Novel leucine ureido derivatives as inhibitors of aminopeptidase N (APN)

Article abstract of DOI:10.1016/j.bmc.2013.01.068

Aminopeptidase N (APN/CD13) over expressed on tumor cells, plays a critical role in tumor invasion, metastasis, and tumor angiogenesis. Here we described the design, synthesis and preliminary activity studies of novel leucine ureido derivatives as aminopeptidase N (APN/CD13) inhibitors. The results showed that compound 8c had the most potent inhibitory activity against APN with the IC ₅₀ value to 0.06 ± 0.041 μM, which could be used for further anticancer agent research.

Full text of DOI:10.1016/j.bmc.2013.01.068

Bioorganic & Medicinal Chemistry 21 (2013) 1621–1627
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel leucine ureido derivatives as inhibitors
of aminopeptidase N (APN)
⇑
Chunhua Ma, Kang Jin, Jiangying Cao, Lei Zhang, Xiaoguang Li, Wenfang Xu
Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University 44, West Culture Road, Jinan 250012, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Aminopeptidase N (APN/CD13) over expressed on tumor cells, plays a critical role in tumor invasion,
metastasis, and tumor angiogenesis. Here we described the design, synthesis and preliminary activity
studies of novel leucine ureido derivatives as aminopeptidase N (APN/CD13) inhibitors. The results
showed that compound 8c had the most potent inhibitory activity against APN with the IC50 value to
Received 31 December 2012
Revised 30 January 2013
Accepted 31 January 2013
Available online 9 February 2013
0.06 0.041
l
M, which could be used for further anticancer agent research.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Aminopeptidase N
Inhibitors
Leucine ureido derivatives
Synthesis
1. Introduction
compound. In our series of L-arginine derivatives, we found that
the compounds contained ortho-substituted phenyl groups have
better activities than those contained unsubstituted.17 And we also
found that a substituent in meta-position such as chlorine, fluorine,
bromine, or methane could enhance the potency of inhibitors in
our series of indoline-2,3-dione derivatives.18 Based on these rea-
sons, a series of novel leucine ureido derivatives which containing
different meta- or ortho-substituents on the phenyl group of 4k
have been synthesized, while, some analogues with substituted
benzyl groups also have been obtained (Fig. 1). The activity results
and analysis of structure–activity relationship (SAR) were also
shown in this article.
Aminopeptidase N (APN; EC 3.4.11.2) is a zinc-dependent type
II membrane-bond ectopeptidase,1 which preferentially releases
neutral and basic amino acids from the N-terminus of oligopep-
tides.2 Scientific research has been found that APN is over-ex-
pressed on tumor cells and plays key roles in extracellular matrix
degradation, tumor cell invasion and tumor angiogenesis.3,4 There-
fore, APN was regarded as an attractive target for anti-cancer drug
design.
Since 1976, several natural and small molecule inhibitors of
APN have been found, for example, Bestatin,5 Probestin,6 Amasta-
tin,7 Actinonin,8 Phebestin,9 Lapstatin,10 AHPA-Val,11 Leuhistin,12
and so on. Among them, Bestatin has been launched and is widely
employed clinically as an anti-tumor agent. Our group has previ-
ously reported several kinds of synthetic APN inhibitors, such as
2. Chemistry
L
-lysine derivates,13 1,3,4-thiadiazole derivates,14
L-iso-glutamine
The target compounds 8a–8g and 10a–10d were synthesized
efficiently via the route outlined in Scheme 1. Starting from com-
mercially available L-leucine, the key intermediate leucine isocya-
derivatives,15 chloramphenicol amine derivatives,16
L-arginine
derivatives,17
derivatives.19
indoline-2,3-dione
derivates18
and ureido
nate was obtained via esterification and isocyanate, and then
coupled with the corresponding ortho-substituted anilines or ben-
zylamines. Without further purification, they were directly trans-
formed into hydroxamic acids as the target products. The main
synthetic methods of target compounds 9a–9g and 11a–11b are
shown in Scheme 2. With corresponding meta-substituted anilines
or benzylamines, as the raw materials, they were converted into
isocyanates, and then coupled with leucine methyl esters, to obtain
the ureido linker. Finally the methyl ester was transformed into
hydroxamic acids as the target compounds.
In our previous work, a series of ureido derivatives was reported
as potent APN inhibitors. Some of them showed potential inhibi-
tory activities against APN. Especially, compound 4k, whose IC50
was 2.7 lM compared with 9.1 l
M of Bestatin.20 Herein, in order
to find better APN inhibitors, we used compound 4k as the leading
⇑
Corresponding author. Tel./fax: +86 531 88382264.
E-mail address: wfxu@yahoo.cn (W. Xu).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.068

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C. Ma et al. / Bioorg. Med. Chem. 21 (2013) 1621–1627
O
O
O
R
H
N
H
N
HO
HO
N
H
N
H
HO
N
H
N
H
O
O
R
O
O
H
N
HO
N
H
N
H
O
H
O
H
N
N
R
HO
N
H
N
N
H
N
H
R
H
O
4k
R=F, Cl, Br, CH3,
OCH3, CF3, OH
Figure 1. The new APNIs derived from compound 4k.
