Facile fabrication of promising protein tyrosine phosphatase (PTP) inhibitor entities based on 'clicked' serine/threonine-monosaccharide hybrids

Article abstract of DOI:10.1016/j.bmc.2011.05.049

Protein tyrosine phosphatases (PTPs) are well-validated therapeutic targets for many human major diseases. The development of their potent inhibitors has therefore become a main focus of both academia and the pharmaceutical industry. We report herein a fa

Full text of DOI:10.1016/j.bmc.2011.05.049

Bioorganic & Medicinal Chemistry 19 (2011) 3892–3900
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Facile fabrication of promising protein tyrosine phosphatase (PTP) inhibitor
entities based on ‘clicked’ serine/threonine–monosaccharide hybrids
Xiao-Peng He a,c, Qiong Deng a, Li-Xin Gao b, Cui Li a, Wei Zhang b, Yu-Bo Zhou b, Yun Tang a,
b,
a,
a,b
Xiao-Xin Shi a, Juan Xie c, , Jia Li , Guo-Rong Chen , Kaixian Chen
⇑
⇑
⇑
a Key Laboratory for Advanced Materials & Institute of Fine Chemicals and School of Pharmacy, East China University of Science and Technology, Shanghai 200237, PR China
b National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, Shanghai 201203, PR China
c PPSM, Institut d’Alembert, ENS de Cachan, CNRS UMR 8531, F-94235 Cachan, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Protein tyrosine phosphatases (PTPs) are well-validated therapeutic targets for many human major dis-
eases. The development of their potent inhibitors has therefore become a main focus of both academia
and the pharmaceutical industry. We report herein a facile strategy toward the fabrication of new and
competent PTP inhibitor entities by simply ’clicking’ alkynyl amino acids onto diverse azido sugar tem-
plates. Triazolyl glucosyl, galactosyl, and mannosyl serine and threonine derivatives were efficiently syn-
thesized via click reaction, which were then identified as potent CDC25B and PTP1B inhibitors selective
over a panel of homologous PTPs tested. Their inhibitory activity and selectivity were found to largely lie
on the structurally and configurationally diversified monosaccharide moieties whereon serinyl and thre-
oninyl residues were introduced. In addition, MTT assay revealed the triazole-connected sugar-amino
acid hybrids may also inhibit the growth of several human cancer cell lines including A549, Hela, and
especially HCT-116. On the basis of such compelling evidence, we consider that this compound series
could furnish promising chemical entities serving as new CDC25B and PTP1B inhibitors with potential
cellular activity. Furthermore, the ‘click’ strategy starting from easily accessible and biocompatible amino
acids and sugar templates would allow the modular fabrication of a rich library of new PTP inhibitors effi-
caciously and productively.
Received 8 April 2011
Revised 21 May 2011
Accepted 23 May 2011
Available online 27 May 2011
Keywords:
Sugar template
Click chemistry
Amino acid
PTP inhibitor
MTT assay
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
lung, etc.5b This suggests that the inhibition of CDC25A and/or B
may become a promising strategy in oncology.
The protein tyrosine phosphatases (PTPs) constitute a large
class of functional enzymes governing the pivotal tyrosine phos-
phorylation processes on cellular level.1 A myriad of biochemical
studies indicated that the suppression of certain overexpressed
PTPs with dysfunctions in vivo may lead to the treatment of many
human major diseases.
For instance, the PTP1B-knockout mice model displayed in-
creased insulin sensitivity and enhanced glycemic control, and
was resistant to diet-induced obesity.2,3 Meanwhile, several recent
investigations suggest that PTP1B may qualify as a new therapeutic
target for breast cancer.4 The cell division cycle 25 (CDC25) phos-
phatases A, B, and C regulate cyclin-dependent kinases, the crucial
component of the eukaryotic cell division cycle.5a Among the three
isoforms, CDC25A and B have been identified to be overexpressed
in a wide range of human cancers including breast, colon, cervix,
Consequently, numerous programs have been initiated en route
to the fabrication of potent and bioavailable small-molecule PTP
inhibitors via both academia and the pharmaceutical industry.6,7
The majority of these bioactive compounds are phosphotyrosine
(pTyr) mimetics that competitively inhibit the targeted PTP. For
example, the difluoromethylene phosphonate (DFMP), carboxylic
acid and heterocyclic pTyr surrogates generally constitute the
competitive inhibitor category of PTP1B.8 Furthermore, noncom-
petitive inhibitors that induce the open conformation of WPD loop
have furnished alternative insight for gaining its inhibition.9 On the
other hand, CDC25 phosphatase inhibitors discovered till date fall
principally into quinoids whereas relatively fewer other bioactive
compound sorts were identified.5
Nevertheless, most of the currently characterized PTPs inhibi-
tors encounter limitations such as the unsatisfactory cellular activ-
ity and low bioavailability. In addition, several special compound
series such as the quinonyl derivatives could release reactive oxy-
gen species (ROS) which may uncertainly bring on toxicity to nor-
mal tissues. As a consequence, the discovery of new chemical
⇑
Corresponding authors. Tel.: +86 21 64253016; fax: +86 21 64252758.
E-mail addresses: joanne.xie@ens-cachan.fr (J. Xie), jli@mail.shcnc.ac.cn (J. Li),
mrs_guorongchen@ecust.edu.cn (G.-R. Chen).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.05.049

X.-P. He et al. / Bioorg. Med. Chem. 19 (2011) 3892–3900
3893
entities competent to serve as PTP inhibitors remains quite
desirable.
The O-propynyl serine and threonine a and b were prepared via
a previously reported method in one-pot from commercially avail-
Sugars are pivotal regulators of numerous biological and patho-
logical events in nature.10a These naturally abundant materials
have also demonstrated their crucial roles in drug develop-
ment.10b,c Becker and co-workers emphasized in a recent review
that the introduction of pharmacophores onto sugar templates that
possess dense stereochemical information is an excellent strategy
for the development of bioactive compounds with rich structural
diversity.10b Hence, the validation of a potent synthetic tool for
efficiently constructing such glycoconjugates has become crucial.
Thanks to the definition of click chemistry11a and sequentially
the identification of its representative reaction, the Cu(I)-catalyzed
azide-alkyne 1,3-dipolar cycloaddition11b by Sharpless and co-
workers, numerous triazole-functionalized sugar derivatives with
potential medicinal values were able to be modularly synthesized
in the past decade.12 ‘Click-stitched’ natural products such as
sugar-amino acid hybrids were also independently prepared by
Dondoni’s13a and Rutjes’s13b groups, which have particularly
absorbed our interest.
Owning to its structural similarity to the reported isoxazole acid
that mimics the PTP peptide substrate by Abbott’s laboratory,14 the
triazolyl amino acid residue is envisioned capable to serve as a new
pTyr surrogate (Fig. 1). Moreover, since the pTyr itself is inade-
quate to achieve PTP inhibition, it is noted that the linkage of
hydrophobic functionalities such as benzyl groups to pTyr could
enhance the binding affinity as well as the cellular activity of the
produced compounds.6,8,9b We have hence chosen several benzyl
1- or 6-azido monosaccharide epimers on which propargyl amino
acids could be ‘clicked’. As shown in Figure 1, the subsequently
formed triazolyl amino acids presented on structurally and config-
urationally diverse benzyl sugar templates are deemed to possess
distinct inhibitory potency and specificity on PTPs. Consequently,
we report herein the preparation and biological studies of a new
class of PTP inhibitors based on triazole-linked sugar-amino acid
hybrids.
able Boc-L-serine and Boc-L
-threonine.16
The Cu(I)-catalyzed Huisgen [3+2] cycloaddition between com-
pounds 1–4 and a or b was sequentially performed in a solvent
mixture of CH2Cl2/H2O (1:1, v/v), shown in Scheme 1. To our de-
light, the click reactions of all azido glycosides with the alkynyl
amino acids proceeded smoothly under the promotion of Na ascor-
bate/CuSO4Á5H2O with vigorous stirring over 8 h at rt, affording the
unique 1,4-disubstituted triazole-linked sugar–serine or threonine
hybrids. The glucosyl, galactosyl and mannosyl 6-substituted seri-
nyl and threoninyl triazoles 5–10 as well as the glucosyl 1-substi-
tuted serinyl triazole 11 were obtained in favorable yields of
80–90%. However, the glucosyl 1-substituted threoninyl triazole
12 was given by the click reaction of compound 4 with b less effi-
ciently under the same condition in a moderate yield of 62%.
