Synthesis and evaluation of new tyrosyl-tRNA synthetase inhibitors as antibacterial agents based on a N2-(arylacetyl)glycinanilide scaffold

Article abstract of DOI:10.1016/j.ejmech.2015.08.025

Tyrosyl-tRNA synthetase (TyrRS), an essential enzyme in bacterial protein biosynthesis, is an attractive therapeutic target for finding novel antibacterial agents, and a series of N2-(arylacetyl)glycinanilides has been herein synthesized and identified as TyrRS inhibitors. These efforts yielded several compounds, with IC₅₀ in the low micromolar range against TyrRS from Staphylococcus aureus. Out of the obtained compounds, 3ap is the most active and exhibits excellent activity against both Gram-positive (S. aureus) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacterial strains. In comparison with the parent scaffold 3-arylfuran-2(5H)-one, N2-(arylacetyl)glycinanilide significantly improved the potency against Gram-negative bacterial strains, indicating that this scaffold offers a significant potential for developing new antibacterial drugs.

Full text of DOI:10.1016/j.ejmech.2015.08.025

European Journal of Medicinal Chemistry 102 (2015) 631e638
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and evaluation of new tyrosyl-tRNA synthetase inhibitors as
antibacterial agents based on a N2-(arylacetyl)glycinanilide scaffold
, b,
Zhu-Ping Xiao a *, Wei Wei a, Peng-Fei Wang b, Wei-Kang Shi a, Na Zhu a, Me-Qun Xie a,
Yu-Wen Sun a, Ling-Xia Li a, Yong-Xiang Xie a, Liang-Song Zhu a, Nian Tang a,
,
, b, **
Hui Ouyang a ***, Xian-Hui Li a, Guang-Cheng Wang a, Hai-Liang Zhu a
a College of Chemistry and Chemical Engineering, Jishou University, Jishou 416000, PR China
b State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Tyrosyl-tRNA synthetase (TyrRS), an essential enzyme in bacterial protein biosynthesis, is an attractive
therapeutic target for finding novel antibacterial agents, and a series of N2-(arylacetyl)glycinanilides has
been herein synthesized and identified as TyrRS inhibitors. These efforts yielded several compounds,
with IC50 in the low micromolar range against TyrRS from Staphylococcus aureus. Out of the obtained
compounds, 3ap is the most active and exhibits excellent activity against both Gram-positive (S. aureus)
and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacterial strains. In comparison with
the parent scaffold 3-arylfuran-2(5H)-one, N2-(arylacetyl)glycinanilide significantly improved the po-
tency against Gram-negative bacterial strains, indicating that this scaffold offers a significant potential
for developing new antibacterial drugs.
Received 15 June 2015
Received in revised form
9 August 2015
Accepted 11 August 2015
Available online 14 August 2015
Keywords:
N2-(arylacetyl)glycinanilide
TyrRS inhibitor
Antibacterial agent
Molecular docking
Structureeactivity relationship
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
Structure-based drug design is now widely applied in most
stages of the drug development process, from initial hit identifi-
The aminoacyl-tRNA synthetases (aaRS) belong to a group of
enzymes, which play a central role in the correct translation of
genetic information into proteins [1]. Each aaRS catalyzes a specific
amino acid to attach the cognate tRNA molecule in the presence of
ATP, yielding an aminoacyl-tRNA [2]. Therefore, inhibition of any
enzyme in the cell will lead to protein synthesis inhibition and cell
growth arrest. Furthermore, the topology of the ATP binding
domain and the functions of the human aaRSs differ from those of
bacteria, indicating that the binding site for ATP may provide an
opportunity to selectively inhibit aaRSs in bacteria [3,4]. These
enzymes are therefore considered as attractive targets for anti-
bacterial agents [5,6]. Most aaRS inhibitors bind to the ATP binding
site are analogs of ATP, or aminoacyl-adenylate intermediates, such
as Microcin C, SB-236996 and Leu-AMS [4,7e9].
cation to lead optimization. Our recent efforts have focused on the
development of novel antimicrobial agents, and we applied this
approach to find 3-arylfuran-2(5H)-ones to target tyrosyl-tRNA
synthetase (TyrRS). Since 2011, several new chemical entities
have been determined as potent antibacterial agents in our group
[6,10e14]. Many analogs of compound 1 (Scheme 1) showed good
inhibitory activity against TyrRS and good potency against intact
bacterial cells [6]. Compound 1 could be therefore considered to
have a potential for the development of antibacterials devoid of
cross-resistance with present drugs. To learn more about the
structure-antibacterial activity relationships and to develop com-
pounds as potential antibacterial agents, we undertook an addi-
tional design for improving the present scaffold (1) and for
screening active compounds in animal infection models. Specif-
ically, the furan-2(5H)-one moiety of 1 was bioisosterically replaced
with an acetoxyacetyl group to give a scaffold (2). The ester func-
tional group in 2 was subsequently substituted with an amide
group to obtain 3 for acquiring more stability in physiological
conditions (Scheme 1). On one hand, the acetylglycinamide moiety
as a pharmacophore is found in a large quantities of natural and
synthetic compounds, which possess useful biological activities
* Corresponding author. College of Chemistry and Chemical Engineering, Jishou
University, Jishou 416000, PR China.
** Corresponding author. College of Chemistry and Chemical Engineering, Jishou
University, Jishou 416000, PR China.
*** Corresponding author.
E-mail addresses: xiaozhuping2005@163.com (Z.-P. Xiao), oyhmail@163.com
(H. Ouyang), zhuhl@nju.edu.cn (H.-L. Zhu).
http://dx.doi.org/10.1016/j.ejmech.2015.08.025
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

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Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 102 (2015) 631e638
preserves about equal activity. Modification of the 2- or 3-position
of this phenyl ring seems less fruitful, and further modification on
the phenylacetyl moiety likewise seems less rewarding. Only the 3-
chloro derivative 3ap (IC50 ¼ 0.70 0.03
mM) showed a slight in-
crease in activity, being the most active among all synthesized
compounds. Generally speaking, our efforts of modification fur-
nished several good TyrRS inhibitors with submicromolar potency,
suggesting an opportunity of this scaffold for further exploration.
2.3. Antibacterial activity
Encouraged by the results of TyrRS inhibitory assays, all syn-
thetic compounds were evaluated for their antimicrobial activities
against two Gram-positive bacterial strains (S. aureus ATCC 6538, S.
aureus ATCC 25923) and three Gram-negative bacterial strains
(Escherichia coli ATCC 8739, E. coli ATCC 35218, Pseudomonas aer-
uginosa ATCC 9027), and the results are presented in Table 2. In
general, the antibacterial potency is totally dependent on the
inhibitory activity against TyrRS, and the obtained compounds
showed more sensitivity to Gram-positive bacterial strains than
Gram-negative bacterial strains. Four compounds (3ad, 3ap, 3as
and 3ax) were found to exhibit good activity against both Gram-
positive and Gram-negative bacterial strains with MIC values
Scheme 1. Design of the N2-(arylacetyl)glycinanilide (3).
including antibacterial, antifungal, antitumorigenic and anti-
Hepatitis C virus replication activities [15e19]. On the other hand,
in terms of the energy-minimized conformation and geometrical
dimension, scaffold 3 shows very similarity to the well known
TyrRS inhibitor, SB-284485 (Scheme 1), which indicates that ana-
logs of 3 will display potent inhibition against TyrRS. Based on this
conception, a series of N2-(arylacetyl)glycinanilides (3aa-3be)
were synthesized and evaluated as TyrRS inhibitors. The results
showed that some of these synthesized compounds exhibited
excellent antibacterial activities.
lower than 20
showed the highest potency of antibacterial activity with MIC of
10.1 M against P. aeruginosa ATCC 9027 and 1.84 M against S.
mM, and the most active enzyme inhibitor 3ap also
m
m
2. Results and discussion
aureus ATCC 6538, showing a compared potency to that of the
positive control kanamycin and a 3.9-fold more potency than the
marketed drug penicillin G, respectively.
