A New Series of Salicylic Acid Derivatives as Non-saccharide α-Glucosidase Inhibitors and Antioxidants

Article abstract of DOI:10.1248/bpb.b18-00661

In this study, a series of salicylic acid derivatives were designed and synthesized as novel non-saccharide α-glucosidase inhibitors. Biological evaluation indicated that when compared to acarbose, compounds T9, T10, and T32 exhibited a higher potency of α-glucosidase inhibitory activity with IC₅₀ values of 0.15±0.01, 0.086±0.01 and 0.32±0.02mM, respectively. Evaluation of the inhibition kinetics indicated that T9, T10, T32, and acarbose interacted with α-glucosidase in a mixed non-competitive inhibitory manner. Moreover, T9, T10, and T32 statically quenched the fluorescence of α-glucosidase by formation of an inhibitor-α-glucosidase complex. The docking results showed that hydrogen bonds were generated between the test compounds and α-glucosidase. The antioxidant study revealed that compound T10 exhibited a higher antioxidant activity via scavenging 1,1-diphenyl-2-picrylhydrazyl free radical (DPPH), thereby inhibiting lipid peroxidation and the total reduction capacity. In brief, the salicylic acid derivatives identified in this study were promising candidates for development as novel non-saccharide α-glucosidase inhibitors.

Full text of DOI:10.1248/bpb.b18-00661

Biological and Pharmaceutical Bulletin Advance Publication by J-STAGE
DOI:10.1248/bpb.b18-00661
Advance Publication
November 30, 2018
Biol. Pharm. Bull.
(Regular Article)
A new series of Salicylic acid derivatives as non-saccharide α-glucosidase inhibitors and
antioxidants
Jiangang Chen, Wenfang Lu, Hao Chen, Xiaoli Bian∗ and Guangde Yang∗
College of Pharmacy, Xi’an Jiaotong University, No.76 Yanta West Road, Xi’an, Shaanxi
Province 710061, People’s Republic of China
∗Correspondence e-mail: bianxl@mail.xjtu.edu.cn, jmw52@mail.xjtu.edu.cn.
Ⓒ 2018 The Pharmaceutical Society of Japan

Summary
In this study, a series of salicylic acid derivatives were designed and synthesized as novel
non-saccharide α-glucosidase inhibitors. Biological evaluation indicated that when compared
to acarbose, compounds T9, T10, and T32 exhibited a higher potency of α-glucosidase
inhibitory activity with IC50 values of 0.15 ± 0.01 mM, 0.086 ± 0.01 mM and 0.32 ± 0.02 mM,
respectively. Evaluation of the inhibition kinetics indicated that T9, T10, T32, and acarbose
interacted with α-glucosidase in a mixed non-competitive inhibitory manner. Moreover, T9,
T10, and T32 statically quenched the fluorescence of α-glucosidase by formation of an
inhibitor-α-glucosidase complex. The docking results showed that hydrogen bonds were
generated between the test compounds and α-glucosidase. The antioxidant study revealed that
compound T10 exhibited
a
higher antioxidant activity via scavenging
1,1-diphenyl-2-picrylhydrazyl free radical (DPPH), thereby inhibiting lipid peroxidation and
the total reduction capacity. In brief, the salicylic acid derivatives identified in this study were
promising candidates for development as novel non-saccharide α-glucosidase inhibitors.
Key words salicylic acid derivative; α-glucosidase; antioxidant; fluorescence; docking
Biological and Pharmaceutical Bulletin Advance Publication

As one of the most prevalent diseases worldwide, diabetes has become a serious
public-health problem.1) Type 2 diabetes mellitus (T2DM) is increasing at an alarming rate.
T2DM affects about 173 million individuals worldwide and in recent years has been
associated with vascular complications that result in excess morbidity and mortality. Such
complications include cardiovascular diseases, stroke, nephropathy, retinopathy, renal failure,
and amputations.2,3) It is generally accepted that diabetes belongs to cardiovascular diseases
(CVDs) and that cardiovascular complications are related to prevailing hyperglycemia,
particularly postprandial hyperglycemia (PPHG). In several studies, it has convincingly been
demonstrated that a variation in blood glucose levels as a key player in contributing to
diabetic micro- and macro-vascular complications is more deleterious than chronic sustained
hyperglycemia.4-6) In addition, PPHG has been identified as one of the earliest detectable
abnormalities expressed in patients with diabetes.7) Therefore, it is considered a better
predictor for the progression of diabetes and has been implicated in inducing oxidative stress
that is recognized as a major pathophysiological link between CVD and diabetes.8,9)
Pharmacological agents that specifically decrease PPHG, including α-glucosidase
inhibitors (AGIs), may become therapeutics of utmost importance.10) Food starches contribute
to major postprandial blood glucose levels, and represent an issue that is of significant
relevance to those of Asian descent because of their specific dietary habits. Slowing down the
digestion and absorption of dietary starches has shown great promise in reducing PPHG, the
burden of oxidative stress, and CVD.8,9,11) This can be achieved through dietary manipulation
with low-glycemic index food or by inhibition of the starch-digesting enzyme α-glucosidase,
which is present at the intestinal brush borders.11) Such α-glucosidase inhibitors are effective
for delaying and prolonging carbohydrate digestion, slowing the rate of glucose absorption,
and consequently blunting the postprandial plasma glucose increase.12,13) Several AGIs,
including acarbose, voglibose, and miglitol, have been effectively used in the clinic to treat
T2DM and to prevent CVD in the Asian population presumably because of their specific food
intake.14,15) However, only few AGIs are available commercially and their structures are
similar to those with sugar, which require tedious multi-step processes for preparation. For
instance, acarbose and vogliose are amino sugars, whereas miglitol is an azasugar. Therefore,
Biological and Pharmaceutical Bulletin Advance Publication

there is considerable interest in the design and synthesis of non-saccharide AGIs.16-21) Kavitha
et al. investigated the binding mode of interaction of several sugar/non-saccharide AGIs at the
active site of α-glucosidase and developed a pharmacophore model, which represened the
critical features responsible for α-glucosidase inhibitory activity.17)
In our previous study, we found that several phthalimide derivatives exhibited good
α-glucosidase inhibitory activity.23) Based on the pharmacophore model of phthalimide
compounds for α-glucosidase, the phthalimide scaffold was broken apart and one carbonyl
group within phthalimide was replaced with a hydroxyl group. Thus, a series of salicylic acid
derivatives were explored as novel non-saccharide AGIs (Fig. 1). In the current study, a
salicylic acid moiety was designed as a new scaffold, possessing similar structural
characteristics as the AGIs pharmacophore model described, and a variety of alcohols and
amines were conjugated with 4-(4-methylphenyl)sulfonamido salicylic acid and
4-acetylamido salicylic acid. The inhibitory activities of the designed compounds were
evaluated against yeast α-glucosidase and the structure-activity relationships were discussed.
Compounds with prominent activities against α-glucosidase were selected to further
investigate the inhibition kinetics and anti-oxidation capacities. Moreover, to provide insight
into the structure-activity relationship of the target compounds towards α-glucosidase,
fluorescence quenching analysis of α-glucosidase and docking simulations of the
representative compounds were performed to investigate their binding characteristics with
α-glucosidase.
HBA3
O
HBA2
R
X
HBA1
O
O
S
N
OH
H
HBD
HBD2
HBA1
HBD1
X=O, N
O
H
O
N
S
alkyl
or aryl
N
O
HBA2
HBA2
O
O
HBA1
R
HBA3
O
O
N
OH
H
HBD2
HBD1
Fig. 1. Design of salicylic acid derivatives based on a new α-glucosidase inhibitor of
Biological and Pharmaceutical Bulletin Advance Publication

phthalimide. HBA represent hydrogen bond acceptor, HBD represent hydrogen bond donor.
(Color figure can be accessed in the online version.)
MATERIALS AND METHODS
All materials were obtained from commercial suppliers and were used without further
purification. The progress of the reaction was monitored by TLC using Qingdao Haiyang
Chemical Co. Ltd, HG/T2354-92 silica Gel GF254. Purification of compounds was
performed by flash column chromatography using silica gel (200-300 mesh) of Qingdao
Haiyang Chemical Co. Ltd (Qingdao, China). 1H-NMR (400 MHz) and 13C-NMR (100 MHz)
spectra were recorded with a Bruker AV400 spectrometer (Switzerland), chemical shifts were
recorded in parts per million (ppm), and coupling constants (J) were given in Hertz. Mass
spectra were obtained on a Shimadzu HPLC-MS-QP2010 instrument (Kyoto, Japan). IR
spectra were recorded with a Nicolet AVATAR 330 FT-IR instrument (Madison, America).
General procedure for the synthesis of target compounds.
A general synthetic approach for the proposed compounds T1-T30 is depicted in Chart 1.
As a starting material, 4-amino salicylic acid 1 was treated with methanol in the presence of
concentrated sulfuric acid to generate methyl 4-amino salicylate 2. Next, the phenolic
hydroxyl group on the intermediate 2 was benzyl-protected by treatment with benzyl bromide
to generate methyl 4-amino-2-(benzyloxy)benzoate 3, which further underwent sulfonylation
with para-toluenesulfonic chloride in dichloromethane in the presence of pyridine to generate
methyl 2-(benzyloxy)-4-[(4-methylphenyl)sulfonamido]benzoate 4. This was hydrolyzed
with sodium hydroxide in a mixed solvent of tetrahydrofuran and water to generate the key
intermediate
2-(benzyloxy)-4-[(4-methylphenyl)sulfonamido]benzoic acid
5.
The
corresponding alcohols and amines were conjugated with key intermediate 5 to give rise to
esters or amides, followed by deprotection of the benzyl group with palladium-carbon
catalyst in a hydrogen atmosphere to obtain target compounds T1-T30, except T6, T17, and
T27, for which the benzyl-deprotection was performed by treatment with titanium
tetrachloride in dichloromethane.23)
Biological and Pharmaceutical Bulletin Advance Publication

O
O
O
a
OH
OH
OCH3
OH
OCH3
b
N
H2
N
N
H2
H2
OBn
1
2
3
O
O
c
OCH3
OBn
OH
d
O
O
O
O
S
S
N
N
OBn
H
H
4
5
O
OCH3
O
O
e
S
N
OH
H
T1
O
NH2
OBn
f
O
O
S
N
H
19a
O
O
g
R1
OBn
R
e or h
O
O
O
O
S
S
N
H
N
OH
H
2a-30a
T2-T30
Chart 1. Synthesis of target compounds T1-T30. Reagents and conditions: (a) con. H2SO4,
MeOH, reflux; (b) BnBr, NaH, DMF, 0℃ to rt.; (c) TsCl, pyridine, CH2Cl2, 0℃ to rt.; (d)
NaOH, THF/H2O, rt.; (e) H2, Pd/C, EtOH, rt.; (f) oxalyl chloride, cat. DMF, then NH3·H2O;
(g) alcohol or amine, HOBt, EDCI, NMM, DMF, rt.; (h) TiCl4, DCM.
Prior to conjugation with the intermediate 5, the hydroxyl group of
2-(4-hydroxyphenyl)ethanol
8b
and
2-(3,4-dihydroxyphenyl)ethanol
9b
were
benzyl-protected using benzyl bromide in acetone in the presence of potassium carbonate to
generate the corresponding compounds 8c and 9c as shown in Chart 2.
Biological and Pharmaceutical Bulletin Advance Publication

