High-throughput-screening-based identification and structure-activity relationship characterization defined (S)-2-(1-aminoisobutyl)-1-(3-chlorobenzyl) benzimidazole as a highly antimycotic agent nontoxic to cell lines

Article abstract of DOI:10.1021/jm200571e

Novel nontoxic (S)-2-aminoalkylbenzimidazole derivatives were found to be effective against Candida spp. at low micromolar concentrations using high-throughput screening with infected HeLa cells. A collection of analogues defined the chemical groups relevant for activity. The most active compound was characterized by transcriptional analysis of the response of C. albicans Sc5314. (S)-2-(1-Aminoisobutyl)-1-(3-chlorobenzyl)benzimidazole had a strong impact on membrane biosynthesis. Testing different clinically relevant pathogenic fungi showed the selectivity of the antimycotic activity against Candida species.

Full text of DOI:10.1021/jm200571e

BRIEF ARTICLE
pubs.acs.org/jmc
High-Throughput-Screening-Based Identification and
StructureÀActivity Relationship Characterization Defined
(S)-2-(1-Aminoisobutyl)-1-(3-chlorobenzyl)benzimidazole as a Highly
Antimycotic Agent Nontoxic to Cell Lines
J€org Bauer,† Stephan Kinast,† Anke Burger-Kentischer,‡ Doris Finkelmeier,‡ Gerald Kleymann,§
Walid Abu Rayyan,|| Klaus Schr€oppel,|| Anurag Singh,|| G€unther Jung,† Karl-Heinz Wiesm€uller,†
Steffen Rupp,‡ and Holger Eickhoff*,†
†EMC microcollections GmbH, Sindelfinger Strasse 3, 72070 T€ubingen, Germany
‡Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, Nobelstrasse 12, 70569 Stuttgart, Germany
§Interfaculty Institute for Biochemistry, University of T€ubingen, Hoppe-Seyler-Strasse 4, 72076 T€ubingen, Germany
Interfaculty Institute of Microbiology and Infection Medicine, University of T€ubingen, Elfriede-Aulhorn-Strasse 6,
72076 T€ubingen, Germany
S Supporting Information
b
ABSTRACT: Novel nontoxic (S)-2-aminoalkylbenzimidazole derivatives were found to be effective against Candida spp. at low
micromolar concentrations using high-throughput screening with infected HeLa cells. A collection of analogues defined the
chemical groups relevant for activity. The most active compound was characterized by transcriptional analysis of the response of
C. albicans Sc5314. (S)-2-(1-Aminoisobutyl)-1-(3-chlorobenzyl)benzimidazole had a strong impact on membrane biosynthesis.
Testing different clinically relevant pathogenic fungi showed the selectivity of the antimycotic activity against Candida species.
’ INTRODUCTION
antifungal compound and the determination of its tolerability
by human cells in one step. Among various cell lines tested
(Caco-2, HeLa, A431, and A459), HeLa cells proved to show
robust and reproducible growth behavior and to be susceptible
to C. albicans infections.9 All accessible potential in vitro tar-
gets of the pathogen and the host were covered simulta-
neously, because as readout, the survival of infected host cells
was measured directly instead of detecting growth retardation
of the microorganism or inhibition of enzymatic functions of
the pathogen.10 Thus, critical compounds that show cytotoxi-
city or are unable to enter the host and/or pathogen cells in
general could be excluded at an early stage.11
The collection of 30 000 individual synthetic compounds em-
ployed as a first basis was previously well characterized to be
composed of 99.7% leadlike structures.12 Subsequently further
combinatorial compound collections comprising more than
70 000 individuals have been investigated.
