Quinazolinones and pyrido[3,4-d]pyrimidin-4-ones as orally active and specific matrix metalloproteinase-13 inhibitors for the treatment of osteoarthritis

Article abstract of DOI:10.1021/jm701274v

Quinazolinones 8 and pyrido[3,4-d]pyrimidin-4-ones 9 as orally active and specific matrix metalloproteinase-13 inhibitors were discovered for the treatment of osteoarthritis. Starting from a high-through-put screening (HTS) hit thizolopyrimidin-dione 7, w

Full text of DOI:10.1021/jm701274v

J. Med. Chem. 2008, 51, 835–841
835
Quinazolinones and Pyrido[3,4-d]pyrimidin-4-ones as Orally Active and Specific Matrix
Metalloproteinase-13 Inhibitors for the Treatment of Osteoarthritis
Jie Jack Li,* Joe Nahra,† Adam R. Johnson, Amy Bunker, Patrick O’Brien, Wen-Song Yue, Daniel F. Ortwine, Chiu-Fai Man,
Vijay Baragi, Kenneth Kilgore, Richard D. Dyer, and Hyo-Kyung Han
Department of Chemistry, Michigan Laboratories, Pfizer Global Research and DeVelopment, 2800 Plymouth Road, Ann Arbor, Michigan 48105
ReceiVed October 10, 2007
Quinazolinones 8 and pyrido[3,4-d]pyrimidin-4-ones 9 as orally active and specific matrix metalloproteinase-
13 inhibitors were discovered for the treatment of osteoarthritis. Starting from a high-through-put screening
(HTS) hit thizolopyrimidin-dione 7, we obtained two chemotypes, 8 and 9, using computer-aided drug design
(CADD) and methodical structure–activity relationship (SAR) studies. They occupy the unique S1′-specificity
pocket and do not bind to the Zn2+ ion. Some pyrido[3,4-d]pyrimidin-4-ones, such as 10a, possess favorable
absorption, distribution, metabolism, and elimination (ADME) and safety profiles. 10a effectively prevents
cartilage damage in rabbit animal models of osteoarthritis without inducing musculoskeletal side effects
when given at extremely high doses to rats.
Scheme 1. Synthesis of Alkynyl Quinazolinones 8
Introduction
Gross and Lapiere discovered the first matrix metallopro-
teinase (MMP-1, also known as collagenase 1) from a meta-
morphosing tadpole in 1962.1 Today, matrix metalloproteinases
(MMPs)a are elucidated as a family of more than 28 subtypes
of zinc- and calcium-dependent endopeptidases that are involved
in the degradation of the extracellular matrix (ECM). MMPs
have been implicated with many inflammatory diseases, cancers,
and other illnesses.2 Scientists in both academia and industry
have carried out an enormous amount of investigations into the
use of MMP inhibitors as innovative medicines.3 Many potent
and orally active broad-spectrum MMP inhibitors have been
discovered and investigated in clinical trials. MMP inhibitors 1
(marimastat)4 and 2 (prinomastat)5 were tested against cancers;
MMP inhibitors 3 (cipemastat)6 and 4 (ilomastat)7 were tried
in the clinics for inflammation. Unfortunately, these broad-
spectrum MMP inhibitors have been limited by nonspecificity
and the subsequent nonselective toxicity and dose-limiting
efficacy. Recognizing that the hydroxamic acid in 1–4 could
be a possible culprit for safety issues because of its powerful
chelating ability toward the MMP zinc ion, efforts have been
made to replace it with milder chelating functional groups. One
such group is a carboxylic acid as represented by 5 (tanoma-
stat),8 and another group is a thiol as represented by 6.9
Regrettably, there is still not a single MMP inhibitor that has
emerged on the market thus far. The sole exception is probably
doxycycline, which was marketed as an antibiotic but was later
found to inhibit MMP-2, -8, -9, and -13.10
much attention.11–15 MMP-13 catalyzes the hydrolysis of type-
3
II collagen at a unique site, which results in /4- and 1/4-length
polypeptide products.16–20 MMP-13 is not expressed in normal
adult tissues, but it is found in the joints and articular cartilage
of osteoarthritis (OA) patients. Therefore, it is a compelling
target for the treatment of OA.21–25 An MMP-13 inhibitor has
been shown to block the degradation of explanted human
osteoarthritic cartilage.19 In genetically modified mice, regulated
expression of human MMP-13 induces osteoarthritis.26 On the
basis of these findings, it is likely that MMP-13 causes
irreversible cartilage damage in OA, and therefore, a selective
MMP-13 inhibitor would effectively prevent cartilage degrada-
tion without causing musculoskeletal syndrome (MSS) side
effects that have plagued many MMP broad-spectrum inhibitors
in clinical development.24,26,27
High-through-put screening (HTS) of our compound collec-
tion provided, in addition to all of the broad-spectrum MMP
inhibitors, a unique hit, thiazolopyrimidinedione 7, which
possessed an astounding selectivity for MMP-13 versus other
MMP isoforms (Figure 2). As a matter of fact, it was specific
for MMP-13 for all practical purposes. Cocrystallization of 7
with the MMP13-CD (catalytic domain) revealed an unexpected
nonzinc-binding mode: instead of binding to the Zn2+ cation,
To overcome the nonselective toxicity and dose-limiting
efficacy, an industry-wide effort has been undertaken to seek
selective MMP inhibitors. In particular, MMP-13 has attracted
* To whom correspondence should be addressed: Discovery Chemistry,
Bristol-Myers Squibb Company, 5 Research Parkway, Wallingford, CT
06492. Telephone: (203) 677-7255. E-mail: jie.li1@bms.com.
†
Present address: Discovery Chemistry, Bristol-Myers Squibb Company,
P.O. Box 5400, Princeton, NJ 08543-5400.
a
Abbreviations: ADME, absorption, distribution, metabolism, and
elimination; AUC, area under curve; CADD, computer-aided drug design;
CD, catalytic domain; CIR, confidence in rationale; ECM, extracellular
matrix; HTS, high-through-put screening; IVMN, in Vitro micronucleus;
MMP, matrix metalloproteinase; MSS, musculoskeletal syndrome; OA,
osteoarthritis; SAR, structure–activity relationship.
10.1021/jm701274v CCC: $40.75
2008 American Chemical Society
Published on Web 02/06/2008