O
O
C
O
b
a
HCl
O
O
O
OH
N
NH2
NH2
1
2
3
R
O
R
O
H
H
N
H
H
N
N
N
OH
O
N
H
O
O
c
d
7a-7g
8a-8g
O
O
H
N
H
N
H
N
H
N
OH
O
N
H
R
O
R
O
13a-13d
10a-10d
Scheme 1. Reagents and conditions: (a) acetyl chloride, MeOH, reflux; (b) triphosgene, NaHCO3, DCM, ice-bath; (c) corresponding ortho-substituted anilines or benzylamines,
DCM, room temperature; (d) NH2OK, MeOH, 1 h.
O
O
H
N
c
R2
a
b
O
R2
HO
N
H
N
H
R2 NH2
4
R2
N C O
N
H
N
H
O
O
9a-9g, 11a-11b
L-Leu and DCM, Et3N, room temp, 1–2 h; (c) NH2OK, MeOH, 1 h.
5
6
Scheme 2. Reagents and conditions: (a) triphosgene, toluene, reflux; (b) methyl ester hydrochlorides of
designing of potential APNIs. As the above mentioned selectivity
against APN, the following SARs were mainly discussed about
APN inhibition.
3. Results and discussion
The target compounds were evaluated for their inhibitory activ-
ities toward APN/CD13 and HDACs. Similar to APN, HDACs are
zinc-dependent metalloproteinase as well and associated closely
with the invasion and metastasis of tumor. Thereby the assay
was performed on both of APN and HDACs so as to identify their
selectivity, all the inhibition results are summarized in
Tables 1–3. Bestatin was used as the positive control to APN, while,
SAHA was used for HDACs.
As shown in Tables 1–3, it is worthy to note that these ureido
derivatives displayed a better enzymatic inhibition towards APN
compared with HDACs, with IC50 values lying in micromole level.
These results, to a certain extent, validated our strategy for rational
Among these inhibitors, generally speaking, compounds with
ortho-substituents of phenyl group showed better activity than
the leading compound 4k. The possible reason may be due to the
substituent groups enhancing the interaction with the hydropho-
bic region of the enzyme. Comparing 8a–8d and 10a–10d, we
can find the activity of substituted anilines is better than benzyl-
amines, it is reason may be the length between benzene ring of
aniline to hydroxamic acid is more similar to the length between
hydrophobic region and zinc ion. Comparing 8a–8g and 9a–9g,
we could find that the compounds contained ortho-substituted
phenyl groups have better activities than those contained

C. Ma et al. / Bioorg. Med. Chem. 21 (2013) 1621–1627
1623
Table 1
The structures and inhibitory activities of the target compounds 8a–8g against APN and HDACs
O
H
N
HO
N
H
N
H
O
R
a
a
Compd
R
IC50
(lM) APN
IC50 (lM) HDACs
8a
8b
8c
8d
8e
8f
F
Cl
Br
CH3
OCH3
CF3
OH
0.52 0.064
0.093 0.120
0.06 0.041
0.18 0.032
0.13 0.027
0.79 0.163
11.9 1.078
>100
>100
>100
>100
>100
>100
>100
8g
NH2
O
Beststin
3.40 0.028
>100
>100
OH
N
H
O
OH
4k
H
1.15 0.528
—
O
H
N
OH
SAHA
0.12 0.03
N
H
O
a
Mean values and standard deviations of triplicate experiments are given.
Table 2
The structures and inhibitory activities of the target compounds 9a–9g against APN and HDACs
O
H
N
HO
N
H
N
H
R
O
a
a
Compd
R
IC50
(lM) APN
IC50
(l
M) HDACs
9a
9b
9c
9d
9e
9f
F
Cl
Br
CH3
OCH3
CF3
OH
1.90 0.258
1.66 0.153
0.58 0.060
0.24 0.234
1.36 0.342
1.97 0.324
1.70 1.890
>100
>100
>100
>100
>100
>100
>100
9g
NH2
O
Beststin
3.40 0.028
1.15 0.528
>100
>100
OH
N
H
O
OH
4k
H
O
H
N
OH
SAHA
0.12 0.03
N
H
O
a
Mean values and standard deviations of triplicate experiments are given.
meta-substituted phenyl groups. Comparing compounds 8a–8g
with ortho-substituted at phenyl groups, the APN inhibitory activ-
ities were different with the substitutions on the aromatic ring. The
data shown in Table 1 suggested that the preferred substitutions
against APN were, in decreasing order, halogen-substitution, meth-
oxy-substitution, methane-substitution and hydroxy-substitution.
