Discovery of vinylogous carbamates as a novel class of β-ketoacyl-acyl carrier protein synthase III (FabH) inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.06.048

β-Ketoacyl-acyl carrier protein synthase III (FabH) catalyzes the initial step of fatty acid biosynthesis via a type II fatty acid synthase in most bacteria. The important role of this essential enzyme combined with its unique structural features and ubiq

Full text of DOI:10.1016/j.bmc.2011.06.048

Bioorganic & Medicinal Chemistry 19 (2011) 4454–4459
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of vinylogous carbamates as a novel class of b-ketoacyl-acyl
carrier protein synthase III (FabH) inhibitors
Huan-Qiu Li a, , Yin Luo b, Hai-Liang Zhu b
⇑
a College of Pharmaceutical Science, Soochow University, Suzhou 215123, 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:
b-Ketoacyl-acyl carrier protein synthase III (FabH) catalyzes the initial step of fatty acid biosynthesis via a
type II fatty acid synthase in most bacteria. The important role of this essential enzyme combined with its
unique structural features and ubiquitous occurrence in bacteria has made it an attractive new target for
the development of new FabH inhibitors. We first used a structure-based approach to develop 24 new
vinylogous carbamates (4a–15a, 4b–15b) that target FabH for the development of new antibiotics in this
paper. Potent FabH inhibitory and selective anti- Gram-negative bacteria activities were observed in most
of these vinylogous carbamates. Especially, compound 6a and 7a showed the most potent FabH inhibitory
Received 20 April 2011
Revised 13 June 2011
Accepted 15 June 2011
Available online 21 June 2011
Keywords:
Vinylogous carbamates
FabH
activity with IC50 of 2.6 and 3.3 lM, respectively. Docking simulation was performed to position com-
pound 6a into the Escherichia coli FabH active site and the possible binding conformation of compounds
has been proposed. The biological data and molecular docking indicated that compounds 6a and 7a were
potent inhibitors of E. coli FabH as antibiotics deserving further research.
Ó 2011 Elsevier Ltd. All rights reserved.
Antibiotics
Structure–activity relationship
1. Introduction
also been demonstrated to be essential for organism survival and
it is presented in a wide number of important human pathogens.
Bacteria resistant to known therapies are a growing threat
across the globe. An increasing fraction of bacterial isolates shows
reduced susceptibility to our most trusted antibiotics. In order to
prevent this serious medical problem, the discovery of new types
of antibacterial agents or the expansion of bioactivity of the previ-
ous drugs is a very important task.1 Therefore, in recent years, the
research has been focused on the development of new antibacterial
agents, which may act through structure design and novel targets,
overcoming the problem of acquired resistance. One of the most
attractive biochemical pathways that could be targeted for new
antibacterial agents is the fatty acid biosynthesis (FAS). This path-
way has been demonstrated to be essential for the bacteria cell
survival,2 and differs considerably from human FAS pathway.
While in humans fatty acid synthesis occurs in a homodimeric
multifunctional enzyme,3,4 in bacteria the pathway is composed
of various discrete enzymes and each one can be considered a
putative molecular target. Those features make the type II FAS
pathway a potential target for new antimicrobial agents.
Furthermore, some chemical compounds have shown to inhibit
FabH from diverse microorganisms, including multi-drug resistant
strains.5,6 These facts support the idea that FabH can be used as an
effective molecular target for the development of new antimicrobial
agents. Schiff bases and their metal complexes display several poten-
tially useful biological activities, of which the antibacterial, anti-
fungal, and FabH inhibitory activities has been widely studied.7–9
However, vinylogous carbamates which are tautomers of Schiff
bases were rarely reported for biological activities and they mostly
appeared as chemical intermediates for cyclization.10,11 Due to the
evident chemical structure similarity as Schiff bases, an enamine
should bear relevant biological properties as the corresponding
Schiff base. As a proof for this hypothesis, our recent report showed
that vinylogous carbamates have considerable antibacterial activi-
ties.12 To the continuation of this work and together with aforemen-
tioned hypothesis, we directed our efforts to the investigation of the
FabH inhibitory activity of new vinylogous carbamates. In this pa-
per, we described the synthesis of vinylogous carbamates and the
evaluation of these compounds as FabH inhibitors for the first time.
The antimicrobial activity of these vinylogous carbamates against
two Gram-negative bacterial strains, Escherichia coli (E. coli) and
Pseudomonas fluorescence (P. fluorescence), and two Gram-positive
bacterial strains, Bacillus Subtilis (B. subtilis) and Staphylococcus
aureus (S. aureus) was also determined. Docking simulations were
performed using the X-ray crystallographic structure of the FabH
A key enzyme in this pathway is the b-ketoacyl-acyl carrier pro-
tein synthase III (FabH), which is the responsible enzyme of the first
pathway reaction and play an important regulatory role. FabH has
⇑
Corresponding author. Tel./fax: +86 512 65882092.
E-mail address: huanqiuli@suda.edu.cn (H.-Q. Li).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.048

H.-Q. Li et al. / Bioorg. Med. Chem. 19 (2011) 4454–4459
4455
of E. coli in complex with an inhibitor to explore the binding modes
of these compounds at the active site.
