Vilsmeier reagent initialed sequential one-pot multicomponent synthesis of N,O-disubstituted glycolamides as dipeptidyl peptidase 4 inhibitors

Article abstract of DOI:10.1016/j.tet.2015.07.041

A series of N,O-disubstituted glycolamide derivatives have been successfully synthesized through Vilsmeier reagent initialed sequential one-pot multicomponent procedure from α-chloro N-arylacetamides with formamide/PBr₃ and acid chloride. The t

Full text of DOI:10.1016/j.tet.2015.07.041

Accepted Manuscript
Vilsmeier Reagent Initialed Sequential One-pot Multicomponent Synthesis of N,O-
Disubstituted Glycolamides as Dipeptidyl peptidase 4 inhibitors
Shi-Han Lu, Wan-Ping Yen, Henry J. Tsai, Chien-Shu Chen, Fung Fuh Wong
PII:
S0040-4020(15)01086-8
10.1016/j.tet.2015.07.041
TET 26986
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 25 May 2015
Revised Date: 10 July 2015
Accepted Date: 14 July 2015
Please cite this article as: Lu S-H, Yen W-P, Tsai HJ, Chen C-S, Wong FF, Vilsmeier Reagent Initialed
Sequential One-pot Multicomponent Synthesis of N,O-Disubstituted Glycolamides as Dipeptidyl
peptidase 4 inhibitors, Tetrahedron (2015), doi: 10.1016/j.tet.2015.07.041.
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ACCEPTED MANUSCRIPT
Vilsmeier Reagent Initialed Sequential One-pot
Multicomponent Synthesis of N,O-Disubstituted
Glycolamides as Dipeptidyl peptidase 4 inhibitors
＊
Shi-Han Lu,a,b Wan-Ping Yen,a,c Henry J. Tsai,d Chien-Shu Chen,a Fung Fuh Wonga,b,
aSchool of Pharmacy, China Medical University, No. 91, Hsueh-Shih Rd., Taichung,
Taiwan 40402, R.O.C.
bGraduate Institute of Pharmaceutical Chemistry, China Medical University, No. 91,
Hsueh-Shih Rd., Taichung, Taiwan 40402, R.O.C.
cPh.D. Program for Biotech Pharmaceutical Industry, China Medical University, No.
91, Hsueh-Shih Rd., Taichung, Taiwan 40402, R.O.C.
dDepartment of Health and Nutrition Biotechnology, Asia University, Taichung 41354,
Taiwan
*Corresponding author. Tel.: +886 4 2205 3366 ext. 5603; Fax: +886 4 2207 8083.
E-mail address: wongfungfuh@yahoo.com.tw, ffwong@mail.cmu.edu.tw (F. F.
Wong).
Key words: Glycolamides ester, Formyloxylation, Vilsmeier-type reaction,
Decarbonylation, Esterification
Abstract: A series of N,O-disubstituted glycolamide derivatives have been
successfully synthesized through Vilsmeier reagent initialed sequential one-pot
multicomponent procedure from α-chloro N-arylacetamides with formamide/PBr3 and
acid chloride. The synthesis is straightforward and involved Vilsmeier
formyloxylation reaction, decarbonylation, and esterification three steps. The strategy
was also applicable to α-chloro N-(naphthalenyl)acetamide to prepare the
corresponding N,O-disubstituted glycolamide products. All of N,O-disubstituted
glycolamides were evaluated against dipeptidyl peptidase 4 inhibitory activity. Based
on the inhibitory results, several of O-furan-2-carbonyl and O-quinoline-8-sulfonyl
N-aryl glycolamide compounds possessed the better effectively inhibition of
dipeptidyl peptidase 4.
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Introduction
Dipeptidyl peptidase 4 (DPP-4) inhibitors have emerged as a new therapeutic
strategy for the treatment of type 2 diabetes1–4 due to it specifically cleaves the in
cretin hormone glucagon-like peptide 1 (GLP-1) with a proline or alanine at the
penultimate position of the N-terminus.5 GLP-1 possesses multiple physiological
functions such as stimulating insulin secretion, inhibiting glucagon release, and
retarding gastric emptying.6 Therefore, inhibition of DPP-4 is able to increase the
level of active GLP-1 in the circulation which in turn enhances insulin secretion and
improves the glucose tolerance. On the other hand, several of approved drugs
including Sitagliptin, Saxagliptin, and Vildagliptin have been developed as the DPP-4
inhibitors for the treatment of type 2 diabetes (see Figure 1).7,8 Based on constructions
of them, we developed sequential one-pot multicomponent procedure to synthesize a
series of glycolamide esters with bearing aryl, naphthalenyl, heterocyclic, adamantly,
or naphthalene-1-sulfonyl, quinoline-8-sulfonyl as the potent DPP-4 inhibitors.
F
OH
F
HO
NH2
O
N
N
N
N
H2N
N
H
CN
O
F
N
CN
O
CF3
Sitagliptin
Saxagliptin
Vildagliptin
Figure 1. The structures of Sitagliptin, Saxagliptin, and Vildagliptin
Glycolamide esters play the important role in modern biochemistry, medicinal
chemistry, molecular biology, organic chemistry, and pharmacology.9-15 On the other
hands, promotion the overall lipophilicity of the molecule be able to promote
membrane permeability and oral absorption.13 With respect to prodrugs of a higher
lipophilicity derived from bioactive compounds with carboxylic functions,
glycolamide ester formation represents a well-known approach.11,13 Therefore,
glycolamide esters often were reported as the potentially useful protecting groups of
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amino acid16 or carboxylic acid17,18 for formation of the chemically stable prodrugs or
drugs. Based on the above observations prompted us to evaluate the sequential
one-pot multicomponent synthesis method to efficiently synthesize glycolamide ester
derivatives.
Recently, the sequential one-pot multicomponent reactions are becoming
increasingly prevalent due to their improved efficiency, reduced waste, and rapid
access of structural diversity.19 Based on their high versatility, they have widely
gained a considerable attracted academic, economic, and ecological interest.20
Therefore, we developed an efficient sequential one-pot multicomponent synthesis of
N,O-disubstituted glycolamides by treating α-chloro N-arylacetamides with
formamide/PBr3 and acid chloride. The straightforward strategy involved Vilsmeier
formyloxylation reaction, decarbonylation, and esterification three steps. All
synthesized N,O-disubstituted glycolamide compounds were further evaluated in vitro
for their DPP-4 inhibitory activity.
Result and Discussion
Development of Vilsmeier reagent initiated sequential one-pot multicomponent
synthesis of N,O-disubstituted glycolamides:
Recently, we reported the newly developed Vilsmeier–Haack reagent-promoted
formyloxylation for synthesis of α-formyloxy N-arylacetamides 2 from α-halo
N-phenylacetamides 1 with formamide in presence of PBr3 at 80–90 °C for 4 h (see
Scheme 1).21 The hydrolyzed product α-hydroxy N-phenylacetamide 3 were also
