Thioether acetamides as P3 binding elements for tetrahydropyrido-pyrazole cathepsin S inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.01.103

A series of tetrahydropyrido-pyrazole cathepsin S (CatS) inhibitors with thioether acetamide functional groups were prepared with the goal of improving upon the cellular activity of amidoethylthioethers. This Letter describes altered amide connectivity, in conjunction with changes to other binding elements, resulting in improved potency, as well as increased knowledge of the relationship between this chemotype and human CatS activity.

Full text of DOI:10.1016/j.bmcl.2010.01.103

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2379–2382
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Thioether acetamides as P3 binding elements for tetrahydropyrido-pyrazole
cathepsin S inhibitors
*
Danielle K. Wiener , Alice Lee-Dutra, Scott Bembenek, Steven Nguyen, Robin L. Thurmond,
Siquan Sun, Lars Karlsson, Cheryl A. Grice, Todd K. Jones, James P. Edwards
Johnson & Johnson Pharmaceutical Research & Development, L.L.C., 3210 Merryfield Row, San Diego, CA 92121, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of tetrahydropyrido-pyrazole cathepsin S (CatS) inhibitors with thioether acetamide functional
groups were prepared with the goal of improving upon the cellular activity of amidoethylthioethers. This
Letter describes altered amide connectivity, in conjunction with changes to other binding elements,
resulting in improved potency, as well as increased knowledge of the relationship between this chemo-
type and human CatS activity.
Received 30 October 2009
Revised 15 January 2010
Accepted 20 January 2010
Available online 8 February 2010
Published by Elsevier Ltd.
Cathepsin S (Cat S) is a cysteine protease that is responsible for
the degradation of major histocompatibility complex class II (MHC
II)-associated invariant chain (Ii) as a means of facilitating antigen
presentation to CD4+ T-cells.1 Inhibition of CatS may attenuate
antigen presentation by impeding the removal of Ii from MHC II
molecules, thus regulating immune hyper responsiveness.
Extensive research surrounding tetrahydropyrido-pyrazole core
molecules as non-covalent inhibitors of CatS has been reported by
our laboratories.2 Availability of crystal structures enabled the tar-
geting of specific binding pockets of the human CatS enzyme,2f
leading to the development of various tethered moieties, including
the aminoethylthioether 1 (Fig. 1).2g Mitigating the basicity of this
amine through amide formation (2) resulted in a complete loss of
cellular activity. To continue this investigation, synthesis of amides
with alternate connectivity was pursued.
Nucleophilic displacement of the previously described aryl nitro
intermediate 3 with mercaptoethylacetate afforded the ester 4.
Regioselective alkylation of the core pyrazole, followed by acetal
hydrolysis and reductive amination, yielded the desired intermedi-
ate with 4-piperidinylpyrrolidin-2-one installed as the P5 substitu-
ent. Saponification of the ethyl ester provided the carboxylic acid,
which could then be coupled with various amines, exemplified by
those shown in Table 1. In order to evaluate the activity of amide
moieties as P3 binding elements, the P5 substituent and P4 sulfon-
amide were held constant (Scheme 1).
CF3
CF3
H
N
N
N
N
N
N
N
N
S
S
O
O
OH
O
N
N
N
N
SO2CH3
SO2CH3
1: hCatS IC50 = 0.17 µM
JY Ii IC50 = 0.04 µM
2: hCatS IC50 = 0.13 µM
JY Ii IC50 > 10 µM
Figure 1. Non-covalent CatS inhibitors with thioether substituents.
