Discovery of novel high potent and cellular active ADC type PTP1B inhibitors with selectivity over TC-PTP via modification interacting with C site

Article abstract of DOI:10.1016/j.ejmech.2017.12.064

PTP1B serving as a key negative regulator of insulin signaling is a novel target for type 2 diabetes and obesity. Modification at ring B of N-{4-[(3-Phenyl-ureido)-methyl]-phenyl}-methane-sulfonamide template to interact with residues Arg47 and Lys41 in t

Full text of DOI:10.1016/j.ejmech.2017.12.064

European Journal of Medicinal Chemistry 144 (2018) 692e700
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Discovery of novel high potent and cellular active ADC type PTP1B
inhibitors with selectivity over TC-PTP via modification interacting
with C site
,
,
Yongli Du a *, Yanhui Zhang a, Hao Ling a, Qunyi Li b **, Jingkang Shen c
a School of Chemistry and Pharmaceutical Engineering, Qilu University of Technology (Shandong Academy of Sciences), 3501 Daxue Road, Jinan 250353,
China
b Clinical Pharmacy Laboratory, Huashan Hospital, Fudan University, 12 Wu Lu Mu Qi M Road, Shanghai 200040, China
c State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203,
China
a r t i c l e i n f o
a b s t r a c t
Article history:
PTP1B serving as a key negative regulator of insulin signaling is a novel target for type 2 diabetes and
obesity. Modification at ring B of N-{4-[(3-Phenyl-ureido)-methyl]-phenyl}-methane-sulfonamide tem-
plate to interact with residues Arg47 and Lys41 in the C site of PTP1B by molecular docking aided design
resulted in the discovery of a series of novel high potent and selective inhibitors of PTP1B. The structure
activity relationship interacting with the C site of PTP1B was well illustrated. Compounds 8 and 18 were
shown to be the high potent and most promising PTP1B inhibitors with cellular activity and great
selectivity over the highly homologous TCPTP and other PTPs.
Received 15 March 2017
Received in revised form
5 September 2017
Accepted 17 December 2017
Available online 20 December 2017
Keywords:
© 2017 Elsevier Masson SAS. All rights reserved.
Protein tyrosine phosphatase 1B inhibitors
N-(2,5-diethoxy-phenyl)-methane
sulfonamide
Type 2 diabetes mellitus
PTP1B
1. Introduction
PTP has a critical role in the development of the immune system
and has been identified as a negative regulator of inflammation [7].
Protein tyrosine phosphatase 1B (PTP1B) is a novel target for
type diabetes and obesity [1]. PTP1B-deficient mice have
The TC-PTP KO mice die at 3e5 weeks of age because of impaired B
cell and T cell functions [6]. TC-PTP is the most homologous
phosphatase to PTP1B with 74% sequence identity in the catalytic
domain and identical active sites. Thus discovery PTP1B inhibitors
with selectivity over TCPTP are really important and difficult.
Nevertheless, this well validated therapeutic target PTP1B for dia-
betes and obesity has continued to inspire medicinal chemists to
pursue novel inhibitors with selectivity and oral availability.
Up to now, only two small-molecule PTP1B inhibitors, Ertipro-
tafib [8] and Trodusquemine [9] (Fig. 1), have reached clinical trials.
Several classes of PTP1B inhibitors have been described in the
literature, and the most frequently reported structural features of
PTP1B inhibitors include the isosteric difluoromethylene phos-
phonate (DFMP) containing inhibitors [10,11], dicarboxylic acid
containing inhibitors [12e14], monoacid containing inhibitors
[15e17], 1,2,5-thiadiazolidin-3-one-1,1-dioxide (TDZ) based in-
hibitors [18,19], isothiazolidin-3-one-1,1-dioxide (IZD) based in-
hibitors [20e24], and arylsulfamic acid containing inhibitors [25],
as exemplified by compounds 1e6 shown in Fig. 1.
2
remarkably low adiposity and are protected from diet-induced
obesity [2]. Studies in cell lines, animal models and clinical trials
on the role of PTP1B have clearly shown that it serves as a key
negative regulator of insulin signaling and is involved in the insulin
resistance associated with Type 2 diabetes [3,4]. However, the
catalytic site of PTP1B is highly conserved, positively charged,
shallow and not deep enough to form a nice pocket, hence PTP1B
enzyme drug design is different compare to a typical enzyme-based
drug design. It is difficult for the discovery of potent cell permeable
and orally bioavailable PTP1B inhibitors [5]. Achieving PTP1B
selectivity over closely associated PTPs, especially T-cell protein
tyrosine phosphatase (TC-PTP) is another major challenge [6]. TC-
* Corresponding author.
** Corresponding author.
E-mail addresses: yldu@qlu.edu.cn (Y. Du), qyli1234@163.com (Q. Li).
https://doi.org/10.1016/j.ejmech.2017.12.064
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

Y. Du et al. / European Journal of Medicinal Chemistry 144 (2018) 692e700
693
In our pursuit to discover novel potent and selective PTP1B in-
hibitors, series of N-{4-[(3-Phenyl-ureido)-methyl]-phenyl}-
compound 7 strongly interacted with the carboxylate group of
residue ASP48.
a
methane-sulfonamide analogues were identified as PTP1B in-
hibitors through fragment-docking-oriented de novel design.
Compound 7 (Fig. 1) was shown to possess potent PTP1B enzyme
inhibition activity (IC50 ¼ 203 nM) and high selectivity against TC-
PTP (no activity) [26]. Following this promising lead, structure ac-
tivity relationship (SAR) studies were initiated with the aim to
further improve the potency on PTP1B and remaining selectivity
against TC-PTP. Herein we will describe in detail the discovery of
these novel high potent and selective ADC type PTP1B inhibitors
based on N-{4-[(3-Phenyl-ureido)-methyl]-phenyl}-methane-sul-
fonamide template (compound 7, Fig. 1) through modification at
ring B.
