Design, synthesis, and preliminary studies of the activity of novel derivatives of N-cinnamoyl-l-aspartic acid as inhibitors of aminopeptidase N/CD13

Article abstract of DOI:10.1016/j.bmc.2009.07.014

A series of novel derivatives of N-cinnamoyl-l-aspartic acid were designed, synthesized, and assayed for their inhibitory activities against aminopeptidase N. The preliminary biological assay showed that compound 8c has the most potent inhibitory activity

Full text of DOI:10.1016/j.bmc.2009.07.014

Bioorganic & Medicinal Chemistry 17 (2009) 7398–7404
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and preliminary studies of the activity of novel derivatives
of N-cinnamoyl-L-aspartic acid as inhibitors of aminopeptidase N/CD13
Yingzi Liu a,b, Luqing Shang a, Hao Fang a, Huawei Zhu a, Jiajia Mu a, Qiang Wang a, Xuejian Wang a,
Yumei Yuan a, Wenfang Xu a,
*
a Department of Medicinal Chemistry, School of Pharmacy, Shandong University, 44 West Culture Road, 250012 Ji’nan, Shandong, PR China
b Department of Judicial Chemistry, Shandong Police College, Ji’nan, Shandong 250014, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel derivatives of N-cinnamoyl-L-aspartic acid were designed, synthesized, and assayed for
their inhibitory activities against aminopeptidase N. The preliminary biological assay showed that com-
pound 8c has the most potent inhibitory activity against APN with an IC50 of 11.1 0.9 lM, this could be
used as the lead compound in future research on anticancer agents.
Received 14 May 2009
Revised 6 July 2009
Accepted 7 July 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Key words:
N-Cinnamoyl-L-aspartic acid derivatives
Synthesis
Aminopeptidase N inhibitors
0
1. Introduction
B, which contains a zinc ion, and part C (the S1 pocket) is on the
right. The phenyl ring of Bestatin interacts with the S1 pocket,
the hydroxyl group and the carbonyl group chelate with the zinc
ion, and the leucine residue is inserted into the S1 pocket.
Aminopeptidase N (APN), which is identical to the human
lymphocyte surface cluster differentiation antigen, CD13, is a
zinc-dependent membrane metalloprotease in the M1 family of
ectoenzymes.1 It is responsible for the cleavage of the N-amino
group from a polypeptide chain, and APN has been shown to
participate in the degradation of polypeptides and proteins. APN
is over-expressed on tumor cells, and it plays a critical role in
tumorigenesis and the regulation of immunological function.2,3
Over more than three decades, studies have demonstrated
numerous natural and synthetic inhibitors of APN, including
AHPA-Val,4–6 Amastatin,7 Bestatin,8 Probestin,9 Curcumin,10 and
lapstatin.11 Among these inhibitors, Bestatin is the best known
for its application in the clinical treatment of adult acute nonl-
ymphocytic leukemia.
0
In this work, we aimed to develop a novel framework for an
inhibitor of APN. Wang et al.16 had reported that
L
-glutamine deriv-
atives could serve as potential antitumor agents that are directed
toward APN. We chose -aspartic acid as the initial material, and
L
a cinnamic acid moiety was introduced on to the scaffold. Cin-
namic acid is a natural differential inductor that can inhibit the
proliferation of insensitive prostatic carcinomas and can reverse
the transformation and induce differentiation of human lung
cancer cells, liver cancer, and human promyelocytic leukemia
cells.18–21 In addition, different building blocks (such as amines)
0
were introduced to interact with the S1 pocket of APN. The phenyl
ring of cinnamic acid was inserted into the pocket S1, and the car-
boxy group or the hydroxymate served as the zinc-binding group.
In this report, we describe the synthesis and evaluation of the
enzymatic activity of the N-cinnamoyl-L-aspartic acid derivatives,
and docking studies of the interaction will also be discussed.
In recent years, our group has reported several new inhibitors of
APN, such as L
-lysine derivatives,12 AHPA (3-2-hydroxyl-phenyl
butanoic acid) derivatives,13 3-phenylpropane-1,2-diamine deriva-
tives,14 cinnamic acid and caffeic acid with hydroxyproline deriva-
tives,15 and -iso-glutamine derivatives.16 In a previous study, the
L
interaction of an APN inhibitor and the active site of APN was re-
ported. Kiyoshi17 showed the co-crystal complex of APN and Best-
atin. According to the literature, the active site of APN can be
divided into three parts: part A (the S1 pocket) is to the left of part
2. Chemistry
The target compounds were synthesized efficiently, following
the procedures shown in Scheme 1.
The starting material, compound 2, was prepared from L-aspar-
tic acid according to methods described in the literature.22 The key
intermediate 6 was obtained by the anhydration of compound 5.
* Corresponding author. Tel./fax: +86 531 88382264.
E-mail address: wfxu@yahoo.cn (W. Xu).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.07.014

Y. Liu et al. / Bioorg. Med. Chem. 17 (2009) 7398–7404
7399
S1
S1
O
COOH
H
N
H
N
H2N
N
H
R2
OH
O
O
O
R1
S1'
S1'
Zn2+
Zn2+
Bestatin
R1: OH or NH-OH
IC50=3.6
µM
The compound 6 was reactivated with amineõ to yield 7a–7o.
Compounds 7a–7o can be activated with hydroxylamine hydro-
chloride to obtain 8a–8o.
Compound 8c exhibited an inhibitory effect on cell proliferation
with an IC50 value of 0.78 0.11 mM, and showed greater potency
than Bestatin, which had an IC50 value of 0.92 0.12 mM.
The results of the assay of enzyme inhibition showed that when
the R1 group was fixed, the R2 was altered from aliphatic to aro-
matic. The compounds bearing aliphatic amines were found to be
more potent than those bearing aromatic amines. This result may
be owing to the fact that aliphatic amines are flexible and can be
3. Results and discussion
In order to identify the activities of our compounds, preliminary
pharmacological studies were performed against APN and MMP-2
in vitro. Similar to APN, MMP-2 is a zinc-dependent metallopro-
teinase that is associated with malignant tumors. According to
the results of the enzyme assay, most compounds exhibited better
inhibitory activity against APN than MMP-2.
