Tyrosyl-tRNA synthetase inhibitors as antibacterial agents: Synthesis, molecular docking and structure-activity relationship analysis of 3-aryl-4-arylaminofuran-2(5H)-ones

Article abstract of DOI:10.1016/j.ejmech.2011.07.047

Thirty-five 3-aryl-4-arylaminofuran-2(5H)-one derivatives were designed, prepared and tested for their inhibitory activity against tyrosyl-tRNA synthetase. Out of these compounds, 3-(3-bromophenyl)-4-(3,5- dichlorophenylamino)furan-2(5H)-one (35) was the most active with IC ₅₀ of 0.09 ± 0.02 μM. The structure-activity relationship revealed that introduction of chlorine atoms at both meta positions of aniline moiety significantly increased the enzyme inhibitory activity. The results of antibacterial assay revealed that the tested compounds showed good activity against Gram-positive bacteria, with 35 being the most potent with MIC ₅₀ of 0.06 μg/mL against Staphylococcus aureus ATCC 25923. Molecular docking of 35 into S. aureus tyrosyl-tRNA synthetase active site was also performed. The inhibitor snugly fitting the active site may well explain its excellent inhibitory activity.

Full text of DOI:10.1016/j.ejmech.2011.07.047

European Journal of Medicinal Chemistry 46 (2011) 4904e4914
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Tyrosyl-tRNA synthetase inhibitors as antibacterial agents: Synthesis, molecular
docking and structureeactivity relationship analysis of 3-aryl-4-arylaminofuran-
2(5H)-ones
,
b
,
c
,
d
,
e
,
a
d
d
d
d
Zhu-Ping Xiao a
, Tao-Wu Ma , Mei-Lin Liao , Yu-Ting Feng , Xiao-Chun Peng , Jia-Liang Li ,
*
Zhi-Ping Li d, Ying Wu d, Qun Luo d, Yang Deng d, Xiao Liang d, Hai-Liang Zhu d
,e,
**
a Key Laboratory of Plant Resources Conservation and Utilization, College of Hunan Province, Jishou University, Jishou 416000, PR China
b Key Laboratory of Hunan Forest Products and Chemical Industry Engineering, Jishou University, Jishou 416000, PR China
c Key Laboratory of Ecotourism’s Application Technology, Hunan Province, Jishou University, Jishou 416000, PR China
d College of Chemistry and Chemical Engineering, Jishou University, Jishou 416000, PR China
e 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:
Thirty-five 3-aryl-4-arylaminofuran-2(5H)-one derivatives were designed, prepared and tested for their
Received 29 March 2011
Received in revised form
22 July 2011
Accepted 26 July 2011
Available online 4 August 2011
inhibitory activity against tyrosyl-tRNA synthetase. Out of these compounds, 3-(3-bromophenyl)-4-(3,5-
dichlorophenylamino)furan-2(5H)-one (35) was the most active with IC50 of 0.09 ꢀ 0.02
mM. The
structureeactivity relationship revealed that introduction of chlorine atoms at both meta positions of
aniline moiety significantly increased the enzyme inhibitory activity. The results of antibacterial assay
revealed that the tested compounds showed good activity against Gram-positive bacteria, with 35 being
the most potent with MIC50 of 0.06 mg/mL against Staphylococcus aureus ATCC 25923. Molecular docking
of 35 into S. aureus tyrosyl-tRNA synthetase active site was also performed. The inhibitor snugly fitting
the active site may well explain its excellent inhibitory activity.
Keywords:
3-Aryl-4-arylaminofuran-2(5H)-one
Antibacterial agent
Ó 2011 Elsevier Masson SAS. All rights reserved.
Molecular docking
Tyrosyl-tRNA synthetase
Structureeactivity relationship
1. Introduction
binding domain and the functions of the human aaRSs differ from
those of bacteria, thus providing an opportunity to inhibit them
The emergence of resistance to clinically used drugs has
promoted development of novel antimicrobial agents which
selectively inhibit novel bacterial targets [1,2,3]. In this regard,
aminoacyl-tRNA synthetases (aaRSs) have attracted considerable
interest in antibacterial drug discovery [4,5,6]. It is well known that
aaRSs are responsible for faithful translation of nucleic acid
sequence information into proteins [7,8]. Consequently, once these
enzymes are inhibited, cell growth is inhibited due to protein
biosynthesis being halted. Nevertheless, the topology of the ATP
selectively in bacteria [9,10]. These enzymes, therefore, are attrac-
tive targets for antibacterial agents [11,12,13].
Furan-2(5H)-one framework is a part of many natural and
synthetic compounds, which possess useful biological activities
including anti-oxidative, anti-inflammatory and HIV-1 integrase
inhibitory activities [14,15,16]. Therefore, derivatives of furan-
2(5H)-one have been receiving significant attention [17,18].
Recently, Lattmann et al. reported 5-alkoxy-4-aminofuran-2(5H)-
ones showing good antibacterial activity against multiresistant
Staphylococcus aureus [19], which encourages us to synthesize and
evaluate the antibacterial activity of a series of substituted 3-
arylfuran-2(5H)-ones. It was found that some 4-alkylamino-3-
arylfuran-2(5H)-ones are potent inhibitors against tyrosyl-tRNA
synthetase (TyrRS), an aaRS, and showed excellent antibacterial
activity against S. aureus [20]. To learn more about the structur-
eeantibacterial activity relationship of 3-arylfuran-2(5H)-ones, we
herein report the synthesis of 3-aryl-4-arylaminofuran-2(5H)-ones
* Corresponding author. College of Chemistry and Chemical Engineering, Jishou
University, Jishou 416000, PR China. Tel./fax: þ86 743 8563911.
** Corresponding author. State Key Laboratory of Pharmaceutical Biotechnology,
Nanjing University, Nanjing 210093, PR China. Tel.: þ86 25 8359 2572; fax: þ86 25
83592672.
E-mail addresses: xiaozhuping2005@163.com (Z.-P. Xiao), zhuhl@nju.edu.cn
(H.-L. Zhu).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.07.047

Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 46 (2011) 4904e4914
4905
(4e38) as TyrRS inhibitors and antibacterial agents. The results
show that some of the synthesized compounds exhibit excellent
antibacterial activities.
2. Results and discussion
2.1. Chemistry
Scheme 2. The synthesis of 37.
Thirty-five 3-aryl-4-arylaminofuran-2(5H)-ones were designed
and synthesized by the routes outlined in Scheme 1 and Scheme 2.
Amination of 2 with appropriately substituted anilines in toluene
gave 3-aryl-4-arylaminofuran-2(5H)-ones (3e37) in the presence
of p-toluene sulfonic acid, while 3-aryl-4-arylaminofuran-2(5H)-
one 38 was obtained by hydrogenolysis of the corresponding
precursor 3 over Pd/C. The intermediates 2 were synthesized
according to the previously reported method [15,20]. All 3-aryl-4-
arylaminofuran-2(5H)-ones were first reported and were fully
characterized by spectroscopic methods and elemental analysis.
compound 12, which showed a slightly loss in activity with respect
to the corresponding unsubstituted compound (10), suggesting
that substitution at the aniline nitrogen is not tolerated. Finally,
replacement of the aniline ring (10) with an aminopyridine ring
(11) resulted in some attenuation of potency.
We then turned our attention to exploring the SAR profile of the
phenyl group (4e9 and 29e38) in hopes of finding a permissive
region. The removal of bromo group (13, 14, 17 and 18) led to an
over 4-fold loss of activity (4e7). Although substitution of the
bromine atom (27 and 14) with a fluorine atom or a chlorine atom
(8 and 9) maintained the activity against the TyrRS, substitution
with 4-methoxy group (26 vs 37) showed a 4-fold loss. This indi-
cates that compounds with electron-withdrawing groups at posi-
tion 4 showed better inhibitory activities than those with electron-
donating groups. Replacement of the 4-methoxy group (37) with
a hydroxyl group (38) led to a further decrease in activity, which
may suggest that the substituent at the 4-position interacts with
a hydrophobic area of the enzyme. In contrast, the 3-chloro
derivatives (29e31) were about 5-fold more potent than the cor-
responding unsubstituted compounds (4e6). Replacement of the
chlorine atom of compound 33 by a bromine atom (35) further
improved the inhibitory activity by a factor of more than 48.
