Novel potent 2,5-pyrrolidinedione peptidomimetics as aminopeptidase N inhibitors. Design, synthesis and activity evaluation

Article abstract of DOI:10.1016/j.bmcl.2011.12.048

A series of novel aminopeptidase N inhibitors with 2,5-pyrrolidinedione scaffold were chemically synthesized. Their preliminary biological activities in enzyme kinetics and cell assay in vitro and anti-metastasis profile in vivo were also evaluated. The r

Full text of DOI:10.1016/j.bmcl.2011.12.048

Bioorganic & Medicinal Chemistry Letters 22 (2012) 850–853
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel potent 2,5-pyrrolidinedione peptidomimetics as aminopeptidase
N inhibitors. Design, synthesis and activity evaluation
Qianbin Li a, , Hao Fang b, Xuejian Wang b, Gaoyun Hu a, Qiang Wang c, Wenfang Xu b,
⇑
⇑
a Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Central South University, 172 Tongzipo Road, 410013 Changsha, Hu’nan, China
b Institute of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, 44 West Wenhua Road, 250012 Ji’nan, Shandong, China
c School of Pharmacy, South-Central University for Nationalities, 708 Minyuan Road, Wuhan 430074, Hubei, 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 novel aminopeptidase N inhibitors with 2,5-pyrrolidinedione scaffold were chemically synthe-
sized. Their preliminary biological activities in enzyme kinetics and cell assay in vitro and anti-metastasis
profile in vivo were also evaluated. The results indicated that all the compounds displayed potent inhib-
itory activity against aminopeptidase N. Compound 8f inhibited aminopeptidase N activity with IC50
Received 2 September 2011
Revised 26 November 2011
Accepted 9 December 2011
Available online 16 December 2011
value of 1.0
l
M and displayed better activity profile in vivo than that of bestatin.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Aminopeptidase N
Synthesis
Peptidomimetics
2,5-Pyrrolidinedione
Anti-tumor
Aminopeptidase N (APN) is a significantly important biological
and medical marker for the development of many diseases.1–5 It
is expressed as an ectopeptidase with one zinc ion in active site
and plays key roles in protein modification, activation, and degra-
dation as well as in the metabolism of biologically active peptides
in tumor metastasis and leukemia.6–8 Therefore, the design and
synthesis of inhibitors against aminopeptidase N may result in
the discovery of potential therapeutic agents.2–4
In our previous work, we have reported a series of piperidined-
ione-N-acetamide peptidomimetics derivatives with free amine
group.9 The biological characterization for the piperidinedione
analogues revealed that the most potent compound 13f displayed
good inhibitory effect against aminopeptidase N.9 Furthermore, 13f
was also demonstrated to delay the growth of human ovarian car-
cinoma cells (ES-2) xenografts in mice after 2 weeks of vena cauda-
lis injection and to inhibit the metastasis of hepatoma H22 cell to
lung tissue.9–11 These results suggested that 13f has a high inhibi-
tory effect on the growth of OVCA cells via decreasing the activity
and expression of APN and can be treated as a leading compound
for further studies and structure optimization.10,11
ing site of the APN with 13f, is composed of at least four parts: (1)
one zinc binding group (ZBG) which can coordinate with zinc ion in
the active site of enzyme; (2) one S1 hydrophobic pocket which
interact with the phenyl group of bestatin; (3) another hydropho-
bic pocket S02 in deeper cavity which contains several Arg residues
and can form hydrogen bond with the ligand. It has been shown
that Probestin, a tripeptide analogue of bestatin, is 50 times more
active than bestatin against APN because of the existence of two
pro residues which can interact with S02 pocket by hydrogen
bonding.9
To further study the structure–activity relationship of the het-
erocycle ring number in 13f and the binding mode of new scaffold
with the crystal structure of APN, herein we report the design and
synthesis of novel 3-amino-2,5-pyrrolidinedione peptidomimetics
(Fig. 1). The structures were modified based on our previous work
as the following: (i) R1 groups interacting with the S1 pocket of en-
zyme active site were modified to be the side chains of different
amino acids, which can interact the active site differently. (ii)
The amino group was maintained freely in the form of hydrochlo-
ride salt so as to interact with the anion binding site of enzyme
which contain a Glu355 (Human) or Glu264 (E. coli), as reported
previously.9,18 (iii) R2 groups were OH or NHOH, which can form
hydrogen bonding with the S02 active site of enzyme.
The discovery of many potent APN inhibitors benefited from the
availability of X-ray crystal structures for free enzyme and com-
plexes with various inhibitors.12–17 As shown in Figure 1, the bind-
In Scheme 1, chemical synthesis started from (S)-tert-butyl 2,5-
dioxopyrrolidin-3-ylcarbamate (1), which was prepared from
⇑
Corresponding authors. Tel./fax: +86 731 8265 0370 (Q.L.); tel./fax: +86 531
8838 2264 (W.X.).
cyclization reaction of commercially available Boc-L-Asn-OH amino
acid in the presence of NHSu and DCC. The NH group in 2,5-dioxo-
E-mail addresses: qbli@csu.edu.cn (Q. Li), xuwenf@sdu.edu.cn (W. Xu).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.048

