Rational design, synthesis and structure-Activity relationships of a cyclic succinate series of TNF-α converting enzyme inhibitors. Part 1: Lead identification

Article abstract of DOI:10.1016/j.bmcl.2003.09.056

Rational design based on the broad spectrum MMP inhibitor CGS 27023A led to the identification of a novel series of cyclic succinate TACE inhibitors. As a mixture of two enantiomers, the lead compound 17b exhibited potent enzyme activity (IC₅₀=

Full text of DOI:10.1016/j.bmcl.2003.09.056

Bioorganic & Medicinal ChemistryLetters 13 (2003) 4293–4297
Rational Design, Synthesis and Structure–Activity Relationships
of a Cyclic Succinate Series of TNF-ꢀ Converting Enzyme
Inhibitors. Part 1: Lead Identification
Chu-Biao Xue,* Xiaohua He, John Roderick, Ronald L. Corbett, James J.-W. Duan,
Rui-Qin Liu, Maryanne B. Covington, Robert C. Newton, James M. Trzaskos,
Ronald L. Magolda, Ruth R. Wexler and Carl P. Decicco
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543-4000, USA
Received 27 May2003; revised 27 May2003; accepted 27 September 2003
Abstract—Rational design based on the broad spectrum MMP inhibitor CGS 27023A led to the identification of a novel series of
cyclic succinate TACE inhibitors. As a mixture of two enantiomers, the lead compound 17b exhibited potent enzyme activity
(IC50=8 nM) in the inhibition of porcine TNF-a converting enzyme (pTACE) and excellent selectivity over aggrecanase and MMP-
1, -2 and -9.
# 2003 Elsevier Ltd. All rights reserved.
Tumor necrosis factor-a (TNF-a)1 is a keyproin-
flammatory cytokine that plays a pivotal role in a num-
ber of autoimmune diseases such as rheumatoid arthritis
(RA).2 The therapeutic application of anti-TNF-a has
been relentlesslypursued in the past through several
approaches. One of them is the neutralization of TNF-a
activitybyforming biologicallyinactive complex with
the cytokine using macromolecules. Efforts in this
approach have culminated in the discoveryof the
inhibition of TNF-a through inhibition of TACE may
represent an alternative approach in anti-TNF-a ther-
apy. As a result, TACE has emerged as a new potential
therapeutic target for small molecule drug design.7
TACE is a member of the ADAM (a disintegrin and a
metalloprotease domian) subfamilyof the metzincin
superfamilythat also includes the astacins, serralysins,
and matrix metalloproteinases (MMPs). X-raycrystal
structure of human TACE revealed that TACE shows
relativelylow overall sequence homologyto MMPs but
3
monoclonal TNF-a antibodyinfliximab and the solu-
ble TNF p75 receptor fusion protein entanercept.4 The
clinical success of these biologics in RA patients clearly
demonstrates that anti-TNF-a is a valid target for the
treatment of autoimmune pathologies.
8
has significant similarityin the active site. This prob-
ablyexplains whybroad-spectrum MMP inhibitors are
capable of inhibiting TNF-a processing and in turn,
most TACE inhibitors derived from MMP leads inhibit
MMPs broadly.7 Since broad-spectrum MMP inhibitors
have been found to cause side-effects in oncologyclin-
ical trials,9 it is desirable to develop selective TACE inhi-
bitors for the long-term treatment of TNF-a mediated
disorders.
TNF-a exists in two forms, the membrane-bound pro-
form comprising 233 amino acids with a molecular mass
of 26 kDa and the soluble form of 17 kDa comprising
157 nonglycosylated amino acids. It has been shown
recentlythat the shedding of the biologicallyactive
TNF-a from its membrane-anchored proform is medi-
ated bya metalloproteinase called TNF- a converting
enzyme (TACE)5 and inhibition of TACE blocks TNF-
a release.6 These experimental evidences implythat
Efforts directed towards the search for TACE inhibitors
during the past have been focused on rational design
based on two classes of MMP leads, the succinate-based
pseudopeptides and non-peptidic sulfonamides as
represented bymarimastat ( 1)10 and CGS 27023A (2),11
respectively. Intensive studies have led to the identifica-
tion of a number of new templates which are selective
*Corresponding author. Tel.: +1-302-283-7706; fax: +1-302-283-
7855; e-mail: cxue@incyte.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.09.056

