Synthesis, characterization, and biological evaluation of some novel glycosyl 1,3,4-thiadiazole derivatives as acetylcholinesterase inhibitors

Article abstract of DOI:10.3987/COM-14-13134

The corresponding 4-substituted glycosyl thiosemicarbazide derivatives (6a-6l) were obtained by the reaction of glycosyl isothiocyanate 4 with various hydrazides. Further cyclization with the system of p-TsCl/TEA led to the formation of N-glycosyl-5-substituted 1,3,4-thiadiazole-2-amine (7a-7l). Subsequent removal of the acetyl groups were conducted using the system of NaOMe/MeOH. The chemical structures of all new products were confirmed by IR, ¹H NMR and ESI-HRMS. The acetylcholinesterase (AChE) inhibitory activities of those compounds were tested by Ellman's method. Among them, the compound 8h possessed the best acetylcholinesterase-inhibition activity with IC50 of 18.38 ± 0.89 μM.

Full text of DOI:10.3987/COM-14-13134

HETEROCYCLES, Vol. 91, No. 2, 2015
275
HETEROCYCLES, Vol. 91, No. 2, 2015, pp. 275 - 286. © 2015 The Japan Institute of Heterocyclic Chemistry
Received, 20th November, 2014, Accepted, 8th January, 2015, Published online, 19th January, 2015
DOI: 10.3987/COM-14-13134
SYNTHESIS, CHARACTERIZATION, AND BIOLOGICAL EVALUATION
OF SOME NOVEL GLYCOSYL 1,3,4-THIADIAZOLE DERIVATIVES AS
ACETYLCHOLINESTERASE INHIBITORS
Wei-wei Liu,1,2,3* Qu-xiang Li, Da-hua Shi, Zhi-ling Cao,1,3 Feng-chang
2
1,3
Cheng, Chuan-zhou Tao, Long Yin, and Xuan Wang1
2
1,3
1
1School of Chemical Engineering, Huaihai Institute of Technology, Lianyungang
2
2
22005, China. E-mail: liuww2007ly@aliyun.com; China University of Mining
and Technology, Xuzhou 221116, China; 3Jiangsu Marine Resources
Development Research Institute, Lianyungang 222005, P.R. of China
Abstract – The corresponding 4-substituted glycosyl thiosemicarbazide
derivatives (6a-6l) were obtained by the reaction of glycosyl isothiocyanate 4 with
various hydrazides. Further cyclization with the system of p-TsCl/TEA led to the
formation of N-glycosyl-5-substituted 1,3,4-thiadiazole-2-amine (7a-7l). Subsequ-
ent removal of the acetyl groups were conducted using the system of NaOMe/
1
MeOH. The chemical structures of all new products were confirmed by IR, H
NMR and ESI-HRMS. The acetylcholinesterase (AChE) inhibitory activities of
those compounds were tested by Ellman’s method. Among them, the compound
8h possessed the best acetylcholinesterase-inhibition activity with IC50 of 18.38 ±
0.89 µM.
INTRODUCTION
Over the past few years, glycobiology, and carbohydrate chemistry have been paid enormous attention
owing to the remarkable role played in many biological events.1-3 D-Glucosamine, as a natural amino
monosaccharide, widely exists in ocean organisms, and is an indispensable substance of our human
4
beings’ gristle, ligament, muscle tendon. Furthermore, D-glucosamine and its derivatives present many
excellent bioactivities such as antitumor, antibacterial, anti-inflammatory, liver-protecting and improving

276
HETEROCYCLES, Vol. 91, No. 2, 2015
immune function.5-9 Recently, the C1-glycosyl heterocycles have given rise to considerable interest,
mainly due to their role in a multitude of biological and pharmaceutical applications like biological
inhibitors and probes,10-13 antitumor,14,15 antimicrobial, and anti-inflammatory.
16
17
On the other hand, the 1,3,4-thiadiazoles are commonly utilized heterocyclic pharmacophores, which
display a broad spectrum of biological activities including antimicrobial,18,19 anticancer,20,21
antioxidant,22,23 antidepressant,24,25 anticonvulsant, 26,27 antihypertensive, and antifungal activity.
28
29,30
In
particular, the most significant 2-amino-1,3,4-thiadiazole derivatives are used in treatment of glaucoma,31
epileptic seizures,32 hemiplegic migraine,33 cystinuria,34 tuberculosis,35 etc. Moreover, some
1
,3,4-thiadiazoles also exhibit high potential employed as acetylcholinesterase inhibitors for the treatment
of Alzheimer’s disease (AD).36,37
Therefore, it is very interesting for us to report the synthesis of a series of new compounds in which the
glycosyl moieties have been used as carriers for the heterocycles possessing thiadiazole ring.
Following our previous studies in searching of novel AChE inhibititors,38,39 the acetylated GlcNAc of
N-bit modification didn’t show enough inhibitory activity toward AChE. Here, we report a mild and
convenient protocol for the synthesis of glycosyl 1,3,4-thiadiazole derivatives via the link of nitrogen
glycoside evaluated by Ellman’s method in order to screen a novel AChE inhibitor and explore the
influence of GlcNAc unit against AChE-inhibition activity.
RESULTS AND DISCUSSION
Chemistry
The starting material, N-acetyl-tri-O-acetyl-β-D-glucopyranosyl isothiocyanate 4, was prepared from the
corresponding D-glucosamine hydrochloride 1 via acylation, chlorination and S
2 reaction.40,41 Treatment
N
of the isothiocyanate 4 with various hydrazides 5 in ethanol at 78 °C for 2 h afforded the glycosyl
acylthiosemicarbazides 6a-6l with almost quantitative yield. A series of glycosyl 1,3,4-thiadiazoles (7a-7l)
were synthesized by cyclizing system of p-TsCl/TEA in N-methylpyrrolidone (NMP) under mild
conditions from the compounds 6a-6l and gave satisfactory isolated yields (65%-89%). The acetyl
derivatives 7 were efficiently deprotected to the hydroxyl derivatives 8 in the presence of NaOMe/MeOH.
The general synthetic pathway to prepare glycosyl 1,3,4-thiadiazole derivatives was performed in Scheme
1
1
. The chemical structures of all the new compounds were confirmed by IR, H NMR and high resolution
mass spectroscopy (HRMS), which were in agreement with the proposed structures.

