Mono- and disalicylic acid derivatives: PTP1B inhibitors as potential anti-obesity drugs

Article abstract of DOI:10.1016/j.bmc.2007.07.010

A series of compounds containing one or two salicylic acid moieties were synthesized, and their efficacy to inhibit the phosphohydrolase activity of PTP1B examined. Some of the methylenedisalicylic acid derivatives were potent inhibitors of PTP1B. Of those derivatives, 3c exhibited about a 14-fold selectivity against TC-PTP, and this compound was tested in a mouse model for its efficacy to prevent diet-induced obesity. It effectively suppressed the increases in body weight and adipose mass, without any noticeable toxic effect. The compound also prevented increases in the plasma triglyceride, cholesterol, and nonesterified fatty acid concentrations; thus, expanding its therapeutic potential to other related metabolic diseases, such as hyperlipidemia and hypercholesterolemia.

Full text of DOI:10.1016/j.bmc.2007.07.010

Bioorganic & Medicinal Chemistry 15 (2007) 6535–6548
Mono- and disalicylic acid derivatives: PTP1B inhibitors
as potential anti-obesity drugs
Suja Shrestha, Bharat Raj Bhattarai, Keun-Hyeung Lee and Hyeongjin Cho*
Department of Chemistry and Institute of Molecular Cell Biology, College of Natural Sciences, Inha University,
Incheon 402-751, Republic of Korea
Received 23 May 2007; revised 7 July 2007; accepted 10 July 2007
Available online 26 July 2007
Abstract—A series of compounds containing one or two salicylic acid moieties were synthesized, and their efficacy to inhibit the
phosphohydrolase activity of PTP1B examined. Some of the methylenedisalicylic acid derivatives were potent inhibitors of PTP1B.
Of those derivatives, 3c exhibited about a 14-fold selectivity against TC-PTP, and this compound was tested in a mouse model for its
efficacy to prevent diet-induced obesity. It effectively suppressed the increases in body weight and adipose mass, without any notice-
able toxic effect. The compound also prevented increases in the plasma triglyceride, cholesterol, and nonesterified fatty acid concen-
trations; thus, expanding its therapeutic potential to other related metabolic diseases, such as hyperlipidemia and
hypercholesterolemia.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
the gastrointestinal tract. Sibutramine inhibits presynap-
tic reuptake of the neurotransmitters, norepinephrine
and serotonin; thus, decreasing appetite.3 Unlike the
earlier amphetamine-like drugs, sibutramine is not asso-
ciated with primary pulmonary hypertension or valvular
heart diseases. However, as a central nervous system
stimulator, sibutramine increases the heart rate and
blood pressure, which have raised concerns about the
unidentified complications following long-term treat-
ment.4 The drugs for short-term use also suppress appe-
tite via a central mechanism, but also exhibit side effects,
such as palpitations, tachycardia, isomnia, hyperten-
sion, dry mouth, and constipation.2b The adverse effects
and risks of these drugs have limited their therapeutic
potential.
Obesity is a serious health-threatening factor in an
urbanized society, which increases the risk of other dis-
eases, such as type 2 diabetes, cardiovascular disease,
hypertension, and some cancers.1 High energy intake
and low energy expenditure, accompanied by changing
lifestyles, have increased the prevalence of obesity and
this increase is expected to continue. Despite the urgent
need for safe and efficient therapeutics and the potential
size of the market for anti-obesity drugs, the current
status for the development of such drugs is still
unsatisfactory.
Until recently, only two drugs, orlistat and sibutramine,
have been accepted for long-term use in the United
States, with a further three drugs, diethylpropion, phen-
dimetrazine, and phentermine, approved for short-term
use.2 In 2006, rimonabant was approved in Europe as a
novel medicine antagonizing CB1 endocannabinoid
receptor to decrease appetite. All these drugs exert their
anti-obesity effect by decreasing energy intake. Orlistat
prevents the absorption of fat by lipase inhibition in
Aside from the side effects, current therapies target the
short-term machineries that regulate appetite or an indi-
vidual meal. The physiological system for the long-term
regulation of fat mass or energy balance includes leptin
and insulin, but leptin has a more important role in the
central nervous system (CNS) control of body weight.5
Leptin is secreted from adipocytes and its plasma level
is proportional to the mass of adipose tissues in the
body.6 The binding of leptin to the leptin receptors in
the hypothalamus is known to initiate a cascade, which
inhibits food intake and promotes energy expenditure.7
PTP1B was recently identified as a negative regulator
of hypothalamic leptin signaling.8 PTP1B inhibition or
Keywords: Obesity; PTP1B inhibitor; Protein tyrosine phosphatase;
Diet-induced obesity; Salicylic acid; Disalicylic acid; Methylenedisali-
cylic acid.
*
Corresponding author. Tel.: +82 32 860 7683; fax: +82 32 867
5604; e-mail: hcho@inha.ac.kr
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.07.010

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S. Shrestha et al. / Bioorg. Med. Chem. 15 (2007) 6535–6548
a reduction of the cellular abundance of PTP1B in mice
resulted in increased sensitivities to leptin and insulin,
and has also exhibited a protective effect against diet-in-
duced obesity.9 These observations provided the basis
for PTP1B as a therapeutic target for obesity, which is
the topic of this study.
(Scheme 1). For the syntheses of monosubstituted
salicylic acids (1a and 1c–e), appropriately substituted
phenolic compounds were converted to 20-hydroxyace-
tophenone derivatives, which were in turn O-methylated
and subjected to a haloform reaction. The phenolic OH
group was methylated for purification, even though the
haloform reaction can be performed without protection
of this group. The hydroxyacetophenone derivatives,
with an aromatic substituent at C-30 or C-50 (7 or 70),
were brominated at the C-50 or C-30 to obtain the 8 or
80 derivatives, respectively. For the syntheses of 1b, 1f,
and 1g, the haloform reaction was performed with 8
or 80. For the generation of 1h–j, 8 or 80 was subjected
to a Suzuki coupling reaction, using the pinacol ester
of phenylboronic acid and a palladium catalyst. The
phenolic OH was protected before the coupling reaction.
Resulting compounds, 9 and 90, were then subjected to
haloform reaction to obtain compounds 1h–j. For the
dibenzyl derivative, 1k, a second benzyl group was
introduced by benzoylation of 4-benzylphenol, followed
by a Wolff–Kishner reduction of the carbonyl group.
For the generation of 1l, the aldehyde (13) was protected
following bromination, with subsequent Stille coupling.
Compound 1m was prepared from 2-bromophenol.
The syntheses of 3,30-methylenebis(2-hydroxybenzoic
acid) derivatives started with the condensation of methyl
5-bromosalicylate (19) and formaldehyde (Scheme 2).
The condensation product (20) served as a precursor
for the syntheses of 3b–e. Hydrolysis of the ester, 20,
produced 3b. The treatment of 20 or 21 with the pinacol
ester of 2-thiophenyl or phenyl boronic acid, in the
presence of Pd(PPh3)4 catalyst, introduced 2-thiophenyl
or phenyl group, yielding 3e and 3c, respectively. The
basic reaction conditions resulted in concomitant hydro-
lysis of the ester. To synthesize 3d, a fully protected
compound (21) was subjected to a Stille coupling
reaction with 2-(tributylstannyl)furan and palladium
In the design of PTPase inhibitors, the primary concern
is the construction of a structure that mimics the phos-
photyrosine residue, and recent research has produced
various scaffolds, including phosphonates, 2-(oxalylami-
no)-benzoic acids, and O-carboxymethyl salicylic acids,
to list but a few.10 High dose of salicylic acid has been
used, more than a century ago, to reduce the symptoms
related to type II diabetes.11 Similar effects have also
been observed with aspirin treatment.12 More recently,
our and another laboratories have reported that poly-
meric substances, containing multiple salicylic acid moi-
eties, are potent inhibitors of PTPases.13 Prompted by
these observations, salicylate moiety was examined as
a pharmacophore for the inhibition of PTP1B, with a
methylenedisalicylic acid derivative reported to have
an anti-obesity effect in a mouse model.14 In this study,
more diverse compounds were synthesized, containing
one or two salicylic acid moieties, and their inhibitory
potency against PTP1B evaluated. The most potent
PTP1B inhibitor was tested in an animal model for its
efficacy in preventing the obesity induced by a high fat
diet.
2. Results
2.1. Chemical synthesis
The syntheses of compounds 1a-k (Fig. 1) exemplifies
the Fries rearrangement-haloform reaction approach
R
R
R2
R
R
HO
OH
HO2C
CO2H
R1
CO2H
CO2H CO2H
OH
OH
OH
1a-p
2f-g
3a-e
R1
R2
R
R
1a
H
Ph
Ph
H
2f
Ph
3a
3b
3c
F
Br
2g
Bz
1b
1c
1d
1e
1f
Br
Ph
H
Ph
O
Bz
H
3d
1q
Bz
Bz
Br
Bz
Ph
Ph
Bz
CO2H
Br
Bz
Ph
Bz
Ph
Bz
OH
S
1g
1h
1i
3e
OH
CO2H
HO2C
1r
HO
1j
OH
CO2H
CO2H
S
1k
1l
1s
4
2-furyl CHO
Br
O
OH
O
1m
1n
1o
1p
Br
H
H
H
H
F
CHO
O
NH
O
HN
S
SO3H
5, Ertiprotafib
OH
Figure 1. Compounds used or referred in this study.

