Synthesis, characterization and tyrosinase inhibitory properties of benzimidazole derivatives

Article abstract of DOI:10.1134/S1068162014040049

1-Alkylbenzimidazole and 1,3-dialkyl benzimidazolium salts were synthesized and characterized by the data of IR, ¹H NMR, ¹³C NMR spectra and elemental analyses. These compounds were investigated as tyrosinase inhibitors. Tyrosinase h

Full text of DOI:10.1134/S1068162014040049

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2014, Vol. 40, No. 4, pp. 461–466. © Pleiades Publishing, Ltd., 2014.
Synthesis, Characterization and Tyrosinase Inhibitory Properties
of Benzimidazole Derivatives1
Mert Olgun Karatasa, Bulent Alicia, Engin Çetinkayab, Çi
Nahit Gençerc, 2, and Oktay Arslanc
g
dem Bilenc,
aInonu University, Faculty of Arts and Sciences, Department of Chemistry , Malatya, 44280 Turkey
bEge University, Faculty of Science, Department of Chemistry, Izmir, 35100 Turkey
cDepartment of Chemistry, Faculty of Art and Sciences, Balikesir University, Balikesir, 10145 Turkey
Received January 8, 2014; in final form, March 3, 2014
Abstract—1ꢀAlkylbenzimidazole and 1,3ꢀdialkyl benzimidazolium salts were synthesized and characterized
by the data of IR, 1H NMR, 13C NMR spectra and elemental analyses. These compounds were investigated
as tyrosinase inhibitors. Tyrosinase has been purified from banana by affinity chromatography on a Sepharose
4B gel conjugated with
sinase activity. Among the compounds studied, 1,4ꢀdi(1
most active tyrosinase inhibitor (IC50 0.31 mM).
L
ꢀtyrosineꢀ
p
ꢀaminobenzoic acid. All the synthesized compounds inhibited the tyroꢀ
H
ꢀbenzo[d]imidazolꢀ1ꢀyl)butane was found to be the
Keywords: benzimidazole, enzymatic browning, tyrosinase inhibitors
DOI: 10.1134/S1068162014040049
2 1
INTRODUCTION
the high reactivity, quinines could polymerize spontaꢀ
neously to form high molecular weight brown pigꢀ
ments (melanins) or react with amino acids and proꢀ
teins to enhance brown colour of the pigment proꢀ
duced [14, 15]. Previous reports confirmed that
tyrosinase not only was involved in melanising in aniꢀ
mals, but also was one of the main causes of most fruits
and vegetables quality loss during post harvest hanꢀ
dling and processing, leading to faster degradation and
shorter shelf life [16]. Recently, investigation demonꢀ
strated that various dermatological disorders, such as
age spots and freckle, were caused by the accumulaꢀ
tion of an excessive level of epidermal pigmentation
[17, 18]. Tyrosinase has also been linked to Parkinꢀ
son’s and other neurodegenerative diseases [19]. In
insects, tyrosinase is uniquely associated with three
different biochemical processes, including sclerotizaꢀ
tion of cuticle, defensive encapsulation and melanisaꢀ
tion of foreign organism, and wound healing [20].
These processes provide potential targets for develꢀ
oping safer and effective tyrosinase inhibitors as
insecticides and ultimately for insect control. Thus,
the development of safe and effective tyrosinase
inhibitors is of great concern in the medical, agriculꢀ
Benzimidazole consists of the fusion of benzene
and imidazole. Benzimidazole has been widely used as
carbon skeleton for synthesis of Nꢀheterocyclic carꢀ
benes (NHC). Benzimidazole derivatives and their
NHC’s are usually used as ligand for transition metal
complexes and these complexes are also used as cataꢀ
lyst in various organic synthesis [1]. Besides using on
synthesis of NHC’s, various biological activities of
benzimidazole derivatives were reported [2]. The bioꢀ
logic potential of benzimidazole can be traced back to
1944. Wooley reported that benzimidazole can act
similar to purines to give some biological responses
[3]. After this study, biological properties of benzimiꢀ
dazole derivatives were investigated intensively. Benzꢀ
imidazole bearing bioactive compounds were reported
as antihypertensive [4], antiꢀinflammatory [5], antiꢀ
microbial [6], antiviral [7], antioxidant [8], antitumor
[9], lipid modulator [10], anticoagulant [11].
