Design, synthesis and biological evaluation of stilbene derivatives as novel inhibitors of protein tyrosine phosphatase 1B

Article abstract of DOI:10.3390/molecules21121722

By imitating the scaffold of lithocholic acid (LCA), a natural steroidal compound displaying Protein Tyrosine Phosphatase 1B (PTP1B) inhibitory activity, a series of stilbene derivatives containing phenyl-substituted isoxazoles were designed and synthesized. The structures of the title compounds were confirmed by ¹H-NMR, ¹³C-NMR and HRMS. Activities of the title compounds were evaluated on PTP1B and the homologous enzyme TCPTP by using a colorimetric assay. Most of the target compounds had good activities against PTP1B. Among them, compound 29 (IC₅₀ = 0.91 ± 0.33 μM), characterized by a 5-(2,3-dichlorophenyl) isoxazole moiety, exhibited an activity about 14-fold higher than the lead compound LCA and a 4.2-fold selectivity over TCPTP. Compound 29 was identified as a competitive inhibitor of PTP1B with a K_i value of 0.78 μM in enzyme kinetic studies.

Full text of DOI:10.3390/molecules21121722

molecules
Article
Design, Synthesis and Biological Evaluation of
Stilbene Derivatives as Novel Inhibitors of Protein
Tyrosine Phosphatase 1B
Haibing He 1, Yinghua Ge 2, Hong Dai 1,*, Song Cui 1, Fei Ye 1, Jia Jin 2,* and Yujun Shi 1,
*
1
College of Chemistry and Chemical Engineering, Nantong University, Nantong 226019, China;
hehaibing@ntu.edu.cn (H.H.); cuisong123@163.com (S.C.); yefei@zstu.edu.cn (F.Y.)
2
College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, China; geyinghua1992@163.com
Correspondence: dai_hong@aliyun.com (H.D.); aukaukauk@163.com (J.J.); ntushyj@163.com (Y.S.);
Tel.: +86-571-8684-3199 (J.J.); +86-513-8501-2856 (H.D. & Y.S.)
*
Academic Editor: Derek J. McPhee
Received: 26 October 2016; Accepted: 12 December 2016; Published: 16 December 2016
Abstract: By imitating the scaffold of lithocholic acid (LCA), a natural steroidal compound displaying
Protein Tyrosine Phosphatase 1B (PTP1B) inhibitory activity, a series of stilbene derivatives containing
phenyl-substituted isoxazoles were designed and synthesized. The structures of the title compounds
were confirmed by 1H-NMR, 13C-NMR and HRMS. Activities of the title compounds were evaluated
on PTP1B and the homologous enzyme TCPTP by using a colorimetric assay. Most of the target
compounds had good activities against PTP1B. Among them, compound 29 (IC50 = 0.91
±
0.33 µM),
characterized by a 5-(2,3-dichlorophenyl) isoxazole moiety, exhibited an activity about 14-fold higher
than the lead compound LCA and a 4.2-fold selectivity over TCPTP. Compound 29 was identified as
a competitive inhibitor of PTP1B with a Ki value of 0.78 µM in enzyme kinetic studies.
Keywords: protein tyrosine phosphatase 1B (PTP1B); lithocholic acid; stilbene; inhibitor
1. Introduction
Protein tyrosine phosphatases (PTPs) are a superfamily of enzymes involved in crucial cellular
signalling mechanisms by controlling the phosphorylation levels of specific tyrosine residues in
peptides and proteins which regulate many cellular functions, such as cell proliferation, survival,
metabolism, adhesion and migration. Among the PTPs so far identified, the cytosolic protein tyrosine
phosphatase 1B (PTP1B) was the first phosphatase to be isolated and is therefore the most widely
studied [1–4]. A large body of experimental data acquired from in vivo studies on humans and
PTP1B-knockout mice has identified PTP1B as a major negative regulator of both insulin and leptin
signals. Consequently, inhibiting of PTP1B was considered to be a potential therapeutics for treating
Type 2 diabetes and obesity by enhance insulin sensitivity and resistance to obesity [5–8]. Moreover, it
is worth noting that PTPs share a high degree of structural conservation in the active site, especially
for T-cell protein tyrosine phosphatase (TCPTP), which has a sequence identity of about 74% in the
catalytic domains with PTP1B [9]. There is always a challenge for the potent PTP1B inhibitors to keep
good selectivity over TCPTP in the same time [10].
In the past decades, several steroids and their derivatives were found to possess moderate to
good PTP1B inhibitory activities, such as lithocholic acid (LCA,
and trodusquemine ( ) [13], the first PTP1B inhibitor for the treatment of diabetes in clinical trials.
However, risk of the hormonal effect of steroids and their derivatives always cannot be neglected.
Years ago, diethylstilbestrol ( ) was developed as a mimic and alternative drug for estradiol ( ) [14],
which indicated the fact that the stilbene moiety could be an ideal replacement of the steroid skeleton
1, Figure 1) [11], claramine (3) [12]
2
4
5
Molecules 2016, 21, 1722; doi:10.3390/molecules21121722
www.mdpi.com/journal/molecules

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in drug design when use the “scaffold hopping” method [15]. Additionally, stilbene is a common
moiety found in many natural products endowed with antibacterial, antioxidative, antiproliferative or
antidiabetic activities, such as resveratrol (6), pinosylvin (7) and piceatannol (8) [16–19].
Figure 1. Steroids 1~3 reported as PTP1B inhibitors and stilbene derivatives 4~8.
