Structure-activity study on a series of α-glutamic acid scaffold based compounds as new ADAMTS inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.06.009

A series of α-glutamic acid scaffold based 4-(benzamido)-4-(1,3,4- oxadiazol-2-yl) butanoic acids were designed and synthesized as new ADAMTS inhibitors. The compounds dose-dependently inhibited the enzymatic activities of ADAMTS-4 and ADAMTS-5. One of the most active compound 2h potently inhibited ADAMTS-4 and ADAMTS-5 with IC₅₀ values of 1.2 and 0.8 μM, respectively. These inhibitors may serve as new lead compounds for further development of therapeutics to treat osteoarthritis.

Full text of DOI:10.1016/j.bmcl.2011.06.009

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4457–4461
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity study on a series of a-glutamic acid scaffold
based compounds as new ADAMTS inhibitors
Lijie Peng a,b, Lei Duan a, Xiaofeng Liu c, Mengjie Shen a,b, Yingjun Li a,b, Jiajie Yan a,b, Honglin Li c, Ke Ding a,
⇑
a Key Laboratory of Regenerative Biology, Institute of Chemical Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, No. 190,
Kaiyuan Avenue, Science Park, Guangzhou 510530, China
b Graduate School of Chinese Academy of Sciences, #19 Yuquan Road, Beijing 100049, China
c Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 February 2011
Revised 8 May 2011
Accepted 3 June 2011
Available online 16 June 2011
A series of
a-glutamic acid scaffold based 4-(benzamido)-4-(1,3,4-oxadiazol-2-yl) butanoic acids were
designed and synthesized as new ADAMTS inhibitors. The compounds dose-dependently inhibited the
enzymatic activities of ADAMTS-4 and ADAMTS-5. One of the most active compound 2h potently inhib-
ited ADAMTS-4 and ADAMTS-5 with IC50 values of 1.2 and 0.8
serve as new lead compounds for further development of therapeutics to treat osteoarthritis.
Ó 2011 Elsevier Ltd. All rights reserved.
lM, respectively. These inhibitors may
Keywords:
Osteoarthritis
Aggrecanase
ADAMTS
Glutamic acid scaffold
Osteoarthritis (OA) is a degenerative disease of the joints,
resulting from degradation of articular cartilage. The aggrecan is
a major component of the cartilage extracellular matrix. The exten-
sive loss of aggrecan via proteolysis is believed to be the early
event that leads to further deterioration of weigh-bearing proper-
ties and eventually irreversible joint dysfunction.1
zinc-binding group (ZBG, i.e., a hydroxamic acid, carboxylic acid,
or semisquaric acid group) and several hydrophobic moieties (P10
and P20 groups) to interact with the S1, S10, and S20 binding pock-
ets, respectively.11,13 Compound 1 has been reported as a highly
potent aggrecanase inhibitor with an in vitro IC50 value of
1.5 nM.12h The detailed contributions to the binding with aggre-
canase from decomposed moieties of 1 are illustrated in Figure 1.
In light of compound 1 and its corresponding interactions with
Aggrecanase-1 and aggrecanase-2, also known as ADAMTS-42
and ADAMTS-5,3 respectively, are members of the ADAMTS (a dis-
integrin and metalloprotease with thrombospondin motifs) family
of zinc-containing metalloproteases.4 Although the relative
contributions of ADAMTS-4 and ADAMTS-5 in different stages of
human disease remain illusive, they are the only identified
enzymes that cleave the interglobular domain (IGD) of aggrecan at
Glu373-Ala374 in osteoarthritis. In addition, a recent study re-
vealed that ADAMTS-5 knockout mice were physiologically normal
and protected from developing osteoarthritis.5 A short-interfering
RNA study showed that both ADAMTS-4 and ADAMTS-5 were
involved in human cartilage degeneration in OA.6 Therefore,
ADAMTS-4 and ADAMTS-5 have been considered as new potential
drug targets for the development of new therapeutic agents to
treat osteoarthritis.7,8
aggrecanase, we designed and synthesized a series of
a-glutamic
acid scaffold based 4-(benzamido)-4-(1,3,4-oxadiazol-2-yl) buta-
noic acids (2) as new potential ADAMTS inhibitors. In the structure
of compound 2, the carboxylic acid group may serve as a zinc-bind-
ing group; while the biphenyl system and the substituted phenyl
group may mimic the corresponding moieties in compound 1 to
interact with the S10 and S20 pockets, respectively. A highly rigid
and metabolic stable fragment, 1,3,4-oxadiazol-2-yl, was also
introduced to bridge the scaffold (ZBG and P10 group) and the P20
group (2).
