The structures of the new alkaloids were elucidated by extensive spectroscopic methods. The absolute configurations of 4, 6, and 8 were determined for the first time by electronic circular dichroism (ECD) calculations. The cytotoxic activity of all compounds was tested against human malignant melanoma cells A375 by the MTT method, and no antitumor activity was observed.Two new indole alkaloids, Bufotenidine B ( Bufo bufo gargarizans Cantor or B. melanostrictus Schneider +  and 547.2526 [2M+Na]+ . The IR spectrum showed absorptions of NH group (3430 cm−1), carbonyl groups (1631 cm−1), and aromatic ring . The UV spectrum showed maximal absorption wavelength at 204, 232.5 and 312.4 nm. The 1H-NMR  spectrum showed three aromatic proton signals at δH7.31 , 7.15, and 6.63 , combining with eight carbon signals at δC 154.8 (C-5), 133.7 (C-8), 128.9 (C-2), 125.4 (C-9), 118.1 (C-7), 113.6 (C-6), 112.9 (C-4), and 110.7(C-3) in 13C-NMR  spectrum, which indicated there was an indole moiety in 2. The DEPT (135°) displayed three aromatic methine carbon signals at δC 128.9 (C-2), 118.1 (C-7), and 113.6 (C-6), and HSQC showed they correlated with 7.15 , 7.31 , and 6.63 , respectively. Combining with the HMBC correlations between 7.31  and 125.4 (C-9) and 154.8 (C-5); between 7.15 and 133.7 (C-8), 125.4 (C-9) and 110.7(C-3); and between 6.63  and 118.1 (C-7) and 112.9 (C-4), confirmed there was a 3, 4, 5- trisubstituted indole moiety in 2. The DEPT (135°) spectrum disclosed the presence of a pair of coupled methylene groups at δC 23.6 (C-10) and 69.2 (C-11), which correlated with 3.32 , 3.23  in its HSQC spectrum. Combining with the HMBC correlations between 3.32  and 69.2 (C-11), and between 3.23  and 23.6 (C-10) indicated the two methylene groups were directly connected. In addition, the HMBC correlations between 7.15  and 23.6 (C-10); between 3.32  and 125.4 (C-9), 110.7(C-3), and 128.9 (C-2); and between 3.23  and 110.7 (C-3) disclosed a pair of coupled methylene groups was attached to the 3, 4, 5- trisubstituted indole moiety at C-3, thus forming a 5-hydroxytryptamine moiety. The HSQC spectrum showed 2.98  correlated with 54.6 . Meanwhile, the DEPT (135°) displayed 54.6  was a positive peak, and the peak height about three times higher than that of other carbon signals, suggesting that there were three identical methyl groups in 2. Finally, three identical methyl groups were connected to the 5-hydroxytryptamine moiety at N-2 by the HMBC correlations between 3.23  and 54.6 , and between 2.98  and 69.2 (C-11) (Compound 2 (C-11) .13H18N2O which missed a [COO−] fragment compared to the molecular formula C14H18N2O3 of 2 was formed. The 13C-NMR spectrum showed the presence of a carbonyl group at δC 176.9, suggesting the [COO-] fragment was attached to C-4 to form an inner salt. The suggestion was further demonstrated by its UV spectrum, which showed that the maximum absorption wavelength of the indole ring shifted from 275 nm to 312 nm. This indicated that the C-4 substituent could prolong the conjugated system. Based on the above analysis, 2 was unambiguously assigned as 5-hydroxy-3-(2-(trimethylammonio) ethyl) -1H-indole-4-carboxylate, and named bufotenidine B, which is a new compound confirmed by Scifinder investigation.According to the aforementioned information, a structural fragment of C5 was obtained as a brown powder. The molecular formula was established as C12H14N2O3 by high-resolution electrospray ionization mass spectroscopy, which gave ions at m/z 467.19261 [2M-H]− . The IR bands of 5 were shown at 3424, 1631 and 1457 cm−1 for N-H, C=O and aromatic ring, respectively. The UV spectrum showed maximal absorptions of an indole chromophore (222 and 276 nm). In the 1H-NMR  spectrum of 5, signals at 7.15 , 7.08 , 6.84 , and 6.60  indicated a typical 3,5-disubstituted indole moiety. Combined with two methylene signals at 3.07  and 2.91 , it was suggested that 5 is a derivative of serotonin. The 13C-NMR  spectrum showed twelve carbon signals. Ten of them at 124.6 (C-2), 109.3 (C-3), 103.0 (C-4), 150.8 (C-5), 112.5 (C-6), 113.0 (C-7), 131.7 (C-8), 128.3 (C-9), 22.6 (C-10), and 47.8 (C-11) were assigned to serotonin skeleton by comparing with the NMR data of serotonin + , 259.1078 [M-H]−  in its HR-ESI-MS spectrum. Characteristic IR absorption bands indicated the existence of aromatic secondary amine (3404 cm−1), carbonyl (1631 cm−1) and aromatic ring (1457 cm−1) groups. The UV spectrum exhibited maximal absorptions of an indole chromophore (221 and 275 nm). The 13C-NMR  showed fourteen carbon signals for one carbonyl group at δC 175.3 (C-12), five sp3 carbons at δC 51.9 (C-1), 39.2 (C-3), 20.4 (C-4), 28.2 (C-10), 33.9 (C-11), and eight sp2 carbons at δC 134.1 (C-9a), 106.4 (C-4a), 127.3 (C-4b), 102.2 (C-5), 150.6 (C-6), 111.2 (C-7), 111.5 (C-8), and 130.5(C-8a). The 1H-NMR  spectrum indicated the presence of one NH group as a singlet [δH10.54 ], and three aromatic protons , disclosed a 2,3,5-trisubstituted indole moiety, which was supported by the HMBC correlations between 10.54  and 134.1 (C-9a), 130.5(C-8a), 127.3 (C-4b), and 106.4 (C-4a); between 6.69  and 106.4 (C-4a), 150.5(C-6), 111.2 (C-7), and 130.5(C-8a); between 6.55  and 130.5(C-8a), 150.5(C-6), and 102.2 (C-5); and between 7.07  and 102.2 (C-5), 127.3 (C-4b), and 150.6 (C-6). 1H-NMR spectrum indicated two methylene signals at δH 2.67–2.54 , 3.20–2.96 , which, respectively, correlated with 20.4 (C-4), 39.2(C-3) in HSQC spectrum. Combining the HMBC correlations between 2.67–2.54  and 39.2 (C-3), 134,1 (C-9a), and 106.4 (C-4a) and between 3.20–2.96  and 20.4 (C-4) and 106.4 (C-4a), it disclosed a pair of coupled methylene groups was attached to the 2, 3, 5-trisubstituted indole moiety at C-3, thus forming a 5-hydroxytryptamine moiety. 1H-NMR spectrum indicated two methylene signals at δH 2.40–2.21 , 1.90–2.10 , which respectively correlated with 33.9 (C-11) and 28.2 (C-10) in HSQC spectrum. The HMBC gave correlations between 2.40–2.21  and 28.2 (C-10) and 175.3 (C-12) and between 1.90–2.10  and 33.9 (C-11) and 175.3 (C-12), which disclosed the presence of a carboxyethyl group. 1H-NMR spectrum showed a methine signal at δH 4.19 , which correspond to 51.9 (C-1) in HSQC spectrum. In addition, the HMBC spectrum showed two methylene groups 2.40–2.21  and 1.90–2.10  were both associated with 51.9 (C-1), indicating that the carboxyethyl is connect to C-1 (51.9). Further, the HMBC spectrum showed correlations between 4.19 , 1.90-2.10  and 134.1 (C-9a), showing that 51.9 (C-1) is linked to 134.1 (C-9a) to form a tricyclic structure of β-carboline (6 was unambiguously assigned as 6-hydroxy-1-(2′-carboxyethyl)-1,2,3,4-tetrahydro-β-carboline, which is a new compound named bufocarboline A.Compound arboline . Accordi4 and 8 were isolated for the first time from toad venom, and the plane structures of them were confirmed respectively as 6-hydroxy-l-methyltetrahydro-β-carboline-1-carboxylic acid  spectrum indicated the presence of one NH group as a singlet at 10.83 , and four aromatic protons at 7.36 , 7.22 , 7.20 , and 6.97 ], indicated a typical 3,5-disubstituted indole moiety. Combined with two methylene signals at 3.27  and 3.00 , it was suggested that 1 is a derivative of serotonin. In addition, there was two methyl signals at 2.82 ]. The 13C-NMR  spectrum showed twelve carbon signals at 20.6 (C-10), 57.1 (C-11), 109.0 (C-4), 110.3 (C-3), 111.2 (C-6), 116.9 (C-7), 124.1 (C-2), 126.8 (C-9), 133.4 (C-8), 146.6 (C-5), 42.6  were basically consistent with bufotenine + and [M-H]- located at m/z 284.9, 306.9 and 282.6 in positive or negative mode, respectively, which confirmed the molecular weight of 1 was 284 instead of 204 (the molecular weight of bufotenine). Thus, 1 was identified as bufotenine O-sulfate known as bufoviridine. This is the first time to report the NMR data about bufoviridine.Compound 3, 7, 9–12 were identified as dehydrobufothionine + , 547.2526 [2M+Na]+ , C14H18N2O3; IR (KBr) νmax: 3430, 2924, 2170, 1698, 1631,1458, 1402, 1384, 1338, 1272, 1117, 1038, 865, 834, 669, 618 cm−1; UV (H2O) λ max (log ε): 204 (4.01), 232.5 (3.68), 312.4 (3.41) nm. 1H-NMR and 13C-NMR spectral data as shown in Yellow powder; HR-ESI-MS m/z 244.6 [M-H]-, C13H14N2O3; 2O). 1H-NMR and 13C-NMR spectral data are shown in Yellow powder; ESI-MS m/z 234.9[M+H]+, 256.9 [M+Na]+, 232.6 [M-H]−, HR-MS m/z 467.19261[2M-H]− , C12H14N2O3; IR (KBr) νmax: 3424, 2925, 2170, 2852, 1697, 1631, 1457, 1402, 1384, 1272, 1197, 1143, 1039, 865, 834, 704, 668, 618 cm−1; UV (H2O) λ max (log ε): 200.5 (4.21), 221.6 (4.06), 275.7 (3.51) nm. 1H-NMR, 13C-NMR, and HMBC spectral data are shown in Yellow powder; ESI-MS m/z 261.1232[M+H]+ , 283.1053 [M+Na]+, 259.1078 [M-H]- , C14H16N2O3; IR (KBr) νmax: 3404, 2922, 2852, 2170, 1631, 1572, 1457, 1402, 1385, 1274, 1230, 1198, 1149, 1049, 864, 831, 798, 668, 621, 475 cm−1; UV (H2O) λ max (log ε): 204 (4.22), 274.5 (3.52), 221 (3.99) nm. 2O). 1H-NMR and 13C-NMR spectral data are shown in Yellow powder; HR-MS m/z 233.0920 [M+H]+ , C12H12N2O3; 2O); UV (H2O) λ max (log ε): 215.7 (3.52), 274.5 (2.93) nm. 1H-NMR and 13C-NMR spectral data are shown in Yellow powder; HR-MS 4, 6, and 8 based on their known relative configuration, all six possible stereoisomers were employed for the conformational random search using the MMFF94s force field by CONFLEX software package [For the computational details for ECD spectra of compounds  package . The con package . They we package .