And different halogens in aromatic ring also have different levels
of impacts on their activities. The compounds with a bromide
group on the benzene ring (8c) were more potent than others con-
taining a chloride substituent (8b), followed by the fluorinate
inhibitors (8a) at last. The possible reason may be due to the bigger
volume hydrophobic substitution can enhance the interaction with
the hydrophobic region of the enzyme. In another way, compound
8f with trifluoromethyl-group showed worse activity than com-
pound 8c, it seems that too big bulk leads to impaired activity, sug-
gesting there is a space requirement in the binding pockets to
accommodate the suitable substituents. Compound with hydroxy-
substitution (8g) on the aromatic ring show the least APN inhibitory
activities, which may reveal hydrophilic groups diminish activities
of compounds.

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C. Ma et al. / Bioorg. Med. Chem. 21 (2013) 1621–1627
Table 3
The structures and inhibitory activities of the target compounds 10a–10d and 11a–11b against APN and HDACs
O
R
1
H
N
R
2
HO
N
H
N
H
O
a
a
Compd
R1
R2
IC50
(
lM) APN
IC50 (lM) HDACs
10a
10b
10c
10d
11a
11b
F
H
H
H
H
Cl
CH3
1.38 0.246
0.22 0.124
0.13 0.036
0.90 0.235
0.35 0.215
0.44 0.165
>100
>100
>100
>100
>100
>100
Cl
Br
CH3
H
H
NH2
O
Beststin
3.40 0.028
1.15 0.528
>100
>100
OH
N
H
OH
O
4k
H
O
H
N
OH
SAHA
0.12 0.03
N
H
O
a
Mean values and standard deviations of triplicate experiments are given.
The most active compounds are 8c, which had a better inhibi-
tion (IC50 = 0.06 0.041 M) than leading compound and control
Bestatin (IC50 = 3.40 0.028 M). Compounds 8b, 8d, 8e, 10b and
10c also have considerable activities (IC50 = 0.093, 0.18, 0.13, 0.22
and 0.13 M, respectively).
Furthermore, we assessed the ability of some compounds with
the nanomolar range for enzymatic inhibition on the human APN
deriving from cultured ES-2 human ovarian clear cell carcinoma
cells high-expressing APN. Results were presented in Figure 2, from
which we can see that all these compounds were still better than
Compound 8c shows the best effect against ES-2 cells with the
IC50 of 405 0.08 M complying with the enzymatic assay.
l
l
l
Aiming to investigate the interaction between the target com-
pounds and APN, the most active compound 8c, was chosen to
be constructed using a Sybyl/Sketch module and optimized via
Powell’s method by the Tripos force field with convergence crite-
rion set at 0.05 kcal/(Å mol), and assigned with the Gasteiger–Hüc-
kel method. The docking study of 8c and the active site of APN was
performed using Sybyl/FlexX module. The active site was defined
as 10.0 Å radius circles around Bestatin in the co-crystal structure
(PDB code: 4YFR). Other docking parameters utilized in the pro-
gram were remained default. The result showed in Figure 3 a sug-
gested that phenyl group can insert into S1 pocket and the
hydroxamic acid group can chelate with the zinc ion which was
the crucial catalytic factor in active site. In addition, a 2D pattern
of detailed binding mode was created as well (Fig. 3b). The phenyl
group of compound 8c can form hydrophobic interaction with
Phe472 of S1 pocket, while, the ureido group and hydroxamic acid
group can from hydrogen bonds with Gln213, Ala353 and Gln213,
Glu355, Glu411, Tyr477, respectively.
l
Bestatin (20.12 1.265
l
M), some even with more than 10-fold
8c showed the best capability
lM). Similar to the above enzyme inhibitory
improvement,
(IC50 = 0.53 0.116
and
activity, the human APN inhibition of these compounds also pre-
sented consistent trends.
Additionally, compounds 8b, 8c, 8e are detected for their poten-
tial effects on proliferation of two tumor cell lines (ES-2 and U-937
cells) with Bestatin as control via MTT assay. The result depicted in
Table 4 indicated that all compounds’ anti-proliferative effects
against ES-2 cells are better than against U-937 cells mainly owing
to APN’s higher expression level on ES-2 cells than the other.
Although the computed information partially supported our
assumption, the exact binding model of 8c with APN should be ob-
Table 4
The structures and cell proliferation IC50 values of some target compounds toward
ES-2 and U-937
O
H
N
HO
N
H
N
H
O
R
a
a
Compd
R
IC50
(lm) towards ES-2
IC50 (lm) towards U-937
8b
8c
8e
Bestatin
Cl
Br
OCH3
—
615
405
480
620
>1000
>1000
>1000
>1000
Figure 2. Inhibitory activity of compounds 8b, 8c, 8d, 8e, 10b, 10c and Bestatin
against APN on ES-2 cells. Data expressed are mean values of three independent
experiments.
a
Mean values and standard deviations of triplicate experiments are given.

C. Ma et al. / Bioorg. Med. Chem. 21 (2013) 1621–1627
1625
million and J in Hertz. TMS was used as an internal standard.
ESI-MS were determined on an API 4000 spectrometer. Measure-
ments were made in DMSO-d6 solutions. Melting points were
tested using an electrothermal melting point apparatus and were
uncorrected.
