Design, synthesis and biological evaluation of novel amino acid ureido derivatives as aminopeptidase N/CD13 inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.04.035

A series of amino acid ureido derivatives as aminopeptidase N (APN/CD13) inhibitors were synthesized and evaluated for their APN inhibitory activities and anti-cancer effects. The results showed that most of these amino acid ureido derivatives exhibited good inhibition against APN, several of which were better than Bestatin. The most active compound 12j (IC₅₀ = 1.1 μM, compared with Bestatin IC₅₀ = 8.1 μM) not only possessed much better APN inhibitory activity and anti-proliferation effect on cancer cells, but also exhibited significant block effect of human cancer cell invasion compared with the positive control, Bestatin. These amino acid ureido derivatives could be possibly developed as new APN inhibitors for cancer chemotherapy in the future.

Full text of DOI:10.1016/j.bmc.2012.04.035

Bioorganic & Medicinal Chemistry 20 (2012) 3807–3815
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of novel amino acid ureido
derivatives as aminopeptidase N/CD13 inhibitors
Li Su a, Yuping Jia a, Lei Zhang a, Yingying Xu a, Hao Fang a, Wenfang Xu a,b,
⇑
a Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, Ji’nan, Shandong 250012, PR China
b Key Laboratory of Molecule Design and Novel Drug Research, Shandong Province, Jinan, Shandong 250012, 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 amino acid ureido derivatives as aminopeptidase N (APN/CD13) inhibitors were synthesized
and evaluated for their APN inhibitory activities and anti-cancer effects. The results showed that most
of these amino acid ureido derivatives exhibited good inhibition against APN, several of which were bet-
Received 16 February 2012
Revised 17 April 2012
Accepted 18 April 2012
Available online 23 April 2012
ter than Bestatin. The most active compound 12j (IC50 = 1.1 lM, compared with Bestatin IC50 = 8.1 lM)
not only possessed much better APN inhibitory activity and anti-proliferation effect on cancer cells,
but also exhibited significant block effect of human cancer cell invasion compared with the positive con-
trol, Bestatin. These amino acid ureido derivatives could be possibly developed as new APN inhibitors for
cancer chemotherapy in the future.
Keywords:
Aminopeptidase N
Inhibitors
Amino acid ureido derivatives
Synthesis
Ó 2012 Elsevier Ltd. All rights reserved.
Anti-cancer
1. Introduction
Recent reports suggest that APN inhibitors could also have the po-
tential to be cancer chemotherapeutic agents according to their po-
Aminopeptidase N is a zinc-dependent integral transmembrane
peptidase, belonging to M1 family,1 which is distributed in various
tissues and cell types.2 As an exopeptidase, APN is reported to be
involved in the cleavage of neutral amino acids from the N-termi-
nals of biological oligopeptides, including enkephalins, neuroki-
nins,3 angiotensins and some cytokines.4 APN is tightly related to
various human cancer growths, and considered to be a cancer che-
motherapeutic target for long time. Recently, a high level of its
neutral cell surface exopeptidase activity to the degradation of ex-
tra cellular matrix (ECM)5 has been detected on various highly
metastatic human malignant tumor cells lines,6 including mela-
noma, prostate, ovarian, colon, renal and pancreas carcinomas, as
well as in some leukemias and lymphomas,7 indicating that APN
is not only responsible in the progression of cancer proliferation
and cancer induced angiogenesis,8 but also in the process of cancer
invasion and metastasis.9 Recently, anti-metastasis agents become
increasingly attractive as promising agents in cancer treatment,
and several APN inhibitors have been studied for years. Bestatin
(1, Fig. 1) as an immuno-regulator on the market for leukemia che-
motherapy, was reported to display significant block effects on the
invasion of human malignant cancer cells in vitro9 at higher con-
centration.10 Another reported APN inhibitor, Amastatin (2,
Fig. 1), also showed anti-invasion effect by blocking APN activity.11
tent anti-proliferation and anti-invasion effects.12
In our previous studies, we designed and synthesized a series of
L
-lysine ureido derivatives13 exhibiting potent APN inhibitory ef-
fects, several of which were similar to Bestatin. In this study, we kept
the hydroxamate as the zinc-binding group (ZBG),14 and varied the
L
-lysine side with other a-amino acids (R1) and the R2 substitutes, to
make a preliminary exploration about their influence of the APN
inhibitory activity. The ureido group as the ideal bioisostere of
amide was still kept as the linker between R1 and the R2 substitutes,
which would be helpful for keeping the conformation and improv-
ing the stability to digestive enzymes. Carbamate was also intro-
duced in some cases as the counterpart of the ureido linker (Fig. 2).
2. Chemistry
The synthetic method of 4a–4i is shown in Scheme 1. The ure-
ido or carbamate linker were synthesized from the isocyanates of
O
O
O
OH
OH
O
H
N
OH
H2N
N
H
N
H
H2N
N
H
OH
O
O
OH
O
1, Bestatin
2, Amastatin
⇑
Corresponding author. Tel./fax: +86 531 88382264.
E-mail address: xuwenf@sdu.edu.cn (W. Xu).
Figure 1. Structures of reported APN inhibitors: Bestatin and Amastatin.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.04.035

3808
L. Su et al. / Bioorg. Med. Chem. 20 (2012) 3807–3815
H
H
N
H
N
H
N
R
N
X
1
O
R
2
R
HO
O
O
HO
O
N
H
O
N
H
O
R
R
= side chains of amino acids
= t-Butylamine, Benzylamine,
Phenethylamine or amino acids
1
2
R = t-Butylamind, Bezylamine,
Phenethylamine or amino acids
X = NH or O
Target Compounds
L-lysine ureido derivatives
Figure 2. Target compounds.
R1
R1
O
R
O
R
1
O
R1
1
b, c
a, b
a
O
OH
O
R2
O
O
R
NH
HCl
2
H2N
N
H
X
N
N
HCl H2N
O
H
H
O
O
O
O
O
R
1
3a-3i
(for 3a-3g, X=NH;
for 3h and 3i, X=O)
2d and 2f
13a and13b
1a-1g
2a-2g
R1
O
R
O
R
1
R2
1
1
H
R1
-H
-CH3
-CH(CH3)2
-CH2CH(CH3)2
-CH(CH3)CH2CH3
-CH2Ph
-CH2CH2SCH3
-CH2CH(CH3)2
-CH2CH(CH3)2
H
H
c
d
N
R2
N
N
HO
N
X
OH
HO
N
N
-CH CH(CH )
-C(CH3)3
a
b
2
3 2
a
b
c
d
e
f
g
h
i
H
H
H
O
O
O
-CH Ph
-C(CH3)3
-C(CH3)3
-C(CH3)3
-C(CH3)3
-C(CH3)3
-C(CH3)3
-CH2Ph
2
14a and 14b
4a-4i
(for 4a-4g, X=NH;
for 4h and 4i, X=O)
Scheme 3. Reagents and conditions: (a) Triphosgene, NaHCO3, DCM, ice-bath; (b)
methyl ester hydrochlorides of
NH2OK, MeOH, 4–6 h.
L-Leu of L-Phe, TEA and DCM, room temp, 1–2 h; (c)
-CH2CH2Ph
fore, the synthesized amino acid ureido derivatives were assayed
for their enzyme inhibitory activity using two types of APN from
porcine kidney (Microsomal, Sigma) and ES-2 human ovarian can-
cer cell surface respectively. The inhibitors’ selectivity over MMP-2
(Recombinant, Sigma) was also assayed. In the anti-invasion assay,
we only chose the most potent compounds to be tested for their ef-
fects on ES-2 cell proliferation, migration, and invasion, to be com-
pared with Bestatin as the positive control.
