Synthesis of NAG-thiazoline-derived inhibitors for β-N-acetyl-d-hexosaminidases

Article abstract of DOI:10.1016/j.carres.2015.06.004

Abstract β-N-Acetyl-d-hexosaminidases are responsible for the metabolism of glycoconjugates in diverse physiological processes that are important targets for medicine and pesticide development. Fourteen new NAG-thiazoline derivatives were synthesized by cyclization and click reaction using d-glucosamine hydrochloride as the starting material. All the compounds created were characterized by NMR and HRMS spectra. A preliminary bioassay, using four enzymes from two β-N-acetyl-d-hexosaminidase families, showed that most of the compounds synthesized exhibit selective inhibition of GH84 β-N-acetyl-d-hexosaminidase. Among the compounds tested, compounds 5a (IC₅₀=12.6 μM, hOGA) and 5e (IC₅₀=12.5 μM, OfOGA) proved to be a highly selective and potent inhibitor.

Full text of DOI:10.1016/j.carres.2015.06.004

Carbohydrate Research 413 (2015) 135e144
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of NAG-thiazoline-derived inhibitors for
b
-N-acetyl- -hexosaminidases
D
,
Hanchu Kong a, Wei Chen b, Huizhe Lu a *, Qing Yang b, Yanhong Dong a, Daoquan Wang a,
,
*
Jianjun Zhang a
a Key Lab of Pesticide Chemistry and Application Technology, Department of Applied Chemistry, China Agricultural University, Beijing 100193, China
b School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China
a r t i c l e i n f o
a b s t r a c t
Article history:
b
-N-Acetyl-
ological processes that are important targets for medicine and pesticide development. Fourteen new
NAG-thiazoline derivatives were synthesized by cyclization and click reaction using -glucosamine hy-
drochloride as the starting material. All the compounds created were characterized by NMR and HRMS
spectra. A preliminary bioassay, using four enzymes from two -N-acetyl- -hexosaminidase families,
showed that most of the compounds synthesized exhibit selective inhibition of GH84 -N-acetyl-
hexosaminidase. Among the compounds tested, compounds 5a (IC50¼12.6 M, hOGA) and 5e
M, OfOGA) proved to be a highly selective and potent inhibitor.
D-hexosaminidases are responsible for the metabolism of glycoconjugates in diverse physi-
Received 15 March 2015
Received in revised form
28 May 2015
Accepted 1 June 2015
Available online 16 June 2015
D
b
D
b
D-
m
Keywords:
Synthesis
(IC50¼12.5
m
© 2015 Elsevier Ltd. All rights reserved.
b
-N-Acetyl-D-hexosaminidases
Selective inhibitors
NAG-thiazoline derivatives
1. Introduction
andhasproventoa goodtransitionstate (TS)analogue. However, this
inhibitor lacks selectivity between GH84 and GH20 b-N-acetyl-D-
b
-N-Acetyl-
D
-hexosaminidases (EC 3.2.1.52) catalyze the
-glucosamine or N-acetyl- -galac-
hexosaminidases. As a result of its lack of selectivity, this inhibitor
causes complex phenotypes when studying the physiological role of
its targets in cellular and physiological contexts. Further studies have
shown that the methyl group on the thiazoline ring can be modified
by a heteroatom, or longer chains, leading to an increase of inhibitory
potency and selectivity (Scheme 1: 1-2 to 1-4).11,18e20 The majority of
the derivative compounds reported thus farcontain substituted alkyl
chains; no aromatic group has been substituted while aromatic
groups are known to engage in hydrophobic and stacking in-
teractions with amino residues. A novel series of NAG-thiazoline
derivatives were synthesized by introducing substituted aromatic
phenyl (Scheme1:5ae5h)andsubstituted1,2,3-triazoles (Scheme1:
6ae6f) into the thiazoline ring. These compounds were studied to
assess the effect of increasing the size and aromaticity of these sub-
removal of -linked N-acetyl-
b
D
D
tosamine from glycoconjugates in a number of biological processes.
These enzymes are classified into three glycosyl hydrolase family
(GH3, GH20, GH84) based on amino acid sequence similarities.1,2
GH84 b-N-acetyl-D-hexosaminidase has been found to be related
to O-GlcNAc cycling in metazoan cells and dysfunction within this
significant physiological process leads to several human diseases
such as cancer, type II diabetes and Alzheimer's disease.3e7 GH20
b
-N-acetyl-D-hexosaminidase together with the GH18 chitinases,
involved in chitin degradation system in chitin-containing organ-
isms, have been shown to be specific fungicidal and pesticidal
targets because chitin is absent in higher plants and animals.8,9
Thus, the design and synthesis of novel inhibitors for
b-N-acetyl-
D
-hexosaminidases have been an active area of chemical, biological
stitutions on the inhibitory activity and selectivity towards
acetyl- -hexosaminidases.
b-N-
and pharmaceutical research.
D
Many small molecule inhibitors of b-N-acetyl-D-hexosaminidases
have been reported (Scheme 1: 1-4), such as NAG-thiazoline,10,11
PUGNAc,12,13 GlcNAcstatins,14,15 DNGNAc,16,17 and their derivatives.
NAG-thiazoline (Scheme 1: 1-1) is the one of most potent inhibitors,
2. Results and discussion
2.1. Synthesis of 1,2-dideoxy-20-aromatic substituted-
a-D-
glucopyrano-[2,1-d]-D
20-thiazolines
As shown in Scheme 2 (routes aec), key intermediate 1,3,4,6-
tetra-O-acetyl-2-deoxy-
-glucosamine hydrochloride 1021,22
* Corresponding authors. Fax: þ86 10 62732219.
E-mail addresses: luhz@cau.edu.cn (H. Lu), zhangjianjun@cau.edu.cn (J. Zhang).
b-D
http://dx.doi.org/10.1016/j.carres.2015.06.004
0008-6215/© 2015 Elsevier Ltd. All rights reserved.

136
H. Kong et al. / Carbohydrate Research 413 (2015) 135e144
HO
HO
HO
OH
O
O
COOH
HO
N
H
HO
HO
HO
HO
O
N
N
N
NH
S
N
O
O
O
R
CH3
HN
NAG-Thiazoline
1-1 R = Me
1-2 R = CH2CH2CH3
1-3 R = CH2N3
3 GlcNAcstatins
2 PUGNAc
1-4 R = NHCH2CH3
HO
HO
HO
HO
HO
O
OH
O
HO
NH
HO
S
N
S
N
HO
NHAc
N
N
N
R'
R''
4 DNGNAc
5a-5h
6a-6f
Scheme 1. Structure of small molecule inhibitors and target NAG-thiazoline derivatives.
Scheme 2. Synthesis of thiazolines 5ae5h. Reagents and conditions: (a) p-anisaladehyde, NaOH, H2O; (b) Py, Ac2O; (c) acetone, HCl, H2O; 60% over three steps (aec) for 10; (d) ROCl,
CH2Cl2, Et3N, 80e90%; (e) Lawesson's reagent, toluene, 80 ꢀC, 63e83%; (f) NaOMe, MeOH, 90e99%.
was obtained from commercially available
D
-glucosamine hydro-
The structure of thiazoline triacetate was confirmed by
comparing the 1H NMR spectrum between compounds 11 and 12.
chloride 7 in a procedure that used p-anisaldehyde for protection of
NH2, acetylation by Ac2O in Py and removal of the p-methox-
ybenzylidene group with HCl in acetone. It is noteworthy that these
three steps could be carried out easily without chromatographic
purification, which made the large-scale preparation of compound
10 possible with good yield (60%).
Subsequently, compound 10 was reacted with substituted ben-
zoyl chloride in anhydrous dichloromethane, and produced ben-
zamide (11ae11h, Scheme 2) in 80e90% yield. Lawesson's reagent
was then used for the selective formation of the thioamide, which
then cyclized by displacement of acetate to give the thiazoline
triacetates (12ae12h, Scheme 2) with a 63e83% yield.
As an example, Fig.1 shows a characteristic doublet for H-1 signal (
5.83 ppm, J1,2¼8.8 Hz) in compound 11b and the downfield shift
signal (
d
d
6.27 ppm, J1,2¼7.1 Hz) for H-1 in compound 12b. In addi-
tion, the NH signal and one of the four acetyl signals absent in the
1H NMR spectrum of 12b, confirmed the successful construction of
the thiazole ring.
Finally, methanolysis of the acetyls gave the 1,2-dideoxy-20-
aromatic
substituted-a-D-glucopyrano-[2,1-d]-D
20-thiazolines
5ae5h as a hygroscopic off-white solid in high yield (>90%). The 1H
NMR spectrum showed an H-1 signal
(d 6.16e6.47 ppm,
J1,2¼6.8e7.1 Hz) and the 13C NMR spectrum showed a C]N signal

H. Kong et al. / Carbohydrate Research 413 (2015) 135e144
137
Fig. 1. 1H NMR partial enlarged spectrogram of compounds 11b (a) and 12b (b).
(
d
164e176 ppm), characteristic of the structure of the thiazoline
spectrum of 19ae19f when detected in CDCl3. However, this NMR
signal could not be found in the 1H NMR spectrum of 5ae5f
detected in CD3OD. This result reveals that the hydrogen atom on
the methylene could be deuterated in protic solvents, confirming
the imine-to-enamine conversion in these NAG-thiazoline
derivatives.
5ae5h.
2.2. Synthesis of 1,2-dideoxy-20-(4-substituted-1,2,3-triazole-1-
methyl)-a-D-glucopyrano-[2,1-d]-D
20-thiazoline
The Cu(II)-catalyzed azide-alkyne cycloaddition reaction is a
powerful tool for an exclusive formation of the 1,4-disubstituted
1,2,3-triazoles.23 In this study, we introduced the azide group to
the methyl moiety on thiazoline ring, then provided the 1,4-
disubstituted 1,2,3-triazoles by ‘click chemistry’.
2.3. Inhibition studies
The prepared compounds 5ae5h and 6ae6f were assayed
against three different enzymes from two families at 25 mM (GH20:
OfHex from Ostrinia furnacalis and hHex from Homo sapiens; GH84:
OfOGA from O. furnacalis and hOGA from Homo sapiens). As shown
in Fig. 2, NAG-thiazoline is a potent inhibitor of these enzymes but
lacks selectivity, although most compounds show a lower inhibi-
tory activity towards GH84 b-N-acetyl-D-hexosaminidase relative
to the NAG-thiazoline (NGT, in Fig. 2). It is significant that none of
the compounds could exhibit any inhibitory activity towards GH20
1,3,4,6-Tetra-O-acetyl-2-deoxy-b-D-glucosamine hydrochloride
10 was first transformed into azidoacetamide 1324 according to
Scheme 3, however, compound 13 was unable to produce the
thiazoline 14 in toluene in the presence of Lawesson's reagent at
80 ꢀC. Subsequently, a mild condition (Lawesson's reagent/THF, rt)
was used and gave a product 15. The 1H NMR spectrum of com-
pound 15 showed the downfield shift of the azidoacetyl, H-2 and
NH signals compared with the data of compound 13. From this it
could be deduced that the reaction stopped at the thioamide 15 but
could not cyclize.
b
-N-acetyl-
D
-hexosaminidase. This result reveals that the active
-N-acetyl- -hexosaminidase could tolerate a
site pocket of GH84
b
D
larger group than the active site of GH20
minidase, leading to obvious selectivity.
