Novel 3-galloylamido-N′-substituted-2,6-piperidinedione-N-acetamide peptidomimetics as metalloproteinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.12.095

Both of aminopeptidase N (APN) and matrix metalloproteinase (MMP) are essential metallopeptidases in the development of tumor invasion and angiogenesis. Novel potent peptidomimetic inhibitors, containing 3-galloylamido-N′-substituted-2,6-piperidinedione-N

Full text of DOI:10.1016/j.bmcl.2006.12.095

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2935–2938
Novel 3-galloylamido-N0-substituted-2,6-piperidinedione-
N-acetamide peptidomimetics as metalloproteinase inhibitors
Qianbin Li, Hao Fang and Wenfang Xu*
Institute of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, 44 West Wenhua Road,
250012 Ji’nan, Shandong, PR China
Received 23 September 2006; revised 12 December 2006; accepted 26 December 2006
Available online 5 January 2007
Abstract—Both of aminopeptidase N (APN) and matrix metalloproteinase (MMP) are essential metallopeptidases in the develop-
ment of tumor invasion and angiogenesis. Novel potent peptidomimetic inhibitors, containing 3-galloylamido-N0-substituted-2,
6-piperidinedione-N-acetamide, have been designed and synthesized according to the conformational constraint strategy. The pre-
liminary biological test showed that most of the compounds displayed high inhibitory activity against MMP-2 and low activity
against APN except compounds 6 (IC50 = 3.1 lM) and 4l (IC50 = 5.2 lM) which exhibit similar potency to Bestatin (IC50 = 2.4 lM).
Ó 2007 Elsevier Ltd. All rights reserved.
During the process of angiogenesis and metastatic cas-
cade, tumor cells and endothelial cells pass through sev-
eral connective tissue barriers. Proteolytic degradation
of the extracellular matrix (ECM) is an important step
of the process, which involves two classes of zinc-depen-
dent metalloproteinases, amino-peptidase N (APN) and
matrix metalloproteinases (MMP).
tors have been used to suppress the process of tumor cell
invasion.18,19
APN inhibitors exhibited prevention of the spread of
malignant cells20 and prove to be clinically efficacious
for invasion-protecting therapy. To date, several inhibi-
tors of APN, including Bestatin, Amastatin, and Acti-
nonin, have been developed and some of them are
currently investigated for clinical uses.21 All these natu-
ral compounds are pseudodipeptides bearing zinc-che-
lating functionality in the molecule.
APN, also known as CD13, is a homodimeric type II
membrane-bound glycoprotein and belongs to a mem-
ber of M1 family ectopeptidase.1–3 It is widely expressed
on the surface of renal and intestinal brush border cells
and other cells.4,5 APN was also shown to be the major
receptor for the TGEV and HCV229E, and Bacillus
thuringensis Cry1A toxin.6–8 Recently, many experimen-
tal results suggest that APN is involved in the down-reg-
ulation of several biological active molecules, such as
enkephaline, fMLP, IL-8, angiotensin III, and major
histocompatibility complex (MHC) class II mole-
cules.9–14 APN plays an essential role in the entry of
HIV into host cells.15 Furthermore, APN is overexpres-
sed on tumor cells and plays a crucial role in ECM deg-
radation and invasion of tumor cells.16,17 Therefore,
anti-APN/CD13 monoclonal antibody and APN inhibi-
MMP gene family consists of at least 28 structurally
related members, among which MMP-2 and -9 are
proved to be highly correlated with cancer. MMPs
belong to endopeptidase and also play a critical role in
the degradation of ECM and tissue remodeling and
wound healing.22,23 Currently, numerous MMP inhibi-
tors are in various developmental stages for different
symptoms, mostly in cancer and arthritis. Compounds
currently under clinical trials as matrix metalloprotein-
ase inhibitors (MMPIs) include Marimastat, Bay-
129566, AG3340, and CGS27023A. All these com-
pounds are applied to treat different types of cancer.24
However, clinical trials exhibit disappointing results
for most of MMPIs and possible reason is due to the
inappropriate drug design or poor selectivity.
Keywords: Aminopeptidase N; Matrix metalloproteinase; Peptidomi-
metics; Conformational constraint.
In general, peptides have drawbacks for clinical applica-
tion, i.e., proteolytical lability, low bioavailability, rapid
excretion, short duration of action, etc. Therefore, it is
*
Corresponding author. Tel./fax: +86 531 883 82264; e-mail:
drqbli@yahoo.com.cn
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.12.095