HETEROCYCLES, Vol. 91, No. 2, 2015
277
6
6
6
6
a, 7a, 8a : R = Me
b, 7b, 8b: R = C
c, 7c, 8c: R = 4-MeC
d, 7d, 8d: R = 3-MeOC
6e, 7e, 8e: R = 4-FC
6
H
4
6i, 7i, 8i: R = 4-MeCO
6j, 7j, 8j: R = 4-(N, N-di-Me)-C
6k, 7k, 8k: R = 3-C
6l, 7l, 8l: R = 2-C
2 6 4
C H
6
H
5
6f, 7f, 8f: R = 2-ClC
6
H
4
6 4
H
6
H
4
6g, 7g, 8g: R = 4-IC
6
H
4
6 4
H N
6
H
4
6h, 7h, 8h: R = 4-NO
2
C
H
6 4
5 3
H S
Scheme 1: Synthetic pathways of glycosyl 1,3,4-thiadiazoles. Reagents and conditions: (i) Ac
ii) AcCl, rt; (iii) KSCN, (n-C NHSO , MeCN, 4ꢀÅꢀmolecular sieves; (iv) RCONHNH (5) EtOH, reflux; (v) p-TsCl,
TEA, NMP, rt; (vi) NaOMe, MeOH, rt.
2
O, TEA, MeOH, 0 °C;
(
4
H
9
)
4
4
2
In the IR spectra, characteristic absorption bands (the compounds 7) for fragments: C=O and C-O-C are
−1
−1
visible in the ranges of: 1660-1750 cm and 1035-1042 cm , respectively. There are moderate bands in
the region about 3307-3429 cm−1 corresponding to ν (N-H) and band of ν (C-H) appears in the region
−
1
2
3
924-2936 cm . In the products 8, the wide and strong hydroxyl absorption bands in the region 3614-
−1
−1
630 cm appeared. Meanwhile, the strong absorption peaks (C=O) about 1743-1750 cm disappeared.
1
In the H NMR spectra, all the compounds 7 showed a characteristic triplet within 5.20–5.35 ppm region
having a coupling constant (J) in the range of 8.6-9.8 Hz, which indicated sugar ring was β-configuration,
due to the presence of H
glycosyl aliphatic protons were shown in the range 3.86-5.20 ppm. In compounds 8, the presence of only
a single peak (CH ) in the chemical shift of about 2.0 indicated that hydroxy protection groups have been
1
proton of the sugar ring coupled by the C -H and N-H while the signals of other
2
3
removed. However, the chemical shift of H proton on the sugar ring varied little.
In the mass spectra (ESI), the compounds 7 and 8 all show molecular ion peaks and give (M+H)+,
+
+
(
M+Na) and (M+K) peak stating this kind of compounds easily combine with metal ions. The detailed
1
results of IR, H NMR, and ESI-MS are presented in the experimental part.

278
HETEROCYCLES, Vol. 91, No. 2, 2015
Biological activity
The AChE-Inhibition activities of the newly synthesized compounds were evaluated in vitro by Ellman’s
method,42 in which the AChE extracts from Electric eel were used. Their inhibitory potency was
described as the inhibition rate and the half of maximal inhibitory concentration, IC50. Tacrine was
measured as a standard for the comparative purpose. The results were summarized in Table 1.
Table 1. In vitro inhibitory activities of glycosyl 1,3,4-thiadiazoles against AChE
Compound
R
Inhibition
Inhibition (%)a
< 10
20.34
24.89
19.76
< 10
43.67
25.60
64.52
< 10
53.21
27.53
58.76
32.64
23.42
73.11
IC50 (µM)
–
–
–
–
–
–
–
21.91±1.64
–
35.14±2.18
–
28.86±4.24
7
7
7
7
7
7
7
7
7
7
7
7
8
8
8
8
a
b
c
d
e
f
g
h
i
Me
C
4-MeC
3-MeOC
4-FC
2-ClC
4-IC
4-NO
4-MeCO
4-(N,N-di-Me)-C
6
H
5
6
H
4
6
H
4
6
H
4
6
H
4
6
H
4
2
C
6
H
4
2
C
6
H
4
j
k
l
6
H
4
3-C
2-C
6
5
H
H
4
3
N
S
c
d
h
j
4-MeC
3-MeOC
4-NO
4-(N,N-di-Me)-C
6
H
4
6
H
4
2
C
6
H
4
18.38±0.89
30.53±1.32
6
H
4
59.23
18.12
14.96
b
m
c
n
Tacrine
0.26±0.004
a
b
c
The inhibition activities of the compounds at the concentration of 50 µg/mL.
m stands for 5-(4-NO )-1,3,4-thiadiazole-2-amine.
n stands for 5-[4-(N,N-di-Me)-C ]-1,3,4-thiadiazole-2-amine.
2
C
6
H
4
6
H
4
As shown in the Table 1, it was found that 13 of the 16 tested compounds showed the AChE-inhibition
activities. Among them, the compounds 7h, 7l with 4-nitrophenyl and 2-thienyl substituent exhibited a
moderate inhibition on AChE with IC50 = 21.91 µM and 28.86 µM, respectively. Deprotected compound
8h showed the best AChE-inhibition activity with the IC50 value of 18.38 µM. Compared with the
4
3
positive control tacrine (IC50 = 0.26 µM) of the first drug used for the treatment of AD, it presented

