Their structures were determined by extensive spectroscopic analysis, including 1D, 2D nuclear magnetic resonance (NMR), and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), in combination with chemical methods (acid hydrolysis). The neuroinflammation model was established by lipopolysaccharide (LPS)-induced BV2 microglia, and the neuroprotective effects of all compounds (1–29) were evaluated.Five new oleanane-type triterpenoid saponins ( Acanthopanax senticosus (Rupr. & Maxim.) Harms, commonly known as Ci Wu Jia or Siberian Ginseng, is a well-known traditional Chinese medicine widely distributed in the northeast of China. With high medicinal value, A. senticosus is popularly used as an “adaptogen” like Panax ginseng. Modern pharmacology study shows that this plant was used for antifatigue, anti-depression, anxiolytic, anti-irradiation, anticancer, anti-inflammatory, hypolipidemic, etc.  (6–29). Their structures were elucidated mainly by spectroscopic methods including 1D and 2D nuclear magnetic resonance (NMR) experiments in combination with high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and by comparison of their physical and spectral data with literature. Meanwhile, their neuroprotective effects were evaluated by lipopolysaccharide (LPS)-induced BV2 microglia.In the present paper, we continue to further explore the active component triterpenoid saponins from the fruits of ns (1–5) , togethe® Prep T3  with a RID-20 A detector, with flow rates of 3 ml/min. Optical rotation measurements were conducted on a JASCO P-2000 instrument. Gas chromatography-mass spectrometry (GC-MS) analysis was performed on an Agilent 7890A system with a DB-5 capillary column. Absorbance (OD) value was detected on a BioTek Epoch™2 Microplate Reader. The FT-IR data of the new triterpenoid saponins was performed on Thermo Scientific Nicolet iS10. Silica gel column chromatography (CC) and octadecyl silica (ODS) chromatography were used in the separation of extracts.The HR-ESI-MS data of the new triterpenoid saponins were obtained on a Thermo Orbitrap Fusion Lumos Tribrid Mass Spectrometer. The 1D and 2D NMR spectra were acquired on a Bruker DPX-600 spectrometer in Pyridine-d5 using TMS as internal standard. Preparative high-performance liquid chromatography (HPLC)  was performed on Waters AtlantisA. senticosus was collected in October 2018 from the Yichun, Heilongjiang Province. The plant was identified by the Professor Rui-Feng Fan of the Heilongjiang University of Chinese Medicine, and its voucher specimen (NO. 20190330) has been deposited at Heilongjiang University of Chinese Medicine.The fruit of A. senticosus were extracted with 70% EtOH three times, under reflux for 2 h each time to afford a crude extract . The crude extract was extracted with petroleum ether, EtOAc, and n-BuOH successively, and the corresponding extract was obtained after removing the solvent, namely, PE fraction (320.0 g), EtOAc fraction (470.0 g), and n-BuOH fraction (510.0 g). The ethyl acetate layer (360.0 g) was chromatographed on a silica gel column (200–300 mesh) eluted successively with CH2Cl2/MeOH (100:1–0:1) to obtain nine fractions. Fr. VI was separated on a silica gel column (200–300 mesh) eluted successively with CH2Cl2/MeOH (50:1–0:1) to obtain five fractions (Fr. VI 1–5). Fr. VI 4 was purified by ODS chromatography to afford 48 fractions. Fr. VI 4–(46) were purified by semi-preparative HPLC (MeOH/H2O 84%) to afford compounds 14 (5.3 mg), 7 (4.9 mg), 15 (5.0 mg), 8 (37.5 mg), 18 (9.7 mg), 16 (45.9 mg), 10 (7.1 mg), and 11 (5.4 mg). Fr. VII was separated on a silica gel column (200–300 mesh), using solvent system CH2Cl2/MeOH (30:1 to 0:1) to give five fractions (Fr. VII 1–7) based on TLC analysis. Fr. VII 5 was purified by ODS chromatography to afford sixty-three fractions. Fr. VII 5–(54) were purified by semi-preparative HPLC (MeOH/H2O 78%) to afford compounds 5 (5.3 mg) and 6 (3.6 mg). Fr. VIII was separated on a silica gel column , using solvent system CH2Cl2/MeOH (30:1 to 0:1) to give nine fractions (Fr. VIII 1–9) based on TLC analysis. Fr. VIII 5 was purified by ODS chromatography to afford fifty fractions. Fr. VIII 5–(47) were purified by semi-preparative HPLC (MeOH/H2O 84%) to afford compounds 13 (66.4 mg) and 9 (52.8 mg). Fr. VIII 6 was purified by ODS chromatography to afford forty-two fractions. Fr. VIII 6-(24) was purified by semi-preparative HPLC (MeOH/H2O 73%) to afford compound 3 (8.8 mg). Fr. VIII 6–(25) was purified by semi-preparative HPLC (MeOH/H2O 73%) to afford compound 12 (5.3 mg). Fr. VIII 6–(27) were purified by semi-preparative HPLC (MeOH/H2O 68%) to afford compounds 1 (26.0 mg), 4 (4.2 mg), 17 (147.1 mg), and 22 (26.4 mg). Fr. VIII 6–(29) was purified by semi-preparative HPLC (MeOH/H2O 73%) to afford compound 25 (42.0 mg). Fr. VIII 6–(29D) were purified by semi-preparative HPLC (MeOH/H2O 78%) to afford compound 19 (3.6 mg). Fr. VIII 6–(30) were purified by semi-preparative HPLC (MeOH/H2O 70%) to afford compound 23 (28.1 mg). Fr. VIII 6–(30C) were purified by semi-preparative HPLC (MeOH/H2O 80%) to afford compound 2 (8.7 mg). Fr. VIII 6–(32) were purified by semi-preparative HPLC (MeOH/H2O 80%) to afford compound 26 (5.4 mg). Fr. VIII 6–(33) were purified by semi-preparative HPLC (MeOH/H2O 81%) to afford compound 24 (9.1 mg). Fr. VIII 6–(34) were purified by semi-preparative HPLC (MeOH/H2O 82%) to afford compounds 28 (8.2 mg) and 27 (7.6 mg). Fr. VIII 6–(35) were purified by semi-preparative HPLC (MeOH/H2O 84%) to afford compounds 29 (2.8 mg), 20 (7.7 mg), and 21 (23.5 mg).The dry fruits (20 kg) of c = 0.15, MeOH); HR-ESI-MS m/z: 784.4845 [M + NH4]+ . The 1H  and 13C NMR  data are shown in Acasentrioid A (1): amorphous powder; c = 0.32, MeOH); HR-ESI-MS m/z: 856.5043 [M + NH4]+ . The 1H  and 13C NMR  data are shown in Acasentrioid B 2): amorphous powder; : amorphoc = 0.28, MeOH); HR-ESI-MS m/z: 737.4490 [M + H]+ . The 1H  and 13C NMR  data are shown in Acasentrioid C (3): amorphous powder; c = 0.22, MeOH); HR-ESI-MS m/z: 798.4648 [M + NH4]+ . The 1H  and 13C NMR  data are shown in Acasentrioid D (4): amorphous powder; c = 0.23, MeOH); HR-ESI-MS m/z: 663.4121 [M + H]+ . The 1H  and 13C NMR  data are shown in Acasentrioid E (5): amorphous powder; 1–5 (each 1.0 mg) were dissolved in 2 ml of 2 M HCl , and hydrolyzed at 90°C for 3 h. After removing dioxane in a vacuum, the solution was diluted with H2O and extracted with EtOAc (3 × 1 ml). The aqueous layer was evaporated to dryness. The dried residue was dissolved in pyridine (200 μl) and treated with L-cysteine methyl ester hydrochloride (2.0 mg). After stirring the mixture for 1 h at 60°C, 100 μl of N-trimethylsilylimidazole was added, and they were kept at 60°C for 1 h. The reaction mixture was suspended in 1.0 ml H2O and extracted with n-hexane (3 × 1.0 ml). The layer of n-hexane was directly analyzed by GC with a DM-5 column  with the elution of N2 as carrier gas. Other GC conditions are as follows: column temperature: 220–270°C with the rate of 3°C/min; injector and detector temperature: 250°C; split ratio: 10:1; and injection volume:1 μl. The configurations of D-glucose, L-arabinose, D-glucuronic acid, and L-rhamnose in compounds 1–5 were determined by comparison of their retention times with those of standard samples.Monosaccharide was determined by GC . Compoun2 until the cells attached to the wall. A drug-containing medium  was added to each well of the administration group, and an equal volume of medium was also added to the blank group, and they were incubate at 37°C and 5% CO2 for 24 h. After the culture, 10 μl of CCK-8 was added to each well and incubated for 1–4 h at 37°C and 5% CO2. The absorbance (OD) value of each well at 450 nm was detected with a microplate reader, repeating three times, and its IC50 value was calculated.In each well of a 96-well plate, 100 μl of logarithmic growth phase cells (density 1.5 × 105/ml) was inoculated and cultured at 37°C and 5% CO1–29 was evaluated by LPS-induced BV2 microglia reported previously , indicating its molecular formula of C41H66O13. The 1H NMR spectrum displayed characteristic resonances of an olean-12-ene skeleton, namely, six methyls , one oxygenated methylene [δH 3.61 ], one oxygenated methine [δH 3.33 ], one olefin [δH 5.51 ], and two anomeric proton signals [δH 4.74  and 5.39 ] , two olefinic carbons (δC 122.5 and 144.9), two anomeric carbons (δC 107.3 and 106.3), one downfield glycosylation-shifted oxygenated methine (δC 88.5), a hydroxymethyl carbon (δC 73.8), and six methyls  (see 1 might be an oleanane-type triterpenoid saponin.Compound 1″)] see , 2. Coup8.0) see , 2. ThesδH 4.74)/C-3 showed the by ether bond location of the sugar chain at C-3. The HMBC connections of H2-22 (δH 2.16)/C-17, C-28 indicated a carboxy fragment attached at C-17  with C-19, C-20, C-21, and C-30 . To determine the absolute configuration of the arabinopyranose and glucopyranose, compound 1 was hydrolyzed by 2 mM HCl to obtain the sugar, and then, the trimethylsilyl thiazolidine derivatives of the sugar and standards, L-arabinose, and D-glucose were prepared. By comparing the retention times of these three trimethylsilyl thiazolidine derivatives obtained from GC, the absolute configuration of the arabinopyranose and glucopyranose in 1 was determined to be L and D, respectively.The HMBC cross-peaks of the anomeric proton H-1′ /H2-29 indicated that H-3, H-5, H-9, H3-23, H3-27, and H2-29 were α-oriented. Therefore, the structure of compound 1 was elucidated to be 3-O-β-glucopyranosyl-(1→3)-β-arabinopyranosyl-29-hydroxy-olean-12-en-28-oic acid, named acasentrioid A.In the NOESY spectrum , the cor2 was isolated as an amorphous powder. Its molecular formula, C44H70O15, was determined by the negative HR-ESI-MS at m/z 856.5043 [M + NH4]+ . The 1H NMR spectrum displayed a skeleton characteristic similar to compound 1, namely, six methyls , together with one methyl triplet at δH 1.14 , one hydroxymethyl , one oxygenated methine [δH 4.30 ], one olefin [δH 5.41 ], and two anomeric proton signals [δH 5.20  and 6.33 ] , two olefinic carbons (δC 122.9 and 144.1), two anomeric carbons (δC 106.4 and 95.6), one downfield glycosylation-shifted oxygenated methine (δC 82.2), oxygenated methylene (δC 64.3), and seven methyls  (see 2 might be as well an oleanane-type triterpenoid saponin.Compound 1″)] see , 2. Ther3.0) see , 2. ThesδH 5.20)/C-3 and H-1″ (δH 6.33)/C-28 show that sugar is attached to the ether bond of C-3 and C-28, respectively. The HMBC connections of H2-22 /C-17, C-28 indicated a glycosylated carboxyl fragment attached at C-17 (2 was similar to ilexoside XLVIII (δC 14.1 and 61.1) linked to C-6′ (δC 170.2) by ether bond in compound 2, which was supported by the HMBC correlation of the ethyl protons H3-8′ and H-7′ with C-7′ and C-6′, respectively . The absolute configurations of both glucuronopyranosyl and glucopyranosyl were determined to be D by the same chemical methods and GC analysis as 1.The heteronuclear multiple bond coherence (HMBC) cross-peaks of the anomeric protons H-1′ /H3-29 indicated that H-3, H-5, H-9, H2-23, H3-27, and H3-29 were α-oriented. Thus, the structure of compound 2 was defined as 3-O-β-D-6′-ethyl-glucuronopyranosyl-23-hydroxy-olean-12-en-28-β-D-glucopyranosyl, named acasentrioid B.In the