5.2. General procedure for the synthesis of 8a–8g and 10a–10d
To a mixture of L-leucine methyl ester hydrochloride (3.62 g,
20 mmol) in saturated NaHCO3 (72 mL) and DCM (72 mL) was
added triphosgene (1.96 g, 6.6 mmol). The reaction mixture was
vigorously stirred under ice-water bath for 1 h and the organic
layer was collected. The water layer was extracted with DCM for
three times and the organic phase was combined and dried with
MgSO4. After the solvent removed under vacuum, the residue
was dissolved in DCM (20 mL). This solution was then added to
corresponding ortho-substituted anilines or benzylamines
(21 mmol) in DCM (80 mL) under ice bath. The reaction mixture
was stirred at room temperature for 12 h and then the solvent
was removed under low pressure. The residue was taken up with
ethyl acetate (100 mL) and washed with 1 N HCl (30 mL) and brine
(30 mL). The organic phase was dried with MgSO4. After the sol-
vent removed, the residue without purification was directly added
to a solution of potassium hydroxylamine (8.37 g, 56 mmol) in
methanol (20 mL). The reaction mixture was stirred at room tem-
perature for 1 h and then removed methanol under low pressure.
The residue was taken up with 1 N HCl and extracted with ethyl
acetate. The organic phase was washed with brine and dried with
MgSO4. After the solvent removed under low pressure, the residue
was separated by silica gel column chromatography to afford 8a–
8g and 10a–10d, respectively.
5.2.1. (S)-2-[3-(2-Fluoro-phenyl)-ureido]-4-methyl-pentanoic
acid hydroxyamide (8a)
White powder, yield 43%, mp = 172–174 °C; 1H NMR (600 MHz,
DMSO-d6) d 10.70 (s, 1H), 8.76 (s, 1H), 8.35 (s, 1H), 8.10 (t, 1H,
J = 6.0 Hz), 7.15 (m, 1H), 7.06 (d, 1H, J = 6.0 Hz), 6.92 (m, 1H),
4.13 (m, 1H), 1.59 (m, 1H), 1.42 (t, 2H, J = 7.2 Hz), 0.89 (dd, 6H,
Figure 3. (a) The FlexX docking result of 8c with APN (PDB: 4YFR). (b) The docking
result of 8c shown by LIGPLOT
J = 6.6, 10.8 Hz); HRMS (AP-ESI) m/z Calcd for
C13H18FN3O3
[M+H]+ 284.1405. Found, 284.1404.
tained from further X-ray crystal studies, which is under research
in our lab.
5.2.2. (S)-2-[3-(2-Chloro-phenyl)-ureido]-4-methyl-pentanoic
acid hydroxyamide (8b)
4. Conclusions
White powder, yield 51%, mp = 163–164 °C; 1H NMR (600 MHz,
DMSO-d6): d 10.80 (s, 1H), 8.87 (s, 1H), 8.15 (m, 2H), 7.39 (dd, 1H,
J = 1.2, 7.2 Hz), 7.36 (d, 1H, J = 9.0 Hz), 7.23 (t, 1H, J = 7.2 Hz), 6.94
(t, 1H, J = 7.2 Hz), 4.18 (m, 1H), 1.58 (m, 1H), 1.42 (t, 2H,
J = 7.2 Hz), 0.89 (dd, 6H, J = 6.6, 10.8 Hz); HRMS (AP-ESI) m/z Calcd
for C13H18ClN3O3 [M+H]+ 300.1109. Found, 300.1110.
In all, one series of novel potent leucine ureido derivative as
APN inhibitors have been synthesized and evaluated. The prelimin-
ary results showed that most of the target compounds exhibited
better inhibition than the control Bestatin and the leading com-
pound 4k. These results suggest that leucine ureido derivatives
could possess good inhibitory activities after substitution intro-
duced in ortho-position of phenyl group, which could be used as
new lead for further structure transformation in the future APNIs
exploration.
5.2.3. (S)-2-[3-(2-Bromo-phenyl)-ureido]-4-methyl-pentanoic
acid hydroxyamide (8c)
White powder, yield 49%, mp = 182–184 °C; 1H NMR (600 MHz,
DMSO-d6): d 10.81 (s, 1H), 8.88 (s, 1H), 8.06 (d, 1H, J = 7.8 Hz), 7.98
(s, 1H), 7.55 (d, 1H, J = 7.8 Hz), 7.43 (d, 1H, J = 9.0 Hz), 7.27 (t, 1H,
J = 7.8 Hz), 6.89 (t, 1H, J = 7.8 Hz), 4.15 (m, 1H), 1.59 (m, 1H), 1.42
(t, 2H, J = 7.2 Hz), 0.89 (dd, 6H, J = 6.6, 10.8 Hz); HRMS (AP-ESI)
m/z Calcd for C13H18BrN3O3 [M+H]+ 344.0604. Found, 344.0598.
