Synthesis and biological activity of novel
bisquinazolinone derivatives
IF Prinsloo
orcid.org/ 0000-0001-7460-1245
Dissertation submitted in fulfilment of the requirements for the
degree
Masters of Science in Pharmaceutical Chemistry
at
the North West University
Supervisor:
Prof DD N’Da
Examination: February 2020
Student number: 24912085
PREFACE
This dissertation is submitted in an article format in accordance with the General Academic Rules (A.13.7.3) of the North-West University. An article in the form of a manuscript is included in this dissertation:
Chapter 1: Introduction and Problem Statement
Chapter 2: Literature Review Chapter 3: Article for submission
Synthesis and in vitro antileishmanial efficacy of novel quinazolinone derivatives
This article will be submitted to the European Journal of Medicinal Chemistry and was prepared according to the author’s guidelines, available in the author information pack on the Journal’s homepage:
https://www.elsevier.com/journals/european-journal-of-medicinal-chemistry/0223-5234/guide-for-authors
ACKNOWLEDGEMENTS
I hereby wish to express my sincere gratitude to the following individuals and institutions:
• My supervisor Prof. D.D. N’Da and my co-supervisors Dr. J Aucamp and Nonkululeko H. Zuma for their ongoing guidance and/or support throughout my M.Sc degree.
• Prof. A. Wessels for guidance during the final stages of completing my dissertation. • Dr. D. Otto and Dr. J. Jordaan for NMR and HRMS spectroscopy.
• Prof F van der Kooy for HPLC analysis. • The NWU and NRF for financial support.
• To my family and friends for their love and emotional support. • To Niël de Beer, we did it, but why did we do it.
• To Nico van Lingen, you were there when times were hard, I really appreciate it.
ABSTRACT
Leishmaniasis is a vector-borne disease caused by the Leishmania parasite. Although there are 20 species that can infect humans, L. donovani is responsible for the majority of visceral leishmaniasis and L. major is responsible for the majority of cutaneous leishmaniasis. In 2019, the estimated annual number of leishmaniasis cases ranged from 700 000 to 1 million, with a death count of 26 000 to 65 000. Chemotherapy, which is the most effective control strategy for leishmaniasis, is structured around five drugs, namely pentavalent antimonials, amphotericin B, paromomycin, pentamidine and miltefosine. However, these drugs possess undesirable characteristics including toxicity, impractical and inaccessible administration (intramuscular (IM) or intravenous (IV) injections) with the exception of miltefosine; which discourages patient compliance and contributes to the emergence of drug resistant leishmanial parasites. Furthermore, leishmaniasis is a neglected disease with growing area distribution. Consequently, the search for new functional, safe and affordable antileishmanial agents is thus urgent and essential.
In this study, an investigation of febrifugine and bisquinazolinone derivatives as antileishmanial agents was undertaken. Quinazolinones, such as febrifugine possess excellent antiprotozoal activity, which includes antileishmanial activity. Shortcomings such as unfavourable toxicity profiles and structural hindrances, chiral centres causing isomer formation, limit their potential as chemotherapeutic agents. Additionally, although these agents occur in nature, derivatisation is often lengthy and provides poor yields. Therefore, in this study two series of compounds (febrifugine derivatives and bisquinazolinone derivatives) were synthesised in a maximum of three steps (condensation, acylation and nucleophilic substitution) and targeting N-3 as the site of reaction. Target compounds were synthesised by SN2 nucleophilic substitution and isolated by
recrystallisation and column chromatography in poor to average yields. After chemical analysis and structure confirmation, the derivatives were prepared for antileishmanial screening against L. donovani and L. major promastigotes.
All synthesised derivatives had poor activity (growth inhibition <30%) against the Leishmania strains. However, it was noted that the febrifugine derivatives were more potent against L. major (growth inhibition 0 − 35%), while the bisquinazolinone derivatives were more potent against L. donovani (growth inhibition 0 − 30%). Furthermore, cytotoxicity of the derivatives was assessed against mammalian (Vero) cells. The compounds had excellent toxicity profiles (IC50 >100 µM).
Although the antileishmanial activity of the derivatives was inadequate when compared to clinical drugs, the lack of cytotoxicity may be a good starting point for the future development of these agents. Through improved design and modification, these derivatives could be building-blocks for future projects.
OPSOMMING
Leishmaniasis is 'n vektor-oordraagbare siekte veroorsaak deur die Leishmania parasiet. Alhoewel daar 20 spesies is wat mense kan infekteer, is L. donovani verantwoordelik vir die meerderheid van visserale leishmaniasis en L. major verantwoordelik vir die meerderheid van kutane leishmaniasis. In 2019 het die beraamde jaarlikse getal van leishmaniasis gevalle gestrek van 700 000 tot 1 miljoen, met 'n sterfte syfer van 26 000 tot 65 000. Chemoterapie wat die mees effektiewe beheer strategie teen leishmaniasis is, is gestruktureerd rondom 5 middels, naamlik pentavalente antimoonbevattende middels, amfoterisien B, paromomisien, pentamidien en miltefosien. Die middels besit egter ongunstige eienskappe insluitende toksisiteit, onpraktiese en ontoeganklike toediening (intramuskulêre (IM) of intraveneuse (IV) inspuitings) met die uitsondering van miltefosien; dié ontmoedig pasiënt meewerkendheid en lewer bydra tot die opkoms van middel weerstandige leishmania parasiete. Verder is leishmaniasis 'n afgeskeepte siekte met 'n groeiende gebied van verspreiding. Gevolglik is die soektog vir nuwe funksionele, veilige en bekostigbare antileishmaniale middels dringend en noodsaaklik.
Tydens die studie is 'n ondersoek ingestel na febrifugine en bisquinazolinone afgeleides as antileishmaniale middels. Quinazolinone, soos febrifugine, besit uitstekende antiprotozoale aktiwiteit wat antileishmaniale aktiwiteit insluit. Tekortkominge soos ongunstige toksisiteit profiele, strukturele hindernisse en chirale sentrums wat isomeervorming veroorsaak beperk die verbindings se potensiaal as chemoterapeutiese middels. Daarbenewens, hoewel hierdie middels in die natuur voorkom, is derivatisering dikwels langdradig en lewer dit swak opbrengste. Daarom is in hierdie studie twee reekse verbindings (febrifugine afgeleides en bisquinazolinone afgeleides) gesintetiseer in 'n maksimum van drie stappe (kondensasie, asilering en nukleufiliese substitusie) en die fokus op N-3 as die plek van reaksie. Teiken middels was gesintetiseer deur middel van SN2 nukleufiliese substitusie en is geïsoleer deur herkristallisasie en
kolomchromatografie in lae tot gemiddelde opbrengste. Na chemiese ontleding en bevestiging van die strukture, is die afgeleides voorberei vir antileishmaniale siftingstoetse teen L. donovani en L. major promastigote. Al die gesintetiseerde afgeleides het swak aktiwiteit getoon (groei inhibering <30%) teen die Leishmania stamme. Daar is egter opgemerk dat die febrifugine afgeleides meer kragtig was teen L. major (groei inhibering 0 – 35%), terwyl die bisquinazolinone afgeleides meer kragtig was teen L. donovani (groei inhibering 0 – 30%). Verder is die sitotoksisiteit van die afgeleides teen soogdier (Vero) selle getoets. Die verbindings het uitstekende toksisiteit profiele getoon (IC50 >100 µM).