We also found these compounds showed different inhibitory
activity towards GH84 -N-acetyl- -hexosaminidase while the
b-N-acetyl-D-hexosa-
To circumvent this problem, we changed our synthesis strategy
by making a use of the imine-to-enamine conversion in NAG-
thiazoline.19 Compound 10 was acylated by Ac2O and gave 1,3,4,6-
b
D
tetra-O-acetyl-2-N-acetyl-2-deoxy-b-D-glucopyranose 16, then
substituent group with similar size, and several compounds exhibit
thiazoline triacetate 17 (Scheme 3) was made available on a large
scale by treatment of compound 16 with Lawesson's reagent (81%,
two steps).10 Iodination of 17 in buffer solution provided a mono-
substituted compound 18 and replacement of the iodo with the
azido group gave the key intermediate 14 (66%, two steps).19
Coupling of 14 with alkynes in MeOH/H2O (1:1) solvent mixture
using copper(II) sulfate and sodium ascorbate, the 1,4-disubstituted
1,2,3-triazoles 19ae19f could be created in high yield (85e94%).
as a highly selective and potent inhibitor such as compounds 5a
(IC50 ¼ 12.6
m
M, hOGA; IC50 ¼ 323.3
m
M, OfOGA) and 5e (IC50
¼
113.8
m
M, hOGA; IC50 ¼ 12.5
m
M, OfOGA). It reveals that the sub-
stituent group could influence the interactions with residues by
changing the electron distribution on the aromatic group, and an
electron-withdrawing substituent group may prove helpful in
increasing the inhibitor's potency.
Finally, deacetylation of the hydroxyl groups by
methoxide-catalyst gave the target product 1,2-dideoxy-20-(4-
substituted-1,2,3-triazole-1-methyl)- -glucopyrano-[2,1-d]-
20-
a sodium
3. Conclusions
a
-D
D
thiazolines 6ae6f in 85e90% yield. The structure of the 1,2,3-
triazole moiety was identified by the presence of an olefinic pro-
We have introduced substituted aromatic phenyl and
substituted 1,2,3-triazoles into NAG-thiazoline and synthesized a
series of inhibitors for b-N-acetyl-D-hexosaminidase. The structures
ton signal, which appeared as a singlet at
d
7.39e8.43 ppm in the 1H
NMR spectrum. It is important to note that compounds 6ae6f were
unstable in acidic or neutral solutions; a drop of deuterated sodium
methoxide solution could be added to retain basic conditions when
detected by NMR.
of these compounds were confirmed by NMR and HRMS spectra.
Several compounds exhibit selectivity and potent inhibitory activ-
ities for GH84 b-N-acetyl-D-hexosaminidase. It is surprising to find
that the substituent groups on the phenyl group could influence the
interactions between the inhibitor and the enzyme by changing the
electron distribution on the aromatic group. These NAG-thiazoline
Interestingly, the methylene, which links the 1,2,3-triazole to
thiazoline ring showed a signal at
d
5.40 ppm in the 1H NMR

138
H. Kong et al. / Carbohydrate Research 413 (2015) 135e144
Scheme 3. Synthesis of 1,2,3-triazoles 6ae6h. Reagents and conditions: (a) N3CH2COCl, CH2Cl2, Et3N, 81%; (b) Lawesson's reagent, toluene, 80 ꢀC, 83% for 17; (c) Lawesson's reagent,
THF, rt, 94%; (d) CH2Cl2, Et3N, Ac2O, 90%; (e) I2, THF, 73%; (f) NaN3, DMF, 90%; (g) CuSO4, sodium ascorbate, RC^CH, 85e94%; (h) (6) K2CO3, MeOH, 85e90%.
4.2. Chemical synthesis
4.2.1. 2-Deoxy-2-[p-methoxybenzylidene(amino)]-b-D-
glucopyranose (8)21
D
-Glucosamine hydrochloride (25 g, 0.12 mol) and p-anisala-
dehyde (17 mL, 0.14 mmol) were added to a stirred solution of
sodium hydroxide (125 mL). The reaction mixture was stirred for
1 h at 0 ꢀC, a white precipitate was observed, the solid was filtered
and washed with water (100 mL), ethanol (100 mL) to give the
product as a white solid (29.0 g, 81%). [
a]
25 ꢁ63 (c 1.0, DMSO); mp
D
163e164 ꢀC; 1H NMR (300 MHz, DMSO-d6):
d 8.11 (s, 1H, NCHAr),
7.69 (d, J 8.8 Hz, 2H, ArH), 6.99 (d, J 8.8 Hz, 2H, ArH), 6.54 (s, 1H, OH),
4.96 (d, J 5.5 Hz, 1H, OH), 4.84 (s, 1H, OH), 4.69 (d, J1,2 7.7 Hz, H-1),
4.56 (s, 1H, OH), 3.79 (s, 3H, OMe), 3.69e3.73 (m, 1H, H-60),
3.41e3.50 (m, 2H, H-3, H-6), 3.14e3.25 (m, 2H, H-4, H-5), 2.75e2.80
(m, 1H, H-2).
Fig. 2. Inhibition of four b-N-acetyl-D-hexosaminidases by NAG-thiazoline derivatives.
derivatives may prove to be valuable lead compounds for devel-
oping new selective inhibitors.
4.2.2. 1,3,4,6-Tetra-O-acetyl-2-deoxy-b-D-glucosamine
4. Experimental
hydrochloride (10)21,22
To a solution of compound 8 (19.8 g, 67 mmol) in pyridine
(130 mL) was added acetic anhydride (70 mL). The mixture was
allowed to stir overnight, concentrated to give compound 9 as a
yellow solid, the residue was used without further purification.
To compound 9 (12 g, 26 mmol) dissolved in acetone (130 mL),
hydrochloric acid (6.6 mL, 5 N aqueous solution) was added drop-
wise, and the reaction mixture was stirred for 1 h at 0 ꢀC, the
4.1. General methods
All solvents were dried prior to use. 1H and 13C NMR spectra
were recorded on a Bruker AVANCE600 spectrometers in CDCl3 or
CD3OD soln. Flash chromatography under a positive pressure was
performed with a column of silica gel (200e300 mesh). High-res-
olution mass spectra (HRMS) was performed by the Beijing
Launcher Scientific Co. Ltd. Thin-layer chromatography (TLC) was
performed on silica gel HF with detection by charring with 30% (v/
v) H2SO4 in MeOH or by UV detection.
precipitate removed by filtration, dried to give the product as a
25
white solid (7.50 g, 75%). [
a
]
þ30 (c 1.0, MeOH); 1H NMR
D
(300 MHz, D2O):
d
5.90 (d, J1,2 8.8 Hz, 1H, H-1), 5.45 (dd, J2,3 10.6 Hz,
0
J3,4 9.4 Hz, 1H, H-3), 5.15 (t, J3,4, J4,5 9.4 Hz, 1H, H-4), 4.30 (dd, J6,6

H. Kong et al. / Carbohydrate Research 413 (2015) 135e144
139
0
0
13.2 Hz, J 5,6 4.4 Hz, 1H, H-6 ), 4.12e4.17 (m, 2H, H-5, H-6), 3.70 (dd,
J1,2 8.8 Hz, J2,3 10.6 Hz, 1H, H-2), 2.15, 2.07, 2.02(2) (4s, 12H,
4ꢂCH3CO).
CDCl3): d 7.60 (d, J 8.2 Hz, 2H, ArH), 7.14 (d, J 8.0 Hz, 2H, ArH), 6.79
(d, J 9.7 Hz, 1H, NH), 5.82 (d, J1,2 8.8 Hz, 1H, H-1), 5.44 (dd, J2,3
10.4 Hz, J3,4 9.8 Hz, 1H, H-3), 5.22 (t, J3,4, J4,5 9.8 Hz, 1H, H-4), 4.64
0
0
(dd, J1,2 8.8 Hz, J2,3 10.4 Hz, 1H, H-2), 4.30 (dd, J6,6 12.4 Hz, J5,6
4.9 Hz, 1H, H-60), 4.17 (dd, J6,6 12.4 Hz, J5,6 2.0 Hz, 1H, H-6),
3.90e3.94 (m,1H, H-5), 2.36 (s, 3H, ArCH3), 2.10, 2.08, 2.05, 2.00 (4s,
0
4.2.3. Representative procedure for the synthesis of 1,3,4,6-tetra-O-
acetyl-2-aromatic acyl-2-deoxy-b-D-glucopyranoses (11ae11f)
To a solution of compound 10 (2.68 g, 7 mmol) in CH2Cl2 (30 mL)
were added Et3N (3 mL, 21 mmol) and substituted benzoyl chloride
(8 mmol). The mixture was stirred at rt for 2 h, TLC (EtOAc) indi-
cated that the reaction was complete. The reaction mixture washed
with HCl (1 N, 2ꢂ10 mL), water (2ꢂ10 mL), then the organic phase
was dried over Na2SO4. Evaporation and purification by flash col-
umn chromatography (petroleum ether/EtOAc 3:1) afforded com-
pounds 11ae11f.
12H, 4ꢂCOCH3). 13C NMR (75 MHz, CDCl3):
d 171.60, 170.57, 169.49,
169.23, 167.20 (4ꢂCOCH3, CONH), 142.31, 130.73, 129.20, 126.90
(C6H5), 92.65 (C-1), 72.94, 72.87, 68.02, 61.82, 52.87, 21.30, 20.72,
20.60, 20.52, 20.44 (4ꢂCOCH3, ArCH3). HRMS (ESI) calcd for
C
22H28NO10 (MþHþ) 466.17077, found 466.17068.
4.2.3.5. 1,3,4,6-Tetra-O-acetyl-2-N-(3,5-dinitro)-benzoyl-2-deoxy-b-
D
-glucopyranose (11e). White solid (3.25 g, 83%); Rf 0.80 (EtOAc);
25
[a]
þ25 (c 0.5, CHCl3); mp 221e223 ꢀC; 1H NMR (300 MHz,
D
4.2.3.1. 1,3,4,6-Tetra-O-acetyl-2-N-(2-fluoro)-benzoyl-2-deoxy-b-D-
CDCl3): d 9.12 (t, J 2.0 Hz,1H, ArH), 8.95 (d, J 2.1 Hz, 2H, ArH), 7.22 (d,
glucopyranose (11a). White solid (2.79 g, 85%); Rf 0.89 (EtOAc);
J 9.3 Hz, 1H, NH), 5.82 (d, J1,2 8.7 Hz, 1H, H-1), 5.44 (dd, J2,3 10.5 Hz,
J3,4 9.7 Hz, 1H, H-3), 5.26 (t, J3,4, J4,5 9.7 Hz, 1H, H-4), 4.64 (dd, J1,2
25
[
a]
þ85 (c 0.5, CHCl3); mp 222e224 ꢀC; 1H NMR (300 MHz,
D
CDCl3): d 7.95 (dt, J 7.8, 1.7 Hz, 1H, ArH), 7.44e7.49 (m, 1H, ArH), 7.23
0
0
8.7 Hz, J2,3 10.5 Hz, 1H, H-2), 4.31 (dd, J6,6 12.5 Hz, J5,6 5.0 Hz, 1H, H-
60), 4.17 (dd, J6,6 12.5 Hz, J5,6 2.2 Hz, 1H, H-6), 3.96e4.01 (m, 1H, H-
(d, J 7.7 Hz, 1H, NH), 7.08 (dd, J 11.4, 8.3 Hz, 1H, ArH), 6.72 (t, J
10.4 Hz, 1H, ArH), 5.90 (d, J1,2 8.7 Hz, 1H, H-1), 5.39 (dd, J2,3 10.3 Hz,
J3,4 9.7 Hz, 1H, H-3), 5.19 (t, J3,4, J4,5 9.7 Hz, 1H, H-4), 4.53 (dd, J1,2
0
5), 2.12, 2.10, 2.09, 2.04 (4s, 12H, 4ꢂCOCH3). 13C NMR (75 MHz,
CD3OD):
d
170.18, 169.93, 169.42, 169.00, 162.71 (4ꢂCOCH3, CONH),
0
0
8.7 Hz, J2,3 10.3 Hz, 1H, H-2), 4.31 (dd, J6,6 12.3 Hz, J5,6 4.7 Hz, 1H, H-
148.45, 136.37, 127.53, 121.43 (C6H5), 91.74 (C-1), 72.33, 71.91, 68.18,
61.65, 53.29, 20.65, 20.63, 20.54, 20.42 (4ꢂCOCH3). HRMS (ESI)
calcd for C21H27N4O14 ðM þ NHþ4 Þ 559.15183, found 559.15189.
