Lipase-Catalyzed Kinetic Resolution of Novel Antifungal N-Substituted Benzimidazole Derivatives

Article abstract of DOI:10.1002/chir.22591

A series of new N-substituted benzimidazole derivatives was synthesized and their antifungal activity against Candida albicans was evaluated. The chemical step included synthesis of appropriate ketones containing benzimidazole ring, reduction of ketones to the racemic alcohols, and acetylation of alcohols to the esters. All benzimidazole derivatives were obtained with satisfactory yields and in relatively short times. All synthesized compounds exhibit significant antifungal activity against Candida albicans 900028 ATCC (% cell inhibition at 0.25 μg concentration > 98%). Additionally, racemic mixtures of alcohols were separated by lipase-catalyzed kinetic resolution. In the enzymatic step a transesterification reaction was applied and the influence of a lipase type and solvent on the enantioselectivity of the reaction was studied. The most selective enzymes were Novozyme SP 435 and lipase Amano AK from Pseudomonas fluorescens (E > 100).

Full text of DOI:10.1002/chir.22591

CHIRALITY 28:347–354 (2016)
Lipase-Catalyzed Kinetic Resolution of Novel Antifungal N-Substituted
Benzimidazole Derivatives
1
2
2
3,4
*
EDYTA ŁUKOWSKA-CHOJNACKA, MONIKA STANISZEWSKA, MAŁGORZATA BONDARYK, JAN K. MAURIN, AND
MARIA BRETNER1
1Faculty of Chemistry, Institute of Biotechnology, Warsaw University of Technology, Warsaw, Poland
2Independent Laboratory of Streptomyces and Fungi Imperfecti, National Institute of Public Health-National Institute of Hygiene, Warsaw, Poland
3National Centre for Nuclear Research, Otwock, Poland
4National Medicines Institute, Warsaw, Poland
ABSTRACT
A series of new N-substituted benzimidazole derivatives was synthesized and
their antifungal activity against Candida albicans was evaluated. The chemical step included syn-
thesis of appropriate ketones containing benzimidazole ring, reduction of ketones to the racemic
alcohols, and acetylation of alcohols to the esters. All benzimidazole derivatives were obtained
with satisfactory yields and in relatively short times. All synthesized compounds exhibit signifi-
cant antifungal activity against Candida albicans 900028 ATCC (% cell inhibition at 0.25 μg con-
centration > 98%). Additionally, racemic mixtures of alcohols were separated by lipase-
catalyzed kinetic resolution. In the enzymatic step a transesterification reaction was applied
and the influence of a lipase type and solvent on the enantioselectivity of the reaction was stud-
ied. The most selective enzymes were Novozyme SP 435 and lipase Amano AK from Pseudomo-
nas fluorescens (E > 100). Chirality 28:347–354, 2016. © 2016 Wiley Periodicals, Inc.
KEY WORDS: azole; benzimidazole; enzyme; lipase; enzymatic transesterification; kinetic
resolution; antifungal activity
Benzimidazole derivatives exhibit high biological potential
and are widely used in veterinary and human medicine.1–3
Many of them possess antibacterial, antifungal, antiviral,
antiallergic, antioxidant, analgesic, antiinflammatory, antihy-
pertensive, and antidiabetic activity.4–6 Moreover, a benzimid-
azole scaffold is present in many compounds with antitumor
activity against leukemia, melanoma, breast adenocarcinoma
(MCF-7), hepatocellular carcinoma, colon carcinoma, and
other cancer cell lines.7–11 The chemical synthesis of various
biologically active benzimidazole derivatives are widely pub-
lished, but to the best of our knowledge the kinetic resolution
of alcohols containing a benzimidazole moiety is described
only in one article.12 Taking into account that the stereochem-
istry of the compounds often determines their activity,13,14
studies on the synthesis of pure enantiomers is of interest to
the modern pharmaceutical industry. Especially valuable is
enzymatic catalysis: an environmentally friendly, efficient,
and not expensive method. Moreover, a unique feature of
enzymes like broad substrate specificities, chemoselectivity,
regioselectivity, and enantioselectivity make this method very
attractive.15 Reactions can be carried out in the presence of
isolated enzymes or whole cells of microorganisms, which
allows simplification of the synthesis of many commercially
available drugs like simvastatin, atorvastatin, pregabalin, parox-
etine, and levetiracetam.16 Moreover, in enzyme-catalyzed
reactions numerous chiral intermediates, used as building
blocks in the synthesis of important drugs with antianxiety, an-
tidiabetic, antiviral, anticancer, anti-infective, antihypertensive,
and anticholesterol activity were obtained.17–20 Stereoselective
biotransformations are also applied in the synthesis of numer-
ous enzyme inhibitors, receptor antagonists, and other biolog-
ically important molecules.17,18 Among all classes of enzymes,
noteworthy are hydrolases, especially lipases, that are used in
chemoenzymatic synthesis of various pharmaceuticals like
(3S, 4R)-(À)-paroxetine, (S)-(+)-citalopram, (S)-(+)-zopiclone,
© 2016 Wiley Periodicals, Inc.
(S)-dapoxetine, (R)-ramatroban, (S)-rivastigmine, (R)-(+) or
(S)-(À)-promethazine, (R)-(+) or (S)-(À)-ethopropazine, non-
steroidal antiinflammatory drugs, and others.21–26 The synthe-
sis of all these pharmaceuticals was performed by means of a
lipase-catalyzed kinetic resolution of corresponding racemic
mixtures of alcohols ((3S, 4R)-(À)-paroxetine, (S)-(+)-
citalopram, (S)-(+)-zopiclone, (R)-(+) or (S)-(À)-promethazine,
(R)-(+) or (S)-(À)-ethopropazine, (R)-ramatroban, (S)-
rivastigmine), amines ((S)-rivastigmine), β-amino esters ((S)-
dapoxetine) or acids (NSAIDs). This technique was also ap-
plied for resolution of other biologically interesting racemic
mixtures of alcohols and esters containing a heterocyclic moi-
ety, like: indole, benzothiazole, benzoxazole, benzotriazole,
and tetrazole.27–31
Taking into account the biological importance of benzimid-
azole derivatives, we describe our research on the synthesis
of new N-substituted benzimidazole derivatives including
ketones, alcohols, and esters and evaluation of their antifun-
gal activity against Candida albicans 900028 ATCC. The race-
mic mixtures of alcohols were separated in lipase-catalyzed
transesterification and the influence of a lipase type and
solvent on the enantioselectivity of the reaction was studied.