Gram-negative bacterial strains: E. coli and P. fluorescence and
two Gram-positive bacterial strains: B. subtilis and S. aureus by
MTT method. The MICs (minimum inhibitory concentrations) of
the compounds against these bacteria were presented in Table 1.
Standard antibacterial agent kanamycin B was also screened under
identical conditions for comparison. The results revealed that most
of the synthesized vinylogous carbamates exhibited potent anti-
bacterial activity. Of all the compounds tested, 6a, 7a, 10a, 12a,
12b, 13a demonstrated excellent inhibitory activity against
Gram-negative Escherichia coli and Pseudomonas aeruginosa. Espe-
2. Chemistry
The general method for preparing the final compounds 4a–15a
and 4b–15b was outlined in Scheme 1.12 In order to find more
effective vinylogous carbamates and convenient to discuss the dif-
ferent trend in SAR from the former report,12 the substituent on the
para position of compound 1 was changed from previously used
methoxy group to benzyloxy group. The designed vinylogous car-
bamates were prepared by a dehydration reaction of the aldehyde
(2) with different arylamines (3a–3l). Aldehyde 2, which was not
commercially available, was synthesized using modified proce-
dures of Beccalh.13 The crude products consisted of the mixture
of Z- and E-isomers (4a–15a and 4b–15b). Subsequent purification
with flash chromatography, 4a–15a and 4b–15b were obtained as
single isomer with high purity. The structures of the vinylogous
carbamates (4a–15a and 4b–15b) were fully characterized by
spectroscopic methods and elemental analysis.
cially, 6a (MIC = 1.562 and 1.562
1.562 g/mL) showed comparable activities to positive control
kanamycin B.
lg/mL) and 7a (MIC = 0.78 and
l
In general, this series of vinylogous carbamates displayed more
effective antibacterial activity than the former report, this indi-
cated that the different substituent (benzyloxy group instead of
methoxy group) on the para position of benzene ring B was benefit
for the antibacterial activity. As shown in Table 1, all Z-isomers had
better antibacterial activity than E-isomers, which implied that
configuration did affect isomers’ activity, and this trend is opposite
to the former report.12 Compounds with a single substitution at the
4-position on A-ring (Scheme 1) demonstrated higher inhibitory
activity, as shown by the most potent six compounds. Multiple
substitutions or a single substitution at other positions of the A
ring decreased their inhibitory activity. For compounds with single
substitution at 4-position on A-ring, 7a showed the best antibacte-
rial activity followed by 6a. As to compounds 10a, 12a and 13a,
3. Results and discussion
3.1. Antibacterial activity
All the synthesized new vinylogous carbamates (4a–15a and
4b–15b) were screened for antibacterial activity against two
O
OEt
HCO2Et
NaH
O
OEt
R1
A
OBn
1
H
R2
R3
N
B
OBn
R4
R1
OHC
CO2Et
4 - 15 a
R2
R3
NH2
EtOH
+
60 - 80 oC
O
R2
R1
R4
OEt
OBn
R3
A
NH
3a -l
2
B
R4
OBn
4 - 15 b
3a R1 = R2 = R4 = H, R3 = Cl;
3b R1 = R3 = Cl, R2 = R4 = H;
3c R1 = R2 = R4 = H, R3 = Br;
4
5
6
7
8
9
R1 = R2 = R4 = H, R3 = Cl;
R1 = R3 = Cl, R2 = R4 = H;
R1 = R2 = R4 = H, R3 = Br;
R1 = R2 = R4 = H, R3 =CH3;
R1 = R3 = Br, R2 = R4 = H;
R1 = Cl, R2 = R3 = R4 = H;
3d R1 = R2 = R4 = H, R3 =CH3;
3e R1 = R3 = Br, R2 = R4 = H;
3f R1 = Cl, R2 = R3 = R4 = H;
3g R1 = R2 = R4 = H, R3 = F;
3h R1 = R3 = F, R2 = R4 = H;
3i R1 = R2 = R4 = H, R3 = CH(CH3)2;
3j R1 = R2 = R4 = H, R3 = NO2;
3k R1 = F, R2 = R3 = R4 = H;
3l R1 = R2 = R3 = R4 = H;
10 R1 = R2 = R4 = H, R3 = F;
11 R1 = R3 = F, R2 = R4 = H;
12 R1 = R2 = R4 = H, R3 = CH(CH3)2;
13 R1 = R2 = R4 = H, R3 = NO2;
14 R1 = F, R2 = R3 = R4 = H;
15 R1 = R2 = R3 = R4 = H;
An "a" is for Z isomer and a "b" is for E
isomer. For example, 4a is Z isomer and
4b is E isomer.
Scheme 1. Synthetic routes of vinylogous carbamates.

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H.-Q. Li et al. / Bioorg. Med. Chem. 19 (2011) 4454–4459
Table 1
important role in the regulation of chain elongation and substrate
Antibacterial activity of synthetic vinylogous carbamates
binding. Since the alkyl chain of CoA is broken by Cys of the cata-
lytic triad of FabH, interactions between Cys and substrate appear
to play an important role in substrate binding (Fig. 1). Qiu, et al. re-
fined three-dimensional structure of E. coli FabH in the presence or
absence of malonyl-CoA by X-ray spectroscopy. Since the malonyl
moiety is degraded by E. coli FabH, molecular docking studies for
FabH and malonyl-CoA were carried out to identify a plausible
malonyl-binding mode.14 They found that in one of the binding
modes appeared in the lower scored conformations, the malonyl
carboxylate formed hydrogen bonds to the backbone nitrogen of
Phe304. Enlightened by these facts, compound 7a with the most
potent inhibitory activity was hit by pharmacophore map I men-
tioned above. The binding model of compound 7a and E. coli FabH
is depicted in Figure 2. In the binding model, the side chain car-
bonyl group of Gly209 forms a hydrogen bond with N–H group
of compound 7a. The A-ring in compound 7a projects into a hydro-
phobic interaction region, which is comprised of the side chains of
Met207, Asn247, Asn274 and Ile156. The hydrophobic interaction
between 6a and FabH was important for the potent inhibitory
activity of 7a.