Scheme 1. Reagents and conditions: (a) Acetyl chloride, MeOH, reflux; (b)
triphosgene, NaHCO3, DCM, ice-bath; (c) t-butylamine, BzOH, of PhCH2CH2OH,
TEA and DCM, room temperature; (d) NH2OK, MeOH, 4–6 h.
the chosen amino acids,15 then coupled with the corresponding
amino acids, amines or alcohols. Without further purification, they
were directly transformed into hydroxamic acids as the target
products. The main synthetic methods of the amino acid ureido
derivatives 12a–12m are shown in Scheme 2. With benzylamine,
phenethylamine or cyclohexylamine as the start materials, they
were converted into isocyanates, and then coupled with amino
acid methyl esters, to obtain the ureido linker. Finally the methyl
ester was transformed into hydroxamic acids as the target com-
pounds. Ureido derivatives of double amino acids 14a and 14b
were synthesized according to Scheme 3.
3.1. Enzyme inhibition assay
3.1.1. Enzyme inhibitory activity of the amino acid ureido
derivatives towards APN from porcine kidney (Microsomal,
Sigma)
The enzyme inhibitory activities of all the target compounds in
Table 1 were tested as a preliminary screening. The potencies of
the compounds varied depending on the substitutes. Benzyl and
phenethyl groups as R2 showed to be very beneficial for the ureido
derivatives to be potent APN inhibitors (12a–12m), as well as for
the two carbamate derivatives (4h and 4i). And we can see that
3. Result and discussion
In this paper, besides anti-proliferation, we also investigated
the anti-invasion effects of APN inhibitors on cancer cells. There-
R
O
R
R
1
1
2
a
b
O
R
2
R
NH
2
R
N C O
2
-CH Ph
2
-CH Ph
-CH Ph
2
-CH Ph
-CH Ph
2
-CH Ph
-CH CH Ph
-CH CH Ph
-CH CH Ph
-CH CH Ph
-CH CH Ph
-CH CH Ph
-CH CH Ph
cyclohexyl
2
-H
-CH
-CH(CH )
-CH(CH )CH CH
-CH Ph
-CH CH SCH
-H
-CH
-CH(CH )
-CH CH(CH )
-CH(CH )CH CH
-CH Ph
-CH CH SCH
N
N
a
b
c
d
e
f
g
h
i
j
k
l
m
n
H
H
2
3
O
8: R =-CH Ph
2
2
5: R =-CH Ph
2
2
3 2
3
9: R =-CH CH Ph
10; R = cyclohexyl
6: R =-CH CH Ph
7; R = cyclohexyl
2
2
2
11a-11n
2
2
2
2
2
2
3
2
2
2
c
2
2
2
2
2
3
2
2
2
2
3
R
1
O
3 2
H
2
2
3 2
2
N
R
2
HO
N
N
2 2
2
2
3
3
H
H
2
2
2
2
2
O
2
3
3 2
12a-12n
-CH CH(CH )
Scheme 2. Reagents and conditions: (a) Triphosgene, toluene, reflux; (b) methyl ester hydrochlorides of
L-Gly,
L
-Ala,
L
-Val,
L
-Leu, L-IIe, L-Phe, or L-Met, TEA and DCM, room
temp, 1–2 h; (c) NH2OK, MeOH, 4–6 h.

L. Su et al. / Bioorg. Med. Chem. 20 (2012) 3807–3815
3809
Table 1
3.1.2. Enzyme inhibitory activity of 12d, 12e, 12f, 12j and 12l
towards MMP-2 (Recombinant, Sigma)
The structures and IC50 values of the target compounds towards APN from porcine
kidney (Microsomal, Sigma)
Matrix metalloproteinase-2 (MMP-2) is also a zinc dependent
metalloproteinase and proved to be closely related with cancer.
In order to determine the enzyme selectivity, the active com-
pounds 12d, 12e, 12f, 12j and 12l, were test for their inhibitory
activities towards MMP-2. The result showed that the classical
APN inhibitor, Bestatin, exhibited a weak inhibitory potency to-
R1
O
H
N
R2
HO
N
H
X
O
Compounds
R1
R2
X
IC50 (lM)
towards APNa
wards MMP-2 with IC50 value of 156
pounds could not possess obvious inhibition even at the
concentration of 500 M (Table 2).
lM. But all the active com-
4a
4b
4c
4d
4e
4f
4g
4h
–H
–CH3
–C(CH3)3
–C(CH3)3
–C(CH3)3
–C(CH3)3
–C(CH3)3
–C(CH3)3
–C(CH3)3
–CH2Ph
–CH2CH2Ph
–CH2Ph
–CH2Ph
–CH2Ph
–CH2Ph
NH
NH
NH
NH
NH
NH
NH
O
>100
>100
>100
>100
>100
>100
>100
9.1 0.78
18 2.2
32 2.0
21 1.6
17 1.2
4.1 0.51
5.6 0.60
3.8 0.28
52 7.2
21 2.8
>100
l
–CH(CH3)2
–CH2CH(CH3)2
3.1.3. Enzyme inhibitory activity of compound 12d, 12e, 12f, 12j
and 12l towards APN on the surface of ES-2 human ovarian
cancer cells
Before the examination of the inhibitory effect of APN inhibitors
on ES-2 cell proliferation and invasion, their APN inhibitory po-
tency using ES-2 cell was evaluated first. Most of the compounds
showed similar inhibitory activity to both of porcine APN and
human APN, except 12l. Compounds 12d, 12e, 12f, and 12j still dis-
played better activity than Bestatin, among which 12j was the
–CH(CH3)CH2CH3
–CH2Ph
–CH2CH2SCH3
–CH2CH(CH3)2
–CH2CH(CH3)2
–H
4i
O
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
12m
12n
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
–CH3
–CH(CH3)2
–CH(CH3)CH2CH3
–CH2Ph
–CH2Ph
–CH2Ph
–CH2CH2SCH3
–H
most potent one with its IC50 value of 5.1
atin of 40 M (Table 3).
lM, compared with Best-
–CH2CH2Ph
–CH2CH2Ph
–CH2CH2Ph
–CH2CH2Ph
–CH2CH2Ph
–CH2CH2Ph
–CH2CH2Ph
Cyclohexyl
–CH3
l
–CH(CH3)2
–CH2CH(CH3)2
–CH(CH3)CH2CH3
–CH2Ph
1.1 0.12
>100
6.5 0.48
>100
3.2. In vitro cell proliferation assay of 12d, 12e, 12f, and 12j
Furthermore, the anti-proliferation effects of the potent com-
pounds 12d, 12e, 12f, and 12j on cancer cells were tested against
ES-2 cells (MTT method). As shown in Table 4, the most potent
compound 12j showed much better anti-proliferative effect than
–CH2CH2SCH3
–CH2CH(CH3)2
>100
H
N
14a
–CH2CH(CH3)2
NH
>100
OH
O
Table 2
The structures and IC50 values of the target compoundsa towards MMP-2
(Recombinant, Sigma)
R1
O
14b
–CH2Ph
—
NH
—
>100
H
N
H
N
R2
OH
HO
N
H
N
H
O
O
Bestatin
—
8.1 0.60
Compounds
R1
R2
IC50 (lM) towards
a
MMP-2a
All of the compounds were assayed three times, and their inhibition results are
expressed as the mean values with standard deviations.
