Design and synthesis of 2-arylbenzimidazoles and evaluation of their inhibitory effect against Chlamydia pneumoniae

Article abstract of DOI:10.1021/jm1008083

Chlamydia pneumoniae is an intracellular bacterium that responds poorly to antibiotic treatment. Insufficient antibiotic usage leads to chronic infection, which is linked to disease processes of asthma, atherosclerosis, and Alzheimer's disease. The Chlamydia research lacks genetic tools exploited by other antimicrobial research, and thus other approaches to drug discovery must be applied. A set of 2-arylbenzimidazoles was designed based on our earlier findings, and 33 derivatives were synthesized. Derivatives were assayed against C. pneumoniae strain CWL-029 in an acute infection model using TR-FIA method at a concentration of 10 μM, and the effects of the derivatives on the host cell viability were evaluated at the same concentration. Fourteen compounds showed at least 80% inhibition, with only minor changes in host cell viability. Nine most potential compounds were evaluated using immunofluorescence microscopy on two different strains of C. pneumoniae CWL-029 and CV-6. The N-[3-(1H-benzimidazol-2-yl)phenyl]-3-methylbenzamide (42) had minimal inhibitory concentration (MIC) of 10 μM against CWL-029 and 6.3 μM against the clinical strain CV-6. This study shows the high antichlamydial potential of 2-arylbenzimidazoles, which also seem to have good characteristics for lead compounds.

Full text of DOI:10.1021/jm1008083

7664 J. Med. Chem. 2010, 53, 7664–7674
DOI: 10.1021/jm1008083
Design and Synthesis of 2-Arylbenzimidazoles and Evaluation of Their Inhibitory
Effect against Chlamydia pneumoniae
Leena Keurulainen,†,^ Olli Salin,‡,^ Antti Siiskonen,†,^ Jan Marco Kern,§ Joni Alvesalo, Paula Kiuru,† Matthias Maass,§
Jari Yli-Kauhaluoma,*,† and Pia Vuorela*,‡
†Division of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Helsinki, P.O. Box 56 (Viikinkaari 5 E), FI-00014 University of
‡
˚
Helsinki, Finland, Pharmaceutical Sciences, Department of Biosciences, Abo Akademi University, Biocity, Artillerigatan 6 A, FI-20520 Turku,
Finland, §Institute of Medical Microbiology, Hygiene and Infectious Diseases, PMU University Hospital Salzburg, Muellner Hauptstrasse 48,
A-5020 Salzburg, Austria, and Division of Pharmaceutical Biology, Faculty of Pharmacy, University of Helsinki, P.O. Box 56 (Viikinkaari 5 E),
FI-00014 University of Helsinki, Finland. ^ These authors contributed equally to this work.
Received June 30, 2010
Chlamydia pneumoniae is an intracellular bacterium that responds poorly to antibiotic treatment.
Insufficient antibiotic usage leads to chronic infection, which is linked to disease processes of asthma,
atherosclerosis, and Alzheimer’s disease. The Chlamydia research lacks genetic tools exploited by other
antimicrobial research, and thus other approaches to drug discovery must be applied. A set of
2-arylbenzimidazoles was designed based on our earlier findings, and 33 derivatives were synthesized.
Derivatives were assayed against C. pneumoniae strain CWL-029 in an acute infection model using
TR-FIA method at a concentration of 10 μM, and the effects of the derivatives on the host cell viability
were evaluated at the same concentration. Fourteen compounds showed at least 80% inhibition, with
only minor changes in host cell viability. Nine most potential compounds were evaluated using
immunofluorescence microscopy on two different strains of C. pneumoniae CWL-029 and CV-6. The
N-[3-(1H-benzimidazol-2-yl)phenyl]-3-methylbenzamide (42) had minimal inhibitory concentration
(MIC) of 10 μM against CWL-029 and 6.3 μM against the clinical strain CV-6. This study shows the high
antichlamydial potential of 2-arylbenzimidazoles, which also seem to have good characteristics for lead
compounds.
Introduction
this bacterium can infect multiple tissue and cell types and can
be cultivated from most of them.1,8,9 Chronic C. pneumoniae
infection has been linked to asthma,10-12 lung cancer,13
atherosclerosis,14,15 and late onset Alzheimer’s disease,16-18
all of which have inflammatory aspects in disease processes.
The ever growing evidence of the connection between the
C. pneumoniae infection and the development of atherosclero-
sisisreviewedindetailbyWatsonandAlp19 andStassenetal.20
In animal models of atherosclerosis, C. pneumoniae promotes
formation of atherosclerotic plaques21 and increases maximal
intimal thickness of thoracic aortas.22 Similar thickening of
arteria and smooth muscle cell proliferation was seen after
C. pneumoniae infection in an ex vivo setting.23 The main
mechanism for C. pneumoniae to trigger the formation of
atherosclerosis is to cause constant inflammation and promote
foam cell formation from macrophages,24 which is suggested
to be triggered through toll-like receptor 2 (TLR-2).25
Antibiotic treatments have given mixed results in treatment
of atherosclerosis in general and in C. pneumoniae-induced
human atherosclerosis in particular. In most animal models of
C. pneumoniae-induced atherosclerosis, repeated infection
is needed to induce atherosclerosis and early antibiotic treat-
ment prevents this effect.19 In some animal models of
C. pneumoniae infection, the inhibitory effect of antibiotics
depended on the timing and the dose of the treatment.26,27
This might also be the case in human studies, as prolonged
antibiotic treatments have not significantly reduced cardiac
events or relieved symptoms.28,29 Current antibiotics may fail
Acute Chlamydia pneumoniae infection causes usually mild
upper respiratory track symptoms and prolonged coughing,
but it is also estimated to cause 10% of community-acquired
pneumonia and 5% of bronchitis and sinusitis in the adult
population. This intracellular bacterium contributes widely to
human diseases, but no reliable treatments are available as the
effect of primary treatment antibiotics is controversial.1,2
Almost every one of us is estimated to get infected at least
once in a lifetime, as the seroprevalence of the bacteria is
60-70%.3 This percentage is huge considered in the light of
recent evidence suggesting that C. pneumoniae is a fairly new
zoonotically acquired human pathogen.4 Acute infection can
be treated with existing antibiotics when used in timely manner
and in sufficient intracellular concentrations. However, infec-
tion often transforms to persistent form, which is difficult to
eradicate for the human immune system or by administering
more antibiotics.5,6
C. pneumoniae causes inflammation of tissues, and with
chronic infection of C. pneumoniae, the inflammation process
is constant as chronic infection produces equal amounts of
immunopathogenic antigen chlamydial heat shock protein 60
(hsp60) compared to acute infection.7 It has been shown that
*To whom correspondence should be addressed. Biology (P.V.):
phone, þ358
2
215 4267; fax, þ358
2 230 5018; E-mail, pia.
