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Indian Journal of Chemistry Vol. 52B, June 2013, pp 794-801 Expedient synthesis of 1,2,4-triazolin-3-one derivatives as DNA cleavage and antioxidant agents Tasneem Taja, Ravindra R Kamble*a, Pramod P Kattimania & Sudha S Belgurb a Department of Studies in Chemistry, Karnatak University Dharwad 580 003, India b Department of Chemistry, Yuvaraja’s College, University of Mysore, Mysore 590 005, India E-mail: [email protected] Received 24 January 2012; accepted (revised) 5 April 2013 The present study involves the synthesis of fluoren-9-ylidene 5a-e and isoindolin-1,3-dione derivatives 5f-j appended to 1,2,4-triazolin-2-one under solvent and neat conditions. The title compounds have been evaluated for the extent of cell penetration through biological membranes (clogP) and drug score has been calculated and further screened for DNA cleavage and antioxidant activities. Keywords: Sydnone, fluorenone, isoindolin-1,3-dione, DNA cleavage, antioxidant Sydnones are extensively studied mesoionic compounds which have gained importance due to their use as synthons for the ring transformations into various heterocyclic systems1-4. Recent years have witnessed a great deal of interest in the synthesis and characterization of Schiff bases5. Several reports have indicated that the presence of a lone pair of electrons in sp2 hybridized orbital of nitrogen atom of the azomethine group has considerable chemical and biological importance6-12. Benzo[b] fluorenone skeleton characterized by an uncommon diazo subunit belongs to a family of complex natural products of kinamycins13-16. The lomaiviticins are glycosylated dimeric analogues of the kinamycins and were reported to have potent anti-cancer/antibiotic activity. Lomaiviticin A was reported to cleave DNA under reducing conditions17. Many research programmes have been directed towards understanding the mechanism by which the kinamycins/lomaiviticins cleave DNA and subsequently harnessing this information toward the design of simple analogues for the development of potential anti-cancer compounds18,19. Several 4-substituted-isoindolinones were linked through various spacers to adenosine and are reported in literature as weak inhibitor of PARP activity20. Interaction of transition metal complexes with DNA has long been intensively investigated for applications in molecular biology, biotechnology, and medicine21, 22. Since DNA is the intracellular target in treating a wide range of anticancer cells, binding of small molecules with DNA is extremely useful in understanding drug–DNA interactions. Several 2-(2hydroxybenzylideneamino)-isoindoline-1,3-dione transition metal complexes have been reported as DNA cleavage agents23 and pthalidomide (2-(2,6-dioxo-3piperidyl-isoindoline-1,3-dione, Contergan) was developed as a sedative24 and is also effective in cancer treatment25-27. Molecules containing a dithiolane moiety are widely investigated due to their antioxidant properties. The archetypal representative of this class of compounds is lipoic acid and indeed the lipoic acid, dihydrolipoic acid couple is part of the antioxidant defense system of the cell28. The deleterious effects of an imbalance between reactive oxygen species (ROS) production and the available antioxidant defense capacity, termed oxidative stress, as well as its role in the aggravation