臺灣博碩士論文加值系統

Viruses have caused some of the most tragic and deadly diseases in human history and threat to all animals along with plants. Among the all kind of viruses, enterovirus (EV) in the family Picornaviridae, and influenza A virus (IAV) in the family Orthomyxoviridae, and their serotypes consistently observed in recent few years and cause worldwide epidemics as well as pandemics. EVs are the primary causal agent of hand-foot-mouth disease, particularly in children under five years old. This disease is sporadically associated with severe neurological diseases, ranging from aseptic meningitis to fatal encephalitis. IAVs are mostly facilitating respiratory related diseases like pneumonia, bronchitis, sinus and ear infections and cause mild to severe illness, sometimes lead to death. Thus far, no antiviral medications currently approved to treat EVs-associated disease or prevent EV infection. Contrariwise, there are few limitations overcomes for IAVs and EVs, development of drug resistant mutants and varying their effectiveness against the latent virus, suggesting an urgency for new antivirus agents. In a previous study, a chemical library containing more than 20,000 compounds were evaluated for anti-IAV activities in a cell-based assay. Among these a probenecid and itaconic acid containing derivatives C-7 and ITA-01, exhibiting anti-viral effects were discovered. Accordingly, in this study, a series of probenecid analogues and itaconic acid belonging derivatives have been designed and synthesized by rationale design strategy to obtain more potent antiviral agents. The in vitro pharmacological antiviral activity represents compounds C-30 and C-44 exhibit the highest inhibitory activity on EV D68 and EV A71 with EC50 values of 2.49/2.09, and 2.59/2.41 µM respectively, in rhabdomyosarcoma cells with lower cytotoxicity (>400µM) and reveals with a broad spectrum of inhibition to other EV strains. Also, the derivatives C-30 and C-44 perform well stability with half-life 48.39 and 60.26 min. respectively, in 25% rat serum. On the other hand, among all ITA-series derivatives, ITA-04 exhibited an extreme inhibitory potency (EC50 = 0.14 µM) with high selective index (>785) against IAV in Madin-Darby canine kidney cell lines. A further mechanism of action study indicated that the initial hit ITA-01 and its derivative ITA-04, inhibited the IAV replication by blocking distribution of viral nucleoproteins from nucleus to cytosol. Therefore, results suggesting, these synthesized compounds may have high potential for further optimization and development against EV D68, EV A71, and IAV.

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Acknowledgement………………........……........……………………..iii
Abstract...................................................................................................iv
Table of Contents...................................................................................vi
List of Figures…………..........……………......…......…….…..............x
List of Tables……............…………………......…......…….…........xxvii
List of Schemes...........…………………......…......…….…........…..xxix
Chapter 1. Introduction...........................................................................1
1.0 Background and significance............................................................2
1.1 Picornaviridae…………………………………………………......3
1.1.1 Enterovirus (EV)…………….....................………….….4
1.1.2 Enterovirus D68…………..…....…..............……….……..5
1.1.3 Enterovirus A71………….……................…….……...5
1.1.4 Virion and genome structure of EV……….............…….6
1.1.5 Replication cycle and specific targets for inhibition of EV...8
1.1.6 Infection and control……………..……….………................10
1.2 Orthomyxoviridae………………………………………….….......11
1.2.1 Influenza A virus (IAV)........……….………………….........12
1.2.2 Virion and genome structure of IAV…...……..…….….........13
1.2.3 Replication cycle and specific targets for inhibition of IAV….14
1.2.4 Infection and control……………………………...…….........16
Chapter 2. Materials and Methods.......................................................19
2.0 Experimental equipment, instruments and chemicals…………......20
2.1 Retro synthetic analysis……………......………………………........25
2.1.1 Analysis of C-7……………..…...................….…………….25
2.1.2 Analysis of ITA-01………………......………………….......26
2.2 Study design and synthetic strategy…………………………….....27
2.2.1 Design for C-7 analogues……………………………......27
2.2.2 Synthetic strategy for C-7 analogues.…....………………..30
2.2.2.1 Synthesis of intermediates……………………….....30
2.2.2.2 Synthesis of target compounds….………………….32
2.2.3 Design for ITA-01 analogues……………………………..40
2.2.4 Synthetic strategy for ITA-01 analogues……...............…….45
2.3 Protocols for bio-assay……………………………………….……46
2.3.1 Antiviral effectiveness assay (EC50 assay) for enteroviruses....46
2.3.2 EC50 assay for influenza viruses..............................................47
2.3.3 Cytotoxicity assay (CC50 assay) .............................................48
2.3.4 Cytopathic effect detection assay............................................48
2.3.5 Time-of-addition assay..........................................................49
2.3.6 Hemagglutination inhibition assay.........................................49
2.3.7 RNA purification and quantitative reverse transcription-PCR (qRT-PCR) assay............................................................................50
2.3.8 Western immunoblotting assay...............................................50
2.3.9 Indirect immunofluorescence assay......................................51
2.4 Metabolic stability analysis in rat serum………………….……......52
2.5 Molecular docking of compounds C-30 & C-44 in pleconaril binding site of VP1 in EV D68………………………………...….……...........53
2.6 Generation of 3D-QSAR pharmacophore hypothesis.......................54
2.7 Michael-addition characteristics using cysteamine by NMR-based assay…...……………………………………………………………....54
2.8 Molecular docking of compounds ITA-01, 04, 06 and 08 in the binding site of LMB in CRM1……………………………………...…………...54


Chapter 3. Results and Discussions of C-Series...................................56
3.0 Results and discussions of C-7 derivatives.............................57
3.1 Structure activity relationship (SAR) study of C-series compounds against EV D68 and EV A71.....................................................……......57
3.2 Metabolic stability analysis in rat serum...........................................68
3.3 Broad spectrum of anti-enterovirus activity of C-30 & C-44….69
3.4 3D-QSAR pharmacophore model and molecular docking of C-30 & C-44 in the active site of pleconaril in EV D68…………………...........71

Chapter 4. Results and Discussions of ITA-Series.............................74
4.0 Results and discussions of ITA-01 derivatives..................................75
4.1 SAR study of ITA-01 derivatives with different substituents against IAV........................................................................................................75
4.2 A broad spectrum of antiviral activity of ITA-01 & 04......................88
4.3 ITA-01 inhibits cytopathic effects caused by influenza A virus........90
4.4 Evaluation of the period of inhibition of IAV, upon treatment with ITA-01 & 04 using time-of-addition assay.............................................90
4.5 Observation of the effect of ITA-01 & 04 treatments on IAV hemagglutinin (HA) activity..................................................................91
4.6 Effect of ITA-01 on IAV viral RNA and protein synthesis..............92
4.7 Exploration of the effect of ITA-01 on the subcellular distribution of IAV viral proteins by IF assay................................................................93

Chapter 5. Conclusion………......………………………………….…95

References…………………….……………......………………...100


Appendix............................................................................................112
Appendix 1. Influenza A/WSN/33 virus causes cytopathic changes, inhibited by ITA-01…………………………………………..112
Appendix 2. Time-of-addition assay to investigate period of inhibition of influenza A/WSN/33 by ITA-01 & 04 treatments….113
Appendix 3. Hemagglutination inhibition assay, to detect the effect of ITA-01 & 04 on the HA…………………………………115
Appendix 4. Exploration of the effect of ITA-01 treatment on vRNA synthesis using RT-qPCR and Western blotting assay.………116
Appendix 5. The effect on IAV proteins (NP, NS2, PA, and M1) distribution detection, by treatment with ITA-01 using IF staining assay…………………………………………………………118
Appendix 6. The effect on IAV proteins (NP, and M1) distribution detection, by treatment with ITA-04 using IF staining assay…….123
Compounds detail...............................................................................126










List of Figures
Figure 1. The most common symptoms due to enterovirus D68 infection................................................................................................5
Figure 2. Enterovirus virion carton structure...........................................7
Figure 3. Genome structure of enterovirus..............................................9
Figure 4. Enterovirus life cycle and specific targets for inhibition...........9
Figure 5. Influenza A virion carton structure...........................................14
Figure 6. Influenza A virus life cycle and specific targets for inhibition...16
