Optimization of the central linker of dicationic bis-benzimidazole anti-MRSA and anti-VRE agents

Article abstract of DOI:10.1016/j.bmcl.2009.05.061

A series of bis-benzimidazole diamidine compounds containing different central linkers has been synthesized and evaluated for in vitro antibacterial activities, including drug-resistant bacterial strains. Seven compounds have shown potent antibacterial activities. The anti-MRSA and anti-VRE activities of compound 1h were more potent than that of the lead compound 1a and vancomycin.

Full text of DOI:10.1016/j.bmcl.2009.05.061

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3374–3377
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Optimization of the central linker of dicationic bis-benzimidazole
anti-MRSA and anti-VRE agents
Laixing Hu a, Maureen L. Kully b, David W. Boykin a, Norman Abood c,
*
a Department of Chemistry, Georgia State University, Atlanta, GA 30303-3083, USA
b Naeja Pharmaceutical Inc., Edmonton, Alta., Canada T6E 5V2
c Immtech Inc. 150 Fairway Suite 150, Vernon Hills, IL 60061-1860, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of bis-benzimidazole diamidine compounds containing different central linkers has been synthe-
sized and evaluated for in vitro antibacterial activities, including drug-resistant bacterial strains. Seven
compounds have shown potent antibacterial activities. The anti-MRSA and anti-VRE activities of com-
pound 1h were more potent than that of the lead compound 1a and vancomycin.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 3 February 2009
Revised 14 May 2009
Accepted 14 May 2009
Available online 20 May 2009
Keywords:
Antibacterials
Benzimidazoles
Diamidines
The worldwide spread of drug-resistant Gram-positive bacterial
pathogens, including methicillin-resistant Staphylococcus aureus
(MRSA) and vancomycin-resistant Enterococccus faecium (VRE),
has become a serious global clinical problem for the treatment of
various nosocomial and community-acquired infections.1 In order
to overcome these emerging bacterial resistance problems, numer-
ous efforts have focused on discovering novel anti-MRSA and anti-
VRE agents in recent decades.2
During a study seeking to discover novel anti-MRSA agents, we
found a new class of dicationic bis-benzimidazole derivatives
which displayed potent anti-MRSA and anti-VRE activities.3 The
lead compound 1a (Fig. 1) has shown potent activity against
Gram-positive bacterial strains and anaerobic bacterial strains,
including both MRSA and VRE (MIC 6 0.5 lg/mL). A structure–
activity relationship (SAR) study of this series of compounds has
shown that bis-5-N-substituted amidine of 1H-benzimidazole is
very important for achieving potent antibacterial activity. Replace-
ment of one of the 2-phenyl rings of the lead compound 1a by thi-
enyl or pyridyl ring also resulted in loss of activity compared to the
lead compound 1a. The mechanism of action of this class com-
pounds appears to be different from existing antibiotics. Therefore,
we have continued to explore structural modifications of this new
class of dicationic bis-benzimidazole compounds. With the goals of
improving the anti-MRSA activity and to further investigate the
SAR of this system, we designed and synthesized analogues 1b–
m and 2 of lead compound 1a. Replacement of ethylene central lin-
Figure 1. Bis-benzimidazole diamidine compounds.
ker with various one atom or two atoms groups in the lead com-
pound 1a was extensively investigated. In this Letter, we report
the synthesis, in vitro antibacterial activity and SAR of this series
of bis-benzimidazole compounds containing different central
linkers.
We have reported the syntheses 1a, 1b, 1d, and 1m by the con-
* Corresponding author.
E-mail address: nabood@immtechpharma.com (N. Abood).
densation
of
4-(N-isopropylamidino)-1,2-phenylenediamine
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.061

