Synthesis of Novel Chloroquine Derivatives and

Insights into the Interactions of a Resistance Protein.

By

Benita Mujinga Kapuku

A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of

Doctor of Philosophy

Department of Chemistry

McGill University, Montreal

November 2020

© Benita M. Kapuku 2020

To my mother, Espérance Kavira Kakule Kapuku, and father, Jean-

Pierre Kapuku, for showing me unconditional love.

II
Abstract

The human toll caused by malaria remains devastating. In 2018 alone, there were 228 million

cases, which resulted in 405,000 deaths. Malaria is caused by the Plasmodium parasite transmitted

by the Anopheles mosquito. There are five species: P. malariae, P.falciparum, P. vivax, P. ovale

and P. knowlesi, in which P. falciparum is the most common and most deadly. An antimalarial

drug called chloroquine (CQ) was first introduced in the 1940s and is considered the gold-standard

antimalarial drug due to its high efficacy, low-cost, and low-toxicity. It is listed under the World

Health Organization (WHO) essential medicines and showed great promise in their endeavor to

eradicate malaria.

Unfortunately, after two decades of use resistance began to arise, which led to its gradual

withdrawal for P. falciparum cases. The WHO has reported an increase in the number of malaria

cases over the last six years (214, 216, 228 million cases in 2014, 2016 and 2018, respectively),

this is alarming as it greatly hampers the effects of eradication. Therefore, it remains crucial to

invest in the research and development of new antimalarial drug candidates. Recent studies have

reported the re-sensitization of chloroquine in countries where it was once completely banned,

such as Malawi, Zambia and certain regions of The Ivory Coast. This indicates that resistance is

reversible because it requires a fitness deficit. When drug pressure is removed, the frequency of

resistance alleles is reduced but not eliminated. Therefore, the reintroduction of drug pressure will

cause a rapid reemergence of resistance. As CQ was the ideal drug, many derivatives with varying

sidechains and aryl substituents such as amodiaquine and piperaquine have emerged as

commercial drugs. Aminoquinolines (AQs) continue to be prepared and tested, and two AQs were

III
recently undergoing phase II clinical trials, demonstrating the robustness of the AQ core and their

future potential.

My research centres around the repurposing of chloroquine via modification at the 3-position of

the quinoline ring. In chapter 2 I describe the synthesis and optimization of 3-aminochloroquine

(3-NH2CQ). The synthesis of this derivative allows for expansion at the 3-position for the synthesis

of a diverse library of antimalarial candidates.

In chapter 3, I describe the synthesis of novel substituted chloroquine analogs. The structure-

activity relationship (SAR) was determined as I synthesized a library of compounds with varying

steric and electronic effects. It was determined that electron-withdrawing substituents at the para-

position gave the best antimalarial activity. Although the synthesized compounds were not as

effective as CQ, they may be candidates for combination therapy with chloroquine.

In chapter 4, I describe the synthesis of a CQ photoaffinity label with minor modifications. An aryl

azide is installed at the 3rd-position on the quinoline ring, making it the first AQ photoaffinity label

in the literature with the least number of modifications whilst retaining all features of the parent

compound, to our knowledge. Labelling studies were then carried out to determine the lability of

the photoaffinity label.

Lastly, the protein responsible for CQ resistance, Plasmodium falciparum chloroquine resistance

transporter (PfCRT), has been isolated for the first time. In chapter 5, I describe binding studies

carried out with PfCRT. In collaboration with Prof. Fidock's group at Columbia University,

IV
fluorescence and UV-Vis binding studies were carried out to determine binding affinities of heme

and CQ with PfCRT.

V
Résumé

Le bilan humain causé par le paludisme reste dévastateur. Rien qu'en 2018, il y a eu 228 millions

de cas, entraînant 405000 décès. Le paludisme est causé par le parasite Plasmodium transmis par

le moustique Anopheles. Il existe 5 espèces: P. malariae, P. falciparum, P. vivax, P. ovale et P.

knowlesi, dans lesquelles P. falciparum est le plus commun et le plus mortel. Un médicament

antipaludique appelé chloroquine (CQ) a été introduit pour la première fois dans les années 1940

et est considéré comme le médicament antipaludique de référence en raison de sa haute efficacité,

son faible coût et sa faible toxicité. Il est répertorié dans la liste des médicaments essentiels de

l'Organisation Mondiale de la Santé (OMS) et s'est montré très prometteur dans leurs efforts pour

éradiquer le paludisme.

Malheureusement, après deux décennies d'utilisation, une résistance a commencé à apparaître et

cela a conduit à son retrait progressif pour les cas de P. falciparum. L'OMS a signalé une

augmentation du nombre de cas de paludisme au cours des 6 dernières années (214, 216, 228

millions de cas en 2014, 2016 et 2018 respectivement), ce qui est alarmant car cela entrave

considérablement les effets de l'éradication. Par conséquent, il reste crucial d'investir dans la

recherche et le développement de nouveaux médicaments antipaludiques. Des études récentes ont

fait état d’un regain de l’efficacité de la chloroquine dans des pays où elle était autrefois

complètement interdite, comme le Malawi, la Zambie et certaines régions de Côte d'Ivoire. Cela

indique que la résistance est réversible car elle nécessite un déficit de forme physique. Lorsque la

pression médicamenteuse est supprimée, la fréquence des allèles de résistance est réduite mais pas

éliminée. Par conséquent, la réintroduction de la pression médicamenteuse provoquera une

réémergence rapide de la résistance. Comme la CQ était le médicament idéal, de nombreux dérivés

VI
avec des chaînes latérales et des substituants aryle variables tels que l'amodiaquine et la

pipéraquine ont émergé en tant que médicaments commerciaux. Les aminoquinoléines (AQ)

continuent d'être préparées et testées sous forme de deux AQ actuellement en phase II d'essais

cliniques: AQ-13 et ferroquine. Démontrer la robustesse du noyau d'AQ et leur potentiel futur.

Mes recherches sont centrées sur la réutilisation de la chloroquine via une modification en position

3. Dans le chapitre 2 je détaille la synthèse et l'optimisation de la 3-aminochloroquine. La synthèse

de ce dérivé permet une dérivatisation supplémentaire en position 3 pour la synthèse d'une

bibliothèque diversifiée d’antipaludiques.

Au chapitre 3, je décris la synthèse de nouveaux analogues de chloroquine substitués. La relation

structure-activité a été déterminée lorsque j'ai synthétisé une bibliothèque de composés avec divers

effets stériques et électroniques. Il a été déterminé que les substituants attracteurs d'électrons en

position para donnaient la meilleure activité antipaludique. Bien que les composés synthétisés ne

soient pas aussi efficaces que la CQ, ils peuvent être des candidats pour une thérapie combinée

avec la chloroquine.

Dans le chapitre 4, je décris la synthèse d'un marqueur de photoaffinité CQ avec des modifications

mineures. Un aryl azide est installé en position 3, 3-azidochloroquine, ce qui en fait le premier

marqueur de photoaffinité AQ de la littérature avec le moins de modifications tout en conservant

à notre connaissance toutes les caractéristiques du composé parent. Des études de marquage ont

ensuite été menées pour déterminer la labilité du marqueur de photoaffinité.

VII
Enfin, la protéine responsable de la résistance à la CQ Plasmodium falciparum chloroquine

résistance transporter (PfCRT) a été isolée pour la première fois. Dans le chapitre 5, je décris les

études de liaison réalisées avec PfCRT. En collaboration avec le groupe Prof. Fidock de

l'Université de Columbia, des études de fluorescence et de liaison UV-Vis ont été réalisées pour

déterminer les affinités de liaison de l'hème et de la CQ avec PfCRT.

VIII
Acknowledgement

I am very thankful to my supervisor, Prof. Scott Bohle, who has taught me so much about the

beauty of chemistry through his love for science. Thank you for challenging me, for all the

imparted wisdom and interesting facts throughout the years.

I want to thank my research committee members Dr. Elias Georges, Prof. Karine Auclair, Prof.

Hanadi Sleiman and Prof. Gonzalo Cosa, for their interest in my research and input over the years.

I would also like to thank Dr. Robin Stein and Dr. Tara Sprules for their help with NMR

spectroscopy and Dr. Nadim K. Saadeh and Dr. Alex Wahba for carrying out High-Resolution

Mass Spectroscopy.

I thank all the group members along the way: Cassidy VanderSchee, Dr. Danae Guerra, Dr. David

Kuter, Harrison Cassidy, Dr. Ivor Wharf, Dr. Kristopher Rosadiuk, Dr. Munendra Yadav and

Quentin Gaydon for the interesting conversations, solicited distractions, and valuable input. I’d

also like to thank my friends Allie Domínguez, Jane Eiyegbenin, Ifenna Mbaezue, and Martin

Sichinga for their support and laughs along the way. Working alongside such a talented group of

individuals further enriched my experience at McGill.

The decision to pursue a doctorate and to see it through is not an easy one. It requires self-

motivation, determination, mental strength, and plenty of support. The love and joy received from

my support system helped make my journey more worthwhile.

IX
I thank the Kapuku clan for being the best family members I could have ever wished for. Thank

you for a lifetime of unforgettable moments and for helping shape the person that I am today. My

success is our success, and this thesis is for all the Kapuku's.

I thank my sisters Nadine and Gloria for being there to listen. Thank you for the words of

encouragement and for always keeping me smiling. Thank you for being my ride-or-die sisters and

for being such great role models.

I thank my brothers Jeremie, Sam and Medi, for being my hype men. Thank you for the energy

and joy that you bring into my life. I can always rely on you guys for a fun time and the most

interesting conversations.

I thank my partner Hubert for being my cheerleader. Thank you for listening to all the rants and

sitting through all the practice presentations. Thank you for the motivational speeches and your

infectious smile. Thank you for keeping me grounded and for being the sugar in my ice-cream.

Finally, none of this would have been possible without my amazing parents' undying love and

support. I thank my mother, Espérance, and father, Jean-Pierre, for teaching me the values of hard

work and perseverance. Thank you for the endless prayers, words of wisdom and for being my

rocks. Thank you for all the sacrifices made so that I could live my best life. Thank you for

believing in me even when I did not believe in myself. I am so grateful to have been loved and to

be loved by you.

X
Contribution of Authors

The author carried out all experimental design, compound synthesis, structural analysis, binding

studies and all the work detailed in the thesis under the supervision of Prof. Scott Bohle.

Chapter 3: Antiplasmodial assays were carried out by our collaborators Dr. Fadi Baakdah, under

the supervision of Dr. Elias Georges at McGill University Institute of Parasitology.

Chapter 5: PfCRT isoform 7G8, GtrB and empty nanodisc were gifted from Jonathan Kim, under

the supervision of Prof. David Fidock at Columbia University.

XI
Table of Contents
Abstract ....................................................................................................................................... III

Résumé......................................................................................................................................... VI

Acknowledgement....................................................................................................................... IX

Contribution of Authors ............................................................................................................ XI

List of Figures .......................................................................................................................... XVI

List of Schemes ..................................................................................................................... XVIII

List of Tables ............................................................................................................................ XIX

List of Abbreviations ................................................................................................................... 20

1.1 Introduction ........................................................................................................................... 25

1.2 Malaria ................................................................................................................................... 25

1.3 Parasite life cycle ................................................................................................................... 25

1.4 Hemozoin formation .............................................................................................................. 27

1.4.1 Digestive Vacuole ........................................................................................................ 27
1.4.2 Hemoglobin Degradation ............................................................................................. 28
1.4.3 Heme Detoxification .................................................................................................... 30

1.5 Antimalarial Drugs ................................................................................................................ 31

1.5.1 8-Aminoquinolines .......................................................................................................... 32
1.5.1.1 Primaquine ................................................................................................................. 32
1.5.1.2 Tafenoquine ............................................................................................................... 33
1.5.1.3 Bulaquine ................................................................................................................... 34
1.5.2 Acridines .......................................................................................................................... 34
1.5.2.1 Mepacrine .................................................................................................................. 35
1.5.2.2 Pyronaridine .............................................................................................................. 35
1.5.3 4-Aminoquinolines .......................................................................................................... 36
1.5.3.1 Chloroquine ............................................................................................................... 37
1.5.3.2 Hydroxychloroquine .................................................................................................. 43
1.5.3.3 Sontoquine ................................................................................................................. 43
1.5.3.4 Amodiaquine ............................................................................................................. 44
1.5.3.5 Piperaquine ................................................................................................................ 47
1.5.4 AQs in clinical trials ........................................................................................................ 48

XII
 1.5.4.1 Ferroquine.................................................................................................................. 48
1.5.4.2 AQ-13 ........................................................................................................................ 50
1.5.5 Aryl aminoalcohols .......................................................................................................... 51
1.5.5.1 Quinine ...................................................................................................................... 51
1.5.5.2 Mefloquine ................................................................................................................ 52
1.5.6 Non-quinoline antimalarials ........................................................................................... 53

1.6 Antimalarial Resistance ........................................................................................................ 54

1.6.1 Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT) ...................... 54
1.6.2 Plasmodium falciparum multidrug resistance protein 1 (pfmdr1) ................................ 56

1.7 Vaccines .................................................................................................................................. 58

1.7.1 RTS,S................................................................................................................................ 59

1.8 References .............................................................................................................................. 61

2.1 Preamble ................................................................................................................................. 73

2.2 Introduction ........................................................................................................................... 73

2.3 Results and Discussion .......................................................................................................... 76

2.3.1 Optimization of 3-BrCQ synthesis .................................................................................. 76
2.3.2 Optimization of 3-NH2CQ synthesis ............................................................................... 79
2.3.2.1 Optimization using ammonium hydroxide as the nitrogen source ............................ 81
2.3.2.2 Optimization using azidotrimethyl silane as the nitrogen source .............................. 82
2.3.2.3 Optimization using sodium azide as the nitrogen source .......................................... 84
2.3.3 Proposed Ullmann-type coupling mechanisms of product formation ........................... 88

2.4 Conclusion .............................................................................................................................. 91

2.5 Experimental .......................................................................................................................... 93

2.5.1 General Information........................................................................................................ 93
2.5.2 Experimental procedures ................................................................................................ 94

2.6 References .............................................................................................................................. 97

3.1 Preamble ............................................................................................................................... 103

3.2 Introduction ......................................................................................................................... 103

3.3 Results and Discussion ........................................................................................................ 106

3.3.1 3-CQ derivatives ............................................................................................................. 106
3.3.2 3-chlorobenzamidoCQ not a substrate for PfCRT ....................................................... 114

XIII
 3.3.3 Isomerization ................................................................................................................. 115
3.3.4 Fluoride coupling in 1H NMR ...................................................................................... 116
3.3.5 Synthesis of Sulfonamides and Schiff-base derivatives ............................................... 117
3.3.6 pKa studies ...................................................................................................................... 119

3.4 Conclusion ............................................................................................................................ 120

3.5 Experimental ........................................................................................................................ 121

3.5.1 General information ...................................................................................................... 121
3.5.2 pKa Determination ........................................................................................................ 122
3.5.3 Parasite cytotoxicity assays and IC50 estimation .......................................................... 123
3.5.4 Synthesis of 3-substituted CQ Derivatives .................................................................... 123

3.6 References ............................................................................................................................ 134

4.1 Preamble ............................................................................................................................... 138

4.2 Introduction ......................................................................................................................... 139

4.3 Results and Discussion ........................................................................................................ 142

4.3.1 Photoaffinity Properties ................................................................................................ 154
4.3.2 Nitrene insertion ............................................................................................................ 159

4.4 Conclusion ............................................................................................................................ 165

4.5 Experimental ........................................................................................................................ 166

4.5.1 General information ...................................................................................................... 166
4.5.2 Experimental procedure ................................................................................................ 167
4.5.3 X-ray crystallography .................................................................................................... 175
4.5.4 UV-Vis Studies ............................................................................................................... 176
4.5.5 NMR experiments .......................................................................................................... 179

4.6 References ............................................................................................................................ 180

5.1 Preamble ............................................................................................................................... 188

5.2 Introduction ......................................................................................................................... 188

5.3 Results and Discussion ........................................................................................................ 191

5.3.1 Fluorescence Studies ..................................................................................................... 191
5.3.1.1 Fluorescence studies with CQ ................................................................................. 191
5.3.1.2 Fluorescence studies with heme .............................................................................. 200

XIV
 5.3.2 UV-Vis Studies ............................................................................................................... 206
5.3.2.1 UV-Vis Studies with heme ...................................................................................... 206

5.4 Conclusion ............................................................................................................................ 208

5.5 Experimental ........................................................................................................................ 210

5.5.1 General information ...................................................................................................... 210
5.5.2 Fluorescence Spectroscopy Procedure ......................................................................... 210
5.5.3 Ultraviolet-Visible Spectroscopy Procedure ................................................................. 212

5.6 References ............................................................................................................................ 213

6.1 Conclusion ............................................................................................................................ 217

6.2 Future Work ........................................................................................................................ 219

Appendix .................................................................................................................................... 221

Chapter 3 ................................................................................................................................. 222
Chapter 5 ................................................................................................................................. 225
NMR Spectra........................................................................................................................... 228

XV
List of Figures

Figure 1.1. Plasmodium life cycle. Illustration adapted from Wirth.7 ................................... 27
Figure 1.2. Catabolism of Hb and heme detoxification mechanism. Illustration adapted
from Rosenthal and Meshnick.14 ........................................................................................ 29
Figure 1.3. Structure of Fe(III)PPIX. Figure adapted from Egan.18 ...................................... 31
Figure 1.4. 8-Aminoquinolines. .................................................................................................. 34
Figure 1.5. Acridines. .................................................................................................................. 36
Figure 1.6. 4-aminoquinolines. ................................................................................................... 36
Figure 1.7. CQ mechanism of action. ......................................................................................... 38
Figure 1.8. Ga(III)PPIX and CQ complex formed in the presence of methanol.42 ............... 39
Figure 1.9. CQ Structure-Activity Relationship. ...................................................................... 41
Figure 1.10. ADQ analogs. .......................................................................................................... 47
Figure 1.11. Antimalarial drug candidates in clinical trials. ................................................... 48
Figure 1.12. a) Hydrogen-bonding in FQ and b) the structure of methyl FQ. ...................... 49
Figure 1.13. Aryl aminoalcohols................................................................................................. 51
Figure 1.14. Non-quinoline antimalarial drugs. ....................................................................... 53
Figure 1.15. Structure of PfCRT 7G8 isoform. A) Topology showing inverted antiparallel
TM helices. B) Surface representation of the central cavity’s electrostatic potential
with red and blue, indicating negative and positive residues, respectively. Figures are
taken from Kim et al.83 ........................................................................................................ 55
Figure 1.16. K76T mutation removes a positive charge, which allows CQ2+ efflux from its
active site. ............................................................................................................................. 56
Figure 1.17 Structure of pfmdr1 with highlighted mutations: N86Y, Y184F, S1034C,
N1042D, D1246Y and two nucleotide-binding domains. Illustration adapted from
Valderramos and Fidock.91 ................................................................................................. 58
Figure 2.1. Simple AQs that do not inhibit hemozoin formation. ........................................... 74
Figure 2.2. AQs that do not inhibit hemozoin formation. ........................................................ 75
Figure 2.3. Proposed pathways for Cu(I)/ Cu(III) Ullmann-type amination catalytic cycle.
............................................................................................................................................... 89
Figure 2.4. Proposed Cu(I)/ Cu(II) SRN1 mechanism. .............................................................. 90
Figure 2.5. Proposed p-complexation Ullmann-type mechanism............................................ 91
Figure 3.1. Craig Plot groups substituents with similar electronic and hydrophobic effects.
Highlighted are synthesized compounds containing substituents at various locations on
the phenyl ring. .................................................................................................................. 107
Figure 3.2. Synthesized compounds. Colours: grey: the reference compound, blue: electron-
withdrawing and hydrophilic, orange: electron-withdrawing and hydrophobic, purple:
electron-donating and hydrophilic, green: electron-donating and hydrophobic. ....... 108
Figure 3.3. Possible benzamidoCQ conformations. ................................................................ 110
Figure 3.4. Possible fluorobenzamidoCQ conformations. ..................................................... 112
Figure 3.5. Possible chlorobenzamidoCQ conformations. ..................................................... 113
Figure 3.6. Trans and Cis benzamidoCQ isomers. ................................................................. 115
Figure 3.7. Fluorine and hydrogen coupling. .......................................................................... 116
Figure 3.8. Structure of sulfonamide and Schiff-base derivatives ........................................ 117
Figure 3.9. Possible sulfonamide conformations. ................................................................... 118

XVI
Figure 4.1. Known photoaffinity labels. .................................................................................. 139
Figure 4.2. Structure of novel 3-Azidomethylchloroquine in which all important CQ
features are present. .......................................................................................................... 141
Figure 4.3. Overlaying spectra of 3-NH2MeCQ, 3-N3MeCQ and CQ.2H3PO4 for
comparison. ........................................................................................................................ 155
Figure 4.4. Photolysis of 3-N3MeCQ at 254 nm excitation. ................................................... 156
Figure 4.5. Photolysis of 42.97 µM N3 in H2O and 35.57 µM N3 in MeOH at 365 nm. ....... 157
Figure 4.6. Photolysis of 3-N3MeCQ at 0 and 16 minutes in MeOD-d4. ............................... 157
Figure 4.7. Photolysis of 3-N3MeCQ in MeOD-d4 over 16 minutes. ..................................... 158
Figure 4.8. Possible irradiated 3-N3MeCQ products formed. ............................................... 159
Figure 4.9. Photolysis of 21.49 µM 3-N3MeCQ with 22.32 µM GlyGlyGly and 4.29 µM 3-
N3MeCQ with 40.18 µM GlyGlyGly at 365 nm excitation over five minutes. ............. 160
Figure 4.10. Photolysis of 19.56 µM 3-N3MeCQ with 18.77 µM maleimide, 7.11 µM 3-
N3MeCQ with 33.37 µM maleimide and 3.56 µM 3-N3MeCQ and 37.54 µM maleimide
at 365 nm excitation over 5 minutes. ............................................................................... 161
Figure 4.11. Photolysis of 26.7mM 3-N3MeCQ and 26.6mM maleimide at 365 nm in d4-
MeOD.................................................................................................................................. 162
Figure 4.12. Comparison between activated and unactivated N-phenylmaleimide. ........... 163
Figure 4.13. a) Desired product v possible observed photolysis product, b) NMR of
photolysis product formation. .......................................................................................... 164
Figure 5.1. Structure of PfCRT 7G8 isoform. A) Topology showing inverted antiparallel
TM helices. B) Surface representation of the electrostatic potential of the central
cavity with red and blue indicating negative and positive residues, respectively.
Figures adapted from Kim et al.3 ..................................................................................... 189
Figure 5.2. Effect of 4 equivalent of CQ on the emission spectrum of PfCRT at pH 7,
PfCRT at pH 8, GtrB and nanodisc. ................................................................................ 192
Figure 5.3. Stern-Volmer plots for PfCRT, GtrB and nanodisc quenched with up to 4
equivalents of CQ (each dot represents a 0.2 molar addition). ..................................... 194
Figure 5.4. Modified Stern-Volmer plots for PfCRT, GtrB and nanodisc quenched with up
to 4 equivalents of CQ (each dot represents a 0.2 molar addition). .............................. 195
Figure 5.5. Concentration-dependent response of PfCRT to CQ. ........................................ 197
Figure 5.6. Binding plot to determine the number of CQ binding sites and binding constant.
............................................................................................................................................. 199
Figure 5.7. Effect of heme on fluorescence spectrum of PfCRT at pH 7 and 8, GtrB and
nanodisc. ............................................................................................................................. 200
Figure 5.8. Stern-Volmer plot for PfCRT, GtrB and nanodisc quenched with up to 4
equivalents of Heme (each dot represents a 0.2 molar addition). ................................. 202
Figure 5.9. Modified Stern-Volmer plots for PfCRT, GtrB and nanodisc quenched with up
to 4 equivalents of heme (each dot represents a 0.2 molar addition). ........................... 203
Figure 5.10. Binding plot to determine the number of heme binding sites and binding
constant. .............................................................................................................................. 205
Figure 5.11. Changes to MPLME with up to 2 molar addition of heme. ............................. 207

XVII
List of Schemes

Scheme 1.1. CQ metabolism. ...................................................................................................... 42

Scheme 1.2. ADQ metabolism. ................................................................................................... 45
Scheme 2.1. Linear synthesis to 3-AminoCQ synthesis............................................................ 80
Scheme 2.2. Known examples of pyridine amination. .............................................................. 82
Scheme 2.3. Determination of 3-NH2CQ stability. ................................................................... 88
Scheme 3.1. a) Halochloroquine synthesis, b) structure of verapamil. ................................. 104
Scheme 3.2. Amination with 3-BromoCQ as a precursor. ..................................................... 108
Scheme 4.1. Proposed mechanism for arylamine formation. ................................................ 142
Scheme 4.2. Ullman amination of aryl bromide in 30% aqueous solution in the presence of
a ligand and reducing agent.............................................................................................. 143
Scheme 4.3. Azidation of arylamine leads to the formation of a triazole CQ product. ...... 144
Scheme 4.4. Methylation of 2-Amino-5-diethylaminopentane with ethyl formate. ............. 146
Scheme 4.5. A) Potential pathways for DBU reaction with an electrophile. B) Crystal
structure of obtained open DBU adduct. ......................................................................... 149
Scheme 4.6. Increasing the electrophilicity of C-4 with the installation of a triflate. ......... 150
Scheme 4.7. Linear synthesis of 3-azidomethylchloroquine. ................................................. 152
Scheme 4.8. Proposed mechanism for 3-azido-7-chloroquinolin-4-ol formation. ............... 153

XVIII
List of Tables

Table 1.1. Mode of action of non-quinoline based antimalarial drugs. .................................. 54

Table 2.1. Optimization of 3-BrCQ synthesis using microwave-assisted synthesis. .............. 77
Table 2.2. 3-NH2CQ formation with NH4OH as NH2 source. ................................................. 81
Table 2.3. 3-NH2CQ formation with TMSN3 as the nitrogen source. ..................................... 84
Table 2.4. 3-NH2CQ formation with NaN3 as the nitrogen source. ........................................ 85
Table 2.5. Reaction conditions to minimize byproduct formation. ......................................... 87
Table 3.1. Antiplasmodial activities of CQ derivatives in sensitive and resistant parasitic
strains.................................................................................................................................. 109
Table 3.2. Effect of verapamil on compound activity............................................................. 115
Table 3.3. Antiplasmodial activity and pKa of quinolinium nitrogen. ................................. 119
Table 4.1. Methylation of CQ with methyl iodide. ................................................................. 144
Table 4.2. Methylation of CQ. .................................................................................................. 145
Table 4.3. Condensation reaction between DCQ and methylated side chain under basic
conditions............................................................................................................................ 147
Table 4.4. Condensation reaction conditions for 7-chloroquinolin-4-yl
trifluoromethanesulfonate 4.13 and the methylated side chain 4.10. ........................... 150
Table 4.5. Condensation reaction between DCQ 4.11, and methylated side chain 4.10 under
acidic conditions................................................................................................................. 151
Table 5.1. Stern-Volmer quenching constants for CQ. .......................................................... 193
Table 5.2. Modified Stern-Volmer constants for CQ. ............................................................ 196
Table 5.3. Binding constant and number of binding sites for CQ with MPLME................ 198
Table 5.4. Stern-Volmer quenching constants with heme. .................................................... 201
Table 5.5. Modified Stern-Volmer constants for Heme. ........................................................ 203
Table 5.6. Binding constants and number of binding sites for heme with MPLME. .......... 204

XIX
List of Abbreviations

[Q] Quencher concentration

3-BrCQ 3-bromochloroquine
3-ClCQ 3-chlorochloroquine
3-ICQ 3-iodochoroquine
3-N3MeCQ 3-azidomethylchloroquine
3-NH2CQ 3-aminochloroquine
3-NH2MeCQ 3-aminomethylchloroquine
3-NO2CQ 3-nitro chloroquine
3-NO2DCQ 4,7-dichloro-3-nitroquinoline
3,6-BrCQ 3,6-dibromo chloroquine
Å Angstrom
AcOH Acetic acid
ACTs Artemisinin combination therapies
ADQ Amodiaquine
ADQQI Amodiaquine quinone-imine
APCI Atomospheric pressure chemical-ionization
AQ Aminoquinoline
N-[4-[I-hydroxy-2-(dibutylamino) ethyl] quinolin-8-yl]-4-azido-2-
ASA-MQ hydroxybenzamide
N-(l-(l-diethylamino-l-methylbutylamino)quinolin-6-yl)-4-azido-2-
ASA-Q hydroxybenzamide
AzBCQ Perfluorophenylazido biotinylated chloroquine
BHIA b-Hematin inhibition assays
BQ Bulaquine
cm-1 Wavenumber
COSY Correlation spectroscopy
CQ Chloroquine
CQR Chloroquine resistant
CQS Chloroquine sensitive
Cu2SO4 Copper (I) sulfate
DADQ N-desethylamodiaquine
DBU 1,8-diazabicyclo[5.4.0]undec-7-ene
DCM Dichloromethane
DCQ Dichloroquinoline
DIPEA N,N-diisopropylethylamine
DMA Dimethylacetamide
DMEDA Dimethylethylenediamine

20
DMF Dimethylformamide
DMSO Dimethyl sulfoxide
DV Digestive vacuole
equiv. Equivalent
ESI Electrospray ionization
EtOAc Ethyl acetate
EtOH Ethanol
FAQ 4-fluoroamodiaquine
Fe(III)PPIX Iron protoporphyrin IX
FQ Ferroquine
g Gram(s)
G6PD Glucose-6-phosphate dehydrogenase
Gtr glycosyltransferase
H2 O Water
Hb Hemoglobin
HCQ Hydroxychloroquine
Hex Hexanes
HMBC Heteronuclear multiple bond correlation spectroscopy
HRMS High-resolution mass spectroscopy
HSQC Heteronuclear single quantum coherence spectroscopy
Hz Hertz
HZ Hemozoin
IC50 Half maximal inhibitory concentration
J Coupling constant
K2CO3 Potassium carbonate
KSV Stern-Volmer quenching constant
L Litre
LiAlH4 Lithium aluminium hydride
M Molar
MeCN Acetonitrile
MeCQ Methyl chloroquine
MeOH Methanol
mg Milligram(s)
MgSO4 Magnesium sulfate
MHz Megahertz
mL Millilitre
mL Microliter
MP Mepacrine
MPLME Membrane proteins and lipid membrane ensemble
MQ Mefloquine

21
MW Microwave
N2 Nitrogen gas
Na2CO3 Sodium carbonate
NaHCO3 Sodium bicarbonate
NaN3 Sodium azide
NaOH Sodium hydroxide
NBD Nucleotide-binding domain
NBS N-bromosuccinimide
NBu4N3 Tetrabutylammoniumazide
NEt3 Triethylamine
NH4OH Ammonium hydroxide
nM Nanomolar
nm Nanometer
NMR Nuclear magnetic resonance
NOSEY Nuclear overhauser effect spectroscopy
PAL Photoaffinity label
PfCRT Plasmodium falciparum chloroquine resistant transporter
pfmdr1 Plasmodium falciparum multidrug resistance protein 1
PN Pyronaridine
ppm Parts per million
PPQ Piperaquine
PQ Primaquine
QD Quinidine
QN Quinine
RT Room temperature
SAR Structure-activity relationship
SET Single-electron transfer
SNAR Nucleophilic aromatic substitution
SQ Sontoquine
SRN1 Unimolecular radical nucleophilic substitution
tBuOK Potassium tert-butoxide
TCNE Tetracyanoethylene
TEMPO (2,2,6,6-Tetramethylpiperidin-1-yl)oxyl
TFQ Tafenoquine
THF Tetrahydrofuran
TLC Thin-layer chromatography
TM Transmembrane
TMSN3 Azidotrimethyl silane
Trp Tryptophan
UV-Vis Ultraviolet-visible spectroscopy

22
VP Verapamil
WHO World health organization

23
 Chapter 1

Development of Antimalarial Drugs

Chapter 1 Development of Antimalarial Drugs

1.1 Introduction

1.2 Malaria

Malaria is a devastating disease in which over 3 billion people are at risk of contracting the disease.

The most recent report by the World Health Organization (WHO) reported 228 million cases in

2018. Of those, there were 405,000 deaths. Africa bears 93% of all cases, followed by South-East

Asia and the Eastern Mediterranean. Children under the age of 5 are the most vulnerable,

accounting for 67% of deaths globally.1 Pregnant women are also at high risk. Despite the WHO’s

sustained efforts, the number of malaria cases has increased year-over-year from 2015-2019 from

212 million to 228 million cases per year.1,2

Malaria is a vector-borne disease carried by the female Anopheles spp. mosquito. It transmits

parasites of the Plasmodium genus. Five Plasmodium species are known to infect humans: P.

falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi. Plasmodium falciparum is the most

deadly, followed by P. vivax.3

1.3 Parasite life cycle

The parasitic life cycle begins during a blood meal of an infected female Anopheles mosquito,

Figure 1.1. The mosquito transmits parasites in their sporozoite stage from its saliva into the hosts’

bloodstream while feeding. The sporozoites travel to and enter the liver within 60 minutes of

entering the host to evade the hosts’ immune system. The liver-stage is asymptomatic and can last

between 5-16 days. Within the liver, sporozoites develop into a tissue schizont containing several

thousand merozoites.4 The asexual erythrocytic cycle begins when mature tissue schizonts rupture,

25
Chapter 1 Development of Antimalarial Drugs

resulting in thousands of merozoites released into the bloodstream. These merozoites then enter

red blood cells (RBCs), where they develop into blood schizonts. Once matured, the blood schizont

ruptures, releasing up to 32 merozoites that can re-enter RBCs to continue the cycle.5 This constant

invasion and rupture of RBCs cause clinical symptoms to appear, such as fever, fatigue, and chills.

These symptoms usually appear within one week after the commencement of the asexual cycle.

The cycle ends at the intervention of the immune system, administration of prophylactic

medication, or the host’s death. The differentiation of merozoites into hypnozoites occurs only in

P. vivax and P. ovale. Hypnozoites are parasites that can remain dormant in the host hepatocytes

for months or years and are the cause of recrudescence of malaria in cured patients.5

Once in the bloodstream, some merozoites within infected erythrocytes differentiate into male and

female gametocytes, which marks the beginning of the sexual cycle. These gametocytes travel to

skin capillaries where they can be taken up during another blood meal. They remain in the

bloodstream but are non-pathogenic; however, it is unclear how they evade the immune system.

The beginning of sexual reproduction occurs within the mosquito’s midgut, where gametes form

a zygote, which develops into an ookinete and eventually forms an oocyst outside the midgut.

Within the oocyst, sporozoites are formed, and once mature, the oocyst ruptures, releasing

sporozoites, which concentrate in the mosquito salivary glands, ready to restart the life cycle. The

sexual cycle within the mosquito takes 7-10 days.4,6

26
Chapter 1 Development of Antimalarial Drugs

Figure 1.1. Plasmodium life cycle. Illustration adapted from Wirth.7

1.4 Hemozoin formation

1.4.1 Digestive Vacuole

During the trophozoite stage of the asexual cycle, the parasite degrades RBCs as it requires room

to grow and to obtain nutrients via the catabolism of hemoglobin (Hb). Hb has a cytosolic

27
Chapter 1 Development of Antimalarial Drugs

concentration of 5 mM, and it accounts for 95% of parasitic cytosolic proteins.8 Hb is transported

into the acidic digestive vacuole (DV), which has a pH of 5.0 – 5.6, where it is catabolized.9–11 Hb

catabolism releases Fe2+ and globin. Globin is hydrolyzed into amino acids as it is the primary

source of amino acids for the parasite. The parasite incorporates Hb amino acids with its proteins

as a source of nutrients and for de novo synthesis of proteins required for replication. However,

the parasite does not solely rely on Hb amino acids as Hb is deficient in isoleucine, methionine,

glutamic acid, and glutamine.8 Therefore, it is a dual requirement to have host amino acids and the

ability to synthesize amino acids as needed. Studies have shown that when Hb proteolysis is

blocked, parasite development is interrupted.12

1.4.2 Hemoglobin Degradation

Hb proteolysis occurs in the DV. It is during the early trophozoite stage where hemozoin (HZ)

formation becomes microscopically visible; however, HZ is present in most of the parasite life

cycle.13 Plasmepsins and falcipains are the main proteases involved in Hb degradation. More

specifically, cysteine and aspartic proteases are the primary enzymes involved in Hb proteolysis

as they account for 20-40% and 60-80% of globin degrading activity, respectively.8

Hb is denatured before heme is released from the peptide.14 Two aspartic proteases, plasmepsin I

and II, initiate Hb hydrolysis, Figure 1.2. Plasmepsin I and II initiate Hb cleavage by cleaving the

hinges that hold the tetramer together when oxygen is bound. Once the tertiary structure falls apart,

the plasmepsins further cleave Hb.15 Plasmepsin I is primarily synthesized in the early ring stage

but also synthesized in the trophozoite stage. Plasmepsin II is synthesized primarily in the

trophozoite stage. Therefore, plasmepsin I is more involved in Hb degradation than plasmepsin II.

28
Chapter 1 Development of Antimalarial Drugs

Studies show that blocking plasmepsin I leads to parasite death.8 Plasmepsin II is predominantly

active in the hydrolysis of the disassembled Hb units.16 The cysteine protease involved in globin

hydrolysis is the 28 kDa protein falcipain, synthesized in the trophozoite stage. Plasmepsins

hydrolysis Hb into large fragments of globin. Falcipain catabolizes globin into smaller peptide

fragments. Assays in which cysteine protease inhibitors were incubated with trophozoite stage

parasite shows a considerable reduction in parasite development, highlighting the importance of

falcipains in parasite survival.14 There are two primary cysteine proteases that the parasite utilizes

falcipain-2 and falcipain-3. Falcipain-2 exerts 90% of falcipain activity and is predominant in the

early trophozoite stage. Falcipain-3 is only present in the late trophozoite stage, and both cleave

native Hb and denatured globin.17 Amino acids generated from globin hydrolysis are incorporated

into the parasite’s proteins.

Figure 1.2. Catabolism of Hb and heme detoxification mechanism. Illustration adapted from

Rosenthal and Meshnick.14

29
Chapter 1 Development of Antimalarial Drugs

1.4.3 Heme Detoxification

Once Hb has been disassembled by the aspartic and cysteine proteases, heme is released into the

cytosol of the DV. The released free heme is very reactive and toxic to the parasite. Free heme can

cause the release of reactive oxygen species, which leads to oxidative stress, membrane rupture,

and eventual death of the parasite. To circumvent this damage, the parasite transforms free heme

into HZ. Studies have shown that 95% of released heme is converted to HZ.18 Pagola et al. have

shown that HZ is an insoluble biomineral that is identical to synthetic b-hematin. X-ray structures

show that HZ is dimerized Fe(III)PPIX where a propionate group of each molecule coordinates

with the adjacent central Fe(III), and the dimers are linked via hydrogen bonding with the

uncoordinated propionate,19 Figure 1.3.

It has been reported that HZ is completely encapsulated with a neutral lipid body upon its

formation, which is comprised of mono-, di- and tri-acylglycerols. Specifically, neutral lipids

monopalmitoyl- and monostearoyl glycerol promote HZ formation.20 It is believed that these lipid

bodies originate from the inner membrane of endocytotic transport vesicles that transport Hb to

the DV.18 Histidine-rich proteins have also been associated with HZ formation.

30
Chapter 1 Development of Antimalarial Drugs

OH H O

O O N
N
N N FeIII
FeIII N N

N N
O
O
OH
O
O
Cyclic dimer
Free heme
O

N N
FeIII
H O
OH N N
X O
O
O O H N N

N N FeIII
FeIII N N

N N
O
O
OH
O
O
Hematin O
X=OH/H2O
N N
FeIII
N N
O

O H

Hemozoin
(β-Hematin or hematin anhydride)

Figure 1.3. Structure of Fe(III)PPIX. Figure adapted from Egan.18

1.5 Antimalarial Drugs

Powdered cinchona tree bark has been used to treat malaria chills for centuries in South America.

It was later discovered that the cinchona sp. contains several alkaloids, and the active ingredient

for treating malaria was quinine (QN) and quinidine (QD), Figure 1.13, first discovered in 1820

and 1833, respectively. QN was first isolated in 1820, and its first total synthesis was reported in

1944; however, its total synthesis is more laborious than its extraction. Depending on the species,

a cinchona bark contains 6 - 10 % alkaloids, of which 2-8 % is QN, and 0.2 % is QD. The tree also

contains smaller quantities of over 30 other alkaloids.21,22 Despite its effectiveness, there are many

31
Chapter 1 Development of Antimalarial Drugs

concerns about QNs toxicity profile; therefore, its use has been limited for treating severe malaria

in combination with doxycycline when other front-line medications are not available.23

The discovery of QN led to the synthesis of different classes of antimalarial drugs, mostly still in

use today. In an attempt to synthesize QN in 1856, William Henry Perkins accidentally synthesized

the first synthetic dye methylene blue. In 1891 Paul Ehrlich cured malaria patients with the dye

after noticing the dye was selectively taken up by the parasite. Methylene blue inhibits glutathione

reductase, disturbing redox homeostasis of the parasite.24 Modification of methylene blue led to

the synthesis of 3 classes of antimalarial drug families: 8-aminoquinolines (8-AQs), acridines, and

4-AQs.5

1.5.1 8-Aminoquinolines

Modification of methylene blue led to the synthesis 8-AQs, which are used as a radical cure against

malaria. The first 8-AQ, Pamaquine was synthesized in 1925 by the German pharmaceutical

company Bayer but displayed many side effects; therefore, it was not widely used.5 Modification

via the removal of the diethylamino sidechain of pamaquine for an unsubstituted amine gave

primaquine (PQ), Figure 1.4.

1.5.1.1 Primaquine

PQ, synthesized in 1946, has a much-improved safety profile compared to pamaquine and is

currently used in clinics.25 It is effective against hypnozoites in P. vivax and P. ovale and the liver

stage of P. falciparum. Therefore, it is administered as a radical cure against malaria. Until

recently, PQ was the only approved drug for the treatment of liver-stage parasites. It has a short

32
Chapter 1 Development of Antimalarial Drugs

elimination half-life of 4-6 hours and is administered over 14 days at 15 mg/day.26 PQ is

contraindicated in patients deficient in glucose-6-phosphate dehydrogenase (G6PD) as it causes

hemolysis and methemoglobinemia. This deficiency is present in 15 - 20% of the population in

malaria-endemic regions.27,28 5-hydroxyprimaquine and 6-methoxy-8-AQ, Figure 1.4, are PQ

metabolites that are directly responsible for its toxicity (quinone toxicity is discussed in section

1.5.3.4.1). PQ is not safe for pregnant women or children under the age of 4.26 Due to its weak

blood schizonticidal activity, monotherapy is highly discouraged. It is usually given as a co-

therapy with an anti-schizonticide antimalarial to prevent relapse.