R
R
OH
OH
a
HO
BnO
8b R=H
8c R=H
9b R=OH
9c R=OBn
Chart 2. Synthesis of the intermediates 8c and 9c. Reagents and conditions: (a) BnBr, K2CO3,
acetone, reflux.
The intermediate of the alcohol moiety for target compounds T10 and T11 was synthesized
as shown in Chart 3. In brief, caffeic acid 10b was esterified with methanol in the presence of
concentrated sulfuric acid to generate compound 10c, which was reacted with benzyl bromide
to give rise to compound 10d, followed by reduction with aluminium hydride, which was
generated in situ from lithium aluminium hydride and benzyl bromide to generate compound
10e.24) Based on a similar procedure, ferulic acid 11b was converted to compounds 11e.
O
O
O
RO
a
c
RO
HO
RO
HO
RO
OH
b
OH
OCH3
OCH3
BnO
BnO
10b R=H
11b R=CH3
10c R=H
11c R=CH3
10d R=Bn
11d R=CH3
10e R=Bn
11e R=CH3
Chart 3. Synthesis of the intermediates 10e and 11e. Reagents and conditions: (a) con. H2SO4,
MeOH, reflux; (b) BnBr, K2CO3, acetone, reflux; (c) LiAlH4, BnBr, THF, 0℃ to rt.
Target compounds T31 and T32 were synthesized via the synthetic route presented in
Chart 4. In brief, methyl 4-amino-2-(benzyloxy)benzoate 3 was treated with acetyl chloride in
the presence of trimethylamine to generate methyl 4-acetamido-2-(benzyloxy)benzoate 6,
which was hydrolyzed with sodium hydroxide in a mixed solvent of tetrahydrofuran and
water to produce key intermediate 4-acetamido-2-(benzyloxy)benzoic acid 7. After being
condensed with alcohol and deprotection of the benzyl group, target compounds T31 and T32
were obtained.
Biological and Pharmaceutical Bulletin Advance Publication

O
O
O
O
O
O
a
b
OCH3
Bn
O
OCH3
Bn
O
OH
Bn
N
H2
N
N
H
H
3
6
7
O
O
O
d
c
R
R
O
O
Bn
N
N
OH
H
H
31a-32a
T31-T32
Chart 4. Synthesis of target compounds T31-T32. Reagents and conditions: (a) AcCl, TEA,
CH2Cl2, 0℃ to rt.; (b) NaOH, THF/H2O, rt.; (c) alcohol, HOBt, EDCI, NMM, DMF, rt.; (d)
H2, Pd/C, EtOH.
Synthesis of methyl 4-amino-2-hydroxybenzoate (2) Concentrated H2SO4 (5.0 mL) was
slowly added to CH3OH (120.0 mL) and the mixture was stirred at room temperature for 2
hours. Then, 4-amino-2-hydroxybenzoic acid (6.00 g, 39.18 mmol) was added in one portion
and the mixture was heated to reflux under N2 for 20 hours. The mixture was concentrated
under reduced pressure to give rise to a residue, which was first basified to pH 7 with 2.0 M
NaOH, then to pH 8 with saturated NaHCO3. The resulting mixture was extracted with
EtOAc (100.0 mL × 3) and the combined organic layers were washed with brine (50.0 mL ×
2), dried over Na2SO4, concentrated in vacuo to yield crude compound 2 as a light pink solid
that was used for next step without further purification (6.40 g, 97.7%).
Synthesis of methyl 4-amino-2-(benzyloxy)benzoate (3) To a stirred solution of
compound 2 (6.40 g, 38.29 mmol) in anhydrous dimethyl formamide, 60% NaH (1.70 g, 42.5
mmol) was added in portions at 0℃, and the mixture was stirred under N2 for 2 hours.
Subsequently, BnBr (5.00 mL, 42.10 mmol) was added dropwise after which the mixture was
stirred at 0℃ for 30 min, then warmed to room temperature overnight. Water (5.0 mL) was
added to quench the reaction and the mixture was concentrated in vacuo to form a residue,
which was dissolved with EtOAc (300.0 mL), then washed with 4 M HCl (100.0 mL × 6), the
combined aqueous layers were first basified to pH 7 with 2.0 M NaOH, then to pH 8 with
saturated Na2CO3. The resulting mixture was extracted with EtOAc (150.0 mL × 4) and the
combined organic layers were washed with brine (100.0 mL × 3), dried over Na2SO4 and
Biological and Pharmaceutical Bulletin Advance Publication

concentrated under reduced pressure to form a residue that was purified by column
chromatography (petroleum ether/ethyl acetate=4:1) on silica gel to yield compound 4 as a
1
white solid (5.30 g, 53.8%). H-NMR (DMSO-d6) δ: 7.52~7.55 (m, 3H, ArH), 7.38~7.42 (t,
2H, ArH), 7.29~7.33 (t, 1H, ArH), 6.28~6.29 (d, 1H, ArH), 6.15~6.17 (m, 1H, ArH), 5.96 (s,
13
2H, -NH2), 5.07 (s, 2H, -OCH2Ar), 3.68 (s, 3H, -OCH3). C-NMR (DMSO-d6) δ: 51.18,
70.26, 99.08, 101.34, 110.37, 126.83, 127.34, 128.89, 131.43, 136.25, 155.18, 161.69, 165.88.
IR (KBr, cm-1): 3380, 2940, 2878, 1758, 1623, 1536, 1470, 1321,1232, 1036, 835. EI-MS m/z:
257.1 (M+). Anal. Calcd for C15H15NO3: C, 70.02; H, 5.88; N, 5.44. Found: C, 70.04; H, 5.86;
N, 5.45.
Synthesis of methyl 2-(benzyloxy)-4-[(4-methylphenyl)sulfonamido]benzoate (4) To a
stirred solution of compound 3 (5.30 g, 20.60 mmol) and pyridine (3.40 mL, 42.21 mmol) in
dichloromethane (80.0 mL), paratoluensulfonyl chloride (4.32 g, 22.66 mmol) was added in
portions at 0℃. After stirring at 0℃ for 30 min, the mixture was warmed to room
temperature overnight. Then, the mixture was washed with 2.0 M HCl, saturated NaHCO3
and brine, respectively. The organic layer was dried over Na2SO4 and concentrated in vacuo
to yield compound 4 as a white solid (8.20 g, 96.8%).1H-NMR (DMSO-d6) δ: 10.71 (s, 1H,
-SO2NH-), 7.55~7.65 (m, 3H, ArH), 7.47~7.49 (d, 2H, ArH), 7.40~7.43 (m, 2H, ArH),
7.32~7.36 (m, 3H, ArH), 6.97 (s, 1H, ArH), 6.70~6.72 (d, 1H, ArH), 5.11 (s, 2H, -OCH2Ar),
13
3.73 (s, 3H, -OCH3), 2.32 (s, 3H, ArCH3). C-NMR (DMSO-d6) δ: 21.42, 51.16, 70.29,
99.06, 108.29, 110.31, 126.80, 127.38, 128.15, 128.79, 129.12, 131.39, 136.29, 136.53,
137.46, 142.58, 161.64, 165.81. IR (KBr, cm-1): 3272, 2943, 2874, 1756, 1622, 1533, 1473,
1354, 1230, 1141, 1033, 833. EI-MS m/z: 411.1 (M+). Anal. Calcd for C22H21NO5S: C, 64.22;
H, 5.14; N, 3.40; S, 7.79. Found: C, 64.20; H, 5.16; N, 3.42; S, 7.77.
Synthesis of 2-(benzyloxy)-4-[(4-methylphenyl)sulfonamido]benzoic acid (5) To a
solution of compound 4 (8.20 g, 19.93 mmol) in tetrahydrofuran (100.0 mL), an aqueous
solution of 4.0 M NaOH was added, and the mixture was stirred at room temperature for 20
hours. Under reduced pressure, the organic solvent was removed to form a residue that was
extracted with EtOAc to remove impurities. The resulting aqueous layer was acidified to pH
1 with 4.0 M HCl and the precipitate was filtered and dried to yield compound 5 as an
Biological and Pharmaceutical Bulletin Advance Publication

off-white solid (7.70 g, 97.2%). 1H-NMR (DMSO-d6) δ: 12.45 (s, 1H, -COOH), 10.65 (s, 1H,
-SO2NH-), 7.61~7.63 (d, 2H, ArH), 7.55~7.57 (d, 1H, ArH), 7.46~7.48 (m, 2H, ArH),
7.36~7.42 (m, 2H, ArH), 7.31~7.33 (m, 3H, ArH), 6.94~6.95 (d, 1H, ArH), 6.66~6.69 (m, 1H,
13
ArH), 5.10 (s, 2H, -OCH2Ar), 2.33 (s, 3H, ArCH3). C-NMR (DMSO-d6) δ: 21.40, 70.31,
99.02, 108.05, 108.26, 126.77, 127.35, 128.12, 128.75, 129.08, 131.57, 136.26, 136.51,
137.42, 142.78, 161.89, 165.93. IR (KBr, cm-1): 3276, 2946, 2877, 1714, 1619, 1531, 1471,
1358, 1234, 1147, 1035, 928, 831. EI-MS m/z: 397.1 (M+). Anal. Calcd for C21H19NO5S: C,
63.46; H, 4.82; N, 3.52; S, 8.07. Found: C, 63.45; H, 4.84; N, 3.54; S, 8.05.
Synthesis of methyl 4-acetamido-2-(benzyloxy)benzoate (6) To a solution of compound
3 (5.00 g, 19.43 mmol) and trimethylamine (3.93 g, 38.81 mmol) in anhydrous
dichloromethane (200.0 mL), acetyl chloride (1.66 mL, 23.26 mmol) was added at 0℃.
Subsequently, the mixture was stirred under N2 overnight at room temperature. Then, the
mixture was washed with 2.0 M HCl, saturated NaHCO3 and brine, respectively. The organic
layer was dried over Na2SO4 and concentrated in vacuo to yield compound 6 as a light yellow
solid (5.77g, 99.2%) that was used for next step without further purification.
Synthesis of 4-acetamido-2-(benzyloxy)benzoic acid (7) Compound 7 was prepared
following the procedure that was used to generate compound 5. From compound 6 (5.77 g,
1
19.28 mmol), compound 7 was obtained as an off-white solid (4.95 g, 90.1%). H-NMR
(DMSO-d6) δ: 12.36 (s, 1H, -COOH), 10.20 (s, 1H, -CONH-), 7.67~7.69 (d, 1H, ArH),
7.50~7.54 (m, 3H, ArH), 7.37~7.40 (t, 2H, ArH), 7.29~7.33 (m, 1H, ArH), 7.17~7.19 (dd, 1H,
13
ArH), 5.13 (s, 2H, -CH2Ph), 2.05 (s, 3H, CH3CO-). C-NMR (DMSO-d6) δ: 23.85, 70.67,
105.45, 113.06, 113.37, 126.85, 127.19, 128.32, 131.21, 136.25, 144.28, 161.24, 166.26,
168.97. IR (KBr, cm-1): 3294, 2957, 2888, 1692, 1623, 1533, 1472, 1373, 1235, 1038, 925,
836. EI-MS m/z: 285.1 (M+). Anal. Calcd for C16H15NO4: C, 67.36; H, 5.30; N, 4.91. Found:
C, 67.34; H, 5.32; N, 4.93.
General procedure for the synthesis of compounds 8c and 9c To a stirred solution of
compound 8b or 9b in acetone, K2CO3 (2.50 equivalent for each phenolic hydroxyl) and
BnBr (1.05 equivalent for each phenolic hydroxyl) were added. The resulting mixture was
heated to reflux under N2 for 10 hours. The mixture was then filtered and the filtrate was
Biological and Pharmaceutical Bulletin Advance Publication

