Novel cyclic-imide peptidomimetics as aminopeptidase N inhibitors. Structure-based design, chemistry and activity evaluation. II

Article abstract of DOI:10.1016/j.ejmech.2009.12.071

A novel class of potent aminopeptidase N inhibitors with 3-amino-cyclic-imide scaffold is described. The preliminary biological test revealed that all the compounds displayed high specific inhibitory activity against aminopeptidase N compared with previous work because of the existence of free amino group. Compounds containing hydroxamate group are more potent than carboxyl and ester derivatives. Compound 13f potentially inhibited APN activity with IC₅₀ value of 1.8?μM and displayed specific activity profiles in cells assay and in vivo anti-metastasis assay.

Full text of DOI:10.1016/j.ejmech.2009.12.071

European Journal of Medicinal Chemistry 45 (2010) 1618–1626
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel cyclic-imide peptidomimetics as aminopeptidase N inhibitors.
Structure-based design, chemistry and activity evaluation. II
*
Qianbin Li, Hao Fang, Xuejian Wang, Wenfang Xu
Institute of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, 44 West Wenhua 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:
A novel class of potent aminopeptidase N inhibitors with 3-amino-cyclic-imide scaffold is described. The
preliminary biological test revealed that all the compounds displayed high specific inhibitory activity
against aminopeptidase N compared with previous work because of the existence of free amino group.
Compounds containing hydroxamate group are more potent than carboxyl and ester derivatives.
Received 25 June 2009
Accepted 29 December 2009
Available online 14 January 2010
Compound 13f potentially inhibited APN activity with IC50 value of 1.8 mM and displayed specific activity
profiles in cells assay and in vivo anti-metastasis assay.
Keywords:
Aminopeptidase N
Peptidomimetics
Anti-tumor
Ó 2010 Elsevier Masson SAS. All rights reserved.
Cyclic-imide
1. Introduction
characterization for this kind of piperidinedione analogues
revealed that the most potent compound 1 displayed potent
inhibitory effect and can be used as a leading compound for further
structure optimization.
A structure-based approach to design potent and selective
inhibitors is an important component of the modern drug devel-
opment process [1,2]. Aminopeptidase N (EC3.4.11.2, APN, CD13),
expressed as an ectopeptidase with one zinc ion in active site, is of
significant biological and medical importance because of its key
role in protein modification, activation, and degradation as well as
in the metabolism of biologically active peptides in tumor metas-
tasis and leukemia [3–6]. The design and synthesis of inhibitors
against aminopeptidase N may result in potential therapeutic
agents [7,8].
Since 2006, the availability of X-ray crystal structures for APN
and complexes with various inhibitors have been investigated
[9–11]. Both Yoshimoto’s and Matthews’s had reported the binding
site and catalytic domain of APN based on the co-crystal complex of
Escherichia coli APN and bestatin, the only APN inhibitors applied in
clinic [10,11]. The binding site of the APN with bestatin has several
characteristics: part I is a hydrophobic pocket interacting with
phenyl group of bestatin; part II is one zinc binding group (ZBG)
which can coordinate with zinc ion in enzyme active site; part III is
another hydrophobic pocket in deeper cavity which can be divided
into two subsites [8,12,13].
Herein, we report the design and synthesis of a novel cyclic-
imide peptidomimetics. In order to find better APN inhibitors, we
modified the structure as the following, Fig. 1: (i) R1 group cor-
responding to the phenyl group of bestatin can be the side chains
of different amino acids. (ii) Keeping the amino group freely so
that it can interact with the anion binding site of enzyme that
contain a Glu355 (Human) or Glu264 (E. coli), as this residue is of
great importance during the first step for the recognition of
substrates or inhibitors by enzyme [15]. (iii) R2 group can be OH,
OC2H5 or NHOH, which play a role in the strong interaction with
the active site of enzyme. The in vitro inhibitory activity was
measured against APN enzyme. Additionally, potent compound
are also evaluated on HL-60 cell and K562 cell lines, and in vivo
anti-metastasis assay as well.
2. Chemistry
Compounds 5a–l were synthesized efficiently following the
procedures as shown in Scheme 1. The starting material (S)-tert-
butyl 2,6-dioxopiperidin-3-ylcarbamate (2) was prepared from
In our previous work, we have reported a series of piper-
idinedione-N-acetamide
derivatives
[14].
The
biological
L-Glutamine following the reference [16]. The amino group of
cyclic-imide scaffold was alkylated by ethyl bromoacetate followed
by deprotection of Boc group in the existence of TFA to the key free
amine intermediate 4. Compound 4 was combined with different
* Corresponding author. Tel.: þ86 531 883 82264; fax: þ86 863 883 82264.
E-mail address: xuwenf@sdu.edu.cn (W. Xu).
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.12.071

Q. Li et al. / European Journal of Medicinal Chemistry 45 (2010) 1618–1626
1619
O
O
O
NH
O
R
O
O
a
Boc
N
Boc
H3CO
H3CO
N
N
H
O
O
N
H
N
H
NHOH
O
O
2
O
7
OCH3
O
O
1
O
O
.
N
BocHN
N
b
TFA H2N
O
N
H
O
c
O
O
9
8
Glu
O
Zn2+
O
O
O
O
O
O
O
O
O
O
O
HCl . H2N
e
N
Part B
H3N
N
d
BocHN
N
N
OH
O
N
R2
N
OH
H
R
O
H
H
R
O
R1
O
Part A
11
10
f
R2=OH, OEt, NHOH
O
R1=side chain
of amino acid
O
O
O
HCl .
H2N
N
e
BocHN
N
N
NHOH
N
NHOH
H
R
O
H
R
O
Fig. 1. The strategy applied to design cyclic-imide peptidomimetics inhibitors with
13
12
free amino group.
Scheme 2. a. benzyl bromoacetate, TBAI, acetone, 56 ꢁC; b. TFA, DCM; c. N-methyl-
morphiline, THF, isobutyl Chloroformate, Boc-AA-OH; d. cyclohexene, C2H5OH, 5%
Pd/C, 40 ꢁC; e. 3N EtOAc–HCl; f. Et3N, THF, isobutyl Chloroformate.
Boc-amino acid derivatives to provide ethyl ester derivatives 5a–l
using DCC and HOBt as coupling reagents.
The critical intermediate 10a–i were synthesized following the
procedures as shown in Scheme 2. Starting from the same material
2 to Scheme 1, the NH was alkylated instead by benzyl bromoace-
tate to obtain benzyl ester intermediate 7. The carboxyl group of
different amino acid can be activated by isobutyl chloroformate and
N-methylmorpholine and then coupled with compound 8 to yield
9a–i whose benzyl ester can be easily deprotected by catalyzed
transfer hydrogenation to form carboxyl derivatives 10a–z. In
addition, the carboxyl group in 10a–i was transferred to its
hydroxamate acid derivatives 12a–i using the same method and
NH2OH as the base.
fragment for part A. According to reference, the free group is of
great importance for the recognition of substrate by aminopepti-
dases [15]. Fragments for R1 are structurally more diverse than
fragments for part A. Fragments R2 are the building blocks of part B
for the strong inhibitors. Interestingly, the tendency of inhibitory
profile of target compounds with different substituents in R2 is
CONHOH > COOH > COOEt.
In order to obtain further insight into the interaction of target
compound with APN, the most active compound 13f was built and
docked into the active site of APN (PDB code: 2DQM) using Gold4.0,
Fig. 2. The binding studies showed that two carbonyl groups (C]O
The Boc-protecting group can be easily removed by 3N HCl in
ethyl acetate to give the target compounds 6a–l, 11a–i and 13a–i as
hydrochloride salts.
from C-2 on the cyclic-imide ring and L-Phe) bind to the zinc ion
and form a five-membered ring in the active pocket with distance
of 2.34 Å and 2.27 Å, respectively. In addition, the C-2 carbonyl
3. Result and discussion
group, C-6 carbonyl group and NH from L-Phe also interact with the
hydroxyl group of Tyr381, Tyr275 and Glu298 by hydrogen bond,
respectively, which would be benefit to stabilize of the reaction
intermediate with the zinc ion. The free amino group in part A can
bind to anion binding site Glu121 (2.59 Å) and Glu264 (2.08 Å)
which can anchor the inhibitor to the active site of the enzyme. The
The enzymatic inhibition assay of APN was monitored by the
spectrophotometric method as described previously [17]. All the
inhibition results are summarized in Table 1.
