Novel aminopeptidase N (APN/CD13) inhibitors derived from 3-phenylalanyl-N′-substituted-2,6-piperidinedione

Article abstract of DOI:10.1016/j.bmc.2010.06.078

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 APN inhibitors with 3-phenylalanyl-N′-substituted-2,6-piperidinedione scaffold. The results showed that compound 7c had the most potent inhibitory activity against APN with the IC₅₀ value to 5.00 ± 3.17 μM, which could be used as the lead compound in the future for anticancer agent research.

Full text of DOI:10.1016/j.bmc.2010.06.078

Bioorganic & Medicinal Chemistry 18 (2010) 5981–5987
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel aminopeptidase N (APN/CD13) inhibitors derived
from 3-phenylalanyl-N0-substituted-2,6-piperidinedione
Xiaopan Zhang, Hao Fang, Huawei Zhu, Xuejian Wang, Lei zhang, Minyong Li, Qianbin Li, Yumei Yuan,
*
Wenfang Xu
Department of Medicinal Chemistry, School of Pharmacy, Shandong University, 44 West Culture Road, 250012 Ji’nan, Shandong, 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 APN inhibitors with 3-phenylalanyl-N0-substituted-2,6-piperidinedione scaffold. The
results showed that compound 7c had the most potent inhibitory activity against APN with the IC50 value
Received 28 April 2010
Revised 19 June 2010
Accepted 22 June 2010
Available online 1 July 2010
to 5.00 3.17
l
M, which could be used as the lead compound in the future for anticancer agent research.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Aminopeptidase N inhibitors
3-phenylalanyl-N0-substituted-2,6-
piperidinedione derivates
Structure-based drug design
1. Introduction
can be divided into two subsites (S10 pocket and S20 pocket). The
free amino group of Bestatin can interact with Glu 355.15,16
Aminopeptidase N (APN; EC 3.4.11.2), also known as CD13 is a
zinc-dependent type II membrane-bond ectopeptidase, and belongs
to the M1 family of MA clan of peptidase.1,2 This enzyme consists of
967 amino acids, and can preferentially release neutral or basic ami-
no acids from the N-terminal of peptides. APN is widely distributed
in the body of mammalian. It can be expressed on the surface of dif-
ferent cells such as myeloid progenitors and monocytes, epithelial
cells of the intestine and kidney, synaptic membranes in the central
nervous system, fibroblasts, endothelial cells, epithelial cells,3–6 and
so on. APN is highly expressed on tumor cells which play a critical
role in tumor invasion, metastasis and angiogenesis. Therefore, the
design and synthesis of APN inhibitors may be a clinically signifi-
cance for the discovery of anti-cancer agents.
Since 1976, the first APN inhibitor Bestatin was discovered,
many APN inhibitors have been developed such as Probestin,7
Amatatin,8 Prebestin,9 Lapstatin,10 AHPA-Val,11 and so on. The X-
ray crystal structures of APN and its complexes with varies inhib-
itors have been studied in recently years.12–14
The binding site of APN with Bestatin could be divided into
three parts (Fig. 1): part A (S1 pocket) is a hydrophobic pocket
which can interact with the phenyl group of Bestatin; part B con-
tains a zinc ion and can interact with the zinc binding group
(ZBG) such as hydroxyl group and carbonyl group in Bestatin;
and part C is another hydrophobic pocket in deep cavity which
In our previous work, a series of piperidinedione derivatives
was reported as potent APN inhibitors.17,18 Compound 13f showed
even better APN inhibitory effect with the IC50 value to 1.8 lM.
Compound 13f can also significantly inhibit the growth and migra-
tion of human ovarian carcinoma cell ES-219 and hepatocellular
carcinoma (HCC) cell line HuH-7.20 Herein, in order to find better
APN inhibitors, we used compound 13f as the leading compound
and modified the structures as follows (Fig. 2): (1) due to the con-
formationally constrained effect, we maintained the cyclic-imide
scaffold; (2) we maintained the phenylalanine moiety in order to
mimic the 3-amino-2-hydroxyl-4-phenylbutanoic acid (AHPA)
skeleton in Bestatin to interact with S1 pocket (part A) of APN;
(3) we replaced the hydroxamate group with different amino acid
benzyl ester, amino acid or organic amino in order to interact with
S10 pocket (part C) of APN.
APN active site
His392
Glu411
S1
His388
S1'
S2'
Zn
O
Glu355
COOH
N
H3N
H
OH
* Corresponding author. Tel./fax: +86 531 88382264.
E-mail address: wfxu@yahoo.cn (W. Xu).
Figure 1. The binding mode of Bestatin with APN.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.078

5982
X. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 5981–5987
Zn2+
part A
O
O
O
O
N
O
O
N
H
NHOH
N
H
N
NH2
O
NH2
O
part C
R
13f
Glu 355
R= amino acid benzyl ester;
amino acid;
organic amino
Figure 2. Chemical structures of the new designed cyclic-imide APN inhibitors.
H2N
O
O
O
O
b
a
Boc
Boc
N
HN
NH
O
Ph
O
HN
OH
Boc
O
O
HN
O
2
O
3
1
O
O
e
O
O
c, d
HN
HN
N
N
NH
Boc
NH
Boc
OH
O
O
O
Ph
4
5
Scheme 1. Reagents and conditions: (a) DCC, HOSu, THF, 65 °C; (b) K2CO3, KI, acetone, BrCH2COOCH2Ph, 56 °C; (c) TFA, DCM; (d) Boc-
THF, ꢀ20 °C; (e) 10% Pb/C, H2, MeOH.
