Additionally, three compounds, 3, 5 and 9, were further characterized using single X-ray diffraction. The X-ray data suggested that extensive hydrogen bonds affect molecular structure by means of intermolecular interactions. In order to evaluate the explosive properties of these synthesized compounds, detonation pressures and velocities were calculated using EXPLO5 (V6.01). These calculations were carried out utilizing experimental data, including density and heat of formation. Among the explosives tested, compounds 7 and 8 exhibited zero oxygen balance and demonstrated exceptional detonation properties. Compound 7 achieved the highest recorded detonation pressure, at 34.2 GPa, while compound 8 displayed the highest detonation velocity, at 8887 m s−1.The synthesis of the new energetic material 4-amino-3-hydrazino-5-methyl-1,2,4-triazole, which shows excellent performance and reliable safety, has drawn attention recently. To fully characterize this material, a comprehensive analysis was performed using various techniques, including differential scanning calorimetry (DSC), infrared spectroscopy (IR), elemental analysis, and  The synthesis and design of new energetic materials has been ongoing since the discovery of 2,4,6-trinitrotoluene (TNT) in 1863 . TriazolIn order to achieve a balance between detonation performance and safety in energetic materials, researchers have employed various methods, including the synthesis of dense ring compounds, the development of eutectic materials, and the preparation of energetic ionic salts. In recent years, energetic salts have drawn widespread attention as a new type of energetic material . CompareAs a nitrogen-rich compound, 4-amino-3-hydrazino-5-methyl-1,2,4-triazole, which brings methyl and 1,2,4-triazole into a single material, has multiple modification sites. This compound can be chemically modified with different groups, leading to enhanced physical and chemical properties when incorporated into ionic salts. Furthermore, the triazole framework allows for the selection of various anions, including those containing nitro groups, which can improve oxygen balance. In contrast to the precursors reported in the literature, which usually have one monovalent anion, the 4-amino-3-hydrazino-5-methyl-1,2,4-triazole described in this article serves as a precursor for polyvalent ion salts, and can be paired with multiple-ion ligands. Polyvalent ions have higher formation heat compared to those of monovalent ion salts with a similar structure. Therefore, the polyvalent state of this invention is related to significant detonation. Taking into consideration the aforementioned advantages, energetic salts with good explosive performances based on 3-hydrazino-4-amino-5-methyl-1,2,4-triazole are reported and comprehensively characterized.3 reveals that it belongs to the space group Pbca, with a crystal density of 1.435 g cm−3 at 296 K. It can be seen from 3 consists of two chloridions and one hetercocylic cation, with two protonating hydrogen atoms on N1 and N5. There is one intramolecular interaction between the cation and chloridion (N1-H1A…Cl1), with a distance of 3.066(2) Å and an angle of 177(3)°. Notably, it can surprisingly be found that all of the atoms except the H of the cation are in nearly the same plane, which can be verified by the torsion angles N6-N3-C1-N4 = 5.866°, C3-C2-N3-C1 = 178.976°, and N3-C1-N2-N1 = −0.149°. However, there is a slight deviation in the torsion angle of the hydrazine moiety, with the angle of N5-N4-C1-N3 being −165.121°. In terms of bond lengths, the distance of N3-NH2 (1.4013 Å) is shorter than that of N4-NH2 (1.4145 Å), and the distance of C2-CH3 is the longest among those of all of the bonds in compound 3, at 1.4639 Å. Additionally, the packing diagram of compound 3 is shown in The crystal structure of compound 5 is depicted in 5 belongs to the space group P2 (1)/c with a crystal density of 1.513 g cm−3 at 296 K. Each lattice cell contains four molecules (Z = 4) and the molar ratio of the compound 5 is 1:1. The protonating hydrogen atom is on N3. There are three intramolecular interactions in the cation with a distance from 2.5858 Å to 3.1800 Å which can be seen in 5 is close to that of compound 3, as is evident from the corresponding torsion angles: N5-C1-N3-N2 = 176.068°, N7-N4-C2-N2 = −176.188°, and N4-C2-N2-N3 = −0.109°. However, some torsion angles deviate from the plane, such as O4-C3-N6-N5 = 7.352°, N6-N5-C1-N3 = 15.253° and C3-N6-N5-C1 = −96.923°. For the electron absorption effect of nitrate, we can find that the distance of N4-NH2 (1.3993 Å) and the distance of N5-N6 (1.3892 Å) are shorter than those of the compound 3. Additionally, the distance of C3-CH3 (1.4855 Å) is longer than that of C2-CH3 (1.4772 Å). The crystal structure of 4-amino-3-acethydrazino-5-melthy-1,2,4-triazole nitrate energetic compound 9 is presented as (AHMT)+ (NT)−, with a molar ratio of 1:2. Compound 9 belongs to the triclinic crystal system with the P-1 space group and a crystal density of 1.621g·cm−3 at 296K. Each lattice cell contains two molecules (Z = 2). The protonating hydrogen atoms are locked on N11 and N15. One intramolecular interaction (N8-H15A) is shown in 3 and 5. Similarly, the planarity of the cation of compound 9 is similar to those of compounds 3 and 5, indicating a coplanar structure. This can be confirmed by the torsion angles of C5-C4-N11-N12 = −179.696°, N16-N13-C3-N12 = 177.788°, N15-N14-C3-N12 = −13.543°, and C4-N11-N12-C3 = 0.620°. However, the anion NT is not in the same plane as the cation, with an angle between them of N14-N15-N8 = 100.263°. The distance of N13-NH2 (1.3993 Å) is shorter than that of N14-NH2 (1.4160 Å), and the distance of C4-CH3 is 1.4754 Å. It is can be seen from Crystal −1. As shown in 3 exhibits the highest thermal stability, with a decomposition temperature of 213.7 °C. Compounds 5 and 9 also show high thermal decomposition temperatures of 145.7 °C and 173.4 °C, respectively. The observed high thermal stability can be attributed to the extensive hydrogen bonding interactions between the amino groups of the cation and the oxygen atoms of the anion within these compounds. These strong intermolecular interactions contribute to the overall stability of the materials.The thermal stabilities of the above energetic salts were determined using DSC measurements with a scanning rate of 5 °C min3–11 are in the range of 1.435–1.989 g cm−3, that most of them are higher than that of TNT (1.65 g cm−3).Density is one of the most important evaluation parameters used to measure the performance of energetic materials, because it has a huge effect on their detonation performances . The den6 (−12.6%), 7 (0%), 8 (0%) and 10 (−16.4%) are much better than that of TNT (−24.7%) and RDX (−21.6%). Compound 7 and 8 are the best in these salts, where the oxygen balance approaches zero, which means that the combustible element of the energetic materials 7 and 8 can be completely oxidized and release maximum heat.Oxygen balance is a measure of the degree of oxidation in an explosive. Currently, most common explosives have a negative oxygen balance, which means they do not have enough oxidizing agents to completely oxidize the combustible elements. As a result, their detonation performances do not reach their maximum potential . The CO 3 exhibited excellent sensitivity parameters, with an impact sensitivity (IS) exceeding 40 J, meaning it can be classified as an insensitive energetic material. On the other hand, 5-nitrotetrazolate-2N-oxide salt 10 demonstrated high sensitivity to impact, with a recorded value of 6.1 J. In comparison to RDX, it was found that all the salts, except for 5-nitrotetrazolate salt 9 and 5-nitrotetrazolate-2N-oxide salt 10, displayed lower sensitivities. This suggests that the majority of the synthesized salts possess reduced sensitivity to impact when compared to that of RDX, which is a desirable characteristic in terms of safety and stability. The impact sensitivities of the synthesized energetic salts were measured by using a BAM drop hammer . Compoun−1 and the detonation pressures are in the range of 17.0–34.2 GPa. These values indicate significantly improved performance compared to that of TNT  with the exception of compound 3. The oxygen-balanced explosives 7 and 8 show the best performance; 7 has the highest detonation pressure (34.2 GPa) and 8 has the highest detonation velocity (8887 m s−1), which is comparable to that of RDX.EXPLO5 V6.01 was used to calculate the detonation velocities and pressures with the data of heats of formation, formula and density ,20. The 5 and 9 ; elemental analysis (%) Calc. for C3H10N6Cl2 (MW = 201.06 g·mol−1): C, 17.91; H, 4.98; N, 41.79; found C, 17.85; H, 5.05; N, 41.27; IR (KBr): 3182, 2836, 2659, 1943, 1704, 1609, 1569, 1541, 1484, 1423, 1377, 1322, 1263, 1228, 1165, 1141, 1092, 1046, 995, 966, 822, 750, 711, 616, 553 cm−1; 1H NMR (DMSO) δ: 9.92, 9.31; 13C NMR (DMSO) δ: 156.95, 153.45 ppm.For 4-Amino-3-hydrazino-5-methyl-1,2,4-triazolium hydrochloride (4): Compound 3  was dissolved in 30 mL of distilled water. While stirring, 16.8 mL of sodium bicarbonate (10 wt%) was added dropwise. The colorless solutions turned purple. The mixture was stirred for 30 min and evacuated under vacuum to produce a purple mixture. The mixture was dissolved in 25 mL of absolute methanol and the inorganic salt was removed via filtration. After filtering, the solvent was removed under reduced pressure to produce a purple solid of 4 at a 73% (0.934 g) yield. MS m/z (ESI+): 129.1 [C3H9N6+]; elemental analysis (%) calcd. for C3H8N6 (MW = 128.14 g·mol−1): C 28.13, H 6.25, N 65.63; found C 28.07, H 6.37, N 64.73; IR (KBr): 3344, 3215, 3026, 2843, 2661, 1965, 1620, 1585, 1526, 1486, 1254, 1232, 1091, 1051, 997, 835, 742, 654 cm−1; 1H NMR (DMSO) δ: 9.62, 9.15, 8.26 ppm; 13C NMR (DMSO) δ: 174.35, 152.25, 20.0 ppm.4-Amino-3-hydrazino-5-methyl-1,2,4-triazolium  in distilled water (20 mL), or silver 5-nitroiminotetrazolate (5 mmol) in distilled water (10 mL) was added dropwise to the solution of compound 11  in distilled water (20 mL). After stirring at room temperature for 1 h, the precipitate was filtered and rinsed with 10 mL of distilled water. The solvent was evaporated in vacuum and the residue was recrystallized from methanol and dried in air to obtain the target product.6): Yield: 96%, white crystals. MS m/z (ESI+): 129.1 [C3H9N6+]; elemental analysis (%) calcd for C3H10N8O6 (MW = 254.16 g·mol−1): C 14.17, H 3.94, N 44.09; found C 14.21, H 4.15, N 43.38; IR (KBr): 3319, 2847, 2724,1643, 1614, 1558, 1418, 1293, 1234,1159, 1077, 1051, 1036, 966, 947, 867, 815, 720, 702, 637, 618, 566 cm−1; 1H NMR(DMSO) δ: 10.30, 9.77, 6.08 ppm; 13C NMR (DMSO) δ: 169.99, 152.89 ppm.4-Amino-3-hydrazino-5-methyl-1,2,4-triazolium nitrate (7): Yield: 90%, colorless transparent crystals. MS m/z (ESI+): 129.1 [C3H9N6+]; elemental analysis (%) calcd for C3H10N6Cl2O8 (MW = 329.05 g·mol−1): C 10.94, H 3.04, N 25.53; found C 10.89, H 3.12, N 26.27; IR (KBr): 3327, 3131, 2989, 2695, 1645, 1536, 1455, 1421, 1320, 1235, 1185, 1039, 946, 835, 