concentrated to form a residue, which was dissolved with EtOAc, washed with 2.0 M HCl,
saturated NaHCO3 and brine. The organic layer was dried over Na2SO4, and concentrated in
vacuo to form a yellow oil that was purified by column chromatography on silica gel to
generate compound 8c or 9c.
Synthesis of 2-[4-(benzyloxy)phenyl]ethan-1-ol (8c) From compound 8b (2.00 g, 14.47
mmol), compound 8c was obtained as a white solid (3.10 g, 93.8%).
Synthesis of 2-[3,4-bis(benzyloxy)phenyl]ethan-1-ol (9c) From compound 9b (2.27 g,
1
14.72 mmol), 9c was obtained as a white solid (2.70 g, 54.8%). H-NMR (DMSO-d6) δ:
7.30~7.46 (m, 10H, ArH), 6.92~6.94 (t, 2H, ArH), 6.69~6.71 (m, 1H, ArH), 5.07~5.09 (d, 4H,
-CH2Ph), 4.602 (t, 1H, -OH), 3.50~3.54 (t, 2H, -OCH2-), 2.60~2.63 (t, 2H, -CH2Ar).
13C-NMR (DMSO-d6) δ: 38.82, 60.85, 70.62, 111.89, 115.66, 121.74, 126.84, 127.18, 128.59,
130.27, 136.41, 146.59, 149.43. IR (KBr, cm-1): 3339, 2953, 2854, 1623, 1533, 1450, 1368,
1235, 1073, 836. EI-MS m/z: 334.1 (M+). Anal. Calcd for C22H22O3: C, 79.02; H, 6.63. Found:
C, 79.04; H, 6.62.
General procedure for the synthesis of compound 10c and 11c Concentrated H2SO4 (1.0
mL) was added to a solution of caffeic acid 10b or ferulic acid 11b in methanol, and the
resulting mixture was heated to reflux under N2 for 12 hours. The mixture was concentrated
under reduced pressure to form a residue that was purified by column chromatography to
generate compound 10c or 11c.
Synthesis of methyl (E)-3-(3,4-dihydroxyphenyl)acrylate (10c) From compound 10b
(5.00 g, 27.75 mmol), compound 10c was obtained (5.20 g, 96.5%). 1H-NMR (DMSO-d6) δ:
9.61 (s, 1H, ArOH), 9.16 (s, 1H, ArOH), 7.46~7.50 (d, 1H, J=16.0 Hz, -CH=), 7.05 (s, 1H,
ArH), 6.98~7.01 (m, 1H, ArH), 6.74~6.76 (d, 1H, ArH), 6.25~6.29 (d, 1H, J=16.0 Hz, -CH=),
3.68 (s, 3H, -OCH3). 13C-NMR (DMSO-d6) δ: 51.81, 114.73, 114.98, 116.93, 122.85, 127.69,
143.35, 145.57, 146.11, 166.17. IR (KBr, cm-1): 3339, 2946, 2878, 1723, 1624, 1536, 1471,
1368, 1230, 1186, 918, 835. EI-MS m/z: 194.0 (M+). Anal. Calcd for C10H10O4: C, 61.85; H,
5.19. Found: C, 61.86; H, 5.17.
Synthesis of methyl (E)-3-(4-hydroxy-3-methoxyphenyl)acrylate (11c) From compound
11b (6.00 g, 30.90 mmol), compound 11c was obtained (6.15 g, 95.6%).
Biological and Pharmaceutical Bulletin Advance Publication

General procedure for the synthesis of compounds 10d and 11d Compounds 10d and
11d were prepared following the procedure that was used to prepare compounds 8c and 9c.
Synthesis of methyl (E)-3-[3,4-bis(benzyloxy)phenyl]acrylate (10d) From compound
10c (5.20 g, 26.78 mmol), compound 10d was obtained (9.50 g, 94.7%). 1H-NMR (DMSO-d6)
δ: 7.54~7.58 (d, 1H, J=16.0 Hz, -CH=), 7.43~7.50 (m, 5H, ArH), 7.36~7.40 (m, 4H, ArH),
7.30~7.33 (m, 2H, ArH), 7.22~7.24 (m, 1H, ArH), 7.06~7.08 (d, 1H, ArH), 6.52~6.56 (d, 1H,
13
J=16.0 Hz, -CH=), 5.19 (s, 4H, -CH2Ph×2), 3.70 (s, 3H, -OCH3). C-NMR (DMSO-d6) δ:
51.83, 70.68, 113.52, 114.39, 114.87, 122.16, 126.72, 126.95, 127.37, 128.59, 136.40, 143.29,
148.75, 149.28, 166.19. IR (KBr, cm-1): 3013, 2948, 2879, 1727, 1627, 1538, 1471, 1365,
1237, 1188, 1042, 906, 838. EI-MS m/z: 374.1 (M+). Calcd for C24H22O4: C, 76.99; H, 5.92.
Found: C, 76.97; H, 5.93.
Synthesis of methyl (E)-3-[4-(benzyloxy)-3-methoxyphenyl]acrylate (11d) From
compound 11c (6.15 g, 29.54 mmol), compound 11d was obtained (8.15 g, 92.5%). 1H-NMR
(DMSO-d6) δ: 7.57~7.61 (d, 1H, J=16.0 Hz, -CH=), 7.37~7.45 (m, 5H, ArH), 7.30~7.35 (m,
1H, ArH), 7.21~7.23 (dd, 1H, ArH), 7.05~7.07 (d, 1H, ArH), 6.55~6.59 (d, 1H, J=16.0 Hz,
-CH=), 5.13 (s, 2H, -CH2Ph), 3.81 (s, 3H, -OCH3), 3.70 (s, 3H, -OCH3). 13C-NMR
(DMSO-d6) δ: 51.81, 56.23, 70.64, 111.12, 114.45, 114.83, 122.11, 126.83, 126.92, 127.31,
128.57, 136.44, 143.31, 145.15, 149.32, 166.23. IR (KBr, cm-1): 3019, 2940, 2873, 1725,
1621, 1534, 1473, 1366, 1232, 1183, 1039, 911, 831. EI-MS m/z: 298.1 (M+). Anal. Calcd for
C18H18O4: C, 72.47; H, 6.08. Found: C, 72.48; H, 6.05.
General procedure for the synthesis of compounds 10e and 11e To a suspension of
LiAlH4 (1.50 equivalent) in anhydrous THF, a solution of BnBr (1.50 equivalent) in
anhydrous THF was added dropwise at 0℃. After stirring for 30 min at room temperature
under N2, a solution of compound 10d or 11d in anhydrous THF was added dropwise at 0℃.
After stirring at 0℃ for 2 hours, the mixture was warmed to room temperature and stirred for
another 2 hours. When the reactant was completely consumed, the reaction was quenched by
the addition of water (1.0 mL per gram of LiAlH4), 10% NaOH aqueous solution (2.0 mL per
gram of LiAlH4), and water (3.0 mL per gram of LiAlH4) in sequence. The resulting mixture
was filtered and the filtrate was concentrated in vacuo to form a residue that was purified by
Biological and Pharmaceutical Bulletin Advance Publication

column chromatography on silica gel to generate compounds 10e or 11e.
Synthesis of (E)-3-[3,4-bis(benzyloxy)phenyl]prop-2-en-1-ol (10e) From compound 10d
(9.50 g, 25.37 mmol), compound 10e was obtained (7.50 g, 85.3%). 1H-NMR (DMSO-d6) δ:
7.29~7.46 (m, 10H, ArH), 7.16 (d, 1H, ArH), 6.97~6.99 (d, 1H, ArH), 6.88~6.91 (dd, 1H,
ArH), 6.40~6.44 (d, 1H, J=16.0 Hz, -CH=), 6.20~6.24 (m, 1H, J=16.0 Hz, -CH=), 5.11~5.15
(d, 4H, -CH2Ph × 2), 4.80~4.83 (t, 1H, -OH), 4.06~4.08 (m, 2H, -OCH2-). 13C-NMR
(DMSO-d6) δ: 63.19, 70.61, 111.15, 114.41, 119.19, 126.86, 127.21, 127.83, 128.52, 130.43,
131.70, 136.54, 148.64, 149.35. IR (KBr, cm-1): 3460, 1609, 1519, 1237, 1046, 986, 915, 823.
EI-MS m/z: 346.1 (M+). Anal. Calcd for C23H22O3: C,79.74; H, 6.40. Found: C, 79.76; H,
6.39.
Synthesis of (E)-3-[4-(benzyloxy)-3-methoxyphenyl]prop-2-en-1-ol (11e) From
compound 11d (8.15 g, 27.32 mmol), compound 11e was obtained (6.18 g, 83.7%). 1H-NMR
(DMSO-d6) δ: 7.30~7.45 (m, 5H, ArH), 7.06 (d, 1H, ArH), 6.95~6.97 (d, 1H, ArH),
6.87~6.89 (dd, 1H, ArH), 6.44~6.48 (d, 1H, J=16.0 Hz, -CH=), 6.24~6.28 (d, 1H, J=16.0 Hz,
-CH=), 5.06 (s, 2H, -CH2Ph), 4.81~4.84 (t, 1H, -OH), 4.07~4.10 (m, 2H, -OCH2-), 3.78 (s,
13
3H, -OCH3). C-NMR (DMSO-d6) δ: 56.20, 63.21, 70.65, 111.16, 114.43, 119.23, 126.88,
127.25, 127.93, 128.60, 130.46, 131.73, 136.52, 145.34, 149.32. IR (KBr, cm-1): 3454, 2944,
2878, 1601, 1512, 1477, 1372, 1233, 1041, 981, 918, 816. EI-MS m/z: 270.1 (M+). Anal.
Calcd for C17H18O3: C, 75.53; H, 6.71. Found: C, 75.51; H, 6.72.
Synthesis of methyl 2-hydroxy-4-[(4-methylphenyl)sulfonamido]benzoate (T1) To a
solution of compound 4 (500.0 mg, 1.22 mmol) in EtOH (30.0 mL), 10% Pd/C (50.0 mg) was
added. After degassing with H2 for 3 times, the mixture was stirred under H2 at room
temperature for 12 hours. Then, the mixture was filtered through a Celite pad and the filtrate
was concentrated in vacuo to give rise to a yellow residue that was purified by column
chromatography (petroleum ether/ethyl acetate=5:1) to afford compound T1 as an off-white
1
solid (340.0 mg, 86.7%). H-NMR (DMSO-d6) δ: 10.84 (s, 1H, ArOH), 10.57 (s, 1H,
-SO2NH-), 7.70~7.72 (d, 2H, ArH), 7.61~7.63 (d, 1H, ArH), 7.37~7.39 (d, 2H, ArH),
6.66~6.69 (m, 2H, ArH), 3.809 (s, 3H, -OCH3), 2.33 (s, 3H, ArCH3). 13C-NMR (DMSO-d6) δ:
21.40, 51.21, 100.65, 102.47, 108.46, 128.03, 129.08, 131.81, 136.29, 137.20, 143.03, 164.66,
Biological and Pharmaceutical Bulletin Advance Publication

