation of compounds. Their structures were elucidated by spectroscopic analysis and by comparison with published data. The antibacterial activity of extracts and compounds was assessed by evaluating the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against pathogenic bacteria. Thirteen compounds including four alkaloids (veprisine (1), naucleofficine III (2), vepridimerine B (3), and vepridimerine C (4)), three triterpenes (barbinervic acid (5), 3-O-α-L-rhamnopyranosyl quinovic acid (6), and oleanolic acid (7)), one steroid (β-sitosterol-3-O-β-D-glucopyranoside (8)), four phenolic compounds (scopoletin (9), gallic acid (10), quercetin-3-O-β-D-glucopyranoside (11), and kaempferol 3-O-α-L-rhamnopyranoside-7-O-α-L-rhamnopyranoside (12)), and one iridoid (borreriagenin (13)) were isolated from the EtOAc and n-BuOH extracts. These compounds were identified by 1D and 2D NMR combined analysis as well as by melting point comparison. The EtOH, EtOAc, and n-BuOH extracts exhibited significant antibacterial activities  against Staphylococcus aureus (Gram-positive bacterium), Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumonia (Gram-negative bacteria). Among the isolated compounds, scopoletin (9) showed a moderate activity against Klebsiella pneumoniae with MIC and MBC values of 16 μg/mL and 32 μg/mL, respectively. It appears that, chemotaxonomically, some of the isolated compounds have already been obtained from the genus Psychotria but to the best of our knowledge, this is the first report on the phytochemical investigation of P. succulenta. Although many other studies need to be achieved, our results support the use of P. succulenta in traditional medicine to cure infectious diseases particularly those caused by the tested bacteria.The use of natural products for medicinal purposes is becoming more and more common nowadays, as evidenced by the presence in plants of secondary metabolites with different potentials such as antioxidant and antibacterial properties. We evaluated in this work the antimicrobial activities of the extracts and some isolated compounds from the seeds of  Psychotria succulenta Hiern. (Rubiaceae) is a shrub of varying size between 1 and 2 m with yellow fruits of ripeness commonly found in most tropical regions [Psychotria  are commonly used in traditional medicine for treating bronchial and gastrointestinal disorders [Infectious diseases caused by bacteria, viruses, fungi, and other parasites continue to cause enormous damage in the world. Gram-negative or Gram-positive bacteria are able to acquire resistance mechanisms to face environmental aggression  either by the modification of sites of action of anti-infective molecules or by production of degradative enzymes. It is therefore important to develop new drugs using natural plants to fight antibiotic resistance and to limit undesirable side effects. isorders .P. succulenta. Previous works carried out on Psychotria species have shown that the different extracts , the fractions, and some isolated compounds exhibited interesting biological activities such as antibacterial, cytotoxic, antioxidant, antimycobacterial, and antimutagenic properties [P. succulenta, leading to the isolation and structure elucidation of thirteen compounds. Furthermore, the crude EtOH extract, the EtOAc and n-BuOH extracts as well as some of the isolated secondary metabolites were evaluated for their antibacterial activity, and the results are also presented.To the best of our knowledge, no phytochemical nor pharmacological works have been achieved on operties –7. Plantoperties . In the operties , 10, we P. succulenta were collected in October 