Acetylenic TACE inhibitors. Part 3: Thiomorpholine sulfonamide hydroxamates

Article abstract of DOI:10.1016/j.bmcl.2005.12.020

A series of thiomorpholine sulfonamide hydroxamate TACE inhibitors, all bearing propargylic ether P1′ groups, was explored. In particular, compound 5h has excellent in vitro potency against isolated TACE enzyme and in cells, oral activity in a model of TN

Full text of DOI:10.1016/j.bmcl.2005.12.020

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1605–1609
Acetylenic TACE inhibitors. Part 3: Thiomorpholine sulfonamide
hydroxamates
J. I. Levin,a,* J. M. Chen,a L. M. Laakso,a M. Du,a J. Schmid,a W. Xu,c T. Cummons,b
J. Xu,a G. Jin,a D. Baroned 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, MA 02140, USA
dAmgen, Seattle, WA 98101, USA
Received 16 September 2005; revised 5 December 2005; accepted 7 December 2005
Available online 19 January 2006
Abstract—A series of thiomorpholine sulfonamide hydroxamate TACE inhibitors, all bearing propargylic ether P10 groups, was
explored. In particular, compound 5h has excellent in vitro potency against isolated TACE enzyme and in cells, oral activity in a
model of TNF-a production and a collagen-induced arthritis model, was selected as a clinical candidate for the treatment of RA.
ꢀ 2005 Elsevier Ltd. All rights reserved.
The commercial success of biologics, including Enbrelꢁ
O
and Remicadeꢁ, that modulate levels of both mem-
O
brane-bound 26 kDa TNF and the soluble 17 kDa form
of this pro-inflammatory cytokine has led to an intensive
search for orally active small molecules that might be
similarly effective in the treatment of rheumatoid arthri-
tis (RA).1
O
O
HO
N
O
H
N
NH2
HO
N
H
N
N
N
O
H
H
O
1 (BMS-561392)
HO
2 (PKF242-484)
One attractive approach for affecting TNF levels is
the inhibition of TNF-a converting enzyme
(TACE/ADAM-17), the metalloprotease responsible
for the shedding of membrane-bound TNF, thereby
reducing circulating levels of soluble TNF.2 Among
the compounds directed at this mechanism of action
are selective inhibitors of TACE, including the clinical
candidate BMS-561392 (1, Fig. 1),3a,b and those that
inhibit enzymes from the related family of matrix
metalloproteinases (MMPs) in addition to TACE, like
PKF242-484 (2, Fig. 1)3c and clinical candidate Ro 32-
7315 (3, Fig. 1).3d The issue of whether broad spectrum
inhibition of TACE and MMPs, or more selective inhi-
bition of TACE, would be optimal remains to be settled
by RA clinical trials. Many broad spectrum MMP
O
O
O
H
O
HO
N
S
O O2S
N
N
HO
N
H
O
N
H
S
Ph
4 (TMI-1)
3 (Ro 32-7315)
Figure 1. Literature TACE inhibitors.
inhibitors have displayed dose-limiting toxicity in oncol-
ogy and osteoarthritis clinical trials,4 but the inhibition
of MMPs that are overexpressed in synovial tissue and
degrade cartilage in RA joints may provide a more
effective therapeutic.5
We, and others, have previously disclosed several ser-
ies of sulfonamide hydroxamic acid TACE inhibitors
bearing novel propargylic P10 groups, exemplified
by TMI-1 (4, Fig. 1).6 Compound 4 is an excellent
Keywords: TACE inhibitor; Thiomorpholine; Sulfonamide: hydroxa-
mate; Rheumatoid arthritis.
*
Corresponding author. Tel.: +1 845 602 3053; fax: +1 845 602
5561; e-mail: levinji@wyeth.com
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.12.020

1606
J. I. Levin et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1605–1609
OH
O
inhibitor of TACE and many MMPs, with good
potency in human whole blood and potent oral activ-
ity in an animal model of RA.6b Unfortunately, poor
solubility and poor bioavailability in rat and dog pre-
cluded the development of compound 4 as a clinical
candidate. We now report our efforts to optimize
the potency, physical properties, and bioavailability
of the thiomorpholine sulfonamides by varying the
P10 terminus of these inhibitors and appending sub-
stituents on the thiomorpholine ring (5a–y, Table 1),
resulting in a clinical candidate for the treatment
of RA.