712, 668, 555 cm−1; 1H NMR(DMSO) δ: 10.41, 9.55 8.43 ppm; 13C NMR (DMSO) δ: 152.53, 142.34 ppm.4-Amino-3-hydrazino-5-methyl-1,2,4-triazolium perchlorate (8): Yield: 88%, white solid. Elemental analysis (%) calcd for C3H10N12O8 (MW = 342.19 g·mol−1): C 10.53, H 2.92, N 49.12; found C 10.61, H 3.09, N 48.82; IR (KBr): 3326, 3162, 3148, 2412, 1932, 1774, 1565, 1412, 1393, 1318, 1285, 1261, 1219, 1086, 1048, 1005, 929, 885, 848, 757, 731, 671, 639, 557 cm−1; 1H NMR(MeOD) δ: 10.82, 9.87, 6.79 ppm; 13C NMR (CD4O) δ: 153.33, 144.34, 143.33 ppm.4-Amino-3-hydrazino-5-methyl-1,2,4-triazolium dinitramide (9): Yield: 93%, pale yellow solid. MS m/z (ESI+): 129.1 [C3H9N6+]; m/z (ESI−): 114.0 [CN5O2−]; elemental analysis (%) calcd for C5H10N16O4 (MW = 358.24 g·mol−1): C 16.76, H 2.79, N 62.57; found C 17.01, H 3.82, N 61.30; IR(KBr): 3355, 2935, 2692, 2100, 1736, 1667, 1608, 1504, 1450, 1320, 1269, 1228, 1187, 1170, 1148, 1106, 1069, 1056, 1039, 772, 746, 710, 615, 557 cm−1; 1H NMR (DMSO) δ: 9.91, 9.38 ppm; 13C NMR (DMSO) δ: 169.33, 157,28, 153.63, 151.31 ppm.4-Amino-3-hydrazino-5-methyl-1,2,4-triazolium 5-nitrotetra-zolate (10): Yield: 86%, pale yellow solid. MS m/z (ESI+): 129.1 [C3H9N6+]; elemental analysis (%) calcd for C5H10N16O6 (MW = 390.24 g·mol−1): C 15.38, H 2.56, N 57.44; found C 15.89, H 2.61, N 56.91; IR(KBr) ν: 3347, 3256, 3121, 3071, 2932, 2789, 1855, 1692, 1585, 1541, 1437, 1406, 1309, 1249, 1197, 1141, 1082, 1057, 996, 965, 934, 837, 775, 709, 638, 553 cm−1; 1H NMR(DMSO) δ: 10.83, 9.97, 8.28 ppm; 13C NMR(DMSO) δ: 161.63, 156.25, 155.13 ppm.4-Amino-3-hydrazino-5-methyl-1,2,4-triazolium 5-nitrotetra-zolate-2N-oxide (11): Yield: 83%, white solid. MS m/z (ESI+): 129.0 [C3H9N6+]; m/z (ESI−): 127.98 [CN6O2−]; elemental analysis (%) calcd for C4H10N12O2 (MW = 258.2 g·mol−1): C 18.60, H 3.88, N 65.12; found C 18.48, H 3.81, N 64.28; M.p. 164.5 °C; IR(KBr): 3440, 3348, 3277, 3090, 2978, 2901, 2733, 2679, 1709, 1651, 1599, 1540, 1441, 1328, 1268, 1234, 1158, 1063, 1033, 999, 869, 836, 685, 504, 425 cm−1; 1H NMR(DMSO) δ: 10.33, 9.13, 7.83, 7.68 ppm; 13C NMR(DMSO) δ: 157.32, 156.35, 150.37, 25.37 ppm.4-Amino-3-hydrazino-5-methyl-1,2,4-triazolium 5-nitroimino-tetrazolate (3 (AHMT·2HCl). The nitrogen-rich compound 3 was synthesized via the reaction of triaminoguanidine hydrochloride and acetic acid and then hydrolyzed with diluted hydrochloric acid. Via Brønsted acid–base reactions, a series of energetic salts based on compound 3 were synthesized and fully characterized. The structures of 3, 5 and 9 were confirmed via X-ray single-crystal diffraction. Cations can be either polyvalent, monovalent or bivalent. Among all energetic salts based on 4-amino-3-hydrazino-5-methyl-1,2,4-triazole, compound 3 shows the best stability, and dinitramide salt 8 shows excellent properties with a detonation pressure of 33.9 GPa, a detonation velocity of 8887 m s−1 and an optimal oxygen-balance state. Therefore, considering its excellent performance and desirable properties, compound 8 holds great potential for utilization in various areas within the energetic materials field.In this study, we successfully synthesized a new precursor, 4-amino-3-hydrazino-5-methyl-1,2,4- triazolium hydrochloride


Scabiosa L. species, S. caucasica M. Bieb. and S. ochroleuca L. (Caprifoliaceae), has led to the isolation of five previously undescribed oleanolic acid glycosides from an aqueous-ethanolic extract of the roots: 3-O-α-L-rhamnopyranosyl-(1→3)-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester, 3-O-β-D-xylopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester, 3-O-β-D-xylopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid, 3-O-β-D-xylopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-β-D-xylopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester, 3-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester. Their full structural elucidation required extensive 1D and 2D NMR experiments, as well as mass spectrometry analysis. For the biological activity of the bidesmosidic saponins and the monodesmosidic saponin, their cytotoxicity on a mouse colon cancer cell line (MC-38) was evaluated.In the field of research on medicinal plants from the Armenian flora, the phytochemical study of two  Scabiosa L.—especially S. caucasica M. Bieb—is one of the herbs most recognized in traditional Armenian medicine for its ability to heal skin irritation, scabies, and the symptoms of influenza +, in agreement with the molecular formula, C76H124O39. This suggests a molecular mass of 1660.The mass spectrum of compound 1 is composed of eight residues, as revealed by the HSQC correlations at δH/δC 5.38 /94.4, 5.21 (s)/100.1, 5.19 (s)/101.4, 4.55 /104.9, 4.52 /103.8, 4.43 /103.0, 4.42 /101.7, and 4.37 /103.2 ppm. The first sugar unit, linked at the C-3 position of the aglycon, was identified as an Ara, based on the HMBC cross-peaks at δH/δC 4.52 (Ara H-1)/89.2 (C-3) and 3.14  (H-3)/103.8 (Ara C-1). This was proven by the ROE correlation at δH/δH 4.52 (Ara H-1)/3.14 (H-3). To determine the oligosaccharidic chain linked to this Ara, our observations were based on the correlations observed in the HMBC and ROESY spectra. The HMBC spectrum of compound 1 displayed correlations at δH/δC 5.21 (Rha I H-1)/74.9 (Ara C-2), and reverse correlations at 3.79 (Ara H-2)/100.1 (Rha I C-1), 4.55 (Xyl I H-1)/80.7 (Rha I C-3), 4.42 (Glc I H-1)/76.9 (Xyl I C-4), 4.43 (Glc II H-1)/78.9 (Glc I C-4), and 5.19 (Rha II H-1)/82.7 (Glc II C-3). The structure of this chain was verified by ROESY cross-peaks at δH/δH at 5.21 (Rha I H-1)/3.79 (Ara H-2), 4.55 (Xyl I H-1)/3.87 (Rha I H-3), 4.42 (Glc I H-1)/3.72 (Xyl I H-4), 4.43 (Glc II H-1)/3.58 (Glc I H-4), 5.19 (Rha II H-1)/3.53 (Glc II H-3). For the glycosidic part, linked at the ester function at C-28, the HMBC cross-peak at δH/δC 5.38 (Glc III H-1)/178.0 (C-28), revealed the presence of a β-D-glucopyranosyl unit. The deshielded value of the position C-6 of this sugar at 68.1, and the HMBC cross-peak at δH/δC 4.37 (Glc IV H-1)/68.1 (Glc III C-6), suggested the 28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl sequence. This was confirmed by the NOE correlation at δH/δH 4.37 (Glc IV H-1)/3.79, 4.15 (Glc III H2-6). According to this analysis, the structure of compound 1 is established as 3-O-α-L-rhamnopyranosyl-(1→3)-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester (The glycosidic part of compound yl ester .2 in ESIMS (positive mode) showed a quasi-molecular ion peak at m/z 1815.8003 [M + Na]+, suggesting a molecular formula of C81H132O43, and a molecular mass of 1792. The difference of 132 u between 1 and 2 is related to a supplementary pentose moiety in the oligosaccharidic chain. The assignments of all the NMR signals of the molecule 2 revealed a structure almost superimposable to 1, except for the Glc II. In fact, the chemical shifts of the positions 2 and 4 were deshielded at δC/δH 76.7/3.44 and 76.9/3.74, respectively. HMBC correlations between δH/δC 4.38  (Xyl II H-1) and 76.7 (Glc II C-2), and between δH/δC 5.37 (s) (Rha II H-1) and 76.9 (Glc II C-4) proved the 2,4 disubstitution of the Glc II. Therefore, the structure of 2 is established as 3-O-β-D-xylopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester.The mass spectrum of compound 3, a quasi-molecular ion peak at m/z 1491.6949 [M + Na]+ in ESIMS (positive mode) revealed a molecular formula of C69H112O33 and a molecular mass of 1468. The main difference with 2, in the NMR spectra, is the value of δC 180.2 ppm of the carbonyl function at C28, which suggests a carboxylic group instead of an ester group in compound 2. This was confirmed by the lack of NMR signals corresponding to the gentiobiose unit, 28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester, which led to the conclusion of a monodesmosidic saponin, 3-O-β-D-xylopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid.For compound 4 in ESIMS (positive mode) showed a quasi-molecular ion peak at m/z 1785.7940 [M + Na]+, suggesting a molecular formula of C80H130O42, and a molecular mass of 1762. The comparison of the molecular weight of compounds 2 and 4, showed a difference of 30 u, which suggests the presence of a pentosyl group in 4, instead of a hexosyl group in 2. For the structure, the NMR signals of the main part of the molecule corresponding to 3-O-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester, are almost superimposable. The difference is located at the last three sugars of the 3-O-heterosidic chain. For compound 4, two β-D-xylopyranosyl (Xyl II and Xyl III), and one α-L-rhamnopyranosyl (Rha II) moieties were found, identified mainly by TOCSY, COSY, and HSQC spectra. Xyl II is linked to the Glc I moiety at the 4 position, according to the HMBC correlation between δH/δC 4.34  (Xyl II H-1) and 78.8 (Glc I C-4). Moreover, cross-peaks at δH/δC 4.25  (Xyl III H-1)/77.0 (Xyl II C-2), and δH/δC 3.62 (Xyl II H-4)/100.9 (Rha II C-1), were allowed to establish the β-D-xylopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-β-D-xylopyranosyl chain. Based on these conclusions, compound 4 is identified as 3-O-β-D-xylopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-β-D-xylopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester.The mass spectrum of compound 5, this molecule shares the same molecular mass of 1660 as compound 1, corresponding to C76H124O39. When the total assignment of the protons and carbons of compound 5 was achieved by 2D NMR, the only difference was located at the Glc II moiety. A deshielded value of δC 77.0 for Glc II-4, and an HMBC cross-peak at δH/δC 5.40 (s) (Rha II-1)/77.0 (Glc II-4), suggested this specific linkage. Thus, the structure of 5 was established as 3-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester.In the case of compound 1–5, found in S. caucasica and S. ochroleuca, have structural similarities with those found in S. semipapposa 25D-15 . For 1H and 13C NMR data , see m/z: 1683.7553 [M + Na]+ .3-O-β-D-xylopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester (2). White, amorphous powder. Melting point, 238°C. [α]25D-10 . For 1H and 13C NMR data , see m/z: 1815.8003 [M + Na]+ .3-O-β-D-xylopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid (3). White, amorphous powder. Melting point, 250 °C. [α]25D-17 . For 1H and 13C NMR data , see m/z: 1491.6949 [M + Na]+ .3-O-β-D-xylopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-β-D-xylopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester (4). White, amorphous powder. [α]25D-20 . For 1H and 13C NMR data , see m/z: 1785.7940 [M + Na]+ .3-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester (5). White, amorphous powder. Melting point, 226°C. [α]25D-12 . For 1H and 13C NMR data , see m/z: 1683.7553 [M + Na]+ .3COOH (25 mL) for 3 h at 95 °C. After extraction with CH2Cl2 (3 × 15 mL), the aqueous layer was evaporated to dryness with H2O until neutral, to produce the sugar residue (40 mg). Arabinose, glucose, xylose, and rhamnose were identified by comparison with authentic samples by TLC using CH3COOEt/CH3COOH/CH3OH/H2O (65:25:15:15). After purification of