836 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Li et al.
Figure 1. Broad-spectrum MMP inhibitors.
Figure 3. Definition of the specificity loops of peptidases.
toward the N terminus and S1′ is the first specificity subunit
from the catalytic site toward the C terminus (Figure 3). The
MMP catalytic domain consists of a five-stranded ꢀ-sheet and
a 3-R-helices, with the catalytic site defined by ꢀ-strand IV and
the central helix, which imparts significant structural differences
for MMP isoforms. The S1′-specificity pocket in MMP-13 is
an unusually large, open channel, nearly twice the size as that
found in MMP-1, which may explain the high degree of
specificity of 7.
However, 7 is an ester, metabolizing readily in the body, and
the corresponding acid is inactive in MMP-binding assays. Using
computer-aided drug design (CADD) and methodical structure–
activity relationship (SAR) studies, we obtained two series,
quinazolinones 8 and 9 and pyrido[3,4-d]pyrimidin-4-ones 10
(Figure 4), that are potent and specific MMP-13 inhibitors, some
of which are orally bioavailable. These specific MMP-13
inhibitors, occupying the unique S1′-specificity pocket, do not
bind to the Zn2+ ion, effectively preventing cartilage damage
in animal models of osteoarthritis without inducing musculosk-
eletal side effects when given at extremely high doses to rats.
Results and Discussion
Chemistry. Synthesis of alkynyl quinazolinones 8 began with
condensation of commercially available 2-amino-5-iodo-benzoic
acid with formamidine acetate in refluxing ethanol to afford
6-iodo-3H-quinazolin-4-one (11).28,29 The key intermediate 11
enabled convenient SAR investigation at both 3 and 6 positions.
Therefore, alkylation of 11 with benzyl chloride 12 in the
presence of Cs2CO3 gave rise to the 3-N-benzylated products
13. Finally, a Sonogashira reaction between 13 and 3-phenyl-
1-propyne 14 delivered alkynyl quinazolinones 8 (Scheme 1).
Figure 2. Nonzinc-binding mode of 7 from the cocrystal structure
(1.72 Å) with the MMP-13 catalytic domain enzyme, with 7 occupying
the S1′-specificity pocket.
1 binds deep within the S1′-specificity loop of the protein and
extends past this pocket out toward the bulk solvent (Figure 2).
In describing the specificity of peptidases, S1 is the first
specificity subunit from the catalytic site (Zn2+ in this case)

