F. Li et al. / European Journal of Medicinal Chemistry 108 (2016) 486e494
491
ꢁ
1
1
1
0
0
0
.6
.2
.8
.4
.0
ethyl acetate/acetic acid (4/3/3/0.02); IR (KBr): 3184 cm phenolic
*
*
ꢁ
1
OeH stretching vibration, 1733 cm C]O stretching vibration,
256 cm CeOeC stretching vibration, 3371 cm NeH stretching
unimodal vibration, 1656 cm C]O stretching vibration (amide);
ꢁ
1
ꢁ1
1
ꢁ1
1
H NMR (DMSO-d
6
,
d
ppm): 3.86 (s, 3H, -COOCH
3
), 5.17 (s, 2H, -O-
), 7.20 (d, 1H,
CH
2
-Ph'), 7.03 (dd, 1H, J ¼ 8.7 Hz, 2.0 Hz, HeC
5
#
#&&
#
#&&
#
#
J ¼ 2.1 Hz, HeC ), 7.34e7.45 (m, 5H, H-Ph'), 7.71 (d, 1H, J ¼ 8.7 Hz,
3
#
#
HeC
for C16
6
), 10.19 (s, 1H, -NH-), 10.65 (s, 1H, Ph-OH); HRMS (ESI): Calcd
þ
#
#++
H15NO
5
, [M þ H] , 302.1023; Found, 302.1027.
#
#++
4
(b-D
.1.4. Preparation of methyl 4-(N-(benzyloxycarbonyl) amino)-2-
-glucopyranosyl) benzoate (4)
Compound 3 (1.21 g, 4 mmol) and TBAB (0.70 g) were dissolved
in CH Cl (20 ml), and 5% NaOH solution (7.2 ml) was added to it.
After the mixture was stirred at room temperature for 30 min, a
solution of compound 1 (5 mmol) in CH Cl (10 ml) was added
dropwise to the above mixture with constant stirring. The mixture
was refluxed for 5 h at 50 C on a thermostatic magnetic heating
stirrer and then cooled in an ice-water bath. The organic layer was
collected and washed with 5% NaOH solution (2 ꢂ 20 ml) and H
3 ꢂ 20 ml), respectively. Then the organic layer was concentrated
under reduced pressure to give intermediate. The intermediate was
dissolved in methanol (20 ml), and 5% Na CO solution (5 ml) was
added dropwise. After stirring at room temperature for 2 h, the
mixture was neutralized with 3 mol/L HCl. The organic solvent was
removed under reduced pressure and sufficient cold distilled water
2 2
was added, filtered and crude product was washed with CH Cl ,
recrystallized in mixture of methanol and distilled water (1:1 v/v),
dried under high vacuum to give compound 4 (1.01 g, yield: 54%).
2
2
S
se
do
A
S
A
S
lthy
Hea
-
TNB
SASP
4-A
5-A
High
Low-dose
2
2
Middle-dose
ꢀ
Fig. 7. Determination of myeloperoxidase activity after final drug administration for
*
*
n ¼ 6 animals. Data are expressed as mean ± SD. P < 0.01 versus healthy group;
#
#
þþ
&&
P < 0.01 versus TNBS group;
middle-dose group.
P < 0.01 versus SASP group;
P < 0.01 versus
2
O
(
2
3
filtered by Buchner funnel, then the filtrate was collected and
washed with NaHCO
saturated solution (4 ꢂ 20 ml), 0.4 mol/L
Na O (3 ꢂ 20 ml), respectively. The
solution (2 ꢂ 20 ml) and H
organic layer was dried over anhydrous Na SO , filtered and
concentrated under reduced pressure to give crude product. The
crude product was recrystallized from ethyl ether to afford pure
3
2
S
2
O
3
2
2
4
ꢀ
ꢀ
white crystals 1 (7.65 g, yield: 67%). mp: 87e89 C (88e89 C in
ꢀ
20
D
2
0
mp: 148e150 C; ½aꢃ
¼ þ44.8 (c 0.1, MeOH); R
f
¼ 0.73 in
literature [22]); ½aꢃ ¼ þ191.2 (c 0.1, CHCl
3
); R
f
¼ 0.63 in petroleum
D
ꢁ1
dichloromethane/methanol (7/1); IR (KBr): 1719 cm
C]O
ether/diethyl ether/ethyl acetate/acetic acid (4/3/3/0.02), iodine
vapours/UV light as detecting agents.
ꢁ
1
stretching vibration, 1231 cm
CeOeC stretching vibration,
305 cm NeH stretching vibration, 1302 cm CeN stretching
ꢁ1
ꢁ1
3
ꢁ1
ꢁ1
vibration, 1687 cm C]O stretching vibration (amide), 3356 cm
4
.1.2. Preparation of methyl 4-amino-2-hydroxybenzoate (2)
Concentrated sulfuric acid (1.25 ml) was added dropwise to
methanol (6 ml) with cooling on a cryostatic bath. The mixture was
stirred for 1 h at room temperature, and 4-ASA (1.53 g, 10 mmol)
was added to it. After refluxing at 60e70 C for 6 h, the mixture was
adjusted to pH 7e8 with 10% Na CO solution in ice-bath. The
2 3
mixture was filtered to afford crude solid product. The crude
product was recrystallized from hot methanol, followed by cooling
to 0 C. White crystals were collected and dried under high vacuum
ꢁ1
ꢁ1
OeH stretching broad peak, 1086 cm and 1028 cm glycosyl
ꢁ1
CeOeC stretching vibration, 1254 cm
glycoside bond Ph-O-C
ppm): 3.23e3.30 (m,
1
stretching vibration; H NMR (DMSO-d
6
,
d
4
H, HeC
HeC
), 3.62 (dd,1H, J ¼ 6.0 Hz, 4.0 Hz, HeC
OHeC structure), HeC
), 4.79[d, 1H, J ¼ 6.9 Hz (
J ¼ 4.5 Hz, OHeC ), 5.09 (d, 1H, J ¼ 3.2 Hz, OHeC
J ¼ 4.0 Hz, OHeC ), 5.18 (s, 2H, -O-CH
.7 Hz, HeC ), 7.36e7.46 (m, 6H, H-Ph' and HeC
J ¼ 8.6 Hz, HeC ), 10.12 (s, 1H, -NH-); HRMS (ESI): Calcd for
25NO10, [M þ Na] , 486.1371; Found, 486.1373.