O
CF3
NO2
CF3
CF3
N
N
O
HN
HN
N
O
S
a
b
N
O
O
S
O
O
N
SO2CH3
N
N
SO2CH3
SO2CH3
3
4
c
CF3
CF3
N
N
R1
O
N
N
N
N
O
d
S
S
N
O
N
O
N
N
SO2CH3
SO2CH3
P5
P4
Scheme 1. Reagents and conditions: (a) HSCH2CO2CH2CH3, K2CO3, DMF, 70 °C
(90%); (b) 2-(2-bromoethyl)-1,3-dioxolane, Cs2CO3, DMF (95%); (c) (i) 1 N HCl (aq),
acetone, 55 °C; (ii) 1-piperidine-4-yl-pyrrolidin-2-one, acetic acid, NaBH(OAc)3,
CH2Cl2 (29%, two steps); (d) (i) LiOH, 3:1:1 THF/H2O/EtOH (42%); (ii) HOBt, EDCI,
HR1, DMF (40–60%).
Initial results proved promising, with a variety of amides yield-
ing sub-micromolar enzymatic activity (5–13). Secondary amides
are tolerated, with hCatS IC50 = 0.37 and 1.15 lM for compounds
15 and 5, respectively, while small, cyclic amides generally achieve
excellent enzymatic activity between 20 and 210 nM (7–13). Previ-
ously reported amidoethylthioethers, such as compound 2, are
inactive in the JY invariant chain degradation cellular assay.2g
Reversing the amide connectivity, as seen in the direct comparison
* Corresponding author. Tel.: +1 858 320 3384; fax: +1 858 450 2089.
E-mail address: dwiener@its.jnj.com (D.K. Wiener).
of 2 and 15, results in comparable enzymatic potency (0.13 lM vs
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2010.01.103

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D. K. Wiener et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2379–2382
Table 1
Table 2
Results of thioether amide screen
Varying P5 substitutents with morpholino amide P3 binding element
CF3
CF3
O
N
N
R1
N
N
N
S
S
N
R2
O
OH
O
O
N
N
N
SO2CH3
SO2CH3
R1
hCatS IC50
(lM)
JY Ii IC50
(
lM)
Compd
R2
hCatS IC50
(lM)
JY Ii IC50 (lM)
a
a
a
a
Compd
5
6
7
8
9
10
11
12
13
14
15
NHCH3
N(CH3)2
1.15
0.52
0.06
0.07
0.02
0.10
0.09
0.21
0.12
0.74
0.37
ND
ND
O
16
17
0.01
0.01
0.19
0.38
N
Pyrrolidine
Piperidine
Morpholine
2-Methyl-pyrrolidine
Pyrrolidin-3(RS)-ol
4-Methyl-piperidine
Piperidine-4-yl-methanol
N-Methyl-piperazine
N-Benzylamine
1.97
3.92
0.45
ꢀ6
N
N
O
0.52
ꢀ8
O
N
N
18
0.01
0.91
0.34
0.11
ꢀ10
F
19
20
0.01
0.02
3.13
3.46
N
N
N
a
CatS IC50 and JY li degradation IC50 values are the mean of n P 2 runs and
determined as described previously2b; ND = not determined.
N
N
O
O
N
21
22
0.04
0.30
0.28
N
0.37 lM, respectively). Further, both the original amidoethylthioe-
ꢀ10
ther (2) and the reverse linkage thioether acetamide (15) are inac-
tive in the cellular assay. Continued manipulation of the amide
substitution, however, offered various patterns with significantly
improved cellular potency. Consider the pyrrolidine (7) and piper-
idine (8) amides: the addition of hydroxyl group substitution sig-
nificantly improves cellular activity (11, 13), while the addition
of methyl substituents to the same ring systems results in a loss
of cellular activity (10, 12). Further, the incorporation of a second
heteroatom within the amide ring improves cellular activity (9,
14), revealing a very promising lead in morpholine amide 9. Inter-
estingly, the addition of a basic amine, N-methylpiperazine amide
14, results in enhanced potency in the cellular assay versus enzy-
matic inhibition. This difference may correspond to previously re-
ported findings in which lysosomotropism triggers the
accumulation of cathepsin inhibitors in cells.2f,g,3 These data sup-
port the hypothesis that within this series of thioethers removal
of amine basicity in the P3 binding element may eliminate this
effect.
