Anthracycline antitumor antibiotics are one of the promising classes of chemotherapeutic agents for cancer treatment. The main deterrent to their use is high toxicity to a healthy environment, including cumulative cardiotoxicity. In our work, bipharmacophore molecules containing in their structure a fragment of the known anthracycline antibiotics daunorubicin and doxorubicin and natural sesquiterpene lactones were obtained for the first time. When studying the biological activity of the synthesized compounds, it was found that with equal and, in some cases, higher cytotoxicity and glycolysis inhibition by anthracycline antibiotics conjugates with sesquiterpene lactones in comparison with doxo- and daunorubicin, a reduced damaging effect on the functioning of rat heart mitochondria was observed. The results obtained allow us to confirm the assumption that the chemical modification of the anthracycline antibiotics molecules doxo- and daunorubicin by natural sesquiterpene lactones can be a promising strategy for creating potential antitumor chemotherapeutic drugs with a pronounced cytotoxic effect on tumor cells and a reduced damaging effect on healthy cells of the human organism. Anthracycline antibiotics have pronounced antitumor properties. The clinical use of representatives of this class as antineoplastic agents dates to the 60s of the last century. Notable examples are doxorubicin, which is an important component of therapy aimed at the treatment of various solid tumors, soft tissue sarcomas, and aggressive lymphomas, and daunorubicin, which is active against acute lymphoblastic and myeloblastic leukemias . Despite2+ homeostasis disruption triggered by anthracycline antibiotics causes the opening of the mitochondrial membrane permeability transition pore (mPTP) and a decrease in the transmembrane potential along with an increase in the permeability of the outer mitochondrial membrane for the release of apoptotic factors furan-3-yl)methyl)amino)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-8-(2-hydroxyacetyl)-1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione (7a). A red solid : m.p. = 170–175 °C; TLC Rf = 0.39 (CHCl3:MeOH = 10:1); HRMS (ESI): m/z = 792.3252 (M+H+). C42H49NO14. Calculated, m/z = [M+H]+ = 792.3226. 1H-NMR  0.86 , 0.99 , 1.29–1.40 , 1.36 , 1.69–1.87 , 1.93 , 1.93–2.00 , 2.14–2.19 , 2.16 , 2.22–2.33 , 2.37 , 2.55 , 2.62 , 2.81–2.96 , 3.03 , 3.26 , 3.65 , 3.84 , 4.00–4.12 , 4.08 , 4.60 , 4.76 , 5.32 , 5.52 , 5.62 , 7.39 , 7.78 , 8.02 , 13.24 , 13.95 ; 13C-NMR  16.4 (C-42), 17.3 (C-27), 19.4 (C-41), 25.7 (C-35), 26.9 (C-36), 30.3 (C-23), 32.7 (C-37), 34.1 (C-10), 35.3 (C-12), 35.6 (C-32), 42.0 (C-38), 42.9 (C-30), 45.8 (C-28), 52.8 (C-24), 56.8 (C-21), 65.6 (C-20), 66.0 (C-11), 67.2 , 69.6 (C-13), 74.4 (H-39), 77.4 (C-31), 101.0 (C-22), 111.6 (C-7), 111.8 (C-16), 118.6 (C-2), 120.0 (C-4), 121.0 (C-18), 128.1 (C-34), 135.0 (C-33), 135.6 (C-5), 135.9 (C-3), 155.8 (C-8), 156.3 (C-15), 161.2 (C-1), 178.0 (C-40), 186.9 (C-6), 187.2 (C-17), 213.8 (C-19).-10--4--5,8a-Dimethyl-2-oxo-2,3,3a,7,8,8a,9,9a-octahydronaphthofuran-3-yl)methyl)amino)-5-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-6,8,11-trihydroxy-8-(2-hydroxyacetyl)-1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione (8a). A red solid : m.p. = 166–170 °C; TLC Rf = 0.54 (CHCl3:MeOH = 10:1); HRMS (ESI): m/z = 774.3148 (M+H+). C42H47NO13. Calculated, m/z = [M+H]+ = 774.3120. 1H-NMR  1.03 ,1.32 , 1.37 , 1.50 , 1.51 , 1.71 , 1.76 , 2.01 , 2.16 , 2.18 , 2.25 , 2.37 , 2.82 , 2.87 , 2.95 , 2.98 , 3.00 , 3.24 , 3.25 , 3.65 , 4.0–4.09 , 4.07 , 4.75 , 4.77 , 5.21 , 5.31 , 5.52 , 5.58 , 7.39 , 7.77 , 8.01 , 13.22 , 13.24 ; 13C-NMR  17.3 (C-27), 20.3 (C-42), 22.3 (C-35), 24.8 (C-41), 30.6 (C-33), 30.9 (C-23), 34.1 (C-10), 35.6 (C-12), 37.3 (C-34), 38.3 (C-30), 39.6 (C-32), 42.6 (C-28), 45.8 (C-29), 52.4 (C-24), 56.8 (C-21), 65.6 (C-20), 67.2 (C-26), 67.4 (C-25), 69.7 (C-13), 76.8 (C-11), 77.4 (C-31), 101.2 (C-22), 111.6 (C-7), 111.7 (C-16), 113.0 (C-39), 118.6 (C-2), 120.0 (C-4), 121.0 (C-18), 126.9 (C-36), 130.4 (C-37), 133.6 (C-9), 134.0 (C-14), 135.6 (C-5), 135.9 (C-3), 145.0 (C-38), 155.8 (C-8), 156.3 (C-15), 161.2 (C-1), 177.8 (C-40), 186.9 (C-6), 187.2 (C-17), 213.8 (C-19).-8-Acetyl-6,8,11-trihydroxy-10--5-hydroxy-4--4-hydroxy-4a,5-dimethyl-2-oxo-2,3,3a,4,4a,5,6,7,9,9a-decahydronaphthofuran-3-yl)methyl)amino)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione (7b). A red solid : m.p. = 155–162 °C; TLC Rf = 0.78 (CHCl3:MeOH = 10:1); HRMS (ESI): m/z = 776.3291 (M+H+). C42H49NO13. Calculated, m/z = [M+H]+ = 776.3277. 1H-NMR  0.86 , 0.98 , 1.36 , 1.29–1.42 , 1.69–1.77 , 1.90–1.99 , 1.93 , 2.06–2.12 , 2.10 , 2.23–2.32 , 2.35 , 2.42 , 2.53 , 2.66 , 2.86 , 2.91 , 2.96 , 3.22 , 3.65 , 3.83 , 4.03–4.10 , 4.08 , 4.60 , 5.29 , 5.50 , 5.62 , 7.38 , 7.78 , 8.02 , 13.27 , 13.96 ; 13C-NMR  16.4 (C-42), 17.2 (C-27), 19.4 (C-41), 24.9 (C-20), 25.6 (C-35), 26.9 (C-36), 30.4 (C-23), 32.7 (C-32), 33.5 (C-10), 35.0 (C-37), 35.3 (C-12), 42.0 (C-30), 42.9 (C-38), 45.7 (C-28), 45.8 (C-29), 52.8 (C-24), 56.8 (C-21), 67.1 (C-26), 67.3 (C-25), 69.8 (C-13), 74.4 (C-39), 77.0 (C-31), 77.1 (C-11), 100.9 (C-22), 111.4 (C-7), 111.6 (C-16), 118.5 (C-2), 119.9 (C-4), 121.1 (C-18), 128.0 (C-34), 134.4 (C-9), 134.5 (C-14), 135.0 (C-33), 135.7 (C-5), 135.9 (C-3), 156.0 (C-8), 156.5 (C-15), 161.2 (C-1), 178.0 (C-40), 186.9 (C-6), 187.2 (C-17), 211.9 (C-19).-8-Acetyl-10--4--5,8a-dimethyl-2-oxo-2,3,3a,7,8,8a,9,9a-octahydronaphthofuran-3-yl)methyl)amino)-5-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-6,8,11-trihydroxy-1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione (8b). A red solid : m.p. = 152–158 °C; TLC Rf = 0.81 (CHCl3:MeOH = 10:1); HRMS (ESI): m/z = 758.3197 (M+H+). C42H47NO12. Calculated, m/z = [M+H]+ = 758.3171. 1H-NMR  1.03 , 1.36 , 1.32 , 1.50 , 1.71 , 1.75 , 2.00 , 2.10 , 2.18 , 2.25 , 2.35 , 2.41 , 2.82 , 2.89 , 2.94 , 2.93, 2.97 , 3.20 , 3.24 , 3.64 , 4.05–4.10  H-26, 4.07 , 4.75 , 5.20 , 5.28 , 5.52 , 5.57 , 7.38 , 7.77 , 8.01 , 13.26 , 13.94 ; 13C-NMR  17.2 (C-27), 20.3 (C-42), 22.3 (C-35), 24.8 (C-41), 24.9 (C-20), 30.5 (C-33), 30.9 (C-23), 33.5 (C-10), 35.0 (C-12), 37.3 (C-34), 38.4 (C-30), 39.6 (C-32), 42.5 (C-28), 45.9 (C-29), 52.4 (C-24), 56.8 (C-21), 67.0 (C-26), 67.5 (C-25), 70.0 (C-13), 77.0 (C-11), 77.3 (C-31), 101.2 (C-22), 111.4 (C-7), 111.6 (C-16), 113.0 (C-39), 118.5 (C-2), 119.9 (C-4), 121.1 (C-18), 126.8 (C-36), 130.5 (C-37), 134.4 (C-9), 134.5 (C-14), 135.6 (C-5), 135.8 (C-3), 144.9 (C-38), 156.0 (C-8), 156.5 (C-15), 161.2 (C-1), 177.8 (C-40), 186.8 (C-6), 187.1 (C-17), 211.9 (C-19).2 atmosphere (5%).Human cell cultures of tumor origin (A549 (lung carcinoma), HCT116 (colon adenocarcinoma), MCF7 (breast cancer), RD (embryonic rhabdomyosarcoma)), and the line of non-transformed cells HEK293 (embryonic kidney) provided by the Laboratory of Tumor Cell Genetics at the N.N. Blokhin Russian Cancer Research Institute, as well as by the Institute of Cytology of the Russian Academy of Sciences, were grown in DMEM  and MEM  containing fetal calf serum (10% by volume) , L-glutamine (2 mM) , and penicillin-streptomycin (1% by volume) . The cultivation was carried out at 37 °C in a humidified CO4 cells/200 μL of complete culture medium and cultured at 37 °C in a CO2 atmosphere (5%). After 24 h of incubation, various concentrations of the test compounds dissolved in DMSO (≤1%) were added to the cell cultures, the solvent was added to the control wells in an equal volume, and then the cells were cultured under the same conditions for 72 h. After the incubation time, MTT -2,5-diphenyltetrazolium bromide, 5 mg/mL) was added to each well, and the plates were further incubated for 2 h (until a characteristic color appeared). Then, the formazan granules were dissolved in DMSO.Cellular viability was determined by the MTT test . The cel50) was determined from dose-response curves using GraphPad Prism 9.0 software.Using a plate analyzer , the optical density was determined at λ = 530 nm. The concentration value causing 50% inhibition of cell population growth  and HEPES (20 mM) , pH = 7.4, at 4 °C and centrifuged at 1500 rpm to obtain a supernatant.To obtain a brain homogenate, decapitation of rats anesthetized in advance with CORat heart mitochondria were isolated by differential centrifugation using ice-cold buffer containing KCl (120 mM) and HEPES (20 mM), pH = 7.4, and a trypsin inhibitor. The mitochondrial yield ranged from 5 to 10 mg/mL of protein in the heart sediment. Mitochondria were stored in plastic containers at 4 °C.The quantitative determination of protein was carried out according to the standard technique using the microbiuret method .4 × 10H2O). After 30 min of incubation at 37 °C, TBA reagent was added to all samples, incubated for 90 min at 90 °C, and centrifuged at 6000 rpm for 15 min. The optical density of the selected supernatant was measured on a Victor 3 plate analyzer , country) at λ = 540 nm.To study the effect of compounds on the process of lipid peroxidation (LPO) of rat brain homogenate, we used a modified version of the TBA test , which iMitochondrial swelling was recorded by the light transmission of the rat heart mitochondrial suspension at λ = 540 nm using a Victor 3 plate analyzer . The swelling rate was estimated as △A540/min and was calculated as the tangent of the angle of the steepest dependence curve part △A540 versus time.2PO4 (1 mM), pH = 7.4, at the rate of 0.5 mg of protein in 1 mL of medium. Safranin A (5 μM) was added to the suspension immediately before the start of the measurement. The organelles were energized with a solution of potassium succinate and rotenone, against which the studied compounds were added at a concentration of 100 μM. The mitochondrial pore was induced by the addition of CaCl2 (25 μM).The transmembrane potential of rat heart mitochondria was measured by a Victor 3 plate analyzer  using a potential-dependent indicator Safranin A . The mitThe effect of the compounds on the functioning of the electron transport chain complexes was carried out using an Agilent Seahorse XF96e Analyzer  according to their ability to change the rate of oxygen uptake by isolated rat heart mitochondria (10 μg/well). The following modulators were used: activators of complex I of the electron transport chain, NADH dehydrogenase,—glutamate/malate; rotenone (2 μM) —a tissue respiration blocker that stops the transfer of electrons from the reduced form of NADH to cytochrome b, was used as complex I inhibitor; potassium succinate (2 μM) , which is a substrate of complex II of the electron transport chain, made it possible to assess mitochondrial respiration mediated by succinate dehydrogenase; complex III (ubiquinol-cytochrome c-oxidoreductase) inhibitor—a blocker of cellular respiration antimycin A , as well as ascorbate/TMPD (0.5 μM) , allowed us to evaluate the cytochrome-c-oxidase mediated respiration of organelles.The ability of the studied compounds to modulate anaerobic glycolysis was investigated using the Agilent Seahorse XF96e Analyzer  according to the level of hydrogen proton production in the samples on the HeLa cervical tumor cell line by the glycolysis stress test .4/well). After 24 h, the rate of medium extracellular acidification by cells treated with the test compounds was recorded in real time during the sequential injection of modulators: glucose (10 mM) , oligomycin (1 μM) , 2-fluoro-2-deoxy-d-glucose (25 mM) . This allowed us to estimate the glycolysis intensity in cells by three main parameters of glycolytic function: glycolysis by