Synthesis and Biological Evaluation of 2,4,6-Trihydroxychalcone Derivatives as Novel Protein Tyrosine Phosphatase 1B Inhibitors

Article abstract of DOI:10.1111/j.1747-0285.2012.01431.x

A series of 2,4,6-trihydroxychalcone derivatives were synthesized and identified as reversible and competitive protein tyrosine phosphatase (PTP) 1B inhibitors with IC₅₀ values in the micromolar range. Compound 4a had the greatest in vitro inhibition activity against PTP1B (IC₅₀=0.27± 0.01μm) and the best selectivity (6.9-fold) for PTP1B relative to T-cell protein tyrosine phosphatases. The compounds identified herein provide a foundation on which to design specific inhibitors of PTP1B and other PTPs.

Full text of DOI:10.1111/j.1747-0285.2012.01431.x

ª 2012 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2012.01431.x
Chem Biol Drug Des 2012
Research Article
Synthesis and Biological Evaluation of 2,4,
6-Trihydroxychalcone Derivatives as Novel
Protein Tyrosine Phosphatase 1B Inhibitors
Liang-Peng Sun1, Li-Xin Gao2, Wei-Ping Ma2,
Fa-Jun Nan2, Jia Li2,* and Hu-Ri Piao1,*
blood glucose and improved insulin sensitivity (14–17). In addition,
several groups have demonstrated that overexpression of PTP1B is
sufficient to drive tumorigenesis in mice, providing additional support
for the use of PTP1B inhibitors for cancer therapy (18,19). Therefore,
PTP1B seems to be a useful target for the treatment of type 2 diabe-
tes mellitus, obesity, and cancer, and small molecular inhibitors of
PTP1B may be promising candidate drugs for these diseases.
1Key Laboratory of Natural Resources of Changbai Mountain &
Functional Molecules, Ministry of Education, Yanbian University
College of Pharmacy, Yanji 133000, China
2National Center for Drug Screening, State Key Laboratory of Drug
Research, Shanghai Institute of Materia Medica, Shanghai
Institutes for Biological Science, Chinese Academy of Sciences,
Shanghai 201203, China
In recent years, following the elucidation of the protein structure of
PTP1B, many synthetic PTP1B inhibitors with submicromolar or
nanomolar activities have been discovered through high-throughput
screening and structure-based design (20,21). However, the low cell
permeability and low bioavailability of these compounds have limited
their development as effective drugs because of the presence of
highly negatively charged residues (including difluoromethylphospho-
nates, carboxymethylsalicyclic acids, and oxalylaminobenzoic acids)
mimicking the phosphate group in IRS. In addition, because of the
structural homology throughout many families of PTPs, it is challeng-
ing to identify selective inhibitors specific to each PTP. Recently, sev-
eral of the proposed inhibitors were derived from natural products
(22–24), which represented a very diverse library of candidate drugs.
*Corresponding authors: Hu-Ri Piao, piaohuri@yahoo.com.cn; Jia Li,
jli@mail.shcnc.ac.cn
A series of 2,4,6-trihydroxychalcone derivatives
were synthesized and identified as reversible and
competitive protein tyrosine phosphatase (PTP) 1B
inhibitors with IC50 values in the micromolar
range. Compound 4a had the greatest in vitro
inhibition activity against PTP1B (IC50 = 0.27
0.01 lM) and the best selectivity (6.9-fold) for
PTP1B relative to T-cell protein tyrosine phospha-
tases. The compounds identified herein provide a
foundation on which to design specific inhibitors
of PTP1B and other PTPs.