Compounds
Minimum inhibitory concentrations (
Gram-negative Gram-positive
P. fluorescence B. subtilis S. aureus
lg /mL)
E. coli
4a
4b
5a
5b
6a
6b
7a
7b
8a
8b
9a
9b
12.5
25
25
6.25
25
25
25
25
25
25
>50
25
25
25
>50
50
>50
25
>50
25
12.5
>50
25
>50
25
>50
25
>50
25
12.5
>50
12.5
>50
12.5
>50
12.5
12.5
25
>50
>50
25
>50
50
1.56
>50
0.78
>50
12.5
3.13
12.5
25
3.125
6.25
6.25
25
3.125
6.25
3.125
>50
12.5
25
12.5
6.25
3.125
1.56
>50
1.56
>50
12.5
12.5
25
>50
3.125
12.5
12.5
25
3.125
6.25
6.25
>50
25
10a
10b
11a
11b
12a
12b
13a
13b
14a
14b
15a
15b
Kanamycin B
25
25
>50
12.5
25
12.5
>50
>50
>50
>50
25
4. Conclusion
>50
25
In this paper, 24 vinylogous carbamates (4a–15a and 4b–15b)
were synthesized and evaluated for E. coli FabH inhibitory activity
as antibacterial agents for the first time. Among the compounds
being studied, potent and selective anti-Gram-negative bacteria
activities were observed in these vinylogous carbamates. Com-
pounds 6a, 7a, 10a, 12a, 12b and 13a exhibited excellent activities
against Gram-negative E. coli and Pseudomonas aeruginosa. Espe-
cially, compounds 6a and 7a showed the most potent E. coli FabH
inhibitory activity. Molecular docking simulation was performed
to position compound 6a into the E. coli FabH active site to deter-
mine the probable binding conformation. Based on the data ob-
tained from this study, we concluded that compounds 6a and 7a
were potent inhibitors of E. coli FabH as antibiotics deserving fur-
ther research.
6.25
3.125
25
0.39
25
1.56
their inhibitory activity was weaker than 6a and 7a. From antibac-
terial evaluation, it was found that Z-isomers had better activity
than that of E-isomers, and compounds with a single substitution
at 4-position on A-ring showed most potent activity.
3.2. E. coli FabH inhibitory activity
The E. coli FabH inhibitory potency of the representative vinyl-
ogous carbamates (6a–10a, 6b, 12b) was examined and the results
are summarized in Table 2. As shown in Table 2, most of the tested
compounds displayed potent E. coli FabH inhibitory activity.
Among them, compounds 8a, 9a and 6b with lower antibacterial
activities also had less inhibitory activity toward E. coli FabH. In
addition to antibacterial activity against Gram-negative E. coli
and P. aeruginosa, Z-isomers compounds 6a and 7a with a single
substitution at the 4-position on A-ring were also good inhibitors
of E. coli FabH, indicating that 6a and 7a may form a hydrophobic
interaction of E. coli FabH. These biological assays indicated that
compounds 6a and 7a were potent inhibitors of E. coli FabH as
antibiotics.
5 Experimental section
5.1. Antibacterial activity
The antibacterial activity of the synthesized compounds was
tested against E. coli, P. fluorescence, B. subtilis and S. aureus using
MH medium (Mueller-Hinton medium: casein hydrolysate 17.5 g,
soluble starch 1.5 g, beef extract 1000 mL), The MICs (minimum
inhibitory concentrations) of the test compounds were determined
by a colorimetric method using the dye MTT (3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyl tetrazolium bromide). A stock solution of
Molecular docking of compound 7a and E. coli FabH was per-
formed on the binding model based on the E. coli FabH-CoA com-
plex structure (1HNJ.pdb).14 The FabH active site generally
contains a catalytic triad tunnel consisting of Cys-His-Asn, which
is conserved in various bacteria. This catalytic triad plays an
the synthesized compound (100 lg/mL) in DMSO was prepared
and graded quantities of the test compounds were incorporated
in specified quantity of sterilized liquid MH medium. A specified
quantity of the medium containing the compound was poured into
microtitration plates. Suspension of the microorganism was pre-
pared to contain approximately 105 cfu/mL and applied to microt-
itration plates with serially diluted compounds in DMSO to be
tested and incubated at 37 °C for 24 h. After the MICs were visually
Table 2
E. coli FabH inhibitory activity of the selected compounds
a
Compound
E. coli FabH IC50
(lM)
Hemolysis LC30 (mg/ml)
6a
7a
8a
9a
10a
6b
12b
3.3
2.6
24
28
8.2
43
>10
>10
>10
>10
>10
>10
>10
determined on each of the microtitration plates, 50 lL of PBS
(Phosphate Buffered Saline 0.01 mol/L, pH 7.4: Na2HPO4Á12H2O
2.9 g, KH2PO4 0.2 g, NaCl 8.0 g, KCl 0.2 g, distilled water 1000 mL)
containing 2 mg of MTT/mL was added to each well. Incubation
was continued at room temperature for 4–5 h. The content of
8.5
each well was removed, and 100
lL of isopropanol containing
a
5% 1 mol/L HCl was added to extract the dye. After 12 h of
Lytic concentration 30%.

