Design and synthesis of a series of (2R)-N4-hydroxy-2-(3-hydroxybenzyl)-N1- [(1S,2R)-2-hydroxy-2,3-dihydro-1H-inden-1-yl]butanediamide derivatives as potent, selective, and orally bioavailable aggrecanase inhibitors

Article abstract of DOI:10.1021/jm015533c

A pharmacophore model of the P1′ site, specific for aggrecanase, was defined using the specificity studies of the matrix metalloproteinases and the similar biological activity of aggrecanase and MMP-8. Incorporation of the side chain of a tyrosine residue

Full text of DOI:10.1021/jm015533c

J . Med. Chem. 2001, 44, 3347-3350
3347
progression of joint destruction,5 and to delineate its role
in normal and disease states.
Design a n d Syn th esis of a Ser ies of
(2R)-N4-Hyd r oxy-2-(3-h yd r oxyben zyl)-N1-
[(1S,2R)-2-h yd r oxy-2,3-d ih yd r o-1H-in d en -
1-yl]bu ta n ed ia m id e Der iva tives a s
P oten t, Selective, a n d Or a lly Bioa va ila ble
Aggr eca n a se In h ibitor s†
There are several known classes of MMP inhibitors,
distinguished by their zinc-chelating groups such as
hydroxamates, thiols, and N-carboxyalkyls, which have
been extensively reviewed.6,7 Targeted screening of
DuPont’s compound collection for inhibitors of aggre-
canase provided a potent lead 1.8 Although this com-
pound has a Ki of 368 nM against aggrecanase, it is also
potent against other MMPs such as MMP-1, -2, and -9.
(Table 1). In this communication, we describe the
conversion of a broad spectrum MMP inhibitor 1 into a
potent, selective, and orally bioavailable inhibitor of
aggrecanase by a structure-based approach using an
enzyme homology model and by the similarity in
biological activity of aggrecanase and MMP-8.
Resu lts a n d Discu ssion . Our initial screen was
conducted using a partially purified preparation from
IL-1 stimulated bovine nasal cartilage. Thus, we began
the medicinal chemistry effort without having amino
acid sequence information for the enzyme. To assist us
in the rational design of specific aggrecanase inhibitors,
we examined the general patterns for substrate prefer-
ence among the MMPs. We and others9 observed that
human neutrophil collagenase (MMP-8) is able to clip
aggrecan at the specific aggrecanase cleavage site, albeit
with relatively poor efficiency. This result suggested
that MMP-8 and aggrecanase might share some com-
mon residues involved in substrate binding.
Nagase and Fields10 have extensively reviewed speci-
ficity studies of the matrix metalloproteinases using a
collagen sequence-based synthetic six-mer peptide span-
ning the scissle bond. Incorporation of a tyrosine residue
at the P1′ position of the peptide substrate dramatically
increased cleavage efficiency for MMP-8, while either
decreasing or maintaining affinity for MMP-1, -2, and
-9.11 This suggested to us that a P1′ tyrosine residue of
the peptide substrate provided good recognition for the
S1′ site of MMP-8 but is less favorable in the S1′ sites
of MMP-1, -2, and -9.
On the basis of the above specificity studies and on
our hypothesis that aggrecanase and MMP-8 share a
topologically similar active site, we envisioned that in-
corporation of a tyrosine side chain into the broad-spec-
trum MMP inhibitor 1 at the P1′ (i.e. 2) position might
provide some selectivity for aggrecanase over MMP-8.
Compound 2 was prepared and found to have IC50’s
of 332 nM and 0.45 nM against aggrecanase12 and
MMP-8, respectively, very similar to those of 1. This
result supports our hypothesis that the S1′ pockets are
