GIST Repository
College of Life Sciences and Medical Engineering
Department of Life Sciences
4. Theses(Ph.D)
Design, Synthesis and Structure-Activity Relationship Studies for the Discovery of Triazolopyrimidine Derivatives as Selective P2X3 Receptor Antagonists
- Abstract
- The P2X3 receptor (P2X3R), an ATP-gated cation channel predominantly expressed in C- and Aδ-primary afferent neurons, has been proposed as a drug target for neurological inflammatory diseases, e.g., neuropathic pain, and chronic cough. Aiming to develop novel, selective P2X3R antagonists, tetrazolopyrimidine-based hit compound 9 was optimized through structure−activity relationship studies by modifying the tetrazole core as well as side chain substituents. The optimized antagonist 26a, featuring a cyclopropane-substituted triazolopyrimidine core, displayed potent P2X3R-antagonistic activity (IC50 = 54.9 nM), 20-fold selectivity versus the heteromeric P2X2/3R, and high selectivity versus other P2XR subtypes. Noncompetitive P2X3R blockade was experimentally confirmed by calcium influx assays. Cryo-electron microscopy revealed that 26a stabilizes the P2X3R in its desensitized state acting as a molecular barrier to prevent ions from accessing the central pore. In vivo studies in a rat neuropathic pain model (spinal nerve ligation) showed dose- dependent antiallodynic effects of 26a, thus presenting a novel, promising lead structure.
- Issued Date
- 2025
- Type
- Thesis
- Alternative Author(s)
- Ga-Ram Kim
- Department
- 대학원 생명과학부
- Advisor
- Kim, Yong-Chul
- Table Of Contents
- CONTENTS
ABSTRACT i
CONTENTS ii
LIST OF SCHEMES AND TABLES xiii
LIST OF FIGURES xiv
Design, Synthesis and Structure-Activity Relationship Studies for the Discovery of Triazolopyrimidine Derivatives as Selective P2X3 Receptor Antagonists
1. ABSTRACT 2
2. INTRODUCTION 3
3. MATERIALS AND METHODS 6
3.1. Materials 6
3.2. Chemistry 6
3.2.1. 3-Chloro-4-ethoxybenzaldehyde (10a) 6
3.2.2. 3-Chloro-4-isopropoxybenzaldehyde (10b) 7
3.2.3. 4-Ethoxybenzaldehyde (10c) 7
3.2.4. 4-Ethoxy-3-fluorobenzaldehyde (10d) 7
3.2.5. 3-Bromo-4-ethoxybenzaldehyde (10e) 7
3.2.6. 4-Ethoxy-3-iodobenzaldehyde (10f) 7
3.2.7. (S, E)-N-(3-Chloro-4-ethoxybenzylidene)-2-methylpropane-2-sulfinamide (11a) 8
3.2.8. (R, E)-N-(3-Chloro-4-ethoxybenzylidene)-2-methylpropane-2-sulfinamide (11a′) 8
3.2.9. (S, E)-N-(3-Chloro-4-isopropoxybenzylidene)-2-methylpropane-2-sulfinamide (11b) 8
3.2.10. (S, E)-N-(4-Ethoxybenzylidene)-2-methylpropane-2-sulfinamide (11c) 8
3.2.11. (S, E)-N-(4-Ethoxy-3-fluorobenzylidene)-2-methylpropane-2-sulfinamide (11d) 9
3.2.12. (S, E)-N-(3-Bromo-4-ethoxybenzylidene)-2-methylpropane-2-sulfinamide (11e) 9
3.2.13. (S, E)-N-(4-Ethoxy-3-iodobenzylidene)-2-methylpropane-2-sulfinamide (11f) 9
3.2.14. (S, E)-N-(3-Chloro-4-methoxybenzylidene)-2-methylpropane-2-sulfinamide (11g) 9
3.2.15. (S)-N-((R)-1-(3-Chloro-4-ethoxyphenyl)ethyl)-2-methylpropane-2-sulfinamide (12a) 9
