Anti-Parasitic and Anti-Bacterial Agents

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January 2013 Anti-parasitic and anti-bacterial agents: Studies on 1,4-dihydropyridines and 2,4-diaminoquinazolines Kurt Steven Van Horn University of South Florida, [email protected]

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Anti-parasitic and anti-bacterial agents: Studies on 1,4-dihydropyridines and 2,4-

diaminoquinazolines

Kurt Steven Van Horn

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry College of Arts and Sciences University of South Florida

Major Professor: Roman Manetsch, Ph.D. Bill J. Baker, Ph.D. Wayne Guida, Ph.D. Dennis E. Kyle, Ph.D. Lindsey Shaw, Ph.D.

Date of Approval: 11 June 2013

Keywords: Malaria, Leishmaniasis, methicillin-resistant Staphylococcus aureus, Acinetobacter baumannii, antimicrobial, structure-activity relationship, structure-property relationship

Acknowledgements

First and foremost I would like to acknowledge Roman Manetsch. He has been my mentor and friend these last eight years and without him I would never have accomplished what I have. Thank you, Roman, it has been a real pleasure.

Working in a medicinal chemistry lab, it is impossible to get anything done without collaborations. Within the Manetsch lab, Niranjan Namelikonda was a great help and most of the lactone work in Chapter 2 would not have occurred without him. He has also been a great help with chemical discussions and has provided much insight that otherwise would not have been found.

Within the chemistry department at the University of South Florida, the help of Edwin

Rivera and the NMR TA’s including David Badger, Will Tay, Andrii Monastyrskii and

Arthur Maknenko has indeed been beneficial for all of the projects within. Their support and commitment to keeping the instruments serviced and running and providing proper education to the users has enabled the smooth characterization of numerous compounds.

Along this same line, the HPLC facility has been well run by Ted Gautier and most recently Mohanraja Kumar. Without their help much of the reactions run herein would not have been followed as well as has been shown within. Lukasz Wojtas also played an important role, facilitating the X-ray crystallography and allowing for the determination of stereochemistry in the 1,4-dihydropyridine project.

Collaborations at USF began with the lab of Dennis Kyle, with Brian Vesely and

Anuradha Srivastava performing the initial tests for the leishmaniasis project. Without their time and studies the quinazoline class would not have been investigated in as much depth. A great help in the Kyle lab has been Tina Mutka, who has peformed all of the in vitro Plasmodium falciparum assays using the TM90-C2B and W2 strains. Her work has provided numerous points of data that has truly allowed the growth of the 1,4- dihydropyridine project.

Another collaboration at USF has been in the lab of Lindsey Shaw. Whittney Burda has performed the bulk of the work concering the MRSA and Acinetobacter baumannii projects and has been able to provide any and all information that I have requested in regards to the project. Another person in the Shaw lab, Renee Fleeman, has been a great help and was involved early on in the chemistry portion of the quinazoline class so she must be thanked two-fold.

The Ohio State University has provided a highly beneficial collaboration with Kyle

Werbovetz. The Werbovetz lab has led the majority of the testing for the leishmaniasis project and I am extremely grateful to the help of Xiaohua Zhu and Trupti Pandharkar for their enthusiasm for the project and the ability to accomplish their work.

None of the pharmacokinetic studies would have been done without the work of Zhuo

Michael Wang at the University of Kansas. In the Wang group, Siyung Yang performed the work and was able to reliably show PK data for the leishmaniasis project.

The malaria project involving the 1,4-dihydropyridines has been a godsend for me as a highly enjoyable project filled with lofty chemistry goals and great achievements. I

would truly like to thank R. Kiplin Guy for involving us on the project, and the rest of his team at the St. Jude Children’s Research Hospital. Martina Sigal has been my go to person and helped me throughout the project. Julie Clark has performed much of the testing and Amy Matheny has been a leader with oversight on the biological side of the project.

Again, I am truly grateful for all of the collaborations in which I have been able to take part. Without them, my graduate career could not have been what you see written within.

Table of Contents

List of Figures ...... ix

List of Tables ...... xiv

List of Abbreviations ...... xvi

Abstract ...... xix

Chapter 1: Introduction to Anti-microbials...... 1 1.1 Overview ...... 1 1.2 Protozoa ...... 2 1.2.1 Genus Plasmodium ...... 2 1.2.1.1 Anti-Malarials ...... 5 1.2.2 Genus Leishmania ...... 8 1.2.2.1 Anti-leishmanials ...... 11 1.3 ESKAPE Pathogens ...... 12 1.3.1 Staphylococcus aureus ...... 14 1.3.1.1 Staphylococcal Toxins ...... 15 1.3.1.2 Antibiotics for Staphylococcus aureus ...... 17 1.3.2 Acinetobacter baumannii ...... 25 1.3.2.1 Antibiotics for Acinetobacter baumannii...... 26

Chapter 2: 1,4-Dihydropyridines as Anti-Malarials ...... 29 2.1 Hantzsch Pyridine Synthesis ...... 29 2.1.1 Enantioselective Syntheses of 1,4-Dihydropyridines ...... 30 2.1.2 Biological Activity of 1,4-Dihydropyridine Derivatives ...... 36 2.2 1,4-Dihydropyridine Syntheses...... 38 2.2.1 Synthetic Plan ...... 39 2.2.1.1 Comparison of Methylene and Oxygen Derivatives ...... 40

2.2.1.2 Synthetic Scheme with Chiral Auxiliaries ...... 44 2.2.1.3 Synthetic Schemes for Chiral β-Ketolactones ...... 56 2.2.2 Structure-Activity Relationship of 1,4-Dihydropyridines ...... 62 2.2.2.1 Structure-Activity Relationship at 4- and 7-Positions ...... 62 2.2.2.2 Structure-Activity Relationship of the 2- and 3-Positions ...... 66 2.2.2.3 A Few Polyhydroquinoline Derivatives...... 68 2.2.2.4 Role of Stereochemistry in Activity ...... 69 2.2.2.5 Role of Stereochemistry on Solubility and Permeability ...... 70 2.3 Summary ...... 71

Chapter 3: Quinazolines as Anti-Leishmanials...... 73 3.1 Chemistry of the Quinazoline Class ...... 73 3.2 Medicinal Importance of the Quinazoline Class ...... 74 3.2.1 Quinazolines in the Literature as Anti-Leishmanials...... 76 3.3 Anti-Leishmanial Testing ...... 77 3.3.1 Synthetic Chemistry ...... 77 3.3.2 In vitro Anti-Leishmanial Efficacy and Cytotoxicity ...... 78 3.3.2.1 Structure-Activity Relationship Studies...... 79 3.3.2.2 Cytotoxicity...... 83 3.3.3 Mechanism of Action ...... 84 3.3.4 Structure-Property Relationship Studies...... 85 3.3.5 In Vivo Anti-Leishmanial Efficacy Studies ...... 87 3.3.6 Pharmacokinetics of 3.16 and 3.23 ...... 90 3.3.7 Summary ...... 93 3.4 Additional Testing ...... 94 3.4.1 Structure-Activity Relationships ...... 95 3.4.1.1 Structure-Activity Relationship of N2- or N4- Alkyl with N2- or N4- Alkyl/Aryl ...... 95 3.4.1.2 Structure-Activity Relationship of N2,N4-Diaryl Derivatives ...... 96 3.4.1.3 Structure-Activity Relationship of N2,N4-Diaryl Derivatives with Backbone Substitution ...... 98 3.4.2 Structure-Property Relationship Studies ...... 98 3.4.3 Pharmacokinetics of 3.41 and 3.46 After Intraperitoneal Administration in Mice ...... 100

3.4.4 In Vivo Anti-Leishmanial Efficacy Studies of Compounds 3.28, 3.41 and 3.46 ...... 101 3.4.5 Summary ...... 102

Chapter 4: Quinazolines and the ESKAPE Pathogens ...... 104 4.1 Quinazolines as Anti-Bacterial Agents ...... 104 4.2 Quinazolin-2,4-Diamine Activity Against Methicillin-Reisistant Staphylococcus aureus ...... 106 4.2.1 Testing of Quinazolines via Kirby-Bauer Assays ...... 106 4.2.2 Minimum Inhibitory Concentration Assays...... 109 4.2.3 Derivatization Based on Initial SAR ...... 113 4.2.4 Determination of Minimum Bactericidal Concentration for Compounds 4.47 and 4.58 ...... 116 4.2.5 Determining the Minimum Biofilm Eradication Concentration for Compound 4.47 ...... 117 4.2.6 Cytotoxicity and Hemolysis Assays of Selected Compounds ...... 118 4.2.7 Determination of Staphylococcus aureus Resistance to Quinazolines ...... 119 4.2.8 Exploring the Mechanism of Action for 4.45 ...... 121 4.2.9 Galleria mellonella Larval Model ...... 122 4.2.10 In vivo Efficacy Using a Murine Model of Lethal Peritonitis ...... 125 4.2.11 Conclusions ...... 126 4.3 Screening of Quinazolines Against ESKAPE Pathogens ...... 127 4.3.1 Structure-Activity Relationship of Quinazolin-2,4-diamines by Minimum Inhibitory Concentration ...... 128 4.3.2 Extended Minimum Inhibitory Concentration Testing ...... 129 4.3.3 Minimum Bactericidal Concentrations Activity of Frontrunner Quinazolin-2,4-Diamines Against Acinetobacter baumannii ...... 131 4.3.4 Determining the Minimum Biofilm Eradication Concentration for 4.41 and 4.68-4.71 ...... 132 4.3.5 Cytotoxicity of 4.70 ...... 133 4.3.6 In vivo Model of Infection Using Galleria mellonella ...... 133 4.3.7 Conclusions ...... 134

vii

Chapter 5: Future Plans ...... 136 5.1 1,4-Dihydropyridines ...... 136 5.1.1 Synthesis Going Forward ...... 136 5.1.2 Property-Based Synthesis ...... 136 5.1.3 Additional Testing ...... 137 5.1.4 Stereoselective Syntheses ...... 138 5.2 Quinazolines as Anti-Leishmanials ...... 139 5.3 Quinazolines as Antibacterials ...... 140 References Cited ...... 142

viii

List of Figures

Figure 1.1: The malaria parasite life cycle ...... 4

Figure 1.2: Structures of anti-malarials ...... 9

Figure 1.3: Life cycle of the Leishmania parasite...... 10

Figure 1.4: Common anti-leishmanials ...... 12

Figure 1.5: Cell wall of a Gram-positive and a Gram-negative bacterium ...... 13

Figure 1.6: Crystal structure of alpha-hemolysin ...... 16

Figure 1.7: β-Lactam deactivation by penicillinase ...... 18

Figure 1.8: Structure of early antibiotics ...... 21

Figure 1.9: MRSA anti-bacterial drugs approved by the FDA since 1999 ...... 24

Figure 1.10: Gram-negative antibiotics active against Acinetobacter baumannii ...... 28

Figure 2.1: Classical Hantzsch pyridine synthesis ...... 29

Figure 2.2: Synthesis of (+) and (–)-nicardipine via alkaloid resolution ...... 30

Figure 2.3: Stereoselective synthesis of nimodipine...... 31

Figure 2.4: Diastereoselective formation of 4-aryl-1,4-dihydropyridines using a

chiral oxazoline ...... 32

Figure 2.5: Stereoselective synthesis of 4-aryl-1,4-dihydropyridines using chiral

hydrazines ...... 33

Figure 2.6: Selective ester cleavage of symmetrical 1,4-dihydropyridine using

Pseudomonas lipase ...... 33

Figure 2.7: Synthesis of chiral 4-aryl-1,4-dihydropyridines via diastereomeric

intermediates ...... 34

Figure 2.8: Synthesis of a chiral 4-aryl-1,4-dihydropyridine using tartaric acid

resolution...... 35

Figure 2.9: Use of chiral BINOL-phosphoric acid in the enantioselective synthesis

of 4-phenyl polyhydroquinolines ...... 35

Figure 2.10: Calcium channel blockers on the market ...... 36

Figure 2.11: 1,4-Dihydropyridines recently reported with anti-cancer, anti-

tubercular and anti-bacterial properties ...... 38

Figure 2.12: Retroysynthesis of 1,4-dihydropyridine derivatives ...... 40

Figure 2.13: Synthesis of 1,4-dihydropyridine derivatives ...... 41

Figure 2.14: Transesterification attempt with (S)-1,2-isopropylideneglycerol ...... 44

Figure 2.15: Hydrolysis with acid or base ...... 45

Figure 2.16: Hydrolysis of the tert-butyl ester 2.3...... 45

Figure 2.17: Synthesis of the 3-carboxylic acid via β-elimination ...... 46

Figure 2.18: Acid derivatization attempts ...... 46

Figure 2.19: Synthesis of chiral auxiliary acetoacetates ...... 47

Figure 2.20: Analytical RP18 HPLC trace of threonine diastereomers ...... 48

Figure 2.21: Carbon NMR of threonine derivative 2.9-P1 (top) and 2.9-P2

(bottom) between 103 and 171 ppm ...... 49

Figure 2.22: Synthesis of pure 1,4-dihydropyridine stereoisomers via (2S,3R)-2.9-

P2 ...... 50

Figure 2.23: Synthesis of pure 2.1 stereoisomers via the chiral oxazolidinone side

chain ...... 51

Figure 2.24: Analytical RP18 HPLC trace of oxazolidinone diastereomers ...... 52

Figure 2.25: Formation of six 2.10 stereoisomers as three enantiomeric pairs ...... 53

Figure 2.26: Identification of 2.1 ethyl esters of 2.10-P1 and 2.10-P2 as

enantiomers ...... 54

Figure 2.27: Crystal structure of (R)-2.10-P1 with equatorial 7-substituent (top)

and axial 7-substituent (bottom) ...... 55

Figure 2.28: Synthesis of (4S,7S)-2.1-A from (2S,3R)-2.9-P2 and (R)-2.10-P1 ...... 56

Figure 2.29: Example of the formation of a β-ketolactone from a benzaldehyde ...... 57

Figure 2.30: Mukaiyama aldol route using Chan’s diene ...... 58

Figure 2.31: Hantzsch reaction using a chiral lactone derivative 2.16 ...... 59

Figure 2.32: Brown’s allylation steps using a TBS protected benzyl alcohol ...... 60

Figure 2.33: Cross metathesis and Michael addition of the Brown allylation

product ...... 61

Figure 2.34: Ring-closing metathesis route ...... 61

Figure 2.35: Anti-malarial SAR of the 1,4-dihydropyridine ...... 71

Figure 3.1: Electronics of the quinazoline system ...... 73

Figure 3.2: Reactivity of the 2,4-dichloroquinazoline ...... 73

Figure 3.3: Biological role of quinazolines in the literature ...... 75

Figure 3.4: Quinazolines shown to have anti-leishmanial activity ...... 76

Figure 3.5: Hit compounds 1 & 2 and SAR positions of quinazoline ...... 77

Figure 3.6: Synthesis of N2,N4-disubstituted quinazoline-2,4-diamines ...... 78

Figure 3.7: In vitro efficacy of quinazolines, methotrexate (MTX), pyrimethamine

(PYR), and miltefosine (MILT) for axenic amastigotes of Leishmania

donovani in the absence or presence of d,l-folinic acid (FNA) ...... 84

Figure 3.8: In vivo efficacy of quinazolines against LV82 in L. donovani infected

BALB/c mice...... 89

Figure 3.9: Plasma (open circles) and tissue (squares for liver and triangles for

spleen) concentration-time profiles after p.o. (A) and i.p. (B)

administration of 16 in mice at a dose level of 100 μmol/kg (~30 mg/kg) ...... 90

Figure 3.10: Plasma (open circles) and tissue (squares for liver and triangles for

spleen) concentration-time profiles after p.o. (A) and i.p. (B)

administration of 23 in mice at a dose level of 100 μmol/kg (~30 mg/kg) ...... 93

Figure 3.11: Plasma and tissue concentration-time profiles after 10 mg/kg i.p.

administration of 3.41 and 3.46 ...... 100

Figure 3.12: In vivo efficacy of quinazolines in L. donovani infected BALB/c

mice ...... 102

Figure 4.1: Quinazolines with reported anti-bacterial activity ...... 105

Figure 4.2: Positions of the quinazoline ring ...... 106

Figure 4.3: Percent recovery curves of 4.47 and 4.58 ...... 116

Figure 4.4: Percent survival curve for 4.47 against MRSA biofilm ...... 117

Figure 4.5: OD540 of erythrocytes in the presence or absence of 4.47 ...... 119

Figure 4.6: MICs of selected compounds of against the seven mutants formed

from 4.47 ...... 121

xii

Figure 4.7: Wax worm larvae survival post-infection for 4.38 with low and high

inoculation volumes. Low and high refer to the inoculation volumes

discussed earlier in the text. * = p < 0.05 ...... 123

Figure 4.8: Wax worm larvae survival post-infection for 4.45 with low and high

inoculation volumes. Low and high refer to the inoculation volumes

discussed earlier in the text. * = p < 0.05 ...... 124

Figure 4.9: Wax worm larvae survival post-infection for 4.47 and 4.58. * = p <

0.05...... 125

Figure 4.10: Mice survival post-infection. * = p < 0.05 ...... 126

Figure 4.11: Percent recovery curves for A. baumannii 1403 incubated with

quinazolines ...... 131

Figure 4.12: Percent recovery curve for A. baumannii biofilm grown in the

presence of 4.70 ...... 133

Figure 4.13: Wax worm larvae survival post-infection with 5x MIC. * = p < 0.05 ...... 134

Figure 5.1: Derivation at the 2-position ...... 137

xiii

List of Tables

Table 2.1: Comparison of the replacement of -CH2- with -O- at the 6-position ...... 42

Table 2.2: Comparison of oxazolidinone and threonine auxiliaries ...... 56

Table 2.3: 4-phenyl modifications ...... 63

Table 2.4: Aromatic changes at 4-position ...... 64

Table 2.5: 7-Phenyl modifications ...... 65

Table 2.6: 2- and 3-position modifications ...... 67

Table 2.7: Polyhydroquinoline derivatives with C2 and C4 changes ...... 69

Table 2.8: Comparison of activities of 2.1 stereoisomers ...... 70

Table 2.9: Comparison of solubilities and permeabilities of 2.1 stereoisomers ...... 70

Table 3.1: SAR study focusing on 2- and 4-positions ...... 81

Table 3.2: Benzenoid substitution...... 83

Table 3.3: Physicochemical properties of quinazolines ...... 86

Table 3.4: Pharmacokinetic outcomes of 3.16 and 3.23 after p.o. and i.p.

administration to mice...... 91

Table 3.5: N2- or N4- alkyl with N2- or N4- alkyl/aryl substitutions ...... 96

xiv

Table 3.6: N2,N4-diaryl derivatives ...... 97

Table 3.7: 6- and 7-substituted derivatives ...... 98

Table 3.8: Physicochemical properties of quinazolines ...... 99

Table 3.9: Pharmacokinetic outcomes of 3.41 and 3.46 after 10 mg/kg i.p.

administration to mice...... 101

Table 4.1: SAR of N-alkyl-N-furfurylquinazolin-2,4-diames...... 110

Table 4.2: SAR of benzenoid substitutions...... 111

Table 4.3: N2- and N4-substitutions rich in sp3 hybridized carbons ...... 112

Table 4.4: N2- and/or N4-benzyl and/or phenyl substitutions ...... 113

Table 4.5: Combination with 7-chloro or p-substituted benzyl at N2 and/or N4 ...... 114

Table 4.6: Combination with 7-chloro and/or p-substituted benzyl at N2 and/or N4 ...... 115

Table 4.7: A549 EC50 and selectivity indices of select compounds ...... 118

Table 4.8: MICs of N-benzyl-N-methylquinazolin-2,4-diamines against A.

baumannii 1403 strain...... 128

Table 4.9: MICs of active compounds and similar compounds against A.

baumanni 1403 and A-L strains ...... 130

List of Abbreviations

6-APA 6-aminopenicillanic acid Ac acetyl ACN acetonitrile ACT artemisinin-based combination therapy AIDS acquired immune deficiency syndrome AIF apoptosis inducing factor Ar aryl AUC area under the curve BINOL 1,1’-bi-2-napthyl Bn benzyl Bu butyl BuLi butyllithium CA-MRSA community-acquired methicillin-resistant Staphylococcus aureus CDC Centers for Disease Control and Prevention CFU colony forming unit CHDL carbapenem-hydrolyzing class D β-lactamase Cmax maximum concentration CNS central nervous system DBU 1,8-diazabicyclo[5.4.0]-undec-7-ene DCM dichloromethane DEAD diethyl azodicarboxylate DHA dihydroartemisinin DHFR dihydrofolate reductase DHPS dihydropteroate synthase DIA diisopropylamine DIAD diisopropyl azodicarboxylate DMAP 4-dimethylaminopyridine DMF dimethylformamide DMSO dimethylsulfoxide DNA deoxyribonucleic acid EC50 half maximal effective concentration ED50 half maximal effective dose ee enantiomeric excess ESI electrospray ionization ESKAPE Enterococcus faecium, Staphylococcus aureus, Klebsiella pnemoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species Et ethyl EtOAc ethyl acetate EtOH ethanol xvi

FDA Food and Drug Administration FNA d,l-folinic acid GC gas chromatography HAI hospital acquired infection HIV human immunodeficiency virus HPβCD 2-(hydroxypropyl)-β-cyclodextrin HPLC high pressure liquid chromatography HRMS high resolution mass spectrometry HTS high throughput screening i.p. intraperitoneal IC50 half maximal inhibitory concentration iPr isopropyl iPrOH isopropanol kDa kilodalton LC-MS liquid chromatography-mass spectrometry LDA lithium diisopropylamide LDU Leishman-Donovan unit Me methyl MeOH methanol MIC minimum inhibitory concentration MILT miltefosine MMV Medicines for Malaria Venture MRSA methicillin-resistant Staphylococcus aureus MTX methotrexate NC not calculable ND not determined NMR nuclear magnetic resonance Omp38 outer membrane protein 38 p.o. oral administration (per os) PABA para-aminobenzoic acid PAMPA parallel artificial membrane permeability assay PBP penicillin-binding protein Pe permeability PEG polyethylene glycol Ph phenyl PK pharmacokinetics PPh3 triphenylphosphine PPTS pyridinium para-toluenesulfonate Pr propyl prep-HPLC preparative high-pressure liquid chromatography pTSA para-toluenesulfonic acid PTSAg pyrogenic toxic superantigen PVL Panton-Valentine leukocydin PYR pyrimethamine QRDR quinoline resistance-determining region RCM ring-closing metathesis xvii

RNA ribonucleic acid RP reverse phase RT room temperature SAR structure activity relationship SE staphylococcal enterotoxin SI selectivity index SPR structure property relationship TBAF tertrabutylammonium fluoride TBS tributyl silyl tBu tert-butyl t-BuOH tert-butanol TEA triethylamine TFA trifluoroacetic acid THF tetrahydrofuran TLC thin layer chromatography TMS trimethyl silyl TSST-1 toxic shock syndrome toxin-1 USF University of South Florida VISA vancomycin intermediate-resistant Staphylococcus aureus VRE vancomycin resistant Enterococcus faecalis VRSA vancomycin resistant Staphylococcus aureus WHO World Health Organization WRAIR Walter Reed Army Institute of Research ZOI zone of inhibition

xviii

Abstract

Thirty-three 1,4-dihydropyridine diastereomeric pairs were synthesized and the structure- activity relationship studied in a Plasmodium falciparum in vitro model. Twenty-nine of these derivatives contained a 6-position oxygen, with 2.31, 2.32, 2.52 and 2.53 having single and double digit nanomolar activities. This SAR study revealed some insightful information about the 1,4-dihydropyridine substitution pattern. Substitution at the 7- position other than 3,4-dimethoxy severely reduced the activity. 4-phenyl substitution with 2- or 4- halo or methyl formed active compounds while substitution at the 3-position or with methoxy or conjugated aryl systems resulted in inactive compounds. The 2- position was found to majorly affect the activity, with groups larger than methyl being the most active. The other four derivatives contained a 6-position methylene, with 2.1,

2.59 and 2.60 having single nanomolar activities. Lastly, stereochemistry was revealed to play an important role in the activity of 2.1. One stereoisomer, (+)-trans-2.1, had subnanomolar activity in two assays. Another stereoisomer, (4S,7S)-2.1, had nanomolar activity. The other two stereoisomers were inactive.

A series of N2,N4-disubstituted quinazoline-2,4-diamines has been synthesized and tested against Leishmania donovani and Leishmania amazonensis intracellular amastigotes. A structure-activity and structure-property relationship study was conducted in part using the Topliss operational scheme to identify new lead compounds. This study led to the identification of quinazolines with EC50s in the single digit micromolar or high nanomolar range in addition to favorable physicochemical properties. Quinazoline 3.23

xix also displayed efficacy in a murine model of visceral leishmaniasis, reducing liver parasitemia by 37% when given by the intraperitoneal route at 15 mg/kg/day for five consecutive days. Their antileishmanial efficacy, ease of synthesis, and favorable physicochemical properties make the N2,N4-disubstituted quinazoline-2,4-diamine compound series a suitable platform for future development of antileishmanial agents.

A similar series of N2,N4-disubstituted quinazoline-2,4-diamines has been synthesized and tested against methicilin-resistant Staphylococcus aureus (MRSA) and multi-drug resistant strains of Acinetobacter baumannii. Quinazolines with MICs in the single digit micromolar or high nanomolar range were identified via SAR. In a murine model of

MRSA infection, 1x the MIC for quinazoline 4.47 allowed for the survival of all tested mice at the end of a one week study. An in vivo model of A. baumannii was also undertaken using a Galleria mellonella model of infection. Quinazolines 4.68, 4.70 and

4.71 afforded an increased protection of 87.5% when compared to the control experiments, with 70% of the wax worms surviving until day three. The observed potencies of frontrunner compounds in in vivo assays and their ease of synthesis make

N2,N4-disubstituted quinazoline-2,4-diamines a suitable platform for the future development of anti-bacterial agents.

Chapter 1: Introduction to Anti-microbials

1.1 Overview

Microorganisms, or microbes, are microscopic organisms that are found all over the world. Microbes may be prokaryotic or eukaryotic, multicellular or unicellular. This diverse group of organisms has played a huge role in the ecology of Earth. Bacteria participate in the nitrogen cycle in the process of nitrogen fixation to turn inorganic nitrogen gas to usable ammonium and nitrate. Algae and cyanobacteria perform photosynthesis to turn carbon dioxide into sugars and oxygen. There is also the fact that all life on earth evolved from these organisms. Microorganisms also play a more local role in mammalian life. Bacteria within the digestive tract play a pivotal role in the health of the human body. The treatment of sewage is undertaken with the help of a panoply of microorganisms. The synthesis of many and large amounts of antibiotics such as penicillin are only undertaken with the use of bacteria. Most important, a great pastime of food and drink could not be undertaken without the help of yeast.1

Although microorganisms are known to be productive members of this planet, oftentimes they also prey on other life forms. Eukaryotes, such as the tropical disease causing parasites of the genera Leishmania and Plasmodium, and prokaryotes, such as the infection causing bacteria of the genera Acinetobacter and Staphylococcus, both are causes of human infection. Eukaryotic and prokaryotic cells can be mobile or immobile.

In the case of the aforementioned bacteria, immobility is the way of life. The eukaryotic

Leishmania and Plasmodium, on the other hand, both display periods of mobility in their life cycles. Leishmania promastigotes have a flagellum for movement which they lose upon transforming to amastigotes.2 Plasmodium sporozoites are able to travel via gliding, a mechanism characterized by the lack of any cell modification during movement.3 These four human pathogens will be studied in more depth within the coming sections.

1.2 Protozoa

1.2.1 Genus Plasmodium

Malaria is a parasitic disease with half of the world’s population living in affected areas.4

Although only fifteen hundred cases of malaria are reported in the United States annually, nearly one million people die worldwide each year from malaria and an additional two hundred million are infected.4 These fatalities make malaria the fifth highest worldwide cause of death following respiratory infections, HIV/AIDS, diarrheal diseases and tuberculosis.4b Thirty five countries, thirty in sub-Saharan Africa and five in Asia, account for 98% of the annual fatalities, while sub-Saharan Africa alone accounts for

89%.4b

Malaria is a disease that is caused in humans by protozoan parasites of the genus

Plasmodium. Five species have been found to cause malaria in humans: P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. P. falciparum and P. vivax are the two most commonly found although P. falciparum is by far the most deadly with nearly all fatal and severe cases of malaria, resulting from the large population of parasites which grow inside of an infected individual.5 The parasites are transmitted by a mosquito vector of the genus Anopheles, whereby a mosquito ingests a blood meal from an infected

2 individual and the transmission occurs when the mosquito next takes a blood meal and expels the parasites into the new host.4 The World Health Organization estimates that 219 million people were infected by malaria and that 660,000 people died worldwide due to malaria in 2010.6 One of the major reasons for such a large population of people being affected by this disease is that it is definitely increased in impoverished areas. Poorer populations have a higher chance of getting malaria as they tend to live in rural areas, do not have proper protection in place to reduce contact with mosquitoes and do not have access to health facilities which may help them to prevent or cure the infections.6 80% of the cases and 91% of the deaths were accounted for by sub-Saharan Africa. Along with this figure, 86% of the deaths were children <5 years old.6

The parasites are spread by the bite of approximately twenty species of mosquitoes in the genus Anopheles.4 When an infected female mosquito bites an animal for a blood meal,

Plasmodium sporozoites are injected into the epithelial cells. These sporozoites migrate through the body until they end up in hepatocytes in the liver of the infected individual

(Figure 1.1).4 Here the sporozoites mature by replicating organelles and the nucleus and become a schizont, which then divides cellularly to become merozoites. The merozoites are then released to the rest of the body when the hepatocyte ruptures after a period of seven days to several weeks, depending on the species.4 The newly released tens of thousands of merozoites infect erythrocytes in the blood stream where they become schizonts and asexually reproduce until the cells burst, restarting the erythrocyte infection.4 A small number of merozoites transform into male and female gametocytes, a sexual form of the parasite that may infect an uninfected Anopheles mosquito when it takes a blood meal from the infected animal.4b, 5 In P. falciparum the gametocytes form 3 later on, with health of the host deteriorating prior to transmission. In P. vivax, however, gametocytes form much earlier and may be transmitted prior to any clinical manifestation of the disease.7

Inside the stomach of the mosquito, a male gamete combines with a female gamete to form a zygote. The zygote transforms into a mobile ookinete which enters the stomach lining and grows into a non-mobile oocyst. This oocyst is thick-walled and multiplies until sporozoites burst from the cell. The sporozoites migrate to the salivary glands of the mosquito and from here they are injected to a new host when the mosquito takes another blood meal.4

Figure 1.1: The malaria parasite life cycle4b

As merozoites leave the liver and infect the rest of the body, signs of infection appear.

With the asexual replication of the merozoites in the erythrocytes, toxins are released which harm the host.5 Flu like symptoms, fever, headache, chills and vomiting are the first symptoms observed in people who are infected with malaria. Severe symptoms that may develop include impaired consciousness, coma, prostration, acidosis, respiratory distress, organ dysfunction including jaundice, hypoglycemia, renal failure, anemia and abnormal bleeding.5 Early identification of the disease reduces the fatality of the severe symptoms.

1.2.1.1 Anti-Malarials

Through the years, a number of drugs have been used to treat malaria. The cinchona alkaloids, including quinine, may have been the first with the use of teas and extracts from the cinchona bark during the last few centuries in South America and Europe.8 The cinchona alkaloids, cinchonine, cinchonidine, quinine and quinidine, all display anti- malarial properties but quinine became the alkaloid that moved forward because of a higher amount present in the natural resource.8 The use of quinine waned with the onset of new anti-malarials, including chloroquine. In recent years, quinine has returned as a second-line treatment in combination therapy to reduce the incidence of resistance.