In order to furnish the desired carboxylic acid-exposed products
with retained benzyl groups on sugar moieties, a Pd/C/H2 system
was used for the hydrogenolysis within a short timeframe of about
20 min.16a Benzyl esters 5–12 were therefore treated with such
condition, affording selectively the desired free acids 13–20 in
good-to-excellent yields (81–99%, Scheme 1).
2.2. Biological assay
The inhibitory activity of the prepared triazolyl serine– and
threonine–sugar hybrids 13–20 on a panel of PTPs including
N
BocHN
O
N
N
CO2R5
R2
O
R1
5: R1 = R3 = OBn, R2 = R4 = H, R5 = Bn (90 %)
7: R2 = R3 = OBn, R1 = R4 = H, R5 = Bn (84 %)
9: R1 = R4 = OBn, R2 = R3 = H, R5 = Bn (87 %)
13: R1 = R3 = OBn, R2 = R4 = H, R5 = H (85 %)
15: R2 = R3 = OBn, R1 = R4 = H, R5 = H (81 %)
17: R1 = R4 = OBn, R2 = R3 = H, R5 = H (93 %)
BnO
OMe
R3 R4
(i)
(1-3)+(a or b)
(ii)
or
2. Results and discussion
2.1. Synthesis
N
BocHN
CO2R5
O
N
N
R2
Me
As shown inFigure 1, to achieve the click reaction for the forma-
tion of the desired triazoles, the known benzyl 6-azido-6-deoxy-1-
O
R1
6: R1 = R3 = OBn, R2 = R4 = H, R5 = Bn (80 %)
8: R2 = R3 = OBn, R1 = R4 = H, R5 = Bn (85 %)
10: R1 = R4 = OBn, R2 = R3 = H, R5 = Bn (87 %)
14: R1 = R3 = OBn, R2 = R4 = H, R5 = H (99 %)
16: R2 = R3 = OBn, R1 = R4 = H, R5 = H (92 %)
18: R1 = R4 = OBn, R2 = R3 = H, R5 = H (94 %)
BnO
OMe
O-methoxy-a-D-glucoside (1), galactoside (2), mannoside (3), and
1-azido-1-deoxy-b-
D
-glucoside (4) were used as glycodonors.15
R3 R4
PHN
CO2H
N
BnOOC
BnOOC
NHBoc
O
CO2H
OBn
N
N
O
a: Ser
NH2
N
BocHN
CO2R5
O
BnO
BnO
O
N
R
O
NHBoc
O
N
OBn
N
b: Thr
Isoxazole acid
Glycosyl triazolyl
amino acid
(i)
11: R5 = Bn (87 %), 19: R5 = H (93 %)
4+(a or b)
(ii)
or
N3
N3
N3
O
OBn
6
OBn
BnO
O
BnO
BnO
4
BnO
BnO
BnO
BocHN
CO2R5
O
BnO
BnO
O
O
O
1
3
2
BnO
OMe
OMe
OMe BnO
N3
N
Me
OBn
OBn
OBn
OBn
N
OBn
N
2 (Gal-6-N3)
1 (Glc-6-N3)
3 (Man-6-N3 )
4 (Glc-1-N3)
12: R5 = Bn (62 %), 20: R5 = H (94 %)
Figure 1. Heterocyclic acid-based PTPs inhibitors and alkynyl amino acid (a and b)
and azido benzyl sugar templates (1–4) used for click reaction in this study.
Scheme 1. Reagents and conditions: (i) Na ascorbate, CuSO4Á5H2O, CH2Cl2/H2O
(1:1, v/v), rt; (ii) Pd/C, H2, MeOH, rt.

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X.-P. He et al. / Bioorg. Med. Chem. 19 (2011) 3892–3900
CDC25A, CDC25B, PTP1B, TCPTP, SHP-1, SHP-2, and LAR was
sequentially assessed via our previously developed methods at a
the threoninyl precursor with C6-modified monosaccharides may
result in a similar inhibitory pattern toward PTPs. However, the
PTP inhibitory effect of C6-triazoloserinyl glycosides is largely
dependent on the epimeric sugar scaffolds whereon the serinyl
precursor was introduced with the mannosyl derivative being the
most potent.
The structurally varied C1-modified glucosides 19 and 20 bear-
ing one additional benzyl group displayed generally improved
inhibition on the tested PTPs compared with their C6-modified gly-
cosyl analogs. Triazole-linked serinyl glucoside 19 possesses the
best PTP1B and CDC25B inhibitory activities among this series with
compound concentration of 100 l
g/mL.17
Notably, despite the click chemistry has been well employed to
establish triazolyl PTPs inhibitor libraries,7a,18 the use of sugar tem-
plates whereon amino acid precursors were ‘clicked’ toward the
same purpose remains much less explored. To our delight, the de-
signed and prepared triazolyl serine– and threonine–sugar hybrids
13–20 showed micromole-ranged inhibitory activities as well as
specificities on different PTPs, given in Table 1.
Interestingly, all compounds tested displayed preferable inhib-
itory effects on PTP1B and CDC25B, lesser inhibitory potency on
TCPTP and CDC25A and almost no inhibition toward SHP-1,
IC50 values equal to 6
enhanced IC50 values on TCPTP (13
(40 M), and SHP-2 (47 M). In contrast, despite the existence of
lM. Moreover, this compound also showed
l
M), CDC25A (24 M), SHP-1
l
SHP-2, and LAR. Glucosyl 6-triazoloserine 13 (IC50 ꢀ 70
l
M) and
l
l
threonine 14 (IC50 ꢀ 25 M) possess, respectively, almost identical
l
the additional methyl group on the threoninyl residue of glucoside
20 may render its slightly decreased inhibitory potency toward
IC50 values on PTP1B and CDC25B whereas the latter is about
threefold more active than the former. This demonstrates that
the additional methyl group on threonine residue contributes evi-
dently to the inhibitory activity. Moreover, both compounds
showed weak or no inhibitory activities on TCPTP, CDC25A, SHP,
and LAR.
PTP1B (IC50 = 7
lM) and CDC25B (IC50 = 12 lM), the selectivity of
this compound has, however, been increased.
In an attempt to verify whether the benzyl groups on the iden-
tified PTPs inhibitors are crucial toward their corresponding activ-
ity, the debenzylated analog of the most potent inhibitor 19 was
synthesized. As shown in Scheme 2, this click reaction was directly
achieved from the known 1-azido glucoside 2119 and O-alkynyl
The galactosyl 6-triazoloserine 15 (IC50 ꢀ 20
lM) is similarly an
equally potent inhibitor of both PTP1B and CDC25B, which dis-
played around 2.5-fold decreased potency toward TCPTP
Boc-
-serine 2220 under microwave irradiation at 60 °C for a ramp
L
(IC50 = 55
trast, its triazolothreoninyl counterpart 16 appeared to be a more
specific PTP1B (IC50 = 15 M) inhibitor with twofold decreased
activity on CDC25B (IC50 = 31 M) and no inhibitory effects on all
of the rest PTPs tested (IC50 >120 M). The mannosyl 6-triazoloser-
ine 17 (IC50 = 8 M) and threonine 18 (IC50 = 12 M) are better
l
M) and no activity on CDC25A, SHP, and LAR. In con-
time of 5 min and a hold time of 15 min, affording the desired
product 23 in a yield of 60%. This relatively low yield could pre-
sumably be ascribed to the partial conversion of the alkynoic acid
22 into enol lactones under the catalysis of Cu(I).21 As shown in Ta-
ble 1, the successive inhibitory assay showed that compound 23 is
l
l
l
l
l
inactive on both PTP1B and CDC25B (IC50 >120 lM), which posi-
CDC25B inhibitors having approximately 2-, 4-, and 10-fold selec-
tivity over PTP1B, CDC25A and TCPTP, respectively, and do not in-
hibit the catalytic activities of SHP and LAR.
tively demonstrates that the benzyl groups appended with the su-
gar scaffold are significant for enhancing the binding affinity of the
triazole-linked sugar-amino acid hybrids constructed.