Bioassay results indicate that most compounds exhibited
a better inhibitory activity against APN than previous work. This
result possibly resulted from the introduction of free amine group
for the requirement of active site of APN. Of the 30 compounds,
most compounds showed strong APN inhibitory activity with IC50
aromatic ring of L-Phe is more favorable as it can form a p–p
interaction with the Tyr376 located in the active site, which is the
same to that of bestatin, Fig. 2. The similar phenomenon can also be
seen from aromatic ring-containing compounds (6g, 6h and 6j, 11f,
11j, 13j). The hydroxamate acid group in part B can form hydrogen
bond interaction with His297 (2.34 Å), that are the essential amino
acids of the conserved sequence (HEXXHX18E) in the catalytic
domain that is well conserved in peptidase M1 family, and Asp327
and Arg293 as well.
values of 1.8–30
mM in Table 1. The activity of the most potent
compound 13f (IC50 ¼ 1.8
m
M) increased 46 times. Structurally, all
compounds have similar chemical scaffolds with the C-3 free amino
group, indicating that the free amine is the most optimized
The effects of the active compounds on HL-60 cell (with high
APN expression) and K562 cell (with low APN expression) prolif-
eration and survival were tested using MTT assay method. A screen
of six test compounds showed that compound 13f and 13i exhibited
the inhibitory effect of HL-60 cell proliferation with IC50 values of
O
O
O
O
O
a
N
Boc
N
b
H2N
O
Boc
c
NH
TFA .
N
H
O
N
H
O
O
O
2
4
3
105 ꢀ 12
potency than that of 1 (IC50 ¼ 136 ꢀ 14
(IC50 ¼ 245 ꢀ 8 M), Fig. 3a. Furthermore, 13f showed less cyto-
toxicity toward K562 cell than bestatin even the dose is much
higher than 400 g/mL, Fig. 3b. Another interesting result showed
that within lower concentration range (0.5 M and 5 M) 13f is
more active than that of bestatin on HL-60 cell proliferation. The
mM and 147 ꢀ 10
m
M, respectively, which showed better
O
O
O
O
O
O
m
M) and Bestatin
HCl . H2N
N
d
BocHN
N
N
H
O
N
H
O
m
R
O
R
O
5
6
m
Scheme 1. a. Ethyl bromoacetate, TBAI, acetone, 56 ꢁC; b. TFA, DCM; c. Et3N, THF, DCC,
m
m
HOBt; d. 3N EtOAc–HCl.

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Q. Li et al. / European Journal of Medicinal Chemistry 45 (2010) 1618–1626
Table 1
Table 1 (continued )
In vitro enzyme assay results for compounds 6, 11, 13 and Bestatin against APN
Compds
Substituents
R1
IC50
APN
/m
Ma
O
R2
O
O
HCl . H2N
N
N
H
R2
11f
OH
16.9 ꢀ 2.7
14.1 ꢀ 1.7
R1
O
H2N
/m
Ma
11g
OH
OH
Compds
Substituents
R1
IC50
APN
R2
H
N
6a
6b
H
CH3
OC2H5
OC2H5
23.7 ꢀ 1.2
64.3 ꢀ 1.3
11h
11i
13.2 ꢀ 1.9
11.3 ꢀ 1.4
6c
OC2H5
28.8 ꢀ 1.6
OH
HO
6d
OC2H5
30.8 ꢀ 1.3
13a
13b
H
CH3
NHOH
NHOH
9.4 ꢀ 1.6
10.9 ꢀ 1.4
6e
6f
OC2H5
OC2H5
23.3 ꢀ 1.6
38.1 ꢀ 2.8
13c
NHOH
12.5 ꢀ 0.1
5.3 ꢀ 1.2
13d
NHOH
HN
N
13e
NHOH
9.2 ꢀ 0.7
6g
6h
OC2H5
OC2H5
18.9 ꢀ 4.4
42.0 ꢀ 7.6
S
13f
NHOH
NHOH
1.8 ꢀ 0.2
9.4 ꢀ 2.1
H2N
13g
6i
OC2H5
20.7 ꢀ 2.7
N
H
H
N
13h
13i
a
NHOH
NHOH
9.7 ꢀ 0.3
6j
OC2H5
OC2H5
OC2H5
16.9 ꢀ 1.5
41.0 ꢀ 0.4
32.1 ꢀ 1.4
HO
7.3 ꢀ 1.4
HO
H2N
6k
6l
1
3.1 ꢀ 0.7
2.4 ꢀ 0.5
Bestatin
H
N
Values are means of three experiments, standard deviation is given.
11a
11b
H
CH3
OH
OH
15.4 ꢀ 1.4
21.5 ꢀ 1.6
results will be published elsewhere. These results suggested that
compound 13f exhibit the most specific inhibitory activity toward
APN with the IC50 value in sub-micromolar range.
11c
OH
15.8 ꢀ 1.4
To further examine effects of 13f, we measured its ability to
inhibit H22 cell’s location to lung tissue in vivo. As shown in Fig. 4,
the inhibitory rate for compound 13f and 13i is higher than that of
Bestatin (45.37%) with the value of 72.91% and 62.71%, respectively.
This activity profile for 13f can also be demonstrated by another
experiment carried on ES-2 tumor cell bearing nude mice, in which
13f displays equal potency at the dosage of 10 mg/kg to bestatin at
50 mg/kg, with the inhibitory rate value against tumor cell growth
of 30.9% and 36.2%, respectively, the results in detail will be pub-
lished elsewhere. All of these results indicate that compound 13f is
11d
OH
22.1 ꢀ 1.5
11e
OH
16.6 ꢀ 2.3

Q. Li et al. / European Journal of Medicinal Chemistry 45 (2010) 1618–1626
1621
precoated silica gel plates, and the resulting chromatograms were
visualized under UV light at 254 nm or stained by ny. Proton NMR
spectra were recorded on a Bruker DRX spectrometer, measure-
ments were made in CDCl3 or DMSO-d6 solutions. Proton chemical
shifts are reported in relation to tetramethylsilane (TMS) with the
unit of
d in ppm and J in Hertz. ESI-MS was measured on an API
4000 spectrometer. Anhydrous reactions were carried out in over-
dried glassware under a nitrogen atmosphere.
5.1.1. (S)-tert-Butyl 2,6-dioxopiperidin-3-ylcarbamate (2) [16]
To the stirring mixture of Boc-L-Gln (41.6 g, 0.169 mol) and N-
hydroxy-succinimide (NHSu, 19.5 g, 0.170 mol) in 200 mL of THF
was added in dropwise DCC (35.1 g, 0.170 mol) in 100 mL of THF at
0
ꢁC on ice-water bath and keep stirring at room temperature for
2 h. The mixture was heated and refluxed for 12 h and then
evaporated. To the residues was added 50 mL of EtOAc and 1 mL
acetic acid and evaporate. The residues were added 100 mL of
EtOAc and cooled in fridge overnight. The mixture was filtered by
4.0 g of Celite and evaporated to obtain yellow oil. To the residues
was added 100 mL of DCM. The organic phase was washed in turn
with saturated citric acid (10 mL ꢂ 3), 0.5% Na2CO3 (20 mL ꢂ 4), and
brine and dried over anhydrous Na2SO4. Filter and evaporate to 1/3
volume to obtain white crystal solid.
Fig. 2. Docking mode for 13f with APN (PDB code: 2DQM).
a potent molecule which can be used for further pre-clinical studies
and is promising for the application of invasion-preventing therapy.
4. Conclusion
Yield: 75.0%. Mp 211.5–213.8 ꢁC; ESI-MS m/z [M þ H]þ 229.6; 1H
NMR (400 MHz, CDCl3):
(m, 4H), 1.42 (s, 9H).
d 11.06 (s,1H), 7.39 (d,1H), 4.53 (d,1H), 2.10
In conclusion, our studies have shown that cyclic-imide pepti-
domimetics is a novel scaffold for the design of aminopeptidase N
inhibitors. Accurate structure knowledge of the inhibitors bound to
the target made it clear that at least four regions of the active site of
APN are needed to be occupied to establish a consistent binding
orientation: one anion binding site and two hydrophobic pocket
and a zinc binding site.
In this effort, a specific activity profile against APN has been
discovered when 3-amino-cyclic-imide scaffold was introduced in
the structure compared with the previous work. Most of the
compounds possess potent activity toward APN and the most
potent compound, 13f, exhibited good in vitro and in vivo activity.
This feature may also guide the design of new, specific inhibitors of
aminopeptidases with ‘one-zinc’ active site.
