Orally bioavailable small molecule ketoamide-based inhibitors of cathepsin K

Article abstract of DOI:10.1016/j.bmcl.2004.02.085

An orally available series of ketoamide-based inhibitors of cathepsin K has been identified. Starting from a potent inhibitor with poor oral bioavailability, modifications to P¹ and P ^1′ elements led to enhancements in solubility and permeability. These improvements resulted in orally available cathepsin K inhibitors.

Full text of DOI:10.1016/j.bmcl.2004.02.085

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2543–2546
Orally bioavailable small molecule ketoamide-based inhibitors of
cathepsin K
David G. Barrett,a,ꢀ John G. Catalano,a David N. Deaton,a,* Stacey T. Long,b
Larry R. Miller,c Francis X. Tavares,a Kevin J. Wells-Knecht,d Lois L. Wrighte and
Hui-Qiang Q. Zhoua
aDepartment of Medicinal Chemistry, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
bDepartment of World Wide Physical Properties, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
cDepartment of Molecular Pharmacology, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
dDepartment of Research Bioanalysis and Drug Metabolism, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
eDiscovery Research Biology, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
Received 30 January 2004; revised 23 February 2004; accepted 26 February 2004
Abstract—An orally available series of ketoamide-based inhibitors of cathepsin K has been identified. Starting from a potent
inhibitor with poor oral bioavailability, modifications to P1 and P1 elements led to enhancements in solubility and permeability.
These improvements resulted in orally available cathepsin K inhibitors.
0
Ó 2004 Elsevier Ltd. All rights reserved.
Bone resorption and bone formation are tightly coupled
processes that maintain skeletal homeostasis.1 However,
when the pace of bone resorption outstrips that of bone
formation, osteoporosis results, leading to decreased
bone mass and increased susceptibility to fracture.
Osteoclasts resorb bone by secreting acid and proteo-
lytic enzymes that remove the mineral and matrix
components of bone. Cathepsin K, a C1A family cys-
teine protease highly expressed in osteoclasts, is the
major proteolytic enzyme responsible for degradation of
bone matrix.2 As part of a complex with glycosamino-
glycans, it efficiently degrades type I collagen, the major
component of bone matrix.3 Small molecule inhibitors
of cathepsin K have proven efficacious in attenuating
bone resorption in animal models of osteoporosis.4
cathepsin K inhibitor 1 (IC50¼2.5 nM).5 Although
ketoamide 1 is a potent cathepsin K inhibitor that was
able to attenuate bone resorption in a rat calvarial
resorption assay,6;7 it exhibited poor oral bioavailability
in male Han Wistar rats (F ¼ 3:1%). This inhibitor
showed reasonable permeability in the Madin–Darby
Canine Kidney cell monolayer transport assay (MDCK,
PAPP ¼ 57 nm/sec),8 but low solubility in fasted state-
simulated intestinal fluid (FS-SIF, solubility¼0.012 mg/
mL).9 Based on the premise that the oral bioavailability
was absorption limited due to the low aqueous solubility
0
of 1, it was decided to vary the P1 and/or P1 substituents
to try to improve solubility within the series, and test
whether these solubility enhancements would result in
improved oral bioavailability.
As part of a larger program to develop novel cathepsin
O
K inhibitors, researchers from these laboratories
recently reported the discovery of the ketoamide
H
H
O
N
N
Ph
nBu
O
O
1
Keywords: Cathepsin K; Ketoamide; Cysteine protease inhibitors.
* Corresponding author. Tel.: +1-919-483-6270; fax: +1-919-315-0430;
e-mail: david.n.deaton@gsk.com
Two general routes were utilized to synthesize the
a-ketoamides. The first process applied the acyl cyano-
phosphorane oxidative cleavage and amine coupling
procedure of Wasserman and Petersen to generate the
ꢀ
Present address: Lilly Forschung GmbH, Essener Str. 93, 22419
Hamburg, Germany.