vuorela@abo.fi. Chemistry (J.Y.-K.): phone, þ358 9 19159170; fax,
þ358 9 19159556; E-mail, jari.yli-kauhaluoma@helsinki.fi.
r
pubs.acs.org/jmc
Published on Web 10/08/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7665
preventing cardiac events because they fail to erase the
pathogen from human macrophages in vivo, although they
show good effect on the same strains cultured in vitro from
these patients.1 The human studies are also prone to fail if
C. pneumoniae has already established chronic infection, as
the chronic infection is highly refractory to antibiotic treat-
ments but still capable of sustaining infection and inflamma-
tion by expressing chlamydial heat shock protein.30
activity of certain 2-arylbenzimidazoles, and the antichlamy-
dial activities were confirmed with in vitro studies. In the
present study, a new set of compounds was assayed against
C. pneumoniae reference strain CWL-029 with two different
methods using the acute infection model, and the results were
verified using a clinical vascular isolate CV-6, which is known
to cause chronic infection in coronary arteries.48 The set of 33
benzimidazoles was also studied for any effects on host cell
viability by measuring the amount of intracellular adenosine-
50-triphosphate (ATP).
The chlamydiae research was boosted by the complete
genome sequences in 1998 and 1999.31,32 The results based
on the genomic approaches have increased our knowledge on
the genus Chlamydia, but many features of the bacterium
remain an enigma. As we still lack tools for genetic manipula-
tion of chlamydiae, some of the general approaches for
antimicrobial research cannot be included to the research
tools. In chlamydiae research, the problems need to be solved
from different points-of-view: the type three secretion system
(TTSSa) apparatus is known to be present in C. pneumoniae at
allstages, andTTSS is a part of the infectiouselementarybody
(EB) as well as the inclusion membrane that separates the
chlamydiae from host cell.33 Compounds that inhibit TTSS
secretion of Yersinia were also effective against C. pneumoniae
in vitro.34 Also a new fluoroketolide antibiotic was evaluated
against Chlamydia, but the effect did not differ from other
known antibiotics.35 Only few intracellularly active sub-
stances are available to treat chlamydial infection. Develop-
ment of resistance is thus of special concern and may warrant
new treatment strategies when spreading.36 Despite the new
knowledge gained in past 10 years, only a few potential can-
didates for further drug development against C. pneumoniae
have been reported. In this light, the treatment of
C. pneumoniae infection is of utmost importance and new
specific antichlamydial compounds are urgently needed.
In our previous study, a set of benzimidazole derivatives
was found to be effective against the acute C. pneumoniae
infection in vitro.37 Benzimidazole derivatives are known to
possess a wide variety of biological activities, in particular,
antibacterial and antiviral activities.38 Recently benzimida-
zole ureas have been shown to be potent antibacterial agents
targeting DNA gyrase and topoisomerase IV.39 More speci-
fically, 2-arylbenzimidazole derivatives have shown effects as
heparanase inhibitors,40 as inhibitors of hepatitis C virus,41 as
suppressors of key markers of allergy,42 inhibitors of sonic
hedgehog (shh) signaling pathway,43 antibacterial,44,45 and
antiprotozoal compounds.46 In addition, benzimidazole
structure as a privileged scaffold is a reasonable starting point
to drug discovery.47 Thus benzimidazole derivatives possess
good lead compound characteristics for antimicrobial agents.
In this study, we report the design, synthesis, and anti-
chlamydial activity of a set of new 2-arylbenzimidazoles. This
set was synthesized on the basis of the structure-activity
relationship (SAR) data available from our previous study.37
In this previous study, in silico screening in a Bacillus subtilis
RNA methyltransferase homology model revealed possible
Results and Discussion
Chemistry. The preparation of the substituted 2-arylben-
zimidazoles is outlined in Scheme 1. Oxidative methods49-51
were first attempted for the one-step synthesis of 2-arylben-
zimidazole, but they gave low yields probably due to the
presence of deactivating meta-nitro group in the starting
material. Therefore, the synthesis of the 2-arylbenzimidazole
core was performed in two steps. First, 1,2-diaminobenzenes
1a-e were diacylated with the corresponding acyl chlorides
in the presence of 4-(dimethylamino)pyridine (DMAP) in
pyridine to produce the bisamides 2-8. This reaction was
accomplished conveniently under microwave (MW) irradia-
tion, which shortened reaction time to 15 min from the
previously required 4-18 h at room temperature. The
para-nitro-substituted 9 and 2-[(3-nitrobenzoyl)amino]phenyl
3-nitrobenzoate 10 were synthesized with the same procedure.
In case the required 1,2-diaminobenzene was not commercially
available, it was prepared by catalytic hydrogenation of the
corresponding nitro compound in the presence of palladium
catalyst. The subsequent condensation of the bisamides 2-8
was accomplished with para-toluenesulfonic acid (p-TSA) in
refluxing p-xylene52 to yield 2-arylbenzimidazoles 11-17. The
para-nitro-substituted compound 18 and the related 1,3-ben-
zoxazole 19 were prepared with the same procedure from 9 and
10. N1-Methylated 2-arylbenzimidazole 20 was prepared from
the unsubstituted benzimidazole 11 by alkylating with iodo-
methane in the presence of K2CO3 in N,N-dimethylformamide
(DMF). Nitro group of nitroaryl benzimidazole was hydro-
genated in the presence of palladium catalyst (23-24, 26-28,
32-33). Alternatively reduction was carried out with SnCl2
3
2H2O (25, 29-31). Finally, the N-acylated products 34-48,
51-52, 55-59, and 61-64 were prepared from 23-33 using
the corresponding acyl chlorides in the presence of triethyl-
amine (TEA) in tetrahydrofuran (THF). Due to the fact that
final acylation step of the target compounds produced numer-
ous byproducts and proceeded in low yield, the imidazole NH
was protected as an N-tert-butoxycarbonyl (t-Boc) carbamate
at room temperature ((t-Boc)2O, Cs2CO3, acetonitrile
(MeCN)). Acylation and the subsequent deprotection with
trifluoroacetic acid (TFA) in dichloromethane (DCM) yielded
the benzimidazoles 49-50, 53-54, and 60.