168.92. IR (KBr, cm-1): 3340, 2945, 2877, 1760, 1625, 1536, 1475, 1357, 1289, 1143, 834.
EI-MS m/z: 321.0 (M+). Anal. Calcd for C15H15NO5S: C, 56.07; H, 4.71; N, 4.36; S, 9.98.
Found: C, 56.05; H, 4.74; N, 4.35; S, 9.96.
Synthesis of 2-(benzyloxy)-4-[(4-methylphenyl)sulfonamido]benzamide (19a) To a
solution of compound 5 (500.0 mg, 1.26 mmol) in anhydrous dichloromethane (30.0 mL),
oxalyl chloride (1.07 mL, 12.50 mmol) and DMF (3 drops) were added, and the resulting
mixture was stirred under N2 at room temperature for 2 hours. Subsequently, the volatiles
were removed under reduced pressure, and the obtained yellow solid was redissolved in
anhydrous dichloromethane (20.0 mL). The resulting solution was added dropwise to
ammonium hydroxide 10 mL, and the mixture was stirred under N2 at room temperature for 2
hours. The organic solvent was removed and the residue was extracted with EtOAc (30.0 mL
× 3). The combined organic layers were washed with 2.0 M HCl, saturated NaHCO3 and
brine, dried over Na2SO4, and concentrated in vacuo to give a yellow oil, which was purified
by column chromatography (chloroform/ methanol=25:1) on silica gel to afford compound
19a as a white solid (263.0 mg, 52.3%).
General procedure for the synthesis of compound 2a-32a (except for compound 19a).
To a solution of compound 5 or compound 7 in anhydrous DMF, alcohol or amine were
added (1.10 equivalent), HOBt (1.50 equivalent), EDCI (1.50 equivalent) and NMM (2.00
equivalent). After being degassed 3 times with N2, the resulting mixture was stirred overnight
at room temperature under N2. The solvent was evaporated and the residue was redissolved in
EtOAc, washed with 2.0 M HCl, saturated NaHCO3 and brine, respectively. The organic
layer was dried over Na2SO4 and concentrated in vacuo to give a crude product, which was
purified by column chromatography on silica gel to afford compound 2a-32a (except for
compound 19a).
General procedure for the synthesis of compounds T2-T32 (except for T6, T17, and
T27) To a solution of compounds 2a-32a (except for 6a, 17a and 27a) in ethanol, 10% Pd/C
was added (10% weight of the reactant). After being degassed 3 times with H2, the resulting
mixture was stirred at room temperature under H2. The mixture was filtered through a Celite
pad, and the filtrate was concentrated in vacuo to give a crude product, which was purified by
Biological and Pharmaceutical Bulletin Advance Publication

column chromatography on silica gel to afford the corresponding target compounds T2-T32
(except for T6, T17, and T27).
Synthesis of ethyl 2-hydroxy-4-[(4-methylphenyl)sulfonamido]benzoate (T2) From
compound 5 (500.0 mg, 1.26 mmol), compound T2 was obtained (280.0 mg, 67.3%, 2 steps).
1H-NMR (DMSO-d6) δ: 10.83 (s, 1H, ArOH), 10.64 (s, 1H, -SO2NH-), 7.70~7.72 (d, 2H,
ArH), 7.61~7.64 (d, 1H, ArH), 7.37~7.39 (d, 2H, ArH), 6.65~6.70 (m, 2H, ArH), 4.25~4.30
(q, 2H, -OCH2), 2.33 (s, 3H, ArCH3), 1.25~1.29 (t, 3H, -CH3). 13C-NMR (DMSO-d6) δ: 14.22,
21.39, 60.46, 100.63, 102.44, 108.47, 128.05, 129.09, 131.83, 136.32, 137.24, 143.05, 164.67,
169.22. IR (KBr, cm-1): 3336, 2945, 2877, 1758, 1621, 1532, 1472, 1355, 1286, 1143, 830.
EI-MS m/z: 335.0 (M+). Anal. Calcd for C16H17NO5S: C, 57.30; H, 5.11; N, 4.18; S, 9.56.
Found: C, 57.32; H, 5.09; N, 4.20; S, 9.55.
Synthesis of n-propyl 2-hydroxy-4-[(4-methylphenyl)sulfonamido]benzoate (T3) From
compound 5 (500.0 mg, 1.26 mmol), compound T3 was obtained (263.0 mg, 60.0%, 2 steps).
1H-NMR (DMSO-d6) δ: 10.84 (s, 1H, ArOH), 10.65 (s, 1H, -SO2NH-), 7.71~7.73 (d, 2H,
ArH), 7.63~7.65 (d, 1H, ArH), 7.37~7.39 (d, 2H, ArH), 6.66~6.70 (m, 2H, ArH), 4.17~4.20
(q, 2H, -OCH2), 2.33 (s, 3H, ArCH3), 1.62~1.71 (m, 2H, -CH2-), 0.90~0.94 (t, 3H, -CH3).
13C-NMR (DMSO-d6) δ: 10.25, 21.35, 21.84, 68.69, 100.61, 102.43, 108.48, 128.07, 129.10,
131.85, 136.35, 137.26, 143.08, 163.82, 164.82. IR (KBr, cm-1): 3332, 2943, 2873, 1751,
1620, 1530, 1471, 1353, 1283, 1142, 829. EI-MS m/z: 349.1 (M+). Anal. Calcd for
C17H19NO5S: C, 58.44; H, 5.48; N, 4.01; S, 9.18. Found: C, 58.46; H, 5.45; N, 4.03; S, 9.20.
Synthesis of i-propyl 2-hydroxy-4-[(4-methylphenyl)sulfonamido]benzoate (T4) From
compound 5 (500.0 mg, 1.26 mmol), compound T4 was obtained (245.0 mg, 55.6%, 2 steps).
1H-NMR (DMSO-d6) δ: 10.82 (s, 1H, ArOH), 10.71 (s, 1H, -SO2NH-), 7.70~7.72 (d, 2H,
ArH), 7.59~7.61 (d, 1H, ArH), 7.37~7.39 (d, 2H, ArH), 6.67~6.70 (m, 1H, ArH), 6.64~6.65
(d, 1H, ArH), 5.08~5.14 (m, 1H, -CH-), 2.33 (s, 3H, ArCH3), 1.27~1.28 (d, 6H, -CH3 × 2).
13C-NMR (DMSO-d6) δ: 21.39, 21.71, 67.10, 100.62, 102.42, 108.46, 128.08, 129.12, 131.84,
136.38, 137.28, 143.07, 164.78, 165.56. IR (KBr, cm-1): 3330, 2945, 2876, 1749, 1624, 1532,
1470, 1355, 1281, 1144, 831. EI-MS m/z: 349.1 (M+). Anal. Calcd for C17H19NO5S: C, 58.44;
H, 5.48; N, 4.01; S, 9.18. Found: C, 58.42; H, 5.50; N, 4.03; S, 9.15.
Biological and Pharmaceutical Bulletin Advance Publication

Synthesis of n-butyl 2-hydroxy-4-[(4-methylphenyl)sulfonamido]benzoate (T5) From
compound 5 (500.0 mg, 1.26 mmol), compound T5 was obtained (277.0 mg, 60.4%, 2 steps).
1H-NMR (DMSO-d6) δ: 10.84 (s, 1H, ArOH), 10.65 (s, 1H, -SO2NH-), 7.70~7.72 (d, 2H,
ArH), 7.61~7.63 (d, 1H, ArH), 7.37~7.39 (d, 2H, ArH), 6.65~6.70 (m, 2H, ArH), 4.21~4.25 (t,
2H, -OCH2), 2.33 (s, 3H, ArCH3), 1.60~1.67 (m, 2H, -CH2-), 1.34~1.35 (m, 2H, -CH2-),
0.87~0.91 (t, 3H, -CH3). 13C-NMR (DMSO-d6) δ: 13.71, 19.12, 21.41, 31.35, 64.25, 100.60,
102.41, 108.45, 128.06, 129.09, 131.84, 136.34, 137.25, 143.07, 163.80, 164.80. IR (KBr,
cm-1): 3330, 2947, 2878, 1747, 1616, 1528, 1471, 1354, 1280, 1142, 824. EI-MS m/z: 363.1
(M+). Anal. Calcd for C18H21NO5S: C, 59.49; H, 5.82; N, 3.85; S, 8.82. Found: C, 59.48; H,
5.84; N, 3.83; S, 8.85.
Synthesis of phenethyl 2-hydroxy-4-[(4-methylphenyl)sulfonamido]benzoate (T7)
From compound 5 (500.0 mg, 1.26 mmol), compound T7 was obtained (213.0 mg, 41.5%, 2
1
steps). H-NMR (DMSO-d6) δ: 10.84 (s, 1H, ArOH), 10.55 (s, 1H, -SO2NH-), 7.70~7.72 (d,
2H, ArH), 7.55~7.57 (d, 1H, ArH), 7.37~7.39 (d, 2H, ArH), 7.28~7.29 (m, 4H, ArH),
7.20~7.21 (m, 1H, ArH), 6.65~6.68 (m, 2H, ArH), 4.41~4.45 (t, 2H, -OCH2-), 2.97~3.00 (t,
13
2H, -CH2Ph), 2.33 (s, 3H, ArCH3). C-NMR (DMSO-d6) δ: 21.41, 34.12, 64.29, 100.66,
102.45, 108.51, 125.53, 127.46, 128.02, 128.34, 129.03, 131.88, 136.39, 137.28, 137.93,
143.11, 163.83, 164.85. IR (KBr, cm-1): 3335, 2946, 2875, 1744, 1622, 1533, 1470, 1350,
1278, 1140, 828, 748, 686. EI-MS m/z: 411.1 (M+). Anal. Calcd for C22H21NO5S: C, 64.22; H,
5.14; N, 3.40; S, 7.79. Found: C, 64.25; H, 5.12; N, 3.42; S, 7.76.
Synthesis of 4-hydroxyphenethyl 2-hydroxy-4-[(4-methylphenyl)sulfonamido]benzo
ate (T8) From compound 5 (500.0 mg, 1.26 mmol), compound T8 was obtained (223.
1
0 mg, 41.8%, 2 steps). H-NMR (DMSO-d6) δ: 10.83 (s, 1H, ArOH), 10.57 (s, 1H, -
SO2NH-), 9.22 (s, 1H, ArOH), 7.70~7.72 (d, 2H, ArH), 7.57~7.59 (d, 1H, ArH), 7.37
~7.39 (d, 2H, ArH), 7.05~7.07 (d, 2H, ArH), 6.64~6.69 (m, 4H, ArH), 4.33~4.37 (t,
2H, -OCH2-), 2.84~2.88 (t, 2H, -CH2Ph), 2.33 (s, 3H, ArCH3). 13C-NMR (DMSO-d6)
δ: 21.40, 34.14, 64.32, 100.67, 102.47, 108.52, 115.58, 128.04, 129.05, 129.96, 130.53,
131.91, 136.37, 137.26, 143.13, 155.41, 163.86, 164.87. IR (KBr, cm-1): 3340, 2948,
2876, 1742, 1619, 1532, 1469, 1348, 1275, 1140, 828. EI-MS m/z: 427.1 (M+). Anal.
Biological and Pharmaceutical Bulletin Advance Publication

