Non-hydroxamate 5-phenylpyrimidine-2,4,6-trione derivatives as selective inhibitors of tumor necrosis factor-α converting enzyme

Article abstract of DOI:10.1016/j.bmcl.2005.04.039

New inhibitors of tumor necrosis factor-α converting enzyme (TACE) were discovered with a pyrimidine-2,4,6-trione in place of the commonly used hydroxamic acid. These non-hydroxamate TACE inhibitors were developed by incorporating a 4-(2-methyl-4-quinolin

Full text of DOI:10.1016/j.bmcl.2005.04.039

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2970–2973
Non-hydroxamate 5-phenylpyrimidine-2,4,6-trione
derivatives as selective inhibitors of tumor necrosis factor-a
converting enzyme
James J.-W. Duan,* Zhonghui Lu, Zelda R. Wasserman, Rui-Qin Liu,
Maryanne B. Covington and Carl P. Decicco
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543-4000, USA
Received 25 March 2005; revised 21 April 2005; accepted 22 April 2005
Abstract—New inhibitors of tumor necrosis factor-a converting enzyme (TACE) were discovered with a pyrimidine-2,4,6-trione in
place of the commonly used hydroxamic acid. These non-hydroxamate TACE inhibitors were developed by incorporating a 4-(2-
methyl-4-quinolinylmethoxy)phenyl group, an optimized TACE selective P10 group. Several leads were identified with IC50 values
around 100 nM in a porcine TACE assay and selective over MMP-1, -2, -9, -13, and aggrecanase.
Ó 2005 Elsevier Ltd. All rights reserved.
Tumor necrosis factor-a (TNFa), a cytokine produced
mainly by activated macrophages and monocytes, is a
major mediator of inflammatory and immune re-
sponses.1 Clinically, TNF antibodies (infliximab and
adalimumab) and soluble receptor (etanercept) have en-
joyed great success in treatment of diseases, including
CrohnÕs disease, rheumatoid arthritis, juvenile rheuma-
toid arthritis, psoriatic arthritis, ankylosing spondylitis,
and psoriasis. As a result, many approaches to intersect
the TNFa signaling pathway with orally active small
molecules are being pursued.2 One such approach is
through the inhibition of TNFa converting enzyme
(TACE), the enzyme responsible for the release of
TNFa from cells.3
determinant for TACE selectivity. The generality of this
group was demonstrated through the conversion of sev-
eral series of potent MMP inhibitors to inhibitors that
are selective for TACE against MMPs.6
TACE is a membrane-bound zinc endopeptidase. Due
to structure similarities between the active sites of TACE
and matrix metalloproteinases (MMPs), early TACE
inhibitors were derived from MMP inhibitors and con-
sequently suffered from broad spectrum activity against
the MMP family. Recently, inhibitors with selectivity
for TACE against MMPs have been reported.4 One
example from our laboratories is lactam 1.5 The 4-(2-
methylquinolin-4-ylmethoxy)phenyl group in 1 was de-
signed and optimized to bind to the S10 specificity pock-
et of TACE, and was shown to be the critical
To the best of our knowledge, TACE inhibitors in the
literature predominantly rely on hydroxamic acid and
related Ôreverse hydroxamic acidÕ (N-hydroxyforma-
mide) ligands to achieve the desired potency.4 Because
hydroxamic acids are often poorly absorbed in vivo
and carry potential metabolic liabilities (hydrolysis and
glucoronidation), there remains considerable interest in
the discovery of non-hydroxamate TACE inhibitors.
In recent years, there has been great progress to replace
the hydroxamic acid group in MMP inhibitors.7 One
such example is a series of novel pyrimidine-2,4,6-trione
based MMP inhibitors reported by scientists at Roche.8
A crystal structure of a pyrimidinetrione analogue 2 in
*
Corresponding author. Tel.: +1 609 252 4199; fax: +1 609 252
7199; e-mail: james.duan@bms.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.04.039

