Novel benzo[d]imidazole-2(3H)-thiones as potent inhibitors of the α-melanocyte stimulating hormone induced melanogenesis in melanoma B16 cells

Article abstract of DOI:10.1248/cpb.58.918

In order to determine the optimum size of heterocycle of lead compound 1 (6-methyl-3-phenethyl-3,4-dihydro-1H-quinoline-2-thione; IC₅₀=0.8 μM) for inhibition of melanogenesis, we have synthesized and evaluated some benzimdazole-2(3H)-thiones 5a

Full text of DOI:10.1248/cpb.58.918

918
Regular Article
Chem. Pharm. Bull. 58(7) 918—921 (2010)
Vol. 58, No. 7
Novel Benzo[d]imidazole-2(3H)-thiones as Potent Inhibitors of the
a
-Melanocyte Stimulating Hormone Induced Melanogenesis in Melanoma
B16 Cells
Jee-Hyun LEE,a Pillaiyar THANIGAIMALAI,a Ki-Cheul LEE,a Seong-Cheol BANG,a Min-Seok KIM,a
Vinay Kumar SHARMA,a Cheong-Yong YUN,b Eunmiri ROH,b Youngsoo KIM,b and Sang-Hun JUNG*,a
a College of Pharmacy and Institute of Drug Research and Development, Chungnam National University; Daejeon
305–764, Korea: and b College of Pharmacy, Chungbuk National University; Cheongju 361–763, Korea.
Received February 16, 2010; accepted April 1, 2010; published online April 6, 2010
In order to determine the optimum size of heterocycle of lead compound 1 (6-methyl-3-phenethyl-3,4-dihy-
dro-1H-quinoline-2-thione; IC50؍0.8 mM) for inhibition of melanogenesis, we have synthesized and evaluated
some benzimdazole-2(3H)-thiones 5a—e. The preliminary bioassay has shown that the benzimdazole-2(3H)-
thione motif of 5 is essential structural unit for their inhibitory activity. Among all thiones 5a—e, the compound
5d strongly inhibited the formation of melanin with IC50 value of 1.3 mM.
Key words benzimdazole-2(3H)-thione; melanogenesis; inhibitor
Melanin is a heterogeneous biopolymer which is princi-
pally responsible for human phenotypic appearance and has
major role in protecting human skin from harmful effect of
UV-radiation from the sun.1) Melanin is formed by a series of
enzymatic catalyzed reactions in melanocytes.2,3) The biosyn-
thesis of melanin is initiated with oxidation of tyrosine to
dopaquinone catalyzed by tyrosinase.4) This first step is the
Fig. 1. cAMP Dependent Melanogenesis Inhibitor 1, 2, 3 and 1,3-Dihy-
drobenzoimidazole-2-ones 4 and Their Thiones 5
rate limiting step in melanogenesis because the remaining
reaction sequence can proceed spontaneously at a physiologi-
cal pH.5) However, over expression of tyrosinase lead to
induction of hyperpigmentary disorders such as melasma,
freckles, age spot, and post-inflammatory melanoderma.6,7)
It is also reported to be linked to Parkinson diseases and
some other neurodegerative diseases.8,9) Therefore, the regu-
lation of melanin synthesis by inhibiting the tyrosinase en-
zyme is the current research topic in context of preventing
hyperpigmentation. In this regard, diverse tyrosinase in-
hibitors have been actively discovered such as kojic acid,10,11)
arbutin,12) ascorbic acid derivatives,13) hydroxylstilbine deriv-
atives like resveratol14—16) and methyl ester of genistic
acid.17,18) However, according to a recent study kojic acid
has serious adverse effects such as skin cancer and
dermatitis and has been banned as a cosmetic ingredient in
many countries.17) Thus other type of molecules, which in-
hibit cyclic adenosine monophosphate (cAMP) dependent
Reagents and conditions; (i) pyridine or NaOH; (ii) Fe, acetic acid; (iii)
phenylchloroformate, xylene; (iv) H2–Pd, MC and ethanol; (v) phenylchlorofor-
mate, xylene; (vi) xylene, reflux; (vii) Lawesson’s reagent, toluene. Note; nϭ1, 2
or 3. RϭCH3 or Cl. Substituents are located in Table 1.
melanogenesis proteins other than tyrosinase, are gaining
attention. Using bioassay system for measuring the amount
of melanin formed from melanoma B16 cells upon stimula-
tion of a-melanocyte stimulating hormone (a-MSH), we
have screened many different compounds. As a result, 6-
Chart 1. Synthesis of Compounds 4 and 5
methyl-3-phenethyl-3,4-dihydro-1H-quinoline-2-thione (Fig. synthesized and evaluated for their inhibitory activity against
1, IC50ϭ0.8 mM) was discovered as highly potent compound melanin production in melanoma B16 cells under the stimu-
against melanogenesis in melanoma B16 cell line.18) The lation of a-MSH.