adding saturating amounts of glucose to the system, glycolytic capacity by the introduction of oligomycin, and glycolytic reserve by using the inhibitor of this glycolysis 2-fluoro-2-deoxy-d-glucose.HeLa cells in the exponential growth phase were plated into a 96-well cell culture microplate Seahorse (the density of the cell culture was 3 × 10a), and daunorubicin (b) were used as such amines. Before carrying out the aza-Michael reaction, daunorubicin hydrochloride was converted to a base. The resulting amine is more reactive and is easily isolated in pure form by extraction with chloroform from an aqueous solution. Doxorubicin is more hydrophilic, and its base is not extracted from the aqueous solution by chloroform. In this case, to carry out the aza-Michael reaction with sesquiterpene lactones, doxorubicin was used in the form of hydrochloride, and the process was carried out in the presence of a base (triethylamine), under the action of which, in the reaction medium, doxorubicin was released in situ as a free amine and directly reacted with the lactone –(8) react both with daunorubicin and doxorubicin under mild conditions, but at a low rate. As a result, a number of previously undescribed conjugates containing two pharmacophore fragments, natural sesquiterpene lactone and anthracycline antibiotics daunorubicin or doxorubicin, were obtained. The degree of conversion of the starting lactones was monitored by TLC and 1H NMR . The structure of the synthesized compounds was established using a set of physicochemical methods: NMR spectroscopy  and high-resolution mass spectrometry. The 1H NMR spectra contains signals of protons appearing in the products at the C-29 atom in the range of 2.5–2.8 ppm in the form of a quartet with a spin-spin coupling (SSC) constant of 6.6–6.9 Hz. The signals and their multiplicity of the remaining protons of both the initial lactone and daunorubicin or doxorubicin are retained, which corresponds to the proposed structures, as well as the configuration of all asymmetric centers of the initial molecules. In addition, a new chiral center with a certain configuration is formed in the conjugates [Sesquiterpene lactones  was 6.78 ± 0.76 μM, in turn, for its derivatives containing isoalantholactone (1a), alantolactone (2a), and alanthodiene (8a), values of IC50 more than double that of the initial cytostatic agent were shown: 19.82 ± 0.42 μM, 29.83 ± 0.18 μM, and 16.42 ± 1.07 μM, respectively. A similar situation was found for the daunorubicin conjugate with epoxyalantolactone (5b). The IC50 (b) value of 12.31 ± 1.17 μM, (5b) shows a value exceeding 18 μM.According to the obtained data, presented in 6a), the IC50 value of cytotoxicity was 0.25 ± 0.04 μM, and for daunorubicin conjugates (4b) the IC50 value was 0.02 ± 0.001 μM. This is an order of magnitude higher than the activity of the original pharmacophores. Thus, the combination of an anthracycline antibiotic with sesquiterpene lactone in one molecule makes it possible to obtain a cytotoxic effect comparable to the initial anthracyclines in relation to tumor origin cells and a reduced damaging effect on a healthy cell culture (HEK293). To understand this effect, we studied the influence of the synthesized compounds on oxidative stress , mitochondrial function, and glycolysis as the main metabolic pathway for obtaining energy by transformed tumor cells.It is also important to note that lactones themselves do not have a pronounced cytotoxic effect on tumor cells, while for modified doxorubicin derivatives, for example,  in real time. The investigation of the oxygen consumption rate (OCR) was carried out under the influence of tissue respiration modulators.6a and 3b) against respiratory chain complexes II and IV.It was found that both doxorubicin and daunorubicin had a pronounced inhibitory effect on complex II of the electron transport chain, which was expressed as a decrease in OCR by organelles by more than 70% C. A simiThus, the results of studying the effect of the synthesized conjugates on the functional characteristics of rat heart mitochondria suggest that doxo- and daunorubicin modification with natural sesquiterpene lactones carried out by us may be a prerequisite for reducing the toxic effect on healthy cells, in particular, cardiomyocytes, against the background of similar cytotoxicity with the original anthracyclines.As is known, cancerogenesis occurs in a hypoxic microenvironment, while a distinctive feature of neoplastic cells already at the earliest stages of the disease development is a high anaerobic glycolysis rate, characterized by intense glucose absorption followed by conversion to lactic acid in the absence of oxygen . Thus, tIn our work, information about the glycolytic activity of the tumor cell culture HeLa under the action of the studied compounds was obtained by measuring the extracellular acidification rate during the sequential injection of metabolic modulators by the glycolysis stress test.5a) and the daunorubicin conjugate with alantolactone (3b) had the most pronounced ability to reduce the acidification rate of the medium by cells.It was found that all studied substances inhibited the process of anaerobic glycolysis , which iThus, we were the first to obtain bipharmacophore molecules containing in their structure a fragment of the known anthracycline antibiotics—daunorubicin and doxorubicin—with natural sesquiterpene lactones. When studying the biological activity of the synthesized compounds, a reduced damaging effect on the rat heart mitochondria functioning was observed with equal and, in some cases, higher cytotoxicity and glycolysis inhibition by anthracycline antibiotic conjugates with sesquiterpene lactones in comparison with doxo- and daunorubicin. These results allow us to confirm the assumption that the chemical modification of the anthracycline antibiotic doxo- and daunorubicin molecules by sesquiterpene lactones can be a promising strategy for creating potential antitumor chemotherapeutic drugs with a pronounced cytotoxic effect on tumor cells and a reduced damaging effect on healthy cells of the human organism.


Maesa perlarius as an anti-EBOV plant lead. The methanol extract (MPBE) of the stems of this plant showed an inhibitory effect against EBOVpp, with an IC50 value of 0.52 µg/mL, which was confirmed by testing the extract against infectious EBOV in a biosafety level 4 laboratory. The bioassay-guided fractionation of MPBE resulted in three proanthocyanidins (procyanidin B2 (1), procyanidin C1 (2), and epicatechin-(4β→8)-epicatechin-(4β→8)-epicatechin-(4β→8)-epicatechin (3)), along with two flavan-3-ols ((+)-catechin (4) and (−)-epicatechin (5)). The IC50 values of the compounds against pseudovirion-bearing EBOV-GP ranged from 0.83 to 36.0 µM, with 1 as the most potent inhibitor. The anti-EBOV activities of five synthetic derivatives together with six commercially available analogues, including EGCG ((−)-epigallocatechin-3-O-gallate (8)), were further investigated. Molecular docking analysis and binding affinity measurement suggested the EBOV glycoprotein could be a potential molecular target for 1 and its related compounds.Ebola virus disease (EVD), a disease caused by infection with Ebola virus (EBOV), is characterized by hemorrhagic fever and a high case fatality rate. With limited options for the treatment of EVD, anti-Ebola viral therapeutics need to be urgently developed. In this study, over 500 extracts of medicinal plants collected in the Lingnan region were tested against infection with Ebola-virus-pseudotyped particles (EBOVpp), leading to the discovery of  Ebola virus (EBOV) is the causative agent of Ebola virus disease (EVD), a disease associated with hemorrhagic fever and high case fatality rates ,2,3. WitIn the current study, to search for inhibitors of Ebola virus, we used a cell-based assay with replication-incompetent pseudotyped viral particles that express a specific viral glycoprotein on the surface and can be performed in a BSL-2 containment . The devOver 50% of approved drugs are natural products or their derivatives and mimics, which illustrates that plants are an important source for drug discovery . There aMaesa perlarius as an anti-EBOV plant lead, which was further investigated to discover active compounds through bioassay-guided separation.A collection of over 500 medicinal plant extracts from the Lingnan region was tested against infection with Ebola-virus-pseudotyped HIV particles (EBOVpp). This led to the identification of M. perlarius is a 1–3 m tall shrub mainly distributed in China, Thailand, and Vietnam + . The chemical shifts and the number of proton signals in the 1H NMR spectrum of compounds 1 matched the 1H NMR spectrum of procyanidin B2 reported by Makabe et al. + . The spectroscopic data matched those of procyanidin C1 reported by Saito et al. + . The 1H NMR and MS data of compound 3 matched those of a procyanidin tetramer reported by Shoji et al. (3 was determined as epicatechin-(4β→8)-epicatechin-(4β→8)-epicatechin-(4β→8)-epicatechin.Epicatechin-(4β→8)-epicatechin-(4β→8)-epicatechin-(4β→8)-epicatechin  wa4) was isolated as a white amorphous powder, c 0.07, MeOH). UV (MeOH) λmax (log ε) 195 (4.75), 280 (3.73) nm. IR (KBr) νmax 3368, 1627, 1520, 1469, 1373, 1284, 1143 cm−1. HRESIMS m/z 291.0862 [M + H]+ . The retention time, ion peak, and fragmentation pattern of compound 4 in LC-MS matched those of the purchased (+)-catechin standard . Accordingly, compound 4 was identified as (+)-catechin.(+)-Catechin (5) was isolated as a white amorphous powder, c 0.12, MeOH). UV (MeOH) λmax (log ε) 210 (4.74), 280 (3.56) nm. IR (KBr) νmax 3456, 2930, 1627, 1521, 1469, 1262, 1145 cm−1. HRESIMS m/z 291.0861 [M + H]+ . The retention time, ion peak, and fragmentation pattern of 5 in LC-MS matched those of purchased (−)-epicatechin . Compound 5 was therefore identified as (−)-epicatechin.(−)-Epicatechin -epi-catechin, was purchased from Tokyo Chemical Industry . All other reagents were purchased from Sigma-Aldrich or J&K . (−)-Epi-catechin was first selectively protected by Bn-groups as described in the literature . The proR,3R)-5,7-Bis(benzyloxy)-2-phenyl)chroman-3-ol (12) was obtained as a white solid + . 1H NMR  δ 7.45–7.29 , 7.13 , 6.98 , 6.95 , 6.26 , 6.25 , 5.17 , 5.00 , 4.90 , 4.20 , 3.03–2.95 , 2.91  ppm.-5,7-Bis(benzyloxy)-2-phenyl)chroman-3-yl benzoate (13a) was obtained as a yellow oily liquid. Compound 12 (0.07 mmol) and 4-DMAP (0.175 mmol) in CH2Cl2 (dichloromethane) (4 mL) were added dropwisely to a solution of benzoyl chloride (0.14 mmol) in CH2Cl2 (1 mL) at 0 °C. The reaction mixture was stirred at room temperature for 16 h. Saturated sodium bicarbonate (NaHCO3) solution was added into the reaction mixture, and the organic layer was separated. The aqueous layer was further extracted with CH2Cl2. The combined CH2Cl2 layers were then dried over Na2SO4. The dried and evaporated residue was purified over PTLC (n-Hex/EtOAc 3:1) to afford the desired compound 13a as a yellow oily liquid (60%). HRESIMS 755.2950 m/z [M + H]+ . 1H NMR  δ 8.02–7.94 , 7.42–7.28 , 7.19 , 6.99 , 6.89 , 6.35 , 6.31 , 5.68 , 5.17–4.99 , 4.95 , 4.79 , 3.13  ppm.-2--5,7-dihydroxychroman-3-yl benzoate (13b) was obtained as a white powder. Under a hydrogen atmosphere, Pd(OH)2   was added to a solution of compound 13a  in a solvent mixture of THF (tetrahydrofuran)/MeOH (methanol)  under a hydrogen atmosphere. The reaction mixture was stirred at room temperature and was monitored by TLC. When the reaction was completed, the reaction mixture was filtered to remove the catalyst. The product 13b (83%) was purified by PTLC (n-Hex/EA 1:1) to afford a white powder. 