Biological activity of some novel synthesized 2-( 4-methylbenzene-sulphonamido ) pentanedioic acid bis amide derivatives : In vitro and in vivo antineoplastic activity

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Introduction
Cancer is a class of disease in which group of cells display uncontrolled growth, invasion and sometimes metastasis.Cancer affects people at all ages with the risk for most types increasing with age (Cancer Research UK, 2007).Cancer caused about 13% of all human deaths in 2007 (7.6 million) (WHO, 2006;American Cancer Society, 2007).
Glutamic acid (2-amino pentanedioic acid) plays an important role in the biosynthesis of purine and pyrimidine bases of DNA and RNA (Rodwell, 2000).It is metabolized to L-glutamine by L-glutamine synthetase and this metabolic process is essential for normal maintenance of cells.The synthesis of L-glutamine is hindered in neoplastic cells due to lower reactivity of Lglutamine synthetase.Thus antagonists of this enzyme can interfere with the metabolic role of L-glutamine and act as anti-cancer agents (Hartman, 1970).Azaserine and 6-diaza-5-oxo-L-norleucine antagonized the metabolic process involving L-glutamine and exhibited antitumor activity in animal models (Eidinoff, 1958).The importance of non-essential amino acid glutamine in proliferation of human tumor cells was studied exten -sively (Graff et al., 1940;Petit, 1977).All tumor cells studied were found to have a high activity of phosphate -dependent glutaminase utilizing glutamine from the medium during long-term culture (Graff et al., 1940).Beside glucose, glutamine is assumed to be the main energy source in tumor cells (Keren and Stark, 1988).It also plays a central role in multiple metabolic pathways and considered to be the most essential component of tissue/cell culture media (Sugita, 1995) for not only as the nitrogen source but also as the carbon source.After a definite time interval, all cells start mutation in cell culture medium, which is also indicative for the role of

Materials and Methods
Commercially available reagents and starting materials for the synthesis were obtained from E. Merck, India, CDH, s.d.Fine Chem, India and Qualigens, India.Silica gel G used for TLC was obtained from E. Merck.The reaction was monitored by TLC using on 0.25 mm E. Merck silica gel 60F252 precoated plates, which were visualized under UV light.Melting points were determined in an open glass capillary using a Kjeldahl flask containing paraffin and are uncorrected.The proton and carbon magnetic resonance spectra ( 1 H NMR, 13 C NMR) were recorded on a Bruker 400 MHz instrument (Bruker, Germany) in dimethyl sulfoxide-d6 (DMSO-d6) using tetramethylsilane as internal standard.Chemical shifts (d) are expressed in ppm and coupling constants (s) singlet, (d) doublet, (t) triplet, (m) multiplet.Position of carbons described in 13 C NMR interpretation is as per general structure (Figure 1) for all the compounds except Va.The infrared spectra of compounds were recorded in KBr on Fourier Transform (FTIR-8400S, Shimadzu, Japan) infrared spectrophotometer.Mass spectra (FAB) were recorded on LC-MS/ MS (API-4000 TM, Applied BioSystems, MDS SCIEX, Canada).Elemental analyses were performed on a Perkin-Elmer model 240c analyzer (Perkin Elmer, USA).

Synthesis of 4-methylbenzene-1-sulfonyl chloride (2)
Tolu-ene (9.2 mL, 0.1 mol) was taken in a 250 mL three necked round bottom flask placed on an ice bath fitted with a mercury sealed mechanical stirrer, a calcium chloride guard tube and a 100 mL dropping funnel.Chlorosulfonic acid (25 mL, 3 equiv., 0.36 mol) was placed in the dropping funnel and was added drop wise slowly by continuous stirring.The temperature of the ice bath was maintained at 0°C.After the addition of the chlorosulfonic acid completely the mixture was allowed to come to room temperature.Then the product was poured slowly into well stirred crushed ice.The crude 4-methylbenzene-1-sulfonyl chloride (2) was filtered and washed with water to remove the excess acid.The crude product was recrystalized from water.The yield was 15.2 g (84.2%) and m.p.: 155-57°C.
L-(+)-glutamic acid (20 g, 0.1 mole) was taken in a 250 mL conical flask and placed on a water both, fitted with a magnetic stirrer.Sodium hydroxide solution (2N) was added slowly till all the L-glutamic acid dissolved and the mass became distinctly alkaline to phenolphthalein.The water bath was maintained at 60-80°C and 4methylbenzene-1-sulfonyl chloride (2; 36.1 g, 0.2 mol) was added slowly with continuous stirring and simultaneous addition of sodium hydroxide solution (2N) to keep the mass alkaline.The reaction was continued till a clear homogeneous solution results.After the reaction was over, it was allowed to cool, acidified to congored with concentrated hydrochloric acid, saturated with sodium chloride, extracted with chloroform, and allowed to dry overnight with anhydrous magnesium sulfate.Chloroform was removed to yield the crude 2-(4-methylbenzenesulfonamido)pentanedioic acid (3).The crude product was recrystalized from hot water after charcoal treatment.Yield 57.9%,Calc. For C12H15NO6S: 301)