Q. Li et al. / Bioorg. Med. Chem. Lett. 22 (2012) 850–853
851
O
O
O
H3N
N
OH
N
H
N
H
O
13f
R1=side chain of different
amino acids
O
O
S2'
R2
N
S1
HN
R1
O
O
NH2
R2=OH, NHOH
O
O
Glu355
Anion binding site
Zinc Binding Group
Figure 1. The binding mode of 13f and design strategies of pyrrolidinedione peptidomimetics.
O
O
O
O
a
b
c
NH
Boc-L-Asn-OH
N
BocHN
BocHN
O
O
O
1
2
O
O
O
O
O
O
OH
O
O
O
N
d
N
e
N
BocHN
BocHN
N
H
N
H
H N
2
TFA .
O
O
O
R
R
5
3
4
Scheme 1. Reagents and conditions: (a) NHSu, DCC, THF, reflux, 85%; (b) acetone, benzyl bromoacetate, TBAI, 56 °C, 70%; (c) TFA, DCM; (d) NMM, THF, isobutyl chloroformate,
Boc-AA-OH, 80–91%; (e) ethanol, cyclohexene, 5% Pd–C, 40 °C, 82–97%.
pyrrolidine was then alkylated by benzyl bromoacetate using tet-
rabutylammonium iodide (TBAI) as phase transfer catalyst to af-
ford compound 2, which was Boc-deprotected with TFA/DCM to
obtain critical free amine TFA-salt (3) and then coupled with vari-
ous a-substituted amino acids (Boc-AA-OH) in the presence of N-
methylmorpholine (NMM), isobutyl chloroformate/THF to give
4a–4i. Pd/C hydrogenation of 4a–4i in ethanol at 40 °C yield corre-
sponding acid derivatives 5a–5i. In Scheme 2, acid compounds 5a–
5i were converted to N-hydroxyacetamide derivatives 7a–7i in the
presence of isobutylchloroformate, Et3N/THF. Compounds 5a–5i
and 7a–7i were Boc-deprotected with TFA/DCM to give 6a–6i
and 8a–8i as hydrochloride salt in high yield.9,19
⁄⁄
Figure 2. IC50 of target compounds in APN kinetics assays.
indicates P <0.01.
The inhibitory activity was evaluated against APN in vitro en-
zyme kinetic assays by the spectrophotometric method as described
Values are means of three experiments, standard deviation is given.
O
O
O
O
OH
O
OH
N
O
b
R=
N
a
HCl .
H2N
N
H
BocHN
N
H
O
(6a, 8a)
H
R
O
R
O
6
(6b, 8b) CH3-
5
(6c, 8c) (CH3)2CH-
(6d, 8d) (CH3)2CHCH2-
(6e, 8e) C2H5(CH3)CH-
(6f, 8f) C6H5CH2-
O
O
O
O
NHOH
NHOH
O
N
(6g, 8g) H2N(CH2)4-
(6h,8h) pyrrolidine-2-
(6i, 8i) 4-OH-C6H5CH2-
a
N
HCl . H2N
N
H
BocHN
N
O
H
O
R
R
7
8
Scheme 2. Reagents and conditions: (a) 3 N HCl/EtOAc, 85–95%; (b) Et3N, THF, isobutyl chloroformate, NH2OH, 78–92%.

