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Division of Pharmaceutical Chemistry and Technology
Faculty of Pharmacy
University of Helsinki
Finland
SYNTHESIS AND EVALUATION OF
ANTICHLAMYDIAL, ANTILEISHMANIAL AND
ANTIMALARIAL ACTIVITY OF
BENZIMIDAZOLE AND BENZOXADIAZOLE
DERIVATIVES
Leena Keurulainen
ACADEMIC DISSERTATION
To be presented, with the permission of the Faculty of Pharmacy of
the University of Helsinki, for public examination in Auditorium 2, Korona Infocenter
(Viikinkaari 11), in June 26, 2015, at 12 noon.
Helsinki 2015
Supervisors Professor Jari Yli-Kauhaluoma
Division of Pharmaceutical Chemistry and
Technology
Faculty of Pharmacy
University of Helsinki
Helsinki, Finland
Dr. Paula Kiuru
Division of Pharmaceutical Chemistry and
Technology
Faculty of Pharmacy
University of Helsinki
Helsinki, Finland
Reviewers Associate Professor Adam Renslo
School of Pharmacy
University of California San Francisco
San Francisco, USA
Professor Angel Messeguer
The Institute for Advanced Chemistry of Catalonia
Consejo Superior de Investigaciones Científicas
Barcelona, Spain
Opponent Professor Danijel Kikelj
Faculty of Pharmacy
University of Ljubljana
Ljubljana, Slovenia
Leena Keurulainen 2015
ISBN 978-951-51-1239-2 (paperback)
ISBN 978-951-51-1240-8 (PDF)
ISSN 2342-3161 (print)
ISSN 2342-317X (online)
http:/ethesis.helsinki.fi
Helsinki University Printing House
Helsinki 2015
i
ABSTRACT
The aim of this thesis was to synthesize 1H-benz[d]imidazole- and
benzo[c][1,2,5]oxadiazole-derived compounds active against intracellular
bacterium Chlamydia pneumoniae and protozoan parasites Leishmania
donovani, and Plasmodium falciparum, and to find new potent compounds
as hit molecules for further development.
A number of issues dictate the importance of the pursuit of this work.
First, C. pneumoniae contributes to human health by being a widespread
bacterium and causing respiratory infections such as pneumonia. In
addition, atherosclerosis has been shown to be connected to the bacterium’s
persistent form. Second, a neglected tropical disease, visceral leishmaniasis
in turn is caused by a protozoan parasite L. donovani and can be fatal if left
untreated. Its current treatments suffer from toxicity, poor compliance and
prevalent parasite resistance. Third, another tropical disease, malaria is
caused by protozoan parasites belonging to the genus Plasmodium. P.
falciparum resistance to recent antimalarial drugs is an ever growing and
alarming issue, and there is an unmet medical need for new antimalarial
chemotypes targeting the different parasite forms present in various stages of
the Plasmodium life cycle.
Heterocyclic chemical structures are widely used in the early drug
discovery process and in compound screening. At the outset of this study, a
series of 2-arylbenzimidazole derivatives was designed to target C.
pneumoniae and L. donovani. Further development of these 2-
arylbenzimidazoles resulted in a set of 2-aminobenzimidazoles against P.
falciparum. Benzoxadiazole derivatives were designed against L. donovani.
Facile and general synthesis routes for the preparation of both benzimidazole
and benzoxadiazole derivatives were developed.
In order to study structure-activity relationships of the antichlamydial
and antileishmanial 2-arylbenzimidazoles, the left, right and central parts of
the core molecular structure were modified and different substitution
patterns were employed. Antichlamydial, antileishmanial and antimalarial
inhibition activities were related to the different structural modifications
carried out. Antichlamydial and antileishmanial 2-arylbenzimidazoles or
benzoxadiazole derivatives inhibited target pathogens at the micromolar
level.
Furthermore, 2-aminobenzimidazoles were studied as antimalarial
compounds. The best derivative from this study inhibits growth of P.
falciparum (IC50 94 nM) and has a good pharmacokinetic profile. The
compound turned out to be efficacious in vivo against P. falciparum upon
once a day oral administration.
In this study, selectivity of the 2-arylbenzimidazoles against selected
intracellular parasites over free living (planktonic) pathogens e.g.
ii
Escherichia coli was observed. This is a great advantage from the
antimicrobial drug discovery point of view. In spite of the mechanisms of
action of the studied derivatives remaining elusive, it was possible to show in
this study that antimicrobial compound design can be successful even in the
case of unknown macromolecular targets of C. pneumoniae, L. donovani,
and P. falciparum.
iii
ACKNOWLEDGEMENTS
This study was carried out in the Division of Pharmaceutical Chemistry and
Technology at the University of Helsinki in Finland during 2008-2015. A part
of the study was carried out as one-year Open Lab project at
GlaxoSmithKline’s Tres Cantos Medicines Development Campus, Spain in
2012-2013. The author is grateful to the University of Helsinki, Tres Cantos
Open Lab Foundation and Academy of Finland for funding. As this has been
a long trip during these years, there has been plenty of people involved in this
project and I want thank them all.
I am most grateful to my supervisors Professor Jari Yli-Kauhaluoma and
Dr. Paula Kiuru. Jari’s enthusiasm and guidance have made this study
possible. His door has always been open for discussion. Also freedom to learn
and study has been important to me. Paula has guided me through these
years, and without Paula’s practical and detailed help this would have taken
even longer.
I want to thank our collaborators and all co-authors of the publications of
this thesis, without you this study would not be possible. Thanks to Professor
Pia Vuorela (University of Helsinki), Professor Charles L. Jaffe (The Hebrew
University of Jerusalem, Israel), and Félix Calderon (GSK). I would like to
address special thanks to Antti Siiskonen, Olli Salin, Teppo Leino, Mikko
Vahermo, Elena Sandoval-Izquierdo, Margarita Puente-Felipe,
Abedelmajeed Nasereddin, Päivi Tammela, Satu Nenonen and Mikko
Heiskari. Associate Professor Adam Renslo and Professor Angel Messeguer
are acknowledged for reviewing this thesis.
Warm thanks to the JYK group, past and present members, teaching staff,
office mates, Tres Cantos lab mates, especially: Raisa, Titti, Irene, Nenad,
Gusse, Kata, Elisa, Katriina, Kristian, Kirsi, Erik, Martina, Riky, Alexi and
Vânia.
During these years both places, Helsinki and Pohjanmaa, and people
there have been really important to me. I want thank my lovely friends for
encouragement and believing in me. And lastly, the greatest thanks go to my
parents, Annele and Tuomo, and to my sister Maija. They have let me make
my own decisions, believed in me totally and supported me through this
project.
Helsinki, May 2015
Leena Keurulainen
iv
CONTENTS
Abstract .................................................................................................................... i
Acknowledgements ............................................................................................... iii
Contents ................................................................................................................. iv
List of original publications................................................................................... vi
Author’s contributions in original publications ................................................... vii
Abbreviations ....................................................................................................... viii
1 General introduction ..................................................................................... 1
1.1 Target pathogens ................................................................................... 2
1.1.1 Chlamydia pneumoniae .................................................................... 2
1.1.2 Leishmania donovani ........................................................................ 3
1.1.3 Plasmodium falciparum .................................................................... 6
1.2 Screening approaches and properties of antimicrobial and antiparasitic hit compounds ................................................................. 7
1.3 Heterocyclic core structures ................................................................. 9
1.3.1 Benzimidazole .................................................................................... 9
1.3.2 Benzo[c][1,2,5]oxadiazole ................................................................ 11
2 Aims of the study ......................................................................................... 13
3 Experimental ............................................................................................... 14
4 Results and discussion ................................................................................. 15
4.1 Design of the derivatives and general synthesis procedures ............. 15
4.2 Biological activity and structure-activity relationships ..................... 20
4.2.1 2-Arylbenzimidazoles as antichlamydial compounds (Publication I).................................................................................. 20
4.2.2 2-Arylbenzimidazoles as antileishmanial compounds (Publication II) .................................................................................24
v
4.2.3 SAR differences of the 2-arylbenzimidazoles as antichlamydial, antileishmanial and antimalarial agents (Publications I―III and unpublished results) ................................. 27
4.2.4 Efficacy of 2-arylbenzimidazoles against other pathogens (Publications I, II and unpublished results) .................................... 28
4.2.5 2-Aminobenzimidazoles as antimalarial compounds (Publication III) ............................................................................... 29
4.2.6 Benzo[c][1,2,5]oxadiazoles as antileishmanial compounds (Publication IV) ................................................................................ 33
5 Summary and conclusions .......................................................................... 35
References ............................................................................................................ 38
vi
LIST OF ORIGINAL PUBLICATIONS
This thesis is based on the following publications:
I Keurulainen L.*, Salin O.*, Siiskonen A.*, Kern J. M., Alvesalo J.,
Kiuru P., Maass M., Yli-Kauhaluoma J., Vuorela P. Design and
synthesis of 2-arylbenzimidazoles and evaluation of their inhibitory
effect against Chlamydia pneumoniae. J. Med. Chem. 2010, 53, 7664-
7674.
