Trifluoromethylphenyl as P2 for ketoamide-based cathepsin S inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.10.012

The trifluoromethylphenyl P2 motif from previously reported heteroarylnitrile series has been successfully applied for the design and synthesis of highly potent novel ketoamide-based cathepsin S inhibitors. The key in this process is the change of the tor

Full text of DOI:10.1016/j.bmcl.2010.10.012

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6890–6894
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Trifluoromethylphenyl as P2 for ketoamide-based cathepsin S inhibitors
Jiaqiang Cai a, , John Robinson a, Simone Belshaw a, Kathryn Everett a, Xavier Fradera a, Mario van Zeeland b,
⇑
Leon van Berkom c, Peter van Rijnsbergen c, Lucy Popplestone a, Mark Baugh a, Maureen Dempster a,
John Bruin a, William Hamilton a, Emma Kinghorn a, Paul Westwood a, Jennifer Kerr a,
Zoran Rankovic a, Wullie Arbuckle a, D. Jonathan Bennett a, Philip S. Jones a, Clive Long a,
Iain Martin a, Joost C. M. Uitdehaag b, Tommi Meulemans c
a Merck Research Laboratories, MSD, Newhouse, Lanarkshire ML1 5SH, United Kingdom
b Merck Research Laboratories, MSD, 5340BH Oss, The Netherlands
c Mercachem B.V. Kerkenbos 1013, 6546 BB Nijmegen, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
The trifluoromethylphenyl P2 motif from previously reported heteroarylnitrile series has been success-
fully applied for the design and synthesis of highly potent novel ketoamide-based cathepsin S inhibitors.
The key in this process is the change of the torsion angle between the P2 phenyl ring and the attached
secondary amide by adding a small Cl, F, or Me group at the 2-position.
Received 14 September 2010
Revised 4 October 2010
Accepted 5 October 2010
Available online 26 October 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Cathepsin s
Cathepsin k
Ketoamide
Aldehyde
X-ray structure
Cathepsin S has been a target of extensive studies by the phar-
maceutical industry.1 A variety of inhibitor subtypes have been
patented,2 published and reviewed.3 We have recently published
heteroarylnitrile based cathepsin K and S inhibitors with trifluoro-
methylphenyl as a distinctive P2 group, such as compound 1
(Fig. 1).4–9
group was attached, compound 413 showed ca. 1
lM inhibitory
activity against human cathepsin S. With a view to increasing the
torsion angle between the trifluoromethylphenyl ring and the
amide group for a potentially better binding conformation, a chlo-
rine atom was inserted at the 2-position of the phenyl ring to give
compound 5. This chlorine atom boosted the human cathepsin S
inhibitory activity about 30-fold to 31 nM. An X-ray structure of
compound 5 bound to human cathepsin S was obtained (Fig. 2).
As part of an effort for identifying non-heteroarylnitrile based
cathepsin S inhibitors of diminished peptidic elements, we rea-
soned that it might be possible to convert the pyrimidine ring of
compound 1 (Fig. 1) to the corresponding aliphatic nitrile based
inhibitor of compound 2. Disappointingly compound 2 showed
no inhibitory activity against both human cathepsins S and K. Com-
puter-aided docking studies suggest that the trifluoromethyl-
phenyl group is not in the correct conformation while the
cyanomethyl amide part overlays well with known cyanomethyl
amide based cathepsin S inhibitors.10 As the thioimidate formed
between the nitrile war-head and the active site cysteine thiol
adopts a flat sp2 structure, it was hoped that an aldehyde war-head
might fit better to the binding site as the thiol hemiacetal formed
from the covalent binding has a tetrahedral sp3 structure. Again,
compound 3 is inactive. Encouragingly, when a small P111,12 ethyl
O
O
N
F3C
F3C
O
F3C
N
H
CN
N
H
N
CN
H
2
3
1
hCat S IC50: 41nM
hCat S IC50: >10000nM
hCat S IC50: >10000nM
O
O
Cl
F3C
O
F3C
O
H
N
H
N
H
H
5
4
hCat S IC50: 31nM
hCat S IC50: 1071nM
⇑
Corresponding author. Tel.: +44 1698 736133; fax: +44 1698 736187.
E-mail address: jiaqiang.cai@merck.com (J. Cai).
Figure 1. Evolution from arylnitrile to aldehyde based human cathepsin
inhibitors using trifluoromethylphenyl as P2.
