|Benzimidazole; Piperazine; Ammonium formate
reduction; Sodium bisulfate adducts; Drug design
|Substituted benzimidazoles are an important class of heterocycles
that exhibit a broad spectrum of pharmacological properties, for
it assumes the position of a privileged structure in drug discovery
research. Derivatives of 1,2-substituted benzimidazoles have been
reported as antagonists  against prostaglandin D2 and angiotensin II
receptors . Similarly, substituted benzimidazoles have been patented
as dopamine-β-hydroxylase inhibitors . Hori et al.  reported
several modified benzimidazoles, serving as guanine biomimetics that
selectively inhibit endothelial cell growth and suppress angiogenesis in
vitro and in vivo. In nature, the benzimidazole nucleus constitutes an
important part of the vitamin B12 structure.
|In continuation of our efforts in drug design , and especially
in the area of Alzheimer’s disease , we became interested in polar
benzimidazoles as they are more water soluble and potentially less
toxic to human . To increase the water solubility we will have to
incorporate functionally active polar groups in the structure, and in
this case, an ethyl piperidine group at position-2 of the benzimidazole
core. It was shown that a methyl piperidine substituent was able to
increase water solubility of a compound by 2 orders of magnitude
. Since benzimidazole derivatives have been widely used in other
areas such as anti-cancer  and anti-mycobacterial  agents, their
pharmacokinetics are well understood. Apart from that, there is also a
recent report of benzimidazole having potent anti-tumor activity .
In view of the diverse biological applications of benzimidazoles, they
represent a good lead in developing new drugs.
|Materials and Methods
|All chemicals were supplied by Sigma-Aldrich (USA) and Merck
Chemicals (Germany). Purity of the compounds was checked on thin
layer chromatography (TLC) plates (silica gel G) in the solvent system
chloroform-methanol (9:1). The spots were located under short (254
nm)/long (365 nm) UV light. Elemental analyses were performed on
Perkin Elmer 2400 Series II CHN Elemental Analyzer and were within
± 0.4% of the calculated values. 1H and 13C NMR were performed on
Bruker Avance 300 (1H: 300 MHz, 13C: 75 MHz) spectrometer in CDCl3
using TMS as internal standard. Direct-infusion mass spectra were
recorded on Varian 320-MS TQ LC/MS using ESI.
|Preparation of Ethyl-4-fluoro-3-nitrobenzoate (1)
|4-Fluoro-3-nitrobenzoic acid (5 g, 27 mmol) was refluxed in ethanol
(50 mL) and concentrated H2SO4 (2 mL) for 8 hours. After completion
of reaction (as evident from TLC), the solvent was evaporated under
reduced pressure. The aqueous layer was extracted with ethyl acetate
(25 mL×3). The organic layer was dried over Na2SO4 and concentrated
under reduced pressure to yield 1 as cream-colored powder (75%).
|Preparation of N-(3-aminopropyl)imidazole (2)
|N-(2-aminoethyl) piperazine (1.30 mL, 9.90 mmol) and N,NDiisopropylethylamine,
DIPEA (0.49 mL, 2.78 mmol) were mixed in
dichloromethane (10 mL). Ethyl-4-fluoro-3-nitrobenzoate, 1 (0.5 g,
2.34 mmol) was added very slowly over 5 minutes. The reaction mixture
was stirred overnight at room temperature. The reaction mixture was
then washed with water (10 mL×2) followed by 10% Na2CO3 solution
(10 mL). The organic layer was dried over Na2SO4 and concentrated
under reduced pressure to afford 2 as yellow solid (92%).