of a plethora of pathological conditions are also documented in the literature29. A variable sensitivity to this excessive oxidative state is observed between tissues and organs, the brain being the most vulnerable due to its high oxygen consumption, high content of unsaturated membrane fatty acids and low level of antioxidant enzymes or vitamins30. In the last decade much effort has been directed towards finding molecules capable of opposing the oxidative stress challenge31. Many studies have been focused on the design of analogues of fluorenones and isoindoline-1,3-dione which possess DNA cleavage ability of pathogens and TAJ et al.: SYNTHESIS OF 1,2,4-TRIAZOLIN-3-ONE 795 O O O CH3 N N N HB HX H A R N N N CH3 6a-e X O R O N N HX O O CH3 NNH2 N N HB HA N CH3 N N N 3 CH3 HX HA R N HB N CH3 2a-j 5a-e 4 O O CH3 R = H, p-OH, o-OH, p-NO2, p-OCH3 O O N N HX R HB HA N N N N CH3 3= O 4= O O 5f-j O Scheme I — Synthesis of Schiff bases of fluorenone 5a-e and isoindolin-1,3-dione derivatives 5f-j in solvent CH3 6a-e X 5a-e O 3 O N N NH2NH2.H2O R HX O N HB N 4 CH3 NH2NH2.H2O 5f-j HA 1a-j Scheme II — Synthesis of Schiff bases of fluorenone 5a-e and isoindolin-1,3-dione derivatives 5f-5j under neat condition also which act as anti-oxidants. Owing to the above literature survey and our zeal to explore newer fluorenone-9-ylidene 5a-e and isoindoline-1,3-diones 5f-j, we herein report the synthesis, in vitro DNA cleavage, antioxidant evaluations and drug score values. Results and Discussion Starting materials viz., 3-(4-(1-acetyl-4,5-dihydro5-aryl-1H-pyrazol-3-yl)phenyl)-5-methyl-1,3,4-oxadiazol-2-(3H)-one 1a-j and 2-[4-(1-acetyl-5-aryl-4,5dihydro-1H-pyrazol-3-yl)-phenyl]-4-amino-5-methyl2,4-dihydro-[1,2,4]triazol-3-one 2a-j were prepared according to reported method32. The title compounds 5a-j were synthesized by two methods. First method involved the reaction of 2-[4-(1-acetyl-5-aryl-4,5dihydro-1H-pyrazol-3-yl)-phenyl]-4-amino-5-methyl2,4-dihydro-[1,2,4]-triazol-3-one 2a-j with fluorenone 3 and/or phthalic anhydride 4 in solvents viz., ethanol and dry butanol respectively to yield the title compounds 5a-j (Scheme I). By this method moderate yield of 62-70% was observed. In an alternative method, under neat condition 3-(4(1-acetyl-4,5-dihydro-5-aryl-1H-pyrazol-3-yl)phenyl)5-methyl-1,3,4-oxadiazol-2(3H)-one 1a-j was reacted with hydrazine hydrate, fluorenone 3 and/or phthalic anhydride 4 (as the case may be) in a single flask operation to get directly the final compounds 5a-j (Scheme II). Interestingly, an appreciable yield of 73-81% was observed. Both the methods differ in INDIAN J. CHEM., SEC B, JUNE 2013 796 Table I — Characterization data of compounds 5a-j Compd Mol. formula m.p. (°C) Yield (g) (%) Method 1* Yield (g) (%) Method 2# 5a C33H26N6O2 103-104 0.530 65 0.577 78 5b C33H26N6O3 123-24 0.539 67 0.583 79 5c C33H26N6O3 143-44 0.504 62 0.549 75 5d C33H25N7O4 135-36 0.480 70 0.515 72 5e C34H28N6O3 155-56 0.510 68 0.535 74 5f C28H22N6O4 123-25 0.482 70 0.511 73 5g C28H22N6O5 132-33 0.481 63 0.519 75 5h C28H22N6O5 143-44 0.495 65 0.526 76 5i C28H21N7O6 125-27 0.513 69 0.515 81 5j C29H24N6O5 138-39 0.468 70 0.500 73 Elemental analysis Found % (Calcd) C H N 73.59 (73.56 71.47 (71.45 71.47 (71.44 67.92 (67.89 71.82 (71.80 66.40 (66.38 64.36 (64.34 64.36 (64.35 60.98 (60.95 64.92 (64.89 4.87 4.85 4.73 4.71 15.60 15.62) 15.15 15.12) 4.73 4.74 