In a previous report we disclosed that the introduction of phenyl-substituted heterocyclic rings,
such as pyrazole, oxazole, isoxazole and thiazoles, into the ring A on LCA would improve the
inhibitory activity against PTP1B. On the other hand, it has also been verified that the 23-COOH of
LCA was irreplaceable for maintaining the activities against PTP1B [11]. Based on the views above, we
tentatively designed a class of stilbene derivatives, as shown in Figure 2, which was characterized by a
phenyl-substituted isoxazole linked to the stilbene scaffold by an ether bond. In order to search for the
optimized phenyl isoxazole moiety, at the very beginning, we designed and synthesized four mimics
(compounds 15, 16, 17 and 18), containing 3-phenyl-4-isoxazolyl-, 5-methyl-3-phenyl-4-isoxazolyl-,
5-phenyl-4-isoxazolyl and 5-phenyl-3-isoxazolyl groups, respectively, and evaluated their inhibitory
effects against PTP1B. As a result, compound 18 was found to have the highest activity to PTP1B
(IC50 = 6.33
ring C was synthesized and evaluated.
±
1.02 µM). Then a series of its derivatives 19~30 with different groups on the phenyl
Figure 2. The design of the title compounds.
2. Results and Discussion
2.1. Chemistry
As shown in Scheme 1, target compounds 15
intermediate 14 and the corresponding chloromethyl isoxazole 15c
acid-binding agent, followed by hydrolysis in KOH solution and acidification with hydrochloric acid.
Starting from 3-methylbenzoate ( ), the key intermediate 14 was synthesized in five steps including
~30 were all prepared by condensation of the stilbene
~30c in DMSO with Cs2CO3 as the
9
bromination [20], Arbuzov reaction, Wittig-Horner reaction [21], demethylation [22] and selective
methylation. At the beginning, by using PPh3, a Wittig reaction was employed to synthesize the

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compound 12, giving a mixture of cis- and trans-products with a ratio of about 1:2. Alternatively,
the Wittig-Horner reaction gave a satisfactory result, unexpectedly accompanied by the hydrolysis
of methyl ester due to the strong basicity of t-BuOK. In addition, intermediate 10 was obtained
conveniently through a routine method using NBS as the brominating agent and AIBN as the radical
initiator. Since deprotection of the methyl of 12 in BBr3/DCM led to a mass of by-products, the reaction
was carried out successfully in a mixture of AlCl3 and Et3N. Before the condensation of phenolic
hydroxyl and the chlorinated intermediates 15c~30c, the carboxyl group on the phenyl A ring of 13
was selectively protected by MeI. Slight methylation of the phenolic hydroxyl group occured in the
same time.
Compounds 15c
reduction by LiAlH4 and chlorination with SOCl2 in DCM. Intermediates from the above two steps
were used directly without any purification. Compounds 15a 16a and 17a were prepared according to
the reported methods [23 25]. By reference to Kamal’s report, compounds 18a 30a were synthesized
from the corresponding substituted acetophenones [26].
~30c were synthesized from the corresponding methyl carboxylates 15a~30a via
,
–
~
Scheme 1. Synthesis of compounds 10
~
30
.
Reagents and conditions: (
a) CCl4, NBS, reflux, 6 h;
(b) P(OEt)3, 160 ◦C; (
c
) t-BuOK, THF, 0 ◦C~r.t., overnight; (
d
) AlCl3, Et3N, 60 ◦C~90 ◦C, 18 h;
(e) MeI, K2CO3, acetone, 60 ◦C, 15 h; (
f
) Cs◦2CO3, DMSO, 65 ◦C, 8 h; (
g
) KOH, H2O, 65 ◦C, 6 h;
◦
(h) LiAlH4, 0 C~r.t., 4 h; (i) SOCl2, DCM, 45 C, 8 h.
2.2. In Vitro Biological Evaluation
All of the title compounds were assayed on the enzyme PTP1B in vitro. In compounds 15~18,
the phenyl position on the isoxazole ring caused huge differences in the inhibition against PTP1B.
As shown in Table 1, both compounds 15 and 17, characterized by a 3-phenyl-4-isoxazole moiety,
exhibit moderate activities against the enzyme. Meanwhile, the 5-phenyl-4-isoxazole compound 16
(IC50 = 16.2
higher activity (IC50 = 6.33
phenyl B ring. This result inspired us to further investigate the substituents on phenyl C and their
effects on the activities against PTP1B. On the other hand, as expected, methylation of the carboxyl
of 18 led to an obvious decrease on PTP1B inhibition.
±
4.52
µ
M) also has similar activity, while another 5-phenyl compound, 18, exhibits a much
±
1.02 M) due to the different position of the isoxazole ring linked to the
µ
Derivatives of 18 and their activities against PTP1B and TCPTP are listed in Table 2. For substituents
at the 2-position of phenyl C, the electron-withdrawing chlorine atom (compound 19) was beneficial to
the activity when compared to an electron-donating methoxy (compound 20). 3-subtituents also affect
the activities remarkably. Compounds containing 3-Cl (21) and 3-Br (23) exhibit obvious inhibition to
PTP1B, whereas a 3-F leads to a dramatic decrease in activity. NO2 (25, IC50 = 2.05
±
0.54 µM) seems to
be the optimal 3-substituent among the synthesized compounds, giving an activity 3-fold higher than
compound that of 18. In addition, when the 3-position is occupied by a methyl group, the resulting
compound 24 still shows good inhibitory effect against the enzyme (IC50 = 3.68 ± 1.12 µM).

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Table 1. The structures of compounds 15, 16, 17, 18, 18d and their IC50 values against PTP1B.
Compound
15
X
R1
IC50 (µM) a
20.7 ± 3.83
H
16
17
H
H
16.2 ± 4.52
15.7 ± 2.15
6.33 ± 1.02
18
H
18d
Me
23.7 ± 3.62
2.96 ± 0.35
Oleanolic acid b
a
IC50 values were determined by regression analyses and expressed as means
Positive control.