2.1. Chemistry
Thirty-one N2-(arylacetyl)glycinanilides were designed and
synthesized by the route outlined in Scheme 2. Briefly, substituted
phenylacetic acids 4ael were subjected to condensation with
methyl glycinate hydrochloride in the presence of TBTU to yield the
corresponding phenylacetamides 5ael, which were then further
amidated in toluene with an appropriated aniline to give the
desired products 3aaebg (Scheme 2).
2.4. Molecular docking
To interpret the enzyme inhibitory activities at the molecular
level, we performed molecular docking studies. The most potent
inhibitor 3ap was docked into the active site of TyrRS based on the
binding model of SB-239629-TyrRS complex structure (1jij.pdb)
[21], where SB-239629 is a typical TyrRS inhibitor. The docking
model obtained in this study indicates that 3ap docks favorably into
the active site of TyrRS (Figs. 1 and 2). Upon analyzing the docking
studies, the binding affinity could be associated to hydrogen-
bonding as well as hydrophobic interactions (DS ¼ ꢀ8.63 kcal/
mol). As shown in Fig. 2, 4-nitrophenyl moiety is of primary
importance for its hydrogen-bonding interactions network formed
between nitro group and two amino-acid residues Tyr-170 and Gln-
174. Specifically, NH2 of Gln-174 as the donor coordinates nitro
group in a quadridentate fashion with H/O bond length in the
range of 1.847e3.364 Å. By contrast, a relatively weak contribution
to the binding affinity is observed between OH of Tyr-170 and nitro
group, which establishes a hydrogen-bond with longer distance
(3.918 Å). These findings indicated that the removal of nitro group
at position 4 (3ai, DS ¼ ꢀ5.87 kcal/mol) or replacement with a weak
2.2. Inhibitory activities of N2-(arylacetyl)glycinanilides against
tyrosyl-tRNA synthetase from Staphylococcus aureus
In the process of discovering anti-infection agents, we prepared
and evaluated a series of 3-arylfuran-2(5H)-one derivatives as
tyrosyl-tRNA synthetase (TyrRS) inhibitors. The chain analogs, N2-
(arylacetyl)glycinanilides (3aaebg), were synthesized for a further
understanding of structureeactivity relationships (SAR), and all
were evaluated for the inhibitory activity against TyrRS. As shown
in Table 1, the compound with 4-nitro substituent (3ad) in the
anilino moiety (Scheme 1) affords a 22-fold improvement over the
unsubstituted analog (3ai), while 4-bromo, 4-methyl or 4-methoxy
group significantly reduces the activity, and the 4-chloro analog
Scheme 2. Preparation of compounds 3aaebe.

Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 102 (2015) 631e638
633
Table 1
In vitro inhibitory activity data of synthesized compounds against S. aureus TyrRS.
Compound
Structure
cLog Pb
IC50
(
m
M)
DS (kcal/mol)
R1
EP [20]
R2
EP [20]
3aa
3ab
3ac
3ad
3ae
3af
3ag
3ah
3ai
H
H
H
H
H
H
H
H
2.10
2.10
2.10
2.10
2.10
2.10
2.10
2.10
2.10
2.10
2.10
3.16
3.16
3.16
3.16
3.16
3.16
3.16
3.16
2.96
3.98
3.98
3.98
3.98
2.86a
2.86a
2.82a
2.82a
2.82a
2.82a
2.82a
3.16
2.29a
4-Br
2.96
3.04
1.99
1.99
1.85
2.01
2.55
2.88
3.69
1.91
2.03
2.88
4.40
2.70
2.70
3.75
2.56
2.70
3.75
2.56
3.90
2.13
2.00
2.13
2.00
1.65
ꢀ1.60
1.32
1.19
1.32
1.19
1.27
3.13
ꢀ0.76
50.6 4.1
82.0 5.0
47.8 4.3
0.87 0.05
162.1 8.1
13.1 0.9
16.6 0.8
132.8 13.5
19.3 2.0
205 27
ꢀ4.72
ꢀ4.24
ꢀ5.07
ꢀ8.35
ꢀ3.23
ꢀ6.51
ꢀ6.19
ꢀ3.61
ꢀ5.87
ꢀ2.72
ꢀ3.54
ꢀ2.49
ꢀ4.42
ꢀ4.52
ꢀ6.62
ꢀ8.63
ꢀ2.95
ꢀ4.32
ꢀ7.72
ꢀ4.63
ꢀ3.83
ꢀ7.49
ꢀ4.06
ꢀ8.02
ꢀ1.30
ꢀ4.17
ꢀ1.95
ꢀ6.57
ꢀ2.09
ꢀ7.08
ꢀ1.72
ꢀ5.74
ꢀ4.95
4-OMe
3-NO2
4-NO2
3,5-Dimethoxyl
3,5-Difluoro
4-Cl
3,5-Dichloro
H
2-Cl
2.86a
3.31a
3.31a
2.86a
3.16
3.16
3.16
2.10
3.16
H
H
H
3aj
3ak
3al
3-Cl
3.16
144.0 11.5
254 23
4-Cl
4-Cl
3-Cl
3-Cl
3-Cl
2-Cl
2-Cl
2-Cl
3-Br
4-F
3,5-Dichloro
4-OMe
4-OMe
4-Br
4-NO2
4-OMe
4-Br
3.16
3am
3an
3ao
3ap
3aq
3ar
3as
3at
3au
3av
3aw
3ax
3ay
3az
3ba
3bb
3bc
3bd
3be
3bf
3bg
2.86a
2.86a
2.96
70.1 4.9
66.5 4.7
8.36 0.74
0.70 0.03
189.3 15.2
77.8 7.7
2.52 0.15
58.4 3.1
112.4 12.4
5.25 0.26
92.7 8.4
1.14 0.06
556 56
88.5 6.2
319 26
10.2 0.6
279 25
6.41 0.26
478 57
21.4 2.3
44.6 3.7
3.31a
2.86a
2.96
4-NO2
4-Br
3.31a
2.96
4-OMe
4-NO2
4-OMe
4-NO2
4-OMe
4-NO2
4-OMe
4-NO2
4-OMe
4-NO2
4-OMe
4-Me
2.86a
3.31a
2.86a
3.31a
2.86a
3.31a
2.86a
3.31a
2.86a
3.31a
2.86a
2.29a
3.31a
4-F
3-F
3-F
3,4-OMe
3,4-OMe
4-OH
4-OH
3-OH
3-OH
2-OH
3-Cl
4-Me
4-NO2
EP: electronegativity parameter.
DS: docking score.
a
Data are from the Ref. [20].