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.085

2544
D. G. Barrett et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2543–2546
Ph
H
N
O
C-
O
O
H
H
R
N+
O
O
H
N
H
H
N
H
H
O
O
N
N
3a-3j
O
CN
PPh3
O
N
N
H
+
R
Ph
10
11
12
H
N
O
Ph
O
Ph
O
O
O
Ph
Ph
a
nBu
nBu
4a-4j
O
O
O
a
H
N
O
O
2
O
Scheme 1. (a) O3, CH2Cl2, )78 °C; N2; 3a–j, )78 °C to rt; AgNO3,
THF, H2O, 16–67%.
OH
OH
H
N
H
N
H
OH
O
H2N
N
O
O
Ph
O
15
ketoamide functionality.10 As displayed in Scheme 1,
ozonolysis of the phosphorus–carbon double bond
in the known phosphorane 211 resulted in the in situ
generation of an acyl nitrile. Displacement of cyanide by
a P1 amine 3a–j generated the desired ketoamides 4a–j.
H
H
N
NH2
N
b, c
d, e
f
13
14
O
0
OH
O
H
N
H
N
H
H
N
O
O
N
0
The P1 amines, 2-(aminomethyl)pyridine 3a, 3-(amino-
O
O
Ph
O
O
Ph
H
N
H
N
H
N
H
N
methyl)pyridine 3b, and 4-(aminomethyl)pyridine 3c,
were commercially available, whereas the amines, 2-
(aminomethyl)thiazole 3f,12 5-(aminomethyl)thiazole
3g,13 4-(aminomethyl)thiazole 3h,14 and (3S)-3-amino-2-
piperidinone 3j,15 were known in the literature. The
amines, 2-(aminomethyl)oxadiazole 3d and (1R)-2-(1-
aminoethyl)oxadiazole 3e, were synthesized as depicted
in Scheme 2. The commercially available esters 5a and
5b were heated at reflux with hydrazine to provide the
hydrazides 6a and 6b.16 Subsequent heating with tri-
ethylorthoformate at reflux afforded the oxadiazoles 7a
16
17
g
O
Scheme 4. (a) PhCOOH, CH2Cl2, 49%; (b) LiOHÆH2O, dioxane, H2O,
99%; (c) H2/Pd–C, EtOH, H2O, 89%; (d) MeN@C@O, THF, 86%; (e)
HCl, EtOAc, 99%; (f) 15, 1.93 M COCl2 in PhMe, THF, 0 °C to rt; 14,
iPr2NEt, MeOH, 79%; (g) DMSO, (COCl)2, CH2Cl2, )60 °C; 16;
NEt3, )60 °C to rt, 69%.
and 7b,17 which were deprotected with hydrogen chlo-
0
The Passerini reaction was exploited as a second tech-
nique to generate the required ketoamide functional-
ity.20 As shown in Scheme 4, the known aldehyde 1021
was reacted with the isonitrile 115 and benzoic acid to
give the a-acyloxyamides 12. Hydrolysis of the esters
12, followed by hydrogenation of the benzyl carba-
mates afforded the /-amino-a-hydroxyamides 13. Cou-
pling of the amines 13 with methyl isocyanate, followed
by acid catalyzed cleavage of the tert-butyl carbamates
provided the b-amino-a-hydroxyamides 14.22 These
amines 14 were then coupled to the chloroformate,
generated from alcohol 15 and phosgene, to give car-
bamates 16. Oxidation of alcohols 16 afforded the
ketoamide 17.
ride to give the P1 amines 3d and 3e.