Calcd for C22H21NO6S: C, 61.82; H, 4.95; N, 3.28; S, 7.50. Found: C, 61.83; H, 4.9
4; N, 3.26; S, 7.53.
Synthesis of 3,4-dihydroxyphenethyl 2-hydroxy-4-[(4-methylphenyl)sulfonamido]
benzoate (T9) From compound 5 (500.0 mg, 1.26 mmol), compound T9 was obtained (237.0
mg, 43.0%, 2 steps). 1H-NMR (DMSO-d6) δ: 10.83 (s, 1H, ArOH), 10.57 (s, 1H, -SO2NH-),
8.78 (s, 1H, ArOH), 8.70 (s, 1H, ArOH), 7.70~7.72 (d, 2H, ArH), 7.58~7.60 (d, 1H, ArH),
7.37~7.39 (d, 2H, ArH), 6.61~6.69 (m, 4H, ArH), 4.48~4.51 (dd, 1H, ArH), 4.31~4.35 (t, 2H,
13
-OCH2-), 2.78~2.81 (t, 2H, -CH2Ph), 2.34 (s, 3H, ArCH3). C-NMR (DMSO-d6) δ: 21.42,
34.22, 64.36, 100.68, 102.48, 108.54, 115.61, 116.18, 122.46, 128.06, 129.07, 131.11, 131.94,
136.39, 137.28, 143.18, 144.13, 145.29, 163.88, 164.89. IR (KBr, cm-1): 3342, 2945, 2871,
1745, 1626, 1537, 1472, 1347, 1279, 1138, 830. EI-MS m/z: 443.1 (M+). Anal. Calcd for
C22H21NO7S: C, 59.59; H, 4.77; N, 3.16; S, 7.23. Found: C, 59.61; H, 4.74; N, 3.14; S, 7.25.
Synthesis of 3-(3,4-dihydroxyphenyl)propyl 2-hydroxy-4-[(4-methylphenyl)
sulfonamido]benzoate (T10) From compound 5 (521.0 mg, 1.31 mmol), compound T10 was
obtained (185.1 mg, 28.7%, 2 steps). 1H-NMR (DMSO-d6) δ: 10.87 (s, 1H, ArOH), 10.64 (s,
1H, -SO2NH-), 8.76 (s, 1H, ArOH), 8.68 (s, 1H, ArOH), 7.71~7.73 (d, 2H, ArH), 7.61~7.65
(d, 1H, ArH), 7.38~7.40 (d, 2H, ArH), 6.68~6.71 (m, 1H, ArH), 6.65~6.66 (d, 1H, ArH),
6.60~6.62 (d, 1H, ArH), 6.57~6.58 (m, 1H, ArH), 6.42~6.44 (dd, 1H, ArH), 4.17~4.20 (t, 2H,
-OCH2-), 3.34~3.35 (t, 2H, -CH2Ar), 2.34 (s, 3H, ArCH3), 1.87~1.90 (m, 2H, -CH2-).
13C-NMR (DMSO-d6) δ: 21.37, 29.85, 32.58, 64.24, 100.67, 102.46, 108.55, 115.83, 116.38,
119.89, 128.06, 129.08, 131.89, 134.67, 136.34, 137.20, 143.03, 144.23, 145.45, 163.78,
164.75. IR (KBr, cm-1): 3340, 2946, 2873, 1750, 1622, 1534, 1474, 1348, 1282, 1140, 828.
EI-MS m/z: 457.1 (M+). Anal. Calcd for C23H23NO7S: C, 60.38; H, 5.07; N, 3.06; S, 7.01.
Found: C, 60.36; H, 5.09; N, 3.08; S, 7.00.
Synthesis of 3-(4-hydroxy-3-methoxyphenyl)propyl 2-hydroxy-4-[(4-methylphenyl)
sulfonamido]benzoate (T11) From compound 5 (500.0 mg, 1.26 mmol), compound T11 was
obtained (164.2 mg, 28.1%, 2 steps). 1H-NMR (DMSO-d6) δ: 10.87 (s, 1H, ArOH), 10.64 (s,
1H, -SO2NH-), 8.73 (s, 1H, ArOH), 7.71~7.73 (d, 2H, ArH), 7.64~7.66 (d, 1H, ArH),
7.38~7.40 (d, 2H, ArH), 6.75~6.76 (d, 1H, ArH), 6.65~6.71 (m, 3H, ArH), 6.57~6.59 (dd, 1H,
Biological and Pharmaceutical Bulletin Advance Publication

ArH), 4.19~4.22 (t, 2H, -OCH2-), 3.70 (s, 3H, -OCH3), 2.56~2.60 (t, 2H, -CH2Ar), 2.34 (s,
3H, ArCH3), 1.90~1.99 (m, 2H, -CH2-). 13C-NMR (DMSO-d6) δ: 21.40, 29.82, 32.56, 56.20,
64.21, 100.65, 102.45, 108.52, 113.21, 115.28, 119.66, 128.09, 129.10, 131.85, 134.48,
136.35, 137.22, 143.08, 145.63, 147.35, 163.82, 164.78. IR (KBr, cm-1): 3342, 2949, 2875,
1747, 1620, 1530, 1476, 1347, 1277, 1141, 827. EI-MS m/z: 471.1 (M+). Anal. Calcd for
C24H25NO7S: C, 61.13; H, 5.34; N, 2.97; S, 6.80. Found: C, 61.15; H, 5.32; N, 2.96; S, 6.82.
Synthesis of 2-hydroxyethyl 2-hydroxy-4-[(4-methylphenyl)sulfonamido]benzoate
(T12) From compound 5 (500.0 mg, 1.26 mmol), compound T12 was obtained (263.1 mg,
1
59.4%, 2 steps). H-NMR (DMSO-d6) δ: 10.83 (s, 1H, ArOH), 10.59 (s, 1H, -SO2NH-),
7.68~7.72 (t, 3H, ArH), 7.37~7.39 (d, 2H, ArH), 6.68~6.70 (dd, 1H, ArH), 6.65~6.66 (d, 1H,
ArH), 4.90~4.93 (t, 1H, -OH), 4.23~4.26 (t, 2H, -OCH2-), 3.63~3.67 (q, 2H, -CH2O-), 2.33 (s,
13
3H, ArCH3). C-NMR (DMSO-d6) δ: 21.38, 60.76, 67.13, 100.58, 102.40, 108.43, 128.03,
129.06, 131.77, 136.30, 137.21, 142.99, 163.84, 164.65. IR (KBr, cm-1): 3358, 2951, 2876,
1750, 1624, 1536, 1475, 1350, 1252, 1143, 1046, 830. EI-MS m/z: 351.0 (M+). Anal. Calcd
for C16H17NO6S: C, 54.69; H, 4.88; N, 3.99; S, 9.12. Found: C, 54.67; H, 4.89; N, 3.97; S,
9.15.
Synthesis of 3-hydroxypropyl 2-hydroxy-4-[(4-methylphenyl)sulfonamido]benzoate
(T13) From compound 5 (500.1 mg, 1.26 mmol), compound T13 was obtained (276.2 mg,
1
60.8%, 2 steps). H-NMR (DMSO-d6) δ: 10.83 (s, 1H, ArOH), 10.61 (s, 1H, -SO2NH-),
7.70~7.72 (d, 2H, ArH), 7.62~7.64 (d, 1H, ArH), 7.37~7.39 (d, 2H, ArH), 6.67~6.70 (dd, 1H,
ArH), 6.65~6.66 (d, 1H, ArH), 4.58~4.60 (t, 1H, -OH), 4.28~4.31 (t, 2H, -OCH2-), 3.48~3.52
13
(q, 2H, -CH2O-), 2.33 (s, 3H, ArCH3), 1.79~1.83 (m, 2H, -CH2-). C-NMR (DMSO-d6) δ:
21.41, 31.24, 58.57, 59.98, 100.61, 102.42, 108.47, 128.05, 129.09, 131.82, 136.34, 137.26,
143.07, 163.75, 164.70. IR (KBr, cm-1): 3355, 2952, 2877, 1754, 1626, 1539, 1477, 1352,
1253, 1146, 1045, 832. EI-MS m/z: 365.0 (M+). Anal. Calcd for C17H19NO6S: C, 55.88; H,
5.24; N, 3.83; S, 8.77. Found: C, 55.87; H, 5.26; N, 3.84; S, 8.75.
Synthesis of 4-hydroxybutyl 2-hydroxy-4-[(4-methylphenyl)sulfonamido]benzoate
(T14) From compound 5 (500.0 mg, 1.26 mmol), compound T14 was obtained (280.9 mg,
1
58.9%, 2 steps). H-NMR (DMSO-d6) δ: 10.84 (s, 1H, ArOH), 10.64 (s, 1H, -SO2NH-),
Biological and Pharmaceutical Bulletin Advance Publication

7.70~7.72 (d, 2H, ArH), 7.62~7.64 (d, 1H, ArH), 7.37~7.39 (d, 2H, ArH), 6.67~6.70 (dd, 1H,
ArH), 6.65~6.66 (d, 1H, ArH), 4.43~4.46 (t, 1H, -OH), 4.23~4.26 (t, 2H, -OCH2-), 3.39~3.43
(q, 2H, -CH2O-), 2.33 (s, 3H, ArCH3), 1.67~1.71 (m, 2H, -CH2-), 1.47~1.51 (m, 2H, -CH2-).
13C-NMR (DMSO-d6) δ: 21.39, 25.27, 28.76, 62.84, 65.23, 100.65, 102.46, 108.50, 128.07,
129.10, 131.85, 136.36, 137.29, 143.13, 163.67, 164.76. IR (KBr, cm-1): 3358, 2952, 2875,
1757, 1627, 1537, 1476, 1353, 1256, 1146, 1046, 833. EI-MS m/z: 379.1 (M+). Anal. Calcd
for C18H21NO6S: C, 56.98; H, 5.58; N, 3.69; S, 8.45. Found: C, 56.99; H, 5.56; N, 3.67; S,
8.48.
Synthesis of 2,3-dihydroxypropyl 2-hydroxy-4-[(4-methylphenyl)sulfonamido]
benzoate (T15) From compound 5 (500.0 mg, 1.26 mmol), compound T15 was obtained
1
(158.1 mg, 33.0%, 2 steps). H-NMR (DMSO-d6) δ: 10.82 (s, 1H, ArOH), 10.56 (s, 1H,
-SO2NH-), 7.68~7.72 (m, 3H, ArH), 7.37~7.39 (d, 2H, ArH), 6.65~6.70 (dd, 1H, ArH),
6.65~6.66 (d, 1H, ArH), 5.02~5.04 (d, 1H, -OH), 4.70~4.73 (t, 1H, -OH), 4.25~4.29 (dd, 1H,
-CH2O-), 4.11~4.16 (dd, 1H, -CH2O-), 3.70 ~3.77 (m, 1H, -OCH-), 3.37~3.45 (m, 2H,
13
-OCH2-), 2.33 (s, 3H, ArCH3). C-NMR (DMSO-d6) δ: 21.41, 64.16, 66.07, 70.64, 100.69,
102.49, 108.56, 128.09, 129.13, 131.90, 136.38, 137.31, 143.18, 163.61, 164.80. IR (KBr,
cm-1): 3341, 2950, 2874, 1751, 1622, 1532, 1475, 1350, 1250, 1142, 1043, 828. EI-MS m/z:
381.0 (M+). Anal. Calcd for C17H19NO7S: C, 53.54; H, 5.02; N, 3.67; S, 8.41. Found: C,
53.55; H, 5.01; N, 3.64; S, 8.43.
Synthesis of 6-hydroxyhexyl 2-hydroxy-4-[(4-methylphenyl)sulfonamido]benzoate
(T16) From compound 5 (500.0 mg, 1.26 mmol), compound T16 was obtained (480.1 mg,
1
64.6%, 2 steps). H-NMR (DMSO-d6) δ: 10.84 (s, 1H, ArOH), 10.64 (s, 1H, -SO2NH-),
7.70~7.72 (d, 2H, ArH), 7.61~7.63 (d, 1H, ArH), 7.37~7.39 (d, 2H, ArH), 6.67~6.70 (dd, 1H,
ArH), 6.65~6.66 (d, 1H, ArH), 4.34~4.36 (t, 1H, -OH), 4.20~4.24 (t, 2H, -OCH2-), 3.37~3.38
(m, 2H, -CH2O-), 2.33 (s, 3H, ArCH3), 1.63~1.67 (m, 2H, -CH2-), 1.28~1.42 (m, 6H,
13
-CH2CH2CH2-). C-NMR (DMSO-d6) δ: 21.38, 25.58, 26.16, 32.07, 32.64, 63.19, 65.27,
100.68, 102.49, 108.54, 128.09, 129.11, 131.90, 136.42, 137.33, 143.17, 163.61, 164.70. IR
(KBr, cm-1): 3356, 2953, 2875, 1758, 1629, 1538, 1478, 1353, 1258, 1147, 1045, 835.
EI-MS m/z: 407.1 (M+). Anal. Calcd for C20H25NO6S: C, 58.95; H, 6.18; N, 3.44; S, 7.87.
Biological and Pharmaceutical Bulletin Advance Publication