mechanism of action of compound 1 is very much unique
and important due to its suppression of the melanogenesis Chemistry
without affecting the tyrosinase activity. The preliminary
Benzo[d]imidazol-2(3H)-ones 4 and benzo[d]imidazole-
study on the structure–activity relationship (SAR) of 1 has 2(3H)-thiones 5 were synthesized with the synthetic proce-
revealed quinazolidine-2-thione in 1 as an essential motif.18) dure as shown in Chart 1. In the first step, commercially
In order to explore the necessity of 6-membered cyclic available amine 6 was treated with appropriate alkyl bromide
thiourea scaffold in 1, benzo[d]imidazol-2(3H)-ones 4 (Fig. using pyridine or sodium hydroxide to give an intermediate
1) containing 5-membered cyclic thiourea were designed, 7.18,19) In case of benzylated amines 7, the reduction reaction
∗ To whom correspondence should be addressed. e-mail: jungshh@cnu.ac.kr.
© 2010 Pharmaceutical Society of Japan

July 2010
919
Table 1. Inhibitory Activity of 4 and 5 on Melanogenesis of Melanoma B
16 Cells
was carried out with iron powder in acetic acid at 50—70 °C
temperature for 2 h to yield 8.20) To get the another important
intermediate 10, the phenyl chloroformate was reacted with 7
under refluxing condition for 7 h to achieve 9,21) which was
subsequently subjected to reduction using catalytic hydro-
genation at ambient temperature to produce 10.22) Further the
diamine 8 was treated with phenyl chloroformate using xy-
lene at refluxing temperature for 33 h to give title compound
4.21) On the other hand, 10 in xylene underwent cyclization at
reflux temperature to produce 4.21) Finally, benzo[d]imidazol-
2(3H)-ones 4 in toluene treated with Lawesson’s reagent at
reflux temperature to obtain the corresponding thiones 5.23)
Inhibition
% at 10 mM
C log Pc)
mM
Compound
R1
n
X
IC50 values
4a
4b
4c
4d
4e
5a
5b
5c
5d
CH3
Cl
CH3
CH3
Cl
CH3
Cl
CH3
CH3
Cl
1
1
2
3
2
1
1
2
3
2
O
O
O
O
O
S
S
S
S
S
Ͻ10
Ͻ10
Ͻ10
Ͻ10
Ͻ10
Ͼ100
Ͼ100
Ͼ100
Ͼ100
Ͼ100
Ͼ100
40
Ͼ100
Ͼ10
Ͼ10
Ͼ10
Ͼ10
1.7
1.9
1.5
1.3
2.0
3.881
4.305
4.210
4.589
4.634
3.521
3.735
3.850
4.229
4.064
4.289
2.381
2.940
Results and Discussion
For all the synthesized compounds 4 and 5 the ability to
inhibit formation of melanin from melanoma B-16 cells was
determined under stimulus of a-MSH (100 nM) for 3 d incu-
bation. Melanoma B16 cell (CRL6323) were obtained from
ATCC (Manassas, U.S.A.). Amounts of melanin released into
the culture media were determined by measuring absorbance
values at 405 nm with synthetic melanin as the standard.24)
Data for % inhibition at 10 mM and IC50 values are mean val-
ues from 3 to 5 separate experiments as shown in Table 1.
Among the synthesized compounds the benzimidazole-
2(3H)-thiones 5 shows promising inhibitory activity against
melanin production in melanoma B16 cells as compared to
5e
1a)
0.8
Ͼ10
2.9
2b)
3b)
93
Arbutin
Kojic acid
—
—
180
70
a) The activity associated with compound 1 was reported in ref. 18. b) The ac-
tivity associated with compounds 2 and 3 was reported in ref. 24. c) C log P values
were calculated by Chemdraw ver. 9.0.
kojic acid (IC50ϭ70 mM) and arbutin (IC50ϭ180 mM). Inter- Conclusion
estingly, these compounds 5 have almost comparable activity
Taken our studies together, the benzimidazole-2(3H)-
to 1 (Ͼ100% inhibition at 10 mM, IC50ϭ0.8 mM). The potent thione motif of 5 is an essential structural unit for their in-
compound 5c (Ͼ100% inhibition at 10 mM, IC50ϭ1.5 mM) has hibitory activity. Among all thiones (5a—e), the compound
almost similar structure to that of 1 with an exception of one 5d (IC50ϭ1.3 mM) most strongly inhibits the melanin forma-
methylene unit on heterocyclic motif. This result intimates tion. Therefore benzo[d]imidazole-2(3H)-thiones 5 could be
that the activities of both quinazolidine-2-thione 1 and benz- quite promising compound for the treatment of hyperpig-
imidazolidine-2-thione 5 are not dependent on variation of mentation.