Chloroquine was introduced for clinical use in 1947 and has since then been used as a staple for treatment and prevention of malaria (Figure 1.2). As a 4-aminoquinoline, chloroquine’s mechanism of action involves the disruption of the formation of hemozoin, a crystalline polymer made from the byproduct of hemoglobin consumption by the parasite.9 A chloroquine-heme complex is incorporated within the polymer, capping and

5 preventing the elongation of the crystal within the parasite lysosomal food vacuole.9 This capping of the polymer allows the heme to build up in the parasite, leading to cell death most likely via oxidative damage and disruption of the cell membrane.9 Chloroquine resistance was first seen in the 1950’s and can be caused by an efflux mechanism.10 The

PfCRT protein is an efflux protein located in the food vacuole membrane and is a cause for chloroquine resistance as it actively removes quinoline molecules from the organelle.10c, d

Pyrimethamine is a 2,4-diaminopyrimidine that began clinical use in 1953 (Figure 1.2). It is given in combination with the sulfonamide sulfadoxine. Pyrimethamine is a dihydrofolate reductase (DHFR) inhibitor and sulfadoxine is a dihydropteroate synthase

(DHPS) inhibitor and in combination are effective inhibitors of the synthesis of tetrahydrofolate, a cofactor for thymidilate, purine nucleotides and certain amino acid synthesis.11 Resistance is encountered when DHFR and/or DHPS mutations occur that result in reduced binding and inhibition by each drug.11

Mefloquine is a quinoline derivative quite similar to quinine that was developed in the

1960’s by the Division of Experimental Therapeutics at Walter Reed Army Institute of

Research (WRAIR) and approved for use by the FDA in 1989 (Figure 1.2).12 Resistance was first seen in Southeast Asia and it is no longer an effective treatment in certain areas.11c, 12-13 Halofantrine is a phenanthrene that, like mefloquine, was created in the

1960’s at WRAIR and FDA approved in 1992. It was initially used in areas with mefloquine resistance; however, it was found that it was much less effective in these areas.11c, 12

Atovaquone is a naphthoquinone that began clinical use in 1992 (Figure 1.2).

Atovaquone interrupts the electron transport chain in the cytochrome bc1 complex of mitochondria which blocks the biosynthesis of pyrimidine and it also collapses the electropotential across the mitochondrial membrane, resulting in parasite death.12, 14

Atovaquone is generally given in combination therapy with proguanil, a DHFR inhibitor, as atovaquone resistance generally occurs readily when given on its own.12, 14 Resistance

14-15 has been seen via a mutation in the gene coding for the cytochrome bc1 complex.

In the late 1960’s the Chinese government invested heavily in finding anti-malarial compounds. In 1971 the natural product artemisinin was discovered to have potent anti- malarial properties through the help of ancient medicinal texts (Figure 1.2).16 In the early

1980’s this great discovery was shared with the rest of the world. A few derivatives of artemisinin were found to have better properties and activity including dihydroartemisinin (DHA), artemether and artesunate.16-17 These derivatives are given in combination known as artemisinin-based combination therapies (ACTs) as first line treatments. Resistance to the artemisinins are hoped to be reduced this way and extend the life of the ACTs. Current ACTs in use include artemether with lumefantrine, dihydroartemisinin with piperaquine, and artesunate with either amodiaquine, mefloquine or sulfadoxine-pyrimethamine.17-18 In 2006 artemisinin resistance was reported in

Southeast Asia and ACT resistance has been noted with artesunate-mefloquine in this region.18-19 ACTs are currently the most relied on anti-malarial in use and resistance to these combinations will be highly detrimental to many countries.

P. ovale and P. vivax often cause relapse in patients months or years after they are clinically cured of malaria. These two species form hypnozoites in the liver, a form of the parasite that is dormant and unaffected by the previously shown drugs which generally kill the erythrocytic stage of the parasites.20 Primaquine is an 8-aminoquinoline that was first used in World War II and is able to kill these dormant parasites (Figure 1.2). It is currently used primarily as a relapse preventer in cases of P. ovale and P. vivax in combination with drugs that kill blood stage parasites.12, 20

Although many drugs are available to treat malaria, many are largely cost prohibitive and this makes their use in poor endemic areas where malaria is present a large financial burden. Current research is aimed at the production of less expensive, one time drugs that will allow for cost effectiveness and non-extended periods of drug therapy.

1.2.2 Genus Leishmania

Leishmaniasis is a debilitating disease that is prevalent across the globe. More than twenty species of parasite in the genus Leishmania are known to cause the disease in humans.21 The parasites are transferred from host to host by about thirty species of female sandfly vectors of the genera Phlebotomus and Lutzomyia that infect the host with promastigotes when taking a blood meal (Figure 1.3).22 The flagella containing promastigotes infect the macrophages of the mammalian host, where the flagellum is lost and the promastigote becomes an amastigote. The amastigotes multiply within the macrophages, increasing the parasite burden on the host and infecting new cells when the macrophage bursts. Infected macrophages are ingested by a sandfly when a bloodmeal is taken, resulting in a new infected vector that can transmit the disease to a new host.

Figure 1.2: Structures of anti-malarials

Symptoms of leishmaniasis include unsightly spontaneously healing ulcers on the skin when cutaneous leishmaniasis presents, non-healing lesions in the mucosa when mucocutaneous leishmaniasis is the affliction, and chronic, debilitating infection of the reticuloendothelial system which is fatal if left untreated due to visceral leishmaniasis. 23

The majority of cases of visceral leishmaniasis, also known as kala-azar, are caused by L. 9 donovani in East Africa and Asia, L. infantum in the Mediterranean region, and L. chagasi in Latin America.21 L. infantum and L. chagasi mainly affect children and immunocompromised individuals and are zoonotic parasites with canines being a major reservoir.23 L. donovani, on the other hand, is an anthroponotic parasite and affects a broad range of ages.23 Throughout the world, 350 million people in 88 countries are at risk of acquiring leishmaniasis.24 The World Health Organization (WHO) estimates that

1-1.5 million people acquire cutaneous leishmaniasis and five hundred thousand others are afflicted with visceral leishmaniasis annually, with over 50,000 deaths per year.22, 24

Figure 1.3: Life cycle of the Leishmania parasite

1.2.2.1 Anti-leishmanials

For over one hundred years, antimonials have been the drug of choice to combat leishmaniasis. In 1912, Gaspar Vianna first reported the use of the trivalent antimonial tartar emetic for the treatment of cutaneous leishmaniasis caused by L. braziliensis in

Brazil.25 Pentavalent antimonials such as meglumine antimoniate and sodium stibogluconate are currently the first line drugs for treatment in many areas (Figure

1.4).23, 26 Treatment involves daily injections for up to a thirty day period.8 Problems of using this treatment includes a high rate of resistance that has been encountered in India, especially the state of Bihar, where up to 60% of infected individuals do not improve with treatment.26a, 27 Since the 1960’s, amphotericin B has been the second line of treatment for visceral leishmaniasis.8 It has a cure rate of over ninety percent, but is often accompanied by severe side effects such as nephrotoxicity that require administration in a hospital setting.26a, 28 Depending on the dose and formulation, the treatment regimen varies from 3-5 days to eight weeks of administration on alternate days.21, 26a Miltefosine is the first oral drug to be released for leishmaniasis and is currently available in India,

Germany, and Colombia.8 Miltefosine is not recommended for women who are pregnant or may become pregnant because it is teratogenic.23, 26a Miltefosine resistance has been demonstrated in vitro, and its long half-life in the body, the twenty-eight day treatment regimen, as well as it previously being available over the counter in India leads to concerns of clinical resistance.23, 26a, 29 A recent study of 567 individuals in the Bihar state of India has been performed to determine the efficacy of miltefosine since its induction in 2002.30 The six month cure rate was found to be roughly 90% and gastrointestinal intolerance was encountered in 64.5% of the cases with two deaths

11 related to drug toxicity.30 Patients who did not improve with treatment were cured using amphotericin B. The authors of this study concluded that the failure rate of miltefosine has increased in the ten years since its introduction for the treatment of visceral leishmaniasis in India.

Figure 1.4: Common anti-leishmanials 1.3 ESKAPE Pathogens

Enterococcus faecium, Staphylococcus aureus, Klebsiella species, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species make up an infamous group of bacteria known as the ESKAPE pathogens. These microbes are of import due to resistance to common anti-bacterials and the fact that they are often hospital acquired 12 infections (HAIs).31 Both Gram-positive and Gram-negative bacteria are found in the

ESKAPE pathogens. Most bacteria form a cell well outside of their plasma membrane and the make-up of this cell wall is what determines whether they are Gram-positive or negative (Figure 1.5).1

Figure 1.5: Cell wall of a Gram-positive and a Gram-negative bacterium

Peptidoglycan is a polymer of sugar cross-linked by short polypeptides and is found in bacterial cell walls. Gram-positive bacteria have a comparatively large amount of peptidoglycan and are less complex.1 The higher complexity in Gram-negative bacteria is due to a lipid membrane outside the peptidoglycan that contains anionic lipopolysaccharides.1 These lipopolysaccharides can be toxic and the outer membrane creates an extra barrier that protects from host attacks and increases the difficulty for antibiotics, especially the hydrophobic variety, to enter the cell.1 Many antibiotics inhibit

13 the cross-linking of peptidoglycan and because of the smaller amount of peptidoglycan and the presence of an outer membrane they will not affect Gram-negative species as well.

1.3.1 Staphylococcus aureus

Bacteria of the genus Staphylococcus are Gram-positive facultative anaerobes with a 0.5 to 1 m diameter that form clusters like grapes.32 These bacteria are pathogens of mammals, including man, and cause infections of the skin, urinary tract, blood and respiratory system among others.

S. aureus is the leading cause of staphylococcal infection in humans. It is estimated that

20% of the world population are persistent carriers of this species in the nasal cavity and axillae while 60% of the population are intermittent carriers.32-33 In the United States the population of carriers is around 30%.34 S. aureus is frequently found as a nosocomial infection (HAI), with numerous infections being encountered from hospital stays and long term healthcare facilities every year. This bacterium causes a wide range of infections, from minor skin infections such as pimples, impetigo, boils and other abscesses to severe infections including osteomyelitis, pneumonia, meningitis, toxic shock syndrome, phlebitis, mastitis and endocarditis. The severe infections are often seen by hospital patients if they become infected during their stay. Community-acquired infections (CA-MRSA) are caused by strains of S. aureus that are distinct from the hospital acquired strains. Whereas the HAIs are opportunistic of unhealthy, immunocompromised people, CA-MRSA infections can be found in typically healthy hosts with no risk factors. CA-MRSA infections are oftentimes overwhelming, tissue

14 destructive infections such as necrotizing fasciitis and fulminant, necrotizing pneumonia.35

1.3.1.1 Staphylococcal Toxins

Numerous toxins and proteins are released by S. aureus and are the root of disease in a host. These proteins include enzymes such as nucleases, proteases, collagenase, hyaluronidase and lipases and the toxins that can be found are alpha-, beta-, gamma- and delta- hemolysins, toxic shock syndrome toxin-1 (TSST-1), staphylococcal enterotoxins

(SEs), exfoliative toxins and Panton-Valentine leukocidin, most of which are released in the post exponential growth phase.36 At this point of growth the bacteria have divided until the amount of nutrients has reduced. Many of the toxins are believed to diminish the immune response of the host and the proteins break down tissues into nutrients for bacterial growth.

The staphylococcal enterotoxins and TSST-1 fall into a category of toxins known as pyrogenic toxin superantigens (PTSAgs). The PTSAg class of toxins display at least three properties: pyrogenicity, superantigenicity and the ability to increase the lethality of endotoxin in rabbits up to 100,000 fold.36-37 Staphylococcal enterotoxins are also potent emetic agents whereas TSST-1 can cross mucosal surfaces and reactivate bacterial cell wall-induced arthritis.38 The genes for the PTSAgs are stored in pathogenicity islands such as plasmids, bactereophages and heterologous genetic fragments.39 The mature

PTSAg has a molecular weight between 20 and 30 kiloDaltons (kDa) when released from the cell and is a nonglycosilated polypeptide. The PTSAg toxins are found in people

15 having menstrual (TSST-1) and nonmenstrual (SEs) toxic shock syndrome, sudden infant death syndrome, food poisoning and Kawasaki syndrome.36

Figure 1.6: Crystal structure of alpha-hemolysin40

Alpha-hemolysin is a pore forming toxin that is found in numerous strains of S. aureus and because of this is one of the most studied toxins produced.36 The mature polypeptide has a mass of 33 kDa and is composed of 65% -sheets and 10% -helices (Figure 1.6).

It is formed in the cell during the late exponential phase of cell growth. When released from the cell, alpha-hemolysin incorporates into host cells as oligomers, forming 1-2 nm pores in the cell membranes as heptamers that may cause the eukaryotic cells to lyse.36, 41 16

This pore allows efflux of K+ and influx of Na+, Ca2+ and small molecules less than 1 kDa, resulting in rupture of the cell due to osmotic swelling.36, 42 Alpha-hemolysin is able to affect numerous cells including epithelial cells, erythrocytes, fibroblasts and monocytes.36, 42

β-Hemolysin is formed by a large number of S. aureus strains, but its role in disease is not known. Gamma-hemolysin and Panton-Valentine leukocydin (PVL), however, are known well as affecting mammalian cells and causing disease.36 PVL is made in less than

5% of strains while gamma-hemolysin is synthesized in nearly every strain. These toxins are similar in that they are composed of two protein subunits and affect neutrophils and macrophages. PVL forms pores in host leukocytes, and at high concentration will cause lytic cell death. At lower concentrations it will activate neutrophils to release granule contents via exocytosis and secrete leukotrine B4 and interleukin 8 which causes inflammation.35d, 36, 43 PVL is thought to be a major cause of the virulency and tissue destruction found in CA-MRSA.44

Delta-hemolysin is a 3 kDa peptide that also causes membrane damage and cell lysis. It is believed that delta-hemolysin acts as a surfactant in cell membranes with the release of larger molecules as longer exposure time to the toxin is experienced. This toxin is able to lyse erythrocytes and other cells as well as internal organelles.36

1.3.1.2 Antibiotics for Staphylococcus aureus

Alexander Fleming’s remarkable discovery of Penicillin in 1928 and the following work by Florey, Chain and coworkers of its isolation led to a revolution in modern day medicine.45 Previously fatal infections were curable by a simple infusion of this new

17 antibiotic. It wasn’t until late 1942 and early 1943 that the new drug was mass produced, with the British and American governments recognizing the use of antibiotics in the war effort. In 1940 Abraham and Chain first identified and named penicillinase, a source of penicillin resistance in S. aureus.46 Shortly thereafter penicillin resistant infections were encountered in hospitals.35d, 47

The penicillins are a group of antibiotics that are secondary metabolites of certain species of the fungi in the genus Penicillium. The major features of the penicillins are the β- lactam and thiazolidine fused rings (Figure 1.8). β-Lactams inhibit the penicillin-binding proteins (PBPs) that are necessary for peptioglycan biosynthesis.45 Penicillinase hydrolyzes the β-lactam ring of the penicillins, causing deactivation (Figure 1.7). The gene coding for penicillinase was found in a plasmid within the resistant bacteria.35d

Figure 1.7: β-Lactam deactivation by penicillinase

The late 1940’s and early 1950’s brought the discovery of cephalosporin C, a β-lactam fused to a dihydrothiazine ring that was inactive to the penicillinase that hampered the penicillins (Figure 1.8). The activity of this compound was much lower than the penicillins and it wasn’t until analogues were made in the 1960’s that the cephalosporins made headway as antibiotics.45

In the late 1950’s after penicillin resistance was a major factor, the Beecham Research

Laboratories made semi-synthetic penicillin analogues that stemmed from 6- aminopenicillanic acid (6-APA) (Figure 1.8). The idea was to add bulky side chains to the amide functional group in order to prevent the β-lactamase enzyme from opening the

β-lactam that caused the penicillins to be ineffective. In 1959 they made methicillin, 6- amino-N6-(2,6-dimethoxybenzoyl)penicillanic acid, which remained an effective antibiotic in the presence of penicillinase.45 Cloxacillin, oxacillin, flucloxacillan and dicloxacillan were similar analogues that displayed better blood concentrations in vivo.

Ampicillin, a penicillin derivative bearing a benzyl amine residue, was released in 1961 and is of some import as it was one of the first broad spectrum antibiotics, with antibiotic effects in gram negative bacteria, even though it was still hydrolyzed by β-lactamases.

Amoxicillin is an analogue of ampicillin bearing a phenol that increases the oral- bioavailability of the β-lactam and was released in 1972.48

In 1959, methicillin was released as a S. aureus killer that was unaffected by β-lactamase.

In 1961, however, the first strains of methicillin resistant Staphylococcus aureus (MRSA) were encountered.35d, 49 Unlike the β-lactamase deactivation, hydrolysis of the β-lactam ring did not occur and resistance to methicillin conferred resistance to all β-lactams.45, 50

The new strains made a new penicillin binding protein, PBP2a, that was encoded by the mecA gene and had low affinity to β-lactams. PBPs cross link peptidoglycan chains in the cell wall and the new membrane-bound protein is unaffected by high concentrations of all

β-lactams.51

In the early to mid-1950’s Eli Lilly isolated a glycopeptide from a soil sample containing bacterial growth. This glycopeptide was vancomycin, a polypeptide that displayed activity against Gram-positive bacteria (Figure 1.8).48 Due to poor bioavailability and side effects, vancomycin was not used as a first treatment option, especially with the advent of non-penicillin antibiotics. This, combined with the low resistance from susceptible bacteria, has allowed for vancomycin to be a last resort for bacterial infections since its FDA approval in 1958.48 Teicoplanin is another glycopeptide that has similar use to vancomycin. The glycopeptides interrupt cell wall synthesis by blocking cross linking between peptidoglycan.45 Due to increased multi-drug resistance across bacterial strains, glycopeptides have been used at a much higher rate in the last 25 years, resulting in the first report of vancomycin intermediate-resistant S. aurues (VISA/GISA) in 1997 from a MRSA strain in Japan.52 Since then, VISA and vancomycin resistant S. aureus (VRSA) have become more prevalent across the globe.53 VRSA isolates have been found to contain the vanA operon from vancomycin resistant Enterococcus faecalis

(VRE), showing that resistance has been transferred via plasmid between orders.51

Broad spectrum fluoroquinolones, such as ciprofloxacin, were introduced in the mid

1980’s (Figure 1.8). Fluoroquinolones act by inhibiting two enzymes that unwind bacterial DNA, DNA gyrase and topoisomerase IV.45, 51 By 1990 mild resistance was seen via efflux with an over expression of the multidrug NorA pump.45, 51 Nucleic acid mutations have also resulted in amino acid substitution in the target enzymes that reduced the binding affinity of the quinolones.51

Figure 1.8: Structure of early antibiotics

The ability of bacteria to acquire resistance to so many drugs is multifold. The multitude of bacteria present in an infection allows for chromosomal mutations that create numerous phenotypes that may through randomization create a bacterium that is not susceptible to a specific antibiotic. When bacteria are part of the normal fauna, as in the

20% of human population who are persistent carriers of S. aureus in the nasal cavities, antibiotics that are used to treat bacterial or viral infections may affect the fauna in lower than therapeutic doses, creating an environment with the high potential for resistance.51

Improper use of antibiotics, such as a patient not finishing a prescription or the improper prescribing of antibiotics for viral infections, has also played a part in resistance. Once resistance has been found in any species of bacteria, it is possible for that resistance to be transferred via plasmid or bacteriophage, as is the belief with VRE transfer to VRSA.51, 53

One last cause of resistance may be the ability for bacteria to from biofilms. Biofilms are a type of bacterial growth where aggregates come together and form a layer on surfaces that allows for protection during growth in hostile environments.54 The formation of biofilms give bacteria an inherent resistance to any attack, be it from a host immune system, amoeba or antibiotics. Attempts to combat an infection caused by a biofilm will increase the chance that cells in the biofilm will grow resistance to the antibiotic in use, since a lower concentration of drug will affect each cell.

Quinupristin and dalfopristin are two semisynthetic pristinamycins that are used in combination and are active against MRSA and VRSA strains and FDA approved in 1999

(Figure 1.9).55 The combination works by inhibiting cytochrome P450 and prevents the synthesis of important biochemical molecules. Resistance has been seen via efflux mechanisms and mutation of the enzyme.55-56

Linezolid is an oxazolidinone that is active against Gram-positive bacteria and was approved for use by the FDA in 2000 (Figure 1.9). Linezolid works by preventing protein synthesis via binding the ribosomal 23S rRNA at doman V and preventing binding of the initiator fMet-tRNA.57 Although serious adverse affects can be encountered, linezolid can be substituted for vancomycin which also has serious side effects. In 2001 the first clinical isolates were found bearing linezolid resistance.58 Resistance has been found

22 from a few mechanisms, including point amino acid mutations and methylation of the

23S rRNA by a horizontally transferred methyltransferase encoded by the plasmid encoded crf gene.57a, 59

Daptomycin is a cyclic lipopeptide that was FDA approved in 2003 (Figure 1.9). It kills bacteria by disrupting membrane depolarization by insertion into the cell membrane resulting in the release of potassium ions and the cessation of biochemical functions in the cell.60 By 2005 resistance had been encountered.61

In 2005 the FDA approved the broad spectrum tetracycline drug tigecycline for use against Gram-postive and Gram-negative bacteria (Figure 1.9). This compound is of note as tetracycline resistance has long been shown in Gram-positive and Gram-negative bacteria.62 Tetracycline resistance is conferred by ribosomal protections and efflux mechanisms and it is believed that tigecycline is able to overcome this due to a bulky substituent on the 9-position, although activity is mildly reduced in the presence of multidrug efflux pumps.62-63 The mechanism of action of tigecycline is the binding of the

30S ribosomal subunit, causing the inhibition of protein synthesis.62

Telavancin, a semi-synthetic lipoglycopeptide, was FDA approved in 2009 (Figure 1.9).

Telavancin has the same mechanism of action as other glycopeptides, but still displays activity against vancomycin resistant strains, although the activity is less than that for

MRSA strains.55, 64

Figure 1.9: MRSA anti-bacterial drugs approved by the FDA since 1999

Ceftaroline fosamil is a broad-spectrum cephalosporin that was FDA approved in 2010

(Figure 1.9). Ceftaroline has a mechanism of action similar to other β-lactams wherein it binds to PBPs, including PBP2a which caused resistance among β-lactams following methicillin resistance in the 1960’s.65 It is also active against vancomycin resistant strains of S. aureus and is generally prescribed for infections associated with CA-MRSA, including pneumonia and severe skin infections.

Ceftobioprole is another cephalosporin that is also active against MRSA and VRSA strains but has not gained FDA approval.66 Dalbavancin is also awaiting FDA approval and is a lipoglycopeptide that is active against MRSA but not against VRSA strains.55

Another glycopeptide is the semi-synthetic oritavancin that displays activity against

MRSA and VRSA strains. Clinical trials are being repeated for this compound.55

1.3.2 Acinetobacter baumannii

Acinetobacter is a genus of Gram-negative coccobacilli bacteria that is strictly aerobic, catalase positive and oxidase negative.67 Bacteria of this genus can be found in water, soil, and as normal fauna of animals, including human skin.68

A. baumannii is a cause of infection in the human populace. It has few virulence factors with most infections in immunocompromised persons.67a, 68-69 Most infections encountered include nosocomial pneumonia, meningitis, urinary tract infections, wound infections such as surgical site infections, and bloodstream infections from central line infections.68 People who are at risk for A. baumannii infections are those who are elderly, are immunocompromised, have had invasive procedures, have catheters, use mechanical

25 ventilation and those who have had major trauma, especially burn victims.67a One reason for the high rate of manifestations in the hospital is that A. baumannii is able to survive long periods of time in a dry environment, meaning that contaminated surfaces may leave viable bacteria for several months.67a, 69-70

Along with the lipopolysaccharide of the outer membrane, one of the known virulence factors in A. baumannii is the outer membrane protein 38 (Omp38, also known as

OmpA), a 38 kDa porin.71 Omp38 has been found to localize at the mitochondria of epithelial cells causing mitochondrial release of apoptosis inducing factor (AIF) and cytochrome c and resulting in apoptosis of the eukaryotes.71

1.3.2.1 Antibiotics for Acinetobacter baumannii

Since the early 1970’s, nosocomial infections of A. baumannii have shown antibiotic resistance to compounds that it was previously susceptible such as gentamicin, minocycline, ampicillin, carbenicillin, chloramphenicol and nalidixic acid.72 Sulbactam and group 2 carbapenems have been the drugs of choice for multidrug resistance strains.67a In the mid 1980’s a gene was found that encodes a carbapenem-hydrolyzing class D -lactamase (CHDL or carbapenemase), called OXA-23.73 This enzyme is transferred via plasmid but can be found within the chromosome and is known to hydrolyze penicillins and some cephalosporins as well.73 This enzyme and its variants are not very effective, but upregulation of the gene may occur resulting in resistance.67a, 74 On its own or in collaboration with CHDLs, carbapenem resistance has also been seen by loss of certain outer-membrane proteins that facilitate the influx of these -lactams.74 The study of a French strain of A. baumannii has revealed the presence of resistance genes

26 transferred from numerous other bacteria.75 AbaR1, an 86 kb resistance island, was found to have 88 open reading frames that came from other organisms, 39 from Pseudomonas spp., 30 from Salmonella spp. and 15 from Eschericia coli.75 45 resistance genes were found within the AbaR1 resistance island.75

Carbapenems that are used as common treatments include imipenem and meropenem

(Figure 1.10). Imipenem, approved in 1985, is usually given as combination therapy with cilastitin, an inhibitor of dehydropeptidase I that hydrolyzes imipenem.76 Meropenem was released in 1996 and is not susceptible to this hydrolysis. The mechanism of action for carbapenems is the same as for other -lactams, but in Gram-negative bacteria the antibiotic enters the periplasmic space between the cell wall and outer cell membrane via porins.76

Colistin is a polymyxin that became available for clinical use in 1959 (Figure 1.10). Its use prior to recent years has been minimal due to nephrotoxicity and neurotoxicity side effects.77 Polymyxins are cyclic polyamine peptides with a fatty acid side chain that disrupt the cell membrane by the interaction of the many primary ammonium functionalities with the anionic lipopolysaccharide on the outer membrane of Gram- negative bacteria.78 The displacement of Mg2+ and Ca2+ that form salt bridges between the lipopolysaccharide reduces the stability of the membrane, along with the insertion of the fatty acid side chain into the hydrophibic lipid layer, resulting in the escape of intracellular components and finally cell death.67a, 78 Resistance to polymyxins has been observed and the mechanism is thought to be a change in the outer membrane with the possible reduction of lipopolysaccharides.77

Tigecycline has shown promising results against multidrug resistant A. baumannii, although it has a low blood serum concentration resulting in poor efficacy against bloodstream infections.62, 67a Tigecycline is also a poor candidate for the treatment of urinary tract infections as it is not cleared from the body by the kidneys.67a A few cases have been reported of tigecycline resistance occurring during therapy for these types of A. baumannii infections.79

Figure 1.10: Gram-negative antibiotics active against Acinetobacter baumannii

Chapter 2: 1,4-Dihydropyridines as Anti-Malarials

2.1 Hantzsch Pyridine Synthesis

The Hantzsch pyridine synthesis is an old reaction that still has applicability in today’s chemistry.80 Classical Hantzsch syntheses employ two equivalents of β-ketoesters with one equivalent of amine and aldehyde to form the symmetrical 1,4-dihydropyridine derivatives (Figure 2.1). This reaction, from the late 19th century, is still used as a reliable method for forming an assortment of 1,4-dihydropyridine products. Asymmetric compounds may be made by using two different β-ketoesters or one β-ketoester with a β- diketone compounds.

Figure 2.1: Classical Hantzsch pyridine synthesis

This 1,4-dihydropyridine system has six  electrons delocalized over 5 atoms of the pyridine ring, and this delocalization is extended to the carbonyls at the 3- and 5- positions as well. 81 The oxidation of this system to the aromatic pyridine occurs readily and even contact with atmospheric oxygen will cause this conversion, making the study of the properties and characterization of dihydropyridines a difficult maneuver.81a

Nevertheless, 1,4-dihydropyridines with biological activity have been identified and developed into FDA approved drugs.

2.1.1 Enantioselective Syntheses of 1,4-Dihydropyridines

Recent developments in this reaction involved the formation of chiral 1,4- dihydropyridines. In the early 1980’s the development of these compounds occurred with their introduction as calcium channel blockers for the treatment of hypertension. In 1980

Shibanuma et al. reported the synthesis of (+) and (–)-nicardipine via the resolution of an intermediate carboxylic acid (Figure 2.2).82 Aminal formation allowed for the demethylation of one ester using sodium and 1-dimethylamino-2-propanol. Chiral resolution occurred by recrystallization using the chiral alkaloids cinchonine or cinchonidine to form diastereomeric salts and repeated until constant optical rotation was achieved. Washing with 1 M HCl removed the alkaloid and left the free acid, which could be esterified to nicardipine following the formation of the acid chloride using phosphorous pentachloride.82 This method has also been used in the synthesis of barnidipine.83

Figure 2.2: Synthesis of (+) and (–)-nicardipine via alkaloid resolution

In 1981 a group at Bayer reported the enantioselective synthesis of nimodipine using a chiral auxiliary (Figure 2.3).84 The synthesis involved the Michael addition of (S)-2- methoxy-2-phenylethyl 3-aminocrotonate 2.A with the condensation product of isopropyl acetoacetate and 3-nitrobenzaldehyde 2.B. With this chiral auxiliary, the product formed

2.C was enantiomerically pure as the 4S isomer. Selective transesterification gave the pure (S)-nimodipine.84

Figure 2.3: Stereoselective synthesis of nimodipine

Meyers and Oppenlaender reported in 1986 the use of a chiral oxazoline 2.D to induce the diastereoselective addition of aryl lithium to a pyridine system, yielding a diastereomeric excess (de) of 78-90% (Figure 2.4).85 The same system was used again in

1996, with Cheng et al. reporting that the aryl lithium preferentially added to the methyl ester, which could be circumvented by using a tert-butyl ester 2.E.86 This addition proceeded with a de of 67%.

Figure 2.4: Diastereoselective formation of 4-aryl-1,4-dihydropyridines using a chiral

oxazoline

In 1988, D. Enders et al. reported an enantioselective synthesis of 4-aryl-1,4- dihydropyridines using chiral hydrazines (Figure 2.5).87 An asymmetric Michael addition of metalated chiral alkyl acetoacetate hydrazones 2.F to Knoevenagel acceptors 2.G and subsequent cleavage of the nitrogen-nitrogen bond resulted in products with enantiomeric excess (ee) between 84% and 98%.

Figure 2.5: Stereoselective synthesis of 4-aryl-1,4-dihydropyridines using chiral

hydrazines

Lipases from Pseudomonas were shown in 1991 to selectively cleave one of two ester groups of a symmetrical 1,4-dihydropyridine, providing an enantiomerically pure product

(2.H, Figure 2.6).88

Figure 2.6: Selective ester cleavage of symmetrical 1,4-dihydropyridine using

Pseudomonas lipase

Initial reactions were carried out in buffer at a pH of 5 giving the best ee of 15%.

Utilizing an n-butanol and water mixture in a ten to one ratio without buffer resulted in an optimal ee of 97% in 31% yield. A higher yield was obtained by a shorter reaction time

33 with a loss of enantiomeric excess. The side product of this reaction is the di-acid and the yield for this compound increased over time.

In 1994 Kosugi et al. reported the synthesis of optically pure 1,4-dihydropyridine derivatives via the diastereomeric separation of Hantzsch intermediates containing an

(R)-1-phenylethylamino group (Figure 2.7).89 Similar to what Enders reported in 1988,

Michael addition followed by the removal of the chiral auxiliary and cyclization gave the desired 4-aryl-1,4-dihydropyridine (2.I).

Figure 2.7: Synthesis of chiral 4-aryl-1,4-dihydropyridines via diastereomeric

intermediates

The resolution of 4-aryl-1,4-dihydropyridine derivatives bearing isothioureido groups off the 2-methyl was described in 2006 by Moshtaghi et al. (Figure 2.8).90 Bromination of the methyl group followed by substitution with the thiourea resulted in the racemic product that was resolved using dibenzoyl-tartaric acid.

Figure 2.8: Synthesis of a chiral 4-aryl-1,4-dihydropyridine using tartaric acid resolution

The use of chiral BINOL-phosphoric acid catalysts was reported by Evans and Gestwicki in 2009 resulting in up to >99% ee in 4-phenyl polyhydroquinoline derivatives (2.J,

Figure 2.9).91 The cyclohexa-1,3-dione dimedone (2.K) was used with ethyl acetoacetate, ammonium acetate and various benzaldehydes in the presence of 10 mol% of catalyst in acetonitrile at room temperature to give a selection of substituted 4-aryl-1,4- dihydropyridine derivatives in at least 87% ee.

Figure 2.9: Use of chiral BINOL-phosphoric acid in the enantioselective synthesis of 4-

phenyl polyhydroquinolines

Although a few methods exist for enantioselective synthesis of a 1,4-dihydropyridine system, chiral auxiliaries or chiral chromatography remain a mainstay in the syntheses of

35 these molecules.85-89, 91-92 Polyhydroquinolines that are formed via the Hantzsch dihyropyridine synthesis generally have only one stereocenter. Thus, a technique allowing for the stereoselective synthesis of 1,4-dihydropyridine derivatives with a second stereocenter would be a novel endeavor.

