MINISTRY OF HEALTH HANOI UNIVERSITY OF PHARMACY NGUYEN NHAT MINH SYNTHESIS AND BIOEVALUATION OF NOVEL 2-MERCAPTOQUINAZOLINE- BASED N-HYDROXYHEPTANAMIDE AS ANTI-CANCER AGENTS PHARMACIST GRADUATION THESIS HANOI – 2025 MINISTRY OF HEALTH HANOI UNIVERSITY OF PHARMACY NGUYEN NHAT MINH Student ID: 2001726 SYNTHESIS AND BIOEVALUATION OF NOVEL 2-MERCAPTOQUINAZOLINE- BASED N-HYDROXYHEPTANAMIDE AS ANTI-CANCER AGENTS PHARMACIST GRADUATION THESIS Supervisors: 1. Dao Thi Kim Oanh 2. Le Thi Thao Conducted at: 1. Department of Pharmaceutical Chemistry Faculty of Pharmaceutical Chemistry and Technology HANOI – 2025 ACKNOWLEDGEMENTS Before presenting my graduation thesis, I want to sincerely thank all people who are always there to inspire and support me throughout my scientific research process over the past three years.
First of all, I would like to express my gratitude to my brilliant and passionate teachers, Prof. Nguyen Hai Nam, Assoc. Dao Thi Kim Oanh, Dr. Duong Tien Anh and M.
Le Thi Thao - Department of Pharmaceutical Chemistry - Faculty of Pharmaceutical Chemistry and Technology – Hanoi University of Pharmacy for all the useful advices and guidances, as well as the motivation you all gave me whenever I was in deep water. You set an example for me to try my best every day and gave me the best opportunity to study, practice, and finish my graduation thesis. I am incredibly grateful for the help from all the teachers and technicians of Department of Pharmaceutical Chemistry - Hanoi University of Pharmacy, Department of Chemistry – University of Science – Vietnam National University, Vietnam Academy of Science and Technology and Department of Pharmacy - Chungbuk National University, Cheongju, Korea. I extremely appreciate the indispensable support of my teammates in the laboratory of Department of Pharmaceutical Chemistry - Hanoi University of Pharmacy, especially my big teammates Vu Anh Minh, Vu Ha Vy, Ngo Quoc Dat, Tran Thi Thu Huyen, Nguyen Thi Nga and Nguyen Duc Tu, my friends Vo Thi Minh Huyen, Nguyen Phuong Ngan, Hoang Kim Ngoc and Nguyen Duc Thien and my younger pals Nguyen Thi Hieu Anh, Nguyen Trang Nhung and Nguyen Thi Phuong Thao and other teammates.
Lastly, I would like to express my appreciation to my family, my friend Nguyen Tran Mai Phuong and her mother Mrs Tran Thi Lan Huong and my classmate Nguyen Thu Thao for always be there for me and inspire me to finish my graduation thesis. Hanoi, March 17th, 2025 Student Nguyen Nhat Minh TABLE OF CONTENTS Page ABBREVIATIONS 4 LIST OF TABLES 6 LIST OF FIGURES 7 LIST OF SCHEMES 8 LIST OF CHARTS 9 INTRODUCTION 1 Chapter 1. QUINAZOLINE DERIVATIVES AS ANTI-CANCER AGENTS 2 1. Synthesis of quinazolin-4(3H)-one derivatives 2 1.
Anticancer activity of quinazolin-4(3H)-one derivatives 4 1. Overview of histone deacetylase 6 1. Active site’s structure 7 1. HISTONE DEACETYLASE INHIBITORS 7 1.
HDAC inhibitors’ role in carcinogenesis 9 1. DIRECTION FOR STRUCTURAL DESIGN 11 Chapter 2. Evaluation of in vitro anticancer activity 13 2. Purify assessment methods 14 2.
