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

REFERENCES

(1) Silver, L. L. Challenges of Antibacterial Discovery. Clin. Microbiol. Rev. 2011, 24, 71–109.

(2) Pink, R.; Hudson, A.; Mouriès, M.-A.; Bendig, M. Opportunities and Challenges in Antiparasitic Drug Discovery. Nat. Rev. Drug Discov. 2005, 4, 727–740.

(3) Sykes, M. L.; Avery, V. M. Approaches to Protozoan Drug Discovery: Phenotypic Screening. J. Med. Chem. 2013, 56, 7727–7740.

(4) Gilbert, I. H. Drug Discovery for Neglected Diseases: Molecular Target-Based and Phenotypic Approaches. J. Med. Chem. 2013, 56, 7719–7726.

(5) Barker, J. J. Antibacterial Drug Discovery and Structure-Based Design. Drug Discov. Today 2006, 11, 391–404.

(6) Swinney, D. C. Phenotypic vs. Target-Based Drug Discovery for First-in-Class Medicines. Clin. Pharmacol. Ther. 2013, 93, 299–301.

(7) Eder, J.; Sedrani, R.; Wiesmann, C. The Discovery of First-in-Class Drugs: Origins and Evolution. Nat. Rev. Drug Discov. 2014, 13, 577–587.

(8) Neglected Tropical Diseases. http://www.who.int/neglected_diseases/diseases/en/ (accessed 16.02.2015).

(9) World Malaria Report 2014 http://apps.who.int/iris/bitstream/10665/144852/2/9789241564830_eng.pdf (accessed 16.02.2015).

(10) GSK Announces EU Regulatory Submission for Malaria Vaccine Candidate RTS,S. http://www.gsk.com/en-Gb/media/press-releases/2014/gsk-Announces-Eu-Regulatory-Submission-for-Malaria-Vaccine-Candidate-Rtss (accessed 4.2.2015).

(11) Taylor, R. D.; MacCoss, M.; Lawson, A. D. G. Rings in Drugs. J. Med. Chem. 2014, 57, 5845–5859.

(12) Hammerschlag, M. R. The Intracellular Life of Chlamydiae. Semin. Pediatr. Infect. Dis. 2002, 13, 239–248.

(13) Hogan, R. J.; Mathews, S. A.; Mukhopadhyay, S.; Summersgill, J. T.; Timms, P. Chlamydial Persistence: Beyond the Biphasic Paradigm. Infect. Immun. 2004, 72, 1843–1855.

(14) Beatty, W. L.; Morrison, R. P.; Byrne, G. I. Persistent Chlamydiae: From Cell Culture to a Paradigm for Chlamydial Pathogenesis. Microbiol. Rev. 1994, 58, 686–699.

(15) Saikku, P.; Mattila, K.; Nieminen, M. S.; Huttunen, J. K.; Leinonen, M.; Ekman, M.-R.; Mäkelä, P. H.; Valtonen, V. Serological Evidence of an Association of a Novel Chlamydia, TWAR, with Chronic Coronary Heart Disease and Acute Myocardial Infarction. Lancet 1988, 332, 983–986.

(16) Volanen, I.; Järvisalo, M. J.; Vainionpää, R.; Arffman, M.; Kallio, K.; Anglé, S.; Rönnemaa, T.; Viikari, J.; Marniemi, J.; Raitakari, O. T.; Simell, O. Increased Aortic Intima-Media Thickness in 11-Year-Old Healthy Children With Persistent Chlamydia pneumoniae Seropositivity. Arterioscler. Thromb. Vasc. Biol. 2006, 26, 649–655.

(17) Rosenfeld, M. E.; Campbell, L. A. Pathogens and Atherosclerosis: Update on the Potential Contribution of Multiple Infectious Organisms to the Pathogenesis of Atherosclerosis: Thromb. Haemost. 2011, 106, 858–867.

(18) Hahn, D. L.; Peeling, R. W.; Dillon, E.; McDonald, R.; Saikku, P. Serologic Markers for Chlamydia pneumoniae in Asthma. Ann. Allergy. Asthma. Immunol. 2000, 84, 227–233.

(19) Cosentini, R.; Tarsia, P.; Canetta, C.; Graziadei, G.; Brambilla, A. M.; Aliberti, S.; Pappalettera, M.; Tantardini, F.; Blasi, F. Severe Asthma

39

Exacerbation: Role of Acute Chlamydophila pneumoniae and Mycoplasma pneumoniae Infection. Respir. Res. 2008, 9, 48.

(20) Blasi, F.; Tarsia, P.; Aliberti, S. Chlamydophila pneumoniae. Clin. Microbiol. Infect. 2009, 15, 29–35.

(21) Papaetis, G. S.; Anastasakou, E.; Orphanidou, D. Chlamydophila pneumoniae Infection and COPD: More Evidence for Lack of Evidence? Eur. J. Intern. Med. 2009, 20, 579–585.

(22) Balin, B. J.; Gérard, H. C.; Arking, E. J.; Appelt, D. M.; Branigan, P. J.; Abrams, J. T.; Whittum-Hudson, J. A.; Hudson, A. P. Identification and Localization of Chlamydia pneumoniae in the Alzheimer’s Brain. Med. Microbiol. Immunol. 1998, 187, 23–42.

(23) Boelen, E.; Steinbusch, H. W. M.; van der Ven, A. J. A. M.; Grauls, G.; Bruggeman, C. A.; Stassen, F. R. M. Chlamydia pneumoniae Infection of Brain Cells: An in Vitro Study. Neurobiol. Aging 2007, 28, 524–532.

(24) Gérard, H. C.; Dreses-Werringloer, U.; Wildt, K. S.; Deka, S.; Oszust, C.; Balin, B. J.; Frey, W. H.; Bordayo, E. Z.; Whittum-Hudson, J. A.; Hudson, A. P. Chlamydophila (Chlamydia) pneumoniae in the Alzheimer’s Brain. FEMS Immunol. Med. Microbiol. 2006, 48, 355–366.

(25) Laurila, A. L.; Anttila, T.; Läärä, E.; Bloigu, A.; Virtamo, J.; Albanes, D.; Leinonen, M.; Saikku, P. Serological Evidence of an Association between Chlamydia pneumoniae Infection and Lung Cancer. Int. J. Cancer 1997, 74, 31–34.

(26) Senn, L.; Hammerschlag, M. R.; Greub, G. Therapeutic Approaches to Chlamydia Infections. Expert Opin. Pharmacother. 2005, 6, 2281–2290.

(27) Hammerschlag, M. R.; Kohlhoff, S. A. Treatment of Chlamydial Infections. Expert Opin. Pharmacother. 2012, 13, 545–552.

(28) Gieffers, J.; Rupp, J.; Gebert, A.; Solbach, W.; Klinger, M. First-Choice Antibiotics at Subinhibitory Concentrations Induce Persistence of Chlamydia pneumoniae. Antimicrob. Agents Chemother. 2004, 48, 1402–1405.

(29) Kutlin, A.; Roblin, P. M.; Hammerschlag, M. R. Effect of Prolonged Treatment with Azithromycin, Clarithromycin, or Levofloxacin on Chlamydia pneumoniae in a Continuous-Infection Model. Antimicrob. Agents Chemother. 2002, 46, 409–412.