5.1.2. (S)-Ethyl-2-(3-(tert-butoxycarbonylamino)-2,6-
dioxopiperidin-1-yl)acetate (3)
To a 1L of flask charged with 2 (14.6 g, 0.064 mol), K2CO3 (10.6 g,
1.2 eq), tetrabutylammonium iodide (TBAI, 2.5 g, 0.1 eq) and
500 mL dry ketone was added ethyl 2-bromoacetate (11.0 mL,
1.5 eq). The mixture was then refluxed for 12 h and filtered and
evaporated to obtain yellow oil, which was added 100 mL of EtOAc
and then washed in turn with 0.5% Na2CO3 (10 mL ꢂ 3), saturated
citric acid (10 mL ꢂ 2) and brine. The organic phase was dried over
anhydrous Na2SO4. Filter and evaporate, and then crystallize with
EtOAc–Ethyl ether to obtain 16.0 g of 3 in white crystals.
Yield: 80.0%. Mp 134.5–136.3 ꢁC; ESI-MS m/z [M þ H]þ 315.4; 1H
NMR (400 MHz, CDCl3):
d 5.36 (br s, 1H), 4.51 (dd, 2 H), 4.39 (m,
5. Experimental section
1 H), 4.19 (q, 2H, J ¼ 6.90), 2.3 2(m, 4H), 1.46 (s, 9 H), 1.27 (t, 3 H,
J ¼ 6.90).
5.1. Chemistry
Unless otherwise stated, materials were obtained from
commercial suppliers and purified according to the methods of
chemical reagents. Melting points were determined on Boetius
apparatus and were not corrected. Column chromatography was
carried out on silica gel (200–300 mesh). TLCs were performed on
5.2. General procedure for the preparation of compound (5)
To the solution of 3 (10 mmol) in 40 mL of DCM was added 10 mL
of TFA and stirred at room temperature for 3 h and 4 was obtained
as yellow oil after evaporation. To the stirring mixture of 4 (1.5 g,
Fig. 3. In vitro HL-60 cell (a) and K562 cell (b) assay for active compounds.

1622
Q. Li et al. / European Journal of Medicinal Chemistry 45 (2010) 1618–1626
5.2.5. Ethyl-2-((S)-3-((2S,3R)-2-(tert-butoxycarbonylamino)-3-met-
hylpentanamido)-2,6-dioxopiperidin-1-yl) acetate (5e)
Yield: 89.0%. Mp 164.6–165.1 ꢁC; ESI-MS m/z [M þ H]þ 428.5; 1H
NMR (300 MHz, CDCl3):
d 6.70 (br s, 1H), 4.98 (br s, 1H), 4.64 (m,
1H), 4.51 (dd, 2H, J ¼ 34.2, 16.8), 4.19 (q, 2H, J ¼ 7.2), 4.01 (m, 1H),
2.86 (m, 2H), 2.55 (m, 1H), 1.90 (m, 2H), 1.45 (s, 9H), 1.27 (t, 3H,
J ¼ 7.2), 1.18 (m, 2H), 0.96 (dd, 6H).
5.2.6. Ethyl-2-((S)-3-((S)-2-(tert-butoxycarbonyl-amino)-3-phenyl
propanamido)-2,6-dioxopiperidin-1-yl)acetate (5f)
Yield: 91.3%. Mp 164.1–164.2 ꢁC; ESI-MS m/z [M þ H]þ 462.6;
1H NMR (300 MHz, CDCl3):
d 7.25 (m, 5H), 6.84 (br d, 1H), 4.94
(br s, 1H), 4.48 (m, 4H), 4.17 (q, 2H, J ¼ 7.2), 3.11 (d, 2H, J ¼ 6.3),
2.86 (m, 2H), 2.53 (m, 1H), 1.80 (m, 2H), 1.45 (s, 9H), 1.28 (t, 3H,
J ¼ 7.2).
5.2.7. Ethyl-2-((S)-3-((S)-2-(tert-butoxycarbony lamino)-3-(1H-
imidazol-4-yl)propanamido)-2,6-dioxopiperidin-1-yl)acetate (5g)
Yield: 64.5%. Mp 153.2–155.7 ꢁC; ESI-MS m/z [M þ H]þ 462.6; 1H
Fig. 4. In vivo H22 cell metastasis assay for active compounds.
NMR (300 MHz, CDCl3): d 8.00 (s,1H), 7.17 (s,1H), 4.47 (m, 4H), 4.18
(q, 2H, J ¼ 0.2), 3.00 (m, 4H), 2.50 (m, 1H), 1.97 (m, 1H), 1.60 (s, 9H),
7 mmol) in 50 mL anhydrous DCM at 0 ꢁC was added dropwise Et3N
(2.86 mL, 20 mmol) under N2, and then active ester of Boc-amino
acid derivative (1.18 mL, 10 mmol) prepared in advance (Boc-amino
acid and 1.1 eq of DCC and 1.2 eq of HOBt) was added in dropwise.
After 1.5 h, remove the ice bath and keep for another 3–12 h at
room temperature. The reaction was detected by TLC (EtOAc:cy-
clohexane, 2:1(V/V)) or (DCM:CH3OH-10:1).The mixture was
washed by 0.5% Na2CO3, brine to neutral and dried over Na2SO4.
Filter and condense by rotary evaporation to obtain crude target
compounds which were purified by recrystallization with anhy-
drous C2H5OH or chromatography column.
1.45 (s, 9H), 1.28 (t, 3H, J ¼ 7.2).
5.2.8. Ethyl-2-((S)-3-((S)-2-(tert-butoxycarbonylamino)-4-
(methylthio)butanamido)-2,6-dioxopiperidin-1-yl)acetate (5h)
Yield: 82.4%. Mp 147.9–148.5 ꢁC; ESI-MS m/z [M þ H]þ 446.6; 1H
NMR (300 MHz, CDCl3):
d 7.03 (br d, 1H), 5.20 (br s, 1H), 4.67 (m,
1H), 4.40 (dd, 2H), 4.18 (q, 2H, J ¼ 7.2), 2.85 (m, 2H), 2.60 (m, 2H),
2.49 (m, 1H), 2.12 (s, 3H), 2.08 (m, 1H), 1.94 (m, 2H), 1.45(s, 9H), 1.28
(t, 3H, J ¼ 7.2).
5.2.9. Ethyl-2-((S)-3-((S)-2-(tert-butoxycarbonyl-amino)-3-(1H-
indol-3-yl)propanamido)-2,6-dioxopiperidin-1-yl)aceþtate (5i)
Yield: 82.6%. Mp 133.1–134.2 ꢁC; ESI-MS m/z[M þ H] 501.5; 1HNMR
The following intermediates are prepared according to the
general procedure.
(300 MHz, CDCl3): d8.16(s,1H), 7.65 (d,1H), 7.35 (d,1H), 7.15 (m, 3H), 6.67
5.2.1. (S)-Ethyl-2-(3-(2-(tert-butoxycarbonyl-amino)acetamido)-
2,6-dioxopiperidin-1-yl)acetate (5a)
(s,1H), 5.13(s,1H), 4.37(m, 4H), 4.15(q, 2H, J¼ 7.2), 3.20 (m,1H), 2.79 (m,
2H), 2.39(m, 1H), 1.67 (m, 2H), 1.43 (s, 9H), 1.25 (t, 3H, J ¼ 7.2).
Yield: 95.8%. Mp 149.2–152.8 ꢁC; ESI-MS m/z [M þ H]þ 372.4; 1H
NMR (300 MHz, CDCl3):
d
6.93(br s, 1H), 4.65 (m, 1H), 4.51 (dd, 2H,
5.2.10. Ethyl-2-((S)-3-((S)-2-(tert-butoxycarbonyl-amino)-3-(4-
(tert-butoxycarbonyloxy)-phenyl)propanamido)-2,6-
dioxopiperidin-1-yl)acetate (5j)
J ¼ 30.3, 16.8), 4.17 (q, 2H, J ¼ 7.2), 3.87 (m, 2H), 2.65 (m, 3H), 1.90
(m, 1H), 1.46 (s, 9H), 1.27 (t, 3H, J ¼ 7.2).
Yield: 93.6%. Mp 179.3–180.8 ꢁC; ESI-MS m/z [M þ H]þ 578.5; 1H
5.2.2. Ethyl-2-((S)-3-((S)-2-(tert-butoxycar bonylamino)
propanamido)-2,6-dioxopiperidin-1-yl)acetate (5b)
NMR (300 MHz, CDCl3):
d
7.20 (d, 2H, J ¼ 8.4), 7.10 (d, 2H, J ¼ 8.4),
6.77 (d, 1H), 4.94 (m, 1H), 4.48 (m, 3H), 4.18 (q, 2H, J ¼ 7.2), 3.10 (d,
2H, J ¼ 6.3), 2.83 (m, 2H), 2.52 (m, 1H), 1.82 (m, 1H), 1.55 (s, 9H), 1.42
(s, 9H), 1.26 (t, 3H, J ¼ 7.2).
