Diastereoselective synthesis, activity and chiral stability of cyclic alkoxyketone inhibitors of cathepsin K

Article abstract of DOI:10.1016/S0960-894X(00)00627-2

The diastereoselective synthesis of a novel class of cathepsin K inhibitors together with their cathepsin K affinity and stability towards aqueous buffer is reported.

Full text of DOI:10.1016/S0960-894X(00)00627-2

Bioorganic & Medicinal Chemistry Letters 11 (2001) 199±202
Diastereoselective Synthesis, Activity and Chiral Stability of
Cyclic Alkoxyketone Inhibitors of Cathepsin K
Ashley E. Fenwick,a Andrew D. Gribble,a Robert J. Ife,a Nichola Stevensb
and Jason Witheringtona,*
aDepartment of Medicinal Chemistry, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park,
Third Avenue, Harlow, Essex, CM19 5AD, UK
bDepartment of Analytical Sciences, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park,
Third Avenue, Harlow, Essex, CM19 5AD, UK
Received 23 June 2000; revised 31 October 2000; accepted 2 November 2000
AbstractÐThe diastereoselective synthesis of a novel class of cathepsin K inhibitors together with their cathepsin K anity and
stability towards aqueous bu€er is reported. # 2001 Published by Elsevier Science Ltd.
Cathepsin K (EC 3.4.22.38), a cysteine protease of the
papain superfamily, is selectively expressed in osteo-
clasts and has been implicated in the process of bone
resorption.1 Selective inhibitors of cathepsin K therefore
could be promising therapeutic agents for the treatment
of diseases characterised by excessive bone loss, such as
osteoporosis.
Although we had a route towards the single diastereomers
of the cyclic alkoxyketones we were interested in develop-
ing a synthesis to provide either diastereomer with known
absolute stereochemistry. To this end, desymmetrisation
of meso-epoxide 4 employing the (R,R)-salen catalyst 65
and TMS-N3 in ether at 25 ꢀC provided the (3S,4R)-
azido silyl alcohol 7 in 98% ee.6 Employing the (1S,2S)-
salen catalyst likewise provided the corresponding
(3R,4S)-azido alcohol, again in 90% yield and 97% ee.
Removal of the silyl group with camphor sulfonic acid in
methanol at room temperature and subsequent reduc-
tion of the azido moiety with hydrogen in the presence of
Pd/C a€orded the amino-alcohol 8. Selective amide
bond formation with the mixed anhydride of Cbz-Leu-
OH and subsequent hydrogenolysis of the Cbz group
a€orded the amine 9. Amide bond formation and sub-
sequent Dess±Martin oxidation of the secondary alcohol
provided the 4S diastereomers 10 (Scheme 2).
Recently we reported the design2 and solid-phase
synthesis3 of a new class of cyclic alkoxyketones 1 as
selective reversible inhibitors of cathepsin K (Fig. 1).
To enable the investigation of both the activity and
chiral stability of these novel inhibitors, we required a
diastereoselective synthesis, as all attempts to separate
the individual diastereomers employing preparative
chromatography proved unsuccessful.
Our initial approach towards the preparation of the sin-
gle diastereomers of these cyclic ketones involved the
preparation of the key enantiomerically enriched amino
alcohols 3. Desymmetrisation of meso-epoxide 4 with
(S)-( )-a-methylbenzylamine4 provided the correspond-
ing amino:alcohols 5 as a 1:1 mixture. Formation of the
hydrochloride salt and fractional crystallisation from eth-
anol provided a single diastereomer of unknown stereo-
chemistry (Scheme 1). Attempts to obtain an X-ray
structure, enabling the absolute chirality of the ring ste-
reochemistry to be established, proved unsuccessful.
The cathepsin K activity for the individual diaster-
eomers showed that activity resided in the 4S isomer
with the 4R isomer typically showing up to a 40-fold
reduction in potency (Table 1).
The cyclic alkoxyketones were also examined for their
stability towards epimerisation in a series of aqueous
bu€ers (hepes, phosphate and acetate). In these studies,
compound concentration was l mM and the ®nal solution
contained 20% THF to aid solubility. Analysis of samples
was carried out after extraction into dichloromethane,
using chiral HPLC.8 Signi®cant epimerisation over a per-
iod of several hours was evident in both diastereomers.
*Corresponding author. Tel.: +44-01279-672832; fax: +44-01279-
627841; e-mail: jason_witherington-1@sbphrd.com
0960-894X/01/$ - see front matter # 2001 Published by Elsevier Science Ltd.
PII: S0960-894X(00)00627-2

