Synthesis of Some Novel Quinazoline Derivatives Having Anti-Cancer Activity

Cancer is the uncontrolled growth and spread of cells. It can affect any part of the body. The growths often invade surrounding tissue and can metastasize to distant sites [1]. Majority of the cancer (90%-95%) are caused by frequent use of tobacco, obesity or over weight, chronic infections and prolonged exposure to radiations and a notable percentage (5%-10%) are due to heredity. Most cancer can be treated by radiology, chemo-therapy and surgery [2]. According to WHO, in 2012 approximately 14 million new cases and 8.2 million cancer related deaths worldwide. The number of new cases is projected to upsurge by about 70% over the next 2 decades. Among men, the most common sites of cancer diagnosed in 2012 were lung, prostate, colorectum, stomach and liver cancer; among women the most common sites diagnosed were breast, colorectum, lung, cervix and stomach cancer.


Introduction
Cancer is the uncontrolled growth and spread of cells. It can affect any part of the body. The growths often invade surrounding tissue and can metastasize to distant sites [1]. Majority of the cancer (90%-95%) are caused by frequent use of tobacco, obesity or over weight, chronic infections and prolonged exposure to radiations and a notable percentage (5%-10%) are due to heredity. Most cancer can be treated by radiology, chemo-therapy and surgery [2]. According to WHO, in 2012 approximately 14 million new cases and 8.2 million cancer related deaths worldwide. The number of new cases is projected to upsurge by about 70% over the next 2 decades. Among men, the most common sites of cancer diagnosed in 2012 were lung, prostate, colorectum, stomach and liver cancer; among women the most common sites diagnosed were breast, colorectum, lung, cervix and stomach cancer.
Tobacco use is the most important cause for around 20% of global cancer deaths and around 70% of global lung cancer deaths. Viral infections caused by HBV/HCV and or HPV are responsible for around 20% of cancer deaths in low-and middle-income countries. Over and above 60% of world's total new annual cases occur in Africa, Asia and Central and South America, these regions account for 70% of the world's cancer deaths. The annual cancer cases expected to rise from 14 million in 2012 to 22 within the next 2 decades [3].

General scheme [4]
The pasty mass obtained was diluted with 50ml of water and treated with aqueous sodium bicarbonate solution. When the effervescence ceased, the precipitate obtained was filtered and washed with water. The crude benzoxazine obtained was dried and recrystallized from diluted ethanol [4][5][6].  To a cold solution of 2-[4-Chloro phenyl]-4H-benzo[d] [1,3] oxazin-4-one(0.05mol) in anhydrous pyridine, (20ml) added a solution of hydrazine hydrate (0.1mol) in anhydrous pyridine (25ml) drop wise with constant stirring. When the addition was complete, the reaction mixture was stirred vigorously for 30min at room temperature and subsequently heated under reflux for 6h under anhydrous reaction conditions. The reaction mixture was allowed to cool at room temperature and poured into ice cold water containing dilute hydrochloric acid. On standing for 1h solidification occurred which was allowed to settle down. It was filtered off, washed repeatedly with water and dried, recrystallized from dilute ethanol [4,6].

In-Vitro anticancer screening
The human cervical cancer cell line (HeLa), NIH 3T3 mouse embryonic fibroblasts were obtained from National Center for cell science (NCCS), Pune. The HeLa cells were grown in Eagles Minimum Essential Medium containing 10% fetal bovine serum (FBS) and NIH 3T3 fibroblasts were grown in Dulbeccos Modified Eagles Medium(DMEM) containing 10%FBS [8][9][10]. For the screening experiment, the cells were seeded into 96-well plates in 100µl of respective medium containing 10% FBS , at a plating density of 10,000 cells/well and incubated at 37 °C, 5%CO 2 , 95% air and 100% relative humidity for 24hrs prior to addition of samples. The extracts were solubilised in DMSO and diluted in respective medium containing 1%FBS. After 24h, the medium was replaced with the respective medium containing the samples at various concentrations and incubated at 37 °C, 5% CO 2 , 95% air and 100% relative humidity for 48h.
Triplicate was maintained and the medium without samples were served as control [8,9]. After 48h, 10µl of MTT (5mg/ml) in phosphate buffered saline was added to each well and incubated at 37 °C for 4h. The medium with MTT was then flicked off and the formed crystals were solubilised in 100 µl of DMSO and measured the absorbance at 570nm using microplate reader [10]. The percentage cell inhibition and IC50 were calculated and are given in Table 1.
The synthesized compounds purity and its structural elucidation were done by various analytical techniques such as Melting point, Thin Layer Chromatography (TLC), Ultra Violet Spectroscopy (UV), Infrared Spectra (IR) and H 1 Nuclear magnetic resonance (H 1 NMR).