accompanied to form as the minor product in the formyloxylation reaction. Moreover,
all of α-chloro N-arylacetamides 1 were prepared as the starting materials by treating
substituted aniline with 2-chloroacetyl chloride via our previous published
procedure.21
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O
O
O
O
OH
O
H
Cl
HN
HN
HN
H
NH2, PBr3
O
+
80-90 oC for 4 h
Y
Y
Y
2. major product
3. minor product
1
Scheme 1. Our previously reported synthesis method for α-formyloxy
N-arylacetamides
2
and α-hydroxy N-arylacetamides
3
from α-halo
N-phenylacetamides 1
O
O
O
O
W
Cl
NH2, PBr3, 80-90 oC
HN
HN
1. H
O
2. THF, 60-70 oC for 1.0-3.0 h
O
3.
W
Cl, NEt3
1a
5a
O
O
Esterification
Formylation
H
NH2, PBr3
W
O
Cl, NEt3
OH
Z
O
O
H
O
H
N
HN
HN
Decarbonylation
NH
O
THF, 60-70 oC
for 1.0-3.0 h
4a. Z = OCH=N+H2
2a
3a
Scheme 2. The new sequential one-pot multicomponent synthesis strategy of
N,O-disubstituted glycolamides
In this work, the typical sequential one-pot multicomponent strategy for
synthesis of N,O-disubstituted glycolamide 5a were designed and shown in Scheme 2,
which were involved Vilsmeier formyloxylation reaction, decarbonylation, and
esterification three steps. Initially, α-chloro N-phenylacetamide 1a were preliminarily
treated with Vilsmeier reagent (HC(O)NH2 + PBr3) to convert the intermediate 4a by
following our published procedure.21 While intermediate 4a was worked-up in
aqueous solution, α-formyloxy N-phenylacetamides 2a can be conceived as the
hydrolysis equivalent product. Subsequently, we attempted to probe the feasibility of
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sequential synthesis of N,O-disubstituted glycolamide 5a without the isolation of
intermediate 4a. After the first-step formyloxylation of α-chloro N-arylacetamide 1a
was completed (monitored by TLC), the co-solvent THF was added in the resulting
solution. Sequentially, the key step of decarbonylation reaction was examined at the
different reaction temperature and time.
Table 1. The study of reaction temperature and time of decarbonylation for sequential
o
ne-pot multicomponent synthesis
O
Z
O
O
O
Cl
O
H
O
H
OH
HN
N
HN
HN
H
NH2, PBr3
Decarbonylation
NH
O
80-90 oC for 4 h
formamide/THF,
30-90 oC for 1.0-
3.0 h
4a. Z = OCH=N+H2
2a
1a
3a
Reaction temperature of
decarbonylation (ºC)
Reaction time of
Yields of α-hydroxy
Entry
decarbonylation (h) N-phenylacetamide 3a (%)
1
2
3
4
5
6
7
8
9
~35
~45
~55
~65
~75
~85
~65
~65
~65
1.0
1.0
1.0
1.0
1.0
1.0
0.5
1.5
2.0
66
68
72
74
71
67
64
79
73
To optimize decarbonylation condition, we preliminarily focus on the reaction
temperature. The further decarbonylation of intermediate 4a (α-formyloxy acetamide
2a) was performed at various reaction temperature including ~35, 45, 55, 65, and
85°C in formamide/THF (1:1) solution for 1.0 h. When the reaction temperature
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ACCEPTED MANUSCRIPT
controlled below 70°C, the corresponding decarbonylation α-hydroxy
N-phenylacetamide products 3a were obtained in 66–74% yields (see entry 1–6 of
Table 1). In the other hands, the decomposition of intermediate 4a to form aniline or
other by-products were observed at the higher reaction temperature (> 70°C, see entry
5–6 of Table 1). Since, the better reaction temperature was set between 60°C and
70°C. Furthermore, we studied the decarbonylation reaction time from 0.5 to 2.0 h by
monitoring TLC, the best reaction time was regarded about 1.5 h (see Table 1).
With good formyloxylation and decarbonylation conditions in hand, we set out to
conceive the final-step esterification. As illustrated in Scheme 2, the sequential
furan-2-carbonyl chloride and NEt3 was added to the resulting solution and stirred at
reflux for 4 h.22 When the reaction was completed, the mixture was quenched with
water, extracted with CH2Cl2, and purified with silica gel column chromatography to
give the corresponding desired N,O-disubstituted glycolamide 5a in 74% yield (see
Scheme 2 and Table 2). Based on controlled and optimized experimental, we believed
the best reliable procedure for the sequential one-pot multicomponent synthesis of
glycolamide ester was involved the treatment of α-chloro N-arylacetamide 1 with 3.0
equivalent of PBr3 in formamide solution at 80–90 °C for 4 h. When the
formyloxylation reaction was completed, the resulting mixture was added with
anhydrous THF solvent (formamide/THF = 1/1) then heated to reflux between 60°C
and 70°C for 0.5–2.0 h (monitored by TLC). Subsequently, acid chloride (1.5
equivalent) and NEt3 (3.0 equivalent) was added to the resulting solution and stirred at
reflux for 4 h. After work-up and purification, the corresponding N,O-disubstituted
glycolamide was readily prepared in good yield (see Scheme 2).
Following the optimization procedure in Scheme 2, this one-pot multicomponent
synthesis protocol worked toward a range of α-chloro N-arylacetamides 1a–f bearing
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ACCEPTED MANUSCRIPT
o-, m-, p-F-Ph, p-OMe-Ph, and p-CF3-Ph with furan-2-carbonyl chloride in the
presence of NEt3 to give the corresponding N,O-disubstituted glycolamide products
5a–f in 66–77% yields (see the entry 1–6 of Table 2). Furthermore, we extended this
same methodology to α-chloro N-naphthalenylacetamide compound 6. The
corresponding glycolamide ester product 7 was also successfully afforded in 68%
yield (see the entry 7 of Table 2). All of N,O-disubstituted glycolamides 5a–f and 7
were fully characterized by spectroscopic methods. For example, compound 5a
presented one singlet peaks at δ 4.97 ppm for methylene group 1H NMR. In 13C NMR
spectrum, compound 5a possessed characterization absorptions at δ 156.8 ppm
for –O(13C=O), at δ 164.7 ppm for NH(13C=O), and at δ 63.2 ppm for –13CH2C=O. Its
IR absorptions showed peaks at 1678 cm–1 for stretching of the –NH(C=O) group and
1734 cm–1 for stretching of the –O(C=O) group.
Table 2. The results of the sequential one-pot multicomponent synthesis of
N,O-disubstituted glycolamides 5a–f and 7
O
O
O
o
1. H
NH2, PBr3, 80-90 C
Cl
O
HN
Ar
HN
Ar
O
2. THF, 60-70 oC for 1.0-3.0 h
3. furan-2-carbonyl chloride/NEt3
O
1a-f and 6
5a-f and 7
Reaction time of
Entry
1a–f and 2 (Ar)
Yields (%) of 5a–f and 7
decarbonylation (h)
1
2
3
4
5
6
7
Ph
0.5
74
71
74
72
77
66
68
1a
1b
1c
1d
1e
1f
6
5a
5b
5c
5d
5e
5f
7
o-F-Ph
1.0
1.0
1.5
1.5
2.0
2.0
m-F-Ph
p-F-Ph
p-OMe-Ph
p-CF3-Ph
Naphthalenyl
7