5-(Aminomethyl)isoxazole 3i was synthesized as shown
in Scheme 3. Treatment of the known bromide 818 with
sodium azide gave the azide 9. Subsequent reduction of
the azide 9 with triphenyl phosphine afforded the desired
amine 3i.19
O
O
H
H
O
N
O
N
NH
O
a
b
O
R
NH2
O
R
0
As shown in Table 1, replacement of the phenyl P1
5a R = H
6a R = H
5b R = Me
6b R = Me
moiety of inhibitor 1 (IC50¼ 2:5 nM) by heterocycles
0
resulted in a loss in inhibitory activity. The pyridine P1
analogs 4a–c, bearing a positive charge at lysosomal pH,
were approximately an order of magnitude less potent
than the starting ketoamide 1. Whereas the more neutral
oxadiazole 4d, the thiazoles 4f–h, and the isoxazole
4i also apparently suffered slight reductions in activity,
the lactam 4j (IC50¼1.0 nM) maintained similar inhibi-
tory potency to ketoamide 1. Since cathepsins contain a
conserved tryptophan (184Trp in cathepsin K) in
their active site,23 lactam 4j could potentially form a
favorable hydrogen bond to the indole NH of 184Trp,
rationalizing its enhanced potency relative to the other
heterocyclic analogs tested. In the absence of an inter-
N
O
N
N
N
H
O
N
H2N
O
c
O
R
R
7a R = H
3d R = H
7b R = Me
3e R = Me
Scheme 2. (a) H2NNH2, EtOH, reflux, 63–83%; (b) HC(OEt)3, reflux,
24–57%; (c) HCl, EtOAc, 60–70%.
N
N
N
O
O
O
b
a
8
9
3i
Br
N3
NH2
10
action with 184Trp, the S subsite seems to prefer the
hydrophobic phenyl group to more hydrophilic hetero-
cycles.
Scheme 3. (a) NaN3, MeOH/H2O (9:1), 44%; (b) PPh3, THF, H2O,
72%.

D. G. Barrett et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2543–2546
2545
0
Table 1. Inhibition of human cathepsin K by P1 analogs
substitution is also permissible in the ketoamide-based
inhibitors. This is not surprising, since an X-ray crystal
structure of one of the aldehyde inhibitors revealed that
the S1 subsite of cathepsin K is a shallow groove.24
Thus, long P1 residues should be completely exposed to
solvent at their termini.
O
H
O
N
R
nBu
O
O
Although these ketoamides suffered attenuation in
cathepsin K inhibitory activity, they were still suitable to
test the hypothesis that increased aqueous solubility
would result in improved oral bioavailability. As shown
in Table 2, these inhibitors did indeed exhibit ꢀ2–30-
fold improvements in solubility in fasted state-simulated
intestinal fluid at pH ¼ 6:8. The calculated octanol/
#
R
IC50 nMa
H
N
1
2.5
water partition coefficients (c log P) for the nonionized
H
N
N
0
P1 analogs are also included in Table 2 for comparison
4a
4b
44
23
with the measured solubilities. The decrease in c log P
from 7.3 to 3.4–5.5 is further evidence of the increased
0
N
hydrophilicity of these heterocyclic P1 analogs. The
apparent MDCK permeability coefficients also im-
H
N
proved. However, these small enhancements resulted in
0
little increase in oral bioavailabilities for the pyridine P1
analogs 4a (F ¼ 10%) and 4c (F ¼ 5:2%). The P1 lysine
derivative 17 showed a small apparent improvement in
oral bioavailability (F ¼ 10%). Notably, the oxazole
analog 4d (F ¼ 16%) and the lactam derivative
4j (F ¼ 20%) showed significant increases in bioavail-
ability. Furthermore, both the thiazole compound
4g (F ¼ 55%) and the isoxazole 4i (F ¼ 32%) exhib-
ited an order of magnitude improvement in bioavaila-
bilities.
H
N
N
4c
4d
30
19
N
H
N
N
N
O
N
H
N
4e
59
O
S
H
N
N
The decrease in hydrophobicity of the analogs resulted
in an apparent decrease in the steady state volume of
distribution (VSS ¼ 90–1600 mL/kg) compared to the
starting ketoamide 1 (VSS ¼ 1900 mL/kg). Also, the total
clearance for most of the inhibitors increased (analogs
4d, 4g, 4i, 4j, 17 C1 ¼ 24–33 mL/min/kg), indicating
increased rates of elimination. These combined changes
resulted in attenuation of the terminal half-lifes for most
of these analogs (analogs 4a, 4d, 4g, 4i t1=2 ¼ 21–
150 min). Furthermore, it is likely that the increased rate
of elimination for these analogs, reflected in the increase
in total clearance as compared to ketoamide 1, func-
tioned to lower the oral bioavailability via enhanced
first-pass metabolism. This, at least partly, negated any
gains made by improving the adsorption of compounds
within the series.
