Novel N-substituted 2-phenyl-1-sulfonylamino-cyclopropane carboxylates as selective ADAMTS-5 (Aggrecanase-2) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.02.024

A series of N-substituted sulfonylamino-alkanecarboxylate ADAMTS-5 (Aggrecanase-2) inhibitors has been synthesized and the in vitro enzyme SAR is discussed. This report is the first example of carboxylate-based ADAMTS-5 inhibitors which show strong potenc

Full text of DOI:10.1016/j.bmcl.2009.02.024

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1575–1580
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel N-substituted 2-phenyl-1-sulfonylamino-cyclopropane carboxylates
as selective ADAMTS-5 (Aggrecanase-2) inhibitors
a
a
a
b
b
Makoto Shiozaki a, , Katsuya Maeda , Tomoya Miura , Yosuke Ogoshi , Julia Haas , Andrew M. Fryer ,
*
Ellen R. Laird b, Nicole M. Littmann b, Steven W. Andrews b, John A. Josey b, Takayuki Mimura a,
Yuichi Shinozaki a, Hiromi Yoshiuchi a, Takashi Inaba a,
*
a Central Pharmaceutical Research Institute, Japan Tobacco Inc., 1-1, Murasaki-cho, Takatsuki, Osaka 569-1125, Japan
b Array BioPharma Inc., 3200 Walnut Street, Boulder, CO 80301, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of N-substituted sulfonylamino-alkanecarboxylate ADAMTS-5 (Aggrecanase-2) inhibitors has
been synthesized and the in vitro enzyme SAR is discussed. This report is the first example of carboxyl-
Received 27 November 2008
Revised 3 February 2009
Accepted 6 February 2009
Available online 11 February 2009
ate-based ADAMTS-5 inhibitors which show strong potency of IC50 < 0.1
over MMP-1 and TACE.
lM with excellent selectivity
Ó 2009 Elsevier Ltd. All rights reserved.
Keyword:
ADAMTS-5 inhibitors
Human osteoarthritis (OA) is a progressive disease of the joints
characterized by degradation of articular cartilage, which appears
to be a result of uncontrolled proteolytic extracellular matrix
destruction. A major component of the cartilage extracellular
matrix is aggrecan, and its loss from cartilage is believed to be
the early event that leads to further breakdown of extracellular
matrix macromolecules.1
ing group (ZBG). While this ZBG group imparts high levels of
potency to these molecules due to the strong binding affinity to
the Zn ion, the enzyme selectivities of these types of inhibitors
tend to be problematic. For this reason, the use of milder ZBGs,
Aggrecanases are members of the ADAMTS (A Disintegrin and
Metalloprotease with Thrombospondin Motifs) family of zinc
metalloproteases, and comprised of two isozymes including
ADAMTS-4 (Aggrecanase-1) and ADAMTS-5 (Aggrecanase-2). They
are known to cleave the aggrecan interglobular domain at the
Glu373-Ala374 peptide bond,2 and are 1000-fold more efficient
than any other MMP enzymes at cleaving aggrecan.
Until recently, it has been unclear which ADAMTS isozyme is
the primary source of devastating aggrecanase activity in cartilage.
In 2005, two independent groups reported that ADAMTS-5 knock-
out mice showed protective effects against the erosion of cartilage
and the occurrence of arthritis.3,4 With these reports, ADAMTS-5
has begun to receive greater attention as an important target for
the prevention of OA. Since the first discovery of ADAMTS-4 inhib-
itors in 2001,5 intensive efforts have been made by several groups
to target inhibition of this isozyme.6–8 By contrast, there have been
only a few examples of ADAMTS-5 inhibitors reported to date.9–11
Historically, the vast majority of Zn metalloprotease inhibitors
including ADAMTS inhibitors contain a hydroxamic acid zinc-bind-
Figure 1. Proposed binding mode for compound 1. The inhibitor was docked
manually into the X-ray structure of ADAMTS-5 (PDB accession code 3B8Z12).
Compound 1 is depicted in green, the solvent-accessible surface of the protein is
violet, and selected side chains are shown in white. The zinc ion is portrayed as a
blue sphere. Electronic contacts are shown as yellow dashed lines.
* Corresponding authors.