Figure 7. Structure and EC50 values of two lead agents C-7 and ITA-01..18
Figure 8. Structural units of the lead compound C-7................................27
Figure 9. Structural units of the lead compound ITA-01….....................43
Figure 10. The structural units of the lead compound ITA-01 and Leptomycin B (LMB)............................................................................43
Figure 11. Structural units of the lead agent C-7.....................................58
Figure 12. In-vitro metabolic stability analysis and half-life (t1/2) determination of compounds C-30, C-31, C-40, C-43, and C-44 in 25% rat serum.................................................................................................68
Figure 13. Molecular docking of C-30 and C-44 in the binding sites of pleconaril in EV D68.............................................................................72
Figure 14. The pharmacophore model of bioactive probenecid derivatives to VP1 in EV D68.....................................................................73
Figure 15. The 2D inter-molecular interaction of the compounds ITA-01 and ITA-06 in the LMB binding site of CRM1…………………….......77
Figure 16. Molecular docking between ligands (ITA-04 & ITA-08) and protein (CRM1) in the binding sites of LMB..................................81
Figure 17. The compounds ITA-01 and ITA-14 with its Michael addition characteristics.........................................................................................85
Figure 18. Michael addition characteristics of ITA-01 and ITA-14 using cysteamine by NMR-based assay.........................................................86
1H-NMR and 13C-NMR spectra of all target compounds
Figure 19: 1H-NMR Spectrum of C-7 (CDCl3, 400MHz)......................194
Figure 20: 13C-NMR Spectrum of C-7 (CDCl3, 100MHz).....................194
Figure 21: 1H-NMR Spectrum of C-14 (Acetone-d6, 400MHz)............195
Figure 22: 13C-NMR Spectrum of C-14 (Acetone- d6, 100MHz)..........195
Figure 23: 1H-NMR Spectrum of C-15 (DMSO-d6, 400MHz).............196
Figure 24: 13C-NMR Spectrum of C-15 (DMSO-d6, 100MHz)............196
Figure 25: 1H-NMR Spectrum of C-16 (DMSO-d6, 400MHz).............197
Figure 26: 13C-NMR Spectrum of C-16 (DMSO-d6, 100MHz)............197
Figure 27: 1H-NMR Spectrum of C-17 (DMSO-d6, 400MHz).............198
Figure 28: 13C-NMR Spectrum of C-17 (DMSO-d6, 100MHz)............198
Figure 29: 1H-NMR Spectrum of C-18 (DMSO-d6, 400MHz).............199
Figure 30: 13C-NMR Spectrum of C-18 (DMSO-d6, 100MHz)............199
Figure 31: 1H-NMR Spectrum of C-19 (DMSO-d6, 400MHz).............200
Figure 32: 13C-NMR Spectrum of C-19 (DMSO-d6, 100MHz)............200
Figure 33: 1H-NMR Spectrum of C-20 (DMSO-d6, 400MHz).............201
Figure 34: 13C-NMR Spectrum of C-20 (DMSO-d6, 100MHz)............201
Figure 35: 1H-NMR Spectrum of C-22 (DMSO-d6, 400MHz).............202
Figure 36: 13C-NMR Spectrum of C-22 (DMSO-d6, 100MHz)............202
Figure 37: 1H-NMR Spectrum of C-23 (CDCl3, 400MHz)....................203
Figure 38: 13C-NMR Spectrum of C-23 (CDCl3, 100MHz)...................203
Figure 39: 1H-NMR Spectrum of C-24 (DMSO-d6, 400MHz).............204
Figure 40: 13C-NMR Spectrum of C-24 (DMSO-d6, 100MHz)............204
Figure 41: 1H-NMR Spectrum of C-25 (DMSO-d6, 400MHz).............205
Figure 42: 13C-NMR Spectrum of C-25 (DMSO-d6, 100MHz)............205
Figure 43: 1H-NMR Spectrum of C-26 (DMSO-d6, 400MHz).............206
Figure 44: 13C-NMR Spectrum of C-26 (DMSO-d6, 100MHz)............206
Figure 45: 1H-NMR Spectrum of C-27 (CDCl3, 400MHz)....................207
Figure 46: 13C-NMR Spectrum of C-27 (CDCl3, 100MHz)...................207
Figure 47: 1H-NMR Spectrum of C-29 (Acetone-d6, 400MHz)............208
Figure 48: 13C-NMR Spectrum of C-29 (Acetone-d6, 100MHz)...........208
Figure 49: 1H-NMR Spectrum of C-30 (Acetone-d6, 400MHz)............209
Figure 50: 13C-NMR Spectrum of C-30 (Acetone-d6, 100MHz)...........209
Figure 51: 1H-NMR Spectrum of C-31 (CDCl3, 400MHz)....................210
Figure 52: 13C-NMR Spectrum of C-31 (CDCl3, 100MHz)...................210
Figure 53: 1H-NMR Spectrum of C-33 (DMSO-d6, 400MHz).............211
Figure 54: 13C-NMR Spectrum of C-33 (DMSO-d6, 100MHz)............211
Figure 55: 1H-NMR Spectrum of C-34 (Acetone-d6, 400MHz)............212
Figure 56: 13C-NMR Spectrum of C-34 (Acetone-d6, 100MHz)...........212
Figure 57: 1H-NMR Spectrum of C-36 (Acetone-d6, 400MHz)............213
Figure 58: 13C-NMR Spectrum of C-36 (Acetone-d6, 100MHz)...........213
Figure 59: 1H-NMR Spectrum of C-37 (CDCl3, 400MHz)....................214
Figure 60: 13C-NMR Spectrum of C-37 (CDCl3, 100MHz)...................214
Figure 61: 1H-NMR Spectrum of C-38 (CDCl3, 400MHz)....................215
Figure 62: 13C-NMR Spectra of C-38 (CDCl3, 100MHz)......................215
Figure 63: 1H-NMR Spectrum of C-40 (Acetone-d6, 400MHz)...........216
Figure 64: 13C-NMR Spectrum of C-40 (Acetone-d6, 100MHz)..........216
Figure 65: 1H-NMR Spectrum of C-43 (CDCl3, 400MHz)....................217
Figure 66: 13C-NMR Spectrum of C-43 (CDCl3, 100MHz)...................217
Figure 67: 1H-NMR Spectrum of C-44 (CDCl3, 400MHz)....................218
Figure 68: 13C-NMR Spectrum of C-44 (CDCl3, 100MHz)...................218
Figure 69: 1H-NMR Spectrum of C-45 (Acetone-d6, 400MHz)............219
Figure 70: 13C-NMR Spectrum of C-45 (Acetone-d6, 100MHz)...........219
Figure 71: 1H-NMR Spectrum of C-46 (Acetone-d6, 400MHz)............220
Figure 72: 13C-NMR Spectrum of C-46 (Acetone-d6, 100MHz)...........220
Figure 73: 1H-NMR Spectrum of C-48 (Acetone-d6, 400MHz)............221
Figure 74: 13C-NMR Spectrum of C-48 (Acetone-d6, 100MHz)...........221
Figure 75: 1H-NMR Spectrum of C-49 (Acetone-d6, 400MHz)............222
Figure 76: 13C-NMR Spectrum of C-49 (Acetone-d6, 100MHz)...........222
Figure 77: 1H-NMR Spectrum of C-50 (Acetone-d6, 400MHz)............223
Figure 78: 13C-NMR Spectrum of C-50 (Acetone-d6, 100MHz)...........223
Figure 79: 1H-NMR Spectrum of C-51 (Acetone-d6, 400MHz)............224
Figure 80: 13C-NMR Spectrum of C-51 (Acetone-d6, 100MHz)...........224
Figure 81: 1H-NMR Spectrum of C-52 (CDCl3, 400MHz)....................225
Figure 82: 13C-NMR Spectrum of C-52 (CDCl3, 100MHz)...................225
Figure 83: 1H-NMR Spectrum of ITA-01 (Acetone-d6, 400MHz).......226
Figure 84: 13C-NMR Spectrum of ITA-01 (Acetone- d6, 100MHz)…..226
Figure 85: 1H-NMR Spectrum of ITA-02 (CDCl3, 400MHz)...............227
Figure 86: 13C-NMR Spectrum of ITA-02 (CDCl3, 100MHz)..............227
Figure 87: 1H-NMR Spectrum of ITA-03 (Acetone-d6, 400MHz).......228
Figure 88: 13C-NMR Spectrum of ITA-03 (Acetone- d6, 100MHz)......228
Figure 89: 1H-NMR Spectrum of ITA-04 (CDCl3, 400MHz)...............229
Figure 90: 13C-NMR Spectrum of ITA-04 (CDCl3, 100MHz)..............229
Figure 91: 1H-NMR Spectrum of ITA-05 (CDCl3, 400MHz)................230
Figure 92: 13C-NMR Spectrum of ITA-05 (CDCl3, 100MHz)..............230
Figure 93: 1H-NMR Spectrum of ITA-06 (Acetone-d6, 400MHz).......231
Figure 94: 13C-NMR Spectrum of ITA-06 (Acetone- d6, 100MHz)......231
Figure 95: 1H-NMR Spectrum of ITA-07 (CDCl3, 400MHz)................232
Figure 96: 13C-NMR Spectrum of ITA-07 (CDCl3, 100MHz)..............232
Figure 97: 1H-NMR Spectrum of ITA-08 (CDCl3, 400MHz)...............233
Figure 98: 13C-NMR Spectrum of ITA-08 (CDCl3, 100MHz)..............233
Figure 99: 1H-NMR Spectrum of ITA-09 (CDCl3, 400MHz)................234
Figure 100: 13C-NMR Spectrum of ITA-09 (CDCl3, 100MHz)............234
Figure 101: 1H-NMR Spectrum of ITA-10 (CDCl3, 400MHz)..............235
Figure 102: 13C-NMR Spectrum of ITA-10 (CDCl3, 100MHz)............235
Figure 103: 1H-NMR Spectrum of ITA-11 (CDCl3, 400MHz).............236
Figure 104: 13C-NMR Spectrum of ITA-11 (CDCl3, 100MHz)............236
Figure 105: 1H-NMR Spectrum of ITA-12 (CDCl3, 400MHz)..............237
Figure 106: 13C-NMR Spectrum of ITA-12 (CDCl3, 100MHz)............237
Figure 107: 1H-NMR Spectrum of ITA-13 (CDCl3, 400MHz)..............238
Figure 108: 13C-NMR Spectrum of ITA-13 (CDCl3, 100MHz)............238
Figure 109: 1H-NMR Spectrum of ITA-14 (Acetone- d6, 400MHz).....239
Figure 110: 13C-NMR Spectrum of ITA-14 (Acetone- d6, 100MHz)....239
Figure 111: 1H-NMR Spectrum of ITA-15 (Acetone-d6, 400MHz).....240
Figure 112: 13C-NMR Spectrum of ITA-15 (Acetone- d6, 100MHz)....240
Figure 113: 1H-NMR Spectrum of ITA-01A (Acetone- d6, 400MHz)..241
Figure 114: 13C-NMR Spectrum of ITA-01A (Acetone- d6, 100MHz)..241
Figure 115: 1H-NMR Spectrum of ITA-02A (Acetone- d6, 400MHz)...242
Figure 116: 13C-NMR Spectrum of ITA-02A (Acetone- d6, 100MHz)..242
Figure 117: 1H-NMR Spectrum of ITA-03A (Acetone- d6, 400MHz)..243
Figure 118: 13C-NMR Spectrum of ITA-03A (Acetone- d6, 100MHz)..243
Figure 119: 1H-NMR Spectrum of ITA-04A (Acetone- d6, 400MHz)..244
Figure 120: 13C-NMR Spectrum of ITA-04A (Acetone- d6, 100MHz)..244
Figure 121: 1H-NMR Spectrum of ITA-05A (Acetone- d6, 400MHz)..245
Figure 122: 13C-NMR Spectrum of ITA-05A (Acetone- d6, 100MHz)..245
Figure 123: 1H-NMR Spectrum of ITA-06A (Acetone- d6, 400MHz)..246
Figure 124: 13C-NMR Spectrum of ITA-06A (Acetone- d6, 100MHz)..246
Figure 125: 1H-NMR Spectrum of ITA-07A (Acetone- d6, 400MHz)..247
Figure 126: 13C-NMR Spectrum of ITA-07A (Acetone- d6, 100MHz)..247
Figure 127: 1H-NMR Spectrum of ITA-08A (Acetone- d6, 400MHz)..248
Figure 128: 13C-NMR Spectrum of ITA-08A (Acetone- d6, 100MHz)..248