±
SD of three replications; b
Table 2. The structures of compounds 18~30 and their IC50 values against PTP1B and TCPTP.
IC50 (µM) a
TCPTP/PTP1B b
Compound
R
PTP1B
TCPTP
18
19
20
21
H
2-Cl
2-OMe
3-Cl
3-F
6.33 ± 1.33
1.75 ± 0.82
7.66 ± 1.05
5.37 ± 1.53
>20
9.58 ± 0.33
36.15 ± 3.82
>40
1.5
20.7
5.3
7.58 ± 1.32
1.4
c
22
>40
-
23
24
25
26
27
28
29
30
3-Br
9.85 ± 2.04
3.68 ± 1.12
2.05 ± 0.54
3.56 ± 0.28
2.23 ± 0.51
3.34 ± 0.81
0.91 ± 0.33
1.22 ± 0.48
12.54 ± 2.51
2.71 ± 0.19
>40
4.1
5.1
6.5
2.2
3.6
1.6
4.2
4.9
1.7
2.3
3-Me
3-NO2
4-Me
4-OMe
4-F
18.91 ± 1.18
13.25 ± 1.70
7.67 ± 0.35
8.02 ± 0.99
5.19 ± 0.31
3.78 ± 0.22
6.02 ± 1.13
20.95 ± 3.66
6.12 ± 0.15
3,4-Cl2
3,4-F2
Lithocholic acid
Oleanolic acid d
a
IC50 values were determined by regression analyses and expressed as means
TCPTP/PTP1B, the ratio of IC50 of TCPTP and PTP1B; “-”, none accurate value was calculated;
Positive control.
±
SD of three replications;
b
c
d
The derivatives present good tolerance of 4-substituents on the phenyl C ring, since compounds 26
(4-Me), 27 (4-OMe) and 28 (4-F) have similar activities against PTP1B. Amazingly, the di-substituted

Molecules 2016, 21, 1722
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compounds, either dichloro (29) or difluoro (30) exhibited the highest inhibitory activities against the
enzyme among all the title compounds. Most of all, compound 29 has an activity (IC50 = 0.91 ± 0.33 µM)
about 14-fold higher than the lead compound LCA, which prompts us to further study on its
structure modification and biological activity. However, as shown in Table 2, it is unfortunate that
most of the compounds also exhibit some activities against TCPTP, except for compounds 20
and 23 The selectivities against PTP1B and TCPTP vary a lot, from 1.4 to 22.9. Among the five
compounds most potent against PTP1B (19 25 27 29 and 30), compound 19 has the best selectivity
TCPTP/PTP1B = 20.7) while compound 29 has about a 4-fold selectivity over TCPTP. These two
, 22
.
,
,
,
(
compounds are promising for further study as anti-diabetes drugs.
2.3. Enzyme Kinetic Study
In order to determine the inhibition mode of the title compounds, the most active compound 29
was selected for a PTP1B enzyme kinetic study using the Michaelis-Menten equation. As shown in
Figure 3, the Vmax value remained constant while the Km value increased with the mounting compound
concentration, indicating that 29 was a competitive PTP1B inhibitor, which was further confirmed by a
Lineweaver–Burk plot (Figure 3c). The calculated inhibitory constant Ki of compound 29 was 0.78 µM.
6
4
2
0
100
80
60
40
20
0
0.0
0.6
1.2
1.8
2.4
0.0
0.6
1.2
[I] μM
(b)
1.8
2.4
[I] μM
(a)
6
4
2
y = 1.668x + 1.301
0.20
0
0.6
1.2
1.8
2.4
0.15
0.10
0.05
-0.6
0.6
1.2
1.8
2.4
-0.8 -0.4 -0.0
-0.05
0.4
0.8
1.2
1.6
-2
1/[pNPP] (L/mmol)
(c)
[I] μM
(d)
Figure 3. Characterization of 29 to PTP1B. (
a,
b) At various fixed concentrations of 29 the initial velocity
was determined with various concentrations of pNPP; (c) Typical competitive inhibition of 29 shown
by Lineweaver–Burk plot; (d) Linear replot of Km versus the inhibitor (29) concentration [I].
3. Experimental Section
3.1. Chemsitry
3.1.1. General Information
All reagents were chemically pure and solvents were dried according to standard methods.
The 1H-NMR and 13C-NMR spectra were obtained on an AV400 spectrometer (400 MHz, 1H; 100 MHz,
13C, Bruker, Billerica, MA, USA) in CDCl3 with tetramethylsilane as the internal standard. The melting
points were determined on an X-4 binocular microscope melting point apparatus (Beijing Tech

Molecules 2016, 21, 1722
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Instrument Co., Beijing, China) and are uncorrected. High-resolution mass data were obtained
on a Micromass TOF II spectrometer (Micromass, Cary, NC, USA). The reactions were monitored by
analytical thin-layer chromatography (TLC) carried out on silica gel GF254 plates with ultraviolet (UV)
light detection.
3.1.2. Synthesis of Methyl 3-((Diethoxyphosphoryl)methyl)benzoate (11)
A solution of methyl 3-methylbenzoate (47 g, 0.31 mol) in CCl4 (200 mL) was heated to
reflux followed by addition of AIBN (2.6 g) in one portion. After that, NBS (67 g, 0.38 mol) was
added carefully to the mixture during 2 h, and then the reaction was refluxed for an additional
5 h. After cooling to r.t. the mixture was filtered and evaporated under vacuum to give methyl
3-(bromomethyl)benzoate (10, 67 g, Yield: 94%) as a yellowish oil. The obtained oil was then dissolved
◦
in triethyl phosphate (130 g, 0.71 mol), heated to 160 C and kept at this temperature for an additional
10 h. The mixture was concentrated on a rotary evaporator and then purified by flash chromatography
(PE:EA = 5:1~PE:EA = 1:3) to give 11 as a yellow oil, which was used without any further purification.