|Preparation of Ethyl 4-(2-(piperazin-1-yl)ethylamino)-3-
|N-(3-aminopropyl)imidazole, 2 (0.322 g, 1 mmol), ammonium
formate (0.189 g, 3 mmol) and Pd/C (50 mg) were mixed in ethanol
(10 mL). The reaction mixture was refluxed until completion (solution
turned colorless). The reaction mixture was then filtered through Celite
545. The filtrate was evaporated under reduced pressure. It was resuspended
in ethyl acetate and washed with water, dried over Na2SO4
and evaporated to dryness to yield 3 (85%) which was used without
|General procedure for the preparation of sodium bisulfite
addcuts of 4-substituted benzaldehyde (4a-e)
|Appropriate benzaldehyde (10 mmol) was dissolved in ethanol
(20 mL). Sodium metabisulfite (15 mmol) in 5 mL water was added
in portion over 5 minutes. The reaction mixture was stirred at room
temperature for 1 hour and subsequently stirred at 4°C overnight. The
precipitate formed was filtered and dried to afford sodium bisulfite
|General procedure for the preparation of 2-substituted
benzimidazole derivatives (5a-5e)
|Ethyl 4-(2-(piperazin-1-yl)ethylamino)-3-aminobenzoate, 3 (1
mmol) and various sodium bisulfite adducts, 4a-e (1.5 mmol) were
dissolved in DMF (5 mL). The reaction mixture was stirred at 90°C
under N2 atmosphere for 24-48 hours. After completion of reaction
(evident by TLC), the reaction mixture was diluted in ethyl acetate
(25 mL) and washed with water (10 mL×3). The organic layer was
collected, dried over Na2SO4 and evaporated under reduced pressure to
afford compounds 5a-5e in 77-89% yields.
|This compound was obtained as yellow oil. Yield: 87%. 1H NMR
(CDCl3, 300 MHz): δH=1.43 (t, 3H, J=7.2 Hz); 2.24 (t, 4H, J=4.8 Hz);
2.78 (t, 2H, J=6.9 Hz); 3.11 (t, 4H, J=4.8 Hz); 3.50 (t, 2H, J=6.9 Hz);
4.35 (q, 2H, J=7.2 Hz); 7.20-7.80 (m, 6H); 8.05 (dd, 1H, J1=8.4 Hz,
J2=1.5 Hz); 8.55 (s, 1H) ppm. 13C NMR (CDCl3, 75 MHz): δC=14.38,
42.79, 53.90, 55.49, 60.93, 62.01, 109.72, 122.44, 124.57, 124.73, 125.36,
129.15, 131.00, 132.08, 138.77, 142.73, 154.62, 167.00 ppm. LC-MS ESIMS:
m/z 380.3 [M+H]+. Anal Calc for C22H25N3O3: C, 69.82%; H, 6.92%;
N, 14.80%. Found: C, 69.62%; H, 6.97%; N, 14.95%.
|This compound was obtained as light brown powder. Yield: 89%.
1H NMR (CDCl3, 300 MHz): δH=1.44 (t, 3H, J=7.2 Hz); 2.21 (t, 4H,
J=4.8 Hz); 2.76 (t, 2H, J=6.9 Hz); 3.10 (t, 4H, J=4.8 Hz); 3.48 (t, 2H,
J=6.9 Hz); 4.34 (q, 2H, J=7.2 Hz); 6.85 (d, 2H, J=8.4 Hz); 7.39 (d,
2H, J=8.4 Hz); 7.87 (d, 1H, J=8.4 Hz); 8.01 (dd, IH, J1=8.4 Hz, J2=1.5
Hz); 8.54 (s, 1H) ppm. 13C NMR (CDCl3, 75 MHz): δC=14.38, 24.54,
42.79, 51.76, 53.85, 57.50, 61.12, 109.97, 122.49, 125.57, 125.82, 127.35,
127.76, 129.63, 130.03, 138.48, 142.50, 154.19, 167.05 ppm. LC-MS ESIMS:
m/z 396.3 [M+H]+. Anal Calc for C22H25N3O3: C, 66.99%; H, 6.64%;
N, 14.20%. Found: C, 66.75%; H, 6.84%; N, 14.33%.
|This compound was obtained as brown oil. Yield: 77%. 1H NMR
(CDCl3, 300 MHz): δH=1.44 (t, 3H, J=7.2 Hz); 2.23 (t, 4H, J=4.8 Hz);
2.45 (s, 3H); 2.77 (t, 2H, J=6.9 Hz); 3.11 (t, 4H, J=4.8 Hz); 3.49 (t, 2H,
J=6.9 Hz); 4.36 (q, 2H, J=7.2 Hz); 6.87 (d, 2H, J=8.4 Hz); 7.39 (d, 2H,
J=8.4 Hz); 7.88 (d, 1H, J=8.4 Hz); 8.01 (dd, 1H, J1= 8.4 Hz, J2=1.5 Hz);
8.55 (s, 1H) ppm. 13C NMR (CDCl3, 75 MHz): δC=14.38, 42.80, 51.75,
53.90, 57.54, 61.12, 109.97, 116.49, 118.73, 122.49, 124.30, 126.41,
128.50, 129.63, 130.06, 138.49, 142.48, 154.17, 159.07, 167.00 ppm. LCMS
ESI-MS: m/z 394.3 [M+H]+. Anal Calc for C22H25N3O3: C, 70.38%;
H, 7.19%; N, 14.27%. Found: C, 70.08%; H, 7.40%; N, 14.26%.