J. J.-W. Duan et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2970–2973
2971
MMP-3 revealed that the pyrimidine-2,4,6-trione not
only served as a ligand for the catalytic zinc but also
was involved in several hydrogen-bonding interactions
in the vicinity of the active site.8c Additionally, the 4-
phenoxyphenyl group was projected into the S10 pocket
of MMP-3. We were intrigued by this binding mode
observation and proceeded to explore the utility of the
pyrimidinetrione as an alternative Zn ligand to the
hydroxamate TACE inhibitors. Based on the structure
of 2/MMP-3 complex, we proposed that the 4-phenoxy-
phenyl group could be replaced with 4-(2-methylquino-
Scheme 2. Reagents and conditions: (a) 4-chloromethyl-2-methylquin-
oline, Cs2CO3, DMSO (72%); (b) NaH, Me2CO3, THF, at reflux
(78%); (c) NaOMe, urea, MeOH, at reflux; (d) K2CO3, Br2, 1-Boc-
piperazine (45% for two steps); (e) CF3CO2H, CH2Cl2 (100%); (f) n-
C5H11CHO, NaBH(OAc)3, i-Pr2NEt, ClCH2CH2Cl (63%).
lin-4-ylmethoxy)phenyl group to derive
a TACE
inhibitor (3). Compound 3 was modeled and found to
fit into the active site of TACE, with the 4-(2-methyl-
quinolin-4-ylmethoxy)phenyl group occupying the deep
S10 site, while keeping the pyrimidinetrione in a binding
mode similar to that observed in the MMP-3 structure.
This letter discloses the synthesis and evaluation of 3
and related compounds as TACE inhibitors.
The synthesis of 3 started from phenylacetate 4 (Scheme
1). Carbomethoxylation using NaH and dimethyl car-
bonate provided the desired diester, which was alkylated
with MeI and NaH to give di-substituted malonate 5
after removal of the benzyl group. The phenol moiety
in 5 was treated with 4-chloromethyl-2-methylquinoline
and Cs2CO3 in DMSO to yield 6. Reaction of 6 with
urea and NaOMe in methanol at reflux provided pyrim-
idine-2,4,6-trione 3. Several analogues with different R
groups were synthesized using a similar sequence.
Scheme 3. Reagents and conditions: (a) NaClO2, KH2PO4, 2-methyl-
2-butene, t-BuOH, THF, H2O (91%); (b) DPPA, Et3N, benzene, NH3,
at reflux (85%); (c) H2, Pd(OH)2/C, MeOH (100%); (d) 4-chloro-
methyl-2-methylquinoline, Cs2CO3, DMSO (28%).
A series of 5-(piperazin-1-yl)pyrimidine-2,4,6-triones
was synthesized using a route highlighted in Scheme 2.
Phenylacetate 7 was first converted to the corresponding
quinolinylmethyl ether using the Cs2CO3 conditions.
Carbomethoxylation provided diester 8, which was con-
verted to pyrimidinetrione with urea and NaOEt. Bro-
mination and displacement of the resulting bromide in
situ was effected with Br2 and mono-Boc-protected
piperazine in the presence of K2CO3. The Boc protecting
group was then removed and an n-hexyl group was
incorporated by reductive alkylation with hexanal to
complete the synthesis of 10. Pyrimidinetrione 9 was
used as a common intermediate to synthesize a number
of functionalized piperazine analogues.
reported previously,5 was oxidized to a carboxylic acid,
and converted to urea 12 after a Curtius rearrangement
in the presence of ammonia. The benzyl protecting
group was removed via hydrogenolysis, and the result-
ing phenol reacted with 4-chloromethyl-2-methylquino-
line and Cs2CO3 in DMSO. Notably, the basic
conditions required for the etherification reaction also
induced a concomitant intramolecular cyclization to af-
ford the desired pyrimidinedione 13 in one pot in 28%
yield (not optimized).
The inhibitory activity was evaluated using porcine
TACE (pTACE), as a result of its availability and
homology to human TACE.9 Selectivity was evaluated
using MMP-2, -9, and -13 as representatives with deep
S10 pockets and MMP-1 as a representative with shal-
low S10.9 Compounds were also assayed against aggre-
canase, a member of ADAMTS family.10 We were
encouraged to find that combination of 4-(2-methyl-
quinolin-4-ylmethoxy)phenyl group with the pyrimidin-
etrione resulted in a 1 lM inhibitor of pTACE (3,
Table 1). Compound 3 was inactive at 10 lM in the
MMP-1, -2, -9, and -13 assays and at 1 lM in the agg-
recanase assay, probably because the 4-(2-methylquino-
lin-4-ylmethoxy)phenyl group is too wide to fit the S10
pockets of these enzymes. In contrast, compound 2,
possessing the phenoxyphenyl group (a P10 well-known
to favor MMP binding), was reported to have activity
under 100 nM for MMP-2, -9, and -13.8 Replacing
the 2-methylquinolin-4-ylmethoxy group in 3 with 4-hy-
A pyrimidine-2,4-dione analogue (13) was also prepared
(Scheme 3). Aldehyde 11, the synthesis of which was
Scheme 1. Reagents and conditions: (a) NaH, Me2CO3, THF, at reflux
(80%); (b) NaH, MeI, THF, at reflux (81%); (c) H2, Pd(OH)2/C,
MeOH (100%); (d) 4-chloromethyl-2-methylquinoline, Cs2CO3,
DMSO (95%); (e) NaOMe, urea, MeOH, at reflux (20%).

