Synthesis of isatin , 5-chloroisatin and their ∆ 2-1 , 3 , 4 oxadiazoline derivatives for comparative cytotoxicity study on brine shrimp

Isatin (3a), isatin 3-carbohydrazone (4a), 5-spiro (isatin) 2-(N-acetyl hydrazino) 4-(N-acetyl)-∆2-1,3,4 oxadiazoline (5a), and 5-spiro (isatin) 2-hydrazino∆2-1,3,4 oxadiazoline (6a) had been synthesized from the unsubstituted oximinoacetanilide (2a). 4-Chlorooximinoacetanilide (2b), 5-chloroisatin (3b), 5-chloroisatin 3-carbohydrazone (4b) and 5-spiro (5-chloroisatin) 2-(N-acetyl hydrazino) 4-N-acetyl ∆2-1,3,4 oxadiazoline (5b) compounds had been synthesized from p-chloroaniline. The structures of the products had been characterized from the spectral analysis and comparative cytotoxicity study of them was studied. Article Info Received: 15 December 2006 Accepted: 1 February 2007 Available Online: 3 January 2008 DOI: 10.3329/bjp.v2i1.494 Cite this article: Islam MR, Mohsin M. Synthesis of isatin, 5-chloroisatin and their ∆2-1, 3, 4 oxadiazoline derivatives for comparative cytotoxicity study on brine shrimp. Bangladesh J Pharmacol. 2007; 2: 7-12. Synthesis of isatin, 5-chloroisatin and their ∆2-1, 3, 4 oxadiazoline derivatives for comparative cytotoxicity study on brine shrimp Md. Rabiul Islam and Mohammad Mohsin Department of Chemistry, Jahangirnagar University, Savar, Dhaka 1342, Bangladesh. This work is licensed under a Creative Commons Attribution 4.0 License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor. (14.7 mL) with dry oximinoacetanilide (4 g; 0.024 mole) at 70-80°C the solution was cooled to room temperature and poured upon 10-12 times of its volume of crushed ice. The orange red crude solid product of 3a was separated from the solution after half an hour which was filtered, washed well with cold water and dried in a vacuum desiccators. The orange red pure product of 3a was re-crystallized from ethyl acetate having m.p. 180-182°C, yield 2.3 g (65%); Rf 0.4 (PE : EA ; 3 : 2). IR: : 3189 (br,nNH; amide), 3106 (w,nCH, aromatic), 1726 (sh,nC=O, keto), 1614 (sh,nC=O, lactam), 1460 (sh,nC-C, aromatic). 1H-NMR (DMSO-d6): d ppm: 11.02 (s, 1H; NH); 6.90 (d, J=7.8 Hz, 1H, C1-H); 7.12 (t, 1H, C2-H); 7.56 (t, 1H; C3) ( 1 max  cm KBr  8 Bangladesh J Pharmacol 2007; 2: 7-12


Introduction
Isatin, possessing an indole nucleus having both the keto and lactam moiety has aroused tremendous curiosity due to its diverse biological and pharmacological studies.From literature survey it is well known that isatin heterocycles exhibit manifold importance in the field of medicinal chemistry as a potent chemotherapeutic agent.Recently Islam et. al., (1992, 2001), in collaboration with National Cancer Institute (NCI) of USA, it was observed that acylated ∆ 2 -1,3,4 thiadiazoline derivatives of isatin showed effective anti-cancer activity against a number of cancer cells especially for breast cancer.This eventful observation encouraged us to devotion of further research on isatin heterocycles reacting with carbohydrazide, especially for spiro 1, 3, 4 oxadiazoline derivatives.We report herein the synthesis of compounds shown in the scheme 1 following previous literature (Islam et al., 2001).The synthesized compounds were characterized by spectral analysis of IR, 1 H-NMR and mass spectrometry.The result of the systematic study on cytotoxicity of the synthesized compounds on brine shrimp has also been discussed.

Materials and Methods
The melting points of the synthesized compounds were recorded by thin disc method on a FISCHER JOHN'S electro-thermal melting point apparatus.TLC was used for the monitoring the progress of reactions.Infrared spectra were recorded on DR-8001, SHIMADZU FT-IR spectrophotometer as a solid which was finely grounded in a small agate mortar in KBr disc. 1 H-NMR spectra were measured by WP 200-NMR spectrometer using TMS (tetramethyl silane) as an internal standard and DMSO-d6 (dimethyle sulphoxide) as a solvent.Mass spectra were recorded on a high resolution mass spectrometer, KARATAS MS-25 using DH-88 data system.
Synthesis of isatin, 3a: Following the previous literature (Islam et al., 2001) oximinoacetanilide was prepared in 75% yield.After addition of concentrated sulphuric acid (14.7 mL) with dry oximinoacetanilide (4 g; 0.024 mole) at 70-80°C the solution was cooled to room temperature and poured upon 10-12 times of its volume of crushed ice.The orange red crude solid product of 3a was separated from the solution after half an hour which was filtered, washed well with cold water and dried in a vacuum desiccators.The orange red pure product of 3a was re-crystallized from ethyl acetate having m.p. 180-182°C, yield 2.3 g (65%); Rf 0.4 (PE : EA ; 3 : 2).

