Pyrazole-based arylalkyne cathepsin S inhibitors. Part II: Optimization of cellular potency

Article abstract of DOI:10.1016/j.bmcl.2009.09.013

Basic lipophilic substituents dramatically improved the cellular potency of a previously disclosed series of pyrazole-based arylalkyne cathepsin S inhibitors. The incorporation of substituted benzylamines in the para position of the arylalkyne maintained

Full text of DOI:10.1016/j.bmcl.2009.09.013

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6135–6139
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyrazole-based arylalkyne cathepsin S inhibitors. Part II: Optimization
of cellular potency
*
Michael K. Ameriks , Hui Cai, James P. Edwards, Damara Gebauer, Elizabeth Gleason, Yin Gu, Lars Karlsson,
Steven Nguyen, Siquan Sun, Robin L. Thurmond, Jian Zhu
Johnson & Johnson Pharmaceutical Research and 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:
Received 23 July 2009
Revised 2 September 2009
Accepted 4 September 2009
Available online 10 September 2009
Basic lipophilic substituents dramatically improved the cellular potency of a previously disclosed series
of pyrazole-based arylalkyne cathepsin S inhibitors. The incorporation of substituted benzylamines in the
para position of the arylalkyne maintained enzymatic activity (hCatS IC50 = 80–420 nM) and imparted
cellular potency (IC50 = 0.8–4.0 lM). Further refinement of the morpholine portion of the pharmacophore
enabled the identification of bicyclic piperidines with enhanced affinity for CatS (IC50 = 10–30 nM) and
sub-micromolar cellular potency (JY Ii IC50 = 200–720 nM).
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
Cathepsin
Protease
Invariant chain
Lysosomotropism
The cysteine protease cathepsin S (CatS) resides in the lysosome
of certain antigen presenting cells, as well as the extracellular ma-
trix of activated macrophages. Due to its central role in both anti-
gen and invariant chain (li) processing, CatS is an important
mediator of MHC class II related immune responses.1 CatS knock-
out mice exhibit impaired invariant chain degradation, as well as
a reduced susceptibility to collagen-induced arthritis compared
to wild type animals.2 This suggests that inhibition of CatS may
prove beneficial for the treatment of certain immunological disor-
ders. Aberrant CatS activity has been implicated in other diseases
as well.3 Many CatS inhibitors are peptidic compounds that cova-
lently bind to the active site cysteine, but recently several non-
covalent inhibitors have been disclosed as well.4–7
ically improve the cellular potency of this series of inhibitors with-
out compromising enzymatic activity.
Although a morpholine ring was initially selected as a solubiliz-
ing group for alkyne 1, its modest enzymatic activity and lack of cel-
lular potency prompted us to consider alternate heterocycles. Since
earlier work related to non-alkyne pyrazole-based CatS inhibitors
established that substituted piperazine7b or piperidine7e groups in
the P5 region of the pharmacophore enhanced enzymatic and cellu-
lar activity, we evaluated these six-membered rings as potential
morpholine replacements within the alkyne series (Scheme 1). To
enable rapid evaluation of the left-hand side of the molecule, cyclic
amineswere introduced via a two-step process involving regioselec-
tive alkylation of pyrazole 27f with epichlorohydrin, followed by
We have previously reported pyrazole-based CatS inhibitors
containing arylalkynes as P1 binding elements.7f A representative
example from this series, 4-chlorophenyl alkyne 1 (Fig. 1), demon-
strated activity against both human and mouse forms of CatS,
exhibited selectivity for CatS over several closely related cysteine
proteases, and reached systemic circulation following oral dosing
in rats (% F = 21). However, this compound and numerous closely
related alkyne analogs failed to display any appreciable activity
P5
Cl
P1
N
N
N
O
Cl
N
(JY Ii IC50 >10 lM) in a secondary cellular assay measuring invari-
SO2CH3
ant chain degradation in human JY cells. In this Letter, we describe
how the incorporation of lipophilic basic substituents on either the
P1 alkyne or P5 morpholine portions of the molecule can dramat-
1
hCatS = 200 nM; mCatS = 690 nM
hCatB, hCatF, hCatL > 20 µM
JY Ii IC50 > 10 µM
* Corresponding author.
E-mail address: mameriks@its.jnj.com (M.K. Ameriks).
Figure 1. Profile of 1.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.013