Obviously, the 3-position is more suitable for further modification.
Out of the obtained compounds, 35 was the most potent.
2.2. Inhibitory activities of 3-aryl-4-arylaminofuran-2(5H)-ones
against TyrRS from S. aureus
All synthesized compounds (4e38) were tested for inhibitory
activity against TyrRS from S. aureus and the IC50 values are pre-
sented in Table 1. In the initial phase of our SAR studies, we focused
our efforts on substitution of the aniline ring (10e28). 3,5-Dichloro-
substituted compound (26), with IC50 of 0.39 mM, was the most
potent member of this series, and substitution with bromo, fluoro
or methoxy groups led to a significant decrease in activity. In
comparison with 10, introduction of chloro, nitro or methoxy
substituents at the 2-position (13, 17 and 22) led to a small (lower
than 4-fold) reduction in potency. Modification at the 2-position
therefore seems to be detrimental to enzyme inhibition.
Changing the position of the chloro group from the 2-position (13)
to the 3-position (14) significantly enhanced the level of TyrRS
inhibition. However, over 6-fold loss of enzyme inhibitory activity
was observed when the 3-chloro group was replaced by nitro or
hydroxyl groups (18 and 20). On the other hand, the exchange of
the chlorine atom from the 2- to the 4-position resulted in an
equally potent compound (15), and modification at the 4-position
with hydroxyl, methoxy, nitro or methyl groups led to a 2- to 5-
fold loss of potency. Methylation of 10 at nitrogen gave
2.3. Antibacterial activity
All synthesized compounds (4e38) were also tested against
representative Gram-positive bacteria (Bacillus subtilis ATCC 6633,
S. aureus ATCC 25923), a Gram-negative bacterium (Escherichia coli
ATCC 35218) and a fungus (Candida albicans ATCC 10231), and the
results are presented in Table 2. In general, many of the evaluated
Scheme 1. Synthetic route of 3-aryl-4-arylaminofuran-2(5H)-ones.

4906
Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 46 (2011) 4904e4914
Table 1
In vitro inhibitory activity data of the synthesized compounds against S. aureus TyrRS.
Structure
IC50 (mM)
Compound
R1
H
R2
H
R3
H
Ar
4
5
6
7
89.9 ꢀ 11.4
20.8 ꢀ 7.1
>100
H
H
H
H
H
H
H
H
H
44.6 ꢀ 10.5
8
9
H
H
F
H
H
9.2 ꢀ 0.9
2.4 ꢀ 0.1
Cl
10
11
12
H
H
H
Br
Br
Br
H
16.6 ꢀ 2.6
25.6 ꢀ 6.7
29.4 ꢀ 5.9
H
Me
13
14
H
H
Br
Br
H
H
21.0 ꢀ 3.5
1.7 ꢀ 0.2
15
16
H
H
Br
Br
H
H
15.2 ꢀ 2.7
11.4 ꢀ 1.4

Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 46 (2011) 4904e4914
4907
Table 1 (continued )
Structure
IC50 (mM)
Compound
R1
R2
Br
R3
Ar
17
H
H
67.3 ꢀ 15.2
18
19
20
H
H
H
Br
Br
Br
H
H
H
10.3 ꢀ 1.5
63.3 ꢀ 9.7
15.4 ꢀ 2.8
21
22
H
H
Br
Br
H
H
85.2 ꢀ 16.7
36.8 ꢀ 13.2
23
24
H
H
Br
Br
H
H
97.6 ꢀ 21.5
>100
25
26
27
H
H
H
Br
Br
Br
H
H
H
6.3 ꢀ 1.1
0.39 ꢀ 0.06
13.6 ꢀ 3.8
17.5 ꢀ 6.5
28
H
Br
H
(continued on next page)

4908
Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 46 (2011) 4904e4914
Table 1 (continued )
Structure
IC50 (mM)
Compound
R1
R2
R3
Ar
29
30
Cl
Cl
H
H
H
H
15.7 ꢀ 5.2
4.3 ꢀ 1.0
31
32
Cl
Cl
H
H
H
H
49.7 ꢀ 16.8
32.5 ꢀ 11.3
33
34
35
Cl
Br
Br
H
H
H
H
H
H
2.9 ꢀ 0.8
5.4 ꢀ 1.2
0.09 ꢀ 0.02
36
37
Br
H
H
H
H
1.9 ꢀ 0.5
1.6 ꢀ 0.9
OMe
38
H
OH
H
3.5 ꢀ 0.8
compounds showed good activities against Gram-positive bacteria,
especially against S. aureus, and all of them completely lost anti-
bacterial activity against Gram-negative bacteria (E. coli) in
comparison to the open-ring derivatives [21,22]. The antibacterial
activities of new synthetic compounds against S. aureus were
significantly improved. Two 3-aryl-4-arylaminofuran-2(5H)-ones
displayed levels of inhibition better than marketed antibiotics, with
compound 35 achieving MIC50 >18-fold lower than that exhibited
by the commercial antibiotic (kanamycin). When MIC50 values
against Gram-positive bacteria (especially against S. aureus) were

Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 46 (2011) 4904e4914
4909
Table 2
Inhibitory activity (MIC50) of the synthetic compounds against microbes.
Compound
MIC50
A
(mg/mL)
B
C
D
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
>50
>50
>50
>50
38.6
10.9
41.7
>50
>50
>50
11.3
>50
>50
>50
28.6
>50
>50
>50
>50
>50
>50
19.4
2.2
>50
>50
47.5
11.7
>50
>50
13.4
31.6
1.2
8.8
9.6
9.8
0.42
>50
32.1
>50
>50
10.2
3.3
27.8
39.0
45.8
34.6
2.5
>50
41.8
>50
>50
28.6
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
e
9.7
37.5
>50
>50
49.3
9.0
25.7
41.8
>50
26.3
>50
>50
>50
>50
>50
>50
21.7
5.8
49.2
>50
46.8
12.2
>50
>50
10.1
18.7
5.4
8.5
12.6
23.1
>50
10.8
>50
25.2
>50
>50
>50
>50
7.1
0.42
23.5
>50
21.5
4.6
>50
49.4
2.9
6.5
0.06
2.3
1.8
2.9
1.1
36
37
38
7.2
10.2
Kanamycin
Penicillin
Ketoconazole
e
e
e
e
e
e
2
4.1
e
Note: A, Bacillus subtilis ATCC 6633; B, Staphylococcus aureus ATCC 25923; C, Candida
albicans ATCC 10231; D, Escherichia coli ATCC 35218.
Fig. 1. a. Binding mode of compound 35 with TyrRS from Staphylococcus aureus. For
clarity, only interacting residues were labeled. Hydrogen-bonding interactions are
shown in dash. This figure was made using PyMol. b. Binding mode of compound 35
with TyrRS. The enzyme is shown as surface; while 35 docked structures are shown as
sticks. This figure was made using PyMol.
compared with the corresponding IC50 values against TyrRS,
a positive correlation was found in almost all cases (Table 1 and
Table 2), which suggests that the antibacterial activity of 3-aryl-4-
arylaminofuran-2(5H)-ones may be due to their inhibition of TyrRS.
2.4. Molecular docking
(Fig. 1a). In addition to these relatively strong hydrogen bonds,
there are five weak hydrogen-bonding interactions with bond
length from 2.688 to 3.937 Å to anchor the ligand to this enzyme
(note: 3.937 Å is the distance between hydrogen donor and
acceptor). Three of them occur between a Cl atom in aniline moiety
and amino acid residues of Asn124 and Thr75, while the other two
occur between the Br atom in benzene-ring and amino acid resi-
dues of Cys37 and Gly38. This indicates that Asp40, His50, Cys37,
Gly38, Thr75 and Asn124 play the relatively important roles in
binding potency. Furthermore, hydrophobic interactions are also
evident in the binding of 35 with TyrRS. The benzene-ring portion
of the molecule is found to match the weak hydrophobic residue
Gly192. While the aniline moiety and the other attached Cl atom
involve the hydrophobic interactions with the strong hydrophobic
residues Leu70 and Ile200 respectively. Our modeling results
revealed that 1,3-dichloro-substituent pattern of aniline moiety
have an important influence on the interactions of the pro-
teineligand complex and is crucial to the potency of TyrRS inhibi-
tory activity. Therefore, removing the two chlorine atom (26 vs 10)
significantly reduced the enzyme inhibitory activity.
