Synthesis and Evaluation of Antioxidant Activities of Some Novel Isatin ...

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Asian J. Res. Pharm. Sci. 2011; Vol. 1: Issue 4, Pg 140-143

ISSN- 2231–5640 (Print) www.asianpharmaonline.org ISSN- 2231–5659 (Online) 0974-3618 RESEARCH ARTICLE

Synthesis and Evaluation of Antioxidant Activities of Some Novel Isatin Derivatives and Analogs C.R. Prakash1*, S. Raja1, G. Saravanan2, P. Dinesh Kumar3 and T. Panneer Selvam4 1

Department of Pharmaceutical Chemistry, DCRM Pharmacy College, Inkollu. Andhra Pradesh, India. Medicinal Chemistry Research Laboratory, Bapatla College of Pharmacy, Bapatla-522 101, (A.P), India. 3 Dep. of Pharmaceutics, Rahul Institute of Pharmaceutical Science and Research, Chirala-523157, (A.P.), India. 4 Department Pharmaceutical Chemistry, Srinivas College of Pharmacy, Mangalore-574142, Karnataka, India. *Corresponding Author E-mail: 2

ABSTRACT:

In the present study, a series of novel Schiff bases of isatin were synthesized by condensation of imesatin with different aromatic aldehydes. The imesatins were synthesized by reaction of isatin with p-phenylenediamine. The chemical structures of the synthesized compounds were confirmed by means of IR, 1H-NMR, mass spectroscopy, and elemental analysis. These compounds were screened for antioxidant activity by DPPH radical scavenging activity. In this method, the compound 3-(4-(4-dimethylaminobenzylideneamino) phenylimino) indoline-2-one (5c) showed highest antioxidant activity because of the presence of electron donating group.

KEYWORDS: antioxidant, isatin; Schiff base. 1. INTRODUCTION:

The aerobic organisms require oxygen to survive. However, during normal metabolism oxygen produces reactive oxygen species such as free radicals and related reactants, or oxidants for brevity, some of which are highly toxic and deleterious for cells and tissues. The oxidants that are not directly scavenged, or in other words not metabolized, attack cellular components producing harmful molecular debris and sometimes causing cellular death (B. Halliwell 1999) to protect the cells from the damage caused by oxidants, the organisms have evolved several antioxidant defense mechanisms for rapid and efficient removal of reactive oxygen species from the intracellular environment. In normal circumstances, there is a balance between antioxidants and oxidants. When the equilibrium between oxidants and antioxidant defense systems is imbalanced in favor of the oxidants, the condition is known as oxidative stress (B. Halliwell 1999).

Received on 13.11.2011 Accepted on 10.12.2011 © Asian Pharma Press All Right Reserved

Asian J. Res. Pharm. Sci. 1(4): Oct.-Dec. 2011; Page 140-143

There is abundant evidence that the oxidative stress triggers various undesired processes at cellular, tissue and organism levels and plays a major role in the pathogenesis of many human diseases like ischemia/reperfusion syndrome, atherosclerosis, chronic renal failure (CRF), etc. It has been found that the oxidative stress plays a key role in the development of various complications during continual hemodialysis (HD) therapy of CRF patients (J. Herrera 2001 and G. Sener 2004). Antioxidants play a significant role in several important biological processes such as immunity, protect ion against tissue damage, reproduction and growth or development. They preserve adequate function of cells against homeostatic disturbances such as those caused by septic shock, aging and, in general, processes involving oxidative stress. These substances are classified according to their mode of action. Important antioxidants include the chainbreaking or scavenging substances (vitamins E, C and A, bilirubin), preventative (albumin, lactoferrin, haptoglobin) and enzyme antioxidants (catalase and glutathione peroxidase) (V.M. Victor 2006). They reduce damage to cells and biochemicals caused by free radicals, which are normal products of metabolism. Antioxidants can prevent cardiovascular disease, cancer, cataracts and various other ailments associated with aging (K. Sudha 2004 and B.

