2-Piperidin-4-yl-benzimidazoles with broad spectrum antibacterial activities

Article abstract of DOI:10.1016/S0960-894X(03)00661-9

A series of 2-piperidin-4-yl-benzimidazoles were synthesized and evaluated for antibacterial activities. Certain compounds inhibit bacterial growth with low micromolar minimal inhibitory concentration (MIC). These benzimidazoles are effective against both

Full text of DOI:10.1016/S0960-894X(03)00661-9

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3253–3256
2-Piperidin-4-yl-benzimidazoles with Broad Spectrum
Antibacterial Activities
Yun He,* Baogen Wu, Jun Yang, Dale Robinson, Lisa Risen, Ray Ranken,
Lawrence Blyn, Suzie Sheng and Eric E. Swayze
Ibis Therapeutics, A Division of Isis Pharmaceuticals, Inc., 2292 Faraday Av., Carlsbad, CA 92008, USA
Received 20 March 2003; revised 6 June 2003; accepted 24 June 2003
Abstract—A series of 2-piperidin-4-yl-benzimidazoles were synthesized and evaluated for antibacterial activities. Certain com-
pounds inhibit bacterial growth with low micromolar minimal inhibitory concentration (MIC). These benzimidazoles are effective
against both Gram-positive and Gram-negative bacteria of clinical importance, particularly entercococci, and represent a new class
of potential antibacterial agents.
# 2003 Elsevier Ltd. All rights reserved.
Almost all the major classes of antibiotics have
encountered resistance in clinical applications.1À5 The
emergence of bacterial resistance to b-lactam anti-
biotics, macrolides, quinolones, and vancomycin is
becoming a major worldwide health problem.4À8 In
particular, antibiotic resistance among Gram-positive
bacteria (staphylococci, enterococci, and streptococci) is
becoming increasingly serious.9À13 Entercococci, which
are frequently resistant to most antibiotics including
penicillin, cephalosporin and aminoglycosides, are often
treated with either a combination of two antibiotics or
vancomycin. However, with the recent increased use of
vancomycin in methicillin-resistance Staphylococcus
aureus (MRSA) infections and colitis due to Clostridium
difficile, multiple resistant Entercoccus faecium has been
spreading.14 As such, the last resort for anti-infective
diseases, the Vancomycin family of antibiotics, has
now been gravely challenged in recent years due to
the emergence of Vancomycin resistance in clinical
practice.11,15
for active leads. Herein, we report on the initial struc-
ture–activity relationship (SAR) studies of a series of
benzimidazoles that have been discovered to possess
broad-spectrum antibacterial activities.
The synthesis of this class of benzimidazoles is shown in
Scheme 1. Treatment of commercially available 4,5-
dichloro-1,2-phenylenediamine (1) and N-Boc-iso-
nipecotic acid (2) with EDC in the presence of catalytic
amount of DMAP led to the formation of the corre-
sponding amide. The crude mixture was then refluxed in
aqueous sodium hydroxide solution to give cyclized
intermediate 3, which was reacted with various alkyl,
benzyl and aryl halides to give 4b–4i. Treatment of
compound 4g with various amines or nitrogen-contain-
ing heterocylces provided 6a–r. Deprotection of the Boc
group with anhydrous hydrogen chloride (HCl, 4.0 M)
in dioxane at room temperature for 30 min formed
benzimidazoles 7a–r. In a similar manner, 3, 4b–4i were
treated with hydrogen chloride to give benzimidazoles
5a–i.
In order to overcome these emerging resistance prob-
lems, there is an urgent need to discover novel anti-
bacterial agents in structural classes distinct from
existing antibiotics. In an effort to identify such com-
pounds, we continuously screen our in-house libraries
In the initial assays, all the benzimidazoles were tested
for activity against S. aureus and Eschericia coli.16 Their
minimal inhibitory concentrations (MIC) are listed in
Table 1. The preliminary data showed that all the amine
analogues (7a–r) exhibited strong ability to inhibit S.
aureus with most of the MICs in the low micromolar
range. Various nitrogen substitutions are accepted includ-
ing straight alkyl chain amines and diamines (7a–g), poly-
amine (7h), cyclic diamines (7i, 7j, 7n–p), heterocyclics
(7k–m, 7q, 7r). These benzimidazoles are also effective
*Corresponding author at current address: Department of Medicinal
Chemistry, Genomics Institute of the Novartis Research Foundation
(GNF), 10675 John Jay Hopkins Drive, San Diego, CA 92121, USA.
Tel.:+1-858-332-4706; fax:+1-858-812-1651; e-mail: yhe@gnf.org
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00661-9