3.1.3. Synthesis of (E)-3-(4-Methoxystyryl)benzoic Acid (12)
To an ice-cooled solution of compound 11 (5 g, about 17.5 mmol) in THF (30 mL) was added a
mixture of t-BuOK (3.9 g, 34.8 mmol) and THF (40 mL) during 30 min. After stirring for additional
30 min, a solution of 4-methoxybenzaldehyde (17.5 mmol) in THF (20 mL) was added and then the
reaction mixture was warmed to room temperature, and stirred for another 8 h before quenching
with 2 N HCl. The mixture was then evaporated to remove the THF and precipitation occurred
meanwhile. The precipitate was then filtered and dried to give 12 as a yellow solid without any
additional purification. Yield: 4.8 g (46%); 1H-NMR (DMSO-d6)
δ: 12.93 (brs, 1H), 8.12 (s, 1H), 7.81
(d, J = 7.6 Hz, 2H), 7.58 (d, J = 8.6 Hz, 2H), 7.49 (t, J = 7.8 Hz, 1H), 7.28 (d, J = 16.5 Hz, 1H), 7.18
(d, J = 16.5 Hz, 1H), 6.96 (d, J = 8.6 Hz, 2H), 3.78 (s, 3H).
3.1.4. Synthesis of (E)-3-(4-Hydroxystyryl)benzoic Acid (13)
Anhydrous aluminium trichloride (30 g) was added to triethylamine (200 mL) in portions during
◦
30 min and then heated to 60 C for 3 h before adding compound 12 (16.5 g, 65.0 mmol) in one portion.
The resultant mixture was then heated to 80 ◦C and kept for another 16 h. After evaporating most
of the triethylamine, the reaction was quenched by addition of iced hydrochloric acid. The water
phase was extracted by ethyl acetate (80 mL
evaporated to give crude 13, which was then recrystallized from ethyl acetate to give 13 as a yellowish
solid. Yield: 7.8 g (48%); 1H-NMR (DMSO-d6)
: 12.98 (brs, 1H), 9.63 (s, 1H), 8.09 (s, 1H), 7.78~7.81 (m,
×
3), dried over anhydrous Na2SO4, filtered and then
δ
2H), 7.48~7.49 (m, 3H), 7.13~7.15 (m, 2H), 6.78 (d, J = 8.7 Hz, 2H).
3.1.5. Synthesis of Methyl (E)-3-(4-Hydroxystyryl)benzoate (14)
Anhydrous K2CO3 (0.58 g, 4.2 mmol) was added to a solution of 13 (1.68 g, 7 mmol) in acetone
(15 mL), and the mixture was then stirred for 2 h at room temperature before adding MeI (1.07 g,
4.2 mmol) in one portion. The reaction was then stirred for an additional 16 h. After that, the mixture
was filtered, evaporated and purified by column chromatography (PE:EA = 3:1) to give 14 as a
yellowish solid. Yield: 0.29 g (51%); 1H-NMR (DMSO-d6)
δ: 9.64 (s, 1H), 8.10 (s, 1H), 7.79~7.85 (m, 2H),
7.46~7.50 (m, 3H), 7.25 (d, J = 16.5 Hz, 1H), 7.12 (d, J = 16.5 Hz, 1H), 6.78 (d, J = 8.5 Hz, 2H), 3.88 (s,
3H); 13C-NMR (DMSO-d6)
δ: 166.2, 157.6, 138.2, 130.3, 130.1, 129.7, 129.1, 128.1 (2C), 127.7, 127.4, 126.6,
123.9, 115.5 (2C), 52.1.
3.1.6. Synthesis of (3-Phenylisoxazol-4-yl)methanol (15b)
On an ice bath, to a solution of 15a (5 mmol) in anhydrous THF (20 mL) was added LiAlH4
(5 mmol) in portions during 30 min. After that, the mixture was stirred for 2 h and quenched by 1 mL

Molecules 2016, 21, 1722
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H2O added dropwise. After adding anhydrous Na2SO4 (5 g), the reaction was filtered and evaporated
to give 15b, which was used in the next step without any purification. Intermediates 16b
synthesized according to methods described above for the synthesis of 15b.
~30b were
3.1.7. Synthesis of 4-(Chloromethyl)-3-phenylisoxazole (15c)
To a solution of 15b (3 mmol), DCM (20 mL) and DMF (1~2 drops), SOCl2 (10 mmol) was added
and heated to 45 ◦C. After keeping at this temperature for 8 h, the solution was evaporated to give
15c as yellow oil, which was used in the next step without any purification. Intermediates 16c~30c
were synthesized according to the above method for the synthesis of 15c and purified by column
chromatography (PE:EA = 5:1~10:1) if necessary.
