Discovery and evaluation of a non-Zn chelating, selective matrix metalloproteinase 13 (MMP-13) inhibitor for potential intra-articular treatment of osteoarthritis

Article abstract of DOI:10.1021/jm201152u

Osteoarthritis (OA) is a nonsystemic disease for which no oral or parenteral disease-modifying osteoarthritic drug (DMOAD) is currently available. Matrix metalloproteinase 13 (MMP-13) has attracted attention as a target with disease-modifying potential because of its major role in tissue destruction associated with OA. Being localized to one or a few joints, OA is amenable to intra-articular (IA) therapy, which has distinct advantages over oral therapies in terms of increasing therapeutic index, by maximizing drug delivery to cartilage and minimizing systemic exposure. Here we report on the synthesis and biological evaluation of a non-zinc binding MMP-13 selective inhibitor, 4-methyl-1-(S)-({5-[(3-oxo-3,4-dihydro-2H-benzo[1,4]oxazin-6-ylmethyl)carbamoyl] pyrazolo[1,5-a]pyrimidine-7-carbonyl}amino)indan-5-carboxylic acid (1), that is uniquely suited as a potential IA-DMOAD: it has long durability in the joint, penetrates cartilage effectively, exhibits nearly no detectable systemic exposure, and has remarkable efficacy.

Full text of DOI:10.1021/jm201152u

Article
pubs.acs.org/jmc
Discovery and Evaluation of a Non-Zn Chelating, Selective Matrix
Metalloproteinase 13 (MMP-13) Inhibitor for Potential Intra-articular
Treatment of Osteoarthritis
Christian Gege,†,§ Bagna Bao,‡ Harald Bluhm,† Jurgen Boer,† Brian M. Gallagher, Jr.,‡ Brian Korniski,‡
̈
Timothy S. Powers,‡,∥ Christoph Steeneck,† Arthur G. Taveras,‡,⊥ and Vijaykumar M. Baragi*,‡
†Alantos Pharmaceuticals AG, Im Neuenheimer Feld 584, D-69120 Heidelberg, Germany
‡Alantos Pharmaceuticals Inc., Riverside Technology Center, 840 Memorial Drive, Cambridge, Massachusetts 02139, United States
ABSTRACT: Osteoarthritis (OA) is a nonsystemic disease for which no oral
or parenteral disease-modifying osteoarthritic drug (DMOAD) is currently
available. Matrix metalloproteinase 13 (MMP-13) has attracted attention as a
target with disease-modifying potential because of its major role in tissue
destruction associated with OA. Being localized to one or a few joints, OA is
amenable to intra-articular (IA) therapy, which has distinct advantages over oral therapies in terms of increasing therapeutic
index, by maximizing drug delivery to cartilage and minimizing systemic exposure. Here we report on the synthesis and biological
evaluation of a non-zinc binding MMP-13 selective inhibitor, 4-methyl-1-(S)-({5-[(3-oxo-3,4-dihydro-2H-benzo[1,4]oxazin-6-
ylmethyl)carbamoyl]pyrazolo[1,5-a]pyrimidine-7-carbonyl}amino)indan-5-carboxylic acid (1), that is uniquely suited as a
potential IA-DMOAD: it has long durability in the joint, penetrates cartilage effectively, exhibits nearly no detectable systemic
exposure, and has remarkable efficacy.
preparations of IA viscosupplements based on hyaluronate as
new devices for the treatment of pain in OA of the knee. It is
reported that pain relief is usually obtained by 8−12 weeks after
the first injection and can last up to six months. The IA
hyaluronate products are believed to work through viscosup-
plementation of the compromised synovial fluid in the OA
diseased joint.4
INTRODUCTION
■
Osteoarthritis (OA) is characterized by progressive loss of
articular cartilage resulting in chronic pain and disability. The
disease is nonsystemic by nature and is commonly restricted to
few joints. In addition to the loss of cartilage, there is an
observed reduction in the viscous properties of the synovial
fluid.
There is now new evidence for development of potential
disease-modifying osteoarthritis drugs (DMOADs), arising
from a fuller understanding of joint metabolism. There is
overwhelming evidence to demonstrate that matrix metal-
loproteinases (MMPs), specifically MMP-13, have a major role
in tissue destruction associated with OA. MMPs are a family of
zinc-dependent endopeptidases involved in the degradation of
extracellular matrix and tissue remodeling.5 MMPs have long
been considered as attractive therapeutic targets for treatment
of OA;6 however, broad-spectrum MMP inhibitors developed
for treatment of arthritis have failed in clinical trials due to
painful, joint-stiffening side effects termed musculoskeletal
syndrome (MSS).7,8 It is believed that MSS is caused by
nonselective inhibition of multiple MMPs other than MMP-
13.8−10 MMP-13 is the major collagenase in OA cartilage and
the most efficient type II collagen-degrading MMP.11 There-
fore, current drug development strategies for treatment of OA
are focused on inhibition of MMP-13 with high isoform
selectivity.12,13
There is no cure for OA; disease management is limited to
treatments that are palliative at best and do little to address the
underlying cause of disease progression. Therapeutic inter-
ventions in OA have been hindered by the difficulty of targeting
drugs to articular cartilage. Because articular cartilage is an
avascular and alymphatic tissue, traditional routes of drug
delivery (oral, intravenous, intramuscular) ultimately rely on
transsynovial transfer of drugs from the synovial capillaries to
cartilage by passive diffusion. Thus, in the absence of a
mechanism for selectively targeting drug to the cartilage,
traditional routes of delivery have found it necessary to expose
the body systemically to high concentrations of drugs to
achieve a sustained, intra-articular (IA) therapeutic dose. As a
consequence of high systemic exposures, most of the traditional
therapies for OA have been plagued by serious toxicities.
