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

¹Department of Pharmaceutical Chemistry, DCRM Pharmacy College, Inkollu. Andhra Pradesh, India.

²Medicinal Chemistry Research Laboratory, Bapatla College of Pharmacy, Bapatla-522 101, (A.P), India.

³Dep. of Pharmaceutics, Rahul Institute of Pharmaceutical Science and Research, Chirala-523157, (A.P.), India.

⁴Department Pharmaceutical Chemistry, Srinivas College of Pharmacy, Mangalore-574142, Karnataka, India.

*Corresponding Author E-mail:

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).

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 chain-breaking 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. 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).

2. MATERIALS AND METHODS:

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.

Scheme – 1

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).

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 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

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-1-picrylhydrazyl (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.

3. RESULTS AND DISCUSSION:

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): δ 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; H, 4.85; N, 11.90.

3.1.2. 3-(4-(4-chlorobenzylideneamino) phenylimino) indoline-2-one 5b:

Pale yellow crystals; Yield: 75%; mp. 346-348 °C; IR : 3130 (N-H), 2988 (Ar-CH), 1613 (C=N), 1700 (C=O), 1599 (C=C), 744 (C-Cl) cm^-1; ¹H-NMR (DMSO): δ 8.25 (s, 1H, -N=CH-), 7.92 (s, 1H, -NH-), 7.03-7.60 (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); EI-MS (m/z, %): 362(M+2), 360(M⁺, 20), 264(22), 91(100), 77(22), 69(44); (Calcd. for C₂₁H₁₄ClN₃O: 359.80); Anal. Calcd. for C₂₁H₁₄ClN₃O: C, 70.10; H, 3.92; N, 11.68; Found: C, 70.15; H, 3.95; N, 11.72.

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): δ 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, H-2', H-3', H-5', H-6' Ar-H), 6.61 (dd, J=7.2 Hz, 2H, H-3'', H-5''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.

3.1.4. 3-(4-(4-methoxybenzylideneamino) phenylimino) indoline-2-one 5d:

Lemon yellow crystals; Yield: 79%; mp. 326-328 °C; IR: 3146 (N-H), 3079 (Ar-CH), 1688 (C=O), 1647 (C=C), 1567 (C=N), 1270 (C-O-C) cm^-1; ¹H-NMR (DMSO): δ 8.39 (s, 1H, -N=CH-), 8.01(s, 1H, -NH-), 7.51(d, J=6.3 H_Z,1H, C-6'' Ar-H), 7.47 (d, J=5.9 Hz_,1H, C-2'' Ar-H), 6.99-7.31 (m, 8H, H-4, H-5_,H-6, H-7, H-2', H-3', H-5^', H-6' Ar-H), 6.81 (d, J=7.2 Hz_,1H, H-5'' Ar-H), 6.77(d, J=6.5 Hz, 1H, H-3'' Ar-H), 3.70 (s, 3H, -OCH₃); EI-MS (m/z, %): 355(M⁺, 18), 282(20), 121(100), 91(42), 55(94); (Calcd. for C₂₂H₁₇N₃O₂: 355.38); Anal. Calcd. for C₂₂H₁₇N₃O₂: C, 74.35; H, 4.82; N, 11.82; Found: C, 74.36; H, 4.80; N, 11.78.

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): δ 8.22 (s, 1H, -N=CH-), 7.06-7.67 (m, 8H, H-4, H-5_,H-6, H-7, H-2', H-3', H-5^', H-6' Ar-H), 6.75-7.40 (m, 4H, H-3'', H-4'', H-5'' and H-6'' Ar-H), 6.01 (s, 1H, -NH-), 5.20 (s,1H, Ar-OH); 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.

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): δ 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.

The IR, ¹H-NMR, ¹³C-NMR, Mass spectroscopy and Elemental analysis for the new compound is in accordance with the assigned structures. The IR spectra of all synthesized compounds show bands at 3150-3245 cm^-1, 1680-1700 cm^-1 and weak bond at 1600-1630 cm^-1 which can be assignable to N-H, C=O and C=N (azomethine linkage) vibrations of the isatin ring respectively. The proton magnetic resonance spectra of imesatin and their corresponding Schiff base derivatives have been recorded in DMSO-d6. The following conclusions can be derived by comparing the spectra of imesatin and their corresponding Schiff base. (a) The signal because of N-H group of the isatin ring at appear δ 8.0 in the spectra of imesatin and their corresponding Schiff base derivative.(b) Imesatin and their corresponding Schiff base derivatives show a multiplet for the aromatic ring at δ 6.99-7.70. (c) A signal because of N=CH appear at δ 7.51-8.39 in all the final compounds and absence of the same signal in imesatin clearly indicates the formation of Schiff base through primary amino group of imesatin. The EI-Mass Spectra of compounds showed molecular ions of different intensity which confirmed their molecular weight. The major fragmentation pathway involved the cleavage of the endocyclic NH-CO bond of isatin ring.

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) at three different concentrations at 20–60 min. Antioxidants can react with DPPH and produce 1,1-diphenyl-2-picryl-hydrazine 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 presence of hydroxyl group in o- position and electron withdrawing chloro group in p-position respectively (Figure - 1).

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Received on 13.11.2011 Accepted on 10.12.2011

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