Novel cyclic-imide peptidomimetics as aminopeptidase N inhibitors. Design, chemistry and activity evaluation. Part I

Article abstract of DOI:10.1016/j.ejmech.2009.07.022

A series of aminopeptidase inhibitors with cyclic-imide scaffold are described. The biological characterization for the piperidinedione analogues revealed that most compounds displayed high inhibitory activity against APN. Among which 4l and 6 showed pote

Full text of DOI:10.1016/j.ejmech.2009.07.022

European Journal of Medicinal Chemistry 44 (2009) 4819–4825
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. Design,
chemistry and activity evaluation. Part I
,
Qianbin Li a, Hao Fang a, Xuejian Wang a, Liping Hu b, Wenfang Xu a
*
a Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, 44 West Wenhua Road, 250012 Ji’nan, Shandong, PR China
b Department of Internal Neurology, Qilu Hospital of Shandong University, 107 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 series of aminopeptidase inhibitors with cyclic-imide scaffold are described. The biological charac-
terization for the piperidinedione analogues revealed that most compounds displayed high inhibitory
activity against APN. Among which 4l and 6 showed potent inhibition against APN with the IC50 value of
Received 23 January 2009
Received in revised form
20 July 2009
Accepted 23 July 2009
Available online 30 July 2009
5.2 mM and 3.1 mM, respectively. In addition, 6 also displayed good activity in HL-60 cell assay and in vivo
anti-metastasis assay. This interesting activity profile may also guide the design of new, specific inhib-
itors of target mammalian aminopeptidases with ‘one-zinc’ active site.
Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords:
Aminopeptidase N
Cyclic-imide
Peptidomimetics
Anti-tumor
1. Introduction
domain of APN based on the co-crystal complex of Escherichia coli
APN and bestatin [8,10]. The binding site of the APN with bestatin
Aminopeptidase N (APN, EC3.4.11.2, CD13), an ectopeptidase
expressed widely by monocytes, myeloid, epithelial cells of the
intestine and kidney, fibroblasts, endothelial cells and tumor cells,
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
metastasis and leukemia [1–4]. The design and synthesis of inhib-
itors for aminopeptidase N may result in potential therapeutic
agents.
Since 1976, several excellent reviews on natural and synthetic
small molecule inhibitors of APN have been published [5,6]. Among
these inhibitors, the best well known is bestatin, which was iso-
lated by Umezawa et al. in a search for low molecular weight
inhibitors for hydrolytic enzymes on the surfaces of cells [7].
In recent years, the availability of X-ray crystal structures for
APN and complexes with various inhibitors has been investigated
[8–10]. Our group had reported some new APN inhibitors, such as
has several characteristics: part I is a hydrophobic pocket inter-
acting with phenyl group of bestatin; part II is one zinc ion which
can interact with the zinc binding group in the structure of inhib-
itors (for bestatin, they are 2-hydroxyl group and 1-carbonyl group
of amide); part III is another hydrophobic pocket in deeper cavity
which can be divided into two subsites [5,11,16].
In our previous work, we have reported a series of L-iso-gluta-
mine derivatives [12]. The biological characterization revealed that
most compounds displayed good inhibitory activity against APN.
Herein, in order to find better APN inhibitors, we modified the
structure as the following: the g-carboxyl group and the nitrogen of
the amide were cyclized to explore new scaffold of cyclic-imide; the
R2 group can be OH, OCH3 and NHOH or some amino acid deriva-
tives to form tri-peptidomimetics, Fig. 1. To improve the bioactivity,
the gallic acid moiety was kept because of its anti-tumor and anti-
oxidative activities [17,18] and R1 group can be either H or CH3. The
in vitro inhibitory activity was measured against APN enzyme.
Additionally, potent compounds are also evaluated on HL-60 cell
line and in vivo anti-metastasis assay.
L-lysine derivates [11], L-iso-glutamine derivatives [12], AHPA
(b-amino-a-hydroxyl-phenylbutanoic acid) derivates [13] and
3-phenylpropane-1,2-diamine derivates [14,15]. Both Yoshimoto’s
and Matthews’s groups had reported the binding site and catalytic
2. Chemistry
The critical intermediate 3 was synthesized following the
procedures as shown in Scheme 1. The starting material
(S)-2-(3,4,5-trimethoxybenzamido)pentanedioic acid (1) was
* 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 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.07.022

4820
Q. Li et al. / European Journal of Medicinal Chemistry 44 (2009) 4819–4825
O
O
O
R1O
R1O
HO
HO
N
H3CO
H3CO
Part A
OH or OCH3
N
H
O
N
H
O
H
O
a
N
O
O
R2
COOH
R
N
O
O
N
O
O
OR1
O
Part B
OH
OCH3
H3CO
H3CO
N
R
R
H
O
COOCH3
side chain of
various amino acids
N
H
3a R=OH
R=NHCH2COOH
3
R=OH
4a R=NHCH2COOCH3
OCH3
R'
7a
OH, OCH3, NHOH
7b R=NHCH(CH2CH(CH3)2)COOH
4b R=NHCH(CH2CH(CH3)2)COOCH3
7c
Fig. 1. Strategy for the design of cyclic-imide derivatives.
R=NHCH(CH2Ph)COOH
4c
R=NHCH(CH2Ph)COOCH3
6a
R=NHOH
6 R=NHOH
Scheme 2. a. (i) BBr3, DCM, ꢂ78 ꢀC; (ii) H2O.
prepared from
carboxyl groups of
L
-Glutamine following Reference [12]. The two
-glutamine were cyclized with acetic acid
L
anhydride as dehydration reagent at 55–60 ꢀC to afford
compound 2. Compound 3 was obtained using one-pot reaction
from compound 2 and glycine, during which process microwave
heating was applied. All the target compounds 4a–r were
obtained in good yield by condensation of carboxyl group of 3 and
appropriate amino acid derivatives with EDCI as the coupling
reagent.
