1,2,4-Oxadiazole Moiety Molecules Synthesis for Cancer

1,2,4-Oxadiazole Moiety Molecules Synthesis for Cancer2.4. Synthesis of 3-(4-(6,7-dihydro-5H-pyrrolo1,2-cimidazol-5-yl)-3-fluorophenyl)-5-substituted-1,2,4-oxadiazole derivatives for their MTT assay utilise MCF-7 thorax cancer cellphone line and degradation of deoxyribonucleic acid in EAT cells2.4.1. INTRODUCTIONIn the biological and pharmacological importance, heterocycles plays a significance role. Oxadiazole molecules maneuver biologically activity includes angiogenesis inhibitor 246 and overly HIV inhibitor 247, tyrosine kinase inhibition 45, histamine H3 antagonism 48, muscarinic agonism 49, potent histamine H2 receptor antagonists 50, 51, muscarinic receptor antagonists 53, 54, interleukin-8 (IL-8) receptor antagonists 65, cytotoxic activities 68, monoamine oxidase inhibition 66, potent therapeutic agents for prostate gland cancer 72, anticonvulsant activity 67, neoplasm-selective and apoptosis-inducing agents 70, 71, antitumor 4f and apoptosis-inducing anticancer agents 73, 74.Breast cancer is a most terrifying disease in which cells in breast tissue paper grow and divide with issue normal control. This type of growth of cells without control forms a lump called tumor. In breast cancer, tumors are called benign or malignant. Malignant tumors willing grow by eating food. They get the food by forming new blood vessels in a process called angiogenesis. These blood vessels are the main tenability to promote the growth of the tumors. After this tumor growing it will spread to nearby tissue, which is called as invasion.The breakage of main tumor cells will spread into otherwise parts of the body and it will lead to metastatic breast cancer. This happens through blood stream or lymphatic system and this process is called metastasis. The main injury of the malignant breast cancer is dividing and grows out of control which leads to form number of new tumors. If those new tumors are in other parts of the body, then also we call those as breast cancer.Esp ecially in women, breast cancer leading to the cause of cancer related death. In developing and developed countries, breast cancer is the second most common malignancy type diagnosed disease in women. In India breast cancer is the most discussing problem in the up-to-date health problem (248). By the survey conceded by the Indian Council of Medical Research (ICMR), the percentage of breast cancer patients has been nearly doubled. In the past a few(prenominal) years nearly one lakh new patients are being detected from 1985 to 2001 (249, 250). It has been estimated that the breast cancer in 2004 is nearly 90,273 and they predicted that in 2015 the patients number may be nearly 1, 12,680 (251).Due to the damage in deoxyribonucleic acid, normal cells will become cancer cells. DNA is present in all cell and it directs to all its actions. When DNA gets damaged in normal cells, the cell either repairs the damage or it dies. But in the cancer cells, damaged DNA is not repaired. The dama ged cell undergoes splitting. As a result cell goes on making new cells that the body doesnt need and those cells have identical damaged DNA as the first cells does. This conjecture the design and synthesis of new anticancer drugs, and drug combination and treatment modalities is still the need for effective treatment of breast cancer patients 252.1,2,4-Oxadiazole moiety molecules show signs of vide variety of biological activities 40, 253-255. In connection to the above studies, our molecules are subjected to the angiogenesis victimization MCF-7 breast cancer cell lines and degradation of DNA studies using in EAT cells.2.4.2. MATERIALSMelting points were recorded (uncorrected) on a Buchi Melting Point B-545 instrument. Infrared (IR) spectra were recorded using a Jasco FTIR-4100 series. every last(predicate) reagents and solvents used were commercially procured and used as received. 