2.1.2 Biological Activity of 1,4-Dihydropyridine Derivatives

1,4-Dihydropyridines have long been known to have biological activity with the first observed activities being analgesic and muscle-relaxing properties.93 In the early 1970’s

1,4-dihydropyridines were found to be calcium channel blockers (Figure 2.10).

Figure 2.10: Calcium channel blockers on the market

These calcium antagonists reduce the influx of Ca2+ in the cells of the contractile system resulting in the direct damping of myocardial work-load metabolism, an increase in blood supply to the coronary vessels and a reduction in arterial flow resistance.84 The reduction of arterial flow resistance has made these compounds effective drugs in the treatment of hypertension and angina pectoris. The enantiomers of these compounds have been found to have opposite effects, with one being an agonist and one being an antagonist.85, 92a, 94 If racemic mixtures are used, one enantiomer should be more active than the other.

Sirisha et al. have shown 1,4-dihydropyridine derivatives that display in vitro anti-cancer, anti-bacterial and anti-tubercular activities (Figure 2.11, 2.L, 2.M and 2.N).95 Anti-cancer activities for the most active compounds (2.L and 2.M) were on par with methotrexate in the single digit g/mL range against cancer cells from breast (MCF-7), colorectal (HT-

29) and skin (A431) cell lines. For compound (2.N), anti-tubercular activity was reported at 12.5 g/mL. Zones of inhibition against both gram positive and negative bacteria were also reported for this compound. Mehta and Verma have also shown 1,4- dihydropyridines with zones of inhibition against both gram positive and negative bacteria.96

1,4-Dihydropyridines have also been found to have anti-inflammatory properties via the inhibition of PDE4 enzymes,97 multidrug resistance reversal properties in anti-cancer drugs via the inhibition of P-glycoprotein98 and have even shown single digit micromolar activity in in vitro anti-leishmanial and anti-trypanosomal assays.99

Figure 2.11: 1,4-Dihydropyridines recently reported with anti-cancer, anti-tubercular and

anti-bacterial properties

2.2 1,4-Dihydropyridine Syntheses

In 2010 a major high-throughput screening (HTS) was undertaken at St. Jude Children’s

Research Hospital.100 This screen started with 309,474 unique compounds tested at 7 M against the 3D7 strain of Plasmodium falciparum. Approximately 1300 compounds displayed >80% activity with 561 having EC50s less than 2 M and a selectivity index of greater than ten when compared against two mammalian cell lines. Forty percent of these compounds were re-purchased and tested and 172 were re-confirmed by three separate laboratories to have anti-malarial activity.

In early 2012, the groups from St. Jude Children’s Research Hospital, Novartis and

GlaxoSmithKline published a review with results from independent anti-malarial HTS screenings.101 In an attempt to involve more research groups in the search for novel anti- malarials, these results were published with the hope that drugs would become available

38 to further progress the elimination of malaria. As part of this testing, 4- arylpolyhydroquinoline derivatives of asymmetric 1,4-dihydropyridines were identified as hits and it was decided that we would begin work on this chemotype.

The synthesis of the 1,4-dihydropyridine derivatives began with 1,4,5,6,7,8- hexahydroquinolines 2.O that were monosubstituted with phenyl derivatives at carbons 4 and 7 (Figure 2.12). This substitution pattern leads to compounds with two stereocenters, with the formation of four stereoisomers. The presence of four stereoisomers required the identification of which stereoisomer is biologically active. The stereoselective synthesis of each stereoisomer was also investigated. Finally, a detailed structure-activity relationship (SAR) was envisioned to identify the most potent derivatives in combination with the most active stereoisomers.

2.2.1 Synthetic Plan

Chiral auxiliaries were used in order to add a preset chirality center to the molecules. It was believed that the formation of four diastereomers would facilitate a straightforward separation, allowing for the testing and crystallization of pure stereoisomers. Alongside this, it was thought that the six position methylene could be replaced with an oxygen, allowing for the formation of 1,4-dihydropyridine derivatives with structures of 4,5,7,8- tetrahydro-1H-pyrano[4,3-b]pyridines 2.P (Figure 2.12). These derivatives could be formed from 6-phenyldihydro-2H-pyran-2,4(3H)-diones 2.Q, lactones that could potentially have chirality built in. The designed usage of the lactone for chiral synthesis led to the idea of utilizing a lactone library for the SAR. While the chiral syntheses were taking place, a racemic library would also be synthesized to determine the most active

39 compounds. The methylene and oxygen derivatives would have to be compared to determine if there was a loss or gain of activity between the two.

Figure 2.12: Retroysynthesis of 1,4-dihydropyridine derivatives

2.2.1.1 Comparison of Methylene and Oxygen Derivatives

The replacement of a methylene with an oxygen could potentially create drastic changes between molecules. These isosteres have electronic differences that can play a significant role in solubility, molecular interactions and lipophilicity, among other properties. In the case of the 1,4-dihydropyridines, the syntheses of the two 6-position derivatives occurs via the same route. Ethyl acetoacetate, benzaldehyde, ammonium acetate and either 5- phenylcyclohexan-1,3-dione 2.R or 6-phenyldihydro-2H-pyran-2,4(3H)-dione 2.Q can be heated at 120ºC in acetonitrile within a sealed tube. Cyclization gives the asymmetric 1,4- dihydropyridine as the major product (Figure 2.13). The diastereomers of 1,4,5,6,7,8- hexahydroquinolines 2.O and 4,5,7,8-tetrahydro-1H-pyrano[4,3-b]pyridines 2.P can be separated via chromatography, with the derivatives of 2.P being more polar and more

40 easily separated via traditional flash chromatography. The 2.O derivatives were not readily separable by flash chromatography, but can be separated using preparative high- pressure liquid chromatography (prep-HPLC). This ease of separability in the oxygen derivatives was a bonus for the use of these derivatives in the SAR. The diastereomeric pairs are labeled A and B, with A being the pair that moves more slowly on normal-phase silica and more quickly on reverse-phase silica. Crystal structure of 2.1 revealed that 2.1-

A is the 4,7-cis isomer and that 2.1-B is the 4,7-trans isomer (Figure 2.27). The crystal structure can also be used to explain why the trans isomer is formed in a slightly larger amount, as an edge-face interaction can be seen between the 4- and 7- aryl substituents.

This interaction would lead to steric hindrance that results in the higher production of the trans stereoisomers.

Figure 2.13: Synthesis of 1,4-dihydropyridine derivatives

Initial 1,4-dihydropyridines utilized a 4-(2-chlorophenyl) and 7-(3,4-dimethoxyphenyl) substituent pattern. Comparison of the anti-malarial properties reveals that the activities are generally diminished when an oxygen replaces the methylene at the 6-position (Table

2.1). Comparison of the A diastereomeric pairs cis-2.1 and 2.2-A show that the methylene version is between 7.9 and 14 times more active than the oxygen version across the four tested strains. The B diastereomers trans-2.1 and 2.2-B are significantly 41 more active than the A diastereomers cis-2.1 and 2.2-A with the methylene derivative again more active, although the W2 parasite was affected similarly by the two derivatives. The methylene version is between 1.2 and 19 times more active than the oxygen version across the four tested strains.

Table 2.1: Comparison of the replacement of -CH2- with -O- at the 6-position

3D7 K1 W2 TM90-C2B J774A.1 Compound X A/B EC50 n EC50 n EC50 n EC50 n EC50  cis 165 141 53.2 128 >20 2.1 CH2 trans 13.5 15.7 40 4.9 >20 mix 17.1 28 99 24.3 >20 A 2330 1390 418 1190 >20 2.2 O B 260 244 48.8 91.2 >20 mix 372 323 66 180 >20

The P. falciparum strains 3D7 and K1were provided by the MR4 Unit of the American

Type Culture Collection (ATCC, Manassas, VA). Those two strains were the chloroquine sensitive strain 3D7 and the chloroquine resistant strain K1. Asynchronous parasites were maintained in culture based on the method of Trager.102 Parasites were grown in presence of fresh group O-positive erythrocytes (Key Biologics, LLC, Memphis, TN) in

Petri dishes at a hematocrite of 4-6% in RPMI based media (RPMI 1640 supplemented with 0.5% AlbuMAX II, 25 mM HEPES, 25 mM NaHCO3 (pH 7.3), 100 µg/mL hypoxanthine, and 5 µg/mL gentamycin). Cultures were incubated at 37°C in a gas mixture of 90% N2, 5% O2, 5% CO2. For EC50 determinations, 20 µL of RPMI 1640 with

5 µg/mL gentamycin were dispensed per well in an assay plate (Corning 384-well mircotiter plate, clear bottom, tissue culture treated, catalog no. 8807BC). An amount of

40 nL of compound, previously serial diluted in a separate 384-well white polypropylene

42 plate (Corning, catalog no. 8748BC), was dispensed to the assay plate by hydrodynamic pin transfer (FP1S50H, V&P Scientific Pin Head) and then an amount of 20 µL of a synchronized culture suspension (1% rings, 4% hematocrite) was added per well, thus making a final hematocrite and parasitemia of 2% and 1%, respectively. Assay plates were incubated for 72 h, and the parasitemia was determined by a method previously described:103 Briefly, an amount of 10 µL of the following solution in PBS (10X Sybr

Green I, 0.5% v/v triton, 0.5 mg/ml saponin) was added per well. Assay plates were shaken for 1 minute, incubated in the dark for 90 minutes, then read with the Envision spectrophotomer at Ex/Em of 485/535 nm. IC50s were calculated with the robust investigation of screening experiments (RISE) with four-parameter logistic equation.

All synthesized 1,4-dihydropyridines were routinely tested against the clinically relevant multidrug resistant malarial strains W2 (chloroquine and pyrimethamine resistant) and

TM90-C2B (chloroquine, mefloquine, pyrimethamine, and atovaquone resistant). The human malaria parasite P. falciparum has been grown in vitro in dilute human erythrocytes in RPMI 1640 media containing 10% heat inactivated plasma.104

Concentration response data have been analyzed by a nonlinear regression logistic dose response model, and the 50% inhibition for each compound against the individual strains has been calculated as the effective concentrations (EC50). Generally, the 1,4- dihydropyridines do not display signs of cytotoxicity at less than 20 μM, rendering cytotoxicity indices (CI = EC50(J774)/EC50(TM90-C2B)) of 1000 or more for the most active compounds. These results indicate that the 1,4-dihydropyridines are selective and nontoxic agents.

2.2.1.2 Synthetic Scheme with Chiral Auxiliaries

Initial synthetic strategy for isolation of pure stereoisomers occurred with compound 2.1

(SJ000025107, initial compound number from St. Jude Children’s Reseach Hospital100) after the separation of the diastereomeric pairs, cis-2.1 and trans-2.1. The enantiomers were thought to be separated by the transesterification of the ethyl ester with a chiral auxiliary, allowing for the separation of the newly formed diastereomers.

Transesterification back to the ethyl ester would allow for the isolation of all four pure stereoisomers of 2.1.

Transesterification was first attempted using (S)-1,2-isopropylideneglycerol in tetrahydrofuran (THF) at high temperature within a sealed tube (Figure 2.14). No reaction occurred over five hours, so 16 equivalents of acetic acid were added in the hopes of having an acid catalyzed reaction occur. Heating for an additional 15 hours resulted in no reaction, with compound 2.1 remaining intact.

Figure 2.14: Transesterification attempt with (S)-1,2-isopropylideneglycerol

Next, hydrolysis to the acid was attempted using 2 equivalents of lithium hydroxide in

THF and water at 65ºC for 24 hours and separately with 10 equivalents of aqueous hydrochloric acid in THF and water at 65ºC for 36 hours (Figure 2.15).105 Both hydrolysis

44 attempts resulted in no reaction occurring, showing that the reactivity of the 3- carboxylate carbon is reduced in this system.

Figure 2.15: Hydrolysis with acid or base

Hydrolysis of the ester was thought to occur more readily by the use of an ester group that was more easily removed. tert-Butyl acetoacetate was used in place of ethyl acetoacetate in a trial run to form the tert-butyl ester 2.3 (Figure 2.16). The stable tert- butyl carbocation was believed to be formed via the reaction with acid, leaving the free carboxylic acid. Reaction with 15 equivalents of hydrochloric acid in water at 100ºC for 3 hours resulted in no reaction.

Figure 2.16: Hydrolysis of the tert-butyl ester 2.3

As formation of the carboxylic acid did not readily occur, treating the carboxylate as a leaving group was the next option. (9H-fluoren-9-yl)methyl acetoacetate 2.4 was synthesized and used in the formation of the fluorenylmethyl ester 2.5. Treatment of this compound with 1.5 equivalents of 1,8-diazabicyclo[5.4.0]-undec-7-ene (DBU) at room temperature resulted in the β-elimination of 9-methylene-9H-fluorene and the release of the acid 2.6. At long last the free carboxylic acid was formed. 45

Figure 2.17: Synthesis of the 3-carboxylic acid via β-elimination

In order to derivatize the acid to form the chiral esters and amides, a carbodiimide coupling was attempted using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) and 4-dimethylaminopyridine (DMAP) in dichloromethane (DCM) or dimethylformamide (DMF) to couple the acid with the primary alcohol (S)-1,2- isopropylideneglycerol, the secondary amide (S)-4-benzyl-2-oxazolidinone or the secondary alcohol (S)-mandelic acid (Figure 2.18).

Figure 2.18: Acid derivatization attempts

No reactions were observed so a Mitsunobu reaction was attempted with the same acid

2.6 and primary alcohol with diisopropyl azodicarboxylate (DIAD) and triphenylphosphine in THF, again with no reaction. Chlorination of the acid to the acyl

46 chloride with thionyl chloride was ineffective, as reactions in DCM or DMF followed by quenching with even the small primary alcohol methanol resulted in no formation of the ester.82-83, 89, 106 As a final derivatization effort, we attempted to synthesize the anhydride using an acid chloride. (S)-Mosher’s acid chloride was added to the acid in THF following the addition of 1.2 equivalents of sodium hydride. A reaction did occur with these conditions, although oxidation of the 1,4-dihydropyridine was believed to occur with decomposition of the side chain carboxylate.

Synthesis then proceeded with the formation of chiral acetoacetate derivatives with the idea that if a chiral auxiliary could not be added to the 1,4-dihydropyridine acid 2.6, it may be possible to add it prior to the cyclization. Two chiral auxiliaries were employed: a threonine derivative 2.7 and an oxazolidinone derivative 2.8 (Figure 2.19).

Figure 2.19: Synthesis of chiral auxiliary acetoacetates

These chiral auxiliary containing acetoactetates and their enantiomers were employed in the cyclization to form diastereomeric sets of polyhydroquinolines and were utilized due to literature reports describing transesterification possibilities and were incorporated into

47 the acetoacetate portion of the molecule.107 The L-threonine derivative (2S,3R)-2.7 was cyclized via the Hantszch reaction and formed three peaks (2R,3S)-2.9 by HPLC (Figure

2.20).107c These three peaks, 2.9-P1, 2.9-P2 and 2.9-P3, were separated using prep-HPLC

(RP18) with an isocratic system utilizing 55:45 acetonitrile to water and 0.05% trifluoroacetic acid (TFA). A note to the reader, the stereochemistry changes between 2.7 and 2.9 due to a change in IUPAC numbering system of the threonine side chain.

Figure 2.20: Analytical RP18 HPLC trace of threonine diastereomers

After separation of the three peaks using preparative HPLC, analytical chiral HPLC using a Lux column confirmed that each peak was a single isomer. NMR of each threonine

48 derivative, however, gave a surprising twist to what was believed to be three different compounds (Figure 2.21).

Figure 2.21: Carbon NMR of threonine derivative 2.9-P1 (top) and 2.9-P2 (bottom)

between 103 and 171 ppm

The NMR of 2.9-P1 and 2.9-P3 were similar to each other, but comparison with 2.9-P2 revealed that there were an abnormally large number of peaks present. This led to the 49 conclusion that 2.9-P2 was a mixture of two diastereomers that could not be separated by

RP18 prep-HPLC. 2.9 was synthesized from both D- and L- threonine, resulting in either

(2S,3R)-2.9 or (2R,3S)-2.9.

An immediate concern was that upon transesterification the ethyl ester versions of 2.9-P1 and 2.9-P3 would reveal that they were enantiomers and that this method would not allow for the separation of all four stereoisomers. Luckily, transformation of 2.9-P2 to the ethyl ester 2.1 resulted in the formation of a pair of diastereomers that could be separated by RP18 HPLC, showing that all four stereoisomers were indeed formed and could be separated (Figure 2.22).

Figure 2.22: Synthesis of pure 1,4-dihydropyridine stereoisomers via (2S,3R)-2.9-P2

Formation of the ethyl ester 2.1 from the threonine ester 2.9 occurred via two steps. First, a β-elimination with DBU removed the threonine portion, leaving the free carboxylic acid

2.6. Next, the acid was reacted with ethyl iodide and potassium carbonate to give the ethyl ester 2.1.

X-ray crystallography is an excellent technique for determination of crystal packing structure of molecules and also for the identification of stereochemistry of unknown stereocenters. In order for this technique to be used, crystals of an adequate size and quality need to be grown. As identification of the unknown stereochemistry of the 2.1 derivatives was a high priority, crystallization with ethanol, methanol, isopropanol,

DCM, DMF, acetonitrile, toluene and acetone was attempted via evaporation of solvent.

Unfortunately, using these conditions no crystals of 2.1, 2.9-P1 or 2.9-P3 could be formed.

A second chiral acetoacetate was used for the preparation of diastereomeric 1,4- dihydropyridines. The oxazolidinone 2.8 also cyclized via the Hantszch reaction and formed three peaks by HPLC (Figure 2.23).

Figure 2.23: Synthesis of pure 2.1 stereoisomers via the chiral oxazolidinone side chain

These three 2.10 peaks were separated using RP18 prep-HPLC under isocratic conditions with 1:1 acetonitrile to water and 0.05% trifluoroacetic acid (TFA) (Figure 2.24). These three 2.10 peaks correlated to three of the possible stereoisomers, unlike 2.9 where one of the peaks consisted of two diastereomers. NMR of each 2.10 peak revealed each peak as one isomer.

Figure 2.24: Analytical RP18 HPLC trace of oxazolidinone diastereomers

Formation of the ethyl ester 2.1 occurred at 150ºC in a sealed tube with ethanol and titanium (IV) ethoxide with a poor yield of 28% (Figure 2.20).107a, b This esterification 52 was attempted without the addition of the lewis acid with no reaction occurring, indicating that the strongly acidic nature of the titanium (IV) is necessary for any reaction to proceed. The poor reaction with the strong lewis acid again underlines the poor reactivity of the 3-carboxylate of this 1,4-dihydropyridine system.

The use of both oxazolidinone enantiomers, (R)-2.8 and (S)-2.8, ensured the formation of all four 2.1 stereoisomers. Each 2.8 enantiomer results in the cis compounds (4S,7S)-2.1 and (4R,7R)-2.1 and one trans-2.1. The oxazolidinone enantiomers (R)-2.8 and (S)-2.8 each react to form three respective diastereomers, (R)-2.10 and (S)-2.10 (Figure 2.25).

Figure 2.25: Formation of six 2.10 stereoisomers as three enantiomeric pairs

Analytical RP18 HPLC analysis of the ethyl esters 2.1 proved that 2.10-P1 and 2.10-P2 are 2.1-A enantiomers (Figure 2.26). 2.10-P3 corresponds to one 2.1-B enantiomer and is formed in the lowest amount from 2.8, as seen by HPLC (Figure 2.24). Crystallization with ethanol, methanol, isopropanol, DMF, acetonitrile, toluene and acetone was attempted with 2.10 via evaporation of solvent. Although the alcohols, acetonitrile and

DMF all gave crystals, none were of a high enough quality for X-ray identification, thus

53 vapor diffusion technique was implemented using dimethyl sulfoxide (DMSO) as the solution solvent and an ethanol/water mix as the diffusion solvents.

Figure 2.26: Identification of 2.1 ethyl esters of 2.10-P1 and 2.10-P2 as enantiomers

Using this technique a crystal of both (R)-2.10-P1 and (S)-2.10-P1 were formed and X- ray crystallography allowed the assignation of absolute stereochemistry for the 2.1-A enantiomers. The 2.1-A 1,4-dihydropyridines are cis dihydropyridines and (R)-2.10-P1 had (4S,7S) stereochemistry while (S)-2.10-P1 had (4R,7R) stereochemistry. The crystal structure of 2.10-P1 had two independent conformations in the crystal lattice (Figure

2.27). The 4-aryl substituent resides in a pseudoaxial conformation with the 7-aryl being either axial or equatorial.

Analytical RP18 HPLC using a chiral Lux column allowed for the comparison of 2.1-A enantiomers formed from 2.10 with the 2.1-A enantiomers from 2.9. This method proved that 2.1-A from (2S,3R)-2.9-P2 has (4S,7S) stereochemistry and 2.1-A from (2R,3S)-2.9-

P2 has (4R,7R) stereochemistry, the same as (R)-2.10-P1 and (S)-2.10-P1, respectively

(Figure 2.28).

Figure 2.27: Crystal structure of (R)-2.10-P1 with equatorial 7-substituent (top) and axial

7-substituent (bottom)

Utilizing these two chiral auxiliaries, we were able to determine the absolute configuration of the 2.1-A enantiomers, determine the relative stereochemistry of the 2.1-

B enantiomers as trans conformers and isolate all four stereoisomers of 2.1 in pure form.

The use of the 2.7 threonine derivative provided a higher yielding method than the use of the 2.8 oxazolidinone derivative (Table 2.2).

Figure 2.28: Synthesis of (4S,7S)-2.1-A from (2S,3R)-2.9-P2 and (R)-2.10-P1

While the synthesis requires three more steps, a 27.4% overall yield was obtained compared to a 10.5% yield with the oxazolidinone. Separation using the preparative

HPLC also occurred more easily with the threonine derivative, all four stereoisomers are formed with the use of one auxiliary enantiomer and more than 50% of the product formed is the more active 4,7-trans conformer. Crystal structures were found using 2.8, but in all other ways 2.7 was the better auxiliary.

Table 2.2: Comparison of oxazolidinone and threonine auxiliaries Oxazolidinone Threonine Cost of auxiliary (Sigma) (R) - $117/5 g – (S) - $95/5 g L - $22/10 g – D - $33/5 g Number of Steps 3 6 Total Yield 10.5% 27.4% Stereoisomers formed 3 4 Prep-HPLC Separation Peak overlap No overlap Amount of B <25% of total >50% of total

2.2.1.3 Synthetic Schemes for Chiral β-Ketolactones

The reasoning behind the use of the lactone was the setting of one stereocenter prior to formation of the 1,4-dihydropyridine core. If the stereochemistry of the 7-position could be set, isolation of pure stereoisomers would depend solely on separation of two

56 diastereomers. As shown in Table 2.1, the replacement of a methylene with an oxygen at the 6-position reduced the activity significantly. It was believed that the easier formation of pure stereoisomers would make up for the reduced activity and the performed SAR would at the least lead to more active compounds that could be reevaluated with the methylene unit.

Synthesis of the racemic lactone 2.11 began with the formation of the dianion of ethyl acetoacetate prepared from the consecutive additions of sodium hydride and n-butyl lithium to the acetoacetate in THF between -20 and 0ºC (Figure 2.27). This was followed by the addition of a benzaldehyde derivative, yielding the secondary benzyl alcohol 2.12.

This alcohol was cyclized with KOH or p-toluenesulfonic acid (pTSA) to yield the β- ketolactone 2.11.108

Figure 2.29: Example of the formation of a β-ketolactone from a benzaldehyde

With a usable synthesis for the racemic lactone 2.11, our efforts turned toward finding a highly stereoselective synthesis of a chiral lactone. The idea was that if the benzyl alcohol 2.12 could be formed with high enantioselectivity then the cyclization would yield the chiral lactone.

Initial efforts began using the Mukaiyama aldol route using Chan’s diene 2.13, the di- trimethylsilyl (TMS) protected methyl acetoacetate. Addition of Chan’s diene to the

57 aldehyde in the presence of (R)-BINOL with lithium chloride and titanium (IV) isopropoxide yielded the TMS protected benzyl alcohol (Figure 2.28).109 Deprotection of the TMS group with pyridinium para-toluenesulfonate (PPTS) resulted in a low conversion. Optical rotation of the isolated alcohol 2.14 showed a low enantiomeric excess (ee) when compared with the reported literature value.

Figure 2.30: Mukaiyama aldol route using Chan’s diene

Keck allylation was attempted using allyltributylstannane, (R)-BINOL and titanium (IV) isopropoxide with 3,4-dimethoxybenzaldehyde in dichloromethane (DCM) with molecular sieves at -20ºC (Figure 2.29).110 After 4 days, less than 20% yield was obtained with 98% ee. Mitsunobu reaction was attempted to determine if one Keck allylation could yield both enantiomers, but the reduction of ee to 20% rendered that idea obsolete. A more electron deficient benzaldehye was used with the hopes of enhancing the nucleophilic attack on the aldehyde. Keck allylation of 4-bromobenzaldehyde to form

2.15 followed by oxidative cleavage to the β-hydroxyaldehyde 2.16, C-H insertion using ethyl diazoacetate and tin (II) chloride to form 2.17, cyclization to the β-ketolactone 2.18 and asymmetric Hantzsch reaction resulted in a 40% ee of 2.19 that was unacceptable. At this point it was believed that the 2,6-lutidine was causing a drop in ee.

Figure 2.31: Hantzsch reaction using a chiral lactone derivative 2.16

The next route began with an allylation using Brown’s reagent.111 This reaction proceeded at -90ºC in diethyl ether with the addition of (+)-B- allyldiisopinocampheylborane to the 3,4-dimethoxybenzaldehyde (Figure 2.30). After thirty minutes a 75% yield and 99% ee was obtained and the compound proceeded through the same steps as the Keck allyation product, minus the addition of lutidine. With this reaction sequence, and especially in the oxidative cleavage step, many inseparable impurities were formed. This led to cyclization with pTSA giving the styrene elimination product 2.20 as the major product. When less pTSA was used the reaction was stagnant and low yielding. At this point it was believed that protecting the benzyl alcohol 2.21 would help to increase the yield and allow for easier purification. With a tributylsilyl

(TBS) protecting group, the yield from the benzyl alcohol 2.21 to the β-ketoester 2.22 dramatically increased from 20% to 52%, even with the extra protection step involved.

The deprotection step was troublesome where the use of HCl, PPTS, HF or tetrabutylammonium fluoride (TBAF) resulted in an unclean reaction, again yielding a large amount of the elimination product 2.20 (Figure 2.30).

Figure 2.32: Brown’s allylation steps using a TBS protected benzyl alcohol

Due to the large amounts of styrene elimination product 2.20 being formed, we decided to add the β-ketone last in order to eliminate a source of conjugation with the alkene.

Cross metathesis using Grubb’s second generation catalyst of the benzyl alcohol 2.21 obtained from Brown’s allylation with ethyl acrylate provided a Michael acceptor 2.25 that could have an oxygen placed in the correct location for oxidation to the ketone

(Figure 2.31).112 Reaction with potassium tert-butoxide and benzaldehyde gave a six- membered acetal 2.26 that was added to the Michael acceptor in the fashion hoped for.112

Using pTSA monohydrate in methanol, it was expected that the acetal would be hydrolyzed to the diol and that the benzyl alcohol would lactonize to give the desired lactone that could be oxidized to the β-ketolactone 2.11. In reality, the conditions gave a methanol addition product 2.27 that eliminated the compound for further use.

Dichloromethane in the place of methanol resulted in a low conversion rate with the diol as a minor product and no cyclization. The use of acetic acid and water in a four to one ratio resulted in no reaction.

Figure 2.33: Cross metathesis and Michael addition of the Brown allylation product

One more attempt using Brown’s allylation product involved a ring closing metathesis

(RCM). The benzyl alcohol product 2.21 was reacted with acryloyl chloride in DCM with either triethylamine (TEA) or pyridine, giving a compound 2.28 that was primed for the

RCM (Figure 2.32). Using Grubb’s first generation catalyst, the six membered alkene

2.29 was formed, albeit in 82% ee when TEA was used in the previous step and 88% ee when pyridine was used.113

Figure 2.34: Ring-closing metathesis route

After multiple attempts to form the chiral lactone derivative, it was decided that the benzyl position was not stable enough to maintain a stereocenter with high ee. As the

SAR was already well underway at this point, the racemic lactone syntheses would continue in the attempts to find more active substituents for the 1,4-dihydropyridine.

Isolation of a pure stereoisomer could then proceed via a chiral auxiliary route.

2.2.2 Structure-Activity Relationship of 1,4-Dihydropyridines

2.2.2.1 Structure-Activity Relationship at 4- and 7-Positions

Synthetic manipulations of the 1,4-dihydropyridine began with ortho-, meta- and para- substitutions of the 4-phenyl ring with chloro-, methyl- or methoxy- substituents to determine the effect of electronics and sterics on activity. As can be seen by compounds

2.2B and 2.32-B, chloro and methyl substitutions at the ortho position result in submicromolar activity, with the methyl derivative displaying better activity against the

3D7 and K1 strains and the chloro derivative displaying better activity against the W2 and TM90-C2B strains (Table 2.3). Submicromolar activity was not seen with any meta substitutions. Substitution at the para position resulted in submicromolar activities with chloro and methyl substituted compounds 2.31 and 2.34-B, with the para chloro derivative having better activity across the board.

Table 2.3: 4-phenyl modificationsa

3D7 K1 W2 TM90-C2B J774A.1 Compound X R A/B EC50 n EC50 n EC50 n EC50 n EC50  cis 165 141 53.2 128 >20 2.1 CH2 2-Cl trans 13.5 15.7 40 4.9 >20 mix 17.1 28 99 24.3 >20 A 2330 1390 418 1190 >20 2.2 O 2-Cl B 260 244 48.8 91.2 >20 mix 372 323 66 180 >20 A >10000 >10000 3930 >5000 >20 2.30 O 3-Cl B >10000 >10000 1640 >5000 >20 mix >10000 >10000 2700 4330 >20 A 769 633 470 822 >20 2.31 O 4-Cl B 47.6 53.8 49.0 65.6 >20 mix 91.8 160 87.0 102 >20 A 1270 2050 1720 4030 >20 2.32 O 2-CH3 B 38.5 59.7 56.9 189 >20 mix 72.7 123 144 300 >20 A >10000 >10000 3810 4070 >20 2.33 O 3-CH3 B >10000 >10000 2640 >5000 >20 mix >10000 >10000 4210 >5000 >20 A >10000 >10000 2500 4220 >20 2.34 O 4-CH3 B 638 605 230 327 >20 mix 1250 1610 534 995 >20 A >5600 >5600 >5000 >5000 n/d 2.35 O 2-OCH3 B 2050 >5500 732 >5000 n/d mix >5500 >5500 1290 >5000 n/d A >10000 >10000 2980 2710 >20 2.36 O 3-OCH3 B >10000 >10000 2920 2930 >20 mix >10000 >10000 >5000 >5000 >20 A >10000 >10000 3150 >5000 >20 2.37 O 4-OCH3 B >10000 >10000 1780 2710 >20 mix >10000 >10000 2400 3120 >20

a Chloroquine (EC50K1 = 1360 ± 960 nM, EC503D7 = 48 ± 25 nM, EC50W2 = 96.7 ± 56.3 ng/mL, EC50TM90-C2B = 55.5 ± 27.8 ng/mL), mefloquine (EC50K1 = 31 ± 18 nM, EC503D7 = 62 ± 31), atovaquone (EC50W2 = 0.574 ± 0.306 ng/mL, EC50TM90-C2B = 7.54 ± 4.65 µg/mL) and dihydroartemisin (EC50W2 = 1.41 ± 0.29 ng/mL, EC50TM90-C2B = 1.76 ± 0.12 ng/mL) are the internal controls for the in vitro anti-malarial activity assays. Podophyllotoxin is the internal control for the in vitro cytotoxicity assay with EC50 = 250 ± 10 nM against J774A.1.

