Design, synthesis and SAR investigation of thienosultam derivatives as ADAMTS-5 (aggrecanase-2) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.01.120

A series of 1,1-dioxothieno[2,3-d]isothiazole (thienosultam) derivatives were designed and synthesized as novel ADAMTS-5 inhibitors for an investigation into a side chain of thienosultam for the S1′ pocket. The resulting compounds (19 and 24) show high AD

Full text of DOI:10.1016/j.bmcl.2013.01.120

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2111–2116
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and SAR investigation of thienosultam derivatives
as ADAMTS-5 (aggrecanase-2) inhibitors
⇑
Masakazu Atobe , Naomi Maekawara, Masashi Kawanishi, Hiroko Suzuki, Eiichi Tanaka, Shiro Miyoshi
Pharmaceutical Research Center, Asahi Kasei Pharma Corporation, 632-1 Mifuku, Izunokuni-shi, Shizuoka 410-2321, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 1,1-dioxothieno[2,3-d]isothiazole (thienosultam) derivatives were designed and synthesized
as novel ADAMTS-5 inhibitors for an investigation into a side chain of thienosultam for the S10 pocket.
The resulting compounds (19 and 24) show high ADAMTS-5 inhibition and other MMP selectivity, and
these compounds show good oral bioavailability.
Received 27 November 2012
Revised 24 January 2013
Accepted 28 January 2013
Available online 9 February 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Osteoarthritis
Aggrecanase-2
ADAMTS-5
Thienosultam
S10 pocket
Osteoarthritis (OA) is characterized by loss of joint cartilage and
thickening of bone, and may lead to bony outgrowths. Joint carti-
lage functions to absorb impacts when pressure is applied to the
joint or the joint is put into motion.1 The pathogenesis of OA is
poorly understood, but a major feature is the loss of the two most
important components of cartilage extracellular matrix (ECM);
Type II collagen and aggrecan.2
In 2005, Glasson et al.7 and Stanton et al.8 reported that in a
mouse genetically modified to show resistance to cleavage in the
interglobular domain of aggrecan, surgically induced OA showed
reduced loss of aggrecan; thus, destruction of cartilage is inhibited
and regeneration of cartilage is promoted. ADAMTS-5 may there-
fore be a suitable target for the development of novel drugs de-
signed to inhibit cartilage destruction in OA.
Aggrecan, a major glycoprotein that is expressed in cartilage tis-
sues, is highly hydrated due to the presence of long, negatively
chargedpolysaccharidechains, thusprovidingcartilagewithitsabil-
itytoresistcompressiveloads.3 Inaddition,aggrecanhasaprotective
effectagainst collagendegradation. Mechanistically, Type II collagen
is exposed due to degradation of aggrecan, the exposed collagen be-
comes an easy target for enzymatic degradation by collagenase, a
proteolytic enzyme specific for collagen chains, and absorptivity of
the cartilage matrix is reduced by loss of aggrecan, resulting in re-
duced elasticity of the matrix and increased mechanical stress on
the collagen chain, thus causing collagen chain breakdown.4
Aggrecanase is a protease that cleaves the aggrecan molecule.
Specifically, it cleaves the peptide bonds between Glu373 and
Ala374 that are present in the aggrecan interglobular domain.5
Two types of aggrecanase, aggrecanase-1 and aggrecanase-2, are
found in joint tissue. These enzymes belong to the family of A Dis-
integrin and Metalloprotease with ThromboSpondin motifs
(ADAMTS), and are also referred to as ADAMTS-4 and ADAMTS-5,
respectively.6
To date, small-molecule ADAMTS-5 inhibitors have been de-
scribed from hydroxamic acid analogues9 or non-hydroxamic acid
analogues,10 no doubt due to the presence of a zinc atom at the ac-
tive site of these enzymes. More recently, Shiozaki et al. reported a
series of sulfonamide compounds bearing cyclopropane carboxyl-
ate to show high ADAMTS-5 inhibition and high selectivity over
other Matrix Metalloproteases (MMPs).11 To design a novel class
of ADAMTS-5 inhibitors, we explored 1,1-dioxothieno[2,3-d]iso-
thiazole (thienosultam) derivatives, and aimed to create an original
structure and to give different profile compound by fixing the con-
formation of sulfonamide 1a (Fig. 1).