1H NMR  δ 7.87 , 7.55–7.48 , 7.39 , 6.96 , 6.79 , 6.68 , 5.99 , 5.97 , 5.58 , 5.08 , 3.04 , 2.91  ppm + . 1H NMR  δ 7.95 , 7.42–7.28 , 7.13 , 7.03 , 6.98 , 6.88 , 6.33 , 6.30 , 5.65 , 5.14–4.99 , 4.97 , 4.84 , 3.10  ppm. 13C NMR  δ 167.1 (C-4′), 164.7164.7 (C-15), 158.9 (C-5), 158.0 (C-8a), 155.7 (C-7), 149.0 (C-11), 148.9 (C-12), 137.2, 137.1, 136.9, 136.9 , 132.5 (C-1′), 132.4, 131.1, 128.7, 128.7, 128.6, 128.6, 128.5, 128.1, 128.0, 127.9, 127.8, 127.7, 127.4, 127.3, 127.3, 126.3 , 119.9 (C-14), 115.7 (C-7), 115.5 (C-3′ and C-6′), 114.8 (C-13), 113.7 (C-10), 100.9 (C-4a), 94.8 (C-8), 94.0 (C-6), 77.5 (C-2), 71.4, 71.3, 70.2, 70.0 , 68.8 (C-3), 26.2 (C-4) ppm.-2--5,7-dihydroxychroman-3-yl 4-fluorobenzoate (14b). Under a hydrogen atmosphere, Pd(OH)2  was added to a solution of compound 14a  in a solvent mixture of THF/MeOH  under a hydrogen atmosphere. The reaction mixture was stirred at room temperature and was monitored by TLC. When the reaction was completed, the reaction mixture was filtered to remove the catalyst. The product 14b (52%) was purified by PTLC (n-hexane /EtOAc 1:1). HRESIMS 413.1036 m/z [M + H]+ . 1H NMR  δ 8.32 , 8.13 , 7.98–7.92 , 7.87–7.81 , 7.23–7.17 , 7.08 , 6.89 , 6.76 , 6.07 , 6.05 , 55.62 , 5.20–5.16 , 3.10 , 2.98  ppm. 13C NMR  δ 165.1 (C-15), 157.9 (C-7), 157.5 (C-8a), 157.0 (C-5), 145.7 (C-12), 145.6 (C-11), 133.1 (C-6′), 133.0 (C-1′), 131.3 (C-9), 127.7 (C-4′), 118.9 (C-14), 116.5 (C-5′), 116.3 (C-3′), 115.7 (C-13), 114.7 (C-10), 98.7 (C-4a), 96.6 (C-6), 95.8 (C-8), 77.8 (C-2), 70.4 (C-3), 26.5 (C-4) ppm.-5,7-Bis(benzyloxy)-2-phenyl)chroman-3-yl 4-methoxybenzoate (15a). A solution of compound 12 (0.07 mmol) and 4-DMAP (0.175 mmol) in CH2Cl2 (4 mL) was added dropwisely to a solution of 4-methyloxybenzoyl chloride (0.14 mmol) in CH2Cl2 (1 mL) at 0 °C. The reaction mixture was stirred at room temperature for 16 h. Saturated NaHCO3 solution was added into the reaction mixture, and the organic layer was separated. The aqueous layer was further extracted with CH2Cl2. The combined CH2Cl2 layer was then dried over Na2SO4. The dried and evaporated residue was purified over PTLC (n-Hex/EtOAc 3:1) to afford compound 15a (49%). HRESIMS 785.2936 m/z [M + H]+ . 1H NMR  δ 7.96–7.91 , 7.41–7.29 , 7.16 , 6.96 , 6.87 , 6.86–6.82 , 6.33 , 6.29 , 5.65 , 5.10–5.00 , 4.92 , 4.74 , 3.77 , 3.13–3.07  ppm. 13C NMR  δ 165.4 (C-4′), 163.5 (C-15), 158.8 (C-5), 158.1 (C-8a), 155.8 (C-7), 149.1 (C-11), 148.9 (C-12), 137.3 , 137.2, 137.0, 136.9, 132.0, 131.3, 128.7, 128.6, 128.6, 128.5, 128.4, 128.1, 128.0, 127.8, 127.7, 127.5, 127.33, 127.28 , 122.5 (C-1′), 120.1 (C-11), 114.8 (C-13), 113.7 (C-10), 101.1 (C-4a), 94.8 (C-8), 93.9 (C-6), 77.8 (C-2), 71.3, 70.2, 70.0 , 68.2 (C-3), 55.5 (-OCH3), 26.3 (C-4) ppm.-2--5,7-dihydroxychroman-3-yl 4-methoxybenzoate (15b) was obtained as a white solid. Under a hydrogen atmosphere, Pd(OH)2  was added to a solution of compound 15a  in a solvent mixture of THF/MeOH  under a hydrogen atmosphere. The reaction mixture was stirred at room temperature and was monitored by TLC. When the reaction was completed, the reaction mixture was filtered to remove the catalyst. The product 15b was purified by PTLC (n-hexane/EtOAc 1:1) (50%) to afford a white solid. HRESIMS 425.1235 m/z [M + H]+ . 1H NMR  δ 8.25 , 8.06 , 7.83–7.87 , 7.85 , 7.85 , 7.08 , 6.95 , 6.90 , 6.76 , 6.06 , 6.04 , 5.58 , 5.17 , 3.84 , 3.08 , 2.97  ppm. 13C NMR  δ 165.7 (C-4′), 164.4 (C-15), 157.8 (C-7), 157.4 (C-8a), 157.0 (C-5), 145.6 (C-12), 145.5 (C-11), 132.2 (C-2′ and C-6′), 131.3 (C-9), 123.4 (C-1′), 119.0 (C-14), 115.6 (C-13), 114.8 (C-3′ and C-5′), 114.5 (C-10), 98.8 (C-4a), 96.4 (C-6), 95.7 (C-8), 77.9 (C-2), 69.7 (C-3), 55.8 (-OCH3), 26.5 (C-4) ppm.-5,7-Bis(benzyloxy)-2-phenyl)chroman-3-yl cyclopentanecarboxylate (16a) was obtained as a white solid. A solution of compound 12 (0.07 mmol) and 4-DMAP (0.175 mmol) in CH2Cl2 (4 mL) was added dropwisely to a solution of cyclopentanecarbonyl chloride (0.14 mmol) in CH2Cl2 (1 mL) at 0 °C. The reaction mixture was stirred at room temperature for 16 h. Saturated NaHCO3 solution was added into the reaction mixture, and the organic layer was separated. The aqueous layer was further extracted with CH2Cl2. The combined CH2Cl2 layer was then dried over Na2SO4. The dried and evaporated residue was purified over PTLC (n-Hex/EtOAc 3:1) to afford compound 16a (95.7%) as a white solid. HRESIMS 747.3079 m/z [M + H]+ . 1H NMR  δ 7.47–7.32 , 7.14 , 6.98 , 6.93 , 6.31 , 6.29 , 5.47–5.42 , 5.17 , 5.05–5.00 , 3.02 , 2.98 , 2.57 , 1.36–1.75  ppm. 13C NMR  δ 176. (C-15)1, 158.7 (C-5), 158.0 (C-8a), 155.5 (C-7), 148.9 (C-11), 148.7 (C-12), 137.3 , 137.0 , 131.2 (C-9), 128.7, 128.6, 128.5, 128.5, 128.1, 128.0, 127.9, 127.8, 127.7, 127.5, 127.3, 127.2 , 119.7 (C-14), 114.9 (C-13), 113.7 (C-10), 101.0 (C-4a), 94.7 (C-8), 93.9 (C-6), 77.2 (C-2), 71.6, 71.4, 70.2, 69.9 , 43.8 (C-1′), 30.3 (C-5′), 29.7 (C-2′), 26.0 (C-4), 25.8 (C-4′), 25.7 (C-3′) ppm.-2--5,7-dihydroxychroman-3-yl cyclopentanecarboxylate (16b). Under a hydrogen atmosphere, Pd(OH)2  was added to a solution of compound 16a  in a solvent mixture of THF/MeOH  under a hydrogen atmosphere. The reaction mixture was stirred at room temperature and was monitored by TLC. When the reaction was completed, the reaction mixture was filtered to remove the catalyst. The product 16b was purified by PTLC (n-hexane/EtOAc 1:1) (50%). HRESIMS 387.1440 m/z [M + H]+ . 1H NMR  δ 8.09 , 7.02 , 6.86–6.76 , 6.06 , 5.97 , 5.37 , 5.05 , 2.94 , 2.79 , 2.60 , 1.78–1.52 , 1.52–1.43  ppm. 13C NMR  δ 176.0 (C-15), 157.8 (C-7), 157.4 (C-8a), 157.0 (C-5), 145.6 (C-12), 145.5 (C-11), 131.3 (C-9), 119.0. (C-14), 115.6 (C-13), 114.9 (C-10), 98.8 (C-4a), 96.4 (C-6), 95.8 (C-8), 77.7 (C-2), 68.9 (C-3), 44.5 (C-1′), 26.3 (C-3′ and C-4′), 26.3 (C-2′ and C-5′), 26.2 (C-4) ppm.-5,7-Bis(benzyloxy)-2-phenyl)chroman-3-yl cyclopropanecarboxylate (17a) was obtained as a white solid + . 1H NMR  δ 7.48–7.32 , 7.17 , 6.97 , 6.93 , 6.31 , 6.30 , 5.44 , 5.22–5.14 , 5.06–4.97 , 3.01 , 2.99 , 1.50 , 0.89–0.68  ppm [(2te solid . A solut-3′) ppm .R,3R)-2--5,7-dihydroxychroman-3-yl cyclopropanecarboxylate (17b) was obtained as a white solid. Under a hydrogen atmosphere, Pd(OH)2  was added to a solution of compound 17a  in a solvent mixture of THF/MeOH  under a hydrogen atmosphere. The reaction mixture was stirred at room temperature and was monitored by TLC. When the reaction was completed, the reaction mixture was filtered to remove the catalyst. The filtrate was evaporated and purified by PTLC (n-hexane/EtOAc 1:1) to afford compound 17b (75%) as a white solid. 1H NMR  δ 6.93 , 6.73–6.76 , 5.95 , 5.92 , 5.32 , 4.98–4.93 , 2.91 , 2.76 , 1.60–1.48 , 0.90–0.65  ppm.(22. 293T (CRL-3216) and A549 (CRM-CCL-185) cells were obtained from the American Type Culture Collection (ATCC) and were grown in Dulbecco’s modified Eagle medium  containing glutamine, supplemented with 10% fetal bovine serum (FBS). The cells were cultured at 37 °C in a humidified incubator with 5% COv/v) DMSO was introduced to the 96-well plate. An amount of 10 μL of 10% (v/v) DMSO was used as negative control, and paclitaxol was used as a positive control. After the treated cells were incubated at 37 °C for 72 h, 50 μL cold trichloroacetic acid  was added into each well. The plates were incubated at 4 °C for an additional 1 h to fix the cells. The plates were then rinsed with low-running tap water four times and dried at room temperature. An amount of 50 μL of SRB solution  was then added to each well. The plates were incubated at room temperature for 10 min and were washed with 1% (v/v) acetic acid to remove the unbound dye. The plates were allowed to dry. The protein-bound dye in each well was dissolved with 100 μL of Tris base solution , followed by shaking on an orbital shaker for at least 30 min. The optical density (OD) values were measured at 515 nm in a microplate reader. The CC50 (the concentration of an agent to cause a by 50% reduction in cell viability) values were calculated by GraphPad Prism 5.0 .A sulforhodamine B (SRB) assay was used to evaluate the cytotoxicity of the samples using A549 cells with modifications [Human embryonic kidney 293T cells were transiently transfected with either 1.875 µg VSV-G envelope-expression plasmid or 1.875 µg Ebola glycoprotein-expression plasmid  and 13.125 µg Env-deficient HIV vector (pNL4-3. Luc. R.E.) ,56 in a The host A549  cells were seeded at 4000 cells per well (96-well plate) in complete DMEM. Plant extracts, fractions, or compounds (10 µL) and the pseudovirus (90 µL) were incubated with host cells. Seventy-two hours post-infection, cell lysates were collected for luciferase assay using a Neolite Reporter gene assay system (PerkinElmer). The luciferase signals were measured in a Perkin Elmer Envision 2104 multilabel reader.Samples were serially diluted to a ratio of 1:10, with 100 μM being the highest concentration tested. For determination of cell viability with CellTiter-Glo, HeLa cells were incubated for 48 h with samples, after which time an equal volume of CellTiter-Glo reagent was added. Luminescence values were determined for control untreated cells and were used as a reference for sample-treated cells. The anti-infectious EBOV assay was performed following the published protocol by Cui at et al. .n = 4 wells per plate). Given that a 35% infection rate is more common than what was seen in our group, the higher infection rate was used to determine experimental infection rates. Experimental values are expressed as a percentage of the control infection.To evaluate whether the samples inhibit infection, 40 μL of a sample was added to each well of VERO E6 cells. The EBOV inoculum was delivered in 10 μL (MOI = 1). After adding virus, the cells were incubated for 1 h at 37 °C, after which time they received another 50 μL of sample. After 48 h, cells were fixed with 10% formalin, and infection was measured. EBOV-infected cells were detected with human Ab KZ52. An anti-human secondary conjugated to Alexafluor 488 was used to visualize infected cells that were counterstained with DAPI. Wells receiving only virus and media were used to establish a “control infection.” Control infection rates varied between plates, from approximately 11% to 35% . The default root, rotatable bonds, and torsions of the ligand were set by the TORSDOF utility in AutoDock Tools.https://www.rcsb.org/structure/5JQ7) (accessed on 30 December 2021). All bound waters, cofactors, and the original ligand were removed from the protein. Geisteger charges were computed, and polar hydrogen atoms were added subsequently. The AutoDock atom types were defined using AUTODOCK Tools 1.5.6, the graphical user interface of AUTODOCK, supplied by MGL Tools. The grid parameters were adjusted to cover the binding site of the original ligand in the protein crystal structure.The target protein, Ebola glycoprotein, was retrieved from the Protein Data Bank  and the pCMV-negative control  were transfected into 293T cells in a separate 90 mm dish using lipofectamine 3000. Forty-eight hours post-transfection, the transfected cells were washed by PBS twice, followed by the addition of M-PER lysis buffer supplemented with protease inhibitor cocktail  to collect the cell lysates.Kd values were determined by fitting the data with the Kd model.MTS experiments were performed according to the NanoTemper Technologies protocol in a Monolith NT.115 (red/blue) instrument . In the