Figure 129: 1H-NMR Spectrum of ITA-09A (Acetone- d6, 400MHz)..249
Figure 130: 13C-NMR Spectrum of ITA-09A (Acetone- d6, 100MHz)..249
Figure 131: 1H-NMR Spectrum of ITA-10A (Acetone- d6, 400MHz)..250
Figure 132: 13C-NMR Spectrum of ITA-10A (Acetone- d6, 100MHz)..250
Mass spectra of all compounds
Figure 133: ESI-MS of C-7...................................................................251
Figure 134: ESI-MS of C-14.................................................................251
Figure 135: ESI-MS of C-15..................................................................252
Figure 136: ESI-MS of C-16.................................................................252
Figure 137: ESI-MS of C-17………….................................................253
Figure 138: ESI-MS of C-18.................................................................253
Figure 139: ESI-MS of C-19………….................................................254
Figure 140: ESI-MS of C-20………….................................................254
Figure 141: ESI-MS of C-22.................................................................255
Figure 142: ESI-MS of C-23.................................................................255
Figure 143: ESI-MS of C-24………….................................................256
Figure 144: ESI-MS of C-25.................................................................256
Figure 145: ESI-MS of C-26.................................................................257
Figure 146: ESI-MS of C-27.................................................................257
Figure 147: ESI-MS of C-29.................................................................258
Figure 148: ESI-MS of C-30.................................................................258
Figure 149: ESI-MS of C-31.................................................................259
Figure 150: ESI-MS of C-33.................................................................259
Figure 151: ESI-MS of C-34.................................................................260
Figure 152: ESI-MS of C-36.................................................................260
Figure 153: ESI-MS of C-37.................................................................261
Figure 154: ESI-MS of C-38.................................................................261
Figure 155: ESI-MS of C-40.................................................................262
Figure 156: ESI-MS of C-43.................................................................262
Figure 157: ESI-MS of C-44.................................................................263
Figure 158: ESI-MS of C-45.................................................................263
Figure 159: ESI-MS of C-46.................................................................264
Figure 160: ESI-MS of C-48.................................................................264
Figure 161: ESI-MS of C-49.................................................................265
Figure 162: ESI-MS of C-50.................................................................265
Figure 163: ESI-MS of C-51.................................................................266
Figure 164: ESI-MS of C-52.................................................................266
Figure 165: ESI-MS of ITA-01.............................................................267
Figure 166: ESI-MS of ITA-02.............................................................267
Figure 167: ESI-MS of ITA-03.............................................................268
Figure 168: ESI-MS of ITA-04.............................................................268
Figure 169: ESI-MS of ITA-05.............................................................269
Figure 170: ESI-MS of ITA-06.............................................................269
Figure 171: ESI-MS of ITA-07.............................................................270
Figure 172: ESI-MS of ITA-08.............................................................270
Figure 173: ESI-MS of ITA-09.............................................................271
Figure 174: ESI-MS of ITA-10.............................................................271
Figure 175: ESI-MS of ITA-11.............................................................272
Figure 176: ESI-MS of ITA-12.............................................................272
Figure 177: ESI-MS of ITA-13.............................................................273
Figure 178: ESI-MS of ITA-14.............................................................273
Figure 179: ESI-MS of ITA-15.............................................................274
Figure 180: ESI-MS of ITA-01A..........................................................274
Figure 181: ESI-MS of ITA-02A..........................................................275
Figure 182: ESI-MS of ITA-03A..........................................................275
Figure 183: ESI-MS of ITA-04A..........................................................276
Figure 184: ESI-MS of ITA-05A..........................................................276
Figure 185: ESI-MS of ITA-06A..........................................................277
Figure 186: ESI-MS of ITA-07A..........................................................277
Figure 187: ESI-MS of ITA-08A..........................................................278
Figure 188: ESI-MS of ITA-09A..........................................................278
Figure 189: ESI-MS of ITA-10A..........................................................279
Figure 190: HRESIMS of C-30.............................................................280
Figure 191: HRESIMS of C-31.............................................................280
Figure 192: HRESIMS of C-36.............................................................280
Figure 193: HRESIMS of C-37.............................................................281
Figure 194: HRESIMS of C-38.............................................................281
Figure 195: HRESIMS of C-44.............................................................281
Figure 196: HRESIMS of C-45.............................................................282
Figure 197: HRESIMS of C-46.............................................................282
Figure 198: HRESIMS of C-51.............................................................282
Figure 199: HRESIMS of C-52.............................................................282
Figure 200: HRESIMS of ITA-02.........................................................283
Figure 201: HRESIMS of ITA-03.........................................................283
Figure 202: HRESIMS of ITA-04.........................................................283
Figure 203: HRESIMS of ITA-05.........................................................284
Figure 204: HRESIMS of ITA-06.........................................................284
Figure 205: HRESIMS of ITA-07.........................................................284
Figure 206: HRESIMS of ITA-08.........................................................284
Figure 207: HRESIMS of ITA-09.........................................................285
Figure 208: HRESIMS of ITA-10.........................................................285
Figure 209: HRESIMS of ITA-11.........................................................285
Figure 210: HRESIMS of ITA-12........................................................285
Figure 211: HRESIMS of ITA-13........................................................286
Figure 212: HRESIMS of ITA-14........................................................286
Figure 213: HRESIMS of ITA-15........................................................286
Figure 214: HRESIMS of ITA-01A.....................................................286
Figure 215: HRESIMS of ITA-02A.....................................................287
Figure 216: HRESIMS of ITA-03A.....................................................287
Figure 217: HRESIMS of ITA-04A.....................................................287
Figure 218: HRESIMS of ITA-05A.....................................................287
Figure 219: HRESIMS of ITA-06A.....................................................288
Figure 220: HRESIMS of ITA-07A.....................................................288
Figure 221: HRESIMS of ITA-08A.....................................................288
Figure 222: HRESIMS of ITA-10A.....................................................288
UV spectra of all compounds
Figure 223: The uv spectrum of C-7.....................................................289
Figure 224: The uv spectrum of C-14...................................................289
Figure 225: The uv spectrum of C-15...................................................289
Figure 226: The uv spectrum of C-16...................................................289
Figure 227: The uv spectrum of C-17...................................................289
Figure 228: The uv spectrum of C-18...................................................289
Figure 229: The uv spectrum of C-19...................................................289
Figure 230: The uv spectrum of C-20...................................................289
Figure 231: The uv spectrum of C-22...................................................289