L-Phe, isobutyl chloroformate, NMM,
O
O
O
O
O
O
b
N
O
HCl
R
N
O
N
H
N
H
NH2
O
O
a
NH
Boc
O
O
N
H
R
N
H
7a-h
O
O
6a-h
O
O
N
O
c
HCl
R1
N
H
b
N
O
N
O
N
H
NH
Boc
O
N
R1
O
OH
NH2
H
NH
Boc
N
H
OBzl
N
H
OBzl
5
9a-g
O
d
8a-g
O
O
O
HCl
R1
O
N
O
b
N
N
H
N
H
NH2
O
R1
N
OH
NH
O
H
O
e
N
H
OH
Boc
11a-g
O
10a-g
O
O
O
O
O
N
O
b
HCl
O
N
N
H
N
H
NH2
O
N
H
NHOH
NH
Boc
O
O
N
NHOH
H
O
13a
12a
Scheme 2. Reagents and conditions: (a) RNH2, isobutyl chloroformate, NMM, THF, ꢀ20 °C; (b) 3 N HCl–EtOAc; (c) benzyl ester of amino acids, isobutyl chloroformate, NMM,
THF, ꢀ20 °C; (d) 10% Pb/C, H2, MeOH; (e) H2NOHꢁHCl, isobutyl chloroformate, NMM, THF, ꢀ20 °C.
from optically pure Boc-L-Gln (1), by using DCC and N-hydroxysuc-
2. Chemistry
cinimide (HOSu) in THF at elevated temperature gives 2,6-dioxo-
pyridine derivative (2), which is on treatment with benzyl
bromoacetate gives compound (3). And then remove the Boc pro-
The target compounds were synthesized following the proce-
dures as shown in Schemes 1 and 2. The key intermediate com-
pound (5) was easily prepared as our early report.18 Starting
tecting group of compound (3) and coupled with Boc-L-Phe gives

X. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 5981–5987
5983
Table 1
compound (4), which was then catalytically hydrogenated using
10% Pd/C to afford the key intermediate (5).
The structures and in vitro APN inhibitory activities of the target compounds and
positive control Bestatin
Subsequently compound (5) was activated with isobutyl chloro-
formate and N-methylmorpholine (NMM) and then coupled with
different substituted aromatic amine to yield 6a–h. The Boc group
can be easily removed by 3 N HCl in anhydrous ethyl acetate to
give hydrochloride salts 7a–h. Using the same procedure, com-
pound (5) was coupled with different benzyl ester of amino acids
to provide 8a–g which can be easily converted to 9a–g after
removing the Boc protecting groups. Catalytically hydrogenating
8a–g, and then deprotecting the Boc group gives compounds
11a–g. In addition, the carboxyl group in compound 10a can be
easily converted to its hydroxamate acid derivative (compound
13a) by using the same method with NH2OH as the base.
O
O
N
N
H
NH2
O
R
O
N
H
a
Compound
R
IC50
(l
M)
APN
MMP-2
7a
7b
252.14 2.60
178.21 2.10
122 28.2
F
>1000
O
H3C
3. Results and discussion
7c
5.00 3.17
6.92 0.67
8.27 0.63
CH3
The newly synthesized piperidinedione derivatives were as-
sayed for the enzymatic inhibitions on both APN and MMP-2 and
the results are listed in Table 1. Similar to APN, MMP-2 is also a
zinc-dependent metalloproteinase which is involved in the process
of tumor invasion and metastasis. The difference between them is
that MMP-2 is an endopeptidase while APN is an exopeptidase. In
order to observe the selectivity of the target compounds against
the two enzymes, all the target compounds were assayed for the
inhibitory activities on APN and MMP-2.
As shown in the results, most compounds exhibited a better
APN inhibitory activity than that of MMP-2, with the IC50 values ly-
ing in micromolar level. The results to a certain extent conformed
our strategy for designing APN special inhibitors. The results may
be explained by the difference between the structures of the activ-
ity site of APN and MMP-2. The activity site of APN is deeper than
that of MMP-2, which may be suitable for most of the compounds.
On the other hand, APN is an exopeptidase which can release the
N-terminal amino acid from the peptide, and the N-terminal amino
acid is the neutral or basic amino acids such as Phe, Tyr, Ala, and
Cl
7d
60.4 0.88
F
7e
7f
245.36 1.21
74.00 1.54
62.7 9.6
71.6 4.97
131 14.2
Br
F
Cl
F
7g
169.85 13.03
7h
53.43 8.03
46.9 0.73
F
9a
9b
9c
9d
9e
9f
9g
11a
11b
11c
11d
11e
11f
11g
13a
Bestatin
-Gly-OCH2Ph
-Ala-OCH2Ph
-Val-OCH2Ph
-Leu-OCH2Ph
-b-Ala-OCH2Ph
-Phe-OCH2Ph
-Glu-(OCH2Ph)2
-Gly-OH
-Ala-OH
-Val-OH
-Leu-OH
-b-Ala-OH
-Phe-OH
13.45 3.33
91.61 1.48
30.14 3.08
30.17 3.31
49.70 1.23
6.48 0.33
91.36 10.91
38.74 2.03
109.84 7.46
13.33 0.03
8.42 0.56
12.75 0.42
66.40 1.35
12.61 1.24
304.21
454 51
-
-
-
L
L
L
129 12.7
18.5 2.38
121 22.7
127 5.65
12 0.12
272 50.82
107 11.7
155 0.75
>1000
>1000
22.7 1.36
315.65 27.08
>1000
Leu. The target compounds we designed all have the L-Phe residue
and could be well recognized by APN, so the compounds may be
suitable for APN inhibitors. And so the following structure–activity
relationships were mainly focused on APN inhibition.
-
-
L
L
Comparing compounds 7a–7h with aromatic side chains as the
hydrophobic side chain, the APN inhibitory activities were different
with the substitutions on the aromatic ring. General speaking, com-
poundswith di-substitutiononthe aromaticring (compounds 7c, 7h
except 7g) showed better APN inhibitory activities than compounds
with mono-substitution on the aromatic ring. Comparing com-
poundswithdi-substitutiononthearomaticring, compound7cwith
two methyl groups on the aromatic ring showed better APN inhibi-
-
-
-
L
L
L
-
-
L
L
-Glu(OH)-OH
-Gly-NHOH
141 28.3
156.98 2.4
4.18
a
Values are means of three experiments, standard derivation is given.
tory activity (IC50 = 5.00 3.17
gens such as compounds 7h (IC50 = 169.85 13.03
(IC50 = 53.43 8.03 M). When it comes to compounds with
lM) than compounds with two halo-
lM) and 7g
l
Compounds 11a–11g, of which the R group was different amino
mono-substitution on the aromatic ring, to some extent, the substi-
acid. Similar to compounds 9a–9g, these compounds also showed
tution at the meta-position showed better activity such as com-
moderate APN inhibitory activity with IC50 values from
pound 7d with the IC50 value to 6.92 0.67
lM.
8.42 0.56 lM to109.84 7.46 lM. The APN inhibitory activity is
Comparing compounds 9a–9g, of which the R group was differ-
ent amino acid benzyl ester. These compounds (except compound
9f) showed moderate APN inhibitory activity with IC50 values from
also associated with the volume of hydrophobic side chain of ami-
no acid. Compounds with bigger volume hydrophobic side chain of
amino acid had better APN inhibitory activities (11d > 11c > 11a).