Notably, galactosyl triazoloserine 15 is almost 3.5-fold more po-
tent than its glucosyl epimer 13, which implies that the axial ben-
zyl group on C4-position of galactoside is privileged in
configuration over the C4-equatorial benzyl group of glucoside to-
ward PTP1B and CDC25B inhibition. However, no obvious IC50 def-
icit is observed between threoninyl sugar C4-epimers 14 and 16.
Similarly, whereas only unapparent inhibitory difference was
emerged between the threoninyl C2-epimers 14 and 18, the serinyl
mannoside 17 owns around eight and fourfold increased inhibitory
potency comparing to its glucosyl C2-epimer 13 on CDC25B and
PTP1B, respectively. Moreover, the specificity of compound 17 to-
ward CDC25B over other PTPs tested is also much enhanced than
that of compound 13. These data suggest that the click ligation of
For evaluating the inhibitory modality of this compound series,
the most active inhibitor 19 was selected for enzymatic kinetic
study on PTP1B and CDC25B, respectively. The effect of compound
19 on PTP1B- and CDC25B-catalyzed reaction was studied at five
different concentrations and the resulting Eadie–Hofstee plots17
are given in Figure 2 (19-PTP1B) and 3 (19-CDC25B). Clearly, the
diagrams in Figure 2 confirm that compound 19 is a competitive
PTP1B inhibitor as the Vmax value (Fig. 3B) retained invariable
while the Km value (Fig. 3A) increased with the increasing com-
pound concentration. In contrast, the plots in Figure 3 are consis-
tent with a mixed-type inhibition pattern of 19 on CDC25B due
to the increasing Km value (Fig. 4A) and concomitantly decreasing
Vmax value (Fig. 4B) upon the gradually increased compound con-
centration. The Ki value of the competitive PTP1B inhibitor 19 is as-
signed to 3.9 lM.
Table 1
The MTT assay was then performed to preliminarily assess the
Inhibitory activity of compounds 13–20 on PTPs
cellular activity of the PTP inhibitors. As shown in Table 2, except
the galactosyl serine 15 (IC50 >120 lM), all compounds showed
micromolar activity on the selected human cancer cell lines includ-
ing A549 (lung), Hela (cervix) and HCT-116 (colon). The glycosyl
C6-triazoloserine and threonine 13, 14, and 16–18 are less toxic
on A549 with the glucosyl threonine 14 and mannosyl threonine
Compd
IC50
CDC25B
(
l
M)a,b,c,d
TCPTP
PTP1B
CDC25A
13
14
15
16
17
18
19
20
23
67.8 4.9
22.3 5.0
20.9 1.7
15.3 0.5
18.5 2.0
26.0 6.7
5.9 0.4
7.1 1.0
>120
67.5 7.5
27.1 5.0
20.6 7.1
31.1 6.6
8.2 3.8
12.2 4.8
6.2 3.0
11.6 1.8
>120
>120
>120
118.7 16.1
>120
80.7 5.6
54.9 10.0
>120
89.2 7.3
105.3 6.2
12.5 1.1
38.6 8.3
n.d.e
>120
26.8 25.1
51.6 10.4
24.3 1.5
53.0 1.8
n.d
OH
O
OH
O
BocHN
BocHN
COOH
O
COOH
HO
HO
HO
HO
(i)
+
a
O
Except compound 19, all other compounds herein showed no inhibitory activity
on SHP-1, SHP-2 (IC50 >120 M).
The IC50 values of compound 19 on SHP-1 and SHP-2 are 40.4 1.4 and
47.6 8.9 M, respectively.
N
N3
l
b
OH
N
OH
N
l
23 (60 %)
21
22
c
All compounds herein showed no inhibitory activity on LAR (IC50 >120
Values are means of three experiments.
n.d. = not determined.
lM).
d
e
Scheme 2. Reagents and conditions: (i) Na ascorbate, CuSO4Á5H2O, CH2Cl2/H2O
(1:1, v/v), microwave irradiation (60 °C).

X.-P. He et al. / Bioorg. Med. Chem. 19 (2011) 3892–3900
3895
Figure 2. Inhibition of PTP1B catalyzed pNPP hydrolysis by compound 19. Experiments were performed at 30 °C and pH 6.5.
Figure 3. Inhibition of CDC25B-catalyzed pNPP hydrolysis by compound 19. Experiments were performed at 30 °C and pH 6.5.
Figure 4. Plausible binding mode of compound 19 with (A) PTP1B and (B) CDC25B. The compound was shown as green stick and residues in PTP shown as light gray line.
Nitrogen atoms are in blue and oxygen atoms in red. Hydrogen bonds were shown as yellow dashed lines and nonpolar interactions were not shown.
18 being around twofold more active than the rest. Their glucosyl
C1-substituted analogs 19 and 20 exhibited similarly increased
cytotoxicity.
The activities of the C6-modified glucosyl serine 13, galactosyl
threonine 16 and mannosyl serine 17 were almost twofold en-
hanced toward Hela, whereas those of the glucosyl threonine 14
and mannosyl threonine 18 remained unchanged. Likewise, the
glucosyl C1-serine 19 and threonine 20 own enhanced cytotoxicity
compared to their C6-modified analogs. Finally, these compounds
showed further improved potency on HCT-116. Parallel IC50 values
were observed among glycosyl C6-substituted derivatives with the
glucosyl serine 13 being slightly less potent. However, their
C1-substituted counterparts 19 and 20 further exhibited approxi-
mately threefold improved activity, demonstrating that these
triazolyl sugar-amino acid hybrids are more specific toward
HCT-116 inhibition.

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X.-P. He et al. / Bioorg. Med. Chem. 19 (2011) 3892–3900
Table 2
4. Experimental section
Cytotoxicity of compounds 13–20 on cancer cell lines
Compd
IC50
(
l
M)a
All purchased chemicals and reagents are of high commercially
available grade. Solvents were purified by standard procedures. 1H
and 13C NMR spectra were recorded on a Bruker AM-400 MHz
spectrometer in CDCl3, D2O or DMSO-d6 solutions. Optical rota-
tions were measured using a Perkin–Elmer 241 polarimeter at
room temperature. Analytical thin-layer chromatography was per-
formed on E. Merck aluminum percolated plates of Silica Gel 60F-
254 with detection by UV and by spraying with 6 N H2SO4 and
heating at 300 °C. The microwave-assisted reaction was performed
in a Yalian (YL8023B1) system. High resolution mass spectra
(HRMS) were recorded on an MA1212 instrument using standard
conditions (ESI, 70 eV).
A549
Hela
HCT-116
13
14
15
16
17
18
19
20
119.5 6.4
62.76 4.8
>120
107.56 11.6
97.3 16.2
57.2 3.1
35.2 3.2
25.3 3.4
75.4 12.4
69.5 11.3
>120
56.4 7.8
54.2 5.3
53.7 5.7
25.3 2.4
31.5 2.8
46.3 4.2
33.5 2.1
>120
36.6 3.1
32.3 2.3
32.2 3.0
12.8 1.3
12.0 1.2
a
Values are means of three experiments.