H.-Q. Li et al. / Bioorg. Med. Chem. 19 (2011) 4454–4459
4457
Figure 1. FabH-catalyzed initiation reaction of fatty acid biosynthesis.
5.4. Chemistry
All chemicals (reagent grade) used were purchased from Aldrich
(U.S.A). Separation of the compounds by column chromatography
was carried out with Silica gel 60 (200–300 mesh ASTM, E. Merck).
The quantity of silica gel used was 50–100 times the weight
charged on the column. Then, the eluates were monitored using
TLC. Melting points were determined on a XT4 MP apparatus (Taike
Corp., Beijing, China). ESI mass spectra were obtained on a Mariner
System 5304 mass spectrometer, and 1H NMR spectra were re-
corded at DPX500 on a Bruker spectrometer. Elemental analyses
were performed on a CHN-O-Rapid instrument and were within
0.4% of the theoretical values.
5.5. General method of synthesis for vinylogous carbamates
Figure 2. Binding model of compound 7a and E. coli FabH.
incubation at room temperature, the optical density (OD) was
measured with a microplate reader at 550 nm.
The starting materials (aldehyde 2) for the synthesis of vinylo-
gous carbamates have been previously published. Equimolar of
the appropriate substituted aromatic amines and the aldehyde
(2) in absolute alcohol (30 mL) were heated at 80ꢀfor 2–4 h. The ex-
cess solvent was removed under reduced pressure. The residue was
purified by a flash chromatography with chloroform/EtOAc/petro-
leum ether to afford two fractions. The first fraction gave a Z-iso-
mer, and the second fraction gave an E-isomer.12
5.2. E. coli FabH purification and activity assay
Native E. coli FabH protein was overexpressed in E. coli DH10B
cells using the pET30 vector and purified to homogeneity in three
chromatographic steps (Q-Sepharose, MonoQ, and hydroxyapatite)
at 4 °C. The selenomethionine- substituted protein was expressed
in E. coli BL21(DE3) cells and purified in a similar way. Harvested
cells containing FabH were lysed by sonication in 20 mM Tris, pH
7.6, 5 mM imidazole, 0.5 M NaCl and centrifuged at 20000 rpm
for 30 min. The supernatant was applied to a Ni-NTA agarose col-
umn, washed, and eluted using a 5–500 mM imidazole gradient
over 20 column volumes. Eluted protein was dialyzed against
20 mM Tris, pH 7.6, 1 mM DTT, and100 mM NaCl. Purified FabD
and FabHs were concentrated up to 2 mg/mL and stored at
À80 °C in 20 mM Tris, pH 7.6, 100 mM NaCl, 1 mM DTT, and 20%
glycerol for enzymatic assay.
5.6. (Z)-Ethyl 2-(4-(benzyloxy)phenyl)-3-(4-chlorophenyl-
amino)acrylate (4a)
Yield: 0.78 g (38%). Shallow yellow powder. Mp 92–93ꢀ. 1H NMR
(DMSO-d6): 1.20 (t, J = 7.2 Hz, 3H); 4.17(q, J = 7.2 Hz, 2H); 5.11 (s,
2H); 6.95 (d, J = 8.8 Hz, 2H); 7.26 (d, J = 8.8 Hz, 2H); 7.31–7.47 (m,
9H); 7.53 (d, J = 12.7 Hz, 1H); 10.2 (d, J = 12.7 Hz, 1H). MS (ESI):
408.9 (C24H22ClNO3, [M+H]+). Anal. calcd for C24H22ClNO3: C,
70.67; H, 5.44; N, 3.43. Found: C, 70.79; H, 5.63; N, 3.67.
In a final 20
0.25 mMMgCl2, and 2.5
and H2O was added to 15
ture of 25 M acetyl-CoA, 0.5 mMNADH, and 0.5 mM NADPH was
added for FabH reaction for 25 min. The reaction was stopped by
adding 20 L of ice-cold 50% TCA, incubating for 5 min on ice,
and centrifuging to pellet the protein. The pellet was washed with
10% ice-cold TCA and resuspended with 5 L of 0.5 M NaOH. The
incorporation of the 3H signal in the final product was read by li-
quid scintillation. When determining the inhibition constant
(IC50), inhibitors were added from a concentrated DMSO stock such
that the final concentration of DMSO did not exceed 2%.
