Acetylenic TACE inhibitors. Part 1. SAR of the acyclic sulfonamide hydroxamates

Article abstract of DOI:10.1016/S0960-894X(03)00514-6

The SAR of a series of potent sulfonamide hydroxamate TACE inhibitors, all bearing a butynyloxy P1′ group, was explored. In particular, compound 5j has excellent in vitro potency against isolated TACE enzyme and in cells, good selectivity over MMP-1 and MMP-9, and oral activity in an in vivo model of TNF-α production and a collagen-induced arthritis model.

Full text of DOI:10.1016/S0960-894X(03)00514-6

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2799–2803
Acetylenic TACE Inhibitors. Part 1. SAR of the Acyclic
Sulfonamide Hydroxamates
J. I. Levin,a,* J. M. Chen,a K. Cheung,a D. Cole,a C. Crago,a E. Delos Santos,a X. Du,a
G. Khafizova,a G. MacEwan,a C. Niu,a E. J. Salaski,a A. Zask,a T. Cummons,b A. Sung,b
J. Xu,a Y. Zhang,c W. Xu,c S. Ayral-Kaloustian,a G. Jin,a R. Cowling,a D. Barone,d
K. M. Mohler,d R. A. Blackd and J. S. Skotnickia
aWyeth Research, 401 N. Middletown Rd., Pearl River, NY 10965, USA
bWyeth Research, PO Box CN-8000, Princeton, NJ 08543, USA
cWyeth Research, 200 Cambridge Park Drive, Cambridge, MA02140, USA
dImmunex Corporation, Seattle, WA98101, USA
Received 18 February 2003; accepted 16 April 2003
Abstract—The SAR of a series of potent sulfonamide hydroxamate TACE inhibitors, all bearing a butynyloxy P10 group, was
explored. In particular, compound 5j has excellent in vitro potency against isolated TACE enzyme and in cells, good selectivity over
MMP-1 and MMP-9, and oral activity in an in vivo model of TNF-a production and a collagen-induced arthritis model.
# 2003 Elsevier Ltd. All rights reserved.
Small molecule mediators of tumor necrosis factor-a
(TNF-a) levels are sought after agents for the treatment
of several inflammatory diseases including rheumatoid
arthritis (RA) and Crohn’s disease.1 EnbrelTM, a soluble
TNF receptor-Fc dimer, modulates TNF-a by interact-
ing with both the 26 kDa membrane-bound form of
TNF-a and 17 kDa soluble TNF-a and is a highly
effective RA therapy.2 However, EnbrelTM must be
parenterally dosed and an orally bioavailable agent
would therefore be highly desirable.
synovial tissue and have been implicated in the destruc-
tion of cartilage in RA joints the optimal MMP/TACE
selectivity profile for a drug to treat rheumatoid arthritis
is at present unresolved.8
We have previously disclosed a series of anthranilic acid
based sulfonamide hydroxamic acid TACE inhibitors
bearing novel propargylic P10 groups, exemplified by
compound 4.7 A number of these propargyl ethers are
excellent inhibitors of TACE in a cell-free enzyme assay
and also potently inhibit the LPS-induced release of
An alternative paradigm for affecting TNF-a levels is
via the inhibition of TNF-a converting enzyme (TACE/
ADAM-17), the enzyme primarily responsible for the
shedding of membrane-bound TNF-a to provide its
soluble form.3 A number of small molecule TACE inhi-
bitors possessing excellent enzyme and cellular potency
have recently been disclosed (Fig. 1).4ꢀ7 Some of these
are selective for TACE over most MMPs (e.g., 1, IK-
6824), others are broad-spectrum inhibitors of TACE
and MMPs (e.g., 2, SDZ 242–4845) and some display
selectivity over MMP-1 (e.g., 36 and 47). Since a variety
of MMPs have been found to be over-expressed in RA
*Corresponding author. Tel.: +1-845-602-3053; fax: +1-845-602-
5561; e-mail: levinji@wyeth.com
Figure 1. Literature TACE inhibitors.
0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00514-6