The interactions between PTP1B and its ligand (PDB entry 2CNE)
in the C site included the multiple hydrogen bonding interactions
with the residues Arg47 and Lys41 [27]. We envisioned that
introducing suitable hydrogen bond acceptors to the B ring of
compound 7 to form hydrogen bonding interactions with the res-
idues Arg47 and Lys41 maybe greatly improve the PTP1B inhibition
activities. Docking simulation results suggested that introducing
-COOMe group as hydrogen bond acceptors at the meta-position
with suitable group -OR at para-position of ureido group on ring
B in compound 7 gave compound 8 and analogues forming strong
new hydrogen bonding interactions with the residue Arg47, as
shown in Fig. 2, based on the binding mode of compound 7. Com-
pounds 8-10 containing eCOOMe group (shown in Fig. 3) were
designed for exploring the optimal interactions with residue Arg47.
Then -COOMe group was substituted with -CONHR group for
improving the structural stability of the designed compounds and
also for detecting the structure activity relationship. To form
interaction with the distant residue Lys41, -CONHR group was
further converted to substituted -CONHPh groups for exploring the
positions and varieties of the substituents on the new phenyl
group. Docking results demonstrated compound 18 with 3,4-di-
MeO-Ph-groups replaced the small -CONHR group on meta-
position of the ring B in compound 8 formed effective hydrogen
bonding interactions with residues Arg47 and Lys41 (shown in
Fig. 2) based on the binding mode of compound 8. Thus compounds
11-18 (shown in Fig. 3) were designed to detect the detailed SAR in
the C site of PTP1B.
2. Inhibitors design
Docking simulations of compound 7 and all other designed in-
hibitors within the ADC site of PTP1B (PDB entry 2CNE) have been
performed using geom docking program (sybyl X 2.0) with the
default setting. The key features of the binding mode of compound
7 are summarized as follows (shown in Fig. 2): First, the two sul-
fonyloxygens of ring A in compound 7 bond with the residues
ILe219, Gly220, and Arg221 in the A site of PTP1B through hydrogen
bonding interactions. Second, the 2-ethoxy group pointed to and
interacted with the D site [26] (lined with polar and charged resi-
dues Tyr46, Glu115, Lys120, Asp181, and Ser216) and the 5-ethoxy
group interacted the hydrophobic residues (Val49, Phe52, Ile219
and Met258). Third, the 1,3-double-NHs of the urea group in
Fig. 1. Representative examples of PTP1B inhibitors previously reported.

694
Y. Du et al. / European Journal of Medicinal Chemistry 144 (2018) 692e700
4. Results and discussion
4.1. PTP1B enzyme inhibitory activity and structureeactivity
relationship
The designed compounds 8-18 (Table 1) were synthesized and
evaluated, respectively. Benzoic acid methyl ester analogues 8-10
with 2-alkoxy groups displayed high potent PTP1B inhibitory ac-
tivities. Compound 8 with 2-ethoxy group on B ring was identified
to possess 10.9 fold more potent inhibitory activity (IC50 ¼ 18.6 nM)
against the PTP1B enzyme than compound 7 (IC50 ¼ 203 nM). The
high potent activity of compound 8 suggested the design to form
strong new hydrogen bonding interactions with the residue Arg47
was rational, important and benefit for improving inhibition ac-
tivity against PTP1B enzyme. Replacement of the 2-ethoxy group on
B ring in compound 8 with more bulky 2-n-propoxy group on B ring
gave compound 9 (IC50 ¼ 68.2 nM) and resulted in about 3.7-fold
decrease in PTP1B enzyme inhibition. Similarly, converting 2-n-
propoxy group to 2-n-butoxy group on B ring gave compound 10
(IC50 ¼ 710.9 nM) and made the inhibition activity against PTP1B
enzyme drop in more than 10-fold when compared with the ac-
tivity of compound 9. These results suggested that the size of 2-
alkoxy group greatly influenced the inhibition activity against
PTP1B enzyme. Smaller 2-ethoxy group in compound 8 was more
suitable than bulky 2-n-propoxy in compound 9 and 2-n-butoxy
group in compound 10 for PTP1B enzyme inhibition.
To improve the structural stability and further detect the
structure activity relationship, 2-ethoxy group in compound 8 was
kept and eCOOMe group on ring B in compound 8 was substituted
with a series of eCONHR groups. Compound 11 (IC50 ¼ 165.9 nM)
with eCONHMe group on ring B displayed potent PTP1B enzyme
inhibition activity, dropped by 8.9 times when compared with high
potent compound 8. Converting eCONHMe group to more bulky
eCONHEt group gave compound 12 (IC50 ¼ 169.2 nM) demon-
strating slightly weaker inhibition activity. Replacement of the
eCONHEt group in compound 12 with larger eCONH-n-Pr group in
compound 13 resulted in further decrease in PTP1B enzyme inhi-
bition (IC50 ¼ 184.1 nM). So the preliminary optimization experi-
ments (11, 12, 13) above suggested that more bulky or hydrophobic
groups were detrimental for PTP1B enzyme inhibition. Compound
14 (IC50 > 25000 nM) with a eCONHPh group and compound 16
(IC50 > 25000 nM) with a eCONH-(4-MeO-Ph) group substituting
eCONHMe group in compound 8 led to a dramatic decrease in
PTP1B enzyme inhibition. These probably meant that significant
changes in the binding mode of compounds 14 and 16 with PTP1B
ADC sites emerged, influenced by phenyl group in compound 14 or
4-MeO-Ph-group in compound 16 compared with the binding
Fig. 2. Superimposition of docking model of compound
7 (yellow), compound 8
(green) and compound 18 (pink) on the ADC site of PTP1B (PDB code 2CNE). Hydrogen
bonding interactions were shown with dotted yellow lines. These images were
generated using the Sybyl-x program. (For interpretation of the references to colour in
this figure legend, the reader is referred to the Web version of this article.)