Additionally, the carboxyl group of compound 3 was converted
to its methyl ester in the existence of SOCl2 and CH3OH to afford
compound 5 and can also be activated by isobutyl chloroformate
and N-methylmorpholine and then coupled with NH2OH to yield its
hydroxamate acid derivatives.
and 6a appeared the most tightly bound with the IC50 values an
order of magnitude lower than the ones calculated for counterpart
acid 3 and ester 5 (3.1 mM and 4.3 mM vs. 50–60 mM). The deriva-
tives extended with an additional amino ester residue showed
further interesting structure–activity relationships. The presence of
an aliphatic side chain portion was not yet discriminating for the
efficiency of studied compounds. The analogues of Val, Ala, Leu and
Ile (4a–f) displayed similar to each other, moderate potency (IC50 in
the range of 20–50
mM), only slightly improved in comparison to
non-substituted Gly 4a (IC50 ¼ 55.4
m
M). Introduction of residues
containing heteroatoms (for example: Arg, Asp, Lys, Cys, etc.) did
not change the situation. However, arylalkyl Phe analogue exhibi-
In addition, as shown in Scheme 2, the methyl group of 3,4,5-
trimethoxybenzoic acid part was deprotected to free the phenyl
group with BBr3 as reagent to study the interaction between free
gallic acid with the active site of enzyme.
ted enhanced activity, with 5-fold preference for natural
L
isomer
(IC50 ¼ 11.0
mM vs. 46.2 mM for the D form). Finally, the most
Table 1
3. Result and discussion
In vitro enzyme assay results for compounds 3–7 and bestatin against APN.
O
The preliminary pharmacological studies of all the target
compounds have been investigated on enzymatic inhibition of APN
and the results are listed in Table 1. Compared with those
compounds in the previous work, preliminary result showed that
most of target compounds displayed improved inhibitory activity
than those reported before [12].
R1O
N
H
R1O
O
N
O
O
OR1
R2
As shown in the results, the activity of piperidinedione inhibi-
tors toward APN was strongly dependent on the nature of the
moiety present at R2. Among simpler carboxylate analogues 3, 5
and 6 (R2 ¼ OH, OMe or NHOH, respectively), hydroxamic acids 6
No
Substituents
R1
IC50
APN
/m
Ma
R2
3
CH3
H
OH
OH
Gly–OCH3
Val–OCH3
Leu–OCH3
Ile–OCH3
49.8 ꢃ 5.5
47.3 ꢃ 6.7
55.4 ꢃ 3.5
21.4 ꢃ 2.6
46.2 ꢃ 3.2
49.3 ꢃ 2.9
53.7 ꢃ 8.4
18.9 ꢃ 4.4
42.0 ꢃ 7.6
20.7 ꢃ 2.7
38.1 ꢃ 3.8
44.8 ꢃ 5.2
43.1 ꢃ 3.2
5.2 ꢃ 2.1
3a
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
O
O
H3CO
H3CO
H3CO
COOH
COOH
N
H
O
N
H
a
H3CO
O
O
b-Ala–OCH3
OCH3
Ala–OCH3
Arg (NO2)–OCH3
Met–OCH3
Lys (Z)–OCH3
Tyr–OCH3
Trp–OCH3
OCH3
1
2
O
O
H3CO
H3CO
H3CO
N
N
H
O
c
H
O
b
N
O
His–OCH3
H3CO
N
O
O
OCH3
O
R
D
-Phe–OCH3
Thr–OCH3
-Phe–OCH3
46.2 ꢃ 6.4
51.9 ꢃ 3.9
11.0 ꢃ 3.0
50.3 ꢃ 4.3
50.0 ꢃ 4.2
21.7 ꢃ 2.1
63.4 ꢃ 3.2
3.1 ꢃ 0.7
OCH3
3
4a-r HN
OH
L
d
e
Cys–OCH3
2-Cl-Ala–OCH3
Asp–(OCH3)2
–OCH3
–NHOH
–NHOH
Gly–OH
Leu–OH
L-Phe–OH
O
O
H3CO
H3CO
N
H
H3CO
H3CO
N
5
6
H
O
O
N
O
O
N
O
O
OCH3
OCH3
6a
7a
7b
7c
Bestatin
4.3 ꢃ 0.4
HN
H
H
H
63.2 ꢃ 1.3
55.4 ꢃ 2.7
10.3 ꢃ 1.4
2.4 ꢃ 0.5
6
O
OH
5
Scheme 1. a. Ac2O, 55–60 ꢀC, 60%; b. Glycine, DMF, Microwave, 110 ꢀC, 1 h, 57%; c.
Methyl ester of amino acids, EDCI, HOBt, DCM/DMSO, 56–85%; d. SOCl2, CH3OH, 98%; e.
Et3N, isobutyl chloroformate, NH2OHꢁHCl, 60%.
a
Values are means of three experiments, standard deviation is given.

Q. Li et al. / European Journal of Medicinal Chemistry 44 (2009) 4819–4825
4821
Fig. 2. Docking modes for 4l (a) and 6 (b) with APN (PDB code: 2DQM).
favorable appeared heteroaromatic system of histidine derived
compound 4l. The latter one (IC50 ¼ 5.2 M) together with
hydroxamate displayed similar efficiency to bestatin
(IC50 ¼ 2.4 M). Interestingly, neither deprotection of the methoxy
sequence (HEXXHX18E) in the catalytic domain that is well
conserved in peptidase M1 family.
To investigate the biological function of target compounds, cells
cytostatic activity assay was carried out in human leukemia HL-60
cell lines, which highly express APN, using MTT assay (see Experi-
mental section). The concentrations inducing a 50% inhibition of
m
6
m
groups in the gallic acid part, nor their simultaneous removal
together with this at the C-termini, caused any significant change in
activity in comparison to blocked counterparts (compare 3 vs. 3a, 6
vs. 6a, 4a vs. 7a, etc.).
cell growth (IC50) in
mM are reported. The data indicated that
several compounds have the cytostatic activity, among which 4g
and 6 displayed potent inhibitory activity, Fig. 3, which are more
effective than the positive control (the IC50 for bestatin was
In order to obtain further insight into the interaction of target
compound with APN, the most active compounds 4l and 6 were
built and docked into the active site of APN (PDB code: 2DQM)
using Gold4.0. The binding studies showed that the aromatic ring of
245 mM). Although the purpose of our research is to design and
synthesize enzyme inhibitors, interestingly there were still several
other compounds induce the growth of HL-60 cells (results will be
published later).
