Design and optimization of a series of novel 2-cyano-pyrimidines as cathepsin K inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.01.100

Morphing structural features of HTS-derived chemotypes led to the discovery of novel 2-cyano-pyrimidine inhibitors of cathepsin K with good pharmacokinetic profiles, for example, compound 20 showed high catK potency (IC₅₀ = 4 nM), >580-fold selectivity over catL and catB, and oral bioavailability in the rat of 52%.

Full text of DOI:10.1016/j.bmcl.2010.01.100

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1524–1527
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and optimization of a series of novel 2-cyano-pyrimidines as cathepsin
K inhibitors
a,
a
a
a
a
Zoran Rankovic a, , Jiaqiang Cai , Jennifer Kerr , Xavier Fradera , John Robinson , Ashvin Mistry ,
Emma Hamilton a, George McGarry a, Fiona Andrews a, Wilson Caulfield a, Iain Cumming a,
Maureen Dempster a, John Waller a, Paul Scullion a, Iain Martin a, Ann Mitchell a, Clive Long a, Mark Baugh a,
Paul Westwood a, Emma Kinghorn a, John Bruin a, William Hamilton a, Joost Uitdehaag b,
Mario van Zeeland b, Dominique Potin c, Laurent Saniere c, Andre Fouquet c, François Chevallier c,
Hortense Deronzier c, Cecile Dorleans c, Eric Nicolai c
*
*
a Schering-Plough Corporation, Newhouse, Lanarkshire, ML1 5SH Scotland, United Kingdom
b Molecular Pharmacology, Schering-Plough Corporation, 5340BH Oss, The Netherlands
c Cerep, 19 Avenue du Quebec, 91951 Courtaboeuf Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Morphing structural features of HTS-derived chemotypes led to the discovery of novel 2-cyano-pyrimi-
dine inhibitors of cathepsin K with good pharmacokinetic profiles, for example, compound 20 showed
high catK potency (IC50 = 4 nM), >580-fold selectivity over catL and catB, and oral bioavailability in the
rat of 52%.
Received 3 December 2009
Revised 15 January 2010
Accepted 16 January 2010
Available online 25 January 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Cathepsin K
Cysteine protease
Osteoporosis
Cathepsin K is a lysosomal cysteine protease of the papain
family. Eleven members of this family are expressed in the human
genome, of which cathepsins L, S and V are most closely related to
cathepsin K.1 The selective and abundant expression of cathepsin K
in osteoclasts, cells involved in bone resorption, indicates its
potentially critical role in bone remodeling. A large body of evi-
dence accumulated over the last decade points towards cathespin
K as an important therapeutic target for the treatment of osteopo-
rosis.2 As such, considerable interest has been generated in the
development of selective cathepsin K inhibitors.3 These compounds
have been shown to display ex-vivo efficacy in osteoclast-based
assays of bone resorption and in vivo efficacy in rodent4 and
primate models of bone resorption.5
This compound exhibited high potency against cathepsin K
(IC50 = 1 nM) and selectivity over cathepsins (158 nM),
(1711 nM) and B (520 nM). However, although 1 showed good sta-
S
L
bility in human microsomes (t >120 min) and hepatocytes
½
(t = 118 min), it was rapidly cleared in rodent microsomes (rat
½
t
½
= 23 min; mouse t = 4 min) and hepatocytes (rat t = 6 min).
½
½
P2
CN
CN
P2
O
N
N
N
N
F3C
N
N
H
N
N
O
NH
Most recently, clinical data have been disclosed for selective
cathepsin K inhibitors showing a reduction of biochemical markers
of bone resorption and increases in bone mineral density (BMD).6
A rapid SAR exploration around a hit obtained from high
throughput screening of the corporate compound collection
against cathepsin K, led to identification of triazine 17 (Fig. 1).
2
1
CN
N
N
F3C
3
* Corresponding authors. Tel./fax: +44 0 1698736157.
E-mail addresses: zoran.rankovic@spcorp.com (Z. Rankovic), jiaqiang.cai@
spcorp.com (J. Cai).
Figure 1. Morphing structures of two HTS-derived inhibitors, triazine 1 and dioxy-
triazine 2, led to the design of pyrimidine 3, a novel cathepsin K inhibitor with
improved metabolic stability.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.100

Z. Rankovic et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1524–1527
1525
In order to explore SAR around the P2 aryl moiety of 3, ana-
logues were prepared according to either of the routes depicted
in Scheme 2. The results of this study are shown in Table 1.