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M. K. Ameriks et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6135–6139
Cl
I
moiety in the morpholine series. Since previous SAR for the aryl-
Cl
I
alkynes established that substituents in the para position of the
aromatic ring were preferred for optimal enzymatic activity, a vari-
ety of polar groups (i.e., alcohols, amines, acids, etc.) were intro-
duced in the 4-position and evaluated in our cellular assay (data
not shown). In addition to altering the lipophilicity and solubility
of the alkyne inhibitors, it was anticipated that these functional
groups would provide a synthetic handle upon which to append
further substitution. However, among all of these motifs, deriva-
tized benzylamines 15 demonstrated the only hint of cellular activ-
ity (Table 2).
N
N
b
HN
N
a
O
NH
X
R
N
N
SO2CH3
SO2CH3
3
2
Cl
I
c
N
N
N
X
OH
R
Initially, the substituted benzylamines 15 were prepared using
one of two routes (Schemes 2 and 3). The parent benzylamine 14
was assembled from the previously described terminal alkyne
117f by a two-step process involving Sonogashira coupling with
(4-iodo-benzyl) carbamic acid tert-butyl ester 12 and subsequent
acid-mediated deprotection (Scheme 2). Although sulfonylation
and acylation of benzylamine 14 proceeded smoothly under stan-
dard conditions, reductive aminations provided only moderate
yields of the desired mono-alkylated benzylamines 15 due to
extensive over-alkylation of the amine. In an attempt to circum-
vent this problem and also access unsymmetrical N,N-disubsti-
tuted benzylamines, benzaldeyhde 17 was prepared and
subjected to reductive aminations with primary and secondary
amines (Scheme 3). Although it was possible to isolate the desired
benzylamines 15, reduction of the alkyne was frequently a compet-
ing side reaction.
As shown in Table 2, derivatization of primary amine 14 had a
minimal effect on enzymatic activity. Both sulfonamide 15a and
amide 15b retained affinity for CatS but did not inhibit invariant
chain degradation in the cellular assay. Although a small alkyl sub-
stituent 15c was not favored, the addition of aromatic groups
15d–15f was tolerated. More importantly, despite their modest
enzymatic activity, the benzyl and phenethyl amines 15d and 15f
displayed a glimmer of cellular activity, and provided a clue that
the key to optimizing cellular potency might lie in balancing the ba-
sicity and lipophilicity of the substituents appended to the amine.
In order to test this theory and further improve the cellular activ-
ityof benzylamine 15d, we profiled a small set of substitutedbenzyl-
amines 19 (Table 3). Given the problems associated with the
previous synthetic routes, most of these compounds were con-
structed using an alternate method in which terminal alkynes 18
were first assembled via a reductive amination between functional-
ized benzylamines and 4-ethynylbenzaldehyde, and then coupled to
iodoarene 16 using conventional Sonogashira conditions (Scheme
4). Gratifyingly, reductive aminations with 4-ethynylbenzaldehye
N
4
SO2CH3
Cl
N
N
N
X
OH
R
Cl
N
SO2CH3
5-10
Scheme 1. Reagents and conditions: (a) epichlorohydrin, Cs2CO3, DMF (73%); (b)
EtOH, 80 °C (68–89%); (c) 4-chloroethynylbenzene, 10% Pd(PPh3)2Cl2, 10% CuI, Et3N,
THF, rt (68–83%).
thermal ring-openings of racemic epoxide 3 with the desired
amines. Late-stage incorporation of the 4-chlorophenylalkyne P1
moiety under standard Sonogashira conditions provided the tar-
geted heterocyclic analogs 5–10 of morpholine 1.
As shown in Table 1, the presence of a hydroxyl group in the lin-
ker had a minimal effect on enzymatic activity, as morpholine 5
exhibited comparable activity to its des-hydroxy analog 1 (CatS
IC50 = 140 and 200 nM, respectively). Although piperazine replace-
ments 6–7 for the morpholine group did not improve enzymatic
activity, piperidine 8 demonstrated a twofold enhancement in CatS
affinity. In general, substitution at the 4-position of the piperidine
ring was tolerated, and methyl ester 9 exhibited an eightfold
improvement in enzymatic activity compared to the parent piper-
idine 8. Thus, it is clear that small changes to the P5 region of the
pharmacophore can dramatically affect CatS affinity. Unfortu-
nately, enhanced enzymatic activity did not translate to an
improvement in cellular potency, and suggested that the lack of
cellular activity for this class of inhibitors might be attributed to
poor cellular permeability.8
Concurrent with our work on left-hand side modifications, we
attempted to improve cellular activity by altering the P1 arylalkyne
Table 2
Table 1
Effect of amine substitution on enzymatic and cellular activity
SAR of morpholine/piperazine/piperidine CatS inhibitorsa
Cl
Cl
N
N
N
N
N
N
R1
N
X
OH
O
R
R2
Cl
N
N
SO2CH3
SO2CH3
Compound
X
R
CatS IC50
(
l
M)
JY Ii IC50 (lM)
Compound
R1
R2
H
SO2Ph
COPh
CH3
CH2Ph
CH2Ph
CH2CH2Ph
Route
CatS IC50
(
lM)
JY Ii IC50 (lM)
5
6
7
8
9
O
N
N
CH
CH
CH
—
Boc
H
H
CO2Me
CONH2
0.14
0.15
0.38
0.08
0.02
0.11
>10
>10
>10
>10
>10
>10
14
H
H
H
H
H
CH3
H
A
A
A
B
A
B
B
0.49
0.16
0.35
1.30
0.42
0.60
0.58
10
15a
15b
15c
15d
15e
15f
>10
>10
>10
4.2
>10
5.7
10
a
CatS IC50 and JY Ii degradation IC50 values are the mean of n P 2 runs and
determined as described previously.6a
a
See Table 1, footnote a for details.

