Preparation of triazole-linked glycosylated α-ketocarboxylic acid derivatives as new PTP1B inhibitors

Article abstract of DOI:10.1016/j.carres.2010.10.023

The synthesis of triazole-linked glycosyl acetophenone, benzoic acid, and α-ketocarboxylic acid derivatives was readily achieved via Cu(I)-catalyzed azide-alkyne cycloaddition ('click' reaction) in excellent yields of 93-97%. Among the synthesized glycoco

Full text of DOI:10.1016/j.carres.2010.10.023

Carbohydrate Research 346 (2011) 140–145
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Preparation of triazole-linked glycosylated
as new PTP1B inhibitors
a
-ketocarboxylic acid derivatives
b,
Zhuo Song a, Xiao-Peng He c,d, Cui Li d, Li-Xin Gao b, Zhao-Xia Wang a, Yun Tang d, Juan Xie c, , Jia Li
⇑
⇑
,
Guo-Rong Chen a,
⇑
a Key Laboratory for Advanced Material and Fine Chemicals, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, PR China
b National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, PR China
c PPSM, Institut d’Alembert, ENS de Cachan, CNRS UMR 8531, 61 Avenue du Pt Wilson, F-94235 Cachan, France
d School of Pharmacy, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of triazole-linked glycosyl acetophenone, benzoic acid, and
tives was readily achieved via Cu(I)-catalyzed azide–alkyne cycloaddition (‘click’ reaction) in excellent
a-ketocarboxylic acid deriva-
Received 19 September 2010
Received in revised form 21 October 2010
Accepted 26 October 2010
Available online 30 October 2010
yields of 93–97%. Among the synthesized glycoconjugates, the triazolyl -ketocarboxylic acids were iden-
a
tified as the most potent protein tyrosine phosphatase 1B (PTP1B) inhibitors with micromole-ranged IC50
values and moderate-to-good selectivity over a panel of homologous PTPs including TCPTP (4.6-fold), LAR
(>30-fold), SHP-1 (>30-fold) and SHP-2 (>30-fold). Moreover, a docking simulation was conducted to pro-
Keywords:
pose a plausible binding mode of the glucosyl
a
-ketocarboxylic acid triazole with the enzymatic target.
a-Ketocarboxylic acid
Ó 2010 Elsevier Ltd. All rights reserved.
Glycoconjugate
Click reaction
PTP1B inhibitor
Protein tyrosine phosphatases (PTPs) are regulators of tyrosine
phosphorylation-dependent cellular events that govern numerous
critical physiological processes.1 Among this large superfamily,
protein tyrosine phosphatase 1B (PTP1B) represents one of the best
therapeutically characterized enzymatic members and is related to
several major human diseases. For example, PTP1B-knockout mice
could lead to the increase of insulin sensitivity and resistance to
diet-induced obesity while treatment with PTP1B antisense oligo-
nucleotides resulted in the improvement of hyperglycemia in dia-
betes mice models.2,3a In addition, more recent reports indicated
that PTP1B also performs as an oncogene in the context of breast
cancer.3b These data suggest that inhibition of PTP1B may repre-
sent a practical strategy for the treatment of type 2 diabetes, obes-
ity, as well as breast cancer.
have provided considerable leading compounds for the structure or
fragment based approaches to derive other novel and potent PTP1B
inhibitors. The
a-ketocarboxylic acid, bearing a fundamental car-
boxylic acid structure and one additional ketone group, was recog-
nized as a promising pTyr mimetic and its derivatives have
recently been identified as favorable PTP inhibitors.11
Carbohydrate-based drug discovery has lately attracted consid-
erable interest due to its structural diversity, natural abundance,
and high biocompatibility.12 In addition, numerous glycosylated
bioactive triazolyl compounds formed via click chemistry have
been reported in the past decade.13 With a continuing interest in
preparing sugar-based inhibitors of negative enzymatic regulators
associated with major human diseases,14 we sought to synthesize
triazole-linked glycosyl
a
-ketocarboxylic acid derivatives as new
Consequently, increasing efforts have been made toward the
development of PTP1B inhibitors by both academia and the phar-
maceutical industry.4 Most of the reported bioactive compounds
fall into isosteric phosphotyrosine (pTyr) surrogates that competi-
tively target the PTP1B active site.5 These nonhydrolyzable
phosphonate and carboxylic acid mimetics including difluoro-
methylene phophonates (DFMP),6 N-aryl oxamic acid,7 isoxazole
acid,8 2-oxalylamino benzoic acid,9 and tyrosine acid derivatives10
OH
O
N
N
N
O
A
: IC50 = 84 µM (PTP1B)
⇑
Corresponding authors. Tel./fax: +86 21 64715047 (G.-R.C.).
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).