12d
12e
12f
12j
12l
–CH(CH3)CH2CH3
–CH2Ph
–CH2CH(CH3)2
–CH2Ph
–CH2Ph
—
–CH2Ph
–CH2Ph
–CH2CH2Ph
–CH2CH2Ph
–CH2CH2Ph
—
>500
>500
>500
>500
>500
156
among 12a–12m, compounds with benzyl seemed to be better
than the ones with phenethyl. Compounds with t-butyl (4a–4g)
and cyclohexyl group (12n) as R2 displayed quite low inhibitory
activity. For the compounds with two amino acid hydroxamates
(14a and 14b) linked together with ureido group only had signifi-
cantly decreased inhibitory activity. Besides, the amino acid resi-
dues also influenced the inhibitory potency. Among 12a–12m,
the ones with
onine residues showed better activities than the ones with
cine, -Alanine and -Valine residues. It demonstrated that the
Bestatin
a
The compounds were assayed three times, and the standard deviations are <10%
of the means.
Table 3
L
-Leucine,
L
-Isoleucine,
L-Phenylalanine and
L
-Methi-
The structures and IC50 values of some target compounds towards APN on the surface
of ES-2
L-Gly-
L
L
R1
O
H
N
amino acid residues with a comparatively larger side chains might
be more beneficial, while 12k and 12m were much less potent.
Among them L-Leucine residue seemed to be the best one, because
R2
HO
N
H
N
H
O
12j showed to be better than others. As for the linkers, carbamates
(4h and 4i) were not as effective as their ureido counterparts, but
still showed similar activity to Bestatin in low micromolar range.
Compound 12j was the most active one in all of those compounds
Compounds
R1
R2
IC50 (lM) towards APN
on the surface of ES-2a
12d
12e
12f
12j
12l
–CH(CH3)CH2CH3
–CH2Ph
–CH2CH2SCH3
–CH2CH(CH3)2
–CH2Ph
–CH2Ph
–CH2Ph
–CH2Ph
–CH2CH2Ph
–CH2CH2Ph
—
39 4.0
16 1.3
22 3.0
5.1 0.41
110 8.1
40 3.8
(IC50 = 1.1 lM), near to eightfold better than Bestatin.
In this preliminary screening, compound 12d, 12e, 12f, 12j, and
12l showed better activity than Bestatin as the positive control.
Hence, their APN inhibitory activities were tested towards MMP-
2 (Recombinant, Sigma) and human APN (hAPN) on the surface
of intact ES-2 human ovarian clear cell carcinoma cells.
Bestatin
—
a
All of the compounds were assayed three times, and their inhibition results are
expressed as the mean values with standard deviations.

3810
L. Su et al. / Bioorg. Med. Chem. 20 (2012) 3807–3815
Table 4
formed on transwell chambers coating with Matrigel. The results
shown in Figure 4 demonstrated that ES-2 cells freely invaded
Matrigel and passed through the chamber. Bestatin, and 12j could
moderately inhibit ES-2 invasion at lower concentration and signif-
icantly blocked ES-2 cell invasion at higher concentration. Com-
pound 12j performed more significant inhibitory effect than
Bestatin (P <0.01), yielding 55% inhibition rate versus the control.
From those assays, compound 12j was the most potent com-
pound with much better inhibitory activities towards both types
of APN from porcine kidney and ES-2 cell surface. Compound 12j
exhibited more potent anti-proliferation effect against ES-2 cell.
Besides, without obvious influence on ES-2 cell migration, 12j
was able to significantly block ES-2 cell invasion in a dose-depen-
dent manner.
The structures and cell proliferation IC50 values of some target compounds towards
ES-2
R1
O
H
N
R2
HO
N
H
N
H
O
Compounds
R1
R2
IC50 (lM)
towards ES-2a
12d
12e
12f
12j
–CH(CH3)CH2CH3
–CH2Ph
–CH2Ph
–CH2Ph
–CH2Ph
–CH2CH2Ph
—
770
510
>1000
96
–CH2CH2SCH3
–CH2CH(CH3)2
—
Bestatin
490
a
The compounds were assayed three times, and the standard deviations are <10%
of the means.
3.4. Statistical analysis
Bestatin. 12d and 12e showed moderate activity, similar to Besta-
tin. There was only slight anti-proliferation observed in 12f at the
assay concentration.
The significance of differences between means was assessed by
Student’s t test. P Values of <0.05 were considered as statistically
significant.
3.3. Anti-invasion assay
4. Conclusion
3.3.1. Cell migration assay of 12d, 12e, and 12j
A series of novel potent amino acid ureido derivative APN inhib-
itors with low micromolar enzyme inhibitory activity were de-
signed and synthesized, twelve of which were identified to have
the IC50 values in a low micromolar range. Among them, compound
Cancer cell migration as one step of the cancer cell invasion pro-
cess, was firstly tested using transwell chambers without Matrigel
coating. From Figure 3 we found that compounds 12d, 12e, 12j as
well as the positive control Bestatin, had no significant inhibitory
effects on ES-2 cell migration, although 12d even had some in-
crease tendency.
We then examined whether the selected APN inhibitors would
affect ES-2 cell migration using transwell chambers without Matri-
gel coating. In this assay, 12d, 12e, and 12j were selected, and we
found that those compounds as well as the positive control Bestatin,
had no significant inhibitory effect on ES-2 cell migration, although
12d even had some increase tendency, as shown in Figure 3.
12j showed the most potent APN inhibitory effects (IC50 = 1.1 lM).
It also displayed significant anti-proliferation and anti-invasion ef-
fects in vitro, even more potent than Bestatin. These results suggest
that the amino acid ureido derivatives as APN inhibitors could be
potential lead compounds to be modified and developed as anti-
cancer proliferation and metastasis agents.
5. Experimental part
5.1. Chemistry
3.3.2. Anti-invasion assay of 12d and 12k
Based on the results of enzyme activity assay towards APN on the
ES-2 cell surface, we selected the most potent compounds 12j to be
assayed for anti-invasion effect on ES-2 cell. This assay was per-
The chemical materials were purchased from commercial sup-
pliers and used without further purification. Solvents were dried
A
B
C
D
E
Migration Rates %
180
160
140
120
100
80
60
40
20
0
G
control
Bestatin
12j
12d
12e
Figure 3. The effects of 12d, 12e, and 12j on the migration of ES-2 cells in vitro. (A) Control, (B) Bestatin 100
lg/mL, (C) 12d 100
l
g/mL, (D) 12e 100 lg/mL, (E) 12j 100 lg/mL,
and (F) the migration rates of the target compounds on ES-2. Each column represents the mean values with SD for three independent experiments.

L. Su et al. / Bioorg. Med. Chem. 20 (2012) 3807–3815
3811
A
B
C
Inhibition Rates %
100
80
60
40
20
0
Bestatin
Bestatin
12j
12j
(10 µg/mL) (100 µg/mL) (10 µg/mL) (100 µg/mL)
D
Figure 4. The inhibitory effects of 12d and 12k on the invasion of ES-2 cells in vitro. (A) Control, (B) Bestatin 100
lg/mL, (C) 12j 100
l
g/mL, and (D) the invasion inhibition
rates of the target compounds on ES-2. Each column represents the mean values with SD values for three independent experiments. ⁄⁄P <0.01, versus the control. #P <0.05,
versus Bestatin treated groups.
over MgSO4 or distilled prior to use and flash chromatography was
performed using silica gel (60 Å, 200 300 mesh). The yields are fi-
nal purified yields. Melting points are uncorrected. NMR spectra
were recorded on a Bruker DRX-300 or a Bruker DRX-600 spec-
trometer. Chemical shifts are in parts per million (ppm). ESI-MS
were determined on an API 4000 spectrometer.