1.5.1.2 Tafenoquine

Tafenoquine (TFQ), developed by GlaxoSmithKline and Medicines for Malaria Venture, is a more

lipophilic derivative of PQ that has a longer elimination half-life of 2 weeks. It was first developed

in 1978 by the Walter Reed Army Institute of Research. In 2018 TFQ was approved by the USA’s

Food and Drug Administration as a prophylactic treatment of P. vivax.29 Due to its long elimination

half-life TFQ is administered orally at 200 mg once a day for three days and then 200 mg once a

week for eight weeks.30 TFQ is effective against liver-stage, asexual stage and sexual stage parasite

in P. vivax and P. falciparum. Like PQ, TFQ is highly effective against hypnozoites. Although it

is better tolerated, TFQ cannot be given to patients with a G6PD deficiency or pregnant women.

TFQ is more potent than PQ against blood-stage parasites, but its activity is low; therefore, it is

given in combination with other blood schizonticidal agents.28,31

33
Chapter 1 Development of Antimalarial Drugs

1.5.1.3 Bulaquine

Bulaquine (BQ) is a PQ prodrug approved outside of North America. It was developed by the

Central Drug Research Institute in India for the treatment of P. vivax,32 and it is given in

combination with chloroquine (CQ). BQ prevents relapse in P. vivax cases and has

gametocytocidal activity. BQ can be used as a safe alternative to PQ. It is currently under Phase II

clinical trial with a recommended dose of 25 mg/day for five days.25

O O O

N N O N
HN HN NH2
NH2 OH

Primaquine 5-hydroxyprimaquine 6-methoxy-8-aminoquinoline

F3C O
O O
O

N O
N O
HN HN
NH2 N

Tafenoquine Bulaquine

Figure 1.4. 8-Aminoquinolines.

1.5.2 Acridines

Another class of antimalarial drugs that arose from methylene blue modifications is acridines,

Figure 1.5. Acridines were first synthesized in the 1930s, and their primary mode of action is on

blood-stage parasites.5 However, their use was short-lived because shortly after their introduction,

CQ was synthesized. As CQ was a significantly better drug, due to its low toxicity and high

34
Chapter 1 Development of Antimalarial Drugs

potency, acridine use was reduced. When CQ resistance became more widespread in the 1960s,

the focus returned to acridines to produce non-resistant antimalarial drugs.

1.5.2.1 Mepacrine

Mepacrine (MP) was first synthesized in 1930; however, its use has not been widespread due to

its many side effects. It accumulates in the liver, kidney, and spleen and has a long elimination

half-life of up to 14 days. MP (also known as Quinacrine) has also been shown to accumulate in

lysosomes and bind to nucleic acids. It is an effective inhibitor of parasitic nucleic acid synthesis,

and it has been shown that low concentrations of MP inhibited the incorporation of 80% of P32

into erythrocytic DNA and RNA of rat P. berghei.33 Another study showed MP inhibits the

incorporation of 3H adenosine triphosphate in P. berghei.34 Despite this antimalarial activity, MP

causes skin and tissue discolouration, dermatitis, diarrhea, and vertigo, to name a few. For these

reasons, MP is no longer in use.

1.5.2.2 Pyronaridine

Pyronaridine (PN) was synthesized in China in 1970 by the Institute of Parasite Diseases. Similar

to MP, PN contains an aza-acridine core; however, dissimilarly, it contains an aminophenol with

two Mannich base side chains. Its mechanism of action remains unknown, but it is speculated that

it interferes with HZ formation. It is highly effective against CQ resistant (CQR) and CQ sensitive

(CQS) parasite strains. There is a possibility of cross-resistance / susceptibility between PN, and

CQ.35 PN is well tolerated; however, it can be metabolized to the quinone imine, a compound toxic

to neutrophils, Figure 1.5. It is currently administered as combination therapy with the artemisinin

artesunate to treat uncomplicated malaria for P. falciparum and P. vivax.5,25

35
Chapter 1 Development of Antimalarial Drugs

N N

OH O

N N N
HN HN HN
O N O N O

Cl N Cl N Cl N
Mepacrine Pyronaridine Pyronaridine quinone imine

Figure 1.5. Acridines.

1.5.3 4-Aminoquinolines

The most important class of compounds to arise from methylene blue modifications are the 4-AQs,

Figure 1.6. There have been countless derivatives and reviews on this group of compounds, which

gave rise to the most crucial antimalarial ever synthesized, CQ, considered the gold standard of

antimalarials due to its low cost and high efficacy.

OH
N N N
HN HN HN

Cl N Cl N Cl N
Chloroquine Hydroxychloroquine Sontoquine

OH N N
N
HN N N

Cl N Cl N N Cl
Amodiaquine Piperaquine

Figure 1.6. 4-aminoquinolines.

36
Chapter 1 Development of Antimalarial Drugs

1.5.3.1 Chloroquine

CQ was first synthesized in 1934 by Bayer Pharmaceuticals. However, its first safety profile

determined that it was too toxic. Interest in CQ was reignited in 1945 when its development was

handed over to the USA.36 15 years after its introduction, there was a high occurrence of CQR

parasite strains, and in 2001 the molecular biomarker of CQ resistance was discovered. It is a

highly effective blood schizonticide in sensitive parasites with an elimination half-life of 1 - 2

months.37

1.5.3.1.1 Mode of action

CQ is a lipophilic, weak diprotic base, with pKas of the quinoline nitrogen and the dialkylamino

side chain of 8.1 and 10.2, respectively. CQ accumulates in the DV, where it is primarily active.

Unprotonated CQ is membrane permeable and easily enters the DV down its pH gradient. The DV

has a pH of ~ 5.2. Once CQ is within the vacuole, it becomes diprotonated and membrane-

impermeable, thus trapped within its target organelle.38 Once within the DV, it has been reported

that CQ interferes with the parasite’s detoxification mechanism of HZ formation, Figure 1.7. CQ

forms a complex with Fe(III)PPIX, thus preventing the propagation of HZ.39 This leads to the

buildup of free heme and the eventual death of the parasite.

37
Chapter 1 Development of Antimalarial Drugs

Figure 1.7. CQ mechanism of action.

1.5.3.1.2 Complex formation

CQ inhibits b-hematin formation by forming a complex with Fe(III)PPIX. This complex may

inhibit crystal nucleation, block the fastest growing face of the HZ crystal,19 or essentially bind to

any stage of HZ formation to block propagation of growth.40 It has been suggested that this

complex is driven by coordination to the Fe(III) centre, H-bonding, and p – p interactions between

the porphyrin and the quinoline ring. The association constant between the porphyrin and quinoline

ring is dependent on the quinoline ring substituents. Lipophilic, electron-withdrawing groups form

a stronger association than electron-donating groups. A stronger association is directly correlated

with greater b-hematin inhibition.41

There has not yet been a universal consensus reached on the structure of the Fe(III)PPIX - CQ

complex formed. With the use of a gallium protoporphyrin IX (Ga(III)PPIX) model, Dodd and

Bohle were able to mimic the potential complex formed both in solution and in the solid-state,42

38
Chapter 1 Development of Antimalarial Drugs

Figure 1.8. They show that CQ forms a distinct, well-defined complex with [Ga(III)PPIX]2 and

highlights the significance of alkoxide coordination and H-bonding as a stabilizing feature. Other

publications focus on !-oxo dimer formation and the reliance on " – " stacking interactions to

explain complex formation.12,43,44 However, there is no evidence of the presence of !-oxo dimer

formation in vivo, though it is expected to form under slightly acidic conditions pH 5.5 – 6.0.

Lower pH’s promote the aggregation of the dimer and may accelerate HZ formation.12,44

Figure 1.8. Ga(III)PPIX and CQ complex formed in the presence of methanol.42

1.5.3.1.3 CQ structure-activity relationship

CQ activity is highly dependent on each component of its structure, Figure 1.9. Structure-Activity

Relationship (SAR) analyses have been carried out to determine the role of each structural

component. As mentioned, CQ accumulates within the DV via the protonation of the two basic

amines. The removal of the quinolinium amine reduces the accumulation of the compound 100 –

39
Chapter 1 Development of Antimalarial Drugs

fold compared to compounds containing a basic side chain.45 As accumulation within the active

site is reduced, so is the antiplasmodial activity. Vippagunta et al. synthesized a CQ analogue in

which the only modification was the absence of the tertiary alkylamine and found that it is 4 – fold

less potent than CQ in CQS strain NF54.46 Egan et al. were able to demonstrate that CQ analogs

lacking a basic side chain retained their ability to inhibit b-hematin formation (discussed in chapter

2); however, they displayed poor antiplasmodial activity, with IC50 values between 3.8 – 5 µM.45

It has been shown that the length of the side chain has a significant impact on the antiplasmodial

activity of the compound. Shortening the chain appears to increase potency, whereas increasing

the length has a negative effect on activity. This is evident in the antimalarial drug candidate

currently undergoing clinical trials, which contains a shortened chain, AQ13, Figure 1.11.

Furthermore, the length of the side chain has an impact on the stability of the quinoline-

Fe(III)PPIX complex formed. Increasing the length by more than three carbons may force the

chain outside the Fe(III)PPIX rim. A chain too short may lower van der Waals contributions,

Figure 1.8.47

A key feature of CQ’s activity is its ability to accumulate within the DV and inhibit b-hematin

formation. The nature of the substituent at the 7-position determines the compound’s ability to

inhibit b-hematin. This is because substituents on the quinoline ring will affect the compound’s

lipophilicity and the pKa of the quinoline nitrogen, which determines the association constant with

Fe(III)PPIX and its ability to accumulate with the DV. Studies have shown that the 7-chloro

substituent is essential for inhibition of b-hematin formation.48 The use of electron-withdrawing

lipophilic groups such as Cl, CF3, and NO2 lowers the pKa of the quinolinium nitrogen and

40
Chapter 1 Development of Antimalarial Drugs

increases the compound’s lipophilicity its association constant. Thus, the Cl moiety shows the

highest inhibition of b-hematin formation.48 The use of electron-donating groups such as NH2 and

OCH3 raises the quinolinium pKa, decreases the lipophilicity of the compound and its association

constant relative to CQ. Substituents on the quinoline ring also affect the pKa of the tertiary

alkylamine; however, the effects on the side chain vary with varying side chain lengths. Hawley

et al. reported the correlation between drug accumulation and b-hematin inhibition,49 and Egan et

al. have reported a linear correlation between b-hematin inhibition and antiplasmodial activity.

Therefore, CQ activity is dependent on its accumulation within the DV and its ability to inhibit b-

hematin formation, which are both influenced by the nature of the substituent at the 7-position.

Antiplasmodial activity

N NH

Weak bases required

for vacular accumulation
via pH trapping
N
Cl
Inhibition of β-hematin
formation Interacts with Fe(III)PPIX

Figure 1.9. CQ Structure-Activity Relationship.

41
Chapter 1 Development of Antimalarial Drugs

1.5.3.1.4 Metabolism

N HN H 2N
NH NH NH NH2

Cl N Cl N Cl N Cl N
Chloroquine Desethylchloroquine Bisdesethylchloroquine 4-amino-7-
chloroquinoline

Scheme 1.1. CQ metabolism.

Following oral administration, CQ is metabolized into desethylCQ, bisdesethylCQ and to a lesser

extent 4-amino-7-CQ by the kidney and liver, as shown in Scheme 1.1. DesethylCQ is the major

metabolite that is found in high concentrations in the blood and plasma.50 DesethylCQ is as active

against CQS strains as CQ but is less active than CQ in CQR strains.47,51 Furthermore, CQ is

primarily excreted through urine and to a lesser extent in feces.52

1.5.3.1.5 Side effects

CQ is well tolerated; it is safe for use in children and pregnant women. However, it does have

some side effects, which include nausea, dizziness, and headaches. CQ can cross the blood-retina

wall and deposit into melanin-rich tissue. Long-term use of over five years can cause retinopathy,

leading to blurred vision to complete vision impairment.53,54 Retinopathy is irreversible, but early

detection can prevent disease progression. An important contraindication of CQ is cardiotoxicity.

The K+ ion channel encoded by the human ether-a-go-go related gene (hERG), which regulates

cardiac action potential, is inhibited by CQ. Inhibition of this channel can cause prolonged QTc

intervals, arrhythmia and, in some cases, heart failure.41,55 Therefore, it is crucial to identify

patients with a history of retinopathy and arrhythmia to avoid adverse effects.

42
Chapter 1 Development of Antimalarial Drugs

1.5.3.2 Hydroxychloroquine

Hydroxychloroquine (HCQ) was first synthesized in 1946. It is a more polar derivative with a

hydroxy group on the alkyl side chain. The pKa of the quinolinium nitrogen is similar to that of

CQ; however, tertiary amine salt has a lowered pKa of 9.7.56 HCQ is less lipophilic therefore does

not easily diffuse across cell membranes, resulting in less accumulation in the DV. HCQ is 1.6 –

fold less potent than CQ in CQS strains and 8.8 - fold less potent than CQ in CQR strains.56 For

these reasons, HCQ is not commonly used as an antimalarial drug.

HCQ is the safer alternative to CQ as it does not readily cross the blood-retina barrier; therefore,

it deposits less onto melanin-rich tissue. Thus, retinopathy is only observed after continuous long-

term usage of HCQ. HCQ can also cross the placental as levels in maternal cells are equivalent to

that of the fetus. Interestingly, to date, there has been no evidence of toxicity to the fetus, no

congenital abnormalities, and no harm to newborns via breastfeeding. Therefore, HCQ is safe for

pregnant and breastfeeding women.57 The presence of the hydroxy group facilitates phase II

conjugation allowing for bile excretion.52 HCQ causes immunomodulatory effects such as

inhibition of phagocytosis and proteolysis, interference with lysosomal acidification, and toll-like

receptor signalling inhibition. In the USA, HCQ is commonly used to treat systemic lupus

erythematosus and rheumatoid arthritis.57

1.5.3.3 Sontoquine

The 3-methyl CQ analogue sontoquine (SQ) was synthesized in 1936 by Bayer Pharmaceuticals.

Its synthesis was fueled by the initial incorrect classification of CQ as too toxic for use. SQ is

slightly less potent than CQ in CQS and CQR strains and was relatively safe. Initial human trials

began in 1937 in Cameroon to treat P. vivax and P. falciparum. This led to further trials in 1942

43
Chapter 1 Development of Antimalarial Drugs

in Tunisia by German and French scientists for the development of SQ. In 1943 research data was

handed over to the USA for analysis, which led to the resynthesis of CQ.36 The rediscovery of CQ

showed that it was non-toxic and more potent antimalarial. SQ development was discontinued in

favour of CQ development.

1.5.3.4 Amodiaquine

Amodiaquine (ADQ), first synthesized in 1948, is a Mannich base 4-AQ with a 4-aminophenol. It

is a more potent and more lipophilic derivative of CQ. ADQ is a weaker base than CQ with pKas

of 7.1 and 8.1 for the quinolinium nitrogen and alkyl nitrogen, respectively. That being said, AQ

accumulation is 2- to 3- fold greater than CQ in CQS strain (HB3). Indicating that its accumulation

is energy and pH-dependent, as only 15% of ADQ accumulation can be attributed to its weak base

properties.58 Due to the hydroxy and diethylamino moieties’ proximity, ADQ can form

intramolecular hydrogen bonds at physiological pH. This potentially plays a role in its increased

potency in CQR parasites. ADQ analogues with hydrogen bonding groups display greater in vitro

activity than those unable to H-bond.

1.5.3.4.1 Metabolism

ADQ is quickly metabolized in the liver and has an elimination half-life of 5.2 hours, Scheme 1.2.

Its metabolic products are slowly excreted from the body and have an elimination half-life >4 days.

N-desethylamodiaquine (DADQ) is the primary ADQ metabolite mediated by cytochrome

P4502C8. AQ is 3 - fold more potent than DADQ, but due to its high concentration, DADQ

contributed to the observed antiplasmodial activity.59

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The presence of the 4-hydroxyanilino group is a cause of toxicity to the compound. The toxic

metabolite ADQ quinone-imine (ADQQI) is formed in the presence of cytochrome P450. This

metabolite is then susceptible to nucleophilic attack by glutathione to give ADQ-GS, which is non-

toxic. However, long-term use or high dosage of ADQ leads to the depletion of glutathione levels,60

leading to liver toxicity due to the increased concentration of ADQQI. ADQQI is also susceptible

to nucleophilic attack by thiol-containing enzymes that can cause hepatotoxicity. They are also

highly immunogenic and lead to agranulocytosis. Hepatoxicity is observed within three weeks to

10 months of continuous use, and agranulocytosis is observed after five to 14 weeks of continuous

use.5 Thus, prophylactic use of ADQ is not recommended. The WHO lists ADQ use only for

combination therapy with the artemisinin artesunate.23

HN N N

HO HO O

NH CYP2C8 NH P450 N

Cl N Cl N Cl N
Desethyl amodiaquine Amodiaquine Amodiaquine quinoneimine

P450

H 2N N

HO HO

Protein
NH S NH

Cl N Cl N
Bisdesthethyl amodiaquine Amodiaquine-protein adduct

Scheme 1.2. ADQ metabolism.

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Chapter 1 Development of Antimalarial Drugs

1.5.3.4.2 Detoxification

Although ADQ is effective in CQR strains, its side effects are a significant deterrent. In an attempt

to reduce its toxicity, several analogs with good antimalarial profiles have been synthesized.

Incorporating a fluorine group can lead to increased lipophilicity, increased oxidation potential,

stronger H-bonding, and changes in pKas. O’Neill et al. substituted the 4-hydroxy group for a 4-

fluoro group, Figure 1.10, to improve the stability of the aryl ring to oxidation, thus blocking

bioactivation to the quinone imine. The 4-fluoroamodiaquine (FAQ) analog shows retention of

antiplasmodial activity with no bioactivation.61

The exchange of the methylene N-diethyl with the hydroxy group gives rise to isoquine. This

exchange completely inhibits the formation of ADQQI, and it is more potent than ADQ in both

CQS and CQR P. falciparum strains. However, the desethyl metabolic product displays

hepatotoxicity.50 To combat the formation of ADQQI and dealkylation of FAQ4, tert-

butylisoquine was synthesized. Tert-butylisoquine contains an N-tert-butyl group, which cannot

be dealkylated due to steric hindrance, and bioactivation is inhibited due to the positioning of the

hydroxy group. This compound also retains its antiplasmodial activity and is less toxic than ADQ.

It is currently in clinical trials at GlaxoSmithKline. FAQ4 contains the N-tert-butyl moiety, and

the 4-hydroxy is replaced with fluorine. Dealkylation is blocked due to steric hindrance, and there

is no observed quinone-imine formed because of the 4-fluoro group. FAQ4 is active in CQS and

CQR strains in P. falciparum. This compound is also currently under clinical trials with Medicines

for Malaria Venture.47

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Chapter 1 Development of Antimalarial Drugs

N HN
OH OH
F F
N N
H
NH NH NH NH

Cl N Cl N Cl N Cl N
Fluoroamodiaquine (FAQ) Isoquine Tert-butylisoquine 4’-fluoro-N-tert-butylamodiaquine
(FAQ4)

Figure 1.10. ADQ analogs.

1.5.3.5 Piperaquine

Piperaquine (PPQ) was first synthesized in the 1960s by a group in China and France

independently. It is a bis-4-AQ linked by an alkyl chain. It was synthesized as a replacement for

CQ after the rise of resistance and used exclusively in China. It was phased out in the late 1980s

due to resistance. PPQ is a less toxic CQ analog that is equipotent to CQ in CQS strains and is 6 -

fold more active in CQR strains than CQ. It has a quinolinium pKa of 8.9, and it is highly lipid-

soluble. It has a similar elimination half-life to that of CQ of 1-2 months. PPQ is believed to exert

its activity similarly to CQ; however, there is insufficient research on its mode of action. PPQ

contains more protonation sites than CQ, which may allow for greater accumulation in CQR

strains. Molecular modelling shows that both quinoline nuclei are capable of forming a complex

with b-hematin.62

PPQ is relatively safe and well-tolerated with minor side effects such as dizziness, vomiting and

mild headache. A rare significant side effect is the increase in blood pressure. PPQ is not

recommended for pregnant women or children under the age of two, as there is insufficient

evidence of safety for these at-risk groups.63 PPQ has been reintroduced in the Chinese market as

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Chapter 1 Development of Antimalarial Drugs

a combination therapy with the artemisinin derivative dihydroartemisinin. It is worth noting that

PPQ resistance has not been observed in high transmission areas such as Sub-Saharan Africa as it

is not widely used.

1.5.4 AQs in clinical trials

HN N HN N

Fe

Cl N Cl N
AQ-13 Ferroquine

Figure 1.11. Antimalarial drug candidates in clinical trials.

1.5.4.1 Ferroquine

Ferroquine (FQ) is a 4-amino-organometalo-quinoline. It was first designed in 1993, and its first

published synthesis was in 1997.64 FQ is the first organometallic antimalarial, with an elimination

half-life of 16 days, see Figure 1.11. It completed Phase II clinical trials in 2011 by Sanofi-Aventis

and has not entered Phase III trials (ClinicalTrials.gov Identifier: NCT00988507). The presence of

1,2-unsymmetrically substituted ferrocene gives the molecule planar chirality. Experiments have

shown no significant difference in activity between the enantiomerically pure S (-)-FQ and R (+)-

FQ; therefore, it is prepared and administered as a racemate. FQ is highly effective against CQS

and CQR strains of P. falciparum, and there is little chance of cross-resistance with CQ. FQ is a

weaker base than CQ with pKas of 7.0 and 8.5 for the quinolinium nitrogen and tertiary nitrogen.

Furthermore, FQ is 100 - times more lipophilic than CQ at cytosolic pH and is more lipophilic

than CQ at vacuolar pH. FQ accumulates 50 times more than CQ at pH 565 despite being a weaker

base.

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Chapter 1 Development of Antimalarial Drugs

Unlike CQ, neutral FQ can easily form a strong intramolecular H-bond, as shown in Figure 1.12.

A 2.17 Å bond is formed between the anilino (N11) and tertiary amino (N24), resulting in a folded

conformation. In contrast, diprotonated FQ adopts an open conformation to allow H-bonding with

the solvent. The folded conformation results in the increase of hydrophobicity and the protrusion

of the ferrocenyl moiety towards the outside. It is believed that the hydrophobic ferrocenyl moiety

interacts with lipid structures in the membrane, and the quinoline stacks with Fe(III)PPIX. This

reversible open/folded conformation allows for the transport of FQ between membranes and is

believed to facilitate FQs ability to accumulate in the DV and to evade resistance transport

system.65,66

a)
Open conformation Folded conformation
(Hydrophilic) (Hydrophobic)

H
O
H H N Fe
24
Fe N
H b) N N
O H H
N N Fe
H 2.17Å 11
Cl N
Cl N Cl N FQ-Me
H

O
H H

Figure 1.12. a) Hydrogen-bonding in FQ and b) the structure of methyl FQ.

To determine the importance of H-bonding in FQ, a 4-methyl analog was synthesized with a

methyl group on the anilino nitrogen. FQ-Me is a more lipophilic and slightly more basic analog

with pKas of 7.1 and 8.8 for quinolinium and tertiary nitrogen, respectively. FQ-Me can inhibit β-

hematin formation; however, its antiplasmodial activity is considerably reduced in CQS and CQR

strains.67 Thus, the H-bond characteristic is required for effective antiplasmodial activity.

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Chapter 1 Development of Antimalarial Drugs

FQ is considered to be relatively non-toxic, with no significant side effects reported. Experiments

to induce resistance under drug pressure shows a low chance of cross-resistance of FQ with CQ

and other antimalarials and a low possibility of the emergence of resistance. Due to its high

antimalarial potency and low change of resistance, FQ could be administered as monotherapy.

However, Phase II clinical trials for combination therapy with artefenomel were recently

terminated due to a lack of efficacy.

1.5.4.2 AQ-13

AQ-13 synthesis was first published in 1948;68 however, its development in clinical trials was not

pursued until 1996.69 It is a CQ analog in which the isopentyl chain is replaced with a propyl chain,

Figure 1.11. AQ-13 was found to be effective against P. vivax and CQS and CQR strains of P.

falciparum. It has similar antiplasmodial activity to CQ in CQS strains and is more potent than CQ

in CQR strains. Like CQ, it is safe for human use with a low toxicity profile. AQ-13 is less

bioavailable than CQ and has a shorter elimination half-life of 13 days. The N-diethyl moieties are

susceptible to oxidative dealkylation. The metabolic products that result in mono- and di-

dealkylation are active in CQS strains but not on CQR P. falciparum.70

Side effects are similar to that of CQ: headache, nausea and diarrhea, and major side effect such

as QTc interval prolongation was observed only in high dosage patients.70 These effects are

avoided when drug concentration is reduced. AQ-13 is currently under phase II clinical trials

sponsored by Tulane University Health Sciences Centre.

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Chapter 1 Development of Antimalarial Drugs

1.5.5 Aryl aminoalcohols

HO N HO N HO
N
H
MeO MeO

N N N CF3
Quinine Quinidine CF3
Mefloquine

Figure 1.13. Aryl aminoalcohols

1.5.5.1 Quinine

QN is extracted from cinchona bark and was the first antimalarial discovered. It was first isolated

in 1820 by Pettetier and Caventon,71 and its first total synthesis was carried out in 1944 by

Woodward and Doering, Figure 1.13.72 As it currently stands, it is more economical to extract QN

than to synthesize it. QN was the front-line antimalarial used until the discovery and synthesis of

more potent AQ antimalarials. QN is a weaker base than CQ with the quinolinium pKa of 5.173

versus 8.2 for CQ. Therefore, QN accumulates in the DV in its monoprotonated form. QN is a fast-

acting schizonticide effective in P. falciparum and P. vivax. Moreover, it is also active against

gametocytes of P. vivax and P. malariae. It has an elimination half-life of 11-18 hours and is used

to treat severe malaria.

Most patients undergoing QN treatment experience a condition called cinchonism. Cinchonism

symptoms include tinnitus, nausea, headaches and slight hearing loss, which is reversible when

drug dosage is reduced. More severe symptoms include vertigo, diarrhea, vision and hearing loss.71

The WHO currently recommends that QN only be used for cases of severe malaria and in

combination with doxycycline.23

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Chapter 1 Development of Antimalarial Drugs

1.5.5.2 Mefloquine

Mefloquine (MQ) contains a single piperidine ring and two trifluoromethyl groups at the 2- and 8-

position, Figure 1.13. MQ was first synthesized during World War II and used as a CQ

replacement in the 1980s. Like CQ, it is a weak diprotic base, and at physiological pH, it can form

an intramolecular H-bond between the hydroxyl group and protonated tertiary amino group.24 MQ

is a blood schizonticide that is active against P. falciparum and P. vivax. Its mechanism of action

is unknown; however, it is believed to act similarly to CQ and QN. MQ can cause severe side

effects, particularly related to the central nervous system, such as neuropsychiatric effects. It is

contraindicated in patients with hypersensitivity to QN-related compounds, and it is not

recommended for pregnant women and children weighing less than 15 kg.74

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Chapter 1 Development of Antimalarial Drugs

1.5.6 Non-quinoline antimalarials

N
N
Cl
HO
OH O O OH OH
HO Cl
H2N
Cl H H
Cl O
N OH
CF3 Cl

Halofantrine Lumefantrine Doxycycline

H
O O
H
O HO
O O
H
HO O
O
O H
HO
O O
OH
O
O Cl

Artesunate Artemether Atovaquone

Cl
H2N
O
H N
N O
S
O2 H2N N NH2
N N

Sulfadoxine Pyrimethamine

Figure 1.14. Non-quinoline antimalarial drugs.

We have discussed various quinoline-based antimalarial drugs as they are the most extensively

studied classes. Other classes of antimalarial drugs and their modes of action are listed in Table

1.1. These antimalarials are often used in combination with schizonticides to ensure the

clearance of the parasite and to reduce the occurrence of resistance.

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Chapter 1 Development of Antimalarial Drugs

Table 1.1. Mode of action of non-quinoline based antimalarial drugs.

Drug Class Mode of action

Halofantrine Aryl aminoalcohol Inhibit hemozoin formation.75
Lumefantrine Aryl aminoalcohol Inhibit hemozoin formation.76
Doxycycline Antibiotic Inhibition of apicoplast.77
Artesunate Sesquiterpene lactone Membrane disruption via free radical production.78
Artemether Sesquiterpene lactone Membrane disruption via free radical production.78
Parasitic mitochondrial membrane disruption via
Atovaquone Naphthoquinone
inhibition of electron transport chain.5
Sulfadoxine Sulfonamide Antifolate: inhibition of dihydropteroate synthase.79
Pyrimethamine Diaminopyrimidine Antifolate: inhibition of dihydrofolate reductase.79

1.6 Antimalarial Resistance

CQ resistance first appeared in the late ’50s and early ’60s in Southeast Asia and South America.

Resistance is a result of sustained drug pressure in a geographical region. Resistance may also

arise due to monotherapy, incorrect dosage, self-medication, and the administration of cheap

counterfeit drugs.

1.6.1 Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT)

In the year 2000, the primary determinant for CQ resistance was identified.80,81 PfCRT is a 48 kDa

protein located on the membrane of the DV. It is a member of the drug – metabolite transporter

(DMT) superfamily, Figure 1.15.80,82 It is a monomeric transmembrane (TM) protein that consists

of 10 TM helices and two juxtamembrane helices: one is located parallel to the parasite cytosol

and the other parallel to the membrane in the DV.

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Chapter 1 Development of Antimalarial Drugs

a) b)

Figure 1.15. Structure of PfCRT 7G8 isoform. A) Topology showing inverted antiparallel TM

helices. B) Surface representation of the central cavity’s electrostatic potential with red and blue,

indicating negative and positive residues, respectively. Figures are taken from Kim et al.83

The TM domains form 5 helical pairs, and TM1-4 and 6-9 form a central cavity of around 3,300

Å3. The point mutation K76T has become a molecular marker for resistance. This mutation

replaces the positively charged lysine with a neutral threonine at the 76 position in the

transmembrane domain.80 The presence of a neutral residue removes the cation – cation repulsion

between protonated CQ and the lysine residue, speculated to prevent CQ efflux from the DV,

Figure 1.16. Consequently, the mutation allows CQ to diffuse out of the DV down its

concentration gradient and reduces CQ accumulation in its active site.84 The cavity has a net

negative charge influenced by three aspartate residues, suggesting possible substrates for the

transporter could be positively charged, Figure 1.15.83

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Chapter 1 Development of Antimalarial Drugs

Figure 1.16. K76T mutation removes a positive charge, which allows CQ2+ efflux from its active

site.

It has been proposed that PfCRT functions as a channel or transporter that mediates the efflux of

CQ out of the DV. In the channel model, PfCRT allows protonated CQ to passively diffuse out the

DV down its concentration gradient, Figure 1.16. However, in the transporter model, PfCRT

functions as an energy coupled transporter that actively effluxes CQ out of the DV.85,86 It is

believed that at low external concentrations of CQ, CQR parasites accumulate up to 10 – fold less

CQ than CQS parasites. This is achieved through the enhanced efflux capacity of the resistance

parasite resulting in higher survival rates.85,87

1.6.2 Plasmodium falciparum multidrug resistance protein 1 (pfmdr1)

Another protein supposedly involved in CQ resistance is Plasmodium falciparum multidrug

resistance protein (pfmdr1). pfmdr1 is a 162 kDa protein that belongs to the ABC (ATP – binding

cassette) superfamily. This protein is structurally similar to the mammalian multidrug resistance

transporter P-glycoprotein. It comprises two homologous halves, which contain six TM domains

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Chapter 1 Development of Antimalarial Drugs

with one nucleotide-binding domain (NBD), Figure 1.17.88 pfmdr1 is localized to the DV

membrane and is believed to behave like an energy - coupled transporter that regulates drug influx

into the vacuole. 89,90

It contains point mutations N86Y, Y184F, S1034C, N1042D, and D1246Y associated with

resistance to AQs and aryl aminoalcohol antimalarial drugs. These mutations arose as a

consequence of drug pressure, and their expression varies with geographical region. For instance,

N84Y is mainly found in Southeast Asian isolates, and the remaining mutations are primarily

found in South American isolates.88 Although these mutations are associated with antimalarial

resistance, it is not believed that they are the sole cause of resistance. It has been shown that the

level of expression of pfmdr1 is correlated to the degree of CQ resistance, which consequently

results in decreased sensitivity to MQ and vice versa. Cross-resistance is also observed with

halofantrine, lumefantrine and artemether. Therefore, the overexpression of pfmdr1 and the

presence of mutations results in decreased sensitivity of the parasite to certain antimalarial

drugs.84,91 Mutations may reduce pfmdr1s ability to transport drugs into the DV, resulting in the

possible reduction of drug accumulation in their active site.

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Chapter 1 Development of Antimalarial Drugs

Figure 1.17 Structure of pfmdr1 with highlighted mutations: N86Y, Y184F, S1034C, N1042D,

D1246Y and two nucleotide-binding domains. Illustration adapted from Valderramos and

Fidock.91

1.7 Vaccines

Malaria vaccine development is a long sorry saga of disappointment. Naturally acquired immunity

from the disease arises after sustained repeated exposure to the parasite to maintain immunity.92

Vaccine development has been complicated because the immune response to P. falciparum

infection is not well understood. There are 5,300 parasitic antigens that elicit an immune response;

however, it is unclear which antigens are responsible for a protective response.92 Vaccine

development is currently prioritizing administration to children under the age of five, as they

represent 67% of all malaria related deaths.

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Chapter 1 Development of Antimalarial Drugs

1.7.1 RTS,S

In 1985 GlaxoSmithKline and the Walter Reed Army Institute of Research began testing and

developing a pre-erythrocytic vaccine named RTS,S which protects against sporozoites. The DNA

of a pre-erythrocytic antigen circumsporozoite protein is expressed in yeast and fused with

hepatitis B surface antigen DNA to produce RTS. RTS binds hepatitis B surface antigens (S) to

form RTS,S particles. Then a proprietary adjuvant system AS01 (a mixture of deacylated

monophosphoryl lipid A and an emulsion QS21) is mixed with the particles to form a vaccine

concoction that is given intramuscularly over several months.93,94

Clinical trials carried out in Mozambique, Kenya and Tanzania on children aged 5 – 17 months

show that RTS,S/AS01 only provides partial immunity. Up to four doses reduced clinical cases by

56% and severe malaria cases by 47%.95 However, vaccine efficacy decreases over time. In a trial

involving children 6 – 12 weeks old, the vaccine has an initial efficacy of 33%.4 Gambian adults

given RTS,S with adjuvant AS02 over 15 weeks showed a 34% efficacy, but most importantly,

efficacy is high at 71% in the first nine weeks, but it drops to 0% in the following six weeks.92,93

It is believed that the vaccine’s low and unsustainable efficacy is because it does not induce a

memory T cell immune response for long-term protection.94 Despite the lack of long-term

immunity RTS,S is the first pre-erythrocytic vaccine that protects P. falciparum.

In 2019 the Malaria Vaccine Implementation Program launched in Ghana, Malawi, and Kenya.

Children aged 6 – 24 months are given a dose at 6, 7, 9 and 24 months in Kenya and Ghana. In

Malawi children aged 5 – 22 months are given a dose at 5, 6, 7 and 22 months.96–98 In the following

years we will discover the effectiveness of this implementation program.

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Chapter 1 Development of Antimalarial Drugs

Over the last century, there have been significant advances in the development of antimalarial

drugs. However, the rise of resistance has hindered progress towards the eradication of the disease.

More than ever, we require more tools for treating and preventing malaria as the number of cases

and deaths increases year over year. We need to develop effective drugs that can circumvent

resistance for known targets and develop drugs for new targets. Vaccine development has been

challenging, however successful implementation of RTS,S along with ACT treatment can help

drive down infection rates. The use of insecticide-treated mosquito nets has proven successful,

therefore ensuring that a greater proportion of at-risk populations have access to these can help

reduce the spread of the disease. The road to eradicating malaria has been long and strenuous;

however, we must sustain research efforts if we are to meet this ambitious goal.

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1.8 References

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C.; Wells, T. N. C. Malaria. Nat. Rev. Dis. Prim. 2017, 3, 17050.

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Development, Currently Used Therapeutics, and Drugs in Clinical Development.

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(6) Josling, G. A.; Llinás, M. Sexual Development in Plasmodium Parasites: Knowing When

It’s Time to Commit. Nat. Rev. Microbiol. 2015, 13 (9), 573–587.

(7) Wirth, D. F. Biological Revelations. Nature 2002, 419 (6906), 495–496.

(8) Francis, S. E.; Sullivan, D. J.; Goldberg, and D. E. Hemoglobin Metabolism In The Malaria

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(9) Spiller, D. G.; Bray, P. G.; Hughes, R. H.; Ward, S. A.; White, M. R. . The PH of the

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for Antimalarial Cytostatic versus Cytocidal Activities. J. Med. Chem. 2013, 56 (13), 5231–

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(13) Sullivan, D. J. Hemozoin: A Biocrystal Synthesized during the Degradation of Hemoglobin.

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& Co. KGaA: Weinheim, Germany, 2005; pp 129–137.

(14) Rosenthal, P.; Meshnick, S. R. Hemoglobin Catabolism and Iron Utilization by Malaria

Parasites. Mol. Biochem. Parasitol. 1996, 83 (2), 131–139.

(15) Ursos, L. M. B.; Roepe, P. D. Chloroquine Resistance in the Malarial Parasite,Plasmodium

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(16) Eggleson, K. K.; Duffin, K. L.; Goldberg, D. E. Identification and Characterization of

Falcilysin, a Metallopeptidase Involved in Hemoglobin Catabolism within the Malaria

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(17) Rosenthal, P. J. Falcipains and Other Cysteine Proteases of Malaria Parasites. Adv. Exp.

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(18) Egan, T. J. Haemozoin Formation. Mol. Biochem. Parasitol. 2008, 157 (2), 127–136.

(19) Pagola, S.; Stephens, P. W.; Bohle, D. S.; Kosar, A. D.; Madsen, S. K. The Structure of

Malaria Pigment β-Haematin. Nature 2000, 404 (6775), 307–310.

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de Dios, A. C. Antimalarial Drugs and Heme in Detergent Micelles: An NMR Study. J.

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2010, 111–129.

(22) Sullivan, D. J. Cinchona Alkaloids: Quinine and Quinidine. In Treatment and Prevention

of Malaria; Springer Basel: Basel, 2011; pp 45–68.

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as Antimalarials. Eur. J. Med. Chem. 2010, 45 (8), 3245–3264.

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Classical Drugs for Malaria. Chem. Rev. 2014, 114 (22), 11164–11220.

(26) Vale, N.; Moreira, R.; Gomes, P. Primaquine Revisited Six Decades after Its Discovery.

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(27) Staines, H. M.; Krishna, S. Treatment and Prevention of Malaria: Antimalarial Drug

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Treatment and Prevention of Malaria; Springer Basel: Basel, 2011; pp 69–94.

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treatment.

(30) Kitchener, S.; Nasveld, P.; Edstein, M. D. Short Report: Tafenoquine for the Treatment of

Recurrent Plasmodium Vivax Malaria. Am. J. Trop. Med. Hyg. 2007, 76 (3), 494–496.

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Chapter 1 Development of Antimalarial Drugs

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 Chapter 2

Optimization of 3-Aminochloroquine synthesis for

Chloroquine Derivatization.

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Chapter 2 Optimization of 3-Aminochloroquine synthesis for Chloroquine Derivatization

2.1 Preamble

In this chapter, we explore the synthesis of CQ functionalized at the 3-position with an amine.

There are only a few reports on the syntheses of 3-substituted CQ analogs in the literature for

malaria treatment.1–4 Most reported examples of 3-substituted CQ were for Leishmania spp. and

ocular melanoma treatment.5,6 The shortage of examples of such compounds might be due to their

lack of synthetic access and preceding literature that reported a decrease in antimalarial activity

due to quinoline substitution at the 3-position. However, we remained interested in functionalizing

this position because previous work carried out in our lab showed promising results in the

combination therapy between CQ and 3-ICQ.7,8 Moreover, substitution at this position will also

result in the synthesis of a new class of antimalarials drug candidates.

2.2 Introduction

The quinoline scaffold has been one of the most significant in antimalarial drug history.9–12 Some

of the most important antimalarial drugs are quinolines, such as CQ, PPQ and PQ. These AQs have

two substituents on the quinoline ring: a secondary amine group and a chloro- or methoxy group.

It was proposed that modifications to the quinoline ring, such as replacing the 7-chloro group

(present in CQ and PPQ) for an electron-withdrawing or an electron-donating group impacts, its

binding affinity to b-hematin.13 The presence of an amine group at the 3-, 5-, 6- or 8-position

decreased activity, and these simple AQs do not form a significant association with b-hematin,

Figure 2.1. However, 2-aminoquinolines (AQs) form a complex with b-hematin but do not inhibit

b-hematin formation14, Figure 2.2.

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Chapter 2 Optimization of 3-Aminochloroquine synthesis for Chloroquine Derivatization

NH2
NH2

N N N

-Does not strongly associate with Fe(III)PIX

-Does not inhibit hemazoin formation

H2N

N N
NH2

Figure 2.1. Simple AQs that do not inhibit hemozoin formation.

These findings deterred other research groups from synthesizing compounds with such

modifications. It is important to note that the aforementioned AQs do not include the 4-amino and

7-chloro moieties required for antimalarial activity. Tim Egan’s group has shown that the 7-chloro

group is crucial for efficient b-hematin association and inhibition.13 Furthermore, the secondary 4-

amino group is essential for antiplasmodial activity as its tautomeric form contributes to the drug’s

activity.15,16 One must also keep in mind that although modifications on the ring will impact the

compound’s association constant with b-hematin, research also shows no direct correlation

between b-hematin inhibition and antiplasmodial activity.15 Such compounds interact with b-

hematin in varying conformations. Furthermore, it is highly probable that b-hematin is not the sole

target of these drugs. As discussed in chapter 1, antiplasmodial activity is dependent on the

compound’s ability to accumulate in the DV and to inhibit b-hematin formation, which are both

dependent on the nature of the substituents on the quinoline ring.

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Chapter 2 Optimization of 3-Aminochloroquine synthesis for Chloroquine Derivatization

-Does strongly associate with Fe(III)PIX

N NH2 N N -Does not inhibit hemazoin formation
H

Figure 2.2. AQs that do not inhibit hemozoin formation.

As will be discussed in the next chapter, 3-haloCQ derivatives have been synthesized previously

in our group. These derivatives contain a halogen group at the 3-position of the quinoline ring. The

3-haloCQ derivatives are less potent than CQ in CQ-sensitive (CQS) and CQ-resistant (CQR)

parasites, they inhibit b-hematin formation but to a lesser extent than CQ. Interestingly, it was

found that of the 3-haloCQs synthesized, 3-ICQ was the most potent. When given as a combination

treatment with CQ in CQR parasites, 3-ICQ resensitizes the parasite to CQ. Thus, combination

treatment in vitro is more effective than monotherapy of CQ alone.7

For these reasons, we decided to explore further how functionality at the 3-position affects activity.