HETEROCYCLES, Vol. 91, No. 2, 2015
279
promising inhibition activity against AChE, potentially useful in the treatment of AD. In addition, the
compounds m and n showed weak inhibitory against AChE compared with compound 7 and 8, indicating
that the presence of GlcNAc unit improved activities of 1,3,4-thiadiazole compounds. And compound 8c,
d, h, j all revealed higher potency as compared with 7c, d, h, j, suggesting that the removal of hydroxyl
protection groups also improved activities in a certain extent. This may be due to the removal of acetyl
groups leading to better water solubility of the compound 8. Compound 8h inhibited AChE with a
dose-dependent relationship (Figure. 1).
1
00
8
6
4
2
0
0
0
0
0
0.005
0.05
0.5
5
50
Concentration (μg/mL)
Figure 1. Dose-dependent inhibition of compound 8h against AChE. Values are means ± SD, n = 3.
EXPERIMENTAL
Chemistry
All chemicals and solvents were purchased from commercial sources and used without further
purification unless otherwise stated. Melting points were measured on a Yanaco melting point apparatus
1
and were uncorrected. IR spectra were recorded on a Bruker Tensor 27 spectrometer with KBr pellets. H
NMR spectra were recorded on a Bruker Avance 400 MHz instrument using DMSO-d
6
or D O as solvents
2
and tetramethylsilane (TMS) as an internal standard. Chemical shifts are expressed in ppm and coupling
constants (J) are reported in Hz. HRMS (ESI) analysis was performed on a Agilent 6230 Q-TOF mass
spectrometer. The purity of the compounds was checked by TLC on plates precoated with silica gel
GF254. Flash column chromatography was performed on silica 200-300 mesh. The compounds 2, 3 and 4

280
HETEROCYCLES, Vol. 91, No. 2, 2015
were prepared in this laboratory according to the procedure reported in literature.40,41
General procedure for the preparation of glycosyl acylthiosemicarbazide (6a−6l)
Glycosyl isothiocyanate 4 (0.388 g, 1 mmol) was added in one portion to a stirred solution of hydrazide 5
(
1 mmol) in EtOH (10 mL). The reaction mixture was reflux for 1-2 h, and then the solvent was removed
via a rotary evaporator. The residue was recrystallized from aqueous EtOH to obtain the desired product.
The characterization of the representative compound 6a was only presented in this paper.)
N-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-N'-methyl-acylthiosemicarbazide
6a)
(
(
−1
Yield 84%, white solid, mp 148-149 °C. IR (KBr) ν (cm ): 3320 (NH), 2972 (C-H), 1749 (CH C=O),
3
1
1
8
4
667 (NHC=O), 1044 (C-O-C). H NMR (400 MHz, DMSO) δ: 9.79 (br, 1H, NH), 9.72 (br, 1H, NH),
.21 (d, J = 8.9 Hz, 1H, NH), 7.84 (d, J = 7.3 Hz, 1H, NH), 5.32 (m, 1H, H
1
), 5.15 (d, J = 9.4 Hz, 1H, H
3
),
.82 (t, J = 9.8 Hz, 1H, H
4
), 4.19-3.81 (m, 4H, H
2
, H
5
, H
6
, H6'), 1.99-1.77 (5s, 15H, 5CH ). ESI-HRMS
3
+
(
m/z): Calcd for C17
H
26
N
4
O
9
SNa (M+Na) : 485.1313; Found: 485.1326.
General procedure for the preparation of glycosyl 1,3,4-thiadiazole (7a−7l)
Tosyl chloride (0.21 g, 1.1 mmol) was added to a stirred solution of acylthiosemicarbazide 6 (1 mmol)
and triethylamine (0.29 mL, 2.1 mmol) in NMP (5 mL). The solution was stirred at room temperature for
4
-6 h (TLC), and extracted with EtOAc (2×10 mL) and distilled water (2×10 mL). After the aqueous
layer removed, the aqueous layer was re-extracted thrice with EtOAc (3×10 mL). The organic phases
were combined and dried over anhydrous MgSO . The solid was filtered out and the solution was
evaporate to afford the crude product, which was purified by column chromatography on silica gel
MeOH/DCM) to give the pure product.
N-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-5-methyl-1,3,4-thiadiazole-2-amine
4
(
−1
(
7a): Yield 81%, White solid, mp 209-210 °C. IR (KBr) ν (cm ): 3328 (NH), 2928 (C-H), 1749
1
(
CH
3
C=O), 1662 (NHC=O), 1040 (C-O-C). H NMR (400 MHz, DMSO) δ: 8.33 (d, J = 9.3 Hz, 1H, NH),
8
1
1
.06 (d, J = 9.0 Hz, 1H, NH), 5.20 (t, J = 8.6 Hz, 1H, H
H, H ), 4.20 (dd, J = 12.4, 4.4 Hz, 1H, H ), 3.97-3.86 (m, 3H, H
.24 (s, 3H, CH ). ESI-HRMS (m/z): Calcd for C17
1
), 5.15 (t, J = 8.9 Hz, 1H, H ), 4.85 (t, J = 9.8 Hz,
3
4
6
2
, H
5
, H6'), 1.98-1.74 (4s, 12H, COCH ),
3
+
3
H
24
N
4
O
8
SNa (M+Na) : 467.1207; Found: 467.1202.
N-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-5-phenyl-1,3,4-thiadiazole-2-amine
−1
(
7b): Yield 84%, White solid, mp 225-226 °C. IR (KBr) ν (cm ): 3358 (NH), 2928 (C-H), 1744