Data are calculated by using ChemDraw 11.0.
b
hydrogen bond acceptor results in a reduced binding affinity (3aa,
DS ¼ ꢀ4.72 kcal/mol; 3ab, DS ¼ ꢀ4.24 kcal/mol and 3ag,
DS ¼ ꢀ6.19 kcal/mol), and is consistent with those observed in SAR
study.
in a considerable decrease in potency. This docking model also
revealed that change of 3-Cl to 4-Cl may cause a steric hindrance to
the residue Val-224, leading reduction of binding affinity. Similarly,
introduction of 3,4-dimethoxyl group (3ay, DS ¼ ꢀ1.30 kcal/mol
and 3az, DS ¼ ꢀ4.17 kcal/mol) may cause steric hindrances to both
Leu-52 and Val-224, resulting dramatic decrease in potency. For a
definite conclusion, modification at the 3-position of phenylaceto
moiety with a hydrophobic group having suitable steric hindrance
would improve the antibacterial activity.
Acetylglycinamide moiety, the linkage between two benzene
rings, also furnishes a considerable contribution to the binding
affinity, which packs in a hydrophilic cleft, forming three strong
hydrogen-bonding interactions together with a relatively weak
one. The aniline moiety by using NH group bonds with the residue
His-50, and the distance between the NH group and the nitrogen
atom of the imidazole ring in His-50 is 3.624 Å. With the respect to
aminoaceto group, the carbonyl group forms a typical hydrogen
bond with the NH group of Gly-193 (O/H, 2.031 Å), while the NH
group of aminoaceto moiety has no significant interactions to be
considered as a contribution for binding. The carbonyl group of the
phenylaceto moiety as an acceptor establishes two hydrogen bonds
with His-50 and His-47, which confirmed that this carbonyl group
is also crucial for binding.
The 3-chlorophenyl group of 3ap drops completely into a hy-
drophobic pocket built by Leu-14, Leu-52, Pro-53, Ile-221, Leu-223,
Val-224, Phe-232 and Phe-217, indicating a detriment of a hydro-
philic substituent on this ring. Therefore, the substitution of a hy-
droxyl group (3baebe, DS from ꢀ7.08 to ꢀ1.72 kcal/mol) for a
halogen atom (3aleax, DS from ꢀ8.63 to ꢀ2.49 kcal/mol) resulted
3. Conclusions
Given the global health threat posed by multiantibiotic resistant
bacteria, there is considerable interest in the discovery of antibac-
terial agents with novel skeleton or/and antibacterial mechanism.
Derivatives of N2-(arylacetyl)glycinanilide, chain analogs of 3-
arylfuran-2(5H)-ones, were determined as TyrRS inhibitors,
showing promise as antibacterial agents with novel mechanism in
terms of clinical drugs. The bioactivity assays demonstrated that
some N2-(arylacetyl)glycinanilides showed good potency against
both extracted enzyme and intact cells. In comparison with the
parent scaffold 3-arylfuran-2(5H)-one, N2-(arylacetyl)glycinanilide
not only maintained the potency against Gram-positive bacterial
strains, but also significantly improved the potency against Gram-

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Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 102 (2015) 631e638
Table 2
assays and molecular studies revealed that 4-nitro group of aniline
moiety and a hydrophobic group at 3-position with a suitable steric
hindrance of phenylaceto moiety are quite important for improving
activity. Consequently, the N2-(arylacetyl)glycinanilide scaffold
offers a significant potential for the discovery of a new class of
antibacterial drugs, and a further chemical pharmacomodulation is
undergoing.
Inhibitory activity (MIC) of the synthetic compounds against microbes.
Compound
MIC (
A
mM)
B
C
D
E
3aa
3ab
3ac
3ad
3ae
3af
3ag
3ah
3ai
3aj
3ak
3al
3am
3an
3ao
3ap
3aq
3ar
113.2
>160
103.2
2.81
>160
40.5
49.7
>160
52.7
>160
>160
>160
>160
148.2
20.9
1.84
>160
69.7
6.51
97.2
>160
14.1
>160
3.14
>160
>160
>160
28.6
>160
15.2
>160
64.9
80.7
>160
>160
156.2
3.67
>160
47.7
69.2
>160
73.5
>160
>160
>160
>160
148.2
24.9
>160
>160
>160
11.2
>160
>160
>160
15.8
>160
>160
>160
18.2
>160
120.1
123.2
>160
133.9
>160
>160
>160
>160
>160
62.4
>160
>160
158.9
>160
>160
>160
>160
>160
>160
>160
83.4
>160
148.3
>160
>160
>160
>160
>160
>160
>160
>160
96.1
4. Experiments
4.1. Preparation of the tyrosyl-tRNA synthetase and enzyme assay
S. aureus tyrosyl-tRNA synthetase was over-expressed in E. coli
and purified to near homogeneity (~98% as judged by SDS-PAGE)
using standard purification procedures [22]. Tyrosyl-tRNA synthe-
tase activity was measured by aminoacylation using modifications
to previously described methods [23]. The assays were performed
at 37 ꢁC in a mixture containing (final concentrations) 100 mM Tris/
Cl pH 7.9, 50 mM KC1, 16 mM MgCl2, 5 mM ATP, 3 mM DTT, 4 mg/ml
2.36
6.80
9.50
10.1
>160
103.4
8.41
>160
>160
17.0
>160
4.11
>160
>160
>160
34.1
>160
>160
13.5
>160
>160
42.3
>160
12.3
>160
>160
>160
86.9
>160
47.1
>160
155.4
>160
1.26
>160
>160
19.7
>160
>160
75.8
>160
>160
34.0
>160
>160
65.6
E. coli MRE600 tRNA (Roche) and 10
3,5-3H] tyrosine (PerkinElmer, Specific activity: 1.48e2.22 TBq/
mmol), 10 M carrier). Tyrosyl-tRNA synthetase (0.2 nM) was
mM L-tyrosine (0.3 mM L-[ring-
3as
3at
3au
3av
3aw
3ax
3ay
3az
3ba
3bb
3bc
3bd
3be
3bf
m
preincubated with a range of inhibitor concentrations for 10 min at
room temperature followed by the addition of pre-warmed
mixture at 37 ꢁC. After specific intervals, the reaction was termi-
nated by adding aliquots of the reaction mix into ice-cold 7% tri-
chloroacetic acid and harvesting onto 0.45 mm hydrophilic
Durapore filters (Millipore Multiscreen 96-well plates) and counted
by liquid scintillation. The rate of reaction in the experiments was
linear with respect to protein and time with less than 50% total
tRNA acylation. IC50s correspond to the concentration at which half
of the enzyme activity is inhibited by the compound. All com-
pounds were tested in triplicate, and the results were presented in
Table 1.
>160
19.6
>160
18.2
>160
>160
>160
143.8
>160
89.4
>160
>160
>160
4.35
>160
>160
>160
134.0
>160
72.0
>160
>160
>160
7.16
>160
18.4
>160
91.1
3bg
Kanamycin
132.1
e
e
Penicillin G
7.13
9.28
e
e
e
Note: A, S. aureus ATCC 6538; B, S. aureus ATCC 25923; C, E. coli ATCC 8739; D, E. coli
ATCC 35218; E, P. aeruginosa ATCC 9027.