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Y. He et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3253–3256
Table 1. Inhibitory effects of benzimidazoles on S. aureus and E. coli
growth and bacterial transcription/translation17
Compd
S. aureus
MIC (mM)
E. coli
MIC (mM)
T/T IC50
MIC (mM)
5a
5b
5c
5d
5e
5f
5g
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
>100
>100
50–100
50–100
>100
50–100
>100
6–12
3–6
6–12
12–25
6–12
6–12
6–12
3–6
6–12
12–25
6–12
12–25
12–25
6–12
6–12
6–12
>100
>100
50–100
50–100
>100
>100
>100
12–25
6–12
12–25
50–100
25–50
12–25
6–12
12–25
12–25
12–25
12–25
12–25
12–25
12–25
12–25
50–100
12–25
6–12
>100
>100
>100
>100
>100
>100
>100
>100
>100
12
20
50
>100
>100
10
>100
>100
25
>100
>100
>100
>100
60
>100
>100
>100
0.56
7p
7q
7r
12–25
6–12
0.75–1.56
1–3
Ciprofloxacin
Paromomycin
0.75–1.56
3–6
be partially due to the inhibition of bacterial transcrip-
tion and/or translation. However, most of the IC50
values are much higher than the corresponding MICs
for S. aureus and E. coli, it is unlikely that the antibacterial
activities for most of these compounds are due to inhibi-
tion of either bacterial transcription or translation.
To test effectiveness of these benzimidazoles against
other bacteria, the active compounds from the pre-
liminary screening were screened against additional four
strains of Gram-positive and four strains of Gram-
negative bacteria, and the results are shown in Table 2.
Again, these compounds exhibited higher potencies
against Gram-positive bacteria (S. aureus 13709, Enter-
ococcus hirae 29212, Streptococcus pyogenes 49399, and
Streptococcus pneumoniae 6303) as compared to Gram-
negative bacteria (E. coli 25922, Proteus vulgaris 8427,
Klebsiella pneumoniae 13383, Pseudomonas aeruginosa
25416). Several benzimidazoles, in particular 7b, 7f and
7g, showed interesting activities against E. hirae. These
compounds were screened against seven additional clini-
cally important Enterococcus strains, and the results are
shown in Table 3. In addition to their original activities,
several compounds (7a, 7b and 7r) displayed strong
inhibitory activities against all eight Enterococcus
strains. It appears that relatively hydrophobic terminal
moieties could enhance the antibacterial activities
against Enterococcus. Further studies are necessary to
fully understand their potent antibacterial activities. To
study the selectivity for inhibition of bacterial growth,
these compounds were also screened against yeast cell
line Candida albicans 10231 (Table 2). Certain com-
pounds were significantly less inhibitory to yeast growth
Scheme 1. Synthesis of benzimidazoles: (a) EDC, DMAP; (b) NaOH,
H2O; (c) RX, NaH or K2CO3; (d) for amines (RH), DMF 25 ꢀC, 2 h;
for heterocycles (RH), K2CO3 DMF, 25 ꢀC, 2 h; (e) 4.0 M HCl/diox-
ane, 0 ꢀC, 0.5 h.
in inhibiting E. coli growth, albeit with somewhat
reduced potency. Nevertheless, some compounds pos-
sess MICs approaching low micromolar concentrations.
In comparison, these benzimidazoles are two or more
folds more effective against S. aureus than E. coli. In
contrast, compound 5a, which has no substitution at N1
position, and compounds 5b–i, which have various
alkyl, benzyl or aryl substitutions at N1 positions did
not exhibit any appreciable ability to inhibit bacterial
growth. This suggests the crucial role of a basic nitrogen
distinct from the benzimidazole core.
All benzimidazoles were also screened for their ability to
inhibit bacterial translation and transcription using a
coupled assay.16 Several compounds (7c, 7d, 7h, 7k)
were found to posses low micromolar IC50s. Since these
compounds have similar inhibitory activities against
S. aureus and E. coli, their antibacterial activities could