2936
Q. Li et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2935–2938
O
important to design and synthesize peptidomimetic
derivatives. The concept of conformationally con-
strained derivatives plays an important role in the design
of peptidomimetics in the drug development process due
to its powerful ability for probing the bioactive confor-
mations of peptide. In addition, steric effect resulting
from conformationally constrained structures could pre-
vent the degradation of hydrolases and minimize possi-
bility of binding to non-target receptors. For example,
cyclic peptides can reduce the rate of degradation by
peptidases and other enzymes and meanwhile improve
the selectivity and affinity to the targets.25–27
O
H3CO
H3CO
H3CO
H3CO
COOH
N
H
O
N
H
a
COOH
O
O
OCH3
OCH3
1
2
O
O
H3CO
H3CO
H3CO
H3CO
N
N
H
O
c
H
O
b
N
O
O
N
O
O
OCH3
OCH3
HN
3
4a-r
OH
R
d
O
O
H3CO
H3CO
H3CO
H3CO
N
N
H
O
e
H
O
N
O
O
N
O
O
In our previous work, Wang et al. have reported that the
novel L-iso-glutamine derivatives can serve as a poten-
tial antitumor agent and possess potent inhibitory activ-
ity toward APN, but the selectivity between APN and
MMP is low.28 In our ongoing work, the strategy of
conformational constraint was used to cyclize the car-
boxyl group with the nitrogen of the amide hoping for
exploring new metalloproteinase inhibitors with anti-
tumor activity (Fig. 1). To improve the bioactivity, the
gallic acid moiety was introduced its derivatives possess
anti-tumor and anti-oxidative activities.29,30 In this
study, we report the preparation and in vitro inhibitory
activity assay of 3-galloylamido-N0-substituted-2,6-pipe-
ridinedione-N-acetamide peptidomimetic derivatives.
OCH3
OCH3
O
HN
OH
5
6
Scheme 1. Reagents and conditions: (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) NH2OH, KOH, CH3OH; 60%.
Table 1. In vitro enzyme assay (APN and MMP-2) results for
compounds 3–6 and Bestatin
O
H3CO
N
H
H3CO
O
N
O
O
OCH3
In order to study the SAR of these novel peptidomimetic
compounds, different amide compounds 4a–r and
hydroxamate derivatives 6 were designed and synthe-
sized via the route outlined in Scheme 1. The synthesis
of N0-substituted-2,6-piperidinedione-N-acetamide pep-
tidomimetic derivatives was started from readily avail-
able dicarboxylic acid 1.28 Compound 2 was obtained
by dehydration of 1 and then reacted with glycine under
microwave irradiation to generate piperidinedione 3.
This was followed by coupling with various amino acid
methyl esters using EDCI to give target compounds
4a–r.31 In addition, compound 3 reacted with thionyl
chloride in methanol to yield methyl ester 5. Finally
hydroxymate 6 was prepared by the reaction of 5 with
NH2OH.
HN
R
a
Compound R
IC50 (lM)
IC50 (APN)/
IC50 (MMP-2)
APN
MMP-2
3
49.8 5.5 4.0 0.5 12.45
55.4 3.5 4.4 1.1 12.59
21.4 2.6 1.0 0.2 21.40
46.2 3.2 3.8 0.5 12.16
49.3 2.9 4.3 0.4 11.47
53.7 8.4 2.2 0.6 24.41
18.9 4.4 1.2 0.3 15.75
4a
4b
4c
4d
4e
4f
4g
4h
4i
Gly-OMe
Val-OMe
Leu-OMe
Ile-OMe
b-Ala-OMe
Ala-OMe
Arg (NO2)-OMe 42.0 7.6 0.3 0.1 140.00
Met-OMe
Z-Lys-OMe
Tyr-OMe
Trp-OMe
His-OMe
20.7 2.7 1.2 0.3 17.25
38.1 3.8 0.6 0.2 63.50
44.8 5.2 0.3 0.1 149.33
43.1 3.2 1.2 0.4 35.92
4j
4k
4l
5.2 2.1 13.0 2.9
0.40
The target compounds were evaluated for inhibitory
activity toward APN and MMP-2. APN inhibitory
activity has been evaluated by measuring p-nitroanilide
liberated from L-Leu-p-nitroanilide32 and MMP assay
was performed according to the literature.33 All the inhi-
bition results are summarized in Table 1.
4m
4n
4o
4p
4q
4r
5
D-Phe-OMe
Thr-OMe
L-Phe-OMe
Cys-OMe
46.2 6.4 1.4 0.5 33.00
51.9 3.9 1.8 0.6 28.83
11.0 3.0 1.3 0.3
50.3 4.3 1.7 0.5 29.59
3.21
21.7 2.1 0.5 0.1 43.40
8.46
2-Cl-Ala-OMe 50.0 4.2 15.6 2.3
Asp-(OMe)2
63.4 3.2 10.6 2.1
3.1 0.7 2.2 0.6
2.4 0.5 3.4 0.6
5.98
1.41
0.71
The inhibition results showed that all the compounds
display excellent potency toward MMP-2 with IC50 val-
ues lying in micromolar level. Comparing 4g, 4i, 4j, 4r,
6
–OH
Bestatin
a Values are means of three experiments, standard deviation is given.
we could confirm that the length of side chains of R
was positively related with the inhibitory activities. This
could be due to its bulky side chain exhibiting good
interaction with the enzymes, hydrophobic domains.
Conformational
constraint
O
O
COOH
N
H
O
R1
O
N
N
R
R1
H
NH
R
O
Cyclize
In addition, as for the inhibitory activity toward APN,
most of compounds showed low affinity except
Figure 1. Cyclization of L-iso-glutamine derivatives of metalloprotein-
ase inhibitors.