Synthesis of the target compounds 67-68 is outlined in
Scheme 2. Their common starting material 2-(nitroaryl)-
imidazole 65 was formed in the reaction between the corre-
sponding nitrile and aminoacetaldehyde diethyl acetal,53
followed by catalytic hydrogenation and acylation to give
the final products.
The A-ring substitution of the benzimidazole core gave
rise to the 1,3-tautomers of the compounds, and they could
be seen in some NMR spectra of the 2-arylbenzimidazoles in
d6-dimethyl sulfoxide (DMSO-d6). Equivalence and fast
exchange of the 4,7-protons and 5,6-protons accounted for
a Abbreviations: ATCC, American Type Culture Collection; ATP,
adenosine-50-triphosphate; t-Boc, N-tert-butoxycarbonyl; DCM, di-
chloromethane; DMF, N,N-dimethylformamide; DMAP, 4-(dimethyl-
amino)pyridine; DMSO, dimethyl sulfoxide; EB, elementary body; HL,
human line; IF, immunofluorescence; IFU, inclusion forming unit;
MCC, minimum chlamydiocidal concentration; MIC, minimum inhibi-
tory concentration; MOI, multiplicity of infection; MW, microwave;
PBS, phosphate buffered saline; SAR, structure-activity relationship;
TEA, triethylamine; TFA, trifluoroacetic acid; THF, tetrahydrofuran;
TR-FIA, time-resolved fluorometric immunoassay; p-TSA, para-tolue-
nesulfonic acid; TTSS, type three secretion system.
1
the broad signals of the aromatic protons in the H NMR

7666 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Keurulainen et al.
Scheme 1a,b
a Reagents and conditions: (a) DMAP, pyridine, 60 ꢀC, MW, 15 min (yield 91-99%); (b) p-TSA, p-xylene, 140 ꢀC, 18 h (yield 31-94%); (c) CH3I,
K2CO3, DMF, 19 h (yield 94%); (d) Cs2CO3, (t-Boc)2O, MeCN, 1 h (yield 82-94%); (e) SnCl2 2H2O, EtOH, 0.5 h, reflux (yield 41-93%) or H2, 10%
3
Pd/C, EtOH/EtOAc, 5 h (yield 71-100%); (f) RCOCl, TEA (yield 14-71%) or RCOCl, TEA and TFA, DCM (yield 6-36%).
b R4: see Tables 2-4.
Scheme 2a,b
a Reagents and conditions: (a) (i) 30% NaOMe, MeOH, 17 h, room temperature, (ii) H2NCH2CH(OEt)2, 50 ꢀC, 1 h, (iii) 6 M HCl, reflux 5 h; (b) H2,
10% Pd/C, EtOH; (c) RCOCl, TEA, THF (yield 22-36%).
b R: see Table 4.
spectra and also for broad and even missing signals of the
same aromatic carbon atoms in the 13C NMR spectra.54,55
The assignment of the 1H and 13C NMR signals of the
representative 2-arylbenzimidazole (34) is based on the
heteronuclear multiple quantum correlation (HMQC) and
heteronuclear multiple bond correlation (HMBC) spectra
and shown in Table 1.
TR-FIA Results and SAR. Thirty-three benzimidazole
derivatives (Tables 2-4) were synthesized to investigate the
SAR of the inhibitory effect of these compounds against
C. pneumoniae strain CWL-029. All compounds were as-
sayed for C. pneumoniae inhibition at a concentration of
10 μM using TR-FIA method56 and for label binding proper-
ties and autofluorescence to exclude false positive signals. In
this study, no false negative inhibition was found (data not
shown). The biological activity results obtained from the new
2-arylbenzimidazoles were compared to the corresponding
activity of 34, known to inhibit the growth of C. pneumoniae
in low micromolar concentration.37 The minimal inhibitory
concentration (MIC) for rifampicin was determined to
be 0.012 μM (0.010 μg/mL) with the TR-FIA method, and
this concentration was used as a control in the TR-FIA
experiments.
The fused 2-arylbenzimidazole structure was found to be
essential to activity because the corresponding 2-arylimida-
zole heterocycles 67 and 68 showed no activity. In addition,
the N-methylated benzimidazole 61 was inactive and the
related benzoxazole 63 showed only moderate activity. The
A-ring-substituted compounds were all active. Methyl and
halogen moieties in the benzimidazoles 55-57 were slightly
better than methoxy 54. Especially the methyl substitution at
the 5-position of compound 55 turned out to be slightly
better for antichlamydial activity than the corresponding
4-position in the compound 57.
Modification of the C-ring substitution (compounds
58-59) did not improve the antichlamydial activity, and

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7667
Table 1. Assignment of 1H and 13C NMR Signals of Compound 34
and divided into three subgroups (A-C) based on the
similarities in the chemical structures and tested at smaller
concentrations with traditional immunofluorescence (IF)
labeling (Figure 1). The 2- and 3-thiophene derivatives 34
and 35 (group A) showed high activity against the bacterium
at a 10 μM concentration and prevented C. pneumoniae
growth almost completely with the highest concentration
used. The same type of effect was seen in A-ring-substituted
compounds 55, 56, and 60 in group C, where almost com-
plete eradication was achieved at a 40 μM concentration.
D-ring-modified benzimidazole derivatives, group B, were
the only compounds eradicating C. pneumoniae strain
CWL-029 from the cell cultures at used concentrations.
