Discovery of Cell-Permeable O-GlcNAc Transferase Inhibitors via Tethering in Situ Click Chemistry

Article abstract of DOI:10.1021/acs.jmedchem.6b01237

O-GlcNAc transferase (OGT) is a key enzyme involved in dynamic O-GlcNAcylation of nuclear and cytoplasmic proteins similar to phosphorylation. Discovery of cell-permeable OGT inhibitors is significant to clarify the function and regulatory mechanism of O-GlcNAcylation. This will establish the foundation for the development of therapeutic drugs for relevant diseases. Here, we report two cell-permeable OGT inhibitors (APNT and APBT), developed from low-activity precursors (IC₅₀ > 1 mM) via “tethering in situ click chemistry (TISCC)”. Both of them were able to inhibit O-GlcNAcylation in cells without significant effects on cell viability. Unusual noncompetitive inhibition of OGT was helpful to discover novel inhibitors and explore the regulatory mechanism of OGT. The development of these molecules validates that TISCC can be utilized to discover novel lead compounds from components that exhibited very weak binding to the target.

Full text of DOI:10.1021/acs.jmedchem.6b01237

Article
pubs.acs.org/jmc
Discovery of Cell-Permeable O‑GlcNAc Transferase Inhibitors via
Tethering in Situ Click Chemistry
Yue Wang,*,†,‡ Jingjing Zhu,‡ and Lianwen Zhang§
†School of Chemistry and Chemical Engineering, University of Chinese Academy of Sciences, Beijing 100049, China
‡State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China
§College of Pharmacy, State Key Laboratory of Medicinal Chemical Biology and Tianjin Key Laboratory of Molecular Drug Research,
Nankai University, Tianjin 300071, China
S
* Supporting Information
ABSTRACT: O-GlcNAc transferase (OGT) is a key enzyme
involved in dynamic O-GlcNAcylation of nuclear and
cytoplasmic proteins similar to phosphorylation. Discovery of
cell-permeable OGT inhibitors is significant to clarify the
function and regulatory mechanism of O-GlcNAcylation. This
will establish the foundation for the development of therapeutic
drugs for relevant diseases. Here, we report two cell-permeable
OGT inhibitors (APNT and APBT), developed from low-
activity precursors (IC50 > 1 mM) via “tethering in situ click
chemistry (TISCC)”. Both of them were able to inhibit O-
GlcNAcylation in cells without significant effects on cell
viability. Unusual noncompetitive inhibition of OGT was
helpful to discover novel inhibitors and explore the regulatory
mechanism of OGT. The development of these molecules validates that TISCC can be utilized to discover novel lead
compounds from components that exhibited very weak binding to the target.
about 50% after treatment.17 All of these were used at
millimolar/micromolar concentrations in cells. Therefore,
O-GlcNAc transferase (OGT), an essential mammalian
development of OGT inhibitors with novel scaffolds is
enzyme, catalyzes the transfer of N-acetylglucosamine from
extremely urgent.
INTRODUCTION
■
UDP-N-acetylglucosamine (UDP-GlcNAc) to hydroxyl groups
Target-guided synthesis (TGS) is a promising methodology
of serines and threonines (Ser/Thr) in nuclear, cytoplasmic,
in drug discovery, which can be divided into dynamic
and mitochondrial proteins,1,2 such as numerous transcription
combinatorial chemistry and kinetically controlled TGS.
factors,3 tumor suppressors, kinases,4 phosphatases,5 and
Tethering and in situ click chemistry are representative
histone-modifying proteins.6 This kind of protein post-
strategies, respectively. The former is a capture method based
translational modification is involved in the regulation of
on thiol−disulfide exchange, where a free thiol on the protein
many cellular signaling pathways and closely associated with the
surface can react with a disulfide-containing small fragment to
occurrence and development of numerous critical illnesses
form a disulfide;20,21 the latter utilizes an irreversible click
including insulin resistance,7 diabetic complications,8 cancer,9
reaction to combine two reactive building blocks into a
cardiovascular disease,10 and Alzheimer’s disease.11 Due to the
potential inhibitory compound.22−24 Combining the advantages
importance of OGT, various small molecules have been
of both approaches, the more general strategy named “tethering
reported to inhibit OGT activity in cells,12 including alloxan
in situ click chemistry (TISCC)” is employed herein to discover
(a uracil mimic),13 BADGP (a N-acetylgalactosamine deriva-
OGT inhibitors.
tive),14 Ac4-5SGlcNAc (a metabolic precursor),15 benzoxazo-
linone (BZX) derivative (a neutral diphosphate mimic),16 and
RESULTS
OSMI-1 (a quinolinone-6-sulfonamide derivative).17 However,
■
In our strategy, one kind of building block is designed with dual
reactive groups X (ICH2CO) and A (N3) and the other one
contains the reactive group Y (CCH). First, the target
protein is incubated with the former building blocks and
therefore tethered with reactive group A (N3) at several sites.
most of these compounds were not shown to inhibit OGT
effectively or selectively. Alloxan also inhibited O-GlcNAcase
(OGA), which removed O-GlcNAc from proteins.18 BADGP
induced abnormal O-glycosylation of mucins.19 Ac4-5SGlcNAc
affected several types of cell surface glycans containing mucin-
type O-glycans.17 BZX was an irreversible inhibitor of OGT,
which was proven too reactive to employ as a selective OGT
inhibitor in cells,16 and OSMI-1 decreased cell viability by
Received: August 26, 2016
© XXXX American Chemical Society
A
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Figure 1. Tethering in situ click chemistry (TISCC): (a) screening of building blocks to discover the new candidate; (b) synthesis of the new
candidate for activity evaluation.
Second, the latter building blocks are added to the mixture
without separation and then classical in situ click chemistry is
performed at proper sites. Finally, the protein is separated from
small molecules with SDS−PAGE, digested with trypsin, and
then determined with LC/MS/MS to find the modified
peptide. The structure of the new candidate can be deduced
from the exact mass of modification (Figure 1a). After that, the
new candidate was synthesized by copper-/ruthenium-catalyzed
azide−alkyne cycloaddition (CuAAC/RuAAC) for activity
evaluation (Figure 1b). Compared with tethering and classical
in situ click chemistry, our strategy employs more general
building blocks and detects the protein fragments instead of the
entire protein or biligand compound itself. Therefore, several
binding pockets can be screened at the same time.
Iodoacetamide is an alkylating agent used for peptide
mapping purposes.25 It covalently binds with the thiol group
of cysteine to prevent the protein from forming disulfide
bonds.26,27 Iodoacetamide inhibits all cysteine peptidases and
most of thiol-dependent enzymes irreversibly via nonselective
alkylation of the catalytic cysteine residue. Therefore,
iodoacetamide was selected as the reactive group X to tether
the target protein in our strategy. To maintain proper molecular
rigidity, phenyl was used to link the reactive groups X
(ICH2CO) and A (N3). p-Azidoiodoacetanilide (1) was chosen
to be the difunctional building block.28
amino group. According to the native cysteine, the exact mass
increases for the expected triazole products were different
enough to be distinguished by MS. It seemed to be inefficient
to screen 3−4 building blocks for one assay, but nonetheless, it
was attractive to screen 17 cysteine residues of OGT at the
same time. On the basis of the foregoing procedure, alkynes
were added to the tethering reaction mixtures without
separation and the resulting mixtures were kept at 37 °C for
another 6 h (Figure S2) before being quenched. Fourteen
different modifications were found on only four cysteine
residues including Cys590, Cys758, Cys921, and Cys927
(Chart 1).
Chart 1. Structures of Building Blocks and Relevant
a
Triazoles
At first, OGT (10 μM) in Tris-HCl buffer solution
containing DTT (1 mM) was incubated with 1 (4 mM) at
37 °C for 2 h. After that, Laemmli sample buffer containing 50
mM DTT was added to quench the reaction. OGT was
separated from other reagents with SDS−PAGE, digested with
trypsin, and then determined with LC/MS/MS. The results
showed most of the cysteine residues in OGT (1−1046 aa)
were tethered including Cys44, Cys68, Cys199, Cys267,
Cys297, Cys315, Cys323, Cys417, Cys531, Cys590, Cys620,
Cys758, Cys845, Cys921, Cys927, Cys962, Cys965 (Figure S1
in Supporting Information). Especially, Cys921 and Cys927,
located near the substrate binding site, were tethered with azido
groups, and this result encouraged us to perform in situ click
chemistry sequentially.
Sixty-one alkyne building blocks were collected including
alcohol, ether, acid, amine, steroid, and aromatic compounds
and divided into 16 groups (Table S1). Each group was
composed of 3−4 components. All the compounds in the same
group had an identical functional group as far as possible. For
example, every building block in the second group possessed an
a
Modification sites of OGT found with LC/MS/MS shown in
paratheses.