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C.-B. Xue et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4293–4297
TACE inhibitors with moderate to potent cellular
activity.12 The discoveryof a series of succinate-based
macrocyclic hydroxamic acids containing biphe-
nylmethyl residues at P10 (e.g., 3) in our laboratoryhas
provided important information that TACE has an S10
pocket different to MMPs and accordingly, selective
TACE inhibitors can be achieved byelaboration at
P10.12a Unfortunately, those macrocycles have low oral
bioavailability, probably due to high molecular weight
(Fig. 1).
such as an aniline derivative (I in Fig. 2). To constrain
the orientation of the aniline P10 residue into the S10
pocket, the 3-position of the succinate could be sub-
stituted with an R2 group (II in Fig. 2) and the resulting
R2 group could be cyclized with the R1 group at the 2-
position to form a five- or six-membered carbocycle or
heterocycle. To maintain the three key binding interac-
tions observed in CGS 27023A, the two carboxylates of
the five- or six-membered cycle should assume a trans
configuration (III in Fig. 2).
To identifysmaller molecule TACE inhibitors, we elec-
ted to explore new templates using CGS 27023A (2) as
our starting point for structure-based rational design.
The crystal structure of CGS 27023A bound to MMP-3
revealed that the hydroxamic acid which chelates to the
catalytic zinc, the pro-R oxygen of the sulfonyl group
which is hydrogen-bonded to the NH group of Leu-164,
To test our design hypothesis, a series of trans-cyclo-
pentanedicarboxylate and trans-cyclohexanedicarboxylate
derivatives were synthesized as depicted in Scheme 1. The
4-(quinolinyloxymethyl)aniline residues were prepared
starting from 4-aminobenzyl alcohol 4 (Scheme 1A).
Boc protection followed bymeslyation provided the
mesylate 5. Alkylation of 5 with 4-, 5- or 6-hydroxy-
quinoline using K2CO3 at an elevated temperature pro-
vided the ether derivative 6 which was treated with an
acid to give the amine 7. 4-(3,5-Dichlorobenzyloxy)-
aniline 10 was prepared byBoc protection of 4-amino-
phenol 8 followed byalkylation of the resulting phenol
with 3,5-dichlorobenzyl bromide and subsequent
removal of the Boc group (Scheme 1B).
and the 4-methoxyphenyl which binds in the S10 pocket
11b,c
are three keystructure features for binding.
Model-
ing studies suggested that since onlyone of the sulfonyl
oxygens in CGS 27023A is involved in hydrogen bond-
ing, the sulfonyl could be replaced with a carbonyl. To
3
compensate for the alteration in geometryfrom SP
hybridization for sulfonyl to SP2 hybridization for car-
bonyl, the amino a to the hydroxamate in 2 needs to be
replaced with an SP3 methylene. Thus we selected a
succinate as the keystructural component in our design.
One of the carboxylate of the succinate would be
derived to a hydroxamic acid as the chelator and the
other would form an amide bond with a P10 residue
Figure 1.
Scheme 1. Reagents and conditions: (a) (Boc)2O, THF, DIEA; (b)
MeSO2Cl, DIEA, CH2Cl2, 85%; (c) 4-, 5- or 6-hydroxyquinoline,
K2CO3, DMF, 60–80 ꢀC; (d) 4 N HCl in dioxane; (e) 3,5-dichloro-
benzyl bromide, K2CO3, DMF, 60 ꢀC, 80%; (f) trimethylsilylacetyl-
ene, Pd(PPh3)4, CuI, DIEA, THF, 75%; (g) K2CO3, MeOH, 90%; (h)
6-trifluoromethanesulfonyloxyquinoline, Pd(PPh3)4, CuI, DIEA,
THF, 65%; (i) an aniline derivative (0.5ꢁ), BOP (0.55ꢁ), DIEA,
DMF; (j) hydroxylamine hydrochloride, BOP, DIEA, DMF.
Figure 2.