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For investigation the acid chloride functional effect in esterification reaction, we
sought to further extend the scope of this practical approach by replacing various acid
chlorides including m-fluorobenzoyl chloride, p-fluorobenzoyl chloride, and
isoxazole-5-carbonyl chloride with α-chloro N-phenylacetamides 1a. Upon the
completion of sequential one-pot multicomponent synthesis, the corresponding
desired N-phenyl-O-substituted glycolamides 8–11 were also successfully obtained in
good yields (71–84% yields, see Scheme 3 and Table 3).
O
O
O
Cl
O
W
HN
HN
1. H
NH2, PBr3
O
2. THF, 60-70 oC for 1.5 h
O
3.
W
Cl, NEt3
1a
8. W = m-FPh, 9. W = p-FPh,
10. W = isoxazolyl,11. W = adamantyl
Scheme 3. Synthesis of N-phenyl-O-substituted glycolamide derivatives 8–11
Table 3. The results of synthesis of N-phenyl-O-substituted glycolamide derivatives
8–11
O
O
Yields (%) of
glycolamide
ester 8–11
Reaction time of
Cl
O
W
Entry
HN
Ph
HN
Ph
decarbonylation (h)
O
F
O
1
0.5
1.0
84
81
1a
1a
8
O
HN
Ph
O
F
O
O
2
9
HN
Ph
O
O
O
N
3
4
1.0
2.0
71
68
1a
1a
10
HN
Ph
O
O
O
O
11
HN
Ph
O
8

ACCEPTED MANUSCRIPT
Recently, many compounds containing the adamantyl group were presented in
current clinical use or the potential therapeutics such as Amantadine was initially
developed as an antiviral agent to treat influenza A and Vildagliptin was developed a
target for agents against type 2 diabetes mellitus according to adamantyl group is
more lipophilic to promote membrane permeability and oral absorption.23 Therefore,
we also introduced adamantly functional group to general the corresponding
N-phenyl-O-adamantylglycolamide 11 in 68% yield by following the same standard
procedure of sequential one-pot multicomponent synthesis method (see Scheme 3 and
Table 3).
On the other hands, many pharmacological agents were grafted with
naphthalene-1-sulfonyl and quinoline-8-sulfonyl moieties in order to confer water
solubility.24 Thus, we were enthusiastically interested to introduce
quinoline-8-sulfonyl moiety to N,O-disubstituted glycolamides. By applying the
standard procedure of sequential one-pot synthesis, α-chloro N-arylacetamide 1a–e
was preliminarily converted to α-hydroxy N-arylacetamide 3a–e. Consequentially, the
esterification was carried out toward quinoline-8-sulfonyl chloride. Due to
quinoline-8-sulfonyl possessed the steric hindrance effect, the corresponding
glycolamide 12a was obtained in the lower yields (53%, see Scheme 4). Since
methoxy- and fluorine-containing25 compounds are well known to play an important
role in wide fields including biochemistry and agrochemistry, we also prepared a
series of N-(o-, m-, or p-F-Ph)-O-quinoline-8-sulfonylglycolamide esters 12b–d
(65–81%) and N-(p-MeO-Ph)-O-quinoline-8-sulfonylglycolamide ester 12e (78%) to
evaluate the structure–activity relationship study (see Scheme 3).
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O
O
O
O
O S
O
Cl
HN
1. H
NH2, PBr3
HN
2. THF, 60-70 oC for 2.0 h
O
Y
X
X
S Cl, NEt3
3.
O
Y
1a. X = H, 1b. X = o-F
1c. X = m-F, 1d. X = p-F
1e. X = OMe
12a. X = H, Y = N (53%)
12b. X = o-F, Y = N (73%)
12c. X = m-F, Y = N (65%)
12d. X = p-F, Y = N (81%)
12e. X = OMe, Y = N (78%)
13a. X = m-F, Y = C (69%)
13b. X = OMe, Y = C (74%)
Y = N or C
Scheme 4. Synthesis of N-aryl-O-quinoline-8-sulfonyl glycolamide esters 12a–e and
N-aryl-O-naphthalene-1-sulfonyl glycolamide esters 13a–b
For further investigation of the substitution effect, the same reaction condition
was applied to naphthalene-1-sulfonyl chloride. The reaction also smoothly gave the
corresponding 13a–b in 69–74% yields (see Scheme 4). All of
N-aryl-O-quinoline-8-sulfonyl
glycolamide
esters
12a–e
and
N-aryl-O-naphthalene-1-sulfonyl glycolamide esters 13a–b were fully characterized
by spectroscopic methods. For example, compound 12e presented two singlet peaks at
δ 3.77 ppm and 4.93 ppm for p-MeO-Ph and α-carbon methylene (–CH2–) in 1H NMR.
13
In C NMR spectrum, compound 12e possessed characteristic absorptions at δ 55.4
ppm for –O13CH3, at δ 66.2 ppm for methylene carbon –13CH2–, and at δ 157.1 ppm
for glycolamide carbon N–(13C=O)–. The structure of compound 12e was also
charactered by X-ray crystallographic analyses and the single-crystal X-ray diffraction
study (ORTEP) was presented in Figure 2.
10

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Figure 2. The ORTEP diagram of N-(p-MeO-Ph)-O-quinoline-8-sulfonylglycolamide
ester 12e
The dipeptidyl peptidase 4 inhibitory activity of α-Formyloxy N-phenylacetamides and
N,O-disubstituted glycolamides:
A preliminary screening of new α-formyloxy N-phenylacetamides 3a–f and
N,O-disubstituted glycolamides compounds 5a–f, 7–11, 12a–e, and 13a–b was
evaluated in vitro against a panel of standard DPP-4. The inhibitory activity was
executed using the disc diffusion method and the measurement of the zone of growth
inhibition for a concentration 1.0 µmol/mL (DMSO) of each α-formyloxy
N-phenylacetamides 3a–f and N,O-disubstituted glycolamide compounds 5c–e, 12a–e,
and 13a–b. The most active compounds will be selected for the further screening. At
first, α-formyloxy N-phenylacetamides 3a–f were prepared by following our previous
published literature procedure21 as the comparison model for the inhibitory activity
study (see Scheme 1 and Table 4).26 Based on the dipeptidyl peptidase 4 inhibitory
study, all of α-formyloxy N-phenylacetamide compounds 3a–f presented the poor
inhibitory activity (< 23.8%, see Table 4). To compare with structure of Sitagliptin,
we modified α-formyloxy N-phenylacetamide 3a–f to prepare N,O-disubstituted
glycolamide derivatives 5a–f, 7–11, 12a–e, and 13a–b by grafting the functional
cyclic or polycyclic framework in α-carbon ester. All N,O-disubstituted glycolamide
11

