The synthesis of dicationic extended bis-benzimidazoles

Article abstract of DOI:10.3390/90300158

The synthesis of extended dicationic bis-benzimidazoles starting from trans-1,2-bis(4-cyanophenyl)ethene and trans-1,2-bis(4-cyanophenyl)cyclopropane is reported. The target diamidines show significant in vitro activity against B. subtilis.

Full text of DOI:10.3390/90300158

Molecules 2004, 9, 158-163
molecules
ISSN 1420-3049
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The Synthesis of Dicationic Extended Bis-Benzimidazoles
1
2
1,*
Zhijan Kang , Christine C. Dykstra and David W Boykin
1
Department of Chemistry Georgia State University, Atlanta, Georgia 30303, USA. Tel. +1-(404)-
51-3798, Fax +1-(404) 651-1416.
Department of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, Alabama
6849, USA.
6
2
3
*
Author to whom correspondence should be addressed; e-mail dboykin@gsu.edu
Received: 14 January 2004; in revised form: 26 January 2004 / Accepted: 27 January 2004 /
Published: 28 February 2004
Abstract: The synthesis of extended dicationic bis-benzimidazoles starting from
trans-1,2-bis(4-cyanophenyl)ethene and trans-1,2-bis(4-cyanophenyl)cyclopropane is
reported. The target diamidines show significant in vitro activity against B. subtilis.
Keywords: DIBAL reduction, benzimidazoles, amidines, Bacillus subtilis.
Introduction
Aromatic diamidines have a long history as antimicrobial agents [1]. Bis-benzimidazoles bearing
amidino groups have been reported to show activity against a number of microorganisms. Flexible
alkyl linked dicationic bis-benzimidazoles [2] and more ridged aryl linked bis-benzimidazoles are
active in vivo against Pneumocystis carinii pneumonia in an immunosupressed rat model [3]. The
later type molecules also show promising in vitro activity against several fungal organisms [4,5]. As
part of a general program of development of aromatic diamidines as antimicrobial agents [1,6] we
report the synthesis and preliminary evaluation of new bis-benzimidazoles which are linked by
diphenylethene and diphenylcyclopropane units that provide extended semi-rigid analogs.

Molecules 2004, 9
159
Results and Discussion
The synthesis of the new bis-benzimidazoles , outlined in Scheme 1, begins with DIBAL reduction
of the appropriate bis-nitriles (1a and 1b) to provide the corresponding bis-aldehydes (2a and 2b) in
moderate yields. The bis-aldehydes 2a and 2b were converted into the target molecules 3a-3c by 1,4-
benzoquinone facilitated oxidative coupling of the aldehydes with the appropriate 3,4-diamino-
benzamidines in 60-70% yields.
Scheme 1.
i)
ii)
N
N
NC
L
CN
OHC
L
CHO
L
A
A
N
N
H
3
H
2
a
b
1a
L =
L =
a
b
L =
L =
L =
A = -C=NH(NH2)
2
1
b
3
A = -C=NH(NH-i-Pr)
3
c
A = -C=NH(NH2)
Reagents and conditions: i) DIBAL, CH Cl
2
2
ii) 3,4-diaminobenzamidine or 3,4-diamino-N-isopropylbenzamidine, 1,4-benzoquinone, CH Cl -EtOH
2
2
Compounds 3a-3c were screened against B. subtilis N10 and E. coli AB1157. The compounds
were inactive against E. coli, but showed good anti-Bacillus activity as indicated in Table 1. The
compounds were also tested for mammalian cell in vitro cytotoxicity using CV1 Vero cells with the
XTT cytotoxicity test (Table 1) [7]. A desirable therapeutic index (2 log differential) was noted for 3c.
Table 1. Antimicrobial Screening Results
MIC/MBC
B. subtilis N10
<0.4/ND
Cell toxicity
CV1 IC50
ND
Therapeutic Index
Compound
(IC50/MIC)
3
a
b
-
20
3
0.4/11
8.0
3
c
0.4/>100
80.0
200
All values are in µM. ND = not done