3.1.8. Synthesis of (E)-3-(4-((3-Phenylisoxazol-4-yl)methoxy)styryl)benzoic Acid (15)
A mixture of 14 (1 mmol), 15c (1 mmol) and Cs2CO3 (2 mmol) in DMSO (5 mL) was heated to
85 ◦C and kept at this temperature for 8 h, then the reaction mixture was cooled to 60 ◦C followed
by the addition of 20% KOH solution (2 mL). After stirring at 60 ◦C for 4 h, the reaction mixture was
cooled, acidified by 1 N hydrochloric acid diluted with water to give a mass of precipitate of the crude
product, which was then recrystallized from methanol to give the purified 15. Yellow solid; Yield: 63%;
1H-NMR (DMSO-d6)
7.27 (t, J = 7.6 Hz, 1H), 7.18 (d, J = 16.5 Hz, 1H), 7.13 (d, J = 16.5 Hz, 1H), 7.04 (d, J = 8.8 Hz, 2H), 5.13
(s, 2H); 13C-NMR (DMSO-d6)
: 169.2, 161.3, 159.2, 157.7, 136.7, 131.0, 130.6, 129.6 (2C), 129.4, 128.7,
128.6, 128.4 (2C), 128.3 (2C), 128.0, 127.8, 127.4, 127.3, 115.6 (2C), 114.6, 59.1; HRMS (ESI) calcd for
C25H18NO4 [M 30
H]− 396.1236, found 396.1265 (see Supplementary Materials). Compounds 16
were synthesized according to the above method for the synthesis of 15.
δ: 13.12 (brs, 1H), 9.22 (s, 1H), 8.07 (s, 1H), 7.77~7.79 (m, 2H), 7.48~7.59 (m, 7H),
δ
−
~
1
(E)-3-(4-((5-Phenylisoxazol-4-yl)methoxy)styryl)benzoic acid (16): Yellow solid; Yield: 47%; H-NMR
(DMSO-d6) : 13.15 (brs, 1H), 9.22 (s, 1H), 8.13 (s, 1H), 7.77~7.80 (m, 4H), 7.60 (d, J = 8.7 Hz, 2H),
7.52~7.54 (m, 4H), 7.31 (d, J = 16.5 Hz, 1H), 7.22 (d, J = 16.5 Hz, 1H), 7.07 (d, J = 8.7 Hz, 2H), 5.14 (s,
2H); 13C-NMR (DMSO-d6)
: 169.5, 161.3, 159.1, 158.1, 138.4, 131.0, 130.6, 130.5, 129.7, 129.6, 129.5 (2C),
δ
δ
129.4, 128.6 (2C), 128.4 (2C), 128.2, 127.8, 127.3, 125.9, 115.6 (2C), 114.6, 59.1; HRMS (ESI) calcd for
C25H18NO4 [M − H]− 396.1236, found 396.1212.
(E)-3-(4-((5-Methyl-3-phenylisoxazol-4-yl)methoxy)styryl)benzoic acid (17): Yellow solid; Yield: 42%;
1H-NMR (DMSO-d6)
δ: 13.04 (brs, 1H), 8.13 (s, 1H), 7.77 (d, J = 7.2 Hz, 1H), 7.69~7.72 (m, 2H),
7.62 (d, J = 7.8 Hz, 1H), 7.58 (d, J = 8.6 Hz, 1H), 7.49~7.52 (m, 3H), 7.36 (t, J = 7.7 Hz, 1H), 7.23 (d,
J = 16.5 Hz, 1H), 7.12 (d, J = 16.5 Hz, 1H), 7.02 (s, 1H), 7.10 (d, J = 8.6 Hz, 2H), 5.00 (s, 2H), 2.53 (s, 3H);
13C-NMR (DMSO-d6)
δ: 170.4, 168.3, 162.6, 158.1, 137.3, 130.9, 130.4, 129.5 (2C), 129.1, 129.0, 128.7, 128.6,
128.4, 128.3 (2C), 128.2 (2C), 127.4, 126.9, 115.7 (2C), 109.9, 59.5, 11.3; HRMS (ESI) calcd for C26H20NO4
[M − H]− 410.1392, found 410.1388.
(E)-3-(4-((5-Phenylisoxazol-3-yl)methoxy)styryl)benzoic acid (18): Yellow solid; Yield: 42%; H-NMR
1
(DMSO-d6)
2H), 7.62 (d, J = 8.8 Hz, 2H), 7.52~7.58 (m, 3H), 7.47 (t, J = 7.7 Hz, 1H), 7.29 (d, J = 16.4 Hz, 1H), 7.22 (d,
J = 16.4 Hz, 1H), 7.20 (s, 1H), 7.10 (d, J = 8.8 Hz, 2H), 5.29 (s, 2H); 13C-NMR (DMSO-d6)
: 170.0, 168.2,
δ: 13.15 (brs, 1H), 8.12 (s, 1H), 7.90 (dd, J1 = 8.0 Hz, J2 = 1.8 Hz, 2H), 7.80 (t, J = 7.5 Hz,
δ
161.8, 158.1, 138.0, 131.1, 130.8, 130.3, 129.8 (2C), 129.3, 129.2, 128.5 (2C), 128.4, 127.4, 127.1, 126.3, 126.1
(2C), 115.6 (2C), 100.5, 61.8; HRMS (ESI) calcd for C25H18NO4 [M − H]− 396.1236, found 396.1219.
(E)-3-(4-((5-(2-Chlorophenyl)isoxazol-3-yl)methoxy)styryl)benzoic acid (19): Yellow solid; Yield: 51%;
1H-NMR (DMSO-d6)
δ: 13.02 (s, 1H), 8.12 (s, 1H), 7.99 (d, J = 8.4 Hz, 1H), 7.97 (d, J = 5.5 Hz, 1H),
7.78~7.85 (m, 3H), 7.62 (d, J = 8.4 Hz, 2H), 7.46~7.51 (m, 2H), 7.40 (t, J = 8.7 Hz, 2H), 7.30 (d, J = 16.4 Hz,
1H), 7.22 (d, J = 16.4 Hz, 1H), 7.18 (s, 1H), 7.09 (d, J = 8.4 Hz, 2H), 5.28 (s, 2H); 13C-NMR (DMSO-d6)
δ
: 168.6, 167.3, 161.3, 157.6, 137.7, 131.2, 130.2, 128.9, 128.2, 128.1, 128.1, 128.0 (2C), 127.9, 126.9, 124.1,

Molecules 2016, 21, 1722
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−
H] 430.0846,
125.6, 116.5, 116.3, 115.5, 115.1 (2C), 99.9, 61.3; HRMS (ESI) calcd for C25H17ClNO4 [M
found 430.0831.