2018 in Foreke-Dschang , West Region of Cameroon, and identified at the National Herbarium of Cameroon by comparison to the voucher specimen deposited under the reference no. 42155/HNC.The seeds of P. succulenta were dried at room temperature and then crushed in fine powder to give 2.8 kg. Two kilograms (2 kg) of this powder was extracted with ethanol (3 × 12 L) for 72 hours to yield 287.2 g of crude ethanol extract after evaporation of the solvent under reduced pressure. A part of this crude extract (250 g) was suspended in distilled water (600 mL), then extracted with EtOAc and n-BuOH, respectively. After evaporation of each solvent under reduced pressure, 168 g and 20 g of EtOAc and n-BuOH extracts were obtained, respectively.The seeds of n-hexane-EtOAc (95 : 5 → 20 : 80), then with EtOAc-MeOH (95 : 5 → 80 : 20) to yield five major fractions A-E. Fraction B (18 g) was purified by column chromatography on silica gel eluted with n-hexane-EtOAc (80-20) to yield compounds 1 (9 mg), 5 (25 mg), 6 (27 mg), and 7 (20 mg). Fraction C (26 g) was subjected to a silica gel column chromatography eluted with n-hexane-EtOAc (30-70) followed by Sephadex LH-20 (Eluted with MeOH) to afford compound 9 (35 mg). Treatment of fraction D (40 g) with column chromatography on silica gel by using n-hexane-EtOAc (50-50) as eluent gave compounds 2 (18 mg), 3 (28 mg), and 4 (23 mg). Fraction E (8 g) was chromatographed on Sephadex LH-20 gel column using MeOH as eluent followed by column chromatography on silica gel eluted by EtOAc-MeOH (95-5) to give compounds 8 (33 mg), 10 (22 mg), and 11 (11 mg). An amount of the n-BuOH fraction (17 g) was first chromatographed on Sephadex column eluted with MeOH to yield two subfractions coded F and G. The subfraction G was submitted to a silica gel column eluted with EtOAc-MeOH-H2O (9-1-1) to give compounds 12 (21 mg) and 13 (15 mg).One hundred and sixty grams (160 g) of the EtOAc extract was subjected to silica gel column chromatography eluted with 254 (Merck) plates developed with different solvents and mixture of hexane-EtOAc, EtOAc-MeOH, MeOH-H2O, and EtOAc-MeOH-H2O. Detection was done by using UV light (254 and 365 nm) and by spraying with 10% H2SO4 followed by heating at 100°C.Column chromatography was carried out on Merck silica gel 60 (70–230 mesh) and gel permeation on Sephadex LH-20, while TLC was carried out on precoated silica gel 60 F1H and 13C-NMR spectra were recorded on a Bruker Avance III 500 spectrometer equipped with a cryoplatform (1H at 500 MHz and 13C at 125 MHz). 2D NMR experiments were achieved using standard Bruker microprograms (Xwin-NMR version 2.1 software). All chemical shifts (δ) are given in parts per million (ppm) with the solvent signal as reference relative to TMS as internal standard, while the coupling constants (J) are given in hertz (Hz). Deuterated solvents such as methanol (methanol-4d), dimethyl sulfoxide (DMSO-d6), and chloroform (CDCl3) were used as solvents.The Staphylococcus aureus ATCC 25923 (Gram-positive bacterium), Pseudomonas aeruginosa ATCC 76110, Escherichia coli ATCC 25922, and Klebsiella pneumonia 22 (Gram-negative bacteria). These microorganisms were taken from the Research Unit of Microbiology and Antimicrobial Substances. The different bacterial species were maintained at +4°C and activated on BBL® nutrient agar  for 24 h before any antibacterial testing.The extracts and some isolated compounds were tested for their antibacterial activities against four bacterial strains, namely, 8 CFU/mL. This suspension was again diluted to 1/100 and adjusted to obtain an absorbance of 0.100 at 600 nm corresponding to a bacterial concentration of 106 CFU/mL. Microtiter plates (96 microwells) were made, and each well received 85 μL of Mueller Hinton broth and 5 μL of inoculum. 