4.32 4.30 4.96 4.94 15.15 15.17) 16.80 16.78) 14.78 14.75) 4.38 4.35 4.24 4.22 4.24 4.23 16.59 16.62) 16.08 16.05) 16.08 16.07) 3.84 3.82 4.51 4.53 17.78 17.75) 15.66 15.63) *Triazole, #Oxadiazole the reaction condition and reaction time. The yields observed in both methods were given in Table I. It was observed that the neat condition exhibited excellent yields. However, attempts to synthesize azetidinone derivatives from Schiff bases 5a-e were failed which is due to steric hindrance of bulkier fluorenone ring. The structures of compounds 5a-i were confirmed by elemental analysis, IR, 1H and 13C NMR and mass spectra. In case of IR spectral analysis, the title compounds 5a-j showed weak bands for CH stretching around 2924-2853, 2930-2855 cm-1, C=N stretching for the pyrazoline ring in the range 1630-1646 cm-1. Compounds 5b, 5c, 5g, and 5h showed two broad medium intensity bands in the range 3400 cm-1 due to OH group. The 1H NMR spectra of the pyrazoline ring (methine and methylene protons) have shown a characteristic feature and form an ABX pattern. The methylene protons can be assigned as HA, HB and the methine proton as Hx. HA and HB are diastereotopic and also anisochronous as they differ in chemical shifts and since this difference is not large as they are identified as AB protons. The methine proton on adjacent carbon with larger shift downfield is the Hx proton and hence all together form ABX pattern. The HA and HB protons appear as doublet due to geminal and vicinal coupling. These HA and HB differ in coupling with the Hx and hence they are also anisogamous. The HA proton appears as doublet of doublet in the range δ 3.29-3.65 with two coupling constants, JAB = 17.6 Hz and JAx = 4.5Hz. HB also appears as doublet of doublet at δ 3.83-4.25 where JBA is 17.6 Hz and JBX = 11.8 Hz. The Hx always appeared as four line spectrum with JXA = 4.49 Hz and JXB = 11.84 in the range δ 4.25-5.50. A broad singlet for OH in the compounds 5b, 5c, 5e and 5g appeared at δ 5.385.49 (D2O exchangeable). The aromatic protons appear around δ 6.85-8.99. The compounds 5e and 5j showed a singlet at δ 3.72-3.77 due to methoxy protons. The 13 C NMR spectral analysis of the title compounds showed the number of signals consistent with number of carbons which are magnetically different and the mass spectra of the title compounds exhibited molecular ion peaks at their respective molecular weights which confirmed their formation. Pharmacological Evaluation Molecular Osiris property For a new molecule to qualify as a drug candidate, it has to be analyzed for the parameters set by Lipinski’s rule of five using Osiris property explorer33-35. TAJ et al.: SYNTHESIS OF 1,2,4-TRIAZOLIN-3-ONE Lipinski’s rule of five is thumb rule to evaluate drug score or determine if a new molecule with a certain pharmacological or biological activity has properties that would make it a likely orally active drug in humans. The molecular properties are important for a drug pharmacokinetics in human body, including their absorption, distribution, metabolism and excretion (ADME). The rule is important for drug development where pharmacologically active lead structure is optimized step wise for increased activity and selectivity, as well as drug like properties as described by Lipinski rule. The modification of the molecular structure often leads to drugs with higher molecular weight, more