852
Q. Li et al. / Bioorg. Med. Chem. Lett. 22 (2012) 850–853
previously.9 As shown in Figure 2, most compounds (the total num-
ber is 18) showed potent APN inhibitory activity with IC50 values of
arranged a ‘U-like’ conformation which render the molecule with
higher energy (43.061 kcal/mol) and lower docking T-score (4.13)
compared with what for the hypothesized docking mode (41.856
and 6.82 kcal/mol). In fact, because of the overall impact of mole-
cule, hydroxamic acid is likely to interact with other sub-site of
the target, which can be demonstrated by J. Su and co-worker’s
previous work. The co-crystal structure of engineered hAPN and
1.0–21
l
M, which is more active than those reported previously.
M) increased
The activity of the most potent one 8f (IC50 = 1.0
l
2.4 times compared with bestatin. Structurally, all compounds pos-
sess a similar chemical scaffold, which is C-3 free amino group, indi-
cating that the free amine is still the successful optimization for the
inhibition of APN. And what is necessary to be emphasized is that
the existence of free group still maintains the high affinity toward
the enzyme, which is consistent with reference and suggest the
importance of amino group in the recognition of inhibitors by APN.
As reported previously, the tendency of inhibitory profile for
target compounds with different substituents in R2 against APN
is –C(@O)NHOH>COOH. In order to obtain further insight into
the interaction of target compound with APN, the most active com-
pound 8f was built and docked into the active site of APN (PDB
code: 2DQM) using Sybyl-X and the 2D-interaction was plotted
by Ligplot.20 The binding study results shown in Figure 3 indicated
that two carbonyl groups (C@O from C2 on the cyclic imide ring
L-Lys hydroxamic acid derivatives (24D) displayed that the –NHOH
group interact with Tyr244 and Gly352 instead of zinc ion.21
To compare the difference between series 8 and series 6, car-
boxylic acid compound (6f) was also docked into the active site
of APN with the same parameters. The results indicated that the
–COOH group interacts weakly with the zinc ion through only
one hydrogen bond with Arg293. However, the –C(@O)NHOH
group displays higher affinity due to the existence of four hydrogen
bonds with Arg293 (see Supplementary data).
As mentioned before, R1 groups interact with the S1 hydropho-
bic pocket of APN, in which Tyr376 is a critical residue. Therefore,
the existence of aromatic ring in R1 group (8f and 8j) displayed
and
the active pocket with distance of 2.53 and 2.06 Å, respectively.
In addition, the C2 carbonyl group and NH from -Phe also interact
with the hydroxyl group of Tyr381, His297 and Glu298 by hydro-
gen 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 (2.90 Å), Glu264
(2.61 Å) and Glu320 (2.76 Å) which anchor the inhibitor to the ac-
L
-Phe) bind to the zinc ion and form a five-member ring in
favorable affinity toward the target through p–p stacking interac-
tion of two phenyl rings, which is the true case for bestatin, the co-
ligand in the crystal complex. Compounds with other side chain
interact weakly with S1 pocket resulting in poor activity against
APN other than compound 8d, which showed identical potency
to bestatin. The possible reason may be due to the small size of
t-butyl group, which can enter the deeper inside of the active site.
The t-butyl group in 8d resembles the corresponding part in the
structure of bestatin, which interacts with another hydrophobic
pocket and displays different binding mode with enzyme.
L
tive site of the enzyme. The aromatic ring of
L-Phe is more favor-
able as it can form a interaction with the Tyr376 located in
p–p
the active site, which is similar to that of bestatin. The similar
phenomenon can also be seen from aromatic ring-containing
compounds (6f, 6j and 8j). The hydroxyl group in N-hydroxyaceta-
mide part can form hydrogen bond interaction with Glu382
(3.10 Å), Arg293 (2.94, 3.26 Å), which is the essential amino acid
residues of for the interaction of potent inhibitors with peptidase
M1 family.9
To further evaluate the effects of the active compounds on cell
proliferation and survival, HL-60 cell was treated with compound
8d, 8f and bestatin and the inhibitory activity was evaluated using
MTT assay method.22 The results displayed that compound 8f
exhibited the inhibitory effect on cell proliferation with IC50 values
of 149
lM, which showed better potency than bestatin
(IC50 = 245
l
M) (Fig. 4).
Theoretically, –C(@O)NHOH is the strongest binding group for
zinc and the flexible docking results also indicated that the
hydroxamic acid in the structure of 8f can also interact with the
metal ion with the distance between NHOH and the zinc of
1.82 Å (see Supplementary data). However, the whole molecule is
To obtain further insight into the effects of 8f in vivo, anti-
metastasis ability of compound against hepatoma H22 cell’s loca-
tion into lung tissue was evaluated using the method reported pre-
viously.23 The inhibitory rate for compound 8f is much higher than
that for Bestatin (45.37%) with the value of 54.21% (Fig. 4). All of
Figure 3. Docking mode for 8f with APN (PDB code: 2DQM) plotted by SYBYL-X and Ligplot.