4.2. Antimicrobial activity
The antibacterial activities of the synthesized compounds was
tested against two Gram-positive bacterial strains (S. aureus ATCC
6538, S. aureus ATCC 25923, penicillin G as positive control) and
three Gram-negative bacterial strains (E. coli ATCC 8739, E. coli
ATCC 35218, P. aeruginosa ATCC 9027, kanamycin as positive con-
trol) using LB medium. The MICs of the test compounds were
determined by a colorimetric method using the dye MTT [24,25]. A
negative bacterial strains. Compound 3ap was identified as the
most potent antibacterial agent with 3-fold more potency than
penicillin G against S. aureus ATCC 6538 (MIC50 ¼ 0.52
compared potency to kanamycin against P. aeruginosa ATCC 9027
(MIC50 ¼ 3.37 M), which deserves further investigation. Enzyme
mM) and
m
stock solution of the synthesized compound (1000 mg/ml) in DMSO
was prepared and graded quantities of the test compounds were
incorporated in specified quantity of sterilized liquid medium (50%
(v/v) of DMSO in PBS). A specified quantity of the medium con-
taining the test compound was poured into 96-well plates. Sus-
pension of the microorganism was prepared to contain
approximate 105 cfu/mL and applied to 96-well plates with serially
diluted compounds to be tested and incubated at 37 ꢁC for 24 h.
Fifty
Incubation was continued at room temperature for 4e5 h, and
100 L of 10% sodium dodecyl sulfate containing 5% isopropanol
mL of PBS containing 2 mg of MTT/mL was added to each well.
m
and 1 mol/L HCl was added to extract the dye. After 12 h of incu-
bation at room temperature, the optical density (OD) was measured
with a microplate reader at 550 nm. The observed MICs were
presented in Table 2.
4.3. Protocol of docking study
The automated docking studies were carried out using Auto-
Dock version 4.2. First, AutoGrid component of the program pre-
calculates a three-dimensional grid of interaction energies based
Fig. 1. Binding mode of compound 3ap with TyrRS. The enzyme is shown as surface;
while 3ap docked structures are shown as sticks.

Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 102 (2015) 631e638
635
Fig. 2. Binding mode of compound 3ap with TyrRS. For clarity, only interacting residues were labeled. Hydrogen bonding interactions are shown in dash. The figure was made using
PyMol.
on the macromolecular target using the AMBER force field. A grid
box of 46 ꢂ 58 ꢂ 48 Å size (x, y, z) with a spacing of 0.375 Å and grid
maps were created representing the catalytic active target site re-
gion where the native ligand was embedded. Then automated
docking studies were carried out to evaluate the binding free en-
ergy of the inhibitor within the macromolecules. The genetic al-
gorithm with local search (GA-LS) was chosen to search for the best
conformers. The parameters were set using the software ADT
(AutoDockTools package, version 1.5.4) on PC which is associated
with AutoDock 4.2. Default settings were used with an initial
population of 50 randomly placed individuals, a maximum number
of 7.5 ꢂ 106 energy evaluations, and a maximum number of
2.7 ꢂ 104 generations. A mutation rate of 0.02 and a crossover rate
of 0.8 were chosen. Results differing by less than 0.5 Å in positional
root-mean-square deviation (RMSD) were clustered together and
the results of the most favorable free energy of binding were
selected as the resultant complex structures.
was taken up in ethyl acetate. The organic phase was washed with
saturated aqueous solutions of sodium bicarbonate and sodium
chloride. The organic layer was dried over sodium sulfate. The
solvent was removed under reduced pressure and the crude
product was purified by chromatography using ethyl acetate/pe-
troleum as eluents (3:1 to 6:1), the pure product (5ael) was
therefore obtained.
4.4.2. General procedure for preparation of compounds 3aaebg
A mixture of a selected compound 5ael (0.5 mmol), an appro-
priately substituted aniline (0.6 mmol) and p-toluene sulphonic
acid (TsOH, 3.4 mg, 0.02 mmol) was heated to 90 ꢁC for 10 min. Five
milliliters toluene was then added and refluxed for 3e7 h. After
toluene was removed under reduced pressure, the residue was
purified by column chromatography on silica gel, eluting with
EtOAc/petroleum ether.
4.4.2.1. N-(4-Bromophenyl)-N2-(phenylacetyl)glycinamide
(3aa).
Colorless crystal, 57%, mp 228e230 ꢁC, 1H NMR (400 MHz, DMSO-
d6): 3.55 (s, 2H); 3.95 (d, J ¼ 5.2 Hz, 2H); 7.16e7.31 (m, 6H);
7.39e7.50 (m, 4H); 9.60 (s, 1H); EIMS m/z 346 (Mþ). Anal. Calcd for
4.4. Chemistry
All chemicals (reagent grade) used were purchased from Aldrich
(U.S.A) and Sinopharm Chemical Reagent Co., Ltd (China). Separa-
tion of the compounds by column chromatography was carried out
with silica gel 60 (200e300 mesh ASTM, E. Merck). The quantity of
silica gel used was 30e70 times the weight charged on the column.
Then, the eluates were monitored using TLC. Melting points (un-
corrected) were determined on a XT4 MP apparatus (Taike Corp.,
Beijing, China). ESI mass spectra were obtained on a Agilent 6400
mass spectrometer, and 1H NMR spectra were recorded on a Bruker
AV 300, 400 or 500 spectrometer at 25 ꢁC with TMS and solvent
signals allotted as internal standards. Chemical shifts were reported
C
16H15BrN2O2: C, 55.35; H, 4.35; Br, 23.01; N, 8.07; found: C, 55.44;
H, 4.35; Br, 22.98; N, 8.08.
4.4.2.2. N-(4-Methoxyphenyl)-N2-(phenylacetyl)glycinamide (3ab).
Colorless crystal, 49%, mp 187e188 ꢁC, 1H NMR (400 MHz, DMSO-
d6): 3.51 (s, 2H); 3.71 (s, 2H); 3.87 (d, J ¼ 5.6 Hz, 2H); 6.88 (d,
J ¼ 8.8 Hz, 2H); 7.20e7.31 (m, 5H); 7.49 (d, J ¼ 8.8 Hz, 2H); 8.41 (s,
1H); 9.89 (s, 1H); EIMS m/z 298 (Mþ). Anal. Calcd for C17H18N2O3: C,
68.44; H, 6.08; N, 9.39; found: C, 68.36; H, 6.07; N, 9.40.
in ppm (d). Elemental analyses were performed on a CHNeO-Rapid
instrument and were within 0.4% of the theoretical values.
4.4.2.3. N-(3-Nitrophenyl)-N2-(phenylacetyl)glycinamide
(3ac).
Colorless crystal, 52%, mp 192e193 ꢁC, 1H NMR (400 MHz, DMSO-
d6): 3.62 (s, 2H); 4.03 (s, 2H); 7.23e7.33 (m, 5H); 7.47 (t, J ¼ 7.8 Hz,
1H); 7.86e7.94 (m, 3H); 8.58 (s, 1H); 10.23 (s, 1H); EIMS m/z 313
(Mþ). Anal. Calcd for C16H15N3O4: C, 61.34; H, 4.83; N, 13.41; found:
C, 61.41; H, 4.83; N, 13.40.