(30) Fauvart, M.; Groote, V. N. D.; Michiels, J. Role of Persister Cells in Chronic Infections: Clinical Relevance and Perspectives on Anti-Persister Therapies. J. Med. Microbiol. 2011, 60, 699–709.

(31) Lewis, K. Platforms for Antibiotic Discovery. Nat. Rev. Drug Discov. 2013, 12, 371–387.

(32) Payne, D. J.; Gwynn, M. N.; Holmes, D. J.; Pompliano, D. L. Drugs for Bad Bugs: Confronting the Challenges of Antibacterial Discovery. Nat. Rev. Drug Discov. 2007, 6 , 29–40.

(33) O’Shea, R.; Moser, H. E. Physicochemical Properties of Antibacterial Compounds: Implications for Drug Discovery. J. Med. Chem. 2008, 51, 2871–2878.

(34) Brown, D. G.; May-Dracka, T. L.; Gagnon, M. M.; Tommasi, R. Trends and Exceptions of Physical Properties on Antibacterial Activity for Gram-Positive and Gram-Negative Pathogens. J. Med. Chem. 2014, 57, 10144–10161.

(35) Hanski, L.; Vuorela, P. M. Recent Advances in Technologies for Developing Drugs against Chlamydia pneumoniae. Expert Opin. Drug Discov. 2014, 9, 791–802.

(36) Mueller, K. E.; Plano, G. V.; Fields, K. A. New Frontiers in Type III Secretion Biology: The Chlamydia Perspective. Infect. Immun. 2014, 82, 2–9.

(37) Bailey, L.; Gylfe, Å.; Sundin, C.; Muschiol, S.; Elofsson, M.; Nordström, P.; Henriques-Normark, B.; Lugert, R.; Waldenström, A.; Wolf-Watz, H.;

40

Bergström, S. Small Molecule Inhibitors of Type III Secretion in Yersinia Block the Chlamydia pneumoniae Infection Cycle. FEBS Lett. 2007, 581, 587–595.

(38) Erkkilä, L.; Jauhiainen, M.; Laitinen, K.; Haasio, K.; Tiirola, T.; Saikku, P.; Leinonen, M. Effect of Simvastatin, an Established Lipid-Lowering Drug, on Pulmonary Chlamydia pneumoniae Infection in Mice. Antimicrob. Agents Chemother. 2005, 49, 3959–3962.

(39) Azenabor, A. A.; Chaudhry, A. U.; Yang, S. Macrophage L-Type Ca2+

Channel Antagonists Alter Chlamydia pneumoniae MOMP and HSP-60 mRNA Gene Expression, and Improve Antibiotic Susceptibility. Immunobiology 2003, 207, 237–245.

(40) Control of the Leishmaniases: Report of a Meeting of the WHO Expert Committee on the Control of Leishmaniases, Geneva, 22-26 March 2010, WHO Technical Report Series 949. http://whqlibdoc.who.int/trs/WHO_TRS_949_eng.pdf?ua=1 (accessed 16.02.2015).

(41) Sustaining the Drive to Overcome the Global Impact of Neglegted Tropical Diseases, Second WHO Report of Neglegted Diseases; 2013. http://www.who.int/neglected_diseases/9789241564540/en/ (accessed 16.02.2015).

(42) Leishmaniaisis. http://www.who.int/mediacentre/factsheets/fs375/en/ (accessed 28.02.2015).

(43) Chappuis, F.; Sundar, S.; Hailu, A.; Ghalib, H.; Rijal, S.; Peeling, R. W.; Alvar, J.; Boelaert, M. Visceral Leishmaniasis: What Are the Needs for Diagnosis, Treatment and Control? Nat. Rev. Microbiol. 2007, 5, 873–882.

(44) Seifert, K. Structures, Targets and Recent Approaches in Anti-Leishmanial Drug Discovery and Development. Open Med. Chem. J. 2011, 5, 31–39.

(45) Sundar, S.; More, D. K.; Singh, M. K.; Singh, V. P.; Sharma, S.; Makharia, A.; Kumar, P. C. K.; Murray, H. W. Failure of Pentavalent Antimony in Visceral Leishmaniasis in India: Report from the Center of the Indian Epidemic. Clin. Infect. Dis. 2000, 31, 1104–1107.

(46) Thakur, C. P.; Sinha, G. P.; Pandey, A. K.; Kumar, N.; Kumar, P.; Hassan, S. M.; Narain, S.; Roy, R. K. Do the Diminishing Efficacy and Increasing Toxicity of Sodium Stibogluconate in the Treatment of Visceral Leishmaniasis in Bihar, India, Justify Its Continued Use as a First Line Drug? An Observational Study of 80 Cases. Ann. Trop. Med. Parasitol. 1998, 92, 561–569.

(47) Sundar, S.; Chakravarty, J. Leishmaniasis: An Update of Current Pharmacotherapy. Expert Opin. Pharmacother. 2012, 14, 53–63.

(48) Croft, S. L.; Sundar, S.; Fairlamb, A. H. Drug Resistance in Leishmaniasis. Clin. Microbiol. Rev. 2006, 19, 111–126.

(49) Alvar, J.; Aparicio, P.; Aseffa, A.; Boer, M. D.; Cañavate, C.; Dedet, J.-P.; Gradoni, L.; Horst, R. T.; López-Vélez, R.; Moreno, J. The Relationship between Leishmaniasis and AIDS: The Second 10 Years. Clin. Microbiol. Rev. 2008, 21, 334–359.

(50) Desjeux, P.; Alvar, J. Leishmania/HIV Co-Infections: Epidemiology in Europe. Ann. Trop. Med. Parasitol. 2003, 97, 3–15.

(51) Sundar, S.; Sinha, P. K.; Rai, M.; Verma, D. K.; Nawin, K.; Alam, S.; Chakravarty, J.; Vaillant, M.; Verma, N.; Pandey, K.; Kumari, P.; Lal, C. S.; Arora, R.; Sharma, B.; Ellis, S.; Strub-Wourgaft, N.; Balasegaram, M.; Olliaro, P.; Das, P.; Modabber, F. Comparison of Short-Course Multidrug Treatment with Standard Therapy for Visceral Leishmaniasis in India: An Open-Label, Non-Inferiority, Randomised Controlled Trial. The Lancet 2011, 377, 477–486.

(52) Sundar, S.; Rai, M.; Chakravarty, J.; Agarwal, D.; Agrawal, N.; Vaillant, M.; Olliaro, P.; Murray, H. W. New Treatment Approach in Indian Visceral

41

Leishmaniasis: Single-Dose Liposomal Amphotericin B Followed by Short-Course Oral Miltefosine. Clin. Infect. Dis. 2008, 47, 1000–1006.

(53) Melaku, Y.; Collin, S. M.; Keus, K.; Gatluak, F.; Ritmeijer, K.; Davidson, R. N. Treatment of Kala-Azar in Southern Sudan Using a 17-Day Regimen of Sodium Stibogluconate Combined with Paromomycin: A Retrospective Comparison with 30-Day Sodium Stibogluconate Monotherapy. Am. J. Trop. Med. Hyg. 2007, 77, 89–94.

