الفهرس 
IntroductionOnly 14 pages are availabe for public view
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Abstract
Dihydrofolate reductase (DHFR) is an enzyme that catalyzes the reduction of folic acid or dihydrofolic acid into tetrahydrofolic acid which then converted into N5, N10-methylenetetrahydrofolate. This later compound functions as the source of the methyl group to convert dUMP into dTMP. Inhibition of DHFR has long been an attractive goal for the development of agents against bacterial and parasitic infections as well as cancer.
Thiazole derivatives constitute an important class of heterocyclic compounds; many of them possessed a wide range of biological activities as anti-inflammatory, analgesic, antifungal, antibacterial, anticancer and anticonvulsant activities. Accordingly, New series of thiazolo[4,5-d]pyridazin and imidazo [2’,1’:2,3]thiazolo[4,5-d]pyridazin analogues were designed, synthesized and evaluated for their in vitro DHFR inhibition, antimicrobial and antitumor activities.
Compounds 90, 115 and 139 proved to be the most active inhibitors with IC50 0.05, 0.06 and 0.06 M respectively, merely comparable to MTX (5, IC50 0.08 M). Compounds 89, 90, 108, 109, 111 and 139 showed GI values of 26.1, 43.8, 27.6, 29.4, 28.7 and 87.4 % against HL-60(TB) leukemia cell line, while compounds 88, 89, 108, 109 showed GI values of 40.4, 40.4, 31.4 and 31.7 % against NCI-H522 non-small cell lung cancer, respectively. Compounds 90 and 139 proved to be the most active antitumor members of this study, the compounds showed growth inhibition potency against most of the tested tumor cell lines. Compound 115 proved lethal to HS 578T breast cancer cell line while compound 139 proved lethal to OVCAR-3 Ovarian cancer and MDA-MB-435 Melanoma. Only compounds 111 and 112 showed moderate activity against the Gram-positive bacteria Staphylococcus aureus with 30.5 and 33.5% inhibition, respectively. Compounds 117 and 145 proved active against Candida albicans with 49.2 and 82.8 % inhibition, respectively; while compounds 113, 145 and 147 showed 88.5, 92.2 and 93.5 % inhibition, respectively against Candida Neoformans. Comparing the potency of the active antibacterial compounds and their DHFR inhibition revealed that compounds 111, 145 and 147 (IC50 values of 2.5, 0.11 and 0.15, respectively) might exert their activity through DHFR inhibition.
Compounds 108-117 and 139-150 formed key interactions with hDHFR pocket. A hydrogen bond was observed between N1-nitrogen of the pyrdazine ring with Glu30. Another H-bond was also formed between the carbonyl group of pyridazine ring with Trp24, Arg70 or Lys64, while the thiazolopyridazine moiety and imidazo-thiazolo-pyridazine moiety were both oriented in the deep hydrophobic pocket at the back of the ATP binding site. In addition -cation interaction with Arg22 and - interaction with Phe31 residues were observed in most of the targeted compounds.
This thesis consists of the following parts:
1. Introduction:
This section contains a brief description of cancer, cancer treatments, folate pathway, dihydrofolate reductase enzyme and dihydrofolate inhibitors. Moreover, literature review presents the various biological and pharmacological activities of DHFR inhibitors.
2. Research Objectives and Rational Design:
It includes the rationale upon which the newly synthesized compounds were designed.
3. Results and Discussion
3.1 Chemistry
It deals with the discussion of the various experimental methods, mechanisms and conditions of reactions adopted for the synthesis of the prepared compounds and the confirmation of their structures by brief data of mass, 1H-NMR and 13C-NMR spectra.
3.2 Biological Evaluation
3.2.1 Dihydrofolate Reductase (DHFR) Inhibition Assay
The synthesized compounds were evaluated for their in vitro DHFR inhibition activity at the Pharmacology and Biochemistry Department, Faculty of Pharmacy; Future University in Egypt. The results revealed that, Compounds 90, 115 and 139 proved to be the most active inhibitors with IC50 0.05, 0.06 and 0.06 M, respectively
3.2.2 Antitumor Screening
The synthesized compounds 88-90, 108-117, 121-123 and 139-150 were tested for their antitumor activity at In vitro antitumor testing was conducted at the NCI’s disease-oriented human cell lines assay facility, Bethesda, MD, USA. Cell Cycle analysis of the effect of compound 115 on HS 578T breast cancer cell line and compound 139 on OVCAR-3 Ovarian cancer and MDA-MB-435 Melanoma was performed at the confirmatory diagnostic unit, VACSERA, Egypt.
3.2.3 Antimicrobial Activity
The synthesized compounds were tested for their in vitro Antimicrobial screening by CO-ADD (The Community for Antimicrobial Drug Discovery), funded by the Wellcome Trust (UK) and The University of Queensland (Australia).” Only compounds 111 and 112 showed moderate activity against the Gram-positive bacteria Staphylococcus aureus with 30.5 and 33.5% inhibition, respectively. Compounds 117 and 145 proved active against Candida albicans with 49.2 and 82.8 % inhibition, respectively; while compounds 113, 145 and 147 showed 88.5, 92.2 and 93.5 % inhibition, respectively against Candida Neoformans.
3.3 Structure Activity Relationship
SAR study was discussed to focus on the important group that affect the activity.
3.4 Molecular Modeling
This part includes accurate picture of the biologically active molecule at the atomic level. Moreover, Computer docking technique that plays a major role in the mechanistic study by placing a molecule into the binding site of the target enzyme macromolecule. Also an ADMET study was made to study the calculation of certain parameters.