II Keurulainen L.*, Siiskonen A.*, Nasereddin A., Sacerdoti-Sierra N.,
Kopelyanskiy D., Leino T., Yli-Kauhaluoma J., Jaffe C. L., Kiuru P.
Synthesis and biological evaluation of 2-arylbenzimidazoles targeting
Leishmania donovani. Bioorg. Med. Chem. Lett. 2015, 25, 1933-1937.
III Keurulainen L., Vahermo M., Puente-Felipe M., Sandoval-Izquierdo
E., Crespo-Fernández B., Guijarro-López L., Huertas-Valentín L., de
las Heras-Dueña L., Leino T., Siiskonen A., Ballell-Pages L., Sanz L.
M., Castañeda-Casado P., Jiménez-Díaz M. B., Martínez-Martínez M.
S., Viera S., Kiuru P., Calderón F., Yli-Kauhaluoma J. A developability-
focused optimization approach allows identification of in vivo fast-
acting antimalarials: N-[3-[(benzimidazol-2-yl)amino]propyl]amides.
J. Med. Chem. 2015, DOI: 10.1021/acs.jmedchem.5b00114.
IV Keurulainen L., Heiskari M., Nenonen S., Nasereddin A., Kopelyanskiy
D., Yli-Kauhaluoma J., Jaffe C. L., Kiuru P. Synthesis of
carboxyimidamide-substituted benzo[c][1,2,5]oxadiazole and their
analogs, and evaluation of biological activity against Leishmania
donovani. Submitted.
The publications are referred to in the text by their Roman numerals. The
supporting information of the Publications I-IV is not included in this thesis
book. This material is available from the author or via Internet
http://pubs.acs.org for Publication I and III, and
http://dx.doi.org/10.1016/j.bmcl.2015.03.027 for Publication II.
Related publications:
Siiskonen, A.*, Keurulainen L.*, Salin O.*, Kiuru P., Pohjala L.,
Vuorela P., Yli-Kauhaluoma J. Conformation study of 2-
arylbenzimidazoles as inhibitors of Chlamydia pneumoniae growth.
Bioorg. Med. Chem. Lett. 2012, 22, 4882-4886.
* Equal contribution
vii
AUTHOR’S CONTRIBUTIONS IN ORIGINAL PUBLICATIONS
I The author synthesized and characterized the benzimidazole
derivatives. Dr. Paula Kiuru synthesized few compounds. The
author wrote the publication with Dr. Olli Salin and the co-
authors. The publication has been included as one of the
required publications in Dr. Olli Salin’s academic dissertation as
well.
II The author synthesized and characterized compounds. Few
compounds were synthesized by Teppo Leino and Dr. Paula
Kiuru. The author wrote the publication with Antti Siiskonen
and the co-authors.
III The author synthesized and characterized compounds together
with Mikko Vahermo. The author wrote the publication together
with the co-authors.
IV The author synthesized and characterized compounds together
with Satu Nenonen. General synthesis route has been developed
in collaboration with Dr. Paula Kiuru and Mikko Heiskari. The
author wrote the publication with the co-authors.
viii
ABBREVIATIONS
AB aberrant body
Ac acetyl
AUC area under the curve
Boc tert-butyloxycarbonyl
Cmax maximum concentration
Cl clearance
CLND chemiluminescent nitrogen detection
CV-6 C. pneumoniae artery strain 6
CWL-029 C. pneumoniae strain CWL-029
Cyt P450 cytochrome P450 enzymes
dba dibenzylideneacetone
DCC N,N′-dicyclohexylcarbodiimide
DCM dichloromethane
DDQ 2,3-dichloro-5,6-dicyanobenzoquinone
DME 1,2-dimethoxyethane
DMF N,N-dimethylformamide
DMAP 4-(dimethylamino)pyridine
EB elementary body
EDC N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride
Et ethyl
F bioavailability
FaSSIF fasted state simulated intestinal fluid
HepG2 human liver hepatocellular carcinoma cell line
hERG the human ether-à-go-go-related gene
HIV human immunodeficiency virus
HL human cell line (human lung)
HOBt 1-hydroxybenzotriazole hydrate
HSA human serum albumin
HTS high throughput screening
IC50 concentration yielding 50% inhibition
IF immunofluorescence microscopy
Me methyl
MCC minimum chlamydiocidal concentration
MIC minimum inhibitory concentration
MW microwave
Ph phenyl
PPA polyphosphoric acid
PPB plasma protein binding
p-TSA p-toluenesulfonic acid
RB reticulate body
rt room temperature
ix
SAR structure-activity relationship
SI selectivity index
t1/2 half life
TCAMS Tres Cantos Antimalarial Set
TCDI 1,1'-thiocarbonyldiimidazole
TEA triethylamine
TFA trifluoroacetic acid
TFAA trifluoroacetic anhydride
THF tetrahydrofuran
THP-1 human leukaemia monocyte cell line
TMS trimethylsilyl
TR-FIA time-resolved fluorometric immunoassay
Vss volume of distribution
1
1 GENERAL INTRODUCTION
Increasing resistance to antibiotic and antiparasitic agents emphasizes the
need for new antimicrobial and antiparasitic discovery. Antibacterial
resistance can be addressed by finding new antibacterial targets and/or by
modifying existing antibacterial agents to overcome specific resistance
mutations. Antibacterial drug discovery is challenging, as it might be easy in
some cases to find compounds that kill bacteria, but a compound which is
worthy of development might prove challenging to find.1 There has been
discovery void of new antimicrobial substances during recent years.
Moreover, many antiparasitic drugs have been first discovered to some other
indication and less novel agents to treat antiparasitic diseases have been
used.2
In order to find biologically active antimicrobial and antiprotozoal small
molecular compounds, high-throughput screening and structure-based
approaches have been used at early stages of drug discovery.3–5 From
screening approaches, phenotypic and target-based screening are used
successfully.6,7
Structurally and mechanistically new antibiotics are needed, malaria
control is one of the highest priorities on the international health agenda
currently9, and leishmaniasis is a pathogen target categorized as a neglected
disease by The World Health Organization8. Diverse antiparasitic research
efforts have increased in recent years around a consensus of necessity for
such work and increased funding from many non-profit foundations.
Antimalarial work has reached a couple of milestones as malaria mortality
rates are significantly decreasing and, for example, a malaria vaccine has
progressed to phase III clinical studies.9,10 There are numerous publications
of the antimalarial agents, whereas research for novel antichlamydial
compounds consists of just few publications during recent years. Target
pathogens Chlamydia pneumoniae, Leishmania donovani, and Plasmodium
falciparum are briefly introduced in this chapter together with some points
of challenges in the early drug discovery process of parasitic diseases.
Heterocycles are widely used and found in biologically active small
molecules in diverse therapeutic areas.11 Heterocycles are used as a starting
point of molecules discussed in this thesis. In this literature introduction,
core structures of the work, 1H-benz[d]imidazoles and
benzo[c][1,2,5]oxadiazoles (referred to as benzimidazole and benzoxadiazole,
respectively), are discussed in brief.
2
1.1 TARGET PATHOGENS
1.1.1 Chlamydia pneumoniae
Chlamydia pneumoniae is a widespread intracellular Gram-negative
bacterium causing approximately 10% of community-acquired pneumonia
and 5% of bronchitis and sinusitis among the adult population. It has a
unique life cycle, which consists of a metabolically active intracellular form
(reticulate body) and an infective extracellular form (elementary body)
(Figure 1).12 Chlamydia infection can also enter a chronic stage (specific
intracellular non-replicating form, aberrant body).13,14 The majority of
studies support the association of increased risk of atherosclerosis with C.
pneumoniae infection.15–17 C. pneumoniae infection can contribute to
atherosclerosis by indirect and direct mechanisms. C. pneumoniae infection
is also associated with other severe diseases and inflammatory processes like
asthma18–20, chronic obstructive pulmonary disease21, Alzheimer’s disease22–
24 and lung cancer25, although not totally without controversial opinions.
Figure 1 C. pneumoniae life cycle. EB elementary body, RB reticulate body, AB aberrant body.
Chlamydial infection is treated with tetracyclines, macrolides, and
fluoroquinolones, antibiotics which affect protein synthesis and DNA
replication, using a long treatment time (Figure 2).26,27 In case of persistent
(chronic) infections, however, this treatment modality can fail .28,29
Figure 2 Drugs used to treat C. pneumoniae infection.
3
In general, antibacterial agents can target a broader or more specified group
of bacteria. Chronic infections and the so-called persister and dormant cells
also warrant the need for new approaches in the drug discovery process, due
to the lack of response to most antibiotics, which are primarily effective
against actively dividing cells.30,31 Chemical diversity is needed in antibiotic
drug discovery.1,32 Usually, the chemical structures of antibacterial
compounds differ from the other orally administrated drugs by being more
hydrophilic and slightly larger in molecular size.32,33 For example, in
commercially available chemical libraries consisting of compounds fulfilling
the criteria for drug-likeness, this might lead to a situation where potential
antibacterial compounds are excluded. In the search for new antibacterial
compounds, natural product screening is encouraged, because of their vast
structural and molecular diversity.33,34
In antichlamydial drug discovery, the bacterium life-cycle causes
challenges in eradicating the infection.35 The antichlamydial compound has
to penetrate the host cell plasma membrane, inclusion membrane
surrounding the bacterium in its replicating form, and finally the outer
membrane of Gram-negative bacterium in order to be able to affect the
metabolically active form of the bacterium. Another option is to try to affect
other cellular components of C. pneumoniae, for example those that do not
reside inside the reticulate body membrane.