Demonstration of cytotoxicity:
The cytotoxicity study of the synthesized compounds -3a, 4a, 5a, 6a, 2b, 3b, 4b and 5b was investigated on brine shrimp as a test organism for convenience.1.6 mg of each of the compounds was taken in the corresponding sample vials with 1.6 mL of dimethyl sulfoxide to prepare stock solution.From this stock solution 33, 99 and 132 ppm of each compounds were placed in separate test tubes by micro syringe 1 mL of extra dimethyl sulfoxide was given in each test tube with 10-12 brine shrimp (Microwell, 1997).After 1, 2, 3 and 4 hours the test tubes were observed and the number of survived naupli in each test tube was counted and results were noted.From this the percentage of lethality of brine shrimp naupli was calculated at each concentration for each sample.Then graphs were drawn by plotting percentage of lethality of brine shrimp versus doses (in ppm) of the synthesized compounds which gave rise to the LD50 values of the corresponding compounds.
The LD50 values of the synthesized compounds are given in Table I.
Reaction of 3a with carbohydrazide in glacial acetic acid gave 4a, whose mass spectrum showed molecular ion peak at m/z 219 which is consistent with the structure of the compound.Acetylation of 4a with acetic anhydride afforded 5a.IR spectrum of 4a indicates the presences of acetyl group (-COCH3) by the band appearing at 1785 cm -1 . 1 H-NMR spectrum confirms the presence of two acetyl groups by two singlets at d 2.2 for NH-COCH3 and at d 2.6 due to -NCOCH3.The expected molecular ion peak (M + ) was not found due to less of fifteen mass unit.Two NH protons were not observed in the 1 H-NMR spectrum due to rapid proton-deuteron exchange reaction in deuterated dimethyl sulfoxide solvent.
Reduction of 5a by hydrazine hydrate stirring at room temperature afforded 6a.The IR spectrum indicated the absence of any acetyl group which confirmed the reduction of 5a has taken place.The bands at 3358 cm -1 and 1686 cm -1 indicate the presence of NH and C=O (lactam) groups respectively.In 1 H-NMR a broad singlet appears at d11.84 due to NH proton of lactam, whereas a sharp singlet at d 11.28 and a broad singlet at d 10.84 indicate the presence of NH proton of N2-H and NH proton of N3-H respectively.No signal of two NH2 protons was observed due to rapid proton-deuteron exchange reaction in deuterated dimethyl sulfoxide solvent.In mass spectrum the molecular ion peak M + was not observed due to loss of fifteen mass unit from 6a instead the peak appearing at m/z 204 (M + -15) is consistent with the structure of 6a.
The IR bands of 2b at 3303 cm -1 , 3203 cm -1 , 1663 cm -1 and 1623 cm -1 indicates the presence of -OH, -NH, C=O (amide) and C=N groups respectively.The 1 H-NMR shows a doublet at d 7.7 due to the effect of neighboring electronegative chlorine of aromatic proton H-3 and a doublet at d 7.4 due to aromatic proton H-4.Two singlets appear at d 12.2 and at d 10.2 due to OH and NH proton respectively.In mass spectrum the molecular ion peak at m/z 198/200 (3:1) which is consistent with the isotopic pattern of chlorine and structure of 2b.
The IR spectrum of 3b showed bands at 3097 cm -1 , 1704 cm -1 and 1617 cm -1 correspond to NH, C=O (keto) and C=O (lactam) groups respectively.The 1 H-NMR shows a broad peak at d 11.1 due to NH proton.Aromatic proton H-2 undergoes coupling with H-3 and meta coupling with H-1 simultaneously and appears as a triplet at d 7.55-7.61.A doublet appears at d 6.91-6.95due to aromatic proton of H-3.The molecular ion peak at m/z 181/183 (3:1) is characteristic of isotopic pattern of chlorine and consistent with the structure of 3b.
In IR spectrum of compound 4b, the bands appear at 3562 cm -1 , 1786 cm -1 and 1620 cm -1 indicate the presence of -NH, C=O (keto) and C=N groups respectively.The 1 H-NMR shows a sharp singlet at d 11.5 due to NH proton of lactam and a singlet at d 8.5 due to two NH protons but two NH2 protons were not observed due to rapid proton deuteron exchange reaction in deuterated dimethyl sulfoxide solvent.The mass spectrum did not show the expected molecular ion peak M + due to loss of thirty mass unit and showed the peak at m/z 223/225 (M + -30, 3:1) which is characteristic of isotopic pattern of chlorine and consistent with the structure of 4b.
Acetylation of 4b with freshly distilled acetic anhydride gave 5b.The IR spectrum of 4b shows bands at 1780 cm -1 indicating presence of acetyl group.The 1 H-NMR spectrum of 5b confirms the presence of two acetyl group groups by two sharp singlets at d 2.2 and at d 2.6 due to three protons of -NHCOCH3 and -NCOCH3 respectively.A broad singlet appears at d 12.1 represents three NH protons of H-4, H-5 and H-6.In mass spectrum the molecular ion peak M + was not observed due to loss of fifteen mass unit instead of that a peak appeared at m/z 322/324 (3:1), which is characteristic of isotopic pattern of chlorine and consistent with the structure of 5b.
On the basis of cytotoxicity study of the synthesized compounds it was found that -carbohydrazido group containing unsubstituted isatin activates the cytotoxic effect of the compound, than that of isatin itself.Spiro 1, 3,4 oxadiazoline derivatives of isatin (5b) having chlorine atom showed cytotoxic effect on brine shrimp more significantly than 5a without chlorine atom.