Discovery of novel hydantoins as selective non-hydroxamate inhibitors of tumor necrosis factor-α converting enzyme (TACE)

Article abstract of DOI:10.1016/j.bmcl.2006.11.089

A series of novel hydantoins was designed and synthesized as structural alternatives to hydroxamate inhibitors of TACE. 5-Mono- and di-substituted hydantoins exhibited activity with IC₅₀ values of 11-60 nM against porcine TACE in vitro and excellent selectivity against other MMPs.

Full text of DOI:10.1016/j.bmcl.2006.11.089

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1413–1417
Discovery of novel hydantoins as selective non-hydroxamate
inhibitors of tumor necrosis factor-a converting enzyme (TACE)q
James E. Sheppeck, II,* John L. Gilmore, Anle Yang, Xiao-Tao Chen, Chu-Biao Xue,
John Roderick, Rui-Qin Liu, Maryanne B. Covington,
Carl P. Decicco and James J.-W. Duan
Bristol-Myers Squibb Pharmaceutical Research Institute, Discovery Chemistry, PO Box 4000, Princeton, NJ 08543-4000, USA
Received 14 September 2006; revised 21 November 2006; accepted 30 November 2006
Available online 2 December 2006
Abstract—A series of novel hydantoins was designed and synthesized as structural alternatives to hydroxamate inhibitors of TACE.
5-Mono- and di-substituted hydantoins exhibited activity with IC50 values of 11–60 nM against porcine TACE in vitro and excellent
selectivity against other MMPs.
Ó 2006 Elsevier Ltd. All rights reserved.
Tumor necrosis factor-a (TNF-a) is a well-established
pro-inflammatory cytokine, primarily produced by acti-
vated macrophages and T-cells, that is implicated in a
variety of autoimmune disorders such as rheumatoid
arthritis, inflammatory bowel disease, and psoriasis.1
Anti-TNF-a drugs such as RemicadeÒ, EnbrelÒ, and
HumiraÒ have clinically validated the approach of
sequestering TNF-a with antibodies and binding pro-
teins to block its action at the TNF-a receptor (TNF-
R) and are a significant advance in treating these diseas-
es.2 However, the high cost of manufacturing these
agents and the need to administer them parenterally
have led to a strong interest to develop orally active mol-
ecules that suppress TNF-a activity.
it from activating TNF-R and short-circuiting the
inflammatory signal transduction cascade.4 The develop-
ment of TACE inhibitors has grown out of the much
larger effort directed at inhibiting the dozens of related
matrix metalloproteases (MMPs). Synonymous with
Zn-metalloprotease inhibition is the hydroxamic acid
structural element found in the vast majority of inhibi-
tors (Fig. 1).5 X-ray co-crystal structures of hydroxamate
inhibitors and MMPs show five strong non-covalent
interactions in the active site alone which largely
account for the typical nM Ki’s observed for this class.
An alternative approach to modulate the action of
TNF-a is to use selective small molecule inhibitors of
TNF-a converting enzyme (TACE, ADAM-17). TACE
is a membrane-bound zinc metalloprotease that is pri-
marily responsible for proteolytically processing the
26-kDa membrane-bound form of pro-TNF-a to its
17 kDa soluble form.3 In principle, TACE inhibitors
would halt the processing of TNF-a thereby precluding
Keywords: TACE; TACE inhibitor; Non-hydroxamate; MMP; Matrix
metalloprotease; Matrix metalloproteinase; TNF.
q
Presented at the 228th National ACS Meeting, Philadelphia, PA,
August 2004; MEDI-274.