Virulence factors have been considered as antichlamydial small molecule
targets. Type 3 secretion system (T3SS) is an essential contributor to
chlamydial virulence known to be present in C. pneumoniae36, and
compounds inhibiting Yersinia pseudotuberculosis T3SS have been shown to
affect also C. pneumoniae growth.37 Also some non-conventional
antimicrobial agents are studied as antichlamydial compounds. Ca2+ channel
blockers, such as nifedipine, have been shown to enhance antibiotic
susceptibility of C. pneumoniae infection, and lipid lowering drug
(simvastatin) has been shown to have antichlamydial affect by decreasing the
viable chlamydial count.38,39 Compounds with new mechanisms of action
might turn out to be a viable solution to improve the current antichlamydial
treatment.
1.1.2 Leishmania donovani
Leishmaniasis is a neglected tropical disease caused by intracellular parasites
belonging to the kinetoplastid genus Leishmania.40 Four different clinical
forms are specified: visceral, cutaneous, mucocutaneous, and diffuse
cutaneous leishmaniasis (post kala-azar leishmaniasis). Leishmania is
transmitted via a bite from the Phlebotomus and Lutzomyia sand flies.
Leihsmaniasis is a strongly poverty-associated disease and approximately 1.3
million new cases occur annually, 300 000 of which are visceral
leishmaniasis.41 A majority of visceral leishmaniasis cases (90%) occur in six
4
countries (Bangladesh, Brazil, Ethiopia, India, South Sudan, and Sudan).42
Visceral leishmaniasis caused by L. donovani (in the Indian subcontinent
and East Africa) or L. infantum (L. chagasi) (in the Mediterrean basin,
Central, and South America) is fatal within two years if left untreated,
causing 20-40,000 deaths per annum. Symptoms of the infection occur after
2-6 months of incubation time. These symptoms include fever, weakness and
weight loss. The parasite also invades the spleen and liver, causing
enlargement of these organs.43
Figure 3 Amphotericin B and its lipid formulation, miltefosine and paromomycin are studied as combination therapies to treat visceral leishmaniasis.
54
Current drugs in use are pentavalent antimonials, amphotericin B and its
lipid formulations, miltefosine, paromomycin, and pentamidine.44 The
existing treatments suffer from toxicity, parasite resistance and poor patient
compliance.45–47 Furthermore, drug sensitivities towards different
Leishmania species vary strongly.48 Coinfection with HIV is known to affect
drug efficacy,48,49 and the number of cases is increasing alarmingly.50
Multidrug therapy (Figure 3) is probably a solution to these problems in the
treatment of visceral leishmaniasis, as several combination therapies studied
have given promising results.51–54
Leishmania has a digenetic life cycle.55 Leishmania parasite has two
morphological forms (Figure 4), a non-motile amastigote form in the
mammalian host and a motile promastigote in vector.
5
Figure 4 Life cycle of L. donovani.
Antileishmanial-drug discovery is focused on treatments which present an
acceptable cost, short duration, and oral administration. In general,
treatments are sought that are feasible in circumstances where leishmaniasis
is usually found.2 Four clinical forms and seventeen different species of
Leishmania are the cause for an urgent need of novel drugs with different
pharmacokinetic profiles as topical and oral drugs are used for treating
different forms of leishmaniasis. Visceral leishmaniasis, which is the variant
that causes mortality, is studied in the most detail.56 Varying sensitivities of
the strain and species causes challenges in testing antileishmanial drugs, as
there are regional differences in responses to drugs.57 A lot of research has
been carried out to find new and potent antileishmanial lead compounds and
clinical candidates.58,59
Both promastigote and amastigote forms of Leishmania are used to assay
antileishmanial compounds. Assays using axenic amastigotes afford a simple
and rapid approach for screening compounds, but antileishmanial activity
should be confirmed by using the intracellular amastigote model, because the
axenic amastigote screens do not necessarily correlate to intracellular
amastigote model.60,61 An intracellular amastigote model (mouse peritoneal
macrophages) is recommended as “the golden standard” for antileishmanial
drug screening, and the intracellular amastigote assay (in macrophages) is
generally regarded to be a good in vitro model for predicting clinical activity. 62 Alternatively an ex vivo model using a splenic explant culture system from
hamsters infected with luciferase-transfected L. donovani can also be used to
screen antileishmanial compounds.63
6
1.1.3 Plasmodium falciparum
Associated strongly to poverty, malaria still kills approximately 600,000
people per year, mostly in the African continent.9 Malaria is caused by
protozoan parasites belonging to the genus Plasmodium. P. falciparum, P.
vivax, P. malariae, and P. knowlesi are known to infect humans: P.
falciparum and P. vivax are the main types responsible for mortality.
Children under the age of five (78% of malaria deaths) and pregnant women
are under the highest risk. P. falciparum resistance to antimalarial drugs is
an ever growing issue. Nowadays also artemisinin resistant strains have been
discovered in addition to chloroquine and pyrimethamine resistant
strains.64,65 Although artemisinin-based combination therapies (Figure 5) are
the recommended way of treating plasmodial infections, there is an unmet
need for novel antimalarial chemotypes.
Figure 5 Examples of used compounds in artemisinin-based combination therapies. Used combinations are AM+LF, DHA+PYR, AS+AQ, AS+MQ.
66
Figure 6 P. falciparum life cycle. It consists of stages in human and in vector.
7
There are numerous transitions and stages in P. falciparum life cycle. (Figure
6).67 The life cycle begins with the bite of an infected Anopheles mosquito.
Asymptomatic liver stage, symptomatic asexual blood stage and sexual
gametocytes constitute the P. falciparum life cycle in the host.
The characteristics considered to be important in antimalarial drug-
discovery include acceptable costs, short treatment duration, and oral
administration. New antimalarial compounds should have an effect on drug-
resistant parasites and cover four different malaria-causing species.2,66 More
specifically, new treatments are needed to address blood stage malaria, block
parasite transmission, and kill dormant hypnozoites (liver form of P.
vivax).68 Many promising antimalarial lead compounds and candidates have
been found through rational antimalarial design and HTS (high throughput
screening) screening.69
Although most antimalarial testing is concentrated on the asexual blood
stage, testing against gametocytes and liver stages can also be done.70–73
Murine models typically utilize rodent parasite P. berghei, but also P.
falciparum is used nowadays to confirm compound activity against the
relevant human parasite.74,75 This model relies on the use of immunodeficient
mice with human erythrocytes.
1.2 SCREENING APPROACHES AND PROPERTIES OF ANTIMICROBIAL AND ANTIPARASITIC HIT COMPOUNDS
Screening approaches
Relevant advantages and disadvantages in cases of antibacterial and
antiparasitic screening can be focused on the following topics. In the case of
parasitic disease there might be only a limited number of validated molecular
targets to use for target-based approaches.4 In target-based screening, the
macromolecular target of the pathogenic microbe is already known, but
compounds’ activities need to be confirmed by using various cell-based
assays. Also, structure-based drug discovery can be less successful due to the
lack of available macromolecular structures, although a growing number of
structure-based targets and methods promise a higher success-rate in the
future.5
The major advantage of phenotypic screening is its focus on the
compound’s activity in the whole cell. This means that issues like cell
penetration, and cell uptake or efflux are captured in the assay, as are all the
targets and biological pathways that one might want to target.4,76,77 The
major challenges in phenotypic screening include maintaining biological
cultures as well as the target identification. However, there are many ways to
investigate macromolecular pathogen-targets, including: affinity
8
chromatography, expression cloning, and protein microanalysis.78 The
compounds found by using phenotype screening can target multiple
microbial macromolecules, but this also means that the chemical
optimization of antimicrobial activity may not be driven by one target only.3
Properties of the hit and lead compound
The hit compound and, later on, the resulting lead compound, should possess
some characteristic properties to increase the probability of its procession to
the next stage of drug discovery, lead optimization. Examples of these
properties are shown in Table 1.
Table 1. Examples of essential properties of experimental compounds in antimicrobial and antiparasitic drug discovery at the hit/lead phase. Collected examples deal with antimalarial, antiparasitic and overall antimicrobial studies.
2,68,79
Efficacy 10 nM - 10 µM / parasitic IC50 1 µg/mL
Cytotoxicity / Selectivity At least ten-fold selectivity over mammalian cell line
Cyt P450 IC50 > 10 µM
hERG IC50 >10 µM
Solubility >200 µM
Intrinsic clearance <3 mL/min/kg
HSA binding <95%
In vitro activity against resistant strains
Lead in vivo active against parasite ≤100 mg/kg, candidate <10 mg/kg per oral
Lead not overtly toxic in animals at efficacious dose
Desired properties are listed in a so-called target product profile. Each case is
considered separately, but efficacy, safety and metabolic stability are very
important. These also include the potential to interfere with the metabolic
cytochrome P450 enzymes to predict potential drug-drug interactions,
human-serum-albumin binding, which reflects on the free fractions of the
drug and has implication for drug function in vivo,80 and finally hERG (the
human ether-à-go-go-related gene), which is associated to possible
cardiotoxicity, a major concern of drug safety81–83. Blockage of hERG K+
channels causes abnormality of cardiac muscle repolarization, which results
in prolongation of the QT interval. hERG cardiotoxicity has resulted in
several withdrawals of drugs from the market.