To examine the efficiency of TISCC, anti- and syn-triazoles
(4a−n) were synthesized by CuAAC and RuAAC, respec-
tively.29,30 Instead of iodine (1), chloride (3) was employed to
reduce off-target effects. All the compounds were produced
successfully except syn-4l,m,n and anti-4n, probably because
the nitrogen atom present in alkynes 2l, 2m, and 2n bound to
the ruthenium/copper atom suppressing its catalytic activity.31
B
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All the compounds were then evaluated for OGT inhibition
activity with HPLC assay that monitored the transfer of
GlcNAc to an OGT acceptor peptide (CKII). Eight of these 22
compounds inhibited OGT > 40% at 100 μM, while the
precursors 1 and 3 showed no significant inhibitory effect at
this concentration (Figure S3). To identify the cysteine sites
that reacted with these eight candidates, tryptic digests of
OGTs were analyzed following treatment with the candidates,
respectively. The modifications of cysteines were determined at
the Cys590 or adjacent Cys531/Cys620 except anti-4e and -f.
Therefore, we focused on anti-4e, -f, and -g for further
investigation. An inactivation time course showed that OGT
inhibition of anti-4g was time-dependent; however, OGT
preincubated with anti-4e or -f kept most of the enzyme
activity during the period of measurement (Figure S4). IC50
values were determined for the positive control (BZX), the
precursors (1, 2e−g, and 3) and candidates (anti-4e, -f, and -g)
(Table 1, entries 1−9 and Figure S5). IC50 values of three
candidates were dramatically lower than those of the precursors
(1, 2e−g, and 3).
COS-7 cells were treated for 4 h in glucose starvation
medium with varying concentrations of APNT or APBT
ranging from 12.5 to 100 μM. After that, glucosamine (2 mM)
and PUGNAc (100 μM) were added to the cells and incubated
for another 3 h. Finally, cell lysates were detected with the O-
GlcNAc antibody (RL-2). APNT and APBT reduced global O-
GlcNAcylation (Figures 3 and S8) in a dose-dependent manner
(Figure S9) with the maximal effect being accomplished at 25
μM. As a result of the limited aqueous solubility of APNT and
APBT, higher concentrations of APNT or APBT did not
decrease O-GlcNAc levels further. Although APNT and APBT
showed weaker inhibition of OGT than BZX in vitro, they were
comparable with BZX in vivo. In addition to the reduction of
global O-GlcNAcylation in cells, the effects of APNT and
APBT on specific cellular markers of OGT inhibition were
investigated. It was found that APNT and APBT, like BZX,
reduced cellular OGA, which decreased when cellular O-
GlcNAcylation was blocked. However, OGT levels remain
unchanged (Figure 3).17 Therefore, we concluded that APNT
and APBT inhibited OGT activity in cells.
We used lectins to probe cell surface glycans following the
treatment of cells with APNT and APBT. Eight commercially
available biotinylated lectins (ConA, LCA, Jacalin, PHA-E,
ECL, GSL-I, PNA, and DBA), which recognize different
features of N- and O-glycans, were employed to probe the
glycan composition of COS-7 cells treated with 100 μM APNT
or APBT for 7 h. For positive control (BZX), APNT and
APBT, all of the ConA, PNA, and DBA levels were kept
between 80% and 120% compared with negative control
(DMSO). For BZX, GSL-I level increased by over 20% while
PHA-E level decreased by over 20%. For APNT and APBT,
LCA, Jacalin, ECL and GSL-I levels increased by over 20%.
Especially, Jacalin and ECL levels changed dramatically (136−
173%), which are relative to Galβ3GalNAc and Galβ4GlcNAc,
respectively. This phenomenon implies that some kind of
galactosyltransferase is likely to be regulated by O-GlcNAcy-
lation (Figures 4 and S10).
Table 1. IC50 Values of Several Compounds
entry
compd
IC50 (μM)
11.9 1.1
1
BZX
1
2
865 43
a
b
3
2e
2f
>4000 (88 1%)
>1000 (79 2%)
1964 13
4
5
2g
3
6
3967 209
71.7 1.0
7
anti-4e
8
anti-4f
91.2 1.9
9
anti-4g
89.3 1.8
a
10
11
12
13
5
>4000 (64% 1%)
66.7 0.8
anti-6e (APNT)
anti-6f (APBT)
anti-6g
139 14
1001
8
a
b
Furthermore, we found no obvious effects on cell viability
following the treatment of cells with APNT and APBT (Figure
S11).
Enzyme activity in the presence of 4000 μM inhibitor. Enzyme
activity in the presence of 1000 μM inhibitor.
DISCUSSION AND CONCLUSIONS
We next examined the ability of three candidates (anti-4e, -f,
and -g) to inhibit global O-GlcNAcylation in COS-7 cells.
Unfortunately, all of the three candidates showed strong
cytotoxicity probably arising from the chloroacetamide group.
Therefore, corresponding acetamide derivatives (anti-6e, -f,
and -g) were synthesized from p-azidoacetanilide (5) via similar
procedures. OGT inhibition assay showed that anti-6e (APNT)
and anti-6f (APBT) kept intrinsic activities; nevertheless, anti-
6g presented less inhibition activity (Table 1, entries 11−13,
and Figure S6). The removal of the activated leaving group
(Cl) in anti-6g and resulting loss of inhibition activity
confirmed that anti-4g was an irreversible inhibitor. With the
commercially available UDP-Glo glycosyltransferase assay, the
mode of inhibition was determined for APNT and APBT, both
of which were found to be noncompetitive with respect to
UDP-GlcNAc (Km = 10.3 0.3 μM, Ki,APNT = 28.6 0.5 μM,
■
In summary, we developed cell-permeable OGT inhibitors
(APNT and APBT) via TISCC and follow-up chemistry.
Traditional in situ click chemistry always utilized building
blocks with a high affinity for the target (IC50 ≤ 6.4 μM). When
the IC50 of the precursor had a micromolar concentration, the
newly produced inhibitor expressed (5−700)-fold increase in
the inhibitory activity.33−35 Click chemistry was also employed
to discover fucosyltransferase inhibitors from GDP-alkyne (Ki =
47 μM), following in situ screening without product isolation.36
However, the diphosphate bridge, which contributed consid-
erably to binding affinity, prevented the resultant inhibitor from
entering the cells. Here, APNT (IC50 = 66.7 0.8 μM) was
composed of two neutral building blocks 2e and 5, which
exhibited very weak binding to OGT (IC50 > 4000 μM) (Table
1, entries 3, 10, and 11). The newly produced inhibitor showed
more than 60-fold increase in the inhibitory activity, the effect
of which was comparable with that of BZX in vivo. Therefore,
TISCC could be used to screen precursors with poor activity,
the IC50 of which was around several millimolar.
Ki,APBT = 92.0 3.0 μM) or CKII peptide (Km = 230 16 μM,
Ki,APNT = 59.8 2.8 μM, Ki, APBT = 310 17 μM) (Figure 2).
In addition, we tested the effect of APNT and APBT on
ppGalNAcT2.32 Neither of them inhibited ppGalNAcT2
remarkably (IC50,APNT = 696 69 μM, IC50,APBT = 430 35
μM) (Figure S7).
Different from most OGT inhibitors, APNT and APBT were
noncompetitive with respect to UDP-GlcNAc or CKII peptide.
C
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Figure 2. Noncompetitive inhibition of OGT by APNT and APBT with respect to UDP-GlcNAc or CKII peptide. (a, c) Double reciprocal plot
showed noncompetitive inhibition of OGT by APNT/APBT at saturating CKII concentrations. Reactions were performed in the presence of
APNT/APBT at saturating peptide concentrations while varying UDP-GlcNAc levels (conditions: 25 nM OGT, 3 mM CKII, UDP-GlcNAc varied
from 2.5 to 40 μM and APNT/APBT at the indicated, fixed concentrations; 1 h incubation at room temperature). (b, d) Double reciprocal plot
showed noncompetitive inhibition of OGT by APNT/APBT at saturating UDP-GlcNAc concentrations. Reactions were performed in the presence
of APNT/APBT at saturating UDP-GlcNAc concentrations while varying CKII concentrations (conditions: 50 nM OGT, 1 mM UDP-GlcNAc,
CKII varied from 50 to 800 μM and APNT/APBT at the indicated, fixed concentrations; 1 h incubation at room temperature). Each point was
determined in triplicate experiments.
Figure 3. Inhibition of OGT by APNT and APBT in cells. Lysates from COS-7 cells, untreated or treated with inhibitors at 100 μM, were detected
for global O-GlcNAc, OGA, and OGT levels. Accompanied with the reduction of global O-GlcNAc levels, APNT and APBT decreased OGA levels
without affecting OGT levels.
mixtures were kept at 37 °C for another 6 h. Then reactions were
quenched by adding Laemmli sample buffer (Biorad) containing 50
mM DTT and boiling samples at 96 °C for 10 min. SDS−PAGE was
performed using precast Any kD Mini-PROTEAN TGX Gel (Biorad),
followed by staining with GelCode Blue stain reagent (Thermo
Scientific) for 1 h and destaining with distilled water for 1 h.