S
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.012

J. Cai et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6890–6894
6891
Table 1
Compounds inhibitory activity against human cathepsins S and K
Compds
R1
R2
IC50 (nM)
CatS
CatK
33
1
2
3
4
5
41
>10,000
>10,000
1070
31
36
18
>10,000
>10,000
na
32
na
na
na
na
H
H
Cl
F
Me
MeO
Cl
H
Et
Et
Et
Et
Et
Me
9
10
11
12
692
110
For assay conditions see Ref. 7; na = not available.
R2
O
Cl
O
Cl
O
R2
O
a
F3C
R1
F3C
R1
OH
N
O
H2N
O
H
OH
OH
14
Figure 2. Crystal structure of compound 5 covalently bound to human cathepsin S
enzyme, solved at 2 Å resolution (PDB code: 3OVX and RCSB code: rcsb061638).
15
13
The Cl atom interacts with the backbone of Gly69, Met71, and
Asn165, and the torsion angle between the phenyl ring and the
amide is 70°, much larger than the one expected for the unsubsti-
tuted phenyl (normally <40°). This suggests that the function of the
chlorine might be to increase binding to the protein but also to
keep the trifluoromethylphenyl ring in an optimal orientation for
binding to S2. The X-ray structure also shows the ethyl group inter-
acting with the backbone of Asn67-Gly68.
O
R2
O
Cl
O
R2
O
Cl
Cl
R3
c
b
F3C
R1
F3C
R1
N
H
OH
N
N
H
H
OH
OH
17
16
O
R2
O
R3
d
F3C
R1
N
H
N
H
O
Compounds 2–5 and further aldehyde analogs were synthesized
according to Scheme 1. Direct amide coupling between acyl chlo-
rides 6 and amines 7 gave compound 2 and other alcohol interme-
diates 8. Oxidation of alcohols 8 with Dess–Martin periodinane
gave the expected aldehydes in high yield. The cathepsin inhibitory
results are summarized in Table 1.
18-32
Scheme 2. Reagents and conditions: (a) HBTU, TEA, DCM, 5 h, 70–90%; (b) LiOH,
THF–H2O (1:1), 3 h, 100%; (c) HATU, DIPEA, R3NH2, rt, 3 h; (d) EDCI, DMSO–toluene
(1:1), rt, 3 h, 20–80%.
As shown in Table 1, compounds 9 and 10 with F, Me at 2-posi-
tion of the phenyl ring are as active as compound 5. However com-
pound (11) is much less potent, presumably because an
intramolecular H-bond between the methoxy oxygen and amide
(H) could alter the required active conformation. The methoxy
group could also be too big to fit in the S2 pocket. Methyl as P1
(12) is slightly less active than the corresponding ethyl group.
With these results in hand, we turned our attention to the more
drug-like ketoamide as war-head using the newly identified 2-
chloro-3-trifluoromethylphenyl as the P2 group. It should also be
possible that interactions with the S10 pocket from this ketoamide
could afford higher binding affinity. The general synthesis of the
ketoamide inhibitors is shown in Scheme 2. Amide coupling be-
tween carboxylic acids 13 and b-aminoesters 14 gave intermedi-
ates 15 which were then converted to the corresponding
carboxylic acids 16 in high yields. Another amide formation be-
tween 16 and amines using HATU as coupling reagent gave the sec-
ondary alcohol intermediates 17 which were then converted to the
desired final ketoamides 18–32 by treating with EDCI in a mixed
solvent of DMSO and toluene.
Cl
Cl
Cl
F3C
O
NO2
a
F3C
F
NO2
b
F3C
F
33
N
34
35
Cl
Cl
c
F3C
O
CN
F3C
NH2
d
N
N
O
36
37
Cl
O
F3C
O
e
OH
N
38
Scheme 3. Reagents and conditions: (a) HNO3–H2SO4, 0 °C to rt, 1 h, 80%; (b)
2-pyridylmethanol, NaH, THF, rt, 5 h, 90%; (c) SnCl2, EtOH, rt, 3 h, 90%; (d) (i) NaNO2,
H2SO4 (aq 10%); (ii) CuCN, NaCN, Na2CO3, 80 °C, 2 h, 30%; (e) NaOH (aq 1 M), 120 °C,
3 days, 100%.
of compound 35 with tin(II) chloride provided aniline compound
36 which was then converted to the arylnitrile 37 by means of a
Sandmeyer reaction. The nitrile group of compound 37 was then
hydrolyzed to the carboxylic acid 38 by heating with 1 M sodium
hydroxide at 120 °C for 3 days.