Yield: 93.3%. Mp 198.2–199.9 ꢁC; ESI-MS m/z [M þ H]þ 386.7; 1H
NMR: (300 MHz, CDCl3):
d 6.92 (br s, 1H), 4.97 (br s, 1H), 4.63 (m,
1H), 4.50 (dd, 2H, J ¼ 30.9, 16.8), 4.18 (q, 3H, J ¼ 7.2), 2.87 (m, 2H),
2.52(m, 1H), 1.90 (dq, 1H, J ¼ 12.9, 5.4), 1.45 (s, 9H), 1.39 (d, 3H,
J ¼ 6.9), 1.27 (t, 3H, J ¼ 7.2).
5.2.11. Ethyl-2-((S)-3-((S)-2,6-bis(tert-butoxy-carbonyl-
amino)hexanamido)-2,6-dioxopi peridin-1-yl)acetate (5k)
Yield: 86.6%. Mp 115.8–117.3 ꢁC; ESI-MS m/z [M þ H]þ 543.7; 1H
5.2.3. Ethyl-2-((S)-3-((S)-2-(tert-butoxy-carbonylamino)-3-methyl
butanamido)-2,6-dioxopiperidin-1-yl)acetate (5c)
NMR (300 MHz, CDCl3): d 6.96 (br s,1H), 5.15 (br s,1H), 4.62 (m,1H),
4.40 (dd, 2H, J ¼ 30.3, 16.8), 4.17 (q, 2H, J ¼ 7.2), 3.11 (m, 2H), 2.85
(m, 2H), 2.50 (m,1H),1.89 (m, 3H), 1.68 (m, 2H),1.50 (m, 2H), 1.44 (s,
18H), 1.28 (t, 3H, J ¼ 7.2).
Yield: 89.0%. Mp 199.3–201.8 ꢁC; ESI-MS m/z [M þ H]þ 414.7; 1H
NMR (300 MHz, CDCl3):
d 6.72 (s, 1H), 5.01 (br s, 1H), 4.64 (m, 1H),
4.50 (dd, 2H, J ¼ 33.6, 16.8), 4.18 (q, 2H, J ¼ 7.2), 3.98 (m, 1H), 2.87
(m, 2H), 2.54 (m, 1H), 2.18 (m, 1H), 1.92 (m, 1H), 1.45 (s, 9H), 1.27 (t,
3H, J ¼ 7.2), 0.96 (dd, 6H).
5.2.12. (S)-tert-Butyl-2-((S)-1-(2-ethoxy-2-oxoethyl)-2,6-
dioxopiperidin-3-ylcarbamoyl)pyrrolidine-1-carboxylate (þ5l)
1
Yield: 83.7%. Mp 151.9–152.7 ꢁC; ESI-MS m/z [M þ H] 412.6; H
5.2.4. Ethyl-2-((S)-3-((S)-2-(tert-butoxy-carbonylamino)-4-methyl
pentanamido)-2,6-dioxopiperidin-1-yl) acetate (5d)
NMR (300 MHz, CDCl3): d 4.53 (m, 3H), 4.15 (m, 3H), 3.46 (m, 2H), 2.86
(m, 2H), 2.50 (m, 1H), 1.89 (m, 4H), 1.46 (s, 9H), 1.26 (t, 3H, J ¼ 7.2).
5.3. General procedure for the preparation of compound (6)
Yield: 86.3%. Mp 176.6–179.2 ꢁC; ESI-MS m/z [M þ H]þ 428.7; 1H
NMR (300 MHz, CDCl3):
d 6.84 (br s, 1H), 4.85 L(br s, 1H), 4.63 (m,
1H), 4.50 (dd, 2H, J ¼ 31.5, 16.8), 4.19 (q, 3H, J ¼ 7.2), 2.84 (m, 2H),
2.53 (m, 1H), 1.92 (dq, 1H, J ¼ 12.9, 4.8), 1.67 (m, 2H), 1.51 (q, 1H,
J ¼ 9.1), 1.45(s, 9H), 1.28 (t, 3H, J ¼ 7.2), 0.95 (dd, 6H).
To the stirring solution of 5 (0.5 g) in 10 mL anhydrous ethyl
acetate was added dropwise 5 mL 3N EtOAc–HCl and after 2–3 h,

Q. Li et al. / European Journal of Medicinal Chemistry 45 (2010) 1618–1626
1623
target compounds 6 were obtained in the form of hydrochloride
salt. Filter quickly and dry the cake in vacuum to obtain dried white
solid with high yield.
The following compounds are prepared according to the general
procedure:
1H), 4.37 (dd, 2H, J ¼ 20.7, 16.8), 4.11 (q, 2H, J ¼ 7.2), 3.92 (m, 1H),
2.98 (m, 1H), 2.78 (m, 1H), 2.60 (m, 2H), 2.05 (m, 6H), 1.66 (m, 1H),
1.18 (t, 3H, J ¼ 6.9).
5.3.9. Ethyl-2-((S)-3-((S)-2-amino-3-(1H-indol-3-yl)
propanamido)-2,6-dioxopiperidin-1-yl)acetate hydrochloride (6i)
5.3.1. (S)-Ethyl-2-(3-(2-aminoacetamido)-2,6-dioxopiper idin-1-yl)
acetate hydrochloride (6a)
Yield: 80.2%. Mp 158.6–159.7 ꢁC; ESI-MS m/z [M þ H]þ
401.6; 1H NMR (300 MHz, DMSO-d6):
d 11.08 (s, 1H), 9.29 (d,
Yield: 89.7%. Mp 218.5–220.4 ꢁC; ESI-MS m/z [M þ H]þ 272.6;
1H), 8.25 (br s, 3H), 7.78 (d, 1H), 7.37 (d, 1H), 7.27 (s, 1H), 7.08
(t, 1H), 7.00 (t, 1H), 4.86 (m, 1H), 4.38 (m, 2H), 4.08 (m, 3H),
3.19 (m, 2H), 3.00 (m, 1H), 2.78 (m, 1H), 2.01 (m, 2H), 1.19 (t,
3H, J ¼ 7.2).
1H NMR (300 MHz, DMSO-d6):
d 8.97 (d, 1H), 8.18 (br s, 3H),
4.82 (m, 1H), 4.38 (dd, 2H, J ¼ 20.7, 16.8), 4.10 (q, 2H, J ¼ 6.9),
3.62 (s, 2H), 3.96 (m, 1H), 2.72 (m, 1H), 1.98(m, 2H), 1.18 (t, 3H,
J ¼ 6.9).
5.3.10. Ethyl-2-((S)-3-((S)-2-amino-3-(4-hydroxyphenyl)-
propanamido)-2,6-dioxopiperidin-1-yl)acetate hydrochloþride (6j)
Yield: 83.4%. Mp 179.9–181.2 ꢁC; ESI-MS m/z [M þ H] : 378.6; 1H
5.3.2. Ethyl-2-((S)-3-((S)-2-aminopropanamido)-2,6-
dioxopiperidin-1-yl)acetate hydrochloride (6b)
Yield: 82.3%. Mp 224.8–225.4 ꢁC; ESI-MS m/z [M þ H]þ 286.4; 1H
NMR (300 MHz, DMSO-d6): d 9.39 (s, 1H), 9.16 (d, 1H), 8.18 (br s,
NMR (300 MHz, DMSO-d6):
d
8.95 (d, 1H), 8.27 (br s, 3H), 4.83 (m,
3H), 7.14 (d, 2H, J ¼ 8.4), 6.71 (d, 2H, J ¼ 8.1), 4.82 (m, 1H), 4.40 (s,
2H), 4.11 (m, 2H), 3.99 (t,1H, J ¼ 6.3), 3.10 (m,1H), 2.96 (m, 2H), 2.78
(m, 1H), 2.00 (m, 2H).
1H), 4.38 (dd, 2H, J ¼ 20.7, 16.8), 4.11 (q, 2H, J ¼ 7.2), 3.87 (q, 1H,
J ¼ 6.9), 2.99 (m, 1H), 2.78 (m, 1H), 2.01 (m, 2H), 1.42 (d, 3H, J ¼ 6.9),
1.18 (t, 3H, J ¼ 6.9).