experiment, the cell lysate expressing His-tagged proteins was labeled with a Monolith His-Tag Labeling Kit RED-tris-NTA (NanoTemper Technologies), as described by the manufacturer. The final concentration of target protein in the cell lysate was kept constant at 25 nM. PBST/M-PER (1:1) was used as the assay buffer. A small-molecule DMSO stock solution (10 µL) was diluted to a ratio of 1:10 with assay buffer and then diluted to a ratio of 1:1 in 10 µL assay buffer to make a 16-sample dilution series . Labelled cell lysate (10 µL) was added to the diluted drug solution, and the mixture was incubated for 20 min in the dark. The mixtures were then loaded to standard Monolith NT.115 capillaries. The experiments were performed on a Monolith NT.115 Red system using medium MST power and 30% LED power at 25°C. The data acquired with MO. Control (NanoTemper Technologies) were analyzed using MO. Affinity Analysis 3.0.5 (NanoTemper Technologies), and the Ebola virus disease (EVD) is associated with fulminant hemorrhagic fever and high case mortality rates. Researchers are making efforts to develop anti-EBOV drugs. However, biosafety level 4 laboratories are required to handle EBOV, which constrains EBOV-related studies. A pseudotyped virion system that consists of envelope-deficient core from a virus, luciferase reporter genes, and envelope glycoproteins from another virus provides a convenient alternative platform for antiviral research. In the present study, we established a pseudotyped screening system involving two types of pseudovirions, EBOVpp and VSVpp, to evaluate the anti-EBOV activity of a sample. By comparing the inhibitory-effect difference of a sample against the two pseudovirions, an antiviral agent that specifically targets EBOV can be determined.M. perlarius as an anti-EBOV lead. Prior phytochemical and pharmacological studies of Maesa plants mainly focused on triterpenoid saponins. Our bioassay-guided fractionation study of the methanol extract of the stems of M. perlarius resulted in the discovery of a number of flavan-3-ol oligomers as potent EBOV inhibitors. In particular, we identified B-type procyanidins belonging to a class of condensed tannins as a new category of anti-EBOV phytochemicals. Furthermore, procyanidin B2 (1) was docked to fit the binding pocket of EBOV-GP, which was confirmed in MTS experiments by comparing it with the known EBOV-GP ligand toremifene. Thus, the current study presents flavan-3-ol monomers and oligomers as lead molecules that can be used as scaffolds for a target-oriented synthesis of additional analogues possessing improved anti-EBOV potency.Natural resources are reservoirs for druggable compounds. The considerable resources of plants in the Lingnan region provide an abundant potential to discover lead molecules. We screened 500 plant extracts made from medicinal plants collected in this region, which led to the discovery of


H-isoindole-1,3(2H)-dione derivatives and five potential pharmacophores were obtained in good yield (47.24–92.91%). The structure of the new imides was confirmed by the methods of elemental and spectral analysis: FT–IR, H NMR, and MS. Based on the obtained results of ESI–MS the probable path of the molecules decay and the hypothetical structure of the resulting pseudo-molecular ions have been proposed. The physicochemical properties of the new phthalimides were determined on the basis of Lipiński’s rule. The biological properties were determined in terms of their cyclooxygenase (COX) inhibitory activity. Three compounds showed greater inhibition of COX-2, three compounds inhibited COX-1 more strongly than the reference compound meloxicam. From the obtained results, the affinity ratio COX-2/COX-1 was calculated. Two compounds had a value greater than that of meloxicam. All tested compounds showed oxidative or nitrosan stress (ROS and RNS) scavenging activity. The degree of chromatin relaxation outside the cell nucleus was lower than the control after incubation with all test compounds. The newly synthesized phthalimide derivatives showed no cytotoxic activity in the concentration range studied (10–90 µM). A molecular docking study was used to determined interactions inside the active site of cyclooxygenases.Isoindoline-1,3-dione derivatives constitute an important group of medicinal substances. In this study, nine new 1 The search for new, safe, and effective synthetic drugs is a constant need. Heterocyclic compounds play an important role as the basis of many bioactive molecules. The isoinoline-1,3-dione moiety is of interest to many teams seeking biologically active compounds as candidates for new drugs. It has been proven that the phthalimide ring does not cause the side effects characteristic of glutarimide derivatives, and phthalimides are the basis of many products with proven biological activity, including analgesics, anti-inflammatories, and cholinesterases inhibitors used in Alzheimer’s disease ,2,3,4,5.The mentioned directions of action of isoindoline-1,3-dione derivatives are the result of several mechanisms for various biological purposes. For example, benzylamino-2-hydroxyalkylphthalimide derivatives are described as moderate and balanced multipotent anti-Alzheimer’s agents capable of inhibiting acetcholinesterase, symptomatic target, and binding modifying target (aggregation of β-secretase and β-amyloid) ,5.While browsing the literature, one can find information about the influence of isoinoline-1,3-dione derivatives on many groups of pro-inflammatory factors, including induced nitric oxide synthase (iNOS), nitric oxide (NO), cyclooxygenase 2 (COX-2), tumor necrosis factor (TNF-), interleukin-1 (IL-1), and interleukin-6 (IL-6). At the same time, they enhanced the expression of anti-inflammatory factors such as arginase-1 and interleukin-10 (IL-10), and inhibited p65 subunit phosphorylation induced by LPS kappa B (NF-B) in macrophages pyridine-1,3 was obtained, which are analogs of the previously described 3,4-pyridinedicarboximide derivatives ,20. MoreCompounds have been designed with potential analgesic and anti-inflammatory effects by blocking cyclooxygenase (COX) activity. It is an enzyme that catalyzes the conversion of arachidonic acid to prostanoids: prostaglandins, prostacyclin, and thromboxane . Two majIn our research, molecular docking of the obtained phthalimide derivatives to cyclooxygenase and protein isoforms was performed, which was compared with the results of affinity for enzymes using the in vitro method. The next step will be to compare the activities of 3,4-pyridinedicarboximide analogs and phthalimides in animal studies. This will allow us to understand the role of the imide moiety in the appearance of analgesic properties of new derivatives.A), 1-(2-methoxyphenyl)piperazine (B), 1-(3-trifluoromethphenyl)piperazine (C), and as a supplement 4-benzhydrylpiperazine/4-diphenylmethyl-1-piperazine (D) + with m/z = 350.1479 being the main peak, which corresponds to the molecular weight of this compound. The spectrum also includes additional peaks, derived from adducts of the molecule with the sodium ion [L + Na]+ m/z = 372.1300 and the dimer of the molecule with the sodium ion [2L + Na]+ m/z = 721.2724. With the help of the DataAnalysis Program +m/z = 402.1391 (H), 402.1419 (I) and the dimer of the molecule with the sodium ion [2L + Na]+ m/z = 781.2925 (G), 781.2912 (H), and 781.2959 (I). The proposed course of the degradation of the methoxy derivative molecule is shown in The structure of the methoxyphenyl derivatives m/z found and the calculated m/z was determined. The above MS spectral analysis data for the compound is presented in the Using the DataAnalysis Program , a compum/z = 307; m/z = 375), for some, they have not been confirmed in the literature -1H-isoindole-1,3(2H)-dione:1H NMR w CDCl3 δ [ppm]: 2.71–3.0 2-); 3.07–3.35 2-N-C6H5); 4.73 ; 6.89–7.25 ; 7.74–7.77 ; 7.87–7.9 .B: N-[(4-(2-methoxyphenyl)-1-piperazinyl)methyl]-1H-isoindole-1,3(2H)-dione:1H NMR w CDCl3 δ [ppm] of A: 2.82–3.35 2-) + -(C2H)2-N-C6H4 –OCH3); 3.81 , 4.76 ; 6.81–6.92 ; 7.75–7.77 ; 7.87–7.9 .C: N-[(4-(3-trifluoromethyl)phenyl)-1-piperazinyl)methyl]-1H-isoindole-1,3(2H)-dione:1H NMR w CDCl3 δ [ppm]: 2.75 -3.0 2-); 3.19–3.45 2-N-C6H4 –mCF3); 4.74 ; 7.0–7.07 ; 7.29–7.35 ; 7.74–7.76 ; 7.87–7.9 .D: N-[(4-(diphenyl-methyl)-1-piperazinyl)methyl]-1H-isoindole-1,3(2H)-dione:1H NMR w CDCl3 δ [ppm]: 2.30–2.55 2-); 2.65–2.87 2-N-CH-(C6H5)2); 4.20 2); 4.67 ; 7.15–7.37 ; 7.79–7.80 ; 7.89–7.90 .1) / 1-(3-trifluoromethyl)piperazine (2) /1-piperazine  and triethylamine  were dissolved in 100 mL of diethyl ether. Chloroacetyl chloride  in 35 mL of diethyl ether was added slowly over the course of 1 h. The mixture was allowed to stir at room temperature for 30 min; after this time the solvent was evaporated completely under reduced pressure, and the residue was dissolved in 120 mL of CHCl3 and 20 mL of water. The organic layer was separated, dried (Na2SO4), filtered gravitationally using a corrugated filter paper and concentrated. The residue was crystallized from ethanol . The oil residue (2) was purified by a chromatography column . The structure of intermediates 1–5 obtained was confirmed by elemental analysis  and 1H NMR spectral, MS-MS.1-Phenyl-4-piperazine (1): 2-Chloro-1-(4-phenyl-1-piperazinyl)ethan-1-on ; yield 68.76%; 1H-NMR , δ (ppm) = 3.19–3.32 2-); 3.71–3.88 2-N-C6H4-), 4.13 ; 6.94–7.07 ; 7.27–7.46 ; MS-MS[ppm] = [L + Na+] 261.0765, found 261.0753 (4.60); [2L + Na+] 499.1638, found 499.1639 (0.2);C2): 2-Chloro-1-[4-(3-fluoromethyl)phenyl-1-piperazinyl]ethan-1-on (13H14N2OF3Cl (MW 306.71); yield 80.72%; 1H-NMR , δ (ppm) = 3.24–3.38 2); 3.71–3.88 2-N-C6H4-); 4.14 , 7.10–7.20 ; 7.37–7.44 . MS-MS[ppm] = [L + H]+ 307.0820, found 307.0823 (0.98); [L + Na]+ 329.0645, [L+K]+ = 344.1590;C3): 2-Chloro-1-[4-(2-methoxy)phenyl-1-piperazinyl]ethan-1-on ; yield 46.27%; 1H-NMR , δ (ppm) = 3.07–3.21 2-); 3.63–3.89 2-N-C6H4-); 3.90 ; 4.13 ; 6.90–6.98 ; MS-MS[ppm] = [L + H]+ 269.1051, found 269.1049 (0.74); [L + Na]+ 291.0871, found 291.0862 (3.09), [2L + Na]+ = 559.1849, found 599.1843 (1.07).C4): 2-Chloro-1-[4-(3-methoxy)phenyl-1-piperazinyl]ethan-1-on ; yield 71.62%; 1H-NMR , δ (ppm) =3.03–3.30 2-); 3.68–3.80 2-N-C6H4-); 3.91 ; 4.13 ; 6.89–7.00 ; 7.04–7.12 ; MS-MS [ppm] = [L + H]+ 269.1051, found 269.1049 (0.74); [L + Na]+ 291.0871, found 291.0862 (3.09), [2L+Na]+ = 559.1849, found 599.1843 (1.07).C5): 2-Chloro-1-[4-(4-methoxy)phenyl-1-piperazinyl]ethan-1-on ; yield 46.64%; 1H-NMR , δ (ppm) = 3.07–3.20 2-); 3.70–3.88 2-N-C6H4-); 3.91 ; 4.13 ; 6.85–7.01 ; MS-MS[ppm] = [L + H]+ 269.1051, found 269.1052 (0.37); [L + Na]+ 291.0871, found 291.0859 (4.12), [2L+Na]+ = 559.1849, found 599.1756 (16.63).CH-isoindole-1,3(2H)-dione was dissolved in 40 mL of acetonitrile, and 0.008 mol of anhydrous potassium carbonate and 0.0077 mol of 2-chloro-1-[4-(arylophenyl)-1-piperazinyl]ethanon (1–5) were added. The reaction mixture was refluxed for 5 h. After this time, the inorganics were filtered on corrugated filter paper. The filtrate was completely evaporated under reduced pressure. The residue was crystallized from ethanol. The properties of obtained compounds E–I are shown below in A 0.007 mol amount of 1E: 2-[2-oxo-2-(4-phenyl-1-piperazinyl)ethyl]-1H-isoindole-1,3(2H)-dione:1H NMR (300MHz) w CDCl3 δ [ppm] of E: 3.20–3.30 2-); 3.71–3.90 2-N-C6H4-); 4.57 ; 6.96–7.08 ; 7.27–7.46 , 7.74–7.76 ; 7.89–7.92 . MS-MS[ppm] = [L + H]+ = 350.1499 found 305.1479 (5.71); [L + Na]+ = 372.1319, found 372.1300 (5.11), [2L+Na]+ = 721.2745, found 721.2724 (2.91).F: 