3. Chemistry
The syntheses of the 1-(2,5-Diethoxy-benzyl)-3-phenyl-urea
containing compounds 8-18 were synthesized as shown in Scheme
1. Starting from material a [28], intermediates 8b-10b were syn-
thesized through alkylation with bromoethane [29]. Intermediates
8b-10b were sequentially subjected to nitro reduction condition to
obtain intermediates 8c-10c. Intermediate 8d was obtained from
intermediate 8c [28] through hydrolyzation reaction with KOH in
MeOH at 80 ꢀC. Then intermediate 8d was converted to interme-
diate 8e after treated with SOCl2 and DMF in solvent DCM. The
intermediate 8e effectively reacted with various amines HR2 to give
intermediates 11f-18f which were sequentially subjected to nitro
reduction to obtain intermediates 11g-18g. The intermediate h
effectively reacted with various amines to give the corresponding
N-benzyl urea intermediates 8i-18i in excellent yields [27,30]. In-
termediates 8j-18j were prepared through the reduction of nitro
intermediates [27,31]. Methanesulfonyl chloride was condensed
with intermediates 8j-18j separately to give compounds 8-18.
Fig. 3. The design process of designed compounds 8-18.

Y. Du et al. / European Journal of Medicinal Chemistry 144 (2018) 692e700
695
Scheme 1. Reagents and conditions for the synthesis of intermediates 11-18: (a) DMF, K2CO3, R1Br, 80 ꢀC, 4 h; (b) NiCl2$6H2O, NaBH4, MeOH, 2 h, then 10%HCl; (c)MeOH, KOH, 80 ꢀC,
3 h, then 10%HCl; (d)SOCl2, DMF, DCM, rt, 6 h; (e) DCM, Et3N, HR2, 3 h; (f) NiCl2$6H2O, NaBH4, MeOH, 2 h, then 10%HCl; (h) Et3N, dioxane, amine intermediate, 80 ꢀC,8 h; (i)
NiCl2$6H2O, NaBH4, MeOH, rt, 0.5 h; (j) methanesulfonyl chloride, DCM, N2, pyridine, rt, 8 h.
mode of compound 8. However, compound 15 (IC50 ¼ 209.1 nM)
with a corresponding -CONH-(4-Me-Ph) group and compound 17
(IC50 ¼ 981 nM) with -CONH-(3-MeO-Ph) on ring B showed potent
and weak PTP1B enzyme inhibition separately, probably because
compound 15 and 17 with the 4-Me- group and 3-MeO- group on
the new phenyl ring C respectively remained beneficial binding
modes to interact with the residue Arg47 but possessed disad-
vantageous hydrophobic interactions with the deep C site of PTP1B.
Excitingly, compound 18 (IC50 ¼ 66.2 nM) with a -CONH-(3,4-di-
MeO-Ph) group on ring B displayed high potent activity again. The

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Y. Du et al. / European Journal of Medicinal Chemistry 144 (2018) 692e700
Table 1
Inhibitory activity of compounds 8-27 against PTP1B.a
5. Conclusion
The novel high potent and selective ADC type PTP1B inhibitors
based on N-{4-[(3-Phenyl-ureido)-methyl]-phenyl}-methane-sul-
fonamide template were discovered through further design on the
ring B to form effective hydrogen interactions with the residues
Arg47 and Lys41. Synthesized compounds 8-18 were evaluated
against PTP1B on the inhibition of enzyme and cellular activities
and the selectivity over other PTPs. Structure activity relationships
of these compounds interacting with C site of PTP1B and residues
Arg47 and Lys41 were illuminated. All the tested compounds
demonstrated excellent selectivity for PTP1B over TCPTP and other
assayed PTPs. Compounds 8 and 18 were discovered to possess high
potent PTP1B enzyme inhibition and significantly increase insulin-
Compound
IC50 (nM) mean SEM
Oleanolic acid
7
8
9
10
11
12
13
14
15
16
17
18
2058 202
203
18.6 5.5
68.2 3.2
710.9 70.6
165.9 37.9
169.2 29.1
184.1 51.8
NAb
209.1 78.1
NA
981 287
66.2 7.9
stimulated glucose uptake at 0.5, 2.5 and 10 mM. These novel
a
Values represent triplicate determinations.
NA: Inhibition rate <50% at 25 mM concentration.
b
compounds reported in this study will provide new insights into
the design and development of potent and selective ADC PTP1B
inhibitors.
greatly improvement of the activity for PTP1B inhibition of com-
pound 18 compared to its analogues 14-17 may be derived from
well interaction binding mode in C site of PTP1B and effective
hydrogen bonding interactions with residues Arg47 and Lys41. 3-
MeO- group probably helped 4-MeO- group to form powerful
hydrogen bonding interaction with residue Lys41 in compound 18
when compared its inhibition activity for PTP1B enzyme with that
of analogues 16 and 17 with just 4-MeO- or 3-MeO- group.
The SAR from compounds 8-18 above suggested that the well
binding mode in the C site of PTP1B and strong hydrogen bonding
interactions with residues Arg47 and Lys41 could greatly improve
the activity against PTP1B, but the increase of the activities of the
inhibitors needed the proper structural cooperating from the 2-
alkoxy groups on B ring and from the substituent groups on new
C phenyl ring.
6. Experimental section
6.1. Chemistry
6.1.1. General synthetic methods
Unless noted otherwise, all starting material, reagents and Sol-
vents were analytical grade and procured commercially without
subsequent purification. And the solvents used were redistilled and
dried by standard methods according to the need.