ring size. In our previous studies24) we observed that imida-
Experimental
zolidine-2-thione analog 2 (42% inhibition at 10 mM, IC50ϭ
General Procedures Melting points (mp) were determined on Electro
Ͼ10 mM) exhibited very low activity unlike six membered
thermal 1A 9100 MK2 apparatus and are uncorrected. All commercial
chemicals were used as obtained and all solvents were purified by the stan-
dard procedures prior to use. Thin layer chromatography was performed on
E Merck silica gel GF-254 per-coated plates and the identification was done
with UV light and colorization with spray 10% phosphomolybdic acid fol-
lowed by heating. Flash column chromatography was performed with E
Merck silica gel (230—400 mesh). IR spectra were recorded with Jasco
IR-Report-100 IR spectrometer in cmϪ1 and corrected against peak at 1601
cmϪ1 of polystyrene. NMR spectra were measured against the peak of
tetramethylsilane by Varian Unity Inova 400 NMR (400 MHz) spectrome-
ters. High resolution-mass spectrum (HR-MS) was recorded on API2000
mass spectrometer (PE Sciex, Toronto, Canada).
cyclic thiourea 3 (93% inhibition at 10 mM, IC50ϭ2.9 mM) as
shown in Fig. 1. Thus, the benzene ring fused with imidazoli-
dine-2-thione of 5 is an essential motif for their inhibitory
activity. Moreover, different position of phenylalkyl sub-
stituent on cyclic thiourea unit in 1 and 5 does not affect the
activity.
Methyl and chloro substituents at position 5 of benzimida-
zole-2-thione of 5 do not change activity as shown in the
activity of 5a (Ͼ100% inhibition at 10 mM, IC50ϭ1.7 mM,
C log Pϭ3.521) and 5b (Ͼ100% inhibition at 10 mM, IC50ϭ
1.9 mM, C log Pϭ3.735). Likewise, compound 5c (Ͼ100%
inhibition at 10 mM, IC50ϭ1.5 mM, C log Pϭ3.850), 5d (Ͼ
100% inhibition at 10 mM, IC50ϭ1.3 mM, C log Pϭ4.229) and
5e (Ͼ100% inhibition at 10 mM, IC50ϭ2.0 mM, C log Pϭ
4.064) show similar level of activity. This indicates that the
size of side chain and the lipophilicity of compounds 5 do
not have much influence on their inhibitory activity.
Unlike benzimidazole-2(3H)-thiones 5, their counterpart
benzo[d]imidazol-2(3H)-ones 4a—e gave a very weak in-
hibitory activity (their IC50 values were more than 10 mM and
inhibition was less than 30% at 10 mM). Thus thiourea unit of
5 is very critical for the activity, which was also observed in
our previous studies with analogs of 1.25)
Synthesis of N-Benzyl-4-methyl-2-nitrobenzenamine (7a) 4-Methyl-
2-nitroaniline (6a, 3 g, 19.0 mmol) and pyridine (4.78 ml, 59.0 mmol) were
dissolved in methylenechloride (150 ml). After stirred for 10 min, benzyl
bromide (3.52 ml, 29.0 mmol) was added. The resulting reaction mixture
was refluxed for 16 h. After cooled, the reaction mixture was washed with
water three times. The organic layer was dehydrated with anhydrous sodium
sulfate and solvent was removed under vacuum. The desired compound 7a
was isolated by column chromatography. Brown solid. Yield 90.3%. mp
1
87.4—88.5 °C. Rfϭ0.53 (hexane : ethyl acetateϭ5 : 1). H-NMR (CDCl3) d:
8.33 (br, 1H), 7.99 (s, 1H), 7.34—7.17 (m, 6H), 6.72 (d, 1H, Jϭ8.8 Hz),
4.54 (d, 2H, Jϭ5.6 Hz), 2.25 (s, 3H). IR (KBr) cmϪ1: 3400, 1635, 1530,
1510. HR-MS Calcd for C14H14N2O2: 242.1055. Found: 242.1049.
Synthesis of N-Benzyl-4-chloro-2-nitrobenzenamine (7b) Using the
same reaction condition for the preparation of 7a, 4-chloro-2-nitroaniline
(6b, 3 g, 17.0 mmol) was treated with benzylbromide (2.48 ml, 20.0 mmol)
in the presence of pyridine (1.69 ml, 20.0 mmol) for the preparation of 7b.