(54) Van Griensven, J.; Balasegaram, M.; Meheus, F.; Alvar, J.; Lynen, L.; Boelaert, M. Combination Therapy for Visceral Leishmaniasis. Lancet Infect. Dis. 2010, 10, 184–194.

(55) Burchmore, R. J. S.; Barrett, M. P. Life in Vacuoles – Nutrient Acquisition by Leishmania Amastigotes. Int. J. Parasitol. 2001, 31, 1311–1320.

(56) Sharlow, E. R.; Grogl, M.; Johnson, J.; Lazo, J. S. Anti-Leishmanial Drug Discovery: Rising to the Challenges of a Highly Neglected Disease. Mol. Interv. 2010, 10, 72–75.

(57) Croft, S. L.; Olliaro, P. Leishmaniasis Chemotherapy—challenges and Opportunities. Clin. Microbiol. Infect. 2011, 17, 1478–1483.

(58) Richard, J. V.; Werbovetz, K. A. New Antileishmanial Candidates and Lead Compounds. Curr. Opin. Chem. Biol. 2010, 14, 447–455.

(59) Nagle, A. S.; Khare, S.; Kumar, A. B.; Supek, F.; Buchynskyy, A.; Mathison, C. J. N.; Chennamaneni, N. K.; Pendem, N.; Buckner, F. S.; Gelb, M. H.; Molteni, V. Recent Developments in Drug Discovery for Leishmaniasis and Human African Trypanosomiasis. Chem. Rev. 2014, 114, 11305–11347.

(60) Rycker, M. D.; Hallyburton, I.; Thomas, J.; Campbell, L.; Wyllie, S.; Joshi, D.; Cameron, S.; Gilbert, I. H.; Wyatt, P. G.; Frearson, J. A.; Fairlamb, A. H.; Gray, D. W. Comparison of a High-Throughput High-Content Intracellular Leishmania donovani Assay with an Axenic Amastigote Assay. Antimicrob. Agents Chemother. 2013, 57, 2913–2922.

(61) Shimony, O.; Jaffe, C. L. Rapid Fluorescent Assay for Screening Drugs on Leishmania Amastigotes. J. Microbiol. Methods 2008, 75, 196–200.

(62) Vermeersch, M.; Luz, R. I. da; Toté, K.; Timmermans, J.-P.; Cos, P.; Maes, L. in Vitro Susceptibilities of Leishmania donovani Promastigote and Amastigote Stages to Antileishmanial Reference Drugs: Practical Relevance of Stage-Specific Differences. Antimicrob. Agents Chemother. 2009, 53, 3855–3859.

(63) Osorio, Y.; Travi, B. L.; Renslo, A. R.; Peniche, A. G.; Melby, P. C. Identification of Small Molecule Lead Compounds for Visceral Leishmaniasis Using a Novel ex Vivo Splenic Explant Model System. PLoS Negl Trop Dis 2011, 5, e962.

(64) Global Report on Antimalarial Efficacy and Drug Resistance: 2000-2010, 2010, WHO.

(65) Tun, K. M.; Imwong, M.; Lwin, K. M.; Win, A. A.; Hlaing, T. M.; Hlaing, T.; Lin, K.; Kyaw, M. P.; Plewes, K.; Faiz, M. A.; Dhorda, M.; Cheah, P. Y.; Pukrittayakamee, S.; Ashley, E. A.; Anderson, T. J. C.; Nair, S.; McDew-White, M.; Flegg, J. A.; Grist, E. P. M.; Guerin, P.; Maude, R. J.; Smithuis, F.; Dondorp, A. M.; Day, N. P. J.; Nosten, F.; White, N. J.; Woodrow, C. J. Spread of Artemisinin-Resistant Plasmodium falciparum in Myanmar: A Cross-Sectional Survey of the K13 Molecular Marker. Lancet Infect. Dis. 2015.

(66) Wells, T. N. C.; Alonso, P. L.; Gutteridge, W. E. New Medicines to Improve Control and Contribute to the Eradication of Malaria. Nat. Rev. Drug Discov. 2009, 8, 879–891.

(67) Greenwood, B. M.; Fidock, D. A.; Kyle, D. E.; Kappe, S. H. I.; Alonso, P. L.; Collins, F. H.; Duffy, P. E. Malaria: Progress, Perils, and Prospects for Eradication. J. Clin. Invest. 2008, 118, 1266–1276.

42

(68) Burrows, J. N.; Leroy, D.; Lotharius, J.; Waterson, D. Challenges in Antimalarial Drug Discovery. Future Med. Chem. 2011, 3, 1401–1412.

(69) Barnett, D. S.; Guy, R. K. Antimalarials in Development in 2014. Chem. Rev. 2014, 114, 11221–11241.

(70) Mazier, D.; Landau, I.; Druilhe, P.; Miltgen, F.; Guguen-Guillouzo, C.; Baccam, D.; Baxter, J.; Chigot, J. P.; Gentilini, M. Cultivation of the Liver Forms of Plasmodium vivax in Human Hepatocytes. Nature 1984, 307, 367–369.

(71) Dembélé, L.; Franetich, J.-F.; Lorthiois, A.; Gego, A.; Zeeman, A.-M.; Kocken, C. H. M.; Le Grand, R.; Dereuddre-Bosquet, N.; van Gemert, G.-J.; Sauerwein, R.; Vaillant, J.-C.; Hannoun, L.; Fuchter, M. J.; Diagana, T. T.; Malmquist, N. A.; Scherf, A.; Snounou, G.; Mazier, D. Persistence and Activation of Malaria Hypnozoites in Long-Term Primary Hepatocyte Cultures. Nat. Med. 2014, 20, 307–312.

(72) Lelièvre, J.; Almela, M. J.; Lozano, S.; Miguel, C.; Franco, V.; Leroy, D.; Herreros, E. Activity of Clinically Relevant Antimalarial Drugs on Plasmodium falciparum Mature Gametocytes in an ATP Bioluminescence “Transmission Blocking” Assay. PloS One 2012, 7, e35019.

(73) March, S.; Ng, S.; Velmurugan, S.; Galstian, A.; Shan, J.; Logan, D.; Carpenter, A.; Thomas, D.; Lee Sim, B. K.; Mota, M. M.; Hoffman, S. L.; Bhatia, S. N. A Microscale Human Liver Platform That Supports the Hepatic Stages of Plasmodium falciparum and vivax. Cell Host Microbe 2013, 14, 104–115.

(74) Jiménez-Díaz, M. B.; Mulet, T.; Viera, S.; Gómez, V.; Garuti, H.; Ibáñez, J.; Alvarez-Doval, A.; Shultz, L. D.; Martínez, A.; Gargallo-Viola, D.; Angulo-Barturen, I. Improved Murine Model of Malaria Using Plasmodium falciparum Competent Strains and Non-Myelodepleted NOD-Scid IL2Rγnull Mice Engrafted with Human Erythrocytes. Antimicrob. Agents Chemother. 2009, 53, 4533–4536.

(75) Angulo-Barturen, I.; Jiménez-Díaz, M. B.; Mulet, T.; Rullas, J.; Herreros, E.; Ferrer, S.; Jiménez, E.; Mendoza, A.; Regadera, J.; Rosenthal, P. J.; Bathurst, I.; Pompliano, D. L.; Gómez de las Heras, F.; Gargallo-Viola, D. A Murine Model of Falciparum-Malaria by in Vivo Selection of Competent Strains in Non-Myelodepleted Mice Engrafted with Human Erythrocytes. PLoS One 2008, 3, e2252.

