Potent and selective aggrecanase inhibitors containing cyclic P1 substituents

Article abstract of DOI:10.1016/S0960-894X(03)00124-0

Anti-succinate hydroxamates with cyclic P1 motifs were synthesized as aggrecanase inhibitors. The N-methanesulfonyl piperidine 23 and the N-trifluoroacetyl azetidine 26 were the most potent aggrecanase inhibitors both having an IC₅₀=3 nM while

Full text of DOI:10.1016/S0960-894X(03)00124-0

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1297–1300
Potent and Selective Aggrecanase Inhibitors Containing
Cyclic P1 Substituents
Robert J. Cherney,* Ruowei Mo, Dayton T. Meyer, Li Wang, Wenqing Yao,
Zelda R. Wasserman, Rui-Qin Liu, Maryanne B. Covington, Micky D. Tortorella,
Elizabeth C. Arner, Mingxin Qian, David D. Christ, James M. Trzaskos,
Robert C. Newton, Ron L. Magolda and Carl P. Decicco
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543-4000, USA
Received 24 October 2002; accepted 17 January 2003
Abstract—Anti-succinate hydroxamates with cyclic P1 motifs were synthesized as aggrecanase inhibitors. The N-methanesulfonyl
piperidine 23 and the N-trifluoroacetyl azetidine 26 were the most potent aggrecanase inhibitors both having an IC50=3 nM while
maintaining >100-fold selectivity over MMP-1, -2, and -9. The cyclic moieties were also capable of altering in vivo metabolism,
hence delivering low clearance compounds in both rat and dog studies as shown for compound 14.
# 2003 Elsevier Science Ltd. All rights reserved.
An early step in degenerative joint disease is cartilage
degradation resulting from the proteolysis of aggrecan
within the interglobular domain (IGD).1 This pro-
teoglycan cleavage occurs between the G1 and G2
domains of the IGD at the Glu373-Ala374 bond.2 The
aggrecanases are the proteolytic enzymes responsible for
this cleavage, which has been observed in the synovial
fluid of patients with osteoarthritis, inflammatory joint
disease, and joint injury.3,4 This aggrecanase cleavage
site is distinct from the matrix metalloproteinase
(MMP) site which occurs between residues Asn341 and
Phe342 within the IGD.5 Two human aggrecanases
(aggrecanase-1 and aggrecanase-2) have been isolated,
cloned and expressed.6,7 They are members of the
ADAM-TS (a disintegrin and metalloproteinase with
thrombospondin motifs) sub-family which is part of the
adamalysin/reprolysin family contained within the met-
zincins (which includes the MMPs).8 Studies indicate
that aggrecanase-1 (ADAM-TS-4) and aggrecanase-2
(ADAM-TS-5) are responsible for the aggrecan degra-
dation within osteoarthritic cartilage.9,10 As a result of
this unique proteoglycan cleavage, there has been much
interest in the design of aggrecanase inhibitors.
Recently, we described the discovery of a-amino
hydroxamate 1 as a potent and selective aggrecanase
inhibitor.11 In order to optimize the potency and phar-
macokinetic profile of this inhibitor, we investigated
hydroxamates 2 with cyclic P1 substituents and report
the results herein (Fig. 1).
A general synthesis of the cyclic P1 hydroxamates is
illustrated in Scheme 1 using compounds 14 and 18 as
examples. The essential anti-succinate core was synthe-
sized using Evans chemistry starting from the sub-
stituted acetic acid 3 (or R=a ring).12,13 The requisite
imide 4 was formed via the coupling of 3 and R-4-ben-
*Corresponding author. Tel.: + 1-609-252-3066; fax: +1-609-252-
6601; e-mail: robert.cherney@bms.com
Figure 1. a-Amino (1) and cyclic P1 (2) inhibitors of aggrecanase.
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00124-0