MATERIALS AND METHODS
Chemicals and Reagents
All solvents and chemicals were purchased from POCH (Poland), Merck
(Germany), and Sigma-Aldrich (Germany) and were used without further
Contract grant sponsor: National Science Centre-Poland; Contract grant num-
ber: DEC-2011/03/D/NZ7/06198.
*Correspondence to: E. Łukowska-Chojnacka, Faculty of Chemistry, Institute
of Biotechnology, Warsaw University of Technology, Noakowskiego St. 3, 00-
664 Warsaw, Poland. E-mail: elukowska@ch.pw.edu.pl
Received for publication 4 December 2015; Accepted 29 January 2016
DOI: 10.1002/chir.22591
Published online 29 February 2016 in Wiley Online Library
(wileyonlinelibrary.com).

348
ŁUKOWSKA-CHOJNACKA ET AL.
purification. Amano AK (native lipase from Pseudomonas fluorescens), Amano
PS (lipase from Burkholderia cepacia, earlier Pseudomonas cepacia), Amano
PS-IM (lipase from Burkholderia cepacia immobilized on diatomite) were
purchased from Sigma-Aldrich (Germany). Lipozyme (lipase from Mucor
miehei) and Novozyme SP 435 (lipase from Candida antarctica-B
200.98. HRMS [M+H]+ m/z calcd for C11H13N2O+ 189.1028, found
189.0733.
Synthesis of 4-(1H-benzimidazol-1-yl)butan-2-one (4c) and
4-(2-methyl-1H-benzimidazol-1-yl)butan-2-one (4d)
immobilized on
a macroporous acrylic resin) were purchased from
A solution of appropriate benzimidazole 1–2 (50.8 mmol), methyl vinyl
ketone (8.5 mL, 101.6 mmol), and Et3N (8.5 mL, 101.6 mmol) in 2-
propanol (150 mL) was stirred at reflux. The progress of the reaction
was monitored by TLC with chloroform:methanol (9:1 v/v) as the eluent.
After the appropriate time (Table 1), the reaction mixture was cooled to
room temperature and the solvent was evaporated under reduced pres-
sure. The product was purified on a column chromatography on silica
gel with chloroform:methanol (15:1 v/v) as the eluent.
Novozyme (Denmark). The 4,5,6,7-tetrabromo-1H-benzimidazole was ob-
tained by bromination of benzimidazole according to the previously reported
method.32
Strains and Growth Conditions
Candida albicans quality control strain ATCC90028 was obtained from
the American Type Culture Collection (Rockville, MD).33 Prior to the re-
spective examinations, the strain was stored on ceramic beads in
Microbank tubes at À70 °C. Routine culturing of strain for growth was
conducted at 30 °C for 18 h in YEPD.34
4-(1H-benzimidazol-1-yl)butan-2-one (4c). Yield 82%, colorless
crystals, m.p. 62–64 °C. IR (nujol, cmÀ1): 1707 (C = O), 1605 (C = N).
1H NMR (CHCl3, 400 Hz,) δ ppm: 2.11 (s, 3H, CH3), 2.96–2.99 (m, 2H,
CH2CO), 4.42–45 (m, 2H, CH2N), 7.25–7.29 (m, 2H, Ar), 7.35–7.37 (m,
1H, Ar), 7.77–7.79 (m, 1H, Ar), 7.96 (s, 1H, CH). 13C NMR (CDCl3,
100 Hz) δ ppm: 30.25, 38.89, 42.72, 109.15, 120.32, 122.05, 122.82,
133.16, 143.42, 143.54, 204.91. HRMS [M+H]+ m/z calcd for
C11H13N2O+ 189.10.28, found 189.0922.
Apparatus and Materials
1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded
on a Varian Mercury 400 MHz spectrometer (Palo Alto, CA) in CDCl3
or DMSO-d6 solution; chemical shifts (δ) are reported in ppm; coupling
constants (J) values are given in hertz (Hz). IR spectra were recorded
on a Carl Zeiss Specord M80 instrument (Germany). HRMS spectra were
recorded on a Micro-mass ESI Q-ToF Premier instrument (Micromass
Manchester UK). The Xcalibur-R κ-axis single crystal diffractometer
equipped with a CCD detector from Oxford Diffraction was used to collect
X-ray diffraction data. Graphite monochromatized CuKα radiation was
applied in experiments. Enantiomeric excess (ee) of the alcohols 5a,d,
e and ester 6e were determined on a Shimadzu HPLC apparatus (Japan)
consisting of an LC-20 AD chromatograph, CTO-10AS oven, SPD-20 A UV
detector, and Chiralcel OD-H column (in hexane:2-propanol 9:1; 0.8 mL/
min λ = 225 nm for alcohols 5a,e and ester 6e, hexane:ethanol 99:1;
1.2 mL/min for alcohol 5d) using the corresponding racemic com-
pounds as references. The retention times (tR/min) were for alcohols:
5a 17.72 (S) and 24.15 (R), 5d 96.30 (S) and 101.73 (R), 5e 30.86 (S)
and 38.86 (R), and for ester 6e 58.92 (S) and 75.51 (R). Optical rotations
were measured with an ATAGO AP-300 automatic polarimeter (Japan).
Melting points were determined in open capillaries and are uncorrected.
The reaction progress was monitored by thin-layer chromatography
(TLC) aluminum plates with silica gel Kieselgel 60F254 (0.2 mm thick-
ness film, Merck, Germany) using UV light for visualization. Column
chromatography was performed using Kieselgel 60 (0.040–0.063 mm,
Merck, Germany).