1H-proton magnetic resonance spectras were recorded on Shimadzu AMX-400-Bruker with 400 MHz with TMS as internal standard. The 13C NMR spectra were examined on a Bruker DPX-400 at 100.6 MHz. The mass spectra were recorded on a JEOL JMS-AX505HA mass spectrometer.2.4.3. EXPERIMENTAL2.4.3.1. ChemistryGeneral procedure for synthesis of (Z)-4-(6,7-dihydro-5H-pyrrolo1,2-cimidazol-5-yl)-3-fluoro-N-hydroxybenzimidamide (2).A solution of hydroxylamine hydrochloride (1.529 g, 22.004 mmol) (2.5eq) and sodium carbonate (1.492 g, 14.082 mmol) (1.6eq) was taken in a round bottom flask. Stir for 10min to dissolve completely, then to this mixture 4-(6,7-dihydro-5H-pyrrolo1,2-cimidazol-5-yl)-3-fluorobenzonitrile (1) (2.0 g, 8.801 mmol) (1.0 eq) is dissolved with ethanol was added. Then the mixture is heated to 60 0C about 5-6 hr. After that the steps forward of the reaction fusion was examined by the thin social class chromatography (TLC). After reaction completion, the solvent and the product was disconnected in vacuum pump under reduced pressure. Then the product was poured to piss and ext racted with ethyl ethanoate. The fundamental degree was process 2-3 times with distilled water. The organic layer was washed 2-3 times with distilled water. The extracted ethyl ethanoate layer was dried over sodium sulphate (anhydrous) and the solvent was evaporated to get (Z)-4-(6,7-dihydro-5H-pyrrolo1,2-cimidazol-5-yl)-3-fluoro-N-hydroxybenzimidamide (2).2.4.3.2. Synthesis of 3-(4-(6,7-dihydro-5H-pyrrolo1,2-cimidazol-5-yl)-3-fluorophenyl)-5-substituted-1,2,4-oxadiazole 4(a-f) derivatives.(Z)-4-(6,7-dihydro-5H-pyrrolo1,2-cimidazol-5-yl)-3-fluoro-N-hydroxybenzimidamide (2) (1.0 eq) is dissolved in dry methylene chloride and cooled to 0-5 0C in ice bath. Then N,N-diisopropylethylamine (1.1 eq) was added to cold reaction mixture and stirred for 10 minutes, then different aromatic acid chlorides (3a-e) (1 eq) were added. The reaction mixture was allowed to inhabit temperature under stirring for 5-6 hr. After that the steps forward of the reaction fusion was examined by the thin la yer chromatography (TLC). After reaction completion, the solvent and the product was separated in vacuum pump under reduced pressure. Then the product was poured to water and extracted with ethyl ethanoate. The organic layer was washed 2-3 times with distilled water. The organic layer was washed 2-3 times with distilled water. The extracted ethyl ethanoate layer was dried over sodium sulphate (anhydrous) and the product was purified with the overhaul of column chromatography over silica mousse (60-120 mesh) using hexane and ethyl acetate (11).Scheme 1.Reagents and conditions (i) Sodium carbonate, water, ethanol, 60 0C, 6 h (ii) dichloromethane, N,N-diisopropylethylamine, 0-5 0C, 6 h 3(a-e) Where 3a = 4-chloro benzoyl chloride 3b = 4-Fluoro benzoyl chloride 3c = 4-(trifluoromethyl)benzoyl chloride 3d = 4-Fluoro-3-Nitrobenzoyl chloride 3e = 4-EthylPhenylbenzoyl chloride.2.4.3.2.1. Synthesis of 5-(4-chlorophenyl)-3-(4-(6,7-dihydro-5H-pyrrolo1,2-cimidazol-5-yl)-3-fluorophenyl)-1,2,4-o xadiazole (4a)Pale yellow likeness from (Z)-4-(6,7-dihydro-5H-pyrrolo1,2-cimidazol-5-yl)-3-fluoro-N-hydroxybenzimidamide (2) (0.1 g, 0.384 mmol), 4-chlorobenzoylchloride (3a) (0.067 g, 0.384 mmol) and N,N-diisopropylethylamine (0.049 g, 0.461 mmol). 