4f
18
14
N
H
N
4g
S
H
N
N
S
N
4h
4i
13
14
H
N
O
H
N
O
NH
4j
1.0
In summary, this report highlights improvements in oral
bioavailability of a potent series of cathepsin K inhibi-
a Inhibition of recombinant human cathepsin K activity in a fluores-
cence assay using 10 lM Cbz-Phe-Arg-AMC as substrate in 100 mM
NaOAc, 10 mM DTT, 120 mM NaCl, pH ¼ 5.5. The IC50 values are
the mean of two or three inhibition assays, individual data points in
each experiment were within a 3-fold range of each other.
tors. Starting from a ketoamide cathepsin K inhibitor 1,
0
the P1, and P1 substituents were replaced with more
hydrophilic moieties. The resulting general decline in
potency was compensated by overall enhancements in
aqueous solubility and membrane permeability com-
pared to the ketoamide lead 1. In addition, these chan-
ges resulted in improvements in oral bioavailability with
analogs 4g and 4i being 55% and 32% orally bioavailable
in male Han Wistar rats, respectively. Subsequent
reports will detail efforts to further improve the drug
properties of these cathepsin K ketoamide-based inhib-
itors.
Researchers from these laboratories had previously
disclosed a series of aldehyde cathepsin K inhibitors.24
Structure–activity relationships had revealed that the P1
norleucine could be replaced by ornithine or lysine
derivatives without attenuating activity. The lysine
derived ketoamide 17 (IC50¼4.3 nM) shows that this

2546
D. G. Barrett et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2543–2546
Table 2. Pharmacokinetics of combination analogs
b
d
f
#
MDCKa PAPP
(nm/sec)
c Log P
Sol. FS-SIFc
(mg/mL)
t1=2 (min)
Cle (mL/min/kg) VSS (mL/kg)
F g (%)
1
57
510
––
7.3
––
0.012
0.028
0.062
0.15
290
77h
240i
21j
150j
52j
220k
260j
8.7
5.2
7.1
1900
240
3.1
10
4a
4c
4d
4g
4i
––
90
980
5.2
16
510
480
410
310
––
3.4
5.3
5.0
4.4
5.5
33
0.051
0.063
0.35
28
29
44
24
1300
1200
1600
1500
55
32
4j
17
20
10
0.032
a Madin–Darby Canine Kidney cell monolayer transport assay.
b PAPP is the apparent permeability coefficient for apical to basolateral flux in nanometers per second. The error in the assay averaged 10%.
c FS-SIF is the equilibrium solubility in fasted state-simulated intestinal fluid @ pH ¼ 6.8. The values are the mean of two measurements.
d t1=2 is the iv terminal half-life dosed as a solution in male Han Wistar rats. All in vivo pharmacokinetic values are the mean of two experiments.
e Cl is the total clearance.
f VSS is the steady state volume of distribution.
g F is the oral bioavailability.
h Dosed as a solution in sulfobutylether-7-cyclodextrin/methane sulfonic acid, pH ¼ 3.0.
i Dosed as a solution in sulfobutylether-7-cyclodextrin/citrate, pH ¼ 3.3.
j Dosed as a solution in 40% hydroxypropyl-b-cyclodextrin, pH ¼ 8.9.
k Dosed as a solution in 15% solutol/citrate, pH ¼ 3.5.
11. Tavares, F. X.; Boncek, V.; Deaton, D. N.; Hassell, A. M.;
Long, S. T.; Miller, A. B.; Payne, A. A.; Miller, L. R.;
Shewchuk, L. M.; Wells-Knecht, K.; Willard, D. H., Jr.;
Wright, L. L.; Zhou, H.-Q. J. Med. Chem. 2004, 47, 588.
12. Irako, N.; Hamada, Y.; Shioiri, T. Tetrahedron 1995, 51,
12731.
Acknowledgement
The authors would like to thank Robert B. McFadyen
for critical reading of the manuscript.
