LIST OF SYMBOLS AND ABBREVIATIONS
3. REVIEW OF LITERATURES
4.1 Experiment Envisaged .1 Synthesis scheme 57-59
N
Synthesis and anticancer activity of some novel benzimidazole nanoparticles Page 36 4.1.1.1 Synthesis of benzimidazolyl chalcones (3a-e)
N potassium hydroxide solution was taken in a round bottom fask to this add 0.012 molar of various aromatic aldehyde (2a-e) namely, benzadehyde (2a) , pyridine aldehyde (2b) , 3-chloro-4- methoxy benzadehyde (2c), 3,4-dimethoxy benzadehyde (2d), anthracene aldehyde (2e) separately, with stirring then refux the mixture for 2 hours, cool to room temperature and pour in to a beaker containing ice cold water, with stirring to get a product.
The precipitated chacolnes was filtered with ice cold water, washed dried and recryataized from ethanol. The chemical names of above synthesized benzimidazoly chalcones as follows: (3a-e).
1-(1H-benzo[d]imidazole-2-yl)-3-phenylprop-2-en-1-one (3a)
1-(1H-benzo[d]imidazole-2-yl)-3-(pyridine-4-yl)prop-2-en-1-one (3b)
1-(1H-benzo[d]imidazole-2-yl)-3-(3-chloro-4-methoxyphenyl)prop-2-en-1-one (3c)
1-(1H-benzo[d]imidazole-2-yl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one (3d)
3-(anthracen-10-yl)1-(1H-benzo[d]imidazole-2-yl)prop-2-en-1-one (3e)
Synthesis and anticancer activity of some novel benzimidazole nanoparticles Page 37 4.1.1.2 Synthesis of 1-methyl-benzimidazolyl chalcones (3f-j)
N
Weighed 0.01 molar of 1-methyl-2-acteyl benzimidazole (1b) and 10m 30
% potassium hydroxide solution was taken in a round bottom fask to this add 0.012 molar of various aromatic aldehyde (2a-e) namely, benzadehyde (2a) , pyridine aldehyde (2b) , 3-chloro-4- methoxy benzadehyde (2c), 3,4-dimethoxy benzadehyde (2d), anthracene aldehyde (2e) separately, with stirring then refux the mixture for 2 hours, cool to room temperature and pour in to a beaker containing ice cold water, with stirring to get a product.
The precipitated chacolnes was filtered with ice cold water, washed dried and recryataized from ethanol. The chemical names of above synthesized benzimidazoly chalcones as follows: (3f-j).
1-(1-methyl-1H-benzo[d]imidazole-2-yl)-3-phenylprop-2-en-1-one (3f)
1-(1-methyl-1H-benzo[d]imidazole-2-yl)-3-(pyridine-4-yl)prop-2-en-1-one
Synthesis and anticancer activity of some novel benzimidazole nanoparticles Page 38 4.1.1.3 Synthesis of benzo[d]imidazol-2-yl pyrimidine derivatives (4a-e)
N guninidine hydrochloride (0.001 mol) in presences of potassium hydroxide (0.002 mol) in absolute ethanol (5ml) at refux on a water bath for 3 hours.
The reaction mixture was cooled at room temperature and poured in ice cold water. Solid product was filtered and washed with water, dried and recrystallized to give 4(a-e), respectively.
4-(1H-benzo[d]imidazol-2-yl)-6-phenylpyrimidin-2-amine (4a)
4-(1H-benzo[d]imidazol-2-yl)-6-(pyridin-4-yl)pyrimidin-2-amine (4b)
Synthesis and anticancer activity of some novel benzimidazole nanoparticles Page 39 4.1.1.4 Synthesis of 1-methyl-benzo[d]imidazol-2-yl pyrimidinederivatives (4f-j) hydrochloride (0.001 mol) in presences of potassium hydroxide (0.002 mol) in absolute ethanol (5ml) at refux on a water bath for 3 hours.
The reaction mixture was cooled at room temperature and poured in ice cold water.
Solid product was filtered and washed with water, dried and recrystallized to give 4(f-j), respectively.
4-(1-methyl-1H-benzo[d]imidazol-2-yl)-6-phenylpyrimidin-2-amine (4f)
4-(1-methyl-1H-benzo[d]imidazol-2-yl)-6-(pyridin-4-yl)pyrimidin-2-amine
Synthesis and anticancer activity of some novel benzimidazole nanoparticles Page 40 4.1.1.5 Synthesis of (pyrazol-3-yl)-benzo[d]imidazole derivatives (5a-e)
N
Solid product was filtered and washed with water, dried and recrystallized to give 5(a-e), respectively.
2-(1,5-diphenyl-1H-pyrazol-3-yl)-1H-benzo[d]imidazole (5a)
2-(1-phenyl-5-(pyridin-4-yl)-1H-pyrazol-3-yl)-1H-benzo[d]imidazole (5b)
Synthesis and anticancer activity of some novel benzimidazole nanoparticles Page 41
2-(1,5-diphenyl-1H-pyrazol-3-yl)-1-methyl-1H-benzo[d]imidazole (5f)
Synthesis and anticancer activity of some novel benzimidazole nanoparticles Page 42 4.2 In-Vitro Cytotoxicities studies 60
In-vitro anti-cancer studies (MTT - assay) for synthesized novel benzimidazole derivatives by using CaCo-2 colon cancer cell line and MCF-7 breast cancer cell line.