M. K. Ameriks et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6135–6139
6137
I
Cl
Cl
NHBoc
N
N
TFA/CH2Cl2, rt
76%
N
N
12
N
N
O
10% Pd(PPh3)2Cl2, CuI
Et3N, THF, rt
78%
O
NHBoc
N
N
SO2CH3
SO2CH3
13
11
Cl
Cl
N
N
N
N
N
N
R'-CHO, Na(OAc)3BH, AcOH, CH2Cl2 (54%)
O
O
or R'SO2Cl, pyr, CH2Cl2
(82-88%)
H
N
NH2
or R'COCl, pyr, CH2Cl2
R
N
N
SO2CH3
SO2CH3
15
14
Scheme 2. Route A: general synthesis of N-substituted benzylamines 15.
Cl
Cl
Cl
N
N
N
N
N
N
N
N
N
I
b
a
O
R1
N
O
O
R2
CHO
N
N
N
15 SO2CH3
SO2CH3
SO2CH3
17
16
Scheme 3. Route B: alternate synthesis of N-substituted benzylamines 15. Reagents and conditions: (a) 4-ethynylbenzaldehyde, 10% Pd(PPh3)Cl2, 10% CuI, Et3N, THF, rt (92%);
(b) R1R2NH, Na(OAc)3BH, AcOH, CH2Cl2 (11–68%).
Table 3
SAR of N,N-dibenzyl cathepsin S inhibitorsa
Cl
N
N
N
O
R2
H
N
N
R1
SO2CH3
Compound
R1
R2
H
4-CH3
4-OCH3
4-Cl
3,4-diCl
H
Route
CatS IC50
(lM)
JY Ii IC50 (lM)
15d
19a
19b
19c
19d
19e
19f
H
H
H
H
A
B
C
C
C
C
C
C
C
0.42
0.35
0.23
0.14
0.08
0.19
0.10
0.10
0.12
4.2
1.1
0.82
1.5
1.8
1.0
1.7
>10
>10
H
CH3 ( )
CH2OH (R)
CO2CH3 (R)
CO2CH3 (S)
H
H
H
19g
19h
a
See Table 1, footnote a for details.
were consistently cleaner than those involving aldehyde 17. Pre-
sumably the absence of a second electron-withdrawing aromatic
ring attenuates the susceptibility of the terminal alkyne towards
reduction.
In order to assess whether the position of the basic nitrogen
substituent is essential for cellular potency, we revisited the P5 mor-
pholine replacements identified in the 4-chlorophenylalkyne series
(see Table 1). Since substituted piperidines demonstrated encourag-
ing enzymatic activity, an additional piperidine ring was installed at
the4-positionto yieldabicyclicreplacement forthemorpholinering
(Scheme 5). It was anticipated that further derivatization of the ter-
minal nitrogen would provide an opportunity to tune the basicity
and lipophilicity of the piperidine, and hopefully impart cellular po-
tency to this series. To this end, enantiopure bicyclic piperidine 22
was prepared in a manner similar to that described earlier for piper-
idine 8 (see Scheme 1), except that pyrazole 2 was alkylated with
(2S)-3-(2-nitrobenzenesulfonyloxy-1,2-epoxypropane 20 (>98%
ee)insteadof epichlorohydrin. Thereactivenosylgroupwasselected
As shown in Table 3, lipophilic groups 19a–d consistently im-
proved both enzymatic and cellular activity relative to the unsubsti-
tuted benzylamine 15d, with each compound typically exhibiting a
5–10-fold difference in activity between the two assays. Substitu-
ents adjacent to the nitrogen 19e–h also improved CatS affinity,
and the enzyme did not exhibit a preference for a particular anti-
pode. Interestingly, despitetheirencouragingenzymaticactivity, es-
ters 19g and 19h failed to demonstrate any inhibition of invariant
chain degradation, further highlighting the potential dependence
of cellular potency on the basicity of the benzylamine nitrogen.