60), 4.15 (dd, J6,6 12.3 Hz, J5,6 2.1 Hz, 1H, H-6), 3.88e3.93 (m, 1H, H-
0
5), 2.10, 2.09, 2.03, 2.00 (4s, 12H, 4ꢂCH3CO). 13C NMR (75 MHz,
CDCl3):
d
170.73, 170.61, 169.32(2), 163.64 (JCeF 2.6 Hz) (4ꢂCOCH3,
CONH), 160.33 (JCeF 246.7 Hz), 133.65 (JCeF 9.3 Hz), 131.68 (JCeF
2.0 Hz), 124.84 (JCeF 3.2 Hz), 120.71 (JCeF 11.9 Hz), 116.65 (JCeF
24.1 Hz) (C6H5), 92.48 (C-1), 72.95, 72.19, 68.02, 61.74, 53.21, 20.77,
20.68, 20.54, 20.47 (4ꢂCOCH3). HRMS (ESI) calcd for C21H28FN2O10
ðM þ NHþ4 Þ 487.17225, found 487.17215.
4.2.3.6. 1,3,4,6-Tetra-O-acetyl-2-N-(1-Naphthaleneacetyl)-2-deoxy-
b-D-glucopyranose (11f). White solid (2.87 g, 77%); Rf 0.89 (EtOAc);
25
[a
]
þ77 (c 0.5, CHCl3); mp 202e204 ꢀC; 1H NMR (300 MHz,
D
CDCl3): d 7.33e7.95 (m, 7H, ArH), 5.66 (d, J 9.2 Hz, 1H, NH), 5.55 (d,
J1,2 8.8 Hz, 1H, H-1), 5.01e5.05 (m, 2H, H-4, H-3), 4.09e4.22 (m, 2H,
H-60, H-2), 4.03 (dd, J6,6 12.5 Hz, J5,6 2.2 Hz, 1H, H-6), 3.93 (s, 2H,
0
4.2.3.2. 1,3,4,6-Tetra-O-acetyl-2-N-(4-methoxy)-benzoyl-2-deoxy-b-
AreCH2), 3.69e3.72 (m, 1H, H-5), 2.04, 1.97, 1.85, 1.70 (4s, 12H,
D
-glucopyranose (11b). White solid (2.97 g, 88%); Rf 0.86 (EtOAc);
4ꢂCOCH3). 13C NMR (75 MHz, CDCl3):
d 170.88, 170.52, 170.48,
25
[
a]
þ80 (c 0.5, CHCl3); mp 197e199 ꢀC; 1H NMR (300 MHz,
D
169.16, 169.03 (4ꢂCOCH3, CONH), 133.95, 131.88, 130.52, 128.84,
128.58, 128.16, 126.76, 126.11, 125.61, 123.50 (ArC), 92.15 (C-1),
72.71, 71.86, 67.81, 61.58, 53.04, 41.86 (AreCH2), 20.59, 20.47, 20.43,
20.10 (4ꢂCOCH3). HRMS (ESI) calcd for C26H33N2O10 ðM þ NHþ4 Þ
533.21297, found 533.21295.
CDCl3): d 7.74 (d, J 8.8 Hz, 2H, ArH), 6.90 (d, J 9.7 Hz,1H, NH), 6.82 (d,
J 8.8 Hz, 2H, ArH), 5.83 (d, J1,2 8.8 Hz, 1H, H-1), 5.45 (t, J2,3, J3,4
10.3 Hz, 1H, H-3), 5.19 (t, J3,4, J4,5 10.3 Hz, 1H, H-4), 4.64 (dd, J1,2
0
0
8.8 Hz, J2,3 10.3 Hz, 1H, H-2), 4.30 (dd, J6,6 12.5 Hz, J5,6 4.9 Hz, 1H, H-
60), 4.11e4.20 (m, 1H, H-6), 3.91e3.99 (m, 1H, H-5), 3.81 (s, 1H,
OCH3), 2.10, 2.09, 2.05, 2.01 (4s, 12H, 4ꢂCOCH3). 13C NMR (75 MHz,
4.2.3.7. 1,3,4,6-Tetra-O-acetyl-2-N-(4-phenyl)-benzoyl-2-deoxy-b-D-
CDCl3):
d
171.63, 170.56, 169.45, 169.27, 166.81 (4ꢂCOCH3, CONH),
glucopyranose (11g). White solid (2.85 g, 87%); Rf 0.85 (EtOAc);
162.38,128.77,125.83,113.70 (C6H5), 92.63 (C-1), 72.97, 72.87, 68.13,
61.88, 55.24 (OCH3), 52.81, 20.70, 20.57, 20.52, 20.43 (4ꢂCOCH3).
HRMS (ESI) calcd for C22H28NO11 (MþHþ) 482.16569, found
482.16565.
25
[a
]
þ46 (c 0.5, CHCl3); mp 232e234 ꢀC; 1H NMR (300 MHz,
D
CDCl3): d 8.64 (d, J 9.2 Hz, 1H, NH), 7.39e7.88 (m, 9H, ArH), 5.95 (d,
J1,2 8.8 Hz,1H, H-1), 5.41 (dd, J2,3 10.2 Hz, J3,4 9.9 Hz,1H, H-3), 5.03 (t,
J3,4 J4,5 9.9 Hz, 1H, H-4), 4.24e4.33 (m, 2H, H-60, H-2), 4.00e4.08 (m,
2H, H-5, H-6), 2.05, 2.03, 2.02, 1.90 (4s, 12H, 4ꢂCOCH3). 13C NMR
4.2.3.3. 1,3,4,6-Tetra-O-acetyl-2-N-(4-ethoxy)-benzoyl-2-deoxy-b-D-
(75 MHz, CDCl3):
d
170.43,170.05, 169.66, 169.24, 166.60 (4ꢂCOCH3,
glucopyranose (11c). White solid (3.12 g, 90%); Rf 0.90 (EtOAc);
25
CONH), 143.58, 139.56, 139.21, 129.41, 128.48, 128.18, 127.29, 127.10
(ArC), 92.34 (C-1), 72.84, 72.15, 68.47, 61.92, 53.04, 20.87 (2), 20.78,
20.64 (4ꢂCOCH3). HRMS (ESI) calcd for C27H30NO10 (MþHþ)
528.18642, found 528.18671.
[a]
þ65 (c 0.5, CHCl3); mp 207e209 ꢀC; 1H NMR (300 MHz,
D
CDCl3):
d 7.66 (dd, J 6.9, 1.9 Hz, 2H, ArH), 6.84 (d, J 9.9 Hz, 1H, NH),
6.80 (d, J 8.9 Hz, 2H, ArH), 5.82 (d, J1,2 8.8 Hz, 1H, H-1), 5.44 (dd, J2,3
10.5 Hz, J3,4 9.5 Hz, 1H, H-3), 5.22 (t, J3,4, J4,5 9.5 Hz, 1H, H-4), 4.64
0
(dd, J1,2 8.8 Hz, J2,3 10.5 Hz,1H, H-2), 4.30 (dd, J6,6 12.4 Hz, J5,6 5.0 Hz,
1H, H-60), 4.17 (dd, J6,6 12.4 Hz, J5,6 2.1 Hz, 1H, H-6), 4.02 (q, J 6.9 Hz,
4.2.3.8. 1,3,4,6-Tetra-O-acetyl-2-N-(indole-3-acetyl)-2-deoxy-b-D-
0
2H, OCH2), 3.92e3.99 (m, 1H, H-5), 2.10, 2.09, 2.05, 2.01 (4 s, 12H,
glucopyranose (11h). White solid (2.92 g, 80%); Rf 0.93 (EtOAc);
25
4ꢂCOCH3). 13C NMR (75 MHz, CDCl3):
d
171.65, 170.56, 169.48,
[a
]
þ95 (c 0.5, CHCl3); mp 187e189 ꢀC; 1H NMR (300 MHz,
D
169.24, 166.81 (4ꢂCOCH3, CONH), 161.80, 128.74, 125.59, 114.17
(C6H5), 92.66 (C-1), 72.98, 72.91, 68.07, 63.54 (OCH2), 61.86, 52.80,
20.72, 20.58, 20.53, 20.43 (4ꢂCOCH3),14.54 (CH3). HRMS (ESI) calcd
for C23H30NO11 (MþHþ) 496.18134, found 496.18134.
CDCl3): d 8.51 (s, 1H, NCH]C), 7.08e7.49 (m, 5H, ArH), 5.88 (d, J
9.3 Hz, 1H, NH), 5.58 (d, J1,2 8.8 Hz, 1H, H-1), 5.00e5.10 (m, 2H, H-4,
H-3), 4.11e4.23 (m, 2H, H-60, H-2), 4.06 (dd, J6,6 12.4 Hz, J5,6 2.1 Hz,
0
1H, H-6), 3.69e3.72 (m, 1H, H-5), 3.66 (s, 2H, AreCH2), 2.06, 1.97,
1.93, 1.80 (4s, 12H, 4ꢂCOCH3). 13C NMR (75 MHz, CDCl3):
d 171.57,
4.2.3.4. 1,3,4,6-Tetra-O-acetyl-2-N-(4-methyl)-benzoyl-2-deoxy-
b
-
D
-
170.62 (2), 169.25, 169.25 (4ꢂCOCH3, CONH), 136.44, 126.85, 123.57,
122.77, 120.17, 118.45, 111.45, 108.51 (ArC), 92.30 (C-1), 72.77, 72.00,
67.90, 61.63, 53.06, 33.59 (AreCH2), 20.66, 20.61, 20.49, 20.24
glucopyranose (11d)25. White solid (2.64 g, 81%); Rf 0.92 (EtOAc);
25
[
a]
þ68 (c 0.5, CHCl3); mp 210e212 ꢀC; 1H NMR (300 MHz,
D

140
H. Kong et al. / Carbohydrate Research 413 (2015) 135e144
(4ꢂCOCH3). HRMS (ESI) calcd for C24H32N3O10 ðM þ NHþ4 Þ
69.38, 68.37, 63.25, 21.43 (AreCH3), 20.91, 20.74, 20.63 (3ꢂCOCH3).
HRMS (ESI) calcd for C20H24NO7S (MþHþ) 422.12680, found
422.12653.
522.20822, found 522.20856.