To evaluate the ability of target compounds to inhibit the
metastasis of cancer cells in vivo, anti-metastasis assay of H22
hepatic cells was carried out. The results are shown in Fig. 4.
Compound 6 displayed higher inhibitory activity (P < 0.001) than
that of bestatin with the inhibitory rate of 81.56% and 60.25%,
respectively, which means that it is of great value for further
preclinical research.
gallic acid for part A is 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 oxygen atoms from trimethoxyl group
interact with Arg825, Gln821 and Asn373. Two carbonyl groups
(C]O from C-2 on the cyclic-imide ring and gallic acid) bind to the
zinc ion and form a five-membered ring in the active pocket with
distance of 2.45 Å and 2.04 Å, respectively. In addition, the C-2
carbonyl group also interacts with the hydroxyl group of Tyr381 by
hydrogen bond which would be benefit to stabilize of the reaction
intermediate with the zinc ion. As for part B, Tyr275, Arg293 and
Asp327 in the active site form hydrogen bond with either 4l (the
methyl ester part, Fig. 2a) or 6 (hydroxamate acid group, Fig. 2b).
Additionally, NHOH of 6 can form hydrogen bond interaction with
His297 (2.03 Å) that is the essential amino acids of the conserved
Fig. 4. In vivo anti-metastasis assay of H22 hepatic cells. (The difference for the
number of metastasized nodes on lung surface between drug groups and negative
control group is analyzed using one-way ANOVA method. **P < 0.01 or ***P < 0.001
indicates significant differences from control group.)
Fig. 3. In vitro cytostatic activity of the compound in HL-60 cells.

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Q. Li et al. / European Journal of Medicinal Chemistry 44 (2009) 4819–4825
4. Conclusion
(3-dimethylaminopropyl)carbodiimide
hydrochloride
(EDCI)
(1.5 equiv) at 0 ꢀC. To the clear solution were added amino acid
derivatives and Et3N (3.5 equiv) and stirred for 10–15 h at 30 ꢀC. The
mixturewasmonitoredbyTLC.Thereactionmixturewasdilutedwith
DCM, washed successively with 0.5% Na2CO3, 1 N HCl and brine, and
dried over anhydrous Na2SO4. Filtration and concentration in vacuum
gave 4 as a crude solid and then purified by column chromatography.
In summary, our studies have shown that cyclic-imide is a novel
scaffold for the design of aminopeptidase N inhibitors. Most of the
compounds possess potent APN inhibitory activity and the most
potent compounds, 4l and 6, exhibited good enzymatic inhibition
against APN. With the combination of the requirement of the active
siteofAPN,thisinterestingactivityprofilemayalsoguidethedesignof
new, specific inhibitors of aminopeptidases with ‘one-zinc’ active site.
5.1.4.1. (S)-Methyl 2-(2-(2,6-dioxo-3-(3,4,5-trimethoxybenzamido)
piperidin-1-yl)acetamido)acetate (4a). The compound was
prepared from 3 (1.9 g, 5.0 mmol) and (S)-methyl 2-aminoacetate
hydrochloride (0.75 g, 6.0 mmol) according to the general
procedure to yield 4a as white solid (1.41 g, 62.7%): mp 95.7–
5. Experimental
5.1. Chemistry
100.6 ꢀC, 1H NMR (400 MHz, DMSO-d6):
d 7.05 (s, 2H), 4.84 (m,
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
precoated silica gel plates, and the resulting chromatograms were
visualized under UV light at 254 nm. Proton NMR spectra were
recorded on a Bruker DRX spectrometer operating at 400 MHz.
Measurements were made in CDCl3 or DMSO-d6 solutions. Proton
chemical shifts are reported in relation to tetramethylsilane (TMS)
1H), 4.37 (dd, 2H, J ¼ 25.5, 15.6), 4.06 (s, 2H,), 3.91 (s, 6H), 3.89 (s,
3H), 3.77 (s, 3H), 2.93 (m, 2H), 2.63 (m, 1H), 2.07 (m, 1H); ESI-MS
m/z [M þ H]þ 452.5.
5.1.4.2. (S)-Methyl 2-(2-((S)-2,6-dioxo-3-(3,4,5-trimethoxybenzamido)-
piperidin-1-yl)acetamido)-3-methylbutanoate (4b). The compound
was prepared from 3 (0.76 g, 2.0 mmol) and (S)-methyl 2-amino-3-
methylbutanoate hydrochloride (0.40 g, 2.38 mmol) according to the
general procedure to yield 4b as white solid (0.75 g, 79.8%): mp 110.5–
112.0 ꢀC, 1H NMR: (400 MHz, DMSO-d6):
d
8.81 (d, 1H, J ¼ 8.3 Hz), 8.22
used as internal standard with the unit of
d
in ppm and J in Hertz.
(d, 1H, J ¼ 8.0 Hz,), 7.210 (s, 2H), 4.93 (m, 1H), 4.34 (s, 2H), 4.16 (s, 1H),
3.83 (s, 6H), 3.71 (s, 3H), 3.67 (s, 3H), 3.30 (s, 3H), 3.09 (m,1H), 2.75 (m,
2H), 2.03 (m, 2H), 0.86 (d, 6H); ESI-MS m/z [M þ H]þ 494.5.
ESI-MS were measured on an API 4000 spectrometer.
5.1.1. (S)-2-(3,4,5-Trimethoxybenzamido)pentanedioic acid (1) [12]
The key intermediate N-(3,4,5-trimethoxybenzoyl)-glutamicacid
anhydride (2) was prepared starting from 3,4,5-trihydroxybenzoic
acid via a three-step reaction according to the literature.