L. Hu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3374–3377
3375
hydrochloride with the corresponding bis-benzaldehydes in pres-
ence of benzoquinone as oxidative reagent.4 The analogs 1c and
1e–l were prepared from the bis-benzaldehydes 3c and 3e–l by fol-
lowing this procedure as shown in Scheme 1. The bis-benzalde-
hydes 3h and 3l are commercially available. The intermediate 3c
was prepared by Sonogoshira coupling of 4-ethynylbenzaldehyde
4 with 4-bromobenzaldehyde in reasonable yield (Scheme 2).5
The starting material 4-bromomethylbenzonitrile 5 was converted
into the benzaldehyde 6 by reduction with diisobutylaluminum
hydride (DIBAL-H) in toluene (Scheme 3).6 4-Bromomethylbenzal-
dehyde 6 was reacted with 4-hydroxybenzaldehyde to yield the
bis-benzaldehyde 3e by using potassium carbonate in the presence
of tetrabutylammonium iodide (TBAI) in DMF.7 The bis-benzalde-
hydes 3f, 3g, and 3i–k were obtained from the corresponding cya-
no compounds by DIBAL-H reduction, as shown in Schemes 4–7.4a
trile (Scheme 4). 4,40-Dibromodiphenylmethane 9 on reaction
with cuprous cyanide in DMF gave the 4,40-dicyanodiphenylme-
thane 10 (Scheme 5).8 The dicyano compound 12 was prepared
from 4-chlorobenzonitrile 11 using sodium sulfide under reflux
condition in DMF (Scheme 6).9 4,40-Dicyanodiphenylamine 14a
was readily prepared by the reaction of 4-aminobenzonitrile 13
with 4-fluorobenzonitrile using potassium tert-butoxide as a base
(Scheme 7).10 The dinitrile compound 14a was converted to N-
methylamino derivative 14b by methyl iodide in the presence of
sodium hydride in DMF. The thioxanthene dicationic bis-benz-
imidazole 2 was prepared from 3,6-dicyanothioxanthene 1511 in
two steps by using a procedure similar to that reported for the
preparation of lead compound 1a (Scheme 8).
All of the bis-benzimidazole amidine hydrochloride compounds
1b–m and 2 were screened for their potential antibacterial activi-
ties in vitro against 10 selected Gram-positive bacterial strains and
two anaerobic bacterial strains, including MRSA, multi-drug resis-
tant S. aureus (MDRSA), methicillin-resistant Staphylococcus epide-
rmidis (MRSE) and VRE strains compared to the lead compound 1a.
Penicillin G (Pen G), ciprofloxacin (CPLX) and vancomycin (VCM)
were used as reference standards. The minimum inhibitory con-
centration (MIC) results for the test compounds are shown in Table
1. Of the 13 compounds tested against the Gram-positive aerobic
strains, seven compounds 1b, 1d, 1g, 1h, and 1j–l showed potent
antibacterial activities comparable to or better than lead com-
The sulfonamide compound
8 was prepared from 4-cya-
nobenzenesulfonylchloride 7 by reaction with 4-aminobenzoni-
pound 1a, including anti-MRSA (MIC value 6 1
VRE activity (MIC value 6 0.5 g/mL); two compounds 1c, 1e were
found to have good anti-MRSA activity (MIC value 6 4 g/mL); and
lg/mL) and anti-
l
l
Scheme 1. Reagents and conditions: (a) 4-(N-isopropylamidino)-1,2-phenylenedi-
amine hydrochloride, 1,4-benzoquinone, EtOH, reflux, 62–85%.
Scheme 5. Reagents and conditions: (a) CuCN, DMF, reflux, 38%; (b) DIBALH,
toluene, 0 °C, 61%.
Scheme 2. Reagents and conditions: (a) 4-bromobenzaldehyde, Cul, PdCl2(PPh3)2,
(i-Pr)2NEt, THF, 50 °C, 69%.
Scheme 6. Reagents and conditions: (a) Na2S, DMF, 48%; (b) DIBALH, toluene, 0 °C,
55%.
Scheme 3. Reagents and conditions: (a) DIBALH, toluene, 0 °C, 81%; (b) 4-
hydroxybenzaldehyde, TBAI, K2CO3, DMF, 98%.
Scheme 4. Reagents and conditions: (a) 4-aminobenzonitrile, Py, CH2Cl2, 49%; (b)
DIBALH, toluene, 0 °C, 35%.
Scheme 7. Reagents and conditions: (a) 4-fluorobenzonitrile, BuOK, DMF, 91%; (b)
Mel, NaH, DMF, 87%; (c) DIBALH, toluene, 0 °C, 21% or 28%.