Tyrosinase (monophenol or oꢀdiphenol, oxygen
oxidoreductase, EC 1.14.18.1), also known as
polyphenol oxidase (PPO), is a copperꢀcontaining
monooxygenase that is widely distributed in microorꢀ
ganisms, animals, and plants [12]. Tyrosinase could tural, and cosmetic industries. However, only a few
catalyze two distinct reactions involving molecular such as kojic acid, arbutin, tropolone, and 1ꢀphenylꢀ
oxygen in the hydroxylation of monophenols to
ꢀdiphenols (monophenolase) and in the oxidation of
2ꢀthiourea are used as therapeutic agents and cosꢀ
metic products [18, 21].
o
oꢀdiphenols to oꢀquinones (diphenolase) [13]. Due to
In this study we synthesized 1ꢀalkylbenzimidazoles
and 1,3ꢀdialkyl benzimidazolium salts and their inhibꢀ
itory properties on PPO activity were evaluated. Imiꢀ
1
2
The article is published in the original.
Corresponding author: eꢀmail: ngencer@balikesir.edu.tr.
461

462
MERT OLGUN KARATAS et al.
dazolium salts similar to we synthesized in this study
were reported as antimicrobial agents [22] but PPO
inhibitory properties of ionic benzimidazole derivaꢀ
tives were investigated first time in this study.
Benzimidazolium
1
8
9
5
10
4
RESULTS AND DISCUSSION
The synthetic procedures employed to obtain the
target compounds (4a–m) are depicted in Scheme 1.
All new synthesized compounds were characterized by
Fig. 1. Anthracene skeleton.
1
13
IR, H NMR, C NMR spectroscopic methods and characteristic aromatic proton signals were obtained
elemental analyses. In the IR spectra of compounds between 8.95–8.86 ppm as singlet.13C NMR signals
(
4a–m), it was possible to see the absorption between and number of peaks were compatible with structure of
13
1593 and 1553 cmꢀ1 belong to N–C–N bond. In the synthesized compounds. In C NMR spectra, charꢀ
1H NMR it was possible to see characteristic NC
HN
acteristic signals were obtained between 141.6
NCN
signals between 9.52–9.01 ppm as singlet. Beside these and 162.8 ppm. Furthermore, elemental analysis datas
signals, in anthracene skeleton, at position 10 (Fig. 1), were compatible with synthesizes compounds.
R
R
R
Compound
(2a), (4a)
N
N
i
Cl–
+
–CH3
–CH2CH=CH2
–CH2CH2CH2CH3
–CH2CH2OCH3
N
N
(
(
(
(
(
(
(
(
(
2b), (4b
2c), (4c
2d), (4d
2e), (4e
)
)
)
)
(
2a–j
)
–CH2C6H5
H
N
–CH2C6H4(CH3)ꢀ2
–CH2C6H4(CH3)ꢀ4
–CH2C6(CH3)5ꢀ2,3,4,5,6
–CH2C6H2(OCH3)3ꢀ3,4,5
–CH2C10H7ꢀ2
2f), (4f)
2g), (4g
)
(
4a–j)
2h), (4h
)
Cl
N
2i), (4i
2j), (4j
)
)
1
Compound (3);
(CH2)n
N
N
N
N
N
n
4
5
Compound
N
N
N
(
2k), (4k
)
(2l), (4l)
(2k, l
)
(2m)
ii
ii
Cl–
Cl–
Cl–
Cl–
N
N
(CH2)n
N
N
N
N
N
N
(
4k, l
)
(4m)
Scheme 1. Synthesized Compounds. Reagents and Conditions: (i) Ethanol, KOH, RX, 8h, reflux (ii) compound (