these sugars, by prep-TLC in the same solvent, the optical rotation of each purified sugar was measured as follows: L-arabinose: Rf = 0.54, [α]25D + 170 , D-glucose: Rf = 0.50, [α]25D + 110 , D-xylose: Rf = 0.60, [α]25D + 85 , and L-rhamnose: Rf = 0.69, [α]25D + 10 .An aliquot (150 mg) of a fraction-rich saponin was hydrolyzed with 2N aqueous CF® ENH204-FP) were seeded at the density of 2 × 104 cells in 96 well plates , and cultivated for 24 h before treatments in an RPMI 1640 medium  with 10% FBS . Cells were then treated with the isolated compounds at the concentrations from 1 to 50 µM, for 48 h in an RPMI 1640 medium without FBS (200 µL per well). Viability was measured using an MTS colorimetric assay , following the manufacturer’s recommendations. Briefly, 20 µL of MTS reagent was added into each well, and optical density at 490 nm was read using a microplate reader , after 1 h incubation at 37 °C, 5% CO2, in the dark. The results were expressed as concentrations of compounds producing 50% toxicity . Fluorouracil (5-FU) was used as positive control and exhibited IC50 values of 11.7 µM. The experiment was conducted in quadruplicate.MC-38 cells -α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester. These glycosides have structural similarities with those found not only in other Scabiosa species from the Dipsacoideae subfamily of the Caprifoliaceae, but also with Weigela species, from the Diervilloideae. From a chemotaxonomic point of view, we need to continue our investigations to determine whether this type of sequence could represent a chemotaxonomic marker of these two subfamilies. Compound 3 exhibits potent cytotoxicity on a mouse colon cancer cell line (MC-38), with an IC50 of 4.37 µM, highlighting the key role of the lack or the presence of an esterification of the C-28 position.The saponins,


Increasing awareness of the structure of microtubules has made tubulin a relevant target for the research of novel chemotherapies. Furthermore, the particularly high sensitivity of glioblastoma multiforme (GBM) cells to microtubule disruption could open new doors in the search for new anti-GBM treatments. However, the difficulties in developing potent anti-tubulin drugs endowed with improved pharmacokinetic properties necessitates the expansion of medicinal chemistry campaigns. The application of an ensemble pharmacophore screening methodology helped to optimize this process, leading to the development of a new tetrazole-based tubulin inhibitor. Considering this scaffold, we have synthesized a new family of tetrazole derivatives that achieved remarkable antimitotic effects against a broad panel of cancer cells, especially against GBM cells, showing high selectivity in comparison with non-tumor cells. The compounds also exerted high aqueous solubility and were demonstrated to not be substrates of efflux pumps, thus overcoming the main limitations that are usually associated with tubulin binding agents. Tubulin polymerization assays, immunofluorescence experiments, and flow cytometry studies demonstrated that the compounds target tubulin and arrest cells at the G2/M phase followed by induction of apoptosis. The docking experiments agreed with the proposed interactions at the colchicine site and explained the structure–activity relationships. Targeting tubulin is a well-validated strategy for the development of anticancer drugs , as demoTubulin is the main constituent of microtubules, which are part of the cytoske-leton of eukaryotic cells and have an essential role in cell division. The polymerization–depolymerization equilibria of microtubules are the basis of the function of the mitotic spindle, acting during cell mitosis for the accurate segregation of chromosomes between the two daughter cells . TubulinAlthough several binding sites have been described in tubulin ,22, onlyZ isomer to the less potent E isomer. However, the most promising representatives of colchicine site ligands have not succeeded and have failed to reach clinical applications mainly due to high toxicity  , low potThe high potency of CA-4 has encouraged researchers to search for non-isomerizable and aqueous soluble analogs of CA-4 . The solH-tetrazol-1-yl)-1-methyl-1H-indole (50 of 45 nM), showing the ability to cause mitotic arrest and microtubule network disruption pyridine ligand  were dried and stored using molecular sieves. TLC was performed on precoated silica gel polyester plates (0.25 mm thickness) through the use of a UV fluorescence indicator 254 (Polychrom SI F254). Chromatographic separations were performed on silica gel columns via flash  chromatography. The compound/stationary phase ratio was 1g of compound/50 g of silica gel.  signals . Chemica signals . A hybri12–15).Method A. General synthetic procedure for the preparation of carbaldehyde derivatives (compounds 3 (6 eq) was added to dry DMF (3 mL) at 0 °C and stirred for 30 min under N2 atmosphere. Then, 1 eq of the indole derivative  was added and the mixture was stirred at rt for 24 h. Then, the reaction was poured into ice water saturated with sodium acetate (10 mL) and kept at 4 °C overnight until precipitation. The resulting powder was filtered, dissolved in CH2Cl2 (15 mL), and dried over anhydrous Na2SO4. The solvent was evaporated under vacuum to obtain the carbaldehyde derivative that was purified by crystallization.POCl16–19).Method B. General synthetic procedure for the preparation of oxime derivatives  NH2OH·HCl (10 eq) and pyridine (10 eq) was heated in refluxing methanol (20–30 mL) for 24 h under N2 atmosphere. Then, the methanol was evaporated under vacuum, and CH2Cl2 (20–30 mL) was added. The organic layer was washed with NaCl saturated aqueous solution  until neutral pH. The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated under vacuum to obtain a mixture of both oximes (E+Z). The products were purified via flash column chromatography.A solution consisting of the aldehyde derivative (compound 20–23).Method C. General synthetic procedure for the preparation of carbonitrile derivatives  was dissolved in pyridine (4 mL), and an excess of acetic anhydride was added (18 eq). The mixture was refluxed for 48 h. The reaction was quenched by pouring onto ice, and the mixture was extracted with CH2Cl2 . The organic layer was washed with 1 M HCl , 5% NaHCO3 , NaCl saturated aqueous solution , dried over anhydrous Na2SO4, filtered, and evaporated under reduced pressure. The products were purified via flash column chromatography.The mixture of 1, 3–6, and 8 [2 [Compounds 6, and 8  and compand 8 [2  were preN-(1-methyl-1H-indol-5-yl)pyridine-3-carboxamide (7). A solution of 2 , EDC , and DMAP  in CH2Cl2 (30 mL) was stirred for 1 h at room temperature (rt). Then, compound 5  was added, and the mixture was stirred under reflux for 24 h. Subsequently, the mixture was poured into cold water (30 mL), and the organic layer was washed with 1 M HCl , NaHCO3 , NaCl saturated aqueous solution , dried over anhydrous Na2SO4, filtered and the solvent was evaporated under vacuum. The crude was purified via column chromatography (1:3–1:1 EtOAc-Hexane) and crystallized in EtOAc-Hexane to obtain 1.71 g of pure product . Rf = 0.25 (1:1 EtOAc-Hexane). M. p.: 187–189 °C. IR (KBr): 3435, 1651, 1604, 1547, 1493 cm−1. 1H-NMR : δ 3.81 , 3.93 , 6.46 , 7.07 , 7.28–7.33 , 7.69 , 7.91 . 13C-NMR : δ 33.0 (CH3), 54.8 (CH3), 101.6 (CH), 108.6 (CH), 110.6 (CH), 113.4 (CH), 115.3 (CH), 116.6 (CH), 129.4 (C), 130.9 (CH), 131.5 (C), 135.3 (C), 149.5 (C), 149.6 (C), 162.3 (C), 165.3 (C). HRMS (C16H14N3O2Cl) m/z: calculated 338.0663 (M + Na+); found 338.0666 (M + Na+).2-chloro-6-methoxy-H-tetrazol-1-yl)-1-methyl-1H-indole (9). A solution consisting of compound 7  and sodium azide  was prepared in acetonitrile (7 mL). Silicon tetrachloride  was added dropwise, and the mixture was stirred under reflux for 48 h. Then, it was cooled down in an ice bath and the reaction was quenched by dilution with CH2Cl2 (14 mL) and 5% NaHCO3 (20 mL) and vigorously stirred for 30 min. The aqueous layer was extracted with EtOAc , and the organic layers were washed with brine , then dried over anhydrous Na2SO4, filtered, and evaporated under reduced pressure. The product was purified via column chromatography (1:3–1:1 EtOAC-Hexane) and crystallized in MeOH to obtain 413 mg of pure product . Rf = 0.52 (1:1 EtOAc-Hexane). M. p.: 149–150 °C. IR (KBr): 1613, 1551, 1493, 1240, 1155 cm−1. 1H-NMR : δ 3.86 , 3.88 , 6.56 , 6.72 , 7.12 , 7.17 , 7.23 , 7.44 , 7.62 . 13C-NMR : δ 33.3 (CH3), 54.5 (CH3), 102.1 (CH), 108.7 (CH), 110.5 (CH), 115.2 (CH), 118.2 (CH), 118.3 (CH), 125.6 (C), 128.6 (C), 131.7 (CH), 136.2 (C), 137.1 (C), 149.5 (C), 150.1 (C), 163.9 (C). HRMS (C16H13N6OCl) m/z: calculated 341.0908 (M + H+); found 341.0912 (M + H+).5-(5-(2-chloro-6-methoxypyridin-4-yl)-1H-tetrazol-1-yl)-1-methyl-1H-indole (10). Sodium methanethiolate  and KOH  were added to a solution of compound 8  in dry DMF (20 mL), and the mixture was refluxed for 24 h. Then, the mixture was cooled down, and the solvent was evaporated under vacuum. The crude was dissolved in EtOAc (20 mL), and the organic layer was washed with 1 M HCl (20 mL) and NaCl saturated aqueous solution , dried over anhydrous Na2SO4, filtered, and evaporated under reduced pressure. The product was purified via column chromatography (7:3 EtOAC-Hex) and crystallized in MeOH to obtain 332 mg . Rf = 0.57 (1:1 EtOAc-Hexane). M. p.: 167–168 °C. IR (KBr): 2922, 1541, 1433, 1301 cm−1. 1H-NMR : δ 2.44 , 3.90 , 6.60 , 7.14 , 7.15 , 7.24 , 7.26 , 7.46 , 7.65 . 13C-NMR : δ 13.5 (CH3), 33.3 (CH3), 102.3 (CH), 110.6 (CH), 117.6 (CH), 118.2 (CH), 118.3 (CH), 118.5 (CH), 125.4 (C), 128.7 (C), 131.8 (CH), 134.1 (C), 137.2 (C), 151.0 (C), 151.7 (C), 162.7 (C). HRMS (C16H13N6SCl) m/z: calculated 357.0683 (M + H+); found 357.0684 (M + H+).5-(5-(2-chloro-6-(methylsulfanyl)pyridin-4-yl)-1H-tetrazol-1-yl)-1-methyl-1H-indole (11). Compound 11 was synthesized following the same methodology as compound 10, i.e., by using a solution consisting of compound 10  in dry DMF (30 mL), sodium methanethiolate , and KOH . The product was purified via column chromatography (7:3 EtOAC-Hexane) and crystallized in MeOH to obtain 524 mg of compound 11 . Rf = 0.61 (1:1 EtOAc-Hexane). M. p.: 160–161 °C. IR (KBr): 2926, 1593, 1434, 1312, 1300 cm−1. 1H-NMR : δ 2.46 , 3.89 , 6.58 , 6.99 , 7.15 , 7.23 , 7.45 , 7.65 . 13C-NMR : δ 13.5 (2CH3), 33.3 (CH3), 102.2 (CH), 110.4 (CH), 115.0 (2CH), 118.3 (CH), 118.4 (CH), 125.9 (C), 128.6 (C), 131.6 (CH), 131.7 (C), 137.1 (C), 151.7 (C), 161.0 (2C). HRMS (C17H16N6S2) m/z: calculated 369.0944 (M + H+); found 369.0950 (M + H+).5-pyridin-4-yl)-1H-tetrazol-1-yl)-1-methyl-1H-indole-3-carbaldehyde (12). Obtained as described in method A from DMF (3 mL), POCl3 , and compound 8 . The crude was crystallized in Et2O to obtain 451 mg of pure product . M. p.: 198–199 °C. IR (KBr): 2925, 1661, 1538, 1434 cm−1. 1H-NMR : δ 3.93 , 7.46 , 7.63 , 7.78 , 8.33 , 8.45 , 9.89 . 