Extended aromatic systems at the 4-position, including 2- and 3-naphthyl and 4-(4- biphenyl) (Table 2.4, 2.40-2.42), decrease the activity of the 1,4-dihydropyridine system, with no submicromolar activities. The 4-bromophenyl 2.38 fared better but was less active than the 4-chlorophenyl 2.31. The 4-nitrophenyl 2.39-B has submicromolar activity against W2 and TM90-C2B only. 63

Table 2.4: Aromatic changes at 4-positiona

3D7 K1 W2 TM90-C2B J774A.1 Compound R A/B EC50 n EC50 n EC50 n EC50 n EC50  A 1450 3370 2350 >5000 >20 2.38 4-bromophenyl B 59 341 170 285 >20 mix 213 634 375 725 >20 A >10000 >10000 2610 3000 >20 2.39 4-nitrophenyl B 2710 2200 317 386 >20 mix >10000 >10000 >5000 >5000 >20 A >10000 >10000 2850 2130 >20 2.40 1-naphthyl B >10000 >10000 3180 3270 >20 mix >10000 >10000 >5000 >5000 >20 A >10000 >10000 4110 >5000 >20 2.41 2-naphthyl B >10000 >10000 3040 >5000 >20 mix >10000 >10000 1620 >5000 >20 A >10000 >10000 >5000 >5000 >20 2.42 4-(4-biphenyl) B >10000 >10000 1640 1990 >20 mix >10000 >10000 2220 >5000 >20 a Chloroquine (EC50K1 = 1360 ± 960 nM, EC503D7 = 48 ± 25 nM, EC50W2 = 96.7 ± 56.3 ng/mL, EC50TM90-C2B = 55.5 ± 27.8 ng/mL), mefloquine (EC50K1 = 31 ± 18 nM, EC503D7 = 62 ± 31), atovaquone (EC50W2 = 0.574 ± 0.306 ng/mL, EC50TM90-C2B = 7.54 ± 4.65 µg/mL) and dihydroartemisin (EC50W2 = 1.41 ± 0.29 ng/mL, EC50TM90-C2B = 1.76 ± 0.12 ng/mL) are the internal controls for the in vitro anti-malarial activity assays. Podophyllotoxin is the internal control for the in vitro cytotoxicity assay with EC50 = 250 ± 10 nM against J774A.1.

Next, the 7-phenyl ring was looked at synthetically with the same ortho-, meta- and para- substitutions with chloro-, methyl- or methoxy- substituents. Substitution at the para position resulted in one compound with submicromolar activity in the 3D7 and K1 assays, the chloro derivative 2.45-A (Table 2.5). The 2.45-B and 4-methoxy derivative

2.51-B gave single digit micromolar activity in these assays, with the 4-methyl derivative

2.48-B giving single digit micromolar activity against K1 only. Submicromolar activity was seen in the W2 assay with the 4-methyl 2.48-B and the 4-methoxy 2.51-B. Single digit micromolar activity in the TM90-C2B assay was found with these compounds. Meta substitution again gave one compound with submicromolar activity in the 3D7 and K1 assays with single digit micromolar activity in the W2 and TM90-C2B assays, the

64 methoxy derivative 2.50-B. The 2.50-A derivative had single digit micromolar activity against the all strains besides K1. W2 was the only strain to show activity when ortho substitution was present, with the o-methyl 2.46-B having submicromolar activity.

Table 2.5: 7-Phenyl moficationsa

3D7 K1 W2 TM90-C2B J774A.1 Compound R A/B EC50 n EC50 n EC50 n EC50 n EC50  A >5500 >5500 2250 >5000 n/d 2.43 2-Cl B >5500 >5500 >5000 >5000 n/d mix >6000 >6000 3070 >5000 n/d A >6000 >6000 >5000 >5000 n/d 2.44 3-Cl B >6000 >6000 2050 4030 n/d mix >6000 >6000 3260 4130 n/d A 510 560 2060 2030 n/d 2.45 4-Cl B 2860 2160 1150 4060 n/d mix >5500 >5500 1720 2260 n/d A >5000 >5000 4610 4370 n/d 2.46 2-CH3 B >5000 >5000 711 4080 n/d mix >5000 >5000 4430 4130 n/d A >5000 >5000 >5000 >5000 n/d 2.47 3-CH3 B >6000 >6000 2110 4350 n/d mix >5000 >5000 3250 >5000 n/d A >5000 >5000 >5000 >5000 n/d 2.48 4-CH3 B >6000 1583 923 3310 n/d mix >5000 >5000 1870 >5000 n/d A >6000 >6000 >5000 >5000 n/d 2.49 2-OCH3 B >5500 >5500 4050 3840 n/d mix >6000 >6000 4050 4240 n/d A 2133 >7000 2260 2280 n/d 2.50 3-OCH3 B 626 363 1490 2260 n/d mix 2417 >6000 2100 2650 n/d A >7000 >7000 4480 4240 n/d 2.51 4-OCH3 B 2155 1960 871 2730 n/d mix >7000 >7000 1670 3830 n/d a Chloroquine (EC50K1 = 1360 ± 960 nM, EC503D7 = 48 ± 25 nM, EC50W2 = 96.7 ± 56.3 ng/mL, EC50TM90-C2B = 55.5 ± 27.8 ng/mL), mefloquine (EC50K1 = 31 ± 18 nM, EC503D7 = 62 ± 31), atovaquone (EC50W2 = 0.574 ± 0.306 ng/mL, EC50TM90-C2B = 7.54 ± 4.65 µg/mL) and dihydroartemisin (EC50W2 = 1.41 ± 0.29 ng/mL, EC50TM90-C2B = 1.76 ± 0.12 ng/mL) are the internal controls for the in vitro anti- malarial activity assays. Podophyllotoxin is the internal control for the in vitro cytotoxicity assay with EC50 = 250 ± 10 nM against J774A.1.

As can be seen by Tables 2.3 and 2.4, changes to the 4-aryl position can increase the activity of the new compound or severely limit it. Phenyl substitution of 4-halo, 4-methyl,

2-chloro or 2-methyl results in compounds with submicromolar activities, with the best activities found with a 4-chloro or a 2-methyl, compounds 2.31-B and 2.32-B. Extended aromatic systems such as naphthyl or biphenyl completely remove the activity from 3D7 or K1 and may display low micromolar activity in W2 and TM90-C2B.

Substitution of the 7-phenyl system also plays a distinct role on activity (Table 2.5). Most of the mono-substituted compounds are inactive against 3D7 and K1, with the exceptions of the 4-chloro 2.45-A and the 3-methoxy 2.50-B having submicromolar activities. The same can be said for W2 and TM90-C2B, as no compounds displayed submicromolar activity against TM90-C2B while the 2-methyl 2.46-B, 4-methyl 2.48-B and the 4- methoxy 2.51-B had submicromolar activity against W2. Comparision of Table 2.4 with compound 2.2 highlights the importance of a disubstituted phenyl substituent at the 7- position.

2.2.2.2 Structure-Activity Relationship of the 2- and 3-Positions

To test the importance of the ester functional group, a number of derivatives were made by varying the ester substituent at the 3-position. The benzyl ester 2.57 did not have good activity (Table 2.6). A tert-butyl 2.56-B or n-propyl 2.55-B ester had similar activities against K1, W2 and TM90-C2B, while the tert-butyl had no activity against the 3D7 strain. The ethyl ester (Table 2.2, 2.2-B) was more active than either ester. Changing the group at C2 revealed that extending the chain increases the activity, with n-propyl 2.52-B enhancing the activity against 3D7 and K1 while keeping in the range of the methyl against W2 and TM90-C2B. Also, a 2-ethoxy-2-oxoethyl 2.53-B increased activity against 3D7 and K1 with a mild decrease against W2 and TM90-C2B. The 2.53-A

66 diastereomers have a much better activity against all four strains when compared to the

2.2-A diastereomers and has activity better than or the same as 2.53-B. A 2-methoxyethyl group with a methyl ester 2.54-B had better activity against 3D7 and K1 and lower activity against W2 and TM90-C2B compared to 2.2-B. Attempts to place a phenyl group at the 2-position resulted in no 1,4-dihydropyridine formation, thus the activity for an aromatic group could not be seen.

Table 2.6: 2- and 3-position modificationsa

3D7 K1 W2 TM90-C2B J774A.1 Compound R1 R2 A/B EC50 n EC50 n EC50 n EC50 n EC50  A 811 418 >5000 >5000 >20 2.52 Et nPr B 16.1 9.2 48.7 114 >20 mix 29 15.5 261 718 >20 A 38.1 13.6 133 312 >20 2-ethoxy-2- 2.53 Et B 119 77.6 124 346 >20 oxoethyl mix 58.7 22.2 346 671 >20 A 2440 >10000 1690 4190 >20 2-methoxy 2.54 Me B 86.6 124 180 746 >20 ethyl mix 237 315 432 962 >20 A 2360 >7000 2400 >5000 >20 2.55 nPr Me B 458 332 205 1370 >20 mix 1670 1630 62.1 1210 >20 A >13000 >13000 1230 2810 >20 2.56 tBu Me B >13000 272 302 950 >20 mix >13000 2650 510 2520 >20 A >11800 >11800 >5000 >5000 >20 2.57 Bn Me B >9100 >9100 3230 >5000 >20 mix >7500 >7500 1070 >5000 >20 a Chloroquine (EC50K1 = 1360 ± 960 nM, EC503D7 = 48 ± 25 nM, EC50W2 = 96.7 ± 56.3 ng/mL, EC50TM90-C2B = 55.5 ± 27.8 ng/mL), mefloquine (EC50K1 = 31 ± 18 nM, EC503D7 = 62 ± 31), atovaquone (EC50W2 = 0.574 ± 0.306 ng/mL, EC50TM90-C2B = 7.54 ± 4.65 µg/mL) and dihydroartemisin (EC50W2 = 1.41 ± 0.29 ng/mL, EC50TM90-C2B = 1.76 ± 0.12 ng/mL) are the internal controls for the in vitro anti-malarial activity assays. Podophyllotoxin is the internal control for the in vitro cytotoxicity assay with EC50 = 250 ± 10 nM against J774A.1.

Changing of the alkyl substituent on the 3-position ester did not form a compound with better activity than the original ethyl ester, 2.2. The benzyl ester had no activity while a tert-butyl ester or n-propyl ester each showed submicromolar activities in three of the

67 four assays (compounds 2.55-2.57). The ethyl ester is clearly a better compound than these other three.

Substitution at the 2-position did, however, create some interesting compounds.

Elongation of the methyl 2.2 to a propyl 2.52 formed a compound with better activity in half of the assays. Replacement with some oxygenated ethyl derivatives resulted in some

B compounds 2.53-B and 2.54-B with better activity in half of the assays and worse in the other half, while the 2.53-A oxygenated ethyl derivative fared much better than the methyl 2.2A.

2.2.2.3 A Few Polyhydroquinoline Derivatives

The synthesis of a 2-phenylpolyhydroquinoline derivative 2.58 allowed for biological screening with a 2-aryl substituent. The activity of this molecule was less than that for the methyl derivative, 2.1 (Table 2.7). The 4-(4-chlorophenyl) derivative 2.59-B was slightly more active than the 2-chloro version 2.1-B in all assays except for the TM90-C2B, where 2.1-B is 12 times more active. Replacing the 2-position methyl of 2.59-B with a 2- ethoxyethyl 2.60-B creates a compound with better across the board activity.

Table 2.7: Polyhydroquinoline derivatives with C2 and C4 changesa

3D7 K1 W2 TM90-C2B J774A.1 Compound R1 R2 R3 A/B EC50 n EC50 n EC50 n EC50 n EC50  A 3720 >10900 2260 3990 >20 2.58 Et phenyl 2-Cl B 40.5 46.7 119 256 >20 mix 176 92.3 274 570 >20 A 264 221 284 511 >20 2.59 Et methyl 4-Cl B 10.3 9.3 27.3 60.8 >20 mix 16.3 18.9 70.4 103 >20 2- A 145 130 191 434 >20 2.60 Me ethoxy 4-Cl B 2.5 15 3.9 7.1 >20 ethyl mix 6.3 11 29.4 20 >20 a Chloroquine (EC50K1 = 1360 ± 960 nM, EC503D7 = 48 ± 25 nM, EC50W2 = 96.7 ± 56.3 ng/mL, EC50TM90-C2B = 55.5 ± 27.8 ng/mL), mefloquine (EC50K1 = 31 ± 18 nM, EC503D7 = 62 ± 31), atovaquone (EC50W2 = 0.574 ± 0.306 ng/mL, EC50TM90-C2B = 7.54 ± 4.65 µg/mL) and dihydroartemisin (EC50W2 = 1.41 ± 0.29 ng/mL, EC50TM90-C2B = 1.76 ± 0.12 ng/mL) are the internal controls for the in vitro anti-malarial activity assays. Podophyllotoxin is the internal control for the in vitro cytotoxicity assay with EC50 = 250 ± 10 nM against J774A.1. 2.2.2.4 Role of Stereochemistry in Activity

Pure stereoisomers of 2.1 were formed via the chiral auxiliaries. Table 2.1 already showed that the trans isomer was more active. As seen in Table 2.8, stereochemistry plays a large role in the activity of 1,4-dihydropyridines in P. falciparum. (-)-Trans-2.1 is completely inactive to all four strains while its enantiomer (+)-trans-2.1 has activity in the picomolar range. The same trend is seen with the cis isomers. The (4R,7R)-2.1 enantiomer is inactive while the (4S,7S)-2.1 enantiomer has double digit nanomolar activity against 3D7 and K1 and triple digit nanomolar activity against W2 and TM90-

C2B.

Table 2.8: Comparison of activities of 2.1 stereoisomersa

4,7- 3D7 K1 W2 TM90-C2B J774A.1 Compound Stereochemistry EC50 n EC50 n EC50 n EC50 n EC50  2.1 4S,7S >6000 >6000 2130 2110 n/d 2.1 4R,7R 17 10.5 200 514 n/d 2.1 (+)-trans <0.3 <0.3 18.1 49.1 n/d 2.1 (-)-trans >6000 >6000 >5000 >5000 n/d a Chloroquine (EC50K1 = 1360 ± 960 nM, EC503D7 = 48 ± 25 nM, EC50W2 = 96.7 ± 56.3 ng/mL, EC50TM90-C2B = 55.5 ± 27.8 ng/mL), mefloquine (EC50K1 = 31 ± 18 nM, EC503D7 = 62 ± 31), atovaquone (EC50W2 = 0.574 ± 0.306 ng/mL, EC50TM90-C2B = 7.54 ± 4.65 µg/mL) and dihydroartemisin (EC50W2 = 1.41 ± 0.29 ng/mL, EC50TM90-C2B = 1.76 ± 0.12 ng/mL) are the internal controls for the in vitro anti- malarial activity assays. Podophyllotoxin is the internal control for the in vitro cytotoxicity assay with EC50 = 250 ± 10 nM against J774A.1.

2.2.2.5 Role of Stereochemistry on Solubility and Permeability

Solubility at pH 7.4 was determined using Biomek FX lab automation workstation with pION µSOL evolution software as reported previously.114 Passive transcellular permeability was assessed in a standard parallel artificial membrane permeability assay

(PAMPA) at pH 7.4. Solubilities of the four stereoisomers ranged from 0.2 µM for (+)- trans-2.1 to 2.9 µM for (4S,7S)-2.1 (Table 2.9). Membrane permeabilities for all four stereoisomers were in a suitable range.

Table 2.9: Comparison of solubilities and permeabilities of 2.1 stereoisomersa

4,7- Permeability pH 7.4 Compound Solubility pH 7.4 µM Stereochemistry Pe 106 cm/s R% 2.1 4R, 7R 1.3 1180 ± 130 80 ± 2 2.1 4S, 7S 2.9 872 ± 105 85 ± 3 2.1 (+)-trans 0.2 846 ± 102 86 ± 0 2.1 (-)-trans 1.3 1110 ± 460 80 ± 4 aAlbendazole (2.4 ± 0.8 μM, 352 ± 35 cm/s, 2.4 ± 0.8%), carbamazepine (91.8 ± 1.2 μM, 111 ± 30 cm/s, 38 ± 4%), ranitidine HCl (81.1 ± 1.2 μM, 4 ± 1 cm/s, 4 ± 1%) and verapamil HCl (74.9 ± 1.7 μM, 1950 ± 240 cm/s, 59 ± 12%) are the internal controls for the solubility and permeability assays. 70

2.3 Summary

Thirty-three 1,4-dihydropyridine diastereomeric pairs were synthesized and the structure- activity relationship studied. Twenty-nine of these derivatives contained a 6-position oxygen, with 2.31, 2.32, 2.52 and 2.53 having single and double digit nanomolar activities. This SAR study revealed some insightful information about the 1,4- dihydropyridine substitution pattern (Figure 2.35). A few combinations would be interesting to synthesize to see how the activities combine. At the 4-position, a 4-chloro-

2-methylphenyl derivative could be expected to increase the activity. A 4-chloro-3- methoxyphenyl at the 7-position can be tested to see how it compares with the 3,4- dimethoxyphenyl substituent.

Figure 2.35: Anti-malarial SAR of the 1,4-dihydropyridine

Using the SAR from the 6-position oxygen derivatives, 6-methylene derivatives with 4-

(4-chlorophenyl) 2.59 and 2.60 were synthesized that displayed better activity than the original 4-(2-chlorophenyl) 2.1 derivative.

Lastly, stereochemistry was revealed to play an important role in the activity of 2.1. One stereoisomer, (+)-trans-2.1, had subnanomolar activity. Another stereoisomer, (4S,7S)-

2.1, had nanomolar activity. The other two stereoisomers were inactive.

1,4-Dihydropyridines have been shown to have potential in the treatment of malaria. The performed SAR has given incredible insight for this chemotype. The high selectivity of these compounds between P. falciparum and human cells is an encouragement for their continued study. Based on the activities of the previous compounds it is recommended that in vivo studies are begun to study the efficacy of these compounds in mammals.

Chapter 3: Quinazolines as Anti-Leishmanials

3.1 Chemistry of the Quinazoline Class

Quinazolines are a class of compounds characterized by two fused six membered aromatic rings, a benzene and a pyrimidine. The electronics of the quinazoline system have two large dipoles, with increased electron density on the two nitrogens and electron deficiency on the 2- and 4-carbons (Figure 3.1).115

Figure 3.1: Electronics of the quinazoline system

Electronically, the quinazoline nitrogens act like electron-withdrawing nitro groups. This electron deficient property causes an activation of reactivity for halo-substituted quinazolines 3.A when the halogen is at the 2- or 4-position, causing nucleophilic aromatic substitutions to occur readily (Figure 3.2).115-116

Figure 3.2: Reactivity of the 2,4-dichloroquinazoline

The halogen at position 4 is more reactive than at position 2, allowing for reactions such as aminolysis to occur under minimal conditions at the 4-positon 3.B and then the 2-

73 position 3.C with more vigorous conditions.115-116 Substitution of the 4-halogen with an amine again reduces the activity at the 2-position, as an electron-donating group is now directly attached to the system.

3.2 Medicinal Importance of the Quinazoline Class

The quinazoline class of compounds displays a wide range of biological activity. They have been shown in literature to have properties as anti-cancer, sedative-hypnotic, alpha blocker, anti-fungal, anti-inflammatory, anti-convulsant, anti-malarial, anti-mutagenic, anti-coccidial, anti-leukemic, CNS depressant, anti-leishmanial and anti-bacterial agents

(Figure 3.3).117 Pandeya et al. showed moderate anti-bacterial and anti-fungal activity with Schiff bases of isatin containing quinazolinone derivatives.118 They tested the anti-

HIV properties of the compounds as well but did not find activity lower than the cytotoxicity ceiling. Gawad et al. tested 3-arylquinazolin-4(3H)-ones in a growth inhibition test against tumor cell lines and displayed results up to 80% inhibition of growth at 10 M.119 Fluorinated hydroquinazoline derivatives were reported by Ghorab et al. displaying anti-fungal activity with minimum inhibitory concentrations as low as 40

g/mL against Aspergillus ochraceus, Penicillium chrysogenum, A. flavus and Candida albicans.120 Aly et al. reported 3-arylquinazolin-4(3H)-ones displaying anti-convulsant, analgesic, anti-fungal against Fuzarium species yet no anti-bacterial activity.121 2-

Styrlquinazolin-4(3H)-ones displayed up to 88% growth inhibition of L-1210 cell lines at

1 g/mL in an anti-leukemic assay reported by Raffa et al.122 Werbel and Degnan reported the reduction of in vivo malarial and in vitro leukemia cell activity with amine substitution of quinazolin-2,4-diamine derivatives.123 Febrifugine derivatives were found to have anti-coccidial activity in an in vivo chicken assay reported by Ye et al., with 74 protection at 9 mg/kg.124 Kumar et al. tested mice and male albino rats for the anti- inflammatory, analgesic and ulcerogenic activity of 2,3,6-trisubstitutedquinazolinones.125

The in vivo properties were thought to be favorable with subcutaneous or intraperitoneal injections up to 100 mg/kg. Balakumar et al. showed imidazo[1,2-c]quinazoline derivatives with anti-inflammatory activity in the same range as indomethacin, a standard used for in vivo studies.126 Sedative-hypnotic and CNS depressant activities were reported by Jatav et al. in in vivo screens of rats and mice with 3-thiadiazol-2-styrylquinazolin-

4(3H)-one derivatives.127 Doxazosin is a quinazolin-2,4-diamine -adrenoreceptor blocker that is sold by Pfizer to treat hypertension.128As shown in the previous studies, numerous in vivo studies have been conducted with quinazoline derivatives.

Figure 3.3: Biological role of quinazolines in the literature

3.2.1 Quinazolines in the Literature as Anti-Leishmanials

Due to increased parasite resistance, cytotoxicity issues of current treatments, and long treatment regimens, new drugs are necessary to have an effective strategy for treating visceral leishmaniasis. Quinazolines are a class of compounds that have been studied and have shown potential as anti-leishmanials. Berman et al. reported a class of 2,4- diaminoquinazolines 3.D with ED50 as low as 0.04 nM against L. major amastigotes in human monocyte-derived macrophages, however the development of this compound series was abandoned due to toxicity issues (Figure 3.4).129 Bhattacharjee et al. 3.E, Ram et al. 3.F, and Shakya and Gupta et al. 3.G and 3.H have also recently tested quinazolines as anti-leishmanials with activities below 100 ng/mL in some cases.130 This class of compound has been reported as being dihydrofolate reductase inhibitors, although another mechanism of action may be involved with Leishmania.129, 131

Figure 3.4: Quinazolines shown to have anti-leishmanial activity

3.3 Anti-Leishmanial Testing

Recently, we tested a small library of structurally diverse compounds, originally designed as potential anti-cancer probes, for anti-leishmanial activity in a L. mexicana axenic amastigote assay. Among this library were N2,N4-disubstituted quinazolines 3.1 and 3.2, which are structurally different from quinazoline series A-E. Anti-leishmanial testing of quinazolines 3.1 and 3.2 against L. mexicana axenic amastigotes revealed EC50 values in the single digit micromolar range, motivating us to investigate whether quinazolines structurally related to the hits 3.1 and 3.2 have potential to display potent anti-leishmanial activity (Figure 3.5). Herein, we report a detailed structure-activity relationship (SAR) study focusing on the 2-position, the 4-position, and the quinazoline’s benzenoid ring. All compounds were initially examined in a L. donovani and L. amazonensis intracellular amastigote assay to preselect quinazoline candidates. Promising compounds have subsequently been tested for efficacy in a murine model of visceral leishmaniasis.

Figure 3.5: Hit compounds 1 & 2 and SAR positions of quinazoline

3.3.1 Synthetic Chemistry

The compounds were synthesized following known procedures (Figure 3.6).132

Commercially available anthranilic acids 3.I were cyclized with urea and the resulting 77 quinazoline-2,4-dione 3.J was reacted with phosphorous oxychloride to give the 2,4- dichloroquinazoline 3.K. Substitution with amines occurred selectively at position 4 yielding 4-amino-2-chloroquinazoline 3.L followed by substitution at position 2 to give the 2,4-diaminosubstituted quinazoline 3.M. In this synthetic sequence, only 4-amino-2- chloroquinazoline 3.L and the final N2,N4-disubstituted quinazoline-2,4-diamine 3.M have been purified and characterized.

Figure 3.6: Synthesis of N2,N4-disubstituted quinazoline-2,4-diamines

3.3.2 In vitro Anti-Leishmanial Efficacy and Cytotoxicity

All target compounds were tested against L. donovani and L. amazonensis intracellular amastigotes to identify molecules that are broadly active against these medically important Leishmania species. The testing involved murine peritoneal macrophages as host cells, since compounds with potential for clinical use must be able to penetrate the infected macrophage in a human host. Anti-leishmanial activity was determined in an assay using transgenic parasites expressing a β-lactamase gene as outlined previously.133

Concentration response data for each compound was fitted by a nonlinear regression

78 model and the concentration that induces 50% inhibition was calculated as the effective concentration EC50 (L. donovani or L. amazonensis.). Additionally, cytotoxicity against the macrophage cell line J774A.1 was determined as the effective concentration EC50

(J774A.1) and the selectivity index SI was calculated as the ratio of EC50 value for

J774A.1 and the value for L. donovani (SI = EC50 (J774A.1)/EC50 (L. donovani)).

3.3.2.1 Structure-Activity Relationship Studies.

To validate and optimize the anti-leishmanial activity of N2,N4-disubstituted quinazoline-

2,4-diamines, two compound subseries were prepared and tested. The first subseries focused mainly on the optimization of the N2- and N4-moieties (Table 3.1), whereas the second subseries was designed to investigate whether analogues being substituted at the quinazoline's benzenoid ring display improved anti-leishmanial activity (Table 3.2). In vitro antileishmanial potency of compounds against intracellular L. donovani133b and L. amazonensis132a parasites was measured using the colorimetric assay as described previously.

Starting from hit compound 3.2, N4-furfuryl-analogues 3.3 and 3.4 were prepared in which the N2-iso-propyl group was replaced by a short trifluoroalkyl- or hydroxyalkyl- group. While the 2,2,2-trifluoroethyl-substituted quinazoline 3.3 was slightly less potent than compound 3.2, alcohol 3.4 lost potency against L. donovani and L. amazonensis by a factor of 10 and >13, respectively. Testing of a small set of quinazoline-2,4-diamines 3.5-

3.9 with N4-monosubstituted or N4-disubstituted with alkyl groups differing in size and polarity did not identify a particular structural motif improving the potency over 3.2.

Replacement of the furfuryl and iso-propyl groups in 3.2 by two benzyl or two n-butyl groups yielded quinazolines 3.10 and 3.11, of which both analogues were more potent 79 than reference 3.2. Bis-benzyl-substituted quinazoline 3.10 displayed EC50s of 667 nM against L. donovani and 1.41 μM against L. amazonensis, whereas analogue 3.11 was approximately twofold less potent in comparison to compound 3.10. Consequently, a following set of six quinazolines 3.12-3.17 was designed in which one of the 2- and 4- positions was substituted by one benzyl amine, while the remaining position was derivatized with an aniline, n-butylamine, or methylamine. Interestingly, with the exception of the N4-benzyl-N2-methyl-quinazoline-2,4-diamine 3.12, all of these compounds demonstrated sub-micromolar EC50s against L. donovani. Among these six quinazolines tested, the N2-benzyl-quinazoline-2,4-diamines 3.15-3.17 appeared to be at least twofold more potent against L. donovani than the N4-benzyl counterparts 3.12-3.14 suggesting that an N2-benzyl is more favorable than an N4-benzyl for anti-leishmanial activity. This observation is similar to previous results with quinazolines 3.2-3.11.

Compound 3.15 was the most potent compound with an EC50 of 149 nM against L. donovani. For L. amazonensis, the set of 3.12-3.17 displayed a similar activity trend, with

N2-benzyl-quinazolines 3.15 and 3.16 being the most potent compounds with submicromolar or single digit micromolar EC50s.

Table 3.1: SAR study focusing on 2- and 4-positionsa

L. amazonensis 1 2 L. donovani J774A.1 Compound R R SI EC50  EC50  EC50 

3.2 2.5 ± 0.4 3.7 ± 1.6 17 ± 6 6.8

3.3 3.6 ± 1.1 5.8 ± 2.3 23 ± 10 6.4

3.4 25 ± 18 > 50 > 33 > 1.3

3.5 3.7 ± 0.3 7.6 ± 3.2 > 33 > 8.9

3.6 4.3 ± 1.2 5.1 ± 0.9 20 ± 6 4.7

3.7 6.9 ± 1.8 20 ± 3 > 33 4.8

3.8 >50.0 > 50.0 n.d. n.d.

3.9 8.9 ± 1.0 19 ± 3 > 33 > 3.7

3.10 0.67 ± 0.27 1.4 ± 0.5 5.5 ± 1.4 8.2

3.11 1.8 ± 0.4 2.1 ± 1.1 5.4 ± 1.4 3.0

3.12 1.5 ± 0.5 13 ± 3 18 ± 8 12

3.13 0.65 ± 0.10 1.8 ± 0.2 5.1 ± 1.5 7.8

3.14 0.64 ± 0.10 2.6 ± 0.7 6.8 ± 0.2 11

3.15 0.15 ± 0.02 0.90 ± 0.27 15 ± 1 100

3.16 0.34 ± 0.12 1.4 ± 0.2 4.9 ± 1.3 14

3.17 0.26 ± 0.15 2.2 ± 0.3 5.2 ± 1.3 20

a Amphotericin B is the internal control for the in vitro anti-leishmanial activity assay with EC50 = 40 ± 9 nM against L. donovani and EC50 = 89 ± 16 nM against L. amazonensis. Podophyllotoxin is the internal control for the in vitro cytotoxicity assay with EC50 = 250 ± 10 nM against J774A.1.

4- 2 N Furfuryl-N -iso-propyl-substituted quinazoline 3.2, with EC50s of 2.50 M against L. donovani and 3.71 μM against L. amazonensis, was considered to be well suited as the

81 key scaffold for a SAR study focusing on the benzenoid moiety of the quinazoline core.

In a systematic approach following the Topliss operational scheme, compound 3.2 was mono-substituted with either a chlorine atom, a methyl group, or a methoxy group in the

5-, 6-, 7-, and 8-positions to probe the benzenoid ring for steric and electronic effects.134

Overall, substitution on the benzenoid ring provided compounds with EC50s in the single digit micromolar or sub-micromolar range against L. donovani. Among the chloro- substituted subseries, 5- and 6-substituted analogues 3.18 and 3.19 were less potent by a factor of two or more compared to reference 3.2, while the 7- and 8-substituted quinazolines 3.20 and 3.21 displayed an activity on par with compound 3.2. In contrast, the majority of the methyl- and methoxy-substituted quinazolines demonstrated a modest potency improvement over quinazoline 3.2. For the methoxy-substituted quinazolines, potency increased according to substitutions in 8- < 7- < 5- ≈ 6-position, whereas a potency dependence in the order of 8- ≈ 6- < 7- ≈ 5-positions was observed for the methyl-substituted compounds. 7-Methoxy-quinazoline 3.28 with an EC50 of 740 nM against L. donovani was the most potent analogue of the compound subseries substituted at the benzenoid ring. In contrast, the testing of the benzenoid ring substituted quinazolines against L. amazonensis gave insight into a SAR, which differed from the one observed for L. donovani. 8-Methoxy-substituted quinazoline 3.29 was the only analogue which was marginally more potent than reference compound 3.2, whereas all of the other analogues were equally or less potent than quinazoline 3.2.

Table 3.2: Benzenoid substitution

L. amazonensis 1 2 3 4 L. donovani J774A.1 Compound R R R R SI EC50  EC50  EC50  3.2 -H -H -H -H 2.5 ± 0.4 3.7 ± 1.6 17 ± 6 6.8 3.18 -H -H -H -Cl 4.4 ± 1.3 18 ± 6 > 33 > 7.5 3.19 -H -H -Cl -H 9.0 ± 3.7 23 ± 5 >100 > 11 3.20 -H -Cl -H -H 2.6 ± 1.9 25 ± 4 > 33 > 13 3.21 -Cl -H -H -H 2.3 ± 0.6 12 ± 3 25 ± 3 11

3.22 -H -H -H -CH3 4.2 ± 2.1 4.4 ± 1.9 > 33 > 7.9

3.23 -H -H -CH3 -H 0.83 ± 0.32 4.1 ± 1.2 > 33 > 40

3.24 -H -CH3 -H -H 4.7 ± 2.5 15 ± 6 > 33 > 7.0

3.25 -CH3 -H -H -H 0.95 ± 0.27 3.6 ± 1.5 20 ± 9 21

3.26 -H -H -H -OCH3 3.2 ± 1.2 7.3 ± 2.5 30 ± 5 9.4

3.27 -H -H -OCH3 -H 1.2 ± 0.5 10 ± 2 16 ± 2 13

3.28 -H -OCH3 -H -H 0.74 ± 0.37 9.6 ± 3.0 14 ± 1 19

3.29 -OCH3 -H -H -H 0.97 ± 0.26 2.7 ± 1.4 18 ± 6 19 a Amphotericin B is the internal control for the in vitro anti-leishmanial activity assay with EC50 = 40 ± 9 nM against L. donovani and EC50 = 89 ± 16 nM against L. amazonensis. Podophyllotoxin is the internal control for the in vitro cytotoxicity assay with EC50 = 250 ± 10 nM against J774A.1.