S. Shrestha et al. / Bioorg. Med. Chem. 15 (2007) 6535–6548
6537
1c (R1 = Ph)
1f (R1 = Bz)
1e (R1 = Bz)
c, d, e, f, g
d, e, f, g
O
O
O
Br
R2
R2
d, e, f, g
d, e, f, g
a, b
a, b
h, i
h, i
j
1h
OH
OH
OH
OCH3
OCH3
R2
CO2H
OH
R1
6
R1
7
R1
8
R1
9
O
O
O
R1
1i, 1j
R2
R2
R2
j
OH
OCH3
OCH3
Br
8'
R1
9'
1h (R1 = Bz, R2 = Ph)
1i (R1 = Ph, R2 = Bz)
1j (R1 = Ph, R2 = Ph)
6'
7'
c, d, e, f, g
d, e, f, g
1a (R2 = Ph)
1d (R2 = Bz)
1b (R2 = Ph)
1g (R2 = Bz)
Bz
Bz
Bz
Bz
d, e, f, g
k, l
a, b, c
O
CO2H
OH
OH
CH3O
OH
11
1k
10
12
O
O
O O
CHO
CHO
h, m
n
o, g
O
O
CO2CH3
CO2H
Br
CO2CH3
CO2CH3
OH
OH
OH
OH
15
1l
13
14
p, q
i, r
s, t
Br
Br
Br
Br
CO2H
OH
16
OH
17
OCH3
18
OH
1m
Scheme 1. Synthetic strategy for the syntheses of 1a–m. Reagents and conditions: (a) CH3COCl, Et3N, ether; (b) AlCl3, heat; (c) (CH3)2SO4, NaOH,
EtOH, reflux; (d) Br2, aq KOH; (e) CH3OH, HCl, reflux; (f) BBr3; (g) aq NaOH, heat; (h) Br2; (i) CH3I, K2CO3, acetone, reflux; (j) ArB(OR)2,
Pd(PPh3)4, aq K3PO4, DMF, heat; (k) benzoyl chloride, AlCl3, CS2, 50 ꢁC; 20% NaOH, 1,4-dioxane, 70 ꢁC; (l) H2NNH2, KOH, (CH2OH)2, 120 ꢁC–
reflux; (m) (CH2OH)2, pTsOH, reflux; (n) 2-(tributylstannyl)furan, Pd(PPh3)2Cl2, 1,4-dioxane, 100 ꢁC; (o) 1 M HCl, CHCl3, reflux; (p) allyl bromide,
K2CO3, acetone, reflux; (q) 200 ꢁC, microwave; (r) aq KOH, EtOH, reflux; (s) KMnO4, acetone; aq NaHSO3; aq HCl; (t) PhSH, K2CO3, NMP,
190 ꢁC.
catalyst. The resulting furanylated compound was
then treated with thiophenol and K2CO3 to remove
the protecting groups. A similar condensation reac-
tion of 5-flourosalicylic acid and paraformaldehyde
yielded 3a.
icylates, the 1 series. Compounds 1a, 1c, and 1d, with a
single phenyl or benzyl substitution of the salicylic acid
moiety, did not improve the inhibitory potency. Bromine
substitution resulted in a slightly increased potency, as
with compounds 1b, 1f, and 1g. Compounds 1h–k, with
double substitutions at the C-3 and C-5 positions, exhib-
ited lower IC50values. No significant difference was ob-
served between the phenyl and benzyl substitutions.
2.2. In vitro enzyme assays
The series of compounds synthesized in this study were
tested for their ability to inhibit PTP1B, using p-nitro-
phenyl phosphate (pNPP) as the substrate. The enzyme
and compounds were preincubated for 10 min prior to
the initiation of the enzyme reaction by addition of the
substrate. The IC50 values for the compounds deter-
mined under these conditions are shown in Table 1. Ki
values were also determined for selected compounds.
In the series of compounds 3a–e, two salicylic acid moi-
eties were flanked by a methylene group. The haloge-
nated derivatives, 3a and 3b, were not good inhibitors
of PTP1B (IC50 > 100 lM). Compound 3c proved to
be the most potent inhibitor of PTP1B (Ki = 6.5 lM).
In 3d and 3e, where the phenyl group in 3c was replaced
with heterocyclic furan or thiophene moieties, respec-
tively, 1.5–3.5-fold decreases in the inhibitory potencies
were observed. Similar results were previously obtained
for the 2 series of compounds.14
As presented in Table 1, the disalicylic acid derivatives
were generally found to be more potent than the monosal-

6538
S. Shrestha et al. / Bioorg. Med. Chem. 15 (2007) 6535–6548
Br
Br
Br
Br
Br
a
b
CO2CH3
H3CO2C
e
CO2CH3
H3CO2C
CO2CH3
OH
19
OH
OH
OCH3 OCH3
20
21
f, g
O
O
h
c, d, e
Br
Br
HO2C
CO2H
HO2C
CO2H
CO2H
HO2C
OH
OH
OH
OH
S
S
3b
3d
HO2C
CO2H
OH
OH
OH
OH
3e
3c
Scheme 2. Synthetic strategy for the syntheses of methylenedisalicylic acid derivatives, 3. Reagents and conditions: (a) 37% aq CH2O, H2SO4,
CH3OH, H2O; (b) (CH3)2SO4, K2CO3, acetone, reflux; (c) (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzene, Pd(PPh3)4, K3PO4, DMF, 80 ꢁC; (d)
BBr3, CH2Cl2; (e) 20% NaOH, 1,4-dioxane, 80 ꢁC; (f) 2-(tributylstannyl)furan, Pd(PPh3)2Cl2, 1,4-dioxane, 100 ꢁC; (g) PhSH, K2CO3, NMP, 190 ꢁC;
(h) 2-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)thiophene, Pd(PPh3)4, K3PO4, DMF, 80 ꢁC.
Table 2. Inhibition of PTPases by selected compounds
Table 1. Inhibitory effect of the compounds on PTP1B
IC50 (lM)a
Ki (lM)b
Compound
IC50 (lM)a (Ki, lM)b
Compound
PTP1B LAR-D1 TC-PTP SHP-1 cat YPTP1
1a
1b
1279 320
351 56
859 30
1180 343
403 63
148 39
153 32
1i
3c
65
19
9
1
>2000
>2000
540 48 129 15
271 48
86
1c
1d
156
[89]
5
37
(9.7)
3
2
[6.5]
1e
a Values are means standard deviations of two or more experiments.
b Values are from a single measurement.
1f
1g
1h
1i
57
65
84
8
9
8
and excellent selectivity against LAR-D1. Less selectiv-
ity was observed over the catalytic domain of SHP-1
(SHP-1cat). YPTP, a yeast PTPase, was tested to com-
pare the PTPases of different origin, but this exhibited
only a modest selectivity.
1j
1k
91 15
380 56
>1000
1l
1m
1n
1o
>1000
>1000
2.3. In vivo experiment
1p
1q
>1000
850 150
>1000
Of the PTP1B inhibitors, 3c was selected for evaluation of
obesity controlling effect in mice susceptible to diet-in-
duced obesity (C57BL/6J Jms Slc male). There is much
evidence of diet-induced obese (DIO) mice being fre-
quently used as a mice model in the determination of com-
pound efficacy for the reduction of body weight.15 Before
feeding the test drug, mice were fed either LFD or HFD
ad libitum for 8 weeks. The LFD-fed lean control mice
showed much lower body weight gain compared to the
HFD-fed obese control group. The HFD-fed mice were
separated into two groups. Each of the groups was then
given HFD or HFD plus 3c for 4 weeks. The compound
3c was administered as mixtures with the food (5 g 3c
per kg of diet). The daily uptake of 3c was approximated
as 13 mg/day/mouse, which is equivalent to 400 mg/day/
kg of mouse weight. For lean control group, LFD was
fed throughout the test period. As shown in Figure 2,
the feeding of 3c significantly reduced the body weight
gain compared to that of the HFD control group
(p < 0.005).
1r
1s
>1000
2f
2g
20 1c
15 4c
215 11
149 45
9.4c
6.3c
3a
3b
3c
3d
19
67
27
1
5
5
6.5
3e
4
5 (Ertiprotafib)
500 100c
1.4 0.1c
102c
1.5c
a Values are means standard deviations of two or more experiments.
b Values are from a single measurement.
c Data reproduced from our previous publication.14
Because 1i and 3c exhibited the lowest IC50 values of the
mono- and disalicylic acid derivatives, respectively, their
inhibitory activities against other PTPases, including
TC-PTP, were tested in order to verify the PTP1B
specificity of these compounds, the results of which are
summarized in Table 2. Compound 3c demonstrated a
14-fold greater selectivity over TC-PTP, the most
homologous with PTP1B among the human PTPases,
A reduction of adiposity was observed by the reduction
of epididymal and retroperitoneal fat pad weights of the

S. Shrestha et al. / Bioorg. Med. Chem. 15 (2007) 6535–6548
6539
the values for these parameters in the obese control
group being significantly higher than those of the lean
control group (p < 0.05). The feeding of 3c together with
the diet significantly suppressed the increases in the cir-
culating TG, cholesterol, and NEFA (p < 0.05).
HFD
34
32
30
28
26
24
HFD + 3C
LFD
In addition, there was no significant change in physical
activities on the administration of either compound,
indicating that the treatment did not cause any behav-
ioral effects in mice. At the end of the experiment, the
obese group mice were physically distinguishable from
the remaining two groups. The outer appearance of
the liver showed no overt toxicity on treatment with
these compounds (data not shown).
0
5
10
15
20
25
30
Time (days)
Figure 2. The effect of LFD, HFD, and HFD + 3c on body weight of
DIO mice. Data points represent means SEM; n = 8–9/group.
p < 0.005 for comparisons against obese control group. Mice fed with
LFD served as a reference group of lean control.
mice treated with 3c as well as 2f (Table 3). As depicted
in Table 3, both fat pad weights were significantly lower
than those of obese control mice (p < 0.001). A gross
carcass appearance of a mouse also revealed the signifi-
cantly decreased epididymal fat pad after drug treat-
ment (Fig. 3) Differences in the liver weights, however,
were not significant between any of the groups
(p > 0.05) (Table 3). There was a modest decrease in
food intake (< 8 days, p < 0.05) when the food was
changed from HFD to HFD plus the drug (Fig. 4). After
8 days of treatment, the drug-treated mice continued to
eat comparable amounts of food to that of the obese
control mice (p > 0.05). The ratio of weight gain to con-
sumed calories is described as the feed efficiency and re-
flects the metabolic efficiency. As shown in Figure 5, the
drug-treated group revealed a feeding efficiency compa-
rable to that of the lean control group, but significantly
lower than that of the obese control group (p < 0.001).
These results suggest that the significant loss of body
fat and decreased body weight gain could be the result
of increased energy expenditure.
Plasma was analyzed for glucose, triglyceride (TG), total
cholesterol, and nonesterified fatty acids (NEFA) (Table
4). The concentrations of glucose and TG were only
slightly higher in the obese control mice than those of
the lean control mice (p > 0.05). However, HFD affects
the concentrations of total cholesterol and NEFA, with
Figure 3. The gross carcass appearance of a mouse from each group at
the end of the study. Five-week-old mice were fed LFD (A) or HFD
(B–D) for 8 weeks and then treated for 4 weeks with (A) LFD, (B)
HFD, (C) HFD + 3c or (D) HFD + 2f.
Table 3. Study of 28 days treatment of 2f and 3c on body weight, liver, and adipose tissue weighta
Body weight (g)
Final
Liver weight (g)
Adipose tissue weight (g)
Initial
Epididymal
Retroperitoneal
LFD
HFD
25.2 1.4
27.9 2.4
27.9 2.9b
27.9 2.0
26.2 1.4c
33.4 2.8
29.2 2.5b,c
28.9 1.7c
0.99 0.10
1.14 0.12
1.03 0.13
1.07 0.09
0.52 0.09c
1.80 0.49
0.86 0.24b,c
0.83 0.34c
0.15 0.04c
0.51 0.11
0.26 0.08b,c
0.26 0.07c
HFD + 2f
HFD + 3c
a Data expressed as means SEM. n = 8 for LFD, n = 9 for others.
b Data reproduced from the previous publication in our laboratory.14
c Significantly different from the HFD (p < 0.005).