200
A. E. Fenwick et al. / Bioorg. Med. Chem. Lett. 11 (2001) 199±202
Figure 1. The evolution of cyclic alkoxyketones.
Scheme 1. Desymmetrisation employing (S)-( )-a-methyl benzylamine.
Scheme 2. Diastereoselective synthesis of ®ve membered cyclic alkoxyketones.
Table 1. Diastereomeric activity of tetrahydrofuranone inhibitors
Having measured the chiral stability and activity for the
®ve membered cyclic alkoxyketones, we wished to assess
the stability of the related six membered cyclic alkoxy-
ketones which also emerged as potent cathepsin K inhi-
bitors.9 Again, attempted separation of the individual
diastereomers employing chiral HPLC proved unsuc-
cessful. However, column chromatography of the related
ketal analogues 11 followed by deketalisation a€orded
the diastereomers 12 in typically 95% d.e., albeit of
unknown stereochemistry (Scheme 3).
Entry
R
S isomer, Ki
(nM)7
R isomer, Ki
(nM)
10a
10b
10c
2-Benzo[b]thiophenyl
2-Naphthy1
2-Quinoly1
7
14
15
68
470
590
Again, cathepsin K activity of the ketones 12 resided in
predominately one, stereochemically unde®ned, diaster-
eomer (Table 2).
Attempts to quantify epimerisation employing chiral
HPLC analysis proved unsuccessful due to incomplete
baseline separation. We were able, however, to employ
1H NMR analysis for this purpose. Using the same
protocol as for the ®ve membered ketones, we observed
However, the rate of epimerisation appears di€erent in the
various bu€ers, being fastest in hepes pH 6.8 bu€er and
slowest in acetate pH 5.5, indicating the epimerisation is
catalysed by hydroxide anion (Fig. 2).

A. E. Fenwick et al. / Bioorg. Med. Chem. Lett. 11 (2001) 199±202
201
Figure 2. Diastereomeric epimerisation of 10b in the acetate bu€er, pH 5.5.
Scheme 3. Diastereoselective synthesis of six membered cyclic alkoxyketones.
Figure 3. Ab initio calculations of cyclic alkoxyketones.
Table 2. Diastereomeric activity of tetrahydropyranone inhibitors
potency, the six membered ketones may o€er signi®cant
development advantages due to their enhanced chiral
stability.
Acknowledgements
The authors would like to acknowledge Thaddeus Tho-
maszek for determination of cathepsin K inhibition.
Entry
R
Diastereomer-1,
Ki (nM)
Diastereomer-2,
Ki (nM)
12a
12b
12c
2-Benzo[b]thiophenyl
2-Naphthyl
3,4-Dimethoxybenzyl
8
32
10
70
380
770
References and Notes
1. (a) Drake, F. H.; Dodds, R. A.; James, I. E.; Connor, J. R.;
Debouck, C.; Richardson, S.; Lee-Rykaczewski, E.; Coleman,
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Biol. Chem. 1996, 271, 12511. (b) Bossard, M. J.; Tomaszek,
T. A.; Thompson, S. K.; Amergadzie, B. Y.; Hanning, C. R.;
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2. Marquis, R. W.; Ru, Y.; Zeng, J.; Trout, R. E. L.; Gribble,
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3. Fenwick, F.; Gamier, B.; Gribble, A.; Ife, R.; Rawlings, A.;
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no signi®cant levels of epimerisation in a series of aqu-
eous bu€ers (hepes, phosphate and acetate). We hypo-
thesised that the epimerisation di€erences observed
between the ®ve and six membered cyclic alkoxyketones
could be due to the energy barrier between the ketol and
enolic forms. Indeed, ab initio calculations in SPAR-
TAN using 3-21G(*) indicate a larger energy di€erence
(ÁE) between the two forms for the 6-ring system relative
to the 5-ring (Fig. 3).
In summary we have completed the diastereoselective
synthesis of a novel class of cathepsin K inhibitors and
examined their stability to a range of bu€er systems.
Whilst the more potent diastereomer of the ®ve and six
membered cyclic alkoxyketones show comparable
6. Optical rotations of 7 and 8 were identical to those reported
by Jacobsen.5

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A. E. Fenwick et al. / Bioorg. Med. Chem. Lett. 11 (2001) 199±202
7. Enzyme inhibition was evaluated as previously described:
Votta, B.; Levy, M. A.; Badger, A.; Bradbeer, J.; Dodds, R. A.;
James, I. E.; Thompson, S.; Bossard, M. J.; Can, T.; Connor,
J. R.; Tomaszek, T. A.; Szewczuk, L.; Drake, F. H.; Veber,
D. F.; Gowen, M. J. Bone Miner. Res. 1997, 12, 1396.
8. Separation was achieved using a Chiralpak AD column
(250 mmÂ4.6 mm) using a mobile phase of 50:50 hexane:
IPA.
9. Gribble, A. D.; Fenwick, A. E.; Marquis, R. W.; Veber,
D. F.; Witherington, J. WO 9850533.