Pyrazole-based cathepsin S inhibitors with improved cellular potency

Article abstract of DOI:10.1016/j.bmcl.2007.08.038

High potency pyrazole-based noncovalent inhibitors of human cathepsin S (CatS) were developed by modification of the benzo-fused 5-membered ring heterocycles found in earlier series of CatS inhibitors. Although substitutions on this heterocyclic framework

Full text of DOI:10.1016/j.bmcl.2007.08.038

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5525–5528
Pyrazole-based cathepsin S inhibitors with improved
cellular potency
Jianmei Wei, Barbara A. Pio, Hui Cai, Steven P. Meduna, Siquan Sun, Yin Gu,
Wen Jiang, Robin L. Thurmond, Lars Karlsson and James P. Edwards*
Johnson & Johnson Pharmaceutical Research & Development, L.L.C., 3210 Merryfield Row, San Diego, CA 92121, USA
Received 3 July 2007; revised 13 August 2007; accepted 15 August 2007
Available online 22 August 2007
Abstract—High potency pyrazole-based noncovalent inhibitors of human cathepsin S (CatS) were developed by modification of the
benzo-fused 5-membered ring heterocycles found in earlier series of CatS inhibitors. Although substitutions on this heterocyclic
framework had a moderate impact on enzymatic potency, dramatic effects on cellular activity were observed. Optimization afforded
indole- and benzothiophene-derived analogues that were high affinity CatS inhibitors (IC50 = 20–40 nM) with good cellular potency
(IC50 = 30–340 nM).
Ó 2007 Elsevier Ltd. All rights reserved.
N
Cathepsin S (CatS) is a cysteine protease of the papain
family that is involved in the presentation of antigens
to the cell surface of certain antigen-presenting cells
(APCs) for recognition by CD4+ T-cells. The main
APC target of the proteolytic activity of CatS is the
invariant chain (Ii), a chaperone molecule for major his-
tocompatibility complex class II molecules (MHCII).1–3
Inhibition of CatS activity impedes the removal of Ii
from MHCII molecules, and thus attenuates antigen
presentation to CD4+ T-cells. Other pharmacologically
relevant activities have also been attributed to CatS.4
Numerous selective CatS inhibitor chemotypes have
been reported, most relying upon covalent modification
of the active site cysteine to achieve good activity.5 Re-
cently, potent noncovalent inhibitors have emerged
from these laboratories6 and from researchers at
Novartis.7,8
I
CN
N
N
N
O
O
NH2
N
NH2
1
O
N
Cl
N
N
O
N
N
Me
Cl
N
SO2Me
2
N
CF3
O
N
N
OH
N
O
N
SO2Me
3
We have previously reported high potency noncovalent
inhibitors of human CatS based on a tetrahydropyri-
do-pyrazole heterocycle. Representative analogues with
different aryl- and heteroaryl-substituted piperazine
and piperidine groups are shown below.
While compounds 1–3 all inhibit human CatS with
IC50’s in the 10–20 nM range, they are much less active
in a secondary cellular assay measuring Ii degradation in
(human) JY cells (IC50 = 0.8–1.2 lM). We had earlier
used to advantage the tolerance of CatS inhibitory po-
tency to structural variability in the aryl/heteroaryl
groups found in the left-hand portion of the pharmaco-
phore to address issues of selectivity and oral bioavail-
ability.6c,d This letter describes a series of benzo-fused
heterocycles found to have greatly improved cellular
potencies.
Keywords: Cathepsin S; Antigen processing; Enzyme inhibitor.
*
Corresponding author. Tel.: +1 858 450 2024; fax: +1 858 784
3116; e-mail: jedward7@prdus.jnj.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.08.038

5526
J. Wei et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5525–5528
Following our previously described route, the known9
benzofurans 4a–b and benzothiophenes 4c–d were pre-
pared and coupled with epoxides 5a and 5b to afford
the racemic target molecules 6–11 (Scheme 1). These
molecules were moderately potent inhibitors of CatS
(Table 1). Replacement of oxygen with sulfur had at best
a minimal positive impact on CatS potency (6 vs 7; 8 vs
9), while the carboxylate analogues showed a trend to-
ward increased potency (6 vs 8; 7 vs 9). Replacing the
acetamide on the tetrahydropyridine ring with sulfon-
amide had little to no impact on potency (7 vs 10; 9 vs
11). Although CatS activity was relatively insensitive
to changes to the ‘head group’ heterocycle, the most po-
tent analogue, compound 11, provided a convenient
handle to interrogate a previously unexplored region
of the pharmacophore.
