Discovery of γ-lactam hydroxamic acids as selective inhibitors of tumor necrosis factor α converting enzyme: Design, synthesis, and structure-activity relationships

Article abstract of DOI:10.1021/jm0255670

New γ-lactam TACE inhibitors were designed from known MMP inhibitors. A homology model of TACE was built and examined to identify the S1′ site as the key area for TACE selectivity over MMPs. Rational exploration of the P1′-S1′ interactions resulted in the discovery of the 3,5-disubstituted benzyl ether as a TACE-selective P1′ group. Further optimization led to the discovery of IK682 as a selective and orally bioavailable TACE inhibitor.

Full text of DOI:10.1021/jm0255670

4954
J . Med. Chem. 2002, 45, 4954-4957
Discover y of γ-La cta m Hyd r oxa m ic Acid s
a s Selective In h ibitor s of Tu m or Necr osis
F a ctor r Con ver tin g En zym e: Design ,
Syn th esis, a n d Str u ctu r e-Activity
Rela tion sh ip s
J ames J .-W. Duan,* Lihua Chen,
Zelda R. Wasserman,† Zhonghui Lu, Rui-Qin Liu,‡
Maryanne B. Covington,‡ Mingxin Qian,§
Karl D. Hardman,† Ronald L. Magolda,‡
Robert C. Newton,‡ David D. Christ,§
Ruth R. Wexler, and Carl P. Decicco
Discovery Chemistry, Bristol-Myers Squibb Company,
Experimental Station, Wilmington, Delaware 19880-0500
Received August 1, 2002
F igu r e 1. Representative MMP inhibitors (1 and 2) and
targets considered during the discovery of γ-lactams (3-5).
Abstr a ct: New γ-lactam TACE inhibitors were designed from
known MMP inhibitors. A homology model of TACE was built
and examined to identify the S1′ site as the key area for TACE
selectivity over MMPs. Rational exploration of the P1′-S1′
interactions resulted in the discovery of the 3,5-disubstituted
benzyl ether as a TACE-selective P1′ group. Further optimiza-
tion led to the discovery of IK682 as a selective and orally
bioavailable TACE inhibitor.
are a class of substrate-based inhibitors, represented
by marimastat (1, Figure 1)7 and have been investigated
extensively in the MMP field. A new series of non-
peptidic inhibitors was subsequently reported by scien-
tists at Novartis, typified by CGS 27023A (2).8 The
hydroxamate group of 2 binds to the catalytic zinc of
MMP-3 in a fashion similar to that of the succinate-
based inhibitors.8b,c Interestingly, only one oxygen (pro-
R) of the SO2 group is involved in H-bonding with
MMP-3 (Leu164), which in turn projects the 4-methoxy-
phenyl group into the S1′ site. The picolyl group of 2 is
projected toward solvent and does not appear to be
critical for binding. Comparison of the binding mode of
1 and 2 revealed a zinc ligand (blue), a hydrogen-bond
acceptor (red), and a P1′ group (pink) as three common
features, which were used as key focal points in the
design of a new scaffold.
Because only the pro-R oxygen of the sulfonyl group
in 2 was involved in binding, we first attempted to dock
benzamide 3 into MMP-3. However, it is impossible to
maintain the three key interactions of 3 with the protein
because the carbon is not only smaller than the sulfur
but also has a planar rather than a tetrahedral geom-
etry. To compensate for the planar and smaller car-
boxamide group, we inserted a methylene between the
carbonyl and phenyl to create phenylacetamide 4. It was
gratifying to find that when modeled in MMP-3, 4 can
be arranged in a conformation with the hydroxamate,
acetamide oxygen, and phenyl group overlapping the
corresponding groups of 2, keeping the three key
interactions we set out to achieve. In this model, the
phenyl group is orientated perpendicular to the aceta-
mide plane, placing the pro-S hydrogen of the benzylic
methylene (Ha) and the methyl group of the tertiary
amide in proximity. We envisioned that replacing Ha
with a methylene and cyclization with the methyl group
would enforce the conformation required for binding.