As in the dioxo-triazine series,9 substitution in the 3-position of
the P2 aryl group proved to be critical. For example, the CF3 dele-
tion analogue 5, as well as both the 2- and 4-positional isomers
6 and 7, showed several orders of magnitude lower potency in
the cathepsin
K assay (IC50 values 985 nM, 4074 nM, and
1000 nM, respectively). Small 4-substituents, in addition to the
obligatory 3-CF3 group, were generally tolerated (e.g., 4-Cl
analogue 8, IC50 = 34 nM), whereas 2-substitution was found to
be detrimental (e.g., 2-Cl analogue 9, IC50 = 1000 nM). The size
and lipophilicity of the 3-substitutent was found to be more
important than its ability to increase or reduce electron density
in the aromatic ring. For instance, the tert-butyl analogue 10
showed a threefold higher potency (IC50 = 11 nM), whereas corre-
sponding analogues with a slightly larger e.g. cyclopentyl (11) or
methylsulfonyl (12) or smaller, for example, Me (13) or CN (14)
substituent in this position, showed 6–10-fold lower potency when
compared to the parent compound 3. Interestingly, whilst the 3,4-
dimethyl analogue 15 displayed only around threefold lower
cathepsin K potency (96 nM), its selectivity over cathepsin S was
significantly higher than that shown by the compound 3 (catS
IC50 >10,000 nM vs 60 nM). Similarly, replacement of the aryl moi-
ety with a cycloheptyl group produced inhibitor 16 with moderate
cathepsin K potency (IC50 = 84 nM), but a significantly improved
selectivity over cathepsin S (IC50 = 1300 nM). Analogue 17, with a
slightly larger cyclooctyl P2 group, showed further improved
cathepsin K potency (IC50 = 11 nM) and over 200-fold selectivity
over cathepsin S (IC50 = 2239 nM), whereas cyclohexyl derivative
18 displayed almost an order of magnitude lower potency for both
enzymes (IC50 catK = 680 nM and catS >10,000 nM). Although
cathepsin S inhibition was not considered detrimental, possibly
even beneficial for this programme,11 a high selectivity over
cathepsins L and B was required. Gratifyingly, the majority of
cathepsin K inhibitors in this series were shown to be inactive in
both cathepsin L and B assays, for example, cat L and B IC50 values
were >10,000 nM for all compounds in Table 1 (data not shown).
As in the dioxo-triazine series,9 incorporation of an amine group
such as the piperidinyl of compound 19, resulted in not only
improved solubility (from <1 mg/L in 3 to 379 mg/L in 19), but
Figure 2. Co-crystal structure of triazine 1 with cathepsin K (1.8 Å resolution; PDB
3KW9). A reversible covalent thioimidate bond is formed between the nitrile and
Cys25, which is similar to the previously reported binding mode of an aliphatic
nitrile group (PDB structure 2F7D). A molecule of trifluoroacetic acid and a water
molecule make additional contacts to the ligand in the prime side.
Similarly, good stability of 1 in human plasma (t >150 min) was
½
contrasted by its poor rodent plasma stability (e.g., rat t <17 min).
½
Consequently, the compound demonstrated a poor pharmacoki-
netic (PK) profile in rat (iv 2 mpk; po 10 mpk), with CL = 159 ml/
min/kg, AUCinf = 64 ng h/ml, t = 2.6 h and oral bioavailability = 6%.
½
Metabolite identification studies indicated that the uniformly
poor rodent plasma stability of compounds in this series was a
result of hydrolysis of the nitrile group into the corresponding acid
and primary amide. This then might account for the clearance
being higher than hepatic blood flow in the rat. Consequently, such
poor rodent PK was considered prohibitive for further develop-
ment of this chemotype, despite the acceptable human in vitro
profile. At this point, in order to improve stability of the nitrile
warhead in 1 we considered reducing its electrophilicity by replac-
ing the triazine with a pyrimidine core.8
The most prominent features of a high resolution X-ray struc-
ture of 1 bound in the active site of cathepsin K are the thioimidate
moiety formed by covalent interaction between the nitrile war-
head and Cys25 in the cathepsin K catalytic site, and the cyclohexyl
group deeply buried into the hydrophobic S2 pocket. The pipera-
zine moiety is bound to the S10 pocket, with the basic amino group
exposed to solvent (Fig. 2).
