2-O-Carboxymethylpyrogallol derivatives as PTP1B inhibitors with antihyperglycemic activity

Article abstract of DOI:10.1016/j.bmcl.2007.08.019

2-O-Carboxymethylpyrogallol derivatives (4-17) were synthesized, with their in vitro inhibitory activities against PTP1B and in vivo antihyperglycemic effects examined. Compound 14, the most potent among the series, showed a K_i value of 1.1 μM

Full text of DOI:10.1016/j.bmcl.2007.08.019

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5357–5360
2-O-Carboxymethylpyrogallol derivatives as PTP1B inhibitors
with antihyperglycemic activity
Bharat Raj Bhattarai,a Suja Shrestha,a Seung Wook Ham,b Kwang Rok Kim,c
Hyae Gyeong Cheon,c Keun-Hyeung Leea and Hyeongjin Choa,*
aDepartment of Chemistry and Institute of Molecular Cell Biology, Inha University, Incheon 402-751, Republic of Korea
bDepartment of Chemistry, Chung-Ang University, Seoul 156-756, Republic of Korea
cCenter for Metabolic Syndrome Therapeutics, Drug Discovery Division, Korea Reasearch Institute of Chemical Technology,
P.O. Box 107, Jang-Dong 100, Yuseong-Gu, Daejeon, 305-343, Republic of Korea
Received 23 May 2007; revised 3 August 2007; accepted 6 August 2007
Available online 15 August 2007
Abstract—2-O-Carboxymethylpyrogallol derivatives (4–17) were synthesized, with their in vitro inhibitory activities against PTP1B
and in vivo antihyperglycemic effects examined. Compound 14, the most potent among the series, showed a Ki value of 1.1 lM
against PTP1B, 7-fold lower than that against TC-PTP. When compound 14 was fed to a high-fat diet-induced diabetic mouse mod-
el, significant improvements were observed in both the fasting glucose level and glucose tolerance.
Ó 2007 Elsevier Ltd. All rights reserved.
Type 2 diabetes mellitus (T2DM) is a serious health
threat in modern society.1 Characterized by hyperglyce-
mia, T2DM is the cause of many complications, includ-
ing cardiovascular disease, blindness, renal failure, and
peripheral nerve damage.2 Increasing concern about
T2DM as health risk factor has led to an intensive
research for the development of an efficient treatment.3
with only one compound, Ertiprotafib, having pro-
gressed to phase 2 clinical trials, before it was stopped
due to insufficient efficacy and dose limiting side effects.7
As for the antidiabetic effect of Ertiprotafib, multiple
points of action have been proposed; PPARa, c and
IKK-b were reported as possible targets.8
Insulin resistance is an important factor in the patho-
genesis of T2DM and, therefore, insulin sensitizers, such
as TZD class molecules, have been used for the treat-
ment of this disease.4 The recent identification of protein
tyrosine phosphatase (PTP) 1B as an enzyme responsi-
ble for the dephosphorylation of insulin receptors has
raised the possibility that small molecule inhibitors of
PTP1B could act as insulin sensitizers and, thus, as anti-
diabetic drugs.5 This conception was validated in mouse
models; increased insulin sensitivity was observed when
the PTP1B gene was disrupted or when its expression
was suppressed by PTP1B antisense nucleotides.6
HO
O
O
O
OH
NH O
HN
S
O
OH
S
2
Br
1, Ertiprotafib
O
O
OH
O
O
O
O
HO
The development of PTP1B inhibitors exhibiting favor-
able pharmacological properties has been a challenge,
O
OCH3
OH
O
N
O
NH
3b
H
NH
Keywords: 2-O-Carboxymethylpyrogallol; PTP1B inhibitor; Diabetes;
Glucose tolerance; Hyperglycemia; Protein tyrosine phosphatase.
O
3a
*
Corresponding author. Tel.: +82 32 860 7683; fax: +82 32 867
5604; e-mail: hcho@inha.ac.kr
Figure 1.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.08.019