NOESY spectrum , the cor3 was also obtained as an amorphous powder with the molecular formula of C40H64O12 as indicated by the molecular ion peak m/z 737.4490 [M + H]+  in the HR-ESI-MS spectra. The 1H NMR spectra gave seven angular methyl signals at δH 0.82 , 0.99 , 1.06 , 1.17 , 1.26 , 1.58 , and 1.63 ; one oxygenated methine at δH 3.25 ; one olefin at δH 5.53 ; and two anomeric proton signals at δH 4.91  and 6.16  , 17.0 (C-24), 17.3 (C-26), 18.5 (C-6″), 26.0 (C-27), 28.0 (C-23), and 25.7 (C-30); an oxygen-substituted methine signal at δC 88.7 (C-3); oxygen-substituted quaternary carbon signal at δC 69.8 (C-20); two olefinic carbon signals at δC 122.6 (C-12) and 144.3 (C-13); and one carboxyl carbon signal at δC 180.0 (C-28), along with two anomeric carbon signals at δC 101.7 (C-1″) and 104.9 (C-1′) as determined by the HSQC and DEPT spectra. All these NMR data were characteristic resonances of olean-12-ene skeleton triterpenes. The NMR data of 3 resembled those of 17, and the major difference was the substituent C-29 is changed from methyl to hydroxyl in compound 3, which was supported by the chemical shift δC 69.8 (C-20) and the HMBC correlation of the anomeric protons of H3-30 (δH 1.58) with C-19, C-20, and C-21 (2-22 (δH 2.07) and H-18 (δH 3.35)/C-28 indicated a carboxy fragment attached at C-17  established the α-arabinopyranosyl linkage in 3. In the NOESY spectrum -α-L-arabinopyranosyl)oxy]-20α,30-dihydroxy-norolean-12-en-28-oic acid, named acasentrioid C.Compound -1″) see , 2. Accoand C-21 . The HMB at C-17 . The absspectrum , the cor4 was obtained as an amorphous powder. Its molecular formula C41H64O14 was established by the HR-ESI-MS spectrum m/z 798.4648 [M + NH4]+  and was supported by the 13C NMR spectroscopic data. The 13C NMR spectrum of 4 displayed 41 carbons, of which 30 were assigned to the aglycone part and the remaining 11 were assigned to two sugar units comprising one pentose and one hexose. The NMR spectra showed signals for six angular methyl at δH 0.80, 0.96, 1.00, 1.18, 1.25, and 1.55 , and their corresponding carbons at δC 15.3 (C-25), 17.2 (C-26), 16.6 (C-24), 28.0 (C-23), 25.9 (C-27), and 19.9 (C-30); an olefinic group at δH 5.51  and δC 123.0 (C-12) and 144.1 (C-13); one oxygenated methine at δH 3.17  and δC 88.7 (C-3); two anomeric proton signals at δH 4.93  and 5.17  and their corresponding carbons at δC 104.6 (C-1′) and 105.7 (C-1″); and two carboxy groups at δC 180.0 (C-28) and 181.1 (C-29), which were characteristic for the triterpenoid saponin with oleanane skeleton. Two-dimensional NMR data of 4 resembled those of 20, and the major difference was the substituent C-29 is changed from methyl to carboxyl in compound 3, which was supported by the chemical shift δC 181.1 (C-29) and the HMBC correlation of H-19 (δH 2.57)/C-29 and H3-30 (δH 1.55)/C-20, C-21, and C-29  and J = 7.8 Hz  established the α-arabinopyranosyl and β-glucopyranosyl linkage in 4. In the NOESY spectrum -α-L-arabinopyranosyl)oxy]olean12-ene-28,29-dioic acid, named acasentrioid D.Compound and C-29 . The absspectrum , the cor5 was obtained as an amorphous powder. The HR-ESI-MS indicated a precise [M + H]+ ion at m/z 663.4121 , indicating an empirical molecular formula of C41H64O14. In the 1H NMR spectrum, six quaternary methyl group protons at δH 0.83, 0.89, 0.92, 1.02, 1.04, and 1.11 ; a methoxy group proton at δH 3.69 ; an olefinic proton at δH 5.27 ; an oxygenated methine proton at δH 4.32 , hydroxymethyl protons at δH 3.70  and δH 4.34 ; and an anomeric proton at δH 5.22  along with six quaternary methyl group carbons at δC 17.3 (C-25), 15.2 (C-27), 13.3 (C-23), 16.2 (C-26), 29.2 (C-30), and 30.7 (C-29); methoxy group carbons at δC 51.9 (C-7′); an oxygen-bearing methine carbon at δC 82.0 (C-3); a set of olefinic carbons at δC 138.9 (C-18) and 131.9 (C-19); a hydroxymethyl carbon at δC 64.1 (C-24); a carboxyl carbon at δC 179.4 (C-28); an ester group carbon at δC 170.8 (C-6′); and an anomeric carbon at δC 106.4 (C-1′) in its 13C NMR suggested the aglycone belongs to oleanane-type triterpene  established the β-glucuronopyranoside in 5. In the NOESY spectrum  (O-α-L-arabinopyranoside (7) (O-β-D-glucuronopyranoside (8) (9)  (11) (12) (O-β-D- glucuronopyranoside 6′-O-methyl ester (13) (14) (O-6′-O-methyl-β-D-glucuronopy-ranoside of gypsogenin (15) (O-β-D-(6′-O-methyl-glucuronopyranosyl) oleanolic acid (16) (O-α-rhamnopyranose- (1→2)-α-arabinopyranosyl-29-hydroxy-olean-12-en-28-oic acid (17) (α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl] oleanolic acid (18) (19) (20) (β-D-glucopyranosyl (1→3)-α-L-arabinopyranoside (21) (22), lucyoside H (23) (β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl oleanolic acid (24) (O-β-D-glucuronopyranosyl methyl ester-28-O-β-D-glucopyranoside (25) (α-L-rhamnopyranosyl(1→2)-α-L-arabinopyranosyl-28-O-β-D-glucopyranosyl ester (26) (27) (α-arabinopyranosyl-(1→2)-β-glucopyranoside-30-norolean-12,20(29)-dien-28-oic acid (28) (β-D-glucopyranosyl-(1→3)-α-L-arabinopyranosyl)oxy]-30- noroleana-12,20(29)-dien-28-oic acid (29)  , hederagside (7) , oleanolside (8) , HN-sapo (8) (9) , 3-O-β-Dcid (10) , gypsoge10) (11) , elatosi11) (12) , hederagter (13) , tragopo13) (14) , 3-O-6′-nin (15) , 3-O-β-Dcid (16) , 3-O-α-rcid (17) , 3-O-[α-cid (18) , HN-sapo18) (19) , saponin19) (20) , oleanolide (21) , lucyosie H (23) , 3-O-β-Dcid (24) , 3-O-β-Dide (25) , oleanolter (26) , paritri26) (27) , 3-O-α-acid (28) , and 3-[cid (29)  by compacid (29) . Compoun1–29 on BV2 microglia was determined by the CCK-8 assay, and the results are listed in 1–29) in vitro was evaluated. Unfortunately, the results of the evaluation were not ideal. Compounds 5, 10, 12, 13, and 16 had moderate inhibitory effects on neuroinflammation, as indicated in The cytotoxicity of compounds 1–5), together with twenty-four known saponins (6–29), were isolated from the fruit of A. senticosus. The structures of all compounds were elucidated by extensive spectroscopic analysis, including 1D, 2D NMR, and HR-ESI-MS, in combination with chemical methods (acid hydrolysis). The neuroinflammation model was established by LPS-induced BV2 microglia, and the neuroprotective effects of all compounds (1–29) were evaluated. Unfortunately, the results of the evaluation were not ideal. Compounds 5, 10, 12, 13, and 16 had moderate inhibitory effects on neuroinflammation, while other compounds have no anti-neuroinflammatory activity.In summary, five previously undescribed oleanane-type triterpenoid saponins (


N′-(2-oxoindolin-3-ylidene)propanehydrazide and -1,1′-)bis(5-oxo-N′-(2-oxoindolin-3-ylidene)pyrrolidine-3-carbohydrazide) were identified as the most active compounds against HT-29 in 2D and 3D cell cultures. The same compounds showed the highest antioxidant activity among the synthesized compounds screened by ferric reducing antioxidant power assay (FRAP). Their antioxidant activity is on par with that of a well-known antioxidant ascorbic acid. A series of novel mono and bishydrazones each bearing a 2-oxindole moiety along with substituted phenylaminopropanamide, pyrrolidin-2-one, benzimidazole, diphenylmethane, or diphenylamine fragments were synthesized, and their anticancer activities were tested by MTT assay against human melanoma A375 and colon adenocarcinoma HT-29 cell lines. In general, the synthesized compounds were more cytotoxic against HT-29 than A375. 3-((4-Methoxyphenyl)(3-oxo-3-(2-(2-oxoindolin-3-ylidene)hydrazinyl)propyl)amino)- Cancer is a malignant disease characterized by rapid and uncontrolled cell proliferation . The earH-indole-2,3-dione) is a versatile heterocyclic compound which has been attracting the attention of medicinal chemists worldwide as a synthetic building block due to the various biological activities of its derivatives. Among the latter, compounds bearing an oxindole scaffold constitute a group of promising compounds endowed with significant biological activities, such as anticonvulsant, antimicrobial, antitubercular phenyl}methyl)phenyl]-5-oxopyrrolidine-3-carboxylate (33) was synthesized by the classical esterification reaction of a corresponding diacid in methanol in the presence of H2SO4 as a catalyst. Afterwards, diester 33 was dissolved in DMSO and treated with hydrazine hydrate to afford dihydrazide 34, which was subjected to a reaction with isatin in the molar ratio 1:2.4. Double intensities of the corresponding proton signals in pyrrolidinone and 2-oxindole moieties in the 1H NMR spectra for 33–35 prove the structures of the target molecules as having two 5-oxo-N′-(2-oxoindolin-3-ylidene)pyrrolidine-3-carbohydrazide “arms“ attached to the diphenylmethane core (The next step required the introduction of the pyrrolidin-2-one moiety into the structure of the target compound ane core .36), which was first converted to ester 37 and then to hydrazide 38 according to the classical synthesis procedures (38 was treated with isatin in methanol at 65 °C to afford 5-oxo-N′-(2-oxoindolin-3-ylidene)-1-(4-(phenylamino)phenyl)pyrrolidine-3-carbohydrazide (39).Reaction of 4-aminodiphenylamine with itaconic acid at the reflux temperature of the reaction mixture provided 5-oxo-1-(4-(phenylamino)phenyl)pyrrolidine-3-carboxylic acid  and colon adenocarcinoma (HT-29) cell lines at 100 µM. This concentration was chosen based on our previous experience and the observations from other studies. It has been shown that 100 µM allows distinguishing the most active compounds from a group better than 10 µM +, found 309.1346.Prepared from 3-(phenylamino)propanehydrazide (1)  (0.89 g)N′-(2-oxoindolin-3-ylidene)-3-(p-tolylamino)propanehydrazide (13)p-tolylamino)propanehydrazide (2) +, found 323.1503.Prepared from 3-(zide (2) ,77 (0.97N′-(2-oxoindolin-3-ylidene)propanehydrazide (14)3-((4-Methoxyphenyl)amino)-Prepared as described in .N′-(2-oxoindolin-3-ylidene)propanehydrazide (15)3-((4-Ethoxyphenyl)amino)-4) +, found 353.1608.Prepared from 3-((4-ethoxyphenyl)amino)propanehydrazide (4)  (1.12 g)N′-(2-oxoindolin-3-ylidene)propanehydrazide (16)3-((4-Bromophenyl)amino)-5) +, found 387.0451.Prepared from 3-((4-bromophenyl)amino)propanehydrazide (5)  (1.29 g)N′-(2-oxoindolin-3-ylidene)propanehydrazide (17)3-((2-Methyl-5-nitrophenyl)amino)-6) +, found 368.1353.Prepared from 3-((2-methyl-5-nitrophenyl)amino)propanehydrazide (6) ,80 (1.2 2O mixture.To a solution of corresponding hydrazide (5 mmol) in methanol (25 mL), isatin (12 mmol) and glacial acetic acid (5 drops) were added. The reaction mixture was stirred at 65 °C for 15 min. Precipitate was filtered off, washed with methanol, and recrystallized from DMF/HN′-(2-oxoindolin-3-ylidene)propanehydrazide (18)3-((3-Oxo-3-(2-(2-oxoindolin-3-ylidene)hydrazinyl)propyl)(phenyl)amino)-7) +, found 524.2041.Prepared from 3,3′-(phenylazanediyl)di(propanehydrazide) (7)  (1.33 g)p-tolyl)amino)-N′-(2-oxoindolin-3-ylidene)propanehydrazide (19)3-((3-Oxo-3-(2-(2-oxoindolin-3-ylidene)hydrazinyl)propyl)(p-tolylazanediyl)di(propanehydrazide) (8) +, found 538.2197.Prepared from 3,3′-(ide) (8)  (1.40 g)N′-(2-oxoindolin-3-ylidene)propanehydrazide (20)3-((4-Methoxyphenyl)(3-oxo-3-(2-(2-oxoindolin-3-ylidene)hydrazinyl)propyl)amino)-9) +, found 554.2146.Prepared from 3,3′-((4-methoxyphenyl)azanediyl)di(propanehydrazide) (9)  (1.48 g)N′-(2-oxoindolin-3-ylidene)propanehydrazide (21)3-((4-Ethoxyphenyl)(3-oxo-3-(2-(2-oxoindolin-3-ylidene)hydrazinyl)propyl)amino)-10) +, found 568.2308.Prepared from 3,3′-((4-ethoxyphenyl)azanediyl)di(propanehydrazide) (10)  (1.7 g).N′-(2-oxoindolin-3-ylidene)propanehydrazide (22)3-((4-Chlorophenyl)(3-oxo-3-(2-(2-oxoindolin-3-ylidene)hydrazinyl)propyl)amino)-11) +, found 559.1706.Prepared from 3,3′-((4-chlorophenyl)azanediyl)di(propanehydrazide) (11)  (1.58 g)p-tolyl)pyrrolidine-3-carbohydrazide (23) +, found 363.1453.Prepared from 5-oxo-1-(ide (23)  (1.17g) 24) +, found 379.1400.Prepared from 1-(4-methoxyphenyl)-5-oxopyrrolidine-3-carbohydrazide (24)  (1.25 g)25) +, found 379.1400.Prepared from 1-(3-methoxyphenyl)-5-oxopyrrolidine-3-carbohydrazide (25) ,87 (1.25H-benzo[d]imidazol-1-yl)acetohydrazide (29) +, found 536.1645.Prepared from 2-(2-(1-(4-chlorophenyl)-5-oxopyrrolidin-3-yl)-1ide (29)  (1.92 g)31) +, found 629.2619.Prepared from 3,3′-)bis(azanediyl))di(propanehydrazide) (31)  (0.85 g)2O mixture. Yield 47% (8.95 g); white crystals; m. p. 166–167 °C; IR (KBr) νmax (cm−1): 1688, 1727 (C=O); 1H NMR : δ 2.67–2.85 , 3.41–3.48 , 3.67 , 3.89 , 3.92–4.06 , 7.21 , 7.54 ; 13C NMR : 34.95 (2CHCO), 34.98 (CH), 39.84 (CH2), 49.77 (NCH2), 52.19 (CH3), 119.67, 128.88, 137.10, 137.25 (CAr), 171.35, 173.15 (C=O); HRMS (ESI): m/z calcd for C25H26N2O6 468.1856 [M+H2O]+, found 468.2130.A mixture of 1,1′-)bis(5-oxopyrrolidine-3-carboxylic acid) 18 g, 43 mmol), methanol (200 mL), and sulfuric acid (10 mL) was heated at reflux for 1 h (until formation of precipitate). Precipitate was filtered off and recrystallized from DMF/H33)  in hot DMSO (30 mL), hydrazine hydrate  was added. Already after 10 min crystals started forming. The reaction mixture was cooled down. The precipitate was filtered off, washed with diethyl ether, and recrystallized from DMF/H2O mixture. Yield 67% (6.05 g); white crystals; m.p. >300 °C decomposes. IR (KBr) νmax (cm−1): 1644, 1728 (C=O), 2820–3433 (NH); 1H NMR : δ 2.57–2.89 , 3.39 , 3.78–4.13 , 7.21 , 7.53 , 9.79 , 11.35 ; 13C NMR : δ 33.97 (2CHCO), 35.41 (CH), 39.8 (CH2), 39.94 (CH2), 50.33 (NCH2), 119.85, 129.06, 137.22, 137.48 (CAr), 171.49, 172.03 (C=O); Anal. Calcd for C23H26N6O4: C, 61.31; H, 5.82; N, 18.66, found: C, 61.47; H, 5.67; N, 18.83 %.To a solution of methyl 1-[4-({4-[4-(methoxycarbonyl)-2-oxopyrrolidine-1-il]phenyl}methyl)phenyl]-5-oxopyrrolidine-3-carboxylate (34) (2.25 g) according to the synthesis procedure for compounds 18–22. Yield 54% (1.9 g); yellow crystals; m.p. 191–192 °C; IR (KBr) νmax (cm−1): 1665, 1694, 1734 (C=O), 3398, 3451 (NH); 1H NMR : δ 2.54–2.64 , 3.44 , 3.67 , 3.89–4.16 , 6.87–6.97 , 7.02–7.12 , 7.21 , 7.37 , 7.45–7.66 , 10.19, 10.82, 11.03, 11.26, 12.59, 13.07 ; 13C NMR : δ 32.34 (2CHCO), 34.94, 34.96 (CH), 39.83, 40.43 (CH2), 49.75, 52.15 (NCH2), 111.13, 112.19, 117.82, 119.65, 120.85, 122.57, 122.75, 124.68, 128.84, 131.65, 137.08, 137.14, 137.22, 138.36, 142.49, 150.71 (CAr), 159.37, 162.47, 171.30, 173.10 (C=O); HRMS (ESI): m/z calcd for C39H32N8O6 712.2761 [M+4H]+, found 712.234.Prepared from 1,1′-)bis(5-oxopyrrolidine-3-carbohydrazide) (max (cm−1): 1677, 1696 (C=O), 3284 (NH); 1H NMR : δ 2.60–2.81 ; 3.29–3.37 , 3.87–4.05 , 6.79 , 6.98–7.12 , 7.21 , 7.49 , 8.17 , 12.76 ; 13C NMR : δ 35.08 (2CHCO), 35.25 (CH), 50.26 (NCH2), 116.23, 117.18, 119.43, 121.08, 129.22, 131.76, 139.85, 143.66 (CAr), 171.15, 174.39 (C=O); HRMS (ESI): m/z calcd for C17H16N2O3 297.1240 [M+H]+, found 297.1242.To a solution of itaconic acid  in water (250 mL), 4-aminodiphenylamine  was added and the reaction mixture was heated at reflux for 24 h. Precipitate was filtered off and dissolved in an aqueous 20% NaOH solution. The solution was heated at reflux with activated carbon for 5 min, filtered, and acidified with conc. HCl to pH 2. Yield 87% (51.30 g); grey crystals; m.p. 168–169 °C; IR (KBr) ν36)  in methanol (120 mL), H2SO4 (2 mL) was added dropwise. The reaction mixture was heated at reflux for 24 h. Aqueous 15% Na2CO3 solution was added until pH 8, and the precipitate was filtered off and dried. Yield 72 % (38.58 g); grey crystals; m.p. 80–81 °C; IR (KBr) νmax (cm−1): 1651, 1687 (C=O), 3213 (NH); 1H NMR : δ 2.64–2.82 , 3.40–3.50 , 3.68 , 3.90–4.06 , 6.79 , 6.99–7.12 , 7.21 , 7.48 , 8.14 ; 13C NMR : δ 34.87 (2CHCO), 35.00 (CH), 50.02 (NCH2), 52.20 (CH3), 116.25, 117.17, 119.44, 121.12, 129.21, 131.62, 139.92, 143.62 (CAr), 170.87, 173.26 (C=O); HRMS (ESI): m/z calcd for C18H18N2O3 311.1396 [M+H]+, found 311.1404.To a solution of 5-oxo-1-(4-(phenylamino)phenyl)pyrrolidine-3-carboxylic acid (37)  in propan-2-ol (25mL), hydrazine hydrate  was added dropwise, and the reaction mixture was heated at reflux for 22 h. Precipitate was filtered off and recrystallized from propan-2-ol. Yield 82 % (8.21 g); grey crystals; m.p. 163–164 °C; IR (KBr) νmax (cm−1): 3299–3028 (NH); 1680, 1644 (C=O); 1H NMR : δ 2.55–2.72 , 3.090–3.21 , 3.77–3.85 , 3.89–3.97 , 4.31, 4.47 , 6.79 , 6.99–7.12 , 7.21 , 7.49 , 8.13 , 9.27 ; 13C NMR : δ 34.16 (CH), 35.57 (NCO2CH), 50.92 (N2CHCH), 116.21, 117.19, 119.38, 120.97, 121.17, 121.22, 129.17, 131.80, 139.77, 143.66 (CAr), 171.34, 171.63 (C=O); HRMS (ESI): m/z calcd for C17H18N4O2 311.1508 [M+H]+, found 311.1503.To a solution of methyl 5-oxo-1-(4-(phenylamino)phenyl)pyrrolidine-3-carboxylate (38) according to the synthesis procedure for compounds 12–17. Yield 89% (1.95 g); yellow crystals; m.p. 243–244 °C; IR (KBr) νmax (cm−1): 1640, 1695, 1725 (C=O), 3175, 3282, 3310 (NH); 1H NMR : δ 2.74–2.94 , 3.37 , 3.96–4.18 , 6.79 , 6.90–6.96 , 7.03–7.10 , 7.21 , 7.36–7.62 , 8.14 , 10.83 , 11.27, 11.37 , 12.60, 13.09 ; 13C NMR : δ 34.50 (CH), 35.47 (NCO2CH), 50.01 (N2CHCH), 110.63, 111.14, 115.21, 116.23, 117.18, 119.40, 120.83, 121.17, 121.22, 121.69, 122.57, 126.13, 129.17, 131.66, 131.71, 139.89, 139.92, 142.49, 143.64, 143.86 (CAr), 162.48, 164.56, 171.10 (C=O); HRMS (ESI): m/z calcd for C25H21N5O3 440.1723 [M+H]+, found 440.1718.Prepared from 5-oxo-1-(4-(phenylamino)phenyl)pyrrolidine-3-carbohydrazide . Human foreskin fibroblasts (HF) CRL-4001 were originally obtained from ATCC and kindly provided by Prof. Helder Santos . A375, HT-29, and HF were cultured in Dulbecco’s modified eagle’s GlutaMAX medium ). Medium was supplemented with 10,000 U/mL penicillin, 10 mg/mL streptomycin (Gibco), and 10% fetal bovine serum (Gibco). Cell cultures were grown at 37 °C in a humidified atmosphere containing 5% CO3 cells/well). After 24 h of incubation, the cells were treated with 100 μM of tested compounds. After 72 h, the cells were exposed to the reagent MTT for 4 h. Then the medium was aspirated and the formed formazan crystals were dissolved in 100 μL DMSO (Sigma-Aldrich Co.). The absorbance was measured at 570 and 630 nm using a multi-detection microplate reader. The compound’s effect on cell viability was calculated using a formula:A—mean of absorbance of tested compound; A0—mean of absorbance of blank ; NCA—mean of absorbance of negative control .The effects of the isatin derivatives on cell viability were studied using 3--2,5-diphenyltetrazolium bromide  assays, as described elsewhere ,89. Brie50 values of the most active compounds, 17, 18, 20, and 35, were established by the same MTT procedure; only the compound serial dilutions from 50 to 1.56 µM were made in a medium and added to the cells in triplicate repeats. Each EC50 value, which represents the concentration of a compound causing a 50% reduction of cancer cells’ metabolic activity, has been calculated using the Hill equation.The EC17, 18, 20, and 35, on cell survival and proliferation via forming colonies, as described elsewhere [2 cells/well) and grown in the previously described cell culture medium at 37 °C in a humidified atmosphere containing 5% CO2. After 24 h, the fresh media containing compounds 17, 18, 20, and 35 at concentrations representing 50% of calculated EC50 values were added to cells. Then, cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2 for the next 7–8 days. Cells treated with medium containing 0.5% DMSO served as a negative control. After incubation, the colonies were stained with a 0.1% crystal violet (Sigma-Aldrich Co.) solution. First, the media were removed from cells, and the cells were washed with sterile PBS. Then, the cells were fixed in a 4% formaldehyde (Thermo Scientific) solution and washed with PBS two times to remove the fixative, and stained with crystal violet for 20 min. After the stain had been removed, the remaining stain residues were washed three times with sterile water. The plates were dried overnight and imaged with the SYNGENE G:BOX gel doc system, using Gen Sys software. Quantification was performed using Gene tools software.Clonogenic assays were used to evaluate the inhibitory effects of the most active compounds, lsewhere . Briefly2. After nanoparticles were taken up by cells, they were trypsinized, centrifuged, and seeded into ultra-low attachment 96-well plate in a volume of 100 µL (1.5 × 103 cancer cells and 1.5 × 103 human fibroblasts/well). The plate was placed on a magnetic drive and incubated for 2 days at 37 °C in a humidified atmosphere containing 5% CO2. Then the fresh medium containing 20 or 50 µM of tested compound was added to the wells. The spheroids were captured every two days using the Olympus IX73 inverted microscope (OLYMPUS CORPORATION). The quantitative analysis of compound anticancer activity in spheroids was performed using ImageJ  and Microsoft Office Excel software).Cancer cell spheroids were formed by using the magnetic 3D Bioprinting method, as described elsewhere . Briefly3+-2,4,6-tripyridyl-s-triazine complex to the intensely blue Fe2+-2,4,6-tripyridyl-s-triazine complex in acidic medium. FRAP values are calculated from increasing absorbances measured at 593 nm. The FRAP reagent contained 2.5 mL of a 10 mM TPTZ  solution in 40 mM HCl, along with 2.5 mL of FeCl3 (20 mM) and 25 mL of acetate buffer . For each compound, 100 μL of it (20 mM) was mixed with 3 mL of the FRAP reagent. The absorbance of the reaction mixture at 593 nm was measured spectrophotometrically. For comprising the calibration curve, five concentrations of FeSO4∙7H2O  were used, and the absorbances of the samples’ solutions were then measured [The FRAP (ferric reducing antioxidant power) assay is based on the reduction of the colorless Femeasured . Each exp < 0.05.All biological experiments were repeated at least three times. The mean and standard deviation are reported. The data were processes using Microsoft Office Excel 2016 software . Statistical analysis was performed by using Student’s t-tests. The level of significance was set as N′-(2-oxoindolin-3-ylidene)propanehydrazide) 20, bearing the electron-donating methoxy group at the p-position of the benzene ring, and diphenylmethane derivative 35, bearing two 5-oxo-N′-(2-oxoindolin-3-ylidene)pyrrolidine-3-carbohydrazide “arms” at p-positions of the benzene rings, were the most active among all compounds synthesized. These compounds reduced the colony-forming abilities of both cell lines, and also inhibited the growth and viability of colon cancer and melanoma spheroids.In conclusion, a series of novel hydrazone-isatin derivatives were synthesized and evaluated for their anticancer and antioxidant properties. In the anticancer activity assay, the colon adenocarcinoma HT-29 cell line appeared to be more sensitive to the treatment with the hydrazone-isatin derivatives, compared to the malignant melanoma A375 cell line. Bis(hydrazone-isatins) were found to be more active than their mono analogues. Bis possessed the highest ferric reducing antioxidant power. These promising results suggest that variously substituted bis(hydrazone-isatins) based on an