Unrestricted compound 1a was previously reported by O’Brien
et al.12 and Hu et al.13 This compound has ADAMTS-5 inhibitory
activity, but was low selectivity against ADAMTS-4, MMPs (Ta-
ble 1).14 MMP-2,15 -316 and -1417 knockout mice studies have been
conducted reported, and inhibition of MMP-2, -3 and -14 may facil-
itate proteoglycan degradation. In addition, an investigation in
ADAMTS-4 knockout mice confirmed that ADAMTS-4 is not
responsible for aggrecan degradation in murine osteoarthritis.18
Therefore, we reasoned that an ADAMTS-5 selective inhibitor
would constitute an ideal OA therapeutic agent.
We prepared thienosultam 2 bearing a phenyl group, and found
⇑
Corresponding author. Tel.: +81 558 76 8493; fax: +81 558 76 5755.
E-mail address: atobe.mb@om.asahi-kasei.co.jp (M. Atobe).
that 2 has moderate ADAMTS-5 inhibitory activity (Table 1).19
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.120

2112
M. Atobe et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2111–2116
Figure 1. Strategy for the design 1,1-dioxothieno[2,3-d]isothiazole.
Table 1
Activity of selected sultams (1–5)
Compounds
R1
R2
ADAMTS-5
ADAMTS-4
MMP-2 (
0.040c
0.03
lM)
MMP-3 (
0.038c
5.1
lM)
MMP-14
ND
1a
2
8.2
2.1
l
Ma
3.0
1.1
l
Mb
–CONHOH
–COOH
lM
lM
1.2 lM
3
4
5
ꢀ3% (10
ꢀ3% (10
l
M inh)
M inh)
ꢀ1.7% (10
ꢀ1.7% (10
ꢀ1.6% (10
l
l
l
M inh)
M inh)
M inh)
16
12
20
12
>30
<30
<30
l
M
M
M
–CONHOH
–COOH
l
0.3
1.8
l
l
2.3 lM
ND = not determined.
a
In house data.
b
Assay details for the inhibition of ADAMTS-4 is described in Ref. 13.
Assay details for the inhibition of MMP-2 is described in Ref. 12.
c
Unfortunately, 2 showed lower selectivity against both MMP-2
(30 nM) and MMP-14 (1.2 M), while carboxylic acid 3 lost ADAM-
Table 2
Activity of linker (5–11)
l
TS-5 inhibitory activity. However, thienosultam 4 bearing a biphe-
nyl group showed reduced MMP-2 inhibitory activity, and
carboxylic acid 5 had enhanced anti-ADAMTS-5 activity.
These results suggested that pocket size of the side chain occu-
pying the S10 pocket provided good selectivity over both MMP-2
and MMP-3.20 The crystal structures of both ADAMTS-5 and
MMP-2 have been reported.21,22 X-ray structures co-crystallized
with different ligands show that the S10 pocket of ADAMTS-5 is
deep, as a result of a compact S10-loop. In contrast, the MMP-2
S10 pocket is entirely enclosed in the S10-loop, as if in a box, and
longer groups led to decreased activity.
Compounds R1
ADAMTS-5
MMP-2
M)
MMP-2/
ADAMTS5
(l
5
6
2.3
3.2
l
M
M
1.8
2.0
0.78
l
0.63
Based on this analysis, our initial investigation focused on the
side chain of thienosultam for the S10 pocket. We report on the en-
zyme activity in a series of analogs of 5 prepared in an effort to in-
crease selectivity by taking advantage of differences between
ADAMTS-5 and MMPs.