*
Corresponding author. Tel.: +1 609 252 3211; fax: +1 609 252
3993; e-mail: jim.sheppeck@bms.com
Figure 1. Precedented zinc ligands found in MMP inhibitors.7
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.11.089

1414
J. E. Sheppeck et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1413–1417
Although we6 and others have exploited the intrinsic
potency of the hydroxamic acid for TACE and MMPs,
there remains considerable interest to discover a
non-hydroxamate inhibitor. The advantages of a non-
hydroxamate metalloprotease inhibitor include the
potential for improved pharmacokinetics and avoiding
the possibility of high clearance due to hydroxamate
hydrolysis or proteolysis in vivo to the corresponding
acid and toxic hydroxylamine.8 A variety of non-
hydroxamate MMP inhibitors have been developed that
bear zinc-ligating functional groups (Fig. 1) but many
suffer from inherent liabilities such as metabolic instabil-
ity, poor bioavailability or cellular penetration, and
cytotoxic functionality.7 Of these hydroxamate replace-
ments, we were intrigued by the potency of the rhoda-
nines (thiazolidinediones) which showed IC50 values of
1–5 nM against MMP-13 despite their simple struc-
tures.7e All efforts to transpose the rhodanines into
TACE inhibitors failed. Attempts to add chemical sta-
bility to the rhodanines led to the synthesis of hydantoin
isosteres which when substituted with a TACE-selective
4-[(2-methyl-4-quinolinyl)methoxy]-phenyl P10 group,9
provided the first leads (Table 1). The synthesis of hyd-
antoins is most efficiently achieved from ketones using
the Bucherer–Bergs reaction. The simple acyclic hydan-
toins 1–7 (Table 1) were prepared using literature
procedures.10
Spirocyclic hydantoins possessing ester-containing side
chains were synthesized from a-substituted cyclopenta-
none or cyclohexanone again applying the Bucherer–
Bergs conditions11 (Scheme 1). Diastereo-selectivity
varied as shown. Acid hydrolysis of the ester followed
by standard peptide coupling with the TACE-selective
4-[(2-methyl-4-quinolinyl)-methoxy]aniline P10 group
gave the final substituted spirocyclopentyl and spiro-
cyclohexyl products 14–16.
Spirocyclic hydantoins that contain a ‘reversed’ amide
side chain were prepared using the complimentary ap-
proach outlined in Scheme 2. Commercially available
racemic 1,2-aminocyclopentanol and 1,2-aminocyclo-
Table 1. In vitro potency of hydantoins for porcine TACE, MMP-1, -2, -9, and -13
R1
R2
m
n
Kia (nM)
a
pTACE IC50 (nM)
Compound
MMP-1
MMP-2
MMP-9
MMP-13
(rac)-1b
H
H
1
—
—
—
—
—
—
—
—
—
0
210
3700
170
150
>100000
>100000
98
>4946
>4946
2472
>3333
>3333
567
>2128
>2128
1000
>5025
>5025
4120
(5S)-1b
H
H
1
(5R)-1b
H
H
1
(5R)-2b
H
H
2
>4946
>4946
>4946
>4946
>4946
>4946
988
>3333
>3333
>3333
>3333
>3333
>3333
307
>2128
>2128
>2128
>2128
>2128
>2128
179
>5025
>5025
>5025
>5025
>5025
>5025
392
(rac)-3b
H
Me
H
1
(rac)-4b
Me
H
1
(5R)-5b
H
—
—
—
0
(rac)-6b
Me
H
H
29
28
(rac)-7b,c
Me
—
—
—
—
—
—
—
—
—
—
—
—
—
(rac)-trans-14d
(rac)-cis-14d
(rac)-cis-15d
(rac)-trans-15d
(5R,6S)-trans-15d,e,f
(5S,6R)-trans-15d,f
(rac)-cis-16d
(rac)-trans-19d
(rac)-cis-19d
(rac)-trans-20d
(5R,6R)-trans-22ad,f
(5R,6R)-trans-22bd,f
(5R,6R)-trans-22cd,f
—
—
—
—
—
—
—
—
—
—
—
—
—
230
64
0
0
>4946
>4946
>4946
>4946
>4946
>4946
>4946
>4946
>4946
>4946
>4946
—
>3333
>3333
>3333
>3333
>3333
>3333
>3333
>3333
>3333
>3333
>3333
>3333
>2128
>2128
>2128
>2128
>2128
>2128
>2128
>2128
>2128
>2128
>2128
—
>5025
>5025
>5025
>5025
>5025
>5025
>5025
>5025
>5025
>5025
>5025
>5025
1
0
7400
17
1
0
1
0
11
1
0
900
270
54
0
1
—
—
—
—
—
—
0
0
5500
24
1
—
—
—
13
25
24
a pTACE IC50 and MMP Ki data are from a single determination.
