Discovery of 3,3-dimethyl-5-hydroxypipecolic hydroxamate-based inhibitors of aggrecanase and MMP-13

Article abstract of DOI:10.1016/j.bmcl.2005.03.105

A series of pipecolic hydroxamate inhibitors of MMP-13 and aggrecanase was discovered based on screening known inhibitors of TNF-α converting enzyme (TACE). Potency versus aggrecanase was optimized by modification of the benzyloxyarylsulfonamide group. In

Full text of DOI:10.1016/j.bmcl.2005.03.105

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2808–2811
Discovery of 3,3-dimethyl-5-hydroxypipecolic
hydroxamate-based inhibitors of aggrecanase and MMP-13
Mark C. Noe,* Vijayalakshmi Natarajan, Sheri L. Snow, Peter G. Mitchell,
Lori Lopresti-Morrow, Lisa M. Reeves, Sue A. Yocum, Thomas J. Carty,
John A. Barberia, Francis J. Sweeney, Jennifer L. Liras, Marcie Vaughn, Joel R. Hardink,
Joel M. Hawkins and Christopher Tokar
Pfizer Global Research and Development Groton Laboratories, Eastern Point Road, Groton, CT 06340, USA
Received 31 January 2005; revised 7 March 2005; accepted 25 March 2005
Available online 28 April 2005
Abstract—A series of pipecolic hydroxamate inhibitors of MMP-13 and aggrecanase was discovered based on screening known
inhibitors of TNF-a converting enzyme (TACE). Potency versus aggrecanase was optimized by modification of the benzyloxyaryl-
sulfonamide group. Incorporation of geminal alkyl substitution at the 3-position of the piperidine ring improved metabolic stability,
presumably by increasing steric hindrance around the metabolically labile hydroxamic acid. This modification also resulted in dra-
matic improvement of aggrecanase activity with a slight reduction in selectivity versus MMP-1. Synthesis, structure activity relation-
ships, and strategies to reduce metabolic clearance are described.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Osteoarthritis is a disease of progressive cartilage degra-
dation that leads to pain and reduced mobility in
affected joints. Approximately 21 million patients are
afflicted with some form of the disease in the United
States, and current treatments are limited to symptom-
atic relief with NSAIDs or COXIBs (selective COX-2
inhibitors), intra-articular injections of hyaluronic acid
conjugates (Synvisc) or surgical joint replacement.1,2
The efficacy of nutritional supplements (glucosamine
and chondroitin sulfate) is somewhat controversial,
and there are currently no treatments available that re-
tard or reverse the gradual destruction of cartilage asso-
ciated with the disease.3–5 The pathogenesis of
osteoarthritis can be attributed to multiple factors that
affect the degeneration of proteoglycan and disruption
of the type II collagen network in cartilage.6 These crit-
ical components of cartilage maintain its structural
integrity and impart mechanical properties associated
with normal joints. Aggrecan monomers interact with
hyaluronic acid and link proteins to form high molecu-
lar weight networks with ordered hydration that pro-
vides cartilage with resilience and compressibility.
Type II collagen is a triple-helical protein that forms a
highly organized three-dimensional structure responsi-
ble for tissue stiffness and integrity. Both aggrecan and
type II collagen can be degraded by proteolytic enzymes
that have elevated expression in osteoarthritic joints.
Aggrecanase refers to a collection of proteases that
cleave aggrecan and belong to the ADAM-TS (A Disin-
tegrin and Metalloprotease possessing Thrombospondin
domains) family of zinc containing metalloproteases.7
On the other hand, collagenases are the principle en-
zymes that cleave type II collagen and are members of
the MMP (matrix metalloprotease) family.8 MMP-13
is particularly efficient at cleaving type II collagen; its
expression is regulated by pro-inflammatory mediators
and elevated in osteoarthritic joints.9 We sought to dis-
cover compounds that possessed inhibitory activity ver-
sus both MMP-13 and aggrecanase and could therefore
serve as therapeutically useful agents for the treatment
of this disease.
Unfortunately, the vast majority of broad-spectrum
MMP inhibitors that have advanced to clinical trials ex-
hibit musculoskeletal side effects characterized by joint
stiffness and pain, particularly in the hands and shoul-
ders.8,10 In rodents, joint fibroplasia and accumulation
of type I collagen in the affected joints accompanies
the observed side effect.11 Others have speculated that
sheddase inhibition (specifically inhibition of TACE)
*
Corresponding author. Tel.: +1 860 441 8825; fax: +1 860 715
2467; e-mail: mark_c_noe@pfizer.com
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.03.105