43.2%
(10 M inh)
7
8
>30
>30
l
ꢀ9% (10
lM
inh)
We evaluated various thienosultam linkers between the thieno-
sultam and the terminal phenyl group. As shown in Table 2, the
linkers for phenyl 5 and olefin-linkered 6 enhanced potency, but
these linkers were nearly equipotent against ADAMTS-5 and
MMP-2. The corresponding pyrazolyl 7 or piperazinyl 8 were not
beneficial for ADAMTS-5 potency, while phenyl-olefin-linkered 9
enhanced ADAMTS-5 potency and showed 2.79-fold greater
selectivity.
9
1.4
l
M
3.9
2.79
10
11
4.0
2.3
l
M
M
>30
>30
>7.5
>13
l
However, alkyne-linkered 10 showed significantly better selec-
tivity over MMP-2 (7.5-fold). In addition, the terminal phenyl sub-
stituent (5) was indispensable for potency, as loss of MMP-2
potency was observed with the replacement of the 3-thiophene
11. Thus, we next investigated the effects of terminal ring replace-
ment of other heterocyclic systems.
pared compounds bearing 2-thiophene 12, 1-N-Methyl 6-indole 13
and 1-N-Methyl 6-indazole 14, and found that these compounds
had increased ADAMTS-5 activity and retained good selectivity
for MMP-2. These compounds lead to higher lipophilicity; thus,
we synthesized nitrogen-containing heterocyclic compounds. But
pyridine 15 and pyrazole 16 lost ADAMTS-5 activity. To exploit
further activity, we prepared an additional set of substituted
pyridines, including dimethylamino group 17 and N-Benzyl-N-
Starting from compound 11, modification of the terminal ring
replacement was subsequently investigated (Table 3). We had pre-

M. Atobe et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2111–2116
2113
Table 3
Table 4
Activity of selected sultams (11–19)
Activity of selected sultams (8, 20–24)
Compounds R5
ADAMTS-5
M)
MMP-2
M)
MMP-2/
ADAMTS5
Compounds R4
ADAMTS-5 MMP-2
M) M)
MMP-2/
ADAMTS5
(
l
(
l
(
l
(
l
8
4.0
9.3
>30
>30
>7.5
11
12
2.3
1.3
>30
>30
>13
>23
20
>3.23
21
22
8.9
4.9
>30
>30
>3.37
>6.12
13
14
1
16.5
4
16.5
13.3
0.3
23
24
1.5
1.5
>30
>30
>20
>20
15
16
>10
>10
>30
>30
17
18
19
7
>30
>30
>30
4.5
reduction of 27 as primary alcohol 28, bromination of primary
alcohol 28 afforded the corresponding bromide 29, which under-
went NaH-mediated cyclization at 60 °C to give sultam derivative
30. Alkylation of 30 with NaH and bromide was converted 31,
which upon hydrolysis formed carboxylic acid 3. Hydroxamic acid
formation was formed by two-step sequence via amidation, fol-
lowed by hydrolysis with HCl to give 2 (R6 = H). Suzuki–Miyaura
coupling of compound 31 (R6 = Br) was used to construct the sec-
ond aromatic ring or vinyl moiety, followed by hydrolysis to give
5, 9 or 11–19. Hydroxamic acid formation was formed by above
condition to give 4 (R6 = Ph).
In the case of methyl 2-{5-bromo-1,1-dioxothieno[2,3-d]iso-
thiazole-2(3H)-yl}butyrate, the synthetic route started from the
commercially available methyl 3-chlorosulfonyl-2-thiophene car-
boxylate 32 (Scheme 2), which was first amidated to the corre-
sponding sulfamoyl 33 with NH4OH aq and then reduced with
DIBAL-H to give primary alcohol 34. After protection of 34 as
TBDPS ether 35, lithiation of the 2-position of thiophene and sub-
sequent quenching with CBr4 resulted in the formation of 2-bromo
1
>30
>75
0.4
methyl amino group 18. These compounds had enhanced activity
and 18 showed improved selectivity for MMP-2 (>30-fold). When
the pyrazole in compound 16 was induced by an N-1-benzyl group
(19), potency increased significantly and retained selectivity over
MMP-2 (>75-fold). These results suggested that long groups, such
as the benzyl group, may form additional hydrophobic interactions
with the ADAMTS-5 S10 pocket.