3376
L. Hu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3374–3377
showed dramatically decreased anti-anaerobic bacterial activity.
The antibacterial potency of c-propyl analog 1d was almost equal
to the lead compound 1a. It is noteworthy that these dicationic
bis-benzimidazole compounds 1a–d generally decrease in anti-
MRSA and anti-VRE activity as the relative electronegativities of
these two-atom linker groups increase: ethylene < c-pro-
pyl < vinyl < ethynyl. A similar effect was found on replacement
of the ethylene group with the more electronegative –CH2O– group
to give compound 1e which showed lower antibacterial activity
compared to that of lead compound 1a. Replacement of the ethyl-
ene group by sulfonamide group led to poor antibacterial activity.
The significant difference in activity of the sulfonamide 1f relative
to the other compounds with two-atom linkers may be due to the
larger size and unique geometry of the sulfonamide linkage.12
These results indicated that the central linker is well tolerated by
vinyl and c-propyl group, however, ethynyl and –CH2O– groups re-
sult in some loss of potency and the sulfonamide group is not
tolerated.
Replacement of ethylene group of bis-benzimidazole lead com-
pound 1a by one-atom groups yielded bis-benzimidazole amidine
compounds 1g–l. The methylene compound 1g showed a slight
loss of activity compared to the lead compound 1a. Interestingly,
when the ethylene group of the lead compound 1a was replaced
by oxygen to yield the ether compound 1h the antibacterial activ-
ity was enhanced compared to the lead compound 1a. The activi-
Scheme 8. Reagents and conditions: (a) DIBALH, toluene, 0 °C, 62%; (b) 4-(N-
isopropylamidino)-1,2-phenylenediamine hydrochloride, 1,4-benzoquinone, EtOH,
reflux, 75%.
four compounds 1f, 1i, 1m, and 2 showed moderate or poor anti-
bacterial activity. Four compounds 1b, 1d, 1g and 1h also showed
good activity against two anaerobic bacterial strains (MIC va-
lue 6 2 lg/mL).
To investigate the effect of ethylene central linker of the lead
compound 1a on antibacterial activity, we first evaluated the anti-
bacterial activities of analogs in which the ethylene group of 1a has
been replaced by other two-atom linkers (1b–f). Antibacterial
activity of the vinyl and ethynyl compounds 1b and 1c were com-
parable to that of the lead compound 1a. However, compound 1c
Table 1
In vitro antibacterial activity of benzimidazole amidine compoundsa
Strain/compound
MICs (
l
g/mL)
1a
1b
1c
1d
1e
1f
1g
1h
1i
L = CH2CH2
CH@CH
C@C
c-Pr
CH2O
SO2NH
CH2
O
S
S. aureus ATCC 29213
S. aureus BAA-39b
0.25–0.5
0.5
0.25–0.5
<0.06
0.12
<0.06
0.25–0.5
0.12
0.12
0.12
0.5–1
0.25–0.5
1
1
0.5
2
4
2
1
0.5
1
4
0.5
>32
16
16
2
4
1
2
4
2
2
1–2
1
0.25
0.5
>32
32
16
8
16
>32
>32
16
16
8
S. aureus ATCC 33591c
S. epidermidis ATCC 12228
S. epidermidis ATCC 51625d
S. pneumoniae ATCC 6301
E. faecalis ATCC 51575e
E. faecium ATCC 700221e
B. subtils ATCC 23857
B. cereus ATCC 11778
B. fragilis ATCC 23745
C. perfringens ATCC 10388
0.25
60.06
0.12
60.06
0.5
0.12
0.12
0.12
0.5
0.25–0.5
0.12
0.25
<0.06
0.25–0.5
0.12
0.25
0.25
1-2
0.12
<0.06
<0.06
<0.06
0.12
<0.06
<0.06
<0.06
2
0.12
0.25
0.25
0.5
0.25
0.25
0.25
0.5–2
0.5–1
0.12
0.25
60.06
32
0.5
4
0.5
>32
4
60.06
0.25
60.06
1
0.12
0.12
0.12
32
2
4
>32
>32
0.25
16
1-2
0.5
MICs (
l
g/mL)
1j
1k
1l
1m
2
Pen G
CPLX
VCM
NH
NMe
NPh
2,5-Furyl
S. aureus ATCC 29213
S. aureus BAA-39b
0.25
0.25
0.25
0.12
0.25
0.12
0.25
0.12
0.12
0.12
>32
2
1
0.5
0.5
1
0.5
0.5
>32
32
2
4
2
0.12
>32
2
2
2
16
8
2
1
>32
>32
32
32
<0.06
4
0.5
8
60.12
60.12
60.12
0.5
0.5
>64
60.12
60.12
0.5
0.5
1
1
1
1
1
>64
>64
0.12–0.5
1–60.12
4–8
S. aureus ATCC 33591c
S. epidermidis ATCC 12228
S. epidermidis ATCC 51625d
S. pneumoniae ATCC 6301
E. faecalis ATCC 51575e
E. faecium ATCC 700221e
B. subtils ATCC 23857
B. cereus ATCC 11778
B. fragilis ATCC 23745
C. perfringens ATCC 10388
0.12
0.12
0.12
0.25
60.06
0.12
0.25
>32
1
0.25
0.25
0.12
0.5
0.12
60.06
0.12
4
2
0.5
0.12
16
0.5
1
1
>32
0.5
>32
<0.06
4–>32
4–8
>32
1
1
60.06–0.12
0.25
0.12–0.25
a
NCCLS guidelines M11-A6 and M7-A6 followed.
b
c
MDRSA.
MRSA.
MRSE.
VRE.
d
e

