Chen et al. Chemistry Central Journal 2011, 5:37 http://journal.chemistrycentral.com/content/5/1/37 RESEARCH ARTICLE Open Access Simple isatin derivatives as free radical scavengers: Synthesis, biological evaluation and structure-activity relationship Gang Chen1, Ye Wang2, Xiaojiang Hao2*, Shuzhen Mu2 and Qianyun Sun2 Abstract To develop more potent small molecules with enhanced free radical scavenger properties, a series of N-substituted isatin derivatives was synthesized, and the cytoprotective effect on the apoptosis of PC12 cells induced by H2O2 was screened. All these compounds were found to be active, and N-ethyl isatin was found with the most potent activity of 69.7% protective effect on PC12 cells. Structure-activity relationship analyses showed the bioactivity of N- alkyl isatins decline as the increasing of the chain of the alkyl group, furthermore odd-even effect was found in the activity, which is interesting for further investigation. Background activity [13-15]. Isatin has a distinct and discontinuous Oxidative stress has been implicated as a major role in distribution in rat brain and other tissues; the highest the onset and progression of a vast variety of clinical concentrations in the brain are found in the hippocam- abnormalities including neurodegenerative disorders. pus and cerebellum [10]. Many Isatin derivatives, such Free radicals play important roles in many physiological as isatin hydrazono, isatin Mannich bases, isatin based and pathological conditions [1]. In general, the genera- spiroazetidinones and 3-(methylene)indolin-2-ones, have tion and scavenging of oxygen free radicals is balanced also been reported to possess neuroprotection activity and any imbalance or excessive amounts of active radi- [16-19]. cals may contribute to disease development. It has been To develop more potent small molecules with found that free radical reactions can produce deleterious enhanced free radical scavenger properties, a series of modifications in membranes, proteins, enzymes, and N-substituted isatin derivatives was synthesized by sub- DNA [2], increasing the risk of diseases such as cancer stitution reactions (as shown in Scheme 1), and the [3], Alzheimer’s[4],Parkinson’s [5], angiocardiopathy cytoprotective effect on the apoptosis of PC12 cells [6], arthritis [7], asthma [8], diabetes [9], and degenera- induced by H2O2 was screened. tive eye disease [10]. Therefore, it is important to find effective scavengers of free radicals for prevention and Results and Discussion treatment of such disorders. Chemistry Isatin is an endogenous indole present in mammalian The N-substituted isatin derivatives were synthesized by tissues and fluids [11]. The substance was initially dis- reactions of substitution reaction. The reaction between covered as a component of endogenous monoamine oxi- isatin and halohydrocarbon has been reported being car- dase (MAO) inhibitory activity, tribulin, and ried out in the presence of NaOEt using EtOH as sol- subsequently identified as a selective inhibitor of MAO vent or in the presence of NaH using DMF as solvent B [12]. Further investigations have shown that isatin [16]. The reactants and the solvents involved in the acts as an antagonist of both atrial natriuretic peptidesti- reactions must be anhydrous. To develop a simple mulated and nitric oxide-stimulated guanylate cyclase method to synthesize N-substituted isatin derivatives, we firstly screened the effect of the base and solvent on the * Correspondence: [email protected] yield of the reaction of isatin and bromoethane 2 Key Laboratory of Chemistry for Natural Products of Guizhou Province and (C H Br), and the results was shown in Table 1. Chinese Academy of Sciences, Guiyang 550002, PR China 2 5 Full list of author information is available at the end of the article © 2010 Chen et al Chen et al. Chemistry Central Journal 2011, 5:37 Page 2 of 5 http://journal.chemistrycentral.com/content/5/1/37 Table 2 Synthesis of N-substituted isatin derivatives Compound R Time (h) Yield (%) 1 H__ 2 CH3 487 Scheme 1 Synthesis of N-substituted isatin derivatives. 