l
L reaction, 20 mM Na2HPO4, pH 7.0, 0.5 m MDTT,
M holo-ACP were mixed with 1 nM FabH,
L. After 1 min incubation, a 2 L mix-
5.7. (E)-Ethyl 2-(4-(benzyloxy)phenyl)-3-(4-chlorophenyl-
amino)acrylate (4b)
l
l
l
l
Yield: 0.88 g (43%). White powder. Mp 114–115ꢀ. 1H NMR
(DMSO-d6): 1.18 (t, J = 5.0 Hz, 3H); 4.17(q, J = 7.1 Hz, 2H); 5.12 (s,
2H); 7.03 (d, J = 8.7 Hz, 2H); 7.15–7.49 (m, 11H); 7.95 (d,
J = 12.9 Hz, 1H); 8.57 (d, J = 12.9 Hz, 1H). MS (ESI): 408.9
(C24H22ClNO3, [M+H]+). Anal. calcd for C24H22ClNO3: C, 59.03; H,
4.68; N, 3.43. Found: C, 70.74; H, 5.71; N, 3.64.
l
l
5.8. (Z)-Ethyl 2-(4-(benzyloxy)phenyl)-3-(2,4-dichlorophenyl-
amino)acrylate (5a)
5.3. Docking simulations
Yield: 1.12 g (51%). White powder. Mp 98–99ꢀ. 1H NMR (DMSO-
d6): 1.20 (t, J = 7.2 Hz, 3H); 4.20(q, J = 7.1 Hz, 2H); 5.12 (s, 2H); 6.97
(d, J = 8.7 Hz, 2H); 7.08–7.68(m, 11H); 7.71 (d, J = 12.3 Hz, 1H);
10.73 (d, J = 12.3 Hz, 1H). MS (ESI): 442.1 (C24H21Cl2NO3, [M+H]+).
Anal. calcd for C24H21Cl2NO3: C, 65.17; H, 4.79; N, 3.17. Found: C,
65.41; H, 4.90; N, 3.25.
Molecular docking of compound 7a into the three-dimensional
X-ray structure of E. coli FabH (PDB code: 1HNJ) was carried out
using the AutoDock software package (version 4.0) as imple-
mented through the graphical user interface AutoDock Tools
(ADT 1.4.6) .15

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H.-Q. Li et al. / Bioorg. Med. Chem. 19 (2011) 4454–4459
5.9. (E)-Ethyl 2-(4-(benzyloxy)phenyl)-3-(2,4-dichlorophenyl-
(C24H21Br2NO3, [M+H]+). Anal. calcd for C24H21Br2NO3: C, 54.26;
amino)acrylate (5b)
H, 3.98; N, 2.64. Found: C, 54.37; H, 4.07; N, 2.83.
Yield: 0.75 g (34%). White powder. Mp 64–65ꢀ. 1H NMR (DMSO-
d6): 1.19 (t, J = 7.2 Hz, 3H); 4.12(q, J = 7.0 Hz, 2H); 5.14 (s, 2H); 7.10
(d, J = 8.4 Hz, 2H); 7.23 (d, J = 8.4 Hz, 2H); 7.36–7.42 (m, 9H); 7.47
(d, J = 12.5 Hz, 1H); 8.02 (d, J = 12.5 Hz, 1H). MS (ESI): 442.1
(C24H21Cl2NO3, [M+H]+). Anal. calcd for C24H21Cl2NO3: C, 65.17;
H, 4.79; N, 3.17. Found: C, 65.41; H, 4.90; N, 3.25.
5.16. (Z)-Ethyl 2-(4-(benzyloxy)phenyl)-3-(2-chlorophenyl-
amino)acrylate (9a)
Yield: 1.12 g (55%). Shallow yellow powder. Mp 70–72ꢀ. 1H NMR
(DMSO-d6): 1.21 (t, J = 7.0 Hz, 3H); 4.20(q, J = 7.0 Hz, 2H); 5.12 (s,
2H); 6.97 (d, J = 8.79 Hz, 2H); 7.22–7.65 (m, 11H); 7.63(d,
J = 12.2 Hz, 1H); 10.71 (d, J = 12.2 Hz, 1H). MS (ESI): 408.9
(C24H22ClNO3, [M+H]+). Anal. calcd for C24H22ClNO3: C, 70.67; H,
5.44; N, 3.43. Found: C, 70.72; H, 5.55; N, 3.62.
5.10. (Z)-Ethyl 2-(4-(benzyloxy)phenyl)-3-(4-bromophenyl-
amino)acrylate (6a)
Yield: 1.02 g (45%). Shallow yellow powder. Mp 91–92ꢀ. 1H NMR
(DMSO-d6): 1.20 (t, J = 7.2 Hz, 3H); 4.07(q, J = 7.2 Hz, 2H); 5.11 (s,
2H); 6.95 (d, J = 8.8 Hz, 2H); 7.22–7.49 (m, 11H); 7.53 (d, J =
12.7 Hz, 1H); 10.19 (d, J = 12.9 Hz, 1H). MS (ESI): 453.3
(C24H22BrNO3, [M+H]+). Anal. calcd for C24H22BrNO3: C, 63.73; H,
4.90; N, 3.10. Found: C, 63.82; H, 5.05; N, 3.21.
5.17. (E)-Ethyl 2-(4-(benzyloxy)phenyl)-3-(2-chlorophenyl-
amino)acrylate (9b)
Yield: 0.61 g (30%). White powder. Mp 78–80ꢀ. 1H NMR (DMSO-
d6): 1.20 (t, J = 7.2 Hz, 3H); 4.19(q, J = 7.1 Hz, 2H); 5.12 (s, 2H);
7.00 (d, J = 8.7 Hz, 2H); 7.23–7.59 (m, 11H); 7.90 (d, J = 12.9 Hz,
1H); 8.17 (d, J = 12.9 Hz, 1H). MS (ESI): 408.9 (C24H22ClNO3,
[M+H]+). Anal. calcd for C24H22ClNO3: C, 59.03; H, 4.68; N, 3.43.