A variety of IA agents, including corticosteroids and several
hyaluronate products, are currently available for symptomatic
relief of OA.1 Corticosteroid clinical trials data suggest that for
most patients the treatment results in a slightly better
improvement in symptoms than placebo and that the effects
do not last more than 4 weeks.2,3 The precise mechanism by
which corticosteroids exert their beneficial effects in an OA
joint is not known. They are believed to act as broad-spectrum
anti-inflammatory agents. The FDA has designated several
MMPs have an extended zinc-binding motif, which contains
three histidines bound to zinc and a glutamate that acts as the
Received: August 29, 2011
Published: December 18, 2011
© 2011 American Chemical Society
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catalytic site for substrate hydrolysis.12 Most small molecule
inhibitors described to date gain activity by interacting with this
catalytic site via a strong zinc-chelating moiety (e.g.,
hydroxamate) and by tight binding within the largely
hydrophobic S1′ pocket.13 The S1′ pocket is in part formed
by the selectivity loop, which varies in length and amino acid
sequence for different MMP isoforms. These S1′ differences
between MMP family members have been utilized to design
MMP inhibitors with different selectivity profiles.12,13 After the
discovery of highly selective nonzinc-chelating MMP-13
inhibitors by Engel et al. (e.g., A in Figure 1),14 which bind
(>20,000-fold over tested MMPs), and highly potent MMP-13
inhibitor and its biological evaluation following IA admin-
istration to rats.
RESULTS
■
Chemistry. Target compound 1 was prepared according to
the synthetic route outlined in Scheme 1. Thus, 1H-pyrazol-3-
a
Scheme 1. Synthesis of Compound 1
Figure 1. Reported nonzinc binding, highly selective MMP-13
inhibitors (A,14 B,16e C,10,22 and PF15218).
a
Reagents and conditions: (a) 1H-pyrazol-3-amine, MeOH, reflux,
to an additional S1′ side pocket, the structural basis for the high
selectivity was apparent.14,15 Several groups with various
structural classes exploited these findings (e.g., B in Figure
1),16,17 and the preclinical experiments in various animal
models demonstrated that these highly selective MMP-13
inhibitors are efficacious and at the same time can overcome
safety problems associated with MMP inhibitors developed so
far for the potential treatment of OA.9,10,18 Thus, there is
compelling data demonstrating the potential of MMP-13
inhibitors as disease-modifying osteoarthritis drugs.
Although corticosteroids as well as nonsteroidal agents
including hyaluronate products are being widely used to treat
signs and symptoms of OA,19 to date, there is no agentoral
or IAon the market that has proven to be disease/structure
modifying. While there have been efforts to evaluate potential
DMOADs including MMP-13 inhibitors via an oral route of
administration, very little information is available on potential
IA DMOADs. The IA route of administration has a distinct
advantage over oral therapies in terms of increasing therapeutic
index, by maximizing drug delivery to target cartilage and
minimizing systemic exposure.
66%; (b) SeO2, TBHP, 50 °C; (c) oxone, rt, 63% (two steps); (d) 5,
HATU, HOAt, NMM, 86%; (e) aqueous LiOH, quant; (f) CuCN,
NMP, 250 °C, 24%; (g) NaBH4, Boc2O, cat. NiCl2, 41%; (h) 4 N HCl
in dioxane, quant; (i) EDCI, NMM, 94%; (j) HCOOH, 95%.
amine was reacted with methyl acetopyruvate 2 in refluxing
methanol, giving compound 3 in 66% yield as the major isomer,
which crystallized from MeOH upon cooling of the reaction
mixture. Oxidation of the methyl group was carried out in two
steps using selene dioxide and tert-butyl hydroperoxide
(TBHP) to afford the aldehyde intermediate, which was
subsequently oxidized with oxone to acid 4 in 63% overall yield.
No migration of the methyl ester moiety was observed under
these reaction conditions. Reaction of amine 520 with acid 4
using O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluro-
nium-hexafluorophosphate (HATU) and 1-hydroxy-7-azaben-
zotriazole (HOAt) as powerful coupling reagents afforded
methyl ester 6 in 86% yield. Saponification with aqueous
lithium hydroxide afforded acid 7 in quantitative yield. The
benzoxazinone building block was prepared by reaction of
chloride 8 with copper(I) cyanide in N-methyl-pyrrolidin-2-one
(NMP) at 250 °C overnight. After aqueous workup,
crystallization from EtOAc/toluene afforded nitrile 9. Reduc-
tion of the nitrile moiety with sodium borohydride and
nickel(II) chloride as catalyst in the presence of di-tert-butyl
We hypothesized that a small molecule MMP-13 inhibitor
with low solubility could be used to clarify the role of MMP-13
in pathological OA, when applied via IA injection. Reported
herein is the synthesis of a low soluble, highly selective
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a
Table 1. Selectivity and Potency of Compound 1 and Compound C
IC50 (nM) vs catalytic domain
compd no.
MMP-1
MMP-2
MMP-3
MMP-7
MMP-8
MMP-9
MMP-12
MMP-13
MMP-14
TACE
Agg 1
1
>20000
>20000
>20000
>20000
8200
>20000
>20000
3200
>20000
>20000
655
0.03
1.3
>20000
>20000
>20000
>20000
2400
7000
C
15000
>20000
>20000
a
Errors are in the range 5−10% of the reported value.
dicarbonate (Boc2O)21 furnished the Boc-protected derivative
10, which was finally deprotected with hydrochloride in
dioxane to give benzoxazinone building block 11. Final
coupling of amine 11 and acid 7 using 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride (EDCI) and
N-methylmorpholine (NMM) as base furnished intermediate
12, which was deprotected in formic acid at 45 °C to give target
compound 1 in high overall yield.
