Dioxo-triazines as a novel series of cathepsin K inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.01.116

A novel dioxo-triazine series of cathepsin K inhibitors was identified from HTS. A rapid exploratory programme led to the discovery of potent and selective cathepsin K inhibitors, typified by compound 24 which displayed IC₅₀ values of 17 nM against catK and >10,000 nM in catL, catB and catS assays.

Full text of DOI:10.1016/j.bmcl.2010.01.116

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1488–1490
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Dioxo-triazines as a novel series of cathepsin K inhibitors
a,
a
a
a
a
Zoran Rankovic a, , Jiaqiang Cai , Xavier Fradera , Maureen Dempster , Ashvin Mistry , Ann Mitchell ,
Clive Long a, Emma Hamilton a, Angela King a, Sylviane Boucharens a, Craig Jamieson a, Jonathan Gillespie a,
Iain Cumming a, Joost Uitdehaag b, Mario van Zeeland b
*
*
a Schering-Plough Corporation, Newhouse, Lanarkshire, ML1 5SH, Scotland, United Kingdom
b Schering-Plough Corporation, 5340BH Oss, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel dioxo-triazine series of cathepsin K inhibitors was identified from HTS. A rapid exploratory pro-
gramme led to the discovery of potent and selective cathepsin K inhibitors, typified by compound 24
which displayed IC50 values of 17 nM against catK and >10,000 nM in catL, catB and catS assays.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 3 December 2009
Revised 20 January 2010
Accepted 21 January 2010
Available online 25 January 2010
Keywords:
Cathepsin K
Cysteine protease
Osteoporosis
Osteoporosis is a systemic, degenerative skeletal condition char-
acterised by low bone mass and micro architectural deterioration of
bone, leading to reduced bone strength.1 It most commonly affects
postmenopausal women, but also occurs in premenopausal women,
andinmen, oftenbeinginducedbyavarietyofsecondarycauses. The
resultant low bone mineral density (BMD) makes osteoporotic pa-
tients prone to fractures, which can lead to serious complications
and fatality. The abundant and selective expression of cathepsin K
in osteoclasts, cells involved in bone resorption, indicates its poten-
tially critical role in bone remodelling.2 Indeed, a number of cathep-
rodent6 and primate models of postmenopausal osteoporosis7
and most recently in patients within clinical settings.8
High throughput screening of the corporate compound file
against cathepsin K led to the identification of 6-cyano-2-(30-trifluo-
romethyl-phenyl)-3,5-dioxo-1,2,4-triazine 1. Despite 1 being a sin-
gleton hit with poor catK potency (IC50 = 2000 nM) and low
aqueous solubility (<1 mg/L), this was an interesting discovery since
at the time only peptidic catK inhibitors were known in the litera-
ture.9 Hence, an exploratory programme was instigated to evaluate
progressability of this chemotype.
sin
K
mutations have been found to be associated with
The synthesis of the dioxo-triazine 1 is described in Scheme 1.
pycnodysostosis, a recessive bone sclerosing disorder characterised
by increased bone density (osteopetrosis), dwarfism and skeletal
abnormalities. Cathepsin K knockout mice display similar skeletal
abnormalities, including osteopetrosis, further corroborating the
key role of cathepsin K in bone remodelling.3 Cathepsin K is a lyso-
somal cysteine protease from the papain family which contains a
highly conserved catalytic triad Cys25, His159 and Asn175 within
its active site (papain numbering).
Eleven members of this family are known to be expressed in the
human genome, of which cathepsins L, S and V are most closely re-
lated to cathepsin K.4 Development of selective cathepsin K inhib-
itors for treatment of osteoporosis attracted considerable attention
in recent years.5 Such compounds displayed ex vivo efficacy in
osteoclast-based assays of bone resorption and in vivo efficacy in
Br
Br
O
O
ii
N
N
i
N
NH
HN
NH
O
O
2a
2b
CN
CN
6
O
O
iii
N
N
N 2
4
N
NH
CF3
N
O
O
2c
1
* Corresponding authors. Tel./fax: +44 1698736157 (Z.R.).
E-mail addresses: zoran.rankovic@spcorp.com (Z. Rankovic), jiaqiang.cai@sp
corp.com (J. Cai).
Scheme 1. Reagents and conditions: (i) KHCO3, n-PrI, DMSO, rt, 1 h; (ii) tetramethyl
urea, CuCN, 145 °C, 3 h; (iii) Cu(OAc)2, 3-CF3PhB(OH)2, CH2Cl2, rt, 18 h.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.116