similar, and that the residues comprising the S1′ pocket
of aggrecanase and MMP-8 could accept this inhibitor
without requiring energetically demanding movement
of the enzyme. This result also suggests that the 4-hy-
droxylbenzyl group (P1′) of 2 might be involved in a
specific hydrogen-bond interaction within the S1′ pocket
of aggrecanase and MMP-8. X-ray structural studies of
MMP-8 revealed that its S1′ pocket projects several
carbonyl groups such as that of Tyr 237, which could
serve as a hydrogen bond acceptor.13 As expected, 2 was
significantly less potent than 1 against MMP-1, -2, and
Wenqing Yao,*,‡ Zelda R. Wasserman,‡
Michael Chao,‡ Gade Reddy,‡ Eric Shi,‡ Rui-Qin Liu,
Maryanne B. Covington, Elizabeth C. Arner,
Michael A. Pratta, Micky Tortorella,
Ronald L. Magolda, Robert Newton,
MingXin Qian,§ Maria D. Ribadeneira,§
David Christ,§ Ruth R. Wexler,‡ and Carl P. Decicco‡
The DuPont Pharmaceuticals Company, Chemical and
Physical Sciences, Inflammatory Diseases Research,
Drug Metabolism and Pharmacokinetics Division,
Experimental Station, Wilmington, Delaware 19880-0500
Received May 9, 2001
Abstr a ct: A pharmacophore model of the P1′ site, specific
for aggrecanase, was defined using the specificity studies of
the matrix metalloproteinases and the similar biological
activity of aggrecanase and MMP-8. Incorporation of the side
chain of a tyrosine residue into compound 1 as the P1′ group
provided modest selectivity for aggrecanase over MMP-1, -2,
and -9. A cis-(1S)(2R)-amino-2-indanol scaffold was incorpo-
rated as a tyrosine mimic (P2′) to conformationally constrain
2. Further optimization resulted in compound 11, a potent,
selective, and orally bioavailable inhibitor of aggrecanase.
Degenerative joint diseases are commonly character-
ized by destruction of the cartilage extracelluar matrix
where loss of aggrecan from cartilage is believed to be
the early event that leads to breakdown of extracellular
matrix macromolecules.1 The loss of aggrecan can be
attributed to proteolytic cleavage of the core protein
within the interglobular domain (IGD). Two significant
cleavage sites have been identified within the IGD: one
between residue Asn341 and Phe342 and the other
between Glu373 and Ala374. Many matrix metallopro-
teinases (MMP-1, 2, 3, 7, 8, 9, and 13) have been shown
to cleave aggrecan in vitro at the Asn341-Phe342 site.2
G-1 fragments resulting from this cleavage site have
been identified within articular cartilage bound to
hyaluronic acid.2b Recently, aggrecanase, an ADAM-TS
of the reprolysin family, was isolated, cloned, and
expressed by Arner and co-workers.3 This enzyme
effectively cleaves at the Glu373-Ala374 bond of the IGD.
Aggrecan fragments resulting from this cleavage have
been identified in the synovial fluid of patients with
osteoarthritis, inflammatory joint disease, and joint
injury, suggesting that aggrecanase plays a pivotal role
in the catabolism of aggrecan in human arthritic
disease.4 There has been substantial interest in devel-
oping a selective aggrecanase inhibitor to prevent the
† Dedicated to Professor Ralph F. Hirschmann on the occasion of
his 79th birthday.
* E-mail: Wenqing.yao@dupontpharma.com. Phone: (302) 695-
4401. Fax: (302) 695-1173.
‡ Chemical and Physical Sciences.
Inflammatory Diseases Research.
§ Drug Metabolism and Pharmacokinetics Division.
10.1021/jm015533c CCC: $20.00 © 2001 American Chemical Society
Published on Web 09/19/2001