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Halliwell 2002). The studies suggest that supplementation with antioxidants may be useful in the prevention and treatment of Parkinson’s disease (K. Asplund 2002 and K.N. Prasad 1999). Oxidative stress is also important in the pathogenesis of Alzheimer’s disease. The studies suggest that supplementation with vitamin E might delay the development of Alzheimer’s disease (A. Kontush 2004 C.J. Foy 1999).

for 8 h. After standing for approximately 24 – 48 h at room temperature the product of different substituted derivatives of isatin (5a-5f) which separated out as a mixture of isomers was filtered, dried and recrystallised from absolute ethanol. Scheme - 2

HON

CCl3CHO.H2O

2. MATERIALS AND METHODS:

1

2.1 Materials: The melting points were taken in open capillary tube and are uncorrected. The IR spectra of the compounds were recorded on ABB Bomem FT-IR spectrometer MB 104 with KBr pellets. The 1H (400 MHz) spectra were recorded on a Bruker 400 NMR spectrometer (with TMS as internal references). Mass spectroscopy was recorded on Shimadzu GC MS QP 5000. Microanalyses were obtained with an elemental analyses system GmbH VarioEL V300 element analyzer. The purity of the compounds was checked by TLC on pre-coated SiO2 gel (HF254, 200 mesh) aluminium plates (E Merk) using ethyl acetate: n-hexane (2:3) and visualized in UV chamber. IR, 1H-NMR, 13C-NMR, Mass spectroscopy and elemental analysis were consistent with the assigned structures.

NH2

NH2OH.HCl Na2SO4

N 2H 60-800C

NH2 CH3COOH C2H5OH

H 2N

N

N H 4

4

N

O2N

NH

RH NO2

O2N

9

5

RO

6

8

7

NO2 6" R=

NO2 DPPH 517 nm

NO2

HC

HC

3

1''

Equimolar quantities of (0.01 mol) of isatin and p – Phenylenediamine, were dissolved in sufficient quantity of methanol in presence of acetic acid and refluxed for 1 h then kept aside for 2 h, the product which separated out was filtered, dried and recrystallised from absolute ethanol. Equimolar quantities (0.01 mol) of imesatin 4 and various aromatic aldehydes were dissolved in ethanol and refluxed

O

R-CHO

N

6'

5'

2'

3'

1' 4'

N

C H

R

N 2 O H 1 ( 5a-5f ) CH 3

,

Cl

3''

OCH3

N

,

CH 3

5c

5b

5a

5d

NH2

5'' 4''

2''

2.2 Synthetic methods: In the present study, aniline 1 was treated with chloral hydrate to form isonitrosoacetanilide 2. Then this intermediate undergoes to cyclization with sulphuric acid to form isatin 3 (C.S.Marvel, G.S. Hiers 1941). Which further reacted with p – Phenylenediamine, resulting in the formation of imesatin 4 [S.K.Sridhar, A.Ramesh 2001). The compound 4 was subjected to react with various aromatic aldehydes in presence of ethanol as a solvent to form Schiff bases (5a-5f) Scheme–2. All the synthesized compounds were soluble in dimethylformamide (DMF).

O

N H 3

C2H5OH

N0

Con.H2SO4 O

Scheme – 1

N

O

,

, HO

5e

CH3

5f

2.3 Antioxidant Method: 2.3.1 DPPH radical assay: A total antioxidant capacity assay was carried out using DPPH as radical. The experimental procedure was adapted from the literature, only with slight modification (N. Nenadis 2002 and A. Torres 2007). Briefly, 2,2-diphenyl-1picrylhydrazyl (DPPH) radical in ethanol (250 mM, 2 mL) was added to 2 mL of an ethanolic solution of the test compounds. The final concentration of the test compounds in the reaction mixtures was 50 mM. Each mixture was then shaken vigorously and held for 30 min at room temperature in the dark. The decrease in absorbance of DPPH at 517 nm was then measured. Ethanol was used as a blank and a DPPH solution (2 mL) in ethanol (2 mL) as the control solution. All tests were performed in triplicate.