4-(2-methyl-1H-benzimidazol-1-yl)butan-2-one (4d). Yield 73%,
colorless crystals, m.p. 68–70 °C. IR (nujol, cmÀ1): 1705 (C = O), 1620
(C = N). 1H NMR (CHCl3, 400 Hz) δ ppm: 2.13 (s, 3H, CH3), 2.63 (s,
3H, CH3), 2.92–2.96 (m, 2H, CH2CO), 4.36–4.39 (m, 2H, CH2N), 7.21–
7.23 (m, 2H, Ar), 7.27–7.28 (m, 1H, Ar), 7.65–7.68 (m, 1H, Ar), 13C
NMR (CDCl3, 100 Hz) δ ppm: 13.76, 30.27, 37.95, 42.36, 108.97, 119.05,
122.04, 122.15, 134.45, 142.40, 151.52, 205.32. HRMS [M+H]+ m/z calcd
for C12H15N2O+ 203.1184, found 203.1724.
Synthesis of 4-(4,5,6,7-tetrabromo-1H-benzimidazol-1-yl)
butan-2-one (4e)
A solution of benzimidazole 3 (3 g, 6.9 mmol) and methyl vinyl ketone
(1.44 mL, 18 mmol) in acetone (180 mL) was stirred at reflux and the
progress of the reaction was monitored by TLC with chloroform:methanol
(9:1 v/v) as the eluent. After 120 h the product was filtered off and the sol-
vent was evaporated under reduced pressure. The residue and the earlier
obtained solid were recrystallized from methanol.
4-(4,5,6,7-tetrabromo-1H-benzimidazol-1-yl)butan-2-one (4e).
Yield 66%, colorless crystals, m.p. 177–179 °C. IR (nujol, cmÀ1): 1705
(C = O), 1580 (C = N). 1H NMR (CHCl3, 400 Hz) δ ppm: 2.14 (s, 3H,
CH3), 3.04–3.07 (m, 2H, CH2CO), 4.74–4.77 (m, 2H, CH2N), 8. 09 (s,
1H, CH). 13C NMR (CDCl3, 100 Hz) δ ppm: 30.27, 41.12, 44.90, 105.76,
117.61, 121.88, 123.59, 130.87, 143.96, 147.78, 204.51. HRMS [M+H]+
m/z calcd for C11H9Br4N2O+ 504.7407, found 504.7268.
Synthesis of 1-(1H-benzimidazol-1-yl)propan-2-one (4a)
and 1-(2-methyl-1H-benzimidazol-1-yl)propan-2-one (4b)
A solution of appropriate benzimidazole 1–2 (50 mmol), chloroacetone
(4 mL, 50 mmol), and Et3N (5 mL, 55 mmol) in acetone (100 mL) was
stirred at reflux. The progress of the reaction was monitored by TLC
using chloroform:methanol (9:1 v/v) as the eluent. After 24 h, the reac-
tion mixture was cooled to room temperature and the solvent was evapo-
rated. The product was purified by a column chromatography on silica gel
with chloroform:methanol (15:1 v/v) as the eluent.
General Procedure for Reduction of Ketones 4a-e to
Alcohols ( )-5a-e
To the cooled and stirred mixture of appropriate ketone 4a-e (20 mmol)
in methanol (90 mL), sodium borohydride (1.52 g, 40 mmol) was added
portionwise and the reaction mixture was stirred at room temperature.
The progress of the reaction was monitored by TLC, using chloroform:
methanol (9:1 v/v) as the eluent. After 24 h the solvent was evaporated
and 80 mL of H2O was added. The alcohol 5a-e was extracted with
CH2Cl2 (4 x 50 mL) and the organic layer was washed with water (3 x
50 mL), dried over anhydrous MgSO4, and evaporated.
1-(1H-benzimidazol-1-yl)propan-2-one (4a). Yield 54%, colorless
crystals, m.p. 120–122 °C. IR (nujol, cmÀ1): 1718 (C = O), 1610 (C = N).
1H NMR (CHCl3, 400 Hz) δ ppm: 2.17 (s, 3H, CH3), 4.88 (s, 2H, CH2),
7.19–7.21 (m, 1H, Ar), 7.28–7.30 (m, 2H, Ar), 7.81–7.83 (m, 1H, Ar),
7.87 (s, 1H, CH). 13C NMR (CDCl3, 100 Hz) δ ppm: 26.98, 53.82, 108.98,
120.43, 122.44, 123.37, 133.67, 143.15, 143.24, 200.71. HRMS [M+H]+
m/z calcd for C10H11N2O+ 175.0871, found 175.0603.
( )-1-(1H-benzimidazol-1-yl)propan-2-ol (5a). Yield 71%, colorless
crystals, m.p. 72–74 °C. IR (nujol, cmÀ1): 3230 (OH), 1610 (C = N). 1H
NMR (CHCl3, 400 Hz) δ ppm: 1.32 (d, 3H, CH3, J = 6.4 Hz), 3.96 (dd,
1H, CHaHb, JHaCH = 8.4 Hz, JHaHb = 14.4 Hz), 4.11 (dd, 1H, CHaHb,
JHbCH = 2.8 Hz), 4.21–4.25 (m, 1H, CH), 4.84 (s, 1H, OH), 7.03–7.07 (m
1H, Ar), 7.15–7.19 (m, 1H, Ar), 7.30–7.38 (m, 2H, Ar), 7.66 (s, 1H, CH).
13C NMR (CDCl3, 100 Hz) δ ppm: 20.79, 52.67, 65.57, 109.53, 119.29,
121.98, 122.69, 133.36, 142.38, 143.25. HRMS [M+H]+ m/z calcd for
1-(2-methyl-1H-benzimidazol-1-yl)propan-2-one (4b). Yield 52%,
colorless crystals, m.p. 148–150 °C. IR (nujol, cmÀ1): 1715 (C = O),
1605 (C = N). 1H NMR (CHCl3, 400 Hz) δ ppm: 2.15 (s, 3H, CH3), 2.49
(s, 3H, CH3C = N) 4.78 (s, 2H, CH2), 7.08–7.10 (m 1H, Ar), 7.21–7.24
(m, 2H, Ar), 7.69–7.71 (m, 1H, Ar). 13C NMR (CDCl3, 100 Hz) δ ppm:
13.78, 27.09, 52.87, 108.30, 119.21, 122.23, 122.44, 134.94, 142.28. 151.43,
C
10H13N2O+ 177.1028, found 177.0733.