2.3. Docking study
4.1. General procedure for the conventional click reaction
Eventually, the plausible complex of compound 19 with, respec-
tively, PTP1B and CDC25B was tentatively proposed via docking
simulation, illustrated in Figure 4. The most likely binding manner
of the competitive inhibitor 19 with PTP1B is shown in Figure 4A. A
densely functionalized hydrogen-bonding network is generated
between the deeply inserted carboxylic acid precursor of the triaz-
oloacid with amino acid residues including Ser216, Ala217, Gly218,
Ile219, and Gly220 of the catalytic site cavity. This is similar to the
reported co-crystallization of PTP1B with the structurally analo-
gous isoxazole acids.14
In addition, the adjacent NHBoc group also made three hydro-
gen bonds with Arg221 and Gln266 while same interactions con-
currently occurred between the oxygen atoms on glycosyl ring
and C6-benzyl group with Lys116, and two nitrogen atoms on tri-
azole ring with Lys116 and Lys120, respectively. Hydrophobic con-
tacts were subsequently observed between the C2-distal benzene
group with Tyr46 and Val49, and the C6-distal benzene moiety
with Phe182 on WPD loop.
The plausible complex of the mixed-type inhibitor 19 with
CDC25B was then elaborated in Figure 4B. Similarly, a series of
hydrogen bonds were made between the carboxylic acid and the
NHBoc group with Tyr428, Arg479, Arg482, and Arg544 in the cata-
lytic area of CDC25B, while hydrogen-bonding interactions were
also observed between the oxygen atoms of glucosyl ring and benzyl
group with Arg544. Moreover, strong nonpolar contacts were gen-
erated between the C6-distal benzene group with Leu540 while a
To a biphasic solution of the azide (1 equiv) and the alkyne
(1 equiv) in CH2Cl2 (8 mL) and water (8 mL), sodium ascorbate
(6 equiv) and CuSO4Á5H2O (3 equiv) were added. This mixture
was stirred vigorously at rt for 8 h and was then directly diluted
with CH2Cl2, washed with brine and dried over MgSO4. The com-
bined organic layer was filtered and concentrated in vacuum to
give
a crude residue which was then purified by column
chromatography.
4.1.1. (R)-Benzyl-2-((tert-butoxycarbonyl)amino)-3-((1-
(((2R,3R,4S,5R,6S)-3,4,5-tris(benzyloxy)-6-methoxytetrahydro-
2H-pyran-2-yl)methyl)-1H-1,2,3-triazol-4-
yl)methoxy)propanoate (5)
Compound 1 (226 mg, 0.5 mmol) was treated with a (169 mg,
0.5 mmol) according to the general procedure. Purification by
chromatography (EtOAc/Petroleum ether; 1:10–1:2) gave 341 mg
(90%) of 5 as a colorless oil. Rf = 0.6 (EtOAc/Petroleum ether; 1:1).
[a]
D = À3.7 (c 0.2, CH3OH); 1H NMR (400 MHz, CDCl3): d = 7.41 (s,
1H), 7.36–7.26 (m, 20H), 5.37 (d, J = 7.6 Hz, 1H), 5.23 (d,
J = 12.4 Hz, 1H), 5.11 (d, J = 12.4 Hz, 1H), 4.98 (d, J = 10.8 Hz, 1H),
4.91 (d, J = 10.8 Hz, 1H), 4.81 (d, J = 10.8 Hz, 1H), 4.74 (t,
J = 12.4 Hz, 2H), 4.63–4.29 (m, 8H), 4.00 (t, J = 9.2, Hz, 1H), 3.92
(br s, 2H), 3.72 (dd, J = 2.4, 9.2 Hz, 1H), 3.43 (dd, J = 3.6, 10.0 Hz,
1H), 3.16 (d, J = 3.6 Hz, 3H), 1.44 (s, 9H); 13C NMR (100 MHz,
CDCl3): d = 170.4, 155.5, 144.2, 138.5, 138.0, 135.6, 128.5, 128.4,
128.2, 128.0, 127.9, 127.7, 124.1, 99.7, 81.8, 80.0, 79.9, 78.0, 75.7,
74.9, 73.3, 70.2, 69.1, 66.9, 64.8, 55.3, 54.1, 50.6, 28.3; HRMS (m/
z): calcd for C46H54N4O10+H: 823.3918, found: 823.3918.
p–p stacking of C3-distal benzene with the benzene residue of
Phe475 might contribute largely to the binding affinity.
Since there is no available crystal structure of triazolyl sugar-
amino acid hybrids with either PTP1B or CDC25B, the above-
depicted binding modes are preliminary whereas further
crystallographic evidence is desirable.
4.1.2. (2S,3S)-Benzyl-2-((tert-butoxycarbonyl)amino)-3-((1-
(((2R,3R,4S,5R,6S)-3,4,5-tris(benzyloxy)-6-methoxytetrahydro-
2H-pyran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)
butanoate (6)
3. Conclusion
Compound 1 (379 mg, 0.8 mmol) was treated with b (268 mg,
0.8 mmol) according to the general procedure. Purification by
chromatography (EtOAc/Petroleum ether; 1:10–1:2) gave 528 mg
(80%) of 6 as a colorless oil. Rf = 0.6 (EtOAc/Petroleum ether; 1:1).
In summary, we have shown in this study that the facilely
‘clicked’ triazolyl sugar-amino acid hybrids could serve as compe-
tent PTP1B and CDC25B inhibitors with micromole-ranged inhibi-
tory activity. The introduction of triazolo-serinyl or threoninyl
precursors onto different monosaccharide templates may result
in distinct inhibitory potency and selectivity of the constructed
products. Furthermore, MTT assay identified these compounds
are toxic toward several cancer cell lines with specificity toward
HCT-116, indicating their potential value of being active on cellular
level. We hence consider that the modular ‘clicking’ of natural ami-
no acids onto structurally and configurationally diverse sugar tem-
plates could probably represent a concise strategy toward the
efficient acquisition of new promising PTP inhibitor entities in a
productive way.
[a]
D = À0.07 (c 0.1, CH3OH); 1H NMR (400 MHz, CDCl3): d =
7.35–7.29 (m, 21H), 5.23 (d, J = 9.2 Hz, 1H), 5.17 (d, J = 12.4 Hz,
1H), 5.07 (d, J = 12.4 Hz, 1H), 4.98 (d, J = 10.4 Hz, 1H), 4.90 (d,
J = 10.8 Hz, 1H), 4.80 (d, J = 10.8 Hz, 1H), 4.73 (t, J = 12.4 Hz, 2H),
4.62–4.32 (m, 8H), 4.15 (d, J = 6.0, Hz, 1H), 4.00 (t, J = 9.2, Hz,
1H), 3.91 (t, J = 7.2, Hz, 1H), 3.43 (dd, J = 3.2, 10.6 Hz, 1H), 3.17 (s,
3H), 1.44 (s, 9H), 1.23 (d, J = 8.8 Hz, 3H); 13C NMR (100 MHz,
CDCl3): d = 170.8, 156.1, 144.7, 138.3, 138.0, 135.5, 128.5, 128.2,
128.0, 127.7, 124.0, 98.0, 81.8, 79.9, 78.0, 75.7, 74.9, 73.4, 69.1,
67.0, 62.6, 58.4, 55.3, 51.6, 28.3, 16.3; HRMS (m/z): calcd for
C46H54N4O10+H: 823.3918, found: 823.4067.