2-{2-oxo-2-[4-(3-trifluoromethyl)-phenyl-1-piperazinyl]ethyl}-1H-isoindole-1,3(2H)-dione:1H NMR (300MHz) w CDCl3 δ [ppm] of F: 3.21–3.42 2-); 3.72–3.80 2-N-C6H4-); 4.56 ; 7.14–7.26 ; 7.38–7.44 ; 7.73–7.56 , 7.88–7.90 . MS-MS[ppm] = [L + H]+ = 418.1373 found 418.1357 (3.83); [L + Na]+ = 440.1192, found 440.1176 (3.64); [2L + Na]+ = 857.2493, found 857.2485 (0.93).G: 2-{2-oxo-2-[4-(2-methoxy)-phenyl-1-piperazinyl]ethyl}-1H-isoindole-1,3(2H)-dione:1H NMR (300MHz) w CDCl3 δ [ppm] of G: 3.02–3.21 2-); 3.68–3.87 2-N-C6H4-); 3.89 ; 4.51 ; 6.89–6.95 ; 7.71–7.74 ; 7.87–7.90 ; MS-MS[ppm] = [L + H]+ = 380.1605 found 380.1575 (18.41); [2L+Na]+ = 781.2956, found 781.2925 (3.97).H: 2-{2-oxo-2-[4-(3-methoxy)-phenyl-1-piperazinyl]ethyl}-1H-isoindole-1,3(2H)-dione:1H NMR (300MHz) w CDCl3 δ [ppm] of H: 3.08–3.60 2-); 3.72–3.90 2-N-C6H4-); 3.92 ; 4.57 ; 6.92–6.99 ; 7.04–7.12 ; 7.73–7.76 ; 7.89–7.92 ; MS-MS[ppm] = [L + H]+ = 380.1605 found 380.1573 (8.42); [L + Na]+ = 402.1424, found 402.1391 (5.63); [2L + Na]+ = 781.2956, found 781.2912 (5.63).I: 2-{2-oxo-2-[4-(4-methoxy)-phenyl-1-piperazinyl]ethyl}-1H-isoindole-1,3(2H)-dione1H NMR (300MHz) w CDCl3 δ [ppm] of I: 3.02–3.21 2-); 3.66–3.73 2-N-C6H4-); 3.79 , 4.54 ; 6.85–6.93 ; 7.71–7.74 ; 7.86–7.89 ; MS-MS[ppm] = [L + H]+ = 380.1605 found 380.1603 (0.53); [L + Na]+ = 402.1424, found 402.1419 (1.24); [2L + Na]+ = 781.2956, found 781.2959 (0.38).2/95% air atmosphere incubator. The cell culture was examined under the microscope twice a week, and cells were passaged if confluence was greater than 70% or the medium was replaced with fresh medium.The NHDF cells obtained from ATCC  were incubated with DMEM without phenol red supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1.25 µg/mL amphotericin B and 100 µg/mL gentamicin at 37 °C in a humidified 5% COThe 10 µM stock solutions were prepared by dissolving compounds in DMSO and then stored at −20 °C for up to 6 months. Before the biological experimental, the working solution was dissolved in the medium in the concentration range of 10, 50, and 100 µM. 50. To evaluated cell viability, an MTT assay was performed. After incubation of cells with the tested compounds for 24 h, the supernatant was removed, and 1 mg/mL MTT solution was applied for 2 h. Next, the formazan was dissolved with propanol for 30 min, and absorbance was measured at 570 nm using a microplate reader. The results are presented as ICThe ROS and RNS were evaluated using DCF–DA assay and Griess reagents, respectively. First, the cells were incubated with 50 µM LPS for 24 h to induce cell stress. Then, after the cells were washed, the 100 µM tested compounds were added for 1 h. Finally, the 50 µL supernatant was transferred to a new plate and added 50 µL of mixture A (1% sulfanilamide in 5% phosphoric acid) and B (0.1% N-(1-Naphthyl)ethylenediamine dihydrochloride) Griess reagents in a volume ratio 1:1 for 20 min in the dark. After that time, the absorbance was measured at 540 nm using a microplate reader. Simultaneously, the rest of the supernatant was removed from the above cell cultures, and 25 µM DCF–DA solution in MEM (without serum and phenol red) was added and left at 37 °C for 1 h and finally, determined fluorometrically with excitation at 485 nm and emission at 535 nm using a microplate reader. However, the cells were detached at the surface after 1h of incubation with the tested compound for evaluating DNA damage. The cells were then subjected to an analogous procedure as described in Grajzer et al. .The NO level was presented as concentration calculated using a standard curve . The ROS level was shown as ratio E/E0, where E0 is fluorescence with the positive control (cells incubated with 50 µM LPS) and E is a fluorescence with tested compound at 100 µM. Finally, the DNA damage was calculated as the ratio of cell nucleus diameter to halo diameter (a measure of DNA damage). Cayman kit No. 701050 was used to evaluate the COX peroxidase activity of all compounds at a concentration of 100 µM. Absorbance was measured with a microplate reader at 590 nm. Concentrations at which 50% inhibition of enzyme activity occurred were calculated separately for COX-1 and COX-2 (IC50). Ratios of IC50 values (COX-2/COX-1) were also calculatedp < 0.05.All statistical analysis performed were compared to the control—healthy cells (MTT assay), and positive control (incubated with 50 µM LPS) for DCF–DA, Griess, and DNA damaged. The data have a normal distribution and equality of variance, so ANOVA and post-hoc Scheffe methods were performed. The significance level was set at https://www.3ds.com/, accessed on 3 June 2021)The ground state geometric optimizations were calculated using density functional theory (DFT) with Becke’s three-parameter hybrid exchange function with the Lee-Yang–Parr gradient corrected correlation (B3LYP) ,46,47 fuB and D (1.10 and 0.96) have a value greater than that of meloxicam (0.71), while the value for imides G, C, and E  is comparable to meloxicam. All compounds tested showed both ROS and NO scavenging activity. It is noteworthy that this also applies to derivatives containing a methoxy substituent on phenyl, which have cell toxicity of 0.9 to 0.92 µM. All of them also cause chromatin relaxation outside the cell nucleus. The obtained results, similar for both types of derivatives: Mannich bases (A–D) and derivatives with an ethan-1-one linker (E–I), indicate a high affinity of N-aryl piperazine-alkyl derivatives cyclooxygenase. The in vitro study results and the analysis of molecular docking of compounds to cyclooxygenase, a constitutive form of COX-1 and inflammation-induced COX-2, are consistent and comparable to the reference drug values determined in the same test. Therefore, it can be assumed that the new phthalimide derivatives are endowed with the expected analgesic and anti-inflammatory activity.New phthalimide derivatives were obtained, their structure was verified and the theoretical bioavailability was tested on the basis of descriptors according to Lipiński’s five rule. The new imides showed an affinity for both isoforms of cyclooxygenase and were non-toxic in the tested concentration range of 10–90 µM. Based on the obtained results, the COX-2/COX-1 ratio was calculated, compounds


Herein, we report on the reaction of nitro-substituted azidobenzofuroxans with 1,3-dicarbonyl compounds in basic media. The known reactions of benzofuroxans and azidofuroxans with 1,3-dicarbonyl compounds in the presence of bases are the 1,3-dipolar cycloaddition and the Beirut reaction. In contrast with this, azidonitrobenzofuroxan reacts with 1,3-carbonyl compounds through Regitz diazo transfer, which is the first example of this type of reaction for furoxan derivatives. This difference is seemingly due to the strong electron-withdrawing effect of the superelectrophilic azidonitrobenzofuroxan, which serves as the azido transfer agent rather than 1,3-dipole in this case. Benzofuroxans have been proposed as monoamine oxidase inhibitors [N-oxide cycle. Benzofuroxans are capable of entering not only substitution reactions [c]oxadiazole 1-oxide quickly rearranging into benzo[c]oxadiazole 3-oxideviathe open dinitroso form) [Derivatives of benzo[c],2,5oxadihibitors ,4, and phibitors ,7,8,9, ahibitors ,11, antihibitors ,13,14, bhibitors ,16,17, vhibitors ,19. Moreeactions ,21, but eactions ,23,24. Aso form) , which oso form)  and makeOrganic azides are valuable building blocks in synthetic organic chemistry due to the well-known ability of azides to undergo 1,3-dipolar cycloaddition reactions. We speculated that the presence of the azide moiety in the benzofuroxan molecule would allow for easy installation of various biologically relevant fragments via a “click” reaction. Surprisingly, the literature survey revealed that the chemistry of the azidobenzofuroxans is still unexplored. The only reaction of the furoxan-based azide with 1,3-dicarbonyl compounds resulting in the 3+2 cycloaddition in the basic media was reported by Fershtat and coworkers  Scheme A. Notabl2 via the SNAr reaction of chloro derivative 1 with sodium azide was observed, as well as the range of the fragmentation peaks (147 [M+H2O-C2H3]+ (19%), 129 [M+H2O-C2H5O]+ (100%), 102 [M+H2O-C3H4O2]+ (43%), and 74 [M+H2O-C3H4N2O2]+ (82%); One of these products was identified as ethyl 2-diazo-3-oxobutanoate hed data . The mas4a may be explained by the diazo transfer reaction described by Regitz oxadiazole 1-oxide 1  and 4-(butylamino)-6-chloro-5-nitrobenzo[c]oxadiazole 1-oxide 5 .The following compounds were prepared following the literature procedures indicated: 4,6-dichloro-5-nitrobenzo[c]oxadiazole 1-oxide 2.The synthesis of the 4-azido-6-chloro-5-nitrobenzooxadiazole 1-oxide 3A and 4-amino-6-chloro-5-nitrobenzo[c]oxadiazole 1-oxide 3B.The synthesis of the 7-amino-5-chloro-6-nitrobenzo[Method 1:c]oxadiazole 1-oxide 2 (0.66 mmol) in 5 mL of CH3CN or CHCl3, triethylamine (0.16 mmol) and ethyl 3-oxobutanoate/pentane-2,4-dione/trifluoromethyl-3-oxobutanoic acid methyl ester (0.66 mmol) were added at room temperature. The reaction was carried out at room temperature and under magnetic stirring, and the conversion was monitored through TLC analysis . The mixture was stirred at room temperature overnight; the solvent was evaporated under vacuum (0.06 mm Hg) at 40 °C to constant weight. The crude product was purified by column chromatography on silica gel (eluent chloroform) to give target compound 3 as the orange powder, with a yield of 56%.To a solution of 4-azido-6-chloro-5-nitrobenzo+ (75), 184 [M-NO2]+ (37), 168 [M-O-NO-NH2]+ (9), and 154 [M-O-2NO]+ (42). Anal. calcd (%) for C6H3ClN4O4:C, 31.26; H, 1.31; Cl, 15.38; N, 24.30. Found: C, 31.28; H, 1.37; Cl, 15.34; and N, 24.29.Orange powder, yield 70%. M.p.: 79–80 °C. IR (d6 experiment (the percentage of each tautomer is given in parentheses).Acetone-c]oxadiazole 1-oxide : δ 7.16  and 7.93 . 13C{1H} NMR : δ 151.3 (C7), 137.6 (C5), 133.8 (C3a), 124.2 (C6), 109.0 (C7a), and 105.3 (C4).7-amino-5-chloro-6-nitrobenzooxadiazole 1-oxide : δ 6.91  and 7.99 . 13C{1H} NMR : δ 148.0 (C4), 137.7 (C6), 129.0 (C3a), 127.8 (C5), 112.8 (C7a), and 100.3 (C7).4-amino-6-chloro-5-nitrobenzooxadiazole 1-oxide (3A) (89%). 1H NMR : δ 6.02  and 6.02 . 13C{1H} NMR : δ 150.5 (C7), 136.3 (C5), 134.3 (C3a), 124.6 (C6), 108.0 (C7a), and 105.6 (C4).7-amino-5-chloro-6-nitrobenzo[c]oxadiazole 1-oxide (3B) (11%). As the concentration of the second tautomer wassmall, it practically did not show up in the 13C{1H} NMR. The chemical shifts of parts of the carbons were determined from the HMBC spectra.1H NMR : δ 5.77  and 5.24 . 13C{1H} NMR  δ 146.5 (C4), n/o (C6), 129.4 (C3a), 127.7 (C5), n/o (C7a), and 101.8 (C7).4-amino-6-chloro-5-nitrobenzo[4d experiment (the percentage of each tautomer is given in parentheses).Methanol-c]oxadiazole 1-oxide (3A) (40%). 1H NMR : δ 7.004  and n/o . 13C{1H} NMR : δ 151.9 (C7), 137.8 (C5), 134.7 (C3a), 125.0 (C6), 109.4 (C7a), and 105.6 (C4).7-amino-5-chloro-6-nitrobenzo[c]oxadiazole 1-oxide (3B) (60%). 1H NMR : δ 6.761  and n/o . 13C{1H} NMR  δ 148.6 (C4), 138.4 (C6), 130.1 (C3a), 128.3 (C5), 113.5 (C7a), and 100.2 (C7).4-amino-6-chloro-5-nitrobenzo[Crystal Data for C6H3ClN4O4 (M = 230.57 g/mol): orthorhombic, space group P212121 (no. 19), a = 5.8860(2) Å, b = 11.2467(3) Å, c = 24.7322(7) Å, V = 1637.22(8) Å3, Z = 8, T = 100.0(4) K, μ(Cu Kα) = 4.246 mm−1, Dcalc = 1.871 g/cm3, 8428 reflections measured (7.148° ≤ 2Θ ≤ 153.52°), 3305 unique , which were used in all calculations. The final R1 was 0.0439 (I > 2σ(I)) and wR2 was 0.1199 .