5358
B. R. Bhattarai et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5357–5360
Table 1. Compounds used in this study
with dose control, might be the strategy required for
drug development targeting PTP1B.1a
R2
OR1
O
OH
Another concern in the design of PTP1B inhibitors is the
cell permeability of the compounds. Negative charges
inherent in simple phosphotyrosine mimetics are unfa-
vorable factors for the translocation of the compounds
across the plasma membrane of the cell. Recently, 3a,
containing (2-hydroxyphenoxy)acetic acid as the core
structure, was reported to have a high level of cell per-
meability, and the equilibrium between the acid (3a)
and the lactone form (3b) has been suggested as a possi-
ble explanation for the improved membrane penetration
(Fig. 1).12 Previous studies on the design of PTP1B
inhibitors in our laboratory have indicated that a bulky
substituent (such as –OR1 in Table 1) in the ortho-posi-
tion of the phosphate mimetic group may contribute to
the inhibitor–enzyme complex formation.15 Based on
these premises, 2-O-carboxymethylpyrogallol deriva-
tives, with a benzyl or a substituted benzyl group at
O-1, were synthesized, and their in vitro PTP1B inhibi-
tory activities and in vivo antidiabetic effects examined.
The syntheses of compounds 4–17 are summarized in
Scheme 1. For the synthesis of 14, 2,6-dimethoxyphenol
(4a) was brominated, and then treated with ethyl bro-
moacetate to introduce a carboxymethyl functionality
to the central hydroxyl group. The palladium-catalyzed
coupling reaction of 6b with 3,5-di(trifluorometh-
yl)phenylboronic acid produced 14a. Removal of the
methyl protecting groups from 14a, followed by con-
trolled alkylation of one of the hydroxyl groups, by
reaction with 4-(trifluoromethyl)benzyl bromide, affor-
ded compound 14.17 Other compounds were synthesized
using similar strategies to that shown in Scheme 1.
OH
O
4-17
Compound
R1
PhCH2
R2
4
5
H
4-CF3PhCH2
PhCH2
H
Br
6
7
4-CF3PhCH2
H
PhCH2
Br
Ph
8
9
Ph
Ph
10
11
12
13
14
15
16
17
4-CF3PhCH2
PhCH2
3-CF3Ph
3-CF3Ph
3,5-(CF3)2Ph
3,5-(CF3)2Ph
4-CF3Ph
4-CF3Ph
Ph
4-CF3PhCH2
PhCH2
4-CF3PhCH2
PhCH2
4-CF3PhCH2
3,5-(CH3O)2PhCH2
Selective inhibition of PTP1B among >100 human PTP
family enzymes, especially the differentiation of PTP1B
and TC-PTP, have been major concerns in the design
of small molecule PTP1B inhibitors. The homology of
amino acid residues between PTP1B and TC-PTP
reaches 94% at the active site, and achieving PTP1B
selectivity has been a challenge.9 However, recent studies
have raised the possibility that the discrimination of the
two enzymes may not be crucial.9b,1a The insulin sensi-
tivity exhibited by heterozygous PTP1B+/ꢀ mice sug-
gests that partial inhibition of PTP1B might afford a
therapeutic effect.6a The healthy survival of TC-PTP+/ꢀ
mice indicates that partial inhibition of TC-PTP might
be tolerable.10 Furthermore, TC-PTP is known to be in-
volved in the regulation of insulin signaling.11 Taken to-
gether, a rather lenient therapeutic window, coupled
The 2-O-carboxymethylpyrogallol derivatives were eval-
uated for their inhibitory activities against PTP1B, with
p-nitrophenyl phosphate (pNPP) used as the substrate.
The enzyme and compounds were preincubated for
O
OEt
O
O
OEt
O
O
OH
O
OH
R2
b
c
MeO
OMe
a
MeO
OMe
HO
OH
HO
O
d
R1
R1
R1
4a
4b (R1 = H)
4c (R1 = H)
4 - 7
b
6b (R1 = Br)
6c (R1 = Br)
OH
e
MeO
OMe
O
OEt
O
OEt
O
OH
O
Br
O
O
R2
6a
c
d
MeO
OMe
HO
OH
HO
O
R3
R3
R3
9 - 17
8a - 17a
8, 9b - 17b
Scheme 1. Synthetic strategy for the syntheses of 2-O-carboxymethylpyrogallol derivatives (4–17). Reagents and conditions: (a) N-bromosuccin-
imide, CHCl3, C2H5OH, NaH, ꢀ75 °C ! rt ! 65 °C, 66%; (b) BrCH2CO2Et, K2CO3, DMF, rt, 6 h, 95%; (c) (i) BBr3, CH2Cl2, ꢀ75 °C ! rt, 1–3 h;
(ii) C2H5OH, H2SO4, 80 °C, overnight, 35–50% overall; (d) R2PhCH2Br, K2CO3, acetone, 75 °C, 3–4 h, 25–41%; (e) R3PhB(OH)2, Pd(PPh3)4,
toluene, C2H5OH, 80 °C, overnight, 75–81%.