4.4.1. General procedure for preparation of compounds 5
To a solution of a substituted phenylacetic acid (4ael) in
dichloromethane at room temperature, were successively added 3
equivalents of triethylamine, 1.2 equivalents of methyl glycinate
hydrochloride and 1.1 equivalents of 2-(1H-benzotriazol-1-yl)-
1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU). The reaction
mixture was well stirred overnight at room temperature. The sol-
vent was removed under reduced pressure and the crude material
4.4.2.4. N-(4-Nitrophenyl)-N2-(phenylacetyl)glycinamide
(3ad).
Light yellow crystal, 58%, mp 227e229 ꢁC, 1H NMR (400 MHz,
DMSO-d6): 3.52 (s, 2H); 3.96 (d, J ¼ 5.6 Hz, 2H); 7.22e7.30 (m, 5H);
7.83 (d, J ¼ 9.1 Hz, 2H); 8.23 (d, J ¼ 9.0 Hz, 2H); 8.50 (bs, 1H); 10.66

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(s, 1H); EIMS m/z 313 (Mþ). Anal. Calcd for C16H15N3O4: C, 61.34; H,
4.83; N, 13.41; found: C, 61.39; H, 4.82; N, 13.40.
J ¼ 8.5 Hz, 2H); 7.47 (d, J ¼ 9.0 Hz, 2H); 8.39 (t, J ¼ 5.6 Hz, 1H); 9.83
(s, 1H); EIMS m/z 332 (Mþ). Anal. Calcd for C17H17ClN2O3: C, 61.36;
H, 5.15; Cl, 10.65; N, 8.42; found: C, 61.42; H, 5.14; Cl, 10.64; N, 8.41.
4.4.2.5. N-(3,5-Dimethoxyphenyl)-N2-(phenylacetyl)glycinamide
(3ae). White powder, 71%, mp 162e164 ꢁC, 1H NMR (300 MHz,
CDCl3): 3.69 (s, 2H); 3.79 (s, 6H); 4.03 (d, J ¼ 5.5 Hz, 2H); 6.26 (s,
2H); 6.72 (s, 2H); 7.32e7.40 (m, 5H); 8.16 (bs, 1H); EIMS m/z 328
(Mþ). Anal. Calcd for C18H20N2O4: C, 65.84; H, 6.14; N, 8.53; found:
C, 65.77; H, 6.15; N, 8.54.
4.4.2.14. N2-[(3-Chlorophenyl)acetyl]-N-(4-methoxyphenyl)glycina-
mide (3an). White powder, 49%, mp 170e172 ꢁC, 1H NMR
(400 MHz, DMSO-d6): 3.55 (s, 2H); 3.72 (s, 3H); 3.88 (d, J ¼ 5.8 Hz,
2H); 6.90 (d, J ¼ 9.0 Hz, 2H); 7.25e7.39 (m, 4H); 7.49 (d, J ¼ 9.0 Hz,
2H); 8.47 (t, J ¼ 5.5 Hz, 1H); 9.88 (s, 1H); EIMS m/z 332 (Mþ). Anal.
Calcd for C17H17ClN2O3: C, 61.36; H, 5.15; Cl,10.65; N, 8.42; found: C,
61.22; H, 5.15; Cl, 10.68; N, 8.43.
4.4.2.6. N-(3,5-Difluorophenyl)-N2-(phenylacetyl)glycinamide (3af).
White powder, 79%, mp 201e203 ꢁC, 1H NMR (400 MHz, DMSO-d6):
3.52 (s, 2H); 3.90 (d, J ¼ 5.6 Hz, 2H); 6.89e6.94 (m, 1H); 7.22e7.30
(m, 7H); 8.46 (bs, 1H); 10.40 (s, 1H); EIMS m/z 304 (Mþ). Anal. Calcd
for C16H14F2N2O2: C, 63.15; H, 4.64; F, 12.49; N, 9.21; found: C,
63.06; H, 4.64; F, 12.50; N, 9.22.
4.4.2.15. N-(4-Bromophenyl)-N2-[(3-chlorophenyl)acetyl]glycina-
mide (3ao). White powder, 72%, mp 182e183 ꢁC, 1H NMR
(400 MHz, DMSO-d6): 3.55 (s, 2H); 3.91 (d, J ¼ 5.8 Hz, 2H);
7.24e7.38 (m, 4H); 7.50 (d, J ¼ 8.9 Hz, 2H); 7.57 (d, J ¼ 8.9 Hz, 2H);
8.51 (t, J ¼ 5.7 Hz, 1H); 10.17 (s, 1H); EIMS m/z 380 (Mþ). Anal. Calcd
for C16H14BrClN2O2: C, 50.35; H, 3.70; Br, 20.94; Cl, 9.29; N, 7.34;
found: C, 50.44; H, 3.70; Br, 20.89; Cl, 9.28; N, 7.35.
4.4.2.7. N-(4-Chlorophenyl)-N2-(phenylacetyl)glycinamide
(3ag).
White powder, 82%, mp 220e222 ꢁC, 1H NMR (400 MHz, DMSO-d6):
3.55 (s, 2H); 3.95 (d, J ¼ 5.2 Hz, 2H); 7.14e7.27 (m, 6H); 7.43e7.45
(m, 4H); 9.56 (s, 1H); EIMS m/z 302 (Mþ). Anal. Calcd for
4.4.2.16. N2-[(3-Chlorophenyl)acetyl]-N-(4-nitrophenyl)glycinamide
(3ap). White powder, 48%, mp 226e228 ꢁC, 1H NMR (400 MHz,
DMSO-d6): 3.56 (s, 2H); 3.98 (d, J ¼ 5.6 Hz, 2H); 7.25e7.39 (m, 4H);
7.84 (d, J ¼ 8.8 Hz, 2H); 8.24 (d, J ¼ 8.7 Hz, 2H); 8.57 (t, J ¼ 5.2 Hz,
1H); 10.67 (s,1H); EIMS m/z 347 (Mþ). Anal. Calcd for C16H14ClN3O4:
C, 55.26; H, 4.06; Cl, 10.19; N, 12.08; found: C, 55.32; H, 4.05; Cl,
10.16; N, 12.05.
C
16H15ClN2O2: C, 63.47; H, 4.99; Cl, 11.71; N, 9.25; found: C, 63.53;
H, 4.99; Cl, 11.70; N, 9.24.
4.4.2.8. N-(3,5-Dichlorophenyl)-N2-(phenylacetyl)glycinamide
(3ah). White powder, 58%, mp 293e295 ꢁC, 1H NMR (400 MHz,
DMSO-d6): 3.51 (s, 2H); 3.90 (d, J ¼ 5.6 Hz, 2H); 7.22e7.30 (m, 6H);
7.65 (s, 2H); 8.47 (bs, 1H); 10.35 (s, 1H); EIMS m/z 336 (Mþ). Anal.
Calcd for C16H14Cl2N2O2: C, 56.99; H, 4.18; Cl, 21.03; N, 8.31; found:
C, 56.91; H, 4.19; Cl, 21.05; N, 8.30.