Chalcones, originally isolated from natural sources and considered
as precursors of flavonoids and isoflavonoids, are abundant in edi-
ble plants. Chalcones are open-chain flavonoids in which two aro-
matic rings are joined by a three carbon a, b-unsaturated carbonyl
system (1,3-diphenyl-2-propen-1-ones). As a minority subgroup of
the flavonoid family, like other members, the compounds with a
chalcone backbone have been reported to be responsible for a vari-
ety of biological activities, including anticancer (25,26), antimicrobial
(27,28), anti-inflammatory (29,30), antioxidative (31), antimalarial
(32), antiproliferative (33), antidepressant (34), and tyrosinase inhibi-
tory activities (35). Hence, chalcones are considered to be a class
with important therapeutic potential. Recently, several chalcones
derived from natural products and their derivatives have been iden-
tified as PTP1B inhibitors (Figure 1). For example, Chen et al. (36)
reported that brousso-chalcone A (I) isolated from Broussonetia
papyrifera exhibited moderate inhibitory effects against PTP1B. Yoon
et al. (37) reported that several chalcones (II, III) isolated from
Glycyrrhiza inflata and semisynthetic licochalcone A derivatives also
exhibited moderate inhibitory effects against PTP1B. Subsequently,
Liu et al. reported that bromo-retrochalcones based on licochalcone
A and E (IV) showed low micromolar IC50 values against PTP1B
(38). In addition, Hoang et al. reported that three chalcone-derived
Diels–Alder compounds isolated from Morus bombycis showed
Key words: 2,4,6-trihydroxychalcone, diabetes, inhibitors, protein
tyrosine phosphatase 1B, structure–activity relationship
Received 29 February 2012, revised 20 April 2012 and accepted for pub-
lication 5 June 2012
Protein tyrosine phosphatases (PTPs) play essential roles in intracellu-
lar signal transduction by regulating the cellular level of tyrosine
phosphorylation. Cellular processes controlled by PTPs include
growth, differentiation, metabolism, migration, gene transcription, ion
channel activity, immune responses, apoptosis, and bone development
(1–4). Unregulated PTP activity is responsible for several human dis-
eases including cancer, diabetes, obesity, and dysfunction of the
immune system (5–7). One member of the PTP family, PTP1B, acti-
vates c-Src in human breast cancer and also downregulates insulin
signaling by dephosphorylating the insulin receptor (IR), insulin recep-
tor substrate-1 (IRS-1), and insulin receptor substrate-2 (IRS-2) (8–11).
Studies from two laboratories have shown that PTP1B-knockout mice
exhibit enhanced insulin sensitivity, improved glucose tolerance and
resistance to diet-induced obesity (12,13). Furthermore, treatment of
diabetic mice with PTP1B antisense oligonucleotides reduced the
expression level of the enzyme and subsequently normalized the
1

Sun et al.
(E)-1-(3-Hydroxyphenyl)-3-phenylprop-2-en-1-one
(1b)
Yield, 75.3%; mp, 192–193 ꢀC; IR (KBr) per cm: 3438 (OH), 1645
(C=O); 1H-NMR (300 MHz, DMSO–d6) d: 6.90–8.17 (m, 9H, Ar–H),
7.87 (d, 1H, J = 15.9 Hz, CH=CH, a–H), 8.01 (d, 1H, J = 15.9 Hz,
CH=CH, b–H); MS m ⁄ z 225 (M + 1).
OH
OH
OH
HO
HO
OCH3
O
OH
O
II Licochalcone A
IC50 = 19.1 0.1 µM
I
IC50 = 36.8 µM
(E)-1-(4-Hydroxyphenyl)-3-phenylprop-2-en-1-one
(1c)
Br
OH
OH
Yield, 72.4%; mp, 180–181 ꢀC; IR (KBr) per cm: 3430 (OH), 1636
(C=O); 1H-NMR (300 MHz, DMSO–d6) d: 6.94–8.22 (m, 9H, Ar–H),
7.75 (d, 1H, J = 15.6 Hz, CH=CH, a–H), 7.96 (d, 1H, J = 15.6 Hz,
CH=CH, b–H); MS m ⁄ z 225 (M + 1).
HO
H3CO
OCH3
OCH3
O
O
IV IC50 = 1.9 0.1 µM
III Licochalcone E
IC50 = 20.7 1.8 µM
Figure 1: Structures of reprted PTP1B inhibitors.
