Discovery and SAR of novel pyrazole-based thioethers as cathepsin S inhibitors: Part 1

Article abstract of DOI:10.1016/j.bmcl.2010.01.108

A series of pyrazole-based thioethers were prepared and found to be potent cathepsin S inhibitors. A crystal structure of 13 suggests that the thioether moiety may bind to the S3 pocket of the enzyme. Additional optimization led to the discovery of aminoethylthioethers with improved enzymatic activity and submicromolar cellular potency.

Full text of DOI:10.1016/j.bmcl.2010.01.108

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2370–2374
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and SAR of novel pyrazole-based thioethers as cathepsin S inhibitors:
Part 1
a
a
a
a
Alice Lee-Dutra a, , Danielle K. Wiener , Kristen L. Arienti , Jing Liu , Neelakandha Mani ,
Michael K. Ameriks a, Frank U. Axe a, , Damara Gebauer a,à, Pragnya J. Desai a, Steven Nguyen a,
Mike Randal b,§, Robin L. Thurmond a, Siquan Sun a, Lars Karlsson a, James P. Edwards a, Todd K. Jones a,
Cheryl A. Grice a
*
a Johnson & Johnson Pharmaceutical Research & Development, L.L.C., 3210 Merryfield Row, San Diego, CA 92121, USA
b Sunesis Pharmaceuticals, 341 Oyster Point Boulevard, South San Francsico, CA 94080, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of pyrazole-based thioethers were prepared and found to be potent cathepsin S inhibitors. A crys-
tal structure of 13 suggests that the thioether moiety may bind to the S3 pocket of the enzyme. Additional
optimization led to the discovery of aminoethylthioethers with improved enzymatic activity and submi-
cromolar cellular potency.
Received 30 October 2009
Revised 15 January 2010
Accepted 20 January 2010
Available online 28 January 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Cathepsin
Protease
Thioether
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 target of the proteolytic activity of CatS is the
invariant chain (Ii), a chaperone molecule for major histocompati-
bility complex class II molecules (MHC II).1 Inhibition of CatS activ-
ity slows the removal of Ii and attenuates antigen presentation to
CD4+ T-cells, resulting in immunosuppression with specificity for
these T-cells.
We have previously reported our efforts to identify novel non-
covalent inhibitors of CatS based on a tetrahydropyrido-pyrazole
heterocycle (Fig. 1).2 Much of this work focused on the SAR of sub-
stituent variations of the ‘headgroup’ as well as the P4 group, as
exemplified by compounds 1 and 2. More recently, we became
interested in exploring substitution that would access S1 binding,
ultimately discovering novel arylalkynes, such as compound 3, that
bind to this region of the enzyme.2f Concurrent efforts to explore
alternative binding elements led to the preparation of analogs con-
taining aryl thioethers as a replacement for these arylalkynes.
Preliminary investigation of such thioethers was conducted
using aryl iodide intermediates 4 and 5, which were synthesized
as previously reported.2f Metal-mediated coupling with commer-
cially available thiols afforded the desired thioether products
(Scheme 1).3
As seen in Table 1, these initial compounds exhibited promising
activity. Compounds 6 and 7 share similarly moderate enzymatic
activity, indicating that the elongation of the methylene tether is
not detrimental to activity. Replacement of the phenyl group with
a methyl group (8) appears to decrease activity, whereas the incor-
poration of a phenyl ether (9) leads to submicromolar inhibition.
Exchanging the para-chloro moiety with a trifluoromethyl group
maintains activity (9 vs 10). However, substituting the morpholine
headgroup with 1-piperidin-4-yl-pyrrolidin-2-one led to threefold
improved activity for compound 11.
Re-design of the synthesis route enabled more facile access to
thioether variations through aromatic substitution rather than pal-
ladium-mediated coupling (Scheme 2). In this instance, initial reac-
tion with mercaptoethanol was followed by alkylation with 2-(2-
bromoethyl)-1,3-dioxolane. Unmasking the aldehyde under acidic
conditions and subsequent reductive amination provided hydrox-
yethylthioether 12, which was found to be equipotent to ether ana-
log 11.
* Corresponding author. Tel.: +1 858 320 3317; fax: +1 858 450 2089.
E-mail address: alee7@its.jnj.com (A. Lee-Dutra).
Present address: Axe Consulting Services, 14595 Surrey Junction Lane, Sutter
Creek, CA 95685, USA.
A crystal structure was obtained for a close analog of alcohol 12
(compound 13, Fig. 2).4 Notably, the hydroxyethylthioether of 13
points toward the S3 region of the active site rather than toward
à
Present address: Cambridge Research & Instrumentation, Inc., 35B Cabot Road,
Woburn, MA 01801, USA.
§
Retired.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.108