(76) Zheng, W.; Thorne, N.; McKew, J. C. Phenotypic Screens as a Renewed Approach for Drug Discovery. Drug Discov. Today 2013, 18, 1067–1073.

(77) Eggert, U. S. The Why and How of Phenotypic Small-Molecule Screens. Nat. Chem. Biol. 2013, 9, 206–209.

(78) Hart, C. P. Finding the Target after Screening the Phenotype. Drug Discov. Today 2005, 10, 513–519.

(79) Young, R. J.; Green, D. V. S.; Luscombe, C. N.; Hill, A. P. Getting Physical in Drug Discovery II: The Impact of Chromatographic Hydrophobicity Measurements and Aromaticity. Drug Discov. Today 2011, 16, 822–830.

(80) Trainor, G. L. The Importance of Plasma Protein Binding in Drug Discovery. Expert Opin. Drug Discov. 2007, 2, 51–64.

(81) Ponti, D. F. D.; Poluzzi, E.; Cavalli, A.; Recanatini, M.; Montanaro, N. Safety of Non-Antiarrhythmic Drugs That Prolong the QT Interval or Induce Torsade de Pointes. Drug Saf. 2012, 25, 263–286.

(82) Recanatini, M.; Cavalli, A.; Masetti, M. Modeling hERG and Its Interactions with Drugs: Recent Advances in Light of Current Potassium Channel Simulations. ChemMedChem 2008, 3, 523–535.

(83) Jamieson, C.; Moir, E. M.; Rankovic, Z.; Wishart, G. Medicinal Chemistry of hERG Optimizations: Highlights and Hang-Ups. J. Med. Chem. 2006, 49, 5029–5046.

43

(84) Spasov, A. A.; Yozhitsa, I. N.; Bugaeva, L. I.; Anisimova, V. A. Benzimidazole Derivatives: Spectrum of Pharmacological Activity and Toxicological Properties (a Review). Pharm. Chem. J. 1999, 33, 232–243.

(85) Bansal, Y.; Silakari, O. The Therapeutic Journey of Benzimidazoles: A Review. Bioorg. Med. Chem. 2012, 20, 6208–6236.

(86) Kus, C.; Sözüdönmez, F.; Altanlar, N. Synthesis and Antimicrobial Activity of Some Novel 2-[4-(Substituted Piperazin-/Piperidin-1-ylcarbonyl)phenyl]-1H-Benzimidazole Derivatives. Arch. Pharm. 2009, 342, 54–60.

(87) Özden, S.; Atabey, D.; Yıldız, S.; Göker, H. Synthesis, Potent Anti-Staphylococcal Activity and QSARs of Some Novel 2-Anilinobenzazoles. Eur. J. Med. Chem. 2008, 43, 1390–1402.

(88) Bandyopadhyay, P.; Sathe, M.; Ponmariappan, S.; Sharma, A.; Sharma, P.; Srivastava, A. K.; Kaushik, M. P. Exploration of in Vitro Time Point Quantitative Evaluation of Newly Synthesized Benzimidazole and Benzothiazole Derivatives as Potential Antibacterial Agents. Bioorg. Med. Chem. Lett. 2011, 21, 7306–7309.

(89) Puratchikody, A.; Nagalakshmi, G.; Doble, M. Experimental and QSAR Studies on Antimicrobial Activity of Benzimidazole Derivatives. Chem. Pharm. Bull. 2008, 56, 273–281.

(90) Huigens III, R. W.; Reyes, S.; Reed, C. S.; Bunders, C.; Rogers, S. A.; Steinhauer, A. T.; Melander, C. The Chemical Synthesis and Antibiotic Activity of a Diverse Library of 2-Aminobenzimidazole Small Molecules against MRSA and Multidrug-Resistant A. baumannii. Bioorg. Med. Chem. 2010, 18, 663–674.

(91) Torres-Gómez, H.; Hernández-Núñez, E.; León-Rivera, I.; Guerrero-Alvarez, J.; Cedillo-Rivera, R.; Moo-Puc, R.; Argotte-Ramos, R.; Carmen Rodríguez-Gutiérrez, M. del; Chan-Bacab, M. J.; Navarrete-Vázquez, G. Design, Synthesis and in Vitro Antiprotozoal Activity of Benzimidazole-Pentamidine Hybrids. Bioorg. Med. Chem. Lett. 2008, 18, 3147–3151.

(92) Hernández-Luis, F.; Hernández-Campos, A.; Castillo, R.; Navarrete-Vázquez, G.; Soria-Arteche, O.; Hernández-Hernández, M.; Yépez-Mulia, L. Synthesis and Biological Activity of 2-(trifluoromethyl)-1H-Benzimidazole Derivatives against Some Protozoa and Trichinella spiralis. Eur. J. Med. Chem. 2010, 45, 3135–3141.

(93) Katiyar, S. K.; Gordon, V. R.; McLaughlin, G. L.; Edlind, T. D. Antiprotozoal Activities of Benzimidazoles and Correlations with Beta-Tubulin Sequence. Antimicrob. Agents Chemother. 1994, 38, 2086–2090.

(94) Ramachandran, S.; Hameed P., S.; Srivastava, A.; Shanbhag, G.; Morayya, S.; Rautela, N.; Awasthy, D.; Kavanagh, S.; Bharath, S.; Reddy, J.; Panduga, V.; Prabhakar, K. R.; Saralaya, R.; Nanduri, R.; Raichurkar, A.; Menasinakai, S.; Achar, V.; Jiménez-Díaz, M. B.; Martínez, M. S.; Angulo-Barturen, I.; Ferrer, S.; Sanz, L. M.; Gamo, F. J.; Duffy, S.; Avery, V. M.; Waterson, D.; Lee, M. C. S.; Coburn-Flynn, O.; Fidock, D. A.; Iyer, P. S.; Narayanan, S.; Hosagrahara, V.; Sambandamurthy, V. K. N-Aryl-2-Aminobenzimidazoles: Novel, Efficacious, Antimalarial Lead Compounds. J. Med. Chem. 2014, 57, 6642-6652.

(95) Saify, Z. S.; Azim, M. K.; Ahmad, W.; Nisa, M.; Goldberg, D. E.; Hussain, S. A.; Akhtar, S.; Akram, A.; Arayne, A.; Oksman, A.; Khan, I. A. New Benzimidazole Derivatives as Antiplasmodial Agents and Plasmepsin Inhibitors: Synthesis and Analysis of Structure–activity Relationships. Bioorg. Med. Chem. Lett. 2012, 22, 1282–1286.

(96) Skinner-Adams, T. S.; Davis, T. M. E.; Manning, L. S.; Johnston, W. A. The Efficacy of Benzimidazole Drugs against Plasmodium falciparum in Vitro. Trans. R. Soc. Trop. Med. Hyg. 1997, 91, 580–584.

44

(97) Alp, M.; Göker, H.; Brun, R.; Yıldız, S. Synthesis and Antiparasitic and Antifungal Evaluation of 2′-Arylsubstituted-1H,1′H-[2,5′]bisbenzimidazolyl-5-Carboxamidines. Eur. J. Med. Chem. 2009, 44 (5), 2002–2008.