Chirality DOI 10.1002/chir

KINETIC RESOLUTION OF BENZIMIDAZOLE DERIVATIVES
349
( )-1-(2-methyl-1H-benzimidazol-1-yl)propan-2-ol (5b). Yield 87%,
colorless crystals, m.p. 188–190 °C. IR (nujol, cmÀ1): 3130 (OH), 1612
(C = N). H NMR (CHCl3, 400 Hz) δ ppm: 1.38 (d, 3H, CH3, J = 6.4 Hz),
2.47 (s, 3H, CH3C = N), 3.97 (dd, 1H, CHaHb, JHaCH = 8.4 Hz,
JHaHb = 14.4 Hz), 4.04 (dd, 1H, CHaHb, JHbCH = 3.6 Hz), 4.28–4.34 (m,
1H, CH), 6.99–7.03 (m 1H, Ar), 7.09–7.19 (m, 1H, Ar), 7.20–7.28 (m,
2H, Ar). 13C NMR (CDCl3, 100 Hz) δ ppm: 14.11, 20.97, 51.54, 65.79,
109.15, 118.20, 121.92, 134.57, 141.24, 152.00. HRMS [M+H]+ m/z calcd
for C11H15N2O+ 191.1184, found 191.1236.
(m, 1H, Ar), 7.92 (s, 1H, CH). 13C NMR (CDCl3, 100 Hz) δ ppm: 17.64,
21.11, 48.94, 68.78, 109.61, 120.19, 122.28, 123.14, 133.84, 143.17, 169.93,
174.07. HRMS [M+H]+ m/z calcd for C12H15N2O2+ 219.1134, found
219.0617.
1
(À)-(R)-4-(1H-benzimidazol-1-yl)butan-2-yl acetate (6c). Yield
79%, oil. [α]2D6.3 = À3.97 (c 3.67 in MeOH, ee = nd%). IR (film, cmÀ1):
1730 (C = O), 1605 (C = N). 1H NMR (CHCl3, 400 Hz) δ ppm: 1.26 (d, 3H,
CH3, J = 6.4 Hz), 2.015 (s, 3H, CH3CO), 2.10–2.20 (m, 2H, CH2), 4.23–
4.26 (m, 2H, CH2N), 4.96–5.99 (m, 1H, CH), 7.29 (m, 2H, Ar), 7.37–7.39
(m, 1H, Ar), 7.81–7.83 (m, 1H, Ar), 8.01 (brs, 1H, CH). 13C NMR (CDCl3,
100 Hz) δ ppm: 20.11, 21.14, 35.82, 41.73, 68.38, 109.50, 120.27, 122.45,
123.17, 133.40, 142.63, 170.54. HRMS [M+H]+ m/z calcd for
C13H17N2O+2 233.1290, found 233.0756.
( )-4-(1H-benzimidazol-1-yl)butan-2-ol (5c). Yield 87%, colorless
crystals, m.p. 106–108 °C. IR (nujol, cmÀ1): 3250 (OH), 1602 (C = N).
1H NMR (CHCl3, 400 Hz) δ ppm: 1.21 (d, 3H, CH3, J = 6 Hz), 1.87–1.93
(m, 1H, CH2), 1.97–2.03 (m, 1H, CH2), 3.65–3.69 (m, 2H, CH, OH), 4.30
(ddd, 1H, CHaHbN, J = 4.4 Hz, J = 6.8 Hz, J = 14 Hz), 4.44 (ddd, 1H,
CHaHbN, J = 6.4 Hz, J = 9.6 Hz), 7.25–7.29 (m, 2H, Ar), 7.43–7.46 (m,
1H, Ar), 7.77–7.79 (m, 1H, Ar), 7.95 (s, 1H, CH). 13C NMR (CDCl3,
100 Hz) δ ppm: 24.30, 38.18, 41.74, 63.71, 109.74, 119.92, 122.07, 122.82,
133.46, 143.19, 143.23. HRMS [M+H]+ m/z calcd for C11H15N2O+
191.1184, found 192.1287.
(À)-(R)-4-(2-methyl-1H-benzimidazol-1-yl)butan-2-yl
acetate
(6d). Yield 60%, colorless crystals, m.p. 80–82 °C. [α]2D4.5 = À1.66 (c
3.02 in MeOH, ee = 43%). IR (nujol, cmÀ1): 1731 (C = O), 1610 (C = N).
1H NMR (CHCl3, 400 Hz) δ ppm: 1.27 (d, 3H, CH3, J = 6 Hz), 1.95–2.08
(m, 2H, CH2), 2.02 (s, 3H, CH3CO), 2.59 (s, 3H, CH3C = N), 4.14 (t,
2H, CH2N, J = 7.6 Hz), 4.95–4.99 (m, 1H, CH), 7.20–7.25 (m, 3H, Ar),
7.66–7.69 (m, 1H, Ar). 13C NMR (CDCl3, 100 Hz) δ ppm: 13.65, 20.08,
21.13, 35.50, 40.31, 68.58, 108.85, 119.04, 121.97, 122.13, 134.67, 142.33,
151.05, 170.49. HRMS [M+H]+ m/z calcd for C14H19N2O+2 247.1447,
found 247.0987.
( )-4-(2-methyl-1H-benzimidazol-1-yl)butan-2-ol (5d). Yield 87%,
colorless crystals, m.p. 83–85 °C. IR (nujol, cmÀ1): 3170 (OH), 1610
1
(C = N). H NMR (CHCl3, 400 Hz) δ ppm: 1.23 (d, 3H, CH3, J = 6.4 Hz),
1.82–1.86 (m, 1H, CH2), 1.93–1.98 (m, 1H, CH2), 2.60 (s, 3H, CH3), 3.11
(brs, 1H, OH), 3.76–3.81 (m, 1H, CH), 4.23–4.31 (m, 2H, CH2N), 7.19–
7.22 (m, 2H, Ar), 7.34–7.37 (m, 1H, Ar), 7.64–7.67 (m, 1H, Ar). 13C
NMR (CDCl3, 100 Hz) δ ppm: 13.64, 24.23, 38.35, 40.51, 64.40, 109.33,
118.69, 121.86, 122.01, 134.90, 142.09, 151.61. HRMS [M+H]+ m/z calcd
for C12H17N2O+ 205.1341, found 205.1875.