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3. RESULTS AND DISCUSSION:

3.1.3. 3-(4-(4-dimethylaminobenzylideneamino) phenylimino) indoline-2-one 5c: Yellow crystals; Yield: 80%; mp. 322-324 °C; IR : 3150 (N-H), 3055 (Ar-CH), 3019 (C-H), 1698 (C=O), 1613 (C=C), 1568 (C=N) cm-1; 1H-NMR(DMSO): 8.21 (s, 1H, -N=CH-), 8.02 (s, 1H, -NH-), 7.42 (dd, J=5.9 Hz, 2H, H-2'' and H-6'' Ar-H), 7.03-7.68 (m, 8H, H-4, H-5, H-6, H-7, H2', H-3', H-5', H-6' Ar-H), 6.61 (dd, J=7.2 Hz, 2H, H-3'' , H5'' Ar-H), 2.85 (s, 6H, -N[CH3]2); EI-MS (m/z, %): 368(M+, 6), 324(14), 242(38), 133(100), 91(20). (Calcd. for C23H20N4O: 368.43); Anal. Calcd. C23H20N4O: C, 74.98: H, 5.47; N, 15.21; Found: C, 74.95; H, 5.49; N, 15.22.

3.1. Chemistry: 3.1.1. 3-(4-(3-phenylallylideneamino) phenylimino) indoline-2-one 5a: Creamy crystals; Yield: 67%; mp. 310-312 °C; IR : 3168 (N-H), 3090 (Ar-CH), 1700 (C=O), 1591 (C=N), 1498 (C=C) cm-1; 1H-NMR (DMSO): 8.01 (s, 1H, -NH-), 7.51 (s, 1H, -N=CH-), 6.99-7.32 (m, 13H, H-4, H-5, H-6, H-7, H-2', H-3', H-5', H-6', H-2'', H-3'', H-4'', H-5'', H-6'' Ar-H), 6.62 (d, 1H, J=7.1 Hz; C6H5-CH=CH-), 5.63 (d, 1H, J=8.2 Hz, C6H5-CH=CH-); EI-MS (m/z, %): 351(M+,26), 300(24), 243(10), 221(8), 179(18), 109(100), 60(32); (Calcd. for C23H17N3O: 351.40); Anal. Calcd. for C23H17N3O: C, 78.61; H, 4.88; N, 11.96; Found: C, 78.59; 3.1.4. 3-(4-(4-methoxybenzylideneamino) phenylimino) indoline-2-one 5d: H, 4.85; N, 11.90. Lemon yellow crystals; Yield: 79%; mp. 326-328 °C; IR: (C=O), 1647 (C=C), 1567 3.1.2. 3-(4-(4-chlorobenzylideneamino) phenylimino) 3146 (N-H), 3079 (Ar-CH),-1 1688 (C=N), 1270 (C-O-C) cm ; 1H-NMR (DMSO): 8.39 (s, indoline-2-one 5b: Pale yellow crystals; Yield: 75%; mp. 346-348 °C; IR : 1H, -N=CH-), 8.01(s, 1H, -NH-), 7.51(d, J=6.3 HZ, 1H, C-6'' 6.99-7.31 (m, 3130 (N-H), 2988 (Ar-CH), 1613 (C=N), 1700 (C=O), 1599 Ar-H), 7.47 (d, J=5.9 Hz, 1H, C-2'' Ar-H), ' (C=C), 744 (C-Cl) cm-1; 1H-NMR (DMSO): 8.25 (s, 1H, - 8H, H-4, H-5, H-6, H-7, H-2', H-3', H-5 , H-6' Ar-H), 6.81 N=CH-), 7.92 (s, 1H, -NH-), 7.03-7.60 (m, 12H, H-4, H-5, (d, J=7.2 Hz, 1H, H-5'' Ar-H), 6.77(d, J=6.5 Hz, 1H,+ H-3'' H-6, H-7, H-2', H-3', H-5', H-6', H-2'', H-3'', H-5'', H-6'', Ar- Ar-H), 3.70 (s, 3H, -OCH3); EI-MS (m/z, %): 355(M , 18), H); EI-MS (m/z, %): 362(M+2), 360(M+, 20), 264(22), 282(20), 121(100), 91(42), 55(94); (Calcd. for C22H17N3O2: 91(100), 77(22), 69(44); (Calcd. for C21H14ClN3O: 359.80); 355.38); Anal. Calcd. for C22H17N3O2: C, 74.35; H, 4.82; N, Anal. Calcd. for C21H14ClN3O: C, 70.10; H, 3.92; N, 11.68; 11.82; Found: C, 74.36; H, 4.80; N, 11.78. Found: C, 70.15; H, 3.95; N, 11.72.