|This compound was obtained as light brown powder. Yield: 86%. 1H
NMR (CDCl3, 300 MHz): δH=1.43 (t, 3H, J=7.2 Hz); 2.21 (t, 4H, J=4.8
Hz); 2.75 (t, 2H, J=6.9 Hz); 3.09 (t, 4H, J=4.8 Hz); 3.48 (t, 2H, J=6.9
Hz); 3.87 (s, 3H); 4.36 (q, 2H, J=7.2 Hz); 6.86 (d, 2H, J=8.4 Hz); 7.37 (d,
2H, J=8.4 Hz); 7.80 (d, 1H, J=8.4 Hz); 8.00 (dd, 1H, J1=8.4 Hz, J2=1.5
Hz); 8.53 (s, 1H) ppm. 13C NMR (CDCl3, 75 MHz): δC=14.39, 42.81,
51.75, 53.90, 56.19, 57.54, 61.12, 110.04, 116.52, 118.73, 122.49, 124.30,
126.41, 128.50, 129.65, 130.06, 138.49, 142.48, 154.16, 159.33, 167.02
ppm. LC-MS ESI-MS: m/z 410.3 [M+H]+. Anal Calc for C22H25N3O3: C,
67.63%; H, 6.91%; N, 13.72%. Found: C, 67.50%; H, 7.02%; N, 13.86%.
|This compound was obtained as yellow oil. Yield: 85%. 1H NMR
(CDCl3, 300 MHz): δH=1.43 (t, 3H, J=7.2 Hz); 2.31 (t, 4H, J=4.8 Hz);
2.73 (t, 2H, J=6.9 Hz); 2.95 (t, 4H, J=4.8 Hz); 4.40 (q, 2H, J=7.2 Hz);
4.41 (t, 2H, J=6.9 Hz); 7.48 (d, 1H, J=8.4 Hz); 7.82 (d, 2H, J=8.4 Hz),
7.97 (d, 2H, J=8.4 Hz), 8.09 (dd, 1H, J1=8.4 Hz, J2=1.5 Hz); 8.55 (s, 1H)
ppm. 13C NMR (CDCl3, 75 MHz): δC=14.39, 42.76, 51.75, 53.87, 57.48,
61.10, 109.97, 122.49, 124.91, 125.57, 125.82, 125.85, 125.88, 129.73,
130.00, 138.68, 142.50, 154.16, 167.03 ppm. LC-MS ESI-MS: m/z 448.2
[M+H]+. Anal Calc for C23H25N4O2F3: C, 61.87%; H, 5.64%; N, 12.55%.
Found: C, 61.75%; H, 5.38%; N, 12.79%.
|Results and Discussion
|The sequence for the formation of the novel benzimidazole
derivatives is proposed and summarized in Scheme 1.
|Our synthetic study into polar benzimidazoles started with
4-fluoro-3-nitro benzoic acid which was esterified in the presence of
catalytic sulfuric acid in ethanol by refluxing for 8 hours to afford the
ethyl-4-fluoro-3-nitrobenzoate 1 in 75% yield. The ethylbenzoate 1.