O
NH
N
F
CF3
H
N
+
a
S
CO2Me
N
12
4c
SO2Me
N
N
N
N
CF3
F
S
CO2Me
SO2Me
13
Scheme 2. Reagents and conditions: (a) NaBH(OAc)3, AcOH, CH2
Cl2, rt (60%).
obtained. As shown in Table 2, changes to the C2 posi-
tion of the benzothiophene impacted both enzymatic
and cellular potency.
Initial attempts to transform the ester moiety of 11 to
amide derivatives were thwarted by intramolecular cap-
ture of activated ester derivatives by the C20 linker hy-
droxyl. We had previously observed6c that this
hydroxyl substituent was unimportant for CatS potency,
so we prepared the des-hydroxy analogue of 11, com-
pound 13 (Scheme 2). Thus, treatment of aldehyde 12
with piperidine 4c in the presence of NaBH(OAc)3 affor-
ded the requisite ester 13 in good yield.
Although hydrolysis of the ester 13 to the acid 14 had no
effect on CatS potency, cellular activity was abolished.
Conversion of the methyl ester to the dimethyl amide
(15) resulted in reduced CatS potency. Interestingly the
ethyl amide 16, isomeric with 15, was 10-fold more po-
tent (37 nM vs 370 nM), perhaps indicative of a favor-
able hydrogen bond interaction with the protein in this
region of the pharmacophore. Compound 16 main-
tained good cellular activity (IC50 = 1.2 lM). Although
encouraged by the activity of these new molecules, aque-
ous solubility was uniformly poor. The 2-aminoethyl
analogue 17 and 2-(1-morpholino)ethyl analogue 18
were thus prepared. Although the solubility was little
changed (data not shown), both 17 and 18 were potent
CatS inhibitors. The addition of the amino moiety to
16 resulted in reduced cellular potency for 17, while
the morpholine analogue 18 was 3-fold more potent
than 16 in the JY assay. Preliminary rat PK experiments
on several analogues demonstrated low levels of oral
bioavailability, however, and this series was not pursued
further.
Functional group manipulation of the ester moiety of 13
afforded a series of amide analogues, and the CatS enzy-
matic potency and cellular data for these inhibitors were
NH
N
F
CF3
N
O
+
a
X
R
N
P
4a: X = O; R = CO2Me
4b: X = O; R = H
4c: X = S; R = CO2Me
4d: X = S; R = H
5a: P = Ac
5b: P = MeSO2
N
N
N
CF3
F
OH
X
N
P
R
6-11
Scheme 1. Reagent and conditions: (a) EtOH, 60–70 °C (50–75%).
Table 2. Carboxylate/carboxamide CatS inhibitorsa,b
Table 1. Benzofuran/thiophene-based CatS inhibitorsa
N
N
N
CF3
F
N
N
N
CF3
F
OH
S
R
N
SO2Me
O
X
N
P
R
Compound
R
CatS IC50 (nM) JY Ii IC50 )lM)
Compound
X
R
P
CatS IC50 (nM)
13
14
15
16
17
18
OMe
OH
130
100
370
37
1.8
>10
ND
1.2
6
7
O
S
H
H
Ac
Ac
Ac
Ac
360
290
220
140
180
130
NMe2
NHEt
8
O
S
CO2Me
CO2Me
H
NHCH2CH2NH2 47
NHCH2CH2R*
39
5.5
0.34
9
10
11
S
MeSO2
MeSO2
S
CO2Me
a CatS IC50 and JY Ii degradation assay IC50 values are means of
n P 3 runs and determined as described previously. All IC50’s were
within a 3-fold range.6a
a CatS IC50 and JY Ii degradation assay IC50 values are means of
n P 3 runs and determined as described previously. All IC50’s were
within a 3-fold range.6a ND, not determined.
b R*, 1-morpholino.