From D-ring-modified derivatives, meta-substituted com-
pounds 42 and 43 inhibited C. pneumoniae with lower con-
centration than ortho-substituted 48 or cyclohexyl derivative
53. Derivatives 42 and 43 were the most effective antic-
hlamydial compounds, having IC50 values of 2 and 3 μM,
respectively, and both of them yielded over 80% inhibition at
a 5 μM concentration. With all studied benzimidazoles, a
clear trend emerged where increasing concentration of com-
pounds produced significantly smaller but still detectable
inclusions and only a few compounds reached complete
inhibition of C. pneumoniae growth. Overall, IF inhibition
of the benzimidazole derivatives were partly lower than
TR-FIA inhibition and smaller inclusion size offers explana-
tion for this phenomenon as the TR-FIA method measures
the overall amount of C. pneumoniae formed whereas IF
method measures the number of inclusions.
IF Measurement of Strain CV-6: First and Second Passage.
The compounds with most potential and the reference anti-
biotic erythromycin were tested in an acute infection model
using the cardiovascular strain of C. pneumoniae CV-6,
which is a human coronary artery-derived clinical isolate
known to cause chronic infections (Table 5).48 The results
verified the effect of the compounds against C. pneumoniae in
general, but small differences in sensitivities were observed.
We found the CV-6 strain to be more sensitive than the
CWL-029 strain to most of the compounds. Differences
between the antichlamydial efficiencies of the compounds
became more obvious as the minimal chlamydiocidal con-
centration (MCC) ranged from 3.2 to >196 μM (Table 5).
The most active compounds against CV-6 and CWL-029
were 42 and 43. Overall, the results with the CV-6 strain were
in line with the results from the CWL-029 experiments.
Compounds 42, 48, 53, 55, and 60 were able to inhibit the
growth of the CV-6 strain completely at the reported MIC
within the first passage, and growth was also prevented at the
same concentrations in the subcultures in antibiotic-free
medium indicating a chlamydiocidal effect. However, com-
pound 43, which was highly effective at the first passage with
a reported MIC value of 3.2 μM, displayed an MCC of
50 μM. Thus, the effect of 42 is chlamydiocidal and the effect
of 43 is chlamydiostatic at low concentrations, although both
are able to inhibit C. pneumoniae growth. Due to the
chlamydiocidal effect of 42, it is considered to be a better
lead compound than 43 and will be used as the lead in future
studies to enhance potency without increasing toxicity and
to improve physicochemical properties.
δ1Hppm
12.94
δ13Cppm
δ1Hppm
δ13Cppm
1
10
130.6
118.7
139.3
121.5
129.3
121.5
2
151.1
20
8.64
3
30
40
3a
4
135.1
118.9
122.6
121.7
111.4
143.8
7.90-7.87
7.56-7.51
7.90-7.87
10.44
7.67
500
5
7.27-7.19
7.27-7.19
7.56-7.51
600
100
200
3
6
7
7a
160.0
139.9
129.3
128.2
132.1
400
500
600
8.12
7.27-7.19
7.90-7.78
the further modifications were directed to the D-ring. An
amide bond linking the C- and D-rings seemed to be bene-
ficial in the meta position, and the D-ring was essential for
the antichlamydial activity because the amide bond in the
para position in compound 64 and a methyl group in place of
the D-ring (compound 51) led to completely inactive com-
pounds. On the basis of our original finding37 that thiophene
ring was present in the most active compounds, other five-
membered heterocyclic derivatives were synthesized. Among
the furan and thiophene series of compounds, 2-positioned
thiophene 34 was more potent than the corresponding
3-thiophene 35, and 3-furan 38 was more potent than 2-furan
37. The compound (39) with the N-methylated pyrrole as the
D-ring moiety showed significant inhibition activity. This
finding was in line with activities of the meta and ortho-
substituted 42 and 48, respectively.
According to our previous screening study, para-substitu-
tion of the D-ring caused lack of activity.37 In the present
study, substituting the meta-position of the D-ring with
various small substituents such as methyl (42), trifluoro-
methyl (43), chloro (44), and methoxy (46) proved to yield
potent antichlamydial compounds, although activity differ-
ences to unsubstituted compound 41 were small. In the case
of the meta-fluoro substituent in the D-ring (45), the ob-
served antichlamydial effect was moderate. Compound 47
with the methoxycarbonyl functionality showed significant
activity but was not selected to further testing because of its
possible metabolic degradation via hydrolysis. The three best
meta substituents, methyl, trifluoromethyl, and methoxy,
were also tested in the ortho position in the 2-arylbenzimi-
dazoles 48-50. The substitution in compound 50 resulted in
a significant decrease of antichlamydial activity. Taken
together, meta-substitution in the D-ring seemed to result
in good antichlamydial activity. The cycloalkane derivative,
the cyclohexanoyl amide 53, was highly active at a concen-
tration of 10 μM, being among the most potent compounds
determined by the screening method, but further testing
showed superiority of the other derivatives (vide infra). In
total, 14 of the synthesized compounds showed at least 80%
inhibition activity at a concentration of 10 μM.
Host Cell Viability. All of the plates during the study were
controlled under microscope for abnormalities. In micro-
scopic analysis, visible changes in cell monolayer and cell
death was seen only at the concentration of 40 μM and
only with compounds 43, 56, and 60, otherwise the cell
IF Measurement of Strain CWL-029: SAR. Nine of the
most potential compounds were selected for further studies

7668 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Keurulainen et al.
Table 2. Chemical Structures of the Benzimidazoles 34-53, Their Inhibition of C. pneumoniae Growth at a Concentration of 10 μM, and Their
Effect on the Viability of the Host Cells at 10 μM
monolayers were comparable to the controls. The effect of all
2-arylbenzimidazoles on the host cell viability was studied
after 72 h of incubation with a 10 μM concentration. The
host cell viability percentage was assessed by the amount of
intracellular ATP present in 2-arylbenzimidazole-treated
cells compared to cells treated with DMSO-adjusted me-
dium. The ATP concentration is considered to be a delicate
indicator of cell viability.57 Thirty-one compounds were
determined to be nontoxic for HL cells, as the viability of
the HL cells remained above the threshold of 80% set prior
to the experiment (Tables 2-4). The selected nine com-
pounds were evaluated for effects on host cell viability at
four concentrations ranging from 0.625 to 40 μM (Figure 1).
