Synthesis and biological activity of piperazine-Based dual MMP-13 and TNF-α converting enzyme inhibitors

Article abstract of DOI:10.1016/S0960-894X(03)00666-8

A series of novel MMP-13 and TNF-α converting enzyme inhibitors based on piperazine 2-hydroxamic acid scaffolds are described. The TACE, MMP-1 and MMP-13 activity of these inhibitors as well as the effect of substitution of the piperazine nitrogen and the P-1′ benzyloxy tailpiece is discussed. Moderate in vivo activity is observed with several members of this group.

Full text of DOI:10.1016/S0960-894X(03)00666-8

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3243–3246
Synthesis and Biological Activity of Piperazine-Based Dual
MMP-13 and TNF-ꢀ Converting Enzyme Inhibitors
Michael A. Letavic,* John T. Barberia, Thomas J. Carty, Joel R. Hardink, Jennifer Liras,
Lori L. Lopresti-Morrow, Peter G. Mitchell, Mark C. Noe, Lisa M. Reeves,
Sheri L. Snow, Ethan J. Stam, Francis J. Sweeney, Marcie L. Vaughn and Chul H. Yu
Pfizer Global Research and Development, Groton Laboratories, Eastern Point Road, Groton, CT 06340, USA
Received 28 March 2003; revised 10 June 2003; accepted 24 June 2003
Abstract—A series of novel MMP-13 and TNF-a converting enzyme inhibitors based on piperazine 2-hydroxamic acid scaffolds are
described. The TACE, MMP-1 and MMP-13 activity of these inhibitors as well as the effect of substitution of the piperazine
nitrogen and the P-10 benzyloxy tailpiece is discussed. Moderate in vivo activity is observed with several members of this group.
# 2003 Elsevier Ltd. All rights reserved.
The pro-inflammatory cytokine tumor necrosis factor-a
(TNF-a) is produced by monocytes/macrophages and
other cell types. In a variety of diseases including rheu-
matoid arthritis (RA) and Crohn’s disease (CD), the
overproduction of TNF-a plays a key role in inducing
the recruitment of inflammatory cells and in stimulating
the production of other mediators of pain and cartilage
breakdown.1 Support for the therapeutic utility of
molecules that inhibit the pro-inflammatory effects of
TNF-a includes results of recent human clinical trials
for two biologics, etanercept, a soluble TNF-a receptor-
Fc dimer (Immunex/Amgen), and infliximab, an anti-
TNFa antibody (Centocor/Johnson and Johnson), in
RA and CD.2 Both of these biologics are currently
marketed for the treatment of rheumatoid arthritis and
both have been shown to decrease signs and symptoms
and to reduce joint destruction in RA patients.
TNF-a is synthesized as a 26 kDa membrane protein
that is shed from the cell following a proteolytic clea-
vage carried out by a 90 kDa membrane-bound zinc
metalloprotease,
TNF-a
Converting
Enzyme
(TACE).3ꢀ5 Once cleaved, sol-TNF-a is liberated from
the cell and it is this soluble form of TNF-a that is
thought to be responsible for much of the inflammation
associated with RA and other inflammatory diseases.
*Corresponding author. Tel.: +1-860-441-1803; fax: +1-860-715-
2467; e-mail: michael_a_letavic@groton.pfizer.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00666-8