Figure 1. Aryl a
-ketocarboxylic acid-based PTP1B inhibitor A.11
0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.10.023

Z. Song et al. / Carbohydrate Research 346 (2011) 140–145
141
PTP1B inhibitors. Meanwhile, glycosyl acetophenone and benzoic
acid derivatives were also prepared for SAR (structure–activity
glycosyl esters in excellent yields of 95% (12), 97% (13), 94% (16)
and 94% (17), respectively. Saponification with LiOH finally gave
the free acids 14, 15, 18, and 19. Notably, the relatively low yields
relationship) study. Recently, an
with a distal biphenyl moiety (Compound A, Fig. 1) has been re-
ported to possess moderate PTP1B inhibitory activity (84
M).11
a-ketocarboxylic acid triazole
of the obtained glycosyl
a-ketocarboxylic acids (52% for 15 and
l
50% for 19) was most likely rendered by partial conversion of the
Meanwhile, it is also well-noted that aryl moieties covalently cou-
pled with the active site-oriented precursor may simultaneously
generate hydrophobic interactions with periphery enzyme surface
(such as the aryl second phosphotyrosine site or YRD motif) of
PTP1B.14a,15 We consequently chose to use benzylated 1-O-propar-
gyl or 6-O-propargyl glucosides as glycodonors to comparatively
investigate whether such an aryl sugar scaffold would lead to
enhanced affinity.
ketoacids into carboxylic acids in basic condition.19
Next, the inhibitory activity of the prepared glycosides (10–19)
as well as the azido
PTP1B. The recombinant human PTP1B was first expressed and
purified. In a typical 100 L assay, a mixture containing 50 mM
a-ketocarboxylic acid 7 was assayed toward
l
MOPS, pH 6.5, 2 mM pNPP and recombinant enzymes, PTP1B activ-
ities were continuously monitored on a SpectraMax 340 microplate
reader at 405 nm for 2 min at 30 °C and the initial rate of the hydro-
lysis was determined using the early linear region of the enzymatic
reaction kinetic curve. IC50 was calculated from the nonlinear curve
fitting of percent inhibition (inhibition (%)) versus inhibitor concen-
tration [I] by using the following equation: inhibition (%) = 100/
{1 + (IC50/[I])k}, where k is the Hill coefficient.20a
As shown in Scheme 1, 1-(4-azidophenyl)ethanone 4,16 methyl
4-azidobenzoate 516 and methyl 2-(4-azidophenyl)-2-oxoacetate
611 were used as the azido reactants while the O-propargyl glyco-
syl donors 8 and 9 were prepared according to the known litera-
ture reports.17 Furthermore, the 2-(4-azidophenyl)-2-oxoacetic
acid 711 was also synthesized for inhibitory assay.
As listed in Table 1, the azido ketoacid 7 (entry 1) was first ob-
served to have moderate inhibitory activity with IC50 va-
The Cu-catalyzed azide–alkyne cycloaddition (click reaction)18
was achieved in the presence of sodium ascorbate and CuSO4Á5H2O
in CH2Cl2/H2O (1:1, v/v) solvent mixture. As illustrated in Scheme
1, azide 4 was first coupled with propargyl 8 and 9, which favor-
ably yielded the desired triazole-linked glycoconjugates 10 and
11 in yields of 96% and 93%, respectively. The click reaction of azido
esters 5 and 6 with alkynes 8 and 9 was then realized to afford the
lue = 30.2
11 (entry 3), lacking the carboxylic acid functionality, weakly
inhibited PTP1B (about 50% inhibition at 100 g/mL). In addition,
lM. The glycosylated acetophenones 10 (entry 2) and
l
both glucosides substituted on 6 position by triazolyl benzoic ester
12 (entry 4) and benzoic acid 14 (entry 6) showed weak inhibition
whereas the glucoside 6-substituted by
a-keto ester 13 (entry 5)
N
N
N
O
O
R
4: R = CH3
O
O
5: R = OCH3
a
O
O
4 + BnO
BnO
BnO
BnO
6: R = CO2CH3
7: R = CO2H
BnO
BnO
N3
OMe
OMe
8
10
(96%)
N
N
N
OBn
OBn
O
a
O
BnO
BnO
BnO
BnO
4 +
O
O
O
OBn
OBn
11
9
(93%)
R
O
R
N
N
N
N
N
N
O
O
a
O
b
5 or 6 + 8
O
BnO
O
BnO
BnO
BnO
BnO
BnO
OMe
OMe
12: R = OCH3 (95%)
13: R = CO2CH3 (97%)
14: R = OH (88%)
15: R = CO2H (52%)
N
N
N
N
N
N
OBn
O
OBn
O
a
b
5 or 6 + 9
BnO
BnO
R
BnO
BnO
R
O
O
OBn
O
OBn
O
16
17
: R = OCH3 (94%)
: R = CO2CH3 (94%)
18
: R = OH (88%)
19: R = CO2H (50%)
Scheme 1. Reagents and conditions: (a) Na ascorbate, CuSO4Á5H2O in CH2Cl2/H2O; (b) LiOH in H2O/MeOH.

