Synthesis and biological activity of selective pipecolic acid-based TNF-α converting enzyme (TACE) inhibitors

Article abstract of DOI:10.1016/S0960-894X(02)00183-X

A series of novel, selective TNF-α converting enzyme inhibitors based on 4-hydroxy and 5-hydroxy pipecolate hydroxamic acid scaffolds is described. The potency and selectivity of TACE inhibition is dramatically influenced by the nature of the sulfonamide

Full text of DOI:10.1016/S0960-894X(02)00183-X

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1387–1390
Synthesis and Biological Activity of Selective Pipecolic
Acid-Based TNF-ꢀ Converting Enzyme (TACE) Inhibitors
Michael A. Letavic,* Matt Z. Axt, John T. Barberia, Thomas J. Carty,
Dennis E. Danley, Kieran F. Geoghegan, Nadia S. Halim, Lise R. Hoth,
Ajith V. Kamath, Ellen R. Laird, Lori L. Lopresti-Morrow, Kim F. McClure,
Peter G. Mitchell, Vijayalakshmi Natarajan, Mark C. Noe, Jayvardhan Pandit,
Lisa Reeves, Gayle K. Schulte, Sheri L. Snow, Francis J. Sweeney,
Douglas H. Tan and Chul H. Yu
Pfizer Global Research and Development, Groton Laboratories, Eastern Point Road, Groton, CT 06340, USA
Received 1 January 2002; accepted 5 March 2002
Abstract—A series of novel, selective TNF-a converting enzyme inhibitors based on 4-hydroxy and 5-hydroxy pipecolate hydrox-
amic acid scaffolds is described. The potency and selectivity of TACE inhibition is dramatically influenced by the nature of the
sulfonamide group which interacts with the S10 site of the enzyme. Substituted 4-benzyloxybenzenesulfonamides exhibit excellent
TACE potency with >100ꢀ selectivity over inhibition of matrix metalloprotease-1 (MMP-1). Alkyl substituents on the ortho
position of the benzyl ether moiety give the most potent inhibition of TNF-a release in LPS-treated human whole blood. # 2002
Elsevier Science Ltd. All rights reserved.
Tumor necrosis factor-a (TNF-a) is a pro-inflammatory
cytokine produced by monocytes/macrophages and
other cell types. In diseases such as rheumatoid arthritis
(RA) and Crohn’s disease (CD), dysregulated cellular
release of TNF-a participates in the recruitment of
inflammatory cells and stimulation of the production of
other mediators of pain and cartilage breakdown (e.g.,
prostaglandins and MMPs, respectively).1 In addition,
data from human clinical trials have supported the
therapeutic utility of two anti-TNF biologics, etanercept,
a soluble TNF-a receptor-Fc dimer (Immunex), and
infliximab, an anti-TNFa antibody (Centocor), in RA
and CD.2 TNF-a is synthesized as a membrane-
anchored 26 kDa precursor. Proteolysis of the Ala76-
Val77 peptide bond leads to the mature cytokine being shed
from the cell as a homotrimer of the 17 kDa C-terminal
fragment. This processing step is performed by a 85 kDa
membrane-anchored zinc metalloprotease, TNF-a
converting enzyme (TACE).3ꢁ5 As one of several strategies
to intervene in the production of TNF-a, we sought a
small molecule inhibitor of TACE to control the level of
extracellular TNF-a. The following describes our efforts
using a series of pipecolates containing a hydroxamate
moiety to form a bidentate ligand to the catalytic zinc
atom of the enzyme, thus inhibiting the TACE-mediated
release of soluble TNF-a from the cell.
During a search for novel MMP inhibitors we dis-
covered a series of endocyclic hydroxamic acids that are
potent TACE inhibitors with good selectivity over
MMP-1. Thus, starting from hydroxamic acids such as
1 that have only weak TACE activity (IC50=9600 nM)6
and significant MMP-1 activity (19 nM), we prepared
compound 2 where the methoxy tailpiece in 1 is replaced
with a 4-fluorophenoxy substituent. Compound 2 shows
*Corresponding author. Tel.: +1-860-441-1803; fax: +1-860-715-2467;
e-mail: michael_a_letavic@groton.pfizer.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00183-X