Investigation of the alkyne linker (Table 4) revealed that analogs
bearing pyridine 20, cyclohexanemethyl 21 and 3,5-di-(trifluoro-
methyl)phenyl group 22 displayed lower anti-ADAMTS-5 potency.
However, analogues bearing biphenyl group 23 and 4-N,N-dimeth-
ylamino group 24 showed increased anti-ADAMTS-5 activity and
displayed no MMP-2 inhibitory effect.
Table 5
Compounds 19 and 24 were tested for ability to inhibit ADAM-
TS-4, MMP-2, MMP-3 and MMP-14. As listed in Table 5, each com-
pound showed excellent ADAMTS-5 selectivity over the four other
Zn metalloproteases.
The pharmacokinetics of compounds 19 and 24 were examined
via two different routes of administration, intravenous (iv) and oral
(po), to male Sprague–Dawley rats. Animals received a single iv bo-
lus of 1 mpk and a p.o. dose of 5 mpk. Compound 19 exhibited low
clearance (16.7 mL/min/kg), and oral bioavailability was 47%. Most
strikingly, compound 24 had a clearance of 3.5 mL/min/kg and the
best oral bioavailability (179%) (Table 6).
Synthesis of thienosultam derivatives is shown in Schemes 1–3.
All compounds are racemate. As shown in Scheme 1, sulfonylation
of methyl 3-amino-5-phenyl-2-thiophencarboxylate 25, as a start-
ing material, under the Sandmeyer condition gave sulfonyl chlo-
ride 26, which yielded sulfonamide 27 on aminolysis. After
Activity of selected sultams (5, 19, 24)
Compounds ADAMTS-5
M)
ADAMTS-4
M)
MMP-2
M)
MMP-3
M)
MMP-14
(lM)
(l
(
l
(
l
(l
5
19
24
2.1
0.4
1.5
1.1
8
>10
0.03
>30
>30
1.2
>30
>30
5.1
>30
>30
Table 6
Pharmacokinetics dataa for compounds 19 and 24
Compounds
AUCb
Cmax
T1/2 (h)
Vss
Cl
F (%)
(lg h/mL)
(mg/mL)
(L/kg)
(L/h/kg)
19
24
4.73
106
3.09
29.5
1.8
2.8
0.33
0.34
16.7
3.5
47
179
a
Dose at 1 mg/kg (iv) and 5 mg/kg (p.o.).
AUC of 0 ? 1.
b

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M. Atobe et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2111–2116
Scheme 1. Reagents and conditions: (a) NaNO3, SO2, 20% HCl, then CuCl, AcOH, 0 °C (26a: 96%, 26b: 81%); (b) 0.5 M NH3–dioxane, rt (27a: 48%, 27b: 59%); (c) LiAlH4, THF, 0 °C
(28a: 77%, 28b: 77%); (d) PPh3, CBr4, CH2Cl2–THF (3:1), rt (29a: 82%, 29b: 88%); (e) NaH, DMF, 55 °C (30a: 38%, 30b: 31%); (f) methylbutanoate, NaH, DMF, 60 °C (31a: 71%,
31b: 71%); (g) 1 M NaOH aq, MeOH, rt (12–84%); (h) 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole hydrate, Et3N, DMF, rt; (i) 2 M
HCl aq, MeOH, rt, 2 steps (2: 57%, 4: 67%); (j) boronic acid, Pd2dba3, (o-tol)3P, K3PO4, CMF, 140 °C microwave irradiation (16–42%).