We have previously observed a very similar binding mode in a
dioxo-triazine series of cathepsin K inhibitors.9 This suggested that
morphing the structural features of triazine 1 and dioxo-triazine 2
into a pyrimidine chemotype could be a feasible medicinal chem-
istry strategy towards novel cathepsin K inhibitors with improved
metabolic stability. This was indeed confirmed by a hybrid 2-cya-
no-pyrimidine 3 with moderate cathepsin K potency (IC50 = 33
nM), Fig. 1.10 The synthesis of pyrimidine 3 is shown in Scheme 1.
enhanced cathepsin
K potency, too (IC50 = 4 nM). Increased
potency against the other cathepsins was also observed although,
importantly, the high selectivity over catL and catB was main-
tained (Table 2). Interestingly, the potency enhancing effect of
amine group incorporation was not observed for pyrimidine ana-
logues with a cycloalkyl P2 group. For example, piperidinyl 22
showed similar catK potency and selectivity profile to the parent
cycloheptyl analogue 16.
The binding mode similarity of pyrimidine 19 (Fig. 3) and an
analogous inhibitor from the dioxo-triazine series9 co-crystallized
with cathepsin K explain the observed SAR similarities between
these two chemical series.
O
O
O
O
OH
N
NH
O
F3C
F3C
F3C
CN
a
b
4a
4b
4c
Cl
N
N
N
N
F3C
F3C
c
d
4d
3
Scheme 1. Reagents and conditions. (a) NaNH2, Et2O, 60 °C; (b) urea, HCl/EtOH, 80 °C; (c) POCl3, 100 °C; (d) CuCN, NMP, 200 °C.

1526
Z. Rankovic et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1524–1527
SH
N
S
S
O
N
O
N
N
N
N
N
O
b
c
a
HO
e
HO
Cl
4e
4f
4g
4h
SO2
SO2
N
CN
N
N
N
d
N
f
R
Cl
4j
R
4i
g
h
CN
N
SO2
N
5-18
N
N
f
Me3Sn
Me3Sn
4k
4l
Scheme 2. Reagents and conditions. (a) thiourea, NaOMe, MeOH, 0–100 °C; (b) KOH, MeOH, MeI; (c) POCl3, 100 °C; (d) mCPBA, DCM; (e) Pd[(Ph3)P]4, K2CO3, aryl boronic acid,
DME, 100 °C; (f) NaCN, DMSO; (g) (SnMe3)2, Pd[(Ph3)P]4, LiCl, 2,6-di-tert-butyl-4-methylphenol, dioxane, 100 °C; (h) Pd(Ph3P)2Cl2, aryl bromide, DMF, 150 °C.
Table 1
Table 2
Optimization of the P2 group of 3
Optimization of the prime side of 3
CN
CN
CN
N
N
N
N
N
N
F3C
NRR'
NRR'
R
a
Compds
R
IC50 (nM)
22
19-21
K
S
a
Compds
NRR0
IC50 (nM)
L
3
5
3-CF3-phenyl-
phenyl-
33
60
985
4074
1000
34
1000
11
457
148
181
293
96
>10,000
>10,000
1259
28
K
S
B
ROCb
22
6
7
2-CF3-phenyl-
4-CF3-phenyl-
19
20
Piperidinyl-
4
23
47
>10,000
4200
HN
8
9
4-Cl-3-CF3-phenyl-
2-Cl-3-CF3-phenyl-
4
6760
2340
6457
34
1047
191
155
93
1300
191
10
11
12
13
14
15
16
17
18
3-tert-Butyl-phenyl-
3-Cyclopentyl-phenyl-
3-Methylsulfonyl-phenyl-
3-Methyl-phenyl-
3-Cyano-phenyl-
3,4-Dimethyl-phenyl-
Cycloheptyl-
21
22
NH-tert-Butyl-
Piperidinyl-
10
100
20
9772
>10,000
56
ND
>10,000
>10,000
a
Inhibition of recombinant human cathepsin K, S, L and B in a fluorescence assay,
employing Z-Phe-Arg-MCA as synthetic substrates. Data represent means of two
experiments performed in duplicate.