4.2.4. Representative procedure for the synthesis of 3,4,6-tri-O-
acetyl-1,2-dideoxy-20-aromatic substituted-
a-D
-glucopyrano-[2,1-
4.2.4.5. 3,4,6-Tri-O-acetyl-1,2-dideoxy-20-(3,5-dinitro)-phenyl-
a-D-
d]-D
20-thiazolines (12ae12f)
Lawesson's reagent (1.20 g, 3.0 mmol) was added to compounds
glucopyrano-[2,1-d]-
63%); Rf 0.68 (petroleum ether/EtOAc 1:1); [
1H NMR (300 MHz, CDCl3):
D
20-thiazoline (12e). Yellow syrup (1.57 g,
a
]
25 þ10 (c 0.3, CHCl3);
D
11ae11f (5 mmol) in toluene (20 mL). The mixture was stirred at
80 ꢀC for 4 h, TLC (petroleum ether/EtOAc 1:1) indicated that the
reaction was complete. Concentrated under reduced pressure and
purification by flash column chromatography (petroleum ether/
EtOAc 3:1) afforded compounds 12ae12f.
d
9.17 (t, J 2.1 Hz, 1H, ArH), 8.99 (d, J
2.1 Hz, 2H, ArH), 6.56 (d, J1,2 7.2 Hz, 1H, H-1), 5.72 (dd, J2,3 3.3 Hz, J3,4
1.4 Hz, 1H, H-3), 5.03 (dt, J4,5 9.2 Hz, J3,4 1.4 Hz, 1H, H-4), 4.84e4.87
(m,1H, H-2), 4.14 (d, J 4.5 Hz, 2H, H-6, H-60), 3.54e3.60 (m,1H, H-5),
2.21, 2.09, 2.08 (3s, 9H, 3ꢂCOCH3). 13C NMR (75 MHz, CDCl3):
d
170.43, 169.31, 169.28 (3ꢂCOCH3), 164.52 (N]CS), 148.65, 135.86,
4.2.4.1. 3,4,6-Tri-O-acetyl-1,2-dideoxy-20-(2-fluoro)-phenyl-
a
-
D
-glu-
128.08, 120.90 (C6H5), 89.61 (C-1), 77.17, 70.13, 69.08, 69.05, 63.09,
copyrano-[2,1-d]-
0.55 (petroleum ether/EtOAc 1:1); [
(300 MHz, CDCl3):
D
20-thiazoline (12a). Yellow syrup (1.62 g, 76%); Rf
20.87, 20.72, 20.63 (3ꢂCOCH3). HRMS (ESI) calcd for C19H20N3O11
S
a
]
D
25 þ27 (c 0.3, CHCl3); 1H NMR
(MþHþ) 498.08131, found 498.08127.
d
7.15e7.93 (m, 4H, ArH), 6.34 (d, J1,2 7.2 Hz, 1H,
H-1), 5.74 (dd, J2,3 3.3 Hz, J3,4 1.4 Hz, 1H, H-3), 5.00 (dt, J4,5 9.3 Hz, J3,4
1.4 Hz, 1H, H-4), 4.68 (dd, J1,2 7.2 Hz, J2,3 3.3 Hz, 1H, H-2), 4.13 (d, J
4.5 Hz, 2H, H-6, H-60), 3.59e3.63 (m, 1H, H-5), 2.18, 2.09, 2.08 (3s,
4.2.4.6. 3,4,6-Tri-O-acetyl-1,2-dideoxy-20-(1-menaphthyl)-
a-D-glu-
copyrano-[2,1-d]-
0.59 (petroleum ether/EtOAc 1:1); [
(300 MHz, CDCl3):
D
20-thiazoline (12f). Yellow syrup (1.96 g, 83%); Rf
a]
25 þ30 (c 0.3, CHCl3); 1H NMR
D
9H, 3ꢂCOCH3). 13C NMR (75 MHz, CDCl3):
d
170.51, 169.47, 169.28
d
7.44e8.11 (m, 7H, ArH), 6.11 (d, J1,2 7.1 Hz, 1H,
(3ꢂCOCH3), 163.49 (JCeF 4.8 Hz) (N]CS), 160.53 (JCeF 253.8 Hz),
133.09 (JCeF 8.8 Hz), 130.28 (JCeF 2.1 Hz), 124.30 (JCeF 3.5 Hz), 120.81
(JCeF 11.0 Hz), 116.50 (JCeF 22.0 Hz) (C6H5), 87.57 (JCeF 4.0 Hz) (C-1),
75.55, 70.45, 69.33, 68.57, 63.37, 20.94, 20.78, 20.67 (3ꢂCOCH3).
HRMS (ESI) calcd for C19H21FNO7S (MþHþ) 426.10173, found
426.10165.
H-1), 5.61 (dd, J2,3 3.2 Hz, J3,4 1.5 Hz,1H, H-3), 4.91 (dt, J4,5 9.6 Hz, J3,4
1.5 Hz, 1H, H-4), 4.51e4.54 (m, 1H, H-2), 4.42 (dd, J 15.4 Hz, 1.5 Hz,
1H, AreCH), 4.25 (dd, J 15.4, 2.2 Hz, 1H, AreCH0), 3.91e3.93 (m, 2H,
H-6, H-60), 3.37e3.43 (m, 1H, H-5), 2.21, 2.07, 1.95 (3s, 9H,
3ꢂCOCH3). 13C NMR (75 MHz, CDCl3):
d 172.35, 170.32, 169.39,
169.16 (3ꢂCOCH3, N]CS), 133.81, 131.97, 131.78, 128.71, 128.37,
127.81, 126.29, 125.87, 125.45, 123.71 (ArC), 87.93 (C-1), 76.41, 70.57,
69.26, 68.18, 62.86, 39.18 (AreCH2), 20.84, 20.74, 20.53 (3ꢂCOCH3).
4.2.4.2. 3,4,6-Tri-O-acetyl-1,2-dideoxy-20-(4-methoxy)-phenyl-
a-D-
glucopyrano-[2,1-d]-
80%); Rf 0.70 (petroleum ether/EtOAc 1:1); [
1H NMR (300 MHz, CDCl3):
D
20-thiazoline (12b). Yellow syrup (1.75 g,
HRMS (ESI) calcd for
472.14218.
C
24H26NO7S (MþHþ) 472.14245, found
a
]
D
25 þ22 (c 0.3, CHCl3);
d
7.74 (dd, J 6.8, 2.1 Hz, 2H, ArH),
6.85e6.89 (m, 2H, ArH), 6.27 (d, J1,2 7.1 Hz, 1H, H-1), 5.63 (dd, J2,3
3.3 Hz, J3,4 1.4 Hz, 1H, H-3), 4.92 (dt, J4,5 9.4 Hz, J3,4 1.4 Hz, 1H, H-4),
4.62e4.66 (m, 1H, H-2), 4.03e4.05 (m, 2H, H-6, H-60), 3.79 (s, 3H,
OCH3), 3.53e3.56 (m, 1H, H-5), 2.10, 2.01, 1.99 (3s, 9H, 3ꢂCOCH3).
4.2.4.7. 3,4,6-Tri-O-acetyl-1,2-dideoxy-20-biphenyl-
a-D-glucopyrano-
[2,1-d]-
(petroleum ether/EtOAc 1:1); [
(300 MHz, CDCl3):
D
20-thiazoline (12g). Yellow syrup (1.94 g, 80%); Rf 0.63
25
a]
þ25 (c 0.3, CHCl3); 1H NMR
D
d
7.37e7.95 (m, 9H, ArH), 6.38 (d, J1,2 7.1 Hz, 1H,
13C NMR (75 MHz, CDCl3):
d
170.53, 169.52, 169.33 (3ꢂCOCH3),
H-1), 5.01 (dt, J4,5 9.4 Hz, J3,4 1.5 Hz, 1H, H-4), 4.74e4.78 (m, 1H, H-
167.48 (N]CS), 162.42, 130.27, 125.38, 113.91 (C6H5), 87.84 (C-1),
77.00, 70.84, 69.45, 68.41, 63.30, 55.43 (OMe), 20.97, 20.80, 20.69
(3ꢂCOCH3). HRMS (ESI) calcd for C20H24NO8S (MþHþ) 438.12171,
found 438.12140.
2), 4.12e4.14 (m, 2H, H-6, H-60), 3.61e3.67 (m, 1H, H-5), 2.18, 2.08,
2.07 (3s, 9H, 3ꢂCOCH3). 13C NMR (75 MHz, CDCl3):
d 171.88, 170.88,
170.69 (3ꢂCOCH3), 169.33 (N]CS), 145.96, 141.29, 132.81, 130.42,
130.30, 129.44, 128.63, 128.51 (ArC), 89.25 (C-1), 78.39, 72.12, 70.83,
69.88, 64.68, 22.33, 22.17, 22.01 (3ꢂCOCH3). HRMS (ESI) calcd for
4.2.4.3. 3,4,6-Tri-O-acetyl-1,2-dideoxy-20-(4-ethoxy)-phenyl-
a
-
D
-
C
25H26NO7S (MþHþ) 484.14245, found 484.14221.
glucopyrano-[2,1-d]-
75%); Rf 0.65 (petroleum ether/EtOAc 1:1); [
1H NMR (300 MHz, CDCl3):
D
20-thiazoline (12c). Yellow syrup (1.70 g,
a]
25 þ33 (c 0.3, CHCl3);
4.2.4.8. 3,4,5-Tri-O-acetyl-1,2-dideoxy-20-3-methylindole-
a-D-gluco-
D
d
7.80e7.82 (m, 2H, ArH), 6.90e6.95 (m,
pyrano-[2,1-d]-
0.60 (petroleum ether/EtOAc 1:1); [
(300 MHz, CDCl3):
D
20-thiazoline (12h). Yellow syrup (1.68 g, 73%); Rf
2H, ArH), 6.34 (d, J1,2 7.1 Hz, 1H, H-1), 5.70 (dd, J2,3 3.3 Hz, J3,4 1.4 Hz,
1H, H-3), 4.99 (dt, J4,5 9.4 Hz, J3,4 1.4 Hz, 1H, H-4), 4.69e4.72 (m, 1H,
H-2), 4.05e4.12 (m, 4H, H-6, H-60, OCH2), 3.59e3.65 (m, 1H, H-5),
2.17, 2.08, 2.06 (3s, 9H, 3ꢂCOCH3). 13C NMR (75 MHz, CDCl3):
a]
25 þ43 (c 0.3, CHCl3); 1H NMR
D
d
8.47 (s, 1H, NCH]C), 7.06e7.64 (m, 5H, ArH),
6.13 (d, J1,2 7.1 Hz, 1H, H-1), 5.64 (dd, J2,3 3.2 Hz, J3,4 1.4 Hz, 1H, H-3),
4.96 (dt, J4,5 9.6 Hz, J3,4 1.4 Hz, 1H, H-4), 4.53e4.56 (m, 1H, H-2),
3.93e4.15 (m, 4H, H-6, H-60, AreCH2), 3.48e3.54 (m, 1H, H-5), 2.12,
d
170.48, 169.47, 169.28 (3ꢂCOCH3), 167.45 (N]CS), 161.77, 130.22,
125.13, 114.33 (C6H5), 87.77 (C-1), 76.97, 70.81, 69.41, 68.35, 63.66
(OCH2), 63.26, 20.93, 20.77, 20.66 (3ꢂCOCH3), 14.61 (CH3). HRMS
(ESI) calcd for C21H26NO8S (MþHþ) 452.13736, found 452.13718.
2.08, 1.97 (3s, 9H, 3ꢂCOCH3). 13C NMR (75 MHz, CDCl3):
d 173.13,
170.49, 169.52, 169.28 (3ꢂCOCH3, N]CS), 136.24, 126.94, 123.14,
122.28, 119.51, 118.70, 111.24, 109.80 (ArC), 87.85 (C-1), 76.50, 70.71,
69.27, 68.18, 62.97, 31.22 (AreCH2), 20.85, 20.71, 20.55 (3ꢂCOCH3).