Found: C, 58.93; H, 6.20; N, 3.45; S,7.85.
Synthesis of 2-(dimethylamino)ethyl 2-hydroxy-4-[(4-methylphenyl)sulfonamido]
benzoate (T18) From compound 5 (500.0 mg, 1.26 mmol), compound T18 was obtained
1
(202.1 mg, 42.3%, 2 steps). H-NMR (DMSO-d6) δ: 7.70~7.72 (d, 2H, ArH), 7.60~7.62 (d,
1H, ArH), 7.37~7.39 (d, 2H, ArH), 6.64~6.69 (m, 2H, ArH), 4.32~4.35 (t, 2H, -OCH2-),
2.62~2.65 (t, 2H, -NCH2-), 2.34 (s, 3H, ArCH3), 2.23 (s, 6H, -N(CH3)2). 13C-NMR
(DMSO-d6) δ: 21.39, 46.89, 61.57, 63.03, 100.66, 102.48, 108.52, 128.07, 129.09, 131.87,
136.39, 137.26, 143.11, 163.55, 164.71. IR (KBr, cm-1): 3340, 2950, 2874, 1744, 1621, 1533,
1473, 1345, 1247, 1180, 827. EI-MS m/z: 378.1 (M+). Anal. Calcd for C18H22N2O5S: C, 57.13;
H, 5.86; N, 7.40; S, 8.47. Found: C, 57.15; H, 5.84; N, 7.43; S, 8.45.
Synthesis of 2-hydroxy-4-[(4-methylphenyl)sulfonamido]benzamide (T19) From
compound 19a (260.1 mg, 0.66 mmol), compound T19 was obtained (145.2 mg, 71.7%).
1H-NMR (DMSO-d6) δ: 13.15 (s, 1H, ArOH), 10.63 (s, 1H, -SO2NH-), 8.19 (s, 1H, -NH2),
7.75 (s, 1H, -NH2), 7.69~7.71 (d, 2H, ArH), 7.64~7.66 (d, 1H, ArH), 7.36~7.38 (d, 2H, ArH),
6.55~6.58 (m, 2H, ArH), 2.33 (s, 3H, ArCH3). 13C-NMR (DMSO-d6) δ: 21.10, 100.65, 105.13,
108.34, 127.48, 128.02, 128.91, 135.75, 136.67, 141.78, 159.38, 171.24. IR (KBr, cm-1):
3348, 3195, 2948, 2872, 1652, 1530, 1470, 1341, 1136, 825. EI-MS m/z: 306.0 (M+). Anal.
Calcd for C14H14N2O4S: C, 54.89; H, 4.61; N, 9.14; S, 10.47. Found: C, 54.87; H, 4.63; N,
9.16; S, 10.45.
Synthesis of 2-hydroxy-N-methyl-4-[(4-methylphenyl)sulfonamido]benzamide (T20)
From compound 5 (500.0 mg, 1.26 mmol), compound T20 was obtained (193.0 mg, 47.8%, 2
1
steps). H-NMR (DMSO-d6) δ: 12.84 (s, 1H, ArOH), 10.62 (s, 1H, -SO2NH-), 8.58~8.62 (q,
1H, -NH-), 7.68~7.70 (d, 2H, ArH), 7.61~7.64 (d, 1H, ArH), 7.36~7.38 (d, 2H, ArH), 6.59 (s,
2H, ArH), 2.74~2.75 (d, 3H, -NHCH3), 2.33 (s, 3H, ArCH3). 13C-NMR (DMSO-d6) δ: 21.08,
27.23, 100.55, 104.94, 108.20, 127.39, 127.91, 128.72, 135.70, 136.61, 141.59, 159.24,
166.87. IR (KBr, cm-1): 3309, 2946, 2870, 1646, 1548, 1467, 1340, 1134, 823. EI-MS m/z:
320.0 (M+). Anal. Calcd for C15H16N2O4S: C, 56.24; H, 5.03; N, 8.74; S, 10.01. Found: C,
56.25; H, 5.01; N, 8.72; S, 10.03.
Biological and Pharmaceutical Bulletin Advance Publication

Synthesis of 2-hydroxy-N,N-dimethyl-4-[(4-methylphenyl)sulfonamido]benzamide
(T21) From compound 5 (500.0 mg, 1.26 mmol), compound T21 was obtained (240.0 mg,
1
57.1%, 2 steps). H-NMR (DMSO-d6) δ: 10.31 (s, 1H, -SO2NH-), 9.93 (s, 1H, ArOH),
7.66~7.68 (d, 2H, ArH), 7.35~7.37 (d, 2H, ArH), 6.92~6.95 (d, 1H, ArH), 6.71~6.72 (d, 1H,
ArH), 6.53~6.55 (dd, 1H, ArH), 2.81 (s, 6H, -N(CH3)2), 2.34 (s, 3H, ArCH3). 13C-NMR
(DMSO-d6) δ: 21.09, 37.96, 100.27, 108.23, 109.98, 127.34, 127.88, 128.45, 135.63, 136.52,
139.84, 158.77, 170.93. IR (KBr, cm-1): 3306, 2944, 2869, 1652, 1610, 1525, 1466, 1338,
1132, 820. EI-MS m/z: 334.1 (M+). Anal. Calcd for C16H18N2O4S: C, 57.47; H, 5.43; N, 8.38;
S, 9.59. Found: C, 57.45; H, 5.44; N, 8.37; S, 9.61.
Synthesis of N-ethyl-2-hydroxy-4-[(4-methylphenyl)sulfonamido]benzamide (T22)
From compound 5 (500.0 mg, 1.26 mmol), compound T22 was obtained (190.1 mg, 45.1%, 2
1
steps). H-NMR (DMSO-d6) δ: 12.90 (s, 1H, ArOH), 10.63 (s, 1H, -SO2NH-), 8.62~8.65 (t,
1H, -NH-), 7.65~7.71 (m, 3H, ArH), 7.36~7.38 (d, 2H, ArH), 6.59~6.61 (m, 2H, ArH),
3.24~3.29 (m, 2H, -NCH2-), 2.33 (s, 3H, ArCH3), 1.07-1.08 (t, 3H, -CH3). 13C-NMR
(DMSO-d6) δ: 15.61, 21.10, 35.09, 100.73, 105.36, 108.55, 127.43, 127.98, 128.97, 135.75,
136.69, 141.70, 159.43, 165.74. IR (KBr, cm-1): 3307, 2945, 2868, 1642, 1607, 1544, 1466,
1338, 1131, 820. EI-MS m/z: 334.1 (M+). Anal. Calcd for C16H18N2O4S: C, 57.47; H, 5.43; N,
8.38; S, 9.59. Found: C, 57.48; H, 5.41; N, 8.40; S, 9.56.
Synthesis of N,N-diethyl-2-hydroxy-4-[(4-methylphenyl)sulfonamido]benzamide (T23)
From compound 5 (500.0 mg, 1.26 mmol), compound T23 was obtained (176.0 mg, 38.6%, 2
1
steps). H-NMR (DMSO-d6) δ: 10.24 (s, 1H, -SO2NH-), 9.79 (s, 1H, ArOH), 7.64~7.66 (d,
2H, ArH), 7.34~7.36 (d, 2H, ArH), 6.86~6.88 (d, 1H, ArH), 6.70~6.71 (d, 1H, ArH),
6.51~6.53 (dd, 1H, ArH), 3.09 (bs, 4H, -N(CH2)2-), 2.33 (s, 3H, ArCH3), 0.98 (s, 6H,
-CH3×2). 13C-NMR (DMSO-d6) δ: 12.95, 21.12, 45.37, 100.13, 108.36, 107.84, 127.41,
128.27, 129.07, 135.71, 136.59, 137.91, 158.28, 166.88. IR (KBr, cm-1): 3304, 2942, 2866,
1648, 1607, 1521, 1464, 1332, 1127, 816. EI-MS m/z: 362.1 (M+). Anal. Calcd for
C18H22N2O4S: C, 59.65; H, 6.12; N, 7.73; S, 8.85. Found: C, 59.67; H, 6.10; N, 7.71; S, 8.88.
Synthesis of 2-hydroxy-4-[(4-methylphenyl)sulfonamido]-N-(n-propyl)benzamide
(T24) From compound 5 (500.0 mg, 1.26 mmol), compound T24 was obtained (414.1 mg,
Biological and Pharmaceutical Bulletin Advance Publication

1
93.3%, 2 steps). H-NMR (DMSO-d6) δ: 12.87 (s, 1H, ArOH), 10.63 (s, 1H, -SO2NH-),
8.64~8.67 (t, 1H, -NH-), 7.68~7.70 (m, 3H, ArH), 7.35~7.38 (d, 2H, ArH), 6.58~6.60 (m, 2H,
ArH), 3.15~3.20 (q, 2H, -NCH2-), 2.33 (s, 3H, ArCH3), 1.46~1.51 (m, 2H, -CH2-), 0.82~0.86
13
(t, 3H, -CH3). C-NMR (DMSO-d6) δ: 11.54, 21.07, 23.67, 42.13, 100.59, 105.18, 108.38,
127.31, 127.85, 128.79, 135.67, 136.62, 141.48, 159.29, 165.42. IR (KBr, cm-1): 3305, 2944,
2866, 1640, 1606, 1542, 1465, 1336, 1130, 820. EI-MS m/z: 348.1 (M+). Anal. Calcd for
C17H20N2O4S: C, 58.60; H, 5.79; N, 8.04; S, 9.20. Found: C, 58.59; H, 5.81; N, 8.06; S, 9.18.
Synthesis of N-(n-butyl)-2-hydroxy-4-[(4-methylphenyl)sulfonamido]benzamide (T25)
From compound 5 (500.0 mg, 1.26 mmol), compound T25 was obtained (185.2 mg, 40.7%, 2
1
steps). H-NMR (DMSO-d6) δ: 12.86 (s, 1H, ArOH), 10.60 (s, 1H, -SO2NH-), 8.58~8.60 (t,
1H, -NH-), 7.64~7.70 (m, 3H, ArH), 7.35~7.38 (d, 2H, ArH), 6.57~6.60 (m, 2H, ArH),
3.21~3.22 (q, 2H, -NCH2-), 2.33 (s, 3H, ArCH3), 1.44~1.48 (m, 2H, -CH2-), 1.25~1.30 (m,
13
2H, -CH2-), 0.84~0.88 (t, 3H, -CH3). C-NMR (DMSO-d6) δ: 14.02, 20.04, 21.11, 32.48,
39.76, 100.50, 105.12, 108.29, 127.26, 127.81, 128.67, 135.61, 136.49, 141.41, 159.17,
165.33. IR (KBr, cm-1): 3306, 2943, 2866, 1638, 1603, 1540, 1463, 1336, 1131, 818.
EI-MS m/z: 362.1 (M+). Anal. Calcd for C18H22N2O4S: C, 59.65; H, 6.12; N, 7.73; S, 8.85.
Found: C, 59.67; H, 6.11; N, 7.71; S, 8.86.
Synthesis of 2-hydroxy-4-[(4-methylphenyl)sulfonamido]-N-phenylbenzamide (T26)
From compound 5 (500.0 mg, 1.26 mmol), compound T26 was obtained (170.1 mg, 35.4%, 2
1
steps). H-NMR (DMSO-d6) δ: 12.01 (s, 1H, ArOH), 10.69 (s, 1H, -SO2NH-), 10.18 (s, 1H,
-NHPh), 7.80~7.82 (d, 1H, ArH), 7.71~7.73 (d, 2H, ArH), 7.61~7.63 (d, 2H, ArH), 7.32~7.39
(m, 4H, ArH), 7.09~7.12 (m, 1H, ArH), 6.74~6.75 (d, 1H, ArH), 6.66~6.69 (dd, 1H, ArH),
2.34 (s, 3H, ArCH3). 13C-NMR (DMSO-d6) δ: 21.07, 100.33, 108.17, 109.46, 121.83, 127.20,
128.17, 128.29, 128.61, 129.16, 135.53, 136.44, 138.17, 141.19, 158.98, 161.83. IR (KBr,
cm-1): 3315, 2949, 2873, 1648, 1613, 1551, 1471, 1343, 1136, 825, 755, 690. EI-MS m/z:
382.1 (M+). Anal. Calcd for C20H18N2O4S: C, 62.81; H, 4.74; N, 7.33; S, 8.38. Found: C,
62.80; H, 4.76; N, 7.35; S, 8.36.
Synthesis of 2-hydroxy-N-(4-methoxyphenyl)-4-[(4-methylphenyl)sulfonamido]
benzamide (T28) From compound 5 (500.0 mg, 1.26 mmol), compound T28 was obtained
Biological and Pharmaceutical Bulletin Advance Publication