2972
J. J.-W. Duan et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2970–2973
Table 1. In vitro potency of 2, 3, and 14–16 in pTACE, MMP-1, -2, -9, -13, and aggrecanase
R
pTACE
IC50, lM
MMP-1
Ki, lM
MMP-2
Ki, lM
MMP-9
Ki, lM
MMP-13 Ki, lM
Aggrecanase
% inh at 1 lM
3
(2-Methylquinolin-4-yl)methyl
Phenyl
1.03
a
—
>4.95
a
>3.33
0.08b
2.53
>2.13
0.052b
0.85
>5.02
0.065b
>5.02
4.02
0
—
a
2
—
>4.95
>4.95
14
15
16
H
Benzyl
(2-Methylquinolin-4-yl)methyl
>100
>100
>100
0
0
1
0.60
>3.33
>2.13
a
—
a
—
a
—
a Not tested.
b Reported data (Ref. 8a).
droxy and 4-benzyloxy groups resulted in a loss of
pTACE activity and restored some MMP activity (14
and 15). The sum of these data is consistent with our
TACE model. Attempt to replace the central benzene
ring in 3 with a 2,6-naphthalene (16) resulted in loss
of TACE activity.
inhibitors with similar potency, whereas a more rigid
4-nitrophenyl analogue 26 is less active (2.8 lM). Amide
(27 and 28), sulfonamide (29), and carbamate (30) ana-
logues were found to offer no advantage. Unlike the
selectivity observed with the majority of analogues, 24
and 26 displayed sub-micromolar KiÕs for MMP-2, -9,
and -13. A plausible explanation is that the symmetrical
pyrimidinetrione core can rotate 180° around the urea
carbonyl to project the phenethyl and 4-nitrophenyl
groups into the deep S10 pockets of the three MMPs.11
The other R0 groups in Table 2 are probably not as com-
plementary to the MMP S10 sites.
Based on the SAR of pyrimidinetrione-derived MMP
inhibitors8 and our computer model of TACE, we
sought to improve TACE potency of 3 by replacing
the methyl group at the 5-position of the pyrimidinetri-
one with a variety of substituents (Table 2). Piperidine
(17), morpholine (18), and piperazine (19) analogues
gave essentially no improvement in pTACE potency.
Methylation on the second nitrogen of the piperazine
(20) resulted in a 91 nM inhibitor of pTACE, a 12-fold
increase compared to the des-Me analogue 19. Com-
pound 20 still maintained selectivity over the 4 MMPs
and aggrecanase. Many other alkyl groups, including
isopropyl, n-hexyl, neopentyl, benzyl, 2-phenylethyl,
and 3-phenylpropyl groups (10, 21–25), gave TACE
We also briefly investigated the effects of other heterocy-
cles in place of the pyrimidinetrione. Compared to
pyrimidinetrione 3, hydantoin analogue 3112 was about
3-fold less active for pTACE (Table 3). Cyanuric acid
3213 was even less effective, resulting in a 7-fold loss of
activity compared to 3. Pyrimidinedione 13 is approxi-
mately 4-fold less active than 3. The reduced activity
of the pyrimidinedione could be due to the higher
Table 2. In vitro potency of 10 and 17–30 in pTACE, MMP-1, -2, -9, -13, and aggrecanase
R0
pTACE
IC50, lM
MMP-1
Ki, lM
MMP-2
Ki, lM
MMP-9
Ki, lM
MMP-13
Ki, lM
Aggrecanase
% inh at 1 lM
17
18
19
20
21
10
22
23
24
25
26
27
28
29
30
Piperidin-1-yl
Morpholin-4-yl
Piperazin-1-yl
0.855
0.535
1.10
>4.95
>4.95
>4.95
>4.95
>4.95
>4.95
>4.95
>4.95
>4.95
>4.95
4.35
>3.33
>3.33
>3.33
>3.33
>3.33
>3.33
>3.33
>3.33
0.083
3.10
>2.13
>2.13
>2.13
>2.13
>2.13
>2.13
>2.13
>2.13
0.085
2.04
>5.02
>5.02
>5.02
>5.02
>5.02
>5.02
>5.02
>5.02
0.095
>5.02
0.356
>5.02
>5.02
>5.02
>5.02
0
1
0
4-Me-piperazin-1-yl
4-i-Pr-piperazin-1-yl
4-n-Hex-piperazin-1-yl
4-neo-Pent-piperazin-1-yl
4-Bn-piperazin-1-yl
4-[Ph(CH2)2]-piperazin-1-yl
4-[Ph(CH2)3]-piperazin-1-yl
4-(4-NO2-Ph)-piperazin-1-yl
4-Ac-piperazin-1-yl
4-Piv-piperazin-1-yl
4-Ms-piperazin-1-yl
4-Boc-piperazin-1-yl
0.091
0.096
0.081
0.160
0.195
0.084
0.110
2.80
37
0
12
10
5
38
12
0
0.116
>3.33
>3.33
>3.33
>3.33
0.138
>2.13
>2.13
>2.13
>2.13
0.620
0.540
0.275
0.160
>4.95
>4.95
>4.95
>4.95
8
0
5
10