Z. Rankovic et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1488–1490
1489
SelectiveN4-propylationof the6-bromo-triazine-3,5-dione2ato
obtain corresponding 4-propyl-triazinedione 2b was achieved using
n-propyl iodide and potassium bicarbonate at room temperature in
DMSO. Bromo displacement with copper (I) cyanide in tetramethyl
urea provided 6-cyano-4-propyl-triazinedione 2c. Compound 1
was obtained by copper (II) acetate promoted N-arylation of 2c with
the appropriate arylboronic acid. The same synthetic route was em-
ployed to prepare aryl derivatives of 1 (4–11), whereas the aliphatic
analogues 12–15 were obtained by alkylation of intermediate 2c
with the corresponding alkyl bromide (KHCO3, DMSO, 60 °C, 48 h).
The SAR around the N4 substituent of dioxo-triazine 1 was ex-
plored using the synthetic route outlined in Scheme 2.
proved potency (IC50 = 72 nM and 288 nM). Further increase in the
size of the substituent in this position was not tolerated, for exam-
ple, 3-biphenyl analogue 9 was inactive in catK assay. Similarly,
replacement of the CF3 group with more polar groups such as the
methoxy in 10, and hydroxymethyl in 11, proved detrimental for
catK potency. Substitution in position 2 of the aromatic ring gener-
ally resulted in loss of potency (data not shown). Considering the
relatively tight SAR around the phenyl ring, it was interesting to
find that replacement with a saturated ring system is tolerated
(12, IC50 = 870 nM). Enlargement of the aliphatic ring proved ben-
eficial, for example, cyclohexyl analogue 13 showed catK an IC50
197 nM against catK. Further homologation of the ring yielded
one of the most potent inhibitors in the series, cycloheptyl ana-
logue 14 (IC50 = 38 nM). By comparison, insertion of a methylene
linker between the two rings yielding 15, was not optimal
(IC50 = 1100 nM).
Intermediate 3c was synthesized in two high-yielding steps fol-
lowing a literature method.10 Alkylation using sodium hydride as a
base produced derivatives 16–27 (31 was prepared by the same
route starting from 3-isopropylaniline).
SAR exploration around the aryl moiety of 1 showed that the
substituent in position 3 is critical for cathepsin K potency. For
example, both CF3 positional and deletion analogues (4 and 5,
respectively) were inactive, whereas methyl derivative 6 showed
catK potency similar to 1 (Table 1). Interestingly, the slightly larger
iso-propyl 7 and tert-butyl 8 analogues displayed considerably im-
At this point we turned our attention to the propyl group of 1. The
chainlengthvariationsin16–18demonstratedthatthepropylgroup
is optimal for this region (Table 2). Interestingly, a larger cyclopentyl
group was tolerated (19, IC50 = 2000 nM), whereas the correspond-
ing phenyl analogue 20 showed a sixfold loss in potency compared
to 1. Although the benzyl analogue 21 showed a slight improvement
CN
Table 2
CN
O
Effect of N4 substitution on Cat K potency
O
O
NH
N
CF3
H2N
O
NH
CF3
HN
CN
6
CN
O
CN
O
ii
O
O
O
1
N
N
N
N
O
N
N
i
4
CF3
N
N
N
3a
3b
R1
R1
R1
O
O
CN
CN
16 - 27
28 - 30
31
O
O
HN
N
N
iii
R1
Cathepsin K inhibition IC50 (nM)
a
Compound
CF3
N
N
CF3
N
R1
16
17
18
19
20
21
22
23
CH3(CH2)3–
CH3CH2–
CH3–
Cyclopentyl–
Phenyl–
PhCH2–
Ph(CH2)2–
(CH3)2N(CH2)3–
7400
2500
4780
2000
12,000
1000
4460
100
O
O
3c
16-27
Scheme 2. Reagents and conditions: (i) NaNO2, HCl, 2 h; (ii) xylene, 140 °C, 1 h; (iii)
NaH, R1Br, KI, DMSO, 70 °C, 1 h.
Table 1
Effect of N2 substitution on Cat K potency
N
24
17
CN
O
N
N
2
N
25
26
182
810
4
N
R
(CH3)2N(CH2)2–
O
a
O
N
Compound
R
Cathepsin K inhibition IC50 (nM)
27
28
112
110
1
4
5
6
7
3-CF3-phenyl-
4-CF3-phenyl-
Phenyl-
3-Methyl-phenyl-
3-iso-Propyl-phenyl-
3-tert-Butyl-phenyl-
3-Biphenyl-
3-Methoxy-phenyl-
3-Hydroxymethyl-phenyl- >10,000
2000
>10,000
>10,000
3500
72
288
(CH3)2N(CH2)3–
N
29
30
31
100
500
38
8
9
>10,000
>10,000
10
11
12
13
14
O
N
Cyclopentyl-
Cyclohexyl-
Cycloheptyl-
870
197
38
N
15
1100
a
a
Inhibition of recombinant human cathepsin
K in a fluorescence assay,
Inhibition of recombinant human cathepsin
K in a fluorescence assay,
employing Z-Phe-Arg-MCA as a synthetic substrate. Data represent means of two
experiments performed in duplicate.
employing Z-Phe-Arg-MCA as a synthetic substrate. Data represent means of two
experiments performed in duplicate.