Quinazolinones and Pyrido[3,4-d]pyrimidin-4-ones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 837
Scheme 2. Synthesis of Amide Quinazolinones 9
Scheme 3. Synthesis of Amides Pyrido[3,4-d]pyrimidin-4-ones
10
Synthesis of the amide quinazolinones 9 was similar to that
of the alkynyl quinazolinones 8 (Scheme 2). Instead of a
Sonogashira coupling reaction, a palladium-catalyzed carbony-
lation of 13 was followed by hydrolysis to produce carboxylic
acid 14. A Mukaiyama amide formation with benzylamine 15
then delivered the desired amide quinazolinones 9.
To block the metabolism site at the C-7 position, efforts were
made to replace the C-H with a nitrogen atom as amide
pyrido[3,4-d]pyrimidin-4-ones 10. Therefore, adduct 17 was
produced from alkylation of the known chloropyrido[3,4-
d]pyrimidin-4(3H)-one (16)29 with benzyl bromide 12. Pal-
ladium-catalyzed carbonylation of 17 afforded methyl ester 18.
After Weinreb’s one-step amidation protocol,30–32 ester 18 was
then treated with the dimethylaluminum amide generated from
AlMe3 and benzylamine 15 to deliver the final amides 10
(Scheme 3).
Structural-Activity Relationship. When the synthetic route
delineated in Scheme 1 was applied, a series of alkynyl
quinazalinones were synthesized with variations at both the
solvent end and the S1′-specificity pocket. For the solvent end,
as shown in entries 1–10, tetrazole 8a and carboxylic acid 8b
were the most potent substituents for the binding of both
catalytic domain (CD) and full-length collagen.33 The potency
was eroded somewhat for other less acidic analogues, such as
2,6-diflurophenol 8c, carboxamide 8d, and sulfonamide 8e. For
derivatives without an acidic moiety (entries 6–9, 8f–8i), the
potency suffered even further. In all, the potency is closely
associated with the pKa of the fragment aligned in the solvent
cavity (thereby the hydrogen-bond-donating ability).
Because carboxylic acid provided the best potency, the solvent
end was kept constant as a carboxylic acid in the process of
investigating the SAR of the S1′-specificity pocket. The nature
of the substituent of the compound binding to the S1′-specificity
pocket has a significant impact on the potency as well. For
instance, when the S1′-specificity pocket was occupied by an
aromatic ring, such as entry 2 (Table 1), compound 8b was
very potent (IC50 ) 1.54 nM). However, the corresponding
allene analogue 8k (entry 11) was over 100-fold less potent
(IC50 ) 194 nM) possibly because its steric alignment did not
fit in the S1′-specificity pocket as well. The two aliphatic cyclic
amine derivatives 8l and 8m fit in the binding pocket tightly,
implying the importance of the π-stacking in achieving binding
potency. Indeed, heteroaryl substituent at the S1′-specificity
pocket, such as imidazole, restored the potency as demonstrated
Figure 4. Progression of the SAR: from thiazolopyrimidinedione 7 to
quinazolinones 8 and 9 and to pyrido[3,4-d]pyrimidin-4-ones 10.