Figure 232: The uv spectrum of C-23...................................................289
Figure 233: The uv spectrum of C-24...................................................290
Figure 234: The uv spectrum of C-25...................................................290
Figure 235: The uv spectrum of C-26...................................................290
Figure 236: The uv spectrum of C-27...................................................290
Figure 237: The uv spectrum of C-29....................................................290
Figure 238: The uv spectrum of C-30....................................................290
Figure 239: The uv spectrum of C-31...................................................290
Figure 240: The uv spectrum of C-33....................................................290
Figure 241: The uv spectrum of C-34....................................................290
Figure 242: The uv spectrum of C-36....................................................290
Figure 243: The uv spectrum of C-37....................................................291
Figure 244: The uv spectrum of C-38....................................................291
Figure 245: The uv spectrum of C-40....................................................291
Figure 246: The uv spectrum of C-43....................................................291
Figure 247: The uv spectrum of C-44....................................................291
Figure 248: The uv spectrum of C-45....................................................291
Figure 249: The uv spectrum of C-46....................................................291
Figure 250: The uv spectrum of C-48....................................................291
Figure 251: The uv spectrum of C-49....................................................291
Figure 252: The uv spectrum of C-50....................................................291
Figure 253: The uv spectrum of C-51....................................................292
Figure 254: The uv spectrum of C-52....................................................292
Figure 255: The uv spectrum of ITA-01................................................293
Figure 256: The uv spectrum of ITA-02................................................293
Figure 257: The uv spectrum of ITA-03................................................293
Figure 258: The uv spectrum of ITA-04................................................293
Figure 259: The uv spectrum of ITA-05................................................293
Figure 260: The uv spectrum of ITA-06................................................293
Figure 261: The uv spectrum of ITA-07................................................293
Figure 262: The uv spectrum of ITA-08................................................293
Figure 263: The uv spectrum of ITA-09................................................293
Figure 264: The uv spectrum of ITA-10................................................293
Figure 265: The uv spectrum of ITA-11...............................................294
Figure 266: The uv spectrum of ITA-12...............................................294
Figure 267: The uv spectrum of ITA-13...............................................294
Figure 268: The uv spectrum of ITA-14...............................................294
Figure 269: The uv spectrum of ITA-15...............................................294
Figure 270: The uv spectrum of ITA-01A............................................294
Figure 271: The uv spectrum of ITA-02A............................................294
Figure 272: The uv spectrum of ITA-03A............................................294
Figure 273: The uv spectrum of ITA-04A............................................294
Figure 274: The uv spectrum of ITA-05A............................................294
Figure 275: The uv spectrum of ITA-06A............................................295
Figure 276: The uv spectrum of ITA-07A............................................295
Figure 277: The uv spectrum of ITA-08A............................................295
Figure 278: The uv spectrum of ITA-09A............................................295
Figure 279: The uv spectrum of ITA-10A............................................295
IR spectra of all compounds
Figure 280: IR spectrum of C-7.............................................................296
Figure 281: IR spectrum of C-14...........................................................296
Figure 282: IR spectrum of C-15...........................................................296
Figure 283: IR spectrum of C-16...........................................................296
Figure 284: IR spectrum of C-17...........................................................296
Figure 285: IR spectrum of C-18...........................................................296
Figure 286: IR spectrum of C-19...........................................................296
Figure 287: IR spectrum of C-20...........................................................296
Figure 288: IR spectrum of C-22...........................................................297
Figure 289: IR spectrum of C-23...........................................................297
Figure 290: IR spectrum of C-24...........................................................297
Figure 291: IR spectrum of C-25...........................................................297
Figure 292: IR spectrum of C-26..........................................................297
Figure 293: IR spectrum of C-27..........................................................297
Figure 294: IR spectrum of C-29..........................................................297
Figure 295: IR spectrum of C-30..........................................................297
Figure 296: IR spectrum of C-31..........................................................298
Figure 297: IR spectrum of C-33..........................................................298
Figure 298: IR spectrum of C-34..........................................................298
Figure 299: IR spectrum of C-36..........................................................298
Figure 300: IR spectrum of C-37..........................................................298
Figure 301: IR spectrum of C-38..........................................................298
Figure 302: IR spectrum of C-40..........................................................298
Figure 303: IR spectrum of C-43..........................................................298
Figure 304: IR spectrum of C-44..........................................................299
Figure 305: IR spectrum of C-45..........................................................299
Figure 306: IR spectrum of C-46..........................................................299
Figure 307: IR spectrum of C-48..........................................................299
Figure 308: IR spectrum of C-49..........................................................299
Figure 309: IR spectrum of C-50..........................................................299
Figure 310: IR spectrum of C-51..........................................................299
Figure 311: IR spectrum of C-52...........................................................299
Figure 312: IR spectrum of ITA-01.......................................................300
Figure 313: IR spectrum of ITA-02.......................................................300
Figure 314: IR spectrum of ITA-03.......................................................300
Figure 315: IR spectrum of ITA-04.......................................................300
Figure 316: IR spectrum of ITA-05.......................................................300
Figure 317: IR spectrum of ITA-06.......................................................300
Figure 318: IR spectrum of ITA-07.......................................................300
Figure 319: IR spectrum of ITA-08.......................................................300
Figure 320: IR spectrum of ITA-09.......................................................301
Figure 321: IR spectrum of ITA-10.......................................................301
Figure 322: IR spectrum of ITA-11.......................................................301
Figure 323: IR spectrum of ITA-12.......................................................301
Figure 324: IR spectrum of ITA-13.......................................................301
Figure 325: IR spectrum of ITA-14.......................................................301
Figure 326: IR spectrum of ITA-15.......................................................301
Figure 327: IR spectrum of ITA-01A....................................................301
Figure 328: IR spectrum of ITA-02A....................................................302
Figure 329: IR spectrum of ITA-03A....................................................302
Figure 330: IR spectrum of ITA-04A....................................................302
Figure 331: IR spectrum of ITA-05A....................................................302
Figure 332: IR spectrum of ITA-06A....................................................302
Figure 333: IR spectrum of ITA-07A....................................................302
Figure 334: IR spectrum of ITA-08A....................................................302
Figure 335: IR spectrum of ITA-09A....................................................302