4.2.1 In-vitro cytotoxic evaluation against MCF-7 and CaCo-2 cell line61 4.2.1.1 Cell culture
MCF-7 (Human breast cancer cell line) and CaCo-2 (Human colon cancer cell line) were obtained from NCCS Pune.
It was maintained in Roswell Park Memorial Institute (RPMI) supplemented with 10% fetal bovine serum (FBS), 2mM glutamine, amphotericin (3 g/ml), gentamycin (400 g/ml), streptomycin (250
g/ml), penicillin (250 units/ml) and 1mg/ml insulin in the incubator at 5% CO2, 37°C was obtained from NCCS Pune.
4.2.1.2 Preparation of media and other reagents required for cell culture 4.2.1.3 RPMI medium
Roswell Park Memorial Institute (RPMI) medium was prepared as follows:
The powdered media was dissolved in 900 ml of sterile glass distilled or Millipore water in an autoclaved glass conical flask under sterile conditions.
The antibiotics were added in the concentration as mentioned above and stirred well. 3.7 g of Sodium bicarbonate was added into the flask and stirred until it gets dissolved completely. 10% FBS was added and mixed well.
The liquid was slowly poured into the upper portion of a media sterilisation unit and filtered through a 0.2 filter under negative pressure.
The medium was immediately stored at 4oC.
Synthesis and anticancer activity of some novel benzimidazole nanoparticles Page 43 4.2.1.4 Saline: Trypsin: Versene (STV)
10X Saline A: 8g NaCl, 0.4g KCl, 1.0g D-Glucose and 0.35 g NaHCO3
(Tissue Culture Grade) were dissolved in 100 ml water and stored at 4oC.
Versene: 1g EDTA (Tissue Culture grade) was added into 90 ml distilled water. Then 5N NaOH was added drop wise until it gets dissolved.
The solution was filter sterilizes and stored at 4oC. 100 ml of STV was prepared by adding 25 mg of trypsin in a mixture of 10 ml of 10X Saline A and 2.5 ml of Versene Double distilled water was added to make upto 100 ml, sterile filtered, liquated, and frozen at –20oC to –70oC.
4.2.1.5 Maintaining and storage of cell lines
MCF-7 and CaCo-2 cells show a steady growth rate with a doubling time approximately 33 hours. The cells reached confluency in 3 to 4 days and these cells were passaged to get the cells for the experiments and also to store in liquid nitrogen.
The culture medium was removed from the T25 culture flask by decanting into a clean container inside the laminar airflow chamber, and cells were rinsed with medium without serum, to remove traces of serum, which may inhibit action of trypsin. 2 ml of STV solution was added to the flask containing cells and incubated at 37oC for a few minutes. As soon as cells started dislocating from the surface, flask was rinsed with 5ml of serum-containing medium to arrest the trypsinisation.
The suspension of cells was collected in a sterile 15 ml centrifuge tube and the cells were pelleted at 1500 rpm for 5 minutes.
The cell pellet was resuspended in fresh medium with serum and a part of the cells were seeded back into the flask.
The remaining cells were used for experiment or pelleted as earlier and resuspended in cryopreservative medium (syntha freeze) in a cryovial and frozen at -70oC for a day then transferred to liquid nitrogen.
Synthesis and anticancer activity of some novel benzimidazole nanoparticles Page 44 4.2.1.6 Drug Preparation
All the compounds were dissolved in DMSO to give a stock concentration of 50μg/μl.
The stock solution was then serially diluted with culture medium.
The test concentration were 0.1,0.3,1,3,10,30 μM.
The concentration of DMSO never exceeded 1% in any of the 96 wells.
4.2.1.7MTT assay
Cell lines were maintained in RPMI supplemented with 10% FBS, 2mM glutamine, amphotericin (3 g/ml), gentamycin (400 g/ml), streptomycin (250 g/ml) and penicillin (250 units/ml) in a carbon dioxide incubator at 5% CO2.
Approximately 2 X 104 cells/well were seeded in 96 well plate using culture medium,the viability was tested using trypan blue dye with help of haemocytometer and 95% of viability was confirmed.After 24hrs, the fresh medium with the extracts were added at respective wells and kept incubation for 72 hrs. After incubation the following assays were performed.
After 72hrs of the drug treatment the fresh medium was changed again for all groups and 10 μl of MTT (5 mg/ml stock solution) was added and the plates were incubated for an additional 4 h. The medium was discarded and the formazan blue, which was formed in the cells, was dissolved with 100μl of DMSO.
The optical density was measured at 570nm.
The percentage toxicity was calculated by using following formula. Graph pad prism software was used to calculated IC50 of the extracts.
% Toxicity = 1- treated cells/untreated cells x 100 4.2.1.8 Apoptosis Studies
Apoptosis studies were performed with a staining method utilizing acridine orange (AO) and ethidium bromide (EB).
Synthesis and anticancer activity of some novel benzimidazole nanoparticles Page 45
CaCo-2 cells were incubated in the absence or presence of the test compound at 37°C and 5% CO2 for 48 h. After 48 h, cells were stained with AO/EB solution (100 μg/mL AO, 100 μg/mL EB).
The cells were observed under a fluorescence microscope.