9
1.3 HETEROCYCLIC CORE STRUCTURES
1.3.1 Benzimidazole
Figure 7 Benzimidazole structure with its tautomer.
Benzimidazole structure is given in Figure 7. Benzimidazole derivatives have
shown a wide variety of biological activities.84,85 Antihypertensive, anti-
inflammatory, antimicrobial, antiviral, anthelmintic, antioxidant, and
antitumor benzimidazole-based compounds are known. In addition, the
benzimidazole core is present in some psychoactive agents, lipid modulators,
anticoagulants, and antidiabetic agents. The benzimidazole-derived
compounds, more relevant for this study, have been reported to possess
antibacterial,86–90 general antiprotozoal,91–93 antimalarial94–102 and
antileishmanial activities103–105. Some structurally interesting examples of
benzimidazole-based compounds are shown in Figure 8. Benzimidazole-
containing drugs are also currently in use (Figure 9), and the benzimidazole
structure can be found in nature, as a subunit of the complex structure of
vitamin B12. This diversity of pharmacological activities suggests the
benzimidazole ring as a privileged structure in medicinal chemistry.106,107 The
term describes an inherent tendency of certain molecular scaffolds to display
biological activity. Usually these scaffolds with versatile biological activities
and binding properties to various macromolecular targets are small ring
systems.
Figure 8 Structurally interesting benzimidazole-derived leads and their selected properties. Hedgehog signaling pathway inhibitor
108 and antimalarial compound
109 as
representative examples.
10
Figure 9 Examples of benzimidazole-derived drugs currently in use. Calculated logP values.
110
Since benzimidazoles have been studied during the past years intensively,
many related synthesis procedures have also been reported. Examples of
representative synthesis methods of benzimidazoles are described in Scheme
1. Generally benzimidazoles have been synthesized via a cyclocondensation
reaction by treating 1,2-diaminobenzene starting material with carboxylic
acids under harsh dehydrating reaction conditions, such as HCl111,112 or
polyphosphoric acid (PPA)114. Nowadays these methods can also be used
under microwave-assisted reaction conditions.115,116
Scheme 1 Representative examples of methods (A116, B117, C115, D118, E119 and F120
) for facile
synthesis of the benzimidazole moiety.
Benzimidazoles can also be synthesized from ortho-aminobenzamides.119,121–
123 Acidic reaction conditions are used in the ring closure step. Another
widely used method for benzimidazole formation is the condensation of 1,2-
diaminobenzene and an appropriate aldehyde, followed by oxidation. This
method has been carried out as a one-pot procedure or through isolating the
intermediate Schiff base. A great number of reagents can be used, for
example Na2S2O5104,117,124,125, FeCl2
126, 1,4-benzoquinone104, Me2S+BrBr-127,
11
H2O2/ceric ammonium nitrate128, DowexTM129, Oxone®130, nitrobenzene131,132,
Pd(OAc)4133, H2O2/HCl134, benzofuroxan135, Ba(MnO4)2
136, DDQ137, Darco®
KB138, CuSO4139, TMSCl140 and even air without other reaction promoting
agents141.
Carboxylic acid derivatives such as nitriles142,143, orthoesters144, esters145,
and lactones146 can be used as a starting material. An alternative method is to
use 2-nitroaniline as a starting material.116,125,147 2-Aminobenzimidazoles can
be synthesized, for example, by using BrCN112,120,148 or isothiocyanate149 as a
starting material.
Palladium(0)-catalyzed reactions are used in benzimidazole synthesis. For
example, (o-bromophenyl)amidine118 or diamines and aromatic halides150
can be used as starting materials.
1.3.2 Benzo[c][1,2,5]oxadiazole
Figure 10 Benzo[c][1,2,5]oxadiazole structure.
The structure of benzo[c][1,2,5]oxadiazole (2,1,3-benzoxadiazole), known
also as benzofurazan, is given in Figure 10. Benzoxadiazoles show a wide
variety of biological activities and they have been studied against tumors,
pathogenic bacteria, and parasites as well as in the treatment of neuronal
disorders.151,152 More relevant to this study, antiparasitic153,154,
antibacterial155–157 and antimalarial158 activities have been found from
benzoxadiazoles and their N-oxides. The benzoxadiazole structure is not
widely used in the current drugs, an example is shown in Figure 11.
Figure 11 Benzoxadiazole-derived drug currently in use. Calculated clogP value.110
Benzoxadiazoles are synthesized through benzoxadiazole N-oxides
(benzofuroxans) mainly in two ways. The first option is a synthesis through
the azide intermediate. Cyclocondensation of an azide in boiling toluene or
acetic acid gives the intermediate benzoxadiazole oxide.156,159–161 Another
method is to use 2-nitroaniline as a starting material and alkaline
hypochlorite oxidation (KOH in ethanol and sodium hypochlorite).160,162–164
The subsequent reductive cleavage of the N-oxide is carried out, for example
12
by using Ph3P to produce the corresponding benzoxadiazoles. In Scheme 2,
representative examples of benzoxadiazole syntheses are given.
Scheme 2 Examples of methods (A165
and B164
) to synthesize benzo[c][1,2,5]oxadiazoles.
13
2 AIMS OF THE STUDY
The objective of the current study was to synthesize various heterocyclic,
mainly benzimidazole- and benzo[c][1,2,5]oxadiazole-derived compounds
against intracellular pathogens, Chlamydia pneumoniae, Leishmania
donovani, and Plasmodium falciparum, the causative microbes of such
diseases as lung chlamydia, leishmaniasis, and malaria, respectively.
Molecules found by virtual screening166 were used as starting points for the
design of antichlamydial, antileishmanial, and antimalarial compounds. The
library of derivatives was designed to cover enough molecular varieties to
study relationships between their chemical structures and antimicrobial
activities. General synthesis routes were developed for each molecular
scaffold. The ultimate aim was to identify, find, and characterize compounds
with micro- or nanomolar antimicrobial activities. In addition, the most
promising compounds were expected to have acceptable properties from the
medicinal chemistry point of view, to enable continuing with the compound
optimization and further development.
The more specific aims of the research were
To design and synthesize 2-arylbenzimidazole derivatives against
Chlamydia pneumoniae or Leishmania donovani, and study their
structure-activity relationships (I, II)
To design and synthesize 2-aminobenzimidazoles as antimalarial
compounds, study their structure-activity relationships and find a
developable lead molecule (III)
To design and synthesize benzoxadiazoles as antileishmanial
compounds and study their structure-activity relationships (IV)
14
3 EXPERIMENTAL
A detailed presentation of the materials, synthetic, and analytical methods
can be found in the original Publications I-IV and in the associated
supporting information.
The supporting information for original publications I-IV is not included
in this thesis book. This material is available from the author or via Internet.
15
4 RESULTS AND DISCUSSION
The main findings of the original publications are described in this chapter,
with some additional unpublished data. More detailed results can be found in
the original Publications I-IV. Reaction conditions have not been
systematically optimized for any of the synthesis routes described, as the
main objective of the synthetic chemistry part was to produce derivatives for
various biological tests and antimicrobial assays.
4.1 DESIGN OF THE DERIVATIVES AND GENERAL SYNTHESIS PROCEDURES
2-Arylbenzimidazoles as antichlamydial and antileishmanial agents
(Publications I and II)
Design of the 2-arylbenzimidazoles is based on the previous finding of our
research group that certain 2-arylbenzimidazoles possess micromolar activity
against Chlamydia pneumoniae.166 Due to the lack of protein structures of C.
pneumoniae, a related homology model was used. C. pneumoniae
dimethyladenosine transferase has high active site homology with Bacillus
subtilis RNA methyltransferase. They are also known to bind their ligands
similarly.167 Dimethyladenosine transferase is known to be related to
functions of ribosomal structure. By using a homology model of C.
pneumoniae dimethyladenosine transferase, a total of 3×105 molecules were
screened virtually.166 This study revealed the activities of 2-
arylbenzimidazoles and benzo[c][1,2,5]oxadiazoles against C. pneumoniae,
when selected compounds were tested in vitro.
Of the selected 2-arylbenzimidazoles, phenyl and 2-thiophene derivatives
showed antichlamydial activity, and the para-substituted phenyl derivatives
were inactive (Figure 12).166 As small modifications of the molecular
structure may change antichlamydial activity dramatically, design of the first
generation antichlamydial 2-arylbenzimidazoles was focused on small
variations, and the basic 2-arylbenzimidazole core structure was kept intact.
Figure 12 Active and inactive benzimidazole derivatives against C. pneumoniae, found using virtual screening.