In-Gel Trypsin Digestion. Each OGT lane was cut into 10 pieces
and reduced with 25 mM DTT, but there was no need to alkylate with
iodoacetamide. Then, in-gel digestion was carried out with sequencing
grade modified trypsin (Promega) in 50 mM ammonium bicarbonate
at 37 °C overnight. Tryptic peptides were extracted twice with 1%
trifluoroacetic acid in 50% acetonitrile aqueous solution for 30 min.
The extractions were then centrifuged in a SpeedVac to reduce the
volume.
According to the results of TISCC, APNT was likely to bind to
the pocket around Cys590. Docking APNT with OGT-UDP-
CKII complex (PDB code 3PE4), we found that APNT bound
the cleft between the TPR domain and the N-terminal domain
of the catalytic region (Figure S12). This information can be
used to design novel OGT inhibitors and understand regulatory
mechanisms of OGT. For further investigation, improvement of
aqueous solubility of APNT and APBT is urgent and the
second round of in situ click chemistry will be performed on
the basis of APNT and APBT.
EXPERIMENTAL SECTION
■
Tethering in Situ Click Chemistry. Reaction mixtures containing
10 μM OGT, 4 mM p-azidoiodoacetanilide (1), and buffer (150 mM
NaCl, 1 mM DTT, 25 mM Tris-HCl, pH = 7.4) were incubated at 37
°C for 2 h. Without separation, alkynes were added at the final
concentration of 1 mM for each building block, and the resulting
LC/MS/MS Analysis. The digestion products were separated by an
85 min gradient elution at a flow rate of 0.250 μL/min with an
Ultimate 3000 HPLC system (Thermo Scientific), which was directly
interfaced with a Orbitrap Q Exactive mass spectrometer (Thermo
D
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Figure 4. Effect of OGT inhibitors on cell surface glycans. Lysates from COS-7 cells, untreated or treated with inhibitors at 100 μM, were probed
with lectins. For APNT and APBT, LCA, Jacalin, ECL, and GSL-I levels increased by over 20% while ConA, PHA-E, PNA and DBA levels were kept
between 80% and 120% compared with negative control (DMSO). According to data sheet of Vector Laboratories, preferred sugar specificity of each
lectin appears to be as follows: concanavalin A (Con A) and Lens culinaris (LCA) recognize αMan and αGlc; Jacalin recognizes Galβ3GalNAc;
Phaseolus vulgaris erythroagglutinin (PHA-E) recognizes Galβ4GlcNAcβ2Manα6(GlcNAcβ4)(GlcNAcβ4Manα3)Manβ4; Erythrina cristagalli (ECL)
recognizes Galβ4GlcNAc; Griffonia (Bandeiraea) simplicifolia I (GSL-I) recognizes αGal and αGalNAc; peanut agglutinin (PNA) recognizes
Galβ3GalNAc; Dolichos biflorus (DBA) recognizes αGalNAc.
E
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1
Scientific). The analytical column was a homemade fused silica
capillary column (75 μm × 150 mm; Upchurch) packed with C-18
resin (300 Å, 5 μm; Varian). Mobile phase A consisted of 0.1% formic
acid in H2O and mobile phase B consisted of 0.1% formic acid in
MeCN. The Q Exactive mass spectrometer was operated in the data-
dependent acquisition mode using Xcalibur 2.1.2 software, and there
was a single full-scan mass spectrum in the orbitrap (400−1 800 m/z,
60 000 resolution) followed by 10 data-dependent MS/MS scans at
27% normalized collision energy (HCD).
The MS/MS spectra from each LC/MS/MS run were searched
against the human.fasta from UniProt (release date of March 19, 2014;
68 406 entries) using an in-house Proteome Discoverer (version
PD1.4, Thermo Scientific). The search criteria were as follows: full
tryptic specificity was required; one missed cleavage was allowed; the
oxidation (Met) and expected modifications (Cys) (for example, the
increase of exact mass is 174.0542 or 148.0637 according to the native
cysteine) were set as the variable modifications; precursor ion mass
tolerances were set at 10 ppm for all MS data acquired in an orbitrap
mass analyzer; and the fragment ion mass tolerance was set at 20 mmu
for all MS2 spectra acquired.
OGT Activity Assay with HPLC. Reaction mixtures containing
200 μM CKII peptide (KKKYPGGSTPVSSANM M), 1 mM UDP-
GlcNAc, 100 nM ncOGT, 12.5 mM MgCl2, buffer (150 mM NaCl, 1
mM EDTA, 2.5 mM tris(hydroxypropyl)phosphine, 20 mM Tris-HCl,
pH 7.4), and inhibitor (if necessary) were incubated at 37 °C for 30
min (for determination of OGT stability) or 60 min (for inhibitor
screening and IC50 determination). After being quenched by adding an
equal volume of methanol, the reaction mixtures were centrifuged at
12 000g for 30 min. The supernatants (40 μL) were loaded onto an
Agilent 1260 Infinity HPLC system to quantify the yield based on the
integrated areas of glycopeptide product and peptide substrate. The
reverse-phase chromatographic column was a Zorbax SB-C18
StableBond analytical column (4.6 mm × 250 mm, 5 μm; Agilent),
preceded by a Zorbax SB-C18 analytical guard column (4.6 mm × 12.5
mm, 5 μm; Agilent). Mobile phase A consisted of 0.1% TFA in H2O,
and mobile phase B consisted of 0.1% TFA in MeCN. The
components were eluted using a gradient (flow rate at 1 mL/min; at
0 min elution solvent mixture A/B = 90/10; at 20 min elution solvent
mixture A/B = 70/30; wavelength = 214 nm). Each reaction was
repeated twice or three times.
on silica gel (200−300 mesh, Tsingdao Haiyang). H NMR and 13C
NMR spectra were recorded on a Bruker AVANCE III 400
spectrometer at 25 °C. Chemical shifts (in ppm) of H NMR were
1
calibrated with tetramethylsilane (δ = 0 ppm) or solvent residual peak
(δ = 2.50 ppm for CD3SOCD2H). Chemical shifts (in ppm) of 13C
NMR were calibrated with solvent peak (δ = 77.16 ppm for CDCl3
and δ = 39.52 ppm for CD3SOCD3). Mass spectra were recorded
using a Waters Xevo G2 Q-TOF spectrometer (ESI) or a Thermo
Finnigan TRACE 2000/Waters GCT Premier (EI). Elemental analysis
was performed with a Vario EL III analyzer. Percentages of purity were
determined with HPLC for the compounds, the purity of which was
not verified with elemental analysis. All the compounds possessed the
purity of at least 95%.
p-Azidochloroacetanilide (3) and p-Azidoacetanilide (5).
Following a literature procedure,37 the mixture of p-iodoaniline
(10.95 g, 50.0 mmol), sodium azide (6.50 g, 100 mmol, 2 equiv),
sodium ascorbate (0.55 g, 2.78 mmol, 5.6%), and DMSO/H2O (V/V
= 5/1, 110 mL) was placed in a two-neck round-bottom flask. Argon
was passed through the solution for 5 min. CuI (0.95 g, 5.0 mmol,
10%) and N,N′-dimethylethylenediamine (0.85 mL, 7.9 mmol, 16%)
were added to the reaction mixture under argon atmosphere. The
reaction was stirred at room temperature for 1 h, and TLC analysis
showed complete consumption of the starting material. The reaction
mixture was subsequently diluted with water (150 mL) and extracted
with ethyl acetate (3 × 150 mL). The organic layers were combined,
washed with brine (3 × 50 mL), dried over Na2SO4, filtered, and
evaporated under reduced pressure. The resulting residue was purified
by flash chromatography (ethyl acetate/petrol ether, V/V = 1/4) to
1
yield p-azidoaniline (7) (5.44 g, 40.6 mmol, 81%). H NMR (400
MHz, CDCl3) δ 7.40 (d, J = 8.1 Hz, 2H, phenyl), 6.47 (d, J = 8.2 Hz,
2H, phenyl), 3.67 (s, 2H, NH2).