Figure 336: IR spectrum of ITA-10A....................................................303
HPLC purity analysis profile of all compounds
Figure 337: HPLC profile of C-7, 80%MeOH.......................................304
Figure 338: HPLC profile of C-7, 60%MeCN.......................................304
Figure 339: HPLC profile of C-14, 80%MeOH.....................................304
Figure 340: HPLC profile of C-14, 60%MeCN.....................................304
Figure 341: HPLC profile of C-15, 70%MeOH.....................................304
Figure 342: HPLC profile of C-15, 60%MeCN.....................................304
Figure 343: HPLC profile of C-16, 70%MeOH.....................................304
Figure 344: HPLC profile of C-16, 60%MeCN.....................................304
Figure 345: HPLC profile of C-17, 70%MeOH.....................................305
Figure 346: HPLC profile of C-17, 60%MeCN.....................................305
Figure 347: HPLC profile of C-18, 70%MeOH.....................................305
Figure 348: HPLC profile of C-18, 60%MeCN.....................................305
Figure 349: HPLC profile of C-19, 70%MeOH.....................................305
Figure 350: HPLC profile of C-19, 60%MeCN.....................................305
Figure 351: HPLC profile of C-20, 70%MeOH.....................................305
Figure 352: HPLC profile of C-20, 60%MeCN.....................................305
Figure 353: HPLC profile of C-22, 70%MeOH.....................................306
Figure 354: HPLC profile of C-22, 60%MeCN.....................................306
Figure 355: HPLC profile of C-23, 70%MeOH.....................................306
Figure 356: HPLC profile of C-23, 60%MeCN.....................................306
Figure 357: HPLC profile of C-24, 70%MeOH.....................................306
Figure 358: HPLC profile of C-24, 60%MeCN.....................................306
Figure 359: HPLC profile of C-25, 70%MeOH.....................................306
Figure 360: HPLC profile of C-25, 60%MeCN.....................................306
Figure 361: HPLC profile of C-26, 70%MeOH.....................................307
Figure 362: HPLC profile of C-26, 60%MeCN.....................................307
Figure 363: HPLC profile of C-27, 70%MeOH.....................................307
Figure 364: HPLC profile of C-27, 60%MeCN.....................................307
Figure 365: HPLC profile of C-29, 75%MeOH.....................................307
Figure 366: HPLC profile of C-29, 65%MeCN.....................................307
Figure 367: HPLC profile of C-30, 80%MeOH.....................................307
Figure 368: HPLC profile of C-30, 70%MeCN.....................................307
Figure 369: HPLC profile of C-31, 75%MeOH.....................................308
Figure 370: HPLC profile of C-31, 65%MeCN.....................................308
Figure 371: HPLC profile of C-33, 65%MeOH.....................................308
Figure 372: HPLC profile of C-33, 55%MeCN.....................................308
Figure 373: HPLC profile of C-34, 70%MeOH.....................................308
Figure 374: HPLC profile of C-34, 55%MeCN.....................................308
Figure 375: HPLC profile of C-36, 85%MeOH.....................................308
Figure 376: HPLC profile of C-36, 70%MeCN.....................................308
Figure 377: HPLC profile of C-37, 85%MeOH.....................................309
Figure 378: HPLC profile of C-37, 70%MeCN.....................................309
Figure 379: HPLC profile of C-38, 75%MeOH.....................................309
Figure 380: HPLC profile of C-38, 65%MeCN.....................................309
Figure 381: HPLC profile of C-40, 80%MeOH.....................................309
Figure 382: HPLC profile of C-40, 70%MeCN.....................................309
Figure 383: HPLC profile of C-43, 75%MeOH.....................................309
Figure 384: HPLC profile of C-43, 65%MeCN.....................................309
Figure 385: HPLC profile of C-44, 75%MeOH.....................................310
Figure 386: HPLC profile of C-44, 65%MeCN.....................................310
Figure 387: HPLC profile of C-45, 75%MeOH.....................................310
Figure 388: HPLC profile of C-45, 65%MeCN.....................................310
Figure 389: HPLC profile of C-46, 75%MeOH.....................................310
Figure 390: HPLC profile of C-46, 65%MeCN.....................................310
Figure 391: HPLC profile of C-48, 75%MeOH.....................................310
Figure 392: HPLC profile of C-48, 65%MeCN.....................................310
Figure 393: HPLC profile of C-49, 70%MeOH.....................................311
Figure 394: HPLC profile of C-49, 60%MeCN.....................................311
Figure 395: HPLC profile of C-50, 75%MeOH.....................................311
Figure 396: HPLC profile of C-50, 65%MeCN.....................................311
Figure 397: HPLC profile of C-51, 75%MeOH.....................................311
Figure 398: HPLC profile of C-51, 65%MeCN.....................................311
Figure 399: HPLC profile of C-52, 85%MeOH.....................................311
Figure 400: HPLC profile of C-52, 75%MeCN.....................................311
Figure 401: HPLC profile of ITA-01, 65%MeOH................................312
Figure 402: HPLC profile of ITA-01, 50%MeCN................................312
Figure 403: HPLC profile of ITA-02, 80%MeOH................................312
Figure 404: HPLC profile of ITA-02, 65%MeCN................................312
Figure 405: HPLC profile of ITA-03, 80%MeOH................................312
Figure 406: HPLC profile of ITA-03, 65%MeCN................................312
Figure 407: HPLC profile of ITA-04, 80%MeOH................................312
Figure 408: HPLC profile of ITA-04, 65%MeCN................................312
Figure 409: HPLC profile of ITA-05, 65%MeOH................................313
Figure 410: HPLC profile of ITA-05, 50%MeCN................................313
Figure 411: HPLC profile of ITA-06, 65%MeOH................................313
Figure 412: HPLC profile of ITA-06, 50%MeCN................................313
Figure 413: HPLC profile of ITA-07, 80%MeOH................................313
Figure 414: HPLC profile of ITA-07, 65%MeCN................................313
Figure 415: HPLC profile of ITA-08, 80%MeOH................................313
Figure 416: HPLC profile of ITA-08, 65%MeCN................................313
Figure 417: HPLC profile of ITA-09, 65%MeOH................................314
Figure 418: HPLC profile of ITA-09, 50%MeCN................................314
Figure 419: HPLC profile of ITA-10, 75%MeOH................................314
Figure 420: HPLC profile of ITA-10, 55%MeCN................................314
Figure 421: HPLC profile of ITA-11, 65%MeOH................................314
Figure 422: HPLC profile of ITA-11, 50%MeCN................................314
Figure 423: HPLC profile of ITA-12, 70%MeOH................................314
Figure 424: HPLC profile of ITA-12, 60%MeCN................................314
Figure 425: HPLC profile of ITA-13, 85%MeOH................................315
Figure 426: HPLC profile of ITA-13, 70%MeCN................................315
Figure 427: HPLC profile of ITA-14, 65%MeOH................................315
Figure 428: HPLC profile of ITA-14, 50%MeCN................................315
Figure 429: HPLC profile of ITA-15, 65%MeOH................................315
Figure 430: HPLC profile of ITA-15, 50%MeCN................................315
Figure 431: HPLC profile of ITA-01A, 50%MeOH+0.05%TFA.........316
Figure 432: HPLC profile of ITA-01A, 40%MeCN+0.05%TFA.........316
Figure 433: HPLC profile of ITA-02A, 50%MeOH+0.05%TFA.........316
Figure 434: HPLC profile of ITA-02A, 40%MeCN+0.05%TFA.........316
Figure 435: HPLC profile of ITA-03A, 50%MeOH+0.05%TFA.........316
Figure 436: HPLC profile of ITA-03A, 40%MeCN+0.05%TFA.........316
Figure 437: HPLC profile of ITA-04A, 50%MeOH+0.05%TFA.........316
Figure 438: HPLC profile of ITA-04A, 40%MeCN+0.05%TFA.........316
Figure 439: HPLC profile of ITA-05A, 50%MeOH+0.05%TFA.........317
Figure 440: HPLC profile of ITA-05A, 40%MeCN+0.05%TFA.........317
Figure 441: HPLC profile of ITA-06A, 50%MeOH+0.05%TFA.........317
Figure 442: HPLC profile of ITA-06A, 40%MeCN+0.05%TFA.........317
Figure 443: HPLC profile of ITA-07A, 50%MeOH+0.05%TFA.........317
Figure 444: HPLC profile of ITA-07A, 40%MeCN+0.05%TFA.........317
Figure 445: HPLC profile of ITA-08A, 50%MeOH+0.05%TFA.........317
Figure 446: HPLC profile of ITA-08A, 40%MeCN+0.05%TFA.........317
Figure 447: HPLC profile of ITA-09A, 50%MeOH+0.05%TFA.........318
Figure 448: HPLC profile of ITA-09A, 40%MeCN+0.05%TFA.........318
Figure 449: HPLC profile of ITA-10A, 65%MeOH+0.05%TFA.........318
Figure 450: HPLC profile of ITA-10A, 40%MeCN+0.05%TFA.........318








List of Tables
Table 1. Synthesized all C-series target compounds with their substituents......................................................................................39
Table 2. SAR studies by replacing different halo-groups at m-position..59
Table 3. SAR studies by introducing chain extension/contraction strategy.............................................................................................60