(E)-1-(2,4-Dihydroxyphenyl)-3-phenylprop-2-en-1-
one (1d)
Yield, 42.4%; mp, 163–164 ꢀC; IR (KBr) per cm: 3429 (OH), 1633
(C=O), 1173 (CO); 1H-NMR (300 MHz, DMSO–d6) d: 6.27–7.69 (m,
8H, Ar–H), 7.70 (d, 1H, J = 15.6 Hz, CH=CH, a–H), 7.91 (d, 1H,
J = 15.6 Hz, CH=CH, b–H); MS m ⁄ z 241 (M + 1).
remarkable inhibitory activity against PTP1B, and the structure–
activity relationship (SAR) indicated that the hydroxyl groups of
chalcone play an important role in the inhibition of PTP1B (39).
These reports suggested that hydroxychalcones might be promising
PTP1B inhibitors.
Although chalcones, which are widely expressed in plants, exert vari-
ous biological activities, including PTP1B inhibition, the SAR of chal-
cones against PTP1B inhibition has not been fully examined because
of an insufficient number of molecular compounds to test. Therefore,
in this study, we designed and synthesized a series of 2,4,6-trihydr-
oxychalcone derivatives in which the B ring was substituted with hal-
ogens (F, Cl, and Br) or an alkoxyl. We then evaluated their inhibitory
effects toward PTP1B and elucidated the SARs. Selected compounds
were also subjected to kinetic and selectivity analyses to determine
whether their biological properties made them suitable for further
development. Docking simulations of the compounds into the active
site of PTP1B were also performed to rationalize the results.
The synthesis procedure and spectral data of the compounds 4a,
4e-t were previously described by our laboratory (34).
(E)-3-(4-(Benzyloxy)phenyl)-1-(2,4,6-
trihydroxyphenyl)prop-2-en-1-one (4b)
Yield, 78.3%; mp, 179–180 ꢀC; IR (KBr) per cm: 3432 (OH), 1631
(C=O), 1172 (CO); 1H-NMR (300 MHz, DMSO–d6) d: 5.36 (s, 2H,
CH2), 5.85–7.49 (m, 11H, Ar–H), 7.62 (d, 1H, J = 15.6 Hz, CH=CH,
a–H), 8.01 (d, 1H, J = 15.6 Hz, CH=CH, b–H); MS m ⁄ z 363 (M + 1).
(E)-3-(Benzo[d][1,3]dioxol-6-yl)-1-(2,4,6-
trihydroxyphenyl)prop-2-en-1-one (4c)
Experimental Section
Yield, 62.1%; mp, 216–218 ꢀC; IR (KBr) per cm: 3435 (OH), 1624
(C=O), 1188 (CO); 1H-NMR (300 MHz, DMSO–d6) d: 6.05 (s, 2H,
CH2), 5.98–7.21 (m, 5H, Ar–H), 7.62(d, 1H, J = 15.6 Hz, CH=CH,
a–H), 7.98 (d, 1H, J = 15.6 Hz, CH=CH, b–H); MS m ⁄ z 301 (M + 1).
Material and methods
Melting points were determined in open capillary tubes and are
uncorrected. Reaction courses were monitored by TLC on silica gel-
precoated F254 Merck plates. Developed plates were examined
with UV lamps (254 nm). IR spectra were recorded (in KBr) on a
FTIR1730. 1H-NMR spectra were measured on a Bruker AV-300
spectrometer using TMS as the internal standard. Mass spectra
were measured on an HP1100LC (Agilent Technologies, Santa Clara,
CA, USA). The major chemicals were purchased from Sigma–Aldrich
and Fluka. The synthesis procedure of the compounds 1a–d was
previously described by our laboratory (40).
(E)-3-(4-(Allyloxy)-3-methoxyphenyl)-1-(2,4,6-
trihydroxyphenyl)prop-2-en-1-one (4d)
Yield, 70.9%; mp, 168–170 ꢀC; IR (KBr) per cm: 3451 (OH), 1627
(C=O), 1185 (CO); 1H-NMR (300 MHz, DMSO–d6) d: 3.84 (s, 3H,
OCH3), 4.57–4.59 (d, 2H, CH2), 5.21–6.05 (m, 3H, CH=CH), 5.95–7.15
(m, 5H, Ar–H), 7.67(d, 1H, J = 15.5 Hz, CH=CH, a–H), 8.02 (d, 1H,
J = 15.5 Hz, CH=CH, b–H); MS m ⁄ z 343 (M + 1).