6540
S. Shrestha et al. / Bioorg. Med. Chem. 15 (2007) 6535–6548
The obesity-controlling effect of the selected compound,
100
80
60
40
20
0
LFD
3c, was determined using a DIO mouse model, with the
result compared with those previously obtained for 2f.
The oral administration of 3c (0.5% in diet w/w) signifi-
cantly reduced the body weight gain compared with that
of the HFD control group (p < 0.005). A decrease in adi-
posity was observed by the reduction of epididymal and
retroperitoneal fat pad weights of mice treated with 3cas
well as 2f. In contrast, there was no significant difference
in the amount of food intake between the compound-
treated and control groups. These results prove the
efficacy of these compounds in controlling obesity. The
in vivo effect of 3c, without any noticeable toxic effect,
suggests a potential therapeutic role for 3c in the
management of obesity. The experimental results also
indicated that the anti-obesity effect was largely due to
increased energy expenditure. The compounds also
prevented the increase of the plasma levels of triglycer-
ide, cholesterol, and nonesterified fatty acids, expanding
the therapeutic potential to other related metabolic dis-
eases, such as hyperlipidemia and hypercholesterolemia.
HFD
HFD+2f
HFD+3c
0
4
8
12
16
20
24
28
Time (Days)
Figure 4. The effects of compounds 2f and 3c on the cumulative food
intake of DIO mice. All values are means SEM; n = 8–9/group.
p < 0.05 for comparisons against the obese control group. Mice fed
with LFD served as a reference lean control group.
1.2
1.0
0.8
0.6
0.4
0.2
0.0
4. Experimental
4.1. Materials
Commercial reagents were from Aldrich Chemical Co.
(Milwaukee, WI) and TCI (Tokyo, Japan). Most chemi-
cals and solvents were of analytical grade and used with-
out further purification. Reactions were monitored by
thin-layer chromatography (TLC), using precoated silica
gel plates (Silica gel 60 F254, Merck), and spots were visu-
alized under UV light (254 nm). Column chromatogra-
phy was carried out using silica gel 60 AF-254 (0.063–
0.200 mm, Merck). Melting points (uncorrected) were
determined on a MEL-TEMP Electrothermal apparatus.
1H and 13C NMR spectra were recorded on a Varian
Gemini 2000 (200 MHz) and a Varian Inova 400
(100 MHz) spectrometers, respectively. Chemical shifts
(d) are expressed in parts per million relative to tetrameth-
ylsilane, which was used as an internal standard, coupling
constants (J) are in hertz, and the signals are designated as
follows: s, singlet; d, doublet; t, triplet; q, quartet; m, mul-
tiplet; br s, broad singlet. Mass spectra were obtained at
the Korea Basic Science Institute, Daegu, Korea.
LFD
HFD
HFD+2f
HFD+3c
Figure 5. Effects of 4 week treatment with 2f and 3c on the feeding
efficiency (body weight gain per calories consumed) of mice. All values
are means SEM; n = 8–9/group. p < 0.001 for comparisons against
obese control group. Mice fed with LFD served as a reference lean
control group.
Table 4. Effects of PTP1B inhibitors 2f and 3c on plasma glucose,
triglyceride, total cholesterol, and free fatty acida
Glucose Triglyceride Total cholesterol NEFA
(mg/dL) (mg/dL)
270 10 47
300 40 63 32
(mg/dL)
(mEq/L)
LFD
HFD
8
104 11b
141 10
129 8b
133 14
0.23 0.1b
0.35 0.1
0.24 0.1b
0.14 0.1b
HFD + 2f 300 50 42 7b
Compounds 1n and 1p–s were purchased from Aldrich.
Compounds 1c and 10 were purchased from TCI. The
intermediates, 8 and 80, used for the generation of sali-
cylic acid derivatives, were obtained by Friedel–Crafts
acetylation, with subsequent controlled bromination of
the commercially available phenols, as described previ-
ously.16 A substrate, pNPP, for the PTPase assay was
purchased from Sigma (St. Louis, USA) in the di(Tris)
salt form. The absorbances were measured using a
Novaspec-II spectrophotometer (Amersham Pharmacia)
or a DU 650 spectrophotometer (Beckman–Coulter).
The native form of PTP1B and the catalytic domain of
SHP-1 (SHP-1cat) were expressed in an Escherichia coli
expression systems and purified as described previ-
ously.17 LAR-D1 (membrane-proximal catalytic domain
HFD + 3c 280 40 48
8
a Data expressed as means SEM. n = 8 for LFD, n = 9 for others.
b Significantly different from the HFD (p < 0.05).
3. Discussion
It has been reported that salicylic acid is a weak compet-
itive inhibitor of PTP1B, with an inhibition constant of
19.4 mM.13a In the present study, a series of compounds
containing one or two salicylic acid moieties were syn-
thesized. Of those compounds, disalicylic acid, 3c, was
proved to be a potent inhibitor of PTP1B, and demon-
strated a 14-fold greater selectivity over TC-PTP, the
most homologous with PTP1B of the human PTPases.