13C-NMR : δ 33.9 (CH3), 112.5 (CH), 117.3 (C), 118.9 (CH), 121.0 (CH), 123.1 (2CH), 124.6 (C), 128.1 (C), 138.1 (C), 138.3 (C), 143.8 (CH), 149.9 (2C), 150.0 (C), 184.9 (CH). HRMS (C16H10N6OCl2) m/z: calculated 395.0184 (M + Na+); found 395.0185 (M + Na+).5--1H-tetrazol-1-yl)-1-methyl-1H-indole-3-carbaldehyde (13). Obtained as described in method A from DMF (3 mL), POCl3 , and compound 9 . The crude was crystallized in Et2O to obtain 379 mg of pure product . M. p.: 214–215 °C. IR (KBr): 2944, 1659, 1461, 1433, 1220 cm−1. 1H-NMR : δ 3.79 , 3.95 , 6.83 , 7.26 , 7.50 , 7.79 , 8.33 , 8.48 , 9.91 . 13C-NMR : δ 34.2 (CH3), 54.8 (CH3), 109.8 (CH), 112.8 (CH), 116.1 (CH), 117.6 (C), 119.2 (CH), 121.6 (CH), 124.9 (C), 128.8 (C), 137.6 (C), 138.5 (C), 144.0 (CH), 148.4 (C), 151.6 (C), 163.7 (C), 185.2 (CH). HRMS (C17H13N6O2Cl) m/z: calculated 369.0860 (M + H+); found 369.0861 (M + H+).5-(5-(2-chloro-6-methoxypyridin-4-yl)-1H-tetrazol-1-yl)-1-methyl-1H-indole-3-carbaldehyde (14). Obtained as described in method A from DMF (3 mL), POCl3 , and compound 10 . The crude was crystallized in Et2O to obtain 315 mg of pure product . M. p.: 201–202 °C. IR (KBr): 2938, 1671, 1448, 1435, 1211 cm−1. 1H-NMR : δ 2.48 , 3.89 , 7.59–7.61 , 7.77 , 8.25 , 8.42 , 9.89 . 13C-NMR : δ 13.8 (CH3), 33.9 (CH3), 110.4 (CH), 116.0 (CH), 117.0 (C), 117.6 (CH), 117.8 (CH), 121.2 (CH), 124.7 (C), 128.3 (C), 136.2 (C), 136.8 (C), 143.6 (CH), 149.5 (C), 151.0 (C), 161.8 (C), 184.4 (CH). HRMS (C17H13N6OSCl) m/z: calculated 385.0633 (M + H+); found 385.0634 (M + H+).5-(5-(2-chloro-6-(methylsulfanyl)pyridin-4-yl)-1H-tetrazol-1-yl)-1-methyl-1H-indole-3-carbaldehyde (15). Obtained as described in method A from DMF (3 mL), POCl3 , and compound 11 . The crude was crystallized in Et2O to obtain 456 mg of pure product . M. p.: 207–208 °C. IR (KBr): 2927, 1665, 1440, 1432, 1219 cm−1. 1H-NMR : δ 2.45 , 3.88 , 7.59 , 7.78 , 7.81 , 8.20 , 8.40 , 9.89 . 13C-NMR : δ 13.7 (2CH3), 33.7 (CH3), 111.0 (CH), 115.1 (2CH), 117.2 (C), 119.5 (CH), 121.3 (CH), 125.1 (C), 128.1 (C), 137.5 (C), 137.9 (C), 143.3 (CH), 151.3 (C), 160.5 (2C), 185.3 (CH). HRMS (C18H16N6OS2) m/z: calculated 397.0900 (M + H+); found 397.0904 (M + H+).5-pyridin-4-yl)-1H-tetrazol-1-yl)-1-methyl-1H-indole-3-carbaldehyde oxime (16). Obtained as described in method B from compound 12 , NH2OH·HCl , and pyridine  in MeOH (20 mL). The product was purified via flash column chromatography (1:4–1:1 AcOEt-Hexane) to obtain 221 mg of the Z+E mixture of 16 . Rf = 0.20 (4:3 EtOAc-Hexane). IR (KBr): 3337, 1601, 1541, 1523, 1433 cm−1. 1H-NMR : δ 3.92 , 3.93 , 7.14–7.70 , 7.43–7.60 , 7.67 , 7.88 , 8.24 , , 8.37 . 13C-NMR : δ 34.8 (CH3), 110.3 (C), 112.0 (CH), 118.3 (CH), 120.0 (CH), 123.8 (2CH), 125.8 (C), 128.6 (C), 136.1 (C), 137.0 (C), 137.7 (CH), 148.1 (CH), 151.7 (2C), 152.4 (C). HRMS (C16H11N7O2Cl2) m/z: calculated 388.0475 (M + H+); found 388.0475 (M + H+).5--1H-tetrazol-1-yl)-1-methyl-1H-indole-3-carbaldehyde oxime (17). Obtained as described in method B from compound 13 , NH2OH·HCl  and pyridine  in MeOH (20 mL). The product was purified via flash column chromatography (1:4–1:1 AcOEt-Hexane) to obtain 191 mg of the Z+E mixture of 17 . Rf = 0.17 (4:3 EtOAc-Hexane). IR (KBr): 3272, 1613, 1551, 1522, 1465, 1219 cm−1. 1H-NMR : δ 3.88 , 3.96 , 7.17–7.22 , 7.29 ,7.40–7.56 , 7.66 , 8.22 , 8.28 , 8.37 , 8.62 . 13C-NMR : δ 33.9 (CH3), 54.3 (CH3), 110.4 (C), 111.7 (CH), 112.8 (CH), 117.7 (CH), 119.1 (CH), 121.3 (CH), 124.6 (C), 128.4 (C), 137.0 (CH), 139.0 (C), 139.2 (C), 148.6 (CH), 149.7 (C), 151.1 (C), 162.0 (C). HRMS (C17H14N7O2Cl) m/z: calculated 384.0970 (M + H+); found 384.0974 (M + H+).5-(5-(2-chloro-6-methoxypyridin-4-yl)-1H-tetrazol-1-yl)-1-methyl-1H-indole-3-carbaldehyde oxime (18). Obtained as described in method B from compound 14 , NH2OH·HCl , and pyridine  in MeOH (20 mL). The product was purified via flash column chromatography (1:3–1:1 AcOEt-Hexane) to obtain 224 mg of the Z + E mixture of 18 . Rf = 0.22 (4:3 EtOAc-Hexane). IR (KBr): 3269, 1625, 1548, 1520, 1460, 1225 cm−1. 1H-NMR : δ 3.81 , 3.95 , 7.05 , 7.25–7.35 , 7.49 , 7.95 , 8.11 , 8.66 . 13C-NMR : δ 13.8 (CH3), 33.9 (CH3), 107.4 (CH), 110.8 (C), 111.3 (CH), 118.2 (CH), 119.5 (CH), 120.8 (CH), 125.1 (C), 128.3 (C), 137.4 (CH), 138.6 (C), 139.0 (C), 148.0 (CH), 150.2 (C), 151.7 (C), 163.0 (C). HRMS (C17H14N7OSCl) m/z: calculated 400.0742 (M + H+); found 400.0744 (M + H+).5-(5-(2-chloro-6-(methylsulfanyl)pyridin-4-yl)-1H-tetrazol-1-yl)-1-methyl-1H-indole-3-carbaldehyde oxime (19). Obtained as described in method B from compound 15 , NH2OH·HCl , and pyridine  in MeOH (30 mL). The product was purified via flash column chromatography (1:4–1:1 AcOEt-Hexane) to obtain 267 mg of the Z+E mixture of 19 . Rf = 0.25 (4:3 EtOAc-Hexane). IR (KBr): 3251, 1619, 1545, 1522, 1463, 1219 cm−1. 1H-NMR : δ 2.48 , 3.98 , 7.23–7.47 , 7.49–7.54 , 7.97 , 8.12 , 8.75 . 13C-NMR : δ 13.8 (2CH3), 33.9 (CH3), 110.7 (C), 111.2 (CH), 115.4 (2CH), 119.7 (CH), 120.4 (CH), 125.1 (C), 128.2 (C), 136.6 (CH), 137.1 (C), 139.1 (C), 148.1 (CH), 152.0 (C), 160.6 (2C). HRMS (C18H17N7OS2) m/z: calculated 412.1009 (M + H+); found 412.1013 (M + H+).5-pyridin-4-yl)-1H-tetrazol-1-yl)-1-methyl-1H-indole-3-carbonitrile (20). Obtained as described in method C from compound 16 , acetic anhydride  in pyridine (4 mL). The product was purified via flash column chromatography (1:4–1:1 AcOEt-Hexane) to obtain 72 mg of pure product . Rf = 0.40 (1:1 EtOAc-Hexane). IR (KBr): 2221, 1577, 1548, 1520, 1460 cm−1. 1H-NMR : δ 3.94 , 7.19 , 7.23 , 7.56 , 7.76 , 7.80 . 13C-NMR : δ 33.5 (CH3), 85.1 (C), 112.0 (CH), 115.0 (C), 118.2 (CH), 119.7 (CH), 124.1 (2CH), 126.0 (C), 128.1 (C), 136.2 (C), 136.7 (CH), 138.1 (C), 149.7 (2C), 151.1 (C). HRMS (C16H9N7Cl2) m/z: calculated 392.0189 (M + Na+); found 392.0191 (M + Na+).5--1H-tetrazol-1-yl)-1-methyl-1H-indole-3-carbonitrile (21). Obtained as described in method C from compound 17 , acetic anhydride  in pyridine (4 mL). The product was purified via flash column chromatography (1:4–1:1 AcOEt-Hexane) to obtain 105 mg of pure product . Rf = 0.39 (1:1 EtOAc-Hexane). IR (KBr): 2221, 1589, 1460, 1437, 1222 cm−1. 1H-NMR : δ 3.89 , 3.90 , 6.72 , 7.13 , 7.31 , 7.59 , 7.77 , 7.84 . 13C-NMR : δ 33.5 (CH3), 54.8 (CH3), 85.0 (C), 110.0 (CH), 112.4 (CH), 115.2 (C), 117.0 (CH), 119.4 (CH), 122.2 (CH), 124.3 (C), 128.2 (C), 134.1 (CH), 137.0 (C), 137.4 (C), 151.0 (C), 151.4 (C), 162.2 (C). HRMS (C17H12N7OCl) m/z: calculated 388.0684 (M + Na+); found 388.0688 (M + Na+).5-(5-(2-chloro-6-methoxypyridin-4-yl)-1H-tetrazol-1-yl)-1-methyl-1H-indole-3-carbonitrile (22). Obtained as described in method C from compound 18 , acetic anhydride  in pyridine (4 mL). The product was purified via flash column chromatography (1:4–1:1 AcOEt-Hexane) to obtain 160 mg of pure product . Rf = 0.45 (1:1 EtOAc-Hexane). IR (KBr): 2221, 1590, 1454, 1440, 1211 cm−1. 1H-NMR : δ 2.53 , 4.01 , 7.15 , 7.35 , 7.42 , 7.59 , 7.77 , 7.81 . 13C-NMR : δ 13.7 (CH3), 32.0 (CH3), 85.0 (C), 107.1 (CH), 115.1 (C), 116.3 (CH), 118.7 (CH), 118.8 (CH), 121.3 (CH), 124.3 (C), 128.0 (C), 131.8 (C), 136.6 (C), 137.8 (CH), 151.0 (C), 151.5 (C), 161.4 (C). HRMS (C17H12N7SCl) m/z: calculated 404.0406 (M + Na+); found 404.0408 (M + Na+).5-(5-(2-chloro-6-(methylsulfanyl)pyridin-4-yl)-1H-tetrazol-1-yl)-1-methyl-1H-indole-3-carbonitrile (23). Obtained as described in method C from compound 19 , acetic anhydride  in pyridine (4 mL). The product was purified via flash column chromatography (1:4–1:1 AcOEt-Hexane) to obtain 198 mg of pure product . Rf = 0.49 (1:1 EtOAc-Hexane). IR (KBr): 2217, 1582, 1461, 1438, 1220 cm−1. 1H-NMR : δ 2.48 , 3.77 , 7.21 , 7.51 , 7.68 , 8.01 , 8.21 . 13C-NMR : δ 13.4 (2CH3), 33.0 (CH3), 85.3 (C), 111.2 (CH), 115.2 (C), 115.9 (2CH), 118.0 (CH), 120.7 (CH), 125.3 (C), 128.3 (C), 134.0 (C), 136.1 (CH), 137.9 (C), 151.2 (C), 160.6 (2C). HRMS (C18H15N7S2) m/z: calculated 416.0723 (M + Na+); found 416.0723 (M + Na+).5-pyridin-4-yl)-1The aqueous solubility of the compounds was determined using the saturation shake-flask method . First, v/v) heat-inactivated fetal bovine serum , 2 mM L-glutamine, 100 μg/mL streptomycin, and 100 units/mL penicillin  at 37 °C, 95% humidity, and 5% CO2. The HT-29 and HCT8 (human colon carcinoma) cell lines were grown in Roswell Park Memorial Institute 1640 (RPMI 1640)  supplemented with 10% HIFBS, 100 μg/mL streptomycin, and 100 units/mL penicillin  at 37 °C, 95% humidity, and 5% CO2. The cells were periodically tested for Mycoplasma infection by using a MycoAlert kit , and only Mycoplasma-free cells were used in the experiments.The cell lines were obtained from ATTC . HeLa (human cervix epithelioid carcinoma), MCF7 (human breast adenocarcinoma), U87 MG (human glioblastoma), T98G (TMZ-resistant human glioblastoma), HepG2 (human hepatocellular carcinoma), and HEK-293 (human embryonic kidney) were grown in Dulbecco’s modified Eagle’s medium  supplemented with 10%  or 15,000 cells/mL (HeLa and MCF7 cells). Then, every compound was added (10 µL/well) to a final concentration of 10 µM. Untreated cells were used as negative controls. The antiproliferative activity of compounds was measured 72 h after drug exposure through the MTT assay. The IC50 value (the drug concentration required to inhibit 50% of cell growth with respect to the untreated control) was determined for compounds showing antiproliferative effects in the initial screening at 10 µM. For this purpose, compounds were used at different concentrations, ranging from 10−10 to 10−2 M. Measurements were performed in triplicate, and each experiment was repeated three times. The IC50 values were determined using Origin software . To study the sensitivity to MDR efflux pumps of compounds, the IC50 values were determined following the same procedure but in the presence of verapamil .The effect of the compounds on the proliferation of human tumor cell lines was determined by using a MTT reagent , which was dissolved in PBS at 5 mg/mL in accordance with the manufacturer’s instructions. Cells were incubated in 96-well plates (100 µL/well) in complete DMEM or RPMI 1640 medium (see above) at 37 °C and 5% CO2, 1.5 mM GTP, 1.5 mg/mL of microtubular protein, and the measured ligand (0.1–10 µM). Control samples had the same composition but in absence of the ligand. The samples were incubated at 20 °C for 30 min to allow the ligands to bind to tubulin and cooled in ice for 10 min to make sure that polymerization did not take place at the initial time point. After reaching a stable plateau, the temperature was adjusted to 4 °C before being shifted to 37 °C to facilitate the assembly process of tubulin. Tubulin polymerization, caused when heating the samples, was accompanied by an increase in the absorbance (at 450 nm) of the sample that was monitored by using a Helios α spectrophotometer. The increase in absorbance obtained for the control sample was considered as 100% of tubulin polymerization (0% of inhibition), so a comparison with the increase in absorbance determined for samples containing ligands yielded the degree of tubulin polymerization inhibition (TPI), which is expressed as a percentage value. The IC50 for TPI (the drug concentration required to inhibit 50% of tubulin polymerization