γ-Lactam 5 was designed and shown to accomplish this
goal. A methyl group was introduced at the R-position
of the lactam to enhance the population of conformers
with the phenyl group in a pseudoaxial orientation. A
computer model of a γ-lactam analogue (5a , R ) H) in
MMP-3 revealed that the γ-lactam template could
effectively project the hydroxamate, lactam carbonyl,
In tr od u ction . Rheumatoid arthritis (RA) is an in-
flammatory autoimmune disease affecting more than 2
million people in the United States alone. It is tradi-
tionally treated with nonsteroidal antiinflammatory
drugs (NSAID) or disease-modifying antirheumatoid
drugs (DMARD). Owing to limited effectiveness and/or
side effects of the two drug classes, there has been a
continued search for improved therapeutics. One at-
tractive target is the cytokine tumor necrosis factor R
(TNFR),1 which has been shown to be overproduced in
the joint of RA patients. The clinical success of anti-
TNFR biologics2 has validated TNFR as a drug discovery
target. While they are effective, these proteins are
expensive to make and must be administered parenter-
ally.
An alternative approach would be inhibiting forma-
tion of TNFR. TNFR is processed from its membrane-
bound precursor by the metalloprotease TNFR convert-
ing enzyme (TACE).3 TACE is a member of the reprolysin
family of the metzincin superfamily that also includes
MMPs (matrix metalloproteinases). Some inhibitors of
MMPs have also been shown to block TACE, suggesting
some similarities between the active sites of these
enzymes. We4 and others5 have successfully drawn from
the knowledge base of MMP inhibitor design to derive
inhibitors of TACE. This paper will disclose the discov-
ery of a new series of selective TACE inhibitors using a
γ-lactam scaffold.6
In h ibitor Design . When our program was initiated,
the tertiary structure of TACE was unknown and all
known TACE inhibitors also inhibited MMPs. MMP
inhibitors were therefore used to aid the initial design
of TACE inhibitors. Succinate-derived hydroxamic acids
* To whom correspondence should be addressed. E-mail: james.duan@
bms.com. Phone: (302) 467-6057. Fax: (302) 467-6802.
† Structural Biology and Molecular Design Group.
‡ Department of Inflammatory Disease Research.
§ Department of Metabolism and Pharmacokinetics.
10.1021/jm0255670 CCC: $22.00 © 2002 American Chemical Society
Published on Web 10/15/2002

Letters
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 4955
Sch em e 1. Synthesis of Lactam Analogue IK682a
a
Conditions: (a) NaHMDS, MeI, THF; (b) NaHMDS, allyl
bromide, THF; (c) O3, CH2Cl2, PPh3 (76% for three steps); (d)
D-alanine methyl ester, Zn, HOAc, at reflux (80% yield for both
isomers, ratio 1:1); (e) H2, Pd(OH)2/C, MeOH (quantitative); (f)
Cs2CO3, 4-chloromethyl-2-methylquinoline hydrochloride, NaI,
DMSO (91%); (g) NH2OH, KOH, MeOH (90%).
F igu r e 2. Computer model of 5a in MMP-3. The surface
representation of MMP-3 (white except for nitrogen atoms of
L164 and A165, blue) is truncated to expose backbone atoms
within hydrogen bonding distance of the lactam’s carbonyl.
Oxygen atoms of the hydroxymate coordinate the zinc ion
(orange), and the phenyl ring projects into the S1′ pocket.
Ta ble 1. In Vitro Potency of Lactams 5a -n a
MMP (Ki, nM)
pTACE,
R
IC50, nM
-1
221
4000
92
-2
-9
5a
H
2200
1000
887
108
701
242
586
9
and phenyl group to maintain the three previously
mentioned binding interactions with active site of
MMP-3 (Figure 2).
5b isobutyl
5c methoxy
4.8
5d tert-butoxy
5e allyloxy
5f benzyloxy
5g phenyl
>10000
b
335
b
14
19
82
<2.8
35
38
11
5812
b
97
11
29
35
<2.1
66
39
40
In clinical trials, broad-spectrum MMP inhibitors
have been shown to cause tendonitis,9 which may be
attributed to nonspecific inhibition of MMPs required
for normal physiological matrix turnover. Therefore, it
would be desirable to discover TACE inhibitors having
no effect on MMP activity. At the start of this work, all
of the TACE inhibitors reported in the literature and
from our in-house collection were found to be more
potent against MMPs. As a starting point for TACE
selectivity, a homology model of TACE was built on the
basis of the structure of atrolysin, a related member of
the reprolysin family. An overlay of the TACE model
and MMP-3 crystal structure was examined to identify
chemical opportunities for imparting TACE selectivity
into lactam 5. A striking difference was observed in S1′.