HETEROCYCLES, Vol. 91, No. 2, 2015
281
1
(
CH
3
C=O), 1670 (NHC=O), 1035 (C-O-C). H NMR (400 MHz, DMSO) δ: 8.77 (d, J = 9.1 Hz, 1H, NH),
8
1
3
.12 (d, J = 9.0 Hz, 1H, NH), 7.79 (dd, J = 7.2, 2.3 Hz, 2H, Ar-H), 7.48 (m, 3H, Ar-H), 5.31 (t, J = 9.5 Hz,
H, H ), 5.19 (t, J = 9.9 Hz, 1H, H ), 4.88 (t, J = 9.8 Hz, 1H, H ), 4.22 (dd, J = 12.4, 4.1 Hz, 1H, H ),
, H6'), 1.99-1.76 (4s, 12H, COCH ). ESI-HRMS (m/z): Calcd for C22 S-
1
3
4
6
.99-3.91 (m, 3H, H
2
, H
5
3
H
26
N
4
O
8
+
Na (M+Na) : 529.1364; Found: 529.1365.
N-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-5-(4-methylphenyl)-1,3,4-thiadi-
−1
azole-2-amine (7c): Yield 89%, White solid, mp 207-208 °C. IR (KBr) ν (cm ): 3307 (NH), 2924 (C-H),
1
1
747 (CH
3
C=O), 1660 (NHC=O), 1037 (C-O-C). H NMR (400 MHz, DMSO) δ: 8.70 (d, J = 9.2 Hz, 1H,
NH), 8.11 (d, J = 9.0 Hz, 1H, NH), 7.67 (d, J = 8.1 Hz, 2H, Ar-H), 7.29 (d, J = 8.1 Hz, 2H, Ar-H), 5.29 (t,
J = 9.4 Hz, 1H, H ), 5.18 (t, J = 9.8 Hz, 1H, H ), 4.88 (t, J = 9.7 Hz, 1H, H ), 4.22 (dd, J = 12.4, 4.3 Hz,
H, H ), 3.99-3 .90 (m, 3H, H ), 1.98-1.76 (4s, 12H, COCH ). ESI-HRMS
m/z): Calcd for C23
1
3
4
1
6
2
, H
5
, H6'), 2.35 (s, 3H, CH
3
3
+
(
H
29
N
4
O
8
S (M+H) : 521.1701; Found: 521.1703.
N-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-5-(3-methoxyphenyl)-1,3,4-thiadi-
−1
azole-2-amine (7d): Yield 86%, White solid, mp 208-209 °C. IR (KBr) ν (cm ): 3269 (NH), 2936 (C-H),
1
1
747 (CH
3
C=O), 1668 (NHC=O), 1042 (C-O-C). H NMR (400 MHz, DMSO) δ: 8.77 (d, J = 9.1 Hz, 1H,
NH), 8.10 (d, J = 9.0 Hz, 1H, NH), 7.41 (t, J = 8.0 Hz, 1H, Ar-H), 7.33 (t, J = 4.4 Hz, 2H, Ar-H), 7.06 (m,
H, Ar-H), 5.29 (t, J = 9.4 Hz, 1H, H ), 5.19 (t, J = 9.8 Hz, 1H, H ), 4.88 (t, J = 9.7 Hz, 1H, H ), 4.22 (dd,
J = 12.4, 4.4 Hz, 1H, H ), 3.99-3.91 (m, 3H, H , H6'), 3.82 (s, 3H, CH ), 1.99-1.76 (4s, 12H, COCH ).
ESI-HRMS (m/z): Calcd for C23
1
1
3
4
6
2
, H
5
3
3
+
H
28
N
4
O
9
S (M+H) : 537.1650; Found: 537.1662.
N-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-5-(4-fluorophenyl)-1,3,4-thiadi-
−1
azole-2-amine (7e): Yield 89%, White solid, mp 206-207 °C. IR (KBr) ν (cm ): 3348 (NH), 2957 (C-H),
1
1
743 (CH
3
C=O), 1662 (NHC=O), 1041 (C-O-C). H NMR (400 MHz, DMSO) δ: 8.74 (d, J = 9.1 Hz, 1H,
NH), 8.09 (d, J = 9.0 Hz, 1H, NH), 7.84 (m, 2H, Ar-H), 7.33 (t, J = 8.8 Hz, 2H, Ar-H), 5.30 (t, J = 9.4 Hz,
1
H, H
1
), 5.19 (t, J = 9.8 Hz, 1H, H
3
), 4.88 (t, J = 9.7 Hz, 1H, H
4
), 4.22 (dd, J = 12.4, 4.4 Hz, 1H, H
6
),
4
.00-3.92 (m, 3H, H
2
, H
5
, H6'), 1.99-1.76 (4s, 12H, COCH ). ESI-HRMS (m/z): Calcd for C22
3
H25FN
4
O
8
S-
+
Na (M+Na) : 547.1269; Found: 547.1269.
N-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-5-(2-chlorophenyl)-1,3,4-thiadi-
−1
azole-2-amine (7f): Yield 78%, White solid, mp 198-199 °C. IR (KBr) ν (cm ): 3332 (NH), 2930 (C-H),
1
1
748 (CH
3
C=O), 1663 (NHC=O), 1039 (C-O-C). H NMR (400 MHz, DMSO) δ: 8.78 (d, J = 9.2 Hz, 1H,
NH), 8.10 (d, J = 9.0 Hz, 1H, NH), 7.99 (m, 1H, Ar-H), 7.64 (m, 1H, Ar-H), 7.51 (m, 2H, Ar-H), 5.33 (t, J