R1
O
O O2S
N
O O2S
N
H
N
HO
i
ii, iii, iv
RO
RO
N
H
S
S
S
6 (R = Me, tBu)
7
5a-g
Scheme 1. Reagents: (i) a—4-HOPhSO2Cl, BTSA; b—MeOH; (ii)
R1OH, PPh3, DEAD; (iii) HCl (g) or LiI; (iv) a—(COCl)2, DMF;
b—NH2OH.
In an effort to achieve additional interactions between
the protein and the inhibitor, we also examined alcohol,
amine, and carbamate derivatives of the butynyl P10
moiety that could potentially contact the glutamate
and valine residues in the TACE S30 subsite. The alco-
hols were prepared as shown in Scheme 2. The one car-
bon tether was prepared by direct Mitsunobu alkylation
of 7 with 2-butyn-1,4-diol. Acetylation of the resulting
alcohol gave 8. Ester cleavage of 8 with TFA, and con-
version of the unmasked carboxylic acid to the hydroxa-
mic acid, provided 5h. The two-, three-, and four-carbon
tethers were prepared starting from THP-protected 3-
butyn-1-ol, 4-pentyn-1-ol, and 5-hexyn-1-ol. These alky-
nes were metallated with nBuLi and the resulting anion
Initial attempts at increasing the potency and selectivi-
ty of 4 focused on the size of the alkynyl ether P10 moi-
ety and the position of the carbon–carbon triple bond
(Scheme 1), which had previously been optimized on
an anthranilate-derived scaffold.6a Sulfonylation of thi-
omorpholine methyl or t-butyl ester 67 with in situ sily-
lated 4-hydroxybenzenesulfonyl chloride provided
phenol 7 after work-up with methanol.8 The phenol
next underwent Mitsunobu alkylation with the selected
alcohol, followed by cleavage of the ester and conver-
sion of the resulting carboxylate into the desired
hydroxamic acids 5a–5g.
Table 1. In vitro potency of cyclic sulfonamide hydroxamic acids
O
R1
O O2S
HO
N
N
H
S
R2
5
Compound
R1
R2
TACEa
THPb
MMP-la
MMP-13a
4 (TMI-1)
5a
5b
CH2CCCH3
CH2CCH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
8
19
94/87
91/76
64/36
38/16
80/50
38/11
82/48
88/62
95/89
57/25
51/19
77/52
79/53
37/14
27/5
7
1
3
1
(CH2)2CCH
(CH2)3CCH
32
40
2
1
5c
5d
11
7
8
CH2CHCHCH3
(CH2)3CH3
35
42
2
5e
7
9
5f
5g
CH2CC(CH2)3CH3
CH2CCCCH
23
42
29
6
7
11
8
5h
5i
CH2CCCH2OH
CH2CC(CH2)2OH
CH2CC(CH2)3OH
CH2CC(CH2)4OH
CH2CCCH2NHBoc
CH2CC(CH2)2NHBoc
CH2CC(CH2)3NHBoc
CH2CC(CH2)4NHBoc
CH2CCCH2NH2
CH2CC(CH2)2NH2
CH2CC(CH2)3NH2
CH2CC(CH2)4NH2
CH2CCCH3
20
62
33
35
159
101
26
300
42
87
97
1052
226
92
17
35
21
19
21
33
14
86
17
4
5j
40
53
5k
51
74
5m
5n
112
153
199
67
7
2
5o
5p
70/40
40/10
4/0
4
8
5q
5r
138
28
27
9
22/0
5s
40
20
37/13
89/73
91/76
94/78
91/81
90/74
87/67
7
5t
5u
CH2CO2H
CH2CONH2
CH2NH2
7
CH2CCCH3
CH2CCCH3
42
57
29
24
14
23
48
5v
5w
5x
5y
CH2CCCH3
CH2CCCH3
CH2CCCH3
CH2NHSO2CH3
(CH2)2OH
(CH2)2NH(CH2CH3)2
11
15
143
a IC50 (nM).
b % Inhibition at 3/1 lM.