In this chapter, I will discuss the installation of an amino-functional group, which allows for further

derivatization to benzamides, sulfonamides and Schiff bases. To install the Csp2-N bond at this

position, we carried out Ullmann-type coupling reactions from a 3-BrCQ precursor.

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Chapter 2 Optimization of 3-Aminochloroquine synthesis for Chloroquine Derivatization

2.3 Results and Discussion

2.3.1 Optimization of 3-BrCQ synthesis

Bromination of CQ has been previously reported in our lab using trifluoroacetic acid and N-

bromosuccinimide (NBS) in acetonitrile.8 The preparation involves activation of N-

halosuccinimide with trifluoroacetic acid in acetonitrile at 80℃ for 30 minutes, followed by CQ

addition. The reaction mixture is then stirred at 80℃ for 2 hours to yield 73-81% of 3-BrCQ

following workup. However, the yield is greatly reduced when the reaction is scaled to gram scale

where the yields are consistently below 30%. Thus, we optimized this reaction to achieve

consistently high yields at the gram scale.

We carried the following reactions under microwave-assisted synthesis. Initially, we carried out a

small-scale reaction in 0.5 mL acetic acid, and the reaction mixture is heated at 80℃ for 30 minutes

(Table 2.1, entry 2). This results in an excellent yield of the desired product and the formation of

3,6-dibromochloroquine (3,6-BrCQ) with a regioselectivity ratio of 89:11 mono: di-brominated

product. Moreover, when the reaction is then carried out on the gram scale in 4.5 mL acetic acid

at 85℃ for 40 minutes, 1H NMR analysis shows 32% consumption of starting material with 41:59

mono-: di- selectivity. An additional equiv. of NBS was added, and the reaction is heated at 80℃

for an additional 30 minutes. This results in 100% consumption of starting material with

regioselectivity of 48:52 (mono-: di-) with an isolated yield of 21% of the mono-brominated

product (entry 1). The poor yield and regioselectivity were unsatisfactory, which may be due to

the acidic environment which favours the activation of the 6-position due to the 7-chloro group

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 Chapter 2 Optimization of 3-Aminochloroquine synthesis for Chloroquine Derivatization

with is an ortho-director. Therefore, we carried out the reaction in acetonitrile on a small scale

with 0.5 mL solvent heated at 80℃ for 30 minutes (entry 4).

Table 2.1. Optimization of 3-BrCQ synthesis using microwave-assisted synthesis.

Et2N Et2N Et2N

NH NH NH
O
Br Br Br
N Br
Cl N O Cl N Cl N
O Monobrominated CQ Dibrominated CQ
2.1 N H 2.3
2.2
O

CQ NBS Temperature Time Monobromo

Entry Solvent Mono:Di
(mg) equiv. (oC) (minutes) Yield (%)

1 1,000 2.5 AcOH 85 70a 48:52 21

2 100 1.5 AcOH 80 30 89:11 99
b b
3 1,000 2.5 MeCN 80-100 130 100:0 40
4 100 1.5 MeCN 80 30 96:4 96
c d
5 1,000 1.5 MeCN 100 60 - 64
e e
6 1,000 2 MeCN 100-140 100 100:0 40
Conditions: Chloroquine, NBS (1.5 equiv.) and solvent placed in a microwave vial and heated for
the stated amount of time. a) additional 1 equiv. of NBS added after 40 minutes. b) Additional 1
equiv. of NBS added and temperature increased to 100℃ after 110 minutes. c) Chloroquine
diphosphate salt used instead of the free base. d) Regioselectivity and conversion not measured
before purification. e) Additional 0.5 equiv. of NBS added and the temperature increased to 140℃
after 90 minutes.

1
H NMR analysis shows 64% conversion of starting material with a regioselectivity of 96:4 (mono-

: di-), and we isolated 96% of the desired mono-brominated product. The reaction is then scaled

up to 1 gram with 5 mL acetonitrile in a 5 mL microwave vial heated at 80℃ for 1 hour. After 1

hour, the TLC shows that the starting material is still present; therefore, the vial is returned to the

microwave and set at 85℃ for 40 minutes. Thereafter, 1H NMR shows 39% conversion of the

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Chapter 2 Optimization of 3-Aminochloroquine synthesis for Chloroquine Derivatization

starting material with regioselectivity of 100:0 mono-: di-. An additional equiv. of NBS is added,

and the reaction runs at 100℃ for an additional 30 minutes. The 1H NMR analysis then shows

94% consumption of starting material to only the desired mono-BrCQ. However, we got an

isolated yield of 40%. The lower than expected yield may be due to side product formation

stemming from the high reaction temperature. As the yields were still low for the gram scale

reactions, we decided to carry out a gram-scale reaction with CQ diphosphate rather than the free

base (entry 5). The reaction with CQ salt is carried out at 100℃ for 1 hour to give an isolated yield

of 64%. The 1H NMR conversion and regioselectivity for this reaction were not measured before

purification; however, only one product spot is observed on the TLC. It is important to note that

the salt does not fully dissolve in acetonitrile at 100℃, and the starting material is visible in the

vial after the reaction is removed from the microwave, reducing the potential yield. To determine

the effect of temperature on the reaction, free base CQ is placed in a microwave vial with 1.5

equiv. NBS and 5 mL acetonitrile, set at 100℃ for 1 hour (entry 6). After the time elapsed, the

starting material is still present as verified by TLC and 0.5 equiv. NBS is added, and the reaction

is run at 100℃ for an additional 30 minutes. Finally, in an attempt to further push the reaction to

completion, it is run at 140℃ for 10 minutes; thus, the reaction was carried out from 100-140℃

for a total of 100 minutes. 1H NMR analysis of the crude mixture shows 36% conversion of the

starting material with regioselectivity 100: 0 (mono-: di-), and we are able to isolate 40% of the

product.

This bromination reaction works best on a small scale to form the 3-BrCQ as the major product

with a minor formation of 3,6-BrCQ. Once the reaction is scaled up in acetic acid, we obtain greater

formation of 3,6-BrCQ, with 3-BrCQ as the minor product. However, we get better regioselectivity

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Chapter 2 Optimization of 3-Aminochloroquine synthesis for Chloroquine Derivatization

in acetonitrile. Similarly, small scale reactions in acetonitrile result in a high yield of our desired

product. When CQ.2H3PO4 is used, we are able to get good yields at gram scale with a yield of

64%. Finally, when higher temperatures are used for CQ free-base in acetonitrile, the reaction

yield greatly suffers, and longer reaction times are required.

As we achieved good yields with gram scale bromination, we continued to the synthesis of 3-

aminochloroquine (3-NH2CQ). The formation of the heteroatom- Csp2 bond occurs under Ullmann-

type coupling reaction conditions,17,18 the mechanism for this cross-coupling is described later in

this chapter.

2.3.2 Optimization of 3-NH2CQ synthesis

3-NH2CQ can be synthesized using the linear route displayed in Scheme 2.1. We began with the

nucleophilic aromatic substitution of 4,7-dichloroquinoline 2.4, to make 7-chloroquinolin-4-ol,

2.5. Then nitration at the 3-position with nitric acid to give 2.6, followed by chlorination with

phosphoryl chloride gives 4,7-dichloro-3-nitroquinoline, 2.7.19 Condensation of 2.7 with a diamine

side chain 2.8 gives 3-NO2CQ 2.9, which is then reduced to 3-NH2CQ 2.10 with stannous

chloride.19 This route involves 5 steps carried out over 5 days; thus, there was a need to create a

faster synthetic route to 3-NH2CQ. We achieved this by creating a 2-step synthesis carried out in

1 day, with the bromination of CQ followed by an amination via an Ullmann-type coupling

reaction.

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Chapter 2 Optimization of 3-Aminochloroquine synthesis for Chloroquine Derivatization

Cl OH HNO3, OH
Acetic Acid, Propionic acid,
NO2
125oC, 1h 140oC, 18h

Cl N Cl N Cl N
2.4 2.5 2.6

Phosphoryl Chloride,
115oC, 18h

Et2N SnCl2.2H2O, Et2N MeCN,

NH EtOH, NH DIPEA, Cl
NH2 80oC, 2h NO2 RT, 30min NO2
Et2N
NH2
Cl N Cl N Cl N
2.10 2.9 2.7 2.8

Scheme 2.1. Linear synthesis to 3-AminoCQ synthesis.

The Ullmann-type reaction is a coupling reaction that involves a copper catalyst and an aryl halide

with various heteroatoms to form a heteroatom - Csp2 bond, with Cu(I) as the active species in this

reaction. However, Cu(0) and Cu(II) salts are used with a base or reducing agent for in situ

reduction/oxidation to generate the active Cu(I) species.17 The use of a reducing agent such as

sodium ascorbate was required to obtain higher yields of the amine. The omission of the reducing

agent did not result in azide formation, It is desirable that we use easily accessible, cheap reagents

for this coupling reaction because antimalarial drug candidates used for treatment in developing

countries require cost-efficient production. We first began with ammonium hydroxide as the amine

source under various conditions with/ without the presence of a ligand or base, Table 2.2.

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Chapter 2 Optimization of 3-Aminochloroquine synthesis for Chloroquine Derivatization

2.3.2.1 Optimization using ammonium hydroxide as the nitrogen source

Table 2.2. 3-NH2CQ formation with NH4OH as NH2 source.

Et2N Et2N
NH Cu, Ligand, NH
Br Base NH2
NH4OH
Cl N Cl N
2.2 2.10

Reducing
Catalyst Temperature Yield
Entry Ligand [mol%] agent/base Solvent
[mol%] (oC) (%)
[mol%]
1 Cu [20] - - EtOH 95 -
L-Ascorbic 2M NH3
2 CuI [20] DMEDA [200] 95 Tracea
Acid [200] in EtOH
Cu2O Diethylenetriamine
3 KOH [200] H2 O 100 -
[10] pentaacetic acid [20]
Sodium
Cu2O Trans +/- Diamino 7N NH3
4 ascorbate 95 -
[100] cyclohexane [20] in MeOH
[200]
a
: 4% conversion of starting material to CQ determined by 1H NMR.

Copper-catalyzed amination reactions from aryl halides using ammonium hydroxide have been

well reported in the literature, Scheme 2.2.20–22 Lang et al. reported the amination of 2-

bromopyridine without the need of a ligand or base.21 They report that amination worked best in

an aqueous or alcoholic solvent; however, they see the formation of a C-O byproduct due to

competing hydroxide attack from the solvent. When polar, aprotic solvents were used, they had

low product conversion, which was attributed to ammonia’s poor solubility in such solvents.

Similarly, Jiao et al. carried out the amination reaction on 3- and 4-bromopyridine and achieved

near quantitative yields with copper powder, exposure to air and optional use of a Lewis base.20

Yang et al. were able to successfully carry out amination of 2- and 3-bromopyridine using a ligand

and inorganic base in H2O.22

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Chapter 2 Optimization of 3-Aminochloroquine synthesis for Chloroquine Derivatization

Br Cu, NH2 OR'

NH3/R1OH

N 80oC, N Lang et al.

N
16 h
85% : 95/5

Cu,
H
Br CH3NH2 N

100oC, Jiao et al.

N 12 h N
94%

CuO, Ligand,
Br NH3.H2O NH2

KOH, H2O Yang et al.

N N
100oC, 12 h
72%

Scheme 2.2. Known examples of pyridine amination.

In our experimentation, the use of ammonia did not yield any desired product (Table 2.2, entries

2 and 4). The reaction is monitored by TLC for up to 24 hours, and if no product spots were

detected by TLC, the reaction was deemed unsuccessful. The use of copper powder in degassed

ethanol (EtOH) was unsuccessful, so was the use of copper(I) oxide with a base or reducing agent.

Dimethylethylenediamine (DMEDA) as a ligand and excess L-ascorbic acid as the reducing agent

for CuI results in trace amounts of the desired product (<2% conversion) and interestingly results

in 4% conversion (determined by 1H NMR) to reductive CQ product formation.

2.3.2.2 Optimization using azidotrimethyl silane as the nitrogen source

We then decided to use an organic azide, azidotrimethyl silane (TMSN3), to improve the solubility

of our amine source in organic solvents and increase the reaction yield. As expected, the control

experiment of 3-BrCQ in 1.5 equiv. TMSN3 (Table 2.3, entry 10), heated at 53℃ overnight, did

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Chapter 2 Optimization of 3-Aminochloroquine synthesis for Chloroquine Derivatization

not yield any product. We then used CuI with an excess of potassium carbonate (K2CO3) in 1,4-

dioxane; however, no product spots were detected on TLC (entry 6). The absence of a ligand gives

a low yield of 11% (entry 5), and the yield is not significantly improved when L-proline (entry 4)

or trans +/- diamino cyclohexane (entry 7) are used as ligands. When a stoichiometric amount of

copper powder is used with excess 2-aminoethanol as a ligand, and excess sodium ascorbate (entry

8) as the reducing agent in dimethylacetamide (DMA) are used, we observe a significant increase

in yield.23 The 2-aminoethanol ligand is essential for this reaction as there is no product formation

in its absence after heating at 95℃ for 23 hours when used with stoichiometric amounts of copper

powder (entry 8). We get similar results using catalytic amounts of copper powder with DMEDA

and sodium ascorbate in anhydrous EtOH (entry 2). The use of excess ligand combined with copper

iodide gave 1.7 - fold increase in yield (entry 1). A different organic azide, tetrabutylammonium

azide (NBu4N3), was used as an amine source (entry 11); however, no product was formed from

this reaction.

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 Chapter 2 Optimization of 3-Aminochloroquine synthesis for Chloroquine Derivatization

Table 2.3. 3-NH2CQ formation with TMSN3 as the nitrogen source.

Reducing
Catalyst Temperature Yield
Entry Ligand [mol%] agent/base Solvent
[mol%] (oC) (%)
[mol%]
L-Ascorbic Acid
1 CuI [20] DMEDA [1200] EtOH 95 42
[30]
Sodium Ascorbate
2 CuI [20] DMEDA [50] EtOH 85 20
[20]
L-Ascorbic Acid
3 Cu [20] DMEDA [30] EtOH 95% 85 13
[30]
Sodium Ascorbate
4 CuI [20] L-Proline [30] EtOH 95% 85 13
[30]
Sodium Ascorbate
5 CuI [20] - EtOH 95% 85 11
[30]
1,4-
6 CuI [20] - K2CO3 [200] 95 -
Dioxane
Trans +/- Diamino Sodium Ascorbate
7 CuI [20] EtOH 95 14
cyclohexane [20] [200]
2-Amino ethanol Sodium Ascorbate
8 Cu [200] DMA 95 25
[250] [200]
Sodium Ascorbate
9 Cu [200] - DMA 95 -
[200]
10 - - - - 53 -
CuI Sodium Ascorbate 1,4-
11a DMEDA [20] 95 -
[100] [200] Dioxane
a) NBu4N3 was used as the nitrogen source.

2.3.2.3 Optimization using sodium azide as the nitrogen source

We then decided to carry out the amination reaction with sodium azide as the amine source. The

control reaction does not proceed in dimethylformamide (DMF) (Table 2.4, entry 6).24,25 The use

of copper sulfate with sodium ascorbate did not yield any product (entry 4),26,27 and Cu(I) iodide

with excess potassium carbonate did not yield any product (entry 2).28 When Cu(I) iodide was used

with bidentate ligand DMEDA in a degassed solution of 70% EtOH, 30% H2O at 95℃, we saw

product formation and were able to isolate 29% of the desired product.29–31 We repeated the

experiment in anhydrous EtOH with the addition of sodium ascorbate and were able to isolate 55%

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Chapter 2 Optimization of 3-Aminochloroquine synthesis for Chloroquine Derivatization

of our desired product.27,32 Goriya and Ramana reported the direct amination of aryl halides with

sodium azide under a Cu(II)-ascorbate redox system.27 They reported that arylamine formation can

occur without the presence of copper and that various concentrations of sodium ascorbate with

copper(I) sulfate (Cu2SO4), sodium carbonate (Na2CO3) and L-proline greatly increases product

formation rate and decreases reaction time. However, in our system, we have found that copper is

required for this synthesis and the combination of DMEDA and sodium ascorbate produces the

highest yield.

Table 2.4. 3-NH2CQ formation with NaN3 as the nitrogen source.

Reducing
Catalyst Ligand Temperature Yield
Entry agent/base Solvent
[mol%] [mol%] (oC) (%)
[mol%]
DMEDA EtOH/H2O
1 CuI [20] - 95 29
[1200] (7:3)
2 CuI [20] - K2CO3 [200] EtOH (95%) 95 -
DMEDA Sodium EtOH/H2O
3 CuI [50] 85 55
[50] Ascorbate [50] (7:3)
CuSO4.H2O L-Proline Sodium DMF/H2O -
4 85
[20] [20] Ascorbate [20] (2:1)
Sodium
CuSO4.H2O DMEDA DMF/H2O
5 Ascorbate [30] 85 -
[20] [30] (2:1)
+ Na2CO3 [20]
6 - - - DMF 100 -

Surprisingly, conditions in Table 2.4 entry 3 results in the formation of the desired 3-aminoCQ

product and the reduction of CQ as a product. We then carried out experiments in an attempt to

minimize the reductive formation of CQ. Heating the reaction in an oil bath instead of microwave-

irradiation resulted in 50% conversion of the 3-BrCQ into 3-NH2CQ and 50% to CQ at 85℃. We

then ran the reaction at 75℃ for 2 hours (Table 2.5, entry 4). However, there was little product or

byproduct formation at this temperature. The temperature was then increased to 85℃, heated for

a further 3 hours and we were able to isolate 44% of the desired product. CQ was also formed, but

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Chapter 2 Optimization of 3-Aminochloroquine synthesis for Chloroquine Derivatization

it was not collected during the purification. A longer reaction time of 19 hours (entry 3) did not

improve the selectivity nor the yield of the desired product. The reaction was carried out in the

presence of 2 equiv. of (2,2,6,6-Tetramethylpiperidin-1-yl)oxyl (TEMPO) - a radical trapping

agent. We got lower yields of both 3-NH2CQ and CQ but also slightly greater selectivity for 3-

NH2CQ. When ten equiv. of TEMPO is used, we got no formation of CQ and trace amounts of 3-

NH2CQ. The greatest selectivity and yield are achieved with three equiv. NaN3, 0.5 equiv. CuI,

0.5 equiv. DMEDA, and 0.5 equiv. sodium ascorbate at 85℃ in an oil bath for 3 hours.

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Chapter 2 Optimization of 3-Aminochloroquine synthesis for Chloroquine Derivatization

Table 2.5. Reaction conditions to minimize byproduct formation.

CuI, DMEDA,
Et2N Na ascorbate, Et2N Et2N
NH EtOH/H2O (7:3), NH NH
Br 85oC NH2

Cl N Cl N Cl N
2.2 2.10 2.11

Reaction 3-NH2CQ
Heating Sodium Conversion
Entry time NaN3 CuI DMEDA Isolated Yield
method Ascorbate (NH2:CQ)
(hours) (%)
1 Oil 3 2 0.3 0.4 0.3 - 49
2 Oil 3 2 0.3 0.4 0.3 - 32b
3 Oil 19 2 0.3 0.4 0.3 - 50
4 Oilc 5 2 0.3 0.3 0.3 - 44
5 Oil 4 2 0.3 0.3 0.3 - Traced
6 Oil 3 3 0.5 0.5 0.5 - 55
7 MWe 0.7 3 3 3 2 91 (30:70) 16
8 MWf 0.5 3 3g 3 3 81 (37:63) 19
9 MW 1 3 0.5 0.5 0.5 69 (36:64) 26
10 MW 1 3 0.5 0.5 0.5 84 (51:49) NAh
a) Reactions run at 85℃ in EtOH /H2O (7:3). b) Reaction included 2 equiv. TEMPO. c) Starting
temperature of 75℃ after 2 hours temperature increased to 85℃. d) Reaction included 10 equiv.
TEMPO, starting temperature of 75℃, after 2 hours temperature increased to 85℃. e) Heated at
100℃ for 30 minutes and then at 140℃ for 10 minutes. f) Heated at 100℃ for 30 minutes. g) 3
equiv. of copper powder used. h) Purification not carried out; product contained 5 equiv. of
TEMPO.

We then carried out this coupling reaction via microwave-assisted synthesis (MW) with reduced

reaction times. When we replicated our optimized reaction conditions in the microwave, we saw a

2-fold decrease in yield (entry 9). One might assume that it may be due to reduced reaction times,

but we observe greater conversion to CQ in microwave-assisted synthesis. We then used

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Chapter 2 Optimization of 3-Aminochloroquine synthesis for Chloroquine Derivatization

stoichiometric amounts of our reagents and increased the temperature from 85℃ to 140℃.

However, we obtain lower yields of 3-NH2CQ and greater conversion of starting material to CQ

(entry 7 and 8). We then carried out the reaction in the presence of 5 equiv. of TEMPO and

surprisingly saw 84% conversion of the starting material with a significantly improved selectivity

for 3-NH2CQ. Hence microwave-assisted synthesis is not recommended for these Ullmann-type

coupling reactions as we get lower yields and greater reductive CQ formation.

CuI, DMEDA,
Et2N Na ascorbate, Et2N
NH EtOH/H2O (7:3), NH
NH2 85oC, 3h
NaN3

Cl N Cl N
0%
2.10 2.11

Scheme 2.3. Determination of 3-NH2CQ stability.

We were then curious to determine the stability of the Csp2-N bond. We replicated the reaction

with 3-NH2CQ as our starting material with CuI, DMEDA, sodium ascorbate and sodium azide in

degassed EtOH /H2O (7:3), which is heated at 85℃ for 3 hours. A TLC is taken, and no CQ spots

are detected, and only the 3-NH2CQ starting material is present. This indicates that there is

competition for the formation of CQ and our desired amine, Scheme 2.3.

2.3.3 Proposed Ullmann-type coupling mechanisms of product formation

The reductive formation of the CQ byproduct was unexpected and reduced the yield of the desired

product. We reexamined the proposed catalytic cycle for Ullmann-type coupling reactions to

determine the cause. As shown in Figure 2.3 there are two proposed pathways for this coupling

reaction, with the upper cycle being the most widely accepted.17,33,34 Copper-catalyzed Ullmann-

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Chapter 2 Optimization of 3-Aminochloroquine synthesis for Chloroquine Derivatization

type coupling is believed to occur via a Cu(I) / Cu(III) catalytic cycle, which is initiated by the

coordination of the nucleophile onto the Cu(I) center. This is followed by oxidative addition of the

aryl halide to form an NR2 - Cu(III) - aryl species and the release of the halide anion. The activation

of the aryl halide is believed to be the rate-limiting step. Reductive elimination then occurs to form

the NR2 - Ar coupling product and releases Cu(I) which can then reenter the cycle. The less

probable route is the initial oxidative addition of the aryl halide to form a halide - Cu(III) - aryl

intermediate, followed by coordination of the nucleophile via the release of the halide to form an

NR2 - Cu(III) - aryl species. Reductive elimination follows to release the product and Cu(I). We

believe that both pathways contribute to the formation of our desired 3-NH2CQ product and that

the lower cycle might be the main contributor to the formation of the CQ byproduct. The formed

ligand – Cu(III) – Ar species can undergo proton-coupled electron transfer causing the release of

the reductive CQ product. It is possible that the amines present in the 3-BrCQ side-chain act as

ligands on the Cu(I) center, and if oxidative addition occurs first, then all coordination sites could

be occupied, preventing nucleophile coordination.

Base-H
Ar-X Oxidative
L CuI NR2 Addition
R2NH + Base X-

Reductive elimination Ar
L CuI L CuIII
NR2
Ar-NR2

Ar-X
Ar Base-HX
Oxidative L CuIII
Addition X R2NH + Base
e- + H+
Proton-coupled
Electron Transfer
L-CuI-X
Ar-H

Figure 2.3. Proposed pathways for Cu(I)/ Cu(III) Ullmann-type amination catalytic cycle.

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Chapter 2 Optimization of 3-Aminochloroquine synthesis for Chloroquine Derivatization

The 2-electron catalytic cycle is the most widely accepted mechanism as the reaction is not

inhibited with radical trapping agents or radical initiators.35 We tested this with the use of TEMPO

in both traditional oil heating and with microwave-assisted synthesis. When using an oil bath with

two equiv. of TEMPO we saw a slight reduction in product and byproduct formation. When ten

equiv. of TEMPO were used, we see no formation of byproduct and little formation of the desired

product. When five equiv. of TEMPO is used under microwave-assisted synthesis, we still observe

84% conversion to products. This suggests that the radical trap has little effect on the reaction

process. It is only when a large excess is used that we see an impact on product formation.

The mentioned Cu(I) / Cu(III) cycle is not the only proposed Ullmann-type mechanism. Another

proposed mechanism is a Cu(I) / Cu(II) Single Electron Transfer (SET) mechanism with aryl

radicals that might lead to product formation.36 Through experimentation with iodoarenes, Kim

and Bunnett proposed ‘outer sphere’ electron transfer through a radical chain mechanism involving

SET as the initiation step.36 They performed experiments in the presence of tetraphenylhydrazine

(a radical trapping agent) and proposed a unimolecular radical nucleophilic substitution (SRN1), as

shown in Figure 2.4.

SET
Initator + ArX (ArX) Ar + X

Ar + NH (ArNH2)
2

H+ + (ArNH) + ArX ArNH2 + (ArX)

Figure 2.4. Proposed Cu(I)/ Cu(II) SRN1 mechanism.

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Chapter 2 Optimization of 3-Aminochloroquine synthesis for Chloroquine Derivatization

A proposed mechanism that does not involve a change in oxidation state is the p-complexation of

Cu(I) with the aryl halide, Figure 2.5. This coordination of Cu(I) with the aromatic ring activates

the C - X bond making it more susceptible to nucleophilic substitution.37

PhO OPh OPh K

Cu Cu
K
PhO
Br PhO
Br Rate
Determining
Step

Figure 2.5. Proposed p-complexation Ullmann-type mechanism.

2.4 Conclusion

The 3-substituted quinolines are surprisingly poorly explored as antimalarial drug candidates. We

have previously published papers on the synthesis of 3-haloCQs.7,8 Thus, we sought to

functionalize this position to explore the potential advantages and disadvantages of such a

substitution. In this chapter, the reaction optimization of 3-BrCQ while using fewer reagents and

decreasing reaction times was reported. We were able to scale up the reaction from 300 mg to 1

gram, and regioselectivity for the product was improved.

With 3-BrCQ in hand, we were able to synthesize 3-NH2CQ via Ullmann-type coupling. We

discovered that ammonium hydroxide is not a good amine source for this coupling reaction. We

then tested azidotrimethyl silane as an amine source because of its increased solubility in organic

solvents, and it proved to be a better candidate than NH4.OH. We determined that a base is not

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Chapter 2 Optimization of 3-Aminochloroquine synthesis for Chloroquine Derivatization

required for this reaction; however, the presence of the reducing agent as sodium ascorbate or L-

ascorbic acid in combination with ligand DMEDA gave the best results, although the yields were

still poor. Tetrabutylammonium azide was also tested with DMEDA and sodium ascorbate;

however, no product was formed. Lastly, sodium azide was tested, and we were able to

significantly increase product yield to 55%, although, we observed the formation of CQ as a

byproduct. Therefore, we created a 2-step synthesis to form 3-NH2CQ from CQ in 35% overall

yield over 2 days.

We concluded that the formation of 3-NH2CQ and CQ occurred mainly via two competing

mechanisms. It is possible that amine formation occurs when nucleophile coordination precedes

oxidative addition and vice versa for CQ. Currently, there is no exact proven mechanism for the

Ullmann-type coupling in the literature. There may be several mechanisms through which our

byproduct is formed. As we have synthesized 3-NH2CQ, we can now derivatize the 3 position

further to synthesize novel 3-substituted CQ antimalarial candidates.

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Chapter 2 Optimization of 3-Aminochloroquine synthesis for Chloroquine Derivatization

2.5 Experimental

2.5.1 General Information

All amination reactions were carried out in the presence of N2 gas by standard syringe and septa

techniques. Glassware was taken directly from the oven (120oC) and allowed to cool in a desiccator

before use. Bromination reactions were carried out in 2 mL and 5 mL Biotage Microwave Vials

under air. All solvents and reagents were obtained commercially and used without further

purification unless noted.

Microwave-assisted reactions were carried out in a Biotage Initiator Classic. High-Resolution

Mass Spectroscopy (HRMS) was obtained by positive/negative ESI, or positive/negative APCI on

a Bruker Maxis Impact QTOF, or a Thermo Scientific ExactivePlus Orbitrap, with results reported

as mass/charge ratios (m/z). Thin-layer chromatography (TLC) plates were visualized using ultra-

violet light, 254 nm. Flash column chromatography was carried out manually using 230 - 400 mesh

silica gel (Silicycle) using reagent grade solvents or using Biotage Isolera One system with Biotage

ZIP 30 g columns.

1 13
H and C NMR spectra were recorded at ambient temperature on Bruker AVIIIHD 400 MHz

(1H 400 MHz, 13C 100 MHz), Bruker AVIIIHD 500 MHz (1H 500 MHz, 13C 125 MHz) and Bruker

AVIIIHD 800 MHz (1H 800 MHz, 13C 201 MHz) using tetramethylsilane as the internal standard.

Chemical shifts are reported relative to the residual deuterated solvent peaks. Chemical shifts are

expressed in parts per million (ppm = δ) values and coupling constants (J) in Hertz (Hz). The terms

m, s, d, t, q and p represent multiplicities of 1H NMR resonances: multiplet, singlet, doublet, triplet,

quartet and pent, respectively. For previously unknown compounds, a combination of 2D

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Chapter 2 Optimization of 3-Aminochloroquine synthesis for Chloroquine Derivatization

experiments (COSY, HSQC, HMBC) was often used to complete the assignment of 1H and 13C

signals. 1H NMR signals are described by chemical shift δ (multiplicity, J (Hz), integration). 13C

NMR signals are described by chemical shift δ and are singlets unless otherwise specified.

2.5.2 Experimental procedures

Chloroquine free-base preparation (2.1)

To chloroquine diphosphate (2 g) dissolved in deionized H2O (300 mL) in a 750 mL Erlenmeyer

flask is added sodium hydroxide (1.86 g, 0.16 M) to give a pH of 11/12. At this pH all the drug

has precipitated. This mixture is then transferred into a 1 L separatory funnel, and dichloromethane

(DCM) (200 mL) is added to extract the free base (repeated three times). The combined organic

layers are washed with brine and then dried over anhydrous magnesium sulfate (MgSO4). MgSO4

is filtered off, and the solvent is removed under reduced pressure. To the flask containing the free-

base, which appears as a colourless oil, is added 15 mL of diethyl ether and is swirled. The solvent

is then removed under reduced pressure, and the flask is transferred into a vacuum oven and left

to dry overnight. White solid, 1.05 g, 84%. The 1H and 13

C NMR was consistent with the

literature.38

N4-(3-bromo-7-chloroquinolin-4-yl)-N1,N1-diethylpentane-1,4-diamine

Et2N O Et2N Et2N

NH Acetonitrile or NH NH
Br Acetic acid
N Br Br Br

O MW, 30 min
Cl N Cl N Cl N
Monobrominated CQ Dibrominated CQ

Chloroquine free-base (100 mg, 0.31 mmol), N-Bromosuccinimide (83.5 mg, 0.47 mmol) and 0.5

mL of acetonitrile or acetic acid are placed in a 2 mL Biotage microwave vial. The mixture is

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Chapter 2 Optimization of 3-Aminochloroquine synthesis for Chloroquine Derivatization

subjected to microwave irradiation at 80℃ for 30 minutes. When complete, the reaction is diluted

in ethyl acetate (EtOAc) and basified with saturated sodium hydroxide and extracted with EtOAc

(3 X 50 mL) and washed with brine. The combined organic extracts are then dried over anhydrous

MgSO4, and the solvent is removed under reduced pressure. The crude product is purified by flash

chromatography with silica gel, solvent system: 70% hexanes (Hex), 25% EtOAc, 5%

triethylamine (NEt3) to reveal a brown oil.

N4-(3-bromo-7-chloroquinolin-4-yl)-N1,N1-diethylpentane-1,4-diamine (2.2)

As reported in the literature.7

N4-(3,6-dibromo-7-chloroquinolin-4-yl)-N1,N1-diethylpentane-1,4-diamine (2.3)

Yield: 119.4 mg, 8%. 1H NMR (400 MHz, Chloroform-d) δ 8.68 (s, 1H), 8.29 (s, 1H), 8.08 (s,

1H), 4.57 (d, J = 10.1 Hz, 1H), 4.00 (dq, J = 10.0, 6.2 Hz, 1H), 2.57 (q, J = 7.2 Hz, 4H), 2.47 (d, J

= 7.0 Hz, 2H), 1.63 (dtd, J = 13.2, 6.9, 3.8 Hz, 4H), 1.29 (d, J = 6.3 Hz, 3H), 1.04 (t, J = 7.2 Hz,
13
6H). C NMR (201 MHz, Chloroform-d) δ 153.01, 148.61, 148.14, 135.46, 130.84, 128.19,

121.88, 119.74, 107.54, 55.05, 52.55, 46.92, 36.67, 23.40, 22.39, 11.26. HRMS (ESI) calcd. for

C18H25 Br2ClN3 [M+H]: 476.0098, found: 476.0097

General procedure for 7-chloro-N4-(5-(diethylamino)pentan-2-yl)quinoline-3,4-diamine

(2.4) synthesis:

Et2N Cu, Ligand, Et2N

NH Reducing agent / Base NH
Br Azide N2 NH2

Cl N Cl N

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Chapter 2 Optimization of 3-Aminochloroquine synthesis for Chloroquine Derivatization

Azide (2 equiv.), copper (0.5 equiv.), ligand (0.5 equiv.) and reducing agent (0.5 equiv.) or base

(2 equiv.) are placed in a 10 mL round bottom flask fitted with a septum. A needle connected to

the vacuum/N2 manifold was inserted, the flask is evacuated and backfilled with N2 gas, this

procedure is repeated three times. Then N4-(3-bromo-7-chloroquinolin-4-yl)-N1,N1-

diethylpentane-1,4-diamine (1 equiv.) dissolved in degassed EtOH/ H2O (7:3 v/v) is added to the

flask via syringe through the septum. The reaction mixture is refluxed at 85℃ under N2 for 3 hours

while stirring vigorously. After the time elapses, the reaction mixture is left to cool down to room

temperature (RT). The reaction mixture is then diluted in H2O and basified with 1 M NaOH. It is

then extracted three times with DCM or EtOAc, and the combined organic phases are washed with

brine and dried over MgSO4. The solvent is then removed by reduced pressure and purified by

prep TLC 50% Hex, 40% EtOAc, 5% methanol (MeOH), 5% NEt3 to give an orange oil 331 mg,

55%. The 1H and 13C NMR was consistent with the literature.5

96
Chapter 2 Optimization of 3-Aminochloroquine synthesis for Chloroquine Derivatization

2.6 References

(1) Surrey, A. R.; Cutler, R. A. The Preparation of 3-Halo-4-Dialkylaminoalkylaminoquinoline

Derivatives. J. Am. Chem. Soc. 1946, 68 (12), 2570–2574.

(2) Kinawi, A.; Dietz, M.; Schmidt, R.; Löwe, W. Kurzmitteilung. Bindung von Razemischen

Chloroquin Sowie Einiger Derivate an Humanserumalbumin. Arch. Pharm. (Weinheim).

1995, 328 (1), 93–94.

(3) Pou, S.; Winter, R. W.; Nilsen, A.; Kelly, J. X.; Li, Y.; Doggett, J. S.; Riscoe, E. W.;

Wegmann, K. W.; Hinrichs, D. J.; Riscoe, M. K. Sontochin as a Guide to the Development

of Drugs against Chloroquine-Resistant Malaria. Antimicrob. Agents Chemother. 2012, 56

(7), 3475–3480.

(4) Bass, G. E.; Hudson, D. R.; Parker, J. E.; Purcell, W. P. Mechanism of Antimalarial Activity

of Chloroquine Analogs from Quantitative Structure-Activity Studies. Free Energy Related

Model. J. Med. Chem. 1971, 14 (4), 275–283.

(5) Konstantinović, J.; Videnović, M.; Orsini, S.; Bogojević, K.; D’Alessandro, S.;

Scaccabarozzi, D.; Terzić Jovanović, N.; Gradoni, L.; Basilico, N.; Šolaja, B. A. Novel

Aminoquinoline Derivatives Significantly Reduce Parasite Load in Leishmania Infantum

Infected Mice. ACS Med. Chem. Lett. 2018, 9 (7), 629–634.

(6) van Langevelde, A.; Bakker, C. N. M.; Boer, H.; Ilmer, T. A. M.; Journée-de Korver, J. A.;

Kaspersen, F. M.; Noach, E. L.; Oosterhuis, J. A.; Pauwels, E. K. J. Potential

Radiopharmaceuticals for the Detection of Ocular Melanoma - Part II. Iodoquinoline

Derivatives and 67Ga-Citrate. Eur. J. Nucl. Med. 1986, 12 (2), 96–104.

(7) Edaye, S.; Tazoo, D.; Bohle, D. S.; Georges, E. 3-Iodo-4-Aminoquinoline Derivative

Sensitises Resistant Strains of Plasmodium Falciparum to Chloroquine. Int. J. Antimicrob.

97
Chapter 2 Optimization of 3-Aminochloroquine synthesis for Chloroquine Derivatization

Agents 2016, 47 (6), 482–485.

(8) Edaye, S.; Tazoo, D.; Bohle, D. S.; Georges, E. 3-Halo Chloroquine Derivatives Overcome

Plasmodium Falciparum Chloroquine Resistance Transporter-Mediated Drug Resistance in

P. Falciparum. Antimicrob. Agents Chemother. 2015, 59 (12), 7891–7893.

(9) Kaur, K.; Jain, M.; Reddy, R. P.; Jain, R. Quinolines and Structurally Related Heterocycles

as Antimalarials. Eur. J. Med. Chem. 2010, 45 (8), 3245–3264.

(10) Coatney, G. R. Pitfalls in a Discovery: The Chronicle of Chloroquine *. Am. J. Trop. Med.

Hyg. 1963, 12 (2), 121–128.

(11) Okombo, J.; Chibale, K. Recent Updates in the Discovery and Development of Novel

Antimalarial Drug Candidates. Medchemcomm 2018, 9 (3), 437–453.

(12) Schlitzer, M. Malaria Chemotherapeutics Part I: History of Antimalarial Drug

Development, Currently Used Therapeutics, and Drugs in Clinical Development.

ChemMedChem 2007, 2 (7), 944–986.

(13) Kaschula, C. H.; Egan, T. J.; Hunter, R.; Basilico, N.; Parapini, S.; Taramelli, D.; Pasini,

E.; Monti, D. Structure−Activity Relationships in 4-Aminoquinoline Antiplasmodials. The

Role of the Group at the 7-Position. J. Med. Chem. 2002, 45 (16), 3531–3539.

(14) Egan, T. J. Structure-Function Relationships in Chloroquine and Related 4-Aminoquinoline

Antimalarials. Mini-Reviews Med. Chem. 2001, 1 (1), 113–123.

(15) Egan, T. J.; Hunter, R.; Kaschula, C. H.; Marques, H. M.; Misplon, A.; Walden, J.

Structure−Function Relationships in Aminoquinolines: Effect of Amino and Chloro Groups

on Quinoline−Hematin Complex Formation, Inhibition of β-Hematin Formation, and

Antiplasmodial Activity. J. Med. Chem. 2000, 43 (2), 283–291.

(16) Natarajan, J. K.; Alumasa, J. N.; Yearick, K.; Ekoue-Kovi, K. A.; Casabianca, L. B.; De

98
Chapter 2 Optimization of 3-Aminochloroquine synthesis for Chloroquine Derivatization

Dios, A. C.; Wolf, C.; Roepe, P. D. 4-N-, 4-S-, and 4-O-Chloroquine Analogues: Influence

of Side Chain Length and Quinolyl Nitrogen PKa on Activity vs Chloroquine Resistant

Malaria. J. Med. Chem. 2008, 51 (12), 3466–3479.

(17) Ribas, X.; Güell, I. Cu(I)/Cu(III) Catalytic Cycle Involved in Ullmann-Type Cross-

Coupling Reactions. Pure Appl. Chem. 2014, 86 (3), 345–360.

(18) Sambiagio, C.; Marsden, S. P.; Blacker, A. J.; McGowan, P. C. Copper Catalysed Ullmann

Type Chemistry: From Mechanistic Aspects to Modern Development. Chem. Soc. Rev.

2014, 43 (10), 3525–3550.

(19) Terzić, N.; Konstantinović, J.; Tot, M.; Burojević, J.; Djurković-Djaković, O.; Srbljanović,

J.; Štajner, T.; Verbić, T.; Zlatović, M.; Machado, M.; Albuquerque, I. S.; Prudêncio, M.;

Sciotti, R. J.; Pecic, S.; D’Alessandro, S.; Taramelli, D.; Šolaja, B. A. Reinvestigating Old

Pharmacophores: Are 4-Aminoquinolines and Tetraoxanes Potential Two-Stage

Antimalarials? J. Med. Chem. 2016, 59 (1), 264–281.

(20) Jiao, J.; Zhang, X. R.; Chang, N. H.; Wang, J.; Wei, J. F.; Shi, X. Y.; Chen, Z. G. A Facile

and Practical Copper Powder-Catalyzed, Organic Solvent-and Ligand-Free Ullmann

Amination of Aryl Halides. J. Org. Chem. 2011, 76 (4), 1180–1183.

(21) Lang, F.; Zewge, D.; Houpis, I. N.; Volante, R. . Amination of Aryl Halides Using Copper

Catalysis. Tetrahedron Lett. 2001, 42 (19), 3251–3254.

(22) Yang, B.; Liao, L.; Zeng, Y.; Zhu, X.; Wan, Y. A Simple and Recyclable Copper/DTPA

Catalyst System for Amination of Aryl Halides with Aqueous Ammonia in Water. Catal.

Commun. 2014, 45, 100–103.

(23) Maejima, T.; Shimoda, Y.; Nozaki, K.; Mori, S.; Sawama, Y.; Monguchi, Y.; Sajiki, H.

One-Pot Aromatic Amination Based on Carbon–Nitrogen Coupling Reaction between Aryl

99
Chapter 2 Optimization of 3-Aminochloroquine synthesis for Chloroquine Derivatization

Halides and Azido Compounds. Tetrahedron 2012, 68 (6), 1712–1722.

(24) Dunn, A. D. A Facile Synthesis Of 3-Amino-1(2)H-Pyrazolo[3,4-C]Pyridine. Org. Prep.

Proced. Int. 1997, 29 (5), 577–579.

(25) Peterson, J. R.; Zjawiony, J. K.; Liu, S.; Hufford, C. D.; Clark, A. M.; Rogers, R. D.

Copyrine Alkaloids: Synthesis, Spectroscopic Characterization, and

Antimycotic/Antimycobacterial Activity of A- and B-Ring-Functionalized Sampangines. J.

Med. Chem. 1992, 35 (22), 4069–4077.