3),
90 C, DMF, 3 days. (iii) 9ꢀ(Chlormethyl)anthracene ( ), 90 C, DMF, 5 days.
°
3
°
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SYNTHESIS, CHARACTERIZATION AND TYROSINASE INHIBITORY PROPERTIES
463
IC50 values for the benzimidazole derivatives tested as tyroꢀ
sinase inhibitors
For evaluating the tyrosinase inhibitory activity, all
the synthesized compounds were subjected to tyrosiꢀ
nase inhibition assay with catechol as substrate. The
result showed that all the synthesized compounds
Compound IC50 (mM) Compound IC50 (mM)
(
4a–m) inhibited the tyrosinase enzyme activity. IC50
(1)
0.50
65.07
87.40
11.09
24.61
0.97
0.96
0.92
0.63
2.25
0.50
0.31
0.81
0.79
0.03
(3)
0.71
13.14
0.52
0.66
0.38
0.73
1.22
0.66
0.38
2.56
1.29
0.67
0.90
0.50
values were calculated from inhibition curves obtained
with benzimidazole derivatives. The inhibition values
of analogues (4a–m) against PPO were summarized in
table. We have determined the IC50 values of 0.31–
13.14 mM for the inhibition of banana PPO. Accordꢀ
ing to IC50 values, compound (2k) was the most effective
inhibitor for BPPO (0.31 mM). Except compound (5)
(2a)
(2b)
(2c)
(2d)
(2e)
(2f)
(4a)
(4b)
(4c)
(4d)
(4e)
(4f)
(4g)
(4h)
(4i)
and compounds (2a, 2b, 2c, 2d, 4a), IC50 values for
(2g)
(2h)
(2i)
23 compounds were in the range of 0.31–2.56 mM.
These results show that aliphatic R groups at position
of benzimidazole skeleton decreased PPO inhibitory
activity. Inhibition of PPO was investigated using gallic
acid as a standard inhibitor, which showed the stronꢀ
gest inhibitory activity with IC50 value of 0.03 mM
(Fig. 2). Gallic acid (3,4,5ꢀtrihydroxybenzoate) has
been isolated and identified as a tyrosinase inhibitor
from many plants, and its inhibitory mechanism
together with those of its ester derivatives has been well
studied by Kubo et al. [23–25]. They found that gallic
acid inhibited diphenolase activity of mushroom tyroꢀ
sinase is 100ꢀfold lower than that of kojic acid. The
enzymatic browning by a specific inhibitor may
involve a single mechanism or may be the result of
interplay of two or more mechanisms of inhibitor
action.
Enzymatic browning of plants may be delayed or
eliminated by removing the reactants, such as oxygen
and phenolic compounds, or by using PPO inhibitors.
Complete elimination of oxygen from plants during
drying is difficult because oxygen is ubiquitous [26].
There are a number of inhibitors, such as sodium metꢀ
abisulphite [27], ascorbic acid [28], glutathione [29],
tropolone [30] decreasing the activity of PPO. Aciduꢀ
lants, such as citric acid can inhibit PPO activity by
reducing pH and/or chelating Cu in a food product
[31, 32]. Ascorbic acid can also be considered as an
effective compound at higher concentrations. The
mechanism of ascorbic acid inhibition involves the
reduction of quinones generated by PPO [33]. The
goal of these studies is determine to the best inhibitor
for decreasing the enzymatic browning.
(2j)
(4j)
(2k)
(2l)
(4k)
(4l)
(2m)
Gallic acid
(4m)
13C NMR were recorded in DMSOꢀd6 using a Bruker
AC300P FT spectrometer operating at 300.13 MHz
(1H), 75.47 MHz (13C). Chemical shifts (
δ
) are given in
ppm relatively TMS and coupling constants ( ) are
J
given in Hz. Elementary analysis were done by
IBTAM (Inonu University Scientific and Technologiꢀ
cal Research Central).
Synthesis of 1ꢀalkylbenzimidazole and 1,1'ꢀbisbenzꢀ
imidazole compounds (2a–m). 1ꢀAlkylbenzimidazole
and bisbenzimidazole compounds were synthesized
according to the procedure of Ozdemir et al. [34].
Potassium hydroxide (1 mmol) was added to a solution
of benzimidazole (1 mmol) in ethanol (20 mL), the
mixture was stirred for 1 h at room temperature, and
the corresponding alkyl halides was added dropwise
and heated for 8 h at 76°C. The mixture was diluted
Activity, %
120
y
= –14503x2 – 1384.5
R2 = 0.9756
x + 101.17
100
80
EXPERIMENTAL
60
40
20
All reactions for preparation of benzimidazolium
salts were carried out in standard Schlenkꢀtype flasks.