142
Z. Song et al. / Carbohydrate Research 346 (2011) 140–145
Table 1
A plausible binding mode of the triazole-linked glucosyl a-keto-
Inhibitory activity of 7 and 10–19 on PTP1Ba
carboxylic acid 15 with PTP1B was then proposed via docking sim-
ulation by starting with a crystal structure in complex with a
reference ligand (PDB code: 3EB1, resolution: 2.40 Å).15 Water
was removed from the original structure, and the rest of the pro-
tein was prepared using the ‘Protein preparation wizard’ workflow
in Maestro (Schrödinger, LLC, New York, NY, 2005). Hydrogens
were added, bond orders assigned, and overlaps treated. The im-
pref utility was run to perform restrained optimizations of the pro-
tein and the compound 15 was prepared using Ligprep 2.1
(Schrödinger, LLC, New York, NY, 2005). OPLS2005 Force field were
employed, and Epik used to perform the ionization followed by
chiralities correction. Then the compound 15 were 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 referenced key residues: Phe182, Cys215
and Gly259. The inhibitor was initially docked by Glide, then the
protein–inhibitor complex was minimized by Prime, and the com-
plex structure redocked by Glide within a specified energy of the
lowest-energy structure.
Entry
Compd
PTP1B
Inhibition rate (%)
IC50 (lM)
1
2
3
4
5
6
7
8
9
7
10
11
12
13
14
15
16
17
18
19
A
96.9
44.1
48.2
51.5
99.5
18.5
97.9
25.6
99.5
30.2
>100
>100
>100
12.7
>100
3.2
>100
12.3
11.1
5.6
10
11
12
100
99.3
—
84b
a
Data are means of three experiments at a concentration of 100
This IC50 value is cited in the Ref. 11.
l
g/mL.
b
displayed excellent inhibition (99.5%) and reasonable IC50 value
(12.7 M) on PTP1B. Its acid-free derivative 15 (entry 7) was then
l
As illustrated in Figure 2, the triazolyl
a-ketocarboxylic acid
determined to possess the best inhibitory potency among all
precursor coupled on the C6-position of the glucopyranoside
located well in the active site cleft of PTP1B, adopting a competi-
tive inhibition manner. Densely functionalized hydrogen bonds
were mainly made between the extended carboxylic acid group
and residues including Cys215, Ser216, Ala217, Gly218, Ile219,
and Gly220 of the catalysis pocket which stably fixed the small
molecule. Thus, the limited inhibition of the glycosyl benzoic acid
derivative 14 could be presumably ascribed to the lack of one addi-
tional ketone group toward the extension of carboxylic group to
the catalysis groove. Interestingly, one hydrogen bond was also
made between the nitrogen atom of the triazole and Gly183 while
the benzene moiety of Phe182 of the WPD loop covers the triazole
ring like a lid. This is quite different from the positioning manner of
pTyr substrate where the benzene moiety of Phe182 residue
chooses to cover the phosphorylated phenol moiety of the tyrosine
residue.15 Moreover, hydrophobic interaction of the C2-benzyl
assayed compounds and owns an almost 10-fold enhanced IC50 va-
lue (3.2 lM) comparing to the model compound 7.
Comparing to the glucosides 6-substituted by benzoic moieties
(12 and 14), the 1-substituted ester 16 (entry 8) similarly showed
no inhibitory activity on PTP1B, while the corresponding acid 18
(entry 10) displayed favorable inhibition (100%) with an IC50 value
of 11.1
lM. On the other hand, despite that the 1-substituted
a-ketoester 17 (entry 9) exhibited nearly equal IC50 value to that
of its 6-substituted counterpart (13), the inhibitory activity of the
corresponding 1-substituted acid 19 (entry 11) was slightly lower
(IC50 = 5.6
(IC50 = 3.2
l
l
M) than that of the 6-substituted acid 15
M). Furthermore, ketoacid 19 displayed an approxi-
mately twofold increased inhibitory activity on PTP1B comparing
to the carboxylic acid 18, unambiguously demonstrating that the
a-ketocarboxylic acids are more effective as a PTP1B inhibitor than
the corresponding benzoic acid derivatives.
In addition, by comparing the inhibitory activity of the reported
biphenyl ketoacid triazole (compound A, Table 1, entry 12) with
that of the identified glucosyl ketoacid triazoles 15 and 19, the glu-
cosyl compounds exhibited remarkably 26- and 15-fold increased
inhibitions. This clearly demonstrated that the natural glucosyl
scaffold employed in this study was advantageous as a chiral scaf-
fold for the enhancement of binding affinity toward PTP1B.
The specificity of the triazole-linked glucosyl
a-ketocarboxylic
acid derivatives 15 and 19 over a panel of homologous PTPs includ-
ing TCPTP (74% identity with PTP1B), LAR, SHP-1 and SHP-2 was
sequentially assessed using the method similar to that employed
for PTP1B.20b As shown in Table 2, whereas the 1-substituted gly-
coside 19 (entry 2) displayed almost identical IC50 value
(IC50 = 6.4
glycoside 15 showed 4.6-fold selectivity over TCPTP
(IC50 = 14.5 M on TCPTP and 3.2 M on PTP1B). In addition, glyco-
side 15 exhibited excellent selectivity over LAR, SHP-1, and SHP-2
(IC50 >100 M) while the glycoside 19 simultaneously displayed
low inhibitory activity on SHP-2 (IC50 = 55.4 M) with no inhibi-
M).
lM on TCPTP and 5.6 lM on PTP1B), the 6-substituted
l
l
l
l
tory potency on LAR and SHP-1 (IC50 >100
l
Table 2
Inhibitory activity of 15 and 19 on PTPsa
Figure 2. PTP1B in complex with compound 15. The surface of the protein was
shown as colored in property of electrostatic potential. The compound was shown
as green stick, and residues of the active site in PTP1B were shown as light grey line.