3.2.16. (R)-N-((S)-1-(3-Chloro-4-ethoxyphenyl)ethyl)-2-methylpropane-2-sulfinamide (12a′) 10
3.2.17. (S)-N-((R)-1-(3-Chloro-4-isopropoxyphenyl)ethyl)-2-methylpropane-2-sulfinamide (12b) 10
3.2.18. (S)-N-((R)-1-(4-Ethoxyphenyl)ethyl)-2-methylpropane-2-sulfinamide (12c) 10
3.2.19. (S)-N-((R)-1-(4-Ethoxy-3-fluorophenyl)ethyl)-2-methylpropane-2-sulfinamide (12d) 10
3.2.20. (S)-N-((R)-1-(3-Bromo-4-ethoxyphenyl)ethyl)-2-methylpropane-2-sulfinamide (12e) 11
3.2.21. (S)-N-((R)-1-(4-Ethoxy-3-iodophenyl)ethyl)-2-methylpropane-2-sulfinamide (12f) 11
3.2.22. (S)-N-((R)-1-(3-Chloro-4-methoxyphenyl)ethyl)-2-methylpropane-2-sulfinamide (12g) 11
3.2.23. (R)-1-(3-Chloro-4-ethoxyphenyl)ethan-1-amine hydrochloride (13a) 11
3.2.24. (S)-1-(3-Chloro-4-ethoxyphenyl)ethan-1-amine hydrochloride (13a′) 12
3.2.25. (R)-1-(3-Chloro-4-isopropoxyphenyl)ethan-1-amine hydrochloride (13b) 12
3.2.26. (R)-1-(4-Ethoxyphenyl)ethan-1-amine hydrochloride (13c) 12
3.2.27. (R)-1-(4-Ethoxy-3-fluorophenyl)ethan-1-amine hydrochloride (13d) 12
3.2.28. (R)-1-(3-Bromo-4-ethoxyphenyl)ethan-1-amine hydrochloride (13e) 12
3.2.29. (R)-1-(4-Ethoxy-3-iodophenyl)ethan-1-amine hydrochloride (13f) 13
3.2.30. (R)-1-(3-Chloro-4-methoxyphenyl)ethan-1-amine hydrochloride (13g) 13
3.2.31. (S, E)-2-Methyl-N-(naphthalen-2-ylmethylene)propane-2-sulfinamide (14) 13
3.2.32. (S)-2-Methyl-N-((R)-1-(naphthalen-2-yl)propyl)propane-2-sulfinamide (15a) 13
3.2.33. (S)-2-Methyl-N-((R)-2-methyl-1-(naphthalen-2-yl)propyl)propane-2-sulfinamide (15b) 13
3.2.34. (S)-N-((R)-Cyclopropyl(naphthalen-2-yl)methyl)-2-methylpropane-2-sulfinamide (15c) 14
3.2.35. (S)-N-((R)-Cyclohexyl(naphthalen-2-yl)methyl)-2-methylpropane-2-sulfinamide (15d) 14
3.2.36. (R)-1-(Naphthalen-2-yl)propan-1-amine hydrochloride (16a) 14
3.2.37. (R)-2-Methyl-1-(naphthalen-2-yl)propan-1-amine hydrochloride (16b) 14
3.2.38. (R)-Cyclopropyl(naphthalen-2-yl)methanamine hydrochloride (16c) 15
3.2.39. (R)-Cyclohexyl(naphthalen-2-yl)methanamine hydrochloride (16d) 15
3.2.40. 5,7-Dichlorotetrazolo[1,5-a]pyrimidine (17) 15
3.2.41. (R)-5-Chloro-N-(1-(3-chloro-4-ethoxyphenyl)ethyl)tetrazolo[1,5-a]pyrimidin-7-amine (18a) 15
3.2.42. (S)-5-Chloro-N-(1-(3-chloro-4-ethoxyphenyl)ethyl)tetrazolo[1,5-a]pyrimidin-7-amine (18b) 16
3.2.43. (R)-5-Chloro-N-(1-(3-chloro-4-methoxyphenyl)ethyl)tetrazolo[1,5-a]pyrimidin-7-amine (18c) 16
3.2.44. (R)-5-Chloro-N-(1-(3-chloro-4-isopropoxyphenyl)ethyl)tetrazolo[1,5-a]pyrimidin-7-amine (18d) 16
3.2.45. (R)-5-Chloro-N-(1-(4-ethoxyphenyl)ethyl)tetrazolo[1,5-a]pyrimidin-7-amine (18e) 16
3.2.46. (R)-5-Chloro-N-(1-(4-ethoxy-3-fluorophenyl)ethyl)tetrazolo[1,5-a]pyrimidin-7-amine (18f) 17
3.2.47. (R)-N-(1-(3-Bromo-4-ethoxyphenyl)ethyl)-5-chlorotetrazolo[1,5-a]pyrimidin-7-amine (18g) 17
3.2.48. (R)-5-Chloro-N-(1-(4-ethoxy-3-iodophenyl)ethyl)tetrazolo[1,5-a]pyrimidin-7-amine (18h) 17