Compound 7 (6.57 g, 49.0 mmol), chloroacetic acid (4.63 g, 49.0
mmol, 1.0 equiv), and 1-(3-dimethylaminopropyl)-3-ethylcarbodii-
mide hydrochloride (18.79 g, 98.0 mmol, 2.0 equiv) were dissolved in
dry THF (265 mL). The reaction was stirred at room temperature for
2 h and then evaporated under reduced pressure. The resulting residue
was redissolved in ethyl acetate (700 mL), washed successively with 1
M HCl (3 × 300 mL), aq NaHCO3 (3 × 200 mL) and brine (150
mL), dried over Na2SO4, filtered, and evaporated under reduced
1
pressure to yield 3 (9.85 g, 46.8 mmol, 95%). H NMR (400 MHz,
Western Blot of Inhibitor-Treated COS-7 Cells. COS-7 cells
were maintained in DMEM (Hyclone) supplemented with 10% fetal
bovine serum (Gibco), 100 U/mL penicillin, and 100 μg/mL
streptomycin (MP). Old growth medium was removed when cells
were grown to 70% confluence. After being rinsed twice with PBS,
cells were grown in glucose starvation medium (DMEM-no glucose;
Caisson Labs) supplemented with 1% fetal bovine serum, 50 U/mL
penicillin, and 50 μg/mL streptomycin) in the absence (control) and
presence of inhibitor (100 μM) for 4 h. Glucosamine (2 mM, Sigma)
and PUGNAc (100 μM, Santa Cruz Biotechnology) were added to the
cells and then incubated for another 3 h. Cell lysates were prepared in
RIPA lysis buffer (Beyotime) supplemented with protease inhibitor
cocktail (Pierce). Lysates were centrifuged at 16 000g for 15 min at 4
°C and analyzed by SDS−PAGE (4−12%, Invitrogen). Proteins were
transferred to a PVDF membrane (Millipore), and O-GlcNAcylation
was detected with anti-O-GlcNAc antibody (RL-2, Abcam, used at
1:1 000) and a secondary anti-mouse IgG-HRP antibody (Jackson
ImmunoResearch, used at 1:10 000). OGT, OGA, and actin were
detected with relevant antibodies (Sigma/MDL Biotech, used at
1:2 000) and a secondary anti-rabbit lgG-HRP antibody (Jackson
ImmunoResearch, used at 1:10 000). Cell surface glycans was probed
with biotinylated lectins (Vector Laboratories, used at 1:2 00) and
HRP-conjugated streptavidin (CST, used at 1:2 000).
CDCl3) δ 8.24 (s, 1H, NH), 7.55 (d, J = 8.7 Hz, 2H, phenyl), 7.02 (d,
J = 8.7 Hz, 2H, phenyl), 4.20 (s, 2H, ClCH2CO). 13C NMR (101
MHz, CDCl3) δ 163.89 (CO), 136.97 (C-Ar), 133.75 (C-Ar),
121.78 (CH-phenyl), 119.77 (CH-phenyl), 42.96 (ClCH2CO). HRMS
(EI-TOF) m/z: [M]+ calcd for C8H7N4OCl 210.0308; found
210.0309.
1
Compound 5 was obtained via a similar procedure. H NMR (400
MHz, CDCl3) δ 7.50 (d, J = 8.8 Hz, 2H, phenyl), 7.23 (s, 1H, NH),
6.98 (d, J = 8.8 Hz, 2H, phenyl), 2.18 (s, 3H, CH3CO). HRMS (ESI-
TOF) m/z: [M + H]+ calcd for C8H9N4O 177.0776; found 177.0778.
General Procedure for Copper-Catalyzed Alkyne−Azide
Cycloaddition. Compound 3 or 5 (0.500 mmol) and alkyne
(0.600 mmol, 1.2 equiv) were stirred together in t-butanol/water
(V/V = 2/1, 5.0 mL) under argon. To this suspension was added aq
copper sulfate (0.50 M, 50 μL, 5.0%) and aq sodium ascorbate (0.10
M, 500 μL, 10%). The reaction was stirred at 50 °C until TLC analysis
showed complete consumption of the starting material (3 or 5). The
precipitate was collected by filtration, washed with aq EDTA (0.1 M, 3
× 5 mL), aq NaHCO3 (5 mL), water (3 × 5 mL), and diethyl ether (2
× 5 mL) successively and then dried overnight in a vacuum desiccator
to yield the desired anti-triazole (4 or 6).
1-(4-Chloroacetamidophenyl)-4-phenyl-1H-1,2,3-triazole
1
(anti-4a). H NMR (400 MHz, CD3SOCD3) δ 10.59 (s, 1H, NH),
9.24 (s, 1H, triazole), 8.00−7.88 (m, 4H, phenyl), 7.83 (d, J = 9.0 Hz,
2H, phenyl), 7.50 (t, J = 7.6 Hz, 2H, phenyl-3′/5′), 7.38 (t, J = 7.4 Hz,
1H, phenyl-4′), 4.31 (s, 2H, ClCH2CO). 13C NMR (101 MHz,
CD3SOCD3) δ 164.96 (CO), 147.22 (C-Ar), 138.75 (C-Ar), 132.29
(C-Ar), 130.28 (C-Ar), 128.98 (CH-phenyl-3′/5′), 128.20 (CH-
phenyl-4′), 125.32 (CH-phenyl-2′/6′), 120.75 (CH-phenyl), 120.24
(CH-phenyl), 119.47 (CH-triazole), 43.54 (ClCH2CO). ESI-MS (m/
General Procedures for Chemical Synthesis. All chemicals
were purchased as reagent grade and used without further purification.
Reactions were monitored by analytical thin-layer chromatography on
silica gel 60 F254 precoated on aluminum plates (E. Merck). Spots
were detected under UV (254 nm) and/or by staining with acidic ceric
ammonium molybdate. Solvents were evaporated under reduced
pressure below 40 °C (bath). Column chromatography was performed
F
DOI: 10.1021/acs.jmedchem.6b01237
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
z, relative intensity): 313 ([M + H]+, 100) , 279 (12), 114(11). Anal.
Calcd for C16H13N4OCl: C, 61.45; H, 4.19; N, 17.91. Found: C, 61.29;
H, 4.22; N, 17.91.
1-(4-Chloroacetamidophenyl)-4-(diphenylhydroxymethyl)-
1
1H-1,2,3-triazole (anti-4g). H NMR (400 MHz, CD3SOCD3) δ
10.55 (s, 1H, NH), 8.47 (s, 1H, triazole), 7.90 (d, J = 8.8 Hz, 2H,
phenyl), 7.78 (d, J = 8.8 Hz, 2H, phenyl), 7.43 (d, J = 7.5 Hz, 4H,
phenyl-2′/6′ × 2), 7.31 (t, J = 7.4 Hz, 4H, phenyl-3′/5′ × 2), 7.24 (t, J
= 7.2 Hz, 2H, phenyl-4′ × 2), 6.70 (s, 1H, OH), 4.29 (s, 2H,
ClCH2CO). 13C NMR (101 MHz, CD3SOCD3) δ 164.90 (CO),
155.06 (C-Ar), 146.78 (C-Ar), 138.54 (C-Ar), 132.30 (C-Ar), 127.58
(CH-phenyl-3′/5′), 127.05 (CH-phenyl-2′/6′), 126.79 (CH-phenyl-
4′), 121.05 (CH-triazole), 120.72 (CH-phenyl), 120.13 (CH-phenyl),
75.83 (C-OH), 43.52 (ClCH2CO). HRMS (ESI-TOF) m/z: [M +
H]+ calcd for C23H20N4O2Cl 419.1275; found 419.1269.
1-(4-Chloroacetamidophenyl)-4-(2-(3-chlorophenoxy)-1-hy-
droxyethyl)-1H-1,2,3-triazole (anti-4h). 1H NMR (400 MHz,
CD3SOCD3) δ 10.56 (s, 1H, NH), 8.73 (s, 1H, triazole), 7.89 (d, J =
8.9 Hz, 2H, phenyl), 7.80 (d, J = 8.9 Hz, 2H, phenyl), 7.31 (t, J = 8.2
Hz, 1H, phenyl-5′), 7.07 (t, J = 1.9 Hz, 1H, phenyl-2′), 7.00 (dd, J =
7.9, 1.1 Hz, 1H, phenyl-4′/6′), 6.96 (dd, J = 8.4, 2.0 Hz, 1H, phenyl-
6′/4′), 5.90 (d, J = 4.7 Hz, 1H, OH), 5.17−5.08 (m, 1H, CH), 4.33
(dd, J = 10.1, 4.3 Hz, 1H, CHH′), 4.30 (s, 2H, ClCH2CO), 4.23 (dd, J
= 10.0, 7.0 Hz, 1H, CHH′). 13C NMR (101 MHz, CD3SOCD3) δ
164.94 (CO), 159.47 (C-Ar), 149.20 (C-Ar), 138.62 (C-Ar), 133.70
(C-Ar), 132.35 (C-Ar), 130.86 (CH-phenyl-5′), 120.92 (CH-triazole),
120.67 (CH-phenyl, CH-phenyl-4′/6′), 120.21 (CH-phenyl), 114.71
(CH-phenyl-2′), 113.76 (CH-phenyl-6′/4′), 71.72 (CH2), 64.44
(CH), 43.54 (ClCH2CO). ESI-MS (m/z, relative intensity): 407
([M + H]+, 100), 361 (46), 279 (26), 274 (18), 183 (10). Anal. Calcd
for C18H16N4O3Cl2: C, 53.09; H, 3.96; N, 13.76. Found: C, 52.92; H,
3.80; N, 13.48.