In contrast to compounds 9a–9g, the best APN inhibitor was 11d
13.45 3.33 lM to 91.61 1.48 lM. The APN inhibitory activity is
associated with the volume of hydrophobic side chain of the amino
acid. General speaking, compounds with bigger volume hydropho-
bic side chain of amino acid had better APN inhibitory activities. To
compound 9f, the R group is phenylalanine benzyl ester and the
IC50 value is 6.48 0.33 lM, the possible reason may be due to
the system of the aromatic ring enhancing the interaction with
(IC50 = 8.42 0.56 lM) of which the R group is Leucine.
Comparing all the target compounds (compounds 7, 9, 11, and
13), we can see that compounds with different organic amines in
part C (compound 7) showed less activities than compounds with
different amino acid benzyl esters (compound 9) and amino acids
(compound 11). Compound 13 with a hydroxamate group in part C
showed the least activity.
the hydrophobic region of the enzyme.

5984
X. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 5981–5987
Additionally, the effects of compounds 7c, 7d, 9f, and 11d on the
Although the computed information partially supported our
assumption, the exact binding model of 7c with APN should be ob-
tained from further X-ray crystal studies, which is under research
in our lab (Fig. 4).
proliferation of two tumor cell lines (HL-60 and MDA-MB-231)
compared with Bestatin were further assessed by using MTT assay,
which were shown in Figure 3. The results indicated that the anti-
proliferative effects of the four compounds (7c, 7d, 9f, and 11d)
against HL-60 cell and MDA-MB-231 cell were significantly lower
than the positive control Bestatin, which is consistent with the re-
sults of enzyme inhibition. For each compound, the anti-prolifera-
tive effect against HL-60 cell is better than MDA-MB-231, which
may due to the APN expression level on HL-60 cell is higher than
that of MDA-MB-231. Compound 7c showed the best anti- prolifer-
ative effect against HL-60 cell with the IC50 value of
1.97 0.11 mM.
Compound 7c showed the most activity towards APN. In order
to obtain further insight into the interaction of 7c with APN, the
docking studies were carried out via Sybyl/Sketch module and
optimized using Powell’s method with the Tripos force field with
convergence criterion set at 0.05 kcal/(Å mol), and assigned with
Gasteiger–Hückel method. The docking study performed using
Sybyl/FlexX module, the residues in a radius of 7.0 Å around Best-
atin in the co-crystal structure (PDB code: 2DQM) were selected as
the active-site. Other docking parameters implied in the program
were kept default. The docking result was shown in Figure 4. As
4. Conclusion
In summary, we have described the synthesis and properties of
a series of novel 3-phenylalanyl-N0-substituted-2,6-piperidinedi-
one derivates as APN inhibitors. Most of the target compounds
showed potent inhibitory activities towards APN, but comparing
with the lead compound 13f, the APN inhibitory activities were
not improved apparently. The most effective compound 7c, exhib-
ited excellent enzymatic inhibition activity which could be used as
a lead compound for further development of small molecular pep-
tidomimetic APN inhibitors.
5. Experiment
5.1. Chemistry: general procedures
All the material we used were commercial available. All the sol-
vents 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 chloride ferric. 200–300 mesh
silica gel was used in column chromatography to depurate the end
products. Proton NMR spectra were determined on a Brucker DRX
spectrometer (600 MHz), d in parts per million and J in Hertz, and
TMS was used as an internal standard. Measurements were made
in DMSO-d6 solutions. ESI-MS were determined on an API 4000
spectrometer. HRMS spectrums were conducted by Shandong
Analysis and Test Center. Melting points were determined on an
electrothermal melting point apparatus and were uncorrected.
The purity of the final products was analyzed using a reverse-
diagramed in Figure 4, the carbonyl group of
L-Phe can interact
with the zinc ion. The phenyl group of -Phe moiety can insert into
L
the S1 pocket as the same with Bestatin; the methyl group in the
4th position of 2,4-dimethylaniline can plunge into the S10 pocket
of APN and the other methyl group can interact with the S20 pocket
of APN.
phase Zorbax Eclipse XDB C18 (5
l
m, 100 Å) column (4.6 ꢂ
150 mm) with CH3OH (solvent A)/H2O (solvent B) as mobile phase,
using isocratic conditions at a flow rate of 1.0 ml/min on an Agilent
1200 apparatus (detector DAD G1312, pumps G1312). The eluted
peaks were monitored at 254 nm.
5.1.1. 2-((S)-3-((S)-2-(tert-Butoxycarbonylamino)-3-phenyl-
propanamido)-2,6-dioxopiperidin-1-yl) acetic acid (5)
The title compound was prepared as described by Li et al.18
5.1.2. (S)-2-Amino-N-((S)-1-(2-(4-fluorophenylamino)-2-
Figure 3. Anti-proliferative activities of compounds 7c, 7d, 9f, 11d, and Bestatin
against two tumor cell lines (HL-60 cell and MDA-MB-231 cell). The columns
represent the mean values of three independent experiments.
oxoethyl)-2,6-dioxopiperidin-3-yl)-3-phenylpropanamide (7a)
To a solution of compound (5) (0.87 g, 2 mmol) and N-methyl-
morpholine (0.26 ml, 2.2 mmol) in 30 ml anhydrous THF was added
isobutyl chloroformate (0.30 ml, 2.2 mmol) at ꢀ15 °C. The mixture
was stirred for 30 min at the same temperature. A solution of 4-flu-
orophenylamine (0.23 g, 2 mmol) in THF (5 ml) was added dropwise
to the reaction mixture. Keep the reaction for 1 h at ꢀ15 °C and then
remove the cooling bath. The reaction was continued for 4 h at room
temperature. Filtrated and concentrated with a rotary evaporator.
The residue was dissolved in 50 ml EtOAc and washed with satu-
ratedNaHCO3 (10 ml ꢂ 3), saturatedcitricacid(10 ml ꢂ 3)andbrine
(10 ml ꢂ 2) in turn. The organic phase was dried over anhydrous
Na2SO4 and concentrated with a rotary evaporator to afford crude
product. The crude product was recrystallized by EtOAc to give com-
pound (6a) (0.56 g, yield 53.2%), mp = 165–167 °C; 1H NMR
(600 MHz, DMSO-d6) d 1.23–1.28 (s, 9H), 2.00–2.02 (m, 2H), 2.75–
2.79 (m, 2H), 2.93–2.99 (m, 1H), 3.04–3.07 (m, 1H), 4.20–4.23
(m, 1H), 4.40–4.48 (m, 2H), 4.78–4.82 (m, 1H), 6.91 (m, J = 7.8 Hz, 1H),
7.14 (t, J = 9.0 Hz, 2H), 7.17–7.19 (m, 1H), 7.25–7.30 (m, 4H), 7.55–
7.57 (m, 2H), 8.46 (s, 1H), 10.22 (s, 1H); ESI-MS: m/z: 527.4 [M+H]+.