Brown solid. Yield 67.1%. mp 78.0—78.2 °C. Rfϭ0.46 (hexane : ethyl ac-
1
etateϭ5 : 1). H-NMR (CDCl3) d: 8.42 (br, 1H), 8.20 (m, 1H), 7.47—7.26

920
Vol. 58, No. 7
(m, 6H), 6.77 (m, 1H), 4.55 (d, 2H, Jϭ5.6 Hz). IR (KBr) cmϪ1: 3400, 1622,
1563. HR-MS Calcd for C13H11ClN2O2: 262.0509. Found: 262.0503.
Synthesis of 4-Methyl-2-nitro-N-phenethylbenzenamine (7c) To the
solution of 4-methyl-2-nitroaniline (6a, 5 g, 32 mmol) in toluene, sodium
2H, Jϭ7.6 Hz). IR (KBr) cmϪ1: 3100, 2850, 1730. HR-MS Calcd for
C21H17ClN2O4: 396.0877. Found: 396.0879.
Synthesis of Phenyl N-(4-Methyl-2-aminophenyl)-N-phenylethylcar-
bamate (10a) Compound 9a (1.5 g, 3.0 mol) was dissolved in the co-sol-
hydroxide (64 mmol) was added and the resulting mixture was stirred for vent (30 ml) of ethanol and dichloromethane (1 : 2). After addition of Pd–C
5 min at room temperature. 2-Bromoethylbenzene (6.73 ml, 48.0 mmol) was (0.150 g), the mixture was stirred under hydrogen gas (45 psi) for 12 h at
added drop wise for 20 min. The resulting mixture was heated at 60—70 °C
room temperature. After filtration through celite pad, the filtrate was evapo-
for 3—4 h. After cooled to ambient condition, solvent was removed under rated under vacuum to give pure 10a. Brown solid. Yield 94.0%. mp
vacuum and the residue was dissolved in dichloromethane and washed with 127.3—127.8 °C. Rfϭ0.15 (hexane : ethyl acetateϭ5 : 1). 1H-NMR (acetone-
water. The organic layer was dehydrated with anhydrous sodium sulfate and d6) d: 7.42—6.68 (m, 13H), 4.60 (br, NH2), 3.65 (m, 2H), 2.92 (t, 2H, Jϭ
evaporated under vacuum. The desired compound 7c was isolated by column 8.1 Hz), 2.21(s, 3H). IR (KBr) cmϪ1: 3500, 3330, 3100, 1710, 1620. HR-MS
chromatography. Brown solid. Yield 54.0%. mp 79.4—79.8 °C. Rfϭ0.45 Calcd for C22H22N2O2: 346.1681. Found: 346.1699.
(hexane : ethyl acetateϭ5 : 1). 1H-NMR (CDCl3) d: 7.84 (br, 1H), 7.31—
6.92 (m, 8H), 3.91 (t, 2H, Jϭ7.4 Hz), 3.05 (t, 2H, Jϭ7.4 Hz), 2.44 (s, 3H).
Synthesis of Phenyl N-(4-Methyl-2-aminophenyl)-N-phenylpropylcar-
bamate (10b) Using the same reaction condition for the preparation of
IR (KBr) cmϪ1: 2950, 2830, 1635, 1545. HR-MS Calcd for C15H16N2O2: 10a, compound 10b was obtained from 9b (1 g, 2.0 mmol). Brown oil. Yield
256.1212. Found: 256.1203.
93.5%. Rfϭ0.28 (hexane : ethyl acetateϭ2 : 1). 1H-NMR (acetone-d6) d:
Synthesis of 4-Methyl-2-nitro-N-(3-phenylpropyl)benzenamine (7d) 8.23—6.71 (m, 13H), 4.82 (br, NH2), 4.02—3.21 (m, 2H), 2.66 (t, 2H, Jϭ
Using the same reaction condition for the preparation of 7c, 7d was obtained 7.8 Hz), 2.27 (s, 3H), 2.06 (m, 2H). IR (KBr) cmϪ1: 3470, 3380, 3050, 2940,
from 4-methyl-2-nitroaniline (6a, 3 g, 19.0 mmol) and 1-bromo-3-phenyl-
propane (3.59 ml, 48.0 mmol). Brown solid. Yield 39.8%. mp 94.9—95.2
°C. Rfϭ0.58 (hexane : ethyl acetateϭ5 : 1). 1H-NMR (CDCl3) d: 7.97 (br,
1H), 7.47—7.23 (m, 6H), 6.70 (d, 1H, Jϭ8.8 Hz), 3.29 (m, 2H), 2.77 (t, 2H,
2850, 1715, 1620. HR-MS-ESI Calcd for C23H24N2O2: 360.1838. Found:
360.1827.