rings, more rotatable bonds and higher lipophilicity. The rule states that in general an orally active drug has no more than 5 hydrogen bond donors and not more than 10 hydrogen acceptors, a molecular weight under 500 and partition co-efficient clogP less than 5. Interestingly, it was observed that the title compounds do not violate the Lipinski rule and they fall well in the range as mentioned when evaluated by the Osiris property explorer, and also the drug Table II — Pharmacological parameters for bioavailability Compd clogP Drug Score 5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 6.96 6.66 6.66 6.83 6.85 3.18 2.88 2.88 3.05 3.08 0.06 0.06 0.06 0.06 0.06 0.13 0.13 0.13 0.10 0.12 797 likeliness in the range -12.36 to -0.15 and drug score 0.03-0.39 which will surely lead us to evaluate the compounds experimentally Table II. Promising results were observed for compounds 5f, 5g, 5h and 5j with the drug score of 0.13. All the title compounds were shown as safe drugs without any mutagenic, tumourigenic, irritability and reproductive effects as evaluated in Osiris property explorer. DNA cleavage activity A number of studies have shown that the clinical efficacies of many drugs correlate with their abilities to induce enzyme-mediated DNA cleavage. The inhibitory potency of the test compounds was assessed by comparing the cleavage of DNA with the control used and the title compounds. The relative efficacy of the drugs to stimulate DNA cleavage varies considerably from one congener to another. A detailed comparison between the compounds tested allows us to make some significant observations. The gel electrophoresis was used for the analysis of 5a-j and after treatment, it was observed that the DNA treated with 5i and 5j (isoindolin-1,3-dione derivatives) have cleaved the DNA completely. In both 5i and 5j lane, with p-NO2 and p-OCH3 of the phenyl group, whole band of DNA was disappeared indicating the complete cleavage of DNA. DNA treated with 5j (with p–OCH3 group) showed significant diminishing of the band with a prominent streak indicating DNA cleavage activity of compound. Only partial degradation activity was found with any other sample in this gel with the compounds 5e-h. Among the compounds 5a-d in another gel also showed partial degradation of DNA as shown in the Figure 1. Figure 1 — DNA cleavage activity of compounds 5a-j Note: In the photograph M- Standard DNA molecular weight marker (λ DNAEcoRI digest, Banglore Genei, Bangalore) C- Control DNA (untreated sample) INDIAN J. CHEM., SEC B, JUNE 2013 798 Figure 2 — Comparison of ferric reducing antioxidant power of 5a-j with butylated hydroxy anisole Anti-oxidant activity The FRAP assay was carried out to determine the ferric reducing ability of biological fluids and aqueous solutions of pure compounds. Butylated hydroxy anisole (BHA) was used as a standard. Although the final dilution of the sample in the reaction mixture was high (100 µg), for any possible influence of the solvent on the reaction was checked. Therefore, aqueous solutions of the FeSO4.7H2O standard used for calibration were prepared and analyzed with the FRAP reagent. Ferrous sulfate dissolved well in both solvents and there was no precipitation problem. The values of absorbance at 700 nm compared to BHA with concentrations of 10, 50, 100 µg are represented in Figure 2. The compounds 5c, 5g and 