with respect to the control) was determined by measuring TPI at different concentrations of ligands (0.1–10 µM) and by performing calculations using Origin software . Measurements were performed in triplicate in two independent experiments using microtubular proteins from different preparations.Bovine brain tubulin was isolated as previously described by following a modified version of the Shelanski method . The pro4 cells/mL) were grown on 0.01% poly-L-lysine pre-coated square glass coverslips (22 mm2), deposited on 6-well plates (1 coverslip/well), and incubated in complete DMEM medium (see above) at 37 °C and 5% CO2 atmosphere for 24 h. Then, the cells were incubated for 24 h in the presence or absence (negative controls) of selected compounds  at 100 nM. After drug treatment incubation, medium was removed, and coverslips were washed three times with PBS, fixed with 4% formaldehyde in PBS for 10 min, permeabilized with 0.5% Triton X-100,  in PBS for 90 s at 4 °C, blocked with 10% BSA in PBS for 30 min, and washed with PBS four times. The coverslips were incubated for 1 h with anti-α-tubulin mouse monoclonal antibody , diluted 1:200 in PBS containing 3% BSA, washed four times with PBS, and then incubated in darkness with the fluorescent secondary antibody Alexa Fluor 488 goat anti-mouse IgG  and diluted 1:400 in PBS containing 1% BSA for 1.5 h. The coverslips were washed four times with PBS, and a drop of ProLong™ Gold Antifade Mountant containing DAPI  was added for cell nuclei staining. The samples were analyzed via confocal microscopy, which was performed by using a LEICA SP5 microscope DMI-6000V model coupled to a LEICA LAS AF software computer.HeLa, MCF7, and U87 MG cells  and incubated in complete DMEM Medium (see above) at 37 °C and 5% CO2 atmosphere for 24 h. Then, the cells were treated with selected compounds  at 100 nM, and untreated cells were used as negative controls. Cells were collected 24, 48, or 72 h following treatment and fixed overnight in ethanol/PBS (7:3) at 4 °C. After two washes with PBS, cells were suspended in PBS and incubated in darkness overnight with 0.2 mg/mL RNase A , 50 µg/mL propidium iodide , and Triton 10x at room temperature. A BD Accuri™ C6 Plus Flow Cytometer  cytometer was used to analyze the samples, and BD Accuri™ C6 Software (version 1.0.264.21) was used for data analysis.HeLa, MCF7, or U87 MG cells were seeded in 6-well plates  and incubated in complete DMEM medium (see above) at 37 °C and 5% CO2 atmosphere for 24 h. Then, the cells were treated with selected compounds  at 100 nM, and untreated cells were used as negative controls. Then, 72 h after incubation, the cells were harvested, centrifugated, resuspended in the Annexin V binding buffer, and incubated in darkness with Annexin V-FITC/PI for 15 min at room temperature. The samples were analyzed by using a BD Accuri™ C6 Plus Flow Cytometer (BD Biosciences), and acquired data were analyzed via BD Accuri™ C6 Software (version 1.0.264.21).Following the manufacturer’s guidelines, an Annexin V-FITC/PI apoptosis detection kit  was used to quantify HeLa, MCF7, or U87 MG cells. Cells were seeded in 12-well plates  level using Spartan 08 software package. The indole rings were kept unsubstituted at the 3-position to avoid long-range interactions that might have confounded the conformational searches. The structures were built and subjected to conformational searches at the molecular mechanism (MMFF force field) level. The retrieved conformations were then minimized by using RB3LYP DFT calculations at the 6–31G(D) level, and the lowest energy configurations amongst all the possible outcomes were selected as the most stable forms for every compound.Ensemble docking studies were performed as previously described, with the incorporation of a higher number of protein structures and with and without water molecules. The different tubulin–ligand complexes sampled the protein conformational space and helped measure the protein flexibility ,69. Brie8, 9, 10, 12, 13, and 14) showed nanomolar IC50 values, exhibiting particularly high potencies against the U87 MG glioblastoma cells and even against the TMZ-resistant T98G cells. To the best of our knowledge, this family represents one of the few examples of colchicine-site ligands where the high antimitotic potency is accompanied by high solubility. The synthesized compounds successively overcame the typical limitations of tubulin ligands such as low selectivity toward cancer cells, as evidenced by the fact that they exhibited selectivity indexes from 30 to 200 and the appearance of resistance mechanisms. Different and complementary techniques have been employed to elucidate the mechanism of action of the three representative compounds . The strong correlation between tubulin polymerization inhibition IC50 and the antiproliferative IC50 together with the severe microtubule network disruption of treated cells  proved that the interaction with tubulin is the mechanism at play. The effects on the cell microtubules 24 h after treatment also affected the cell cycle of the cancer cells, as revealed by the cell cycle histograms obtained 24, 48, and 72 h after treatment. The following common pattern was noted in all of the cases: the compounds were able to provoke severe mitotic arrest at an early time point (24 h), and the G2/M population decreases progressively at the expense of the increase in the subG0/G1 population, which reached its maximum value 72 h after treatment, showing good agreement with the results obtained in the antiproliferative experiments. Our studies of the cell death mechanism were consistent with the cell cycle assays and demonstrated apoptotic induction caused by the compounds. The DFT and docking experiments in the colchicine site of tubulin not only predicted a binding mode of synthesized compounds similar to CA-4 but also accounted for the observed structure–activity relationships. The best-performing ligands were those containing a formyl group at the 3-indole position, as they proved that they were able to make hydrogen bonds with the sidechain ammonium group of Lys352β, which is not encountered for oximes and nitriles derivatives. Regarding the effect of the substituents on the pyridine ring, the superiority of the ligands with chlorine atoms can be explained by their ability to make a halogen bond with the carbonyl oxygen of Val238β (H7), which is not possible for the least potent bismethylsulfanyl derivatives. The conformational penalties calculated for the methoxy derivatives account for their weaker interactions in comparison with 2,6-dichloro- and 2-chloro-6-methylsulfanylpyridine analogs, whereas the smaller size of 2,6-dichloropyridines explain their less efficient packing capacities compared to the 2-chloro-6-methylsulfanylpyridine counterparts. Overall, our results further justify the scientific interest in this class of tetrazole derivatives, and further research could be directed toward the development of new anti-GBM therapies targeting tubulin. Future in vivo experiments are necessary to explore the potential of these new molecules. The formyl group at the 3-indole position could help to improve the pharmacokinetic or pharmacodynamic properties of drugs developed for treatment by serving as an anchor for further derivatization. The described compounds are potential candidates for further clinical development, especially for the treatment of difficult-to-treat glioblastomas.Starting from an initial hit from an ensemble pharmacophore search for colchicine site ligands, a new family of diheteroaryl compounds has been designed, synthesized, and evaluated as antitumor compounds. The novel family of tetrazole-1-yl indole derivatives was prepared following an efficient synthetic methodology that is very suitable for the incorporation of further modifications on the two tetrazole-pending heteroaryl fragments. The best-performing candidates were compounds with a formyl group on the 3-indole position and the 2-chloro-6-methylsulfanyl-pyridine derivatives. All the synthesized derivatives were able to exert antiproliferative effects in the submicromolar range against a broad panel of cancer cells, and the most potent compounds (compounds


Two lead compounds (3c and 3d) exhibited a well-balanced multifunctional profile, demonstrating intriguing acetylcholinesterase (AChE) inhibition, promising antioxidant activity assessed by DPPH, ABTS, and FRAP methods, as well as the inhibition of lipid peroxidation in the linoleic acid system. Compound 3n, possessing two indole scaffolds, showed the highest activity against butyrylcholinesterase (BChE) and a high selectivity index (SI = 47.34), as well as a pronounced protective effect in H2O2-induced oxidative stress in SH-SY5Y cells. Moreover, compounds 3c, 3d, and 3n showed low neurotoxicity against malignant neuroblastoma cell lines of human (SH-SY5Y) and murine (Neuro-2a) origin, as well as normal murine fibroblast cells (CCL-1) that indicate the in vitro biocompatibility of the experimental compounds. Furthermore, compounds 3c, 3d, and 3n were capable of penetrating the blood–brain barrier (BBB) in the experimental PAMPA-BBB study. The molecular docking showed that compound 3c could act as a ligand to both MT1 and MT2 receptors, as well as to AchE and BchE enzymes. Taken together, those results outline compounds 3c, 3d, and 3n as promising prototypes in the search of innovative compounds for the treatment of AD-associated neurodegeneration with oxidative stress. This study demonstrates that hydrazone derivatives with melatonin and donepezil are appropriate for further development of new AChE/BChE inhibitory agents.Alzheimer’s disease (AD) is considered a complex neurodegenerative condition which warrants the development of multitargeted drugs to tackle the key pathogenetic mechanisms of the disease. In this study, two novel series of melatonin- and donepezil-based hybrid molecules with hydrazone ( Alzheimer’s disease is a progressive neurodegenerative disorder that primarily affects memory and cognitive functions ,2. The eCurrent approaches in AD pharmacotherapy based on the cholinergic hypothesis aim to increase the level of ACh. This can be achieved by influencing the activity of the cholinesterase enzymes (ChE). There are two isoenzymes which play a key role in hydrolyzing the mediator: ACh–acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). AChE activity is prominent in a healthy brain; however, in the case of AD, it remains unchanged or is slightly lowered, whereas BChE function may increase . The vasIn light of this, researchers continue to make efforts in the design and development of small molecules with good activity towards AChE because of their favorable “drug-like” properties, i.e., low molecular mass and good pharmacokinetic profiles, but also continue the quest for multi-target-directed ligands (MTDLs) with low hepatotoxicity and a good safety profile ,16.MTDLs are organic scaffolds designed to target multiple biological pathways simultaneously with the goal of synergistic enhancement of the