120

100

200 µg/ml

% Inhibition

80

400 µg/ml 60

600 µg/ml 800 µg/ml

40

1000 µg/ml

20

0 Compound Compound Compound Compound Compound Compound Standard5a 5b 5c 5d 5e 5f Vitamin C Figure - 1 Radical scavenging activity of synthesized compound against DPPH test

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3.1.5. 3-(4-(2-hydroxybenzylideneamino) phenylimino) indoline-2-one 5e: Creamy crystals; Yield: 73%; mp. 318-320 °C; IR : 3467(Ar-OH), 3210 (N-H), 3065 (Ar-CH), 1678 (C=O), 1649 (C=C), 1575 (C=N) cm-1; 1H-NMR (DMSO): 8.22 (s, 1H, -N=CH-), 7.06-7.67 (m, 8H, H-4, H-5, H-6, H-7, H2', H-3', H-5', H-6' Ar-H), 6.75-7.40 (m, 4H, H-3'', H-4'', H5'' and H-6'' Ar-H), 6.01 (s, 1H, -NH-), 5.20 (s,1H, ArOH); EI-MS (m/z, %): 341(M+, 36), 282(6), 242(34), 131(100), 89(26), 77(30). (Calcd. for C21H15N3O2: 341.36); Anal. Calcd. for C21H15N3O2: C, 73.89; H, 4.43; N, 12.31; Found: C, 73.91; H, 4.45; N, 12.35.

at three different concentrations at 20–60 min. Antioxidants can react with DPPH and produce 1,1-diphenyl-2-picrylhydrazine scheme -1 (M.S. Blois 1958). Due to its odd electron DPPH give s a strong absorption band at 517 nm. As this electron becomes paired off in the presence of a free radical scavenger, the absorption vanishes and the resulting decolorization is stoichiometric with respect to the number of electrons taken up. The change of absorbance produced in this reaction is assessed to evaluate the antioxidant potential of test samples and this assay is useful as a primary screening system.