5.2.1. 2-(3-tert-Butyl-ureido)-N-hydroxy-acetmaide (4a)
White powder. Yield 65%, Rf = 0.28 (petrol ether/EtOAc, 1:1);
mp = 175–177 °C; 1H NMR (600 MHz, DMSO-d6): d 10.47 (s, 1H),
8.77 (s, 1H), 5.95 (s, 1H), 5.85 (s, 1H), 3.48 (s, 2H), 1.20 (s, 9H);
13C NMR (300 MHz, DMSO-d6): d 167.0, 157.1, 49.0, 40.2, 29.2;
HRMS-ESI (+) calcd for C7H15N3O3Na, 212.1011; found, 212.0991
[M+Na]+.
5.2. General procedure for the synthesis of 4a–4g
5.2.2. (S)-2-(3-tert-Butyl-ureido)-N-hydroxy-propionamide (4b)
White powder. Yield 60%, Rf = 0.32 (petrol ether/EtOAc, 1:1);
mp = 107–109 °C; 1H NMR (300 MHz, DMSO-d6): d 10.57 (s, 1H),
8.79 (s, 1H), 5.87 (s, 2H), 4.03 (q, 1H, J = 7.2 Hz), 1.19 (s, 9H), 1.10
(d, 3H, J = 7.2 Hz); 13C NMR (300 MHz, DMSO-d6): d 170.1, 156.4,
48.9, 45.7, 29.2, 19.8; HRMS-ESI (+) calcd for C8H15N2O2,
171.1133; found, 171.1131 [MÀNHOH]+.
To a mixture of
21 mmol) or the other six amino acid (
Phe, and -Met) methyl ester hydrochlorides (21 mmol), respec-
L
-Glycine methyl ester hydrochloride (2.63 g,
L
-Ala, -Val, -Leu, -Ile, L-
L
L
L
L
tively, in saturated NaHCO3 (80 mL) and DCM (80 mL) was added
triphosgene (2.08 g, 7 mmol). The reaction mixture was vigorously
stirred under ice-water bath for 15 min 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 the mixture of t-
butylamine (1.46 g, 21 mmol) and triethylamine (2.12 g, 21 mmol)
in DCM (80 mL) under ice bath. The reaction mixture was stirred at
room temperature for 30 min and then the solvent was removed
under low pressure. The residue was taken up with ethyl acetate
(40 mL) and washed with 1 N HCl (10 mL) and brine (20 mL). The
organic phase was dried with MgSO4. After the solvent 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 temperature
for 5 h and then removed methanol under low pressure. The resi-
due 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 4a–4g,
respectively.
5.2.3. (S)-2-(3-tert-Butyl-ureido)-N-hydroxy-2-isopropyl-
acetmaide (4c)
White powder. Yield 55%, Rf = 0.38 (petrol ether/EtOAc, 1:1);
mp = 138–140 °C; 1H NMR (600 MHz, DMSO-d6): d 10.58 (s, 1H),
8.79 (s, 1H), 5.91 (s, 1H), 5.78 (d, 1H, J = 9 Hz), 3.80–3.77 (m, 1H),
1.75–1.72 (m, 1H), 1.19 (s, 9H), 0.81 (6H, d, J = 6.6 Hz); 13C NMR
(300 MHz, DMSO-d6): d 168.7, 156.8, 55.2, 48.9, 31.4, 29.2 19.1,
18.2; HRMS-ESI (+) calcd for
C10H22N3O3, 232.1661; found,
232.1653 [M+H]+.
5.2.4. (S)-2-(3-tert-Butyl-ureido)-N-hydroxy-2-(2-methyl)-
propyl-acetmaide (4d)
White powder. Yield 49%, Rf = 0.46 (petrol ether/EtOAc, 1:1);
mp = 124–126 °C; 1H NMR (300 MHz, DMSO-d6): d 10.62 (s, 1H),
8.86 (s, 1H), 5.88 (s, 1H), 5.85–5.83 (m, 1H), 4.01 (dd, 1H,
J = 6.6 Hz, 12.6 Hz), 1.56–1.45 (m, 1H), 1.30–1.27 (m, 2H), 1.19 (s,
9H), 0.85 (d, 6H, J = 6.9 Hz); 13C NMR (300 MHz, DMSO-d6): d

3812
L. Su et al. / Bioorg. Med. Chem. 20 (2012) 3807–3815
169.5, 156.6, 48.9, 48.6, 42.6, 29.2, 24.2, 22.7, 22.3; HRMS-ESI (+)
calcd for C11H25N3O3, 246.1818; found, 246.1830 [M+H]+.
5.3.1. (S)-(1-Hydroxycarbamoyl-3-methyl-butyl)-carbamic acid
benzyl ester (4h)
White powder. Yield 39%, Rf = 0.35 (petrol ether/EtOAc, 2:1);
mp = 86–88 °C; 1H NMR (300 MHz, DMSO-d6): d 10.65 (s, 1H),
8.79 (s, 1H), 8.30 (s, 1H), 7.42–30 (m, 5H), 5.01 (s, 2H), 4.08–4.03
(m, 1H), 1.58–1.38 (m, 3H), 0.90–0.82 (m, 6H); 13C NMR
(300 MHz, DMSO-d6): d 168.8, 155.7, 137.0, 128.3, 127.7, 127.6,
65.3, 50.8, 40.8, 24.1, 22.7, 21.6; HRMS-ESI (+) calcd for
5.2.5. (S)-2-(3-tert-Butyl-ureido)-N-hydroxy-2-(1-methyl)-
propyl-acetmaide (4e)
White powder. Yield 47%, Rf = 0.46 (petrol ether/EtOAc, 1:1);
mp = 110–113 °C; 1H NMR (300 MHz, DMSO-d6): d 10.58 (s, 1H),
d 8.78 (s, 1H), d 5.89–5.86 (m, 2H), d 3.85–3.79 (m, 1H), d 1.55–
1.36 (m, 2H), d 1.19 (s, 1H), d 0.84–0.77 (m, 6H); 13C NMR
(300 MHz, DMSO-d6): d 168.7, 156.7, 54.2, 48.9, 37.7, 29.2, 24.5,
15.3, 11.2; HRMS-ESI (+) calcd for C11H25N3O3, 246.1818; found,
246.1821 [M+H]+.
C
14H21N2O4, 281.1501; found, 281.1487 [M+H]+.
5.3.2. (S)-(1-Hydroxycarbamoyl-3-methyl-butyl)-carbamic acid
phenethyl ester (4i)
White powder. Yield 41%, Rf = 0.40 (petrol ether/EtOAc, 2:1);
mp = 88–90 °C; 1H NMR (300 MHz, DMSO-d6): d 10.61 (s, 1H),
8.78 (s, 1H), 7.32–7.18 (m, 6H), 4.15–4.11 (m, 2H), 3.95–3.85 (m,
1H), 2.87–2.83 (m, 2H), 1.59–1.24 (m, 3H), 0.87–0.82 (m, 6H);
13C NMR (300 MHz, DMSO-d6): d 168.9, 155.8, 138.2, 128.8,
128.3, 126.2, 64.4, 50.7, 40.8, 34.8, 24.1, 22.7, 21.6; HRMS-ESI (+)
calcd for C15H23N2O4, 295.1658; found, 295.1656 [M+H]+.