5. Experiment
5.1. Chemistry: general procedures
All the materials involved were purchased from commercial
suppliers. Solvents were distilled prior to use. All the reactions
were monitored by thin-layer chromatography on 0.25 mm silica
gel plates (60GF-254) and visualized with UV light, or chloride fer-
ric. The products were purified by column chromatography which
was performed using 200–300 mesh silica gel. NMR spectra were
determined on a Brucker Avance 600 spectrometer, d in parts per
5.2.4. (S)-4-Methyl-2-(3-o-tolyl-ureido)-pentanoic acid
hydroxyamide (8d)
White powder, yield 47%, mp = 184–186 °C; 1H NMR (600 MHz,
DMSO-d6): d 10.80 (s, 1H), 8.88 (s, 1H), 7.85 (d, 1H, J = 8.4 Hz), 7.11
(d, 1H, J = 7.2 Hz), 7.07 (t, 1H, J = 7.8 Hz), 6.86 (m, 2H), 4.18 (m, 1H),
2.17 (s, 3H), 1.58 (m, 1H), 1.41 (t, 2H, J = 7.2 Hz), 0.89 (dd, 6H,

1626
C. Ma et al. / Bioorg. Med. Chem. 21 (2013) 1621–1627
J = 6.6, 8.4 Hz); HRMS (AP-ESI) m/z Calcd for C14H21N3O3, [M+H]+
280.1656. Found, 280.1654.
(dd, 6H, J = 6.6, 8.4 Hz); HRMS (AP-ESI) m/z Calcd for C15H23N3O3,
[M+H]+ 294.1812. Found, 294.1811.
5.2.5. (S)-2-[3-(2-Methoxy-phenyl)-ureido]-4-methyl-pentanoic
acid hydroxyamide (8e)
5.3. General procedure for the synthesis of 9a–9g and 11a–11b
White powder, yield 43%, mp = 170–172 °C; 1H NMR (600 MHz,
DMSO-d6): d 10.79 (s, 1H), 8.82 (s, 1H), 8.09 (s, 1H), 8.06 (dd, 1H,
J = 1.2, 7.2 Hz), 7.15 (d, 1H, J = 9.0 Hz), 6.95 (d, 1H, J = 7.2 Hz),
6.86 (t, 1H, J = 7.2 Hz), 6.82 (t, 1H, J = 7.2 Hz), 4.12 (m, 1H), 3.82
(s, 3H), 1.58 (m, 1H), 1.40 (t, 2H, J = 7.2 Hz), 0.89 (dd, 6H, J = 6.6,
Corresponding meta-substituted anilines or benzylamines
(15 mmol) was added to
a solution of triphosgene (2.22 g,
7.5 mmol) in dry toluene (80 mL) in room temperature. The reac-
tion mixture was refluxed for 4 h and then solvents removed under
low pressure. The residue was dissolved in DCM (20 mL) and this
10.8 Hz); HRMS (AP-ESI) m/z Calcd for
C
14H21N3O4, [M+H]+
solution was added to the mixture of L-leucine methyl ester hydro-
296.1605. Found, 296.1604.
chloride (2.72 g, 15 mmol) and triethylamine (1.52 g, 15 mmol) in
DCM (80 mL) under ice-bath. After stirred at room temperature
for 12 min, the reaction mixture was concentrated under vacuum
and then ethyl acetate (20 mL) was added to the residue. The or-
ganic phase was washed with 1 N HCl (10 mL) and saturated brine
(10 mL) and dried with MgSO4. After the solvent removed under
low pressure, the residue without purification was directly added
to a solution of potassium hydroxylamine (8.37 g, 56 mmol) in
methanol (20 mL). The reaction mixture was stirred at room tem-
perature for 1 h and then removed methanol under low pressure.
The residue was taken up with 1 N HCl and extracted with ethyl
acetate. The organic phase was washed with brine and dried with
MgSO4. After the solvent removed under low pressure, the residue
was separated by silica gel column chromatography to afford 9a–
9g and 11a–11b, respectively.
5.2.6. (S)-4-Methyl-2-[3-(2-trifluoromethyl-phenyl)-ureido]-
pentanoic acid hydroxyamide (8f)
White powder, yield 45%, mp = 179–180 °C; 1H NMR (600 MHz,
DMSO-d6): d 10.81 (s, 1H), 8.85 (s, 1H), 7.94 (m, 2H), 7.61 (d, 1H,
J = 7.8 Hz), 7.56 (t, 1H, J = 7.8 Hz), 7.37 (d, 1H, J = 7.8 Hz), 7.18 (t,
1H, J = 7.2 Hz), 4.18 (m, 1H), 1.58 (m, 1H), 1.42 (t, 2H, J = 7.2 Hz),
0.89 (dd, 6H, J = 6.6, 10.8 Hz); HRMS (AP-ESI) m/z Calcd for
C
14H18F3N3O3, [M+H]+ 334.1373. Found, 334.1373.