10 μL of test sample stock solution at a corresponding concentration was then added to each well to reach final concentrations ranging from 0.25 to 256 μg/mL. The positive control was made with the appropriate liquid medium and bacterial suspension only while the negative control was made with 10% DMSO aqueous solution in place of the inoculum. Ciprofloxacin was used as reference antibiotic. The plates were covered and incubated under agitation at 35°C for 24 h. Bacterial growth was determined by introducing 5 μL of a 0.2 mg/mL para-iodonitrotetrazolium solution. Any change in colour from yellow to violet indicates bacterial growth. The minimum inhibitory concentration was defined as the smallest concentration of the substance that prevents this colour change. 10 μL of the contents of each well were aseptically collected and spread separately on the surface of Mueller Hinton agar medium for the purpose of determining the minimum bactericidal concentrations (MBC), which are defined as the smallest concentrations that result in a negative subculture or only one colony. Three replicates were performed for each test sample.The determination of the minimum inhibitory concentration (MIC) was performed using the broth microdilution method . Bacterin-BuOH extracts from the EtOH crude extract of P. succulenta were subjected to repeated column chromatography on silica gel and Sephadex LH-20 to yield thirteen metabolites (1-13). Their structures were elucidated by spectroscopic and spectrometric analysis as well as by comparison with literature data  [2) [3) [4) [5) [β-O-α-L-rhamnoside (6) [9) [10) [O-β-D-glucopyranoside (11) [O-α-L-rhamnopyranoside-7-O-α-L-rhamnopyranoside (12) [13) [7 and 8 were identified by co-TLC with authentic samples and melting point measurement as oleanolic acid [β-sitosterol 3-O-β-D-glucopyranoside [The EtOAc and lable 1) , naucleoolites 1-. Their s [2) [3) , vepridi [3) [4) , barbine [4) [5) , quinoviside (6) , scopole (6) [9) , gallic [9) [10) , quercetide (11) , kaempfe12) [13) . Compounlic acid  and β-siranoside , respectVeprisin (1): yellow amorphous powder; 1H-NMR : δ (ppm) = 7.75 , 7.09 , 6.61 , 5.61 , 3.98 , 3.91 , 3.80 , and 1.51 . 13C-NMR : δ (ppm) = 162.8 (C-1), 156.1(C-7), 155.8 (C-3), 137.0 (C-8), 133.8 (C-9), 125.6 (C-3′), 118.8 (C-5), 116.7 (C-4′), 111.5 (C-4), 108.1 (C-6), 102.9 (C-2), 78.6 (C-3′), 60.4 (OCH3), 54.6 (OCH3), 33.2 (NCH3), and 26.1 (CH3)Naucleofficine III (2): white amorphous powder; 1H-NMR  δ (ppm) = 7.53 , 7.37 , 7.23 , 7.17 , 5.54 , 5.28 , 5.17 , 5.04 , 4.32 , 4.20 , 3.04 , 3.02 , 2.77 , 2.74 , 2.58 , 2.56 , 2.10 , and 1.62 . 13C-NMR : δ (ppm) = 169.1 (C-22), 136.0 (C-13), 133.4 (C-20), 133.0 (C-2), 127.4 (C-8), 122.4 (C-11), 122.2 (C-19), 120.2 (C-10), 118.4 (C-9), 111.1 (C-12), 111.0 (C-7), 93.8 (C-17), 68.0 (C-21), 53.0 (C-3), 48.6 (C-16), 42.0 (C-5), 29.2 (C-15), 28.0 (C-14), 21.0 (C-6), and 12.7 (C-18)Vepridimerine C (3): white amorphous powder; 1H-NMR : δ (ppm) = 8.08 , 7.71 , 6.96 , 6.84 , 3.95 , 3.90 , 3.77 , 3.74 , 3.92 , 3.76 , 3.96 , 3.20 , 2.74 , 2.15 , 1.91 ), 1.72 ), 1.60 , 1.48 , 1.34 , and 1.41 ). 13C-NMR : δ (ppm) = 176.5 (C-17), 163.4 (C-8), 157.7(C-4b), 155.4 (C-2), 155.0 (C-13b), 154.9 (C-10), 137.6 (C-11), 136.7 (C-1), 134.8 (C-18a), 134.1 (C-9a), 122.0 (C-4), 118.8 (C-13), 121.4 (C-4a), 112.7 (C-13a), 112.1 (C-7a), 108.4 (C-3), 107.0 (C-12), 100.1 (C-16b), 84.7 (C-6), 78.5 (C-15), 61.7 (OCH3), 61.3 (OCH3), 56.3 (OCH3), 56.2 (OCH3), 52.3 (C-6a), 39.3 (C-19), 35.8 (NCH3), 33.8 (NCH3), 31.1 (C-16), 29.2 (15-CH3), 28.6 (6-CH3), 25.7 (C-16a), 25.6 (C-7), and 21.0 (6-CH3)Vepridimerine B (4): white amorphous powder; 1H-NMR : δ (ppm) = 7.70 , 7.66 , 6.88 , 6.84 , 3.98 , 3.95 , 3.84 , 3.76 , 3.93 , 3.87 , 3.84 , 3.24 , 2.62 , 2.17 , 1.92 , 1.71 , 1.59 , 1.47 , 1.32 , and 1.39 ). 