therapeutic efficacy . Due to 2+/mitochondria pathways to ameliorate Aβ oligomer-induced neurotoxicity; and improves cognitive function and sleep quality in AD patients  ,35 3a–r 3a–r and sulfonyl hydrazones 5a–l, are provided in 50 as the mean of three measurements ± standard deviation. The enzyme inhibition percentage at 1 mM of the compounds is also given  belonging to the first family showed relatively high IC50 values . The derivative 3d with a p-MeO benzene ring had the lowest IC50 (9.77 ± 0.76) in the family. The presence of the methoxy group on the appropriate position in the benzene ring (3d) lead to more of an increase in inhibitory activity than the o-, p-disubstituted benzene ring (3b) and indole containing hybrids 3a and 3c, increasing the selectivity index for the compound 3d (SI = 20.19). Also, replacing the indole moiety in 3c with a 2,4-dihydroxy benzene ring in compound 3b caused a decrease in the AChE inhibitory activity. It is impressive that the introduction of a methylene group in the connecting hydrazone fragment in compound 3a, compared to the indole-donepezil hybrid 3c with the same moieties, lead to a loss of inhibitory activity towards AchE and an increase in activity towards the butyrylcholinesterase enzyme; the selective index is in favor of BChE (SI BChE = 1.35). The SAR, including molecular modelling studies, disclosed that the benzyl moiety provided interaction with CAS residues, while the indole moiety was oriented within PAS. On the other hand, compound 5a with sulfonyl hydrazone linkage showed the best IC50 (162.83 ± 10.73 μM) from this family, indicating that the sulfonyl hydrazone fragment probably decreased the inhibitory activity.None of the 30 tested compounds proved to be a more potent AChE inhibitor than the positive controls galanthamine and donepezil. The piperidinyl-containing derivatives  with o-, p- dihydroxy and o-p- trihydroxy benzene rings (3e and 3i), it seemed that a combination of two hydroxyl groups at the ortho- and para-position assisted AChE inhibition. The presence of three hydroxyl groups in the o- and para- position of the benzene ring led to increased BChE inhibitory activity (SI BChE = 1.96).Pyrrolidine-containing derivatives tert-butyl-2-hydrazinyl-2-oxoethylcarbamate derivatives from the third family with different substitutions in the indole ring (3j–m) did not show significant inhibitory activity towards both AchE and BchE in the measured concentration, compared with the results obtained from H. Zhang et al., 2022  cation radical decolorization method . The BHT3+)-ligand to the intensely blue ferrous complex (Fe2+) by means of antioxidants in acidic environments. Unlike other methods, the FRAP test is carried out in acidic pH conditions (pH = 3.6) to maintain solubility of iron complexes. Thus, the FRAP assay utilizes an SET (single electron transfer) mechanism to determine the capacity of an antioxidant in the reduction of an oxidant. With respect to the FRAP method, compound 3c has the strongest activity, followed by 5a, 5h, donepezil, 3m and 5j.The Ferric Reducing Antioxidant Power (FRAP) test measures the reduction in the complex of ferric ions  was determined using the FTC method in the linoleic acid system . During  control .3c, which was a promising acetylcholinesterase inhibitor from all new derivatives, showed the best antioxidant activity in the three tested methods FRAP, ABTS, and FTC. As a future perspective, hydrazone 3c will be evaluated in vivo in models of Alzheimer’s disease and melatonin deficiency as well as Aβ (1–42) aggregation.The presented data of direct and indirect antioxidant activities as measured by the DPPH, ABTS, FRAP, and FTC methods  demonstr3a–d, 3n and 5a) were further subjected to an in vitro evaluation of their neuroprotective properties against H2O2-induced oxidative stress damage.Consequently, the prioritized structures  in vitro involves oxidative stress and damage to cellular components. H2O2 can readily cross cell membranes and generate reactive oxygen species (ROS) through its spontaneous decomposition. ROS, including hydroxyl radicals and peroxyl radicals, can cause damage to DNA, proteins, and lipids, leading to cellular dysfunction and injury -2-(1H-indol-3-yl)acetohydrazide, 3a Yield: 68%; m.p. 167–168 °C. 1H NMR : 1:0.83 mixture of conformers; signals for major synperiplanar conformer around the amide bond: δ = 1.42–1.53 , 1.67–1.78 , 1.94–2.04 , 2.17–2.24 , 2.77–2.82 , 3.46 , 3.90 , 6.98 , 7.05 , 7.17 , 7.22–7.33 , 7.35 , 7.44 , 7.53 , 10.84 , 10.85 ; resolved signals for minor antiperiplanar conformer around the amide bond: 3.45 , 3.53 , 6.96 , 7.07 , 7.20 , 10.89 , 11.06 ; 13C NMR : signals for major synperiplanar conformer around the amide bond: δ = 28.85 (CH2), 29.06 (CH2), 38.10 (C-4′), 52.45 (CH2), 62.39 (CH2), 108.22 (C-3), 111.24 (C-7), 118.19 (C-5), 118.65 (C-4), 120.84 (C-6), 120.95 (C-5), 123.83 (C-2), 126.80 (C-4″), 127.14 (C-4a), 127.39 (C-4a), 128.11 (C-3″ and C-5″), 128.73 (C-2″ and C-6″), 135.95 (C-7a), 138.51 (C-1″), 149.21 (CH), 166.66 (C=0); resolved signals for minor antiperiplanar conformer around the amide bond: 152.96 (CH), 172.20 (C=O). HRMS (ESI) m/z: calcd: [M+H]+ 375.217938. Found: [M+H]+ 375.2178.N’-[(E)-(1-benzylpiperidin-4-yl)methylidene]-2,4-dihydroxybenzohydrazide, 3b Yield: 59%; m.p. 201–203 °C. 1H NMR : δ = 1.46  and 1.52 , 1.74 , 2.00–2.05 , 2.22–2.34 , 2.82 , 3.48 , 6.27 , 6.32 , 7.23–7.35 , 7.67 , 7.72 , 10.17 , 11.32 , 12.44 . 13C NMR : δ = 29.00 (C-3′ and C-5′), 38.53 (C-4′), 52.42 (C-2′ and C-6′), 62.36 (CH2), 102.81 (C-3), 105.87 (C-1), 107.19 (C-5), 126.86 (C-4″), 128.13 (C-3″ and C-5″), 128.78 (C-2′’ and C-6″), 129.35 (C-6), 138.41 (C-1″), 155.26 (CH), 162.49 and 162.53 (C-2 and C-4), 165.44 (C=O). HRMS (ESI) m/z: calcd: [M+H]+ 354.181218. Found: [M+H]+ 354.1811.N’-[(E)-(1-benzylpiperidin-4-yl)methylidene]-1H-indole-3-carbohydrazide, 3c Yield: 72%; m.p. 249–250 °C. 1H NMR : δ = 1.51 , 1.57 , 1.79 , 2.09 , 2.23–2.32 , 2.82 , 3.49 , 7.12 , 7.16 , 7.21–7.26 , 7.29–7.32 , 7.44 , 7.55 , 8.12 , 8.17 , 10.53 , 11.36 . 13C NMR : δ = 29.01 (C-3′ and C-5′), 37.97 (C-4′), 52.19 (C-2′ and C-6′), 62.11 (CH2), 108.42 (C-3), 111.44 (C-7), 120.21 (C-5), 120.95 (C-4), 121.73 (C-6), 126.43 (C-4″), 126.63 (C-4a), 127.74 (C-3″ and C-3″), 128.44 (C-2″ and C-6″), 129.27 (C-2), 135.69 (C-7a), 138.34 (C-1″), 150.89 (CH), 162.35 (C=O). HRMS (ESI) m/z: calcd: [M+H]+ 361.202288. Found: [M+H]+ 361.2021.N’-[(E)-(1-benzylpiperidin-4-yl)methylidene]-4-methoxybenzohydrazide, 3d Yield: 82%; m.p. 138–140 °C. 1H NMR : δ = 1.49  and 1.74 , 2.01  and 2.82 , 2.25 , 3.47 , 3.82 , 7.02 , 7.25 , 7.30–7.34 , 7.67 , 7.84 , 11.28 . 13C NMR : δ = 29.14 (C-2′ and C-6′), 38.57 (C-1′), 52.48 (C-3′ and C-5′), 55.37 (OCH3), 62.42 (CH2), 113.60 (C-3 and C-5), 125.58 (C-1), 126.82 (C-4″), 128.12 (C-3″ and C-5″), 128.76 (C-2″ and C-6″), 129.34 (C-2 and C-6), 138.51 (C-1″), 154.21 (CH), 161.80 (C-4), 162.23 (C=O). HRMS (ESI) m/z: calcd: [M+H]+ 352.201953. Found: [M+H]+ 352.20115.1-benzyl-N’-[(E)-(4-hydroxyphenyl)methylidene]pyrrolidine-3-carbohydrazide, 3e Yield: 84%; m.p. 240–241 °C. 1H NMR : 1:0.77 mixture of conformers; signals for major synperiplanar conformer around the amide bond: δ = 1.94–2.03 , 2.40–2.42 , 2.62–2.69 , 2.88–2.91 , 3.55–3.62 , 3.61–3.66 , 6.79 , 7.22–7.26 , 7.30–7.33 , 7.44 , 7.85 , 9.84 , 11.05 ; resolved signals for minor antiperiplanar conformer around the amide bond: 8.04 , 9.87 , 11.10 . 13C NMR : signals for major synperiplanar conformer around the amide bond: δ = 26.95 (C-4′), 41.47 (C-3′), 53.58 (C-5′), 56.43 (C-2′), 59.30 (CH2), 115.65 (C-3 and C-5), 125.37 (C-1), 126.75 (C-4″), 128.11 (C-2″ and C-6″), 128.28 (C-2 and C-6), 128.65 (C-3″ and C-5″), 139.16 (C-1″), 142.74 (CH), 158.99 (C-4), 175.01 (C=O); resolved signals for minor antiperiplanar conformer around the amide bond: 146.34 (CH), 159.22 (C-4), 169.78 (C=O). HRMS (ESI) m/z: calcd: [M+H]+ 324.170653. Found: [M+H]+ 324,1705.1-benzyl-N’-[(E)-methylidene]pyrrolidine-3-carbohydrazide, 3f Yield: 81%; m.p. 224–225 °C. 1H NMR : δ = 1:0.39 mixture of conformers; signals for major synperiplanar conformer around the amide bond: δ = 1.92–2.04 , 2.41–2.48 , 2.66–2.69 , 2.82–2.94 , 3.52–3.57 , 3.57–3.61 , 6.29 , 6.33 , 7.25 , 7.29–7.33 , 8.21 , 9.91 , 11.32 , 11.36 ; resolved signals for minor antiperiplanar conformer around the amide bond: 8.11 , 9.79 , 10.11 , 11.04 . 13C NMR : signals for major synperiplanar conformer around the amide bond: δ = 27.47 (C-4′), 41.23 (C-3′), 53.52 (C-5′), 56.83 (C-2′), 59.17 (CH2), 102.58 (C-3), 107.57 (C-5), 110.41 (C-1), 126.81 (C-4′’), 128.14 (C-2′’ and C-6′’), 128.51 (C-3′’ and C-5′’), 131.16 (C-6), 139.17 (C-1′’), 147.58 (CH), 159.27 (C-2), 160.52 (C-4), 169.58 (C=O); resolved signals for minor antiperiplanar conformer around the amide bond: 141.95 (CH), 157.97 (C-2), 160.22 (C-4), 174.44 (C=O). HRMS (ESI) m/z: calcd: [M+H]+ 340.165568. Found: [M+H]+ 340.1655.1-benzyl-N’-[(E)-methylidene]pyrrolidine-3-carbohydrazide, 3g Yield: 72%; m.p. 199–201 °C. 1H NMR : 1:0.65 mixture of conformers; signals for major synperiplanar conformer around the amide bond: δ = 2.57 , 2.63 , 3.32–3.37 , 3.57 , 3.91 , 4.35  and 4.46 , 6.75 , 6.89 , 7.09 , 7.23–7.25 , 7.27–7.30 , 7.34–7.37 , 7.80 , 9.15 , 9.39 , 11.23 ; resolved signals for minor antiperiplanar conformer around the amide bond: 7.96 , 9.24 , 9.37 , 11.26 . 13C NMR : signals for major synperiplanar conformer around the amide bond: δ = 33.54 (C-3′), 35.41 (C-4′), 45.79 (CH2), 48.98 (C-5′), 49.37 (C-2′), 113.21 (C-2), 116.05 (C-5), 120.37 (C-6), 126.02 (C-1), 127.73 (C-4″), 128.05 (C-2″ and C-6″), 129.06 (C-3″ and C-5″), 137.24 (C-1″), 144.49 (CH), 146.11 (C-3), 148.17 (C-4), 172.83 (C=O); resolved signals for minor antiperiplanar conformer around the amide bond: 147.81 (CH), 146.15 (C-3), 148.43 (C-4), 173.81 (C=O). HRMS (ESI) m/z: calcd: [M+H]+ 340.165568. Found: [M+H]+ 340.1646.1-benzyl-N’-[(E)-methylidene]pyrrolidine-3-carbohydrazide, 3h Yield: 65%; m.p. 215–217 °C. 1H NMR : 1:0.19 mixture of conformers; signals for major synperiplanar conformer around the amide bond: δ = 1.91–2.03 , 2.41–2.47 , 2.66–2.69 , 2.81–2.85 , 2.85–2.91 , 3.57  and 3.60 , 5.81 , 7.23–7.27 , 7.30–7.33 , 8.49 , 9.77 , 10.95 , 11.33 ; resolved signals for minor antiperiplanar conformer around the amide bond: 8.37 , 9.80 , 10.51 , 11.12 . 13C NMR : signals for major synperiplanar conformer around the amide bond: δ = 27.47 (C-3′), 41.19 (CH2), 53.50 (C-4′), 56.81 (C-5′), 59.16 (C-2′), 94.29 (C-3 and C-5), 98.80 (C-1), 126.81 (C-4″), 128.14 (C-2″ and C-6″), 128.51 (C-3″ and C-5″), 139.12 (C-1″), 145.15 (CH), 159.48 (C-2 and C-6), 161.34 (C-4), 169.21 (C=O). HRMS (ESI) m/z: calcd: [M+H]+ 356.160483. Found: [M+H]+ 356.1604.tert-butyl(2-{(2E)-2-[(1-benzyl-1H-indol-3-yl)methylidene]hydrazinyl}-2-oxoethyl)carbamate, 3i1H NMR, 13C NMR and HRMS spectra of compound 3i were published elsewhere [lsewhere .tert-butyl(2-{(2E)-2-[(5-methoxy-1-methyl-1H-indol-3-yl)methylidene]hydrazinyl}-2-oxoethyl)carbamate, 3j Yield: 48%; m.p. 203–206 °C. 1H NMR : 1:0.28 mixture of conformers; signals for major synperiplanar conformer around the amide bond: δ = 1.41 , 3.79 , 4.16 , 6.76 , 6.85 , 7.34 , 7.62 , 7.72 , 8.14 , 11.00 , 11.40 ; resolved signals for minor antiperiplanar conformer around the amide bond: 8.35 , 11.09 , 11.40 . 