13. Alterman, M.; Bjoersne, M.; Muehlman, A.; Classon, B.;
Kvarnstroem, I.; Danielson, H.; Markgren, P.-O.; Nill-
roth, U.; Unge, T.; Hallberg, A.; Samuelsson, B. J. Med.
Chem. 1998, 41, 3782.
References and notes
1. Einhorn, T. A. In Osteoporosis; Marcus, R., Feldman, D.,
Kelsey, J., Eds.; Academic: San Diego, California, 1996; p
3.
14. Oku, T.; Sawada, K.; Kuroda, A.; Inoue, T.; Kayakiri, N.;
Sawada, Y.; Mizutani, T. PCT Int. Appl. WO 9,954,284,
1999; Chem. Abstr. 1999, 131, 310451.
2. Yamashita, D. S.; Dodds, R. A. Curr. Pharm. Des. 2000,
6, 1.
15. Stark, P. A.; Thrall, B. D.; Meadows, G. G.; Abdel-
Monem, M. M. J. Med. Chem. 1992, 35, 4264.
16. Moutevelis-Minakakis, P.; Photaki, I. J. Chem. Soc.,
Perkin. Trans. 1 1985, 2277.
17. Kramer, J. B.; Boschelli, D. H.; Connor, D. T.
J. Heterocycl. Chem. 1994, 31, 1439.
18. DeShong, P.; Cipollina, J. A.; Lowmaster, N. K. J. Org.
Chem. 1988, 53, 1356.
19. Banfi, L.; Guanti, G.; Rasparini, M. Eur. J. Org. Chem.
2003, 1319.
3. Li, Z.; Hou, W.-S.; Escalante-Torres, C. R.; Gelb, B. D.;
Bromme, D. J. Biol. Chem. 2002, 277, 28669.
4. Stroup, G. B.; Lark, M. W.; Veber, D. F.; Bhattacharyya,
A.; Blake, S.; Dare, L. C.; Erhard, K. F.; Hoffman, S. J.;
James, I. E.; Marquis, R. W.; Ru, Y.; Vasko-Moser, J. A.;
Smith, B. R.; Tomaszek, T.; Gowen, M. J. Bone Miner.
Res. 2001, 16, 1739.
5. Catalano, J. G.; Deaton, D. N.; Long, S. T.; McFadyen,
R. B.; Miller, L. R.; Payne, J. A.; Wells-Knecht, K. J.;
Wright, L. L. Bioorg. Med. Chem. Lett. 2004, 14, 719.
6. Hahn, T. J.; Westbrook, S. L.; Halstead, L. R. Endocri-
nology 1984, 114, 1864.
20. Semple, J. E.; Owens, T. D.; Nguyen, K.; Levy, O. E. Org.
Lett. 2000, 2, 2769.
21. Hamada, Y.; Shioiri, T. Chemical & Pharmaceutical
Bulletin 1982, 30, 1921.
7. Conaway, H. H.; Grigorie, D.; Lerner, U. H. J. Endocri-
nol. 1997, 155, 513.
8. Irvine, J. D.; Takahashi, L.; Lockhart, K.; Cheong, J.;
Tolan, J. W.; Selick, H. E.; Grove, J. R. J. Pharm. Sci.
1999, 88, 28.
22. Catalano, J. G.; Deaton, D. N.; Miller, A. B.; Tavares, F.
X. PCT Int. Appl. WO 03,062,192, 2003; Chem. Abstr.
2003, 139, 133839.
23. Somoza, J. R.; Palmer, J. T.; Ho, J. D. J. Mol. Biol. 2002,
322, 559.
9. Dressman, J. B.; Amidon, G. L.; Reppas, C.; Shah, V. P.
Pharm. Res. 1998, 15, 11.
10. Wasserman, H. H.; Petersen, A. K. Tetrahedron Lett.
1997, 38, 953.
24. Catalano, J. G.; Deaton, D. N.; Furfine, E. S.; Hassell, A.
M.; McFadyen, R. B.; Miller, A. B.; Miller, L. R.;
Shewchuk, L. M.; Willard, D. H.; Wright, L. L. Bioorg.
Med. Chem. Lett. 2004, 14, 275.