5.3.11. Ethyl-2-((S)-3-((S)-2,6-diaminohexanamido)-2,6-
dioxopiperidin-1-yl)acetate hydrochloride (6k)
5.3.3. Ethyl-2-((S)-3-((S)-2-amino-3-methylbutanamido)-2,6-
dioxopiperidin-1-yl) acetate hydrochloride (6c)
Yield: 78.5%. Mp 133.5–136.2 ꢁC; ESI-MS m/z [M þ H]þ: 343.6;
Yield: 79.6%. Mp 136.0–138.3 ꢁC; ESI-MS m/z [M þ H]þ 314.5; 1H
1H NMR (300 MHz, DMSO-d6):
d 9.05 (s,1H), 8.08 (br s, 6H), 4.86 (m,
NMR (300 MHz, DMSO-d6):
d
8.90 (d, 1H), 8.19 (br s, 3H), 4.90 (m,
1H), 4.38 (dd, 2H), 4.11 (q, 2H, J ¼ 6.9), 3.80 (t, 1H, J ¼ 6.9), 3.00 (m,
1H), 2.77 (m, 3H), 2.03 (m, 2H), 1.78 (m, 2H), 1.54 (m, 4H), 1.20 (t,
3H, J ¼ 7.2).
1H), 4.38 (dd, 2H, J ¼ 20.7, 16.8), 4.10 (q, 2H, J ¼ 7.2), 3.62 (d, 1H,
J ¼ 5.4), 3.00 (m, 1H), 2.78 (m, 1H), 2.14 (m, 1H), 2.00 (m, 2H), 1.17 (t,
3H, J ¼ 6.9), 1.01 (d, 3H, J ¼ 6.3).
5.3.12. Ethyl-2-((S)-2,6-dioxo-3-((S)-pyrrolidine-2-
5.3.4. Ethyl-2-((S)-3-((S)-2-amino-4-methylpentanamido)-2,6-
dioxopiperidin-1-yl) acetate hydrochloride (6d)
carboxamido)piperidin-1-yl)acetate hydrochloride (6l)
Yield: 64.8%. Mp 179.0–181.1 ꢁC; ESI-MS m/z [M þ H]þ 312.6; 1H
Yield: 73.4%. Mp 141.4–142.6 ꢁC; ESI-MS m/z [M þ H]þ 328.7; 1H
NMR (300 MHz, DMSO-d6): d 9.04 (m, 1H), 4.83 (m, 1H), 4.37 (dd,
NMR (300 MHz, DMSO-d6):
d
9.05 (d, 1H), 8.34 (br s, 3H), 4.86 (m,
2H), 4.22 (m, 1H), 4.10 (q, 2H, J ¼ 6.9), 3.20 (m, 2H), 3.00 (m, 1H),
1H), 4.38 (dd, 2H, J ¼ 20.7, 16.8), 4.08 (q, 2H, J ¼ 6.9), 3.79 (t, 1H,
J ¼ 6.9), 3.00 (m, 1H), 2.78 (m, 1H), 2.02 (m, 2H), 1.79 (m, 1H), 1.62
(m, 2H), 1.19 (t, 3H, J ¼ 7.2), 0.92 (d, 6H, J ¼ 6.6).
2.78 (m, 1H), 2.34 (m, 1H), 1.98(m, 5H), 1.19 (t, 3H, J ¼ 7.2).
5.3.13. (S)-Benzyl-2-(3-(tert-butoxycarbonylamino)-2,6-
dioxopiperidin-1-yl)acetate (7)
5.3.5. Ethyl-2-((S)-3-((2S,3R)-2-amino-3-methylpentan amido)-
2,6-dioxopiperidin-1-yl)acetate hydrochloride (6e)
The procedure is similar to that of 3 using benzyl-2-bromoace-
tate instead of ethyl 2-bromoacetate to obtain white crþystal.
Yield: 67%. Mp 78.0–81.0 ꢁC; ESI-MS m/z [M þ H] 377.6; 1H
Yield: 79.4%. Mp 171.1–172.3 ꢁC; ESI-MS m/z [M þ H]þ 328.7; 1H
NMR (300 MHz, DMSO-d6):
d
8.95 (d, 1H), 8.29 (br s, 3H), 4.88 (m,
NMR (300 MHz, CDCl3): d 7.37 (m, 5H), 5.14 (s, 2H), 4.39 (m, 3H),
1H), 4.38 (dd, 2H, J ¼ 20.7, 16.8), 4.10 (q, 2H, J ¼ 6.9), 3.67 (d, 1H,
J ¼ 5.4), 2.99 (m, 1H), 2.78 (m, 1H), 2.03 (m, 2H), 1.87 (m, 1H), 1.59
(m, 1H), 1.21 (m, 1H), 1.18 (t, 3H, J ¼ 7.2), 0.98 (d, 3H, J ¼ 6.9), 0.88 (t,
3H, J ¼ 6.6).
2.93 (m, 1H), 2.72 (m, 1H), 1.97 (m, 2H), 1.40 (s, 9H).
5.4. General procedure for the preparation of compound 10
To the solution of 7 (5.5 mmol) in 22 mL of DCM was added
5.5 mL of TFA and stirred at room temperature for 3 h and 8 was
obtained as yellow oil, which was dissolved in 20 mL of anhydrous
THF and cooled to ꢃ20 ꢁC. N-methylmorphiline (about 1.3 mL) was
added slowly to control the pH of the mixture at 6.0. To the flask
charged with Boc-amino acid derivatives (5.5 mmol) and 20 mL of
THF at ꢃ20 ꢁC was added dropwise N-methylmorphiline 5.5 mmol
(0.62 mL), and then 5.5 mmol (0.72 mL isobutyl chloroformate after
5 min). The mixture was stirred at ꢃ20 ꢁC for another 10 min and to
the cold mixture was added 8 in THF dropwise and keep stirring for
another 15 min at ꢃ20 ꢁC and then 0 ꢁC for 3 h. The reaction
mixture was filtered with the aid of 2.0 g of Celite. The filtrate was
condensed and to the residues was added 80 mL of EtOAc and then
washed in turn by 0.5% Na2CO3, 5% citric acid and brine. The organic
phase was dried over Na2SO4. After filter and condense to obtain
intermediates 9 with high yield without further purification. To the
clear solution of benzyl ester derivatives 9 (4.0 mmol) in 20 mL
anhydrous EtOH was added 20 mL of cyclohexene as hydrogen
donor and equivalent amount of 5% Pd/C as catalyst. The mixture
5.3.6. Ethyl-2-((S)-3-((S)-2-amino-3-phenylpropan amido)-2,6-
dioxopiperidin-1-yl) acetate hydrochloride (6f)
Yield: 86.4%. Mp 156.3–158.0 ꢁC; ESI-MS m/z [M þ H]þ 362.4; 1H
NMR (300 MHz, DMSO-d6):
d 9.18 (d, 1H), 8.23 (br s, 3H), 7.31 (m,
5H), 4.83 (m, 1H), 4.40 (dd, 2H), 4.10 (m, 3H), 3.21 (m, 1H), 2.99 (m,
2H), 2.78 (m, 1H), 2.02 (m, 2H), 1.20 (t, 3H, J ¼ 7.2).
5.3.7. Ethyl-2-((S)-3-((S)-2-amino-3-(1H-imidazol-4-yl)-
propanamido)-2,6-dioxopiperidin-1-yl)acetate hydrochloride (6g)
Yield: 80.4%. Mp 126.6–127.8 ꢁC; ESI-MS m/z [M þ H]þ 352.5; 1H
NMR (300 MHz, DMSO-d6):
d 14.60 (br s, 1H), 9.30 (d, 1H), 9.06 (s,
1H), 8.63 (br s, 3H), 7.53 (s, 1H), 4.79 (m, 1H), 4.41 (m, 3H), 4.11 (m,
2H), 2.97 (m, 1H), 2.22 (m, 1H), 2.03 (m, 2H), 1.19 (t, 3H, J ¼ 7.2).
5.3.8. Ethyl-2-((S)-3-((S)-2-amino-4-(methylthio)butan amido)-
2,6-dioxopiperidin-1-yl)acetate hydrochloride (6h)
Yield: 65.7%. Mp 139.2–142.1 ꢁC; ESI-MS m/z [M þ H]þ 346.4; 1H
NMR (300 MHz, DMSO-d6): d 9.19 (dd, 1H), 8.47 (br s, 3H), 4.81 (m,

1624
Q. Li et al. / European Journal of Medicinal Chemistry 45 (2010) 1618–1626
was stirred at 45 ꢁC for 1 h and filtered with the aid of 4.0 g of Celite.