B. R. Bhattarai et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5357–5360
Table 2. Inhibition of PTPases by compounds 1–17a
5359
b
Compound
IC50 [Ki, lM] (lM)
PTP1B
TC-PTP
[182d]
SHP-1cat
LAR-D1
YOP
YPTP-1
1, Ertiprotafib
1.4 ( 0.1) [1.5c]
500 ( 100) [102c]
[9.0d]
213
312
4
>1000
>1000
5
6
300 ( 14)
73 ( 5)
>1000
7
8
9
93 ( 5)
17 ( 1)
10
11
12
13
14
15
16
17
2.2 ( 0.2)
5.0 ( 0.4)
5.7 ( 0.4)
2.0 ( 0.1) [1.1e]
13 ( 1)
120 ( 20)
65 ( 3)
37 ( 2)
22 ( 1) [8.2e]
96 ( 4)
44 ( 3)
38 ( 2)
13 ( 1)
>200
>200
>200
33 ( 4)
21 ( 1)
15 ( 1)
6.0 ( 0.5)
34 ( 2)
13 ( 1)
52 ( 4)
17 ( 2)
20 ( 1)
9.0 ( 0.4)
17 ( 2)
23 ( 2)
9.0 ( 0.3)
4.0 ( 0.2)
25 ( 1)
116 ( 8)
>200
>200
5.4 ( 0.3)
40 ( 3)
12 ( 1)
a The enzyme reaction was initiated by the addition of pNPP to the enzyme in assay buffer, preincubated for 10 min with inhibitors dissolved in
DMSO. The final assay mixture contained: 2 mM pNPP, 40 nM PTP1B (or 5 lg/mL for SHP-1cat, 33 U (manufacturer’s definition)/mL for LAR-
D1 and TC-PTP, 50 U (manufacturer’s definition)/mL for YOP, and 15 nM for YPTP-1), 50 mM Hepes, and 5 mM EDTA, at pH 7, plus 10%
enzyme dilution buffer (25 mM Hepes, 5 mM EDTA, 1 mM DTT, and 1 mg/mL bovine serum albumin, at pH 7.3). After incubation at 37 °C for
3 min, 0.5 M NaOH (0.95 mL) was added, and the A405 measured to determine the amount of p-nitrophenol released. The IC50 values of the
inhibitors were determined by measuring the PTPase activity with a range of different inhibitor concentrations. The PTPs used in this study were
obtained as described previously, except the native form of PTP1B was used.14
b Values are means ( standard deviations) of 2–3 experiments. The kinetic data were analyzed using the GraFit 5.0 program (Erithacus Software).
c Reproduced from our previous report.15
d Reproduced from a previous report.12
e The Ki value for 14 was determined using the relationships: Kiapp = ([S] + Km)/(Km/Ki + [S]/aKi) and ts/to = 1/([I]/Kiapp + 1).16
10 min prior to the initiation of the enzyme reaction by
the addition of the substrate. As shown in Table 2, low
micromolar IC50 values were observed for compounds
11–16. Compound 14 was found to be the most potent
of the derivatives, with a Ki value of 1.1 lM, which
was low compared to both 3 (Ki = 9.0 lM) and Ertipro-
tafib (Ki = 1.5 lM).12,15 Table 2 also shows the selectiv-
ity profiles of compounds 11–16 against several
phosphatases. These compounds demonstrated 5- to
13-fold greater PTP1B selectivity against TC-PTP.
Compound 14 showed a 7-fold greater selectivity for
PTP1B versus TC-PTP.
The antihyperglycemic effect of 14 was examined in a
high-fat diet (HFD)-induced diabetic mouse model.18
After 8 wks on HFD, the mice (C57BL/6JCr Slc, male)
were fed HFD or HFD + 14 for a further 4 weeks. The
compound 14 was administered as mixtures with the
food (1.0 g of 14 per kg of diet). The daily uptake of
14 was approximated as 3 mg/day/mouse, which is
equivalent to 85 mg/day/kg of mouse weight. At the
end of the 4 weeks of drug-feeding period, the fasting
glucose level of the drug-fed group was significantly low-
er than the HFD control group (Fig. 2, 0 min). After the
injection of glucose, the drug-fed group exhibited a sig-
nificantly faster decrease in the blood glucose concentra-
tion compared to the HFD control group (Fig. 2). In
contrast to the antihyperglycemic effect, no difference
was observed in body weights between the HFD and
HFD + 14 groups.
Figure 2. Intraperitoneal glucose tolerance test. Five-wk-old mice
(C57BL/6J Cr Slc, male), acclimatized for 1 wk, were fed HFD for 8
wks, and then divided into two groups (7 mice/group). The two groups
were fed HFD (d) or HFD + 14 (h) for 4 weeks. The lean control
group (j) was fed LFD all throughout the entire 12 wk period. The
mice were then fasted for 6 h, starting from the beginning of the light
cycle. Just before the intraperitoneal injection of glucose (1 g/kg body
weight), blood was withdrawn from the tip of the tail, and the baseline
glucose level measured. After the injection of glucose, tail blood was
collected at 20, 40, 60, 90, and 120 min later, with the blood glucose
concentration determined using an Accu-ChekÒ Active diagnostic kit
(Roche, Germany). All values are mean values SEM; n = 7/group.
Statistical comparisons between HFD and HFD + 14 groups were
performed using a 1-way ANOVA, where, *p < 0.01 and **p < 0.001.