S. Shrestha et al. / Bioorg. Med. Chem. 15 (2007) 6535–6548
6541
of LAR) and TC-PTP were purchased from New Eng-
land Biolabs Inc. (Beverly, USA).
successively with water (2· 10 mL) and brine (10 mL),
and then dried (Na2SO4). Concentration of the solvent
under reduced pressure gave a cream-colored solid,
which was recrystallized from ether to give 1b (56 mg).
The mother liquor was concentrated, and the residue
purified by silica gel chromatography (1% acetic acid
in hexane/EtOAc, 4:1, Rf = 0.2), to afford 1bas cream-
colored crystals (41 mg, 87% overall yield): mp 214–
4.2. Chemical synthesis
4.2.1. 5-Bromo-4-hydroxybiphenyl-3-carboxylic acid (1b)
4.2.1.1. Step 1. Bromine (0.2 mL, 3.9 mmol) was
added dropwise to a stirred solution of 20% aqueous
KOH (4 mL) cooled in an ice–salt bath at ꢀ5 to 0 ꢁC.
The resulting yellow solution was added to 3-bromo-2-
methoxy-5-phenylacetophenone (80b) (200 mg, 0.65 mmol)
in 1,4-dioxane (10 mL), at 5–10 ꢁC, over a period
of 5 min, the mixture stirred below 10 ꢁC for a fur-
ther 2 h and stirred overnight at room temperature.
The reaction mixture was diluted with water (20 mL),
acidified with 1 M HCl (40 mL), and extracted with
EtOAc (3· 20 mL). The combined organic extracts were
washed successively with Na2S2O3 (20 mL) and brine
(20 mL), and dried (Na2SO4). Removal of the solvent
gave a cream-colored solid, which upon recrystallization
from EtOAc afforded 5-bromo-4-methoxybiphenyl-3-
carboxylic acid (84 mg). The mother liquor was
concentrated, and the residue subjected to column chro-
matography on silica gel (1% acetic acid in hexane/
EtOAc, 1:1, Rf = 0.5) to yield the acid as white needles
1
216 ꢁC; H NMR (CDCl3, 200 MHz) d 11.07 (s, 1H),
8.13 (d, J = 2.6 Hz, 1H), 8.05 (d, J = 2.2 Hz, 1H),
7.57–7.36 (m, 5H); 13C NMR (DMSO-d6, 100 MHz) d
171.49 (CO2H), 157.13, 137.74, 136.30, 132.26, 129.03,
127.55, 127.39, 126.35, 114.66, 111.02 (Carom); LRMS
m/z 294 (M+, 81Br), 292 (M+, 79Br); HRMS calcd for
C13H9BrO3291.9735 (M+, 79Br), found 291.9732.
4.2.2. 5-Benzyl-2-methoxy-3-phenylacetophenone (90i). A
flame-dried flask was cooled to room temperature under
a nitrogen purge and charged with (4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)benzene (137 mg, 0.68 mmol)
and tetrakis (triphenylphosphine) palladium (8 mg,
9 lmol). After 30 min nitrogen purging, 5-benzyl-3-bro-
mo-2-methoxyacetophenone (80i) (145 mg, 0.45 mmol)
dissolved in dry DMF (5 mL) was added into the flask
via syringe. To this mixture, 2 M K3PO4 (1.1 mL,
2.3 mmol) was added slowly and the mixture was stirred
at 90 ꢁC overnight. After cooling to room temperature,
water (5 mL) and EtOAc (10 mL) were added into the
reaction mixture. It was then stirred for 10 min, and
the aqueous layer was extracted with EtOAc (3·
10 mL). The combined organic extracts were washed
successively with water (2· 10 mL) and brine (10 mL),
dried (Na2SO4), and then concentrated. The crude prod-
uct was purified by column chromatography on silica gel
(hexane/EtOAc, 9:1, Rf = 0.4) to afford 90i as a colorless
1
(92 mg, 88% overall yield): mp 193–195 ꢁC; H NMR
(CDCl3, 200 MHz) d 8.31 (d, J = 2.6 Hz, 1H), 8.03 (d,
J = 2.6 Hz, 1H), 7.60–7.40 (m, 5H), 4.10 (s, 3H, CH3).
4.2.1.2. Step 2. The above-obtained acid (160 mg,
0.52 mmol) was dissolved in MeOH, saturated with
dry HCl (3 mL), and stirred overnight at 60 ꢁC. The
solution was further stirred at room temperature for
30 min, with the solvent evaporated to give a yellow
oil, which was dissolved in EtOAc (15 mL). The EtOAc
solution was washed successively with water (3· 15 mL)
and 5% NaHCO3(15 mL), and then dried (Na2SO4).
Evaporation of solvent under reduced pressure followed
by silica gel column chromatography (hexane/EtOAc
19:1, Rf = 0.2) afforded methyl 5-bromo-4-methoxybi-
phenyl-3-carboxylate as a colorless oil (142 mg, 85%
yield): 1H NMR (CDCl3, 200 MHz) d 7.97 (d, J =
2.2 Hz, 1H), 7.94 (d, J = 2.6 Hz, 1H), 7.57–7.37 (m,
5H), 3.97 (s, 3H, CO2CH3), 3.96 (s, 3H, OCH3).
1
oil (117 mg, 81% yield): H NMR (CDCl3, 200 MHz) d
7.54–7.22 (m, 12H), 3.98 (s, 2H, CH2), 3.38 (s, 3H,
OCH3), 2.65 (s, 3H, COCH3).
4.2.3. 5-Benzyl-2-hydroxybiphenyl-3-carboxylic acid (1i)
4.2.3.1. Step 1. Bromine (0.24 mL, 4.7 mmol) was
added dropwise to an aqueous KOH (20%, 5 mL) with
stirring at ꢀ5 to 0 ꢁC. The resulting yellow solution
was slowly added to a solution of 90i (246 mg,
0.78 mmol) in 1,4-dioxane (10 mL) at 0 ꢁC over a 5-
min period. After the addition was complete, the solu-
tion was allowed to warm to 10 ꢁC, and stirred at the
same temperature for 2 h, during which the color has
changed from yellow to light yellow. The mixture was
further stirred at 80 ꢁC for 1 h, at which point the solu-
tion became colorless. It was cooled, diluted with water
(15 mL), acidified with 1 M HCl (10 mL), and extracted
with EtOAc (3· 15 mL). The organic extracts were com-
bined, washed successively with aqueous Na2S2O3
(25 mL) and brine (25 mL), dried (Na2SO4), and concen-
trated. The crude product was taken to the next step
without further purification. This crude product
(307 mg, 0.96 mmol) was dissolved in MeOH saturated
with dry HCl (8 mL) and stirred at 80 ꢁC overnight.
After 30 min stirring at room temperature, the solvent
was evaporated and the residue was dissolved in EtOAc
(20 mL). The organic solution was then washed succes-
sively with water (2· 10 mL), 5% NaHCO3(10 mL),
4.2.1.3. Step 3. BBr3 (1.0 M in CH2Cl2, 0.95 mL,
0.95 mmol) was added to a stirred solution of methyl
5-bromo-4-methoxybiphenyl-3-carboxylate
(125 mg,
0.38 mmol) in CH2Cl2 (4 mL), over 5 min period, while
cooling the reaction flask in a dry ice-acetone bath. The
reaction mixture was stirred overnight at room temper-
ature under nitrogen atmosphere. The reaction was
quenched with the addition of 1 M HCl (2 mL) and
the aqueous layer then extracted with EtOAc (2·
10 mL). The combined organic layers were washed suc-
cessively with water (2· 10 mL) and brine (10 mL), and
dried (Na2SO4). Removal of the solvent using a rotary
evaporator gave a brown solid, which was further dis-
solved in 1,4-dioxane (1 mL), and then treated with
20% NaOH (0.5 mL) at 60 ꢁC for 3 h. After cooling to
room temperature, the reaction mixture was acidified
with 1 M HCl (5 mL), and extracted with EtOAc (2·
10 mL). The combined organic extracts were washed

6542
S. Shrestha et al. / Bioorg. Med. Chem. 15 (2007) 6535–6548
and brine (10 mL), and dried (Na2SO4). The solvent was
removed using a rotary evaporator, and the residue puri-
fied by column chromatography (hexane/EtOAc, 9:1,
Rf = 0.4) to give methyl 5-benzyl-2-methoxybiphenyl-3-
carboxylate as a colorless oil (217 mg, 84% yield): H
NMR (CDCl3, 200 MHz) d 7.59–7.19 (m, 12 H), 3.99 (s,
2H, CH2), 3.91 (s, 3H, CO2CH3), 3.45 (s, 3H, OCH3).
(Carom), 40.81 (CH2); LRMS m/z 228 (M+); HRMS
calcd for C14H12O3228.0786 (M+), found 228.0789.
4.2.6. 3-Benzyl-2-hydroxybenzoic acid (1e). From 3-ben-
zyl phenol, 1e was obtained as a cream-colored solid in
the same way as 1a. Mp 129–131 ꢁC; 1H NMR (CDCl3,
200 MHz) d 10.70 (s, 1 H), 7.83–7.78 (dd, J = 7.6 Hz,
1.8 Hz, 1H), 7.35–7.19 (m, 6H), 6.89 (t, 1 H), 4.03 (s,
2 H, CH2); 13C NMR (CDCl3, 100 MHz) d 174.95
(CO2H), 160.28, 140.06, 137.50, 129.99, 129.06, 128.93,
128.41, 125.12, 119.11, 110.87 (Carom), 35.37 (CH2);
LRMS m/z 228 (M+); HRMS calcd for
C14H12O3228.0786 (M+), found 228.0789.
1
4.2.3.2. Step 2. BBr3 (1.0 M in CH2Cl2, 2.4 mL,
2.4 mmol) was added to a stirred solution of methyl
5-benzyl-2-methoxybiphenyl-3-carboxylate (200 mg,
0.60 mmol) in dry CH2Cl2 (6 mL) under a nitrogen atmo-
sphere over a 2-min period, while cooling the reaction
flask in dry ice–acetone bath. The resulting orange to dark
red solution was stirred at room temperature overnight.
The reaction was quenched by addition of 1 M HCl
(2 mL), and the aqueous layer was extracted with EtOAc
(2· 15 mL). The organic extracts were combined and
washed successively with water (2· 15 mL) and brine
(15 mL), and dried (Na2SO4). After evaporation of the
solvent under reduced pressure, the brown residue was
dissolved in 1,4-dioxane (2 mL), and aqueous NaOH
(20%, 0.85 mL) was added. The mixture was stirred at
80 ꢁC for 5 h, quenched with 1 M HCl (6 mL), and ex-
tracted with EtOAc (2· 15 mL). The combined organic
extracts were washed successively with water (2· 15 mL)
and brine (15 mL), dried (Na2SO4), and concentrated.
The crude product was recrystallized from EtOAc to give
1i (94 mg), and the mother liquor was concentrated and
purified by silica gel chromatography (1% acetic acid in
hexane/EtOAc, 4:1, Rf = 0.3) to yield 1i as a cream-col-
4.2.7. 3-Benzyl-5-bromo-2-hydroxybenzoic acid (1f). The
Friedel–Crafts acetylated and brominated product of
3-benzyl phenol was subjected to the reaction steps
(haloform reaction, protection, and deprotection steps)
as for 1b to obtain 1f as a cream-colored solid. mp
1
204 ꢁC; H NMR (CDCl3, 200 MHz) d 10.68 (s, 1 H),
7.91 (d, J = 2.2, Hz, 1H), 7.41 (d, J = 2.6 Hz, 1H),
7.30–7.18 (m, 5H), 3.99 (s, 2H, CH2); 13C NMR
(DMSO-d6, 100 MHz)
d 171.20 (CO2H), 158.40,
139.65, 137.95, 132.23, 130.06, 128.68, 128.41, 126.13,
114.40, 109.56 (Carom), 34.24 (CH2); LRMS m/z 308
(M+, 81Br), 306 (M+, 79Br); HRMS calcd for
C14H11BrO3305.9892 (M+, 79Br), found 305.9892.
4.2.8. 5-Benzyl-3-Bromo-2-hydroxybenzoic acid (1g). In
the same way as for 1f, 1g was obtained from 4-benzyl
1
phenol as a cream-colored solid. Mp 166 ꢁC; H NMR
1
ored solid (67 mg, 88% overall yield): mp 171 ꢁC; H
(CDCl3, 200 MHz) d 10.90 (s, 1H), 7.72 (d, J = 2.2 Hz,
1H), 7.62 (d, J = 2.2 Hz, 1H), 7.35–7.15 (m, 5H), 3.92
(s, 2H, CH2); 13C NMR (CDCl3, 100 MHz) d 174.14
(CO2H), 157.06, 140.79, 139.83, 133.53, 130.02, 128.74,
126.58, 111.99, 111.52 (Carom), 40.54 (CH2); LRMS
m/z 308 (M+, 81Br), 306 (M+, 79Br); HRMS calcd for
C14H11BrO3305.9892 (M+, 79Br), found 305.9889.
NMR (CDCl3, 200 MHz) d 10.80 (s, 1H), 7.78 (d,
J = 2.2 Hz, 1H), 7.57–7.19 (m, 11H), 3.97 (s, 2H, CH2);
13C NMR (CDCl3, 100 MHz) d 175.14 (CO2H), 158.01,
140.60, 138.71, 136.89, 132.16, 130.82, 129.95, 129.33,
128.72, 128.62, 128.16, 127.50, 126.32, 111.26 (Carom),
40.95 (CH2); LRMS m/z 304 (M+); HRMS calcd for
C20H16O3304.1099 (Mtextsuperscript+), found 304.1099.
4.2.9. 5-Benzyl-4-hydroxybiphenyl-3-carboxylic acid (1h).
Compound 1h was obtained as a white solid following
the same reaction steps as for 1i. Mp 205 ꢁC; 1H
NMR (CDCl3, 200 MHz) d 10.87 (s, 1H), 8.04 (d,
J = 2.2 Hz, 1H), 7.59–7.20 (m, 11H), 4.10 (s, 2H,
CH2); 13C NMR (DMSO-d6, 100 MHz): d 172.38
(CO2H), 158.82, 140.33, 139.10, 134.47, 129.88, 128.95,
128.66, 128.31, 127.04, 126.15, 125.94, 111.81 (Carom),
34.80 (CH2); LRMS m/z 304 (M+); HRMS calcd for
C20H16O3304.1099 (M+), found 304.1101.
Compounds 1a–j were prepared using similar methods
and conditions.
4.2.4. 4-Hydroxybiphenyl-3-carboxylic acid (1a). Fri-
edel–Crafts acetylation product of 4-phenyl phenol
was subjected to haloform reaction, and proctection
and deprotection as for 1b to obtain 1a as a white solid.
1
mp 214 ꢁC; H NMR (DMSO-d6, 200 MHz) d 8.04 (d,
J = 2.2 Hz, 1H), 7.87–7.81 (dd, 8.8 and 2.4 Hz, 1H),
7.64–7.34 (m, 5H), 7.09 (d, J=8.8 Hz, 1H); 13C NMR
(DMSO-d6, 100 MHz)
d
171.74 (CO2H), 160.55,
4.2.10. 2-Hydroxy-3,5-diphenylbenzoic acid (1j). The sim-
ilar reaction steps for 1i were performed to 2-phenylphe-
nol to obtain 1j as cream-colored crystals. Mp 219–
221 ꢁC; 1H NMR (DMSO-d6, 200 MHz) d 8.08 (d,
J = 2.4 Hz, 1H), 7.84 (d, J = 2.2 Hz, 1H), 7.73–7.35 (m,
10H); 13C NMR (DMSO-d6, 100 MHz) d 172.49
(CO2H), 158.12, 138.98, 136.77, 134.45, 131.19, 130.02,
129.32, 128.97, 128.12, 127.37, 127.29, 127.17, 126.37,
113.44 (Carom).
139.00, 133.89, 131.29, 128.97, 127.93, 127.07, 126.16,
117.81, 113.33 (Carom); LRMS m/z 214 (M+); HRMS
calcd for C13H10O3214.0630 (M+), found 214.0632.
4.2.5. 5-Benzyl-2-hydroxybenzoic acid (1d). In the same
way as 1a, from 4-benzyl phenol, 1d was obtained as
cream-colored recrystallized needles. Mp 139–140 ꢁC;
1H NMR (CDCl3, 200 MHz) d 10.25 (s, 1H), 7.75 (d,
J = 2.2 Hz, 1H), 7.37–7.15 (m, 6H), 6.96 (d,
J = 8.4 Hz, 1H), 3.94 (s, 2H, CH2); 13C NMR (CDCl3,
100 MHz) d 174.79 (CO2H), 160.68, 140.64, 137.86,
132.37, 130.57, 128.75, 128.59, 126.30, 117.99, 110.95
4.2.11. 2,4-Dibenzyl phenol (11)
4.2.11.1. Step 1. A mixture of 4-benzyl phenol (3.00 g,
16.3 mmol) and benzoyl chloride (9.5 mL, 82 mmol) in