4.4.2.17. N2-[(2-Chlorophenyl)acetyl]-N-(4-methoxyphenyl)glycina-
mide (3aq). White powder, 76%, mp 160e161 ꢁC, 1H NMR
(400 MHz, DMSO-d6): 3.69 (s, 2H); 3.72 (s, 3H); 3.90 (d, J ¼ 5.6 Hz,
2H); 6.89 (d, J ¼ 9.0 Hz, 2H); 7.25e7.33 (m, 2H); 7.41e7.44 (m, 2H);
7.49 (d, J ¼ 8.8 Hz, 2H); 8.41 (t, J ¼ 5.6 Hz,1H); 9.86 (s, 1H); EIMS m/z
332 (Mþ). Anal. Calcd for C17H17ClN2O3: C, 61.36; H, 5.15; Cl, 10.65;
N, 8.42; found: C, 61.42; H, 5.15; Cl, 10.62; N, 8.40.
4.4.2.9. N-Phenyl-N2-(phenylacetyl)glycinamide (3ai). White pow-
der, 49%, mp 215e217 ꢁC, 1H NMR (400 MHz, DMSO-d6): 3.53 (s,
2H); 3.92 (d, J ¼ 5.5 Hz, 2H); 6.94e7.12 (m, 5H); 7.20e7.31 (m, 5H);
8.51 (bs, 1H); 10.31 (s, 1H); EIMS m/z 268 (Mþ). Anal. Calcd for
C
16H16N2O2: C, 71.62; H, 6.01; N, 10.44; found: C, 71.51; H, 6.02; N,
10.46.
4.4.2.18. N-(4-Bromophenyl)-N2-[(2-chlorophenyl)acetyl]glycina-
mide (3ar). White powder, 73%, mp 184e185 ꢁC, 1H NMR
(400 MHz, DMSO-d6): 3.69 (s, 2H); 3.93 (d, J ¼ 5.7 Hz, 2H);
7.27e7.32 (m, 2H); 7.40e7.44 (m, 2H); 7.50 (d, J ¼ 8.9 Hz, 2H); 7.57
(d, J ¼ 8.8 Hz, 2H); 8.45 (t, J ¼ 5.8 Hz, 1H); 10.16 (s, 1H); EIMS m/z
380 (Mþ). Anal. Calcd for C16H14BrClN2O2: C, 50.35; H, 3.70; Br,
20.94; Cl, 9.29; N, 7.34; found: C, 50.29; H, 3.70; Br, 20.96; Cl, 9.30;
N, 7.35.
4.4.2.10. N-(2-Chlorophenyl)-N2-(phenylacetyl)glycinamide
(3aj).
White powder, 86%, mp 156e158 ꢁC, 1H NMR (300 MHz, CDCl3):
3.72 (s, 2H); 4.11 (d, J ¼ 5.1 Hz, 2H); 6.16 (s, 1H); 7.08 (t, J ¼ 7.5 Hz,
1H); 7.28e7.41 (m, 7H); 8.28 (double s, 2H); EIMS m/z 302 (Mþ).
Anal. Calcd for C16H15ClN2O2: C, 63.47; H, 4.99; Cl, 11.71; N, 9.25;
found: C, 63.39; H, 5.00; Cl, 11.72; N, 9.25.
4.4.2.11. N-(3-Chlorophenyl)-N2-(phenylacetyl)glycinamide (3ak).
White powder, 81%, mp 177e179 ꢁC, 1H NMR (400 MHz, DMSO-d6):
3.51 (s, 2H); 3.90 (d, J ¼ 5.6 Hz, 2H); 7.11 (d, J ¼ 7.8 Hz, 1H);
7.23e7.36 (m, 6H); 7.43 (d, J ¼ 8.1 Hz,1H); 7.79 (s, 1H); 8.44 (bs,1H);
10.20 (s, 1H); EIMS m/z 302 (Mþ). Anal. Calcd for C16H15ClN2O2: C,
63.47; H, 4.99; Cl, 11.71; N, 9.25; found: C, 63.52; H, 4.98; Cl, 11.70;
N, 9.24.
4.4.2.19. N2-[(2-Chlorophenyl)acetyl]-N-(4-nitrophenyl)glycinamide
(3as). White powder, 52%, mp 228e230 ꢁC, 1H NMR (400 MHz,
DMSO-d6): 3.70 (s, 2H); 4.00 (d, J ¼ 5.6 Hz, 2H); 7.29e7.33 (m, 2H);
7.41e7.43 (m, 2H); 7.84 (d, J ¼ 8.7 Hz, 2H); 8.25 (d, J ¼ 8.8 Hz, 2H);
8.52 (t, J ¼ 5.6 Hz, 1H); 10.66 (s, 1H); EIMS m/z 347 (Mþ). Anal. Calcd
for C16H14ClN3O4: C, 55.26; H, 4.06; Cl, 10.19; N, 12.08; found: C,
55.20; H, 4.06; Cl, 10.20; N, 12.10.
4.4.2.12. N2-[(4-Chlorophenyl)acetyl]-N-(3,5-dichlorophenyl)glyci-
namide (3al). Colorless crystal, 47%, mp 190e192 ꢁC, 1H NMR
(400 MHz, DMSO-d6): 3.52 (s, 2H); 3.90 (d, J ¼ 5.8 Hz, 2H); 7.28 (t,
J ¼ 1.9 Hz, 1H); 7.31 (d, J ¼ 8.5 Hz, 2H); 7.37 (d, J ¼ 8.4 Hz, 2H); 7.64
(d, J ¼ 1.9 Hz, 2H); 8.47 (t, J ¼ 5.7 Hz, 1H); 10.34 (s, 1H); EIMS m/z
370 (Mþ). Anal. Calcd for C16H13Cl3N2O2: C, 51.71; H, 3.53; Cl, 28.62;
N, 7.54; found: C, 51.65; H, 3.53; Cl, 28.64; N, 7.55.
4.4.2.20. N-(4-Bromophenyl)-N2-[(3-bromophenyl)acetyl]glycina-
mide (3at). White powder, 58%, mp 182e184 ꢁC, 1H NMR
(400 MHz, DMSO-d6): 3.55 (s, 2H); 3.92 (d, J ¼ 5.5 Hz, 2H);
7.27e7.30 (m, 2H); 7.42e7.45 (m, 1H); 7.49 (d, J ¼ 8.9 Hz, 2H);
7.50e7.53 (m, 1H); 7.57 (d, J ¼ 8.8 Hz, 2H); 8.51 (t, J ¼ 5.3 Hz, 1H);
10.17 (s, 1H); EIMS m/z 424 (Mþ). Anal. Calcd for C16H14Br2N2O2: C,
45.10; H, 3.31; Br, 37.50; N, 6.57; found: C, 45.17; H, 3.31; Br, 37.47;
N, 6.576.
4.4.2.13. N2-[(4-Chlorophenyl)acetyl]-N-(4-methoxyphenyl)glycina-
mide (3am). Colorless crystal, 55%, mp 172e174 ꢁC, 1H NMR
(400 MHz, DMSO-d6): 3.52 (s, 2H); 3.72 (s, 3H); 3.86 (d, J ¼ 5.8 Hz,
2H); 6.88 (d, J ¼ 9.0 Hz, 2H); 7.31 (d, J ¼ 8.5 Hz, 2H); 7.36 (d,
4.4.2.21. N2-[(4-Fluorophenyl)acetyl]-N-(4-methoxyphenyl)glycina-
mide (3au). White powder, 66%, mp 192e194 ꢁC, 1H NMR
(400 MHz, DMSO-d6): 3.51 (s, 2H); 3.72 (s, 3H); 3.87 (d, J ¼ 5.7 Hz,

Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 102 (2015) 631e638
637
2H); 6.89 (d, J ¼ 9.0 Hz, 2H); 7.13 (t, J ¼ 8.9 Hz, 2H); 7.33 (dd,
J ¼ 8.4 Hz, J ¼ 5.8 Hz, 2H); 7.49 (d, J ¼ 9.0 Hz, 2H); 8.41 (t, J ¼ 5.6 Hz,
1H); 9.87 (s,1H); EIMS m/z 316 (Mþ). Anal. Calcd for C17H17FN2O3: C,
64.55; H, 5.42; F, 6.01; N, 8.86; found: C, 64.48; H, 5.43; F, 6.03; N,
8.88.