838 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Li et al.
Table 1. SAR of Alkynyl Quinazalinones 8 for the Solvent End (Entries 1–9) and the S1′ End (Entries 10–14)
a “MMP-13 CD av. IC50 (nM)” represents the average IC50 value in nanomolars for the binding assay of the compound using the MMP-13 catalytic
domain enzyme. b “MMP-13 Collagen II av. IC50 (nM)” represents the average IC50 value in nanomolars for the binding assay of the compound using the
MMP-13 full-length enzyme.
in entry 14 (IC50 ) 7.82 nM for 8n). 8n has an added benefit
of increased aqueous solubility.
position by making pyrido[3,4-d]pyrimidin-4-ones 10 with
amides at the S1′ end. The SAR trends of amide pyrido[3,4-
d]pyrimidin-4-ones 10 bear a striking resemblance to the
corresponding amide quinazolinones 9, although 10 is generally
less potent than 9.35–37 For instance, carboxylic acid 10a, with
an IC50 of 6.72 nM, is about 4-fold less potent than the
corresponding carboxylic acid 9a, with an IC50 of 1.61 nM.
Pharmacokinetics, Safety, and Efficacy. We further inves-
tigated the absorption, distribution, metabolism, and elimination
(ADME) profiles for MMP-13 inhibitors with IC50 < 10 nM in
the binding assay using truncated enzyme containing the
catalytic domain and IC50 < 100 nM in the binding assay using
the full-length enzyme. Some exceptions to these in Vitro profile
cut-offs were made for representative chemotypes to understand
the correlation between structure and bioavailability. Some of
the alkynyl quinazalinones 8 tested gave favorable oral bio-
availability. For instance, carboxylic acid 8b with 3-phenyl-1-
propyne moiety at the S1′ end has moderate clearance and
moderate volume distribution in rats as shown in Table 3 (entry
1). As a result, 8b has a half-life of 5.4 h, F% of 75%, and area
under curve (AUC) of 12 400 ng h-1 mL-1 (po). The corre-
sponding methoxyl analogue 8j has a similar, although slightly
less favorable, pharmacokinetics profile than that of 8b. These
alkynes were not further pursued because of safety concerns
(Vide supra). As far as amide quinazalinones 9 were concerned,
none of the derivatives in this series possessed desirable oral
bioavailability in rats, as exemplified by amides 9a and 9b in
entries 5 and 6 in Table 3. It was found that oxidative
metabolism of amides 9a and 9b took place on the benzene
ring of the quinazalinone core structure. In an effort to block
While the alkynyl quinazalinones 8 furnished great potency
and bioavailability, our in Vitro adduct formation studies
indicated potential safety issues. Moreover, the ease of isomer-
ization of benzyl alkynes to the corresponding allenes (e.g., 8b
f 8k) under basic conditions added further concerns regarding
the chemical stability of these compounds. In the literature, some
acetylene-containing drugs (e.g., efavirenz) were extensively
metabolized in ViVo from cytochrome P450 interactions and the
activation of the alkyne led to a cysteinylglycine adduct.34 As
a consequence, nephrotoxicity was observed for efavirenz.
Although the mere presence of an alkyne in a molecule is not
sufficient to produce nephrotoxic events from cytochrome P450
interactions, a decision was made to pursue amide as a
bioisostere for the ester instead of the alkyne as in alkynyl
quinazalinones 8 to avoid further complications.
The synthesis was achieved using chemistry delineated in
Scheme 2, and the SAR of amide quinazalinones 9 is shown in
Table 2 (entries 1–4). Similar to the SAR of 8, carboxylic acid
at the solvent end afforded the highest potency (hydroxamic
acid was intentionally avoided because of selectivity concerns).
In agreement with our docking (onto the MMP-13 CD enzyme)
and overlay studies, the amide quinazolinones 9 are found to
be generally less potent than the corresponding alkynyl quinazoli-
nones 8. The most potent amide analogue 9a is 10-fold less
potent than the corresponding alkyne analogue 8j.
Because CYP450 oxidation of 9 was the major metabolism
pathway to give the corresponding phenol at the C-7 position
of the amide quinazolinones, efforts were made to block the