Figure 4. The FlexX docking of compound 7c with the active-site in Escherichia coli
APN (PDB: 2DQM). The protein is represented by molecular surface. Zinc ion is
indicated (atom types: polar H, sky-blue; N, dark-blue; O, red.).

X. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 5981–5987
5985
Compound (6a) (0.3 g, 0.56 mmol) was dissolved in 10 ml HCl–
EtOAc (3 mol/L). After 3 h, the solvent was filtrated and the precip-
itate was washed with EtOAc to get compound (7a) as a white solid
(0.21 g, yield 80.8%), mp = 195–198 °C; 1H NMR (600 MHz, DMSO-
d6) d 2.02–2.06 (m, 2H), 2.77–2.81 (m, 1H), 2.95–3.05 (m, 2H), 3.22
(dd, J = 5.4 Hz, 14.4 Hz, 1H), 4.10 (s, 1H), 4.43–4.51 (m, 2H), 4.82–
4.87 (m, 1H), 7.14 (t, J = 8.4 Hz, 2H), 7.25–7.28 (m, 1H), 7.32 (t,
J = 7.2 Hz, 1H), 7.37 (d, J = 7.2 Hz, 1H), 7.58–7.61 (m, 2H), 8.30 (s,
2H), 9.25 (s, 1H), 10.47 (s, 1H); HPLC tR = 17.5 min (89.40 area%);
HRMS(AP-ESI) m/z: calcd for C22H24FN4O4 [M+H]+ 427.2001, found
427.2009.
tR = 17.05 min (92.58 area%); HRMS(AP-ESI) m/z: calcd for
22H24BrN4O4 [M+H]+ 488.3544, found 488.3535.
C
5.1.8. (2S)-2-Amino-N-(1-(2-(3-chloro-4-fluorophenylamino)-2-
oxoethyl)-2,6-dioxopiperidin-3-yl)-3-phenylpropanamide (7g)
White solid (0.21 g, yield 75.5%), mp = 192–195 °C; 1H NMR
(600 MHz, DMSO-d6) d 2.02–2.06 (m, 2H), 2.77–2.81 (m, 1H),
2.96–3.04 (m, 2H), 3.22 (dd, J = 5.4 Hz, 14.4 Hz, 1H), 4.11 (s, 1H),
4.44–4.53 (m, 2H), 4.83–4.87 (m, 1H), 7.27–7.29 (m, 1H), 7.30–
7.32 (m, 2H), 7.33–7.39 (m, 3H), 7.46–7.49 (m, 1H), 7.90–7.92
(m, 1H), 8.31 (s, 2H), 9.22 (s, 1H), 10.71 (s, 1H); HPLC tR = 16.20 min
(92.60 area%); HRMS(AP-ESI) m/z: calcd for C22H23ClFN4O4 [M+H]+
461.8938, found 461.8920.
The other compounds (7b–7i) were synthesized in the same
procedure as described above.
5.1.3. (2S)-2-Amino-N-(1-(2-(4-methoxyphenylamino)-2-
5.1.9. (2S)-2-Amino-N-(1-(2-(3,4-difluorophenylamino)-2-
oxoethyl)-2,6-dioxopiperidin-3-yl)-3-phenylpropanamide (7h)
White solid (0.23 g, yield 85.4%), mp = 183–185 °C; 1H NMR
(600 MHz, DMSO-d6) d 2.03–2.06 (m, 2H), 2.77–2.81 (m, 1H),
2.96–3.04 (m, 2H), 3.22 (dd, J = 5.4 Hz, 14.4 Hz, 1H), 4.11 (s, 1H),
4.44–4.53 (m, 2H), 4.83–4.88 (m, 1H), 7.26–7.41 (m, 8H), 8.26 (s,
2H), 9.22 (s, 1H), 10.71 (s, 1H); HPLC tR = 16.81 min (93.83 area%);
HRMS(AP-ESI) m/z: calcd for C22H23F2N4O4 [M+H]+ 445.4392,
found 45.4382.
oxoethyl)-2,6-dioxopiperidin-3-yl)-3-phenylpropanamide (7b)
White solid (0.21 g, yield 81%), mp = 179–182 °C; 1H NMR
(600 MHz, DMSO-d6) d 2.02–2.06 (m, 2H), 2.77–2.81 (m, 1H),
2.95–3.04 (m, 2H), 3.22 (dd, J = 5.4 Hz, 14.4 Hz, 1H), 3.71 (s, 3H),
4.10 (s, 1H), 4.40–4.49 (m, 2H), 4.83–4.87 (m, 1H), 6.87 (d,
J = 7.2 Hz, 2H), 7.27 (t, J = 7.2 Hz, 1H), 7.33 (t, J = 7.2 Hz, 2H), 7.37
(d, J = 7.2 Hz, 2H), 7.47 (d, J = 7.2 Hz, 2H), 8.27 (s, 2H), 9.22 (s,
1H), 10.17 (s, 1H); HPLC tR = 17.04 min (95.16 area%); HRMS(AP-
ESI) m/z: calcd for C23H27N4O5 [M+H]+ 440.2119, found 440.2128.
5.1.10. (S)-Benzyl-2-(2-(3-((S)-2-amino-3-phenylpropanamido)-
2,6-dioxopiperidin-1-yl))acetate (9a)
5.1.4. (2S)-2-Amino-N-(1-(2-(2,4-dimethylphenylamino)-2-
oxoethyl)-2,6-dioxopiperidin-3-yl)-3-phenylpropanamide (7c)
White solid (0.22 g, yield 81.5%), mp = 175–177 °C; 1H NMR
(600 MHz, DMSO-d6) d 2.01–2.06 (m, 2H), 2.14 (s, 3H), 2.24 (s, 3H),
2.76–2.80 (m, 1H), 2.94–3.05 (m, 2H), 3.23 (dd, J = 5.4 Hz, 14.4 Hz,
1H), 4.10 (s, 1H), 4.44–4.51 (m, 2H), 4.84–4.89 (m, 1H), 6.95 (d,
J = 7.8 Hz, 1H), 7.02 (s, 1H), 7.18 (d, J = 7.8 Hz, 1H), 7.28 (t, J = 7.2 Hz,
1H), 7.33 (t, J = 7.2 Hz, 2H), 7.38 (m, 2H), 8.26 (s, 2H), 9.19 (s, 1H),
9.48 (s, 1H); HPLC tR = 18.74 min (95.06 area%); HRMS(AP-ESI) m/z:
calcd for C24H29N4O4 [M+H]+ 437.2565, found 437.2575.