Chemicals were purchased from Sigma Aldrich. DMF
used as a solvent in the synthesis of benzimidazolium salt
was dried by P2O5. 9ꢀ(Chloromethyl) anthracene (
3) was
used without further purification. Melting points were
determined using an Electrothermalꢀ9200 melting point
apparatus. FT–IR spectra were recorded on an ATR unit
in the range of 400–4000 cm–1 using a Perkin Elmer
0
0.005 0.010 0.015 0.020 0.025 0.030
Gallic acid, mM
1
Fig. 2. Inhibitory activity of gallic acid on BPPO.
Spectrum 100 Spectrophotometer. H NMR and
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 40
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464
MERT OLGUN KARATAS et al.
with 30 mL of water and extracted with chloroform 131.6, 131.5, 130.9, 130.0, 128.3, 127.3, 127.2, 126.1,
10 mL). The compounds (2a–d) were distilled 123.9, 122.5, 114.7, 114.1, 69.5, 59.5, 46.5, 44.0.
(
3
×
under reduced pressure and the compounds (2e–m
)
1ꢀBenzylꢀ3ꢀ(anthracenꢀ9ꢀylmethyl)benzimidazolium
chloride (4e). Yield 70%, mp 234 235 C. Calculated
for C29H23ClN2 (%): C 80.08, H 5.33, N 6.44. Found:
were recrystallized from ethanolꢀhexane.
–
°
Synthesis of benzimidazolium salts (4a–j). 10 mmol
1ꢀalkylbenzimidazole (2a–j) was dissolved in 5 mL of
dried DMF, then 10 mmol 9ꢀ(chloromeꢀ
thyl)anthracene was added into the solution and the
C 79.71, H 5.42, N 6.53. FTꢀIR (cm–1): 1565 (C–N),
1H NMR: 9.47 (s, 1H, NC
H
N), 8.94 (s, 1H, Ar
7.28 (m, 12H, Ar ), 6.81
2Ant), 5.63 (s, 2H, –C
2Ph). 13C NMR:
H),
8.46–8.27 (m, 5H, ArH), 7.97–
H
mixture was heated for 3 days at 90°C. After cooling
(s, 2H, –C
H
H
the mixture to room temperature, diethyl ether was
added, and the precipitate was collected by filtration.
The crude product was washed with hexane and then
dried under reduced pressure.
142.2, 134.7, 132.4, 131.7, 131.5, 131.4, 131.0, 130.0,
129.3, 129.0, 128.3, 128.2, 127.5, 127.3, 126.1, 123.9,
122.6, 114.9, 114.5, 50.1, 44.0.
1ꢀ(2ꢀMethylbenzyl)ꢀ3ꢀ(anthracenꢀ9ꢀylmethyl)benzimꢀ
1ꢀMethylꢀ3ꢀ(anthracenꢀ9ꢀylmethyl)benzimidazoliꢀ
idazolium chloride (4f). Yield 51%, mp 230–232°C.
Calculated for C30H25ClN2 (%): C 80.25, H 5.61,
um chloride (4a). Yield 81%, mp 255–256°C. Calcuꢀ
lated for C23H19ClN2 (%): C 76.98, H 5.34, N 7.81.
N 6.24. Found: C 79.88, H 5.80, N 6.38. FTꢀIR (cm–1):
Found: C 76.88, H 5.62, N 7.65. FTꢀIR (cm–1): 1563
1560 (C–N), 1H NMR: 9.25 (s, 1H, NC
H
N), 8.93 (s,
), 7.83–6.85 (m,
H2Ant), 5.64 (s, 2H,
1
(C–N). H NMR 9.03 (s, 1H, NC
H
N), 8.93 (s, 1H,
), 6.76 (s, 2H, –C 2Ant),
3). C NMR: 142.0, 132.6, 132.0,
1H, Ar
11H, Ar
–C 2Ph), 2.13 (s, 3H, ArꢀCH3). C NMR: 142.5,
H
H
), 8.47–8.26 (m, 5H, Ar
), 6.83 (s, 2H, –C
H
ArH
), 8.42–7.60 (m, 12H, Ar
3.91 (s, 3H, NꢀC
H
H
13
H
13
H
131.6, 131.5, 130.9, 129.9, 128.3, 127.3, 127.2, 126.1,
124.0, 122.6, 114.6, 114.2, 43.7, 33.7.
132.8, 132.4, 131.9, 131.6, 131.5, 131.1, 131.0, 130.0,
128.8, 128.7, 127.5, 127.3, 126.7, 126.1, 123.9, 122.5,
115.0, 114.5, 48.9, 44.2, 19.0.