None of the nine compounds had an effect on host cell
viability at a 2.5 μM concentration, and at a 40 μM con-
centration, four compounds did not cause over 20% decrease
of the viability. Thus the compounds proved to be well
tolerated by host cells, and from the results it was concluded
that the effect on host cell viability cannot explain the anti-
chlamydial effect of the compounds.
Other Antimicrobial Activities. None of the benzimida-
zoles showed any inhibition of the growth of the Gram-
negative bacteria Escherichia coli at a concentration of
50 μM. In the case of Gram-positive Staphylococcus aureus,
compound 43 showed 85% inhibition of resazurin metabo-
lism, whereas the other compounds had no effect.
As the target of 2-arylbenzimidazoles in Chlamydia is still
an enigma, there are multiple possible explanations why
2-arylbenzimidazoles were not effective against free living
bacteria. At least four reasons are possible: (1) the 2-aryl-
benzimidazoles modify host cell processes, which are non-
essential to host cells but essential to C. pneumoniae survival
in host cells and thus have little effect on free living bacteria,
(2) the compounds are modified by host cells to target bacterial
functions, (3) the free living bacteria may have mechanisms
to compensate the effects of 2-arylbenzimidazoles, (4) the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7669
compounds affect directly the essential components or path-
ways of C. pneumoniae such as the TTSS.
covery are needed.58-60 The chlamydia research does not have
tools to modify the bacterium genetically, and thus other
methods of drug development than design of targeted inhibi-
tors must be applied. On the basis of our previous in silico and
in vitro studies, we synthesized a set of 2-arylbenzimidazoles
and evaluated the SAR of the compounds against the
C. pneumoniae strain CWL-029, the effects on host cell
viability, and verified antichlamydial activity with a clinical
C. pneumoniae isolate CV-6. We have also carried on further
SAR studies based on the conformation of 2-arylbenzimi-
dazoles.61
The benzimidazole derivatives seem to be highly selective
in inhibiting intracellular C. pneumoniae compared to free
living bacteria. The compound 42, N-[3-(1H-benzimidazol-
2-yl)phenyl]-3-methylbenzamide, completely abolished the
C. pneumoniae infection of the strain CWL-029 at a 10 μM
concentration measured by either TR-FIA or IF methods, and
the clinical isolate strain CV-6 at a concentration of 6.3 μMwas
clearly more efficient in inhibiting C. pneumoniae than com-
pound 34 published in our previous study on 2-aryl-
benzimidazoles.37 The same 6.3 μM concentration of 42 also
prevented the CV-6 strain from infecting new host cells at the
second passage of C. pneumoniae, proving that the effect was
chlamydiocidal. Overall, the meta-substituted 42 was the most
prominent candidate for further lead optimization and drug
development, as it was highly effective against C. pneumoniae
and did not decrease host cell viability.
The antimicrobial selectivity was assayed as it is beneficial
to find a highly selective antichlamydial lead compound as it
can help avoiding some of the main issues of antimicrobial
drugs. First, if the compound can be administered via oral
route, it should not affect the normal gut flora and thus the
most common adverse effects of antibiotics can be avoided.
Second, if the effect is selective to C. pneumoniae or to
intracellular bacteria in general, it is more unlikely that
resistance would occur. The likelihood of finding a fast and
complete clinical antichlamydial therapy is small, and thus
compounds that can be used for prolonged times without
effect to the normal microflora are wanted. With possible
prolonged treatments, the importance of other possible
adverse effects must be carefully addressed in further drug
development. However, the time period of any antimicrobial
treatment is normally fairly short (up to six months) com-
pared to treatments of many other diseases that can be
addressed to patients for a lifetime.
Conclusions
Antimicrobial drug development is at its turning point as
high throughput screens have not produced reliable results,
and this is why new approaches to antimicrobial drug dis-
Table 3. Chemical Structures of the Benzimidazoles 54-59, Their
Inhibition of C. pneumoniae Growth at a Concentration of 10 μM,
and Their Effect on the Viability of the Host Cells at 10 μM
In this study, we showed how structure-based approach can
be applied even without an unambiguous target. In the case of
Chlamydia, it is often extremely difficult to determine what the
target is and a highly efficient and predictably behaving
compound is needed to proceed to more complex biological
evaluations, which couldrevealthe target. Further proteomics
studies have already been started to address the potential
mechanism(s) of action.
compd
R1
R2
R3
inhibition % ( SEM viability %
Experimental Section
54
55
56
57
58
59
5-OMe
5-Me
5-Cl
74 ( 6.3
99 ( 0.9
89 ( 3.9
89 ( 3.2
59 ( 4.9
52 ( 7.9
98
95
Materials and Methods. Cultivation of Cells and Infection
Protocol. HL cell line (human line)62 was cultivated and
all cell-based experiments were done in a medium consisting
of RPMI 1640 w/o L-glutamine (Biowhittaker, Lonza, Basel,
Switzerland) supplemented with 7.5% fetal bovine serum (FBS)
92
4-Me
102
87
Me
OMe
101
Table 4. Chemical Structures of the Products 60-64 and 67-68, Their Inhibition of C. pneumoniae Growth at a Concentration of 10 μM, and
Their Effect on the Viability of the Host Cells at 10 μM

7670 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Keurulainen et al.
Figure 1. Differences in chemical structure of benzimidazoles (groups A-C) reflected well the antichlamydial effect (lines) and the effect on
the host cell viability (bars). For chlamydial growth inhibition, n = 12 except for 40 μM, n = 8, error bars þ SEM. The viability was compared
to DMSO-adjusted control. For host cell viability n = 3, error bars þ SD.
(Biowhittaker, Lonza, Basel, Switzerland), 2 mM L-glutamine
(Biowhittaker, Lonza, Basel, Switzerland), and 20 μg/mL gen-
tamicin (Fluka, Buchs, Switzerland). Cells were grown in cell
culture flasks (Greiner bio-one, Frickenhausen, Germany) at
37 ꢀC, 5% CO2, and 95% humidity and dispensed either to
Plates were centrifuged at 550 for 1 h at room temperature (Heraeus
Multifuge 3s, Thermo Fischer Scientific, Vantaa, Finland) and left
to stand for 1 h in the incubator.