X.-P. He et al. / Bioorg. Med. Chem. 19 (2011) 3892–3900
3897
4.1.3. (R)-Benzyl-2-((tert-butoxycarbonyl)amino)-3-((1-
(((2R,3S,4S,5R,6S)-3,4,5-tris(benzyloxy)-6-methoxytetrahydro-
2H-pyran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)
propanoate (7)
chromatography (EtOAc/Petroleum ether; 1:8–1:1) gave 868 mg
(87%) of 10 as a colorless oil. Rf = 0.5 (EtOAc/Petroleum ether;
1:1). [
a]
D = À0.05 (c 0.2, CH3OH); 1H NMR (400 MHz, CDCl3):
d = 7.48 (s, 1H), 7.36–7.26 (m, 20H), 5.24 (d, J = 9.6 Hz, 1H), 5.16
(d, J = 11.6 Hz, 1H), 5.10 (d, J = 12.4 Hz, 1H), 4.96 (d, J = 11.2 Hz,
1H), 4.74 (d, J = 12.0 Hz, 1H), 4.68 (d, J = 12.4 Hz, 1H), 4.67–4.56
(m, 6H), 4.40–4.35 (m, 2H), 4.32 (dd, J = 6.0, 9.2 Hz, 1H), 4.18–
4.10 (m, 1H), 3.92–3.85 (m, 2H), 3.78 (t, J = 2.0 Hz, 1H), 3.66 (t,
J = 9.6 Hz, 1H), 3.07 (s, 3H), 1.45 (s, 9H), 1.22 (d, J = 6.4 Hz, 3H);
13C NMR (100 MHz, CDCl3): d = 170.8, 156.1, 144.5, 138.2, 138.1,
135.6, 128.6, 128.5, 128.3, 128.2, 127.8, 127.7, 126.1, 125.8,
124.2, 99.2, 80.2, 79.8, 75.1, 75.0, 74.7, 74.6, 73.0, 72.1, 70.7, 67.0,
62.5, 60.3, 58.5, 54.9, 51.0, 28.3, 16.3; HRMS (m/z): calcd for
Compound 2 (142 mg, 0.3 mmol) was treated with a (116 mg,
0.3 mmol) according to the general procedure. Purification by
chromatography (EtOAc/Petroleum ether; 1:10–1:2) gave 202 mg
(84%) of 7 as a yellow oil. Rf = 0.6 (EtOAc/Petroleum ether; 1:1).
[a]
D = +0.5 (c 0.3, CH3OH); 1H NMR (400 MHz, CDCl3): d = 7.43–
7.29 (m, 21H), 5.34 (d, J = 12.0 Hz, 1H), 5.23 (d, J = 12.4 Hz, 1H),
4.91 (d, J = 11.6 Hz, 2H), 4.85 (d, J = 12.0 Hz, 1H), 4.77–4.45 (m,
8H), 4.33–4.12 (m, 2H), 4.04 (dd, J = 3.6, 10.0 Hz, 1H), 4.00–3.81
(m, 4H), 3.69 (dd, J = 2.8, 9.2 Hz, 1H), 2.97 (s, 3H), 1.44 (s, 9H);
13C NMR (100 MHz, CDCl3): d = 170.4, 155.5, 144.1, 138.5, 138.3,
138.0, 135.5, 128.7, 128.6, 128.4, 128.3, 128.2, 128.0, 127.9,
127.8, 127.7, 124.1, 98.7, 80.0, 79.2, 78.8, 76.2, 75.1, 74.7, 74.4,
73.9, 73.7, 70.3, 69.5, 67.1, 64.7, 57.1, 55.2, 54.1, 51.2, 28.3; HRMS
(m/z): calcd for C46H54N4O10+H: 823.3918, found: 823.3909.
C47H56N4O10+Na: 859.3894, found: 859.3901.
4.1.7. (R)-Benzyl-2-((tert-butoxycarbonyl)amino)-3-((1-
((2R,3R,4S,5R,6R)-3,4,5-tris(benzyloxy)-6-((benzyloxy)methyl)
tetrahydro-2H-pyran-2-yl)-1H-1,2,3-triazol-4-yl)methoxy)
propanoate (11)
4.1.4. (2S,3S)-Benzyl-2-((tert-butoxycarbonyl)amino)-3-((1-
(((2R,3S,4S,5R,6S)-3,4,5-tris(benzyloxy)-6-methoxytetrahydro-
2H-pyran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)
butanoate (8)
Compound 4 (790 mg, 1.4 mmol) was treated with a (431 mg,
1.3 mmol) according to the general procedure. Purification by
chromatography (EtOAc/Petroleum ether; 1:10–1:2) gave 899 mg
(87%) of 11 as a colorless oil. Rf = 0.7 (EtOAc/Petroleum ether;
Compound 2 (179 mg, 0.4 mmol) was treated with b (129 mg,
0.4 mmol) according to the general procedure. Purification by
chromatography (EtOAc/Petroleum ether; 1:10–1:2) gave 314 mg
(85%) of 8 as a white powder. Rf = 0.6 (EtOAc/Petroleum ether;
1:1). [
a]
D = À1.9 (c 0.2, CH3OH); 1H NMR (400 MHz, CDCl3):
d = 7.42 (s, 1H), 7.36–7.23 (m, 18H), 7.19–7.15 (m, 5H), 6.94–6.92
(m, 2H), 5.53 (d, J = 9.2 Hz, 1H), 5.40 (d, J = 8.8 Hz, 1H), 5.21 (d,
J = 12.4 Hz, 1H), 5.06 (d, J = 12.4 Hz, 1H), 4.92 (t, J = 11.2 Hz, 2H),
4.86 (d, J = 10.4 Hz, 1H), 4.62–4.44 (m, 8H), 4.07 (d, J = 10.4 Hz,
1H), 4.04–4.00 (m, 1H), 3.86–3.79 (m, 2H), 3.75–3.68 (m, 4H),
1.35 (s, 9H); 13C NMR (100 MHz, CDCl3): d = 170.5, 155.5, 144.7,
138.2, 137.7, 137.0, 135.5, 128.6, 128.5, 128.4, 128.2, 128.1,
128.0, 127.9, 127.8, 122.0, 87.6, 85.5, 80.6, 80.1, 78.0, 75.8, 75.3,
74.9, 73.6, 70.6, 68.0, 67.1, 65.0, 54.1, 28.4; HRMS (m/z): calcd for
1:1). [a]
D = À7.2 (c 0.1, CH3OH); 1H NMR (400 MHz, CDCl3):
d = 7.34–7.29 (m, 21H), 5.14 (d, J = 12.4 Hz, 2H), 5.07 (d,
J = 6.0 Hz, 1H), 5.04 (d, J = 5.2 Hz, 1H), 4.90 (d, J = 11.6 Hz, 1H),
4.84 (d, J = 12.0 Hz, 1H), 4.75 (d, J = 11.6 Hz, 1H), 4.70–4.59 (m,
4H), 4.31 (d, J = 9.6 Hz, 1H), 4.14 (d, J = 11.2 Hz, 2H), 4.03 (dd,
J = 3.6, 10.0 Hz, 1H), 3.98 (br s, 1H), 3.92 (dd, J = 2.8, 10.0 Hz, 1H),
3.83 (d, J = 2.0 Hz, 1H), 2.98 (s, 3H), 1.44 (s, 9H), 1.24 (s, 3H); 13C
NMR (100 MHz, CDCl3): d = 170.8, 156.1, 144.5, 138.2, 138.1,
135.6, 128.6, 128.5, 128.3, 128.2, 128.0, 127.7, 126.1, 125.8,
124.2, 99.2, 80.2, 79.8, 75.1, 75.0, 74.7, 74.6, 73.0, 72.1, 70.7, 67.0,
62.5, 60.3, 58.5, 54.9, 51.0, 28.3, 16.3; HRMS (m/z): calcd for
C52H59N4O10+H: 899.4231, found: 899.4205.
4.1.8. (2S,3S)-Benzyl 2-((tert-butoxycarbonyl)amino)-3-((1-
((2R,3R,4S,5R,6R)-3,4,5-tris(benzyloxy)-6-((benzyloxy)methyl)
tetrahydro-2H-pyran-2-yl)-1H-1,2,3-triazol-4-yl)methoxy)
butanoate (12)
C47H56N4O10+Na: 859.3894, found: 859.3898.