Comparison of compounds 7a–7o and 8a–8o showed that the
introduction of a stronger zinc-binding group, hydroxymate, could
significantly increase the inhibitory activities against APN. The
compounds 7a–7o, which had a carboxy zinc-binding group, pos-
sessed inhibitory activities but most of them were of low potency
for the inhibition of APN. As shown in Table 1, compounds 8c–8f
exhibited well activity towards APN among these target com-
pounds. The IC50 values for these four inhibitors of APN ranged be-
0
inserted into the S1 pocket more easily than the aromatic amines.
Moreover, the length of the side chain will partly influence the
activities of the compounds. The R2 group of the most potent com-
pound, 8c, is a butyl group, which is similar to the isobutyl side
chain of Bestatin. When the R2 group was converted to undecane,
the compound 8g, possessed a fortieth of the activity of compound
8c.
In order to investigate the interaction of the target compounds
with APN, the representative compound 8c was constructed with a
Sybyl/Sketch module and optimized using Powell’s method with a
Tripos force field with the convergence criterion set at 0.05 kcal/
(Å mol), and assigned with the Gasteiger–Hückel method. The
docking study was performed using a Sybyl/FlexX module: the res-
idues in a radius of 7.0 Å around Bestatin in the co-crystal structure
(PDB code: 2DQM) were selected as the active site. Other docking
tween 11.11 and 18.55 lM. In addition, the effects of 8c–8f on the
proliferation of HL-60 cells, compared with Bestatin, are shown
in Figure 1. Cell viability was assessed by the MTT method.
O
NH2
O
NH2
a
O
O
HO
OH
O
O
O
1
2
4
O
OH
O
O
O
O
c
b,2
N
H
N
H
OH
O
OH
O
O
3
5
O
O
OH
O
O
O
e
d
f
O
N
N
H
H
R2
O
N
H
6
7a-7o
O
NH
OH
R2
O
N
H
O
N
H
8a-8o
Scheme 1. Reagents and conditions: (a) MeOH, HCl, ice bath; (b) isobutyl chloroformate, N-methylmorpholine, THF, ꢀ15 °C; (c) MeOH, NaOH, HCl; (d) Ac2O, 55–60 °C; (e)
AcOH, Et3N; (f) isobutyl chloroformate, Et3N, THF, MeOH, NH2OH, HCl.

7400
Y. Liu et al. / Bioorg. Med. Chem. 17 (2009) 7398–7404
Table 1
Table 1 (continued)
The structures and IC50 values of the target compounds
a
Compound
R1
R2
IC50
(l
M)
MMP-2
O
O
APN
R1
NH
NH R2
8a
8b
8c
8d
8e
8f
NH–OH
NH–OH
NH–OH
NH–OH
NH–OH
NH–OH
NH–OH
NH–OH
36.0 1.1
65.9 1.5
11.1 0.9
17.4 1.4
18.1 2.2
18.5 2.0
438.2 3.4
191.5 2.7
131.0 2.3
166.6 2.7
232.5 1.8
196.5 2.5
178.6 1.5
285.9 3.0
>1000
O
a
Compound
R1
R2
IC50
(
l
M)
APN
MMP-2
7a
7b
–OH
–OH
9108.6 8.2
2199.7 8.1
>1000
>1000
7c
7d
7e
7f
–OH
–OH
–OH
–OH
–OH
–OH
189.7 2.3
204.3 1.5
387.8 2.4
232.2 2.1
962.0 4.0
348.5 4.9
594.3 3.7
>1000
(CH2)5
(CH2)7
(CH2)5
>1000
8g
8h
(CH2)11
(CH2)7
>1000
7g
7h
176.5 2.1
>1000
196.6 1.8
(CH2)11
Cl
Cl
Cl
Cl
8i
NH–OH
NH–OH
NH–OH
NH–OH
NH–OH
NH–OH
NH–OH
86.6 1.3
1206 2.7
65.1 1.6
115.0 1.2
1440 5.2
189.1 2.1
2050 4.6
3.60 0.6
>1000
Cl
7i
–OH
–OH
–OH
–OH
–OH
263.4 3.6
718.6 3.2
206.8 1.4
232.4 1.2
1781 4.7
>1000
>1000
999 3.2
>1000
>1000
Cl
8j
>1000
7j
8k
447.2 2.8
324.4 2.0
>1000
7k
7l
8l
O2N
O2N
8m
8n
O2N
O2N
7m
>1000
7n
7o
–OH
–OH
273.1 2.0
739.2 6.8
>1000
>1000
8o
>1000
Bestatin
162.0 4.8

Y. Liu et al. / Bioorg. Med. Chem. 17 (2009) 7398–7404
7401
ion with a distance of 1.97 and 1.74 Å, respectively. In addition, the
amino group of compound 8c formed a hydrogen bond with the
oxygen atom of Tyr381 with a distance of 1.84 Å (Fig. 2b). Tyr381
has been reported to be beneficial to the stabilization of the reac-
tion intermediate with the zinc ion.17 The hydroxymate group
can form a hydrogen bond with the carboxyl group of Glu298 with
a spaced length of 1.89 Å. Glu298 is a strong base that is capable of
mediating the attack of a water molecule upon the carbonyl carbon
of the substrate in the catalytic mechanism of APN.
2
4. Conclusion
In conclusion, we have described the synthesis of a series of
derivatives of N-cinnamoyl-L-aspartic acid and their properties as
inhibitors of APN. Most of the target compounds have potent inhib-
itory activities against APN and the most effective compound, 8c,
exhibited excellent enzymatic inhibition activity and selectivity
for APN. Compound 8c could be used as a lead compound for the
development of small molecular peptidomimetic inhibitors of
APN for investigation as potential anticancer agents in the future.