Nitrogen atoms are in blue and oxygen atoms in red. Hydrogen bonds were shown
as yellow dash line and all nonpolar hydrogens were hidden. (For interpretation of
the references to colour in this figure legend, the reader is referred to the web
version of this article.)
Entry
Compd
IC50
(
l
M)
PTP1B
TCPTP
LAR
SHP-1
SHP-2
1
2
15
19
3.2
5.6
14.5
6.4
>100
>100
>100
>100
>100
55.4
a
Data are means of three experiments at a concentration of 100 lg/mL.

Z. Song et al. / Carbohydrate Research 346 (2011) 140–145
143
group on sugar scaffold with Tyr20 of the second phosphotyrosine
site was also possibly generated for enhancing the binding affinity.
In summary, we have efficiently synthesized glycosylated ace-
(EtOAc/petroleum ether, 1:1); [
a
]
À1 (c 0.1 in CH2Cl2); 1H NMR
D
(400 MHz, CDCl3): d 8.03 (s, 1H), 8.02 (d, 2H, J = 8.0 Hz), 7.63 (d,
2H, J = 8.8 Hz), 7.34–7.26 (m, 18H), 7.17–7.15 (m, 2H), 5.09 (d,
1H, J = 13.6 Hz), 5.03 (d, 1H, J = 13.2 Hz), 4.94 (d, 1H, 11.2 Hz),
4.92 (d, 1H, 11.2 Hz), 4.83–4.78 (m, 3H), 4.62 (d, 1H, J = 12.4 Hz),
4.56–4.52 (m, 3H), 3.77 (dd, 1H, J = 1.6, 10.8 Hz), 3.71 (dd, 1H,
J = 4.8, 10.8 Hz), 3.66–3.63 (m, 2H), 3.53 (t, 1H, J = 8.0 Hz), 3.51–
3.48 (m, 1H), 2.64 (s, 3H); 13C NMR (100 MHz, CDCl3): d 196.6,
146.3, 140.0, 138.5, 138.0, 136.7, 130.0, 128.5, 128.4, 128.0,
127.9, 127.8, 127.7, 121.0, 119.9, 102.8, 84.7, 82.3, 77.8, 75.8,
75.1, 74.9, 74.8, 73.5, 69.0, 63.1, 26.7; ESIMS m/z: [M+H]+ calcd
for C45H45N3O7: 740.3336; found: 740.3333.
tophenone, benzoic acid, and
via Cu(I)-catalyzed 1,3-dipolar cycloaddition in excellent yields.
The glycosyl -ketocarboxylic acids were identified as promising
a-ketocarboxylic acid derivatives
a
sugar-based PTP1B inhibitors for the first time with at least several
fold selectivities over a panel of homologous PTPs. In addition,
docking study plausibly proposed a typical competitive inhibitory
manner of ketoacid 15 with the enzymatic target, suggesting that
both the triazolyl
a-ketocarboxylic acid precursor and the benzy-
lated glucosyl scaffold being the contributors. Our work may pro-
vide new insight toward the preparation of sugar scaffold-based
PTP1B inhibitors.
1.2.3. Methyl 2,3,4-tri-O-benzyl-6-O-[1-(4-(methoxycarbonyl)-
phenyl)-1H-1,2,3-triazol-4-ylmethyl]
a-
D-glucopyranoside (12)
From compound (71 mg, 0.40 mmol) and
0.40 mmol), column chromatography (EtOAc/petroleum ether,
5
8
(201 mg,
1. Experimental
1.1. General
1:2) afforded 12 as a yellow powder (260 mg, 95%). Rf = 0.35
(EtOAc/petroleum ether, 1:1); [a]
+40 (c 0.1 in CH2Cl2); 1H NMR
D
Solvents were purified by standard procedures. 1H and 13C NMR
spectra were recorded on Bruker JEOL DX-400 spectrometers in
CDCl3 solution. Optical rotations were measured using a Perkin–El-
mer 241 polarimeter at room temperature and a 10-cm 1-mL cell.
Analytical thin-layer chromatography was performed on E. Merck
aluminum percolated plates of Silica Gel 60F-254 with detection
by UV and by spraying with 6 N H2SO4 and heating about 2 min
at 300 °C. High resolution mass spectra (HRMS) were recorded on
a Waters LCT Premier XE spectrometer using standard conditions
(ESI, 70 eV). Analytical HPLC was measured using Agilent 1100 Ser-
ies equipment.