(98) Howarth, J.; Hanlon, K. Novel N-Ferrocenylmethyl, N′-Methyl-2-Substituted Benzimidazolium Iodide Salts with in Vitro Activity against the P. falciparum Malarial Parasite Strain NF54. Tetrahedron Lett. 2001, 42, 751–754.

(99) Skerlj, R. T.; Bastos, C. M.; Booker, M. L.; Kramer, M. L.; Barker, R. H.; Celatka, C. A.; O’Shea, T. J.; Munoz, B.; Sidhu, A. B.; Cortese, J. F.; Wittlin, S.; Papastogiannidis, P.; Angulo-Barturen, I.; Jimenez-Diaz, M. B.; Sybertz, E. Optimization of Potent Inhibitors of P. falciparum Dihydroorotate Dehydrogenase for the Treatment of Malaria. ACS Med. Chem. Lett. 2011, 2, 708–713.

(100) Roman, G.; Crandall, I. E.; Szarek, W. A. Synthesis and Anti-Plasmodium Activity of Benzimidazole Analogues Structurally Related to Astemizole. ChemMedChem 2013, 8, 1795–1804.

(101) Reker, D.; Seet, M.; Pillong, M.; Koch, C. P.; Schneider, P.; Witschel, M. C.; Rottmann, M.; Freymond, C.; Brun, R.; Schweizer, B.; Illarionov, B.; Bacher, A.; Fischer, M.; Diederich, F.; Schneider, G. Deorphaning Pyrrolopyrazines as Potent Multi-Target Antimalarial Agents. Angew. Chem. Int. Ed. 2014, 53, 7079–7084.

(102) Mayence, A.; Vanden Eynde, J. J.; Kaiser, M.; Brun, R.; Yarlett, N.; Huang, T. L. Bis(oxyphenylene)benzimidazoles: A Novel Class of Anti-Plasmodium falciparum Agents. Bioorg. Med. Chem. 2011, 19, 7493–7500.

(103) Shaukat, A.; Mirza, H. M.; Ansari, A. H.; Yasinzai, M.; Zaidi, S. Z.; Dilshad, S.; Ansari, F. L. Benzimidazole Derivatives: Synthesis, Leishmanicidal Effectiveness, and Molecular Docking Studies. Med. Chem. Res. 2013, 22, 3606–3620.

(104) Ismail, M. A.; Batista-Parra, A.; Miao, Y.; Wilson, W. D.; Wenzler, T.; Brun, R.; Boykin, D. W. Dicationic near-Linear Biphenyl Benzimidazole Derivatives as DNA-Targeted Antiprotozoal Agents. Bioorg. Med. Chem. 2005, 13, 6718–6726.

(105) Oh, S.; Kim, S.; Kong, S.; Yang, G.; Lee, N.; Han, D.; Goo, J.; Siqueira-Neto, J. L.; Freitas-Junior, L. H.; Song, R. Synthesis and Biological Evaluation of 2,3-dihydroimidazo[1,2-a]benzimidazole Derivatives against Leishmania donovani and Trypanosoma cruzi. Eur. J. Med. Chem. 2014, 84, 395–403.

(106) DeSimone, R. W.; Currie, K. S.; Mitchell, S. A.; Darrow, J. W.; Pippin, D. A. Privileged Structures: Applications in Drug Discovery. Comb. Chem. High Throughput Screen. 2004, 7, 473–493.

(107) Evans, B. E.; Rittle, K. E.; Bock, M. G.; DiPardo, R. M.; Freidinger, R. M.; Whitter, W. L.; Lundell, G. F.; Veber, D. F.; Anderson, P. S. Methods for Drug Discovery: Development of Potent, Selective, Orally Effective Cholecystokinin Antagonists. J. Med. Chem. 1988, 31, 2235–2246.

(108) Munchhof, M. J.; Li, Q.; Shavnya, A.; Borzillo, G. V.; Boyden, T. L.; Jones, C. S.; LaGreca, S. D.; Martinez-Alsina, L.; Patel, N.; Pelletier, K.; Reiter, L. A.; Robbins, M. D.; Tkalcevic, G. T. Discovery of PF-04449913, a Potent and Orally Bioavailable Inhibitor of Smoothened. ACS Med. Chem. Lett. 2011, 3, 106–111.

(109) Ramachandran, S.; Hameed P., S.; Srivastava, A.; Shanbhag, G.; Morayya, S.; Rautela, N.; Awasthy, D.; Kavanagh, S.; Bharath, S.; Reddy, J.; Panduga, V.; Prabhakar, K. R.; Saralaya, R.; Nanduri, R.; Raichurkar, A.; Menasinakai, S.; Achar, V.; Jiménez-Díaz, M. B.; Martínez, M. S.; Angulo-Barturen, I.; Ferrer, S.; Sanz, L. M.; Gamo, F. J.; Duffy, S.; Avery, V. M.; Waterson, D.; Lee, M. C. S.; Coburn-Flynn, O.; Fidock, D. A.; Iyer, P. S.; Narayanan, S.; Hosagrahara, V.; Sambandamurthy, V. K. N-Aryl-2-

45

Aminobenzimidazoles: Novel, Efficacious, Antimalarial Lead Compounds. J. Med. Chem. 2014, 57, 6642–6652.

(110) Calculated Using Advanced Chemistry Development (ACD/Labs) Software V11.02 (© 1994-2015 ACD/Labs).

(111) Phillips, M. A. The Formation of 2-Substituted Benziminazoles. J. Chem. Soc. 1928, 2393–2399.

(112) Andrzejewska, M.; Yépez-Mulia, L.; Cedillo-Rivera, R.; Tapia, A.; Vilpo, L.; Vilpo, J.; Kazimierczuk, Z. Synthesis, Antiprotozoal and Anticancer Activity of Substituted 2-Trifluoromethyl- and 2-Pentafluoroethylbenzimidazoles. Eur. J. Med. Chem. 2002, 37, 973–978.

(113) Hein, D. W.; Alheim, R. J.; Leavitt, J. J. The Use of Polyphosphoric Acid in the Synthesis of 2-Aryl- and 2-Alkyl-Substituted Benzimidazoles, Benzoxazoles and Benzothiazoles1. J. Am. Chem. Soc. 1957, 79, 427–429.

(114) Wang, R.; Lu, X.; Yu, X.; Shi, L.; Sun, Y. Acid-Catalyzed Solvent-Free Synthesis of 2-Arylbenzimidazoles under Microwave Irradiation. J. Mol. Catal. Chem. 2007, 266, 198–201.

(115) Lu, J.; Yang, B.; Bai, Y. Microwave Irradiation Synthesis of 2-Substituted Benzimidazoles Using PPA as a Catalyst Under Solvent-Free Conditions. Synth. Commun. 2002, 32, 3703–3709.

(116) VanVliet, D. S.; Gillespie, P.; Scicinski, J. J. Rapid One-Pot Preparation of 2-Substituted Benzimidazoles from 2-Nitroanilines Using Microwave Conditions. Tetrahedron Lett. 2005, 46, 6741–6743.