4. Conclusion
5. Experimental
This part presents the practical procedures used for the synthesis of new intermediates, as well as new final compounds. Also, it includes their physical and spectral data.
The following new compounds were synthesized
• Ethyl 2-[3-4-(chloro-phenyl)-thioureido]thiazole-4-carboxylate (88).
• Ethyl 2-[3-(4-methoxy-phenyl)-thioureido]thiazole-4-carboxylate (89).
• Ethyl 2-[3-(4-phenoxy-phenyl)-thioureido]thiazole-4-carboxylate (90).
• 1-(4-Chloro-phenyl)-3-(4-oxo-7-phenyl-4,5-dihydro-thiazolo[4,5-d]pyridazin-2-yl)thiourea (108).
• 1-(4-Methoxy-phenyl)-3-(4-oxo-7-phenyl-4,5-dihydro-thiazolo[4,5-d]pyridazin-2-yl)thiourea (109).
• 1-(4-Chloro-phenyl)-3-(7-(4-methoxy-phenyl)-4-oxo-4,5-dihydro-thiazolo[4,5-d]pyridazin-2-yl)thiourea (110).
• 1-(4-Methoxy-phenyl)-3-(7-(4-methoxy-phenyl)-4-oxo-4,5-dihydro-thiazolo[4,5-d]pyridazin-2-yl)thiourea (111).
• 1-(4-Chloro-phenyl)-3-(-4-oxo-7-(4-methyl-phenyl)-4,5-dihydro-thiazolo[4,5-d]pyridazin-2-yl)thiourea (112).
• (1-(4-Methoxy-phenyl)-3-(-4-oxo-7-(4-methyl-phenyl)-4,5-dihydro-thiazolo[4,5-d]pyridazin-2-yl)thiourea (113).
• 1-(4-Phenoxy-phenyl)-3-(-4-oxo-7-(4-methyl-phenyl)-4,5-dihydro-thiazolo[4,5-d]pyridazin-2-yl)thiourea (114).
• 1-(7-(4-Bromo-phenyl)-4-oxo-4,5-dihydro-thiazolo[4,5-d]pyridazin-2-yl)-3-(4-chloro-phenyl)thiourea (115).
• 1-(7-(4-Bromo-phenyl)-4-oxo-4,5-dihydro-thiazolo[4,5-d]pyridazin-2-yl)-3-(4-methoxy-phenyl)thiourea (116).
• 1-(7-(4-Bromo-phenyl)-4-oxo-4,5-dihydro=thiazolo[4,5-d]pyridazin-2-yl)-3-(4-phenoxy-phenyl)thiourea (117).
• Ethyl-5-(4-bromo-phenyl)-imidazo[2,1-b]thiazole-3-carboxylate (121).
• Ethyl-5-(4-methyl-phenyl)-imidazo[2,1-b]thiazole-3-carboxylate (122).
• Ethyl-5-(4-methoxy-phenyl)-imidazo[2,1-b]thiazole-3-carboxylate (123).
• 3-(4-Bromo-phenyl)-8-phenyl-imidazo[2’,1’:2,3]thiazolo[4,5-d]pyridazin-5(6H)-one (139).
• 3-(4-Methyl-phenyl)-8-phenyl-imidazo[2’,1’:2,3]thiazolo[4,5-d]pyridazin-5(6H)-one (140).
• 3-(4-Methoxy-phenyl)-8-phenyl-imidazo[2’,1’:2,3]thiazolo[4,5-d]pyridazin-5(6H)-one (141).
• 3-(4-Bromo-phenyl)-8-(4-methoxy-phenyl)-imidazo[2’,1’:2,3]thiazolo[4,5-d]pyridazin-5(6H)-on (142).
• 3-(4-Methyl-phenyl)-8-(4-methoxy-phenyl)-imidazo[2’,1’:2,3]thiazolo[4,5-d]pyridazin-5(6H)-on (143).
• 3-(4-Methoxy-phenyl)-8-(4-methoxy-phenyl)-imidazo[2’,1’:2,3]thiazolo[4,5-d]pyridazin-5(6H)-one (144).
• 3-(4-Bromo-phenyl)-8-(4-methyl-phenyl)-imidazo[2’,1’:2,3]thiazolo[4,5-d]pyridazin-5(6H)-one (145).
• 3-(4-Methyl-phenyl)-8-(4-methyl-phenyl)-imidazo[2’,1’:2,3]thiazolo[4,5-d]pyridazin-5(6H)-one (146).
• 3-(4-Methoxy-phenyl)-8-(4-methyl-phenyl)-imidazo[2’,1’:2,3]thiazolo[4,5-d]pyridazin-5(6H)-one (147).
• 3,8-bis-(4-Bromo-phenyl)-imidazo[2’,1’:2,3]thiazolo[4,5-d]pyridazin-5(6H)-one (148).
• 8-(4-Bromo-phenyl)-3-(4-methyl-phenyl)-imidazo[2’,1’:2,3]thiazolo[4,5-d]pyridazin-5(6H)-one (149).
• 8-(4-Bromo-phenyl)-3-(4-methoxy-phenyl)-imidazo[2’,1’:2,3]thiazolo[4,5-d]pyridazin-5(6H)-one (150)
This part includes also the procedures of DHFR inhibition assay, antitumor activity, antimicrobial activity and molecular modeling study.
6. References:
This part includes 183 references covering the period from 1948-2017.
7. Appendix.
8. Arabic Summary