Q. Li et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2935–2938
2937
compound 4l and compound6. Comparing 5 and 6, it
was shown that introducing strong zinc binding group,
hydroxymate, could significantly enhance the inhibi-
tion against APN and compound 6 (IC50 = 3.1 lM)
tion of the requirement of the active site of APN, novel
conformationally constrained peptidomimetic inhibitors
against APN will also be investigated.
exhibited almost equivalent potency to Bestatin (IC50
=
2.4 lM). The docking results of 6 with the active site of
Escherichia coli APN are shown in Figure 2a. The two
oxygen atoms in hydroxymate (OH) and 6-carbonyl
group of piperidinedione can interact with the metal
Acknowledgments
This work was supported by National Nature Science
Foundation of China (Grant No. 36072541) and the
Ph.D. Programs Foundation of Ministry of Education
of P.R. China (No. 20060422029) and Shandong Nature
Science Foundation of China (Grant No. Y2004C02).
˚
˚
ion with the distance of 1.03 A and 1.88 A, respectively.
Compound 4l also displays potent activity against APN
with the IC50 of 5.2 lM. The docking results of 4l with
E. coli APN show that sp2 nitrogen of the imidazole ring
can be coordinated with the zinc ion of APN (Fig. 2b,
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1
and H NMR spectra that exhibit no discernible impuri-
ties. Compound 4a: 1H NMR (400 MHz, DMSO-d6): d
9.04 (1H, d), 8.88 (1H, t), 7.17 (2H, s), 4.53 (1H, m), 4.37
(2H, s), 4.16 (2H, s), 3.83 (6H, s), 3.71 (3H, s), 3.67 (3H, s),
2.91 (2H, m), 2.18 (1H, m), 2.11 (1H, m); MS (ESI+) m/z
452.6 [M+1]+; mp 98.7–100.6 °C.