was then treated with N-(3-aminopropyl) piperazine and DIPEA in
dry dichloromethane at room temperature to yield 2. However, our
initial effort to synthesize ethyl 4-(2-(piperazin-1-yl) ethylamino)-3-
nitrobenzoate 2 gave only 40% yield. The low yield of the product 2
led us to probe the reaction. Upon further analyses, we identified two
side products from the reaction which were ethyl 4-(4-(2-aminoethyl)
piperazin-1-yl)-3-nitrobenzoate, 2a and the diester product, 2b (Scheme
|This clearly showed that the reaction between a fluoro phenyl and
amine happened at a very fast rate where no complete selectivity for
primary amine over secondary amine was observed. This indirectly
also implied that the probability of collision between the ethyl ester 1
and the reactant will determine the product(s) formed. Optimization
to increase the yield of 2 was carried out and the results are presented
in Table 1. No further efforts were made to determine the kinetics of
|The amino compound 2 was reduced to ethyl 4-(2-(piperazin-1-yl)
ethylamino-3-aminobenzoate 3 using ammonium formate and 10%
Pd/C for 1 hour to give 85% yield. We also tested the reduction reaction
using sodium borohydride. However, comparatively, the yield obtained
from NaBH4 was far lower (57%). This proved that palladium-catalysed
transfer hydrogenation is an excellent method in reducing nitrobenzene
to aminobenzene. This method is convenient, economical and uses
a stable nonpyrophobic catalyst. The phenylenediamine 3 was then
refluxed with various substituted bisulfite adduct of aromatic aldehydes 4a-e in DMF overnight to afford benzimidazole derivatives 5a-e in
good to excellent yields. Among the literature reports available for the
synthesis of benzimidazoles by the reaction of phenylenediamine with
acid chloride , aldehyde  and acid , we found that access
into benzimidazole derivatives via this metabisulfite route is efficient,
environmental friendly and afforded good yield of the benzimidazoles.
|The 1H NMR spectrum of benzimidazole 5a showed a singlet at
δ 1.43 ppm due to the -CH3 from the ethyl group. The N-methylene
protons on position-2 connected to the piperazine ring appeared as a
double triplet at δ 2.78 and 3.50 ppm while the N-methylene protons
from the piperazine also appeared as a double triplet at δ 2.24 and 3.11
ppm. The O-methylene protons (from the ester group) appeared as
a quartet at δ 4.35 ppm. Similar 1H patterns were obtained for other
substituted benzimidazoles derivatives 5b-e. The 13C NMR spectrum of
5a which resonated at δ 154.62 and 167.00 ppm are assigned to imine
(C=N) and ester carbonyl carbon respectively.
|The novel benzimidazole derivatives were subsequently assayed
for their cholinesterase inhibition potency by Ellman’s method .
Results for their acetylcholinesterase (AChE) inhibition potentials are
shown in Table 2. Rivastigmine was used as reference in the assays. We
observed that electron withdrawing substituents at the R position in the
phenyl ring are important for good activities as shown by 5e. The best
inhibition was achieved by 5e with IC50 of 31.40 μM which was better
than the standard drug rivastigmine.
|LogP/CLogP values  of the newly synthesized compounds
5a-e are shown in Table 3. Basically all of them fall in good range for
prediction of drug activity and moderate toxicity. The tolerable toxicity
of the compounds 5a-e was confirmed by the cytotoxicity test (IC50) in
VERO cells at concentrationsup to 50 μM. After 72 hours of exposure,
viability was assessed on the basis of cellular conversion of MTS into
a formazan product using the Promega Cell Titer 96 Non-radioactive
Cell proliferation method according to manufacturer’s protocol. All the compounds were found to be non-toxic up to 50 μM.
|A series of novel polar benzimidazoles was successfully synthesized under mild reaction condition in good to excellent yield. Piperazinyl
ethylbenzimidazole derivatives were derived from ethyl 4-(2-(piperazin-
1-yl) ethylamino-3-aminobenzoate with various substituted bisulfite
adduct of benzaldehyde under reflux conditions. The synthesized
novel polar benzimidazoles have potential biological applications
such as therapeutics for Alzheimer’s disease in view of their good
bioavailability. The bioactivity studies as well as quantitative structureactivity
relationship of the newly synthesized polar benzimidazoles are
on-going in our laboratory and would be published in the future.
|The authors wish to express their gratitude and appreciation to
Pharmacogenetics and Novel Therapeutics Research Cluster, Institute for
Research in Molecular Medicine, Universiti Sains Malaysia, Penang for supporting
this work. This work was funded through Research Grant No.RUC (1001/
PSK/8620012) and HiCoE research Grant No (311.CIPPM.4401005).
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- Values derived from ChemDraw Ultra 11.0.1, CambridgeSoft Corporation, Cambridge.