Synthesis and in vitro activity of dicationic bis-benzimidazoles as a new class of anti-MRSA and anti-VRE agents

Article abstract of DOI:10.1016/j.bmcl.2009.01.075

A new class of novel bis-benzimidazole diamidine compounds have been synthesized and evaluated for in vitro antibacterial activities, including drug-resistant bacterial strains. Anti-MRSA and anti-VRE activities of the most potent compound 1 were more act

Full text of DOI:10.1016/j.bmcl.2009.01.075

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1292–1295
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and in vitro activity of dicationic bis-benzimidazoles as a new class
of 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 Pharmaceuticals, Inc. 150 Fairway Drive, 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 new class of novel bis-benzimidazole diamidine compounds have been synthesized and evaluated for
in vitro antibacterial activities, including drug-resistant bacterial strains. Anti-MRSA and anti-VRE activ-
ities of the most potent compound 1 were more active than Vancomycin. The mechanism of action for
this class of compounds appears to be different from existing antibiotics. Bis-benzimidazole diamidine
compounds have potential for further investigation as a new class of potent anti-MRSA and anti-VRE
agents.
Received 4 January 2009
Revised 22 January 2009
Accepted 22 January 2009
Available online 7 February 2009
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
Antibacterials
Benzimidazoles
Diamidines
Since methicillin-resistant Staphylococcus aureus (MRSA) was
firstly identified in 1961,1 it has been the leading cause of morbid-
ity and mortality in various nosocomial and community-acquired
infections.2 With the increasing use of Vancomycin, as the last line
of defense against MRSA in recent decades, vancomycin-resistant
Enterococccus faecium (VRE) has emerged and can lead to bactere-
mia and death.3 Therefore, a tremendous need exists to discover
novel anti-MRSA and anti-VRE agents for overcoming these emerg-
ing bacterial resistance problems.
Benzimidazole derivatives have received much interest in drug
discovery, since they display diverse biological activities, such as
antihelminth, antiulcer, antiviral, anticancer, and antihistaminic
activity.4–6 Some benzimidazole-containing compounds, including
2-phenyl-1H-benzimidazole monoamidine derivatives, have
shown interesting anti-MRSA activity.7,8 We have previously re-
ported several series of benzimidazole diamidine compounds,
which have displayed diverse biological activities against parasites,
fungi and Bovine Viral Diarrhea virus (BVDV).9 Screening our benz-
imidazole dicationic compounds led to the discovery that com-
pound 1 (Fig. 1), DB325, is a potent anti-MRSA agent. Thus, we
designed and synthesized analogues of lead compound 1 to inves-
tigate the structure–activity relationship (SAR) of this series of bis-
benzimidazole amidine compounds. In this letter, we report the
synthesis, in vitro antibacterial activity and initial SAR of these
compounds as novel potent anti-MRSA and anti-VRE agents.
We have reported the synthesis of lead compound 1 by the con-
densation of 4-(N-isopropylamidino)-1,2-phenylenediamine
hydrochloride with 1,2-bis-(4-formylphenyl)ethane in presence
of benzoquinone as oxidative reagent.9a The analogs 5a–g of lead
compound 1 were prepared by following this procedure as shown
in Scheme 1. The cyano group of starting material 3,4-diam-
inobenzonitrile 2 was converted into the imidate ester 3 by using
the Pinner method.10 Then, the imidate ester was used directly to
react with suitable commercially available amines to yield the 4-
(N-substituted amidino)-1,2-phenylenediamine 4a–g. Condensa-
tion of these derivatives with 1,2-bis-(4-formylphenyl)ethane in
the presence of benzoquinone as oxidative reagent afforded the
corresponding bis-benzimidazole amidine hydrochloride salts
5a–g. 1,2-Bis-(4-formylphenyl)ethane was prepared in two steps
from 2,3-bis-(4-bromophenyl)propionic acid by the action of cop-
per(I) cyanide in DMF, and reduction with diisobutylaluminum hy-
dride (DIBAL-H), followed by as reported in the preparation of lead
compound 1.9a
Thienyl bis-benzimidazole amidines 12a and 12b were ob-
tained using a similar procedure to that of lead compound 1 in
six steps, as shown in Scheme 2. 4-Bromophenylpropionic acids
* Corresponding author.