13C NMR : signals for major synperiplanar conformer around the amide bond: δ = 28.22 (CH3), 41.39 (CH2), 55.05 (OCH3), 77.88 (OC), 103.48 (C-4), 111.10 (C-3), 112.16 (C-7), 112.53 (C-6), 124.63 (C-3a), 130.50 (C-2), 131.93 (C-7a), 140.68 (CH), 154.37 (C-5), 155.89 (OC=O), 169.74 (C=O). HRMS (ESI) m/z: calcd: [M+H]+ 347.171382. Found: [M+H]+ 347.1712.tert-butyl(2-{(2E)-2-[(5-methoxy-1H-indol-3-yl)methylidene]hydrazinyl}-2-oxoethyl)carbamate, 3k Yield: 83%; m.p. 197–199 °C. 1H NMR : 1:0.33 mixture of conformers; signals for major synperiplanar conformer around the amide bond: δ = 1.41 , 3.78 , 3.80 , 4.16 , 6.76 , 6.92 , 7.41 , 7.62 , 7.71 , 8.11 , 11.07 ; resolved signals for minor antiperiplanar conformer around the amide bond: 8.32 , 11.00 , 13C NMR : signals for major synperiplanar conformer around the amide bond: δ = 28.01 (CH3), 32.91 (NCH3), 41.38 (CH2), 55.11 (OCH3), 77.88 (OC), 103.66 (C-4), 109.95 (C-3), 111.05 (C-7), 112.10 (C-6), 125.01 (C-3a), 132.65 (C-7a), 134.03 (C-2), 140.20 (CH), 154.65 (C-5), 155.89 (OC=O), 169.72 (C=O). HRMS (ESI) m/z: calcd: [M+H]+ 361.187032. Found: [M+H]+ 361.1869.tert-butyl(2-{(2E)-2-[(1-methyl-1H-indol-3-yl)methylidene]hydrazinyl}-2-oxoethyl)carbamate, 3l Yield: 54%; m.p. 178–179 °C. 1H NMR : 1:0.40 mixture of conformers; signals for major synperiplanar conformer around the amide bond: δ = 1.41 , 3.82 , 4.12 , 6.83 , 7.22 , 7.28 , 7.51 , 7.77 , 8.08 , 8.13 , 11.06 ; resolved signals for minor antiperiplanar conformer around the amide bond: 8.34 , 11.02 . 13C NMR : signals for major synperiplanar conformer around the amide bond: δ = 28.24 (CH3), 32.76 (NCH3), 41.42 (CH2), 77.87 (OC), 110.27 (C-7), 110.38 (C-3), 120.83 (C-5), 121.55 (C-4), 122.66 (C-6), 124.43 (C-3a), 133.90 (C-2), 137.56 (C-7a), 140.22 (CH), 155.96 (OC=O), 169.78 (C=O). HRMS (ESI) m/z: calcd: [M+H]+ 331.176467. Found: [M+H]+ 331.1764.tert-butyl(2-{(2E)-2-[(1-benzyl-1H-indol-3-yl)methylidene]hydrazinyl}-2-oxoethyl)carbamate, 3m Yield: 90%; m.p. 207–208 °C. 1H NMR : 1:0.40 mixture of conformers; signals for major antiperiplanar conformer around the amide bond: δ 11.10 , 8.15 , 8.08 , 7.95 , 7.50 , 7.30 , 7.26–7.22 , 7.21–7.18 , 7.24 , 7.20 , 6.85 , 5.44 , 4.10 , 1.40 ; resolved signals for minor antiperiplanar conformer around the amide bond: 7.05 , 8.21 , 8.34 , 3.62 . 13C NMR : signals for major antiperiplanar conformer around the amide bond: δ 170.03 (C=O), 156.16 (O-C=O), 140.39 (CH), 137.68 (i-Ph), 137.02 (C-7a), 133.54 (C-2), 128.79 (C-o), 127.72 (p-Ph), 127.27 (m-Ph), 124.85 (C-3a), 123.03 (C-5), 121.87 (C-4), 121.18 (C-6), 111.15 (C-3), 110.93 (C-7), 78.12 (C), 49.45 (CH2), 42.56 (CH2), 28.38 (CH3); resolved signals for minor synperiplanar conformer around the amide bond: 143.22 (CH), 122.29 (C-2), 78.27 (C), 41.57 (CH2), 28.35 (CH3). HRMS (ESI) m/z: calcd: [M+H]+ 407.207767. Found: [M+H]+ 407.2078.2-(1H-indol-3-yl)-N’-[(E)-(5-methoxy-1H-indol-3-yl)methylidene]acetohydrazide, 3n1H NMR, 13C NMR and HRMS spectra of compound 3n were published elsewhere [lsewhere .N’-[(E)-methylidene]-1H-indole-3-carbohydrazide, 3o Yield: 78%; m.p. 267–270 °C. 1H NMR : δ = 3.83 , 3.84 , 7.03 , 7.14 , 7.18 , 7.20 , 7.35 , 7.48 , 8.21 , 8.26 , 10.98 , 11.52 . 13C NMR : δ = 55.48 (CH3), 55.56 (CH3), 108.45 (C-3), 109.14 (C-2′), 111.45 (C-5′), 112.05 (C-7), 120.22 (C-4), 120.74 (C-5), 120.89 (C-6′), 121.73 (C-6), 126.53 (C-1′), 127.66 (C-3a), 129.17 (C-2), 135.69 (C-7a), 144.03 (CH), 149.15 (C-3′), 150.35 (C-4′), 162.13 (C=O). HRMS (ESI) m/z: calcd: [M+H]+ 324.134268. Found: [M+H]+ 324.13358.N’-[(E)-methylidene]-2-(1H-indol-3-yl)acetohydrazide, 3p Yield: 92%; m.p. 177–180 °C. 1H NMR : 1:0.80 mixture of conformers; signals for major antiperiplanar conformer around the amide bond: δ = 3.80 , 3.81 , 4.05 , 6.95 , 7.01 , 7.06 , 7.17 , 7.28 , 7.35 , 7.36 , 7.60 , 7.91 , 10.87 , 11.18 ; resolved signals for minor antiperiplanar conformer around the amide bond: 8.15 , 10.91 , 11.40  13C NMR : signals for major antiperiplanar conformer around the amide bond: δ = 29.24 (CH2), 55.40 (OCH3), 108.22 (C-2′), 108.25 (C-3), 111.30 (C-5′), 111.56 (C-7), 118.28 (C-4), 118.66 (C-5), 120.97 (C-6′), 121.59 (C-6), 123.86 (C-2), 127.13 (C-1′), 127.39 (C-3a), 135.98 (C-7a), 142.46 (CH), 148.99 (C-3′), 150.56 (C-4′), 172.52 (C=O). HRMS (ESI) m/z: calcd: [M+H]+ 338.149918. Found: [M+H]+ 338.14902.N’-[(E)-(4-hydroxy-3-methoxyphenyl)methylidene]-2-(1H-indol-3-yl)acetohydrazide, 3q Yield: 72%; m.p. 246–247 °C. 1H-NMR : 0.54: 0.46 mixture of two conformers; signals for major conformer: δ = 3.81 , 4.04 , 6.82 , 6.96–7.00 , 7.02–7.09 , 7.22 , 7.29 , 7.33 , 7.59 , 7.87 , 9.46 , 10.86 , 11.10 ; Signals for minor conformer: δ(ppm): 3.61 , 3.79 , 6.81 , 6.93–6.97 , 7.02–7.09 ,7.24 , 7.35 , 7.59 , 8.11 , 9.48 , 10.90 , 11.33 . 13C-NMR : 0.54:0.46 mixture of two conformers; signals for major conformer: δ = 29.20 (CH2), 55.51 (CH3), 108.33 (C-3), 109.10 (C-2′), 111.30 (C-7), 115.37 (C-5′), 118.27 (C-5), 118.68 (C-4), 120.88 (C-6′), 121.11 (C-6), 123.86 (C-2), 125.82 (C-1′), 127.42 (C-4a), 135.98 (C-7a), 142.81 (CH), 147.97 (C-3′), 148.51 (C-4′), 172.43 (C=O); signals for minor conformer: δ(ppm): 31.69 (CH2), 55.51 (CH3), 108.31 (C-3), 108.93 (C-2′), 111.35 (C-7), 115.52 (C5′), 118.36 (C-5), 118.68 (C-4), 121.00 (C-6′), 121.87 (C-6), 123.86 (C-2), 125.73 (C-1′), 127.14 (C-4a), 136.10 (C-7a), 146.65 (CH), 147.97 (C-3′), 148.78 (C-4′), 166.79 (C=O). HRMS (ESI) m/z: calcd: [M+H]+ 324.134268. Found: [M+H]+ 324.1341.2,4-dihydroxy-N’-[(E)-(4-hydroxy-3-methoxyphenyl)methylidene]benzohydrazide, 3r Yield: 76%; m.p. 242–243 °C. 1H NMR : 3.36 , 6.31 , 6.37 , 6.85 , 7.10 , 7.32 , 7.80 , 8.33 , 9.56 , 10.19 , 11.54 , 12.45 . 13C NMR : 55.55 (CH3), 102.86 (C-3), 106.20 (C-1), 107.33 (C-5), 108.99 (C-2′), 115.45 (C-6′), 122.27 (C-5′), 125.59 (C-1′), 129.49 (C-6), 148.05 (C-4′), 148.61 (CH), 149.09 (C-3′), 162.31 (C-4), 162.57 (C-2), 165.28 (C=O). HRMS (ESI) m/z: calcd: [M+H]+ 303.097548. Found: [M+H]+ 303.0974.1a, 1i, 1j, 1k, 1l, 1o) in 10 mL of absolute ethanol was mixed with a hot solution of 20 mmol (60 °C) benzenesulfonohydrazide (4a) or 4-methylbenzenesulfonohydrazide (4b) and 4-methoxylbenzenesulfonohydrazide (4c) in 10 mL of absolute ethanol and stirred for 1–3 h. The obtained crystalline precipitates were filtered, washed with ethanol-ether, recrystallized from ethanol.The solution of 20 mmol of the corresponding carbonyl compounds (N’-[(E)-(1-benzylpiperidin-4-yl)methylidene]benzenesulfonohydrazide, 5a Yield: 59%; m.p. 110–111 °C. 1H NMR : δ = 1.26–1.36 , 1.54–1.58 , 1.91–1.97 , 2.05–2.14 , 2.65–2.68 , 3.40 , 7.19 , 7.21–7.27 , 7.29–7.33 , 7.59–7.63 , 7.65–7.69 , 7.78–7.81 , 10.93 .13C NMR (DMSO) : δ = 27.62 (CH2), 36.94 (CH), 51.11 (CH2), 61.25 (CH2), 125.85 (C-4″), 126.12 (C-2′ and C-6′), 127.11 (C-3″ and C-5″), 127.75 (C-2′’ and C-6′’), 128.07 (C-3′ and C-5′), 131.86 (C-4′), 137.28 (C-1′’), 137.94 (C-1′), 153.47 (CH).HRMS (ESI) m/z: calcd: [M+H]+ 358.158373. Found: [M+H]+ 358.1581.4-methoxy-N’-[(E)-(5-methoxy-1-methyl-1H-indol-3-yl)methylidene]benzenesulfonohydrazide, 5b Yield: 78%; m.p. 145–146 °C. 1H NMR : δ = 3.74 , 3.77 , 3.80 , 6.86 , 7.11 , 7.36 , 7.47 , 7.64 , 7.85 , 8.05 , 10.75 . 13C NMR : δ = 32.90 (NCH3), 55.22 (OCH3), 55.64 (OCH3), 103.31 (C-4), 109.71 (C-3), 111.01 (C-7), 112.54 (C-6), 114.20 (C-3′ and C-5′), 124.90 (C-3a), 129.49 (C-1′), 130.83 (C-2′ and C-6′), 132.52 (C-7a), 134.20 (C-2), 145.01 (CH), 154.66 (C-4), 162.45 (C-4′). HRMS (ESI) m/z: calcd: [M+H]+ 374.11597. Found: [M+H]+ 374.1169.4-methoxy-N’-[(E)-(5-methoxy-1H-indol-3-yl)methylidene]benzenesulfonohydrazide, 5c Yield: 53%; m.p. 159–163 °C. 1H NMR : δ = 3.76 , 3.80 , 6.80 , 7.11 , 7.29 , 7.46 , 7.66 , 7.85 , 8.08 , 10.76 , 11.37 .13C NMR : δ = 55.15 (OCH3), 55.63 (OCH3), 103.12 (C-4), 110.85 (C-3), 112.48 (C-7), 112.59 (C-6), 114.19 (C-3′ and C-5′), 124.49 (C-3a), 129.48 (C-1′), 130.73 (C-2′ and C-6′), 130.84 (C-7a), 131.78 (C-2), 145.46 (CH), 154.35 (C-4), 162.43 (C-4′). HRMS (ESI) m/z: calcd: [M+H]+ 360.10125. Found: [M+H]+ 360.10037.N’-[(E)-(1-benzyl-1H-indol-3-yl)methylidene]benzenesulfonohydrazide, 5d Yield: 89%; m.p. 146–149 °C. 1H NMR : δ = 5.41 , 7.15 , 7.18 , 7.21 , 7.24 , 7.30 , 7.48 , 7.59–7.65 , 7.90 , 7.93 , 7.97 , 8.10 , 11.01 .13C NMR : δ = 49.27 (CH2), 110.65 (C-3), 110.67 , 120.89 (C-5), 121.78 (C-4), 122.79 (C-6), 124.61 (C-3a), 127.09 (C-2′ and C-6″), 127.28 (C-2″ and C-6″), 127.51 (C-4′), 128.59 (C-3′ and C-5′), 129.05 (C-3″ and V-5″), 132.82 (C-4″), 133.50 (C-2), 136.75 (C-7a), 137.44 (C-1′), 139.13 (C-1″), 144.60 (CH). HRMS (ESI) m/z: calcd: [M+H]+ 390.127073. Found: [M+H]+ 390.12604.4-methoxy-N’-[(E)-(1-methyl-1H-indol-3-yl)methylidene]benzenesulfonohydrazide, 5e Yield: 66%; m.p. 170–175 °C. 1H NMR : δ = 3.78 , 3.79 , 7.11 , 7.17 , 7.24 , 7.46 , 7.69 , 7.84 , 7.97 , 8.05 , 10.76 . 13C NMR : δ = 32.73 (NCH3), 55.61 (OCH3), 110.11 (C-3), 110.17 (C-7), 114.18 (C-3′ and H-5′), 120.74 (C-5), 121.66 (C-4), 122.62 (C-6), 124.40 (C-3a), 129.48 (C-2′ and C-6′), 130.81 (C-2′ and C-6′), 133.89 (C-1′), 137.42 (C-7a), 144.44 (CH), 162.44 (C-4′).HRMS (ESI) m/z: calcd: [M+H]+ 344.106338. Found: [M+H]+ 344.1055.N’-[(E)-(5-methoxy-1-methyl-1H-indol-3-yl)methylidene]benzenesulfonohydrazide, 5f1H NMR, 13C NMR and HRMS spectra of compound 5f were published elsewhere [lsewhere .N’-[(E)-(1-benzyl-1H-indol-3-yl)methylidene]-4-methylbenzenesulfonohydrazide, 5g Yield: 79%; m.p. 145–146 °C.1H NMR : δ = 2.34 , 5.41 , 7.13–7.32 , 7.39 , 7.48 , 7.81 , 7.89 , 7.99 , 8.08 , 10.92 .13C NMR : δ = 20.95 (CH3), 49.26 (CH2), 110.66 (C-7), 110.72 (C-3), 120.88 (C-5), 121.83 (C-4), 122.78 (C-6), 124.63 (C-4a), 127.08 (C-2″ and C-6″), 127.33 (C-2′ and C-6′), 127.51 (C-4″), 128.59 (C-3″ and C-5″), 129.47 (C-3′ and C-5′), 133.41 (C-2), 136.26 (C-1′), 136.74 (C-7a), 137.46 (C-1″), 143.16 (C-4′), 144.32 (C-H). HRMS (ESI) m/z: calcd: [M+H]+ 404.142723. Found: [M+H]+ 404.1425.N’-[(E)-methylidene]-4-methylbenzenesulfonohydrazide, 5h1H NMR, 13C NMR and HRMS spectra of compound 5h were published elsewhere [lsewhere .N’-[(E)-methylidene]benzenesulfonohydrazide, 5i1H NMR, 13C NMR and HRMS spectra of compound 5i were published elsewhere [lsewhere .N’-{(E)-[5-(benzyloxy)-1H-indol-3-yl]methylidene}-4-methylbenzenesulfonohydrazide, 5j Yield: 67%; m.p. 222–223 °C. 1H NMR : δ = 2.28 , 5.03 , 6.88 , 7.30 , 7.34 , 7.36 , 7.43 , 7.53 , 7.57 , 7.68 , 7.81 , 8.09 , 10.84 , 11.40 . 