J. J.-W. Duan et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2970–2973
Table 3. In vitro potency of 13, 31, and 32 in pTACE
2973
4. For a recent review of TACE inhibitors, see: Skotnicki, J.
S.; Levin, J. I. Annu. Rep. Med. Chem. 2003, 38, 153.
5. Duan, J. J.-W.; Chen, L.; Wasserman, Z. R.; Lu, Z.; Liu,
R.-Q.; Covington, M. B.; Qian, M.; Hardman, K. D.;
Magolda, R. L.; Newton, R. C.; Christ, D. D.; Wexler, R.
R.; Decicco, C. P. J. Med. Chem. 2002, 45, 4954.
6. (a) Wasserman, Z. R.; Duan, J. J.-W.; Voss, M. E.; Xue,
C.-B.; Cherney, R. J.; Nelson, D. J.; Hardman, K. D.;
Decicco, C. P. Chem. Biol. 2003, 10, 215; (b) Cherney, R.
J.; Duan, J. J.-W.; Voss, M. E.; Chen, L.; Wang, L.;
Meyer, D. T.; Wasserman, Z. R.; Hardman, K. D.; Liu,
R.-Q.; Covington, M. B.; Qian, M.; Mandlekar, S.; Christ,
D. D.; Trzaskos, J. M.; Newton, R. C.; Magolda, R. L.;
Wexler, R. R.; Decicco, C. P. J. Med. Chem. 2003, 46,
1811; (c) Duan, J. J.-W.; Lu, Z.; Xue, C.-B.; He, X.; Seng,
J. L.; Roderick, J. J.; Wasserman, Z. R.; Liu, R.-Q.;
Covington, M. B.; Magolda, R. L.; Newton, R. C.;
Trzaskos, J. M.; Decicco, C. P. Bioorg. Med. Chem. Lett.
2003, 13, 2035.
A
pTACE IC50, lM
31a
4.70
32
7.30
3.70
13a
7. For a review of MMP inhibitors, see: Skiles, J. W.;
Gonnella, N. C.; Jeng, A. Y. Curr. Med. Chem. 2001, 8,
425.
a Tested as a racemic mixture.
8. (a) Foley, L. H.; Palermo, R.; Dunten, O.; Wang, P.
Bioorg. Med. Chem. Lett. 2001, 11, 969; (b) Grams, F.;
Brandstetter, H.; DÕAlo, S.; Geppert, D.; Krell, H.-W.;
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enthalpic cost to enolize, as the pyrimidinetrione was
shown to exist as an enol tautomer when binding to
MMPs.8
In summary, using a quinoline P10 group that was pre-
viously optimized in our laboratories for TACE, we suc-
cessfully converted pyrimidine-2,4,6-trione MMP
inhibitors to TACE inhibitors. Modification of the
5-phenylpyrimidine-2,4,6-trione template led to identifi-
cation of several inhibitors with IC50s of 100 nM for
porcine TACE and selectivity against MMP-1, -2, -9,
-13, and aggrecanase.
9. For description of assay protocols, see: Xue, C.-B.; Voss,
M. E.; Nelson, D. J.; Duan, J. J.-W.; Cherney, R. J.;
Jacobson, I. C.; He, X.; Roderick, J.; Chen, L.; Corbett,
R. L.; Wang, L.; Meyer, D. T.; Kennedy, K.; DeGrado,
W. F.; Hardman, K. D.; Teleha, C. A.; Jaffee, B. D.; Liu,
R.-Q.; Copeland, R. A.; Covington, M. B.; Christ, D. D.;
Trzaskos, J. M.; Newton, R. C.; Magolda, R. L.; Wexler,
R. R.; Decicco, C. P. J. Med. Chem. 2001, 44, 2636.
10. Miller, J. A.; Liu, R.-Q.; Davis, G. L.; Pratta, M. A.;
Trzaskos, J. M.; Copeland, R. A. Anal. Biochem. 2003,
314, 260.
Acknowledgments
The authors thank Bin Jiang for assistance in synthesis
of compound 16, John Giannaras and Paul Strzemienski
for assistance in enzymatic assays, and Dr. James E.
Sheppeck II for a critical review of the manuscript.
References and notes
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12. Compound 31 was isolated as a byproduct during
conversion of 8 to 9, presumably because only one of
the ester groups reacted with urea in the urea/NaOEt step
and subsequently cyclized under K2CO3/Br2 conditions.
13. Synthesis of 32: (a) 4-(hydroxymethyl)phenol, 4-chloro-
methyl-2-methylquinoline, Cs2CO3, DMSO (81%); (b)
cyanuric acid, DEAD, PPh3, DMF (14%).