Found: C, 70.79; H, 5.78; N, 3.65.
5.11. (E)-Ethyl 2-(4-(benzyloxy)phenyl)-3-(4-bromophenyl-
amino)acrylate (6b)
Yield: 0.84 g (37%). White powder. Mp 108–109ꢀ. 1H NMR
(DMSO-d6): 1.18 (t, J = 7.0 Hz, 3H); 4.10(q, J = 7.1 Hz, 2H); 5.12 (s,
2H); 7.03 (d, J = 8.8 Hz, 2H); 7.11–7.49 (m, 11H); 7.94 (d,
J = 13.2 Hz, 1H); 8.57 (d, J = 13.2 Hz, 1H). MS (ESI): 453.3
(C24H22BrNO3, [M+H]+). Anal. calcd for C24H22BrNO3: C, 63.73; H,
4.90; N, 3.10. Found: C, 63.92; H, 5.12; N, 3.25.
5.18. (Z)-Ethyl 2-(4-(benzyloxy)phenyl)-3-(4-fluorophenyl-
amino)acrylate (10a)
Yield: 1.13 g (58%). Shallow yellow powder. Mp 112–113ꢀ. 1H
NMR (DMSO-d6): 1.20 (t, J = 6.9 Hz, 3H); 4.11(q, J = 6.9 Hz, 2H);
5.13 (s, 2H); 6.99 (d, J = 6.6 Hz, 2H); 7.15–7.48 (m, 11H); 7.52 (d,
J = 12.7 Hz, 1H); 10.2 (d, J = 12.7 Hz, 1H). MS (ESI): 392.4
(C24H22FNO3, [M+H]+). Anal. calcd for C24H22FNO3: C, 73.64; H,
5.66; N, 3.58. Found: C, 73.79; H, 5.88; N, 3.75.
5.12. (Z)-Ethyl 3-(p-toluidino)-2-(4-(benzyloxy)phenyl)acrylate
(7a)
Yield: 1.06 g (55%). Shallow yellow powder. Mp 75–76ꢀ. 1H NMR
(DMSO-d6): 1.20 (t, J = 7.0 Hz, 3H); 4.17(q, J = 6.9 Hz, 2H); 5.11 (s,
2H); 6.95 (d, J = 8.6 Hz, 2H); 7.10 (d, J = 8.6 Hz, 2H); 7.15 (d,
J = 8.6 Hz, 2H); 7.25 (d, J = 8.6 Hz, 2H); 7.30–7.47 (m, 5H); 7.53 (d,
J = 12.8 Hz, 1H); 10.2 (d, J = 12.8 Hz, 1H). MS (ESI): 388.18
(C25H25NO3, [M+H]+). Anal. calcd for C25H25NO3: C, 77.49; H,
6.50; N, 3.61. Found: C, 77.38; H, 6.67; N, 3.80.
5.19. (Z)-Ethyl 2-(4-(benzyloxy)phenyl)-3-(4-fluorophenyl-
amino)acrylate (10b)
Yield: 0.63 g (32%). White powder. Mp 114–115ꢀ. 1H NMR
(DMSO-d6): 1.20 (t, J = 6.9 Hz, 3H); 4.10(q, J = 6.9 Hz, 2H); 5.13 (s,
2H); 7.03 (d, J = 6.6 Hz, 2H); 7.19–7.49 (m, 11H); 7.57 (d, J =
13.8 Hz, 1H); 8.00 (d, J = 13.8 Hz, 1H). MS (ESI): 392.4 (C24H22FNO3,
[M+H]+). Anal. calcd for C24H22FNO3: C, 73.64; H, 5.66; N, 3.58.
Found: C, 73.78; H, 5.70; N, 3.69.
5.13. (Z)-Ethyl 3-(p-toluidino)-2-(4-(benzyloxy)phenyl)acrylate
(7b)
5.20. (Z)-Ethyl 2-(4-(benzyloxy)phenyl)-3-(2,4-difluorophenyl-
amino)acrylate (11a)
Yield: 0.54 g (28%). White powder. Mp 95–96ꢀ. 1H NMR (DMSO-
d6): 1.18 (t, J = 7.0 Hz, 3H); 4.08(q, J = 7.0 Hz, 2H); 5.12 (s, 2H); 7.03
(d, J = 8.8 Hz, 2H); 7.01–7.49 (m, 11H); 7.98 (d, J = 10.1 Hz, 1H);
8.35 (d, J = 10.5 Hz, 1H). MS (ESI): 388.18 (C25H25NO3, [M+H]+).
Anal. calcd for C25H25NO3: C, 77.49; H, 6.50; N, 3.61. Found: C,
77.32; H, 6.64; N, 3.78.