Although still remarkably selective toward MMP-13, MMPs
with similar size and shape of the S1′ pocketnamely MMP-3,
-8, and -1217are targeted to some extent by compounds in
this class. Comparison with other compounds of the same class
(e.g., C in Figure 1) indicated that to a major extent the
benzoxazinone moiety is responsible for this effect.10
Remarkably, the fragment 6-methyl-4H-benzo[1,4]oxazin-3-
one alone is reported to inhibit MMP-12 with an IC50 of 355
nM.26
Inhibition of In Vitro Cartilage Degradation. To
characterize the functional activity of compound 1, its ability
to block collagen degradation was studied in vitro in bovine
articular cartilage explants by measuring the interleukin-1α (IL-
1α)/oncostatin-M (OSM)-induced release of C1,2C, the
product of collagen degradation.22 Compound 1 was effective
in blocking collagen degaradation in a dose-dependent manner
with an IC50 of approximately 20 nM (Table 2).
PHARMACOKINETICS
■
Systemic Exposure. Little or no systemic exposure in rats
was observed up to 100 mg/kg oral or intraperitonial doses, or
0.5 mg/kg IA dose. Only a minimal exposure with a rapid
clearance of compound 1 from the systemic circulation was
seen in the 1.0 mg/kg intravascular dose (data not shown).
However, the studies on the microsomal stability of compound
1 indicated 95% and 96% of the unmetabolized compound in
the rat and human microsomes, respectively, after 60 min of
incubation.
Table 2. Results from in Vitro Cartilage Degradation Assay
a
and Ex Vivo Model of Cartilage Degradation
Safety, Tolerability, and Pharmacokinetics Following
Intra-articular Administration. Following IA injection of the
right knee joint of rats with the test compound 1, synovial
lavage, cartilage, and plasma samples were collected at various
time points for a period of 8 weeks and analyzed for compound
concentrations using LC/MS/MS methods. Compound
concentrations ranging from 0.30 0.02 to 3.40 1.28 μM
(mean SE) were measured in the synovial lavage and from
4.09 0.57 to 7.10 0.66 μM in the cartilage for a period of
about 21 days post a single injection of 0.5 mg of compound/
joint. However, by day 56, the concentration of compound 1 in
the synovial lavage was below the level of detection, whereas
concentrations of ≥2.0 μM were maintained in the cartilage. In
comparison, plasma concentrations remained near or below the
level of detection for the entire duration of the study. On day
56, the joints were scored for behavioral signs of potential
musculoskeletal side effects. None of the six animals in the
compound-treated group displayed any signs of impaired use of
their limbs. There was no difference between the control and
drug treated groups in terms of the total score received (total
score = 0) for behavioral signs of toxicity, as assessed by the
ability to use their hind legs.
ex vivo model of cartilage
degradation (% inhibition)
compd cat. MMP-13
bovine cartilage
explant (nM)
no.
IC50 (nM)
24 h
7 d
14 d 21 d
1
0.03
1.3
∼20
∼5
100
95
100
60
100
0
100
n.t.
C
a
n.t. = not tested.
Activity in the Ex Vivo Model of Rat Articular
Cartilage Degradation. To gain insight into the functional
activity of the compound in vivo, the extent and duration of
chondroprotective effects of the compound in the rat knee joint
was assessed. Following a single injection (0.5 mg/joint) of
compound 1, rats were sacrificed at various times and cartilage
obtained from knee joints was challenged with human MMP-13
ex vivo and the resulting collagen loss was measured using
C1,2C ELISA. Compound 1 was effective in blocking 100% of
MMP-13-induced release of C1,2C at 1, 7, 14, and 21 days
post-IA injection (Table 2).
DISCUSSION AND CONCLUSION
■
Therapeutic interventions in OA have been hindered by the
difficulty of targeting drugs to articular cartilage. Because
articular cartilage is an avascular and alymphatic tissue,
traditional routes of drug delivery (oral, intravenous, intra-
muscular) ultimately rely on trans-synovial transfer of drugs
from the synovial capillaries to cartilage by passive diffusion.
Thus, in the absence of a mechanism for selectively targeting
drug to the cartilage, traditional routes of delivery have found it
necessary to expose the body systemically to high concen-
trations of drugs to attain therapeutic concentrations in the
joint. As a consequence of high systemic exposures, most of the
traditional therapies for OA have been plagued by serious
toxicities. Thus, given the side-effect profiles of current oral
PHARMACOLOGY
■
Potency and Selectivity. The potency (IC50) of
compound 1 against the catalytic domain of recombinant
human MMP-13 was determined22 to be 0.03 nM (Table 1).
Compared to other derivatives of the same compound
class,10,23 the introduction of the 4H-benzo[1,4]oxazin-3-one-
6-ylmethyl moiety improved potency by at least ten times that
seen with structurally simpler moieties (e.g., 3-trifluoromethyl-
4-fluorobenzyl as in compound C of Table 1). This was also
observed in the compound classes with different core
structures.24,25 Compound 1 is >20000-fold selective over all
the MMPs, TACE and Aggrecanase 1, tested (Table 1).