5.2.6. (S)-2-(3-tert-Butyl-ureido)-N-hydroxy-2-benzyl-
acetmaide (4f)
White powder. Yield 52%, Rf = 0.50 (petrol ether/EtOAc, 1:1);
mp = 107–109 °C; 1H NMR (600 MHz, DMSO-d6): d 10.61 (s, 1H),
8.82 (s, 1H), 7.27–7.16 (m, 5H), 5.95–5.94 (m, 2H), 5.86 (s, 1H),
4.21 (d, 1H, J = 7.2 Hz), 2.83 (dd, 1H, J = 7.2 Hz, 13.5 Hz), 2.68 (d,
1H, J = 7.2 Hz, 13.5 Hz), 1.16 (s, 9H); 13C NMR (300 MHz, DMSO-
d6): d 168.6, 156.5, 137.7, 129.2, 128.0, 126.1, 51.5, 48.9, 29.2;
HRMS-ESI (+) calcd for C14H21N3O3Na, 302.1481; found, 302.1461
[M+Na]+.
5.3.3. (S,S)-2-[3-(1-Hydroxycarbamoyl-3-methyl-butyl)-ureido]-
4-methyl-pentanoic acid hydroxyamide (14a)
White powder. Yield 24%, Rf = 0.32 (petrol ether/EtOAc, 1:2);
mp = 170–173 °C; 1H NMR (300 MHz, DMSO-d6): d 10.65 (s, 1H),
8.80 (s, 1H), 6.20 (d, 1H, J = 5.7 Hz), 4.07–3.99 (m, 1H), 1.54–1.32
(m, 3H), 0.88–0.83 (m, 6H); 13C NMR (300 MHz, DMSO-d6): d
169.7, 154.4, 52.6, 23.9, 23.0, 21.4; HRMS-ESI (+) calcd for
5.2.7. (S)-2-(3-tert-Butyl-ureido)-N-hydroxy-2-(2-
methylsulfanyl)ethyl-acetmaide (4g)
White powder. Yield 51%, Rf = 0.45 (petrol ether/EtOAc, 1:1);
mp = 125–127 °C; 1H NMR (600 MHz, DMSO-d6): d 10.68 (s, 1H),
8.88 (s, 1H), 6.00 (s, 1H), 5.91 (s, 1H), 4.09 (dd, 1H, J = 4.2 Hz,
14.4 Hz), 2.56–2.38 (m, 2H), 2.08 (s, 3H), 1.81–1.77 (m, 1H),
1.71–1.67 (m, 1H), 1.21 (s, 9H); 13C NMR (300 MHz, DMSO-d6): d
168.8, 156.6, 49.5, 49.0, 33.5, 29.4, 29.2, 14.7; HRMS-ESI (+) calcd
for C10H21N3O3SNa, 286.1201; found, 286.1203 [M+Na]+.
C
13H27N4O5, 319.1982; found, 319.1993 [M+H]+.
5.3.4. (S,S)-N-Hydroxy-2-[3-(1-hydroxycarbamoyl-2-phenyl-
ethyl)-ureido]-3-phenyl-propionamide (14b)
White powder. Yield 24%, Rf = 0.38 (petrol ether/EtOAc, 1:2);
mp = 155–158 °C; 1H NMR (300 MHz, DMSO-d6): d 10.71 (s, 1H),
10.56 (s, 1H), 8.93 (s, 1H), 8.81 (s, 1H), 7.32–7.07 (m, 10H), 6.38
(s, 1H), 6.35 (s, 1H), 4.66–4.60 (m, 2H), 4.18–4.16 (s, 1H), 4.10–
4.05 (m, 1H), 3.37–3.14 (m, 4H); 13C NMR (300 MHz, DMSO-d6):
d 173.4, 168.3, 164.9, 155.8, 137.6, 136.3, 129.2, 129.0, 128.2,
128.0, 126.5, 126.4, 57.3, 52.2, 37.7, 33.4; HRMS-ESI (+) calcd for
5.3. General procedure for the synthesis of 4h, 4i, 14a and 14b
To a mixture of
L-Leucine methyl ester hydrochloride (3.81 g,
21 mmol, for 4h, 4i, and 14a) or
L-Phenylalanine methyl ester
hydrochlorides (4.52 g, 21 mmol, for 14b), respectively, in satu-
rated NaHCO3 (80 mL) and DCM (80 mL) was added triphosgene
(2.08 g, 7 mmol). The reaction mixture was vigorously stirred
under ice-water bath for 15 min and the organic layer was col-
lected. 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 the mixture of ben-
zyl alcohol (2.16 g, 21 mmol, for 4h), phenylethanol (2.46 g,
C
19H23N4O5, 387.1668; found, 387.1656 [M+H]+.
5.4. General procedure for the synthesis of 12a–12f
Benzylamine (1.61 g, 15 mmol).was added to a solution of tri-
phosgene (2.22 g, 7.5 mmol) in dry toluene (80 mL) in room tem-
perature. The reaction mixture was refluxed for 4 h and then
solvents removed under low pressure. The residue was dissolved
in DCM (20 mL) and this solution was added to the mixture of
Glycine methyl ester hydrochloride (2.64 g, 15 mmol) or the other
amino acid ( -Ala, -Val, -Ile, -Phe, and -Met) methyl ester hydro-
L-
21 mmol, for 4i),
21 mmol, for 14a), or
L
-Leucine methyl ester hydrochloride (3.81 g,
-Phenylalanine methyl ester hydrochlorides
L
L
L
L
L
L
(4.52 g, 21 mmol, for 14b), respectively, and triethylamine (2.12 g,
21 mmol) in DCM (80 mL) under ice bath. The reaction mixture
was stirred at room temperature for 30 min and then the solvent
was removed under low pressure. The residue was taken up with
ethyl acetate (40 mL) and washed with 1 N HCl (10 mL) and brine
(20 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 5 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 4h,
4i, 14a and 14b, respectively.
chlorides (15 mmol), respectively, and triethylamine (1.52 g,
15 mmol) in DCM (80 mL) under ice-bath. After stirred at room
temperature for 30 min, the reaction mixture was concentrated
under vacuum and then ethyl acetate (20 mL) was added to the
residue. The organic 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 temperature for 5 h and then removed methanol
under low pressure. The residue was taken up with 1 N HCl and ex-
tracted 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 chro-
matography to afford 12a–12f, respectively.

L. Su et al. / Bioorg. Med. Chem. 20 (2012) 3807–3815
3813
5.4.1. 2-(3-Benzyl-ureido)-N-hydroxy-acetmaide (12a)
White powder. Yield 68%, Rf = 0.26 (petrol ether/EtOAc, 1:1);
mp = 137–139 °C; 1H NMR (300 MHz, DMSO-d6): d 10.49 (s, 1H),
8.78 (s, 1H), 7.33–7.19 (m, 5H), 6.61 (t, 1H, J = 7.5 Hz), 6.18–6.02
(t, 1H, J = 5.7 Hz), 4.20 (d, 2H, J = 7.5 Hz), 3.57 (d, 2H, J = 5.7 Hz);
13C NMR (300 MHz, DMSO-d6): d 166.9, 157.9, 140.6, 128.2,
127.0, 126.5, 42.9, 40.6; HRMS-ESI (+) calcd for C10H24N3O3,
224.1035; found, 224.1034 [M+H]+.
temperature. The reaction mixture was refluxed for 4 h and then
solvents removed under low pressure. The residue was dissolved
in DCM (20 mL) and this solution was added to the mixture of
L
-Glycine methyl ester hydrochloride (2.64 g, 15 mmol) or the
other amino acid ( -Ala, -Val, -Leu, -Ile, -Phe, and -Met)
L
L
L
L
L
L
methyl ester hydrochlorides (15 mmol), respectively, and trieth-
ylamine (1.52 g, 15 mmol) in DCM (80 mL) under ice-bath. After
stirred at room temperature for 30 min, the reaction mixture was
concentrated under vacuum and then ethyl acetate (20 mL) was
added to the residue. The organic 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 with-
out purification was directly added to a solution of potassium
hydroxylamine (8.37 g, 56 mmol) in methanol (20 mL). The reac-
tion mixture was stirred at room temperature for 5 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 12h–12n,
respectively.