Alhoewel die antileishmaniale aktiwiteit van hierdie afgeleides onvoldoende is in vergelyking met kliniese middels, kan die gebrek aan sitotoksisiteit 'n goeie beginpunt wees vir toekomstige ontwikkeling van hierdie middels. Deur verbeterde ontwerp en modifikasie kan hierdie afgeleides boustene wees vir toekomstige projekte.
Sleutelwoorde: Leishmania, quinazolinone, toksisiteit, weerstandigheid, febrifugine, bisquinazolinone.
TABLE OF CONTENTS
SOLEMN DECLARATION ...i
TURNITIN REPORT ... ii PREFACE ... iii ACKNOWLEDGEMENTS ... iv ABSTRACT ...v OPSOMMING ... viii LIST OF TABLES ... xi
LIST OF FIGURES ... xii
LIST OF SCHEMES ... xiv
LIST OF ABBREVIATIONS ... xv
CHAPTER 1 Introduction and Problem Statement ... 1
1.1 Introduction ... 1
1.2 Aims and objectives ... 7
References ... 9
CHAPTER 2 Literature Review ... 14
2.1 Introduction ... 14
2.2 Epidemiology ... 16
2.3 Diagnosis and symptoms ... 18
2.4 Disease control ... 20
2.5 Clinical forms of leishmaniasis ... 20
2.5.1 Mucocutaneous leishmaniasis ... 20 2.5.2 Cutaneous leishmaniasis ... 20 2.5.3 Visceral leishmaniasis ... 21 2.6 Leishmaniasis therapy ... 21 2.6.1 Physical therapy ... 21 2.6.2 Leishmaniasis chemotherapy ... 22 2.6.3 Drug resistance ... 27
2.6.4 Drug combination therapy ... 27
2.7 Novel antileishmanial drug discovery ... 28
2.7.2 Bisquinazolinones ... 33
2.7.3 Bioactive thiazine and benzothiazine derivatives ... 33
References ... 36
CHAPTER 3 ARTICLE FOR SUBMITTION ... 47
Synthesis and in vitro antileishmanial efficacy of novel quinazolinone derivatives ... 48
Highlights ... 49
Abstract ... 50
1. INTRODUCTION ... 52
2. MATERIAL AND METHODS ... 54
2.1. Materials ... 54 2.2. General procedures ... 55 2.3. Synthesis ... 56 2.4. Biological evaluation ... 66 2.4.1 Antileishamanial activity ... 66 2.4.2 Cytotoxicity assay ... 67
3. RESULTS AND DISCUSSION ... 68
3.1. Chemistry ... 68 3.1.1. Febrifugine derivatives ... 68 3.1.2. Bisquinazolinones ... 71 3.2. Biological activity ... 73 4. CONCLUSION ... 76 Acknowledgments ... 76 Disclaimer ... 77 References ... 78
CHAPTER 4 Summary and Conclusion ... 81
References ... 84
ADDENDUM A... 86
LIST OF TABLES
Table 1: Acetylamide substituted of quinazolinones 2a-g and 3a-d... 70 Table 2: Bisquinazolinones 4a-f and 5a-c ... 72 Table 3: Leishmania promastigote growth inhibition (%) at 10 μM and
LIST OF FIGURES
Figure 1.1: Limitations of clinical antileishmanial chemotherapy ... 2
Figure 1.2: Structure of febrifugine, the coloured/highlighted area indicates chirality ... 3
Figure 1.3: The interconversion of febrifugine ... 4
Figure 1.4: Isomerisation of febrifugine into isofebrifugine ... 4
Figure 1.5: Structure of febrifugine, the coloured/highlighted area indicates the acetonyl tether and the piperidine ring... 5
Figure 1.6: Structure of febrifugine, the coloured/highlighted area indicates the methylene group on the acetonyl linker ... 6
Figure 1.7: Structure of 1,4-benzothiazine ... 7
Figure 1.8: Chemical structures of methyl 5,9-dioxo-3,4,5,9-tetrahydro-2H-thieno(2',3':4,5)benzo(1,2-b)(1,4)thiazine-7-carboxylate 1,1-dioxide and thiaplakortone A ... 7
Figure 2.1: Status of endemicity of cutaneous leishmaniasis worldwide, 2016 ... 14
Figure 2.2: Status of endemicity of visceral leishmaniasis worldwide, 2016 ... 15
Figure 2.3: Life cycle of Leishmania ... 16
Figure 2.4: Different species of Leishmania of the old and new world that infect humans ... 17
Figure 2.5: Classification of Leishmaniasis based on clinical appearance ... 19
Figure 2.6: Chemical structures of sodium stibogluconate (1) and meglumine antimoniate (2) ... 23
Figure 2.7: Chemical structure of Amphotericin B (3) ... 24
Figure 2.8: Structure of Paromomycin (4) ... 25
Figure 2.9: Chemical structure of Pentamidine (5) ... 25
Figure 2.10: Chemical structure of Miltefosine (6) ... 26
Figure 2.11: Adverse effects of antileishmanial therapies... 27 Figure 2.12: Chemical structure of 4(3H)-quinazolinone (7) and
2-cyano-3,4-Figure 2.13: Chemical structure of Raltitrexed (9) ... 30 Figure 2.14: Isomerisation of febrifugine (10) into isofebrifugine (11) ... 30 Figure 2.15: Chemical structure of halofuginone ... 31 Figure 2.16: Chemical structure of
6-chloro-3-[3-(3-hydroxypiperidin-2-yl)-2-oxopropyl]-7-(pyridin-4-yl)quinazolin-4(3H)-one (13) and
(S)-6,7-difluoro-3-[3-(3-oxopiperidin-2-yl)propyl]quinazolin-4(3H)-one (14) ... 32 Figure 2.17: The interconversion of febrifugine ... 33 Figure 2.18: Chemical structure of
2,2’-{1,1’-[3,3’-dimethyl-(1,1’-biphenyl)-4,4’-diyl]bis(4-oxoazetidine-2,1-diyl)}bis[3-methylquinazolin-4(3H)-one] (15) (Reddy et al., 2010) and
N,N’-[2,2’-(ethane-1,2-diyl)bis(4-oxoquinazoline-3,2(4H)-diyl)]bis(N-acetylacetamide) (16) ... 34 Figure 2.19: Chemical structure of thiazine (17) and 1,4-benzothiazine (18) ... 35 Figure 2.20: Chemical structure of methyl
5,9-dioxo-3,4,5,9-tetrahydro-2H-thieno(2',3':4,5)benzo(1,2-b)(1,4)thiazine-7-carboxylate 1,1-dioxide
(19) ... 35 Figure 1: Current antileishmanial drugs in clinics ... 53 Figure 2: Structures of methaqualone, febrifugine, halofuginone and
bisquinazolinone ... 54 Figure 4.1: Graphical summary ... 83
LIST OF
SCHEMES
Scheme 1: Synthesis of compounds 1a-b, 2a-g and 3a-d ... 69 Scheme 2: Synthesis of compounds 4a-f and 5a-c ... 72
LIST OF ABBREVIATIONS
CAC Chloroacetyl chloride
CL Cutaneous leishmaniasis
DCM Dichloromethane
DNA Deoxy ribonucleic acid
DMF Dimethylformamide
DMSO Dimethyl sulfoxide
ELISA Enzyme-linked immunosorbent assay
ESI Electrospray ionisation
HRMS High resolution mass spectrometry IC50 50% inhibitory concentration
IIFA Indirect immunofluorescence assay
IM Intramuscular
IR Infrared spectroscopy
IV Intravenous
MCL Mucocutaneous leishmaniasis
mp Melting point
NMR Nuclear magnetic resonance spectroscopy PCR Polymerase chain reaction
PKDL Post-kala-azar dermal leishmaniasis
QZ Quinazolinone
spp Species
TEA Triethylamine
TMS tetramethylsilane
VL Visceral leishmaniasis
CHAPTER 1
Introduction and Problem Statement
1.1 Introduction
Leishmania spp. are grouped as part of a diverse collection of organisms in the Kinetoplastida order (de Souza et al., 2009). These Leishmania spp. are vector borne parasites that opportunistically infect animal and human hosts. Protozoan species can spread relatively effectively, causing tremendous suffering for millions of people across the world (Morrissette & Sibley, 2002). It is estimated that 700 000 to 1 000 000 new cases and 26 000 to 65 000 deaths occur annually, making leishmaniasis a very concerning disease (WHO, 2019).