MMP-3 is known to have a straight and deep S1′ pocket,
while the TACE model predicts an L-shaped S1′ pocket.
One basis for the difference is a tyrosine (Tyr223), which
in MMP-3 forms one side of the S1′ pocket and is
conserved across the MMP family. In our TACE model,
this residue is replaced by the much smaller alanine.
These observations suggested that selectivity could be
achieved by suitable modification of P1′ such that the
designated P1′ group should be able to adopt a bent
conformation.
Syn th esis. The synthesis of a γ-lactam analogue
IK682 (5s) is depicted in Scheme 1. Phenylacetate 6 was
reacted sequentially with iodomethane and allyl bro-
mide under basic conditions. Ozonolysis of the crude
material provided aldehyde 7 in 76% overall yield.
Treatment of 7 with D-alanine methyl ester and Zn dust
in HOAc at reflux facilitated reductive amination and
cyclization in one pot. The two lactam diastereomers,
epimeric at the quaternary center, were separated by
chromatography, and the desired isomer 8 was deben-
zylated to give 9. The structure of 9 was confirmed by
single-crystal X-ray analysis. Phenol 9 was reacted with
4
>5000
2288
143
>5000
>5000
>5000
4747
13000
185
4
5h phenoxy
5i 2-nitrobenzyloxy
5j 3-nitrobenzyloxy
5k 4-nitrobenzyloxy
5l 3,5-di-Me-benzyloxy
5m 3,5-di-MeO-benzyloxy
5n 3,5-bis-CF3-benzyloxy
6
6
4
7
1634
>2000
>2000
>5000 >3000
>5000 >3000
2
a
pTACE IC50 and MMP Ki values are from a single determi-
b
nation. Not tested.
4-chloromethyl-2-methylquinoline in the presence of Cs2-
CO3 and converted to IK682 using NH2OH and KOH.
Other lactam analogues were synthesized from either
appropriately substituted phenylacetate in place of 6 or
phenol intermediate 9.
Resu lts a n d Discu ssion . Lactam 5a was found to
have a submicromolar affinity for MMP-1, -2, and -910
(Table 1), validating the lactam model. In the porcine
TACE (pTACE) assay,11 5a is about 10-fold less potent.
Docking and examination of 5a in the TACE model
suggested that the phenyl group of 5a occupies the
upper section of the S1′ pocket. From this projection,
we anticipated that the TACE potency would be en-
hanced by substitution in the para position of the phenyl
group. The isobutyl and methoxy analogues (5b,c)
yielded only 2-fold improvement in affinity for pTACE.
However, the methoxy compound 5c increased MMP-2
and -9 potency by more than 20-fold over 5a , indicating
a divergent SAR for the two enzyme classes. A bulky
tert-butoxy group was not tolerated, as evidenced by the
loss of activity for pTACE (5d ). The first encouraging
result was obtained with an allyloxy group (5e), which
boosted pTACE potency to 97 nM (>20-fold over 5a ).
More dramatically, the benzyloxy substitution (5f)
resulted in a 4 nM inhibitor, a 20-fold enhancement
compared to 5e, while MMP-2 and -9 activity remained

4956 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Letters
Ta ble 2. In Vitro Potency of 5o-s in pTACE and WBAa
pTACE,
IC50, nM
WBA,b
IC50, nM
R
5o
5p
5q
5r
(pyridin-4-yl)methoxy
21
16
3
5.9
8.1
6.6
2.25
0.35
(2,6-di-Me-pyridin-4-yl)methoxy
(2,6-di-Me-pyridin-4-yl)methoxy
(quinolin-4-yl)methoxy
1.8
5s
(2-Me-quinolin-4-yl)methoxy
1c
a
b
See footnotes a and b in Table 1. Inhibition of TNFR release
in WBA was determined with three donors. c Detection limit of
the assay.