The synthesis of designed compounds 2a–2n was outlined in
Scheme 1.14,15 Briefly, hydrazide 3 coupled with Boc-Glu(Ochx)-
OH to afford the diacylhydrazide 4. Cyclodehydration of 4 with
Ph3P, CBr4 and triethylamine provided oxadiazole 5. With interme-
diate 5 in hands, compound 6 was readily prepared by selectively
removing the N-Boc group via TFA and following acylation with
different carboxylic acids. Finally, compounds 2a–2n were easily
obtained by hydrolysis of 6.
Since the first discovery of the ADAMTS-4 inhibitors in 2001,9
several classes of small-molecular inhibitors of ADAMTS have been
identified.10–12 Structural feature analysis of these aggrecanase
inhibitors suggested that almost all the compounds possessed a
⇑
The designed compounds were preliminarily screened for their
inhibitory activities against ADAMTS-4 at 1.0 and 10.0 lM by using
Corresponding author. Tel.: +86 20 32015276; fax: +86 20 32015299.
E-mail address: ding_ke@gibh.ac.cn (K. Ding).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.009

4458
L. Peng et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4457–4461
was threefold more potent than the parental compound 2b. (Table
P1' moiety
P1' moiety
1) The corresponding tri-substituted compound 2h based on 2a
displayed the best inhibitory activity against ADAMTS-4 with
IC50 value of 1.2 lM (Table 1).
O
O
Y
X
To investigate the selectivity, the inhibitory potencies of four
most potent compounds 2b, 2h, 2k, and 2n against ADAMTS-5
were also evaluated by using a similar biochemical assay.16
Although compounds 2b and 2k displayed modest selectivity over
ADAMTS-4 (five to sixfold lower potency against ADAMTS-5), com-
pounds 2h or 2n showed almost equal potency against ADAMTS-4
O
O
NH
O
O
HO
ZBG
O
HO
N
H
N
HN
N
A
R
n
ZBG
HO
P2' moiety
and ADAMTS-5 (1.2
4.3 M for compound 2n).
Molecular docking study was performed to investigate the
lM vs 0.8 lM for compound 2h; 3.3 lM vs
l
P2' moiety
2
1
binding modes of compound 2h with ADAMTS-4 and ADAMTS-5
using Glide.17 The results are illustrated in Figure 2A and B. Since
the active sites of ADAMTS-4 and ADAMTS-5 shared highly con-
served residues with similar configurations,13 2h bound to the pro-
teins with similar modes. The carboxylic acid of 2h chelated with
the zinc ions as expected, and the rigid biphenyl group protruded
into the S10 pocket and made extensive hydrophobic contacts with
the residues like His361, Ala392, Ala396, and Val398 in ADAMTS-4
(His410, Ile442, Leu443, and ILE446 in the case of ADAMTS-5).
Moreover, the amide group formed hydrogen bonds with the main
chain residues on the S20-loops (Leu330 in ADAMTS-4 and Leu379
in ADAMTS-5). The significant improvement of the inhibitory po-
tency of 2h can be attributed to the hydrogen bonds formed be-
tween the methoxyl groups on the phenyl ring and the Met395
in ADAMTS-4 (the Ser375 in ADAMTS-5). Overall, the low selectiv-
ity of the most potent compounds 2h may also stem from the puta-
tive similar binding modes in ADAMTS-4 and ADAMTS-5. Not
surprisingly, the inactive compound 2e could not bind to the pock-
ets of ADAMTS-4 or ADAMTS-5 properly. Due to the steric hin-
drance caused by the dimethyl group between the oxadiazol and
phenyl rings, the P20 moiety of 2e was not able to fully occupy
the S20 binding sites in the proteins. Moreover, lacking of polar
substituents on the P20 phenyl also prohibits forming hydrogen
bonds with the S20 site and further decreases the potency of 2e
(as shown in the Supplementary data).