In order to give an explanation and understanding of the
excellent activity observed, molecular docking of the most potent
inhibitor 35 into the ligand binding site of TyrRS from S. aureus was
performed on the binding model based on the TyrRS complex
structure (1jij.pdb) [7]. The binding model of compound 35 and
TyrRS is depicted in Fig. 1a and the enzyme surface model was
shown in Fig. 1b, which revealed that the molecule is well filled in
the active pocket.
Several hydrogen-bonding interactions together with some
hydrophobic interactions anchoring 35 to the active site tightly
might explain its excellent inhibitory activity. In brief, the
furanone-ring system of 35 is oriented toward the entrance cavity
surrounded by Asp40, His50 and Asp80. There are two important
interactions considered as hydrogen bonds: one is in between the
backbone amino groups of His50 and carbonyl O atom of the
furanone-ring with H$O bond length of 1.937 Å, while the other is
observed between the Asp40 backbone NH group and the carbonyl
O atom of the furanone-ring with H$O bond length of 1.886 Å

4910
Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 46 (2011) 4904e4914
3. Conclusions
quantity of sterilized liquid medium (50% (v/v) of DMSO in PBS). A
specified quantity of the medium containing the test compound
was poured into 96-well plates. Suspension 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. In the case of fungi, plates were incu-
Thirty-five derivatives of 3-aryl-4-arylaminofuran-2(5H)-one
were prepared and tested for their inhibitory activity against TyrRS
from S. aureus. Compound 35, 3-(3-bromophenyl)-4-(3,5-
dichlorophenylamino)furan-2(5H)-one, showed the most potent
inhibitory activity with IC50 of 0.09 ꢀ 0.02
mM against this enzyme.
bated at 27 ꢁC for 48 h. 50
added to each well. Incubation was continued at room temperature
for 4e5 h. The content of each well was removed, and 100 L of 10%
mL of PBS containing 2 mg of MTT/mL was
Modification of aniline moiety confirmed that substitution at both
meta positions with chlorine atoms significantly increased the
enzyme inhibitory activity and substitution with other groups was
detrimental to the inhibitory activity. On the other hand, the SAR
analysis of the benzene-ring disclosed that the 3-bromo derivatives
were the most potent members in their own series. Molecular
dockings of the most potent compound 35 into S. aureus TyrRS
active site were performed. Many interactions, including two
relatively strong hydrogen-bonding interactions, anchoring the
ligand to the active site of the enzyme tightly might explain its
excellent inhibitory activity. The antibacterial activity assay
revealed that the tested compounds showed good activity against
Gram-positive bacteria, especially against S. aureus, and all of them
completely lost the antibacterial activities against Gram-negative
bacteria (E. coli) in comparison to the open-ring derivatives. It is
to be noted that 35 is also the most potent antibacterial agent
m
sodium dodecyl sulfate containing 5% isopropanol and 1 mol/L HCl
was added to extract the dye. After 12 h of incubation at room
temperature, the optical density (OD) was measured with a micro-
plate reader at 550 nm. The observed MIC50s were presented in
Table 2.
4.3. Protocol of docking study
The automated docking studies were carried out using Auto-
Dock version 4.2, and Try36, Ala39, Asp40, His50, Asp80, Gln174
and Gln196 were selected as flexible residues. First, AutoGrid
component of the program pre-calculates a three-dimensional grid
of interaction energies based on the macromolecular target using
the AMBER force field. The three-dimensional structure of
compound 35 was constructed using Chem. 3D ultra 11.0 software
[Chemical Structure Drawing Standard; Cambridge Soft corpora-
tion, USA (2009)], then it was energetically minimized by using
MOPAC with 100 iterations and minimum RMS gradient of 0.10. The
Gasteiger-Hückel charge of the ligand was assigned. 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 region
where the native ligand was embedded. Then automated docking
studies were carried out to evaluate the binding free energy of the
inhibitor within the macromolecules. The genetic algorithm 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 genera-
tions. 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.
among the synthetic compounds with MIC50 of 0.06 mg/mL against
S. aureus. This indicates that 35 would be a potential antibacterial
agent for further research.
4. Experiments
4.1. Preparation of the tyrosyl-tRNA synthetase and enzyme assay
S. aureus TyrRS was over-expressed in E. coli and purified to near
homogeneity (w98% as judged by SDS-PAGE) using standard
purification procedures [7]. TyrRS activity was measured by ami-
noacylation using modifications to previously described methods
[8]. 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 E. coli MRE600 tRNA (Roche)
and 10
m
M
L
-tyrosine (0.3
m
M
L
-[ring-3,5-3H] tyrosine (Per-
M carrier).
kineElmer, Specific activity: 1.48e2.22TBq/mmol), 10
m
TyrRS (0.2 nM) was pre-incubated 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 terminated by adding aliquots of the reaction mix
into ice-cold 7% trichloroacetic 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 concentra-
tion at which half of the enzyme activity is inhibited by the
compound. The results were presented in Table 1.
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
(uncorrected) were determined on an XT4 MP apparatus (Taike
Corp., Beijing, China). ESI mass spectra were obtained on a Mariner
System 5304 mass spectrometer, and 1H NMR and 13C NMR spectra
were recorded on a Bruker DPX400 or DPX300 spectrometer at
25 ꢁC with TMS and solvent signals allotted as internal standards.
4.2. Antimicrobial activity
The antibacterial activities of the synthesized compounds were
tested against two Gram-positive bacterial strains (B. subtilis ATCC
6633, S. aureus ATCC 25923, kanamycin as positive control) and
a Gram-negative bacterial strain (E. coli ATCC 35218, penicillin as
positive control) using LB medium, The antifungal activities of the
compounds was tested against C. albicans ATCC 10231 (ketocona-
zole as positive control) using RPMI-1640 medium. The MIC50s of
the test compounds were determined by a colorimetric method
using the dye MTT [23,24]. A stock solution of the synthesized
Chemical shifts were reported in ppm (d). Elemental analyses were
performed on a CHNeO-Rapid instrument and were within ꢀ0.4%
of the theoretical values.
4.4.1. General procedure for preparation of compounds 1aeh
An appropriately substituted phenylacetic acid (50 mmol) was
added to a solution of sodium ethoxide (60 mmol) in ethanol
compound (1000
mg/mL) in DMSO was prepared and graded
quantities of the test compounds were incorporated in specified

Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 46 (2011) 4904e4914
4911
(70 mL) and the mixture was stirred at 25 ꢁC for 10 min. Ethyl
bromoacetate (55 mmol) was added and the stirred mixture was
heated under reflux temperature for 4e6 h. The precipitated salt
was filtered off and the filtrate concentrated under reduced pres-
sure. The resulted oil was dissolved in EtOAc and washed with
water and brine. Then the solution was dried over MgSO4 and
concentrated under reduced pressure. The oily residue was then
purified by column chromatography on silica gel to give 1aeh in
yields of 75%e88%.
4.4.3.5. 4-(3,5-Dibromophenylamino)-3-(4-fluorophenyl)furan-2(5H)-
one (8). Colorless crystal, 79%, mp 125e127 ꢁC, 1H NMR (DMSO-d6):
4.99 (s, 2H); 7.06 (s, 2H); 7.14 (t, J ¼ 9.0 Hz, 2H); 7.25 (s, 1H); 7.36 (dd,
J ¼ 8.8 Hz, J ¼ 5.7 Hz, 2H); 9.50 (s, 1H). 13C NMR (DMSO-d6): 172.82;
163.98; 158.74; 140.66; 134.12; 131.37; 129.23; 128.78; 123.40;
122.88; 97.46; 66.42. EIMS m/z 427 (Mþ). Anal. Calcd for
C16H10Br2FNO2: C, 45.00; H, 2.36; Br, 37.42; F, 4.45; N, 3.28; found: C,
45.08; H, 2.34; Br, 37.35; F, 4.46; N, 3.27.