J. I. Levin et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1605–1609
1607
O
OH
O
O
O O2S
N
O O2S
N
NH2
O2S
N
i, ii
HO
O
i, ii, iii
tBuO
RO
CO2tBu
AcO
S
MeO
2
S
S
S
8
7a (R = tBu)
7b (R = Me)
v
12
2
11
iii, iv
iv
O
O
(n = 2-4)
O
O
O O2S
O O2S
N
O O2S
N
Ref 10
O O2S
N
vi, vii, viii
N
S
HO
HO
MeO
N
N
MeO
THPO
H
HO
n
n
H
R
S
CO2tBu
S
S
9
5h-k
5t-y
13
Scheme 2. Reagents: (i) HOCH2CCCH2OH, PPh3, DEAD; (ii) Ac2O;
(iii) HCl (g); (iv) a—(COCl)2, DMF; b—NH2OH; (v)
HOCH2CC(CH2)nOTHP, PPh3, DEAD; (vi) LiI, EtOAc; (vii) a—
(COCl)2, DMF or EDC, HOBT; b—NH2OH; (viii) PPTS.
Scheme 4. Reagents: (i) a—HCl (g); b—SOCl2, MeOH; (ii) 4-
butynyloxybenzene sulfonyl chloride, TEA; (iii) BrCH2CH@CHCO2t-
Bu, K2CO3; (iv) Bu3P, THF, H2O.
was quenched with formaldehyde. Mitsunobu alkylation
of 7b then gave 9, followed by ester cleavage and
hydroxamate formation, and subsequent removal of
the THP moiety to give 5i–5k.
acid-based TACE inhibitors.9 The preparation of these
compounds, 5t–5y, from D-penicillamine disulfide (11)
utilizing a key diastereoselective intra-molecular Mi-
chael reaction to construct the thiomorpholine ring of
13 from 12, has been reported elsewhere and is summa-
rized briefly in Scheme 4.10
The Boc carbamates 5l–5o and the primary amine deriv-
atives 5p–5s were prepared from the protected alcohol
intermediates 8 and 9 according to Scheme 3. Thus, after
deprotection of 8 and 9 the alcohols were converted into
the propargylic bromides, followed by displacement
with sodium azide, reduction of the resulting azide to
the primary amine, and Boc protection to give 10. The
ester moiety was next converted into the desired
hydroxamate in two steps to give carbamates 5l–5o.
Removal of the Boc group then afforded the primary
amines 5p–5s as TFA or HCl salts.
All of the sulfonamide hydroxamic acids were tested
in vitro11 for their ability to inhibit MMP-1, MMP-13,
and TACE.12 Activity against MMP-1 was assessed
since the inhibition of this enzyme had been postulated
to contribute to musculoskeletal side effects seen for
MMP inhibitors.4 The inhibition of MMP-13 was mea-
sured since this may help prevent the degradation of car-
tilage in RA.5 The ability of compounds to inhibit TNF
production in LPS-stimulated THP-1 cells was also as-
sessed, although IC50s were determined for only the
most active analogs.13 The in vitro potencies for the
cyclic sulfonamide hydroxamic acid analogs bearing a
variety of P10 groups are shown in Table 1.
Finally, analysis of computer models of compound 4
bound to the active site of TACE indicated that the
6-position (see R2 in 5, Table 1) of the thiomorpholine
is solvent exposed, and that substituents affecting
the physical properties of the molecule might be placed
there with minimal loss of enzyme inhibitory activity.
A similar strategy has been used on a series of pipecolic
As for the analogs of anthranilate-based TACE inhibi-
tors,6a the shorter propargyl ether P10 analog 5a is sub-
stantially more active than butynyl ether derivative 4
against MMP-1, while slightly less active against TACE.