282
HETEROCYCLES, Vol. 91, No. 2, 2015
=
9.4 Hz, 1H, H
1
), 5.19 (t, J = 9.8 Hz, 1H, H
3
), 4.89 (t, J = 9.7 Hz, 1H, H
4
), 4.22 (dd, J = 12.4, 4.4 Hz, 1H,
H
6
), 4.00-3.93 (m, 3H, H
2
, H
5
, H6'), 1.99-1.76 (4s, 12H, COCH ). ESI-HRMS (m/z): Calcd for
3
+
C
22
H25ClN
4
O
8
SNa (M+Na) : 563.0974; Found: 563.0970.
N-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-5-(4-iodophenyl)-1,3,4-thiadiazole-
−
1
2
-amine (7g): Yield 82%, White solid, mp 232-233 °C. IR (KBr) ν (cm ): 3348 (NH), 2927 (C-H), 1743
1
(
CH
3
C=O), 1661 (NHC=O), 1041 (C-O-C). H NMR (400 MHz, DMSO) δ: 8.81 (d, J = 9.0 Hz, 1H, NH),
8
.11 (d, J = 9.0 Hz, 1H, NH), 7.86 (d, J = 8.6 Hz, 2H, Ar-H), 7.59 (d, J = 8.6 Hz, 2H, Ar-H), 5.31 (t, J =
.4 Hz, 1H, H ), 5.19 (t, J = 9.7 Hz, 1H, H ), 4.89 (t, J = 9.9 Hz, 1H, H ), 4.22 (dd, J = 12.4, 4.4 Hz, 1H,
), 4.01-3.92 (m, 3H, H , H , H6'), 1.99-1.76 (4s, 12H, COCH ). ESI-HRMS (m/z): Calcd for
SNa (M+Na) : 655.0330; Found: 655.0316.
N-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-5-(4-nitrophenyl)-1,3,4-thiadiazole-
9
1
3
4
H
6
2
5
3
+
C
22
H25IN
4
O
8
−1
2
-amine (7h): Yield 83%, Yellow solid, mp 117-118 °C. IR (KBr) ν (cm ): 3422 (NH), 2927 (C-H),
1
1
749 (CH
3
C=O), 1662 (NHC=O), 1041 (C-O-C). H NMR (400 MHz, DMSO) δ: 9.01 (d, J = 9.0 Hz, 1H,
NH), 8.33 (m, 2H, Ar-H), 8.08 (m, 3H, NH, Ar-H), 5.35 (t, J = 9.3 Hz, 1H, H ), 5.20 (t, J = 9.8 Hz, 1H,
1
H
3
), 4.90 (t, J = 9.7 Hz, 1H, H
4
), 4.22 (dd, J = 12.4, 4.4 Hz, 1H, H
6
), 4.02-3.96 (m, 3H, H
2
, H , H6'),
5
+
1
.99-1.76 (4s, 12H, COCH
3
). ESI-HRMS (m/z): Calcd for C22
H
25
N
5
O
10SNa (M+Na) : 574.1214; Found:
5
74.1208.
N-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-5-(4-acetoxyphenyl)-1,3,4-thiadi-
−1
azole-2-amine (7i): Yield 76%, White solid, mp 116-117 °C. IR (KBr) ν (cm ): 3382 (NH), 2936 (C-H),
1
1
750 (CH
3
C=O), 1666 (NHC=O), 1040 (C-O-C). H NMR (400 MHz, DMSO) δ: 8.75 (d, J = 9.1 Hz, 1H,
NH), 8.09 (d, J = 9.1 Hz, 1H, NH), 7.84 (m, 2H, Ar-H), 7.26 (m, 2H, Ar-H), 5.31 (t, J = 9.4 Hz, 1H, H
1
),
), 3.99-3.91 (m,
). ESI-HRMS (m/z): Calcd for
5
3
.19 (t, J = 9.8 Hz, 1H, H
3
), 4.89 (t, J = 9.7 Hz, 1H, H
4
), 4.22 (dd, J = 12.4, 4.4 Hz, 1H, H
6
H, H
2
, H
5
, H6'), 2.31 (s, 3H, CH
3
), 1.99-1.76 (4s, 12H, COCH
3
+
C
24
H
28
N
4
O
10SNa (M+Na) : 587.1418; Found: 587.1410.
N-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-5-(4-N,N-dimethylphenyl)-1,3,4-
−
1
thiadiazole-2-amine (7j): Yield 88%, White solid, mp 197-198 °C. IR (KBr) ν (cm ): 3429 (NH), 2927
1
(
C-H),1748 (CH
3
C=O), 1662 (NHC=O), 1042 (C-O-C). H NMR (400 MHz, DMSO) δ: 8.50 (d, J = 9.2
Hz, 1H, NH), 8.09 (d, J = 8.9 Hz, 1H, NH), 7.58 (d, J = 8.9 Hz, 2H, Ar-H), 6.76 (d, J = 9.0 Hz, 2H, Ar-H),
5
4
.26 (t, J = 9.4 Hz, 1H, H
1
), 5.16 (m, 1H, H
3
), 4.87 (dd, J = 11.5, 8.5 Hz, 1H, H
), 1.99-1.75 (4s, 12H, COCH
4
), 4.21 (m, 1H, H
6
),
.00-3.93 (m, 3H, H , H , H6'), 2.98 (s, 6H, CH
2
5
3
3
). ESI-HRMS (m/z):