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M. A. Letavic et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1387–1390
moderate TACE activity (IC50=120nM)6 while retaining
potent MMP-1 activity. Further improvements in
TACE activity were observed upon installation of a
4-fluorobenzyloxy substituent (3, IC50=5 nM against
rTACE). The addition of the benzyloxy substituent in
3 also led to the erosion of MMP-1 activity (IC50=
1600 nM). In order to optimize the in vitro and in vivo
activity of these endocyclic hydroxamic acids we
have explored the biological properties of the related
4-hydroxy and 5-hydroxy pipecolic acid based hydro-
xamic acids 4 and 5 (Schemes 1 and 2). The hydroxy
substituents on the pipecolic acid moiety were installed
to lower the overall logP and potentially improve the
physicochemical properties of the molecule (the addi-
tion of the hydroxy group in 4b lowers the mlogP of the
molecule from 2.1 to 1.3). We installed various benzyl-
oxy tailpieces to these novel endocyclic hydroxamic
acids in order to assess the TACE and MMP-1 activity
in relation to changes at this site of the inhibitor.
For the purpose of preparing a wide range of benzyloxy
analogues of the 4-hydroxypipecolate scaffold 4, phenol
6 was prepared from 4-hydroxypiperidine-2-carboxylic
acid7 by treating the amine with 4-(benzyloxy)phenyl-
sulfonyl chloride in the presence of triethylamine
followed by hydrogenation of the resulting benzyloxy-
ether. Alkylation of the phenol with the appropriate
benzyl halide in the presence of cesium carbonate gave the
required benzyloxylactones 7, which were subsequently
converted to hydroxamic acids 4 upon treatment with
hydroxylamine in methanol.
The preparation of the corresponding 5-hydroxy pipe-
colates 5 is shown in Scheme 2. The intermediate diazo-
ketone 8 was generated on treating an ether solution of
the methyl ester of N-Boc-pyroglutamic acid with the
anion of TMS-diazomethane at ꢁ100 ꢂC.8 Rhodium
catalyzed cyclization9 of 8 afforded the 5-oxopipecolic
acid derivative 9, which was stereoselectively reduced
with methanolic sodium borohydride. Concomitant
removal of the Boc group and lactonization of the
hydroxy ester, followed by sulfonylation with 4-benzyl-
oxyphenylsulfonyl chloride afforded the pipecolate lac-
tone 10. Hydrogenolysis of the benzyl ether of 10 using
palladium adsorbed on activated charcoal afforded the
5-hydroxy pipecolate scaffold phenol 11, which could be
transformed into the corresponding pipecolic hydrox-
amic acids 5 as described in Scheme 1.
Figure 1 depicts the X-ray co-crystal structure of 5k
bound to TACE.10 The binding mode of the hydrox-
amic acid and sulfonamide moieties are typical for this
class of metalloprotease inhibitors;11 the hydroxamic
acid chelates to the catalytic zinc, and a sulfonamide
oxygen accepts a hydrogen-bond from the main chain.
Scheme 1. (a) H2, Pd/C, MeOH; (b) 6 N HCl; (c) 4-(benzyloxy)-phe-
nylsulfonyl chloride, triethylamine, DMF, 80%; (d) H2, Pd/C, MeOH,
84%; (e) Cs2CO3, ArCH2X, DMF, 58–99%; (f) NH2OH, MeOH,
60 ꢂC, 36–94%.
Scheme 2. (a) Trimethylsilyldiazomethane, n-butyllithium, Et2O,
ꢁ100 ꢂC, 96%; (b) Rh2(OAc)4, benzene, reflux, 97%; (c) NaBH4,
MeOH, 96%; (d) 6 M HCl, reflux, 100%; (e) 4-benzyloxy-
phenylsulfonyl chloride, Et3N, DMF, 57%; (f) H2, Pd/C, MeOH,
99%; (g) K2CO3, ArCH2X, DMF; (h) NH2OH, MeOH, reflux, 30–
47%.
Figure 1. X-ray crystal structure of 5k complexed with rTACE. The
local solvent-accessible surface is rendered in greenblue, with the cata-
lytic zinc portrayed as a violet CPK sphere. Atom colors are as follows:
carbon, yellow; nitrogen, blue; oxygen, red; sulfur, green; iodine, gray.