Q. Li et al. / Bioorg. Med. Chem. Lett. 22 (2012) 850–853
853
Figure 4. In vitro cell assays (left) and in vivo anti-metastasis of H22 cell assay (right) for active compounds.
5. Ansorge, S.; Bank, U.; Heimburg, A.; Helmuth, M.; Koch, G.; Tadje, J.; Lendeckel,
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these results indicate that compound 8f is a potent molecule which
can be applied for further pre-clinical studies and is promising for
the application of invasion-preventing therapy.
In summary, based on the achievement in our previous work,
which is the existence of 3-amino group is essential for the inhibi-
tion of aminopeptidases. In this effort, a specific activity profile
against aminopeptidase N has been discovered when the heterocy-
cle ring was converted from six-member (piperidinedione) to five-
member (pyrrolidinedione). Our studies have shown that 3-amino-
2,5-pyrrolidinedione is an another novel scaffold for the discovery
of potent aminopeptidase N inhibitors and most compounds dis-
played potent inhibitory activity. The most potent compound, 8f,
exhibited good activity profiles both in vitro and in vivo and wor-
thy of further investigation.
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Acknowledgments
This work was supported by National Nature Science Founda-
tion of China (Grant Nos. 21102184; 21102185; 30772654;
36072541), Freedom Explore Program of Central South University
(Grant No. 201012200087), Science and Technology Project of Hu-
nan Province (Grant No. 2010FJ3004), the Ph.D. Programs Founda-
tion of Ministry of Education of China (No. 20060422029), National
High Technology Research and Development Program of China
(863 project; Grant No. 2007AA02Z314).
19. General procedure for the preparation compound 8: To the clear solution of
HClꢀNH2OH (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 5 (2.0 mmol) were
dissolved in 20 ml THF and cooled to ꢁ20 °C. To the mixture was added
dropwise 2.0 equiv of Et3N, and then 1.01 equiv of isobutyl chloroformate after
5 min. The mixture was stirred at ꢁ20 °C for another 10 min and the MeOH
solution with NH2OH was added dropwise, after which the mixture was stirred
at ꢁ20 °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 7 and
purified by column of silica gel. To the stirring solution of 7 (0.5 g) in 10 ml
anhydrous ethyl acetate was added dropwise 5 ml 3 N EtOAc–HCl and after 2–
3 h, target compounds 8 were obtained in the form of hydrocholoride salt.
Filter quickly and dry the cake in vaccum to obtain dried white solid with high
yield. Compound 8f: yield 91.7%. Mp 155.8–157.2 °C; ESI-MS m/z [M+1]+ 335.3;
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.12.048.
½
a 2D5
ꢂ
ꢁ40.0 (c 1.00, MeOH); 1H NMR (300 MHz, DMSO-d6): d 10.75 (s, 1H), 9.48
(s, 1H), 8.36 (s, 3H), 7.36–7.26 (s, 5H), 4.73–4.66 (m, 1H), 4.15–3.68 (m, 2H),
3.11–2.98 (m, 3H), 2.47–2.41 (dd, 1H, J = 17.7 Hz, 9.0).
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