S. Shrestha et al. / Bioorg. Med. Chem. 15 (2007) 6535–6548
6543
CS2 (12 mL) was added into ice-cooled suspension of
AlCl3 (6.5 g, 49 mmol) in CS2 (20 mL) over a 15-min
period. After stirring at 50 ꢁC for 8 h, the flask was
cooled in an ice bath and 1 M HCl (30 mL) was added
(white precipitate formed). It was then extracted with
EtOAc (3· 50 mL), and the organic layers were com-
bined, washed successively with 10% NaHCO3(50 mL),
water (2· 50 mL), and brine (50 mL), dried (MgSO4),
and concentrated. The crude product was dissolved in
1,4-dioxane (80 mL), treated with 20% NaOH (40 mL),
and allowed to stir at 70 ꢁC for 2 h. The reaction was
cooled, quenched by acidifying with 1 M HCl
(230 mL), and aqueous layer was extracted with EtOAc
(3· 60 mL). The organic extracts were combined,
washed successively with 5% NaOH (60 mL), water
(2· 60 mL), and brine (60 mL), and dried (MgSO4).
Evaporation of solvent and purification by silica gel col-
umn chromatography (hexane/EtOAc, 2:1, Rf = 0.4)
afforded C-benzoylated intermediate as a white solid
(4.044 g, 86% yield): mp 133 ꢁC; 1H NMR (CDCl3,
200 MHz) d 7.81–7.72 (m, 4H), 7.58–7.43 (m, 4H),
7.30 (s, 1H), 7.09 (d, J = 8.4 Hz, 2H), 6.81–6.77 (m,
2H), 3.99 (s, 2H, CH2).
lated intermediate (298 mg). The mother liquor was
evaporated, and the residue purified by silica gel column
chromatography (benzene/EtOAc, 9:1, Rf = 0.3), to ob-
tain a yellow solid (58 mg). Overall, 356 mg (71% yield)
of solid product was obtained. Methylation of this acet-
ylated intermediate (219 mg, 0.69 mmol) in EtOH
(4 mL), 10 M NaOH (0.26 mL), and dimethyl sulfate
(0.19 mL, 2.1 mmol) gave 12 as white crystals (214 mg,
1
94% yield): mp 79 ꢁC; H NMR (CDCl3, 200 MHz) d
7.90 (s, 1H), 7.85 (s, 1H), 7.29–7.25 (m, 3H), 7.11–7.07
(m, 5H), 6.84 (d, J = 8.8 Hz, 2H), 3.99 (s, 2H, CH2),
3.89 (s, 2H, CH2), 3.78 (s, 3H, OCH3), 2.57 (s, 3H,
COCH3).
4.2.13. 3,5-Dibenzyl-2-hydroxybenzoic acid (1k). Com-
pound 1k was obtained as a cream-colored solid in
90% yield from 3,5-dibenzyl-2-methoxyacetophenone
(12) following the similar steps as for 1b. Mp 208–
210 ꢁC; 1H NMR (CDCl3, 200 MHz) d 9.16 (s, 1H),
7.86 (s, 1H), 7.82 (s, 1H), 7.34 (s, 1H), 7.30 (s, 1H),
7.11 (s, 4H), 6.99 (s, 1H), 6.96 (s, 1H), 6.67 (d,
J = 8.4 Hz, 2H), 3.95 (s, 2H, CH2), 3.76 (s, 2H, CH2);
13C NMR (DMSO-d6, 100 MHz) d 167.19 (CO2H),
155.47, 146.65, 139.82, 137.87, 131.38, 129.53, 129.50,
128.79, 128.74, 128.67, 128.51, 115.11 (Carom), 40.13,
38.88 (CH2); LRMS m/z 318 (M+); HRMS calcd for
C21H18O3318.1256 (M+), found 318.1255.
4.2.11.2. Step 2. Hydrazine hydrate (2.8 mL, 90 mmol)
and KOH (780 mg, 14 mmol) were added to the interme-
diate (2.00 g, 6.94 mmol) in ethylene glycol (6 mL). The
reaction mixture was stirred at 120 ꢁC for 2 h and allowed
to reflux overnight. After cooling the mixture to room
temperature, it was diluted with benzene (70 mL), washed
with water (2· 80 mL), and then dried (MgSO4). The
benzene solution was concentrated under reduced pres-
sure and the purification of resulting residue by silica
gel column chromatography (hexane/EtOAc, 4:1,
Rf = 0.3) gave 11 as white shiny crystals (1.38 g, 73%
4.2.14. Methyl 3-bromo-5-(1,3-dioxolan-2-yl)-2-hydrox-
ybenzoate (14)
4.2.14.1. Step 1. Bromine (1 M in CH2Cl2, 55 mL,
55 mmol) was added dropwise over a 3-h period into
the ice-cooled solution of methyl 5-formylsalicylate
(4.95 g, 27.47 mmol) in CH2Cl2 (200 mL) with occa-
sional TLC check of the reaction progress. After the
reaction was complete, saturated NaHCO3 (200 mL)
was added to the reaction mixture and it was allowed
to stir for 30 min. Aqueous layer was extracted with
CH2Cl2 (100 mL). The combined organic layers were
washed successively with saturated NaHCO3 (100 mL),
water (2· 100 mL) and brine (100 mL), dried (Na2SO4),
and then concentrated. The crude product was dissolved
in EtOAc (80 mL) and adsorbed on 7 g of silica. Re-
moval of EtOAc under reduced pressure gave a powder
which, after purification on silica gel (hexane/EtOAc,
4:1, Rf = 0.3), gave methyl 3-bromo-5-formyl-2-hydrox-
ybenzoate as a light yellow solid (4.72 g, 67% yield): mp
1
yield): mp 89 ꢁC; H NMR (CDCl3, 200 MHz) d 7.25–
7.02 (m, 11H), 6.76 (d, J = 8.4 Hz, 2H), 4.67 (s, 1H,
OH), 3.94 (s, 2H, CH2), 3.87 (s, 2H, CH2).
4.2.12. 3,5-Dibenzyl-2-methoxyacetophenone (12). Acety-
lation of 11 (2.45 g, 8.93 mmol) with acetyl chloride
(2.5 mL, 36 mmol) and Et3N (7.5 mL, 54 mmol) in dry
ether (50 mL) gave 2,4-dibenzyl O-acetylated intermedi-
ate as a white solid (2.426 g, 89% yield). A mixture of
this intermediate (500 mg, 1.58 mmol) and acetyl chlo-
ride (0.34 mL, 4.7 mmol) in CS2 (1.5 mL) was added
into ice-cold suspension of AlCl3 (420 mg, 3.2 mmol)
in CS2 (2 mL) over a 5-min period. The reaction mixture
was stirred at 60 ꢁC for 5 h. After cooling with an ice
bath, the reaction was quenched by addition of 1 M
HCl (8 mL, yellow precipitate). It was then extracted
with EtOAc (2· 20 mL) and the combined organic ex-
tracts were washed successively with 10% NaH-
CO3(20 mL), water (2· 20 mL) and brine (20 mL),
dried (MgSO4), and then concentrated to yield a yellow
solid. The residue was dissolved in 1,4-dioxane (4 mL),
treated with 20% NaOH (1 mL), and stirred at 70 ꢁC
for 2 h. It was cooled and acidified with 1 M HCl
(8 mL, brown precipitate), and extracted with EtOAc
(3· 20 mL). The organic extracts were combined,
washed successively with water (2· 20 mL) and brine
(20 mL), dried (MgSO4), and concentrated. The residue
was recrystallized from EtOAc to give dibenzyl C-acety-
1
114 ꢁC; H NMR (CDCl3, 200 MHz): d 12.09 (s, 1H),
9.85 (s, 1H), 8.37 (d, J = 2.2 Hz, 1H), 8.28 (d,
J = 1.8 Hz, 1H), 4.05 (s, 3H).
4.2.14.2. Step 2. Ethylene glycol (2.53 mL, 45.5 mmol)
was added to a solution of methyl 3-bromo-5-formyl-2-
hydroxybenzoate (2.35 g, 9.1 mmol), p-toluenesulfonic
acid (94 mg, 5 mol%), and anhydrous copper sulfate
(5.8 g) in benzene (47 mL) and the resulting mixture
was refluxed overnight. After cooling to ambient temper-
ature, the reaction mixture was filtered, the residue was
washed with EtOAc, and the filtrate was treated with
1 M NaOH (20 mL). The organic solution was washed
with water (2· 100 mL) and brine (100 mL), dried
(Na2SO4), and concentrated. The crude product was sub-
jected to silica gel column chromatography (hexane/