These compounds are easily accessible in pure E-configuration by aldol condensation or by oxidative coupling/elimination. The α-methyl groups make these compounds potentially reactive with singlet oxygen following the gem-rule that predicts 1O2 regioselectivity. Even after long irradiations times in the presence of the singlet oxygen sensitizer tetraphenylporphyrin (TPP) and oxygen, however, no oxygenation products were detected. Under these conditions, all substrates were converted into 9:1 E/Z-mixtures despite the use of low-energy light that does not allow direct or sensitized excitation of the substrates 7. Additionally, chalcone 7a reduced the singlet oxygen reactivity of the tiglic ketone 3a by about a factor of two, indicating substantial physical quenching of singlet oxygen by the α-methylated chalcones 7a–7e. Thus, a singlet oxygen-induced E/Z-isomerization involving 1,2-dioxatetra-methylene biradicals that leads to triplet oxygen and thermodynamic E/Z-mixtures is postulated and supported by quantum chemical (DFT)-calculations.The α-methylated chalcones  It is easily generated from air oxygen (ground-state electronic triplet state) by energy transfer from a vast number of electronically excited state dye molecules + 281.11744 (+ 078 ppm), [M + Na]+ 303.09933 (+ 0.53 ppm); UV/Vis (10−5 M in CH3CN): λabs (max) = 289 nm (ε = 24.117 M−1cm−1).(E)-Methyl-4-(3-(4-methoxyphenyl)-2-methyl-3-oxoprop-1-en-1-yl)benzoate (7e): According to GP 1 4.93 g  4-methoxypropiophenone (5b) and 4.92 g  methyl 4-formylbenzoate (6c) were converted to methanol. After column chromatographic purification  the product 7e was recovered as a colorless solid. Yield: 0.79 g , Rf: 0.33 , 1H-NMR : 8.07 , 7.82 , 7.47 , 7.08 , 6.96 , 3.93 , 3.88 , 2.25 .13C-NMR 198.0 , 166.8 , 163.1 , 140.6 , 139.0 , 138.2 , 132.2 , 130.3 , 129.8 , 129.7 , 129.5 , 113.7 , 55.6 , 52.4 , 15.3 . FT-IR (ATR): ṽ (cm−1) = 1722.0 (s), 1634.4 (m), 1595.4 (s), 1507.3 (m), 1458.0 (w), 1437.5 (m), 1416.1 (m), 1357.2 (w), 1312.3 (m), 1284.6 (s), 1191.5 (m), 1182.3 (s), 1168.7 (s), 1143.2 (s), 1111.5 (s), 1014.4 (s), 1003.4 (m), 967.8 (w), 933.7 (m), 918.0 (m), 840.4 (m), 802.5 (w), 771.0 (s), 754.2 (w), 693.1 (m), 623.4 (s), 612.9 (s), 601.8 (s), 529.3 (m), 511.7 (w), HR-MS (ESI): m/z [M + H]+ 311.12802 (+ 077 ppm), [M + Na]+ 333.10973 (+ 0.00 ppm); UV/Vis (10−5 M in CH3CN): λabs. (max) = 291 nm (ε = 31.299 M−1cm−1).−4 M solutions in CH3CN in 1 cm quartz cuvettes; (b) singlet oxygen conditions were investigated using 200 mM stock solutions of chalcones and 5 × 10−4 mol/L sensitizer (TPP) in CDCl3. As reaction vessels, 2 mL pyrex NMR tubes were irradiated with three white LEDs  under constant oxygen bubbling. As results of the direct (350 nm) and indirect (1O2-induced) E/Z-isomerizations, the following set of photostationary and induced isomerization states resulted  UV-irradiations of chalcones for analysis of the E/Z-photostationary states were performed in a LuzChem CCP-4V photochemical reactor  using 16 UV-A lamps  and 10resulted  45]::−4 M solAll computations are performed with GAUSSIAN 16 Revision B.01 . The str


This bioactivity is due to the simultaneous polypharmacological stimulation of receptors PPARα, PPARγ, and GPR40 and the enzyme inhibition of aldose reductase (AR) and protein tyrosine phosphatase 1B (PTP-1B). The nine compounds share the same four pharmacophore elements: an acid moiety, an aromatic ring, a bulky hydrophobic group, and a flexible linker between the latter two elements. Addition and substitution reactions were performed to obtain molecules at moderated yields. In silico pharmacological consensus analysis (PHACA) was conducted to determine their possible modes of action, protein affinities, toxicological activities, and drug-like properties. The results were combined with in vivo assays to evaluate the ability of these compounds to decrease glucose levels in diabetic mice at a 100 mg/kg single dose. Compounds 6 (a phenylpropanoic acid derivative) and 9  ameliorated the hyperglycemic peak in a statically significant manner in a mouse model of type 2 diabetes. Finally, molecular dynamics simulations were executed on the top performing compounds to shed light on their mechanism of action. The simulations showed the flexible nature of the binding pocket of AR, and showed that both compounds remained bound during the simulation time, although not sharing the same binding mode. In conclusion, we designed nine acid bioisosteres with robust in vivo antihyperglycemic activity that were predicted to have favorable pharmacokinetic and toxicological profiles. Together, these findings provide evidence that supports the molecular design we employed, where the unified pharmacophores possess a strong antidiabetic action due to their multitarget activation.Substituted phenylacetic ( Hyperglycemia and insulin resistance are hallmarks of diabetes; the prevalence of diabetes is almost 425 million people globally, but this number would be twice that if it considered the people who are currently undiagnosed . Inactiv10), and isovanillin (12). In the case of the ferulic acid, we decided to perform a reduction of the double bond using catalytic hydrogenation to improve the free rotation of the final compound and to decrease the reactivity and the intrinsic toxicity of the α,β—unsaturated carbonyl, obtaining Compound 11 (12) and thiazolidine-2,4-dione using a Dean–Stark apparatus to remove the water formed in the reaction . MD simulations of the enzymes AR and PTP-1B were performed in the Molecular docking was performed with the enzymes PTP-1B and AR. In order to incorporate the flexibility of the receptor into the analysis, an ensemble docking approach was performed using the six most representative structures of the MD trajectories. All the compounds tested were shown to interact with both receptors, although with putative different affinity, shown both in terms of docking score and ligand efficiency. We considered the bioisosteric groups to be deprotonated at pH 7.4.1 and 4–9 showed interactions similar to the cocrystallized ligand, such as those established with Tyr48, Leu300, and Trp111. The latter contact has been reported as one of the main binding residues in previous AR inhibitors acetic acid (10), K2CO3, and 1-(chloromethyl)naphthalene (16) was stirred and heated at 80 °C for 9 h. Recrystallized from EtOH, yield = 70%; mp = 114.8–116.3 °C; 1H-NMR  δ: 3.49 , 5.52 , 7.04 , 7.19 , 7.51 , 7.54–7.59 , 7.67 , 7.93 , 7.98 , 8.09 ; 13C-NMR  δ: 40.49 (CH2), 68.23 (OCH2), 115.06 , 124.31 (C-8″), 125.8 (C-3″), 126.39 (C-6″), 126.8 (C-4″), 127.03 (C-7″). 127.8 (C-1′), 128.9 (C-2″), 129.06 (C-5″), 130.85 , 131.55 (C-4″a), 133.71 (C-8″a), 133.02 (C-1″), 157.62 (C-4′), 173.4 (C-1) ppm. EIMS: m/z = 292.3 [M+], 157.9 [M-135], 141.0 [M-151].The mixture of (4-hydroxyphenyl)acetic acid (2){4-[(2′-cyanobiphenyl-4-yl)methoxy]phenyl}acetic acid  was stirred and heated at 80 °C for 15 h. Recrystallization from EtOH was performed to afford colorless crystals of 2. Yield = 60%; mp = 164.9–166.8 °C; 1H-NMR  δ: 3.48 , 5.17 , 6.99 , 7.19 , 7.59 , 7.64 , 7.79 , 7.95 ; 13C-NMR  δ: 39.68 (CH2), 68.74 (OCH2), 110.59 (C-2″’), 114.97 , 118.99 (C-4), 125.8 (C-3″), 126.39 (C-6″), 127.82 (C-6″’). 128.32 , 128.68 (C-4″’), 129.24 , 130.56 (C-1′), 130.86 , 133.98 (C-3″’), 134.24 (C-5″’), 137.68 (C-4″), 138.27 (C-1″), 144.65 (C-1″’), 157.48 (C-4′). 173.40 (C-1) ppm. EIMS: m/z = 343.3 [M+], 326 [M-17], 193.0 [M-151].The mixture of (4-hydroxyphenyl)acetic acid (10), K3)[4-(biphenyl-3-ylmethoxy)phenyl]acetic acid (10) acid, K2CO3, and 3-phenylbenzyl bromide (18) was stirred and heated at 80 °C for 7 h. Recrystallization from EtOH was performed to afford crystals of 3. Yield = 75%; mp = 141.9–143.8 °C. 1H-NMR  δ: 3.48 , 5.16 , 6.98 , 7.18 , 7.37 , 7.44 , 7.49–7.46 , 7.62 , 7.67 , 7.72 ; 13C-NMR  δ: 39.68 (CH2), 68.74 (OCH2), 110.59 (C-2″’), 114.24 , 125.58 (C-2″), 125.76 (C-6″), 126.29 (C-4″’), 126.35 , 126.87 (C-5″), 127.17 (C-4″), 128.58 , 128.70 (C-1′). 130.02 , 137.54 (C-3″), 139.57 (C-1″), 139.95 (C-1″’), 172.55 (C-1). EIMS: m/z = 318.3 [M+], 241 [M-77], 165 [M-153].The mixture of (4-hydroxyphenyl)acetic (4)3-[3-methoxy-4-(1-naphthylmethoxy)phenyl]propanoic acid naphthalene (16) was stirred and heated for 8 h. Recrystallization from EtOH was performed to afford 4. Yield = 64%; mp = 135.6–136.5 °C; 1H-NMR  δ: 2.52 , 2.77 , 3.72 , 5.46 , 6.74 , 6.88 , 7.08 , 7.50 , 7.56 , 7.64 , 7.93 , 7.95 , 8.11 , 12.13 . 13C-NMR  δ: 30.03 , 35.5 , 55.4 (O-CH3), 68.6 (O-CH2), 112.57 (C-2), 114 (C-5), 119.9 (C-6), 124 (C-8′), 125.9 (C-3′), 126.3 (C-6′), 126.6 (C-7′), 128.2 (C-5′), 128.5 (C-4′), 131.1 (C-8a), 131.2 (C-1), 133.3 , 146.1(C-4), 149 (C-3), 173.8 (COOH). EIMS: m/z = 336.3 [M+], 156.0 [M-207], 127.0.The mixture of 5)3-{4-[(2′-cyanobiphenyl-4-yl)methoxy]-3-methoxyphenyl}propanoic acid (11 and 4′-bromomethyl-2-biphenylcarbonitrile (17) was stirred and heated at 80 °C for 2 h. Recrystallization from EtOH was performed to afford white crystals of 5. Yield = 60%; mp = 133.7–134.9 °C; 1H-NMR  δ: 2.51 , 2.76 , 3.77 , 5.12 , 6.72 , 6.80 , 6.96 , 7.59 , 7.64 , 7.95 . 13C-NMR  δ: 29.5 , 35.1 , 55.1 , 69.2 (C-11 OCH2), 109.7 (C-2″), 112.2 (C-2), 117.3 , 118.2 (C-12), 119.5 (C-6), 127.5 , 127.8 (C-4′), 128.3 , 129.7 (C-4″), 133.4 , 133.6 (C-5″), 137.7 (C-1′), 143.8 (C-4), 145.7 (C-1″), 148.6 (C-3), 173.4 (C-9). EIMS: m/z = 387.4 [M+].The mixture of 6)3-[4-(biphenyl-3-ylmethoxy)-3-methoxyphenyl]propanoic acid (11 and 3-phenylbenzyl bromide (18) was stirred and heated at 80 °C for 12 h. Recrystallization from EtOH was performed to afford crystals of 6. Yield = 84%; mp = 119.5–120.0 °C. 1H NMR  δ: 2.50 , 2.75 , 3.75 , 5.11 , 6.70 , 6.87 , 6.95 , 7.37 , 7.42 , 7.47 , 7.61 , 7.65  12.09 . 