13C-NMR : δ (ppm) = 164.3 (C-17), 163.3 (C-8), 155.8 (C-4b), 155.0 (C-1), 154.9 (C-10), 154.6 (C-13b), 136.8 (C-2), 136.5 (C-11), 134.2 (C-18a), 134.0 (C-9a), 119.0 (C-4), 118.8 (C-13), 112.8 (C-4a), 112.7 (C-13a), 112.2 (C-7a), 107.6 (C-3), 107.0 (C-12), 105.4 (C-16b), 81.9 (C-6), 78.5 (C-15), 61.7 (OCH3), 61.6 (OCH3), 56.3 (OCH3), 56.2 (OCH3), 33.8 (NCH3), 33.3 (NCH3), 52.3 (C-6a), 39.7 (C-19), 31.1 (C-16), 29.3 (15-CH3), 28.5 (6-CH3), 26.3 (C-16a), 25.5 (C-7), and 21.0 (6-CH3)Barbinervic acid (5): brown powder; 1H-NMR : δ (ppm) = 5.30 , 3.62 , 3.55 , 3.32 , 2.52 , 1.35 , 1.21 , 0.99 , 0.94 , 0.81 , and 0.72 . 13C-NMR : δ (ppm) = 180.8 (C-28), 138.7 (C-13), 128.1 (C-12), 72.6 (C-3), 72.2 (C-19), 66.1 (C-24), 53.8 (C-18), 45.5 (C-17), 47.4 (C-5), 47.1 (C-9), 41.8 (C-4), 41.6 (C-20), 41.2 (C-14), 39.6 (C-8), 38.1 (C-1), 37.6 (C-22), 36.5 (C-10), 32.3 (C-7), 28.1 (C-15), 26.0 (C-2), 25.9 (C-21), 25.7 (C-29), 24.9 (C-16), 23.4 (C-27), 23.2 (C-11), 17.9 (C-6), 16.1 (C-26), 15.1 (C-30), 14.8 (C-25), and 11.3 (C-23)Quinovic acid 3β-O-α-L-rhamnopyranoside (6): brown powder; 1H-NMR : δ (ppm) = 5.61 , 3.07 , 2.28 , 1.00 , 0.94 , 0.92 , 0.91 , 0.81 , and 0.78 . L-rhamnose: 4.75 , 3.84 , 3.65 , 3.35 , 3.71 , and 1.20 . 13C-NMR : δ (ppm) = 180.1 (C-28), 177.5 (C-27), 132.5 (C-13), 129.0 (C-12), 103.0 (C-1′), 88.9 (C-3), 72.6 (C-4′), 71.4 (C-3′), 71.1 (C-2′), 68.4 (C-5′), 55.8 (C-14), 55.3 (C-5), 54.1 (C-18), 47.9 (C-17), 46.6 (C-9), 39.3 (C-8), 38.9 (C-19), 38.6 (C-4), 38.4 (C-1), 36.8 , 36.4 (C-22), 36.2 (C-7), 29.8 (C-21), 27.3 (C-24), 25.3 , 25.1 (C-16), 22.4 (C-11), 20.1 (C-30), 18.0 (C-6), 16.6 , 17.6 (C-6′).15.4 (C-23), and 15.4 (C-25)Scopoletin (9): yellow needle; 1H-NMR : δ (ppm) = 7.80 , 7.12 , 6.80 , 6.21 , and 3.91 . 13C-NMR : δ (ppm) = 162.9 (C-2), 151.4 (C-8a), 150.0 (C-7), 145.6 (C-6), 144.7 (C-4), 111.3 (C-4a), 111.2 (C-3), 108.4 (C-5), 102.7 (C-8), and 55.6 (O-CH3)Gallic acid (10): white amorphous powder; 1H-NMR : δ (ppm) = 7.07 . 13C-NMR : δ (ppm) = 168.9 (C = 0), 145.3 (C-3), 138.2 (C-4), 120.5 (C-1), and 108.8 (C-2/C-6)Quercetin 3-O-β-D-glucopyranoside (11): yellow amorphous powder; 1H-NMR : δ (ppm) = 7.72 ,7.61 , 6.89 , 6.43 , 6.23 , 5.27 , 3.72 , 3.58 , 3.50 , 3.44 , 3.37 , and 3.28 . 13C-NMR : δ (ppm) = 179.1 (C-4), 165.7 (C-7), 162.7 (C-5), 161.5 (C-2), 158.5 (C-9), 149.2 (C-4′), 145.6 (C-3′), 135.3 (C-3), 122.4 (C-1′), 122.8 (C-6′), 116.9 (C-2′), 116.5 (C-5′), 105.3 (C-10), 103.8 (C-1″), 99.3 (C-6), 94.2 (C-8), 78.0 (C-5″), 77.6 (C-3″), 62.3 (C-6″), 75.2 (C-2″), and 70.7 (C-4″)Kaempferol 3-O-α-L-rhamnopyranoside-7-O-α-L-rhamnopyranoside (12): yellow amorphous powder; 1H-NMR : δ (ppm) = 7.77 , 6.94 , 6.70 , 6.45 , 5.54 , 5.40 , 4.26 , 4.05 , 3.86 , 3.74 , 3.62 , 3.50 , 3.34 , 3.33 , 1.28 , and 0.94 . 13C-NMR : δ (ppm) = 178.5 (C-4), 162.0 (C-7), 161.5 (C-5), 160.1 (C-4′), 158.5 (C-2), 156.8 (C-9), 135.2 (C-3), 130.7 (C-2′/C-6′), 121.1 (C-1′), 115.4 (C-3′/C-5′), 106.5 (C-10), 102.0 (C-1″), 99.5 (C-6), 98.3 (C-1‴), 94.4 (C-8), 72.3 (C-4′″), 71.9 (C-4″), 70.8 (C-3‴), 70.4 (C-2″), 70.6 (C-4‴), 70.2 (C-2″′), 70.6 (C-5″), 69.8 (C-5′″), 17.1 (C-6″′), and 16.6 (C-6″)Borreriagenin (13): yellow oil; 1H-NMR : δ (ppm) = 5.85 , 5.41 , 4.20 , 3.92 , 3.87 , 3.77 , 3.59 , 3.52 , 3.34 , 3.11 , and 2.97 . 