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S. Shrestha et al. / Bioorg. Med. Chem. 15 (2007) 6535–6548
EtOAc, 4:1, Rf = 0.3) to give 14 as a light yellow solid
(1.60 g, 58% yield): mp 72–73 ꢁC; 1H NMR (CDCl3,
200 MHz): d 11.54 (s, 1H), 7.92 (d, J = 2.2 Hz, 1H),
7.86 (d, J = 2.2 Hz, 1H), 5.72 (s, 1H), 4.12 (m, 2H),
4.05 (m, 2H), 3.98 (s, 3H).
6.68 (m, 1H); 13C NMR (DMSO-d6, 100 MHz): d
191.16 (CHO), 171.94 (CO2H), 161.94, 147.60, 142.92,
131.41, 129.63, 127.63, 119.76, 113.97, 112.25, 111.34;
LRMS m/z 232 (M+); HRMS calcd for
C12H8O5232.0372 (M+), found 232.0368.
4.2.15. Methyl 5-(1,3-dioxolan-2-yl)-3-(furan-2-yl)-2-hydrox-
ybenzoate (15). A flame-dried flask was cooled to room
temperature under a nitrogen purge and charged
with dichlorobis(triphenylphosphine)palladium (104 mg,
3 mol%), 1,4-dioxane (3 mL), and 14 (1.5 g, 4.97 mmol)
in 1,4-dioxane (5 mL) successively. After nitrogen purg-
ing for 10 min, 2-(tributylstannyl)furan (2.34 mL,
7.45 mmol) was added via syringe and the reaction mix-
ture was stirred at 100 ꢁC for 16 h. The reaction mixture
was then cooled to room temperature and quenched by
addition of 10% ammonium hydroxide (25 mL). It was
extracted with EtOAc (3· 100 mL) and the combined
organic solution was washed successively with water
(2· 100 mL) and brine (100 mL), dried (Na2SO4), and
concentrated. The brown oil was subjected to silica gel
column chromatography (hexane/EtOAc, 2:1, Rf = 0.5)
to give 15 as a white solid (1.14 g, 79% yield): mp 105–
4.2.17. 2-Bromo-6-(2-propenyl)phenol (17). To a solution
of 2-bromophenol (13.43 g, 77.6 mmol) in acetone
(90 mL) were added K2CO3 (19.1 g, 138.15 mmol) and
allyl bromide (14.83 g, 122.62 mmol). The mixture was
refluxed for 4 h and cooled to ambient temperature.
The inorganic salt was removed by filtration and the or-
ganic solution was evaporated to dryness. Resulting
dark brown solution was passed through a cotton plug
and taken to the next step. The crude product obtained
above was irradiated with microwave (Samsung, 700 W)
for 66 min maintaining the temperature 200 ꢁC. Com-
pound 17 was obtained as a dark-colored liquid
(16.5 g, 100% yield): 1H NMR (CDCl3, 200 MHz): d
7.33 (d, J = 8 Hz, 1H), 7.08 (d, J = 8 Hz, 1H), 6.77 (t,
J = 8 Hz, 1H), 6.05 (m, 1H), 5.59 (s, 1H), 5.13 (m,
2H), 3.44 (d, J = 6.6 Hz, 2H).
1
108 ꢁC; H NMR (CDCl3, 200 MHz): d 11.73 (s, 1H),
4.2.18. 3-Bromo-2-methoxy-1-(1-propenyl)benzene (18).
Methyl iodide (14.5 mL, 232 mmol) was added to the
mixture of 17 (16.5 g, 77.5 mmol) and K2CO3 (16 g,
116 mmol) in acetone (130 mL) and the mixture was
heated to reflux for 4.5 h. After cooling to room temper-
ature, inorganic salt was removed by filtration and the
solvent was evaporated to dryness. The crude product
was passed through a silica gel pad to obtain brown li-
quid, which was dissolved in EtOH (62 mL) and mixed
with KOH (27 g, 0.48 mol) in H2O (9 mL). The resulting
mixture was stirred at 90 ꢁC for 5 h and then cooled to
room temperature. EtOH was removed under reduced
pressure and the brown aqueous solution was diluted
with water (140 mL). It was extracted with EtOAc (2·
100 mL), and the combined extracts were washed with
water (2· 100 mL) and brine (100 mL), dried (Na2SO4),
and then concentrated to afford 18 as a brown oil
(13.3 g, 75% yield): 1H NMR (CDCl3, 200 MHz): d
7.40 (d, J = 7.6 Hz, 2H), 6.96 (t, J = 7.6 Hz, 1H), 6.69
(m, 1H), 6.32 (m, 1H), 3.79 (s, 3H), 1.94 (dd, J = 1.4,
6.6 Hz, 3H).
8.15 (d, J = 2.2 Hz, 1H), 7.90 (d, J = 2.2 Hz, 1H), 7.48
(d, J = 1.4 Hz, 1H), 7.15 (d, J = 3.4 Hz, 1H), 6.53 (m,
1H), 5.79 (s, 1H), 4.18 (m, 2H), 4.08 (m, 2H), 3.98 (s,
3H).
4.2.16. 5-Formyl-3-(furan-2-yl)-2-hydroxybenzoic acid
(1l)
4.2.16.1. Step 1. HCl (1M, 27 mL) was added to a
solution of compound 15 (1.11 g, 3.83 mmol) in chloro-
form (27 mL). The mixture was heated to reflux for 18 h.
The organic layer was separated and the aqueous solu-
tion was extracted with CHCl3 (2· 50 mL). The com-
bined organic layers were washed successively with 5%
NaHCO3 (3· 50 mL), water (2· 50 mL), and brine
(50 mL), and dried over Na2SO4. The solvent was
evaporated and the residue was recrystallized from
EtOAc to provide methyl 5-formyl-3-(furan-2-yl)-2-
hydroxybenzoate as brown needles (902 mg, 96% yield):
1
mp 160–161 ꢁC; H NMR (CDCl3, 200 MHz): d 12.25
(s, 1H), 9.93 (s, 1H), 8.54 (d, J = 2.2 Hz, 1H), 8.32 (d,
J = 2.2 Hz, 1H), 7.53 (d, J = 1.4 Hz, 1H), 7.19 (d, J
= 3.6 Hz, 1H), 6.57 (m, 1H), 4.03 (s, 3H).
4.2.19. 3-Bromosalicylic acid (1m)18
4.2.19.1. Step 1. Potassium permanganate (3.45 g,
21.81 mmol) was added to the solution of 18 (1.5 g,
6.61 mmol) in acetone (80 mL) while cooling the reac-
tion flask at 0 ꢁC. After stirring the heterogeneous mix-
ture at 0 ꢁC for 3 h, solvent was removed under reduced
pressure. The dark brown solid was mixed with H2O
(17 mL) and NaHSO3 (4 g) and the resulting mixture
was stirred for 1 h at room temperature. It was filtered
by passing through Celite and the filtrate was acidified
with concd HCl (1 mL) and additional 1 M HCl
(2 mL). The white precipitate was separated and dis-
solved in 2 M NaOH (15 mL). The pale yellow solution
was washed with CH2Cl2 (2· 6 mL) and the organic
washings were discarded. The aqueous solution was
neutralized with concd HCl (1.5 mL) and acidified with
1 M HCl (5.3 mL). White precipitate was filtered,
washed with 0.1 M HCl (3 mL), and dissolved in EtOAc
4.2.16.2. Step 2. Methyl 5-formyl-3-(furan-2-yl)-2-
hydroxybenzoate (190 mg, 0.77 mmol) was dissolved in
methanol (15 mL) and mixed with 1 M NaOH (2 mL).
The mixture was heated to reflux for 1 day. After cool-
ing to room temperature, the reaction mixture was acid-
ified with 1 M HCl (2.5 mL), and solvent was
evaporated to remove methanol. The residue was dis-
solved in EtOAc (50 mL), and washed successively with
water (2· 30 mL) and brine (30 mL), and dried
(Na2SO4). The solvent was evaporated under reduced
pressure to give brownish solid (153 mg, 85% yield). It
could be further purified by recrystallization from
EtOAc to provide 1l as brown needles: mp 208–210 ꢁC
1
(dec); H NMR (DMSO- d6, 200 MHz): d 9.96 (s, 1H),
8.42 (d, J = 2.2 Hz, 1H ), 8.33 (d, J = 2.2 Hz, 1H),
7.86 (d, J = 1.8 Hz, 1H ), 7.15 (d, J = 3.6 Hz, 1H ),