Ta ble 3. Selectivity Profile of IK682
F igu r e 3. Model of 5f in MMP-3 (left) and the TACE
homology model (right). Nitrogen atoms are blue, oxygens red,
and carbon atoms yellow and light-blue in the MMP-3 and
TACE models, respectively.
enzyme
Ki,a nM
pTACEb
MMP-1 (collagenase-1)
0.56
30000
2050
141
259
257
10340
1417
15872
3997
1599
MMP-2 (gelatinase A)
MMP-3 (stremolysin-1)
MMP-7 (matrilysin)
MMP-8 (collagenase-2)
essentially unchanged. The decreased affinity for MMP-1
was anticipated because the 4-(benzyloxy)phenyl group
is too long to fit into the now well-established shallow
S1′ pocket of MMP-1.12 Docking 5f in the TACE and
MMP-3 active sites suggested that the flexible 4-(ben-
zyloxy)phenyl group can adopt a bend predicted to be
necessary for binding in the curved S1′ pocket of TACE,
as well as a more linear staggered conformation when
binding to MMPs (Figure 3). To further validate the
models, phenyl and phenoxy analogues were synthe-
sized. As shown in Table 1, as the linearity and rigidity
of P1′ increased from phenoxyphenyl (5h ) to biphenyl
(5g), a more detrimental effect on pTACE potency was
observed (40-fold and 3000-fold loss of potency, respec-
tively, compared to 5f). MMP-2 and -9 potency remained
essentially unchanged for 5g but increased considerably
for 5h , consistent with a deep but more linear S1′ pocket
of these proteins.
Attempts to further improve selectivity for TACE by
monosubstitution of the benzyl group of 5f proved to be
unsuccessful. For example, 5i-k gave selectivity profiles
almost identical to that of 5f. The effect of a second
substitution, specifically the 3,5-disubstitution, was
dramatic. Dimethyl compound 5l maintained pTACE
potency and, more importantly, exhibited exquisite
selectivity over MMP-1, -2, and -9 (1100-, 1400-, and
400-fold, respectively). The selectivity appears largely
due to steric interactions because both electron-donating
(OMe) and electron-withdrawing (CF3) groups yielded
inhibitors with comparable selectivity (5m and 5n ). The
sum of these data suggested that the S1′ pocket of
MMP-2 and -9 is narrower, while TACE appears to have
a more spacious S1′.
Despite their potency for pTACE, 5f and 5i-n were
determined to be weak inhibitors of TNFR production
in the LPS-stimulated human whole blood assay (WBA;
IC50 > 10 µM).11 It has been proposed that most of pro-
TNFR processing occurs intracellularly,13 indicating that
compounds that lack cell membrane penetration are
likely to have weak activity in the WBA. Protein binding
could also adversely affect the potency in WBA. In our
Caco-2 assay, 5f had an excellent Papp value (49 × 10-6
cm/s), indicating that cell membrane penetration may
not be an issue for the series. However, the series was
found to be highly protein-bound (0.84% free for 5l). To
increase the free fraction, several polar bioisosteres of
the benzyl group were introduced. The 4-picolyl ether
5o increased the free fraction to 32%, and most signifi-
cantly, the WBA potency improved to 5.9 µM (8-fold)
MMP-9 (gelatinase B)
MMP-13 (collagenase-3)
MMP-14 (membrane type-1 MMP)
MMP-15 (membrane type-2 MMP)
MMP-16 (membrane type-3 MMP)
a
b
n ) 3. Generated with a tight binding inhibition equation.
despite the 5-fold loss of affinity for pTACE compared
to 5l (Table 2). The 2,6-dimethyl- and 2,6-dichloro-4-
picolyl analogues 5p and 5q maintained comparable
potency in the WBA to 5o. Analogous to the SAR in the
benzyl ether series, 5p and 5q restored selectivity over
MMP-2 and -9 (data not shown). The 4-quinolinyl-
methoxy analogue 5r further improved affinity to 2.25
µM in the WBA but suffered from MMP-2 and -9 activity
(384 and 506 nM, respectively). 2-Methylquinoline
analogue IK682 (5s) not only reestablished selectivity
over MMP-2 and -9 but also resulted in a 6-fold increase
in the WBA potency (0.35 µM). The WBA activity of
IK682 can be partially contributed to the improved
potency for pTACE (vide infra) and the relatively high
free fraction in human serum (3.6%).