The HeLa cell line was simultaneously incubated with
C. albicans in the presence of the individual compounds (∼20 μM)
for 5 days. After 18 h of incubation viability of HeLa cells was
analyzed microscopically and after 5 days quantified using fluo-
rescein diacetate (FDA) which is metabolized to a colorimetric
detectable fluorophore by vital cells. Additionally, individual wells
were visually scanned for growth of C. albicans. Thus, a fluo-
rescence signal of >40% per well in comparison to the positive
control (untreated HeLa cells ∼100%) and negative control
In the treatment of invasive fungal infections several thera-
peutic problems remain, in particular toxicity, variable drug bio-
availability, lack of oral or intravenous preparations, and sig-
nificant drug interactions for some agents.1 Previously rare fungal
species emerge, and moreover, most known strains develop
resistance against marketed antifungals.2 Invasive aspergillosis
and candidosis still account for high rates of nosocomial infec-
tions and mortality, in particular for immunocompromised
patients. Amphotericin B remains a standard therapy for many
life threatening mycoses despite its high nephrotoxicity.3 Keto-
conazole is more and more replaced for its low selectivity and
hepatic side effects.4 Numerous triazole based antimycotic thera-
peutics are continuously optimized and introduced in clinical
practice.1a,5 Generally, the azole class of antifungals acts by
binding to the active site of 14-lanosterol demethylase (14R-
sterol demethylase or P-450DM) inhibiting the biosynthesis of
the fungal cell-wall component ergosterol.6 However, a gradual
rise in triazole resistance7 and reproductive and hepatic toxicity
of triazole based antifungals is observed. The ability of triazoles to
inhibit CYP-dependent enzymes raises concerns about triazole
effects on hormone synthesis and drug metabolism.8 Therefore,
we also included compound collections of the more favorable
imidazole type for our antifungal HTS approach.
’ RESULTS AND DISCUSSION
ActivityÀSelectivity Assay for Antifungal Screening. An
Received: December 22, 2010
Published: June 29, 2011
assay has been set up allowing the identification of a potent
r
2011 American Chemical Society
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|

Journal of Medicinal Chemistry
BRIEF ARTICLE
Table 1. In Vitro Antifungal Activities (IC50), Cytotoxicity
(CC50), and Resulting Selectivities (SI) of Selected Com-
pounds 15Xx against Candida albicans
Figure 1. Hit compound 1a, MTPA-derivative (R,S)-2a, (R)-enantio-
mer 1b, and NAc derivative 3.
(untreated infected HeLa cells ∼0%) was the prerequisite for a
hit compound and indicated growth inhibition of C. albicans
without significantly impairing the vitality of the host cells. The
total hit rate of the HTS assay amounts to 1:1000 compounds
(0.1%) and thus was found to be in a typical and acceptable range
for a robust homogeneous assay. The primary hit compounds
belonged to different classes of heterocyclic compounds, but only
the benzimidazoles were promising in terms of high activity and
low cytotoxicity. All primary hit compounds were resynthesized,
and dose related responses were determined. The antimycotic
activity was measured as half maximal inhibitory concentration
IC50. Half maximal cytotoxic concentrations were determined as
CC50. The resulting selectivity index (SI) was measured as the
ratio CC50/IC50.
Chemistry and Strategy for SAR Studies. Among the initial
hits from the primary HTS the 1,2-disubstituted benzimidazole
1a (Figure 1) showed high antifungal activity and low cytotoxi-
city (IC50 = 0.75 μM, CC50 = 97.5 μM) resulting in a high
selectivity index (CC50/IC50) SI = 130 (Table 1). The N-acetyl-
ated analogue 3 and the (S)-enantiomer 1b were completely
inactive. The integrity of the stereochemistry has been confirmed
by derivatization to the respective Mosher’s amide 2a (Figure 1).
Obviously, the potency of 1a entirely depended on the config-
uration similar to that described for the dioxolan-based antifungal
drugs ketoconazole and itraconazole which are still administered
to patients as racemic mixtures of differently active cis/trans
isomers.13,14 Next we constructed compound collections of (S)-
2-aminoalkylbenzimidazoles by systematically varying three re-
gions of the core structure without affecting the essential (S)-
configuration and without changing the free primary amino
group. The synthesis procedures of 1a and analogues are sum-
marized in Scheme 1.