Scheme 2. Reagents and conditions: (a) 25%, NH4OH aq, dioxane, rt (67%); (b) DIBAL-H, CH2Cl2, 0 °C (86%); (c) TBDPSCl, imidazole, DMF, rt (32%); (d) LDA (4 equiv), THF,
ꢁ78 °C to rt (82%); (e) TBAF, THF, rt (99%); (f) PBr3, pyridine, CHCl3, ꢁ20 °C (99%); (g) NaH, DMF, 55 °C (58%); (h) methyl 2-bromobutyrate, NaH, DMF, 50 °C (44%).
thiophene 36. After deprotection of the TBDPS group with TBAF,
bromination of primary alcohol 37 afforded the corresponding bro-
mide 38, which underwent NaH-mediated cyclization at 60 °C to
give the sultam derivative 39. Alkylation of 39 with NaH and bro-
mide was converted 40, as a common intermediate.
Suzuki–Miyaura coupling of compound 40 was used to prepare
the olefin moiety and the pyrazole-ring moiety (Scheme 3), fol-
lowed by hydrolysis to give 6 or 7. Sonogashira coupling of com-
pound 40 was used to construct the alkyne moiety, followed by
hydrolysis to give 10 or 20–24. Buchwald N-arylation of compound

M. Atobe et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2111–2116
2115
Scheme 3. Reagents and conditions: (a) (E)-(4-(trifluoromethyl)styryl)boronic acid, Pd2dba3, (o-tol)3P, K3PO4, DMF, 140 °C microwave irradiation (53%); (b) 1 M-NaOH aq,
MeOH, rt (41–49%); (c) (1-benzyl-1H-pyrazol-3-yl)boronic acid, Pd(PPh3)4, 2 M Na2CO3 aq, DMF, 100 °C (38%); (d) alkyne, PdCl2(PPh3)2, CuI, Et3N, PPh3, THF, 80 °C (22%); (e) 1-
phenylpiperazine, Cs2CO3, xanphos, Pd2dba3, dioxane, 100 °C (22%).
M.; Tokar, C. Bioorg. Med. Chem. Lett. 2005, 15, 2808; (d) Xiang, J. S.; Hu, Y.;
Rush, T. S.; Thomason, J. R.; Ipek, M.; Sum, P.-E.; Abrous, L.; Sabatini, J. J.;
Georgiadis, K.; Reifenberg, E.; Majumdar, M.; Morris, E. A.; Tam, S. Bioorg. Med.
40 was used to give the piperazine-ring moiety, followed by hydro-
lysis to give 8.
In summary, a series of thienosultam derivatives were designed
and synthesized as new ADAMTS-5 inhibitors in an investigation
focusing on the side chain of thienosultam for the S10 pocket. The
resulting compounds 19 and 24 showed better ADAMTS-5 inhibi-
tion and MMP selectivity; these compounds also showed good oral
bioavailability. These inhibitors may serve as lead compounds for
further development of disease-modifying OA drugs.
Chem. Lett. 2006, 16, 1; (f) Sum, P.-E.; How, D. B.; Sabatini, J. J.; Xiang, J. S.; Ipec,
M.; Feyfant, E. PCT Int. Appl. WO2007008994, 2007.; (g) Shiozaki, M.; Maeda,
K.; Miura, T.; Ogoshi, Y.; Haas, J.; Fryer, A. M.; Laird, E. R.; Littmann, N. M.;
Andrews, S. W.; Josey, J. A.; Mimura, T.; Shinozaki, Y.; Yoshiuchi, H.; Inaba, T.
Bioorg. Med. Chem. Lett. 2009, 19, 1575; (h) Hopper, D. W.; Vera, M. D.; How, D.;
Sabatini, J.; Xiang, J. S.; Ipek, M.; Thomason, J.; Hu, Y.; Feyfant, E.; Wang, Q.;
Georgiadis, K. E.; Reifenberg, E.; Sheldon, R. T.; Keohan, C. C.; Majumdar, M. K.;
Morris, E. A.; Skotnicki, J.; Sum, P.-E. Bioorg. Med. Chem. Lett. 2009, 19, 2487; (i)
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