J. Wei et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5525–5528
5527
Whilst pursuing the benzofuran and benzothiophene
analogues described above, we simultaneously prepared
the analogous indole derivatives. Although the parent 4-
(indol-3-yl)piperidine was commercially available,
substituted analogues had to be synthesized (Scheme
3). Accordingly, condensation of an indole 19 with 1-
tert-butoxycarbonyl-4-piperidone 20 afforded the tetra-
hydropyridines 21. Reduction of the olefin by transfer
hydrogenation and subsequent treatment with trifluoro-
acetic acid afforded the requisite indole-substituted pip-
eridines 23 in excellent yields. Conversion to the target
molecules proceeded as shown in Scheme 1.
had no impact on potency (32 and 33), nor did the addi-
tion of a methyl group at C2 of the indole (compound
34). These observations are similar to what was ob-
served in our earlier series of ketobenzimidazole
derivatives.6c
Although the indoles 24–34 all displayed excellent enzy-
matic potency, the cellular data were quite inconsistent.
For example, compound 24 had an IC50 of 2.3 lM in
our JY Ii assay, while the chloro analogue 25 was inac-
tive at concentrations up to 10 lM, as were many of the
other indoles (e.g., 28, 29, and 32). Of the analogues
tested, only the ethyl ester 31 had an IC50 below one
micromolar in the JY Ii assay (IC50 = 410 nM).
The indol-3-yl piperidines demonstrated uniformly
excellent CatS inhibition (Table 3). The parent analogue
24 had an IC50 of 72 nM that was only moderately af-
fected by substitutions on the indole core. For example,
the addition of a chlorine atom to C50 (25) had no im-
pact on CatS potency, while replacement of the chlorine
atom in the sulfonamide analogue 27 with methoxy,
methyl, cyano or ethyl carboxylate afforded potencies
in a very tight range (21–58 nM; compounds 28–31).
The position of a chlorine atom on the indole nucleus
The excellent enzymatic potency of the indoles combined
with the high sensitivity of cellular activity to changes in
functionality displayed in both the indole and benzothi-
ophene series prompted us to consider further modifica-
tions of the indole core to improve cellular potency.
Thus, we prepared azaindole analogues to gauge the im-
pact of reduced lipophilicity of the indole moiety on activ-
ity. The requisite piperidines were prepared from all four
azaindole isomers following the route depicted in Scheme
3, and the data obtained on the CatS inhibitors derived
therefrom are tabulated in Table 4.
R1
Boc
R1
Boc
N
N
+
a
O
HN
19
All four azaindoles were well tolerated by the enzyme,
although the 4-aza analogue 35 was 2- to 3-fold less po-
tent than the other isomers 36–38. A much larger impact
on cellular potency was observed. Although the weakest
inhibitor of the enzyme, compound 35 had an IC50 of
95 nM in the cellular assay. The 5-aza analogue 36
and 6-aza analogue 37 were also quite potent at inhibit-
ing p10 degradation in this assay, with IC50’s of 25 nM
and 38 nM, respectively. The anomaly in this set of
molecules was the 7-aza derivative 38. Although a very
potent inhibitor of the enzyme (IC50 = 27 nM), com-
pound 38 was only moderately active in JY cells
(IC50 = 860 nM). The reasons for the decreased cellular
potency of 38 relative to the isomers 35–37 are not readily
R2
HN
21
R2
20
b
Boc
R1
R1
NH
N
c
HN
23
HN
22
R2
R2
Scheme 3. Reagents and conditions: (a) KOH, MeOH, reflux (85–
95%); (b) Pd/C, NH4HCO2, MeOH, reflux (85–95%); (c) TFA,
CH2Cl2, rt, (95+%).