166
16
The same set of 2-arylbenzimidazole derivatives was also tested against L.
donovani, because structurally similar benzoxazoles, i.e. N-(2-benzoxazole-
2-ylphenyl)benzamides have been shown previously to possess
antileishmanial activity.168 Extension of the benzimidazole set was planned to
cover more modifications of the various parts of the molecular scaffold for
the structure-activity relationship (SAR) study. First and second generation
modifications of the core structure were the following: substitution of the A-
and C-rings, alkylation of the B-ring, modification and position of the amide
linker and variation of the linker, modification and substitution of the D-ring
(Figure 13).
Figure 13 Modification sites of the 2-arylbenzimidazoles as antichlamydial and antileishmanial compounds.
The 2-arylbenzimidazole core structure was synthesized in two steps
(Scheme 3). First, 1,2-diaminobenzene 6 was diacylated with the
corresponding benzoyl chlorides in the presence of 4-
(dimethylamino)pyridine (DMAP) in pyridine to produce the bisamides 7
under MW irradiation. The subsequent cyclization of the bisamides was
accomplished with p-toluenesulfonic acid (p-TSA) refluxing in p-xylene121 to
yield 2-arylbenzimidazoles 8 in good yield, except in the case of C-ring-
substituted derivatives (R3≠H), which were obtained in lower yields. Direct
oxidation methods117,128,130 to synthesize m-nitro-2-arylbenzimidazole were
tested, but those failed or gave poor yields probably due to the presence of an
deactivating m-nitro moiety. Thus, the original two-step synthesis route was
found to be convenient and better yielding. The nitro group of nitroaryl
benzimidazole was hydrogenated in the presence of palladium(0) catalyst or,
alternatively, reduction of the A-ring halogenated intermediates (R1= Cl, Br)
was carried out with SnCl2∙2H2O. Finally, the N-acylated products 10 were
formed from the corresponding acyl chlorides in the presence of
triethylamine (TEA) in tetrahydrofuran (THF).
17
Scheme 3 General synthesis route for 2-arylbenzimidazoles.
Benzimidazole derivatives as antimalarial agents (Publication III)
Our interest in benzimidazole derivatives as antimalarial compounds was
raised by the recent finding that certain benzimidazole-structured derivatives
displayed promising antimalarial activity (Figure 14).169 These compounds
were reported by GSK’s Tres Cantos unit, and with these (and other)
scaffolds they invited the scientific community to embark on antimalarial
discovery and to continue developing these scaffolds further.
Figure 14 Benzimidazole-derived scaffolds with antimalarial activity.169
2-Arylbenzimidazoles with antichlamydial and antileishmanial propertiesI,II
were tested against P. falciparum (unpublished results, 66 compounds).170
The derivative 11 showed the best antimalarial activity, and the initial
modification was started from this derivative (Figure 15). The first line
modification was to replace the C-ring with a non-aromatic ring or chain to
decrease the compound’s lipophilicity. This modification plan was supported
by the set of antimalarial compounds from TCAMS (Tres Cantos Antimalarial
Set). The second line modification plan focused on the substitution (or other
modifications) of the rings A/B and D.
18
Figure 15 Medicinal chemistry plans to modify the original 2-arylbenzimidazole scaffold (compound 11).
The D-ring modified derivatives were synthesized by using a two-step
synthetic route starting from 2-chlorobenzimidazole 13 (Scheme 4). A 1.5-
day heating of a mixture of 2-chlorobenzimidazole and 1,3-diaminopropane
gave N1-(1H-benzimidazol-2-yl)propane-1,3-diamine 15. It was then coupled
to the corresponding benzoic acids to yield the derivatives 16.
Scheme 4 General synthesis procedures for the D-ring modified 2-aminobenzimidazole derivatives.
The A-ring modification can be carried out by using a four-step synthetic
route with formation of the benzimidazole core, starting with the standard
amidation of 3,5-dichlorobenzoyl chloride 17 (Scheme 5). The subsequent
reactions of the -aminopropionamide 19 with 1,1'-thiocarbonyldiimidazole
(TCDI) yielded the corresponding isothiocyanate 20, which on treating with
a substituted o-diaminobenzene in the presence of N,N′-
dicyclohexylcarbodiimide (DCC) or N-(3-dimethylaminopropyl)-N′-
ethylcarbodiimide hydrochloride (EDC) gave the desired benzimidazole
derivatives 22.
19
Scheme 5 The general synthesis procedures for the A-ring modified 2-aminobenzimidazole derivatives.
Benzo[c][1,2,5]oxadiazoles as antileishmanial agents (Publication IV)
Certain benzoxadiazole derivatives have been found to have activity against
Chlamydia penumoniae166 (Figure 16) in a previous study by our group. We
expected to also find antileishmanial activity among the benzoxadiazoles,
because, in our former studies, benzimidazoles had revealed both
antichlamydial and antileishmanial activities.I, II A set of benzoxadiazoles was
designed, and the synthetic modifications were directed to substitution of the
right-hand side phenyl ring, replacement of benzoxadiazole moiety with
other heterocycles, and modification/simplification of the chain structure
(Figure 16).
Figure 16 Two benzoxadiazole derivatives having antichlamydial activity,166
as a basis for the design of new derivatives. Figure presents a simplified general structure for the benzoxadiazole derivatives and related compounds designed as antileishmanial agents.
Synthesis of benzo[c][1,2,5]oxadiazoles derivatives started from the
commercially available 4-amino-3-nitrobenzoic acid 25 (Scheme 6). It was
converted to benzo[c][1,2,5]oxadiazole-5-carboxylic acid 26 by using a two-
step procedure via the N-oxide intermediate that was produced in the
presence of sodium hypochlorite in an alkaline EtOH-H2O solution. The
subsequent reduction with P(OEt)3 gave the benzoxadiazolecarboxylic acid.171
Next, the carboxylic acid was converted to the primary amide 28 via reaction
of the corresponding acyl chloride 27 with aqueous ammonia. The reaction of
the obtained primary amide with trifluoroacetic anhydride and TEA in
THF172 gave the corresponding nitrile 29, which was converted to the
amidoxime by using hydroxylamine hydrochloride 30 in the presence of TEA
in EtOH.173 The final step to obtain the N'-[(phenylcarbamoyl)oxy]-
20
benzo[c][1,2,5]oxadiazole-5-carboximidamides 31 was carried out in the
presence of the substituted phenyl isocyanates in THF or CHCl3.
Scheme 6 A general synthesis route to the benzo[c][1,2,5]oxadiazole derivatives.
4.2 BIOLOGICAL ACTIVITY AND STRUCTURE-ACTIVITY RELATIONSHIPS
4.2.1 2-Arylbenzimidazoles as antichlamydial compounds (Publication
I)
The inhibitory effect of 2-arylbenzimidazoles against C. pneumoniae was
evaluated in Publication I. Compounds were assayed for C. pneumoniae
(strain CWL-029) at a concentration of 10 μM using TR-FIA (time-resolved
fluorometric immunoassay) method174 and for label binding properties as
well as autofluorescence to exclude false positive signals. The effect of all 2-
arylbenzimidazoles on host-cell (HL, human line175) viability was studied
after 72 h of incubation with a 10 μM concentration.
Figure 17 SAR of 2-arylbenzimidazoles as antichlamydial compounds. Order of the substituent and structural parts in figure are based on C. pneumoniae % inhibition values (TR-FIA).
From this set of 2-arylbenzimidazoles (33 compounds), 14 derivatives
showed at least 80% inhibition activity at a concentration of 10 µM. The SAR
is reviewed in Figure 17. SAR studies showed that benzimidazole structure is
essential to activity, and the nature of the D-ring and its substitution is
21
affecting antichlamydial activity. Methylation of the benzimidazole,
sulfonamide replacement of the amide linker, and moving of the amide
connecting structure to the para position instead of meta resulted in
completely inactive compounds.
Host cell (HL) viability was over 80% to the majority of the tested
compounds (exceptions 34, 38, 39), which indicates that 2-
arylbenzimdazole structure is not primarily associated with host cell toxicity
in vitro.
From the set of benzimidazoles, nine of the most promising compounds
(Figure 18) were selected, and antichlamydial activity was evaluated using a
traditional immunofluorescence labeling (IF) method. Figure 19 presents the
IF results, including host-cell viability of the selected compounds.
Figure 18 The nine potent compounds selected for further testing as antichlamydial agents.
Compound 33 and 34 inhibited growth of C. pneumoniae, also at low
concentrations, but derivative 34 had an effect on viability of the HL cells at
higher concentrations. Interestingly, there was a clear trend that increasing
concentration of compounds resulted in smaller but still detectable
chlamydial inclusions. TF-FIA method measures the overall amount of C.
pneumoniae, whereas IF method measures the number of chlamydial
inclusions. This explains that, overall, IF inhibition results were partly lower
than TR-FIA. Only few of the compounds were able to inhibit C. pneumoniae
completely.
22
Figure 19 Chlamydial growth inhibition (%) and host cell viability in different concentrations of the nine selected compounds. Compounds are divided into the structural group A (thiophene derivatives), B (D-ring modified, phenyl or cyclohexane derivatives), and C (A-ring substituted).