Structures determining methods 14 2. Anticancer activity evaluating methods 15 Chapter 3. EXPERIMENTS, RESULTS & DICUSSION 17 3. Bioactivity evaluation 35 Chapter 4.
CONCLUSION AND RECOMMENDATION 38 4. RECOMMENDATION 38 REFERENCES 1 APPENDIX 5 ABBREVIATIONS 13 C-NMR Carbon-13 Nuclear Magnetic Resonance Spectroscopy 1 H-NMR Proton Nuclear Magnetic Resonance Spectroscopy AcOH Acetic acid ADR Adriamycin AR Analytical reagents B16 Murine tumor cell line used for research as a model for human skin cancers Bak Bcl-2 homologous antagonist killer Bax Bcl-2-associated X protein Bcl-2 B-cell lymphoma 2 Bcl-xL B-cell lymphoma-extra large c-Myc Gene causing cancer when mutated CTCL Cutaneous T cell lymphoma DCM Dicloromethane DISC Death-inducing signal complex DMD Duchenne muscular dystrophy DMF Dimethylformamide DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid EA Ethyl acetate ESI Electrospray ionization Fas-L Fas ligand FDA U. Food and Drug Administration GATA Family of proteins characterized by zinc finger DNA-binding domains HAT Histone acetyltransferase HCT116 The human colorectal cancer cell line HDAC Histone deacetylase HDACi Histon deacetylase inhibitor HIV Human immunodeficiency viruses HRMS High Resolution Mass Spectrometry IC50 The half maximal inhibitory concentration IR Infrared J Coupling constant (Hz) MCF-7 The human breast cancer cell line MDA-MB-231 The human breast cancer cell line MeOH Methanol MM Multiple myeloma MRC-5 Human fetal lung fibroblast cells MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MW Molecular weight NAD+ Nicotinamide adenine dinucleotide OD Optical density p21 Cyclin-dependent kinase inhibitor p53 Tumor protein p53 PTCL Peripheral T cell lymphoma ROS Reactive oxygen species Rf Retention factor SAHA Suberoylanilide hydroxamic Acid Smad7 Negative regulator of the transforming growth factor beta signaling pathway Stat3 Protein that plays an important role in many cellular processes such as cell growth, differentiation SW620 The human colorectal cancer cell line TLC Thin layer chromatography TMS Tetramethylsilane TNF Tumor necrosis factor TRAIL Tumor necrosis factor-related apoptosis-inducing ligand TRAIL-R1 TRAIL receptor 1 TRAIL-R2 TRAIL receptor 2 TSA Trichostatin A UV Ultraviolet ν Wavenumber VEGF Vascular endothelial growth factor ZBG Zinc binding group δ (ppm) Chemical shift value (ppm) LIST OF TABLES Table 1. Bioactivity of compounds Ia and Ib 5 Table 1.
Bioactivity of compound II 5 Table 3. Physical status and overall yields of synthesis procedure of compounds 4a- d 22 Table 3. Rf values and melting points of compounds 4a-d 23 Table 3. Analysis results of IR spectroscopy of compounds 4a-d 23 Table 3.
Analysis results of HRMS of compounds 4a-d 24 Table 3. Analysis results of 1H-NMR of compounds 4a-d 25 13 Table 3. Analysis results of C-NMR of compounds 4a-d 26 Table 3. Cytotoxicity assessment results against cancer cell lines (Viability) 27 LIST OF FIGURES Figure 1.
Structures of quinazoline, quinazoline’s isomeric forms and quinazolinone 2 Figure 1. Structures of quinazolin-4(3H)-one derivatives having anticancer activity 4 Figure 1. HDAC inhibitors’ pharmacophore model 8 Figure 1. Classes of HDAC inhibitors based on structures 9 Figure 1.
Structural design of targeting compounds 11 Figure 2. Structures of four targeting compounds 13 Figure 3. IR spectrograph of compound 4d 30 Figure 3. High-resolution mass spectrograph of compound 4a 31 1 Figure 3.