(400 MHz, CDCl3): d 8.17 (d, 2H, J = 8.4 Hz), 7.96 (s, 1H), 7.75 (d,
2H, J = 8.4 Hz), 7.36–7.20 (m, 15H), 4.98 (d, 1H, J = 10.8 Hz), 4.86
(d, 1H, J = 11.2 Hz), 4.83–4.77 (m, 3H), 4.69 (d, 1H, J = 12.4 Hz),
4.66 (d, 1H, J = 12.4 Hz), 4.62 (d, 1H, J = 3.6 Hz), 4.56 (d, 1H,
J = 10.8 Hz), 4.00 (t, 1H, J = 9.2 Hz), 3.95 (s, 3H), 3.83 (dd, 1H,
J = 3.6, 10.4 Hz), 3.79–3.74 (m, 2H), 3.62 (t, 1H, J = 9.2 Hz), 3.55
(dd, 1H, J = 3.6, 9.6 Hz), 3.38 (s, 3H); 13C NMR (100 MHz, CDCl3):
d 165.9, 146.2, 140.0, 138.8, 138.3, 138.2, 131.3, 130.2, 128.5,
128.4, 128.0, 127.9, 127.7, 127.6, 120.5, 119.9, 98.3, 82.1, 79.9,
75.8, 75.0, 73.4, 70.0, 69.1, 64.8, 55.3, 52.5; ESIMS m/z: [M+H]+
calcd for C39H41N3O8: 680.2972; found: 680.2984.
1.2.4. Methyl 2,3,4-tri-O-benzyl-6-O-[1-(4-(2-methoxy-2-
oxoacetyl)phenyl)-1H-1,2,3-triazol-4-yl methyl] a-D-
glucopyranoside (13)
1.2. General procedure for Cu(I)-catalyzed 1,3-dipolar
cycloaddition
From compound
0.40 mmol), column chromatography (EtOAc/petroleum ether,
1:2) afforded 13 as a yellow powder (274 mg, 97%). Rf = 0.42
(EtOAc/petroleum ether, 1:1); [a]
6 (82 mg, 0.40 mmol) and 8 (200 mg,
To a biphasic solution of alkynyl sugar (1 equiv) and azide
(1 equiv) in CH2Cl2 (5 mL) and H2O (5 mL), Na ascorbate (4 equiv)
and CuSO4Á5H2O (2 equiv) were added. Then the mixture was stir-
red vigorously at rt. for 6 h. After completion of the reaction, the
resulting mixture was diluted with CH2Cl2, washed with water,
dried over MgSO4, filtered, and evaporated to give a crude residue
which was purified by column chromatography.
+55 (c 0.1 in CH2Cl2); 1H NMR
D
(400 MHz, CDCl3): d 8.20 (d, 2H, J = 8.4 Hz), 8.00 (s, 1H), 7.84 (d,
2H, J = 8.8 Hz), 7.35–7.22 (m, 15H), 4.99 (d, 1H, J = 10.8 Hz), 4.87
(d, 1H, J = 11.2 Hz), 4.82–4.77 (m, 3H), 4.70 (d, 1H, J = 12.0 Hz),
4.66 (d, 1H, J = 12.0 Hz), 4.62 (d, 1H, J = 3.6 Hz), 4.56 (d, 1H,
J = 11.2 Hz), 4.00 (t, 1H, J = 9.2 Hz), 4.00 (s, 3H), 3.84 (dd, 1H,
J = 4.0, 10.8 Hz), 3.80–3.74 (m, 2H), 3.62 (t, 1H, J = 9.2 Hz), 3.55
(dd, 1H, J = 3.6, 9.6 Hz), 3.38 (s, 3H); 13C NMR (100 MHz, CDCl3):
d 184.1, 163.3, 146.5, 141.2, 138.8, 138.3, 138.2, 132.2, 132.0,
128.5, 128.4, 128.1, 128.0, 127.9, 127.7 (1), 127.7 (2), 127.6,
120.6, 120.1, 98.3, 82.1, 79.9, 75.8, 74.9, 73.4, 70.0, 69.2, 64.8,
55.3, 53.1; ESIMS m/z: [M+H]+ calcd for C40H41N3O9: 708.2921,
found: 708.2927.