5.1.4.3. (S)-Methyl 2-(2-((S)-2,6-dioxo-3-(3,4,5-trimethoxybenzamido)-
piperidin-1-yl)acetamido)-4-methylpentanoate (4c). The compound
was prepared from 3 (1.9 g, 5.0 mmol) and (S)-methyl 2-amino-4-
methylpentanoate hydrochloride (1.01 g, 5.6 mmol) according to the
general procedure to yield 4c as white solid (1.72 g, 67.8%): mp 85.3–
19.7 g (0.105 mol) of glutamic acid and 19.2 g (0.181 mol) of
sodium carbonate were dissolved in 150 ml water. The mixture is
cooled to 0–2 ꢀC on ice–water bath and to the mechanically stirred
solution was added dropwise 0.1 mol 3,4,5-trimethoxybenzoyl
chloride in 150 ml anhydrous benzene over 2 h and kept stirring for
about 5 h. The aqueous phase was acidified by concentrated
hydrochloride to pH 4 and extracted by 100 ml of chloroform. The
aqueous phase was acidified further and the product precipitated in
a great quantity. Filter and wash to neutrality with ice–water. The
crude product was recrystallized in EtOAc to obtain 23.7 g (yield
69.5%) white crystal.
87.4 ꢀC, 1H NMR (400 MHz, DMSO-d6):
d 8.82 (d, 1H), 8.40 (d, 1H), 7.22
(s, 2H), 4.91 (m, 1H), 4.30 (m, 3H), 3.83 (s, 6H), 3.71 (s, 3H), 3.61 (s, 3H),
3.09 (m, 1H), 3.03 (m, 1H), 2.14 (m, 1H), 2.03 (m, 1H), 1.86 (m, 1H), 1.60
(m, 2H), 0.85 (dd, 6H); ESI-MS m/z [M þ H]þ 508.5.
5.1.4.4. (2S)-Methyl 2-(2-((S)-2,6-dioxo-3-(3,4,5-trimethoxybenza-
mido)piperidin-1-yl)acetamido)-3-methylpentanoate
(4d). The
compound was prepared from 3 (0.76 g, 2.0 mmol) and (S)-methyl
2-amino-3-methylpentanoate hydrochloride (0.73 g, 4.0 mmol)
according to the general procedure to yield 4d as white solid
(0.75 g, 79.8%): mp 84.0–85.7 ꢀC, 1H NMR (400 MHz, DMSO-d6):
5.1.2. (S)-N-(2,6-Dioxo-tetrahydro-2H-pyran-3-yl)-3,4,5-
trimethoxybenzamide (2)
d
7.05 (s, 2H), 4.81 (m, 1H), 4.56 (s, 3H), 3.91 (s, 6H), 3.89 (s, 3H),
Compound 1 (11.4 g, 33.4 mmol) was suspended in 80 ml Ac2O.
After stirring for 6 h at 55–60 ꢀC, the resulting mixture was filtered,
and to the filtrate was added equal volume of ethyl ether:petroleum
ether (2:1) and cool in fridge overnight. 5.6 g (yield 52%) white
crystal solid was obtained by filtrating.
3.74 (s, 3H), 2.88 (m, 3H), 2.64 (m, 1H), 2.04 (m, 1H), 1.90 (m, 1H),
1.43 (m,1H),1.23 (m,1H), 0.91 (m, 6H); ESI-MS m/z [M þ H]þ 508.5.
5.1.4.5. (S)-Methyl 3-(2-(2,6-dioxo-3-(3,4,5-trimethoxybenzamido)-
piperidin-1-yl)acetamido)propanoate (4e). The compound was
prepared from 3 (0.76 g, 2.0 mmol) and (S)-methyl 3-amino-
propanoate hydrochloride (0.56 g, 4.0 mmol) according to the
general procedure to yield 4e as white solid (0.80 g, 87.6%): mp
5.1.3. (S)-2-(2,6-Dioxo-3-(3,4,5-trimethoxybenzamido)piperidin-1-
yl)acetic acid (3)
8.7 g (27 mmol) of compound 2 and 2.03 g (27 mmol) of glycine
weresuspendedin70 mlDMF. Themixturewasheatedbymicrowave
at1000 W,110 ꢀC forabout 3 h. Coolthemixturetoroom temperature
andequalvolumeofwaterwasadded,thesolutionwasacidifiedtopH
3 and cooled in fridge overnight. 5.7 g (yield 55.7%) of white solid
were obtained. mp 246.5 ꢀC (decomp).1H NMR (400 MHz, DMSO-d6):
139.3–140.5 ꢀC, 1H NMR (400 MHz, DMSO-d6):
d 8.82 (d, 1H,
J ¼ 8.3 Hz), 8.16 (d, 1H, J ¼ 8.0 Hz), 7.22 (s, 2H), 4.93 (m, 1H), 4.23 (s,
2H), 3.84 (s, 6H), 3.72 (s, 3H), 3.60 (s, 3H), 3.29 (m, 2H), 2.96 (m,
1H), 2.75 (m, 1H), 2.45 (t, 2H), 2.24 (m, 1H), 2.03 (m, 1H); ESI-MS
m/z [M þ H]þ 466.6.
d
12.34 (s,1H), 8.88 (d,1H, J ¼ 8.3 Hz), 7.17 (s, 2H), 4.53 (m,1H), 4.42 (s,
5.1.4.6. (S)-Methyl 2-(2-((S)-2,6-dioxo-3-(3,4,5-trimethoxybenzamido)-
piperidin-1-yl)acetamido)propanoate (4f). The compound was
prepared from 3 (1.9 g, 5.0 mmol) and (S)-methyl 2-aminopropanoate
hydrochloride (0.84 g, 6 mmol) according to the general procedure to
yield 4f as white solid (1.65 g, 70.9%): mp 98.8–101.6 ꢀC, 1H NMR
2H), 3.83 (s, 6H), 3.71 (s, 3H), 2.18 (m, 2H), 2.11 (m, 2H).