A. Lee-Dutra et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2370–2374
2371
P1
substituent
linker
OR
I
CF
Cl
3
N
N
N
CN
N
N
O
N
N
N
N
N
OH
O
N
O
Cl
N
N
N
P4
substituent
NH
2
SO CH
SO CH
2
3
2
3
O
P5 substituent
"headgroup"
2
3
1: R = CONH
2
Figure 1. Previous CatS inhibitors with tetrahydropyrido-pyrazole core.
Table 1
R
Effect of thioether substitution on cathepsin S activitya
N
N
amine
I
a
R
N
1
amine
N
SR
N
SO2CH3
N
SO CH
4: R = Cl
2
3
5: R = CF
R1
CatS IC50
(lM)
3
Compd
Amine
R
6
7
8
9
10
Morpholine
Morpholine
Morpholine
Morpholine
Morpholine
Cl
Cl
Cl
Cl
CH2Ph
CH2CH2Ph
CH2CH2CH3
CH2CH2OPh
CH2CH2OPh
1.65
1.47
3.60
0.60
0.74
R
N
amine
N
SR1
CF3
O
N
11
CF3
CH2CH2OPh
0.22
SO2CH3
N
O
N
N
6-9, 13: R = Cl
10-11: R = CF
12
13
CF3
Cl
CH2CH2OH
CH2CH2OH
0.20
0.96
N
3
Morpholine
Scheme 1. Reagents and conditions: (a) HSR1, Pd2(dba)3, dppf, Et3N, DMF or NMP,
70–80 °C (36–95%). For HSCH2CH2OH: CuI, neocuproine, NaOt-Bu, toluene, 110 °C
(43%). R1 is defined in Table 1.
a
CatS IC50 values are the mean of n P 2 runs and determined as described
previously.2b
CF
S
3
CF
3
3
N
N
OH
R
N
a, b, c
N
HN
NO
2
O
4
R
3
4
N
N
12: NR R =
N
N
SO CH
2
3
SO CH
2
3
a, b, d
d
5
5
CF
CF
S
3
3
R
N
R
N
O
3
c
N
N
R
6
6
O
N
S
N
N
R
R
4
R
N
N
SO CH
SO CH
2 3
2
3
14-31
Scheme 2. Reagents and conditions: (a) HSCH2CH2OH, K2CO3, DMF, 90 °C (75–89%); (b) 2-(2-bromoethyl)-1,3-dioxolane, Cs2CO3, DMF (95%); (c) (i) 1 N HCl (aq), acetone,
55 °C; (ii) HNR3R4, acetic acid, NaBH(OAc)3, CH2Cl2 (6–51%, two steps); (d) (i) CH3SO2Cl, Et3N, CH2Cl2; (ii) HNR5R6, EtOH/DCE, 60 °C (33–65%, two steps). NR3R4 and NR5R6 are
defined in Tables 2 and 3.