Table 4. SAR studies of compounds with their bio-isostere derivatives..61
Table 5. SAR studies by substituting a hetero (N) group on p-position of unit-I.......................................................................................................62
Table 6. SAR studies of pyrrolidine and diethylamine containing derivatives..................................................................................63
Table 7. SAR studies of morpholine containing derivatives....................64
Table 8. SAR studies by substituting methyl group to amide proton........65
Table 9. SAR studies by replacing dipropyl amine to dipropene- and dipropargyl- amine group.................................................................66
Table 10. SAR studies by substituting Chloro-group on different positions of Unit-I site……………………………………………………….…...67
Table 11. Antiviral activity of C-30 and C-44 against various EV strains in RD cells…………………………………………………………70
Table 12. SAR studies by change in fluoro-position..........................76
Table 13. SAR studies by introducing different EWGs to replace fluoro-substituents........................................................................................79
Table 14. SAR studies by conjugating highly EWGs to replace m & p-fluoro groups.......................................................................................80
Table 15. SAR studies by conjugating highly EDGs to replace m & p-fluoro groups.........................................................................................82
Table 16. SAR studies by conjugating highly EWGs to replace o-F groups………………………………………………………................83
Table 17. SAR studies by introducing chain extension strategy............85
Table 18. SAR studies by introducing different EWGs at unit-I site to replace fluoro-groups...........................................................................87
Table 19. Inhibition spectrum of ITA-01 and 04 against different virus strains.....................................................................................89
Table 20. Structures and EC50 of all synthesized C-series compounds...126
Table 21. Structure and activity profile of all Synthesized ITA-series compounds………………...................................................................129
Table 22. HPLC purity analysis data of all C-series synthetics..............131
Table 23. HPLC purity analysis of all ITA-01 derivatives....................132
Table 24. HPLC purity analysis of ITA-01A and its derivatives...........133
Table 25. In vitro data of C-7 Analogues against EV D68 and EV A71..134
Table 26. Itaconic acid derivatives (ITA-01~15) and the anti-IAV activity profile..................................................................................135
Table 27. Itaconic acid derivatives (ITA-01A~10A) and the anti-IAV activity profile……………………………………………………136












List of Schemes
Scheme 1. Retro synthetic analysis of the lead agent C-7.........................26
Scheme 2. Retro synthetic analysis of the lead agent ITA-01..................27
Scheme 3. Chemical modification strategy of the compound C-7...........29
Scheme 4. Reaction protocol for the all target compounds of C-7 analogues...............................................................................................30
Scheme 5. Synthetic procedure of 4-[(dialkylamino)sulfonyl]benzoic acid analogues...........................................................................................31
Scheme 6. Synthetic protocol for C-21 and C-32.....................................31
Scheme 7. Mechanism for the synthesis of sulfonamides........................32
Scheme 8. Mechanism of acid to acid chloride formation........................33
Scheme 9. Mechanism for the formation of peptide bond.......................34
Scheme 10. Synthetic protocol for the compounds C-7, C-14, C-15, C-16, C-17, C-18, C-19, C-20, C-22, C-23, C-29, C-30, C-31, C-36, C-37 and C-40..................................................................................................35
Scheme 11. Synthetic protocol for the compounds C-24, C-25, C-26, C-27, C-33, and C-34.................................................................................36
Scheme 12. Synthetic protocol for the compounds C-38, C-43, C-44, C-45, and C-46………………………………………………………37
Scheme 13. Synthetic protocol for the compounds C-48, C-49, C-50, C-51, and C-52…………………………………………………….…38
Scheme 14. Chemical modification strategy of compound ITA-01.........44
Scheme 15. Reaction protocol for the all ITA-01 derivatives..................45

1. Mack, A.; Choffnes, E. R. Emerging Viral Diseases: The One Health Connection: Workshop Summary. National Academies Press. 2015, 1-5. Workshop Overview. Available from: http://www.ncbi.nlm.nih.gov/books/NBK284993/
2. Al Hajjar, S.; McIntosh, K. The first influenza pandemic of the 21st century. Ann. Saudi Med. 2010, 30(1), 1–10.
3. Taubenberger, J. K.; Morens, D. M. The pathology of influenza virus infections. Annu. Rev. pathol-Mech. 2008, 3, 499-522.
4. Lin, J. Y.; Shih, S. R. Cell and tissue tropism of enterovirus 71 and other enteroviruses infections. J. Biomed. Sci. 2014, 21(1), 1-6.
5. Owatanapanich, S.; Wutthanarungsan, R.; Jaksupa, W. Risk factors for severe hand, foot and mouth disease. S. E. Asian J. Trop. Med. 2015, 46(3), 449-459.
6. Midgley, C. M.; Watson, J. T.; Nix, W. A.; Curns, A. T.; Rogers, S. L.; Brown, B. A.; Hassan, F. Severe respiratory illness associated with a nationwide outbreak of enterovirus D68 in the USA (2014): a descriptive epidemiological investigation. Lancet Respir. Med. 2015, 3(11), 879-887.
7. Ho, M.; Chen, E. R.; Hsu, K. H.; AbuBakar, S.; Chee, H. Y.; Al-Kobaisi, M. F.; Lam, S. K. Outbreaks of enterovirus 71 infection. N. Engl. J. Med. 2000, 2000(342), 355-356.
8. Jiang, P.; Liu, Y.; Ma, H. C.; Paul, A.V.; Wimmer, E. Picornavirus morphogenesis. Microbiol Mol Biol Rev. 2014, 78(3), 418-437.
9. Knipe D. M.; Howley P. M. Fields Virology, 5th ed. Wolters Kluwer, Lippincott Williams & Wilkins: Philadelphia. 2013, pp 796-811.

10. Tapparel, C.; Siegrist, F.; Petty, T. J.; Kaiser, L. Picornavirus and enterovirus diversity with associated human diseases. Infect. Genet. Evol. 2013, 14, 282-293.
11. Ulferts, R.; de Boer, S. M.; van der Linden. L.; Bauer, L.; Lyoo, H. R.; Maté, M. J.; Lichière J.; Canard B.; Lelieveld D.; Omata W.; Egan, D.; Coutard B.; Kuppeveld F. J. M. van. Screening of a library of FDA-approved drugs identifies several enterovirus replication inhibitors that target viral protein 2C. Antimicrob. Agents Chemother. 2016, 60(5), 2627-2638.
12. Kim, Y.; Kankanamalage, A. C. G.; Damalanka, V. C.; Weerawarna, P. M.; Groutas, W. C.; Chang, K. O. Potent inhibition of enterovirus D68 and human rhinoviruses by dipeptidyl aldehydes and α-ketoamides. Antiviral Res. 2016, 125, 84-91.
13. King, A. M. Virus taxonomy: classification and nomenclature of viruses; Ninth Report of the International Committee on Taxonomy of Viruses, Elsevier, 2012, 9. 855–880.
14. Slade, T. A.; Klekamp, B.; Rico, E.; Mejia-Echeverry, A. Measles outbreak in an unvaccinated family and a possibly associated international traveler-Orange County, Florida, December 2012-January 2013. MMWR Morb. Mortal. Wkly. Rep. 2014, 63(36), 781-4.
15. Schieble, J. H.; Fox, V. L.; Lennette, E. H. A probable new human picornavirus associated with respiratory disease. Am. J. Epidemiol. 1967, 85(2), 297-310.
16. Oberste, M. S.; Maher, K.; Schnurr, D.; Flemister, M. R.; Lovchik, J. C.; Peters, H.; Sessions, W.; Kirk, C.; Chatterjee, N.; Fuller, S.; Hanauer, J. M. Enterovirus 68 is associated with respiratory illness and shares biological features with both the enteroviruses and the rhinoviruses. J. Gen. Virol. 2004, 85(9), 2577-2584.
17. Enterovirus D68, Centers for Disease Control and Prevention, 2016. http://www.cdc.gov/non-polio-enterovirus/about/ev-d68.html
18. Lau, S. K.; Yip, C. C.; Zhao, P. S. H.; Chow, W. N.; To, K. K.; Wu, A. K.; Yuen, K. Y.; Woo, P. C. Enterovirus d68 infections associated with severe respiratory illness in elderly patients and emergence of a novel clade in Hong Kong. Sci. Rep. 2016, 6 (25147), 1-9.
19. Oermann, C. M.; Schuster, J. E.; Conners, G. P.; Newland, J. G.; Selvarangan, R.; Jackson, M. A. Enterovirus d68, A focused review and clinical highlights from the 2014 US Outbreak. Ann. Am. Thorac. Soc. 2015, 12(5), 775-781.
20. Holm-Hansen, C. C.; Midgley, S. E.; Fischer, T. K. Global emergence of enterovirus D68: a systematic review. Lancet Infect. Dis. 2016, 16(5), e64-e75.
21. Huang, Y. P.; Lin, T. L.; Lin, T. H.; Wu, H. S. Molecular and epidemiological study of enterovirus D68 in Taiwan. J. Microbiol. Immunol. Infect. 2017, 50(4), 411-417.
22. Lin, T. Y.; Twu, S. J.; Ho, M. S.; Chang, L. Y.; Lee, C. Y. Enterovirus 71 outbreaks, Taiwan: occurrence and recognition. Emerg. Infect. Dis. 2003, 9(3), 291-293.
23. Zeng, M.; El Khatib, N. F.; Tu, S.; Ren, P.; Xu, S.; Zhu, Q.; Altmeyer, R. Seroepidemiology of enterovirus 71 infection prior to the 2011 season in children in Shanghai. J. Clin. Virol. 2012, 53(4), 285-289.
24. Huang, W.; Wang, G.; Zhuge, J.; Nolan, S. M.; Dimitrova, N.; Fallon, J. T. Whole-genome sequence analysis reveals the Enterovirus D68 isolates during the United States 2014 outbreak mainly belong to a novel clade. Sci. Rep. 2015, 5 (15223), 1-11.