3.3.2.2 Cytotoxicity

An important quality of the quinazolines is their selectivity to inhibit parasite growth over mammalian cells. Generally, the majority of the quinazolines 3.2-3.29 exhibited EC50s of

20 μM or higher against J774A.1 (Tables 1 and 2). Quinazolines 3.18-3.20 and 3.22-3.24, whose benzenoid ring is substituted with one chloride or one methyl in 5-, 6- or 7- position, were significantly less toxic than their reference 3.2. The quinazolines displaying potent anti-leishmanial activity, especially the N2-benzyl-quinazoline-2,4- diamines 3.15-3.17, were shown to have respectable SI values of 10 and larger indicating that they are relatively selective, nontoxic chemotypes. Toxicity of compounds against

83 the murine macrophage cell line J774A.1 was conducted using the 3-(4,5)- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as previously described135 except that cells were incubated with compounds for 72 h.

3.3.3 Mechanism of Action

We investigated the possibility that quinazolines inhibit folate metabolism in the parasite.

L. donovani axenic amastigotes and J774A.1 (mouse macrophage cell line) were adapted to grow in media deficient in p-aminobenzoic acid (PABA) and folic acid prior to susceptibility testing. Susceptibility to quinazolines, methotrexate, pyrimethamine, and miltefosine were assessed in the presence or absence of folinic acid (Figure 3.7).

Figure 3.7: In vitro efficacy of quinazolines, methotrexate (MTX), pyrimethamine (PYR), and miltefosine (MILT) for axenic amastigotes of Leishmania donovani in the absence or

presence of d,l-folinic acid (FNA)

The presence of folinic acid dramatically increased the EC50 values of each of the quinazolines as well as the methotrexate and pyrimethamine. For example, quinazoline

3.23 was 6.4 fold less potent in the presence of 488 nM folinic acid than in completely deficient media. Conversely, efficacy of miltefosine was not affected by addition of folinic acid to the media. Interestingly, we saw no antagonism of activity of quinazolines 84

3.10, 3.12, 3.13, 3.15, 3.16 or 3.23 for the mammalian cell line (J774A.1) in the presence of folinic acid. For example, the EC50 of quinazoline 3.10 was 84.8 ± 2.2 μM and 84.2 ±

2.8 μM in the presence or absence of 488 nM folinic acid, respectively. In contrast, the efficacy of methotrexate and pyrimethamine were antagonized significantly by folinic acid in J774A.1. These results are consistent with the hypothesis that the quinazolines interfere with folate metabolism in L. donovani.

3.3.4 Structure-Property Relationship Studies.

In parallel to testing the compounds for anti-leishmanial activity in vitro, a structure- property relationship (SPR) study focusing on log D, aqueous solubility, and permeability has been conducted with all compounds to assess potential physicochemical liabilities

(Table 3.3). Log D3.0 and Log D7.4, the distribution coefficient between octanol and water at pH 3.0 and pH 7.4, were experimentally determined via a previously described HPLC- based method.136 Solubility at pH 7.4 was determined using Biomek FX lab automation workstation with pION µSOL evolution software as reported previously and at pH 2.0 using an in-house HPLC assay based on UV absorption.114 Passive transcellular permeability was assessed in a standard parallel artificial membrane permeability assay

(PAMPA) at pH 7.4 and pH 4.0. Generally, the aqueous solubility, the distribution coefficient Log D, and the permeability of all quinazolines display a pH dependence

(exemplified on Log D or permeability in Table 3.3). The permeability is enhanced at neutral pH while the aqueous solubility and Log D are better at lower pH ranges.

However, since the aqueous solubility, the distribution coefficient, and the permeability are within the acceptable ranges (solubility > 20 M, Pe > 10 x 10-6 cm·s-1, 1 < Log D <

4), the quinazoline compound series is considered to be suitable for the development of bioavailable anti-leishmanial compounds.

Table 3.3: Physicochemical properties of quinazolines -6 a Permeability Pe ·10 Solubility b Log D Compound (cm/s) pH 7.4 pH 2.0 pH 7.4 pH 4.0 pH 7.4 pH 3.0 3.2 ***** ***** 883 62.2 3.15 2.18 3.3 *** ***** 979 67.7 4.20 2.02 3.4 **** ***** 1130 26.4 2.19 1.48 3.5 ***** ***** 780 19.7 3.19 2.40 3.6 **** ***** 1480 28.8 3.02 2.06 3.7 **** ***** 744 38.8 2.54 1.73 3.8 **** ***** 783 112 4.69 3.56 3.9 *** ***** 1120 72.3 3.13 1.64 3.10 ** **** 383 312 3.82 2.82 3.11 *** ***** 880 485 3.89 2.91 3.12 ***** ***** n/d n/d 3.14 2.11 3.13 *** ***** 1250 161 3.80 2.84 3.14 * ** 1480 271 4.17 2.78 3.15 *** ***** 1720 57.1 2.96 2.00 3.16 *** ***** 1020 341 3.84 2.86 3.17 * ***** 663 412 4.25 2.67 3.18 ** ***** 1330 67.6 3.96 2.38 3.19 **** ***** 1380 103 3.91 2.51 3.20 **** ***** 1630 170 3.99 2.60 3.21 *** ***** 1240 169 4.44 2.62 3.22 **** ***** 666 45.9 3.55 1.57 3.23 **** ***** 798 67.2 3.61 1.58 3.24 **** ***** 789 67.2 3.63 1.55 3.25 *** ***** 812 45.9 3.55 2.44 3.26 **** ***** 1060 38.4 3.40 2.99 3.27 **** ***** 1330 59.2 3.70 3.11 3.28 ***** ***** 1010 80.2 3.58 2.95 3.29 **** ***** 624 87.0 3.35 2.53 a (*) for solubility ≤ 5 μM, (**) for 5 μM < solubility ≤ 10 μM, (***) for 10 μM < solubility ≤ 20 μM, (****) for 20 μM < solubility ≤ 30 μM, (*****) for solubility ≥ 30 μM. n/d: not determined. b For the determination of the Pe values, the following internal controls were utilized: carbazepine pH 4.0 permeability Pe = 108·10-6 (cm/s) and pH 7.4 permeability Pe = 130·10-6 (cm/s); ranitidine·HCl pH 4.0 permeability Pe = 5.2·10-6 (cm/s) and pH 7.4 permeability Pe = 2.2·10-6 (cm/s); verapamil·HCl pH 4.0 permeability Pe = 20.6·10-6 (cm/s) and pH 7.4 permeability Pe = 1360·10-6 (cm/s).

3.3.5 In Vivo Anti-Leishmanial Efficacy Studies

For the selection of the candidates for in vivo efficacy against L. donovani, only compounds which displayed submicromolar in vitro activity against L. donovani with a

SI value greater than ten and a balanced combination of good physicochemical properties were chosen. Among the benzyl-substituted quinazolines 3.12-3.17, 3.15 and 3.16 appeared to be the best candidates for in vivo evaluation due to their combination of potency, selectivity, and favorable physicochemical properties. From the compound subseries substituted at the benzenoid ring, the physicochemical properties were not discriminatory and hence compound 3.23 was chosen as a viable candidate due to its submicromolar EC50 against L. donovani as well as the outstanding SI value of this compound (> 40).

The three compounds mentioned above were dissolved in an appropriate vehicle (3.15 and 3.23 dissolved in 0.5% methyl cellulose and 0.1% Tween80 in water, 3.16 dissolved in 45% (w/v) (2-hydroxypropyl)-β-cyclodextrin (HPβCD) solution) and administered to uninfected BALB/c mice intraperitoneally to determine a tolerated dose for in vivo efficacy studies. While 3.15 was well tolerated when given at 30 mg/kg ip for five consecutive days, 3.16 (slowed breathing) and 3.23 (hypoactivity) were toxic to animals when given at the same dosing regimen. Considering the toxicity of 3.16 and 3.23 when given at 30 mg/kg i.p., lower doses of these compounds were administered in subsequent in vivo efficacy studies in a murine visceral leishmaniasis model (Figure 3.8). When tested at 5 × 15 mg/kg ip, 3.23 inhibited liver parasitemia by 37% compared to the vehicle control. However, 3.15 did not show significant anti-leishmanial efficacy when tested at 5 × 30 mg/kg i.p. There was also no significant difference in the parasite burden 87 between mice in the group treated with compound 3.16 (5 × 10 mg/kg i.p.) and the vehicle control group (P > 0.05). As expected, the 45% HPβCD vehicle used to solubilize

3.16 itself resulted in 18% inhibition of liver parasitemia, consistent with a previous report.133b

The in vivo antileishmanial efficacy of compounds was evaluated in a murine model of visceral leishmaniasis by the general method described earlier132a with minor modifications. Briefly, BALB/c mice were inoculated with LV82 promastigotes on day 0, and then weighed and marked individually on day 6 in order to calculate the volume of solution to be administered to each mouse. In these studies, 45% (w/v) (2- hydroxypropyl)-β-cyclodextrin (HPβCD) vehicle was used to formulate 3.16 and 0.5% methyl cellulose/0.1% Tween80 in water (MC) was used to dissolve 3.15 and 3.23; all compounds and vehicles were administered intraperitoneally. After 5 consecutive days of treatment from days 7 to 11, animals were sacrificed on day 14. Liver smear slides from each animal were made and stained, and the liver parasitemia expressed in Leishman-

Donovan units (LDU) was determined by microscopy.132a The efficacy of each compound was calculated according to the method published previously.132a

Results are presented as the liver parasitemia (LDU) for each mouse (♦) and the average

LDU in each group (-) determined by microscopy (n = 4). (A), LDU for mice treated with

3.16 and 3.23; (B), LDU for mice treated with 3.15. All treatments were given by the i.p. route. Compounds 3.15 and 3.23 were dissolved in 0.5% methyl cellulose and 0.1%

Tween80 (MC), while compound 3.16 was dissolved in 45% (w/v) (2-hydroxypropyl)-β- cyclodextrin solution (HPβCD). (*): p < 0.05, compared with untreated control.

(A) 4000

3500 (*) (*) 3000 (*) 2500

2000

1500

1000

500 (*) 0

Leishman - Donovan Units (LDU) Units Donovan - Leishman Untreated MC HPCD Miltefosine 16 23 control control control 10 mg/kg 10 mg/kg 15 mg/kg

5000 (B)

4000

3000

2000

1000

(*) 0

Leishman - Donovan Units (LDU) Units Donovan - Leishman Untreated Miltefosine 15 control 10 mg/kg 30 mg/kg

Figure 3.8: In vivo efficacy of quinazolines against LV82 in L. donovani infected

BALB/c mice.

3.3.6 Pharmacokinetics of 3.16 and 3.23

Pharmacokinetic studies were conducted to determine the systemic and target tissue exposures of 3.16 and 3.23. The mean plasma and tissue concentration–time profiles of

3.16 and 3.23 after p.o. and i.p. administration are shown in Figure 3.9 and Figure 3.10, respectively. The relevant pharmacokinetic outcomes are listed in Table 3.4. After p.o. administration at 100 µmol/kg (or approximately 30 mg/kg), both compounds were absorbed from the gastrointestinal tract of mice and plasma concentration reached a Cmax of 0.44 and 0.25 μM for 3.16 and 3.23, respectively.

Figure 3.9: Plasma (open circles) and tissue (squares for liver and triangles for spleen)

concentration-time profiles after p.o. (A) and i.p. (B) administration of 16 in mice at a

dose level of 100 μmol/kg (~30 mg/kg)

Symbols and error bars in Figure 3.9 and Figure 3.10 represent the mean and standard error of triplicate determinations, except those labeled with asterisks where only one or two determinations were obtained due to sample loss. 3.23 was below the detection limit

(0.1 μM) in the liver and spleen 12 and 24 h after i.p. administration.

The systemic and tissue exposure of 3.16 (AUC and Cmax) were considerably greater than

those of 3.23 (Table 3.4). After i.p. administration, plasma concentration reached a Cmax

of 5.2 and 2.7 μM for 3.16 and 3.23, respectively, before decreasing rapidly in the first 4

h, followed by a slower elimination process until 24 h. The rapid decline in plasma

concentration was likely due to extensive tissue redistribution after absorption, as

indicated by the high target tissue concentrations. The plasma and tissue exposures after

i.p. administration were markedly greater than those after p.o. administration (Table 3.4),

suggesting significant first-pass metabolism and/or partial gastrointestinal absorption

after oral administration of 3.16 and 3.23.

Table 3.4: Pharmacokinetic outcomes of 3.16 and 3.23 after p.o. and i.p. administration to mice p.o. i.p. Outcomes plasma liver spleen plasma liver spleen

Cmax (μM) 0.44 15.8 7.0 5.21 148 163

Tmax (h) 4 1 1 1 1 1

a 16 AUClast (μM•h) 7.8 110 68 11 500 620

b c t1/2 (h) NC ND ND 20 ND ND

d Mic t1/2 (min) 27

Cmax (μM) 0.25 5.2 0.9 2.67 48 45.8

Tmax (h) 1 1 1 1 1 1

a 23 AUClast (μM•h) 2.5 29 7.4 4.6 42 71

t1/2 (h) 24 ND ND 5.4 ND ND

d Mic t1/2 (min) 9.4 a AUC(0-24h) was calculated for plasma using noncompartmental analysis, whereas AUC(1-24h) was calculated for tissues using trapezoid rule. AUClast, AUC from the time of dose to the last measurable concentration; Cmax and Tmax, b maximum concentration and time to reach Cmax; t1/2, terminal half-life. NC, not calculable due to lack of a data point. c ND, not determined. d In vitro mouse liver microsomeal half-life.

The terminal elimination half-life ranged from 5 to 20 h. Minor reversible overt toxicity

(hypoactivity) was observed after i.p. administration of 3.16, however more severe toxicity was observed, unexpectedly, after a single i.p. administration of 3.23, which warranted euthanization of some mice within 15 min post dose. As efficacy was evaluated at lower doses than 30 mg/kg to avoid toxicity and dose linearity for pharmacokinetic outcomes were unknown, it is not possible to directly correlate drug exposure with anti-leishmanial activity in mice. In addition, it is worth pointing out that the terminal half-life of 3.23 after i.p. administration was considerably shorter than that after p.o. administration (5 h vs. 20 h; Table 3.4), and the liver concentration of 3.23 was detectable at 12 and 24 h after p.o. administration, whereas it was below detection limit after i.p. administration (Figure 3.10). These observations suggest flip-flop pharmacokinetics, where the rate of gastrointestinal absorption is slower than the rate of elimination due to dosage formulations (e.g. sustained-release), excipients, physicological factors (e.g. intestinal mobility and pH), and/or drug characteristics.137 The dramatic decrease (~12-fold) in the permeability of 3.23 from neutral to acidic pH (Table 3.3) could contribute to the slowed absorption of this compound in the upper gastrointestinal tract. In contrast, the permeability of 3.16 decreased only 3-fold from neutral to acidic pH

(Table 3.3). Furthermore, 3.23 was metabolized quickly in the mouse liver microsomes

(t1/2 = 9.4 min; Table 3.4), suggesting that it is possible that the rate of elimination was greater than the rate of gastrointestinal absoption for 23.

Figure 3.10: Plasma (open circles) and tissue (squares for liver and triangles for spleen)

concentration-time profiles after p.o. (A) and i.p. (B) administration of 23 in mice at a

dose level of 100 μmol/kg (~30 mg/kg)

3.3.7 Summary

N2,N4-disubstituted quinazoline-2,4-diamines 3.1 and 3.2 were found to display anti- leishmanial activity in the single digit micromolar range. Subsequently a total of 28 molecules have been synthesized systematically by varying the substitutions in the 2-, 4-,

5-, 6-, 7-, and/or 8-positions. All quinazolines have been tested with the aim to further optimize hit compounds 3.1 and 3.2 and to conduct a detailed SAR study against L. donovani and L. amazonensis. The most potent activities with EC50s in the submicromolar range against L. donovani were obtained with quinazoline-2,4-diamine scaffolds bearing either a N2-benzyl-N4-alkyl/phenyl or a N2-ispropyl-N4-furfuryl substituent combination. Furthermore, although the benzenoid ring of the quinazoline-

2,4-diamine scaffold has been identified to play a secondary role for efficacy, quinazolines substituted at the 5- or 6-position with one methyl or one methoxy group have also been identified to possess submicromolar EC50s against L. donovani. The best 93 quinazolines were shown to display cytotoxicity against the macrophage cell line

J774A.1 at 10 M or higher concentrations yielding SI values larger than 10. In addition, assessment of key physicochemical properties confirmed that the quinazoline's aqueous solubility, distribution coefficient, and passive transcellular permeability were in acceptable ranges. These promising results led to efficacy testing of the lead compounds

3.15, 3.16 and 3.23 in an in vivo murine visceral leishmaniasis model. While compounds

3.15 and 3.16 did not have activity translate from in vitro to in vivo, quinazoline 3.23 reduced parasitemia by 37% when 15 mg/kg/day were given via the intraperitoneal route for five consecutive days. Pharmocokinetic studies of compound 3.23 revealed a maximum plasma concentration that was threefold higher than the EC50 and a terminal half-life of 5 hours after i.p. administration. Thus, the observed potencies of frontrunner compounds 3.15, 3.16 and 3.23 in conjunction with favorable physicochemical and pharmacokinetic properties make N2,N4-disubstituted quinazoline-2,4-diamines a suitable platform for the future development of anti-leishmanial agents.

3.4 Additional Testing

Following the initial study concluding with in vivo data showing a reduction of parasitemia by 37%, a terminal half-life of 5 hours after i.p. administration and a plasma

Cmax 3.25 times the L. donovani EC50 for compound 3.23, an additional twenty-nine compounds were synthesized with the hopes of improving the in vivo efficacy. These compounds mostly carried aromatic N2- and/or N4- substituents as these were the substituents that showed the highest activity in the previous study. A small sampling of

N2,N4 dialkyl substitutions were also included to see if a broader sampling would show activity. 94

3.4.1 Structure-Activity Relationships

3.4.1.1 Structure-Activity Relationship of N2- or N4- Alkyl with N2- or N4- Alkyl/Aryl

As seen previously in Table 3.1, dialkyl substitution of the quinazolin-2,4-diamine resulted in compounds with single digit micromolar activity against intracellular L. donovani amastigotes (Table 3.5, compounds 3.30-3.35, 3.37). N4- phenyl or benzyl with

N2-isopropyl resulted in compounds 3.34 and 3.35 with activities at 2 M, while N4- furfuryl-N2-methyl 3.36 lost all potency. Another N4-furfuryl compound, 3.38 with N2- cyclopentyl, also lost all potency whereas the N2-cyclohexyl derivative displayed single digit micromolar activity. The only compound from this set displaying submicromolar activity was the N2-benzyl 3.41, the N4-isopropyl derivative of compound 3.15 from

Table 3.1. Replacing the N4-methyl of 3.15 with N4-isopropyl resulted in a drop of activity from 150 nM to 380 nM, but the cytotoxicity stayed in the same range at 14 M.

Table 3.5: N2- or N4- alkyl with N2- or N4- alkyl/aryl substitutions

L. donovani J774A.1 Compound R1 R2 SI EC50  EC50 

3.30 6.3 ± 1.9 n/d

3.31 6.1 ± 2.8 n/d

3.32 2.2 ± 0.5 n/d

3.33 8.9 ± 0.1 n/d

3.34 1.9 ± 0.2 n/d

3.35 2.0 ± 0.1 n/d

3.36 >25 n/d

3.37 4.4 ± 1.2 n/d

3.38 >25 n/d

3.39 4.0 ± 2.5 n/d

3.40 12 ± 9 n/d

3.41 0.38 ± 0.09 14 ± 1 37

3.42 20 ± 5 n/d

3.43 11 ± 3 n/d

3.4.1.2 Structure-Activity Relationship of N2,N4-Diaryl Derivatives

The diarylquinazolin-2,4-diamines generally had activity in the single digit micromolar or triple digit nanomolar range, with the exception of the N2-furfuryl derivative 3.44 96

(Table 3.6). Derivatives of compound 3.10 having benzyl substitutions at the para position with chloro, methyl or methoxy at N2 and/or N4 had activities between 0.4 and 6

M with selectivity indexes around ten. The most active derivatives 3.46 and 43.7 were substituted with N2-4-chlorobenzyl and N4-4-methylbenzyl respectively, with a benzyl on the opposite amine. Compounds 3.49 and 3.52 were disubstituted with N2,N4-di(4- methylbenzyl) and N2,N4-di(4-methoxybenzyl), respectively, and had potencies above 1

M.

Table 3.6: N2,N4-diaryl derivatives

L. donovani J774A.1 Compound R1 R2 SI EC50  EC50 

3.44 14 ± 6 n/d

3.45 0.82 ± 0.23 8.4 ± 0.5 10

3.46 0.61 ± 0.12 5.8 ± 0.8 9.5

3.47 0.39 ± 0.16 4.8 ± 2.1 12

3.48 0.89 ± 0.10 6.9 ± 0.5 7.8

3.49 1.1 ± 0.5 n/d

3.50 2.5 ± 0.2 n/d

3.51 0.72 ± 0.10 6.8 ± 0.4 9.4

3.52 6.0 ± 2.9 n/d

3.4.1.3 Structure-Activity Relationship of N2,N4-Diaryl Derivatives with Backbone

Substitution

Derivatives of compound 3.15 were made with 6-methoxy, 6-chloro or 7-chloro derivation (compounds 3.53-3.55, table 3.7). The 6-methoxy derivative, 3.53, was the only compound of the three to have submicromolar activity, although with a modest selectivity index of 14. Three 7-chloro derivatives of 3.10 were also made with all three having activities less than 0.5 M (compounds 3.56-3.58). The trichloro 3.58 displayed the highest potency at 260 nM with the monochloro 3.56 next with 370 nM and a selectivity index of 38.

Table 3.7: 6- and 7-substituted derivatives

L. donovani J774A.1 Compound R1 R2 R3 SI EC50  EC50 

3.53 6-MeO 0.35 ± 0.06 4.8 ± 2.2 14

3.54 6-Cl 2.5 ± 0.3 n/d

3.55 7-Cl 1.4 ± 0.2 9.7 ± 4.0 6.9

3.56 7-Cl 0.37 ± 0.8 14 ± 1 38

3.57 7-Cl 0.26 ± 0.15 n/d

3.58 7-Cl 0.47 ± 0.05 5.1 ± 1.0 11

3.4.2 Structure-Property Relationship Studies

A structure-property relationship study was conducted for the new compounds as described for the previous compounds (Table 3.8). As can be seen in the lower half of the 98 table, the aryl substituted quinazolines have lower aqueous solubility and membrane permeability.

Table 3.8: Physicochemical properties of quinazolines -6 a Permeability Pe ·10 Solubility b Log D Compound (cm/s) pH 7.4 pH 7.4 pH 4.0 pH 7.4 3.30 *** 1490 13.6 2.45 3.31 *** 1300 421 3.04 3.32 *** 1190 42.1 3.14 3.33 **** 744 38.8 n/d 3.34 **** 1790 124 3.62 3.35 **** 1340 159 3.50 3.36 **** 744 4.51 2.61 3.37 *** 1690 22.8 2.88 3.38 **** 2090 116 3.54 3.39 **** 854 176 4.18 3.40 **** 788 46.1 4.66 3.41 **** 436 71.1 3.54 3.42 *** 963 10.5 2.59 3.43 *** 543 n/d 3.23 3.44 ** 288 72.2 3.25 3.45 n/d n/d n/d n/d 3.46 * n/d n/d n/d 3.47 **** 854 67.3 n/d 3.48 * 1070 160 n/d 3.49 * 1 4 n/d 3.50 * 1350 246 n/d 3.51 * 1750 415 n/d 3.52 * 441 14.6 n/d 3.53 n/d n/d n/d n/d 3.54 n/d n/d n/d n/d 3.55 n/d n/d n/d n/d 3.56 * n/d 153 n/d 3.57 * 0.3 n/d n/d 3.58 * 0.5 n/d n/d a (*) for solubility ≤ 5 μM, (**) for 5 μM < solubility ≤ 10 μM, (***) for 10 μM < solubility ≤ 20 μM, (****) for 20 μM < solubility ≤ 30 μM, (*****) for solubility ≥ 30 μM. n/d: not determined. b For the determination of the Pe values, the following internal controls were utilized: carbazepine pH 4.0 permeability Pe = 108·10-6 (cm/s) and pH 7.4 permeability Pe = 130·10-6 (cm/s); ranitidine·HCl pH 4.0 permeability Pe = 5.2·10-6 (cm/s) and pH 7.4 permeability Pe = 2.2·10-6 (cm/s); verapamil·HCl pH 4.0 permeability Pe = 20.6·10-6 (cm/s) and pH 7.4 permeability Pe = 1360·10-6 (cm/s).

3.4.3 Pharmacokinetics of 3.41 and 3.46 After Intraperitoneal Administration in Mice

Pharmacokinetic studies were conducted to determine the systemic and target tissue exposures of 3.41 and 3.46. The mean plasma and tissue concentration–time profiles of

3.41 and 3.46 after i.p. administration are shown in Figure 3.11.

Figure 3.11: Plasma and tissue concentration-time profiles after 10 mg/kg i.p.

administration of 3.41 and 3.46

The relevant pharmacokinetic outcomes are listed in Table 3.9. After i.p. administration, plasma concentration reached a Cmax of 2.15 and 1.40 μM for 3.41 and 3.46, respectively.

The rapid decline in plasma concentration was likely due to extensive tissue redistribution after absorption, as indicated by the high target tissue concentrations

(Figure 3.11). The tissue concentration of 3.41 drastically declined over 12 hours while the decline was much slower for 3.46 with 10 μM concentrations still found at 12 hours in the liver and spleen. The terminal elimination half-life was 2 hours and 9 hours for

3.41 and 3.46, respectively.

100

Table 3.9: Pharmacokinetic outcomes of 3.41 and 3.46 after 10 mg/kg i.p. administration to mice i.p. Outcomes plasma

Cmax (μM) 2.15

Tmax (h) 0.25 3.41 AUClast (μM•h) 7.14

t1/2 (h) 2.05

Cmax (μM) 1.40

Tmax (h) 0.50 3.46 AUClast (μM•h) 15.7

t1/2 (h) 9.31

3.4.4 In Vivo Anti-Leishmanial Efficacy Studies of Compounds 3.28, 3.41 and 3.46

For the selection of the candidates for in vivo efficacy against L. donovani, only compounds which displayed submicromolar in vitro activity against L. donovani were chosen, with the rationale for selections based on other data we had acquired. Compound

3.28 was reintroduced because of its best-in-class activity among the original benzenoid substitutions and excellent SPR data. Compound 3.41 was chosen as an analogue to 3.15.

It was believed that 3.15 lost all activity in the in vivo assay due to the presence of the N4- methyl substitution and enzymatic demethylation in vivo. 3.41, with the N4-isopropyl substitution, was hoped to circumvent this problem and maintain excellent activity in the murine model. Compound 3.46, with admittedly poor SPR data, was chosen based on the high concentration-time profiling found during the pharmacokinetic study.

101

The three compounds 3.28, 3.41 and 3.46 were dissolved in a vehicle consisting of 5%

DMSO, 50% PEG 400, 10% ethanol and 35% water. When tested at 5 × 10 mg/kg ip,

3.28 inhibited liver parasitemia by 17%, 3.41 inhibited liver parasitemia by 24%, and

3.46 inhibited liver parasitemia by 12% compared to the vehicle control (Figure 3.12).

4000

3500

3000

2500 Mouse 1 Mouse 2 2000

Mouse 3 Donovan Units (LDU)

- 1500 Mouse 4 1000 Average

Leishman 500

0 Untreated Miltefosine 10 mg/kg 10 mg/kg 10 mg/kg Control 3.28 3.41 3.46

Figure 3.12: In vivo efficacy of quinazolines in L. donovani infected BALB/c mice

3.4.5 Summary

Following the original study of 28 synthesized molecules with various substitutions in the

2-, 4-, 5-, 6-, 7-, and/or 8-positions, an additional 29 were synthesized and tested. The most potent activities with EC50s in the submicromolar range against L. donovani were obtained with quinazoline-2,4-diamine scaffolds bearing either a N2,N4-dibenzyl or a N2- benzyl-6-methoxy-N4-methyl substituent combination. Enhancement of activity was encountered with quinazolines substituted at the 7-position with a chloro group. The best quinazolines were shown to display SI values larger than 10 with few compounds having cytotoxicity values of 10 M or higher concentrations against the macrophage cell line

102

J774A.1. These promising results led to efficacy testing of the lead compounds 3.28, 3.41 and 3.46 in an in vivo murine visceral leishmaniasis model. Of the three, quinazoline 3.41 reduced parasitemia the most by 24% when 10 mg/kg/day were given via the intraperitoneal route for five consecutive days. Pharmocokinetic studies of compound

3.41 revealed a maximum plasma concentration that was 2.3 times higher than the EC50.

For compound 3.46, the concentration was 5.7 times higher than the EC50.

The observed potencies of frontrunner compounds 3.28, 3.41 and 3.46 in conjunction with the earlier data from 3.15, 3.16 and 3.23 make N2,N4-disubstituted quinazoline-2,4- diamines a possible platform for the future development of anti-leishmanial agents. In vivo efficacy needs to be improved in order for a hit to become a lead and this may occur via the study of new substituents. Complex secondary amines will be looked at as well as some of the untested compounds from this study, such as 3.47, that have displayed decent activities with better physicochemical properties.

103

Chapter 4: Quinazolines and the ESKAPE Pathogens

4.1 Quinazolines as Anti-Bacterial Agents

Quinazolines are a class of compounds that have been studied and have shown potential as anti-bacterial agents. Huband et al. have reported substituted quinazolin-2,4-dione 4.A as bacterial gyrase and topoisomerase inhibitors (Figure 4.1).138 They report a minimum inhibitory concentration against 90% of the tested strains (MIC90) of 0.5 µg/mL versus staphylococci, 0.06 µg/mL versus streptococci, 2 µg/mL versus enterococci, and 0.5

µg/mL versus Moraxella catarrhalis, Haemophilus influenzae, Listeria monocytogenes,

Legionella pneumophila and Neisseria species. Compound 4.A was used in a S. aureus in vivo murine acute lethal infection model, where an oral dose of 2.5 ± 1.8 mg/kg of 4.A was found to protect 50% of mice infected with MRSA strain SA-1417.

Indolo[1,2-c]quinazoline 4.B and its analogues have been shown to have anti-bacterial and anti-fungal activity by Rohini et al. (Figure 4.1).139 These compounds had zones of inhibition (ZOI) in Kirby-Bauer tests, and their MICs were evaluated against Gram- positive and negative bacteria, including Staphylococcus aureus, Bacillus subtilis,

Streptococcus pyogenes, Salmonella typhimurium, Escherichia coli and Klebsiella pneumonia, and also against the fungi Aspergillus niger, Candida albicans and

Trichoderma viridae. 4.B had MICs between 2 and 5 µg/mL against all tested bacteria and between 2 and 10 µg/mL for fungal isolates.

104

Figure 4.1: Quinazolines with reported anti-bacterial activity Mycobacterial growth inhibition and dihydrofolate reductase inhibition was reported by

DeGraw et al. with a series of 2,4-diaminoquinazolines 4.C (Figure 4.1).140 An MIC of

1.8 µM was reported for 4.C and a marked synergistic effect was found with diaminodiphenyl sulfone. Although activity was found with Mycobacterium, 4.C was found to be inactive against Staphylococcus aureus, Enterococcus faecalis,

Corynebacterium acnes, Pseudominas aeruginosa, Erysipelothrix insidiosa, Escherichia coli, Proteus vulgaris and Salmonella chloraesuis.

In vitro activity against E. coli, S. aureus and K. pneumonia was seen from 4.D at MICs of 2 µM, 12 µM and 12 µM, respectively, as reported by Kung et al. (Figure 4.1).141 In an in vivo murine model, 4.D afforded a 40% survival rate when injected subcutaneously at

100 mg/kg in mice infected with K. pneumonia.

Gottasová et al. reported the in vitro activity of 4-aniloquinazoline 4.E against B. subtilis

142 and S. aureus (Figure 4.1). An MIC of 10 µg/mL and an EC50 of 0.8 µg/mL for 4.E

105 was found against S. aureus. 4.E also had an MIC of 1 µg/mL and an EC50 of 0.7 µg/mL in assays using B. subtilis.

4.2 Quinazolin-2,4-Diamine Activity Against Methicillin-Reisistant Staphylococcus aureus

Recently, we tested a small library of quinazolines, structurally different from previously reported antibacterial quinazolines and originally designed as potential anti-leishmanials, for anti-bacterial activity. The results were promising as several compounds had zones of inhibition against methicillin-resistant Staphylococcus aureus (MRSA) in Kirby-Bauer tests. The Kirby-Bauer test is an introductory assay that allows for the quick identification of compounds that inhibit the growth of bacteria. A larger library was then tested and minimum inhibitory concentrations (MICs) were determined for those compounds having zones of inhibition. Herein, we report a detailed structure-activity relationship (SAR) study focusing on the 2-position, the 4-position, and the quinazoline’s benzenoid ring. Furthermore, we report the selection process leading to frontrunner compounds displaying low mutation frequencies and promising in vivo efficacy in murine models of infection.