Figure 1. The designed 4-(benzamido)-4-(1,3,4-oxadiazol-2-yl) butanoic acids 2.
a well established fluorimetric assay (SensoLyte 520 aggrecanase-1
assay kit).16 Although (S)-4-(5-benyl-1,3,4-oxadiazol-2-yl)-4-
biphenyl-4-ylcarbosamido butanoic acid (2a) did not show obvious
inhibition against ADAMTS-4 at 10.0
dependently inhibited the enzymatic activity of ADAMTS-4 with
inhibitory rates of 30% or 59% at 1.0 or 10.0 M, respectively. Fur-
ther evaluation revealed that compound 2b possessed an IC50 value
of 9.7 M against ADAMTS-4 (Table 1). However, when the termi-
lM, compound 2b dose-
l
l
nal phenyl group in compound 2b was replaced with 3-pyridinyl or
4-pyridinyl group (compounds 2c and 2d, respectively), obviously
lower bioactivity than that of original compound 2b was observed.
The structure–activity study revealed that although the intro-
duction of stereo hindrant dimethyl group at
a or b position of
the 2-phenyl-ethyl group in compound 2b did not obviously affect
its ADAMTS-4 inhibition activity (Table 1, compounds 2i and 2j),
when the benzyl group in compound 2a was substituted with
a,a-dimethyl or cyclopropyl group, significantly lower ADAMTS-4
inhibitory potency was observed (Table 1, compounds 2e and
2f). Further investigations demonstrated that the ADAMTS-4 inhib-
itory activity was barely affected by a mono-substitution at R posi-
tion (Table 1 and Figure 1, compounds 2g, and 2k–2m).
Interestingly, when the 3,4,5-trimethoxyl groups were intro-
duced to the bottom phenyl ring (ring A in compound 2a or 2b,
Figure 1and Table 1), the potency against ADAMTS-4 was obviously
improved. For instance, the 3,4,5-trimethoxyl substituted com-
Very recently, the S10 loop of ADAMTS-5 was proved flexible
upon binding with compound 1 by creating a novel pocket to
accommodate the rigid and hydrophobic P10 moiety, which might
be crucial for designing new potent and selective aggrecanase
inhibitors.18 In light of this new site flexibility, we also modeled
the binding poses of 2a and 2h using this new structure (altered
S10 pocket, PDB ID: 3LJT). The biphenyl moiety of 2h could occupy
the same S10 pocket induced by the phenylbenzodioxole of the
compound 1 and make variant hydrophobic contacts with the res-
idues in S10 like Phe406, Leu438, and Ile446 (as shown in Figure
2C). The carboxylic acid still chelated with the zinc ion and the ring
B protruded deeper into the S20 site with slightly different orienta-
tions from the binding pose in the conventional S10 pocket. Simi-
larly, the improvement of the inhibitory potency of 2h over 2a
could be attributed to the hydrogen bond formed between the
m-methoxyl group on the phenyl ring and Thr444 (as shown in
the Supplementary data). However, it is difficult to decide the ac-
tual binding modes of the inhibitors just from the docking results,
because the docking scores for 2h in the conventional and altered
S10 pockets are very close (GScores are ꢀ11.34 and ꢀ10.85, respec-
tively). Further designs of the new inhibitors based on two differ-
ent molecular docking studies are undergoing and will be
reported in due course.
pound 2n had an IC50 value of 3.3 lM against ADAMTS-4, which
O
O
N
O
O
H
N
H
N
a
R
b
NH2
BocHN
BocHN
NH
N
O
O
O
O
R
R
3
4
5
c,d
O
OH
O
O
O
e
O
N
N
H
N
R
N
O
N
H
N
R
O
Y
X
Y
X
2a-n
6
In summary, a series of 4-(benzamido)-4-(1,3,4-oxadiazol-2-yl)
butanoic acids were designed and synthesized as new dual inhibi-
tors of ADAMTS-4 and ADAMTS-5. These compounds dose-depen-
dently inhibited the enzymatic activities of both ADAMTS-4 and
ADAMTS-5. One of the most potent compound 2h potently inhib-
Scheme 1. Reagents and conditions: (a) Boc-Glu(Ochx)-OH, HATU, DIPEA, CH2Cl2,
rt, 65–75%; (b) PhP3, CBr4, Et3N, CH2Cl2, rt, 60–70%; (c) TFA, CH2Cl2, rt, 98%; (d)
diarylcarboxylic acid, EDCI, HOBT, Et3N, CH2Cl2, rt, 85%; (e) LiOH, THF/MeOH/H2O,
rt, 99%.