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derivatives 5a–f, 7–11, 12a–e, and 13a–b were also evaluated in vitro for their DDP-4
inhibitory activity.
Table 4. The DPP-4 inhibitoryry activity of α-formyloxy N-phenylacetamides 3a–f
and N,O-disubstituted glycolamide compounds 5a–f, 7–11, 12a–e, and 13a–b
Entry
1
N,O-Disubstituted glycolamides 5a–f and 7–11
DPP-4% Inh@1µMa
O
O
H
23.5
3a
3b
HN
Ph
O
O
O
O
H
H
HN
HN
2
3
19.9
18.7
F
O
O
3c
O
F
O
O
H
HN
O
4
5
20.9
11.6
3d
F
O
O
H
HN
O
3e
OMe
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O
O
H
HN
O
6
23.8
3f
CF3
O
O
7
8
35.5
84.3
5a
5b
HN
Ph
O
O
O
O
O
HN
HN
O
O
F
O
O
9
100.0
94.4
5c
O
F
O
O
O
O
HN
O
O
O
O
10
5d
F
O
HN
O
11
12
99.7
44.1
5e
5f
OMe
O
HN
O
CF3
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O
O
HN
O
13
42.1
7
O
F
O
14
15
65.4
56.5
8
9
O
HN
Ph
O
F
O
O
HN
Ph
O
O
O
N
16
17
30.4
85.6
10
11
HN
Ph
O
O
O
O
HN
Ph
O
O
O
O S
O
HN
18
100.0
12a
N
aConcentration of each used compound is 1.0 µmol L-1 (DMSO)
Introduction of furan-2-carbonyl into the α-carbon position of α-hydroxy
N-arylacetamides 3a–f and α-hydroxy N- naphthalenyl acetamide bearing Ph, o-, m-,
p-F-Ph, p-OMe-Ph, p-CF3-Ph, and naphthalenyl group in N-1 position led to
N,O-disubstituted glycolamides 5a–f and 7. Most of target compounds 5a–f and 7
showed the improved DPP-4 inhibition activity in comparison with compounds
against 3a–f (> 35.5%, see Table 4). In particular, compounds 5c–e with m-, p-F-Ph,
p-OMe-Ph, and p-CF3-Ph groups in N-1 position possessed the significant inhibitory
activity against DDP-4 (94.4–100 %, see Table 4).
14

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To further evaluate the impact of substituents on α-carbon position of α-hydroxy
N-phenylacetamides 3a on inhibition activity, the m-fluorobenzoyl, p-fluorobenzoyl,
isoxazole-5-carbonyl and adamantyl-1-carbonyl were introduced into α-carbon
position to give the corresponding glycolamide ester products 8–11. Most of
synthesized compounds 8–11 showed the slightly increase DPP-4 inhibition activity in
comparison with compound 5a, except for compounds 11 with
O-adamantyl-1-carbonyl group (> 85.6%).
Based on the above structure–activity relationship study, we found that
compounds 5c–e with m-, p-F-Ph, p-OMe-Ph, and p-CF3-Ph groups in N-1 position
possessed the significant inhibitory activity against DDP-4 (> 94.4%). In the other
hands, we tried to graft naphthalene-1-sulfonyl or quinoline-8-sulfonyl moiety for the
improvement of solubility.24 Thus, we modified and converted compounds 5c–e to
N,O-disubstituted glycolamides 12a–e and 13a–b, which were maintained
N-substituted groups including m-, p-F-Ph, p-OMe-Ph, and p-CF3-Ph and
simultaneously introduced naphthalene-1-sulfonyl and quinoline-8-sulfonyl groups in
O-substituted
position
of
glycolamide
esters.
However,
most
of
O-quinoline-8-sulfonyl
N-aryl
glycolamide
esters 12b–e
and
O-naphthalene-1-sulfonyl N-aryl glycolamide esters 13a–b possessed the poor
solubility in DMSO solution, except for compound 12a. Since, the DPP-4 inhibitory
activities of glycolamide esters 12b–e and 13a–b were not accurately detectable.
To analysis the DPP-4 inhibitory activity data of all of N,O-disubstituted
glycolamides compounds 5a–f, 7–11, and 12a, we found that compounds 5c–e and
12a presented the better inhibitory activity for DPP-4. The order of activity into the
series of the N,O-disubstituted glycolamides was 12a (100%) = 5c (100%) > 5e
(99.7%) > 5d (94.4%) > 11 (85.6%) > 5b (84.3%) >> 8 (65.4%) > 9 (56.5%) > 5f
15

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(44.1%) > 7 (42.1%) > 5a (100%) > 10 (30.4%). The most active compounds 5c and
12a were selected for further screening against DPP-4 cell line in vitro. The IC50
value indicates that compounds 5c and 12a were 72.2 µM and 46.0 µM, respectively.
Based on the above results, compounds 12a possessed the best inhibitory activity and
quinoline-8-sulfonyl group as O-substituents was regarded as the best active
bioisostere for construction of the potent lead compounds.
Docking study of N-aryl-O-quinoline-8-sulfonyl glycolamide esters 12a into the active
site of DPP-4
To predict the possible binding mode of N-aryl-O-quinoline-8-sulfonyl
glycolamide esters 12a in the active site of DPP-4, we performed molecular docking
study using the docking program GOLD 5.0 and the X-ray crystal structure of DPP-4
(PDB ID 2AJL). As depicted in Figure 3, the docking model reveals that compound
12a can form four hydrogen bonds with residues Tyr547, Tyr666, and Arg669.
Compound 12a makes hydrophobic interactions with some surrounding residues,
including Phe357, Val656, Tyr662, Tyr666, and Val711. Based on these observations,
we believed that O-quinoline-8-sulfonyl N-aryl glycolamide ester compounds formed
more hydrogen bonds. As a result, they possessed the better DPP-4 inhibitory activity.
16

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Figure 3. Docking model of compound 12a into the active site of DPP-4. Some
representative amino acid residues (cyan) interacting with compound 12a (yellow)
was shown as stick structures. The red dashed lines indicate hydrogen-bonding
interactions.
Conclusion
A new sequential one-pot multicomponent method was developed for
preparation of N,O-disubstituted glycolamides from α-chloro N-arylacetamides via
Vilsmeier formyloxylation reaction, decarbonylation, and esterification three steps.
All of synthesized glycolamide ester derivatives were also evaluated the DPP-4
inhibitory activity. Based on results of the structure–activity relationship and
inhibitory activity study, the synthesized glycolamide ester derivatives 5c–e and 12a
containing O-furan-2-carbonyl, O-naphthalene-1-sulfonyl, and O-8-quinoline-sulfonyl
moieties, would be able to conceive as potential DPP-4 inhibitors for therapeutic type
2 diabetes mellitus. For the further IC50 screening in vitro, compounds 12a possessed
the best inhibitory activity and quinoline-8-sulfonyl group was regarded as the best
active O-substituted bioisostere for construction of the potent lead compounds.
Experimental Section
All chemicals were reagent grade and used as purchased. All reactions were carried
17