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therapies for OA, a chronic but nonlethal disease, there exists
an opportunity for alternative OA therapies. OA being localized
to a few large joints, there is a competitive advantage to deliver
a small molecule locally to the affected joint. IA delivery would
ideally minimize structural damage and inflammation and
provide symptomatic relief of OA, while minimizing systemic
exposure. In addition, the agent should minimize toxicity and
provide cost-of-goods savings and convenience of dosing at 4 to
6 injections/year. Clearly, the IA route of administration, by
maximizing drug delivery to cartilage, the site where it is most
needed, and minimizing systemic exposure, potentially offers
the distinct advantage over traditional therapies in terms of
increasing therapeutic index.
exhibited no significant systemic exposure. The compound
showed no risk of joint damage: no joint irritation or adverse
effects in both acute (48 h) and chronic (3 weeks) studies in
rats (data not shown). It also showed no gross changes in the
joint or signs of MSS in an 8 week durability study (0.5 mg/
joint for 8 weeks). The compound is amenable to IA delivery
and has sufficient durability to allow for a minimal number of
injections annually. Taken together, these findings demonstrate
that compound 1 represents a promising first-in-class IA-
DMOAD candidate.
EXPERIMENTAL SECTION
■
1
Chemistry. H NMR spectra were recorded on a Bruker Avance
A variety of IA agents, including corticosteroids and several
hyaluronate products, are currently available for short-term
symptomatic relief of OA. IA corticosteroid injections are
effective in providing symptomatic relief for weeks and
sometimes months. However, because of concerns of damage
to the cartilage, corticosteroids are not used for long-term
treatment of OA. There are five preparations of IA hyaluronate
marketed in the US for treatment OA knee pain. They are
generally reported to provide short-term positive effects on
pain and function after a series of 3−5 injections. However, the
mechanisms of their effect are not known, and most have a
synovial half-life of only 2−8 days. There are currently no FDA
approved oral or IA therapies that alter joint structure in OA.
Compound 1 is uniquely suited as a potential IA-DMOAD
candidate. It is the most potent and selective MMP-13 inhibitor
reported so far (IC50: 0.03 nM). As we have previously
reported, MMP-13 specific inhibitors are without the
musculoskeletal liability that has been associated with broad-
spectrum MMP inhibitors.10 Compound 1 is distinguished for
its remarkable durability in the joint. A single IA injection of
compound 1 was sufficient to sustain high-levels of the
compound in cartilage (2 μM) for up to 8 weeks. Combined
with this outstanding durability in the joint, compound 1 has a
PK profile that minimizes systemic exposure. Negligible or no
levels of the compound were detected in the plasma following
100 mg/kg oral and intraperitoneal routes, the 1 mg/kg IV
route, or the 0.5 mg/kg IA route of administration of the
compound. Consistent with this joint durability and systemic
PK profile, compound 1 showed excellent in vitro and ex vivo
efficacy while demonstrating no clinical signs of MSS in the
animals or gross changes in the joint in an 8 week IA durability
study. In vitro, compound 1 inhibited IL-1 and oncostatin M-
stimulated collagen loss from bovine articular cartilage with an
IC50 ∼ 20 nM. IA administration of compound 1 demonstrated
dose-dependent chondroprotection in rats with ex vivo
challenge of cartilage with hMMP-13 (IC50 = 0.09 mg/joint,
data not shown). Taken together, these findings demonstrate
that the compound represents a promising new class of
potential IA-DMOADs. To date, there is no agentoral nor
IAon the market that has proven to be disease/structure
modifying.27
(250 MHz) instrument at 300 K in CDCl3 or DMSO-d6. Chemical
shifts are reported in δ values (ppm); the hydrogenated residues of
deuterated solvent were used as internal standard (CDCl3: δ 7.26 and
DMSO: δ 2.50). Signals are described as s, d, t, dd, m, and b for
singlet, doublet, triplet, double−doublet, multiplet, and broad,
respectively. Mass spectra (LC-MS) were measured on a Ion Trap
Esquire 3000+ instrument. Chemical names follow IUPAC nomen-
clature. Starting materials were used without purification. Column
chromatography was performed using silica gel (40−63 μm), and the
reaction progress was determined by thin layer chromatography
analyses on Merck silica gel plastic plates 60F254. The purity of tested
compounds was established by LC/MS and combustion analysis to be
>95%.
7-Methyl-pyrazolo[1,5-a]pyrimidine-5-carboxylic Acid
Methyl Ester (3). To a solution of 1H-pyrazol-3-amine (2.50 g,
30.1 mmol) in MeOH (90 mL) was added methyl acetopyruvate
(4.42 g, 30.7 mmol). The mixture was heated to reflux for 5 h and then
cooled to room temperature overnight. The precipitated yellow
needles were collected by filtration and recrystallized from MeOH to
afford the major isomer 3 (3.80 g, 66%) as off-white needles (TLC
(cyclohexane/ethyl acetate =2:1): Rf = 0.21 for 3 in comparison to
1
Rf = 0.13 for the regioisomer). H NMR (DMSO-d6): δ 8.40 (d, J =
2.4 Hz, 1H, CH), 7.56 (d, J = 0.9 Hz, 1H, CH), 6.99 (d, J = 2.4 Hz,
1H, CH), 3.93 (s, 3H, CH3), 3.32 (s, 3H, CH3). C9H9N3O2; MW:
191; MS 192 [M + H]+.
Pyrazolo[1,5-a]pyrimidine-5,7-dicarboxylic Acid 5-Methyl
Ester (4). To a suspension of SeO2 (44.4 g, 400 mmol) in dry 1,4-
dioxane (200 mL) was added tert-butyl hydroperoxide (200 mL of a
5−6 M solution in decane), and this solution was placed in a
preheated oil bath (50 °C) and vigorously stirred at this temperature
until a clear colorless solution was obtained (approximately 30 min).