The nine selected potent compounds were also tested using the CV-6 strain
in an acute in vitro infection model (Table 2). These results verify
antichlamydial activity. The CV-6 strain is a cardiovascular strain, which is a
human coronary artery-derived clinical isolate known to cause chronic
infections.176 Overall, results were in line with the result using strain CWL-
29, except compounds 2, 32, and 38 did not inhibit chlamydial growth (MIC,
minimum inhibition concentration, 196 µM). The detected minimum
chlamydiocidal concentration (MCC) revealed a range from 3.2 to >196 µM.
Derivative 33 had the lowest MCC. Growth was also prevented in the
23
subcultures in antibiotic-free medium using the lowest concentration,
indicating the effect to be chlamydiocidal.
Table 2. MIC and MCC of the selected derivatives using C. pneumoniae strain CV-6.
µM
Compound MIC MCC
2 196 >196
32 >196 >196
33 3.2-6.3 3.2-6.3
34 3.2 50.4
35 6.1-48.9 24.5
36 24.5 24.5
37 12.6 12.6
38 196 >196
39 12.6 12.6
Erythromycin 0.17 0.17
The best compound 33 in this study has a minimal inhibitory concentration
of 10 µM against CWL-029, and 6.3 µM against CV-6. The mechanism of
action of these compounds is unknown and remains elusive. As derivatives
affect the growth of C. pneumoniae and they do not show cytotoxicity, it is
very likely that they are able to cross the host cell membrane.
The importance of conformational effects on the structure–activity
relationship of these 2-arylbenzimidazoles was studied.177 Minimized energy
and energy differences were calculated using simplified N-phenylbenzamides
to describe actual derivatives. It was found that there is a correlation between
antichlamydial inhibition activity and energy of the D-ring to adopt a 45
degree angle compared to the C-ring (Figure 20). Compounds which could
more easily adopt a non-coplanar conformation show higher bioactivity. This
might indicate that preferred binding conformation to the target is non-
coplanar.
24
Figure 20 Calculated energy difference between the global minimum energy conformation and the conformation wherein the C-ring dihedral is forced to an angle of 45 degrees (∆E45). Also presented in the table is antichlamydial activity at 10 µM (TR-FIA) for the same analogs. At right is an overlay of the conformations of 2-methyl and 2-(trifluoromethyl)phenyl analogs.
177
4.2.2 2-Arylbenzimidazoles as antileishmanial compounds
(Publication II)
Antileishmanial activity of the 2-arylbenzimidazole derivatives (56
derivatives, including compounds tested against C. pneumoniae in
Publication I and extension of the set) was evaluated in Publication II. The
antileishmanial activity was detected by using a fluorescent viability
microplate assay with L. donovani (MHOM/SD/1962/1S-Cl2d) axenic
amastigotes and alamarBlue61 using three concentrations (50, 15, 5 µM) of
the compounds.
Figure 21 Antileishmanial SAR based on the axenic amastigote assay. Order of the substituent and structural parts in the figure is based on L. donovani inhibition %.
Parent compound
Structure of the D-ring ∆E45 (kcal/mol)
Chlamydial inhibition at 10 µM
35 2-methylphenyl 0.0 99
- 2-(trifluoromethyl)phenyl 0.0 96
1 phenyl 0.6 99
32 3-thiophenyl 1.2 68
2 2-thiophenyl 1.4 89
- 3-furanyl 1.5 78
- N-methylpyrrol-2-yl 1.7 80
- 2-fluorophenyl 2.0 0
- 2-methoxyphenyl 2.1 0
- 2-furanyl 3.5 45
- 1,3-thiazol-4-yl 4.3 39
- pyridin-2-yl 4.4 36
25
The SAR based on L. donovani axenic amastigote results are summarized in
Figure 21. Modifications were studied starting from the right-hand side of the
molecule. Different aromatic, heteroaromatic, non-aromatic rings, and alkyl
moieties are tolerated in the place of D-ring. Urea and the original amide
structures constitute the best options for the linker modifications. The
position of the linker can be meta or ortho. Modification of 2-
arylbenzimidazole was studied using the 2-thiophene moiety as the D-ring of
the molecule. The A-ring substitution is tolerated as well as alkylation of the
benzimidazole NH. Finally, substitution of the D-phenyl ring was also
studied. Many substitution patterns are tolerated, but in this set of new
derivatives, significantly more active derivatives were not found.
The importance of the right-hand-side part of the molecule for the activity
was observed in the case of structurally related N-(2-benzoxazol-2-ylphenyl)
benzamides.168 A substitution of the pyridine ring was significantly affecting
antileishmanial activity, when the compounds were assayed against axenic
amastigotes.
Figure 22 Ten benzimidazole derivatives selected for further antileishmanial testing.
Ten derivatives (Figure 22), which showed over 30% inhibition of axenic
amastigotes at a concentration of 5 µM, were selected for further evaluation.
To confirm actual activity of the compounds on infected macrophages they
were evaluated using THP-1 (a human leukemia monocyte cell line) cells.
First, cytotoxicity of these derivatives was determined at a concentration of
15 µM (Table 3). Second, for the seven non-cytotoxic derivatives the
percentage inhibition in L. donovani-infected THP-1 cells was measured. L.
donovani axenic amastigote results of the selected derivatives did not vary
significantly but antileishmanial inhibition values of these derivatives varied
when they were assayed by using infected THP-1 cells. Interestingly, the
position of the methylation seems to affect antileishmanial activity
significantly (compounds 41 and 42, Table 3). To further characterize the
most active benzimidazole derivatives, their IC50 values on axenic
amastigotes and fibroblasts (cytotoxicity, murine cell line) were determined.
Overall IC50 values on axenic amastigotes were lower than expected based on
initial screening at 5 µM, and this has impact on selectivity index. The
26
cytotoxicity of benzimidazole derivatives in fibroblasts varied between 15 µM
and 300 µM, indicating that this set of compounds also includes cytotoxic
compounds. This is why, cytotoxicity liabilities must be considered in the
future studies. The best compound of the study (42) inhibited L. donovani-
infected THP-1 cells ~ 50% at 5 µM, without signs of cytotoxicity against the
THP-1 cell line (at concentration of 15 µM) or murine fibroblasts cell line.
Table 3. Cytotoxicity on THP-1 macrophages and NIH/3T3 fibroblasts, and percent inhibition of amastigotes in L. donovani-infected macrophages, IC50 on axenic amastigotes, and selectivity indices for selected benzimidazole-derived compounds.
THP-1 cells IC50 (µM)
Cmpd
Cytotoxicity 15 µM
± s.e.
% Inhibition on
L.donovani infected cells
5 µM ± s.e.
Cytotoxicity in
NIH/3T3 fibroblasts
Inhibition of
axenic
amastigotes
SI
40 -0.6 ± 4.2 21.7 ± 4.9 15 25.1 0.60
41 14.1 ± 2.2 4.1 ± 1.4 16.7 10.5 1.59
42 5.3 ± 4.3 46.4 ± 3.5 >300 17.8 >16.8
43 -12.9 ± 3.6 27.7 ± 2.4 61.3 22.1 2.77
44 23.8 ± 0.6 19.8 ± 2.8 22.2 8.8 2.52
45 54.1 ± 4.5
46 88.7 ± 1.5
47 -11.9 ± 2.6 16.0 ± 5.3 15.4 23.4 0.66
48 62.8 ± 6.4
49 -4.4 ± 4.9 22.2 ± 1.2 42.2 7.6 5.55
Amphotericin B 96.7 ± 0.7a
Structurally related benzoxazoles, which provided the original inspiration to
test these 2-arylbenzimidazoles as possible antileishmanial compounds, have
unsatisfactory results in the mouse peritoneal macrophage assay, probably
due to their poor cell penetration.168 In the case of our 2-arylbenzimidazole
derivatives, their mechanisms of action are not yet known, but these
compounds are possibly able to penetrate cell membranes as they retain their
antileishmanial activity on THP-1 cells. Two-fold increase in their inhibition
values would benefit, and advance greatly, further study of these compounds.
27
4.2.3 SAR differences of the 2-arylbenzimidazoles as antichlamydial,
antileishmanial and antimalarial agents (Publications I―III and
unpublished results)
Synthesized 2-arylbenzimidazole-derived compounds were evaluated as
antichlamydial, antileishmanial, or antimalarial compounds.I,II,170,177,178 Partly
the same set of derivatives was tested against three intracellular parasites, C.
pneumoniae, L. donovani and P. falciparum. Antichlamydial testing was
carried out with the extension set of antileishmanial benzimidazole
derivatives in addition to the Publication I compounds. Finally, antimalarial
testing was carried out with the nearly whole set of benzimidazole-derived
compounds.
Differences in their SAR are described in Table 4. The main differences
are the preferred position of the amide linker and substitutions of the D-ring.
In addition, alkylation of the benzimidazole NH is allowed in antileishmanial
compounds. The phenyl ring of the benzimidazole (A-ring) was essential for
all activities studied.
Antimalarial testing was carried out at low concentration, and most of the
tested compounds were inactive. Testing at higher concentrations would
have probably given more differences in the inhibition activities of the
compounds as well as more variable SAR data.