Leishmaniasis is a disease transmitted by the unicellular parasites of the Leishmania genus, of which there are more than 20 species known to infect humans. The disease is transmitted through the bites of phlebotomine sand-flies and present in three clinically distinct forms, the first being the cutaneous form, second the mucocutaneous form and thirdly the visceral form. The visceral leishmaniasis is the most dangerous, causing excessive internal damage with a mortality rate of 95 % if left untreated (Uliana et al., 2018; WHO, 2019). Leishmaniasis is a vastly neglected tropical disease, affecting the poor and disadvantaged populations in more than 98 countries around the world. The disease is slowly developing drug resistance against the current antileishmanial drugs with an ever-increasing area of distribution and no sign of it scaling down (Alvar et al., 2012; WHO, 2018a; 2018b; 2019).
Current antileishmanial chemotherapies consists of the following drugs: pentavalent antimonials, amphotericin B, miltefosine, paromomycin and pentamidine. Miltefosine is the only drug administered orally while the intravenous route is preferred to administer the others (WHO, 2010). Each of these clinical antileishmanial drugs suffer from various limitations (Figure 1.1) and efficacy issues. Efficacy of pentamidine, paromomycin, miltefosine and pentavalent antimonials is highly varied based on geographical areas. Paromomycin is more than 94 % effective in Asia, but the efficacy in Africa varies between 46 to 85 % (Zulfiqar et al., 2017). Pentavalent antimonials efficacy in India has slowly deteriorated over years of use and is now no longer used as monotherapy in India (Haldar et al., 2011). However, amphotericin B still possesses high efficacy throughout its distribution zone (Zulfiqar et al., 2017).
The reduced efficacy of antileishmanial drug has prompted the implementation of combination therapies, to maintain efficacy and hinder resistance against these drugs. Currently, only two combination therapies are in use; pentavalent antimonials with paromomycin and miltefosine with amphotericin B (Uliana et al., 2018). Thus, it is clear that the use of current clinical antileishmanial drugs are under threat, which is further importunate with the lack of new clinical drugs. Thus, there is an urgent demand for new, safe and affordable antileishmanial drugs.
Figure 1.1: Limitations of clinical antileishmanial chemotherapy (Freitas-Junior et al., 2012; Rajasekaran & Chen, 2015; Zulfiqar et al., 2017)
Quinazolinones (QZ) are versatile compounds with a broad spectrum of biological activities, including anticancer (Chandrika et al., 2008), anti-inflammatory (Alagarsamy et al., 2009), antimicrobial (Kuyper et al., 1996), antimalarial (Verhaeghe et al., 2008) and antileishmanial (Romero et al., 2019) activity (Khan et al., 2014). Two well-known antiprotozoal QZ are febrifugine and its analogue halofuginone (Jain et al., 2017). Febrifugine is an isomeric quinazolinone alkaloid reported to present with excellent antiprotozoal activity and analogues thereof have presented with noticeable activity against Leishmania (Bule et al., 2017; Jain et al., 2017; Zhu et al., 2012). Febrifugine is extracted from the Chinese medicinal plant Dichroa febrifuga. This indigenous plant has been used to treat malaria symptoms for over 2000 years (Burns, 2008; Jiang et al., 2005). Pandey and colleagues (2017) analysed febrifugine and its analogues through molecular docking as potential trypanothione reductase inhibitors, the researchers then identified a few febrifugine analogues as potential targetable antileishmanial agents (Pandey et al., 2017).
Limitations Heat instability Hospitalisation needed for administration High cost Drug Amphotericin B Clinical antileishmanial drugs Drug Paromomycin Drug Pentamidine Drug Pentavelent antimonials Drug Miltefosine Limitations Resistance reported in India Compliance issues (painful administration and lengthy treatment) Limitations Long half-life encourages resistance Cannot be administered to pregnant patients Limitations Varying efficacy based of geographical location of the disease Limitations Potential for resistance due to varying efficacy between different strains of Leishmania
This presents the quinazolinone scaffold as a good platform to investigate for potential novel antiprotozoal drugs (Pandey et al., 2017). Febrifugine does not adhere to the standards of commercial antiparasitic drugs due to several side effects associated with its use, including nausea, vomiting and strong liver toxicity (McLaughlin et al., 2014). These unwanted effects may significantly impact patient compliance if used as therapeutic drugs, leading to below standard therapeutic dosages and subsequent drug resistance development (WHO, 2003). Thus, febrifugine’s development as a novel antiparasitic drug has been hindered and adaptation is required for possible future usage (Jiang et al., 2005; van Ooij, 2016; Zhu et al., 2012).
Furthermore, the stereochemistry of febrifugine presents a challenge for its synthesis and development into an effective and affordable antiprotozoal drug. This is clear due to studies reporting between 7 and 13 steps to synthesise febrifugine (McLaughlin et al., 2014; Smullen & Evans, 2017; Zhang et al., 2017). Indeed, febrifugine structurally contains two distinct chiral centres both located on the piperidine ring (Figure 1.2, red and blue) which results in four potential stereoisomers (two trans- and two cis isomers) during its synthesis (McLaughlin et al., 2014).
N N O ∗ O ∗ H N HO
Figure 1.2: Structure of febrifugine, the coloured/highlighted area indicates chirality
The trans-a isomer (Figure 1.3, blue) has the highest potency and provides the desired antiprotozoal activity when compared to the other three. Consequently, a lower total antiprotozoal activity is obtained when all four isomers are present in a combination (McLaughlin et al., 2014).
O H N HO O H N HO O H N HO O H N HO N N N N trans-a trans-b cis-a cis-b N N N N O O O O
Figure 1.3: The interconversion of febrifugine
Furthermore, the tautomer of febrifugine, isofebrifugine (Figure 1.3), is produced in co-occurrence during the synthesis. Isomerisation takes place between these isomers to maintain an equilibrium (Figure 1.4) (Barringer et al., 1973; McLaughlin et al., 2014). This process takes place under acidic conditions (as those prevailing in gastric medium) between the hydroxy group on the piperidine ring and the carbonyl (Figure 1.2, purple) (McLaughlin et al., 2014).