13C-NMR  δ: 29.6 , 35.1 , 55.2 (OCH3), 69.7 (OCH2), 112.2(C-2), 113.5 (C-5), 119.6 (C-6), 126.3 , 127.2 (C-6′), 128.6 , 133.58 (C-1), 137.2 , 139.5 , 139.8 , 145.7 (C-4), 148.69 (C-3), 173.4 (COOH). EIMS: m/z = (% int. rel): 362.4 [M+].The mixture of 12) as precursor. A mixture of 1.1 eq of 12 and 2.2 eq of K2CO3 was added to a round-bottom flask and was mechanically stirred using acetonitrile as a solvent. After 1 h, the respective arylmethylbromide or chloride (1 eq) was added to the mixture and was set in reflux under a nitrogen atmosphere to form the respective substitution product. The crude product was filtered in vacuo. Cold water was added to wash the crude product and was then filtered, thus obtaining a solid. Recrystallization from the appropriate solvent was performed to afford colorless crystals of each compound.The compounds were synthesized using isovanillin (13)4-methoxy-3-(1-naphthylmethoxy)benzaldehyde (16) was stirred and heated at 80 °C for 2 h. Recrystallization from EtOH was performed to afford colorless crystals of 13. Yield = 81%; mp = 114.8–116.3 °C; 1H-NMR  δ: 3.92 , 5.60 , 7.01 , 7.45 , 7.49 , 7.51–7.53 , 7.55 , 7.59 , 7.62 , 7.84 , 7.88 , 8.08 , 9.84 . EIMS: m/z = 292.1 [M+].The mixture of isovanillin and 1-(chloromethyl)naphthalene (14)4′−[(5-formyl-2-methoxyphenoxy)methyl]biphenyl-2-carbonitrile (17) was stirred and heated at 80 °C for 4 h. Recrystallization from EtOH was performed to afford colorless crystals of 14. Yield = 82%; mp = 157.4–158.3 °C; 1H-NMR  δ: 3.97 , 5.24 , 7.01 , 7.43 , 7.49 , 7.58 , 7.63 , 7.76 , 9.84 . EIMS: m/z = 343.2 [M+].The mixture of isovainillin and 4′-bromomethyl-2-biphenylcarbonitrile (15)3-(biphenyl-3-ylmethoxy)-4-methoxybenzaldehyde (18) was stirred and heated at 80 °C for 2 h. Recrystallization from EtOH was performed to afford colorless crystals of 15. Yield = 66%; mp = 82.1–83.9 °C. 1H-NMR  δ: 3.96 , 5.25 , 6.99 , 7.35 , 7.42–7.45 , 7.46–7.48 , 7.49 , 7.53–7.55 , 7.59 , 7.68 , 9.82 . EIMS: m/z = 318.1 [M+].The mixture of isovanillin and 3-phenylbenzyl bromide (13–15 (1 eq) were collocated in a round-bottom flask, and 1.1 eq of thiazolidine-2,4-dione was added. The mixture was stirred using toluene as the solvent and was then heated to reflux, after which 0.3 eq of piperidine and 0.3 eq of benzoic acid were added and coupled with a Dean–Stark system. The mixture was maintained in reflux for 4 h. The crude reaction was filtered in vacuo and was washed with water to afford the respective Compounds 7–9.Compounds 7)(5 Z)-5-[4-methoxy-3-(1-naphthylmethoxy)benzylidene]-1,3-thiazolidine-2,4-dione  δ: 3.81 , 5.59 , 7.15–7.16 , 7.21 , 7.43 , 7.52 , 7.56 , 7.59 , 7.67 , 7.75 , 7.94 , 7.98 , 8.12 ; 13C-NMR  δ: 55.74 (OCH3), 68.55 (OCH2), 112.44 (C-5), 115.53 (C-2), 120.59 (C-9), 123.88 (C-8′), 124.08 (C-3′), 125.36 (C-6), 125.7 (C-8), 125.98 (C-6′), 126.43 (C-1), 126.67 (C-4′), 128.44 (C-7′), 128.74 (C-2′), 131.11 (C-4′a), 132.03 (C-5′), 132.19 (C-1′), 133.26 (C-8′a), 147.89 (C-3), 151.24 (C-4), 167.36 (C-11), 167.93 (C-10). EIMS: m/z: 391.2 [M+].General procedure was performed obtaining a yellow solid of 8)4′-({5-[(Z)-methyl]-2-methoxyphenoxy}methyl) biphenyl-2-carbonitrile  δ: 3.83 , 5.21 , 7.12–7.14 , 7.17–7.19 , 7.26–7.27 , 7.55–7.57 , 7.58–7.61 , 7.71–7.72 , 7.65 , 7.75–7.78 , 7.92–7.93 . 13C-NMR  δ: 56.22 (OCH3), 69.98 (OCH2), 110.61 (C-2″), 112.85 (C-5), 115.31 (C-2), 124.49 (C-6), 126.38 (C-8), 128.54 , 128.69 (C-6″), 129.27 , 130.56 (C-4″), 131.43 (C-1), 133.96 (C-3″), 134.27 (C-5″), 137.78 (C-1″), 137.88 (C-4′), 144.63 (C-1′), 148.25 (C-3), 151.38 (C-4), 169.22 (C-11), 169.45 (C-10). EIMS, m/z: 442.2 [M+].General procedure was performed obtaining a yellow solid of 9)(5 Z)-5-[3-(biphenyl-3-ylmethoxy)-4-methoxybenzylidene]-1,3-thiazolidine-2,4-dione  δ: 3.85 , 5.23 , 7.14–7.15 , 7.20–7.21 , 7.27–7.28 , 7.36–7.39 , 7.45–7.50 , 7.62–7.63 , 7.66–7.67 , 7.71 , 7.76 ; 13C-NMR  δ: 55.77 (OCH3), 69.96 (OCH2), 112.41 (C-5), 114.87 (C-2), 120.69 (C-9), 124.41 (C-4′), 125.57 (C-6), 126.14 (C-8), 126.29 (C-2′), 126.69 , 126.79 (C-1), 127.54 (C-5′), 128.93 , 129.11 (C-4″), 131.97 (C-6′), 137.41 (C-1′), 139.88 (C-1″), 140.35 (C-3′), 147.81 (C-3), 151.19 (C-4), 167.40 (C-11), 167.92 (C-10). EIMS, m/z: 417.4 [M+].General procedure was performed obtaining a yellow solid of ICR male mice weighing 25 ± 5 g were housed in animal cages with 12 h periods of light and 12 h periods of dark. Animals were allowed to acclimatize to the laboratory conditions. The animals were maintained in a 25 °C environment, and water and food were provided ad libitum. All animal experiments were conducted according to protocols that were approved by the Mexican government NOM-065-ZOO-1999 and NOM-033-ZOO-2014 .Induction of diabetes was performed according to previous works ,36. AnimWe performed this assay according to a previous methodology that was used in our lab ,37. BrieThe crystallographic data of the proteins PTP-1B and AR were downloaded from PDB  with a convergence criterion of 50 kcal mol−1 Å−2; for the second stage, backbones were constrained with a 10 kcal mol−1 Å−2 force constant using a convergence criterion of 10 kcal mol−1 Å−2 for 2000 steps ; for the third stage, the systems were minimized with no restraints for 1000 steps  with a convergence criterion of 1 kcal mol−1 Å−2. Equilibration was carried out in several steps. At first, Brownian dynamics for 250 ps were determined with the Berendsen thermostat. Simulation on the NVT ensemble was then performed, slowly heating from 10 to 300 K over 3000 ps. At this stage, constraints were enforced on solute heavy atoms, using a constant of 50 kcal mol−1. Finally, equilibration on NPT ensemble used the Berendsen thermostat and Langevin barostat for additional 250 ps was done.MD studies of the protein–ligand complexes were performed using Desmond  with the OPLS3e forcefield . The comWith the system ready for production, 300 ns of simulation were performed, under an NPT ensemble at 1 bar and 300 K using the Martyna–Tuckerman–Klein (MTK) barostat and the Nosé–Hoover thermostat. Electrostatic forces were calculated with the smooth PME method using a 9 Å cut-off, while constraints were enforced with the M-SHAKE algorithm. Integration was done every 2 fs, with a recording interval of 500 ps. The quality analysis of the simulation and other trajectory analyses were carried out with the tools implemented in the Maestro 12.1  and with VMD .holo state  were clustered based on the backbone and sidechain RMSD, and the first six clusters, covering ~60% of the sampled conformational space, were recovered. The obtained conformations were stripped from waters and ions and subjected to a restrained minimization of the side chains. With the nine compounds, and the corresponding cocrystallized inhibitor, docking procedures were performed on the six protein conformations. All docking procedures were performed with Glide 8.4 with the XP methodology [The simulations of the two receptors in the hodology  and the hodology . A grid After the docking calculations were performed for the six protein conformations, the results for each individual compound were pooled and clustered following the ligand heavy atoms. For data fusion in ensemble docking, multiple approaches have been proposed . For ourFrom the total number of poses, the percentage of poses that were included in the top cluster was also included in the report table. Validation of the docking protocol used was carried out by redocking the respective cocrystallized ligands. It was observed that the cocrystallized inhibitor docked adequately in the six conformers (not more than 2.0 Å of RMSD) and that the clustering of all poses also generated a most-populated cluster with the correct crystallographic pose within the 2.0 Å of tolerance.An a priori pharmacological consensus analysis (PHACA) was performed with the results that were obtained from several computational tools. The pharmacodynamics properties were calculated from the output score that was obtained with Glide XP. The following pharmacokinetic properties were acquired using AdmetSAR and SwissADME software: consensus logP, water solubility class, human intestinal absorption, blood–brain permeability, P-glycoprotein substrate, bioavailability score, and inhibition of CYP3A4. Toxicological profiles  were obtained from the web server AdmetSAR and ACD/Tox Suite version 2.95 ,23,44. Tp values were <0.05.The data were analyzed by two-way ANOVA followed by Dunnett’s post hoc test. The results are expressed as the means ± S.E.M. Statically significance was assumed when 6 and 9 are interesting bioactive compounds for the treatment of diabetes due to their antihyperglycemic effect. Further studies are needed to identify other possible targets in the body [In this work, we reported the design, synthesis, and in vivo evaluation of nine compounds that were developed for the experimental treatment of diabetes, and we aimed to complement a unified antidiabetic pharmacophore with multitarget action. An a priori analysis of the compounds employing a pharmacological consensus analysis (PHACA) showed highly satisfactory results for five compounds, and their in vivo evaluation confirmed their biological activity. The molecules controlled the blood glucose levels without exacerbating the effect, in contrast to glibenclamide, which had a robust hypoglycemic effect. Moreover, molecular dynamics analysis shed light on the molecular recognition process and provided an explanation for the experimental results obtained. Together, these findings provide a rationale for the molecular design we employed in our lab, as we found the unified pharmacophore to possess a strong antidiabetic activity. Moreover, the use of an acid bioisostere surrogate, such as thiazolidine-2,4-dione or carboxylic acid, resulted in an interaction similar to those presented by the original cocrystallized inhibitors. Indeed, Compounds the body , in orde