(26) Liu, Y.; Xiao, Q.; Liu, Y.; Li, Z.; Qiu, Y.; Zhou, G.-B.; Yao, Z.-J.; Jiang, S. Biological

Evaluation of New Mimetics of Annonaceous Acetogenins: Alteration of Right Scaffold by

Click Linkage with Aromatic Functionalities. Eur. J. Med. Chem. 2014, 78, 248–258.

(27) Goriya, Y.; Ramana, C. V. The [Cu]-Catalyzed SNAR Reactions: Direct Amination of

Electron Deficient Aryl Halides with Sodium Azide and the Synthesis of Arylthioethers

under Cu(II)–Ascorbate Redox System. Tetrahedron 2010, 66 (38), 7642–7650.

(28) Altman, R. A.; Buchwald, S. L. Cu-Catalyzed Goldberg and Ullmann Reactions of Aryl

Halides Using Chelating N- and O-Based Ligands. Nat. Protoc. 2007, 2 (10), 2474–2479.

(29) Markiewicz, J. T.; Wiest, O.; Helquist, P. Synthesis of Primary Aryl Amines Through a

Copper-Assisted Aromatic Substitution Reaction with Sodium Azide. J. Org. Chem. 2010,

75 (14), 4887–4890.

(30) Lanke, S. R.; Bhanage, B. M. Copper Bis(2,2,6,6-Tetramethyl-3,5-Heptanedionate)–

Catalyzed Coupling of Sodium Azide with Aryl Iodides/Boronic Acids to Aryl Azides or

Aryl Amines. Synth. Commun. 2014, 44 (3), 399–407.

(31) Sklorz, J. A. W.; Hoof, S.; Rades, N.; De Rycke, N.; Könczöl, L.; Szieberth, D.; Weber, M.;

Wiecko, J.; Nyulászi, L.; Hissler, M.; Müller, C. Pyridyl-Functionalised 3 H -1,2,3,4-

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Chapter 2 Optimization of 3-Aminochloroquine synthesis for Chloroquine Derivatization

Triazaphospholes: Synthesis, Coordination Chemistry and Photophysical Properties of

Low-Coordinate Phosphorus Compounds. Chem. - A Eur. J. 2015, 21 (31), 11096–11109.

(32) Messaoudi, S.; Brion, J.-D.; Alami, M. An Expeditious Copper-Catalyzed Access to 3-

Aminoquinolinones, 3-Aminocoumarins and Anilines Using Sodium Azide. Adv. Synth.

Catal. 2010, 352 (10), 1677–1687.

(33) Sung, S.; Braddock, D. C.; Armstrong, A.; Brennan, C.; Sale, D.; White, A. J. P.; Davies,

R. P. Synthesis, Characterisation and Reactivity of Copper(I) Amide Complexes and

Studies on Their Role in the Modified Ullmann Amination Reaction. Chem. - A Eur. J.

2015, 21 (19), 7179–7192.

(34) Sperotto, E.; Van Klink, G. P. M.; Van Koten, G.; De Vries, J. G. The Mechanism of the

Modified Ullmann Reaction. Dalt. Trans. 2010, 39 (43), 10338–10351.

(35) Cohen, T.; Cristea, I. Kinetics and Mechanism of the Copper(I)-Induced Homogeneous

Ullmann Coupling of o-Bromonitrobenzene. J. Am. Chem. Soc. 1976, 98 (3), 748–753.

(36) Kim, J. K.; Bunnett, J. F. Evidence for a Radical Mechanism of Aromatic “Nucleophilic”

Substitution. J. Am. Chem. Soc. 1970, 92 (25), 7463–7464.

(37) Weingarten, H. Mechanism of the Ullmann Condensation. J. Org. Chem. 1964, 29 (12),

3624–3626.

(38) Sinha, M.; Dola, V. R.; Soni, A.; Agarwal, P.; Srivastava, K.; Haq, W.; Puri, S. K.; Katti,

S. B. Synthesis of Chiral Chloroquine and Its Analogues as Antimalarial Agents. Bioorg.

Med. Chem. 2014, 22 (21), 5950–5960.

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Synthesis of Novel Substituted Chloroquine Derivatives

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Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

3.1 Preamble

Following the optimization of 3-NH2CQ synthesis, we wanted to derivatize the 3-position further.

The installed arylamine serves as a precursor for various derivatives such as secondary amines,

benzamides, sulfonamides, triazoles, and Schiff-bases. We were interested in the synthesis of such

derivatives as possible combination therapy partners for CQ. Additionally, b-hematin may not be

their only target. 3-substituted CQs may target other stages of the parasite's life cycle. As we know,

8-AQs such as PQ are active against liver-stage hypnozoites. We discovered that 3-haloCQs when

used in combination with CQ, resensitize resistance strains of the parasite to CQ. Furthermore, the

potencies of 3-haloCQ are unaffected by VP, which suggests that they are not substrates for mutant

PfCRT. This was an exciting discovery that inspired us to create a library of novel 3-substituted

CQs. We are interested in their drug targets and their ability to resensitize resistant parasites to

CQ. Artemisinin combination therapies (ACTs) are the recommended treatments for malaria,

which are beginning to fail in Asian countries. Insights into new combination therapies, especially

ones that utilize a cheap drug, would be very valuable.

3.2 Introduction

As illustrated in chapter 1, the 4-aminoquinolines are the most studied of all the classes of

antimalarial drugs. Although the quinoline structure has been extensively modified, it still has

great potential, as seen with antimalarial drug candidates FQ and AQ-13. Both antimalarial

candidates were in phase II clinical trials but have not yet advanced to phase III.

Previous results from our group included the synthesis of 3-halo CQs: 3-ClCQ, 3-BrCQ and 3-

ICQ.1 Their preparation involves activation of N-halosuccinimide with trifluoroacetic acid in

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Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

acetonitrile at 80oC for 30 minutes, followed by CQ addition. The reaction mixture is then stirred

at 80oC for 2 hours. Yields ranged between 73% - 81%. In vitro assays were carried out and it was

determined that each derivative is equally potent against CQS and CQR P. falciparum strains;

however, they were ~8.5 - fold less potent than CQ.1 These results are in-line with previous

structure-activity relationship (SAR) reports which showed that modifications to the quinoline ring

resulted in decreased antiplasmodial activity.2,3 In CQS strain 3D7, 3-haloCQ had a 19 - fold

decrease in potency compared to CQ. The IC50 values range between 367 nM - 747 nM in CQS

3D7 strain and 623 nM – 1163 nM in CQR strain Dd2, Scheme 3.1. Of the three derivatives

synthesized, 3-ICQ was the most potent with IC50 values of 367 nM and 623 nM in 3D7 and Dd2,

respectively.4

Et2N Trifluoro- Et2N

NH O acetic acid, NH
X
N Acetonitrile X

a) O 80oC, 2h
Cl N Cl N X= Cl, Br, I
Chloroquine 3-halo chloroquine

O
N O
b)
O N O

Verapamil

Scheme 3.1. a) Halochloroquine synthesis, b) structure of verapamil.

Furthermore, a b-hematin inhibition assay was carried out to determine 3-ICQ's ability to inhibit

b-hematin formation, and it was found that 3-ICQ does inhibit b-hematin formation but to a lesser

extent than CQ. This is possibly due to the presence of the electron-withdrawing iodine that lowers

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Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

the pKa of the quinolinium nitrogen (Table 3.3) and may reduce the compound's affinity to

Fe(III)PIX. These results are not surprising or unexpected as substituents on the quinoline ring

affect the drug's ability to interact with heme.5 A large, electron-withdrawing group such as iodine

can reduce affinity for heme.

Verapamil (VP), Scheme 3.1, is a chemosensitizer that presumably inhibits PfCRT allowing CQ

to remain in its active site and exert its potency. When resistant parasites are treated with 3-ICQ

and VP, there was no improvement of activity, suggesting that 3-ICQ may not be a substrate for

mutant PfCRT, as its activity is independent of the presence of VP. A surprising and significant

finding of these results is that combination treatment of CQ and 3-ICQ was more potent than either

drug alone against CQR parasites. This chemosensitization effect suggests that 3-ICQ may act like

VP to resensitize the parasite to CQ.4

With these promising results in hand, we wanted to explore the functionality of the 3-position

further. In this chapter, we describe the synthesis of CQ derivative, their antiplasmodial activity

and their structure-activity relationships. We will also describe pKa studies carried out to determine

the effect of substituents on the quinolinium pKa.

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Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

3.3 Results and Discussion

3.3.1 3-CQ derivatives

Previous work in our lab demonstrated the chemosensitization effect that 3-ICQ had on CQ.4 It

was evident that 3-ICQ sensitizes CQR parasites to CQ, suggesting it may not be a substrate for

mutant PfCRT. The possibility of synthesizing drugs for combination therapy was attractive as

that is the basis of ACT. We wanted to further explore modifications at the 3rd position of the

quinoline to find other drug candidates that modulate CQ activity.

We began with the functionalization of the 3rd position of the quinoline, with the installation of an

amine functional group (discussed in chapter 2), Scheme 3.2. With the amine installed, we were

then able to carry out the final step of the synthesis. We explored three classes of derivatives:

amides, sulfonamides and Schiff-bases. The formation of amides was straightforward as benzoyl

chlorides are widely available and relatively inexpensive, Figure 3.2. Amides add rigidity to the

molecule as well as the introduction of an H-bond acceptor. The resulting derivatives were then

tested for their antiplasmodial activity in vitro.

While designing the new CQ-derivatives, we used Lipinski's rule of 5 as a guide to possibly make

the compounds more drug-like for oral administration.6,7 The rules state that a drug should: i) have

a molar mass of less than 500 g/mol, ii) have a partition coefficient of less than 5, iii) have no more

than 5 H-bond donors and iv) have no more than 10 H-bond acceptors. As a starting point, we

began with the synthesis of benzamides and used the Craig Plot 8,9 as a guide to determine which

substituents to place on the ring, Figure 3.1.

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Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

+s - p +s +p

-s -p -s +p

Figure 3.1. Craig Plot groups substituents with similar electronic and hydrophobic effects.

Highlighted are synthesized compounds containing substituents at various locations on the phenyl

ring.

The Craig Plot groups various substituents based on their electronic and hydrophobic properties

using benzoic acid as the reference point, the center of the graph. This helps identify simple

changes to make on a compound to possibly increase activity and/or lipophilicity. We then

experimented with substitution patterns on the benzoyl ring to get a complete SAR profile.

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Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

Et2N Cu(I)I, NaN3 Et2N

NH DMEDA, NH
Br Sodium ascorbate NH2
EtOH/H2O
85oC
Cl N Cl N
55%
3.1 3.2

Scheme 3.2. Amination with 3-BromoCQ as a precursor.

Our collaborators, Dr. Fadi Baakdah, supervised by Dr. Elias Georges at the McGill University

Institute of Parasitology, carried out in vitro antiplasmodial assays to identify which substituents

showed the greatest potency, the results are displayed in Table 3.1.

Et2N
NH
H
N R

O
Cl N

H CN NO2 Cl F

3.3 3.4 3.5 2 Cl: 3.6 2 F: 3.9

3 Cl: 3.7 3 F: 3.10
4 Cl: 3.8 4 F: 3.11

NH2 OMe SMe NMe2 Me

3.12 3.13 3.14 3.15 3.16

Figure 3.2. Synthesized compounds. Colours: grey: the reference compound, blue: electron-

withdrawing and hydrophilic, orange: electron-withdrawing and hydrophobic, purple: electron-

donating and hydrophilic, green: electron-donating and hydrophobic.

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Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

Table 3.1. Antiplasmodial activities of CQ derivatives in sensitive and resistant parasitic strains

IC50 (nM) CQS IC50 (nM) CQR

Entry R % Yield
3D7 Dd2

1 H 85 50.5 ± 2.3 1,192.0 ± 232.0

2 4-NH2 41 10.4 ± 0.8 1,564.0 ± 53.0
3 4-Me 59 265.1 ± 10.1 564.4 ± 11.4
4 4-OMe 75 114.2 ± 18.6 963.0 ± 8.5
5 4-SMe 61 79.5 ± 9.1 205.5 ± 21.6
6 4-NMe2 31 280.4 ± 25.4 619.9 ± 89.0
7 4-CN 66 391.3 ± 37.2 808.4 ± 85.0
8 4-NO2 76 278.6 ± 6.0 870.9 ± 99.8
9 2-F 66 502.9 ± 19.9 1,010.0 ± 11.8
10 3-F 64 255.1 ± 2.0 733.9 ±79.0
11 4-F 60 8.7 ± 0.4 667.0 ± 60.4
12 2-Cl 59 69.8 ± 8.3 918.0 ± 3.0
13 3-Cl 61 84.0 ± 9.7 497.2 ± 36.7
14 4-Cl 51 7.0 ± 0.4 311.1 ± 63.5
a
15 11 266.2 ± 33.5 1,007.0 ± 457.0
b
16 17 105.8 ± 12.2 404.0 ± 42.7
3-NH2CQ - - 297.0 ± 52.0 720.0 ± 107.0
3-ICQ - - 367.0 ± 24 623.5 ± 72.5
CQ - - 20.5 ± 4.5 169.9 ± 16.6
a) 4-Cl sulfone derivative b) 4-Cl Schiff-base derivative, Figure 3.8

The vast majority of the synthesized drug candidates show some antiplasmodial activity; however,

most are less potent than CQ in CQS 3D7 strain and CQR Dd2 strain. The benzamido-analog, 3.3,

was 2.5 - fold less potent than CQ in CQS 3D7 strains, and was 7 - fold less potent than CQ in

CQR Dd2 strains. We believe this decrease in activity is due to decreased affinity to heme, similar

to 3-ICQ, which is correlated to antiplasmodial activity.5 For the Dd2 strain, the mutations might

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Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

cause reduced accumulation of the benzamidoCQ in the DV, it was reported that accumulation is

correlated to antiplasmodial activity.10

Et2N R Et2N
NH NH H
H
N N
O
O
Cl N Cl N
1 Et2N
2
R

Et2N O O
NH R HN R
N N
H H
Cl N 3 Cl N 4

Figure 3.3. Possible benzamidoCQ conformations.

Halogen substituents such as fluoride and chloride had varying activity depending on their position

on the benzoyl ring. We discovered that the para-position gave the best activity for both halogens,

3.8 and 3.11. They were more potent than the benzamidoCQ compound; 7.2 and 5.8 - fold better

in CQS strain than benzamidoCQ for chloro- and fluoro-respectively. The activity at the meta-

position, 3.7 and 3.10, was in between that of the ortho- and para- positions. The ortho-position

gave the worst activity for both substituents, 3.6 and 3.9, with the chloro- being more potent in

CQS strain. They were more potent than benzamidoCQ in CQR strains but not as effective on CQS

strains. Overall, the para-chloro-substituent, 3.8, was the most potent.

Electron-withdrawing, hydrophobic nitro-substituent, 3.5, was 5.5 - fold less potent than the

reference compound in CQS strain and was 1.4 – fold more potent than the reference compound

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Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

in CQR strain. The decrease in activity in CQS strain is likely due to an increase in the size of the

moiety. Furthermore, the rigidity of this substituent might play a role in its reduced activity.

The addition of an electron-withdrawing, hydrophilic cyano group, 3.4, was 7.7 - fold weaker in

CQS strain and 1.5 - fold better in CQR than the benzamidoCQ. The decrease in activity in 3D7

strains might be due to the addition of an H-bond acceptor. The lack of flexibility at the 4th position

might reduce the compound's ability to enter the target / active site. In Dd2 strains, the activity is

essentially the same; thus, the reduced activity compared to CQ might be due to decreased

accumulation.

The electron-donating, hydrophilic amino group, 3.12, was 5 - fold more potent than

benzamidoCQ in CQS but 1.3 – fold weaker in the CQR parasite. In contrast, the methoxy, 3.13,

was 2 - fold weaker in CQS and 1.2 – fold stronger in the CQR parasite than the benzamidoCQ.

The addition of an H-bond donor NH2 group significantly improves the antiplasmodial activity in

CQS strains. This increase in activity might be due to a slight increase in accumulation as a small

percentage of the amine will be protonated at lysosomal pH. Furthermore, the ability to form strong

hydrogen bonds might further stabilize the compound.11 In contrast, the methoxy derivative's

reduced potency might result from reduced H-bonding ability and increased size of the methoxy

in CQS strains. In resistant strains, however, the H-bonding ability of the amino group is a

disadvantage, this is a trend in all our compounds. Therefore, we observe that the methoxy group,

which does not H-bond as strongly as the amine, in CQR strains, is 1.6 - fold more potent than the

amine counterpart.

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Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

We found that electron-donating, hydrophobic methyl, 3.16, and dimethylamino-substituents,

3.15, have similar activity in CQS and CQR strains. They were 5 - fold less potent than

benzamidoCQ, 3.3, in CQS parasites but ~2.3 - fold more potent in resistant parasites. However,

the methylthio-substituent, 3.14, was 1.6 – fold less potent than benzamidoCQ in CQS strains but

was 5.8 - fold more potent in CQR strains. Although these substituents belong within the same

quadrant, it is important to note that there is a difference in electronegativity and steric bulk. The

methylthio-substituent is 3.4 - fold more potent than the methyl and dimethylamino-substituent in

CQS strains. The methylthio-derivative is also 3 - fold more potent than these derivatives in CQR

strains. This might be due to the favorable electronics and size of the methylthio over the simple

methyl and H-bond accepting dimethylamino-substituents.

F
Et2N O O
Et2N F Et2N
NH NH NH
H
N N N
H H
O F
Cl N 1 Cl N 2 Cl N 3

F
O F Et2N Et2N O
Et2N NH
NH H NH
N N N F
H H
O
Cl N 4 Cl N Cl N 6
5

Figure 3.4. Possible fluorobenzamidoCQ conformations.

When looking at the possible configurations to explain differences in activity, one can see how

electronic effects and steric hindrance may impact activity (see Figure 3.3 to Figure 3.5). The

ortho-position which showed the worst antiplasmodial activity exhibits lone pair / lone pair

repulsion with the carbonyl oxygen. These forces may be unfavourable for activity. However,

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Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

these conformers also possibly exhibit intramolecular H-bonding, which could constrain the steric

effects. However, the repulsion might be stronger than H-bonding stabilization. The meta-position

contains conformers that possibly exhibit intramolecular H-bonding. This reduced repulsion might

contribute to an increase of antiplasmodial activity in both CQS and CQR strains. The para-fluoro-

substituent exhibits the least steric effects with the possible H-bond of the carbonyl and secondary

amine, further stabilizing the compound and contributing to increased activity in CQS strains.

However, it was clear that these factors do not enhance activity in CQR strains. As mentioned

above, H-bonding ability is disadvantageous in resistant strains.

Cl
Et2N O O
Et2N Cl Et2N
NH NH NH
H
N N N
H H
O Cl
Cl N 1 Cl N 2 Cl N 3

Cl
O Cl Et2N Et2N O
Et2N NH
NH H NH
N N N Cl
H H
O
Cl N 4 Cl N Cl N 6
5

Figure 3.5. Possible chlorobenzamidoCQ conformations.

In contrast, the chloro-substituent shows better antiplasmodial activity across the board in CQS

strains. A likely explanation is the reduced electronegativity of the chloride and longer C - X bond,

which minimizes the " - delocalization. Furthermore, the chloride is unable to H – bond, explaining

its better antiplasmodial activity in CQR strains.

To summarize, in 3D7 strains, the best substituents are chloro, 3.8, fluoro, 3.11, methylthio, 3.14,

and amino 3.12. These are relatively compact or can form strong hydrogen bonds. In the case of

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Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

Dd2 strains, H-bonding substituents are disfavoured. The best substituents are chloro, 3.8, and

methylthio-substituent 3.14. These are relatively small or flexible and do not form H-bonds. The

optimal substituent positioning is the para-position. This position is void of possible steric effects,

which are favourable for activity.

3.3.2 3-chlorobenzamidoCQ not a substrate for PfCRT

To determine whether the synthesized derivatives were substrates for PfCRT, in vitro assays were

carried out in CQS and CQR strains in the presence of VP. VP is believed to act as a

chemosensitizer that works on resistant strains by inhibiting mutant PfCRT's ability to transport

drugs out of the DV. Assays of CQ with VP in Dd2 show a significant increase in activity as

mutant PfCRT is inhibited. When 3-ICQ is assayed with VP in 3D7 and Dd2, no significant

increase in activity is observed. This suggests that 3-ICQ is not a possible substrate for wild-type

or mutant PfCRT.4 Similarly, VP does not modulate 3.16 activity in Dd2 strains. Our next objective

would be to determine whether 3-chlorobenzamidoCQ displays synergistic or antagonistic effects

when used in combination with CQ. Interestingly, VP increases the IC50 value of wild-type PfCRT,

Table 3.2. There is currently no evidence of VP interacting with wild-type PfCRT; furthermore,

there are only a few reports on the speculated normal function(s) of wild-type PfCRT.

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Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

Table 3.2. Effect of verapamil on compound activity.

IC50 CQS (nM) IC50 CQR (nM)

Compound 1 !M verapamil
3D7 Dd2
- 20.5 ± 4.5 169.9 ± 16.6
CQ
+ 16.2 ± 0.6 21.0 ± 4.5
- 367.0 ± 24.0 623.5 ± 72.5
3-ICQ
+ 335.4 ± 46.1 621.2 ± 23.3
3-chloro - 7.0 ± 0.4 311.1 ± 63.5
benzamidoCQ + 146.3 ± 21.5 282.1 ± 50.0

3.3.3 Isomerization

The synthesized derivatives can adapt two main isomeric forms: the cis or trans-isomers, Figure

3.6. These isomers can be easily distinguished in an NMR spectrum as major or minor contributors.

However, we found that all of the compounds were isolated as one isomer at RT, according to the
1
H NMR. As the rotational barrier of rotamers is high, it was assumed that they were isolated as

one rotamer. The expected major isomer is the trans-amide because it exhibits the least steric

effects. This is confirmed as the NOESY NMR does not show evidence of there being the E isomer.

We do not observe the expected 2 - H / phenyl interaction associated with the E isomer.

Et2N R Et2N
NH NH
H H
N N O

O
Cl N Cl N
trans-amide cis-amide
R

Not observed on NOESY

Figure 3.6. Trans and Cis benzamidoCQ isomers.

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Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

3.3.4 Fluoride coupling in 1H NMR

O
d
N c
H
b
c
d F
a
a 3
b

O
h
N g
H
e f

e,h F
f 2
g

O
i
N j
H
F j
i 1

8.25 8.20 8.15 8.10 8.05 8.00 7.95 7.90 7.85 7.80 7.75 7.70 7.65 7.60 7.55 7.50 7.45 7.40 7.35 7.30 7.25 7.20 7.15
f1 (ppm)

Figure 3.7. Fluorine and hydrogen coupling.

For the fluoride-containing derivatives, 1H NMR analysis was carried out with H-F coupled mode.

Evident in the spectra, Figure 3.7, is the extend of fluoride coupling on the benzoyl ring. We

observe up to 4J coupling in all derivatives, as evidenced by the splitting patterns. The coupling

constant for proton i, a triplet is 6.5 Hz, while the coupling constant for proton j, a triplet is 8.4

Hz. Similarly, it is evident the extent of proton-fluorine coupling in proton b, which is a triplet of

double of doublets (tdd) where J = 1.9, 5.3 and 7.2 Hz and proton d, which is a triplet of doublets

(td) where J = 5.6 and 8 Hz. The multiplicity of proton a, is a double of double of doublets (ddd)

pattern with J = 1.1, 3.9 and 8.3 Hz.

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Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

3.3.5 Synthesis of Sulfonamides and Schiff-base derivatives

Et2N Cl Et2N Cl
NH NH
H
N N
S
O2
Cl N Cl N
3.17 3.18

Figure 3.8. Structure of sulfonamide and Schiff-base derivatives

Once we discovered that the 4-chlorobenzamidoCQ, 3.8, was the most potent compound, we

synthesized sulfonyl, 3.17, and Schiff-base, 3.18, equivalents to determine the effect of the

carbonyl group on activity, as shown in Figure 3.8. We found that the addition of the sulfone has

a negative impact on the activity of the compound. This might be due to the increased mass by 36

g/mol, which breaks the Lipinski rule that states a drug should have a molar mass less than 500

g/mol and the introduction of an additional H-bond acceptor, Figure 3.9. This further reinforces

the notion that H-bonding groups are disfavoured in resistant strains. Furthermore, the sulfone

group might sterically hinder localization in the active site. The Schiff-base was 15 - fold less

potent in the 3D7 strain and 1.3 - fold less potent in the Dd2 strain than the 4-chlorobenzamide.

The decrease in activity in the sensitive strain might indicate the need for some electron density in

the active site. Therefore, the electronegativity of the carbonyl is beneficial to activity. In Dd2, the

activity is similar, further reinforcing that H-bonding is not beneficial in mutant parasites.

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Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

Et2N Et2N R
NH NH
H O H
N N
S R S
O O O
Cl N Cl N
1 2
R

Et2N O O Et2N O
NH S NH S O
N R N
H H
Cl N Cl N
3 4

Figure 3.9. Possible sulfonamide conformations.

Due to the size of these compounds and the positioning of the modifications, b-hematin inhibition

assays (BHIA) were not carried out. It is known that modification on the quinoline ring reduces

the affinity of heme - CQ complex formation. These compounds are not expected to be able to

prevent b-hematin formation to any significant extent. For this reason, BHIA was not performed.

Furthermore, although the current widely accepted mechanism of action for 4-aminoquinolines is

b-hematin inhibition, it may not be the only way these compounds exert their activity.12–16

Although these derivatives may not be as potent as CQ, their activity may be linked to a different

mechanism. For this reason, the next step of this project would be to carry out assays in which

parasites are given both CQ and our derivatives to determine if they behave as 3-ICQ in a

synergistic manner.

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Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

3.3.6 pKa studies

CQ is a weak base that can accumulate within its target site via the protonation of its tertiary alkyl

nitrogen and quinoline nitrogen.17 At pH 7.4, CQ is 17% mono-protonated and still lipid-soluble

and can cross cell membranes. At lysosomal pH of 5.2, it is entirely diprotonated and no longer

membrane permeable.15 Thus, the pKas of the molecule plays a crucial role in its localization,

Table 3.3.

Table 3.3. Antiplasmodial activity and pKa of quinolinium nitrogen.

Drug IC50 (3D7) nM IC50 (Dd2) nM Quinolinium pKa

CQ 20.5 ± 4.5 169.9 ± 16.6 8.2

3-ChloroCQ 747.2 ± 65.0 1163.3 ± 46.0 5.3
3-BromoCQ 712.4 ± 89.6 987.8 ± 48.5 5.4
3-IodoCQ 367.0 ± 24 623.5 ± 72.5 5.5
NH2-CQ 297.0 ± 52.0 720.0 ± 107.0 6.3

The presence of the electron-withdrawing halogens at the 3-position has a significant effect on the

quinolinium pKa. 3-halo CQs are 2 - 3 log units lower than CQ; therefore, they are not expected

to be extensively diprotonated at physiological or lysosomal pH. Halogen substitution increases

the overall lipophilicity of the molecule, which may promote membrane permeability; however,

due to the decreased pKa, it is not expected to become acid trapped within the DV as seen with

CQ. This lack of accumulation in the target site may contribute to the observed decreased

antiplasmodial activity. Furthermore, acid trapping is not believed to be the only method used for

accumulation within the DV.2 Unsurprisingly, the presence of an electron-donating amine

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Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

substituent does not lower the quinolinium pKa as much as the halogens. This log unit difference

in pKa means a greater portion of 3-NH2CQ will be protonated at physiological and lysosomal pH.

Interestingly, although the pKa is higher for 3-NH2-CQ, the IC50 values are similar to 3-ICQ in

CQS and CQR strains; this might suggest a similar mode of action for 3-substituted CQ's.

3.4 Conclusion

CQ has not been extensively modified at the 3-position in prior studies. A possible reason for this

is the reduced antiplasmodial activity resulting from the lowered pKa of the quinolinium nitrogen

as a result of such a modification. However, we have observed a resensitization effect of the

parasite to CQ when 3-substituted CQs derivatives are used in combination with CQ. We were

able to further derivatize 3-substituted CQs via the installation of an amine. A library of derivatives

was successfully synthesized represented across in the Craig Plot. Derivatives contain varying

electronic and hydrophobic properties, which allow us to compare the effectiveness of substituents

and the significance of regioselectivity. We determined that the hydrophilic and electron-

withdrawing chloro-substituent was the most effective; furthermore, the para-position gives the

best activity. Although the derivatives were active towards the parasite, they were on average 8 –

fold less effective than CQ in sensitive strains and 6 – fold less effective than CQ in resistant

strains. A sulfone and Schiff-base derivative were also synthesized; however, they were less potent

than 4-chlorobenzamidCQ.

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Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

3.5 Experimental

3.5.1 General information

Glassware was taken directly from the oven (120oC) and let cool in a desiccator before use. All

reactions were carried out under N2 pressure. Anhydrous DCM was obtained from MBraun MB-

SPS solvent purification system into an oven-dried flask on the day of use. All solvents and

reagents were obtained commercially and used without further purification unless noted.

High-Resolution Mass Spectroscopy (HRMS) was obtained by positive/negative ESI, or

positive/negative APCI on a Bruker Maxis Impact QTOF, or a Thermo Scientific ExactivePlus

Orbitrap, with results reported as mass/charge ratios (m/z). Thin-layer chromatography plates were

visualized using ultra-violet light, 254nm. Flash column chromatography was carried out manually

using 230 - 400 mesh silica gel (Silicycle) using reagent grade solvents or using Biotage Isolera

One system with Biotage ZIP 30 g columns. Cole Parmer Microcomputer pH-Vision Model

05669-20 pH Meter used with Sigma-Aldrich micro pH combination electrode, glass body. The

UV-Vis spectra were measured on Agilent 8453.

1 13
H and C NMR spectra were recorded at ambient temperature on Bruker AVIIIHD 400 MHz

(1H 400 MHz, 13C 100 MHz) and Bruker AVIIIHD 500 MHz (1H 500 MHz, 13C 125 MHz) using

tetramethylsilane as the internal standard. Chemical shifts are reported relative to the residual

deuterated solvent peaks. Chemical shifts are expressed in parts per million (ppm = δ) values and

coupling constants (J) in Hertz (Hz). The terms m, s, d, t and q represent multiplicities of 1H NMR

resonances: multiplet, singlet, doublet, triplet and quartet, respectively. For previously unknown

compounds, a combination of 2D experiments (COSY, HSQC, HMBC) was often used to

121
Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

complete the assignment of 1H and 13C signals. 1H NMR signals are described by chemical shift δ
13
(multiplicity, J (Hz), integration). C NMR signals are described by chemical shift δ and are

singlets unless otherwise specified.

3.5.2 pKa Determination

A 0.01M stock solution of XH3PO4.haloCQ was made in a 1 mL volumetric flask. In a 4 mL

cuvette, 1,990 µL of deionized H2O was added, and into this cuvette was added 10 µL of the 0.01

M stock solution to make a final concentration of 50 µM XH3PO4.haloCQ and a total volume in

the cuvette of 2,000 µL. The pH of the solution was measured on a calibrated pH meter. The pH

should be acidic. Base (NaOH) will be added to the cuvette to increase the pH. Solutions of 10mM

and 1mM NaOH and HCl were made. 5-20 µL of NaOH was added to increase the pH by 0.2 units.

If the pH increases were too large, HCl was added to lower the pH. After each addition of NaOH,

the solution was well mixed, the pH probe was fully submerged into the cuvette, and the pH was

measured. The pH, incremental volume increase and absorbance at the lmax were recorded. NaOH

was added until a pH > 11/12 was reached or until the Absorbance versus pH graph plateaus. The

graph was adjusted for dilution with the formula:

0)$'$1% 2(%&3- + 5('1% 2(%&3-!"#

#$%&'$() +(,,-.'$()!"# =
0)$'$1% 2(%&3-

The measured absorbance was then adjusted for dilution at pHx with the following formula:

+(,,-.'-6 789(,81).- = :-19&,-6 189(,81).- × #$%&'$() +(,,-.'$()

The pH and corrected absorbances were plotted on a non-linear curve fit graph on Origin Pro or

GraphPad Prism 7. The equation used to fit absorbance values to produce a pKa value is as follows:

1 1
7 = <1 − @ 7'( + < @7 !
1 + 10!"$!%& 1 + 10!"$!%& '(

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Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

Where ACQ is the absorbance of neutral CQ, ACQ+ is the absorbance of protonated CQ. Where

AB1 = 7.35 ± 0.1.

3.5.3 Parasite cytotoxicity assays and IC50 estimation

Parasite proliferation assays using CQ or CQ-derivatives were tested on CQS 3D7 and CQR Dd2

Plasmodium falciparum lab strains. Briefly, 0.5% ring-stage parasites were incubated in a 0.2%

hematocrit in 96 black well clear bottom plates in triplicates (Corning Inc.; ref: 3603) for 72hrs in

an incubator maintaining 37°C and atmospheric conditions of 3% O2, 5% CO2 and 92% N2. Later,

the plates were frozen at -80°C and thawed at RT. Then they were treated with a lysis-developing

solution (Tris; 20 mM pH 7.5, 5 mM EDTA; 0.008% w/v saponin, 0.08% v/v Triton X-100, 0.2

μL /ml of 10,000x SYBR® Green I Nucleic Acid gel stain dye) and kept away from light for 1

hour. Fluorescence was measured using Synergy H4 plate reader with Ex 485 nm / Em 535 nm.

The resulting data were analyzed using GraphPad Prism version 8.4.0 (671) to obtain the 50%

inhibitory concentration (IC50).

3.5.4 Synthesis of 3-substituted CQ Derivatives

To a stirring solution of 3-NH2CQ (1 equiv.) in anhydrous DCM at 0oC is added benzoyl chloride

(1.5 equiv.) under nitrogen followed by NEt3 (3 equiv.). The solution is stirred and allowed to

warm to RT. The reaction progress is monitored by TLC. When complete, the reaction is poured

into saturated NaHCO3 solution and extracted with DCM (3 X 20 mL) and washed with brine. The

combined organic extracts are then dried over anhydrous MgSO4, and the solvent is removed under

reduced pressure. The crude product is purified by prep-TLC with silica gel, solvent system: 90%

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Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

EtOAc, 5% MeOH, 5% NEt3 or with C18 prep-TLC in 50% H2O, 50% acetonitrile, 1%

trifluoroacetic acid (unless otherwise stated) to give an off-white powder.

Et2N
NH
H
N

O
Cl N

N-(7-chloro-4-((5-(diethylamino)pentan-2-yl)amino)quinolin-3-yl)benzamide (3.3)

Yield: 53.3 mg, 85%. 1H NMR (800 MHz, Methanol-d4) δ 8.56 (d, J = 9.2 Hz, 2H), 8.07 (dt, J =

7.1, 1.3 Hz, 2H), 7.94 (d, J = 2.1 Hz, 1H), 7.76 (dd, J = 9.1, 2.1 Hz, 1H), 7.69 (td, J = 7.3, 1.3 Hz,

1H), 7.61 (t, J = 7.8 Hz, 2H), 4.57 (dt, J = 12.4, 7.0 Hz, 1H), 3.10 (tq, J = 12.5, 5.8, 5.1 Hz, 4H),

3.07 – 3.02 (m, 1H), 3.00 – 2.94 (m, 1H), 1.82 (dq, J = 12.1, 7.6, 6.7 Hz, 1H), 1.70 – 1.62 (m, 3H),

1.43 (d, J = 6.5 Hz, 3H), 1.20 (q, J = 6.9 Hz, 6H). 13C NMR (201 MHz, Methanol-d4) δ 169.09,

153.47, 151.04, 145.62, 139.46, 138.61, 132.71, 132.69, 128.77, 127.55, 125.29, 119.60, 117.39,

111.52, 51.57, 51.02, 33.27, 20.79, 20.20, 7.70, 7.64. HRMS (ESI) calcd. for C25H31ClN4O

[M+H]: 439.2259, found: 439.2247.

Et2N CN
NH
H
N

O
Cl N

N-(7-chloro-4-((5-(diethylamino)pentan-2-yl)amino)quinolin-3-yl)-4-cyanobenzamide (3.4)

Yield: 45.6 mg, 66%, mixture of isomers. 1H NMR (500 MHz, Chloroform-d) δ 8.85 (s, 1H), 8.20

(d, J = 7.9 Hz, 2H), 7.99 (dd, J = 3.8, 2.1 Hz, 1H), 7.88 (dd, J = 9.2, 7.5 Hz, 2H), 7.80 (d, J = 8.0

Hz, 2H), 7.42 (ddd, J = 8.5, 6.1, 2.2 Hz, 1H), 4.32 (s, 1H), 3.72 (s, 1H), 2.52 (q, J = 7.1 Hz, 4H),

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Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

2.39-2.35 (m, 2H), 1.60 – 1.40 (m, 4H), 1.24 (d, J = 9.0 Hz, 3H), 0.99 (t, J = 7.2 Hz, 6H). 13C

NMR (126 MHz, Chloroform-d) δ 164.96, 150.39, 150.26, 148.53, 148.15, 145.96, 145.17,

143.09, 137.64, 135.05, 134.73, 132.72, 130.94, 129.70, 129.08, 129.05, 128.57, 127.59, 126.51,

126.47, 124.07, 123.76, 121.50, 121.26, 119.82, 119.01, 118.07, 115.85, 53.62, 53.51, 52.72,

52.60, 46.82, 46.73, 36.71, 23.78, 23.40, 22.40, 22.26, 21.69, 11.19, 10.57. HRMS (ESI) calcd.

for C26H31ClN5O [M+H]: 464.2212, found: 464.2206.

Et2N NO2
NH
H
N

O
Cl N

N-(7-chloro-4-((5-(diethylamino)pentan-2-yl)amino)quinolin-3-yl)-4-nitrobenzamide (3.5)

Yield: 109.5 mg, 76%. 1H NMR (500 MHz, Methanol-d4) δ 8.60 (s, 1H), 8.58 (d, J = 9.3 Hz, 1H),

8.44 (d, J = 8.5 Hz, 2H), 8.29 (d, J = 8.7 Hz, 2H), 7.95 (d, J = 2.1 Hz, 1H), 7.76 (dd, J = 9.2, 1.9

Hz, 1H), 4.65 – 4.53 (m, 1H), 3.14 (q, J = 7.3 Hz, 4H), 3.10 – 3.03 (m, 2H), 1.90 – 1.81 (m, 1H),

1.77 – 1.66 (m, 3H), 1.43 (d, J = 6.5 Hz, 3H), 1.23 (t, J = 7.3 Hz, 6H). 13C NMR (126 MHz,

Methanol-d4) δ 167.24, 153.71, 150.34, 145.10, 139.63, 138.53, 138.33, 129.02, 127.57, 125.62,

123.60, 119.42, 117.04, 111.31, 51.94, 51.09, 46.94, 33.30, 20.81, 20.25, 7.62. HRMS (ESI) calcd.

for C25H30ClN5O3 [M+H]: 484.2110, found: 484.2105.

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Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

Et2N Cl
NH
H
N

O
Cl N

2-chloro-N-(7-chloro-4-((5-(diethylamino)pentan-2-yl)amino)quinolin-3-yl)benzamide (3.6)

Yield: 42 mg, 59%. 1H NMR (500 MHz, Methanol-d4) δ 8.60 (s, 1H), 8.55 (d, J = 9.2 Hz, 1H),

7.95 (s, 1H), 7.79 – 7.70 (m, 2H), 7.63 – 7.48 (m, 3H), 4.73 (q, J = 6.7, 5.9 Hz, 1H), 3.14 (q, J =

7.3 Hz, 4H), 3.10 – 3.02 (m, 2H), 1.93 – 1.84 (m, 1H), 1.77 (dq, J = 12.1, 7.6, 6.0 Hz, 3H), 1.45

(d, J = 6.4 Hz, 3H), 1.22 (q, J = 7.0 Hz, 6H). 13C NMR (126 MHz, Methanol-d4) δ 170.67, 154.26,

147.24, 140.60, 136.11, 133.34, 132.14, 131.59, 130.39, 128.90, 128.66, 126.75, 121.75, 119.26,

52.65, 52.58, 34.84, 22.16, 21.95, 9.08, 8.99. HRMS (ESI) calcd. for C25H31Cl2N4O [M+H]:

473.1869, found: 473.1869.

Cl
Et2N
NH
H
N

O
Cl N

3-chloro-N-(7-chloro-4-((5-(diethylamino)pentan-2-yl)amino)quinolin-3-yl)benzamide (3.7)

Yield: 43.3 mg, 61%. 1H NMR (500 MHz, Chloroform-d) δ 8.87 (s, 1H), 8.10 (s, 1H), 8.01 (d, J

= 8.7 Hz, 1H), 7.91 (d, J = 9.0 Hz, 1H), 7.56 (d, J = 8.1 Hz, 1H), 7.47 (t, J = 7.8 Hz, 1H), 7.42 (d,

J = 9.6 Hz, 1H), 4.31 (d, J = 11.2 Hz, 1H), 3.73 (d, J = 13.1 Hz, 2H), 2.57 (t, J = 7.5 Hz, 5H), 2.42

(d, J = 6.7 Hz, 2H), 1.52 (dt, J = 10.6, 6.8 Hz, 1H), 1.26 (d, J = 7.3 Hz, 7H), 1.02 (t, J = 7.2 Hz,
13
7H). C NMR (126 MHz, Chloroform-d) δ 165.40, 150.70, 148.64, 145.89, 135.46, 135.18,

134.86, 132.37, 130.24, 129.15, 128.37, 126.37, 126.02, 124.30, 121.36, 119.41, 53.58, 52.48,

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Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

46.72, 36.69, 22.84, 22.52, 10.09. HRMS (ESI) calcd. for C25H31Cl2N4O [M+H]: 473.1869, found:

473.1874.

Et2N Cl
NH
H
N

O
Cl N

4-chloro-N-(7-chloro-4-((5-(diethylamino)pentan-2-yl)amino)quinolin-3-yl)benzamide (3.8)

Yield: 36.4 mg, 51%. 1H NMR (400 MHz, Chloroform-d) δ 9.35 (s, 1H), 8.83 (s, 1H), 8.11 (d, J

= 8.2 Hz, 2H), 8.04 – 7.97 (m, 1H), 7.90 (d, J = 9.0 Hz, 1H), 7.48 (dd, J = 8.4, 2.1 Hz, 2H), 7.41

(dd, J = 9.0, 2.0 Hz, 1H), 4.35 (s, 1H), 3.76 (s, 1H), 2.63 (q, J = 7.2 Hz, 4H), 2.47 (t, J = 7.4 Hz,

2H), 1.62 (d, J = 9.3 Hz, 1H), 1.58 – 1.44 (m, 3H), 1.27 (d, J = 6.4 Hz, 3H), 1.05 (t, J = 7.2 Hz,
13
6H). C NMR (126 MHz, Chloroform-d) δ 165.91, 151.05, 148.71, 146.63, 138.64, 134.81,

131.99, 129.66, 129.09, 129.04, 126.22, 124.53, 121.23, 119.38, 53.49, 52.30, 46.58, 36.58, 22.61,

22.30, 9.54. HRMS (ESI) calcd. for C25H31Cl2N4O [M+H]: 473.1869, found: 473.1872.