Synthesis of 2-(4-methylbenzenesulfonamido)pentanedioic acid bis amide derivatives (5a-l)
The 2-(4-methylbenzene-sulfonamido)pentanedioyl dichloride (4) formed was dissolved in dry benzene (10 mL) and the whole mass was cooled by dipping in ice water.Aniline and its derivatives, previously cooled was added to it and mixed well.The whole mass was transferred in a mortar and pestle and triturated when the product was obtained.It was then acidified with dilute hydrochloric acid.The precipitate obtained was filtered and washed with distilled water to remove excess acid.The residue was dried and recrystallized from dilute ethanol after charcoal treatment.C, 43.63; H, 4.97; N, 18.17; found C, 43.53; H, 5.05; N, 18.19%.

MTT assay
The MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) assay (Mosmann, 1983) was modified and used to determine the inhibitory effects of test compounds on cell growth in vitro.In brief, the trypsinized cells from T-25 flask were seeded in each well of 96-well flat bottomed tissue culture plate at a

In vitro anti-cancer activity (IC50) of synthesized compounds against human breast cancer (MCF-7), leukemia (K-562), ovarian cancer (OVCAR-3), human colon adenocarcinoma (HT-29) and human kidney carcinoma (A-498)
Com Tamoxifen 8.2 ± 0.0 a 10.4 ± 0.0 a 9.3 ± 0.0 a 14.3 ± 0.0 a 12.2 ± 0.0 a Compounds with IC50 >100 µM were considered not active; a p<0.05 compared to tamoxifen (standard) density of 5x10 3 cells/well in growth medium and cultured at 37°C in 5% CO2 to adhere.After 48 hours incubation, the supernatant was discarded and the cells were pretreated with growth medium and were subsequently mixed with different concentrations of both standard (tamoxifen) and test compounds 5a-l (8, 16, 32, 64, 128 and 256 µg/mL) in triplicates to achieve a final volume of 100 µL and then cultured for 48 hours.The compound was prepared as 1.0 mg/mL concentration stock solutions in PBS.Culture medium and solvent are used as controls.Each well then received 5 µL of fresh MTT (0.5 mg/mL in PBS) followed by incubation for 2 hours at 37°C.The supernatant growth medium was removed from the wells and replaced with 100 µL of DMSO to solubilize the colored formazan product.
After 30 min incubation, the absorbance (OD) of the culture plate was read at a wavelength of 570 nm on an ELISA reader, Anthos 2020 spectrophotometer.Both standard and test maintained in triplicate.The IC50 value refers to the drug concen-tration that produces a 50% reduction in cellular growth when compared to untreated control cells (Holbeck, 2004).

In vivo anti-cancer assay
The animal experiments were performed following the approval of study protocols by the Institutional Animal Ethics Committee (BCRCP/IAEC/7/2012).The synthesized compounds were biologically screened against Ehrlich ascites carcinoma (EAC) in female Swiss Albino mice using tumor weight and cell count as activity parameters.Amongst various evaluation systems in vogue, this method has been standardized and numbers of screening results have been reported earlier (De and Pal, 1975;De and Pal, 1977;Ray and De, 2009).Two groups of Swiss Albino mice, each containing five healthy mice of the same sex (female in this case), approximately of same age and body weight (18-20 g), were selected at random and kept in two different cages under identical condition.One of these two groups served as control while the other as test.Ehrlich ascites carcinoma (EAC) cells were collected from the donor mice and were suspended in sterile isotonic solution (0.9% w/v NaCl).The numbers of tumor cells per ml of this suspension were counted under microscope with the help of haemocytometer.A definite number (about 2 x 10 6 /0.2 mL) of these living viable cells was injected or implanted into the peritoneal cavity of each mouse.In this instance, the tumor cells multiplied relatively freely with in the peritoneal cavity and ascites developed.A day of incubation was allowed to establish the disease in the body before the administration of synthesized compounds.From the second day of transplantation up to the eight day a suitable dose (0.2 mmol/kg body weight) of the drug solution/suspension in sterile phosphate buffer (pH 7.2) was injected intraperitoneally to each mouse in the test group at 24 hours interval.Thus, seven doses of the drug were administered to each mouse in the test group.On the ninth day food and water were withheld or withdrawn 6 hours before the testing operation started.The weight of all the animals was recorded before they were sacrificed.The peritoneal cavity was dissected and the ascites fluid was drawn by a syringe to a suitable volume with sterile ice cold saline and preserved in ice bath.The total number of living cells per milliliter in the peritoneal fluid of the three mice in a group was calculated.

Statistical analysis
Values are expressed as mean ± SEM and data was analyzed by ANOVA followed by Dunnet's test.p<0.05 was considered as significant.