b Synthesized as described in Ref. 10.
c Compound 7 is diastereomerically pure but its relative stereochemistry was not determined.
d All relative stereochemistry was determined using NOESY 1H NMR.
e Compound trans-15 was resolved by chiral HPLC and the absolute stereochemistry was tentatively assigned (5R,6S) based on analogy to the TACE
activity of (5R)-1, (5R)-2, (5R)-5, and (5R,6R)-22a–c.
f Absolute and relative stereochemistry was determined via X-ray crystal structure of a CSA-salt of a 22 synthetic intermediate that had been resolved
by chiral HPLC and was used to make (5R,6R)-trans-22a–c.

J. E. Sheppeck et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1413–1417
1415
Scheme 3. Reagents and conditions: (a) 4-[(2-methyl-4-quinoli-
nyl)methoxy]benzoyl chloride, 10% NaHCO3, DCM; (b) Dess–Martin
periodinane, DCM (40%, two steps); (c) (NH4)2CO3, KCN, EtOH/
H2O, sealed tube 80 °C, (48%); (d) 2:1 TFA/DCM (100%); (e)
(iPrCO)2O, DIEA, DCM (70%).
Scheme 1. Reagents and conditions: (a) (NH4)2CO3, KCN, EtOH/
H2O, sealed tube 80 °C; (b) 4 M HCl (37%, 34%, 32% for two steps);
(c) 4-[(2-methyl-4-quinolinyl)methoxy]anilineÆHCl, HATU or PyBOP,
DIEA, DMSO (8%, 35%, 32%).
of the synthesized hydantoins was evaluated using
porcine TACE (pTACE) because of its availability and
high homology to human TACE.14 MMP-1, -2, -9, and
-13 were used as a representative selectivity panel that
possess both shallow and deep S10 pockets.6
The hydantoins shown in Table 1 were the first leads
discovered to have pTACE activity. Compound 1, pre-
pared from racemic aspartic acid, had an IC50 of
210 nM against pTACE and was selective against
MMP-1, -2, -9, and -13. When the same compound
was made discretely from (S) and (R) aspartic acid, it
was the 5-(R) stereochemistry that accounted for the
pTACE activity. The one methylene longer homolog
(R)-2 (prepared from R–Glu) was marginally more ac-
tive at 150 nM. Independent N-methylation of the
hydantoin nitrogens (compounds 3 and 4) established
that they must be unsubstituted to retain activity. Rever-
sal of the pendant amide side chain led to additional
improvement in pTACE activity such as compound 5
(Table 1) which had an IC50 of 98 nM against pTACE.
Addition of a methyl substituent in the 5- or 6-position
to bias the side-chain conformation gave compounds 6
Scheme 2. Reagents and conditions: (a) (Boc)2O, MeOH; (b) TPAP,
NMO, DCM (90%, 88% for two steps); (c) (NH4)2CO3, KCN, EtOH/
H2O, sealed tube 80 °C (25%, 81%); (d) 2:1 TFA/DCM (100%, 100%);
(e) 4-[(2-methyl-4-quinolinyl)-methoxy]benzoic acid, BOP, iPr2NEt,
DMSO (18%, 12%).
hexanol were Boc-protected and the alcohol oxidized to
the ketone using Ley’s catalyst or Dess–Martin period-
inane.12 Bucherer–Bergs reaction gave a 4:1 trans/cis
mixture of hydantoins for cyclopentane and 8:1 trans/
cis for cyclohexane. Acid deprotection of the Boc group
followed by BOP coupling to 4-[(2-methyl-4-quinoli-
nyl)methoxy]benzoic acid—a TACE-selective P10 group
that we had developed in our hydroxamate work6 gave
the final ‘reversed’ amide hydantoins 19 and 20.
and 7 with improved IC50s of 29 and 28 nM,
respectively.