IK682 was found to be a potent inhibitor of pTACE
with a Ki of 0.56 nM (Table 3). It exhibited excellent
selectivity (>2000-fold) for pTACE relative to MMP-1,
-2, -9, -13, -14, -15, and -16. Even though it has a
submicromolar Ki for MMP-3, -7, and -8, IK682 is still
more than 200-fold selective for pTACE.
The pharmacokinetics of IK682 administered intra-
venously and orally to Sprague Dawley rats and Beagle
dogs was studied in a cassette-dose fashion, and the
plasma samples were analyzed by a LC-MS-MS as-
say.14 After iv administration, the systemic clearance,
volume of distribution at steady state, and terminal
half-life were 2.1 L h-1 kg-1, 2 L kg-1, and 11.8 h in rat
and 0.9 L h-1 kg-1, 0.9 L kg-1, and 7.1 h in dog,
respectively (Table 4). Oral absorption was rapid in both
species with peak concentration occurring within 0.5 h.
Oral bioavailability was good in both species, 41% (rat)
and 32% (dog). The favorable oral bioavailability and
long terminal half-life were likely due to the low to
moderate clearance and good absorption consistent with
the excellent Caco-2 Papp value (17.3 × 10-6 cm/s).
In conclusion, a novel lactam series of potent, selec-
tive, and orally bioavailable TACE inhibitors were
designed using computer models, crystal structure of
MMP-3, and known MMP inhibitors. To understand and

Letters
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 4957
Ta ble 4. Pharmacokinetic Parameters of IK682a
Design, Synthesis, and Structure-Activity Relationships of
Macrocyclic Hydroxamic Acids That Inhibit Tumor Necrosis
Factor-R Release in Vitro and in Vivo. J . Med. Chem. 2001, 44,
2636-2660. (b) Xue, C.-B.; He, X.; Corbett, R. L.; Roderick, J .;
Wasserman, Z. R.; Liu, R.-Q.; J affee, B. D.; Covington, M. B.;
Qian, M.; Trzaskos, J . M.; Newton, R. C.; Magolda, R. L.; Wexler,
R. R.; Decicco, C. P. Discovery of Macrocyclic Hydroxamic Acids
Containing Biphenylmethyl Derivatives at P1′, a Series of
Selective TNF-R Converting Enzyme Inhibitors with Potent
Cellular Activity in the Inhibition of TNF-R Release. J . Med.
Chem. 2001, 44, 3351-3354.
PK parameters
iv
rat
dog
dose (mg/kg)
t1/2 (h)
4
11.8
2.1
4
7.1
Cl (L h-1 kg-1
)
0.94
0.93
10211
Vss (L kg-1
)
2.0
AUC (nM h)
4171
po
dose (mg kg-1
tmax (h)
)
8
8
(5) (a) Rabinowitz, M. H.; Andrews, R. C.; Becherer, J . D.; Bickett,
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0.38
41
0.38
32
F (%)
a
Determination of two for each dosing group.
optimize TACE selectivity, a homology model of TACE
was built, and the S1′ site was identified as a key area
for selectivity. Rational exploration of the P1′-S1′
interactions using the lactam template resulted in
potent and selective TACE inhibitors, exemplified by
IK682. Discovery of these selective molecules should
enable more precise studies delineating the biological
roles of TACE and other MMP inhibitors in inflamma-
tory, cardiovascular, and oncologic models of human
disease and perhaps offer new therapeutic agents for
the treatment of rheumatoid arthritis.
Ackn owledgm en t. We thank John Giannaras, Sher-
rill Nurnberg, and Paul Strzemienski for assistance in
in vitro assays, Maria Ribadeneira for Caco-2 assay, Will
Marshall for the crystal structure of 9, and Matthew
Voss and Chu-Biao Xue for discovery of the (2-methyl-
4-quinolinyl)methoxy group in a different series, which
will be reported in a separate communication.
(6) During the course of this work, a patent application disclosing
unrelated lactam-based MMP inhibitors was published: J acob-
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work, another series of unrelated lactam-based MMP inhibitors
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Structure-Based Design and Synthesis of a Potent Matrix
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Su p p or tin g In for m a tion Ava ila ble: Experimental de-
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at http://pubs.acs.org.
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(10) Selectivity profile was assessed using MMP-1 as a representative
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S1′ pockets. Details of the assays are described in ref 4a.
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