N N O ∗ O ∗ H N HO H+ ∗ ∗ H N O HO N N O Febrifugine +(-) Isofebrifugine isomerization
Figure 1.4: Isomerisation of febrifugine into isofebrifugine
Although isofebrifugine also possesses antiprotozoal activity, it is lower than that of febrifugine. Thus, the combination of all four stereoisomers and isofebrifugine ultimately reduces the total amount of antiprotozoal activity produced. Therefore, the most pharmacologically active stereoisomer must be separated from the rest.
However, this proves to be as challenging as the normal synthesis of febrifugine (Barringer et al., 1973; McLaughlin et al., 2014; Smullen & Evans, 2017; Zhu et al., 2010). Thus, strategies have been devised to eliminate either the chirality and/or the tautomerisation as this will result in a less tedious synthesis. Since the hydroxypiperidine moiety presents the primary synthetic challenge (two chiral centres) of febrifugine, its modification would solve the problematic isomerisation while possibly retaining or improving the antiprotozoal activity. However, there is no research consensus on the importance of the modification in regards to its impacts on the antiprotozoal activity. Some literature reportedly states that modification of the acetonyl tether (Figure 1.5, burgundy) and/or the piperidine ring (Figure 1.5, green) results in a complete loss of antiprotozoal activity, suggesting, the hydroxyl group and nitrogen of the piperidine ring to be necessary for antiprotozoal activity (Jiang et al., 2005).
N N O O H N HO
Figure 1.5: Structure of febrifugine, the coloured/highlighted area indicates the acetonyl tether and the piperidine ring
Others assign a decrease in toxicity and an increase in antiprotozoal activity to modification of the piperidine ring, demonstrating that this is well-tolerated for modification (Fernández-Álvaro et al., 2016). Furthermore, the methylene group in the acetonyl linker (Figure 1.6, pink) is reported to have no influence on the antiprotozoal activity of febrifugine while the QZ moiety is required for activity (Chien & Cheng, 1970). Thus, there is a clear controversy with regards to the impact that the modification of the piperidine ring and acetonyl linker has on the antiprotozoal activity of febrifugine. Subsequently, only investigation of further derivatives devoid of these structural features will allow assessment of their importance. A possible avenue is to replace the problematic piperidine ring with another ring devoid of an alcohol group and non-chiral, such as another quinazolinone ring attached in its place.
N N O O H N HO methylene group
Figure 1.6: Structure of febrifugine, the coloured/highlighted area indicates the methylene group on the acetonyl linker
By replacing this piperidine ring with 4(3H)-quinazolinone to form a bisquinazolinone derivative, the synthetic limitations of febrifugine could be resolved. Quinazolinone derivatives have been reported to possess antileishmanial activity and, by linking two QZ moieties while keeping the acetonyl tether, this is synthetically achievable and may result in improved antiprotozoan activity (Romero et al., 2019; Sahu et al., 2017). Literature on antiprotozoal activity of bisquinazolinones is scanty. However, these derivatives have shown antifungal, antimicrobial and antifeedant activities, thus giving an indication of pesticidal activity linked to these compounds (Reddy et al., 2002; 2010). Thus, in this study, bisquinazolinones wherein a 1,4-quinozolinone replaces the piperidine ring of febrifugine to remove all synthetic challenges, were synthesised and the antileishmanial activity assessed.
Over many years 1,4-benzothiazine (Figure 1.7) derivatives have extensively been investigated with many presenting promising hits for further development. The 1,4-benzothiazine derivatives present with a wide range of biological properties, including antibacterial, antiviral, antimalarial and antifungal activity (Lam et al., 2013). Thiaplakortone A and methyl 5,9-dioxo-3,4,5,9-tetrahydro-2H-thieno(2',3':4,5)benzo(1,2-b)(1,4)thiazine-7-carboxylate 1,1-dioxide (Figure 1.8) are both similar to 1,4-benzothiazine derivatives and have shown good antiprotozoal activity. These studies have shown that thiazine derivatives can be incorporated into different structures and still retain antiprotozoal activity (Aswathy et al., 2016; Davis et al., 2013; Lam et al., 2013). Thus, replacing the quinazolinone ring of febrifugine with a 1,4-benzothiazine scaffold may produce compounds with potent antileishmanial activity and reduced toxicity.
N S
Figure 1.7: Structure of 1,4-benzothiazine
N H S O O O O S O O N H S N H O O O O NH2 Thiaplakortone A Methyl 5,9-dioxo-3,4,5,9-tetrahydro-2H-thieno(2',3':4,5)benzo(1,2-b)(1,4)thiazine-7-carboxylate 1,1-dioxide
Figure 1.8: Chemical structures of methyl
5,9-dioxo-3,4,5,9-tetrahydro-2H-thieno(2',3':4,5)benzo(1,2-b)(1,4)thiazine-7-carboxylate 1,1-dioxide and thiaplakortone A
In summary, there is an important need for new antileishmanial treatments. Febrifugine provides a promising scaffold to work from. However, toxicity is a major drawback for febrifugine. The synthesis of modified febrifugine analogues is a possible solution to the problem.
1.2 Aims and objectives
The aim of this study was to investigate novel analogues of N-3 substituted bisquinazolinone and related quinazolinone compounds (Figure 1.6, orange) in the search for new, effective, safe and affordable antileishmanial agents.
The objectives of this study were:
• To synthesise (in two or three steps) novel quinazolinone, bisquinazolinone and thiazine derivatives through nucleophilic substitution at N-3 of the 4-(3H)quinazolinone and
confirm their structures using IR (infrared spectroscopy), NMR (nuclear magnetic resonance spectroscopy) and HRMS (high-resolution mass spectrometry) techniques. • To assess in vitro the antileishmanial activity of the quinazolinone, bisquinazolinone and
thiazine derivatives against various strains of Leishmania parasite.
• To assess in vitro cytotoxicity profiles of the antileishmanial active compounds against mammalian cell lines.
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CHAPTER 2
Literature Review
2.1 Introduction
Parasites are a diverse collection of organisms that have troubled human societies for centuries. A class of parasites that includes Leishmania spp. (agents for leishmaniasis), called Kinetoplastida parasites, is of significant interest. Kinetoplastida parasites bear a peculiar single mitochondrion that exhibits a condensed network of DNA at the flagellar basal body. This arrangement of the DNA is called the kinetoplast and it presents a new target for drug development towards highly neglected diseases, such as leishmaniasis (de Souza et al., 2009; Rao et al., 2018).
Leishmaniasis is known to affect poor societies across the world, and is associated with malnutrition, lack of resources and weak immune systems (WHO, 2018b; 2019a). The disease is caused by Leishmania, a unicellular parasite with ancient origins, dating back to 1500-2500 BCE (Steverding, 2017). Leishmaniasis is a vector-borne disease that spreads effectively and thus hinders disease management.
The female sand-fly requires mammalian blood for reproduction purposes, thus it is the vector responsible for the transmission of the disease. Consequently, Leishmania has a digenetic life cycle (Figure 2.3), the first stage occurs inside the sand-fly (the promastigote form) and the second inside the mammalian host (amastigote form). The promastigote is the extracellular, flagellated and motile form of the parasite responsible for infection of the mammalian host. The amastigote is the clinically relevant, non-flagellated and non-motile form that multiplies inside the macrophages of the mammalian host (Singh et al., 2012).