5.4.2. (S)-2-(3-Benzyl-ureido)-N-hydroxy-propoinamide (12b)
White powder. Yield 67%, Rf = 0.32 (petrol ether/EtOAc, 1:1);
mp = 108–111 °C; 1H NMR (300 MHz, DMSO-d6): d 10.59 (s, 1H),
8.80 (s, 1H), 7.33–7.19 (m, 5H), 6.48 (t, 1H, J = 6.0 Hz),,6.16 (d,
1H, J = 8.1), 4.19 (d, 2H, J = 6.0 Hz), 4.14–4.04 (m, 1H), 1.14 (d,
J = 6.9 Hz); 13C NMR (300 MHz, DMSO-d6): d 175.2, 157.5, 140.6,
128.2, 126.9, 126.5, 48.1, 42.7, 18.3; HRMS-ESI (+) calcd for
C
11H27N3O3, 238.1192; found, 238.1181 [M+H]+.
5.4.3. (S)-2-(3-Benzyl-ureido)-N-hydroxy-2-isopropyl-
acetamide (12c)
White powder. Yield 68%, Rf = 0.36 (petrol ether/EtOAc, 1:1);
mp = 180–183 °C; 1H NMR (300 MHz, DMSO-d6): d 10.61 (s, 1H),
8.80 (s, 1H), 7.33–7.19 (m, 5H), 6.50 (t, 1H, J = 6.0 Hz), 6.16 (d,
1H, J = 9.3 Hz), 4.20 (d, 2H, J = 6.0 Hz), 3.87 (dd, 1H, J = 7.2 Hz,
9.3 Hz), 1.85–1.74 (m, 1H), 0.85 (d, 6H, 6.9 Hz); 13C NMR
(300 MHz, DMSO-d6): d 168.5, 157.6, 140.7, 128.2, 126.9, 126.5,
55.8, 42.8, 31.3, 19.1, 18.2; HRMS-ESI (+) calcd for C13H19N3O3Na,
288.1324; found, 288.1330 [M+H]+.
5.5.1. 2-(3-Phenethyl-ureido)-N-hydroxy-acetmaide (12g)
White powder. Yield 63%, Rf = 0.28 (petrol ether/EtOAc, 1:1);
mp = 125–128 °C; 1H NMR (300 MHz, DMSO-d6): d 9.53 (s, 2H),
7.32–7.17 (m, 5H), 6.22–6.12 (m, 1H), 6.10–5.98 (m, 1H), 3.86 (d,
2H, J = 4.5 Hz), 3.21 (dd, 2H, J = 6.9 Hz, 12.9 Hz), 2.66 (t, 2H,
J = 6.9 Hz); 13C NMR (300 MHz, DMSO-d6): d 166.7, 157.9, 139.7,
128.6, 128.2, 125.9, 41.0, 40.8, 36.1; HRMS-ESI (+) calcd for
5.4.4. (S)-2-(3-Benzyl-ureido)-N-hydroxy-2-(1-methyl)-propyl-
acetamide (12d)
C
11H16N3O3, 238.1192; found, 238.1189 [M+H]+.
White powder. Yield 55%, Rf = 0.42 (petrol ether/EtOAc, 1:1);
mp = 148–150 °C; 1H NMR (300 MHz, DMSO-d6): d 10.63 (s, 1H),
8.80 (s, 1H), 7.33–7.10 (m, 5H), 6.52–6.45 (m, 1H), 6.18–6.10 (m,
1H), 4.20 (d, 1H, J = 6.0 Hz), 4.06–4.00 (m, 1H), 1.34–1.19 (m,
2H), 1.10–1.08 (m, 1H), 0.92–0.79 (m, 6H); 13C NMR (300 MHz,
DMSO-d6): d 168.8, 157.5, 140.7, 128.2, 126.9, 126.5, 54.8, 42.8,
29.3, 25.7, 24.4; HRMS-ESI (+) calcd for C14H22N3O3, 280.1661;
found, 280.1662 [M+H]+.
5.5.2. (S)-2-(3-Phenethyl-ureido)-N-hydroxy-propoinamide
(12h)
White powder. Yield 64%), Rf = 0.32 (petrol ether/EtOAc, 1:1);
mp = 137–139 °C; 1H NMR (300 MHz, DMSO-d6): d 10.59 (s, 1H),
8.82 (s, 1H), 7.32–7.18 (m, 5H), 6.13 (d, 1H, J = 8.1 Hz), 6.03 (t,
1H, J = 5.4 Hz), 4.10–4.00 (m, 1H), 3.24–3.17 (m, 2H), 2.65 (t, 2H,
J = 7.2 Hz), 1.10 (d, 3H, J = 6.9 Hz); 13C NMR (300 MHz, DMSO-d6):
d 169.9, 157.1, 139.6, 128.6, 128.2, 125.9, 46.2, 40.8, 36.1, 19.7;
HRMS-ESI (+) calcd for C12H17N3O3Na, 274.1168; found, 274.1154
[M+Na]+.
5.4.5. (S)-2-(3-Benzyl-ureido)-N-hydroxy-2-benzyl-acetmaide
(12e)
White powder. Yield 45%, Rf = 0.46 (petrol ether/EtOAc, 1:1);
mp = 140–142 °C; 1H NMR (300 MHz, DMSO-d6): d 10.64 (s, 1H),
8.82 (s, 1H), 7.31–7.14 (m, 10H), 6.43 (t, 1H, J = 6.0 Hz), 6.24 (d,
1H, J = 5.8 Hz), 4.34–4.22 (m, 1H), 4.15 (d, 2H, J = 6.0 Hz), 2.88
(dd, 1H, J = 6.0 Hz, 15.0 Hz), 2.72 (dd, 1H, J = 6.0 Hz, 15.0 Hz); 13C
NMR (300 MHz, DMSO-d6): d 168.8, 157.2, 140.6, 137.7, 129.2,
128.1, 128.0, 126.8, 126.5, 126.2, 52.1, 42.7; HRMS-ESI (+) calcd
for C17H20N3O3, 314.1505; found, 314.1492 [M+H]+.
5.5.3. (S)-2-(3-Phenethyl-ureido)-N-hydroxy-2-isopropyl-
acetamide (12i)
White powder. Yield 47%, Rf = 0.32 (petrol ether/EtOAc, 1:1);
mp = 87–90 °C; 1H NMR (300 MHz, DMSO-d6): d 8.19 (s, 1H),
7.31–7.17 (m, 1H), 3.90–3.88 (m, 1H), 3.67–3.49 (m, 2H), 2.87–
2.82 (m, 2H), 1.98–1.90 (m, 1H), 0.93–0.65 (m, 7H); 13C NMR
(300 MHz, DMSO-d6): d 173.4, 157.0, 138.1, 128.6, 128.3, 126.3,
61.2, 38.6, 33.2, 29.5, 18.4, 15.7; HRMS-ESI (+) calcd for
5.4.6. (S)-2-(3-Benzyl-ureido)-N-hydroxy-2-(2-methylsulfanyl)
ethyl-acetmaide (12f)
C
14H19N2O2, 247.1446; found, 247.1448 [MÀNHOH]+.