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M. K. Ameriks et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6135–6139
R2
H2N
R1
a
R2
H
Cl
N
CHO
R1
18
N
N
N
b
O
R2
H
N
Cl
I
N
N
R1
N
N
SO2CH3
19
O
N
SO2CH3
16
Scheme 4. Route C: optimized synthesis of N,N-disubstituted benzylamines 19. Reagents and conditions: (a) Na(OAc)3BH, AcOH, CH2Cl2 (60–90%); (b) 10% Pd(PPh3)2Cl2, 10%
CuI, Et3N, THF, rt (>80%).
N–H was not required for cellular potency. However, the presence
of electron-withdrawing groups 22 and 24 completely abrogated
Cl
Cl
ONs
N
cellular activity in a manner consistent with the trends observed
for the P1 alkynylbenzylamines in the morpholine series (see Table
2). Thus, it appears that a balance of nitrogen basicity and lipophilic-
ity is crucial for achieving activity in the cellular assay, but the posi-
tion of the basic nitrogen atom is not essential.
N
O
b,c
HN
I
20
N
I
O
a
N
N
SO2CH3
SO2CH3
2
21
The ability of basic, lipophilic substituents to enhance inhibition
of lysosomal proteases in cellular assays was first reported by
researchers at Merck during their studies on cathepsin K inhibi-
tors.10 In a series of elegant experiments, they demonstrated that
this phenomenon can be attributed to lysosomotropism, the intra-
cellular accumulation of drug in acidic lysosomes, and can result in
enhanced cellular inhibition of CatS as well. We have previously re-
ported a series of pyrazole-based CatS inhibitors that appear to dis-
play lysosomotropic properties,7e but the compounds described in
this Letter are distinctly different in that the IC50 values obtained
from the cellular assay are always 5–10-fold higher than the corre-
sponding IC50 observed in the purified enzyme assay. Thus,
although it is difficult to rule out lysosomal trapping as the source
of the enhanced cellular activity of pyrazole-based arylalkynes
bearing basic, lipophilic substituents, additional factors may need
to be considered as well (i.e., active cationic transport into cells,
intracellular aggregation and/or membrane binding, etc.).11
In conclusion, a variety of basic, lipophilic substituents dramati-
cally improved the cellular potency of a series of pyrazole-based
arylalkyne cathepsin S inhibitors. The incorporation of substituted
benzylamines in the para position of the P1 arylalkyne maintained
enzymatic activity and imparted cellular potency. Insight gleened
fromtheSARforthisserieswasusedto refinetheP5morpholinepor-
tion of the pharmacophore and enabled the identification of bicyclic
piperidines with enhanced affinity for CatS (IC50 = 10–30 nM) and
sub-micromolar cellular potency (IC50 = 200–720 nM).
Cl
22, R = Boc
N
d
N
N
23, R = H
OH
e or f
RN
Cl
N
24-25
SO2CH3
Scheme 5. Reagents and conditions: (a) Cs2CO3, DMF (70%); (b) N-Boc-4,40-
bipiperidine, EtOH, 80 °C (90%); (c) 4-chloroethynylbenzene, 10% Pd(PPh3)2Cl2,
10% CuI, Et3N, THF, rt (80%); (d) TFA, CH2Cl2 (68%); (e) AcCl, pyridine (61%); (f) aq
CH2O (3 equiv), Na(OAc)3BH, DCE (45%).
Table 4
SAR of piperidine-based cathepsin S inhibitorsa
Cl
N
N
N
OH
N
Cl
N
R
SO2CH3
Compound
R
CatS IC50
(
lM)
JY Ii IC50 (lM)
22
24
23
25
CO2tBu
COCH3
H
0.01
0.03
0.01
0.01
>10
10
0.20
0.72
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CH3
a
See Table 1, footnote a for details.
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M. K. Ameriks et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6135–6139
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