3.2.49. (R)-5-Chloro-N-(1-(naphthalen-2-yl)ethyl)tetrazolo[1,5-a]pyrimidin-7-amine (18i) 17
3.2.50. (R)-1-(4-(7-((1-(3-Chloro-4-ethoxyphenyl)ethyl)amino)tetrazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)ethan-1-one (19a) 17
3.2.51. (S)-1-(4-(7-((1-(3-Chloro-4-ethoxyphenyl)ethyl)amino)tetrazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)ethan-1-one (19b) 18
3.2.52. (R)-1-(4-(7-((1-(3-Chloro-4-methoxyphenyl)ethyl)amino)tetrazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)ethan-1-one (19c) 18
3.2.53. (R)-1-(4-(7-((1-(3-Chloro-4-isopropoxyphenyl)ethyl)amino)tetrazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)ethan-1-one (19d) 18
3.2.54. (R)-1-(4-(7-((1-(4-Ethoxyphenyl)ethyl)amino)tetrazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)ethan-1-one (19e) 19
3.2.55. (R)-1-(4-(7-((1-(4-Ethoxy-3-fluorophenyl)ethyl)amino)tetrazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)ethan-1-one (19f) 19
3.2.56. (R)-1-(4-(7-((1-(3-Bromo-4-ethoxyphenyl)ethyl)amino)tetrazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)ethan-1-one (19g) 19
3.2.57. (R)-1-(4-(7-((1-(4-Ethoxy-3-iodophenyl)ethyl)amino)tetrazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)ethan-1-one (19h) 20
3.2.58. (R)-1-(4-(7-((1-(Naphthalen-2-yl)ethyl)amino)tetrazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)ethan-1-one (19i) 20
3.2.59. 5,7-Dichloro-[1,2,4]triazolo[1,5-a]pyrimidine (20a) 20
3.2.60. 5,7-Dichloro-2-methyl-[1,2,4]triazolo[1,5-a]pyrimidine (20b) 21
3.2.61. 5,7-Dichloro-2-ethyl-[1,2,4]triazolo[1,5-a]pyrimidine (20c) 21
3.2.62. 5,7-Dichloro-2-isopropyl-[1,2,4]triazolo[1,5-a]pyrimidine (20d) 21
3.2.63. 5,7-Dichloro-2-cyclopropyl-[1,2,4]triazolo[1,5-a]pyrimidine (20e) 21
3.2.64. 5,7-Dichloro-2-cyclobutyl-[1,2,4]triazolo[1,5-a]pyrimidine (20f) 21
3.2.65. 5,7-Dichloro-2-cyclopentyl-[1,2,4]triazolo[1,5-a]pyrimidine (20g) 21
3.2.66. 5,7-Dichloro-2-cyclohexyl-[1,2,4]triazolo[1,5-a]pyrimidine (20h) 22
3.2.67. 5,7-Dichloro-2-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine (20i) 22
3.2.68. 5,7-Dichloro-2-(4-fluorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine (20j) 22
3.2.69. (R)-5-Chloro-N-(1-(naphthalen-2-yl)ethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (21a) 22
3.2.70. (R)-5-Chloro-2-methyl-N-(1-(naphthalen-2-yl)ethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (21b) 22
3.2.71. (R)-5-Chloro-2-ethyl-N-(1-(naphthalen-2-yl)ethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (21c) 23
3.2.72. (R)-5-Chloro-2-isopropyl-N-(1-(naphthalen-2-yl)ethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (21d) 23
3.2.73. (R)-5-Chloro-2-cyclopropyl-N-(1-(naphthalen-2-yl)ethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (21e) 23
3.2.74. (S)-5-Chloro-2-cyclopropyl-N-(1-(naphthalen-2-yl)ethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (21e′) 24
3.2.75. (R)-5-Chloro-2-cyclobutyl-N-(1-(naphthalen-2-yl)ethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (21f) 24