Et2N
NH
H
N

O F
Cl N

N-(7-chloro-4-((5-(diethylamino)pentan-2-yl)amino)quinolin-3-yl)-2-fluorobenzamide (3.9)

Yield: 45.1 mg, 66%. 1H NMR (500 MHz, Methanol-d4) δ 8.58 (d, J = 9.2 Hz, 1H), 8.56 (s, 1H),

7.99 – 7.91 (m, 2H), 7.73 (dd, J = 9.2, 1.7 Hz, 1H), 7.68 (tdd, J = 7.4, 5.1, 1.8 Hz, 1H), 7.41 (t, J

= 7.6 Hz, 1H), 7.36 (dd, J = 11.3, 8.4 Hz, 1H), 4.71 (h, J = 6.9, 6.0 Hz, 1H), 3.13 (q, J = 7.3 Hz,

4H), 3.09 – 3.00 (m, 2H), 1.87 (dt, J = 13.5, 8.2 Hz, 1H), 1.73 (dq, J = 12.4, 7.8, 6.1 Hz, 3H), 1.43

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Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

(d, J = 6.5 Hz, 3H), 1.22 (t, J = 7.3 Hz, 6H). 13C NMR (126 MHz, Methanol-d4) δ 167.59, 154.51,

147.18, 140.74, 140.26, 135.60, 135.53, 132.13, 128.89, 126.77, 126.23, 126.20, 122.98, 122.88,
19
121.20, 52.71, 52.52, 48.34, 34.71, 22.19, 21.73, 9.02. F NMR (377 MHz, Methanol-d4) δ -

115.14. HRMS (ESI) calcd. for C25H3oClFN4O [M+H]: 457.2165, found: 457.2162.

F
Et2N
NH
H
N

O
Cl N

N-(7-chloro-4-((5-(diethylamino)pentan-2-yl)amino)quinolin-3-yl)-3-fluorobenzamide (3.10)

Yield: 43.7 mg, 64%. 1H NMR (500 MHz, Chloroform-d) δ 8.89 (s, 1H), 8.75 (s, 1H), 8.00 (d, J

= 2.2 Hz, 1H), 7.88 (d, J = 9.0 Hz, 1H), 7.80 (dd, J = 28.6, 8.5 Hz, 2H), 7.50 (td, J = 8.0, 5.6 Hz,

1H), 7.42 (dd, J = 9.0, 2.1 Hz, 1H), 7.29 (dd, J = 8.0, 2.3 Hz, 1H), 4.28 (d, J = 9.5 Hz, 1H), 3.72

(s, 1H), 2.50 (q, J = 7.1 Hz, 4H), 2.40 – 2.32 (m, 2H), 1.59 – 1.42 (m, 4H), 1.22 (d, J = 6.4 Hz,
13
3H), 0.97 (t, J = 7.2 Hz, 6H). C NMR (126 MHz, Chloroform-d) δ 165.24, 164.04, 162.07,

150.30, 148.37, 145.43, 136.10, 136.04, 134.86, 130.72, 130.66, 129.14, 126.50, 123.87, 123.15,

121.43, 119.51, 119.46, 119.34, 115.25, 115.06, 53.55, 52.62, 46.77, 36.69, 23.51, 22.33, 10.82.
19
F NMR (377 MHz, Chloroform-d) δ -111.57. HRMS (ESI) calcd. for C25H31ClFN4O [M+H]:

457.2165, found: 457.2160.

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Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

Et2N F
NH
H
N

O
Cl N

N-(7-chloro-4-((5-(diethylamino)pentan-2-yl)amino)quinolin-3-yl)-4-fluorobenzamide (3.11)

Yield: 41 mg, 60%. 1H NMR (500 MHz, Chloroform-d) δ 8.86 (s, 1H), 8.74 (s, 1H), 8.07 (t, J =

6.7 Hz, 2H), 7.99 (d, J = 2.1 Hz, 1H), 7.88 (d, J = 9.0 Hz, 1H), 7.41 (dd, J = 9.0, 2.2 Hz, 1H), 7.18

(t, J = 8.4 Hz, 2H), 4.28 (d, J = 9.4 Hz, 1H), 3.71 (s, 1H), 2.49 (q, J = 7.2 Hz, 4H), 2.40 – 2.30 (m,

1H), 1.59 – 1.37 (m, 2H), 1.21 (d, J = 6.4 Hz, 3H), 0.97 (t, J = 7.2 Hz, 6H). 13C NMR (126 MHz,

Chloroform-d) δ 166.20, 165.35, 164.18, 150.25, 148.20, 145.37, 134.64, 130.09, 130.02, 129.78,

129.76, 128.95, 126.28, 123.79, 121.27, 119.52, 115.98, 115.81, 53.40, 52.45, 46.61, 36.50, 23.27,
19
22.19, 10.57. F NMR (377 MHz, Chloroform-d) δ -107.08. HRMS (ESI) calcd. for

C25H31ClFN4O [M+H]: 457.2165, found: 457.21.

Et2N NH2
NH
H
N

O
Cl N

4-amino-N-(7-chloro-4-((5-(diethylamino)pentan-2-yl)amino)quinolin-3-yl)benzamide (3.12)

Yield: 53.8 mg, 41%. 1H NMR (400 MHz, Methanol-d4) δ 8.31 (s, 1H), 8.26 (d, J = 9.1 Hz, 1H),

7.84 (d, J = 2.2 Hz, 1H), 7.83 – 7.78 (m, 2H), 7.48 (dd, J = 9.1, 2.2 Hz, 1H), 6.76 – 6.70 (m, 2H),

2.48 (q, J = 7.1 Hz, 4H), 2.39 (d, J = 8.0 Hz, 2H), 1.64 (d, J = 7.2 Hz, 1H), 1.52 – 1.38 (m, 3H),

1.26 (d, J = 6.4 Hz, 3H), 0.96 (t, J = 7.2 Hz, 6H). 13C NMR (126 MHz, Methanol-d4) δ 169.78,

154.45, 154.12, 148.95, 148.71, 136.26, 130.65, 127.88, 126.72, 125.18, 121.94, 121.23, 115.33,

129
Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

114.70, 53.36, 52.16, 47.64, 36.67, 23.79, 21.90, 10.92. HRMS (ESI) calcd. for C25H33ClN5

[M+H]: 454.2368, found: 454.2369.

Et2N O
NH
H
N

O
Cl N

N-(7-chloro-4-((5-(diethylamino)pentan-2-yl)amino)quinolin-3-yl)-4-methoxybenzamide

(3.13)

Yield: 52.2 mg, 75%. 1H NMR (500 MHz, Chloroform-d) δ 8.92 (s, 1H), 8.34 (s, 1H), 7.99 (d, J

= 2.2 Hz, 1H), 7.97 (d, J = 8.6 Hz, 2H), 7.87 (d, J = 9.0 Hz, 1H), 7.40 (dd, J = 9.0, 2.2 Hz, 1H),

6.99 (d, J = 8.8 Hz, 2H), 4.25 (d, J = 9.7 Hz, 1H), 3.88 (s, 3H), 3.73-3.67 (m, 1H), 2.46 (q, J = 7.1

Hz, 4H), 2.33 (t, J = 6.7 Hz, 2H), 1.62 – 1.40 (m, 4H), 1.18 (d, J = 6.3 Hz, 3H), 0.95 (t, J = 7.2

Hz, 6H). 13C NMR (126 MHz, Chloroform-d) δ 165.98, 162.96, 150.28, 148.13, 145.14, 134.61,

129.52, 129.05, 126.40, 125.98, 123.80, 121.55, 120.07, 114.20, 55.64, 53.55, 52.73, 46.81, 36.71,

23.80, 22.25, 11.22. HRMS (ESI) calcd. for C26H34ClN4O2 [M+H]: 469.2365, found: 469.2369.

Et2N SMe
NH
H
N

O
Cl N

N-(7-chloro-4-((5-(diethylamino)pentan-2-yl)amino)quinolin-3-yl)-4(methylthio)benzamide

(3.14)

Yield: 47 mg, 61%. 1H NMR (500 MHz, Chloroform-d) δ 8.80 (s, 1H), 8.17 (d, J = 8.0 Hz, 2H),

8.01 (d, J = 2.2 Hz, 1H), 7.95 (d, J = 9.0 Hz, 1H), 7.41 (dd, J = 9.0, 2.2 Hz, 1H), 7.34 (d, J = 8.6

Hz, 2H), 4.41 (d, J = 10.2 Hz, 1H), 3.80 (s, 1H), 2.77 – 2.67 (m, 4H), 2.58-2.45 (m, 5H), 1.74-1.65

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Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

(m, 1H), 1.57 – 1.44 (m, 3H), 1.33 (d, J = 6.4 Hz, 3H), 1.11 (t, J = 7.3 Hz, 6H). 13C NMR (126

MHz, Chloroform-d) δ 166.45, 151.45, 148.80, 144.30, 137.55, 134.50, 129.25, 128.81, 128.73,

125.80, 125.29, 124.96, 121.01, 119.38, 53.31, 51.95, 46.32, 36.39, 22.70, 21.15, 14.95, 8.37.

HRMS (ESI) calcd. for C26H34ClN4OS [M+H]: 485.2136, found: 485.2146.

Et2N NMe2
NH
H
N

O
Cl N

N-(7-chloro-4-((5-(diethylamino)pentan-2-yl)amino)quinolin-3-yl)-4-

(dimethylamino)benzamide (3.15)

Yield: 22.3 mg, 31%. 1H NMR (800 MHz, Dimethyl sulfoxide-d6) δ 9.71 (s, 1H), 8.40 (d, J = 9.1

Hz, 1H), 8.29 (s, 1H), 7.91 (d, J = 8.9 Hz, 2H), 7.84 (d, J = 2.3 Hz, 1H), 7.50 (dd, J = 8.9, 2.3 Hz,

1H), 6.78 – 6.74 (m, 2H), 5.97 (d, J = 9.1 Hz, 1H), 3.98 (dq, J = 8.8, 6.4 Hz, 1H), 3.00 (s, 9H),

2.38 (s, 4H), 2.28 (s, 2H), 1.60 – 1.54 (m, 1H), 1.36 (ddt, J = 12.6, 9.8, 6.1 Hz, 1H), 1.29 (t, J =

7.9 Hz, 2H), 1.12 (d, J = 6.4 Hz, 3H), 0.85 (t, J = 7.1 Hz, 6H). 13C NMR (201 MHz, Dimethyl

sulfoxide-d6) δ 165.98, 154.20, 152.42, 147.67, 146.07, 133.05, 129.05, 127.50, 124.85, 124.61,

120.32, 119.96, 114.71, 110.84, 51.99, 50.41, 46.06, 39.99, 34.97, 22.61, 21.51, 7.16. HRMS

(ESI) calcd. for C27H36ClN5O [M+H]: 482.2681, found: 482.2675.

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Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

Et2N
NH
H
N

O
Cl N

N-(7-chloro-4-((5-(diethylamino)pentan-2-yl)amino)quinolin-3-yl)-4-methylbenzamide

(3.16)

Yield: 38.1 mg, 59%. 1H NMR (500 MHz, Chloroform-d) δ 8.95 (s, 1H), 8.34 (s, 1H), 8.01 (d, J

= 2.2 Hz, 1H), 7.89 (dd, J = 11.0, 8.2 Hz, 3H), 7.41 (dd, J = 8.9, 2.2 Hz, 1H), 7.31 (d, J = 7.7 Hz,

2H), 4.25 (d, J = 9.8 Hz, 1H), 3.75 – 3.64 (m, 1H), 2.51 – 2.40 (m, 7H), 2.33 (t, J = 6.6 Hz, 2H),
13
1.61 – 1.41 (m, 4H), 1.19 (d, J = 6.4 Hz, 3H), 0.96 (t, J = 7.2 Hz, 6H). C NMR (126 MHz,

Chloroform-d) δ 166.42, 150.35, 148.23, 145.26, 143.02, 134.66, 130.93, 129.67, 129.09, 127.66,

126.40, 123.88, 121.54, 119.95, 53.53, 52.68, 46.80, 36.67, 23.57, 22.30, 21.69, 10.99. HRMS

(ESI) calcd. for C26H34ClN4O [M+H]: 453.2416, found: 453.2417.

To a stirring solution of 3-NH2CQ (1 equiv.) in anhydrous EtOH at 0oC is added 4-

chlorobenzenesulfonyl chloride (1 equiv.) under nitrogen followed by NEt3 (1 equiv.). The

solution is stirred and allowed to warm to RT. The reaction progress is monitored by TLC. After

24 hours the solvent is removed under reduced pressure. The residue is then diluted with DCM,

and the solution is poured into a flask containing saturated NaHCO3 solution and is extracted with

DCM (3 X 20 mL) and washed with brine. The combined organic extracts are then dried over

anhydrous MgSO4 and the solvent removed under reduced pressure. The crude product is purified

by prep-TLC with silica gel, solvent system: 70% Hex, 20% EtOAc, 5% MeOH, 5% NEt3 to give

a bright yellow powder. Due to the low-yielding nature of the reaction, we were unable to obtain

full NMR characterization, but preliminary data is consistent with the expected product.

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Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

Et2N Cl
NH
H
N
S
O2
Cl N

4-chloro-N-(7-chloro-4-((5-(diethylamino)pentan-2-yl)amino)quinolin-3-

yl)benzenesulfonamide (3.17)

HRMS (ESI) calcd. for C24H30Cl2N4O2S [M+H]: 509.1539, found: 509.1520.

To a stirring solution of 3-NH2CQ (1 equiv.) in anhydrous EtOH at 0oC is added 4-

chlorobenzaldehyde (1.5 equiv.) under nitrogen followed by NEt3 (3 equiv.). The solution is stirred

and allowed to warm to RT. The reaction progress is monitored by TLC. After 24 hours the solvent

is removed under reduced pressure. The residue is then diluted with DCM, and the solution is

poured into a flask containing saturated NaHCO3 solution and is extracted with DCM (3 X 20 mL)

and washed with brine. The combined organic extracts are then dried over anhydrous MgSO4 and

the solvent removed under reduced pressure. The crude product is purified by prep-TLC with silica

gel, solvent system: 70% Hex, 20% EtOAc, 5% MeOH, 5% NEt3 to give a bright yellow powder.

Et2N Cl
NH
N

Cl N

(E)-N4-(7-chloro-3-((4-chlorobenzylidene)amino)quinolin-4-yl)-N1,N1-diethylpentane-1,4-

diamine (3.18)

HRMS (ESI) calcd. for C25H30Cl2N4 [M+H]: 457.1920, found: 457.1921.

133
Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

3.6 References

(1) Edaye, S.; Tazoo, D.; Bohle, D. S.; Georges, E. 3-Halo Chloroquine Derivatives Overcome

Plasmodium Falciparum Chloroquine Resistance Transporter-Mediated Drug Resistance in

P. Falciparum. Antimicrob. Agents Chemother. 2015, 59 (12), 7891–7893.

(2) Egan, T. J. Structure-Function Relationships in Chloroquine and Related 4-Aminoquinoline

Antimalarials. Mini-Reviews Med. Chem. 2001, 1 (1), 113–123.

(3) Bass, G. E.; Hudson, D. R.; Parker, J. E.; Purcell, W. P. Mechanism of Antimalarial Activity

of Chloroquine Analogs from Quantitative Structure-Activity Studies. Free Energy Related

Model. J. Med. Chem. 1971, 14 (4), 275–283.

(4) Edaye, S.; Tazoo, D.; Bohle, D. S.; Georges, E. 3-Iodo-4-Aminoquinoline Derivative

Sensitises Resistant Strains of Plasmodium Falciparum to Chloroquine. Int. J. Antimicrob.

Agents 2016, 47 (6), 482–485.

(5) Kaschula, C. H.; Egan, T. J.; Hunter, R.; Basilico, N.; Parapini, S.; Taramelli, D.; Pasini,

E.; Monti, D. Structure−Activity Relationships in 4-Aminoquinoline Antiplasmodials. The

Role of the Group at the 7-Position. J. Med. Chem. 2002, 45 (16), 3531–3539.

(6) Lipinski, C. A. Lead- and Drug-like Compounds: The Rule-of-Five Revolution. Drug

Discov. Today Technol. 2004, 1 (4), 337–341.

(7) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Experimental and

Computational Approaches to Estimate Solubility and Permeability in Drug Discovery and

Development Settings. Adv. Drug Deliv. Rev. 1997, 23 (1–3), 3–25.

(8) Craig, P. N. Interdependence between Physical Parametess and Selection of Substituent

Groups for Correlation Studies. J. Med. Chem. 1971, 14 (8), 680–684.

(9) Ertl, P. Craig Plot 2.0: An Interactive Navigation in the Substituent Bioisosteric Space. J.

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Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

Cheminform. 2020, 12 (1), 10–15.

(10) Hawley, S. R.; Bray, P. G.; Mungthin, M.; Atkinson, J. D.; O’Neill, P. M.; Ward, S. A.

Relationship between Antimalarial Drug Activity, Accumulation, and Inhibition of Heme

Polymerization in Plasmodium Falciparum In Vitro. Antimicrob. Agents Chemother. 1998,

42 (3), 682–686.

(11) Madrid, P. B.; Liou, A. P.; DeRisi, J. L.; Guy, R. K. Incorporation of an Intramolecular

Hydrogen-Bonding Motif in the Side Chain of 4-Aminoquinolines Enhances Activity

against Drug-Resistant P. Falciparum (Journal of Medical Chemistry (2006) 49 (4540)). J.

Med. Chem. 2008, 51 (3), 699.

(12) Egan, T. J.; Marques, H. M. The Role of Haem in the Activity of Chloroquine and Related

Antimalarial Drugs. Coord. Chem. Rev. 1999, 190–192, 493–517.

(13) Nsumiwa, S.; Kuter, D.; Wittlin, S.; Chibale, K.; Egan, T. J. Structure–Activity

Relationships for Ferriprotoporphyrin IX Association and β-Hematin Inhibition by 4-

Aminoquinolines Using Experimental and Ab Initio Methods. Bioorg. Med. Chem. 2013,

21 (13), 3738–3748.

(14) Thomé, R.; Lopes, S. C. P.; Costa, F. T. M.; Verinaud, L. Chloroquine: Modes of Action of

an Undervalued Drug. Immunol. Lett. 2013, 153 (1–2), 50–57.

(15) Al-Bari, M. A. A. Chloroquine Analogues in Drug Discovery: New Directions of Uses,

Mechanisms of Actions and Toxic Manifestations from Malaria to Multifarious Diseases.

J. Antimicrob. Chemother. 2015, 70 (6), 1608–1621.

(16) Hänscheid, T.; Egan, T. J.; Grobusch, M. P. Haemozoin: From Melatonin Pigment to Drug

Target, Diagnostic Tool, and Immune Modulator. Lancet Infect. Dis. 2007, 7 (10), 675–685.

(17) Schlesinger, P. H.; Krogstad, D. J.; Herwaldt, B. L. Antimalarial Agents: Mechanisms of

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Chapter 3 Synthesis of Novel Substituted Chloroquine Derivatives

Action. Antimicrob. Agents Chemother. 1988, 32 (6), 793–798.

136
 Chapter 4

Synthesis and Reactivity of a Compact Chloroquine

Photoaffinity label.

137
Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

4.1 Preamble

In drug discovery protein:compound interactions can be detected by covalently linking the drug to

the potential active site of the protein of interest. This technique is known as photoaffinity labelling

and facilitates the determination of a compound’s mechanism of action. The formation of a label

generally involves installing a photoreactive aryl azide or an aryl/ alkyl diazirine onto the

compound. Cross-linking between the compound and protein of interest occurs following the

irradiation of the photoreactive azide or diazirine. Historically, installation of the photoreactive

moiety is generally accompanied with the installation of large functional groups that may impact

the activity of the parent compound and interact with surrounding proteins. Photoaffinity labels

(PAL) can mimic the biological mechanism of the parent compound, but one cannot ignore the

influence of any additional functional group. With this in mind, we wanted to synthesize a PAL

for CQ that required the least amount of modifications to the CQ structure. With our 3-NH2CQ

precursor, we decided that it was the ideal location to install an azide PAL. We discovered that the

secondary amine at the 4-position is labile and subject to cyclization to form a triazole product

during the installation of the azide. We were able to prevent cyclization via the placement of a

methyl group to give a tertiary amine at the 4-position. All modifications resulted in the addition

of 2 small functional groups, a methyl and azide, where the methyl is unreactive. The resulting

PAL is easily activated under UV irradiation. The successful installation of a PAL with minimal

structural modifications will allow for a more precise mechanistic determination of the substrate-

binding site. The PAL is currently being used for labelling studies at the Fidock group at Columbia

University.

138
Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

4.2 Introduction

Quinoline-based antimalarials have been in use for centuries. Despite the long tenure of AQs on

the commercial market, their mechanism of action is still a matter of debate.1–5 There is a consensus

that blood-stage AQs interact with heme to inhibit the parasite’s proliferation; however, it is not

believed to be their sole function. The mechanism of quinoline accumulation into its active site

and its targets are still being investigated. Thus, to determine the site and identity of the proteins

with which the antimalarial drugs interact, several PALs have been previously synthesized, Figure

4.1. These labels are useful because they form strong covalent bonds with the surrounding proteins

and allow for the isolation and identification of the ligated product.

NEt2 NEt2
O HN HN
NH OMe

N3 OH N N3 N
ASA-Q 3-Azido-mepacrine

F
N3
F
F
O
HO
N F
O
I
N3 O O
N HN
N
NH HN N NH
H
S
OH O
ASA-MQ Cl N AzBCQ

Figure 4.1. Known photoaffinity labels.

When designing a PAL, one wants to mimic the biochemical binding sites of the parent compound

as closely as possible. This means that one must minimize the interactions of the label with

139
Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

surrounding residues to avoid unnecessary interaction. Therefore, it is best to avoid adding reactive

functional groups to PALs. Known PALs of AQs and MP are shown in Figure 4.1 and include: N-

(l-(l-diethylamino-l-methylbutylamino)quinolin-6-yl)-4-azido-2-hydroxybenzamide (ASA-

Q),6 3-azido-9-[[4-(diethylamino)-1-methylbutyl]amino]-7-methoxyacridine (3-azido-

mepacrine),7 N-[4-[I-hydroxy-2-(dibutylamino) ethyl] quinolin-8-yl]-4-azido-2-

hydroxybenzamide (ASA-MQ),8 and perfluorophenylazido biotinylated CQ (AzBCQ).9 Although

the PALs mentioned above can cross-link to proteins of interest, their design omits certain core

functionalities of the parent compound. ASA-Q completely removes the 7-chloro group found in

CQ, which is essential for inhibiting hemozoin formation. It introduces an amide, hydroxy and

phenyl group, which can hydrogen bond and form additional p-stacking interactions. These are

unfavourable modifications as they can interact with the surrounding environment. 3-Azido-

mepacrine replaces the 7-chloro group found in MP for the photoreactive aryl azide, this is

unfavourable because, as mentioned above, the chloro group is required for hemozoin inhibition.

ASA-MQ, an MQ PAL, does a poor job of retaining the parent compound’s core functions.

Desneves et al. claim that ASA-MQ is 10-fold more potent than mefloquine and that ASA-MQ is

inactive in the CQR Kl Mef2 strain which is selected in vitro for resistance to MQ. AzBCQ is a

CQ PAL that contains a biotin tag and a perfluorophenylazido group, and the quinoline ring

remains unmodified. However, there is the addition of large groups on the side chain.

In this chapter, I describe the synthesis of the first PAL, which contains the least modifications,

where all the essential features for CQ activity remain unmodified, Figure 4.2. Photolysis studies

were carried out with the PAL to determine the optimal wavelength for its activation. Examples of

labelling partners will also be described.

140
Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

Figure 4.2. Structure of novel 3-Azidomethylchloroquine in which all important CQ features are
present.

141
Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

4.3 Results and Discussion

We have synthesized a CQ PAL derivative with minimal modification of the quinoline structure.

The aryl azide is placed at the 3-position, a position that our group has previously utilized.10,11

Installation of the aryl azide can occur via Ullman coupling to form the Csp2 - N bond.12 This is

generally carried out in the presence of air, with a copper catalyst and ligand in a polar solvent,

with or without the presence of a base.13–15 In our experimentation, the azidation reaction from an

aryl bromide did not yield any trace of aryl azide. Rather an arylamine was the only product

observed. A further search of the literature shows that direct amination of aryl bromide with

sodium azide as an amine source is a common observation.16–18 Initially, Thatcher et al. suggest

this to be a result of thermal decomposition.19 Then Alami et al. proposed a mechanism of amine

formation via a nitrene intermediate, Scheme 4.1.17 However, they were unable to gather

experimental evidence of azide formation, but believe that the aryl azide is first formed and through

Cu(I) catalyzed thermo-initiation, N2 is liberated to form a nitrene, which abstracts protons and

electrons from the solvent.

Br NaN3, Cu(I), N3 N NH2

Base, Ligand N2 2H+

N EtOH, 100oC N N N
Intermediate

Scheme 4.1. Proposed mechanism for arylamine formation.

However, Helquist et al. later disproved the notion of amine formation due to azide thermal

decomposition by heating an aryl azide in DMSO for 72 hrs and found that the azide remained

intact.16 They then heated an aryl bromide in the presence of a copper catalyst and DMEDA and

only recovered an azo compound. They determined that amine formation from an azide precursor

142
Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

is dependent on the presence of a copper catalyst and excess sodium azide (see Table 2.2 – 2.4).

We did not isolate the desired azido product in our experiments, nor did we isolate any azo product;

only aryl amine was detected and isolated, Scheme 4.2. As shown in Chapter 2, regardless of the

reaction conditions the arylamine is the only isolated product.

Et2N CuI, DMEDA, Et2N Et2N

NH Sodium Ascorbate, NH NH
Br NaN3, N3 NH2
EtOH/H2O (7:3),
85oC
Cl N Cl N Cl N
0% 55%
4.1 4.2 4.3

Scheme 4.2. Ullman amination of aryl bromide in 30% aqueous solution in the presence of a ligand

and reducing agent.

With the arylamine in-hand, we continued to form the aryl azide via a route that first involves

forming a diazonium salt with sodium nitrite in the presence of sulfuric acid. Following the

diazonium’s formation, sodium azide is added to the reaction mixture to form the aryl azide.

However, the aryl azide was not isolated; rather, a cyclized triazole compound was isolated, as

depicted in Scheme 4.3. NMR and HRMS confirmed this structure. Formation of the triazole

indicates reactivity of the secondary amine at the 4-position. The reactive amine had to be

permanently blocked to prevent cyclization because if we were to temporarily protect the

secondary amine and then deprotect after azide installation, there remains a high probability of

cyclization still occurring.

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

Et2N Et2N Et2N

NH NH N N
1) H2SO4, NaNO2, 0oC
NH2 2) NaN3, 0oC N3 N

Cl N Cl N Cl N
0% 49%
4.3 4.3 4.4

Scheme 4.3. Azidation of arylamine leads to the formation of a triazole CQ product.

Initially, the goal of this project was to synthesize a PAL with the least possible modifications of

the parent compound. To remain true to the objective, we opted to methylate the secondary amine.

Methylation allows for the addition of an unreactive functional group. The first attempt of

methylation involves the use of methyl iodide for direct methylation of CQ, Table 4.1.20,21

However, this method was low yielding and in some instances resulted in a dimethylated product.

Dimethylation occurs at the tertiary amine and the secondary amine.

Table 4.1. Methylation of CQ with methyl iodide.

Et2N Et2N
NH N

Cl N Cl N
0%
4.5 4.6

Methylating Temperature
Base Solvent Yield (%)
agent (oC)
NaH DMF MeI -20 to 0 23
NaH DMF MeI Room temp. 15
NaH DMF MeI -20 9
NaH DMF MeI -20 N.D.
NaH MeCN MeI -20 -

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

Varying reaction conditions did not improve product yield or selectivity, and the HRMS

continuously shows the formation of mono and dimethylated CQ. We then attempted methylating

the secondary amine via the formation of a formamide, followed by a reduction of the formamide

to produce the desired methyl CQ (MeCQ), Table 4.2.

Table 4.2. Methylation of CQ.

O
Et2N Et2N Et2N
NH N H N
LiAlH4, THF,
0oC - RT

Cl N Cl N Cl N
4.5 4.7 4.6

Formulating
Solvent Temperature (oC) Catalyst 4.7 Yield (%)
agent

Formic acid - 70-84 - -

Formic acid - 70 I2 -
Sodium Ethoxide CHCl3 55 - -

Rahman et al. reported the 60% yield of N-formamide formation from diphenylamine with formic

acid in the absence of solvent.22 Kim and Jang reported 82% formation of N-formamide from N-

methylaniline from formic acid with I2 as a catalyst,23 and Shastri et al. report the formation of N-

formamide with the use of sodium ethoxide in chloroform via the Riemer– Teimann reaction.24

However, in our experiments only starting material was present. Following these attempts, we

deduced that the formylation of the aniline was not viable.

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

Ethyl formate, O LiAlH4, THF,

70oC, 18 h 0oC - RT
Et2N Et2N Et2N
NH2 N H N
H H
97%
4.8 4.9 4.10

Scheme 4.4. Methylation of 2-Amino-5-diethylaminopentane with ethyl formate.

It was decided that methylation of the side chain followed by a nucleophilic aromatic substitution

(SNAr) reaction would be the most efficient method for the synthesis of MeCQ, Scheme 4.4.

Formylation of the primary amine with formic acid and acetic anhydride proved to be unsuccessful.

This is likely due to the insufficient formation of acetic formic anhydride before the addition of

primary amine. However, the reaction with a primary amine and formic acid produced the desired

formamide in a 57% yield.22 Reactivity is improved using ethyl formate to give 97% of a product

and easy recovery with a simple acid and base workup.25 When ethyl formate, is used the side

chain does not get protonated; as is the case with formic acid, the primary amine is more

nucleophilic resulting in our higher yield. Once the formamide is formed, the final step in the

formation of 4.10 is a reduction with lithium aluminum hydride,26 which results in a 65% yield.

Lastly, to form our desired MeCQ, we performed a condensation reaction between 4,7-

dichloroquinoline (DCQ), 4.11 and 4.10, Table 4.3. Generally, this type of condensation reaction

occurs under basic conditions. As the alkyl side chain contains a basic secondary amine, we opted

to carry out the reaction with the amine acting as the base and/or solvent.

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

Table 4.3. Condensation reaction between DCQ and methylated side chain under basic conditions.

Et2N
Cl N
Et2N
N
H
Cl N Cl N
4.11 4.10
4.3

Yield (%)
Base Solvent Temperature (oC) Side product % Yield of 4.3

- - 85 -
- - 120-125 6
- - 120a -

- Ethylene glycol 120a 6

NEt3 - 125 -
NEt3 - 85a -
NEt3 EtOH 0-40 -
DBU - 80a 10
DBU - 125 82b -
DIPEA DMSO 80 -
DIPEA DMSO 120 -
DIPEA MeCN 85 -
t
BuOK DMSO 80-120 Decomp.
t
BuOK THF RT-80oC -
c
1,4-dioxane 85 8
a) Microwave-assisted reaction, b) 1-(3-((7-chloroquinolin-4-yl)amino)propyl)azepan-2-one
formed, see Scheme 4.4 b, c) Pd(OAc)2, DPEPhos, K3PO4.

There are numerous examples of thermal activation for this type of condensation reaction with

primary amines that give moderate to excellent yields.27–29 However, in our experiments, diamine

used as a base results in low yields of our product, even at elevated temperatures. The use of a

polar solvent such as ethylene glycol did not improve the yield. Therefore, it became clear that the

diamine was not nucleophilic enough to initiate the SNAr reaction; thus, we used stronger bases

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

such as triethylamine (NEt3), 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), N,N-

Diisopropylethylamine (DIPEA) and potassium tert-butoxide (tBuOK). NEt3 did not result in

product formation, nor did DIPEA with various polar solvents. DBU gave 10% product yield at

80℃; however, when the temperature was increased to 125oC, we get 82% formation of a ring-

opened DBU adduct, as illustrated in Scheme 4.5. Such adducts of ring-opened amidine bases

have been observed in the literature, formed in the presence of an inorganic base.30,31 A recent

publication from Merck scientists details their discovery of a ring-opened DBU adduct during the

development of a hepatitis C drug. Hyde et al. hypothesized that the hydrolysis of DBU occurs in

trace amounts of H2O and high temperatures or during an aqueous workup.32 When examining the

reactivity of electrophilic reactions in the presence of DBU they proposed two pathways to the

observed ring-opened DBU adduct. In the first pathway, the amidine base is hydrolyzed generating

a primary amine, which reacts with an electrophile. In the second pathway, a cationic intermediate

is formed via a reversible interaction of an amidine and an electrophile. This then reacts with trace

amounts of H2O or an aqueous workup that results in the ring-opened product. In our experiments,

while monitoring the reaction with TLC, the product is formed before quenching the reaction; thus,

trace amounts of H2O are responsible for the hydrolysis of DBU.

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

NH2
H2O Path 1 E
Hydrolysis first O

N N
E
N N
H

H2O
E
N

N
E
Path 2
a) Electrophile addition first

b)

Scheme 4.5. A) Potential pathways for DBU reaction with an electrophile. B) Crystal structure of

obtained open DBU adduct.

When potassium tert-butoxide is used, it leads to the decomposition of the reaction mixture and

palladium acetate catalyzed condensation was unsuccessful, Table 4.3.

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

Tf Et2N Et2N
Cl OH O N N
H
4.10

Cl N Cl N Cl N Cl N
4.11 4.12 4.13 4.6

Scheme 4.6. Increasing the electrophilicity of C-4 with the installation of a triflate.

We then concluded that the C-4 was not electrophilic enough; therefore, we opted for a better

leaving group than the chloride, Scheme 4.6. Triflic acid has a pKa value of -14, whereas

hydrochloric acid has a pKa of -8; therefore, we opted for the weaker conjugate base being the

triflate. Installation of the triflate involves nucleophilic substitution with acetic acid to give 7-

chloroquinolin-4-ol, 4.12. With the hydroxy in hand, we then carried out an electrophilic carbonyl

reaction to afford the triflate quinoline, 4.13.33 The final step to obtaining our desired MeCQ, 4.6,

was the condensation reaction with the methylated side chain, 4.10, Table 4.4.

Table 4.4. Condensation reaction conditions for 7-chloroquinolin-4-yl trifluoromethanesulfonate

4.13 and the methylated side chain 4.10.

Base Solvent Temperature (oC) % Yield of 4.6

- - 80-125 24
DIPEA DMSO 80-125 trace
DIPEA Acetonitrile RT-40 15
DIPEA - 125 26
NEt3 - 80-125 22
NEt3 EtOH 0-40 -

Initially, the reactions were placed in an 80oC oil bath, and the reaction was monitored for one

hour. When monitoring the reaction, if no product formed after one hour, the flask was then

transferred into a 125oC oil bath and heated for a further hour before quenching. It is evident from

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

the results that using a weaker base as a leaving group more than doubled the product yield, thus

proving the hypothesis for a need to make C-4 more electrophilic. However, although yields were

improved, they were still low.

We then decided to carry out the reaction under acidic conditions. There are examples in the

literature of this reaction being carried out in weakly acidic conditions with the use of phenol and

examples in strongly acidic conditions such as hydrochloric acid.34–37 The acid protonates the

secondary amine, which forms a quaternary ammonium cation, which would be slightly less basic

than NEt3, Table 4.5.

Table 4.5. Condensation reaction between DCQ 4.11, and methylated side chain 4.10 under
acidic conditions.

Et2N
Cl N

Et2N
N
Cl N H Cl N
4.10
4.11 4.6
Temperature Yield (%)
Acid Solvent Yield (%)
(oC) Side product
Phenola - 125 36b 14
Phenol - 125c 16
p-toluenesulfonic acid DMSO 125 -

p-toluenesulfonic acid - 120c 63

a) Reaction mixture includes 1.2 eq NEt3. b) 36% of 7-chloro-4-phenoxyquinoline formed. c)
microwave-assisted reaction.

Phenol is a weak acid with a pKa value of 10. It did not improve the reaction yield compared to

the use of strong bases. We observed a 7-chloro-4-phenoxyquinoline side product formation in the

reaction mixture containing phenol and NEt3. This side product has been reported by other research

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 Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

groups, formed in the absence of NEt3.38 As phenol did not optimize the reaction, we decided to

use a stronger organic acid p-toluenesulfonic acid with a pKa of -3, Table 4.5. The reaction

containing DMSO did not yield any product, which might be due to hydrogen bonding between

the sulfonic acid and the sulfoxide when prevented the protonation of the amine. Pan et al. reported

similar reaction conditions between primary and secondary amines with 4-chloro-2-

methylquinoline.39 Under traditional thermal conditions, a reaction mixture containing acid, a

cyclic secondary amine, quinoline, DMF and high temperatures give yields ranging 25 – 52%.

However, they demonstrated that they could produce good to excellent yields, 55 – 75%, under

microwave-assisted conditions and yields were improved to 81% when the solvent was removed

from the reaction.39 In our experiments, a microwave-assisted system without the use of solvent

improved the yield of the reaction to 63%. With this improved result we were satisfied with the

yield and proceeded to the final stage of the synthesis to form 3-azidomethylchloroquine (3-

N3MeCQ), 4.17.

Cl OH HNO3, OH Phosphoryl Chloride, Cl

Acetic Acid, Propionic acid, 115oC, 18h
NO2 NO2
125oC, 1h 140oC, 18h
Et2N
N
Cl N Cl N Cl N Cl N H
4.12 4.13 4.14 4.10
4.11
DIPEA,
MeCN,
85oC, 2h
>99%

Et2N Et2N SnCl2.2H2O, Et2N

OH N 1) H2SO4 (3.2 M in H2O), N N
EtOH,
N3 N3 NaNO2 (2.5 M in H2O), 0oC NH2 80oC, 2h NO2

Cl N N 2) NaN3 (4.5 M in H2O), 0oC Cl N Cl N

Cl
37% 28% 4.16 4.15
4.18 4.17

Scheme 4.7. Linear synthesis of 3-azidomethylchloroquine.

Once 4,7-dichloro-3-nitroquinoline, 4.14, is synthesized, we carried out the condensation reaction

to form the 3-NO2MeCQ, 4.15, see Scheme 4.7. Remarkably, in the case of 4.14, the optimal

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 Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

reaction conditions are with the use of DIPEA in acetonitrile. This reaction occurs very quickly

and gives quantitative yields. An explanation for this observation is the proximity of the electron-

withdrawing nitro group, which greatly increases the electrophilicity of the C-4 position. The nitro

group is then reduced with stannous chloride to afford 3-aminomethylchloroquine (3-NH2MeCQ),

4.16. The final step is converting the amine to azide via a diazonium salt with sodium nitrite and

sulfuric acid. The diazotization reaction occurs immediately following the addition of sodium

nitrite. However, a 3-azido-7-chloroquinolin-4-ol, 4.18 side product begins to form just as rapidly;

thus, the addition of sodium azide followed by quenching the reaction with sodium carbonate must

be done within 10 minutes of starting the reaction. A proposed pathway for side product formation

is shown below, Scheme 4.8. Once the diazonium ion forms, it can form an iminium salt that

results in an electrophilic C-4. This is then subject to nucleophilic attack by H2O. The methylated

side chain is then expulsed to give 4.22. The formation of 4.18 occurs after the addition of sodium

azide. The side product is insoluble in the acidic media and is filtered out of the solution to allow

the collection of the desired 4.17 product.

Et2N Et2N H NEt2 H

N N H N O OH
N N O H O N N
N N H N N N3

Cl N Cl N Cl N Cl N Cl N
4.19 4.20 4.21 4.22 4.18

Et2N
N
4.10 H

Scheme 4.8. Proposed mechanism for 3-azido-7-chloroquinolin-4-ol formation.

To reduce side product formation, we carried out the reaction in an organic solvent to minimize

H2O concentration. All reagents used have a degree of solubility in MeOH which was the chosen

solvent. The strong organic acid p-toluenesulfonic acid was chosen, and this reaction was carried

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

out at 0oC to RT and stirred for 18 hours. TLC showed minimal product conversion. When the

time elapsed and purification was performed only 1.4% of the product was isolated with no side

product formation. Factors affecting product formation for the new method included partial

solubility of sodium nitrite and sodium azide, therefore a lower rate of formation of the diazonium

intermediate. Another factor affecting the formation of the product is the pH of the solution. For

the nitrosonium ion to form an acidic environment is needed, which is not the case with p-

toluenesulfuric acid in MeOH.

4.3.1 Photoaffinity Properties

With the desired 3-N3MeCQ in hand, we have synthesized the first known quinoline-based PAL

with the least modifications to the parent compound. In doing so, we are able to retain all the

necessary functionality for antimalarial activity.

We first determined the UV-Vis spectroscopic difference between CQ, 3-NH2MeCQ and 3-

N3MeCQ. The spectroscopic profile for CQ diphosphate salt was prepared in H2O and measured

at pH 7.3. The samples for 3-NH2MeCQ and 3-N3MeCQ were prepared in MeOH. As expected,

CQ has a very different profile to its derivatives, whereas there are several similarities between 3-

NH2MeCQ and 3-N3MeCQ. As shown in Figure 4.3, there is a 6 nm bathochromic shift in the

lmax of the amine compared to the azide from 237 nm to 243 nm. Both compounds have weak

p®p* bands at 278 nm and 282 nm for the amine and azide, respectively, and weaker n®p* bands

at 357 nm and 358 nm for the amine and azide, respectively. CQ diphosphate is a colourless salt

and this is proven in the spectra where the absorbances are in the ultraviolet region. 3-NH2MeCQ

and 3-N3MeCQ have a yellow tint, this is due to the tailed absorbance at 400 nm.

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

3-NH2MeCQ v 3-N3MeCQ v CQ.2H3PO4

3-MeN3CQ
3-MeNH2CQ
Absorbance
CQ.2H3PO4

0
200 300 400
wavelength

Figure 4.3. Overlaying spectra of 3-NH2MeCQ, 3-N3MeCQ and CQ.2H3PO4 for comparison.

We then determined the excitation wavelength for the photolabile moiety. It has been reported that

aryl azides can be irradiated at 254 nm without causing damage to the compound; therefore, we

decided to test its stability at that excitation wavelength, Figure 4.4.40,41 The azide was first

exposed to 254 nm using a UV mercury penlight in the dark, exposure lasted 1 hour, and the spectra

were taken at different time intervals. As shown in the spectra, photoexcitation/ decomposition is

rapid as the p®p* band disappears within one minute. However, photodecomposition begins to

occur within 30 minutes, with the compound having little UV-Vis signature after one hour.

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

Azide w/254nm light

2.0
No Light

1.5
1 minute
Absorbance

3 minutes
1.0 8 minutes
18 minutes
0.5 30 minutes
1 hour
0.0
200 300 400
Wavelength (nm)

Figure 4.4. Photolysis of 3-N3MeCQ at 254 nm excitation.