Following the infection, the infection medium was removed
from the wells and samples were added in 0.5 μg/mL cyclohex-
imide-supplemented medium, 1 mL/well on a 24-well plate, and
200 μL/well on a 96-well plate. The compounds were added as
three parallel replicates on the 24-well plate and as six parallel
replicates on the 96-well plate. On each plate, there were
noninfected and infected controls as well as infected controls
treated with the antibiotic rifampicin 0.012 μM (BioChemika
>97.0% HPLC; Fluka, Buchs, Switzerland). Plates were in-
cubated at 37 ꢀC, 5% CO2, and 95% humidity for 72 h, after
which wells were washed with 1 mL/well or 300 μL/well of
24-well plates (Cellstar, Greiner bio-one, Frickenhausen, Germany)
5
€
with coverslips (Menzel-Glaser, Braunschweig, Germany) 4 ꢀ 10
cells/well or to 96-well plate with clear wells and white matrix
(Wallac Isoplate 1450-516, PerkinElmer, Waltham, MA, USA)
6 ꢀ 104 cells/well. Twenty-four h after plating, the cells were infected
with 0.2 multiplicity of infection (MOI) on 24-well and 96-well plates
with C. pneumoniae sequenced American Type Culture Collection
(ATCC) reference strain CWL-029 in medium supplemented with
0.5 μg/mL cycloheximide (Sigma-Aldrich, St. Louis, MO, USA).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7671
Table 5. The Overall Pattern of Efficacy against C. pneumoniae Re-
mained the Same with the CV-6 Strain Compared to the CWL-029
Strain (n = 2 except with 42 and 48 n = 4)
MCC was subsequently determined by removing the drug-
containing medium, washing the wells with 6.7 mM PBS (pH
7.4), and adding fresh medium without antibiotic supplementa-
tion. The infected monolayer was then disrupted, passed onto
fresh monolayers, and incubated and stained as above. The
MCC was the lowest drug concentration that inhibited the
production of IFU in the antibiotic-free passage. All titrations
were carried out in duplicate. The compounds 42 and 48 were
tested twice, as they were first tested separately and then retested
among the other seven selected compounds.
Host Cell Viability Measurement. In the viability measure-
ment, a commercial kit “CellTiter-Glo Luminescent Cell Viabi-
lity Assay” (Promega, Madison, WI, USA) was used. The 96-
well plate was seeded with HL cells as described above, but one
column was left empty for blank measurement. Twenty-six
hours after seeding the plate, the compounds were added to
three wells each, according to the infection protocol, as neither
infection nor centrifugation was needed. Cells were incubated
for 72 h and handled according to the instructions of the kit with
one exception. Approximately 200 μL of old medium was
removed and 100 μL of fresh medium was applied just before
adding “CellTiter-Glo” reagents. The plate was shaken on the
plate shaker on low intensity for 2 min to induce complete cell
lysis. After this, the signal was allowed to stabilize for 10 min and
luminescence was measured by VarioSkan flash plate reader
(Thermo Fischer Scientific, Vantaa, Finland). Viability percen-
tage was calculated by comparing the ATP concentration of
cells treated with DMSO-adjusted medium to the ATP concen-
tration of cells treated with 2-arylbenzimidazoles.
Other Antimicrobial Activities. To determine general antimi-
crobial effects of the benzimidazoles, they were tested against
Staphylococcus aureus ATCC-25923 (Gram-positive) and
Escherichia coli ATCC-25922 (Gram-negative). Both microbes
were first grown overnight in 5 mL of nutrient broth at 37 ꢀC and
repassaged by adding 0.1 mL of the bacterial suspension to
10 mL of nutrient broth and grown overnight at 37 ꢀC in a
shaker at the speed of 100 rpm. Of this suspension, 0.1 mL was
suspended to 100 mL of nutrient broth and suspension was
mixed thoroughly. Then 130 μL of formed bacterial suspension
was added to the wells of 96-well plate (Wallac Isoplate
1450-516, PerkinElmer, Waltham, MA, USA) excluding nega-
tive growth and background wells, where plain nutrient broth
was added. To all wells were added either 50 μL of 0.01% (w/v)
resazurin (cat. no. R-2127, Sigma-Aldrich, St. Louis, MO, USA)
solution in sterile water or plain sterile water, and 50 μL of
assayed compounds or control compound ampicillin (cat. no.
A-6140, Sigma-Aldrich, St. Louis, MO, USA) in nutrient broth
or plain nutrient broth. The end concentration of benzimida-
zoles and ampicillin were 50 and 25 μM (10 μg/mL), respectively.
Plates were incubated at 37 ꢀC for 18 h, and the amount
of resazurin was measured by photometric method with
VarioSkan flash plate reader at the wavelength of 600 nm.
Synthesis. All reagents were commercially available and were
acquired from Fluka (Buchs, Switzerland), Aldrich (Schnelldorf,
Germany), Mallinckrodt Baker (Phillipsburg, NJ, USA), Riedel-de
MIC range
μM (μg/mL)
196
MCC range
compd
μM
(μg/mL)
34
35
42
43
48
53
55
56
60
(64)
>196
>196
3.2-6.3
50.4
(>64)
(>64)
(1-2)
(16)
>196
3.2-6.3
3.2
(>64)
(1-2)
(1)
6.1-48.9
24.5
12.6
(2-16)
(8)
(4)
24.5
24.5
(8)
(8)
12.6
(4)
196
12.6
(64)
(4)
>196
12.6
0.17
(>64)
(4)
(0.125)
erythromycin (control)
0.17
(0.125)
6.7 mM PBS (pH 7.4) solution (Biowhittaker, Lonza, Basel,
Switzerland) and fixed with 1 mL/well or 300 μL/well of
methanol (Mallinckrodt Baker, Phillipsburg, NJ, USA) for
10 min. The coverslips were removed from the 24-well plates
and the methanol from the 96-well plates, and fixed cell layers
were left to dry for at least 24 h.