1
(224.0 mg, 43.2%, 2 steps). H-NMR (DMSO-d6) δ: 12.20 (s, 1H, ArOH), 10.67 (s, 1H,
-SO2NH-), 10.09 (s, 1H, -NHAr), 7.80~7.82 (d, 1H, ArH), 7.71~7.73 (d, 2H, ArH), 7.50~7.53
(d, 2H, ArH), 7.37~7.39 (d, 2H, ArH), 6.90~6.92 (d, 2H, ArH), 6.65~6.71 (m, 2H, ArH), 3.73
(s, 3H, -OCH3), 2.34 (s, 3H, ArCH3). 13C-NMR (DMSO-d6) δ: 21.14, 56.02, 100.25, 108.10,
109.30, 113.96, 122.01, 127.24, 128.36, 128.76, 128.95, 135.59, 136.57, 141.03, 158.47,
158.79, 161.53. IR (KBr, cm-1): 3317, 2950, 2874, 1651, 1616, 1555, 1448, 1344, 1138,
828.EI-MS m/z: 412.1 (M+). Anal. Calcd for C21H20N2O5S: C, 61.15; H, 4.89; N, 6.79; S,
7.77. Found: C, 61.17; H, 4.88; N, 6.81; S, 7.75.
Synthesis of 2-hydroxy-N-(2-hydroxyethyl)-4-[(4-methylphenyl)sulfonamido]
benzamide (T29) From compound 5 (500.0 mg, 1.26 mmol), compound T29 was obtained
1
(187.0 mg, 33.8%, 2 steps). H-NMR (DMSO-d6) δ: 12.70 (s, 1H, ArOH), 10.61 (s, 1H,
-SO2NH-), 8.60~8.63 (t, 1H, -NH-), 7.68~7.70 (d, 3H, ArH), 7.36~7.38 (d, 2H, ArH),
6.58~6.62 (m, 2H, ArH), 4.75~4.78 (t, 1H, -OH), 3.45~3.49 (q, 2H, -OCH2-), 3.27~3.32 (m,
13
2H, -NCH2-), 2.34 (s, 3H, ArCH3). C-NMR (DMSO-d6) δ: 21.11, 41.85, 61.47, 100.86,
105.51, 108.67, 127.46, 128.13, 129.20, 135.81, 136.77, 141.83, 159.62, 165.98. IR
(KBr, cm-1): 3350, 2946, 2870, 1645, 1609, 1546, 1469, 1340, 1132, 1038, 823. EI-MS m/z:
350.0 (M+). Anal. Calcd for C16H18N2O5S: C, 54.85; H, 5.18; N, 8.00; S, 9.15. Found: C,
54.83; H, 5.19; N, 8.02; S, 9.14.
Synthesis of 2-hydroxy-N-(2-hydroxyethyl)-N-methyl-4-[(4-methylphenyl)
sulfonamido]benzamide (T30) From compound 5 (500.0 mg, 1.26 mmol), compound T30
1
was obtained (115.0 mg, 25.0%, 2 steps). H-NMR (DMSO-d6) δ: 10.27 (s, 1H, -SO2NH-),
9.86 (s, 1H, ArOH), 7.65~7.67 (d, 2H, ArH), 7.34~7.36 (d, 2H, ArH), 6.91~6.93 (d, 1H, ArH),
6.69~6.70 (d, 1H, ArH), 6.51~6.54 (dd, 1H, ArH), 4.66~4.68 (t, 1H, -OH), 3.41~3.44 (m, 2H,
13
-OCH2-), 3.19~3.25 (m, 2H, -NCH2-), 2.84 (s, 3H, -NCH3)2.33 (s, 3H, ArCH3). C-NMR
(DMSO-d6) δ: 21.07, 35.87, 54.98, 59.14, 100.32, 108.26, 110.14, 127.41, 128.11, 129.03,
135.74, 136.75, 139.19, 159.37, 170.16. IR (KBr, cm-1): 3348, 2943, 2868, 1650, 1608, 1525,
1467, 1328, 1130, 1035, 820. EI-MS m/z: 364.1 (M+). Anal. Calcd for C17H20N2O5S: C, 56.03;
H, 5.53; N, 7.69; S, 8.80. Found: C, 56.05; H, 5.51; N, 7.67; S, 8.82.
Biological and Pharmaceutical Bulletin Advance Publication

Synthesis of 4-hydroxyphenethyl 4-acetamido-2-hydroxybenzoate (T31) From
compound 7 (500.0 mg, 1.75 mmol), compound T31 was obtained (214.0 mg, 39.0%, 2
1
steps). H-NMR (DMSO-d6) δ: 10.62 (s, 1H, ArOH), 10.24 (s, 1H, -CONH-), 9.24 (s, 1H,
ArOH), 7.66~7.68 (d, 1H, ArH), 7.37 (s, 1H, ArH), 7.04~7.11 (m, 3H, ArH), 6.68~6.70 (d,
2H, ArH), 4.39~4.42 (t, 2H, -OCH2-), 2.89~2.92 (t, 2H, -CH2Ar), 2.07 (s, 3H, CH3CO-).
13C-NMR (DMSO-d6) δ: 24.51, 34.87, 64.61, 106.84, 109.07, 114.63, 116.16, 130.35, 131.11,
132.08, 144.73, 156.02, 164.83, 165.15, 170.22. IR (KBr, cm-1): 3330, 2917, 2850, 1728,
1690, 1606, 1518, 1465, 1370, 1262, 836. EI-MS m/z: 315.1 (M+). Anal. Calcd for
C17H17NO5: C, 64.75; H, 5.43; N, 4.44. Found: C, 64.77; H, 5.42; N, 4.46.
Synthesis of 3,4-dihydroxyphenethyl 4-acetamido-2-hydroxybenzoate (T32) From
compound 7 (500.0 mg, 1.75 mmol), compound T32 was obtained (188.0 mg, 32.7%, 2
1
steps). H-NMR (DMSO-d6) δ: 10.61 (s, 1H, ArOH), 10.24 (s, 1H, -CONH-), 8.81 (s, 1H,
ArOH), 8.72 (s, 1H, ArOH), 7.66~7.68 (d, 1H, ArH), 7.36~7.37 (d, 1H, ArH), 7.03~7.06 (dd,
1H, ArH), 6.67~6.68 (d, 1H, ArH), 6.63~6.65 (d, 1H, ArH), 6.51~6.54 (dd, 1H, ArH),
4.36~4.39 (t, 2H, -OCH2-), 2.82~2.85 (t, 2H, -CH2Ar), 2.06 (s, 3H, CH3CO-). 13C-NMR
(DMSO-d6) δ: 24.45, 35.22, 64.65, 106.71, 108.89, 114.49, 116.30, 116.78, 123.18, 131.83,
132.08, 144.60, 145.11, 146.06, 164.69, 165.01, 170.13. IR (KBr, cm-1): 3334, 2944, 2875,
1731, 1692, 1607, 1521, 1466, 1264, 839. EI-MS m/z: 331.1 (M+). Anal. Calcd for
C17H17NO6: C, 61.63; H, 5.17; N, 4.23. Found: C, 61.65; H, 5.16; N, 4.25.
General procedure for the synthesis of compounds T6, T17, and T27 To a solution of
compound 6a, 17a or 27a in anhydrous dichloromethane, TiCl4 was added (1.1 equivalent of
reactant). The mixture was stirred under N2 at room temperature for 30 min. Ethanol 0.5 mL
was added to quench the reaction and the mixture was concentrated in vacuo to give a residue
that was purified by column chromatography on silica gel to afford target compound.
Synthesis of Benzyl 2-hydroxy-4-[(4-methylphenyl)sulfonamido]benzoate (T6) From
compound 5 (500.0 mg, 1.26 mmol), compound T6 was obtained (258.1 mg, 52.0%, 2 steps).
1H-NMR (DMSO-d6) δ: 10.85 (s, 1H, ArOH), 10.57 (s, 1H, -SO2NH-), 7.71~7.73 (d, 2H,
ArH), 7.65~7.68 (d, 1H, ArH), 7.42~7.44 (m, 2H, ArH), 7.36~7.39 (m, 4H, ArH), 7.31 (s, 1H,
ArH), 6.68~6.70 (m, 2H, ArH), 5.31 (s, 2H, -OCH2Ar), 2.33 (s, 3H, ArCH3). 13C-NMR
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(DMSO-d6) δ: 21.40, 66.08, 100.70, 102.60, 108.63, 126.79, 127.42, 128.11, 128.56, 129.17,
131.95, 135.72, 136.39, 137.33, 143.15, 164.80, 166.96. IR (KBr, cm-1): 3336, 2945,
2877,1755, 1618, 1530, 1472, 1357, 1283, 1147, 828, 761, 697. EI-MS m/z: 397.1 (M+). Anal.
Calcd for C21H19NO5S: C, 63.46; H, 4.82; N, 3.52; S, 8.07. Found: C, 63.48; H, 4.81; N, 3.54;
S, 8.06.
Synthesis of (E)-cinnamyl 2-hydroxy-4-[(4-methylphenyl)sulfonamido]benzoate (T17)
From compound 5 (500.0 mg, 1.26 mmol), compound T17 was obtained (250.1 mg, 58.5%, 2
steps). 1H-NMR (DMSO-d6) δ: 10.85 (s, 1H, ArOH), 10.60 (s, 1H, - SO2NH-), 7.68~7.72 (m,
3H, ArH), 7.46~7.48 (d, 2H, ArH), 7.32~7.39 (m, 5H, ArH), 6.74~6.78 (d, 1H, J=16Hz,
=CH), 6.68~6.70 (m, 2H, ArH), 6.42~6.46 (d, 1H, J=16Hz, =CH), 4.92~4.93 (t, 2H, -OCH2-),
2.33 (s, 3H, ArCH3). 13C-NMR (DMSO-d6) δ: 21.39, 64.88, 100.75, 102.57, 108.63, 120.97,
127.51, 127.96, 128.12, 128.46, 128.94, 131.93, 133.57, 135.98, 136.39, 137.27, 143.15,
163.90, 166.24. IR (KBr, cm-1): 3345, 2948, 2875, 1756, 1625, 1538, 1351, 1288, 1143, 944,
831, 765, 702. EI-MS m/z: 423.1 (M+). Anal. Calcd for C23H21NO5S: C, 65.23; H, 5.00; N,
3.31; S, 7.57. Found: C, 65.22; H, 5.02; N, 3.29; S, 7.58.
Synthesis of 2-hydroxy-4-[(4-methylphenyl)sulfonamido]-N-(4-nitrophenyl)
benzamide (T27) From compound 5 (500.0 mg, 1.26 mmol), compound T27 was obtained
1
(235.1 mg, 44.0%, 2 steps). H-NMR (DMSO-d6) δ: 11.61 (s, 1H, ArOH), 10.72 (s, 1H,
-NHAr), 10.62 (s, 1H, -SO2NH-), 8.21~8.24 (d, 2H, ArH), 7.91~7.95 (d, 2H, ArH), 7.71~7.75
(m, 3H, ArH), 7.37~7.39 (d, 2H, ArH), 6.81~6.82 (d, 1H, ArH), 6.67~6.70 (dd, 1H, ArH),
2.34 (s, 3H, ArCH3). 13C-NMR (DMSO-d6) δ: 21.09, 100.51, 108.30, 109.62, 118.98, 123.61,
128.22, 129.19, 129.36, 135.59, 136.48, 141.40, 142.96, 143.69, 156.16, 162.27. IR (KBr,
cm-1): 3319, 2951, 2875, 1652, 1556,1482, 1322, 1139, 829, 762, 697. EI-MS m/z: 427.0
(M+). Anal. Calcd for C20H17N3O6S: C, 56.20; H, 4.01; N, 9.83; S, 7.50. Found: C, 56.22; H,
4.00; N, 9.81; S, 7.51.
α-Glucosidase inhibition studies The inhibitory activities of compounds T1-T32 against
yeast α-glucosidase were evaluated based on protocols previously described in the literature
with slight modifications.21) Briefly, α-glucosidase (G0660-750UN, from saccharomyces
cerevisiae, Sigma Aldrich, St. Louis, MO, USA) was dissolved in phosphate buffer (pH 6.86)
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to a final concentration of 10 U/mL. The enzyme solution (15 μL) and 60 μL of the phosphate
buffer (pH 6.86) were preincubated with 15 μL of the target compounds at varying
concentrations in DMSO at 37℃ for 30 min. The reaction was triggered by the addition of
30 μL of 4-nitrophenyl-α-D-glucopyranoside (α-PNPG, final concentration 5.3 mM) and
stopped after 30 min by the addition of 75 μL of 1.0 M Na2CO3. The amount of released
4-nitrophenol from α-PNPG was measured by determining the absorbance of the solution at
405 nm. A blank control (15 μL of phosphate buffer instead of enzyme solution and 15 μL of
DMSO instead of test solution) and negative control (15 μL of DMSO instead of test solution)
were prepared. In each set of experiments the compound was run in triplicate. The inhibition
ratio was calculated as follows:
Inhibition (%) = (Anegtive－Asample)／(Anegtive－Ablank) × 100
Where Anegtive was the absorbance of negative control, Asample was the absorbance of sample,
Ablank was the absorbance of blank control.
Inhibition kinetics of α-glucosidase studies The inhibition type of compounds T9, T10,
and T32 was determined from Lineweaver-Burk plots, using an approach that has been
previously described.25,26) Typically, two different concentrations of each compound around
the IC50 value were chosen. At each concentration, the α-glucosidase activity was determined
by varying the concentration of α-PNPG. The enzyme reaction was performed under the
above-mentioned reaction condition. The enzyme solution (40 μL) and 20 μL of the inhibitor
were mixed with 1880 μL phosphate buffer (pH 6.86), and pre-incubated at 37℃ for 30 min.
After the addition of 60 μL α-PNPG at different concentrations, the enzymatic reaction was
performed at 37℃ for 60s and the increase in absorbance was measured at 405 nm.
Inhibition types were determined by double-reciprocal plots.27,28) Binding constants were
calculated based on secondary plots of the slopes of the straight lines or vertical intercept
versus the different concentration of inhibitor, respectively.
Fluorescence quenching analysis Fluorescence spectra were performed on a Shimadzu
RF-5310pc fluorescence spectrophotometer (Shimadzu, Kyoto, Japan) equipped with a 1.0
cm quartz cuvette. Fluorescence measurements were performed at 298 K, and the excitation
wavelength was set at 232 nm. The excitation and emission slit widths were set at 5.0 nm and
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the mission spectra were recorded from 300 to 400 nm. Briefly, 10 mL of a diluted
α-glucosidase stock solution (0.6 μM) was titrated by 3.97 mM T9, 2.49 mM T10, 6.76 mM
T32, or 15.5 mM acarbose and mixed for 5 min. Subsequently, the solutions were transferred
to quartz cuvettes in which the emission spectra were measured.
The fluorescence quenching data were analyzed by the Stern-Volmer equation as
follows (1):29)
F F = 1+ Kqt0 Q = 1+ K
Q
[ ]
SV
[ ]
(1)
0
where F0 and F represented the fluorescence intensities before and after addition of
quencher (T9, T10, T32, or acarbose), respectively. Kq represented the bimolecular
quenching constant, τ0 represents the lifetime of the fluorophore in the absence of quencher
(τ0 ≈ 10-8 s), KSV represents the Stern-Volmer quenching constant, and [Q] represents the
concentration of quencher. Next, Eq. (1) was applied to determine the KSV by linear
regression of a plot of F0/F against [Q].
When small molecules independently bind to a set of equivalent sites on a macromolecule,
the equilibrium between free and bound molecules is calculated by equation (2). The binding
constant (K) and the number of binding sites (n) between the quencher and α-glucosidase
were calculated.30)
F − F