HETEROCYCLES, Vol. 91, No. 2, 2015
283
+
Calcd for C24
H
31
N
5
O
8
SNa (M+Na) : 572.1786; Found: 572.1776.
N-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-5-(3-pyridyl)-1,3,4-thiadiazole-2-
−
1
amine (7k): Yield 71%, Yellow solid, mp 113-114 °C. IR (KBr) ν (cm ): 3426 (NH), 2930 (C-H), 1749
1
(
CH
3
C=O), 1663 (NHC=O), 1040 (C-O-C). H NMR (400 MHz, DMSO) δ: 8.98 (d, J = 2.1 Hz, 1H,
Pyridine), 8.89 (d, J = 9.0 Hz, 1H, NH), 8.65 (dd, J = 4.8, 1.5 Hz, 1H, Pyridine), 8.18 (m, 1H, Pyridine),
.13 (d, J = 9.0 Hz, 1H, NH), 7.53 (dd, J = 7.9, 4.8 Hz, 1H, Pyridine), 5.33 (t, J = 9.3 Hz, 1H, H ), 5.19 (t,
J = 9.9 Hz, 1H, H ), 4.89 (t, J = 9.8 Hz, 1H, H ), 4.23 (dd, J = 12.4, 4.3 Hz, 1H, H ), 4.00-3.93 (m, 3H,
, H , H6'), 1.99-1.76 (4s, 12H, COCH ). ESI-HRMS (m/z): Calcd for C21
SNa (M+Na)+:
30.1316; Found: 530.1320.
N-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-5-(2-thienyl)-1,3,4-thiadiazole-2-
8
1
3
4
6
H
2
5
3
H
25
N
5
O
8
5
−1
amine (7l): Yield 65%, White solid, mp 201-202 °C. IR (KBr) ν (cm ): 3418 (NH), 2926 (C-H), 1745
1
(
CH
3
C=O), 1668 (NHC=O), 1037 (C-O-C). H NMR (400 MHz, DMSO) δ: 8.76 (d, J = 9.1 Hz, 1H, NH),
8
.10 (d, J = 9.0 Hz, 1H, NH), 7.68 (dd, J = 5.1, 1.1 Hz, 1H, Thiophene), 7.50 (dd, J = 3.7, 1.1 Hz, 1H,
Thiophene), 7.15 (dd, J = 5.1, 3.7 Hz, 1H, Thiophene), 5.27 (t, J = 9.4 Hz, 1H, H ), 5.18 (t, J = 9.8 Hz,
1
1
1
5
H, H
3
), 4.88 (t, J = 9.8 Hz, 1H, H
4
), 4.22 (dd, J = 12.4, 4.5 Hz, 1H, H
6
), 4.00-3.91 (m, 3H, H
2
, H , H6'),
5
+
.99-1.76 (4s, 12H, COCH
3
). ESI-HRMS (m/z): Calcd for C20
H
24
N
4
O
8
S
2
Na (M+Na) : 535.0928; Found:
35.0930.
General procedure for deprotection of O-acetyl-substituted compounds 7 to hydroxy derivatives 8
A solution of compound 7 (0.5 mmol) in MeOH (5 mL) was treated with sodium methoxide (1 M in
MeOH, 0.2 mL). The mixture was stirred at ambient temperature for 2 h and then neutralized with
Amberlite IR 120 H+ resin and filtered. The filtrate was concentrated to afford the deprotected product 8.
Selected products 8c, d, h, j are characterized below.
N-(2-Acetamido-2-deoxy-β-D-glucopyranosyl)-5-(4-methylphenyl)-1,3,4-thiadiazole-2-amine (8c):
−1
Yield 90%, White solid, mp 156-157 °C. IR (KBr) ν (cm ): 3414 (OH), 2928 (C-H), 1626 (NHC=O),
1
1
074 (C-O-C). H NMR (400 MHz, D
2
O) δ: 7.66 (d, J = 8.1 Hz, 2H, Ar-H), 7.29 (d, J = 8.0 Hz, 2H,
), 3.66-3.15 (m, 6H, H , H , H , H , H ), 2.34 (s, 3H, CH ), 1.81 (s, 3H,
). ESI-HRMS (m/z): Calcd for C17 SNa (M+Na) : 417.1203; Found: 417.1202.
N-(2-Acetamido-2-deoxy-β-D-glucopyranosyl)-5-(3-methoxyphenyl)-1,3,4-thiadiazole-2-amine (8d):
Ar-H), 4.81 (t, J = 8.7 Hz, 1H, H
1
2
3
4
5
6
3
+
CH
3
H
22
N
4
O
5
−1
Yield 88%, White solid, mp 101-102 °C. IR (KBr) ν (cm ): 3416 (NH), 2934 (C-H), 1629 (NHC=O),
1
1
044 (C-O-C). H NMR (400 MHz, D
2
O) δ: 7.39 (m, 1H, Ar-H), 7.32 (m, 2H, Ar-H), 7.03 (m, 1H, Ar-H),