Figure 4.2: Positions of the quinazoline ring

4.2.1 Testing of Quinazolines via Kirby-Bauer Assays

Assessment of the quinazolin-2,4-diamine structure shows three locales for substitution patterns: N2-substitution, N4-substitution, and benzenoid substitution at positions C5-8 106

(Figure 4.2). Initial studies focused on the amine substitutions as shown in Table 4.1.

Based on Kirby-Bauer assays, zones of inhibitions were determined. S. aureus CBD-635 was struck out onto plates and incubated over night. A single colony was used to inoculate broth and again allowed to grow overnight. The overnight culture was diluted

1:1000 into 5 mL of molten overlay agar and mixed, before being used to inoculate growth plates. After plates were allowed to dry, 3 sterile filter disks were added per plate and then inoculated with 10 µL of test compound. The stock concentration for each test compound was 5 mg mL-1 suspended in DMSO. Bacterial plates were incubated for 24 hours and zones of inhibition (ZOI) were measured in millimeters, with the assay being performed in triplicate.

First, a subset of N2,N4-substituted quinazolines were tested, in which N4 was modified by a furfuryl moiety, while the N2-group was varied (Table 4.1). Quinazolines 4.1 and 4.2 with a N2-methyl or N2-ethyl did not have ZOIs in the Kirby-Bauer assays. Branched alkyl groups on N2 furnished compounds 4.3 and 4.4 displaying inhibition while unbranched polar substituents in 4.5 and 4.6 lacked any activity. Cycloalkyl groups on N2 varied in potency based on size: N2-cyclopropyl and N2-cyclobutyl quinazolines 4.7 and

4.8 were inactive while analogues 4.9 and 4.10 with a N2-cyclopentyl and a N2- cyclohexyl respectively created ZOIs. Next, compounds were tested with a furfuryl group at N2. Quinazoline 4.11 with N4-methyl was active while derivative 4.12 with a polar alcohol functionality remained inactive. Conclusions from this set showed that when a furfuryl was substituted at N4 activity would be seen when a bulky alkyl substituent was at N2 while small substituents were tolerated at N4 with N2 furfuryl.

107

A second subset of quinazolines was prepared to probe whether antibacterial activity can be improved via steric and electronic effects on the quinazoline benzenoid ring (Table

4.2). N4-furfuryl-N2-isopropyl-substituted quinazoline 4.3 was selected as a reference compound, to which analogues 4.13-4.24 were compared with mono-substitution at positions C5-C8 with either a chloro, a methyl or a methoxy group. Quinazolines 4.13-

4.16 with one chloro substitutent suppressed the growth of bacteria. The analogues 4.17-

4.24 substituted with a methyl or a methoxy group were less potent with inhibition of growth observed in 25% and 50% of the samples, respectively. The trend noted for this set are that chloro- substitution is well tolerated while methoxy- is less so and methyl- is even less.

With the idea to reduce the aromaticity, a subset of compounds was designed to test whether N2- and N4-residues rich in sp3-hybridized carbons are tolerated without compromising activity (Table 4.3). Analogues 4.25-4.40 were synthesized in which one of the two N2,N4-substitutents was a methyl, an isopropyl, or a 2-hydroxyethyl group, whereas the other consisted of a branched, an unbranched, or a saturated cycloalkyl moiety. None of these analogues were particularly active with the exceptions of the N2- cyclopentyl-N4-isopropyl derivative 4.32 and N4-cyclohexyl-N2-isopropyl derivative 4.38, which both inhibited bacterial growth.

A final focus was compounds with aromatic substitution (Table 4.4). Analogues 4.41-

4.54, bearing phenyl and/or benzyl derivation at N2 and/or N4 were successful in inhibiting the growth of S. aureus in Kirby-Bauer tests.

108

4.2.2 Minimum Inhibitory Concentration Assays

After the identification of active compounds by Kirby-Bauer testing, the minimum inhibitory concentration (MIC) for these compounds was assessed to provide a more in- depth structure-activity relationship. The minimum inhibitory concentrations (MIC) were determined for compounds which displayed antimicrobial activity against S. aureus

CBD-635. Broth cultures were prepared as described above. These overnight cultures were diluted 1:1000 into fresh media and 200 µL was transferred to a sterile 96-well plate. Relevant concentrations of test compounds were applied to each well and samples were mixed by pipetting before being incubated at 37°C overnight. MICs were determined by visual inspection for the minimum concentration of drug that produced no bacterial growth (as determined by a lack of turbidity). The assay was performed in triplicate to verify MICs.

The N2-isopropyl derivative 4.3 is more active than the N2-tert-butyl 4.4 derivative (Table

4.1). N2-cyclopentyl 4.9 and N2-cyclohexyl 4.10 display similar MICs and are comparable to the isopropyl. N4-isopropyl derivative 4.12 had an MIC ten times lower than the N4-methyl 4.11, with MICs of 35 and 390 , respectively.

109

Table 4.1: SAR of N-alkyl-N-furfurylquinazolin-2,4-diames

Compound Structure ZOI MIC Compound Structure ZOI MIC (mm) ( (mm) (

4.1 none n/d 4.7 none n/d

4.2 none n/d 4.8 none n/d

4.3 10 177 4.9 12 162

4.4 9 337 4.10 10 155

4.5 none n/d 4.11 10 393

4.6 none n/d 4.12 12 35.4

C6-, C7- and C8- chloro substituted 4.13-4.15 had MICs 5-6 times more active than the

5-chloro 4.16 or unsubstituted benzenoid 4.3 (Table 4.2). The 8-methyl 4.17, the 5- methoxy 4.24 and the 6-methoxy 4.23 were less active than the original unsubstituted benzenoid compound.

110

Table 4.2: SAR of benzenoid substitutions

Compound Structure ZOI MIC Compound Structure ZOI MIC (mm) ( (mm) (

4.3 10 177 4.19 none n/d

4.13 10 31.6 4.20 none n/d

4.14 12 31.6 4.21 none n/d

4.15 11 31.6 4.22 none n/d

4.16 9 158 4.23 10 240

4.17 10 337 4.24 9 320

4.18 none n/d

The two lone active non-aromatic 2,4-diaminoquinazolines displayed quite different

MICs. The N2-cyclopentyl-N4-isopropyl 4.32 had an MIC comparable to the N4-furfuryl-

N2-cycloalkyl 4.9 and 4.10 or N4-furfuryl-N2-isopropyl 4.3. Compound 4.38, N4- cyclohexyl-N2-isopropylquinazolin-2,4-diamine with an MIC of 3.5 µM, was the only analogue of the entire series displaying single digit micromolar activity. This lone highly active dialkyl 2,4-diaminoquinazoline makes for an interesting point in the table.

111

Table 4.3: N2- and N4-substitutions rich in sp3 hybridized carbons

ZOI MIC ZOI MIC Compound Structure (mm) ( Compound Structure (mm) (

4.25 none n/d 4.33 none n/d

4.26 none n/d 4.34 none n/d

4.27 none n/d 4.35 none n/d

4.28 none n/d 4.36 none n/d

4.29 none n/d 4.37 none n/d

4.30 none n/d 4.38 13 3.52

4.31 none n/d 4.39 none n/d

4.32 8 185 4.40 none n/d

N2-benzyl substituted 4.41 and 4.42 mimic the N2-furfuryl substituted quinazolines 4.11 and 4.12 (Table 4.4). N4-benzyl 4.47 displays a better activity than N4-furfuryl 4.53, however, with respective MICs of 5.9 and 30 . Phenyl and benzyl substituted 4.48-

4.50 have a wide range of MICs, from 30 to 1300 . N-butyl derivatives of N-benzyl

4.43 and 4.45 and N-phenyl 4.44 and 4.46 have MICs ranging from 6.5 to 160  with

N4-benzyl-N2-butyl 4.45 having an MIC of 6.5 .

112

Table 4.4: N2- and/or N4-benzyl and/or phenyl substitutions

ZOI MIC ZOI MIC Compound Structure (mm) ( Compound Structure (mm) (

4.41 11 189 4.48 10 32.0

4.42 13 34 4.49 10 1300

4.43 12 163 4.50 10 30.6

4.44 12 34.2 4.51 10 28.5

4.45 12 6.53 4.52 10 28.5

4.46 14 34.2 4.53 11 30.3

4.47 13 5.88

4.2.3 Derivatization Based on Initial SAR

The three most active quinazolines found in the SAR were 4.38, 4.45 and 4.47.

Compound 4.38 was the lone highly active quinazoline without an aromatic side chain. It was decided that analogues would not be synthesized based on mutation frequency, data shown later (section 4.2.7). As seen in Table 4.4, all tested compounds with aromatic side chains were active. Table 4.2 allowed for the determination that benzenoid chloro substitution could increase the activity of new derivatives. Thus, analogues were synthesized by combining the benzenoid 7-chloro group with a variety of N2- and N4- benzyl substitutions and also by using para substituted benzyl amines. 113

In comparison to reference compounds 4.45 and 4.47, 7-chloro-quinazolines 4.54 and

4.55 displayed slightly reduced MICs (Table 4.5).

Table 4.5: Combination with 7-chloro or p-substituted benzyl at N2 and/or N4

MIC MIC Compound Structure ( Compound Structure (

4.45 6.53 4.59 5.64

4.47 5.88 4.60 70.5

4.54 5.34 4.61 5.43

4.55 2.93 4.62 5.40

4.56 5.34 4.63 67.5

4.57 5.34 4.64 35.0

4.58 0.61

The para positions of the N2- and N4-benzyl moieties were considered to be ideal sites to introduce chloro, methyl and methoxy groups. Mono N2-4-chlorobenzyl 4.56, N2-4- methylbenzyl 4.59 and N2-4-methoxybenzyl 4.62, as well as the mono N4-4-chlorobenzyl

4.57, were as active as the parent 4.47. The mono N4-4-methylbenzyl 4.60 and N4-4- methoxybenzyl 4.63 were less active than 4.47. The N2,N4-di-(4-methylbenzyl) 4.61 was

114 as active as 4.47, N2,N4-di-(4-methoxybenzyl) 4.64 was less active, and N2,N4-di-(4- chlorobenzyl) 4.58 was nearly ten times more active with an MIC of 0.61 .

The MIC of less than 1 µM for compound 4.58 was exciting and seen as a target to be improved upon. Benzenoid chloro substitution was applied without success in compounds 4.65 and 4.66 (Figure 4.6). A one hundred fold activity decrease from the dichloro 4.58 to the trichloro 4.66 was observed. The N2-(p-methoxybenzyl) 4.62 was also thought to be improved with 7-chloro substituted 4.65, but a four-fold drop in activity was observed. A combination 7-chloro-N4-(p-chlorobenzyl)-N2-(p- methoxybenzyl) 4.67 was tested and displayed better activity than the trichloro compound, but was still not near the original activity that was hoped to be replicated.

Table 4.6: Combination with 7-chloro and/or p-substituted benzyl at N2 and/or N4

MIC MIC Compound Structure ( Compound Structure (

4.47 5.88 4.65 22.2

4.54 5.34 4.66 67.6

4.58 0.61 4.67 51.2

4.62 5.40

115

4.2.4 Determination of Minimum Bactericidal Concentration for Compounds 4.47 and

4.58

An important distinction of antibiotics is whether they are bactericidal or bacteriostatic.

Bactericidal agents kill bacteria while bacteriostatic agents prevent their reproduction.

The minimum bactericidal concentrations (MBC) were determined from MIC samples.

The MIC samples were serial diluted and plated onto TSA plates. The plates were then incubated overnight at 37°C. MBCs were determined by calculating the CFU per mL for each of the concentrations of compounds tested. The data is represented as the percent killing by comparing the drug containing samples to samples that contain no drug.

Quinazoline 4.47 was not found to be bactericidal when tested up to two and a half times the MIC, with a percent recovery over 100% (Figure 4.3). 4.58, on the other hand, was found to be bactericidal with a 95% recovery at four times the MIC and a percent recovery of 1.0% at eight times the MIC. The MBC50 for 4.58 was 3.6 µM and was six times the MIC at 0.61 µM.

180 100 160 80 140 120 60 100 80 40

60 PercentRecovery 20 40 PercentRecovery 20 0 0 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 -20 Concentration of 4.47 (M) Concentration of 4.58 (M)

Figure 4.3: Percent recovery curves of 4.47 and 4.58

116

4.2.5 Determining the Minimum Biofilm Eradication Concentration for Compound

4.47

Quinazoline 4.47 was also tested to determine the minimum biofilm eradication concentration (MBEC). Determining the MBEC demonstrates if a compound is able to target cells forming biofilms, which is a common problem in healthcare settings.

Quinazoline 4.47 was not found to eliminate biofilms when tested up to two and a half times the MIC, with a percent survival over 100% (Figure 4.4).

200 180 160

140 120 100 80

Percent Survival Percent 60 40 20 0 0 2 4 6 8 10 12 14 16 Concentration of 4.47 (M)

Figure 4.4: Percent survival curve for 4.47 against MRSA biofilm

The MBEC is determined based on the recovered CFU per milliliter compared to the control with no added compound. Briefly, S. aureus biofilms were grown in a 96-well plate, at 37°C for 24 hours. After this time, media was aspirated leaving a biofilm that is adherent to the walls of the 96-well plate, and replaced with fresh media containing 0X,

0.2X, 0.5X, 1X, 2.5X, 5X or 10X the MIC of relevant compounds. Samples were incubated at 37°C overnight, before gently being washed twice with sterile PBS, being careful not to disrupt the biofilm. Buffer was then removed by aspirating and biofilms 117 were disrupted by the addition of 200 μL of PBS and pipetting samples repeatedly until all cells were in suspension. This was then serially diluted, and plated for viable colony forming units. MBEC values were determined by comparison to untreated controls.

4.2.6 Cytotoxicity and Hemolysis Assays of Selected Compounds

The cytotoxicity of front runner compounds was determined against adenocarcinomic human alveolar basal epithelial cells (A549 cells, Table 4.7). Front runner compounds

4.38, 4.45 and 4.47 had EC50s of approximately 20 µM against the A549 cells and selectivity indices (EC50(A549)/MIC) between 3.0 and 6.3. The designed compound 4.58 had a selectivity index of 10 with a lower EC50 of 6.1 µM.

Table 4.7: A549 EC50 and selectivity indices of select compounds

Compound EC50 (M) SI (EC50/MIC) 4.38 22.2 ± 4.2 6.3 4.45 19.3 ± 2.6 3.0 4.47 25.6 ± 2.1 4.4 4.58 6.11 ± 1.47 10

As another cytotoxicity test, compound 4.47 was tested in a hemolysis assay to determine the effect of this quinazoline against blood cells at the MIC. An optical density curve at

540 nm shows the free hemoglobin that is released from lysed cells. Over a four hour period there was no significant difference between the standard solution with no compound and the test solution with 4.47 at the minimum inhibition concentration

(Figure 4.5).

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8 540

OD 6 No Drug MIC 4.47 4

0 0 1 2 3 4 Time (hours)

Figure 4.5: OD540 of erythrocytes in the presence or absence of 4.47

4.2.7 Determination of Staphylococcus aureus Resistance to Quinazolines

An important attribute of potential anti-microbials is that spontaneous resistance is not easily obtained. Spontaneous resistance occurs when point mutations results in the ability of bacteria to gain resistance to antibiotics. Thus the spontaneous mutation frequencies for a few of the frontrunner compounds were determined. Spontaneous mutation frequencies were tested for compounds 4.38, 4.45 and 4.47 by plating 1 x 109 cells of S. aureus CBD-635 on agar containing 2.5 X the MIC of each compound. For compound

4.38 a bacterial lawn was obtained, indicating that resistance to this compound was encountered quite readily. Seven spontaneous mutants were found for bis-benzyl- substituted quinazoline 4.47 over ten replicates with a combined inoculum of 2.31 x 1010, yielding a mutation frequency of 3.03 x 10-10. N2-butyl-N4-benzyl 4.45 grew no mutants with a combined inoculum of 2.31 x 1010, resulting in a mutation frequency of less than 1 x 10-11. These results are very promising considering that generally a mutation frequency

119 smaller than 10-8 is desired for the development of anti-bacterial agents. The seven mutants formed using 4.47 were then tested against compounds 4.54-4.64.

4.47 lost all activity against the mutant strains, with MICs greater than 590 µM, compared with 5.9 µM against the original USA 635 (Figure 4.6). 4.54, with an original activity of 5.34 µM, had activity drop to 26 µM for M1 and M2 while it increased to 2.6

µM against M3-7. Activity for 4.55 was found to be 2.93 µM against the mutants and original strain alike. Activity was reduced from 5.34 µM to 26 µM against M1 for 4.56, while the activity against M2-M7 was increased to 2.6 µM. 4.57 saw the activity increase from 5.34 µM to 2.6 µM against all seven mutants. 4.58 was only tested against M1 and

M2, and had activity drop from 0.61 µM to 24 µM. 4.59, with an original activity of 5.64

µM, had activity drop to 28 µM for M1 while it increased to 2.8 µM against M2-7.

Activity was reduced from 5.43 µM to 27 µM against M1 and M2 for 4.61, while the activity against M3-M7 was increased to 2.7 µM. 4.62 had activity jump from 5.40 µM to

27 µM against M1-3 and to 270 µM against M4, while the activity increased to 2.7 µM in

M5-M7. In all mutant strains, 4.64 had a reduction of activity from 35 µM against the original strain. Against strains M5 and M6 the activity was 125 µM and for the rest of the strains it was greater than 250 µM. Compounds 4.60 (original MIC 70.5 ) and 4.63

(original MIC 67.5 ) were found to be more active against the mutants than the original strain. Against M1 and M2 4.60 had MICs of 2.8 µM whereas the MIC for M3-7 was 28 µM. 4.63 had MICs of 2.7 µM in M4 and M6 and 27 µM in the rest of the mutants. This data shows that although mutants are formed, they do not have broad spectrum resistance to the class of quinazolines.

120

600

500 USA 635

M1 400 M2

300 M3 M4

200 M5 Concentration (µM)Concentration M6 100 M7 0 4.47 4.54 4.55 4.56 4.57 4.58 4.59 4.60 4.61 4.62 4.63 4.64

Figure 4.6: MICs of selected compounds of against the seven mutants formed from 4.47

4.2.8 Exploring the Mechanism of Action for 4.45

DNA sequence analysis of the mutants formed from quinazoline analogue 4.45 was employed to determine if a common mechanism of action could be determined from the mechanism of resistance. The Quinolone Resistance-Determining Regions (QRDR) for the genes encoding DNA gyrase (gyrA and gyrB) and topoisomerase IV (grlA and grlB) were sequenced for the mutants and determined to be identical to the parent strain, using the method reported previously by Horri et al.143 As quinazolines have been shown to be inhibitors of dihydrofolate reductase (DHFR), sequencing analysis of the gene coding for this enzyme was also undertaken.144 Again, the mutant strain was found to be identical to the parent strain. These results allow for the conclusion that the mechanism of resistance for the few mutant strains was not via a change in primary enzyme structure.

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4.2.9 Galleria mellonella Larval Model

Galleria mellonella larvae, also known as the wax moth, were used as an initial in vivo model to determine efficacy of front runner compounds 4.38, 4.45, 4.47 and 4.58 (Figure

4.6). Uninfected larvae were injected with DMSO to determine the effect of the injection solvent. Larvae were infected by injection with between 1.78 x 109 and 4.63 x 109 colony forming units (CFUs) of S. aureus USA 300 FPR strain. One hour post-infection 5x or

10x the MIC for each compound was injected, or the larvae were left untreated.

30% of uninfected larvae died within twenty-four hours post DMSO injection while the remaining 70% lived to day three (p = 0.057), p values were calculated using Graph pad.

Mortality was measured using a log rank and chi-square test with 1 degree of freedom.

The significance level was set at 0.05. All untreated infected larvae were dead within forty-eight hours following infection. Compound 4.38 was tested at two inoculations,

2.89 x 109 and 4.63 x 109 CFUs (Figure 4.7). Within the first twenty-four hours, 50% and

20% of larvae died after doses of 4.38 of 5x and 10x the MIC, respectively, with 40% from each surviving after day two from 2.89 x 109 CFUs. For 4.63 x 109 CFUs, 40% died within twenty-four hours with 10% additional dying every twenty-four hours until day three at a dose of 5x the MIC (p = 0.0526). At 10x the MIC, 30% died within twenty-four hours and the remaining 70% survived until day three (p = 0.157).

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10 9

7 Untreated 6 DMSO only 5 5 x 4.38 low 4 10 x 4.38 low

3 5 x 4.38 high Number of living ofliving Numberlarvae 2 10 x 4.38 high 1 0 Day 0 Day 1 Day 2 Day 3

Figure 4.7: Wax worm larvae survival post-infection for 4.38 with low and high inoculation volumes. Low and high refer to the inoculation volumes discussed earlier in

the text. * = p < 0.05

Quinazoline 4.45 was tested at two inoculations, 2.89 x 109 and 4.63 x 109 CFUs (Figure

4.8). In the 5x MIC test at 2.89 x 109 CFUs, 50% of larvae died within twenty-four hours and 40% survived past day two (p = 0.156). 30% of larvae died within twenty-four hours and the rest survived until day three in the 10x MIC test (p = 0.0268). For 4.63 x 109

CFUs, 80% died within twenty-four hours with 10% additional dying before day three at

5x the MIC (p = 0.156). At 10x the MIC, 40% died the first day, 10% the second and

30% the third (p = 0.0268).

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10 9

7 Untreated 6 DMSO only 5 5 x 4.45 low 4 10 x 4.45 low*

3 5 x 4.45 high Number of living ofliving Numberlarvae 2 10 x 4.45 high* 1 0 Day 0 Day 1 Day 2 Day 3

Figure 4.8: Wax worm larvae survival post-infection for 4.45 with low and high inoculation volumes. Low and high refer to the inoculation volumes discussed earlier in

the text. * = p < 0.05

Compound 4.47 was tested at 1.78 x 109 CFUs (Figure 4.9). In the 5x MIC test, 10% of larva died within twenty-four hours and an additional 40% died in the next twenty-four hours, leaving 50% alive at day three (p = 0.0035). In contrast, 30% of larvae died within twenty-four hours and an additional 40% died in the next twenty-four hours, leaving 30% alive at day three in the 10x MIC test (p = 0.0573).

Compound 4.58 was tested at 2.89 x 109 CFUs (Figure 4.9). In the 5x MIC assay, 50% of larvae died within twenty-four hours and the remaining 50% survived until day three (p =

0.0779). 30% of larvae died within twenty-four hours and an additional 10% died in the next twenty-four hours, leaving 60% alive at day three in the 10x MIC test (p = 0.0147).

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10 9 8 7 Untreated 6 DMSO only 5 5 x 4.47* 4 10 x 4.47* 3 5 x 4.58 2 10 x 4.58* 1 0 Day 0 Day 1 Day 2 Day 3

Figure 4.9: Wax worm larvae survival post-infection for 4.47 and 4.58. * = p < 0.05

4.2.10 In vivo Efficacy Using a Murine Model of Lethal Peritonitis

The efficacy of compounds 4.45 and 4.47 were determined in a murine model of lethal S. aureus peritonitis (Figure 4.10). Groups of six female 6 week old CD1 mice were injected intraperitoneally with 1 x 108 CFUs of S. aureus USA 300. After one hour the groups of mice were given intravenous injections with either 45% w/v (2- hydroxypropyl)--cyclodextrin (HPCD) in water, 5 mg/kg or 10 mg/kg of vancomycin,

5x the MIC of 4.45 in 45% w/v HPCD in water and 1x, 5x, or 10x the MIC of 4.47 in

45% w/v HPCD in water. At 5 mg of vancomycin, three mice died after the first day and the rest survived to the end of the week (p = 0.264). At 10 mg of vancomycin, one mouse died after the first day and the rest survived to the end of the week (p = 0.027). For compound 4.45, one mouse died immediately following the 5x MIC injection and the other five survived to the end of the test at day 5 (p = 0.352). For compound 4.47, all six mice survived 5 days at 1x the MIC (p = 0.0058) while one mouse died immediately

125 following the 5x injection (p = 0.0589) and five died immediately following the 10x injection. With injection of 45% w/v HPCD in water all six mice were dead by day three (p = 0.53).

5 mg/kg Vancomycin 4 10 mg/kg Vancomycin* 3 HPbCD 5x 4.45* 2

1x 4.47* Number of Living ofLiving NumberMice 1 5x 4.47 10x 4.47 0 Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Time (days)

Figure 4.10: Mice survival post-infection. * = p < 0.05

4.2.11 Conclusions

A total of 67 N2,N4-disubstituted quinazoline-2,4-diamines, many displaying anti- bacterial activities, have been synthesized systematically by varying the substitutions in the 2-, 4-, 5-, 6-, 7-, and/or 8-positions. The most potent in vitro activities with MICs in the single digit to sub-micromolar range against S. aureus were obtained with quinazoline-2,4-diamines 4.38, 4.45, 4.47, 4.54-4.59 and 4.61-4.62 bearing a N4- cyclohexyl-N2-isopropyl or N4-benzyl and either N2-benzyl or N2-n-butyl substituent combination. Furthermore, the benzenoid ring of the quinazoline-2,4-diamine scaffold has been identified to play a secondary role for efficacy, with quinazolines substituted at the 6-, 7- or 8-position with one chloro group possessing lower MICs against S. aureus.

126

These promising results led to efficacy testing of the lead compounds 4.38, 4.45, 4.47 and

4.58 in an in vivo wax moth larva S. aureus model and compounds 4.45 and 4.47 in an in vivo murine S. aureus model.

All compounds fared better in the wax moth larva study than the untreated versions. On average, out of ten, 3.83 larvae treated with 5x the MIC survived to the end of the study while 4.83 treated with 10x the MIC survived. This results in a 55% and 69% respective survival rate when compared to the DMSO standard.

In the murine model, all treated mice also fared better than the untreated versions. 1x the

MIC for compound 4.47 gave the best results with all mice surviving to the end of the one week study. 5x the MIC for both compounds 4.45 and 4.47 had five of six mice survive to the end of the week, giving the next best results and the same as 10 mg/kg of vancomycin. 10x the MIC for compound 4.47 resulted in the immediate death of five of the six mice. Additional experiments are required to determine whether these deaths have been caused due to toxicity or limited solubility during administration of compounds 4.45 and 4.47.

The observed potencies of frontrunner compounds 4.38, 4.45, 4.47 and 4.58 in in vivo assays make N2,N4-disubstituted quinazoline-2,4-diamines a suitable platform for the future development of anti-bacterial agents, with a strong focus being on methicillin- resistant Staphylococcus aureus.

4.3 Screening of Quinazolines Against ESKAPE Pathogens

With the activity of the quinazoline class of compounds against MRSA, we decided to test the library of quinazolines against the rest of the ESKAPE pathogens (Enterococcus 127 faecium, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and

Enterobacter species) in a high-throughput screen. Testing of 67 quinazolines was performed with the remaining pathogens (e.g. not MRSA) using MIC assays as described above. We determined that compound 4.41 from this class was active against A. baumannii with an MIC of 18.9 µM. The rest of the compounds had MICs >56 µM and all were inactive to the rest of the ESKAPE pathogens.

4.3.1 Structure-Activity Relationship of Quinazolin-2,4-diamines by Minimum

Inhibitory Concentration

As compound 4.41 was the only active compound, derivatives of this quinazoline were tested. N4-isopropyl derivative 4.42 and the N4-n-butyl 4.43 were inactive, showing that small alkyl substituents other than methyl are not active. Thus, a variety of N-benzyl-N- methylquinazolin-2,4-diamines were synthesized and tested (Table 4.8).

Table 4.8: MICs of N-benzyl-N-methylquinazolin-2,4-diamines against A. baumannii 1403 strain

MIC MIC MIC Structure Structure Structure (M (M (M

18.9 33.5 3.40 4.41 4.69 4.71

18.9 1.67 4.68 4.70

The N4-benzyl-N2-methyl derivative 4.68 displayed the same activity as 4.41, showing that N-benzyl-N-methyl derivation leads to activity against A. baumannii. The N2-n-butyl

4.45 was also inactive in the original assay. Backbone substituted derivatives of 4.41, however, were found to be active. 7-chloro derivative 4.69 had activity reduced nearly

128 two-fold, while 6-derivation to methoxy 4.71 and chloro 4.70 increased the activity greater than five-and-a-half- and eleven-fold, respectively.

4.3.2 Extended Minimum Inhibitory Concentration Testing

After the initial tests against A. baumanni strain 1403, the active compounds 4.41 and

4.68-4.71 and some similar analogues (4.1, 4.11, 4.14, 4.15 and 4.43) were tested for

MICs against other drug resistant strains of A. baumannii to determine if activity was shown across a wide range of isolates (Table 4.9). A. baumanni strains A-L were provided courtesy of Paul Dunman from the University of Rochester Medical Center. The five original hits 4.41 and 4.68-4.71 were active against the majority of the additional strains. The 6- and 7-substituted derivatives 4.69-4.71 were active against all thirteen strains with MICs below 34 µM. Compound 4.71 displayed the best across the board activity with MICs of 3.4 µM for all strains except against K and L, with MICs of 17 µM.

Three of the compounds (4.11, 4.42 and 4.43) that were inactive against the 1403 strain were active against at least one of the other twelve strains. Compound 4.11 was active against the A and B strains with MICs of 39 µM, 4.42 was active against the B strain with an MIC of 34 µM and 4.43 was active against A, B and D strains with MICs of 3.3

µM, 3.3 µM and 33 µM, respectively.

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Table 4.9: MICs of active compounds and similar compounds against A. baumanni 1403 and A-L strains MIC (M

Structure 1403 A B C D E F G H I J K L

>98 >98 >98 >98 >98 >98 >98 >98 >98 >98 >98 >98 >98 4.1

>98 39 39 >98 >98 >98 >98 >98 >98 >98 >98 >98 >98 4.11

>79 >79 >79 >79 >79 >79 >79 >79 >79 >79 >79 >79 >79 4.14

>79 >79 >79 >79 >79 >79 >79 >79 >79 >79 >79 >79 >79 4.15

18.9 19 38 3.8 38 38 38 38 38 3.8 95 >95 >95 4.41

>86 >86 34 >86 >86 >86 >86 >86 >86 >86 >86 >86 >86 4.42

>82 3.3 3.3 >82 33 >82 >82 >82 >82 >82 >82 >82 >82 4.43

18.9 38 3.8 3.8 95 >95 95 95 38 95 >95 95 95 4.68

33.5 6.7 6.7 6.7 3.4 6.7 6.7 5.0 30 6.7 6.7 6.7 3.4 4.69

1.67 17 17 8.4 0.3 8.4 8.4 3.3 17 8.4 17 34 8.4 4.70

3.40 3.4 3.4 3.4 3.4 3.4 3.4 3.4 3.4 3.4 3.4 17 17 4.71

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4.3.3 Minimum Bactericidal Concentrations Activity of Frontrunner Quinazolin-2,4-

Diamines Against Acinetobacter baumannii

MBC testing of 4.41, 4.42, 4.68, 4.70 and 4.71 was performed to determine whether the compounds were bactericidal or bacteriostatic against A. baumannii 1403 (Figure 4.11).

All tested compounds reduced the percent recovery of cells as the concentration increased and were thus found to be bactericidal, including compound 4.42 which was completely inactive against A. baumannii 1403 when the MIC was tested.

120 100

100 80

80 60 4.41 60 40 4.42 40 4.68 PercentRecovery 20 PercentRecovery 4.71 20 0 0 0 50 100 150 200 0 2 4 6 8 10 -20 Concentration of 4.70 (M) Concentration (M)

Figure 4.11: Percent recovery curves for A. baumannii 1403 incubated with quinazolines

For 4.41, we observed 0.06% recovery of cells at the MIC of 18.9 µM with an MBC50 value of 3.6 µM and a MBC90 value of 4.3 µM. For 4.42, we observed <6% recovery of cells at the MIC of >86 µM with an MBC50 value of 2.5 µM and a MBC90 value of 40

µM. For 4.68, we observed 12.67% recovery of cells at the MIC of 18.9 µM with an

MBC50 value of 2.2 µM and a MBC90 value of 5.0 µM. For 4.70, we observed 30% recovery of cells at the MIC of 1.57 µM with an MBC50 value of 0.85 µM and a MBC90 value of 5.9 µM. Finally, for 4.71, we observed 6.8% recovery of cells at the MIC of 3.40

µM and an MBC50 value of 1.8 µM and a MBC90 value of 9.1 µM. These demonstrate

131 that all of the tested N-benzyl-N-methyl-2,4-diaminoquinazolines are broadly bactericidal in action towards A. baumannii with the MBC50 values for all compounds less than their

MIC.