3348 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21
Letters
Ta ble 1. In Vitro Binding Data of Inhibitors 1-11 against Aggrecanase, MMP-8, MMP-1, MMP-2, and MMP-9
Ki (nM)a,b
IC50 (nM)a
aggrecanase
compd
R
MMP-8
MMP-1
MMP-2
MMP-9
1
2
3
4
5
6
7
8
9
368
332
182
0.70
0.45
70
37
303
15300
9.2
8600
1192
40000
171
<1
<1
<1
10.7
4.3
3.2
4-OH-Bn
i-butyl
946
82
408
73
166
162
1700
13333
1309
16.4
64
3139
790
12
1087
75000
1163
30953
95500
50000
33160
1533
31600
52
1507
6600
35000
6300
4200
21200
203
3324
15500
22000
4468
4-OMe-Bn
3-OH-Bn
3-OMe-Bn
10
11
1,3-benzodioxol-5-ylmethyl
a
b
Values are (SD of three determinations unless otherwise noted. The IC50 values on aggrecanase and Ki values on MMPs were
determined as previously described.18 All of the hydroxamic acids studies here were assumed to act as competitive inhibitors of the enzyme,
binding to the active site Zn atom as previously demonstrated by crystallographic and NMR studies of MMP complexed with related
hydroxamic acids.19 On the basis of the assumption of competitive inhibition, the IC50 values were converted to Ki values from the equation,
Ki ) IC50/(1 + [S]/Km), where IC50 is the concentration of competing compound producing 50% inhibition of the enzymatic activity at the
substrate concentration [S]. Km is the substrate concentration that provides a reaction velocity that is half the maximal velocity obtainable
under saturating substrate conditions.
Ch a r t 1
was also demonstrated by testing compound 6 against
aggrecanase, which was found to show only modest
activity (IC50 ) 1309 nM, Table 1). Molecular modeling
studies show that this hydroxyl group mimics the
carbonyl oxygen of the tyrosine residue of 2.
The 4-methoxybenzyl derivative 7 maintains similar
potency for aggrecanase and MMP-8, and has IC50s of
1163 nM, 52 nM, and 203 nM against MMP-1, -2, and
-9, respectively. The 10-fold potency increases for 7 over
3 against aggrecanase can be rationalized by two expla-
nations. The methoxy group of 7 might act as a hydro-
gen bond acceptor. However, the lower potency of 3
could also suggest that there may be a binding energy
liability, perhaps due to the desolvation of the hydro-
philic hydroxyl group in a highly hydrophobic S1′
pocket.
The SAR investigation around positional isomers of
the phenol hydroxyl group reveals that the 3-hydroxyl-
benzyl analogue 8 is significantly more potent than 3
against aggrecanase (Table 1). A different and possibly
unique hydrogen-bonding interaction between the hy-
droxyl phenol of 8 and residues in the S1′ pocket of
aggrecanase was proposed, different from that between
the 4-hydroxylbenzyl group of 2 and residues of the S1′
pocket of MMP-8 and aggrecanase. Additionally, com-
pound 8 is more than 2 orders of magnitude selective
for aggrecanase over other MMPs (Table 1) and shows
no inhibitory activity against MMP-8. This observation
suggests that although the active sites of MMP-8 and
aggrecanase are quite similar as described earlier, there
exist exploitable differences.
-9, (Table 1). The tight SAR is consistent with Fields
and co-worker’s results and suggests that the S1′ sites
of MMP-1, -2, and -9 are less tolerant of polar functional
groups such as the 4-hydroxylbenzyl of 2.
It is well-known that restricting the flexibility of a
molecule can enhance the potency and selectivity of an
inhibitor. To enhance potency against our target en-
zyme, we next studied conformational restraint on com-
pound 2. cis-1-Amino-2-indanol has been reported as a
phenylglycine mimetic and has been successfully em-
ployed in the development of Crixivan, an HIV protease
inhibitor.14 We replaced the 4-methoxy-tyrosine moiety
of compound 2 with cis-(1S)(2R)-1-amino-2-indanol,
which led to compound 3 (Chart 1). Compound 3 was
found to be about 2-fold more potent than 2 in the
aggrecanase inhibitory assay with an IC50 of 182 nM.
More importantly, the structural change significantly
reduced the potency against MMP-1, -2, -9 and MMP-8
(Table 1).
After much of the work described here was completed,
the aggrecanase protein sequence became available to
us. Noting the active site sequence similarity with those
of atrolysin C15 and adamalysin II,16 we constructed a
homology model of aggrecanase. Docking compound 8
into the active site of the model17 revealed a unique
threonine residue in the aggrecanase S1′ pocket at a
The stereochemistry of cis-(1S)(2R)-1-amino-2-indanol
moiety is important for aggrecanase activity. Compound
5, a distereomer of cis-(1S)(2R)-1-amino-2-indanol ana-
logue 4, was found to be inactive in the aggrecanase
assay. The importance of the indanol hydroxyl group

