2-Phenyl-9H-purine-6-carbonitrile derivatives as selective cathepsin S inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.06.049

Starting from previously disclosed equally potent cathepsin K and S inhibitor 4-propyl-6-(3-trifluoromethylphenyl)pyrimidine-2-carbonitrile 1, a novel 2-phenyl-9H-purine-6-carbonitrile scaffold was identified to provide potent and selective cathepsin S inhibitors.

Full text of DOI:10.1016/j.bmcl.2010.06.049

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4447–4450
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2-Phenyl-9H-purine-6-carbonitrile derivatives as selective cathepsin
S inhibitors
a
a
a
a
Jiaqiang Cai a, , D. Jonathan Bennett , Zoran Rankovic , Maureen Dempster , Xavier Fradera ,
Jonathan Gillespie a, Iain Cumming a, William Finlay a, Mark Baugh a, Sylviane Boucharens a, John Bruin a,
Kenneth S. Cameron a, William Hamilton a, Jennifer Kerr a, Emma Kinghorn a, George McGarry a,
John Robinson a, Paul Scullion a, Joost C. M. Uitdehaag b, Mario van Zeeland b, Dominique Potin c,
Laurent Saniere c, Andre Fouquet c, Francois Chevallier c, Hortense Deronzier c, Cecile Dorleans c,
Eric Nicolai c
*
a Merck Research Laboratories, MSD, Newhouse, Lanarkshire ML1 5SH, UK
b Merck Research Laboratories, MSD, 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:
Starting from previously disclosed equally potent cathepsin K and S inhibitor 4-propyl-6-(3-trifluoro-
methylphenyl)pyrimidine-2-carbonitrile 1, a novel 2-phenyl-9H-purine-6-carbonitrile scaffold was iden-
tified to provide potent and selective cathepsin S inhibitors.
Received 12 May 2010
Revised 7 June 2010
Accepted 8 June 2010
Available online 15 June 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Cathepsin S
Transition state
Purine-6-carbonitrile
Lysosomotropism
Eleven members of the cysteine cathepsin family proteases
have been identified in the human genome (cathepsins B, C, H, F,
K, L, O, S, V, W, and X).1 Two of these, cathepsins K and S have been
the subject of extensive effort in the pharmaceutical industry.1–3
Cathepsin S is highly expressed in antigen presenting cells and
plays a major role in the degradation of the invariant peptide chain
associated with the major histocompatibility complex and affects
antigen presentation. Selective cathepsin S inhibitors should then
be useful therapeutics for autoimmune disorders, for example,
rheumatoid arthritis (RA) and multiple sclerosis (MS). More re-
cently cathepsin S has also been indicated for neuropathic pain.4
Several cathepsin K inhibitors have progressed into human clinical
trials for osteoporosis,5 but as yet there are no reports of any
cathepsin S inhibitors in advanced stages of human clinical trials.
Several different cathepsin S inhibitor chemotypes have been re-
ported in the literature, most of which contain some kind of pep-
tide feature within the molecule and pose some challenges in
terms of pharmacokinetic property optimization.2
We6,7 and others8–17 have recently reported non-peptide hetero-
aryl nitriles as cathepsin K and S inhibitors. Here we describe our
latest effort in identifying selective cathepsin S inhibitors through
P2 residue optimization and scaffold hopping starting from recently
disclosed non-selective cathepsin S and K inhibitor 4-propyl-6-(3-
trifluoromethylphenyl)pyrimidine-2-carbonitrile (Fig. 1).7
There are several major differences between the highly homol-
ogous cathepsin S and K binding sites. The cathepsin K S3 pocket
contains an aspartic acid residue (Asp61) against a lysine for
cathepsin S (Lys64). This major difference has been applied suc-
cessfully for identifying selective cathepsin K inhibitors.8 With
the cathepsin S enzyme, the S2 pocket could be regarded as the
CN
N
N
F3C
1
hCat S IC50: 41nM
hCat K IC50: 33nM
* Corresponding author. Tel.: +44 1698736133; fax: +44 1698736187.