Synthesis of Phenyl N-(4-Chloro-2-aminophenyl)-N-phenylethylcar-
bamate (10c) Using the same reaction condition for the preparation of
Jϭ7.4 Hz), 2.25 (s, 3H), 2.10 (m, 2H). IR (KBr) cmϪ1: 3400, 1635, 1541. 10a, compound 10c was obtained from 9c (1 g, 2.0 mmol). Brown solid.
HR-MS Calcd for C16H18N2O2: 270.1368. Found: 270.1361.
Yield 78.6%. mp 119.0—120.8 °C. Rfϭ0.53 (hexane : ethyl acetateϭ2 : 1).
Synthesis of 4-Chloro-2-nitro-N-phenethylbenzenamine (7e) Using
1H-NMR (acetone-d6) d: 7.35—6.55 (m, 13H), 5.01 (br, NH2), 4.11—3.42
the same reaction condition for the preparation of 7c, 7e was obtained from (m, 2H), 2.96 (t, 2H, Jϭ7.9 Hz). IR (KBr) cmϪ1: 3450, 3350, 3240, 2940,
4-chloro-2-nitroaniline (6b, 5 g, 29.0 mmol) and 1-bromo-2-phenylethane
1720, 1670, 1630. HR-MS Calcd for C21H19ClN2O2: 366.1135. Found:
(5.94 ml, 43.0 mmol). Orange solid. Yield 38.2%. mp 83.2—84.2 °C. Rfϭ 366.1126.
0.51 (hexane : ethyl acetateϭ5 : 1). 1H-NMR (CDCl3) d: 8.14 (br, 1H),
Synthesis of 1-Benzyl-5-methyl-1H-benzo[d]imidazol-2(3H)-one (4a)
7.42—7.20 (m, 6H), 6.79 (d, 1H, Jϭ9.2 Hz), 3.51 (m, 2H), 3.00 (t, 2H, Jϭ To the solution of diamine 8a (0.500 g, 2.0 mmol) in xylene (10 ml),
7.1 Hz). IR (KBr) cmϪ1: 3350, 3100, 3020, 2950, 2830, 1620. HR-MS Calcd phenylchloroformate (0.325 ml, 2.2 mmol) was added in drop wise. The re-
for C14H13ClN2O2: 276.0666. Found: 276.0659.
sulting mixture was refluxed for 33 h. After cooling, xylene was removed
under vacuum. The residue was dissolved in dichloromethane (100 ml) and
Synthesis of N1-Benzyl-4-methylbenzene-1,2-diamine (8a) Compound
7a (2 g, 8.0 mmol) and Fe powder (4.6 g, 8.0 mmol) were added to the glacial extracted with water (70 ml) twice. The organic layer was dehydrated over
acetic acid (30 ml) the resulting mixture was allowed to heated at 50—70 °C anhydrous sodiumsulfate and evaporated under vacuum. The residue was
for 2 h. The resulting mixture was filtered and the filter cake was washed subjected to column chromatography for isolation of 4a. White solid. Yield
with hot ethanol (50 ml) pre-heated at 60—70 °C. The filtrate was evapo- 52.5%. mp 191.2—192.4 °C. Rfϭ0.076 (hexane : ethyl acetateϭ2 : 1). 1H-
rated under vacuum and the residue was subjected to column chromatogra- NMR (CDCl3) d: 9.33 (br, 1H), 7.46—6.66 (m, 8H), 5.07 (s, 2H), 2.34 (s,
phy for separation of 8a. Violet solid. Yield 51.4%. mp 111.9—112.4 °C. 3H). IR (KBr) cmϪ1: 3250, 2900, 1705, 1675. HR-MS Calcd for C15H14N2O:
Rfϭ0.30 (hexane : ethyl acetateϭ5 : 1). 1H-NMR (CDCl3) d: 7.41—7.13 (m, 238.1106. Found: 238.1101.
5H), 6.38 (m, 2H), 5.87 (d, 1H, Jϭ7.9 Hz), 4.21 (s, 2H), 3.67 (br, 1H), 2.17
Synthesis of 1-Benzyl-5-chloro-1H-benzo[d]imidazol-2(3H)-one (4b)
(s, 3H). IR (KBr) cmϪ1: 3450, 3360, 3050, 2940, 1710, 1620. HR-MS-elec- Using the same reaction condition for the preparation of 4a, compound 4b
trospray ionization (ESI) Calcd for C14H16N2: 212.1313. Found: 212.1307.
