Potent, selective pyrimidinetrione-based inhibitors of MMP-13

Article abstract of DOI:10.1016/j.bmcl.2006.08.066

Using SAR from two related series of pyrimidinetrione-based inhibitors, compounds with potent MMP-13 inhibition and >100-fold selectivity against other MMPs have been identified. Despite high molecular weights, c log Ps, and polar surface areas, the compo

Full text of DOI:10.1016/j.bmcl.2006.08.066

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5822–5826
Potent, selective pyrimidinetrione-based inhibitors of MMP-13
Lawrence A. Reiter,a,* Kevin D. Freeman-Cook,a Christopher S. Jones,a
Gary J. Martinelli,a Amy S. Antipas,a Martin A. Berliner,a Kaushik Datta,b
James T. Downs,a James D. Eskra,a Michael D. Forman,a Elaine M. Greer,a
Roberto Guzman,b Joel R. Hardink,a Fouad Janat,a Nandell F. Keene,a
Ellen R. Laird,a Jennifer L. Liras,a Lori L. Lopresti-Morrow,a Peter G. Mitchell,a
Jayvardhan Pandit,a Donald Robertson,b Diana Sperger,a
Marcie L. Vaughn-Bowser,a Darra M. Wallera and Sue A. Yocuma
aPfizer Global Research & Development, Groton Laboratories, Eastern Point Road, Groton, CT 06340, USA
bPfizer Global Research & Development, Ann Arbor Laboratories, 2800 Plymouth Road, Ann Arbor, MI 48105, USA
Received 18 July 2006; revised 11 August 2006; accepted 14 August 2006
Available online 30 August 2006
Abstract—Using SAR from two related series of pyrimidinetrione-based inhibitors, compounds with potent MMP-13 inhibition and
>100-fold selectivity against other MMPs have been identified. Despite high molecular weights, clogPs, and polar surface areas, the
compounds are generally well absorbed and have excellent pharmacokinetic (PK) properties when dosed as sodium salts. In a rat
fibrosis model, a compound from the series displayed no fibrosis at exposures many fold greater than its MMP-13 IC50.
Ó 2006 Elsevier Ltd. All rights reserved.
We and others have reported the design of pyrimidine-
trione-based inhibitors of matrix metalloproteases
(MMP) and related proteases.1–7 These proteases, hav-
ing been implicated in an array of diseases and/or path-
ological states, have been the subject of intense research
over the past two decades since inhibition of one or a
small set of them may be sufficient to significantly alter
the course of a specific disease or pathology. Our focus
has primarily been on discovering disease modifying
therapeutic agents for the treatment of osteoarthritis
(OA).8 In this regard, MMP-13 appears to be a prime
target in that it is present in human OA cartilage tis-
sue,9,10 turns over type II collagen,11 is co-localized with
cleaved type II collagen in OA cartilage tissue,12 and
degrades cartilage.13
MSS is characterized by a stiffening of various joints
and may derive from a lack of normal turnover of extra-
cellular matrix due to inappropriate inhibition of one or
more non-targeted MMPs.14–17 The postulate that
MMP-1 inhibition alone is responsible for the side ef-
fects was dispelled by clinical trials with CP-544439, a
hydroxamic acid-based MMP-13 inhibitor that spared
MMP-1 but which was otherwise non-selective.18 This
compound still produced MSS despite its lack of activity
versus MMP-1. MMP-14 inhibition has also been pro-
posed to cause MSS,1 but this idea has not yet been clin-
ically evaluated. In our most recent work we adopted a
more conservative—but operationally more challeng-
ing—approach, seeking to identify a compound that
potently inhibits MMP-13 while sparing other MMPs
available to us (MMPs -1, -2, -3, -8, -9, -12, and -14).
Of course, selectivity over this set did not guarantee uni-
versal selectivity for MMP-13. However, since the set
contains representatives of both shallow- and deep-
pocket S01 proteases, we were confident that a compound
selective against this set would have broad selectivity for
MMP-13 over other MMPs.
