Synthesis, Characterization, and In Vitro Anticancer Evaluation of Novel 2,5-Disubstituted 1,3,4-Oxadiazole Analogue

In this series, we have synthesised a new 2,5-disubstituted 1,3,4-oxadiazole in search of potential therapeutics for cancer. The anticancer activities were evaluated on a panel of 60 cell lines by the National Cancer Institute according to its own screening protocol. Out of the 24 compounds, 11 were selected and evaluated via single high dose (10−5 M). In the next phase, two compounds have been selected for five-dose assay. The compounds 3-(5-benzyl-1,3,4-oxadiazol-2-yl)quinolin-2(1H)-one 18 (NSC-776965) and 3-[5-(2-phenoxymethyl-benzoimidazol-1-ylmethyl)-[1,3,4]oxadiazol-2-yl]-2-p-tolyloxy-quinoline 27 (NSC-776971) showed mean growth percentage of 66.23 and 46.61, respectively, in one-dose assay and their GI50 values ranging between 1.41–15.8 μM and 0.40–14.9 μM, respectively, in 5-dose assay.


Introduction
Cancer is primarily an environmental disease with 90-95% cases being related to environmental factors and 5-10% to genetics [1]. Common environmental factors causing cancer are tobacco (25-30%), diet and obesity (30-35%), infections (15-20%), radiation (both ionizing and nonionizing, up to 10%), stress, lack of physical activity, and environmental pollutant [2]. Death rates for cancer have continued to decline for both men and women of all racial and ethnic groups and have decreased by 1.5% per year from 2000-2009 for both sexes [3]. Trends in cancer death rates continue to decline; however, increase in incidence rates for some HPV associated cancer and low vaccination coverage among adolescents emphasize need for prevention in HPV associated cancer as well as to increase the coverage of vaccine. The development of new anticancer therapeutic agents is one of the fundamental goals in medicinal chemistry [4]. Medicinal chemists have great interest in research and development for the search of newer and safer anticancer agents. Epidermal growth factor receptor (EGFR) family of tyrosine kinase (TK) play a vital role in cancer proliferation and it is suggested that any agent would inhibit the TK activity and may have a considerable role in cancer treatment. N-containing heterocyclic specially 1,3,4oxadiazole ring are of great interest for researchers as they are found in natural products and are used frequently in medicinal and pharmaceutical chemistry [5]. Oxadiazole has a furan ring with two methane (-CH=) groups and is replaced by two pyridine types of nitrogen (-N=) atoms. Four types of isomers are possible in oxadiazole nucleus depending on the position of nitrogen present in the ring [6]. (See Scheme 1).

Material and Method
2.1. Chemistry. The chemicals used for experimental work were commercially procured from various chemical units, namely, E. Merck India Ltd., CDH and S.D. Fine chem. and Qualigens. These solvent and reagents were of LR grade and were purified before use. The silica gel G (160-120 mesh) used for analytical chromatography (TLC) was obtained from E. Merck India Ltd. The solvent system used was benzene : acetone (9 : 1) and (8 : 2) and toluene : ethyl acetate : formic acid (5 : 4 : 1). Ashless Whattman number 1 filter paper was used for vacuum filtration. Melting points were determined in open glass capillary using melting point apparatus and are uncorrected. The proton nuclear magnetic resonance ( 1 HNMR) spectra were recorded on Bruker 300 MHz instrument in DMSO-d 6 /CDCl 3 using tetramethylsilane [(CH 3 ) 4 Si] as internal standard. The infrared spectra of the compound were recorded in KBr on Perkin-Elmer FTIR Spectrometer, and the iodine chamber and U.V.-lamp were used for visualisation of TLC spots. Mass spectra were recorded on API 2000 LC/MS/MS system and elemental analyses were performed on Perkin-Elmer 2400. The commercially available grades of solvents and reagents were found to be of adequate purity. However, the presence of undesirable impurities and others was likely to be used for experimental work for purification.

Synthesis of 2-Chloroquinoline-3-carbaldehyde (2). 2-
Chloroquinoline-3-carbaldehyde was synthesized from acetanilide via a Vilsmeier-Haack reaction or by traditional methods. To a solution of acetanilide (5 mmole) in dry DMF (15 mmole) at 0-5 ∘ C with stirring, phosphorous oxychloride (60 mmole) was added dropwise and the mixture was stirred at 80-90 ∘ C for time ranges between 4 and 15 hr. The mixture was poured into crushed ice and stirred well and the resulting solid substance was filtered, washed well with cold water, and dried.