E-mail address: makoto.shiozaki@ims.jti.co.jp (M. Shiozaki).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.024

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M. Shiozaki et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1575–1580
O
O
HCl
H
Cl
H2N
S N
O
S
O
a
Cl
Cl
O
O
O
O
9
10
11
O
O
R
O
O
O
R
Cl
Cl
S
N
S
N
b
c
O
HO
O
12
1-8
Scheme 1. Reagents and conditions: (a) Et3N, DCM, rt, 1 h (98%); (b) RX, K2CO3, DMF, 60 °C, 5 h or ROH, DIAD, PPh3, THF, rt, 2 h (75–98%); (c) TFA, DCM, rt, 2 h (56–99%).
Table 2
such as the carboxylic acid, the hydantoin, and the 2-thioxothiaz-
In vitro ADAMTS-5 inhibitory activity of constrained analogues of compound 8
olidin-4-one, has become a popular approach in recent ADAMTS
inhibitor programs.9–11
O
O
O
O
N
N
Cl
S N
Cl
S N
O
O
HO
HO
O
O
O
O
H
Cl
S
N
13
14
HO
O
Compounds
Enantiomer
% Inhibition at 10
0.10
l
M
Agg-2 IC50
0.20
(lM)
13
14
(1R, 2S)
1
R
As a starting point for our research efforts toward ADAMTS-5
inhibitors, we focused on weaker carboxylic acid ZBG based inhib-
itors, reasoning that these would allow us to obtain good selectiv-
ity over other MMPs. From an initial carboxylic acid focused
combinatorial library, we found (R)-phenylalanine derivative 1 to
be a moderate ADAMTS-5 inhibitor. We were attracted to this
Table 3
Modification of the substituent on the N-atom of N-substituted 2-phenyl-1-
sulfonylamino-cyclopopanecarboxylate
O
O
N
R
S N
O
HO
Table 1
O
In vitro ADAMTS-5 inhibitory activity of N-substituted 2-[4-(4-Cl-phenyl)phenyl]sul-
fonylamino-3-phenyl-propionates
Compounds
R
% Inhibition at
10
Agg-2 IC50
M)
l
M
(l
O O
S N
R
Cl
13
15
0.20
0.88
Cl
Cl
HO
*
*
O
Compounds
Enantiomer
R
% Inhibition
at 10
Agg-2 IC50 (lM)
N
l
M
H
TFA
1
R
3.2
8.0
N
*
16
17
1.1
6.6
Cl
*
*
Me
Me
2
3
R
S
*
*
*
TFA
ꢀ0.12
N
4
5
6
7
R
R
R
R
46
H
N
*
18
4.5
*
*
22
30
O
TFA
19
20
21
18
N
*
HO
7.3
1.6
ꢀ1.5
*
*
*
O
2.9
N
8
R
*
O

M. Shiozaki et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1575–1580
1577
structure because we believed the synthetic flexibility of the
amino acid derived sulfonamide would allow us to rapidly evaluate
the ADAMTS-5 SAR.
a
-
of the zinc by the carboxylic acid and deep association of the biaryl
moiety in the hydrophobic S10 pocket. Additionally, the benzyl
group rests above the surface formed by the imidazole ring of
His414.