Increasing the length of the linker between the alkyne
and the P10 ether oxygen (5b–5c) progressively decreased
MMP-1 and TACE activity, and activity against LPS-
stimulated TNF production in THP-1 cells. The trans-
olefin analog 5d is 4-fold less potent than 4 against
cell-free TACE and also slightly less active than 4 in
THP-1 cells. This trend continues with the fully saturat-
ed butyl ether P10 derivative 5e, which has dramatically
less cellular activity than 4 and 5d, though it is essential-
ly equipotent to 5d against cell-free TACE. Lengthening
of the butynyl ether to the heptynyl ether 5f or the rigid
diyne 5g results in a slight loss of activity against both
cell-free TACE and in cells. That analogs 5f and 5g do
not increase selectivity for TACE over MMP-1, though
the P10 groups of both can clearly extend past the
arginine side chain that normally forms the base of the
shallow MMP-1 S10 pocket, confirms that this residue
O
O
O O2S
N
O O2S
N
i-v
RO
RO
R1O
BocHN
n
n
S
S
8 (n=1, R1=Ac)
10
9 (n=2-4, R1=THP)
vi, vii
O
O
O O2S
O O2S
N
viii
HO
HO
N
N
N
H
H2N
H
BocHN
n
n
S
S
5p-s
5l-o
Scheme 3. Reagents: (i) MeOH, K2CO3 (for 8), PPTS (for 9) (ii) PPh3,
CBr4; (iii) NaN3; (iv) Bu3P, THF, H2O; (v) Boc2O; (vi) LiI; (vii) a—
HOBt, EDC; b—NH2OH; (viii) HCl (g) or TFA.

1608
J. I. Levin et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1605–1609
is mobile and can move to accommodate larger P10 sub-
stituents on the ligand.14 All of compounds 5a–5g re-
main potent inhibitors of MMP-13.
is tolerated at this position, as was expected. Thus, the
carboxylic acid 5t is only 2-fold less active than 4
against cell-free TACE and slightly less active than 4
in THP cells at 1 lM. The corresponding primary
amide 5u and the primary amine 5v are at least 5-fold
less active than 4 in the FRET assay, but still sub-mi-
cromolar in cells. Sulfonamide 5w and alcohol 5x are
the most potent of the 6-substituted thiomorpholines
in the TACE FRET assay, similar in potency to 4,
and both are sub-micromolar inhibitors in THP cells.
Diethylamine 5y, like the other analog bearing a basic
amine at the thiomorpholine 6-position (5v), loses sig-
nificant enzyme activity in the FRET assay, 18-fold
compared to 4, but is still reasonably potent in THP
cells, affording 67% inhibition of LPS-stimulated
TNF production at 1 lM. We have previously noted
that discrepancies between the level of enzyme activity
in the TACE FRET assay and in THP cells are
frequently not attributable to cell permeability and
protein binding issues.16
The alcohols 5h–5k were all less active than the butynyl
analog 4 in the TACE FRET assay, with the shortest
analog, 5h, being the most potent. TACE activity does
not fall off substantially with increasing length, but
activity in THP cells decreases dramatically in going
from 5h (89% inhibition at 1 lM, IC50 = 140 nM) to
the pentynyl and hexynyl alcohols 5i and 5j (<25% inhi-
bition at 1 lM). Interestingly, the heptynyl alcohol 5k
recovers substantial cell activity (52% inhibition at
1 lM), relative to 5i–j. The same general trend is seen
for carbamates 5l–5o. Despite their unimpressive activi-
ty against TACE enzyme, at least 80-fold less potent
than 4, both 5l and 5o display modest cellular activity
(P40%) at 1 lM, while 5m and 5n are only weakly ac-
tive even at 3 lM. The amines 5p and 5q are essentially
equivalent in activity to their corresponding carbamates,
5l and 5m, against TACE, but are far less potent in the
THP cellular assay. Furthermore, the hexynyl and
heptynyl amines, 5r and 5s, are both approximately 5-
fold more active in the TACE FRET assay than the
analogous carbamates, 5n and 5o, but are less active in
cells. Although all of the amines are only weakly active
in cells, the butynyl and heptynyl analogs are again the
most potent, as in the alcohol and carbamate series. All
of the alcohols, carbamates, and amines, 5h–5s, main-
tain excellent activity against MMP-13, with little or
no selectivity for TACE over MMP-1.