L. Peng et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4457–4461
4459
Table 1
4-(Benzamido)-4-(1,3,4-oxadiazol-2-yl) butanoic acids as new AMDAMTS inhibitorsa
O
OH
N
O
N
H
N
O
R
X
Y
2
Compd
X
Y
R
ADAMTS-4 inhibition
ADAMTS-5 inhibition (IC50, lM)
@ 1.0
lM
@ 10.0
l
M
IC50, lM
2a
2b
CH
CH
CH
CH
17
26
—
—
30
12
8
59
39
18
9.7
—
57.3
2c
CH
N
N
—
—
2d
CH
—
2e
2f
CH
CH
CH
CH
-5
5
5
—
—
—
—
20
2g
2h
CH
CH
CH
CH
5
21
86
—
—
MeO
MeO
47
1.2
0.8
MeO
OMe
2i
2j
CH
CH
CH
CH
23
19
47
45
—
—
—
—
2k
2l
CH
CH
CH
CH
CH
CH
35
27
22
66
56
51
4.9
—
24.3
—
MeO
Me
2m
—
—
HO
(continued on next page)

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L. Peng et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4457–4461
Table 1 (continued)
Compd
X
Y
R
ADAMTS-4 inhibition
ADAMTS-5 inhibition (IC50, lM)
@ 1.0
lM
@ 10.0
lM
IC50
3.3
, lM
MeO
MeO
2n
CH
CH
35
74
4.3
OMe
a
The data were reported as the means of at least three independent experiments, standard deviation are 15% of the data presented.
Figure 2. Docking poses of compound 2h into the active sites of (A) ADAMTS-4, (B) conventional S10 site of ADAMTS-5, and (C) altered S10 site of ADAMTS-5. The zinc ions are
depicted as violet spheres and the predicted inter-molecule hydrogen bonds were shown as yellow dash lines. The binding sites of the ADAMTS-5 structures with different S10
pockets conformations were superposed, featuring the pocket of the altered S10 site structure shown in orange mesh and S10 loop colored in cyan. The binding pose predicted
in the altered S10 pocket is colored in cyan and the binding pose in the conventional S10 pocket as well as the conventional S10 loop are shown in reference colored in green.
The three hydrophobic residues at the bottom of the altered S10 in contact with the biphenyl moiety of 2h are shown in green sticks.
6. Song, R. H.; Tortorella, M. D.; Malfait, A. M.; Alston, J. T.; Yang, Z.; Arner, E. C.;
ited ADAMTS-4 and ADAMTS-5 with IC50 values of 1.2 and 0.8 lM,
respectively. These inhibitors might serve as new lead compounds
for further development of therapeutics to treat osteoarthritis.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.06.009.
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16. The biological evaluation was performed following the manufactory
instruction from the ANASPEC (www.anaspect.com).
17. Docking protocol: Prior to docking, compound 1h was prepared to create
meaningful protonation states and cover potential tautomers (LigPrep,
Schrödinger Inc., www.schrodinger.com). Crystal structures of ADAMTS-4
(2RJP) and ADAMTS-5(2RJQ) were selected for docking (Glide, Schrödinger Inc.,
www.schrodinger.com). Amino acid residues located within 14 Å from the
complexed inhibitors were defined as part of the binding site and prepared
with the Protein Preparation Wizard of Schrödinger to add hydrogen atoms
and appropriate protonation states without further energy minimization. The
Glide XP mode was applied for docking with default settings and the top
ranked configurations were observed as the predicted binding poses. Results
are presented in Supplementary data.
18. Shieh, H.-S.; Tomasselli, A. G.; Mathis, K. J.; Schnute, M. E.; Woodard, S. S.;
Caspers, N.; Williams, J. M.; Kiefer, J. R.; Munie, G.; Wittwer, A.; Malfait, A.-M.;
Tortorella, M. D. Protein Sci. 2011, 20, 735.