Perhaps the most significant impediment to the develop-
ment of an MMP inhibitor for the treatment of any dis-
ease is musculoskeletal syndrome (MSS), a side effect
clinically observed with non-selective MMP inhibitors.
Keywords: Pyrimidinetrione; Matrix metalloprotease; MMP-13;
Collagenase-3.
*
Two observations made during the course of our inves-
tigation of the pyrimidinetrione series led us to believe
Corresponding author. Tel.: +1 860 441 3681; e-mail:
lawrence.a.reiter@pfizer.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.08.066

L. A. Reiter et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5822–5826
5823
that the strategy posed above could be a success. First,
as we recently reported,1 a P01 group containing a termi-
nal heterocyclic group, and particularly an oxadiazole or
oxazole, can lead to compounds with good MMP-13 poten-
cy and excellent selectivity (>100-fold) over MMPs -1 and
-14. Second, in pursuing inhibitors containing P01 termi-
nal amides, we observed that reasonable levels of selec-
tivity (>50-fold) over MMPs -2, -8, and -12 could be
obtained with certain substitution patterns (see
Table 1).19,20 For example, the N-phenyl amide 1 is a
potent MMP-13 inhibitor with 200-fold selectivity ver-
sus MMP-8 and the N-(3-fluorobenzyl) amide 4 displays
120-fold selectivity for MMP-13 versus MMP-12. The
N-(3-pyridyl) amide 7 was among the most interesting
of this set in that it displays >100-fold selectivity for
MMP-13 versus both MMPs -8 and -12 and >50-fold
selectivity over MMP-2.
Accordingly, we sought to prepare heterocyclic variants
of the N-aryl and/or N-benzyl amides and to this end,
prepared seven different heterocyclic oxa-azole systems
containing phenyl substituents on the heterocycles (see
Table 2).19,21
Three of these systems, 15–17, were not broadly pursued
owing to their lower potency against MMP-13. Another,
14, was not pursued owing to its relative lack of selectiv-
ity versus MMPs -2 and -12. The other series, represent-
ed by 11–13, display good potency and promising
selectivity. Two of these, the 4-aryloxazol-2-yl and 3-ar-
yl-1,2,4-oxadiazol-5-yl systems (e.g., 12 and 13, respec-
tively), were investigated thoroughly with particular
attention to the effects of substituents on the pendant
aryl ring (see Table 3).19,22
In general the oxazoles are more potent than the oxadia-
zoles; however, one oxadiazole, the 4-pyridyl analog 23,
displayed potency and selectivity of the order that we
sought. However, in vitro metabolism studies revealed
this analog to have unacceptable turnover in micro-
somes and hepatocytes. In contrast, nearly all of the
oxazoles displayed the potency sought (<1.0 nM); some,
however, did not meet the selectivity criteria of being
>100-fold more potent against MMP-13 versus the off-
target MMPs. For example, the three pyridyl analogs
36–38 and the 3-cyano derivatives 32–33 did not meet
the criterion with respect to MMP-2. Much of the
SAR in the oxazole series was probed with both meth-
oxyethyl and ethoxyethyl sides chains on the pyrimidin-
etrione. Although the potencies of the resulting pairs of
analogs are generally very similar, the methoxyethyl
analogs are somewhat more selective. This observation,
plus the advantage that the methoxyethyl analogs have
lower MW and clogPs, lead us to select two methoxy-
ethyl analogs (28 and 34) for further studies.
Although we were unable to identify an amide possess-
ing the potency and selectivity attributes that we desired
(IC50 6 1 nM with >100-fold potency over MMPs in
our panel), the activity of compounds in the series sug-
gested that compounds meeting our criteria might be
found by combining structural features from the earlier
reported heterocyclic and the amide series. The impor-
tant features of the heterocyclic series were believed to
be the position of the heteroatoms while in the amide
series the overall length of the P01 group and the presence
and position of heteroatoms therein were deemed to be
critical to the selectivity observed. The importance of the
overall length of the P10 group was reinforced by the
X-ray crystal structure of 10 in MMP-13 (Fig. 1). This
revealed significant space below the terminal fluoro-
phenoxy group that could be occupied by a large, planar
amide such as that in 7. Furthermore, the selectivity ob-
served in the amide series was likely due to the terminal
residue contacting the loop at the base of the S01 pocket,
a region that is varied among the various MMPs with re-
spect to the number and identity of the residues being
present. This gave support to the premise that interac-
tions with this region, both favorable and unfavorable,
play an important role in determining selectivity.