Compound 4 (623 mg, 1.1 mmol) was treated with b (320 mg,
0.9 mmol) according to the general procedure. Purification by
chromatography (EtOAc/Petroleum ether; 1:8–1:2) gave 512 mg
(62%) of 12 as a colorless oil. Rf = 0.3 (EtOAc/Petroleum ether;
4.1.5. 2-(tert-Butoxycarbonylamino)-3-((1-(((2R,3R,4S,5S,6S)-
3,4,5-tris(benzyloxy)-6-methoxytetrahydro-2H-pyran-2-
yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)propanoate benzyl
ether (9)
1:4). [
a]
D = À3.4 (c 0.1, CH3OH); 1H NMR (400 MHz, CDCl3):
Compound 3 (483.4 mg, 1.0 mmol) was treated with a (316 mg,
1.0 mmol) according to the general procedure. Purification by
chromatography (EtOAc/Petroleum ether; 1:10–1:2) gave 646 mg
(87%) of 9 as a colorless oil. Rf = 0.6 (EtOAc/Petroleum ether; 1:1).
d = 7.34–7.25 (m, 21H), 7.20–7.17 (m, 3H), 6.94–6.90 (m, 2H),
5.51 (d, J = 9.2 Hz, 1H), 5.23 (d, J = 10.0 Hz, 1H), 5.15 (d,
J = 12.4 Hz, 1H), 5.03 (d, J = 12.0 Hz, 1H), 4.95–4.89 (m, 2H), 4.86
(d, J = 10.4 Hz, 1H), 4.61–4.32 (m, 7H), 4.08 (d, J = 10.8 Hz, 1H),
4.00 (t, J = 9.2 Hz, 1H), 3.86–3.79 (m, 2H), 3.74–3.66 (m, 4H), 1.44
(s, 9H), 1.25 (d, J = 6.4 Hz, 3H); 13C NMR (100 MHz, CDCl3):
d = 170.9, 156.2, 145.3, 138.2, 137.7, 137.0, 135.5, 128.6, 128.4,
128.2, 128.1, 128.0, 127.9, 127.8, 126.0, 125.8, 121.8, 87.5, 85.5,
80.6, 80.0, 78.0, 75.8, 75.3, 75.2, 74.8, 73.5, 68.0, 67.1, 62.7, 60.4,
[a]
D = +0.05 (c 0.1, CH3OH); 1H NMR (400 MHz, CDCl3): d = 7.59
(s, 1H), 7.37–7.32 (m, 20H), 5.36 (d, J = 8.0 Hz, 1H), 5.23 (d,
J = 12.4 Hz, 1H), 5.12 (d, J = 12.4 Hz, 1H), 4.95 (d, J = 11.2 Hz, 1H),
4.75 (d, J = 12.0 Hz, 1H), 4.69 (d, J = 11.6 Hz, 1H), 4.67–4.45 (m,
9H), 3.92 (dd, J = 6.4, 8.8 Hz, 2H), 3.90–3.87 (m, 1H), 3.78 (br s,
1H), 3.71–3.62 (m, 2H), 3.08 (d, J = 6.0 Hz, 3H), 1.45 (s, 9H); 13C
NMR (100 MHz, CDCl3): d = 170.4, 155.5, 144.0, 138.2, 138.1,
135.5, 128.6, 128.5, 128.4, 128.2, 127.8, 127.7, 124.3, 99.2, 80.2,
80.0, 75.1, 75.0, 74.9, 74.6, 73.0, 72.9, 72.1, 70.6, 70.5, 70.1, 67.1,
64.8, 64.7, 64.8, 54.1, 51.0, 29.7, 29.6, 28.3; HRMS (m/z): calcd for
58.3, 28.4, 16.2; HRMS (m/z): calcd for
C53H60N4O10+Na:
913.4388, found: 913.4393.
4.2. Preparation of (R)-2-((tert-butoxycarbonyl)amino)-3-((1-
((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)
tetrahydro-2H-pyran-2-yl)-1H-1,2,3-triazol-4-yl)methoxy)
propanoic acid (23)
C46H54N4O10+H: 823.3918, found: 823.3922.
4.1.6. (2S,3S)-Benzyl 2-((tert-butoxycarbonyl)amino)-3-((1-
(((2R,3R,4S,5S,6S)-3,4,5-tris(benzyloxy)-6-methoxytetrahydro-
2H-pyran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)
butanoate (10)
Compound 3 (586 mg, 1.2 mmol) was treated with b (421 mg,
1.2 mmol) according to the general procedure. Purification by
To a well-stirred biphasic solution of sugar azide 21 (226 mg,
1.1 mmol) and alkynyl amino acid 22 (205 mg, 1.0 mmol) in
tBuOH/THF/H2O (8/8/8 mL), sodium ascorbate (0.2 equiv) and
CuSO4Á5H2O (0.1 equiv) was added. Then the mixture was trans-
ferred to the microwave oven at 60 °C for a ramp time of 5 min

3898
X.-P. He et al. / Bioorg. Med. Chem. 19 (2011) 3892–3900
and hold time of 15 min. Upon completion, the resulting mixture
was diluted with CH2Cl2, washed with brine, dried over MgSO4, fil-
tered and evaporated to give a crude residue. Purification by chro-
matography (EtOAc/EtOH; 1:8–1:4) gave 267 mg (60%) of 23 as a
(100 MHz, DMSO-d6): d = 171.9, 155.3, 143.4, 138.7, 138.6, 138.5,
128.2, 128.1, 128.0, 127.6, 127.5, 127.4, 127.3, 124.8, 97.5, 78.2,
77.7, 75.3, 74.9, 74.0, 71.6, 69.2, 69.0, 63.4, 54.2, 53.9, 50.5, 28.1;
HRMS (m/z): calcd for C39H49N4O10+H: 733.3449, found: 733.3451.
white powder. Rf = 0.5 (EtOAc/CH3OH; 10:1). [
a
]
D = À10.9 (c 0.1,
CH3OH); 1H NMR (400 MHz, D2O): d = 8.21 (s, 1H), 5.71 (d,
J = 9.2 Hz, 1H), 4.71–4.64 (m, 2H), 4.32 (br s, 1H), 3.96 (t,
J = 9.2 Hz, 1H), 3.87 (d, J = 11.2 Hz, 2H), 3.80 (dd, J = 3.6, 10.4 Hz,
1H), 3.76 (d, J = 5.2 Hz, 1H), 3.37–3.66 (m, 3H), 3.59 (t, J = 9.2 Hz,
1H), 1.38 (s, 9H); 13C NMR (100 MHz, D2O): d = 176.6, 173.8,
160.0, 146.5, 126.8, 89.9, 84.0, 81.3, 78.4, 74.8, 70.1, 65.8, 62.9,
59.9, 56.7, 56.5, 32.1, 30.1, 19.3; HRMS (m/z): calcd for
4.3.4. (2S,3S)-2-((tert-Butoxycarbonyl)amino)-3-((1-
(((2R,3S,4S,5R,6S)-3,4,5-tris(benzyloxy)-6-methoxytetrahydro-
2H-pyran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)butanoic
acid (16)
From compound 8 (113 mg, 0.1 mmol) afforded 16 as a white
powder (101 mg, 92%). Rf = 0.7 (EtOAc/EtOH; 8:1). [
a
]
D = À0.3
(c 0.1, CH3OH); 1H NMR (400 MHz, DMSO-d6): d = 8.00 (s, 1H),
7.41–7.29 (m, 15H), 6.26 (d, J = 8.4 Hz, 1H), 4.90 (d, J = 11.2 Hz,
1H), 4.78 (t, J = 12.0, 1H), 4.66–4.62 (m, 1H), 4.58–4.43 (m, 3H),
4.15 (s, 1H), 4.07 (dd, J = 3.2, 9.6 Hz, 1H), 3.98 (d, J = 8.0, 2H),
3.91–3.84 (m, 3H), 3.53 (dd, J = 8.0, 9.6 Hz, 1H), 3.45 (dd, J = 6.8,
14.0 Hz, 1H), 3.33 (br s, 4H), 2.93 (s, 3H), 1.39 (s, 9H), 1.09 (d,
J = 5.6, 3H); 13C NMR (100 MHz, DMSO-d6): d = 172.1, 155.6,
144.1, 138.7, 138.6, 128.2, 128.1, 127.5, 127.4, 127.3, 124.5, 97.6,
78.3, 77.8, 75.5, 75.0, 74.0, 71.6, 69.2, 61.8, 58.0, 54.3, 50.5, 28.1,
16.4; HRMS (m/z): calcd for C40H51N4O10+H: 747.3605, found:
747.3608.