HRMS (ESI) calcd for C22H25N2O7S (MþHþ) 461.13770, found
461.13766.
4.2.4.4. 3,4,6-Tri-O-acetyl-1,2-dideoxy-20-(4-methyl)-phenyl-
a-D-
glucopyrano-[2,1-d]-
82%); Rf 0.66 (petroleum ether/EtOAc 1:1); [
1H NMR (300 MHz, CDCl3):
D
20-thiazoline (12d). Yellow syrup (1.73 g,
a
]
D
25 þ37 (c 0.3, CHCl3);
d
7.75 (d, J 8.2 Hz, 2H, ArH), 7.25 (d, J
4.2.5. Representative procedure for the synthesis of 1,2-dideoxy-20-
aromatic substituted-a-D-glucopyrano-[2,1-d]-D
20-thiazolines
(5ae5f)
A solution of compounds 12ae12f (1 mmol) in 5 mL of methanol
was treated with sodium methoxide (0.1 mL of a 1 M solution in
methanol) and the reaction mixture was stirred at rt for 30 min. The
reaction mixture was neutralized to pH 7 by Amberlyst Hþ resin,
8.0 Hz, 2H, ArH), 6.35 (d, J1,2 7.1 Hz, 1H, H-1), 5.72 (dd, J2,3 3.3 Hz, J3,4
1.4 Hz, 1H, H-3), 5.00 (dt, J4,5 9.4 Hz, J3,4 1.4 Hz, 1H, H-4), 4.71e4.75
(m, 1H, H-2), 4.10e4.12 (m, 2H, H-6, H-60), 3.58e3.64 (m, 1H, H-5),
2.44 (s, 3H, AreCH3), 2.17, 2.07, 2.05 (3s, 9H, 3ꢂCOCH3). 13C NMR
(75 MHz, CDCl3):
CS), 142.25, 129.90, 129.23, 128.47 (C6H5), 87.68 (C-1), 76.95, 70.68,
d
170.45, 169.45, 169.25 (3ꢂCOCH3), 168.14 (N]

H. Kong et al. / Carbohydrate Research 413 (2015) 135e144
141
concentrated and chromatographed with 1:20 methanol/EtOAc to
afford compounds 5ae5f.
4.2.5.6. 1,2-Dideoxy-20-(1-menaphthyl)-
a
-
D
-glucopyrano-[2,1-d]-
D
20-thiazoline (5f). Hygroscopic off-white solid (0.31 g, 90%); Rf
25
0.18 (EtOAc-methanol 10:1); [
(300 MHz, CD3OD):
a
]
D
þ44 (c 0.5, MeOH); 1H NMR
d
7.30e8.00 (m, 7H, ArH), 6.16 (d, J1,2 7.1 Hz, 1H,
4.2.5.1. 1,2-Dideoxy-20-(2-fluoro)-phenyl-
a-D-glucopyrano-[2,1-d]-
H-1), 4.20e4.29 (m, 1H, H-2), 4.00 (app t, J2,3 4.6 Hz, 1H, H-3), 3.55
D
20-thiazoline (5a). Hygroscopic off-white solid (0.28 g, 93%); Rf
(dd, J6,6 12.1 Hz, J5,6 2.7 Hz, 1H, H-60), 3.44e3.52 (m, 2H, H-6, H-4),
3.21e3.25 (m, 1H, H-5). 13C NMR (75 MHz, CD3OD):
175.82 (N]
0
0
25
0.15 (EtOAc/methanol 10:1); [a]
(300 MHz, CD3OD): d 7.18e7.90 (m, 4H, ArH), 6.47 (d, J1,2 7.0 Hz, 1H,
þ40 (c 0.5, MeOH); 1H NMR
D
d
CS), 135.42, 133.49, 133.10, 129.76, 129.43, 129.03, 127.55, 126.99,
126.55, 124.92 (ArC), 90.17 (C-1), 79.83, 79.46 (AreCH2), 76.34,
74.37, 71.08, 63.19. HRMS (ESI) calcd for C18H20NO4S (MþHþ)
346.11076, found 346.11078.
H-1), 4.54 (dd, J1,2 7.0 Hz, J2,3 4.8 Hz, 1H, H-2), 4.29 (t, J2,3 4.8 Hz, 1H,
H-3), 3.75 (dd, J6,6 12.1 Hz, J5,6 2.6 Hz, 1H, H-60), 3.59e3.65 (m, 2H,
H-6, H-4), 3.34e3.42 (m, 1H, H-5). 13C NMR (75 MHz, CD3OD):
0
0
d
164.28 (JCeF 4.4 Hz) (N]CS), 159.96 (JCeF 252.1 Hz), 132.75 (JCeF
8.6 Hz), 129.80 (JCeF 2.2 Hz), 123.88 (JCeF 3.5 Hz), 120.62 (JCeF
11.4 Hz),115.68 (JCeF 21.9 Hz) (C6H5), 88.20 (JCeF 3.2 Hz) (C-1), 78.20,
74.99, 72.48, 69.71, 61.81. HRMS (ESI) calcd for C13H15FNO4S
(MþHþ) 300.07003, found 300.07004.
4.2.5.7. 1,2-Dideoxy-20-biphenyl- 20-thia-
a-D-glucopyrano-[2,1-d]-D
zoline (5g). Hygroscopic off-white solid (0.33 g, 93%); Rf 0.16
(EtOAc/methanol 10:1); [
a
]
25 þ54 (c 0.5, DMSO); 1H NMR (300 MHz,
D
DMSO-d6):
d
7.39e7.89 (m, 9H, ArH), 6.47 (d, J1,2 6.8 Hz, 1H, H-1),
5.33 (d, J 4.6 Hz,1H, OH), 5.08 (d, J 5.2 Hz,1H, OH), 4.67 (t, J2,3 4.4 Hz,
1H, H-3), 4.53 (dd, J1,2 6.8 Hz, J2,3 4.7 Hz, 1H, H-2), 4.14 (dd, J 8.6 Hz,
4.4 Hz, 1H, OH), 3.55e3.61 (m, 1H, H-60), 3.45e3.48 (m, 2H, H-6, H-
4.2.5.2. 1,2-Dideoxy-20-(4-methoxy)-phenyl-
d]-
0.13 (EtOAc/methanol 10:1); [
(300 MHz, CD3OD):
8.8 Hz, 2.1 Hz, 2H, ArH), 6.44 (d, J1,2 6.9 Hz, 1H, H-1), 4.52 (dd, J1,2
6.9 Hz, J2,3 4.6 Hz, 1H, H-2), 4.22 (t, J2,3 4.6 Hz, 1H, H-3), 3.83 (s, 3H,
a-D-glucopyrano-[2,1-
D
20-thiazoline (5b). Hygroscopic off-white solid (0.30 g, 95%); Rf
25
þ45 (c 0.5, MeOH); 1H NMR
4), 3.17e3.24 (m, 1H, H-5). 13C NMR (75 MHz, DMSO-d6):
d 164.79
a
]
D
d
7.79 (dd, J 8.8 Hz, 2.1 Hz, 2H, ArH), 6.96 (dd, J
(N]CS),143.05,139.18,132.13,129.21,128.79,128.29,127.06,126.96
(ArC), 88.75 (C-1), 81.13, 75.94, 72.99, 70.14, 61.91. HRMS (ESI) calcd
for C19H20NO4S (MþHþ) 358.11076, found 358.11090.
0
0
0
OCH3), 3.75 (dd, J6,6 12.1 Hz, J5,6 2.5 Hz,1H, H-6 ), 3.58e3.65 (m, 2H,
H-6, H-4), 3.40e3.45 (m, 1H, H-5). 13C NMR (75 MHz, CD3OD):
4.2.5.8. 1,2-Dideoxy-20-3-methylindole-
a-D-glucopyrano-[2,1-d]-
20-thiazoline (5h). Hygroscopic off-white solid (0.32 g, 95%); Rf
d
168.69 (N]CS), 162.62, 129.74, 125.55, 113.57 (C6H5), 88.49 (C-1),
D
þ54 (c 0.5, MeOD); 1H NMR
25
79.66, 75.15, 73.23, 70.06, 62.03, 54.57 (OCH3). HRMS (ESI) calcd for
0.13 (EtOAc/methanol 10:1); [a]
(300 MHz, CD3OD): d 10.37 (s, 1H, NCH]C), 6.97e7.55 (m, 5H, ArH),
D
C
14H18NO5S (MþHþ) 312.09002, found 312.09018.
6.24 (d, J1,2 7.0 Hz,1H, H-1), 4.33 (m, 1H, H-2), 4.10 (m, 1H, H-3), 3.99
0
0
0
4.2.5.3. 1,2-Dideoxy-20-(4-ethoxy)-phenyl-
a-D-glucopyrano-[2,1-d]-
(d, J 4.9 Hz, 2H, AreCH2), 3.69 (dd, J6,6 12.1 Hz, J5,6 2.6 Hz, 1H, H-6 ),
3.54e3.61 (m, 2H, H-6, H-4), 3.36e3.40 (m, 1H, H-5). 13C NMR
D
20-thiazoline (5c). Hygroscopic off-white solid (0.32 g, 97%); Rf
25
0.20 (EtOAc/methanol 10:1); [a]
(300 MHz, CD3OD):
þ53 (c 0.5, MeOH); 1H NMR
(75 MHz, CD3OD):
d 174.87 (N]CS), 136.60, 126.99, 123.36, 121.11,
D
118.51, 117.93, 110.82, 108.60 (ArC), 88.42 (C-1), 78.73, 74.74, 73.03,
69.68, 61.74, 30.83 (AreCH2). HRMS (ESI) calcd for C18H20NO4S
(MþHþ) 335.10593, found 335.10600.
d 7.76 (dt, J 9.7 Hz, 2.8 Hz, 2H, ArH), 6.94 (dt, J
9.7, 2.8 Hz, 2H, ArH), 6.42 (d, J1,2 6.9 Hz, 1H, H-1), 4.49e4.53 (m, 1H,
H-2), 4.22 (t, J2,3 4.5 Hz, 1H, H-3), 4.07 (q, J 7.0 Hz, 2H, OCH2), 3.75
(dd, J6,6 12.1 Hz, J5,6 2.5 Hz, 1H, H-60), 3.58e3.65 (m, 2H, H-6, H-4),
3.39e3.44 (m, 1H, H-5), 1.39 (t, J 7.0 Hz, 3H, CH3). 13C NMR (75 MHz,
0
0
4.2.6. 1,3,4,6-Tetra-O-acetyl-2-N-azidoacetyl-2-deoxy-b-D-
glucopyranose (13)24 and compound (15)
CD3OD):
d 168.53 (N]CS), 161.87, 129.71, 125.47, 114.01 (C6H5),
To a solution of compound 10 (2.68 g, 7 mmol) in CH2Cl2 (30 mL)
were added Et3N (3 mL, 21 mmol) and azidoacetyl chloride (0.95 g,
8 mmol). The mixture was stirred at rt for 2 h, TLC (petroleum
ether/EtOAc 1:1) indicated that the reaction was complete. The
reaction mixture washed with HCl (1 N, 2ꢂ10 mL), water
(2ꢂ10 mL), then the organic phase was dried over Na2SO4. Evapo-
ration and purification by flash column chromatography (petro-
88.47 (C-1), 79.78, 75.13, 73.25, 70.07, 63.40 (OCH2), 62.03, 13.60
(CH3). HRMS (ESI) calcd for C15H20NO5S (MþHþ) 326.10567, found
326.10574.