Reactions were monitored by thin-layer chromatography (TLC)
on silica gel plates. A Bruker AVANCE II 400 MHz NMR Fourier
transform spectrometer was used to record1 HNMR (400 MHz) and
13C (101 MHz) NMR. The chemical shifts were given in
d
(ppm) refer
77.16, 13C NMR) and the
d
39.52, 13C NMR).
to the signal of CDCl3 (
d d
7.26, 1H NMR and
signal of (CD3)2SO (
d
2.54, 1H NMR and
4.2. The selectivities of compounds 8, 9, 11, 13, 15 and 18 over TCPTP,
SHP-1, SHP-2, LAR
6.1.2. 5-[3-(2,5-Diethoxy-4-methanesulfonylamino-benzyl)-
ureido]-2-ethoxy-benzoic acid methyl ester (8)
The mixture of 2-hydroxy-5-nitro-benzoic acid methyl ester (a)
(1.0 g, 5.07 mmol) and K2CO3 (1.39 g, 10.1 mmol) in N,N-
dimethylformamide (DMF) was heated at 80ꢀC for 10 min, then
the bromo-ethane (710 mg, 6.59 mmol) was added dropwise to the
solution followed by stirring at 80 ꢀC for 4 h. DMF was removed by
distillation and the residue was dissolved in dichloromethane and
extracted with water (3 ꢁ 200 ml). The combined organic phases
were dried with Na2SO4 and concentrated under reduced pressure.
The remaining solid was recrystallized from methanol to give the
pale yellow solid (8b, 933 mg, yield 83%).
According to the PTP1B enzyme inhibition activity, compounds
8, 9, 11, 13, 15 and 18 were selected to test against a panel of PTPs
including homologous TCPTP, SHP1, SHP2 and the receptorelike
PTPs LAR. As shown in Table 2, all these inhibitors demonstrated
excellent PTP1B selectivity over TCPTP, SHP1, SHP2 and LAR.
Compounds 8, 9, 11 and 13 displayed more than ten times selec-
tivity for PTP1B over the highly homologous TCPTP. Among them,
compounds 8 and 11with high PTP1B enzyme inhibition activities
demonstrated 36-fold and 45.9-fold selectivity for PTP1B over
TCPTP respectively. The selectivity for PTP1B over TCPTP of com-
pound 18 was 5.9 times. All the tested compounds possessed
obviously better selectivity for PTP1B over SHP1, SHP2 and LAR
than over TCPTP.
To a solution of 2-ethoxy-5-nitro-benzoic acid methyl ester (8b,
889 mg, 3.95 mmol) in MeOH (15 ml) was added NiCl2$6H2O
(1.61 g, 6.8 mmol), NaBH4 (0.51 g, 13.6 mmol), and the mixture was
stirred at room temperature for 2 h. The mixture was made acidic
with
a 10% HCl solution and extracted with ethyl acetate
(3 ꢁ 150 ml), the organic layer was washed with a saturated NaCl
solution (3 ꢁ 100 ml), and the combined organic phases were dried
with Na2SO4, filtered, concentrated, and purified by flash column
chromatography to give 5-amino-2-ethoxy-benzoic acid methyl
ester (8c, 680 mg, yield 88.6%) as a white solid.
(2,5-diethoxy-4-nitro-benzyl)-carbamic acid phenyl ester (h,
0.4 g, 1.11 mmol) was dissolved in dioxane after addition of 5-
amino-2-ethoxy-benzoic acid methyl ester (8c, 0.215 g, 1.1 mmol)
and triethylamine (1.12 g, 11.1 mmol). The mixture was heated at
80 ꢀC for 8 h. Then the mixture was cooled to room temperature
and dioxane was removed by distillation. The residue was dissolved
in dichloromethane and extracted with water (3 ꢁ 100 ml). The
combined organic phases were dried with Na2SO4, and
4.3. Cellular activities of selected compounds
Because PTP1B inhibition resulted in a marked improvement in
insulin sensitivity and glucose tolerance [32], we examined the
PTP1B inhibition exerted by compounds 8, 18 and resulted in
enhanced insulin-stimulated glucose uptake. As shown in Fig. 4,
insulin-stimulated glucose uptake was significantly increased in L6
myotubes treated with compound 8, and this increase was 16.2%,
22.4% and 22.9% at 0.5, 2.5 and 10 mM, respectively. When treated
with compound 18, as shown in Fig. 5, insulin-stimulated glucose
uptake was significantly increased in L6 myotubes and the increase
was 6.6%, 18.7% and 25.8% at 0.5, 2.5 and 10 mM, respectively.

Y. Du et al. / European Journal of Medicinal Chemistry 144 (2018) 692e700
697
Table 2
The inhibitory activity of select compounds 8, 9, 11, 13, 15 and 21as PTP1B inhibitors against a panel ofPTPs, IC50
a
.
Compound
PTP1B (nM)
1536 267
TCPTP (
m
M)
SHP1 (
m
M)
SHP2 (
m
M)
LAR (
m
M)
TCPTP/PTP1B
oleanolic acid
5.46 0.74
Na3VO4
8
9
11
13
15
18
14.19 2.63
1.09 0.13
3.45 0.61
NA
8.63 1.19
2.94 0.56
0.85 0.11
11.62 2.84
0.91 0.12
7.76 0.93
NA
NA
NA
22.31 4.67
5.36 0.83
NAb
NA
NA
18.6 5.5
68.2 3.2
165.9 37.9
184.1 51.8
209.1 78.1
66.2 7.9
0.67 0.05
1.18 0.14
7.62 0.83
2.02 0.33
0.87 0.15
0.39 0.06
36
17.3
45.9
10.9
4.1
6.95 1.31
7.34 1.22
1.59 0.16
5.9
a
ThepNPP assay. IC50 values were determined by regression analysis and expressed as means SD of three replications.