−
(E)-3-(4-((5-(2-Methoxyphenyl)isoxazol-3-yl)methoxy)styryl)benzoic acid (20): Yellow solid; Yield: 47%;
1H-NMR (DMSO-d6)
: 13.05 (brs, 1H), 8.13 (s, 1H), 7.79~7.83 (m, 2H), 7.62 (d, J = 8.8 Hz, 2H), 7.46~7.52
(m, 3H), 7.26 (d, J = 16.4 Hz, 1H), 7.20 (d, J = 16.4 Hz, 1H), 7.10~7.14 (m, 2H), 6.99 (s, 1H), 6.79 (d,
J = 8.8 Hz, 2H), 5.29 (s, 2H), 3.96 (s, 3H); 13C-NMR (DMSO-d6)
: 167.8, 166.2, 161.5, 158.2, 138.4, 138.2,
δ
δ
131.7, 130.7, 130.5, 130.0, 129.4, 129.4, 128.6 (2C), 128.2, 127.4, 127.2, 126.1, 124.6, 121.3, 115.6 (2C), 103.6,
61.8, 56.3; HRMS (ESI) calcd for C26H20NO5 [M − H]− 426.1341, found 426.1376.
(E)-3-(4-((5-(3-Chlorophenyl)isoxazol-3-yl)methoxy)styryl)benzoic acid (21): Yellow solid; Yield: 40%;
1H-NMR (DMSO-d6)
δ
: 13.12 (brs, 1H), 8.13 (s, 1H), 8.01 (s, 1H), 7.78~7.82 (m, 2H), 7.62 (d, J = 8.6 Hz,
2H), 7.58~7.59 (m, 2H), 7.46~7.50 (m, 2H), 7.33 (s, 1H), 7.30 (d, J = 16.4 Hz, 1H), 7.22 (d, J = 16.4 Hz, 1H),
7.10 (d, J = 8.6 Hz, 2H), 5.30 (s, 2H); 13C-NMR (DMSO-d6)
: 168.5, 167.9, 161.9, 158.1, 138.2, 134.5, 131.7,
δ
130.8, 130.6, 130.0, 129.4, 129.0, 128.5, 127.4, 126.2 (2C), 125.8, 124.7, 124.6, 115.6 (2C), 101.7; HRMS (ESI)
calcd for C25H17ClNO4 [M − H]− 430.0846, found 430.0857.
(E)-3-(4-((5-(3-Fluorophenyl)isoxazol-3-yl)methoxy)styryl)benzoic acid (22): Yellow solid; Yield: 48%;
1H-NMR (DMSO-d6)
δ
: 13.05 (brs, 1H), 8.13 (s, 1H), 7.76~7.84 (m, 4H), 7.58~7.64 (m, 3H), 7.49 (t,
J = 7.7 Hz, 1H), 7.29~7.40 (m, 3H), 7.22 (d, J = 16.4 Hz, 1H), 7.10 (d, J = 8.7 Hz, 2H), 5.30 (s, 2H);
13C-NMR (DMSO-d6)
: 168.7, 167.7, 162.9 (d, J = 145.6 Hz), 161.9, 158.1, 138.2, 132.1, 132.0, 131.7,
δ
130.7, 129.4, 129.1, 129.0, 128.5 (2C), 128.4, 127.4, 126.2, 122.3, 118.2 (d, J = 21.2 Hz), 115.6 (2C), 113.0 (d,
J = 23.7 Hz), 101.6, 61.7; HRMS (ESI) calcd for C25H17FNO4 [M − H]− 414.1142, found 414.1133.
(E)-3-(4-((5-(3-Bromophenyl)isoxazol-3-yl)methoxy)styryl)benzoic acid (23): Yellow solid; Yield: 51%;
1H-NMR (DMSO-d6)
δ: 13.13 (brs, 1H), 8.14 (s, 1H), 8.12 (s, 1H), 7.89~7.93 (m, 1H), 7.79 (d, J = 7.6 Hz,
1H), 7.71~7.73 (m, 1H), 7.61 (d, J = 8.7 Hz, 2H), 7.49~7.58 (m, 2H), 7.43 (t, J = 7.6 Hz, 1H), 7.27 (d,
J = 16.5 Hz, 1H), 7.20 (d, J = 16.5 Hz, 1H), 7.19 (s, 1H), 7.09 (d, J = 8.7 Hz, 2H), 5.29 (s, 2H); 13C-NMR
(DMSO-d6) δ:170.0, 168.4, 161.8, 158.0, 137.8, 133.7, 131.9, 131.0, 130.9, 129.8, 129.1, 128.9, 128.7, 128.5
(2C), 127.4, 126.6, 126.1, 125.0, 123.0, 115.6 (2C), 100.5, 61.7; HRMS (ESI) calcd for C25H17BrNO4
[M − H]− 474.0341, found 474.0371.