Melting point
The melting point is an important physical property of a compound and it can be used to identify a substance and its purity. The melting point of solid is the temperature at which the solid exists in equilibrium with its liquid under an external pressure of one atmosphere [11]. A synthesized compounds melting point and its reactants melting point were recorded by open capillary tube method [4,12], which are uncorrected. A reactant and products melting point were differing from each other. It clearly indicates that the formation of a new chemical entities. The melting point values of the synthesized compounds are presented in Table 2.

Compound HeLa (IC50)
Compound S1 93.30mM Compound S3 6.90mM Data are the mean of three or more experiment and are reported as mean±standard error of the mean (SEM).

Thin layer chromatography
Thin layer chromatography (TLC) is a very expedient and effective technique for the separation and identification of inorganic ions. It permits selective separations, simple detection and easy manipulation of the mobile phase. As a result, numerous sorbents and even better number of mobile phases have been developed for attaining the improved chromatographic performance in terms of selectivity, resolution, rapidity and reproducibility [13]. Thin-layer chromatography can be used to monitor the progress of a reaction, identify compounds present in a given mixture, and determine the purity of a substance [14]. Thin layer chromatography techniques were performed for all synthesized compound as well as the parent compounds, all synthesized compounds gave a single spot whose Rf values are different from their reactants. It ultimately shows that the compound's purity and completion of the reaction. The Rf value are presented in Table 3.

Infra-red spectra
Infrared spectroscopy is one of the classical methods for structure determination of small molecules. This standing is due to its sensitivity to the chemical composition and architecture of molecules [15]. Infra-Red spectroscopy was taken for all the synthesized compounds. The characteristic absorption peaks were observed for all relevant groups. Aromatic acid chlorides strongly absorb at 1800-1770cm -1 which was not observed in spectrum, it clearly gives a chemical entity in step I. The absorption peaks around 1600-1500cm -1 indicates that the formation of C=N Schiff bases. C=O stretching vibration around 1870cm -1 was not appeared in spectrum. It shows that there was no impurities like aldehyde/ketone., C-C, C=C & C=N ring stretching vibration at 1050-1200,754cm-1 also appeared and aromatic amide N-H was observer at 3100-3500cm -1 and all other relevant groups absorption were observed for all the synthesized compounds.

H 1 Nuclear magnetic resonance
NMR or nuclear magnetic resonance spectroscopy is a technique used to determine a compound's unique structure. It identifies the carbon-hydrogen framework of an organic compound. Using this method and other instrumental methods including infrared and mass spectrometry, scientists are able to determine the entire structure of a molecule [16]. 1 H nuclear magnetic spectra were taken for all the synthesized compounds. Aromatic protons were observed 6.68-8.13δppm, Amide N-H proton was observed at 6.05-6.40δppm, for all the synthesized compounds.

Anti-cancer activity
Cancer is a multi-step disease incorporating physical, environmental, metabolic, chemical and genetic factors, which play a direct and/or indirect role in the induction and deterioration of cancers [17]. Cancer has a major impact on society in the United States and across the world. Cancer statistics describe what happens in large groups of people and provide a picture in time of the burden of cancer on society [18]. Hence, some of the synthesized compounds were tested for in-vitro anticancer activity. Among the synthesized derivatives compound S1 and S3 were screened for in-vitro cytotoxicity effect. The results indicate that the synthesized compound possesses cytotoxicity against both cancer cells as well as normal cells. It proves that suitable structural modifications will have to be carried out to get novel compounds having potent anticancer activity with least effect on normal cells [19][20][21].

Conclusion
In summary, a new series of quinazoline derivatives (s1-s10) have been synthesized and evaluated for anti-cancer as well as anti-bacterial activities. Among the synthesized compounds S1, S3 were the potent compounds with good results for anti-cancer activity. These quinazoline derivatives S1, S3 represent promising structures for the development of new compounds with better activity results and for future in-vivo analysis.