Quinazolinones and Pyrido[3,4-d]pyrimidin-4-ones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 839
Table 2. SAR of Amide Quinazalinones 9 and Amide Pyrido[3,4-d]pyrimidin-4-ones 10
a “MMP-13 CD av. IC50 (nM)” represents the average IC50 value in nanomolars for the binding assay of the compound using the MMP-13 catalytic
domain enzyme. b “MMP-13 Collagen II av. IC50 (nM)” represents the average IC50 value in nanomolars for the binding assay of the compound using the
MMP-13 full-length enzyme.
Table 3. Pharmacokinetics Data for Selected MMP-13 Inhibitors
AUC (0 f ∞)
entry
compound
CL (mL min-1 kg-1
)
Vdss (L/kg)
(ng h-1 mL-1) (po)
T1/2 (h)
F%
solubility (µg/mL)
1
2
3
4
5
6
7
8
9
10
8b
8c
8j
8n
9a
9b
10a
10b
10l
19a
4.9
39.4
5.3
0.6
2.8
1
1.9
0.4
1.5
0.9
0.5
2
12 400
41
5160
570
5.4
0.7
75
2
40
19
9
48
73
24
3
<3
4.5
>60
89.6
1
31
0.9
3.5
1.4
3.9
2.3
20.0
23.5
7.6
11.2
20.6
16.4
377
1720
13 400
1810
1010
1470
60
60
0.5
2.1
40
41
this oxidation, a fluorine was installed at the C-7 position to
give 19a. Regrettably, not only was it less potent with an IC50
of 31.1 nM in the MMP-13 CD assay (entry 18 in Table 2),
19a also had low oral bioavailability in rats (entry 10 in Table
3). Gratifyingly, when the C-H moiety was replaced with a
nitrogen atom, amide pyrido[3,4-d]pyrimidin-4-ones 10 had both
an increase in potency and an increase in solubility, as well as
elevated bioavailability in some cases. Moreover, 10a has a
moderate clearance and moderate volume distribution in rats
as shown in Table 3 (entry 7). As a result, its half-life is 3.9 h,
MMP-14CD, and MMP-17CD. Possibly thanks to its remarkable
specificity, similar to the outcome of the vehicle, no undesirable
MSS was observed for 10a, while a broad-spectrum MMP
inhibitor caused MSS for 12 out of 12 rats treated with it.
Therefore, in the rat model, the MMP-13-specific inhibitor 10a
is completed devoid of MSS, a side effect that has plagued the
majority of broad-spectrum MMP inhibitors in the clinics.
Compound 10a also tested negative in both mini-Ames and in
Vitro micronucleus (IVMN) tests. Furthermore, the in ViVo
toxicity study involving two species (rat and rabbit) did not show
any noticeable toxicity issues (Table 4).
F% of 73%, and AUC (po) of 13400 ng h-1 mL-1
.
Because of the favorable ADME profile of 10a, it was further
characterized more thoroughly. It is a potent MMP-13 inhibitor
with an IC50 of 6.72 nM in the MMP-13 CD assay but does not
inhibit all other MMP isozymes tested, including MMP-1,
MMP-2, MMP-3CD, MMP-7, MMP-8CD, MMP-9, MMP-12,
More significantly, a cartilage protection in ViVo study was
carried out using the NZW male rabbits that were inflicted with
OA surgically. On day 35, gross morphology (lesion area and
severity) indicated that, at 10 mg/kg dose, 10a inhibited the
degeneration both of tibial plateau and femoral condyle cartilage

840 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Li et al.
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Conclusion
In summary, the HTS hit thiazolopyrimidinedione 7 is a
MMP-13-specific inhibitor, which imparts its specificity by
binding the unique S1′-specificity pocket. Our extensive SAR
investigation resulted in amide pyrido[3,4-d]pyrimidin-4-ones
10 as MMP-13-selective inhibitors (specific versus other MMPs)
that bind to the unique S1′-specificity pocket and do not bind
to the Zn2+ ion. Some derivatives, such as 10a, possess
favorable ADME and safety profiles. More significantly, our
positive cartilage protection in ViVo study in rabbits using 10a
provided increased CIR for this approach of using MMP-13-
specific inhibitors to treat OA.
Supporting Information Available: 1H NMR spectra and data
and MS(APCI) data of all products and melting points and CHN
elemental analysis data for all solid samples. This material is
available free of charge via the Internet at http://pubs.acs.org.
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