N-arylenamine C-N bond. The study showed that silica gel-bound Cu(II)–enaminone complexes are readily available and suitable heterogeneous catalysts for the synthesis of 6,7-dihydro-1H,5H-pyrazolopyrazoles.A series of 12 silica gel-bound enaminones and their Cu(II) complexes were prepared and tested for their suitability as heterogeneous catalysts in azomethine imine-alkyne cycloadditions (CuAIAC). Immobilized Cu(II)–enaminone complexes showed promising catalytic activity in the CuAIAC reaction, but these new catalysts suffered from poor reusability. This was not due to the decoordination of copper ions, as the use of enaminone ligands with additional complexation sites resulted in negligible improvement. On the other hand, reusability was improved by the use of 4-aminobenzoic acid linker, attached to 3-aminopropyl silica gel via an amide bond to the enaminone over the more hydrolytically stable  Bicyclic pyrazolidinones exhibit antibiotic pyrazole scaffold, which is accessible by [3 + 2] cycloaddition of 3-oxopyrazolidin-1-ium-2-ides to acetylenes pyrazoles in a regio- and stereoselective manner under mild conditions that are compliant with requirements of “click” chemistry  . The mosPfDHODH) ,5. Theseyrazolo1,-apyrazolyrazolo1,-apyrazoletylenes ,14,15,16hemistry  17,18,1,18,1apyrhemistry .N-, C-, and O-nucleophiles take place under mild conditions by substitution of the dimethylamino group to give β-functionalized propenoates. With ambident nucleophiles, enaminones undergo cyclization into different heterocyclic systems pyrazoles, and would complement well the known examples of heterogeneous Cu(0)- pyrazole-2-carboxylates.In recent years, an important part of our ongoing research on the chemistry of 3-pyrazolidinones  has beens Cu(0)- , Cu(I)- s Cu(0)- ,67,68,69s Cu(0)-  in the C2a–g were prepared from active methylene compounds 1a–g by treatment with N,N-dimethylformamide dimethylacetal (DMFDMA) or tert-butoxy-bis(dimethylamino)methane (TBDMAM) at 20–110 °C following literature procedure pyrazoles via [3 + 2] cycloaddition of 3-pyrazolidinone-derived azomethine imines to terminal ynones.Transamination of enaminones 1H NMR, 13C NMR, and 2D NMR spectra were recorded in CDCl3 and DMSO-d6 as solvents using Me4Si as the internal standard on a Bruker Avance III UltraShield 500 plus instrument  at 500 MHz for 1H and at 126 MHz for 13C nucleus, respectively. IR spectra were recorded on a Bruker FTIR Alpha Platinum spectrophotometer . Microanalyses were performed by combustion analysis on a Perkin-Elmer CHN Analyzer 2400 II . Mass spectra were recorded on an Agilent 6224 Accurate Mass TOF LC/MS . Parallel stirring was carried out on a Tehtnica Vibromix 313 EVT orbital shaker  . Flash column chromatography was performed on silica gel .All solvents and reagents were used as received. Melting points were determined on SRS OptiMelt MPA100—Automated Melting Point System . The 1a–g, 3-aminopropyl silica gel (3) , 4-aminobenzoic acid (12), Cu(OAc)2·H2O, N,N-dimethylformamide dimethylacetal , tert-butoxy-bis(dimethylamino)methane , and 1,1′-carbonyldiimidazole (CDI) are commercially available (Sigma-Aldrich). Enaminones 2a 1a–g, 3-a 1a–g, 3-a 1a–g, 3-a 1a–g, 3-a + requires 280.0968; .Enaminone 13  in acetonitrile (5 mL) and the mixture was stirred at room temperature for 1 h. Then, 3-aminopropyl silica gel (3)  was added and the suspension was stirred at room temperature for 120 h. Ethanol (2 mL) was added and the insoluble material was collected by filtration using a short column with fritted bottom  and the functionalized silica gel 14 was washed with EtOH-MeCN , EtOH (2 × 5 mL), DMF (2 × 5 mL), EtOH (3 mL), and Et2O (2 × 5mL) and air-dried. Brown powder ; FT-IR (ATR): νmax 1603 (C = O/C = N) cm−1.1,1′-Carbonyldiimidazole  was added to a stirred suspension of carboxylic acid 14  was added to a solution of Cu(OAc)2·H2O  in methanol (10 mL) and the mixture was stirred at 20 °C for 48 h. The insoluble material was collected by filtration, washed carefully with methanol until the filtrate was colorless (around 5 × 5 mL), and air-dried to give the copper(II) catalyst 15. Brown powder ; FT-IR (ATR): νmax 1603 (C = O/C = N) cm−1.3-Enaminopropyl silica gel 3-Cu, 5, 11, or 15 (10–30 mg) was added to a mixture of azomethine imine 6a–f (25–37 mg pyrazole-2-carboxylate (8b). Prepared from azomethine imine 6b , methyl propiolate (7) , and CH2Cl2 (4 mL). Yellow oil ; νmax/cm−1 (ATR) 2955, 1696 (C = O), 1599, 1511, 1444, 1408, 1371, 1323, 1200, 1173, 1099, 1031, 959, 824, 727; δH : 1.14 , 1.22 , 2.38 , 2.86 , 3.62 , 3.79 , 5.43 , 6.87 , 7.35 , 7.48 ; δC : 19.1, 25.1, 49.6, 51.6, 55.3, 64.1, 64.4, 113.9, 117.1, 129.0, 129.3, 134.2, 159.3, 164.3, 166.5; HRMS (ESI): MH+, found 317.1493 (MH+). [C17H21N2O4]+ requires 317.1496.7,7-Dimethyl-1-(4-methylphenyl)-5-oxo-6,7-dihydro-1H,5H-pyrazolopyrazole-2-carboxylate (8c). Prepared from azomethine imine 6c , methyl propiolate (7) , and CH2Cl2 (4 mL). Yellow solid ; mp 152–155 °C; νmax/cm−1 (ATR) 3082, 2946, 1731 (C = O), 1687 (C = O), 1601, 1514, 1323, 1275, 1225, 1192, 1120, 1099, 1039, 1007, 947, 818, 733; δH : 1.12 , 1.19 , 2.33 , 2.38 , 2.82 , 3.58 , 5.40 , 7.12 , 7.29 , 7.46 ; δC : 19.1, 21.3, 25.1, 49.5, 51.6, 64.4, 64.6, 117.0, 127.8, 129.2, 129.5, 137.6, 139.1, 164.3, 166.7; HRMS (ESI): MH+, found 301.1546 (MH+). [C17H21N2O3]+ requires 301.1547.8a,d–f were obtained in the following isolated yields: compound 8a (92%), 8d (89%), 8e (84%), and 8f (88%). Spectral data for compounds 8a [8d [8e [8f [Following the above Procedure C, also known cycloadducts ounds 8a ,79, 8d [s 8a [8d ,84, 8e [ [8d [8e , and 8f  [8e [8f ,79 were


Among them, the new isolated withanolides, withaperoxidins A–D (1–4) have an unusual six-membered cyclic peroxide moiety on the withasteroid skeleton as a structural novelty. Their structures have been elucidated by means of spectroscopic analyses, including 2D NMR experiments. In addition, extensive structure–activity relationships and in silico ADME studies were employed to understand the pharmacophore and pharmacokinetic properties of this series of withasteroids. Compounds 15, 16, and 22 together with withaferin A (14) were identified as having improved antiproliferative effect (IC50 ranging from 0.2 to 0.7 μM) on human leukemia HL-60 cell lines compared with the reference drug, etoposide. This cytotoxic potency was also coupled with good selectivity index (SI 33.0–9.2) on non-tumoral Vero cell line and in silico drug likeness. These findings revealed that these natural withasteroids are potential candidates as chemotherapeutic agents in the treatment of leukemia.Leukemia is a blood or bone marrow cancer with increasing incidence in developed regions of the world. Currently, there is an ongoing need for novel and safe anti-leukemic agents, as no fully effective chemotherapy is available to treat this life-threatening disease. Herein, are reported the isolation, structural elucidation, and anti-leukemic evaluation of twenty-nine withanolide-type steroids ( Leukemia is a blood or bone marrow cancer characterized by an uncontrolled hematopoiesis process . Acute mNatural products represent a rich source of drug leads, and they have contributed greatly to anticancer drugs discovery and development ,8. In thWithania aristata (Ait.) Pauq. (Solanaceae), an endemic medicinal plant from the Canary Islands , two oxymethylene protons , and two vicinal vinyl protons , as the most upshift signals were observed. The 1H-NMR spectrum in combination with a 1H-1H COSY experiment allowed the identification of spin systems: H-2/H-3/H-4 in ring A, H-6/H2-7 in ring B, and H-20/H3-21, H-20/H-22, and H-22/H2-23 in the side chain.Compound spectrum  showed s13C-NMR spectrum of 1 , two oxygenated tertiary carbons (δC 84.2 and 84.9), two vinyl methines (δC 126.5 and 141.4), and two olefinic quaternary carbons (δC 125.1 and 154.3), together with two carbonyl carbons assigned to a δ-lactone (δC 167.1) and a ketone (δC 206.0). The aforementioned data suggest that 1 possesses a common tetracyclic withanolide-type steroid skeleton with a ketone, an α,β-unsaturated-δ-lactone, one disubstituted double bond, two oxygenated quaternary groups, two oxymethine groups, and one oxymethylene group  and the signals at δC 78.6 (C-2), 84.2 (C-5), 67.1 (C-6), and 48.2 (C-10), and correlation of the proton resonance at δH 1.31 (H3-19) and signals at δC 206.0 (C-1), 84.2 (C-5), 42.1 (C-9), and 48.2 (C-10), locating the ketone at C-1, a double bond at C-3/C-4, and the oxygenated groups at C-2, C-4, C-5, and C-6. Correlations observed from methyl resonances at δH 1.06 (H3-21) and 0.88 (H3-18) to the signal at δC 84.9 (C-17) confirmed the structure of the D ring, whereas the signal at δH 2.05 (H3-28) was linked to the resonance at δC 32.9 (C-23), 154.3 (C-24), and 125.1 (C-25), establishing the structure of the α,β-unsaturated-δ-lactone on the side chain. All these data accounted for nine out of 10 degrees of unsaturation established by its HRESIMS spectrum, indicating that compound 1 has an additional ring in the withanolide skeleton. The presence of a 1,2-dioxane system is the only option that satisfies this chemical shift requirements, which can be attributed to a six- or four-membered cyclic peroxide ring system at C-2/C-5 or C-5/C-6. This naturally occurring cycloendoperoxide would arise from a [4 + 2] or [2 + 2] cycloaddition reaction between the cyclohexadiene system ∆2,4 or double bond ∆5 on the withonolide skeleton and in situ formed singlet oxygen , a primary hydroxyl group , three oxymethine protons , two tertiary hydroxyl groups [δC 84.6 (C-5) and 85.0 (C-17)], and an α,β-unsaturated δ-lactone moiety . These NMR data show 2 to be similar to 1. The most significant differences were the downfield shift of H3-19 (Δδ 0.30) and those of C-2 (Δδ 1.4) and C-6 (Δδ 2.0), and an upfield shift of H-4 (Δδ 0.46) and C-19 (Δδ 3.4) in the 1H and 13C-NMR