ACCEPTED MANUSCRIPT
out under argon or nitrogen atmosphere and monitored by TLC. Flash column
chromatography was carried out on silica gel (230–400 mesh). Analytical thin-layer
chromatography (TLC) was performed on precoated plates (silica gel 60 F-254)
purchased from Merck Inc. Mixtures of ethyl acetate and hexanes were used as
eluants. Infrared (IR) spectra were measured on a Bomem Michelson Series FT-IR
spectrometer. The wavenumbers reported are referenced to the polystyrene absorption
at 1601 cm–1. Absorption intensities are recorded by the following abbreviations: s,
strong; m, medium; w, weak. Proton NMR spectra were obtained on a Bruker (200
MHz or 500 MHz) spectrometer by use of CDCl3 as solvent. Multiplicities are
recorded by the following abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet; J, coupling constant (Hz). Carbon-13 NMR spectra were obtained on a
Bruker (50 MHz or 125 MHz) spectrometer by used of CDCl3 as solvent. Carbon-13
chemical shifts are referenced to the center of the CDCl3 triplet (δ 77.0 ppm).
High-resolution mass spectra were obtained from a JEOL JMS-HX110 mass
spectrometer.
General Procedure for Sequential One-pot Multicomponent Synthesis of
N,O-Disubstituted Glycolamides 5a–f, 7–11, 12a–e, and 13a–b.
The reliable procedure involved the treatment of the treatment of 4 h α-chloro
N-arylacetamide 1a–f or α-chloro N-(naphthalenyl)acetamide 2 (1.0 equiv) with PBr3
(~3.0 equiv) in formamide solution (2 mL) at 80–90 °C within 4 h. When the
formyloxylation reaction was completed (monitored by TLC), the resulting mixture
was added with anhydrous THF solvent (2.0 mL, formamide/THF = 1/1) then heated
to reflux between 60°C and 70°C for 0.5–2.0 h (monitored by TLC) to give α-hydroxy
N-arylacetamide or N-(naphthalenyl)acetamide intermediates. Sequentially, acid
chloride (1.5 equiv) including furan-2-carbonyl chloride, m-fluorobenzoyl chloride,
18

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p-fluorobenzoyl chloride, isoxazole-5-carbonyl chloride, adamantane-1-carbonyl
chloride, or naphthalene-1-sulfonyl chloride (1.5 equiv) and quinoline-8-sulfonyl
chloride (1.5 equiv), was added to the resulting solution in the presence of NEt3 (3.0
equiv) and stirred at reflux for 4 h. When the sequential one-pot multicomponent
synthesis reaction was completed, the reaction mixture was added to saturate sodium
bicarbonate (15 mL) and extracted with dichloromethane (15 mL × 2). The combined
organic layer was washed saturated aqueous NaHCO3 (15 mL), dried over MgSO4,
filtered, and concentrated under reduced pressure. The residue solution was purified
by column chromatography on silica gel to give the corresponding N,O-disubstituted
glycolamides 5a–f, 7–11, 12a–e, and 13a–b in 66–84 % yields.
1
2-Furan-2-oxo-2-(phenylamino)ethyl ester (5a): white solid; mp 127–129 °C; H
NMR (CDCl3, 400 MHz) δ 4.97 (s, 2 H, CH2), 6.66 (d, J = 1.6 Hz, 1 H, furan-H),
7.22 (dd, J = 14.8, 7.4 Hz, 1 H, ArH), 7.39–7.43 (m, 3 H, ArH + furan-H), 7.60 (d, J =
7.60 Hz, 2 H, ArH), 7.72 (d, J = 1.6 Hz, 1 H, furan-H), 7.99 (br, 1 H, NH); 13C NMR
(CDCl3, 100 MHz) δ 63.18, 112.38, 119.67, 120.27 (2 × CH), 125.07, 129.11 (2 ×
CH), 136.62, 143.42, 147.17, 156.84, 164.73; IR (KBr) 3265 (br, NH), 3026, 1734 (s,
C=O), 1678, 1548, 759 cm–1; EIMS m/z: 245 (M+, 20), 229 (22), 121 (22), 105 (68),
95 (100), 93 (36), 91 (19), 77 (58), 57 (42); HRMS Calcd for C13H11NO4: 245.0688;
Found: 245.0678.
2-Furan-2-oxo-[(2-fluorophenyl)amino]ethyl ester (5b): white solid; mp 122–124
°C; 1H NMR (CDCl3, 400 MHz) δ 4.91 (s, 2 H, CH2), 6.58 (dd, J = 8.4, 8.4 Hz, 1 H,
furan-H), 7.06–7.15 (m, 3 H, ArH), 7.33 (d, J = 3.2 Hz, 1 H, furan-H), 7.67 (d, J =
14.4 Hz, 1 H, furan-H), 8.25 (br, 1 H, NH), 8.32 (dd, J = 16.0, 8.0 Hz, 1 H, ArH); 13C
NMR (CDCl3, 100 MHz) δ 63.09, 112.33, 114.88 (d, J = 20.11 Hz), 114.97, 119.55,
121.70, 124.70, 125.01, 143.29, 147.36, 148.48, 164.75, 165.82 (d, J = 241.92 Hz);
IR (KBr) 3255 (br, NH), 2360, 2337, 1735 (s, C=O), 1674, 1543, 759 cm–1; EIMS
19

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m/z: 263 (M+, 11), 153 (35), 125 (10), 111 (16), 95 (100) ,83 (9); HRMS Calcd for
C13H10FNO4: 263.0594; Found: 263.0599.
2-Furan-2-oxo-[(3-fluorophenyl)amino]ethyl ester (5c): white solid; mp 119–121
°C; 1H NMR (CDCl3, 400 MHz) δ 4.89 (s, 2 H, CH2), 6.58 (dd, J = 4.8, 3.2 Hz, 1 H,
furan-H), 6.83 (dd, J = 16.4, 8.4 Hz, 1 H, ArH), 7.18 (d, J = 8.0 Hz, 1 H, ArH), 7.28
(dd, J = 14.8, 8.2 Hz, 1 H, ArH), 7.34 (d, J = 3.6 Hz, 1 H, furan-H), 7.49 (d, J = 10.4
13
Hz, 1 H, ArH), 7.63 (d, J = 16.2 Hz, 1 H, furan-H), 7.98 (br, 1 H, NH); C NMR
(CDCl3, 100 MHz) δ 63.14, 107.74 (d, J = 26.0 Hz), 111.94, 112.43, 113.67 (d, J =
351.0 Hz), 119.74 (d, J = 20.0 Hz), 130.24, 138.18(d, J = 10.0 Hz), 143.32, 147.19(d,
J = 10.0 Hz), 156.83, 161.75, 164.87; IR (KBr) 3267 (br, NH), 2360, 2337, 1759 (s,
C=O), 1681, 1546, 775 cm–1; EIMS m/z: 263 (M+, 11), 153 (35) , 138 (3), 125
(11) ,111 (15), 95 (100), 83 (8); HRMS Calcd for C13H10FNO4: 263.0594; Found:
263.0602.
2-Furan-2-oxo-[(4-fluorophenyl)amino]ethyl ester (5d): white solid; mp 127–129
1
°C; H NMR (CDCl3, 400 MHz) δ 4.89 (s, 2 H, CH2), 6.58 (d, J = 1.6 Hz, 1 H,
furan-H), 7.02 (dd, J = 17.2, 8.6 Hz, 2 H, ArH), 7.29 (d, J = 3.6 Hz, 1 H, furan-H) ,
7.50 (dd, J = 13.6, 8.8 Hz, 2 H, ArH), 7.63 (d, J = 10.0 Hz, 1 H, furan-H), 7.91 (br, 1
H, NH); 13C NMR (CDCl3, 100 MHz) δ 63.14, 112.21 (2 × CH), 112.42, 115.83 (d, J
= 23.0 Hz), 119.82 (2 × CH), 122.18 (d, J = 8.0 Hz), 143.89, 147.26, 162.65, 163.14
(d, J = 251.25 Hz), 164.87; IR (KBr) 3197 (br, NH), 2360, 2341, 1789 (s, C=O), 1635,
1543, 756 cm–1; EIMS m/z: 263 (M+, 11), 153 (30), 125 (8), 111 (18) ,95 (100), 83 (8);
HRMS Calcd for C13H10FNO4: 263.0594; Found: 263.0592.
2-Furan-2-oxo-[(4-methoxyphenyl)amino]ethyl ester (5e): yellow solid; mp
1
125–127 °C; H NMR (CDCl3, 400 MHz) δ 3.75 (s, 3 H, OCH3), 4.85 (s, 2 H, CH2),
6.54 (dd, J = 4.8, 3.2 Hz, 1 H, furan-H), 6.83 (d, J = 8.8 Hz, 2 H, ArH), 7.31 (d, J =
3.6 Hz, 1 H, furan-H), 7.40 (d, J = 8.8 Hz, 2 H, ArH), 7.61 (d, J = 4.8 Hz, 1 H,
20