P1 (bearing hydrogen substitution) was the most potent derivative against MOLT-4 with an IC50 value of 7.1 ± 1.1 μM, whereas P11 (bearing phenyl substitution) demonstrated considerable cytotoxicity against MCF-7 with an IC50 value of 15.4 ± 2.9 μM. Compounds P7, P8, P14 and P15 exhibited moderate cytotoxic effects. Furthermore, to confirm the potential DNA intercalation and Bcl-2 inhibitory activities of phenanthro-triazine scaffolds, molecular docking analysis was performed. Molecular docking studies indicated that these compounds not only bind to DNA by intercalation mainly through stacking interactions but also are well accommodated in the active site of Bcl-2. Therefore, P1 and P11 having phenanthro-triazine-3-thiol scaffold could be presented as cytotoxic agents with dual DNA intercalation and Bcl-2 inhibitory activities. Novel phenanthro-triazine-3-thiol derivatives were designed as potential DNA intercalators and Bcl-2 inhibitors. After being synthesized, the compounds were evaluated for their cytotoxic activity against MOLT-4 (human acute lymphoblastic leukemia) and MCF-7 (human breast adenocarcinoma) cells by MTT assay.  Despite the availability of various cancer therapeutic agents, we continue to face complications and a lack of efficacy of available drugs. The Discovery of potential novel chemotherapeutics with improved efficacy and fewer side effects is an enormous challenge , 2. DNA is still one of the most important targets for anticancer drug discovery , 4. DNA etc.). All Bcl-2 family proteins contain a BH3 domain; one of four BH domains involves in interactions between these proteins. The anti-apoptotic and pore-forming proteins contain all four BH domains (multi-BH domain proteins) and adopt a highly conserved tertiary structure forming a hydrophobic BH3 domain-binding groove that acts as a receptor for BH3 domains of other family members. The BH3-only proteins are subdivided into activator and sensitizer proteins, containing only the BH3 domain except for Bid are unstructured in solution  family proteins are important regulators of apoptosis which are generally overexpressed in many cancer cells. Therefore, another trend for the treatment of cancer is finding inhibitors targeting the Bcl-2 proteins. The Bcl-2 family is divided into three groups based on their primary function 1) anti-apoptotic proteins , 2) pro-apoptotic pore-formers  and 3) pro-apoptotic BH3-only proteins  demonstrated cytotoxic effects by DNA intercalation mechanism with an IC50 of 10-7–10-8 M against diverse cancer cell lines and introduced compound II as a promising Bcl-2 inhibitor -acenaphtho-1,2,4-triazine analogs, IV, exhibited moderate to good cytotoxic activities and suggested that these compounds could be potential Bcl-2 inhibitors by molecular docking analysis  are reported as ppm. IR spectra were obtained on a Perkin-Elmer spectrometer (KBr disk) . Mass spectra were recorded with an Agilent spectrometer . All commercially available solvents and reagents were of analytical grade and obtained from Merck and Aldrich.Melting points were measured on a hot stage apparatus  and were uncorrected. Procedure for the synthesis of phenanthro triazine-3-thiol (P1)oC with stirring for about half an hour. Thiosemicarbazide  was added, and the mixture was refluxed for about three hours. The reaction mixture cooled to room temperature and then poured into ice-cold water. The solid product was filtered and washed with cold water. Yield: 67.5%, Rf = 0.56 (EtOAc/ PetEther 1:1), mp: 122-124 °C. 1H NMR : δH 14.65 ppm , 8.74-8.39 , 8.19-8.06 , 7.78-7.37 , 6.72 . MS (EI) m/z (%) 263 (38.6), 235 (77.2), 176.1 (100), 150 (10.53). IR (KBr): υ = 3145 (CH-aromatic), 3413 (NH), 3242 cm-1 (SH).The phenanthrene-9,10-dione  was added to 80 mL of acetic acid, and the mixture was heated to about 100 General procedure for the synthesis of phenanthrotriazine-3-thiol derivatives (P2-P16)The phenanthrotriazine-3-thiol  was added to a stirred hot ethanolic solution of sodium hydroxide (0.1 M). After cooling to room temperature, corresponding alkyl/benzyl halide was added, and the mixture was stirred for about 2-24 h at 25 °C. Completion of the reaction was checked by thin-layer chromatography (TLC). The solvent was reduced under vacuum; the precipitate was filtered and washed with ethanol and cold water. Finally, the dried precipitate was purified by recrystallization or chromatography to give the final pure derivative .3-(methylthio)phenanthrotri-azine (P2)P2 was prepared by the described procedure using methyl iodide  and the obtained precipitate was recrystallized from ethyl acetate. Yield: 36.1%, Rf = 0.67 (EtOAc/ PetEther 1:1), mp: 157-160 °C. 1H NMR : δH 9.36 , 9.15 , 8.53 , 7.88-7.72 , 2.89 ppm . MS (EI) m/z (%) 277.1 (43.9), 249.2 (79), 235 (46.5), 203.1 (7), 176.1 (100), 150.1 (14). IR (KBr): υ = 3060 (CH-aromatic), 2927 cm-1 .Compound 3-(ethylthio)phenanthrotri-azine (P3)P3 was prepared by the described procedure using ethyl iodide  and the obtained precipitate was recrystallized from ethyl acetate. Yield: 54.91%, Rf = 0.67 (EtOAc/ PetEther 1:1), mp: 131-133 °C. 1H NMR : δH 9.4 , 9.2 , 8.6 , 7.92-7.74 , 3.5 , 1.6 ppm . MS (EI) m/z (%) 291.1 (40.35), 263.1 (28), 235.1 (100), 203.1 (10), 176.1 (75.44), 150.1 (10.5). IR (KBr): υ = 3065 (CH-aromatic), 2928 cm-1 .Compound 3-(propylthio)phenanthrotri-azine (P4)P4 was prepared by the described procedure using 1-bromopropane , the obtained precipitate was dissolved in appropriate solvent and purified using silica gel column chromatography with petroleum ether-ethyl acetate as the mobile phase. Yield: 49.12%, Rf = 0.69 (EtOAc/ PetEther 3:7), mp: 118-120 °C. 1H NMR : δH 9.37 , 9.15 , 8.55 , 7.90-7.30 , 3.48 , 2.0 , 1.22 ppm . MS (EI) m/z (%) 305.1 (28), 279.1 (36.9), 263.1 (47.4), 235.1 (100), 203.1 (14), 176.1 (77.2), 150.1 (14). IR (KBr): υ = 3071 (CH-aromatic), 2960, 2929 cm-1 . Compound 3-(butylthio)phenanthrotri-azine (P5)P5 was prepared by the described procedure using 1-bromobutane , the obtained precipitate was purified using silica gel column chromatography with petroleum ether-ethyl acetate as the mobile phase and then recrystallized from chloroform. Yield: 14%, Rf = 0.66 (EtOAc/ PetEther 3:7), mp: 81-83 °C. 1H NMR : δH 9.31 , 9.06 , 8.47 , 7.85-7.67 , 3.48 2-CH3), 1.96 , 1.66 , 1.07 ppm . MS (EI) m/z (%) 319.2 (50.9), 291.1 (19.3), 263.1 (52.6), 235.1 (100), 203.1 (64.9), 176.1 (75.4), 150.1 (14). IR (KBr): υ = 3071 (CH-aromatic), 2963 cm-1 .Compound 3-(isobutylthio)phenanthrotri-azine (P6)P6 was prepared by the described procedure using 1-bromopropane2-methylpropan , the obtained precipitate was purified using silica gel column chromatography with petroleum ether-ethyl acetate as the mobile phase and then recrystallized from chloroform. Yield: 17.8%, Rf = 0.67 (EtOAc/ PetEther 2:8), mp: 93-96 °C. 1H NMR : δH 9.4 , 9.2 , 8.6 , 7.93-7.75 , 3.4 2)), 2.3-2.1 2)), 1.23 ppm 2). MS (EI) m/z (%) 319.2 (10.5), 291.1 (17.5), 263.1 (75.4), 235.1 (100), 203.1 (10.5), 176.1 (49), 150.1 (8.8). IR (KBr): υ = 3060 (CH-aromatic), 2963, 2926 cm-1 .Compound 3-propanenitrile (P7)P7 was prepared by the described procedure using 3-bromopropionitrile  and the obtained precipitate was recrystallized from ethyl acetate. Yield: 25.3%, Rf = 0.27 (EtOAc/ PetEther 3:7), mp: 128-130 °C. 1H NMR : δH 9.4 , 9.17 , 8.62 , 7.93-7.75 , 3.76 , 3.15 ppm . MS (EI) m/z (%) 316.2 (33.3), 288.1 (100), 260.1 (17.5), 235.1 (21.9), 201.1 (4.4), 176.1 (73.7), 150.1 (12.3). IR (KBr): υ = 3064 (CH-aromatic), 2923 , 2245 cm-1 (CN).Compound 3-((2-methoxyethyl)thio)phenanthrotriazine (P8)P8 was prepared by the described procedure using 2-methoxyethyl chloride , the obtained precipitate was purified using silica gel column chromatography with petroleum ether-ethyl acetate as the mobile phase and then recrystallized from ethyl acetate. Yield: 16.2%, Rf = 0.49 (EtOAc/ PetEther 3:7), mp: 100-102 °C. 1H NMR : δH 9.25 , 9.0 , 8.42-8.39 , 7.82-7.63 , 3.91 , 3.69 , 3.5 ppm . MS (EI) m/z (%) 321 (10.5), 293 (7), 263 (98.3), 23 (100), 203 (73.7), 176 (63.2), 150 (17.5). IR (KBr): υ = 3065 (CH-aromatic), 2962, 2927 cm-1 .Compound 3-(cyclopentylthio)phenanthrotriazine (P9)P9 was prepared by the described procedure using cyclopentyl bromide , the obtained precipitate was purified using silica gel column chromatography with petroleum ether-ethyl acetate as the mobile phase and then recrystallized from ethyl acetate. Yield: 11.6%, Rf = 0.73 (EtOAc/ PetEther 2:8), mp: 170-172 °C. 1H NMR : δH 9.34 , 9.11 , 8.50 , 7.87-7.69 , 4.41 , 2.51-2.46 , 1.95-1.82 ppm . MS (EI) m/z (%) 331.1 (29.9), 303.1 (22.8), 279.1 (8.8) 262.1 (29.9), 235. 1(100), 208.1 (91.2), 176.1 (63.2), 149 (52.6). IR (KBr): υ = 3032 (CH-aromatic), 2959 cm-1 .Compound 3-(cyclohexylthio)phenanthrotriazine (P10)P10 was prepared by the described procedure using cyclohexyl bromide , the obtained precipitate was purified using silica gel column chromatography with petroleum ether-ethyl acetate as the mobile phase and then recrystallized from ethyl acetate. Yield: 15.8%, Rf = 0.49 (EtOAc/ PetEther 2:8), mp: 144-147 °C. 1H NMR : δH 9.27 , 9.05 , 8.45 , 7.78-7.61 , 4.13 , 1.84-1.80 , 1.65-1.50 ppm , 1.37-1.18 ppm . MS (EI) m/z (%) 345.2 (28), 317.2 (14), 264.1 (31.6), 235.1 (100), 203.1 (17.5), 176.1 (42), 150.1 (7). IR (KBr): υ = 3060 (CH-aromatic), 2962, 2924 cm-1 .Compound 3-(benzylthio)phenanthrotri-azine (P11)P11 was prepared by the described procedure using benzyl bromide , the obtained precipitate was purified using silica gel column chromatography with petroleum ether-ethyl acetate as the mobile phase and then recrystallized from ethyl acetate. Yield: 41.4%, Rf = 0.65 (EtOAc/PetEther 1:4), mp: 167-170 °C. 1H NMR : δH 9.122 , 8.887 , 8.275-8.245 , 7.655-7.172 , 4.608 ppm . MS (EI) m/z (%) 353.1 (58.2), 190.1 (100), 91.1 (61.8). IR (KBr): ν 3025 (CH-aromatic), 2963 cm-1 .Compound 3-((4-bromobenzyl)thio)phenanthrotriazine (P12)P12 was prepared by the described procedure using 4-bromobenzyl bromide , the obtained precipitate was purified using silica gel column chromatography with petroleum ether-ethyl acetate as the mobile phase and then recrystallized from ethyl acetate. Yield: 48.8%, Rf = 0.62 (EtOAc/PetEther 1:4), mp: 171-175 °C. 1H NMR : δH 9.304-9.273 , 9.065 , 8.508-8.461 , 7.841-7.627 , 7.389 , 4.598 ppm . MS (EI) m/z (%) 90.1 (18.3), 190.1 (100), 170.9 (57.9), 433.0 (56.2). IR (KBr): ν 3062 (CH-aromatic), 2960 cm-1 .Compound 3-((4-nitrobenzyl)thio)phenanthrotriazine (P13)P13 was prepared by the described procedure using 4-nitrobenzyl bromide . The obtained precipitate was purified using silica gel column chromatography with petroleum ether-ethyl acetate as the mobile phase and then recrystallized from ethyl acetate. Yield: 16.6%, Rf = 0.46 (EtOAc/PetEther 1:4), mp: 198-202 °C.1H NMR : δH 9.356 , 9.105 , 8.589-8.540 , 8.196 , 7.896-7.702 , 4.787 ppm . MS (EI) m/z (%) 398.1 (44.3), 