0
Bes
8c
8d
8e
8f
Compound
Figure 1. Effects of Bestatin and compounds 8c–8f on proliferation of the HL-60 cell
line. Each column represents the mean and SE of five independent experiments.
parameters available in the program were maintained as the de-
faults. The docking studies showed that the phenyl group of the
cinnamic acid moiety was inserted into the S1 pocket, and the butyl
5. Experimental
5.1. APN inhibition assay
0
group plunged into the pocket S1 (Fig. 2a). The carbonyl group and
the oxygen atoms in hydroxymate (OH) can interact with the zinc
IC50 values against APN were determined as previously de-
scribed, with the use of
microsomal aminopeptidase from Porcine Kidney microsomes
(Sigma) as the enzyme, in 50 M PBS, pH 7.2, at 37 °C. The hydro-
L-Leu-p-nitroanilide as a substrate and
l
lysis of the substrate was monitored by following the change in the
absorbance measured at 405 nm with a UV–vis spectrophotome-
ter, Pharmacia LKB, Biochrom 4060. All the solutions of the inhib-
itors were prepared in the assay buffer, and the pH was adjusted to
7.5 by the addition of 0.1 M HCl or 0.1 M NaOH. All the inhibitors
were preincubated with APN for 30 min at room temperature.
The assay mixture, which contained the inhibitor solution (the
concentration of which was dependent on the inhibitor), the en-
zyme solution (4 mg/ml final concentration), and the assay buffer,
was adjusted to 200 ll.
5.2. MMP inhibition assay
Gelatinase A (MMP-2) and TNBS were purchased from Sigma,
and the substance was synthesized as described by Vijaykumar
et al. The gelatinase, substance, and inhibitor were dissolved in so-
dium borate (pH 8.5, 50 mmol/l) and incubated for 30 min at 37 °C.
Subsequently, 0.03% TNBS was added and incubated for another
20 min: the resulting solution was investigated using a wavelength
of 450 nm to measure absorption.
5.3. MTT assay
HL-60 cells were grown in RPMI1640 medium containing 10%
FBS at 37 °C in a humidified incubator with 5% CO2. Cell prolifera-
tion was determined by the MTT (3-[4,5-dimethyl-2-thiazolyl]-
2,5-diphenyl-2H-tetrazolium bromide) assay. Briefly, cells were
plated in a 96-well plate at 10,000 cells per well, cultured for 4 h
in complete growth medium, then treated with 1600, 800, 400,
200, or 100 lg/ml of the compounds for 48 h. Following this,
0.5% MTT solution was added to each well. After further incubation
for 4 h, the formazan formed from MTT was extracted by adding
DMSO and mixing for 15 min. The optical density was read with
an ELISA reader at 570 nm.
Figure 2. (a) The FlexX docking result of 8c with APN; (b) The FlexX docking result
of 8c shown by PyMOL.

7402
Y. Liu et al. / Bioorg. Med. Chem. 17 (2009) 7398–7404
5.4. Chemistry: general procedures
was added some anhydrous diethyl ether and this was placed in
a refrigerator to obtain the white crystal: 4.75 g (Compound 6),
All the materials we used were commercially available. All the
solvents were distilled before use. All reactions were monitored
by thin-layer chromatography on 0.25-mm silica gel plates
(60GF-254) and visualized with UV light or ferric chloride. Silica
gel of 200–300 mesh size, was used in column chromatography
to depurate the end products. The proton NMR spectra were deter-
mined on a Brucker DRX spectrometer (300 MHz), d in parts per
million and J in Hertz, and TMS was used as an internal standard.
ESI-MS were determined on an API 4000 spectrometer. Measure-
ments were made in CD3OD solutions. Melting points were deter-
mined using an electrothermal melting point apparatus, and were
uncorrected.
yield: 51%, mp: 102–105 °C; ½a D25
ꢁ
+36.3 (c l, MeOH); ESI-MS: m/z:
245.3 [M+H]+; 1H NMR (CD3OD) d 3.10–3.45 (m, 2H), 5.09–5.21
(m, 1H), 6.70 (d, J = 16, 1H), 7.35–7.44 (m, 3H), 7.59–7.63 (m,
3H). Anal. Calcd for C13H11NO4: C, 63.67; H, 4.52; N, 5.71. Found:
C, 63.86; H, 4.49; N, 5.74.
5.4.5. 2-Cinnamamido-4-oxo-4-(propylamino) butanoic acid
(7a)
Compound 6 (2.45 g, 10 mmol) and n-propylamine (0.65 g,
11 mmol) were suspended in glacial acetic acid (30 ml). The sus-
pension was stirred at room temperature. The reaction was moni-
tored by thin-layer chromatography in order to ensure complete
reaction. The product solution was rotary evaporated under re-
duced pressure. To the residue was added some water, and the
product was left in a refrigerator to obtain the white crystal, which
was filtered, dried and weighed: 2.45 g (7a), yield: 80.59%, mp:
5.4.1. Dimethyl
L-aspartate hydrochloride (2)
L-Aspartic acid (1) (5 g, 37.6 mmol) was suspended in methanol
(300 ml) and the suspension was stirred in an ambient ice bath un-
der a chloride hydrogen atmosphere until the suspension became
clear. The product solution was rotary evaporated under reduced
pressure. Methanol was then added and rotary evaporated to re-
move the hydrogen chloride. A mass of white solid was obtained
and was crystallized in diethyl ether to produce 5.73 g (yield:
81.4%) of white crystal, mp: 114–117 °C.
116–118 °C; ½a 2D5
ꢁ
19.8 (c l, MeOH); ESI-MS: m/z: 320.4 [M+H]+;
1H NMR (CD3OD) d 0.89–0.95 (m, 3H), 1.48–1.58 (m, 2H), 2.73–
2.93 (m, 2H), 3.13–3.21 (m, 2H), 4.84–4.92 (m, 1H), 6.70 (d,
J = 16, 1H), 7.38–7.44 (m, 3H), 7.54–7.61 (m, 3H). Anal. Calcd for
C16H20N2O4: C, 63.14; H, 6.62; N, 9.20. Found: C, 62.95; H, 6.59;
N, 9.27.
Compounds 7b–7o were synthesized following the procedure
described above.
5.4.2. Dimethyl 2-cinnamamidosuccinate (4)
N-Methylmorpholine (1.23 ml, 11.2 mmol) in THF (30 ml) was
added isobutyl chloroformate (1.44 ml, 11.2 mmol) at ꢀ15 °C.
The mixture was stirred for 30 min at the same temperature. A
solution of the compound dimethylster 2-aspartic acid hydrochl-
orate (3.02 g, 15.3 mmol) in THF (20 ml) was added dropwise to
the reaction mixture. The stirring was continued for 1 h at
ꢀ15 °C and then the mixture was removed from the cooling bath.