Molecules 2004, 9
160
Conclusions
We have presented a facile route for the synthesis of the benzimidazoles 3a-3c and demonstrated
that these molecules show significant in vitro activity versus B. subtilis.
Experimental
General
Melting points were recorded using a Thomas-Hoover (Uni-Melt) capillary melting point apparatus
1
13
and are uncorrected. H and C NMR spectra were recorded employing a Varian GX400 instrument
and chemical shifts (δ) are in ppm relative to TMS used as internal standard. Mass spectra were
recorded on a VG analytical 70-SE spectrometer. Elemental analyses were obtained from Atlantic
Microlab Inc., Norcross, GA. All chemicals and solvents were purchased from Aldrich Chemical Co.
or Fisher Scientific.
trans-1,2-Bis(4-formylphenyl)ethane (2a). Diisobutyl aluminum hydride (12 mL, 12 mmol, 1M in
dichloromethane) was slowly added under nitrogen to a stirred solution of trans-1,2-bis(4-cyano-
phenyl)ethene (1a) [8] (0.92 g, 4.0 mmol) in anhydrous dichloromethane (100 mL). The mixture was
heated at reflux for 4 h, then treated with a mixture of ice and 10% sulfuric acid and extracted with
dichloromethane. The organic layer was dried over sodium sulfate and the solvent was evaporated. The
crude product was purified by flash chromatography using ethyl acetate and hexane (1:2) as the eluent.
The solid obtained was crystallized from ethanol to yield 0.56 g (59 %) of 2a, mp 167-169 °C (lit.[9]
+
1
mp 165-170°C); MS: m/z: 236 (M ); H-NMR (DMSO-d
6
) δ 10.01 ( s, 2Η), 7.93 (d, 4H, J = 8.1 Hz),
.86 (d, 4H, J = 8.1 Hz), 7.54( s, 2H); C-NMR (DMSO-d ) δ 192.4, 142.4, 135.1, 130.7, 130.0,
27.4; Anal. Calcd. for C16 : C, 81.33; H, 5.12. Found: C, 81.09; H, 54.36.
1
3
7
1
6
H
12
O
2
trans-1,2-Bis[4-(5-amidino-2-benzimidazolyl)phenyl]ethane hydrochloride (3a). A solution of trans-
,2-Bis(4-formylphenyl)ethane (2a) (0.21 g, 0.90 mmol) in dichloromethane (10 mL) was stirred until
all the solid dissolved. Absolute ethanol (60 mL), 3,4-diaminobenzamidine hydrochloride [10] (0.34 g,
.8 mmol), and 1,4-benzoquinone (0.20 g, 1.8 mmol) were added to this solution. The mixture was
1
1
heated at reflux for 4 h under nitrogen. After cooling to 0 °C anhydrous ethyl ether was added and the
resulting precipitate was filtered and washed with dry ether. The solid was dissolved in absolute
ethanol (25 mL) and absolute ethanol saturated with hydrogen chloride gas (35 mL), and the mixture
was heated at gentle reflux for 2 h. After cooling to 0 °C, addition of dry ether yielded a precipitate.
The solid was filtered, washed with dry ether, and crystallized from ethanol to yield 0.40 (69 %) of
1
desired product 3a, mp > 300 °C; MS for C H N (free base): 497.1 (M+H); H-NMR (DMSO-d )
3
0
24
8
6
δ 8.47 (d, 4H, J = 7.6 Hz), 8.27 (s, 2H), 7.92 (d, 4H, J = 7.6 Hz), 7.92 (d, 2H, J = 7.6 Hz), 7.82 (d, 2H,
1
3
J = 7.6 Hz), 7.57 (s, 2H); C-NMR (DMSO-d ) δ 162.8, 150.2, 146.3, 140.8, 139.1, 138.6, 129.5,
6