In an effort to elucidate the bound conformation of
these acyclic inhibitors, the 5- and 6-positions were
annulated to make the conformationally constrained
5,5-spirocyclic hydantoins. These analogs have the add-
ed benefit of circumventing racemization that 5-mono-
substituted hydantoins (compounds 1–5 and 7) are
prone to. The observation that methyl substitution in
the 5 and 6 positions (compounds 6 and 7) enhanced
pTACE activity suggested that such an annulation strat-
egy might further increase potency.
The synthesis of spiropyrrolidinyl hydantoins was per-
formed starting from racemic compound 2113 as shown
in Scheme 3. The primary amine was coupled to the
TACE-selective P10 group previously mentioned fol-
lowed by oxidation of the hydroxyl group to a ketone
with Dess–Martin periodinane and hydantoin formation
to furnish 22a. Boc-deprotection of 22a with TFA/DCM
provided 22b which was then coupled to isovaleric anhy-
dride to form the pyrrolidinyl amide 22c.
Compound 14 (the spirocyclopentyl version of com-
pound 1) indeed showed an improvement in pTACE
activity from 210 to 64 nM (comparing racemates, Ta-
ble 1). Although trans-14 had some activity with
diminished selectivity, cis-14 proved to be the more
active diastereomer. When the side chain of 14 was
homologated by one carbon to give cis/trans-15 (the
spirocyclopentyl version of compound 2), the active
stereoisomer reversed with trans-15 having pTACE
activity of 17 nM, whereas cis-15 had an IC50 of
7400 nM. This apparent paradox is easily rationalized
The chemistry used to synthesize compounds 14–22 gave
variable diastereoselectivity, which allowed each diaste-
reomer to be isolated and assayed independently. All rel-
ative stereochemistry was determined by NOESY 1H
NMR and the absolute stereochemistry of compounds
1, 2, 5, and 22 was uniformly (5R), originating from the
chiral pool or purified by chiral HPLC and elucidated
by small molecule crystallography. The in vitro activity

1416
J. E. Sheppeck et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1413–1417
3. Black, R. A.; Rauch, C. T.; Kozlosky, C. J.; Peschon, J. J.;
Slack, J. L.; Wolfson, M. F.; Castner, B. J.; Stocking, K.
L.; Reddy, P.; Srinivasan, S.; Nelson, N.; Bolani, N.;
Schooley, K. A.; Gerhart, M.; Devis, R.; Fitzner, J. N.;
Johnson, R. S.; Paxton, R. J.; March, C. J.; Cerretti, D. P.
Nature 1997, 385, 729.
4. (a) Moss, M. L.; White, J. M.; Lambert, M. H.; Andrews,
R. C. Drug Discovery Today 2001, 6, 417; (b) Skotnicki, J.
S.; Martin, J.; Levin, J. I. Curr. Opin. Drug Discovery Dev.
2003, 6, 742.
by comparing the 3-dimensional structures of cis-14
and trans-15 which both project the side-chain amide
carbonyl in the same orientation with different cyclo-
pentane puckering (see accompanying paper). Racemic
trans-15 was separated into its enantiomers using chi-
ral HPLC and the putative (5R,6S)-trans-15 enantio-
mer accounted for the pTACE activity at 11 nM.
Reversing the amide side chain in the spirocyclic hyd-
antoins (akin to compound (5R)–5) produced an even
more conformationally constrained analog, compound
19, as a pair of diastereomers. Like its isosteric analog
trans-15, trans-19 proved to be the more active diaste-
reomer albeit 3-fold less active for pTACE at 54 nM.