M. C. Noe et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2808–2811
2809
could be responsible for the observed side effects, possi-
bly through the failure to downregulate receptor signal-
ing by proteolytic release of the extracellular domain.12
We therefore desired to spare both MMP-1 inhibition
and TACE inhibition (as measured by suppression of
TNF-a release in human blood stimulated with LPS).
improved diastereoselectivity.15,16 We were gratified to
find that treatment of 5 with concentrated aqueous
HCl at reflux afforded the desired six-membered lactone
6 in 97% yield. Sulfonylation with p-toluenesulfonyl
chloride in pyridine was followed by spontaneous cycliz-
ation to form the bicylic intermediate 7. Removal of the
a-methylbenzyl group by hydrogenolysis, followed by
sulfonylation with the appropriate arylsulfonyl chloride
and triethylamine afforded the bicyclic sulfonamide 9 in
good yield. For the preparation of analogs, the unsubsti-
tuted benzyloxysulfonamide 9 (R = Ph) was treated with
hydrogen in the presence of Pd–C and methanol, afford-
ing the intermediate phenol, which was then alkylated
with the appropriate benzyl halide in the presence of ce-
sium carbonate and acetonitrile to afford the desired
derivative 9 in good yield. Opening of the lactone under
WeinrebÕs conditions using O-allylhydroxylamine
hydrochloride, followed by removal of the allyl group
afforded the desired compounds in moderate yield.17
Substituted 4-benzyloxy aryl sulfonamides were pre-
pared to probe structure activity relationships about this
group, which is thought to bind in the S10 pocket by
analogy with the previously discovered pipecolic
hydroxamate TACE inhibitors. The biological data ob-
tained for each of these compounds are shown in Table
1.18,19
Previous MMP and TNF-a converting enzyme (TACE)
programs at Pfizer led to the discovery of pipecolic acid
based inhibitors such as 1.13 Screening these com-
pounds in a cell based aggrecanase assay revealed sev-
eral analogs with modest activity. Only compounds
possessing an ortho-methyl or halo group exhibited
potent inhibitory activity (<500 nM), with 2 providing
the most potent inhibition of both MMP-13 and aggrecan-
ase activities. Compounds from this series generally
exhibit poor pharmacokinetic properties due to high
metabolic clearance (Rat PK data for 2: Clp:
86 mL minꢀ1 kgꢀ1; T1/2: 1.7 h; bioavailability: 12%).
Therefore, we incorporated additional substituents at
the piperidine 3-position with the intent of improving
the metabolic stability of the hydroxamate group by
increasing steric hindrance, thereby limiting access to
metabolic enzymes (Fig. 1).
The synthesis of the 3,3-dimethyl-5-hydroxy pipecolic
hydroxamates is shown in Scheme 1. The first chiral cen-
ter is established through a highly diastereoselective
Strecker reaction of 2,2-dimethyl-4-pentenal with potas-
sium cyanide and (R)-a-methylbenzylamine-hydrochlo-
ride.14 The resulting a-aminonitrile can be purified to
homogeneity by recrystallization from aqueous metha-
nol. Attempted hydrolysis of the nitrile by treatment
with concentrated acid or base in refluxing aqueous
methanol was unsuccessful, presumably due to extreme
steric hindrance imparted by the geminal dimethyl and
bulky amine substituents. Reasoning that derivatization
of the nitrile might be more easily effected by intramo-
lecular lactonization with a hydroxy substituent, we
decided to functionalize the olefin first. Reaction of 4
with catalytic OsO4 and NMO in aqueous acetone afford-
ed the diol product as a 1:1 mixture of diastereomers.
Asymmetric dihydroxylation using the DHQD-PHN
ligand under standard conditions afforded 5 with
The 3,3-dimethyl-5-hydroxypipecolic hydroxamates gen-
erally had improved aggrecanase activity and reduced
selectivity for MMP-13 over MMP-1 compared to the
corresponding 5-hydroxy pipecolic hydroxamates (com-
pare 21 with 2, other data not shown). As observed for
the 5-hydroxypipecolic hydroxamates, incorporation of
an ortho-substituted benzyl ether on the sulfonamide
moiety results in potent aggrecanase inhibition. The re-
duced potency observed for the isopropyl substituted
analog 18 relative to the ortho-methyl (11), bromo (14),
or ethyl (17) analogs suggests a size limitation for this
substituent. MMP-13 inhibition is also sensitive to the
presence of a large ortho-substituent, and a 10-fold
reduction in MMP-13 activity is observed for 18 relative
to 11. Interestingly, the opposite effect is observed for the
inhibition of TNF-a release in LPS-stimulated human
whole blood. This observation is consistent with the pre-
viously noted influence of ortho-alkyl substitution on
TACE potency, the pipecolic hydroxamate series 1.13
The presence of a second halogen substituent in the
meta- or para-positions results in enhanced potency for
ortho-alkyl or halo substituted analogs. The addition of
a para-substituent erodes selectivity for MMP-1 (e.g.,
compounds 13, 16, and 20). The 2,4-disubstituted com-
pounds provide the best potency, with the 2-chloro-4-flu-
oro analog 22 displaying nanomolar inhibitory activity
and >100· selectivity for MMP-13 and aggrecanase over
MMP-1.
Several substituted heterocyclic analogs were prepared
with the intent of reducing lipophilicity while retaining
good MMP-13 and aggrecanase activity. Biological data
for these analogs are shown in Table 2. Aggrecanase po-
tency is strongly influenced by the position of the hetero-
atoms on the terminal heterocycle, despite the presence
of ortho-substitution in each case tested. The same is
Figure 1.