To a solution of compound (5) (0.87 g, 2 mmol) and N-methyl-
morpholine (0.26 ml, 2.2 mmol) in 30 ml anhydrous THF was
added isobutyl chloroformate (0.30 ml, 2.2 mmol) at -15 °C. The
mixture was stirred for 30 min at the same temperature. A solution
of glycine benzyl ester p-toluenesulfonate salt (0.67 g, 2 mmol) in
DCM (5 ml) (which was previous neutralized with N-methylmor-
pholine) was added dropwise to the reaction mixture. Keep the
reaction for 1 h at ꢀ15 °C and then remove the cooling bath. The
reaction was continued for another 4 h at room temperature. Fil-
trated and concentrated with a rotary evaporator. The residue
was dissolved in 50 ml EtOAc and washed with saturated NaHCO3
(10 ml ꢂ 3), saturated citric acid (10 ml ꢂ 3) and brine (10 ml ꢂ 2)
in turn. The organic phase was dried over anhydrous Na2SO4 and
concentrated with a rotary evaporator to afford crude product.
The crude product was recrystallized by EtOAc/diethyl ether to
give compound (8a) (0.87 g, yield 75.4%), mp = 154–156 °C; 1H
NMR (600 MHz, DMSO-d6) d 1.25 (s, 9H), 1.99 (s, 1H), 2.70–2.81
(m, 2H), 2.86–2.92 (m, 1H), 2.96–3.08 (m, 1H), 3.91 (d, J = 5.7 Hz,
2H), 4.21–4.24 (m, 1H), 4.30 (d, J = 6.6 Hz, 2H), 4.74–4.83 (m,
1H), 5.12 (s, 2H), 6.93 (d, J = 8.7 Hz, 1H), 7.19–7.21 (m, 1H), 7.24–
7.29 (m, 4H), 7.31–7.37 (m, 5H), 8.43 (d, J = 8.7 Hz, 1H), 8.52
(t, J = 5.7 Hz, 1H); ESI-MS: m/z: 581.6 [M+H]+.
Compound (8a) (0.1 g, 0.17 mmol) was dissolved in 10 ml HCl–
EtOAc (3 mol/L). After 3 h, the solvent was filtrated and the precip-
itate was washed with EtOAc to get compound (9a) as a white solid
(0.08 g, yield 89.1%), mp = 158–161 °C; 1H NMR (600 MHz, DMSO-
d6) d 2.01 (s, 2H), 2.74–2.77 (d, 1H), 2.91–2.96 (m, 1H), 3.01–3.05
(m, 1H), 3.23 (dd, J = 4.8 Hz, 14.4 Hz, 1H), 3.92 (d, J = 5.4 Hz, 2H),
4.10 (s, 1H), 4.27–4.37 (m, 2H), 4.81–4.85 (m, 1H), 5.12 (s, 2H),
7.27 (t, J = 7.2 Hz, 1H), 7.33 (t, J = 7.2 Hz, 3H), 7.37 (m, 6H), 8.31
(s, 2H), 8.61 (s, 1H), 9.26 (s, 1H); HPLC tR = 16.32 min (90.67 area%);
HRMS(AP-ESI) m/z: calcd for C25H29N4O6 [M+H]+ 481.5210, found
481.5201.
5.1.5. (2S)-2-Amino-N-(1-(2-(3-chlorophenylamino)-2-
oxoethyl)-2,6-dioxopiperidin-3-yl)-3-phenylpropanamide (7d)
White solid (0.21 g, yield 78.3%), mp = 186–188 °C; 1H NMR
(600 MHz, DMSO-d6) d 2.02–2.06 (m, 2H), 2.77–2.81 (m, 1H),
2.96–3.02 (m, 2H), 3.20 (dd, J = 5.4 Hz, 14.4 Hz, 1H), 4.07 (s,
1H), 4.44–4.53 (m, 2H), 4.83–4.87 (m, 1H), 7.12 (d, J = 7.8 Hz,
1H), 7.27 (t, J = 7.2 Hz, 1H), 7.31–7.36 (m, 2H), 7.40 (d, J = 7.8 Hz, 1H),
7.78 (s, 1H), 8.09 (br s, 2H), 9.16 (s, 1H), 10.61 (s, 1H); HPLC
tR = 17.59 min (90.73 area%); HRMS(AP-ESI) m/z: calcd for
C
22H24ClN4O4 [M+H]+ 443.1486, found 443.1495.
5.1.6. (2S)-2-Amino-N-(1-(2-(2-fluorophenylamino)-2-
oxoethyl)-2,6-dioxopiperidin-3-yl)-3-phenylpropanamide (7e)
White solid (0.23 g, yield 88.7%), mp = 172–175 °C; 1H NMR
(600 MHz, DMSO-d6) d 2.02–2.06 (m, 2H), 2.77–2.81 (m, 1H),
2.95–3.05 (m, 2H), 3.23 (dd, J = 5.4 Hz, 14.4 Hz, 1H), 4.11 (s, 1H),
4.51–4.58 (m, 2H), 4.83–4.87 (m, 1H), 7.13–7.17 (m, 2H), 7.25–
7.28 (m, 2H), 7.32 (t, J = 7.2 Hz, 1H), 7.38 (d, J = 7.2 Hz, 1H), 7.86
(s, 1H), 8.27 (s, 2H), 9.27 (s, 1H), 10.10 (s, 1H); HPLC tR = 18.87 min
(90.82 area%); HRMS(AP-ESI) m/z: calcd for C22H24FN4O4 [M+H]+
427.2001, found 427.2009.