spectra  gave a molecular formula C28H38O7, indicating an oxygen atom less than 1. Its 1H and 13C-NMR data  in 1 by a methine group [ and ] in 3, indicating that 3 is the 17-deoxy analogue of 1. Analysis of 2D NMR data allowed the complete and unambiguous assignment of chemical shifts, regiosubstitution, and relative configuration of compound 3. The HMBC experiment showed as the most relevant three-bond correlations those of the proton resonances of H3-18 (δH 0.79) and H3-21 (δH 1.05) with the signal of C-17 (δC 51.9). Accordingly, the structure of withaperoxidin C (3) was defined as 6β,27-dihydroxy-2α,5α-dioxan-1-oxo-witha-3,24-dien-26,22-olide.The HRESIMS of withaperoxidin C , determined by HR-ESIMS, revealed 18 mass units less than the parent 1. A comparison of their NMR spectra showed the replacement of the signals assigned to the aliphatic H2-16 and the tertiary hydroxyl group at C-17 in compound 1 by resonances for a trisubstituted double bond  in 4. Even so, it should be noted that a set of 2D NMR spectra was performed for 4 with the aim of gaining the full and unequivocal assignment of the 1H and 13C-NMR resonances (4) as 6β,27-dihydroxy-2α,5α-dioxan-1-oxo-witha-3,16,24-trien-26,22-olide.Compound sonances . The reg5)  are given in 10−1 deg cm2 g−1. UV spectra were obtained on a JASCO V-560 spectrophotometer , and IR (film) spectra were measured on a Bruker IFS 55 spectrophotometer . NMR experiments were performed on a Bruker Avance 400 spectrometer , and chemical shifts are shown in δ (ppm) with tetramethylsilane (TMS) as internal reference. EIMS and HREIMS were recorded on a Micromass Autospec spectrometer, and ESIMS and HRESIMS (positive mode) were measured on an LCT Premier XE Micromass Electrospray spectrometer . Purification was performed using silica gel 60 μM for column chromatography (particle size 15–40 and 63–200 μm), POLYGRAM SIL G/UV254 used for analytical and preparative TLC, and HPTLC-platten Nano-Sil 20 UV254 were purchased from Panreac . Sephadex LH-20 for exclusion chromatography was obtained from Pharmacia Biotech . The spots in the TLC were visualized by UV light and heating silica gel plates sprayed with H2O-H2SO4-AcOH (1:4:20). All solvents used were analytical grade from Panreac . Reagents were purchased from Sigma Aldrich  and used without further purification.Optical rotations were measured on a Perkin Elmer 241 automatic polarimeter  in CHCl2 and 98% humidity.HL-60 (human promyelocytic leukemia cell line) and Vero (African green monkey kidney) cell lines from American Type Culture Collection (ATCC-LGC) were each grown in Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L glucose (Sigma-Aldrich), supplemented with 10% fetal bovine serum (Gibco), 1% of a penicillin–streptomycin mixture , and 200 mM L-glutamine. Cells were maintained at 37 °C in 5% CO4 /200 μL/well) in log-phase of growth seeded in DMEM, supplemented with 5% fetal bovine serum in microtiter well plates  were added to compounds predissolved in dimethylsulphoxide (DMSO) at different concentrations. After 48 h of incubation, 20 μL/well of the MTT solution  were added and plates incubated for 4 h at 37 °C. Subsequently, the medium/MTT solutions were removed carefully by aspiration and replaced with 150 μL/well of DMSO to dissolve the formazan crystals. Absorbance was measured in a microplate reader  at a wavelength of 550 nm. Solvent control cultures were considered 100% and percentage of viable treated cells were plotted against compound concentrations. The 50% cell viability value (IC50) was calculated from the curve. Each experiment was performed at least three times in triplicates. Data are given in arithmetic means ± SD. Selectivity ratio was defined as IC50 value for the tumoral cell line divided by IC50 value for the Vero cell line.Viable cells were assessed using the colorimetric MTT + (100); HRESIMS m/z 525.2454 .6β,17α,27-Trihydroxy-2α,5α-dioxan-1-oxo-witha-3,24-dien-26,22-olide . Colorless lacquer; [α]1. To a solution of 1  in dry dichloromethane (2 mL) was added triethylamine  and acetic anhydride . The reaction mixture was stirred for 72 h at room temperature until all starting material was consumed. The mixture was evaporated to dryness. The residue was then purified by preparative TLC  to give 1a : Colorless lacquer; [α]D20 + 56.0 ; UV λmax 223 nm; IR νmax 2925, 1697, 1454, 1393, 1255, 1063, 834 cm−1; 1H-NMR δ 0.88 , 1.05 , 1.14 , 1.21 , 1.39 , 1.52 , 1.61 , 1.63 , 1.68 , 1.69 , 1.71 , 1.72 , 1.73 , 1.76 , 1.98 , 2.00 , 2.09 , 2.36 , 2.59 , 4.54 , 4.68 , 4.89, 4.93 , 5.38 , 6.62 , 7.00 , OAc-27 [2.09 ], OAc-6 [2.16 ]; 13C-NMR δ 9.4 (CH3-21), 15.0 (CH3-18), 18.0 (CH3-19), 20.6 (CH3-28), 21.8 (CH2-11), 23.6 (CH2-15), 29.8 (CH-8), 31.9 (CH2-16), 32.0 (CH2-12), 33.1 (CH2-23), 36.5 (CH2-7), 42.6 (CH-20), 43.5 (CH-9), 47.1 (C-10), 48.3 (C-13), 49.1 (CH-14), 58.0 (CH2-27), 69.8 (CH-6), 78.7 (CH-2), 79.1 (CH-22), 85.0 (C-5), 85.9 (C-17), 121.3 (C-25), 130.9 (CH-3), 142.9 (CH-4), 158.6 (C-24), 165.5 (C-26), 205.4 (C-1), OAc-6 , OAc-27 ; ESI/MS m/z % 609 [M + Na]+ (100); HRESIMS m/z 609.2679 .Acetylation of 2). Colorless lacquer; [α]D20 + 31.9 ; UV λmax 214 nm; IR νmax 3430, 2929, 2855, 1689, 1457, 1394, 1013, 754 cm−1; 1H and 3C-NMR data (see m/z % 525 [M + Na]+ (100); HRESIMS m/z 525.2465 .6β,17α,27-Trihydroxy-2β,5β-dioxan-1-oxo-witha-3,24-dien-26,22-olide . Colorless lacquer; [α]D20 − 3.9 ; UV λmax 217 nm; IR νmax 3444, 2927, 1700, 1458, 1393, 1130, 1024, 754 cm−1; 1H and 3C-NMR data (see m/z % 509 [M + Na]+ (100); HRESIMS m/z 509.2508 .6β,27-Dihydroxy-2α,5α-dioxan-1-oxo-witha-3,24-dien-26,22-olide . Colorless lacquer; [α]D20 − 4.0 ; UV λmax 214 nm; IR νmax 3410, 2930, 1681, 1461, 1380, 1131, 1078, 755 cm−1; 1H and 3C-NMR data (see m/z % 507 [M + Na]+ (100); HRESIMS m/z 507.2355 .6β,27-Dihydroxy-2α,5α-dioxan-1-oxo-witha-3,16,24-trien-26,22-olide  properties of the compounds were predicted using the QikProp program   in Fast W. aristata. Cyclic-endoporoxide natural products are very rare, and this is the first report of withasteroid-endoperoxides from Withania genus, suggesting that the enzymatic system in W. aristata is quite different to other Withania species. Biological evaluation and in silico ADME exploration revealed that four compounds from this series are promising anti-leukemic agents, showing higher effectiveness than the known chemotherapeutic drug etoposide. This potency is also coupled with a good selectivity index on non-tumoral cells and drug-likeness profile. SAR analysis provided new, valuable information on the pharmacophore for withanolide-type compounds, suggesting that chemical optimization of B, D, and lactone rings will be helpful in the rational design of anti-leukemic drugs. Therefore, the compounds identified deserve further studies to unravel their potential as a therapeutic alternative against leukemia. These results reinforce the potential of Withania species as a source of lead compounds and chemical diversity.In the current study, and as a continuation of our efforts to find new drug candidates for leukemia treatment, four new withanolide-type steroids, named withaperoxidins A–D, with an unusual cyclic peroxide moiety, and twenty-five known compounds were identified from


N-Hydroxy-6--1H-benzo[d]imidazol-1-yl)hexanamide (11b) is the most interesting compound, with IC50 values of 0.66; 1.46 and 3.67 µM. for HDAC6; HDAC1 and CK2; respectively. Cellular assays on different cancer cell lines rendered promising results for N-Hydroxy-8--1H-benzo[d]imidazol-1-yl)octanamide (11d). This inhibitor presented the highest cytotoxic activity, proapoptotic capability, and the best mitochondria-targeting and multidrug-circumventing properties, thus being the most promising drug candidate for further in vivo studies.The design of multitarget drugs (MTDs) has become an innovative approach for the search of effective treatments in complex diseases such as cancer. In this work, we communicate our efforts in the design of multi-targeting histone deacetylase (HDAC) and protein kinase CK2 inhibitors as a novel therapeutic strategy against cancer. Using tetrabromobenzotriazole (TBB) and 2-dimethylamino-4,5,6,7-tetrabromo-benzimidazole (DMAT) as scaffolds for CK2 inhibition, and a hydroxamate to coordinate the zinc atom present in the active site of HDAC , new multitarget inhibitors have been designed and synthesized. According to the in vitro assays,  The development of new efficient anticancer drugs still represents a challenge for medicinal chemists. Although significant progress has been made, single-target drugs still have significant drawbacks, such as induced toxicity and drug-resistance. Combination cancer therapy, a modality that combines two or more agents acting on different pharmacological targets, has expanded the treatment options. These therapies achieve the desirable results in a concerted manner through different mechanisms of action ,2,3. NevProtein kinase CK2 is a pleiotropic and ubiquitous serine/threonine protein kinase, which is composed of tetrameric complexes consisting of two catalytic subunits  and a dimer of the regulatory β subunits ,15. CK2 Histone deacetylases (HDAC) are a type of epigenetic enzymes that in recent years have aroused great interest in oncology. Its aberrant activity has been related to cell proliferation in a variety of human pathologies, especially solid tumors and myeloid neoplasia ,39,40,4150 = 4.4, 2.4 and 15.7 nM, respectively), and Fimepinostat (CUDC-907), which is an orally active pan PI3K/HDAC inhibitor  and in DMSO-d6 [calibrated at 2.50 ppm (1H) and 39.5 ppm (13C)]. Data are presented as follows (1H): chemical shift (ppm), multiplicity , coupling constant J (Hz) and integration. Data for 13C-NMR are reported in terms of chemical shifts (ppm). Mass spectra (MS) were determined on a Bruker Esquire 3000 spectrometer (ionizing voltage = 70 eV). Analytical purity of the tested compounds was determined by using an Agilent 1260 Infinity II HPLC system. Melting points (uncorrected) were taken in open-end capillary tubes and were determined on a Stuart Scientific SMP3 apparatus. Thin-layer chromatography (TLC) was run on Merck silica gel 60 F254 plates. Column chromatography was performed using silica gel Merk-60 (230–400 mesh).Unless stated otherwise, purchased starting materials used were high-grade commercial products. NMR spectra were recorded at 400 MHz (H-benzo[d]imidazole (4a)2,4,5,6,7-Pentabromo-1-(prop-2-yn-1-yl)-13 imidazole (4b)2,4,5,6,7-Pentabromo-1-(but-3-yn-1-yl)-13 imidazol-2-amine (4c)4,5,6,7-tetrabromo-4a  in metanol (5 mL), dimethylamine  was added. The reaction mixture was placed in a sealed tube and refluxed at 110 °C for 48 h. After removing the solvent under reduced pressure, the crude material was purified by column chromatography on silica gel (hexane/EtOAc 8:2) to afford compound 4c  as white solid (m.p. 194-195 °C). 