2810
M. C. Noe et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2808–2811
Scheme 1. Synthesis of 3,3-dimethyl-5-hydroxypipecolic-3-hydroxamic acids from 2,2-dimethyl-4-pentenal.
Table 1. Aggrecanase and MMP-13 inhibitory activity of benzyl ether analogs of 10
Compds
R
Aggrecanase
IC50 (nM)a
MMP-13
IC50 (nM)
MMP-1
IC50 (nM)
rTACE
IC50 (nM)
TNF HWB
IC50 (nM)b
11
12
13
14
15
16
17
18
19
20
21
22
23
2-Methylphenyl
3-Methylphenyl
38
>100
19%
16
14
2.8
1.2
8
690
773
198
715
ND
100
1500
2300
679
255
790
592
430
7.3
12
4900
25,850
19,150
8900
4-Methylphenyl
2-Bromophenyl
11
11
3-Bromophenyl
4-Bromophenyl
2-Ethylphenyl
57%
>100
67
ND
1.4
36
15
10
18,950
7700
1100
6.7
12
2-Isopropylphenyl
2-Methyl-3-fluorophenyl
2-Methyl-4-fluorophenyl
2-Methyl-5-fluorophenyl
2-Chloro-4-fluorophenyl
2,4-Dichlorophenyl
78%
3.9
2.7
188
4.3
3.5
6.5
4.1
3.0
1800
2875
17
5.2
7.3
ND
19
5483
3450
40
0.5
2.1
10,200
31,000
aValues expressed as a percentage represent percent inhibition of enzyme activity at 500 nM.
b HWB = human whole blood.
Table 2. Aggrecanase and MMP-13 inhibitory activity of heteroarylmethyl ether analogs of 10
Compds
R
Aggrecanase
IC50 (nM)a
MMP-13
IC50 (nM)
MMP-1
IC50 (nM)
rTACE
IC50 (nM)
TNF HWB
IC50 (nM)b
24
25
26
27
28
29
3-Methylisothiazol-4-yl
4-Methylthiazol-5-yl
4-Isoquinolinyl
76%
55%
>100
15
ND
ND
53
ND
12
16
5200
4950
2425
950
290
170
10
4-Quinolinyl
2-Chloro-4-pyridinyl
2-Methyl-3-pyridinyl
7.8
19
ND
ND
6.7
18
91
1100
ND
10,150
2000
ND
aValues expressed as a percentage represent percent inhibition of enzyme activity at 500 nM.
b HWB = Human whole blood.
observed for MMP-1 and TNF-a inhibition, with the
4-quinolinyl derivative 27 showing the greatest potency
against all of the endpoints.
(Clb = 44 mL minꢀ1 kgꢀ1 and 34 mL minꢀ1 kgꢀ1, respec-
tively).21 The markedly lower metabolic clearance with
this compound compared to the original lead 2, is attrib-
uted to increased steric hindrance proximal to the meta-
bolically labile hydroxamic acid as well as the
replacement of the ortho-methyl substituent on the ter-
minal aromatic ring with a chlorine. Oral bioavailability
in rat was 50% when dosed as a suspension in 0.5%
aqueous methylcellulose.
We chose compound 23 for further profiling based on its
excellent aggrecanase and MMP-13 potency, selectivity
versus MMP-1, and poor potency against TNF-a release
in LPS treated whole blood. Compound 23 showed
moderate clearance in rat20 (Clp = 44 mL minꢀ1 kgꢀ1
,
T1/2 = 2 h) and dog (Clp = 25 mL minꢀ1 kgꢀ1, T1/2
=
2 h) that was consistent with predicted extraction ratios
based on incubations with rat and dog hepatocytes
We have discovered a novel series of MMP inhibitors
structured around a 3,3-dimethyl-5-hydroxy pipecolic