4.3.4 Determining the Minimum Biofilm Eradication Concentration for 4.41 and 4.68-

4.71

As biofilm formation by A. baumannii is a factor that increases pathogenicity and innate antibiotic resistance, the active quinazolines 4.41 and 4.68-4.71 were tested for their capacity to destroy biofilms. Briefly, A. baumannii biofilms were grown in a 96-well plate, at 37°C for 24 hours. After this time, media was aspirated leaving a biofilm that is adherent to the walls of the 96-well plate, and replaced with fresh media containing 0X,

0.2X, 0.5X, 1X, 2.5X, 5X or 10X the MIC of relevant compounds. Samples were incubated at 37°C overnight, before gently being washed twice with sterile PBS, being careful not to disrupt the biofilm. Buffer was then removed by aspirating and biofilms were disrupted by the addition of 200 μL of PBS and pipetting samples repeatedly until all cells were in suspension. This was then serially diluted, and plated for viable colony forming units. MBEC values were determined by comparison to untreated controls.

Compounds 4.41, 4.68, 4.69 and 4.71 were completely ineffective against biofilms at all tested concentrations, with greater than 100% recovery of cells at all tested concentrations. Quinazoline 4.70, however, showed the ability to eradicate cells within biofilms. At the MIC of 1.67 µM, 46.2% of the cells from the original culture were recovered while 38.5% of the cells were recovered at the lowest tested concentration

(0.2X the MIC) of 0.33 µM and 15.4% recovery at 8.4 µM (5X the MIC). The MBEC50

132 of 0.3 µM and MBEC90 of 3.0 µM show this compound to have anti-biofilm activity at activities comparable to the MIC.

100

80 70 60 50 40 30

Percent RecoveryPercentof Cells 20 10 0 0 2 4 6 8 10 Concentration (M)

Figure 4.12: Percent recovery curve for A. baumannii biofilm grown in the presence of

4.70

4.3.5 Cytotoxicity of 4.70

The cytoxicity of 4.70 was determined against A549 cells and was found to be 16.7 ± 6.0

µM. This gave a selectivity index of 10 when compared to the MIC, showing that this compound may have therapeutic use in vivo.

4.3.6 In vivo Model of Infection Using Galleria mellonella

The G. mellonella model was used as an initial in vivo study to test the efficacy of frontrunner quinazolines against A. baumannii. Ten larvae per compound were infected with 1.0 x 107 A. baumannii. One hour post-infection, 5x the MIC of each compound or the DMSO vehicle was administered to the worms. An additional group of uninfected larvae was tested for survivability with DMSO. All infected worms subjected to 133 treatment with DMSO were dead within two days, while 80% of the uninfected worms survived to day 3 (p = 0.0012, Figure 4.10).

Compound 4.41 had 50% of worms survive past the first day (p = 0.0056), while compound 4.69 had 40% survive (p = 0.214). Compounds 4.68, 4.70 and 4.71 resulted in

70% surviving until day three, with compound 4.68 having 10% die after the first day and

20% after the second (p = 0.0003), while 4.70 (p = 0.0061) and 4.71 (p = 0.002) had all

30% die after the first day.

8 DMSO (uninfected)* 7 6 DMSO (infected) 5 4.41* 4 4.68* 3 4.69

2 Number of Living ofLiving NumberWorms 1 4.70* 0 4.71* 0 1 2 3 Day

Figure 4.13: Wax worm larvae survival post-infection with 5x MIC. * = p < 0.05

A murine model of infection is currently being studied to determine the efficacy of these compounds in mammalian systems.

4.3.7 Conclusions

Testing of the 67 quinazolines in an in vitro model of A. baumannii infection revealed one compound 4.41 that displayed activity, a surprisingly low number when compared to the activity of these agents with MRSA. Screening of analogues of 4.41 revealed five

134 quinazolines that were broadly effective against thirteen strains of A. baumannii, with compound 4.71 displaying activity most consistently. While the other four compounds were inactive, 4.70 was also found to eradicate biofilms, showing the ability to remove cells that are generally impervious to antibiotics. The in vivo G. mellonella model of infection produced results that suggested good in vivo activity for these compounds.

Compounds 4.68, 4.70 and 4.71 had 70% of the wax worms survive, which corresponds to an 87.5% cure rate when compared to the DMSO model. In summary, the N-benzyl-N- methylquinazolin-2,4-diamines display excellent in vitro and in vivo efficacy for A. baumannii treatment as bactericidal agents. This data strongly suggests that these compounds should be studied further as anti-bacterials.

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Chapter 5: Future Plans

5.1 1,4-Dihydropyridines

5.1.1 Synthesis Going Forward

Analysis of the structure-activity relationship revealed logical compounds to be synthesized for the further optimization of activity. At the 4- and 7- positions, the use of unsubstituted phenyl derivatives will allow for the determination of whether or not substitution is necessary. Derivatives with a 7-O that were highly active, such as 2.31,

2.32, 2.52 and 2.53, will be synthesized as well to see if a corresponding increase in activity is encountered with the 7-CH2 derivative.

SAR of the 3-position revealed that a smaller alkyl chain has the best activity. Since an ethyl group was the smallest derivative formed, two new sets will be tested, one with a methyl ester and the second with the free acid to determine if a smaller ester or the free acid can be as active as the ethyl ester.

5.1.2 Property-Based Synthesis

Determination of the solubility properties of the pure stereoisomers of 2.1 showed that the compounds have low aqueous solubilities. A higher aqueous solubility is desirable for potential therapeutic use and oral bioavailability. As discussed previously, the free acid at the 3-position will be synthesized. If this molecule retains in vitro activity it should have an increased solubility compared to an ester derivative.

136

The 2-position appears to be an excellent place to attempt modifications based on properties as derivatives with larger groups have been more active than a simple methyl group. Determination of the solubilities of oxygen containing derivatives 2.53 and 2.54 may reveal compounds that already have desirable solubilities in place. The solubilites of compounds 2.1 and 2.2 will also be compared to determine how much of an effect on solubility the 6-O has on the molecule. As well as these compounds, derivatives can be synthesized with functional groups that are well known for increasing solubility such as morpholine. Synthesis of these derivatives can be undertaken from the 1,4- dihydrohypyridine by halogenation at the 2-position followed by substitution with a proper group (Figure 5.1). Derivatives will also be tested baring heteroaryl functionality at the 4- and 7- positions to see the effects of different aromatic systems, such as pyridyl.

Figure 5.1: Derivation at the 2-position

5.1.3 Additional Testing

Additional property testing should be performed to determine the potential for these compounds to be successful therapeutic treatments. Pharmacokinetics (PK) and pharmacodynamics (PD) are important to study because they are used to decipher what the body does to a compound (PK) and what a compound does to the body (PD).

137

Common PK tests are the ADME properties: absorption, distribution, metabolism and excretion. These properties are used to determine how well a compound acts in the body and are an excellent description of the potential a compound has for in vivo testing.

Microsomal testing should be performed to see how the compounds can be metabolized in the body. Identifying sites of oxidation or bond cleavage will allow for modifications to the structure that will provide for a longer active effect for the compound in the body, preventing the excretion of a metabolized compound from a body. Oftentimes aryl rings can be oxidized to phenols by an enzyme and the specific sites can be blocked by replacing a hydrogen atom with a fluorine with minimal change in activity. Esters and ethers can often times be cleaved, and if this can be identified early on the functional groups can be changed, such as replacing the methoxy groups on the 7-phenyl.

Identification of more highly soluble compounds and compounds displaying good PK properties will lead to compounds that will be valuable as in vivo leads. A murine model of infection for P. falciparum has been found to be an effective test for initial studies. If this test leads to good results then further in vivo tests will be performed in a primate model to determine whether the 1,4-dihydropyridines will make a good platform for clinical studies.

5.1.4 Stereoselective Syntheses

The stereoselective synthesis of the 1,4-dihydropyridines can be studied as well. The trans isomer is generally formed in a slight excess due to steric hindrance between the 4- and 7- positions, and varying substituents can be tested to see their effect on the ratio.

138

Bulkier substituents would reveal whether it is strictly a steric observation while using strongly electronegative atoms would reveal whether an electronic repulsion is observed.

A revisit of a few stereoselective processes will also occur. Chiral lactone synthesis will be attempted again, with the replacement of the benzyl proton with a methyl group with the hopes of isolating enantiopure compounds. The α-β unsaturated lactone may also try to be resynthesized, as there was a noted difference in ee when pyridine was used as the base instead of triethylamine. It may be that a suitable base can be found to increase the ee into the upper ninety percent.

Enantioselective syntheses of the 1,4-dihydropyridine is another method that will be reevaluated. A chiral hydrazine could be used to set the stereochemistry via the nitrogen of the pyridine ring. As Enders et al. performed stereoselective syntheses with one stereocenter, their method may be applied to a series with a second stereocenter.87

Several conditions will be used to see the effects that solvent and temperature have on the reaction.

5.2 Quinazolines as Anti-Leishmanials

As limited success has been found with the quinazolin-2,4-diamines in in vivo murine models, studies need to be conducted on the properties of these compounds to determine if better efficacy can be attained by the synthesis of compounds with more favorable properties. The microsomal stability should be studied especially since the furan substituent is a moiety that is well known at being metabolized. Isosteres may need to be synthesized that keep the biological activity but prevent degradation and increase

139 solubility as the active compounds without a furan are generally more hydrophobic with less favorable aqueous solubility.

5.3 Quinazolines as Antibacterials

As a large number of compounds have been tested, a qualitative structure-activity relationship (QSAR) study may be appropriate to have a computational method to show compounds that would have better in vitro activity. QSAR may be able to explain the lack of highly active compounds bearing dialkyl amine substitutions and shed light on the more active aryl substituted compounds.

The most active 2,4-diaminoquinazolines in the MRSA assay bore benzyl substituted amines. These compounds are highly lipophilic and do not have a high aqueous solubility. Increasing the aqueous solubility should be a priority; derivatives can be made with more soluble substituents such as hydroxyl or carboxyl substituted benzyl groups.

Pyridyl substitution may also serve to increase the aqueous solubility.

In vivo testing of the lead quinazolines revealed that protection was provided in both a murine model of infection and a wax worm model of infection. The murine model followed the test subjects for one week, while the wax worm model observed for three days. An extended study may provide more data via the observation of whether the protection will last for a longer period of time. The addition of test compound may be changed to a time further out than one hour post infection, allowing for the infection to proceed throughout the animal prior to therapy.

As compound 4.58 was found to be bactericidal at eight times the MIC and was also the most active quinazoline in vitro, a murine model with this compound should be 140 undertaken to determine the efficacy of this compound. Since it is bactericidal, it may provide more protection than the previously tested quinazolines in vivo.

The only active compounds in the A. baumannii test bore N-benzyl and N-methyl substitution. The solubility of these compounds are higher than that of the dibenzyl derivatives in the MRSA study, but there is room for improvement. Aqueous solubility could be improved via functionality on the benzyl substituent, with testing of the in vitro properties showing whether the analogues retain activity.

The bactericidal and anti-biofilm activities of the active quinazolin-2,4-diamines reveal a strong potential for these compounds. A murine model of infection will provide data as to whether these compounds have potential efficacy in mammals. To go along with these in vivo tests, PK tests and microsomal stability can help to determine how effective they can be in vivo. Compound 4.41 (3.15) was tested previously in a murine model of infection and found to be inactive against L. donovani. The PK and microsomal stability tests can determine whether the lack of activity is due to the body rendering the compounds inactive.

141

References Cited

1. Campbell, N. A.; Reece, J. B., Biology, Sixth Edition. Benjamin Cummings: San Francisco, CA, 2002.

2. Garnham, P. C., The genus Leishmania. Bulletin of the World Health Organization 1971, 44 (4), 477-489.

3. Ménard, R., Gliding motility and cell invasion by Apicomplexa: insights from the Plasmodium sporozoite. Cellular Microbiology 2001, 3 (2), 63-73.

4. (a) Malaria. World Health Organization www.who.int/tropics/malaria/, accessed 13 May 2013; (b) Malaria. Center for Disease Control and Prevention www.cdc.gov/malaria/, accessed 13 May 2013.

5. Nadjm, B.; Behrens, R. H., Malaria: An Update for Physicians. Infectious Disease Clinics of North America 2012, 26 (2), 243-259.

6. World Malaria Report. World Health Organization http://www.who.int/malaria/publications/world_malaria_report_2012/en/index.ht ml, accessed 13 May 2013.

7. Miller, L. H.; Baruch, D. I.; Marsh, K.; Doumbo, O. K., The pathogenic basis of malaria. Nature 2002, 415 (6872), 673-679.

8. Achan, J.; Talisuna, A. O.; Erhart, A.; Yeka, A.; Tibenderana, J. K.; Baliraine, F. N.; Rosenthal, P. J.; D’Alessandro, U., Quinine, an old anti-malarial drug in a modern world: role in the treatment of malaria. Malar J 2011, 10 (1), 1-12.

9. (a) Sullivan, D. J.; Matile, H.; Ridley, R. G.; Goldberg, D. E., A Common Mechanism for Blockade of Heme Polymerization by Antimalarial Quinolines. Journal of Biological Chemistry 1998, 273 (47), 31103-31107; (b) Sullivan, D. J.; Gluzman, I. Y.; Russell, D. G.; Goldberg, D. E., On the molecular mechanism of chloroquine's antimalarial action. Proceedings of the National Academy of Sciences 1996, 93 (21), 11865-11870.

142

10. (a) Wellems, T. E.; Plowe, C. V., Chloroquine-Resistant Malaria. Journal of Infectious Diseases 2001, 184 (6), 770-776; (b) Payne, D., Spread of chloroquine resistance in Plasmodium falciparum. Parasitology Today 1987, 3 (8), 241-246; (c) Fidock, D. A.; Nomura, T.; Talley, A. K.; Cooper, R. A.; Dzekunov, S. M.; Ferdig, M. T.; Ursos, L. M. B.; bir Singh Sidhu, A.; Naudé, B.; Deitsch, K. W.; Su, X.-z.; Wootton, J. C.; Roepe, P. D.; Wellems, T. E., Mutations in the P. falciparum Digestive Vacuole Transmembrane Protein PfCRT and Evidence for Their Role in Chloroquine Resistance. Molecular Cell 2000, 6 (4), 861-871; (d) Sidhu, A. B. S.; Verdier-Pinard, D.; Fidock, D. A., Chloroquine Resistance in Plasmodium falciparum Malaria Parasites Conferred by pfcrt Mutations. Science 2002, 298 (5591), 210-213.

11. (a) Olliaro, P., Mode of action and mechanisms of resistance for antimalarial drugs. Pharmacology & Therapeutics 2001, 89 (2), 207-219; (b) Sibley, C. H.; Hyde, J. E.; Sims, P. F. G.; Plowe, C. V.; Kublin, J. G.; Mberu, E. K.; Cowman, A. F.; Winstanley, P. A.; Watkins, W. M.; Nzila, A. M., Pyrimethamine– sulfadoxine resistance in Plasmodium falciparum: what next? Trends in Parasitology 2001, 17 (12), 582-588; (c) Krostad, D. J., Malaria as a Reemerging Disease. Epidemiologic Reviews 1996, 18 (1), 77-89.

12. Kitchen, L. W.; Vaughn, D. W.; Skillman, D. R., Role of US Military Research Programs in the Development of US Food and Drug Administration–Approved Antimalarial Drugs. Clinical infectious diseases 2006, 43 (1), 67-71.

13. Dassonville-Klimpt, A.; Jonet, A.; Pillon, M.; Mullié, C.; Sonnet, P., Mefloquine derivatives : synthesis, mechanisms of action, antimicrobial activities. In Science against Microbial Pathogens: Communicating Current Research and Technological Advances, Mendez-Vilas, A., Ed. Formatex Research Center: 2011; Vol. 3.

14. Baggish, A. L.; Hill, D. R., Antiparasitic Agent Atovaquone. Antimicrobial Agents and Chemotherapy 2002, 46 (5), 1163-1173.

15. Vaidya, A. B.; Mather, M. W., Atovaquone resistance in malaria parasites. Drug Resistance Updates 2000, 3 (5), 283-287.

16. (a) Tu, Y., The discovery of artemisinin (qinghaosu) and gifts from Chinese medicine. Nature Medicine 2011, 17 (10), 1217-1220; (b) Klayman, D., Qinghaosu (artemisinin): an antimalarial drug from China. Science 1985, 228 (4703), 1049-1055.

143

17. Mutabingwa, T. K., Artemisinin-based combination therapies (ACTs): Best hope for malaria treatment but inaccessible to the needy! Acta Tropica 2005, 95 (3), 305-315.

18. Noedl, H., Combination Therapy in Light of Artemisinin Resistance. In Treatment and Prevention of Malaria, Staines, H. M.; Krishna, S., Eds. Springer Basel: 2012.

19. Noedl, H.; Se, Y.; Schaecher, K.; Smith, B. L.; Socheat, D.; Fukuda, M. M., Evidence of Artemisinin-Resistant Malaria in Western Cambodia. New England Journal of Medicine 2008, 359 (24), 2619-2620.

20. Baird, J. K.; Hoffman, S. L., Primaquine Therapy for Malaria. Clinical infectious diseases 2004, 39 (9), 1336-1345.

21. Pearson, R. D.; Sousa, A. Q., Clinical spectrum of Leishmaniasis. Clinical Infectious Diseases 1996, 22 (1), 1-13.

22. Desjeux, P., Leishmaniasis. Public health aspects and control. Clinics in dermatology 1996, 14 (5), 417-423.

23. Chappuis, F.; Sundar, S.; Hailu, A.; Ghalib, H.; Rijal, S.; Peeling, R. W.; Alvar, J.; Boelaert, M., Visceral leishmaniasis: what are the needs for diagnosis, treatment and control? Nature Reviews Microbiology 2007, 5 (11, Suppl.), S7- S16.

24. (a) Carroll, W. A.; Agrios, K. A.; Altenbach, R. J.; Buckner, S. A.; Chen, Y.; Coghlan, M. J.; Daza, A. V.; Drizin, I.; Gopalakrishnan, M.; Henry, R. F.; Kort, M. E.; Kym, P. R.; Milicic, I.; Smith, J. C.; Tang, R.; Turner, S. C.; Whiteaker, K. L.; Zhang, H.; Sullivan, J. P., Synthesis and Structure-Activity Relationships of a Novel Series of Tricyclic Dihydropyridine-Based KATP Openers That Potently Inhibit Bladder Contractions in Vitro. Journal of Medicinal Chemistry 2004, 47, 3180-3192; (b) Leishmaniasis. World Health Organization http://www.who.int/leishmaniasis/en/ accessed 7 October 2009.

25. (a) Rey, L., Gaspar Vianna and the discovery of the treatment of leishmaniasis by antimonials. Revista do Instituto de Medicina Tropical de São Paulo 1962, 4, 47- 52; (b) Miranda, E. S.; Miekeley, N.; De-Carvalho, R. R.; Paumgartten, F. J. R., Developmental toxicity of meglumine antimoniate and transplacental transfer of antimony in the rat. Reproductive Toxicology 2006, 21 (3), 292-300. 144

26. (a) Maltezou, H. C., Drug resistance in visceral leishmaniasis. Journal of Biomedicine & Biotechnology 2010, 2010, Article ID 617521; (b) Frezard, F.; Martins, P. S.; Barbosa, M. C. M.; Pimenta, A. M. C.; Ferreira, W. A.; de Melo, J. E.; Mangrum, J. B.; Demicheli, C., New insights into the chemical structure and composition of the pentavalent antimonial drugs, meglumine antimonate and sodium stibogluconate. Journal of Inorganic Biochemistry 2008, 102 (4), 656- 665.

27. Sundar, S.; More, D. K.; Singh, M. K.; Singh, V. P.; Sharma, S.; Makharia, A.; Kumar, P. C.; Murray, H. W., Failure of pentavalent antimony in visceral leishmaniasis in India: report from the center of the Indian epidemic. Clinical infectious diseases 2000, 31 (4), 1104-1107.

28. Guerin, P. J.; Olliaro, P.; Sundar, S.; Boelaert, M.; Croft, S., L.; Desjeux, P.; Wasunna, M., K.; Bryceson, A., D. M., Visceral leishmaniasis: current status of control, diagnosis, and treatment, and a proposed research and development agenda. Lancet Infectectios Diseases 2002, 2 (8), 494-501.

29. Perez-Victoria, F. J.; Sanchez-Canete, M. P.; Seifert, K.; Croft, S. L.; Sundar, S.; Castanys, S.; Gamarro, F., Mechanisms of experimental resistance of Leishmania to miltefosine: implications for clinical use. Drug Resistance Updates 2006, 9 (1- 2), 26-39.

30. Sundar, S.; Singh, A.; Rai, M.; Prajapati, V. K.; Singh, A. K.; Ostyn, B.; Boelaert, M.; Dujardin, J.-C.; Chakravarty, J., Efficacy of Miltefosine in the Treatment of Visceral Leishmaniasis in India After a Decade of Use. Clinical infectious diseases 2012, 55, 543-550.

31. (a) Boucher, H. W.; Talbot, G. H.; Bradley, J. S.; Edwards, J. E.; Gilbert, D.; Rice, L. B.; Scheld, M.; Spellberg, B.; Bartlett, J., Bad Bugs, No Drugs: No ESKAPE! An Update from the Infectious Diseases Society of America. Clinical infectious diseases 2009, 48 (1), 1-12; (b) Pendleton, J. N.; Gorman, S. P.; Gilmore, B. F., Clinical relevance of the ESKAPE pathogens. Expert Review of Anti-infective Therapy 2013, 11 (3), 297-308.

32. Foster, T., Staphylococcus. In Medical Microbiology, 4th Edition, Baron, S., Ed. University of Texas Medical Branch at Galveston: Galveston, TX, 1996.

145

33. (a) Williams, R. E. O., Healthy Carriage of Staphylococcus aureus: Its Prevalence and Importance. Bacteriological Reviews 1963, 27 (1), 56-71; (b) Kluytmans, J.; van Belkum, A.; Verbrugh, H., Nasal carriage of Staphylococcus aureus: epidemiology, underlying mechanisms, and associated risks. Clinical Microbiology Reviews 1997, 10 (3), 505-520.

34. Gorwitz, R. J.; Kruszon-Moran, D.; McAllister, S. K.; McQuillan, G.; McDougal, L. K.; Fosheim, G. E.; Jensen, B. J.; Killgore, G.; Tenover, F. C.; Kuehnert, M. J., Changes in the Prevalence of Nasal Colonization with Staphylococcus aureus in the United States, 2001–2004. Journal of Infectious Diseases 2008, 197 (9), 1226- 1234.

35. (a) Gonzalez, B. E.; Hulten, K. G.; Dishop, M. K.; Lamberth, L. B.; Hammerman, W. A.; Mason, E. O.; Kaplan, S. L., Pulmonary Manifestations in Children with Invasive Community-Acquired Staphylococcus aureus Infection. Clinical infectious diseases 2005, 41 (5), 583-590; (b) Francis, J. S.; Doherty, M. C.; Lopatin, U.; Johnston, C. P.; Sinha, G.; Ross, T.; Cai, M.; Hansel, N. N.; Perl, T.; Ticehurst, J. R.; Carroll, K.; Thomas, D. L.; Nuermberger, E.; Bartlett, J. G., Severe Community-Onset Pneumonia in Healthy Adults Caused by Methicillin- Resistant Staphylococcus aureus Carrying the Panton-Valentine Leukocidin Genes. Clinical infectious diseases 2005, 40 (1), 100-107; (c) Kallen, A. J.; Brunkard, J.; Moore, Z.; Budge, P.; Arnold, K. E.; Fosheim, G.; Finelli, L.; Beekmann, S. E.; Polgreen, P. M.; Gorwitz, R.; Hageman, J., Staphylococcus aureus Community-Acquired Pneumonia During the 2006 to 2007 Influenza Season. Annals of emergency medicine 2009, 53 (3), 358-365; (d) Henry, F. C.; Frank, R. D., Waves of resistance: Staphylococcus aureus in the antibiotic era. Nature Reviews Microbiology 2009, 7 (9), 629-641.

36. Dinges, M. M.; Orwin, P. M.; Schlievert, P. M., Exotoxins of Staphylococcus aureus. Clinical Microbiology Reviews 2000, 13 (1), 16-34.

37. (a) Bohach, G. A.; Fast, D. J.; Nelson, R. D.; Schlievert, P. M., Staphylococcal and Streptococcal Pyrogenic Toxins Involved in Toxic Shock Syndrome and Related Illnesses. Critical Reviews in Microbiology 1990, 17 (4), 251-272; (b) Marrack, P.; Kappler, J., The Staphylococcal Enterotoxins and Their Relatives. Science 1990, 248 (4956), 705-711.

38. Hamad, A. R. A.; Marrack, P.; Kappler, J. W., Transcytosis of Staphylococcal Superantigen Toxins. The Journal of Experimental Medicine 1997, 185 (8), 1447- 1454.

146

39. (a) Zhang, S.; Iandolo, J. J.; Stewart, G. C., The enterotoxin D plasmid of Staphylococcus aureus encodes a second enterotoxin determinant (sej). FEMS Microbiology Letters 1998, 168 (2), 227-233; (b) Lindsay, J. A.; Ruzin, A.; Ross, H. F.; Kurepina, N.; Novick, R. P., The gene for toxic shock toxin is carried by a family of mobile pathogenicity islands in Staphylococcus aureus. Molecular Microbiology 1998, 29 (2), 527-543.

40. (a) Song, L.; Hobaugh, M. R.; Shustak, C.; Cheley, S.; Bayley, H.; Gouaux, J. E., Structure of Staphylococcal α-Hemolysin, a Heptameric Transmembrane Pore. Science 1996, 274 (5294), 1859-1865; (b) PDB ID: 7AHL.

41. (a) Belmonte, G.; Cescatti, L.; Ferrari, B.; Nicolussi, T.; Ropele, M.; Menestrina, G., Pore formation by Staphylococcus aureus alpha-toxin in lipid bilayers. Eur Biophys J 1987, 14 (6), 349-358; (b) Gouaux, J. E.; Braha, O.; Hobaugh, M. R.; Song, L.; Cheley, S.; Shustak, C.; Bayley, H., Subunit stoichiometry of staphylococcal alpha-hemolysin in crystals and on membranes: a heptameric transmembrane pore. Proceedings of the National Academy of Sciences 1994, 91 (26), 12828-12831.

42. Bhakdi, S.; Tranum-Jensen, J., Alpha-toxin of Staphylococcus aureus. Microbiological Reviews 1991, 55 (4), 733-751.

43. (a) König, B.; Prévost, G.; Piémont, Y.; König, W., Effects Of Staphylococcus Aureus Leukocidins On Inflammatory Mediator Release From Human Granulocytes. Journal of Infectious Diseases 1995, 171 (3), 607-613; (b) Colin, D. A.; Mazurier, I.; Sire, S.; Finck-Barbançon, V., Interaction of the two components of leukocidin from Staphylococcus aureus with human polymorphonuclear leukocyte membranes: sequential binding and subsequent activation. Infection and Immunity 1994, 62 (8), 3184-3188.

44. Lina, G.; Piémont, Y.; Godail-Gamot, F.; Bes, M.; Peter, M.-O.; Gauduchon, V.; Vandenesch, F.; Etienne, J., Involvement of Panton-Valentine Leukocidin— Producing Staphylococcus aureus in Primary Skin Infections and Pneumonia. Clinical infectious diseases 1999, 29 (5), 1128-1132.

45. Smith, A., Bacterial resistance to antibiotics. In Hugo and Russell's Pharmaceutical Microbiology, 7th Edition, Denyer, S. P., Hodges, N., Gorman, S. P., Ed. Blackwell Science Ltd: Ashford Colour Press, Gosport, United Kingdom, 2004.

147

46. Abraham, E. P.; Chain, E., An Enzyme from Bacteria Able to Destroy Penicillin. Reviews of Infectious Diseases 1988, 10 (4), 677-678.

47. Kirby, W. M. M., Extraction of a Highly Potent Penicillin Inactivator from Penicillin Resistant Staphylococci. Science 1944, 99 (2579), 452-453.

48. Greenwood, D., Antimicrobial Drugs: Chronicle of a twentieth century medical triumph. Oxford University Press, USA: 2008.

49. (a) Jevons, M. P., “Celbenin” - resistant Staphylococci. British Medical Journal 1961, 1 (5219), 124-125; (b) Barber, M., Methicillin-resistant staphylococci. Journal of Clinical Pathology 1961, 14 (4).

50. Hallander, H. O.; Laurell, G., Identification of Cephalosporin-Resistant Staphylococcus aureus with the Disc Diffusion Method. Antimicrobial Agents and Chemotherapy 1972, 1 (5), 422-426.

51. Lowy, F. D., Antimicrobial resistance: the example of Staphylococcus aureus. The Journal of Clinical Investigation 2003, 111 (9), 1265-1273.

52. Hiramatsu, K.; Hanaki, H.; Ino, T.; Yabuta, K.; Oguri, T.; Tenover, F. C., Methicillin-resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibility. Journal of Antimicrobial Chemotherapy 1997, 40 (1), 135-136.

53. Staphylococcus aureus Resistant to Vancomycin. United States, 2002. MMWR 2002, 51, 565-567.

54. Costerton, J. W.; Stewart, P. S.; Greenberg, E. P., Bacterial Biofilms: A Common Cause of Persistent Infections. Science 1999, 284 (5418), 1318-1322.

55. Lentino, J. R.; Narita, M.; Yu, V. L., New antimicrobial agents as therapy for resistant gram-positive cocci. Eur J Clin Microbiol Infect Dis 2008, 27 (1), 3-15.

56. Hershberger, E.; Donabedian, S.; Konstantinou, K.; Zervos, M. J.; Eliopoulos, G. M., Quinupristin-Dalfopristin Resistance in Gram-Positive Bacteria: Mechanism of Resistance and Epidemiology. Clinical Infectious Diseases 2004, 38 (1), 92-98.

148

57. (a) Meka, V. G.; Pillai, S. K.; Sakoulas, G.; Wennersten, C.; Venkataraman, L.; DeGirolami, P. C.; Eliopoulos, G. M.; Moellering, R. C.; Gold, H. S., Linezolid Resistance in Sequential Staphylococcus aureus Isolates Associated with a T2500A Mutation in the 23S rRNA Gene and Loss of a Single Copy of rRNA. Journal of Infectious Diseases 2004, 190 (2), 311-317; (b) Aoki, H.; Ke, L.; Poppe, S. M.; Poel, T. J.; Weaver, E. A.; Gadwood, R. C.; Thomas, R. C.; Shinabarger, D. L.; Ganoza, M. C., Oxazolidinone Antibiotics Target the P Site on Escherichia coli Ribosomes. Antimicrobial Agents and Chemotherapy 2002, 46 (4), 1080-1085; (c) Shinabarger, D., Mechanism of action of the oxazolidinone antibacterial agents. Expert Opinion on Investigational Drugs 1999, 8 (8), 1195- 1202.

58. Tsiodras, S.; Gold, H. S.; Sakoulas, G.; Eliopoulos, G. M.; Wennersten, C.; Venkataraman, L.; Moellering Jr, R. C.; Ferraro, M. J., Linezolid resistance in a clinical isolate of Staphylococcus aureus. Lancet 2001, 358 (9277), 207-208.

59. Morales, G.; Picazo, J. J.; Baos, E.; Candel, F. J.; Arribi, A.; Peláez, B.; Andrade, R.; de la Torre, M.-Á.; Fereres, J.; Sánchez-García, M., Resistance to Linezolid Is Mediated by the cfr Gene in the First Report of an Outbreak of Linezolid- Resistant Staphylococcus aureus. Clinical Infectious Diseases 2010, 50 (6), 821- 825.

60. (a) Silverman, J. A.; Perlmutter, N. G.; Shapiro, H. M., Correlation of Daptomycin Bactericidal Activity and Membrane Depolarization in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 2003, 47 (8), 2538-2544; (b) Steenbergen, J. N.; Alder, J.; Thorne, G. M.; Tally, F. P., Daptomycin: a lipopeptide antibiotic for the treatment of serious Gram-positive infections. Journal of Antimicrobial Chemotherapy 2005, 55 (3), 283-288.

61. Mangili, A.; Bica, I.; Snydman, D. R.; Hamer, D. H., Daptomycin-Resistant, Methicillin-Resistant Staphylococcus aureus Bacteremia. Clinical Infectious Diseases 2005, 40 (7), 1058-1060.