The reaction was continued for 4 h and the mixture was filtrated.
After filtration, the filtrate was concentrated with a rotary evapora-
tor. The residue was dissolved in EtOAc and washed with 5% NaH-
CO3, 10% citric acid, and saturated brine in turn. The EtOAc solution
was dried over Na2SO4 and concentrated with a rotary evaporator
to afford the crude product. The crude product was recrystallized
by EtOAc to afford pure title compound 4, 2.00 g, yield: 67.54%,
mp: 123–125 °C. ESI-MS: m/z: 292.3 [M+H]+; 1H NMR (CD3OD) d
2.89 (s, 2H), 3.75 (s, 6H), 4.97–5.10 (m, 1H), 6.71 (d, J = 16,1H),
7.28–7.44 (m, 4H), 7.61–7.66 (m, 2H). Anal. Calcd for C15H17NO5:
C, 61.85; H, 5.88; N, 4.81. Found: C, 61.67; H, 5.85; N, 4.85.
5.4.6. 2-Cinnamamido-4-(isopropylamino)-4-oxobutanoic acid
(7b)
2.51 g, yield: 82.57%, mp: 130–133 °C, ½a D25
ꢁ
+31.2 (c l, MeOH);
ESI-MS: m/z: 320.4 [M+H]+; 1H NMR (CD3OD) d 0.99 (s, 6H),
2.67–2.74 (m, 2H), 3.85–3.89 (m, 1H), 4.78–4.82 (m, 1H), 6.72 (d,
J = 16, 1H), 7.41–7.43 (m, 3H), 7.52–7.59 (m, 3H). Anal. Calcd for
C16H20N2O4: C, 63.14; H, 6.62; N, 9.20. Found: C, 62.96; H, 6.57;
N, 9.26.
5.4.7. 4-(Butylamino)-2-cinnamamido-4-oxobutanoic acid (7c)
2.66 g, yield: 83.65%, mp: 124–126 °C, ½a D25
ꢁ
+41.3 (c l, MeOH);
ESI-MS: m/z: 319.3 [M+H]+; 1H NMR (CD3OD) d 0.91 (t, J = 9,3H),
1.26–1.47 (m, 4H), 2.57–2.79 (m, 2H), 3.13–3.29 (m, 2H), 4.83–
4.89 (m, 1H), 6.69 (d, J = 16, 1H), 7.38–7.41 (m, 3H), 7.56–7.62
(m, 3H). Anal. Calcd for C17H22N2O4: C, 64.13; H, 6.97; N, 8.80.
Found: C, 64.32; H, 7.01; N, 8.75.
5.4.8. 2-Cinnamamido-4-oxo-4-(pentylamino) butanoic acid
(7d)
5.4.3. 2-Cinnamamidosuccinic acid (5)
To a stirred solution of compound 4 (2 g, 6.9 mmol) in MeOH
(20 ml) was added 1 M NaOH (10 ml). The reaction mixture was
stirred for 3 h. The pH of the reaction mixture was adjusted to 5–
6 with 1 M HCl. After removal of solvents, the residue was parti-
tioned between EtOAc and 10% citric acid. The aqueous phase
was extracted twice with EtOAc. The combined organic phase
was washed with water and brine, dried over Na2SO4, filtered,
and concentrated with a rotary evaporator to give 1.74 g of crude
oil. The crude oil was purified by flash column chromatography
on silica gel (CH2Cl2/MeOH. 50/1) to afford compound 5, 1.47 g,
2.75 g, yield: 82.83%, mp: 124–125 °C, ½a D25
ꢁ
+32.4 (c l, MeOH);
ESI-MS: m/z: 333.4 [M+H]+; 1H NMR (CD3OD) d 0.93 (t, J = 7.5,
3H), 1.27–1.35 (m, 4H), 1.49–1.55 (m, 2H), 2.56–2.74 (m, 2H),
3.06–3.12 (m, 2H), 4.80–4.87 (m, 1H), 6.469 (d, J = 16, 1H), 7.40–
7.43 (m, 3H), 7.56–7.60(m, 3H). Anal. Calcd for C18H24N2O4: C,
65.04; H, 7.28; N, 8.43. Found: C, 64.85; H, 7.22; N, 8.36.
5.4.9. 2-Cinnamamido-4-(hexylamino)-4-oxobutanoic acid (7e)
2.80 g, yield: 80.92%, mp: 112–114 °C, ½a D25
ꢁ
+20.8 (c l, MeOH);
ESI-MS: m/z: 347.3 [M+H]+; 1H NMR (CD3OD) d 0.94 (t, J = 9, 3H),
1.25–1.37 (m, 6H), 1.50–1.54 (m, 2H), 2.59–2.76 (m, 2H), 3.12–
3.17 (m, 2H), 4.80–4.86 (m, 1H), 6.71 (d, J = 16, 1H), 7.35–7.43
(m, 3H), 7.57–7.64 (m, 3H). Anal. Calcd for C19H26N2O4: C, 65.87;
H, 7.56; N, 8.09. Found: C, 65.66; H, 7.60; N, 8.13.
yield: 81.2%, mp: 102–105 °C; ½a D25
ꢁ
+23.8 (c l, MeOH); ESI-MS: m/
z: 264.2 [M+H]+; 1H NMR (CD3OD) d 2.90–2.93 (m, 2H), 4.89 (d,
J = 16,1H), 7.39–7.41 (m, 3H), 7.55–7.61 (m, 3H). Anal. Calcd for
C13H13NO5: C, 59.31; H, 4.98; N, 5.32. Found: C, 59.49; H, 4.94;
N, 5.35.