L. Hu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3374–3377
3377
ties of 1h against MRSA ATCC 33591, MRSE ATCC 51625, VRE ATCC
51575 and 700221 were twofold more active than that of the lead
compound 1a. However, compound 1i with sulfur as a linker
showed a dramatic loss of antibacterial activity. Antibacterial
activity of the compounds with –NH, –NMe and –NPh linkers
(1j–l) were similar to that of the lead compound 1a. However,
the compounds 1j–l showed decreased anti-anaerobic bacterial
activity. These results were in contrast to that for those compounds
1a–e with two-atom linkers: the greater the electronegativity of
the central one-atom linker group (the order of relative electroneg-
ativities: –O– > –NH– > –CH2–), the greater the antibacterial activ-
ity of the compounds 1h, 1j and 1g. Replacement of oxygen with
less electronegative sulfur in the compound 1h resulted in loss of
antimicrobial activity. These results suggest that the antibacterial
activity of the dicationic bis-benzimidazole compounds is related
to the electronegativity of the central linkers, however, the central
linkers of one-atom and two-atoms showed opposite effects on
activity. This result may be related to the difference in geometry
of the two series since the single-atom linker produces a much
more bent molecule than the two-atom linker. This may suggest
different binding modes for the two series. Replacement of ethyl-
ene group with 2,5-furyl ring in lead compound 1a to yield the
bis-benzimidazole amidine compound 1m resulted in significant
loss of antibacterial activity. When the diphenyl ethylene of the
lead compound 1a is replaced by a thioxanthenyl ring, the antibac-
terial potency is reduced compared to the lead compound 1a. How-
ever, it is noteworthy that compound 2 showed better activity than
the sulfur compound 1i. The order of anti-MRSA and anti-VRE
activity of these dicationic bis-benzimidazole compounds contain-
strains. The SAR study of this series compounds has shown that
the antibacterial activity of the dicationic bis-benzimidazole
compounds can be related to the relative electronegativity of
the central linkers, however, the central linkers of one-atom
and two-atoms showed opposite effects on activity. The most
potent compound 1h is more active than the lead compound
1a against MRSA, MRSE and VRE strains. The anti-MRSA or
anti-MDRSA activity of the most potent compound 1h as well
as the lead compound 1a was equivalent to that of anti-MSSA
(methicillin-susceptible S. aureus) activity, which was clearly dif-
ferent from that of the reference antibiotics Pen G and CPLX.
The anti-VRE activity of compound 1h was more active by
512 times than that of VCM. These results suggest that the
mechanism of action of the compound 1h might be different
from the reference antibiotics. The compound 1h merits further
investigation as a new lead of this class of dicationic bis-benz-
imidazole anti-MRSA and anti-VRE agents.
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ing different central linkers was: lh > 1a, 1d,
1j–l > 1b,
1g > 1e > 1c > 2 > 1f > 1m > 1i. These results clearly demonstrate
that the central linker of the dicationic bis-benzimidazole com-
pounds is very important for achieving potent antibacterial
activity.
In conclusion, we have synthesized and evaluated the anti-
bacterial activities of analogues of lead compound 1a by
replacement of the central ethylene linker with various two-
atom or one-atom groups for probing the SAR of this system.
Seven compounds have shown potent antibacterial activities
against Gram-positive bacteria, including drug-resistant bacterial
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