3 C2H5 12 89 4 (CH2)2CH3 12 89 5 (CH2)3CH3 24 93 In this reaction, the protons transfers from N-H (a 6 (CH2)4CH3 24 90 Brösted acid) to a Brösted or Lewis base via the hydro- 7 (CH2)5CH3 24 95 gen-bonded covalent and ionic complexes [20], produ- 8 CH2CH = CH2 12 93 cing the isatin anion which is the nucleophilic reactant 9 CH2C6H5 12 90 to the halohydrocarbon. Higher solvent polarity can pro- 10 CH2COOC2H5 12 93 mote the proton-transfer equilibrium and leads to the 11 C2H4Cl 24 79 higher yield [20]. From this table, it can be found that 12 C2H4Br 24 83 K2CO3-DMF system was an effective promotion for this reaction and other base-solvent systems were not effec- μ a tive with the yield no more than 60%. The possible rea- g/ml, which were more effective than VE ((±) -Toco- son might be that weak base can not help the proton phreol with the percentage of 22.5%). There is a note- transfer at the beginning effectively, but the too strong worthy phenomenon that the activities of all compounds μ bases will lead to the substitution reaction between bro- at the condensation of 2 g/ml are more potent than - μ moethane and OH . DMF exhibits the highest yield of that at the condensation of 20 g/ml, and the mechan- ism will be interesting for the further investigation. 89% with K2CO3 for its highest solvent polarity, so the Compound 3 and 8 exhibited the most potent activity K2CO3-DMF was selected as the reactant reaction sys- tem in the following synthesis, and the results were with the protective effect of 69.8% and 69.5% at the con- μ shown in Table 2. densation of 2 g/ml respectively, which are more potent than that at the condensation of 20 μg/ml. Bioactivity Almost all of these compounds were weakly cytotoxic μ The chemical modification of lead compound 1,focus- to PC12 cells at the concentrations of 2-20 g/ml except ing on the N-substituent, was carried out to further compound 11 and 12. Almost all compounds are cyto- μ improve the free scavenging ability. A series of new N- toxic to PC12 cells at the concentrations of 200 g/ml, substituted isatin derivatives (compounds 2-12)was the PC12 cells inhibitory effects are more than 40%. synthesized. The free radical scavenging properties of Based on the factors, we can conclude the addition of 11 these derivatives were evaluated to elucidate structure- halogenous atom in the substituents (compound and activity relationships. The protective effect on the apop- tosis of PC12 cells induced by H2O2 by free radical Table 3 Inhibitory and protective effects of N-substituted scavenging of these compounds against H2O2 were eval- isatin derivatives uated by cell survival assay in PC12 cells using a a b Compound Inhibitory effect/% Protective effect/% reported method [21]. The results were given in Table 3. 200 20 2 200 20 2 From the table, we can find almost all of the com- μg/ml μg/ml μg/ml μg/ml μg/ml μg/ml pounds showed potent activity at the condensation of 2 1 92.0 0.0 2.6 - 20.1 40.4 2 60.6 2.7 5.0 - 31.4 50.9 Table 1 The substitution reaction between isatin and 3 45.6 0.4 7.0 - 38.2 69.8 bromoethane 4 43.0 3.3 0.0 - 10.1 39.7 No. Solvent Base Time (h) Yield (%) 5 53.3 0.0 0.0 - 30.0 60.8 6 51.9 0.0 0.0 - 8.2 24.1 1 DMF Na2CO3 24 33 7 50.4 0.0 0.0 0 15.5 20.9 2 DMF K2CO3 12 89 8 54.0 2.1 0.0 - 14.8 69.5 3 DMF NaOH 12 40 9 63.1 0.0 0.0 - 9.3 51.3 4 DMF KOH 12 41 10 67.9 0.0 5.3 - 46.6 54.5 5 DMF TEA 24 60 11 63.5 20.5 5.6 - 0 25.1 6 MeOH K2CO3 24 12 12 61.6 24.1 12.3 - 0 62.1 7 THF K2CO3 24 25 VE 0.0 22.5 8 DCM K2CO3 24 19 a) Inhibition of PC12 cell growth; b) protective effect on the apoptosis of 9 Acetonitrile K2CO3 24 15 PC12 cells induced by H2O2. Chen et al. Chemistry Central Journal 2011, 5:37 Page 3 of 5 http://journal.chemistrycentral.com/content/5/1/37 12) enhance the cytotoxicity at the concentrations of 2- Experimental 20 μg/ml. All starting materials and solvents (A.R. grade) were The substitution reaction between isatin and halohy- commercially available and were used without further drocarbon (C1 to C6) gave compounds 2-7, which pro- purification. NMR spectra were recorded using a Bruker vided the appropriate material for the structure-activity Drx-400 spectrometer operating at 400 MHz for 1H. relationship analyses. The cytoprotective activities of N- Mass spectra were recorded on a Micromass Platform substituted isatin derivatives with the alkyl group con- spectrometer using a direct-inlet system operating in the taining one to six carbon atoms were shown in Figure 1. electron impact (EI) mode at 75 eV. Elemental analyses Theactivityapproximatelydeclinesastheincreaseof were obtained using a Carlo Erba 1106 elemental the chain of the alkyl group. With a further analysis, it analyzer. was found that there was a clear odd-even effect in General synthesis of N-alkyl substituted isatin derivatives these activities. The activities of N-substituted isatin Isatin (1 mmol) and halohydrocarbon (1.2 mmol) were derivatives with odd carbon atoms alkyl group (one, dissolved in DMF (20 ml), and 3 mmol anhydrous three and five carbon atoms, corresponding compound K2CO3 was added.