E-mail address: nabood@immtechpharma.com (N. Abood).
Figure 1. Bis-benzimidazole diamidine lead compound 1 (DB325).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.075

L. Hu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1292–1295
1293
Scheme 3. Reagents and conditions: (a) 6-bromo-3-pyridinecarboxaldehyde or 5-
bromopyridine-2-carboxaldehyde, Cul, PdCI2(PPh3)2, (i-Pr)2NEt, THF, 50 °C, 79% or
56%; (b) 4-(N-isopropylamidino)-1,2-phenylenediamine hydrochloride, 1,4-benzo-
quinone, EtOH, reflux, 59% or 65%; (c) H2, Pd(OH)2, EtOH, 67% or 60%.
Scheme 1. Reagents and conditions: (a) HCI (gas), EtOH; (b) corresponding amines,
EtOH, reflux, 62–90% in two steps; (c) 1,2-bis-(4-formylphenyl)ethane, 1,4-benzo-
quinone, EtOH, reflux, 53–86%.
Scheme 4. Reagents and conditions: (a) 1,2-diaminobenzene, 1,4-benzoquinone,
EtOH, refulx, 30%; (b) 4-iodobenzaldehyde, Cul, PdCI2(PPh3)2, NEt3, DMF, 50 °C, 91%;
(c) 4-(N-isopropylamidino)-1,2-phenylenediamine hydrochloride, 1,4-benzoqui-
none, EtOH, reflux, 98%; (d) H2, Pd(OH)2, EtOH, 68%.
ira coupling, shown in Scheme 3–5.11 4-Ethynylbenzaldehyde 13
was coupled with bromopyridinyl aldehyde to obtain the dialde-
hyde intermediates 14a and 14b using Sonogoshira conditions.12
Then, the dialdehyde intermediates were condensed with 4-(N-iso-
propylamidino)-1,2-phenylenediamine hydrochloride followed by
benzoquinone oxidation to yield the corresponding benzimidaz-
oles 15a and 15b. The final compounds 16a and 16b were obtained
by hydrogenation of the acetylene group using the Pearlman’s cat-
alyst in DMF.13 The synthesis of bis-benzimidazole monoamidine
20 (Scheme 4) was carried out by a similar procedure for that of
16a and 16b. 4-Ethynylbenzaldehyde 13 was condensed with
1,2-diaminobenzene to prepare 2-(4-ethynylphenyl)-1H-benz-
imidazole 17. The target compound 20 was prepared by Sonogosh-
ira coupling, condensation and hydrogenation in three steps
similar to the prodedure of 11a and 11b. The preparation of
mono-benzimidazole diamidine compound 25 (Scheme 5) was
achieved by using a similar procedure in five steps from ethynyl-
benzonitrile 21 as starting material.
Scheme 2. Reagents and conditions: (a) 5 or 4-bromothiophene-2-aldehyde, 5 N
NaOH, MeOH, 63% or 70%; (b) NaBH4, Pyridine, MeOH, 85% or 82%; (c) 20% NaOH,
EtOH, reflux, 79% or 85%; (d) CuCN, N-methyl-2-pyrrolidone,73% or 68%; (e) DIBAL,
CH2CI2, 65% or 62%; (t) 4-(N-isopropylamidino)-1,2-phenylenediamine hydrochlo-
ride, 1,4-benzoquinone, EtOH, reflux, 58% or 67%.
9a and 9b were prepared from 4-bromophenylacetonitrile 6 by
condensation with thiophene-2-aldehydes 7a and 7b and reduc-
tion with sodium borohydride in two steps.
Pyridinyl bis-benzimidazole amidines 16a and 16b, bis-benz-
imidazole monoamidine 20 and mono-benzimidazole diamidine
25 were prepared in an alternative approach employing Sonogosh-
All the benzimidazole amidine hydrochloride compounds pre-
pared herein were screened for their potential antibacterial activi-

1294
L. Hu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1292–1295
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 according to the NCCLS guidelines.
aerobic strains, five compounds 1, 5b, 5d, 12a, and 12b showed
good antibacterial activities, including potent anti-MRSA (MIC va-
lue 61 lg/mL) and anti-VRE activity (MIC value 62 lg/mL); six
compounds 5c, 5e, 5f, 16a, 16b, and 25 were found to have mod-
erate activity; and three compounds 5a, 5 g, and 20 showed poor or
no antibacterial activity. Four compounds 1, 5b, 12a, and 12b also
showed good activity against two anaerobic bacterial strains (MIC
Penicillin
G (Pen G), Ciprofloxacin (CPLX), and Vancomycin
(VCM)) were used as reference standards. The minimum inhibitory
concentration (MIC) results for the test compounds are shown in
Table 1. Of the 14 compounds tested against the Gram-positive
value 64 lg/mL).
