Synthesis and biological evaluation of novel N-(alkoxyphenyl)-aminocarbonylbenzoic acid derivatives as PTP1B inhibitors

Article abstract of DOI:10.1016/j.cclet.2010.07.005

Based on the fact that petroselinic acid showed good inhibitory activity (IC₅₀=6.99μmol/L) against protein tyrosine phophatase 1B(PTP1B) in vitro, a series of novel N-(alkoxyphenyl)-aminocarbonylbenzoic acid derivatives were designed and synthesized. The results indicated that most of the derivatives showed more potent activities against PTP1B. Especially, compound 13 had obvious activity with an IC₅₀ of 106nmol/L in vitro.

Full text of DOI:10.1016/j.cclet.2010.07.005

Available online at www.sciencedirect.com
Chinese Chemical Letters 21 (2010) 1415–1418
www.elsevier.com/locate/cclet
Synthesis and biological evaluation of novel
N-(alkoxyphenyl)-aminocarbonylbenzoic
acid derivatives as PTP1B inhibitors
Yuan Feng Tong a, Pei Zhang a, Feng Chen a, Ling Hua Hao a, Fei Ye b,
Jin Ying Tian b, Song Wu a,
*
a Department of New Drug Research and Development (Key Laboratory of Biosynthesis of Natural Products, Ministry of Health of PRC), Institute
of Materia Medica, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing 100050, China
b Department of Pharmacology, Institute of Materia Medica, Peking Union Medical College & Chinese Academy
of Medical Sciences, Beijing 100050, China
Received 8 April 2010
Abstract
Based on the fact that petroselinic acid showed good inhibitory activity (IC50 = 6.99 mmol/L) against protein tyrosine
phophatase 1B(PTP1B) in vitro, a series of novel N-(alkoxyphenyl)-aminocarbonylbenzoic acid derivatives were designed and
synthesized. The results indicated that most of the derivatives showed more potent activities against PTP1B. Especially, compound
13 had obvious activity with an IC50 of 106 nmol/L in vitro.
# 2010 Song Wu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: Petroselinic acid; PTP1B; N-(Alkoxyphenyl)-aminocarbonylbenzoic acid derivatives; Synthesis; Diabetes
Type II diabetes mellitus (T2DM) is a metabolic disease that is characterized by insulin resistance, resulting in
impaired glucose homeostasis [1]. An effective way of treating this disease is enhancing insulin action and lowering
plasma glucose to regulate glucose homeostasis [2,3]. Recent studies indicate that protein tyrosine phophatase 1B
(PTP1B), which is a negative regulator of insulin, play a key role in developing insulin resistance [4,5]. Therefore,
PTP1B is regarded as a novel drug target for T2DM [6,7]. Up to now, many small molecule inhibitors targeting PTP1B
have been reported [8–12].
In general, natural products are an important source of lead compounds. Petroselinic acid (Fig. 1), a natural product,
was found to show good PTP1B inhibitory activity (IC50: 6.99 mmol/L) in vitro, but not in vivo (unpublished data).
Based on this result, we supposed that petroselinic acid may be easily metabolized to lose its activity through the b-
oxidation route in vivo because it is a fatty acid.
Because of the novelty structure as a PTP1B inhibitor, petroselinic acid was chosen as a lead compound for further
modification. Through analyzing the structure of petroselinic acid, three structure-types compounds were designed as
* Corresponding author.
E-mail address: ws@imm.ac.cn (S. Wu).
1001-8417/$ – see front matter # 2010 Song Wu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
doi:10.1016/j.cclet.2010.07.005

[(Fig._1)TD$FIG]
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Y.F. Tong et al. / Chinese Chemical Letters 21 (2010) 1415–1418
Fig. 1. Structure of petroselinic acid.
[(Scheme_1)TD$FIG]
Scheme 1. Design from the structure of petroselinic acid.
described in Scheme 1. At first, an oxygen atom was introduced to substitute the b-position carbon atom to investigate
its effects on the activity. Because of the structure with a 6,7-Z double bond, 1,2-position substituted benzene ring was
introduced to keep the retention of configuration. Next, catechol and o-aminophenol derivatives were designed.
Considering the importance of the long aliphatic chain to the activity, we used alkyl bromide with the same number of
carbon atom reacting with hydroxy group to obtain the long chain. Lastly, because the carboxyl group might be very
important to the activity, the designed structure should have a carbolic acid.
The results of biological evaluation confirmed that the aminocarbonylbenzoic acid derivatives showed good
inhibitory activity against PTP1B. Further studies resulted in the discovery of 2-(2-tetradecanoxyphenyl)-
aminocarbonyl-6-nitrobenzoic acid (4, Fig. 2), which was active both in vitro and in vivo. The results indicated that
those series of derivatives may be novel and effective PTP1B inhibitors.
Previous studies had indicated that the substituent in the 2-position (such as –OC14H29 in Fig. 2), or the other
substituent in the ortho-position (such as NO2) may contribute to the inhibitor-enzyme complex formation. To
examine the structure–activity relationships (SAR), a variety of compounds with different substituted positions (such
as meta or para position) or different chain-length alkoxy groups (such as OCH3, OC4H9, and so on) on the two
benzene rings were synthesized. In addition, the benzoic acid of the structure was replaced by pyridinecarbolic acid to
evaluate the activities.
The synthesis of compounds 4–33 is summarized in Scheme 2. Alkylation of nitrophenol 1 with alkyl bromide in
the presence of anhydrous potassium carbonate in acetone afforded compound 2 [13]. The nitro group of compound 2
[(Fig._2)TD$FIG]
Fig. 2. IC50 of compound 4: 0.44 mmol/L.
[(Scheme_2)TD$FIG]
Scheme 2. Reagents and conditions: (a) R1Br, K2CO3, CH3COCH3; (b) 10%Pd/C, H2, EtOH; and (c) anhydride, CH3COCH3.