M. C. Noe et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2808–2811
2811
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hydroxamate template. The aggrecanase structure activ-
ity relationships that have emerged from this study are
reminiscent of those seen for TACE inhibition with
the earlier less-substituted pipecolate hydroxamate ser-
ies 1 (requirement of an ortho-substituted benzyl ether
for optimum cellular activity). Many of these com-
pounds possess potent inhibitory activity versus aggre-
canase and MMP-13 with selectivity versus MMP-1
and TACE (as measured by TNF-a release in LPS-stimu-
lated human blood) and could provide a useful starting
point for the discovery of agents for the treatment of
osteoarthritis.
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1
MS analysis.
References and notes
19. TACE, MMP, and whole blood TNF-a release assays
were conducted as described in Ref. 13. Aggrecanase
activity was assessed using a cell based assay: Porcine
chondrocytes were plated in 48 well tissue culture plates.
Glycosaminoglycan (GAG) chains were labeled by includ-
ing 35S sulfate (5 mCi/mL) in the culture medium. Unin-
corporated label was washed out and aggrecan
degradation stimulated by the addition of IL-1a (5 ng/
mL). Ten hours later, aggrecan degradation was quanti-
fied by scintillation counting of 35S content of both the
conditioned medium and cell layers.
20. Male Sprague–Dawley rats were obtained with jugular
vein catheters from Charles River Labs, Wilmington, MA.
I.V. bolus doses were administered in glycerol formal
(5 mg/kg). Blood samples (600 lL) were collected from the
jugular vein (t = 5, 15, 30 min, 1, 2, 4, 6, 8, and 24 h).
Plasma was diluted with an equal volume of water and
extracted with MTBE. Drug levels were determined by
LC–MS/MS analysis. Pharmacokinetic parameters were
calculated using the non-compartmental method in Win-
Nonlin v2.1 (Pharsight, Mountain View, CA).
21. In vitro extraction ratios were calculated from predicted
blood clearances (CLb) according to the following equa-
tion ER = Clb/Q where 70 and 35 mL/min/kg were
assumed as hepatic blood flow (Q) for rat and dog,
respectively. Predicted clearances were obtained from the
disappearance of drug in cryopreserved rat and dog
hepatocytes according to the half-life method of Shibata,
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