Some of these ranitidine analogs also possessed potent inhibitory activities of acetylcholinesterase (AChE), which is another therapeutic target in AD. These ranitidine analogs, by addressing both Aβ aggregation and AChE, offer insight into the key chemical features of a new type of multi-target directed ligands for the pharmaceutical treatment of AD. The aggregation of amyloid β (Aβ) peptides and deposition of amyloid plaques are implicated in the pathogenesis of Alzheimer’s disease (AD). Therefore, blocking Aβ aggregation with small molecules has been proposed as one therapeutic approach for AD. In the present study, a series of ranitidine analogs containing cyclic imide isosteres were synthesized and their inhibitory activities toward Aβ aggregation were evaluated using in vitro thioflavin T assays. The structure–activity relationship revealed that the 1,8-naphthalimide moiety provided profound inhibition of Aβ aggregation and structural modifications on the other parts of the parent molecule (compound  HRMS: Obs. M+H, 429.1039. Calc. M+H, 429.1039.6-Chloro-2-(2-(((5-((dimethylamino)methyl)furan-2-yl)methyl)thio)ethyl)-1H-benzo [de]isoquinoline-1,3(2H)-dione(9) mp: 116–118 °C; 1H NMR  δ 2.35 2), 2.83 , 3.77 , 3.97 2NCH2), 4.42, 7.28 , 7.77 , 8.23 , 8.62 . HRMS: Obs. M+H, 412.1152. Calc. M+H, 412.1153. 2-(2-((2-((Dimethylamino)methyl)thiazol-4-yl)methylthio)ethyl)-1H-benzo[de]iso quinoline-1,3(2H)-dione (10) mp: 124–125 °C; 1H NMR  δ 2.36 2), 2.84 , 3.78 , 3.96 2NCH2), 4.44 , 7.29 , 8.23 , 8.28 , 8.82 , 8.87 , 9.35 . HRMS: Obs. M+H, 456.5409. Calc. M+H, 456. 5409.2-(2-(((2-((Dimethylamino)methyl)thiazol-4-yl)methyl)thio)ethyl)-5-nitro-1H-benzo[de]isoquinoline-1,3(2H)-dione (11. Dimethylamine hydrochloride (1.2 mmol) and compound 11 (0.6 mmol) were added to a stirred 37% formaldehyde solution (0.12 mol) under a nitrogen atmosphere. The solution was heated from 25 °C to 90 °C for 20 hours and then was allowed to cool to room temperature, transferred to a separatory funnel, and then extracted with 20 mL of dichloromethane. The aqueous layer was stirred with a Na2CO3-saturated solution (20 ml). The mixture was extracted with dichloromethane  in toluene (50 mL), a solution of 2-furan-2-yl-ethanolamine (4 mmol) in ethanol was added dropwise. The reaction mixture was refluxed with a condenser and a Dean–Stark trap for 6 hours and then was evaporated under reduced pressure. The resulting residue was purified by column chromatography. The silica gel was first conditioned with dichloromethane, and then the product was eluted with 50:1 dichloromethane/methanol. Evaporation of the solvent yielded compound 11) mp: 85–87 °C; 1H NMR δ 2.24 2, δ 3.08, 3.392NCH2), 4.49, 5.99, 6.06, 7.94, 8.43, 8.76, 9.13, 9.29. HRMS: Obs. M+H, 394.1404. Calc. M+H, 394.1403. 2-(2-(5-((Dimethylamino)methyl)furan-2-yl)ethyl)-5-nitro-1H-benzo[de]isoquino line-1,3(2H)-dione (12) mp:182–183 °C; 1H NMR δ 3.10, 4.49, 6.09, 6.83, 7.31, 7.95, 8.82, 8.77, 9.14, 9.31. HRMS: Obs. M+H, 336.0747. Calc. M+H, 336.0746.2-(2-(furan-2-yl)ethyl)-5-nitro-1H-benzo[de]isoquinoline-1,3(2H)-dione (6 (0.5 mmol) in glacial acetic acid (20 mL) was treated with 35% hydrogen peroxide (38 µL) in ice bath. The mixture was stirred while warming to room temperature overnight and then was evaporated under reduced pressure. The resulting residue was purified by flash column chromatography using dichloromethane/methanol mobile system. Evaporation of the solvent yielded products compound 13 and 15. Compound 14 was synthesized from compound 3 using the same procedure.A solution of compound 13) mp: 106.5–108 °C; 1H NMR  δ 2.28 2), 3.10 , 3.51 , 4.17 2NCH2), 4.68 , 6.24 , 6.39 , 7.95 , 8.45 , 8.80 , 9.16 , 9.34 . HRMS: Obs. M+H, 456.1232. Calc. M+H, 456.1229.2-(2-(((5-((Dimethylamino)methyl)furan-2-yl)methyl)sulfinyl)ethyl)-5-nitro-1H-benzo[de]isoquinoline-1,3(2H)-dione(14) mp: 117–119 °C; 1H NMR  δ 2.22 2), 3.09 , 3.43 , 4.18 2NCH2), 4.64 , 6.19 , 6.39 , 7.76 , 8.24 , 8.62 . HRMS: Obs. M+H, 411.1382. Calc. M+H, 411.1378. 2-(2-(((5-((Dimethylamino)methyl)furan-2-yl)methyl)sulfinyl)ethyl)-1H-benzo[de] isoquinoline-1,3(2H)-dione(15) mp: 138–40 °C; 1H NMR  δ 3.18 2), 3.47 , 4.47 2NCH2), 4.58 , 4.71 , 6.69 , 7.98 , 8.47 , 8.82 , 9.17 , 9.34 . HRMS: Obs. M+H, 488.1141. Calc. M+H, 488.1127.N,N-Dimethyl-1--yl)ethyl) sulfonyl)methyl)furan-2-yl)methanamine oxide(−1 cm−1 at 276 nm. Purified Aβ monomer samples were used fresh or stored at 4 ℃ for up to 4 days.Lyophilized Aβ peptide was reconstituted in 50 mM NaOH at a concentration of 2 mg/mL and purified via size-exclusion chromatography (SEC) on a Superdex 75 HR 10/300 column  using a running buffer of 40 mM Tris-HCl (pH 8.0). Bovine serum albumin (BSA) (2 mg/mL) was employed as a pretreatment to reduce nonspecific Aβ interaction with the dextran matrix of the column. The elution profile showed a characteristic peak corresponding to monomeric Aβ. The concentrations of eluted Aβ monomer fractions were determined with a calculated extinction coefficient of 1450 Mv/v) DMSO in the final reaction solution. DMSO (2% v/v) was added to control reactions run in parallel.All compounds were dissolved in DMSO at 5 mM and stored at −20 °C. Immediately prior to experimentation, stock solutions were diluted into DMSO such that subsequent dilution to 100 μM ranitidine analog yielded 2% (SEC-purified Aβ monomer was diluted to 40 μM in 40 mM Tris-HCl (pH 8.0) and agitated  without (control) or with 100 µM ranitidine analogs. Periodically, a 20-μL aliquot was removed and combined with 140 μL of 10 μM ThT. Fluorescence  was evaluated using a Perkin-Elmer LS-45 luminescence spectrometer . Fluorescence values were calculated as the integrated area under the emission curve with baseline  subtraction and plotted vs. aggregation time. Each compound was evaluated using at least three independent experiments. 50 values were determined by nonlinear regression analysis of the concentration–response curves generated. Each compound was evaluated using at least three independent experiments.The AChE inhibition assay was modified based on the method of Ellman et al.  in a 24-In this study, a series of multi-target ranitidine analogs containing cyclic imide isosteres was explored through rationally designed structural modifications. Of particular note, the analogs with 1,8-naphthamide resulted in potent inhibition of Aβ aggregation. While improving the Aβ aggregation inhibition, these structural modifications also retained high efficacy as AChE inhibitors. These results imply that this series of ranitidine analogs represents a new type of MTDLs with inhibitory activities of both Aβ aggregation and AChE. The structural elements in these analogs can be further explored to develop more effective MTDLs for the pharmaceutical treatment of AD.


The structures of the natural products 1–9 have been assigned by MS, as well as IR, 1D NMR , and 2D NMR  spectroscopic methods. The compounds 2 and 4–7 represent new chemical structures. Acacetin (8) and L-proline anhydride (9) have been obtained from C. mongolica for the first time.The seven pyridine alkaloids  Caryopteris mongolica Bunge is a deciduous shrub and belongs to the Verbenaceae family. C. mongolica which is widely distributed in the mountainous and Gobi regions of Mongolia 1. In fact only this species of Caryopteris is growing in Mongolia, whereas about 16 species of this genus occur all over the world. In traditional Mongolian medicine, the aerial parts of this plant have been prepared as decoction and used for haemorrhage, increasing muscle strength, urinary excretion, pulmonary windy oedema and chronic bronchitis2. In Chinese folk medicine, Caryopteris terniflora has been used as antipyretic, detoxifying, expectorant, and anti-inflammatory agent and for the treatment of cold, tuberculosis and rheumatism3.Caryopteris mongolica showed the presence of mono- and sesquiterpenoids4, hypolaetin-7-glucoside5, iridoid glucosides and steroids6. From the roots of this plant, the abietane ortho-quinone caryopteron A and derivatives were isolated7. In our previous phytochemical investigation of the aerial parts of C. mongolica, we have identified some phenolic compounds such as acacetin, apigenin, luteolin, and caffeic acid8. From other species of Caryopteris steroidal and iridoid glucosides, phenylethanoids, diterpenoids, α-caryopterone, a new pyranojuglone, clandonoside and its acetylated derivatives, flavonoids and sterols have been isolated13. In the present paper, we describe the isolation of pyridine monoterpene alkaloids, isoquinoline alkaloids, a dipyrrolopyrazine, and a flavonoid (acacetin) from the aerial parts of C. mongolica and their structure elucidation using MS, as well as IR, 1H NMR, 13C NMR, and 2D NMR spectroscopic methods.Previous chemical investigations of 254 were used. Spots detected on the TLC plates under the UV lamp at 254 or 365 nm were visualized by spraying Dragendorff’s reagent. The EI-MS were recorded on a Finnigan MAT-95 mass spectrometer (70 eV) and the ESI-MS on an Bruker Esquire LC with an ion trap detector. Positive and negative ions were detected. ESI-HRMS were recorded on a Waters Xevo G2-XS QTOF mass spectrometer. The 1D and 2D NMR spectra (600 MHz 1H NMR and 150 MHz 13C NMR) were measured on a Bruker AVANCE III 600 MHz spectrometer using CDCl3 or CD3OD as solvents. The chemical shifts δ are reported in ppm using either CHCl3/CDCl3  or CH3OH/CD3OD  as internal standard relative to TMS. Coupling constants J are given in Hz. The complete assignment of 1H and 13C NMR signals was confirmed by 2D NMR spectroscopic methods .Flash column chromatography (FCC) was performed using silica gel 200–300 mesh from Acros Organics occasionally on a Büchi Sepacore system equipped with a UV monitor. For thin layer chromatography (TLC), silica gel plates Merck 60 FCaryopteris mongolica Bunge were collected at Baruun kharaa of Selenge province Bayantsogt Mountain, during the flowering period in July 2014. The plant was identified by Prof. E. Ganbold and Dr. B. Mandahk, Institute of Botany, MAS, and voucher specimen were deposited at the Herbarium of Laboratory of Natural Products Chemistry, ICCT, Research Institute of the Mongolian Academy of Sciences, in Ulaanbaatar (voucher numbers: UBA-0004020 and UBA-0004021). The authors confirm that the present study complies with the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora.The aerial parts of Caryopteris mongolica were extracted three times at room temperature with 95% aqueous ethanol. The ethanol was removed and the residue treated with 2.5% HCl (pH = 1–2). The aqueous acidic solution was extracted successively with n-hexane and chloroform. Then, the residual aqueous solution was adjusted to pH = 9–10 by addition of aqueous NH4OH (25%), extracted with CHCl3 and dried over Na2SO4. Finally, CHCl3 was evaporated under reduced pressure to give 12.02 g of crude total alkaloid extract (0.096%).The air-dried and powdered aerial parts (12.5 kg) of C. mongolica was purified by FCC  (175 g silica gel). The elution with CHCl3, CHCl3–CH3OH mixtures ranging from 90:10, 85:15, 80:20, 75:25, and 50:50 (1000–1500 mL), and pure CH3OH afforded 11 sub-fractions from A to K: A (108 mg), B (69.6 mg), C (1.50 g), D (851 mg), E and F (218 mg), G (399 mg), H (1.34 g), I (161 mg), J (274 mg), and K (85.0 mg). The fractions were monitored by TLC, compounds detected as described by Dragendorff’s reagent, and checked by ESI-MS.The crude total alkaloid extract (12.02 g) isolated from 3–CH3OH mixtures ranging from 99:1 to 95:5 to afford seven fractions from A1 to G1: A1 (8.7 mg), B1 (46.0 mg), C1 (55.0 mg), D1 (46.9 mg), E1/F1 (234 mg), and G1 (183 mg). From fraction B1, compound 3 (aucubinine B)15 (13.2 mg) was isolated 13 (20.1 mg) and 2  have been obtained from fraction G1. Fraction C1 provided by crystallization from CHCl3 colorless needles which were identified as compound 8 (acacetin)19 (23.6 mg). The purified fraction E1/F1 afforded a mixture of the compounds 4 and 5 . Finally, we have combined all fractions (139 mg) exhibiting in their ESI–MS a peak at m/z = 296.2 [M + H]+, subjected them to FCC (25 g silica gel), and eluted with CHCl3–CH3OH mixtures ranging from 99:1 to 95:5 to provide compound 4 (2.7 mg) and compound 5 (7.7 mg) as shown by their NMR spectra  was subjected to FCC (25 g silica gel), and eluted with CHCl.50 g wasa Figure .Figure 13–CH3OH mixtures ranging from 99:1 to 95:5 to afford 5 fractions from A2 to E2: A2 (27.2 mg), B2 (137 mg), C2 (172 mg), D2 (84.5 mg), and E2 (37.8 mg). After evaporation of the solvent, fraction B2 afforded a colorless residue which has been purified to provide a mixture of compounds 6 and 7 (137 mg) showing in the ESI-MS a peak at m/z = 293.2 [M + H]+. Finally, we further purified a fraction