H-NMR spectrograph of compound 4a 32 Figure 3. 13C-NMR spectrograph of compound 4a 34 LIST OF SCHEMES Scheme 1. Synthesis of quinazolin-4(3H)-one derivatives from anthranilic acids 3 Scheme 1. Synthesis of quinazolin-4(3H)-one derivatives from anthranilic acids and isothiocyanates 3 Scheme 1.
Synthesis of quinazolin-4(3H)-one derivatives from anthranilamide 3 Scheme 1. Synthesis of quinazolin-4(3H)-one derivatives from 2-methyl benzoxazin-4-one 3 Scheme 1. Synthesis of quinazolin-4(3H)-one derivatives from o-nitrobenzamide 4 Scheme 3. General synthesis procedure of compounds 4a-d 17 Scheme 3.
Synthesis of intermediate 2a 17 Scheme 3. Synthesis of intermediate 3a 18 Scheme 3. Synthesis of compound 4a 18 Scheme 3. Synthesis of compound 4b 19 Scheme 3.
Synthesis of compound 4c 20 Scheme 3. Synthesis of compound 4d 21 Scheme 3. A proposed mechanism for the quinazoline ring forming reaction 28 Scheme 3. A proposed mechanism for the S-alkylation reaction 29 Scheme 3.
A proposed mechanism for the hydroxamic acid forming reaction 30 LIST OF CHARTS Chart 3. Bioactivity of synthesized compounds against colorectal cancer cell line (SW620) 35 Chart 3. Bioactivity of synthesized compounds against breast cancer cell line (MDA-MB-231) 36 INTRODUCTION Causing three in ten global premature deaths due to noncommunicable diseases [1], cancer has been a dangerous threat to public health and the amount of new cases is expected to rise 77% by 2050 in comparison with the number of 2022 [2], raising the need for drug therapies for cancer patients. However, present drug treatments for cancer are either causing severe side effects or being resisted [3], therefore, the development of newly potent drugs against tumor cells has caught the attention of many scientists.
Molecular targeted compounds is one of the most common strategies in developing novel anticancer drugs with many target such as Bcl-2, vascular endothelial growth factor (VEGF) or tumor necrosis factor (TNF) [4]. Most HDAC inhibitors follow a pharmacophore model including three parts: cap group, linker and zinc binding group [6], [7]. Four out of five approved HDAC inhibitors have hydroxamic group as zinc binding group, which suggest this moiety’s potent in term of inhibiting HDAC. To develop new HDAC inhibitors, modifications to the linker and the cap group are made in order to find potential effective compounds [8].
Quinazoline ring has been integrated into many substances with different bioactivities such as antimicrobial, anti-HIV, antituberculosis, antiviral, anti- inflammatory and anticancer [9], [10]. Additionally, recent research evaluated HDACs inhibitory and anticancer activity of this scaffold, suggesting potential effect of quinazoline heterocycle in research and development of novel anticancer drugs by inhibiting HDACs [11], [12]. Based on above studies, research topic “Synthesis and bioevaluation of novel 2-mercaptoquinazoline-based N-hydroxyheptanamide as anti-cancer agents” is carried out with two objectives: 1. Synthesizing four 2-mercaptoquinazoline-based N-hydroxyheptanamide derivatives.
Assessing the in vitro cytotoxicity of synthesized compounds against several cancer cell lines. QUINAZOLINE DERIVATIVES AS ANTI-CANCER AGENTS 1. Quinazoline derivatives Quinazoline ring is a nitrogen heterocycle that have been applied in different fields by its ease of synthesis and structural modification, as well as its diverse potential bioactivites [13]. Quinazoline ring has four isomeric forms distinguished by the position of nitrogen atoms, i.
quinazoline, quinoxaline, cinnoline and phthalazine [10]. In medicinal chemistry, quinazoline derivatives have been synthesized over the years, showing various bioactivities such as antimicrobial, anti-HIV, antituberculosis, antiviral, anti-inflammatory and anticancer [9]. Structures of quinazoline, quinazoline’s isomeric forms and quinazolinone Formed by adding carbonyl groups to quinazoline ring, there are two isomeric forms of quinazolinone, i. Several bioactive compounds have quinazolinone in their structure, for instance, proquazone used as a non-steroidal anti- inflammatory drug [9], raltitrexed used to treat large intestine cancer or albaconazole used as an antifungal drug [10].