The lead compound 1 exhibited potent activity against all se-
lected Gram-positive bacterial strains and anaerobic bacterial
strains, including MRSA, MRSE and VRE (MIC value <1 lg/mL).
Anti-MRSA activity of the lead compound 1 was more potent than
that of VCM by two times and anti-VRE activity was more active by
512 times than that of VCM. It is noteworthy that anti-MRSA or
anti-MDRSA activity of the lead compound 1 was equivalent to that
of anti-MSSA activity. These results suggested that the mechanism
of action of the lead compound 1 could be different from existing
antibiotics.
To investigate the effect of N-substituent of 5-amidine of 1H-
benzimidazole on antibacterial activity, we designed and prepared
the N-substituted analogs 5a–g of the lead compound 1. It is note-
worthy that the parent diamidine compound 5a exhibited almost
no antibacterial activity. Antibacterial activity of the sec-butyl
and c-pentyl compounds 5b and 5d were comparable to that of
lead compound, however, compound 5d decreased anti-anaerobic
bacterial activity. N-butyl and c-propyl substituents 5c and 5e
led to some loss of antibacterial potency. Replacement of the iso-
propylamino group of the amidine with a pyrrolidinyl group
yielded the compound 5f which showed similar anti-S. aureus
and anti-S. epidermidis activities and but was less active against
the other bacterial strains compared to that of lead compound 1.
However, replacement of 5-N-isopropylamidine substituent of
benzimidazole by imidazoline led to total loss of antibacterial
Scheme 5. Reagents and conditions: (a) 4-iodobenzaldehyde, Cul, PdCI2(PPh3)2,
NEt3, DMF, 50 °C, 78%; (b) 4-(N-isopropylamidino)-1,2-phenylenediamine hydro-
chloride, 1,4-benzoquinone, EtOH, refulx, 92%; (c) HCI (gas), EtOH; (d) i-PrNH2,
EtOH, reflux, 46% in two steps; (e) H2, Pd(OH)2, EtOH, 54%.
Table 1
In vitro antibacterial activity of benzimidazole amidine compoundsa
Strain/compound
MICs (lg/mL)
1
5a
5b
5c
5d
5e
5f
5g
12a
S. aureus ATCC 29213
0.25–0.5
0.5
0.25–0.5
<0.06
0.125
<0.06
0.25–0.5
0.12
0.12
0.12
0.5–1
0.25–0.5
>32
>32
>32
4
1
0.5
4
4
2
0.25
0.5
1
32
2
1
4
32
0.5
1
1
8
16
8
0.12
0.25
<0.06
32
1
0.5
0.5
0.25
0.25
0.25
>32
16
2
2
1
2
2
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
1
0.5
b
S. aureus BAA-39
S. aureus ATCC 33591c
0.25
<0.06
0.12
0.12
2
0.5
0.25
0.5
0.5
<0.06
0.12
0.25
2
0.25
1
0.5
>32
4
0.5
0.12
0.12
60.06
0.5
60.06
0.5
0.25
4
S. epidermidis ATCC 12228
S. epidermidis ATCC 51625
S. pneumoniae ATCC 6301
E. faecalis ATCC 51575
E. faecium ATCC 700221
B. subtils ATCC 23857
B. cereus ATCC 11778
B. fragilis ATCC 23745
C. perfringens ATCC 10388
d
32
>32
>32
32
e
e
32
1
>32
>32
>32
0.25
32
1
2
0.5
0.25
12b
16a
16b
20
25
DB788
Pen-G
CPLX
VCM
S. aureus ATCC 29213
S. aureus BAA-39b
1
1
16
8
8
2
4
8
4
16
32
8
4
8
4
0.25
1
0.12
2
1
>32
>32
>32
4
4
8
4
0.25
1
0.12
2
1
4
8
4
4
4
8
32
8
4
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
S. aureus ATCC 33591c
S. epidermidis ATCC 12228
0.25
60.06
0.25
0.12
1
0.25
0.5
0.25
1
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
8
>32
>32
16
>32
32
>32
1
1
<0.06
4->32
4–8
0.12–0.5
1-60.12
4–8
0.5
32
8
0.5
32
>32
4
>32
16
16
0.5
>32
1
0.25
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) 1292–1295
1295
activity. These results clearly indicated that N-substituent of the
amidine of 1H-benzimidazole is very important for antibacterial
activity.
