Synthesis and anti-melanogenic activity of hydroxyphenyl benzyl ether analogues

Article abstract of DOI:10.1016/j.bmc.2011.02.044

In order to develop potent skin whitening agents, we have synthesized 17 hydroxyphenyl benzyl ether compounds and tested their melanin synthesis inhibitory activity, DPPH free radical scavenging activity and tyrosinase inhibitory activity. Compounds 32, 35 and 36 possessing 4-hydroxyphenyl benzyl ether structure showed excellent inhibitory capacity with almost 50-fold than arbutin used as a reference in the inhibition test of α-MSH stimulated melanin synthesis in B-16 cells. 4-Hydroxyphenyl benzyl ether compounds also showed good antioxidant activity in the DPPH free radical scavenging test. The tyrosinase function was effectively inhibited by 3,5-dihydroxyphenyl benzyl ether analogues, especially compounds 18, 22, and 24.

Full text of DOI:10.1016/j.bmc.2011.02.044

Bioorganic & Medicinal Chemistry 19 (2011) 2168–2175
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and anti-melanogenic activity of hydroxyphenyl benzyl ether
analogues
Kiran Sapkota a, Eunmiri Roh b, Eunyoung Lee c, Eun-Mi Ha a, Jae-Ho Yang d, Eung-Seok Lee e,
f,
Youngjoo Kwon c, Youngsoo Kim b, , Younghwa Na
⇑
⇑
a College of Pharmacy, Catholic University of Daegu, Gyeongsan, Gyeongbuk, Republic of Korea
b College of Pharmacy, Chungbuk National University, Cheongju 361-763, Republic of Korea
c College of Pharmacy, Ewha Womans University, Seoul 120-750, Republic of Korea
d The Marine Biotechnology Research Center, Catholic University of Daegu, Gyeongsan, Gyeongbuk, Republic of Korea
e College of Pharmacy, Yeungnam University, Gyeongsan, 712-749, Republic of Korea
f College of Pharmacy, Cha University, Pochon, 487-010, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to develop potent skin whitening agents, we have synthesized 17 hydroxyphenyl benzyl ether
compounds and tested their melanin synthesis inhibitory activity, DPPH free radical scavenging activity
and tyrosinase inhibitory activity. Compounds 32, 35 and 36 possessing 4-hydroxyphenyl benzyl ether
structure showed excellent inhibitory capacity with almost 50-fold than arbutin used as a reference in
Received 14 December 2010
Revised 20 February 2011
Accepted 23 February 2011
Available online 26 February 2011
the inhibition test of a-MSH stimulated melanin synthesis in B-16 cells. 4-Hydroxyphenyl benzyl ether
compounds also showed good antioxidant activity in the DPPH free radical scavenging test. The tyrosi-
nase function was effectively inhibited by 3,5-dihydroxyphenyl benzyl ether analogues, especially com-
pounds 18, 22, and 24.
Keywords:
a-MSH induced melanin synthesis
Tyrosinase
Ó 2011 Elsevier Ltd. All rights reserved.
DPPH free radical scavenging
4-Hydroxyphenyl benzyl ether
3,5-Dihydroxyphenyl benzyl ether
1. Introduction
Tyrosinase is a multifunctional copper containing glycoprotein
and induces the browning of fruits, vegetables and mammalian
Melanin is one of the most widely distributed pigments and
found in bacteria, fungi, plants and animals.1 Melanin pigments
are known to be biosynthesized in a lysosome-like organells,
termed melanosomes, of a specialized pigment producing cells
called melanocytes, through the process of melanogenesis. Natural
melanin is a mixed-type copolymer of black–brown eumelanin and
red–yellow sulfur-containing pheomelanin. The type and amount
of melanin synthesized by melanocytes and its distribution in the
surrounding keratinocytes determines the actual color of the skin.2
Various dermatological disorders result in the accumulation of
excessive levels of epidermal pigmentation. These hyperpigmented
lentigines include melasma, age spots or liver spots, and sites of
actinic damage.3,4
melanogenesis. This is also known as polyphenol oxidase. A number
of studies have been conducted on targeting tyrosinase to relieve
melanogenesis since its discovery.5,6 Tyrosinase, known to be the
most critical and rate limiting enzyme during melanin biosynthesis,
exists ubiquitously from microorganisms to plants and animals.7
It catalyzes first two steps in the melanin biosynthesis, converting
L-tyrosine to dopa and then dopaquinone (o-quinone type). Due to
the essential role of tyrosinase, the approach to inhibit tyrosinase
function is most common to achieve skin depigmentation.8
Kojic acid (1)9 and arbutin (2)10 are well-known hypopigment-
ing agents. Recently, bibenzyl analogues (3) are reported to have
potent anti-tyrosinase activity with almost 20-fold stronger than
kojic acid.11
OGlucose
HO
OH
HO
O
OH
O
HO
OH
O
OH
Bibenzyl analogue (3)
OH
Kojic acid (1)
Arbutin (2)
Monobenzone (4)
⇑
Corresponding authors. Tel.: +82 2 555 4076; fax: +82 2 3468 3373 (Y.K.); tel.: +82 43 261 2823; fax: +82 43 268 2732 (Y.N.).
E-mail addresses: yna7315@cha.ac.kr (Y. Kim), youngsoo@chungbuk.ac.kr (Y. Na).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.02.044

K. Sapkota et al. / Bioorg. Med. Chem. 19 (2011) 2168–2175
2169
OCH3
A
OH
OH
HO
O
i
ii
OCH3
HO
OH
AcO
AcO
OAc
OR
OR
5
6 R = Ac
7 R = H
8 R = 3,5-Dimethoxybenzyl
HO
OR
B
ii
OAc
OH
9 R = 4-Fluorobenzyl
11 R = 2-Chlorobenzyl
13 R = 3-Chlorobenzyl
18 R = 4-Fluorobenzyl
20 R = 2-Chlorobenzyl
22 R = 3-Chlorobenzyl
24 R = 3,4-Dichlorobenzyl
OH
15 R = 3,4-Dichlorobenzyl
i
AcO
OAc
OR
OR1
5
HO
AcO
ii
OR
OR2
10 R1, R2 = 4-Fluorobenzyl
12 R1, R2 = 2-Chlorobenzyl
14 R1, R2 = 3-Chlorobenzyl
16 R1, R2 = 3,4-Dichlorobenzyl
19 R = 4-Fluorobenzyl
21 R = 2-Chlorobenzyl
23 R = 3-Chlorobenzyl
25 R = 3,4-Dichlorobenzyl
17 R1 = 3,4-Dichlorobenzyl, R2 = H
Scheme 1. Synthesis of hydroxyphenyl benzyl ether analogues. Reagents and conditions: (A) (i) Ac2O, pyridine, reflux; (ii) 3,5-dimethoxybenzyl bromide, K2CO3, acetone,
reflux. (B) (i) Benzyl halide, K2CO3, acetone, reflux; (ii) 1.0 M KOH, ethanol, reflux.
Hydroquinone is known to inhibit tyrosinase enzyme and thus stop
conversion of DOPA to melanin.12 Monobenzone, a monobenzyl
ether of hydroquinone (MBEH), has similar biological property to
hydroquinone, which is subjected to selective uptake by melano-
cytes and is metabolized into reactive free radicals, resulting in
permanently destroying melanocytes.13
As an effort to develop potential skin whitening agents we de-
signed and synthesized 3,5-dihydroxyphenyl or 4-hydroxyphenyl
benzyl ether analogues possessing halogens or methoxy groups
on the aromatic ring in benzyl part. We chose halogens and meth-
oxy groups as mild electron withdrawing and donating groups to
evaluate the effect of electron disposition in the benzyl part. Com-
pounds prepared were tested for the tyrosinase inhibitory and
anti-melanogenesis activities. Since anti-oxidation can be a meth-
od to reduce the free radical formation and auto-oxidation, we also
conducted anti-oxidation activity of compounds using DPPH free
radical scavenging test.