3.2.76. (R)-5-Chloro-2-cyclopentyl-N-(1-(naphthalen-2-yl)ethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (21g) 24
3.2.77. (R)-5-Chloro-2-cyclohexyl-N-(1-(naphthalen-2-yl)ethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (21h) 24
3.2.78. (R)-5-Chloro-N-(1-(naphthalen-2-yl)ethyl)-2-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (21i) 25
3.2.79. (R)-5-Chloro-2-(4-fluorophenyl)-N-(1-(naphthalen-2-yl)ethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (21j) 25
3.2.80. (R)-5-Chloro-2-cyclopropyl-N-(1-(naphthalen-2-yl)propyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (21k) 25
3.2.81. (R)-5-Chloro-2-cyclopropyl-N-(2-methyl-1-(naphthalen-2-yl)propyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (21l) 25
3.2.82. (R)-5-Chloro-2-cyclopropyl-N-(cyclopropyl(naphthalen-2-yl)methyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (21m) 26
3.2.83. (R)-5-Chloro-N-(cyclohexyl(naphthalen-2-yl)methyl)-2-cyclopropyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (21n) 26
3.2.84. (R)-1-(4-(7-((1-(Naphthalen-2-yl)ethyl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)ethan-1-one (22a) 26
3.2.85. (R)-1-(4-(2-Methyl-7-((1-(naphthalen-2-yl)ethyl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)ethan-1-one (22b) 27
3.2.86. (R)-1-(4-(2-Ethyl-7-((1-(naphthalen-2-yl)ethyl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)ethan-1-one (22c) 27
3.2.87. (R)-1-(4-(2-Isopropyl-7-((1-(naphthalen-2-yl)ethyl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)ethan-1-one (22d) 27
3.2.88. (R)-1-(4-(2-Cyclopropyl-7-((1-(naphthalen-2-yl)ethyl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)ethan-1-one (22e) 28
3.2.89. (R)-1-(4-(2-Cyclobutyl-7-((1-(naphthalen-2-yl)ethyl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)ethan-1-one (22f) 28
3.2.90. (R)-1-(4-(2-Cyclopentyl-7-((1-(naphthalen-2-yl)ethyl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)ethan-1-one (22g) 28
3.2.91. (R)-1-(4-(2-Cyclohexyl-7-((1-(naphthalen-2-yl)ethyl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)ethan-1-one (22h) 29
3.2.92. (R)-1-(4-(7-((1-(Naphthalen-2-yl)ethyl)amino)-2-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)ethan-1-one (22i) 29
3.2.93. (R)-1-(4-(2-(4-Fluorophenyl)-7-((1-(naphthalen-2-yl)ethyl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)ethan-1-one (22j) 29
3.2.94. (R)-1-(4-(2-Cyclopropyl-7-((1-(naphthalen-2-yl)propyl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)ethan-1-one (22k) 30
3.2.95. (R)-1-(4-(2-Cyclopropyl-7-((2-methyl-1-(naphthalen-2-yl)propyl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)ethan-1-one (22l) 30
3.2.96. (R)-1-(4-(2-Cyclopropyl-7-((cyclopropyl(naphthalen-2-yl)methyl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)ethan-1-one (22m) 31
3.2.97. (R)-1-(4-(7-((Cyclohexyl(naphthalen-2-yl)methyl)amino)-2-cyclopropyl-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)ethan-1-one (22n) 31