In our hamster model of MMP-13 inhibition,23 a model
that assesses the distribution of the compound into the
knee joint and its ability to inhibit MMP-13 within the
synovial fluid milieu, the 4-cyano derivative 34 was
Table 1. MMP IC50s of P01 terminal amides
O
HN
HN
O
O
O
O
OEt
NHR
O
Compound
R
MMP-13
Fold selectivity
IC50 (nM)
MMP-2
MMP-8
MMP-12
1
2
3
4
5
6
7
8
9
–Ph
–CH2Ph
0.84
8.0
58
21
55
74
17
27
63
17
26
200
ND
27
50
27
–CH2-(2-F–Ph)
–CH2-(3-F–Ph)
–CH2-(4-F–Ph)
–2-Pyridyl
1.1
80
0.85
3.4
25
120
11
ND
15
3.9
27
–3-Pyridyl
–CH2–(2-pyridyl)
–CH2–(3-pyridyl)
0.40
6.1
120
ND
ND
190
30
2.6
27

5824
L. A. Reiter et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5822–5826
A
O
O
HN
HN
O
O
O
OEt
F
10
B
Figure 1. (A) Compound 10 (yellow) bound in MMP-13 (variable region in red) (PDB: 1YOU). (B) Loop sequence alignment of MMPs (variable
region in red).
Table 2. MMP IC50s of oxa-azole-based inhibitors
O
HN
HN
O
O
O
O
OEt
het
Compound
Heterocyclic system
MMP-13
Fold selectivity
MMP-8
IC50 (nM)
MMP-2
165
MMP-12
309
O
11
12
13
14
15
16
17
1.7
212
115
208
571
43
Ph
N
N
2.4
3.4
1.6
7.5
12
58
54
259
138
38
O
Ph
N
O
Ph
Ph
Ph
Ph
Ph
N
N
11
O
N
N
N
O
O
N
144
66
103
126
37
98
N
O
15
27
ND
found to give an ED50 of ꢀ1 mg/kg while the 4-fluoro
derivative 28 was about 10-fold less active. Compound
high clogP of 3.9, and a polar surface area of 153, high
absorption (80%) was observed when 34 was dosed as a
spray dried dispersion. Compound 34 was also effective
in preventing cartilage degradation (as measured by
hydroxyproline release) from bovine nasal cartilage with
34
(Clp 0.63 mL/min/kg, Vdss 0.34 L/kg, t1/2 6.9 h) in rats.
also
displayed
excellent
pharmacokinetics
Despite the compound having a MW > 500, a relatively

L. A. Reiter et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5822–5826
Table 3. MMP IC50s of 3-Ar-1,2,4-oxadiazoles (X = N) and 4-aryloxazoles (X = CH)
5825
O
HN
HN
O
O
O
O
OR
N
O
Ar
X
Compound
R
X
Ar
MMP-13
Fold selectivity
MMP-2
IC50 (nM)
MMP-8
MMP-12
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
Et
Et
N
2-F–Ph
3-F–Ph
4-F–Ph
3.3
7.8
167
95
102
88
176
56
N
Et
Et
N
6.1
1.0
210
271
320
555
149
270
152
133
338
243
516
569
140
155
394
233
57
80
61
114
71
N
2-Pyridyl
3-Pyridyl
4-Pyridyl
2-F–Ph
Et
Et
N
2.0
134
291
288
562
239
292
356
455
173
142
29
504
232
605
868
1313
906
960
573
342
178
181
134
619
373
158
124
530
N
0.54
1.4
Me
Et
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
2-F–Ph
3-F–Ph
0.93
0.46
0.36
0.57
0.68
0.73
0.88
0.63
0.53
0.36
0.45
1.0
Me
Et
3-F–Ph
4-F–Ph
4-F–Ph
Me
Et
Me
Et
2-CN–Ph
2-CN–Ph
3-CN–Ph
3-CN–Ph
4-CN–Ph
4-CN–Ph
2-Pyridyl
3-Pyridyl
4-Pyridyl
Me
Et
31
Me
Et
397
429
74
Et
Et
0.34
0.40
265
250
38
70
Et
an IC50 of 40 nM.24 This potency level is somewhat bet-
ter than that observed for the MMP-1-sparing, broad
spectrum, hydroxamic acid-based MMP-13 inhibitor,
CP-544439, which gave an IC50 of 70 nM in the same
assay.25
MWs, clogPs, and polar surface areas. Molecules such
as 34 should allow a conclusive assessment of whether
an MMP-13 inhibitor can escape the spectre of MSS
and provide protection to type II collagen in articular
cartilage and relief to OA patients.