E-mail address: jiaqiang.cai@merck.com (J. Cai).
Figure 1.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.049

4448
J. Cai et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4447–4450
Table 1
most important binding site for inhibitor design. There are five sig-
nificant differences between cathepsin S and K in this site18 namely
Phe70, Gly137, Val162, Gly165, and Phe211 for cathepsin S and
Tyr67, Ala133, Leu157, Ala160, and Leu209 for cathepsin K. Two
smaller glycine residues at the bottom of the pocket provide
cathepsin S with a slightly deeper (wider) S2 pocket than the cor-
responding S2 pocket for cathepsin K. The Phe211 sits at the end of
the S2 pocket. The side chain of Phe211 is quite mobile, but is pref-
erentially found in an ‘open’ conformation which increases the
space available at the end of the S2 pocket by opening a narrow
passage between Phe70 and Phe211. The equivalent residues for
cathepsin K, Tyr67 and Leu209, are quite close and do not afford
any additional space between them. Val162 sits on side of the
cathepsin S S2 pocket, and makes the S2 pocket for cathepsin S
slightly narrower in that region. As reported previously, the trifluo-
romethylphenyl group of the compound 1 analogs binds to the
cathepsin K S2 pocket. Considering the high overall and binding
site similarity between cathepsin K and S, it is reasonable to be-
lieve that this group also binds to the S2 pocket in cathepsin S.
For this reason, we envisaged that further optimization of the tri-
fluoromethylphenyl region could improve activity against cathep-
sin S and also selectivity over cathepsin K.
Inhibitory activity of arylpyrimidine compounds against human cathepsin S and K
a
Compound
R1
R2
R3
R4
R5
IC50 (nM)
Cat S
Cat K
1
H
H
CF3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CF3
H
H
H
Cl
H
H
H
CF3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CF3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
41
>10,000
>10,000
1259
148
174
447
93
33
1122
4070
977
60
4a
4b
4c
4d
4e
4f
4g
4h
4i
Br
44
MeCO
MeSO2
Me
316
148
182
71
166
56
950
83
Et
4j
4k
4l
n-Pr
i-Pr
i-Bu
t-Bu
CF3O
c-Pr
c-Bu
c-Pen
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
410
195
224
191
115
200
63
155
51
1047
13
28
10
15
6
32
58
398
11
4m
4n
4o
4p
4q
4r
4s
4t
4u
4v
4w
4x
4y
4z
661
195
145
457
44
H
H
H
F
1000
32
Cl
34
17
The analogs of 1 were synthesized by applying our previously
reported chemistry7 as shown in Scheme 1. The results of this
study are shown in Table 1.
Me
MeO
EtO
CF3O
i-PrO
151
40
Identical to that recently reported for cathepsin K,7 the meta-sub-
stitution of the P2 phenyl group also proved to be critical for cathep-
sin S inhibition. Both the unsubstituted 4a and ortho-trifluoromethyl
substituted 4b compounds show no activity against human cathep-
sin S. The para-substitution by CF3 group provided compound 4c 30
times less active than the corresponding meta-substituted analog 1.
The trifluoromethyl substitution at the meta-position of the P2 phe-
nyl group also appeared to be optimal for cathepsin S inhibitionas all
other compounds 4d–4q are less active. A second substitution at po-
sition 5 (R4) of the phenyl ring is well tolerated, such as compound
4r, which has a similar activityand selectivityprofile to compound 1.
A chlorine substitution at position 6 4s (R5) of the P2 phenyl ring ap-
peared to be detrimental for both cathepsin S and K inhibition, prob-
ably due to an unfavorable torsion angle between pyrimidine and
benzene rings. A second substitution on position 4 (R3) enhances
cathepsin S inhibition activity (compounds 4t–4y) except com-
pound 4z with 4-ethoxy substitution (4x) as the most active human
cathepsin S inhibitor in the series. Although only a small improve-
ment in most cases, the trend of these results is clear and it was
expected from our computer aided docking studies. These groups
on position 4 were expected to interact with Phe70, Val162, and
Phe211 as shown in Figure 2.