5.2.7. (S)-2-[3-(2-Hydroxy-phenyl)-ureido]-4-methyl-pentanoic
acid hydroxyamide (8g)
White powder, yield 48%, mp = 146–148 °C; 1H NMR (600 MHz,
DMSO-d6): d 10.65 (s, 1H), 9.70 (s, 1H), 8.73 (s, 1H), 8.02 (s, 1H),
7.84 (d, 1H, J = 7.8 Hz), 7.04 (d, 1H, J = 7.8 Hz), 6.72 (m, 3H), 4.12
(m, 1H), 1.61 (m, 1H), 1.42 (t, 2H, J = 7.2 Hz), 0.89 (dd, 6H, J = 6.6,
10.8 Hz); HRMS (AP-ESI) m/z Calcd for
C
14H19N3O4, [M+H]+
5.3.1. (S)-2-[3-(3-Fluoro-phenyl)-ureido]-4-methyl-pentanoic
acid hydroxyamide (9a)
282.1448. Found, 282.1449.
White powder, yield 43%, mp = 186–187 °C; 1H NMR (600 MHz,
DMSO-d6): d 10.81 (s, 1H), 8.88 (s, 1H), 8.78 (s, 1H), 7.43 (m, 1H),
7.24 (q, 1H, J = 7.8 Hz), 6.97 (d, 1H, J = 8.4 Hz), 6.71 (m, 1H),
6.411 (t, 1H, J = 8.4 Hz), 4.14 (m, 1H), 1.59 (m, 1H), 1.42 (t, 2H,
J = 7.2 Hz), 0.89 (dd, 6H, J = 6.6, 10.8 Hz); HRMS (AP-ESI) m/z Calcd
for C13H18FN3O3, [M+H]+ 284.1405. Found, 284.1402.
5.2.8. (S)-2-[3-(2-Fluoro-benzyl)-ureido]-4-methyl-pentanoic
acid hydroxyamide (10a)
White powder, yield 51%, mp = 164–166 °C; 1H NMR (600 MHz,
DMSO-d6): d 10.68 (s, 1H), 8.80 (s, 1H), 7.58 (d, 1H, J = 7.8 Hz), 7.36
(m, 1H), 7.29 (d, 1H, J = 6.0 Hz), 7.19 (t, 1H, J = 7.2 Hz), 6.48 (t, 1H,
J = 6.0 Hz), 6.30 (d, 1H, J = 9.0 Hz), 4.22 (d, 2H, J = 6.0 Hz), 4.08 (m,
1H), 1.54 (m, 1H), 1.36 (t, 2H, J = 7.2 Hz), 0.86 (dd, 6H, J = 6.6,
10.8 Hz); HRMS (AP-ESI) m/z Calcd for C14H20FN3O3, [M+H]+
298.1561. Found, 298.1563.
5.3.2. (S)-2-[3-(3-Chloro-phenyl)-ureido]-4-methyl-pentanoic
acid hydroxyamide (9b)
White powder, yield 47%, mp = 178–180 °C; 1H NMR (600 MHz,
DMSO-d6): d 10.76 (s, 1H), 8.85 (s, 1H), 8.72 (s, 1H), 7.61 (s, 1H),
7.20 (t, 1H, J = 7.8 Hz), 7.09 (d, 1H, J = 9.0 Hz), 6.90 (d, 1H,
J = 7.8 Hz), 6.39 (d, 1H, J = 9.0 Hz), 4.09 (m, 1H), 1.54 (m, 1H),
1.37 (t, 2H, J = 7.2 Hz), 0.86 (dd, 6H, J = 5.4, 6.6 Hz); HRMS (AP-
5.2.9. (S)-2-[3-(2-Chloro-benzyl)-ureido]-4-methyl-pentanoic
acid hydroxyamide (10b)
White powder, yield 46%, mp = 170–172 °C; 1H NMR (600 MHz,
DMSO-d6): d 10.68 (s, 1H), 8.80 (s, 1H), 7.42 (d, 1H, J = 7.2 Hz), 7.31
(m, 2H), 7.27 (m, 1H), 6.48 (t, 1H, J = 6.0 Hz), 6.27 (d, 1H, J = 9.0 Hz),
4.26 (d, 2H, J = 6.0 Hz), 4.08 (m, 1H), 1.54 (m, 1H), 1.34 (t, 2H,
J = 7.2 Hz), 0.86 (dd, 6H, J = 6.6, 11.4 Hz); HRMS (AP-ESI) m/z Calcd
for C14H20ClN3O3, [M+H]+ 314.1266. Found, 314.1263.
ESI) m/z Calcd for
C
13H18ClN3O3, [M+H]+ 300.1109. Found,
300.1105.