General Method for the Synthesis of 2-(4-Methylphenoxy)-3-(5-substituted-1,3,4-oxadiazol-2-yl)quinoline (37-44). To an ethanolic solution of substituted-(2-p-tolyloxy-quinolin-3ylmethylene)-hydrazide (0.01 mole) (29-36)
and chloramin-T (0.01 mole) was added. The solution was refluxed for 4 h, and sodium chloride which separated out during the course of reaction was filtered off. Excess ethanol was completely removed from the filtrate by distillation under reduced pressure, leaving behind a solid mass which was crystallized from ethanol to give the desired compound. Recrystallize from ethyl alcohol.     [35][36][37][38][39][40]. There were eleven compounds of the series, selected and screened for their anticancer activity for one-dose assay and among that two compounds were selected for 5-dose assay, after one-dose assay done by the National Cancer Institute (NCI) on leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate, and breast cancers cell lines, nearly 60 in number according to their screening protocol. All the synthesised compounds and structure of the compounds were submitted online to the official site of NCI for anticancer screening. Among 24 compounds only 11 compounds were selected for anticancer screening. NCI has its own selection procedure of the compounds for anticancer screening based on the novelty of heterocyclic ring system, drug-like properties utilizing the concept of privileged scaffolds, structure based on computeraided drug design, and so forth, while the chemical structures containing some problematic linkages or functional groups. The anticancer screening was carried out as per the NCI US protocol. All compounds submitted to the NCI 60 Cell screen were tested initially at a single high dose (10 −5 M) on leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate, and breast cancers cell lines, nearly 60 in number. The one-dose assay data was reported as a mean graph of the percent growth of treated cells. The number reported for the one-dose assay is the growth relative to the nodrug control and relative to the time zero number of cells. Using the absorbance measurements (time zero (Tz), control growth (C), and test growth in the presence of drug at the five concentration levels (Ti)) the percentage growth was calculated at each of the drug concentration levels.
Three dose response parameters are calculated for each experimental agent. The growth inhibition of 50% (GI50) is calculated from [(Ti − Tz)/(C − Tz)] × 100 = 50, which is the drug concentration resulting in a 50% reduction in the net protein increase (as measured by SRB staining) in control cells during the drug incubation. The drug concentration resulting in total growth inhibition (TGI) is calculated from Ti = Tz. The LC50 (concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning) indicating a net loss of cells following treatment is calculated from [(Ti − Tz)/Tz] × 100 = −50. Values are calculated for each of these three parameters if the level of activity is reached; however, if the effect is not reached or is exceeded, the value for that parameter is expressed as greater or less than the maximum or minimum concentration tested or if the effect exceeded the level of activity, the value of parameter was expressed as greater than the maximum concentration tested.   Log GI50, log TGI, and log LC50 are the logarithm molar concentrations producing 50% growth inhibition (GI50), a total growth inhibition (TGI), and a 50% cellular death (LC50), respectively.     pared in the presence of 70% acetic acid, and 2-(p-tolyloxy)quioline-3-carbaldehyde 4 was prepared from 2 by using -cresol and K 2 CO 3 in the presence of DMF. In the step 1, 2- In general the 1H NMR spectra of the compound show one singlet between 5 to 6 CH 2 O. The Schiff bases explained the presence of -CONH ( 10-12) and -N=CH from the presence of one singlet from 8 to 9. The next step of the synthesis of 1,3,4-oxadiazole, the disappearance of -CONH and -N=CH peaks confirm the formation of 1,3,4-oxadiazole (Scheme 2).

Anticancer
Activity. Eleven compounds have been evaluated for their anticancer activity by NCI, USA. The observed anticancer screening of the compounds is given in Table 1. The two compounds were selected for five-dose assay; these compounds are given in The result of anticancer activity of the five-dose assay of the selected compounds amongst their respected series is given in Table 2

Conclusion
The novel series of 1,3,4-oxadiazole analogues have been synthesized in good yields and the anticancer activity showed good results in two compounds, that is, 18, 27 (NSC 776965 and NSC 776971). The present studies reveal that compound 27 is a potent lead compound for anticancer drug discovery and further research. The 1,3,4-oxadiazole anlogue provides a valuable new therapeutic intervention for the treatment of cancer disease.