65
91
a
Inhibition of recombinant human cathepsin S, K, L, and B in a fluorescence
assay, employing synthetic substrates. Data represents means of two experiments
in duplicate. All compounds shown do not have inhibitory activity against human
cathepsin B and L (IC50 >10,000 nM).
Although all pyrimidine analogs tested showed no or very little
activity against human cathepsin B and L, the selectivity over human
cathepsin K is less than 10-fold in almost all cases. One of the reasons
for this poor selectivity might be due to the high reactivity of the
thio-trapping nitrile war-head of the pyrimidine-2-carbonitriles
on top of the high level of binding site homology between human
cathepsin K and S. Visualizing all the X-ray structures of aryl nitrile
inhibitors complexed to the human cathepsin K protein, one feature
is that the cys25 attacks the nitrile war-head from the P2 side of
Figure 2. Compound 4x (EtO at position 4 of the P2 phenyl ring) docked into
cathepsin S active site showing hydrophobic interactions between Et and Phe70
(top), Phe211 (left), and Val162 (bottom).
H
H+
N
N+
Cys
P2
Cys
P2
S
N
S
N
H
H
N
N
N
H
N
CN
b
a
R1
CN
N
R1
R4
N
N
R2
R3
Br
R5
R2
R3
N
Figure 3. Covalent interactions between thio-trapping war-head nitrile group with
active site cysteine thiol group.
R5
a
Me3Sn
R4
4a-4x
3
2
the molecule to form a planar thio-imidate with the NH on the prime
side of the molecule (Fig. 3a). We envisaged that a purine template
Scheme 1. Reagents and condition: (a) Pd(Ph3P)2Cl2, aryl bromide, DMF, 150 °C.

J. Cai et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4447–4450
4449
Table 2
could be an ideal replacement for the pyrimidine core as N7 (from
imidazole ring) could potentially form an intramolecular hydrogen
bond with thio-imidate NH (Fig. 3b) to further stabilize the transi-
tion state. This purine scaffold could potentially offer a more stable
thio-trapping nitrile war-head.
Inhibitory activity of purine compounds 11a–12c against human cathepsin S, K and
cellular activity in human JY cells (Lip10)
Lip10b
a
Compound
R
R0
IC50 (nM)
Cat S
Cat K
IC50 (nM)
The purine compounds were synthesized according to Scheme 2.
Displacement of 6-chlorine of 2,6-dichloro-9H-purine with a ben-
zylthio group followed by THP protection of the purine NH provided
compound 7. Suzuki coupling of compound 7 with a suitable boronic
acid or ester gives compound 8. Oxidation of sulfide by oxone pro-
vided sulfone with the concomitant deprotection of the THP group
which was then re-installed to afford compound 9. Replacement of
the sulfone with cyano-group gives compound 10. Removal of THP
group delivers the desired compound 11a–11c. Compound 11c
was further modified to afford compounds 12a–12c. Biological
results of compound 11a–11c and 12a–12c are shown in Table 2.
The purine-6-carbonitrile core appeared to be a good scaffold
for cathepsin S inhibition. In comparison with pyrimidine com-
pound 1, compound 11a roughly maintained the cathepsin S inhib-
itory activity while it is 15 times less active against human
cathepsin K. The SAR in the P2 aryl region appeared to be in agree-
ment with that observed in the pyrimidine series with EtO > -
MeO > H for cathepsin S inhibition. These 4-ethoxy analogs 11c,
12a, and 12b all have >50-fold selectivity over human cathepsin
K and no detectable activity against human cathepsin B and L at
11a
11b
11c
12a
12b
12c
H
H
H
H
Et
60
23
3.9
7.2
4.5
6.9
470
630
1072
2291
282
na
na
>10,000
na
509
59
MeO
EtO
EtO
EtO
EtO
HO(CH2)2
Me2N(CH2)3
117
a
Inhibition of recombinant human cathepsin S, K, L, and B in a fluorescence
assay, employing synthetic substrates. Data represents means of two experiments
in duplicate. All compounds shown do not have inhibitory activity against human
cathepsin B and L (IC50 >10,000 nM).
