ANTI-CANCER ACTIVITY OF SOME NOVEL DERIVATIVES OF 2-AMINO BENZOTHIAZOLES: IN VITRO AND IN VIVO STUDIES.
M. Pharm Dissertation Protocol
Submitted to the
Rajiv Gandhi University of Health Sciences, Karnataka, Bengaluru
BY
MR. ASHWIN SANGAM M
B. Pharm.
UNDER THE GUIDANCE OF
PROF. ITTAGI SHANMUKHA
M. Pharm., (Ph.D).
P. G. DEPARTMENT OF PHARMACOLOGY
S. C. S. COLLEGE OF PHARMACY,
HARAPANAHALLI-583131
2013-14
Rajiv Gandhi University of Health Sciences, Karnataka, Bengalore.
Annexure – II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
01 / Name and Address of the Candidate /ASHWIN SANGAM M
S/O MANJUNATHA S GGAJJUGANAHALLI,THIMMAPANAHALLI(POST),CHALLAKERE(Tq),CHITRADURGA(DIST)-577543.
S02 / Name of the Institution / T. M. A. E. Society’s
S. C. S. College of Pharmacy,
Harapanahalli – 583 131
Davangere - dist
Karnataka
03 / Course of the Study
Branch / M. Pharm.
(Pharmacology)
04 / Date of Admission to course / 05-08-2013
05 / Title of the Topic / ANTI-CANCER ACTIVITY OF SOME NOVEL DERAVITIVES OF 2-AMINO BENZOTHIAZOLES: IN VITRO AND IN VIVO STUDIES.
06 /
Brief resume of the intended work
6.1. Need for the Study /Enclosure – I
6.2. Review of the Literature / Enclosure – II6.3. Objective of the Study
/ Enclosure – III07 /
Materials and Methods
7.1. Source of data /Enclosure – IV
7.2. Methods of collection of data
/ Enclosure – V7.3. Does the study require any
Investigations on animals?
If yes give details / Enclosure – VI
7.4. Has ethical clearance been
obtained form your institution
in case of 7.3. / Yes, Registration No: 157/PO/C/ 1999/ CPCSEA
(Copies enclosed)
08 /
List of References (About 4 – 6)
/ Enclosure – VII09 /
Signature of the Candidate
/ (ASHWIN SANGAM M)10 /
Remarks of the Guide
/ The present research work is original and not published in any of the journals. This work can be carried out in our Pharmacology laboratory.11 /
Name and Designation of
(in Block Letters)11.1. Guide
11.2.Signature
11.3.Co-Guide (if any)
11.4.Signature
11.5. Head of the Department
11.6. Signature / Prof. ITTAGI SHANMUKHA
M. PHARM.,(PhD)
P.G. Dept. of Pharmacology
S.C.S. College of Pharmacy
Harapanahalli-583 131.
Davangere. (Dist.)
Karnataka
------
Prof. A. VEERANA GOUDA
M. Pharm.
Head of the dept. of pharmacology
S.C.S. College of Pharmacy
Harapanahalli-583 131.
Davangere. (Dist.)
Karnataka
12 /
Remarks of the Principal
12.1. Signature / The present study is permitted to perform in the Pharmacology laboratory of our institution and the study protocol has been approved by IAEC.Dr.R.Nagendra Rao
Principal
ENCLOSURE-I
06. Brief resume of Intended Work
6.1 Need for the study.
Cancer is a group of diseases caused by loss of cell cycle control. Cancer is associated with abnormal uncontrolled cell growth.1 It is caused by both external factors (tobacco, chemicals, radiation and infectious organisms) and internal factors (inherited mutations, hormones, immune conditions, and mutations that occur from metabolism). It is a widespread life-threatening disease that attacks people at all ages, especially those over 65 years. It is significant worldwide health problem generally due to the lack of widespread and comprehensive early detection methods, the associated poor prognosis of patients diagnosed in later stages of the disease and its increasing incidence on a global scale. Indeed, the struggle to combat cancer is one of the greatest challenges of mankind.2 It is an environmental disease related to lifestyle, environmental factors and less commonly genetic factors. Various remedies have been reported for the treatment of this disease, but the development of suitable therapeutic is still a major challenge. Many substances are in clinical trial or used in practice to treat cancer. However, they have major drawbacks; their high cost and adverse effects.3,4 Therefore, our target is to look for alternative ways to develop novel drug candidates with fewer side effects and less cost.