4.4.3.6. 3-(4-Chlorophenyl)-4-(3-chlorophenylamino)furan-2(5H)-
one (9). White powder, 75%, mp 142e144 ꢁC, 1H NMR (DMSO-d6):
5.02 (s, 2H); 7.05 (s,1H); 7.07 (t, J ¼ 8.6 Hz,1H); 7.10 (d, J ¼ 8.4 Hz,1H);
7.24 (d, J ¼ 8.8 Hz,1H); 7.26 (d, J ¼ 7.9 Hz, 2H); 7.46 (d, J ¼ 8.2 Hz, 2H);
9.60 (s, 1H). 13C NMR (DMSO-d6): 172.79; 158.85; 140.62; 137.66;
136.58; 134.11; 132.63; 130.67; 129.74; 123.85; 122.93; 122.68; 97.51;
66.42. EIMS m/z 319 (Mþ). Anal. Calcd for C16H11Cl2NO2: C, 60.02; H,
3.46; Cl, 22.15; N, 4.37; found: C, 60.11; H, 3.43; Cl, 22.12; N, 4.38.
4.4.2. General procedure for preparation of compounds 2aeh
A dropwise solution of compound 1 (10 mmol) in dry THF
(10 mL) was added to a suspension of NaH (24 mmol) in dry THF
(20 mL) in an ice-cold bath and the stirring was maintained at room
temperature for 5e8 h (monitored by TLC). Water (30 mL) was
added and the solution was extracted twice with ethyl ether. The
aqueous phase was cooled to 0 ꢁC and then acidified with
concentrated hydrochloric acid to give a solid precipitate. Filtration
and washing with water furnished compounds 2. This material was
used without further purification.
4.4.3.7. 3-(4-Bromophenyl)-4-(phenylamino)furan-2(5H)-one (10).
Yellow crystal, 62%, mp 209e211 ꢁC, 1H NMR (DMSO-d6): 4.98 (s,
2H); 7.07e7.15 (m, 3H); 7.25 (d, J ¼ 7.2 Hz, 1H); 7.28 (d, J ¼ 7.4 Hz,
1H); 7.34 (d, J ¼ 8.6 Hz, 2H); 7.49 (d, J ¼ 8.4 Hz, 2H); 9.52 (s, 1H). 13C
NMR (DMSO-d6): 172.68; 158.80; 140.65; 134.04; 132.03; 131.91;
131.36; 130.78; 130.30; 123.77; 97.53; 66.42. EIMS m/z 329 (Mþ).
Anal. Calcd for C16H12BrNO2: C, 58.20; H, 3.66; Br, 24.20; N, 4.24;
found: C, 58.27; H, 3.66; Br, 24.24; N, 4.23.
4.4.3. General procedureforpreparationof3-aryl-4-arylaminofuran-
2(5H)-ones (3e37)
A
mixture of compound 2 (0.5 mmol), an appropriately
substituted aniline (0.6 mmol) and p-toluene sulfonic acid (TsOH,
3.4 mg, 0.02 mmol) was heated to 90 ꢁC for 10 min. 5 mL 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.
4.4.3.8. 3-(4-Bromophenyl)-4-(pyridin-2-ylamino)furan-2(5H)-one
(11). Colorlesscrystal, 45%, mp184e186 ꢁC,1HNMR(DMSO-d6):4.91
(s, 2H); 7.01e7.11 (m, 4H); 7.36 (d, J ¼ 8.4 Hz, 2H); 7.50 (d, J ¼ 8.6 Hz,
2H); 9.43 (s, 1H). 13C NMR (DMSO-d6): 172.87; 161.04; 148.90;
138.35; 136.07; 134.10; 131.33; 130.49; 123.66; 118.25; 94.35; 65.94.
EIMS m/z 329 (Mþ). Anal. Calcd for C15H11BrN2O2: C, 54.40; H, 3.35;
Br, 24.13; N, 8.46; found: C, 54.49; H, 3.34; Br, 24.14; N, 8.44.
4.4.3.1. 4-(2-Chlorophenylamino)-3-phenylfuran-2(5H)-one
(4).
Colorless crystal, 51%, mp 109e110 ꢁC, 1H NMR (DMSO-d6): 4.80
(s, 2H); 7.04e7.24 (m, 8H); 7.45 (d, J ¼ 7.5 Hz, 1H); 9.29 (s, 1H). 13C
NMR (DMSO-d6): 172.74; 158.89; 140.68; 134.08; 132.98; 130.93;
130.51; 128.67; 127.51; 123.23; 122.25; 120.72; 97.44; 66.39. EIMS
m/z 285 (Mþ). Anal. Calcd for C16H12ClNO2: C, 67.26; H, 4.23; Cl,
12.41; N, 4.90; found: C, 67.32; H, 4.22; Cl, 12.43; N, 4.92.
4.4.3.9. 3-(4-Bromophenyl)-4-(methyl(phenyl)amino)furan-2(5H)-
one (12). Colorless crystal, 51%, mp 157e158 ꢁC, 1H NMR (DMSO-
d6): 3.27 (s, 3H); 4.97 (s, 2H); 6.84 (d, J ¼ 8.4 Hz, 2H); 7.03e7.15 (m,
5H); 7.19 (d, J ¼ 8.4 Hz, 2H). 13C NMR (DMSO-d6): 172.68; 158.82;
140.66; 134.04; 132.04; 131.90; 131.36; 130.78; 130.32; 123.75;
97.53; 66.44; 39.85. EIMS m/z 343 (Mþ). Anal. Calcd for
C17H14BrNO2: C, 59.32; H, 4.10; Br, 23.21; N, 4.07; found: C, 59.41; H,
4.09; Br, 23.22; N, 4.04.
4.4.3.2. 4-(3-chlorophenylamino)-3-phenylfuran-2(5H)-one
(5).
Colorless crystal, 89%, mp 146e148 ꢁC, 1H NMR (DMSO-d6): 5.04 (s,
2H); 7.02e7.09 (m, 3H); 7.19e7.26 (m, 2H); 7.29e7.39 (m, 4H); 7.54
(s, 1H). 13C NMR (DMSO-d6): 172.72; 158.86; 140.69; 134.08;
134.00; 131.16; 130.51; 128.64; 127.73; 123.20; 122.24; 120.69;
97.47; 66.41. EIMS m/z 285 (Mþ). Anal. Calcd for C16H12ClNO2: C,
67.26; H, 4.23; Cl, 12.41; N, 4.90; found: C, 67.29; H, 4.25; Cl, 12.40;
N, 4.91.
4.4.3.10. 3-(4-Bromophenyl)-4-(2-chlorophenylamino)furan-2(5H)-
one (13). Colorless crystal, 69%, mp 153e155 ꢁC, 1H NMR (DMSO-
d6): 4.78 (s, 2H); 7.07e7.22 (m, 5H); 7.38 (d, J ¼ 8.4 Hz, 2H); 7.46 (d,
J ¼ 7.4 Hz, 1H); 9.42 (s, 1H). 13C NMR (DMSO-d6): 172.79; 158.91;
140.68; 134.10; 132.96; 132.01; 131.88; 126.79; 126.03; 125.85;
125.25; 122.66; 97.53; 66.43. EIMS m/z 365 (Mþ). Anal. Calcd for
C16H11BrClNO2: C, 52.70; H, 3.04; Br, 21.91; Cl, 9.72; N, 3.84; found:
C, 52.64; H, 3.04; Br, 21.95; Cl, 9.73; N, 3.85.
4.4.3.3. 4-(2-Nitrophenylamino)-3-phenylfuran-2(5H)-one (6). Orange
crystal, 79%, mp 169e170 ꢁC, 1H NMR (CDCl3): 3.65 (s, 2H); 5.54 (dd,
J ¼ 8.4 Hz, J ¼ 1.1 Hz, 1H); 5.61 (td, J ¼ 8.4 Hz, J ¼ 1.2 Hz, 1H); 5.85 (td,
J ¼ 8.3 Hz, J ¼ 1.4 Hz, 1H); 5.91e6.04 (m, 5H); 6.68 (dd, J ¼ 8.4 Hz,
J ¼ 1.6 Hz, 1H); 8.59 (s, 1H). 13C NMR (DMSO-d6): 172.70; 158.82;
140.71; 136.86; 134.05; 131.73; 130.51; 130.14; 127.87; 125.65; 122.25;
120.68; 97.41; 66.48. EIMS m/z 296 (Mþ). Anal. Calcd for C16H12N2O4: C,
64.86; H, 4.08; N, 9.46; found: C, 64.82; H, 4.09; N, 9.44.