5.1.4. General procedure for the preparation of 4a–r
To a mixture of 3 and 1-hydroxy-1H-benzotriazole (HOBt)
(1.5 equiv) in DCM (20 ml) and DMSO (3 ml) was added 1-ethyl-3-
(400 MHz, DMSO-d6): d 8.84 (d, 1H), 8.49 (d, 1H), 7.22 (s, 2H), 4.93 (m,

Q. Li et al. / European Journal of Medicinal Chemistry 44 (2009) 4819–4825
4823
1H), 4.29 (m, 3H), 3.83 (s, 6H), 3.71 (s, 3H), 3.60 (s, 3H), 2.95 (m, 1H),
2.75 (m, 1H), 2.17 (m, 1H), 1.98 (m, 1H), 1.27 (d, 3H); ESI-MS m/z
[M þ H]þ 466.4.
methyl 2-amino-3-(1H-imidazol-4-yl)propanoate hydrochloride
(0.97 g, 4.0 mmol) according to the general procedure to yield 4l as
white solid (0.70 g, 65.9%): mp 111.7–113.5 ꢀC, 1H NMR (400 MHz,
DMSO-d6):
d
13.4 (d, 1H), 8.84 (d,1H, J ¼ 8.3 Hz), 8.73 (d, 1H), 8.32 (d,
5.1.4.7. (S)-Methyl 2-(2-((S)-2,6-dioxo-3-(3,4,5-trimethoxybenzamido)-
piperidin-1-yl)acetamido)-5-(3-nitroguanidino)pentanoate (4g). The
compound was prepared from 3 (0.76 g, 2.0 mmol) and (S)-methyl 2-
1H, J ¼ 8.0 Hz), 7.66 (s,1H), 7.23 (s, 2H), 4.96 (m,1H), 4.52 (m,1H), 4.34
(s, 2H), 3.83 (s, 6H), 3.71 (s, 3H), 3.63 (s, 3H), 3.20 (m, 3H), 2.76 (m,1H),
2.14 (m, 1H), 2.03 (m, 1H); ESI-MS m/z [M þ H]þ532.6.
amino-5-(3-nitroguanidino)pentanoate
hydrochloride
(0.81 g,
3.0 mmol) according to the general procedure to yield 4g as white
5.1.4.13. (R)-Methyl 2-(2-((S)-2,6-dioxo-3-(3,4,5-trimethoxybenz-
amido)piperidin-1-yl)acetamido)-3-phenylpropanoate (4m). The
compound was prepared from 3 (0.76 g, 2.0 mmol) and (R)-methyl
2-amino-3-phenylpropanoate hydrochloride (0.86 g, 3.0 mmol)
according to the general procedure to yield 4m as white solid
solid (0.79 g, 66.3%): mp 53.9–55.1 ꢀC, 1H NMR (400 MHz, DMSO-d6):
d
8.82 (d, 1H, J ¼ 8.3 Hz), 8.47 (d, 1H, J ¼ 8.0 Hz), 7.83 (d, 1H), 7.22 (s,
2H), 4.92 (m, 1H), 4.32 (s, 2H), 4.27 (s, 1H,), 3.83 (s, 6H), 3.71 (s, 3H),
3.69 (s, 3H), 3.14 (m, 2H), 2.93 (m,1H), 2.75 (m,1H), 2.186 (m,1H), 2.03
(m, 1H), 1.62 (m, 4H); ESI-MS m/z [M þ H]þ596.6.
(0.74 g, 68.3%): mp 69.3–71.2 ꢀC, 1H NMR (400 MHz, CDCl3):
d 7.25
(m, 7H), 4.82 (m, 2H), 4.48 (dd, 2H) 3.90 (s, 6H), 3.87 (s, 3H), 3.73
(s, 3H), 3.14 (d, 2H), 2.94 (m, 2H), 2.62 (m, 1H), 2.03 (m, 1H); ESI-
MS m/z [M þ H]þ542.6.
5.1.4.8. (S)-Methyl 2-(2-((S)-2,6-dioxo-3-(3,4,5-trimethoxybenzamido)-
piperidin-1-yl)acetamido)-4-(methylthio)butanoate
compound was prepared from 3 (0.76 g, 2.0 mmol) and (S)-methyl
2-amino-4-(methylthio)butanoate hydrochloride (0.80 g,
(4h). The
5.1.4.14. (S)-Methyl 2-(2-((S)-2,6-dioxo-3-(3,4,5-trimethoxybenz-
amido)piperidin-1-yl)acetamido)-3-hydroxypropanoate (4n). The
compound was prepared from 3 (0.76 g, 2.0 mmol) and (S)-
methyl 2-amino-3-hydroxypropanoate hydrochloride (0.62 g,
4.0 mmol) according to the general procedure to yield 4n as white
solid (0.59 g, 61.3%): mp 158.9–160.1 ꢀC, 1H NMR (400 MHz,
4.0 mmol) according to the general procedure to yield 4h as white
solid (0.67 g, 63.7%): mp 91.4–93.8 ꢀC, 1H NMR (400 MHz, DMSO-
d6):
d
8.82 (d, 1H, J ¼ 8.3 Hz), 8.15 (d, 1H, J ¼ 8.0 Hz), 7.23 (s, 2H),
4.93 (m, 1H), 4.39 (m, 1H), 4.31 (s, 2H), 3.83 (s, 6H), 3.71 (s, 3H), 3.63
(s, 3H), 2.95 (m, 1H), 2.78 (m, 1H), 2.46 (m, 2H), 2.11 (m, 1H), 2.02 (s,
3H), 1.93 (m, 3H); ESI-MS m/z [M þ H]þ526.6.
CDCl3):
d
8.82 (d, 1H, J ¼ 8.3 Hz), 8.43 (d, 1H, J ¼ 8.0 Hz), 7.23 (s,
2H), 5.08 (m, 1H), 4.92 (m, 1H), 4.35 (m, 2H), 3.83 (s, 6H), 3.71 (s,
3H), 3.67 (m, 1H), 3.63 (s, 3H), 3.60 (m, 1H), 2.96 (m, 1H), 2.78 (m,
1H), 2.17 (m, 1H), 2.03 (m, 1H); ESI-MS m/z [M þ H]þ482.6.