1H-NMR : δ 4.95 , 3.53 , 3.07 . 13C-NMR : δ 161.9, 142.9, 133.4, 120.2, 118.9, 112.5, 105.7, 79.2, 76.9, 41.4, 36.9.To a stirred solution of N,N-dimethyl-1H-benzo[d]imidazol-2-amine (4d)4,5,6,7-Tetrabromo-1-(but-3-yn-1-yl)-4b  and dimethylamine , and after purification by column chromatography on silica gel (hexane/EtOAc 8:2), compound 4d  was obtained as white solid (m.p. 190–192 °C). 1H-NMR : δ 4.41 , 2.99 , 2.78 , 2.49–2.45 . 13C-NMR : δ 163.1, 143.5, 132.8, 120.2, 119.0, 112.9, 105.9, 79.7, 73.3, 44.5, 41.7, 19.2.Following the same procedure as before, starting from H-benzo[d]imidazol-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-N-((tetrahydro-2H-pyran-2-yl)oxy)butanamide (6a)4--14c  and azide 5b imidazol-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-N-((tetrahydro-2H-pyran-2-yl)oxy)pentanamide (6b)5--14c , azide 5c imidazol-1-yl)ethyl)-1H-1,2,3-triazol-1-yl)-N-((tetrahydro-2H-pyran-2-yl)oxy)propanamide (6c)3--14d , azide 5a imidazol-1-yl)ethyl)-1H-1,2,3-triazol-1-yl)-N-((tetrahydro-2H-pyran-2-yl)oxy)butanamide (6d)4--14d , azide 5b imidazol-1-yl)methyl)-1H-1,2,3-triazol-1-yl)butanamide (7a)6a  in MeOH (5 mL), a pre-cooled solution of AcCl  in MeOH (1 mL) was slowly added over 30 minutes at 0 °C. The reaction mixture was stirred at RT for 15 minutes. Then, NaHCO3 (saturated solution) was added until neutral pH. After extraction with EtOAc (3 × 20 mL), the combined organic layers were dried over MgSO4. The solvent was evaporated under reduced pressure, and the resulting crude material was purified by column chromatography (DCM/MeOH 98:2) to give 7a  as a white solid (mp 191–193 °C). 1H-NMR : δ 10.37 , 8.71 , 7.99 , 5.55 , 4.28 , 2.97 , 1.97–1.89 . MS (ESI) m/z 682 [M+ + 23]. HPLC purity 99%.To a solution of N-Hydroxy-5--1H-benzo[d]imidazol-1-yl)methyl)-1H-1,2,3-triazol-1-yl)pentanamide (7b)6b  and AcCl , and after purification by column chromatography (DCM/MeOH 98:2), 7b  was obtained as a white solid (mp 203–205 °C). 1H- NMR : δ 10.33 , 8.66 , 7.95 , 5.55 , 4.27 , 2.98 , 1.93 , 1.74–1.67 , 1.39–1.32 . MS (ESI) m/z 696 [M+ + 23], 674 [M+ + 1]. HPLC purity 95%.Following the same procedure as before, starting from N-Hydroxy-3--1H-benzo[d]imidazol-1-yl)ethyl)-1H-1,2,3-triazol-1-yl)propanamide (7c)6c  and AcCl  and after purification by column chromatography (DCM/MeOH 98:2), 7c  was obtained as a white solid (mp 168-170 °C). 1H- NMR : δ 10.49 , 8.83 , 7.73 , 4.53–4.45 , 2.92 , 2.87 , 2.56-2.52 . MS (ESI) m/z 682 [M+ + 23], 660 [M+ + 1]. HPLC purity 95%.Following the same procedure as before, starting from N-Hydroxy-4--1H-benzo[d]imidazol-1-yl)ethyl)-1H-1,2,3-triazol-1-yl)butanamide (7d)6d  and AcCl , and after purification by column chromatography (DCM/MeOH 98:2), 7d  was obtained as a white solid (159–161 °C). 1H- NMR : δ 10.39 , 8.73 , 7.74 , 4.53 , 4.23–4.27 , 2.91 , 2.89 , 2.60 . MS (ESI) m/z 696 [M+ + 23], 674 [M+ + 1]. HPLC purity 98%.Following the same procedure as before, starting from H-benzo[d]imidazol-1-yl)pentanoate (8a).Methyl 5--1DMAT imidazol-1-yl)hexanoate (8b)Ethyl 6--1DMAT imidazol-1-yl)heptanoate (8c)Ethyl 7--1DMAT imidazol-1-yl)octanoate (8d)Methyl 8--1DMAT imidazol-1-yl)pentanoic acid (9a)5--18a  in THF/H2O (2.5 mL/1 mL), LiOH  was added and the reaction mixture was stirred at RT for 2 h. After removing solvents under reduced pressure, H2O (20 mL) was added and EtOAc (20 mL) to wash. The aqueous phase was acidified with HCl (1 M) and extracted with EtOAc (2 × 20 mL). Combined organic layers were washed with brine, dried over MgSO4, filtered and the solvent was evaporated under reduced pressure to afford compound 9a  as a white oil. 1H-NMR : δ 12.01 , 4.26 , 2.98 , 2.17 , 1.57–1.50 , 1.28–1.23 . 13C-NMR : δ 174.1, 162.6, 143.3, 132.9, 120.0, 119.1, 112.9, 106.0, 45.9, 41.8, 32.9, 29.1, 21.0.To a solution of H-benzo[d]imidazol-1-yl)hexanoic acid (9b)6--18b  and LiOH , 9b  was obtained as a white solid. 1H-NMR : δ 4.24 , 2.98 , 2.12 , 1.53–1.42 , 1.11–1.07 . 13C- NMR : δ 174.3, 162.8, 143.3, 132.9, 119.9, 119.0, 112.9, 105.8, 46.0, 41.8, 33.6, 29.4, 25.0, 24.0.Following the same procedure as before, and starting from H-benzo[d]imidazol-1-yl)heptanoic acid (9c)7--18c  and LiOH , 9c  was obtained as a white solid. 1H- NMR : δ 4.24 , 2.98 , 2.15–2.11 , 1.53–1.45 , 1.44–1.36 , 1.25–1.17 , 1.09–1.02 . 13C- NMR : δ 174.4, 163.0, 143.4, 133.0, 120.0, 119.0, 112.9, 105.9, 46.1, 41.8, 33.5, 29.5, 27.9, 25.2, 24.3.Following the same procedure as before, and starting from H-benzo[d]imidazol-1-yl)octanoic acid (9d)8--18d  and LiOH , 9d  was obtained as a colorless oil. 1H- NMR : δ 11.96 , 4.24 , 2.98 , 2.15 , 1.52–1.48 , 1.43–1.40 , 1.20–1.15 , 1.05–1.02 .Following the same procedure as before, and starting from H-benzo[d]imidazol-1-yl)-N-((tetrahydro-2H-pyran-2-yl)oxy)pentanamide (10a)5--19a  in DMF (10 mL) were successively added HOBt , NMM , NH2OTHP  and EDCI . The resulting mixture was stirred at RT overnight. H2O (20 mL) and EtOAc (3 × 20 mL) were added. After extractions, combined organic layers were washed with brine, dried over MgSO4, filtered and evaporated under reduced pressure. The crude obtained was purified by column chromatography on silica gel (EtOAc/hexane 8:2) affording compound 10a  as a white solid. 1H-NMR : δ 10.86 , 4.71 , 4.24 , 3.84 , 3.40 , 2.98 , 1.93 , 1.64–1.47 , 1.30–1.26 . 13C- NMR : δ 168.6, 162.9, 143.4, 132.9, 120.0, 119.0, 112.9, 106.0, 100.8, 61.3, 46.0, 41.8, 31.6, 29.2, 27.7, 24.7, 21.6, 18.3.To a solution of H-benzo[d]imidazol-1-yl)-N-((tetrahydro-2H-pyran-2-yl)oxy)hexanamide (10b)6--19b , HOBt , NMM , NH2OTHP  and EDCI , 10b  was obtained as a white solid. 1H- NMR : δ 8.25 , 4.91 , 4.23 , 3.93–3.89 , 3.63–3.60 , 3.02 , 2.09–1.98 , 1.81–1.76 , 1.67–1.55 , 1.19–1.14 . 13C- NMR : δ 170.0, 163.0, 143.6, 132.8, 121.0, 120.3, 114.0, 105.9, 102.5, 62.5, 46.3, 42.3, 32.9, 29.9, 27.9, 25.6, 24.9, 24.6, 18.5.Following the same procedure as before, and starting from H-benzo[d]imidazol-1-yl)-N-((tetrahydro-2H-pyran-2-yl)oxy)heptanamide (10c)7--19c , HOBt , NMM , NH2OTHP  and EDCI , 10c was obtained  as a white solid. 1H- NMR : δ 8.19 , 4.92 , 4.23 , 3.95–3.89 , 3.63–3.60 , 3.03 , 2.05 , 1.80–1.77 , 1.62–1.52 , 1.30–1.25 , 1.17–1.12 . 13C- NMR : δ 170.0, 163.0, 143.6, 132.9, 121.0, 120.3, 114.0, 105.9, 102.5, 62.6, 46.5, 42.4, 42.3, 33.1, 30.1, 28.5, 28.0, 25.9, 25.0, 18.5.Following the same procedure as before, and starting from H-benzo[d]imidazol-1-yl)-N-((tetrahydro-2H-pyran-2-yl)oxy)octanamide (10d)8--19d , HOBt , NMM , NH2OTHP  and EDCI , 10d  was obtained as a white solid. 1H- NMR : δ 4.92 , 4.22–4.17 , 3.92–3.87 , 3.56–3.54 , 3.03 , 2.09–2.06 , 1.79–1.69 , 1.57–1.51 , 1.18–1.14 , 1.08–1.13 .Following the same procedure as before, and starting from N-Hydroxy-5--1H-benzo[d]imidazol-1-yl)pentanamide (11a)7a, starting from 10a  and AcCl , and after purification by column chromatography (EtOAc/Hexane 4:1), 11a  was obtained as a white solid (m.p.168 - 170 °C). 1H- NMR : δ 10.30 , 8.67 , 4.25 , 2.98 , 1.89 , 1.50–1.46 , 1.29–1.25 . MS (ESI) m/z 615 [M+ + 23], 593 [M+ + 1]. HPLC purity 98%.Following the same procedure that we used for the synthesis of N-Hydroxy-6--1H-benzo[d]imidazol-1-yl)hexanamide (11b)7a, starting from 10b , and AcCl  and after purification by column chromatography (EtOAc/Hexane 4:1), 11b  was obtained as a white solid (m.p. 167–169 °C). 1H- NMR : δ 10.29 , 8.64 , 4.23 , 2.99 , 1.87 , 1.50–1.44 , 1.08–1.06 . MS (ESI) m/z 629 [M+ + 23], 607 [M+ + 1]. HPLC purity 97%.Following the same procedure that we used for the synthesis of N-Hydroxy-7--1H-benzo[d]imidazol-1-yl)heptanamide (11c)7a, starting from 10c  and AcCl , and after purification by column chromatography (DCM/MeOH 98:2), 11c  was obtained as a white solid (m.p. 120-122 °C). 1H- NMR : δ 10.3 , 8.62 , 4.23 , 2.99 , 1.89 , 1.51–1.48 , 1.42–1.38 , 1.21–1.17 , 1.08–1.04 . MS (ESI) m/z 643 [M+ + 23], 621 [M++ 1]. HPLC purity 98%.Following the same procedure that we used for the synthesis of N-Hydroxy-8--1H-benzo[d]imidazol-1-yl)octanamide (11d)7a, starting from 10d  and AcCl , and after purification by column chromatography (DCM/MeOH 98:2), 11d  was obtained as a white solid (m.p. 118 °C). 1H- NMR : δ 10.3 , 8.64 , 4.24 , 2.99 , 1.89 , 1.51–1.48 , 1.43–1.40 , 1.19–1.13 , 1.06–1.02 . MS (ESI) m/z 657 [M+ + 23], 635 [M++ 1]. HPLC purity 95%.Following the same procedure that we used for the synthesis of H-benzo[d]triazol-2-yl)methyl)benzoate (12a) and methyl 4-methyl)benzoate (12b)Methyl 4-((tetrabromo-2TBB triazol-1-yl)methyl)benzoic acid (13b)4-, 13b  was obtained as a white solid. 1H-NMR  δ 7.89 , 7.21 , 6.29 .Following the same procedure as before and starting from H-benzo[d]triazol-2-yl)methyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)benzamide (14a)4-, HOBt , NMM , NH2OTHP  and EDCI , 14a  was obtained, after column chromatography on silica gel (EtOAc/hexane 3:7), as a white solid. 