With the current chemotherapy, lack of selectivity of chemotherapeutic agents against cancerous cells is a significant problem. Receptor tyrosine kinases (RTKs) are high affinity cell surface receptors that bind polypeptide growth factors, cytokines and hormones. They have been shown to be key regulators of normal cellular processes and additionally play a critical role in the development and progression of many types of cancer.5 Protein tyrosine kinases occupy a central position in the control of cellular proliferation. Over expression of certain RTKs show association with promotion and maintenance of malignancies. Thus, inactivation of the specific tyrosine kinases those are responsible for the malignant phenotype of certain cancers represent a potential approach for design of antiproliferative drugs.6,7
Benzothiazole type compounds have attracted considerable attention to anticancer research and several attempts were made for modifying the benzothiazole nucleus to improve their anti-tumor activities. Modifications on the benzothiazole nucleus have resulted in a large number of compounds having diverse pharmacological activities. Among them imidazo benzothiazoles as well as polymerized benzothiazoles and other substituted benzothiazoles such as 2-(3,4-dimethoxyphenyl)-5-fluorobenzothiazole (PMX 610) has been shown to exhibit exquisitely potent (GI50 < 0.1 nM) and selective in vitro antitumor properties in human cancer cell lines (e.g., colon, non small e cell lung and breast subpanels) of the National Cancer Institute (NCI) 60 human cancer cell line screen8
and also exhibited remarkable antitumor activity against malignant cell lines.9 2-(4-aminophenyl)-benzothiazole (CJM 126) and its analogs comprise a novel mechanistic class of antitumor agents.10,11 These nucleuses come from the related structure polyhydroxylated 2-phenylbenzothiazoles, flavone quercetin and the isoflavone genistein, which are tyrosine kinase inhibitors bearing potent antitumor activity.12,13 The isoflavone, genistein and the flavone, quercetin are competitive inhibitors at the ATP-binding site of kinases.14,15 As the crystal structure of 5,6-dimethoxy-2-(4-methox- yphenyl) benzothiazole was solved, the preliminary analysis based on comparisons between polyhydroxylated 2-phenyl- benzothiazoles and the adenine fragment of ATP suggested that suitably substituted benzothiazoles might mimic the ATP competitive binding of genistein and quercetin at tyrosine kinases. Hence in continuation of our efforts on the design and synthesis of novel anti-cancer agents 16-18 and keeping in mind the medicinal importance of benzothiazole moiety. The research aimed at further exploration of the SAR of this novel benzothiazoles core has led to discovery of highly selective compounds that target tyrosine kinases. The novel compounds synthesized were meant to act via competing with ATP for binding at the catalytic domain of tyrosine kinase. Taking inspiration from above mentioned facts. The proposed study is planned to evaluate the newly synthesized benzothiazoles for their anti cancer potential using In vitro and In vivo models.
Chemistry
The compounds were synthesized using various isothiocyanates 2(a-e), which was prepared from different aromatic primary amines 1(a-e). Prepared isothiocyanates 2(a-e) yielded thioureas 3(a-e) on condensation with 2,6-dimethyl and 2,6-dichloro aniline. Oxidative cyclization of 3(a- e) by bromine resulted in the synthesis of proposed compounds 4(a-j).