With its excellent selectivity profile (as shown in Table 3
plus MMP-1: 2400-fold, MMP-3: 17,000-fold, MMP-9:
540-fold, and MMP-14: 540-fold) and pharmacokinetics
much superior to the hydroxamic acid-based inhibitors
that we previously examined,18 34 was well-suited to test
the hypothesis that selective inhibition of MMP-13
would eliminate the MSS side effect seen with other
MMP inhibitors. To this end, 34 was examined in the
rat fibroplasia model, a surrogate for producing MSS
in humans.26 At doses of 30, 100, and 300 mg/kg bid
for 14 days the compound was well tolerated and at
the 100 mg/kg dose, a Cmax of 198 lg/mL (ꢀ370 lM)
and AUC of 3840 (lg h/mL) were obtained.27 This Cmax
exceeds by >50-fold the rat MMP-13 IC50 of 7.2 lM
(determined in rat plasma).28 Even at the Cmin of
129 lg/mL (ꢀ240 lM) the rat MMP-13 IC50 is exceeded
>30-fold. Despite exposures well above the rat MMP-13
IC50, no fibroplasia was observed.
References and notes
1. Blagg, J. A.; Noe, M. C.; Wolf-Gouveia, L. A.; Reiter,
L. A.; Laird, E. R.; Chang, S.-P. P.; Danley, D. E.;
Downs, J. T.; Elliott, N. C.; Eskra, J. D.; Griffiths, R.
J.; Hardink, J. R.; Haugeto, A. I.; Jones, C. S.; Liras, J.
L.; Lopresti-Morrow, L. L.; Mitchell, P. G.; Pandit, J.;
Robinson, R. P.; Subramanyam, C.; Vaughn-Bowser,
M. L.; Yocum, S. A. Bioorg. Med. Chem. Lett. 2005, 15,
1807.
`
2. Grams, F.; Brandstetter, H.; D’Alo, S.; Geppert, D.;
Krell, H.-W.; Leinert, H.; Livi, V.; Menta, E.; Oliva, A.;
Zimmermann, G. Biol. Chem. 2001, 382, 1277.
3. Foley, L. H.; Palermo, R.; Dunten, P.; Wang, P. Bioorg.
Med. Chem. Lett. 2001, 11, 969.
4. Dunten, P.; Kammlott, U.; Crowther, R.; Levin, W.; Foley,
L. H.; Wang, P.; Palermo, R. Protein Sci. 2001, 10, 923.
5. Brandstetter, H.; Grams, F.; Glitz, D.; Lang, A.; Huber,
R.; Bode, W.; Krell, H.-W.; Engh, R. A. J. Biol. Chem.
2001, 276, 17405.
6. Kim, S.-H.; Pudzianowski, A. T.; Leavitt, K. J.; Barbosa,
J.; McDonnell, P. A.; Metzler, W. J.; Rankin, B. M.; Liu,
R.; Vaccaro, W.; Pitts, W. Bioorg. Med. Chem. Lett. 2005,
15, 1101.
7. Duan, J. J.-W.; Lu, Z.; Wasserman, Z. R.; Liu, R.-Q.;
Covington, M. B.; Decicco, C. P. Bioorg. Med. Chem.