Y.F. Tong et al. / Chinese Chemical Letters 21 (2010) 1415–1418
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was hydrogenated into an amino group under the catalysis of 10% Pd/C in ethanol. Finally, treatment of 3 with the
corresponding anhydride in acetone gave the target compounds 4–33 [14].
PTP1B inhibition assay: The recombinant GST-hPTP1B (gluthathione S-transferase-human protein tyrosine
phosphatase 1B) bacteria pellets were purified by a GST bead column. The dephosphorylation of para-nitrophenyl
phosphate ( p-NPP) was catalyzed to para-nitrophenol by PTP1B. Enzyme activity involving an end-point assay,
which intensifies the yellow color, was measured at a wavelength of 405 nm [12]. All compounds were dissolved in
100% dimethyl sulfoxide (DMSO), and reactions, including controls, were performed at a final concentration of 10%
DMSO. Selected compounds were first evaluated for their ability to inhibit the PTPase reaction at a 10 mmol/L
concentration at 30 8C for 10 min, in a reaction system with 3 mmol/L p-NPP in HEPES assay buffer (pH 7.0). The
reaction was initiated by addition of the enzyme and quenched by addition of 1 mol/L NaOH. The amount of p-
nitrophenol produced was determined at 405 nm using a microplate spectrophotometer (uQuant, Bio-tek). IC50 values
were evaluated using a sigmoidal dose-response (variable slope) curve-fitting program of GraphPad Prism 4.0
software [15]. The results of biological activities of N-(alkoxyphenyl)-aminocarbonylbenzoic acid derivatives against
PTP1B in vitro were described in Table 1.
Table 1
Inhibitory activities of N-(alkoxyphenyl)-aminocarbonylbenzoic acid derivatives against PTP1B.
[TD$INLE]
.
a
b
Compounds
R1
R2
R3
X
%
IC50
4
5
2-n-C14H29
2-CH3
6-NO2
H
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
N
N
N
N
N
N
97.8
NA
0.442
–
H
H
6
2-n-C4H9
H
H
38.3
26.9
88.3
98.9
97.5
98.9
53.3
99.7
98.8
98.1
99.0
61.5
98.4
99.8
98.6
99.4
97.9
54.8
97.1
99.9
98.9
80.8
99.1
100.3
82.8
79.3
88.1
98.5
–
7
2-n-C10H21
2-n-C11H23
2-n-C12H25
2-n-C12H25
2-n-C14H29
2-n-C14H29
2-n-C14H29
2-n-C14H29
2-n-C18H37
2-n-C14H29
3-n-C10H21
3-n-C11H23
3-n-C12H25
3-n-C12H25
3-n-C11H23
3-n-C14H29
4-n-C10H21
4-n-C11H23
4-n-C12H25
4-n-C14H29
4-n-C10H21
2-n-C14H29
2-n-C16H33
2-n-C18H37
2-n-C22H45
3-n-C12H25
4-n-C12H25
H
H
–
8
H
H
0.598
3.543
0.857
0.598
–
9
H
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
H
4-NO2
H
H
5-F
H
6-F
H
0.106
0.601
0.407
0.180
–
H
5-Br
H
H
3,4,5,6-Tetrachloro
H
H
H
H
H
1.368
2.614
1.291
0.372
0.976
–
H
H
3-NO2
H
3,4,5,6-Tetrachloro
H
H
4-OCH3
H
H
H
H
3.757
1.271
0.370
5.331
0.390
0.140
0.367
–
H
H
H
3-Cl
H
6-NO2
H
H
H
H
H
H
H
H
H
H
3.239
1.928
H
H
a
% Inhibition at 10 mmol/L, NA means no inhibition at 10 mmol/L.
IC50 values in mmol/L with calculating inhibition more than 80%.
b

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Y.F. Tong et al. / Chinese Chemical Letters 21 (2010) 1415–1418
The results of activities demonstrated that those compounds 13, 16 and 29 had more potent activities against PTP1B
compared with the lead compound. The preliminary structure–activity relationships can be summarized as follows.
Firstly, the length of the chain was very important to the activities. While the number of carbon atom was not more than
ten, the activity was weaker. Secondly, the substituted group was the same importance on the phenyl of benzoic acid.
On the basis of the results, the activities of the compounds with the electron-withdrawing groups were better than those
compounds without them. Lastly, the substituted position on another phenyl group had little influence on the activity.
In the same, the replacement of the benzoic acid with pyridinecarbolic acid had little effect on the inhibitory activity.
In conclusion, based on the lead compound petroselinic acid, a series of novel N-(alkoxyphenyl)-
aminocarbonylbenzoic acid derivatives were designed and synthesized. The results of biological activities indicated
that most of new derivatives exhibited more potent activities against PTP1B. Among these compounds, the compound
13 displayed good potency, with an IC50 of 106 nmol/L. More activities evaluations are in progress in vivo.
Acknowledgments
Financial support was provided by the National Natural Science Funds for Young Scholar (No. 30901841), the
Research Fund for the Doctoral Program of Higher Education of China (No. 200800231145), and the Major Scientific
and Technological Special Project for ‘‘Significant New Drugs Innovation’’ (No. 2009ZX09301-003).
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