13C-NMR : δ (ppm) = 179.6 (C-11), 151.9 (C-8), 123.6 (C-7), 86.7 (C-6), 63.0 (C-1), 61.4 (C-3), 53.9 (C-10), 48.6 (C-9), 44.6 (C-4), and 42.6 (C-5)in vitro activities of the EtOH, n-BuOH, and EtOAc extracts as well as some isolated compounds against pathogenic bacteria are presented in n-BuOH and EtOAc extracts showed antibacterial activity against Gram-positive and Gram-negative bacteria  whereas the EtOH extract was active only on Gram-negative bacteria . The antibacterial activity of the plant extracts can be classified as significant (MIC < 100 μg/mL), moderate (100 < MIC ≤ 625 μg/mL), and weak (MIC > 625 μg/mL) [n-BuOH extract was the most active with a lowest MIC value of 32 μg/mL against Pseudomonas aeruginosa ATCC 76110, Staphylococcus aureus ATCC 25923, and Klebsiella pneumoniae 22 and of 64 μg/mL against Escherichia coli ATCC 25922 followed by the EtOH extract which displayed a MIC value of 32 μg/mL on K. pneumoniae 22 and of 64 μg/mL on P. aeruginosa ATCC 76110, S. aureus ATCC 25923, and K. pneumoniae 22. The isolated secondary metabolites showed inhibition ranging from moderate to weak according to the scale which states that antimicrobial activity of pure compounds can be classified as significant (MIC < 10 μg/mL), moderate (10 < MIC ≤ 100 μg/mL), and weak (MIC > 100 μg/mL) [9) exhibited a moderate activity with a MIC value of 16 μg/mL against S. aureus ATCC 25923, K. pneumoniae 22, and a MIC value of 32 μg/mL against P. aeruginosa ATCC 76110 while barbinervic acid (5) showed a moderate activity against P. aeruginosa ATCC 76110 and K. pneumoniae 22 with a MIC value of 32 μg/mL.The results of 5 μg/mL) . Accordi0 μg/mL) . ScopoleP. succulenta which led to the isolation and structure elucidation of thirteen compounds including four alkaloids (veprisine (1), naucleofficine III (2), vepridimerine B (3), and vepridimerine C (4)), three triterpenes (barbinervic acid (5), 3-O-α-L-rhamnopyranosyl quinovic acid (6), and oleanolic acid (7)), one steroid (β-sitosterol-3-O-β-D-glucopyranoside (8)), four phenolic compounds (scopoletin (9), gallic acid (10), quercetin-3-O-β-glucopyranoside (11), and kaempferol 3-O-α-L-rhamnopyranoside-7-O-α-L-rhamnopyranoside (12)), and one iridoid (borreriagenin (13)). Although all these compounds are isolated from P. succulenta for the first time, some of them have already been obtained from other Psychotria species. It is the case of barbinervic acid (5) previously found in P. stachyoides [7) and β-sitosterol-3-O-β-D-glucopyranoside (8) isolated from P. viridis [9) obtained from P. vellosiana and P. stachyoides [Psychotria since according to Calixto et al. (2016), they are characterized as an abundant source of indole, monoterpene indole, quinoline, and isoquinoline alkaloids as well as flavonoids. Furthermore, approximately 52% of the metabolites reported were characterized as alkaloids, followed by triterpenes (12%) and flavonoids (6%) along with constituents of other classes [2) isolated during our investigation has already been obtained from the stems of Nauclea officinalis [P. succulenta of a metabolite belonging to this class of compounds reported to derive biosynthetically from the coupling of tryptophan, and the iridoid seccolaganin is not surprising since many congeners have been isolated from other Psychotria species [The present study reports the first phytochemical investigation of chyoides , oleanol viridis , and scochyoides , 27. The classes . The monriagenin ). Althou species –30. Furt species .1), vepridimerine B (3), and vepridimerine C (4) are quinolone-terpene alkaloids occurring mainly in plants of the Rutaceae family [Araliopsis soyauxii (Rutaceae) [Melodinus yunnanensis (Apocynaceae) [Alstonia scholaris (Apocynaceae) [Clausena lansium (Rutaceae) [O-β-glucopyranoside (11) and kaempferol 3-O-α-L-rhamnopyranoside-7-O-α-L-rhamnopyranoside (12) from a plant of the Psychotria genus although these secondary metabolites have already been found in the family Rubiaceae, precisely in Hedyotis diffusa and Hedyotis verticillata, respectively [13) obtained during this work has already been isolated from some plants of the Rubiaceae family including Borreria verticillata [Morinda longifolia [Psychotria.Veprisine  , Alstoniynaceae) , and Clautaceae) . This fuutaceae) . This seectively . The iriicillata  and Moringifolia , but to P. succulenta seeds. This suggests that P. succulenta contains several active principles with different polarities as shown by the nature of the isolated compounds. Indeed, the antibacterial activities of medicinal plants are correlated with the presence in their extracts of one or more bioactive secondary metabolites [P. succulenta contains also polar antibacterial compounds. These differences in antibacterial activities from different solvents had also been observed [2, 5, and 9) that are most active toward the tested bacteria from P. succulenta. The ethyl acetate is a semipolar solvent and could effectively extract active principles with semipolar properties such as alkaloids, sterols, terpenoids, flavonoids, and glycosides from the plant [The findings of the present study showed differences between the antibacterial activities of extracts from abolites . The n-Bobserved , 11. Henhe plant .Psychotria are commonly used in traditional medicines for treating bronchial and gastrointestinal disorders such as cough, bronchitis, ulcer, and stomachache [Psychotria species like P. microlabastra , P. gardineri (branches and leaves), and P. nigra (branches and leaves) have shown that methanol, dichloromethane, and hexane extracts exhibit antibacterial activities [P. succulenta in traditional medicine but also to approve the literature data.Different parts  of plants of the genus machache , 40. Themachache . Previoutivities . Our resP. succulenta seeds have a bactericidal effect on the sensitive bacteria [The findings of the present study showed that the MBC values are in general fourfold lesser than the MIC values on the corresponding bacteria; suggesting that the extracts and some isolated compounds from bacteria .P. succulenta seeds are in agreement with those of the literature. Indeed, veprisine isolated from the root wood of Teclea maniensis (Rutaceae) exhibited moderate to higher antimycobacterial activity against two mycobacterial strains, namely, Mycobacterium madagascariense DSM 44641 and Mycobacterium indicus pranii DSM 45239 with the MIC values of 657.9 μM and 2.63 × 103 μM, respectively [Miconia species displayed antibacterial effect with MIC values ranging from 30 μg/mL to 70 μg/mL [Lasianthus lucidus Blume (Rubiaceae) proved to be effective against Pseudomonas aeruginosa ATCC 27853 (AmpC β-lactamase producing strain) and P. aeruginosa DMSC 37166 [P. aeruginosa, E. coli, S. aureus, and Lysteria monocytogenes through hydrophobicity changes, decrease of negative surface charge, and occurrence of local rupture or pore formation in the cell membranes with consequent leakage of essential intracellular constituents [Hedyotis pilulifera (Rubiaceae) showed antibacterial activity against Staphylococcus aureus, with an MIC value of 100 μg/mL [O-α-L-rhamnopyranosyl quinovic acid, quercetin-3-O-β-D-glucopyranoside, kaempferol 3-O-α-L-rhamnopyranoside-7-O-α-L-rhamnopyranoside, and borreriagenin from P. succulenta seeds. The overall study emphasizes the potential of P. succulenta seeds as a sustainable source of broad spectrum antibacterial