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J. I. Levin et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2799–2803
soluble TNF in THP-1 cells. Selectivity for TACE over
MMP-1 and potent inhibitory activity in an in vivo
model of TNF-a production has also been demon-
strated. We now disclose the extension of the use of the
butynyloxy P10 group to a series of sulfonamide hydroxa-
mates, 5, derived from acyclic amino acids (Fig. 2).
removal of the Fmoc, sulfonylation with 6 and cleavage
of the linker with TFA gave the desired hydroxamates 5.
Variants of the butynyloxy P10 group were available
from glycine according to the routes described in
Scheme
2.
Initial
formation
of
4-fluoro-
phenylsulfonamide 9a was followed by formation of the
t-butyl ester and alkylation of the sulfonamide to give
10a. Displacement of the fluorine with propargylamine
led to 11a, while reaction with potassium ethyl xanthate
and reduction of the resulting disulfide, followed imme-
diately by S-alkylation with 1-bromo-2-butyne, led to
11b. Conversion of 11a–b into hydroxamates 12a–b
proceeded via the acids, with HOBT/EDC and
hydroxylamine. Nitrile analogue 12c resulted from a
Heck coupling of 4-bromophenylsulfonamide 10b with
acrylonitrile and subsequent hydrogenation to give
intermediate 11c, followed by hydroxamate formation.
Sulfonamide hydroxamic acids 5a–i (Table 1) were pre-
pared as shown in Scheme 1. Thus, 4-butynyloxy-
benzene sulfonyl chloride, 6, was readily available from
the reaction of 1-bromo-2-butyne with 4-hydroxy-
benzenesulfonic acid, followed by treatment with oxalyl
chloride. Sulfonylation of various amino-esters with 6
gave sulfonamides 7 in good yield. These N-H sulfona-
mides could then be converted into the corresponding
hydroxamates, or first N-alkylated to afford 8 followed
by hydroxamate formation. The cysteine and penicill-
amine derivatives 5k–n resulted from initial S-alkylation
of the amino acids, and subsequent sulfonylation and
hydroxamate formation according to Scheme 1. Deriva-
tive 5o was available by appending a Boc protected
N-methyl ethanolamine sidechain on Fmoc protected
4-hydroxyphenylglycine methyl ester via Mitsunobu
reaction prior to Fmoc removal and sulfonylation with
6. Compound 5p required sulfonylation of TBS-pro-
tected 4-hydroxyphenylglycine methyl ester, followed by
N-methylation of the sulfonamide, desilylation and
functionalization of the phenol. Alternatively, N-H
sulfonamides, including 5j, could be obtained via solid
phase synthesis. Coupling of Fmoc protected amino
acids with a hydroxylamine-linked resin9 followed by
b-Amino acid derivatives 13a–b were prepared by sul-
fonylation of b-alanine t-butyl ester and subsequent
conversion into the desired NH- or N-methyl sulfonamide
hydroxamates similarly to Scheme 1.
Scheme 1. (i) 1-Bromo-2-butyne, NaOH; (ii) (COCl)2, DMF; (iii)
amino-ester; (iv) R2X, K2CO3 or NaH; (v) NaOH; (vi) (a) (COCl)2,
DMF or EDC/HOBT; (b) NH2OH.
Figure 2. Acyclic a-sulfonamide hydroxamates.
Table 1. In vitro potency of substituted a-sulfonamide hydroxamic acids
Compd
D/L
R1
R2
TACEa
THPb
MMP-1a
MMP-9a
MMP-13a
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
D
D
D
D
D
D
D
D
D
H
H
H
CH3
H
CH3
CH2-3-Py
H
CH3
H
CH3
H
H
H
H
CH3
H
CH3
H
5
7
7
4
5
10
15
17
13
10
3
6
6
54
18
26
15
22
45
40
60
58
79
78
9
50
56
80
49
52
48
87
47% (10)
1895
1156
4031
330
2965
259
880
10,000
2471
1908
61
753
309
40
796
68
185
99
20
195
28
18
38
41
396
86
28
5
CH3
CH(CH3)2
CH(CH3)2
C(CH3)3
(CH3)2
42
11
224
1377
777
160
14
130
4
349
11
D
CH(CH3)OH
CH2SCH2-3-Py
CH2SCH2-3-Py
C(CH3)2SCH2-3-Py
C(CH3)2SCH2-3-Py
Ph-4-O(CH2)2NHCH3
Ph-4-O(CH2)2NHCH3
D,L
D,L
D,L
D,L
D
476
9
10,000
927
8
2
146
27
D
CH3
aIC50, nM or % inhibition (mM).
b% Inhibition at 1 mM.