(E)-1-(2-Hydroxyphenyl)-3-phenylprop-2-en-1-one
(1a)
Yield, 82.6%; mp, 88–89 ꢀC; IR (KBr) per cm: 3435 (OH), 1643
(C=O); 1H-NMR (300 MHz, DMSO–d6) d: 7.01–8.32 (m, 9H, Ar–H),
7.90 (d, 1H, J = 15.3 Hz, CH=CH, a–H), 8.08 (d, 1H, J = 15.3 Hz,
CH=CH, b–H); MS m ⁄ z 225 (M + 1).
(E)-3-(3-(Trifluoromethyl)phenyl)-1-(2,4,
6-trihydroxyphenyl)prop-2-en-1-one (4u)
Yield, 57%; mp, 147–148 ꢀC; IR (KBr) per cm: 3356 (OH), 1685
(C=O), 1231 (CO); 1H-NMR (300 MHz, DMSO–d6) d: 5.85–8.01 (m,
6H, Ar–H), 7.65 (d, 1H, J = 15.3 Hz, CH=CH, a–H), 8.16 (d, 1H,
J = 15.3 Hz, CH=CH, b–H); MS m ⁄ z 325 (M + 1).
2
Chem Biol Drug Des 2012

2,4,6-trihydroxychalcones as PTP1B inhibitors
Biological assays for PTP1B and related PTPs
The enzymatic assays for PTP1B and related PTPs were performed
as described elsewhere (41). Briefly, the enzymatic activity of the
PTP1B catalytic domain was determined at 30 ꢀC by monitoring the
hydrolysis of pNPP. Dephosphorylation of pNPP generates the prod-
uct pNP, which was monitored at an absorbance of 405 nm using
an EnVision multilabel plate reader (PerkinElmer Life Sciences, Bos-
ton, MA, USA). Assays were performed in a total volume of 100 lL
containing 50 mM 3-[N-morpholino]propane-sulfonic acid (MOPs), pH
6.5, 2 mM pNPP, and 30 nM recombinant PTP1B, as well as the indi-
cated concentrations of the inhibitor. The production of pNP was
continuously monitored, and the initial rate of the hydrolysis was
determined from the early linear region of the enzymatic reaction
kinetic curve.
of PTP1B was regarded as the binding site. Compound 4p was
docked in a flexible manner, and conformations are determined by
the stochastic Monte Carlo conformation search method (maximum,
50 000 trials).
Results and Discussion
Chemistry
The synthetic pathways 1a–d and 4a–u are illustrated in
Schemes 1 and 2, respectively. The preparation of compounds 1a–
d was carried out via Claisen–Schmidt condensation of the appro-
priate acetylbenzene with benzaldehyde (Scheme 1). The compounds
4a–u were synthesized according to Scheme 2. 1-(2,4,6-trihydroxy-
phenyl)-ethanone was treated with chloromethyl methyl ether and
potassium carbonate in acetone at room temperature to produce 1-
(2-hydroxy-4,6-bis-methoxymeth-oxy-phenyl)-ethanone 2. Intermedi-
ates 3a–u were prepared by Claisen–Schmidt condensation of 2
with appropriate aromatic aldehydes or hydroxyaromatic aldehydes,
protected as chloromethyl methyl ether. Then, demethoxymethyla-
tion of 3a–u was carried out in 5M HCl in methanol at reflux tem-
perature obtaining products 4a–u in good yield (43). The spectral
Enzyme kinetic analysis
The enzyme kinetic assays of compound 4p were performed as previ-
ously (41,42). Assays were performed in a total volume of 100 lL con-
taining 50 mM MOPS, pH 6.5, 30 nM PTP1B, pNPP in twofold dilutions
from 80 mM, and different concentrations of the inhibitor. In the pres-
ence of the competitive inhibitor, the Michaelis–Menten equation is
described as 1 ⁄ v = (Km ⁄ [Vmax[S]])(1 + [I] ⁄ Ki) + 1 ⁄ Vmax, where v = ini-
tial rate, Vmax = maximum rate, and [S] = substrate concentration.