the aromatic ring, whereas only minor modifications in the
aliphatic residue R3 were made (13Xx, 14Xx, 15Xx). The latter
was restricted to various aliphatic R-amino acids 9aÀf to diversify
the R3 group. Therefore, a focused collection of 90 benz-
imidazoles (R1 Â R2 Â R3) = (3 Â 5 Â 6) was synthesized
(Figure 2). The results of the bioassay show that 13À15 with
5-CF3 and 5,6-diCl substitution of the benzimidazole moiety
are inactive. Modifications of the benzylic substitution pattern in
the R2 group are tolerated without a general loss of antifungal
activity. Interestingly, a variation of the aliphatic R3 branch is also
tolerated in a relatively broad range. Further modifications re-
sulted in a loss of the antifungal activity (see Supporting Infor-
mation). The purely aliphatic substituted compounds 15Xa and
15Xf showed high antifungal activity almost in the range of that
To delineate the structural requirements essential for biologi-
cal activity, we altered the substitution pattern of 13À15 on the
benzimidazole core (Figure 2). Trifluoromethyl, chloro, and
hydrogen substituents as R1 were introduced via nitroanilines
5aÀc, leading to the scaffolds 13 (R1 = 5-CF3), 14 (R1 = 5,6-
diCl), and 15 (R1 = H). The further diversification of the benzylic
R2 group consisted of halo, methyl, and methoxy substitution on
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Journal of Medicinal Chemistry
BRIEF ARTICLE
Scheme 1a
a Reagents and conditions: (a) 3 equiv of NaH, DMF, 2.5 equiv of nitroaniline 5aÀc, 12 h; (b) 5 equiv of alkyl bromide AÀE, 8 equiv of LiOtBu, THF/
DMSO (1:1, v/v), 12 h; (c) 1 M SnCl2 in DMF, 12 h; (d) 2.5 equiv of Fmoc-amino acid 9aÀf, 5 equiv of DIEA, 2.5 equiv of PyBrOP, NMP, 12 h;
(e) 25% TFA/DCM, 1 h; (f) HOAc, reflux, 12 h; (g) 5% piperidine, DCM, 2 h.
of amphotericin B (IC50 = 0.5 μM), the reference compound for
antifungal treatment.
Since none but hydrogen substitution of 15Xx on the benzim-
idazole core was connected to activity, we performed a “scaffold
hop” to the corresponding non-benzo-anellated 2-aminoalkylim-
idazoles which resulted in a complete loss of antimycotic activity.
Selected structural benzoanellated analogues of 15Da = 1a and their
respective activities are summarized in the supplement including
respective IC50, CC50, and SI. Particularly the linkage to the phenyl
substituent and the position and nature of the halogen therein are
crucial for activity. The essential benzene ring in the benzimidazole
core acts most likely as a hydrophobic interaction site. The highest
activity and selectivity was determined for the initial hit 15Da which
in turn was studied further. Only the 3-fluorobenzyl substituted
analogue 15Ea showed comparable activity (Table 1).
Identification of the Potential Molecular Target by Tran-
scriptional Profiling. To identify the mode of action of 15Da,
transcriptional profiling was performed. For this purpose, the C.
Figure 2. Compound collection 13Xx, 14Xx, 15Xx.
albicans strain Sc5314 was incubated in rich medium in the
presence or absence of 15Da (IC50 = 0.75 μM). The cultures
were analyzed using DNA microarrays as described elsewhere.9 A
significant number of genes that were found to be up-regulated at
least 2.0-fold belong to the Erg pathway, are connected to cell wall
biogenesis, or are potential membrane proteins. This indicates that
the novel (S)-2-aminoalkylbenzimidazole identified has a strong
impact on membrane biosynthesis via the ergosterol pathway,
similar as thishas been shown earlier for the azoles.15 Incontrast to
the antifungal benzimidazole benomyl or the anthelmintic me-
bendazol, 15Da does not affect microtubules.9
Determination of Minimal Inhibitory Concentrations
(MIC) and Comparison to a Reference Antifungal Com-
pound with Proven Activity. To compare the results from the
activityÀselectivity assay to standard MIC determination, the
MICs of 15Da and related compounds were assessed using the
M27 standard protocol, which included a comparison of the
activities to a reference compound with proven activity (CLSI
Reference Method for Broth Dilution Antifungal Susceptibility
Testing of Yeasts, Approved Standard M27-A3).16 MICs were
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Journal of Medicinal Chemistry
BRIEF ARTICLE