S. Shrestha et al. / Bioorg. Med. Chem. 15 (2007) 6535–6548
6545
1
(30 mL). The EtOAc solution was then washed with
brine (1 mL), dried (Na2SO4), and concentrated to ob-
tain 3-bromo-2-methoxybenzoic acid as a white solid
(846 mg, 57% yield): mp 119–121 ꢁC; 1H NMR (CDCl3,
200 MHz): d 10.01 (br s, 1H, exchangeable with D2O),
8.03 (dd, J = 1.8, 8 Hz, 1H), 7.82 (dd, J = 1.8, 8 Hz,
1H), 7.14 (t, J = 8 Hz, 1H), 4.02 (s, 3H).
233 ꢁC; H NMR (CDCl3, 200 MHz) d 11.04 (s, 2H),
7.86 (d, J = 2.6 Hz, 2H), 7.47 (d, J = 1.8 Hz, 2H), 3.97
(s, 2H, CH2), 3.95 (s, 6H, CH3); 13C NMR (CDCl3,
100 MHz) d 169.83 (CO2Me), 158.73, 138.93, 130.53,
130.16, 113.49, 110.54, (Carom), 52.62 (CH3), 28.80
(CH2).
4.2.22. 5,50-Dibromo-3,30-dicarboxy-2,20-dihydroxydiphe-
nylmethane (3b).20 To a solution of 20 (100 mg,
0.21 mmol) in 1,4-dioxane (1 mL) was added 10% aque-
ous NaOH (2.0 mL) and the resulting solution was stir-
red at 80 ꢁC for 40 min. The reaction mixture was cooled
to room temperature and acidified by addition of 1 M
HCl (3 mL). The white precipitate was dissolved in
EtOAc (10 mL) and aqueous layer was extracted with
EtOAc (2· 10 mL). The combined organic extracts were
washed with water (2· 10 mL) and brine (10 mL), dried
(Na2SO4), and concentrated under reduced pressure to
give 3b as a white solid (91 mg, 97% yield): mp 281–
286 ꢁC (dec), rep 290–291 ꢁC; 1H NMR (CDCl3,
200 MHz) d 11.17 (s, 2H), 7.88 (d, J = 2.6 Hz, 2H),
7.56 (d, J = 2.4 Hz, 2H), 4.01 (s, 2H, CH2); LRMS m/z
446 (M+, 81Br), 444 (M+, 79Br); HRMS calcd for
C15H10Br2O6443.8844 (M+, 79Br), found 443.8848.
4.2.19.2. Step 2. A mixture of 3-bromo-2-methoxy-
benzoic acid (800 mg, 3.46 mmol), PhSH (0.35 ml,
3.46 mmol), and K2CO3 (29 mg, 0.207 mmol) in anhy-
drous NMP (3.46 mL) was heated at 190 ꢁC for
40 min under nitrogen atmosphere. The reaction mix-
ture was cooled, diluted with saturated aqueous
NaHCO3solution (35 mL), and extracted with EtOAc
(3· 30 mL, discarded). The aqueous layer was acidified
with 6 M HCl (10 mL) and extracted with EtOAc (3·
30 mL). The combined organics were washed with water
(2· 10 mL) and brine (20 mL), dried (Na2SO4), and con-
centrated to afford a yellow oil. The crude product was
subjected to silica gel column chromatography 2%
AcOH in hexane/EtOAc, 2:1, to obtain pure 1m as a
white solid (480 mg, 64% yield): mp 179–182 ꢁC; 1H
NMR (CDCl3, 200 MHz): d 11.01 (s, 1H), 7.94 (d,
J = 8 Hz, 1H), 7.81 (d, J = 8 Hz, 1H), 6.86 (t,
J = 8 Hz, 1H); 13C NMR (Acetone-d6, 100 MHz) d
172.47 (CO2H), 159.64, 140.03, 130.83, 121.04, 114.68,
111.45, (Carom); LRMS m/z 218 (M+, 81Br), 216 (M+,
79Br); HRMS calcd for C7H5BrO3215.9422 (M+, 79Br),
found 215.9421.
4.2.23. 5,50-Dibromo-3,30-dicarbomethoxy-2,20-dimetho-
xydiphenylmethane (21). In a 250-mL flask fitted with a
reflux condenser and CaCl2 tube was added 20 (2.28 g,
4.81 mmol) in anhydrous acetone (100 mL), K2CO3
(2.0 g, 14 mmol), and dimethyl sulfate (2.3 mL,
24 mmol). After refluxing overnight, the solvent was
evaporated under reduced pressure, and the solid was
partitioned between water (100 mL) and EtOAc
(100 mL). The organic layer was separated to save and
the aqueous layer was extracted with EtOAc (2·
100 mL). The combined organic extracts were washed
successively with water (2· 50 mL) and brine (50 mL),
and dried (Na2SO4). After concentration, the crude
product was column chromatographed (hexane/EtOAc,
9:1) to give 21 as a white solid (2.35 g, 97% yield): mp
4.2.20. 3,30-Dicarboxy-5,50-difluoro-2,20-dihydroxydiphe-
19
nylmethane (3a). To a suspension of 5-fluorosalicylic
acid (500 mg, 3.20 mmol) in 1,4-dioxane (1 mL) was
added conc. H2SO4 (3 mL) dropwise while cooling the
reaction flask in water bath. Paraformaldehyde
(97 mg) was added and the mixture was vigorously stir-
red at 65 ꢁC for 9 h. The reaction mixture was poured on
crushed ice (18 g). The precipitate was filtered and sub-
jected to column chromatography (3% AcOH in hexane/
EtOAc, 1:1, Rf = 0.3) to give 3a as a white solid (300 mg,
81 ꢁC; 1H NMR (CDCl3, 200 MHz)
d 7.85 (d,
J = 2.6 Hz, 2H), 7.35 (d, J = 2.6 Hz, 2H), 4.02 (s, 2H,
CH2), 3.93 (s, 6H, CO2CH3), 3.78 (s, 6H, OCH3).
1
58% yield). ): mp >300 ꢁC (dec); H NMR (Acetone-d6,
200 MHz) d 11.57 (br s, 2 H), 7.49 (dd, J = 8.8 Hz,
3.4 Hz, 2H), 7.27 (dd, J = 9.0 Hz, 3.4 Hz, 2H), 4.03 (s,
2H); 13C NMR (Acetone-d6, 100 MHz) d 172.27
(CO2H), 157.60, 154.36, 130.83, 124.74, 114.44, 113.14,
30.49 (CH2); LRMS m/z 324 (M+); HRMS calcd for
C15H10F2O6324.0445 (M+), found 324.0449.
4.2.24. 3,30-Dicarboxy-2,20-dihydroxy-5,50-diphenyldiph-
enylmethane (3c)
4.2.24.1. Step 1. A flame-dried flask was cooled to
room temperature under a nitrogen purge and charged
with (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)ben-
zene (463 mg, 2.27 mmol) and tetrakis(triphenylphos-
phine)palladium (34 mg, 0.03 mmol). After nitrogen
purging for 20 min, compound 22 (245 mg, 0.77 mmol)
dissolved in dry DMF (16 mL) was added into the flask
via syringe. Then 2 M K3PO4 (3.7 mL, 7.6 mmol) was
slowly added and the mixture was stirred at 80 ꢁC over-
night. The reaction mixture was allowed to cool to room
temperature, diluted with water (20 mL) and EtOAc
(20 mL), and stirred for 10 min. The aqueous layer
was extracted with EtOAc (2· 50 mL), and the com-
bined organic layers were washed successively with
water (2· 50 mL) and brine (50 mL), dried (Na2SO4),
and concentrated. The black oil was purified by silica
gel column chromatography (hexane/EtOAc, 2:1,
4.2.21. 5,50-Dibromo-3,30-dicarbomethoxy-2,20-dihydroxy-
diphenylmethane (20). 20 To a stirred suspension of
methyl 5-bromosalicylate (725 mg, 3.14 mmol) in water
(0.31 mL) was added concentrated H2SO4 (12.5 mL)
over a 20-min period while the temperature was main-
tained between ꢀ5 and 0 ꢁC. Then 1:1 mixture of 37%
aqueous HCHO and MeOH (3.1 mL) was added to
the reaction mixture over a 15-min period. The reaction
mixture was then stirred at 0 ꢁC for 2 h and at room
temperature for 22 h, and poured into crushed ice
(100 g). White precipitate was filtered, washed with
water, and dried to give a brown solid. The crude prod-
uct was column chromatographed on silica gel (CHCl3)
to obtain 20 as a white solid (304 mg, 41% yield): mp