(À)-(R)-4-(4,5,6,7-tetrabromo-1H-benzimidazol-1-yl)butan-2-yl ac-
etate (6e). Yield 50%, colorless crystals, m.p. 138–140 °C. [α]D26.5
=
À7.50 (c 0.4 in MeOH, ee = 80%). IR (nujol, cmÀ1): 1711 (C = O), 1580
(C = N). 1H NMR (CHCl3, 400 Hz) δ ppm: 1.28 (d, 3H, CH3, J = 6 Hz),
2.05 (s, 3H, CH3CO), 2.11–2.17 (m, 2H, CH2), 4.47–4.60 (m, 2H, CH2N),
4.98–5.05 (m, 1H, CH), 7.95 (s, 1H, CH). 13C NMR (CDCl3, 100 Hz) δ
ppm: 20.34, 21.32, 38.29, 44.03, 68.10, 106.02, 117.57, 121.97, 123.83,
131.04, 144.00, 146.61, 170.35. HRMS [M+H]+ m/z calcd for
C13H13Br4N2O+2 548.7670, found 548.8654.
( )-4-(4,5,6,7-tetrabromo-1H-benzimidazol-1-yl)butan-2-ol (5e).
Yield 86%, colorless crystals, m.p. 223–225 °C. IR (nujol, cmÀ1): 3340
(OH), 1580 (C = N). 1H NMR (DMSO-d6, 400 Hz) δ ppm: 1.07 (d, 3H,
CH3, J = 6 Hz ), 1.77–1.85 (m, 2H, CH2), 3.56 (brs, 1H, OH), 4.44–4.52
(m, 1H, CH2N), 4.57–4.64 (m, 1H, CH2N), 4.67–4.68 (m, 1H, CH), 8.43
(s, 1H, CH). 13C NMR (DMSO-d6, 100 Hz) δ ppm: 23.70, 40.53, 43.92,
63.14, 106.52, 116.47, 120.27, 122.26, 131.33, 143.65, 148.94. HRMS
[M+H]+ m/z calcd for C11H11Br4N2O+ 506.7564, found 506.7310.
Synthesis of Racemic Esters 6a-e
A mixture of the alcohol 5a-e (2 mmol), N,N′-dicykloheksylokarbodiimid
(DCC, 0.45 g, 2.2 mmol), glacial acetic acid (0.12 mL, 2.2 mmol) and cata-
lytic amount of 4-dimethylaminopyridine (DMAP) in dry CH2Cl2
(100 mL) was stirred at room temperature for the time indicated in Table 3.
The progress of the reaction was monitored by TLC with chloroform:meth-
anol (9:1 v/v) as the eluent. The solid was filtered off and the solvent was
evaporated under reduced pressure. The crude products were purified on
General Procedure for Enzymatic Kinetic Resolution of
Alcohols 5a-e
An alcohol 5a-d (2 mmol) or 5e (0.6 mmol) was dissolved in 30 mL or
18 mL of appropriate solvent (Table 4), respectively. Then, methyl vinyl
ketone 2 mmole (0.184 mL in the case of alcohols 5a-d) or 0.6 mmole
(0.06 mL in the case of alcohol 5e) and molecular sieves 4 Ä (300 mg)
were added. The mixture was stirred on a shaker at 200 rpm at room tem-
perature (20–25 °C). The progress of the reaction was monitored by TLC
with chloroform:methanol (9:1 v/v) as the eluent. After the appropriate
time (Table 4), the molecular sieves and enzyme were filtered off. The
solvent was evaporated under reduced pressure and products were sepa-
rated by column chromatography on silica gel using a mixture of chloro-
form:methanol (20:1 v/v) as the eluent. The specific rotations were
measured in MeOH solution and for enantiomerically enriched alcohols
are as follows:
a
column chromatography on silica gel with chloroform methanol
(20:1 v/v) as the eluent.
( )-1-(2-methyl-1H-benzimidazol-1-yl)propan-2-yl acetate (6b).
Yield 86%, oil. IR (film, cmÀ1): 1735 (C = O), 1610 (C = N). 1H NMR (CHCl3,
400 Hz) δ ppm: 1.32 (d, 3H, CH3, J = 6.4 Hz), 1.91 (s, 3H, CH3CO), 2.65 (s,
3H, CH3C = N), 4.17 (dd, 1H, CHaHb, J = 4.8 Hz, J = 14.8 Hz), 4.25 (dd, 1H,
CHaHb, J = 7.6 Hz), 5.22–5.27 (m, 1H, CH), 7.22–2.25 (m, 2H, Ar), 7.34–7.36
(m, 1H, Ar), 7.66–7.69 (m, 1H, Ar). 13C NMR (CDCl3, 100 Hz) δ ppm: 13.91,
17.82, 20.94, 47.88, 68.83, 109.38, 118.94, 122.20, 122.36, 135.13, 141.94,
151.66, 169.99. HRMS [M+H]+ m/z calcd for C13H17N2O+2 233.1290, found
233.0872.
(+)-(S)-1-(1H-benzimidazol-1-yl)propan-2-ol (5a): [α]2D5 = + 45.06 (c 1.7 in
MeOH, ee = 97%)
Susceptibility Testing of Candida to Benzimidazole
Derivatives 4a-d, 5a-d and 6a-d
(+)-(S)-4-(1H-benzimidazol-1-yl)butan-2-ol (5c): [α]2D6.6 = + 25.00 (c 0.5 in
MeOH, ee = nd%)
(+)-(S)-4-(2-methyl-1H-benzimidazol-1-yl)butan-2-ol (5d): [α]2D5 = + 27.59
(c 1.45 in MeOH, ee = 99%)
Susceptibility was conducted for C. albicans 90028 using the method M27-
A3 (CLSI).33 The final inoculum: from 0.5x102 to 2.5x103 cells/mL saline
was prepared in synthetic RPMI medium (Sigma, St. Louis, MO). The com-
pound test wells (CTW) were prepared with stock solution of the tested
compounds (1600 μg/mL) dissolved in water 33% DMSO. Subsequently,
serial 2-fold dilutions were done with RPMI. Then the fungal blastoconidial
suspension and the compounds (final dilution 1:100) were dispensed into
96-well microplates. The compounds were tested at seven concentrations
that ranged from 0.25 to 16 μg/mL. The DMSO was tested at final concen-
trations ≤0.3% (v/v) in all the experiments. DMSO at these concentrations
(+)-(S)-4-(4,5,6,7-tetrabromo-1H-benzimidazol-1-yl)butan-2-ol (5e): [α]D26.2
=
+ 25.00 (c 0.2 in MeOH, ee = 99%)
(À)-(R)-1-(1H-benzimidazol-1-yl)propan-2-yl acetate (6a). Yield
50%, oil. [α]2D5 = À3.98 (c 2.26 in MeOH, ee = 87%). IR (film, cmÀ1): 1735
(C = O), 1605 (C = N). 1H NMR (CHCl3, 400 Hz) δ ppm: 1.28 (d, 3H,
CH3, J = 6.4 Hz), 1.97 (s, 3H, CH3CO), 4.21–4.31 (m, 2H, CH2), 5.20–5.25
(m, 1H, CH), 7.28–7.32 (m, 2H, Ar), 7.43–7.45 (m, 1H, Ar), 7.79–7.81
Chirality DOI 10.1002/chir

350
ŁUKOWSKA-CHOJNACKA ET AL.
