Anthranilate sulfonamide hydroxamate TACE inhibitors. Part 2: SAR of the acetylenic P1′ group

Article abstract of DOI:10.1016/S0960-894X(02)00136-1

The SAR of a series of potent sulfonamide hydroxamate TACE inhibitors bearing novel acetylenic P1′ groups was explored. In particular, compound 4t bearing a butynyloxy P1′ moiety has excellent in vitro potency against isolated TACE enzyme and in cells, go

Full text of DOI:10.1016/S0960-894X(02)00136-1

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1199–1202
Anthranilate Sulfonamide Hydroxamate TACE Inhibitors.
Part 2: SAR of the Acetylenic P10 Group
J. I. Levin,a,* J. M. Chen,a M. T. Du,a F. C. Nelson,a L. M. Killar,b S. Skala,b A. Sung,b
G. Jin,a R. Cowling,a D. Barone,c C. J. March,c K. M. Mohler,c R. A. Blackc
and J. S. Skotnickia
aWyeth-Ayerst Research, 401N. Middletown Road, Pearl River, NY 10965, USA
bWyeth-Ayerst Research, PO Box CN-8000, Princeton, NJ 08543, USA
cImmunex Corporation, Seattle, WA 98101, USA
Received 10 December 2001; accepted 4 February 2002
Abstract—The SAR of a series of potent sulfonamide hydroxamate TACE inhibitors bearing novel acetylenic P10 groups was explored. In
particular, compound 4t bearing a butynyloxy P10 moiety has excellent in vitro potency against isolated TACE enzyme and in cells, good
selectivity over MMP-1 and oral activity in an in vivo model of TNF-a production. # 2002 Elsevier Science Ltd. All rights reserved.
Tumor necrosis factor-a (TNF-a) is a pro-inflammatory
cytokine that exists in two forms, a 26 kDa membrane-
bound form and a soluble non-covalently bound
homotrimer of 17 kDa units.1 The enzyme responsible
for the cleavage of 26 kDa TNF into soluble TNF is
TNF-a converting enzyme (TACE) a member of the
ADAM family of enzymes.2 It has been postulated that
agents that inhibit TACE, and thereby reduce levels of
soluble TNF-a, might offer an effective treatment for
rheumatoid arthritis.3 To that end a variety of small
molecule TACE inhibitors have been reported to
date,3a,4 including the reverse hydroxamates exemplified
by 1,5 the hydroxamic acid hydrazide 2,6 and lactam
hydroxamate 3 (Fig. 1).7
class of TACE inhibitors with acetylenic P10 groups (4,
Fig. 2, R1=CH2CCCH3).11 We nowwish to report
further on the results of our investigation into the SAR
of the anthranilate hydroxamic acids in both a TACE
enzyme assay and a cellular assay. Compounds have
been identified which are highly active against TACE and
We have previously disclosed several anthranilic acid
based sulfonamide hydroxamic acid TACE inhibitors
with different MMP inhibition profiles.8,9 Although the
optimization of TACE activity within the anthranilate
hydroxamate series led us to several potent inhibitors of
this enzyme, including some that were also selective over
MMP-1, MMP-9, and MMP-13, none of these com-
pounds had significant (<3 mM) activity as inhibitors of
TNF-a production in a THP-1 cellular assay.10
Figure 1. Literature TACE inhibitors.
In the preceding communication, we have reported the
structure-based design and initial discovery of a new
*Corresponding author. Tel.: +1-914-732-3053; fax: +1-914-732-
5561; e-mail: levinji@war.wyeth.com
Figure 2. Anthranilate hydroxamic acids.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00136-1