The range of mammalian hosts include humans, marsupials, canids and rodents (Steverding, 2017). There are more than 20 known Leishmania spp. that infect humans. Leishmaniasis presents in three main clinical forms, namely: i) cutaneous (Figure 2.1), which is the most common; ii) mucocutaneous (also known as espundia) and iii) visceral leishmaniasis (Figure 2.2) (also known as kala-azar), which is the most serious (fatality rate of 95 % if left untreated) of the three forms (Chappuis et al., 2007; WHO, 2019a). Infection is separated by geographical distribution into Old World and New World leishmaniasis (Figure 2.4). Old World refers to infection by species found in Africa, the Middle East and the Mediterranean basin. New World refers to the species found in Central America, South America and Mexico (David & Craft, 2009).
Figure 2.3: Life cycle of Leishmania (CDC, 2018)
2.2 Epidemiology
Leishmaniasis is endemic to 98 countries and is among the most neglected and devastating tropical diseases in the world (Alvar et al., 2012; Zulfiqar et al., 2017).
The disease is the third biggest cause of parasite-related deaths in the world, with malaria being the first and schistosomiasis the second (WHO, 2017). As of March 2019, the estimated annual number of new leishmaniasis cases was 700 000 to 1 million, of which 50 000 to 90 000 were visceral leishmaniasis cases. Furthermore, the annual death count of leishmaniasis is estimated between 26 000 to 65 000 (WHO, 2019a). The annual increase in the number of cases (27 000 new cutaneous cases in 2018 compared to 2017 and a total increase of 173 000 annual cases compared to 1998) is concerning for the affected people and the WHO (WHO, 2019b).
Figure 2.4: Different species of Leishmania of the old and new world that infect humans (WHO, 2010)
The duration of which the parasite remains in the vector varies from 4 to 18 days, depending on temperature. High temperatures shorten this time period and vice versa (Torres-Guerrero et al., 2017). After this short duration within the vector, the parasite is transmitted to the human host and starts the incubation proses. This incubation period will differ for each Leishmania species (section 2.5) (WHO, 2010).
The Leishmania parasite is climate sensitive, resulting in strong ecological effects with the smallest change of climate. Global warming may drastically affect the epidemiology of leishmaniasis (WHO, 2019a) by increasing surface area of distribution to areas where the disease had previously not been endemic. This, combined with human migration, illustrates that Leishmania evolution is intrinsically tied with human activity (Steverding, 2017; WHO, 2010).
Furthermore, it emphasises the role that humans play in increasing the area of distribution and drug resistance of Leishmania (Zarlenga et al., 2014). These factors, combined with the increase in treatment failures (predominately due to drug resistance), are alarming (Uliana et al., 2018).
2.3 Diagnosis and symptoms
Leishmaniasis is diagnosed using serological and/or parasitological tests that can be combined with clinical symptoms to ascertain the infecting species. Parasitological tests use either microscopic examinations of tissue samples to observe the parasite or polymerase chain reaction (PCR) to detect parasite DNA within the samples (WHO, 2010).
Figure 2.5: Classification of Leishmaniasis based on clinical appearance (WHO, 2019a)
Parasitological tests are predominantly used for the diagnosis of visceral and cutaneous leishmaniasis, and they provide good sensitivity (93 – 99% in the spleen, 53 – 86% in bone marrow and 53 – 65% in lymph nodes) (WHO, 2010). Serological tests predominately use either the indirect immunofluorescence assay (IIFA) or enzyme-linked immunosorbent assays (ELISAs). ELISAs measure the amount of anti-α-galactosyl (antibody) that, on average, is nine times higher in infected people. IIFA uses compound stains to detect antibodies in the serum.
The stain emits colour under ultraviolet light which is used for detection (Burza et al., 2018; Chatzis et al., 2014; Goto & Lindoso, 2010; WHO, 2010).
The symptoms of this disease differ between the three clinical forms. Cutaneous leishmaniasis mainly presents as ulcers and skin lesions, while mucocutaneous leishmaniasis causes a partial or total loss of the mucous membranes inside the throat, mouth and nose. Visceral leishmaniasis is characterised by an enlarged spleen and liver, as well as anaemia, weight loss and irregular fevers (WHO, 2019a). Thus, distinguishing between the three clinical forms is quite simple (Figure 2.5).
2.4 Disease control
Vector control is generally achieved through insect repellents, insecticides, bed nets and the removal of stagnant water in the surrounding areas. It is quite straightforward but labour intensive and expensive. Thus, disease control strategies mainly focus on the treatment of the disease in humans and not prevention (Reithinger et al., 2007). Prophylaxis consists of wearing thick and long-sleeved clothes and pants that can be impregnated with insecticides or repellents ( Torres-Guerrero et al., 2017). These methods have been quite successful in the geographical areas of leishmaniasis, but treatment with drug therapy remains the most effective method used against leishmaniasis (Monzote, 2011; Uliana et al., 2018; WHO, 2019a).
2.5 Clinical forms of leishmaniasis
2.5.1 Mucocutaneous leishmaniasis
Mucocutaneous leishmaniasis is predominantly caused by L. braziliensis and L. panamensis, but any Leishmania species can cause this form of leishmaniasis. This disease form is mainly reported in Brazil, Bolivia, Ethiopia and Peru, and is clinically characterised by lesions in the mucosa of the nose and mouth (WHO, 2010; 2019a). The best treatment for mucocutaneous leishmaniasis remains chemotherapy (section 2.6.2.1) (Sundar & Chakravarty, 2015).
2.5.2 Cutaneous leishmaniasis
L. major is responsible for the majority of cutaneous leishmaniasis infections, the easiest form to diagnose and treat (Figure 2.4). In 2017, 95% of new cases occurred in six countries: Afghanistan, Algeria, Brazil, Colombia, Iran and Iraq (WHO, 2019a).
Clinical features differ between and within geographical areas, but consist of a lesion which starts as a nodule or papule at the site of inoculation. This grows slowly within the period of a week and eventually develops a central crust which could fall away and leave an exposed ulcer. Incubation periods differ between Leishmania species, but mainly range from 2 − 8 months (WHO, 2010). Local wound care is indicated if patients have fewer than four lesions and/or if the lesions are less than 5 cm in diameter. If local therapy is required, the options are as follows: cryotherapy, thermotherapy and chemotherapy (Sundar & Chakravarty, 2015).
2.5.3 Visceral leishmaniasis
L. donovani is one of two strain of Leishmania that causes visceral leishmaniasis (also known as black fever) (Figure 2.4) and is one of the main strains to be studied in detail by researchers. This Leishmania species is generally found in India, Sudan and Ethiopia (Burza et al., 2018). If left untreated, significant internal damage can occur and invariably leads to the death of the patient. Incubation periods for visceral leishmaniasis varies in range from 10 days to 1 year and tends to be chronic in nature and predominately affects children (WHO, 2010). Common signs include fevers, chills and weight loss (Figure 2.5). Treatment varies for visceral leishmaniasis caused by L. donovani, but pentavalent antimonials (section 2.6.2.1) and amphotericin B (section 2.6.2.2) are the two preferred therapies in use (Murray et al., 2005; Sundar & Chakravarty, 2015).