62. Pankey, G. A., Tigecycline. Journal of Antimicrobial Chemotherapy 2005, 56 (3), 470-480.

149

63. (a) Hirata, T.; Saito, A.; Nishino, K.; Tamura, N.; Yamaguchi, A., Effects of Efflux Transporter Genes on Susceptibility of Escherichia coli to Tigecycline (GAR-936). Antimicrobial Agents and Chemotherapy 2004, 48 (6), 2179-2184; (b) Petersen, P. J.; Jacobus, N. V.; Weiss, W. J.; Sum, P. E.; Testa, R. T., In Vitro and In Vivo Antibacterial Activities of a Novel Glycylcycline, the 9-t- Butylglycylamido Derivative of Minocycline (GAR-936). Antimicrobial Agents and Chemotherapy 1999, 43 (4), 738-744; (c) Connell, S. R.; Tracz, D. M.; Nierhaus, K. H.; Taylor, D. E., Ribosomal Protection Proteins and Their Mechanism of Tetracycline Resistance. Antimicrobial Agents and Chemotherapy 2003, 47 (12), 3675-3681.

64. Krause, K. M.; Renelli, M.; Difuntorum, S.; Wu, T. X.; Debabov, D. V.; Benton, B. M., In Vitro Activity of Telavancin against Resistant Gram-Positive Bacteria. Antimicrobial Agents and Chemotherapy 2008, 52 (7), 2647-2652.

65. (a) Zhanel, G.; Sniezek, G.; Schweizer, F.; Zelenitsky, S.; Lagacé-Wiens, P. S.; Rubinstein, E.; Gin, A.; Hoban, D.; Karlowsky, J., Ceftaroline. Drugs 2009, 69 (7), 809-831; (b) Laudano, J. B., Ceftaroline fosamil: a new broad-spectrum cephalosporin. Journal of Antimicrobial Chemotherapy 2011, 66 (suppl 3), iii11- iii18.

66. Anderson, S. D.; Gums, J. G., Ceftobiprole: An Extended-Spectrum Anti– Methicillin-Resistant Staphylococcus aureus Cephalosporin. The Annals of Pharmacotherapy 2008, 42 (6), 806-816.

67. (a) Pogue, J. M.; Mann, T.; Barber, K. E.; Kaye, K. S., Carbapenem-resistant Acinetobacter baumannii: epidemiology, surveillance and management. Expert Review of Anti-infective Therapy 2013, 11 (4), 383-393; (b) Bouvet, P. J. M.; Grimont, P. A. D., Taxonomy of the Genus Acinetobacter with the Recognition of Acinetobacter baumannii sp. nov., Acinetobacter haemolyticus sp. nov., Acinetobacter johnsonii sp. nov., and Acinetobacter junii sp. nov. and Emended Descriptions of Acinetobacter calcoaceticus and Acinetobacter lwoffii. International Journal of Systematic Bacteriology 1986, 36 (2), 228-240.

68. Peleg, A. Y.; Seifert, H.; Paterson, D. L., Acinetobacter baumannii: Emergence of a Successful Pathogen. Clinical Microbiology Reviews 2008, 21 (3), 538-582.

69. Wendt, C.; Dietze, B.; Dietz, E.; Rüden, H., Survival of Acinetobacter baumannii on dry surfaces. Journal of Clinical Microbiology 1997, 35 (6), 1394-1397.

150

70. Kramer, A.; Schwebke, I.; Kampf, G., How long do nosocomial pathogens persist on inanimate surfaces? A systematic review. BMC Infectious Diseases 2006, 6, 130.

71. (a) McConnell, M. J.; Actis, L.; Pachón, J., Acinetobacter baumannii: human infections, factors contributing to pathogenesis and animal models. FEMS Microbiology Reviews 2013, 37 (2), 130-155; (b) Choi, C. H.; Lee, E. Y.; Lee, Y. C.; Park, T. I.; Kim, H. J.; Hyun, S. H.; Kim, S. A.; Lee, S.-K.; Lee, J. C., Outer membrane protein 38 of Acinetobacter baumannii localizes to the mitochondria and induces apoptosis of epithelial cells. Cellular Microbiology 2005, 7 (8), 1127- 1138.

72. Bergogne-Bérézin, E.; Towner, K. J., Acinetobacter spp. as nosocomial pathogens: microbiological, clinical, and epidemiological features. Clinical Microbiology Reviews 1996, 9 (2), 148-165.

73. Scaife, W.; Young, H.-K.; Paton, R. H.; Amyes, S. G. B., Transferable imipenem- resistance in Acinetobacter species from a clinical source. Journal of Antimicrobial Chemotherapy 1995, 36 (3), 585-586.

74. Poirel, L.; Nordmann, P., Carbapenem resistance in Acinetobacter baumannii: mechanisms and epidemiology. Clinical Microbiology and Infection 2006, 12 (9), 826-836.

75. Fournier, P.-E.; Vallenet, D.; Barbe, V.; Audic, S.; Ogata, H.; Poirel, L.; Richet, H.; Robert, C.; Mangenot, S.; Abergel, C.; Nordmann, P.; Weissenbach, J.; Raoult, D.; Claverie, J.-M., Comparative Genomics of Multidrug Resistance in Acinetobacter baumannii. PLoS Genetics 2006, 2 (1), e7.

76. Hellinger, W. C.; Brewer, N. S., Carbapenems and Monobactams: Imipenem, Meropenem, and Aztreonam. Mayo Clinic Proceedings 1999, 74 (4), 420-434.

77. Falagas, M. E.; Kasiakou, S. K.; Saravolatz, L. D., Colistin: The Revival of Polymyxins for the Management of Multidrug-Resistant Gram-Negative Bacterial Infections. Clinical Infectious Diseases 2005, 40 (9), 1333-1341.

151

78. (a) Peterson, A. A.; Hancock, R. E.; McGroarty, E. J., Binding of polycationic antibiotics and polyamines to lipopolysaccharides of Pseudomonas aeruginosa. Journal of Bacteriology 1985, 164 (3), 1256-1261; (b) Dixon, R. A.; Chopra, I., Leakage of periplasmic proteins from Escherichia coli mediated by polymyxin B nonapeptide. Antimicrobial Agents and Chemotherapy 1986, 29 (5), 781-788.

79. (a) Peleg, A. Y.; Potoski, B. A.; Rea, R.; Adams, J.; Sethi, J.; Capitano, B.; Husain, S.; Kwak, E. J.; Bhat, S. V.; Paterson, D. L., Acinetobacter baumannii bloodstream infection while receiving tigecycline: a cautionary report. Journal of Antimicrobial Chemotherapy 2007, 59 (1), 128-131; (b) Anthony, K. B.; Fishman, N. O.; Linkin, D. R.; Gasink, L. B.; Edelstein, P. H.; Lautenbach, E., Clinical and Microbiological Outcomes of Serious Infections with Multidrug-Resistant Gram- Negative Organisms Treated with Tigecycline. Clinical infectious diseases 2008, 46 (4), 567-570; (c) Reid, G. E.; Grim, S. A.; Aldeza, C. A.; Janda, W. M.; Clark, N. M., Rapid Development of Acinetobacter baumannii Resistance to Tigecycline. Pharmacotherapy 2007, 27 (8), 1198-1201.

80. Hantzsch, A., Synthesis of pyridine derivatives from ethyl acetoacetate and aldehydammonia. Liebigs Annalen der Chemie 1882, 215, 1-82.

81. (a) Abramovitch, R. A., Pyridine and Its Derivatives: Supplement Part one. John Wiley & Sons, Inc.: 1974; Vol. 14; (b) Klingsberg, E., Pyridine and Its Derivatives: Part one. Interscience Publishers: New York, 1960.

82. Shibanuma, T.; Iwanani, M.; Okuda, K.; Takenaka, T.; Murakami, M., Synthesis of Optically Active 2-(N-Benzyl-N-methylamino)ethyl Methyl 2,6-Dimethyl-4- (m-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (Nicardipine). Chemical & Pharmaceutical Bulletin 1980, 28 (9), 2809-2812.

83. Tamazawa, K.; Arima, H.; Kojima, T.; Isomura, Y.; Okada, M.; Fujita, S.; Furuya, T.; Takenaka, T.; Inagaki, O.; Terai, M., Stereoselectivity of a potent calcium antagonist, 1-benzyl-3-pyrrolidinyl methyl 2,6-dimethyl-4-(m- nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate. Journal of Medicinal Chemistry 1986, 29 (12), 2504-2511.

84. Bossert, F.; Meyer, H.; Wehinger, E., 4-Aryldihydropyridines, a New Class of Highly Active Calcium Antagonists. Angewandte Chemie International Edition 1981, 20 (9), 762-769.

152

85. Meyers, A. I.; Oppenlaender, T., An asymmetric synthesis of chiral nifedipine analogues. Journal of the Chemical Society, Chemical Communications 1986, (12), 920-921.

86. Cheng, C.-Y.; Chen, J.-Y.; Lee, M.-J., An asymmetric synthesis of 4-aryl-1,4- dihydopyridines. Heterocycles 1996, 43 (11), 2425-2434.

87. Enders, D.; Müller, S.; Demir, A. S., Enantioselective hantzsch dihydropyridine synthesis via metalated chiral alkyl acetoacetate hydrazones. Tetrahedron Letters 1988, 29 (49), 6437-6440.

88. Holdgrün, X. K.; Sih, C. J., A chemoenzymatic synthesis of optically-active dihydropyridines. Tetrahedron Letters 1991, 32 (29), 3465-3468.

89. Kosugi, Y.; Hori, M.; Nagasaka, T., Synthesis of Optically Pure 1,4- Dihydropyridine Derivatives by Means of Diastereomeric Separation of the Hantzsch Intermediates Bearing (R)-1-Phenylethylamino Group. Heterocycles 1994, 39 (2), 591-602.

90. Moshtaghi, Z. A.; Raisossadat, O. M.; Sadeghi, S. Q., Synthesis of novel unsymmetrically substituted 1,4-dihydropyridines and separation of the enantiomers of racemic 1,4-dihydropyridine containing isothioureido group. Arkivoc 2006, 14, 15-21.

91. Evans, C. G.; Gestwicki, J. E., Enantioselective Organocatalytic Hantzsch Synthesis of Polyhydroquinolines. Organic Letters 2009, 11 (14), 2957-2959.

92. (a) Dai, Z.; Pittman, C. U.; Li, T., Enantiomeric Separation of Racemic 4-Aryl- 1,4-Dihydropyridines and 4-Aryl-1,2,3,4-Tetrahydropyrimidines on a Chiral Tetraproline Stationary Phase. Chirality 2013, 25 (4), 238-242; (b) Schade, D.; Lanier, M.; Willems, E.; Okolotowicz, K.; Bushway, P.; Wahlquist, C.; Gilley, C.; Mercola, M.; Cashman, J. R., Synthesis and SAR of b-Annulated 1,4- Dihydropyridines Define Cardiomyogenic Compounds as Novel Inhibitors of TGFβ Signaling. Journal of Medicinal Chemistry 2012, 55 (22), 9946-9957.

93. Phillips, A. P., Hantzsch's Pyridine Synthesis. Journal of the American Chemical Society 1949, 71 (12), 4003-4007.

153

94. Shan, R.; Velazquez, C.; Knaus, E. E., Syntheses, Calcium Channel Agonist−Antagonist Modulation Activities, and Nitric Oxide Release Studies of Nitrooxyalkyl 1,4-Dihydro-2,6-dimethyl-3-nitro-4-(2,1,3-benzoxadiazol-4- yl)pyridine-5-carboxylate Racemates, Enantiomers, and Diastereomers. Journal of Medicinal Chemistry 2004, 47 (1), 254-261.

95. Sirisha, K.; Achaiah, G.; Reddy, V. M., Facile Synthesis and Antibacterial, Antitubercular, and Anticancer Activities of Novel 1,4-Dihydropyridines. Archiv der Pharmazie 2010, 343 (6), 342-352.

96. Mehta, P.; Verma, P., Antimicrobial Activity of Some Derivatives of 1,4- Dihydropyridines. Journal of Chemistry 2013, 2013, 4.

97. Poondra, R. R.; Nallamelli, R. V.; Meda, C. L. T.; Srinivas, B. N. V.; Grover, A.; Muttabathula, J.; Voleti, S. R.; Sridhar, B.; Pal, M.; Parsa, K. V. L., Discovery of novel 1,4-dihydropyridine-based PDE4 inhibitors. Bioorg Med Chem Lett 2013, 23 (4), 1104-1109.

98. (a) Baumert, C.; Günthel, M.; Krawczyk, S.; Hemmer, M.; Wersig, T.; Langner, A.; Molnár, J.; Lage, H.; Hilgeroth, A., Development of small-molecule P-gp inhibitors of the N-benzyl 1,4-dihydropyridine type: Novel aspects in SAR and bioanalytical evaluation of multidrug resistance (MDR) reversal properties. Bioorgan Med Chem 2013, 21 (1), 166-177; (b) Kawase, M.; Shah, A.; Gaveriya, H.; Motohashi, N.; Sakagami, H.; Varga, A.; Molnár, J., 3,5-Dibenzoyl-1,4- dihydropyridines: Synthesis and MDR Reversal in Tumor Cells. Bioorgan Med Chem 2002, 10 (4), 1051-1055.

99. Reimão, J. Q.; Scotti, M. T.; Tempone, A. G., Anti-leishmanial and anti- trypanosomal activities of 1,4-dihydropyridines: In vitro evaluation and structure– activity relationship study. Bioorgan Med Chem 2010, 18 (22), 8044-8053.

100. Guiguemde, W. A.; Shelat, A. A.; Bouck, D.; Duffy, S.; Crowther, G. J.; Davis, P. H.; Smithson, D. C.; Connelly, M.; Clark, J.; Zhu, F.; Jiménez-Díaz, M. B.; Martinez, M. S.; Wilson, E. B.; Tripathi, A. K.; Gut, J.; Sharlow, E. R.; Bathurst, I.; Mazouni, F. E.; Fowble, J. W.; Forquer, I., Chemical genetics of Plasmodium falciparum. Nature 2010, 465 (7296), 311-315.

101. Guiguemde, W. A.; Shelat, Anang A.; Garcia-Bustos, Jose F.; Diagana, T. T.; Gamo, F.-J.; Guy, R. K., Global Phenotypic Screening for Antimalarials. Chemistry & Biology 2012, 19 (1), 116-129.

154

102. Trager, W.; Jensen, J. B., Human malaria parasites in continuous culture. Science 1976, 193 (4254), 673-5.

103. Smilkstein, M.; Sriwilaijaroen, N.; Kelly, J. X.; Wilairat, P.; Riscoe, M., Simple and inexpensive fluorescence-based technique for high-throughput antimalarial drug screening. Antimicrobial Agents and Chemotherapy 2004, 48 (5), 1803-6.

104. Desjardins, R. E.; Canfield, C. J.; Haynes, J. D.; Chulay, J. D., Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrobial Agents and Chemotherapy 1979, 16 (6), 710-718.

105. Zhang, B.-l.; He, W.; Shi, X.; Huan, M.-l.; Huang, Q.-j.; Zhou, S.-y., Synthesis and biological activity of the calcium modulator (R) and (S)-3-methyl 5-pentyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate. Bioorg Med Chem Lett 2010, 20 (3), 805-808.

106. Carroll, W. A.; Agrios, K. A.; Altenbach, R. J.; Buckner, S. A.; Chen, Y.; Coghlan, M. J.; Daza, A. V.; Drizin, I.; Gopalakrishnan, M.; Henry, R. F.; Kort, M. E.; Kym, P. R.; Milicic, I.; Smith, J. C.; Tang, R.; Turner, S. C.; Whiteaker, K. L.; Zhang, H.; Sullivan, J. P., Synthesis and Structure−Activity Relationships of a Novel Series of Tricyclic Dihydropyridine-Based KATP Openers That Potently Inhibit Bladder Contractions in Vitro. Journal of Medicinal Chemistry 2004, 47 (12), 3180-3192.

107. (a) Evans, D. A.; Ellman, J. A.; Dorow, R. L., Asymmetric halogenation of chiral imide enolates. A general approach to the synthesis of enantiomerically pure α- amino acids. Tetrahedron Letters 1987, 28 (11), 1123-1126; (b) Kramp, G. J.; Kim, M.; Gais, H.-J.; Vermeeren, C., Fully Stereocontrolled Total Syntheses of the Prostacyclin Analogues 16S-Iloprost and 16S-3-Oxa-Iloprost by a Common Route, Using Alkenylcopper-Azoalkene Conjugate Addition, Asymmetric Olefination, and Allylic Alkylation. Journal of the American Chemical Society 2005, 127 (50), 17910-17920; (c) Shan, R.; Howlett, S. E.; Knaus, E. E., Syntheses, Calcium Channel Agonist−Antagonist Modulation Activities, Nitric Oxide Release, and Voltage-Clamp Studies of 2-Nitrooxyethyl 1,4-Dihydro- 2,6- dimethyl-3-nitro-4-(2-trifluoromethylphenyl)pyridine-5-carboxylate Enantiomers. Journal of Medicinal Chemistry 2002, 45 (4), 955-961.

155

108. (a) Xu, C.; Yuan, C., Candida Rugosa lipase-catalyzed kinetic resolution of β- hydroxy-β-arylpropionates and δ-hydroxy-δ-aryl-β-oxo-pentanoates. Tetrahedron 2005, 61 (8), 2169-2186; (b) Erichsen, M. N.; Huynh, T. H. V.; Abrahamsen, B.; Bastlund, J. F.; Bundgaard, C.; Monrad, O.; Bekker-Jensen, A.; Nielsen, C. W.; Frydenvang, K.; Jensen, A. A.; Bunch, L., Structure−Activity Relationship Study of First Selective Inhibitor of Excitatory Amino Acid Transporter Subtype 1: 2- Amino-4-(4-methoxyphenyl)-7-(naphthalen-1-yl)-5-oxo-5,6,7,8-tetrahydro-4H- chromene-3-carbonitrile (UCPH-101). Journal of Medicinal Chemistry 2010, 53 (19), 7180-7191.

109. Iqbal, M.; Mistry, N.; Clarke, P. A., An asymmetric Maitland–Japp reaction: a highly enantioselective synthesis of tetrahydropyran-4-ones. Tetrahedron 2011, 67 (27–28), 4960-4966.

110. Keck, G. E.; Tarbet, K. H.; Geraci, L. S., Catalytic asymmetric allylation of aldehydes. Journal of the American Chemical Society 1993, 115 (18), 8467-8468.

111. Racherla, U. S.; Brown, H. C., Chiral synthesis via organoboranes. 27. Remarkably rapid and exceptionally enantioselective (approaching 100% ee) allylboration of representative aldehydes at -100o under new, salt-free conditions. The Journal of Organic Chemistry 1991, 56 (1), 401-404.

112. Smith, C. M.; O'Doherty, G. A., Enantioselective Syntheses of Cryptocarya Triacetate, Cryptocaryolone, and Cryptocaryolone Diacetate. Organic Letters 2003, 5 (11), 1959-1962.

113. Reddy, M. V. R.; Yucel, A. J.; Ramachandran, P. V., An Efficient Asymmetric Synthesis of Tarchonanthuslactone1. The Journal of Organic Chemistry 2001, 66 (7), 2512-2514.

114. Zhang, Y.; Clark, J. A.; Connelly, M. C.; Zhu, F.; Min, J.; Guiguemde, W. A.; Pradhan, A.; Iyer, L.; Furimsky, A.; Gow, J.; Parman, T.; El, M. F.; Phillips, M. A.; Kyle, D. E.; Mirsalis, J.; Guy, R. K., Lead Optimization of 3-Carboxyl-4(1H)- Quinolones to Deliver Orally Bioavailable Antimalarials. Journal of Medicinal Chemistry 2012, 55, 4205-4219.

115. Armarego, W. L. F., Quinazolines. In Advances in Heterocyclic Chemistry, Katritzky, A. R., Ed. Academic Press Inc.: New York, 1963; Vol. 1, pp 253-309.

156

116. Brown, D. J., Halogenoquinazolines. In Chemistry of Heterocyclic Compounds, Taylor, E. C.; Weissberger, A., Eds. John Wiley & Sons, Inc.: New York, 1996; pp 181-218.

117. Rajput, R.; Mishra, A. P., A Review on Biological Activity of Quinazolinones. International Journal of Pharmacy and Pharmaceutical Sciences 2012, 4 (2), 66- 70.

118. Pandeya, S. N.; Sriram, D.; Nath, G.; De Clercq, E., Synthesis, antibacterial, antifungal and anti-HIV evaluation of Schiff and Mannich bases of isatin derivatives with 3-amino-2-methylmercapto quinazolin-4(3H)-one. Pharmaceutica Acta Helvetiae 1999, 74 (1), 11-17.

119. Abdel Gawad, N. M.; Georgey, H. H.; Youssef, R. M.; El-Sayed, N. A., Synthesis and antitumor activity of some 2, 3-disubstituted quinazolin-4(3H)-ones and 4, 6- disubstituted- 1, 2, 3, 4-tetrahydroquinazolin-2H-ones. European Journal of Medicinal Chemistry 2010, 45 (12), 6058-6067.

120. Ghorab, M. M.; Abdel-Gawad, S. M.; El-Gaby, M. S. A., Synthesis and evaluation of some new fluorinated hydroquinazoline derivatives as antifungal agents. Il Farmaco 2000, 55 (4), 249-255.

121. Aly, M. M.; Mohamed, Y. A.; El-Bayouki, K. A. M.; Basyouni, W. M.; Abbas, S. Y., Synthesis of some new 4(3H)-quinazolinone-2-carboxaldehyde thiosemicarbazones and their metal complexes and a study on their anticonvulsant, analgesic, cytotoxic and antimicrobial activities – Part-1. European Journal of Medicinal Chemistry 2010, 45 (8), 3365-3373.

122. Raffa, D.; Daidone, G.; Maggio, B.; Cascioferro, S.; Plescia, F.; Schillaci, D., Synthesis and antileukemic activity of new 3-(5-methylisoxazol-3-yl) and 3- (pyrimidin-2-yl)-2-styrylquinazolin-4(3H)-ones. Il Farmaco 2004, 59 (6), 451- 455.

123. Werbel, L. M.; Degnan, M. J., Antimalarial drugs. 63. Synthesis and antimalarial and antitumor effects of 2-amino-4-(hydrazino and hydroxyamino)-6- [(aryl)thio]quinazolines. Journal of Medicinal Chemistry 1987, 30 (11), 2151- 2154.

157

124. Ye, C.; You, J.; Li, X.; You, R.; Weng, Y.; Li, J.; Wang, Y., Design, synthesis and anticoccidial activity of a series of 3-(2-(2-methoxyphenyl)-2-oxoethyl) quinazolinone derivatives. Pesticide Biochemistry and Physiology 2010, 97 (3), 194-198.

125. Kumar, A.; Sharma, S.; Archana; Bajaj, K.; Sharma, S.; Panwar, H.; Singh, T.; Srivastava, V. K., Some new 2,3,6-trisubstituted quinazolinones as potent anti- inflammatory, analgesic and COX-II inhibitors. Bioorgan Med Chem 2003, 11 (23), 5293-5299.

126. Balakumar, C.; Lamba, P.; Pran Kishore, D.; Lakshmi Narayana, B.; Venkat Rao, K.; Rajwinder, K.; Raghuram Rao, A.; Shireesha, B.; Narsaiah, B., Synthesis, anti-inflammatory evaluation and docking studies of some new fluorinated fused quinazolines. European Journal of Medicinal Chemistry 2010, 45 (11), 4904- 4913.

127. Jatav, V.; Mishra, P.; Kashaw, S.; Stables, J. P., Synthesis and CNS depressant activity of some novel 3-[5-substituted 1,3,4-thiadiazole-2-yl]-2-styryl quinazoline-4(3H)-ones. European Journal of Medicinal Chemistry 2008, 43 (1), 135-141.

128. (a) Kario, K.; Schwartz, J. E.; Pickering, T. G., Changes of Nocturnal Blood Pressure Dipping Status in Hypertensives by Nighttime Dosing of α-Adrenergic Blocker, Doxazosin: Results from the HALT Study. Hypertension 2000, 35 (3), 787-794; (b) Van Kleef, E. M.; Smits, J. F. M.; Schwartz, S. M.; Daemen, M. J. A. P., Doxazosin blocks the angiotensin II-induced smooth muscle cell DNA synthesis in the media, but not in the neointima of the rat carotid artery after balloon injury. Cardiovascular Research 1996, 31 (2), 324-330.

129. Berman, J. D.; King, M.; Edwards, N., Antileishmanial activities of 2,4- diaminoquinazoline putative dihydrofolate reductase inhibitors. Antimicrobial Agents and Chemotherapy 1989, 33 (11), 1860-1863.

158

130. (a) Bhattacharjee, A. K.; Skanchy, D. J.; Jennings, B.; Hudson, T. H.; Brendle, J. J.; Werbovetz, K. A., Analysis of stereoelectronic properties, mechanism of action and pharmacophore of synthetic indolo[2,1-b]quinazoline-6,12-dione derivatives in relation to antileishmanial activity using quantum chemical, cyclic voltammetry and 3-D-QSAR CATALYST procedures. Bioorgan Med Chem 2002, 10 (6), 1979-1989; (b) Ram, V. J.; Goel, A.; Verma, M.; Kaul, I. B.; Kapil, A., Latent leishmanicidal activity of quinazolinones and 1,2,4-triazoloquinazolinones. Bioorg Med Chem Lett 1994, 4 (17), 2087-2090; (c) Agarwal, K. C.; Sharma, V.; Shakya, N.; Gupta, S., Design and synthesis of novel substituted quinazoline derivatives as antileishmanial agents. Bioorg Med Chem Lett 2009, 19 (18), 5474- 5477; (d) Kumar, S.; Shakya, N.; Gupta, S.; Sarkar, J.; Sahu, D. P., Synthesis and biological evaluation of novel 4-(hetero)aryl-2-piperazinoquinazolines as anti- leishmanial and anti-proliferative agents. Bioorg Med Chem Lett 2009, 19 (9), 2542-2545.

131. Gilbert, I. H., Inhibitors of dihydrofolate reductase in leishmania and trypanosomes. Biochimica et Biophysica Acta-Molecular Basis of Disease 2002, 1587 (2-3), 249-257.

132. (a) Feng, J.; Zhang, Z.; Wallace, M. B.; Stafford, J. A.; Kaldor, S. W.; Kassel, D. B.; Navre, M.; Shi, L.; Skene, R. J.; Asakawa, T.; Takeuchi, K.; Xu, R.; Webb, D. R.; Gwaltney, S. L., II, Discovery of Alogliptin: A Potent, Selective, Bioavailable, and Efficacious Inhibitor of Dipeptidyl Peptidase IV. Journal of Medicinal Chemistry 2007, 50 (10), 2297-2300; (b) Ife, R. J.; Brown, T. H.; Blurton, P.; Keeling, D. J.; Leach, C. A.; Meeson, M. L.; Parsons, M. E.; Theobald, C. J., Reversible Inhibitors of the Gastric (H+/K+)-ATPase. 5. Substituted 2,4- Diaminoquinazolines and Thienopyrimidines. Journal of Medicinal Chemistry 1995, 38 (14), 2763-2773; (c) Kanuma, K.; Omodera, K.; Nishiguchi, M.; Funakoshi, T.; Chaki, S.; Semple, G.; Tran, T.-A.; Kramer, B.; Hsu, D.; Casper, M.; Thomsen, B.; Sekiguchi, Y., Lead optimization of 4- (dimethylamino)quinazolines, potent and selective antagonists for the melanin- concentrating hormone receptor 1. Bioorg Med Chem Lett 2005, 15 (17), 3853- 3856.

133. (a) Buckner, F. S.; Wilson, A. J., Colorimetric assay for screening compounds against Leishmania amastigotes grown in macrophages. Am J Trop Med Hyg 2005, 72 (5), 600-605; (b) Zhu, X.; Pandharkar, T.; Werbovetz, K., Identification of new antileishmanial leads from hits obtained by high-throughput screening. Antimicrobial Agents and Chemotherapy 2012, 56, 1182-1189.

134. Topliss, J. G., Utilization of operational schemes for analog synthesis in drug design. Journal of Medicinal Chemistry 1972, 15 (10), 1006-1011.

159

135. Yamada, T.; Goto, M.; Punj, V.; Zaborina, O.; Kimbara, K.; Das, G. T. K.; Chakrabarty, A. M., The bacterial redox protein azurin induces apoptosis in J774 macrophages through complex formation and stabilization of the tumor suppressor protein p53. Infection and Immunity 2002, 70, 7054-7062.

136. Donovan, S. F.; Pescatore, M. C., Method for measuring the logarithm of the octanol-water partition coefficient by using short octadecyl-poly(vinyl alcohol) high-performance liquid chromatography columns. Journal of Chromatography, A 2002, 952, 47-61.

137. (a) Zornoza, T.; Cano-Cebrian, M. J.; Hipolito, L.; Granero, L.; Polache, A., Evidence of a flip-flop phenomenon in acamprosate pharmacokinetics: an in vivo study in rats. Biopharmaceutics and Drug Disposition 2006, 27 (7), 305-311; (b) Yanez, J. A.; Remsberg, C. M.; Sayre, C. L.; Forrest, M. L.; Davies, N. M., Flip- flop pharmacokinetics--delivering a reversal of disposition: challenges and opportunities during drug development. Therapeutic delivery 2011, 2 (5), 643- 672.

138. Huband, M. D.; Cohen, M. A.; Zurack, M.; Hanna, D. L.; Skerlos, L. A.; Sulavik, M. C.; Gibson, G. W.; Gage, J. W.; Ellsworth, E.; Stier, M. A.; Gracheck, S. J., In Vitro and In Vivo Activities of PD 0305970 and PD 0326448, New Bacterial Gyrase/Topoisomerase Inhibitors with Potent Antibacterial Activities versus Multidrug-Resistant Gram-Positive and Fastidious Organism Groups. Antimicrobial Agents and Chemotherapy 2007, 51 (4), 1191-1201.

139. Rohini, R.; Muralidhar Reddy, P.; Shanker, K.; Hu, A.; Ravinder, V., Antimicrobial study of newly synthesized 6-substituted indolo[1,2-c]quinazolines. European Journal of Medicinal Chemistry 2010, 45 (3), 1200-1205.

140. DeGraw, J. I.; Brown, V. H.; Colwell, W. T.; Morrison, N. E., Potential antileprotic agents. 3. Inhibition of mycobacterial dihydrofolic reductase by 2,4- diamino-5-methyl-6-alkylquinazolines. Journal of Medicinal Chemistry 1974, 17 (7), 762-764.

141. Kung, P.-P.; Casper, M. D.; Cook, K. L.; Wilson-Lingardo, L.; Risen, L. M.; Vickers, T. A.; Ranken, R.; Blyn, L. B.; Wyatt, J. R.; Cook, P. D.; Ecker, D. J., Structure−Activity Relationships of Novel 2-Substituted Quinazoline Antibacterial Agents. Journal of Medicinal Chemistry 1999, 42 (22), 4705-4713.

142. Gottasová, R.; Kubíková, J.; Čipák, L., Antibacterial Effect of Some 2,6- Disubstituted 4-Aniloquinazolines. Folia Microbiologica 1998, 43 (6), 679-682. 160

143. Horii, T.; Suzuki, Y.; Monji, A.; Morita, M.; Muramatsu, H.; Kondo, Y.; Doi, M.; Takeshita, A.; Kanno, T.; Maekawa, M., Detection of mutations in quinolone resistance-determining regions in levofloxacin- and methicillin-resistant Staphylococcus aureus: effects of the mutations on fluoroquinolone MICs. Diagnostic Microbiology and Infectious Disease 2003, 46 (2), 139-145.

144. (a) Ashton, W. T.; Walker, F. C.; Hynes, J. B., Quinazolines as inhibitors of dihydrofolate reductase. 1. Journal of Medicinal Chemistry 1973, 16 (6), 694-697; (b) Cody, V.; Luft, J. R.; Pangborn, W.; Gangjee, A.; Queener, S. F., Structure determination of tetrahydroquinazoline antifolates in complex with human and Pneumocystis carinii dihydorfolate reductase: correlations between enzyme selectivity and stereochemistry. Acta Crystallographica: Section D 2004, 60 (4), 646-655; (c) Whitlow, M.; Howard, A. J.; Stewart, D.; Hardman, K. D.; Chan, J. H.; Baccanari, D. P.; Tansik, R. L.; Hong, J. S.; Kuyper, L. F., X-ray Crystal Structures of Candida albicans Dihydrofolate Reductase: High Resolution Ternary Complexes in Which the Dihydronicotinamide Moiety of NADPH Is Displaced by an Inhibitor. Journal of Medicinal Chemistry 2001, 44 (18), 2928- 2932.

161