With this observation, we decided that photolysis at 254 nm was too energetic and tried

photoexcitation at 365 nm. There are also many examples of azide irradiation above 300 nm which

result in insertion reactions.6,42,43 We carried out stability tests in H2O and MeOH, and the aryl

azide remains intact after longer than 18 hours with exposure to 365 nm of light, Figure 4.5.

Due to the high reactivity of the nitrene, the below spectra are the results of product formation

from the nitrene reacting with the polar solvent, Figure 4.5. These results show that the aryl azide

is indeed photolabile; however, it is also a cause of concern that the reaction with the solvent will

occur faster than insertion with a chosen compound, making detection of azide-compound

insertion difficult with UV-Vis spectroscopy. Furthermore, binding regioselectivity is important

as the photo label should ideally insert at the binding site.

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

42.97uM N3 in H2O 35.57uM N3 in MeOH

245
1.0 219 220 245
0 seconds
0 seconds
259 10 seconds
10 seconds

20 seconds 1.0 20 seconds

30 seconds 30 seconds
285 1 minute 1 minute
2 minutes 287 2 minutes
Absorbance

3 minutes 3 minutes

Absorbance
4 minutes 4 minutes
0.5
5 minutes 5 minutes

0.5

363
360

0.0
250 300 350 400 450 0.0
Wavelength (nm) 250 300 350 400 450
Wavelength (nm)

Figure 4.5. Photolysis of 42.97 µM N3 in H2O and 35.57 µM N3 in MeOH at 365 nm.

We then carried out the same reaction in an NMR tube with a 365 nm penlight in the dark in

deuterated MeOH.

0 minutes l g
k g e
N
i
h
e l
a f N
j
bd c
N3 k
c b
j h, i
Cl N a f h’
d
16 minutes

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5
f1 (ppm)

Figure 4.6. Photolysis of 3-N3MeCQ at 0 and 16 minutes in MeOD-d4.

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

0 minutes

2 minutes

6 minutes

11 minutes

16 minutes

9.2 9.1 9.0 8.9 8.8 8.7 8.6 8.5 8.4 8.3 8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1
f1 (ppm)

Figure 4.7. Photolysis of 3-N3MeCQ in MeOD-d4 over 16 minutes.

Irradiation of the aryl azide with 365 nm shows an apparent change in the structure, Figure 4.6.

Changes become evident after 2 minutes of exposure. As seen in the UV spectra, the p ® p* band

disappears, in the NMR spectra we can see the aromatic protons decrease in intensity as additional

peaks arise. Additional photodecomposition occurs with longer exposure times, Figure 4.7, the

aromatic region is more affected than the aliphatic regions as one would expect. Therefore, when

carrying out photolysis experiments exposure times did not exceed 10 minutes. High-Resolution

Mass Spectroscopy was carried out for the azide samples in H2O and MeOH. We discovered the

formation of 3 consistent molecular ions with their possible structures shown below, Figure 4.8.

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

The formation of nitroso-methylCQ occurs in the presence of H2O and MeOH when irradiated

at 365 nm for 5 minutes, this might be a result of hydroxyl insertion. Interestingly, only in the

presence of H2O do we get the formation of a nitroso-chloroquine derivative that has lost a methyl

group. The formation of this product may also result from H2O insertion and the HRMS data

leads us to believe the shown structure is the most probable product. Not-so surprisingly, we have

the formation of an intramolecular insertion product; however, it only formed in the presence of

MeOH. Formations of such azirines are well documented for phenyl azide, which exists in

resonance with the corresponding ketenimine.44–46

N N N
N NH N
N
N N
O O
Cl N Cl N Cl N
Formed in the presence Formed only in the Formed only in the
of water and methanol presence of water presence of methanol

Figure 4.8. Possible irradiated 3-N3MeCQ products formed.

4.3.2 Nitrene insertion

We carried out experiments to determine if the expected singlet nitrene intermediate that forms

inserts into C-H, C-N or C=C bonds. To achieve this, we first used a simple tripeptide

GlyGlyGly. A UV-Vis spectrum of the azide and GlyGlyGly was taken at different

stoichiometries, Figure 4.9, and the change in the UV-Vis signature is evident at the different

concentrations. From the UV-Vis spectra it is clear that there is a change in the aromatic system

of the aryl azide when mixed with the peptide. To confirm that insertion occurred with the peptide

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 Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

we carried out HRMS for the resulting product. However, there were no peaks related to an

insertion product detected by HRMS.

21.49uM N3 and GlyGlyGly in H2O (1:1) 4.29uM N3 and GlyGlyGly in H2O (1:9.4)
0.08
0 secs 0 secs
245 10 secs 10 secs
20 secs 245 20 secs
0.4 30 secs 30 secs
0.06
1 minute 1 minute
2 minutes 2 minutes
285 3 minutes 3 minutes
285

Absorbance
Absorbance

4 minutes 4 minutes
0.04
5 minutes 5 minutes

0.2

0.02
358
358

0.00
0.0

250 300 350 400 450 250 300 350 400 450
Wavelength (nm) Wavelength (nm)

Figure 4.9. Photolysis of 21.49 µM 3-N3MeCQ with 22.32 µM GlyGlyGly and 4.29 µM 3-

N3MeCQ with 40.18 µM GlyGlyGly at 365 nm excitation over five minutes.

We then decided to carry out UV-Vis and NMR studies to trap the nitrene with strong electron

acceptors. First, we used N-phenylmaleimide with 3-N3MeCQ in the dark exposed to 365 nm of

light, Figure 4.10. At higher concentrations, the absorbance associated with N-Phenylmaleimide

begin to obscure that of the quinoline; however, it seems to follow a similar pattern to that of the

control experiment. The HRMS did not detect any molecular ions associated with a possible

insertion product.

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 Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

19.56uM N3 and N-Phenylmaleimide in MeOH (1:1) 3.56uM N3 and N-Phenylmaleimide in MeOH (1:10.6)
0.8 218
220 0 secs 0 secs
1.0
10 secs 10 secs
20 secs 20 secs
30 secs 30 secs
0.6
1 minute 1 minute
2 minutes 2 minutes
3 minutes 3 minutes

Absorbance
Absorbance

4 minutes 4 minutes
0.4
286 5 minutes 5 minutes
0.5

286
0.2
354

0.0 0.0
250 300 350 400 450 250 300 350 400 450
Wavelength (nm) Wavelength (nm)

Figure 4.10. Photolysis of 19.56 µM 3-N3MeCQ with 18.77 µM maleimide, 7.11 µM 3-N3MeCQ

with 33.37 µM maleimide and 3.56 µM 3-N3MeCQ and 37.54 µM maleimide at 365 nm excitation

over 5 minutes.

We then carried out the same experiment in an NMR tube to detect product formation at higher

concentrations of both reagents. As shown in Figure 4.11, doublets appear at 6.53 ppm and 6.29

ppm. To further investigate N-phenylmaleimide, was irradiated alone in MeOH for a total of nine

minutes, and we saw the appearance of the vinyl protons indicating independent activation of the

maleimide, Figure 4.12.

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

3-N3MeCQ

3-N3MeCQ + N-Phenylmaleimide
365 nm for 6 minutes

6.53 4
6.29

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
f1 (ppm)

Figure 4.11. Photolysis of 26.7mM 3-N3MeCQ and 26.6mM maleimide at 365 nm in d4-MeOD.

Hott and Heusinger (1977) investigated the photolysis of maleimides in polar solutions using

electron spin resonance.47 They note the detection of radical anions of maleimides in alcohol

solutions. They determined that radical anions are the primary ion species. Kiselev et al. have

reported the slow hydrolysis of N-phenylmaleimide to the acid in the presence of H2O.48 It is

possible that the radical anion formation speeds up the hydrolysis of the maleimide, resulting in

the ring-opened carboxylic acid.

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

No light
O
N
2
O

365mn for 9 minutes O

OH
N
H
O

8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 6.2 6.1 6.0
f1 (ppm)

Figure 4.12. Comparison between activated and unactivated N-phenylmaleimide.

We then decided to use the stronger electron acceptor tetracyanoethylene (TCNE). Murata et al.

reported the photochemical reaction of mesityl azide with TCNE with 360 nm ± 15 nm light

produced two adducts formed via singlet nitrene trapping.49 Irradiation of our aryl azide and TCNE

for 9 minutes shows the formation of a minor product (23% conversion); however, the HRMS of

the mixture did not detect the formation of the expected photolysis product but rather corresponds

to a formal HCN insertion product.

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

CN
N NC N
N CN N
H
N N CN
CN

Cl N Cl N
Expected product Possible product
a) 23% conversion

9.3 9.2 9.1 9.0 8.9 8.8 8.7 8.6 8.5 8.4 8.3 8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0
b) f1 (ppm)

Figure 4.13. a) Desired product v possible observed photolysis product, b) NMR of photolysis

product formation.

The exact mass and chemical formula obtained from HRMS ([M+H+] m/z: 374.21) led us to the

proposed structure, Figure 4.13. This product was not isolated and longer excitation times leads

to the decomposition of the spectroscopically detected intermediate.

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

4.4 Conclusion

We have synthesized the first photolabile CQ derivative with minimal modifications of the parent

compound. The modification involved installing a methyl group on the 4-AQ to prevent

cyclization to a triazole derivative. In order to install the amine we first methylated the side chain

and then carried out a condensation reaction with 4,7-dichloro-3-nitroquinoline. Once the desired

azide was obtained we carried out UV-Vis stability tests to determine the best-suited

photoexcitation energy. We found that irradiation at 254 nm was too energetic and led to the

decomposition of the aromatic system and 365 nm of light was the most suitable as it activated the

azide, causing a release of nitrogen gas to form a new compound. The reactive nitrene intermediate

species forms an adduct with the surrounding solvent which was detected by HRMS. We then

carried out photolysis studies with a GlyGlyGly, N-phenylmaleimide and tetracyanoethylene and

discovered that the electron acceptor and the solvent determine the conditions for insertion. We

could not detect the expected insertion product with TCNE in which the nitrene intermediate is

inserted into the C-N triple bond; however, we detected a possible HCN insertion product. The

PAL is currently being used for photoaffinity labelling on PfCRT with the Fidock group at

Columbia University.

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

4.5 Experimental

4.5.1 General information

Glassware was taken directly from the oven (120oC) and let cool in a desiccator before use. NMR

experiments were carried out in New Era NMR routine grade tubes 5 mm X 8”. All solvents and

reagents were obtained commercially and used without further purification unless noted.

UV irradiation was carried out using Analytik Jena UVP Pen-Ray UV mercury lamp 254 nm and

365 nm. High-Resolution Mass Spectroscopy (HRMS) was obtained by positive/negative ESI, or

positive/negative APCI on a Bruker Maxis Impact QTOF, or a Thermo Scientific ExactivePlus

Orbitrap, with results reported as mass/charge ratios (m/z). Thin-layer chromatography plates were

visualized using ultra-violet light, 254nm. Flash column chromatography was carried out manually

using 230 - 400 mesh silica gel (Silicycle) using reagent grade solvents or using Biotage Isolera

One system with Biotage ZIP 30 g columns. Cole Parmer Microcomputer pH-Vision Model

05669-20 pH Meter used with Sigma-Aldrich micro pH combination electrode and glass body.

The UV-Vis spectra were measured on Agilent 8453.

1 13
H and C NMR spectra were recorded at ambient temperature on Bruker AVIIIHD 400 MHz

(1H 400 MHz, 13C 100 MHz) and Bruker AVIIIHD 500 MHz (1H 500 MHz, 13C 125 MHz) using

tetramethylsilane as the internal standard. Chemical shifts are reported relative to the residual

deuterated solvent peaks. Chemical shifts are expressed in parts per million (ppm = δ) values and

coupling constants (J) in Hertz (Hz). The terms m, s, d, t and q represent multiplicities of 1H NMR

resonances: multiplet, singlet, doublet, triplet and quartet, respectively. For previously unknown

compounds, a combination of 2D experiments (COSY, HSQC, HMBC) was often used to

complete the assignment of 1H and 13C signals. 1H NMR signals are described by chemical shift δ

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

13
(multiplicity, J (Hz), integration). C NMR signals are described by chemical shift δ and are

singlets unless otherwise specified.

4.5.2 Experimental procedure

N
N N
N
2H3PO4
Cl N

4-(7-chloro-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-N,N-diethylpentan-1-amine diphosphate

(4.4)

A solution of 7-chloro-N4-(5-(diethylamino)pentan-2-yl)quinoline-3,4-diamine (50 mg, 0.14

mmol) in of 3.2 M sulfuric acid (98%), is cooled to 0oC and a 1 mL solution of 2.5 M sodium

nitrite is added dropwise. The reaction mixture is stirred at this temperature for 15 minutes. After

15 minutes, 1 mL of 4.5 M sodium azide is added dropwise to the reaction mixture at 0oC. Addition

of azide results in visible N2 gas evolution. The reaction mixture is left to stir for a further 1 hour

and the mixture is allowed to warm from 0oC to RT. Once complete, the reaction mixture is treated

with saturated Na2CO3 solution and the compound is extracted with DCM (3 X 10 mL). The

combined organic layers are rinsed with brine and dried over anhydrous MgSO4. The solvent is

removed under reduced vacuum to reveal a bright orange oil. The oil was purified by flash

chromatography on silica gel with the solvent system: 55% Hex, 40% DCM, 5% NEt3 to give a

bright yellow oil 24 mg, 46% yield.

The free-base is then converted to a phosphate salt with phosphoric acid as follows: To a

solution of the free base 4-(7-chloro-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-N,N-diethylpentan-1-

amine (24 mg, 0.069 mmol) in 1 mL of MeOH is added, with cooling, a phosphoric acid solution

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

made from 0.14 g of 85% phosphoric acid and 3 mL of MeOH. Isopropyl alcohol is then added to

separate the salt from the solution as an oil. The oil is triturated with ether and stirred until it

solidifies. The mixture was then filtered, the diphosphate washed with ether and quickly placed in

a vacuum oven overnight. White powder 26.3 mg, 70 %.

Yield: 26.3 mg, 70%. 1H NMR (400 MHz, Deuterium Oxide-d2) δ 9.26 (s, 1H), 8.26 (d, J = 9.0

Hz, 1H), 7.78 (d, J = 2.1 Hz, 1H), 7.69 (dd, J = 8.9, 2.2 Hz, 1H), 5.43 (p, J = 6.6 Hz, 1H), 3.19 –

3.09 (m, 6H), 2.47 – 2.34 (m, 1H), 2.28 – 2.15 (m, 1H), 1.81 (d, J = 6.6 Hz, 3H), 1.79 – 1.63 (m,

2H), 1.22 – 1.18 (m, 6H). 13C NMR (126 MHz, Deuterium Oxide-d2) δ 144.70, 142.82, 139.06,

135.94, 133.40, 129.23, 126.81, 123.73, 113.20, 57.99, 50.95, 47.23, 32.16, 19.96, 19.89, 8.07 (d,

J = 4.6 Hz). 31P NMR (162 MHz, Deuterium Oxide-d2) δ 0.03. HRMS (ESI) calcd. for C18H25ClN5

[M+H]: 346.1798, found: 346.1793.

N
N

Cl N

N4-(7-chloroquinolin-4-yl)-N1,N1-diethyl-N4-methylpentane-1,4-diamine (4.6)

The mixture of 4,7-dichloroquinoline (500 mg, 2.5 mmol) and N1,N1-diethyl-N4-methylpentane-

1,4-diamine (652.6 mg, 3.8 mmol, 1.5 equiv.) are placed in a microwave flask with p-

toluenesulfonic acid (816 mg, 4.3 mmol, 1.7 equiv.). This mixture was heated at 120oC for 1.5

hours. Once complete, the reaction is let cool to RT. The mixture is poured into ice H2O, and then

1 M NaOH is added. This is extracted with EtOAc (3 X 100 mL), washed with brine, and the

organic layers are then dried over anhydrous MgSO4. The solvent is removed under reduced

vacuum to reveal an orange oil. The oil was purified by flash chromatography on silica gel with

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

the solvent system: 50% Hex, 50% to 100% EtOAc and 5 - 10% NEt3 to give an orange oil 534.5

mg, 63% yield.

Yield: 534.5mg, 63%. 1H NMR (500 MHz, Chloroform-d) δ 8.60 (d, J = 5.2 Hz, 1H), 7.99 (d, J

= 2.2 Hz, 1H), 7.88 (d, J = 9.0 Hz, 1H), 7.35 (dd, J = 9.0, 2.2 Hz, 1H), 6.75 (d, J = 5.2 Hz, 1H),

3.85 (h, J = 6.8 Hz, 1H), 2.85 (s, 3H), 2.43 (qd, J = 7.1, 1.6 Hz, 4H), 2.34 – 2.29 (m, 2H), 1.76 –

1.65 (m, 1H), 1.56 – 1.47 (m, 1H), 1.46 – 1.31 (m, 0H), 1.25 (d, J = 6.5 Hz, 3H), 0.94 (t, J = 7.1

Hz, 6H). 13C NMR (126 MHz, Chloroform-d) δ 157.55, 151.53, 150.78, 134.64, 128.90, 125.98,

125.22, 121.86, 109.02, 58.80, 52.87, 46.91, 32.49, 32.35, 24.64, 17.10, 11.70. HRMS (APCI)

calcd. for C19H29ClN3 [M+H+]: 334.2045, found: 334.2046.

O
N
N H
H

N-(5-(diethylamino)pentan-2-yl)formamide (4.9)

A mixture of 2-Amino-5-diethylaminopentane (5 g, 31.6 mmol) and ethyl formate (7.7 mL, 3

equiv.) is heated at 70°C for 18 hours in a round-bottom flask with molecular sieves. After

completion the mixture was cooled to RT and EtOAc was added. The resulting solution was

washed with 1 M HCl and extracted with EtOAc (3 X 150 mL). The organic layer was then basified

with 1 M NaOH and extracted with EtOAc (3X 200 mL) then rinsed with brine and dried over

anhydrous magnesium sulfate. The solvent was evaporated under reduced pressure to get N-(5-

(diethylamino)pentan-2-yl) formamide product, used without further purification to give an orange

oil, 5.689 g, 97% yield.

Yield: 5.689 g, 97%. Major trans rotamer: 1H NMR (500 MHz, Chloroform-d) δ 8.10 (s, 1H),

6.55 (s, 1H), 4.08 – 3.97 (m, 1H), 2.53 (dd, J = 7.2, 4.7 Hz, 4H), 2.45 – 2.35 (m, 2H), 1.50 (dd, J

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

= 6.2, 3.8 Hz, 4H), 1.15 (d, J = 6.6 Hz, 3H), 1.04 – 1.00 (m, 6H). 13C NMR (126 MHz, Chloroform-

d) δ 160.68, 53.02, 46.77, 44.01, 34.80, 23.14, 20.84, 11.42. Minor cis rotamer: 1H NMR (500

MHz, Chloroform-d) δ 8.07 (d, J = 12.0 Hz, 1H), 5.93 (s, 1H), 3.49 (s, 1H), 2.53 (dd, J = 7.2, 4.7

Hz, 4H), 2.45 – 2.35 (m, 2H), 1.50 (dd, J = 6.2, 3.8 Hz, 4H), 1.21 (d, J = 6.6 Hz, 3H), 0.99 (s,
13
6H). C NMR (126 MHz, Chloroform-d) δ 163.78, 52.65, 48.37, 46.86, 36.00, 23.58, 22.79,

11.62. HRMS (APCI) calcd. for C10H23ON2 [M+H]: 187.18049, found: 187.18033.

N
N
H

N1,N1-diethyl-N4-methylpentane-1,4-diamine (4.10)

To a suspension of 2 M LiAlH4 in THF, N-(5-(diethylamino)pentan-2-yl)formamide (4.5 g, 24.2

mmol), which is dissolved and degassed in anhydrous THF, is added portion-wise under stirring

and cooling with an ice bath. The mixture is let stir at 0oC for 30 minutes, and then the reaction

mixture is refluxed for 24 hours under nitrogen. After completion 1 M NaOH is added dropwise

to hydrolyze any remaining LiAlH4. All precipitates are filtered off and washed with THF. The

filtrate is then placed in a separatory funnel and the organic layer separates. The organic layer is

then acidified with 1 M HCl and extracted with DCM (3 X 200 mL). The combined organic layers

are then basified with sat. NaOH and extracted with DCM (3 X 300 mL). The organic layers are

then rinsed with brine and dried over anhydrous MgSO4 and the solvent is removed under reduced

pressure to reveal pure N1,N1-diethyl-N4-methylpentane-1,4-diamine, which was used without

further purification. Orange oil, 2.685 g, 65% yield.

Yield: 2.685 g, 65% yield. 1H NMR (500 MHz, Chloroform-d) δ 2.52 (q, J = 7.2 Hz, 4H), 2.40

(d, J = 6.5 Hz, 5H), 1.51 – 1.42 (m, 3H), 1.34 – 1.23 (m, 1H), 1.05 (d, J = 6.3 Hz, 3H), 1.01 (t, J

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

= 7.2 Hz, 6H). 13C NMR (126 MHz, Chloroform-d) δ 55.07, 53.32, 46.87, 34.91, 33.79, 23.68,

19.79, 11. HRMS (APCI) calcd. for C10H25N2 [M+H]: 173.20123, found: 173.20097.

N NH

Cl N

1-(3-((7-chloroquinolin-4-yl)amino)propyl)azepan-2-one

4,7-dichloroquinoline (126 mg, 0.64 mmol), N1,N1-diethyl-N4-methylpentane-1,4-diamine (100

mg, 0.58 mmol) and DBU (0.43 mL, 2.9 mmol) are placed in a round bottom flask and heated to

125oC. The mixture was let stir for 18 hours. The brown mixture is then cooled to RT and washed

with sat. NaOH and extracted with EtOAc (3 X 100 mL). The combined organic layers are then

rinsed with H2O and dried over anhydrous MgSO4 and the solvent was removed under reduced

pressure. The oil was purified by flash chromatography on silica gel with the solvent system: 85%,

EtOAc, 10% MeOH and 5% NEt3 to give a white solid, 173.8 mg, 82% yield.

Yield: 173.8 mg, 82%.1H NMR (500 MHz, Chloroform-d) δ 8.46 (d, J = 5.5 Hz, 1H), 8.00 (d, J

= 9.0 Hz, 1H), 7.95 (d, J = 2.2 Hz, 1H), 7.39 (dd, J = 9.0, 2.2 Hz, 1H), 7.02 (s, 1H), 6.39 (d, J =

5.6 Hz, 1H), 3.53 – 3.47 (m, 2H), 3.41 – 3.33 (m, 4H), 2.64 – 2.58 (m, 2H), 1.85 – 1.70 (m, 6H),

1.67 (p, J = 5.6 Hz, 2H). 13C NMR (126 MHz, Chloroform-d) δ 177.42, 151.23, 150.33, 148.73,

135.16, 127.91, 125.47, 122.25, 117.65, 98.09, 49.88, 45.11, 38.47, 37.17, 29.93, 28.44, 25.63,

23.46. HRMS (ESI) calcd. for C18H23ClN3O [M+H]: 332.1524, found: 332.1520.

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

N
N
NO2

Cl N

N4-(7-chloro-3-nitroquinolin-4-yl)-N1,N1-diethyl-N4-methylpentane-1,4-diamine (4.15)

To a solution of 4,7-dichloro-3-nitroquinoline (500 mg, 2.1 mmol) in acetonitrile is added N1,N1-

diethyl-N4-methylpentane-1,4-diamine (709 mg, 4.1 mmol, 2 equiv.) and N,N-

diisopropylethylamine (0.54 mL, 1.5 equiv.) which is stirred until it formed a homogenous

solution. This mixture is let stir at RT for up to 10 minutes. The reaction mixture is then transferred

to an 85oC oil bath and let stir for 30 minutes. Once complete the reaction mixture is evaporated

to dryness under reduced pressure. The residue is diluted with EtOAc and H2O and basified with

1 M NaOH. The resulting solution is extracted with EtOAc (3 X 100 mL). The combined organic

layers were then rinsed with brine and dried over anhydrous MgSO4. The solvent is removed under

reduced pressure. The crude product is purified by flash silica column chromatography with 85%

Hex, 10% EtOAc, 5% NEt3 to reveal a bright orange solid, 719.7mg, 92%.

Yield: 719.7 mg, 92%. 1H NMR (500 MHz, Chloroform-d) δ 8.93 (s, 1H), 8.06 (d, J = 2.2 Hz,

1H), 8.02 (d, J = 9.2 Hz, 1H), 7.50 (dd, J = 9.1, 2.2 Hz, 1H), 4.03 – 3.95 (m, 1H), 2.85 (s, 3H),

2.51 (d, J = 7.3 Hz, 4H), 2.42 (d, J = 7.7 Hz, 2H), 1.89 – 1.81 (m, 1H), 1.65 (dq, J = 13.5, 8.0 Hz,

1H), 1.47 (q, J = 7.8 Hz, 2H), 1.43 (d, J = 6.6 Hz, 3H), 1.00 (t, J = 7.1 Hz, 6H). 13C NMR (126

MHz, Chloroform-d) δ 151.13, 150.70, 147.90, 137.63, 137.04, 129.68, 127.55, 127.18, 123.82,

60.99, 52.76, 46.94, 33.91, 32.73, 24.46, 18.19, 11.42. HRMS (APCI) calcd. for C19H27ClN4O2

[M-H+]: 378.1828, found: 378.1822.

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N
N
NH2

Cl N

7-chloro-N4-(5-(diethylamino)pentan-2-yl)-N4-methylquinoline-3,4-diamine (4.16)

N4-(7-chloro-3-nitroquinolin-4-yl)-N1,N1-diethyl-N4-methylpentane-1,4-diamine (500 mg, 1.3

mmol) and stannous chloride (1.49 g, 5 equiv.) are placed in a round bottom flask. EtOH is added

and the mixture was let stir at 80oC for 2 hours. Once complete the mixture is let cool to RT. Once

cool, the solvent is removed by reduced pressure. The residue is diluted with EtOAc and basified

with sat. NaOH and extracted with EtOAc (3 X 50 mL). The combined organic layers are washed

with brine and dried over anhydrous MgSO4. The solvent is removed under reduced pressure to

reveal a dark orange oil. This was purified by column chromatography with the solvent system:

EtOAc (100→90%)/ MeOH (0→10%)/ NEt3 (5%) to give an orange oil, 313 mg, 68% yield.

Yield: 313 mg, 68%. 1H NMR (400 MHz, Dimethyl sulfoxide-d6) δ 9.40 (s, 1H), 8.62 (s, 1H),

7.97 (d, J = 9.2 Hz, 1H), 7.92 (d, J = 2.2 Hz, 1H), 7.53 (dd, J = 9.2, 2.2 Hz, 1H), 3.56 – 3.47 (m,

1H), 3.10 – 2.99 (m, 3H), 2.93 (s, 5H), 1.66 – 1.48 (m, 4H), 1.18 (d, J = 6.5 Hz, 3H), 1.12 (t, J =
13
7.2 Hz, 6H). C NMR (101 MHz, Dimethyl sulfoxide-d6) δ 140.47, 139.38, 138.79, 138.67,

130.61, 127.23, 126.25, 125.55, 124.01, 55.24, 50.92, 46.45, 46.30, 35.02, 31.32, 20.25, 17.77,

8.55 (d, J = 1.7 Hz). HRMS (ESI) calcd. for C19H30ClN4O2 [M+H-]: 349.2153, found: 349.2143.

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

N
N
N3

Cl N

N4-(3-azido-7-chloroquinolin-4-yl)-N1,N1-diethyl-N4-methylpentane-1,4-diamine (4.17)

7-chloro-N4-(5-(diethylamino)pentan-2-yl)-N4-methylquinoline-3,4-diamine (330 mg, 0.95 mmol)

is dissolved in a solution of concentrated sulfuric acid 98% and H2O (3.2 M). The resultant solution

is cooled to 0°C; to this, a sodium nitrite solution in H2O (2.5 M) is added dropwise with stirring.

The solution was stirred at this temperature for 5 min. Then a solution of sodium azide in H2O (4.5

M) is added to it with vigorous stirring. The mixture was stirred for 5 minutes at 0°C. The reaction

mixture is then quenched with sat. NaCO3 and extracted with EtOAc (3 X 25 mL). The combined

extracts are rinsed with brine and dried over anhydrous MgSO4 and solvent is removed under

reduced pressure to reveal a dark orange oil. This oil is purified by column chromatography with

the solvent system: 65% EtOAc, 35% Hex, 5% NEt3 to reveal a dark orange oil, 101 mg, 28%.

Yield: 101 mg, 28%. 1H NMR (500 MHz, Chloroform-d) δ 8.67 (s, 1H), 8.00 (d, J = 2.2 Hz, 1H),

7.98 (d, J = 9.0 Hz, 1H), 7.42 (dd, J = 9.0, 2.2 Hz, 1H), 3.49 (q, J = 6.4 Hz, 1H), 2.49 (q, J = 7.2

Hz, 4H), 2.38 (t, J = 7.3 Hz, 2H), 1.69 – 1.61 (m, 1H), 1.53 – 1.43 (m, 3H), 1.21 (d, J = 6.5 Hz,
13
3H), 0.98 (t, J = 7.1 Hz, 6H). C NMR (126 MHz, Chloroform-d) δ 147.93, 146.13, 145.08,

134.30, 128.91, 128.14, 127.48, 125.96, 125.91, 58.61, 53.02, 46.94, 35.52, 32.74, 24.11, 17.90,

11.54. IR (cm-1) 2966.6, 2933.2, 2110.6, 1559.3, 1451.4, 1381.8, 1319.9, 1073.4, 915.8. HRMS

(ESI) calcd. for C19H28ClN6 [M+H+]: 375.2072, found: 375.2074.

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

OH
N3

Cl N

3-azido-7-chloroquinolin-4-ol (4.18)

See procedure for 4.17 and 4.18 was a precipitate that was filtered, washed with H2O and dried

under house vacuum overnight to give a brown solid, 81.1 mg, 39%.

Yield: 81.1 mg, 39%. 1H NMR (500 MHz, Dimethyl sulfoxide-d6) δ 12.23 (d, J = 6.2 Hz, 1H),

8.11 (d, J = 8.7 Hz, 1H), 7.95 (d, J = 6.3 Hz, 1H), 7.63 (d, J = 2.0 Hz, 1H), 7.38 (dd, J = 8.7, 2.0

Hz, 1H). 13C NMR (126 MHz, Dimethyl sulfoxide-d6) δ 171.72, 139.35, 136.45, 130.19, 127.04,

124.10, 122.37, 120.30, 117.71. IR (cm-1) 3075.6, 2124.7, 1627.9, 1558.2, 1512.3, 1460.4, 1350.8,

1188.7, 1075.2, 812.8. HRMS (ESI) calcd for C9H5ClN4O [M+Na+]: 243.0044, found: 243.0043.

4.5.3 X-ray crystallography

Crystals for the ring-opened DBU adduct 1-(3-((7-chloroquinolin-4-yl)amino)propyl)azepan-2-

one are grown from DCM and Hex. A large 0.1 mm x 0.1 mm x 0.06 mm prism is mounted on a

glass fibre with epoxy resin. Single-crystal X-ray diffraction is then measured at RT with a

BRUKER SMART CCD or BRUKER APEX-II CCD diffractometer by using graphite-

monochromated MoKα radiation (λ = 0.71073 Å). The crystal crystallizes in the monoclinic space

group P21/c with the unit cell parameters a = 10.65(2); b = 16.68(3); c = 10.68(2) Å and α = γ =

90 and β = 116.34(3) ° which corresponds to V = 1700.0(6) Å3 and Z = 4. SAINT is used for

integration of the intensity reflections and scaling and SADABS for absorption correction. A

combination of intrinsic phasing and direct methods were used for solving the structure which

were subsequently anisotropically refined after all non-hydrogen atoms were located by difference

Fourier maps and final solution refinements are solved by full-matrix least-squares method on F2

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

of all data, by using SHELXTL software. The hydrogen atoms are placed in calculated positions.

The final refinement has R1 = 0.0340 and Sgof = 1.167.

4.5.4 UV-Vis Studies

A 2.4 mg of 3-N3MeCQ is dissolved in 30 mL of MeOH to make a 213.4 µM stock solution. Then

2.9 mg of 3-N3MeCQ is dissolved in 1 mL of MeOH and the resulting mixture is diluted with 29

mL of H2O to make a 257.8 µM stock solution. A 3.8 mg of GlyGlyGly is dissolved in 30 mL

deionized H2O to make a 669.6 µM stock solution. Then 6.5 mg of N-Phenylmaleimide was

dissolved in 30 mL of MeOH to make a 1,251.2 µM stock solution.

General procedure

A 2 mL blank solution of deionized H2O or MeOH in a 4 mL quartz cuvette is taken in an Agilent

UV-Vis Spectrophotometer for the area 190 nm – 1100 nm. To a different 4 mL quartz cuvette is

added 333 µL of the 257.8 µM stock solution and 1,667 µL of deionized H2O to make a 42.97 µM

3-N3MeCQ solution. This is thoroughly mixed using a micropipette to make a homogeneous

solution. A stir bar is placed in the cuvette and the cuvette is capped and placed in a cuvette holder

fitted with a 365 nm penlight and the setup is then covered with aluminum foil. The control

spectrum is measured with the light off. The experiment is then started in kinetics mode for a

spectrum to be taken every 10 seconds for 300 seconds whilst the light is on and the solution is

stirring. When the experiment is finished the resulting solution is transferred into a 2mL Eppendorf

tube for mass spectroscopy.

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

To a different 4 mL quartz cuvette the stock 3-N3MeCQ solution is diluted with deionized H2O or

MeOH to make the desired concentrated solution. This is thoroughly mixed using a micropipette

to make a homogeneous solution. A stir bar is placed in the cuvette and the cuvette is capped and

placed in a cuvette holder fitted with a 365 nm penlight and the setup is then covered with

aluminum foil. The control spectrum is measured with the light off. The experiment is then started

in kinetics mode for a spectrum to be taken every 10 seconds for 300 seconds whilst the light is on

and the solution is stirring. When the experiment is finished the resulting solution is transferred

into a 2 mL Eppendorf tube for mass spectroscopy.

Insertion experiments

21.49 µM 3-N3MeCQ and GlyGlyGly in H2O (1:1)

5,000 µL of the 257.8 µM 3-N3MeCQ stock solution is further diluted into 30 mL of deionized

H2O to make a 42.97 µM solution, and 1,000 µL of this solution is then transferred into a 4 mL

quartz cuvette. 2,000 µL of the 669.6 µM GlyGlyGly stock solution is further diluted into 30 mL

of deionized H2O to make a 44.64 µM and 1,000 µL of this solution is added into the above 4 mL

cuvette to give final concentrations of 21.49 µM and 22.32 µM of 3-N3MeCQ and GlyGlyGly

respectively. The kinetics experiment is run as mentioned above.

4.29 µM 3-N3MeCQ and GlyGlyGly in H2O (1:9.4)

200 µL of the freshly made 42.97 µM 3-N3MeCQ solution is transferred into a 4 mL quartz cuvette.

To this is added 1,800 µL of the freshly made 44.64 µM GlyGlyGly solution to give final

concentrations of 4.29 µM and 40.18 µM of 3-N3MeCQ and GlyGlyGly, respectively. The kinetics

experiment is run as mentioned above.

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

19.56 µM 3-N3MeCQ and N-Phenylmaleimide in MeOH (1:1)

5,000 µL of the 213.4 µM 3-N3MeCQ stock solution is further diluted into 30 mL of MeOH to

make a 35.57 µM solution and 1,100 µL of this solution is then transferred into a 4 mL quartz

cuvette. 1,000 µL of the 1,251.2 µM N-Phenylmaleimide stock solution is further diluted into 30

mL of MeOH to make a 41.71 µM solution and 900 µL of this solution is added into the above 4

mL cuvette to give final concentrations of 19.56 µM and 18.78 µM of 3-N3MeCQ and N-

Phenylmaleimide respectively. The kinetics experiment is run as mentioned above.

7.11 µM 3-N3MeCQ and N-Phenylmaleimide in MeOH (1:4.7)

400 µL of the freshly made 35.57 µM 3-N3MeCQ solution is transferred into a 4 mL quartz cuvette.

To this is added 1,600 µL of the freshly made 41.71 µM N-Phenylmaleimide solution to give final

concentrations of 7.11 µM and 33.37 µM of 3-N3MeCQ and N-Phenylmaleimide, respectively.

The kinetics experiment is run as mentioned above.

3.56 µM 3-N3MeCQ and N-Phenylmaleimide in MeOH (1:10.6)

200 µL of the freshly made 35.57 µM 3-N3MeCQ solution is transferred into a 4 mL quartz cuvette.

To this is added 1,800 µL of the freshly made 41.71 µM N-Phenylmaleimide solution to give final

concentrations of 3.56 µM and 37.54 µM of 3-N3MeCQ and N-Phenylmaleimide, respectively.

The kinetics experiment is run as mentioned above.

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

4.5.5 NMR experiments

General procedure

In a 0.5-dram vial is added 3-N3MeCQ, the electron acceptor and 500 µL of deuterated MeOH.

This is thoroughly mixed to make a homogenous solution. This mixture is then transferred into an

8” NMR tube fitted with a stir bar. A 365 nm penlight is positioned 5 cm from the NMR tube. In

a dark fume hood with the setup covered in aluminum foil, the solution is let stir exposed to 365

nm for 6 to 9 minutes. Once the time elapses the stir bar is removed and a 1H NMR spectrum is

measured.

3-N3MeCQ and N-Phenylmaleimide

5 mg of 3-N3MeCQ and 2.3 mg of N-Phenylmaleimide were used and irradiated for 6 minutes.

N-Phenylmaleimide

2.3 mg of N-Phenylmaleimide used and irradiated for 9 minutes.

3-N3MeCQ and Tetracyanoethylene

5 mg of 3-N3MeCQ and 2.3 mg of Tetracyanoethylene were used and irradiated for 9 minutes.

179
Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

4.6 References

(1) Slater, A. F. G. Chloroquine: Mechanism of Drug Action and Resistance in Plasmodium

Falciparum. Pharmacol. Ther. 1993, 57 (2–3), 203–235.

(2) Drinkwater, N.; McGowan, S. From Crystal to Compound: Structure-Based Antimalarial

Drug Discovery. Biochem. J. 2014, 461 (3), 349–369.

(3) Sullivan, D. J. Quinolines Block Every Step of Malaria Heme Crystal Growth. Proc. Natl.

Acad. Sci. 2017, 114 (29), 7483–7485.

(4) Kapishnikov, S.; Staalsø, T.; Yang, Y.; Lee, J.; Pérez-Berná, A. J.; Pereiro, E.; Yang, Y.;

Werner, S.; Guttmann, P.; Leiserowitz, L.; Als-Nielsen, J. Mode of Action of Quinoline

Antimalarial Drugs in Red Blood Cells Infected by Plasmodium Falciparum Revealed in

Vivo. Proc. Natl. Acad. Sci. U. S. A. 2019, 116 (46), 22946–22952.

(5) Bray, P. G.; Park, B.; Asadollaly, E.; Biagini, G.; Jeyadevan, J.; Berry, N.; Ward, S.; O’

Neill, P. A Medicinal Chemistry Perspective on 4-Aminoquinoline Antimalarial Drugs.

Curr. Top. Med. Chem. 2006, 6 (5), 479–507.

(6) Foley, M.; Deady, L. W.; Ng, K.; Cowman, A. F.; Tilley, L. Photoaffinity Labeling of

Chloroquine-Binding Proteins in Plasmodium Falciparum. J. Biol. Chem. 1994, 269 (9),

6955–6961.

(7) Mueller, D. M.; Hudson, R. A.; Lee, C.-P. Azide Photoaffinity Analogs for Acridine Dye

Binding Sites. J. Am. Chem. Soc. 1981, 103 (7), 1860–1862.

(8) Desneves, J.; Thorn, G.; Berman, A.; Galatis, D.; La Greca, N.; Sinding, J.; Foley, M.;

Deady, L. W.; Cowman, A. F.; Tilley, L. Photoaffinity Labeling of Mefloquine-Binding

Proteins in Human Serum, Uninfected Erythrocytes and Plasmodium Falciparum-Infected

Erythrocytes. Mol. Biochem. Parasitol. 1996, 82 (2), 181–194.

180
Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

(9) Lekostaj, J. K.; Natarajan, J. K.; Paguio, M. F.; Wolf, C.; Roepe, P. D. Photoaffinity

Labeling of the Plasmodium Falciparum Chloroquine Resistance Transporter with a Novel

Perfluorophenylazido Chloroquine †. Biochemistry 2008, 47 (39), 10394–10406.

(10) Edaye, S.; Tazoo, D.; Bohle, D. S.; Georges, E. 3-Iodo-4-Aminoquinoline Derivative

Sensitises Resistant Strains of Plasmodium Falciparum to Chloroquine. Int. J. Antimicrob.

Agents 2016, 47 (6), 482–485.

(11) Edaye, S.; Tazoo, D.; Bohle, D. S.; Georges, E. 3-Halo Chloroquine Derivatives Overcome

Plasmodium Falciparum Chloroquine Resistance Transporter-Mediated Drug Resistance in

P. Falciparum. Antimicrob. Agents Chemother. 2015, 59 (12), 7891–7893.

(12) Ribas, X.; Güell, I. Cu(I)/Cu(III) Catalytic Cycle Involved in Ullmann-Type Cross-

Coupling Reactions. Pure Appl. Chem. 2014, 86 (3), 345–360.

(13) Andersen, J.; Bolvig, S.; Liang, X. Efficient One-Pot Synthesis of 1-Aryl 1,2,3-Triazoles

from Aryl Halides and Terminal Alkynes in the Presence of Sodium Azide. Synlett 2005,

2005 (19), 2941–2947.

(14) Lanke, S. R.; Bhanage, B. M. Copper Bis(2,2,6,6-Tetramethyl-3,5-Heptanedionate)–

Catalyzed Coupling of Sodium Azide with Aryl Iodides/Boronic Acids to Aryl Azides or

Aryl Amines. Synth. Commun. 2014, 44 (3), 399–407.

(15) Zhu, W.; Ma, D. Synthesis of Aryl Azides and Vinyl Azides via Proline-Promoted CuI-

Catalyzed Coupling Reactions. Chem. Commun. 2004, No. 7, 888–889.

(16) Markiewicz, J. T.; Wiest, O.; Helquist, P. Synthesis of Primary Aryl Amines Through a

Copper-Assisted Aromatic Substitution Reaction with Sodium Azide. J. Org. Chem. 2010,

75 (14), 4887–4890.

(17) Messaoudi, S.; Brion, J.-D.; Alami, M. An Expeditious Copper-Catalyzed Access to 3-

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

Aminoquinolinones, 3-Aminocoumarins and Anilines Using Sodium Azide. Adv. Synth.

Catal. 2010, 352 (10), 1677–1687.

(18) Goriya, Y.; Ramana, C. V. The [Cu]-Catalyzed SNAR Reactions: Direct Amination of

Electron Deficient Aryl Halides with Sodium Azide and the Synthesis of Arylthioethers

under Cu(II)–Ascorbate Redox System. Tetrahedron 2010, 66 (38), 7642–7650.