2
0
e5
0
), 3.77 (s, 3H, -COOCH
3
), 3.73 (dd, 1H, J ¼ 7.2 Hz, 1.8 Hz,
ꢀ
0
0
), 4.47 (t,1H, J ¼ 5.9 Hz,
6
6
0
b
0
], 5.03 (d, 1H,
0
), 5.13 (d, 1H,
6
1
0
4
3
0
-Ph'), 7.30 (dd, 1H, J ¼ 8.6 Hz,
2
2
ꢀ
1
5
3
), 7.67 (d, 1H,
ꢀ
to give compound 2 (1.29 g, yield: 77%). mp: 119e121 C; R
in petroleum ether/diethyl ether/ethyl acetate/acetic acid (4/3/3/
f
¼ 0.73
6
þ
22
C H
ꢁ
1
0
3
1
.02); IR (KBr): 3260 cm
phenolic OeH stretching vibration,
ꢁ1
ꢁ1
474 cm
and 3380 cm
NeH stretching bimodal vibration,
4
.1.5. Preparation of methyl 4-amino-2-( -glucopyranosyl)
b-D
ꢁ
1
ꢁ1
638 cm NeH bending vibration; 1662 cm C]O stretching
benzoate (5)
ꢁ
1
1
vibration, 1284 cm CeOeC stretching vibration; H NMR (DMSO-
ppm): 3.79 (s, 3H, -COOCH ), 6.13
), 6.00 (d,1H, J ¼ 2.1 Hz, HeC
), 6.14 (s, 2H, Ph-NH ), 7.46 (d, 1H,
Protected 4-ASA glucose conjugate (1.67 g, 3.6 mmol) was dis-
solved in methanol (150 ml), and then was catalytically hydroge-
nated under 0.4 MPa with 10% Pd/C (0.20 g) in a high pressure
d
(
6
,
d
3
3
dd, 1H, J ¼ 8.7 Hz, 2.2 Hz, HeC
5
2
J ¼ 8.7 Hz, HeC
6
), 10.77 (s, 1H, Ph-OH); HRMS (ESI): Calcd for
ꢀ
apparatus. After stirring at 50 C for 24 h, the solution was filtered
þ
C
8
H
9
NO
3
, [M þ H] , 168.0655; Found, 168.0655.
and concentrated under reduced pressure to give crude product
which was purified by silica gel column chromatography using the
solvent system ethyl acetate/methanol (5/1 v/v) to give compound
4
.1.3. Preparation of methyl 4-(N-(benzyloxycarbonyl) amino)-2-
hydroxybenzoate (3)
2
0
5 (0.98 g, yield: 83%). ½aꢃ ¼ þ66.7 (c 0.1, MeOH); R ¼ 0.35 in
f
D
ꢁ
1
Methyl-4-ASA (1.67 g, 10 mmol) was dissolved in a mixture of
dichloromethane/methanol (7/1); IR (KBr): 1688 cm
C]O
ꢁ
stretching vibration, 1200 cm
1
CH
1.9 ml) was added dropwise. After stirring at room temperature for
h, the solution was concentrated under reduced pressure to about
5e20 ml. Sufficient cold distilled water was added, filtered and
2
Cl
2
(20 ml) and pyridine (5 ml). Then benzylchloroformate
CeOeC stretching vibration,
OeH and NeH stretching vibration (broad peak),
1608 cm NeH bending vibration, 1073 cm glycosyl CeOeC
stretching vibration, 1258 cm glycoside bond Ph-O-C stretching
ꢁ1
(
4
1
3365 cm
ꢁ
1
ꢁ1
ꢁ
1
1
crude product was obtained. The crude product was recrystallized
by dissolving in ethanol and cooling to 0 C. Purified white crystals
were dried under high vacuum to give compound 3 (2.86 g, yield:
5%). mp: 142e144 C; R
vibration; H NMR (DMSO-d
3.70 (s, 3H, -COOCH
), 3.75 (dd, 1H, J ¼ 11.2 Hz, 4.8 Hz, HeC
(dd, 1H, J ¼ 11.2 Hz, 4.4 Hz, HeC ), 4.57 (s, 1H, OHeC ), 4.73[d, 1H,
J ¼ 7.1 Hz ( structure), HeC ], 5.06e5.10 (m, 3H, OHeC ), 5.97
6
,
d
ppm): 3.28e3.31 (m, 4H, HeC
2 e5
0 0
),
ꢀ
0
6
), 3.59
3
0
0
6
6
ꢀ
9
f
¼ 0.83 in petroleum ether/diethyl ether/
b
1
0
0 0
2 ~4