The Ki value was determined using a linear replot of the apparent
Km ⁄ Vmax slope from the primary reciprocal plot versus inhibitor con-
centration [I] according to the equation Km ⁄ Vmax = 1 + [I] ⁄ Ki.
1
data obtained from infrared spectroscopy, H-nuclear magnetic reso-
nance, and mass spectrometry were consistent with the structures
proposed.
Biological results and discussion
The inhibitory activities of the synthesized compounds against
PTP1B were measured using p-nitrophenyl phosphate (pNPP) as a
substrate. The results are summarized in Table 1. Ursolic acid, a
known PTP1B inhibitor (IC50 = 3.20 € 0.31 lM), was used as a posi-
tive control (44).
Molecular docking
Molecular docking was performed using Discovery Studio 2.1 (Ac-
celrys, San Diego, CA, USA). The crystal structure of PTP1B (Protein
Data Bank, 1NL9) was defined as the receptor, and the active site
As shown in Table 1, 22 compounds of 25 tested showed good
PTP1B inhibitory activity at a concentration of 20 lg ⁄ mL and dose
dependently inhibited PTP1B with IC50 values ranging from 0.27 to
19.52 lM. Compounds 4a, 4d, 4e, 4f, 4g, and 4j showed IC50
values <1 lM. Of these, 4a, the most potent among the series, had
an IC50 value of 0.27 € 0.01 lM, about 12-fold better than that of
the positive control, ursolic acid.
a
R
R
O
O
1a-d
1b: 3-OH
1d: 2,4-(OH)2
R= 1a: 2-OH
1c: 4-OH
Firstly, several hydroxychalcones (monohydroxychalcones 1a–c, 2,4-
didroxychalcone 1d) were synthesized to examine the impact of
Scheme 1: Reagents and conditions: (a) benzaldehyde, H3BO4,
ethylene glycol, 120 ꢀC.
Scheme 2: Reagents and con-
ditions: (a) MOMCl, K2CO3, acetone
0 ꢀC – rt; (b) corresponding benzal-
dehyde, 10% KOH, ethanol ⁄ H2O,
rt; (c) 5 M HCl, MeOH ⁄ THF, reflux.
Chem Biol Drug Des 2012
3

Sun et al.
Table 1: Inhibitory activity of compounds 1a–d and 4a–u
against PTP1B
Table 2: Inhibitory activity of selected compounds against
related protein tyrosine phosphatases (PTPs)
Compounds
PTP1B IC50 (lM)a
Compounds
PTP1B IC50 (lM)
IC50 (lM)a
1a
1b
1c
1d
4a
4b
4c
4d
4e
4f
NAb
NA
NA
4j
4k
4l
0.92 € 0.12
7.83 € 0.56
3.55 € 0.16
6.21 € 1.81
3.42 € 0.17
4.74 € 1.05
1.15 € 0.06
1.96 € 0.46
3.18 € 1.19
3.66 € 0.23
3.63 € 0.46
1.71 € 0.19
Compounds TCPTP
CDC25B
LAR SHP-1
SHP-2
4a
4d
4e
4f
1.85 € 0.13 0.36 € 0.03 NAb
9.30 € 0.73
9.66 € 2.22
3.53 € 0.32 0.93 € 0.05 NA 19.47 € 1.37 22.95 € 1.34
2.31 € 0.17 1.88 € 0.31 NA 20.83 € 2.90 22.90 € 2.72
3.79 € 0.28 0.75 € 0.03 NA 16.39 € 1.17 14.37 € 2.72
2.41 € 0.15 1.07 € 0.07 NA 27.11 € 2.37 29.37 € 1.40
5.25 € 0.51 2.46 € 0.30 NA 68.79 € 6.09 69.90 € 5.67
19.52 € 2.78
0.27 € 0.01
2.17 € 0.18
1.44 € 0.28
0.37 € 0.04
0.58 € 0.03
0.55 € 0.07
0.58 € 0.06
1.41 € 0.13
1.26 € 0.03
4m
4n
4o
4p
4q
4r
4g
4p
TCPTP, T-cell protein tyrosine phosphatase; LAR, leukocyte antigen-related
phosphatase.