13C NMR : δ = 20.90 (CH3), 69.68 (CH2), 104.82 (C-4), 110.81 (C-3), 112.50 (C-6), 113.02 (C-7), 124.48 (C-4a), 127.37 (C-2′ and C-6′), 127.84 (C-4″), 127.93 (C-2″ and C-6″), 128.46 (H-3″ and H-5″), 129.41 (C-3′ and C-5′), 130.96 (C-2), 131.98 (C-7a), 136.33 (C-1′), 137.35 (C-1″), 143.08 (C-4′), 145.68 (CH), 153.38 (C-5). HRMS (ESI) m/z: calcd: [M+H]+ 404.142723. Found: [M+H]+ 420.1373.N’-((E)-[5-(benzyloxy)-1H-indol-3-yl]methylidenebenzenesulfonohydrazide, 5k1H NMR, 13C NMR and HRMS spectra of compound 5k were published elsewhere [lsewhere .N’-[(E)-(4-chlorophenyl)methylidene]benzenesulfonohydrazide, 5l1H NMR, 13C NMR and HRMS spectra of compound 5l were published elsewhere piperidine (E20) and co-factor 2-acetamido-2-deoxy-beta-D-glucopyranose (NAG), retrieved from Protein Data Bank ((2)BChE, complexed with butyl-[(2~(S))-1-(2-cycloheptylethylamino)-3-(1~(H)-indol-3-yl)-1-oxidanylidene-propan-2-yl]azanium (HUN) and NAD again (PDB ID 6QAA).Selection of PDB structures: The following crystallographic structures of human acetylcholinesterase (AChE) and human butyrylcholinesterase (BChE) have been chosen for molecular docking studies:The choice of aforementioned crystallographic structures followed previous investigations of Alov et al. , who, ai3e–3i were used in docking. These forms correspond to the extended conformations of the compounds, thus fitting better to the deep and narrow binding cavity of the cholinesterases. The PDB complexes were prepared using the “Quick preparation” procedure in MOE. In addition to adding hydrogens and assigning protonation states of titratable protein groups by the Generalized Born electrostatics model at physiologically relevant conditions , tethers were added to the receptor heavy atoms and distant atoms were fixed. Following this, the system refinement was performed applying Amber10: EHT force field and the default RMS gradient of 0.1 kcal/mol/Å2.Structures preparation: The 3D structures of the investigated compounds were built and optimized with AMBER10:EHT in MOE. The R enantiomers  of compounds Prediction of the protonation state of the investigated structures. The “Protomer” panel in MOE was used to explore the possible ionization states of the molecules that can be populated at physiological pH. The resulting ionization states were sorted in order of decreasing population and the ionization state with the highest % of population at the physiological pH was used for docking.Docking simulations: Molecular docking studies were performed in the “Docking” module in MOE. The following settings were applied in the docking protocol: (I) in the set of atoms defining the receptor, the receptor and solvent atoms were included. (II) “Triangle Matcher” placement to generate poses by aligning ligand triplets of atoms on triplets of alpha spheres in the most systematic way; the London dG scoring function was used to rank the poses (30 placement poses). (III) Subsequent refinement using “Rigid receptor” or “Induced fit” (flexible side chains) tools based on London dG and GBVI/WSA scoring functions . London dG is the default scoring function in MOE that estimates the free energy of binding of the ligand from a given pose combining enthalpy and entropy terms. The GBVI/WSA dG is the default force field-based scoring function that is used after rescoring for the final refinement of the docking poses. The protein–ligand interactions in the active site of the complexes were visualized using the “Ligand Interactions” MOE tool.We prepared a model of human melatonin receptors MT1 and MT2 which we employed as docking templates in order to propose the possible molecular mechanism of action of our ligands. XRD structures of human MT1 and MT2 receptors were selected for our modeling on the base of resolution and completeness of crystallized deposited data in Protein Data Bank.If more than one copy of the receptor structure in the PDB existed, we used all of them in further analysis.In all selected PDB structures, the minor structural improprieties were corrected, followed by removal of all nonprotein species. For the purpose of attaining the right protonation state at 7.0 pH, which was the physiological one, we used Labute’s protonate 3D algorithm (Labute 2008) at 300K and 0.154 M/l salt concentration as it is implemented in MOE software package.The codes of selected PDB for MT1 were 6ME2, 6ME3, 6ME4, 6ME5, 7DB6, 7VGY, 7VGZ.Although only 2 amino acids in the active site differ between these structures ALA104 in 6ME2, 6ME3, 6ME4, 6ME5 is GLY104 in 7DB6, 7VGY, 7VGZ and PHE251 in 6ME2, 6ME3, 6ME4, 6ME5 is TRP251 in 7DB6, 7VGY, 7VGZ, we prepared our model for docking by homology modeling the sequence of NCBI NP005949.1 melatonin receptor type 1A [Homo Sapiens] on consensus model formed by all of the PDB structures. The above-mentioned differences are due to the point mutations for increased thermostability of the protein introduced by Stauch et al.  on consensus model formed by all of the selected PDB structures.Fortunately, all of the selected XRD structures were crystallized with their natural-like ligands in the cavity, therefore presenting the active state of MT1 and MT2 receptors.All newly synthesized ligands were protonated according to their protonation state at 7 pH. Due to the low amount of rotatable bonds, the systematic approach was used in formation of ligand library. Every generated structure was further energy minimized using AMBER12EHT [MOE] force field in gas phase. All unique conformational structures within 10 kcal/mol from the lowest structure of every ligand were used for further docking.All conformations of all ligands were docked in active site of the MT1 and MT2 receptors using AlphaPMI algorithm [MOE] for initial placement of every structure. These poses were scored by London dG [MOE] function, which estimates the free energy of binding of the ligand from a given pose and consists of terms that estimate average gain/loss of rotational and translational entropy, measures the geometric imperfections of hydrogen bonds and the desolvation energy of atoms.The best 100 poses for every ligand for every pocket were further optimized with Induced Fit methodology using AMBER12EHT force field and Generalized Born solvation model with optimization cutoff of 5A from the ligand. The GBVI/WSA dG [MOE] was used as a rescoring function and the best 30 poses were collected for the next coming analysis.In all cases, the original ligands from the PDB files were also used in our docking study as they were subjected to the same ligand preparation methodology as our ligands. Placement of the original ligands in the receptor cavities from the placement algorithm in a position similar to that in the intact PDB was used as a criterion for the adequacy of our docking methodology.RMSD of alpha carbons of the active site residues between aligned chains of selected PDB structures for MT1 and MT2 structures are presented in Z and E isomers in some of the ligands discussed in the text was calculated using B3LYP/6–311++G** hybrid functional and basis set as they are implemented in GAUSSIAN 09 software suite.The difference between As a verification of our docking study, we included in our ligand set the original ligands from the used pdb files. These ligands were treated in the same way as our structures and were used to expand the ligand set for docking. Analysis of their interaction energies shows that all the structures studied have better interaction energies than the original ligands and the docking procedure used positions the original ligands in the receptor cavities in the same way as they are in the original PDB files.https://www.sib.swiss, accessed on 20 April 2022).In silico ADME studies were performed by using the online tool SwissADME/Tox of the Swiss Institute of Bioinformatics .The webserver was used to predict BBB permeability of compounds, SwissADME (−6 cm/s) of the compounds was calculated and presented as −logPe. Samples were analyzed in triplicate and average values were reported. Highly permeable compounds were indicated by values of −logPe < 5, the medium permeable by −logPe between 5 and 6, and if −logPe > 6, the compound was considered as low permeable [The blood–brain barrier permeabilities were measured by PAMPA Permeability Analyzer . The tested compounds and reference standards were prepared as 10 mM stock solutions in DMSO and further diluted in Prisma HT buffer pH 7.4. The experiment followed the BBB Protocol: 200 μL sample’s aliquots in the donor compartment, Brain Sink Buffer aliquots (200 μL) in the acceptor wells, BBB-1 lipid for coating the permeation membranes, “sandwich” assembly with the acceptor plate on top of the donor one, and incubation at temperature 25 °C for 4 h under humidity control without stirring. For permeability assessment, UV spectra of the blank, reference, donor, and acceptor plates were collected at wavelength 250–500 nm. Effective permeability Pe , while compound 3n, containing melatonin, exhibited notable BChE inhibitory activity (21.12 ± 1.48 μM).Alzheimer’s disease (AD) is a significant concern for chemistry researchers seeking new multi-target compounds to address this complex neurodegenerative condition. Focusing on melatonin/donepezil hybrids as potential candidates for AD treatment, we synthesized 30 new derivatives. Among them, compounds 5k, 5j, 5g, and hydrazone 3r revealed the highest DPPH activity and hydrazones 3i, 3d, 3c, and 5h showed the lowest values of IC50 in the ABTS test. With respect to the FRAP method, compound 3c has the strongest activity, followed by 5a, 5h, donepezil, 3m, and 5j. In the FTC method, the highest significant diminution was demonstrated by 3a followed by 3c, 5h, and 5j, compared with BHT. Thus, compound 3c, a promising acetylcholinesterase inhibitor among the new derivatives, demonstrated very good antioxidant activity across the three tested methods . Additionally, in vitro studies revealed that the melatonin derivative 3n efficiently prevents oxidative stress-induced injury in SH-SY5Y cells, exhibiting the best BChE inhibitory activity. As a future perspective, we plan to evaluate hydrazones 3c, 3d, 3a, 3n, and 5a in vivo using models of Alzheimer’s disease and melatonin deficiency, as well as Aβ (1–42) aggregation.The in vitro antioxidant activities for the most promising molecules were investigated by the DPPH, FRAP, ABTS, and FTC methods. All tested compounds hold potential as therapeutic neuroprotective agents for neurodegenerative disorders. The sulfonylhydrazones 3a–d , 3k (130.8 μM), and 5a (142.0 μM) exhibited the best bioavailability, with IC50 values twice as high as the reference donepezil (79.3 ± 6.2 μM) in the human neuroblastoma cell line SH-SY5Y and three times higher in normal mouse fibroblasts. Notably, the most active compounds 3a–d and 5a displayed negligible cytotoxic activity in the mouse neuroblastoma cell culture Neuro-2a (IC50 > 300 μM); making them promising structures for further study in AD. An in silico pharmacokinetics analysis predicted that all tested hybrids could be well absorbed, metabolized, and excreted, with most of them capable of crossing the BBB. Additionally, the most promising compounds were measured by PAMPA for blood–brain barrier permeabilities.Importantly, the new series of melatonin derivatives containing the donepezil fragment showed low cytotoxicity and a good in vitro safety profile. Compounds 3c in complex diseases such as major depression and AD, which involve targeting AChE and/or BChE enzymes and melatonin MT1 and MT2 receptors.Finally, docking studies suggest potential future applications of the MTDL