of this mixture (20.3 mg) by preparative thin layer chromatography  using the solvent system CHCl3–CH3OH (9:1). The PTLC afforded a mixture of the compounds 6 and 7 (3.2 mg) and compound 7 (17.2 mg) along with compounds 1 and 2 as impurities.Fraction D (851 mg) was subjected to FCC (25 g of silica gel), eluted with CHClCaryopteris mongolica Bunge, the structures of the compounds 1–9  was isolated as colorless needles (mp 81.3–90 °C). The ESI-MS (m/z = 148.1 [M + H]+) and EI-MS (m/z = 147 [M+]) correspond to the molecular formula C9H9NO. The 1H NMR spectrum of 1 exhibits nine proton signals -7-Ethoxy-6,7-dihydro-7-methyl-5H-cyclopenta[c]pyridin-5-ol.2 (10.1 mg) was obtained as a brown residue. The ESI-MS (m/z = 194.2 [M + H]+) corresponds to the molecular formula C11H15NO2. The 1H NMR spectrum of 2 exhibits nine proton signals  indicated a cis-arrangement for the hydroxy and the ethoxy group. The 2D NMR correlations supporting the stereochemical assignment for compound 2 are shown in the Supplementary Information -7-ethoxy-6,7-dihydro-7-methyl-5H-cyclopenta[c]pyridin-5-ol and represents a new compound obtained from C. mongolica for the first time. However, it cannot be excluded that compound 2 has been formed by electrophilic addition at 1 on treatment with ethanol/HCl during the isolation process.Compound 2 0.1 mg wa Figures –S17-1. CCompound 3: 6,7-Dihydro-7-methylcyclopenta[c]pyridine-5-one (aucubinine B).3 (10.1 mg) was obtained as a brown residue. The ESI-MS (m/z = 148.1 [M + H]+) corresponds to the molecular formula C9H9NO. The 1H NMR spectrum of compound 3 exhibits three signals for the pyridine protons, a quartet of doublet of doublets for the methine group (H-7), two doublets of doublets for the two methylene protons at C-6, and one doublet at 1.46 ppm for the methyl group  (3). Compound 3 was obtained from C. mongolica for the first time.Compound 3 0.1 mg wa4 and 5 were obtained as pure compounds and as a mixture -10,11a-Dimethyl-6,7,9,10,11,11a-hexahydro-5H-6,11-epoxycyclopentaazulenopyridin-5,8(5aH)-dione.4 (30.8 mg) was isolated as a colorless solid. The ESI-MS (m/z = 296.2 [M + H]+) and EI-MS (m/z = 295 [M+]) correspond to the molecular formula C18H17NO3; ESI-HRMS calcd. for C18H18NO3+ ([M + H]+): 296.1281, found: 296.1286. The 1H NMR spectrum exhibits 13 signals for 17 protons -10,11a-dimethyl-6,7,9,10,11,11a-hexahydro-5H-6,11-epoxycyclopentaazulenopyridin-5,8(5aH)-dione. Compound 4 is a new pyridine derivative that has been isolated from C. mongolica for the first time.A series of longe-range HMBC correlations has been used to confirm the connectivity of the structural moieties in compound 4 Fig. . The sigCompound 5: -10-Hydroxy-7a,11a-dimethyl-5a,6,7,7a,8,11a-hexahydro-5H-6,8-epoxycyclopentaazuleno pyridin-5-one.5 has been obtained as a mixture with compound 9 as a colorless solid. The ESI-MS (m/z = 296.2 [M + H]+) and EI-MS (m/z = 295 [M+]) correspond to the molecular formula C18H17NO3; ESI-HRMS calcd. for C18H18NO3+ ([M + H]+): 296.1281, found: 296.1287. Thus, it is isomeric to compound 4. The 1H NMR spectrum of 5 displayed 12 proton signals ; H-5a with C-10a, C-11b, and C-13; H-13 (Me) with C-1, C-5a, C-11, and C-11b; H-6 with C-5a and C-10a; H-11 with C-5a, C-7a, C-9, C-10a, and C-11a; H-7, H-8, and H-14 (Me) with C-10a -10-hydroxy-7a,11a-dimethyl-5a,6,7,7a,8,11a-hexahydro-5H-6,8-epoxycyclopentaazulenopyridin-5-one. Compound 5 is a new pyridine derivative that has been obtained from C. mongolica for the first time.The assignment of the NMR signals and the elucidation of the framework of compound 6 and 7. While compound 6 was isolated as a pure substance, compound 7 was obtained only as a mixture, either together with the compounds 6, 1, and 2, or together with the compounds 1 and 2 (Supplementary Information). All isolated fractions of either pure 6 or mixtures containing 7 showed the same ESI-MS peak at m/z = 293.2 ([M + H]+) that corresponds to a molecular formula of C18H16N2O2. However, we obtained two different sets for the 1H NMR and 13C NMR signals of both compounds (Table 6 and for the by-product 7 which has the same molecular formula but a different constitution (see below).We also performed detailed NMR studies of the compounds Compound 6: -5-Hydroxy-10b,11-dimethyl-5,5a,10b,11-tetrahydro-6H-5,11-methanopyridocyclopentaisoquinolin-6-one.6 (3.2 mg) was isolated as a colorless solid. The ESI-MS (m/z = 293.2 [M + H]+) and EI-MS (m/z = 292 [M+]) correspond to the molecular formula C18H16N2O2. The 1H NMR spectrum exhibits 11 signals for 16 protons , one methine group (at 66.89 ppm in CD3OD), three quaternary carbon atoms, one methylene group (64.12 ppm in CD3OD), and two methyl groups.Compound 6 .2 mg was6 .2 mg was6 were confirmed by the following characteristic long-range HMBC correlations: H-4 with C-5; H-5a with C-5, C-6 (carbonyl group), C-10b, C-6a, C-10a, C-4a, and C-13; H-7 with C-6 (carbonyl group); H-10 with C-10a; H-12 with C-5, C-11, C-5a, C-4a, C-10b, and C-11a; H-13 with C-5a, C-10a, C-10b, and C-11; and H-14 with C-10b, C-11, C-11a, and C-12  with H-10, H-5a, and one of the protons from the methylene group (H-12a); H-12a with H-5a; and H-14 (Me) with H-1 and H-10 -5-hydroxy-10b,11-dimethyl-5,5a,10b,11-tetrahydro-6H-5,11-methanopyridocyclopentaisoquinolin-6-one. Compound 6 is a new compound that has been obtained from C. mongolica for the first time.The assignments of the NMR signals and the framework of compound Compound 7: -6a-Hydroxy-11,11a-dimethyl-6,6a,11,11a-tetrahydro-5H-6,11-methanopyridocyclopentaisoquinolin-5-one.7 is a colorless solid and was obtained as a small by-product during the extraction of compound 6. The ESI-MS (m/z = 293.2 [M + H]+) corresponds to the molecular formula C18H16N2O2. The 1H NMR spectrum of 7 exhibited 11 signals for 16 protons -6a-hydroxy-11,11a-dimethyl-6,6a,11,11a-tetrahydro-5H-6,11-methanopyridocyclopentaisoquinolin-5-one. Compound 7 is a new compound that has been obtained from C. mongolica for the first time.The assignment of the NMR signals and the structural elucidation of compound tra Fig. . The COSCompound 8: 5,7-Dihydroxy-2-(4-methoxyphenyl)-4H-chromen-4-one .8 (23.6 mg) was obtained as colorless needles. The ESI-MS (m/z = 285.1 [M + H]+) corresponds to the molecular formula C16H12O5. The 1H NMR spectrum shows signals for 10 protons including seven aromatic protons and a methoxy group at δ 3.88 ppm. The 13C NMR spectrum exhibits signals for 16 carbon atoms: seven aromatic methine (CH) groups, six aromatic carbon atoms, one olefinic carbon atom, a methoxy group at 55.94 ppm, and a carbonyl group at 181.20 ppm. The assignment was additionally supported by 2D NMR spectroscopy. Thus, the structure of compound 8 was confirmed as 5,7-dihydroxy-4'-methoxyflavone (acacetin)17. This natural product was isolated for the first time from the aerial parts of C. mongolica.Compound 1H NMR : δ 7.90 , 7.07 , 6.45 , 6.16 , 5.98 , 3.88 .13C NMR, : δ 181.20 (C-4), 163.73 (C-2), 162.75 (C-7), 161.76 (C-4'), 160.91 (C-5), 156.79 (C-9), 129.02 , 122.30 (C-1'), 115.51 , 104.05 (C-6), 103.19 (C-10), 102.99 (C-3), 97.97 (C-8), 55.94 (OCH3).Compound 9: Octahydro-5H,10H-dipyrrolopyrazin-5,10-dione (L-proline anhydride).9 (2.4 mg) was obtained as a colorless amorphous solid. The ESI-MS (m/z = 195.2 [M + H]+) and EI-MS (m/z = 194 [M+]) correspond to the molecular formula C10H14N2O2. The 1H NMR spectrum displays six proton signals for six methylene groups and two methine protons. The 13C NMR spectrum exhibits five signals for six methylene groups, two methine groups and two carbonyl groups. Therefore, compound 9 has been assigned as octahydro-5H,10H-dipyrrolopyrazine-5,10-dione (L-proline anhydride). Compound 9 was isolated for the first time from the aerial parts of C. mongolica.Compound 1H NMR : δ 4.18 , 3.53 , 2.31 , 2.18 , 2.02 , 1.92 .13C NMR : δ 166.2 (C-5), 60.54 (C-4), 45.21 (C-1), 27.69 (C-3), 23.35 (C-2).1–7), the flavonoid acacetin (8), and L-proline anhydride (9) from the aerial parts of Caryopteris mongolica Bunge. The chemical structures of these compounds were elucidated by MS as well as by 1H NMR, 13C NMR and 2D NMR  spectroscopic methods. -7-Ethoxy-6,7-dihydro-7-methyl-5H-cyclopenta[c]pyridin-5-ol (2), -10,11a-dimethyl-6,7,9,10,11,11a-hexahydro-5H-6,11-epoxycyclopentaazulenopyridin-5,8(5aH)-dione (4), -10-hydroxy-7a,11a-dimethyl-5a,6,7,7a,8,11a-hexahydro-5H-6,8-epoxycyclopentaazulenopyridin-5-one (5), -5-hydroxy-10b,11-dimethyl-5,5a,10b,11-tetrahydro-6H-5,11-methanopyridocyclopentaisoquinolin-6-one (6), -6a-hydroxy-11,11a-dimethyl-6,6a,11,11a-tetrahydro-5H-6,11-methanopyridocyclopentaisoquinolin-5-one (7), 5,7-dihydroxy-2-(4-methoxyphenyl)-4H-chromen-4-one  (8), and octahydro-5H,10H-dipyrrolopyrazine-5,10-dione (L-proline anhydride) (9) have been obtained from C. mongolica for the first time.We have isolated seven pyridine alkaloids , the compounds 2, 4, 5, 6, and 7 (Fig. Most likely, the cyclopenta[1H NMR, 150 MHz 13C NMR, COSY, HSQC, HMBC, and NOESY spectra of the compounds 1–9.600 MHz Supplementary Figures.


H-thienoimidazole-4,6-diyl)bis(thiophene-2-carboxylic acid)  was synthesized in one step from previously reported 4,6-di(thiophen-2-yl)-1H-thienoimidazole , and their photophysical properties were studied and compared accordingly. Under solvothermal conditions, reacting 5 with Mn(OAc)2 yielded a new three-dimensional metal-organic framework  which was structurally defined by single-crystal X-ray diffraction. In 6, all Mn(II) ions octahedrally bind to carboxylate-O atoms to form a linear Mn3 secondary building unit (SBU) that contains three distinct coordination modes. Importantly, 6 exhibits dual functional properties of ligand-based emission and metal-based magnetic behaviors.A new terthiophene-based imidazole luminophore 5,5’-(1 Once functionalized with dicarboxylic acids, 5 (TIBTCH2) still possesses interesting photoluminescent properties; therefore, MOFs based on this linker were expected to possess functionalities stemming from both the organic linker and the metal SBUs. Herein, we describe the dual solid-state functions of a novel material, 6, featuring terthiophene-based imidazole emissive linkers with linear Mn3 SBUs. Material 6 is a porous crystalline material with both emissive and magnetic functionalities.Terthiophene-based N-fused heterocyclic molecules such as 5,7-di(2-thienyl)thienopyrazine derivatives   Scheme  are of i4, DTTI) was prepared by following the previously reported method by our group imidazole (ur group . Yellow ructures . Close-rructures . The dicod yield . We were4 and 5 were first studied in dimethyl sulfoxide (DMSO) at room temperature  for the formation of a Mn(II)-based MOF (6). The new MOF, with empirical formula 2H·[Mn3(TIBTC)4]·3H2O, was obtained as a red needle-like polycrystalline material by treating a suspension of 5 (0.04 mmol) with Mn(OAc)2·4H2O (0.1 mmol) in a solution of DMF containing H2O (10 vol%) at 85 °C for 10 days. The atomic structure was determined by a single-crystal X-ray diffraction analysis, and the composition was confirmed by elemental analysis ((C60H32Mn3N8O19S12): Calcd. C, 41.94%; H, 1.88%; N, 6.52%; S, 22.39%. Found. C, 41.96%; H, 1.41%; N, 6.52%; S, 22.46%). Material 6 crystallizes in the orthorhombic Pcca space group and has an infinite 3-D structure comprising linear [Mn3]6+ units as the SBUs  compounds (5.8–6.1 eV) and is significantly larger than the range of both Mn(III) (4.6–5.4 eV) and Mn(IV) (4.5–4.7 eV) compounds + (C15H9N2O4S3+), calcd. 376.9719, found: 376.9728. m.p.: 205–208 °C.TCH2): 4  4]·3H2O): 5  and Mn(OAc)2·2H2O  were suspended in a mixture of H2O and N,N-dimethylformamide  in a 20 mL scintillation vial, which was surpersonicated for 20 min before heating by submersion in a pre-heated graphite thermal bath and held at 85 °C for 7–10 days. The crystals of 6 were isolated by washing and decanting any impurities away using a 10% water in N,N-dimethylformamide mixture. The solid was then filtered using a Büchner funnel and thoroughly washed with the same solvent. The crystalline solid was dried in the air. Average yield, 3.2 mg . FT-IR (cm−1): as-synthesized: 3062 (w), 2071 (m), 2021 (w), 1994 (w),1871 (s), 1675 br/(m), 1585 (w), 1477 (m), 1432 (m), 1306 (w), 1260 (w), 1183 (w), 1158 (w), 1088 (m), 1068 (w), 1025 (m), 998 (w), 801 (w), 740 (s), 691 (s), 624 (m), 609 (m), 573 (s), 509 (s), 493 (s), 446 (m), 416 (s). Activated: 3482 (w), 3275 (m), 3237 (m), 3157 (m), 2955 (m), 2929 (m), 2881 (m), 1641 (w), 1584 (m), 1522 , 1483 (m), 1438 (m), 1356 (m), 1328 (m), 1274 (m), 1152 (m), 1100 (m), 1015 (s), 978 (m), 905 (w), 764 (m), 655 (s), 583 (w), 516 (s), 490 (s), 447 (m). Elemental analysis (C60H32Mn3N8O19S12): Calcd. C, 41.94%; H, 1.88%; N, 6.52%; S, 22.39%. Found. C, 41.96%; H, 1.41%; N, 6.52%; S, 22.46%.