Then 3 (38.2 g, 200 mmol) was added under vigorous stirring until
complete consumption (approximately 19 h). The clear yellow to
orange solution was cooled to room temperature and diluted with 1,4-
dioxane (800 mL) and water (120 mL), and oxone (123 g, 200 mmol)
was added successively. The resulting orange suspension was stirred at
room temperature until the intermediate aldehyde was consumed
(approximately 68 h). The yellow suspension was filtered through a
frit (P2), and the filter cake was washed with 1,4-dioxane (100 mL)
and air-dried. The remaining gluey yellow solid residue was washed
with CHCl3/methanol (95:5, 2 × 400 mL) and air-dried. The obtained
yellow powdery solid was transferred into an extraction thimble and
placed in a Soxhlet extractor (500 mL) and continuously extracted
with refluxing CHCl3 (1.6 L) until a beige solid remained in the
extraction thimble (approximately 48 h). The dark yellow CHCl3
extract was concentrated to dryness under reduced pressure, ground,
and suspended in methanol (200 mL), filtered through a frit (P3),
washed with methanol (100 mL), and dried under reduced pressure
In summary, compound 1 is the most potent MMP-13
selective inhibitor reported so far. Mechanistically, given the
compelling role of MMP-13 in cartilage degradation associated
with OA, compound 1 has the disease-modifying potential. It is
uniquely suited for IA delivery: It is able to penetrate and attain
high, sustained concentrations (≥2 μM for 8 weeks) in
cartilage. Correlating with its long durability in the joint, the
compound demonstrated sustained efficacy (100% inhibition
for 3 weeks). Despite outstanding efficacy, the compound
1
for 15 h to obtain the product (27.8 g, 63%) as a yellow powder. H
NMR (DMSO-d6): δ 8.44 (s, 1H, CH), 7.47 (s, 1H, CH), 6.98 (s, 1H,
CH), 3.93 (s, 3H, CH3). C9H7N3O4; MW: 221; MS 222 [M + H]+.
7-(5-tert-Butoxycarbonyl-4-methyl-indan-1-(S)-ylcarbamo-
yl)-pyrazolo[1,5-a]pyrimidine-5-carboxylic Acid Methyl Ester
(6). Acid 4 (43.7 g, 198 mmol) and 1-(S)-amino-4-methyl-indan-5-
carboxylic acid tert-butyl ester 5 (44.5 g, 180 mmol) and 1-hydroxy-7-
azabenzotriazole (26.8 g, 197 mmol) were dissolved in dry DMF
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(1 L), and then subsequently N-methylmorpholine (22 mL) and O-(7-
azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium-hexafluorophos-
phate (90 g, 237 mmol) were added at room temperature. After 1 h,
precipitation occurred and the black suspension was allowed to stand
overnight and was evaporated under reduced pressure. To the residue
was added 10% citric acid, and the precipitate was filtered, washed with
water, and absorbed on silica. Purification by flash chromatography
using cyclohexane/ethyl acetate 7/3 to 3/7 as eluent afforded the
product (70.0 g, 86%) as yellow solid. 1H NMR (DMSO-d6): δ 10.36
(d, J = 7.9 Hz, 1H, NH), 8.57 (d, J = 2.5 Hz, 1H, CH), 8.10 (s, 1H,
CH), 7.61 (d, J = 7.9 Hz, 1H, CH), 7.33 (d, J = 7.9 Hz, 1H, CH), 7.26
(d, J = 2.5 Hz, 1H, CH), 5.68 (q, 1H, CH), 4.01 (s, 3H, CH3), 3.15−
2.61 (m, 3H, CH2, CHH), 2.44 (s, 3H, CH3), 2.11−1.98 (m, 1H,
CHH), 1.58 (s, 9H, 3 CH3). C24H26N4O5; MW: 450; MS 451 [M +
H]+.
4-Methyl-1-(S)-({5-[(3-oxo-3,4-dihydro-2H-benzo[1,4]oxazin-
6-ylmethyl)-carbamoyl]pyrazolo[1,5-a]pyrimidine-7-
carbonyl}amino)indan-5-carboxylic Acid tert-Butyl Ester (12).
To a solution of 7 (25.4 g, 58.2 mmol), amine 11 (15.0 g, 70 mmol),
1-hydroxy-7-azabenzotriazole (8.7 g, 64 mmol), and N-methylmorpho-
line (13 mL) in dry DMF (1 L) was added N-ethyl-N′-(3-
dimethylaminopropyl)carbodiimide hydrochloride (22.3 g, 116
mmol) at room temperature. After 15 min, precipitation occurred.
The suspension was stirred over a weekend, diluted with DMF (1 L),
and heated to 40 °C, and then the solution was filtered over Celite and
evaporated under reduced pressure. The residue was slurried in 5%
aqueous citric acid (∼5 L), filtered, and washed with 5% aqueous citric
acid and then water. Drying at 40 °C under reduced pressure afforded
1
the product (32.7 g, 94%) as a yellow solid. H NMR (DMSO-d6): δ
10.72 (s, 1H, NH), 10.37 (d, J = 8.1 Hz, 1H, NH), 9.66 (t, J = 6.2 Hz,
1H, NH), 8.54 (d, J = 2.5 Hz, 1H, CH), 8.14 (s, 1H, CH), 7.61 (d, J =
7.7 Hz, 1H, CH), 7.33 (d, J = 7.9 Hz, 1H, CH), 7.13 (d, J = 2.4 Hz,
1H, CH), 6.98−6.91 (m, 3H, 3 CH), 5.69 (q, 1H, CH), 4.56 (s, 2H,
CH2), 4.45 (d, J = 6.3 Hz, 2H, CH2), 3.13−2.67 (m, 3H, CH2, CHH),
2.44 (s, 3H, CH3), 2.12−1.97 (m, 1H, CHH), 1.58 (s, 9H, 3 CH3).