The conformational effects of spirocyclohexane ana-
logs were also investigated. Compound cis-16 was
4-fold less active than the cyclopentyl analog 14, pre-
sumably due to a less than optimal orientation of the
equatorial P10 side chain. Nevertheless, reversing the
amide in the cyclohexyl series gave trans-20 which
had good pTACE activity at 24 nM. In an effort to
elaborate on the good pTACE activity of compound
19, the spirocyclopentane was replaced with a pyrrol-
idine ring to give compounds 22a–c. The pTACE
activity of these analogs was good with IC50s of 13,
25, and 28 nM, respectively, with excellent selectivity.
5. (a) Skiles, J. W.; Gonnella, N. C.; Jeng, A. Y. Curr. Med.
Chem. 2001, 8, 425; (b) Whittaker, M.; Floyd, C. D.;
Brown, P.; Gearing, A. J. H. Chem. Rev. 1999, 99, 2735.
6. 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, and
references therein.
7. For an excellent review that discusses hydroxamate
shortcomings and non-hydroxamate inhibitors, see: (a)
Breuer, E.; Trant, J.; Reich, R. Expert Opin. Ther. Patents
2005, 15, 253, and ref. 5. Also; (b) Michaelides, M. R.;
Dellaria, J. F.; Gong, J.; Holms, J. H.; Bouska, J. J.;
Stacey, J.; Wada, C. K.; Heyman, H. R.; Curtin, M. L.;
Guo, Y.; Goodfellow, C. L.; Elmore, I. B.; Albert, D. H.;
Magoc, T. J.; Marcotte, P. A.; Morgan, D. W.; Davidsen,
S. K. Bioorg. Med. Chem. Lett. 2001, 11, 1553; (c)
Schroder, J.; Henke, A.; Wenzel, H.; Brandstetter, H.;
Stammler, H. G.; Stammler, A.; Pfeiffer, W. D.; Tsche-
sche, H. J. Med. Chem. 2001, 44, 3231; (d) Grams, F.;
Brandstetter, H.; D’Alo, S.; Geppert, D.; Krell, H.-W.;
Leinert, H.; Livi, V.; Menta, E.; Oliva, A.; Zimmerman,
G. Biol. Chem. 2001, 382, 1277; (e) Miyata, K.; Kimura,
H.; Ishikawa, T.; Yamamoto, K. WO2001/72718, 2001; (f)
Kawamura, N.; Yamashita, T.; Takizawa, M.; Yoshim-
ura, K. JP 143650, 2000.
8. In vivo hydrolysis or proteolysis of hydroxamates is highly
variable and structure-dependent. It should be noted that
hydroxylamine exposure in humans causes methemoglo-
binemia—an acute toxicity apparently not observed dur-
ing safety studies of several hydroxamates that have
passed phase II clinical trials. Nevertheless, many
hydroxamates have shown human toxicity which has been
conjectured to arise from broad-spectrum metalloprotease
inhibition, e.g., the muscle toxicity caused by marimistat.