In case of the D-ring, antichlamydial activity was related to the non-
planar orientation of the D-ring, whereas antileishmanial activity seems to be
more related to the electronic nature of that moiety.I,II,177 In cases of
antimalarial compounds, 3,5-disubstitution of the D-ring clearly increased
the antiplasmodial activity of the compounds.
28
Table 4. SAR comparison of 2-arylbenzimidazoles as antileishmanial, antichlamydial and antimalarial compounds. These results are based on TR-FIA and unpublished IF results (C. pneumoniae)
I,177,178, a fluorescent viability microplate assay with axenic
amastigotes (L. donovani)II, and whole cell assay at 2 µM (P. falciparum,
unpublished results)170
. The table only describes relative activity change between derivatives against specific pathogens. The precise amount of activity changes cannot be compared between different pathogens.
4.2.4 Efficacy of 2-arylbenzimidazoles against other pathogens
(Publications I, II and unpublished results)
Some 2-arylbenzimidazoles have shown inhibition activity against
intracellular Gram-negative pathogen C. pneumoniae as well as protozoan
parasites L. donovani and P. falciparum. A set of benzimidazoles
(Publication I and selected compounds in II) were also tested against other
bacteria or fungi. Tested benzimidazoles showed no activity against Gram-
Activity change*
Sites and modifications C. pneumoniae L. donovani P. falciparum
1 Replacement with carbon - - - -
2 Amide structure with aryl D-ring at ortho position
+ + + - - -
3 Replacement of amide structure with urea
+ + - -
4 Replacement of D-ring group with various heterocycles
- - - / n.e. -
4 Replacement of D-ring with alkyl - - - n.e. - -
4 Substitution at para position - - + - -
5 3,5-Disubstitution of the D-ring ++ + + + + +
6 Ortho substitution -- n.e. +/- -
7 N-alkylation - - - - - -
8 Amide structure with aryl D-ring at para position
- - - + - -
9 N-alkylation - - - n.e. - - -
9 Replacement with oxygen - - - - - -
10 Removal of aryl A-ring - - - - - - - - -
*+++ major increase, ++ moderate increase, + minor increase, n.e. no effect/irrelevant, - minor decrease, --moderate decrease, - - - major decrease
29
negative bacterium Escherichia coli (at a concentration of 50 µM), Gram-
positive bacteria Staphylococcus aureus (50 µM) (except 34, which showed
also cytotoxicity)I, nor fungus Candida albicans (50 µM)II. A small set of the
2-arylbenzimidazole derivatives has also been tested against the tuberculosis
causing Mycobacterium tuberculosis to demonstrate no apparent inhibition
activity (unpublished data).179 Similarly, no inhibition activity was revealed
when testing a small set of compounds against non-pathogenic
Mycobacterium smegmatis and Corynebacterium glutamicum.179
Selectivity is essential and important, especially critical in the case of
benzimidazole-derived compounds, because the benzimidazole core has been
used as a chemical scaffold to target many biological functions, and some
benzimidazole-structured derivatives might have protein-assay interfering
properties.180 Our 2-arylbenzimidazoles have shown activity in whole cell-
based assays and selectivity over free living bacteria.
4.2.5 2-Aminobenzimidazoles as antimalarial compounds (Publication
III)
2-Aminobenzimidazole derivatives (72 compounds) were evaluated as
antimalarial compounds in Publication III. IC50 determinations were carried
out following the standard methods using the 3H-hypoxanthine
incorporation assay.181 Cytotoxicity was studied by using mammalian HepG2
cell line182 and hERG inhibition values of all the compounds were tested
using hERG channel by IonWorks® electrophysiology183–185. The identified
antimalarial initial hit compound was 3,5-dichlorobenzoyl derivative 11
(Figure 23). It displayed moderate antimalarial activity against P.
falciparum, 3D7A in vitro (Table 5).
Figure 23 Selected 2-aminobenzimidazole derivatives.IV
First, the C-ring of the initial hit compound 11 was replaced with various
non-aromatic rings and functionalized chains. A total of 15 derivatives were
30
synthesized. Replacement of the central aromatic ring with an N-(3-
aminopropyl)amide linker (Figure 23) resulted in a significant increase in
antimalarial potency (12, Table 5). Compound 12 gave an IC50 of 94 nM,
lower cytotoxicity, and significantly better solubility than the initial hit
compound.
Table 5. Selected 2-aminobenzimidazole derivatives and their inhibition activities, hERG inhibition, HepG2 toxicity, solubility, and selectivity index (Pf 3D7A IC50 / HepG2 Tox50). Chloroquine and pyrimethamine were used as antimalarial reference compounds.
IV
Cmpd Pf IC50
3D7A
Pf IC50
W2
Pf IC50
V1/S
hERG
inhibition
HepG2
Tox50 CLND solubility SI
µM
11 1.52 >14.79 26.3 64 17.3
12 0.094 0.410 1.029 1.17 36.31 239 386
50 0.25 22.39 >100 349 >400
51 2.30 >50.1 >100 322 >43.5
52 0.90 >50.1 >100 393 >111
53 0.15 >50.1 25.7 29 171
Chloroquine 0.036 >1 >1
Pyrimethamine 0.068 >20 >20
The D-ring-modified derivatives (49 compounds) and the A-and B-ring-
modified derivatives (8 compounds) were synthesized. The main structure-
activity relationships of the compounds are summarized in Figure 24.
Changes in substitution are tolerated in the D-ring of the benzimidazole
scaffold, but the derivative 12 remained the most active. Trifluoromethyl-
substituted isonicotinic derivative 50 displayed better cytotoxicity profile
than the derivative 12. Furthermore, its hERG activity was reduced slightly,
although its antimalarial efficacy was decreased by over two-fold (Table 5).
The pyrrolidine derivative 51 serves as another example of this series of
compounds. It shows neither cytotoxicity nor hERG activity, but its
antimalarial activity is decreased to an unacceptably low level. Non-aromatic
derivatives showed moderate potency or loss of potency. The piperidine
derivative 52 was the most active from this group of compounds, but had
only moderate antimalarial potency. The A-ring-modified methoxycarbonyl
derivative 53 showed good antimalarial activity, but suffered from a very
poor aqueous solubility. The derivative 12 was chosen to undergo a more
extensive study to verify the potential of this derivative and its chemotype as
an antimalarial agent.
31
Figure 24 SAR of the 2-aminobenzimidazoles and related derivatives as antimalarial agents. Order of the substituent and structural parts in figure are based on P. falciparum IC50.
IV
When tested against chloroquine resistant and multiresistant strains, W2
and V1/S, the potency of compound 12 was slightly reduced (Table 5)
compared to the non-resistant strain. Pharmacokinetic parameters of the
derivative 12 were determined and they are highly promising for further
development, as half-life and clearance of 2-aminobenzimidazole derivative
12 are moderate and its volume of distribution is high (Table 6).
Furthermore, the compound 12 has good solubility and cell membrane
permeability. Plasma protein binding is high, but does not prevent
continuing.
Parasite reduction rate describes how fast the compound is able to kill
parasites compared to the known antimalarial drug. Derivative 12 can be
regarded as a fast/moderate affecting antimalarial compound, like
dihydroartemisinin and piperaquine. Finally, 2-aminobenzimidazole 12 was
brought forward for in vivo testing to confirm its in vivo efficacy in the
humanized mouse model of malaria.
Table 6. Physicochemical and in vitro profile of 12 as well as its pharmacokinetic parameters estimated on CD-1 mice (dose iv 1 mg/kg, po 10 mg/kg).
Profile of compound 12
Molecular weight 363 Cl (mL/min/kg) 39.7
clogP 3.4 Vss (L/kg) 6.35
Solubility FaSSIF (µg/mL) 125 t1/2 (h) iv 1.15
Artificial membrane permeability nm/s 380 F % 81
PPB, human 99.6 AUC(0-8) (ng·h/mL) 3447 ± 254
Ratio blood/plasma 0.72 Cmax (ng/mL) 1663 ± 176
Parasite reduction rate186
Fast/Moderate
32
In vivo efficacy of compound 12 against P. falciparum was studied by using
mice engrafted with human erythrocytes (Figure 25).74 The compound 12
was proved to be efficacious in vivo against P. falciparum upon once a day
oral administration (100 mg/kg). Compound 12 was able to reduce the
parasitemia under the limit of detection after two doses, showing a parasite
clearance rate consistent with the known fast-acting antimalarials. The
microscopic observations suggest that 12 induces in vivo rapid killing of P.
falciparum erythrocyte stages, because pyknotic parasites emerge two days
after the initiation of treatment.
Figure 25 Efficacy of 12 in the P. falciparum (3D70087/N9
) mouse model. Data shows individual parasitemia for two mice (M1 and M2) at 100 mg/kg or vehicle.