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J. I. Levin et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1199–1202
selective over MMP-1, with vastly improved cellular
activity. One of these compounds has shown potent inhi-
bitory activity in an in vivo model of TNF-a production.
alkylation of phenol 6b with propargyl alcohol (Scheme 2).
Mannich alkylation with formaldehyde and diethylamine
in the presence of CuCl then afforded amine 9b. Reac-
tion of 9b with cyanogen bromide then gave the pro-
pargylic bromide 9c. Displacement of the bromide with
methanolic sodium hydroxide to give 10a was followed
by conversion of the ester into the hydroxamate as
before to give 4m. Bromide displacement by amines
provided 10b–d, which was followed by protection of
the resulting propargylic amine with di-t-butyl dicar-
bonate, conversion of the ester into the corresponding
hydroxamate and removal of the Boc group to form the
hydrochloride salts 4n–p.
Chemistry
Sulfonamide hydroxamic acids 4a–c (Table 1) were pre-
pared as described previously.8,9 Compounds 4d–k were
prepared from phenol 6b and the desired propargylic
alcohol, as shown in Scheme 1, according to the method
described in the preceding paper.11 Alcohol 4l was
derived in this manner starting from mono-THP
protected 2-butyn-1,4-diol.
Piperazine derivative 4t was also synthesized starting
from phenol 6b as shown in Scheme 2. Protection of the
phenol as its TBS-ether (6c) was followed by NBS-bro-
mination of the benzylic methyl group. Displacement of
the benzylic bromide with 1-methylpiperazine occurred
with concomitant desilylation to give phenol 8a.
Mitsunobu alkylation of the phenol with 2-butyn-1-ol
and subsequent conversion of the ester into the desired
hydroxamic acid via the carboxylic acid gave 4t. The
3-methylpiperazine-5-phenyl anthranilate derivative, 4u,
was prepared in a similar manner from 7a, via silyl ether
7b and piperazine 8b, with incorporation of the phenyl
group accomplished using a Suzuki coupling with
phenylboronic acid.
Biology
All of the anthranilate hydroxamic acids were tested in
vitro12 for their ability to inhibit MMP-1, MMP-9,
Scheme 1. (i) 4-F-PhSO2Cl, TEA; (ii) CH3I, K2CO3; (iii) (a)
CH3CCCH2OH, NaH, DMF, 80 ꢀC; (b) HCl, H2O; (iv) R1OH, PPh3,
DEAD; (v) NaOH; (vi) (a) (COCl)2, DMF; (b) NH2OH.
Propargylic ether 4m and amines 4n–p were prepared
from the terminal alkyne 9a, available from Mitsunobu
Table 1. In vitro potency of substituted anthranilate hydroxamic acids
Compd
R1
R2
R3
R5
MMP-1a
MMP-9a
MMP-13a
TACEa
THPb
4a
4b
4c
4d
4e
4f
4g
4h
4i
CH3
CH3
CH2-3-C5H4N
(CH2)3CH3
CH2CCH
CH(CH3)CCH
CH2CCCH3
CH2CCCH2CH3
CH2CC(CH2)2CH3
CH2CC(CH2)3CH3
CH2CCPh
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
CH3
CH2[(CH2)2]2NCH3
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
H
114
194
42% (10)
2488
113
1534
1616
1228
2364
11
2
28% (1)
21
15
455
304
800
232
389
857
477
387
643
1205
2827
–
21
5
48% (1)
68
52
433
154
289
358
701
321
83
233
255
908
2377
416
32
26
28
67
11
122
16
12
47
34
66
7
11
58
29
346
28
84
27
25
42
14
42
9
0
55
9
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
84
48
11
16
20
88
46
15
14
5
55
7
32
94
95
4j
4k
4l
53% (10)
3815
3203
CH2CCCH2OH
CH2CCCH2OCH3
CH2CCCH2NHCH3
CH2CCCH2NH(CH2)3N(CH3)2
CH2CCCH2N(CH2CH3)2
CH2CCCH3
4m
4n
4o
4p
4q
4r
4s
4t
7803
26% (10)
32% (10)
21% (10)
85% (10)
6% (10)
35% (10)
1658
CH2CCCH3
CH2CCCH3
CH2CCCH3
CH2CCCH3
H
H
Br
Ph
–
–
166
28
42% (10)
61% (10)
252
CH3
CH3
CH3
H
CH2[(CH2)2]2NCH3
CH2[(CH2)2]2NCH3
4u
1923
47
aIC50 (nM) or % inhibition (mM).
b% Inhibition at 3 mM.