(5%) (Scheme 1A). In the 1H NMR spectrum, compound 6 showed
a singlet peak at d 2.24 corresponding to acetyl protons. Further
coupling reaction of compound 5 with corresponding benzyl halide
under the same condition in Scheme 1A provided monobenzyl (9,
11, 13, 15) and dibenzyl (10, 12, 14, 16) ether compounds at the
same time. In the reaction of 3,4-dichlorobenzyl chloride reaction,
compound 17 was also isolated from product mixture, which lost
one acetyl group in the course of reaction. Subsequent alkaline
hydrolysis of acetyl protecting group gave desired benzyl ether
compounds 18–25 in 62–99% reaction yields (Scheme 1B). All the
spectral data were consistent with proposed structures.
2.1.2. Synthesis of 4-hydroxyphenyl benzyl compounds 32–36
Compound 26 was selected as protected starting compound for
preparation 4-hydroxyphenyl benzyl ether compounds 32–36 It
was prepared by acetylation of hydroquinone with acetic anhy-
dride in pyridine. Williamson ether reaction of compound 26 with
corresponding benzyl halide in the presence of K2CO3 in acetone
afforded acetyl protected benzyl ether compounds 27–31 in 61–
71% reaction yields. Subsequent alkaline hydrolysis of compounds
gave desired benzyl ether compounds 32–36 in 86–96% reaction
yields (Scheme 2). All the spectral data were consistent with pro-
posed structures.
2. Results and discussion
2.1. Chemistry
2.1.1. Synthesis of 3,5-dihydroxyphenyl benzyl ether
compounds 6–8, and 18–25
2.2. Pharmacological evaluation
Synthetic methods are straightforward. Compound 5 was selected
as starting compound for preparation of 3,5-dihydroxyphenyl
benzyl ether compounds 6–25. It was prepared by di-acetylation
of phloroglucinol with acetic anhydride in pyridine. Williamson
ether synthesis of compound 5 with 3,5-dimethoxybenzyl bromide
with K2CO3 in acetone provided compound 6 (14%), 7 (33%) and 8
2.2.1.
a-MSH-induced melanin synthesis inhibitory activity
in vitro
Melanin synthesis inhibition was studied in mouse melanoma
B-16 cells. Most 3,5-dihydroxyphenyl benzyl ether compounds

2170
K. Sapkota et al. / Bioorg. Med. Chem. 19 (2011) 2168–2175
OH
OH
OH
OR
OR
iii
i
ii
HO
AcO
OAc
32 R = 3,5-dimethoxy
33 R = 4-Fluorobenzyl
34 R = 2-Chlorobenzyl
35 R = 3-Chlorobenzyl
36 R = 3,4-Dichlorobenzyl
27 R = 3,5-dimethoxy
28 R = 4-Fluorobenzyl
29 R = 2-Chlorobenzyl
30 R = 3-Chlorobenzyl
31 R = 3,4-Dichlorobenzyl
26
Scheme 2. Synthesis of compounds 32–36 from hydroquinone. Reagents and conditions: (i) Ac2O, pyridine, reflux; (ii) benzyl halide, K2CO3, acetone, reflux; (iii) 1.0 M KOH,
ethanol, reflux.
were not active to
induced B-16 cells at 10
a
-melanocyte-stimulating hormone (
a-MSH)
Table 2
DPPH free radical scavenging activity of compounds
lM concentration. But 4-hydroxyphenyl
benzyl compounds showed better melanin synthesis inhibitory
activities than 3,5-dihydroxyphenyl benzyl ether compounds
without cell toxicity within tested concentration range. The results
are listed in Table 1. Of the compounds, 32, 35 and 36 were potent
a
Compound
% Inhbition at 100
l
M
EC50 (lM)
Vitamin C
6
7
8
93.9
1.7
26.2 1.5
NDb
128.7 25.7
ND
140.0 85.9
ND
165.4 7.7
ND
46.8
27.4
46.6
21.2
42.8
15.6
49.0
22.1
8.00
37.3
17.0
54.1
48.2
46.5
54.7
33.2
for inhibiting melanin synthesis without cell toxicity up to 10
Compounds 33 and 34 showed good melanin synthesis inhibitory
activity at 10 M concentration, but they were toxic at 30 M con-
lM.
18
19
20
21
22
23
17
24
25
32
33
34
35
36
l
l
centration. From a view of structural point, the results showed that
the substitution of chlorine group (compounds 34, 35, and 36)
might enhance the melanin synthesis inhibitory activity than that
of fluorine (compound 33). Location of chlorine also affected the
activity of these series of compounds (compound 35 vs. 34). These
observations imply that 4-hydroxyphenyl benzyl ether moiety
possessing proper types and location of substituents is very impor-
tant to inhibit melanin synthetic pathways.
119.4 14.7
ND
ND
ND
ND
76.3 3.9
101.7 7.2
113.1 3.3
76.8 3.20
ND
2.2.2. DPPH Free radical scavenging activity
a
Concentration for scavenging of 50% of DPPH free radical was expressed in
Polyphenol compounds are well known with their antioxidant
property. Since our compounds also have phenolic hydroxyl group,
we intended to test anti-oxidative activity of compounds whether
antimelanogenic activity of the compounds is induced by antioxi-
dant function. The anti-oxidative activity of compounds was tested
by measuring their ability to convert DPPH (1,1-diphenyl-2-picryl
hydrazyl) free radical to DPPH-H (1,1-diphenyl-2-picryl hydra-
zine). It was monitored by measuring the change in light absorp-
tion at 517 nm using microplate reader. The results are indicated
in Table 2. 3,5-Dihydroxyphenyl benzyl ether compounds were
mean value standard deviation obtained in triplicate.
b
Not determined.
weak anti-oxidants. But most 4-hydroxyphenyl benzyl compounds
showed moderate antioxidant activity except 36. These results are
parallel with melanin synthesis inhibitory activity stream. Both
compounds 32 and 35 were most active among the series of com-
pounds. From the results, it seems that 4-hydroxyphenyl moiety is
important for the anti-oxidation activity in these compounds.
2.2.3. Tyrosinase inhibitory activity
Compounds were evaluated for tyrosinase inhibitory activity
using L-tyrosine as a substrate. The percentage inhibition is pre-
Table 1
Inhibitory activity of compounds on a-MSH-induced melanogenesis
a
Compound
% Inhibition at 10
lM
IC50
(
lM)
sented in Table 3. The test results of compounds were contradic-
tory to that of melanin synthesis inhibition. 4-Hydroxyphenyl
benzyl compounds were all inactive to tyrosinase function (data
not shown). However some of 3,5-dihydroxyphenoxy benzyl
compounds 18, 22 and 24 showed superior tyrosinase inhibitory
activities to that of arbutin. The results suggest that 3,5-dihydroxy-
phenyl moiety with proper species and location of substituents
in this series is necessary for inhibiting tyrosinase function.