1H NMR (400 MHz, CDCl3) 8.32 (d, 1H, Ar-H), 7.75 (dd, 2H, Ar-H), 7.70, (d, 1H, imid-H), 7.55 (d, 1H, Ar-H), 7.50 (dd, 2H, Ar-H), 7.35 (d, 1H, imid-H), 7.30 (d, 1H, Ar-H), 5.05 (d, 1H, pyrrole-H), 2.56-2.30 (d, 4H, pyrrole-H) MS (ESI) m/z 381.081 (100.0%), Anal. calcd. for C20H14ClFN4O (in %) C- 63.08, H- 3.71, N- 14.71.2.4.3.2.2. Synthesis of 3-(4-(6,7-dihydro-5H-pyrrolo1,2-cimidazol-5-yl)-3-fluorophenyl)-5-(4-fluorophenyl)-1,2,4-oxadiazole (4b)Orange food wringing from (Z)-4-(6,7-dihydro-5H-pyrrolo1,2-cimidazol-5-yl)-3-fluoro-N-hydroxybenzimidamide (2) (0.1 g, 0.384 mmol), 4-Fluoro benzoyl chloride (3b) (0.060 g, 0.384 mmol)and N,N-diisopropylethylamine (0.049 g, 0.461 mmol). 1H NMR (400 MHz, CDCl3) 8.31 (d, 1H, Ar-H), 7.30 (dd, 2H, Ar-H), 7.72, (d, 1H, imid-H), 7.56 (d, 1H, Ar-H), 7.34 (d, 1H, imid-H), 7.31 (d, 1H, Ar-H), 7.29 (dd, 2H, Ar-H), 5.02 (d, 1H, pyrrole-H), 2.58-2.31 (d, 4H, pyrrole-H) MS (ESI) m/z 365.114 (100.0%), Anal. calcd. for C20H14F2N4O (in %) C- 65.93, H- 3.87, N- 15.38.2.4.3.2.3. Synthesis of 3-(4-(6,7-dihydro-5H-pyrrolo1,2-cimidazol-5-yl)-3-fluorophenyl)-5-(4-(trifluoromethyl)phenyl)-1,2,4-oxadiazole (4c)Dark brown color from (Z)-4-(6,7-dihydro-5H-pyrrolo1,2-cimidazol-5-yl)-3-fluoro-N-hydroxybenzimidamide (2) (0.1 g, 0.384 mmol), 4-(trifluoromethyl)benzoyl chloride (3c) (0.080 g, 0.384 mmol) and N,N-diisopropylethylamine (0.049 g, 0.461 mmol). 1H NMR (400 MHz, CDCl3) 8.33 (d, 1H, Ar-H), 8.10 (dd, 2H, Ar-H), 7.74 (d, 1H, imid-H), 7.70 (dd, 2H, Ar-H), 7.58 (d, 1H, Ar-H), 7.37 (d, 1H, imid-H), 7.33 (d, 1H, Ar-H), 5.06 (d, 1H, pyrrole-H), 2.59-2.29 (d, 4H, pyrrole-H) MS (ESI) m/z 415.110 (100.0%), Anal. calcd. for C21H14F4N4O (in %) C- 60.87, H- 3.41, N- 13.52.2.4.3.2.4. Synthesis of 3-(4-(6,7 -dihydro-5H-pyrrolo1,2-cimidazol-5-yl)-3-fluorophenyl)-5-(4-fluoro-3-nitrophenyl)-1,2,4-oxadiazole (4d)Pale yellow color from (Z)-4-(6,7-dihydro-5H-pyrrolo1,2-cimidazol-5-yl)-3-fluoro-N-hydroxybenzimidamide (2) (0.1 g, 0.384 mmol), 4-Fluoro-3-Nitrobenzoyl chloride (3d) (0.078 g, 0.384 mmol)and N,N-diisopropylethylamine (0.049 g, 0.461 mmol). 1H NMR (400 MHz, CDCl3) 8.71 (d, 1H, Ar-H), 8.65 (d, 1H, Ar-H), 8.34 (d, 1H, Ar-H), 7.74 (d, 1H, imid-H), 7.61 (dd, 1H, Ar-H), 7.58 (d, 1H, Ar-H), 7.37 (d, 1H, imid-H), 7.33 (d, 1H, Ar-H), 5.06 (d, 1H, pyrrole-H), 2.59-2.29 (d, 4H, pyrrole-H) MS (ESI) m/z 410.099 (100.0%), Anal. calcd. for C20H13F2N5O3 (in %) C- 58.68, H- 3.20, N- 13.52.2.4.3.2.5. Synthesis of 3-(4-(6,7-dihydro-5H-pyrrolo1,2-cimidazol-5-yl)-3-fluorophenyl)-5-(5-ethyl-1,1-biphenyl-2-yl)-1,2,4-oxadiazole (4e).White color from (Z)-4-(6,7-dihydro-5H-pyrrolo1,2-cimidazol-5-yl)-3-fluoro-N-hydroxybenzimidamide (2) (0.1 g, 0.384 mmol), 4-EthylPhenylbenzoyl chloride (3e) (0.094 g, 0.384 mmol) and N,N-diisopropylethylamine (0.049 g, 0.461 mmol). 1H NMR (400 MHz, CDCl3) 8.31 (d, 1H, Ar-H), 7.95 (d, 1H, Ar-H), 7.80 (dd, 2H, Ar-H), 7.75 (d, 1H, Ar-H), 7.72, (d, 1H, imid-H), 7.53 (dd, 2H, Ar-H), 7.56 (d, 1H, Ar-H), 7.45 (d, 1H, Ar-H), 7.34 (d, 1H, imid-H), 7.30 (d, 1H, Ar-H), 7.31 (d, 1H, Ar-H), 5.03 (d, 1H, pyrrole-H), 2.65 (q, 2H, -CH2), 2.58-2.31 (d, 4H, pyrrole-H), 1.27 (t, 3H, -CH3), MS (ESI) m/z 451.186 (100.0%), Anal. calcd. for C28H23FN4O (in %) C- 74.65, H- 5.15, N- 12.44.2.4.4. Biology2.4.4.1. Culture of MCF-7 cellsMCF-7 cells were nuanced with minor modification in Minimal Essential medium (Invitrogen) supplemented with 10% foetal bovine serum, 