class of potent anti-MRSA and anti-VRE agents is underway and
will be reported in due course.
Replacement of one of the 2-phenyl rings of bis-benzimidazole
lead compound 1 by 2-thien-5-yl, 2-thien-4-yl, 2-pyridin-5-yl or 5-
pyridin-2-yl ring yielded bis-benzimidazole amidine compounds
12a, 12b and 16a, 16b. Thienyl compounds 12a and 12b showed
a slight loss of activity compared to lead compound 1. However,
pyridinyl compounds 16a and 16b exhibited only moderate anti-
bacterial activity compared to the lead compound 1.
References and notes
1. Jevons, M. P. Br. Med. J. 1961, 1, 124.
2. (a) Barrett, J. F. Expert Opin. Ther. Targets 2005, 9, 253; (b) Loffler, C. A.;
MacDougall, C. Expert Rev. Anti-Infect. Ther. 2007, 5, 961; (c) Bryskier, A. Expert
Rev. Anti-Infect. Ther. 2005, 3, 505.
3. (a) Levy, S. B.; Marshell, B. Nat. Med. 2004, 10, S122; (b) Waglehner, F. M. E.;
Naber, K. G. Int. J. Antimicro. Agents 2004, 24S, S39; (c) Mitchell, M. O. Anti-Infect.
Agents Med. Chem. 2007, 6, 243.
Removal one of 5-N-isopropylamidino group in the lead com-
pound 1 led to much loss of antibacterial activity for 20. This result
is consistent with the SAR results for the N-substituents of 5-ami-
dine of 1H-benzimidazole analogues and further demonstrates that
N-substituent of 5-amidine of 1H-benzimidazole is important for
achieving potent antibacterial activity. Keeping both isopropylam-
idino groups and removal of one of benzimidazole rings of lead
compound 1 yielded compound 25 which gave reduced anti-MRSA
and anti-anaerobic bacterial activities. Replacement of the 5-N-iso-
propylamidino group with 4-methoxy group on the phenyl ring of
the compound 25, the previously reported DB788 exhibited similar
activities against MRSA and anaerobic bacteria.9d However, DB788
showed decreased activities against other bacterial strains, such as
S. epidermidis and VRE, compared to the 5-isopropylamidino com-
pound 25. These results suggest that bis-5-N-substitued amidino
groups of 1H-benzimidazole is necessary for potent antibacterial
activity.
In conclusion, we have identified the bis-benzimidazole diami-
dine lead compound 1 as a potent anti-MRSA and anti-VRE agent
and prepared a number of analogues for probing the SAR of this
system. Five compounds 1, 5b, 5d, 12a, and 12b have shown good
antibacterial activities against Gram-positive bacteria, including
drug-resistant bacterial strains. The SAR study of this series of
compounds has shown that bis-5-N-substitued amidine of 1H-
benzimidazole is very important for achieving potent antibacterial
activity. The mechanism of action of the lead compound 1 could be
different from existing antibiotics. Further structural optimization,
study of the mechanism of action and in vivo efficacy of this new
4. Spasov, A. A.; Yozhitsa, I. N.; Bugaeva, L. I.; Anisimova, V. A. Pharma. Chem. J.
1999, 33, 232.