4.4.3.11. 3-(4-Bromophenyl)-4-(3-chlorophenylamino)furan-2(5H)-
one (14). Colorless crystal, 83%, mp 148e150 ꢁC, 1H NMR (DMSO-
d6): 5.03 (s, 2H); 7.02e7.12 (m, 3H); 7.24 (d, J ¼ 8.6 Hz, 1H); 7.27 (d,
J ¼ 8.6 Hz, 2H); 7.49 (d, J ¼ 8.6 Hz, 2H); 9.61 (s, 1H). 13C NMR
(DMSO-d6): 172.65; 158.83; 140.66; 136.80; 134.04; 132.84; 131.91;
131.69; 124.55; 123.96; 123.40; 122.92; 97.41; 66.37. EIMS m/z 365
(Mþ). Anal. Calcd for C16H11BrClNO2: C, 52.70; H, 3.04; Br, 21.91; Cl,
9.72; N, 3.84; found: C, 52.77; H, 3.03; Br, 21.93; Cl, 9.71; N, 3.83.
4.4.3.4. 4-(3-Nitrophenylamino)-3-phenylfuran-2(5H)-one
(7).
Yellow crystal, 47%, mp 203e205 ꢁC, 1H NMR (DMSO-d6): 3.32 (s,
2H); 4.91 (s, 1H); 5.40 (d, J ¼ 8.9 Hz, 2H); 5.53 (s, 1H); 5.80 (t,
J ¼ 7.3 Hz, 1H); 5.93 (t, J ¼ 7.5 Hz, 2H); 5.99 (d, J ¼ 7.5 Hz, 2H); 6.64
(d, J ¼ 8.9 Hz, 1H). 13C NMR (DMSO-d6): 172.38; 158.83; 158.74;
140.63; 134.09; 132.92; 131.67; 130.90; 130.51; 127.57; 122.21;
120.68; 97.47; 66.45. EIMS m/z 296 (Mþ). Anal. Calcd for
C16H12N2O4: C, 64.86; H, 4.08; N, 9.46; found: C, 64.89; H, 4.09;
N, 9.43.
4.4.3.12. 3-(4-Bromophenyl)-4-(4-chlorophenylamino)furan-2(5H)-
one (15). Colorless crystal, 49%, mp 203e205 ꢁC, 1H NMR (DMSO-
d6): 4.99 (s, 2H); 7.11 (d, J ¼ 8.9 Hz, 2H); 7.29 (d, J ¼ 8.6 Hz, 2H); 7.31

4912
Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 46 (2011) 4904e4914
(d, J ¼ 8.6 Hz, 2H); 7.50 (d, J ¼ 8.6 Hz, 2H); 9.57 (s, 1H). 13C NMR
(DMSO-d6): 172.88; 158.93; 140.69; 134.11; 131.99; 131.72; 131.07;
128.70; 126.84; 123.95; 97.49; 66.44. EIMS m/z 365 (Mþ). Anal.
Calcd for C16H11BrClNO2: C, 52.70; H, 3.04; Br, 21.91; Cl, 9.72; N,
3.84; found: C, 52.73; H, 3.03; Br, 21.88; Cl, 9.74; N, 3.84.
2H); 9.07 (s, 1H). 13C NMR (DMSO-d6): 172.62; 160.26; 158.94;
140.70; 131.90; 131.05; 129.10; 125.82; 123.51; 123.07; 122.90;
114.77; 97.50; 66.41; 55.39. EIMS m/z 359 (Mþ). Anal. Calcd for
C17H14BrNO3: C, 56.69; H, 3.92; Br, 22.18; N, 3.89; found: C, 56.74; H,
3.91; Br, 22.15; N, 3.90.
4.4.3.13. 3-(4-Bromophenyl)-4-(4-bromophenylamino)furan-2(5H)-
one (16). White powder, 37%, mp 174e176 ꢁC, 1H NMR (DMSO-d6):
5.00 (s, 2H); 7.06 (d, J ¼ 8.8 Hz, 2H); 7.32 (d, J ¼ 8.4 Hz, 2H); 7.43 (d,
J ¼ 8.8 Hz, 2H); 7.51 (d, J ¼ 8.6 Hz, 2H); 9.56 (s,1H). 13C NMR (DMSO-
d6): 172.85; 158.90; 140.69; 134.09; 132.80; 132.05; 131.48; 127.61;
123.08; 120.46; 97.47; 66.41. EIMS m/z 409 (Mþ). Anal. Calcd for
C16H11Br2NO2: C, 46.98; H, 2.71; Br, 39.07; N, 3.42; found: C, 46.90;
H, 2.71; Br, 39.09; N, 3.43.
4.4.3.20. 3-(4-Bromophenyl)-4-(4-methoxyphenylamino)furan-2(5H)-
one(23). Colorless crystal, 53%, mp 204e206 ꢁC,1H NMR (DMSO-d6):
3.73 (s, 3H); 4.84 (s, 2H); 6.85 (d, J ¼ 8.8 Hz, 2H); 7.12 (d, J ¼ 8.8 Hz,
2H); 7.37 (d, J ¼ 8.4 Hz, 2H); 7.50 (d, J ¼ 8.6 Hz, 2H); 9.36 (s, 1H). 13C
NMR (DMSO-d6): 172.65; 160.97; 158.82; 140.70; 132.11; 131.95;
132.18; 128.33; 123.35; 120.02; 97.48; 66.43; 55.62. EIMS m/z 359
(Mþ). Anal. Calcd for C17H14BrNO3: C, 56.69; H, 3.92; Br, 22.18; N,
3.89; found: C, 56.66; H, 3.91; Br, 22.20; N, 3.88.
4.4.3.14. 3-(4-Bromophenyl)-4-(2-nitrophenylamino)furan-2(5H)-
one (17). Red crystal, 69%, mp 192e195 ꢁC, 1H NMR (DMSO-d6):
5.04 (s, 2H); 7.23 (d, J ¼ 8.6 Hz, 2H); 7.25e7.33 (m, 2H); 7.44e7.52
(m, 3H); 8.03 (dd, J ¼ 8.6 Hz, J ¼ 1.6 Hz, 1H); 9.78 (s, 1H). 13C NMR
(DMSO-d6): 172.88; 158.92; 140.70; 138.35; 134.14; 132.75; 131.90;
131.17; 126.36; 122.77; 120.25; 117.89; 97.55; 66.42. EIMS m/z 374
(Mþ). Anal. Calcd for C16H11BrN2O4: C, 51.22; H, 2.96; Br, 21.30; N,
7.47; found: C, 51.31; H, 2.95; Br, 21.33; N, 7.48.
4.4.3.21. 3-(4-Bromophenyl)-4-(4-methylphenylamino)furan-2(5H)-
one (24). White powder, 74%, mp 155e157 ꢁC, 1H NMR (DMSO-d6):
2.08 (s, 3H); 4.92 (s, 2H); 7.04 (d, J ¼ 8.6 Hz, 2H); 7.09 (d, J ¼ 8.4 Hz,
2H); 7.37 (d, J ¼ 8.6 Hz, 2H); 7.50 (d, J ¼ 8.6 Hz, 2H); 9.43 (s, 1H). 13C
NMR (DMSO-d6): 172.85; 161.06; 136.08; 134.13; 130.90; 130.55;
130.31; 129.47; 122.71; 119.02; 94.29; 65.96; 20.45. EIMS m/z 343
(Mþ). Anal. Calcd for C17H14BrNO2: C, 59.32; H, 4.10; Br, 23.21; N,
4.07; found: C, 59.40; H, 4.09; Br, 23.23; N, 4.06.