3.2.98. (R)-2-Cyclopropyl-5-(4-methylpiperazin-1-yl)-N-(1-(naphthalen-2-yl)ethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (22o) 31
3.2.99. (R)-2-Cyclopropyl-5-(4-ethylpiperazin-1-yl)-N-(1-(naphthalen-2-yl)ethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (22p) 32
3.2.100. (R)-Cyclopropyl(4-(2-cyclopropyl-7-((1-(naphthalen-2-yl)ethyl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)methanone (22q) 32
3.2.101. tert-Butyl (R)-4-(2-cyclopropyl-7-((1-(naphthalen-2-yl)ethyl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate (23a) 32
3.2.102. tert-Butyl (S)-4-(2-cyclopropyl-7-((1-(naphthalen-2-yl)ethyl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate (23a′) 33
3.2.103. tert-Butyl (R)-4-(2-cyclobutyl-7-((1-(naphthalen-2-yl)ethyl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate (23b) 33
3.2.104. tert-Butyl (R)-4-(2-cyclopentyl-7-((1-(naphthalen-2-yl)ethyl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate (23c) 33
3.2.105. tert-Butyl (R)-4-(2-cyclohexyl-7-((1-(naphthalen-2-yl)ethyl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate (23d) 34
3.2.106. (R)-2-Cyclopropyl-N-(1-(naphthalen-2-yl)ethyl)-5-(piperazin-1-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (24a) 34
3.2.107. (S)-2-Cyclopropyl-N-(1-(naphthalen-2-yl)ethyl)-5-(piperazin-1-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (24a′) 34
3.2.108. (R)-2-Cyclobutyl-N-(1-(naphthalen-2-yl)ethyl)-5-(piperazin-1-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (24b) 35
3.2.109. (R)-2-Cyclopentyl-N-(1-(naphthalen-2-yl)ethyl)-5-(piperazin-1-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (24c) 35
3.2.110. (R)-2-Cyclohexyl-N-(1-(naphthalen-2-yl)ethyl)-5-(piperazin-1-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (24d) 35
3.2.111. (R)-1-(4-(2-Cyclopropyl-7-((1-(naphthalen-2-yl)ethyl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)propan-1-one (25a) 35
3.2.112. Methyl (R)-4-(2-cyclopropyl-7-((1-(naphthalen-2-yl)ethyl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate (25b) 36
3.2.113. (R)-1-(4-(2-Cyclopropyl-7-((1-(naphthalen-2-yl)ethyl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)-2,2,2-trifluoroethan-1-one (25c) 36
3.2.114. (R)-4-(2-Cyclopropyl-7-((1-(naphthalen-2-yl)ethyl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxamide (26a) 37
3.2.115. (S)-4-(2-Cyclopropyl-7-((1-(naphthalen-2-yl)ethyl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxamide (26a′) 37
3.2.116. (R)-4-(2-Cyclobutyl-7-((1-(naphthalen-2-yl)ethyl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxamide (26b) 37
3.2.117. (R)-4-(2-Cyclopentyl-7-((1-(naphthalen-2-yl)ethyl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxamide (26c) 38
3.2.118. (R)-4-(2-Cyclohexyl-7-((1-(naphthalen-2-yl)ethyl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxamide (26d) 38
3.3. Biological Methods 38