Table 2. Activity of Five Selected EMC Compounds and Drug References on a Set of 21 Test Strainsa
test compd, MIC (μg/mL)
test strain
15Cf
15Df
15Ea
15Ca
fluconazole
15Da
ATCC Control Strains
C. albicans DSMZ 11949
8
4
8
4
4
4
16
8
16
0.25
1
2
2
C. glabrata ATCC 90030
C. parapsilosis 22019
64
0.125
1
0.125
2
128
128
128
16
16
16
C. parapsilosis 90018
1
0.25
0.25
0.5
0.5
Issatchenkia orientalis ATCC 6258
1
Clinical Isolates
C. glabrata AN 8626
64
4
8
16
0.125
32
4
0.125
2
C. glabrata CG 7
64
64
4
C. albicans CA20
1
0.125
0.125
0.125
16
0.125
0.5
32
0.125
0.125
0.5
0.125
128
0.5
8
0.125
0.125
16
C. albicans CA21
64
0.125
0.5
8
C. albicans MY 2902/2008
C. albicans SCS 71865 L
C. albicans Jg 32570
32
128
128
64
16
32
64
128
8
128
4
128
16
16
C. albicans AM2001/0007
C. albicans AN 1699
0.25
32
128
128
128
128
128
128
128
128
70.66
72.88
16
16
8
16
64
C. albicans AN 1994
2
32
128
128
1
16
C. tropicalis AN 1946
32
8
4
C. albicans AN 562
16
1
16
32
16
C. albicans VB 1723
4
32
0.5
8
0.25
16
C. albicans AN 3156
16
8
16
32
C. parapsilosis AN 5485
C. parapsilosis AV 7675
av MIC [μg/mL] (geometric mean) for 21 strains
av MIC [μg/mL] (geometric mean) for 16 clinical isolates
16
2
0.25
2
1
128
5.38
5.66
128
10.42
9.51
128
6.78
11.31
8
1.49
2.18
2.00
2.83
a C. albicans DSMZ 11949, C. glabrata ATCC 90030, C. parapsilosis ATCC 22019, C. parapsilosis 90018, and Issatchenkia orientalis ATCC 6258 are
standard reference strains for in vitro susceptibility testing of yeast. The 16 clinical isolates were previously cultured from specimens of infected patients
and selected at random for this study.
measured for type strains, and significant clinical isolates of
several Candida spp. (Table 2), C. albicans DSMZ 11949,
C. glabrata ATCC 90030, C. parapsilosis 22019, C. parapsilosis
90018, and Issatchenkia orientalis ATCC 6258 were used as
reference control strains. Fluconazole was used as a control for
antifungal activity of the test compounds. The average minimal
inhibitory concentration (MICav) of 15Da for a set of 16 strains
was 2.83 μg/mL, which is within the same concentration as
the one of fluconazole (MICav, 2.18 μg/mL) (Table 2). Inter-
estingly, three of the clinical isolates (C. albicans Jg 32570,
C. albicans AN 1994, and C. tropicalis AN 1946) were resistant
to fluconazole, but 15Da showed antifungal activity.
Candida albicans SC5314, (S)-2-(1-aminoisobutyl)-1-(3-chloro-
benzyl)benzimidazole 15Da indicated a strong impact on mem-
brane biosynthesis pathways of the pathogen. These results give
rise to our assumption that primarily the inhibition of the fungal
cytochrome P450 oxidase-mediated synthesis of ergosterol is the
putative mode of action of (S)-2-aminoalkylbenzimidazoles.
Furthermore, we examined the activity against a set of 21
fungal strains including different type strains and significant
clinical isolates of Candida spp. and confirmed the selective anti-
mycotic activity of (S)-2-aminoalkylbenzimidazoles. The most
active compounds showed potencies equal to that of flucon-
azole. Three Candida strains that are resistant to fluconazole
showed a better susceptibility to 15Da. Consequently, the novel
nontoxic (S)-2-aminoalkylbenzimidazoles represent promising
candidates for future developments in antimycotic drug discovery.
’ CONCLUSION
In summary, a high throughput based screening approach
allowed the simultaneous in vitro determination of cytotoxicity in
host cells and antifungal activity of a compound against Candida
spp. Novel nontoxic antimycotic (S)-2-aminoalkylbenzimidazoles
were identified as antifungal substances, and a structureÀactivity
based characterization was performed including the reduction
of the active benzimidazole core to the corresponding non-
active imidazole scaffold. Interestingly, only the (S)-stereo-
isomers of 2-aminoalkylbenzimidazoles were found to be effica-
cious. By transcription profiling analysis of the response of
’ EXPERIMENTAL SECTION
Chemistry. General Methods. The (S)-2-aminoalkylbenzimida-
zoles were prepared by solid-phase chemistry (Scheme 1). The purity of
the key compounds was > 95% (HPLC-MS, RP-18, GromSil 80, ODS-7
pH, 4 μm, 40 mm  2 mm; gradient, eluent A (0.1% aqueous TFA) to
eluent B (0.1% TFA in acetonitrile) over 8 min; flow rate 0.6 mL/min;
MS: Waters Micromass ZQ quadrupole mass spectrometer).