>10,000
1300
2239
>10,000
Rabbit osteoclasts assay.13
b
84
11
Cyclooctyl-
Cyclohexyl-
680
a
Inhibition of recombinant human cathepsin K and S in a fluorescence assay,
employing Z-Phe-Arg-MCA as synthetic substrates. Data represent means of two
experiments performed in duplicate.
Pyrimidine 19 was found covalently bound to Cys25 in the
cathepsin K active site, with the aryl-CF3 group deeply buried into
the S2 pocket where it makes extensive hydrophobic contacts with
the protein, as in the previously reported dioxo-triazine structure.9
The propyl-piperidine moiety of 19 is angled towards the solvent ex-
posed prime side where the basic nitrogen interacts via a sulfate
molecule, an artifact of the crystallization, with Trp184 and Gln19.
A similar interaction in situ via a bridging water molecule together
with additional hydrophobic contacts may account for the positive
effect of amine group incorporation into this region of the molecule.
The X-ray structure of 22 (Fig. 4) revealed the cycloheptyl ring
bound in the S2 pocket, but not as deeply as the 3-CF3-phenyl in
19, which may explain the potency difference between the two
compounds although as a more voluminous group, it forms tighter
contacts to both sides of the pocket. Interestingly, the S2 pocket is
slightly narrower in catS (there is a ꢀ0.7 Å shift between cat K and
cat S residues Leu160, Asn161, and His162), and deeper (Ala134
replaced by Gly in catS) which may explain the greater catS
selectivity displayed by 22 (IC50 >10,000 nM).
Figure 3. Co-crystal structure of pyrimidine 19 with cathepsin K (1.9 Å resolution;
PDB 3KWZ). The sulfate molecule in the prime side is likely to be an artifact of the
crystallization.
The crystal structure of 19 also explains our findings that SAR is
highly flexible around the ‘prime side’ binding region of the mole-
cule, which was shown to tolerate a wide range of amine substitu-

Z. Rankovic et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1524–1527
1527
pyrimidine inhibitors of cathepsin K with good rat PK properties.
Compounds in this series showed high catK potency and efficacy
in the rabbit osteoclast bone resorption assay, as well as selectivity
over catL and catB, but were not progressed due to their high hERG
potency. Results of hERG optimization efforts and efficacy of com-
pounds from this series on bone resorption markers in primates
will be reported in future publications.
Acknowledgements
We thank Han Kok and Wim Koot for running fermentors to
produce cathepsin K protein.
References and notes
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(b) Deal, C. Curr. Opin. Rheum. 2009, 21, 380.
Figure 4. Co-crystal structure of cycloheptyl analogue 22 with cathepsin K (1.7 Å
resolution; PDB 3KX1).
4. Lark, M. W.; Stroup, G. B.; James, I. E.; Dodds, R. A.; Hwang, S. M.; Blake, S.
M.; Lechowska, B. A.; Hoffman, S. J.; Smith, B. R.; Kapadia, R.; Liang, X.;
Erhard, K.; Ru, Y.; Dong, X.; Marquis, R. W.; Veber, D.; Gowen, M. Bone
2002, 30, 746.
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D. J.; Hwang, S.-M.; Tomaszek, T.; Yamashita, D. S.; Marquis, R. W.; Oh, H.;
Jeong, J. U.; Veber, D. F.; Gowen, M.; Lark, M. W.; Stroup, G. Bone 2007, 40,
122; (b) Stroup, G. B.; Lark, M. W.; Veber, D. F.; Bhattacharyya, A.; Blake, S.;
Dare, L.; Erhard, K. F.; Hoffman, S. J.; James, I. E.; Marquis, R. W.; Ru, Y.;
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Table 3
Pharmacokinetic properties in male Sprague–Dawley rats (iv 2 mpk; po 10 mpk) and
hERG potency
Compds
Vss (L/kg)
CL (ml/min/kg)
t
(h)
F (%)
hERGa IC50
(lM)
½
19
20
21
3.3
3.5
12
13.3
13.5
30
4.1
3.5
5.7
23
52
61
0.16
0.32
1.1
a
Inhibition of [3H]-Dofetilide binding to hERG K+ channel in HEK293 cells fol-
6. Stoch, S. A.; Zajic, S.; Stone, J.; Miller, D. L.; Van Dyck, K.; Gutierrez, M. J.; De
Decker, M.; Liu, L.; Liu, Q.; Scott, B. B.; Panebianco, D.; Jin, B.; Duong, L. T.;
Gottesdiener, K.; Wagner, J. A. Clin. Pharmacol. Ther. 2009, 86, 175.