C17H29N4O10ÀH: 447.1727, found: 447.1735.
4.3. General procedure for the debenzylation
To a solution of benzyl ester in MeOH (15 mL), was added 10%
Pd/C (5 wt %). The mixture was stirred vigorously under hydrogen
atmosphere for 20 min. Then the mixture was filtered and concen-
trated in vacuum to afford the unique product without further
purification.
4.3.1. (R)-2-((tert-Butoxycarbonyl)amino)-3-((1-
(((2R,3R,4S,5R,6S)-3,4,5-tris(benzyloxy)-6-methoxytetrahydro-
2H-pyran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)
propanoic acid (13)
4.3.5. (R)-2-((tert-Butoxycarbonyl)amino)-3-((1-
(((2R,3R,4S,5S,6S)-3,4,5-tris(benzyloxy)-6-methoxytetrahydro-
2H-pyran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)
propanoic acid (17)
From compound 5 (121 mg, 0.2 mmol), afforded 13 as a white
powder (92 mg, 85%). Rf = 0.7 (EtOAc/EtOH; 8:1). [
a
]
D = À15.7
From compound 9 (134 mg, 0.2 mmol) afforded 17 as a white
(c 0.1, CH3OH); 1H NMR (400 MHz, DMSO-d6): d = 1.38 (s, 9H),
3.04 (s, 3H), 3.35–3.30 (m, 3H), 3.48–3.43 (m, 3H), 3.63 (d, J = 5.2,
3H), 3.80 (t, J = 9.6 Hz, 1H), 3.87 (t, J = 8.0 Hz, 1H), 4.08 (br s, 1H),
4.63–4.49 (m, 3H), 4.88–4.78 (m, 2H), 6.78 (d, J = 7.6 Hz, 1H),
7.35–7.30 (m, 15H), 8.03 (s, 1H); 13C NMR (100 MHz, DMSO-d6):
d = 28.1, 50.3, 53.8, 54.3, 63.5, 68.8, 69.0, 71.4, 74.0, 74.5, 78.2,
78.4, 79.4, 81.0, 96.7, 125.0, 127.4, 127.5, 127.6, 127.8, 128.1,
128.2, 128.3, 138.1, 138.3, 138.5, 143.5, 155.3, 171.9; HRMS
(m/z): calcd for C39H48N4O10+H: 733.3449, found: 733.3446.
powder (111 mg, 93%). Rf = 0.7 (EtOAc/EtOH; 8:1). [
a
]
D = À5.9
(c 0.2, CH3OH); 1H NMR (400 MHz, DMSO-d6): d = 8.06 (s, 1H),
7.39–7.23 (m, 15H), 6.83 (d, J = 8.0 Hz, 1H), 4.87 (d, J = 11.2 Hz,
1H), 4.78 (t, J = 12.0 Hz, 1H), 4.64–4.50 (m, 3H), 4.13–4.09 (m,
1H), 3.89 (t, J = 2.8 Hz, 1H), 3.84–3.76 (m, 2H), 3.63–3.58 (m, 4H),
3.43 (dd, J = 6.8, 14.0 Hz, 2H), 3.33 (br s, 5H), 3.01 (s, 3H), 1.38 (s,
9H); 13C NMR (100 MHz, DMSO-d6): d = 171.9, 155.3, 143.4,
138.3, 138.2, 128.2, 128.1, 127.6, 127.5, 125.1, 98.0, 79.0, 78.2,
75.0, 74.1, 74.0, 71.9, 70.4, 70.0, 69.0, 63.5, 56.1, 54.1, 53.8, 50.4,
28.1, 27.8; HRMS (m/z): calcd for C39H49N4O10+H: 733.3449, found:
733.3453.
4.3.2. (2S,3S)-2-((tert-Butoxycarbonyl)amino)-3-((1-
(((2R,3R,4S,5R,6S)-3,4,5-tris(benzyloxy)-6-methoxytetrahydro-
2H-pyran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)butanoic
acid (14)
4.3.6. (2S,3S)-2-((tert-Butoxycarbonyl)amino)-3-((1-
(((2R,3R,4S,5S,6S)-3,4,5-tris(benzyloxy)-6-methoxytetrahydro-
2H-pyran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)butanoic
acid (18)
From compound 6 (111 mg, 0.1 mmol) afforded 14 as a white
powder (98 mg, 99%). Rf = 0.7 (EtOAc/EtOH; 8:1). [
a
]D = À11.7
(c 0.1, CH3OH); 1H NMR (400 MHz, DMSO-d6): d = 7.99 (s, 1H),
7.38–7.26 (m, 15H), 6.31 (d, J = 8.4 Hz, 1H), 4.89–4.78 (m, 2H),
4.64–4.47 (m, 3H), 3.99 (d, J = 6.8 Hz, 2H), 3.89–3.84 (m, 1H),
3.80 (t, J = 9.2 Hz, 1H), 3.48–3.42 (m, 5H), 3.35–3.30 (m, 5H), 3.05
(s, 3H), 1.39 (s, 9H), 1.07 (dd, J = 6.0, 12.8 Hz, 3H); 13C NMR
(100 MHz, DMSO-d6): d = 172.1, 155.7, 144.1, 138.5, 138.3, 138.1,
128.3, 128.2, 128.1, 127.8, 127.6, 127.5, 127.4, 124.8, 96.7, 81.0,
79.4, 78.3, 74.5, 74.0, 73.9, 71.4, 68.8, 61.7, 58.0, 56.0, 54.4, 50.3,
28.1, 16.4; HRMS (m/z): calcd for C40H51N4O10+H: 747.3605, found:
747.3608.
From compound 10 (128 mg, 0.2 mmol) afforded 18 as a white
powder (105 mg, 92%). Rf = 0.5 (EtOAc/EtOH; 6:1). [
a
]
D = À0.6
(c 0.1, CH3OH); 1H NMR (400 MHz, DMSO-d6): d = 8.00 (s, 1H),
7.41–7.29 (m, 15H), 6.26 (d, J = 8.4 Hz, 1H), 4.86 (d, J = 11.2 Hz,
1H), 4.78 (s, 1H), 3.88 (s, 1H), 4.68 (d, J = 10.8 Hz, 1H), 4.64 (t,
J = 5.2, 3H), 4.57–4.51 (m, 3H), 4.44 (d, J = 12.4 Hz, 1H), 3.98 (d,
J = 6.4 Hz, 2H), 3.83–3.75 (m, 2H), 3.62–3.37 (m, 4H), 3.02 (s, 3H),
1.38 (s, 9H), 1.08 (d, J = 5.6 Hz, 3H); 13C NMR (100 MHz, DMSO-
d6): d = 172.2, 155.7, 144.1, 138.3, 138.2, 128.2, 128.1, 127.8,
127.6, 127.5, 124.8, 98.0, 80.0, 78.3, 75.0, 74.0, 73.9, 71.8, 70.4,
70, 61.7, 58.1, 54.2, 50.4, 28.1, 16.4; HRMS (m/z): calcd for
4.3.3. (R)-2-((tert-Butoxycarbonyl)amino)-3-((1-
(((2R,3S,4S,5R,6S)-3,4,5-tris(benzyloxy)-6-methoxytetrahydro-
2H-pyran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)
propanoic acid (15)
C40H51N4O10+H: 747.3605, found: 747.3608.