was obtained from compound 8b (0.300 g, 0.1.2 mmol) and phenyl chloro-
Synthesis of N1-Benzyl-4-chlorobenzene-1,2-diamine (8b) Using the
formate (0.17 ml, 1.26 mmol). White solid. Yield 54.3%; mp 185.2—
1
same reaction condition for the preparation of 8a, compound 8b was ob- 187.2 °C. Rfϭ0.15 (hexane : ethyl acetateϭ2 : 1). H-NMR (CDCl3) d: 10.2
tained from 7b (2 g, 7.0 mmol). Dark brown solid. Yield 65.1%. mp 115.3—
(br, 1H), 7.31—6.70 (m, 8H), 5.08 (s, 2H). IR (KBr) cmϪ1: 3250, 2680,
1
117.5 °C. Rfϭ0.24 (hexane : ethyl acetateϭ5 : 1). H-NMR (CDCl3) d: 7.32 1740—1650, 1620, 1600. HR-MS Calcd for C14H11ClN2O: 258.0560.
(m, 5H), 6.42 (m, 2H), 5.81 (m, 1H), 4.21 (s, 2H), 3.77 (br, 1H). IR (KBr)
cmϪ1: 3350, 3070, 3030, 2970, 2920, 2880, 2850, 1595. HR-MS-ESI Calcd
for C13H13ClN2: 232.0767. Found: 232.0752.
Found: 258.0545.
Synthesis of 5-Methyl-1-phenethyl-1H-benzo[d]imidazol-2(3H)-one
(4c) To the solution of compound 10a (0.500 g, 1.4 mmol) in toluene
Synthesis of Phenyl N-(4-Methyl-2-nitrophenyl)-N-phenylethylcarba- (20 ml), sodium hydroxide (0.115 g, 2.8 mmol) and water (0.5 ml) were
mate (9a) To the mixture of compound 7c (2 g, 7.0 mmol) in xylene added. The resulting mixture was refluxed for 3 h. After cooled, toluene was
(20 ml) was added phenylchloroformate (2.94 ml, 21.0 mmol). The resulting removed under vacuum. The residue was dissolved in dichloromethane
reaction mixture was refluxed for 7 h. After cooling, xylene was removed (100 ml) and extracted with water (70 ml) twice. The organic layer was
under vacuum and the residue was dissolved in dichloromethane and washed dehydrated with anhydrous sodiumsulfate and evaporated under vacuum.
with water. The organic layer was dehydrated over anhydrous sodium sulfate The residue was subjected to column chromatography for isolation of 10c.
and evaporated under vacuum. The residue was purified by column chro- Pale pink solid. Yield 74.4%. mp 139.9—140.7 °C. Rfϭ0.15 (hexane : ethyl
matography to give desired compound 9a. Pale brown solid. Yield 93.8%. acetateϭ2 : 1). 1H-NMR (CDCl3) d: 10.6 (br, 1H), 7.24—6.08 (m, 8H), 4.09
mp 110.1—110.3 °C. Rfϭ0.18 (hexane : ethyl acetateϭ5 : 1). 1H-NMR (t, 2H, Jϭ7.6 Hz), 3.04 (t, 2H, Jϭ7.6 Hz), 2.35 (s, 3H). IR (KBr) cmϪ1
:
(CDCl3) d: 7.62—6.55 (m, 13H), 3.80 (t, 2H Jϭ7.6 Hz), 2.99 (t, 2H, Jϭ 3250, 2600, 1740, 1600. HR-MS Calcd for C16H16N2O: 252.1263. Found:
7.6 Hz), 2.27 (s, 3H). IR (KBr) cmϪ1: 3100, 2850, 1730. HR-MS Calcd for 252.1247.
C22H20N2O4: 376.1423. Found: 376.1418.
Synthesis of Phenyl N-(4-Methyl-2-nitrophenyl)-N-(3-phenylpropyl) one (4d) Using the same reaction condition for the preparation of 4c, com-
carbamate (9b) Using the same reaction condition for the preparation of pound 4d was obtained from compound 10b (0.250 g, 6.94 mmol). Pale
9a, compound 9b was obtained from 7d (1 g, 3.7 mmol). Yellow solid. Yield brown solid. Yield 33.5%. mp 132.1—132.5 °C. Rfϭ0.13 (hexane : ethyl ac-
Synthesis of 5-Methyl-1-(3-phenylpropyl)-1H-benzo[d]imidazol-2(3H)-
1
93.7%. mp 104.6—105.3 °C. Rfϭ0.22 (hexane : ethyl acetateϭ5 : 1). 1H-
etateϭ2 : 1). H-NMR (CDCl3) d: 9.66 (br, NH), 7.26 (m, 6H), 6.93—6.86
NMR (CDCl3) d: 7.83—7.04 (m, 13H), 3.78 (m, 2H), 2.68 (t, 2H, Jϭ (m, 2H), 3.91 (t, 2H, Jϭ7.2 Hz), 2.72 (t, 2H, Jϭ7.8 Hz), 2.37 (s, 3H), 2.08
8.5 Hz), 2.44 (s, 3H), 2.07 (m, 2H). IR (KBr) cmϪ1: 3050, 2920, 2850, 1730.
HR-MS Calcd for C23H22N2O4: 390.1580. Found: 390.1572.