O
N
a
CatS IC50 and JY li degradation IC50 values are the mean of n P 2 runs and
determined as described previously2b; all compounds tested as racemic mixtures.
Table 3
Varied thioether-linked amides with preferred P5 substituent
CF3
N
R3
N
S
N
OH
O
N
N
SO2CH3
R3
hCatS IC50
(l
M)
JY Ii IC50 (lM)
a
a
Compd
17
23
24
25
26
Morpholine
Pyrrolidine
Pyrrolidin-3(R)-ol
Pyrrolidin-3(S)-ol
Piperidine
0.01
0.38
0.47
0.03
0.03
1.54
0.001
0.007
0.007
0.01
Utilizing the encouraging potency of the morpholine thioether
acetamide 9, evaluation of the overall binding motif through alter-
ation of the P5 element proceeded. In order to readily probe this re-
gion of the molecule, a modified synthesis was established in
which the mercaptoethyl acetate intermediate 4 was converted di-
rectly to the morpholine amide, by way of microwave heating
(Scheme 2). Alkylation with epichlorohydrin yielded 20:1 pyrazole
N-atom regioselectivity with the morpholine amide intermediate.
Surprisingly, this transformation was not regioselective in the
presence of mercaptoethyl acetate, though good selectivity was ob-
a
See Table 2 for details.
served with the amide moiety elaborated. Access to an intermedi-
ate with the morpholine amide in place allowed for more facile
analog synthesis through ring-opening of the epoxide using vari-
ous amine P5 substituents (Table 2).
While changes to the P5 region result in enzymatic potency
comparable to the parent morpholine amide 9, the cellular potency
varies widely. The 4-aryl-piperidine P5 elements (16, 17) proved
most effective in this investigation; and disappointingly, truncat-
ing the P5 region of the molecule results in a complete loss of cel-
lular potency (22). Direct comparison of molecules with and
without the hydroxyl group in the linker between the core and
the P5 binding element display no significant difference in activity
(data not shown).
CF3
CF3
N
O
N
a
HN
N
HN
O
S
S
O
O
N
N
SO2CH3
SO2CH3
4
b
Incorporation of an aryl-piperidine P5 element to thioether
amides of interest from Table 1 yielded excellent results (Table
3), with pyrrolidinol analogs 24 and 25 reaching single digit nano-
molar activity in the hCatS enzymatic assay. Pyrrolidine amide
analogs such as 23 consistently maintain cellular potency despite
the lack of additional solubilizing features such as oxygen-contain-
ing heterocycles or hydroxyl-substitution, which may improve cell
permeability.
CF3
CF3
O
N
O
N
c
R2
N
N
N
N
S
S
O
OH
O
O
N
N
SO2CH3
SO2CH3
Scheme 2. Reagents and conditions: (a) Morpholine, 220 °C, microwave 30 min
(80%); (b) epichlorohydrin, Cs2CO3, DMF (75%); (c) HR2, EtOH, 80 °C (35–85%).

D. K. Wiener et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2379–2382
2381
CF3
CF3
CF3
N
N
N
R3
HN
HN
HN
O
NO2
S
S
a
b
O
O
N
N
N
Boc
Boc
Boc
c
CF3
CF3
N
R3
N
N
N
R3
d
R2
S
S
O
O
OH
O
N
N
Boc
Boc
e
CF3
N
R3
R2
N
S
OH
O
N
R4
Scheme 3. Reagents and conditions: (a) HSCH2CO2CH2CH3, K2CO3, DMF, 70 °C
(67%); (b) HR3, 170 °C microwave, 90 min (41–48%); (c) epichlorohydrin, Cs2CO3,
DMF (77–84%); (d) HR2, EtOH; (e) (i) 4 N HCl/dioxane, CH2Cl2; (ii) Dowex 550A
resin; (iii) HOR4, HATU, HOAt, DIEA, DMF, 70 °C; or ClR4, CH2Cl2; or HR4,
NaBH(OAc)3, MeOH (11–31% three steps).