0
lg
= lg K + n lg Q
(2)
[ ]
F
where F0 and F represent the fluorescence intensities before and after addition of quencher
(T9, T10, T32, or acarbose), respectively. K represents the binding constant to a site, n
represents the number of binding sites per α-glucosidase molecule, and [Q] represents the
concentration of the quencher.
Molecular docking studies Docking studies were carried to investigate the molecular
binding pattern of target compounds T9, T10, and T32 with the active site pocket of crystal
structure of α-glucosidase (PDB ID: 3L4V), which was downloaded from the RCSB protein
Data Bank. The water molecules in α-glucosidase were removed, and Gasteiger-Huckel
charges and polar hydrogen atoms were added to the macromolecule. Prior to the docking
studies, the structure of all inhibitors was optimized. For comparison purposes, molecular
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docking studies of the standard inhibitor acarbose were also performed.
Antioxidant activity The antioxidant activity of target compounds T9, T10, and T32 was
evaluated towards the scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical,
inhibiting lipid peroxidation, and total reduction capacity.
a) DPPH radical scavenging activity DPPH radical is based on the nitrogen atom in the
center of the structure and can be stably existed in organic reagents.31) The absorbance of the
DPPH radical solution after addition of anti-oxygen was reduced at a wavelength of 517 nm.
A 1.00 mL aliquot of 0.08 g/mL DPPH solution was combined with 1.00 mL of the sample
solution. After addition of each component, the solution was mixed well and placed at room
temperature for 30 min in the dark. The control was prepared as above using 1.00 mL
anhydrous ethanol instead of anti-oxygen. The absorbance was monitored at 517 nm against a
blank control and the results were expressed as follows:
DPPH radical scavenging activity (%) = [(A0－A1)/A0] × 100
Where A0 represents the absorbance of solvent control reaction, and A1 represents the
absorbance of samples with varying concentrations. All tests were performed in triplicate.
b) Anti-lipid peroxidation activity The anti-lipid peroxidation activity of compounds T9,
T10, and T32 was determined based on a previously described procedure with slight
modifications.32,33) In brief, 0.10 mL of sample solution, 0.20 mL of yolk suspension, 0.20
mL of 25 mM FeSO4 solution, and 1.50 mL of 0.1 M phosphate buffer (Na2HPO4-NaH2PO4,
pH 7.45) were mixed well and incubated in water bath at 37℃ for 30 min. The reaction was
stopped by the addition of 1.00 mL 20% trichloroacetic acid (TCA). The solutions were
shaken well and centrifuged at 3500 rpm for 15 min. Next, 2.00 mL of the supernatant was
removed and 1.00 mL of 0.8% TBA was added to the removed supernatant. The mixture was
heated in a water bath at 100℃ for 15 min, then cooled to room temperature. The absorbance
was measured at 532 nm. Experiments were performed in triplicate and ascorbic acid was
used as the standard. The percentage of lipid peroxidation inhibition was calculated as
follows:
Anti-lipid peroxidation activity (%) = (A0－As)/A0 × 100
where A0 represents the absorbance of the control reaction, and As represents the
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absorbance of the sample reaction.
c) Total reduction capacity A modified reported method was utilized to study the total
reduction capacity of T9, T10 and T32.34) In brief, 0.20 mL of sample solution, 1.00 mL of
0.2 M phosphate buffer (NaH2PO4-Na2HPO4, pH 6.6), and 1.00 mL of potassium ferric
cyanide (K3Fe(CN), 1%) were mixed well and incubated at 50℃ for 20 min. After being
cooled to room temperature, 1.00 mL of 10% trichloroacetic acid (TCA) was added to stop
the reaction, and the mixture was centrifuged at 3000 rpm for 10 min. A 1.00 mL aliquot
taken from the upper layer of the solution was mixed with 1.00 mL of distilled water and 0.20
mL of 0.1% ferric chloride, and the absorbance was measured at 700nm. The increase in
absorbance of the reaction mixture indicated an increase in reducing power. Experiments
were performed in triplicates and ascorbic acid was used as the standard. The concentration
of anti-oxygen when absorbance was increased by an optical density of 0.5 was selected to
compare the reduction capacity.
RESULTS AND DISCUSSION
α-Glucosidase inhibition studies In the past, yeast α-glucosidase has been mostly used for
screening and evaluation of α-glucosidase inhibitors due to its simple and convenient
method.35) In this study, the α-glucosidase inhibitory activity of the synthesized compounds
was determined using yeast α-glucosidase. The values of inhibitor concentration required for
50% inhibition of the α-glucosidase activity (IC50) are listed in Table 1, and acarbose was
used as the standard. The IC50 values indicated that the synthesized salicylic acid derivatives
T1-T32 exhibited different levels of inhibitory activity against yeast α-glucosidase. The
efficiency of the inhibitory activity was influenced by the variation of the substitute group of
the salicylic acid derivatives.
Based on the sidechain of 4-(4-methylphenyl)sulfonamido salicylic acid derivatives, the
target compounds T1-T30 included two series: i) compounds T1-T18, the salicylate of
various alcohols; ii) compounds T19-T30, the salicyamide of various amines. The different
structure of the ester or amide greatly affected the inhibitory activity. Taken together, our
studies indicated that salicylate was more efficient when compared to salicyamide. The
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activity increased with the increase of the carbon chain. When aryl was introduced into the
sidechain, the activity increased. Among compounds T1-T30, the inhibitory activity of
compounds T9 and T10 was significantly higher when compared to that of acarbose (IC50 =
0.45 mM), with IC50 values of 0.15 mM and 0.086 mM, respectively, which were 3-fold and
5.2-fold higher than that of acarbose.
To investigate the effect of the substituent group on amino, a tosyl group was replaced by
an acetyl group, and compounds T31 and T32 were designed and synthesized. Compound
T32 exhibited the IC50 value of 0.32 mM, which was 1.41-fold higher compared to that of
acarbose. When comparing compounds T8 and T31, we concluded that a tosyl group on
amino had an advantage over the acetyl group due to its stronger electron withdrawing
properties. The same conclusion could be drawn from the comparison between compounds
T9 and T32.
Table 1. IC50 values (mM) of the inhibition of α-glucosidase by the target compounds.a
O
R2
OH
R1
N
H
Compound
R1
Ts
Ts
Ts
R2
O
IC50 / mM
1.73 ± 0.14
0.99 ± 0.02
0.51 ± 0.03
T1
T2
T3
O
O
O
T4
T5
T6
Ts
Ts
Ts
0.89 ± 0.04
0.35 ± 0.02
0.47 ± 0.02
O
O
O
T7
T8
T9
Ts
Ts
Ts
0.44 ± 0.03
0.20 ± 0.01
0.15 ± 0.01
O
OH
O
OH
OH
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