NA: Inhibition rate <50% at 25 mM concentration.
b
amino-2,5-diethoxy-benzyl)-ureido]-2-ethoxy-benzoic
acid
methyl ester (8j) 320 mg (yield 85.6%), white solid. 5-[3-(4-amino-
2,5-diethoxy-benzyl)-ureido]-2-ethoxy-benzoic acid methyl ester
(8j, 300 mg, 0.70 mmol) was dissolved in dichloromethane after
addition of pyridine (60.8 mg, 0.77 mmol). The solution was stirring
at room temperature for 10 min and methanesulfonyl chloride
(80 mg, 0.70 mmol) was added dropwise to the solution, followed
by stirring at room temperature for 8 h. Dichloromethane was then
removed by distillation. The solid residue was dissolved in
dichloromethane (50 mL) and washed with a saturated NaCl solu-
tion (3 ꢁ 100 ml). The combined organic phases were dried with
Na2SO4 and concentrated under reduced pressure. The crude
product was purified by flash column chromatography to give pure
product (8)190 mg (yield 53.7%) of white solid.
Fig. 4. Effects of compound 8 on insulin-stimulated glucose uptake in L6 myotubes. L6
cells were serum starved for 2 h and then incubated with varying concentration of
compound 8 (0, 0.5, 2.5 and 10 mM). After 4 h, insulin (10 nM) stimulated glucose
1H NMR (400 MHz, DMSOd6):
d 8.84 (s, 1H), 8.58 (s, 1H), 7.72 (d,
J ¼ 2.6 Hz, 1H), 7.44 (dd, J ¼ 8.9, 2.6 Hz, 1H), 7.01 (d, J ¼ 9.0 Hz, 1H),
6.93 (s, 1H), 6.87 (s, 1H), 6.33 (t, J ¼ 5.7 Hz, 1H), 4.20 (d, J ¼ 5.7 Hz,
2H), 4.04e3.94 (m, 6H), 3.75 (s, 3H), 2.92 (s, 3H), 1.30 (dt, J ¼ 14.5,
uptake was evaluated using 2-NBDG as described in Methods. Values are presented as
mean SEM. n ¼ 3. #P < .05vs the control group,**P < .01vs the insulin-treated group.
6.8 Hz, 9H). 13C NMR (101 MHz, DMSOd6):
d 166.08, 155.20, 152.11,
149.56, 145.42, 133.37, 126.17, 125.00, 122.90, 120.46, 120.00, 114.84,
113.47, 110.40, 64.58, 64.52, 64.01, 51.71, 40.09, 38.11, 14.68, 14.63,
14.62.
6.1.3. 5-[3-(2,5-Diethoxy-4-methanesulfonylamino-benzyl)-
ureido]-2-propoxy-benzoic acid methyl ester (9)
Compound 9 (white solid, 220 mg) was prepared by a procedure
similar to that described for the synthesis of compound 8.
1H NMR (400 MHz, DMSOd6):
d 8.84 (s, 1H), 8.57 (s, 1H), 7.72 (d,
J ¼ 2.4 Hz, 1H), 7.45 (dd, J ¼ 8.9, 2.4 Hz, 1H), 7.01 (d, J ¼ 9.0 Hz, 1H),
6.93 (s, 1H), 6.87 (s, 1H), 6.32 (t, J ¼ 5.6 Hz, 1H), 4.18 (t, J ¼ 13.8 Hz,
2H), 4.03e3.94 (m, 4H), 3.90 (t, J ¼ 6.2 Hz, 2H), 3.76 (s, 3H), 2.92 (s,
3H), 1.74e1.60 (m, 2H), 1.32 (dd, J ¼ 12.7, 6.6 Hz, 6H), 0.96 (t,
Fig. 5. Effects of compound 18 on insulin-stimulated glucose uptake inL6 myotubes. L6
cells were serum starved for 2 h and then incubated with varying concentration of
compound 18 (0, 0.5, 2.5 and 10 mM). After 4 h, insulin (10 nM) stimulated glucose
J ¼ 7.3 Hz, 3H). 13C NMR (101 MHz, DMSOd6):
d 166.16, 155.21,
152.32, 149.56, 145.41, 133.28, 126.18, 125.02, 122.95, 120.28, 120.05,
114.57, 113.49, 110.58, 70.22, 64.53, 64.02, 51.69, 40.09, 38.11, 22.09,
14.68, 14.62, 10.34.
uptake was evaluated using 2-NBDG as described in Methods. Values are presented as
mean SEM. n ¼ 3. #P < .05 vs the control group,**P < .01vs the insulin-treated group.
concentrated under reduced pressure. The remaining solid was
recrystallized from methanol to give pure product 5-[3-(2,5-
diethoxy-4-nitro-benzyl)-ureido]-2-ethoxy-benzoic acid methyl
ester (8i) 0.44 g (yield 86.7%), pale yellow solid.
6.1.4. 2-Butoxy-5-[3-(2,5-diethoxy-4-methanesulfonylamino-
benzyl)-ureido]-benzoic acid methyl ester (10)
Compound 10 (white solid, 200 mg) was prepared by a pro-
cedure similar to that described for the synthesis of compound 8.