6546
S. Shrestha et al. / Bioorg. Med. Chem. 15 (2007) 6535–6548
Rf = 0.5) to obtain 3,30-dicarbomethoxy-2,20-dime-
thoxy-5,50-diphenyldiphenylmethane as a pale yellow
solid (125 mg, 33% yield): mp 147 ꢁC; 1H NMR (CDCl3,
200 MHz) d 7.94 (d, J = 1.6 Hz, 2H), 7.52–7.31 (m,
12H), 4.23 (s, 2H, CH2), 3.95 (s, 6H, CO2CH3), 3.84
(s, 6H, OCH3).
4.2.25.2. Step 2. A mixture of 3,30-dicarbomethoxy-
5,50-difuran-2-yl-2,20-dimethoxydiphenylmethane (248 mg,
0.52 mmol), PhSH (0.21 ml, 2.1 mmol), and K2CO3
(140 mg, 1.0 mmol) in anhydrous NMP (20 mL) was
heated at 190 ꢁC for 40 min under nitrogen atmosphere.
The reaction mixture was cooled and saturated aqueous
NaHCO3 (25 mL) was added. It was extracted with
EtOAc (3· 100 mL) and the organic layers discarded.
The aqueous layer was acidified with 1 M HCl (40 mL)
and extracted with EtOAc (3· 50 mL). Combined organic
extracts were washed with water (2· 50 mL) and brine
(50 mL), dried (Na2SO4), and concentrated to afford
yellow solid (215 mg). It was washed with EtOAc to
obtain pure 3d as a yellow solid (165 mg, 75% yield): mp
>300 ꢁC (dec);1H NMR (DMSO-d6, 400 MHz) d 7.98
(d, J = 2.4 Hz, 2H), 7.68 (d, J = 2.4 Hz, 2H), 7.65 (d,
J = 2 Hz, 2H), 6.77(d, J = 3.2 Hz, 2H), 6.52 (m, 2H),
4.01 (s, 2H); 13C NMR (DMSO-d6, 100 MHz) d 172.18
(CO2H), 158.84, 152.17, 142.42, 131.03, 128.31, 123.16,
121.47, 112.76, 111.99, 104.57, 38.89 (CH2); LRMS m/z
420 (M+); HRMS calcd for C23H16O8420.0845 (M+),
found 420.0844.
4.2.24.2. Step 2. BBr3 (1.0 M in CH2Cl2, 2.0 mL,
2.0 mmol) was added to a stirred solution of 3,30-dicar-
bomethoxy-2,20-dimethoxy-5,50-diphenyldiphenylme-
thane (125 mg, 0.20 mmol) in dry CH2Cl2 (5 mL) under
a nitrogen atmosphere over 2 min period with cooling
the reaction mixture in dry-ice acetone base. The result-
ing orange to dark red solution was stirred at room tem-
perature overnight. The reaction was quenched by
addition of 1 M HCl (4 mL) and the aqueous layer
was extracted with EtOAc (3· 20 mL). The organic ex-
tracts were combined, washed successively with water
(2· 20 mL) and brine (20 mL), and then dried (Na2SO4).
After evaporation of the solvent under reduced pressure,
the brown residue was dissolved in 1,4-dioxane (4 mL)
and mixed with 20% aqueous NaOH (0.74 mL). The
reaction mixture was stirred at 80 ꢁC for 4 h. After evap-
oration of the solvent, the brown solution was diluted
with water (2 mL) and washed with EtOAc (2· 4 mL).
The aqueous layer was acidified with 1 M HCl (4 mL),
and the precipitate formed was dissolved in 100 mL
EtOAc. The organic layer was washed successively with
water (2· 20 mL) and brine (30 mL), and then dried
(Na2SO4). Evaporation of the solvent under reduced
pressure gave 3c as a yellowish brown solid (86 mg,
98% yield): mp >300 ꢁC (dec); 1H NMR (DMSO-d6,
200 MHz) d 11.82 (s, 2H, OH), 7.93 (d, J = 2 Hz, 2H),
7.73 (d, J = 2 Hz, 2H), 7.57–7.32 (m, 10H), 4.09 (s,
2H, CH2); 13C NMR (DMSO-d6, 100 MHz): d 172.37
(CO2H), 159.06, 139.23, 134.46, 130.65, 129.01, 128.33,
127.04, 126.14, 112.75, (Carom), 40.14 (CH2); LRMS
m/z 440 (M+); HRMS calcd for C27H20O6440.1260
(M+), found 440.1259.
4.2.26. 3,30-Dicarboxy-2,20-dihydroxy-5,50-dithiophen-2-
yl-diphenylmethane (3e). In a similar way with the
synthesis of 3c, 3e was obtained from 20 (300 mg,
0.63 mmol), tetrakis(triphenylphosphine)palladium
(29 mg), 2.0 M K3PO4 (3.16 mL, 6.33 mmol), 2-(4,4,5,
5-tetramethyl-1,3,2-dioxaborolan-2-yl)thiophene (332 mg,
1.58 mmol), and DMF (9 mL). Compound 3e was ob-
tained as a white solid (72 mg, 25% yield) along with
the monosubstituted product (37 mg, 13% yield): mp
1
>300 ꢁC (dec); H NMR (DMSO-d6, 400 MHz) d 7.89
(d, J = 2.4 Hz, 2H), 7.67 (d, J = 2.4 Hz, 2H), 7.45 (m,
2H), 7.32 (m, 2H), 7.08 (m, 2H), 4.01 (s, 2H, CH2);
13C NMR (DMSO-d6, 100 MHz) d 172.13 (CO2H),
158.98, 142.44, 133.15, 128.52, 128.41, 124.98, 124.83,
124.65, 122.93, 112.82, 38.88 (CH2); LRMS m/z 452
(M+); HRMS calcd for C23H16O6S2452.0388 (M+),
found 452.0387.
4.2.25. 3,30-Dicarboxy-5,50-difuran-2-yl-2,20-dihydroxy
diphenylmethane (3d)
4.3. IC50 determination
4.2.25.1. Step 1. A flame-dried flask was cooled to
room temperature under a nitrogen purge and charged
with dichlorobis(triphenylphosphine)palladium (51 mg,
0.07 mmol) and 1,4-dioxane (0.5 mL). Compound 21
(455 mg, 0.91 mmol) in 1,4-dioxane (10 mL) was then
added. After 10 min nitrogen purging, 2-(tributylstan-
nyl)furan (1.14 mL, 3.62 mmol) was added via syringe
and the reaction mixture was stirred at 100 ꢁC for 7 h.
The reaction mixture was then cooled to room tempera-
ture and quenched by addition of 10% ammonium
hydroxide (40 mL). It was extracted with EtOAc (3·
50 mL) and the combined organic solution was washed
successively with water (2· 50 mL) and brine (50 mL),
dried (Na2SO4), and concentrated. The brown solid was
subjected to silica gel column chromatography to give
3,30-dicarbomethoxy-5,50-difuran-2-yl-2,20-dimethoxydi-
phenylmethane as a yellow solid (248 mg, 58% yield): mp
107 ꢁC; 1H NMR (CDCl3, 200 MHz) d 8.01 (d, J = 2.2 Hz,
2H), 7.57 (d, J = 2.2 Hz, 2H), 7.43 (d, J = 1.8 Hz, 2H), 6.58
(d, J = 3.2 Hz, 2H), 6.45 (m, 2H), 4.15 (s, 2H, CH2), 3.95 (s,
6H, CO2CH3), 3.79 (s, 6H, OCH3).
The PTPase activities were assayed in buffer A (100 mM
Hepes, 5 mM EDTA, pH 7.0) at 37 ꢁC in a final pNPP
concentration of 2 mM. For this assay, the enzyme
was diluted with enzyme dilution buffer (25 mM Hepes,
5 mM EDTA, 1 mM DTT, and 1 mg/mL bovine serum
albumin, pH 7.3). The absorbance at 405 nm was mea-
sured to determine the amount of p-nitrophenol re-
leased. The IC50 values of the inhibitors were
determined by measuring the PTPase activity with a
range of different inhibitor concentrations. A typical
50-lL reaction system contained 5 lL pNPP, 5 lL en-
zyme, 5 lL inhibitor dissolved in DMSO, 10 lL 5· buf-
fer A, and 25 lL H2O. After the mixture, without pNPP,
had been incubated at 37 ꢁC for 10 min, the enzyme
reaction was initiated by the addition of pNPP. After
3 min at 37 ꢁC, the reaction was quenched by the addi-
tion of 0.5 M NaOH (0.95 mL), with the absorbance
at 405 nm measured to quantify the p-nitrophenol pro-
duced. The concentrations of PTPases in the assay mix-
tures were 2.1 lg/mL for PTP1B, 5 lg/mL for SHP-1cat,

S. Shrestha et al. / Bioorg. Med. Chem. 15 (2007) 6535–6548
6547
30 ng/mL for YPTP1 and 33 U (manufacturer’s defini-
tion)/mL for LAR and TC-PTP. The kinetic data were
analyzed using the GraFit 5.0 program (Erithacus
Software).
determine the NEFA concentration, 25 lL of plasma
sample and 1.5 mL of NEFA C reagent were incubated
at 37 ꢁC for 20 min, and then A550 measured.
Liver, epididymal fat depots, and retroperitoneal fat de-
pots were excised immediately following blood collec-
tion, washed in cold physiological (isotonic) saline,
gently blotted, and then weighed. Tissues were immedi-
ately frozen in liquid nitrogen and stored at ꢀ75 ꢁC for
further analysis.
4.4. Mouse experiment for obesity
Twenty-six C57BL/6J Jms Slc mice (4-week-old, male,
17–19 g) were purchased from Japan SLC Inc., Haruno
Breeding branch. The mice were individually housed
and maintained in a 12-h light/dark cycle at 22 2 ꢁC.
Food and water were available ad libitum. The experi-
mental diets, high fat diet (HFD, D12451) and low fat
diet (LFD, D10012G) containing 45% and 16% of the
calories from fat, respectively, were obtained from Re-
search Diets (New Brunswick, NJ). The diets were either
in pellet or powder form.
4.5. Statistical analysis
The data for the mice were expressed as means SEM.
Data were analyzed using a 1-way ANOVA with the
SPSS version 11.5 statistical package for windows (SPSS
Inc., Chicago, Illinois). Differences were considered sig-
nificant at p < 0.05.
All mice were acclimatized for 1 week (LFD), with 18
mice fed HFD for first 8 weeks of the study for the
development of obesity; the remaining eight were fed
LFD. The mice assigned to the LFD group were main-
tained on this diet throughout the study, as a lean con-
trol group. At week 8, all the HFD-fed mice were
assigned to one of two groups containing nine mice
each. The first group remained on HFD throughout
the study, as an obese control group. The remaining
group was fed HFD containing the PTP1B inhibitor,
3c, for 4 weeks. The concentration of 3c in the diets
was 5 g/kg of diet (0.5% w/w).
Acknowledgments
This work was supported by a Grant (C00344) from the
basic research program of the Korean Research Foun-
dation. B.R. Bhattarai was a recipient of a BK21
fellowship.
References and notes
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