J. I. Levin et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2799–2803
2801
S30 channel of TACE is confirmed by the X-ray struc-
ture of 5d (Fig. 3). A potential polar interaction between
the sulfonamide NH of 5d, and its analogues, and the
backbone carbonyl of an active-site proline residue in
both MMP-1 and TACE may serve to reduce the con-
formational entropy of these ligands. This would
adversely affect the ability of the butynyl moiety of the
NH-sulfonamides to fit in the S10 pocket of MMP-1,
and effectively increase selectivity for TACE over
MMP-1. The difference in selectivity between the NH
and N-methyl sulfonamide series might then be ration-
alized by the observation, through NMR analysis, of
multiple binding conformers of compound 5g bound to
MMP-1.13
Scheme 2. (i) 4-Br or 4-F-PhSO2Cl; (ii) DMF-t-butyl acetal; (iii) MeI,
K2CO3; (iv) H2NCH2CCH, DMSO, 80 ꢁC; (v) (a) C2H5OCS2K,
DMSO, 100 ꢁC; (b) PPh3, HCl, H2O; (c) 1-Bromo-2-butyne, NaH; (vi)
(a) CH2=CHCN, (PPh3)2PdCl2, TEA; (b) Pd/C, NH4CO2H (vii)
TFA, TES (for 11a–b); LiOH (for 11c) (viii) HOBT/EDC/NH2OH.
In contrast to selectivity, activity in the THP-1 cellular
assay14 is improved for the N-methyl sulfonamides
relative to the NH-derivatives, with the exception of the
glycine (5a, 5b) and penicillamine (5m, 5n) series. Cel-
lular potency also improves with increasing steric bulk
of the alkyl amino acids in both the NH and N-methyl
sulfonamide series, but selectivity over MMP-1 decrea-
ses as substitution on the amino acid b-carbon increases
(compare 5d, 5f, and 5h), presumably due in part to
beneficial interactions with the S20 pocket of the MMPs.
An exception is the geminal dimethyl analogue 5i, which
has poor cell activity, although it has >100-fold selc-
tivity for TACE over both MMP-1 and MMP-9.
All of the sulfonamide hydroxamic acids were tested in
vitro10 for their ability to inhibit MMP-1, MMP-9,
MMP-13 and TACE.11 The inhibition of these enzymes
may help prevent cartilage degradation in RA, and
therefore be therapeutically desirable.8 However, inhi-
bitors of TACE with various MMP inhibition profiles
were also sought in order to gain insight into the possi-
ble source of musculoskeletal side effects seen in clinical
trials of some broad-spectrum MMP inhibitors.12
In search of a boost in cellular potency several analo-
gues were prepared with polar functionality incorpo-
rated in the inhibitor P1, or P20 groups. Appending a
picolyl group to the sulfonamide nitrogen in the glycine
series (5c) does not affect enzyme potency, but increases
cell activity relative to the corresponding NH and N-
methyl analogues (5a–b). A threonine residue (5j) pro-
vides good selectivity over each of the MMPs tested
along with moderate cellular potency. Compounds 5k–
p, with basic amines in the P1 substituent, also offered
no distinct advantage over the alanine and valine ana-
logues in terms of their potency in the THP assay.
The in vitro potencies for a series of acyclic sulfonamide
hydroxamic acid analogues bearing butynyl P10 groups
are shown in Table 1.
All of the a-amino acids used to prepare the sulfon-
amide hydroxamates in Table 1 afforded potent inhibi-
tors of TACE enzyme in a cell-free assay. The d-a-
amino acids were generally greater than 50-fold more
potent than the analogous l-a-amino acid derivatives
(not shown). For each series the NH-sulfonamides (5a,
5d, 5f, 5h–k, 5m, and 5o) are far more selective for
TACE over MMP-1 than the analogous N-methyl sul-
fonamides (5b, 5e, 5g, 5l, 5n, and 5p), with selectivity
ratios ranging from 80- to 2000-fold. That the butynyl
group of these analogues does in fact reside in the S10-
To investigate whether the butynyloxy P10 group, opti-
mal for TACE enzyme and cell activity in the anthrani-
late sulfonamide series,7 was preferred for the a-amino
hydroxamate series the analogues in Table 2 were pre-
pared. The propargyl amine 12a and thioether 12b are
both less potent than butynyl ether 5b in the enzyme
and cellular assays. Surprisingly, nitrile 12c is poorly
Table 2. In vitro potency of P10 variants
Compd
Y
O
Z
TACEa THPb MMP-1a MMP-9a MMP-13a
5b
CCH3
7
120
38
33
8
14
1895
ꢂ10,000
4948
309
1511
111
74
99
751
84
12a
12b
12c
NH CH
S
CH2
CCH3
N6% (1)
7
>10,000
207
aIC50, nM or % inhibition (mM).
b% Inhibition at 3 mM.
Figure 3. X-ray structure of compound 5d bound to TACE.