was not able to inhibit the growth of C. albicans cells. Growth control wells
(GCW) (containing medium, inoculum, the same amount of DMSO as used
in CTW, but compound-free) and sterility control wells (SCW) (sample,
medium, and sterile water replacing inoculum) were included in the study.
The optical density (OD) of the plates was determined after 48 h with the In-
finite M200 PRO NANOQuant (Tecan Group, Austria). The endpoint was
calculated as a 100% reduction in OD405 as compared to the growth in the
control well. Reduction of growth for each compound concentration was cal-
culated as follows: % of inhibition = 100 – (OD405 CTW – OD405 SCW)/
(OD405 GCW – OD405 SCW). Amphotericin B (Sigma-Aldrich) was diluted
in DMSO (1600 μg/mL) to be subsequently used in the assay as a reference
antimycotic at the concentration of 0.5 μg/mL (100% cell inhibition). MICs of
the compound were read visually after 18 and 48 h. Two endpoint criteria for
MIC determination were used: 1) total inhibition (MICTI), the lowest
concentration of compounds that yielded no visible growth (a clear well);
and 2) partial inhibition (MICPI), the lowest concentration that produced
a prominent decrease in turbidity compared to that of the drug-free con-
trol (visual assessment).35 All tests were carried out in triplicate.
Scheme 1. Synthesis of ketones 4a-e. Route A: ClCH2COCH3, Et3N,
CH3COCH3, reflux. Route B: CH3COCH=CH2, Et3N, 2-propanol, reflux.
Reactions were performed in the presence of Et3N, in ace-
tone at reflux. The progress of reactions were monitored by
TLC. Ketones 4c-e were obtained by Michael addition of
benzimidazole 1 and its derivatives 2 and 3 to methyl vinyl
ketone (route B, Scheme 1). The synthesis of ketones 4c-d
was carried out in the presence of Et3N at reflux in 2-
propanol, whereas ketone 4e was obtained in the absence
of amine. All products were obtained with satisfactory yields
(Table 1).
Analyzing the results presented in Table 1 it can be con-
cluded that N-alkylation reactions took place in lower yields
than Michael additions. Additionally, in the case of Michael
additions the presence of substituents at the benzimidazole
moiety strongly influences the reaction time. The reactions
were slower and the time extended from 2 h (for ketone
4c) to 120 h (for ketone 4e).
All synthesized ketones 4a-e were reduced (with the use of
NaBH4) to the racemic mixtures of alcohols ( )-5a-e
(Scheme 2). The progress of reactions was monitored on
TLC plates. In all cases the complete conversion of substrate
was reached after 24 h of stirring at room temperature. All al-
cohols were obtained with high yields (Table 2).
The last step of chemical synthesis was an acetylation of all
racemic mixtures of alcohols ( )-5a-e. First, the acetylation of
alcohol 5a was examined, using an excess of acetic anhy-
dride and pyridine. The reaction was carried out at room tem-
perature for 24 h. The product 6a was obtained in moderate
yield, 55%. In order to increase the yield, the reaction was
Assignment of Absolute Configuration of (À)-4-(4,5,6,7-
tetrabromo-1H-benzimidazol-1-yl)butan-2-yl acetate (6e)
Good quality crystal of dimensions 0.49 × 0.13 × 0.03 mm was
mounted on the diffractometer. After initial determination of unit cell
constants the suitable strategy of data collection was chosen. To enable
determination of an absolute structure all Friedel pairs were measured.
In all, 15,399 reflections were collected of which 3090 were classified as
independent after merging. Data were corrected for Lorenz-polarization
effects and for absorption and then were used for solving crystal struc-
ture. To solve the structure the direct methods from the SHELXS-97
program were used.36 The obtained model was then refined anisotrop-
ically by the least-squares treatment using SHELXL-97 software.36 Dur-
ing the refinement correctness of the molecule configuration was
verified using the Flack parameter37: the near-zero value (À0.01(3))
correspond to a proper absolute structure and hence a proper enantio-
mer of the compound. The conformation of the molecule is shown in
Figure 1. The detailed structural data can be found under number
CCDC 1436634.38
RESULTS AND DISCUSSION
Herein we described our studies on the chemoenzymatic
synthesis of various N-substituted benzimidazole deriva-
tives. The starting ketones were synthesized in two types
of reactions: N-alkylation and Michael type addition.
Ketones 4a-b were obtained by N-alkylation of benzimid-
azole 1 and its derivative 2 with chloroacetone (route A,
Scheme 1).
TABLE 1. The reaction times and yields of synthesis of ketones
4a-e
Compound
R
X
n
Time (h)
Yield (%)
4a
4b
4c
4d
4e
H
CH3
H
CH3
H
H
H
H
H
Br
1
1
2
2
2
24
24
2
24
120
54
52
82
73
66
Fig. 1. Conformation of the (À)-(R)-4-(4,5,6,7-tetrabromo-1H-benzimidazol-
1-yl)butan-2-yl acetate ((À)-(R)-6e) with the numbering scheme. The
nonhydrogen atoms are shown as 30% probability ellipsoids.
Scheme 2. Synthesis of racemic mixtures of alcohols ( )-5a-e and esters
( )-6a-e.