3.3.1. Cell-based calcium influx assays at human P2X3 and P2X2/3 receptors 38
3.3.2. Cell-based calcium influx assays at human P2X1, P2X2, P2X4, and P2X7 receptors 39
3.3.3. Metabolic stability test 41
3.3.4. Parallel artificial membrane permeability assay (PAMPA) 41
3.3.5. In vivo efficacy assessment in neuropathic pain animal model 42
3.3.6. Protein expression and purification 42
3.3.7. Grid preparation and Cryo-EM data acquisition 43
3.3.8. Cryo-EM data processing 44
3.3.9. Model building and refinement 44
3.3.10. Statistical analysis 45
4. RESULTS AND DISCUSSION 46
4.1. Strategies of SAR analysis and optimization of hit compound 9 46
4.2. Chemistry 48
4.3. Biology 53
4.3.1. Structure-activity relationship (SAR) of the triazolopyrimidine derivatives as P2X3R antagonists 53
4.3.2. P2XR selectivity profile of compound 26a 55
4.3.3. Mechanism of antagonism of compound 26a 56
4.3.4. Cryo-electron microscopy (cryo-EM) structure of the human P2X3R in complex with compound 26a 56
4.3.5. In vivo antiallodynic effects of compound 26a in a neuropathic pain model 57
5. CONCLUSION 70
6. REFERENCES 71
APPENDIX. Discovery of Novel 1H-benzo[d]imidazole-4,7-dione Based Transglutaminase 2 Inhibitors as p53 Stabilizing Anticancer Agents in Renal Cell Carcinoma
1. ABSTRACT 78
2. INTRODUCTION 79
3. MATERIALS AND METHODS 81
3.1. Materials 81
3.2. Chemistry 81
3.2.1. 4,7-Dimethoxy-2-methyl-1H-benzo[d]imidazole (7a) 81
3.2.2. 4,7-Dimethoxy-2-(trifluoromethyl)-1H-benzo[d]imidazole (7b) 82
3.2.3. 2-Cyclopropyl-4,7-dimethoxy-1H-benzo[d]imidazole (7c) 82
3.2.4. 2-Cyclobutyl-4,7-dimethoxy-1H-benzo[d]imidazole (7d) 82
3.2.5. 2-Cyclopentyl-4,7-dimethoxy-1H-benzo[d]imidazole (7e) 82
3.2.6. 2-Cyclohexyl-4,7-dimethoxy-1H-benzo[d]imidazole (7f) 83
3.2.7. 4,7-Dimethoxy-2-phenyl-1H-benzo[d]imidazole (7g) 83
3.2.8. 2-(3,4-Difluorophenyl)-4,7-dimethoxy-1H-benzo[d]imidazole (7h) 83
3.2.9. 2-(4-Chloro-3-fluorophenyl)-4,7-dimethoxy-1H-benzo[d]imidazole (7i) 83
3.2.10. 2-(4-Bromo-3-fluorophenyl)-4,7-dimethoxy-1H-benzo[d]imidazole (7j) 84
3.2.11. 2-(3-Chloro-4-fluorophenyl)-4,7-dimethoxy-1H-benzo[d]imidazole (7k) 84
3.2.12. 2-(3-Bromo-4-fluorophenyl)-4,7-dimethoxy-1H-benzo[d]imidazole (7l) 84
3.2.13. 2-(3,4-Dichlorophenyl)-4,7-dimethoxy-1H-benzo[d]imidazole (7m) 84
3.2.14. 2-(3-Bromo-4-chlorophenyl)-4,7-dimethoxy-1H-benzo[d]imidazole (7n) 84
3.2.15. 5,6-Dichloro-2-methyl-1H-benzo[d]imidazole-4,7-dione (8a) 85
3.2.16. 5,6-Dichloro-2-(trifluoromethyl)-1H-benzo[d]imidazole-4,7-dione (8b) 85
3.2.17. 5,6-Dichloro-2-cyclopropyl-1H-benzo[d]imidazole-4,7-dione (8c) 85
3.2.18. 5,6-Dichloro-2-cyclobutyl-1H-benzo[d]imidazole-4,7-dione (8d) 85
3.2.19. 5,6-Dichloro-2-cyclopentyl-1H-benzo[d]imidazole-4,7-dione (8e) 85
3.2.20. 5,6-Dichloro-2-cyclohexyl-1H-benzo[d]imidazole-4,7-dione (8f) 86
3.2.21. 5,6-Dichloro-2-phenyl-1H-benzo[d]imidazole-4,7-dione (8g) 86
3.2.22. 5,6-Dichloro-2-(3,4-difluorophenyl)-1H-benzo[d]imidazole-4,7-dione (8h) 86