4.4.3.15. 3-(4-Bromophenyl)-4-(3-nitrophenylamino)furan-2(5H)-
one (18). Yellow crystal, 72%, mp 224e226 ꢁC, 1H NMR (DMSO-d6):
5.08 (s, 2H); 7.22 (d, J ¼ 8.6 Hz, 2H); 7.43e7.53 (m, 4H); 7.74 (s, 1H);
7.85 (dt, J ¼ 7.0 Hz, J ¼ 2.3 Hz, 1H); 9.89 (s, 1H). 13C NMR (DMSO-d6):
172.80; 158.91; 157.87; 140.71; 134.10; 132.42; 131.74; 130.08;
129.78; 123.36; 120.31; 117.55; 97.48; 66.40. EIMS m/z 374 (Mþ).
Anal. Calcd for C16H11BrN2O4: C, 51.22; H, 2.96; Br, 21.30; N, 7.47;
found: C, 51.29; H, 2.95; Br, 21.28; N, 7.47.
4.4.3.22. 3-(4-Bromophenyl)-4-(3,5-difluorophenylamino)furan-2(5H)-
one (25). Colorless crystal, 48%, mp 174e176 ꢁC, 1H NMR (DMSO-d6):
5.12 (s, 2H); 6.78 (dd, J ¼ 9.3 Hz, J ¼ 2.2 Hz, 2H); 6.90 (tt, J ¼ 9.3 Hz,
J ¼ 2.4 Hz, 1H); 7.31 (d, J ¼ 8.4 Hz, 2H); 7.54 (d, J ¼ 8.4 Hz, 2H); 9.72 (s,
1H). 13C NMR (DMSO-d6): 172.92; 132.77; 158.98; 140.74; 134.13;
131.90; 131.73; 123.55; 110.58; 97.42; 95.29; 66.42. EIMS m/z 365
(Mþ). Anal. Calcd for C16H10BrF2NO2: C, 52.48; H, 2.75; Br, 21.82; F,
10.38; N, 3.83; found: C, 52.56; H, 2.75; Br, 21.79; F, 10.37; N, 3.83.
4.4.3.16. 3-(4-Bromophenyl)-4-(4-nitrophenylamino)furan-2(5H)-
one (19). Light yellow crystal, 53%, mp 253e255 ꢁC, 1H NMR
(DMSO-d6): 5.20 (s, 2H); 7.20 (d, J ¼ 9.1 Hz, 2H); 7.32 (d, J ¼ 8.6 Hz,
2H); 7.54 (d, J ¼ 8.4 Hz, 2H); 8.08 (d, J ¼ 9.1 Hz, 2H); 10.07 (s,1H). 13C
NMR (DMSO-d6): 172.85; 158.93; 140.76; 138.25; 134.13; 131.68;
130.94; 126.60; 123.77; 122.81; 97.43; 66.36. EIMS m/z 374 (Mþ).
Anal. Calcd for C16H11BrN2O4: C, 51.22; H, 2.96; Br, 21.30; N, 7.47;
found: C, 51.20; H, 2.96; Br, 21.31; N, 7.48.
4.4.3.23. 3-(4-Bromophenyl)-4-(3,5-dichlorophenylamino)furan-
2(5H)-one (26). Colorless crystal, 59%, mp 204e205 ꢁC, 1H NMR
(DMSO-d6): 5.07 (s, 2H); 7.03 (d, J ¼ 1.8 Hz, 2H); 7.23 (s, 1H); 7.24 (d,
J ¼ 8.5 Hz, 2H); 7.51 (d, J ¼ 8.6 Hz, 2H); 9.72 (s,1H). 13C NMR (DMSO-
d6): 172.69; 158.72; 141.11; 134.06; 130.90; 130.61; 130.10; 122.96;
119.97; 119.72; 97.84; 66.52. EIMS m/z 399 (Mþ). Anal. Calcd for
C16H10BrCl2NO2: C, 48.16; H, 2.53; Br, 20.02; Cl, 17.77; N, 3.51;
found: C, 48.11; H, 2.53; Br, 20.04; Cl, 17.79; N, 3.50.
4.4.3.17. 3-(4-Bromophenyl)-4-(3-hydroxyphenylamino)furan-2(5H)-
one (20). Colorless crystal, 33%, mp 166e168 ꢁC, 1H NMR (DMSO-
d6): 3.60 (s, 2H); 6.44 (d, J ¼ 8.0 Hz,1H); 6.94 (d, J ¼ 8.4 Hz,1H); 7.05
(t, J ¼ 8.0 Hz, 1H); 7.15 (s, 1H); 7.28 (d, J ¼ 8.4 Hz, 2H); 7.51 (d,
J ¼ 8.4 Hz, 2H); 9.32 (s, 1H); 10.00 (s, 1H). 13C NMR (DMSO-d6):
172.69; 159.82; 158.84; 140.66; 134.02; 131.97; 131.08; 130.85;
122.89; 120.01; 114.23; 109.75; 97.47; 66.41. EIMS m/z 345 (Mþ).
Anal. Calcd for C16H12BrNO3: C, 55.51; H, 3.49; Br, 23.08; N, 4.05;
found: C, 55.44; H, 3.50; Br, 23.10; N, 4.05.
4.4.3.24. 3-(4-Bromophenyl)-4-(3,5-dibromophenylamino)furan-2(5H)-
one (27). Colorless crystal, 84%, mp 212e214 ꢁC, 1H NMR (DMSO-d6):
5.03 (s, 2H); 7.05 (d, J ¼ 1.6 Hz, 2H); 7.24 (s, 1H); 7.26 (d, J ¼ 8.6 Hz, 2H);
7.53 (d, J ¼ 8.6 Hz, 2H); 9.81 (s, 1H). 13C NMR (DMSO-d6): 172.80;
158.92; 140.70; 134.06; 131.65; 130.81; 127.48; 127.01; 126.55; 115.20;
97.49; 66.40. EIMS m/z 487 (Mþ). Anal. Calcd for C16H10Br3NO2: C,
39.38; H, 2.07; Br, 49.12; N, 2.87; found: C, 39.35; H, 2.06; Br, 49.18; N,
2.87.
4.4.3.25. 3-(4-Bromophenyl)-4-(3,5-dimethoxyphenylamino)furan-
2(5H)-one (28). Colorless crystal, 77%, mp 196e198 ꢁC, 1H NMR
(CDCl3): 3.76 (s, 6H); 4.95 (s, 2H); 6.14 (s, 2H); 6.29 (s, 1H); 6.93 (s,
1H); 7.43 (d, J ¼ 8.8 Hz, 2H); 7.57 (d, J ¼ 8.9 Hz, 2H). 13C NMR
(DMSO-d6): 172.81; 164.60; 158.91; 140.69; 134.07; 131.84; 130.98;
123.21; 97.49; 94.51; 90.27; 66.45; 55.74. EIMS m/z 389 (Mþ). Anal.
Calcd for C18H16BrNO4: C, 55.40; H, 4.13; Br, 20.48; N, 3.59; found: C,
55.49; H, 4.12; Br, 20.46; N, 3.59.
4.4.3.18. 3-(4-Bromophenyl)-4-(4-hydroxyphenylamino)furan-2(5H)-
one (21). Colorless crystal, 31%, mp 216e218 ꢁC, 1H NMR (DMSO-
d6): 4.78 (s, 2H); 6.66 (d, J ¼ 8.8 Hz, 2H); 6.99 (d, J ¼ 8.8 Hz, 2H); 7.37
(d, J ¼ 8.6 Hz, 2H); 7.49 (d, J ¼ 8.6 Hz, 2H); 9.28 (s, 1H); 9.43 (s, 1H).
13C NMR (DMSO-d6): 172.64; 158.82; 158.64; 140.60; 131.77; 131.49;
130.62; 123.06; 120.95; 120.04; 97.50; 66.43. EIMS m/z 345 (Mþ).
Anal. Calcd for C16H12BrNO3: C, 55.51; H, 3.49; Br, 23.08; N, 4.05;
found: C, 55.59; H, 3.48; Br, 23.06; N, 4.06.