Yield: 0.65 g (32%). Shallow yellow powder. Mp 149–150ꢀ. 1H
NMR (DMSO-d6): 1.18 (t, J = 6.9 Hz, 3H); 4.22(q, J = 6.9 Hz, 2H);
5.13 (s, 2H); 6.97 (d, J = 8.6 Hz, 2H); 7.21–7.63 (m, 11H); 7.53 (d,
J = 12.7 Hz, 1H); 10.37 (d, J = 11.3 Hz, 1H). MS (ESI): 410.2
(C24H21F2NO3, [M+H]+). Anal. calcd for C24H21F2NO3: C, 70.41; H,
5.17; N, 3.42. Found: C, 70.59; H, 5.28; N, 9.60.
5.14. (Z)-Ethyl 2-(4-(benzyloxy)phenyl)-3-(2,4-dibromophenyl-
amino)acrylate (8a)
5.21. (E)-Ethyl 2-(4-(benzyloxy)phenyl)-3-(2,4-difluorophenyl-
amino)acrylate (11b)
Yield: 1.35 g (51%). Shallow yellow powder. Mp 88–90ꢀ. 1H NMR
(DMSO-d6): 1.20 (t, J = 7.0 Hz, 3H); 4.21(q, J = 6.9 Hz, 2H); 5.13 (s,
2H); 6.97 (d, J = 8.6 Hz, 2H); 7.28–7.67 (m, 5H); 7.83 (d,
J = 12.1 Hz, 1H); 10.69 (d, J = 12.1 Hz, 1H). MS (ESI): 532.2
(C24H21Br2NO3, [M+H]+). Anal. calcd for C24H21Br2NO3: C, 54.26;
H, 3.98; N, 2.64. Found: C, 54.32; H, 3.97; N, 2.80.
Yield: 0.86 g (42%). White powder. Mp 97–98ꢀ. 1H NMR (DMSO-
d6): 1.22 (t, J = 7.2 Hz, 3H); 4.10(q, J = 7.2 Hz, 2H); 5.14 (s, 2H); 7.00
(d, J = 8.6 Hz, 2H); 7.05 (d, J = 8.6 Hz, 2H); 7.18–7.60 (m, 9H); 7.72
(d, J = 11.3 Hz, 1H); 10.37 (d, J = 11.3 Hz, 1H). MS (ESI): 410.2
(C24H21F2NO3, [M+H]+). Anal. calcd for C24H21F2NO3: C, 70.41; H,
5.17; N, 3.42. Found: C, 70.59; H, 5.28; N, 9.60.
5.15. (E)-Ethyl 2-(4-(benzyloxy)phenyl)-3-(2,4-dibromophenyl-
amino)acrylate (8b)
5.22. (Z)-Ethyl 2-(4-(benzyloxy)phenyl)-3-(4-isopropylphenyl-
amino)acrylate (12a)
Yield: 0.74 g (28%). Shallow yellow powder. Mp 92–94ꢀ. 1H NMR
(DMSO-d6): 1.18 (t, J = 7.0 Hz, 3H); 4.16(q, J = 7.0 Hz, 2H); 5.13 (s,
2H); 7.03 (d, J = 8.8 Hz, 2H); 7.09–7.59 (m, 11H); 7.79 (d,
J = 12.9 Hz, 1H); 8.35 (d, J = 12.9 Hz, 1H). MS (ESI): 532.2
Yield: 1.21 g (58%). Shallow yellow powder. Mp 88–89ꢀ. 1H NMR
(DMSO-d6): 1.20 (t, J = 6.9 Hz, 3H); 4.11(q, J = 6.9 Hz, 2H); 5.13 (s,

H.-Q. Li et al. / Bioorg. Med. Chem. 19 (2011) 4454–4459
4459
2H); 7.00 (d, J = 6.6 Hz, 2H); 7.17 (d, J = 6.6 Hz, 2H); 7.28–7.47 (m,
9H); 7.57 (d, J = 13.5 Hz, 1H); 10.22 (d, J = 13.5 Hz, 1H). MS (ESI):
416.5 (C27H29NO3, [M+H]+). Anal. calcd for C27H29NO3: C, 78.04;
H, 7.03; N, 3.37. Found: C, 78.19; H, 5.15; N, 3.47.
9H); 7.57 (d, J = 13.5 Hz, 1H); 8.00 (d, J = 13.5 Hz, 1H). MS (ESI):
392.4 (C24H22FNO3, [M+H]+). Anal. calcd for C24H22FNO3: C, 73.64;
H, 5.66; N, 3.58. Found: C, 73.74; H, 5.78; N, 3.65.
5.28. (Z)-Ethyl 2-(4-(benzyloxy)phenyl)-3-(phenylamino)
5.23. (E)-Ethyl 2-(4-(benzyloxy)phenyl)-3-(4-isopropylphenyl-
acrylate (15a)
amino)acrylate (12b)
Yield: 0.99 g (63%). White powder. Mp 95–96ꢀ. 1H NMR (DMSO-
d6): 1.18 (t, J = 7.2 Hz, 3H); 4.09(q, J = 7.2 Hz, 2H); 5.12 (s, 2H); 6.95
(d, J = 6.9 Hz, 2H); 7.26 (d, J = 8.8 Hz, 2H); 7.02–7.38 (m, 11H); 7.50
(d, J = 13.5 Hz, 1H); 10.35 (d, J = 13.5 Hz, 1H). MS (ESI): 374.4
(C24H23NO3, [M+H]+). Anal. calcd for C24H23NO3: C, 77.19; H,
6.21; N, 3.75. Found: C, 77.32; H, 6.33; N, 3.87.