Post-kala-azar dermal leishmaniasis (PKDL) appears in areas endemic to L. donovani and could appear concurrently or after the apparent cure of visceral leishmaniasis. It usually manifests between 6 months to a year after the initial visceral infection (WHO, 2010). Hypopigmented or erythematous macules on any part of the body may become nodular, specifically the face. It may also appear on the conjunctiva, buccal and genital mucosa (WHO, 2010). In east Africa the disease predominately heals over time without any treatment, but this is not the case in India where treatment application is needed (Burza et al., 2018).
2.6 Leishmaniasis therapy
2.6.1 Physical therapy
Local treatment consists mainly of the methods, thermotherapy or cryotherapy. Thermotherapy is based on in vitro laboratory studies which has shown Leishmania parasite not to multiply at temperatures above 39°C. Consequently, it was adopted as a form of leishmaniasis treatment
Thermotherapy is a low-cost treatment, requires fewer treatment intervals, provides minimal scarring and improves patient compliance (Chakravarty & Sundar, 2019). It is a very effective treatment and a good alternative option for patients with contraindications towards systemic treatments. Cryotherapy has mainly been used against old world CL and is performed by applying liquid nitrogen (-195°C) to a localised infected area of a patient. This causes the formation of intracellular ice crystals that causes ischemic necrosis at the infected site (Chakravarty & Sundar, 2019).
2.6.2 Leishmaniasis chemotherapy
Conventional treatment used against leishmaniasis consists of either one or a combination of the following drugs; pentavalent antimonials, amphotericin B, paromomycin, pentamidine and miltefosine (WHO, 2010). These drugs have been in use for extended periods, which consequently led to the development of signs of resistance and higher toxicity profiles. Additionally, the current rate of hit discovery in leishmaniasis is far behind that of other neglected diseases (Balana-Fouce et al., 2019). Thus, until new drugs are introduced into the market, the current antileishmanial agents are all that are available to fight an ever-growing disease (Uliana
et al., 2018). This highlights the importance of research into the discovery and development of new antileishmanial drugs.
2.6.2.1 Pentavalent antimonials
Pentavalent antimonials were the first class of drugs used for mucocutaneous, cutaneous and visceral leishmaniasis treatment (WHO, 2010; Zulfiqar et al., 2017). For decades, these drugs played an enormous role as first line drugs in antileishmanial chemotherapy. Drugs in this class include sodium stibogluconate (Figure 2.6) and meglumine antimoniate (Figure 2.6). Their molecular and cellular mechanisms of action are not yet fully understood (Mishra et al., 2007). It is also not clear if Sb(III) or Sb(V) is the final active form that interacts with Leishmania (Haldar et
al., 2011). For example, one suggested mode of action is the inhibition of DNA topoisomerases, but no exact mechanism has been reported (Lucumi et al., 1998).
O Sb O Sb O OH HO O O OH OH OH O O O O O Na Na Na H N OH OH OH OH OH Sb OH O O
Pentostam (sodium stibogluconate) Glucantime (meglumine antimoniate)
1 2
Figure 2.6: Chemical structures of sodium stibogluconate (1) and meglumine antimoniate (2)
These drugs were demoted to second line drugs due to increased resistance cases when used in monotherapy. Subsequently, the WHO has recommended the use of pentavalent antimonials in combination with amphotericin B (section 2.6.2.2) (Uliana et al., 2018; WHO, 2010). It is thus rarely used as monotherapy. An additional motivation for the recommended combination is the fact that pentavalent antimonials possess some severe adverse effects which include; cardiotoxicity, pancreatitis and anaemia (Figure 2.12) (Burza et al., 2018). Moreover, the requirement for hospitalisation to administer these drugs further restricts their usage (Palumbo, 2010). Furthermore, widespread drug misuse has contributed to the emergence of resistant Leishmania strains, possibly limiting the future use of pentavalent antimonials in antileishmanial therapies (Croft et al., 2006).
2.6.2.2 Amphotericin B
First isolated from Streptomyces nodosus in 1955, amphotericin B presents excellent antileishmanial activity (Figure 2.7) and is a good alternative for the treatment of resistant leishmanial infections with antimonials (Jha et al., 1995; Kleinberg, 2006). However, toxicity is the biggest drawback with amphotericin B (Figure 2.11), which is reduced by using lipid formulations (Lemke et al., 2005). Amphotericin B is active by causing pore formation in the cell membrane, resulting in a fatal constituent efflux (Le Pape, 2008; Monzote, 2011). It is currently a first line treatment against visceral leishmaniasis with no documented drug resistance (WHO, 2010; Zulfiqar et al., 2017).
O O OH OH HO O OH OH OH OH OH O OH NH2 HO OH O 3
Figure 2.7: Chemical structure of Amphotericin B (3)
2.6.2.3 Paromomycin
This antibiotic (Figure 2.8), also known as aminosidine, is used in parenteral formulations for the treatment of visceral leishmaniasis, with topical and parenteral formulations used for the treatment of cutaneous leishmaniasis (Sundar & Chakravarty, 2015). Paromomycin is reported to act by binding to the 30S ribosome subunit of Leishmania and inhibiting protein synthesis. Additional research is required on paromomycin to confirm the exact mechanism of action against the Leishmania parasite (WHO, 2010). This drug has the advantage of being a low-cost alternative treatment with limited drug resistance, making it a promising drug in combination therapy, specifically when used with pentavalent antimonials (Uliana et al., 2018). The adverse effects of paromomycin includes ototoxicity, pain and oedema (Figure 2.11) (Zulfiqar et al., 2017).
O O O O O O H2N OH OH OH NH2 H2N HO NH2 HO HO H2N OH HO 4
Figure 2.8: Structure of Paromomycin (4)
2.6.2.4 Pentamidine
Pentamidine (Figure 2.9) affects the DNA synthesis of the Leishmania parasite, promoting the fragmentation of the mitochondrial membrane and, in turn, leading to cell death (de Almeida Machado et al., 2019). The biggest advantages of pentamidine include no documented resistance and shorter dosage regimens that improves patient compliance. However, it does possess some severe adverse effects which include; headache, hyperglycaemia, hypotension and vomiting (Figure 2.11) (Zulfiqar et al., 2017). The drug is mainly used for the treatment of visceral leishmaniasis and is administered either by the intravenous or intramuscular route (WHO, 2010). This is a disadvantage due to the pain, induration and sterile abscess that forms at the site of injection and subsequently lowers patient compliance (Sundar & Chakravarty, 2015).
O O H2N NH NH2 NH 5
2.6.2.5 Miltefosine
Miltefosine (Figure 2.10) was the first drug used as oral treatment against visceral leishmaniasis (Dorlo et al., 2012). Advantages of miltefosine include high cure rates among the old world leishmaniasis, convenient and practical logistics, as well as less severe adverse effects (de Almeida Machado et al., 2019). These include nausea, vomiting and diarrhoea, all which are much simpler to treat when compared to the adverse effects of other antileishmanial drugs (Figure 2.11) (David & Craft, 2009). Miltefosine acts by inhibiting kinase B (Akt protein), an important protein for intracellular signalling and protozoan cell survival (Dorlo et al., 2012).