The filtrate was condensed and dissolved in 60 mL EtOAc and then
washed in turn by 5% citric acid and brine. The organic phase was
dried over Na2SO4. Compound 10 was further purified by column to
obtain white solid.
5.4.9. 2-((S)-3-((S)-2-(tert-Butoxycarbonylamino)-3-(4-(tert-
butoxycarbonyloxy)phenyl)propanamido)-2,6-dioxopiperidin-1-
yl)acetic acid (10i)
Yield: 91.4%. Mp 89.7–90.4 ꢁC; ESI-MS m/z [M þ H]þ 550.6; 1H
NMR (300 MHz, CDCl3):
2H, J ¼ 8.4), 5.30 (d,1H) 4.51 (m, 4H), 2.88 (m, 4H), 2.34 (m,1H),1.74
d
7.29 (d, 1H), 7.20 (d, 2H, J ¼ 8.4), 7.08 (d,
The following intermediates are prepared according to the
general procedure.
(m, 1H), 1.54 (s, 9H), 1.37 (s, 9H).
5.4.1. (S)-2-(3-(2-(tert-Butoxycarbonylamino)acetamido)-2,6-
5.5. General procedure for the preparation of compound 11
dioxopiperidin-1-yl)acetic acid (10a)
Yield: 98.3%. Mp 171–172.9 ꢁC; ESI-MS m/z [M þ H]þ 344.3; 1
H
The carboxyl acid derivatives 10 (0.3–0.5 g) were dissolved in 5 mL
3N HCl–EtOAc and stirred at room temperature for 2–3 h to obtain
target compound 11 in the form of hydrochloride salt. Filter quicklyand
dry the cake in vacuum to obtain dried white solid with high yield.
The following compounds are prepared according to the general
procedure.
NMR (300 MHz, DMSO-d6):
d 8.90 (d, 1H), 8.09 (br s, 3H), 4.82 (m,
1H), 4.30 (dd, 2H, J ¼ 21.0, 16.8), 3.63 (m, 2H), 2.85 (m, 2H), 1.99 (m,
2H), 1.43 (s, 9H).
5.4.2. ((S)-3-((S)-2-(tert-Butoxycarbonylamino)propanamido)-2,6-
dioxopiperidin-1-yl)acetic acid (10b)
Yield: 73.8%. Mp 177.7–178.9 ꢁC; ESI-MS m/z [M þ H]þ 428.7; 1H
5.5.1. (S)-2-(3-(2-Aminoacetamido)-2,6-dioxopiperidin-1-yl)acetic
NMR (300 MHz, DMSO-d6):
d
12.96 (br s,1H), 8.89 (d,1H), 8.22 (br s,
acid hydrochloride (11a)
1
3H), 4.83 (m, 1H), 4.29 (dd, 2H, J ¼ 20.4, 17.1), 3.87 (m, 1H), 2.86 (m,
Yield: 85.9%. Mp 170.1–172.1 ꢁC; ESI-MS m/z [M þ H]þ 244.2; H
2H), 2.10 (m, 2H), 1.44 (s, 9H), 1.41 (d, 3H, J ¼ 6.9).
NMR (300 MHz, DMSO-d6): d 8.90 (d, 1H), 8.09 (br s, 3H), 4.82 (m, 1H),
4.30 (dd, 2H, J ¼ 21.0, 16.8), 3.63 (m, 2H), 2.85 (m, 2H), 1.99 (m, 2H).
5.4.3. 2-((S)-3-((S)-2-(tert-Butoxycarbonylamino)-3-
methylbutanamido)-2,6-dioxopiperidin-1-yl)acetic acid (10c)
5.5.2. 2-((S)-3-((S)-2-Aminopropanamido)-2,6-dioxopiperidin-1-yl)
acetic acid hydrochloride (11b)
Yield: 89.5%. Mp 95.8–96.8 ꢁC; ESI-MS m/z [M þ H]þ 386.4; 1H
NMR (300 MHz, CDCl3):
d
7.22 (d,1H), 5.35 (s,1H), 4.79 (m,1H), 4.54
Yield: 96.9%. Mp 121.5–122.0 ꢁC; ESI-MS m/z [M þ H]þ258.2; 1H
(dd, 2H, J ¼ 30.0, 17.1), 3.97 (m, 1H), 2.85 (m, 2H), 2.36 (m, 1H), 2.00
NMR (300 MHz, DMSO-d6): d 12.96 (br s,1H), 8.89 (d,1H), 8.22 (br s,
(m, 2H), 1.43 (s, 9H), 0.96 (t, 6H).
3H), 4.83 (m, 1H), 4.29 (dd, 2H, J ¼ 20.4, 17.1), 3.87 (m, 1H), 2.86 (m,
2H), 2.10 (m, 2H), 1.41 (d, 3H, J ¼ 6.9).
5.4.4. 2-((S)-3-((S)-2-(tert-Butoxycarbonylamino)-4-
methylpentanamido)-2,6-dioxopiperidin-1-yl)acetic acid (10d)
5.5.3. 2-((S)-3-((S)-2-amino-3-methylbutanamido)-2,6-
dioxopiperidin-1-yl)acetic acid hydrochloride (11c)
Yield: 91.4%. Mp 91.2–92.0 ꢁC; ESI-MS m/z [M þ H]þ 400.4;
1H NMR (300 MHz, DMSO-d6):
d
9.03 (d, 1H), 8.41 (br s, 3H),
Yield: 91.8%. Mp 119.6–121.4 ꢁC; ESI-MS m/z [M þ H]þ 286.3; 1H
4.84 (m, 1H), 4.29 (dd, 2H, J ¼ 20.4, 17.1), 3.78 (t, 1H), 2.89 (m,
2H), 2.02 (m, 2H), 1.78 (m, 1H), 1.43 (s, 9H), 0.97 (d, 6H,
J ¼ 6.9).
NMR (300 MHz, DMSO-d6): d 8.84 (s, 1H), 8.22 (br s, 3H), 4.83 (m,
1H), 4.29 (dd, 2H, J ¼ 20.4, 17.1), 3.87 (m, 1H), 2.87 (m, 2H), 2.10 (m,
4H), 1.00 (d, 3H, J ¼ 6.6).
5.4.5((S)-3-((2S,3R)-2-(tert-Butoxycarbonylamino)-3-
5.5.4. 2-((S)-3-((S)-2-Amino-4-methylpentanamido)-2,6-
dioxopiperidin-1-yl)acetic acid hydrochloride (11d)
methylpentanamido)-2,6-dioxopiperidin-1-yl)acetic acid þ(10e)
Yield: 84.5%. Mp 73.5–75.3 ꢁC; ESI-MS m/z [M þ H] 400.4; 1H
Yield: 95.2%. Mp 156.5–158.1 ꢁC; ESI-MS m/z [M þ H]þ 300.3; 1H
NMR (300 MHz, CDCl3):
d
7.32 (d,1H), 5.37 (s,1H), 4.79 (m,1H), 4.53
NMR (300 MHz, DMSO-d6): d 9.05 (d, 1H), 8.40 (br s, 3H), 4.85 (m,
(dd, 2H, J ¼ 35.1, 17.1), 3.99 (m, 1H), 2.84 (m, 2H), 2.34 (m, 1H),
1.95 (m, 1H), 1.74 (m, 1H), 1.54 (m, 1H), 1.43 (s, 9H), 1.15 (m, 1H), 0.91
(dt, 6H).
1H), 4.29 (dd, 2H, J ¼ 20.4, 17.1), 3.78 (t, 1H), 2.89 (m, 2H), 2.02 (m,
2H), 1.78 (m, 1H), 1.01 (d, 6H, J ¼ 6.9).
5.5.5. 2-((S)-3-((2S,3R)-2-Amino-3-methylpentanamido)-2,6-
dioxopiperidin-1-yl)acetic acid hydrochloride (11e)
5.4.6. 2-((S)-3-((S)-2-(tert-Butoxycarbonylamino)-3-
phenylpropanamido)-2,6-dioxopiperidin-1-yl)acetic acid þ(10f)
Yield: 83.3%. Mp 143.3–145.7 ꢁC; ESI-MS m/z [M þ H]þ 300.3; 1H
Yield: 73.8%. Mp 89.2–91.5 ꢁC; ESI-MS m/z [M þ H] 434.5; 1H
NMR (300 MHz, DMSO-d6): d 12.96 (br s,1H), 8.92 (d,1H), 8.26 (br s,
NMR (300 MHz, CDCl3):
d
7.23 (m, 5H), 5.26 (s, 1H), 4.54 (m, 4H),
3H), 4.87 (m, 1H), 4.29 (dd, 2H, J ¼ 20.4, 17.1), 3.66 (d, 1H), 2.84 (m,
2H), 2.04 (m, 2H),1.88 (m,1H),1.59 (m,1H),1.16 (m,1H), 0.98 (d, 3H,
J ¼ 6.9), 0.83 (d, 3H, J ¼ 7.2).