agents.The results of the antibacterial activity of some isolated compound from ectively . Oleanol70 μg/mL . A phenoP. succulenta led to isolation and characterization of thirteen compounds, namely, veprisine (1), naucleofficine III (2), vepridimerine B (3), vepridimerine C (4), barbinervic acid (5), quinovic acid 3β-O-α-L-rhamnoside (6), oleanolic acid (7), β-sitosterol-3-O-β-D-glucopyranoside (8), scopoletin (9), gallic acid (10), quercetin 3-O-β-D-glucopyranoside (11), kaempferol 3-O-α-L-rhamnopyranoside-7-O-α-L-rhamnopyranoside (12), and borreriagenin (13). All these secondary metabolites were isolated from this plant species for the first time although some of them have already been isolated from plants of the genus Psychotria. Our results clearly showed that P. succulenta has a close chemotaxonomic relationship with other plants of the genus Psychotria. Furthermore, the extracts and some isolated compounds showed antibacterial activity against pathogenic bacteria, confirming the use of P. succulenta in traditional medicine to cure infectious diseases. Barbinervic acid (5) and scopoletin (9) were the most antibacterial principles of P. succulenta.In conclusion, the phytochemical investigation of the seeds of


Their structures, including absolute configurations, were elucidated by analyses of NMR spectroscopic HRESIMS data and electronic circular dichroism (ECD) calculations. Compounds 5, 6, 9, and 14 exhibited weak cytotoxicities against four human cancer cell lines, with IC50 values ranging from 22.1 to 100 μM. Pestaloketide A (1) is an unusual polyketide, featuring a rare 5/10/5-fused ring system. Pestaloketides A (1) and B (2) exhibited moderately inhibited LPS-induced NO production activity, with IC50 values of 23.6 and 14.5 μM, respectively, without cytotoxicity observed. Preliminary bioactivity evaluations and molecular docking analysis indicated that pestaloketides A (1) and B (2) had the potential to be developed into anti-inflammatory activity drug leads.Five undescribed polyketide derivatives, pestaloketides A–E ( Analysis of 1H NMR and HSQC spectrum , 1.20, s, H3-12, 1.32, d , 0.97, d , 1.19, s, H3-17, and 1.29, d ), two oxygenated methines , 4.93, td ), and two methylenes . The 13C NMR and HSQC spectra of 1 exhibited 20 carbon signals, including six methyls, eight methines, two methylenes, and four oxygenated quaternary carbons. The above NMR data revealed the structure of 1 as a polyketide derivative, which was supported again by key HMBC correlations from H-19 (δH 2.88) to C-18 (δC 179.6), C-20 (δC 11.6), C-10 (δC 49.5), and C-9 (δC 44.0); and from H-14 (δH 2.70) to C-13 (δC 181.1), C-6 (δC 81.4), C-5 (δC 54.0), C-4 (δC 50.6), and C-15 (δC 18.3). The key HMBC correlations from H-2 (δH 2.16) to C-1 (δC 81.0), C-4 (δC 50.6), and C-17 (δC 23.8); and from H-7 (δH 2.02/2.21) to C-9 (δC 44.0), C-5 (δC 54.0), C-6 (δC 81.4), and C-12 (δC 23.8), the COSY correlations H-20/H3-19/H-10/H-9/H3-11/H-1/H-2 (fragment Ⅰ) and H3-16/H-4/H-5/H-14/H3-15/H-6/H-7 (fragment Ⅱ) -1 and -1, calculations of the ECD spectrum of 1 were performed. As a result, the calculated spectrum of -1 coincided well with its experimental data   indicateelations . The crotal data , suggest2 was yielded as a white solid. Its molecular formula C21H32O10 was established by 13C NMR data together with HRESIMS at m/z 443.1923, [M-H]-. Analysis of NMR spectra of 2 showed six methyl protons , one methylene protons , and one olefinic protons at δH 6.53 . The DEPT and 13C NMR spectra revealed 21 resonances including six methyl , one methylene (δC 45.7), six methines , seven nonprotonated carbons, one carbonyl group (δC 180.4), and