1490
Z. Rankovic et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1488–1490
of up to 10 lM. However, poor chemical and plasma stability (hu-
man and rat t1/2 <5 min) which proved to be intrinsic to this chem-
otype prevented further progression of compounds in this series.
LCMS studies identified the nitrile hydrolysis to corresponding pri-
mary amide as the major route of degradation in plasma.
In summary, a novel non-peptidic dioxo-triazine seriesof cathep-
sin K inhibitors was identified from HTS. Potent and highly selective
inhibitors were identified, that is, compound 24 displayed IC50 val-
ues of 17 nM in catK and >10,000 nM in catL, catB and catS assays.
Intrinsically poor chemical and plasma stability precluded further
progression of this series. However, the discovery of new S2 pocket
bindinggroups as well as the SAR and biostructural informationgen-
erated around this chemotype proved invaluable in the design of no-
vel cathepsin K inhibitors with excellent pharamacokinetic profile,
as we describe in the following article in this issue.
Acknowledgements
Figure 1. Co-crystal structure of dioxo-triazine 24 with cathepsin K (2 Å resolution;
PDB entry 3KWB). The 3-CF3-phenyl group is bound to the hydrophobic S2 pocket,
whereas the propyl-pyperidine interacts with the enzyme prime side largely
exposed to solvent. The core N1 atom forms a hydrogen bond with the conserved
water in the S1 pocket (key interactions with water molecules are indicated with
dashed yellow lines).
We thank Han Kok and Wim Koot for running fermentors to
produce CatK protein.
References and notes
(IC50 = 1000 nM), further extension of the linker proved not to be
beneficial for cathepsin K potency (22, IC50 = 4460 nM). Most grati-
fyingly, incorporation of dimethylamine group led to a 20-fold in-
crease in potency (23, IC50 = 100 nM). Another fivefold improve
ment was achieved by replacing the dimethylamine with a piperi-
dine group in 24 (IC50 = 17 nM). The n-propyl spacer was found to
be optimal, as demonstrated by around 8–10-fold loss in potency
of ethyl-linked analogues 25 and 26. More polar amines such as mor-
pholine 27 displayed uniformly lower potency in comparison to 24.
Interestingly, combinations of the best R and R1 groups were not
additive. For example, in contrast to the profound effect observed in
the 3-CF3-phenyl series, incorporation of an amine group into cyclo-
heptyl 13 or 3-isopropy-phenyl 6 analogues was either deleterious
(28–30)11 or neutral (31) with respect to catK potency.
Considering the structural novelty of this chemotype we were
pleased to be able to obtain a high resolution X-ray structure of
inhibitor 24 bound in the active site of cathepsin K (Fig. 1). The struc-
ture showed that a covalent thioamidate bond is formed between
the nitrile and Cys25, similarly to previously reported aliphatic ni-
trile inhibitors (PDB structure 2F7D). C5 Carbonyl group of the di-
oxo-triazine core forms a hydrogen bond with an extensive water
network in the prime side, whilst the other carbonyl moiety is ex-
posed to solvent. A conserved water molecule in the S1 pocket forms
a bridge between the NH of Gly 66 and a N1 atom of the dioxo-tri-
azine core. The aryl ring binds into the unprime side of the active site,
with the CF3 group deeply buried into the hydrophobic S2 pocket,
formed by the side chains of Tyr67, Met68, Ala134, Ala163 and
Leu209 (papain numbering). The S2 pocket, the only true binding
pocket within a relatively shallow active site of cathepsin K, is the
primary determinant of enzyme specificity which favours small
hydrophobic groups.5 This could rationalise the observed preference
for small hydrophobic aryl or cyclic aliphatic rings in N2 position of
the dioxo-triazine core (Table 1). The propyl-piperidine moiety
binds in the prime side, and makes a number of hydrophobic con-
tacts in the S20/S30, as well as an interaction with a conserved water
in the S10 pocket. The piperidine amino group is at 3.9 Å distance to a
conserved water in S10 pocket, indicating possibility of a weak
interaction.
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11. Synthesis of the cycloheptyl derivatives 28–30 is shown below:
CN
CN
O
O
i
N
N
HN
N
HN
NH
O
O
32
33
CN
O
N
ii
N
N
R1
O
28-30
Inhibitors in this series were generally found highly selective
over closely related anti targets such as cathepsins B and L, as well
as the less critical cathepsin S. For example, compounds 24–26
were shown inactive in catB, catL and catS assays at concentrations
Reagents and conditions: (i) KHCO3, c-heptylBr, KI, DMSO, 70 °C, 2.5 h; (ii) NaH, R1Br,
KI, DMSO, 70 °C, 1 h.