Synthesis , Spectroscopy , Computational and Anticancer Evaluation of Some Novel Isatin Derivatives

A series of Isatin derivatives was synthesized using potassium 2-cyano-3-oxo-3-(2-(2-oxoindolin-3ylidene)hydrazinyl)-1-(phenylamino)prop-1-ene-1-thiolate (2) as starting material. Compound (2) reacts with various reagents under different conditions to give the corresponding thiazol, thiophen and pyridine derivatives, which were characterized by elemental analysis, spectroscopy (1H-NMR, IR and Mass spectra). The anticancer activities of the newly synthesized compounds were studied against colon carcinoma cells by using the Minimum Inhibition Concentration (MIC) method. Compounds belonging to 8, 12 and 13 series produced a high anti-cancer reactivity.


Introduction
Isatin or 1 H-indole-2,3-dione is an indole derivative.The compound was first obtained [1,2] as a product from the oxidation of indigo dye by nitric acid and chromic acid.Isatin forms a blue dye if it is mixed with sulfuric acid and crude benzene.The formation of the blue indophenin was long believed to be a reaction with benzene.Victor Meyer was able to isolate the substance responsible for this reaction from benzene.This new heterocyclic compound was thiophene [3].Isatin is exerting a broad spectrum of biological activity like antipyretic activity, analgesic effect anticonvulsant activity; few compounds were also reported as psychotropic agents and Monoamine Oxidase (MAO) inhibitors [3].
Isatins are an important group of heterocyclic compounds which are biologically active and of significant importance in medicinal chemistry.A literature survey identified several Isatin derivatives in the development phase as potential new drugs.Isatin ( 1 H-indole-2,3-Dione) and its derivatives exhibit various biological activities such as anticancer [4], anticonvulsant [5], anti-inflammatory [6], antimicrobial, antiviral [7] and anti neo-plastic activities [8].These compounds are versatile building blocks for the synthesis of a large variety of heterocyclic compounds such as indoles, isotopic anhydride, quinolines, spirooxin-doles, and etc.The unique structural array of these compounds has made them attractive synthetic targets in chemistry.Isatins are capable of crossing the blood-brain-barrier [9].Isatin, a heterocyclic compound was identified in animals as a major component of the endogenous MAO inhibitors.The various substituents at 3rd position of the Isatin which were reported various substituted phenyl ring moieties, heterocyclic rings and aliphatic system.Isatin ( 1 H-Indole-2,3-dione) is one of the most promising new class of heterocyclic molecules having many interesting activity profiles and well-tolerated in human subjects.As a continuation of our efforts [10][11][12][13][14][15][16][17][18][19] to identify new condition that may be of value in designing new, potent, selective, less toxic antimicrobial agents, we report here the synthesis of some new heterocycles incorporating an indole moiety starting from cyanoacetohydrazide and Isatin, we found that 2-cyano-N'-(2-oxoindolin-3-ylidene) acetohydrazide (1) [20] is a highly active against tumor cells and useful building for the synthesis of a variety of phenylthiazolidin, phenylthiazol and thiophene derivatives incorporating satin moiety of potential biological activity.

Instruments
All melting points (m.p.) are recorded in Gallenkamp electric m.p. apparatus and are uncorrected.The IR spectra νcm -1 (KBr) were recorded in Perkin Elmer Infrared Spectrophotometer Model 157, Grating.The 1 H-NMR spectra were run on Varian Spectrophotometer at 300 and 75 MHz, respectively, using Tetramethylsilane (TMS) as an internal reference and DMSO-d 6 as solvent.The mass spectra (EI) were recorded on 70 eV with Kratos MS equipment and/or a Varian MAT 311 A Spectrometer at Micro analytical Unit, Faculty of Science, Cairo University and Al-Azhar University, Cairo, Egypt.Elemental analyses (C, H and N) were carried out at the micro analytical center of Cairo University, Giza, Egypt, the results were found to in good agreement (± 0.3%) with the calculated values.Antimicrobial screening for the selected new compounds was carried out in the regional center for mycology and biochemistry, Al-Azhar University, Cairo, Egypt.(1) with phenyl isothiocyanate in the presence of potassium hydroxide in Dimethylformamide (DMF) to give a potassium 2-cyano-3-Oxo-3-(2-(2-oxoindolin-3-ylidene)hydrazinyl)-1-(phenylamino)prop-1-ene-1thiolate (2) was prepared as previously described [21].