Molecules 2004, 9
161
1
1
28.4, 127.6, 126.9, 122.8, 120.1, 115.2; Anal. Calcd. for C30
6.74. Found: C, 53.86; H, 4.79; N, 16.49.
H
28
N
8
Cl
4
•1.5H
2
O: C, 53.82; H, 4.67; N,
trans-l,2-Bis[4-(5-N-isopropylamidino-2-benzanidazolyl)phenyl]ethene hydrochloride (3b). Solutions
of trans-1,2-bis(4-formylphenyl)ethene (2a, 0.21 g, 0.90 mmol) in CH Cl (10 mL) and 3,4-diamino-
2
2
N-isopropylbenzamidine hydrochloride [11] (0.42 g, 1.8 mmol) in ethanol (60mL) and 1,4-benzo-
quinone (0.20 g, 1.8 mmol) were allowed to react and then worked up as described above for 3a to
o
1
yield 0.46 (71 %) mp > 300 C; MS for C H N (free base): 581.4 (M+H); HNMR (DMSO-d ) δ
3
6
36
8
6
8
=
.50 (d, 4H, J = 8.4 Hz), 8.14 (s, 2H), 7.94 (d, 4H, J = 8.4 , Hz), 7.91 (d, 2H, J = 8.4 Hz), 7.71 (d, 2H, J
8.4 Hz), 7.61 (s, 2H), 4.11 (m, 2H), 1.31 (d, 12H, J = 6.4 Hz); C-NMR (DMSO-d6) δ 162.3, 153.7,
1
3
1
52.2, 149.6, 141.5, 138.8, 129.0, 128.5, 127.2, 127.0, 122.4, 121.9, 115.6, 44.9, 21.1; Anal. Calcd. for
C H N C : C, 59.51; H, 5.55; N, 15.42. Found: C, 59.34; H, 5.64; N, 14.98.
3
6
40
8
14
trans-1,2-Bis(4-formylphenyl)cyclopropane (2b). A solution of trans-1,2-bis(4-bromophenyl)cyclo-
propane [12] (1.0 g, 2.8 mmol) and copper cyanide (2.1 g, 22 mmol) in N, N-dimethylformamide (10
mL) was heated at reflux for 24 h under nitrogen. The mixture was poured onto ice. The precipitate
was filtered and then extracted three times with boiling chloroform (3x30 mL). The combined organic
extracts were washed two times with hydrochloric acid (2x50 mL) and three times with water (3x50
mL), and dried over sodium sulfate. After evaporation of solvent, the bis-nitrile trans-1,2-bis(4-cyano-
phenyl)cyclopropane (0.30 g, 44 %) was obtained by crystallization from ethanol, mp 124-126 °C; MS:
+
1
m/z: 244 (M ); H-NMR (acetone-d ) δ 7.69 (d, 4H, J = 8.1 Hz), 7.43 (d, 4H, J = 8.1 Hz), 2.48 (t, 2H, J
6
1
3
=
1
7.8 Hz), 1.72 (t, 2H, J = 7.8 Hz); C-NMR (DMSO-d
9.9. Anal. Calcd. for C17 : C, 83.58; H, 4.95; N, 11.47. Found: C, 83.81; H, 4.90; N, 11.35.
The bis-nitrile (0.49 g, 2.0 mmol) in dry methylene chloride (50 mL) and diisobutylaluminum
hydride (6.0 mL, 6.0 mmol, 1 M in dichloromethane) were allowed to react and worked up as
described for 2a to yield 0.32 g (64 %) of 2b, mp = 145-146 °C; MS: m/z: 250 (M ); H-NMR
acetone-d ) δ 10.00 (s, 2H), 7.86 (d, 4H, J = 8.0 Hz), 7.44 (d, 4H, J = 8.0 Hz), 2.50 (t, 2H, J = 7.2 Hz),
6
) δ 147.9, 132.1, 126.5, 118.9, 108.4, 28.7,
H
12
N
2
+
1
(
1
6
1
3
.74 (t, 2H, J = 7.2 Hz); C-NMR (acetone-d ) δ 192.4, 150.5, 135.9, 130.6, 127.3, 30.0, 20.6; Anal.
6
Calcd. for C17
H
14
O
2
: C, 81.58; H, 5.64. Found: C, 81.51; H, 5.72.
trans-l,2-Bis[4-(5-amidino-2-benzimidazolyl)phenyl]cyclopropane hydrochloride (3c). Solutions of
trans-1,2-bis(4-formylphenyl)cyclopropane (2b, 0.38 g, 1.5 mmol) in dichloromethane (10 mL),
3
,4-diaminobenzamidine hydrochloride [10] (0.56 g, 3.0 mmol) in ethanol (80mL) and 1,4-benzo-
quinone were allowed to react and worked up as describe for 3a to yield 0.80 g (82 %) of 3c, mp > 300
1
°
C; MS for C H N (free base): 511.1 (M+H); H-NMR (DMSO-d ) δ 8.42 (d, 4H, J = 8.0 Hz), 8.28
3
1
26
8
6
(
s, 2H), 7.94 (d, 2H, J = 8.8 Hz), 7.87 (d, 2H, J = 8.8 Hz), 7.54 (d, 4H, J = 8.0 Hz), 2.55 (t, 2H, J = 7.2
1
3
Hz), 1.80 (t, 2H, J = 7.2 Hz); C-NMR (DMSO-d
24.1, 123.6, 122.5, 115.2, 114.4, 29.0, 20.0; Anal. Calcd. for C31
N, 16.84. Found: C, 55.94; H, 4.73; N, 16.66.
6
) δ 165.6, 152.3, 147.6, 137.8, 134.5, 128.2, 126.5,
1
H
30
N
8
Cl •0.5H O: C, 55.95; H, 4.70;
4
2