322.3 (66.9), 190.0 (100). IR (KBr): ν 3076 (CH-aromatic), 2923 , 1524, 1354 (ArNO2), 872 cm-1 (C-NO2). Compound 3-((4-fluorobenzyl)thio)phenanthrotriazine (P14)P14 was prepared by the described procedure using 4-fluorobenzyl chloride , the obtained precipitate was purified using silica gel column chromatography with petroleum ether-ethyl acetate as the mobile phase and then recrystallized from ethyl acetate. Yield: 61.2%, Rf = 0.60 (EtOAc/PetEther 1:4), mp: 157-160 °C. 1H NMR : δH 9.171-9.141 , 8.910 , 8.326-8.288 , 7.692-7.438 , 6.976-6.919 , 4.581 ppm . MS (EI) m/z (%) 371.1 (79.3), 343.1 (35.1), 190.1 (73.9), 109.0 (100) IR (KBr): ν 3070 (CH-aromatic), 2963 cm-1 .Compound 3-((4-methylbenzyl)thio)phenanthrotriazine (P15)P15 was prepared by the described procedure using 4-methylbenzyl chloride , the obtained precipitate was purified using silica gel column chromatography with petroleum ether-ethyl acetate as the mobile phase and then recrystallized from ethyl acetate. Yield: 34.7%, Rf = 0.65 (EtOAc/PetEther 1:4), mp: 138-141 °C. 1H NMR : δH 9.013-8.990 , 8.766-8.724 , 8.112-8.085 , 7.574-7.409 , 4.561 & 4.526 , 2.227 ppm . MS (EI) m/z (%) 91.1 (32.1), 105.5 (100), 190.1 (96.2), 367.1 (89.1) IR (KBr): ν 3074 (CH-aromatic), 2926 cm-1 .Compound 3-((4-methoxybenzyl)thio)phenanthrotriazine (P16)P16 was prepared by the described procedure using 4-methoxybenzyl chloride , the obtained precipitate was purified using silica gel column chromatography with petroleum ether-ethyl acetate as the mobile phase and then recrystallized from ethyl acetate. Yield: 31.2%, Rf = 0.57 (EtOAc/PetEther 1:4), mp: 152-154 °C. 1H NMR : δH 9.230-9.199 , 9.006-8.975 , 8.371-8.334 , 7.777-7.572 , 7.501-7.472 , 6.891-6.862 , 4.646 , 3.777 ppm . MS (EI) m/z (%) 383.1 (44.8), 190.0 (31.1), 121.0 (100). IR (KBr): ν 3068 (CH-aromatic), 2952 , 1249, 1045 cm-1 (C-O-C).Compound Biological sectionCell lines and cell culture2. RPMI-1640, FBS, trypsin and phosphate-buffered saline (PBS) were purchased from Biosera . 3--2,5-diphenyltetrazolium bromide (MTT) was obtained from Sigma-Aldrich . Standard chemotherapeutic agents, dimethylsulfoxide (DMSO) and penicillin/streptomycin were purchased from EBEWE Pharma , Merck  and Invitrogen , respectively. This study used two cell lines, including MOLT-4 (human acute lymphoblastic leukemia) and MCF-7 (human breast adenocarcinoma). MOLT-4 was grown in suspension, while MCF-7 was grown in monolayer culture. The MOLT-4 cell line was purchased from the National Cell Bank of Iran, Pasteur Institute, Tehran, Iran, while MCF-7 cells were obtained from the Iranian Biological Resource Center (IBRC), Tehran, Iran. The cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 100 units/mL penicillin-G and 100 µg/mL streptomycin at 37 °C in humidified air containing 5% COMTT assay4 cells/ml (100 µl in each well) and incubated for 24 h at 37 °C. Afterward, the synthesized compounds were added to each well (in triplicates) at three different concentrations. Doxorubicin and cisplatin were used as positive controls. The plates were further incubated for 72 h, and then 80 μL of the medium was removed, and the same volume of an MTT solution was added at a final concentration of 0.5 mg/mL. Plates were incubated for 4 h at 37 °C to allow the formazan crystals to be formed. Then,200 μL DMSO was added to each well to dissolve the formazan crystals. Absorbance was determined at 570 nm with background correction at 655 nm using a Bio-Rad microplate reader (Model 680). The percentage of viability inhibition compared to control was assessed for each concentration of the compound, and IC50 values were computed with CurveExpert software version 1.34 for Windows.Cells were seeded into 96-well plates with a density of 5×10Molecular docking analysisComputational docking, an extremely useful tool to achieve deep insight into drug-target interactions, has played a crucial role in drug discovery. The synthesized derivatives were subjected to molecular docking studies using AutoDock Tools and AutoDock software in this study.Molecular docking studies with DNAThe X-ray crystal structure of B-DNA (PDB ID: 1Z3F) was obtained from the Protein Data Bank. The ligand structures were drawn and minimized under Semi-empirical AM1 methods using HyperChem 7. Molecular docking studies were done using the AutoDock Tools 1.5.7rc1 and AutoDock 4.2.5.1 docking programs. The energy calculations were made using genetic algorithms (LGA) with the number of GA runs set to 100. Molecular docking studies with Bcl-2 proteinhttp://www.rcsb.org).Innate ligand and waters were removed, hydrogen atoms were added, non-polar hydrogens were merged, and Gasteiger charges were added using AutoDock Tools to prepare the protein for docking. 3D structures of ligands were sketched and minimized under Molecular Mechanics MM+ and then Semi-empirical AM1 methods using HyperChem software. A grid of 60, 60, and 60 points in x = -17.788, y = -12.682 and z = 10.891 directions with 0.375 Å grid spacing was built. The docking process was performed by AutoDock 4.2. To find the conformers with the lowest binding energy, Lamarckian genetic algorithm (LGA) was used, and the number of GA runs was set to 100. The other parameters were left at program default values. Docking procedure validity was examined by re-docking the structure co-crystallized ligand into the receptor (self-docking) using the protocol mentioned above.The crystal structure of Bcl-2 (PDB ID: 3ZLN) was acquired from RCSB Protein Data Bank . Substitution reaction of different halides with intermediate P1 in an ethanolic solution of sodium hydroxide furnished phenanthro-triazine-3-thiol derivatives (P2-P16). The chemical structures were characterized by IR, MS, 1H NMR analysis.The synthetic routes of Cytotoxicityin-vitro cytotoxicity against MOLT-4 (human acute lymphoblastic leukemia) and MCF-7 (human breast adenocarcinoma) cell lines using the MTT assay. MOLT-4 and MCF-7 are frequently used cell lines for screening anticancer agents included in the NCI-60 panel of cell lines suggested by the National Cancer Institute for in-vitro tests. In addition, these cell lines express easily detectable levels of Bcl-2 and Bcl-xl and hence are valuable tools for the study of the inhibitors of these targets  demonstrated good cytotoxic activities against MCF-7 with IC50 values of 15.4 ± 2.9, while P15 (bearing 4-methylphenyl substitution) had an IC50 of 19.4 ± 2.5 μM against MOLT-4 cells. Compounds P7, P8 and P14, containing propanenitrile, 2-methoxyethyl, and 4-fluorophenyl moieties, respectively, exhibited moderate cytotoxic activities against MCF-7 cells with IC50 values in the range of 34.3-39.7 μM.The compounds were tested for their  targets -24. The Molecular docking studiesMolecular docking studies with DNA duplex with the sequence of d(CGATCG)22. 3D representations of docking results of compounds P1, P11 and P15 with DNA are depicted in P1, the hydrogen atom attached to the sulfur atom provided a hydrogen bond interaction with deoxyguanosine 6 ( DG6) while P15, 4-methylphenyl substitution established an additional Pi-Pi interaction with deoxycytidine 5 (DC5).A molecular docking study was employed to identify the binding interactions and estimate the binding affinity of the most active derivatives to DNA duplex with sequence d(CGATCG)Molecular docking studies with Bcl-2 proteinIn-silico molecular docking was performed to gain information on the binding modes of the most active compounds  in the Bcl-2 active site. All the three phenanthro-triazine-3-thiol derivatives were well accommodated into the active site of Bcl-2 . It is also referred that the same trend is predicted for P15 by forming Pi-Pi stacking, Pi-alkyl and alkyl-alkyl interactions between its methylphenyl moiety and Phe146, Leu130 and Ala149 which lead to better binding free energy . By analyzing the contributed residues, it can be concluded that hydrophobic residues primarily contribute to the binding of three derivatives with Bcl-2. Result for re-docking the co-crystallized ligand into the receptor (self-docking) is depicted in P1, P11 and P15.P1, P11 and P15 were found to be the most potent cytotoxic agents. The docking study revealed that these derivatives unwounded the DNA double helix mainly via hydrophobic interactions with the GC base pairs. Additionally, the active compounds were well accumulated in the active site of Bcl-2 through interactions with Leu108, Ser106, Phe105, Ala142, Leu30, Ala149 and Phe146. Therefore, P1, P11, and P15 could be proposed as effective cytotoxic agents with potential DNA intercalation and Bcl-2 inhibitory activities. Further biological evaluations are necessary to confirm our findings.Phenanthro-triazine-3-thiol derivatives have been synthesized and evaluated for their cytotoxic activity against MOLT-4 and MCF-7 cancer cell lines. Compounds


S. aureus, L. monocytogenes, E. coli and S. typhimirium and three resistant strains MRSA, E. coli and P. aeruginosa in order to improve activity of previously evaluated 6-OCF3-benzothiazole-based thiazolidinones. The evaluation of minimum inhibitory and minimum bactericidal concentration was determined by microdilution method. As reference compounds ampicillin and streptomycin were used. Results: All compounds showed antibacterial activity with MIC in range of 0.12–0.75 mg/mL and MBC at 0.25–>1.00 mg/mL The most active compound among all tested appeared to be compound 18, with MIC at 0.10 mg/mL and MBC at 0.12 mg/mL against P. aeruginosa. as well as against resistant strain P. aeruginosa with MIC at 0.06 mg/mL and MBC at 0.12 mg/mL almost equipotent with streptomycin and better than ampicillin. Docking studies predicted that the inhibition of LD-carboxypeptidase is probably the possible mechanism of antibacterial activity of tested compounds. Conclusion: The best improvement of antibacterial activity after modifications was achieved by replacement of 6-OCF3 substituent in benzothiazole moiety by 6-Cl against S. aureus, MRSA and resistant strain of E. coli by 2.5 folds, while against L. monocytogenes and S. typhimirium from 4 to 5 folds.Background: Infectious diseases still affect large populations causing significant morbidity and mortality. Bacterial and fungal infections for centuries were the main factors of death and disability of millions of humans. Despite the progress in the control of infectious diseases, the appearance of resistance of microbes to existing drugs creates the need for the development of new effective antimicrobial agents. In an attempt to improve the antibacterial activity of previously synthesized compounds modifications to their structures were performed. Methods: Nineteen thiazolidinone derivatives with 6-Cl, 4-OMe, 6-CN, 6-adamantan, 4-Me, 6-adamantan substituents at benzothiazole ring were synthesized and evaluated against panel of four bacterial strains  Amalgamations of drugs endowed with different medicinal activities have been dispensed to patients for decades. It is known that an adapted adjustment of different targets may offer an improved medicinal aspect and an advantageous side effect in contrast with the effect of a ligand that has a unique mode of action . In compCharacterization of new therapies for both antifungal and antibacterial disorders that