1.2.1. Methyl 2,3,4-tri-O-benzyl-6-O-[1-(4-acetylphenyl)-1H-
1,2,3-triazol-4-ylmethyl]
From compound
a
-
D
-glucopyranoside (10)
4
(58 mg, 0.36 mmol) and
8 (150 mg,
0.30 mmol), column chromatography (EtOAc/petroleum ether,
1:2) afforded 10 as a yellow powder (191 mg, 96%). Rf = 0.52
(EtOAc/petroleum ether, 1:1); [a]
+12 (c 0.1 in CH2Cl2); 1H NMR
D
(400 MHz, CDCl3): d 8.10 (d, 2H, J = 8.8 Hz), 7.97 (s, 1H), 7.79 (d,
2H, J = 8.8 Hz), 7.36–7.20 (m, 15H), 4.98 (d, 1H, J = 10.8 Hz), 4.87
(d, 1H, J = 10.8 Hz), 4.82–4.77 (m, 3H), 4.70 (d, 1H, J = 14.4 Hz),
4.66 (d, 1H, J = 12.4 Hz), 4.61 (d, 1H, J = 3.6 Hz), 4.55 (d, 1H,
J = 11.2 Hz), 4.00 (t, 1H, J = 9.2 Hz), 3.83 (dd, 1H, J = 3.6, 10.4 Hz),
3.79–3.74 (m, 2H), 3.62 (t, 1H, J = 9.2 Hz), 3.55 (dd, 1H, J = 3.2,
9.6 Hz), 3.38 (s, 3H), 2.66 (s, 3H); 13C NMR (100 MHz, CDCl3):
d = 196.3, 146.2, 140.0, 138.3, 138.2, 136.8, 130.1, 128.5, 128.4,
128.0, 127.9, 127.7, 127.6, 120.6, 120.0, 98.3, 82.1, 79.9, 75.8,
75.0, 73.4, 70.0, 69.2, 64.8, 55.3, 26.7; ESIMS m/z: [M+H]+ calcd
for C39H41N3O7: 664.3023; found: 664.3038.
1.2.5. 1-(4-(Methoxycarbonyl)phenyl)-1H-1,2,3-triazol-4-
ylmethyl 2,3,4,6-tetra-O-benzyl
From compound (55 mg, 0.31 mmol) and
0.26 mmol), column chromatography (EtOAc/petroleum ether,
1:2) afforded 16 as a white powder (196 mg, 94%). Rf = 0.33
(EtOAc/petroleum ether, 1:1); [
(400 MHz, CDCl3): d 8.11 (d, 2H, J = 8.4 Hz), 8.01 (s, 1H), 7.60 (d,
2H, J = 8.4 Hz), 7.34–7.26 (m, 18H), 7.17–7.15 (m, 2H), 5.09 (d,
1H, J = 13.2 Hz), 5.03 (d, 1H, J = 13.2 Hz), 4.93 (d, 2H, J = 12.0 Hz),
4.83–4.80 (m, 3H), 4.62 (d, 1H, J = 12.0 Hz), 4.54 (m, 3H), 3.96 (s,
3H), 3.76–3.69 (m, 2H), 3.67–3.61 (m, 2H), 3.52 (t, 1H, J = 8.0 Hz),
3.50–3.48 (m, 1H); 13C NMR (100 MHz, CDCl3): d 165.9, 146.2,
140.0, 138.4, 137.9, 131.2, 130.0, 128.5, 128.4, 128.0, 127.9,
127.8, 127.7, 120.9, 119.7, 102.7, 84.7, 82.3, 77.8, 75.0, 74.8, 73.5,
a
-D
-glucopyranoside (16)
(150 mg,
5
9
a]
À4 (c 0.3 in CH2Cl2); 1H NMR
D
1.2.2. 1-(4-Acetylphenyl)-1H-1,2,3-triazol-4-ylmethyl 2,3,4,6-
tetra-O-benzyl
a-
D-glucopyranoside (11)
From compound
4
(24 mg, 0.15 mmol) and
9 (80 mg,
0.14 mmol), column chromatography (EtOAc/petroleum ether,
1:2) afforded 11 as a white powder (95 mg, 93%). Rf = 0.36

144
Z. Song et al. / Carbohydrate Research 346 (2011) 140–145
68.9, 63.0, 52.4; ESIMS m/z: [M+Na]+ calcd for C45H45N3O8:
778.3104; found: 778.3111.
ded 18 as a white powder (48 mg, 88%). Rf = 0.73 (CH2Cl2/MeOH,
7:1); [
a]
À16 (c 0.1 in CH2Cl2); 1H NMR (400 MHz, CDCl3): d
D
8.08 (d, 2H, J = 8.8 Hz), 7.96 (s, 1H), 7.55 (d, 2H, J = 8.8 Hz), 7.26–
7.17 (m, 18H), 7.10–7.07 (m, 2H), 5.04 (d, 1H, J = 13.6 Hz), 4.98
(d, 1H, J = 13.6 Hz), 4.88 (d, 1H, J = 11.2 Hz), 4.86 (d, 1H,
J = 11.2 Hz), 4.76–4.72 (m, 3H), 4.55 (d, 1H, J = 12.0 Hz), 4.47 (d,
1H, J = 12.0 Hz), 4.50–4.44 (m, 2H), 3.70 (d, 1H, J = 9.6 Hz), 3.66
(d, 1H, J = 8.8 Hz), 3.63–3.56 (m, 2H), 3.45 (t, 1H, J = 8.0 Hz),
3.45–3.42 (m, 1H); 13C NMR (100 MHz, CDCl3): d 168.8, 145.2,
139.4, 137.4, 137.3, 136.9, 136.8, 130.8, 128.3, 127.5, 127.4,
127.3, 126.9, 126.8, 126.7, 126.6, 120.0, 118.7, 101.6, 83.7, 81.3,
76.8, 74.7, 74.0, 73.9, 73.7, 72.5, 67.9, 61.9; ESIMS m/z: [M+H]+
calcd for C44H43N3O8: 742.3128, found: 742.3126.