5j respectively with groups like o-OH and p-OCH3, have shown very good activity compared to BHA, at all the concentrations tested. The antioxidant activity of these compounds may be related to their redox properties, which allow them to act as reducing agents or hydrogen atom donors, their ability to chelate metals, inhibit lipoxygenase and scavenge free radicals. Such antioxidants function as free-radical scavengers and chain breakers, complexers of prooxidant metal ions and quenchers of singlet-oxygen formation. Compounds 5a, 5e, 5f with phenyl and -OCH3 (isoindolin-1,3-dione) substituents have shown moderate activity compared to the standard. Compounds 5b, 5d, 5h and 5i have shown least activity compared to standard BHA. Experimental Section Melting points of the synthesized compounds were determined in open capillaries. The TLC was performed on alumina silica gel 60 F254 (Merck). The mobile phase was hexane and ethyl acetate (1:1) and detection was made using UV light. IR spectra were recorded using KBr pellets on Nicolet 5700 FTIR instrument. The 1H and 13C NMR were recorded on Bruker Avance-300 (300 MHz) model spectrophotometer in CDCl3 and DMSO-d6 as solvents and TMS as internal standard with 1H resonance frequency of 300 MHz. The chemical shifts were measured in ppm downfield from internal standard TMS at δ = 0. Mass spectra were recorded on Shimadzu Japan QP2010 S model spectrometer and elemental analyses were carried out using Heraus CHN rapid analyzer. The resulting compounds were purified by recrystallization with either methanol or ethanol. All the compounds gave C, H and N analysis within +/- 0.5% of the theoretical values. DNA cleavage and antioxidant evaluations were carried out at Biogenics, Research and training centre in Biotechnology, Hubli, Karnataka, India. The clog p, drug likeliness and drug scores have been evaluated in Osiris property explorer software for the structural analogues of the synthesized compounds and are uncorrected. General procedure Preparation of 4-(9H-fluoren-9-ylideneamino)-2(4-(1-acetyl-4,5-dihydro-5-aryl-1H-pyrazol-3-yl)phenyl)-5-methyl-2H-1,2,4-triazol-3(4H)-one, 5a-e Method 1: Compound 2a (0.5 g, 1.38 mmol), fluorenone 3 (0.237 g, 1.32 mmol) and ethanol (5 mL) were refluxed at 98-100°C for about 6 hr on a water bath. The reaction mixture after cooling was poured into ice cold water and the solid mass separated was recrystallized in ethanol to get pale yellow needles of compound 5a. (Scheme I) Method 2: Compound 1a (0.5 g, 1.32 mmol), fluorenone 3 (0.234 g, 1.38 mmol) and hydrazine TAJ et al.: SYNTHESIS OF 1,2,4-TRIAZOLIN-3-ONE hydrate (5 mL) were refluxed at 130-35°C for 5 hr. The reaction mixture was extracted with ether (3×5 mL), washed with brine solution and water. The ethereal layer was dried over anhyd. Na2SO4, ether was evaporated and the separated solid obtained was recrystallized using ethanol to get the compound 5a. (Scheme II). Similarly, the compounds 5b-e were prepared according to this procedure. 4-(9H-Fluoren-9-ylideneamino)-2-(4-(1-acetyl4,5-dihydro-5-phenyl-1H-pyrazol-3-yl)phenyl)-5methyl-2H-1,2,4-triazol-3(4H)-one, 5a: Pale yellow solid; IR (KBr): 2961, 2923, 2848 (CH), 1712, 1653 (C=O), 1603 (C=N) cm-1; 1H NMR (CDCl3): δ 2.35 (s, 3H, CH3), 3.06 (s, 3H, CH3), 3.62 (dd, 1H, CH2), 4.25 (dd, 1H, CH2), 5.50 (m, 1H, CH), 6.87-8.94 (m, 17H, Ar-H); 13C NMR (CDCl3): δ 22.80 (CH3), 23.54 (CH3), 39.90 (CH2), 58.68 (CH), 118.18, 118.35, 120.71, 121.34, 124.72, 125.93, 128.05, 129.29, 129.47, 129.73, 134.54 (Ar-C), 145.10 (C=N), 144.82, 194.34 (C=O); MS: m/z 538 (M+). 