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M. A. Letavic et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3243–3246
now describe the activity of a novel series of piperazine
based hydroxamic acids that are dual TACE/MMP-13
inhibitors with minimal MMP-1 inhibition. We also
report in vivo efficacy in an acute model of inflamma-
tion and pharmacokinetic data in rats for several of
these inhibitors.
The preparation of the piperazines 5 was completed in
one of two ways. Scheme 1 describes the initial route to
these compounds, which utilizes an asymmetric amino-
hydroxylation14 of t-butyl crotonate to form the
aminoalcohol 6. The alcohol of 6 is then converted to
the mesylate upon treatment with methansulfonyl
chloride. Subsequent cyclization in the presence of
potassium t-butoxide provides the aziridine in moderate
Scheme 1. (a) (DHQD)2PHAL, K2OsO2(OH)4, CBZNHCl, t-BuOCl,
60%; (b) methanesulfonyl chloride, Et3N,CH2Cl2, 78%; (c) KOt-Bu,
30–40%; (d) H2, Pd/C, EtOH, 86%; (e) ArSO2Cl, Et3N, DMF, 96%;
(f) 2-aminoethanol, CH2Cl2, 47%; (g) Ph3P, DEAD, 90%; (h) acetyl
chloride, Et3N, DMF, 100%; (i) TFA, CH2Cl2, 100%; (j) O-allyl-
hydroxylamine, EDC, HOBt, DIPEA, 50–68%; (k) Pd(Ph3P)4,
HCO2H/Et3N, 21–55%.
Therefore we,6 and others,7ꢀ9 have had an interest in
molecules that have the potential to inhibit TACE in
vivo, thereby reducing the circulating levels of sol-TNF-
a. Our group at Pfizer has also had a long-standing
interest in the use of matrix metalloprotease inhibitors
(MMP inhibitors) for the treatment of arthritis and a
variety of other disease states.10 More specifically, we
were interested in molecules that are potent MMP-13
inhibitors as MMP-13 has been reported to be expressed
in human osteoarthritic cartilage and recombinant
MMP-13 has been shown to efficiently cleave type II
collagen in vitro.11 We were also seeking compounds
with diminished activity against MMP-1 hypothesizing
that this might eliminate some of the side effects repor-
ted to be associated with broad spectrum MMP inhibi-
tion. Based on the above information we began a
program to discover small molecule inhibitors of TACE
and MMP-13 that were devoid of MMP-1 activity with
the idea that these molecules should be useful for the
treatment of a variety of disorders. Several examples of
dual MMP/TACE inhibitors have been reported in the
literature recently, including GW3333,9 the benzodiaze-
pines12 and the anthranilates7,8 shown below. In addi-
tion to the structures shown, we recently disclosed the
discovery of a series of pipecolic acid-based TACE
inhibitors6 and Procter and Gamble has recently repor-
ted on a series of broad-spectrum piperazine-based
MMP inhibitors.13 Following up on these efforts, we
Scheme 2. (a) Benzyl chloroformate, then cesium carbonate, 100%;
(b) 3-bromomethyl-3-methyloxetane, DMF, 78%; (c) BF3–Et2O, 36%;
(d) H2, Pd/C, EtOH, 100%; (e) ArSO2Cl, Et3N, DMF, 87%; (f) Ph3P,
DEAD; (g) 2-aminoethanol; (h) Ph3P, DEAD; (i) R1-X, R-NCO, R-
C(O)Cl or R-SO2Cl; (j) trifluoroacetic acid then Cs2CO3, 50–70%,
(k) O-allylhydroxylamine, EDC, HOBt, DIPEA, 40–68%;
(l) Pd(Ph3P)4, HCO2H:Et3N, 22–55%.