5.3.3. (S)-2-[3-(3-Bromo-phenyl)-ureido]-4-methyl-pentanoic
acid hydroxyamide (9c)
White powder, yield 60%, mp = 168–170 °C; 1H NMR (600 MHz,
DMSO-d6): d 10.81 (s, 1H), 8.85 (s, 1H), 8.74 (s, 1H), 7.79 (s, 1H),
7.17 (m, 2H), 7.07 (d, 1H, J = 6.6 Hz), 6.42 (d, 1H, J = 8.4 Hz), 4.16
(m, 1H), 1.57 (m, 1H), 1.42 (t, 2H, J = 7.2 Hz), 0.89 (dd, 6H, J = 5.4,
5.2.10. (S)-2-[3-(2-Bromo-benzyl)-ureido]-4-methyl-pentanoic
acid hydroxyamide (10c)
White powder, yield 56%, mp = 173–174 °C; 1H NMR (600 MHz,
DMSO-d6): d 10.68 (s, 1H), 8.80 (s, 1H), 7.28 (m, 2H), 7.17 (m, 2H),
6.42 (t, 1H, J = 6.0 Hz), 6.19 (d, 1H, J = 9.0 Hz), 4.24 (d, 2H,
J = 6.0 Hz), 4.08 (m, 1H), 1.54 (m, 1H), 1.34 (t, 2H, J = 7.2 Hz), 0.89
(dd, 6H, J = 6.6, 10.8 Hz); HRMS (AP-ESI) m/z Calcd for
6.6 Hz); HRMS (AP-ESI) m/z Calcd for
C
13H18BrN3O3, [M+H]+
344.0604. Found, 344.0599.
C
14H20BrN3O3, [M+H]+ 358.0761. Found, 358.0763.
5.3.4. (S)-4-Methyl-2-(3-m-tolyl-ureido)-pentanoic acid
hydroxyamide (9d)
White powder, yield 61%, mp = 160–162 °C; 1H NMR (600 MHz,
DMSO-d6): d 10.70 (s, 1H), 8.78 (s, 1H), 8.41 (s, 1H), 7.14 (m, 3H),
6.71 (d, 1H, J = 7.8 Hz), 6.27 (d, 1H, J = 7.8 Hz), 4.14 (m, 1H), 2.25
(s, 3H), 1.59 (m, 1H), 1.42 (t, 2H, J = 7.2 Hz), 0.90 (dd, 6H, J = 6.6,
5.2.11. (S)-4-Methyl-2-[3-(2-methyl-benzyl)-ureido]-pentanoic
acid hydroxyamide (10d)
White powder, yield 49%, mp = 162–164 °C; 1H NMR (600 MHz,
DMSO-d6): d 10.68 (s, 1H), 8.80 (s, 1H), 7.18 (m, 4H), 6.29 (t, 1H,
J = 5.4 Hz), 6.09 (d, 1H, J = 9.0 Hz), 4.17 (d, 2H, J = 5.4 Hz), 4.09
(m, 1H), 2.25 (s, 1H), 1.54 (m, 1H), 1.34 (t, 2H, J = 7.2 Hz), 0.86
10.8 Hz); HRMS (AP-ESI) m/z Calcd for
C
14H21N3O3, [M+H]+
280.1656. Found, 280.1653.

C. Ma et al. / Bioorg. Med. Chem. 21 (2013) 1621–1627
1627
5.3.5. (S)-2-[3-(3-Methoxy-phenyl)-ureido]-4-methyl-pentanoic
acid hydroxyamide (9e)
5.4.2. In vitro HDACs inhibition assay
In vitro HDACs inhibitory activity assay was determined by
using Boc-Lys (acetyl)-AMC as substrate and Hela nuclear extract
(containing HDAC1, HDAC3, HDAC5 and HDAC8) as enzymes in
White powder, yield 55%, mp = 172–174 °C; 1H NMR (600 MHz,
DMSO-d6): d 10.79 (s, 1H), 8.89 (s, 1H), 8.55 (s, 1H), 7.13 (m, 2H),
6.81 (d, 1H, J = 7.8 Hz), 6.48 (dd, 1H, J = 1.8, 6 Hz), 6.32 (d, 1H,
J = 9.0 Hz), 4.12 (m, 1H), 3.69 (s, 1H), 1.57 (m, 1H), 1.39 (t, 2H,
J = 7.2 Hz), 0.89 (dd, 6H, J = 6.6, 10.8 Hz); HRMS (AP-ESI) m/z Calcd
for C14H21N3O4, [M+H]+ 296.1605. Found, 296.1599.
15 mM Tris–HCl (PH 8.0) at 37 °C. First, 10
was added to tested compounds solutions at various concentra-
tions (50 L) and incubated for 5 min at 37 °C. Then 40 L of sub-
strate was added and the mixture continued to incubate for
another 30 min in the same environment. Finally, 100 L of devel-
lL of enzymes solution
l
l
l
5.3.6. (S)-4-Methyl-2-[3-(3-trifluoromethyl-phenyl)-ureido]-
pentanoic acid hydroxyamide (9f)
oper which containing trypsin and TSA was putted into the mix-
ture. Twenty minutes later, fluorescence intensity was measured
at 390 nm excitation and 460 nm emission wavelengths with a
microplate reader.