The in vivo activity of the compounds in Table 1 with
the greatest activity in the THP assay was initially mea-
sured by their ability to inhibit the LPS-stimulated pro-
duction of TNF-a after oral dosing in a mouse.13 Of
compounds 5a–5y, several compounds equaled or
exceeded the level of activity of 4 in this model at a
50 mg/kg po dose. When dosed orally at 50 mg/kg, 5h,
5v, 5x, and 5y each provide P95% inhibition of TNF-
a levels one hour after administration of LPS. The most
potent analog in this model is propargylic alcohol deriv-
ative 5h with an ED50 of 5 mg/kg po. Analogs 5h and 5v
were also examined in a mouse prophylactic collagen-in-
duced arthritis (CIA) efficacy model,13 using an LPS
boost, and both gave statistically significant (p 6 0.05)
reductions in clinical severity scores at 10 mg/kg bid
po. In fact, in this model a 10 mg/kg po dose of 5h pro-
vides a > 50% reduction in clinical severity score
(p 6 0.05, n = 15), comparable to a 30 lg qd dose of En-
brelꢁ. Also, in human whole blood 5h inhibits LPS-stim-
ulated TNF production with an IC50 of 300 nM.
Analysis of in vitro stability of 5h in liver microsomes
from rat, dog, monkey, and human shows diminished
levels of metabolites resulting from loss of the P10 moi-
ety, as compared to 4. In addition, 5h is permeable in
Caco-2 cells (Mean Papp = 2.2 · 10ꢀ6 cm/s), is not highly
protein bound (ꢁ50%), and has vastly improved solubil-
ity in simulated gastric and intestinal fluids (>1.5 mg/
mL) as compared to 4 (<0.08 mg/mL). Perhaps as a re-
sult of these properties, bioavailability of 5h at 10 mg/kg
(mouse: 24%, rat: 8%, and dog: 36%) is in general better
than that of 4 (mouse: 71%, rat: 5%, and dog: 0%),
particularly in dogs.
An X-ray crystal structure has been obtained for 5p
bound to the active site of TACE (Fig. 2).15 It is interest-
ing to note that the amine moiety of 5p is within hydro-
gen bonding distance to both a valine residue and a
glutamate residue in the S30 pocket of TACE, yet it is
less active against the enzyme than 4, which makes
neither of these contacts.
The in vitro activity of the 6-substituted thiomorpho-
lines 5t–5y demonstrates that a variety of functionality
In summary, we have expanded a series of thiomorpho-
line sulfonamide hydroxamates bearing a propargylic
P10 group as inhibitors of MMPs and TACE. One of
these compounds, 5h, has activity equivalent to that of
compound 4 in an animal model of RA, but with im-
proved solubility and PK properties, and has been ad-
vanced to clinical trials. The complete pharmacological
profile of propargylic alcohol 5h (TMI-005) will be
reported in due course.17
Figure 2. X-ray structure of compound 5p bound to TACE.

J. I. Levin et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1605–1609
1609
(c) Tsukida, T.; Moriyama, H.; Inoue, Y.; Kondo, H.;
Yoshino, K.; Nishimura, S. Bioorg. Med. Chem. Lett.
2004, 14, 1569; (d) Moriyama, H.; Tsukida, T.; Inoue, Y.;
Yokota, K.; Yoshino, K.; Kondo, H.; Miura, N.;
Nishimura, S.-I. J. Med. Chem. 2004, 47, 1930.
Acknowledgments
We thank A. Sung, S. Skala, and R. Mulvey for technical
support in the MMP and THP assays, and R. A. Black, R.
Cowling, L. Killar, K. M. Mohler, and Y. Zhang for their
insight and guidance during the course of this work. We
also thank U. Jain and the PCC group for physical data,
and C. Huselton and group for PK data.
7. Bender, S.L.; Melnick, M. J. U.S. Patent 5,753,653, 1998;
Chem. Abstr. 1998, 129, 16140.
8. Levin, J. I.; Du, M. T.; Park, K. Synthetic Commun. 2004,
34, 2773.
9. Letavic, M. A.; Axt, M. Z.; Barberia, J. T.; Carty, T. J.;
Danley, D. E.; Geoghegan, K. F.; Halim, N. S.; Hoth, L.
R.; Kamath, A. V.; Laird, E. R.; Lopresti-Morrow, L. L.;
McClure, K. F.; Mitchell, P. G.; Natarajan, V.; Noe, M.
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10. Laakso, L. M.; Levin, J. I.; Du, M.; Jin, G.; Cowling, R.;
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S. Abstracts of Papers, 226th National Meeting of the
American Chemical Society, New York, NY, Sept 7–11,
2003; MEDI 116. Manuscript in preparation.
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