Novel agonists and potent antagonists at P2Y_1tn1_1tn1 purinergic receptors: synthesis and biological testing [Elektronische Ressource] = Neue Agonisten und potente Antagonisten an P2Y_1tn1_1tn1-Purinergic-Rezeptoren: Synthese und biologische Untersuchung / vorgelegt von Sophi Damayanti

De
Novel Agonists and Potent Antagonists at P2Y Purinergic Receptors: 11Synthesis and Biological Testing Neue Agonisten und potente Antagonisten an P2Y Purinergen Rezeptoren: 11 Synthese und Biologische Untersuchung Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf vorgelegt von Sophi Damayanti aus Bandung, Indonesien Düsseldorf, 2008 Aus dem Institut für Pharmazeutische und Medizinische Chemie der Heinrich-Heine-Universität-Düsseldorf „Gedruckt mit Unterstützung der Islamic Development Bank (IDB)“ Gedruckt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf Referent: Prof. Dr. Matthias U. Kassack Koreferent: Prof. Dr. Thomas Kurz Tag der mündlichen Prüfung: 26.11.2008 ii Erklärung Ehrenwörtlich erkläre ich hiermit, dass ich die vorgelegte Dissertation mit dem Titel: “Novel agonists and potent antagonists at P2Y purinergic receptors: 11Synthesis and biological testing” selbst angefertigt und ohne fremde Hilfe verfasst habe.
Publié le : mardi 1 janvier 2008
Lecture(s) : 24
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Source : DOCSERV.UNI-DUESSELDORF.DE/SERVLETS/DERIVATESERVLET/DERIVATE-10229/DISSERTATION_SOPHI%20DAMAYANTI%20PDFA1B.PDF
Nombre de pages : 292
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Novel Agonists and Potent Antagonists
at P2Y Purinergic Receptors: 11
Synthesis and Biological Testing


Neue Agonisten und potente Antagonisten
an P2Y Purinergen Rezeptoren: 11
Synthese und Biologische Untersuchung






Inaugural-Dissertation
zur
Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakultät
der Heinrich-Heine-Universität Düsseldorf










vorgelegt von
Sophi Damayanti
aus Bandung, Indonesien




Düsseldorf, 2008



Aus dem Institut für Pharmazeutische und Medizinische Chemie
der Heinrich-Heine-Universität-Düsseldorf

„Gedruckt mit Unterstützung der Islamic Development Bank (IDB)“
































Gedruckt mit der Genehmigung
der Mathematisch-Naturwissenschaftlichen Fakultät
der Heinrich-Heine-Universität Düsseldorf

Referent: Prof. Dr. Matthias U. Kassack
Koreferent: Prof. Dr. Thomas Kurz

Tag der mündlichen Prüfung: 26.11.2008





ii
Erklärung

Ehrenwörtlich erkläre ich hiermit, dass ich die vorgelegte Dissertation mit dem
Titel: “Novel agonists and potent antagonists at P2Y purinergic receptors: 11
Synthesis and biological testing” selbst angefertigt und ohne fremde Hilfe
verfasst habe. Quellen und Hilfsmittel sind vollständig angegeben. Weiterhin
erkläre ich, dass ich bisher keine erfolglosen Promotionsversuche
unternommen habe.


Düsseldorf, 27.10.2008
Sophi Damayanti
































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For my husband and my parents