M. A. Letavic et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3243–3246
Table 1. Biological data for piperazine analogues, 5
3245
Compd
R1
Ar
rTACE (IC50, nM)
Whole blood (IC50, mM)
MMP-13 (IC50, nM)
MMP-1 (IC50, nM)
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
H
H
Ph
2-MePh
2-MePh
2-EtPh
3,5-FPh
4-FPh
25
19
6
7
6
18
2
1
0.6
1
2
11
19
3
40
20
3
12,000
12,000
1600
6700
3800
1700
1900
10,000
6400
830
H3CC(O)
H3CC(O)
H3CC(O)
H3CC(O)
H3CC(O)
H3CC(O)
H3CC(O)
H3CC(O)
H3CC(O)
MeSO2
4-MePhSO2
Me
PhCH2
PhNHC(O)
H3COC(O)
i-PrNHC(O)
EtOC(O)
6
7
2-Me, 5-FPh
2-CF3Ph
4-Isoquinoline
4-Quinoline
3-(2-Me)-pyridyl
2-MePh
1
2
7
11
ND
ND
5
127
98
17
8
0.4
0.3
0.8
1
26
33
33
12
2
3400
1300
ND
6a
4
2-MePh
2-MePh
2-MePh
2-MePh
2-MePh
2-MePh
2-MePh
32a
31a
68a
26a
14a
34b
28b
ND
ND
ND
ND
4
ND
17,000
2200
1500
1400
1500
3
3
4
6
aAs assayed using a rTACE assay.
bAs assayed using a partially purified TACE membrane preparation.
yield. Hydrogenation to give aziridine 7 is followed by
formation of the sulfonamide 8. Reaction with
2-aminoethanol provides a mixture of aminoalcohols 9
and 10. Chromatographic separation followed by the
treatment of 9 with PPh3/DEAD gives the piperazine 11
which can be converted to the corresponding piperazine
hydroxamic acid by treatment with an appropriate
electrophile, removal of the t-butyl ester, formation of
the allyl hydroxamate and palladium mediated removal
of the allyl moiety. This route has a number of issues.
First, the sequence is rather lengthy, second, aziridine 7 is
volatile and therefore difficult to handle and third, the
ring opening reaction of 8 proceeds with little selectivity
for the desired product 9. In order to address these issues,
a second route to these molecules was developed.
In vitro and human whole-blood data for a number
of piperazines 5 is shown in Table 1. Variations of
the substituents on the benzyl tailpiece and the
piperazine nitrogen were examined. Most of the
compounds shown are potent TACE inhibitors (<50
nM).15
With
the
exception
of
the
2-tri-
fluoromethylphenyl and 4-isoquinoline compounds 5i–
j, all of the compounds shown also have good MMP-
13 activity.16 The human whole-blood activity of
these analogues varies considerably. This variation is
likely caused by a number of factors, including protein
binding and permeability. However, the ortho sub-
stituted aryl groups such as 5c–d and 5k and the 4-iso-
quinoline and 4-quinoline derivatives 5i–j are clearly the
most potent compounds in whole blood, with several
compounds less than 500 nM. As expected from pre-
vious modeling6 the benzyl tailpiece on these analogues
is too large to fit in the smaller P-10 pocket of MMP-1
and therefore none of the compounds shown has sig-
nificant MMP-1 activity.
In the modified route (Scheme 2) d-threonine is con-
verted to the N-CBZ protected 4-methyl-2,6,7-trioxa-
bicyclo[2.2.2]oct-1-yl ester (OBO ester) in a four-step
procedure that provides 12 in moderate yield. Conver-
sion of 12 to the aziridine 13 is straightforward and
provides a very sterically hindered aziridine to perform
the ring opening reaction with 2-aminoethanol. Regio-
selective ring opening followed by reaction with PPh3/
DEAD gives the desired piperazine 14 in 65% overall
yield. Acylation of the piperazine nitrogen is followed by
removal of the OBO ester (TFA then Cs2CO3). The
synthesis of analogues 5 is then completed as was described
in Scheme 1. All of the compounds in Table 1 were pre-
pared using slight modifications of these two procedures.
Compounds 5b, 5c and 5l were examined in an acute rat
LPS-induced model of systemic TNF-a production.17 The
iv pharmacokinetic parameters for these compounds were
also determined in a rat.18 The data are shown in Table
2. Of the compounds shown 5c is the most potent in
vivo (ED50=17 mg/kg). The moderate in vivo activity
observed for these compounds is very likely due the
their short half-life in rat which appears to be primarily
due to high clearance.
In summary, these piperazine-based hydroxamic acids
represent a new sub-type of potent dual TACE/MMP-
13 inhibitors with potent TNF activity in human whole
blood. The moderate in vivo activity of these analogues
is likely due to high clearance in the rat. Further opti-
mization of the in vivo properties of these and related
TACE/MMP-13 inhibitors will be reported in due
course.
Table 2. In vivo data for piperazine analogues, 5
Compd
Rat TNF
ED50 (mg/kg)
Rat Clp Vdss
(mL/min/kg) (L/kg)
iv half-life
(h)
5c
5b
5l
17
39% inhib@30 mg/kg
55% inhib@30 mg/kg
54
83
98
0.6
1.1
2.1
0.27
0.24
0.21

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M. A. Letavic et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3243–3246
Leenitzer, M. A.; Mcdougald, D. L.; Han, B.; Hedeen, K.;
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