These results were inconsistent with previous observation14
which implied the importance of p-hydroxyphenyl group for
the tyrosinase inhibitory process due to its structural resemblance
Arbutin
6
7
NDb
ND
19
6
ND
14
12
11
33
18
21
15
4
99.0
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
1.93
ND
ND
1.83
1.87
8
17
18
19
20
21
22
23
24
25
32
33
34
35
36
to L-tyrosine, a substrate for tyrosinase during early stage of
melanogenesis.
>100
42
55
78
87
3. Conclusion
We have synthesized 17 hydroxyphenylbenzyl ether compounds
and tested their melanin synthesis inhibitory activity, DPPH free
radical scavenging activity and tyrosinase inhibitory activity. In the
a
The IC50 value was expressed as the mean of 50% inhibitory concentrations of
triplicate determinations.
b
Not determined.

K. Sapkota et al. / Bioorg. Med. Chem. 19 (2011) 2168–2175
2171
Table 3
(0.040–0.063 mm). All solvents used for chromatography were di-
rectly used without distillation. NMR spectra were recorded on
Varian AS 400 (1H NMR at 400 MHz and 13C NMR at 100 MHz) with
tetramethylsilane (TMS) as an internal standard. Chemical shift (d)
values are expressed in ppm and coupling constant (J) values in
hertz (Hz). GC mass data were recorded using Agilent-5975C
MSD (USA) and Shimadzu GCMS-QP2010 (Japan).
Tyrosinase inhibitory activity of compounds
Compound
% Inhbition at 100
l
M
IC50 (lM)
Arbutin
6
7
NDa
ND
27.5
0
ND
55.5
5.5
45.5
3.5
70.5
1.5
66.0
0
1550
ND
ND
ND
ND
90.0
ND
ND
ND
54.7
ND
66.4
ND
ND
ND
ND
ND
ND
8
17
18
19
20
21
22
23
24
25
32
33
34
35
36
4.2. General procedure 1
To the solution of acetoxyphenol in acetone were added
sequentially potassium carbonate and benzyl halide under nitro-
gen at rt. The mixture was allowed to reflux overnight. After cool-
ing to rt, the mixture was filtered and washed with acetone. The
filtrate was concentrated under reduced pressure and purified of
by SGC to afford acetoxyphenylbenzyl ether compound.
0
1.5
0
1.0
9.5
4.3. General procedure 2
a
Not determined.
To the acetylated compound were added 1.0 M KOH (aq.) and
ethanol in 1:1 (v/v) ratio. The reaction mixture was refluxed
(1 h). After cooling to rt, the mixture was neutralized with 1.0 M
HCl solution. Solid formed was collected by filtration, washed with
water and dried to give a desired product. In some cases, where
there was no precipitation, reaction mixture was extracted with
EtOAc and the organic layer was washed with brine and dried over
anhydrous Na2SO4. Solvent was removed under reduced pressure.
Wherever necessary, it was further purified by SGC to yield the
hydroxyphenyl benzyl ether compound.
inhibition test of
a-MSH-induced melanin synthesis using B-16
cells, compounds 32, 35 and 36 possessing 4-hydroxyphenyl
benzyl ether structure moiety showed excellent inhibitory capac-
ity with almost 50-fold than arbutin used as a reference. The
structure-activity relationship analysis revealed that species and
location of substituents are also important factor for the inhibi-
tion of melanogenesis. 4-Hydroxyphenyl benzyl ether compounds
also showed moderate antioxidant activity in DPPH free radical
scavenging test. But 3,5-dihydroxyphenyl benzyl ether analogues
were mostly weaker than 4-hydroxyphenyl benzyl ether com-
pounds. The tyrosinase function was effectively inhibited by 3,5-
dihydroxyphenyl benzyl ether analogues, especially compounds
18, 22, and 24. These three compounds possess 3,5-dihydroxy-
phenyl moiety, which may reflect the importance of this core for
tyrosinase inhibitory activity in the hydroxyphenyl benzyl ether
analogues. The results, however, was discrepant with those of the
previous DPPH free radical scavenging test and tyrosinase inhibi-
tory activity of 4-hydroxy-20-nitrodiphenyl ether analogues.14 At
this point, although we don’t have a clear clue for the discrepancy,
we suspect that the electron disposition property between benzyl
and nitrophenyl groups affect the biological functions of the 4-
hydroxyphenyl benzyl ether and 4-hydroxy-2’-nitrodiphenyl ether
analogues. Because there was no clear correlation between inhibi-
tion of melanongenesis and tyrosinase function of the compounds
32, 35, and 36, anti-melanogenic activity of the compounds might
be derived from affecting on the other pathways including anti-
oxidation during the melanin biosynthesis process. Further mech-
anism study will enhance the possibility of the compounds to be
developed as potential candidate for skin whitening agent.
4.4. 3,5-Diacetoxy phenol (5)
To a solution of phloroglucinol (4.00 g, 31.72 mmol) in pyridine
(50 mL) was added acetic anhydride (8.09 g, 79.30 mmol) under
stirring. The reaction mixture was refluxed overnight (120–
130 °C) and then pyridine was removed under reduced pressure.
The residue was purified by SGC (EtOAc/n-hexane = 1:5) to afford
compound 5 (4.95 g, 74.5%) as a white solid. Rf = 0.56 (EtOAc/n-
hexane = 1:1); mp 110–112 °C; 1H NMR (400 MHz, CDCl3) d 2.28
(s, 6H), 6.44 (d, 2H, J = 2.0 Hz), 6.45 (t, 1H, J = 2.0 Hz); 13C NMR
(100 MHz, CDCl3) 21.4, 107.2, 107.4, 151.7, 157.4, 169.8 ppm.
4.5. 5-(3,5-Dimethoxybenzyloxy)benzene-1,3-diol (6), 3-(3,5-
dimethoxybenzyloxy)-5-hydroxyphenyl acetate (7) and 3,5-
bis(3,5-dimethoxybenzyloxy)phenol (8)
Using the general procedure 1, a reaction mixture of compound
5 (1.07 g, 5.06 mmol), 3,5-dimethoxy benzyl bromide (1.17 g,
5.06 mmol) and K2CO3 (1.40 g, 10.14 mmol) in acetone (25 mL)
was refluxed (15 h). Purification by SGC (EtOAc/n-hexane = 1:4)
provided compound 6 (240 mg, 14.2%) as a light brown viscous li-
quid, compound 7 (462 mg, 33.1%) and compound 8 (98 mg, 4.5%)
as light brown viscous liquid. Compound 6: Rf = 0.56 (EtOAc/n-hex-
ane = 1:1); 1H NMR (400 MHz, CDCl3) d 2.24 (s, 3H), 3.75 (s, 6H),
4.86 (s, 2H), 6.16 (t, 1H, J = 2.0 Hz), 6.25 (t, 1H, J = 2.0 Hz), 6.27 (t,
1H, J = 2.0 Hz), 6.39 (t, 1H, J = 2.0), 6.52 (d, 2H, J = 2.0, Hz); 13C
NMR (100 MHz, CDCl3) 21.3, 55.5, 70.2, 100.3, 100.9, 102.4,
105.5, 139.1, 152.1, 157.7, 160.4, 161.1, 170.2 ppm; EI-MS (m/z)
318.1 [M]+. Compound 7: Rf = 0.39 (EtOAc/n-hexane = 1:1); 1H
NMR (400 MHz, CDCl3) d 3.77 (s, 6H), 4.86 (s, 2H), 5.94 (t, 1H,
J = 2.0 Hz), 6.04 (d, 2H, J = 2.4 Hz), 6.40 (t, 1H, J = 2.0 Hz), 6.54 (d,
2H, J = 2.0, Hz); 13C NMR (100 MHz, CDCl3) 55.6, 70.2, 95.6, 96.3,
100.1, 105.6, 139.4, 157.6, 160.9, 161.1 ppm; EI-MS (m/z) 276.0
[M]+. Compound 8: Rf = 0.22 (CH2Cl2); 1H NMR (400 MHz, CDCl3)
d 3.78 (s, 12H), 4.90 (s, 4H), 5.86 (brs, 1H), 6.10 (s, 2H), 6.22 (s,
1H), 6.42 (s, 2H), 6.57 (s, 4H); 13C NMR (100 MHz, CDCl3) 55.5,
4. Experimental
4.1. General
Most of the chemicals and reagents used were purchased from
Aldrich Chemical Co. and some were from other companies like
Junsei, Acros Organics, Tokyo Chemicals. Melting points were mea-
sured in open capillaries with Barnstead Electrothermal melting
point apparatus, Manual MEL-TEMP (Model No: 1202D) without
correction. Chromatographic separations were monitored by
thin-layer chromatography (TLC) using commercially available
pre-coated Merck Kieselgel 60 F254 plate (0.25 mm) and detected
by visualizing under UV at 254 and 365 nm. Silica gel column
chromatography (SGC) was carried out with Merck Kieselgel 60

2172
K. Sapkota et al. / Bioorg. Med. Chem. 19 (2011) 2168–2175
70.1, 95.0, 95.6, 100.1, 105.5, 139.3, 157.6, 160.8, 161.1 ppm; EI-MS
128.4, 130.1, 134.7, 138.6, 152.4, 160.1, 169.4 ppm; EI-MS (m/z)
(m/z) 426.1 [M]+.