4s
4t
4g
4h
4i
UAc
aThe pNPP assay. IC50 values were determined by regression analyses and
expressed as means € SD of three replications.
bNot active at 20 lg ⁄ mL concentration.
4u
3.20 € 0.31
aThe pNPP assay. IC50 values were determined by regression analyses and
expressed as means € SD of three replications.
bNot active at 20 lg ⁄ mL concentration.
ring significantly increased the PTP1B inhibitory activity. Com-
pared to 4e, replacing the methoxy group with a benzyloxy
group as in compound 4b resulted in about a fourfold loss of
activity. This seemed to indicate increasing the bulkiness in the
B ring was not well tolerated and caused loss of activity. Fur-
thermore, the position of the substituent (F, Cl) on the B ring
significantly influenced the PTP1B inhibitory activities, with an
order of activity of m-F > p-F > o-F for fluorinated compounds
and p-Cl > 2,4-Cl2 > o-Cl > m-Cl for chlorinated compounds. For
the brominated compounds, the impact of the substituent posi-
tion was not evident.
cPositive control.
hydroxyl groups on the A ring for PTP1B inhibitory activity. Mono-
hydroxychalcones (i.e., 1a–c) showed no activity at 20 lg ⁄ mL, but
interestingly, introduction of two hydroxyl groups to compound 1 at
the 2- and 4-position of the A ring (1d) dramatically improved the
PTP1B inhibitory activity with IC50 values of 19.52 € 2.78 lM. Sur-
prisingly, when the third hydroxyl group was introduced at the 6-
position of the A ring of 1d (i.e., 4j), a more remarkable enhance-
ment in activity was achieved (IC50 = 0.92 € 0.12 lM). The previous
results suggest that increasing the number of hydroxyl groups on
the A ring in chalcones leads to stronger binding and improves
potential inhibitory effects against PTP1B. It was thought that
increasing the number of hydroxyl groups might stabilize the com-
plex with the enzyme through H-bond interactions within the active
site and surrounding subpockets, as this has often been observed
in PTP1B ⁄ inhibitor complexes (21).
In addition to potency improvements, we investigated the selectivity
of the representative compounds 4a, 4d, 4e, 4f, 4g, and 4p
against other PTPs. As shown in Table 2, homogeneous T-cell pro-
tein tyrosine phosphatase (TCPTP) inhibitory activities were investi-
gated simultaneously by the same method (44). These compounds
showed four- to 10-fold greater selectivity for PTP1B than for
TCPTP. Besides TCPTP, there are several other critical PTPs nega-
tively regulating insulin dephosphorylation such as cell division cycle
25 homolog B (CDC25B), leukocyte antigen-related phosphatase
(LAR), src homology phosphatase-2 (SHP-2), and src homology phos-
phatase-1 (SHP-1). Cell division cycle 25 homolog B (CDC25B), a
dual-specificity phosphatase, is an attractive potential therapeutic
antitumor target for small molecule intervention because of its cen-
tral role in controlling malignant cell proliferation by regulating
cyclin-dependent kinases and its high expression in many human
tumors (45). Thus, we tested the inhibitory activity of these com-
pounds on the purified recombinant human enzymes CDC25B, LAR,
SHP-1, and SHP-2 (20,41). As shown in Table 2, we concluded that
these compounds had no visible activities against receptor-like
transmembrane LAR and possessed about 30- to 60-fold and 26- to
62-fold selectivity for PTP1B over SHP-1 and SHP-2, respectively.
The compounds demonstrated a one- to threefold selectivity for
PTP1B over CDC25B.