(117) Göker, H.; Kuş, C.; Boykin, D. W.; Yildiz, S.; Altanlar, N. Synthesis of Some New 2-Substituted-Phenyl-1H-Benzimidazole-5-Carbonitriles and Their Potent Activity against Candida Species. Bioorg. Med. Chem. 2002, 10, 2589–2596.

(118) Brain, C. T.; Steer, J. T. An Improved Procedure for the Synthesis of Benzimidazoles, Using Palladium-Catalyzed Aryl-Amination Chemistry. J. Org. Chem. 2003, 68, 6814–6816.

(119) Büttner, A.; Seifert, K.; Cottin, T.; Sarli, V.; Tzagkaroulaki, L.; Scholz, S.; Giannis, A. Synthesis and Biological Evaluation of SANT-2 and Analogues as Inhibitors of the Hedgehog Signaling Pathway. Bioorg. Med. Chem. 2009, 17, 4943–4954.

(120) Charifson, P. S.; Grillot, A.-L.; Grossman, T. H.; Parsons, J. D.; Badia, M.; Bellon, S.; Deininger, D. D.; Drumm, J. E.; Gross, C. H.; LeTiran, A.; Liao, Y.; Mani, N.; Nicolau, D. P.; Perola, E.; Ronkin, S.; Shannon, D.; Swenson, L. L.; Tang, Q.; Tessier, P. R.; Tian, S.-K.; Trudeau, M.; Wang, T.; Wei, Y.; Zhang, H.; Stamos, D. Novel Dual-Targeting Benzimidazole Urea Inhibitors of DNA Gyrase and Topoisomerase IV Possessing Potent Antibacterial Activity: Intelligent Design and Evolution through the Judicious Use of Structure-Guided Design and Stucture−Activity Relationships. J. Med. Chem. 2008, 51, 5243–5263.

(121) DeLuca, M. R.; Kerwin, S. M. The Para-Toluenesulfonic Acid-Promoted Synthesis of 2-Substituted Benzoxazoles and Benzimidazoles from Diacylated Precursors. Tetrahedron 1997, 53, 457–464.

(122) Huang, S.-T.; Hsei, I.-J.; Chen, C. Synthesis and Anticancer Evaluation of Bis(benzimidazoles), Bis(benzoxazoles), and Benzothiazoles. Bioorg. Med. Chem. 2006, 14, 6106–6119.

(123) Heravi, M. M.; Tajbakhsh, M.; Ahmadi, A. N.; Mohajerani, B. Zeolites. Efficient and Eco-Friendly Catalysts for the Synthesis of Benzimidazoles. Monatsh. Chem. 2005, 137, 175–179.

(124) Ridley, H. F.; Spickett, R. G. W.; Timmis, G. M. A New Synthesis of Benzimidazoles and Aza-Analogs. J. Heterocycl. Chem. 1965, 2, 453–456.

(125) Yang, D.; Fokas, D.; Li, J.; Yu, L.; Baldino, C. M. A Versatile Method for the Synthesis of Benzimidazoles from o-Nitroanilines and Aldehydes in One Step via a Reductive Cyclization. Synthesis 2005, 47–56.

46

(126) Singh, M. P.; Sasmal, S.; Lu, W.; Chatterjee, M. N. Synthetic Utility of Catalytic Fe(III)/Fe(II) Redox Cycling Towards Fused Heterocycles: A Facile Access to Substituted Benzimidazole, Bisbenzimidazole and Imidazopyridine Derivatives. Synthesis 2000, 1380–1390.

(127) Das, B.; Holla, H.; Srinivas, Y. Efficient (bromodimethyl)sulfonium Bromide Mediated Synthesis of Benzimidazoles. Tetrahedron Lett. 2007, 48, 61–64.

(128) Bahrami, K.; Khodaei, M. M.; Naali, F. Mild and Highly Efficient Method for the Synthesis of 2-Arylbenzimidazoles and 2-Arylbenzothiazoles. J. Org. Chem. 2008, 73, 6835–6837.

(129) Mukhopadhyay, C.; Tapaswi, P. K. Dowex 50W: A Highly Efficient and Recyclable Green Catalyst for the Construction of the 2-Substituted Benzimidazole Moiety in Aqueous Medium. Catal. Commun. 2008, 9, 2392–2394.

(130) Beaulieu, P.; Haché, B.; von Moos, E. A Practical Oxone®-Mediated,High-

Throughput, Solution-Phase Synthesis of Benzimidazolesfrom 1,2-Phenylenediamines and Aldehydes and Its Application to Preparative Scale Synthesis. Synthesis 2003, 1683–1692.

(131) Yadagiri, B.; Lown, J. W. Convenient Routes to Substituted Benzimidazoles and Imidazolo[4,5-b]pyridines Using Nitrobenzene as Oxidant. Synth. Commun. 1990, 20, 955–963.

(132) Tawar, U.; Jain, A. K.; Dwarakanath, B. S.; Chandra, R.; Singh, Y.; Chaudhury, N. K.; Khaitan, D.; Tandon, V. Influence of Phenyl Ring Disubstitution on Bisbenzimidazole and Terbenzimidazole Cytotoxicity: Synthesis and Biological Evaluation as Radioprotectors. J. Med. Chem. 2003, 46, 3785–3792.

(133) Stephens, F. F.; Bower, J. D. 626. The Preparation of Benziminazoles and Benzoxazoles from Schiff’s Bases. Part I. J. Chem. Soc. 1949, 2971–2972.

(134) Bahrami, K.; Khodaei, M. M.; Kavianinia, I. H2O2/HCl as a New and Efficient System for Synthesis of 2-Substituted Benzimidazoles. J. Chem. Res. 2006, 783–784.

(135) Pätzold, F.; Zeuner, F.; Heyer, T.; Nielas, H.-J. Dehydrogenations Using Benzofuroxan as Oxidant. Synth. Commun. 1992, 22, 281–288.

(136) Srivastava, R. G.; Venkataramani, P. S. Barium Manganate Oxidation in Organic Synthesis: Part III: Oxidation of Schiff’s Bases to Benzimidazoles Benzoxazoles and Benzthiazoles. Synth. Commun. 1988, 18, 1537–1544.

(137) Vanden Eynde, J. J.; Delfosse, F.; Lor, P.; Van Haverbeke, Y. 2,3-Dichloro-5,6-Dicyano-1,4-Benzoquinone, a Mild Catalyst for the Formation of Carbon-Nitrogen Bonds. Tetrahedron 1995, 51, 5813–5818.

(138) Kawashita, Y.; Nakamichi, N.; Kawabata, H.; Hayashi, M. Direct and Practical Synthesis of 2-Arylbenzoxazoles Promoted by Activated Carbon. Org. Lett. 2003, 5, 3713–3715.

(139) Jain, R.; Agarwal, D. D.; Sahu, P. K.; Selvam, D. T.; Sharma, Y.; Gupta, R.; Prakash, A. Mild and Highly Efficient Copper (II) Sulfate Catalyzed One Pot Synthesis of 2-Aryl Benzimidazole Using Atmospheric Air as an Oxidant and Its Antibacterial Study. Med. Chem. Res. 2013, 22, 1788–1794.