Yield: 0.52 g (25%). Shallow yellow powder. Mp 179–180ꢀ. 1H
NMR (DMSO-d6): 1.20 (t, J = 6.9 Hz, 3H); 4.17(q, J = 6.9 Hz, 2H);
5.13 (s, 2H); 7.03 (d, J = 6.9 Hz, 2H); 7.15–7.49 (m, 11H); 7.52 (d,
J = 13.5 Hz, 1H); 8.00 (d,
J = 13.5 Hz, 1H). MS (ESI): 416.5
(C27H29NO3, [M+H]+). Anal. calcd for C27H29NO3: C, 78.04; H,
7.03; N, 3.37. Found: C, 78.19; H, 5.15; N, 3.47.
5.29. (E)-Ethyl 2-(4-(benzyloxy)phenyl)-3-(phenylamino)
5.24. (Z)-Ethyl 2-(4-(benzyloxy)phenyl)-3-(4-nitrophenyl-
acrylate (15b)
amino)acrylate (13a)
Yield: 0.58 g (43%). White powder. Mp 89–90ꢀ. 1H NMR (DMSO-
d6): 1.18 (t, J = 6.6 Hz, 3H); 4.10 (q, J = 6.6 Hz, 2H); 5.12 (s, 2H); 6.94
(d, J = 6.9 Hz, 2H); 7.03–7.49 (m, 11H); 7.99 (d, J = 13.5 Hz, 1H);
8.57 (d, J = 13.5 Hz, 1H). MS (ESI): 374.4 (C24H23NO3, [M+H]+). Anal.
calcd for C24H23NO3: C, 77.19; H, 6.21; N, 3.75. Found: C, 77.32; H,
6.33; N, 3.87.
Yield: 0.75 g (57%). Yellow powder. Mp 179–180ꢀ. 1H NMR
(DMSO-d6): 1.18 (t, J = 7.2 Hz, 3H); 4.15(q, J = 7.2 Hz, 2H); 5.10 (s,
2H); 6.92 (d, J = 8.4 Hz, 2H); 7.14–7.53 (m, 11H); 8.16 (d,
J = 12.3 Hz, 1H); 10.18 (d, J = 12.3 Hz, 1H). MS (ESI): 419.4
(C24H22N2O5, [M+H]+). Anal. calcd for C24H22N2O5: C, 68.89; H,
5.30; N, 6.69. Found: C, 68.96; H, 5.43; N, 6.85.
Acknowledgments
5.25. (E)-Ethyl 2-(4-(benzyloxy)phenyl)-3-(4-nitrophenyl-
amino)acrylate (13b)
The work was financed by a grant (Project SBK200930597) from
Natural Science Foundation of Jiangsu Province, and China Postdoc-
toral Science Special Foundation (Project 200902513).
Yield: 0.26 g (20%). Yellow powder. Mp 130–131ꢀ. 1H NMR
(DMSO-d6): 1.20 (t, J = 7.2 Hz, 3H); 4.12(q, J = 7.2 Hz, 2H); 5.13 (s,
2H); 7.05 (d, J = 8.1 Hz, 2H); 7.18 (d, J = 8.1 Hz, 2H); 7.33–7.49 (m,
8H); 8.02 (d, J = 12.9 Hz, 1H); 8.13 (d, J = 8.7 Hz, 1H); 9.14 (d,
J = 12.9 Hz, 1H). MS (ESI): 419.4 (C24H22N2O5, [M+H]+). Anal. calcd
for C24H22N2O5: C, 68.89; H, 5.30; N, 6.69. Found: C, 68.96; H,
5.43; N, 6.85.
References and notes
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5.26. (Z)-Ethyl 2-(4-(benzyloxy)phenyl)-3-(2-fluorophenyl-
amino)acrylate (14a)
6. Asheka, A.; Cho, S. J. Bioorg. Med. Chem. 2006, 14, 1474.
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72, 4276.
Yield: 0.44 g (36%). White powder. Mp 124–125ꢀ. 1H NMR
(DMSO-d6): 1.18 (t, J = 7.2 Hz, 3H); 4.15(q, J = 7.2 Hz, 2H); 5.11 (s,
2H); 6.93 (d, J = 8.4 Hz, 2H); 7.14–7.45 (m, 11H); 7.52 (d,
J = 12.9 Hz, 1H); 10.49 (d, J = 12.9 Hz, 1H). MS (ESI): 392.4
(C24H22FNO3, [M+H]+). Anal. calcd for C24H22FNO3: C, 73.64; H,
5.66; N, 3.58. Found: C, 73.74; H, 5.78; N, 3.65.
11. Prabhakaran, E. N.; Nugent, B. M.; Williams, A. L.; Nailor, K. E.; Johnston, J. N.
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4212.
5.27. (E)-Ethyl 2-(4-(benzyloxy)phenyl)-3-(2-fluorophenyl-
amino)acrylate (14b)
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1145.
Yield: 0.55 g (45%). Shallow yellow powder. Mp 75–77ꢀ. 1H NMR
(DMSO-d6): 1.19 (t, J = 6.9 Hz, 3H); 4.11(q, J = 6.9 Hz, 2H); 5.13 (s,
2H); 7.00 (d, J = 6.6 Hz, 2H); 7.05 (d, J = 6.6 Hz, 2H); 7.14–7.54 (m,