4.2.5.4. 1,2-Dideoxy-20-(4-methyl)-phenyl-
a-D-glucopyrano-[2,1-d]-
D
20-thiazoline (5d). Hygroscopic off-white solid (0.30 g, 99%); Rf
25
0.15 (EtOAc/methanol 10:1); [a]
(300 MHz, CD3OD): d 7.72 (d, J 8.0 Hz, 2H, ArH), 7.26 (d, J 8.0 Hz, 2H,
þ62 (c 0.5, MeOH); 1H NMR
D
leum ether/EtOAc 3:1) afforded compound 13 (2.43 g, 81%) as a
25
white solid. Rf 0.45 (petroleum ether/EtOAc 1:1); [
a
]
D
þ10 (c 0.3,
ArH), 6.45 (d, J1,2 6.9 Hz, 1H, H-1), 4.52e4.56 (m, 1H, H-2), 4.23 (t,
CHCl3); mp 201e203 ꢀC; 1H NMR (300 MHz, CDCl3):
d 6.68 (d, J
J2,3 4.5 Hz, 1H, H-3), 3.75 (dd, J6,6 12.1 Hz, J5,6 2.6 Hz, 1H, H-60),
3.58e3.63 (m, 2H, H-6, H-4), 3.39e3.44 (m, 1H, H-5), 2.37 (s, 3H,
0
0
9.4 Hz, 1H, NH), 5.82 (d, J 8.7 Hz, 1H, H-1), 5.31 (dd, J2,3 10.4 Hz, J3,4
9.6 Hz, 1H, H-3), 5.14 (t, J3,4, J4,5 9.6 Hz, 1H, H-4), 4.25e4.32 (m, 2H,
CH3). 13C NMR (75 MHz, CD3OD):
d170.69 (N]CS), 143.60, 131.72,
H-2, H-60), 4.14 (dd, J6,6 12.5 Hz, J5,6 2.3 Hz, 1H, H-6), 3.91 (s, 2H,
0
139.30, 129.36 (C6H5), 89.87 (C-1), 81.13, 76.58, 74.57, 71.45, 63.43,
21.46 (CH3). HRMS (ESI) calcd for C14H18NO4S (MþHþ) 296.09511,
found 296.09512.
N3CH2), 3.86e3.90 (m, 1H, H-5), 2.12, 2.10, 2.06, 2.05 (4s, 12H,
4ꢂCOCH3).
Lawesson's reagent (1.20 g, 3.0 mmol) was added to compound
13 (2.15 g, 5 mmol) in THF (20 mL), and stirred at rt for 4 h, TLC
(petroleum ether/EtOAc 1:1) indicated that the reaction was com-
plete. Concentrated under reduced pressure and purification by
flash column chromatography (petroleum ether/EtOAc 3:1) affor-
ded compound 15 (2.10 g, 94%) as a yellow solid. Rf 0.55 (petroleum
4.2.5.5. 1,2-Dideoxy-20-(3,5-dinitro)-phenyl-
d]-
0.12 (EtOAc/methanol 10:1); [
(300 MHz, CD3OD):
a-D-glucopyrano-[2,1-
D
20-thiazoline (5e). Hygroscopic off-white solid (0.34 g, 92%); Rf
25
a
]
þ62 (c 0.5, MeOH); 1H NMR
D
d
9.09 (t, J 2.1 Hz, 1H, ArH), 8.97 (d, J 2.1 Hz, 2H,
ArH), 6.66 (d, J1,2 7.0 Hz, 1H, H-1), 4.70 (dd, J1,2 7.0 Hz, J2,3 4.6 Hz, 1H,
ether/EtOAc 1:1); [
(300 MHz, CDCl3):
a
d
]
25 þ25 (c 0.3, CHCl3); mp 204e206 ꢀC; 1H NMR
D
0
0
H-2), 4.32 (t, J2,3 4.6 Hz, 1H, H-3), 3.74 (dd, J6,6 12.1 Hz, J5,6 2.6 Hz,
8.20 (d, J 9.1 Hz,1H, NH), 5.87 (d, J 8.1 Hz,1H, H-
1H, H-60), 3.59e3.65 (m, 2H, H-6, H-4), 3.33e3.39 (m, 1H, H-5). 13C
1), 5.19e5.37 (m, 3H, H-2, H-3, H-4), 4.35 (s, 2H, N3CH2), 4.31 (dd,
0
0
0
NMR (75 MHz, CD3OD):
d 165.81 (N]CS), 150.08, 137.52, 128.85,
J6,6 12.5 Hz, J5,6 4.7 Hz, 1H, H-60), 4.17 (dd, J6,6 12.5 Hz, J5,6 2.4 Hz,
1H, H-6), 3.87e3.91 (m, 1H, H-5), 2.11, 2.10, 2.06, 2.01 (4s, 12H,
4ꢂCOCH3).
121.71 (C6H5), 91.96 (C-1), 81.72, 76.81, 74.28, 71.39, 63.45. HRMS
(ESI) calcd for C13H14N3O8S (MþHþ) 372.04961, found 372.04999.

142
H. Kong et al. / Carbohydrate Research 413 (2015) 135e144
4.2.7. 3,4,6-Tri-O-acetyl-1,2-dideoxy-20-methyl-
a-
D-glucopyrano-
4.2.9.1. 3,4,6-Tri-O-acetyl-1,2-dideoxy-20-(4-t-butyl-1,2,3-triazole-1-
methyl)- -glucopyrano-[2,1-d]- (19a).
20-thiazoline
[2,1-d]-
D
20-thiazoline (17)10
a-D
D
Compound 10 (3.83 g, 10 mmol) was dissolved in CH2Cl2
(50 mL), Et3N (4.3 mL, 30 mmol) and Ac2O (11 mL) were added,
after 1 h, TLC analysis showed complete conversion. The solution
was washed with NaHCO3 (satd aq), 1 M HCl, dried (Na2SO4),
concentrated and purification by flash column chromatography
(petroleum ether/EtOAc 1:1) afforded compound 16 as a white solid
Colourless oil (0.42 g, 90%); Rf 0.45 (petroleum ether/EtOAc 1:1);
25
[a
]
þ5 (c 0.3, CHCl3); 1H NMR (300 MHz, CDCl3):
d 7.39 (s, 1H,
D
NeCH]C), 6.32 (d, J1,2 7.2 Hz, 1H, H-1), 5.55 (dd, J2,3 3.4 Hz, J3,4
2.0 Hz, 1H, H-3), 5.38 (dd, J 5.1 Hz, 1.8 Hz, 2H, NCH2), 4.94 (d, J4,5
9.3 Hz, 1H, H-4), 4.56e4.60 (m, 1H, H-2), 4.09e4.12 (m, 2H, H-6, H-
60), 3.48e3.52 (m, 1H, H-5), 2.19, 2.09, 2.07 (3s, 9H, 3ꢂCOCH3), 1.36
(3.5 g, 90%). Rf 0.30 (petroleum ether/EtOAc 1:1); [
a
]
d
23 þ2.9 (c 1.0,
(s, 9H, 3CH3). 13C NMR (75 MHz, CDCl3):
d 170.47, 169.38, 169.26
D
CHCl3); mp 188e189 ꢀC; 1H NMR (300 MHz, CDCl3):
5.75 (d, J 9.6,
(3ꢂCOCH3), 166.64 (N]CS), 158.44, 119.20 (NC]CH), 89.04 (C-1),
76.19, 70.19, 68.98, 68.85, 63.10, 51.54 (NCH2), 30.79 (C(CH3)3),
30.26 (3, C(CH3)3), 20.88, 20.80, 20.66 (3ꢂCOCH3). HRMS (ESI) calcd
for C20H29N4O7S (MþHþ) 469.17515, found 469.17468.
1H, NH), 5.70 (d, J1,2 8.8 Hz, 1H, H-1), 5.13e5.21 (m, 2H, H-4, H-3),
4.24e4.33 (m, 2H, H-60, H-2), 4.13 (dd, J6,6 12.4 Hz, J5,6 2.2 Hz,1H, H-
0
6), 3.80e3.85 (m, 1H, H-5), 2.12, 2.09, 2.05, 2.04, 1.94 (5s, 15H,
5ꢂCOCH3).
Lawesson's reagent (1.36 g, 3.3 mmol) was added to compound
16 (2.00 g, 5.1 mmol) in toluene (20 mL). The mixture was stirred at
80 ꢀC for 4 h, TLC indicated that the reaction was complete.
Concentrated under reduced pressure and purification by flash
column chromatography (petroleum ether/EtOAc 3:1) afforded
4.2.9.2. 3,4,6-Tri-O-acetyl-1,2-dideoxy-20-(4-phenyl-1,2,3-triazole-1-
methyl)-
a-D-glucopyrano-[2,1-d]-
D
20-thiazoline
(19b).
Colourless oil (0.41 g, 85%); Rf 0.48 (petroleum ether/EtOAc 1:1);
25
[
a
]
þ20 (c 0.3, CHCl3); 1H NMR (300 MHz, CDCl3):
d 7.93 (s, 1H,
D
NCH]C), 7.35e7.87 (m, 5H, ArH), 6.34 (d, J1,2 7.2 Hz, 1H, H-1), 5.54
(dd, J2,3 3.5 Hz, J3,4 2.3 Hz, 1H, H-3), 5.47 (d, J 1.9 Hz, 2H, NCH2), 4.93
(d, J4,5 9.3 Hz,1H, H-4), 4.56e4.61 (m,1H, H-2), 4.08e4.11 (m, 2H, H-
6, H-60), 3.52e3.56 (m, 1H, H-5), 2.13, 2.04, 2.00 (3s, 9H, 3ꢂCOCH3).
compound 17(1.47 g, 83%) as a yellow oil. Rf 0.50 (petroleum ether/
23
EtOAc 1:1); [
d
a]
ꢁ28.0 (c 0.4, CHCl3); 1H NMR (300 MHz, CDCl3):
D
6.25 (d, J1,2 7.1 Hz,1H, H-1), 5.57 (dd, J2,3 3.1 Hz, J3,4 1.7 Hz,1H, H-3),
4.96 (d, J4,5 9.5 Hz, 1H, H-4), 4.47e3.49 (m, 1H, H-2), 4.12 (d, J 4.4 Hz,
2H, H-6), 3.52e3.58 (m, 1H, H-5), 2.32 (d, J 2.2 Hz, 3H, oxazoline
Me), 2.14, 2.10, 2.09 (3s, 9H, 3ꢂCOCH3).
13C NMR (75 MHz, CDCl3):
d
170.53, 169.41, 169.29 (3ꢂCOCH3),
166.32 (N]CS), 148.49, 130.21, 128.87, 128.42, 125.80, 120.39 (C6H5,
NC]CH), 89.41 (C-1), 76.20, 70.28, 69.04, 68.96, 63.18, 51.76
(NCH2), 20.87, 20.72, 20.56 (3ꢂCOCH3). HRMS (ESI) calcd for
4.2.8. 3,4,6-Tri-O-acetyl-1,2-dideoxy-20-azidomethyl-
glucopyrano-[2,1-d]-
20-thiazoline (14)18
a
-D-
C
22H25N4O7S (MþHþ) 489.14385, found 489.14365.