25. Imamura, T.; Oshitani, H. Global re-emergence of enterovirus D68 as an important pathogen for acute respiratory infections. Rev. Med. Virol. 2015, 25(2), 102-114.
26. Patick, A. K.; Potts, K. E. Protease inhibitors as antiviral agents. Clin. Microbiol. Rev. 1998, 11(4), 614-627.
27. Lin, J. Y.; Chen, T. C.; Weng, K. F.; Chang, S. C.; Chen, L. L.; Shih, S. R. Viral and host proteins involved in picornavirus life cycle. J. Biomed. Sci. 2009, 16(1), 1-14.
28. Gao, Y.; Sun, S. Q.; Guo, H. C. Biological function of Foot-and-mouth disease virus non-structural proteins and non-coding elements. Virol. J. 2016, 13(1), 107-123.
29. Watanabe, T.; Watanabe, S.; Kawaoka, Y. Cellular networks involved in the influenza virus life cycle. Cell Host Microbe. 2010, 7(6), 427-439.
30. Shih, S. R.; Stollar, V.; Li, M. L., Host factors in enterovirus 71 replication. J. Virol. 2011, 85(19), 9658-9666.
31. Liu, Y.; Sheng, J.; Baggen, J.; Meng, G.; Xiao, C., Thibaut, H. J.; Rossmann, M. Sialic acid-dependent cell entry of human enterovirus D68. Nat. commun. 2015, 6 (8865), 1-7
32. De Chassey, B.; Meyniel-Schicklin, L.; Vonderscher, J.; André, P.; Lotteau, V. Virus-host interactomics: new insights and opportunities for antiviral drug discovery. Genome Med. 2014, 6(11), 1-14.
33. Ruiz, A.; Russell, S. J. A new paradigm in viral resistance. Cell Res. 2012, 22(11), 1515-1517.
34. Tan, C. S.; Cardosa, M. J.; High-titred neutralizing antibodies to human enterovirus 71 preferentially bind to the N-terminal portion of the capsid protein VP1. Arch. Virol. 2007, 152(6), 1069-1073.
35. Ku, Z.; Shi, J.; Liu, Q.; Huang, Z. Development of murine monoclonal antibodies with potent neutralization effects on enterovirus 71. J. Virol. Methods. 2012, 186(1), 193-197.
36. Paul, A. V.; Peters, J.; Mugavero, J.; Yin, J.; van Boom, J. H.; Wimmer, E. Biochemical and genetic studies of the VPg uridylylation reaction catalyzed by the RNA polymerase of poliovirus. J. Virol. 2003, 77(2), 891-904.
37. Whitton, J. L.; Cornell, C. T.; Feuer, R. Host and virus determinants of picornavirus pathogenesis and tropism. Nat. Rev. Microbiol. 2005, 3(10), 765-776.
38. Centers for Disease Control and Prevention; Pregnancy & Non-Polio Enterovirus Infection, 2016, Available from: http://www.cdc.gov/non-polio-enterovirus/pregnancy.html
39. De Clercq, E. Antiviral drugs in current clinical use. J. Clin. Virol. 2004, 30(2), 115-133.
40. Zhou, Y.; Li, J. X.; Jin, P. F.; Wang, Y. X.; Zhu, F. C. Enterovirus 71: a whole virion inactivated enterovirus 71 vaccines. Expert Rev. Vaccines. 2016, 15(7), 803-813.
41. Sun, L.; Meijer, A.; Froeyen, M.; Zhang, L.; Thibaut, H. J.; Baggen, J.; George, S.; Vernachio, J.; van Kuppeveld, F. J. M.; Leyssen, P.; Hilgenfeld, R.; Neyts, J.; Delang, L. Antiviral activity of broad-spectrum and enterovirus-specific inhibitors against clinical isolates of enterovirus D68. Antimicrob. Agents Chemother. 2015, 59(12), 7782-7785.
42. Wang, Y.; Li, G.; Yuan, S.; Gao, Q.; Lan, K.; Altmeyer, R.; Zou, G. In vitro assessment of combinations of enterovirus inhibitors against enterovirus 71. Antimicrob. Agents Chemother. 2016, 60(9), 5357-5367.
43. Murray, J. S.; Mirch, M. C.; Amin, P. M., Enterovirus D68: What pediatric healthcare professionals need to know. J. Spec. Pediatr. Nurs. 2015, 20(2), 131-135.
44. World Health Organization Enterovirus Surveillance Guidelines. Guidelines for Enterovirus Surveillance in Support of the Polio Eradication Initiative, 2015, 1-46.
45. Calder, L. J.; Wasilewski, S.; Berriman, J. A.; Rosenthal, P. B. Structural organization of a filamentous influenza A virus. Proc. Natl. Acad. Sci. U.S.A. 2010, 107(23), 10685-10690.
46. Barberis, I.; Myles, P.; Ault, S. K.; Bragazzi, N. L.; Martini, M. History and evolution of influenza control through vaccination: from the first monovalent vaccine to universal vaccines. J. Prev. Med. Hyg. 2016, 57(3), p. E115.
47. Franci, G.; Palomba, L.; Falanga, A.; Zannella, C.; D’Oriano, V.; Rinaldi, L.; Galdiero, S.; Galdiero, M. Influenza virus infections: clinical update, molecular biology, and therapeutic options. Microbiol. Resp. Sys. Infect. 2016, 1-32.
48. Boyd, M.; Clezy, K.; Lindley, R.; Pearce, R. Pandemic influenza: clinical issues. Med. J. Aus. 2006, 185(10), p. S44.
49. Kilbourne, E. D. Influenza pandemics of the 20th century. Emerg. Infect. Dis. 2006, 12(1), 9-14.
50. Patel, K. K.; Patel, A. K.; Shah, S.; Ranjan, R.; Shah, S.V. Adult patient with novel H1N1 infection presented with encephalitis, rhabdomyolysis, pneumonia and polyneuropathy. J. Glob. Infect. Dis. 2012, 4(3), p.178.
51. Influenza (Flu), Centers for Disease Control and Prevention, 2016. https://www.cdc.gov/flu/about/disease/high_risk.htm
52. Wu, X.; Wu, X.; Sun, Q.; Zhang, C.; Yang, S.; Li, L.; Jia, Z. Progress of small molecular inhibitors in the development of anti-influenza virus agents. Theranostics. 2017, 7(4), p.826.
53. Taubenberger, J. K.; Morens, D. M. Pandemic influenza–including a risk assessment of H5N1. Rev. Sci. Tech. OIE. 2009, 28(1), 187-202.
54. World Health Organization Influenza Fact Sheet 211. In: Centre M, editor. World Health Organization. Geneva. 2016.
55. Davis, A. M.; Chabolla, B. J.; Newcomb, L. L. Emerging antiviral resistant strains of influenza A and the potential therapeutic targets within the viral ribonucleoprotein (vRNP) complex. Virol. J., 2014, 11(1), p.167.
56. Gallagher, J. R.; Torian, U.; McCraw, D. M.; Harris, A. K.; Structural studies of influenza virus RNPs by electron microscopy indicate molecular contortions within NP supra-structures. J. Struct. Biol. 2017, 197(3), 294-307.
57. Medina, R. A.; García-Sastre, A. Influenza A viruses: new research developments. Nat. Rev. Microbiol. 2011, 9(8), p.590.
58. Muramoto, Y.; Noda, T.; Kawakami, E.; Akkina, R.; Kawaoka, Y. Identification of novel influenza A virus proteins translated from PA mRNA. J. Virol. 2013, 87(5), 2455-2462.
59. Matsuoka, Y.; Matsumae, H.; Katoh, M.; Eisfeld, A. J.; Neumann, G.; Hase, T.; Ghosh, S.; Shoemaker, J. E.; Lopes, T. J.; Watanabe, T.; Watanabe, S.; Fukuyama, S.; Kitano, H.; Kawaoka, Y. A comprehensive map of the influenza A virus replication cycle. BMC Sys. Biol. 2013, 7(1), 97-115.
60. Neumann, G.; Noda, T.; Kawaoka, Y. Emergence and pandemic potential of swine-origin H1N1 influenza virus. Nature. 2009, 459(7249), p.931.
61. Shi, Y.; Wu, Y.; Zhang, W.; Qi, J.; Gao, G. F. Enabling the'host jump': structural determinants of receptor-binding specificity in influenza A virus. Nat. Rev. Microbiol. 2014, 12(12), p.822.
62. König, R.; Stertz, S.; Zhou, Y.; Inoue, A.; Hoffmann, H. H.; Bhattacharyya, S.; Alamares, J. G.; Tscherne, D. M.; Ortigoza, M. B.; Liang, Y.; Gao, Q. Human host factors required for influenza virus replication. Nature. 2010, 463(7282), p.813.