5.4.10. 2-Cinnamamido-4-(octylamino)-4-oxobutanoic acid
(7f)
5.4.4. N-(2,5-Dioxotetrahydrofuran-3-yl) cinnamamide (6)
Compound 5 (10 g, 38.0 mmol) was added to 80 ml acetic anhy-
dride. The reaction mixture was stirred at 55–60 °C for 5 h. The
mixture was filtered while the solution was hot, to the filtrate
3.21 g, yield: 85.83%, mp: 108–110 °C, ½a D25
ꢁ
+29.7 (c l, MeOH);
ESI-MS: m/z: 375.5 [M+H]+; 1H NMR (CD3OD) d 0.91 (t, J = 6, 3H),
1.28–1.62 (m, 12H), 2.34–2.56 (m, 2H), 3.15–3.28 (m, 2H),

Y. Liu et al. / Bioorg. Med. Chem. 17 (2009) 7398–7404
7403
4.58–4.67 (m, 1H), 6.69 (d, J = 16, 1H), 7.34–7.38 (m, 3H), 7.40–7.54
5.4.18. 2-Cinnamamido-4-(4-nitrophenylamino)-4-
(m, 3H). Anal. Calcd for C21H30N2O4: C, 67.35; H, 8.07; N, 7.48.
Found: C, 67.56; H, 8.13; N, 7.52.
oxobutanoic acid (7n)
3.04 g, yield: 79.37%, mp: 156–159 °C, ½a D25
ꢁ
+28.9 (c l, MeOH);
ESI-MS: m/z: 384.4 [M+H]+; 1H NMR (CD3OD) d 2.61–2.84 (m,
2H), 4.62–4.90 (m, 1H), 6.70 (d, J = 16, 1H), 7.38–7.42 (m, 3H),
7.56–7.60 (m, 3H), 7.85–7.92 (m, 2H), 8.22–8.25 (m, 2H). Anal.
Calcd for C19H17N3O6: C, 59.53; H, 4.47; N, 10.96. Found: C,
59.41; H, 4.56; N, 11.00.
5.4.11. 2-Cinnamamido-4-(dodecylamino)-4-oxobutanoic acid
(7g)
3.57 g, yield: 83.02%, mp: 82–86 °C, ½a D25
ꢁ
+17.8 (c l, MeOH); ESI-
MS: m/z: 431.3 [M+H]+; 1H NMR (CD3OD) d 0.94 (t, J = 9.6, 3H),
1.23–1.45 (m, 20H), 2.64–2.72 (m, 2H), 3.09–3.16 (m, 2H), 4.60–
4.69 (m, 1H), 6.70 (d, J = 16, 1H), 7.34–7.44 (m, 3H), 7.54–7.57
(m, 3H). Anal. Calcd for C25H38N2O4: C, 69.74; H, 8.90; N, 6.51.
Found: C, 69.51; H, 8.97; N, 6.46.
5.4.19. 2-Cinnamamido-4-(naphthalene-1-ylamino)-4-
oxobutanoic acid (7o)
3.23 g, yield: 83.25%, mp: 188–190 °C, ½a D25
ꢁ
+34.5 (c l, MeOH);
ESI-MS: m/z: 389.3 [M+H]+; 1H NMR (CD3OD) d 2.66–2.86 (m,
2H), 4.86–4.92 (m, 1H), 6.67 (d, J = 16, 1H), 6.91 (d, J = J = 12, 1H),
7.29–7.37 (m, 3H), 7.49–7.54 (m, 3H), 7.56–7.59 (m, 4H), 8.54–
8.61 (m, 2H). Anal. Calcd for C23H20N2O4: C, 71.12; H, 5.19; N,
7.21. Found: C, 71.35; H, 5.24; N, 7.13.
5.4.12. 4-(4-Chlorophenylamino)-2-cinnamamido-4-
oxobutanoic acid (7h)
3.27 g, yield: 87.90%, mp: 152–155 °C, ½a D25
ꢁ
+32.6 (c l, MeOH);
ESI-MS: m/z: 373.2 [M+H]+; 1H NMR (CD3OD) d 2.79–3.02 (m,
2H), 4.89–5.02 (m, 1H), 6.71 (d, J = 16, 1H), 7.30–7.33 (m, 3H),
7.39–7.41 (m, 3H), 7.58–7.63 (m, 4H). Anal. Calcd for
C19H17ClN2O4: C, 61.03; H, 4.60; N, 7.51. Found: C, 59.49; H,
4.63; N, 7.46.
5.4.20. 2-Cinnamamido-N1-hydroxy-N4-propylsuccinamide
(8a)
Compound 7a (1.0 g, 2.4 mmol) was suspended in THF (20 ml)
and isobutyl chloroformate (1.44 ml, 11.2 mmol) and Et3N (2 ml)
were added; the suspension was stirred at room temperature and
the reaction was monitored by thin-layer chromatography in order
to ensure complete reaction. Hydroxylamine hydrochloride (0.41 g,
5.9 mmol) was dissolved in absolute methanol (20 ml) and Et3N
(1.56 ml); the mixture was stirred for 20 min, then added the mix-
ture to the above-mentioned reaction dropwise. The reaction was
monitored by thin-layer chromatography. The product solution
was rotary evaporated under reduced pressure. The residue was dis-
solved in EtOAc (50 ml) and washed with 1 N HCl and distilled water
in turn, dried over Na2SO4, filtered, and concentrated with a rotary
evaporator to give 0.74 g of crude oil. The crude oil was purified by
column chromatography on silica gel (CH2Cl2/MeOH, 50/1) to afford
5.4.13. 4-(2-Chlorophenylamino)-2-cinnamamido-4-
oxobutanoic acid (7i)
3.13 g, yield: 84.14%, mp: 148–151 °C, ½a D25
ꢁ
+20.0 (c l, MeOH);
ESI-MS: m/z: 373.2 [M+H]+; 1H NMR (CD3OD) d 2.76–2.89 (m,
2H), 4.76–4.92 (m, 1H), 6.69 (d, J = 16, 1H), 7.21–7.32 (m, 2H),
7.30–7.34 (m, 3H), 7.39–7.42 (m, 3H), 7.98 (s, 1H). Anal. Calcd for
C19H17ClN2O4: C, 61.03; H, 4.60; N, 7.51. Found: C, 59.53; H,
4.68; N, 7.43.