284
HETEROCYCLES, Vol. 91, No. 2, 2015
4
.85 (t, J = 9.0 Hz, 1H, H
ESI-HRMS (m/z): Calcd for C17
N-(2-Acetamido-2-deoxy-β-D-glucopyranosyl)-5-(4-nitrophenyl)-1,3,4-thiadiazole-2-amine (8h):
1
), 3.76-3.21 (m, 6H, H
2
, H
3
, H
4
, H
5
, H
6
), 3.80 (s, 3H, CH
3
), 1.82 (s, 3H, CH ).
3
+
H
22
N
4
O
6
SNa (M+Na) : 433.1152 ; Found: 433.1150.
−
1
Yield 81%, Yellow solid, mp 175-176 °C. IR (KBr) ν (cm ): 3430 (OH), 2926 (C-H), 1634 (NHC=O),
1
1
1
048 (C-O-C). H NMR (400 MHz, D
2
O) δ: 8.30 (m, 2H, Ar-H), 8.10 (m, 2H, Ar-H), 4.92 (t, J = 8.9 Hz,
H, H
1
), 3.81-3.26 (m, 6H, H
2
, H
3
, H , H , H ), 1.84 (s, 3H, CH ). ESI-HRMS (m/z): Calcd for
4
5
6
3
+
C
16
H
19
N
5
O
7
SNa (M+Na) : 448.0897 ; Found: 448.0895.
N-(2-Acetamido-2-deoxy-β-D-glucopyranosyl)-5-(4-N,N-dimehylphenyl)-1,3,4-thiadiazole-2-amine
−1
(
8j): Yield 84%, White solid, mp 184-185 °C. IR (KBr) ν (cm ): 3412 (OH), 2925 (C-H), 1627
1
(
NHC=O), 1074 (C-O-C). H NMR (400 MHz, D
2
O) δ: 7.63 (d, J = 7.3 Hz, 2H, Ar-H), 6.83 (d, J = 7.2
), 3.73-3.24 (m, 6H, H , H , H , H , H ), 3.02 (s, 6H, CH ),
). ESI-HRMS (m/z): Calcd for C18 SNa (M+Na) : 446.1469; Found: 446.1468.
Hz, 2H, Ar-H), 4.91 (d, J = 9.4 Hz, 1H, H
1
2
3
4
5
6
3
+
1
.90 (s, 3H, CH
3
H
25
N
5
O
5
Biological tests
Acetylcholinesterase (AChE, from Electric eel), acetylthiocholine iodide (ATCI), 5,5-dithiobis-(2-nitro-
benzoic acid) (DTNB) and tacrine were purchased from Sigma-Aldrich (America). AChE activities were
measured by Ellman’s colorimetric method with a slight modification,42 using tacrine as the reference
compounds. In assays, 10 µL of rat cortex homogenate was incubated with 10 µL of tested compounds
and 140 µL of 0.1 M PBS (pH 8.0) for 10 min in 96-well microplates before addition of 20 µL of 3.33
mM DTNB solution and 20 µL of 5.30 mM ATCI solution. After the addition of DTNB and ATCI, the
9
6-well microplates were read at 412 nm with a microplate reader (SPECTRAFLUOR, TECAN, Sunrise,
Austria) for 15 min. One triplicate sample without inhibitors was always present to yield 100% of AChE
activity. One triplicate sample with 20 µM tacrine was always present to yield 100% AChE inhibition (to
obviate the ChE-independent substrate hydrolysis). The reaction rates were compared and the percent
inhibition due to the presence of tested compounds was calculated. Each concentration was assayed in
triplicate. The 50% inhibitory concentration (IC50) was calculated from a dose-response curve obtained by
plotting the percentage of inhibition versus the log concentration with the use of Origin 8.0 software. The
results were described as the mean ± standard deviation (SD).
ACKNOWLEDGEMENTS
We are grateful to the Major Project of Natural Science Research in Colleges and Universities in Jiangsu

HETEROCYCLES, Vol. 91, No. 2, 2015
285
Education Committee (No.10KJA170003), the Industrialization Project of Scientific Research in Colleges
and Universities of Jiangsu Province (No.JHB2012-60), the Science and the Technology Critical Project
Foundation of Lianyungang (No.CG1105), Collegiate Natural Science Fund of Jiangsu Province
(
12KJB350001) and Science and Technology Project of Lianyungang (CG1202).
REFERENCES
1
2
3
4
. Y. E. Tsvetkov, M. Burg-Roderfeld, and G. Loers, J. Am. Chem. Soc., 2011, 134, 426.
. M. Kimura, Y. Masui, and Y. Shirai, Carbohydr. Res., 2013, 374, 49.
. R. M. Schmaltz, S. R. Hanson, and C. H. Wong, Chem. Rev., 2011, 111, 4259.
. A. Varki, D. C. Cummings, J. D. Esko, H. H. Freeze, P. Stanley, C. R. Bertozzi, G. W. Hart, and M. E.
Etzler, Essentials of Glycobiology, 2nd ed. Cold Spring Habor Laboratory Press: New York, 2009.
. Y. Guan, Y. Tian, Y. Li, Z. F. Yang, Y. Y. Jia, and T. J. Hang, J. Pharm. Biomed., 2011, 55, 181.
. B. Liberek, A. Melcer, A. Osuch, and R. Wakiecb, Carbohydr. Res., 2005, 340, 1876.
. S. S. Shivatare, S. H. Chang, T. I. Tsai, C. T. Ren, H. Y. Chuang, C. W. Lin, S. T. Li, C. Y. Wu, and C.
H. Wong, J. Am. Chem. Soc., 2013, 135, 153821.
5
6
7
8
. V. Pozsgay, J. Kubler-Kielb, P. Santacroce, J. B. Robbins, and R. Schneerson, J. Org. Chem., 2012, 77,
5
922.
9
1
. A. Fekete, D. Eszenyi, M. Herczeg, and V. Pozsgay, Carbohydr. Res., 2014, 386, 33.
0. S. Kun, G. Z. Nagy, M. Tóth, L. Czecze, T. Docsa, P. Gergely, M. D. Charavgi, T. Patonay, and L.
Somsák, Carbohydr. Res., 2011, 346, 1427.
1
1
1. S. Bera and R. J. Linhardt, J. Org. Chem., 2011, 76, 3181.
2. A. Chibba, S. Dasgupta, N. Yakandawala, S. Madhyastha, and M. Nitz, J. Carbohydr. Chem., 2011, 30,
5
49.
1
1
3. M. E. Jung, T. A. Dong, and X. Cai, Tetrahedron Lett., 2011, 52, 2533.
4. A. Drozdová, P. Bojarová, K. Křenek, L. Weignerova, B. Hensen, L. Elling, H. Christensen, H. H.
Jensen, H. Pelantova, and M. Kuzma, Carbohydr. Res., 2011, 346, 1599.
1
1
5. K. Du, X. Cao, P. Zhang, and H. Zheng, Bioorg. Med. Chem. Lett., 2014, DOI: 10.1016/ j.bmcl.
2
014.09.042.
6. K. A. Dilmaghani, F. N. Pur, N. H. Jazani, A. Alavi, Z. Niknam, and F. Mirfakhraee, Phosphorus,
Sulfur Silicon Relat. Elem., 2014, 189, 81.
1
1
7. A. R. El-Gazzar, H. N. Hafez, and H. A. S. Abbas, Eur. J. Med. Chem., 2009, 44, 4249.
8. B. Chandrakantha, A. M. Isloor, P. Shetty, H. K. Fun, and G. Hegde, Eur. J. Med. Chem., 2014, 44,
4
249.
1
2
9. K. R. Alagawadi and S. G. Alegaon, Arabian J. Chem., 2011, 4, 465.
0. M. Juszczak, J. Matysiak, M. Szeliga, P. Pozarowski, and W. Rzeski, Bioorg. Med. Chem. Lett., 2012,