D
Compound 17 (0.35 g, 1 mmol) was dissolved in THF (10 mL) at
0 ꢀC. Pyridine was added, followed by triflic acid (0.18 g, 1.2 mmol),
then iodine (0.51 g, 2.0 mmol). The reaction mixture was stirred for
15 min at rt, concentrated and purification by flash column chro-
matography (petroleum ether/EtOAc 3:1) afforded compound 17 as
4.2.9.3. 3,4,6-Tri-O-acetyl-1,2-dideoxy-20-(4-hydroxyethyl-1,2,3-
triazole-1-methyl)-
Colourless oil (0.39 g, 86%); Rf 0.41 (EtOAc); [
1H NMR (300 MHz, CDCl3):
a
-
D
-glucopyrano-[2,1-d]-
D
20-thiazoline
(19c).
a
]
25 þ8 (c 0.3, CHCl3);
D
d
7.56 (s,1H, NCH]C), 6.33 (d, J1,2 7.2 Hz,
1H, H-1), 5.55 (dd, J2,3 3.6 Hz, J3,4 2.1 Hz, 1H, H-3), 5.39 (d, J 1.9 Hz,
a brown oil (0.34 g, 73%). Rf 0.60 (petroleum ether/EtOAc 1:1);
2H, NeCH2), 4.91e4.95 (m, 1H, H-4), 4.54e4.58 (m, 1H, H-2), 4.15
25
[
a]
ꢁ12 (c 0.3, CHCl3); 1H NMR (300 MHz, CDCl3):
d 6.34 (d, J1,2
(dd, J6,6 12.3 Hz, J5,6 2.9 Hz, 1H, H-60), 4.06 (dd, J6,6 12.3 Hz, J5,6
6.2 Hz, 1H, H-6), 3.96 (dd, J 10.7 Hz, 5.3 Hz, 2H, CH2CH2OH),
3.48e3.53 (m, 1H, H-5), 2.98 (t, J 5.7 Hz, 2H, CH2CH2OH), 2.72 (t, J
5.3 Hz, 1H, OH), 2.14, 2.10, 2.06 (3s, 9H, 3ꢂCOCH3). 13C NMR
0
0
0
D
7.1 Hz,1H, H-1), 5.57 (dd, J2,3 3.2 Hz, J3,4 1.5 Hz,1H, H-3), 4.94 (dt, J4,5
0
9.3 Hz, J3,4 1.5 Hz, 1H, H-4), 4.49 (m, 1H, H-2), 4.24 (dd, J6,6 12.0 Hz,
J5,6 2.0 Hz, 1H, H-60), 4.08e4.14 (m, 3H, H-6, ICH2), 3.59e3.65 (m,
0
1H, H-5), 2.15, 2.11, 2.09 (3s, 9H, 3ꢂCOCH3).
(75 MHz, CDCl3):
d
170.61, 169.42, 169.33 (3ꢂCOCH3), 166.31 (N]
Compound 18 (500 mg, 1.1 mmol) was dissolved in DMF (5 mL).
A solution containing sodium azide (690 mg, 11 mmol) in DMF
(5 mL) was added by cannula. After 2 h, the reaction was quenched
with 10% aqueous sodium bicarbonate, concentrated, diluted with
dichloromethane, washed with 5% aqueous sodium bicarbonate
and then dried over sodium sulfate, concentrated and then the
residue was purified by flash column chromatography on a silica gel
column (petroleum ether/EtOAc 3:1) to give compound 14 as a
CS), 146.30, 122.40 (NC]CH), 89.29 (C-1), 76.10, 70.20, 68.95, 68.87,
63.19, 61.40 (CH2CH2OH), 51.61 (NCH2), 28.81 (CH2CH2OH), 20.87,
20.77, 20.65 (3ꢂCOCH3). HRMS (ESI) calcd for C18H25N4O8S (MþHþ)
457.13876, found 457.13867.
4.2.9.4. 3,4,6-Tri-O-acetyl-1,2-dideoxy-20-[4-(1-hydroxy)-isopropyl-
1,2,3-triazole-1-methyl]-
(19d). Colourless oil (0.41 g, 87%); Rf 0.38 (EtOAc); [
CHCl3); 1H NMR (300 MHz, CDCl3):
a-D-glucopyrano-[2,1-d]-D
20-thiazoline
a
]
D
25 þ10 (c 0.3,
yellow oil (550 mg, 90%). Rf 0.59 (petroleum ether/EtOAc 1:1);
d
7.62 (s, 1H, NCH]C), 6.34 (d,
25
[
a]
ꢁ8 (c 0.3, CHCl3); 1H NMR (300 MHz, CDCl3):
d
6.33 (d, J1,2
J1,2 7.3 Hz,1H, H-1), 5.54 (dd, J2,3 3.4 Hz, J3,4 1.8 Hz,1H, H-3), 5.39 (d, J
D
7.2 Hz, 1H, H-1), 5.61 (dd, J2,3 3.3 Hz, J3,4 1.5 Hz, 1H, H-3), 4.96 (dt,
J4,5 9.3 Hz, J3,4 1.5 Hz, 1H, H-4), 4.55e4.59 (m, 1H, H-2), 4.21 (d, J
1.2 Hz, 2H, N3CH2), 4.13e4.15 (m, 2H, H-6, H-60), 3.51e3.57 (m, 1H,
H-5), 2.15, 2.09, 2.08 (3s, 9H, 3ꢂCOCH3).
1.5 Hz, 2H, NCH2), 4.90 (dt, J4,5 9.3 Hz, J3,4 1.8 Hz,1H, H-4), 4.57e4.60
(m, 1H, H-2), 4.17 (dd, J6,6 12.2 Hz, J5,6 2.8 Hz, 1H, H-60), 4.00 (dd,
0
0
0
J6,6 12.2 Hz, J5,6 6.9 Hz, 1H, H-6), 3.43e3.49 (m, 1H, H-5), 3.14 (s, 1H,
OH), 2.15, 2.13, 2.05 (3s, 9H, 3ꢂCOCH3), 1.65 (d, J 15.1 Hz, 6H,
2ꢂCH3). 13C NMR (75 MHz, CDCl3):
d 170.86, 169.45, 169.27
4.2.9. Representative procedure for the synthesis of 3,4,6-tri-O-
(3ꢂCOCH3), 166.15 (N]CS), 156.92, 119.82 (NC]CH), 89.23 (C-1),
76.07, 70.04, 68.96, 68.73, 68.50 (C(CH3)2OH), 63.46, 51.58 (NCH2),
30.56, 30.23 (2ꢂCH3), 20.87, 20.78, 20.64 (3ꢂCOCH3). HRMS (ESI)
calcd for C19H27N4O8S (MþHþ) 471.15441, found 471.15408.
acetyl-1,2-dideoxy-20-[(4-substituted-1,2,3-triazole)-1-methyl]-
a-
D-glucopyrano-[2,1-d]-
D
20-thiazolines (19ae19f)
To a solution of compound 14 (386 mg, 1.0 mmol) in MeOH/H2O
(10 mL, 1:1), alkyne (1.0 mmol) was added followed by sodium
ascorbate (30 mg, 0.15 mmol) and copper(II) sulfate (25 mg,
0.1 mmol). The reaction mixture was stirred at rt for 5 h, concen-
trated and poured into H2O (20 mL), extracted with CH2Cl2
(3ꢂ10 mL). The organic extracts were combined, dried and
concentrated. The crude product was purified by column chroma-
tography (petroleum ether/EtOAc 1:1).
4.2.9.5. 3,4,6-Tri-O-acetyl-1,2-dideoxy-20-(4-hydroxymethyl-1,2,3-
triazole-1-methyl)-
Colourless oil (0.42 g, 94%); Rf 0.35 (EtOAc); [
1H NMR (300 MHz, CDCl3):
a
-
D
-glucopyrano-[2,1-d]-
D
20-thiazoline
(19e).
a
]
25 þ13 (c 0.3, CHCl3);
D
d
7.69 (s,1H, NCH]C), 6.34 (d, J1,2 7.3 Hz,
1H, H-1), 5.54 (dd, J2,3 3.4 Hz, J3,4 1.7 Hz, 1H, H-3), 5.40 (dd, J 5.8 Hz,
2.1 Hz, 2H, NCH2), 4.90 (dt, J4,5 10.1 Hz, J3,4 1.7 Hz, 1H, H-4), 4.81 (d, J

H. Kong et al. / Carbohydrate Research 413 (2015) 135e144
143
0
6.0 Hz, 1H, CH2OH), 4.56e4.60 (m, 1H, H-2), 4.17 (dd, J6,6 12.2 Hz,
(75 MHz, CD3OD):
d
167.92 (N]CS), 146.80, 125.04 (NC]CH), 91.20
J5,6 2.7 Hz, 1H, H-60), 3.95 (dd, J6,6 12.2 Hz, J5,6 7.0 Hz, 1H, H-6),
3.34e3.40 (m, 1H, H-5), 3.16 (s, 1H, OH), 2.14, 2.13, 2.04 (3s, 9H,
(C-1), 80.56, 76.52, 73.86, 71.25, 63.37, 62.00 (CH2CH2OH), 58.31
(NCH2), 29.95 (CH2CH2OH). HRMS (ESI) calcd for C12H19N4O5S
(MþHþ) 331.10707, found 331.10709.
0
0
3ꢂCOCH3). 13C NMR (75 MHz, CDCl3):
d 171.02, 169.46, 169.27
(3ꢂCOCH3), 166.15 (N]CS), 149.07, 122.11 (NC]CH), 89.22 (C-1),
76.08, 70.02, 68.94, 68.93, 63.51, 56.76 (CH2OH), 51.59 (NCH2),
20.86, 20.75, 20.62 (3ꢂCOCH3). HRMS (ESI) calcd for C17H23N4O8S
(MþHþ) 443.12314, found 443.12311.
4.2.10.4. 1,2-Dideoxy-20-[4-(1-hydroxy)-isopropyl-1,2,3-triazole-1-
methyl]-a-D-glucopyrano-[2,1-d]-D
20-thiazoline (6d).
Hygroscopic off-white solid (0.15 g, 85%); Rf 0.15 (EtOAc/methanol
3:2); [
a
]
25 þ30 (c 0.3, CH3OH); 1H NMR (300 MHz, CD3OD):
d 7.95 (s,
D
4.2.9.6. 3,4,6-Tri-O-acetyl-1,2-dideoxy-20-(4-methoxymethyl-1,2,3-
1H, NeCH]C), 6.43 (d, J1,2 7.0 Hz, 1H, H-1), 4.40e4.44 (m, 1H, H-2),
20-thiazoline
(19f).
4.18 (dd, J2,3 4.6 Hz, J3,4 3.5 Hz, 1H, H-3), 3.70 (dd, J6,6 12.1 Hz, J5,6
0
0
triazole-1-methyl)-
a
-D
-glucopyrano-[2,1-d]-
D
Colourless oil (0.40 g, 88%); Rf 0.34 (petroleum ether/EtOAc 1:1);
2.6 Hz,1H, H-60), 3.55e3.63 (m, 2H, H-6, H-4), 3.27e3.30(m,1H,H-5),
25
[
a]
þ3 (c 0.3, CHCl3); 1H NMR (300 MHz, CDCl3):
d
7.70 (s, 1H,
1.57 (s, 6H, 2ꢂCH3). 13C NMR (75 MHz, CD3OD):
d 167.92 (N]CS),
D
NCH]C), 6.34 (d, J1,2 7.2 Hz, 1H, H-1), 5.54 (dd, J2,3 3.4 Hz, J3,4 1.8 Hz,
1H, H-3), 5.43 (d, J 2.0 Hz, 2H, NCH2), 4.93 (dt, J4,5 9.3 Hz, J3,4 1.8 Hz,
1H, H-4), 4.61 (s, 2H, OCH2), 4.54e4.58 (m, 1H, H-2), 4.08e4.11 (m,
2H, H-6, H-60), 3.46e3.51 (m, 1H, H-5), 3.42 (s, 3H, OCH3), 2.14, 2.10,
157.57, 122.96 (NC]CH), 91.19 (C-1), 80.58, 76.49, 73.87, 71.27, 69.02
(C(CH3)2OH), 63.46, 58.32 (NCH2), 30.60, 30.58 (2ꢂCH3). HRMS (ESI)
calcd for C13H21N4O5S (MþHþ) 345.12272, found 345.12266.