TR-FIA Labeling. Assay buffer (PerkinElmer, Waltham,
MA, USA) was supplemented with europium-conjugated chla-
mydial antibody (100 ng/mL), and 100 μL of this assay buffer
was added to the wells of the 96-well plates. Plates were
incubated for 30 min at 37 ꢀC and washed six times with wash
solution (PerkinElmer, Waltham, MA, USA) using a plate
washer (BW 50, Biohit, Helsinki, Finland). Enhancement solu-
tion was added to the wells and plates shaken at low intensity
for 5 min on a plate shaker (Delfia Plateshaker, PerkinElmer
Finland, Turku, Finland), and signals were measured by a
multilabel plate reader Victor2 (PerkinElmer Finland, Turku,
Finland). The TR-FIA method is described in detail elsewhere.56
Autofluorescence of Compounds and Affinity of Europium-
conjugated Chlamydial Antibody to Compounds. The 96-well
plate was handled according to the infection protocol, but no
infection was induced. The compounds were added as in the
antichlamydial TR-FIA assay. The plate was analyzed by
Victor2 plate reader after 72 h of incubation for autofluores-
cence by the compounds. The procedure was repeated but with
labeling the plate as in TR-FIA and time-resolved fluorescence
was measured with Victor2 to detect unspecific binding of the
label.
IF Measurement of Strain CWL-029. The dried coverslips
were stained with Pathfinder Chlamydia Culture Confirmation
System (Bio-Rad, Hercules, CA, USA) containing isothiocya-
nate-conjugated monoclonal antibody. The Pathfinder solution
was distributed to a marked slide, and coverslips were placed on
the slide cells facing down and incubated for 30 min at 37 ꢀC in a
humid environment. The excess label was removed by dipping
the coverslips twice to 6.7 mM PBS (pH 7.4) and once to purified
water (Milli-Q). Coverslips were mounted to objective glasses,
and the chlamydial inclusions were counted under a fluores-
cence microscope (Nikon Eclipse TE300, Tokyo, Japan) in a
magnification of 200ꢀ. Four eye-fields from every coverslips
were counted, totaling 12 measurements per compound.
IF Measurement of Strain CV-6. MIC testing of C. pneumo-
niae was carried out in cell culture with HEp-2 cells (ATCC
CCL-23) grown to confluency in 24-well plates with glass cover-
slips. After a centrifugation step (1700g for 45 min, 35 ꢀC) with
103 inclusion forming units (IFU) per mL, the medium was
replaced with minimal essential medium containing cyclohex-
imide (1 μg/mL) and assayed compounds in serial 2-fold dilu-
tions. Visualization of chlamydial inclusions was performed
after incubation for 72 h at 35 ꢀC and 5% CO2, with a
fluorescein-conjugated monoclonal anti-Chlamydia lipopoly-
saccharide antibody (Oxoid, Ely, UK). The MIC was defined
as the lowest concentration at which no IFU were observed. The
€
Haen (Seelze, Germany), and Alfa Aesar (Karlsruhe, Germany).
THF was distilled over sodium/benzophenone ketyl. Anhydrous
MeCN (Aldrich) was stored under an inert atmosphere of dry
argon. All reactions in anhydrous solvents were performed under an
argon atmosphere. The progress of chemical reaction was mon-
itored by thin-layer chromatography on silica gel 60-F254 plates
acquired from Merck (Darmstadt, Germany). The eluent consisted
of EtOAc and n-hexane or CH2Cl2 and MeOH, and detection was
conducted at 254 or 366 nm. MW-assisted synthesis was conducted
by Biotage SP1MW instrument (Uppsala, Sweden). Flash SiO2
column chromatography was performed with a Merck silica gel 60
(230-400 mesh), or products were purified by flash chromatogra-
phy on a silica gel with a Biotage SP1 purification system (Uppsala,
Sweden) using 25 þ M or 12 þ M cartridges (25 or 12 mL/min flow
rate, detection 254 nm). Melting points were measured using an

7672 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Keurulainen et al.
IA9100 digital melting point apparatus (Electrothermal Engineer-
ing, Essex, UK) and are uncorrected. IR spectra were recorded on a
Bruker Vertex 70 FT-IR spectrometer (Ettlingen, Germany) with
KBr technique. The synthesized compounds were analyzed by
NMR on a Varian Mercury 300 MHz spectrometer (Varian, Palo
Alto, CA). 1H and 13C NMR were recorded as solutions in DMSO-
d6, CDCl3, or CD3OD. Deuterated solvents were purchased from
Aldrich. Chemical shifts (δ) are given in parts per million (ppm)
relative to the NMR solvent signals (DMSO-d6 2.50 and 39.51 ppm,
d6) δ 12.69 (s, 1H), 7.55 (s, br, 2H), 7.42 (t, J 1.8 Hz, 1H), 7.27
(dt, J 7.8, 1.5 Hz, 1H), 7.19-7.14 (m, 3H), 6.67 (ddd, J 8.0, 2.4,
1.0 Hz, 1H), 5.30 (s, 2H). 13C NMR (75 MHz, DMSO-d6)
δ 152.1, 149.1, 143.8, 134.9, 130.7, 129.3, 122.2, 121.4,
118.7, 115.5, 113.9, 111.9, 111.2 (1H NMR and 13C NMR
were in agreement with reported data43). Rf = 0.47 (EtOAc:
n-hexane = 1:1).
General Procedure E. To the solution of compound (23-31,
1.0 mmol, 1 equiv) in anhydrous THF was added the appro-
priate acyl chloride (1.1 mmol, 1.1 equiv) and TEA (210 μL,
1.5 mmol, 1.5 equiv). After the given time, the eluent was
evaporated in vacuo and the residue was partitioned between
water (100 mL) and EtOAc (50 mL). The aqueous layer was
separated and extracted with EtOAc (50 mL), unless otherwise
stated, and the combined organic phases were washed with
saturated NaHCO3 (50 mL) and brine (50 mL), dried over
anhydrous Na2SO4, filtered, and the filtrate was evaporated in
vacuo to give the product.
CDCl3 7.26 and 77.16 ppm, CD3OD 3.31 and 49.00 for 1H and 13
C
NMR, respectively). LC-MS analyses were performed by the use of
an HP1100 instrument with UV detector (λ 210 nm) and Esquire
LC spectrometer (Bruker Daltonik, Bremen, Germany) with ESI
ion source. Signal separation was carried out by use of a Waters
XTerra MS RP18 column (4.6 mm ꢀ 30 mm, 2.5 μm). The eluent
consisted of water (þ0.1% HCO2H) and acetonitrile (þ0.1%
HCO2H) (gradient run 90:10 f 10:90). Purity of all tested com-
pounds was >95%. High resolution mass spectra (HRMS) were
measured on a JEOL MStation JMS-700 (Tokyo, Japan) instru-
ment operating at 70 eV.