4.3.7. (R)-2-((tert-Butoxycarbonyl)amino)-3-((1-
((2R,3R,4S,5R,6R)-3,4,5-tris(benzyloxy)-6-((benzyloxy)
methyl)tetrahydro-2H-pyran-2-yl)-1H-1,2,3-triazol-4-
yl)methoxy)propanoic acid (19)
From compound 7 (105 mg, 0.1 mmol) afforded 15 as a white
powder (70 mg, 81%). Rf = 0.6 (EtOAc/EtOH; 8:1).
[a]
D = À3.3
(c 0.2, CH3OH); 1H NMR (400 MHz, DMSO-d6): d = 8.04 (s, 1H),
7.41–7.28 (m, 15H), 6.66 (d, J = 6.8 Hz, 1H), 4.89 (d, J = 11.2, 1H),
4.78 (t, J = 12.0 Hz, 1H), 4.66–4.61 (m, 1H), 4.51–4.46 (m, 3H),
4.15 (s, 1H), 4.07–4.02 (m, 3H), 3.90–3.86 (m, 2H), 3.69–3.60 (m,
3H), 3.52–3.34 (m, 5H), 2.91 (s, 3H), 1.38 (s, 9H); 13C NMR
From compound 11 (166 mg, 0.2 mmol) afforded 19 as a white
powder (139 mg, 93%). Rf = 0.8 (EtOAc/EtOH; 6:1). [
a
]D = À0.7
(c 0.6, CH3OH); 1H NMR (400 MHz, DMSO-d6): d = 8.49 (s, 1H),
7.32–7.30 (m, 13H), 7.21–7.19 (m, 5H), 6.92 (d, J = 8.0 Hz, 1H),
6.87 (d, J = 2.8 Hz, 2H), 5.92 (d, J = 9.2 Hz, 1H), 4.85 (s, 2H), 4.77

X.-P. He et al. / Bioorg. Med. Chem. 19 (2011) 3892–3900
3899
(d, J = 10.8 Hz, 1H), 4.61–4.56 (m, 3H), 4.52–4.43 (m, 3H), 4.17 (t,
J = 9.2 Hz, 2H), 4.00–3.91 (m, 3H), 3.71–3.49 (m, 7H), 1.35 (s,
9H); 13C NMR (100 MHz, DMSO-d6): d = 172.2, 155.7, 144.1,
138.3, 138.2, 128.2, 128.1, 127.8, 127.6, 127.5, 124.8, 98.0, 80.0,
78.3, 75.0, 74.0, 73.9, 71.8, 70.4, 70.0, 61.7, 58.1, 54.2, 50.4, 28.1;
HRMS (m/z): calcd for C45H53N4O10+H: 809.3762, found: 809.3765.
A range of concentrations of the test compounds were added and
the plate was incubated at 37 °C for 72 h before 40 L MTT
(5 mg/mL)/well was added. After 3 h incubation, the medium was
removed and 100 L DMSO was added to each well. The absor-
bance was measured on SpectraMax 340 microplate reader at
550 nm with a reference at 690 nm. The optical density of the re-
sult in MTT assay was directly proportional to the number of viable
cells.
l
l
4.3.8. (2S,3S)-2-((tert-Butoxycarbonyl)amino)-3-((1-
((2R,3R,4S,5R,6R)-3,4,5-tris(benzyloxy)-6-((benzyloxy)
methyl)tetrahydro-2H-pyran-2-yl)-1H-1,2,3-triazol-4-
yl)methoxy)butanoic acid (20)
4.7. Docking simulation
From compound 12 (125 mg, 0.1 mmol) afforded 20 as a white
The docking simulation was initiated with a crystal structure in
complex with a reference ligand (for PTP1B, PDB code: 3EB19b and
for CDC25B, PDB code: 1QB05c). Water was removed from the ori-
ginal structure and the rest protein was prepared using the Protein
preparation wizard (Schrödinger, LLC, New York, NY, 2005). Then
compound 19 was docked to the active site of the protein using
the Induced Fit Docking workflow (Schrödinger, LLC, New York,
NY, 2005). The center atom was set to be a virtual center of refer-
enced key residues: Phe182, Cys215 and Gly259 (PTP1B), and
Cys473, Glu474, Glu478, and Met531 (CDC25B).
powder (106 mg, 94%). Rf = 0.6 (EtOAc/EtOH; 6:1). [
a
]D = À0.6 (c
0.2, CH3OH); 1H NMR (400 MHz, DMSO-d6): d = 8.00 (s, 1H),
7.34–7.27 (m, 20H), 6.45 (d, J = 9.2 Hz, 1H), 4.86 (d, J = 11.6 Hz,
1H), 4.82 (d, J = 10.8 Hz, 1H), 4.77 (d, J = 3.2 Hz, 1H), 4.73–4.69
(m, 2H), 4.66–4.58 (m, 3H), 4.55–4.46 (m, 3H), 4.03–3.94 (m,
2H), 3.88–3.83 (m, 1H), 3.79 (t, J = 9.2, Hz, 1H), 3.45 (dd, J = 3.6,
9.6 Hz, 1H), 3.04 (s, 3H), 1.35 (s, 9H), 1.10 (s, 3H); 13C NMR
(100 MHz, DMSO-d6): d = 172.1, 155.8, 144.2, 138.3, 128.3, 128.2,
128.1, 127.8, 127.6, 127.5, 124.8, 96.7, 81.0, 79.4, 78.4, 78.3, 74.5,
74.0, 71.4, 68.8, 61.8, 58.1, 54.4, 50.3, 28.1, 16.5.
Acknowledgments
4.4. Inhibitory assay
Project supported by National Natural Science Foundation of
China (Grant Nos. 20876045 and 30801405), National Basic Re-
search Program of China (No. 2007CB914201), Shanghai Science
and Technology Community (No. 10410702700), Chinese Academy
of Sciences (No. KSCX2-EW-R-15) and the Fundamental Research
Funds for the Central Universities (No. WK1013002). X.-P. also
gratefully acknowledges the French Embassy in PR China for a
co-tutored doctoral fellowship.
The recombinant human PTP1B catalytic domain was expressed
and purified according to procedures described previously.17a Enzy-
matic activity of PTP1B was determined at 30 °C by monitoring the
hydrolysis of pNPP. Dephosphorylation of pNPP generates product
pNP, which can be monitored at 405 nm. In a typical 100 lL assay,
mixture containing 50 mM MOPS, pH 6.5, 2 mM pNPP and recombi-
nant enzymes, PTP1B activities were continuously monitored on a
SpectraMax 340 microplate reader at 405 nm for 2 min at 30 °C
and the initial rate of the hydrolysis was determined using the early
linear region of the enzymatic reaction kinetic curve. For calculating
IC50, inhibition assays were performed with 30 nM recombinant en-
zyme, 2 mM pNPP in 50 mM MOPS at pH 6.5, and the inhibitors di-
luted around the estimated IC50 values. IC50 was calculated from
the nonlinear curve fitting of percent inhibition (inhibition (%)) ver-
sus inhibitor concentration [I] by using the following equation: inhi-
bition (%) = 100/{1 + (IC50/[I])k}, where k is the Hill coefficient. To
study the inhibition on other PTP family members, human CDC25A,
CDC25B, TCPTP, SHP-1, SHP-2, and LARD1 were prepared and assays
were performed according to procedures described previously.17b
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.05.049.
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