(19) Qin, Z.; Kastrati, I.; Chandrasena, R. E. P.; Liu, H.; Yao, P.; Petukhov, P. A.; Bolton, J. L.;

Thatcher, G. R. J. Benzothiophene Selective Estrogen Receptor Modulators with Modulated

Oxidative Activity and Receptor Affinity. J. Med. Chem. 2007, 50 (11), 2682–2692.

(20) Gehringer, M.; Forster, M.; Pfaffenrot, E.; Bauer, S. M.; Laufer, S. A. Novel Hinge-Binding

Motifs for Janus Kinase 3 Inhibitors: A Comprehensive Structure-Activity Relationship

Study on Tofacitinib Bioisosteres. ChemMedChem 2014, 9 (11), 2516–2527.

(21) Kaila, N.; Green, N.; Li, H.-Q.; Hu, Y.; Janz, K.; Gavrin, L. K.; Thomason, J.; Tam, S.;

Powell, D.; Cuozzo, J.; Hall, J. P.; Telliez, J.-B.; Hsu, S.; Nickerson-Nutter, C.; Wang, Q.;

Lin, L.-L. Identification of a Novel Class of Selective Tpl2 Kinase Inhibitors: 4-

Alkylamino-[1,7]Naphthyridine-3-Carbonitriles. Bioorg. Med. Chem. 2007, 15 (19), 6425–

6442.

(22) Rahman, M.; Kundu, D.; Hajra, A.; Majee, A. Formylation without Catalyst and Solvent at

80°C. Tetrahedron Lett. 2010, 51 (21), 2896–2899.

(23) Kim, J.-G.; Jang, D. Facile and Highly Efficient N-Formylation of Amines Using a Catalytic

Amount of Iodine under Solvent-Free Conditions. Synlett 2010, 2010 (14), 2093–2096.

(24) Shastri, L. A.; Shastri, S. L.; Bathula, C. D.; Basanagouda, M.; Kulkarni, M. V. Mild,

Simple, and Efficient Method for N -Formylation of Secondary Amines via Reimer–

Tiemann Reaction. Synth. Commun. 2011, 41 (4), 476–484.

182
Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

(25) Collet, J. W.; Ackermans, K.; Lambregts, J.; Maes, B. U. W.; Orru, R. V. A.; Ruijter, E.

Modular Three-Component Synthesis of 4-Aminoquinolines via an Imidoylative

Sonogashira/Cyclization Cascade. J. Org. Chem. 2018, 83 (2), 854–861.

(26) Pajkert, R.; Böttcher, T.; Ponomarenko, M.; Bremer, M.; Röschenthaler, G.-V. Synthesis

and Characterization of Novel Carbene Complexes of Phosphorus(V) Fluorides with

Potential Liquid-Crystalline Properties. Tetrahedron 2013, 69 (42), 8943–8951.

(27) Sánchez-Martín, R.; Campos, J. M.; Conejo-García, A.; Cruz-López, O.; Báñez-Coronel,

M.; Rodríguez-González, A.; Gallo, M. A.; Lacal, J. C.; Espinosa, A. Symmetrical Bis-

Quinolinium Compounds: New Human Choline Kinase Inhibitors with Antiproliferative

Activity against the HT-29 Cell Line. J. Med. Chem. 2005, 48 (9), 3354–3363.

(28) Rodrigues, T.; Guedes, R. C.; dos Santos, D. J. V. A.; Carrasco, M.; Gut, J.; Rosenthal, P.

J.; Moreira, R.; Lopes, F. Design, Synthesis and Structure–Activity Relationships of (1H-

Pyridin-4-Ylidene)Amines as Potential Antimalarials. Bioorg. Med. Chem. Lett. 2009, 19

(13), 3476–3480.

(29) Natarajan, J. K.; Alumasa, J. N.; Yearick, K.; Ekoue-Kovi, K. A.; Casabianca, L. B.; De

Dios, A. C.; Wolf, C.; Roepe, P. D. 4-N-, 4-S-, and 4-O-Chloroquine Analogues: Influence

of Side Chain Length and Quinolyl Nitrogen PKa on Activity vs Chloroquine Resistant

Malaria. J. Med. Chem. 2008, 51 (12), 3466–3479.

(30) Shi, M.; Shen, Y. A Novel Reaction of 1,8-Diazabicyclo[5.4.0]Undec-7-Ene (DBU) or 1,5-

Diazabicyclo[4.3.0]Non-5-Ene (DBN) with Benzyl Halides in the Presence of Water. Helv.

Chim. Acta 2002, 85 (5), 1355.

(31) Kraft, A. Branched Non-Covalent Complexes between Carboxylic Acids and Two

Tris(Amidines). J. Chem. Soc. Perkin Trans. 1 1999, No. 6, 705–714.

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(32) Hyde, A. M.; Calabria, R.; Arvary, R.; Wang, X.; Klapars, A. Investigating the

Underappreciated Hydrolytic Instability of 1,8-Diazabicyclo[5.4.0]Undec-7-Ene and

Related Unsaturated Nitrogenous Bases. Org. Process Res. Dev. 2019, 23 (9), 1860–1871.

(33) Sather, A. C.; Lee, H. G.; De La Rosa, V. Y.; Yang, Y.; Müller, P.; Buchwald, S. L. A

Fluorinated Ligand Enables Room-Temperature and Regioselective Pd-Catalyzed

Fluorination of Aryl Triflates and Bromides. J. Am. Chem. Soc. 2015, 137 (41), 13433–

13438.

(34) Sinha, M.; Dola, V. R.; Soni, A.; Agarwal, P.; Srivastava, K.; Haq, W.; Puri, S. K.; Katti,

S. B. Synthesis of Chiral Chloroquine and Its Analogues as Antimalarial Agents. Bioorg.

Med. Chem. 2014, 22 (21), 5950–5960.

(35) Johnson, W. S.; Buell, B. G. A New Synthesis of Chloroquine. J. Am. Chem. Soc. 1952, 74

(18), 4513–4516.

(36) Gorlitzer, K.; Kramer, C.; Meyer, H.; Walter, R. D.; Jomaa, H.; Wiesner, J. Pyrido[3,2-

b]Indol-4-Yl-Amine – Synthese Und Prufung Auf Wirksamkeit Gegen Malaria. Die Pharm.

- An Int. J. Pharm. Sci. 2004, 59 (4), 243–250.

(37) Liu, Y.; Zhang, Z.; Wu, A.; Yang, X.; Zhu, Y.; Zhao, N. A Novel Process for Antimalarial

Drug Pyronaridine Tetraphosphate. Org. Process Res. Dev. 2014, 18 (2), 349–353.

(38) Elslager, E. F.; Gold, E. H.; Tendick, F. H.; Werbel, L. M.; Worth, D. F. Amodiaquine N-

Oxides and Other 7-Chloro-4-Aminoquinoline n-Oxides. J. Heterocycl. Chem. 1964, 1 (1),

6–12.

(39) Wang, X.; Pan, W.; Cai, Y.; Xie, X.; Huang, C.; Li, J.; Chen, W.; He, M. Microwave-

Assisted Efficient Synthesis of 4-Substituted Amino-2-Methylquinolines Catalyzed by p-

Toluenesulfonic Acid. Heterocycles 2016, 92 (10), 1864.

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

(40) Bayley, H.; Staros, J. V. Photoaffinity Labeling and Related Techniques. In Azides and

Nitrenes; Elsevier, 1984; pp 433–490.

(41) Vezmar, M.; Georges, E. Direct Binding of Chloroquine to the Multidrug Resistance Protein

(MRP). Biochem. Pharmacol. 1998, 56 (6), 733–742.

(42) Baruah, H.; Puthenveetil, S.; Choi, Y.-A.; Shah, S.; Ting, A. Y. An Engineered Aryl Azide

Ligase for Site-Specific Mapping of Protein-Protein Interactions through Photo-Cross-

Linking. Angew. Chemie Int. Ed. 2008, 47 (37), 7018–7021.

(43) Berman, A.; Shearing, L. N.; Ng, K. F.; Jinsart, W.; Foley, M.; Tilley, L. Photoaffinity

Labelling of Plasmodium Falciparum Proteins Involved in Phospholipid Transport. Mol.

Biochem. Parasitol. 1994, 67 (2), 235–243.

(44) Wang, J.; Burdzinski, G.; Platz, M. S. Ultrafast Time-Resolved Studies of the

Photochemistry of Aryl Azides. In Nitrenes and Nitrenium Ions; 2013; pp 1–31.

(45) Vyas, S.; Winter, A. H.; Hadad, C. M. Theory and Computation in the Study of Nitrenes

and Their Excited-State Photoprecursors. In Nitrenes and Nitrenium Ions; John Wiley &

Sons, Inc.: Hoboken, NJ, USA, 2013; pp 33–76.

(46) Bräse, S.; Banert, K. Organic Azides; Brse, S., Banert, K., Eds.; John Wiley & Sons, Ltd:

Chichester, UK, 2009.

(47) Zott, H.; Heusinger, H. Photolysis of Maleic Anhydride and Maleimides in Solution Studied

by Electron Spin Resonance. Radiat. Phys. Chem. 1977, 10 (5–6), 297–302.

(48) Kiselev, V. D.; Kashaeva, H. A.; Potapova, L. N.; Kornilov, D. A.; Latypova, L. I.;

Konovalov, A. I. Hydrophobic Acceleration in the Diels—Alder Reaction of 9-

Hydroxymethylanthracene with N-Phenylmaleimide. Russ. Chem. Bull. 2016, 65 (9), 2202–

2205.

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Chapter 4 Synthesis and Reactivity of a Compact Chloroquine Photoaffinity label.

(49) Murata, S.; Abe, S.; Tomioka, H. Photochemical Reactions of Mesityl Azide with

Tetracyanoethylene: Competitive Trapping of Singlet Nitrene and Didehydroazepine. J.

Org. Chem. 1997, 62 (10), 3055–3061.

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 Chapter 5

The Determination of Heme and Chloroquine Interactions

with Plasmodium falciparum Chloroquine Resistance

Transporter (PfCRT)

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Chapter 5 The Determination of Heme and Chloroquine interactions with PfCRT

5.1 Preamble

In the previous chapter, the synthesis and utilization of our photoaffinity label was described. This

label is expected to cross-link to PfCRT. PfCRT contains several point mutations that have been

directly linked to CQ resistance. There is much debate about this protein’s function, whether it acts

as a transporter or channel and if it is energy coupled.1 There is also evidence of cross-resistance

between different classes of antimalarials, and the mechanism of substrate recognition is also

poorly understood.2 As our photoaffinity label contains minor modifications compared to CQ, this

possibly avoids unnecessary interactions with surrounding proteins. PfCRT has been isolated, and

its cryo-electron microscopy structure was determined for the first time.3 Therefore, before

embarking on photoaffinity labelling on PfCRT, we wanted to determine the binding interactions

and binding affinity of PfCRT with CQ and heme. In collaboration with the Fidock group who

provided PfCRT isoform, we carried out fluorescence and UV-Vis studies. These studies were

carried out in buffers of pH 7.0 – 8.0, which is within the range of cytosolic pH. The challenge

with carrying out this assay is that PfCRT is a transmembrane protein with one side exposed to pH

5.2 and the other at pH 7.4. Therefore we chose to model the cytosolic pH.

5.2 Introduction

CQ, first synthesized in 1934, revolutionized antimalarial therapy. It was viewed as the drug that

would eradicate malaria. Unfortunately, in the early 1960s, resistance to CQ was discovered.4 A

tremendous amount of effort has been made in determining the antimalarial mechanism of CQ and

the origin of its resistance.5–9 The structure of hemozoin which is a biomineralization byproduct

synthesized by the parasite to detoxify free heme in the DV was solved in 2000.10 A lysine to

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Chapter 5 The Determination of Heme and Chloroquine interactions with PfCRT

threonine mutation at position 76 (K76T) on the PfCRT is a biomarker for CQ resistance that was

also discovered in 2000.6 PfCRT has been sequenced, and there has been much speculation on the

mechanism in which the protein recognizes antimalarial drugs. Moreover, for the first time, a

PfCRT isoform of CQ-resistant 7G8 parasites was isolated using single-particle cryo-electron

microscopy and antigen-binding technology in 2019.3 The nanodisc-incorporated PfCRT 7G8

isoform was solved at 3.2 Å.

The isolated PfCRT isoform is a 424 amino acid protein with a molecular weight of 48 kDa. It is

a monomeric transmembrane protein that consists of ten transmembrane (TM) helices and two

juxtamembrane helices. One is exposed to the parasite cytosol and the other to the DV (Figure

5.1). The transmembrane domains form 5 helical pairs, and TM1-4 and 6-9 form a central cavity

of around 3,300 Å3. The K76T mutation is located within the lining of the cavity. The cavity has

a net negative charge influenced by three aspartate residues, suggesting positively charged

substrates in acidic media.3

a) b)

Figure 5.1. Structure of PfCRT 7G8 isoform. A) Topology showing inverted antiparallel TM

helices. B) Surface representation of the electrostatic potential of the central cavity with red and

blue indicating negative and positive residues, respectively. Figures adapted from Kim et al.3

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Chapter 5 The Determination of Heme and Chloroquine interactions with PfCRT

In this chapter, fluorescence spectroscopy and ultraviolet-visible spectroscopy were two

techniques used to determine interactions between CQ and heme with PfCRT are described.

Including two control substrates: empty nanodisc (1D1), which has the same lipid membrane used

for PfCRT filled with lipids and unrelated GtrB from an enterobacteria phage enclosed in a

different nanodisc (1E3D1). This ensemble will be referred to as membrane proteins and lipid

membrane ensemble (MPLME).

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Chapter 5 The Determination of Heme and Chloroquine interactions with PfCRT

5.3 Results and Discussion

In collaboration with Prof. David Fidock’s lab, we were generously gifted PfCRT 7G8 isoform,

nanodisc and GtrB. It was first believed that the cavity with a volume of 3,300 Å3 was large enough

to transport out the CQ - heme complex that forms within the DV. CQ and hemin have approximate

volumes of 396 Å3 and 702 Å3, respectively (as estimated from crystallographic results). We began

with binding studies of the individual drugs with the ensembles to determine possible recognition

of CQ or heme with the transporter protein. To determine the interactions between CQ, heme and

PfCRT, we carried out some fluorescence titration assays.

5.3.1 Fluorescence Studies

5.3.1.1 Fluorescence studies with CQ

Fluorescence spectroscopy is a very useful tool in determining protein-drug interactions.11 It is

proposed that CQ is transported out of the DV by PfCRT, which will entail recognition, binding

and transport.2,6 The observed PfCRT fluorescence primarily originates from tryptophan (Trp) 280

and 316, with the empty nanodisc contains 2 Trp residues and GtrB 3 Trp residues. As Trp

emission is highly sensitive to its microenvironment, changes in fluorescence are used to determine

protein-drug interactions.12 PfCRT’s two Trp residues are located in the juxtamembrane, two and

TM eight, exposed to the DV, Figure 5.1. Fluorescence quenching and shifts in the emission

wavelength typically indicate protein binding or collisional quenching by the drug.

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Chapter 5 The Determination of Heme and Chloroquine interactions with PfCRT

5.92uM PfCRT pH 7 4.98uM PfCRT pH 8

2.0×107
PfCRT PfCRT
8×106 1eq 1eq

Fluorescence Intensity
Fluorescence Intensity

2eq 2eq
1.5×107
3eq 3eq
6×106 4eq
4eq
1.0×107
4×106

2×106 5.0×106

0 0.0
350 400 450 500 350 400 450
Wavelength (nm) Wavelength (nm)
3.16uM GtrB 5.92uM Nanodisc
4×107 1×107
GtrB Nanodisc
1eq 1eq
Fluorescence Intensity

Fluorescence Intensity
2eq 2eq
3×107
3eq 3eq
4eq 4eq
2×107 5×106

1×107

0 0
350 400 450 350 400 450 500
Wavelength (nm) Wavelength (nm)

Figure 5.2. Effect of 4 equivalent of CQ on the emission spectrum of PfCRT at pH 7, PfCRT at

pH 8, GtrB and nanodisc.

We assayed PfCRT at pH 7 and pH 8, GtrB at pH 7 and nanodisc at pH 7 with CQ.2H3PO4 (in

solution as CQH2+). As shown in Figure 5.2, increasing amounts of CQ, up to 4 equivalents, causes

a slight decrease in emission energy, and this decrease is more pronounced in empty nanodisc.

Furthermore, we observe the following shifts in the fluorescence maxima: PfCRT pH 8 10 nm red

shift, GtrB 5 nm red shift and nanodisc 5 nm red shift. The fluorescence quenching constant was

analyzed using the Stern-Volmer equation11 (1):

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Chapter 5 The Determination of Heme and Chloroquine interactions with PfCRT

H)
= 1 + I* J) [L] = 1 + B+, [L]
H

where F0 and F are the fluorescence intensities of the protein in the absence and presence of the

quencher, respectively, kq is the bimolecular quenching constant, τ0 is the protein unquenched

lifetime, KSV is the Stern-Volmer quenching constant and [Q] is the concentration of the quencher.

Table 5.1. Stern-Volmer quenching constants for CQ.

PROTEIN KSV × 104 (M-1)

PfCRT pH 7 0.14 ± 0.14
PfCRT pH 8 0.06 ± 0.31
GtrB 1.54 ± 0.41
Nanodisc 3.55 ± 0.44

Table 5.1 shows the KSV values of our membrane proteins and lipid membrane ensemble

(MPLME) and CQ. The higher the quenching constant, the stronger the association. The data

shows that nanodisc has the highest quenching constant. In general, a linear plot suggests the

occurrence of either static or dynamic quenching, Figure 5.3. Static quenching involves the

formation of nonfluorescent complexes between the fluorophore and the quencher that occur in

the ground state. Dynamic quenching, which occurs in the excited state, involves the diffusion of

the fluorophore during the lifetime of the excited state. The fluorophore then returns to the ground

state upon contact without emission of a photon. Both types of quenching mechanisms occur only

through space contact between the fluorophore and quencher and do not result in a permanent

change to the molecules involved.11

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Chapter 5 The Determination of Heme and Chloroquine interactions with PfCRT

5.92uM PfCRT pH 7 4.98uM PfCRT pH 8

1.20 1.3

1.15
1.2
F0/F (345nm)

F0/F (335nm)
1.10
1.1
1.05
1.0
1.00

0.95 0.9
0.0 5.0×10-6 1.0×10-5 1.5×10-5 2.0×10-5 0.0 5.0×10-6 1.0×10-5 1.5×10-5
[CQ] [CQ]
3.16uM GtrB 5.92uM Nanodisc
1.3 2.0

1.8
1.2
F0/F (340nm)

F0/F (340nm)
1.6
1.1
1.4
1.0
1.2

0.9 1.0
0 2×10-6 4×10-6 6×10-6 8×10-6 1×10-5 0.0 5.0×10-6 1.0×10-5 1.5×10-5 2.0×10-5
[CQ] [CQ]

Figure 5.3. Stern-Volmer plots for PfCRT, GtrB and nanodisc quenched with up to 4 equivalents

of CQ (each dot represents a 0.2 molar addition).

A factor affecting the specific quenching mechanism is the accessibility of the quencher to the

fluorophore. The fraction of accessible fluorophores can be measured using the modified Stern-

Volmer, equation (2):

H) 1 1
= +
H) − H NB& [L] N

Where F0 and F are the fluorescence intensities in the presence and absence of the quencher, α is

the fraction of accessible fluorophores, Ka is the effective quenching constant and [Q] is the

concentration of the quencher.

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Chapter 5 The Determination of Heme and Chloroquine interactions with PfCRT

5.92uM PfCRT pH 7 4.98 uM PfCRT pH 8

60 20

15
40
F0/F0-F

F0/F0-F
10

20
5

0 0
0 2×105 4×105 6×105 8×105 1×106 0 500000 1×106 2×106
1/[CQ] 1/[CQ]
3.16uM GtrB 5.92uM Nanodisc
40 8

30 6
F0/F0-F

F0/F0-F
20 4

10 2

0 0
0.0 5.0×105 1.0×106 1.5×106 2.0×106 0 2×105 4×105 6×105 8×105 1×106
1/[CQ] 1/[CQ]

Figure 5.4. Modified Stern-Volmer plots for PfCRT, GtrB and nanodisc quenched with up to 4

equivalents of CQ (each dot represents a 0.2 molar addition).

The modified Stern-Volmer plots, shown in Figure 5.4, give an N value of 35% or less. In general,

N values less than 100% are a result of fractional accessibility, which is due to the existence of at

least two fluorophore populations with one that is accessible and the other is buried or

inaccessible.13 Each of these fluorophores would have varying KSV values and are differently

accessible to the quencher. The Ka value, in Table 5.2, is the effective quenching constant of

accessible fluorophores, this is in accordance with the KSV values in Table 5.1. CQ in nanodisc is

exposed to the highest fraction of accessible fluorophores as it has the highest KSV value. CQ has

little accessibility to the Trp residues found in PfCRT at both pH 7 and 8.

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Chapter 5 The Determination of Heme and Chloroquine interactions with PfCRT

Table 5.2. Modified Stern-Volmer constants for CQ.

PROTEIN Ka × 105 (M-1) ɑ (%)

PfCRT pH 7 4.92 ± 2.58 5.42
PfCRT pH 8 8.21 ± 3.09 14.16
GtrB 4.35 ± 4.79 13.65
Nanodisc 7.17 ± 2.62 35.51

Interestingly, there appears to be a concentration dependant response from the Stern-Volmer plot

for PfCRT pH 7 and CQ, Figure 5.5. . The data points of 0 - 1 equiv. give KSV of 1.69 × 10-4 M-
1
and 1 - 4 equiv. of 0.19 × 10-4 M-1. This may be due to conformational change following binding

of one equivalent of CQ which reduces the accessibility of fluorophores to additional CQ.

Similarly, for PfCRT pH 8 and CQ, data points of 0 – 2 equiv. give KSV of 1.5 × 10-4 M-1; however,

2 – 4 equiv. of -2.2 × 10-4 M-1,

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5.92uM PfCRT pH 7 0-1 eq. CQ 5.92uM PfCRT pH 7 1-4 eq. CQ

1.15 1.20

1.15
1.10

F0/F (345nm)
F0/F (345nm)

1.10
1.05
1.05

1.00
1.00

0.95 0.95
0 2×10-6 4×10-6 6×10-6 0.0 5.0×10-6 1.0×10-5 1.5×10-5 2.0×10-5
[CQ] [CQ]

4.98uM PfCRT pH 8 0-2 eq. CQ 4.98uM PfCRT pH 8 2-4 eq. CQ

1.3 1.3

1.2 1.2
F0/F (335nm)
F0/F (335nm)

1.1 1.1

1.0 1.0

0.9 0.9
0 2×10-6 4×10-6 6×10-6 8×10-6 1×10-5 6.0×10-6 8.0×10-6 1.0×10-5 1.2×10-5 1.4×10-5
[CQ] [CQ]

Figure 5.5. Concentration-dependent response of PfCRT to CQ.

To determine the number of binding sites for CQ present on the MPLME, we use the following

equation (3)14:

H) − H
log = log B- + ) log [L]
H

where F0 and F are fluorescent intensities in the presence and absence of a quencher, Kb is the

binding constant, n is the number of binding sites and [Q] is the quencher concentration.

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Chapter 5 The Determination of Heme and Chloroquine interactions with PfCRT

Table 5.3. Binding constant and number of binding sites for CQ with MPLME.

PROTEIN logKb n
PfCRT pH 7 0.06 ± 0.83 0.27 ± 0.16
PfCRT pH 8 0.01 ± 0.87 0.18 ± 0.17
GtrB 1.89 ± 0.58 0.54 ± 0.11
Nanodisc 3.05 ± 0.37 0.67 ± 0.07

Stronger binding interactions give higher logKb values, and the values in Table 5.3. are in

accordance with previous KSV and Ka values. Nanodisc displays the strongest binding interactions

with CQ in the ensemble, although with an n value of 0.67, we cannot conclusively say that

nanodisc contains a binding site for CQ, Figure 5.6. PfCRT at both pH’s form weak associations

with CQ and does not have a specific CQ binding site. The control, GtrB, forms stronger

associations with CQ than PfCRT; however, it does not contain a binding site for CQ.

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5.92uM PfCRT pH 7 4.98uM PfCRT pH 8

-0.8 0.0

-1.0
-0.5
Log(F0-F)/F

Log(F0-F)/F
-1.2

-1.4 -1.0

-1.6
-1.5
-1.8

-2.0 -2.0
-6.5 -6.0 -5.5 -5.0 -4.5 -6.5 -6.0 -5.5 -5.0 -4.5
Log[CQ] Log[CQ]
3.16uM GtrB 5.92uM Nanodisc
0.0 0.0

-0.5 -0.2
Log(F0-F)/F

-1.0 Log(F0-F)/F -0.4

-1.5 -0.6

-2.0 -0.8
-7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -6.0 -5.5 -5.0 -4.5
Log[CQ] Log[CQ]

Figure 5.6. Binding plot to determine the number of CQ binding sites and binding constant.

All assays were carried out at 25oC at pH 7, except PfCRT at pH 8. At pH 7, CQ is approximately

65% diprotonated and 35% monoprotonated; however, at pH 8 CQ is approximately 83%

monoprotonated and 17% diprotonated. Thus, the results showing a lower binding affinity for CQ

at pH 8 than pH 7 are reasonable. Furthermore, it is known that the DV has a pH of approximately

5.215, this is the optimal condition for PfCRT activity, therefore it is not surprising that we do not

see strong binding interactions or higher fractions of accessible fluorophores at pH 7. Furthermore,

this supports the belief that PfCRT is responsible for CQ efflux from the DV, and not its influx as

it does not bind CQ at cytosolic pH.4,16

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5.3.1.2 Fluorescence studies with heme

Heme can form reversible non-covalent binding interactions with proteins and these interactions

cause spectral changes that can be measured by UV-Vis and Fluorescence spectroscopy.13 High-

spin Fe3+ porphyrins are fluorescence quenchers due to their low-lying excited states and

paramagnetism allowing for multiple relaxation pathways.17 Therefore, any observed fluorescence

quenching between heme and proteins results from non-covalent complex formation.

5.92uM PfCRT pH 7 4.98uM PfCRT pH 8

1.5×107
PfCRT PfCRT
1eq 1eq
Fluorescence Intensity

2eq Fluorescence Intensity 2eq

3eq 3eq
1.0×107 4eq
4eq
5×106

5.0×106

0 0.0
350 400 450 350 400 450
Wavelength (nm) Wavelength (nm)

3.16uM GtrB 5.92uM Nanodisc

4×107 GtrB Nanodisc
1eq 1×107 1eq
Fluorescence Intensity

Fluorescence Intensity

2eq 2eq
3×107 3eq 3eq
4eq 4eq

2×107 5×106

1×107

0 0
350 400 450 350 400 450
Wavelength (nm) Wavelength (nm)

Figure 5.7. Effect of heme on fluorescence spectrum of PfCRT at pH 7 and 8, GtrB and nanodisc.

As shown in Figure 5.7 in PfCRT and nanodisc, increasing amounts of hemin cause a decrease in

fluorescence intensity. However, in the case of GtrB a decrease in fluorescence is only observed

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Chapter 5 The Determination of Heme and Chloroquine interactions with PfCRT

when at least 4 equiv. of hemin is added. To determine the fluorescence quenching constant for

heme, we used the Stern-Volmer equation (1) as noted above.

Table 5.4. Stern-Volmer quenching constants with heme.

PROTEIN KSV × 104 (M-1)

PfCRT pH 7 3.03 ± 1.00
PfCRT pH 8 17.98 ± 1.08
GtrB -
Nanodisc 9.15 ± 0.86

The KSV values, in Table 5.4, show a strong quenching constant of heme in PfCRT at pH 8 and to

a lesser extent in nanodisc as can be seen in Figure 5.8. we get a negative slope when heme is

added to GtrB, this is attributed to the inaccessibility of heme to GtrB fluorophores. In this

experiment, we can see the effect of pH and protein conformation with heme association. There is

an order of magnitude difference in the Stern-Volmer constant with the pH is lowered by one unit.

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Chapter 5 The Determination of Heme and Chloroquine interactions with PfCRT

5.92uM PfCRT pH 7 4.98uM PfCRT pH 8

2.5 4

F0/F (340nm)
F0/F (340nm)

2.0

1.5
1

1.0 0
0.0 5.0×10-6 1.0×10-5 1.5×10-5 2.0×10-5 0.0 5.0×10-6 1.0×10-5 1.5×10-5
[Heme] [Heme]

3.16uM GtrB 5.92uM Nanodisc

1.2 3

1.1
F0/F (340nm)

F0/F (345nm)
2

1.0

1
0.9

0.8 0
0 2×10-6 4×10-6 6×10-6 8×10-6 0.0 5.0×10-6 1.0×10-5 1.5×10-5 2.0×10-5
[Heme] [Heme]

Figure 5.8. Stern-Volmer plot for PfCRT, GtrB and nanodisc quenched with up to 4 equivalents

of Heme (each dot represents a 0.2 molar addition).

To determine the fraction of accessible fluorophores, we used the modified Stern-Volmer equation

(2), the results are displayed in Table 5.5 and Figure 5.9. The fraction of accessible fluorophores

is greater for PfCRT at pH 8 than pH 7. Whereas in the case of GtrB there are no accessible

fluorophores for heme to interact with. This is in agreement with the null KSV value in Table 5.4.

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Table 5.5. Modified Stern-Volmer constants for Heme.

PROTEIN Ka × 105 (M-1) N (%)

PfCRT pH 7 4.45 ± 2.59 56.82
PfCRT pH 8 2.18 ± 3.05 89.85
GtrB - -
Nanodisc 8.47 ± 2.59 61.05

Lastly, to determine whether the proteins contain binding sites for heme, we used equation (3), the

plots are shown in Figure 5.10 and values are shown in Figure 5.6.

5.92uM PfCRT pH 7 4.98uM PfCRT pH 8

6 8

6
4
F0/F0-F
F0/F0-F

2
2

0 0
0 2×105 4×105 6×105 8×105 1×106 0.0 5.0×105 1.0×106 1.5×106
1/[Heme] 1/[Heme]

3.16uM GtrB 5.92uM Nanodisc

100 4

50 3
F0/F0-F

F0/F0-F

0 2

-50 1

-100 0
0.0 5.0×105 1.0×106 1.5×106 2.0×106 0 2×105 4×105 6×105 8×105 1×106
1/[Heme] 1/[Heme]

Figure 5.9. Modified Stern-Volmer plots for PfCRT, GtrB and nanodisc quenched with up to 4

equivalents of heme (each dot represents a 0.2 molar addition).

GtrB does not contain any binding sites for heme, and we get a negative slope and intercept values

for the Stern-Volmer quenching constants; thus, we can say that heme is not a substrate for GtrB.

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Chapter 5 The Determination of Heme and Chloroquine interactions with PfCRT

PfCRT at pH 8 contains one binding site for heme, this is in accordance with the 90% accessibility

of fluorophores and strong Stern-Volmer quenching constant.

Table 5.6. Binding constants and number of binding sites for heme with MPLME.

PROTEIN logKb n
PfCRT pH 7 2.08 ± 0.39 0.43 ± 0.08
PfCRT pH 8 5.34 ± 0.25 1.02 ± 0.05
GtrB - -
Nanodisc 2.81 ± 0.26 0.54 ± 0.05

The difference of one pH unit has a 1,000 - fold impact as at pH 7 PfCRT does not contain any

binding sites for heme. Consequently, heme has access to a lesser percentage of the fluorophore

resulting in a low quenching constant. Finally, nanodisc does not contain any binding sites for

heme, which is also in accordance with previous results. The same nanodisc is incorporated onto

PfCRT to keep it membrane-bound; thus, these results indicate that at pH 8 heme is truly binding

to the protein not the membrane and that there is a significant conformational change to the protein

at lower pH as heme no longer binds.

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Chapter 5 The Determination of Heme and Chloroquine interactions with PfCRT

5.92uM PfCRT pH 7 4.98uM PfCRT pH 8

0.2 0.5

0.0
Log(F0-F)/F

Log(F0-F)/F
0.0
-0.2

-0.4
-0.5
-0.6

-0.8 -1.0
-6.5 -6.0 -5.5 -5.0 -4.5 -6.5 -6.0 -5.5 -5.0 -4.5
Log[Heme] Log[Heme]

3.16uM GtrB 5.92uM Nanodisc

-1.0 0.4

0.2
-1.2
Log(F0-F)/F

Log(F0-F)/F
0.0
-1.4
-0.2
-1.6
-0.4

-1.8 -0.6
-7.0 -6.5 -6.0 -5.5 -5.0 -6.5 -6.0 -5.5 -5.0 -4.5
Log[Heme] Log[Heme]

Figure 5.10. Binding plot to determine the number of heme binding sites and binding constant.

As mentioned above, it is important to remember that heme is possibly removed out of the DV by

PfCRT at pH 5.2. Secondly, the two PfCRT Trp residues are located on the DV portion of the

membrane; thus, at physiological pH results may differ. It may be the case that PfCRT indeed

contains at least one binding site for CQ and heme at physiological pH. Nonetheless, we were able

to demonstrate that PfCRT binds heme at pH 8.

The PfCRT central cavity has a volume of approximately 3,300 Å3, heme and CQ molecules have

approximate volumes of 702 Å3 and 396 Å3, respectively. Furthermore, the cavity has a max

diameter of 25 Å, with the max diameters for heme and CQ being approximately 14 Å and 8 Å,

respectively. Hence, it is possible that the resistance protein is capable of effluxing both heme and

CQ as individual drugs and efflux the heme:CQ complex.

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Chapter 5 The Determination of Heme and Chloroquine interactions with PfCRT

5.3.2 UV-Vis Studies

5.3.2.1 UV-Vis Studies with heme

The heme Soret, Q-bands and charge transfer bands can be identified by UV-Vis spectroscopy and

are used to determine changes to heme oxidation state and spin state. We can use the observed

changes of these bands to determine whether heme is involved in any binding interactions,

especially those with direct iron-protein interactions.

All heme solutions were made in a 40% DMSO/ aqueous mixture, in this solution, heme is in its

monomeric form.18 As shown in Figure 5.11, for GtrB, there is no observed shift of the Soret band

when two molar equivalents of heme are added. Heme added to the empty nanodisc causes a small

2 nm hypochromic shift (blue shift) from 412 nm to 410 nm and a greater 6 nm blue shift for the

Q band. When heme is added to PfCRT, we observe the greatest change, an 8 nm blue shift, from

403 nm to 395 nm with no change to the Q band. Changes to the Soret peak which are associated

with " → "* transition suggesting the occurrence of heme binding interactions.19

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2.0
2.82µM PfCRT pH 8 2.16µM GtrB
0.6
PfCRT 0.4eq
0.4eq 0.8eq
1.5 0.8eq 1.2eq
0.4
Absorbance

Absorbance
1.2eq 1.6eq
1.6eq 2eq
1.0 2eq
0.2 !max 413￫413
!max 403￫395
0.5
0.0
0.0
300 400 500 600 300 400 500 600
Wavelength (nm) Wavelength (nm)
6µM Nanodisc
0.8
Nanodisc
0.4eq
0.6 0.8eq
Absorbance

1.2eq
1.6eq
0.4 2eq
!max 412￫410
0.2

0.0
300 400 500 600
Wavelength (nm)

Figure 5.11. Changes to MPLME with up to 2 molar addition of heme.

The control protein GtrB does not bind with heme as there is no measured shift in the Soret peak.

This was confirmed with fluorescence assays which showed that GtrB has no binding sites for

heme (Table 5.6). In the case of lipid nanodisc, we observe a small 2 nm shift when two molar

equivalents of heme are added, which we attribute to weak associations of heme with the

membrane. Fluorescence studies showed that heme has a logKb value of 2.81, which is stronger

than with GtrB but less than with PfCRT at pH 8. When heme is added to PfCRT at pH 8 we see

the greatest shift of 8 nm, indicating greater binding interactions between PfCRT - Heme than with

nanodisc and GtrB. Furthermore, these interactions are independent of the nanodisc, which gave a

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2 nm shift. These results are in accordance with fluorescent studies, which show that PfCRT at pH

8 contains one binding site for heme; furthermore, heme has access to 90% of its Trp fluorophores.

5.4 Conclusion

CQ resistance was first discovered in the 1960s, and there has been sustained research into

determining the mechanism(s) of resistance and how to reverse and/or prevent it. A central protein

involved in CQ resistance has for the first time been isolated, this breakthrough will allow for a

better understanding of the resistance mechanism. In collaboration with the Fidock group, we

worked with samples of the PfCRT 7G8 isoform and were able to carry out fluorescence and UV-

Vis studies to determine binding of CQ and heme to the protein (an empty nanodisc membrane in

which PfCRT is embedded in and an unrelated control protein GtrB) at cytosolic pH.

Fluorescence studies with GtrB show that it does not bind CQ or heme. We also determined that

CQ and heme have little to no access to fluorophores located on the protein. Empty nanodisc does

not bind CQ or heme, indicating that these drugs do not get embedded within these membranes.

Additionally, heme has access to a greater fraction of fluorophores than CQ, which highlights the

different interactions between the drugs and the protein. Lastly, PfCRT interactions are pH-

dependent. At pH 7 PfCRT does not bind CQ or heme, this is in accordance with previous reports

that state PfCRT effluxes CQ from the DV into the cytosol, it does not transport CQ into the DV.

Therefore, binding interactions possibly occur in vacuolar pH.

We also carried out UV-Vis experiments of heme with GtrB, nanodisc and PfCRT at pH 8. We

used the change in the Soret peak as an indication of non-covalent binding interactions. There were

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no observed changes in the Soret peak with GtrB and a 2 nm blue shift with nanodisc. We observed

an 8 nm blue shift of the Soret peak in PfCRT. This 8 nm shift indicates significant interactions

between heme and PfCRT.

The results of our fluorescence and UV-Vis studies demonstrate that PfCRT at pH 8, contains one

binding site for heme and at lower pH does not bind heme. At pH 7 and 8, PfCRT does not form

significant binding interactions with CQ. This supports the belief that PfCRT transports CQ from

the DV into the cytosol. Future work would involve performing fluorescence assays at vacuolar

pH to determine binding interactions in an acidic environment.

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Chapter 5 The Determination of Heme and Chloroquine interactions with PfCRT

5.5 Experimental

5.5.1 General information

All solvents and reagents were obtained commercially and used without further purification unless

noted. The UV-Vis spectra were measured on Agilent 8453. The fluorescence measurements are

performed on a Molecular Devices SpectraMax i3x, equipped with a black bottom 96 well plate

obtained from Corning.

The PfCRT 7G8 isoform, nanodisc (MSP1D1 + POPG) and GtrB (encapsulated in MSP1E3D1)

gifted from Prof. David Fidock’s group at Columbia University. PfCRT at pH 7, nanodisc and

GtrB were provided in a buffer solution containing 20 mM HEPES and 150 mM NaCl. PfCRT at

pH 8 was provided in a buffer solution containing 20 mM TRIS pH 8 and 150 mM NaCl. CQ and

hemin solutions were made in the appropriate buffers.

All solutions are freshly prepared and deionized H2O is used in the preparation of all buffer

solutions. All data analysis is performed using Prism 7.0a (GraphPad Software). Cole Parmer

Microcomputer pH-Vision Model 05669-20 pH Meter used with Sigma-Aldrich micro pH

combination electrode, glass body, which is calibrated with standard buffer solutions before use.

1.3 mg of hemin is dissolved in 2 mL 100% DMSO to make a 1 mM stock solution. 10.3 mg of

chloroquine diphosphate is dissolved in 10 mL of buffer to make a 2 mM stock solution. A vortex

mixer was used to ensure complete dissolution.

5.5.2 Fluorescence Spectroscopy Procedure

The emission spectra are then measured at 25.0oC and are recorded in a wavelength range of 305-

500 nm for nanodisc and GtrB following excitation at 280 nm. The emission spectra for PfCRT

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Chapter 5 The Determination of Heme and Chloroquine interactions with PfCRT

are recorded with a wavelength range of 315-500 nm following excitation at 290 nm. The

excitation and emission bandwidths are 5 nm. The initial volume of all proteins in the 96 well

plates is 100 μL. Titrations are done manually using micropipettes.

PfCRT pH 7 and nanodisc experiments: 39 μL of hemin stock solution is diluted into 1 mL 40%

DMSO, 60% buffer mixture to make a 39 μM 40% hemin solution. 39 μL of CQ stock is then

diluted into 2 mL of buffer to make a 39 μM solution. PfCRT is used undiluted as a 5.92 μM

HEPES solution. 19 μL of 31.8 μM Nanodisc is diluted into 81 μL buffer to make a 5.92 μM

solution.

PfCRT pH 8 experiments: 66.2 μL of hemin stock solution is diluted into 2 mL 40% DMSO, 60%

buffer mixture to make a 33.1 μM 40% hemin solution. 82.7 μL of CQ stock is then diluted into 5

mL of buffer to make a 33.1 μM solution. PfCRT is used undiluted as a 4.98 μM TRIS solution.

GtrB experiments: 21.1 μL of hemin stock solution is diluted into 1 mL 40% DMSO, 60% buffer

mixture to make a 21.1 μM 40% hemin solution. 52.8 μL of CQ stock is then diluted into 5 mL of

buffer to make a 21.1 μM solution. GtrB is used undiluted as a 3.16 μM HEPES solution.

Once prepared the solutions are kept on ice in a polystyrene cooler box. CQ or hemin is then

titrated in 3 μL increments which correspond to 0.2 molar equivalents addition. Once four equiv.

of the drug are added subsequent additions are in 15 μL increments until 7 molar equivalents are

added. The equilibrium time between each addition is 10 minutes. During titration the solution is

stirred and kept at a constant temperature of 25oC. The total volume of drug added is 150 μL.

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Chapter 5 The Determination of Heme and Chloroquine interactions with PfCRT

5.5.3 Ultraviolet-Visible Spectroscopy Procedure

At RT, the spectrophotometer is equipped with a 1 cm quartz cuvette of 4 mL capacity. All spectra

are recorded against the appropriate blank buffer solution in the range of 260-800 nm.

Nanodisc: 76 μL of 39.5 μM nanodisc in HEPES pH 7.5 buffer is diluted in 1 mL buffer to make

a 6 μM solution. 400 μL of hemin stock solution is diluted into 1 mL 40% DMSO, 60% buffer

mixture to make a 400 μM 40% hemin solution.

PfCRT: 100 μL of 14.1 μM PfCRT is diluted to 500 μL TRIS pH 8 buffer to make a 2.82 μM

solution. 188 μL of hemin stock solution is diluted into 1 mL 40% DMSO, 60% buffer mixture to

make a 188 μM 40% hemin solution.

GtrB: 340 μL of 3.18 μM GtrB is diluted into 500 μL HEPES pH 7.5 buffer to make a 2.16 μM

solution. 144 μL of hemin stock solution is diluted into 1 mL 40% DMSO, 60% buffer mixture to

make a 144 μM 40% hemin solution.