1.2.6. 1-(4-(2-Methoxy-2-oxoacetyl)phenyl)-1H-1,2,3-triazol-4-
ylmethyl 2,3,4,6-tetra-O-benzyl-
a
-
D
-glucopyranoside (17)
(208 mg,
From compound (80 mg, 0.40 mmol) and
6
9
0.36 mmol), column chromatography (EtOAc/petroleum ether,
1:2) afforded 17 as a yellow powder (264 mg, 94%). Rf = 0.27
(EtOAc/petroleum ether, 1:1); [
a]
À3 (c 0.1 in CH2Cl2); 1H NMR
D
(400 MHz, CDCl3): d 8.09 (d, 2H, J = 8.8 Hz), 8.07 (s, 1H), 7.65 (d,
2H, J = 8.8 Hz), 7.36–7.23 (m, 18H), 7.18–7.15 (m, 2H), 5.09 (d,
1H, J = 13.6 Hz), 5.04 (d, 1H, J = 13.6 Hz), 4.94 (d, 2H, J = 10.8 Hz),
4.84–4.79 (m, 3H), 4.62 (d, 1H, J = 12.0 Hz), 4.56–4.53 (m, 3H),
4.00 (s, 3H), 3.78 (dd, 1H, J = 2.8, 10.8 Hz), 3.72 (dd, 1H, J = 4.8,
10.8 Hz), 3.67 (d, 1H, J = 8.8 Hz), 3.63 (d, 1H, J = 8.8 Hz), 3.54 (t,
1H, J = 8.0 Hz), 3.53–3.49 (m, 1H); 13C NMR (100 MHz, CDCl3): d
184.0, 163.3, 146.5, 138.0, 127.8, 121.0, 119.9, 102.8, 77.8, 74.9,
74.8, 73.5, 69.0, 63.1, 53.1; ESIMS m/z: [M+Na]+ calcd for
C46H45N3O9: 806.3054, found: 806.3051.
1.3.4. 1-(4-(Carboxycarbonyl)phenyl)-1H-1,2,3-triazol-4-
ylmethyl 2,3,4,6-tetra-O-benzyl a-D-glucopyranoside (19)
From compound 17 (96 mg, 0.12 mmol) and LiOH (5 mg,
0.12 mmol), column chromatography (CH2Cl2/MeOH, 10:1) affor-
ded 19 as a white powder (47 mg, 50%). Rf = 0.21 (CH2Cl2/MeOH,
7:1); [
a]
À16 (c 0.1 in CH2Cl2); 1H NMR (400 MHz, CDCl3): d
D
1.3. General procedure for saponification
8.14 (d, 2H, J = 8.4 Hz), 8.03 (s, 1H), 7.62 (d, 2H, J = 8.4 Hz), 7.34–
7.26 (m, 18H), 7.17–7.15 (m, 2H), 5.10 (d, 1H, J = 13.2 Hz), 5.04
(d, 1H, J = 13.6 Hz), 4.94 (dd, 2H, J = 2.4, 11.2 Hz), 4.84–4.79 (m,
3H), 4.62 (d, 1H, J = 12.0 Hz), 4.56–4.52 (m, 3H), 3.77 (dd, 1H,
J = 1.2, 10.8 Hz), 3.71 (dd, 1H, J = 4.8, 10.8 Hz), 3.67–3.62 (m, 3H),
3.53 (t, 1H, J = 8.0 Hz); 13C NMR (100 MHz, CDCl3): d 197.3, 164.7,
146.3, 142.8, 138.5, 138.4, 138.1, 138.0, 128.4, 127.9, 114.1,
102.8, 84.4, 81.6, 77.8, 75.6, 75.0, 73.5, 68.9, 62.7; ESIMS m/z:
[M+H]+ calcd for C45H43N3O9: 770.3078, found: 770.3077. Analyti-
cal HPLC: tR = 4.1 min (solvent: MeOH, 0.6 mL/min over 14 min,
purity 99%).
To a solution of methyl ester in MeOH (5 mL) and water (5 mL)
were added LiOH (1.5 equiv/ester). The mixture was stirred at rt for
6 h, then acidified with resin H+, filtered, and evaporated to give
the free acid and purified by column chromatography.