1H-NMR  δ 8.71 , 7.76 , 7.51 , 5.92 , 5.06 , 4.03–3.89 , 3.69–3.57 , 1.93–1.57 .Following the same procedure that was used for the synthesis of H-benzo[d]triazol-1-yl)methyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)benzamide (14b)4-, HOBt , NMM , NH2OTHP  and EDCI , 14b  was obtained, after column chromatography on silica gel (EtOAc/hexane 2:1 to 1:1), as a white solid. 1H-NMR  δ 9.03 , 7.71 , 7.15 , 6.18 , 5.04 , 3.98–3.90 , 3.72–3.51 , 1.91–1.47 .Following the same procedure that was used for the synthesis of N-Hydroxy-4-methyl)benzamide (15a)7a, starting from 14a  and AcCl , crude reaction was obtained. A precipitate formed after addition of Et2O was filtered and solved in DCM/MeOH (5%) and precipitated again with hexane. The solid was filtered, giving 15a  as a white solid (m.p. 218–220 °C). 1H-NMR  δ 11.23 , 9.07 , 7.75 , 7.46 , 6.12 . MS (ESI) m/z 584.6 [M++1]. HPLC purity 94%.Following the same procedure that we used for the synthesis of N-Hydroxy-4-methyl)benzamide (15b)14b , and AcCl , 15b  was obtained as a white solid (m.p. 139-142 °C). 1H-NMR  δ 11.22 , 7.70 , 7.18 , 6.25 . MS (ESI) m/z 584.6 [M++1]. HPLC purity 97%.Following the same procedure as before starting from H-benzo[d]imidazol-1-yl)methyl)benzoate (16)Methyl 4--1DMAT imidazol-1-yl)methyl)benzamide (19)7a, and starting from 18 , 19  was obtained as a white solid (m.p. 210–213 °C). 1H-NMR  δ 7.67 , 7.07 , 5.57 , 2.94 . MS (ESI) m/z 648.7 [M++23]. HPLC purity 95%.Following the same procedure that we used for the synthesis of To date, 158 crystal structures of the CK2 alpha catalytic subunit have been deposited in the PDB database . Four of them  are bound to TBB derivatives, including compound JRJ that is the most similar to our set of molecules. Interestingly, JRJ is crystallized in two different conformations: in 5CQU the flexible chain points to the ATP phosphate-binding region, whereas in 5CQW it points to the α-D helix. Of these two 3D structures, we selected 5CQU as the protein shows a more open G-loop compared to 5CQW. For HDAC1, despite its biological interest, only two 3D-structures have been deposited in the PDB: 4BKX and 5ICN (UniProt Entry Q13547 Human Histone deacetylase 1). Of those, PDB code 5ICN was selected due to the presence of a hydroxamic derivative bound to the holo structure . For HDAThe Glide Grid  module was used to calculate the grid-box with a 20 Å length, using as box center the ligands JRJ and TSN for CK2 and HDAC6, respectively; and, the catalytic Zn for HDAC1, applying the versatile metal binding restriction to the zinc ion in both HDACs. Note must be taken that when using the ligand as box center, the program automatically deletes the ligand for the grid generation. The set of described ligands were prepared with the LigPrep module of Maestro  considering a pH of 7 ± 4, and to obtain all possible hydroxamic protonation states and the metal binding site identification was also used. Ligands with a protonated hydroxamic acid were used for docking on CK2 whereas ligands with the deprotonated hydroxamate were docked in HDAC1 and HDAC6. The docking calculations were performed with Glide using the XP algorithm , applyinFor the MD simulations of the CK2-ligand complexes, all ligands were prepared as follows: the geometry optimization and charge distributions were calculated quantum mechanically (RHF/6-31+G**) with Gaussian 09 Revision A.1 .The general AMBER force field 2 (GAFF2) was used to assign bonded and nonbonded parameters .For HDAC1 and HDAC6-ligand complexes, binding metal sites and ligands were parametrized with the MCPB module embedded into the MTK++ software package in AMBER16 , as thishttp://ambermd.org/). The classic ff14SB AMBER force field  ATP (specific radioactivity 300–1000 cpm/pmol), 15 mM MgHuman HCT-116 colon carcinoma cells, breast adenocarcinoma cells of MCF-7 line, Jurkat T-leukemia cells, leukemia cells of HL-60 cell line and its drug-resistant sublines HL-60/adr (overexpression of MRP-1) and HL-60/vinc (overexpression of P-glycoprotein) were kindly provided by Prof. Walter Berger, Institute of Cancer Research, Vienna Medical University. Human embryonal kidney cells of HEK293 line were obtained from cell culture collection of R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, National Academy of Sciences of Ukraine .2 humidified atmosphere at 37 °C. Cells were seeded (5 × 105/mL for leukemia cells and 1 × 105/mL for carcinoma cells) into 24-well tissue culture plates  and were permitted to adhere and grow for 24 h before the experiment. Short-term (24 h) cytotoxic effect of antitumor drugs was studied under the Evolution 300 Trino microscope  after cell staining with trypan blue dye (0.1%).Cells were cultured in RPMI-1640 medium  supplemented with 10% fetal calf serum , 50 µg/mL streptomycin , and 50 units/mL penicillin  in 5% COStock solutions (2 mM) of each CK2-HDAC inhibitor in DMSO  were prepared, and additionally dissolved in serum-free culture medium (RPMI for leukemia cells and DMEM for carcinoma cells) prior to addition to cell culture. Final concentration of DMSO in cell culture was 0.5% or less. Cytotoxicity studies revealed no statistically significant toxicity of 0.5% DMSO solution on all cell lines, used in these studies.For cell death analyses, cells were stained with FITC-conjugated annexin V and propidium iodide (PI) using an apoptosis detection kit , according to the manufacturer’s instructions. In particular, 24 h after the addition of various concentration of CK2-HDAC inhibitors, Jurkat T-leukemia cells were centrifuged at 2,000 rpm, washed twice with 1x PBS, and incubated for 15 minutes in Annexin V binding buffer  containing 1/50 volume of FITC-conjugated Annexin V solution and PI (50 µg/mL). Then samples were diluted two times by appropriate volume of Annexin V binding buffer  and immediately measured on FL1/FL2 (FITC-PI) channels of FACScan flow cytometer . Analysis of the obtained results was carried out using the Cytomation Summit Software v3.1 .6 cells were collected, pelleted by spinning at 1,000 rpm, 4 °C for five minutes, resuspended in 1 mL of cold PBS and fixed by adding drop by drop 4 mL of −20 °C absolute ethanol. On the next day, fixed cells were centrifuged again and cell pellets were resuspended in 1 mL of PBS. Then 100 µL of 200 µg/mL DNase-free, RNase A  was added to cell suspension and incubated at 37 °C for 30 minutes. After this 100 µL of 1 mg/mL propidium iodide was added to samples, which were incubated at room temperature for 5–10 min and then analyzed on FACScan flow cytometer . Cell cycle analysis was carried out using the Cytomation Summit Software v3.1 .Cell cycle of the analyzed cells was assessed according to protocol described by Walker et al. . After d6 Jurkat cells were treated for 1, 3, 6, 12 and 24 h with the tested drugs. After PBS washing, cells were incubated for 10 minutes in freshly prepared JC-1 solution (10 mg/mL in culture medium) at 37 °C. Spare dye was removed by PBS washing and cell-associated fluorescence was measured on FL1/FL2 (FITC-PI) channels of FACScan flow cytometer . Analysis of the obtained results was carried out using the Cytomation Summit Software v3.1 .Breakdown of ΔΨm mitochondrial membrane potential was determined by FACS analysis using 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). For this purpose, the Mitochondrial Membrane Potential Detection Kit  was used as described in the manufacturer’s instruction. Briefly, 102O2) and dihydroethidium  in concentrations of 10 μM at 37 °C for 30 minutes. After incubation with the dyes, cells were washed with PBS and immediately analyzed at FL1 (DCFDA) or FL2 channel (DHE) of FACSCalibur flow cytometer .Cellular ROS contents were measured by incubating control or drug-treated cells with fluorescent dyes dihydrodichlorofluorescein diacetate .All experiments were performed in triplicate and repeated three times. Statistical analysis of data was conducted in GraphPad Prism 7.0 Software  using Student’s t test. Statistical significance was set at P ≤ 0.05. Evaluation of LC11b , a DMAT derivative with a five-carbon atom chain connecting the hydroxamate moiety. Furthermore, this compound was found to be even more active against HDAC6 (IC50 = 0.86 µM).In this study we demonstrate that a multitarget approach can provide interesting anticancer lead compounds. In silico studies showed plausible predicted binding modes for all the designed compounds in spite of their different dynamic behaviors. The best result for a dual CK2/HDAC1 inhibitor in the enzymatic assays was found for 50 in the order of 4–23 µM in several tumor cell lines. Studies on the mechanism of cell death induction by 11b showed apoptotic activity and G1/S growth arrest in Jurkat T-leukemia cells. This compound was also able to circumvent multidrug resistance of human leukemia cells, caused by overexpression of MRP-1 and P-glycoprotein, and this phenomenon was accompanied by an increase in superoxide production both in sensitive and MDR cell lines.This compound showed a promising in vitro antiproliferative activity, with LC11d, showing lower enzymatic activity against the three protein targets , unexpectedly provided the best cellular activity with LC50 in 3–8 µM range. This difference in cellular behavior may be attributed to the increase in lipophilicity brought about by the longer seven-membered chain of 11d compared to the five carbon linker of 11b. Moreover, this inhibitor presented the highest cytotoxic activity, proapoptotic capability, and the best mitochondria-targeting and MDR circumventing properties, thus being the most promising drug candidate for further in vivo studies.Compound Altogether, this work further demonstrates that our multitarget HDAC/CK2 approach is a valid strategy for drug discovery in the field of anticancer drug development.