can operate more efficaciously and that are exempt of the side effects related to the present medications continues to be a principal requirement in pharmaceutical research ,3. ApplyThe initial step in investigating for dual-acting molecules is constituted by screening collections of drugs to design pharmacophores’ coupling . This inComputer-aided prediction of pharmacological activity spectra of compounds and drugs based on their structural formulas can be appraised by software Prediction of Activity Spectra for substances (PASS)  to perfoOn the other hand, the thiazolidinone core attracted the interest of researchers owing to its various degrees of pharmacological and medicinal activities ,45,46,473, and 6-Cl keeping the same substitution at position 4 of benzene ring mostly for 6-Cl, 4-OMe and in some cases of 6-CN ([d]thiazole-6-carbonitrile (1–5), 3-(6-(adamantan-1-yl)benzo[d]thiazol-2-yl)-2-(substituted phenyl) thiazolidin-4-one , 3-(6-(adamantan-1-yl)-4-methylbenzo[d]thiazol-2-yl)-2-(substituted phenyl)thiazolidin-4-one , 3-(4-methoxybenzo[d]thiazol-2-yl)-2-(4-substituted phenyl)thiazolidin-4-one (10–14), 3-(6-chlorobenzo[d]thiazol-2-yl)-2-(4-substituted phenyl)thiazolidin-4-one (15–19).Continuing our ongoing research in the field of antimicrobial agents ,46,47 an of 6-CN . Thus, wCompounds were synthesized according to 1H NMR, 13C-NMR and MS for some compounds).All products were obtained as racemates and synthesized compounds were characterized by TLC and spectroscopic methods .The 50) administered to rodents. The reliable estimates are considered to be more than 0.025. All derivatives showed confidence from 0.026 to 0.041 and LD50 of 500–1000 mg/kg or higher belonging to group four according to Globally Harmonized System (GHS) of Labeling and Chemicals’ Classification thiazole-6-carbonitrile (1).Synthesis of 2-(2-(4-nitrophenyl)-4-oxothiazolidin-3-yl)benzothiazole-6-carbonitrile (2).Synthesis of 2--4-oxothiazolidin-3-yl)benzothiazole-6-carbonitrile (3).Synthesis of 2-(2-(2-chloro-6-fluorophenyl)-4-oxothiazolidin-3-yl)benzothiazole-6-carbonitrile (4).Synthesis of 2--4-oxothiazolidin-3-yl)benzothiazole-6-carbonitrile (5).Synthesis of 2-(2-(4-fluorophenyl)-4-oxothiazolidin-3-yl)benzo−1, Nujol): 2364 (=Ν-), 2218 (-CN), 1709 (C=O). 1H-NMR : 3.94 , 4.10 , 6.82 , 7.12–7.22 , 7.81 , 7.92  8.16 . 13C-NMR : δ ppm 33.21 , 64.14, 104.41, 115.45, 117.65, 118.23, 126.72, 129.47, 130.55, 131.09, 134.18, 158.15, 161.25, 164.69, 170.92 .Reaction time: 25 h (A), 30 min (B) respectively. Yield: 52.4%(A), 87.1% (B), respectively. M p.185–186 °C. Rf: 0.68 (petroleum ether/ethyl acetate: 8/2). IR: (cmd]thiazol-2-yl)-2-thiazolidin-4-one (6).Synthesis of 3-(6-(adamantan-1-yl)benzothiazol-2-yl)-2-(2-chloro-6-fluorophenyl) thiazolidin-4-one (7).Synthesis of 3-(6-(adamantan-1-yl)benzothiazol-2-yl)-2-thiazolidin-4-one (8).Synthesis of 3-(6-(adamantan-1-yl)-4-methylbenzothiazol-2-yl)-2-thiazolidin-4-one (9).Synthesis of 3-(6-(adamantan-1-yl)-4-methylbenzo−1, Nujol): 2842 (C-H Ad), 2352 (=Ν-), 1709 (C=O), 1535 (C=C arom), 1383 (-CH3), 1099 (C-F). 1H-NMR : δ ppm 1.73–1.84 , 1.86–1.93 , 1.95–2.03 , 2.54 , 3.86 , 4.14 , 6.85 , 7.25–7.39 , 7.59 , 7.95 . 13C-NMR : δ ppm 16.75 28.91 , 32.74 , 36.41, 36.92, 43.41, 58.86 , 111.51(2C), 112.74, 115.91, 124.12, 124.76, 128.49(2C), 131.01, 142.33 , 162.85, 164.11 , 171.08 . (MS): (m/z) 497 , 482 (7%), 463 (5%), 423 (78%), 244 (100%), 198 (10%), 171 (11%), 152 (18%).Reaction time: 22 h (A), 30 min (B) respectively. Yield: 87.3% (A), 89.6% (B), respectively. M p.188–189 °C. Rf: 0.59 (petroleum ether/ethyl acetate: 8/2). IR: (cmd] thiazol-2-yl) thiazolidin-4-one (10).Synthesis of 2-(4-fluorophenyl) -3-: 2364 (=Ν-), 1709 (C=O), 1587 (C=C arom), 1272 (-OCH3), 1113 . 1H-NMR : δ ppm 3.68–3.86 , 3.91–4.09 , 4.25 , 6.88 , 7.06 , 7.26 , 7.55 , 7.61–7.76 , 8.08–8.26 . 13C-NMR : δ ppm 32.86 , 55.82 , 63.18 , 108.26, 114.11, 115.43(2C), 121.18, 130.33(2C), 131.99, 135.59, 142.31, 150.23, 161.51 , 163.91 , 171.08 . (MS): (m/z) 361 , 343 (42%), 321 (36%), 290 (72%), 288 (31%), 279 (39%), 225 (24%).Reaction time: 6h (A). Yield:51.4%. M p. 251–252 °C.: Rf: 0.73 (petroleum ether/ethyl acetate: 8/2). IR: (cm[d]thiazol-2-yl)-2-(4-nitrophenyl)thiazolidin-4-one (11).Synthesis of 3-(4-methoxybenzof: 0.48 (petroleum ether/ethyl acetate: 8/2). IR: : 2364 (=Ν-), 1709 (C=O), 1585 (C=C arom), 1272 (-OCH3). 1H-NMR : δ ppm 3.77 , 4.03 , 4.25 , 6.93 , 7.04 , 7.26  7.56  7.67  8.17 . 13C-NMR : δ ppm 32.85 , 55.86 , 63.21 , 108.25, 114.12, 121.18, 123.84(2C), 129.66(2C), 131.97, 142.31, 145.26, 146.32, 150.21, 163.95 , 171.02 . (MS): (m/z) 388 , 288 (73%), 272 (3%), 225 (42%), 184 (76%), 136 (42%), 115 (31%), 73 (28%).Reaction time: 10 h (A). Yield: 63.5%.M p: 298–299 °C. Rd]thiazol-2-yl)thiazolidin-4-one (12).Synthesis of 2-(4-chlorophenyl)-3-(4-methoxybenzothiazol-2-yl)-2-(4-methoxyphenyl)thiazolidin-4-one (13).Synthesis of 3-(4-methoxybenzothiazol-2-yl)thiazolidin-4-one (14).Synthesis of 2-(4-hydroxyphenyl)-3-(4-methoxybenzothiazol-2-yl)-2-(4-fluorophenyl)thiazolidin-4-one (15).Synthesis of 3-: 2364 (=Ν-), 1681 (C=O), 1571 (C=C arom), 1101 (C-F Bz). 1H-NMR : δ ppm 3.85 , 4.11 , 6.75 , 7.00–7.03 , 7.30–7.34 , 7.60 , 7.76 . 13C-NMR : δ ppm 32.79 , 63.09 , 115.77, 115.94, 120.83, 122.70, 126.89, 127.49, 127.55, 133.32, 136.12, 146.77, 156.13 , 163.52 , 170.87 . (MS): (m/z) 364 , 343 (10%), 299 (10%), 290 (100%), 279 (9%), 184 (11%), 115 (80%), 88 (9%), 73 (94%).Reaction time: 8 h (A). Yield: 51.3%. M p.: 196–197 °C. Rf: 0.76(petroleum ether/ethyl acetate: 8/2). IR: (cmd]thiazol-2-yl)-2-(4-nitrophenyl)thiazolidin-4-one (16).Synthesis of 3-: 2364 (=Ν-), 1704 (C=O), 1591 (C=C arom). 1H-NMR : δ ppm 3.90 , 4.12 , 6.78 , 7.21–7.30, 7.53 , 7.80 , 8.20 . 13C-NMR : δ ppm 32.86 , 62.55 , 121.09, 122.76, 124.42 (2C), 126.37, 127.20(2C), 130.93, 133.31, 145.21 146.85, 151.28 , 163.55 , 170.48 .Reaction time: 12 h (A), 30 min, 100 °C (B), respectively. Yield: 52.7% (A), 88.3% (B) respectively. M p.: 208–209 °C. Rf: 0.62(petroleum ether/ethyl acetate: 8/2). IR: (cmd]thiazol-2-yl)-2-(4-chlorophenyl)thiazolidin-4-one (17).Synthesis of 3-: 2358(=Ν-), 1709 (C=O), 1591 (C=C arom), 720 (C-Cl bz). 1H-NMR : δ ppm 3.86 , 4.10 , 6.73 , 7.26–7.40 , 7.56 , 7.76 , 8.13 . 13C-NMR : δ ppm 32.84 , 63.11 , 119.02, 120.82, 122.76, 127.98(2C), 129.74(2C), 131.97, 133.30, 135.46, 138.85, 148.06 , 158.04 , 171.19 .Reaction time: 28 h (A), 30 min, 100 °C (B), respectively. Yield: 44.2% (A), 72.1% (B) respectively. M p.: 188–189 °C. Rf: 0.71 (petroleum ether/ethyl acetate: 8/2). IR: (cmd]thiazol-2-yl)-2-(4-methoxyphenyl)thiazolidin-4-one (18).Synthesis of 3-: 2364 (=Ν-), 1709 (C=O), 1571 (C=C arom), 1222 (-OCH3). 1H-NMR : δ ppm 3.76 , 3.91 , 4.10 , 6.72 , 6.82 , 7.12 , 7.23–7.38 , 7.46–7.53 , 8.08 . 13C-NMR : δ ppm 32.88 , 55.27 , 63.47 , 114.20(2C), 120.79, 126.76, 126.79(2C), 127.73, 129.92, 132.28, 133.36, 147.44, 158.89 , 163.59 , 171.01 .Reaction time: 13 h (A). Yield: 62.3%. M p. 220–221 °C: Rf: 0.62 (petroleum ether/ethyl acetate: 8/2). IR: (cmd]thiazol-2-yl)-2-(4-hydroxyphenyl)thiazolidin-4-one (19).Synthesis of : 3198 (-OH), 2358 (=Ν-), 1709 (C=O), 1573 (C=C arom). 1H-NMR : δ ppm 3.86 , 4.10 , 5.35 , 6.73 , 7.26–7.43 , 7.76 , 8.13 . 13C-NMR  δ ppm 33.12 , 63.71 , 115.75 (2C), 118.23, 121.55, 125.14, 129.02, 130.01(2C), 131.52, 132.87, 151.68, 156.71 , 163.77 , 171.12 . (MS): (m/z) 363 , 288 (88%), 272 (9%), 225 (33%), 184 (48%), 146 (7%), 136 (21%), 115 (18%), 73 (17%).Reaction time 18 h (A), 30 min, 100 °C (B), respectively. Yield: 24.1% (A), 72.5% (B) respectively. Mp. 236–237°C °C: Rf: 0.40 (petroleum ether/ethyl acetate: 8/2). IR: .Bacterial strains utilized include Gram-negative: Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 35210), and Gram-positive bacteria: Staphylococcus aureus (ATCC 6538) and Listeria monocytogenes (NCTC 7973) bacteria. Pathogens were provided from the Mycological Laboratory, Institute for Biological Research “Siniša Stankovic” Belgrade. Resistant strains used were MRSA IBRS MRSA 011, E. coli IBRS E003 and P. aeruginosa IBRS P001 obtained as described in Kartsev et al. [v et al. . The MICv et al. ,64.E. coli strain were tested by the disc diffusion method on Mueller Hinton agar with the use of antibiogram discs  and tablets  for the following antibiotics: penicillin, amoxicillin, tetracycline, neomicin, gentamicin, colistin, ceftriaxon, sulfamethaxasole with trimetoprim, enrofloxacin and florfenicol. E. coli strain was resistant to all tested antibiotics with the exception of enrofloxacin, colistin and florfenicol [Sensitivity studies of rfenicol . It is drfenicol .P. aeruginosa strain were tested by the disc diffusion method on Mueller Hinton agar with the use of antibiogram discs  and tablets  for the following antibiotics: penicillin, amoxicillin, tetracycline, neomicin, gentamicin, ceftriaxon, sulfamethaxasole with trimetoprim, enrofloxacin and florfenicol. P. aeruginosa strain was resistant to all tested antibiotics with the exception of enrofloxacin, and florfenicol [Sensitivity studies of rfenicol ,66.This method was performed as described previously ,67 with Aspergillus niger (ATCC 6275), Aspergillus fumigatus (human isolate), Aspergillus versicolor (ATCC 11730), Penicillium funiculosum (ATCC 36839), Trichoderma viride (IAM 5061), Penicillium verrucosum var. cyclopium (food isolate). The organisms were obtained from the Mycological Laboratory, Department of Plant Physiology, Institute for Biological Research ‘Siniša Stankovic’, Belgrade, Serbia. All experiments were performed in duplicate and repeated three times [For the antifungal bioassays, six fungi were used: um ATCC 339, Trichee times .All tests were performed three times and the values were determined as standard deviation (SD) and mean values. One-way ANOVA test was allowed to determine variance analysis with Tukey HSD Test (0.05 levels). Analysis was executed with the help of SPSS statistics software (version 18).http://swissadme.ch, accessed on 28 June 2021) using the ChemAxon’s Marvin JS structure drawing tool.The targeted molecules were appraised for predicting the Drug-likeness based on 5 separate filters namely Egan , Ghose , Muegge E. coli DNA GyrB, Thymidylate kinase, E. coli primase, E. coli MurB, LD-carboxypeptidase,  were retrieved from Brookhaven Protein Data Bank (PDB). The pdb files of proteins were submitted to “Build/check/repair model” to the session “Prepare PDB file for docking programs” and missing side chains were modeled in; water positions and symmetry were corrected, and hydrogen atoms were added. Only chain A of each enzyme of the repaired pdb file was evaluated and passed to AutodockTools (ADT ver. 1.5.6) for pdbqt file preparation. ADT assigned polar hydrogens, water molecules and non-standard residues were removed, only polar hydrogens were maintained, and Gasteiger charges were computed for protein atoms. AutoDock saved the prepared file in PDBQT format.Protein Preparation: X-ray crystal structures of Ligand Preparation: All the molecules were sketched in chemdraw12.0 program. The geometry of built compounds was optimized using the molecular mechanical force fields 94 (MMFF94) energy via program LigandScout, partial charges were also calculated, comformers of each ligand were generated and the best one was