Based on these observations, a series of 2,4,6-trihydroxychalcone
derivatives 4a–u were synthesized and their PTP1B inhibitory
activity was evaluated to examine the impact of substituents on
the B ring. After analyzing the activities of the synthesized com-
pounds 4a–u, the following SARs were obtained. By comparison
with compound 4j (IC50 = 0.92 € 0.12 lM), substitutions (R) with
electron-donating groups on the B ring in 4a–i (IC50 = 0.27–
1.41 lM) showed better or comparative activity, whereas substitu-
tion by the withdrawing groups 4k–u (IC50 = 1.15–7.83 lM)
resulted in a decrease or loss of potency. These results indicated
that electron-donating groups contributed more to the inhibitory
activity of PTP1B than the electron-withdrawing groups. This is
consistent with the docking results where the electron-donating
groups increase the electron density of the B ring and result in
a more affinitive p–p interaction with Tyr46. By comparison with
compound 4i (IC50 = 1.26 € 0.03 lM), the methylation at C-3 of
4i (i.e., 4a), the dimethylation at C-3 and C-4 of 4i (i.e., 4f),
and the methylation at C-3 and O-allyloxylation at C-4 of 4i
(i.e., 4d) contribute more to the inhibitory activity. These results
suggested that substitution of the methoxy group on chalcone B
A kinetic study was performed to investigate the inhibitory mecha-
nism of compound 4p (41,42). As shown in Figure 2A, the inhibition
modality of 4p toward PTP1B exhibited characteristics typical of a
competitive inhibitor, including increased Km values and unchanged
4
Chem Biol Drug Des 2012

2,4,6-trihydroxychalcones as PTP1B inhibitors
A
B
C
Figure 2: Characterization of
4p to PTP1B. (A) At various fixed
concentrations of 4p the initial
velocity was determined with vari-
ous concentrations of pNPP. (B)
Typical competitive inhibition of
4p shown by Lineweaver-Burk
plot. (C) Ki determination.
A
B
Figure 3: Docking result of
compound 4p in the PTP1B cata-
lytic pocket. (A) Binding pose of
4p in PTP1B active site; (B) Key
residues in binding site surrounding
4p.
Vmax values following increases in inhibitor concentration. The
Lineweaver–Burk plot also indicated that 4p is a competitive inhibi-
tor of PTP1B because a fit of the data yielded an intersection of
the best fit lines on the y-axis (Figure 2B). The results indicate that
4p binds to the catalytic pocket of PTP1B and behaves as a com-
petitor to the physiological substrate. The Ki value calculated from
Figure 2C was 0.47 lM.
Conclusion
In conclusion, a series of 2,4,6-trihydroxychalcone derivatives were
synthesized and identified as reversible and competitive PTP1B
inhibitors with IC50s in the micromolar range. After performing sys-
tematic SAR studies of the R substituents of the B ring, we identi-
fied six compounds with IC50 values <1 lM. We also predicted the
mechanisms involved in the interaction between the inhibitors and
PTP1B using docking analysis. Among these, the representative
compounds had no visible activities against receptor-like transmem-
brane LAR and possessed about 30- to 60-fold, 26- to 62-fold, and
one- to threefold selectivity for PTP1B over SHP-1, SHP-2, and
CDC25B, respectively. More importantly, these compounds exhibited
four- to 10-fold selectivity for PTP1B over TCPTP. These results pro-
vide a possible opportunity for the development of novel PTP1B
inhibitors with promising cell permeability, bioavailability, and
improved pharmacological properties.
To understand the inhibitory activity of 4p against PTP1B, we per-
formed molecular docking analysis of 4p using CDOCKER in concert
with Discovery Studio 2.1 (http://www.accelrys.com). The X-ray crys-
tal structure of PTP1B with a resolution of 2.40 ꢀ (Protein Data
Bank, 1NL9) was used for the docking studies. The preferred coordi-
nation modes of 4p are shown in Figure 3. Fragment A of 4p is
bound into the active site not only through H-bond interactions of
2-OH and 4-OH of the A ring, but also through the carbonyl. The 2-
OH of the A ring showed an H-bond interaction with Ser216 and
Ala217. The 4-OH of the A ring showed an H-bond interaction with
Arg221, while the carbonyl showed H-bond interactions with
Gly220. The predicted model also showed the B ring of the ligands
(e.g., the lipophilic 4-chlorophenyl group) extended deep into the
hydrophobic pocket and made a p–p stacking interaction with
Tyr46.
Acknowledgment
This work was supported by the National Natural Science Founda-
tion of China (Grants 20962021 and 81125023).
Chem Biol Drug Des 2012
5

Sun et al.
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