C32H32N6O6; MW: 596; MS 619 [M + Na]+.
7-(5-tert-Butoxycarbonyl-4-methyl-indan-1-(S)-ylcarbamo-
yl)-pyrazolo[1,5-a]pyrimidine-5-carboxylic Acid (7). Compound
6 (69.5 g, 154 mmol) was dissolved in THF (1.2 L) at 40 °C and then
a solution of lithium hydroxide (4.8 g, 200 mmol) in water (200 mL)
was added. The yellow solution turned green and was stirred for 2 h at
room temperature. The solution was adjusted to pH 4 by addition of
1N HCl (180 mL), and then the THF was removed under reduced
pressure. To the remaining suspension was added additional 1N HCl
(30 mL), and then the suspension was cooled to ∼10 °C, filtered, and
extensively washed with water (∼1 L) and dried at 40 °C to afford the
4-Methyl-1-(S)-({5-[(3-oxo-3,4-dihydro-2H-benzo[1,4]oxazin-
6-ylmethyl)carbamoyl]pyrazolo[1,5-a]pyrimidine-7-carbonyl}-
amino)indan-5-carboxylic Acid (1). Compound 12 (72.9 g,
122 mmol) was added in portions to a warm (45 °C) solution of formic
acid (1.6 L) and stirred for 2 h, cooled to room temperature, then
filtered, washed with formic acid and then water, and finally dried on a
lyophilization apparatus for 70 h to afford the product (62.7 g, 95%) as
1
product (68.8 g, quant.) as a yellow solid. H NMR (DMSO-d6): δ
10.38 (d, J = 8.1 Hz, 1H, NH), 8.53 (d, J = 2.5 Hz, 1H, CH), 8.09 (s,
1H, CH), 7.61 (d, J = 7.9 Hz, 1H, CH), 7.33 (d, J = 7.9 Hz, 1H, CH),
7.20 (d, J = 2.7 Hz, 1H, CH), 5.68 (q, 1H, CH), 3.40 (bs, 1H, OH),
3.13−2.64 (m, 3H, CH2, CHH), 2.44 (s, 3H, CH3), 2.12−1.97 (m,
1H, CHH), 1.58 (s, 9H, 3 CH3). C23H24N4O5; MW: 436; MS 437 [M
+ H]+.
3-Oxo-3,4-dihydro-2H-benzo[1,4]oxazine-6-carbonitrile (9).
A suspension of 6-chloro-3-oxo-3,4-dihydro-2H-benzo[1,4]oxazin-3-
one (3.2 g, 17.4 mmol) and CuCN (2.9 g) in dry N-methyl-pyrrolidin-
2-one (15 mL) was placed in a preheated oil bath (∼250 °C) under
argon. After stirring at this temperature overnight, the mixture was
concentrated, diluted with water (200 mL), and extracted with EtOAc
(3 × 200 mL). The combined organic layers were washed with water
(2 × 200 mL) and brine (200 mL), dried over MgSO4, filtered, and
concentrated. The remaining residue crystallized from EtOAc/toluene
to afford the product (720 mg, 24%) as a tan solid. 1H NMR (DMSO-
d6): δ 10.99 (s, 1H, NH), 7.41 (dd, J = 8.4 Hz, J = 1.8 Hz, 1H, CH),
7.21 (d, J = 1.8 Hz, 1H, CH), 7.11 (d, J = 8.4 Hz, 1H, CH), 4.71 (s,
3H, CH3). C9H6N2O2; MW: 174; MS 175 [M + H]+.
1
a pale yellow solid. H NMR (DMSO-d6): δ 12.84 (bs, 1H, COOH),
10.72 (s, 1H, NH), 10.37 (d, J = 7.9 Hz, 1H, NH), 9.66 (t, J = 6.2 Hz,
1H, NH), 8.55 (d, J = 2.7 Hz, 1H, CH), 8.14 (s, 1H, CH), 7.73 (d, J =
7.9 Hz, 1H, CH), 7.33 (d, J = 7.7 Hz, 1H, CH), 7.13 (d, J = 2.5 Hz,
1H, CH), 6.98−6.91 (m, 3H, 3 CH), 5.68 (q, 1H, CH), 4.56 (s, 2H,
CH2), 4.45 (d, J = 6.3 Hz, 2H, CH2), 3.13−2.67 (m, 3H, CH2, CHH),
2.50 (s, 3H, CH3), 2.13−2.01 (m, 1H, CHH). 13C NMR (DMSO-d6):
δ 168.90, 164.85, 162.18, 158.37, 149.41, 147.91, 145.79, 145.62,
143.54, 142.15, 138.59, 134.93, 133.30, 139.48, 129.06, 127.00, 122.25,
120.96, 115.83, 115.03, 106.35, 98.49, 66.66, 54.96, 42.10, 32.37,
28.86, 16.80. C28H24N6O6; MW: 540; MS 541 [M + H]+.