maintained and saved as mol2 files that were passed to ADT for pdbqt file preparation. Polar hydrogens were added to each structure, followed by computing Gasteiger and Kollman charges, and the torsions.Gbinding, kcal/mol) and number in cluster. A preliminary blind docking was performed in order to validate the protocol. The RMSD values were predicted by superimposing each docked co-ligand on its original crystallographic bound conformation. The RMSD of all enzymes were in range of 0.85 to 1.43, which are acceptable.Docking Procedure: Autodock 4 (ver. 4.2.6) was employed for docking simulations. The region of interest, used by Autodock4 for docking runs and by Autogrid4 for affinity grid maps preparation, was defined in such a way to comprise the whole catalytic binding site using a grid of 50 × 50 × 50 points with a grid space of 0.375 Å. All parameters used in docking were default. The translation, quaternion and torsions steps were taken from default values in AutoDock. The Lamarckian genetic algorithm and the pseudo-Solis and Wets methods were applied for minimization using default parameters. The number of docking runs was 100. After docking, the 100 solutions were clustered into groups with RMS lower than 1.0 Ε. The clusters were ranked by the lowest energy representative of each cluster. Upon completion of docking, the best poses were screened by examination of binding energy  and ligPlot+ (ver. 2.2).S. aureus, L. monocytogenes, E. coli and S. Typhimirium and three resistant strains MRSA, E. coli and P. aeruginosa.With the purpose to improve the antibacterial activity of previously synthesized compounds, modifications were performed to the initial structure and nineteen new derivatives with 6-Cl, 4-OMe, 6-CN, 6-adamantan, 4-Me,6-adamantan substituents at benzothiazole ring were synthesized and evaluated in silico and experimentally for their antimicrobial activity against panel of four bacterial strains 18 being the most promising . It should be mentioned that some of compounds exhibited superior/equal activity mostly against P. aeruginosa resistant and non-resistant, E. coli and two of them against the most resistant L. monocytogenes than reference drugs, ampicillin and streptomycin. The most sensitive bacteria appeared to be E. coli.It was observed that among all compounds tested the derivatives of 6-chlorobenzothiazole-based thiazolidinone exhibited higher activity, with compound 19 exhibited higher antibiofilm activity than streptomycin. The comparison of obtained results on antibacterial activity with those of compounds with 6-OCF3 substituent in benzothiazole ring revealed that activity depends on substitutients not only at benzothiazole moiety but also of benzene ring. Thus, in case of 4-F substitution at benzene ring the replacement of 6-OCF3 by 6-Cl improved twice the activity against P. aeruginosa and P. aeruginosa resistant, while the replacement by 4-OMe slightly improve only activity against E. coli (0.15 mg/mL and 0.12mg/mL). Among 4-nitro derivatives the presence of 6-Cl as well as 6-CN substituent at benzothiazole ring appeared to be beneficial compared to 6-OCF3 and 4-OCH3, since activity against S. aureus, MRSA and resistant strain of E. coli increased 2.5 folds, while against L. monocytogenes and S. typhimurium from 4- to 5-folds better results (2–3 times) were obtained in case of the presence of 4-OMe and 4-OH substituents in benzene ring of 6-Cl-benzothiazole derivatives compared to the same substituents at the 4 position of benzene ring of 6-OCF3 derivatives.Compounds with the most promising antibacterial potential were studied for their effect on biofilm formation. It was found that the activity of tested compounds in concentration of MIC did not exceed the activity of reference drugs. On the other hand, in concentration of 0.5 MIC compound E. coli primase, E. coli MurB and E. coli LD carboxypeptidase indicate the probable involvement of the last enzyme in the mechanism of the antibacterial activity of the tested compounds.Docking analysis to DNA Gyrase, thymidylate kinase, 15–19 and especially 18 are promising for further modifications in order to develop new more active antibacterial agents.Antifungal activity was moderate to low, lower than antibacterial. Finally, it can be concluded that in general compounds


Pittosporum tenuifolium (Pittosporaceae) from New Zealand: three acylated barringtogenol C glycosides from the leaves, with two previously undescribed 3-O-β-d-glucopyranosyl-(1→2)-[α-l-arabinopyranosyl-(1→3)]-β-d-glucuronopyranosyl-21-O-angeloyl-28-O-acetylbarringtogenol C, 3-O-β-d-galactopyranosyl-(1→2)-[α-l-arabinopyranosyl-(1→3)]-β-d-glucuronopyranosyl-21-O-angeloyl-28-O-acetylbarringtogenol C, and the known 3-O-β-d-glucopyranosyl-(1→2)-[α-l-arabinopyranosyl-(1→3)]-β-d-glucuronopyranosyl-21-O-angeloyl-28-O-acetylbarringtogenol C (Eryngioside L). From the roots, the known 3-O-β-d-glucopyranosyl-(1→2)-β-d-galactopyranosyl-(1→2)-β-d-glucuronopyranosyloleanolic acid (Sandrosaponin X) was identified. Their structures were elucidated by spectroscopic methods including 1D- and 2D-NMR experiments and mass spectrometry (ESI-MS). According to their structural similarities with gymnemic acids, the inhibitory activities on the sweet taste TAS1R2/TAS1R3 receptor of an aqueous ethanolic extract of the leaves and roots, a crude saponin mixture, 3-O-β-d-glucopyranosyl-(1→2)-[α-l-arabinopyranosyl-(1→3)]-β-d-glucuronopyranosyl-21-O-angeloyl-28-O-acetylbarringtogenol C, and Eryngioside L were evaluated.Four oleanane-type glycosides were isolated from a horticultural cultivar “Green Elf” of the endemic  Pittosporum, which comprises about 200 species, is well known for its horticultural uses. From a phytochemical point of view, these species are rich in triterpene-type glycosides with biological interests, such as antimicrobial, antioxidant, cytotoxic, and antiproliferative activities -GlcA. This was confirmed by the ROESY cross-peaks at δH/δH 4.85 (GlcA-1)/3.30 (H-3), 5.65 (Glc-1)/4.52 (GlcA-2), and 5.38 (Ara-1)/4.40 . The structure of compound 1 was, thus, established as 3-O-β-d-glucopyranosyl-(1→2)-[α-l-arabinopyranosyl-(1→3)]-β-d-glucuronopyranosyl-21-O-angeloyl-28-O-acetylbarringtogenol C.The presence of three sugar moieties in ectively . Complet2 was the same as compound 1, with a molecular formula of C54H84O22. Extensive 2D-NMR analysis /78.8 (GlcA-2) and the ROESY correlation at δH/δH 5.49 /4.48 (GlcA-2) confirmed the 3-O-β-d-galactopyranosyl-(1→2)-[α-l-arabinopyranosyl-(1→3)]-β-d-glucuronopyranosyl sequence. Thus, the structure of 2 was elucidated as 3-O-β-d-galactopyranosyl-(1→2)-[α-l-arabinopyranosyl-(1→3)]-β-d-glucuronopyranosyl-21-O-angeloyl-28-O-acetylbarringtogenol C. Natural similar compounds of 1, 2 with acylated 3-O-glucuronopyranosylbarringtogenol C derivatives have already been isolated from a “variegatum” cultivar of P. tenuifolium -β-d-glucuronopyranosyl-21-O-angeloyl-28-O-acetylbarringtogenol C (1). White amorphous powder. [α]25D = −9.5 . For 1H-NMR  and 13C-NMR  data, see m/z: 1107.5359 [M + Na]+ .3-O-β-d-galactopyranosyl-(1→2)-[α-l-arabinopyranosyl-(1→3)]-β-d-glucuronopyranosyl-21-O-angeloyl-28-O-acetylbarringtogenol C (2). White amorphous powder. [α]25D = −13.7 . For 1H-NMR  and 13C-NMR  data, see m/z: 1107.5359 [M + Na]+ .3-3COOH (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 give the sugar residue (64 mg). Glucuronic acid, glucose, galactose, and arabinose 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: d-galactose, Rf = 0.47, [α]25D + 130 , l-arabinose, Rf = 0.54, [α]25D + 170 , d-glucuronic acid, Rf = 0.24, [α]25D + 15 , and d-glucose, Rf = 0.50, [α]25D + 110 .An aliquot (180 mg) of a rich saponin fraction was hydrolyzed with 2 N aqueous CF2, 5 mM sodium pyruvate, pH 7.4). Test substances were initially solubilized in DMSO, followed by in C1 solution for further dilution, before being subjected to 10 min stimulation on cells. After washing with C1 buffer, cells were stimulated by automatic injection of 300 µM sucralose, and changes in intracellular calcium levels were measured at 510 nm in a Fluorometric Imaging Plate Reader  after excitation at 488 nm. For data analysis of dose–response curves, signals of wells receiving the same treatment were averaged, the fluorescent signal of mock cells was subtracted from receptor-transfected cells, and the net signal was normalized to background . For calculation of the half-maximal effective concentration EC50 and half-maximal inhibitory concentration IC50 values, ΔF/F0 was plotted against concentration of the test substance using a four-parameter logistic equation [f(x) = min + (max − min)/(1 + (x/EC50)nH)] with curves fitting of Sigma Plot software.For functional experiments on the sweet taste receptor, HEK293T cells stably expressing the chimeric G-protein subunit Gα16gust44 were seeded into 96-well plates as previously described -β-d-glucuronopyranosyl-21-O-angeloyl-28-O-acetylbarringtogenol C and 3-O-β-d-galactopyranosyl-(1→2)-[α-l-arabinopyranosyl-(1→3)]-β-d-glucuronopyranosyl-21-O-angeloyl-28-O-acetylbarringtogenol C. The inhibitory activities on the sweet taste TAS1R2/TAS1R3 receptor of an aqueous ethanolic extract of the leaves and roots, a crude saponin mixture, the pure compound 1, and Eryngioside L were evaluated. None of them showed clear activity, but exhibited real toxicity. The lack of toxic effects of gymnemic acids, compared with the toxicity of the molecules isolated from Pittosporum tenuifolium “Green Elf”, could be related to the presence of a secondary alcoholic function at the C-23 position of gymnemagenin.Four oleanane-type glycosides were isolated from a horticultural cultivar “Green Elf” of the endemic