Isocoumarins, isomeric to comarins which act as effective carbonic anhydrase  inhibitors, were investigated for the first time as inhibitors of this enzyme. A series of 3-substituted and 3,4-disubstituted isocoumarins incorporating phenylhydrazone, 1-phenyl-pyrazole and pyrazolo-substituted pyrimidine trione/thioxo-pyrimidine dione moieties were investigated for their interaction with four human (h) CA isoforms, hCA I, II, IX and XII, known to be important drug targets. hCA I and II were not inhibited by these compounds, whereas hCA IX and XII were inhibited in the low micromolar range by the less bulky derivatives. The inhibition constants ranged between 2.7–78.9 µM against hCA IX and of 1.2–66.5 µM against hCA XII. As for the coumarins, we hypothesise that the isocoumarins are hydrolysed by the esterase activity of the enzyme with formation of 2-carboxy-phenylacetic aldehydes which act as CA inhibitors. Isocoumarins represent a new class of CA inhibitors. Thin layer chromatography (TLC) was carried out on Merck silica gel 60 F254 plates.All chemicals and anhydrous solvents were purchased from Sigma-Aldrich, Merck, Across Organics and TCI and used without further purification. Melting points (mp) were determined with SMP30 melting point apparatus in open capillaries and are uncorrected. FT-IR spectra were recorded by using Perkin Elmer Spectrum 100 FT-IR spectrometer. Nuclear Magnetic Resonance (2.2.X(1–5) were dried under vacuum and fully characterised by FT-IR, 1H-NMR, 13C-NMR, and melting points.The methyl ketone (10 mmol) and phenylhydrazine derivative compounds were added to a reaction flask by adding 20 ml EtOH with a catalytic amount of acetic acid and refluxed for 2 h. After the reaction complete, the obtained compounds were filtered off and crystallised from ethanol. The final products 3–(1-(2-phenylhydrazono)ethyl)-1H-isochromen-1-one (X1) Yield: 90%; mp: 202–204 °C; FT-IR (cm−1): 3294 (NH), 1695 (C = O); 1H-NMR : 2.21 , 6.94–7.69 , 8.28 .3–(1-(2–(4-chlorophenyl) hydrazono)ethyl)-1H-isochromen-1-one (X2) Yield: 80%; mp: 238°–240 °C; FT-IR (cm−1): 3287 (NH), 1692 (C = O); 1H-NMR : 2.47 , 7.20–8.11 , 9.79 .4–(2-(1–(1-oxo-1H-isochromen-3-yl)ethylidene)hydrazinyl) benzonitrile (X3) Yield: 82%; mp: 250°–252 °C; FT-IR (cm−1): 3271 (NH), 2222 (C≡N), 1692 (C = O), 1597 (C = N);1H-NMR : 2.45 , 7.29–8.12 , 10.18. 13C-NMR : 12.2, 82.9, 99.3, 101.1, 103.8, 113.8, 120.3, 120.4, 127.2, 129.2, 133.9, 135.7, 137.5, 137.7, 148.9, 152, 161.5, 175.4.4-methyl-3–(1-(2-phenylhydrazono)ethyl)-1H-isochromen-1-one (X4) Yield: 82%; mp: 210°–212 °C; FT-IR (cm−1): 3271 (NH), 1733, 1709 (C = O); 1H-NMR : 2.17 , 2.47 , 7.18–8.17 , 9.57 . 13C-NMR : 13.4, 39.4, 109.3, 112.8, 113.4, 120.2, 124.6, 128.5, 129.3, 129.4, 135.6, 135.9, 138.9, 145.6, 149.6, 161.4.3–(1-(2–(4-chlorophenyl) hydrazono)ethyl)-4-methyl-1H-isochromen-1-one (X5) Yield: 83%; mp: 240°–242 °C; FT-IR (cm−1): 3320 (NH), 1715 (C = O); 1H-NMR : 2.17 , 2.41 , 7.17–8.18 , 9.70 . 13C-NMR : 13.4, 40.6, 109.5, 114.8, 120.2, 123.5, 124.6, 128.6, 129.2, 129.3, 135.7, 136.8, 138.8, 144.6, 149.4, 161.32.3.3  was added dropwise over the reaction mixture by keeping the temperature between 0° and 5 °C. After the completion of adding, the mixture was allowed to stir overnight at room temperature. Next day, the mixture was poured into the ice-water and it was triturated with 10% NaOH solution. The precipitate was filtered off and dried under vacuum at room temperature. The final products X(6–10) were fully characterised by FT-IR, 1H-NMR, 13C-NMR, and melting points.The phenylhydrazone derivatives X (1–5) (10 mmol) and DMF  were placed in a reaction flask and the POCl3–(1-oxo-1H-isochromen-3-yl)-1-phenyl-1H-pyrazole-4-carbaldehyde (X6) Yield: 82%; mp: 228°–230 °C; FT-IR (cm−1): 3124, 2921 (C-H), 1728 (C = O isocoumarin), 1676 ; 1H-NMR : 7.22–8.32 , 8.53 , 10.54 .1–(4-chlorophenyl)-3–(1-oxo-1H-isochromen-3-yl)-1H-pyrazole-4-carbaldehyde (X7) Yield: 80%; mp: 258°–260 °C; FT-IR (cm−1): 3287, 3025 (C-H), 1716 (C = O isocoumarin), 1682 ; 1H-NMR : 7.58–8.55 , 9.03 , 10.58 .4–(4-formyl-3–(1-oxo-1H-isochromen-3-yl)-1H-pyrazol-1-yl) benzonitrile (X8) Yield: 78%; mp: >300oC; FT-IR (cm−1): 3120–3078 (CH), 2228 (C≡N), 1712 (C = O isocoumarin), 1673 ; 1H-NMR : 7.69–8.24 , 9.50 , 10.31 .3–(4-methyl-1-oxo-1H-isochromen-3-yl)-1-phenyl-1H-pyrazole-4-carbaldehyde (X9) Yield: 80%; mp: 201°–203 °C; FT-IR (cm−1): 3117, 2921 (C-H), 1718 (C = O isocoumarin), 1679 ; 1H-NMR : 12.9, 113.5, 119.7, 121.1, 123.9, 124.7, 128.3, 128.8, 129.6, 129.8, 129.9, 134.9, 138.2, 138.8, 142.9, 146.8, 161.4, 185.5.1–(4-chlorophenyl)-3–(4-methyl-1-oxo-1H-isochromen-3-yl)-1H-pyrazole-4-carbaldehyde (X10) Yield:78%; mp: 288°–290 °C; FT-IR (cm−1): 3124, 2911 (C-H), 1715 (C = O isocoumarin), 1679 ; 1H-NMR : 2.41 , 7.65–8.30 , 9.40 , 10.09  .2.4.X(6–10) (10 mmol) was dissolved in acetic acid and the barbituric acid/2-thiobarbituric acid (10 mmol) was added over the mixture and stirred overnight at room temperature. Then, the mixture was filtered off and crystallised from ethanol to yield compounds X(11–20). The obtained final products were dried under vacuum and fully characterised by FT-IR, 1H-NMR, 13C-NMR, and melting points.The aldehyde derivatives 5-((3–(1-oxo-1H-isochromen-3-yl)-1-phenyl-1H-pyrazol-4-yl) methylene) pyrimidine-2,4,6-trione (X11) Yield: 82%; mp: >300 °C; FT-IR (cm−1): 3235, 3081 (NH), 1738, 1722, 1680 (C = O), 1574 (C = N); 1H-NMR : 7.44–8.20 , 8.68 , 9.74 , 12.41 , 12.45 .5-((1–(4-chlorophenyl)-3–(1-oxo-1H-isochromen-3-yl)-1H-pyrazol-4-yl) methylene) pyrimidine-2,4,6-trione (X12) Yield: 81%; mp: >300oC; FT-IR (cm−1): 3238, 3058 (NH), 1735, 1699, 1666 (C = O), 1571 (C = N); 1H-NMR : 7.20–8.20 , 8.62 , 9.69 , 11.29 , 11.36 .4–(3-(1-oxo-1H-isochromen-3-yl)-4--ylidene) methyl)-1H-pyrazol-1-yl) benzonitrile (X13) Yield: 80%; mp: >300oC; FT-IR (cm−1): 3192, 3068 (NH), 2231 (-C≡N), 1735, 1712, 1676 (C = O), 1565 (-C = N); 1H-NMR : 7.37–8.17 , 8.56 , 9.71 , 11.28 , 11.36 .5-((3–(4-methyl-1-oxo-1H-isochromen-3-yl)-1-phenyl-1H-pyrazol-4-yl) methylene) pyrimidine-2,4,6-trione (X14) Yield: 81%; mp: >300oC; FT-IR (cm−1): 3248, 3084 (NH), 1731, 1712, 1686 (C = O), 1568 (C = N); 1H-NMR : 2.46 , 7.59–7.91 , 8.11 , 9.76 , 11.32 , 11.33 . 13C-NMR : 13.3, 114.8, 116, 117.2, 120.3, 120.8, 125, 128.8, 129.6, 130, 130.4, 134.8, 136.1, 137.6, 138.8, 142, 142.1, 150.2, 150.5, 161.1, 162.9, 163.6…5-((1–(4-chlorophenyl)-3–(4-methyl-1-oxo-1H-isochromen-3-yl)-1H-pyrazol-4-yl) methylene) pyrimidine-2,4,6-trione (X15) Yield: 82%; mp: >300oC; FT-IR (cm−1): 3163, 3042 (NH), 1738, 1709, 1666 (C = O), 1575 (C = N); 1H-NMR : 2.46 , 7.63–8.25 , 8.27 , 9.75 , 11.32 , 11.33 .5-((3–(1-oxo-1H-isochromen-3-yl)-1-phenyl-1H-pyrazol-4-yl) methylene)-2-thioxodihydropyrimidine-4,6-dione (X16) Yield: 82%; mp: >300oC; FT-IR (cm−1): 3143, 3055 (NH), 1761, 1715 (C = O), 1565 (C = N); 1H-NMR : 7.42–8.20 , 8.64 , 9.69 , 11.28 , 11. 35 . 13C-NMR : 107.3, 116.3, 116.5, 120.2, 120.6, 127.4, 128.9, 129.4, 129.9, 130.3, 135.5, 135.9, 136.8, 138.6, 143.9, 147.3, 149, 160.7, 161, 162, 162.7, 178.8.5-((1–(4-chlorophenyl)-3–(1-oxo-1H-isochromen-3-yl)-1H-pyrazol-4-yl) methylene)-2-thioxodihydropyrimidine-4,6-dione (X17) Yield: 82%; mp: >300oC; FT-IR (cm−1): 3137, 3075 (NH), 1754, 1712, (C = O), 1558 (C = N); 1H-NMR : 7.42–8.18 , 8.65 , 9.72 , 12.39 , 12. 45 .4–-ylidene) methyl)-3–(1-oxo-1H-isochromen-3-yl)-1H-pyrazol-1-yl) benzonitrile (X18) Yield: 80%; mp: >300oC; FT-IR (cm−1): 3215, 3137 (NH), 2235 (C≡N), 1751, 1715 (C = O), 1574 (C = N); 1H-NMR : 2.46 , 7.47–8.26 , 8.28 , 9.79 , 12.43 .5-((3–(4-methyl-1-oxo-1H-isochromen-3-yl)-1-phenyl-1H-pyrazol-4-yl) methylene)-2-thioxodihydropyrimidine-4,6-dione (X19) Yield: 81%; mp: >300oC; FT-IR (cm−1): 3147, 2902 (NH), 1705, 1666 (C = O), 1568 (C = N); 1H-NMR : 2.46 , 7.61–8.26 , 8.28 , 9.78 , 12.43 .13C-NMR : 13.4, 115, 116.1, 117.4, 120.3, 120.8, 125, 128.9, 129.6, 130, 130.4, 135, 136.1, 137.6, 138.7, 141.9, 143, 150.4, 160.7, 161, 161.9, 172.9, 178.8.5-((1–(4-chlorophenyl)-3–(4-methyl-1-oxo-1H-isochromen-3-yl)-1H-pyrazol-4-yl) methylene)-2-thioxodihydropyrimidine-4,6-dione (X20) Yield: 82%; mp: >300oC; FT-IR (cm−1): 3130, 2915 (NH), 1715, 1669 (C = O), 1568 (C = N); 1H-NMR : 7.47–8.26 , 8.42 , 9.76 , 11.35 .2.5.2SO4 or NaClO4 , following the CA-catalyzed CO2 hydration reaction for a period of 5–10 s. Saturated CO2 solutions in water at 25 °C were used as substrate. Stock solutions of inhibitors were prepared at a concentration of 10 mM  and dilutions up to 0.01 nM done with the assay buffer mentioned above. At least seven different inhibitor concentrations have been used for measuring the inhibition constant. Inhibitor and enzyme solutions were pre-incubated together for 15 min–6 h at 4 °C prior to assay, in order to allow for the formation of the E-I complex. Triplicate experiments were done for each inhibitor concentration, and the values reported throughout the paper is the mean of such results. The inhibition constants were obtained by nonlinear least-squares methods using the Cheng-Prusoff equation, as reported earlier, and represent the mean from at least three different determinations,An SX.18 MV-R Applied Photophysics  stopped-flow instrument has been used to assay the inhibition of various CA isozymes3.3.1.X(1–20) were synthesised according to the general synthetic route shown in B were synthesised as previously described by some of usX(1–5) were obtained by reacting B with substituted hydrazinesX(6–10) were synthesised in high yields by using the Vilsmeier-Haack procedureX(10–20). The chemical structures of the novel isocoumarin-substituted derivatives reported here were confirmed by analytical and spectral data .The structurally diverse isocoumarin derivatives 3.2.per se act as inhibitors, binding at the entrance of the enzyme active site and occluding itCoumarins act as prodrug inhibitors, being hydrolysed by the esterase activity of CAs to the corresponding hydroxy-cinnamic acids which X12, X17 and X20) showed low micromolar inhibitory power against these isoforms, with KIs in the range of 2.7 − 78.9 µM against hCA IX and of 1.2 − 66.5 µM against hCA XII, respectively  present in some of these compounds.Four human (h) CA isoforms, known to be relevant drug targets ectively . It can 4.We investigated here whether isocoumarins, which are isomeric compounds to comarins known to act as effective CAIs, also act as inhibitors of this enzyme. A series of 3-substituted and 3,4-disubstituted isocoumarins incorporating phenyl-hydrazone, 1-phenyl-pyrazole and pyrazolo-substituted pyrimidine trione/thioxo-pyrimidine dione moieties prepared by an original approach were investigated for their interaction with hCA I, II, IX and XII, known to be important drug targets. hCA I and II were not inhibited by these compounds, whereas hCA IX and XII were inhibited in the low micromolar range by the less bulky derivatives. The inhibition constants ranged between 2.7 − 78.9 µM against hCA IX and of 1.2 − 66.5 µM against hCA XII. As for the coumarins, we hypothesise that the isocoumarins are hydrolysed by the esterase activity of the enzyme with formation of 2-carboxy-phenylacetic aldehydes which act as CA inhibitors. Isocoumarins represent a new class of CAIs.