Heme is titrated in 3 μL increments which correspond to 0.4 molar equivalents addition. Heme is

added until 2 molar equiv. are reached, with an equilibrium time of 10 minutes between additions.

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5.6 References

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(3) Kim, J.; Tan, Y. Z.; Wicht, K. J.; Erramilli, S. K.; Dhingra, S. K.; Okombo, J.; Vendome,

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Carragher, B.; Kossiakoff, A. A.; Quick, M.; Fidock, D. A.; Mancia, F. Structure and Drug

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Roepe, P. D.; Wellems, T. E. Mutations in the P. Falciparum Digestive Vacuole

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(7) Djimdé, A.; Doumbo, O. K.; Cortese, J. F.; Kayentao, K.; Doumbo, S.; Diourté, Y.;

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Malaria Pigment β-Haematin. Nature 2000, 404 (6775), 307–310.

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Terzić Jovanović, N. V.; Šolaja, B. A.; Verbić, T. Human Serum Albumin Binding of

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Spectrosc. 2018, 193, 23–32.

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Diphosphate and Phenelzine Sulfate Drugs with Human Serum Albumin and Human

Hemoglobin Proteins by Spectroscopic Techniques. J. Lumin. 2013, 140, 87–94.

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Falciparum-Infected Erythrocytes Using PHluorin. Cell. Microbiol. 2007, 9 (4), 1004–

1013.

(16) Valderramos, S. G.; Fidock, D. A. Transporters Involved in Resistance to Antimalarial

Drugs. Trends Pharmacol. Sci. 2006, 27 (11), 594–601.

(17) Dodd, E. L.; Bohle, D. S. Orienting the Heterocyclic Periphery: A Structural Model for

Chloroquine’s Antimalarial Activity. Chem. Commun. 2014, 50 (89), 13765–13768.

(18) Egan, T. J.; Mavuso, W. W.; Ross, D. C.; Marques, H. M. Thermodynamic Factors

Controlling the Interaction of Quinoline Antimalarial Drugs with Ferriprotoporphyrin IX.

J. Inorg. Biochem. 1997, 68 (2), 137–145.

(19) Dayer, M.; Moosavi-Movahedi, A.; Dayer, M. Band Assignment in Hemoglobin Porphyrin

Ring Spectrum: Using Four-Orbital Model of Gouterman. Protein Pept. Lett. 2010, 17 (4),

473–479.

215
 Chapter 6

Conclusions and Future Work

216
Chapter 6 Conclusions and Future Work

6.1 Conclusion

When I started my Ph.D., the main objective was to synthesize a chloroquine photoaffinity label

with the simple addition of an azide at the 3-position. In pursuit of the azide, the discovery of

undesired products led to the creation of projects that make the body of my thesis. This is a

testament to not dismissing side/byproduct formation. As chemists, we must truly get in the flask

to understand why and what reactions are occurring to the best of our ability. In that sense, is there

truly such a thing as a failed reaction?

In my first chapter, I described the optimization of 3-NH2CQ synthesis. This project was born out

of the formation of the 3-NH2CQ rather than 3-N3CQ. To optimize amine formation, large amounts

of the precursor 3-BrCQ were required. However, the known synthetic route for 3-BrCQ at the

time did not allow large-scale synthesis. Therefore, 3-BrCQ synthesis was optimized using

microwave-assisted synthesis and excess amounts of a brominating agent. We were able to scale

the reaction to gram scale and obtain a 64% yield. With large amounts of my precursor, we were

able to carry out a series of experiments with varying copper catalysts, ligands, solvents, reducing

agents, and nitrogen sources. A moderate yield of 55% with the optimized reaction conditions was

reached.

The formation of 3-NH2CQ opened up the possibility of derivatization, which paved the way for

chapter 2. A quick search of the literature for 4-aminoquinolines reveals compounds that only

contain a 7-chloro substituent on the quinoline ring. Fortunately, previous work done in our lab

showed that 3-ICQ, when used in combination with CQ, resensitizes the parasite to CQ. Therefore,

we decided to exploit the newly formed 3-NH2CQ to synthesize a library of 3-substituted CQ

217
Chapter 6 Conclusions and Future Work

derivatives. These derivatives would then be tested for any synergistic effects they may have in

combination therapy. Derivatives were synthesized with varying electronic and hydrophobic

effects, with guidance from the Craig Plot and consideration of Lipinski’s Rule of 5. Various

benzamides were synthesized, and we determined that para - chloro substituent showed the best

antiplasmodial activity. In CQS 3D7 strains, it was 3 – fold more effective, and in CQR Dd2 strains

it was 1.8 – fold less effective than CQ. The equivalent para – chloro sulfonamide and Schiff base

were less active than the chlorobenzamido derivative.

The precursor for the desired photoaffinity label was 3-NH2CQ. However, as explained in chapter

4, we were unable to synthesize the photoaffinity label with only one modification at the 3-

position. Doing so leads to the creation of a triazole product. To prevent cyclization, we blocked

the reactivity of the 4-amine by installing a methyl to form a tertiary amine. A methyl was chosen

due to its small size and lack of reactivity. This minor modification then allowed the synthesis of

the desired photoaffinity label. Preliminary experiments to test the reactivity of the aryl azide were

carried out.

The final chapter is a result of a collaboration that arose after an oral conference presentation. Prof.

Fidock was interested in the potential of the photoaffinity label and disclosed that they had, for the

first time, resolved the cryo-EM structure of Plasmodium falciparum Chloroquine Resistant

Transporter (PfCRT) isoform. We obtained samples of the isoform and were able to carry out

binding studies. We determined that there was no evidence of CQ binding to PfCRT at pH 7 and

8. We also determined that heme binds to PfCRT at pH 8. As mentioned in chapter 5, binding is

very dependent on pH; therefore, at lower pH, CQ might bind to PfCRT.

218
Chapter 6 Conclusions and Future Work

6.2 Future Work

Regarding the 3-substituted CQ library, future steps involve isobolographic analysis to determine

whether interactions between the derivatives and CQ are synergistic, additive, or antagonistic.

Results from such experiments can give more detailed S.A.R. data about the derivatives and their

targets. The 3-NH2CQ can be further derivatized to expand the library and synthesize different

families of antimalarial compounds. As the synthetic route for 3-N3CQ has been developed. It can

be used as a precursor to a family of triazole derivatives for click chemistry, thus further expanding

the library of compounds.

The photoaffinity label has been synthesized and can be irradiated with UV light of 365 nm. The

synthetic route developed can be used for various 4-aminoquinolines, thus allowing adequate

labelling of many antimalarial drug candidates. More studies need to be carried out to determine

the best conditions required for C-H, C-N, and C-C insertion and the optimal stoichiometry that

will produce the highest conversion and yield. Future work involves using the label with proteins

of interest to determine the insertion of the PAL. Such studies will have a significant impact on

our understanding of CQ’s biological pathway. Furthermore, once successfully incorporated into

the target protein, one can carry out protein digestion for mass spectroscopy. This will allow for

the identification of the insertion product along the protein sequence.

We have demonstrated that PfCRT binds heme at pH 8 and does not bind CQ at pH 7 and 8. The

next area of interest is determining the binding mechanism (static or dynamic) by carrying out

temperature-dependent fluorescence assays. Static quenching is a result of complex formation

between the quencher and the fluorophore in the ground state. Dynamic quenching is a result of

219
Chapter 6 Conclusions and Future Work

interactions between the quencher and fluorophore in the excited state. Quenching emission is

temperature-dependent; therefore, temperature-dependent assays can give valuable information on

the mechanism of interaction between the quencher and fluorophore. Lastly, it would be of great

interest to determine the binding behaviour of PfCRT towards CQ and heme at lower pH such as

pH 5.2.

220
Appendix

221
Appendix

Chapter 3
pH:
CQ titration 5.08
CQ pKa
7.58
4.87 7.71
1.5
5.55 7.92
5.84 8.11 0.8
Absorbance

1.0
6.03 8.49
6.1 8.65

A342
0.5 6.24 8.88 0.6
6.57 9.06
0.0 6.7 9.21
250 300 350 400 6.96 9.54
Wavelength (nm)
7.13
0.4
9.78
7.34 10 6 8 10
pH

pH: 3-ClCQ pKa

3.3 5.64
3-ClCQ titration
3.43 5.78
1.5 3.61 6.05
0.6
3.99 6.13
Absorbance

4.27 6.43
1.0
A342

4.47 6.57
4.81 6.77 0.4
0.5
5 6.94
5.11 7.12
0.0 5.29 7.39
250 300 350 400
Wavelength (nm) 5.45 7.88 0.2
3 4 5 6 7 8
pH

222
Appendix

pH:
3.42 5.69
3-BrCQ pKa
3-BrCQ titration
3.57 5.85
1.5 3.77 6
4.13 6.12
0.6
Absorbance

1.0 4.23 6.27

4.39 6.39

A342
4.62 6.48 0.4
0.5
4.81 6.61
4.98 6.72
0.0
250 300 350 400
5.17 6.89 0.2
Wavelength (nm) 5.26 7.08
5.41 7.58
4 6 8
5.53 8.14
pH

pH:
3-ICQ pKa
3.39 5.8
3-ICQ titration
3.53 5.94
3.73 6.09 0.5
1.5
3.97 6.23
Absorbance

4.42 6.34
1.0 0.4
4.77 6.44 A342
4.83 6.59
0.5
4.95 6.69
0.3
5.02 6.92
0.0 5.21 7.14
250 300 350 400
5.43 7.52 0.2
Wavelength (nm)
5.61 8.04 4 6 8
pH

223
Appendix

pH:
3.58 5.89
3-NH2CQ pKa
3-NH2CQ titration
3.61 6.04 0.40
1.5 3.7 6.14
4
3.9 6.3 0.35
Absorbance

1.0 4.05 6.54

4.25 6.63 0.30

A342
4.42 6.92
0.5
4.72 7.05 0.25
4.81 7.36
0.0 0.20
250 300 350 400 450 4.94 7.65
Wavelength (nm) 5.19 7.91
0.15
5.37 8.24 4 6 8
5.57 8.47
pH
5.7

224
Appendix

Chapter 5

Fluorescence Studies with chloroquine

Linear equation for Stern-Volmer plot:

!!
= 1 + &" '! [)] = 1 + +#$ [)]
!

PROTEIN EQUATION KSV X 104 (M-1) R2

PfCRT pH 7 Y = 1405*X + 1.038 0.14 ± 0.14 0.0536
PfCRT pH 8 Y = 558.9*X + 1.124 0.056 ± 0.31 0.001812
GtrB Y = 15442*X + 1.025 1.54 ± 0.41 0.4437
Nanodisc Y = 35486*X + 1.148 3.55 ± 0.44 0.7811

Linear equation for modified Stern-Volmer plot:

!! 1 1
= +
!! − ! -+% [)] -

PROTEIN EQUATION Ka X 105 (M-1) ⍺ (%) R2

PfCRT pH 7 Y = 3.748e-005*X + 18.44 4.92 ± 2.58 5.42 0.3195
8.21 ± 3.09
PfCRT pH 8 Y = 8.604e-006*X + 7.064 14.16 0.468

GtrB Y = 1.684e-005*X + 7.324 4.35 ± 4.79 13.65 0.7143

Nanodisc Y = 3.925e-006*X + 2.816 7.17 ± 2.62 35.51 0.4535

225
Appendix

Linear equations to determine the number of binding sites:

!! − !
log = log +& + 2 log [)]
!

PROTEIN EQUATION logKb n R2

PfCRT pH 7 Y = 0.2715*X + 0.06151 0.06 ± 0.83 0.27 ± 0.16 0.1424
0.009 ± 0.18 ± 0.17
PfCRT pH 8 Y = 0.1811*X + 0.009318 0.06101
0.87
GtrB Y = 0.5416*X + 1.897 1.89 ± 0.58 0.54 ± 0.11 0.5993
Nanodisc Y = 0.6693*X + 3.047 3.05 ± 0.37 0.67 ± 0.07 0.8366

Concentration-dependent Stern-Volmer plot, with CQ.

PROTEIN EQUATION KSV X 104 (M-1) R2

PfCRT pH 7 0-1 eq Y = 16892*X + 0.9991 1.69 ± 0.44 0.8333

PfCRT pH 7 1-4 eq Y = 1967*X + 1.03 0.19 ± 0.21 0.05661

PfCRT pH 8 0-2 eq Y = 15324*X + 1.061 1.53 ± 0.57 0.4715

PfCRT pH 8 2-4 eq Y = -22032*X + 1.355 -2.2 ± 0.61 0.5949

Fluorescence Studies with heme

Linear equation for Stern-Volmer plot:

!!
= 1 + &" '! [)] = 1 + +#$ [)]
!

PROTEIN EQUATION KSV X 104 (M-1) R2

PfCRT pH 7 Y = 30034*X + 1.513 3.03 ± 1.0 0.3324
PfCRT pH 8 Y = 179807*X + 1.006 17.98 ± 1.08 0.9386
GtrB Y = -26259*X + 1.063 - 0.7085
Nanodisc Y = 91473*X + 1.373 9.15 ± 0.86 0.8622

226
Appendix

Linear equation for modified Stern-Volmer plot:

!! 1 1
= +
!! − ! -+% [)] -

PROTEIN EQUATION Ka X 105 (M-1) ⍺ (%) R2

PfCRT pH 7 Y = 3.956e-006*X + 1.76 4.45 ± 2.59 56.82 0.9076

PfCRT pH 8 Y = 5.116e-006*X + 1.113 2.18 ± 3.05 89.85 0.9414

GtrB Y = 2.575e-005*X - 17.34 - - 0.1494

Nanodisc Y = 1.933e-006*X + 1.638 8.47 ± 2.59 61.05 0.7061

Linear equations to determine the number of binding sites:

!! − !
log = log +& + 2 log [)]
!

PROTEIN EQUATION logKb n R2

2.08 ± 0.39 0.43 ± 0.08
PfCRT pH 7 Y = 0.4313*X + 2.08 0.6413
5.34 ± 0.25 1.02 ± 0.05
PfCRT pH 8 Y = 1.016*X + 5.336 0.9618
- -
GtrB Y = -1.147*X - 8.234 0.9376

Nanodisc Y = 0.539*X + 2.805 2.81 ± 0.26 0.54 ± 0.05 0.8589

227
Appendix

NMR Spectra

228
 9.5
1H
Et2N

9.0
Appendix

Cl
Br
1.00 8.68

8.5
1.09 8.29

2.3
1.00 8.08

8.0
NH
Br

7.5
NMR (400 MHz, Chloroform-d)

7.0
6.5
6.0
4.58
4.56

5.5
4.04
4.02
4.01

5.0
4.00
4.00
1.07 3.99

f1 (ppm)
4.5
3.98
3.96

EtOAc
1.23 2.60

4.0
2.58
2.56
2.54

3.5
2.49
2.48
2.46

3.0
1.68
4.53 1.68
2.5 1.67
2.23
1.66
1.65
EtOAc

1.65
2.0

1.63
1.63
4.69
1.5

1.62
3.53 1.62
6.64 1.61
EtOAc

1.0

1.60
1.30
1.29
0.5

1.06
1.04
1.02
0.0

229
Appendix

153.01
148.61
148.14

135.46
130.84
128.19
121.88
119.74

107.54

55.05
52.55
46.92

36.67

23.40
22.39

11.26
13
C NMR (201 MHz, Chloroform-d)

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10
f1 (ppm)
230
 8.56
8.55

Et2N
8.08

9.0
1H
Cl
Appendix

8.08
8.07
8.07

8.5
8.07
8.07

8.0
7.94
NH

7.94
3.3
N
H

7.76

7.5
7.76
O

7.75
7.75

7.0
7.70
7.70
7.69
NMR (800 MHz, Methanol-d4)

6.5
7.69
7.69
7.68

6.0
7.62
7.62
7.61

5.5
7.60
7.60
4.57

5.0
4.57
3.10
3.09

f1 (ppm)
4.5
3.08
3.07
3.05

4.0
3.05
3.04
3.04

3.5
2.99
2.98
2.98

3.0
2.97
1.83
2.5 1.82
1.82
1.81
2.0

1.66
1.66
1.66
1.5

1.65
1.65
1.64
1.0

1.43
1.43
1.21
0.5

1.21
1.20
1.19
0.0

231
 13

210
Appendix

200
190
180
169.09

170
160
153.47
C NMR (201 MHz, Methanol-d4)

151.04
145.62

150
139.46
138.61

140
132.71
132.69

130
128.77
127.55
125.29

120
119.60
117.39

110
111.52

100
f1 (ppm)
80
70
60 90
51.57

50
51.02
40

33.27
30

20.79
20

20.20

7.70
10

7.64
0
-10

232
 9.5
Et2N
0.32 8.92

9.0
1H
Cl
Appendix

1.00 8.85
8.20

8.5
8.19
2.03 8.00
7.99

N
1.59
NH

8.0
7.99
2.35
7.98
N
H

3.4
2.25
7.89
O

7.5
1.57
7.88
0.77 7.88
7.87

7.0
7.86
7.81
CN

7.80

6.5
NMR (500 MHz, Chloroform-d)

7.43
7.43
7.42

6.0
7.42
7.41

5.5
7.40
7.40
7.32

5.0
7.30

4.5
4.31

f1 (ppm)
1.56 4.27
4.24

4.0
1.66 3.72

3.5
2.54
2.53
2.52

3.0
2.50
4.49 2.48
3.81 2.48

2.5
2.18 2.47
2.45
1.10
2.44
2.0

2.39
7.33 2.38
2.36
1.5

3.38
2.36
1.25
2.34
6.14
1.0

2.33
2.92 1.53
1.53
0.5

1.51
1.50
1.48
0.0

1.47
1.45
233
 13

230
Appendix

220
210
200
190
180
164.96
C NMR (126 MHz, Chloroform-d)

150.39

170
150.26
148.53

160
148.15
145.96

150
143.09
137.64
135.05

140
134.73
132.72

130
130.94
129.70
129.08
129.05

f1 (ppm)
128.57

120 110
127.59
126.51

100
126.47
124.07
123.76

90
121.26
119.01

80
118.07
115.85

70
53.62
53.51
52.72
60

52.60
46.82
50

46.78
46.73
40

36.71
30

29.85
23.78
23.40
20

22.40
22.26
10

21.69
11.19
0

10.57
234
 8.60
8.59

Et2N

9.0
8.57

Cl

1H
Appendix

8.45
0.75 8.43

8.5
1.20 8.30
1.95 8.29

N
2.05 8.28
NH

8.0
1.00 7.96
7.95
N
H

0.99
3.5 7.77
O

7.5
7.77
7.75
7.75

7.0
NO2

NMR (500 MHz, Methanol-d4)

6.5
4.60
4.59
4.58

6.0
4.57
4.57
4.56

5.5
4.55
3.17
3.15

5.0
3.14
3.12
1.02

f1 (ppm)
3.09

4.5
3.07
3.07
3.06

4.0
3.06
3.04
3.04

3.5
1.89
4.06 1.88
2.08 1.86

3.0
1.85
1.84
2.5 1.83
1.81
1.74
2.0

1.73
1.14 1.72
3.09 1.71
1.5

3.00 1.71
6.16 1.70
1.69
1.0

1.67
1.44
1.43
0.5

1.25
1.23
1.22
0.0

235
 13

230
220
Appendix

210
200
190
180
170
C NMR (126 MHz, Methanol-d4)

167.24

160
153.71
150.34
145.10

150
139.63
138.53

140
138.33
129.02

130
127.57
125.62
123.60
119.42
117.04

f1 (ppm)
111.31

120 110 100

90
80
70
60
50 51.94
51.09
46.94
40

33.30
30

20.81
20

20.25
10

7.62
0
-10

236
 9.5
8.56
8.54

Et2N

1H
7.95

9.0
Cl
Appendix

7.78
7.77
7.76

8.5
7.76
7.74

N
NH

7.72

8.0
3.6
7.61
N
H

7.61
Cl

7.5
7.60
7.59
7.59

7.0
7.58
7.57
NMR (500 MHz, Methanol-d4)

7.57

6.5
7.56
7.55
7.54

6.0
7.53
7.52
7.52

5.5
7.51
7.50
4.75

5.0
4.74
4.72
4.71

f1 (ppm)
4.5
3.16
3.16
3.14

4.0
3.13
3.11
3.11

3.5
3.09
3.09
3.07

3.0
1.89
1.88
2.5 1.86
1.80
1.79
2.0

1.78
1.77
1.77
1.5

1.76
1.74
1.46
1.0

1.45
1.44
1.24
0.5

1.22
1.21
1.19
0.0

237
 13

230
220
Appendix

210
200
190
180
170.67
C NMR (126 MHz, Methanol-d4)

170
154.26
147.24

160
140.60
136.11

150
133.34
132.14

140
131.59
130.39

130
128.90
128.66
126.75
121.75
119.26

f1 (ppm)
120 110 100
90
80
70
60 52.65
52.58
50
40

34.84
30

22.16
21.95
20

9.08
10

8.99
0
-10

238
 9.0
Et2N
1.00 8.87

1H
Cl
Appendix

8.10
8.02

8.5
8.00
0.98 7.91
2.17

N
7.90

8.0
NH
1.39 7.57
7.55

3.7
1.10
N
H

1.26
O 7.48

7.5
1.51 7.47
7.45
Cl

7.43

7.0
7.41
NMR (500 MHz, Chloroform-d)

5.0 6.5
6.0
5.5
4.5
f1 (ppm)
4.32
1.04
4.30

4.0
3.78
1.12 3.75
3.72

3.5
3.0
2.60
2.59
4.94 2.57
2.5 3.05 2.56
2.43
2.42
2.0

1.55
1.54
1.52
1.5

4.88 1.52
2.73 1.51
7.33 1.49
1.0

1.27
1.26
1.04
0.5

1.02
1.01
0.99
0.0

239
 13

230
220
Appendix

210
200
190
180
170
C NMR (126 MHz, Chloroform-d)

165.40

160
150.70

150
148.64
145.89
135.46

140
135.18
134.86

130
132.37
130.24
129.15
128.37
126.37

f1 (ppm)
126.02
124.30

120 110 100

121.36
119.41

90
80
70
60
53.58
52.48
50
46.72
40

36.69
30

22.84
22.52
20

10.09
10
0
-10

240
 1H
Et2N

10.0
Cl
Appendix

9.5
0.93 9.35
8.83

9.0
8.12
NH
1.00

3.8
N 8.09
H

8.01

8.5
O 8.01
1.98 8.00
1.03 7.91

8.0
1.06 7.89
7.50
Cl

2.04
NMR (400 MHz, Chloroform-d)

7.5
7.49
1.09
7.47
7.47

7.0
7.42
7.42

6.5
7.40
7.39

5.5 6.0
5.0
f1 (ppm)
4.5
1.05 4.35

4.0
1.11 3.76

3.5
2.66
2.64
2.62

3.0
2.60
4.12 2.49
2.47
2.5
2.28
2.45
2.43
2.0

1.10 1.53
1.52
1.5

3.03
3.74 1.52
1.51
6.08
1.0

1.50
1.28
1.26
0.5

1.07
1.05
1.03
0.0

241
 13

230
220
Appendix

210
200
190
180
170
C NMR (126 MHz, Chloroform-d)

165.91
151.05

160
148.71
146.63

150
138.64
134.81
131.99

140
129.66
129.09

130
129.04
126.22
124.53
121.23
119.38

f1 (ppm)
120 110 100
90
80
70
60
53.49
50 52.30
46.58
40

36.58
30

22.61
22.30
20
10

9.54
0
242
-10
 9.5
Et2N

9.0
1H
Cl
8.59
Appendix

1.04 8.57
8.56

8.5
1.03
7.95
7.95

N
NH

1.89 7.94

8.0
1.00 7.94

3.9
N
H

0.97 7.94
O

7.74

7.5
0.96
7.74
1.00
F

7.73
7.72

7.0
7.70
7.69
NMR (500 MHz, Methanol-d4)

7.68

6.5
7.68
7.67
7.42

6.0
7.41
7.39

5.5
7.37
7.36
7.35

5.0
7.34
1.07 4.72
4.71

f1 (ppm)
4.5
4.70
3.15
3.14

4.0
3.12
3.11
3.09

3.5
3.08
4.07 3.07
1.92 3.06

3.0
3.06
3.04
3.03
2.5
1.88
1.86
1.84
2.0

1.05 1.75
3.10 1.74
1.73
1.5

3.04
1.73
6.02
1.72
1.43
1.0

1.42
1.23
1.22
0.5

1.21
0.0

243
Appendix

167.60
167.58
154.51
147.18
140.74
140.26
135.60
135.53
132.14
132.12
128.89
126.77
126.23
126.20
122.98
122.88
121.20

52.71
52.52
48.34

34.71

22.19
21.73

9.02
13
C NMR (126 MHz, Methanol-d4)

Trifluoroacetic
Trifluoroacetic
acid
acid

230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10
f1 (ppm)
244
Appendix

-115.14
-76.94
Trifluoroacetic
acid

19
F NMR (377 MHz, Methanol-d4)

10 0 -10 -20 -30 -40 -50 -60 -70 -80 -90 -100 -110 -120 -130 -140 -150 -160 -170 -180 -190 -200 -210
f1 (ppm)
245
 9.5
Et2N

1H

9.0
Cl
1.00 8.89
Appendix

0.84 8.00
8.00

8.5
7.89
7.87

N
1.08
7.84
NH

8.0
1.17
7.82
N
H

2.12

3.10
7.78
O
1.10 7.76

7.5
1.18 7.51
F

0.95 7.50
7.49

7.0
7.49
7.47
NMR (500 MHz, Chloroform-d)

6.5
7.43
7.42
7.41

6.0
7.41
7.30
7.30

5.5
7.30
7.29
7.28

5.0
f1 (ppm)
4.5
4.29
1.04
4.27

4.0
1.12 3.72

3.5
2.52
2.51

3.0
2.49
2.48
4.25 2.37
2.5 2.16 2.36
2.36
2.35
2.0

1.54
1.54
1.53
1.5

4.28
1.52
3.02
1.23
6.24 1.22
1.0

0.99
0.97
0.96
0.5
0.0

246
 13

230
Appendix

220
210
200
190
180
C NMR (126 MHz, Chloroform-d)

170
165.24
164.04
162.07

160
150.30

150
148.37
145.42
136.10

140
136.04
134.86

130
130.72
130.66
129.14
126.50

f1 (ppm)
123.87

120 110
123.16
123.14

100
121.44
119.51

90
119.46
119.34
115.25

80
115.06

70
60

53.55
52.62
50

46.77
40

36.69
30

23.51
22.33
20

10.82
10
0

247
Appendix

-111.57
19
F NMR (377 MHz, Chloroform-d)

10 0 -10 -20 -30 -40 -50 -60 -70 -80 -90 -100 -110 -120 -130 -140 -150 -160 -170 -180 -190 -200 -210
f1 (ppm)
248
 9.5
8.86

Et2N

9.0
1H
Cl
8.74
Appendix

1.00
8.08
0.82
8.07

8.5
8.06
7.99
2.19

N
7.98
NH

8.0
1.09 7.88
N
H

1.22 7.87

3.11
O
7.42

7.5
1.14 7.41
2.25 7.40
7.40

7.0
7.20
F

7.18
NMR (500 MHz, Chloroform-d)

7.16

5.0 6.5
6.0
5.5
f1 (ppm)
4.5
4.29
1.12
4.28

4.0
1.20 3.71

3.5
2.51
2.50

3.0
2.48
2.47
4.37 2.37
2.5 2.35
2.43
2.34
2.33
2.0

1.54
1.53
1.5

4.78
1.52
3.35
1.51
6.66
1.0

1.48
1.48
1.22
0.5

1.21
0.98
0.97
0.0

0.95
249
 13

230
220
Appendix

210
200
190
180
166.20

170
C NMR (126 MHz, Chloroform-d)

165.35
164.18

160
150.25

150
148.20
145.37
134.64

140
130.09
130.02

130
129.78
129.76
128.95
126.28
123.79

f1 (ppm)
121.27
119.52

120 110 100

115.98
115.81

90
80
70
60
53.40
50 52.45
46.61
40

36.50
30

23.27
22.19
20

10.57
10
0
-10

250
Appendix

-107.08
19
F NMR (377 MHz, Chloroform-d)

10 0 -10 -20 -30 -40 -50 -60 -70 -80 -90 -100 -110 -120 -130 -140 -150 -160 -170 -180 -190 -200 -210
f1 (ppm) 251
 1.00 8.31
8.27
1.15

Et2N
8.25

1H
Cl

8.0
7.84
Appendix

0.88 7.83
2.02 7.82
1.19 7.80

7.5
7.49

N
NH

7.49
7.47

3.12
N
H

7.0
7.46
O

2.19 6.74
6.72

6.5
NMR (400 MHz, Methanol-d4)
NH2

4.5 6.0
5.5
5.0
4.13
4.11
1.62 4.10

f1 (ppm)
4.0
4.09
4.07

3.5
3.0
2.51
2.49
3.66 2.47

2.5
2.45
2.02
2.40
2.38
2.0

1.65
1.63
1.47
1.41
1.45
1.5

3.24 1.44
3.30 1.42
6.15 1.26
1.0

1.25
0.98
0.96
0.5

0.95
252
0.0
 13

220
210
Appendix

200
190
180
169.78

170
C NMR (126 MHz, Methanol-d4)

160
154.45
154.12
148.95

150
148.71
136.26

140
130.65
127.88

130
126.72
125.18
121.94

120
121.23
115.33
114.69

110 100
f1 (ppm)
80
70
60 90
53.36
52.16
50
40 47.64

36.67
30

23.79
21.90
20

10.92
10
0
-10

253
 9.5
Et2N
8.92

9.0
1H
Cl
Appendix

1.00
8.34
8.00
7.99

8.5
0.94
7.98
1.05

N
7.96
NH

1.97

8.0
7.88
1.13
N
H

7.86

3.13
7.41
O

7.5
1.09 7.41
7.40
7.39
2.25

7.0
6.99
6.98
OMe

NMR (500 MHz, Chloroform-d)

5.0 6.5
6.0
5.5
f1 (ppm)
4.5
4.26
1.05 4.24
3.88

4.0
3.23
1.22 3.73
3.70
3.68

3.5
2.48
2.47

3.0
2.45
2.44
2.5 4.60 2.34
2.33 2.33
2.32
2.30
2.0

1.53
1.51
1.5

4.68
1.50
3.31
1.49
6.80
1.0

1.49
1.47
1.19
0.5

1.18
0.97
0.95
254
0.0

0.94
 13

210
Appendix

200
190
180
170
165.98
162.96
C NMR (126 MHz, Chloroform-d)

160
150.28

150
148.13
145.14
134.61

140
129.52
129.05

130
126.40
125.98
123.80

120
121.55
120.07

110
114.20

f1 (ppm)
90
80
70
60100 55.64
53.55
50

52.73
46.81
40

36.71
30

23.80
22.25
20

11.22
10
0

255
 9.5
Et2N

Cl

1H

9.0
Appendix

8.80
1.00
8.18
8.17

8.5
8.02
1.94

N
8.01
NH

1.02 7.95

8.0
1.17 7.94
N
H

3.14 7.42
O

7.41

7.5
1.15
2.24 7.40
7.40
7.35

7.0
7.33
SMe

NMR (500 MHz, Chloroform-d)

6.5
6.0
4.42
4.40
3.84

5.5
3.80
3.77
2.73

5.0
2.72
2.58
2.54

4.5
f1 (ppm)
1.19
2.54
2.53
2.52

4.0
1.26 2.52
2.51

3.5
2.50
2.48
2.46

3.0
1.71
4.16 1.66
1.65
6.77
2.5 1.55
1.53
1.53
2.0

1.52
2.33 1.50
3.44 1.50
1.5

3.56 1.49
1.48
6.36
1.47
1.0

1.46
1.34
1.32
0.5

1.13
1.11
0.0

1.10
256
 13

230
220
Appendix

210
200
190
180
170
C NMR (126 MHz, Chloroform-d)

166.45
151.45

160
148.80
144.30
137.55

150
134.50
129.25

140
128.81
128.73

130
125.80
125.29
124.96
121.01
119.38

f1 (ppm)
120 110 100
90
80
70
60
53.31
51.95
50
46.32
40

36.39
30

22.70
20

21.15
14.95
10

8.37
0
-10

257
 10.5
Et2N

10.0
1H
Cl
Appendix

1.06 9.71

9.5
9.0
8.40

N
NH

8.39
8.29

8.5
N
H

3.15
1.15 7.92
O

1.00 7.91
7.85

8.0
2.11
7.84
0.98
7.50
1.07

7.5
7.50
7.49
NMe2

7.49
NMR (800 MHz, Dimethyl sulfoxide-d6)

7.0
6.77
2.34
6.76

6.5
5.98
1.14

6.0
5.97

5.5
3.99

f1 (ppm)
5.0
3.98
3.98
3.97

4.5
3.02
3.02
1.41

4.0
3.01
3.01
3.01

3.5
3.00
2.99
6.84 2.98
3.0
2.5 2.98

4.21 2.41
2.28 2.41
2.38
2.0

1.58
1.57
1.93 1.57
1.5

2.02 1.57
3.20 1.36
1.0

6.45 1.35
1.13
1.12
0.5

0.86
0.85
0.84
0.0

258
 13

210
Appendix

200
190
180
170
165.98

160
154.20
152.42

150
147.67
146.07
133.05

140
C NMR (201 MHz, Dimethyl sulfoxide-d6)

129.05
127.50

130
124.85
124.61

120
120.32
119.96
114.71

110
110.84

100
f1 (ppm)
80
70
60 90
51.99

50
50.41
40 46.06
39.99
34.97
30

22.61
21.51
20
10

7.16
0
-10

259
 9.5
8.95

Et2N

9.0
1H
Cl
1.00 8.95
Appendix

8.34
8.01

8.5
8.00
0.93 7.91

N
1.13 7.89
NH

8.0
3.40 7.89
N
H

7.87
3.16
O

7.42

7.5
1.20
7.42
2.39 7.41
7.40

7.0
7.32
7.31
NMR (500 MHz, Chloroform-d)

5.0 6.5
6.0
5.5
f1 (ppm)
4.5
4.26
1.18
4.24

4.0
1.31 2.49
2.47

3.5
2.46
2.44
2.35

3.0
2.33
2.32
8.24 2.32
2.5 1.53
2.64
1.51
1.51
2.0

1.50
1.49
1.49
1.5

4.90
1.48
3.60
1.48
6.98
1.0

1.47
1.47
1.20
0.5

1.18
0.97
0.96
0.0

0.94
260
 13

230
220
Appendix

210
200
190
180
170
C NMR (126 MHz, Chloroform-d)

166.42
150.35

160
148.23
145.26

150
143.02
134.66
130.93

140
129.67
129.09

130
127.66
126.40
123.87
121.53
119.95

f1 (ppm)
120 110 100
90
77.41
77.37

80
77.16
77.05

70
76.90

60
53.53
52.67
50
46.80
40

36.67
30

23.57
22.30
22.28
20

21.69
10.99
10
0
-10

261
Appendix

N
N N
N
2H3PO4
Cl N
4.4
1H NMR (400 MHz, Deuterium Oxide-d2)

262
Appendix

13
C NMR (126 MHz, Deuterium Oxide-d2)

263
Appendix

31
P NMR (162 MHz, Deuterium Oxide-d2)

264
 8.61

1H
8.60
7.99

9.0
Appendix

7.98

N
7.89

Cl
1.00
7.87

8.5
7.36
7.35

4.6
0.94

8.0
7.34

N
N

1.05 7.34
6.75

7.5
6.74
1.02
2.45
2.45

7.0
2.44
NMR (500 MHz, Chloroform-d)

1.05 2.44
2.42

6.5
2.42
2.41
2.41

6.0
2.33
2.33
2.32

5.5
2.32
2.32
2.31

5.0
2.30
2.30
1.72

4.5
f1 (ppm)
1.71
1.70
1.69

4.0
1.08 1.53
1.52

3.5
1.52
1.51
1.50

3.0
1.42
3.09
1.41
1.40
2.5
4.14 1.40
2.06 1.40
1.40
2.0

1.39
1.14 1.38
1.12 1.38
1.5

1.37
2.14
1.37
3.15
1.36
1.0

6.14
1.35
1.26
1.24
0.5

0.96
0.94
0.93
0.0

265
Appendix

157.56
151.53
150.78

134.64
128.90
125.99
125.22
121.86

109.02

58.79
52.87
46.90

32.48
32.34
24.63
17.09
11.69
13
C NMR (126 MHz, Chloroform-d)

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0
f1 (ppm)

266
 9.5
1H
Appendix

9.0
N

8.5
8.10

4.9
1.00
8.08

H
N
0.40

8.0
8.06
O

7.5
NMR (500 MHz, Chloroform-d)

7.0
1.06 6.55

6.5
6.0
0.23 5.93

5.0 5.5
f1 (ppm)
4.5
4.04
4.03
4.02
1.21

4.0
4.02
4.01

0.29

3.5
2.54
2.53
2.52

3.0
2.51
2.42
2.5 6.50 2.42
3.26 2.42
1.51
1.51
2.0

1.50
1.49
6.54
1.49
1.5

0.89
1.21
3.93
1.20
8.98 1.16
1.0

2.28 1.15
1.03
1.02
0.5

1.00
1.00
267
0.0

0.99
Appendix

163.78
160.68

53.02
52.65
48.37
46.86
46.77
44.01
36.00
34.80
23.58
23.14
22.79
20.84
11.62
11.42
13
C NMR (126 MHz, Chloroform-d)

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0
f1 (ppm)

268
 9.5
1H
Appendix

9.0
N

8.5
4.10

8.0
H
N

7.5
7.0
NMR (500 MHz, Chloroform-d)

6.5
6.0
5.5
2.55
2.53

5.0
2.52
2.50

f1 (ppm)
2.43

4.5
2.41
2.40
1.49

4.0
1.48
1.48

3.5
1.48
1.48
1.47

3.0
1.47
1.46
5.13 1.46
2.5
1.45
5.18
1.45
1.45
2.0

1.31
1.31
1.31
3.00
1.5

1.30
1.04
1.30
3.04
1.29
1.0

6.54 1.29
1.06
1.04
0.5

1.03
1.01
1.00
0.0

269
Appendix

55.07
53.32
46.87

34.91
33.79

23.68
19.79

11.70
13
C NMR (126 MHz, Chloroform-d)

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0
f1 (ppm)

270
 9.5
1H

9.0
Appendix

Cl
8.47
N 8.45

8.5
1.00
8.01
1.00 7.99

8.0
7.96
1.00
N 7.95
NH

7.40

7.5
1.01 7.40
7.38
0.97 7.38

7.0
NMR (500 MHz, Chloroform-d)

7.02

6.39

6.5
1.03
6.38

5.0 6.0
5.5
f1 (ppm)
4.5
3.51

4.0
3.50
3.49
2.12 3.39

3.5
4.15 3.38
3.37
3.36

3.0
2.62
2.61
2.14
2.60
2.5 2.60
1.80
1.80
2.0

6.46 1.79
2.31 1.79
1.79
1.5

1.78
1.77
1.0

1.74
1.73
1.72
0.5

1.68
1.67
0.0

271
Appendix

13
C NMR (126 MHz, Chloroform-d)

272
 1H

9.0
8.93

N
1.00
Appendix

Cl

8.5
8.06
8.06
1.00

4.15
8.03

N
N

8.0
1.08
8.01
7.51
1.08 7.50

7.5
NO2

7.49
7.49

7.0
NMR (500 MHz, Chloroform-d)

5.0 6.5
6.0
5.5
4.01
4.00

4.5
f1 (ppm)
4.00
4.00
1.20

4.0
3.99
3.99
3.98

3.5
3.97
2.85
2.52

3.0
2.50
3.07
2.45
4.10
2.45
2.5 2.15 2.43
2.41
2.0

1.36
1.86
1.40
1.84
2.06 1.67
1.5

3.24 1.65
1.48
6.42 1.46
1.0

1.45
1.43
0.5

1.42
1.01
1.00
0.0

0.98
).
273
Appendix

151.14
150.71
147.91
137.64
137.05
129.68
127.56
127.19
123.83

60.99

52.76

46.94

33.91
32.72

24.45
18.18
11.42
13
C NMR (126 MHz, Chloroform-d)

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0
f1 (ppm)

274
 10.0
Et2N

9.5
0.91 9.40

Cl
Appendix

9.0
1.00 8.62

8.5
N
N

4.16
7.98
7.96
7.93

8.0
1.05
NH2

7.92
1.05
7.55
1.07

7.5
7.54
7.52
6

7.52
1H NMR (400 MHz, Dimethyl sulfoxide-d )

5.0 7.0
6.5
6.0
5.5
f1 (ppm)
4.04.5
3.55
3.53
1.78 3.52

3.5
3.51

4.05 3.05

3.0
5.13 3.04
3.03
2.5 2.96
2.95
2.93
2.0

1.59
1.58
4.27 1.56
1.5

1.56
3.31
1.55
6.21
1.54
1.0

1.52
1.19
1.17
0.5

1.14
1.12
0.0

1.10
275
Appendix

140.47
139.38
138.79
138.67
130.61
127.23
126.25
125.55
124.01

55.24
50.92
46.45
46.30

35.02
31.32

20.25
17.77

8.56
8.54
13
C NMR (101 MHz, Dimethyl sulfoxide-d6)

Trifluoroacetic Trifluoroacetic
acid acid

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10
f1 (ppm)

276
 9.5
1H
N

9.0
Appendix

Cl
1.00 8.67

8.5
8.00

4.17
8.00

N
N
1.03

8.0
7.98
1.34
7.97
N3

7.43

7.5
1.21 7.42
7.41
7.41
NMR (500 MHz, Chloroform-d)

5.0 7.0
6.5
6.0
5.5
3.51
3.50
3.48

f1 (ppm)
4.5
3.47
2.93
2.51

4.0
2.50
2.49
1.31 2.47

3.5
2.40
2.38
2.37

3.0
4.12
1.68
1.67
4.48
2.5 1.66
2.05 1.65
1.65
2.0

1.65
1.63
1.37 1.49
1.5

3.30 1.48
3.31 1.48
6.65 1.47
1.0

1.47
1.21
1.20
0.5

1.00
0.98
0.97
0.0

277
Appendix

147.93
146.13
145.08
134.30
128.91
128.14
127.48
125.96
125.91

58.61
53.02
46.94

35.52
32.74

24.11
17.90
11.54
13
C NMR (126 MHz, Chloroform-d)

230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0
f1 (ppm)

278
Appendix

279
Appendix

12.23
12.22

8.12
8.10
7.95
7.94
7.63
7.62
7.40
7.39
7.38
7.37
OH
N3

Cl N
4.18
1H NMR (500 MHz, Dimethyl sulfoxide-d6)
1.06

1.03
1.00
1.00
1.05

12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
f1 (ppm)

280
Appendix

171.72

139.35
136.45
130.19
127.04
124.10
122.37
120.30
117.71
13
C NMR (126 MHz, Dimethyl sulfoxide-d6)

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10
f1 (ppm)

281
Appendix

282
283