2802
J. I. Levin et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2799–2803
active against TACE, although whether it is the carbon
linker or the nitrile that is not tolerated has not been
determined.
Acknowledgements
We thank Stacey Skala, Ruth Mulvey and Thomas
Stratman for technical support in the MMP and THP
assays, and Loran Killar for her insight and guidance
during the course of this work.
The acyclic b-amino acid derivatives, 13a–b, are not as
potent against TACE enzyme as 5a–b, but appear to be
greater than 100-fold selective over MMP-1, -9, and-13
(Table 3). Unfortunately, both compounds are also less
active in the cellular assay at 3 mM. Additional work in
this series aimed at enhanced cellular potency will be
reported in due course.
References and Notes
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measured by their ability to inhibit the LPS-stimulated
production of TNF-a in a mouse.14 Several of these
compounds (5d, 5h, and 5p), dosed at 50 mg/kg po,
provided greater than 75% inhibition of TNF-a levels
one h after administration of LPS, and two (5f and 5j)
gave greater than 50% inhibition at 25 mg/kg po. The
most potent analogue in this model was threonine deri-
vative 5j with an ED50 of 3 mg/kg po. The potential of
these compounds to be effective treatments for RA was
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lagen-induced arthritis (CIA) efficacy model, a standard
model of human RA.15 Compound 5j is reproducibly
active at reducing clinical severity scores in this efficacy
model at 20 mg/kg po bid (n=4) and has shown activity
at 10 mg/kg po bid (n=2). Furthermore, 5j has excellent
bioavailability at 10 mg/kg in the mouse (100%), rat
(46%), dog (75%) and monkey (17%), and inhibits
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blood with an IC50 of 1 mM. Compound 5j is stable in
liver microsomes of several species, including humans.
The glucuronide, observed in CD-1 mouse and
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In summary, we have synthesized a series of butynyl-
oxy-based a-sulfonamide hydroxamate inhibitors of
MMPs and TACE. Many of these compounds are
potent inhibitors of TACE in an isolated enzyme assay
and in THP-1 cells, with several possessing greater than
100-fold selectivity for TACE over MMP-1 and MMP-
9. Oral activity has been demonstrated for several of
these compounds in a mouse model of LPS-stimulated
TNF-a production and one (5j) shows good oral
potency in a prophylactic CIA efficacy model. The
extension of this work to additional inhibitor scaffolds
will be reported in due course.
Table 3. In vitro potency of b-sulfonamide hydroxamates
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Compd
R
TACEa THPb MMP-1a MMP-9a MMP-13a
(%)
(%)
(%)
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13a
13b
H
CH3
145
73
0
9
9 (10)
33 (10)
18 (10)
53 (10)
37 (10)
57 (10)
aIC50, nM or % inhibition (mM).
b% Inhibition at 3 mM.
12. Scrip 1998, 2349, 20–21.
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began on day 18 prior to the onset of arthritis and continued
for 17 days. On day 21, disease was induced by injecting 40 mg
of LPS intraperitoneally.
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