5.1.7. (2S)-2-Amino-N-(1-(2-(4-bromophenylamino)-2-
oxoethyl)-2,6-dioxopiperidin-3- yl)-3-phenylpropanamide (7f)
White solid (0.22 g, yield 74.9%), mp = 195–197 °C; 1H NMR
(600 MHz, DMSO-d6) d 2.02–2.06 (m, 2H), 2.77–2.81 (m, 1H),
2.96–3.04 (m, 2H), 3.22 (dd, J = 5.4 Hz, 14.4 Hz, 1H), 4.09 (s, 1H),
4.43–4.53 (m, 2H), 4.83–4.88 (m, 1H), 7.27 (t, J = 7.2 Hz, 1H), 7.32
(t, J = 7.2 Hz, 2H), 7.36 (d, J = 7.2 Hz, 2H), 7.49 (d, J = 7.2 Hz, 2H),
7.55 (d, J = 7.2 Hz, 2H), 8.27 (s, 2H), 9.22 (s, 1H), 10.55 (s, 1H); HPLC
The other compounds (9b–9g) were synthesized in the same
procedure as described above.
5.1.11. (S)-Benzyl-2-(2-(3-((S)-2-amino-3-phenylpropanamido)-
2,6-dioxopiperidin-1-yl) acetamido)propanoate (9b)
White solid (0.08 g, yield 88.9%), mp = 128–130 °C; 1H NMR
(600 MHz, DMSO-d6) d 1.27 (s, 3H), 2.01 (s, 2H), 2.75–2.78

5986
X. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 5981–5987
(m, 1H), 2.91–2.97 (m, 1H), 3.01–3.05 (m, 1H), 3.23 (dd, J = 5.4 Hz,
14.4 Hz, 1H), 4.10 (t, J = 6.6 Hz, 1H), 4.21–4.33 (m, 2H), 4.37 (d,
J = 14.4 Hz, 1H), 5.08–5.14 (m, 2H), 7.26–7.39 (m, 10H), 8.30 (s,
2H), 8.62 (s, 1H), 9.24 (s, 1H); HPLC tR = 15.22 min (92.37 area%);
HRMS(AP-ESI) m/z: calcd for C26H31N4O6 [M+H]+ 495.5475, found
495.5465.
area%); HRMS(AP-ESI) m/z: calcd for
643.7062, found 643.7055.
C
35H39N4O8 [M+H]+
5.1.17. 2-(2-(3-((S)-2-Amino-3-phenylpropanamido)-2,6-diox-
opiperidin-1-yl)acetamido)acetic acid (11a)
Compound (8a) (0.4 g, 8.02 mmol) and catalytic amount of 10%
Pd/C in methanol (30 ml) was hydrogenated in the presence of H2
at room temperature. After 16 h, the catalyst was filtered and the
solvent was removed under vacuum to give compound (10a) in
white solid (3.38 g, yield 97.2%). Compound (10a) (0.1 g, 0.2 mmol)
was then dissolved in 10 ml HCl–EtOAc (3 mol/L). After 3 h, the sol-
vent was filtrated and the prepicitate was washed with EtOAc to
get compound (11a) as white solid (0.07 g, yield 85%), mp = 178–
180 °C; 1H NMR (600 MHz, DMSO-d6) d 2.01 (s, 2H), 2.73–2.76
(m, 1H), 2.91–2.95 (m, 1H), 3.05 (m, 1H), 3.24 (d, J = 12 Hz, 1H),
3.76 (s, 2H), 4.02–4.10 (m, 1H), 4.25–4.35 (m, 2H), 4.82 (m, 1H),
5.12 (s, 1H), 7.27 (t, J = 7.2 Hz, 1H), 7.32 (t, J = 7.2 Hz, 2H), 7.38
(d, J = 7.2 Hz, 2H), 8.44 (s, 2H), 8.57 (s, 1H), 9.30 (s, 1H); HPLC
tR = 7.51 min (90.86 area%); HRMS(AP-ESI) m/z: calcd for
5.1.12. (S)-Benzyl-2-(2-(3-((S)-2-amino-3-phenylpropanamido)-
2,6-dioxopiperidin-1-yl) acetamido)-3-methylbutanoate (9c)
White solid (0.08 g, yield 85.1%), mp = 134–137 °C; 1H NMR
(600 MHz, DMSO-d6) d 0.86 (s, 6H), 1.98–2.07 (m, 3H), 2.75 (d,
J = 17.4 Hz, 1H), 2.91–2.97 (m, 1H), 3.01–3.05 (m, 1H), 3.22–3.24
(m, 1H), 4.09 (s, 1H), 4.22 (t, J = 7.2 Hz, 1H), 4.31–4.42 (m, 2H),
4.79–4.84 (m, 1H), 5.10–5.17 (m, 2H), 7.27 (t, J = 7.2 Hz, 1H), 7.32
(t, J = 6.6 Hz, 3H), 7.37 (s, 6H), 8.30 (s, 2H), 8.50 (s, 1H), 9.22 (s,
1H); HPLC tR = 17.28 min (90.34 area%); HRMS(AP-ESI) m/z: calcd
for C28H35N4O6 [M+H]+ 523.6007, found 523.6002.
5.1.13. (S)-Benzyl-2-(2-((S)-3-((S)-2-amino-3-phenylpropanam-
ido)-2,6-dioxopiperidin-1-yl)acetamido)-4-methylpentanoate
(9d)
C
18H23N4O6 [M+H]+ 391.3984, found 391.3975.
The other compounds (11b–11g) were synthesized in the same
White solid (0.08 g, yield 82.5%), mp = 120–121 °C; 1H NMR
(600 MHz, DMSO-d6) d 0.81–0.82 (m, 3H), 0.87–0.88 (m, 3H),
1.48–1.64 (m, 3H), 1.97–2.02 (m, 2H), 2.76 (d, J = 17.4 Hz, 1H),
2.91–2.97 (m, 1H), 3.24 (dd, J = 5.4 Hz, 14.4 Hz, 1H), 4.10 (t,
J = 6.0 Hz, 1H), 4.25–4.37 (m, 3H), 4.80–4.84 (m, 1H), 4.31–4.42
(m, 2H), 4.79–4.84 (m, 1H), 5.09–5.14 (m, 2H), 7.27 (t, J = 7.2 Hz,
1H), 7.31–7.36 (m, 5H), 7.36–7.38 (m, 4H), 8.32 (s, 2H), 8.58 (s,
1H), 9.24 (s, 1H); HPLC tR = 17.86 min (92.32 area%); HRMS(AP-
ESI) m/z: calcd for C29H37N4O6 [M+H]+ 537.6273, found 537.6264.
procedure as described above.