Y. He et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3253–3256
3255
Table 2. Minimal inhibitory concentrations (MIC, mM) of benzimidazoles against bacteria and yeasta
Compd
Gram+
GramÀ
Yeast
CA1
SA1
6–12
EH2
SP4
SP6
EC2
PV8
KP1
6–12
6–12
12–25
25–50
25–50
12–25
6–12
12–25
12–25
12–25
6–12
12–25
25–50
25–50
PA2
7a
7b
7c
7d
7e
7f
7g
7h
7i
7k
7n
7o
7p
7r
1–3
1–3
3–6
6–12
3–6
1–3
1–3
3–6
3–6
3–6
3–6
3–6
3–6
3–6
3–6
3–6
6–12
6–12
6–12
3–6
12–25
6–12
12–26
6–12
25–50
12–25
25–50
NT
25–50
25–50
12–25
25–50
25–50
25–50
25–50
12–25
12–25
25–50
12–25
25–50
12–25
12–25
50–100
25–50
>100
>100
>100
>100
>100
>100
50–100
>100
50–100
>100
50–100
50–100
3–6
6–12
12–25
6–12
6–12
6–12
3–6
6–12
6–12
6–12
6–12
6–12
6–12
12–25
25–50
25–50
12–25
6–12
12–25
50–100
25–50
12–25
6–12
12–25
12–25
12–25
12–25
12–25
50–100
6–12
NT
25–50
12–25
25–50
25–50
25–50
12–25
25–50
25–50
25–50
3–6
3–6
6–12
6–12
6–12
6–12
6–12
6–12
12–25
12–25
12–25
12–25
12–25
25–50
6–12
NT, not tested.
aSA1, S. aureus 13709; EH2, E. hirae 29212; SP4, S. pyogenes 49399; SP6, S. pneumoniae 6303; EC2, E. coli 25922; PV8, P. vulgaris 8427; KP1, K.
pneumoniae 13383; PA2, P. aeruginosa 25416; CA1, C. albicans 10231.
Table 3. Minimal inhibitory concentrations (MIC, mM) of benzimidazoles against Enterococcus
Compd
E. faecalis
ATCC 11823
E. faecalis
ATCC 23241
E. faecalis
ATCC 4200
E. faecalis
ATCC 49757
E. faecalis
ATCC 828
E. faecium
ATCC 6569
E. faecium
ATCC 882
E. faecium
ATCC 29212
7a
7b
7c
7d
7e
7g
7I
7l
7n
7o
7p
7r
3–6
3–6
6–12
3–6
12–25
25–50
12–25
NT
12–25
12–25
6–12
12–25
12–25
6–12
6–12
3–6
6–12
3–6
6–12
3–6
3–6
3–6
6–12
3–6
12–25
25–50
12–25
NT
12–25
12–25
6–12
12–25
12–25
6–12
1–3
1–3
3–6
6–12
3–6
1–3
3–6
3–6
3–6
3–6
3–6
3–6
6–12
12–25
12–25
6–12
12–25
6–12
12–25
12–25
12–25
3–6
12–25
25–50
12–25
25–50
12–25
12–25
12–25
12–25
12–25
6–12
12–25
12–25
12–25
50–100
12–25
12–25
12–25
6–12
12–25
12–25
12–25
50–100
12–25
6–12
6–12
12–25
12–25
12–25
12–25
6–12
6–12
6–12
6–12
3–6
6–12
12–25
12–25
6–12
12–25
6–12
NT, not tested.
as compared to bacterial growth, suggesting a selectivity
index for inhibition of bacterial growth.
MD 21702-5014 is the awarding and administering
office. The content of this manuscript does not necessarily
reflect the position or policy of the Government, and no
official endorsement should be inferred.
In summary, we have discovered a novel series of ben-
zimidazoles that exhibit a broad spectrum of anti-
bacterial activities.
These benzimidazoles are
particularly effective against Gram-positive bacteria,
including clinically relevant strains of Enterococcus. The
simplicity of their structures and their high potencies
against different types of bacteria render these benzimi-
dazoles interesting leads for further investigation. The
preliminary SAR has pointed to a high degree of toler-
ance for structure modifications at the N1 of the benzi-
midazole core. The effect of extensive modification
around this core will be the subject of further optimiza-
tion studies, which shall be reported in due course.
References and Notes
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For financial support the authors thank USAMRID
DAMD717-02-2-0023. The U.S. Army Medical Research
Acquisition Activity 820 Chandler Street, Fort Detrick

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17. The assays were carried out in 150 mL volume in duplicate
in 96-well clear flat-bottom plates. The growth bacterial or
yeast suspension from an overnight culture in appropriate
medium was added to a solution of test compound in 2.5%
DMSO in water. Final bacterial or yeast inoculum is approxi-
mately 102–103 CFU/well. The percentage growth of the bac-
teria or yeast in test wells relative to that observed for a
control well containing no compound was determined by
measuring absorbance at 595 nm (A595) after 20–24 h at 37 ꢀC
(bacteria) or 40–48 h (yeast) at 25 ꢀC. The MIC was deter-
mined as a range of concentration where complete inhibition
of growth is observed at the higher concentration and bacter-
ial/yeast cells are viable at the lower concentration.