Chirality DOI 10.1002/chir

KINETIC RESOLUTION OF BENZIMIDAZOLE DERIVATIVES
351
TABLE 2. The reaction times and yields of synthesis of alco-
hols ( )-5a-e
Alcohol
R
X
n
Time (h)
Yield (%)
5a
5b
5c
5d
5e
H
CH3
H
CH3
H
H
H
H
H
Br
1
1
2
2
2
24
24
24
24
24
71
87
87
87
86
Scheme 3. Lipase-catalyzed kinetic resolution of racemic mixtures of alco-
hols ( )-5a-e.
of Novozyme SP 435 and lipase Amano AK differ significantly
and depend on the solvent. The transesterification of alcohol
( )-5a catalyzed by Novozyme SP 435 took place in a shorter
time and with lower enantioselectivity, in comparison to the
reaction carried out in the presence of Amano AK, which is
the most evident in the case of TBME and toluene. The reaction
time extended from 48 h (reactions catalyzed by Novozyme
SP 435) to 120 h (reactions catalyzed by Amano AK). Taking
into account the enantioselectivity of the reaction, lipase
Amano AK appeared to be the most selective enzyme, but con-
sidering the time of the reaction, enantiomerically enriched alco-
hol (+)-(S)-5a and ester (À)-(R)-6a were obtained with high
optical purity (ee = 97% for alcohol and ee = 87% for ester) in a
much shorter time using Novozyme SP 435 as a catalyst. Inter-
esting and worth emphasizing is that the compound ( )-5b,
possessing a small methyl group at C-2 position of benzimid-
azole ring did not undergo the transesterification with vinyl ace-
tate in the presence of both lipases (Novozyme SP 435 and
Amano AK) in all tested solvents. Probably this was caused by
the steric congestion around the hydroxy group, which hinders
the access of the substrate to the active center of the enzymes.
This problem was not observed when similar studies were per-
formed for alcohol ( )-5d, which also possesses methyl substit-
uent at the C-2 position of the benzimidazole ring, but a longer
distance between the benzimidazole moiety and hydroxy group.
Moreover, as can be seen from the data presented in Table 4, the
length of the alkyl chain between the hydroxy group and
the benzimidazole scaffold significantly affects both reaction
time and enantioselectivity. In Novozyme SP 435-catalyzed
transesterification of alcohol ( )-5d, regardless of the solvent,
high conversion (39–77%) was reached after 24 h, which in com-
parison to the data obtained for the kinetic resolution of alcohol
( )-5a shows the higher catalytic efficiency of this enzyme.
The opposite trend was observed in reactions catalyzed by lipase
Amano AK, where in all solvents, except TBME (conversion 37%
after 72 h), significantly longer reaction times were required to
achieve conversion 18% (456 h, THF), 26% (672 h, CH2Cl2), 35%
(336 h, toluene and Et2O), or 35% (335 h, toluene). Knowing that
the substituent at the imidazole ring affects the catalytic activity
of lipases, we investigated if four bromine atoms at the benzene
ring also influence the functionality of enzymes. The enantio-
mers separation of alcohol ( )-5e was performed in the pres-
ence of Novozyme SP 435 and Amano AK. Taking into
account the high molar mass of compound ( )-5e, first the
amount of Novozyme SP 435 was optimized. The reaction
was carried out in TBME at room temperature with 5 mg,
20 mg, and 30 mg of enzyme per 0.2 mmole of substrate.
The observed long reaction times caused that further studies
were performed on a bigger scale with 90 mg of enzyme per
0.6 mmole of ( )-5e, in five solvents including TBME, tolu-
ene, Et2O, CH2Cl2, and THF. In most cases, the obtained
enantioselectivities (Table 4) were very low (E = 1–9). Satisfac-
tory optical purities of products were obtained using THF as a
Chirality DOI 10.1002/chir
performed again in the presence of dicyclohexylcarbodiimide
(DCC) and 4-dimethylaminopyridine (DMAP), in dry
chloromethane with glacial acetic acid (Scheme 2). Product
6a was obtained after 4 h with yield 88%. Taking into account
that the changing reaction conditions significantly improved
the yield and shortened by four times the time of the reaction,
other esters were obtained using glacial acetic acid as an acet-
ylating agent. The results are summarized in Table 3.
The racemic mixtures of alcohols ( )-5a-e were used as
substrates in lipase-catalyzed kinetic resolutions. The influ-
ence of the position of the hydroxy group relative to the
substituted and not substituted benzimidazole ring on the se-
lectivity of lipases was investigated. The separation of enantio-
mers was performed using transesterification reaction and
vinyl acetate as an acyl donor. Preliminary studies were per-
formed for ( )-1-(1H-benzimidazol-1-yl)propan-2-ol 3a
(Scheme 3).
The reaction was carried out in the presence of various com-
mercially available lipases: Amano AK (lipase from Pseudomonas
fluorescens), Amano PS (lipase from Burkholderia cepacia, earlier
Pseudomonas cepacia), Amano PS-IM (lipase from Burkholderia
cepacia immobilized on diatomite), Lipozyme (lipase from
Mucor miehei), and Novozyme SP 435 (lipase from Candida
antarctica-B immobilized on a macroporous acrylic resin). The
control experiment revealed that the reaction did not proceed
in the absence of enzyme. Among the tested enzymes: Amano
PS, Amano PS-IM, and Lipozyme proved to be useless, because
of a low conversion of substrate (2–10%), even after 7 d of carry-
ing out each of the reactions in tert-butyl methyl ether (TBME)
at room temperature. Satisfactory results were obtained when
the reaction was catalyzed by Novozyme SP 435 and lipase
Amano AK. The influence of various organic solvents on their
selectivity was evaluated. Besides TBME, other solvents differ-
ing in the value of logarithm of the partition coefficient between
n-octanol and water (logP) were applied. The reactions were
performed in toluene, diethyl ether, dichloromethane, and tetra-
hydrofuran. The progress of the reactions was monitored on
TLC plates and reactions were stopped when the conversion
reached ~50% or less, in the case of particularly sluggish reac-
tions. Taking into account the results presented in Table 4, it
can be concluded that the catalytic efficiencies and selectivities
TABLE 3. The reaction times and yields of synthesis of esters
( )-6a-e
Ester
R
X
n
Time (h)
Yield (%)
6a
6b
6c
6d
6e
H
CH3
H
CH3
H
H
H
H
H
Br
1
1
2
2
2
3
95
76
85
92
86
48
96
72
48

352
ŁUKOWSKA-CHOJNACKA ET AL.