Table 3. Effect of indole substitution on CatS activitya
Table 4. Enzymatic and cellular potency of azaindole analoguesa
5
N
R1
6
4
R3
N
N
OH
N
X5
R3
N
N
X4
7
X6
OH
HN
R2
N
P
X7
HN
N
Compound R1
R2
R3
P
CatS IC50 (nM)
SO2Me
24
25
26
27
28
29
30
31
32
33
34
H
H
H
H
H
H
H
H
H
H
H
CF3 Ac
CF3 Ac
72
95
Compound X4 X5
X6 X7 R3
CatS JY Ii
IC50
(nM)
5-Cl
5-Cl
5-Cl
IC50 (lM)
Br
MeSO2 54
CF3 MeSO2 44
CF3 MeSO2 29
CF3 MeSO2 58
CF3 MeSO2 21
CF3 MeSO2 27
CF3 MeSO2 29
CF3 MeSO2 34
35
36
37
38
39
40
41
N
H
H
H
H
H
H
H
N
H
H
N
H
H
H
N
H
H
H
H
H
H
H
N
H
N
H
CF3 120
0.095
0.025
0.038
0.86
5-MeO
5-Me
5-CN
5-CO2Et
6-Cl
CF3
CF3
CF3
Br
58
30
27
77
0.15
ND
Br 195
CF3 200
7-Cl
5-Cl
N+–Oꢀ
0.24
Me CF3 MeSO2 37
a CatS IC50 and JY Ii degradation assay IC50 values are means of
n P 3 runs and determined as described previously. All IC50’s were
within a 3-fold range.6a
a CatS IC50 and JY Ii degradation assay IC50 values are means of
n P 3 runs and determined as described previously. All IC50’s were
within a 3-fold range.6a

5528
J. Wei et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5525–5528
4. (a) Chang, W.-S. W.; Wu, H.-R.; Yeh, C.-T.; Wu, C.-W.;
Chang, J.-Y. J. Cancer Mol. 2007, 3, 5; (b) Sukhova, G.
K.; Zhang, Y.; Pan, J.-H.; Wada, Y.; Yamamoto, T.;
Naito, M.; Kodama, T.; Tsimikas, S.; Witztum, J. L.; Lu,
M. L.; Sakara, Y.; Chin, M. T.; Libby, P.; Shi, G.-P. J.
Clin. Invest. 2003, 111, 897; (c) 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, doi:10.1016/
j.pain.2006.11.017.
5. (a) Kim, W.; Kang, K. Expert Opin. Ther. Pat. 2002, 12,
419; (b) Leroy, V.; Thurairatnam, S. Expert Opin. Ther.
Pat. 2004, 14, 301; (c) Link, J. O.; Zipfel, S. Curr. Opin.
Drug Disc. Dev. 2006, 9, 471; (d) Thurmond, R. L.; Sun,
S.; Karlsson, L.; Edwards, J. P. Curr. Opin. Invest. Drugs
2005, 6, 473.
6. (a) Thurmond, R. L.; Sun, S.; Sehon, C. A.; Baker, S.
M.; Cai, H.; Gu, Y.; Jiang, W.; Riley, J. P.; Williams, K.
N.; Edwards, J. P.; Karlsson, L. J. Pharm. Exper. Ther.
2004, 308, 268; (b) Thurmond, R. L.; Beavers, M. P.;
Cai, H.; Meduna, S. P.; Gustin, D. J.; Sun, S.; Almond,
H. J.; Karlsson, L.; Edwards, J. P. J. Med. Chem. 2004,
47, 4799; (c) Gustin, D. J.; Sehon, C. A.; Wei, J.; Cai, H.;
Meduna, S. P.; Khatuya, H.; Sun, S.; Gu, Y.; Jiang, W.;
Thurmond, R. L.; Karlsson, L.; Edwards, J. P. Bioorg.
Med. Chem. Lett. 2005, 15, 1687; (d) Grice, C. A.; Tays,
K.; Khatuya, H.; Gustin, D. J.; Butler, C. R.; Wei, J.;
Sehon, C. A.; Sun, S.; Gu, Y.; Jiang, W.; Thurmond, R.
L.; Karlsson, L.; Edwards, J. P. Bioorg. Med. Chem.
Lett. 2006, 16, 2209.
apparent. Screening of other related proteases (CatB,
CatE, CatL, legumain) showed no inhibition at concen-
trations up to 5 lM, for those analogues tested.