Measured by Western blot using human B lymphoblastoid cells, 500,000/ml,
mouse anti-CD74 Pin. 1 monoclonal antibody, 50% of the maximum activity of LHVS
in the same assay as IC50; na, not available.
b
from our docking studies, the remote ionic interactions with two
carboxylic residues (Asp61 and Glu59) might be the reason for
the lost selectivity. In contrast, there are no negatively charged res-
idues in human cathepsin S binding sites.
Three of the purine compounds were further assessed in the cell
based Lip10 accumulation assay. Although neutral compounds 11c
and 12b have low cellular activities, the basic compound 12c has
an IC50 of 59 nM. This high cellular activity is likely due to the pre-
viously reported lysosomotropic effect.19,20
Although compound 12c possesses good cellular activity, one of
its close analogs21 showed a half life of 17 min in our NMR based
nitrile stability studies. This poor stability prevented this class of
compounds from progressing any further.
10 lM. With a view to improving solubility and cellular activity,
a basic nitrogen side chain was attached to N9 nitrogen to give
compound 12c. This compound maintained cathepsin S inhibitory
activity, however it lost some selectivity over cathepsin K.
Although this basic nitrogen side chain seems to have no direct
interactions with any specific cathepsin K binding site residues
In summary, starting from a non-selective human cathepsins K
and S pyrimidine-2-carbonitrile based inhibitor 1, by using bio-X-
ray structural data and computer aided molecule design, purine-6-
carbonitrile compounds were synthesized to provide a novel class
selective human cathepsin S inhibitor.
Ph
Ph
S
Cl
S
N
b
N
N
N
a
N
N
N
N
Cl
N
N
H
N
H
Cl
N
Cl
O
References and notes
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Ph
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N
N
d, b
c
N
F3C
R
F3C
R
N
N
N
O
N
O
9a: R=H
9b: R=MeO
9c: R=EtO
8a: R=H
8b: R=MeO
8c: R=EtO
CN
N
CN
N
N
N
f
e
F3C
R
F3C
N
H
N
N
O
R
11a: R=H
11b: R=MeO
11c: R=EtO
10a: R=H
10b: R=MeO
10c: R=EtO
CN
N
g
N
F3C
R
N
N
R'
11. Irie, O.; Kosaka, T.; Kishida, M.; Sakaki, J.; Masuya, K.; Konishi, K.; Yokokawa, F.;
Ehara, T.; Iwasaki, A.; Iwaki, Y.; Hitomi, Y.; Toyao, A.; Gunji, H.; Teno, N.;
Iwasaki, G.; Hirao, H.; Kanazawa, T.; Tanabe, K.; Hiestand, P.; Malcangio, M.;
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12a: R=EtO, R'=Et
12b: R=EtO, R'=HO(CH2)2
12c: R=EtO, R'=Me2N(CH2)3
Scheme 2. Reagents and conditions. (a) BnSH, TEA, EtOH, 60 °C, 1.5 h; (b) DHP,
TsOH, EtOAc, 50 °C, 2 h; (c) ArB(OH)2, Pd(Ph3P)4, K2CO3, NMP–H2O, 130 °C, 7 min;
(d) oxone, MeCN–H2O, 16 h; (e) KCN, DMSO, 90 °C, 7 min; (f) MeOH–DCM, TsOH,
2 h; (g) R0OH, Ph3P, DEAD, 1 h.

4450
J. Cai et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4447–4450
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21. Half life (t1/2) was measured by NMR with the final concentration of inhibitor
at 1.6 mM and glutathione at 6.7 mM in 1:1 CD3OD and D2O at 20 °C at pD 7.5.
Structure of the compound measured is shown below which is synthesized
according to Scheme 2.
CN
N
N
F3C
N
N
18. Gauthier, J. Y.; Black, W. C.; Courchesne, I.; Cromlish, W.; Desmarais, S.; Houle,
R.; Lamontagne, S.; Li, C. S.; Masse, F.; Mckay, D. J.; Ouellet, M.; Robichaud, J.;
N