(m, 2H). IR (KBr) cmϪ1: 3260, 2800, 1705, 1650. HR-MS Calcd for
C17H18N2O: 266.1419. Found: 266.1410.
Synthesis of Phenyl N-(4-Chloro-2-nitrophenyl)-N-phenylethylcarba-
mate (9c) Using the same reaction condition for the preparation of 9a,
Synthesis of 5-Chloro-1-phenethyl-1H-benzo[d]imidazol-2(3H)-one (4e)
Using the same reaction condition for the preparation of 4c, compound 4e
compound 9e was obtained from 7e (1.5 g, 5.0 mmol). Pale brown solid. was obtained from compound 10c (0.400 g, 1.0 mmol). Pale brown solid.
Yield 90.2%. mp 114.3—115.8 °C. Rfϭ0.20 (hexane : ethyl acetateϭ5 : 1).
Yield 93.26%. mp 176.1—177.8 °C. Rfϭ0.18 (hexane : ethyl acetateϭ2 : 1).
1H-NMR (CDCl3) d: 7.62—6.55 (m, 13H), 3.80 (t, 2H, Jϭ7.6 Hz), 2.99 (t,
1H-NMR (CDCl3) d: 10.8 (br, 1H), 7.22—6.63 (m, 8H), 4.08 (t, 2H,

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Jϭ7.3 Hz), 3.03 (t, 2H, Jϭ7.3 Hz). IR (KBr) cmϪ1: 3250, 3000, 1705, 1650.
HR-MS Calcd for C15H13ClN2O: 272.0716. Found: 272.0707.
19, 550—571 (2006).
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(2002).
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Ortonne J. P., Oxford University Press, New York, 1998, pp. 760—
766.
7) Urabe K., Nakayama J., Hori Y., “The Pigmentary System: Physiology
and Pathophysiology,” ed. by Norlund J. J., Boissy R. E., Hearing V. J.,
King R. A., Ortonne J. P., Oxford University Press, New York, 1998,
pp. 909—991.
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(2003).
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Biol. Inorg. Chem., 5, 262 (2000).
Synthesis of 1-Benzyl-5-methyl-1H-benzo[d]imidazole-2(3H)-thione
(5a) To the solution of compound 4 (0.050 g, 2.10 mmol) in toluene (5 ml),
Lawesson’s reagent (0.053 g, 1.2 mmol) was added. The resulting mixture
was refluxed for 18 h. After cooling, toluene was removed under vacuum.
The residue was dissolved in dichloromethane (100 ml) and extracted with
water (70 ml) twice. The organic layer was dehydrated with anhydrous
sodium sulfate and evaporated under vacuum. The residue was subjected
to column chromatography for isolation of 5a. Pale yellow solid. Yield
50.9%. mp 212.2—212.8 °C. Rfϭ0.39 (hexane : ethyl acetateϭ2 : 1). 1H-
NMR (CDCl3) d: 10.7 (br, 1H), 7.39—6.91 (m, 8H), 5.52 (s, 2H), 2.38 (s,
3H). IR (KBr) cmϪ1: 3200, 2850, 1475. HR-MS Calcd for C15H14N2S:
254.0878. Found: 254.0866.
Synthesis of 1-Benzyl-5-chloro-1H-benzo[d]imidazole-2(3H)-thione
(5b) Using the same reaction condition for the preparation of 5a, com-
pound 5b was obtained from compound 4b (0.070 g, 2.70 mmol) and Lawes-
son’s reagent (0.065 g, 1.67 mmol). White solid. Yield 30.9%. mp 213.3—
1
214.5 °C. Rfϭ0.68 (hexane : ethyl acetateϭ2 : 1). H-NMR (CDCl3) d: 11.0
(br, 1H), 7.32—6.87 (m, 8H), 5.44 (s, 2H). IR (KBr) cmϪ1: 3200, 3020,
2980, 2900, 2850, 1620. HR-MS Calcd for C14H11ClN2S: 274.0331. Found:
274.0326.
11) Maeda K., Fukuda M., J. Pharmacol. Exp. Ther., 276, 765—769
(1996).
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Nozawa Y., Biosci. Biotechnol. Biochem., 67, 1587—1589 (2003).
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Kin Y. S., Biochem. Biophys. Res. Commun., 243, 801—803 (1998).