416.0 [M]+.
4.6. 5-(4-Fluorobenzyloxy)-1,3-phenylene diacetate (9) and
3,5-bis(4-fluorobenzyloxy)phenyl acetate (10)
4.9. 5-(3,4-Dichlorobenzyloxy)-1,3-phenylene diacetate (15),
3,5-bis(3,4-dichlorobenzyloxy)phenyl acetate (16) and 3-(3,4-
dichlorobenzyloxy)-5-hydroxyphenyl acetate (17)
Using the general procedure 1, a reaction mixture of compound
5 (0.40 g, 1.90 mmol), 4-fluorobenzyl bromide (0.36 g, 1.90 mmol)
and K2CO3 (5.30 g, 3.80 mmol) in acetone (15 mL) was refluxed
(15 h). Purification by SGC (EtOAc/n-hexane = 1:7) yielded
compound 9 (369 mg, 26.2%) as a cream colored solid and
compound 10 (191 mg, 2.6%) as a white solid. Compound 9:
Rf = 0.45 (EtOAc/n-hexane = 1:3); mp 112–114 °C; 1H NMR
Using the general procedure 1, a reaction mixture of compound
(0.23 g, 1.10 mmol), 3,4-dichlorobenzyl chloride (0.22 g,
5
1.10 mmol) and K2CO3 (0.30 g, 2.20 mmol) in acetone (15 mL)
was refluxed (12 h). Purification by SGC (EtOAc/n-hexane = 1:6)
afforded compound 15 (70 mg, 17.3%) as a light gray liquid, com-
pound 16 (60 mg, 11.2%) and compound 17 (60 mg, 16.7%) as
white solids. Compound 15: Rf = 0.39 (EtOAc/n-hexane = 1:3); 1H
NMR (400 MHz, CDCl3) d 2.26 (s, 6H), 4.94 (s, 2H), 6.57 (t, 1H,
J = 2.0 Hz), 6.60 (d, 2H, J = 2.0 Hz), 7.21 (d, 1H, J = 8.0 Hz), 7.43 (d,
1H, J = 8.0 Hz), 7.49 (s, 1H); 13C NMR (100 MHz, CDCl3) 21.3, 69.1,
106.3, 108.7, 126.8, 129.4, 130.8, 132.3, 132.9, 136.6, 151.9,
159.5, 169.0 ppm; EI-MS (m/z) 368.0 [M]+. Compound 16:
Rf = 0.60 (EtOAc/n-hexane = 1:3); mp 166–168 °C; 1H NMR
(400 MHz, CDCl3) d 2.28 (s, 3H), 4.95 (s, 4H), 6.35 (d, 2H,
J = 2.0 Hz), 6.42 (t, 1H, J = 2.0 Hz), 7.22 (dd, 2H, J = 2.0, 8.0 Hz),
7.45 (d, 2H, J = 8.0 Hz), 7.50 (d, 2H, J = 2.0 Hz); 13C NMR
(100 MHz, CDCl3) 21.4, 69.0, 100.0, 101.9, 126.7, 129.4, 130.8,
132.4, 133.0, 136.9, 152.5, 160.0, 169.4 ppm; EI-MS (m/z) 484.0
[M]+. Compound 17: Rf = 0.23 (EtOAc/n-hexane = 1:3); mp
124–126 °C; 1H NMR (400 MHz, CDCl3/CD3OD) d 2.23 (s, 3H),
4.88 (s, 2H), 6.19 (d, 2H, J = 1.2 Hz), 6.28 (t, 1H, J = 1.6 Hz), 7.18
(dd, 1H, J = 1.6, 8.0 Hz), 7.40 (d, 1H, J = 8.4, Hz), 7.46 (d, 1H,
J = 1.6, Hz); 13C NMR (100 MHz, CDCl3) 21.5, 68.7, 100.3, 102.6,
113.0, 126.7, 129.3, 130.7, 132.1, 132.8, 137.1, 152.2, 158.4,
160.0, 170.0 ppm; EI-MS (m/z) 326.0 [M]+.
(400 MHz, CDCl3)
d 2.25 (s, 6H), 4.94 (s, 2H), 6.55 (t, 1H,
J = 1.6 Hz), 6.61 (d, 2H, J = 1.6 Hz), 7.05 (dd, 2H, J = 7.2, 8.4 Hz),
7.35 (dd, 2H, J = 5.6, 8.0, Hz); 13C NMR (100 MHz, CDCl3) 21.2,
69.9, 106.3, 108.3, 115.6, 115.8, 129.6, 132.1, 151.8, 159.8, 161.5,
164.0, 169.1 ppm; EI-MS (m/z) 318.1 [M]+. Compound 10:
Rf = 0.50 (EtOAc/n-hexane = 1:3); mp 132–134 °C; 1H NMR
(400 MHz, CDCl3) d 2.29 (s, 3H), 4.97 (s, 4H), 6.37 (d, 2H,
J = 2.0 Hz), 6.47 (t, 1H, J = 1.6 Hz), 7.08 (dd, 4H, J = 8.0, 8.0 Hz),
7.38 (dd, 4H, J = 6.0, 8.0 Hz); 13C NMR (100 MHz, CDCl3) 21.4,
69.8, 100.0, 101.6, 115.7, 115.9, 129.7, 132.4, 152.4, 160.3, 161.6,
164.0, 169.5 ppm; EI-MS (m/z) 384.1 [M]+.