J. I. Levin et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1199–1202
1201
maintain while modifying the P10 group. Thus, for the
series of linear propargylic ethers 4g–j, going from a
4-carbon chain to a 7-carbon chain, activity in the THP
assay falls off with increasing length of the P10 group
and lipophilicity, although they are all good inhibitors
of TACE enzyme (see Table 1). Addition of a branched
methyl group on propargyl ether 4e gives acetylenic
ether 4f and reduces potency against TACE enzyme and
in cells. Phenyl acetylene 4k, in which the distal phenyl
ring can extend into the S30 pocket of TACE, also
displays reduced potency against TACE enzyme and in
cells relative to butynyl ether 4g.
Analysis of molecular modeling studies of anthranilate
hydroxamates bound to the active site of TACE, mak-
ing use of the X-ray crystal structure of the TACE
catalytic domain,16 suggested that forming a polar
interaction between the ligand and the glutamate resi-
due residing in the S30 pocket of TACE offered an
opportunity for further enhancing binding potency and
selectivity. To that end the propargylic alcohol 4l, ether
4m, and amines 4n–p were prepared. Alcohol 4l proved
to be the most potent inhibitor of TACE in the anthra-
nilate series. Compound 4l also offered selectivity over
both MMP-1 and MMP-9 and cellular activity equal to
butynyl ether 4g. Compound 4l gave 69% inhibition of
TNF-a production at a concentration of 1 mM. The
analogous methyl ether, 4m, has substantially the same
MMP/TACE profile as alcohol 4l, but with diminished
cellular activity. Amine 4n is 5-fold less potent against
TACE than the isosteric methyl ether 4m, and has much
weaker potency in cells. Diamine 4o is the most potent
and selective TACE inhibitor of the propargylic amines,
with a selectivity profile similar to the picolyl analogue
4c. Unfortunately, as with 4c, diamine 4o has only very
lowlevel cellular activity. The more sterically demand-
ing diethylamine, 4p, is still weaker in cells as well as
versus the MMPs and TACE enzyme.
Scheme 2. (i) NBS; (ii) 1-methylpiperazine, K2CO3; (iii) R1OH, PPh3,
DEAD; (iv) NaOH; (v) (a) (COCl)2, DMF; (b) NH2OH; (vi) MeOH,
NaOH; (vii) excess RNH2; (viii) HCl(g).
MMP-13 and TACE.13 Levels of each of these enzymes
have been found to be up-regulated in the synovium of
RA patients and the broad spectrum inhibition of these
enzymes may therefore be therapeutically desirable.14
However, compounds with selectivity over MMP-1 were
also sought in order to gain insight into whether the
inhibition of MMP-1 is a possible source of the muscu-
loskeletal side effects seen in clinical trials of broad
spectrum MMP inhibitors.15
The search for compounds selective for TACE over
MMP-1 and MMP-13 was also addressed by paring
down functionality from the anthranilate hydroxamic
acids that had been required for potency against the
MMPs and TACE prior to the utilization of the acetyl-
enic P10 groups. For example, removal of the 5-bromo
substituent from butynyl analogue 4g to give 4q does
not appreciably affect MMP/TACE activity. Com-
pound 4r, the NH–sulfonamide analogue of 4q is less
potent than 4q against TACE and MMP-1 and MMP-
13. Elimination of both the 5-bromo and 3-methyl sub-
stituents from 4g to provide 4s does not reduce TACE
enzyme activity at all, but does abolish activity against
MMP-1 and MMP-13 to a great extent resulting in a
very selective TACE inhibitor. Each of these analogues
unfortunately suffers from diminished cellular activity
relative to parent compound 4g.
The in vitro potencies for a series of anthranilate
hydroxamic acid analogues containing a variety of P10
groups are shown in Table 1. The simple alkyl ether P10
groups of compounds 4a, 4b, and 4d provide potent
TACE and MMP enzyme inhibition, but little cellular
activity. Compounds 4c–d, with lengthier P10 moieties
have reduced potency against MMP-1, due to the shal-
0
lowS1 pocket of that enzyme, while TACE activity is
minimally diminished. The 3-picolyl derivative, 4c, is
selective for TACE over several MMPs, but again is
poorly active in cells at 3 mM.8
As we have reported in the preceeding paper, anthrani-
late hydroxamic acids 4e, 4g, and 4j, bearing acetylenic
P10 groups are also potent inhibitors of TACE enzyme.
In addition, compounds 4g and 4j are at least 100-fold
selective over MMP-1. To our delight, butynyl ether 4g
also possesses a relatively high level of potency in the
THP cellular assay, providing 84% inhibition of TNF-a
production at 3 mM and 59% inhibition at 1 mM.
However, this increased cellular activity was difficult to
Finally, since previous work8 on the anthranilate
hydroxamates had shown that oral activity is greatly
enhanced by the incorporation of a basic amine moiety
into the inhibitor molecule, we targeted analogues of
piperazine 4b which would also contain the P10 butynyl
moiety. The resulting compounds, 4t and 4u, are the