286
HETEROCYCLES, Vol. 91, No. 2, 2015
2
2, 5466.
2
2
2
1. M. N. Noolvi, H. M. Patel, N. Singh, A. K. Gadad, and A. Badiger, Eur. J. Med. Chem., 2011, 46,
4
411.
2. D. Sunil, A. M. Isloor, P. Shetty, K. Satyamoorthy, and A. S. B. Prasad, Arabian J. Chem., 2010, 3,
11.
2
3. I. Khan, S. Ali, S. Hameed, N. H. Rama, M. T. Hussain, A. Wadood, R. Uddin, Z. Ul-Haq, A. Khan, S.
Ali, and M. I. Choudhary, Eur. J. Med. Chem., 2010, 45, 5200.
2
2
4. F. Clerici, D. Pocar, M. Guido, A. Loche, V. Perlini, and M. Brufani, J. Med. Chem., 2001, 44, 931.
5. S. Jubie, P. N. Ramesh, P. Dhanabal, R. Kalirajana, N. Murugananthamb, and A. S. Antonyc, Eur. J.
Med. Chem., 2012, 54, 931.
2
2
2
6. V. Jatav, P. Mishra, S. Kashaw, and J. P. Stablesb, Eur. J. Med. Chem., 2008, 43, 1945.
7. K. P. Harish, K. N. Mohana, and L. S. Mallesha, Drug Invention Today, 2013, 5, 92.
8. T. Hasui, N. Matsunaga, T. Ora, N. Ohyabu, N. Nishigaki, Y. Imura, Y. Igata, H. Matsui, T. Motoyaji,
N. Habuka, S. Sogabe, and M. Ono, J. Med. Chem., 2011, 54, 8616.
2
3
9. P. Zoumpoulakis, C. Camoutsis, G. Pairas, M. Sokovic, J. Glamoclija, C. Potamitis, and A. Pitsas,
Bioorg. Med. Chem., 2012, 20, 1569.
0. W. M. Xu, S. Yang, P. Bhadury, J. He, M. He, L. L. Gao, and B. A. Song, Pestic. Biochem. Physiol.,
2
011, 101, 6.
3
3
3
3
3
3
1. I. P. Kaur, R. Smitha, D. Aggarwal, and M. Kapil, Int. J. Pharm., 2002, 248, 1.
2. P. Wolf, CNS Neurosci. Ther., 2011, 17, 442.
3. M. B. Russell and A. Ducros, Lancet Neurol., 2011, 10, 457.
4. H. G. Tiselius, Curr. Opin. Urol., 2010, 20, 169.
5. S. Talath and A. K. Gadad, Eur. J. Med. Chem., 2006, 41, 918.
6. A. Skrzypek, J. Matysiak, M. M. Karpinska, and A. Niewiadomy, Enzyme Inhib. Med. Chem., 2013,
2
8, 816.
7. A. Skrzypek, J. Matysiak, A. Niewiadomy, M. Bajda, and P. Szymanski, Eur. J. Med. Chem., 2013, 62,
11.
3
3
3
8. W. W. Liu, D. H. Shi, C. Z. Tao, Y. Q. Zhao, and L. J. Tang, Proceedings of 2009 International
Conference of Natural Products and Traditional Medicine, Xi’An, China, October 16-18, 2009 [C];
Berlin: Springer.
3
4
4
4
4
9. D. H. Shi, W. Huang, C. Li, L.T. Wang, and S. F. Wang, Bioorg. Med. Chem., 2013, 21, 1064.
0. V. R. Bouvet and R. N. Ben, J. Org. Chem., 2006, 71, 3619.
1. M. J. Amarasa, P. Fernandez-Resa, and M. T. Garcia-Lopez, Synthesis, 1984, 509.
2. G. L. Ellman, K. D. Courtney, V. Andres, and R. M. Feather-Stone, Biochem. Pharmacol., 1961, 7, 88.
3. J. L. Cummings, Am. J. Psychiatry, 2000, 157, 4.