4.7. 5-(2-Chlorobenzyloxy)-1,3-phenylene diacetate (11) and
3,5-bis(2-chlorobenzyloxy)phenyl acetate (12)
Using the general procedure 1, a reaction mixture of compound
5 (0.30 g, 1.43 mmol), 4-fluorobenzyl bromide (0.29 g, 1.43 mmol)
and K2CO3 (0.39 g, 2.85 mmol) in acetone (15 mL) was reluxed
(15 h). Purification by SGC (EtOAc/n-hexane = 1:7) yielded com-
pound 11 (250 mg, 52.3%) as an off-white solid and compound
12 (277 mg, 67.6%) as light brown liquid. Compound 11: Rf = 0.47
(EtOAc/n-hexane = 1:3); mp 98–100 °C; 1H NMR (400 MHz, CDCl3)
d 2.27 (s, 6H), 5.12 (s, 2H), 6.57 (t, 1H, J = 2.0 Hz), 6.64 (d, 2H,
J = 1.6 Hz), 7.26–7.32 (m, 2H), 7.40 (dd, 1H, J = 2.0, 7.2 Hz), 7.52
(dd, 1H, J = 2.8, 7.2 Hz); 13C NMR (100 MHz, CDCl3) 21.3, 67.8,
106.4, 108.7, 127.3, 129.1, 129.4, 129.6, 132.8, 134.2, 151.9,
159.8, 169.1 ppm; EI-MS (m/z) 334.0 [M]+. Compound 12:
Rf = 0.60 (EtOAc/n-hexane = 1:3); mp 88–90 °C; 1H NMR
(400 MHz, CDCl3) d 2.28 (s, 3H), 5.12 (s, 4H), 6.40 (d, 2H,
J = 2.0 Hz), 6.52 (t, 1H, J = 2.4 Hz), 7.24–7.31 (m, 4H), 7.39 (dd,
2H, J = 1.6, 7.2 Hz), 7.52 (dd, 2H, J = 2.4, 6.8 Hz); 13C NMR
(100 MHz, CDCl3) 21.4, 67.6, 100.0, 101.8, 127.2, 129.0, 129.3,
129.6, 132.8, 134.4, 152.4, 160.2, 169.4 ppm; EI-MS (m/z) 416.0
[M]+.
4.10. 5-(4-Fluorobenzyloxy)benzene-1,3-diol (18)
Using the general procedure 2, compound
9 (140 mg,
0.44 mmol) was hydrolyzed in1.0 M KOH (7 mL) and ethanol
(7 mL). After extraction with EtOAc (3 ꢀ 30 mL), compound 18
(103 mg, 99.7%) was obtained as a brick red solid. Rf = 0.10
(EtOAc/n-hexane = 1:3); mp 80–82 °C; 1H NMR (400 MHz, CDCl3/
CD3OD) d 4.87 (s, 2H), 5.91 (t, 1H, J = 1.6 Hz), 5.96 (d, 2H,
J = 1.2 Hz), 6.99 (dd, 2H, J = 8.8, 8.8 Hz), 7.31 (dd, 2H, J = 5.6,
8.4 Hz); 13C NMR (100 MHz, CDCl3/CD3OD) 69.4, 94.5, 96.0,
115.4, 115.6, 129.6, 132.9, 158.6, 160.7, 161.4, 163.8 ppm; EI-MS
(m/z) 234.0 [M]+.
4.11. 3,5-Bis(4-fluorobenzyloxy)phenol (19)
4.8. 5-(3-Chlorobenzyloxy)-1,3-phenylene diacetate (13) and
3,5-bis(3-chlorobenzyloxy)phenyl acetate (14)
Using the general procedure 2, compound 10 (99 mg,
0.25 mmol) was hydrolyzed in1.0 M KOH (3 mL) and ethanol
(3 mL). After extraction with EtOAc (2 ꢀ 30 mL), compound 19
(87 mg, 98.8%) was obtained as an off-white solid. Rf = 0.33
(EtOAc/n-hexane = 1:3); mp 82–84 °C; 1H NMR (400 MHz, CDCl3)
d 4.95 (s, 4H), 5.34 (s, 1H), 6.10 (d, 2H, J = 1.6 Hz), 6.20 (t, 1H,
J = 1.6 Hz), 7.07 (dd, 4H, J = 7.6, 8.8 Hz), 7.37 (dd, 4H, J = 5.6,
7.6 Hz); 13C NMR (100 MHz, CDCl3) 69.6, 95.0, 95.7, 115.6, 115.8,
129.6, 132.7, 157.7, 160.8, 161.5, 163.9 ppm; EI-MS (m/z) 342.1
[M]+.
Using the general procedure 1, a reaction mixture of compound
5 (0.40 g, 1.90 mmol), 3-chlorobenzyl bromide (0.39 g, 1.90 mmol)
and K2CO3 (0.53 g, 3.80 mmol) in acetone (15 mL) was refluxed
(12 h). Purification by SGC (EtOAc/n-hexane = 1:6) afforded com-
pound 13 (352 mg, 55.3%) and compound 14 (206 mg, 26.0%) as
cream colored solids. Compound 13: Rf = 0.41 (EtOAc/n-hex-
ane = 1:3); mp 66–68 °C; 1H NMR (400 MHz, CDCl3) d 2.25 (s,
6H), 4.96 (s, 2H), 6.56 (t, 1H, J = 2.0 Hz), 6.61 (d, 2H, J = 2.0 Hz),
7.24–7.30 (m, 3H), 7.39 (s, 1H); 13C NMR (100 MHz, CDCl3) d
21.3, 69.7, 106.3, 108.5, 125.6, 127.6, 128.5, 130.1, 134.7, 138.4,
151.8, 159.7, 169.0 ppm; EI-MS (m/z) 334.0 [M]+. Compound 14:
Rf = 0.48 (EtOAc/n-hexane = 1:3); mp 76–78 °C; 1H NMR
(400 MHz, CDCl3) d 2.27 (s, 3H), 4.95 (s, 4H), 6.36 (d, 2H,
J = 1.6 Hz), 6.45 (t, 1H, J = 2.0 Hz), 7.24–7.30 (m, 6H), 7.39 (s, 2H);
13C NMR (100 MHz, CDCl3) 21.3, 69.6, 100.0, 101.8, 125.6, 127.6,
4.12. 5-(2-Chlorobenzyloxy)benzene-1,3-diol (20)
Using the general procedure 2, compound 11 (146 mg,
0.43 mmol) was hydrolyzed in 1.0 M KOH (6 mL) and ethanol
(6 mL). After extraction with EtOAc (3x30 mL), compound 20
(103 mg, 94.3%) was obtained as a brick red solid. Rf = 0.10
(EtOAc/n-hexane = 1:3); mp 130–132 °C; 1H NMR (400 MHz,

K. Sapkota et al. / Bioorg. Med. Chem. 19 (2011) 2168–2175
2173
CDCl3/CD3OD) d 5.08 (s, 2H), 5.98 (t, 1H, J = 0.8 Hz), 6.05 (d, 2H,
J = 1.2 Hz), 7.22–7.29 (m, 2H), 7.37 (dd, 1H, J = 1.2, 7.2 Hz), 7.51
(dd, 1H, J = 1.2, 7.2 Hz); 13C NMR (100 MHz, CDCl3/CD3OD) 67.2,
94.4, 96.0, 127.0, 128.9, 129.0, 129.3, 132.6, 134.8, 158.5,
160.6 ppm; EI-MS (m/z) 250.0 [M]+.
CD3OD) 68.7, 94.2, 95.8, 126.7, 129.4, 130.7, 132.1, 132.9, 137.3,
158.8, 160.4 ppm; EI-MS (m/z) 443.9 [M]+.