7. Rankovic, Z.; Cai, J.; Cumming, I. WO 2005011703.
lowing a 10 M dose of test compound. Data represent means of two experiments
performed in duplicate.
l
8. Oballa, R. M.; Truchon, J.-F.; Bayly, C. I.; Chauret, N.; Day, S.; Crane, S.;
Berthelette, C. Bioorg. Med. Chem. Lett. 2007, 17, 998.
9. Preceding article in this issue: Rankovic, Z.; Cai, J.; Fradera, X.; Dempster, M.;
Mistry, A.; Mitchell, A.; Long, L.; Hamilton, E.; King, A.; Jamieson, C.; Gillespie,
J.; Cumming, I.; Uitdehaag, J.; van-Zeeland, M. Bioorg. Med. Chem. Lett.,
doi:10.1016/j.bmcl.2010.01.116.
10. Subsequently, structurally related cathepsin K inhibitors have been reported
by other groups: (a) Altmann, E.; Aichholz, R.; Betschart, C.; Buhl, T.; Green, J.;
Irie, O.; Teno, N.; Lattmann, R.; Tintelnot-Blomley, M.; Missbach, M. J. Med.
Chem. 2007, 50, 591; (b) Morley, A. D.; Kenny, P. W.; Burton, B.; Heald, R. A.;
MacFaul, P. A.; Mullett, J.; Page, K.; Porres, S. S.; Ribeiro, L. R.; Smith, P.; Ward,
S.; Wilkinson, T. J. Bioorg. Med. Chem. Lett. 2009, 19, 1658.
11. (a) Barclay, J.; Clark, A. K.; Ganju, P.; Gentry, C.; Patel, S.; Wotherspoon, G.;
Buxton, F.; Song, C.; Ullah, J.; Winter, J.; Fox, A.; Bevan, S.; Malcangio, M. Pain
2007, 130, 225; (b) Clark, A. K.; Yip, P. K.; Grist, J.; Gentry, C.; Staniland, A. A.;
Marchand, F.; Dehvari, M.; Wotherspoon, G.; Winter, J.; Ullah, J.; Bevan, S.;
Malcangio, M. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 10655; (c) Irie, O.; Kosaka,
T.; Ehara, T.; Yokokawa, F.; Kanazawa, T.; Hirao, H.; Iwasaki, A.; Sakaki, J.; Teno,
N.; Hitomi, Y.; Iwasaki, G.; Fukaya, H.; Nonomura, K.; Tanabe, K.; Koizumi, S.;
Uchiyama, N.; Bevan, S. J.; Malcangio, M.; Gentry, C.; Fox, A. J.; Yaqoob, M.;
Culshaw, A. J.; Hallett, A. J. Med. Chem. 2008, 51, 5502.
ents of different size, basicity and structure (data not shown).
However, we focussed mainly on small, secondary amine ana-
logues, such as neopentyl (20) and tert-butyl (21) due to their gen-
erally superior PK profile (Table 3).
As initially predicted, both chemical and metabolic stability
were greatly improved within the pyrimidine series as compared
with the original triazine and dioxo-triazine chemotypes. The high
potency of 19–21 in the cathepsin K biochemical assay was well
translated into efficacy in the functional rabbit osteoclast bone
resorption assay (Table 2). However, these compounds were not
progressed further due to their prohibitively high hERG potency
(Table 3). The significant hERG block is probably related to the rel-
atively high lipophilicity and basicity of these compounds, for
example, 19 c log P = 4 and pKa = 9.19.12
In summary, biostructurally guided morphing of the triazine
and dioxo-triazine cores led to the discovery of novel 2-cyano
12. Jamieson, C.; Moir, E. M.; Rankovic, Z.; Wishart, G. J. Med. Chem. 2006, 49, 5029.
13. Frith, J. C.; Rogers, M. J. J. Bone Miner. Res. 2003, 18, 204.