(140) Ryabukhin, S. V.; Plaskon, A. S.; Volochnyuk, D. M.; Tolmachey, A. A. Synthesis of Fused Imidazoles and Benzothiazoles from (Hetero)aromatic Ortho-Diamines or Ortho-Aminothiophenol and Aldehydes Promoted by Chlorotrimethylsilane. Synthesis 2006, 3715–3726.

(141) Lin, S.; Yang, L. A Simple and Efficient Procedure for the Synthesis of Benzimidazoles Using Air as the Oxidant. Tetrahedron Lett. 2005, 46, 4315–4319.

(142) Höljes, E. L.; Wagner, E. C. Some Reactions of Nitriles as Acid Anammonides. J. Org. Chem. 1944, 31–49.

47

(143) Sluiter, J.; Christoffers, J. Synthesis of 1-Methylbenzimidazoles from Carbonitriles. Synlett 2009, 63–66.

(144) Bastug, G.; Eviolitte, C.; Markó, I. E. Functionalized Orthoesters as Powerful Building Blocks for the Efficient Preparation of Heteroaromatic Bicycles. Org. Lett. 2012, 14, 3502–3505.

(145) Jing, X.; Zhu, Q.; Xu, F.; Ren, X.; Li, D.; Yan, C. Rapid One‐Pot Preparation of 2‐Substituted Benzimidazoles from Esters Using Microwave Conditions. Synth. Commun. 2006, 36, 2597–2601.

(146) Botta, A. Process for Preparing a 2(ω-Aminoalkyl)-1,3-Heterocyclic Compounds , US1976/3972890.

(147) Hanan, E.; Chan, B.; Estrada, A.; Shore, D.; Lyssikatos, J. Mild and General One-Pot Reduction and Cyclization of Aromatic and Heteroaromatic 2-Nitroamines to Bicyclic 2H-Imidazoles. Synlett 2010, 2759–2764.

(148) Kikuchi, K.; Hannak, R. B.; Guo, M.-J.; Kirby, A. J.; Hilvert, D. Toward Bifunctional Antibody Catalysis. Bioorg. Med. Chem. 2006, 14, 6189–6196.

(149) Kelley, J. L.; Mclean, E. W.; Davis, R. G.; Crouch, R. Synthesis of 6-[[(hydroxyimino)phenyl]methyl]-1-[(1-Methylethyl)sulfonyl]-1H-imidazo[4,5-b]pyridin-2-Amine. An Aza Analogue of Enviroxime. J. Heterocycl. Chem. 1990, 27, 1821–1824.

(150) Perry, R. J.; Wilson, B. D. A Novel Palladium-Catalyzed Synthesis of 2-Arylbenzimidazoles. J. Org. Chem. 1993, 58, 7016–7021.

(151) Palumbo Piccionello, A.; Guarcello, A. Bioactive Compounds Containing Benzoxadiazole, Benzothiadiazole, Benzotriazole. Curr. Bioact. Compd. 2010, 6, 266–283.

(152) Cerecetto, H.; Porcal, W. Pharmacological Properties of Furoxans and Benzofuroxans: Recent Developments. Mini Rev. Med. Chem. 2005, 5, 57–71.

(153) Porcal, W.; Hernández, P.; Aguirre, G.; Boiani, L.; Boiani, M.; Merlino, A.; Ferreira, A.; Maio, R. D.; Castro, A.; González, M.; Cerecetto, H. Second Generation of 5-Ethenylbenzofuroxan Derivatives as Inhibitors of Trypanosoma Cruzi Growth: Synthesis, Biological Evaluation, and Structure–activity Relationships. Bioorg. Med. Chem. 2007, 15, 2768–2781.

(154) Cerecetto, H.; Di Maio, R.; González, M.; Risso, M.; Saenz, P.; Seoane, G.; Denicola, A.; Peluffo, G.; Quijano, C.; Olea-Azar, C. 1,2,5-Oxadiazole N-Oxide Derivatives and Related Compounds as Potential Antitrypanosomal Drugs:  Structure−Activity Relationships. J. Med. Chem. 1999, 42, 1941–1950.

(155) Cho, Y.; Ioerger, T. R.; Sacchettini, J. C. Discovery of Novel Nitrobenzothiazole Inhibitors for Mycobacterium tuberculosis ATP Phosphoribosyl Transferase (HisG) through Virtual Screening. J. Med. Chem. 2008, 51, 5984–5992.

(156) Jorge, S. D.; Masunari, A.; Rangel-Yagui, C. O.; Pasqualoto, K. F. M.; Tavares, L. C. Design, Synthesis, Antimicrobial Activity and Molecular Modeling Studies of Novel Benzofuroxan Derivatives against Staphylococcus Aureus. Bioorg. Med. Chem. 2009, 17, 3028–3036.

(157) Galkina, I. V.; Takhautdinova, G. L.; Tudrii, E. V.; Yusupova, L. M.; Falyakhov, I. F.; Pozdeev, O. K.; Shulaeva, M. P.; Kipenskaya, L. V.; Sakhibullina, V. G.; Krivolapov, D. B.; Litvinov, I. A.; Galkin, V. I.; Cherkasov, R. A. Synthesis, Structure, and Antibacterial Activity of Aminobenzofuroxan and Aminobenzofurazan. Russ. J. Org. Chem. 2013, 49, 591–597.

(158) Bendale, P.; Olepu, S.; Suryadevara, P. K.; Bulbule, V.; Rivas, K.; Nallan, L.; Smart, B.; Yokoyama, K.; Ankala, S.; Pendyala, P. R.; Floyd, D.; Lombardo, L. J.; Williams, D. K.; Buckner, F. S.; Chakrabarti, D.; Verlinde, C. L. M. J.; Van Voorhis, W. C.; Gelb, M. H. Second Generation

48

Tetrahydroquinoline-Based Protein Farnesyltransferase Inhibitors as Antimalarials. J. Med. Chem. 2007, 50, 4585–4605.

(159) Aguirre, G.; Boiani, L.; Cerecetto, H.; Di Maio, R.; González, M.; Porcal, W.; Denicola, A.; Möller, M.; Thomson, L.; Tórtora, V. Benzo[1,2-c]1,2,5-Oxadiazole N-Oxide Derivatives as Potential Antitrypanosomal Drugs. Part 3: Substituents-Clustering Methodology in the Search for New Active Compounds. Bioorg. Med. Chem. 2005, 13, 6324–6335.

(160) Ghosh, P. B.; Whitehouse, M. W. Potential Antileukemic and Immunosuppressive Drugs. Preparation and in Vitro Pharmacological Activity of Some 2,1,3-Benzoxadiazoles (benzofurazans) and Their N-Oxides (benzofuroxans). J. Med. Chem. 1968, 11, 305–311.

(161) Sartini, S.; Cosconati, S.; Marinelli, L.; Barresi, E.; Di Maro, S.; Simorini, F.; Taliani, S.; Salerno, S.; Marini, A. M.; Da Settimo, F.; Novellino, E.; La Motta, C. Benzofuroxane Derivatives as Multi-Effective Agents for the Treatment of Cardiovascular Diabetic Complications. Synthesis, Functional Evaluation, and Molecular Modeling Studies. J. Med. Chem. 2012, 55, 10523–10531.

(162) Haroun, M.; Helissey, P.; Giorgi-Renault, S. New Synthesis of 5,10-Dioxyphenazine-2-Carboxylic Acid. Synth. Commun. 2001, 31, 2329–2334.