Synthesis of quinazolin-4(3H)-one derivatives - From anthranilic acids (The Niementowski condensation reaction): At 120˚C, quinazolin-4(3H)-ones was formed by anthranilic acids and excess formamide [14], [15]. Synthesis of quinazolin-4(3H)-one derivatives from anthranilic acids In another variation, anthranilic acids reacted with different isothiocyanates on refluxing in ethanol with triethylamine to create 2-mercapto-quinazolin- 4-one derivatives [16], [17], [11]. Synthesis of quinazolin-4(3H)-one derivatives from anthranilic acids and isothiocyanates - From anthranilamide: In DMSO, 2-arylquinazolin-4(3H)-ones were synthesized from anthranilamide and benzaldehyde derivatives [18], [19], [20]. Synthesis of quinazolin-4(3H)-one derivatives from anthranilamide - From 2-methyl benzoxazin-4-one: 2-methyl benzoxazin-4-one reacted with aniline derivatives to form 3-subtituted quinazolin-4-ones on refluxing in acetic acid [21], [22], [23].
Synthesis of quinazolin-4(3H)-one derivatives from 2-methyl benzoxazin- 4-one 3 - From o-nitrobenzamide: 2-subtituted quinazolin-4(3H)-ones was synthesized from o-nitrobenzamide and different alcohols with palladium as the catalyst in various solvent [24]. Synthesis of quinazolin-4(3H)-one derivatives from o-nitrobenzamide 1. Anticancer activity of quinazolin-4(3H)-one derivatives Until now, many quinazolin-4(3H)-one derivatives have been approved for the treatment of various types of cancer. For instance, raltitrexed has been used in patients with metastatic colorectal cancer and shown efficacy in head and neck cancer, malignant mesothelioma, hepatic cancer, gastric and pancreatic carcinoma [25].
Another example is idelalisib, which was approved by FDA for use in patients with relapsed chronic lymphocytic leukemia in combination with rituximab, relapsed follicular B-cell non- Hodgkin lymphoma and relapsed small-lymphocytic lymphoma [26]. Structures of quinazolin-4(3H)-one derivatives having anticancer activity In recent studies, numerous compounds containing quinazolin-4(3H)-one scaffold have been synthesized and evaluated anticancer activity in different cancer cell lines. Khetmalis et al. synthesized a series of hydroxamic acid derivatives having a quinazolin-4(3H)-one as cap group and tested their ability to inhibit HDAC and antiproliferative activity in several tumor cell lines.
Among all the compounds evaluated, most of them demonstrated sub-micromolar activities on HDAC6 and Ia showed the most selective inhibition with an IC50 of 150 nM. Besides, Ib had the most potent anticancer activity against multiple human cancer cell lines [11]. Bioactivity of compounds Ia and Ib IC50 ± SD (nM) Compound R HDAC4 HDAC6 HDAC8 Ia H 2300 ± 1100 150 ± 10 1400 ± 300 SAHA 27000 86 5300 Trichostatin A 3300 17 360 Tubastatin 4700 ± 1500 160 ± 30 2700 ± 1800 IC50 ± SD (µM) HCT116 MCF7 B16 Ib 4-CH3 12. Emami et al.
synthesized some quinazolin-4(3H)-one derivatives having a thiol group at position 2 of the quinazoline ring and evaluated their bioactivitiy against MCF7 and SW480 cell lines.