Synthesis of the target compounds was readily accomplished through a reaction of acyl hydrazide (1) with carbon disulfide in the presence of alcoholic potassium hydroxide to afford the corresponding intermediate potassium thiocarbamate salt (2), which underwent cyclization reaction in the presence of excess hydrazine hydrate to the corresponding triazole thiol (3). Further cyclisation reaction with substituted benzoyl chloride derivatives in the presence of phosphorous oxychloride afforded the final 6-phenyl-indol-3-yl -triazolo--thiadiazole compounds (5a–l). The novel series showed selective sub-micromolar IC50 growth-inhibitory activity against Bcl-2-expressing human cancer cell lines. The most potent 6- substituted analogue (5k) showed selective IC50 values of 0.31–0.7 µM against Bcl-2-expressing cell lines without inhibiting the Bcl-2-negative cell line (Jurkat). ELISA binding affinity assay (interruption of Bcl-2-Bim interaction) showed potent binding affinity for (5k) with an IC50 value of 0.32 µM. Moreover, it fulfils drug likeness criteria as a promising drug candidate.A series of 3--1H-indoles ( The anti-apoptotic Bcl-2 protein family influences oncogenesis signalling pathways through control of the intrinsic apoptotic process. The parent Bcl-2 protein is an apoptosis suppressor that is found to be frequently overexpressed in cancer cells , and theIndoles are nitrogen-based heterocyclic scaffolds that are found in most common FDA-approved anticancer drugs with the ability to sensitize cancer cells towards apoptosis . Inspire5a–l) The reaction of 1H-indole-3-carboxylic acid hydrazide 1 with carbon disulphide and ethanolic KOH under reflux gave the corresponding potassium thiocarbamate salt 2 in 80% isolated yield, according to the previously reported method . The int4, 5a–l) were found to inhibit the growth of the Bcl-2-expressing human cancer cell lines  with low micromolar IC50 values, except 5c, which was inactive against the KG1A (Acute Myeloid Leukemia) cell line  except compounds 5a and 4. Compounds  showed no activity against Jurkat cells, indicating that Bcl-2 could play a role in mediating the observed anticancer activity that later was confirmed through the ELISA binding assay. Considering the structure–activity relationships, the most active compound was 5k , followed by 5l . These observations reveal that the dimethoxyphenyl substitution was the most favourable for potent activity. In addition, the 4-methoxy 5b, 4-trifluoromethyl 5i and 4-bromo 5e substitutions showed moderately potent inhibitory activity against Bcl-2-expressing cell lines. The different phenyl substitution patterns affected the physiochemical properties, spatial arrangement and orientation within the binding active site. A selection of the most potent and moderate active and selective compounds  were further investigated for their competitive Bcl-2 binding affinity (ELISA assay).All the tested compounds  were further evaluated according to their ability to compete with the pro-apoptotic BH3 domain Bim peptide for binding to His-tagged Bcl-2 protein, as previously described -triazolo--thiadiazol-3-yl]-1H-indole) as a potent Bcl-2 inhibitor, with two-fold lower IC50 value compared to (−)-gossypol positive control. The analysis highlights that the hydrogen bond acceptor (HBA) and negative ionic interactions significantly enhanced the activity. Hence, the potent activity of 5k and 5l could be attributed to the presence of two oxygen pharmacophoric HBA groups and the negatively ionic properties, followed by one HBA of 5b. The 5i compound has a negatively ionic fluorine atom with a weak HBA feature, while 5e compound showed decreased activity with a weak HBA feature and less negatively ionic properties.A competitive Bcl-2 ELISA assay recognizes compound gossypol), we are planning to further expand our study to substantiate the wider anticancer potential and mechanistic features of these active compounds such as 5k and 5l. This further work will include screening within a wider panel of cancer cell lines, including normal cell line controls.The study presented here is focused on early design, synthesis and SAR of novel triazolothiadiazole-based heterocycles as Bcl2-inhibitory anticancer hit compounds. Whilst we provide an early indication that the most active new compounds bind to Bcl-2 (with greater potency than the positive control (−)-0/G1 arrest; and apoptosis induction for gossypol and obatoclax. -triazole-3-thiol  was dissolved in a solution of potassium hydroxide . Carbon disulphide (15 mL) was added dropwise and heated at reflux for 18 h. The reaction mixture was evaporated under reduced pressure, the residue was purified by dissolving in 10% potassium hydroxide solution, and the cold filtrate was acidified with hydrochloric acid. The obtained solid was filtered, washed with water and recrystallized from aqueous ethanol. Yield 70%, mp: 170–172. 1H-NMR  were added to a solution of the appropriate 4-amino-5-(1H-indol-3-yl)-4H--triazole-3-thiol  in phosphorous oxychloride (10 mL), and the mixture was heated at reflux for 8 h. The excess phosphorous oxychloride was removed under reduced pressure, ice water was added, and the precipitate was filtered and washed with aqueous NaHCO5a). Yield 64%, mp: 183–185 °C. 1H-NMR (DMSO-d6): δ 7.21–7.31 , 7.57 , 7.90 , 8.04 , 8.33 , 8.42 , 11.85 . 13C-NMR (DMSO-d6): δ 100.49 (C-3), 100.85 (ArCH), 112.10 (ArCH), 120.65 (ArCH), 121.00 (ArCH), 122.63 (ArCH), 124.25 (ArCH), 126.09 (ArCH), 128.69 (ArC), 128.81 (ArC), 136.25 (ArC), 138.42 (ArC), 143.82 (ArC), 151.54 (ArC), 165.27 (ArC). Calcd mass for C17H10IN5S: 442.87, found : 443.27.3--triazolo--thiadiazol-3-yl]-1H-indole (5b). Yield 73%, mp: 192–194 °C. 1H-NMR (DMSO-d6): δ 4.30 , 7.54–7.77 , 7.98 , 8.46 , 8.75 , 8.81 , 12.24 . 13C-NMR (DMSO-d6): δ 55.66 (OCH3), 100.95 (C-3), 112.09 (ArCH), 115.01 (ArCH), 120.62 (ArCH), 121.00 (ArCH), 121.49 (ArCH), 122.61 (ArCH), 124.24 (ArCH), 126.01 (ArC), 129.07 (ArC), 136.23 (ArC), 143.69 (ArC), 150.91 (ArC), 155.89 (ArC), 162.73 (ArC). Calcd mass for C18H13N5OS: 347.08, found : 348.40.3--triazolo--thiadiazol-3-yl]-1H-indole (5c). Yield 75%, mp: 201–203 °C. 1H-NMR (DMSO-d6) δ 2.56 , 7.24–7.36 , 7.47 , 7.57 , 8.00 , 8.33 , 8.40 , 11.82 . 13C-NMR (DMSO-d6): δ 21.13 (CH3), 100.93 (C-3), 112.10 (ArCH), 115.21 (ArCH), 120.10 (ArCH), 120.63 (ArCH), 121.01 (ArCH), 122.62 (ArCH), 124.25 (ArC), 126.04 (ArC), 127.15 (ArC), 130.14 (ArC), 143.24 (ArC), 151.71 (ArC), 163.79 (ArC). Calcd mass for C18H13N5S: 331.09, found : 332.34.3--1H-indole (5d). Yield 63%, mp: 179–182 °C. 1H-NMR (DMSO-d6): δ 7.21–7.30 , 7.55-7.65 , 7.91 , 8.11 , 8.33 , 8.47 , 11.83 . 13C-NMR (DMSO-d6): δ 100.74 (C-3), 112.14 (ArCH), 120.65 (ArCH), 120.65 (ArCH), 122.62 (ArCH), 122.72 (ArCH), 124.27 (ArCH), 126.36 (ArCH), 126.55 (ArCH), 129.34 (ArCH), 131.33 (ArC), 131.69 (ArC), 135.41 (ArC), 136.26 (ArC), 143.89 (ArC), 151.67 (ArC), 164.39 (ArC). Calcd mass for C17H10BrN5S: 394.98, found : 396.20.3--triazolo--thiadiazol-3-yl]-1H-indole (5e). Yield 67%, mp: 196–198 °C. 1H-NMR (DMSO-d6): δ 7.19–7.29 , 7.6 , 7.85 , 8.09 , 8.35 , 8.49 , 11.94 . 13C-NMR (DMSO-d6): δ 100.84 (C-3), 112.10 (ArCH), 120.64 (ArCH), 121.01 (ArCH), 122.62 (ArCH), 124.26 (ArCH), 126.11 (ArCH), 126.35 (ArCH), 128.43 (ArC), 129.11 (ArC), 132.60 (ArC), 136.25 (ArC), 143.83 (ArC), 151.07 (ArC), 164.93 (ArC). Calcd mass for C17H10BrN5S: 394.98, found : 396.60.3--triazolo--thiadiazol-3-yl]-1H-indole (5f). Yield 64%, mp: 185–187 °C. 1H-NMR (DMSO-d6): δ 7.29–7.35 , 7.59 , 7.67 , 7.79 , 8.07 , 8.22 , 8.36 , 8.41 , 12.13 . 13C-NMR (DMSO-d6): δ 100.77 (C-3), 112.11 (ArCH), 120.63 (ArCH), 121.00 (ArCH), 122.60 (ArCH), 124.27 (ArCH), 126.18 (ArCH), 126.33 (ArCH), 126.60 (ArCH), 131.15 (ArCH), 131.49 (ArC), 132.48 (ArC), 134.36 (ArC), 136.27 (ArC), 143.89 (ArC), 151.64 (ArC), 164.44 (ArC). Calcd mass for C17H10ClN5S: 351.03, found : 352.45.3--triazolo--thiadiazol-3-yl]-1H-indole (5g). Yield 74%, mp: 183–185 °C. 1H-NMR (DMSO-d6): δ 7.15–7.28 , 7.54–7.58 , 7.64–7.78 , 7.93 , 8.15 , 8.47 , 8.58 , 11.87 . 13C-NMR (DMSO-d6): δ 100.75 (C-3), 113.82 (ArCH), 114.01 (ArCH), 119.54 (ArCH), 119.71 (ArCH), 120.62 (ArCH), 121.00 (ArCH), 122.58 (ArCH), 123.67 (ArCH), 124.28 (ArCH), 126.34 (ArC), 131.81 (ArC), 136.29 (ArC), 143.88 (ArC), 151.59 (ArC), 161.40 (ArC), 163.36 . Calcd mass for C17H10FN5S: 335.06, found :336.36.3--triazolo--thiadiazol-3-yl]-1H-indole (5h). Yield 67%, mp: 196–198 °C. 1H-NMR (DMSO-d6): δ 7.22–7.32 , 7.57 , 8.31 , 8.37–8.47 , 11.94 . 13C-NMR (DMSO-d6): δ 100.47 (C-3), 112.17 (ArCH), 117.29 (ArCH), 120.80 (ArCH), 121.00 (ArCH), 122.67 (ArCH), 124.20 (ArCH), 125.56 (ArCH), 126.33 (ArC), 127.07 (ArC), 131.12 (ArC), 140.43 (ArC), 143.18 (ArC), 156.26 (ArC), 163.34 (ArC). Calcd mass for C17H10N6O2S: 362.06, found : 363.03.3--triazolo--thiadiazol-3-yl]-1H-indole (5i). Yield 60%, mp: 194–196 °C. 1H-NMR (DMSO-d6): δ 7.22–7.31 , 7.58 , 8.03 , 8.27–8.33 , 8.44 , 11.88 . 13C-NMR (DMSO-d6): δ 100.60 (C-3), 112.82 (ArCH), 117.01 (ArCH), 120.12 (ArCH), 121.00 (ArCH), 122.28 (ArCH), 125.08 (ArCH), 127.04 (ArCH), 131.85 , 136.29 (ArC), 143.54 (ArC), 151.45 (ArC), 155.09 (ArC), 161.34 (ArC), 163.47 (ArC). Calcd mass for C17H10F3N5S: 385.06, found : 386.45.3--triazolo--thiadiazol-3-yl]-1H-indole (5j). Yield 67%, mp: 196–198 °C.1H-NMR (DMSO-d6): δ 7.22–7.31 , 7.58 , 7.96 , 8.34 , 8.45 , 8.55 , 8.80 , 11.87 . 13C-NMR (DMSO-d6): δ 99.47 (C-3), 112.37 (ArCH), 116.59 (ArCH), 120.19 (ArCH), 120.95 (ArCH), 122.77 (ArCH), 124.01 (ArCH), 125.46 (ArCH), 126.92 (ArCH), 136.39 (ArCH), 140.89 (ArC), 145.11 (ArC), 151.07 (ArC), 156.56 (ArC), 157.37 (ArC), 160.14 (ArC), 161.27 (ArC). Calcd mass for C17H10N6O2S: 362.06, found : 363.11.3--triazolo--thiadiazol-3-yl]-1H-indole (5k). Yield 73%, mp: 178–180 °C. 1H-NMR (DMSO-d6): δ 3.90 , 4.09 , 6.83 , 6.90 , 7.20–7.31 , 7.56 , 8.34-8.36 , 8.42 , 11.80 . 13C-NMR (DMSO-d6): δ 55.86 (OCH3), 56.52 (OCH3), 98.62 (C-3), 101.21 (ArCH), 107.93 (ArCH), 110.04 (ArCH), 112.04 (ArCH), 120.48 (ArCH), 121.07 (ArCH), 122.51 (ArCH), 124.32 (ArCH), 125.77 (ArC), 129.45 (ArC), 136.23 (ArC), 142.99 (ArC), 152.80 (ArC), 158.79 (ArC), 161.42 (ArC), 164.13 (ArC). Calcd mass for C19H15N5O2S: 377.09, found : 377.42.3--triazolo--thiadiazol-3-yl]-1H-indole (5l). Yield 68%, mp: 193–195 °C. 1H-NMR (DMSO-d6): δ 3.90 , 4.19 , 6.84 , 7.19 , 7.21–7.29 , 7.59 , 8.33 , 8.43 , 11.68 . 13C-NMR (DMSO-d6): δ 55.75 (OCH3), 100.84 (C-3), 104.20 (ArCH), 105.24 (ArCH), 112.10 (ArCH), 120.60 (ArCH), 121.02 (ArCH), 122.59 (ArCH), 124.27 (ArCH), 126.17 (ArC), 130.90 (ArC), 136.24 (ArC), 143.80 (ArC), 151.53 (ArC), 161.11 (ArC), 165.62 (ArC). Calcd mass for C19H15N5O2S: 377.09, found : 377.63.3--triazolo--thiadiazol-3-yl]-1H-indole .Human breast cancer (MDA-MB-231) and cervical cancer (HeLa) cells, obtained from the ATCC , were maintained as previously described ,16. Cyto® solution was added, followed by reading of the fluorescence according to our previous protocol [50 values were obtained by using GraphPad Prism 5 software . For each concentration, six independent experiments were carried out.Human acute myeloid leukemia KG1a and acute T-cell lymphocytic leukemia Jurkat cells were cultured and maintained as previously described ,6,7,8. Fprotocol ,6,7,8. I50 for the Bcl-2 inhibition activity.ELISA assay was performed to evaluate the ability of the compounds to compete with Bim protein and inhibit its interaction with Bcl-2 protein. ,6,7,8. Thttps://www.rcsb.org/ obtained on September 2021). The proteins were prepared and refined using the Protein Preparation Wizard Maestro to ensure structural correctness [The 3D crystal structure of human Bcl-2 (PDB ID: 4AQ3) protein was downloaded from the protein data bank , acceptor (A), hydrophobic group (H), negatively ionizable (N), positively ionizable (P) and aromatic ring (R).The R-group mapping analysis was performed in the Schrödinger suite. First, the input LigPrep structure with the associated IC5a–l) as novel inhibitors of Bcl-2 protein. The various aromatic substitutions and spatial orientations of the (5a–l) compounds within the Bcl-2 binding pockets afforded variable activity, while the most potent compound with sub-micromolar IC50 values was (5k), containing a  substitution. Moreover, molecular docking studies showed stable interactions of the (5k) compound within the BH3 domain of Bcl-2. This was confirmed with ELISA assay, which displayed a potent inhibitory IC50 value of 0.3 µM, representing a two-fold greater potency than gossypol as positive control. The predicted ADME properties showed that the compounds fulfil the criteria of drug likeness and preferable properties range. This indicates that they are promising drug candidates for further development as a lead compound against a Bcl-2 anti-apoptotic target.This study rationalized the design, synthesis and evaluation of novel series of -1H-indoles (