White powder, yield 40%, mp = 173–174 °C; 1H NMR (600 MHz,
DMSO-d6): d 10.73 (s, 1H), 8.88 (s, 1H), 8.80 (s, 1H), 7.95 (s, 1H),
7.45 (m, 2H), 7.23 (d, 1H, J = 7.8 Hz), 6.41 (d, 1H, J = 7.8 Hz), 4.16
(m, 1H), 1.44 (m, 1H), 1.39 (t, 2H, J = 7.2 Hz), 0.91 (dd, 6H, J = 6.6,
10.8 Hz); HRMS (AP-ESI) m/z Calcd for C14H18F3N3O3, [M+H]+
334.1373. Found, 334.1372.
5.4.3. MTT assay
ES-2 cell and U-937 cell were grown in RPMI1640 medium with
10 FBS at 37 °C in 5% CO2 humidified incubator. Cell proliferation
was determined by MTT [(3-[4,5-dimethyl-2-thiazolyl]-2,5-diphe-
nyl-2H-tetrazolium bromide)] method. In brief, cells were pated in
96-well plates (5000/well) and cultivated for 4 h, and then differ-
ent concentrations of inhibitors were added. Followed by another
48 h treatment, 1% MTT was added each well. Four hours later,
DMSO was added and mixed for 15 min. Finally, the optical density
values were monitored at 570 nm.
5.3.7. (S)-2-[3-(3-Hydroxy-phenyl)-ureido]-4-methyl-pentanoic
acid hydroxyamide (9g)
White powder, yield 41%, mp = 122–124 °C; 1H NMR (600 MHz,
DMSO-d6): d 10.79 (s, 1H), 9.21 (s, 1H), 8.84 (s, 1H), 8.41 (s, 1H),
6.98 (m, 2H), 6.70 (d, 1H, J = 7.8 Hz), 6.27 (m, 2H), 6.32 (d, 1H,
J = 9.0 Hz), 4.12 (m, 1H), 1.57 (m, 1H), 1.39 (t, 2H, J = 7.2 Hz), 0.89
(dd, 6H, J = 6.6, 10.8 Hz); HRMS (AP-ESI) m/z Calcd for
14H19N3O4, [M+H]+ 282.1448. Found, 282.1448.
Acknowledgments
C
This work was supported by National Science and Technology
Specific Projects of China (No. 2011ZX09401-015) and National
Nature Science Foundation of China (No. 21172134).
5.3.8. (S)-2-[3-(3-Chloro-benzyl)-ureido]-4-methyl-pentanoic
acid hydroxyamide (11a)
White powder, yield 49%, mp = 166–167 °C; 1H NMR (600 MHz,
DMSO-d6): d 10.68 (s, 1H), 8.80 (s, 1H), 7.33 (t, 1H, J = 7.8 Hz), 7.27
(m, 2H), 7.18 (d, 1H, J = 7.8 Hz), 6.48 (t, 1H, J = 6.0 Hz), 6.19 (d, 1H,
J = 8.4 Hz), 4.22 (d, 2H, J = 6.0 Hz), 4.09 (m, 1H), 1.54 (m, 1H), 1.35
(t, 2H, J = 7.2 Hz), 0.86 (dd, 6H, J = 6.6, 8.4 Hz); HRMS (AP-ESI) m/z
Calcd for C14H20ClN3O3, [M+H]+ 314.1266. Found, 314.1269.
References and notes
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5.3.9. (S)-4-Methyl-2-[3-(3-methyl-benzyl)-ureido]-pentanoic
acid hydroxyamide (11b)
White powder, yield 53%, mp = 162–163 °C; 1H NMR (600 MHz,
DMSO-d6): d 10.68 (s, 1H), 8.80 (s, 1H), 7.19 (t, 1H, J = 7.8 Hz), 7.02
(m, 3H), 6.37 (t, 1H, J = 6.0 Hz), 6.11 (d, 1H, J = 9.0 Hz), 4.16 (d, 2H,
J = 6.0 Hz), 4.09 (m, 1H), 2.28 (s, 3H), 1.55 (m, 1H), 1.35 (t, 2H,
J = 7.2 Hz), 0.86 (dd, 6H, J = 6.6, 8.4 Hz); HRMS (AP-ESI) m/z Calcd
for C15H23N3O3, [M+H]+ 294.1812. Found, 294.1815.
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5.4.1. In vitro APN inhibition assay
IC50 values against APN were determined by using L-leu-p-nitro-
anilide as substrate and Microsomal aminopeptidase from Porcine
Kidney Microsomes (Sigma) as enzyme in 50 mM PBS (PH 7.2) or
suspension of ES-2 cells in PBS (1 Â 105/well). After adding the de-
tected compounds, the solution with various concentrations was
incubated with APN at 37 °C for 5 min. Then the solution of sub-
strate was added into the above mixture, which was incubated
for another 30 min at 37 °C. The hydrolysis of the substrate was
measured by following the change in the absorbance monitored
at 405 nm with a plate reader (Varioskan, Thermo, USA).