Lett. 2005, 15, 2970.
In summary, we have described the discovery of a series
of selective MMP-13 inhibitors. Selectivity was obtained
by optimizing the P01 heterocycle and its terminal substi-
tuent, both of which lie deep in the S01 pocket. Generally,
the ionizability of the pyrimidinetrione moiety confers
good solubility to molecules in the series and good
absorption can be obtained despite moderate to high

5826
L. A. Reiter et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5822–5826
8. Tardif, G.; Reboul, P.; Pelletier, J.-P.; Martel-Pelletier, J.
Mod. Rheumatol. 2004, 14, 197.
21. The 1-substituted-4-(4-hydroxyphenoxy)benzenes utilized
to prepare 11–17 were prepared by: (11) a Suzuki reaction
of 5-bromo-2-phenyloxazole and 4-(4-methoxyphen-
oxy)benzeneboronic acid followed by demethylation using
methionine in neat methanesulfonic acid (Ref. 29); (12) a
Negishi reaction of 4-phenyloxazole and 1-iodo-4-(4-
methoxyphenoxy)benzene followed by demethylation.
The iodide was prepared by iodination of 4-(4-methoxy-
9. Mitchell, P. G.; Magna, H. A.; Reeves, L. M.; Lopresti-
Morrow, L. L.; Yocum, S. A.; Rosner, P. J.; Geoghegan,
K. F.; Hambor, J. E. J. Clin. Invest. 1996, 97, 761.
10. Yocum, S. A.; Lopresti-Morrow, L. L.; Reeves, L. M.;
Mitchell, P. G. Ann. NY Acad. Sci. 1999, 878, 583;
Borden, P.; Solymar, D.; Sucharczuk, A.; Lindman, B.;
Cannon, P.; Heller, R. A. J. Biol. Chem. 1996, 271, 23577.
11. Knauper, V.; Lopez-Otin, C.; Smith, B.; Knight, G.;
Murphy, G. J. Biol. Chem. 1996, 271, 1544.
phenoxy)benzene (Ref. 30); (13)
a TBTU-mediated
condensation of 4-(4-hydroxyphenoxy)benzoic acid with
N-hydroxybenzenecarboxamidine (Ref. 31); (14) a Negishi
reaction of 3-phenyl-5-(trifluoromethanesulfonyl)oxyisox-
azole and 4-(4-methoxyphenoxy)benzeneboronic acid
followed by demethylation; (15) a Negishi reaction of
5-phenyloxazole and 1-iodo-4-(4-methoxyphenoxy)ben-
zene followed by demethylation; (16) a condensation of
ethyl 4-(4-methoxyphenoxy)benzoate with hydrazine fol-
lowed by acylation with benzoyl chloride, thionyl chlo-
ride-mediated ring closure, and demethylation; (17) a
reaction of 4-(4-hydroxyphenoxy)benzenecarbonitrile with
hydroxylamine followed by TBTU-mediated condensation
with benzoic acid and demethylation.
12. Freemont, A. J.; Byers, R. J.; Taiwo, Y. O.; Hoyland, J. A.
Ann. Rheum. Dis. 1999, 58, 357.
13. Billinghurst, R. C.; Dahlberg, L.; Ionescu, M.; Reiner, A.;
Bourne, R.; Rorabeck, C.; Mitchell, P.; Hambor, J.;
Diekmann, O.; Tschesche, H.; Chen, J.; Van Wart, H.;
Poole, A. R. J. Clin. Invest. 1997, 99, 1534; Billinghurst, R.
C.; Wu, W.; Ionescu, M.; Reiner, A.; Dahlberg, L.; Chen,
J.; Van Wart, H.; Poole, A. R. Arthritis Rheum. 2000, 43,
664.
14. Hutchinson, J. W.; Tierney, G. M.; Parsons, S. L.; Davis,
T. R. C. J. Bone Joint Surg. (Br) 1998, 80-B, 907.
15. Wojtowicz-Praga, S.; Torri, J.; Johnson, M.; Steen, V.;
Marshall, J.; Ness, E.; Dickson, R.; Sale, M.; Rasmussen,
H. S.; Chiodo, T. A.; Hawkins, M. J. J. Clin. Oncol. 1998,
16, 2150.