5.4.14. 4-(3-Chlorophenylamino)-2-cinnamamido-4-
oxobutanoic acid (7j)
3.29 g, yield: 88.44%, mp: 140–144 °C, ½a D25
ꢁ
+24.6 (c l, MeOH);
ESI-MS: m/z: 373.2 [M+H]+; 1H NMR (CD3OD) d 2.67–2.78 (m,
2H), 4.70–4.75 (m, 1H), 6.68 (d, J = 16, 1H), 7.23–7.47 (m, 3H),
7.40–7.45 (m, 3H), 7.56–7.64 (m, 3H), 7.99 (s, 1H). Anal. Calcd for
C19H17ClN2O4: C, 61.03; H, 4.60; N, 7.51. Found: C, 59.47; H,
4.66; N, 7.56.
compound 8a: 0.69 g, yield: 65.78%. mp: 145–147 °C; ½a D25
ꢁ
+22.3 (c l,
MeOH); ESI-MS: m/z: 320.4 [M+H]+; 1H NMR (CD3OD) d 0.93 (t,
J = 7.5, 3H), 1.51–1.58 (m, 2H), 2.52–2.76 (m, 2H), 3.15–3.17 (m,
2H), 4.85–4.93 (m, 1H), 6.72 (d, J = 16, 1H), 7.34–7.42 (m, 3H),
7.56–7.61 (m, 3H). Anal. Calcd for C16H21N3O4: C, 60.17; H, 6.63; N,
13.16. Found: C, 60.35; H, 6.67; N, 13.08.
5.4.15. 4-(Benzylamino)-2-cinnamamido-4-oxobutanoic acid
(7k)
5.4.21. 2-Cinnamamido-N1-hydroxy-N4-isopropylsuccinamide
2.87 g, yield: 81.53%, mp: 148–151 °C, ½a D25
ꢁ
+30.4 (c l, MeOH);
(8b)
ESI-MS: m/z: 353.4 [M+H]+; 1H NMR (CD3OD) d 2.58–2.71 (m,
2H), 4.33 (s, 2H), 4.85–4.91 (m, 1H), 6.70 (d, J = 16, 1H), 7.21–
7.29 (m, 5H), 7.37–7.42 (m, 3H), 7.55–7.62 (m, 3H). Anal. Calcd
for C20H20N2O4: C, 68.17; H, 5.72; N, 7.95. Found: C, 67.97; H,
5.69; N, 7.90.
0.65 g, yield: 61.96%, mp: 163–164 °C; ½a D25
ꢁ
+34.8 (c l, MeOH);
ESI-MS: m/z: 320.4 [M+H]+; 1H NMR (CD3OD) d 1.24 (d, J = 9.6,
6H), 2.50–2.78 (m, 2H), 3.85–4.01 (m, 1H), 4.76–4.85 (m, 1H),
6.67 (d, J = 16, 1H), 7.30–7.42 (m, 3H), 7.58–7.66 (m, 3H). Anal.
Calcd for C16H21N3O4: C, 60.17; H, 6.63; N, 13.16. Found: C,
60.37; H, 6.59; N, 13.27.
5.4.16. 2-Cinnamamido-4-oxo-4-(phenylamino) butanoic acid
(7l)
5.4.22. N4-Butyl-2-cinnamamido-N1-hydroxysuccinamide (8c)
2.71 g, yield: 80.18%, mp: 166–168 °C, ½a D25
ꢁ
+19.0 (c l, MeOH);
0.71 g, yield: 67.81%, mp: 153–154 °C; ½a D25
ꢁ
+38.6 (c l, MeOH);
ESI-MS: m/z: 339.4 [M+H]+; 1H NMR (CD3OD) d 2.67–2.80 (m,
2H), 4.87–4.96 (m, 1H), 6.69 (d, J = 16, 1H), 7.30–7.34 (m, 3H),
7.37–7.62 (m, 5H), 7.62–7.65 (m, 3H). Anal. Calcd for
C19H18N2O4: C, 67.44; H, 5.36; N, 8.28. Found: C, 67.64; H, 5.41;
N, 8.34.
ESI-MS: m/z: 334.7 [M+H]+; 1H NMR (CD3OD) d 0.95 (t, J = 6.3,
3H), 1.15–1.51 (m, 4H), 2.57–2.79 (m, 2H), 3.16–3.34 (m, 2H),
4.85–4.93(m, 1H), 6.68 (d, J = 16, 1H), 7.40–7.41 (m, 3H), 7.54–
7.59 (m, 3H). Anal. Calcd for C17H23N3O4: C, 61.25; H, 6.95; N,
12.60. Found: C, 61.08; H, 6.89; N, 12.46.
5.4.17. 2-Cinnamamido-4-(3-nitrophenylamino)-4-
oxobutanoic acid (7m)
5.4.23. 2-Cinnamamido-N1-hydroxy-N4-pentylsuccinamide
(8d)
3.11 g, yield: 81.20%, mp: 140–142 °C, ½a D25
ꢁ
+26.4 (c l, MeOH);
0.86 g, yield: 82.30%, mp: 169–170 °C; ½a D25
ꢁ
+30.9 (c l, MeOH);
ESI-MS: m/z: 384.3 [M+H]+; 1H NMR (CD3OD) d 2.67–2.74 (m,
2H), 4.78–4.86 (m, 1H), 6.71 (d, J = 16, 1H), 7.40–7.41 (m, 3H),
7.56–7.61 (m, 3H), 7.76–7.99 (m, 3H), 8.69 (s, 1H). Anal. Calcd for
C19H17N3O6: C, 59.53; H, 4.47; N, 10.96. Found: C, 59.34; H, 4.54;
N, 11.04.
ESI-MS: m/z: 348.5 [M+H]+; 1H NMR (CD3OD) d 0.94 (t, J = 7.5,
3H), 1.23–1.38 (m, 4H), 1.50–1.56 (m, 2H), 2.56–2.81 (m, 2H),
3.15–3.18 (m, 2H), 4.82–4.89 (m, 1H), 6.72 (d, J = 16, 1H), 7.34–
7.43 (m, 3H), 7.58–7.63 (m, 3H). Anal. Calcd for C18H25N3O4: C,
62.23; H, 7.25; N, 12.10. Found: C, 62.42; H, 7.31; N, 12.01.