Potency and Selectivity Assays. MMP-13 activity and the
selectivity assays were performed using the catalytic domains of
recombinant human MMPs (MMP-1, -2, -3, -7, -9, -12: Biomol,
Hamburg, Germany; MMP-8: Calbiochem, Schwalbach, Germany;
MMP-13, -14: Invitek, Berlin, Germany) and the appropriate
fluorogenic peptide substrates. MMP-13 activity was tested using the
specific MMP-13 substrate MCA-Pro-Cha-Gly-Nva-His-Ala-Dpa-NH2
(Calbiochem). MMP-3 was tested using the NFF-3 substrate MCA-
Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(DNP)-NH2 (Calbio-
chem), and all remaining MMPs were tested using OmniMMP
substrate MCA-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2·AcOH (Bio-
mol).28 TACE activity was measured using the recombinant human
TACE enzyme (R&D Systems, Wiesbaden, Germany) and the specific
peptide substrate MCA-Pro-Leu-Ala-Gln-Ala-Val-Dpa-Arg-Ser-Ser-
Ser-Arg-NH2 (Calbiochem).
The MMP-3 assay was performed in 50 mM MES buffer, pH 6.0, 10
mM CaCl2, and 0.05% Brij-35. The aggrecanase-1 assay and all other
MMP assays were performed in 50 mM Tris-HCl buffer, pH 7.5, 150
mM NaCl, 5 mM CaCl2, and 0.05% Brij-35. TACE assay was run in 25
mM Tris-HCl buffer, pH 9, 1.25 μM Zn(OAc)2, and 0.005% Brij-35.
Concentrations of enzymes and substrates were optimized for each
assay and varied between 1 and 10 nM enzyme and 4−10 μM
substrate. The enzyme activity was measured after 10 min
preincubation of the enzyme with varying concentrations of the
inhibitor. Each assay was run at least in duplicate, and the IC50 values
were calculated using the Life Science Workbench (LSW) Data
Analysis Plugin for Microsoft Excel. The data were fitted into the
formula y = Vmax/(1 + [I]/IC50).22
(3-Oxo-3,4-dihydro-2H-benzo[1,4]oxazin-6-ylmethyl)-
carbamic Acid tert-Butyl Ester (10). To an ice cooled solution of
nitrile 9 (700 mg, 4.02 mmol) in dry MeOH (20 mL) were added di-
tert-butyl dicarbonate (1.8 g, 8.25 mmol) and NiCl2·6H2O (40 mg),
followed by the careful portionwise addition of NaBH4 (900 mg, 23.8
mmol). The resulting black mixture was stirred for 20 min at 0−5 °C
and then the ice bath was removed and stirring at room temperature
was continued overnight. Diethylenetriamine (0.5 mL) was added, and
the mixture was concentrated to dryness. The remaining residue was
dissolved in EtOAc and washed subsequently with 10% aqueous citric
acid, saturated aqueous NaHCO3, and brine, dried over MgSO4,
filtered, concentrated, and purified by flash chromatography using
cyclohexane/EtOAc 3/2 to 1/1 as eluent to afford the product (459
1
mg, 41%) as white needles. H NMR (DMSO-d6): δ 10.70 (s, 1H,
NH), 7.33 (t, J = 6.2 Hz, 1H, NH), 6.89−6.75 (m, 3H, 3 CH), 4.52 (s,
2H, CH2), 4.01 (d, J = 6.3 Hz, 2H, CH2), 1.39 (s, 9H, 3 CH3).
C14H18N2O4; MW: 278; MS 301 [M + Na]+.
6-Aminomethyl-4H-benzo[1,4]oxazin-3-one Hydrochloride
(11). To compound 10 (450 mg, 1.62 mmol) was added a 4 M
solution of HCl in 1,4-dioxane (3 mL), and the reaction mixture was
stirred at room temperature for 3 h and then concentrated and dried
under high vacuum to afford the product (350 mg, quant.) as an off-
white solid. 1H NMR (DMSO-d6): δ 10.94 (s, 1H, NH), 8.46 (bs, 3H,
NH3), 7.09−6.95 (m, 3H, 3 CH), 4.57 (d, J = 4.2 Hz, 2H, CH2), 3.89
(s, 2H, CH2). C9H11ClN2O2; MW: 214; MS 162 [M − NH3Cl]+.
Aggrecanase-1 activity was determined using the InviLISA Kit
(Invitek). Human recombinant aggrecanase-1 (0.75 nM) was
incubated with different concentrations of inhibitor and the
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dx.doi.org/10.1021/jm201152u | J. Med. Chem. 2012, 55, 709−716

Journal of Medicinal Chemistry
Article
recombinant interglobular domain of human aggrecan (100 nM
aggrecan-IGD) as substrate for 15 min at 37 °C. After EDTA
termination, the generated aggrecan cleavage neoepitopes (ARGSVIL)
were detected and quantified by ELISA.22
In Vitro Cartilage Degradation Assay. Freshly isolated bovine
articular cartilage (∼3-mm2 pieces) were incubated with 5 ng/mL IL-
1α plus 50 ng/mL OSM in the presence or absence of compounds, for
11 days. Culture medium was refreshed twice weekly. Collagen
degradation products in the conditioned medium were measured using
the C1,2C enzyme-linked immunosorbent assay (Ibex, Montreal,
Quebec, Canada).22
gradient elution. Compound 1 was detected by positive electrospray
ionization in the multiple reaction monitoring (MRM) mode.
The compound concentration in cartilage (ng/mg) was converted
to compound per volume of cartilage (ng/mL) on the basis of the
assumption that the density of the cartilage was the same as that of
water, 1 g/mL. The cartilage compound concentration was converted
to a molar concentration (μM) for comparison across sample types.