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13
furan-H), 7.94 (br, 1 H, NH); C NMR (CDCl3, 100 MHz) δ 55.40, 63.06, 112.29,
114.14 (2 × CH), 119.54, 122.22 (2 × CH), 129.61, 143.88, 147.09, 156.86, 156.95,
164.69; IR (KBr) 3255, 1732 (s, C=O), 1666 (s, C=O), 1546, 763 cm–1; EIMS m/z:
275 (M+, 45), 153 (9), 135 (5), 123 (63), 122 (19), 108 (12), 95 (100); HRMS Calcd
for C14H13NO5: 275.0794; Found: 275.0795.
2-Furan-2-oxo-[(4-trifluorophenyl)amino]ethyl ester (5f): white solid; mp 124–126
°C; 1H NMR (CDCl3, 400 MHz) δ 4.91 (s, 2 H, CH2), 6.59 (dd, J = 4.8, 3.2 Hz, 1 H,
furan-H), 7.35 (d, J = 3.6 Hz, 1 H, furan-H), 7.58 (d, J = 8.4 Hz, 2 H, ArH), 7.66–7.69
13
(m, 3 H, ArH + furan-H), 8.07 (br, 1 H, NH); C NMR (CDCl3, 100 MHz) δ 63.17,
112.48 (2 × CH), 119.85 (2 × CH), 119.94 (2 × CH), 123.51 (q, J = 260.2 Hz), 126.36
(2 × CH), 143.27, 147.29, 156.82, 165.05; IR (KBr) 3498 (br, NH), 1732 (s, C=O),
1643, 1593, 798 cm–1; EIMS m/z: 313 (M+, 8), 296 (14), 269 (10), 265 (12), 186 (12),
153 (43), 105 (15), 95 (100), 77 (15), 70 (21), 57 (13); HRMS Calcd for C14H10F3NO4:
313.0562; Found: 313.0560.
2-Furan-2-oxo-(1-naphthalenylamino)ethyl ester (7): white solid; mp 123–125 °C;
1H NMR (CDCl3, 400 MHz) δ 5.03 (s, 2 H, CH2), 6.60 (d, J = 1.2 Hz, 1 H, furan-H),
7.39 (d, J = 3.2 Hz, 1 H, furan-H), 7.46–7.54 (m, 3 H, ArH), 7.66 (d, J = 1.2 Hz, 1 H,
furan-H), 7.72 (d, J = 8.0 Hz, 1 H), 7.86 (dd, J = 14.0, 7.0 Hz, 2 H), 8.00 (d, J = 7.0
13
Hz, 1 H), 8.49 (br, 1 H, NH); C NMR (CDCl3, 100 MHz) δ 63.56, 112.48, 119.75,
120.17, 120.74, 125.75, 126.11, 126.31, 126.58, 126.90, 128.87, 131.06, 134.09,
143.48, 147.20, 156.88, 165.32; IR (KBr) 3263 (br, NH), 1732 (s, C=O), 1643, 1481,
779 cm–1; EIMS m/z: 295 (M+, 42), 153 (14), 143 (65), 115 (31), 95 (100); HRMS
Calcd for C17H13NO4: 295.0845; Found: 295.0838.
1
3-Fluoro-2-oxo-2-(phenylamino)ethyl ester (8): yellow liquid; H NMR (CDCl3,
400 MHz) δ 4.93 (s, 2 H, CH2), 7.14 (dd, J = 14.4, 7.2 Hz, 1 H, ArH), 7.32 (dd, J =
15.6, 7.8 Hz, 3 H, ArH), 7.45–7.52 (m, 3 H, ArH), 7.76 (dd, J = 8.8, 4.4 Hz, 1 H,
21

ACCEPTED MANUSCRIPT
ArH), 7.89 (d, J = 7.60 Hz, 1 H, ArH); 13C NMR (CDCl3, 100 MHz) δ 63.90, 116.76
(d, J = 23.0 Hz), 120.29 (2 × CH), 121.03 (d, J = 21.0 Hz), 125.13, 125.58, 129.14 (2
× CH), 130.48, 136.55, 161.40, 162.81 (d, J = 256.5 Hz),163.87, 164.80; IR (KBr)
3439 (br, NH), 1722 (s, C=O), 1625, 1591, 734 cm–1; EIMS m/z: 273 (M+, 19), 186
(10), 181 (17), 123 (100), 95 (34), 93 (44), 77 (10), 70 (16); HRMS Calcd for
C15H12FNO3: 273.0801; Found: 273.0809.
1
4-Fluoro-2-oxo-2-(phenylamino)ethyl ester (9): white solid; mp 134–136 °C; H
NMR (CDCl3, 500 MHz) δ 4.88 (s, 2 H, CH2), 7.10–7.14 (m, 3 H, ArH), 7.29 (dd, J =
8.1, 15.8 Hz, 2 H, ArH), 7.49 (d, J = 8.0 Hz, 2 H, ArH), 8.04 (br, 1 H, NH), 8.07–8.10
(m, 2 H, ArH); 13C NMR (CDCl3, 125 MHz) δ 63.64, 115.50 (d, J = 21.3 Hz), 115.88
(d, J = 22.5 Hz), 120.32(2 × CH), 124.99, 125.06 (d, J = 2.5 Hz), 129.01 (2 × CH),
132.43 (d, J = 8.8 Hz), 132.60 (d, J = 10.0 Hz), 136.67, 165.21, 166.17 (d, J = 253.7
Hz); IR (KBr) 3439 (br, NH), 1730 (s, C=O), 1631, 1554, 756 cm–1; EIMS m/z: 273
(M+, 5), 228 (74), 216 (14), 186 (78) ,123 (40), 95 (10), 70 (100), 55 (13); HRMS
Calcd for C15H12FNO3: 273.0801; Found: 273.0804.
5-Isoxazole-2-oxo-2-(phenylamino)ethyl ester (10): yellow solid; mp 120–122 °C;
1H NMR (CDCl3, 400 MHz) δ 4.95 (s, 2 H, CH2), 7.12–7.19 (m, 2 H, ArH+
isoazole-H), 7.34 (dd, J = 15.6, 7.8 Hz, 2 H, ArH), 7.53 (d, J = 7.6 Hz, 2 H, ArH),
7.90 (br, 1 H, NH), 8.43 (d, J = 1.6 Hz, 1 H, isoazole-H); 13C NMR (CDCl3, 100 MHz)
δ 63.93, 110.32, 120.37 (2 × CH), 125.29, 129.17, 129.28, 136.39, 150.91, 154.89,
158.70, 163.63; IR (KBr) 3419 (br, NH), 1726 (s, C=O), 1627, 1552, 711 cm–1; EIMS
m/z: 246 (M+, 41), 188 (8), 154 (6), 126 (9), 120 (12), 96 (49), 93 (100), 77 (21), 65
(12); HRMS Calcd for C12H10N2O4: 246.0641; Found: 246.0633.
1-Adamantyl-2-oxo-(phenylamino)ethyl ester (11): yellow liquid; 1H NMR (CDCl3,
400 MHz) δ 1.64–1.76 (m, 5 H, ArH), 1.96–2.13 (m, 10 H, ArH), 4.64 (s, 2 H, CH2),
7.10 (dd, J = 14.4, 7.2 Hz, 1 H, ArH), 7.30 (dd, J = 15.6, 7.8 Hz, 2 H, ArH), 7.48 (d, J
22