Molecules 2004, 9
162
MIC, MBC and in vitro cell toxicity determinations.
o
B. subtilis N10 and E. coli AB1157 were grown in LB broth at 35 C for liquid culture or plated on
LB plates containing 1.5% agar [13]. In vitro cell toxicity tests were performed as described in the
literature [7] using CV1 cells cultured in Glutamax RPMI medium (Invitrogen) plus 5% fetal bovine
serum (Atlanta Biologicals), 100 U of penicillin G/mL, 100 µg streptomycin/mL and 0.25 µg of
amphotericin B/mL. XTT was from Sigma. A rapid screen was initially performed to evaluate potential
antimicrobial properties of the compounds. Approximately 0.1 mL of overnight cultures of several test
strains grown in LB broth were spread on LB agar plates. After drying, a 2 µL spot of a 10 mM stock
o
solution for the test compounds was placed on the plate. After overnight incubation at 35 C, zones of
inhibition were measured with a ruler; compounds with inhibition zones greater than 3 mm were tested
for their MIC and MBC values.
The minimum inhibitory concentration test (MIC) used a broth microdilution method in 96 well
culture plates by placing 100 µL of LB broth in the wells. Test compounds were placed in the top row
for a final concentration of 100 µM. Three-fold dilutions were performed down the wells of the plate
4
with a final dilution of 0.4 µM. 100 µL of an overnight culture diluted to approximately 1 X 10
o
cells/mL was then placed in each well. After overnight incubation at 35 C, plates were scored for
turbidity. The MIC was scored as the lowest concentration that appeared clear after overnight
incubation.
The minimum bactericidal concentration (MBC) test was performed on wells that appeared clear
after overnight incubation. 50 µL of the culture was spread on a LB agar plate and incubated
overnight. The MBC was scored at the lowest concentration of test compound that resulted in no
growth of the bacterial strain.
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