(E)-3-(4-((5-(m-Tolyl)isoxazol-3-yl)methoxy)styryl)benzoic acid (24): Yellow solid; Yield: 44%; 1H-NMR
(DMSO-d6)
7.41~7.49 (m, 2H), 7.33 (d, J = 16.5 Hz, 1H), 7.25 (d, J = 16.5 Hz, 1H), 7.41 (s, 1H), 7.10 (d, J = 8.8 Hz, 2H),
5.28 (s, 2H), 2.39 (s, 3H); 13C-NMR (DMSO-d6)
: 170.2, 168.1, 161.7, 158.1, 139.2, 138.0, 131.7, 130.7,
δ: 13.05 (brs, 1H), 8.12 (s, 1H), 7.96 (s, 1H), 7.80 (t, J = 7.0 Hz, 2H), 7.61~7.73 (m, 4H),
δ
130.3, 129.7, 129.3, 129.2, 128.5 (2C), 128.4, 127.4, 127.0, 126.5, 126.3, 123.3, 115.6 (2C), 100.4, 61.8, 21.3;
HRMS (ESI) calcd for C26H20NO4 [M − H]− 410.1392, found 410.1388.
(E)-3-(4-((5-(3-Nitrophenyl)isoxazol-3-yl)methoxy)styryl)benzoic acid (25): Yellow solid; Yield: 38%;
1H-NMR (DMSO-d6)
δ
: 13.17 (brs, 1H), 8.35 (s, 1H), 8.13 (s, 1H), 7.78~7.83 (m, 3H), 7.63 (d, J = 8.8 Hz
2H), 7.46~7.49 (m, 4H), 7.28 (d, J = 16.5 Hz, 1H), 7.22 (d, J = 16.5 Hz, 1H), 7.11 (d, J = 8.8 Hz, 2H), 5.33
(s, 2H); 13C-NMR (DMSO-d6)
: 167.7, 162.2, 158.0, 148.9, 138.5, 138.2, 131.7, 130.7, 130.5, 130.0, 129.4,
,
δ
128.6 (2C), 128.4, 127.4, 127.2, 126.2, 125.4, 124.6, 120.7, 115.6 (2C), 102.5, 61.7; HRMS (ESI) calcd for
C25H17N2O6 [M − H]− 441.1087, found 441.1072.
(E)-3-(4-((5-(p-Tolyl)isoxazol-3-yl)methoxy)styryl)benzoic acid (26): Yellow solid; Yield: 55%; H-NMR
1
(DMSO-d6) δ: 12.98 (brs, 1H), 8.13 (s, 1H), 7.82~7.88 (m, 2H), 7.79 (d, J = 8.1 Hz, 1H), 7.62 (d, J = 8.6 Hz,
2H), 7.49~7.54 (m, 1H), 7.35 (d, J = 8.1 Hz, 2H), 7.47 (t, J = 7.7 Hz, 1H), 7.28 (d, J = 16.5 Hz, 1H), 7.23 (d,
J = 16.5 Hz, 1H), 7.10 (d, J = 8.6 Hz, 2H), 7.09 (s, 1H), 5.27 (s, 2H), 2.37 (s, 3H); 170.2, 166.7, 161.7, 158.2,
141.0, 138.4, 131.0, 130.6, 130.3 (2C), 129.7, 129.6, 128.6 (2C), 127.3, 126.1 (2C), 125.9, 124.4, 115.6 (2C),
99.9, 61.8, 21.5; HRMS (ESI) calcd for C26H20NO4 [M − H]− 410.1392, found 410.1373.
(E)-3-(4-((5-(4-Methoxyphenyl)isoxazol-3-yl)methoxy)styryl)benzoic acid (27): Yellow solid; Yield: 52%;
1H-NMR (DMSO-d6)
δ: 13.04 (brs, 1H), 8.12 (s, 1H), 7.77~7.86 (m, 5H), 7.62 (d, J = 8.4 Hz, 2H), 7.46~7.54

Molecules 2016, 21, 1722
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(m, 2H), 7.30 (d, J = 16.5 Hz, 1H), 7.22 (d, J = 16.5 Hz, 1H), 7.09 (d, J = 8.4 Hz, 2H), 7.04 (s, 1H), 5.26
(s, 2H), 3.83 (s, 3H); 13C-NMR (DMSO-d6)
: 167.7, 166.1, 161.5, 158.2, 138.4, 138.2, 131.7, 130.7, 130.7,
δ
130.0, 129.4, 128.6 (2C), 127.7, 127.5, 127.4, 127.2, 126.1, 124.6, 120.0, 115.2 (2C), 100.0, 61.8, 55.7; HRMS
(ESI) calcd for C26H20NO5 [M − H]− 426.1341, found 426.1365.
(E)-3-(4-((5-(4-Fluorophenyl)isoxazol-3-yl)methoxy)styryl)benzoic acid (28): Yellow solid; Yield: 37%;
1H-NMR (DMSO-d6)
δ
: 13.10 (brs, 1H), 8.13 (s, 1H), 7.83~7.95 (m, 5H), 7.64 (d, J = 8.5 Hz, 2H),
7.48~7.55 (m, 2H), 7.31 (d, J = 16.5 Hz, 1H), 7.24 (d, J = 16.5 Hz, 1H), 7.21 (s, 1H), 7.11 (d, J = 8.4 Hz,
2H), 5.33 (s, 2H); 13C-NMR (DMSO-d6)
: 168.1, 166.7, 161.5, 158.8 (d, J = 154.0 Hz, 2C), 158.1, 139.4,
δ
132.5, 132.4 (2C), 130.9,129.9, 128.7 (2C), 128.5, 128.2, 127.9, 127.3, 126.2 (d, J = 26.5 Hz, 2C), 124.6, 115.5
(2C), 104.8, 61.8; HRMS (ESI) calcd for C25H17FNO4 [M − H]− 414.1142, found 414.1159.