4.18. 4-Acetoxy phenol (26)
After dissolving hydroquinone (4.00 g, 36.33 mmol) in pyridine
(50 mL), was added drop wise acetic anhydride (5.56 g, 54.5 mmol)
under stirring. Reaction mixture was allowed to reflux (120–
130 °C) overnight. Pyridine was removed under reduced pressure
before purification by SGC (EtOAc/n-hexane = 1:4) to yield com-
pound 26 (3.45 g, 62.4%) as a white solid. Rf = 0.53 (EtOAc/n-hex-
ane = 1:3); mp 146–148 °C; 1H NMR (400 MHz, CDCl3) d 2.27 (s,
3H), 6.74 (dd, 2H, J = 2.8, 8.8 Hz), 6.89 (dd, 2H, J = 2.8, 8.8 Hz); 13C
NMR (100 MHz, CDCl3) 21.3, 116.3, 122.5, 144.0, 154.0, 170.9 ppm.
4.13. 3,5-Bis(2-chlorobenzyloxy)phenol (21)
Using the general procedure 2, compound 12 (85 mg,
0.20 mmol) was hydrolyzed in 1.0 M KOH (3 mL) and ethanol
(3 mL). Solid formed after neutralization with 1.0 M HCl was col-
lected by filtration and dried to obtain compound 21 (67 mg,
88.0%) as an off-white solid. Rf = 0.43 (EtOAc/n-hexane = 1:3); mp
92–94 °C; 1H NMR (400 MHz, CDCl3) d 4.92 (s, 1H), 5.11 (s, 4H),
6.13 (s, 2H), 6.26 (s, 1H), 7.25–7.29 (m, 4H), 7.38 (d, 2H,
J = 7.6 Hz), 7.52 (d, 2H, J = 6.8 Hz, H-30, 300); 13C NMR (100 MHz,
CDCl3) 67.4, 95.2, 95.7, 127.0, 129.0, 129.2, 129.6, 132.8, 134.7,
157.6, 160.8 ppm; EI-MS (m/z) 374.0 [M]+.
4.19. 4-(3,5-Dimethoxybenzyloxy)phenyl acetate (27)
Using the general procedure 1, a reaction mixture of compound
26 (0.41 g, 2.7 mmol), 3,5-dimethoxy benzyl bromide (0.63 g,
2.7 mmol) and K2CO3 (0.75 g, 5.40 mmol) in acetone (15 mL) was
refluxed (20 h). Purification by SGC (EtOAc/n-hexane = 1:4) was
done to afford compound 27 (494 mg, 60.5%) as a cream colored
semi-solid. Rf = 0.42 (EtOAc/n-hexane = 1:3); 1H NMR (400 MHz,
CDCl3) d 2.25 (s, 3H), 3.77 (s, 6H), 4.96 (s, 2H), 6.41 (t, 1H,
J = 2.4 Hz), 6.57 (d, 2H, J = 2.4 Hz), 6.94 (d, 2H, J = 8.0 Hz), 6.54 (d,
2H, J = 8.4 Hz); 13C NMR (100 MHz, CDCl3) 21.1, 55.4, 70.4, 100.0,
105.3, 115.5, 122.4, 139.3, 144.5, 156.5, 161.1, 169.9 ppm; EI-MS
(m/z) 302.1 [M]+.
4.14. 5-(3-Chlorobenzyloxy)benzene-1,3-diol (22)
Using the general procedure 2, compound 13 (143 mg,
0.43 mmol) was hydrolyzed in 1.0 M KOH (7 mL) and ethanol
(7 mL). Solid formed after neutralization with 1.0 M HCl was col-
lected by filtration and dried to obtain compound 22 (71 mg,
66.3%) as a cream colored solid. Rf = 0.14 (EtOAc/n-hexane = 1:3);
mp 130–132 °C; 1H NMR (400 MHz, CDCl3/CD3OD) d 4.93 (s, 2H),
5.97 (s, 1H), 6.03 (s, 2H), 7.27–7.32 (m, 3H), 7.39 (s, 1H); 13C NMR
(100 MHz, CDCl3/CD3OD) 69.3, 94.8, 96.3, 125.6, 127.6, 128.2,
130.0, 134.6, 139.2, 158.3, 160.7 ppm; EI-MS (m/z) 250.0 [M]+.
4.20. 4-(4-Fluorobenzyloxy)phenyl acetate (28)
4.15. 3,5-Bis-(3-chlorobenzyloxy)phenol (23)
Using the general procedure 1, a reaction mixture of compound
26 (0.29 g, 1.94 mmol), 4-fluorobenzyl bromide (0.36 g,
1.94 mmol) and K2CO3 (0.54 g, 3.88 mmol) in acetone (15 mL)
was refluxed (15 h). Purification by SGC (EtOAc/n-hexane = 1:5)
afforded compound 28 (357 mg, 70.7%) as an off-white solid.
Rf = 0.52 (EtOAc/n-hexane = 1:3); mp 102–104 °C; 1H NMR
(400 MHz, CDCl3) d 2.29 (s, 3H), 5.02 (s, 2H), 6.95 (dd, 2H, J = 1.6,
8.8 Hz), 7.01 (dd, 2H, J = 1.2, 9.2 Hz), 7.08 (dd, 2H, J = 7.2, 8.0 Hz),
7.40 (dd, 2H, J = 5.6, 7.2 Hz); 13C NMR (100 MHz, CDCl3) 21.3,
70.0, 115.6, 115.8, 122.6, 129.6, 132.7, 144.7, 156.5, 161.5,
170.1 ppm; EI-MS (m/z) 260.1 [M]+.
Using the general procedure 2, compound 14 (95 mg,
0.23 mmol) was hydrolyzed in 1.0 M KOH (2 mL) and ethanol
(2 mL). Solid formed after neutralization with 1.0 M HCl was col-
lected by filtration and dried to yield compound 23 (72 mg,
84.6%) as an off-white solid. Rf = 0.34 (EtOAc/n-hexane = 1:3); mp
80–82 °C; 1H NMR (400 MHz, CDCl3) d 4.96 (s, 4H), 4.99 (s, 1H),
6.09 (s, 2H), 6.18 (s, 1H), 7.25–7.29 (m, 4H), 7.40 (s, 2H); 13C
NMR (100 MHz, CDCl3) 69.4, 95.1, 95.7, 125.6, 127.6, 128.4,
130.1, 134.7, 139.0, 157.6, 160.7 ppm; EI-MS (m/z) 374.0 [M]+.
4.16. 5-(3,4-Dichlorobenzyloxy)benzene-1,3-diol (24)
4.21. 4-(2-Chlorobenzyloxy)phenyl acetate (29)
Using the general procedure 2, compound 15 (55 mg,
0.15 mmol) was hydrolyzed in 1.0 M KOH (4 mL) and ethanol
(4 mL). After extraction with EtOAc (2x20 mL), compound 24
(39 mg, 91.8%) was obtained as a cream colored solid. Rf = 0.10
(EtOAc/n-hexane = 1:3); mp 146–148 °C; 1H NMR (400 MHz,
CDCl3/CD3OD) d 4.89 (s, 2H), 5.93 (t, 1H, J = 2.0 Hz), 5.96 (d, 2H,
J = 2.0 Hz), 7.19 (d, 1H, J = 8.4 Hz) 7.39 (d, 1H, J = 8.0 Hz), 7.47 (s,
2H); 13C NMR (100 MHz, CDCl3/CD3OD) 68.6, 94.5, 96.2, 126.7,
129.3, 130.6, 131.9, 132.7, 137.6, 158.6, 160.5 ppm; EI-MS (m/z)
284.0 [M]+.