100units/ml penicillin-G, 100 g/ml streptomycin and 1% sodium bicarbonate (Invitrogen). MCF-7 cells were obtained from booth repository unit of National Center for Cell Sciences (NCCS), Pune, India. All cell lines were maintained at 37C in a humidified atmosphere with 5% CO2 256.2.4.4.2. Culture of EAT cellsAnimals, in vivo tumor extension and imidazole derivatives treatment Six to eight weeks old female mice were acclimated for one week while caged in-group of five. Mice were housed and fed a diet of animal chow and water ad libitum throughout the experiment. All the experiments were approved by the institutional animal care and use committee of the University of Mysore, Mysore, India. Ehrlich Ascites Tumor (EAT) cells (5106 cells/mouse) were injected intraperitoneally. These cells grow in mouse peritoneum forming an ascites tumor with massive abdominal swelling. The animals showed a dramatic increase in body weight over the growth period and the animals succumbed to the tumor burden 1416 days after implantation.2.4.4.2.1. Isolation of EAT cells from mice peritoneal cavity and compound treatment From the peritoneal cavity of tumor-bearing mice the EAT cells were isolated (control and tempered). 2-3 mm of sterile phosphate buffer solution was injected in to the peritoneal cavity of the mice a nd the peritoneal fluid containing tumor cells withdrawn, collect in sterile petri dishes and incubated at 370C for 2 h. The cells of macrophage linage adhered to the bottom of Petri dishes. The non-adherent population was aspirated out gently and washed repeatedly with PBS. Moreover, viability of these cells was assessed and was found to be 95% by trypan blue dye exclusion. The viable EAT cells were processed for throw out experiments. The EAT cells (5 x 106) were treated with or without compounds of 3-(4-(6,7-dihydro-5H-pyrrolo1,2-cimidazol-5-yl)-3-fluorophenyl)-5-substituted-1,2,4-oxadiazole series 4(a-e) and incubated at 370 C for different time interval or for known period of time. After the incubation period the cells were used for the further analysis 258.2.4.4.2.2. Cell count by Trypan blue dye exclusion assay.EAT cells were treated with different concentrations of 3-(4-(6,7-dihydro-5H-pyrrolo1,2-cimidazol-5-yl)-3-fluorophenyl)-5-substituted-1,2,4-oxadiazole compounds 4(a-e ) at various time periods (04 h). Cell viability was assessed by mixing aliquots of cell suspension with 0.4% trypan blue and counted using heamocytometer. Cells that picked up the dye were considered to be dead 259(a).2.4.5. Result and Discussion2.4.5.1. ChemistrySynthesis of the bring out intermediate (Z)-4-(6,7-dihydro-5H-pyrrolo1,2-cimidazol-5-yl)-3-fluoro-N-hydroxybenzimidamide (2) is outlined in Scheme 1. Briefly, hydroxylamine hydrochloride and sodium carbonate was taken in water and stirred. 4-(6,7-dihydro-5H-pyrrolo1,2-cimidazol-5-yl)-3-fluorobenzonitrile (1) was dissolved in ethanol and added to the reaction mixture. The presence of NH2 and =N-OH proton peaks NMR spectra indicates the formation of (Z)-4-(6,7-dihydro-5H-pyrrolo1,2-cimidazol-5-yl)-3-fluoro-N-hydroxybenzimidamide (2). The key intermediate compound (2) was taken in dry dichloromethane and cooled to 0-5 0C, and N,N-diisopropylethylamine was added. Stirred for 10 min, then different aromatic acid chlorides 3(a- e) was added give notice by drop. The reaction mixture was allowed to room temperature under stirring for 5-6 h and after that the steps forward of the reaction fusion was examined by the thin layer chromatography (TLC). After reaction completion, the