4-(9H-Fluoren-9-ylideneamino)-2-(4-(1-acetyl-4, 5-dihydro-5-(4-hydroxyphenyl)-1H-pyrazol-3-yl)phenyl)-5-methyl-2H-1,2,4-triazol-3(4H)-one, 5b: Pale brown solid; IR (KBr): 2965, 2925, 2850 (CH), 1715, 1654 (C=O), 1605 (C=N) cm-1; 1H NMR (CDCl3): δ 2.40 (s, 3H, CH3), 3.10 (s, 3H, CH3), 3.65 (dd, 1H, CH2), 4.20 (dd, 1H, CH2), 5.23 (m, 1H, CH), 5.45 (s, 1H, -OH), 7.01-8.84 (m, 16H, Ar-H); 13C NMR (CDCl3): δ 23.84 (CH3), 24.55 (CH3), 38.90 (CH2), 56.68 (CH), 119.18, 119.32, 120.73, 121.46, 124.87, 125.83, 128.15, 129.30, 129.50, 129.78 (ArC), 134.60 (C-OH), 146.10 (C=N), 147.82, 195.34 (C=O); MS: m/z 554 (M+). 4-(9H-Fluoren-9-ylideneamino)-2-(4-(1-acetyl-4, 5-dihydro-5-(2-hydroxyphenyl)-1H-pyrazol-3-yl)phenyl)-5-methyl-2H-1,2,4-triazol-3(4H)-one, 5c: Brown solid; IR (KBr): 2968, 2925, 2853 (CH), 1712, 1655 (C=O), 1601 (C=N) cm-1; 1H NMR (CDCl3): δ 2.44 (s, 3H, CH3), 3.12 (s, 3H, CH3), 3.63 (dd, 1H, CH2), 4.15 (dd, 1H, CH2), 5.25 (m, 1H, CH), 5.50 (s, 1H, OH), 6.95-8.45 (m, 16H, Ar-H); 13C NMR (CDCl3): δ 22.80 (CH3), 23.54 (CH3), 39.90 (CH2), 58.68 (CH), 118.18, 118.35, 120.71, 121.34, 124.72, 125.93, 128.05, 129.29, 129.47, 129.73, 134.54 (ArC), 137.60 (C-OH), 146.10 (C=N), 147.82, 198.34 (C=O); MS: m/z 554 (M+). 4-(9H-Fluoren-9-ylideneamino)-2-(4-(1-acetyl-4, 5-dihydro-5-(4-nitrophenyl)-1H-pyrazol-3-yl)phenyl)-5-methyl-2H-1,2,4-triazol-3(4H)-one, 5d: Pale yellow solid; IR (KBr): 2963, 2935, 2855 (CH), 1717, 1657 (C=O), 1610 (C=N) cm-1; 1H NMR (CDCl3): δ 799 2.38 (s, 3H, CH3), 3.15 (s, 3H, CH3), 3.68 (dd, 1H, CH2), 4.22 (dd, 1H, CH2), 5.27 (m, 1H, CH), 7.118.99 (m, 16H, Ar-H); 13C NMR (CDCl3): δ 24.84 (CH3), 25.55 (CH3), 39.90 (CH2), 57.68 (CH), 120.18, 120.32, 121.73, 122.46, 125.87, 126.83, 129.15, 130.30, 130.50, 130.78 (Ar-C), 136.60 (C-OH), 147.10 (C=N), 148.82, 197.34 (C=O); MS: m/z 583 (M+). 4-(9H-Fluoren-9-ylideneamino)-2-(4-(1-acetyl-4, 5-dihydro-5-(4-methoxyphenyl)-1H-pyrazol-3-yl)phenyl)-5-methyl-2H-1,2,4-triazol-3(4H)-one, 5e: Yellow solid; IR (KBr): 2961, 2921, 2851, 1711, 1651 (C=O), 1601 (C=N) cm-1; 1H NMR (CDCl3): δ 2.38 (s, 3H, CH3), 3.09 (s, 3H, CH3), 3.64 (dd, 1H, CH2), 3.72 (s, 3H, -OCH3), 4.18 (dd, 1H, CH2), 5.10 (m, 1H, CH), 7.05-8.85 (m, 16H, Ar-H); 13C NMR (CDCl3): δ 22.85 (CH3), 23.57 (CH3), 37.89 (CH2), 57.69 (CH), 118.18, 119.32, 121.73, 122.46, 124.87, 126.83, 127.15, 128.30, 129.50, 129.83 (Ar-C), 133.62 (C-OH), 147.15 (C=N), 149.82, 197.38 (C=O); MS: m/z 568 (M+). Preparation of 2-(1-(4-(1-acetyl-4,5-dihydro-5aryl-1H-pyrazol-3-yl)phenyl)-3-methyl-5-oxo-1H1,2,4-triazol-4(5H)-yl)isoindoline-1,3-dione, 5f-j Method 1: Compound 1a (0.5 g, 1.38 mmol), phthalic anhydride 4 (0.195 g, 1.32 mmol) and dry butanol (5 mL) were refluxed at 120-21°C for 6hr. The reaction mixture after cooling was poured to ice cold water and the separated solid was recrystallized using ethanol to yield the compound 5f (Scheme I). Method 2: A mixture of compound 2a (0.5 g, 1.32 mmol), phthalic anhydride 4 (0.192 g, 1.38 mmol) and hydrazine hydrate (5 mL) were refluxed at 13840°C for 5hr. The reaction mixture after cooling was extracted with DCM (3×5 mL), washed with brine solution and water. The organic layer was dried over anhyd. Na2SO4, DCM was evaporated and the separated solid was recrystallized using ethanol to yield the compound 5f (Scheme II). Similarly, the compounds 5b-e were prepared according to this procedure. 