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J. I. Levin et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1199–1202
most potent anthranilate hydroxamic acids in the THP
cellular assay and have good potency against TACE
enzyme. Both 4t and 4u provide slightly greater than
50% inhibition at 300 nM in THP-1 cells.
4. Moss, M. L.; White, J. M.; Lambert, M. H.; Andrews, R. C.
Drug Discov. Today 2001, 6, 417.
5. (a) Musso, D. L.; Andersen, M. W.; Andrews, R. C.; Aus-
tin, R.; Beaudet, E. J.; Becherer, J. D.; Bubacz, D. G.; Bickett,
D. M.; Chan, J. H.; Conway, J. G.; Cowan, D. J.; Gaul, M. D.;
Glennon, K. C.; Hedeen, K. M.; Lambert, M. H.; Leesnitzer,
M. A.; McDougald, D. L.; Mitchell, J. L.; Moss, M. L.;
Rabinowitz, M. H.; Rizzolio, M. C.; Schaller, L. T.; Stanford,
J. B.; Tippin, T. K.; Warner, J. R.; Whitesell, L. G.; Wiethe,
R. W. Bioorg. Med. Chem. Lett. 2001, 11, 2147. (b) Rabino-
witz, M. H.; Andrews, R. C.; Becherer, J. D.; Bickett, D. M.;
Bubacz, D. G.; Conway, J. G.; Cowan, D. J.; Gaul, M.;
Glennon, K.; Lambert, M. H.; Leesnitzer, M. A.; McDougald,
D. L.; Moss, M. L.; Musso, D. L.; Rizzolio, M. C. J. Med.
Chem. 2001, 44, 4252.
Due to the excellent potency of compound 4t in the
TACE enzyme and THP cellular assays it was tested in
a preliminary in vivo model to measure its ability to
inhibit the LPS stimulated production of TNF-a in a
mouse.10 A 50 mg/kg oral dose of compound 4t
provided 100% inhibition of TNF-a production 1 h after
dosing. The effect lasted at least 6 h post dosing, with a
75% inhibition of TNF-a production at that time point.
6. (a) Broadhurst, M. J.; Johnson, W. H.; Walter, D. S. PCT
Int. Appl. WO0032570, 2000; Chem. Abstr. 2000, 133, 30513.
(b) Exp. Opin. Ther. Pat. 2000, 10, 1617.
7. (a) Duan, J.; Decicco, C. P.; Wasserman, Z. R.; Madus-
kuie, T. P., Jr. PCT Int. Appl., WO9918074, 1999; Chem.
Abstr. 1999, 130, 296611. (b) Decicco, C. Abstracts of Papers,
221st National Meeting of the American Chemical Society,
San Diego, CA, April 1–5, 2001; American Chemical Society:
Washington, DC, 2001; MEDI 189.
8. Levin, J. I.; Chen, J.; Du, M.; Hogan, M.; Kincaid, S.;
Nelson, F.; Venkatesan, A. M.; Wehr, T.; Zask, A.; DiJoseph,
J.; Killar, L. M.; Skala, S.; Sung, A.; Sharr, M.; Roth, C.; Jin,
G.; Cowling, R.; Mohler, K. M.; Black, R. A.; March, C. J.;
Skotnicki, J. S. Bioorg. Med. Chem. Lett. 2001, 11, 2189.
9. Levin, J. I.; Chen, J. M.; Du, M. T.; Nelson, F. C.; Wehr,
T.; DiJoseph, J. F.; Killar, L. M.; Skala, S.; Sung, A.; Sharr,
M. A.; Roth, C. E.; Jin, G.; Cowling, R.; Di, L.; Sherman, M.;
Xu, Z. B.; March, C. J.; Mohler, K. M.; Black, R. A.; Skot-
nicki, J. S. Bioorg. Med. Chem. Lett. 2001, 11, 2975.
10. (a) Mohler, K. M.; Sleath, P. R.; Fitzner, J. N.; Cerretti,
D. P.; Alderson, M.; Kerwar, S. S.; Torrance, D. S.; Otten-
Evans, C.; Greenstreet, T.; Weerawarna, K.; Kronheim, S. R.;
Petersen, M.; Gerhart, M.; Kozlosky, C. J.; March, C. J.;
Black, R. A. Nature 1994, 370, 218. (b) Levin, J. I.; Chen, J.
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150908.
In summary, we have synthesized a series of anthrani-
late-hydroxamic acid inhibitors of MMPs and TACE
with novel propargylic ether P10 substituents. Many of
these compounds are potent inhibitors of TACE in an
isolated enzyme assay. Several of these compounds (4g–
h, 4j, and 4l–o) are greater than 100-fold selective for
TACE over MMP-1. In an assay measuring the inhibi-
tion of TNF-a production in THP-1 cells four members
of the butynyl P10 series of analogues (4g, 4l, and 4t–u)
produced greater than 50% inhibition at concentrations
of 1 mM or less. Most importantly, compound 4t has
been shown to inhibit TNF-a production with good
duration of action on oral dosing in an in vivo murine LPS
model. The application of acetylenic P10 groups to other
TACE inhibitor scaffolds will be reported in due course.
Acknowledgements
J.I.L. dedicates this manuscript to the memory of Mar-
vin H. Levin.
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