5360
B. R. Bhattarai et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5357–5360
Opgenorth, T. J.; Ulrich, R. G.; Crosby, S.; Butler, M.;
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11357.
In summary, a series of 2-O-carboxymethylpyrogallol
derivatives were synthesized in an attempt to improve
the inhibitory potency of (2-hydroxyphenoxy)acetic acid
derivatives against PTP1B. Among the compounds, 14
exhibited a Ki value of 1.1 lM against PTP1B and dis-
played in vivo efficacy, which significantly lowered the
fasting glucose level and improved the glucose tolerance
in an obesity-induced diabetic mouse model.
7. Zhang, Z.-Y.; Lee, S.-Y. Expert Opin. Investig. Drugs
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8. (a) Erbe, D. V.; Wang, S.; Zhang, Y. L.; Harding, K.;
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Acknowledgments
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This work was supported by a Grant (C00344) from the
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dation. B. R. Bhattarai was a recipient of a BK21
fellowship.
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j.bmcl.2007.08.019.
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17. Compound 14: mp 177–179 °C; 1H NMR (acetone-d6,
200 MHz), d 8.23 (s, 2H), 8.00 (s, 1H), 7.81–7.78 (m, 4H),
7.18 (d, J = 2.2 Hz, 1H), 7.00 (d, J = 2.2 Hz, 1H), 5.44 (s,
2H), 4.85 (s, 2H); 13C NMR (acetone-d6, 100 MHz): d
175.32 (CO2H), 153.24, 152.45, 144.20, 142.87, 137.80,
135.67, 132.91, 132.26, 129.05, 128.86, 128.33, 126.44,
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