ACCEPTED MANUSCRIPT
= 7.6 Hz, 2 H, ArH), 7.86 (br, 1 H, NH); 13C NMR (CDCl3, 100 MHz) δ 27.69, 27.74
(2 × CH), 36.22 (2 × CH), 36.32, 38.53, 38.75 (2 × CH), 40.70, 62.71, 119.96 (2 ×
CH), 124.79, 129.00 (2 × CH), 136.70, 165.44, 175.93; IR (KBr) 3321 (br, NH), 2908,
2850, 1732 (s, C=O), 1697, 1546, 752 cm–1; EIMS m/z: 313 (M+, 12), 221 (17), 135
(100), 93 (41), 79 (11); HRMS Calcd for C19H23NO3: 313.1678; Found: 313.1670.
8-Quinolinesulfonyl-2-oxo-(phenylamino) ethyl ester (12a): yellow solid; mp
137–139 °C; 1H NMR (CDCl3, 400 MHz) δ 4.94 (s, 2 H, CH2), 7.15 (dd, J = 15.2, 7.6
Hz, 1 H, ArH), 7.31 (dd, J = 15.6, 7.8 Hz, 2 H, ArH), 7.44 (d, J = 8.0 Hz, 2 H, ArH),
7.55 (dd, J = 12.4, 8.2 Hz, 1 H), 7.74 (dd, J = 15.6, 5.4 Hz, 1 H), 8.18 (d, J = 8.0 Hz,
1 H), 8.30 (dd, J = 9.60, 8.40 Hz, 1 H), 8.59 (d, J = 7.2 Hz, 1 H), 9.03 (dd, J = 5.60,
4.20 Hz, 1 H), 9.35 (br, 1 H, NH); 13C NMR (CDCl3, 100 MHz) δ 66.38, 121.14 (3 ×
CH), 122.75, 125.20, 125.64, 129.02 (2 × CH), 129.22, 132.15, 133.94, 135.64,
136.81, 137.23, 152.17, 164.31; IR (KBr) 3456 (br, NH), 1793, 1629, 1512, 709 cm–1;
EIMS m/z: 342 (M+, 7), 193 (8), 129 (100), 101 (6), 92 (3), 77 (8), 65 (4); HRMS
Calcd for C17H14N2O4S: 342.0674; Found: 342.0670.
8-Quinolinesulfonyl-2-oxo-[(2-fluorophenyl)amino]ethyl ester (12b): yellow solid;
mp 128–130 °C; 1H NMR (CDCl3, 400 MHz) δ 5.03 (s, 2 H, CH2), 7.02–7.10 (m, 3 H,
ArH), 7.52 (dd, J = 12.4, 8.2 Hz, 1 H), 7.70 (dd, J = 15.6, 7.8 Hz, 1 H), 7.96–8.00 (m,
1 H, ArH), 8.16 (dd, J = 8.8, 8.0 Hz, 1 H), 8.27 (dd, J = 10.0, 8.4 Hz, 1 H), 8.58 (dd, J
13
= 8.4, 7.4 Hz, 1 H), 9.00 (d, J = 2.8 Hz, 2 H); C NMR (CDCl3, 100 MHz) δ 66.95,
115.25 (d, J = 19.0 Hz), 122.69, 123.43, 124.50 (d, J = 8.9 Hz), 125.50 (d, J = 33.0
Hz, 1C), 125.83, 125.90, 129.14, 132.55, 133.78, 135.53, 136.93, 143.68, 147.43 (d, J
= 203.7 Hz), 152.28, 164.54; IR (KBr) 3286 (br, NH), 1689, 1535, 1454, 756 cm–1;
EIMS m/z: 360 (M+, 6), 129 (100), 110 (3), 101 (5), 83 (5), 77 (3); HRMS Calcd for
C17H13FN2O4S: 360.0580; Found: 360.0577.
8-Quinolinesulfonyl-2-oxo-[(3-fluorophenyl)amino]ethyl ester (12c): yellow liquid;
23

ACCEPTED MANUSCRIPT
1H NMR (CDCl3, 500 MHz) δ 4.95 (s, 2 H, CH2), 6.80–6.84 (m, 1 H, ArH), 7.13 (dd,
J = 1.1, 9.2 Hz, 1 H, ArH), 7.22–7.26 (m, 1 H, ArH), 7.34–7.37 (m, 1 H, ArH), 7.56
(dd, J = 4.3, 12.6 Hz, 1 H), 7.69–7.72 (m, 1 H), 8.18 (dd, J = 1.3, 9.5 Hz, 1 H), 8.30
(dd, J = 1.7, 10.0 Hz, 1 H), 8.56 (dd, J = 1.4, 8.7 Hz, 1 H), 9.02 (dd, J = 1.7, 6.1 Hz, 1
13
H), 9.47 (s, 1 H, NH); C NMR (CDCl3, 125 MHz) δ 66.18, 108.45 (d, J = 25.0 Hz
1C), 111.84 (d, J = 21.3 Hz, 1C), 116.25, 122.74, 125.61, 129.19, 130.07 (d, J = 8.8
Hz ,1C), 131.98, 133.86, 135.70, 137.31, 138.38 (d, J = 10.0 Hz, 1C), 143.56, 152.04,
162.83 (d, J = 243.8 Hz, 1C), 164.45; IR (KBr) 3456 (br, NH), 1678, 1612, 1176, 786
cm–1; EIMS m/z: 360 (M+, 8), 193 (7), 129 (100), 110 (3), 101 (6), 95 (4), 83 (4);
HRMS Calcd for C17H13FN2O4S: 360.0580; Found: 360.0585.
8-Quinolinesulfonyl-2-oxo-[(4-fluorophenyl)amino]ethyl ester (12d): white solid;
mp 125–127 °C; 1H NMR (CDCl3, 400 MHz) δ 4.95 (s, 2 H, CH2), 7.01 (dd, J = 16.4,
10.4 Hz, 2 H, ArH), 7.41 (dd, J = 13.2, 8.6 Hz, 2 H, ArH), 7.56 (dd, J = 12.8, 8.4 Hz,
1 H), 7.73 (dd, J = 15.6, 7.8 Hz, 1 H), 8.19 (d, J = 8.0 Hz, 1 H), 8.31 (d, J = 8.4 Hz, 1
H), 8.59 (d, J = 7.6 Hz, 1 H), 8.97 (d, J = 4.0 Hz, 1 H), 9.55 (br, 1 H, NH); 13C NMR
(CDCl3, 100 MHz) δ 66.09, 115.75 (d, J = 8.4 Hz, 2 × CH), 115.86, 121.99, 122.74,
123.17 (d, J = 8.0 Hz), 125.68, 129.22, 131.99, 132.83, 133.96, 135.70, 137.35,
143.59, 152.02, 159.62 (d, J = 247.5 Hz, 1C), 164.49; IR (KBr) 3286 (br, NH), 1681,
1508, 1176, 833 cm–1; EIMS m/z: 360 (M+, 5), 193(4), 93 (100), 129 (100), 109 (9),
101 (5), 83 (6); HRMS Calcd for C17H13FN2O4S: 360.0580; Found: 360.0585.
8-Quinolinesulfonyl-2-oxo-[(4-methoxyphenyl)amino]ethyl ester (12e): yellow
solid; mp 135–137 °C; 1H NMR (CDCl3, 400 MHz) δ 3.77 (s, 3 H, OCH3), 4.93 (s, 2
H, CH2), 6.84 (d, J = 8.8 Hz, 2 H, ArH), 7.31 (d, J = 8.8 Hz, 2 H, ArH), 7.54 (dd, J =
12.4, 8.2 Hz, 1 H), 7.71 (dd, J = 15.6, 7.8 Hz, 1 H), 8.18 (d, J = 8.0 Hz, 1 H), 8.30 (dd,
J = 9.6, 8.4 Hz, 1 H), 8.58 (d, J = 7.2 Hz, 1 H), 8.98 (dd, J = 5.6, 4.2 Hz, 1 H), 9.40
(br, 1 H, NH); 13C NMR (CDCl3, 100 MHz) δ 55.44, 66.24, 114.19 (2 × CH), 122.71,
24