Generally, electron donating groups have a good capability to catch free radicals by themselves. The highest scavenger activity observed in compound 5c is probably due to the presence of dimethyl groups at position 4 in aromatic ring. The moderate activity of compound 5d and 5f due to presence of methoxy and methyl group, which is also present at p- position in the aromatic ring, has high electron-releasing properties (Positive mesomeric effect is higher than negative inductive effect) and it activates aromatic ring. Generally halogens groups are electron withdrawing substituents, they deactivate aromatic ring and have no capability to bind the free radicals. So the least activity was observed in compound 5e and 5b because The IR, 1H-NMR, 13C-NMR, Mass spectroscopy and presence of hydroxyl group in o- position and electron Elemental analysis for the new compound is in accordance withdrawing chloro group in p-position respectively with the assigned structures. The IR spectra of all (Figure - 1). synthesized compounds show bands at 3150-3245 cm-1, 1680-1700 cm-1 and weak bond at 1600-1630 cm-1 which 4. REFERENCES: can be assignable to N-H, C=O and C=N (azomethine 1. A .Torres de Pinedo, P. Penalver, J.C. Morales, Food Chem. 103 (2007) 55–61. linkage) vibrations of the isatin ring respectively. The proton magnetic resonance spectra of imesatin and their 2. Kaur, C.; Kapoor, H. C. Antioxidants in fruits and vegetablessthe millennium’s health. Int. J. Food Sci. Technol. (2001), 703-725. corresponding Schiff base derivatives have been recorded in 3. B. Halliwell, Drugs Aging 18 (2001) 685e716; Acc. Chem. Abstr. DMSO-d6. The following conclusions can be derived by 137 (2002) 15048t. comparing the spectra of imesatin and their corresponding 4. B. Halliwell, J.M.C. Gutteridge, Free Radicals in Biology and Medicine, third ed. Oxford University Press, Oxford, (1999). Schiff base. (a) The signal because of N-H group of the isatin ring at appear 8.0 in the spectra of imesatin and 5. C.J. Foy, A.P. Passmore, M.D. Vahidassr, I.S. Young, J.T. Lawson, Q. J.Med. 92 (1999) 39-45. their corresponding Schiff base derivative.(b) Imesatin and 6. C.S. Marvel,G.S. Hiers, Organic syntheses, Coll. Vol. 1 (1941) 327their corresponding Schiff base derivatives show a multiplet 330. for the aromatic ring at 6.99-7.70. (c) A signal because of 7. G. Sener, K. Paskaloglu, H. Toklu, C. Kapucu, G. Ayanoglu-Dulger, A . Kacmaz, A . Sakarcan, J. Pineal Res. 36 (2004) 232–241. N=CH appear at 7.51-8.39 in all the final compounds and absence of the same signal in imesatin clearly indicates the 8. J. Herrera, M. Nava, F. Romero, B. Rodri ´ guez-Iturbe, Am. J. Kidney Dis. 37 (2001) 750. formation of Schiff base through primary amino group of 9. K. Asplund, J. Intern. Med. 251 (2002) 372-392. imesatin. The EI-Mass Spectra of compounds showed 10. K. Sudha, A. Rao, S. Rao, A. Rao, Neurol. India 51 (2003) 60e62; molecular ions of different intensity which confirmed their Acc. Chem. Abstr. 141 (2004) 52141w. molecular weight. The major fragmentation pathway 11. K.N. Prasad, W.C. Cole, B. Kumar, J. Am. Coll. Nutr. 18 (5) (1999) 413-423. involved the cleavage of the endocyclic NH-CO bond of 12. Kontush, S. Schekatolina, Ann. N. Y. Acad. Sci. 1031 (1) (2004) isatin ring. 3.1.6. 3-(4-(4-methylbenzylideneamino) phenylimino) indoline-2-one 5f: Pale yellow crystals; Yield: 77%; mp. 320-322 °C; IR: 3198 (N-H), 3144 (Ar-CH), 1696 (C=O), 1618 (C=C), 1518 (C=N) cm-1; 1H-NMR(DMSO): 8.21 (s, 1H, -N=CH-), 8.01 (s, 1H, -NH-), 7.01-7.50 (m, 12H, H-4, H-5, H-6, H-7, H-2', H-3', H-5', H-6', H-2'',H-3'', H-5'', H-6'' Ar-H), 2.30 (s, 3H, -CH3); EI-MS (m/z, %): 339(M+, 28), 235(40), 222(80), 104(92), 55(100). (Calcd. for C22H17N3O: 339.38); Anal. Calcd. for C22H17N3O: C, 77.86; H, 5.05; N, 12.38; Found: C, 77.84; H, 5.09; N, 12.34.

3.2. Biological results: 3.2.1. DPPH free radicals scavenging activity: The chemical structure-scavenger activity relationship can be made. The antioxidant activity in general of isatin can be explained with the presence of enolic hydroxyl group at the second position due to keto-enol tautomerism between NH and C=O. The reducing abilities of the examined compounds were determined by their interaction with the free stable radical 1,1-diphenyl-2-picryl-hydrazyl (DPPH)

249-262. 13. M.S. Blois, Nature (London) 181 (1958) 1 199. 14. N. Nenadis, M. Tsimidou, J. Am. Oil Chem. Soc. 79 (2002) 1191– 1195. 15. S.K. Sridhar, A. Ramesh, Biol.Bull. 24(10) (2001) 1149-1152. 16. V.M. Victor, K.J. McCreath, M. Rocha, Recent Patents Anti-Infect. Drug Disc. 1 (2006) 17-31.

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