5.1.18. (S)-2-(2-(3-((S)-2-Amino-3-phenylpropanamido)-2,
6-dioxopiperidin-1-yl)acetamido)propanoic acid (11b)
White solid (0.067 g, yield 86.5%), mp = 195–198 °C; 1H NMR
(600 MHz, DMSO-d6) d 1.27 (s, 3H), 1.96–2.01 (m, 2H), 2.73–2.76
(m, 1H), 2.92–2.97 (m, 1H), 3.00–3.04 (m, 1H), 3.22 (dd,
J = 5.4 Hz, 14.4 Hz, 1H), 4.09 (s, 1H), 4.17–4.25 (m, 2H), 4.35 (d,
J = 16.2 Hz, 1H), 4.79–4.83 (m, 1H), 7.28 (t, J = 7.2 Hz, 1H), 7.33 (t,
J = 7.2 Hz, 2H), 7.37 (t, J = 7.2 Hz, 2H), 8.27 (s, 2H), 8.43 (s, 1H),
9.18 (s, 1H), 12.60 (s, 1H); HPLC tR = 6.67 min (92.33 area%);
HRMS(AP-ESI) m/z: calcd for C19H25N4O6 [M+H]+ 405.4250, found
405.4240.
5.1.14. Benzyl-3-(2-((S)-3-((S)-2-amino-3-phenylpropanamido)-
2,6-dioxopiperidin-1-yl)acetamido)propanoate (9e)
White solid (0.083 g, yield 92.2%), mp = 127–128 °C; 1H NMR
(600 MHz, DMSO-d6) d 2.01 (s, 2H), 2.53 (d, J = 6.6 Hz, 2H), 2.74–
2.77 (m, 1H), 2.90–2.96 (m, 1H), 3.02–3.05 (m, 1H), 3.23 (dd,
J = 5.4 Hz, 14.4 Hz, 1H), 3.30 (dd, J = 6.6 Hz, 12.6 Hz, 2H), 4.10 (m,
1H), 4.11–4.27 (m, 2H), 4.80–4.85 (m, 1H), 5.10 (s, 2H), 7.26 (t,
J = 7.2 Hz, 1H), 7.32 (t, J = 7.2 Hz, 3H), 7.37–7.38 (m, 6H), 8.25 (s,
1H), 8.31 (s, 2H), 9.27 (s, 1H); HPLC tR = 16.61 min (89.23 area%);
HRMS(AP-ESI) m/z: calcd for C26H31N4O6 [M+H]+ 495.5475, found
495.5460.
5.1.19. (S)-2-(2-(3-((S)-2-Amino-3-phenylpropanamido)-2,
6-dioxopiperidin-1-yl)acetamido)-3-methylbutanoic acid (11c)
White solid (0.079 g, yield 85%), mp = 178–180 °C; 1H NMR
(600 MHz, DMSO-d6) d 0.87 (s, 6H), 1.96–2.06 (m, 3H), 2.74 (d,
J = 18 Hz, 1H), 2.90–2.97 (m, 1H), 3.01–3.05 (m, 1H), 3.21–3.24
(m, 1H), 4.09 (s, 1H), 4.14–4.16 (m, 1H), 4.30–4.40 (m, 2H), 4.78–
4.82 (m, 1H), 7.27 (t, J = 7.2 Hz, 1H), 7.32 (t, J = 7.2 Hz, 2H), 7.37
(d, J = 7.2 Hz, 2H), 8.31 (s, 2H), 9.22 (s, 1H), 12.68 (s, 1H); HPLC
tR = 9.45 min (94.85 area%); HRMS(AP-ESI) m/z: calcd for
5.1.15. (S)-Benzyl-2-(2-((S)-3-((S)-2-amino-3-phenylpropanam-
ido)-2,6-dioxopiperidin-1-yl)acetamido)-3-phenylpropanoate
(9f)
C
21H29N4O6 [M+H]+ 433.4782, found 433.4775.
5.1.20. (S)-2-(2-((S)-3-((S)-2-Amino-3-phenylpropanamido)-2,6-
dioxopiperidin-1-yl)acet amido)-4-methylpentanoic acid (11d)
White solid (0.083 g, yield 87%), mp = 170–172 °C; 1H NMR
(600 MHz, DMSO-d6) d 0.82–0.83 (m, 3H), 0.88–0.89 (m, 3H),
1.48–1.57 (m, 2H), 1.62–1.64 (m, 1H), 2.00 (s, 2H), 2.75 (d,
J = 17.4 Hz, 1H), 2.90–2.96 (m, 1H), 3.02–3.05 (m, 1H), 3.23 (dd,
J = 4.8 Hz, 14.4 Hz, 1H), 4.06–4.10 (m, 2H), 4.20–4.27 (m, 2H),
4.34 (d, J = 15.6 Hz, 1H), 4.78–4.82 (m, 1H), 4.31–4.42 (m, 2H),
4.79–4.84 (m, 1H), 7.26–7.39 (m, 5H), 8.38 (s, 2H), 8.50 (s, 1H),
9.24 (s, 1H); HPLC tR = 11.21 min (95.80 area%); HRMS(AP-ESI) m/
z: calcd for C22H31N4O6 [M+H]+ 447.5047, found 447.5035.
White solid (0.082 g, yield 82%), mp = 143–145 °C; 1H NMR
(600 MHz, DMSO-d6) d 2.01 (s, 2H), 2.53 (d, J = 6.6 Hz, 2H), 2.74–
2.77 (m, 1H), 2.90–2.96 (m, 1H), 3.02–3.05 (m, 1H), 3.23 (dd,
J = 5.4 Hz, 14.4 Hz, 1H), 3.30 (dd, J = 6.6 Hz, 12.6 Hz, 2H), 4.10 (m,
1H), 4.11–4.27 (m, 2H), 4.80–4.85 (m, 1H), 5.10 (s, 2H), 7.26 (t,
J = 7.2 Hz, 1H), 7.32 (t, J = 7.2 Hz, 3H), 7.37–7.38 (m, 6H), 8.25 (s,
1H), 8.31 (s, 2H), 9.27 (s, 1H); HPLC tR = 16.81 min (93.45 area%);
HRMS(AP-ESI) m/z: calcd for C32H35N4O6 [M+H]+ 571.6435, found
571.6425.