Figure 2. Crystal structure of 36 bound to a Cys25Ser mutant of cathepsin S.
Compound conformation is shown as an average of two enantiomers. PDB
deposition: 3KWN.
ide P4 functionality (35, 36). This finding is particularly interesting
in light of previously reported crystal structures2f depicting P4 sub-
stituent interactions locking the tetrahydropyrido-pyrazole core
into the S2 pocket of the enzyme.
With optimized P3 and P5 substituents we sought to evaluate
the impact of the P4 element, which had been held constant as a
methyl sulfonamide. The synthetic sequence of Scheme 2 was al-
tered to accommodate the use of a Boc-protected tetrahydropyri-
do-pyrazole core (Scheme 3). Lowering the temperature of amide
formation limited premature Boc-removal; however the tempera-
ture of 170 °C used to form the desired amides excluded the use
of enantiopure pyrrolidinol reagents due to potential epimeriza-
tion of the stereogenic center. Analysis of the P4 region was there-
fore conducted using pyrrolidino and morpholino amides. After
Boc-removal under acidic conditions, treatment with Dowex basic
resin allowed for direct manipulation of the amino intermediate to
yield the structures described in Table 4.
This data set depicts encouraging results for this series. Not only
did acetamide and substituted acetamide P4 elements maintain
potency in both the enzymatic and cellular assays (27, 31, 32),
but deletion of the P4 substituent altogether was well tolerated
(29, 34). Again, pyrrolidino amides impart cellular activity where
morpholino amides fail (29 vs 28, 34 vs 33). Additionally, smaller
P5 binding elements maintain activity when coupled with acetam-
An X-ray crystal structure of 36 was obtained (Fig. 2). The
resulting data not only confirmed binding of the thioether acetam-
ide in the S3 pocket of the CatS enzyme, but also revealed a poten-
tial hydrogen bond interaction between the carbonyl of the amide
and a water molecule in the S1 pocket. This additional binding
interaction may account for the improved enzymatic potency of
thioether acetamides when compared to previously reported
aminoethylthioethers.2g,h It may also explain the ability of this ser-
ies of compounds to function without substitution in the P4 region
of the molecule—the new interaction may serve the function of fix-
ing the tetrahydropyrido-pyrazole into the S2 pocket.
In conclusion, thioether acetamides were discovered as potent,
non-covalent CatS inhibitors within the tetrahydropyrido-pyrazole
series. Access to a beneficial hydrogen bond between the carbonyl
of the amide moiety and a conserved water in the binding
pocket allowed the removal of P4 binding elements while main-
taining potency.
Table 4
The effects of varying P4 substitution
CF3
N
N
O
N
N
R3
R2
S
OH
N
O
N
R4
A
B
R2
R3
R4
hCatS IC50
(lM)
JY Ii IC50 (lM)
a
a
Compd
27
28
29
30
31
32
33
34
A
A
A
A
B
B
B
B
Morpholine
Morpholine
Pyrrolidine
Pyrrolidine
Morpholine
Morpholine
Morpholine
Pyrrolidine
C(O)CH2OH
H
H
CH2CH3
C(O)CH2OH
C(O)CH3
H
0.01
0.11
0.06
0.25
0.01
0.02
0.19
0.06
0.16
1.68
0.06
ND
0.48
0.90
1.85
0.12
H
N
35
36
Pyrrolidine
Pyrrolidine
C(O)CH3
C(O)CH3
0.18
0.15
0.08
0.11
HO
N
a
See Table 2 for details.

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D. K. Wiener et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2379–2382
Butler, C. R.; Wei, J.; Sehon, C. A.; Sun, S.; Gu, Y.; Jiang, W.; Thurmond, R. L.;
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