ENCLOSURE-II
6.2 Review of Literature:
The literature survey revealed that modifications on the benzothiazole nucleus have resulted in a large number of compounds having diverse pharmacological activities as follows:
Pyrimido [2,1-b] benzothiazoles were synthesized by conjugation addition to iminonitrogen of 2-aminobenzothiazoles to alkyne β-carbon atom of acytylenic acid followed by ring closure and synthesized compounds are studied for antimicrobial activity against E.coli and Enterobacter as test organisms at conc 100μg per disc using vancomycine and meropenam as standard drug.19
Some newly synthesized benzothiozole and benzisoxazole from 2-amino 5/6-hydroxybenzothiazole, 6-hydroxy-3-methyl-1, 2-benzisoxal and different dihaloalkanes and screened for their antimicrobial activity against Staphylococcus aureus, and E. coli by disc diffusion method and anti fungal activity against Aspergillus flavus,and Candida albicans. Ciprofloxacin (10μg/ml) and fluconazole (10μg/ml) were used as standard drug for antibacterial and antifungal activity respectively.20
Some newly synthesized 2-mercaptobenzothiazoles and coreleted the effect on antimicrobial potency by varying the substituents in benzene part of the benzothiazole ring system.21
Aryl substituted benzothiazoles were synthesized and evaluated them against Human Cervical Cancer cell lines as anticancer drugs.22 Some benzothiazole containing phthalimide were synthesized and studied their anti-cancer activity on human carcinoma cell lines.23
Newly synthesized carbon 11 labeled fluorinated 2-aryl benzothiazoles used for protein emission tomography (PET) to image tyrocinekinese in cancer.24 Fluorinated benzothiazole analogue 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F203, NSC 703786) (1d), exhibit selective and potent anticancer activity. It is the favoured analogue for clinical consideration possessing enhanced efficacy in vitro and superior potencies against human breast and ovarian tumor xenografts implanted in nude mice Its lysylamide prodrug,(Phortress, NSC710305), (1e) is under phase I clinical trials at the United Kingdom.25
Flurobenzothiazole comprising sulfonamide pyrazole derivitives were screened for anthelmentic activity by using earthworms (Peritumaposthum). Albendazole was used as standard drug.The compounds were evaluated by time taken for complete paralysis and death of worms.26
2-amino [5`(4-sulphonylbenzylidine)-2,4-thiazolidnedione]-7-chloro-6-flurobenzothiazole series were synthesized and screened for their antidiabetic activity.27
F-labeled 2-(4′-fluorophenyl)-1-3-benzothiazoles were evaluated as amyloid imaging agent in Alzheimers disease in comparison with [11C]PIB (11C labeled 6-hydroxy-2-(4”-N- [11C] methylaminophenol)-1,3-benzothiazole and showed excellent characteristics comparable with those of [11C]PIB, namely good affinity for amyloid plaques present in human Alzheimers disease.28
ENCLOSURE -Ш
6.3 Objectives of the study:
The objective of study is to evaluate the In vitro and In vivo anti-cancer activity
1. Novel derivatives of 2-amino benzothiazoles (Compound 4a and Compound 4i) are evaluating for their in-vitro cytotoxicity against three human cancer cell lines such as: Lung cancer cell line(A 549),breast cancer cell line (MCF7) and colerectal cancer cell line (HT29) and
2. In Vivo anti-tumor activity against Ehrlich ascites carcinoma (EAC) tumor bearing Swiss albino mice at two different concentrations.
ENCLOSURE – IV
7. Material & methods:
7.1 Method of collection of data:
The data is generated using laboratory experimental techniques. The cancerous tissues are obtained from Radiant Research Bangalore. Culture preparation and development of different human cell-lines are going to be carried out in Radiant Research, Bangalore. In-vivo pharmacological activities will be carried out on Swiss albino mice.
Statistical Analysis
The data are expressed as the mean ± standard error of the mean (SEM). Statistical analysis will be performed by one-way ANOVA, followed by Tukey’s post hoc test using Graph Pad Prism 5.02. Differences were considered significant p<0.05.
ENCLOSURE – V
7.2 Materials and methods
Assessment of in vitro cytotoxic activity:29
The cytotoxicity of the synthesized compounds will be measured using the MTT assay against three human cancer cell lines: Lung cancer cell line (A 549), breast cancer cell line (MCF7) and colerectal cancer cell line (HT29).