Chemistry
: To a stirred solution of potassium hydroxide (0.01 mol) in N,N-DMF (20 ml) was added compound (1) (0.01 mol).After the mixture was stirred for 30 min, phenyl isothiocyanate (0.01 mol) was added to the resulting mixture.Stirring was continued at room temperature for 12 hrs.The reaction mixture was acidified with cold dilute HCl.The solid product that separated was filtered, washed with water and recrystallized from ethanol to give (3) [10].

Anticancer evaluation of cytotoxicity against HCT cell line
Antitumor activity assay: Human colon carcinoma (HCT-116) cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD).The cells were grown on RPMI-1640 medium supplemented with 10% inactivated fetal calf serum and 50 µg/ml gentamycin.The cells were maintained at 37°C in a humidified atmosphere with 5% CO 2 and were subculture two to three times a week.For antitumor assays, the tumor cell lines were suspended in medium at concentration 5 × 10 4 cell/well in Corning® 96-well tissue culture plates, then incubated for 24 hrs.
The tested compounds were then added in 96-well plates (six replicates) to achieve eight concentrations of each compound.Six vehicle controls with media or 0.5% DMSO were run for each 96 well plate as a control.After incubating for 24 hrs, the numbers of viable cells were determined by the MTT test.Briefly, the media was removed from the 96 well plate and replaced with 100 µl of fresh culture RPMI 1640 medium without phenol red, then 10 µl of the 12 mm MTT stock solution (5 mg of MTT in 1 ml of PBS) to each well including the untreated controls (Figure 1).The 96 well plates were then incubated at 37°C and 5% CO 2 for 4 hrs.An 85 µl aliquot of the media was removed from the wells, and 50 µl of DMSO was added to each well and mixed thoroughly with the pipette and incubated at 37°C for 10 min.Then, the optical density was measured at 590 nm with the micro plate reader (SunRise, TECAN, Inc, USA) to determine the number of viable cells and the percentage of viability was calculated as [1-(ODt/ODc)]x 100% where ODt is the mean optical density of wells treated with the tested sample and ODc is the mean optical density of untreated cells [22][23][24].The relation between surviving cells and drug concentration is plotted to get the survival curve of each tumor cell line after treatment with the specified compound.The 50% inhibitory concentration (IC 50 ), the concentration required to cause toxic effects in 50% of intact cells, was estimated from graphic plots of the dose response curve for each conc.The synthesized compounds (5)(6)(7)(8)(9)(10)(11)(12)(13) showed the greater selectivity towards colon carcinoma cells (Table 1).The most active compound shown antiproliferative at low micro molar concentration and also activated the effector capsases in a dose dependent manner.It is interesting that the compound ( 8), ( 12) and ( 13) showed signification growth inhibitory activity on the HCT-116 c, while ( 5) and ( 6) showed the lowest effect on the same tumor cell line (Figure 1).