2-(1-(4-(1-Acetyl-4,5-dihydro-5-phenyl-1H-pyrazol-3-yl)phenyl)-3-methyl-5-oxo-1H-1,2,4-triazol-4(5H)-yl)isoindoline-1,3-dione, 5f: Pale yellow solid; IR (KBr): 2961, 2932, 2871, 1809 (CH), 1722, 1710, 1640 (C=O), 1606 (C=N) cm-1; 1H NMR (DMSO): δ 2.36 (s, 3H, CH3), 2.51 (s, 3H, CH3), 3.29 (dd, 1H, CH2), 3.64 (dd, 1H, CH2), 4.25 (m, 1H, CH), 7.64-8.14 (m, 13H, Ar-H); 13C NMR (DMSO): δ 22.3 (CH3), 23.4 (CH3), 39.9 (CH2), 58.9 (CH), 121.7, 122.0, 126.8, 800 INDIAN J. CHEM., SEC B, JUNE 2013 127.1, 127.4, 127.6, 127.8, 128.6, 128.9, 129.4, 129.6, 130.0, 132.1, 132.5, 132.3, 132.7, 140.1, 143.5 (Ar-C), 151.0 (C=N), 154.0 (C=O), 155.0 (C=N), 164.8, 164.09, 168.3 (C=O); MS: m/z 506 (M+). 2-(1-(4-(1-Acetyl-4,5-dihydro-5-(4-hydroxyphenyl)-1H-pyrazol-3-yl)phenyl)-3-methyl-5-oxo-1H-1, 2,4-triazol-4(5H)-yl)isoindoline-1,3-dione, 5g: Yellow solid; IR (KBr): 3447, 2961, 2924, 2848 (CH), 1800, 1742, 1640 (C=O), 1605 (C=N) cm-1; 1H NMR (CDCl3): δ 2.14 (s, 3H, CH3), 2.55 (s, 3H, CH3), 3.49 (dd, 1H, CH2), 4.23 (dd, 1H, CH2), 4.28 (m, 1H, CH), 5.38 (s, 1H, OH), 7.20-7.82 (m, 12H, Ar-H); 13C NMR (CDCl3): δ 23.2, 25.3 (CH3), 40.9 (CH2), 57.9 (CH), 115.9, 121.8, 127.6, 132.1, 132.2, 136.1, 140.3 (Ar-C), 151.9 (C=N), 154.4 (C=O), 155.4 (C=O), 156.5 (C-OH), 164.8, 165.1, 168.9 (C=O); MS: m/z 523 (M+). 2-(1-(4-(1-Acetyl-4,5-dihydro-5-(2-hydroxyphenyl)-1H-pyrazol-3-yl)phenyl)-3-methyl-5-oxo-1H-1, 2,4-triazol-4(5H)-yl)isoindoline-1,3-dione, 5h: Pale yellow solid; IR (KBr): 3440, 2967, 2927, 2846 (CH), 1807, 1740, 1642 (C=O), 1600 (C=N) cm-1; 1H NMR (CDCl3 d6): δ 2.20 (s, 3H, CH3), 2.56 (s, 3H, CH3), 3.50 (dd, 1H, CH2), 4.25 (dd, 1H, CH2), 4.32 (m, 1H, CH), 5.49 (s, 1H, OH), 7.25-7.92 (m, 12H, Ar-H); 13C NMR (CDCl3): δ 23.5, 25.5 (CH3), 41.9 (CH2), 58.9 (CH), 115.5, 121.9, 127.4, 132.4, 132.8, 136.3, 140.8 (Ar-C), 152.1 (C=N), 154.6 (C=O), 155.7 (C=O), 156.9 (C-OH), 164.9, 165.3, 169.0 (C=O); MS: m/z 523 (M+). 2-(1-(4-(1-Acetyl-4,5-dihydro-5-(4-nitrophenyl)1H-pyrazol-3-yl)phenyl)-3-methyl-5-oxo-1H-1,2,4triazol-4(5H)-yl)isoindoline-1,3-dione, 5i: Pale brown solid; IR (KBr): 2963.8 (CH), 1747, 1715, 1646 (C=O), 1607 (C=N) cm-1; 1H NMR (DMSO): δ 2.09 (s, 3H, CH3), 2.51 (s, 3H, CH3), 3.37 (dd, 1H, CH2), 3.83 (dd, 1H, CH2), 4.24 (m, 1H, CH), 7.218.24 (m, 12H, Ar-H); 13C NMR (DMSO): δ 38.63, 38.84 (CH3), 39.05 (CH2), 39.88 (CH), 106.17, 114.63, 125.54, 125.61, 129.23, 129.89, 129.98 (ArC), 130.23 (C=N), 132.35, 133.07 (C=O), 133.24 (CNO2), 139.84, 148.85, 163.85 (C=O); MS: m/z 551 (M+). 2-(1-(4-(1-Acetyl-4,5-dihydro-5-(4-methoxyphenyl)-1H-pyrazol-3-yl)phenyl)-3-methyl-5-oxo-1H1,2,4-triazol-4(5H)-yl)isoindoline-1,3-dione, 5j: Brown solid; IR (KBr): 2966, 2923, 2854, 2300 (CH), 1739, 1725, 1644 (C=O), 1606 (C=N) cm-1; 1H NMR (CDCl3): δ 2.30 (s, CH3), 2.42 (s, CH3), 3.39 (dd, CH2), 3.77 (s, -OCH3), 3.86 (dd, CH2), 4.32 (m, CH), 6.85-8.05 (m, Ar-H); 13C NMR (CDCl3): δ 23.20, 25.35 (CH3), 41.9 (CH2), 55.90 (-OCH3), 58.9 (CH), 114.93, 122.82, 126.64, 131.14, 131.25, 135.10, 141.30 (Ar-C), 150.9 (C=N), 155.40 (C=O), 154.40 (C=O), 156.52 (C-OH), 164.91, 165.16, 168.94 (C=O); MS: m/z 536 (M+). Biological evaluation assays DNA Cleavage analysis36 Culture media Nutrient broth was used