Anticipating that the pyridine nitrogen of the azaindole
would be a metabolic liability, the pyridine N-oxide of
36, compound 41, was also prepared. This molecule
was significantly less active than 36 in both the enzy-
matic and cellular assays, although the cellular potency
of 240 nM was very competitive with previously re-
ported analogues.6
After this work was completed, a report from Merck-
Frosst demonstrated that certain cathepsin K (CatK)
inhibitors with moderate activity against CatS enzyme
displayed significant levels of CatS inhibitory activity
in cellular assays.10 In an elegant series of experiments,
these investigators demonstrated that covalent, revers-
ible CatK inhibitors with basic moieties (e.g., pipera-
zines) accumulated in lysosomes both in vitro and
in vivo (rat whole-body radiography), thus leading to
a loss in cellular selectivity. Similar experiments have
not been performed on the molecules reported in this let-
ter, although the improved cellular potency of the azain-
doles (Table 4) compared to the indoles (Table 3) may
be indicative of lysosomotropism.
In conclusion, bioisosteric replacement of the head-
group ketobenzimidazole piperidines with 4-(indol-3-
yl) piperidines resulted in a new series of CatS inhibitors
with greatly improved cellular potency. Determining the
reason for the disparity between enzymatic potency and
cellular potency for these noncovalent CatS inhibitors
still requires further investigation. The utility of these
novel CatS inhibitors in elucidating the pharmacology
of CatS, and its potential as a target for immunosup-
pressive therapies, will be reported in due course.
7. (a) Altmann, E.; Renaud, J.; Green, J.; Farley, D.;
Cutting, B.; Jahnke, W. J. Med. Chem. 2002, 45, 2352;
(b) Altmann, E.; Green, J.; Tintelnot-Blomley, M. Bioorg.
Med. Chem. Lett. 2003, 13, 1997.
8. (a) Liu, H.; Tully, D. C.; Epple, R.; Bursulaya, B.; Li,
J.; Harris, J. L.; Williams, J. A.; Russo, R.; Tumanut,
C.; Roberts, M. J.; Alper, P. B.; He, Y.; Karanewsky,
D. S. Bioorg. Med. Chem. Lett. 2005, 15, 4979; (b)
Alper, P. B.; Liu, H.; Chatterjee, A. K.; Nguyen, K. T.;
Tully, D. C.; Tumanut, C.; Li, J.; Harris, J. L.;
Tuntland, T.; Chang, J.; Gordon, P.; Hollenbeck, T.;
Karanewsky, D. S. Bioorg. Med. Chem. Lett. 2006, 16,
1486; (c) Tully, D. C.; Liu, H.; Alper, P. B.; Chatterjee,
A. K.; Epple, R.; Roberts, M. J.; Williams, J. A.;
Nguyen, K. T.; Woodmansee, D. H.; Tumanut, C.; Li,
J.; Spraggon, G.; Chang, J.; Tuntland, T.; Harris, J. L.;
Karanewsky, D. S. Bioorg. Med. Chem. Lett. 2006, 16,
1975.
9. Watanabe, Y.; Yoshiwara, H.; Kanao, M. J. Het. Chem.
1993, 30, 445.
10. Falgueyret, J.-P.; Desmarais, S.; Oballa, R.; Black, W. C.;
Cromlish, W.; Khougaz, K.; Lamontagne, S.; Masse, F.;
Riendeau, D.; Toulmond, S.; Percival, M. D. J. Med.
Chem. 2005, 48, 7535.
References and notes
1. Villandangos, J. A.; Bryant, R. A. R.; Deussing, J.;
Driessen, C.; Lennon-Dumenil, A.-M.; Riese, R. J.; Roth,
W.; Saftig, P.; Shi, G.-P.; Chapman, H. A.; Peters, C.;
Ploegh, H. L. Immunol. Rev. 1999, 172, 109.
2. Nakagawa, T. Y.; Rudensky, A. Y. Immunol. Rev. 1999,
172, 121.
3. Shi, G.-P.; Villadangos, J. A.; Dranoff, G.; Small, C.; Gu,
L.; Haley, K. J.; Riese, R.; Ploegh, H. L.; Chapman, H. A.
Immunity 1999, 10, 197.