IV
Further studies are needed to explore the A- and B-ring modification of the
molecule, and its benzimidazole/guanidine-type moiety for more thorough
structure-activity relationships, especially those related to the cardiotoxicity-
inducing hERG activity. The hERG activity of some compounds was also
tested using the Qpatch method in addition of IonWorks®
electrophysiology.187 Results from these two methods do not correlate
completely, as only part of the compounds have been tested using both
methods and thus clear conclusions in this respect cannot be drawn. In any
case, non-hERG active compounds containing N-[3-[(benzimidazol-2-
yl)amino]propyl]amide structure were found in both methods, which proves
that it is possible to find non-hERG active antimalarial compounds from this
structural group. In addition, increasing the antimalarial potency against
chloroquine and multiresistant strains of the protozoan parasite P.
falciparum warrants more studies.
2-Aminobenzimidazoles have shown antimalarial activity in various
studies recently. N-Aryl-2-aminobenzimidazoles (Figure 8 in Section 1.3.1)
have shown nanomolar antimalarial activity and in vivo efficacy, but also
33
lower inhibition activity to resistant strains as well as possible hERG
issues.109 In another study, analogs structurally related to astemizole were
investigated as antimalarial agents showing nanomolar activity and good
selectivity indices.100 Although the chemical structures of these analogs differ
from those of ours, combined with the fact that 2-aminobenzimidazole is just
one fragment of compounds tested as antimalarial agents, the found
similarity is rather interesting and worth keeping in mind.
4.2.6 Benzo[c][1,2,5]oxadiazoles as antileishmanial compounds
(Publication IV)
The antileishmanial activity of 25 carboxyimidamide-substituted
benzoxadiazole and related derivatives was detected by using a fluorescent
viability microplate assay with L. donovani (MHOM/SD/1962/1S-Cl2d)
axenic amastigotes and alamarBlue. The main SAR findings are described in
Figure 26. Substitution in the right-hand side of the core structure was
studied, and meta-substitution seems to be preferred. The linker part could
be shortened and modified without losing activity. The benzoxadiazole ring is
not essential for antileishmanial activity and could be replaced with other
heterocycles. Even removal of the ring fails to abolish the antileishmanial
activity completely.
Figure 26 SAR of the benzoxadiazoles and related derivatives as antileishmanial compounds. Order of the substituent and structural parts in figure are based on L. donovani % inhibition values (axenic amastigotes).
The best compounds in this study were carboxyimidamide-substituted 24
and N,N’-oxydiamide 54 (Figure 27). They have low IC50 values on axenic
amastigotes and no signs of cytotoxicity against fibroblasts (Table 7). Further
modification of the structure, especially its linker part, could be planned to
study SAR more thoroughly and hopefully to reach nanomolar
antileishmanial activity.
34
Figure 27 The best L. donovani growth inhibiting compound (24, 54) in this study.
Table 7. IC50 on axenic amastigotes, cytotoxicity on NIH/3T3 fibroblasts and selectivity indices of the best derivatives.
Compound Axenic amastigotes
IC50 (µM)
Fibroblasts IC50
(µM) SI
24 4.0 >300 >58
54 5.2 >300 >75
35
5 SUMMARY AND CONCLUSIONS
Resistance to antimicrobial agents is alarmingly increasing, and parasitic
diseases still commonly threaten millions of people. This is why, every effort
to support and facilitate studies of novel antimicrobial agents is beneficial.
The heterocyclic structures, in turn, are the key fragments commonly used in
drug design and found in the chemical structures of the approved drugs and
active pharmaceutical ingredients. This thesis focuses on benzimidazoles and
benzo[c][1,2,5]oxadiazoles. This study enhances knowledge of 2-
arylbenzimidazole, 2-aminobenzimidazole, and benzo[c][1,2,5]oxadiazole
derivatives against intracellular pathogens, Chlamydia pneumoniae,
Leishmania donovani, and Plasmodium falciparum, causative microbes of
such diseases as lung chlamydia, leishmaniasis, and malaria.
The initial finding of 2-arylbenzimidazoles as antichlamydial compounds
was based on in silico screening, and the subsequent study of benzimidazoles
as antimalarial compounds was initiated because of positive benzimidazole
hit compounds found by the phenotypic HTS screening. Design of 2-
arylbenzimidazoles during antichlamydial and antileishmanial studies as well
as 2-aminobenzimidazoles as antimalarial agents were based on the iterative
SAR studies. Derivatives in all studies have been designed to cover enough
structural variations in the compound series to find and identify potent
antimicrobial compounds. We have shown that antimicrobial compound
design can be performed successfully without unambiguous macromolecular
targets.
Various methods to synthesize the benzimidazole moiety were used, and
in every case, the most practical methods were selected. Facile synthesis
routes to prepare benzimidazole and benzo[c][1,2,5]oxadiazole derivatives
were developed.
2-Arylbenzimidazoles were studied as antichlamydial agents. meta-
Methyl derivative 33 inhibited C. pneumoniae CWL-029 (IC50 10 µM)
without affecting host cell viability. In addition, antichlamydial effect was
confirmed by using strain CV-6 (IC50 6.3 µM). A low minimum
chlamydiocidal concentration of the derivative 33 indicates chlamydiocidal
effect. In the antileishmanial study, the best 2-arylbenzimidazole 42
inhibited L. donovani-infected THP-1 cells 46% at concentration of 5 µM.
Overall, according to our study the 2-arylbenzimidazole scaffold cannot be
regarded as firmly associated to cellular cytotoxicity and this is why, careful
attention should be paid to structure-cytotoxicity analyses in further studies.
Antichlamydial, antileishmanial, and antimalarial SAR of the 2-
arylbenzimidazoles differ considerably. The main findings are the following:
tolerance for NH alkylation (L. donovani tolerated), differences in
substitution of the right-hand side ring (para substitution not tolerated in
antichlamydial compound), and differences of the linker (amide) position
36
(antichlamydial and antileishmanial compounds). 3,5-Disubstitution of the
D-ring increased antimalarial activity. The benzimidazole phenyl ring was
shown to be essential for antichlamydial, antileishmanial, and antimalarial
activities of the assayed compounds. In antichlamydial compounds, the high
inhibition activity was related to non-planar orientation between C- and D-
ring in the right-hand side of the molecule, and antileishmanial activity
seems to be more related to the electronic nature of the substituents.
In the study of 2-aminobenzimidazoles as antimalarial compounds,
derivative 12 inhibited growth of P. falciparum 3D7A (IC50 94 nM), although
its activity was reduced against resistant strains. Solubility, cytotoxicity, and
pharmacokinetic parameters were promising for derivative 12, and it was
forwarded to in vivo test to confirm in vivo efficacy of this structural group.
Derivative 12 was efficacious upon once a day oral administration, and was
able to reduce parasitemia under limit of detection with just two doses (100
mg/kg), killing parasite rapidly.
Furthermore, benzoxadiazole derivatives showed antileishmanial activity.
The best compound was the 3-chloro derivative (24) of the carboxy-
imidamide substituted derivatives and N,N'-oxydiamide-benzoxadiazole
derivative 54. They inhibit the growth of L. donovani having IC50 4.0 and 5.6
µM, respectively without any sign of cytotoxicity against fibroblasts.
Selectivity of the 2-arylbenzimidazoles over free living (planktonic)
pathogens e.g. E. coli is an advantage. For example, during an oral
administration regimen, it is a great advantage if normal microbial flora of
the gut is not affected by the antimicrobial agent employed. The observed
selectivity enhances possibilities of the successful development of these
compounds further. Overall, it is known that benzimidazole derivatives have
shown wide variety of biological activities. However, we have shown that the
benzimidazole scaffold can be used due to its feasible chemical modification
protocols as a versatile starting point to study antibacterial and antiparasitic
compounds and their structure-activity relationships.
Mechanisms of action of the derivatives remain elusive, and further
studies should be conducted to clarify those. Our current level of knowledge
is that benzimidazole derivatives can affect all three intracellular target
pathogens studied (Chlamydia pneumoniae, Leishmania donovani, and
Plasmodium falciparum), and possibly penetrate their cell membranes. The
2-arylbenzdimidazole derivatives inhibit growth of C. pneumoniae and affect
the amastigote form of L. donovani. The antimalarial 2-aminobenzimidazole
derivatives clearly affect the blood stage of malaria.
2-Arylbenzimidazoles as antichlamydial compounds still show very
interesting aspects from the medicinal chemistry point of view, as very small
modifications affect the antichlamydial inhibition activity. As antileishmanial
compounds both 2-arylbenzimidazoles and benzoxadiazoles would need
changes to their current chemical structures to reach nanomolar activities
and unequivocal structure-activity relationships. Moreover, possible
37
cytotoxicity issues related to 2-arylbenzimidazoles need to be addressed since
cytotoxicity varies considerably between different derivatives.
The antimicrobial compound design without the knowledge on their
macromolecular targets could be continued, but increase in potency should
be achieved. In addition, most of our current heterocyclic derivatives are still
relatively lipophilic, so further modifications of their chemical structures
would be needed to overcome issues related to lipophilicity and its
implications to other critical physicochemical and pharmacokinetic
parameters. Nanomolar activity of the benzimidazole-derived compound was
reached in the antimalarial study of this thesis. 2-Aminobenzimidazole
derivatives could be studied further. Their properties for further
development are the most promising of all the compounds assayed during
this study.
38
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