M. A. Letavic et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1387–1390
Table 1. Biological data for 4-hydroxy analogues, 4
1389
Compd
Ar
rTACE IC50 (nM)
Whole blood IC50 (mM)
MMP-1 IC50 (nM)
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
Phenyl
4-Fluorophenyl
3-Fluorophenyl
2-Fluorophenyl
4-Chlorophenyl
3-Chlorophenyl
2-Chlorophenyl
4-Methylphenyl
3-Methylphenyl
2-Methylphenyl
3-Methoxyphenyl
2-Methoxyphenyl
2-Trifluoromethylphenyl
4-Cyanophenyl
3-Cyanophenyl
2-Phenylphenyl
9
14
21
67
7
10
36
20
18
6
27
14
16
25
15
67
NDa
12
9
>30
58
>30
40
>30
>30
12
>30
26
16
77
8000
ND
16,000
12,000
6300
6500
29,000
30,000
9000
5000
24,000
2200
ND
30,000
23,000
ND
33
>33
aND, not determined.
Table 2. Biological data for 5-hydroxy analogues, 5
Compd
Ar
rTACE IC50 (nM)
Whole blood IC50 (mM)
MMP-1 IC50 (nM)
5a
5b
5c
5d
5e
5f
5g
5h
5i
Phenyl
4-Trifluoromethyl
3-Trifluoromethylphenyl
2-Trifluoromethylphenyl
3-Cyanophenyl
7
10
15
5
49
39
8
10
11
16
7
14
28
>30
4
63
42
3
30,000
3700
5800
15,000
NDa
15,000
3000
ND
2-Cyanophenyl
2-Methylphenyl
2-Ethylphenyl
2-Isopropylphenyl
1-Naphthyl
2-Iodophenyl
1
1
2
1
ND
ND
ND
5j
5k
aND, not determined.
Despite the elongated benzyloxy P10 group, the binding
conformation permits the two aryl rings to adopt positions
similar to that reported for phenoxyphenyl sub-
stituents.11 The ortho iodo substituent protrudes into a
sub-pocket of S10 that consists of the side chains of
Val440 and Asn447 and the mainchain atoms of Ile438,
Tyr436, Val434, and Tyr433. Comparison with the first
published X-ray structure of TACE (PDB accession
1BKC)12 reveals that accommodation of the iodo sub-
stituent requires significant conformational movement
on the part of the enzyme. In the present structure
Val440 is displaced by 2.4 A relative to the previously
reported structure and the backbone of Gly442 inverts and
moves 5.7 A to accommodate the inhibitor. The selec-
tivity over MMP-1 that is observed with these inhibitors
is attributed to the large P10 substituent that is not
accommodated by the shallow S10 pocket of MMP-1.11
group. Hence the unsubstituted benzyl analogue 4a has
approximately the same potency as 4j (2-Me) and 5a is
roughly equipotent with 5g–i (2-Me, 2-Et and 2-i-Pr).
However, when the substituent on the aryl ring is large
(i.e., 2-phenyl, 4p) there is significant loss of rTACE
activity. Halogens in the ortho position (2-chloro and 2-
fluoro) result in a loss of rTACE activity, but these
substituents are tolerated at the meta and para positions
of the ring.
Substitution at the ortho position of the terminal aryl
ring has a more dramatic effect on the human whole
blood potency. The inactivity of the 2-fluoro and
2-chloro analogues 4d and 4g in human whole blood
might have been expected from the loss of rTACE
activity observed with these compounds. However,
significantly improved potency in human whole blood
was observed, particularly in the 5-hydroxy series 5,
when ortho alkyl groups were introduced. Thus compound
5a is 5- to 14-fold less potent than the substituted ana-
logues 5g–i in human whole blood in spite of the fact
that the rTACE IC50’s for the compounds are nearly
identical. Also, the 5-hydroxypipecolates 5 are generally
more potent in human whole blood than the 4-hydroxy-
pipecolates 4. For example, 4j has an IC50 of 12 mM in
human whole blood, but the corresponding 5-hydroxy-
pipecolate 5g has an IC50 of 3 mM. The 2-ethyl and
2-isopropyl analogues 5h and 5i are even more potent
The hydroxamic acids in Tables 1 and 2 were assayed
for TACE activity using a recombinant TACE assay13
and a human whole blood assay measuring TNF-a.14
Many of the analogues were also screened against
MMP-1.15 No significant MMP-1 activity was observed
with any member of this series.
An examination of the rTACE activity within this series
indicates that an ortho substituent on the benzyloxy ring
is tolerated when the substituent is an electron-donating