4.4.3.26. 3-(3-Chlorophenyl)-4-(2-chlorophenylamino)furan-2(5H)-
one (29). Colorless crystal, 68%, mp 109e111 ꢁC, 1H NMR (DMSO-
d6): 4.80 (s, 2H); 7.08e7.23 (m, 7H); 7.45 (d, J ¼ 7.5 Hz, 1H); 9.51 (s,
1H). 13C NMR (DMSO-d6): 172.72; 158.87; 140.65; 135.77; 134.04;
130.93; 130.50; 128.67; 128.06; 127.60; 126.35; 125.81; 124.29;
4.4.3.19. 3-(4-Bromophenyl)-4-(2-methoxyphenylamino)furan-2(5H)-
one(22). Colorless crystal, 46%, mp 158e159 ꢁC,1H NMR (DMSO-d6):
3.76 (s, 3H); 4.74 (s, 2H); 6.82 (t, J ¼ 7.6 Hz, 1H); 7.03 (t, J ¼ 8.4 Hz,
2H); 7.18 (t, J ¼ 7.8 Hz, 1H); 7.27 (d, J ¼ 8.6 Hz, 2H); 7.43 (d, J ¼ 8.6 Hz,

Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 46 (2011) 4904e4914
4913
121.48; 97.44; 66.39. EIMS m/z 319 (Mþ). Anal. Calcd for
C16H11Cl2NO2: C, 60.02; H, 3.46; Cl, 22.15; N, 4.37; found: C, 59.96;
H, 3.46; Cl, 22.17; N, 4.36.
125.58; 123.79; 123.30; 116.76; 97.47; 66.40. EIMS m/z 487 (Mþ).
Anal. Calcd for C16H10Br3NO2: C, 39.38; H, 2.07; Br, 49.12; N, 2.87;
found: C, 39.46; H, 2.06; Br, 49.09; N, 2.88.
4.4.3.27. 3-(3-Chlorophenyl)-4-(3-chlorophenylamino)furan-2(5H)-
one (30). Colorless crystal, 89%, mp 173e175 ꢁC,1H NMR (DMSO-d6):
5.03 (s, 2H); 7.03e7.14 (m, 3H); 7.23e7.35 (m, 5H); 9.71 (s, 1H). 13C
NMR (DMSO-d6): 172.73; 158.89; 140.66; 135.72; 134.27; 133.96;
130.55; 130.13; 128.20; 127.64; 126.39; 124.80; 124.02; 123.44;
97.47; 66.42. EIMS m/z 319 (Mþ). Anal. Calcd for C16H11Cl2NO2: C,
60.02; H, 3.46; Cl, 22.15; N, 4.37; found: C, 60.07; H, 3.45; Cl, 22.18; N,
4.38.
4.4.3.34. 3-(4-Methoxyphenyl)-4-(3,5-dichlorophenylamino)furan-
2(5H)-one (37). White powder, 63%, mp 206e208 ꢁC, 1H NMR
(DMSO-d6): 3.62 (s, 3H); 5.08 (s, 2H); 7.01 (d, J ¼ 1.8 Hz, 2H); 7.23 (s,
1H); 7.20 (d, J ¼ 8.5 Hz, 2H); 7.49 (d, J ¼ 8.6 Hz, 2H); 9.71 (s, 1H). 13C
NMR (DMSO-d6): 172.71; 158.77; 141.10; 134.11; 130.94; 130.60;
130.13; 122.89; 119.93; 119.75; 97.84; 66.48; 56.66. EIMS m/z 349
(Mþ). Anal. Calcd for C17H13Cl2NO3: C, 58.31; H, 3.74; Cl, 20.25; N,
4.00; found: C, 58.27; H, 3.74; Cl, 20.29; N, 4.01.
4.4.3.28. 3-(3-Chlorophenyl)-4-(3-nitrophenylamino)furan-2(5H)-
one (31). Orange crystal, 65%, mp 159e160 ꢁC, 1H NMR (DMSO-d6):
5.03 (s, 2H); 7.18e7.32 (m, 6H); 7.47 (td, J ¼ 8.8 Hz, J ¼ 1.8 Hz, 1H);
8.02 (dd, J ¼ 8.8 Hz, J ¼ 1.8 Hz, 1H); 9.83 (s, 1H). 13C NMR (DMSO-
d6): 172.80; 158.92; 158.70; 140.68; 134.72; 134.11; 130.74; 130.09;
128.34; 127.36; 126.02; 125.87; 120.18; 114.49; 97.50; 66.43. EIMS
m/z 330 (Mþ). Anal. Calcd for C16H11ClN2O4: C, 58.11; H, 3.35; Cl,
10.72; N, 8.47; found: C, 58.18; H, 3.35; Cl, 10.70; N, 8.46.
4.4.4. The synthesis of 4-(3,5-dichlorophenylamino)-3-(4-hydroxy-
phenyl)furan-2(5H)-one (38)
Under a hydrogen atmosphere, Pd/C (10%, 0.30 g) was added to 3
(0.64 g, 1.5 mmol) dissolved in THF/EtOH (5/15 mL). The reaction
mixture was stirred for 7 h at room temperature. After filtration of
the catalyst and evaporation of the solvent, crystallization from
acetoneepetroleum ether produced 0.47 g (93%) of 38 (colorless
crystal). Mp 219e221 ꢁC, 1H NMR (DMSO-d6): 5.07 (s, 2H); 7.01 (d,
J ¼ 1.8 Hz, 2H); 7.20 (s, 1H); 7.26 (d, J ¼ 8.8 Hz, 2H); 7.55 (d,
J ¼ 8.9 Hz, 2H); 9.24 (s, 1H); 9.85 (s, 1H). 13C NMR (DMSO-d6):
172.68; 163.24; 159.22; 140.97; 134.05; 132.08; 131.83; 122.07;
120.53; 118.79; 97.27; 66.52. EIMS m/z 335 (Mþ). Anal. Calcd for
C16H11Cl2NO3: C, 57.16; H, 3.30; Cl, 21.09; N, 4.17; found: C, 57.24; H,
3.31; Cl, 21.05; N, 4.16.
4.4.3.29. 3-(3-Chlorophenyl)-4-(2-methoxyphenylamino)furan-2(5H)-
one (32). White powder, 68%, mp 92e94 ꢁC, 1H NMR (DMSO-d6):
3.75 (s, 3H); 4.76 (s, 2H); 6.81 (t, J ¼ 7.5 Hz, 1H); 6.99 (d, J ¼ 8.2 Hz,
1H); 7.04 (d, J ¼ 7.9 Hz, 1H); 7.14e7.30 (m, 5H); 9.20 (s, 1H). 13C NMR
(DMSO-d6): 172.69; 161.75; 158.83; 140.63; 134.34; 134.07; 130.64;
130.17; 128.22; 127.83; 126.78; 125.83; 123.77; 122.50; 97.42; 66.37;
55.92. EIMS m/z 315 (Mþ). Anal. Calcd for C17H14ClNO3: C, 64.67; H,
4.47; Cl, 11.23; N, 4.44; found: C, 64.59; H, 4.48; Cl, 11.25; N, 4.43.
Acknowledgments
The work was financed by a Project supported by Hunan
Provincial Natural Science Foundation of China (grant No. 11JJ3113),
by the Key Laboratory of Plant Resources Conservation and Utili-
zation (Jishou University), College of Hunan Province (grant No. JSK
201106), by the Key Laboratory of Hunan Forest Products and
Chemical Industry Engineering of Hunan Province (grant No.
JDZ201102), by the Key Laboratory of Ecotourism’s Application
Technology, Hunan Province (grant No. JDSTLY 01103) and by aid
program for Science and Technology Innovative Research Team
(Chemicals of Forestry Resources and Development of Forest
Products) in Higher Educational Institutions of Hunan Province.
4.4.3.30. 3-(3-Chlorophenyl)-4-(3,5-dichlorophenylamino)furan-
2(5H)-one (33). Colorless crystal, 48%, mp 189e191 ꢁC, 1H NMR
(DMSO-d6): 4.76 (s, 2H); 7.05 (d, J ¼ 1.8 Hz, 2H); 7.10e7.21 (m, 3H);
7.23 (s, 1H); 7.25 (d, J ¼ 8.6 Hz, 1H); 9.68 (s, 1H). 13C NMR (DMSO-
d6): 172.80; 158.93; 140.68; 134.88; 134.07; 131.35; 130.85; 129.02;
128.44; 127.25; 120.36; 118.41; 97.45; 66.39. EIMS m/z 353 (Mþ).