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Journal of Medicinal Chemistry
BRIEF ARTICLE
Exemplary Synthesis of (S)-2-(1-Aminoisobutyl)-1-(3-
chlorobenzyl)benzimidazole (15Da). Immobilized 2-nitrophe-
nylcarbamate 6 was synthesized from o-nitroaniline 5 and p-nitrophenyl
carbonate Wang resin 4 using sodium hydride as base in dry DMF.
Alkylation with 3-chlorobenzyl bromide and lithium tert-butoxide and
subsequent reduction with 1 M stannous chloride dihydrate in DMF led
to 3-chlorobenzyl-(2-aminophenyl)carbamate Wang resin 8D. Acyla-
tion of 8D with Fmoc-valine 9a and PyBroP as coupling reagent in DMF
was followed by cleavage of 10Da from the polymeric support with 25%
TFA in DCM. The collection of lyophilized crude 11Da was cyclized for
16 h in neat glacial acetic acid at 80 °C to produce (S)-2-[1-(N-Fmoc-
amino)isobutyl]-1-(3-chlorobenzyl)benzimidazole 12Da which was pur-
ified by flash chromatography. Removal of the Fmoc protecting group
led to (S)-2-(1-aminoisobutyl)-1-(3-chlorobenzyl)benzimidazole 15Da
which was purified by flash chromatography. Yield: 121.8 mg (89%).
C18H20ClN3, M = 313.83 g/mol. Purity: 98% (HPLCÀESI-MS,
214 nm), [M + H]+ = 314.6 m/z. 1H NMR (400 MHz, CDCl3): δ =
7.88 (d, 1H, Ar-H, 3J = 7.6 Hz), 7.62À7.49 (m, 3H, Ar-H), 7.33À7.26
(m, 2H, Ar-H), 7.18À7.13 (m, 2H, Ar-H), 5.66 (dd, 2H, ArÀCH2, 2J =
to Fluconazole and Voriconazole by Standardized Disk Diffusion
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3
17.0 Hz), 4.75 (d, 1H, CH-NH2, J = 8.7 Hz), 2.60 (m, 1H, CH-
(CH3)2), 1.17 (d, 3H, CH(CH3), 3J = 6.1 Hz), 0.49 (d, 3H, CH(CH3),
3J = 6.3 Hz). 13C NMR (100 MHz, CDCl3): δ = 167.9, 148.7, 135.5,
135.1, 132.1, 130.8, 129.5, 127.4, 127.1, 125.3, 124.7, 116.3, 112.4,
70.8, 51.7, 48.7, 32.3, 18.9, 18.7.
’ ASSOCIATED CONTENT
Supporting Information. Synthesis protocols, 1H NMR
S
b
and 13C NMR data, purification details, yields, purities by
HPLCÀMS and activityÀselectivity assay. This material is
available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: +49 7071 407416. E-mail: eickhoff@microcollections.de.
’ ACKNOWLEDGMENT
This work was supported within the frame of the BioProfile
Program Stuttgart/Neckar-Alb, Grant FKZ: 0313709A, as
well as within the BMBF Program “Basisinnovationen in der
Genombasierten Infektionsforschung“, Grant FKZ 0315221B.
’ ABBREVIATIONS USED
CC, cytotoxic concentration; CYP, cytochrome P450; DCM, di-
chloromethane; FDA, fluorescein diacetate; Fmoc, fluorenylmethox-
ycarbonyl; Caco-2, HeLa, A431, and A459, human cell lines; HTS,
high-throughput screening; MIC, minimal inhibitory concentrations;
PyBroP, bromo-tris-pyrrolidinophosphonium hexafluorophosphate;
RP-HPLC, reverse phase high performance liquid chromatography;
SI, selectivity index; SAR, structureÀactivity relationship; TFA, tri-
fluoroacetic acid
(15) Rupp, S. Transcriptomics of the Fungal Pathogens, Focusing on
Candida albicans. In Human and Animal Relationships, 2nd ed.; Brakhage,
A. A., Zipfel, P. F., Eds.; The Mycota, VI; Springer-Verlag: Berlin, 2008;
pp 187À222.
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