5.1.16. (S)-Dibenzyl-2-(2-((S)-3-((S)-2-amino-3-phenylpropa-
namido)-2,6-dioxopiperidin-1-yl)acetamido)pentanedioate (9g)
White solid (0.09 g, yield 78%), mp = 118–122 °C; 1H NMR
(600 MHz, DMSO-d6) d 1.88–1.93 (m, 1H), 2.01–2.05 (m, 3H),
2.42–2.47 (m, 2H), 2.76 (d, J = 18 Hz, 1H), 2.90–2.95 (m, 1H),
3.02–3.05 (m, 1H), 3.23 (dd, J = 4.2 Hz, 13.8 Hz, 1H), 4.09–4.11
(m, 1H), 4.27–4.30 (m, 1H), 4.32–4.38 (m, 2H), 4.80–4.84 (m,
1H), 5.06–5.09 (m, 2H), 5.11–5.14 (m, 2H), 7.25–7.38 (m, 15H),
8.32 (s, 3H), 8.65 (s, 1H), 9.26 (s, 1H); HPLC tR = 17.23 min (92.78
5.1.21. 3-(2-((S)-3-((S)-2-Amino-3-phenylpropanamido)-2,
6-dioxopiperidin-1-yl)acetamido)propanoic acid (11e)
White solid (0.076 g, yield 86%), mp = 174–176 °C; 1H NMR
(600 MHz, DMSO-d6) d 1.08–1.11 (m, 1H), 1.16–1.19 (m, 1H),
1.99–2.03 (m, 2H), 2.73–2.76 (m, 1H), 2.88–2.95 (m, 1H), 3.01–
3.06 (m, 1H), 3.26–3.28 (m, 2H), 3.39–3.40 (m, 1H), 4.09–4.11
(m, 1H), 4.18–4.26 (m, 2H), 4.77–4.83 (m, 1H), 4.78–4.82 (m,
1H), 7.25–7.29 (m, 1H), 7.33 (t, J = 7.2 Hz, 1H), 7.38 (d, J = 7.2 Hz,

X. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 5981–5987
5987
2H), 8.15–8.21 (m, 2H), 8.47 (s, 1H), 9.27 (s, 1H); HPLC
and then 0.03% TNBS was added and incubated for another 20 min,
the resulting solution was detected under 450 nm wavelength to
gain absorption.
tR = 8.46 min (89.85 area%); HRMS(AP-ESI) m/z: calcd for
C
19H25N4O6 [M+H]+ 405.4250, found 405.4242.
5.1.22. (S)-2-(2-((S)-3-((S)-2-Amino-3-phenylpropanamido)-2,6-
dioxopiperidin-1-yl)acetamido)-3-phenylpropanoic acid (11f)
White solid (0.09 g, yield 88%), mp = 119–120 °C; 1H NMR
(600 MHz, DMSO-d6) d 1.96–2.02 (m, 2H), 2.72–2.76 (m, 1H),
2.90–3.05 (m, 4H), 3.23 (dd, J = 5.4 Hz, 14.4 Hz, 1H), 4.06–4.11
(m, 1H), 4.22–4.33 (m, 2H), 4.38–4.42 (m, 1H), 4.77–4.82 (m,
1H), 7.18–7.24 (m, 3H), 7.25–7.29 (m, 3H), 7.31–7.33 (m, 2H),
7.38 (d, J = 7.2 Hz, 2H), 8.29–8.36 (m, 2H), 8.63 (s, 1H), 9.23 (s,
1H); HPLC tR = 16.99 min (92.32 area%); HRMS(AP-ESI) m/z: calcd
for C25H29N4O6 [M+H]+ 481.5210, found 481.5201.
5.4. MTT assay
HL-60 cell (with high APN expression) and MDA-MB-231 Cell
(with low APN expression) were grown in RPMI1640 medium with
10% fetal bovine serum at 37 °C in 5% CO2 humidified incubator.
Cell proliferation 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, cul-
tured for 4 h in complete growth medium, then treated with
2000, 1000, 500, 250, or 125 lg/ml of the compounds for 72 h. Fol-
lowing this, 0.5% MTT solution was added to each well. After fur-
ther incubation for 4 h, the formazan formed from MTT was
extracted by adding DMSO and mixing for 15 min. The optical den-
sity was read with ELISA reader at 570 nm.
5.1.23. (S)-2-(2-((S)-3-((S)-2-Amino-3-phenylpropanamido)-2,6-
dioxopiperidin-1-yl)acetamido)pentanedioic acid (11g)
White solid (0.086 g, yield 86%), mp = 187–189 °C; 1H NMR
(600 MHz, DMSO-d6) d 1.74–1.80 (m, 1H), 1.91–2.01 (m, 3H),
2.23–2.30 (m, 2H), 2.75 (d, J = 18 Hz, 1H), 2.90–2.97 (m, 1H),
3.00–3.04 (m, 1H), 3.21 (dd, J = 4.8 Hz, 14.4 Hz, 1H), 4.08 (s, 1H),
4.19–4.24 (m, 1H), 4.25–4.36 (m, 2H), 4.79–4.83 (m, 1H), 7.28 (t,
J = 7.2 Hz, 1H), 7.32–7.37 (m, 4H), 8.16 (s, 3H), 8.46 (s, 1H), 9.17
(s, 1H), 12.45 (br s, 2H); HPLC tR = 9.78 min (92.34 area%);
HRMS(AP-ESI) m/z: calcd for C21H27N4O8 [M+H]+ 463.4611, found
463.4609.
Acknowledgments
This work was supported by National High Technology Re-
search and Development Program of China (863 project; Grant
No. 2007AA02Z314) and National Nature Science Foundation of
China (No. 90713041, No. 30772654) and the Doctoral Foundation
of Ministry of Education of the People’s Republic of China (Grant
No. 20060422029).
5.1.24. (2S)-2-Amino-N-(1-(2-(2-(hydroxyamino)-2-oxoethyla-
mino)-2-oxoethyl)-2,6-dioxopiperidin-3-yl)-3-phenylpropana-
mide (13g)
Supplementary data
The title compound was synthesized in the same procedure as
described in the preparation of compound (7a). White solid
(0.075 g, yield 85%), mp = 181–183 °C; 1H NMR (600 MHz, DMSO-
d6) d 2.00 (s, 2H), 2.71–2.81 (m, 1H), 2.91–2.94 (m, 1H), 3.03 (m,
1H), 3.22–3.25 (m, 1H), 4.11 (s, 1H), 4.29–4.37 (m, 2H), 4.81 (m,
1H), 7.20–7.39 (m, 5H), 8.33 (s, 2H), 9.24–9.29 (m, 1H), 10.72 (s,
1H); HPLC tR = 8.79 min (94.34 area%); HRMS(AP-ESI) m/z: calcd
for C18H24N5O6 [M+H]+ 406.4131, found 406.4125.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.06.078. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
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L-Leu-p-nitroanilide as a substrate and
l
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5.3. MMP inhibition assay
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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,