solvent and the product was separated in vacuum pump under reduced pressure. Then the product was poured to water and extracted with ethyl ethanoate. The organic layer was washed 2-3 times with distilled water to get target 3-(4-(3-(1H-imidazol-5-yl)propyl)-3-fluorophenyl)-5-substituted-1,2,4-oxadiazole 4(a-e). Upon completion crude products 4(a-e) were obtained with a good yield of 8193% and which the product was purified with the help of column chromatography over silica gel (60-120 mesh) using hexane and ethyl acetate (11). The absence of CO proton peak in synthesized derivatives in 1H spectra confirmed the identity of the products. The inside information of chemical structures, physical data and integrity of compounds are given i n panel 1.CompoundR1YieldMP (oC)Purity4a90277904b85100934c81110894d82142924e799581Table 1. Chemical structures, physical data and purity of compounds 4(ae)2.4.5.2. Biology2.4.5.2.1. MTT assayThe MTT assay was performed according to the protocol previously reported 257. MCF-7 cells were plated at a density of 1 X 105 cells in 96-well plates. (Subsequently, the 3-(4-(6,7-dihydro-5H-pyrrolo1,2-cimidazol-5-yl)-3-fluorophenyl)-5-substituted-1,2,4-oxadiazole series 4(a-e) were assayed using concentrations from 0.05 to 0.5 mM). After 24 h of incubation, 10 L of 5% 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma Aldrich) (0.05 mg/mL) were added to the culture medium. After 4 h at 370C the culture medium was removed and 200 L of DMSO were added to dissolve the salts of formazan. The absorbance was measured with a 96-wells plate spectrophotometer at 570 nm. The experiments were independently performed three times and each experiment contained double replicates. C ontrol samples containing a complete culture medium devoid of cells or control cells with 0.1% DMSO were also included in each experiment. word form 1. The MTT assay of compounds 4(a-e) in MCF-7 breast cancer cell lines.Sl.No.Name of the compoundIC50 Value1Cisplatin10g24a100ug34b200ug44c100ug54d800ug64e200ugTable 2. Compounds 4(a-e) and their IC50 value (g/ml) on MCF-7 breast cancer cell lines.2.4.5.3. DNA fragmentation assayEAT cells were collected from mice treated with or without 3-(4-(6,7-dihydro-5H-pyrrolo1,2-cimidazol-5-yl)-3-fluorophenyl)-5-substituted-1,2,4-oxadiazole series 4(a-e). Thein vivo and DNA was isolated using the phenolchloroform method. In brief, cells were lysed in a buffer containing 50mM TrisHCl, pH 8.0, and 0.5% SDS, and incubated for 30min at 37C. The cell lysate was subjected to 8M potassium acetate foolhardiness and left for 1h at 4C. The supernatant was subjected to phenol/chloroform/isoamyl alcohol (25241) extraction and once to chloroform extraction. D NA was precipitated by adding 12 volumes of ice-cold ethanol. The precipitated DNA was dissolved in 50L TE buffer (pH 8.0). The DNA was digested with 20g/mL RNase at 37C for 1h. The DNA was quantitated and equal concentration of DNA (25g) was resolved on 1.5% agarose gel, viewed under UV light, and documented using BIORAD gel documentation system Figure 2 259(b).Figure 2. The DNA degradation of compounds 4(a-e) in Ehrlich Ascites Tumor (EAT) cells.ConclusionA series of 3-(4-(6,7-dihydro-5H-pyrrolo1,2-cimidazol-5-yl)-3-fluorophenyl)-5-substituted-1,2,4-oxadiazoles 4(a-e) has been synthesized by using simple synthetic procedures and were screened for their MTT assay using MCF-7 breast cancer cell line and degradation of DNA in Ehrlich Ascites Tumor (EAT) cells activity. All the final compounds exhibited good in all the in-vitro activity.