General Procedure A. To the solution of diamine or amino-
phenol compound (5.5 mmol, 1 equiv) in pyridine (20 mL) was
added 3-nitrobenzoyl chloride (2.14 g, 11.6 mmol, 2.1 equiv),
unless otherwise stated, and DMAP (34 mg, 0.31 mmol, 0.05
equiv). After MW irradiation (15 min, 60 ꢀC) in a sealed tube
(20 mL), the mixture was poured into 2 M HCl solution (30 mL)
on an ice bath. The precipitate was collected and washed with
4 M HCl solution (2 ꢀ 20 mL), H2O (20 mL), 1 M NaOH
solution (20 mL), H2O (20 mL), and Et2O (20 mL) to give a
crude product, which was used for the next step without
purification.
3-Nitro-N-[2-[(3-nitrobenzoyl)amino]phenyl]benzamide (2). Syn-
thesis according to the general procedure A using 1,2-diamino-
benzene (0.59 g, 5.5 mmol) as a starting compound gave com-
pound 2 (2.21 g, 99%) as an off-white powder. 1H NMR
(300 MHz, DMSO-d6) δ 10.37 (s, 2H), 8.78 (t, J 2.0 Hz, 2H),
8.44-8.38 (m, 4H), 7.82 (t, J 8.1 Hz, 2H), 7.71-7.68 (m, 2H),
7.36-7.31 (m, 2H). 13C NMR (75 MHz, DMSO-d6) δ 163.6 (2C),
147.7 (2C), 136.1 (2C), 134.2 (2C), 131.3 (2C), 130.2 (2C), 126.3
(2C), 126.2 (2C), 125.9 (2C), 122.6 (2C). Rf = 0.53 (EtOAc:
n-hexane = 1:1).
General Procedure B. Bisamide compound (5.00 mmol,
1 equiv) and p-TSA (1.52 g, 8.00 mmol, 1.6 equiv) were refluxed
in p-xylene (40 mL) for 18 h. Reaction mixture was partitioned
between EtOAc (100 mL) and 1 M NaOH solution (100 mL),
and the aqueous layer was extracted with EtOAc (2 ꢀ 50 mL).
The combined organic layers were washed with brine, dried over
anhydrous Na2SO4, filtrated, and evaporated in vacuo.
2-(3-Nitrophenyl)-1H-benzimidazole (11). Following the gen-
eral procedure B, the product was synthesized from 2 (2.03 g,
5.00 mmol). Recrystallization from EtOH gave 11 as yellow
crystals (0.86 g, 72%); mp 208 ꢀC (lit. 206-207 ꢀC61). 1H NMR
(300 MHz, DMSO-d6) δ 13.28 (s, H), 9.01, (t, J 1.8 Hz, 1H), 8.61
(d, J 7.8 Hz, 1H), 8.31 (dd, J 8.0, 1.7 Hz, 1H), 7.84 (t, J 8.1 Hz,
1H), 7.66-7.64 (m, 2H), 7.28-7.22 (m, 2H). 13C NMR (75
MHz, DMSO-d6) δ 149.1, 148.3, 132.5, 131.7, 130.7, 124.2,
122.7, 120.8 (1H NMR and 13C NMR were in agreement with
reported data63). LC-MS: [M þ H]þ, m/z 340 (tr = 4.5 min). Rf
= 0.51 (EtOAc: n-hexane = 1:1).
N-[3-(1H-Benzimidazol-2-yl)phenyl]thiophene-2-carboxamide
(34). Following the procedure E mixture of 23 (0.21 g, 1.0
mmol), TEA, and 2-thiophenecarbonyl chloride (120 μL, 1.1
mmol) in THF (10 mL) was stirred for 30 min. The residue was
purified by column chromatography on silica gel (EtOAc: n-
hexane = 1:1 f 3:2) to give 34 as a white powder (0.17 g, 55%);
1
mp 134 ꢀC (recrystallized from MeOH). H NMR (300 MHz,
DMSO-d6) δ 12.94 (s, 1H), 10.44 (s, 1H), 8.64 (t, J 1.8 Hz, 1H),
8.12 (dd, J 1.2, 3.6 Hz, 1H), 7.90-7.87 (m, 3H), 7.67 (d, J 6.3 Hz,
1H), 7.56-7.51 (m, 2H), 7.27-7.19 (m, 3H). 13C NMR (75
MHz, DMSO-d6) δ 160.0, 151.1, 143.8, 139.9, 139.3, 135.1,
132.1, 130.6, 129.3, 129.3, 128.2, 122.6, 121.7, 121.5, 118.9,
118.7, 111.4. LC-MS: [M þ H]þ, m/z 320 (tr = 4.3 min). FT-
IR (KBr, cm-1): 3301, 2962, 1641, 1555, 1262, 742. Rf = 0.22
(EtOAc: n-hexane = 1:1). HRMS (EI): calcd for C18H13N3OS,
319.0779; found, 319.0780.
Acknowledgment. P.V. and O.S. thank the ERA-NET
project ECIBUG (European Initiative to Fight Chlamydial
Infections by Unbiased Genomics) for collaboration and the
Academy of Finland (119804) for funding. This study has also
been supported in part by the Austrian FFG/GEN-AU
initiative within ERA-NET PathoGenoMics ChlamyTrans-
project (M.M.). J.Y.-K. thanks Walter och Lisi Wahls Stif-
€
telse for Naturvetenskaplig Forskning for financial support.
We thank Prof. Emer. Pekka Saikku and Prof. Emer. Maija
Leinonen for the generous gift of C. pneumoniae strain CWL-
029 as well as Dr. Mirja Puolakkainen for expert help on
Chlamydia. Dr. Jorma Matikainen is acknowledged for expert
help in HRMS analyses.
Supporting Information Available: Material describing the
synthesis and characterization data of 1b, 3-22, and 24-68, and
the calculations of cLogP values for all final compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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