As a beginning to our SAR exploration, we investigated substitu-
tion effects on the sulfonamide N-atom (Table 1). Initially, a simple
methyl group was introduced on the sulfonamide nitrogen atom
for both R- and S-phenylalanine derivatives. The R-configuration
was found to be favorable and complete loss of potency was
observed for the corresponding S-isomer 3. This remarkable enan-
tiometric preference led us to use the (R)-phenylalanine as a core
During the course of this work, we utilized homology models
and X-ray crystal structures of other ADAM family members to
guide our compound design and SAR evaluation. Recently, X-ray
structures of ADAMTS-4 and ADAMTS-5 have become available12,13
and we have incorporated them here (PDB code 3B8Z12) to
illustrate refined models of selected compounds. Figure 1 depicts
the expected binding mode of compound 1 in ADAMTS-5; all
of the conventional interactions are present, including chelation
O
O
O
O
O
O
HO
O
S+
+
a
b
I
O
O
O
O
22
23
24
25
O
O
O
O
H
N
H
N
H
N
c
d
e
O
HO
O
HO
O
O
O
O
26
27
28
Y
O
O
O
O
H
N
H
N
X
H2N
S
f
g
h
O
O
O
O
O
O
X = Cl, Br, I
Y = N, CH
29
30
31
Y
O
O
Y
O O
N
N
S
N
R1
X
S
N
i
j
O
O
O
O
O
O
32
33
O
N
O
O
N
O
H
H
N
Cl
S
N
S
O
l
k
30
O
O
O
O
O
34
35
O
N
O
S N
N
Cl
i
O
O
O
36
15-21
m
33 or 36
Scheme 2. Reagents and conditions: (a) NaH, DMSO, 12 h (76%); (b) 4 N NaOH, EtOH/H2O, 12 h (92%); (c) Et3N, DPPA, t-BuOH, 120 °C, 12 h (86%); (d) 4 N NaOH, EtOH/H2O,
110 °C, 6 h (92%); (e) (ꢀ)-cinchonidine, AcOEt/2–propanol, rt, 12 h (31%, 97.7% ee,); (f) Me2NCH(Ot-Bu)2, toluene, 100 °C, 5 h (92%); (g) p-TsOHꢁH2O, MeOH, 12 h (85%); (h)
ArSO2Cl, pyridine, CHCl3, 12 h (59–81%); (i) 1-(2-hydroxyethyl)-pyrrolidine-2-one, DIAD, PPh3, THF, 2 h or 1-(2-bromoethyl)-pyrrolidine-2-one, K2CO3, DMF, 90 °C, 12 h
(68–96%); (j) R1B(OH)2, Pd(PPh3)4, 2 M Na2CO3/DME, 90 °C, 1 h or R2OCH2CCH, PdCl2(PPh3)2, CuI, Et3N, CHCl3, rt, 0.5 h (21–99%); (k) ClSO2NCO, 2-Cl-ethanol, NMM, CH3CN,
0–50 °C, 2 h (56%); (l) 4-(4-Cl-phenyl)-1,2,3,6-tetrahydropyridineꢁHCl, Et3N, 1,4-dioxane, 100 °C, 12 h (40%); (m) TFA, CHCl3, 12 h (43–77%).

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M. Shiozaki et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1575–1580
skeleton for further study. A small set of hydrophobic R-substitu-
ents were introduced with intent to pick up another interaction
in the binding pocket of ADAMTS-5. Although most of the com-
pounds with R-substituents larger than a methyl group (4–6) were
less potent than compound 2, compound 8 showed subtle but sig-
nificant improvement in potency. This discovery was quite encour-
aging since phenyl analogue 7 showed reduced activity. We
hypothesized that the increased potency of 8 may be the result
of a new hydrogen-bonding interaction between the terminal lac-
tam and the enzyme.
Compounds 1–8 were prepared according to Scheme 1.
Phenylalanine t-Bu ester was first sulfonylated with [4-(4-Cl-
phenyl)phenyl]sulfonyl chloride under basic conditions to give
compound 11, which was then alkylated with the corresponding
halides in the presence of base or with the alcohols under Mitsun-
obu conditions to give 12. Finally, ester cleavage by TFA led 12 to
compounds 1–8.
sequence of sulfonylation, alkylation, and subsequent ester cleav-
age gave the desired compounds.
Having identified potent amino acid and sulfonamide nitrogen
substitutions, we next turned our attention to the sulfonamide
S10 moiety. Our SAR investigation of the biphenyl portion is shown
in Table 3. The importance of the internal benzene ring of 13 was
made apparent when a 4-fold reduction of inhibitory activity was
observed by replacement of this ring with the 1,2,3,6-tetrahydro-
pyridine ring or pyridine ring (15 and 16). Additionally, the termi-
nal 4-Cl-phenyl substituent was indispensable for potency as loss
of potency was observed by the replacement of either the chlorine
atom (17 and 18) or the phenyl ring itself (19–21).
The synthesis of compounds 15–21 is shown in Scheme 2.