5.1.4.9. (S)-Methyl 6-(benzyloxycarbonylamino)-2-(2-((S)-2,6-dioxo-
3-(3,4,5-trimethoxybenzamido)piperidin-1-yl)acetamido)hexanoate
(4i). The compound was prepared from 3 (0.76 g, 2.0 mmol)
and (S)-methyl 2-amino-6-(benzyloxycarbonylamino)hexanoate
hydrochloride (0.80 g, 4.0 mmol) according to the general proce-
dure to yield 4i as white solid (0.89 g, 67.8%): mp 79.8–82.3 ꢀC, 1H
5.1.4.15. (S)-Methyl 2-(2-((S)-2,6-dioxo-3-(3,4,5-trimethoxybenz-
amido)piperidin-1-yl)acetamido)-3-phenylpropanoate
(4o). The
compound was prepared from 3 (1.9 g, 5.0 mmol) and (S)-methyl
2-amino-3-phenylpropanoate hydrochloride (1.30 g, 6 mmol)
according to the general procedure to yield 4o as white solid
NMR (400 MHz, DMSO-d6):
d
8.81 (d, 1H), 8.15 (d, 1H, J ¼ 8.0 Hz),
7.29 (m, 5H), 7.22 (s, 2H), 5.05 (s, 2H), 4.99 (s, 2H), 4.95 (s, 1H), 4.31
(dd, 2H,), 4.21 (m,1H), 3.83 (s, 6H), 3.71 (s, 3H), 3.61 (s, 3H), 2.98 (m,
3H), 2.79 (m, 1H), 2.15 (m, 1H), 2.03 (m, 1H), 1.81 (m, 2H), 1.40–1.36
(m, 2H), 1.27 (m, 2H); ESI-MS m/z [M þ H]þ657.6.
(2.6 g, 96.0%): mp 68.5–70.6 ꢀC, 1H NMR (400 MHz, CDCl3):
d 7.25
(m, 7H), 4.81 (m, 2H), 4.48 (dd, 2H) 3.90 (s, 6H), 3.87 (s, 3H), 3.73
(s, 3H), 3.14 (d, 2H), 2.95 (m, 2H), 2.63 (m, 1H), 2.03 (m, 1H); ESI-
MS m/z [M þ H]þ542.6.
5.1.4.10. (S)-Methyl 2-(2-((S)-2,6-dioxo-3-(3,4,5-trimethoxybenz-
amido)piperidin-1-yl)acetamido)-3-(4-hydroxyphenyl)propanoate
(4j). The compound was prepared from 3 (0.76 g, 2.0 mmol) and
(S)-methyl 2-amino-3-(4-hydroxyphenyl)propanoate hydrochlo-
ride (0.92 g, 4.0 mmol) according to the general procedure to yield
4j as white solid (0.72 g, 64.6%): mp 95.6–97.8 ꢀC, 1H NMR
5.1.4.16. (S)-Methyl 2-(2-((S)-2,6-dioxo-3-(3,4,5-trimethoxybenz-
amido)piperidin-1-yl)acetamido)-3-mercaptopropanoate (4p). The
compound was prepared from 3 (0.76 g, 2.0 mmol) and (S)-methyl
2-amino-3-mercaptopropanoate hydrochloride (0.69 g, 4 mmol)
according to the general procedure to yield 4p as white solid
(400 MHz, DMSO-d6):
d
9.20 (s, 1H), 8.81 (d, 1H, J ¼ 8.3 Hz), 8.41 (d,
(0.53 g, 53.3%): mp 88.5–90.0 ꢀC, 1H NMR (400 MHz, CDCl3):
d 8.83
1H, J ¼ 8.0 Hz), 7.22 (s, 2H), 6.96 (d, 2H), 6.61 (d, 2H), 4.96 (m, 1H),
4.36 (m, 1H), 4.28 (s, 2H), 3.83 (s, 6H), 3.71 (s, 3H), 3.56 (s, 3H), 3.23
(m, 1H), 2.67 (m, 5H), 2.02 (m, 1H); ESI-MS m/z [M þ H]þ558.6.
(d, 1H, J ¼ 8.0 Hz), 8.16 (d, 1H, J ¼ 8.4 Hz), 7.23 (s, 2H), 4.93 (m, 1H),
4.49 (m, 1H), 4.33 (s, 2H,), 3.83 (s, 6H), 3.71 (s, 3H), 3.67 (s, 3H),
3.08 (m, 1H), 2.97 (m, 2H), 2.76 (m, 1H), 2.20 (m, 1H), 2.02 (m, 1H);
ESI-MS m/z [M þ H]þ498.5.
5.1.4.11. (S)-Methyl 2-(2-((S)-2,6-dioxo-3-(3,4,5-trimethoxybenz-
amido)piperidin-1-yl)acetamido)-3-(1H-indol-3-yl)propanoate (4k). The
compound was prepared from 3 (0.76 g, 2.0 mmol) and (S)-methyl
5.1.4.17. (S)-Methyl
thoxybenzamido)piperidin-1-yl)acetamido)propanoate
3-chloro-2-(2-((S)-2,6-dioxo-3-(3,4,5-trime-
(4q). The
2-amino-3-(1H-indol-3-yl)propanoate
hydrochloride
(0.76 g,
compound was prepared from 3 (0.76 g, 2.0 mmol) and (S)-methyl
2-amino-3-chloropropanoate hydrochloride (0.52 g, 3.0 mmol)
according to the general procedure to yield 4u as white solid (0.68 g,
3.0 mmol) according to the general procedure to yield 4k as white
solid (0.67 g, 57.7%): mp 91.3–93.2 ꢀC, 1H NMR (400 MHz, DMSO-
d6):
d
10.87 (s, 1H), 8.83 (t, 1H, J ¼ 7.6 Hz), 8.51 (d, 1H, J ¼ 7.5 Hz),
68.0%): mp 116.5–117.4 ꢀC, 1H NMR (400 MHz, CDCl3):
d 8.81 (d, 1H,
7.47 (d, 1H, J ¼ 7.8 Hz), 7.32 (d, 1H, J ¼ 7.9 Hz), 7.23 (s, 2H), 7.13 (s,
1H), 7.05 (t, 1H, J ¼ 7.1 Hz), 6.98 (t, 1H, J ¼ 7.9 Hz), 4.98 (m, 1H), 4.52
(d, 1H, J ¼ 6.56 Hz), 4.31 (s, 2H,), 3.83 (s, 6H), 3.71 (s, 3H), 3.56 (s,
3H), 3.12 (m, 3H), 2.77 (m,1H), 2.18 (m,1H), 2.02 (m,1H); ESI-MS m/
z [M þ H]þ581.5.