I am truly blessed

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LIST OF CONTENTS

1. INTRODUCTION .............................................................. 1
1.1. Purinergic receptors................................................................................ 1
1.1.1. General aspects ........................................................................... 1
1.1.2. P2X receptors............................................................................... 2
1.1.2.1. Role and location ............................................................ 2
1.1.2.2. Ligands at P2X receptors................................................ 4
1.1.3. P2Y receptors 6
1.1.3.1. G protein-coupled receptors............................................ 6
1.1.3.2. Location and role of P2Y receptors................................. 8
1.1.3.3. Ligands at P2Y receptors.............................................. 10
1.2. P2Y receptors..................................................................................... 15 11
1.2.1. General aspects ......................................................................... 15
1.2.2. Role of P2Y receptors.............................................................. 15 11
1.2.3. Ligands at P2Y receptors......................................................... 16 11
2. AIM AND SCOPE OF THE STUDY................................ 19
2.1. Synthesis of novel ligands..................................................................... 19
2.1.1. Synthesis of urea derivatives containing trisodium 7-
naphthalene-1,3,5-trisulfonate substituent and 4-fluoro -3,1-
phenylene-linker......................................................................... 19
2.1.2. Synthesis of urea derivatives containing trisodium 3-(2,4-
disulfonatophenylcarbamoyl)benzoate substituent..................... 21
2.2. Biological evaluation ............................................................................. 21
3. CHEMISTRY................................................................... 22
3.1. Synthesis of urea derivatives containing trisodium 7-naphthalene-
1,3,5-trisulfonate substituent ................................................................. 22
3.1.1. Synthesis of “small urea” derivatives.......................................... 24
3.1.1.1. Trisodium 7-(4-fluoro-3-nitrobenzamido)-naphthalene-
1,3,5-trisulfonate ........................................................... 24
3.1.1.2. Trisodium 7-(3-amino-4-fluorobenzamido)-
naphthalene-1,3,5-trisulfonate ...................................... 33
3.1.1.3. Hexasodium 7,7’- carbonylbis[azanediyl(4-fluoro-3,1-
phenylene) carbonylazanediyl] bis(naphthalene-1,3,5-
trisulfonate) ................................................................... 40
3.1.2. Synthesis of “large urea” derivatives .......................................... 47
3.1.2.1. Trisodium 7-[4-(3-nitrobenzamido)-benzamido]-
naphthalene-1,3,5-trisulfonate ...................................... 47
3.1.2.2. Trisodium 7-[4-(3-aminobenzamido)-benzamido]-isulfonate 50
3.1.2.3. Hexasodium 7,7 - carbonylbis[azanediyl-3,1-
phenylene carbonylazanediyl(4,1-
phenylene)carbonylazl] bis(naphthalene-1,3,5-
trisulfonate) ................................................................... 53
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3.2. Synthesis of urea derivatives containing 4-fluoro-3,1- phenylene-
linker at benzene or naphthalene sulfonates .........................................57
3.2.1. Disodium 2-(4-fluoro-3-nitrobenzamido)benzene-1,4-
disulfonate ..................................................................................58
3.2.2. Disodium 2-(3-amino-4-fluorobenzamido)benzene-1,4-
disulfonate64
3.2.3. Tetrasodium 2,2'- carbonylbis[azanediyl(4-fluoro-3,1-
phenylene)carbonylazanediyl] bis(benzene-1,4-disulfonate) .....69
3.3. Synthesis of urea derivatives containing trisodium 3-(2,4-
disulfonatophenylcarbamoyl)benzoate substituent................................75
3.3.1. Trisodium 3-(2,4-disulfonatophenylcarbamoyl)-5-(4-
nitrobenzamido)benzoate ...........................................................77
3.3.2. l)-5-(4-
aminobenzamido)benzoate.........................................................81
3.3.3. Hexasodium 5,5 -[carbonylbis(azanedyl-4,1-
phenylenecarbonylazanediyl]bis[3-(2,4-
disulfonatophenylcarbamoyl)benzoat] ........................................84
4. PHARMACOLOGY89
4.1. Evaluation of the test system.................................................................89
4.2. Agonist screening of compounds at P2Y receptors ............................91 11
4.2.1. Primary agonist screening of compounds at P2Y receptors.....91 11
4.2.2. Efficacy and potency testing .......................................................94
4.2.3. Concentration-response curves of compounds 8c and 9c ..........96
4.2.4. Schild analysis of compound 9c..................................................99
4.3. Antagonist screening at P2Y receptors .............................................101 11
4.4. Urea derivatives containing trisodium 7-naphthalene-1,3,5-trisulfonate
substituent ...........................................................................................107
4.4.1. Apparent pK value of urea 1c -14c...........................................107 i
4.4.2. Schild analysis of compound 5c................................................117
4.5. Urea derivatives containing 4-fluoro-3,1-phenylene-linker...................118
4.6. Urea derivatives containing trisodium 3(2,4-
disulfonatophenylcarbamoyl)benzoate substituent..............................123
4.7. Selectivity of the test compound..........................................................125
4.7.1. Selectivity test at P2Y receptors..............................................125 1
4.7.2. 127 2
4.7.3. receptors130 4
5. CONCLUSION ..............................................................133
6. ABSTRACT...................................................................137
7. ZUSAMMENFASSUNG ................................................138
8. MATERIALS AND METHODS......................................140
8.1. Chemistry ............................................................................................140
8.1.1. Instruments and analytical methods .........................................140
8.1.1.1. pH stat.........................................................................140
8.1.1.2. Thin layer chromatography..........................................140
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8.1.1.3. High performance liquid chromatography ................... 141
8.1.1.4. UV-visible spectrophotometry ..................................... 142
8.1.1.5. Titration method: NaCl determination ......................... 142
8.1.1.6. Elemental analysis ...................................................... 142
8.1.1.7. Infrared spectroscopy ................................................. 143
8.1.1.8. Nuclear magnetic resonance spectroscopy ................ 143
8.1.1.9. Mass spectrometry 143
8.1.2. Chemical .................................................................................. 144
8.1.3. General reaction procedures (GRP)......................................... 145
8.1.3.1. GRP 1: Preparation of acylchloride............................. 145
8.1.3.2. GRP 2: Synthesis of nitro derivative ........................... 145
8.1.3.3. GRP 3: Synthesis of amine derivative......................... 145
8.1.3.4. GRP 4: Synthesis of urea compound.......................... 146
8.2. Biological testing ................................................................................. 147
8.2.1. Instruments and materials ........................................................ 147
8.2.2. Chemicals................................................................................. 147
8.2.3. Buffers and solutions................................................................ 148
8.2.4. Cell culture method .................................................................. 150
8.2.4.1. General aspects.......................................................... 150
8.2.4.2. Growth medium........................................................... 150
8.2.4.3. Detaching process ...................................................... 151
8.2.4.4. Cryoconservation ........................................................ 151
8.2.4.5. Passaging cells 151
8.2.5. Biological testing technique 152
8.2.5.1. Preparation of the dilution series................................. 152
8.2.5.2. Assay preparation ....................................................... 152
8.2.5.3. Screening of compounds ............................................ 154
8.2.5.4. Assay condition........................................................... 155
8.2.5.5. EC and IC .............................................................. 155 50 50
8.2.5.6. Data analysis 156
8.2.5.7. Schild-analyse............................................................. 157
9. MONOGRAPHS 158
10. REFERENCES 253
11. ABBREVIATIONS ........................................................ 261
12. APPENDIX.................................................................... 263








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