P O O O O N C16H33 6
Figure 2.10: Chemical structure of Miltefosine (6)
Disadvantageously, miltefosine is expensive and associated with teratogenic effects. Together with a long half-life (± 7 days), frequent use of this drug is not advised (Dorlo et al., 2012). However, careful monitoring and its use in combination with amphotericin B reduce the adverse effects and the development of resistance (Sundar & Chakravarty, 2015; WHO, 2010). Efficacy of the drug is still high (<94%) with only laboratory strains reported to have developed resistance (Zulfiqar et al., 2017).
Figure 2.11: Adverse effects of antileishmanial therapies (Bhargava & Singh, 2012; Le Pape, 2008)
2.6.3 Drug resistance
Genes responsible for drug resistance in Leishmania have not been fully identified. This presents a challenge towards predicting in which manner each species of Leishmania will evolve drug resistance. This results in a scarcity of literature coverage on the topic and the prevention of antileishmanial drug resistance (Alonso et al., 2018). The lack of literature, new clinical drugs and alternatives to the current antileishmanial drugs albeit reduced effectiveness, altogether placed these clinical antileishmanial agents under pressure. This results in overuse, which could further raise the spectre of widespread resistance (de Koning, 2017; Pramanik et al., 2019). Consequently, there is an urgent need for new, safe, effective and affordable antileishmanial drugs (Bekhit et al., 2018).
2.6.4 Drug combination therapy
Treatment failures due to sub-therapeutic dosages and the ever-growing antileishmanial drug resistance against monotherapies, express the need for alternative treatment methods.
A suggested solution, which is receiving increasing attention, has been the implementation of drug combination therapies. The rationale behind drug combination therapy is synergism of therapeutic effects when drugs with different pharmaceutical properties are used in combination with the result of increased effectiveness of each individual agent. This method shortens drug therapy periods, lowers dosages and reduces adverse effects of each drug, which ultimately lowers the possibility of drug resistance from emerging and improves patient compliance (Sundar & Chakravarty, 2013; 2015).
Since January 2017, two antileishmanial drug combinations have been approved as effective regimens for the treatment of the disease. First, the combination of pentavalent antimonials and paromomycin. This combination has been proven effective against visceral leishmaniasis in India and Africa, with reduced treatment duration compared to the pentavalent antimonials alone (Uliana et al., 2018). This combination has been advocated for use against visceral leishmaniasis in east Africa and Yemen since 2010 (WHO, 2010). Second, the combination of amphotericin B and miltefosine. This combination showed high efficacy and good tolerability during the investigation of its therapeutic potential against visceral leishmaniasis (Sundar et al., 2011). This combination resulted in a reduced treatment period, toxicity and cost, and an increase in the ease of drug use and protection against drug resistance emergence (Uliana et al., 2018). Thus, current antileishmanial combination therapies signify the importance of combination therapy to curb adverse effects, take advantage of synergistic effects, reduce treatment cost and slow the spread of drug-resistant leishmanial strains.
2.7 Novel antileishmanial drug discovery
The introduction of a new drug to the pharmacological market is painstaking work. The limited number of clinically viable drugs is concerning and thus a need for a new class of antileishmanial compounds is desired (Uliana et al., 2018). Many scaffolds for Leishmania treatment have been explored, including: triazoles (Meinel et al., 2020), flavonols (Borsari et al., 2019), hydrazides (de Lima et al., 2019) and quinazolinones (Jain et al., 2017). Quinazolinone as a scaffold specifically has been an excellent pharmacophore in the discovery of novel therapeutic agents, and as such may be further exploited for the development of new antileishmanials.
2.7.1 Quinazolinones
Quinazolinones (Figure 2.12) are heterocyclic nitrogen compounds and the building blocks for over 150 naturally occurring alkaloids (Mhaske & Argade, 2006).
The first quinazolinone derivative (2-cyano-3,4-dihydro-4-oxoquinazoline) was synthesised in 1869 (Figure 2.12) by P. Griess and was preceded by many other derivatives produced over the years (Griess, 1869). These scaffolds have attracted attention due to their potential to result in novel compounds with diverse pharmacological properties, which include antidiabetic (Malamas & Millen, 1991), anticancer (El-Bordany & Ali, 2018), antihypertensive (Jain et al., 2008), anti-inflammatory (Alagarsamy et al., 2002), antimalarial (Verhaeghe et al., 2008), antileishmanial (Romero et al., 2019), antimicrobial (Kuyper et al., 1996), anticonvulsant (Ugale & Bari, 2014) and antioxidant activities (Kumara et al., 2018).
N NH O N NH O N 7 8 4(3H)-quinazolinone 2-cyano-3,4-dihydro-4-oxoquinazoline
Figure 2.12: Chemical structure of 4(3H)-quinazolinone (7) and 2-cyano-3,4-dihydro-4-oxoquinazoline (8) (Griess, 1869)
The literature is extensive on synthesised quinazolinone derivatives and their possible biological activities. The interest in this class of compounds is evident from the high output of synthetic analogues and the presence of these scaffolds in many marketed drugs. Raltitrexed (Figure 2.13) is a good example of a quinazolinone derivative anticancer drug used in the treatment of colorectal cancer and is well tolerated (Liu et al., 2014) despite its side effects including; anaemia, asthenia, nausea and vomiting (Khan et al., 2016; Khan et al., 2019).
NH N O N S NH HO O O O HO 9
Figure 2.13: Chemical structure of Raltitrexed (9)
Febrifugine (Figure 2.14) is another quinazolinone derivative that possesses potent antiprotozoal activity, but is not clinically used due to adverse effects, including nausea, vomiting and liver toxicity (McLaughlin et al., 2014). Febrifugine and its isomer, isofebrifugine (Figure 2.14) are isolated from the roots (Chang Shan) and leaves (Shuu Chi) of Dichroa febrifuga, a Chinese medicinal plant used locally for over 2000 years to treat malaria fevers (Jiang et al., 2005; Koepfli
et al., 1947).
The combination of good availability, fast-acting effect and no drug resistance makes febrifugine an appealing compound (Bule et al., 2017). However, the adverse effects and difficult synthesis of febrifugine limited its clinical use (Burns, 2008; Jiang et al., 2005). Active febrifugine derivatives, including halofuginone (Figure 2.15) have been synthesised to remedy these adverse effects. However, the results have been unsatisfactory due to most derivatives conserving the side effects of the parent drug, febrifugine (Jiang et al., 2005; Ryley & Betts, 1973).
N N O ∗ O ∗ H N HO H+ ∗ ∗ H N O HO N N O Febrifugine +(-) Isofebrifugine isomerization 10 11
Halofuginone was originally synthesised in the 1960s as a potential antimalarial agent (Figure 2.15). It is now an approved feed additive used worldwide in the commercial poultry industry as an anti-coccidiosis agent (Jiang et al., 2005; Ryley & Betts, 1973). Halofuginone was also previously used to treat cattle against protozoan parasites (Peeters et al., 1993).