C.-B. Xue et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4293–4297
4295
The synthesis of 4-[2-(6-quinolinyl)ethynyl]aniline 13
disubstitution on the phenyl ring.12g The selectivityof
involved two sequential palladium mediated couplings
(Scheme 1C). Boc protection of 11 followed bycoupling
with trimethylsilylacetylene gave rise to intermediate 12.
After desilylation, coupling of the resulting acetylene
with the triflate of 6-hydroxyquinoline followed by acid
removal of the Boc group yielded 13. The final hydrox-
amic acids 16a–e and 17a–e were obtained bycoupling
of an aniline derivative with an excess amount (2
equivalents) of the commerciallyavailable trans-1,2-
cyclopentanedicarboxylic acid 14 or trans-1,2-cyclohex-
anedicarboxylic acid 15 using BOP (1.1 equivalent)
followed bycondensation of the resulting carboxlyic
acid with hydroxylamine hydrochloride using BOP
(Scheme 1D).
16a over MMP-2, -9 and aggrecanase probablystems
from the combination of the ring rigidityof the cyclic
succinate and the optimal projection of the benzyloxy
residue in the TACE S10 pocket but not in the MMP
and aggrecanase S10 pockets. Despite the decent affinity
for pTACE, 16a showed <50% inhibition at 50 mM
concentration in WBA, which is probablycaused by
high protein binding. To improve the polarity, the ben-
zyl residue in 16a was replaced with (4-pyridinyl)methyl
to provide the more polar analogue 16b, which led to a
six-fold loss in pTACE affinity. Removal of the oxygen
between the (4-pyridinyl)methyl and the aniline in 16b
enhanced the pTACE affinityby3-fold but slightly
reduced the selectivityover MMP-2 ( 16c). 3,5-Dichloro-
substitution on the phenyring of the benzyloxyin 16a
or replacement of the benzyloxy in 16a with (4-quinoli-
nyl)oxymethyl improved the pTACE potency by 2–3-
fold but did not affect the cellular activity( 16d and 16e,
IC50>50 mM).
Porcine TACE (pTACE) assaywas used to assess the
affinityfor TACE and LPS-stimulated human whole
blood assay(WBA) was emploeyd to determine the
inhibition of TNF-a production. MMP-1, -2, and -9 and
aggrecanase were selected as the representative enzymes
13
for counterscreen to understand the selectivityprofile.
We next examined the six-membered cyclic succinate
template. Compound 17a exhibited a profile in activity
and selectivitysimilar to its five-membered counterpart
16a (Table 2). With (4-quinolinyl)oxymethyl at the 4-
position of the aniline residue, compound 17b displayed
an IC50 value of 8 nM in the pTACE assayas a mixture
of two enantiomers, which is about 5-fold better than its
five-membered counterpart 16b. In addition, 17b exhib-
ited an excellent TACE selectivityprofile versus MMP-1,
-2, and -9 and aggrecanse but remained inactive in WBA
(IC50>50 mM). Replacement of 4-quinolinyl in 17b with
5-quinolinyl (17c) or 6-quinolinyl (17d) reduced the
pTACE potencyby5- and 7-fold, respectively, and suffered
from a loss in selectivityover MMP-2 and -9. With a linear
As shown in Table 1, with a benzyloxy at the 4-position
of the aniline, the five-membered cyclic succinate deriva-
tive 16a (racemate) displayed a submicromolar affinity
for pTACE with a decent selectivityover MMP-1, -2,
-9 and aggrecanase. While the selectivityof 16a over
MMP-1 was anticipated as MMP-1 possesses a shallow
S10 pocket,12a the selectivityover MMP-2 and-9 which
have a narrow and deep S10 pocket12a was a surprise to
us since it is in striking contrast to what we observed
with the g-lactam series discovered in the same group, in
which the small and deep benzyloxy residue at the P10
position resulted in potent MMP-2 and -9 activityand
selectivityfor TACE over MMP-2 and -9 requires 3,5-
Table 1. In vitro potencyof the five-membered cyclic succinates 16a–eaꢂc
Compd
R
pTACE
IC50, mM
WBA
MMP-1, MMP-2, MMP-9
Ki, nM
16a
16b
16c
16d
16e
Benzyloxy
0.11
>50
>50
>50
>50
>50
>4949
>3333
>2128
(4-Pyridinyl)methoxy
(4-Pyridinyl)methyl
3,5-Dichlorobenzyloxy
(4-Quinolinyl)oxymethyl
0.62
>4949
>3333
>2128
0.21
>4949
675
>2128
0.053
0.039
>4949
>3333
>2128
>4949
>3333
>2128
aAll compounds are racemic.
bAll compounds showed IC50 values of >500 nM in the inhibition of aggrecanase.
cpTACE, WBA, aggrecanase IC50 and MMP Ki values are from single determination.

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C.-B. Xue et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4293–4297
Table 2. In vitro potencyof the six-membered cyclic succinates 17a–eaꢂc
Compd
R
pTACE
WBA
MMP-1, MMP-2, MMP-9
Ki, nM
IC50, mM
17a
17b
17c
17d
17e
Benzyloxy
0.089
0.008
0.045
0.056
7.24
>50
>50
>50
>50
>50
>4949
>3333
>2128
(4-Quinolinyl)oxymethyl
(5-Quinolinyl)oxymethyl
(6-Quinolinyl)oxymethyl
(6-Quinolinyl)ethynyl
>4949
>3333
>2128
>4949
585
1359
>4949
1079
876
>4949
>3333
>2128
aAll compounds are racemic.
bAll compounds showed IC50 values of >500 nM in the inhibition of aggrecanse.
cpTACE, WBA, aggrecanase IC50 and MMP Ki values are from single determination.
and rigid (6-quinolinyl)ethynyl at the 4-position of the
aniline, compound 17e displayed a weak affinity for
pTACE (IC50=7.24 mM), in agreement with our pre-
vious observation from the homologymodel that TACE
has a curved or boomerang-like shape S10 pocket.12a,14
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In summary, rational design based on the broad spec-
trum MMP inhibitor CGS 27023A led to the discovery
of a novel series of cyclic succinate TACE inhibitors.
With 4-[(4-quinolinyl)oxymethyl]anilide as a P10 resi-
due, the six-membered analogue 17b displayed an IC50
of 8 nM for pTACE as a mixture of two enantiomers.
Excellent selectivitywas observed for pTACE relative to
MMP-1, -2, -9 and aggrecanase, which are members of
the related metalloproteinase family. Unfortunately,
compound 17b is ineffective in the inhibition of TNF-a
release, with an IC50 value of >50 mM in WBA. To
optimize the cellular potency, modifications at the
cyclohexyl ring and the quinoline moiety of this lead
compound have been carried out in our laboratory,
leading to the identification of a potent TACE inhibitor
in the cellular assay, which will be presented in the
following communication.
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Acknowledgements
We thank John Giannaras, Sherrill Nurnberg and Paul
Stremienski for carrying out the enzyme and cell assays.
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