ACCEPTED MANUSCRIPT
123.10 (2 × CH), 125.62, 129.19, 129.72, 132.09, 133.92, 135.61, 137.24, 143.61,
152.09, 157.10, 164.25; IR (KBr) 3282 (br, NH), 1728, 1681, 1512, 190 cm–1; EIMS
m/z: 372 (M+, 8), 333 (7), 135 (18), 129 (100), 122 (13), 109 (9), 101 (5); HRMS
Calcd for C18H16N2O5S: 372.0780; Found: 372.0789.
1-Naphthalenesulfonyl-2-oxo-[(3-fluorophenyl)amino]ethyl ester (13a): yellow
solid; mp 121–123 °C; 1H NMR (CDCl3, 400 MHz) δ 4.52 (s, 2 H, CH2), 6.81 (dd, J
= 10.8, 5.4 Hz, 1 H, ArH), 6.94 (d, J = 8.0 Hz, 1 H, ArH), 7.18–7.25 (m, 2 H, ArH),
7.58–7.69 (m, 2 H), 7.78 (dd, J = 15.2, 11.2 Hz, 1 H, ArH), 7.87 (br, 1 H, NH), 7.99
(d, J = 8.4 Hz, 1 H), 8.18 (d, J = 8.0 Hz, 1 H), 8.33 (d, J = 7.6 Hz, 1 H), 8.65 (d, J =
13
8.4 Hz, 1 H); C NMR (CDCl3, 100 MHz) δ 67.15, 107.46, 107.73, 111.97 (d, J =
21.0 Hz), 115.30, 124.02, 124.28, 127.67, 128.16, 129.47, 129.70, 130.10, 130.19,
131.37, 134.32, 136.39, 162.51 (d, J = 242.3 Hz), 163.09; IR (KBr) 3302 (br, NH),
1685 (s, C=O), 1612, 1180, 771 cm–1; EIMS m/z: 359 (M+, 48), 208 (14), 191 (30),
127 (100), 111 (16), 83 (8); HRMS Calcd for C18H14FNO4S: 359.0628; Found:
359.0632.
1-Naphthalenesulfonyl-2-oxo-[(4-methoxyphenyl)amino]ethyl ester (13b): yellow
solid; mp 136–138 °C; 1H NMR (CDCl3, 400 MHz) δ 3.75 (s, 3 H, OCH3), 4.49 (s, 2
H, CH2), 6.79 (d, J = 8.8 Hz, 2 H, ArH), 7.20 (d, J = 9.2 Hz, 2 H, ArH), 7.57–7.67 (m,
2 H), 7.73–7.77 (m, 2 H), 7.98 (d, J = 8.4 Hz, 1 H), 8.17 (d, J = 8.4 Hz, 1 H), 8.32 (d,
13
J = 7.2 Hz, 1 H), 8.65 (d, J = 8.8 Hz, 1 H); C NMR (CDCl3, 100 MHz) δ 55.43,
67.21, 114.15 (2 × CH), 122.01 (2 × CH), 124.10, 124.24, 127.59, 128.19, 129.14,
129.35, 129.38, 129.77, 131.33, 134.28, 136.26, 157.02, 162.79; IR (KBr) 3302 (br,
NH), 1674 (s, C=O), 1512, 1176, 829 cm–1; EIMS m/z: 371 (M+, 77), 220 (74), 156
(43), 127 (54), 84 (30), 78 (45), 57 (100), 55 (34); HRMS Calcd for C19H17NO5S:
371.0827; Found: 371.0826.
25

ACCEPTED MANUSCRIPT
In vitro Dipeptidyl peptidase 4 assay
Preparation of the DPP-4 enzyme
Human recombinant dipeptidyl
peptidase
IV
and
its
substrate,
Gly-Pro-7-amido-4-methylcoumarin (GP- AMC) and its natural inhibitor, Ile-Pro-Ile
tripeptide (diprotin A), were purchased from R&D Systems (Minneapolis, MN). All
other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).
Enzyme-based assay of Dipeptidyl Peptidase IV
The reaction was carried out at pH 7.5 in the presence of 50mM HEPES, 140mM
NaCl, 10mM KCl, and 15 mM of G-P-AMC (the Km for substrate GP- AMC is about
15 mM). The reaction volume is 200 ml with 20 ng of recombinant DPP-IV at 251C.
The reaction rate was monitored by a fluorometer with 380-nm excitation and 460-nm
emission filters, and determined by the linear slope of the fluorescence accumulation.
For inhibition kinetic, substrate and inhibitor concentrations varied and encompassed
the Km and Ki values, respectively, and the data were analyzed by the EZ-FIT
program, which uses a nonlinear regression method to obtain kinetic parameters
.
The reaction was carried out at pH 7.5 in the presence of 50mM HEPES, 140mM
NaCl, 10mM KCl, and 15 mM of G-P-AMC (the Km for substrate GP- AMC is about
15 mM). The reaction volume is 200 ml with 20 ng of recombinant DPP-4 at 251C.
The reaction rate was monitored by a fluorometer with 380-nm excitation and 460-nm
emission filters, and determined by the linear slope of the fluorescence accumulation.
For inhibition kinetic, substrate and inhibitor concentrations varied and encompassed
the Km and Ki values, respectively, and the data were analyzed by the EZ-FIT
program, which uses a nonlinear regression method to obtain kinetic parameters.26
Acknowledgment
26

ACCEPTED MANUSCRIPT
We are grateful to Tsuzuki Institute for Traditional Medicine, and the National
Science Council Taiwan for financial support (NSC-102-2113-M-039-003).
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