(E)-3-(4-((5-(3,4-Dichlorophenyl)isoxazol-3-yl)methoxy)styryl)benzoic acid (29): Yellow solid; Yield: 52%;
1H-NMR (DMSO-d6)
δ
: 13.03 (brs, 1H), 8.22 (s, 1H), 8.12 (s, 1H), 7.80~7.83 (m, 3H), 7.62 (d, J = 8.7 Hz,
2H), 7.45~7.52 (m, 2H), 7.37 (s, 1H), 7.30 (d, J = 16.4 Hz, 1H), 7.23 (d, J = 16.4 Hz, 1H), 7.09 (d, J = 8.7 Hz,
2H), 5.30 (s, 2H); 13C-NMR (DMSO-d6)
: 167.3, 167.1, 161.5, 157.5, 137.7, 133.1, 132.2, 131.6, 131.3,
δ
130.5, 130.3, 130.2, 130.1, 129.1, 128.9, 128.1, 128.0 (2C), 127.5, 127.0, 126.9, 125.7, 115.5, 115.1 (2C), 101.6,
61.2; HRMS (ESI) calcd for C25H16Cl2NO4 [M − H]− 464.0456, found 464.0438.
(E)-3-(4-((5-(3,4-Difluorophenyl)isoxazol-3-yl)methoxy)styryl)benzoic acid (30): Yellow solid; Yield: 45%;
1H-NMR (DMSO-d6)
δ
: 13.09 (brs, 1H), 8.10 (s,1H), 8.03~8.08 (m, 1H), 7.72~7.80 (m, 3H), 7.57~7.67 (m,
3H), 7.43 (t, J = 7.7 Hz, 1H), 7.17~7.29 (m, 3H), 7.08 (d, J = 8.7 Hz, 1H), 6.96 (d, J = 8.5 Hz, 1H), 5.29
(s, 2H); 13C-NMR (DMSO-d6)
: 167.3, 167.2, 159.1, 157.5, 137.9, 137.7, 130.7 (d, J = 97.3 Hz), 130.6 (d,
δ
J = 97.0 Hz), 130.0, 129.5, 128.9, 128.1, 128.0, 127.7, 127.6, 126.9, 126.7, 125.6, 124.1, 115.5, 115.1, 100.9,
61.2; HRMS (ESI) calcd for C25H16F2NO4 [M − H]− 432.1047, found 432.1083.
3.2. Biological Activity against PTP1B and TCPTP
A colorimetric assay to measure inhibition against PTP1B and TCPTP was performed in 96-well
plates. The assay was conducted as described by Zhang et al. [27]. Briefly, the tested compounds
were solubilized in DMSO and serially diluted into concentrations for the inhibitory test. The assays
were carried out in a final volume of 100 mL containing 50 mmol/L MOPS, pH 6.5, 2 mmol/L pNPP,
30 nmol/L GST-PTP1B or GST-TCPTP, and 2% DMSO, and the catalysis of pNPP was continuously
monitored on a SpectraMax 340 microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 405 nm
◦
for 2 min at 30 C. The IC50 value was calculated from the nonlinear curve fitting of the percent
inhibition [inhibition (%)] vs. the inhibitor concentration [I] using the following equation:
Inhibition (%) = 100/[1 + (IC50/[I])·κ],
where κ is the Hill coefficient.
3.3. Characterization of Compound 29
To determine the mode of inhibition of compound 29, an assay was carried out in a 100 µL assay
mixture containing 50 mM MOPS at pH 6.5, 30 nM PTP1B, pNPP in 2-fold dilution up to 80 mM, and
different concentrations of inhibitor as described by Zhang et al. [27]. In the presence of the competitive
inhibitor, the Michaelis–Menten equation is described as 1/ν = (Km/[Vmax[S]])·(1 + [I]/Ki) + 1/Vmax
,
where is the initial rate, Vmax is the maximum rate, and [S] is the substrate concentration. Ki value
ν
was obtained by Lineweaver–Burk plot of apparent Km/Vmax (slope) from primary reciprocal plot
versus the inhibitor concentration [I] according to the equation Km/Vmax = 1 + [I]/Ki.
4. Conclusions
In this paper we report a series of stilbene derivatives containing phenyl-substituted
isoxazoles as inhibitors against protein tyrosine phosphatase 1B (PTP1B). Most of these compounds

Molecules 2016, 21, 1722
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showed moderate to good inhibitory activity against PTP1B. The most potent compound 29
(IC50 = 0.91 ± 0.33 µM) showed 14-fold improved PTP1B inhibitory activity compared to the lead
compound LCA and about a 4-fold selectivity over TCPTP. Our SAR study clarified the effects of the
substituents on the phenyl C ring on the corresponding activities. Compound 29 was identified as
a competitive inhibitor of PTP1B with a Ki value of 0.78 µM in enzyme kinetic studies. These novel
stilbene derivatives could be promising lead compounds for the development of a new class of PTP1B
inhibitors. Further SAR research and evaluation of new derivatives are still in progress.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/
12/1722/s1.
Acknowledgments: This research was financially supported by the Science Fund for Young Scholar of Jiangsu
(BK20140425), Initiating Fund for Introduced Talents of Nantong University (03080694), the National Natural
Science Foundation of China (81402849), Zhejiang Province Natural Science Foundation (LQ14H300003) and
Public Projects of Zhejiang Province (2015C33159 & 20116C31017). Many thanks to Li Jia’s group of National
Center for Drug Screening (Shanghai Institute of Materia Medica Academy of Science) for their help in bioassays.
We also thank the Analysis & Testing Centre of Nantong University for support.
Author Contributions: Y.S. designed the research; H.H., J.J., and S.C. performed the research and analyzed the
data; Y.G. and F.Y. performed the biological test; H.D. wrote the paper. All authors read and approved the
final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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©
2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).