On day 56, the animals from control and drug treated groups were
observed for behavioral signs of potential musculoskeletal side effects
using a procedure reported previously.8 Two observers individually
scored the animals for three categories of resting posture, gait, and
willingness to move when stimulated, and the scores in each category
were averaged from both observers. The averages were then added
together to obtain a total score ranging from 0 to 7.
Ex Vivo Model of Rat Articular Cartilage Degradation.29 Male
Sprague−Dawley (Harlan, Indianapolis, Indiana) rats weighing 175−
200 g, with three animals/group, were used in this study. Rats in the
vehicle group received a single IA injection of 50 μL phosphate
buffered saline (PBS) into their right knee joint. Rats in the inhibitor
groups received a single IA injection (50 μL) of 10 mg/mL of
compound 1 suspended in PBS into their right knee joint. The
compound suspension was sonicated in a water bath for 30−60 min
and vortexed immediately before IA injection into their right knee
joint. The animals were sacrified at 1, 7, 14, and 21 days after the IA
injection via CO2 asphyxiation, and the cartilage from the tibia of the
right joints was collected. Two vehicle groups (Veh 0% and Veh
100%) were sacrificed at each time point.
The susceptibility of cartilage to degradation was assessed by
incubating cartilage sample from each animal with 10 μg/mL solution
of activated MMP-13 (activated with 1 mM 4-aminophenylmercuric
acetate) for 24 h at 37 °C in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl,
5 mM CaCl2 buffer. Samples from the vehicle group were incubated
for the same duration but with or without MMP-13 to represent 100%
(Veh 100%) and 0% inhibition (Veh 0%), respectively. Incubation was
stopped by adding the chelating agent, ethylenediaminetetraacetic acid
to a final concentration of 10 mM. The supernatants were collected
and stored at −40 °C for further analysis. Cartilage degradation was
assessed by measuring the C1,2C release into the supernatant, using an
ELISA (Ibex). The average C1,2C values for the vehicle groups (Veh
100% and Veh 0%) were used to calculate the % inhibition value for
each sample:
AUTHOR INFORMATION
Corresponding Author
■
*Present address: Prasan Pharmaceutical Consultants, 5311
Betheny Circle, Ypsilanti, MI 48198, USA. E-mail:
vijaybaragi05@yahoo.com.
Present Addresses
§Contact for chemistry. E-mail: christian.gege@web.de. Present
address: Phenex Pharmaceuticals AG, Wieblinger Straße 104,
D-69123 Heidelberg, Germany.
∥Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA
91320, USA.
⊥Biogen Idec Inc., 14 Cambridge Center, Cambridge, MA
02142, USA.
ACKNOWLEDGMENTS
The authors acknowledge Gabriele Becher, Maren Burger,
Heike Kheil, Dorothea Piecha, Andreas Timmermann, and
■
Jurgen Weik from Alantos Pharmaceuticals AG for the
̈
determination of the IC50 values, Andreas Jaworski and Maic
Seegel for analytical assistance, and Ralf Biesinger and Rory
Dodd for resynthesis of some intermediates. We thank Sarah
Lively and Roy Black from Amgen Inc. for helpful discussions.
We also thank immensely Keith Dionne from Alantos
Pharmaceuticals, Inc. for his encouragement, support, and
guidance for carrying out this project.
[1 − (C1, 2C
− C1, 2C
)
sample
veh100%,ave
/(C1, 2C
− C1, 2C
)] × 100%
veh0%,ave
Veh100%,ave
Safety, Tolerability, and Pharmacokinetics Following Intra-
articular Administration. The study consisted of 24 male Sprague−
Dawley rats (Harlan, Indianapolis, Indiana) weighing 175−200 g, with
three male animals for each time point. The right knee joint of each rat
was injected once with 50 μL/joint of PBS containing 0.5 mg of
compound 1 or 50 μL/joint of PBS alone (control rats). Animals were
sacrificed on days 1, 7, 14, 21, 28, 42, and 56 days after the IA
injection. Plasma from each animal as well as cartilage and synovial
fluid lavage from both the injected and contra-lateral joints were
collected. Samples were analyzed for compound concentration by LC/
MS/MS. On day 56, animals from PBS and compound 1 were scored
for behavioral signs indicative of potential musculoskeletal side effect
reported for MMP inhibitors.
The cartilage from the tibial articular surface was shaved, weighed,
and digested overnight at 65 °C in 100 μL digestion buffer, containing
6.9 mg/mL NaH2PO4, 74.4 mg/mL EDTA, 0.35 mg/mL cysteine, and
10 U/mL papain (Sigma), pH 6.5. Synovial fluid samples were
collected by lavaging the knee joints with 100 μL of saline. Blood
(400−500 μL) was collected from all study animals at sacrifice into
tubes containing dipotassium ethylenediaminetetraacetic acid and
processed into plasma.
ABBREVIATIONS
■
Boc, tert-butyloxycarbonyl; DMF, N-dimethylformamide;
DMOAD, disease-modifying osteoarthritis drug; EDCI, 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride;
EDTA, ethylenediaminetetraacetic acid; ELISA, enzyme-linked
immunosorbent assay; HATU, O-(7-azabenzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium-hexafluorophosphate; HOAt, 1-
hydroxy-7-azabenzotriazole; IA, intra-articular; IL-1α, interleu-
kin-1α; MMP, matrix metalloproteinase; MSS, musculoskeletal
syndrome; NMM, N-methylmorpholine; NMP, N-methylpyr-
rolidin-2-one; OA, osteoarthritis; OSM, oncostatin-M; TACE,
tumor necrosis factor-α converting enzyme; TBHP, tert-butyl
hydroperoxide; THF, tetrahydrofuran
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