Results and Discussion
The synthetic strategies followed for the preparation of the substituted 2-(4-methylbenzenesulfo-namido)pentanedioic acid bis amide derivatives 5a-l are depicted in Scheme 1. Synthesis was started with chlorosulphonation of toluene, to get 4-methylbenzene-1-sulfonyl chloride 2. This sulphonyl halide proved to be versatile synthon in the subsequent step in the preparation of substituted sulphonyl glutamic acids.With the application of Schotton-Bauman reaction, substituted sulphonyl glutamic acids 3 were prepared by one-step condensation of 4-methylbenzene-1-sulfonyl chloride 2 with L-glutamic acid.In this reaction alkaline medium was maintained to remove the hydrochloric acid which was formed during condensation.Reaction of the resulting intermediates with thionyl chloride afforded corresponding acid chloride followed by the amination with various amines afford the correspond-ding amines 5a-l.
Formation of 2-(4-methylbenzenesulphonamido)pentanedioic acid bis amide derivatives 5a-l were confirmed by recording their IR, NMR, mass spectra and elemental analyses.The IR spectra of compounds 5a-l revealed the presence of absorption bands from 3012.54 to 2542 cm -1 that indicate the presence of OH str. of COOH, from 1696.07 to 1570.8 cm -1 for C=O str., from 1507 to 1577.31 cm -1 for C=C str. of phenyl ring, from 3287.32 to 3397 cm -1 for N-H str., from 1593-1645 cm -1 for N-H bending, from 1280.84-1354.5cm -1 for S=O str.anti-symmetric of SO2N and 1209-1310.18cm -1 for S=O str.symmetric of SO2N vibrations.In addition to proton signals of the functional groups and both aromatic ring present in the respective molecule 1 H NMR spectra of these compounds contained one proton singlet from δ 2.0 to 2.203 ppm which was assigned to -SO2NH-proton and from 2.21 to 2.604 ppm for -CH3 proton.The 1 H NMR spectra of compounds 5a-l showed doublets from δ 7.201 to 7.98 ppm for aromatic protons and triplets from δ 3.54 to 4.1 ppm for -OC-CH-CH2-proton confirming the formation of compounds 5a-l.The 13 C NMR spectra of compounds 5a-l showed peaks from δ 118.57ppm to 156.38 ppm for aromatic protons, from δ 20.12 ppm to 22.31 ppm for -CH3 carbon confirming the formation of compounds 5a-l.The mass spectra of compounds 5a-l showed molecular ion peaks [M +1 ] at m/z corresponding to their respective molecular masses, which is in consistency with their respective molecular formulas.
For the compound 5a, molecular weight 385 is consistent with the molecular formula C9H19N5O6S.The values for the remaining compounds have been presented under the experimental part.
The cytotoxicity of the synthesized compounds 5a-l and the intermediate compound number 3 were studied using the MTT assay in five human cancer cell lines, including human breast cancer (MCF-7), leukemia (K-562), ovarian cancer (OVCAR-3), human colon adenocarci-noma (HT-29) and human kidney carcinoma (A-498) (Table I).showed high cytotoxicity in all cell lines with IC50 concentrations lines, except for the human colon adeno carcinoma (HT-29) cell line.The primary antitumor activity of tamoxifen by inhibition protein kinase C (Gelman, 1997) and also ability to facilitate the apoptosis in cancer cell not expressing estrogen receptor is due to generation of oxidative stress resulting in thiol depletion and activation of the transcriptional factor NF-kappaB.Many clinical studies explain the tamoxifen application in various kinds of maligamant diseases (Ferlini et al., 1999;NCI, 1999).
All the newly synthesized final compounds 5a-l along with compound no. 3 were screened for their anticancer activity against Ehrlich ascites carcinoma is summarized in Table II together with mitomycin-C.Among the synthesized com-pounds, the ureide 5a, anilide 5b, p-nitoanilide 5h and o-bromoanilide 5k derivatives showed encouraging activity in both the parameter, viz., ascetic fluid weight (93.0% for 5a, 91.6% for 5b, 91.5% for 5h and 96.4% for 5k) and ascetic cell count (98.9% for 5a, 98.8% for 5b, 98.7% for 5h and 98.6% for 5k) respectively.Hence, a detailed and prolonged study is necessary to establish their activity in other models.
Thus, twelve new compounds along with an intermediate compound 3 were synthesized, characterized and biologically screened for in vitro and in vivo antitumor activity.It was noticed that final derivative compounds showing better activity than the parent compound and it may be due to the substituents present in those compounds.These observations encourage performing QSAR study in future.
The fluid was sucked by absorbent cotton.The weight of the five mice after sacrifice was recorded.The evaluation of the test drug was made by comparing the cell count and ascetic fluid weight of the test with that of the control.The percentage inhibition of ascetic cell count and ascetic fluid weight was obtained by the following expressions.Percentage inhibition of ascitic cell = (1 -T/C) x 100 Percentage inhibition of ascitic fluid = (1 -T/C) x 100 Where, T= Average number of ascitic cells per mL in test animals, C= Average number of ascitic cells per mL in control animals, T= Average weight of ascitic fluid in test animals and C= Average weight of ascitic fluid in control animals.Mitomycin-C (1 mg/kg body weight) in sterile phosphate buffer (pH 7.2) was used as standard.