Cyclopropanation was first performed with benzylidene malonate
to give 24. The sterically less hindered ester was selectively hydro-
lyzed and the resultant mono-ester was transformed to compound
26 by Curtius rearrangement. After another hydrolysis, an optical
resolution was performed with (ꢀ)-cinchonidine to give desired
enantiomer in over 97% ee. After protection of the carboxylate as
the t-Bu ester, the Boc group was selectively deprotected with
stoichiometric amounts of p-TsOHꢁH2O, then sulfonylation and
Starting from compound 8, modification of the amino acid core
skeleton was investigated next. The transformation of compound 8
into the D-cyclo-phenylalanine analogue 13, by means of a methy-
lene group addition between the alpha position of the carboxylate
and the beta position of 8, caused an increase of the binding affin-
ity for ADAMTS-5 of one order of magnitude (Table 2).
The increased potency of compound 13 can be attributed to
rigidifying the compound into a favorable conformation for bind-
ing in the active site of the enzyme. This hypothesis is supported
by the significant loss of potency observed for the more flexible
analogue 14.
Compounds 13 and 14 were prepared essentially the same way
as described in Scheme 1. Each amino acid portion was commer-
cially available or prepared according to known literature proce-
dures.14,15 Subjecting these amino acids to the earlier described
Table 4
Modification of the substituent on the N-atom of 2-phenyl-1-sulfonylamino-cyclop-
opanecarboxylate: cyclic amide and urethane series
O
O
S N
R
Cl
HO
O
Compounds
R
Agg-2 IC50
0.20
(lM)
N
13
*
O
O
O
37
38
39
40
41
0.31
0.22
0.21
0.18
0.078
N
N
*
*
O
O
N
N
N
*
*
*
O
O
O
O
Figure 2. (A) Proposed binding mode for compound 51. The color scheme is as
described for Figure 1. Residues that contribute to the outer rim of the S10 pocket
are shown in white; for clarity, only the side chain of Leu443 is shown explicitly. (B)
The X-ray crystal structure of TACE in complex with a succinate-based inhibitor
(PDB accession code 1BKC).16 A hydrogen-bond to the main chain NH of Ala439 is
comparable to the contact to Leu443 illustrated in Figure 2A.
O

M. Shiozaki et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1575–1580
1579
R1
O
O
O O
S N
H
Cl
Cl
S
N
a
b
O
O
30
O
O
55
56
Br
R2
O
O
O
O
Cl
Cl
S
N
S
N
d
c
O
O
55
O
O
57
58
R1 or R2
O
O
Cl
S
N
e
HO
56 or 58
O
37-54
Scheme 3. Reagents and conditions: (a) [4-(4-Cl-Phenyl)phenyl]sulfonyl chloride, pyridine, CHCl3, 12 h (90%); (b) R1CH2CH2Br, K2CO3, DMF, 60 °C, 2.5 h or R1CH2CH2OH,
DIAD, PPh3, THF, rt, 2 h (37–99%); (c) 1,2-dibromoethane, K2CO3, DMF, 60 °C, 4 h (89%); (d) R2H, K2CO3, DMF, 80 °C, 12 h (13–62%); (e) 4 N HCl in AcOEt, rt, 12 h or TFA, CHCl3,
rt, 12 h (21–99%).
successive N-alkylation gave compound 32 which was ready for
the following Suzuki- or Sonogashira-coupling. Both couplings
proceeded in moderate to good yield under typical conditions,
and a final acidic ester cleavage gave the target compounds
16–21. Amino intermediate 30 could also be converted to
oxazolidine-2-one-3-sulfonamide 34, which in turn could be re-
acted with 4-(4-chlorophenyl)-1,2,3,6-tetrahydropyridine to give
sulfamide 35. N-Alkylation of 35 followed by the final ester cleav-
age gave compound 15.
The last point of modification was the substituent on the N-
atom of the sulfonamide. According to the SAR of D-phenylalanine
derivatives shown in Table 1, the 2-(2-oxopyrrolidinyl)ethyl was
identified as a favorable substituent at that position, and we envi-
sioned the existence of its putative hydrogen-bonding partner in
the binding pocket of ADAMTS-5.
At this point, we decided to explore this 2-(2-oxopyrrol-
idinyl)ethyl analogues further with the goal of identifying better
binders and to bolster the inhibitory activity. The initial attempts
of the introduction of an extra heteroatom or the ring expansion
were discouraging since virtually no improvement was observed
in potencies (compounds 37–40, Table 4) of the carbonyl contain-
ing ring systems. Replacement of the pendent ring with an aro-
matic ring and subsequent introduction of a methoxy group was
effective to increase the potency as seen in compound 41, which
showed an IC50 of 78 nM.