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R. J. Cherney et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1297–1300
Scheme 1. Reagents and conditions: (a) LiCl, Piv-Cl, R-4-benzyl-2-oxazolidinone THF; (b) NaN(TMS)2, BrCH2CO2tBu, THF, ꢀ78 to ꢀ22 ꢁC; (c)
H2O2, LiOH; (d) LDA, HMPA, 7, THF, ꢀ78 ꢁC to rt; (e) DBU, BnBr, Ph-H; (f) (i) TFA, CH2Cl2; (ii) Boc2O, Et3N, dioxane, H2O; (g) BOP reagent
(1S,2R)-cis-1-amino-2-indanol, DMF; (h) H2, Pd(OH)2, MeOH; (i) BOP reagent, H2NOH–HCl, (iPr)2NEt, DMF; (j) TFA, CH2Cl2; (k) 37%
HCHO, NaBH (OAc)3, DCE; (l) H2, Pd(OH)2, MeOH.
zyl-2-oxazolidinone.14 Asymmetric alkylation of 4 with
tert-butylbromoacetate gave the single diastereomer 5.
Hydrolysis gave the carboxylate 6 which was used in the
next alkylation with benzyl iodide 7. The resulting suc-
cinate 8 was isolated as a mixture of diastereomers (1:1)
and was taken into the next step. A benzyl ester was
installed to afford 9 before the tert-butyl ester was con-
verted to the carboxylate 10. Treatment of 10 with BOP
reagent and (1S,2R)-cis-1-amino-2-indanol gave the
amide 11. The benzyl groups were removed to 12, and
direct hydroxamate formation was accomplished with
BOP reagent and hydroxylamine. The resulting anti-
succinate 13 was isolated via reverse-phase HPLC.
Treatment of 13 with TFA gave 14. Other N-substituted
analogues were synthesized from compound 11 by Boc
removal with TFA to afford 15. Using 18 as an example,
reductive amination of 15 gave the N-Me 16. As above,
the benzyl ester was converted to the target hydrox-
amate 18 through the carboxylate 17. Additional ana-
logues differed only in the functionalization (step k) of
15 as acylation gave 20, sulfonylation gave 23, and car-
bamoylation gave 24. The homologated analogues (21
and 22) and the azetidines (25 and 26) were made in a
similar manner starting, in each case, from the requisite
substituted acetic acid 3.
evaluate these hydroxamates.11 We also used a counter
screen of MMP-1, -2, and -9 to assess initial selectivity.15
It was our desire to evaluate exclusively the aggrecanase
clip site (and its relevance in arthritic disease), hence we
required at least 100-fold selectivity for aggrecanase
Table 1. In vitro evaluation of P1 cyclic compounds
Compd
m
n
X
Agga
IC50 (nM)
MMP-9
Ki (nM)a
1
See Figure 1
12
19
>1000
28
4468
1302
>2128
4000
>2128
>2128
1741
>2128
>2128
2099
13
12
14
18
19
20
21
22
23
24
25
26
0
2
See Scheme 1
Boc
0
0
0
0
1
1
0
0
0
0
2
2
2
2
2
2
2
2
1
1
H
Me
Et
C(O)Me
C(O)Me
C(O)Et
SO2Me
CO2Et
H
210
45
29
114
97
3
7
7
3
The newly synthesized hydroxamates were evaluated as
aggrecanase inhibitors before ADAM-TS-4 and
ADAM-TS-5 were isolated. At the time, a bovine
derived aggrecanase assay (measuring total aggrecanase
activity: ADAM-TS-4 plus ADAM-TS-5) was used to
832
>2128
1490
C(O)CF3
aValues are an average from three determinations.

R. J. Cherney et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1297–1300
Table 2. Rat and dog pharmacokinetic data for 14a
1299
Dog
over the MMPs. The a-amino compound 1 displayed
excellent selectivity over the MMPs as controlled by the
phenol in P10 and the indanol in P20. From our model-
ing studies, the P1 group appeared to project toward the
solvent exposed area of the active site, and hence we
assumed cyclic substituents could be accommodated. As
shown in Table 1, we were gratified to see compound 13
supported this model (aggrecanase IC50=19 nM) with-
out disruption of the excellent MMP selectivity. In fact,
13 displayed greater than 100-fold selectivity over
MMP-1 and -2 while maintaining 68-fold selectivity
over MMP-9 (all the inhibitors of Table 1 displayed
>100-fold selectivity for aggrecanase over MMP-1 and
-2, hence only MMP-9 is shown). The hydroxamate was
required for these inhibitors, as the carboxylate 12 was
inactive. Removal of the Boc group gave the water
soluble 14 with good aggrecanase affinity (IC50=28 nM)
and greater than 100-fold selectivity versus MMP-1, -2,
and -9. The N-Me (18) was detrimental toward aggre-
canase affinity, as was the ethyl (19). Acylation (20) was
equipotent in aggrecanase inhibition to 14; however
selectivity versus MMP-9 was somewhat compromised
(60-fold for MMP-9). Homologation of the P1 ring
away from the extended conformation was unfavorable
as 21 and 22 lost affinity (3-fold) for aggrecanase versus
14. Sulfonylation gave 23 which displayed a 9-fold
(compared to 14) increase in aggrecanase affinity while
still maintaining excellent MMP selectivity. Carbamoy-
lation was also beneficial as 24 displayed excellent
MMP selectivity and was 4-fold more potent versus
aggrecanase than 14. Other rings were explored, and the
azetidine (25) displayed excellent aggrecanase affinity (4-
fold improvement over 14) and MMP selectivity. Again
installation of a carbonyl was beneficial, as the tri-
fluoroacetamide 26 increased the aggrecanase affinity by
2-fold over 25 without affecting the MMP selectivity.
Parameters
Rat
iv
Dose (mg/kg)
Cl (L/h/kg)
t1/2 (h)
5.0
0.5
4.0
0.3
0.5
0.1
3.8
0.2
Vss (L/kg)
po
Dose (mg/kg)
tmax (h)
F%
10.0
1.5
1%
1.0
4.3
20%
aData is averaged from two dosings.
In summary, we have identified anti-succinate hydrox-
amates containing P1 cyclic moieties as potent and
selective aggrecanase inhibitors. Cyclic substituents
containing a properly displayed carbonyl group were
found to be the most potent inhibitors. The cyclic moi-
eties were also effective in creating low clearance com-
pounds that should aid in the development of future
aggrecanase inhibitors.
Acknowledgements
We acknowledge Julian Austin, John V. Giannaras, and
Patty K. Welch for Ki and IC50 determinations. We also
thank Dr. Percy H. Carter for critical reading of this
manuscript.
References and Notes
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but also into our in vivo rat efficacy models for proof of
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