J ¼ 8.1 Hz), 8.15 (d, 1H, J ¼ 8.4 Hz), 7.23 (s, 2H), 4.94 (s, 1H), 4.49 (m,
2H), 4.12 (m, 1H), 3.87 (m, 1H), 3.77 (s, 6H), 3.71 (s, 3H), 3.67 (s, 3H),
3.30 (s, 2H), 2.96 (m, 1H), 2.77 (m, 1H), 2.21 (m, 1H), 2.031 (m, 1H);
ESI-MS m/z [M þ H]þ499.5, [M þ 2]þ501.5.
5.1.4.18. (S)-Dimethyl 2-(2-((S)-2,6-dioxo-3-(3,4,5-trimethoxybenz-
amido)piperidin-1-yl)acetamido)succinate (4r). The compound
was prepared from 3 (0.76 g, 2.0 mmol) and (S)-dimethyl 2-
aminosuccinate hydrochloride (0.59 g, 3.0 mmol) according to
5.1.4.12. (S)-Methyl 2-(2-((S)-2,6-dioxo-3-(3,4,5-trimethoxybenz-
amido)piperidin-1-yl)acetamido)-3-(1H-imidazol-5-yl)propanoate
(4l). The compound was prepared from 3 (0.76 g, 2.0 mmol) and (S)-

4824
Q. Li et al. / European Journal of Medicinal Chemistry 44 (2009) 4819–4825
the general procedure to yield 4r as white solid (0.76 g, 72.6%):
mp 88.8–90.7 ꢀC, 1H NMR (400 MHz, CDCl3):
8.83 (d, 1H,
J ¼ 8.32 Hz), 8.16 (d, 1H, J ¼ 8.44 Hz), 7.23 (s, 2H), 4.921 (m, 1H),
4.63 (m, 1H, J ¼ 6.74 Hz), 4.3 (dd, 2H), 3.83 (s, 6H), 3.71 (s, 3H),
3.61 (s, 6H), 2.96 (m, 1H), 2.74 (m, 3H), 2.18 (m, 1H), 2.01 (m, 1H);
ESI-MS m/z [M þ H]þ524.6.
NMR (300 MHz, DMSO-d6): d 8.64 (d, 1H), 8.22 (d, 1H), 8.04 (s, 3H),
d
6.86 (s, 2H), 4.91 (m, 1H), 4.25 (m, 3H), 2.31 (m, 2H), 1.97 (m, 2H),
1.58 (m, 3H), 0.86 (dd, 6H); ESI-MS m/z [M þ H]þ 452.5.
5.1.7.4. (S)-2-(2-((S)-2,6-Dioxo-3-(3,4,5-trihydroxybenzamido)
piperidin-1-yl)acetamido)-3-phenylpropanoic acid (7c). Yellow solid
0.21 g, yield 43.3%: mp 102.5–104.9 ꢀC; 1H NMR (400 MHz, DMSO-
5.1.5. (S)-Methyl 2-(2,6-dioxo-3-(3,4,5-trimethoxybenzamido)
piperidin-1-yl)acetate (5)
d6): d 8.50 (m, 1H), 8.38 (s, 1H), 7.24 (m, 5H), 6.85 (s, 2H), 4.82 (m,
1H), 4.38 (m, 2H), 4.25 (s, 2H), 3.00 (m, 2H), 1.98 (m, 2H); ESI-MS m/
Compound 3 (3.8 g, 10 mmol) was suspended in 100 ml MeOH.
To the mixture was added SOCl2 (1.5 ml) dropwise. During the
addition, the inner temperature was controlled under 3 ꢀC by ice–
water bath. After 1 h, the reaction mixture was stirred at room
temperature for 10 h and condensed by rotary evaporation to obtain
white solid 3.76 g (yield 95.4%): mp 135.1–137.0 ꢀC, 1H NMR
z [M þ H]þ486.6.
5.1.7.5. (S)-3,4,5-Trihydroxy-N-(1-(2-(hydroxyamino)-2-oxoethyl)-
2,6-dioxopiperidin-3-yl)benzamide (6a). White solid 0.20 g yield
57.1%: mp 210 ꢀC (dec); 1H NMR (400 MHz, DMSO-d6):
d 10.78 (s,
1H), 9.01 (s, 3H), 8.83 (d, 1H), 8.06 (d, 1H), 6.85 (s, 2H), 4.67 (m, 1H),
4.33 (m, 2H), 2.77 (m, 1H), 2.53 (m, 1H), 2.07 (m, 1H), 1.96 (m, 1H);
ESI-MS m/z [M þ H]þ: 354.3.
(300 MHz, DMSO-d6):d 8.81(d,1H), 8.32(d,1H), 7.23(s, 2H), 4.95 (m,
1H,), 4.55 (dd, 2H), 3.83 (s, 6H), 3.71 (s, 3H), 3.65 (s, 3H), 3.02 (m,1H),
2.80 (m, 1H), 2.20 (m, 1H), 2.04 (m, 1H); ESI-MS m/z [M þ H]þ 395.4.
5.2. Biological materials and methods
5.1.6. (S)-N-(1-(2-(Hydroxyamino)-2-oxoethyl)-2,6-dioxpiperidin-
3-yl)-3,4,5-trimethoxybenzamide (6)
5.2.1. Cell line and cell culture
0.2 g of NH2OHꢁHCl was dissolved in 1.5 ml anhydrous MeOH
and to the clear solution was added 0.5 ml of Et3N. The carboxyl
acid derivative 3 (2.0 mmol) was dissolved in 20 ml anhydrous THF
and to control the inner temperature not higher than ꢂ20 ꢀC.