1390
M. A. Letavic et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1387–1390
(IC50’s=1 mM). The reasons for this are not entirely
clear. Presumably the shift in activity observed in the
human whole blood assay as compared to the rTACE
assay is largely due to cell permeability requirements in
combination with plasma protein binding issues.
10. TACE catalytic domain (a 29,267 Da fragment encom-
passing residues 215–474 of human TACE with N264A and
N452A mutations) was crystallized as a complex with 5k.
Protein at 1 mg/mL (35 mM) in 25 mM HEPES, 100 mM NaCl,
pH 7.5 was treated with 5k and then concentrated to 22 mg/
mL. Crystals which diffracted to 2.0 A grew from hanging
drops set up with equal volumes of protein and well solution
The excellent human whole blood potency observed
with these substituted pipecolic acid derivatives has
prompted us to investigate the in vivo properties of the
more potent members of this series. The results of these
studies will be reported in due course.
equilibrated over 26–28% PEG4K, 0.1 M Tris, 0.2 M MgCl
2
(1–2 weeks). The crystals are orthorhombic (space group
P212121, a=77.8, b=81.7, c=85.9 A), and contain two copies
of the protein-inhibitor complex in the crystallographic asym-
metric unit. Data to 2.0 A resolution were collected from a
cryo-cooled crystal on a RAXIS IIC detector mounted on a
Rigaku RU200 X-ray generator. The structure was solved by
molecular replacement, using the coordinates of unliganded
TACE (ref 11, PDB accession code 1BKC), as a starting
search model in the program AmoRe (Navaza, J. Acta Crys-
tallogr. 1994, A50, 157). Difference Fourier maps calculated
after initial refinement of the molecular replacement solution
showed unambiguous electron density at the active site for the
bound inhibitor molecule. The overall protein structure is
essentially superimposable on the coordinates of 1BKC, with
an r.m.s. difference of 0.63 A for 255 C-a atoms.
11. Lovejoy, B.; Welch, A. R.; Carr, S.; Luong, C.; Broka, C.;
Hendricks, R. T.; Campbell, J. A.; Walker, K. A. M.; Martin,
R.; Van Wart, H.; Browner, M. F. Nature Struct. Biol. 1999, 6,
217.
12. Maskos, K.; Fernandez-Catalan, C.; Huber, R.; Bour-
enkov, G. P.; Bartunik, H.; Ellestad, G. A.; Reddy, P.; Wolf-
son, M. F.; Rauch, C. T.; Castner, B. J.; Davis, R.; Clarke,
H. R. G.; Peterson, M.; Fitzner, J. N.; Cerretti, D. P.; March,
C. J.; Paxton, R. J.; Black, R. A.; Bode, W. Proc. Natl. Acad.
Sci. U.S.A. 1998, 95, 3408.
13. Enzyme reaction was comprised of 20 mM Hepes buffer
(pH, 7.5), 20 mM ZnCl2, 10 mM fluorescent quenched sub-
strate, test compound, and rTACE enzyme. The reaction was
started by the addition of substrate and initial rates of clea-
vage were monitored by increase in fluorescence at 530 nm
(excitation at 409 nm) over 30 min. The specificity of the
enzyme cleavage at the amide bond between alanine and valine
was verified by HPLC and mass spectrometry. The fluorogenic
substrate was prepared as described in Geoghegan, K. F. Bio-
conjugate Chem. 1996, 7, 385. Recombinant TACE catalytic
domain was expressed with its prodomain in baculoviral
infected insect (SF9) cells. Active enzyme was secreted into the
extracellular medium and partially purified by anion-exchange
chromatography on Q Sepharose Fast Flow (Pharmacia Bio-
tech).
14. Heparinized human whole blood was stimulated by
Escherichia coli LPS in the presence of test agent in volume of
0.25 mL for 4 h at 37 ꢂC, after which plasma was recovered
and TNF-a measured by ELISA (R&D Systems).
15. Compounds were assayed versus MMP-1 using a quen-
ched fluorescent peptide substrate assay as described in Bick-
ett, D. M.; Green, M. D.; Berman, J.; Dezube, M.; Howe,
A. S.; Brown, P. J.; Roth, J. T.; McGeehan, G. M. Anal. Bio-
chem. 1993, 212, 58.
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3. Black, R. A.; Rauch, C. T.; Kozlosky, C. J.; Peschon, J. J.;
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Reddy, P.; Srinivasan, S.; Nelson, N.; Boiani, N.; Schooley,
K. A.; Gerhart, M.; Davis, R.; Fitzner, J. N.; Johnson, R. S.;
Paxton, R. J.; March, C. J.; Cerretti, D. P. Nature 1997, 385, 729.
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