7404
Y. Liu et al. / Bioorg. Med. Chem. 17 (2009) 7398–7404
5.4.24. 2-Cinnamamido-N4-hexyl-N1-hydroxysuccinamide (8e)
5.4.32. 2-Cinnamamido-N1-hydroxy-N4-(3-nitrophenyl)
succinamide (8m)
0.82 g, yield: 78.62%, mp: 178–180 °C; ½a D25
ꢁ
+22.3 (c l, MeOH);
ESI-MS: m/z: 362.6 [M+H]+; 1H NMR (CD3OD) d 0.94 (t, J = 7.5,
3H), 1.25–1.34 (m, 6H), 1.50–1.56 (m, 2H), 2.55–2.82 (m, 2H),
3.18–3.22 (m, 2H), 4.81–4.88 (m, 1H), 6.71 (d, J = 16, 1H), 7.34–
7.43 (m, 3H), 7.56–7.62 (m, 3H). Anal. Calcd for C19H27N3O4: C,
63.14; H, 7.53; N, 11.63. Found: C, 62.95; H, 7.46; N, 11.51.
0.70 g, yield: 67.37%, mp: 153–155 °C, ½a D25
ꢁ
+23.9 (c l, MeOH);
ESI-MS: m/z: 399.4 [M+H]+; 1H NMR (CD3OD) d 2.71–2.79 (m,
2H), 4.85–4.94 (m, 1H), 6.67 (d, J = 16, 1H), 7.40–7.41 (m, 3H),
7.56–7.61 (m, 3H), 7.74–7.78 (m, 3H), 8.68 (s, 1H). Anal. Calcd for
C19H118N4O6: C, 57.28; H, 4.55; N, 14.06. Found: C, 57.10; H,
4.52; N, 14.14.
5.4.25. 2-Cinnamamido-N1-hydroxy-N4-octylsuccinamide (8f)
0.85 g, yield: 81.73%, mp: 156–158 °C, ½a D25
ꢁ
+32.0 (c l, MeOH);
5.4.33. 2-Cinnamamido-N1-hydroxy-N4-(4-nitrophenyl)
succinamide (8n)
ESI-MS: m/z: 390.4 [M+H]+; 1H NMR (CD3OD) d 0.91 (t, J = 6, 3H),
1.25–1.77 (m, 12H), 2.27–2.50 (m, 2H), 3.27–3.45 (m, 2H), 4.59–
4.69 (m, 1H), 6.70 (d, J = 16, 1H), 7.28–7.35 (m, 3H), 7.40–7.56
(m, 3H). Anal. Calcd for C21H31N3O4: C, 64.76; H, 8.02; N, 10.79.
Found: C, 64.96; H, 8.06; N, 10.72.
0.74 g, yield: 71.22%, mp: 206–207 °C, ½a D25
ꢁ
+31.2 (c l, MeOH);
ESI-MS: m/z: 399.4 [M+H]+; 1H NMR (CD3OD) d 2.61–2.84 (m,
2H), 4.61–4.93 (m, 1H), 6.73 (d, J = 16, 1H), 7.40–7.42 (m, 3H),
7.59–7.62 (m, 3H), 7.87–7.90 (m, 2H), 8.22–8.25 (m, 2H). Anal.
Calcd for C19H118N4O6: C, 57.28; H, 4.55; N, 14.06. Found: C,
57.09; H, 4.50; N, 14.12.
5.4.26. 2-Cinnamamido-N4-dodecyl-N1-hydroxysuccinamide (8g)
0.70 g, yield: 68.43%, mp: 155–157 °C, ½a D25
ꢁ
+19.2 (c l, MeOH);
ESI-MS: m/z: 446.6 [M+H]+; 1H NMR (CD3OD) d 0.86 (t, J = 9, 3H),
1.20–1.35 (m, 20H), 2.60–2.80 (m, 2H), 3.15–3.25 (m, 2H),4.70–
4.80 (m, 1H), 6.72 (d, J = 16, 1H), 7.30–7.42 (m, 3H), 7.52–7.58
(m, 3H). Anal. Calcd for C25H39N3O4: C, 67.39; H, 8.82; N, 9.43.
Found: C, 67.18; H, 8.75; N, 9.51.
5.4.34. 2-Cinnamamido-N1-hydroxy-N4-(naphthalene-1-yl)
succinamide (8o)
0.66 g, yield: 63.52%, mp: 170–172 °C; ½a D25
ꢁ
+36.8 (c l, MeOH);
ESI-MS: m/z: 404.6 [M+H]+; 1H NMR (CD3OD) d 2.66–2.86 (m,
2H), 4.93–4.96 (m, 1H), 6.65 (d, J = 16, 1H), 6.82 (d, J = 12, 1H),
7.21–7.22 (m, 3H), 7.39–7.41 (m, 3H), 7.44–7.56 (m, 4H), 8.64–
8.71 (m, 2H). Anal. Calcd for C23H21N3O4: C, 68.47; H, 5.25; N,
10.42. Found: C, 68.26; H, 5.28; N, 10.49.
5.4.27. N4-(4-Chlorophenyl)-2-cinnamamido-N1-hydroxysuccin-
amide (8h)
0.79 g, yield: 75.96%, mp: 154–156 °C, ½a D25
ꢁ
+29.5 (c l, MeOH);
ESI-MS: m/z: 388.5 [M+H]+; 1H NMR (CD3OD) d 2.67–2.79 (m, 2H),
4.93–5.01 (m, 1H), 6.71 (d, J = 16, 1H), 7.30–7.33 (m, 3H), 7.40–
7.59 (m, 4H), 7.60–7.68 (m, 3H). Anal. Calcd for C19H18ClN3O4: C,
58.84; H, 4.68; N, 10.84. Found: C, 58.66; H, 4.73; N, 10.76.
Acknowledgements
This work was supported by the National Nature Science Foun-
dation of China (Grant Nos. 30772654, 90713041) and the Ph.D.
Programs Foundation of the Ministry of Education of PR China
(No. 20060422029) and the National High Technology Research
and Development Program of China (863 Project; Grant No.
2007AA02Z314).