White powder. Yield 42%, Rf = 0.40 (petrol ether/EtOAc, 1:1);
mp = 153–155 °C; 1H NMR (300 MHz, DMSO-d6): d 10.68 (s, 1H),
8.86 (s, 1H), 7.33–7.19 (m, 5H), 6.47 (t, 1H, J = 6.0 Hz), 6.27 (d,
1H, J = 6.0 Hz), 4.20 (d, 2H, J = 6.0 Hz), 4.16–4.09 (m, 1H), 2.45–
2.34 (m, 2H), 2.03 (s, 3H), 1.85–1.63 (m, 2H); 13C NMR (300 MHz,
DMSO-d6): d 168.6, 157.4, 140.6, 128.2, 126.9, 126.6, 50.0, 42.8,
33.3, 29.4, 14.8; HRMS-ESI (+) calcd for C13H20N3O3S, 298.1225;
found, 298.1230 [M+H]+.
5.5.4. (S)-2-(3-Phenethyl-ureido)-N-hydroxy-2-(2-methyl)-
propyl-acetamide (12j)
White powder. Yield 41%, Rf = 0.38 (petrol ether/EtOAc, 1:1);
mp = 155–157 °C; 1H NMR (300 MHz, DMSO-d6): d 10.65 (s, 1H),
8.78 (s, 1H), 6.06 (d, 1H, J = 9.0 Hz), 5.94 (t, 1H, J = 5.7 Hz), 4.05
(dd, 1H, J = 7.5 Hz, 16.2 Hz), 3.21 (dd, 2H, J = 6.9 Hz, 13.2 Hz), 2.65
(t, 2H, J = 7.2 Hz), 1.56–1.34 (m, 1H), 1.31 (t, 2H, J = 7.5 Hz), 0.86
(t, 6H, J = 6.9 Hz); 13C NMR (300 MHz, DMSO-d6): d 169.5, 157.3,
139.7, 139.6, 128.6, 128.2, 125.9, 49.0, 42.4, 40.8, 24.2, 22.7, 22.3,
5.5. General procedure for the synthesis of 12g–12m
22.1; HRMS-ESI (+) calcd for
C15H24N3O3, 294.1818; found,
Phenethylamine (1.82 g, 15 mmol).was added to a solution of
triphosgene (2.22 g, 7.5 mmol) in dry toluene (80 mL) in room
294.1813 [M+H]+.

3814
L. Su et al. / Bioorg. Med. Chem. 20 (2012) 3807–3815
5.5.5. (S)-2-(3-Phenethyl-ureido)-N-hydroxy-2-(1-methyl)-
propyl-acetamide (12k)
5.7. Biological activity methods. Cell culture
White powder. Yield 40%, Rf = 0.38 (petrol ether/EtOAc, 1:1);
mp = 108–110 °C; 1H NMR (300 MHz, DMSO-d6): d 10.48 (s, 1H),
8.15 (s, 1H), 7.30–7.18 (m, 5H), 4.00–3.92 (m, 1H), 3.66–3.52 (m,
2H), 2.83 (t, 2H, J = 6.6 Hz), 1.73–1.67 (m, 1H), 1.39–1.29 (m, 1H),
1.20–1.08 (m, 1H), 0.87–0.76 (m, 3H), 0.59–0.57 (m, 3H); 13C
NMR (300 MHz, DMSO-d6): d 173.8, 157.1, 138.0, 128.6, 128.3,
126.3, 59.7, 35.9, 33.1, 25.4, 12.8, 11.6; HRMS-ESI (+) calcd for
ES-2 cells were cultured in RPMI-1640 culture medium supple-
mented with 10% (v/v) fetal bovine serum (FBS).
5.7.1. Enzyme inhibitory activity of the amino acid ureido
derivatives towards APN from porcine kidney (Microsomal,
Sigma)
IC50 values against APN were determined by using L-Leu-p-
C
15H21N2O2,261.1603; found, 261.1606 [MÀNHOH]+.
nitroanilide as substrate and Microsomal APN from Porcine Kidney
Microsomes (Sigma) as lymphilized powder 15–25 units/mg pro-
tein. In brief, the assay was performed in 96-well plates in
50 mM PBS, pH 7.2 as the assay buffer, at 37 °C. All solutions of
the inhibitors were prepared in the assay buffer with 0.5% DMSO
in final concentration as the fluxing agent. All inhibitors were
pre-incubated with APN for 5 min at room temperature. The assay
5.5.6. (S)-2-(3-Phenethyl-ureido)-N-hydroxy-2-benzyl-
acetmaide (12l)
White powder. Yield 45%, Rf = 0.45 (petrol ether/EtOAc, 1:1);
mp = 133–135 °C; 1H NMR (300 MHz, DMSO-d6): d 10.62 (s, 1H),
8.81 (s, 1H), 7.30–7.16 (m, 10H), 6.20 (d, 1H, J = 8.7 Hz), 5.99 (t,
1H, J = 5.7 Hz), 4.29–4.21 (m, 1H), 3.20–3.13 (m, 2H), 2.84 (dd,
1H, J = 6.0 Hz, 13.5 Hz), 2.72 (dd, 1H, J = 6.0 Hz, 13.5 Hz), 2.68–
2.60 (m, 2H); 13C NMR (300 MHz, DMSO-d6): d 168.6, 157.1,
139.6, 137.7, 129.2, 128.6, 128.2, 128.0, 126.1, 125.9, 52.0, 40.8,
mixture, which contained 40
tration dependent on the inhibitors), 100
tion (6 g/mL in final concentration),
lL of the inhibitor solution (concen-
l
L of the enzyme solu-
of the substrate
L, then incu-
l
5
lL
solution and the assay buffer, was adjusted to 200
l
bated at 37 °C for 30 min. The hydrolysis of the substrate was mon-
itored by following the change in the absorbance measured at
405 nm with the UV–Vis spectrophotometer Pharmacia LKB, Bio-
chrom 4060. The enzyme activity inhibition rate was calculated
as [(ODc À ODt)/(ODc À ODz)] Â 100. ODc represents the OD values
of the control group, ODt represents the OD values of the treating
groups, and ODz represents the OD values of the zero-setting
groups. The concentration of inhibiting 50% of the enzyme activity
(IC50) was calculated.
36.0; HRMS-ESI (+) calcd for
C18H22N3O3, 328.1661; found,
328.1664 [M+H]+.
5.5.7. (S)-2-(3-Phenethyl-ureido)-N-hydroxy-2-(2-
methylsulfanyl)ethyl-acetmaide (12m)
White powder. Yield 40%, Rf = 0.40 (petrol ether/EtOAc, 1:1);
mp = 138–140 °C; 1H NMR (300 MHz, DMSO-d6): d 10.65 (s, 1H),
8.78 (s, 1H), 7.32–7.17 (m, 5H), 6.21 (d, 1H, J = 8.7 Hz), 6.00 (s,
1H) 4.12–4.06 (m, 1H), 3.22 (dd, 2H, J = 6.9 Hz, 13.0 Hz), 2.66 (t,
2H, J = 7.2 Hz), 2.42–2.31 (m, 2H), 2.03 (s, 3H), 1.82–1.60 (m,
2H); 13C NMR (300 MHz, DMSO-d6): d 168.7, 157.3, 139.6, 128.6,
128.2, 126.9, 50.0, 40.8, 36.0, 33.2, 29.4, 14.6; HRMS-ESI (+) calcd
for C14H22N3O3S, 312.1382; found, 312.1367 [M+H]+.