2.07 (3s, 9H, 3ꢂCOCH3). 13C NMR (75 MHz, CDCl3):
d
170.38, 169.31,
4.2.10.5. 1,2-Dideoxy-20-(4-hydroxymethyl-1,2,3-triazole-1-methyl)-
169.14 (3ꢂCOCH3), 165.95 (N]CS), 145.81, 123.10 (NC]CH), 89.15
(C-1), 76.05, 69.95, 68.81 (2), 65.89 (OCH2), 63.06, 58.24 (OCH3),
51.50 (NCH2), 20.77, 20.62, 20.54 (3ꢂCOCH3). HRMS (ESI) calcd for
a-D
-glucopyrano-[2,1-d]-
D
20-thiazoline (6e). Hygroscopic off-white
25
solid (0.14 g, 90%); Rf 0.10 (EtOAc/methanol 3:2); [
a
]
þ35 (c 0.3,
D
CH3OH); 1H NMR (300 MHz, CD3OD):
d
8.03 (s, 1H, NCH]C), 6.44
C
18H25N4O8S (MþHþ) 457.13876, found 457.13824.
(d, J1,2 7.0 Hz, 1H, H-1), 4.68 (s, 2H, CH2OH), 4.42 (dd, J1,2 7.0 Hz, J2,3
0
4.8 Hz, 1H, H-2), 4.18 (t, J2,3 4.8 Hz, 1H, H-3), 3.72 (dd, J6,6 12.1 Hz,
4.2.10. Representative procedure for the synthesis of 1,2-dideoxy-20-
[(4-substituted-1,2,3-triazole)-1-methyl]- -glucopyrano-[2,1-d]-
20-thiazolines (6ae6f)
A solution of compounds 19ae19f (0.5 mmol) in 5 mL of
methanol was treated with sodium methoxide (0.1 mL of a 1 M
solution in methanol) and the reaction mixture was stirred at rt for
30 min. The reaction mixture was neutralized to pH 7 by Amberlyst
Hþ resin, concentrated and chromatographed with 1:10 methanol/
EtOAc to afford compounds 6ae6f.
J5,6 2.5 Hz, 1H, H-60), 3.55e3.62 (m, 2H, H-6, H-4), 3.27e3.30 (m,
0
a-D
1H, H-5). 13C NMR (75 MHz, CD3OD):
d 164.94 (N]CS), 146.66,
D
122.28 (NC]CH), 88.34 (C-1), 77.62, 73.61, 71.00, 68.29, 60.51,
55.42 (NCH2), 53.59 (CH2OH). HRMS (ESI) calcd for C11H17N4O5S
(MþHþ) 317.09142, found 317.09140.
4.2.10.6. 1,2-Dideoxy-20-(4-methoxymethyl-1,2,3-triazole-1-methyl)-
a-D
-glucopyrano-[2,1-d]-
D
20-thiazoline (6f). Hygroscopic off-white
25
solid (0.15 g, 89%); Rf 0.32 (EtOAc/methanol 10:1); [
a
]
þ48 (c
D
0.3, CH3OH); 1H NMR (300 MHz, CD3OD):
d
8.09 (s, 1H, NCH]C),
4.2.10.1. 1,2-Dideoxy-20-(4-tert-butyl-1,2,3-triazole-1-methyl)-
glucopyrano-[2,1-d]-
20-thiazoline (6a). Hygroscopic off-white
solid (0.15 g, 85%); Rf 0.20 (EtOAc/methanol 10:1); [
0.3, CH3OH); 1H NMR (300 MHz, CD3OD):
7.87 (s, 1H, NCH]C),
6.43 (d, J1,2 7.0 Hz, 1H, H-1), 4.42e4.46 (m, 1H, H-2), 4.20 (dd, J2,3
a
-
D
-
6.44 (d, J1,2 7.0 Hz, 1H, H-1), 4.54 (s, 2H, OCH2), 4.42 (dd, J1,2 7.0 Hz,
0
D
J2,3 4.6 Hz,1H, H-2), 4.17 (t, J2,3 4.0 Hz,1H, H-3), 3.71 (dd, J6,6 12.1 Hz,
25
J5,6 2.6 Hz, 1H, H-60), 3.56e3.68 (m, 2H, H-6, H-4), 3.36 (s, 3H,
0
a]
þ35 (c
D
d
OCH3), 3.25e3.28 (m, 1H, H-5). 13C NMR (75 MHz, CD3OD):
d 167.64
(N]CS), 146.23, 126.17 (NC]CH), 91.27 (C-1), 80.62, 76.54, 73.90,
71.24, 66.22 (OCH2), 63.37, 58.35, 58.32 (NCH2, OCH3). HRMS (ESI)
calcd for C12H19N4O5S (MþHþ) 331.10707, found 331.10706.
0
0
4.5 Hz, J3,4 3.3 Hz, 1H, H-3), 3.70 (dd, J6,6 12.1 Hz, J5,6 2.6 Hz, 1H, H-
60), 3.56e3.61 (m, 2H, H-6, H-4), 3.26e3.29 (m, 1H, H-5), 1.33 (s, 9H,
3ꢂCH3). 13C NMR (75 MHz, CD3OD):
d 167.96 (N]CS), 159.09,
122.23 (NC]CH), 91.10 (C-1), 80.61, 76.41, 73.81, 71.23, 63.41, 58.31
(NCH2), 31.70 (C(CH3)3), 30.64 (3, C(CH3)3). HRMS (ESI) calcd for
4.3. Materials and methods for inhibition study
C
14H23N4O4S (MþHþ) 343.14345, found 343.14346.
4.3.1. Enzymes preparing
The O. furnacalis
b-hexosaminidase (OfHex2) was overexpressed
4.2.10.2. 1,2-Dideoxy-20-(4-phenyl-1,2,3-triazole-1-methyl)-
copyrano-[2,1-d]-
20-thiazoline (6b). Hygroscopic off-white solid
(0.16 g, 88%); Rf 0.15 (EtOAc/methanol 10:1); [
CH3OH); 1H NMR (300 MHz, CD3OD):
8.43 (s, 1H, NCH]C),
a-D-glu-
in Pichia pastoris and then purified as described previously.26 The
D
human placental
Sigma (A6152).
b-hexosaminidase (HsHex) was purchased from
25
a
]
þ45 (c 0.3,
D
d
The gene of human O-GlcNAcase (hOGA, Gene ID: 10724) was
prepared syntheticallyand cloned intothebacterial expressionvector
pET28a (Novagen, Merck, Germany) via the NdeI and XhoI restriction
sites. The gene was intracellularly expressed in Escherichia coli
BL21(DE3) cells (Novagen, Merck, Germany) under the induction by
0.5mMIPTG. After10hofcultivationat25ꢀC,thecellswereharvested
by centrifugation (4000ꢂg,15 min) and resuspended in a suspension
buffer (20 mM sodium phosphate, 500 mM sodium chloride, 20 mM
imidazole, pH 7.4) and lysed by high pressure homogenization
(1000 bar) using a nano homogenize machine (ATS Engineering inc.,
Canada) at 4 ꢀC. After centrifugation (12,000ꢂg, 15min), the super-
natant wasloaded onto HiTrap™ Clelating HP column (GEHealthcare,
USA) according to the manufacturer's instructions. The column was
equilibrated with 20 mM sodium phosphate buffer with 500 mM
sodium chloride and 50 mM imidazole, pH 7.4. The fusion proteinwas
eluted from the column using 20 mM sodium phosphate buffer with
500 mM sodium chloride and 250 mM imidazole, pH 7.4.
7.31e7.83 (m, 5H, ArH), 6.46 (d, J1,2 7.0 Hz, 1H, H-1), 4.45 (dd, J1,2
0
7.0 Hz, J2,3 4.6 Hz,1H, H-2), 4.20 (d, J2,3 4.5 Hz,1H, H-3), 3.72 (dd, J6,6
12.1 Hz, J5,6 2.6 Hz, 1H, H-60), 3.57e3.63 (m, 2H, H-6, H-4),
0
3.31e3.36 (m, 1H, H-5). 13C NMR (75 MHz, CD3OD):
d 166.26 (N]
CS), 147.85, 130.12, 128.56, 128.05, 125.34, 121.93 (C6H5, NC]CH),
89.89 (C-1), 79.16, 75.08, 72.44, 69.80, 62.01, 45.58 (NCH2). HRMS
(ESI) calcd for C16H19N4O4S (MþHþ) 363.11212, found 363.11215.
4.2.10.3. 1,2-Dideoxy-20-(4-hydroxyethyl-1,2,3-triazole-1-methyl)-
-glucopyrano-[2,1-d]-
20-thiazoline (6c). Hygroscopic off-white
solid (0.14 g, 86%); Rf 0.12 (EtOAc/methanol 3:2); [
CH3OH); 1H NMR (300 MHz, CD3OD):
7.91 (s,1H, NCH]C), 6.44 (d,
a-
D
D
25
a
]
þ41 (c 0.3,
D
d
J1,2 7.0 Hz, 1H, H-1), 4.40e4.42 (m, 1H, H-2), 4.18 (dd, J2,3 4.6 Hz, J3,4
3.5 Hz, 1H, H-3), 3.80 (t, J 6.6 Hz, 2H, CH2CH2OH), 3.70 (dd,
0
0
0
J6,6 12.0 Hz, J5,6 2.6 Hz, 1H, H-6 ), 3.58e3.63 (m, 2H, H-6, H-4), 3.34
(m, 1H, H-5), 2.91 (dt, J 6.6 Hz, 0.4 Hz, 2H, CH2CH2OH). 13C NMR

144
H. Kong et al. / Carbohydrate Research 413 (2015) 135e144
The gene of O. furnacalis O-GlcNAcase (OfOGA, GenBank ID:
References and notes
JQ666007.1) was obtained from total RNA extracted from fifth instar
O. furnacalis larvae (5L3). The procedure for the expression and
isolation of OfOGA was analogous to the procedure for hOGA above.
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4.3.2. Inhibitory activity assays
b
-N-Acetylhexosaminidase and O-GlcNAcase activities were
assayed in end-point experiments using p-nitrophenyl 2-
acetamido-2-deoxy- -glucopyranoside (pNP- -GlcNAc; Sigma
Aldrich) as a substrate. The assay components were incubated in a
final volume of 100
L at 25 ꢀC for 30 min in the presence of Brit-
b
-D
b
m
toneRobinson buffer (pH 6.0), dimethylsulfoxide at the final con-
centration of 2%, enzyme, compounds and pNP-GlcNAc. Then,
enzyme reaction was stopped by the addition of 100
dium carbonate solution.
mL 0.5 M so-
Acknowledgements
This work was partially supported by the National Natural
Science Foundation of China (21172257), the National 863 Program
(2011AA10A206) and the National S&T Pillar Program
(2015BAK45B01) of China.
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Supplementary data
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Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.carres.2015.06.004.