two ester carbonyl groups . The key HMBC correlations from H-12 (δH 2.26) to C-13 (δC 43.9), C-18 (δC 14.2), C-11 (δC 80.2), C-16 (δC 74.4), and C-19 (δC 180.4); H-15  to C-20 (δC 22.1), C-13 (δC 43.9), and C-14 (δC 80.0); H-17 (δH 1.35) to C-16 (δC 74.4), and C-1′ (δC 179.5), together with 1H-1H COSY correlation information between H-18/H-12/H-13/H-11 established the fragment A (1H-1H COSY correlation information between H-10 (δH 1.30) and H-6 (δH 4.54), along with the key HMBC correlations from H-4 (δH 6.53) to C-8 (δC 165.9), C-5 (δC 133.7), C-2 (δC 94.1), C-7 (δC 21.0), and C-6 (δC 66.0); and H-2 (δH 4.80) to C-4 (δC 139.5), C-6 (δC 66.0), C-7 (δC 21.0) and C-11 (δC 80.2) established the fragment B. Therefore, the structure of 2 was assigned   . As a re3 was detected as yellow oil, giving the molecular formula of C12H16O5 from the analysis of their 13C NMR data and HRESIMS ) and three methyls . The 13C NMR and HSQC spectroscopic data of 3 exhibited resonances for one lactone carbon (δC 164.8 (C-6)) and three oxygenated carbons . Detailed analysis of these above data of 3 revealed that compound 3 was very similar to those of 12, 3-methyl-2-penten-5  in m/z 179 was attributed to [CH3(CH2)4(CH)2CO(CH)2(CH2)2] cations. These results assisted to reconfirm the configuration of compound 5.In order to further investigate the structure of compound 6–16 were determined to be the known 11-keto-9(E),12(E)-octadecadienoic acid (6) + .Pestaloketide B (2): white powder; − .Pestaloketide C (3): yellow oil; + .Pestaloketide D (4): yellow oil; + .Pestaloketide E (5): colorless oil; - .1–4 were chosen at the B3LYP/6-311+G level. The overall calculation of the ECD was performed using the TDDFT method for the stable conformers of new compounds. The spectra were obtained by SpecDis 1.6.The calculations were applied by the Spartan’14, Gaussian 09 software, and Merck Molecular Force Field (MMFF), respectively. The conformers of 1–16 was conducted according to the one described previously [The method for the assay of cytotoxicity activity of eviously . Positiv1–16 were examined by inhibited NO production in LPS-stimulated RAW. The detailed process of the assay is described in the previously published paper [The activity of compounds ed paper . Positivhttp://www.rcsb.org, accessed on 30 October 2022) [1) and B (2) were docked using Autodock vina [1 and 2 were generated by Grade Web Server . A grid box of a 48.02 Å × 42.58 Å × 33.75 Å size was centered on the catalytic site. All docking parameters were set to default values. The docking results were further analyzed and presented using PyMOL  and LigPlot+ [The three-dimensional structure of INOS (PDB ID:3E6T) was acquired from the Protein Data Bank (er 2022) ,26, for ock vina  and Autoock vina . The geo1–5), together with other eleven known natural products (6–16), were isolated from the fungus Pestalotiopsis sp. SWMU-WZ04-2. Pestaloketide A (1) is an unusual polyketide featuring a rare 5/10/5-fused ring system. Compounds 5, 6, 9, and 14 showed weak cytotoxicities against four human cancer cell lines (IC50: 22.1–100 μM). Other compounds were inactive (100 μM). Anti-inflammatory activities were performed for compounds 1–4, 7–8, 10–13, and 15–16, and Pestalolactones A (1) and B (2) showed moderate inhibitory of NO production with IC50 values of 23.6 and 14.5 μM, respectively, without cytotoxicity observed.” Although the detailed mechanism of action is still undefined for pestaloketides A (1) and B (2), molecular docking analysis showed that both compounds had the potential to be developed into anti-inflammatory activity drug leads.In summary, five new compounds (