A
mixture of 5-[3-(2,5-diethoxy-4-nitro-benzyl)-ureido]-2-
1H NMR (400 MHz, DMSOd6):
d 8.84 (s, 1H), 8.57 (s, 1H), 7.72 (d,
ethoxy-benzoic acid methyl ester (8i, 400 mg, 0.87 mmol),
NiCl2$6H2O (353 mg, 1.48 mmol), NaBH4 (112 mg, 2.96 mmol), and
MeOH was stirring at room temperature for 0.5 h. The mixture was
made acidic with a 10% HCl solution and extracted with Ethyl ac-
etate, the organic layer was washed with a saturated NaCl solution
(3 ꢁ 100 ml), the combined organic phases were dried with Na2SO4,
and concentrated under reduced pressure. The product was puri-
fied by flash column chromatography to give pure product 5-[3-(4-
J ¼ 2.6 Hz, 1H), 7.44 (dd, J ¼ 8.9, 2.6 Hz, 1H), 7.01 (d, J ¼ 9.0 Hz, 1H),
6.93 (s, 1H), 6.87 (s, 1H), 6.32 (t, J ¼ 5.8 Hz, 1H), 4.21 (d, J ¼ 5.7 Hz,
2H), 3.97 (ddd, J ¼ 19.3, 9.7, 5.9 Hz, 6H), 3.75 (s, 3H), 2.92 (s, 3H),
1.73e1.54 (m, 2H), 1.48e1.37 (m, 2H), 1.32 (dd, J ¼ 12.8, 6.7 Hz, 6H),
0.91 (t, J ¼ 7.4 Hz, 3H). 13C NMR (101 MHz, DMSOd6):
d 166.15,
155.21, 152.33, 149.57, 145.41, 133.28, 126.18, 125.04, 122.95, 120.32,
120.06, 114.60, 113.51, 110.57, 68.46, 64.54, 64.04, 51.67, 40.10, 38.12,
30.77, 18.59, 14.68, 14.61, 13.60.

698
Y. Du et al. / European Journal of Medicinal Chemistry 144 (2018) 692e700
6.1.5. 5-[3-(2,5-Diethoxy-4-methanesulfonylamino-benzyl)-
procedure similar to that described for the synthesis of compound
ureido]-2-ethoxy-N-methyl-benzamide (11)
11.
To a solution of 2-ethoxy-5-nitro-benzoic acid methyl ester [24]
(8c, 6 g, 26.7 mmol) in methanol (80 ml) was added KOH (3 g,
53.6 mmol) and the solution was stirred at 80 ꢀC for 3 h. Then the
mixture was made acidic with a 10% HCl solution and the white
solid was precipitated. The solid was collected by filtration and
washed with water, then dried in vacuum over 18 h to give the 2-
ethoxy-5-nitro-benzoic acid (8d, 5.5 g, yield 97.7%). To a stirring
solution of compound 8d (5.5 g, 26.1 mmol) in dichloromethane
was added N,N-dimethylformamide(0.1 ml) followed by thionyl
chloride (6.2 ml, 52.1 mmol), and the mixture was stirred at room
temperature for 6 h. Dichloromethane (3 ꢁ 100 mL) was used as
azeotrope for the residue and the solvent was removed to give 2-
ethoxy-5-nitro-benzoyl chloride (8e) as a yellow oil (0.55 g, yield
97%), which was used without further purification. To a stirring
solution of compound 8e (0.50 g, 2.18 mmol) in dichloromethane
was added methylamine hydrochloride (0.147 g, 2.18 mmol) fol-
lowed by triethylamine(0.484 g, 4.80 mmol), and the mixture was
stirred at room temperature for 3 h. Then water (50 ml) was added
and the mixture was extracted with dichloromethane (3 ꢁ 50 mL).
The combined extracts were dried over Na2SO4, filtered, concen-
trated and purified by flash column chromatography to give pure
product 2-ethoxy-N-methyl-5-nitro-benzamide (11f, 0.371 g, yield
76%) as white solid. To a stirring solution of 11f (0.336 g, 1.50 mmol)
in MeOH (8 ml) was added NiCl2$6H2O (0.707 g, 2.97 mmol) fol-
lowed by NaBH4 (0.226 g, 5.94 mmol), and the mixture was stirred
at room temperature for 2 h.Then the mixture was made acidic
1H NMR (400 MHz, DMSOd6):
d 8.81 (s, 1H), 8.54 (s, 1H), 8.11 (t,
J ¼ 5.5 Hz, 1H), 7.68 (d, J ¼ 2.7 Hz, 1H), 7.54 (dd, J ¼ 8.9, 2.7 Hz, 1H),
7.00 (d, J ¼ 8.9 Hz, 1H), 6.94 (s, 1H), 6.87 (s, 1H), 6.26 (t, J ¼ 5.8 Hz,
1H), 4.21 (d, J ¼ 5.7 Hz, 2H), 4.08 (q, J ¼ 6.9 Hz, 2H), 3.99 (qd, J ¼ 6.9,
2.8 Hz, 4H), 3.23 (dd, J ¼ 12.7, 6.6 Hz, 2H), 2.93 (s, 3H), 1.51 (dd,
J ¼ 14.3, 7.2 Hz, 2H), 1.40e1.28 (m, 9H), 0.90 (t, J ¼ 7.4 Hz, 3H). 13
C
NMR (101 MHz, DMSOd6):
d 164.56, 155.23, 150.79, 149.60, 145.40,
133.75, 126.23, 125.06, 123.14, 121.58, 120.08, 113.68, 113.57, 110.56,
64.63, 64.56, 64.06, 40.70, 40.11, 38.12, 22.24, 14.68, 14.62, 14.54,
11.35.
6.1.8. 5-[3-(2,5-Diethoxy-4-methanesulfonylamino-benzyl)-
ureido]-2-ethoxy-N-phenyl -benzamide (14)
Compound 14 (white solid, 200 mg) was prepared by a pro-
cedure similar to that described for the synthesis of compound 11.