63. Watanabe, T.; Kawakami, E.; Shoemaker, J. E.; Lopes, T. J.; Matsuoka, Y.; Tomita, Y.; Kozuka-Hata, H.; Gorai, T.; Kuwahara, T.; Takeda, E.; Nagata, A. Influenza virus-host interactome screen as a platform for antiviral drug development. Cell host microbe. 2014, 16(6), 795-805.
64. Scorza, F. B.; Tsvetnitsky, V.; Donnelly, J. J. Universal influenza vaccines: Shifting to better vaccines. Vaccine. 2016, 34(26), 2926-2933.
65. Kilic, H.; Kanbay, A.; Karalezli, A.; Hasanoğlu, H. C.; Ateş, C. Clinical characteristics of 75 pandemic H1N1 influenza patients from Turkey; risk factors for fatality. Turk J. Med. Sci. 2015, 45(3), 562-567.
66. Centers for Disease Control and Prevention, Influenza antiviral medications: summary for clinicians. 2017. Available from: https://www.cdc.gov/flu/professionals/antivirals/summary-clinicians.htm
67. Govorkova, E. A.; Webster, R. G. Combination chemotherapy for influenza. Viruses. 2010, 2(8), 1510-1529.
68. Kamali, A., & Holodniy, M. Influenza treatment and prophylaxis with neuraminidase inhibitors: a review. Infect. Drug Resist. 2013, 6, 187-198.
69. Moscona, A. Global transmission of oseltamivir-resistant influenza. N. Engl. J Med. 2009, 360(10), 953-956.
70. Fiore, A. E., Fry, A., Shay, D., Gubareva, L., Bresee, J. S., Uyeki, T. M., & Centers for Disease Control and Prevention (CDC). Antiviral agents for the treatment and chemoprophylaxis of influenza—recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep, 2011, 60(1), 1-24.
71. Mai-Phuong, H. V.; Thach, N. C.; Hang, N. L. K.; Phuongb, N. T. K.; Mai L. Q. Oseltamivir resistance among influenza viruses: surveillance in northern Viet Nam, 2009–2012. Western Pac. Surveill Response J. 2013, 4(2), 25-29.
72. Hinsberger, S.; Hüsecken, K.; Groh, M.; Negri, M.; Haupenthal, J.; Hartmann, R.W. Discovery of novel bacterial RNA polymerase inhibitors: pharmacophore-based virtual screening and hit optimization. J. Med. Chem. 2013, 56(21), 8332-8338.
73. Watanabe, T.; Kawaoka, Y. Influenza virus–host interactomes as a basis for antiviral drug development. Curr. Opin. Virol. 2015, 14, 71-78.
74. Oh, D. Y.; Hurt, A. C. A review of the antiviral susceptibility of human and avian influenza viruses over the last decade. Scientifica. 2014, 2014, 1-10.
75. Mathew, C.; Ghildyal, R. CRM1 inhibitors for antiviral therapy. Front. Microbiol. 2017, 8, p.1171.
76. Sun, Q.; Carrasco, Y. P.; Hu, Y.; Guo, X.; Mirzaei, H.; MacMillan, J.; Chook, Y.M. Nuclear export inhibition through covalent conjugation and hydrolysis of Leptomycin B by CRM1. Proc. Natl. Acad. Sci. U.S.A. 2013, 110(4), 303-1308.
77. Xu, D.; Grishin, N. V.; Chook, Y. M. NESdb: a database of NES-containing CRM1 cargoes. Mol. Biol. Cell, 2012, 23(18), 3673-3676.
78. Nguyen, K. T.; Holloway, M. P.; Altura, R. A. The CRM1 nuclear export protein in normal development and disease. Int. J. Biochem. Mol. Biol. 2012, 3(2), 137-151.
79. Le Sage, V.; Mouland, A. J.; Viral subversion of the nuclear pore complex. Viruses, 2013, 5(8), 2019-2042.
80. Hsu, J. T. A.; Yeh, J. Y.; Lin, T. J.; Li, M. L.; Wu, M. S.; Hsieh, C. F.; Chou, Y. C.; Tang, W. F.; Lau, K. S.; Hung, H. C.; Fang, M.Y. Identification of BPR3P0128 as an inhibitor of cap-snatching activities of influenza virus. Antimicrob. Agents Chemother. 2012, 56(2), 647-657.
81. Hsieh, C. F.; Chen, Y. L.; Lin, C. F.; Ho, J. Y.; Huang, C. H.; Chiu, C. H.; Hsieh, P. W.; Horng, J. T. An extract from Taxodium distichum targets hemagglutinin-and neuraminidase-related activities of influenza virus in vitro. Sci. Rep. 2016, 6, p.36015.
82. Hsieh, C. F.; Lo, C. W.; Liu, C. H.; Lin, S.; Yen, H. R.; Lin, T. Y.; Horng, J. T. Mechanism by which ma-xing-shi-gan-tang inhibits the entry of influenza virus. J. Ethnopharmacol. 2012, 143, 57-67.
83. Hsieh, C. F.; Yen, H. R.; Liu, C. H.; Lin, S. Horng, J. T. Ching-fang-pai-tu-san inhibits the release of influenza virus. J. Ethnopharmacol. 2012, 144, 533-544.
84. Takayama, K.; Mori, K.; Tanaka, A.; Nomura, E.; Sohma, Y.; Mori, M.; Taguchi, A.; Taniguchi, A.; Sakane, T.; Yamamoto, A.; Minamino, N. Discovery of a human neuromedin U receptor 1-selective hexapeptide agonist with enhanced serum stability. J. Med. Chem., 2017, 60 (12), 5228–5234.
85. Gandini, A.; Bartolini, M.; Tedesco, D.; Martinez-Gonzalez, L.; Roca, C.; Campillo, N. E.; Zaldivar-Diez, J.; Perez, C.; Zuccheri, G.; Miti, A.; Feoli, A.; Castellano, S.; Petralla, S.; Monti, B.; Rossi, M.; Moda, F.; Legname, G.; Martinez, A.; Bolognesi, M. L. Tau-Centric multitarget approach for Alzheimer’s disease: development of first-in-class dual glycogen synthase kinase 3β and tau-aggregation inhibitors. J. Med. Chem. 2018, 61(17), 7640-7656.
86. Han, X.; Sun, N.; Wu, H.; Guo, D.; Tien, P.; Dong, C.; Wu, S.; Zhou, H.B. Identification and structure–activity relationships of diarylhydrazides as novel potent and selective human enterovirus inhibitors. J. Med. Chem. 2016, 59(5), 2139-2150.
87. Sławiński, J.; Szafrański, K.; Pogorzelska, A.; Żołnowska, B.; Kawiak, A.; Macur, K.; Belka, M.; Bączek, T. Novel 2-benzylthio-5-(1, 3, 4-oxadiazol-2-yl) benzenesulfonamides with anticancer activity: Synthesis, QSAR study, and metabolic stability. Eur. J. Med. Chem. 2017. 132, 236-248.
88. Liu, Y.; Sheng, J.; Fokine, A.; Meng, G.; Shin, W. H.; Long, F.; Kuhn, R. J.; Kihara, D.; Rossmann, M. G. Structure and inhibition of EV-D68, a virus that causes respiratory illness in children. Science, 2015, 347(6217), 71-74.
89. Sethy, B.; Hsieh, C. F.; Yeh, C.; Horng, J. T.; Hsieh, P. W. Design, synthesis & structure–activity relationships of a new class of antihuman enterovirus D68 & A71 agents. Future Med. Chem. 2018, 10(11), 1333-1347.
90. Sethy, B.; Hsieh, C. F.; Lin, T. J.; Hu, P. Y.; Chen, Y. L.; Lin, C. Y.; Tseng, S. N.; Horng, J. T.; Hsieh, P. W. Design, synthesis, and biological evaluation of itaconic acid derivatives as potential anti-influenza agents. J. Med. Chem. 2019, 62(5), 2390-2403.
91. Zhao, X.; Li, R.; Zhou, Y.; Xiao, M.; Ma, C.; Yang, Z.; Zeng, S.; Du, Q.; Yang, C.; Jiang, H.; Hu, Y.; Wang, K.; Mok, C. K. P.; Sun, P.; Dong, J.; Cui, W.; Wang, J.; Tu, Y.; Yang, Z.; Hu, W. Discovery of highly potent pinanamine-based inhibitors against amantadine-and oseltamivir-resistant influenza A viruses. J. Med. Chem. 2018, 61, 5187-5198.
92. Watanabe, K.; Takizawa, N.; Katoh, M.; Hoshida, K.; Kobayashi, N.; Nagata, K. Inhibition of nuclear export of ribonucleoprotein complexes of influenza virus by leptomycin B. Virus Res. 2001, 77, 31-42.