5.7.2. Enzyme inhibitory activity of 12d, 12e, 12f, 12j and 12l
towards APN on the surface of ES-2 cells
IC50 values against APN were determined by using L-Leu-p-
nitroanilide as substrate and ES-2 human ovarian carcinoma cells
as the enzyme source. In brief, the assay was performed in 96-well
plates in 10 mM PBS, pH 7.4 as the assay buffer, at 37 °C. All solu-
tions of inhibitors were prepared in the assay buffer with 0.1%
DMSO in final concentration as the fluxing agent. Bestatin, 12d,
12e, 12f, 12j and 12l were pre-incubated with ES-2 cell suspension
(2 Â 105 cells per well) for 5 min at room temperature. The assay
5.6. General procedure for the synthesis of 12n
Cyclohexylamine (1.47 g, 15 mmol).was added to a solution of
triphosgene (2.22 g, 7.5 mmol) in dry toluene (80 mL) in room
temperature. The reaction mixture was refluxed for 4 h and then
solvents removed under low pressure. The residue was dissolved
in DCM (20 mL) and this solution was added to the mixture of
mixture, which contained 20
tration dependent on the inhibitors), 70
sion, 10 L of the substrate solution, or the assay buffer, was
adjusted to 100 L, and then incubated at 37 °C for 1 h. The hydro-
l
L of the inhibitor solution (concen-
l
L of the ES-2 cell suspen-
l
L-Leucine methyl ester hydrochloride (2.72 g, 15 mmol) and trieth-
l
ylamine (1.52 g, 15 mmol) in DCM (80 mL) under ice-bath. After
stirred at room temperature for 30 min, the reaction mixture was
concentrated under vacuum and then ethyl acetate (20 mL) was
added to the residue. The organic 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 hydrox-
ylamine (8.37 g, 56 mmol) in methanol (20 mL). The reaction mix-
ture was stirred at room temperature for 5 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 re-
moved under low pressure, the residue was separated by silica
gel column chromatography to afford 12n, white powder, yield
60%, Rf = 0.42 (Petrol Ether/EtOAc, 1:1); mp = 138–141 °C; 1H
NMR (300 MHz, DMSO-d6): d 10.66 (s, 1H), 9.20 (s, 1H), 5.90–
5.86 (m, 2H), 4.08–4.02 (m, 1H), 3.33–3.28 (m, 1H), 1.72–1.47
(m, 6H), 1.33–1.02 (m, 7H), 0.99–0.83 (m, 6H); 13C NMR
(300 MHz, DMSO-d6): d 169.5, 156.6, 48.8, 47.5, 42.6, 33.2, 25.2,
lysis of the substrate in the supernatant liquor after centrifugation
was monitored by following the change in the absorbance mea-
sured at 405 nm with the UV–Vis spectrophotometer Pharmacia
LKB, Biochrom 4060. The enzyme activity inhibition rate was cal-
culated as [(ODc À ODt)/(ODc À ODz)] Â 100. ODc represents the
OD values of the control group, ODt represents the OD values of
the treating groups, and ODz represents the OD values of the
zero-setting groups. The concentration of inhibiting 50% of the en-
zyme activity (IC50) was calculated.
5.7.3. Enzyme inhibitory activity of the compounds towards
MMP-2 (Recombinant, Sigma)
IC50 values against MMP-2 in 96-well microtiter plates using
succinylated gelatin as the substrate. The compounds and the en-
zyme were dissolved in sodium borate buffer (pH 8.5, 50 mM)
and incubated at 37 °C for 10 min. The substrate was added and
incubated at 37 °C for additional 60 min. The control and zero-set-
ting groups were also carried out. Then 0.03% picrylsulfonic acid
solution was added and incubated at room temperature for addi-
tional 20 min. The resulting solutions were measured under
24.3, 24.2, 22.7, 22.2; HRMS-ESI (+) calcd for
C13H26N3O3,
272.1974; found, 272.1969 [M+H]+.

L. Su et al. / Bioorg. Med. Chem. 20 (2012) 3807–3815
3815
450 nm with the UV–Vis spectrophotometer Pharmacia LKB, Bio-
chrom 4060. The inhibitory rates were calculated by [(ODc À ODt)/
(ODc À ODz)] Â 100. ODc represents the OD values of the control
group, ODt represents the OD values of the treating groups, and
ODz represents the OD values of the zero-setting groups. The con-
centration of inhibiting 50% of the enzyme activity (IC50) was
calculated.
1640 culture medium with 10% FBS were added to the lower
chambers. Various concentrations (10 and 100 g/mL) of 12j in
100 L of RPMI-1640 culture medium with 1% FBS (with 0.1%
DMSO in final concentration as the fluxing agents) were added to
the upper chamber at the same time. Cells in 400 L of RPMI-
1640 culture medium with 1% FBS (100,000 cells per well) were
added and allowed to invade for 8 h at 37 °C in a CO2 incubator.
8 h later, matrigel and cells in the upper chamber were removed
with a cotton swab. The remaining cells were fixed, stained with
0.1% crystal violet, and photographs were taken under a micro-
scope. The inhibition rates were quantitated by counting the num-
ber of cells in five random fields (Â100) per insert.
l
l
l
5.7.4. Anti-proliferation assay
The anti-proliferation assay of compound 12d, 12e, 12f, and 12j
were assayed by MTT method. In brief, the ES-2 cells were plated in
a 96-well plate (8000 cells per well) in RPMI-1640 culture medium
with 10% FBS, and allowed to adhere and spread for 10 h. Then the
culture medium was removed and of Bestatin, 12d, 12e, 12f, and
12j at various concentration in RPMI-1640 culture medium with
10% FBS (with 0.1% DMSO as fluxing agent, also in the control
the zero-setting wells) were added, and then the cells were cul-
tured for 48 h at 37 °C in a CO2 incubator. MTT solution (10 lL of
5 mg/mL) was added per well and the cells were cultured for addi-
tional 4 h. Then the medium were removed and the residue were
dissolved in 100 lL DMSO per well. The optical density (OD) values
5.7.7. Statistical analysis
All biological experiments, including the enzyme inhibition as-
says, the in MTT assay, the cell migration assay, and the anti-inva-
sion assay were performed at least three times with triplicates in
each experiment. Representative results are depicted in the report.
Data are presented as means SD, and comparisons were made
using Student’ t test. A probability of 0.05 or less was considered
statistically significant.
were measured at 590 nm. The growth inhibition rate was calcu-
lated as [(ODc À ODt)/(ODc À ODz)] Â 100. ODc represents the OD
values of the control group, ODt represents the OD values of the
treating groups, and ODz represents the OD values of the zero-set-
ting groups. The concentration of inhibiting 50% of the enzyme
activity (IC50) was calculated.
Acknowledgements
This work was supported by National Natural Science Founda-
tion Research of China (Grant Nos. 9071304, 30772654), National
Science and Technology Major Project (Grant No. 2009-ZX09103-
118), Natural Science Foundation for Young Scholars of Shandong
Province (2006BS03021)
5.7.5. Cell migration assay
The Thincert™ Chambers (Costar, Cambridge, MA) with 100
lg/
mL of 12d, 12e, 12f and 12j in 100 L of RPMI-1640 culture med-
l
References and notes
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The BD BioCoat™ Matrigel™ Invasion Chambers (BD Bioscience,
Bedford, MA) were rehydrated with 500
medium with 1% FBS in the upper and lower chambers, respec-
tively, for 2 h. After the medium was removed, 750 L of RPMI-
lL RPMI-1640 culture
l