1ꢀAllylꢀ3ꢀ(anthracenꢀ9ꢀylmethyl)benzimidazolium
chloride (4b). Yield 65%, mp 204–207°C. Calculated
for C25H21ClN2 (%): C 78.01, H 5.50, N 7.31. Found:
1ꢀ(4ꢀMethylbenzyl)ꢀ3ꢀ(anthracenꢀ9ꢀylmethyl)benzꢀ
C 77.92, H 5.68, N 7.11. FTꢀIR (cm–1): 1554 (C–N).
imidazolium chloride (4g). Yield 40%, mp 236–237°C.
Calculated for C30H25ClN2 (%): C 80.25, H 5.61, N 6.24.
1H NMR: 9.14 (s, 1H, NC
H
N), 8.93 (s, 1H, Ar
), 6.77 (s, 2H, –C 2Ant),
=H'H'', JCH –CH 4.8, JCH–H'
H),
Found: C 79.85, H 5.80, N 6.39. FTꢀIR (cm–1):
8.42–7.60 (m, 12H, Ar
5.94 (ddt, 1H, –CH2–C
6.8, CH–H''10.3), 5.27–5.16 (2H, dd, JH–H' 6.8, JH–H'
10.4), 5.03 (d, 2H, –C 2CH=CH'H'', JCH –CH 4.9).
H
H
H
1558 (C–N). 1H NMR: 9.41 (s, 1H, NC
H
N), 8.94 (s,
), 7.95–7.11 (m,
H2Ant), 5.56 (s, 2H,
2
1H, Ar
11H, Ar
–C 2Ph), 2.25 (s, 3H, ArꢀCH3). C NMR: 162.8,
H
H
), 8.44–8.25 (m, 5H, Ar
), 6.79 (s, 2H, –C
H
J
13
H
H
2
13C NMR: 141.9, 132.3, 131.8, 131.7, 131.6, 131.5,
131.0, 130.0, 128.4, 127.4, 127.3, 126.1, 123.9, 122.5,
119.9, 114.8, 114.5, 49.1, 44.0.
142.0, 138.5, 132.4, 131.7, 131.5, 131.5, 131.0, 130.0,
129.8, 128.3, 128.2, 127.4, 127.3, 126.2, 123.9, 122.5,
114.9, 114.6, 49.9, 44.2, 21.1.
1ꢀ(
n
ꢀButyl)ꢀ3ꢀ(anthracenꢀ9ꢀylmethyl)benzimidazoꢀ
1ꢀ(2,3,4,5,6ꢀPentamethylbenzyl)ꢀ3ꢀ(anthracenꢀ9ꢀ
ylmethyl)benzimidazolium chloride (4h). Yield 44%,
mp 256–258°C. Calculated for C34H33ClN2 (%):
C 80.85, H 6.59, N 5.55. Found: C 80.88, H 6.80,
N 5.48. FTꢀIR (cm–1): 1560 (C–N). 1H NMR: 9.01 (s,
lium chloride (4c). Yield 61%, mp. 241
–
243°C. Calꢀ
culated for C26H25ClN2 (%): C 77.89, H 6.29, N 6.9.
Found: C 77.82, H 6.51, N 6.88. FTꢀIR (cm–1): 1564
1
(C–N). H NMR: 9.22 (s, 1H, NC
H
N), 8.92 (s,
), 6.76 (s, 2H,
2CH2CH2CH3, 7),
2CH2CH3, 7), 1.11 (six, 2H,
7), 0.76 (t, 3H, –CH2CH2CH2CH3
7). C NMR : 141.8, 132.2, 131.7, 131.6, 131.5,
1H, NC
Ar ), 6.81 (s, 2H, –C
2.26 (s, 3H, ArꢀC 3ꢀp position), 2.20 (s, 6H, ArꢀC
position), 2.08 (s, 6H, ArꢀC
3ꢀm position). 13C NMR:
H
N), 8.86 (s, 1H, Ar
H
), 8.48–7.48 (m, 12H,
2Ph),
3ꢀo
1H, Ar
–C 2Ant), 4.36 (t, 2H, –C
1.69 (five, 2H, –CH2C
–CH2CH2C 2CH3,
H), 8.42–7.60 (m, 12H, ArH
H
H2Ant), 5.69, (s, 2H, –C
H
H
H
J
H
H
H
J
H
J
,
H
13
141.6, 136.7, 134.0, 133.5, 132.1, 132.0, 131.5, 131.1,
130.8, 130.1, 128.2, 127.2, 127.1, 126.2, 126.1, 123.7,
123.2, 114.6, 114.4, 47.2, 44.5, 17.5, 17.2, 16.8.