The carboxylic acid 38 was synthesized using the route shown
in Scheme 3. Nitration of 2-chloro-6-fluorobenzyltrifluoride 33
gave intermediate 34 which was then treated with 2-pyridylmeth-
anol and sodium hydride in THF to afford compound 35. Reduction
Carboxylic acid 43 was synthesized according to Scheme 4.
Treatment of compound 34 with sodium ethoxide in ethanol gave
intermediate 39 which was then reduced by tin(II) chloride to the
corresponding aniline 40. This aniline was converted to the aryl io-
dide 41 by using the Sandmeyer reaction. The methyl ester 42 was
obtained in high yield by treating the iodide with carbon monoxide
in methanol using palladium acetate as catalyst at 50 °C. The final
R1
O
R2
O
R1
O
R2
R1
H2N R3
R2
a
b
O
F3C
F3C
R3
F3C
N
Cl
N
H
H
H
3-5, 9-12
2, 8
7
6
Scheme 1. Reagents and conditions: (a) NaHCO3, H2O–THF, rt, 1 h; (b) Dess–Martin
periodinane, DCM, rt, 3 h.

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J. Cai et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6890–6894
Cl
carboxylic acid 43 was obtained by hydrolyzing the methyl ester
with aqueous lithium hydroxide.
Cl
Cl
F3C
O
NH2
F3C
O
NO2
F3C
F
NO2
a
b
The b-amino esters of Scheme 2 were synthesized according to
Scheme 5. The Boc protected aldehydes 44 were treated with ace-
tone cyanohydrin and triethylamine in DCM at room temperature
for 20 h to give the cyanohydrin intermediates 45 in quantitative
yield. Heating at reflux of the cyanohydrin intermediates in 5 M
hydrochloride in MeOH for 6 h provided the b-amino esters 14 in
quantitative yield.
40
34
39
Cl
Cl
O
Cl
O
F3C
I
d
F3C
O
e
F3C
O
c
O
OH
O
41
43
42
The biological results of compounds 18–32 are shown in
Table 2.
Scheme 4. Reagents and conditions: (a) EtONa–EtOH, rt, 3 h, 79%; (b) SnCl2, EtOH,
rt, 3 h, 100%; (c) (i) NaNO2, HCl, 30 min; (ii) KI, rt, 8 h, 59%; (d) CO (6 bar), DMF-
MeOH (1:1), TEA, Pd(OAc)2 (cat.), 50 °C, 6 h, 98%; (e) LiOH, THF–H2O, rt, 3 h, 100%.
In comparison with aldehyde 12, the ketoamide compound 18
is slightly more potent. In order to improve interactions with the
S10 pocket, a set of two dozen amides was synthesized. Of these
compounds, only 2-aminopyrazole derivatives 19–21 were identi-
fied to have better cathepsin S inhibitory activity than the original
primary amide 18. As with the aldehyde series, ethyl as P1 is also
better than methyl in this ketoamide series, for example, com-
pound 22 is sixfold better than compound 19. Attachment of an
ethoxy group at the 4-position of the P2 phenyl ring6–8 increases
cathepsin S inhibitory potency two- to five-fold, such as compound
R2
O
R2
a
R2
b
Boc
CN
OH
Boc
O
H2N
O
N
H
N
H
OH
14
44
45
Scheme 5. Reagents and conditions: (a) acetone cyanohydrin, TEA, DCM, rt, 20 h,
100%; (b) HCl/MeOH, reflux, 6 h, 100%.