The MTT assay, based on the conversion of the yellow tetrazolium salt-MTT to purple-formazan crystals by metabolically active cells, provides a quantitative determination of viable cells. Cells are plated on to 96 well plates at a cell density of 2×105 mL-1 per well in 100 µL of RPMI 1640 and allowed to grow in CO2 incubator for 24 h (37˚C, 5 % CO2). The medium is then removed and replaced by fresh medium containing different concentrations of sample for 48 h. The cells are incubated for 24-48 hr (37 ˚C, 5 % CO2). Then, 20µL MTT ([3-(4, 5-dimethylthiazol-yl)-2, 5-diphenyltetrazolium bromide]) stock solution (5 mg/mL in PBS) is added to each well and incubated for 5 h. The medium is removed and 200 µL DMSO is added to each well to dissolve the MTT metabolic product. Then the plate is shaken at 150 rpm for 5 min and the optical density is measured at 560nm. Untreated cells (basal) are used as a control of viability (100 %) and the results are expressed as % viability (log) relative to the control.
In vivo Anti-tumor Activity against Ehrlich’s Ascites Carcinoma (EAC) Model in Mice:30
EAC cells are aspirated from EAC tumor bearing mice using a 23 gauge needle into a sterile syringe. Cells viability is determined by trypan blue exclusion test and cells wiil be counted using hemocytometer. The ascitic fluid is suitably diluted in normal saline or sterile phosphate buffered saline (PBS) to get a concentration of 10× 106 cells/ml of cell suspension. The animals are randomly dividing into 7 groups of ten animals in each group and then 0.25 ml (2.5 million cells) of cell suspension is injected intraperitoneally to each mouse except normal group and treatment with test compounds or vehicle is as follows:
Group 1: The normal mice.
Group 2: The EAC-bearing mice (Control).
Group 3: The EAC-bearing mice treated with Gefitinib 25 mg/kg, i.p.
Groups 4 and 5: The EAC bearing mice treated with Compound-4a at 10 and 20 mg/kg, i.p.
Groups 6 and 7: The EAC bearing mice treated with Compound-4i at 10 and 20 mg/kg, i.p.
After 24 hrs of tumor inoculation, Standard (Gefitinib) and the test compounds are administered intraperitoneally on days 1, 3, 5, 7, 10, 12 and 14 (7doses). The control group is treated with same volume of the vehicle [0.25% CMC (Carboxy Methyl Cellulose)]. Every third day animals are weighed
to assess the tumor growth. Twenty-four hours of last dose and 18hr fasting, five animals from each group will be sacrifice for measurement of tumor volume, tumor cell count, viable and nonviable cells count and rest of animals will be kept with food and water to check percentage increase in lifespan(%ILS) and survival time.
Haematological Parameters:
To study the influence of treatment on haematological parameters, blood will be collected from similar set of animals, on 15th day from retro-orbital plexus. The blood will be collecting into a microcentrifuge tubes containing EDTA and to determine (1) haemoglobin count, (2) erythrocyte count and (3) leukocyte count.
ENCLOSURE – VI
7.3 Does the study require any investigation or interventions to be conducted on patients
or other humans or animals? If so, please describe briefly.
Yes, Albino mice will be used for the evaluation of anti-cancer activity.
7.4 Has ethical clearance been obtained from your institution in case of 7.3?
The present study is applied for permission from Institutional Animal Ethics
Committee.
ENCLOSURE – VII
List of references:
1. Krishnamurthi K. Screening of natural products for anticancer and antidiabetic properties.
Health Administrator 2007; 1&2: 69-75.
2. Divisi D, Di TS, Salvemini S, et al., Diet and cancer. Acta Biomed 2006; 77: 118-123.
3. Sogno I, Vannini N, Lorusso G, Cammarota R, Noonan DM, et al. Antiangiogenic activity of a novel class of chemopreventive compounds: oleanic acid terpenoids. Recent Results Cancer Res 2009;181: 209-12.