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Chem., 277, 6340—6344 (2002).
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V., Hearing V. J. Jr., Dooly T. P., Biochem. Pharmacol., 57, 663—672
(1999).
Synthesis of 5-Methyl-1-(2-phenylethyl)-1H-benzo[d]imidazole-2(3H)-
thione (5c) Using the same reaction condition for the preparation of 5a,
compound 5c was obtained from compound 4c (0.100 g, 0.0396 mmol) and
Lawesson’s reagent (0.096 g, 0.023 mmol). White solid. Yield 48.0%. mp
1
206.6—207.2 °C. Rfϭ0.42 (hexane : ethyl acetateϭ2 : 1). H-NMR (CDCl3)
d: 11.7 (br, 1H), 7.83—6.84 (m, 8H), 4.42 (t, 2H, Jϭ7.6 Hz), 3.08 (t, 2H,
Jϭ7.6 Hz), 2.34 (s, 3H). IR (KBr) cmϪ1: 3200, 2850, 1470. HR-MS Calcd
for C16H16N2S: 268.1034. Found: 268.1030.
Synthesis of 5-Methyl-1-(3-phenylpropyl)-1H-benzo[d]imidazole-2(3H)-
thione (5d) Using the same reaction condition for the preparation of 5a,
compound 5d was obtained from compound 4d (0.030 g, 0.0112 mmol) and
Lawesson’s reagent (0.027 g, 0.0063 mmol). White solid. Yield 53.4%. mp
1
128.4—129.2 °C. Rfϭ0.56 (hexane : ethyl acetateϭ2 : 1). H-NMR (CDCl3)
17) Passi S., Nazzaro-Porro M. B., J. Dermatol., 104, 659—665 (1981).
18) Kim Y., Jung S. H., Min K., Park J., Lee J., Korean Patent KR747042
(2007).
d: 7.29—6.83 (m, 8H), 4.30 (t, 2H, Jϭ7.5 Hz), 2.77 (t, 2H, Jϭ7.6 Hz), 2.39
(s, 3H), 2.17 (m, 2H). IR (KBr) cmϪ1: 3160—2850, 1515. HR-MS Calcd for
C17H18N2S: 282.1191. Found: 282.1178.
19) Samuel A., Brown, Carnelo J. R., Synth. Commun., 26, 4065—4080
(1996).
20) Boyce R. S., Guo H., Mendenhall K. G., Walter A. O., Wang W., Xiu
Y., (Novertis Vaccine and Diagnostic, Inc., U.S.A.) U. S. Patent Appl.
Publ. US 20060420 AI 20060420 (2006).
Synthesis of 5-Chloro-1-phenethyl-1H-benzo[d]imidazole-2(3H)-thione
(5e) Using the same reaction condition for the preparation of 5a, com-
pound 5e was obtained from compound 4e (0.100 g, 0.0366 mmol) and
Lawesson’s reagent (0.089 g, 0.0216 mmol). White solid. Yield 65.2%. mp
1
219.4—220.5 °C. Rfϭ0.54 (hexane : ethyl acetateϭ2 : 1). H-NMR (CDCl3)
21) Gomez-Parra V., Sanchez F., Torres T., Monatshefte fur Chemie, 116,
639—644 (1985).
22) Orjales A., Mosguera R., Rodes R., J. Med. Chem., 40, 586—593
(1997).
d: 11.10 (br, 1H), 7.25—6.72 (m, 8H), 4.46 (t, 2H, Jϭ7.6 Hz), 3.13 (t, 2H,
Jϭ7.6 Hz). IR (KBr) cmϪ1: 3200, 2750, 1620, 1607, 1500. HR-MS Calcd
for C15H13ClN2S: 288.0488. Found: 288.0480.
23) Cava M. P., Levinson M. I., Tetrahedron, 41, 5061—5087 (1985).
24) Thanigaimalai P., Lee K. C., Bang S. C., Lee J. H., Yun C. Y., Roh E.,
Hwang B. Y., Kim Y., Jung S. H., Bioorg. Med. Chem., 18, 1135—
1142 (2010).
Acknowledgment This work was supported by a grant (R01-2007-000-
20099-0) and Priority Research Centers Program (2009-0093815) through
the National Research Foundation of Korea (NRF) funded by the Ministry of
Education, Science and Technology.
25) Thanigaimalai P., Lee K. C., Bang S. C., Lee J. H., Yun C. Y., Roh E.,
Hwang B. Y., Kim Y., Jung S. H., Bioorg. Med. Chem., 18, 1555—
1562 (2010).
References and Notes
1) Francisco S., Stefania B., Mauro P., Ghanem G., Pigment Cell Res.,