Table 2
Compounds inhibitory activity against human cathepsins S, K, B, L, V, and human JY cell cellular activity (Lip10)
Compds
Structure
IC50 (nM)
CatB
Lip10
CatS
30.9
CatK
CatL
na
CatV
na
IC50 (nM)
O
O
Cl
Cl
O
F3C
F3C
18
19
107.2
na
na
na
na
NH2
N
N
H
O
O
O
H
N
O
14.1
51.3
na
na
N
H
N
H
H
N
N
O
Cl
Cl
O
20
21
22
23
10.5
8.5
26.3
33.1
11.7
195
na
na
na
na
F3C
F3C
N
H
N
H
N
N
O
O
na
na
na
na
N
H
N
O
O
H
H
N
O
N
Cl
O
2.3
5754
na
891
na
29.5
na
1300
na
F3C
N
H
N
H
O
Cl
O
F3C
O
NH2
N
11.0
H
O
H
N
O
N
Cl
O
F3C
O
24
25
26
2.9
4.8
1.1
91.2
37.2
8.7
na
na
na
na
na
na
na
na
na
na
N
H
N
H
O
O
O
Cl
Cl
O
O
F3C
O
NH2
N
H
na
N
N
O
O
F3C
O
NH2
N
N
H
1925
N
N
O
O
Cl
Cl
O
O
F3C
O
N
N
H
27
28
1.0
1.1
12.9
20.9
15,136
3236
479
141
36.3
16.6
na
78
H
N
N
O
O
N
F3C
O
N
H
N
H

J. Cai et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6890–6894
6893
Table 2 (continued)
Compds
Structure
IC50 (nM)
Lip10
CatS
2.3
CatK
11.5
CatB
CatL
na
CatV
na
IC50 (nM)
N
N
O
N
N
Cl
O
F3C
N
H
N
H
29
30
na
na
O
N
O
O
O
O
Cl
Cl
Cl
O
O
O
F3C
1.9
47.9
na
na
na
na
N
H
N
H
O
O
O
N
O
F3C
N
H
N
H
31
32
19.5
46.8
>10,000
>10,000
>10,000
>10,000
1122
na
na
N
O
F3C
N
N
H
>10,000
>10,000
>10,000
N
O
For assay conditions see Ref. 7; na = not available.
pairs 23/18 and 24/19. Compounds 25 and 27 with pyridyl-2-
methoxy as the extended P2 are 6- and 9-fold more potent than
the unsubstituted compounds 18 and 21, respectively. These re-
sults indicate that the P2 region shares parallel SAR with the pre-
viously reported purine7 and imidazopyridine6 analogs. The
impact of P1 ethyl relative to the methyl is smaller for compounds
with the extended binding interactions, for example, nearly flat
activity for compounds 27–30. At least one proton is required for
the right-hand amide, for example, compound 32 is totally inactive
against all cysteine cathepsin proteases tested. These results agree
with the previously reported ketoamide-based cathepsin K inhibi-
tors.14 Some selected compounds were also tested for cathepsins K,
B, L, and V inhibitory activities. In general, this type of inhibitor has
low selectivity against human cathepsins K and V. Selectivity over
human cathepsin L is generally over 100-fold. Activity against hu-
man cathepsin B is quite low. Several compounds from this series
were further assessed for cell based Lip10 activity. Most com-
pounds, such as 22 and 26, have IC50 in micromolar level; one of
the most active is compound 28 which has a Lip10 IC50 of 78 nM.
An X-ray structure of compound 26 bound to human cathepsin
K was obtained (Fig. 3a). Based on this structure, compound 26 was
then modeled into the active site of human cathepsin S. This model
suggests that the pyridyl-2-methoxy group occupies the extended
S2 pocket of cathepsin S, interacting with Phe70, Phe211, and
Val162 as reported before.6 2-Chloro-3-trifluoromethylphenyl
binds to the most important S2 pocket with a torsion angle of
approximately 70° between the phenyl ring and the secondary
amide. The ethyl group interacts with the shallow S1 protein sur-
face. The amide of the ligand forms hydrogen bonding interactions
to Gln19, and possibly to solvent, while the hydroxyl group result-
ing from the reaction of the ketoamide group with Cys25 has a
hydrogen bonding interaction to His164 (His162 in cathepsin K).
The cathepsin S structure suggests that there is space available in
the prime side for a secondary amide, and a pyrazole substituent
could potentially have an attractive interaction to Trp186. Tertiary
amides, such as compound 32, are not tolerated, probably because
the extra methyl could disrupt the hydrogen bonding interaction to
His164.
In summary, the trifluoromethylphenyl P2 motif from previ-
ously reported heteroarylnitrile series has been successfully ap-
plied for the design and synthesis of highly potent novel
ketoamide-based cathepsin S inhibitors. The key in this process is
the change of the torsion angle between the P2 phenyl ring and
the attached secondary amide by adding a small Cl, F, or Me group
at the 2-position. The previously reported extended S2 pocket has
also been used to improve human cathepsin S inhibitory potency.
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