22. The 1-substituted-4-(4-hydroxyphenoxy)benzenes utilized
to prepare 18–38 were prepared: (18–23) analogously to 13
using the requisite aryl carboxylic acid; (24–38) by either a
Suzuki or Negishi reaction of the requisite boronic acid
(ester) or organozinc derivative and 2-[4-(4-methoxyphen-
oxy)phenyl]-4-(trifluoromethanesulfonyl)oxyoxazole fol-
lowed by demethylation. The oxazole triflate was prepared
according to the method of Kelly and Lang (Ref. 32).
23. Otterness, I. G.; Bliven, M. L.; Eskra, J. D.; te Koppele, J.
M.; Stukenbrok, H. A.; Milici, A. J. Osteoarthritis
Cartilage 2000, 8, 366.
24. Milner, J. M.; Elliot, S.-F.; Cawston, T. E. Arthritis
Rheum. 2001, 44, 2084.
25. CP-544439: compound 10 in Ref. 18.
26. Renkiewicz, R.; Qiu, L.; Lesch, C.; Sun, X.; Devalaraja,
R.; Cody, T.; Kaldjian, E.; Welgus, H.; Baragi, V.
Arthritis Rheum. 2003, 48, 1742.
27. Drug exposures of animals at the 300 mg/kg dose were
somewhat lower than the exposures at the 100 mg/kg dose.
28. The IC50 for 34 against rat MMP-13 is 4.7 nM. The
compound is highly protein bound (>99%) which leads to
a large shift in potency in rat plasma.
29. Andre, J. D.; Dormoy, J. R.; Heymes, A. Synth. Commun.
1992, 22, 2313.
30. Brewster, R. Q.; Choguill, H. S. J. Am. Chem. Soc. 1939,
61, 2702.
16. Griffioen, A. W. IDrugs 2000, 3, 336.
17. Levitt, N. C.; Eskens, F. A. L. M.; O’Byrne, K. J.;
Propper, D. J.; Denis, L. J.; Owen, S. J.; Choi, L.;
Foekens, J. A.; Wilner, S.; Wood, J. M.; Nakajima, M.;
Talbot, D. C.; Steward, W. P.; Harris, A. L.; Verweij, J.
Clin. Cancer Res. 2001, 7, 1912.
18. Reiter, L. A.; Robinson, R. P.; McClure, K. F.; Jones, C.
S.; Reese, M. R.; Mitchell, P. G.; Otterness, I. G.; Bliven,
M. L.; Liras, J.; Cortina, S. R.; Donahue, K. M.; Eskra, J.
D.; Griffiths, R. J.; Lame, M. E.; Lopez-Anaya, A.;
Martinelli, G. J.; McGahee, S. M.; Yocum, S. A.;
Lopresti-Morrow, L. L.; Tobiassen, L. M.; Vaughn-
Bowser, M. L. Bioorg. Med. Chem. Lett. 2004, 14, 3389.
19. The pyrimdinetriones were prepared from the requisite
1-substituted-4-(4-hydroxyphenoxy)benzenes and either
5-bromo-5-ethoxyethyl- or 5-bromo-5-methoxyethylpyr-
imidinetrione as previously described (Ref. 1).
20. The 4-(4-hydroxyphenoxy)benzamides utilized to prepare
1–5 and 8–9 were prepared through a BOP-mediated
coupling of the requisite amine with 4-(4-hydroxyphen-
oxy)benzoic acid. The 4-(4-hydroxyphenoxy)benzamides
utilized to prepare 6–7 were prepared through a coupling
of 4-(4-benzyloxyphenoxy)benzoyl chloride with the req-
uisite pyridine amine followed by hydrogenolytic cleavage
of the benzyl ether.
31. Poulain, R. F.; Tartar, A. L.; Deprez, B. P. Tetrahedron
Lett. 2001, 42, 1495.
32. Kelly, T. R.; Lang, F. J. Org. Chem. 1996, 61, 4623.