C17H18N2O4: C, 64.96; H, 5.77; N, 8.91; found: C, 64.91; H, 5.78; N,
8.92.
4.4.2.28. N2-[(4-Hydroxyphenyl)acetyl]-N-(4-nitrophenyl)glycina-
mide (3bb). White powder, 51%, mp 240e242 ꢁC, 1H NMR
(400 MHz, DMSO-d6): 3.39 (s, 2H); 3.95 (d, J ¼ 5.8 Hz, 2H); 6.69 (d,
J ¼ 8.4 Hz, 2H); 7.08 (d, J ¼ 8.4 Hz, 2H); 7.83 (d, J ¼ 9.3 Hz, 2H); 8.24
(d, J ¼ 9.2 Hz, 2H); 8.38 (t, J ¼ 5.7 Hz, 1H); 9.25 (s, 1H); 10.65 (s, 1H);
EIMS m/z 329 (Mþ). Anal. Calcd for C16H15N3O5: C, 58.36; H, 4.59; N,
12.76; found: C, 58.32; H, 4.59; N, 12.79.
4.4.2.22. N2-[(4-Fluorophenyl)acetyl]-N-(4-nitrophenyl)glycinamide
(3av). White powder, 70%, mp 246e248 ꢁC, 1H NMR (400 MHz,
DMSO-d6): 3.53 (s, 2H); 3.98 (d, J ¼ 5.7 Hz, 2H); 7.14 (t, J ¼ 8.9 Hz,
2H); 7.33 (dd, J ¼ 8.4 Hz, J ¼ 5.8 Hz, 2H); 7.84 (d, J ¼ 9.2 Hz, 2H); 8.25
(d, J ¼ 9.2 Hz, 2H); 8.52 (t, J ¼ 5.6 Hz, 1H); 10.67 (s, 1H); 13C NMR
(100 MHz, DMSO-d6): 40.42; 43.44; 115.37 (d, J ¼ 20.9 Hz); 119.18;
125.55; 131.38 (d, J ¼ 7.9 Hz); 132.81 (d, J ¼ 3.2 Hz); 142.63; 145.51;
161.50 (d, J ¼ 240.3 Hz); 169.29; 171.15; EIMS m/z 331 (Mþ). Anal.
Calcd for C16H14FN3O4: C, 58.01; H, 4.26; F, 5.73; N, 12.68; found: C,
58.06; H, 4.25; F, 5.72; N, 12.66.
4.4.2.29. N2-[(3-Hydroxyphenyl)acetyl]-N-(4-methoxyphenyl)glyci-
namide (3bc). White powder, 69%, mp 156e157 ꢁC, 1H NMR
(400 MHz, DMSO-d6): 3.42 (s, 2H); 3.72 (s, 3H); 3.87 (d, J ¼ 5.7 Hz,
2H); 6.62 (d, J ¼ 7.5 Hz,1H); 6.71 (d, J ¼ 6.4 Hz,1H); 6.72 (s,1H); 6.89
(d, J ¼ 8.9 Hz, 2H); 7.08 (t, J ¼ 8.0 Hz, 1H); 7.49 (d, J ¼ 8.9 Hz, 2H);
8.33 (t, J ¼ 5.4 Hz, 1H); 9.32 (s, 1H); 9.85 (s, 1H); 13C NMR (100 MHz,
DMSO-d6): 42.58; 43.10; 55.59; 113.79; 114.32; 116.52; 120.23;
121.07; 129.55; 132.47; 137.96; 155.63; 157.65; 167.64; 171.02; EIMS
m/z 314 (Mþ). Anal. Calcd for C17H18N2O4: C, 64.96; H, 5.77; N, 8.91;
found: C, 65.01; H, 5.76; N, 8.89.
4.4.2.23. N2-[(3-Fluorophenyl)acetyl]-N-(4-methoxyphenyl)glycina-
mide (3aw). White powder, 79%, mp 153e155 ꢁC, 1H NMR
(400 MHz, DMSO-d6): 3.56 (s, 2H); 3.72 (s, 3H); 3.89 (d, J ¼ 5.7 Hz,
2H); 6.89 (d, J ¼ 9.0 Hz, 2H); 7.07 (dt, J ¼ 8.6 Hz, J ¼ 2.1 Hz, 1H); 7.15
(t, J ¼ 8.2 Hz, 2H); 7.32e7.38 (m, 1H); 7.49 (d, J ¼ 9.0 Hz, 2H); 9.88 (s,
1H); 13C NMR (100 MHz, DMSO-d6): 42.03; 43.10; 55.59; 113.63 (d,
J ¼ 20.6 Hz); 114.33; 116.31 (d, J ¼ 21.3 Hz); 121.10; 125.75 (d,
J ¼ 2.7 Hz); 130.47 (d, J ¼ 8.3 Hz); 132.46; 139.53 (d, J ¼ 7.9 Hz);
155.64; 162.49 (d, J ¼ 241.4 Hz); 167.59; 170.53; EIMS m/z 316 (Mþ).
Anal. Calcd for C17H17FN2O3: C, 64.55; H, 5.42; F, 6.01; N, 8.86;
found: C, 64.49; H, 5.42; F, 6.03; N, 8.87.
4.4.2.30. N2-[(3-Hydroxyphenyl)acetyl]-N-(4-nitrophenyl)glycina-
mide (3bd). White powder, 60%, mp 197e198 ꢁC, 1H NMR
(400 MHz, DMSO-d6): 3.43 (s, 2H); 3.97 (d, J ¼ 5.7 Hz, 2H); 6.63 (d,
J ¼ 7.8 Hz, 1H); 6.71 (d, J ¼ 6.4 Hz, 1H); 6.72 (s, 1H); 7.09 (t,
J ¼ 8.0 Hz, 1H); 7.84 (d, J ¼ 9.2 Hz, 2H); 8.24 (d, J ¼ 9.1 Hz, 2H); 8.45
(t, J ¼ 5.6 Hz, 1H); 9.33 (s, 1H); 10.66 (s, 1H); 13C NMR (100 MHz,
DMSO-d6): 42.48; 43.45; 113.83; 116.53; 119.18; 120.22; 125.55;
129.57; 137.84; 142.63; 145.52; 157.66; 169.31; 171.22; EIMS m/z
329 (Mþ). Anal. Calcd for C16H15N3O5: C, 58.36; H, 4.59; N, 12.76;
found: C, 58.41; H, 4.58; N, 12.74.