2.90 (m, 4H), 2.35 (m, 1H), 1.89 (m, 1H), 1.36 (s, 9H).
5.4.7. 2-((S)-3-((S)-2,6-Bis(tert-
butoxycarbonylamino)hexanamido)-2,6-dioxopiperidin-1-yl)acetic
acid (10g)
5.5.6. 2-((S)-3-((S)-2-Amino-3-phenylpropanamido)-2,6-
dioxopiperidin-1-yl)acetic acid hydrochloride (11f)
Yield: 79.7%. Mp 93.2–93.7 ꢁC; ESI-MS m/z [M þ H]þ 515.6; 1H NMR
Yield: 93.8%. Mp 135.6–138.2 ꢁC; ESI-MS m/z [M þ H]þ 334.3; 1H
(300 MHz, CDCl3):
d
7.53 (d,1H), 5.55 (s,1H), 4.93 (m,1H), 4.77 (m,1H),
NMR (300 MHz, DMSO-d6): d 9.15 (s,1H), 8.24 (br s, 3H), 7.31 (s, 5H),
4.53 (dd, 2H, J¼ 35.1,17.1), 4.21 (m,1H), 3.08 (m, 2H), 2.97–2.87 (m, 2H),
4.83 (m, 1H), 4.31 (dd, 2H, J ¼ 19.2, 16.8), 4.03 (m, 1H), 3.21 (m, 1H),
2.32 (m, 1H), 2.06 (m, 1H), 1.79 (m, 1H), 1.63 (m, 1H), 1.43 (s, 18H).
2.98 (m, 2H), 2.76 (m, 1H), 2.00 (m, 2H).
5.4.8. 2-((S)-3-((S)-1-(tert-Butoxycarbonyl)pyrrolidine-2-
carboxamido)-2,6-dioxopiperidin-1-yl)acetic acid (10h)
Yield: 71.7%. Mp 91.7–92.5 ꢁC; ESI-MS m/z [M þ H]þ 384.4; 1H
5.5.7. 2-((S)-3-((S)-2,6-Diaminohexanamido)-2,6-dioxopiperidin-
1-yl)acetic acid dihydrochloride (11g)
Yield: 96.3%. Mp 126.8–127.3 ꢁC;ESI-MSm/z[M þ H]þ 315.3; 1HNMR
NMR (300 MHz, CDCl3):
d
7.66 (d,1H), 5.55 (s,1H), 4.94 (m,1H), 4.49
(300 MHz, DMSO-d6): d 12.96 (br s, 1H), 9.11 (d, 1H), 8.37 (br s, 3H), 8.02
(s, 2H), 4.38 (m, 1H), 3.50 (m, 1H), 3.37 (m, 1H), 2.78 (s, 2H), 2.27 (m,
1H), 2.05 (m, 3H), 1.66 (m, 2H), 1.43 (s, 9H).
(br s, 3H), 4.85 (m,1H), 4.30 (dd, 2H, J ¼ 20.4,17.1), 3.83 (m,1H), 2.99 (m,
1H), 2.77 (m, 3H), 2.00 (m, 2H), 1.80 (m, 2H), 1.61 (m, 2H), 1.43 (m, 2H).

Q. Li et al. / European Journal of Medicinal Chemistry 45 (2010) 1618–1626
1625
5.5.8. 2-((S)-2,6-Dioxo-3-((S)-pyrrolidine-2-
5.6.5. (2S,3R)-2-Amino-N-((S)-1-(2-(hydroxyamino)-2-oxoethyl)-
carboxamido)piperidin-1-yl)acetic acid hydrochloride (11h)
2,6-dioxopiperidin-3-yl)-3-methylpentanamide hydrochloride (13e)
Yield: 53.9%. Mp 132.4–135.4 ꢁC; ESI-MS m/z [M þ H]þ 284.3; 1H
Yield: 85.5%. Mp 174.0–175.0 ꢁC; ESI-MS m/z [M þ H]þ 315.3; 1H
NMR (300 MHz, DMSO-d6):
d
12.96 (br s, 1H), 9.49 (br s, 1H), 8.97
NMR (300 MHz, DMSO-d6): d 10.59 (s, 1H), 8.82 (d, 1H), 8.16 (s, 3H),
(br s, 1H), 8.59 (br s, 1H), 7.32 (s, 5H), 4.83 (m, 1H), 4.31 (dd, 2H,
J ¼ 19.2, 16.8), 4.23 (m, 1H), 3.22 (m, 2H), 2.97 (m, 1H), 2.77 (m, 1H),
2.30 (m, 1H), 1.98 (m, 5H).
4.16 (s, 2H), 3.65 (s,1H), 2.92 (m,1H), 2.74 (m,1H), 2.06 (m, 2H),1.85
(m, 1H), 1.56 (m, 1H), 1.21 (m, 1H), 0.98 (d, 3H, J ¼ 6.9), 0.89 (t, 3H,
J ¼ 7.2).
5.5.9. 2-((S)-3-((S)-2-Amino-3-(4-hydroxyphenyl)propanamido)-
2,6-dioxopiperidin-1-yl)acetic acid hydrochloride (11i)
5.6.6. (S)-2-Amino-N-((S)-1-(2-(hydroxyamino)-2-oxoethyl)-2,6-
dioxopiperidin-3-yl)-3-phenylpropanamide hydrochloride (13f)
Yield: 84.9%. Mp 171.1–172.6 ꢁC; ESI-MS m/z [M þ H]þ 349.4; 1H
Yield: 96.9%. Mp 146.6–148.1 ꢁC; ESI-MS m/z [M þ H]þ 350.3; 1H
NMR (300 MHz, DMSO-d6):
d
12.94 (br s, 1H), 9.39 (s, 1H), 9.11 (s,
NMR (300 MHz, DMSO-d6): d 10.62 (s, 1H), 8.86 (s, 1H), 8.24 (s, 1H),
1H), 8.14 (br s, 3H), 7.13 (d, 2H, J ¼ 8.4), 6.72 (d, 2H, J ¼ 8.4), 4.82 (m,
1H), 4.31 (dd, 2H, J ¼ 19.2, 16.8), 4.03 (m, 1H), 3.09 (m, 1H), 2.94 (m,
2H), 2.75 (m, 1H), 1.95 (m, 2H).
7.32 (s, 5H), 4.84 (m, 1H), 4.16 (dd, 2H, J ¼ 20.7, 15.6), 3.21 (m, 1H),
2.78 (m, 2H), 2.72 (m, 1H), 2.00(s, 2H).
5.6.7. (S)-2,6-Diamino-N-((S)-1-(2-(hydroxyamino)-2-oxoethyl)-
2,6-dioxopiperidin-3-yl) hexanamide dihydrochloride (13g)
Yield: 91.1%. Mp 143.5–144.9 ꢁC; ESI-MS m/z [M þ H]þ 330.4; 1H
5.6. General procedure for the preparation of compound 13
To the clear solution of HCl.NH2OH (0.2 g) in 1.5 mL anhydrous
MeOH was added 0.5 mL of Et3N to control the pH value at 7.0. The
carboxyl acid derivatives 10 (2.0 mmol) were dissolved in 20 mL THF
and cooled to ꢃ20 ꢁC. To the mixture was added dropwise 2.0 eq of
Et3N, and then 1.01 eq of isobutyl chloroformate after 5 min. The
mixture was stirred at ꢃ20 ꢁC for another 10 min and the MeOH
solution with NH2OH was added dropwise, after which the mixture
was stirred at ꢃ20 ꢁC for 15 min and then 0 ꢁC for 2 h. The reaction
mixture was filtered with the aid of 2.0 g of Celite. The filtrate was
condensed and added 30 mL of EtOAc and washed in turn with
0.05%NaHCO3, 5% citric acid and brine. The organic phase was dried
over Na2SO4. Remove the solvents to obtain 12 and purified by column.