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A. Lee-Dutra et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2370–2374
Table 2
Cl
Effect of aminoethylthioether substitution on cathepsin S activitya
N
OH
N
N
S
CF3
S
O
N
R
N
N
O
N
N
SO2CH3
N
13
SO2CH3
Compd
R
CatS IC50
,
lM
JY Ii IC50 (lM)
N
14
2.01
ND
15
16
N
1.28
1.06
ND
N
0.13
N
N
H
17
18
0.80
0.70
ND
N
H
0.46
19
20
21
22
23
24
25
0.41
0.34
0.27
0.20
0.17
0.16
0.11
0.08
0.35
0.68
0.95
0.04
0.33
0.81
N
HO
O
N
N
N
Figure 2. Crystal structure of 13 bound to a Cys25Ser mutant of cathepsin S and
interacting with one molecule of water at the S2 pocket.
N
N
S1, as was observed for arylalkynes such as 3. The close proximity
of the S3 pocket as well as the tolerance for the hydroxyl group
suggested that replacement with an amine functionality might
be acceptable. To this end, analogs were prepared using alcohol
12 as an intermediate. Formation of the mesylate and reaction with
various amines led to compounds 14–26. Alternatively, by revers-
ing the final two steps of the sequence, the right-hand side amine
was held constant and the headgroup substitution was investi-
gated, furnishing amines 27–31.
N
N
26
0.055
0.46
a
CatS IC50 and JY Ii degradation IC50 values are the mean of n P 2 runs and
determined as described previously.2b ND = not determined.
The enzymatic data in Table 2 for the right-hand side analogs
suggests a preference for cyclic (19–26) over acyclic (14–15, 17–
18) amine substitution. Neither the display of an additional basic
amine (16) nor truncation of the amine substitution (15, 17) is fa-
vored. Double-digit nanomolar activity was attained through the
incorporation of decahydroisoquinoline (26).
In general, the cellular activity of these analogs, as measured by
the invariant chain degradation in a human JY cell assay, is submi-
cromolar and reasonably corresponds to the enzymatic data. Three
examples (16, 19, and 23) exhibit enhanced cellular versus
enzymatic inhibition. This anomaly has been reported previously
for our tetrahydropyrido-pyrazoles and may be due to
lysosomotropism.2e,5
Variation of the headgroup can also influence the enzymatic
activity, as previously observed between compounds 10 and 11
(Table 1). Compound 23 is included in Table 3 for ease of compar-
ison with these additional analogs. Notably, smaller headgroups
such as morpholine (27), pyrrolidine (29), and piperidine (30) are
less preferred over the parent compound 23. The presence of an
additional basic amine (28) is not favored in this region of the mol-
ecule, though not detrimental to activity as it was for compound
16. Replacement of 1-piperidin-4-yl-pyrrolidin-2-one with the
headgroup from 2 yields 31 and confers equipotent enzymatic
The observed cellular activity for compounds 28–30 closely
matches the enzymatic data. However, both 27 and 31 experience
a drop in their cellular activity with respect to their enzymatic
inhibition, though 31 remains relatively potent compared to the
other analogs with an IC50 = 210 nM.
Concomitantly, amide analogs were also explored using an
alternative thioether intermediate (Scheme 3). The synthesis of
these amides allowed for the incorporation of a 2-hydroxypropyl
linker, which was featured in previous work in the tetrahydropyr-
ido-pyrazoles and is exemplified by compound 2. Though enzy-
matic and cellular activity is usually unchanged, the inclusion of
this hydroxyl group can provide different physicochemical proper-
ties for the molecule. Alkylation with epichlorohydrin proceeded
smoothly with the Boc-protected aminoethylthioether. Following
nucleophilic opening of the epoxide and removal of the protecting
group, standard coupling conditions enabled ready access to race-
mic mixtures of the amide analogs 32–36. An additional deprotec-
tion step provided compound 37. Notably, similar alkylating
protocols were not regioselective with the hydroxyethylthioether
intermediate from Scheme 2; alkylation would occur equally at
either of the nitrogen atoms of the pyrazole ring.
There are few clear trends from the enzymatic data for these
moderately active amides (Table 4). Substitution on the benzamide
does little to affect enzymatic inhibition. In fact, neither the inclu-
inhibition (2: CatS IC50 = 0.023 lM).

A. Lee-Dutra et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2370–2374
2373
Table 3
Table 4
Effect of varying the headgroup on cathepsin S activitya
Effect of amidoethylthioether substitution on cathepsin S activitya
CF3
CF3
S
H
N
N
N
N
R
R
N
S
N
N
O
OH
O
N
N
N
SO2CH3
SO2CH3
Compds
32
R
CatS IC50
0.38
(lM)
Compd
R
Cat S IC50
(lM)
JY Ii IC50
0.04
(lM)
O
23
0.17
N
N
33
0.39
OH
27
28
29
30
O
0.81
0.56
0.52
0.38
3.57
0.32
0.47
0.38
N
34
0.36
F
N
N
N
35
0.53
N
36
37
0.13
0.42
N
O
N
NH
a
O
N
See Table 1 for details. These compounds were tested as racemic mixtures.
31
0.03
0.21
a
aminoethylthioethers are easily influenced by amine choice at
the headgroup or at the right-hand side of the molecule. Efforts
to improve cellular activity within this thioether series will be re-
ported in due course.
See Table 2 for details.
sion of an aniline (35) nor a more basic piperidine group (37) im-
pacts activity. Only the insertion of a methylene group (benzamide
32 vs phenylacetamide 36) results in a threefold increase in CatS
inhibition. Ultimately, additional analogs in this amide series,
including des-hydroxy linker-containing compounds, as well as
further study of individual enantiomers were deprioritized due to
References and notes
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In conclusion, novel thioethers have been discovered as CatS
inhibitors, where the thioether portion of the molecule appears
to serve as a potential S3 binding element. The amidoethylthioe-
thers demonstrate moderate enzymatic inhibition despite varia-
tions of the amide substitution. On the other hand, the
CF3
CF3
N
NHBoc
N
a, b
N
S
HN
NO2
O
N
N
SO2CH3
c, d
SO2CH3
CF3
S
CF3
H
N
H
N
e
R5
R3
R3
N
N
NH
N
N
N
N
S
R4
R4
OH
O
OH
O
5
R =
NBoc
N
N
SO2CH3
SO2CH3
32-36
37
Scheme 3. Reagents and conditions: (a) HSCH2CH2NHBoc, K2CO3, DMF, 70 °C (86%); (b) epichlorohydrin, Cs2CO3, DMF (40%); (c) HNR3R4, EtOH, 90 °C (88%); (d) (i) Et3SiH, TFA,
CH2Cl2; (ii) R5CO2H, HATU, HOAt, (i-Pr)2EtN, DMF (13–58%, two steps); (e) Et3SiH, TFA, CH2Cl2 (30%). NR3R4 and R5 are defined in Table 4.

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