J
130.9, 130.0, 128.3, 127.3, 127.2, 126.1, 123.9, 122.6,
114.7, 114.4, 46.8, 44.0, 31.1, 19.3, 13.6.
1ꢀMethoxyethylꢀ3ꢀ(anthracenꢀ9ꢀylmethyl)benzimꢀ
1ꢀ(3,4,5ꢀTrimethoxybenzyl)ꢀ3ꢀ(anthracenꢀ9ꢀylmeꢀ
thyl)benzimidazolium chloride (4i). Yield 69%,
mp 230–235°C. Calculated for C32H29ClO3N2 (%):
idazolium chloride (4d). Yield 73%, mp 223–226°C.
Calculated for C25H23ClON2 (%): C 74.52, H 5.75,
N 6.95. Found: C 74.42, H 6.03, N 6.89. FTꢀIR (cm–1): C 73.20, H 5.57, N 5.34. Found: C 73.18, H 5.80, N
1563 (C–N). 1H NMR: 9.11 (s, 1H, NC
H
N), 8.93 (s, 5.38. FTꢀIR (cm–1): 1593 (C–N). H NMR: 9.39 (s,
1
1H, Ar
–C 2Ant), 4.56 (t, 2H, –N–C
3.59 (t, 2H, –N–CH2C 2OCH3,
3). C NMR: 142.3, 132.0, 131.9, (s, 3H, PhꢀOCH
H
), 8.43–7.60 (m, 12H, Ar
2CH2OCH3,
5), 3.05 (s, 3H, 2H, –C
H
), 6.80(s, 2H, 1H, NC
5), Ar ), 6.77 (s, 2H, Ar
2Ph), 3.60 (s, 6H, PhꢀOC
3ꢀp position). 13C NMR: 153.4,
H
N), 8.95 (s, 1H, Ar
), 6.69 (s, 2H, –C
3ꢀm positon), 3.58
H
), 8.44–7.64 (m, 12H,
H
H
J
H
H
H2Ant), 5.47 (s,
H
J
H
H
13
–N–CH2CH2OC
H
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SYNTHESIS, CHARACTERIZATION AND TYROSINASE INHIBITORY PROPERTIES
465
141.6, 137.8, 132.3, 131.7, 131.54, 131.51, 131.0,
130.1, 130.0, 128.3, 127.5, 127.3, 126.1, 123.8, 122.4, were carried out at 25
Purification of tyrosinase. All purification steps
. The extraction procedure
°C
114.9, 114.6, 106.1, 60.4, 56.3, 50.2, 44.1.
1ꢀ(2ꢀNaphtylmethyl)ꢀ3ꢀ(anthracenꢀ9ꢀylmethyl)benzꢀ
was adopted from WescheꢀEbeling & Montgomery
[35]. The bananas were washed with distilled water
three times to prepare the crude extract. 50 g of
bananas were cut quickly into thin slices and homogꢀ
enized in a Waring blender for 2 min using 100 mL of
0.1 M phosphate buffer, pH 7.3 containing 5% polyꢀ
ethylene glycol and 10 mM ascorbic acid. The homoꢀ
genate was filtered through muslin, the filtrate was
imidazolium chloride (4j). Yield 64%, mp 223–224°C.
Calculated for C33H25ClN2 (%): C 81.72, H 5.20.