To the stirring solution of 12 (0.5 g) in 10 mL anhydrous ethyl
acetate was added dropwise 5 mL 3N EtOAc–HCl and after 2–3 h,
target compounds 13 were obtained in the form of hydrochloride
salt. Filter quickly and dry the cake in vacuum to obtain dried white
solid with high yield.
NMR (300 MHz, DMSO-d6):
d
10.66 (s, 1H), 9.09 (d, 1H, J ¼ 8.4), 8.37
(s, 3H), 8.03 (s, 3H), 4.86 (m, 1H), 4.17 (dd, 2H, J ¼ 18.0, 15.9), 3.82
(m, 1H), 2.94 (m, 1H), 2.71 (m, 3H), 2.04 (m, 2H), 1.80 (m, 2H), 1.51
(m, 4H).
5.6.8. (S)-N-((S)-1-(2-(Hydroxyamino)-2-oxoethyl)-2,6-
dioxopiperidin-3-yl) pyrrolidine-2-carboxamide hydrochloride
(13h)
Yield: 45.8%. Mp 138.6–139.4 ꢁC; ESI-MS m/z [M þ H]þ 299.3; 1H
NMR (300 MHz, DMSO-d6): d 10.61 (s, 1H), 9.84 (d, 1H), 9.00 (d, 1H,
J ¼ 8.4), 8.58 (s, 1H), 8.03 (s, 3H), 4.82 (m, 1H), 4.45 (m, 1H), 4.17 (dd,
2H, J ¼ 19.5, 15.9), 3.20 (m, 2H), 2.92 (m, 1H), 2.72 (m, 1H), 2.32 (m,
1H), 1.96 (m, 5H).
5.6.9. (S)-2-Amino-N-((S)-1-(2-(hydroxyamino)-2-oxoethyl)-2,6-
dioxopiperidin-3-yl)-3-(4-hydroxyphenyl) propanamide
hydrochloride (13i)
The following compounds are prepared according to the general
procedure.
Yield: 93.1%. Mp 165.5–167.1 ꢁC; ESI-MS m/z [M þ H]þ 365.4; 1H
NMR (300 MHz, DMSO-d6):
d 10.70 (s, 1H), 9.40 (s, 1H), 9.31(s, 1H),
8.21(s, 3H), 7.06 (d, 2H, J ¼ 8.4), 6.70 (d, 2H, J ¼ 8.4), 4.69 (m, 1H),
5.6.1. (S)-2-Amino-N-(1-(2-(hydroxyamino)-2-oxoethyl)-2,6-
dioxopiperidin-3-yl)acetamide hydrochloride (13a)
3.97 (m, 3H), 3.02 (m, 3H), 2.49 (m, 1H), 1.91 (m, 2H).
Yield: 56.6%. Mp 121.6–123.2 ꢁC; ESI-MS m/z [M þ H]þ 259.2; 1H
5.7. Biological materials and methods
NMR (300 MHz, DMSO-d6):
d 10.61 (s, 1H), 8.90 (d, 1H), 8.12 (br s,
3H), 4.82 (m, 1H), 4.16 (dd, 2H, J ¼ 20.4, 17.1), 3.60 (m, 2H), 2.92 (m,
5.7.1. Materials
1H), 2.70 (m, 1H), 2.00 (m, 2H).
5.7.1.1. Chemicals. Chemicals was synthesized as mentioned above
and dissolved in dimethylsulfoxide (DMSO) or PBS and diluted to
the required concentration with culture medium when used.
5.6.2. (S)-2-Amino-N-((S)-1-(2-(hydroxyamino)-2-oxoethyl)-2,6-
dioxopiperidin-3-yl)propanamide hydrochloride (13b)
Yield: 82.0%. Mp 154.3–155.1 ꢁC; ESI-MS m/z [M þ H]þ273.3; 1H
5.7.1.2. Cell line and cell culture. The human leukemia cell line, HL-
60 and K562 were obtained from the Institute of National Cancer
Research of China (Beijing, China). Cells were maintained in RPMI-
1640 supplemented with 10% (v/v) heat-inactivated fetal bovine
NMR (300 MHz, DMSO-d6): d 10.61 (s, 1H), 8.88 (s, 1H), 8.22 (s, 3H),
4.83 (m, 1H), 4.20 (m, 2H), 3.87 (m, 1H), 2.95 (m, 1H), 2.74 (m, 1H),
1.94 (m, 2H), 1.40 (d, 3H).
serum, penicillin–streptomycin (100 IU/ml–100
mg/mL), 2 mM
5.6.3. (S)-2-Amino-N-((S)-1-(2-(hydroxyamino)-2-oxoethyl)-2,6-
dioxopiperidin-3-yl)-3-methylbutanamide hydrochloride (13c)
Yield: 67.3%. Mp 165.6–167.5 ꢁC; ESI-MS m/z [M þ H]þ 301.3; 1H
glutamine, and 10 mM Hepes buffer at 37 ꢁC in a humid atmosphere
(5% CO2–95% air). Cell viability was measured by 3-[4,5-dime-
thylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT, Sigma,
USA) assay as described elsewhere.
NMR (300 MHz, DMSO-d6): d 9.90 (s, 1H), 8.89 (br s, 1H), 8.21 (br s,
3H), 4.88 (m, 1H), 4.63 (m, 1H), 4.15 (dd, 2H, J ¼ 18.9, 15.9), 2.93 (m,
1H), 2.74 (m, 1H), 2.08 (m, 2H), 1.02 (dd, 6H).
5.7.2. In vitro APN enzyme assay [17]
IC50 values against APN were determined as previously
5.6.4. (S)-2-Amino-N-((S)-1-(2-(hydroxyamino)-2-oxoethyl)-2,6-
dioxopiperidin-3-yl)-4-methylpentanamide hydrochloride (13d)
Yield: 79.8%. Mp 176.3–176.9 ꢁC; ESI-MS m/z [M þ H]þ 315.3; 1H
described and using L-Leu-p-nitroanilide as substrate and micro-
somal aminopeptidase from porcine kidney microsomes (Sigma) as
the enzyme in 50 mM PBS, pH 7.2, at 37 ꢁC. The hydrolysis of the
substrate was monitored by following the change in the absorbance
measured at 405 nm with the UV–vis spectrophotometer Phar-
macia LKB, Biochrom 4060. All inhibitors were preincubated with
NMR (300 MHz, DMSO-d6): d 8.89 (br s, 1H), 8.25 (br s, 3H), 4.87 (br
s, 1H), 4.62 (m, 1H), 4.41 (dd, 2H), 3.87 (m, 1H), 3.01 (m, 1H), 2.73
(m, 1H), 2.00(s, 2H), 1.78 (m, 1H), 1.61 (m, 2H), 0.88 (d, 6H).

1626
Q. Li et al. / European Journal of Medicinal Chemistry 45 (2010) 1618–1626
APN for 30 min at room temperature. The assay mixture, which
contained the inhibitor solution (concentration dependent on the
day later, the metastasized nodes on the surface of lungs were
counted.
inhibitor), the enzyme solution (4
mg/mL final concentration), and
the assay buffer, was adjusted to 200
mL.
Acknowledgements
5.7.3. In vitro cytostatic activity [18]
We thank Prof. Xianjun Qu and his graduate students (Institute of
Pharmacology, School of Pharmacy, Shandong University) for in vivo
compounds screenings. We would also like to thank Dr. Philip
Mosier (Department of Medicinal Chemistry, Virginia Common-
wealth University) for docking software GOLD4.0 and training. This
workwas supported byNational NatureScience Foundation of China
(Grant No. 30772654; 36072541) and the Ph.D. Programs Founda-
tion of Ministry of Education of P.R. China (No. 20060422029) and
National High Technology Research and Development Program of
China (863 project; Grant No 2007AA02Z314).
The inhibitors were dissolved in culture medium and diluted to
the required concentration, and the in vitro cytostatic activity was
evaluated by MTT assay. Briefly, the human leukemia cell line HL-60
was maintained in RPMI-1640 medium supplemented with 10%
fetal bovine serum and incubated at 37 ꢁC in a CO2 incubator. Cells
were seeded on a 96-well plate (104 cells per well). After 4 h
incubation, inhibitors were then added to the wells to achieve final
concentration of 800, 600, 400, 200 and 100 mg/mL Control wells
were prepared by addition of culture medium. At same time, the
bestatin (Apeloa Pharma/Kangyu Pharma, China) was used as
positive control. The plate was incubated for 48 h. Upon completion
of the incubation, 1% of 0.5 mg/mL MTT was added to each well and
incubated for an additional 4 h. After centrifugalization, medium
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