2.0 equiv of Et3N was added slowly, and after 5 min, 1.01 equiv of
isobutyl chloroformate was added. The mixture was stirred at
ꢂ20 ꢀC for another 10 min and to the mixture was added slowly
MeOH solution with NH2OH after which the mixture was stirred at
ꢂ20 ꢀC for 15 min and then kept at 0 ꢀC for 2 h. The solid formed
was filtered by 2.0 g celite and the filtrate was condensed and
dissolved in 30 ml of AcOEt. The organic phase was then washed in
turn by 0.05%NaHCO3 (10 ml ꢄ 2), 5% Citric acid (10 ml ꢄ 1), brine
and then dried over Na2SO4. Purified by column chromatography to
obtain 6 0.37 g (47.5%) as white crystal: mp 182.8–185.3 ꢀC; 1H NMR
The human leukemia cell line, HL-60 was 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 serum, penicillin–streptomycin (100 IU/
ml–100 m
g/ml), 2 mM 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-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazo-
lium bromide (MTT, Sigma, USA) assay as described elsewhere.
5.2.2. In vitro APN enzyme assay [19]
IC50 values against APN were determined as previously
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 solutions of inhibitors were
prepared in the assay buffer, and pH was adjusted to 7.5 by the
addition of 0.1 M HCl or 0.1 M NaOH. All inhibitors were pre-
incubated with APN for 30 min at room temperature. The assay
mixture, which contained the inhibitor solution (concentration
(300 MHz, DMSO-d6):
d
12.31 (s,1H), 8.44 (d,1H, J ¼ 7.9 Hz), 8.23 (d,
1H, J ¼ 5.7 Hz,), 7.24 (s, 2H), 4.50 (m, 1H), 3.84 (s, 6H), 3.75 (s, 2H),
2.34 (m, 2H), 2.07 (m, 1H), 1.93 (m, 1H); ESI-MS m/z [M þ H]þ 396.2.
5.1.7. General procedure for the deprotection of methyl group of
3,4,5-trimethoxybenzamide compounds (3, 4a–c, 6)
The round-bottom flask was charged with 1.0 mmol of trime-
thoxybenzamide compounds under N2. 10 ml of anhydrous DCM
was added and cooled at dry-ice acetone bath to ꢂ78 ꢀC and stirred
for 15 min. To the mixture was added dropwise 3.1 mmol of BBr3 in
anhydrous DCM, and kept stirring at ꢂ78 ꢀC for another 10 min and
then 4 h at room temperature. The mixture was quenched by the
addition of saturated NaHSO3 solution and extracted with DCM.
The crude products were recrystallized by EtOH.
dependent on the inhibitor), the enzyme solution (4
mg/ml final
concentration), and the assay buffer, was adjusted to 200
ml.
5.2.3. In vitro cytostatic activity [20]
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, Cyclic-imide peptidomimetic inhibitors were then
added to the wells to achieve final concentration of 800, 600, 400,
5.1.7.1. (S)-2-(2,6-Dioxo-3-(3,4,5-trihydroxybenzamido)piperidin-1-
yl)acetic acid (3a). Yellow solid 0.18 g, yield 53.1%: mp 187.2–
189.4 ꢀC; 1H NMR (400 MHz, DMSO-d6):
d 12.31 (s, 1H), 9.01 (s, 3H),
8.83 (d, 1H), 6.81 (s, 2H), 4.69 (m, 1H), 4.27 (m, 2H), 2.77 (m, 1H),
2.53 (m, 1H), 2.07 (m, 1H), 1.96 (m, 1H); ESI-MS m/z [M þ H]þ 339.3.
200 and 100 mg/ml Control wells were prepared by addition of
culture medium. At same time, the bestatin (Apeloa Pharma/Kan-
gyu Pharma, China) was used as positive control. The plates were
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 was removed and
5.1.7.2. (S)-2-(2-(2,6-Dioxo-3-(3,4,5-trihydroxybenzamido)piper-
idin-1-yl)acetamido)acetic acid (7a). Off-white solid 0.20 g, yield
50.6%: mp 112.8–115.2 ꢀC; 1H NMR (400 MHz, DMSO-d6):
d 8.61 (d,
1H), 8.20 (d,1H), 8.03 (s, 3H), 6.85 (s, 2H), 4.92 (m,1H), 4.27 (m, 3H),
2.31 (m, 2H), 1.97 (m, 2H); ESI-MS m/z [M þ H] 396.4.
100 ml DMSO was added. Absorbance at 570 nm was measured
using an enzyme-linked immunosorbent assay reader (Model 680,
BIO-RAD), and absorbance at 630 nm was used as a reference. The
percent growth inhibitory rate of treated cells was calculated by
(ODdrugꢂfree control ꢂ ODtested)/ODdrugꢂfree control ꢄ 100%, where OD is
5.1.7.3. (S)-2-(2-((S)-2,6-Dioxo-3-(3,4,5-trihydrox-
ybenzamido)piperidin-1-yl)acetamido)-4-methylpentanoic
acid
(7b). Yellow powder 0.29 g, yield 64.4%; mp 127.3–129.4 ꢀC; 1H

Q. Li et al. / European Journal of Medicinal Chemistry 44 (2009) 4819–4825
4825
the mean value calculated using the data from three replicate tests.
The IC50 values were determined by plotting the percentage
viability vs. concentration on a logarithmic graph and reading off
the concentration at which 50% of cells viable relative to the
control. Each experiment was repeated at least three times to get
the mean values. The curves were defined using Origin 7.5 software.
High Technology Research and Development Program of China
(863 project; Grant No 2007AA02Z314).
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We thank Prof. Xianjun Qu and Dr. Xuejian Wang (Institute of
Pharmacology, School of Pharmacy, Shandong University) for in
vivo compounds screenings and HL-60 cells assay. We would also
like to thank Dr. Philip Mosier (Department of Medicinal chemistry,
School of Pharmacy, Virginia Commonwealth University) for the
GOLD4.0 docking software and training. This work was supported
by National Nature Science Foundation of China (Grant Nos.
30772654; 36072541) and the Ph.D. Programs Foundation of
Ministry of Education of PR China (No. 20060422029) and National
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