The discovery of compound 41 possessing a highly functional-
ized heteroaromatic ring prompted us to explore these analogues
further. The pyridine analogues were first synthesized and a 3-pyr-
idyl isomer was found out to be most favorable among the three
regioisomers (42–44). The five-membered azole derivatives were
also synthesized and all the compounds showed good potency
without strict restriction of the number and the position of N
atoms (45–50). Among these five-membered azole derivatives,
imidazole derivative 45 was chosen in consideration of the syn-
thetic flexibility and the ease of further modification. Since the
methoxy group of compound 41 was speculated to be a key
functionality for the observed improvements in potency, analogous
Lewis basic functionalities were introduced on the imidazole ring
of compound 45, and these changes resulted in compounds 51
and 53, with IC50s of 73 and 71 nM, respectively.
Figure 2 illustrates a rationale for the improved potency of com-
pound 51 and other potent analogues: A correctly placed hydro-
gen-bond acceptor is capable of reaching the main chain NH of
Leu443, which forms part of the outer rim of the S10 pocket.
Consideration of X-ray structures for MMPs and ADAM family
members that contain peptide-like inhibitors show that this
hydrogen-bonding interaction is common (Fig. 2B illustrates this
interaction using an X-ray crystal structure of TACE in complex
with a succinate-based inhibitor16). The methoxy group provides
an added hydrophobic contact against the surface created by the
branched side chain of Leu443.
Compounds 37–54 were prepared according to Scheme 3. After
sulfonylation of compound 30, the resultant sulfonamide 55 was
either alkylated with the appropriate alkyl bromides or reacted
with the appropriate alcohols under Mitsunobu conditions to give
compound 56. Since some 2-bromoalky- or 2-hydroxyalkyl-substi-
tuted heterocycles were neither commercially available nor easy to
synthesize, the corresponding substituents were introduced by
way of the ethyl bromide 57 which was easily prepared from com-
pound 55 and 1,2-dibromoethane.
To access our original hypothesis that these carboxylic acid ZBG
ADAMTS-5 inhibitors would maintain high selectivity, we exam-
ined the potency of the most active compounds in MMP-1 and
TACE, representatives of two distinct classes of Zn metalloproteas-
es, the MMP and ADAM family. In fact all the compounds shown in
Tables 4 and 5 had no inhibitory activity against these two en-
zymes below 10 lM (data not shown). This compound series dem-
onstrates a high level of selectivity for inhibition of ADAMTS-5 over
these closely related Zn metalloproteases.
In conclusion, we have discovered a series of novel N-substi-
tuted 2-phenyl-1-sulfonylamino-cyclopropane carboxylates as
ADAMTS-5 inhibitors which are conformationally constrained
phenylalanine analogues. To the best of our knowledge, this was
the first disclosure of carboxylate-based ADAMTS-5 inhibitors

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M. Shiozaki et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1575–1580
Table 5
which show both strong potencies (IC50 < 0.1 lM) as well as good
Modification of the substituent on the N-atom of 2-phenyl-1-sulfonylamino-cyclop-
opanecarboxylate: heteroaromatic series
selectivities against closely related MMPs like MMP-1 and TACE.
The continued efforts to further optimize this series of compounds
are currently ongoing and will be reported in due course.
O
O
S N
R
Cl
Acknowledgments
HO
O
The authors wish to thank our Analytical Research and
Development Laboratories for collecting analytical data and
Dr. Jun-ichi Haruta is also acknowledged for their continuous
encouragement.
Compounds
R
Agg-2 IC50
0.75
(lM)
HCl
N
42
*
N
Supplementary data
43
44
45
46
47
48
49
50
51
0.29
0.82
0.32
0.18
0.19
0.26
0.27
0.27
0.073
*
*
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.02.024.
HCl
N
HCl
HCl
N
References and notes
N
N
N
*
*
*
*
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N
N
N
N
HCl
N
N
N
N
N
N
*
N
N
N
N
N
*
*
O
N
O
HCl
N
HCl
N
52
0.23
*
O
O
N
N
TFA
N
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N
N
53
54
0.071
0.17
*
*
N
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TFA
N
N