4.4.2.24. N2-[(3-Fluorophenyl)acetyl]-N-(4-nitrophenyl)glycinamide
(3ax). White powder, 56%, mp 232e233 ꢁC, 1H NMR (400 MHz,
DMSO-d6): 3.57 (s, 2H); 3.99 (d, J ¼ 5.7 Hz, 2H); 7.07 (dt, J ¼ 8.7 Hz,
J ¼ 2.4 Hz, 1H); 7.13e7.16 (m, 2H); 7.32e7.38 (m, 1H); 7.84 (d,
J ¼ 9.2 Hz, 2H); 8.24 (d, J ¼ 9.2 Hz, 2H); 8.57 (t, J ¼ 5.6 Hz, 2H); 10.68
(s,1H); EIMS m/z 331 (Mþ). Anal. Calcd for C16H14FN3O4: C, 58.01; H,
4.26; F, 5.73; N, 12.68; found: C, 58.06; H, 4.26; F, 5.74; N, 12.70.
4.4.2.31. N2-[(2-Hydroxyphenyl)acetyl]-N-(4-methoxyphenyl)glyci-
namide (3be). White powder, 57%, mp 184e186 ꢁC, 1H NMR
(400 MHz, DMSO-d6): 3.48 (s, 2H); 3.72 (s, 3H); 3.88 (d, J ¼ 5.6 Hz,
2H); 6.75 (t, J ¼ 7.4 Hz, 1H); 6.81 (d, J ¼ 7.9 Hz, 1H); 6.89 (d,
J ¼ 9.0 Hz, 2H); 7.07 (td, J ¼ 7.8 Hz, J ¼ 1.5 Hz, 1H); 7.13 (dd,
J ¼ 7.4 Hz, J ¼ 1.2 Hz, 1H); 7.48 (d, J ¼ 9.0 Hz, 2H); 8.23 (t, J ¼ 5.6 Hz,
1H); 9.67 (s, 1H); 9.81 (s, 1H); EIMS m/z 329 (Mþ). Anal. Calcd for
4.4.2.25. N2-[(3,4-Dimethoxyphenyl)acetyl]-N-(4-methoxyphenyl)
glycinamide (3ay). White powder, 72%, mp 167e168 ꢁC, 1H NMR
(400 MHz, DMSO-d6): 3.44 (s, 2H); 3.72 (s, 3H); 3.73 (s, 3H); 3.75 (s,
3H); 3.87 (d, J ¼ 5.3 Hz, 2H); 6.80 (d, J ¼ 8.2 Hz, 1H); 6.86e6.90 (m,
3H); 6.95 (s, 1H); 7.49 (d, J ¼ 7.2 Hz, 2H); 8.32 (bs, 1H); 9.86 (s, 1H);
13C NMR (100 MHz, DMSO-d6): 42.15; 43.10; 55.59; 55.82; 55.97;
112.15; 113.39; 114.33; 121.03; 121.52; 129.12; 132.50; 147.90;
148.91; 155.62; 167.71; 171.34; EIMS m/z 358 (Mþ). Anal. Calcd for
C17H18N2O4: C, 64.96; H, 5.77; N, 8.91; found: C, 64.88; H, 5.78; N,
8.93.
4.4.2.32. N2-[(3-Chlorophenyl)acetyl]-N-(4-methylphenyl)glycina-
mide (3bf). White powder, 38%, mp 173e174 ꢁC, 1H NMR (500 MHz,
DMSO-d6): 2.25 (s, 3H); 3.55 (s, 2H); 3.90 (d, J ¼ 5.7 Hz, 2H); 7.11 (d,
J ¼ 8.3 Hz, 2H); 7.26 (d, J ¼ 7.4 Hz, 1H); 7.30 (d, J ¼ 8.1 Hz, 1H); 7.34
(t, J ¼ 7.6 Hz, 1H); 7.39 (s, 1H); 7.46 (d, J ¼ 8.3 Hz, 2H); 8.44 (t,
J ¼ 5.6 Hz, 1H); 9.90 (s, 1H); EIMS m/z 316 (Mþ). Anal. Calcd for
C
19H22N2O5: C, 63.67; H, 6.19; N, 7.82; found: C, 63.67; H, 6.19; N,
7.82.
4.4.2.26. N2-[(3,4-Dimethoxyphenyl)acetyl]-N-(4-nitrophenyl)glyci-
namide (3az). White powder, 75%, mp 224e226 ꢁC, 1H NMR
(400 MHz, DMSO-d6): 3.45 (s, 2H); 3.73 (s, 3H); 3.75 (s, 3H); 3.97 (d,
J ¼ 5.8 Hz, 2H); 6.81 (dd, J ¼ 8.2 Hz, J ¼ 1.8 Hz, 1H); 6.88 (d,
J ¼ 8.2 Hz, 1H); 6.94 (d, J ¼ 1.7 Hz, 1H); 7.83 (d, J ¼ 9.2 Hz, 2H); 8.24
(d, J ¼ 9.2 Hz, 2H); 8.42 (t, J ¼ 5.8 Hz, 1H); 10.66 (s, 1H); 13C NMR
(100 MHz, DMSO-d6): 42.03; 42.46; 55.84; 55.98; 112.16; 113.37;
119.16; 121.51; 125.56; 128.99; 142.63; 145.54; 147.91; 148.91;
169.39; 171.51; EIMS m/z 373 (Mþ). Anal. Calcd for C18H19N3O6: C,
57.90; H, 5.13; N, 11.25; found: C, 57.85; H, 5.14; N, 11.27.
C17H17ClN2O2: C, 64.46; H, 5.41; Cl,11.19; N, 8.84; found: C, 64.54; H,
5.41; Cl, 11.17; N, 8.83.
4.4.2.33. N2-[(4-Methylphenyl)acetyl]-N-(4-nitrophenyl)glycinamide
(3bg). Light yellow powder, 47%, mp over 300 ꢁC, 1H NMR
(500 MHz, DMSO-d6): 2.27 (s, 3H); 3.48 (s, 2H); 3.96 (d, J ¼ 5.8 Hz,
2H); 7.11 (d, J ¼ 7.9 Hz, 2H); 7.19 (d, J ¼ 7.9 Hz, 2H); 7.83 (d,
J ¼ 9.2 Hz, 2H); 7.19 (d, J ¼ 9.2 Hz, 2H); 8.42 (t, J ¼ 5.7 Hz, 1H); 10.63
(s, 1H); EIMS m/z 327 (Mþ). Anal. Calcd for C17H17N3O4: C, 62.38; H,
5.23; N, 12.84; found: C, 62.16; H, 5.24; N, 12.87.
4.4.2.27. N2-[(4-Hydroxyphenyl)acetyl]-N-(4-methoxyphenyl)glyci-
namide (3ba). White powder, 53%, mp 216e220 ꢁC, 1H NMR
(400 MHz, DMSO-d6): 3.37 (s, 2H); 3.72 (s, 3H); 3.85 (d, J ¼ 5.8 Hz,
2H); 6.68 (d, J ¼ 8.4 Hz, 2H); 6.89 (d, J ¼ 9.0 Hz, 2H); 7.08 (d,
J ¼ 8.4 Hz, 2H); 7.48 (d, J ¼ 9.0 Hz, 2H); 8.27 (t, J ¼ 5.6 Hz, 1H); 9.34
(bs, 1H); 9.86 (s, 1H); EIMS m/z 314 (Mþ). Anal. Calcd for
Acknowledgments
The work was financed by grants from National Natural Science
Foundation of China (grant No. 21262013), by a Project supported
by Hunan Provincial Natural Science Foundation of China (grant No.

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