5.4.28. N4-(2-Chlorophenyl)-2-cinnamamido-N1-hydroxysuccin-
amide (8i)
0.82 g, yield: 78.85%; mp: 157–159 °C, ½a D25
ꢁ
+23.2 (c l, MeOH);
ESI-MS: m/z: 388.5 [M+H]+; 1H NMR (CD3OD) d 2.67–2.76 (m,
2H), 4.92–5.02 (m, 1H), 6.68 (d, J = 16, 1H), 7.30–7.33 (m, 3H),
7.40–7.59 (m, 4H), 7.60–7.68 (m, 3H). Anal. Calcd for
C19H18ClN3O4: C, 58.84; H, 4.68; N, 10.84. Found: C, 58.68; H,
4.75; N, 10.74.
References and notes
1. Robert, S. D. Heart Fail Rev. 2008, 133, 293–298.
2. Shimizu, T.; Tani, K.; Hase, K.; Ogawa, H.; Huang, L.; Shinomiya, F.; Sone, S.
Arthritis Rheum. 2002, 46, 2330.
3. Sato, Y. Biol. Pharm. Bull. 2004, 772–776.
4. Chung, M. C.; Lee, H. J.; Chun, H. K.; Lee, C. H.; Kim, S. I.; Kho, Y. H. Biosci.
Biotechnol. Biochem. 1996, 5, 898–900.
5. Chung, M. C.; Chun, H. K.; Han, K. H.; Lee, H. J.; Lee, C. H.; Kho, Y. H. J. Antibiot.
(Tokyo) 1996, 491, 99–102.
6. Chung, M. C.; Lee, C. H.; Lee, H. J.; Kho, Y. H.; Chun, H. K. Biotechnol. Lett. 1997,
197, 607–610.
7. Rich, D. H.; Moon, B. J.; Harbeson, S. J. Med. Chem. 1984, 274, 417–422.
8. Burley, S. K.; David, P. R.; Lipscomb, W. N. Proc. Natl. Acad. Sci. U.S.A. 1991, 8816,
6916–6920.
5.4.29. N4-(3-Chlorophenyl)-2-cinnamamido-N1-hydroxysuccin-
amide (8j)
0.78 g, yield: 75.0%, mp: 149–152 °C, ½a D25
ꢁ
+26.1 (c l, MeOH);
ESI-MS: m/z: 388.5 [M+H]+; 1H NMR (CD3OD) d 2.66–2.77 (m,
2H), 4.73–4.78 (m, 1H), 6.70 (d, J = 16, 1H), 7.18–7.39 (m, 3H),
7.40–7.44 (m, 4H), 7.55–7.63 (m, 3H). Anal. Calcd for
C19H18ClN3O4: C, 58.84; H, 4.68; N, 10.84. Found: C, 58.67; H,
4.74; N, 10.78.
9. Aoyagi, T.; Yoshida, S.; Nakamura, Y.; Shigihara, Y.; Hamada, M.; Takeuchi, T. J.
Antibiot. (Tokyo) 1990, 432, 143–148.
5.4.30. N4-Benzyl-2-cinnamamido-N1-hydroxysuccinamide
(8k)
10. Shim, J. S.; Kim, J. H.; Cho, H. Y.; Yum, Y. N.; Kim, S. H.; Park, H. J.; Shim, B. S.;
Choi, S. H.; Kwon, H. J. Chem. Biol. 2003, 108, 695–704.
11. Umezawa, H.; Aoyagi, T.; Suda, H.; Hamada, M. Antibiotics 1976, 29, 97.
12. Li, Q. B.; Fang, H.; Xu, W. F. Bioorg. Med. Chem. Lett. 2007, 17, 2935.
13. Wang, Q.; Chen, M. Y.; Zhu, H. W.; Zhang, J.; Fang, H.; Wang, B. H.; Xu, W. F.
Bioorg. Med. Chem. 2008, 16, 5473.
14. Shang, L. Q.; Maeda, T.; Xu, W. F.; Kishioka, S. J. Enzyme Inhib. Med. Chem. 2008,
23, 198–205.
15. Shang, L. Q.; Wang, Q.; Fang, H.; Mu, J. J.; Wang, X. J.; Yuan, Y. M.; Wang, B. H.;
Xu, W. F. Bioorg. Med. Chem. 2008, 16, 6663.
0.80 g, yield: 76.70%, mp: 136–138 °C, ½a D25
ꢁ
+33.2 (c l, MeOH);
ESI-MS: m/z: 368.5 [M+H]+; 1H NMR (CD3OD) d 2.61–2.70 (m,
2H), 4.45 (s, 2H), 4.88–4.93 (m, 1H), 6.71 (d, J = 16, 1H), 7.23–
7.32 (m, 5H), 7.40–7.41 (m, 3H), 7.55–7.59 (m, 3H). Anal. Calcd
for C20H21N3O4: C, 65.38; H, 5.76; N, 11.44. Found: C, 65.59; H,
5.80; N, 11.36.
16. Wang, J. L.; Xu, W. F. J. Chem. Res., Synop. 2003, 12, 789.
17. Kiyoshi, I.; Yoshitaka, N.; Tadashi, Y., et al J. Biol. Chem. 2006, 281, 33664–
33676.
18. Wang, T.; Wang, S. M. Chin. J. Cancer 2000, 19, 782–785.
19. Fan, T. L.; Xu, P. R.; Yang, J. Shandong Med. 2006, 46334, 8–9.
20. Zhu, W.-Y.; Huang, J.-Q.; Huang, W. Cancer Res. Prev. Treat. 2000, 273, 182–184.
21. Qian, H.; Liu, J. Jiangsu Med. 2001, 27, 17–19.
5.4.31. 2-Cinnamamido-N1-hydroxy-N4-phenylsuccinamide (8l)
0.77 g, yield: 73.75%, mp: 166–168 °C; ½a D25
ꢁ
+21.8 (c l, MeOH);
ESI-MS: m/z: 354.5 [M+H]+; 1H NMR (CD3OD) d 2.66–2.78 (m, 2H),
4.90–4.99 (m, 1H), 6.64 (d, J = 16, 1H), 7.14–7.34 (m, 3H), 7.36–
7.60 (m, 5H), 7.60–7.68 (m, 3H). Anal. Calcd for C19H19N3O4: C,
64.58; H, 5.42; N, 11.89. Found: C, 64.37; H, 5.46; N, 11.81.
22. Jordis, U.; Sauter, F.; Siddiqi, S. M. Ind. J. Chem. Soc. 1989, 28, 294–296.