Persicaria chinensis, a new hydrolyzable tannin called persicarianin was isolated and characterized to be 3-O-galloyl-4,6-(S)-dehydrohexahydroxydiphenoyl-d-glucose. Interestingly, acid hydrolysis of this compound afforded ellagic acid, despite the absence of a hexahydroxydiphenoyl group. From the rhizome of Polygonum runcinatum var. sinense, a large amount of granatin A, along with minor ellagitannins, helioscpoinin A, davicratinic acids B and C, and a new ellagitannin called polygonanin A, were isolated. Based on 2D nuclear magnetic resonance (NMR) spectroscopic examination, the structure of polygonanin A was determined to be 1,6-(S)-hexahydroxydiphenoyl-2,4-hydroxychebuloyl-β-d-glucopyranose. These are the second and third hydrolyzable tannins isolated from Polygonaceous plants. In addition, oligomeric proanthocyanidins of Persicaria capitatum and P. chinensis were characterized by thiol degradation. These results suggested that some Polygonaceous plants are the source of hydrolyzable tannins not only proanthocyanidins.The aim of this study was to characterize hydrolyzable tannins in Polygonaceous plants, as only a few plants have previously been reported to contain ellagitannins. From  O-galloyl-1,6-(S)-HHDP-β-d-glucose  has been isolated from Persicaria capitatum (syn. Polygonum capitatum) , H-4 , and H-6  indicates acylation of these positions. The acyl groups were shown to be a galloyl and a dehydrohexahydroxydiphenoyl (DHHDP) group by 13C NMR spectroscopy, which exhibited signals attributable to an aliphatic methine (δ 43.0), a double bond (δ 151.7 and 130.8), a ketone (δ 192.0), and two acetal carbons (δ 91.5 and 96.5), constructing a hydrated cyclohexenetrione ring of the DHHDP group. The large chemical shift differences of the glucose H-6 methylene proton signals in the 1H NMR spectrum suggested that the DHHDP esters bridges between the C-4 and C-6 hydroxy groups  with the HHDP ester carbonyl carbons . The atropisomerism of the HHDP was shown to be an S configuration by appearance of positive and negative Cotton effects at 242 nm and 262 nm, respectively, in the electronic circular dichroism (ECD) spectrum. The configuration is the same as that of 1, 7, 9, and 10. Coupling constants of the pyranose ring protons  were <2 Hz. This was similar to those of 7, 9, and 10, but different to those of 1 , suggesting that the glucopyranoses of 7, 9, 10, and 11 adopt a 1C4 conformation, whereas the glucopyranose core of 1 with a 2,3,4-trigalloyl structure adopts a boat conformation (11 was composed of 3 carboxyl carbons (δC 172.3 (C-1’), 172.0 (C-6’), 166.1 (C-7’)), an oxygenated tertiary carbon (δC 76.3 (C-4’)), an oxygenated methine (δC 66.3 (C-2’)), a methylene (δC 41.6 (C-5’)), and a benzylic methine (δC 49.2 (C-3’)), along with a trihydroxy benzoyl moiety. Taking the molecular formula C34H26O24 indicated by HR-fast atom bombardment (FAB) MS into account, these building blocks of the 2,4-acyl group suggested that 8 is generated by addition of H2O to the double bond of 9 or 10 and rearrangements of lactone formation. This was supported by HMBC correlations, as illustrated in H 4.84 , 4.90 , 3.28 and 3.43 ; δC 68.5 (C-2’), 47.3 (C-3’), 77.6 (C-4’), 42.5 (C-5’)) are similar to those of 11  and 4.88 , 3.35 and 2.78 ; δC 66.3 (C-2’), 49.2 (C-3’), 76.3 (C-4’), 41.6 (C-5’)). The configuration of C-2’, C-3’, and C-4’ was concluded to be S*, R*, and S* based on the nuclear Overhauser effect spectroscopy (NOESY) correlations between the H-2’, H-3’ and H-5’ of 11. The most stable conformation of 11 obtained by computational calculation along with NOESY correlations is shown in 11 was deduced to be the same as that of the DHHDP group of 7. Interestingly, the glucose H-3 of 11 (δH 5.14) resonated at a much lower field compared to those of 7a (δH 4.58), 9 (δH 4.40) and 10 (δH 4.44). This could be due to the deshielding effect of the ester carbonyl group attached to the glucose C-2 hydroxy group  derivatives of epicatechin and epicatechin-3-O-gallate originating from extension units, accompanied by small peaks of catechin, epicatechin, and epicatechin gallate arising from terminal units. Oligomeric proanthocyanidins of P. chinensis yielded 2-hydroxyethylsulfanyl derivatives of epicatechin, epigallocatechin, epicatechin-3-O-gallate and epigallocatechin-3-O-gallate originating from extension units, along with the free form of epicatechin-3-O-gallate originating from the terminal unit. The results indicates that proanthocyanidins in these two plants belonging the same genus Persicaria are composed of different flavan-3-ol units  operating at 500 MHz and 126 MHz for the 1H and 13C nuclei, respectively. 1H- and 13C-NMR spectra were also recorded on a JEOL JNM-AL400 spectrometer  operating at 400 and 100 MHz for the 1H and 13C nuclei, respectively. Coupling constants (J) were expressed in hertz and chemical shifts (δ) are reported in ppm with the solvent signal used as a standard . FAB-MS data were recorded on a JMS700N spectrometer  using m-nitrobenzyl alcohol or glycerol as the matrix. ESI-TOF-MS data were recorded on a quadrupole (Q)-TOF LC/MS . Column chromatography was performed using Sephadex LH-20 , Diaion HP20PSS , Chromatorex ODS , and Toyopearl butyl-650M  columns. TLC was performed on precoated Kieselgel 60 F254 plates  with CHCl3-MeOH-H2O  and toluene-ethyl formate-formic acid  mixtures being used as the eluents. The spots were detected using UV illumination and by spraying with a 5% H2SO4 solution followed by heating. Analytical HPLC was performed on a Cosmosil 5C18-ARII  column  with a gradient elution of 4–30% (39 min) and 30–75% (15 min) CH3CN in 50 mM H3PO4 at 35 °C .Optical rotations were measured on a JASCO P-1020 digital polarimeter . IR spectra were measured on a JASCO FT/IR 410 spectrophotometer. Ultraviolet (UV) spectra were obtained on a JASCO V-560 UV/VIS spectrophotometer. ECD spectra were measured with a JASCO J-725N spectrophotometer. Persicaria capitata was collected in Shui Cheng, China, in 2014, and in Nagasaki, Japan, in 2017. The aerial part of Persicaria chinensis were collected in Nagasaki, Japan, in 2014. Voucher specimens were deposited at the Nagasaki University Graduate School of Biomedical Sciences. Fresh rhizome of Polygonum runcinatum var. sinense were purchased in a medicinal plant market in Guangxi, China. A voucher specimen was deposited at the Guangxi Institute of Botany. Aerial parts of Reynoutria japonica, Persicaria perfoliata, P. longiseta, P. lapathifolia, P. filiformis, and P. thunbergii were collected in Nagasaki, Japan.The whole plant of Reynoutria japonica, Persicaria perfoliata, P. longiseta, P. lapathifolia, P. capitata (syn. Polygonum capitatum), P. chinensis (syn. Polygonum chinense), P. filiformis, and P. thunbergii were extracted with 60% CH3CN (20 mL) and analyzed by HPLC  of  by HPLC .P. capitata was suspended in 50% MeOH and insoluble material was removed by filtration. The soluble part (132 g) was fractionated by Diaion HP20SS column chromatography (8 cm i.d. × 35 cm) with 0–100% MeOH  to give 8 fractions (fr.): fr. 1 (84.5 g), fr. 2 (2.2 g), fr. 3 (3.9 g), fr. 4 (9.5 g), fr. 1–5 (14 g), fr. 1–6 (4.3 g), fr. 1–7 (6.9 g), and fr. 1–8 (2.1 g). Fr. 3 was subjected to a combination of column chromatography using Sephadex LH-20 (0–100% MeOH), Avicel cellulose (2% AcOH), and Diaion HP20SS (0–50% MeOH) to give 2,3-di-O-galloyl-d-glucose (719 mg) and 2,4-di-O-galloyl-d-glucose (101 mg). Similar separation of fr. 4 by chromatography using Sephadex LH-20 and Chromatorex ODS  to yield 2,3,4-tri-O-galloyl-d-glucose (2.26 g), 6-O-galloyl arbutin (79 mg), 4-hydroxy-3-methoxyphenol 1-O-β-d--glucopyranoside (37 mg), 4-hydroxy-2-methoxyphenol 1-O-β-d--glucopyranoside (46 mg). Fr. 6 was purified by precipitation from water to give davidiin (1) (13 g). Fr. 7 was subjected to a column chromatography on Sephadex LH-20 (60–100% MeOH), Diaion HP20SS, and Chromatorex ODS to give quercetin 3-O-α-rhamnoside (2.5 g), quercetin (719 mg), quercetin 3-O-β-d-glucopyranoside (182 mg), quercetin 3-O-α-L--rhamnoside (6.7 mg). The insoluble part of the extract (68 g) was washed with CHCl3–MeOH  to remove non-polar substances and a part (5 g) of the residue (31.5 g) was subjected to size-exclusion column chromatography using a Sephadex LH-20 column (4 cm i.d. × 45 cm) with 7 M urea:acetone  to give fractions containing oligomeric polyphenols and compounds with low-molecular weight , and Diaion HP20SS column chromatography to afford 8 (990 mg) and a mixture of oligomeric polyphenols detected at origin on TLC plate.The dried root of D +43.8 , IR νmax cm−1: 3412, 1728, 1708, 1614, 1533, 1450, UV λmax (MeOH) nm (log ε): 271 (4.06), 218 (4.59), HRESIMS m/z: 673.0690 , ECD (MeOH) Δε (nm): −25.16 (213), +6.68 (234), −5.47 (283), +2.36 (374). 1H-NMR  δH: 7.19 , 7.18 , 6.76 , 6.38 , 6.37 , 5.63 , 5.42 , 5.40 , 5.32 , 5.01 , 4.94 , 4.88 , 4.65 , 4.63 , 4.25 , 3.95 , 3.89 , 3.66 , 13C NMR  δC: 192.0 , 168.1 , 166.9 , 164.6 , 151.7 , 146.0 , 145.9 , 142.3 , 138.8 , 135.4 , 130.8 , 124.1 , 121.7 , 112.7 , 110.1 , 107.7 (β-DHHDP-3), 107.6 (α-DHHDP-3), 97.8 (β-glc H-1), 96.5 , 93.6 (α-glc C-1), 91.5 , 75.6 (β-glc C-3), 74.1 (α-glc C-4), 74.0 (β-glc C-4), 73.7 (β-glc C-2), 73.3 (α-glc C-3), 71.2 (α-glc C-2), 68.3 (β-glc C-5), 66.9 (α-glc C-6), 66.4 (β-glc C-6), 64.4 (α-glc C-5), 43.0 .Brown amorphous powder, [α]D −51.3° , FABMS m/z: 841 (M + Na)+, 819 (M + H)+, HRFABMS m/z: 819.0891 (M + H)+ , IR νmax cm-1 (neat): 3412, 1719, 1616, 1445, 1313, 1223, 1190. UV λmax (MeOH) nm (log ε): 266 (4.40), 230 (4.70). ECD (MeOH) λmax (Δε): 316 (−2.4), 283 (+12.6), 261 (−8.9), 241 (+17.9), 227 (+0.6), 217 (+5.5). 1H NMR  δH: 7.46  H-3), 6.83 , 6.79 , 5.95 , 5.14 , 5.10 , 4.99 , 4.88 , 4.83 , 4.72 , 4.48 , 4.08 , 3.35 , 2.78 . 13C NMR  δC: 172.3 (HChe-1’), 172.0 (C-6’), 168.2 (HHDP-7’), 166.7 (HChe-7), 166.4 (HHDP-7), 166.1 (HChe-7’), 146.0 (HChe-4), 145.3 (HHDP-4’), 144.5, 144.9 , 143.0 (HChe-6), 144.7 (HHDP-4), 138.5 (HChe-5), 136.9 (HHDP-5), 136.3 (HHDP-5’), 125.9 (HHDP-2), 125.7 (HHDP-2’), 121.0 (HChe-2), 116.7 (HHDP-1), 116.2 (HChe-3), 115.4 (HHDP-1’), 114.5 (HChe-1), 109.8 (HHDP-3), 108.7 (HHDP-3’), 90.4 (glc C-1), 76.3 (HChe-4’), 71.7 (glc C-2), 70.9 (glc C-5), 69.2 (glc C-4), 66.3 (HChe-2’), 64.2 (glc C-6), 59.1 (glc C-3), 49.2 (HChe-3’), 41.6 (HChe-5’).Tan amorphous powder, [α]3) (10 mg) was hydrolyzed by heating in 5% H2SO4 (1 mL) at 105 °C for 5 h in a screw capped vial. The precipitates (2.7 mg) formed in the mixture were collected by filtration. HPLC analysis of the filtrate showed peaks assignable to gallic acid (8.94 min), brevifolin carboxylic acid (21.36 min), and ellagic acid (30.66 min), which were identified by comparison of tR and UV absorption with those of authentic samples. HPLC analysis of the precipitates showed only a peak for ellagic acid. The filtrate was neutralized with saturated Ba(OH)2 and resulting precipitate of BaSO4 was removed by filtration. The filtrate was concentrated and the residue was dissolved in 0.5 mL of pyridine containing L-cysteine HCl (5.0 mg), and heated at 60 °C for 1 h. To the mixture was added o-tolylisothiocyanate (20 μL) and this mixture heated at 60 °C for 1 h. The final mixture was then cooled to ambient temperature and directly analyzed using HPLC. The retention time of the peak at 34.8 min coincided with that of the thiazolidine derivatives of D-glucose (L-glucose: 35. 6 min).Persicarianin . The low-energy conformers within a 6 kcal/mol window were optimized at the B3LYP/6-31G level in acetone (SMD). The vibrational frequencies were also calculated at the same level to confirm their stability, and no imaginary frequencies were found. The magnetic shielding constants (σ) of the low-energy conformers with Boltzmann populations greater than 1% were calculated using the gauge-independent atomic orbital (GIAO) method at the mPW1PW91/6-311+G level in acetone (PCM) and were weight-averaged ,37,38.Polymeric polyphenols obtained from the three plants (5 mg) were dissolved in a solution containing 4% 2-mercaptoethanol and 0.1% HCl in 60% EtOH (1 mL). The mixture was heated at 70 °C for 7 h and analyzed by HPLC. The standard thiol degradation products were obtained from persimmon proanthocyanidins .Persicaria capitatum. In this study, we reinvestigated P. capitatum and showed the presence of minor gallotannins and proanthocyanidin oligomers comprised of epicatechin and epicatechin-3-O-gallate. From Persicaria chinensis, a new hydrolyzable tannin called persicarianin (3) was isolated together with an ellagitannin geraniin and proanthocyanidin oligomers mainly comprising epicatechin, epigallocatechin and their galloyl esters. The rhizome of Polygonum runcinatum var. sinense contained granatin A (7) as the major constituent along with minor ellagitannins, including a new ellagitannin polygonanin A (11). These results prompted us to continue investigations into hydrolyzable tannins in this plant family.Distribution of hydrolyzable tannins in the plant kingdom is much more limited compared with that of proanthocyanidins. In Polygonaceous plants, a hydrolyzable tannin had only been isolated from