Using the general procedure 1, a reaction mixture of compound
26 (0.23 g, 1.48 mmol), 2-chlorobenzyl bromide (0.30 g,
1.48 mmol) and K2CO3 (0.40 g, 2.96 mmol) in acetone (15 mL) re-
fluxed (15 h). Purification by SGC (EtOAc/n-hexane = 1:6) afforded
compound 29 (277 mg, 67.6%) as a light brown liquid. Rf = 0.58
(EtOAc/n-hexane = 1:3); 1H NMR (400 MHz, CDCl3) d 2.26 (s, 3H),
5.14 (s, 2H), 6.96 (dd, 2H, J = 2.4, 9.2 Hz), 7.01 (dd, 2H, J = 2.4,
9.2 Hz), 7.24–7.28 (m, 2H), 7.38 (dd, 1H, J = 2.0, 7.2 Hz), 7.53 (dd,
1H, J = 2.0, 7.2 Hz); 13C NMR (100 MHz, CDCl3) 21.3, 67.7, 115.7,
122.6, 127.2, 129.0, 129.2, 129.6, 132.8, 134.8, 144.8, 156.4,
170.0 ppm; EI-MS (m/z) 276.0 [M]+.
4.17. 3,5-Bis-(3,4-dichlorobenzyloxy)phenol (25)
4.22. 4-(3-Chlorobenzyloxy)phenyl acetate (30)
Using the general procedure 2, compound 16 (40 mg,
0.08 mmol) was hydrolyzed in 1.0 M KOH (2 mL) and ethanol
(2 mL). Solid formed after neutralization with 1.0 M HCl was col-
lected by filtration and dried to obtain compound 25 (24 mg,
66.0%) as an off-white solid. Rf = 0.35 (EtOAc/n-hexane = 1:3); mp
140–142 °C; 1H NMR (400 MHz, CDCl3/CD3OD) d 4.92 (s, 4H),
6.07 (s, 2H), 6.08 (s,1H), 7.21 (dd, 2H, J = 1.6, 8.0 Hz), 7.42 (d, 2H,
J = 8.0 Hz), 7.49 (d, 2H, J = 1.2 Hz); 13C NMR (100 MHz, CDCl3/
Using the general procedure 1, a reaction mixture of compound
26 (0.34 g, 2.23 mmol), 3-chlorobenzyl bromide (0.46 g, 2.23 mmol)
and K2CO3 (0.62 g, 4.46 mmol) in acetone (15 mL) was refluxed
(15 h). Purification by SGC (EtOAc/n-hexane = 1:5) afforded com-
pound 30 (470 mg, 76.0%) as a white crystalline solid. Rf = 0.51
(EtOAc/n-hexane = 1:3); mp 94–96 °C; 1H NMR (400 MHz, CDCl3) d
2.29 (s, 3H,), 5.02 (s, 2H), 6.96 (dd, 2H, J = 2.0, 8.8 Hz), 7.02 (dd, 2H,

2174
K. Sapkota et al. / Bioorg. Med. Chem. 19 (2011) 2168–2175
J = 2.0, 8.8 Hz), 7.27–7.31 (m, 3H, H-4’), 7.43 (s, 1H); 13C NMR
(100 MHz, CDCl3) 21.3, 69.8, 115.7, 122.7, 125.6, 127.6, 128.4,
130.1, 134.7, 139.1, 144.8, 156.4, 170.1 ppm; EI-MS (m/z) 276.0 [M]+.
(100 MHz, CDCl3) 70.2, 116.2, 116.3, 125.6, 127.6, 128.2, 130.0,
134.7, 139.6, 150.1, 152.9 ppm; EI-MS (m/z) 234.0 [M]+.
4.28. 4-(3,4-Dichlorobenzyloxy)phenol (36)
4.23. 4-(3,4-Dichlorobenzyloxy)phenyl acetate (31)
Using the general procedure 2, compound 31 (150 mg,
0.48 mmol) was hydrolyzed in 1.0 M KOH (3 mL) and ethanol
(3 mL). Solid formed after neutralization with 1.0 M HCl was col-
lected by filtration and dried to yield compound 36 (124 mg,
95.6%) as a gray solid. Rf = 0.33 (EtOAc/n-hexane = 1:3); mp 100–
102 °C; 1H NMR (400 MHz, CDCl3) d 4.70 (brs, 1H), 4.94 (s, 2H),
6.76 (d, 2H, J = 8.8 Hz), 6.82 (d, 2H, J = 8.4 Hz), 7.23 (d, 1H,
J = 8.4 Hz), 7.43 (d, 1H, J = 8.0 Hz), 7.51 (s, 1H); 13C NMR
(100 MHz, CDCl3) 69.6, 116.3, 116.4, 126.7, 129.5, 130.7, 132.1,
132.9, 137.8, 150.2, 152.7 ppm; EI-MS (m/z) 268.0 [M]+.
Using the general procedure 1, a reaction mixture of compound
26 (0.39 g, 2.56 mmol), 3,4-dichlorobenzyl chloride (0.50 g,
2.56 mmol) and K2CO3 (0.70 g, 5.12 mmol) in acetone (15 mL)
was refluxed (15 h). Purification by SGC (EtOAc/n-hexane = 1:6)
afforded compound 31 (515 mg, 64.7%) as a white crystalline solid.
Rf = 0.57 (EtOAc/n-hexane = 1:3); mp 128–130 °C; 1H NMR
(400 MHz, CDCl3) d 2.28 (s, 3H), 4.98 (s, 2H), 6.92 (dd, 2H, J = 1.2,
9.2 Hz), 7.01 (dd, 2H, J = 1.2, 8.8 Hz), 7.24 (dd, 1H, J = 1.2, 8.4 Hz),
7.45 (d, 1H, J = 8.0 Hz), 7.52 (s, 1H); 13C NMR (100 MHz, CDCl3)
21.3, 69.2, 115.7, 122.7, 12637, 129.4, 130.8, 132.2, 133.0, 137.3,
144.9, 156.2, 170.0 ppm; EI-MS (m/z) 310.0 [M]+.
4.29. DPPH free radical scavenging assay
4.24. 4-(3,4-Dimethoxybenzyloxy)phenol (32)15
DPPH free radical scavenging assay was carried out as de-
scribed by Bondet et al.18 with slight modification. All com-
pounds used were dissolved in ethanol (Sigma, USA). In order
to determine the capacity to scavenge the stable free radical,
Using the general procedure 2, compound 27 (190 mg,
0.63 mmol) was hydrolyzed with 1.0 M KOH (3 mL) and ethanol
(3 mL). After extraction with EtOAc (3x30 mL), compound 32
(141 mg, 86.3%) was obtained as a dark brown liquid. Rf = 0.23
(EtOAc/n-hexane = 1:3); 1H NMR (400 MHz, CDCl3/CD3OD) d 3.75
(s, 6H), 4.89 (s, 2H), 6.38 (t, 1H, J = 2.4 Hz), 6.55 (d, 2H, J = 2.4 Hz),
6.72 (dd, 2H, J = 2.4, 9.2 Hz), 6.80 (dd, 2H, J = 2.4, 9.2 Hz); 13C NMR
(100 MHz, CDCl3/CD3OD) 55.3, 70.8, 99.86, 105.3, 116.0, 116.1,
139.9, 150.7, 152.3, 160.9 ppm; EI-MS (m/z) 260.1 [M]+.