1-(4-Chloroacetamidophenyl)-4-(1-hydroxy-2-methylprop-
yl)-1H-1,2,3-triazole (anti-4i). 1H NMR (400 MHz, CD3SOCD3) δ
10.54 (s, 1H, NH), 8.54 (s, 1H, triazole), 7.89 (d, J = 8.1 Hz, 2H,
phenyl), 7.78 (d, J = 8.1 Hz, 2H, phenyl), 5.32 (d, J = 3.9 Hz, 1H,
OH), 4.50 (t, J = 4.6 Hz, 1H, CHOH), 4.29 (s, 2H, ClCH2CO), 2.13−
1.94 (m, 1H, CH(CH3)2), 0.90 (d, J = 6.3 Hz, 3H, CH3), 0.86 (d, J =
6.4 Hz, 3H, CH3). 13C NMR (101 MHz, CD3SOCD3) δ 164.89 (C
O), 151.88 (C-Ar), 138.41 (C-Ar), 132.47 (C-Ar), 120.46 (CH-
phenyl), 120.17 (CH-phenyl), 120.08 (CH-triazole), 70.73 (CHOH),
43.53 (ClCH2CO), 33.63 (CH(CH3)2), 18.64 (CH3), 17.71 (CH3).
ESI-MS (m/z, relative intensity): 331 ([M + Na]+, 8), 309 ([M + H]+,
43), 263 (100). Anal. Calcd for C14H17N4O2Cl: C, 54.46; H, 5.55; N,
18.15. Found: C, 54.28; H, 5.70; N, 18.06.
1-(4-Chloroacetamidophenyl)-4-(4-nitrophenyl)-1H-1,2,3-tri-
1
azole (anti-4b). H NMR (400 MHz, CD3SOCD3) δ 10.60 (s, 1H,
NH), 9.50 (s, 1H, triazole), 8.37 (d, J = 8.8 Hz, 2H, phenyl), 8.20 (d, J
= 8.7 Hz, 2H, phenyl), 7.92 (d, J = 8.9 Hz, 2H, phenyl), 7.84 (d, J =
8.9 Hz, 2H, phenyl), 4.31 (s, 2H, ClCH2CO). 13C NMR (101 MHz,
CD3SOCD3) δ 165.00 (CO), 146.82 (C-Ar), 145.27 (C-Ar), 139.03
(C-Ar), 136.68 (C-Ar), 132.00 (C-Ar), 126.09 (CH-phenyl), 124.48
(CH-phenyl), 121.60 (CH-triazole), 120.92 (CH-phenyl), 120.25
(CH-phenyl), 43.55 (ClCH2CO). ESI-MS (m/z, relative intensity):
358 ([M + H]+, 100). Anal. Calcd for C16H12N5O3Cl: C, 53.72; H,
3.38; N, 19.58. Found: C, 53.65; H, 3.44; N, 19.41.
1-(4-Chloroacetamidophenyl)-4-benzyl-1H-1,2,3-triazole
1
(anti-4c). H NMR (400 MHz, CD3SOCD3) δ 10.54 (s, 1H, NH),
8.53 (s, 1H, triazole), 7.84 (d, J = 9.0 Hz, 2H, phenyl), 7.77 (d, J = 9.0
Hz, 2H, phenyl), 7.41−7.26 (m, 4H, phenyl-2′/3′/5′/6′), 7.26−7.15
(m, 1H, phenyl-4′), 4.29 (s, 2H, ClCH2CO), 4.08 (s, 2H, ArCH2). 13
C
NMR (101 MHz, CD3SOCD3) δ 164.89 (CO), 147.06 (C-Ar),
139.28 (C-Ar), 138.47 (C-Ar), 132.39 (C-Ar), 128.52 (CH-phenyl-2′/
6′/3′/5′), 128.44 (CH-phenyl-3′/5′/2′/6′), 126.22 (CH-phenyl-4′),
120.60 (CH-triazole), 120.57 (CH-phenyl), 120.15 (CH-phenyl),
43.52 (ClCH2CO), 31.20 (ArCH2). ESI-MS (m/z, relative intensity):
327 ([M + H]+, 100). Anal. Calcd for C17H15N4OCl: C, 62.48; H,
4.63; N, 17.15. Found: C, 62.20; H, 4.88; N, 16.90.
1-(4-Chloroacetamidophenyl)-4-(cyclohexylmethyl)-1H-
1
1,2,3-triazole (anti-4d). H NMR (400 MHz, CD3SOCD3) δ 10.53
(s, 1H, NH), 8.48 (s, 1H, triazole), 7.84 (d, J = 9.0 Hz, 2H, phenyl),
7.77 (d, J = 9.0 Hz, 2H, phenyl), 4.29 (s, 2H, ClCH2CO), 2.57 (d, J =
6.9 Hz, 2H, ArCH2), 1.76 − 1.55 (m, 6H, cyclohexyl-2a/6a,
cyclohexyl-3a/5a, cyclohexyl-4a, cyclohexyl-1), 1.28−1.05 (m, 3H,
cyclohexyl-3b/5b, cyclohexyl-4b), 1.05 − 0.89 (m, 2H, cyclohexyl-2b/
6b). 13C NMR (101 MHz, CD3SOCD3) δ 164.88 (CO), 146.55 (C-
Ar), 138.36 (C-Ar), 132.50 (C-Ar), 120.44 (CH-phenyl), 120.36 (CH-
triazole), 120.16 (CH-phenyl), 43.53 (ClCH2CO), 37.49 (cyclohexyl-
1), 32.70 (ArCH2), 32.47 (cyclohexyl-2/6), 25.94 (cyclohexyl-4),
25.64 (cyclohexyl-3/5). ESI-MS (m/z, relative intensity): 333 ([M +
H]+, 100). Anal. Calcd for C17H21N4OCl: C, 61.35; H, 6.36; N, 16.83.
Found: C, 61.09; H, 6.27; N, 16.66.
1-(4-Chloroacetamidophenyl)-4-(naphthalen-2-yl)-1H-1,2,3-
triazole (anti-4e). 1H NMR (400 MHz, CD3SOCD3) δ 10.60 (s, 1H,
NH), 9.38 (s, 1H, triazole), 8.51 (s, 1H, naphthalene-1), 8.09 (d, J =
8.6 Hz, 1H, naphthalene-3/4), 8.05 (d, J = 8.6 Hz, 1H, naphthalene-4/
3), 8.01 (d, J = 7.7 Hz, 1H, naphthalene-5/8), 7.96 (d, J = 8.7 Hz, 3H,
phenyl, naphthalene-8/5), 7.85 (d, J = 8.7 Hz, 2H, phenyl), 7.64−7.47
(m, 2H, naphthalene-6/7), 4.32 (s, 2H, ClCH2CO). 13C NMR (101
MHz, CD3SOCD3) δ 164.95 (CO), 147.22 (C-Ar), 138.77 (C-Ar),
133.11 (C-Ar), 132.69 (C-Ar), 132.29 (C-Ar), 128.62 (naphthalene-4/
3), 128.00 (naphthalene-5/8), 127.74 (C-Ar), 127.72 (naphthalene-8/
5), 126.68 (naphthalene-6/7), 126.27 (naphthalene-7/6), 123.70
(naphthalene-1), 123.65 (naphthalene-3/4), 120.75 (CH-phenyl),
120.26 (CH-phenyl), 119.86 (CH-triazole), 43.54 (ClCH2CO). ESI-
MS (m/z, relative intensity): 363 ([M + H]+, 100), 279 (25), 274
(13). Anal. Calcd for C20H15N4OCl: C, 66.21; H, 4.17; N, 15.44.
Found: C, 66.45; H, 4.28; N, 15.25.
1-(4-Chloroacetamidophenyl)-4-(cyclopentylmethyl)-1H-
1
1,2,3-triazole (anti-4j). H NMR (400 MHz, CD3SOCD3) δ 10.54
(s, 1H, NH), 8.50 (s, 1H, triazole), 7.85 (d, J = 8.8 Hz, 2H, phenyl),
7.78 (d, J = 8.8 Hz, 2H, phenyl), 4.29 (s, 2H, ClCH2CO), 2.69 (d, J =
7.3 Hz, 2H, ArCH2), 2.25−2.11 (m, 1H, cyclopentyl-1), 1.83−1.67
(m, 2H, cyclopentyl-2a/5a), 1.67−1.56 (m, 2H, cyclopentyl-3a/4a),
1.56−1.42 (m, 2H, cyclopentyl-3b/4b), 1.30−1.14 (m, 2H, cyclo-
pentyl-2b/5b). 13C NMR (101 MHz, CD3SOCD3) δ 164.87 (CO),
147.52 (C-Ar), 138.35 (C-Ar), 132.49 (C-Ar), 120.43 (CH-phenyl),
120.16 (CH-phenyl), 120.08 (CH-triazole), 43.52 (ClCH2CO), 39.35
(cyclopentyl-1), 31.88 (cyclopentyl-2/5), 31.04 (ArCH2), 24.64
(cyclopentyl-3/4). HRMS (ESI-TOF) m/z: [M + H]+ calcd for
C16H20N4OCl 319.1326; found 319.1333.