(163) Mallory, F. B. Benzofurazan Oxide. 1957, 37, 1. (164) Wang, L.; Zhang, Y.-Y.; Wang, L.; Liu, F.; Cao, L.-L.; Yang, J.; Qiao, C.;

Ye, Y. Benzofurazan Derivatives as Antifungal Agents against Phytopathogenic Fungi. Eur. J. Med. Chem. 2014, 80, 535–542.

(165) Monge, A.; Palop, J. A.; de Cerain, A. L.; Senador, V.; Martinez, F. J.; Sainz, Y.; Narro, S.; Garcia, E.; de Miguel, C. Hypoxia-Selective Agents Derived from Quinoxaline 1,4-Di-N-oxides. J. Med. Chem. 1995, 38, 1786–1792.

(166) Alvesalo, J. K. O.; Siiskonen, A.; Vainio, M. J.; Tammela, P. S. M.; Vuorela, P. M. Similarity Based Virtual Screening:  A Tool for Targeted Library Design. J. Med. Chem. 2006, 49, 2353–2356.

(167) Seppälä, H.; Skurnik, M.; Soini, H.; Roberts, M. C.; Huovinen, P. A Novel Erythromycin Resistance Methylase Gene (ermTR) in Streptococcus pyogenes. Antimicrob. Agents Chemother. 1998, 42, 257–262.

(168) Tipparaju, S. K.; Joyasawal, S.; Pieroni, M.; Kaiser, M.; Brun, R.; Kozikowski, A. P. In Pursuit of Natural Product Leads: Synthesis and Biological Evaluation of 2-[3-Hydroxy-2-[(3-Hydroxypyridine-2-Carbonyl)amino]phenyl]benzoxazole-4-Carboxylic Acid (A-33853) and Its Analogues: Discovery of N-(2-Benzoxazol-2-ylphenyl)benzamides as Novel Antileishmanial Chemotypes. J. Med. Chem. 2008, 51, 7344–7347.

(169) Calderón, F.; Barros, D.; Bueno, J. M.; Coterón, J. M.; Fernández, E.; Gamo, F. J.; Lavandera, J. L.; León, M. L.; Macdonald, S. J. F.; Mallo, A.; Manzano, P.; Porras, E.; Fiandor, J. M.; Castro, J. An Invitation to Open Innovation in Malaria Drug Discovery: 47 Quality Starting Points from the TCAMS. ACS Med. Chem. Lett. 2011, 2 , 741–746.

(170) Unpublished Results, P. falciparum, Tres Cantos Medicines Development Campus, GlaxoSmithKline, Tres Cantos, Madrid, Spain.

(171) Mueller, R.; Street, L. J. Bicyclic Amide Derivatives for Enhancing Glutamatermic Synaptic Responses. US 2012/0122861.

(172) Wallén, E. A. A.; Christiaans, J. A. M.; Forsberg, M. M.; Venäläinen, J. I.; Männistö, P. T.; Gynther, J. Dicarboxylic Acid Bis(l-Prolyl-Pyrrolidine) Amides as Prolyl Oligopeptidase Inhibitors. J. Med. Chem. 2002, 45, 4581–4584.

(173) Käsnänen, H.; Myllymäki, M. J.; Minkkilä, A.; Kataja, A. O.; Saario, S. M.; Nevalainen, T.; Koskinen, A. M. P.; Poso, A. 3-Heterocycle-Phenyl N-Alkylcarbamates as FAAH Inhibitors: Design, Synthesis and 3D-QSAR Studies. ChemMedChem 2010, 5, 213–231.

49

(174) Tammela, P.; Alvesalo, J.; Riihimäki, L.; Airenne, S.; Leinonen, M.; Hurskainen, P.; Enkvist, K.; Vuorela, P. Development and Validation of a Time-Resolved Fluorometric Immunoassay for Screening of Antichlamydial Activity Using a Genus-Specific Europium-Conjugated Antibody. Anal. Biochem. 2004, 333, 39–48.

(175) Kuo, C.; Grayston, J. T. A Sensitive Cell Line, HL Cells, for Isolation and Propagation of Chlamydia pneumoniae Strain TWAR. J. Infect. Dis. 1990, 162, 755–758.

(176) Maass, M.; Bartels, C.; Engel, P. M.; Mamat, U.; Sievers, H.-H. Endovascular Presence of Viable Chlamydia pneumoniae Is a Common Phenomenon in Coronary Artery Disease 1. J. Am. Coll. Cardiol. 1998, 31, 827–832.

(177) 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.

(178) Unpublished Results, Hanski, L. University of Helsinki, Division of Pharmaceutical Biosciences. C. pneumoniae.

(179) Unpublished Results, Schneider, P. Global Health Institute, Lausanne, Switzerland. M. tuberculosis H37Rv, M. smegmatis mc2155, C. glutamicum ATCC13032.

(180) Baell, J. B.; Holloway, G. A. New Substructure Filters for Removal of Pan Assay Interference Compounds (PAINS) from Screening Libraries and for Their Exclusion in Bioassays. J. Med. Chem. 2010, 53, 2719–2740.

(181) Desjardins, R. E.; Canfield, C. J.; Haynes, J. D.; Chulay, J. D. Quantitative Assessment of Antimalarial Activity in Vitro by a Semiautomated Microdilution Technique. Antimicrob. Agents Chemother. 1979, 16, 710–718.

(182) Crouch, S. P. M.; Kozlowski, R.; Slater, K. J.; Fletcher, J. The Use of ATP Bioluminescence as a Measure of Cell Proliferation and Cytotoxicity. J. Immunol. Methods 1993, 160, 81–88.

(183) Bruin, M. L. D.; Pettersson, M.; Meyboom, R. H. B.; Hoes, A. W.; Leufkens, H. G. M. Anti-hERG Activity and the Risk of Drug-Induced Arrhythmias and Sudden Death. Eur. Heart J. 2005, 26, 590–597.

(184) R, W.; D, L.; D, W. Towards a Drug Concentration Effect Relationship for QT Prolongation and Torsades de Pointes. Curr. Opin. Drug Discov. Devel. 2002, 5, 116–126.

(185) Redfern, W. S.; Carlsson, L.; Davis, A. S.; Lynch, W. G.; MacKenzie, I.; Palethorpe, S.; Siegl, P. K. S.; Strang, I.; Sullivan, A. T.; Wallis, R.; Camm, A. J.; Hammond, T. G. Relationships between Preclinical Cardiac Electrophysiology, Clinical QT Interval Prolongation and Torsade de Pointes for a Broad Range of Drugs: Evidence for a Provisional Safety Margin in Drug Development. Cardiovasc. Res. 2003, 58, 32–45.

(186) Sanz, L. M.; Crespo, B.; De-Cózar, C.; Ding, X. C.; Llergo, J. L.; Burrows, J. N.; García-Bustos, J. F.; Gamo, F.-J. P. Falciparum in Vitro Killing Rates Allow to Discriminate between Different Antimalarial Mode-of-Action. PLoS ONE 2012, 7, e30949.

(187) Wood, C.; Williams, C.; Waldron, G. J. Patch Clamping by Numbers. Drug Discov. Today 2004, 9, 434–441.