9. see Ref. 6 and Wasserman, Z. R.; Duan, J. J.-W.; Voss,
M.; Xue, C.-B.; Cherney, R. J.; Nelson, D. J.; Hardman,
K. D.; Decicco, C. P. Chem. Biol. 2003, 10, 215.
In summary, hydantoins have been known for over 100
years and 5,5-diphenylhydantoin (DilantinÒ, phenytoin)
has been used for decades to treat epilepsy. The use of
hydantoins as a privileged scaffold for MMP inhibition
has not been reported in the literature15—remarkable
considering this century old heterocycle, mass produced
in the early 1990s as the first combinatorial libraries,16
has no doubt passed through extensive high-throughput
screening against all varieties of metalloproteases. When
incorporated into a TACE inhibitor, the hydantoin is a
good albeit intrinsically less potent Zn ligand than an
analogous hydroxamate. Our evidence suggests that
the stringent requirement for unsubstituted hydantoin
nitrogens, the unnatural (5R) stereochemistry, and an
H bond acceptor appended to an appropriately func-
tionalized hydrophobic P10 side chain that complements
the MMP S10 pocket are what confer activity to this new
class of molecules.17
10. (a) Nyc, J. F.; Mitchell, H. K. J. Am. Chem. Soc. 1947, 69,
1382; (b) Cescon, L. A.; Day, A. R. J. Org. Chem. 1962,
27, 581; (c) Obrecht, D.; Karajiannis, H.; Lehmann, C.;
Schonholzer, P.; Spiegler, C.; Muller, K. Helv. Chim. 1995,
78, 703; (d) Stratford, E. S.; Curley, R. W., Jr. J. Med.
Chem. 1983, 26, 1463; (e) Sacripante, G.; Edward, J. T.
Can. J. Chem. 1982, 60, 1982; (f) Stratford, E. S.; Curley,
R. W., Jr. J. Med. Chem. 1983, 26, 1463.
Acknowledgment
We thank Tom Scholz for NMR experiments and M.
Galella for X-ray crystallographic analysis of a 22 pre-
cursor which has been deposited to the CCDC.
11. (a) Curry, K.; McLennan, H.; Rettig, S. J.; Trotter, J. Can.
J. Chem. 1993, 71, 76; (b) Oldfield, W.; Cashin, C. H.
J. Med. Chem. 1965, 8, 239.
References and notes
12. (a) Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113,
7277; (b) Ley, S. V.; Norman, J.; Griffith, W. P.; Marsden,
S. P. Synthesis 1994, 639.
13. Hong, C. Y.; Kim, Y. K.; Lee, Y. H.; Kwak, J. H. Bioorg.
Med. Chem. Lett. 1998, 8, 221.
14. For a 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,
1. (a) Aggarwal, B.; Kohr, W.; Hass, P.; Moffat, B.; Spencer,
S.; Henzel, W.; Bringman, T.; Nedwin, G.; Goeddel, D.;
Harkins, R. J. Biol. Chem. 1985, 260, 2345; (b) Bemel-
mans, M.; van Tits, L.; Buurman, W. Crit. Rev. Immunol.
1996, 16, 1.
2. (a) Braun, J.; van der Heijde, D. Exp. Opin. Invest. Drugs
2003, 12, 1097; (b) Mikuls, T. R.; Moreland, L. W. Exp.
Opin. Pharm. 2001, 2, 75.

J. E. Sheppeck et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1413–1417
1417
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.
to, M.; Lundkvist, M.; Munck AF Rosenschold, M.;
Zlatoidsky, P. WO2002/074751, 2002; Lepisto, M.; Munck
AF Rosenschold, M. WO2002/074752, 2002; Eriksson, A.;
Lepisto, M.; Lundkvist, M.; Munck AF Rosenschold, M.;
Zlatoidsky, P. WO2002/074767, 2002.
15. During the course of our studies, several patents appeared
from the Astra-Zeneca group describing the inhibition of
MMP-12 using hydantoins. See: Munck, AF Rosenschold,
M. WO 2002/074748, 2002; Lepisto, M.; Munck AF
Rosenschold, M. WO 2002/074749, 2002; Eriksson, A.;
Lepisto, M.; Lundkvist, M.; Munck AF Rosenschold, M.;
Zlatoidsky, P. WO2002/074750, 2002; Eriksson, A.; Lepis-
16. DeWitt, S. H.; Kiely, J. S.; Stankovic, M. C.; Shroeder, D.
M.; Reynolds, C.; Pavia, M. R. Proc. Natl. Acad. Sci.
U.S.A 1993, 90, 6909.
17. Sheppeck II, J. E. et al. Bioorg. Med. Chem. Lett. 2007,
doi:10.1016/j.bmcl.2006.11.082.