1-(4-Chloroacetamidophenyl)-4-(diethoxymethyl)-1H-1,2,3-
triazole (anti-4k). 1H NMR (400 MHz, CD3SOCD3) δ 10.55 (s, 1H,
NH), 8.70 (s, 1H, triazole), 7.91 (d, J = 8.8 Hz, 2H, phenyl), 7.78 (d, J
= 8.7 Hz, 2H, phenyl), 5.74 (s, 1H, CH-acetal), 4.30 (s, 2H,
ClCH2CO), 3.71−3.48 (m, 4H, CH2 × 2), 1.17 (t, J = 7.0 Hz, 6H,
CH3 × 2). 13C NMR (101 MHz, CD3SOCD3) δ 164.93 (CO),
146.95 (C-Ar), 138.67 (C-Ar), 132.23 (C-Ar), 121.05 (CH-triazole),
120.81 (CH-phenyl), 120.11 (CH-phenyl), 96.06 (CH-acetal), 60.82
(CH2), 43.54 (ClCH2CO), 15.10 (CH3). HRMS (ESI-TOF) m/z: [M
+ Na]+ calcd for C15H19N4O3NaCl 361.1043; found 361.1031.
1-(4-Chloroacetamidophenyl)-4-(((tert-butoxycarbonyl)-
1-(4-Chloroacetamidophenyl)-4-([1,1′-biphenyl]-4-yl)-1H-
1
1,2,3-triazole (anti-4f). H NMR (400 MHz, CD3SOCD3) δ 10.60
(s, 1H, NH), 9.30 (s, 1H, triazole), 8.04 (d, J = 8.4 Hz, 2H, phenyl),
7.94 (d, J = 9.0 Hz, 2H, phenyl), 7.84 (d, J = 9.1 Hz, 2H, phenyl), 7.82
(d, J = 8.4 Hz, 2H, phenyl), 7.74 (d, J = 7.2 Hz, 2H, phenyl-2″/6″),
7.49 (t, J = 7.6 Hz, 2H, phenyl-3″/5″), 7.39 (t, J = 7.3 Hz, 1H, phenyl-
4″), 4.32 (s, 2H, ClCH2CO). 13C NMR (101 MHz, CD3SOCD3) δ
164.97 (CO), 146.90 (C-Ar), 139.79 (C-Ar), 139.52 (C-Ar), 138.78
(C-Ar), 132.29 (C-Ar), 129.37 (C-Ar), 128.99 (CH-phenyl-3″/5″),
127.62 (CH-phenyl-4″), 127.23 (CH-phenyl), 126.56 (CH-phenyl-2″/
6″), 125.86 (CH-phenyl), 120.76 (CH-phenyl), 120.25 (CH-phenyl),
119.56 (CH-triazole), 43.56 (ClCH2CO). HRMS (ESI-TOF) m/z:
[M + H]+ calcd for C22H18N4OCl 389.1169; found 389.1153.
1
amino)methyl)-1H-1,2,3-triazole (anti-4l). H NMR (400 MHz,
CD3SOCD3) δ 10.55 (s, 1H, ArNH), 8.49 (s, 1H, triazole), 7.86 (d, J
= 8.9 Hz, 2H, phenyl), 7.78 (d, J = 8.9 Hz, 2H, phenyl), 7.36 (t, J = 5.2
Hz, 1H, BocNH), 4.29 (s, 2H, ClCH2CO), 4.25 (d, J = 5.5 Hz, 2H,
G
DOI: 10.1021/acs.jmedchem.6b01237
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Article
ArCH2), 1.39 (s, 9H, CH3 × 3). 13C NMR (101 MHz, CD3SOCD3) δ
164.91 (CO), 155.56 (CO), 146.53 (C-Ar), 138.56 (C-Ar),
132.33 (C-Ar), 120.66 (CH-phenyl, CH-triazole), 120.20 (CH-
phenyl), 77.97 (C(CH3)3), 43.52 (ClCH2CO), 35.58 (ArCH2),
28.21 (CH3 × 3). HRMS (ESI-TOF) m/z: [M + H]+ calcd for
C16H21N5O3Cl 366.1333; found 366.1329.
1-(4-Chloroacetamidophenyl)-4-(pyridin-3-ylmethyl)-1H-
1,2,3-triazole (anti-4m). 1H NMR (400 MHz, CD3SOCD3) δ 10.62
(s, 1H, NH), 9.37 (s, 1H, triazole), 9.15 (br. s, 1H, pyridine-2), 8.60
(br s, 1H, pyridine-6), 8.30 (d, J = 7.9 Hz, 1H, pyridine-4), 7.92 (d, J =
8.9 Hz, 2H, phenyl), 7.84 (d, J = 8.9 Hz, 2H, phenyl), 7.55 (br s, 1H,
pyridine-5), 4.32 (s, 2H, ClCH2CO). 13C NMR (101 MHz,
CD3SOCD3) δ 165.01 (CO), 149.22 (CH-pyridine-6), 146.54
(CH-pyridine-2), 144.46 (C-Ar), 138.95 (C-Ar), 132.54 (CH-
pyridine-4), 132.14 (C-Ar), 124.17 (CH-pyridine-5), 120.91 (CH-
phenyl), 120.30 (CH-triazole), 120.27 (CH-phenyl), 43.57
(ClCH2CO). HRMS (ESI-TOF) m/z: [M + H]+ calcd for
C15H13N5OCl 314.0809; found 314.0808.
1-(4-Chloroacetamidophenyl)-5-(4-nitrophenyl)-1H-1,2,3-tri-
1
azole (syn-4b). H NMR (400 MHz, CD3SOCD3) δ 10.60 (s, 1H,
NH), 8.31 (s, 1H, triazole), 8.25 (d, J = 8.9 Hz, 2H, phenyl), 7.76 (d, J
= 8.8 Hz, 2H, phenyl), 7.57 (d, J = 8.9 Hz, 2H, phenyl), 7.43 (d, J =
8.8 Hz, 2H, phenyl), 4.29 (s, 2H, ClCH2CO). 13C NMR (101 MHz,
CD3SOCD3) δ 165.06 (CO), 147.50 (C-Ar), 139.73 (C-Ar), 135.82
(C-Ar), 134.13 (CH-triazole), 132.71 (C-Ar), 131.08 (C-Ar), 129.62
(CH-phenyl), 126.39 (CH-phenyl), 123.92 (CH-phenyl), 119.90
(CH-phenyl), 43.48 (ClCH2CO). HRMS (ESI-TOF) m/z: [M +
H]+ calcd for C16H13N5O3Cl 358.0707; found 358.0703.
1-(4-Chloroacetamidophenyl)-5-benzyl-1H-1,2,3-triazole
1
(syn-4c). H NMR (400 MHz, CD3SOCD3) δ 10.60 (s, 1H, NH),
7.77 (d, J = 8.8 Hz, 2H, phenyl), 7.58 (s, 1H, triazole), 7.48 (d, J = 8.8
Hz, 2H, phenyl), 7.25 (t, J = 7.2 Hz, 2H, phenyl-3′/5′), 7.20 (t, J = 7.2
Hz, 1H, phenyl-4′), 7.05 (d, J = 7.0 Hz, 2H, phenyl-2′/6′), 4.31 (s, 2H,
ClCH2CO), 4.08 (s, 2H, ArCH2). 13C NMR (101 MHz, CD3SOCD3)
δ 165.05 (CO), 139.45 (C-Ar), 137.42 (C-Ar), 136.95 (C-Ar),
133.04 (CH-triazole), 131.27 (C-Ar), 128.54 (CH-phenyl-3′/5′),
128.40 (CH-phenyl-2′/6′), 126.69 (CH-phenyl-4′), 125.95 (CH-
phenyl), 119.75 (CH-phenyl), 43.55 (ClCH2CO), 28.74 (ArCH2).
ESI-MS (m/z, relative intensity): 327 ([M + H]+, 100). Anal. Calcd
for C17H15N4OCl: C, 62.48; H, 4.63; N, 17.15. Found: C, 62.53; H,
4.78; N, 16.95.