Letters
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21 3349
Sch em e 1. Synthesis of Compound 8a
a
Reagents: (a) Bop, DMF, Hunig base (85%); (b) 2-methoxy-
propene, PPTS (cat), CH2Cl2 (91%); (c) nBu-Li, THF, -78 °C,
BrCH2CO2tBu (61%); (d) TFA, CH2Cl2, H2O (96%); (e) BnONH2,
TBTU, DMF; (f) H2, Pd/BaSO4, MeOH.
F igu r e 1. Model of possible binding mode for compound 8 in
the active site of aggrecanase homology model.
Following oral administration of 11 to dogs, substan-
tial absorption was found with an average oral bioavail-
ability of 43%. The terminal half-life was 6 h with a low
systemic clearance of 0.5 L/h/kg.
Ch a r t 2
A representative synthesis of 8 is shown in Scheme
1. Starting from acid 12,22 amide 13 was obtained in
85% by coupling the acid with cis-(1S)(2R)-amino-2-
indanol. The hydroxyl group of compound 13 was
protected as the acetonide. Subsequent alkylation af-
forded the desired product 15 in 61% yield.23 Removal
of the tert-butyl group with TFA, followed by coupling
with O-benzyl hydroxyamine and subsequent hydroge-
nation afforded the desired final product 8.
In summary, we have discovered potent and selective
inhibitors of aggrecanase by a structure-based approach
using an enzyme homology model and the similar
biological activity profile of aggrecanase and MMP-8.
Extensive specificity studies of the matrix metallopro-
teinases using collagen sequence-based synthetic pep-
tide reported by Nagase and Fields provided crucial
information about the active site of MMP-8. This al-
lowed us to incorporate the side chain of a tyrosine
residue into the broad spectrum MMP inhibitor 1 to
achieve modest selectivity for aggrecanase over MMP-
1, -2, and -9. The cis-(1S)(2R)-amino-2-indanol scaffold
was selected and incorporated as a P2′ peptide mimic,
further enhancing the potency and selectivity for ag-
grecanase. Finally, compound 11, a potent and selective
inhibitor of aggrecanase, was synthesized and found to
be orally bioavailable in the dog, with an excellent
pharmacokinetic profile (T1/2 ) 6 h). The discovery of a
selective and orally bioavailable aggrecanase inhibitor
provides an important tool to further study the biological
function of this enzyme and in the development of new
medications for degenerative joint diseases.
position occupied by valine in MMPs 1, 2, 3, 8, and 9
(Figure 1). This finding supported our hypothesis that
the 3-hydroxyl group of inhibitor 8 provides selectivity
through a specific hydrogen-bonding interaction with
this residue in the S1′ pocket. The proof that 8 forms a
specific hydrogen bond to the hydroxyl group of the
threonine in the S1′ pocket of aggrecanase awaits
further enzyme-inhibitor crystallographic evidence.
Compounds 9 and 10 bind only weakly to aggrecanase
with IC50 of 3139 nM and 790 nM, respectively. The
weak activity of 9 and 10 might suggest that the steric
nature of these two P1′ substituents of 9 and 10 possibly
imparts unfavorable interactions in this region, which
prevent these inhibitors from binding to the enzyme.
In an attempt to further improve the binding potency
of 8 for aggrecanase and modify the physicochemical
properties to improve the pharmacokinetic profile, we
investigated a wide selection of substituents at the posi-
tion R to the hydroxamic acid. Hirayama and co-workers
have shown that P1 substitution can significantly in-
crease the potency of these succinate-based hydroxamic
acids.20 Moreover, the introduction of a bulky substitu-
ent R to the hydroxamic acid has been investigated as
a means of preventing the hydrolysis of hydroxamic
acids in vivo, which could result in an improved phar-
macokinetic profile.21 On the basis of this analysis and
the need to have a water-soluble molecule to facilitate
in vivo animal efficacy model studies, an amino func-
tional group was selected and introduced into the P1
position of 8. This analysis and extensive SAR studies
eventually led to the synthesis of molecule 11 (Chart
2). This compound was found to have an IC50 of 12 nM
in the aggrecanase assay and maintained the desired
selectivity for aggrecanase over MMP-1, -2, and -9.
Ack n ow led gm en t. The authors acknowledge J .
Austin, J . Giannaras, and Patty Welch for determina-
tion of IC50 values in the aggrecanase, MMP-1, MMP-
2, MMP-8, and MMP-9 assays and B. Brogdon (animal
resource) and Bioanalytical groups for performing PK
studies reported in this Letter.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails and analytical data for the preparation of compounds 8,
13-15 as well as spectral characteristics of the target com-

3350 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21
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