1,1-diphenyl-2-picryl-hydrazyl (DPPH) (Sigma, USA), 50
each compound solution was added to each well in 96-well plate
containing 50 L of 200 M DPPH stock solution. After incuba-
tion for 30 min. with shaking at room temperature, the absor-
bance was recorded at 517 nm using microplate reader
lL of
l
l
a
(VERSAmax, Molecular Devices). The percent DPPH scavenging
activity was expressed as follow:
DPPH scavenging activity ð%Þ ¼ ½ðAc ꢁ AbÞ ꢁ ðAs ꢁ AbÞꢂ=ðAc ꢁ AbÞ
ꢀ 100
4.25. 4-(4-Fluorobenzyloxy)phenol (33)16
Using the general procedure 2, compound 28 (205 mg,
0.78 mmol) was hydrolyzed with 1.0 M KOH (3 mL) and ethanol
(3 mL). Solid formed after neutralization with 1.0 M HCl was col-
lected by filtration to obtain compound 33 (155 mg, 90.3%) as an
off-white solid. Rf = 0.34 (EtOAc/n-hexane = 1:3); mp 124–126 °C;
1H NMR (400 MHz, CDCl3/CD3OD) d 4.9 (s, 2H), 6.71 (dd, 2H,
J = 2.0, 8.8 Hz), 6.78 (dd, 2H, J = 1.2, 8.8 Hz), 7.01 (dd, 2H, J = 7.6,
7.6 Hz), 7.40 (dd, 2H, J = 5.6, 7.6 Hz); 13C NMR (100 MHz, CDCl3/
CD3OD) 70.4, 115.4, 155.6, 116.0, 116.2, 129.5, 133.2, 151.0,
152.2, 161.3, 163.8 ppm; EI-MS (m/z) 218.1 [M]+.
where Ac was the absorbance of the DPPH solution without
compound, As was that of the DPPH solution with compound,
and Ab was that of the blank. Experiments for each compound were
performed in triplicate and vitamin C was used as a positive con-
trol. For compounds showing more than 40% of DPPH scavenging
activity from screening test with 100 lM concentration in final,
The EC50 values for DPPH radical scavenging activities were calcu-
lated using the data graphing software, TableCurve 2D (Systat Soft-
ware Inc.) with the concentration range from 20 to 200 lM. The
EC50 value was defined as a concentration that could scavenge
50% DPPH free radical.
4.26. 4-(2-Chlorobenzyloxy)phenol (34)
4.30. Tyrosinase inhibition assay in vitro
Using the general procedure 2, compound 29 (184 mg,
0.66 mmol) was hydrolyzed with 1.0 M KOH (3 mL) and ethanol
(3 mL). After extraction with EtOAc (3 ꢀ 30 mL), compound
B16 cells were stimulated with 10 nM a-MSH alone for 48 h and
disrupted by sonication on ice. After centrifugation, supernatants
were dialyzed against 50 mM sodium phosphate buffer (pH 6.8)
and then used as the tyrosinase sources. Enzyme activity was
34(143 mg, 91.6%) was obtained as
a gray crystalline solid.
Rf = 0.33 (EtOAc/n-hexane = 1:3); mp 64–66 °C; 1H NMR
(400 MHz, CDCl3) d 4.79 (brs, 1H), 5.10(s, 2H), 6.77 (dd, 2H,
J = 2.4, 9.2 Hz), 6.88 (dd, 2H, J = 2.4, 9.2 Hz), 7.22–7.30 (m, 2H),
7.39 (dd, 1H, J = 2.0, 7.6 Hz), 7.54 (dd, 1H, J = 2.0, 7.2 Hz); 13C
NMR (100 MHz, CDCl3) 68.2, 116.2, 116.3, 127.1, 129.0, 129.1,
129.6, 132.8, 135.2, 150.1, 153.0 ppm; EI-MS (m/z) 234.0 [M]+.
determined by
ously.19 Briefly, sample was incubated with cell-free tyrosinase
sources and then reacted with 5 mM -tyrosine as a substrate.
L-tyrosine oxidation velocity as described previ-
L
The initial velocity of dopachrome formation was determined by
the increasing rate of absorbance values at 475 nm. One unit of
the enzyme activity was defined as the conversion of 1 nmol
tyrosine to dopachrome per min.
4.27. 4-(3-Chlorobenzyloxy)phenol (35)17
L-
Using the general procedure 2, compound 30 (310 mg,
1.12 mmol) was hydrolyzed in 1.0 M KOH (4 mL) and ethanol
(4 mL). Solid formed after neutralization with 1.0 M HCl was col-
lected by filtration and dried to obtain the compound 35
(242 mg, 92.1%) as an off-white solid. Rf = 0.41 (EtOAc/n-hex-
ane = 1:3); mp 112–114 °C; 1H NMR (400 MHz, CDCl3) d 4.75 (br
s, 1H), 4.99 (s, 2H), 6.77 (dd, 2H, J = 2.4, 9.2 Hz), 7.02 (dd, 2H,
J = 2.4, 9.2 Hz), 7.27–7.30 (m, 3H), 7.43 (s, 1H); 13C NMR
4.32. a-MSH-induced melaninsynthesis inhibition assay in vitro
Melanin production inhibition assay was carried out by the
method of Hosoi et al.20 with slight modification. The mouse mel-
anoma B-16 cells were seeded at a density 2.5 ꢀ 103 cells per well
in Dulbecco’s modified eagle medium (DMEM) supplemented with
10% Fetal bovine serum (FBS) using a 96-well culture plates. The

K. Sapkota et al. / Bioorg. Med. Chem. 19 (2011) 2168–2175
2175
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Hearing, V. J.; Dooley, T. P. Biochem. Pharmacol. 1999, 57, 663.
5. Schallreuter, K. U.; Kothari, S.; Chavan, B.; Spencer, J. D. Exp. Dermatol. 2007, 17,
395.
6. Parvez, S.; Kang, M.; Chung, H. S.; Bae, H. Phytother. Res. 2007, 21, 805.
7. Criton, M.; Le Mellay-Hamon, V. Bioorg. Med. Chem. Lett. 2008, 12, 3607.
8. Solano, F.; Briganti, S.; Picardo, M.; Ghanem, G. Pigment Cell Res. 2006, 19,
550.
9. Ohyama, Y.; Mishima, Y. Fragrance J. 1990, 6, 53.
10. (a) Tokiwa, Y.; Kitagawa, M.; Raku, T.; Yanagitani, S.; Yoshino, K. Bioorg. Med.
Chem. Lett. 2007, 17, 3105; (b) Hori, I.; Nihei, K.-I.; Kubo, I. Phytother. Res. 2004,
18, 475.
11. Oozeki, H.; Tajima, R.; Nihei, K.-i. Bioorg. Med. Chem. Lett. 2008, 18,
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12. Palumb, A.; d’Ischia, M.; Misuraca, G.; Prota, G. Biochem. Biophys. Acta 1991,
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cells were grown in a 5% CO2–95% air atmosphere at 37 °C for
1 day. Then, -MSH, together with various sample concentrations,
a
was added to the wells. The cells were incubated for 3 days at 37 °C
and then equilibrated to ambient temperature and atmosphere.
The absorbance of each well was measured in a reader at
405 nm. The absorbance values were compared with a standard
curve obtained with synthetic melanin (Sigma–Aldrich) which
was dissolved in 0.85 N KOH and diluted in DMEM-10% FBS culture
medium, and distributed in 96-well plates to measure the optical
density (OD) at 405 nm. The inhibitory activity was calculated
from the change from the initial rate of OD.
Acknowledgements
This work was supported by the RIC program of MKE (Ministry
of Knowledge Economy), Korea.
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