1-(4-Acetamidophenyl)-4-(naphthalen-2-yl)-1H-1,2,3-tria-
1
zole (anti-6e). H NMR (400 MHz, CD3SOCD3) δ 10.25 (s, 1H,
NH), 9.36 (s, 1H, triazole), 8.51 (s, 1H, naphthalene-1), 8.08 (d, J =
8.7 Hz, 1H, naphthalene-3/4), 8.04 (d, J = 8.7 Hz, 1H, naphthalene-4/
3), 8.00 (d, J = 7.7 Hz, 1H, naphthalene-5/8), 7.95 (d, J = 7.5 Hz, 1H,
naphthalene-8/5), 7.91 (d, J = 8.3 Hz, 2H, phenyl), 7.83 (d, J = 8.5
Hz, 2H, phenyl), 7.65−7.46 (m, 2H, naphthalene-6/7), 2.10 (s, 3H,
CH3CO). 13C NMR (101 MHz, CD3SOCD3) δ 168.61 (CO),
147.16 (C-Ar), 139.67 (C-Ar), 133.12 (C-Ar), 132.67 (C-Ar), 131.62
(C-Ar), 128.60 (naphthalene-4/3), 128.00 (naphthalene-5/8), 127.79
(C-Ar), 127.72 (naphthalene-8/5), 126.68 (naphthalene-6/7), 126.25
(naphthalene-7/6), 123.67 (naphthalene-1, naphthalene-3/4), 120.65
(CH-phenyl), 119.82 (CH-phenyl), 119.70 (CH-triazole), 24.05
(CH3CO). HRMS (ESI-TOF) m/z: [M + H]+ calcd for C20H17N4O
329.1402; found 329.1404.
1-(4-Chloroacetamidophenyl)-5-(cyclohexylmethyl)-1H-
1
1,2,3-triazole (syn-4d). H NMR (400 MHz, CD3SOCD3) δ 10.61
(s, 1H, NH), 7.81 (d, J = 8.6 Hz, 2H, phenyl), 7.70 (s, 1H, triazole),
7.50 (d, J = 8.6 Hz, 2H, phenyl), 4.31 (s, 2H, ClCH2CO), 2.55 (d, J =
7.0 Hz, 2H, ArCH2), 1.63−1.47 (m, 5H, cyclohexyl-3a/5a, cyclohexyl-
4a, cyclohexyl-2a/6a), 1.47−1.36 (m,1H, cyclohexyl-1), 1.20−0.97 (m,
3H, cyclohexyl-3b/5b, cyclohexyl-4b), 0.80 (q, J = 11.2 Hz, 2H,
cyclohexyl-2b/6b). 13C NMR (101 MHz, CD3SOCD3) δ 165.05 (C
O), 139.41 (C-Ar), 136.91 (C-Ar), 132.48 (CH-triazole), 131.47 (C-
Ar), 126.18 (CH-phenyl), 119.85 (CH-phenyl), 43.53 (ClCH2CO),
36.56 (cyclohexyl-1), 32.25 (cyclohexyl-2/6), 30.10 (ArCH2), 25.65
(cyclohexyl-4), 25.36 (cyclohexyl-3/5). EI-MS (m/z, relative inten-
sity): 332 (M+, 41), 250 (30), 221 (100), 91 (50), 77 (55), 65 (40).
Anal. Calcd for C17H21N4OCl: C, 61.35; H, 6.36; N, 16.83. Found: C,
61.17; H, 6.45; N, 16.78.
1-(4-Chloroacetamidophenyl)-5-(naphthalen-2-yl)-1H-1,2,3-
triazole (syn-4e). 1H NMR (400 MHz, CD3SOCD3) δ 10.57 (s, 1H,
NH), 8.23 (s, 1H, triazole), 7.97 (s, 1H, naphthalene-1), 7.95−7.89
(m, 2H, naphthalene-3/4/5/8), 7.89−7.83 (m, 1H, naphthalene-8/5),
7.73 (d, J = 8.8 Hz, 2H, phenyl), 7.61−7.52 (m, 2H, naphthalene-6/7),
7.43 (d, J = 8.8 Hz, 2H, phenyl), 7.28 (dd, J = 8.5, 1.5 Hz, 1H,
naphthalene-4/3), 4.28 (s, 2H, ClCH2CO). 13C NMR (101 MHz,
CD3SOCD3) δ 164.99 (CO), 139.41 (C-Ar), 137.58 (C-Ar), 133.29
(CH-triazole), 132.60 (C-Ar), 132.49 (C-Ar), 131.60 (C-Ar), 128.34
(CH-naphthalene), 128.08 (CH-naphthalene), 128.00 (CH-naphtha-
lene), 127.61 (CH-naphthalene), 127.19 (CH-naphthalene-6/7),
126.90 (CH-naphthalene-7/6), 126.26 (CH-phenyl), 125.44 (naph-
thalene-4/3), 123.73 (C-Ar), 119.81 (CH-phenyl), 43.48 (ClCH2CO).
HRMS (ESI-TOF) m/z: [M + H]+ calcd for C20H16N4OCl 363.1013;
found 363.1016.
1-(4-Acetamidophenyl)-4-([1,1′-biphenyl]-4-yl)-1H-1,2,3-tri-
1
azole (anti-6f). H NMR (400 MHz, CD3SOCD3) δ 10.25 (s, 1H,
NH), 9.28 (s, 1H, triazole), 8.03 (d, J = 8.0 Hz, 2H, phenyl), 7.89 (d, J
= 8.8 Hz, 2H, phenyl), 7.86−7.78 (m, 4H, phenyl), 7.74 (d, J = 7.6 Hz,
2H, phenyl-2″/6″), 7.49 (t, J = 7.6 Hz, 2H, phenyl-3″/5″), 7.39 (t, J =
7.4 Hz, 1H, phenyl-4″), 2.10 (s, 3H, CH3CO). 13C NMR (101 MHz,
CD3SOCD3) δ 168.59 (CO), 146.80 (C-Ar), 139.73 (C-Ar), 139.65
(C-Ar), 139.51 (C-Ar), 131.60 (C-Ar), 129.40 (C-Ar), 128.95 (CH-
phenyl-3″/5″), 127.58 (CH-phenyl-4″), 127.19 (CH-phenyl), 126.53
(CH-phenyl-2″/6″), 125.82 (CH-phenyl), 120.64 (CH-phenyl),
119.68 (CH-phenyl), 119.49 (CH-triazole), 24.04(CH3CO). HRMS
(ESI-TOF) m/z: [M + H]+ calcd for C22H19N4O 355.1559; found
355.1557.
1-(4-Acetamidophenyl)-4-(diphenylhydroxymethyl)-1H-
1
1,2,3-triazole (anti-6g). H NMR (400 MHz, CD3SOCD3) δ 10.20
(s, 1H, NH), 8.45 (s, 1H, triazole), 7.84 (d, J = 9.0 Hz, 2H, phenyl),
7.76 (d, J = 8.6 Hz, 2H, phenyl), 7.43 (d, J = 7.2 Hz, 4H, phenyl-2′/6′
× 2), 7.31 (t, J = 7.5 Hz, 4H, phenyl-3′/5′ × 2), 7.24 (t, J = 7.2 Hz,
2H, phenyl-4′ × 2), 6.71 (s, 1H, OH), 2.08 (s, 3H, CH3CO). 13C
NMR (101 MHz, CD3SOCD3) δ 168.60 (CO), 155.03 (C-Ar),
146.83 (C-Ar), 139.47 (C-Ar), 131.65 (C-Ar), 127.62 (CH-phenyl-3′/
5′), 127.08 (CH-phenyl-2′/6′), 126.82 (CH-phenyl-4′), 121.03 (CH-
triazole), 120.64 (CH-phenyl), 119.61 (CH-phenyl), 75.86 (C-OH),
24.08(CH3CO). HRMS (ESI-TOF) m/z: [M + H]+ calcd for
C23H21N4O2 385.1665; found 385.1666.
General Procedure for Ruthenium-Catalyzed Alkyne−Azide
Cycloaddition. To the solution of 3 (105 mg, 0.500 mmol) and
alkyne (0.600 mmol, 1.2 equiv) in dioxane (3.6 mL) was added
Cp*RuCl(PPh3)2 (12.0 mg, 0.0150 mmol, 3.0%). The vial was purged
with argon, sealed, and heated at 60 °C until TLC analysis showed
complete consumption of the starting material (3). The reaction
mixture was subsequently diluted with water (20 mL) and extracted
with ethyl acetate (3 × 20 mL). The organic layers were combined,
washed with brine (10 mL), dried over Na2SO4, filtered, and
evaporated under reduced pressure. The resulting residue was purified
by flash chromatography (ethyl acetate/petrol ether) to yield the
desired syn-triazole (4).