1.3.1. Methyl 2,3,4-tri-O-benzyl-6-O-[1-(4-carboxyphenyl)-1H-
1,2,3-triazol-4-ylmethyl] a-D-glucopyranoside (14)
From compound 12 (282 mg, 0.42 mmol) and LiOH (35 mg,
0.84 mmol), column chromatography (CH2Cl2/MeOH, 10:1) affor-
ded 14 as a yellow powder (242 mg, 88%). Rf = 0.62 (CH2Cl2/MeOH,
7:1); [
a]
+19 (c 0.2 in CH2Cl2); 1H NMR (400 MHz, CDCl3): d 8.23
1.4. Inhibitory assay
D
(br s, 2H), 7.97 (s, 1H), 7.76 (br s, 2H), 7.34–7.22 (m, 15H), 4.98 (d,
1H, J = 10.8 Hz), 4.87 (d, 1H, J = 10.8 Hz), 4.81 (d, 1H, J = 10.8 Hz),
4.79 (d, 2H, J = 12.0 Hz), 4.68–4.62 (m, 3H), 4.56 (d, 1H,
J = 10.8 Hz), 4.00 (t, 1H, J = 9.2 Hz), 3.84 (d, 1H, J = 10.0 Hz), 3.80–
3.75 (m, 2H), 3.63 (t, 1H, J = 9.2 Hz), 3.56 (dd, 1H, J = 2.4, 9.2 Hz),
3.38 (s, 3H); 13C NMR (100 MHz, CDCl3): d 168.9, 146.2, 140.4,
138.7, 138.2, 138.1, 131.9, 128.5, 128.4, 128.2, 128.1, 128.0,
127.8, 127.7, 127.6, 120.5, 119.9, 98.3, 82.1, 79.7, 75.8, 75.0, 73.4,
70.0, 69.1 64.8, 63.7, 55.3; ESIMS m/z: [MÀH]À calcd for
C38H39N3O8: 664.2659, found: 664.2651.
Recombinant human PTP1B catalytic domain was expressed
and purified according to procedures described previously.20a
Enzymatic activity of PTP1B was determined at 30 °C by monitor-
ing the hydrolysis of pNPP. Dephosphorylation of pNPP generates
product pNP, which can be monitored at 405 nm. In a typical
100 lL assay, mixtures containing 50 mM MOPS, pH 6.5, 2 mM
pNPP, and recombinant enzymes, PTP1B activities were continu-
ously 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 enzyme, 2 mM pNPP in
50 mM MOPS at pH 6.5, and the inhibitors diluted around the esti-
mated IC50 values. IC50 was calculated from the nonlinear curve fit-
ting of percent inhibition (inhibition (%)) versus inhibitor
concentration [I] by using the following equation: inhibition
(%) = 100/{1 + (IC50/[I])k}, where k is the Hill coefficient.
1.3.2. Methyl 2,3,4-tri-O-benzyl-6-O-[1-(4-
(carboxycarbonyl)phenyl)-1H-1,2,3-triazol-4-ylmethyl] a-D-
glucopyranoside (15)
From compound 13 (170 mg, 0.24 mmol) and LiOH (10 mg,
0.24 mmol), column chromatography (CH2Cl2/MeOH, 10:1) affor-
ded 15 as a white powder (87 mg, 52%). Rf = 0.32 (CH2Cl2/MeOH,
7:1); [a]
+27 (c 0.1 in CH2Cl2); 1H NMR (400 MHz, CDCl3): d
D
8.70 (d, 2H, J = 7.2 Hz), 7.97 (s, 1H), 7.80 (d, 2H, J = 7.6 Hz), 7.32–
7.21 (m, 15H), 4.97 (d, 1H, J = 10.8 Hz), 4.86 (d, 1H, J = 11.2 Hz),
4.82–4.76 (m, 4H), 4.63–4.60 (m, 2H), 4.55 (d, 2H, J = 10.8 Hz),
3.99 (t, 1H, J = 9.2 Hz), 3.75 (t, 2H, J = 10.8 Hz), 3.60 (t, 1H,
J = 9.2 Hz), 3.53 (dd, 1H, J = 3.2, 9.6 Hz), 3.37 (s, 3H); 13C NMR
(100 MHz, CDCl3): d 190.6, 171.2, 138.8, 138.2, 138.1, 131.3,
128.4, 128.3, 128.1, 128.0, 127.9, 127.7, 127.6, 120.5, 98.3, 82.0,
79.9, 75.7, 75.0, 73.3, 70.0, 69.2, 60.4, 55.3; ESIMS m/z: [MÀH]À
calcd for C39H39N3O9: 692.2608; found: 692.2605. Analytical HPLC:
tR = 5.7 min (solvent: MeOH, 0.6 mL/min over 32 min, purity 97%).
To study the inhibition selectivity on other PTP family mem-
bers, human TCPTP, SHP-1, SHP-2, and LARD1 were prepared and
assays were performed according to procedures described
previously.20b
Acknowledgments
This work was supported by the National Natural Science Foun-
dation of China (Grant No. 20876045), the National Science & Tech-
nology Major Project of China ‘Key New Drug Creation and
Manufacturing Program’ (No. 2009ZX09302-001), Shanghai Sci-
1.3.3. 1-(4-Carboxyphenyl)-1H-1,2,3-triazol-4-ylmethyl 2,3,4,6-
ence
and
Technology
Community
(No.
10410702700,
tetra-O-benzyl
a
-
D
-glucopyranoside (18)
08DZ2291300), and the Chinese Academy of Sciences (No.
KSCX2-YW-R-168). X.-P.H. also thanks the French Embassy in Bei-
jing, China for a doctoral fellowship.
From compound 16 (55 mg, 0.07 mmol) and LiOH (6 mg,
0.14 mmol), column chromatography (CH2Cl2/MeOH, 10:1) affor-

Z. Song et al. / Carbohydrate Research 346 (2011) 140–145
145
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