1H NMR (400 MHz, DMSOd6):
d 10.16 (s, 1H), 8.84 (s, 1H), 8.61 (s,
1H), 7.71 (dd, J ¼ 12.2, 5.2 Hz, 3H), 7.55 (dd, J ¼ 8.9, 2.5 Hz, 1H), 7.34
(t, J ¼ 7.8 Hz, 2H), 7.08 (t, J ¼ 8.3 Hz, 2H), 6.95 (s, 1H), 6.87 (s, 1H),
6.32 (t, J ¼ 5.7 Hz, 1H), 4.22 (d, J ¼ 5.6 Hz, 2H), 4.13 (q, J ¼ 6.8 Hz,
2H), 3.99 (qd, J ¼ 6.7, 2.4 Hz, 4H), 2.93 (s, 3H), 1.45e1.25 (m, 9H). 13
C
NMR (101 MHz, DMSOd6):
d 163.66, 155.24, 150.59, 149.60, 145.41,
138.87, 133.89, 128.78 (2C), 126.20, 125.06, 123.79, 123.48, 121.94,
119.72, 119.46 (2C), 113.79, 113.57, 110.58, 64.75, 64.55, 64.06, 40.14,
38.14, 14.69, 14.62, 14.61.
6.1.9. 5-[3-(2,5-diethoxy-4-methanesulfonylamino-benzyl)-
ureido]-2-ethoxy-N-p-tolyl-benzamide (15)
with
a 10% HCl solution and extracted with ethyl ace-
tate(2 ꢁ 150 ml), the organic layer was washed with a saturated
NaCl solution (3 ꢁ 80 ml), the combined organic phases were dried
with Na2SO4, filtered, concentrated and purified by flash column
chromatography to give the pure product 5-amino-2-ethoxy-N-
methyl-benzamide (11g, 0.253 g, yield 87%) as white solid. Then
compound 11 (white solid, 0.170 g) was prepared by a procedure
similar to that described for the synthesis of compound 8 using the
corresponding 5-amino-2-ethoxy-N-methyl-benzamide (11g)
instead of 5-amino-2-ethoxy-benzoic acid methyl ester (8c).
Compound 15 (white solid, 170 mg) was prepared by a proced-
ure similar to that described for the synthesis of compound 11.
1H NMR (400 MHz, DMSOd6):
d 10.08 (s,1H), 8.82 (s,1H), 8.61 (s,
1H), 7.72 (d, J ¼ 2.6 Hz, 1H), 7.56 (dd, J ¼ 14.5, 5.5 Hz, 3H), 7.14 (d,
J ¼ 8.2 Hz, 2H), 7.06 (d, J ¼ 9.0 Hz, 1H), 6.95 (s, 1H), 6.87 (s, 1H), 6.33
(t, J ¼ 5.7 Hz, 1H), 4.22 (d, J ¼ 5.7 Hz, 2H), 4.12 (q, J ¼ 6.8 Hz, 2H),
3.99 (qd, J ¼ 6.8, 3.4 Hz, 4H), 2.92 (s, 3H), 2.26 (s, 3H), 1.41e1.31 (m,
9H). 13C NMR (101 MHz, DMSOd6):
d 163.40, 155.25, 150.57, 149.58,
145.41, 136.38, 133.90, 132.40, 131.52, 129.15, 128.60, 126.21, 125.04,
123.78, 121.87, 119.43, 113.79, 113.54, 110.57, 67.39, 64.97, 64.76,
64.55, 64.06, 40.10, 38.12, 20.41, 14.68, 14.62, 14.61.
1H NMR (400 MHz, DMSOd6):
d 8.81 (s, 1H), 8.54 (s, 1H), 8.06 (d,
J ¼ 4.6 Hz, 1H), 7.70 (d, J ¼ 2.7 Hz, 1H), 7.54 (dd, J ¼ 8.9, 2.7 Hz, 1H),
7.00 (d, J ¼ 9.0 Hz, 1H), 6.94 (s, 1H), 6.87 (s, 1H), 6.26 (t, J ¼ 5.8 Hz,
1H), 4.21 (d, J ¼ 5.7 Hz, 2H), 4.09 (q, J ¼ 6.9 Hz, 2H), 3.99 (qd, J ¼ 6.8,
2.8 Hz, 4H), 2.93 (s, 3H), 2.78 (t, J ¼ 8.7 Hz, 3H), 1.33 (dt, J ¼ 10.1,
6.1.10. 5-[3-(2,5-diethoxy-4-methanesulfonylamino-benzyl)-
ureido]-2-ethoxy-N-(4-methoxy-phenyl)-benzamide (16)
Compound 16 (white solid, 110 mg) was prepared by a proced-
ure similar to that described for the synthesis of compound 11.
6.8 Hz, 9H). 13C NMR (101 MHz, DMSOd6):
d 165.23, 155.22, 150.71,
149.56, 145.42, 133.67, 126.22, 124.99, 123.02, 121.60, 120.08, 113.70,
113.49, 110.58, 64.52 (2C), 64.01, 40.09, 38.12, 26.15, 14.68, 14.62,
14.49.
1H NMR (400 MHz, DMSOd6):
d 10.02 (s,1H), 8.80 (s,1H), 8.58 (s,
1H), 7.73 (d, J ¼ 2.6 Hz, 1H), 7.61 (d, J ¼ 8.9 Hz, 2H), 7.55 (dd, J ¼ 8.9,
2.6 Hz, 1H), 7.05 (d, J ¼ 9.0 Hz, 1H), 6.92 (dd, J ¼ 19.5, 10.6 Hz, 4H),
6.31 (t, J ¼ 5.7 Hz, 1H), 4.22 (d, J ¼ 5.7 Hz, 2H), 4.17e4.04 (m, 3H),
3.99 (qd, J ¼ 6.8, 3.3 Hz, 4H), 3.73 (s, 3H), 2.93 (s, 3H), 1.42e1.29 (m,
6.1.6. 5-[3-(2,5-Diethoxy-4-methanesulfonylamino-benzyl)-
ureido]-2-ethoxy-N-ethyl-benzamide (12)
Compound 12 (white solid, 150 mg) was prepared by a proced-
ure similar to that described for the synthesis of compound 11.