N 5.78. Found: C 81.68, H 5.50, N 5.88. FTꢀIR (cm–1):
1
1557 (C–N). H NMR: 9.52 (s, 1H, NC
H
N), 8.95
), 6.83 (s, 2H,
H
2Naphtalen). 13C NMR:
(s, 1H, ArH), 8.48–7.34 (m, 19H, ArH
–CH2Ant), 5.80 (s,2H, –C
centrifuged at 15000 g for 30 min, and the supernatant
142.3, 133.0, 132.4, 132.2, 131.7, 131.6, 131.5, 131.0,
130.0, 129.1, 128.3, 128.2, 128.1, 127.5, 127.3,
127.23, 127.2, 126.2, 125.5, 124.0, 122.6, 114.9,
114.5, 50.3, 44.3.
was collected. A crude protein precipitate was made by
adding (NH4)2SO4 to 80% saturation. The resulting
precipitate was suspended in a minimum volume of 5
mM phosphate buffer and then dialyzed against the
same buffer overnight. The enzyme solution was then
applied onto the Sepharose 4Bꢀtyrosineꢀpꢀamino
benzoic acid affinity column [36], preꢀequilibrated
with 5 mM phosphate buffer, pH 5.0. The affinity gel
was extensively washed with the same buffer and then the
banana PPO (BPPO) was eluted with 1 M NaCl, 5 mM
phosphate, pH 7.0.
Synthesis of bisbenzimidazolium salts (4k–m).
5
mmol 1,1 ꢀbisbenzimidazole (2k–m) was dissolved in
'
5 mL dried DMF then 10 mmol 9ꢀ(chloromeꢀ
thyl)anthracene was added into the solution and the
mixture was heated for 5 days at 90°C. After cooling to
room temperature, diethyl ether was added to the mixꢀ
ture and precipitate was collected by filtration. Crude
product was washed with acetone and dried under
reduced pressure.
Tyrosinase enzyme activity. Enzyme activity was
determined according to the method Espin et al. [37]
using catechol as a substrate by measuring the increase
in absorbance at 420 nm on a Biotek automated
recording spectrophotometer. All measurements were
performed in duplicate and corrected for the nonꢀ
enzymatic hydrolysis. Enzyme activity was calculated
from the linear portion of the curve. One unit of PPO
activity was defined as the amount of enzyme that
causes an increase in absorbance of 0.001 unit’s min–1
1,1'ꢀBis(anthracenꢀ9ꢀylmethyl)ꢀ3,3'buthylenedibenzꢀ
imidazolium dichloride (4k). Yield 42%, 255–257°C.
Calculated for C48H40Cl2N4 (%): C 77.51, H 5.42, N 7.53.
Found: C 77.48, H 5.80, N 7.68. FTꢀIR (cm–1): 1558
(C–N). 1H NMR: 9.17 (s, 2H, NC
H
N), 8.90 (s, 2H,
), 6.74 (s, 4H,
2CH2), 1.63 (five, 4H,
H2). C NMR: 141.8, 132.1, 131.7, 131.6,
Ar
H
H
), 8.38–7.52 (m, 24H, Ar
2Ant), 4.31 (t, 4H, NC
H
⎯ C
NCH2C
H
13
for 1 mL of enzyme at 25°C.
131.4, 130.1, 130.0, 128.2, 127.3, 127.2, 126.1, 123.9,
122.5, 114.7, 114.3, 46.4, 44.1, 26.1.
Inhibition of tyrosinase enzyme activity. An aliquot
of each inhibitor at various final concentrations was
added to the standard reaction solution immediately
before the addition of enzyme extract. The concentraꢀ
tion of inhibitor (benzimidazole derivatives) producꢀ
ing 50% inhibition (IC50) was determined from a plot
of residual activity against inhibitor concentration
using 10 mM catechol as substrate. The activity withꢀ
out inhibitor was taken as a control.
3,3'ꢀBis(anthracenꢀ9ꢀylmethyl)ꢀ1,1'penthyleneꢀ
dibenzimidazolium dichloride (4l). Yield 33%, mp:
239–242°C. Calculated for C49H42Cl2N4 (%):
C 77.66, H 5.59, N 7.39. Found: C 77.58, H 5.80,
1
N 7.44. FTꢀIR (cm–1): 1553 (C–N). H NMR: 9.22
(s, 2H, NC
24H, Ar ), 6.58 (s, 4H, –C
NC 2CH2CH2–, 7), 2.51 (five, 4H,
NCH2C 2CH2–, 7), 1.66 (five, 2H, NCH2CH2C 2–,
7). 13C NMR: 141.8, 132.1, 131.7, 131.6, 131.4, 130.9,
H
N), 8.90 (s, 2H, Ar
H), 8.42–7.58 (m,
H
H2Ant), 4.25 (t, 4H,
H
J
H
J
H
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129.9, 128.3, 127.3, 127.2, 126.1, 123.9, 122.6, 114.7,
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