1-(4-Chloroacetamidophenyl)-5-([1,1′-biphenyl]-4-yl)-1H-
1
1,2,3-triazole (syn-4f). H NMR (400 MHz, CD3SOCD3) δ 10.62
(s, 1H, NH), 8.19 (s, 1H, triazole), 7.77 (d, J = 8.8 Hz, 2H, phenyl),
7.72 (d, J = 8.4 Hz, 2H, phenyl), 7.69 (d, J = 7.3 Hz, 2H, phenyl-2″/
6″), 7.52−7.41 (m, 4H, phenyl), 7.41−7.31 (m, 3H, phenyl), 4.30 (s,
2H, ClCH2CO). 13C NMR (101 MHz, CD3SOCD3) δ 165.09 (C
O), 140.68 (C-Ar), 139.57 (C-Ar), 138.90 (C-Ar), 137.28 (C-Ar),
133.10 (CH-triazole), 131.68 (C-Ar), 129.02 (CH-phenyl-3″/5″),
128.82 (CH-phenyl), 127.96 (CH-phenyl-4″), 127.02 (CH-phenyl),
126.68 (CH-phenyl-2″/6″), 126.52 (CH-phenyl), 125.27 (C-Ar),
119.88 (CH-phenyl), 43.56 (ClCH2CO). HRMS (EI-TOF) m/z:
[M]+ calcd for C22H17N4OCl 388.1091; found 388.1092.
1-(4-Chloroacetamidophenyl)-5-(diphenylhydroxymethyl)-
1
1H-1,2,3-triazole (syn-4g). H NMR (400 MHz, CD3SOCD3) δ
10.39 (s, 1H, NH), 7.44 (d, J = 8.9 Hz, 2H, phenyl), 7.31−7.18 (m,
10H, phenyl-2′/3′/4′/5′/6′ × 2), 7.15 (s, 1H, triazole), 7.14 (d, J =
8.9 Hz, 2H, phenyl), 6.96 (s, 1H, OH), 4.25 (s, 2H, ClCH2CO). 13C
H
DOI: 10.1021/acs.jmedchem.6b01237
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
NMR (101 MHz, CD3SOCD3) δ 164.82 (CO), 144.67 (C-Ar),
143.96 (C-Ar), 138.72 (C-Ar), 134.51(CH-triazole), 132.74 (C-Ar),
127.81 (CH-phenyl-2′/6′/3′/5′), 127.32 (CH-phenyl/phenyl-4′),
127.28 (CH-phenyl-4′/phenyl), 126.53 (CH-phenyl-3′/5′/2′/6′),
118.23 (CH-phenyl), 74.75 (C-OH), 43.52 (ClCH2CO). EI-MS (m/
z, relative intensity): 418 (M+, 3), 236 (42), 208 (82), 182 (87), 131
(94), 105 (100), 91 (51), 77 (100), 63 (64), 51 (85). Anal. Calcd for
C23H19N4O2Cl: C, 65.95; H, 4.57; N, 13.38. Found: C, 66.18; H, 4.67;
N, 13.21.
ACKNOWLEDGMENTS
■
This work was financially supported by the National Natural
Science Foundation of China (Grant 21302182), China
Postdoctoral Science Foundation (Grants 2013M540133,
2014T70113), and the State Key Laboratory of Natural and
Biomimetic Drugs (Grants K20130211, K20140210,
K20150207). We thank the Protein Chemistry Facility at the
Center for Biomedical Analysis of Tsinghua University for
sample analysis.
1-(4-Chloroacetamidophenyl)-5-(2-(3-chlorophenoxy)-1-hy-
droxyethyl)-1H-1,2,3-triazole (syn-4h). 1H NMR (400 MHz,
CD3SOCD3) δ 10.63 (s, 1H, NH), 8.00 (s, 1H, triazole), 7.82 (d, J
= 8.9 Hz, 2H, phenyl), 7.61 (d, J = 8.8 Hz, 2H, phenyl), 7.27 (t, J = 8.3
Hz, 1H, phenyl-5′), 7.03−6.94 (m, 2H, phenyl-2′, phenyl-4′/6′),
6.92−6.83 (m, 1H, phenyl-6′/4′), 6.08 (s, 1H, OH), 4.95 (t, J = 5.8
ABBREVIATIONS USED
■
APBT, 1-(4-acetamidophenyl)-4-([1,1′-biphenyl]-4-yl)-1H-
1,2,3-triazole; APNT, 1-(4-acetamidophenyl)-4-(naphthalen-2-
yl)-1H-1,2,3-triazole; BADGP, benzyl-2-acetamido-2-deoxy-α-
D-galactopyranoside; BZX, benzoxazolinone; Con A, concana-
valin A; CuAAC, copper-catalyzed azide−alkyne cycloaddition;
DBA, Dolichos biflorus; DMEM, Dulbecco’s modified Eagle
medium; ECL, Erythrina cristagalli; GSL-I, Griffonia (Bandeir-
aea) simplicifolia I; HCD, higher energy collisional dissociation;
HRP, horseradish peroxidase; LCA, Lens culinaris; OGA, O-
GlcNAcase; OGT, O-GlcNAc transferase; PHA-E, Phaseolus
vulgaris erythroagglutinin; PNA, peanut agglutinin; PUGNAc,
(Z)-O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino N-
phenylcarbamate; PVDF, polyvinylidene fluoride; RuAAC,
ruthenium-catalyzed azide−alkyne cycloaddition; TGS, target-
guided synthesis; TISCC, tethering in situ click chemistry
Hz, 1H, CH), 4.32 (s, 2H, ClCH2CO), 4.27−4.15 (m, 2H, CH2). 13
C
NMR (101 MHz, CD3SOCD3) δ 165.09 (CO), 159.02 (C-Ar),
139.63 (C-Ar), 138.55 (C-Ar), 133.72 (C-Ar), 132.33 (CH-triazole),
131.38 (C-Ar), 130.83(CH-phenyl-5′), 126.08 (CH-phenyl), 120.86
(CH-phenyl-4′/6′), 119.85 (CH-phenyl), 114.59 (CH-phenyl-2′),
113.77 (CH-phenyl-6′/4′), 70.62 (CH2), 61.56 (CH), 43.56
(ClCH2CO). HRMS (ESI-TOF) m/z: [M + H]+ calcd for
C18H17N4O3Cl2 407.0678; found 407.0677.
1-(4-Chloroacetamidophenyl)-5-(1-hydroxy-2-methylprop-
1
yl)-1H-1,2,3-triazole (syn-4i). H NMR (400 MHz, CD3SOCD3) δ
10.63 (s, 1H, NH), 7.81 (d, J = 8.7 Hz, 2H, phenyl), 7.80 (s, 1H,
triazole), 7.54 (d, J = 8.8 Hz, 2H, phenyl), 5.62 (d, J = 5.5 Hz, 1H,
OH), 4.32 (s, 2H, ClCH2CO), 4.26 (dd, J = 7.4, 5.5 Hz, 1H, CHOH),
1.91−1.75 (m, 1H, CH(CH3)2), 0.83 (d, J = 6.6 Hz, 3H, CH3), 0.65
(d, J = 6.7 Hz, 3H, CH3). 13C NMR (101 MHz, CD3SOCD3) δ 165.10
(CO), 141.13 (C-Ar), 139.52 (C-Ar), 131.87 (CH-triazole), 131.66
(C-Ar), 126.29 (CH-phenyl), 119.84 (CH-phenyl), 68.25 (CHOH),
43.57 (ClCH2CO), 33.15 (CH(CH3)2), 18.83 (CH3), 18.06 (CH3).
ESI-MS (m/z, relative intensity): 309 ([M + H]+, 100). Anal. Calcd
for C14H17N4O2Cl: C, 54.46; H, 5.55; N, 18.15. Found: C, 54.50; H,
5.60; N, 18.03.
1-(4-Chloroacetamidophenyl)-5-(diethoxymethyl)-1H-1,2,3-
triazole (syn-4k). 1H NMR (400 MHz, CD3SOCD3) δ 10.61 (s, 1H,
NH), 7.85 (s, 1H, triazole), 7.79 (d, J = 8.9 Hz, 2H, phenyl), 7.58 (d, J
= 8.9 Hz, 2H, phenyl), 5.79 (s, 1H, CH-acetal), 4.32 (s, 2H,
ClCH2CO), 3.55−3.38 (m, 4H, CH2 × 2), 1.02 (t, J = 7.0 Hz, 6H,
CH3 × 2). 13C NMR (101 MHz, CD3SOCD3) δ 165.05 (CO),
139.47 (C-Ar), 136.30 (C-Ar), 132.90 (CH-triazole), 131.72 (C-Ar),
125.68 (CH-phenyl), 119.58 (CH-phenyl), 94.28 (CH-acetal), 61.37
(CH2), 43.56 (ClCH2CO), 14.86 (CH3). ESI-MS (m/z, relative
intensity): 339 ([M + H]+, 100), 237 (14). Anal. Calcd for
C15H19N4O3Cl: C, 53.18; H, 5.65; N, 16.54. Found: C, 53.42; H,
5.68; N, 16.30.
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* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.6b01237.
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tion of peptides (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: yuewang@ucas.ac.cn; yuewang@bjmu.edu.cn. Phone:
+86-10-88256092. Fax: +86-10-88256092.
ORCID
Yue Wang: 0000-0002-2574-6306
Notes
The authors declare no competing financial interest.
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