for the growth of the organism. The media (50 mL) was prepared, autoclaved for 15 min at 121°C, 15 lb pressure. The autoclaved media were inoculated with the seed culture and incubated at 37°C for 24 hr. Isolation of DNA DNA was isolated using the procedure mentioned below. The bacterial culture (1.5 mL) was centrifuged to obtain the pellet. The pellet was then dissolved in lysis buffer (0.5 mL, 100 mM tris pH 8.0, 50 mM EDTA, 50 mM lysozyme) followed by addition of saturated phenol (0.50 mL) and incubated at 55°C for 10 min. This mixture was centrifuged at 10,000 rpm for 10 min and to the supernatant liquid equal volume of chloroform: isoamyl alcohol (24:1) and 1/20th volume of 3M sodium acetate (pH 4.8) were added. Then the mixture was centrifuged at 10,000 rpm for 10 min and to the supernatant liquid three volumes of chilled absolute alcohol was added. The precipitated DNA was separated by centrifugation. The pellet was dried and dissolved in Tris buffer (10 mM tris pH 8.0) and stored under cold condition. Treatment of DNA with the samples The synthesized compounds (100 µg) were added separately to the DNA sample. The sample mixtures were incubated at 37°C for 2 hr. Agarose gel electrophoresis Following the treatment of DNA samples, the electrophoresis of the samples was done according to the following procedure. Agarose (200 mg) was dissolved in TAE buffer (25 mL) (4.84 g Tris base, pH 8.0, 0.5 M EDTA/1 L) by boiling. When the gel attained ≈ 55°C, it was poured into the gel cassette fitted with comb. The gel was then allowed to solidify. The comb was carefully TAJ et al.: SYNTHESIS OF 1,2,4-TRIAZOLIN-3-ONE removed and the gel was placed in the electrophoresis chamber flooded with TAE buffer. DNA sample (20 µL, mixed with bromophenol blue dye at 1:1 ratio), was loaded carefully into the wells, along with standard DNA marker and constant 50 V of electricity was passed for around 45 min. The gel was removed and carefully stained with ETBR solution (10 µg/mL) for 10-15 min and the bands were observed under UV transilluminator. Anti-oxidant analysis37 Various concentrations of samples (10µg, 50µg, 100µg) were mixed with 200 mM sodium phosphate buffer (2.5 mL, pH 6.6) and potassium ferricyanide (1%, 2.5 mL). The mixture was incubated at 50ºC for 20 min. Trichloroacetic acid. (10%, 2.5 mL) (w/v) were added; above solution (5.0 mL) was mixed with distilled water (5.0 ml) and ferric chloride (0.1%, 1 mL). The absorbance was measured spectrophotometrically at 700 nm. BHA was used as standard antioxidant. Conclusions A series of novel fluoren-9-ylidene 5a-e and isoindolin-3,4-dione 5a-j with ease under neat conditions were prepared and analysed for their drug likeliness, drug score values. Further, these results were substantiated by DNA cleavage and antioxidant property. In case of DNA cleavage activity, the compounds 5i and 5j cleaved DNA completely. Antioxidant activity revealed the potent activity of the compounds 5c, 5g and 5j as compared to BHA. Acknowledgements The authors are thankful to the University Scientific Instrumentation Centre (USIC), K U Dharwad, for providing spectral as well as analytical data and to Prof. (Mrs) Bharathi V. Badami for the encouragement. One of the authors (TT) thanks UGC, New Delhi for the award of RFSMS fellowship to carry out the present work. 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