9H). 13C NMR (101 MHz, DMSOd6):
d 163.18, 155.41, 155.23, 150.55,
149.59, 145.41, 133.86, 132.06, 126.20, 125.04, 123.85, 121.77, 120.94
(2C), 119.72, 113.94 (2C), 113.76, 113.55, 110.58, 64.73, 64.54, 64.04,
55.17, 40.13, 38.14, 14.69, 14.63, 14.60.
1H NMR (400 MHz, DMSOd6):
d 8.81 (s, 1H), 8.54 (s, 1H), 8.11 (t,
J ¼ 5.3 Hz, 1H), 7.69 (d, J ¼ 2.7 Hz, 1H), 7.54 (dd, J ¼ 8.9, 2.7 Hz, 1H),
7.00 (d, J ¼ 9.0 Hz, 1H), 6.94 (s, 1H), 6.87 (s, 1H), 6.27 (t, J ¼ 5.8 Hz,
1H), 4.21 (d, J ¼ 5.7 Hz, 2H), 4.08 (q, J ¼ 6.9 Hz, 2H), 3.99 (qd, J ¼ 6.9,
2.8 Hz, 4H), 3.27 (dd, J ¼ 13.5, 6.6 Hz, 2H), 2.93 (s, 3H), 1.38e1.28 (m,
6.1.11. 5-[3-(2,5-diethoxy-4-methanesulfonylamino-benzyl)-
ureido]-2-ethoxy-N-(3-methoxy-phenyl)-benzamide (17)
Compound 17 (white solid, 110 mg) was prepared by a proced-
ure similar to that described for the synthesis of compound 11.
9H), 1.11 (t, J ¼ 7.2 Hz, 3H). 13C NMR (101 MHz, DMSOd6):
d 164.39,
155.22, 150.79, 149.58, 145.41, 133.76, 126.22, 125.03, 123.09, 121.58,
120.03, 113.77, 113.52, 110.56, 64.68, 64.54, 64.03, 40.09, 38.13,
33.83, 14.68, 14.62 (2C), 14.49.
1H NMR (400 MHz, DMSOd6):
d 10.14 (s, 1H), 8.82 (s, 1H), 8.59 (s,
1H), 7.72 (d, J ¼ 2.4 Hz, 1H), 7.55 (dd, J ¼ 8.9, 2.5 Hz, 1H), 7.41 (s, 1H),
7.27e7.17 (m, 2H), 7.06 (d, J ¼ 9.0 Hz, 1H), 6.95 (s, 1H), 6.88 (s, 1H),
6.67 (d, J ¼ 7.3 Hz, 1H), 6.32 (t, J ¼ 5.7 Hz, 1H), 5.75 (s, 1H), 4.22 (d,
J ¼ 5.6 Hz, 2H), 4.12 (q, J ¼ 6.8 Hz, 2H), 3.99 (qd, J ¼ 6.8, 2.9 Hz, 4H),
3.74 (s, 3H), 2.93 (s, 3H), 1.42e1.26 (m, 9H). 13C NMR (101 MHz,
6.1.7. 5-[3-(2,5-Diethoxy-4-methanesulfonylamino-benzyl)-
ureido]-2-ethoxy-N-propyl-benzamide (13)
Compound 13 (white solid, 180 mg) was prepared by
a

Y. Du et al. / European Journal of Medicinal Chemistry 144 (2018) 692e700
699
DMSOd6):
d
163.73, 159.56, 155.23, 150.57, 149.59, 145.41, 140.01,
All the designed compounds were docked into the ADC sites of
PTP1B in the open conformation.
133.87, 129.58, 126.19, 125.05, 123.80, 121.95, 119.68, 113.76, 113.56,
111.77, 110.58, 108.84, 105.39, 64.73, 64.55, 64.05, 54.97, 40.12,
38.14, 14.69, 14.62, 14.59.
Acknowledgment
6.1.12. 5-[3-(2,5-diethoxy-4-methanesulfonylamino-benzyl)-
ureido]-N-(3,4-dimethoxy-phenyl)-2-ethoxy-benzamide (18)
Compound 18 (white solid, 115 mg) was prepared by a proced-
ure similar to that described for the synthesis of compound 11.
This work was supported by two Grants from the National
Natural Science Foundation of China (81202389 and 81302853).
Appendix A. Supplementary data
1H NMR (400 MHz, DMSOd6):
d 10.02 (s,1H), 8.82 (s,1H), 8.57 (s,
1H), 7.73 (d, J ¼ 2.6 Hz, 1H), 7.53 (dd, J ¼ 8.9, 2.6 Hz, 1H), 7.43 (d,
J ¼ 2.0 Hz, 1H), 7.19 (dd, J ¼ 8.7, 2.1 Hz, 1H), 7.06 (d, J ¼ 9.0 Hz, 1H),
6.95 (s, 1H), 6.88 (s, 1H), 6.32 (t, J ¼ 5.7 Hz, 1H), 4.22 (d, J ¼ 5.7 Hz,
2H), 4.13 (q, J ¼ 6.9 Hz, 2H), 3.99 (qd, J ¼ 6.8, 2.8 Hz, 4H), 3.73 (d,
J ¼ 6.0 Hz, 6H), 2.93 (s, 3H), 1.44e1.25 (m, 9H). 13C NMR (101 MHz,
Supplementary data associated with this article can be found in
the online version, at https://doi.org/10.1016/j.ejmech.2017.12.064.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
DMSOd6):
d 163.25, 155.23, 150.54, 149.58, 148.62, 145.42, 145.07,
References
133.85, 132.53, 126.20, 125.04, 123.89, 121.79, 119.69, 113.73, 113.55,
112.21,111.38,110.59,104.74, 64.71, 64.54, 64.04, 55.76, 55.38, 40.10,
38.14, 14.69, 14.63, 14.58.
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