5. Alamgir, M.; Black, D. St. C.; Kumar, N. Top. Heterocycl. Chem. 2007, 9, 87.
6. Bhattacharya, S.; Chaudhuri, P. Curr. Med. Chem. 2008, 15, 1762.
7. (a) Kennedy, G.; Viziano, M.; Winders, J. A.; Cavallini, P.; Gevi, M.; Micheli, F.;
Rodegher, P.; Seneci, P.; Zumerle, A. Bioorg. Med. Chem. Lett. 2000, 10, 1751; (b)
Weidner-Wells, M. A.; Ohemeng, K. A.; Nguyen, V. N.; Fraga-Spano, S.;
Macielag, M. J.; Werblood, H. M.; Foleno, B. D.; Webb, G. C.; Barrett, J. F.;
Hlasta, D. J. Bioorg. Med. Chem. Lett. 2001, 11, 1545; (c) Bürli, R. W.; McMinn, D.;
Kaizerman, J. A.; Hu, W.; Ge, Y.; Pack, Q.; Jiang, V.; Gross, M.; Garcia, M.; Tanaka,
R.; Moser, H. E. Bioorg. Med. Chem. Lett. 2004, 14, 1253; (d) He, Y.; Wu, B.; Yang,
J.; Robinson, D.; Risen, L.; Ranken, R.; Blyn, L.; Sheng, S.; Swayze, E. E. Bioorg.
Med. Chem. Lett. 2003, 13, 3253; (e) He, Y.; Yang, J.; Wu, B.; Risen, L.; Swayze, E.
E. Bioorg. Med. Chem. Lett. 2004, 14, 1217.
8. (a) Göker, H.; Alp, M.; Yıldız, S. Molecules 2005, 10, 1377; (b) Göker, H.; Özden, S.;
Yıldız, S.; Boykin, D. W. Eur. J. Med. Chem. 2005, 40, 1062; (c) Özden, S.; Atabey, D.;
Yıldız, S.; Göker, H. Bioorg. Med. Chem. 2005, 13, 1587;(d)Göker, H.; Boykin, D. W.;
Yıldız, S. Bioorg. Med. Chem. 2005, 13, 1707; (e) Ates-Alagöz, Z.; Alp, M.; Kus, C.;
Yıldız, S.; Buyukbingöl, E.; Göker, H. Arch. Pharm. Chem. Life Sci. 2006, 339, 74.
9. (a) Del Poeta, M.; Schell, W. A.; Dykstra, C. C.; Jones, S.; Tidwell, R. R.; Czarny, A.;
Bajic, M.; Bajic, M.; Kumar, A.; Boykin, D.; Perfect, J. R. Antimicrob. Agents
Chemother. 1998, 42, 2495; (b) Del Poeta, M.; Schell, W. A.; Dykstra, C. C.; Jones,
S. K.; Tidwell, R. R.; Kumar, A.; Boykin, D. W.; Perfect, J. R. Antimicrob. Agents
Chemother. 1998, 42, 2503; (c) Hopkins, K. T.; Wilson, W. D.; Bender, B. C.;
McCurdy, D. R.; Hall, J. E.; Tidwell, R. R.; Kumar, A.; Bajic, M.; Boykin, D. W.
J. Med. Chem. 1998, 41, 3872; (d) Givens, M. D.; Dykstra, C. C.; Brock, K. V.;
Stringfellow, D. A.; Kumar, A.; Stephens, C. E.; Göker, H.; Boykin, D. W.
Antimicrob. Agents Chemother. 2003, 47, 2223; (e) Ismail, M. A.; Brun, R.;
Wenzler, T.; Tanious, F. A.; Wilson, W. D.; Boykin, D. W. Bioorg. Med. Chem.
2004, 12, 5405; (f) Ismail, M. A.; Batista-Parra, A.; Miao, Y.; Wilson, W. D.;
Wenzler, T.; Brun, R.; Boykin, D. W. Bioorg. Med. Chem. 2005, 13, 6718.
10. Roger, R.; Neilson, D. G. Chem. Rev. 1961, 61, 179.
11. Khabnadideh, S.; Pez, D.; Musso, A.; Brun, R.; Ruiz Pérez, L. M.; González-
Pacanowska, D.; Gilbert, I. H. Bioorg. Med. Chem. 2005, 13, 2637.
12. Sonogahsira, K.; Tohda, Y.; Hagihara, N. Tetrahedron Lett. 1975, 16, 4467.
13. Harris, N. V.; Smith, C.; Bowden, K. Synlett 1990, 577.