Anal. Calcd for C16H10Cl3NO2: C, 54.19; H, 2.84; Cl, 29.99; N, 3.95;
found: C, 54.27; H, 2.85; Cl, 29.96; N, 3.94.
4.4.3.31. 3-(3-Bromophenyl)-4-(4-bromophenylamino)furan-2(5H)-
one(34). Colorless crystal, 89%, mp 157e159 ꢁC, 1H NMR (DMSO-
d6): 4.97 (s, 2H); 7.08 (d, J ¼ 9.0 Hz, 2H); 7.21e7.28 (m, 3H); 7.32
(d, J ¼ 8.4 Hz, 1H); 7.38 (d, J ¼ 8.2 Hz, 1H); 7.44 (s, 1H); 9.66 (s,
1H). 13C NMR (DMSO-d6): 172.78; 158.78; 140.76; 134.09;
133.45; 131.02; 130.21; 129.88; 127.72; 125.26; 122.21; 120.04;
97.47; 66.38. EIMS m/z 409 (Mþ). Anal. Calcd for C16H11Br2NO2:
C, 46.98; H, 2.71; Br, 39.07; N, 3.42; found: C, 46.91; H, 2.71; Br,
39.09; N, 3.43.
References
[1] H.W. Boucher, G.H. Talbot, J.S. Bradley, J.E. Edwards Jr., D. Gilbert, L.B. Rice,
M. Scheld, B. Spellberg, J. Bartlett, Clin. Infect. Dis. 48 (2009) 1e12.
[2] J.J. Barker, Drug Discov. Today 11 (2006) 391e404.
[3] I. Masip, E. Pérez-Payá, A. Messeguer, Comb. Chem. High T. Scr. 8 (2005)
235e239.
[4] P. Van de Vijver, T. Ostrowski, B. Sproat, J. Goebels, O. Rutgeerts, A. Van Aer-
schot, M. Waer, P. Herdewijn, J. Med. Chem. 51 (2008) 3020e3029.
[5] J.M. Berge, N.J.P. Broom, C.S.V. Houge-Frydrych, R.L. Jarvest, L. Mensah,
D.J. Mcnair, P.J. O’hanlon, A.J. Pope, S. Rittenhouse, J. Antibiot. 53 (2000)
1282e1292.
[6] L.H. Jonathan, B. Christian, J. Dieter, M. Jürgen, É Audrey, P.B. Sébastien,
C. Robert, L. Jacques, Biochemistry 46 (2007) 13190e13198.
[7] X. Qiu, C.A. Janson, W.W. Smith, S.M. Green, P. McDevitt, K. Johanson, P. Carter,
M. Hibbs, C. Lewis, A. Chalker, A. Fosberry, J. Lalonde, J. Berge, P. Brown,
C.S. Houge-Frydrych, R.L. Jarvest, Protein Sci. 10 (2001) 2008e2016.
[8] A.L. Stefanska, N.J. Coates, L.M. Mensah, A.J. Pope, S.J. Ready, S.R. Warr,
J. Antibiot. 53 (2000) 345e350.
4.4.3.32. 3-(3-Bromophenyl)-4-(3,5-dichlorophenylamino)furan-
2(5H)-one (35). Colorless crystal, 92%, mp 200e202 ꢁC, 1H NMR
(DMSO-d6): 5.06 (s, 2H); 7.03 (d, J ¼ 1.8 Hz, 2H); 7.21e7.28 (m,
3H); 7.41 (s, 1H); 7.42 (d, J ¼ 7.8 Hz, 1H); 9.78 (s, 1H). 13C NMR
(DMSO-d6): 172.69; 159.21; 140.98; 134.03; 133.24; 131.06;
129.97; 129.51; 127.51; 123.23; 121.26; 120.16; 97.25; 66.50.
EIMS m/z 399 (Mþ). Anal. Calcd for C16H10BrCl2NO2: C, 48.16; H,
2.53; Br, 20.02; Cl, 17.77; N, 3.51; found: C, 48.21; H, 2.53; Br,
19.98; Cl, 17.78; N, 3.51.
[9] R. Farhanullah, T. Kang, E.-J. Yoon, E.-C. Choi, S. Kim, J. Lee, Eur. J. Med. Chem.
44 (2009) 239e250.
[10] P. Van de Vijver, G.H.M. Vondenhoff, T.S. Kazakov, E. Semenova,
K. Kuznedelov, A. Metlitskaya, A. Van Aerschot, K. Severinov, J. Bacteriol. 191
(2009) 6273e6280.
[11] C. Balg, M. De Mieri, J.L. Huot, S.P. Blais, J. Lapointe, R. Chênevert, Bioorg. Med.
Chem. 18 (2010) 7868e7872.
4.4.3.33. 3-(3-Bromophenyl)-4-(3,5-dibromophenylamino)furan-
2(5H)-one (36). Colorless crystal, 75%, mp 208e210 ꢁC, 1H NMR
(DMSO-d6): 4.98 (s, 2H); 7.05 (d, J ¼ 1.6 Hz, 2H); 7.20e7.29 (m, 3H);
7.43 (s, 1H); 7.45 (d, J ¼ 8.0 Hz, 1H); 9.52 (s, 1H). 13C NMR (DMSO-
d6): 172.70; 158.85; 140.62; 134.05; 131.83; 130.66; 130.37; 128.62;
[12] Y. Wu, K. Yu, B. Xu, L. Chen, X. Chen, J. Mao, A. Danchin, X. Shen, D. Qu,
H. Jiang, J. Antimicrob. Chemother. 60 (2007) 502e509.

4914
Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 46 (2011) 4904e4914
[13] J.G. Hurdle, A.J. O’Neill, I. Chopra, J. Antimicrob. Agents Chemother. 48 (2005)
4821e4833.
[19] E. Lattmann, N. Sattayasai, C.S. Schwalbe, S. Niamsanit, D.C. Billington, Curr.
Drug Discov. Tech. 3 (2006) 125e134.
[14] F. Bailly, C. Queffélec, G. Mbemba, J.F. Mouscadet, N. Pommery, J. Pommery,
J.P. Hénichart, P. Cotelle, Eur. J. Med. Chem. 43 (2008) 1222e1229.
[15] V. Weber, C. Rubat, E. Duroux, C. Lartigue, M. Madesclaire, P. Coudert, Bioorg.
Med. Chem. 13 (2005) 4552e4564.
[20] Z.-P. Xiao, X.-B. He, Z.-Y. Peng, T.-J. Xiong, J. Peng, L.-H. Chen, H.-L. Zhu, Bioorg.
Med. Chem. 19 (2011) 1571e1579.
[21] Z.-P. Xiao, J.-Y. Xue, S.-H. Tan, H.-Q. Li, H.-L. Zhu, Bioorg. Med. Chem. 15 (2007)
4212e4219.
[16] P. Cotelle, N. Cotelle, E. Teissier, H. Vezin, Bioorg. Med. Chem. 11 (2003)
1087e1093.
[22] Z.-P. Xiao, R.-Q. Fang, H.-Q. Li, J.-Y. Xue, Y. Zheng, H.-L. Zhu, Eur. J. Med. Chem.
43 (2008) 1828e1836.
[17] P. Senel, L. Tichotová, I. Votruba, V. Buchta, M. Spulák, J. Kunes, M. Nobilis,
O. Krenk, M. Pour, Bioorg. Med. Chem. 18 (2010) 1988e2000.
[18] B. Nadal, S.A. Thetiot-Laurent, S. Pin, J.P. Renault, D. Cressier, G. Rima, A. Le Roux,
S. Meunier, A. Wagner, C. Lion, T. Le Gall, Bioorg. Med. Chem. 18 (2010) 7931e7939.
[23] C.C. Chiou, N. Mavrogiorgos, E. Tillem, R. Hector, T.J. Walsh, Antimicrob.
Agents Ch. 45 (2001) 3310e3321.
[24] J. Meletiadis, J.F. Meis, J.W. Mouton, J.P. Donnelly, P.E. Verweij, J. Clin.
Microbiol. 38 (2000) 2949e2954.