Chemistry
It was now that the reaction of Isatin with cyanoacetohydrazide in the presence of catalytic amount of triethylamine at room temperature yielded the corresponding C-condensation product (1).Compound (3) was obtained in good yield upon treatment of (1) with phenyl isothiocyanate in KOH/DMF followed by acidification with dilute HCl.The micro analytical and spectroscopic data were in agreement with the proposed structure (3) [10].
Treatment of the Isatin (1) with phenyl isothiocyanate in DMF, in the presence of potassium hydroxide give the intermediate (2) and also, an equal molar amount of 2-chloroacetylchloride and ethyl 2bromoacetate reacted with intermediate (2) give intermediate ( 4(a,b)) furnished, in each case give the same product (5).The intermediate (4a), was established based on IR spectrum, which showed band in the region 3566,3447 and 3061 cm -1 due to three (NH) groups, a strong, sharp band at 2198 cm -1 due to nitrile function and strong absorption band at 1700 cm -1 due to the carbonyl group (C=O).Moreover, its 1 H-NMR showed singlet at δ 4.45 due to methylene in addition to aromatic multiple in the region δ 6.91-7.46 and three single's at δ 10.03, 10.44 and 12.133 due to NH-amid, NH group and NH-hydrazid (Scheme 1).Also, the intermediate (4b) was established based on IR spectrum, which showed band in the region 3316, 3190 and 3109 cm -1 due to three (NH) groups, a strong, sharp band at 2218 cm -1 due to nitrile function and strong absorption band at 1729 cm -1 due to the carbonyl group (C=O).Moreover, its 1 H-NMR showed triplet at δ 1.5 due to methyl group and showed singlet at δ 3.98 due to methylene and also, showed quarterly at δ 4.18 due to methylene in addition to aromatic multiple in the region δ 6.91-7.46 and three single's at δ 10.03, 10.44 and 12.133 due to NH-amid and NH-hydrazid.The reaction products were identified as (2-cyano-2-(4-oxo-3-phenylthiazolidin-2ylidene)-N'-(2-oxoindolin-3-ylidene) acetohydrazide (5).The structures were established based on IR spectrum, which showed band in the region 3327-3193 cm -1 due to two (NH) groups, a strong, sharp band at 2218 cm -1 due to nitrile function and strong absorption band at 1729 cm -1 due to the carbonyl group (C=O).Moreover, its 1 H-NMR showed singlet at δ 3.32 due to methylene in addition to aromatic multiple in the region δ 6.91-7.46 and two single's at δ 10.03 and 11.133 due to NH-amid and NH-hydrazid (Scheme 1).
Reaction of intermediate (2) with chloroacetone containing the intermediate (4c) which the final isolable product 2-cyano-2-(4-methyl-3-phenylthiazol-2(3H)-ylidene)-N'-(2-oxoindolin-3ylidene)acetohydrazide (6).The intermediate (4c) which showed on IR spectrum a strong band in the region 3423, 3327 and 3193 cm -1 due to three (NH) groups, a strong, sharp band at 2179 cm -1 due to nitrile function and strong absorption band at 1709 cm -1 due to the carbonyl group (C=O).Moreover, its 1 H-NMR showed singlet at δ 2.28 due to methyl group and also, showed a singlet at δ 4.03 due to methylene in addition to aromatic multiple in the region δ 6.91-7.46 and three single's at δ 10.03, 10.44 and 12.133 due to NH-amid, -NH and NHhydrazid.The IR spectrum of the product (6) while revealed the presence of 3369, 3193 cm -1 due to two (NH) groups, a presence of 2199 cm -1 due to nitrile function and strong absorption band at 1700 cm -1 due to carbonyl group and the 1 H-NMR spectrum of compound (6) showed three singlet at δ 5.56, 10.03 and 11.133 due to CHthiazole, NH-amid and NH-hydrazide (Scheme 1).8) through the intermediate (4e) which showed on IR spectrum a strong band in the region 3423, 3327 and 3193 cm -1 due to three (NH) groups, a strong, sharp band at 2179 cm -1 due to nitrile function and strong absorption band at 1709 cm -1 due to the carbonyl group (C=O).Moreover, its 1 H-NMR showed singlet at δ 2.28 due to methyl group and also, showed a singlet at δ 4.03 due to methylene in addition to aromatic multiple in the region δ 6.91-7.46 and three single's at δ 10.03, 10.44 and 12.133 due to NH-amid, -NH and NH-hydrazid.The IR spectrum of compound (8) showed the absence of Nitrile band and appearance band in the region 3566-3591 cm -1 due to two (NH) groups, 3421 cm -1 due to secondary amine and strong absorption band at 1706 cm -1 due to the carbonyl group (C=O).Moreover, its 1 H-NMR showed singlet at δ 6.91 due to NH 2 in addition to aromatic multiple in the region δ 6.91-7.59and two single's at δ 8.501, 10.03 and 10.91 due to NH-thiophene, NH-amid and NH-hydrazid (Scheme 2).The IR spectrum showed bands at 3677, 2938 and 2214 cm -1 due to and hydroxyl group, NH 2 and nitrile group, a strong absorption band at 1706 cm -1 due to the carbonyl group (C=O).Moreover, its 1 H-NMR showed singlet at δ 3.83 due to the methoxy group in addition to aromatic multiple in the region δ 6.84-7.59and two single's at δ 6.49, 10.03 due to NH 2 and NH-amid and eat δ 11.30 due to OH (Scheme 3).

Figure 1 :
Figure 1: Activity of compound towards colon carcinoma cells.

Scheme 2 :
Scheme 2: Synthesis and reactions of Thiazole derivatives and Thiophen derivatives.

Scheme 3 :
Scheme 3: Synthesis and reactions of pyridine derivatives.