TABLE 4. Results of lipase-catalyzed transesterification of alcohols ( )-5a-e
b
Alcohol
Enzymee
Solvent
Time (h)
ca (%)
eealk. (%)
eebest. (%)
Ea
Yieldalk. (%)
Yieldest.(%)
5a
Novozyme SP 435
TBME
toluene
Et2O
CH2Cl2
THF
TBME
toluene
Et2O
CH2Cl2
THF
48
48
53
54
59
40
33
37
47
10
30
38
97
94
65
66
44
59
87
10
43
61
87
80
45
97
88
99
99
98
61
31
5
45
43
36
57
62
48
50
86
64
57
50
50
55
35
27
30
43
6
120
120
144
120
120
168
120
144
>100
24
>100
>100
89
>100
>100
Amano AK
99
98
22
33
5b
5c
5d
Novozyme SP 435
Amano AK
Novozyme SP 435
Amano AK
TBME
TBME
TBME
TBME
TBME
toluene
Et2O
CH2Cl2
THF
TBME
toluene
Et2O
CH2Cl2
THF
TBME
toluene
Et2O
CH2Cl2
THF
CH2Cl2
THF
no reaction
no reaction
21
336
24
24
24
24
24
72
336
336
672
456
48
48
48
48
24
384
312
nd
nd
70
77
74
46
39
37
35
35
26
18
77
42
70
54
56
15
26
nd
nd
99
99
98
79
63
55
53
54
34
22
27
31
29
73
99
17
35
nd
nd
43
30
35
92
99
95
99
99
99
99
8
43
12
62
80
95
99
nd
nd
11
8
8
58
>100
68
>100
>100
>100
>100
17
61
28
20
23
50
57
56
60
58
69
74
19
50
25
40
37
45
56
79
30
60
65
66
32
26
21
28
24
16
8
64
38
61
47
50
6
Novozyme SP 435
Amano AK
Novozyme SP 435
Amano AK
5e
1.5
3.3
1.6
9
45
46
>100
13
aConversion (c) and E values were calculated from the enantiomeric excess of substrate (+)-5a,d,e (eealk.) and product (À)-6a,d,e (eeestr.) using the formula:
c = eealk./(eealk. + eeestr.), E = Ln[(1-eealk.)(eeestr./eealk. + eeestr.)]/Ln[(1 + eealk.)(eealk. + eeestr.)].
bDetermined by HPLC analysis using Chiracel OD-H column.
solvent (E = 45). On the basis of these findings, the reaction
was carried out again in CH2Cl2 and THF using lipase Amano
AK as catalyst. As can be seen from the results presented in
Table 4, reactions mediated by lipase Amano AK were slug-
gish and even after 16 (CH2Cl2) and 14 (THF) d of stirring
low conversions were achieved (15% and 26%, respectively).
The progresses of all lipase-catalyzed reactions were moni-
tored on TLC plates and products were separated and purified
by column chromatography. The enantiomeric excesses of al-
cohols (+)-(S)-5a, (+)-(S)-5d, (+)-(S)-5e, and acetate (À)-(R)-
6e were directly determined by HPLC analysis using a chiral
column. Unfortunately, this column was inappropriate for the
detection of enantiomeric excesses of esters: (À)-(R)-6a and
(À)-(R)-6d, therefore they were hydrolyzed to the corre-
sponding alcohols. The reaction was performed in MeOH with
1 M NaOH solution at room temperature. This column was
also inappropriate for the detection of the ee of alcohol (+)-
(S)-5c and ester (À)-(R)-6c, therefore these results are
unknown.
The absolute configuration of ester (À)-6e (ee = 95%, prod-
uct of transesterification catalyzed by Amano AK in CH2Cl2),
was determined by X-ray crystallography.38 The crystal data
indicated that this is the R enantiomer (Fig. 1), which means
that the unchanged alcohol (+)-5e possesses S configuration
on the chiral center. The comparison of the specific rotation
values of other esters ((À)-6a,c,d) and alcohols ((+)-5a,c,d)
Chirality DOI 10.1002/chir
allowed us to state that all esters are R and alcohols S enantio-
mers. This is also in agreement with Kazlauskas rule,39
according to which in lipase-catalyzed transesterification R
enantiomer reacts faster.
For synthesized N-substituted benzimidazole derivatives
4a-d, ( )-5a-d, and ( )-6a-d antifungal activity against Can-
dida albicans wildtype strain 90028 was evaluated. This micro-
organism exists as a component of the normal microflora of
the oral cavity and gastrointestinal, vaginal, and urogenital
tracts of humans. It is recognized as the most common
pathogen among the Candida spp., which can cause chronic,
superficial, and systematic infections, especially dangerous
for immunocompromised patients. Treatment of candidiasis
is difficult because of the limited number of antimycotics
and the rise of Candida resistant to classic antifungals. The
antifungal activity of synthesized benzimidazole derivatives
was evaluated at various concentrations (from 0.25 g/mL to
16 μg/mL), and in all cases over 97% of cell growth inhibition
was observed. Analysis of antifungal activity showed that alco-
hols ( )-5a-d and esters ( )-6a-d are more active against
C. albicans (% of growth reduction ≥99.01 at 16 μg/mL) than
ketones 4a-d (% ≥97.98 at 16 μg/mL). Thus, reduction of
ketones to alcohols followed by their acylation to esters led
to improvement of the anti-Candida activity. In most cases,
distinctly lower values of percent growth reduction were
observed at higher concentrations compared to the lower

KINETIC RESOLUTION OF BENZIMIDAZOLE DERIVATIVES
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This work was financially supported by Warsaw University
of Technology and by the European Regional Development
Fund under the Innovative Economy Operational Programme
2007–2013: “Biotransformations for Pharmaceutical and Cos-
metic Industry” POIG.01.03.01-00-158/09. M.S. and M.B.
received research support from the National Science Centre-
Poland (No. DEC-2011/03/D/NZ7/06198). J.K.M. thanks
the Foundation for Development Diagnostics and Therapy,
Warsaw, Poland, for support. We thank graduate students
Justyna Szyjer and Ewelina Rogala for assistance in the chem-
ical experiments.
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