N N O O H N HO Cl Br 12
Figure 2.15: Chemical structure of halofuginone
Febrifugine and its synthetic derivative halofuginone have garnered much attention due to their excellent potency and pharmacological activities, such as anticancer (Pines & Spector, 2015) and antileishmanial activity (Sheffer et al., 2007). In fact, both quinazolinones possess better antiprotozoan activity when compared to quinine and chloroquine (McLaughlin et al., 2014; Zhu
et al., 2012). Numerous studies covered these compounds. A docking study by Pandey and colleagues (2017) confirmed febrifugine analogue 6-chloro-3-[3-(3-hydroxypiperidin-2-yl)-2-oxopropyl]-7-(pyridin-4-yl)quinazolin-4(3H)-one (Figure 2.16) to be a potential drug candidate to treat L. donovani infections (Pandey et al., 2017). Jain et al. (2017) also demonstrated febrifugine and halofuginone derivatives (such as (S)-6,7-difluoro-3-[3-(3-oxopiperidin-2-yl)propyl]quinazolin-4(3H)-one) to be potent against the L. major parasite and that improved tolerability could be achieved (Figure 2.16). These recent studies provide a strong ground for further investigation into antileishmanial activities of febrifugine derivatives in order to produce new clinical drugs.
N N O N O N H HO Cl 13 N N O H N O F F 14 (S)-6,7-difluoro-3-[3-(3-oxopiperidin-2-yl)propyl]quinazolin-4(3H)-one 6-chloro-3-[3-(3-hydroxypiperidin-2-yl)-2-oxopropyl]-7-(pyridin-4-yl)quinazolin-4(3H)-one
Figure 2.16: Chemical structure of 6-chloro-3-[3-(3-hydroxypiperidin-2-yl)-2-oxopropyl]-7-(pyridin-4-yl)quinazolin-4(3H)-one (13) and
(S)-6,7-difluoro-3-[3-(3-oxopiperidin-2-yl)propyl]quinazolin-4(3H)-one (14) (Prolyl-tRNA synthetase IC50 ± 68 nM)
Structurally, febrifugine has a complex stereochemistry. The structure contains two distinct chiral centres on the piperidine ring (Figure 2.14, red and blue stars), which translate into four possible stereoisomers (Figure 2.17) (McLaughlin et al., 2014). All four isomers possess antiprotozoal activity, but only one (Figure 2.17, trans-a, as purple) possesses the highest antiprotozoal potency. Furthermore, its tautomer, isofebrifugine (Figure 2.14) is produced in co-occurrence during febrifugine synthesis and undergoes isomerisation into febrifugine to maintain an equilibrium (Barringer et al., 1973; McLaughlin et al., 2014).
O H N HO O H N HO O H N HO O H N HO N N N N trans-a trans-b cis-a cis-b N N N N O O O O
Figure 2.17: The interconversion of febrifugine
Several studies with the aim of improving the synthetic routes (shorter synthetic routes or improved synthetic yields) for febrifugine and halofuginone have been conducted (Smullen & Evans, 2017, Xu et al., 2019; Zhang et al., 2017). Procedural limitations include time-costs, increased number of steps ranging from 7 to 13, low yields (2 – 17%) and isomerisation which still takes place (McLaughlin et al., 2014; Smullen & Evans, 2017; Zhang et al., 2017). Ultimately, these attempts at route improvement of the synthesis febrifugine and halofuginone were unsuccessful.
2.7.2 Bisquinazolinones
Limited literature is available on the synthesis and biological activity of bisquinazolinones. Only a handful of bisquinazolinones have been synthesised and very limited biological testing has been conducted on them. A study by Reddy and colleagues showed bisquinazolinone, 2,2’-{1,1’-[3,3’-dimethyl-(1,1’-biphenyl)-4,4’-diyl]bis[4-oxoazetidine-2,1-diyl]}bis[3-methylquinazolin-4(3H)-one] (Figure 2.18) with antimicrobial, antifungal and antifeedant activities (Reddy et al., 2002; Reddy
et al., 2010). Morsy and co-workers synthesised bisquinazolinone compounds from bis-benzoxazin-4-one (Figure 2.22, N,N’-{2,2’-(ethane-1,2-diyl)bis[4-oxoquinazoline-3,2(4H)-diyl]}bis(N-acetylacetamide) (Figure 2.18) which presented with insecticidal activity (Elshahawi et
al., 2016). This shows that quinazolinone retains biological properties, even in bisquinazolinone compounds.
N N O N N O N N O O O O N N N N N N O O O O N,N'-{2,2'-(ethane-1,2-diyl)bis [4-oxoquinazoline-3,2(4H)-diyl]}bis (N-acetylacetamide) 2,2'-{1,1'-[3,3'-dimethyl-(1,1'-biphenyl)-4,4'-diyl]bis [4-oxoazetidine-2,1-diyl]}bis[3-methylquinazolin-4(3H)-one] 15 16
Figure 2.18: Chemical structure of 2,2’-{1,1’-[3,3’-dimethyl-(1,1’-biphenyl)-4,4’-diyl]bis[4-oxoazetidine-2,1-diyl]}bis[3-methylquinazolin-4(3H)-one] (15) (Reddy et al., 2010) and
N,N’-{2,2’-(ethane-1,2-diyl)bis[4-oxoquinazoline-3,2(4H)-diyl]}bis(N-acetylacetamide) (16) (Spodoptera littoralis LC50 147.895 ppm) (Elshahawi et al., 2016)
2.7.3 Bioactive thiazine and benzothiazine derivatives
The antiprotozoal properties of thiazines were first reported in 1891 and since then various studies have been conducted on these compounds (Ehrlich & Guttmann, 1891). More than a century later, it is now known that thiazine and benzothiazine derived compounds (Figure 2.19) have a diverse array of medicinal properties, which in turn provides a wide range of pharmaceutical activities (Badshah & Naeem, 2016). Antipathogenic activities include antimicrobial (Haider & Haider, 2012), antiviral (Kharb et al., 2011), antifungal (Sarmiento et al., 2011), anticancer ( Magd-El-Din et al., 2012; Wang et al., 2012) and antipsychotic (Odin et al., 2013; Blokhina et al., 2014) activity (Choudhary et al., 2018).
N S N H S Thiazine 1,4-benzothiazine 17 18
Figure 2.19: Chemical structure of thiazine (17) and 1,4-benzothiazine (18)
Lam et al. (2013) reported various thiazine derivatives (Figure 2.20, methyl 5,9-dioxo-3,4,5,9-tetrahydro-2H-thieno(2’,3’:4,5)benzo(1,2-b)(1,4)thiazine-7-carboxylate 1,1-dioxide) to have good antiprotozoal activity, including antileishmanial activity (Lam et al., 2013). This infers that benzothiazines (Figure 2.20, green) can be incorporated into cyclic structures without losing antiprotozoal activity (Lam et al., 2013).
N H S O O O O S O O 19
Figure 2.20: Chemical structure of methyl
5,9-dioxo-3,4,5,9-tetrahydro-2H-thieno(2’,3’:4,5)benzo(1,2-b)(1,4)thiazine-7-carboxylate 1,1-dioxide (19) (L. donovani IC50 6.3
μM)
The next chapter, Chapter 3, is a manuscript formatted to be submitted to the European Journal of Medicinal Chemistry, wherein details of the synthesis, analytical and biological assessment of novel febrifugine, benzothiazine and bisquinazolinone derivatives will be covered and discussed. The chapter will discuss chemical and biological properties of these novel quinazolinone derivatives and expand on future endeavours regarding this work.