3.2.23. 5,6-Dichloro-2-(4-chloro-3-fluorophenyl)-1H-benzo[d]imidazole-4,7-dione (8i) 86
3.2.24. 2-(4-Bromo-3-fluorophenyl)-5,6-dichloro-1H-benzo[d]imidazole-4,7-dione (8j) 87
3.2.25. 5,6-Dichloro-2-(3-chloro-4-fluorophenyl)-1H-benzo[d]imidazole-4,7-dione (8k) 87
3.2.26. 2-(3-Bromo-4-fluorophenyl)-5,6-dichloro-1H-benzo[d]imidazole-4,7-dione (8l) 87
3.2.27. 5,6-Dichloro-2-(3,4-dichlorophenyl)-1H-benzo[d]imidazole-4,7-dione (8m) 87
3.2.28. 2-(3-Bromo-4-chlorophenyl)-5,6-dichloro-1H-benzo[d]imidazole-4,7-dione (8n) 88
3.2.29. 5-Chloro-6-((4-fluorophenyl)amino)-2-phenyl-1H-benzo[d]imidazole-4,7-dione (9a) 88
3.2.30. 6-((4-Bromophenyl)amino)-5-chloro-2-phenyl-1H-benzo[d]imidazole-4,7-dione (9b) 88
3.2.31. 5-Chloro-6-((3-chloro-4-fluorophenyl)amino)-2-phenyl-1H-benzo[d]imidazole-4,7-dione (9c) 88
3.2.32. 5-Chloro-6-((3-fluorophenyl)amino)-2-phenyl-1H-benzo[d]imidazole-4,7-dione (9d) 89
3.2.33. 6-((3-Bromophenyl)amino)-5-chloro-2-phenyl-1H-benzo[d]imidazole-4,7-dione (9e) 89
3.2.34. 5-Chloro-2-phenyl-6-(p-tolylamino)-1H-benzo[d]imidazole-4,7-dione (9f) 89
3.2.35. 5-Chloro-2-(3,4-difluorophenyl)-6-methoxy-1H-benzo[d]imidazole-4,7-dione (10a) 89
3.2.36. 2-(4-Bromo-3-fluorophenyl)-5-chloro-6-methoxy-1H-benzo[d]imidazole-4,7-dione (10b) 90
3.2.37. 2-(3,4-Difluorophenyl)-5,6-dimethoxy-1H-benzo[d]imidazole-4,7-dione (10c) 90
3.2.38. 2-(4-Bromo-3-fluorophenyl)-5,6-dimethoxy-1H-benzo[d]imidazole-4,7-dione (10d) 90
3.2.39. 6-Amino-2-(4-bromo-3-fluorophenyl)-5-chloro-1H-benzo[d]imidazole-4,7-dione (10e) 91
3.3. Biological Methods 92
3.3.1. Cell culture and viability 92
3.3.2. TG2 activity assay using radioactive isotopes 92
3.3.3. SRB assay for anti-proliferative activity 92
3.3.4. TG2 activity 93
3.3.5. Cell apoptosis assay 93
3.3.6. Western blotting 94
3.3.7. Co-IP (Co-immunoprecipitation) 94
3.3.8. Surface plasmon resonance (SPR) assay 94
3.3.9. Molecular docking 95
3.3.10. Preclinical xenograft tumor models 95
3.3.11. Immunohistochemistry (IHC) 95
3.3.12. Parallel artificial membrane permeability assay (PAMPA) 96
3.3.13. Statistical analysis 96
4. RESULTS AND DISCUSSION 97
4.1. Design strategy 97
4.2. Chemistry 98
4.3. Biology 99
4.3.1. Structure-activity relationship (SAR) 99
4.3.2. Reversibility assessment of compound 8j (MD102) for TG2 100
4.3.3. TG2 level was positively correlated between TG2 activity and protein expression level of TG2 in cancer cells 101
4.3.4. Structural comparison of TG2 wild type (WT), TG2 β-sandwich deletion, and TG2(C227S)-active site and molecular docking study 101
4.3.5. Compound 8j (MD102) induced apoptosis and disturbance of the interaction between p53 and TG2 through regulating the p53/AKT/mTOR signaling 102
4.3.6. In vivo antitumor effect of compound 8j (MD102) on ACHN xenograft model 102
5. CONCLUSION 116
6. REFERENCES 117
ABSTRACT IN KOREAN 120
ACKNOWLEDGMENTS 121
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