Arylaminoethyl amides as noncovalent inhibitors of cathepsin S. Part 2: Optimization of P1 and N-aryl

Article abstract of DOI:10.1016/j.bmcl.2005.12.056

A systematic study of anilines led to the discovery of a metabolically robust fluoroindoline replacement for the alkoxy aniline toxicophore in 1. Investigations of the P1 pocket resulted in the discovery of a wide tolerance of functionality leading to the discovery of 11 as a potent and selective inhibitor of cathepsin S.

Full text of DOI:10.1016/j.bmcl.2005.12.056

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1486–1490
Arylaminoethyl amides as noncovalent inhibitors of cathepsin S.
Part 2: Optimization of P1 and N-aryl
Phillip B. Alper,* Hong Liu, Arnab K. Chatterjee, KhanhLinh T. Nguyen, David C. Tully,
Christine Tumanut, Jun Li, Jennifer L. Harris, Tove Tuntland, Jonathan Chang,
Perry Gordon, Thomas Hollenbeck and Donald S. Karanewsky
Genomics Institute of the Novartis Research Foundation, 10675 John J. Hopkins Dr., San Diego, CA 92121, USA
Received 2 November 2005; revised 9 December 2005; accepted 12 December 2005
Available online 18 January 2006
Abstract—A systematic study of anilines led to the discovery of a metabolically robust fluoroindoline replacement for the alkoxy
aniline toxicophore in 1. Investigations of the P1 pocket resulted in the discovery of a wide tolerance of functionality leading to
the discovery of 11 as a potent and selective inhibitor of cathepsin S.
Ó 2005 Elsevier Ltd. All rights reserved.
Cathepsin S is a papain-like cysteine protease that is
predominantly expressed in antigen presenting cells.
Cathepsin S plays an important role in the immune re-
sponse by processing the invariant chain (li) of the major
histocompatibility class II complex (MHC II). This pro-
teolytic event is necessary for the loading of antigen
onto the MHC II molecules, which subsequently leads
to antigen presentation to CD4+ T-cells.1 Cathepsin S-
deficient mice show major impairment in the degrada-
tion of the invariant chain in antigen presenting cells
along with resistance to collagen-induced arthritis2 and
autoimmune myasthenia gravis.3 Since these mice ap-
peared normal in all other respects, small molecule
inhibitors of cathepsin S are thought to be good candi-
dates for the treatment of diseases that require modula-
tion of the immune response.4
P3
P1
R1
O
H
H
1
R1 = H
N
N
Me
N
CatS K = 62 nM
CatK K = 43 nM
CatL Ki = 7 nM
i
H
O
i
OMe
P1'
P2
Figure 1. Screening hit for the inhibition of cathepsin S.
series for potency, selectivity, and removal of metabolic
liabilities.8
The methoxyaniline moiety in compound 1 is a known
toxicophore. The phenolic metabolite, as a result of O-
demethylation, is further oxidized to 4-benzoquinone
imine in vivo, which elicits toxicity by GSH depletion
and covalent adducts with cellular macromolecules.
Therefore, suitable replacements were explored.9
Compound 1 (Fig. 1) was reported by Altmann and
coworkers as a noncovalent competitive inhibitor of
cathepsin K but was not reported to be selective.5 A
high-throughput screening effort identified 1 as an inhib-
itor of cathepsin S.6 Our previous report described the
optimization of the P2 and P3 substituents to improve
the potency and selectivity over related cathepsins.7
The subject of this article is our continuing efforts in
hit-to-lead optimization of the P1/P10 moieties of this
Scheme 1 shows the chemistry used to access the initial
group of compounds. Leucine was acylated with m-tol-
uoyl chloride under Schotten–Bauman conditions and
then reacted with ethanolamine using EDC as the cou-
pling agent to afford 2. This material was then oxidized
to the aldehyde with the Dess–Martin periodinane and
reductively aminated with various anilines and indolines
Keywords: Cathepsin S; Noncovalent protease inhibitor; SAR.
*
Corresponding author. Tel.: +1 858 812 1536; fax: +1 858 812
1648; e-mail: palper@gnf.org
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.12.056

P. B. Alper et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1486–1490
1487
compounds 3c, 3d, and 3e showed that the methoxy
unit was not required for inhibitory activity, but
the halogens appeared to be less preferred. HPLC
analysis of a sample of 3c, which was treated with
rat liver microsomes, showed a new peak whose
weight corresponded to the trifluoromethoxy group
being replaced with a hydroxyl group. It has been
reported that para-substituted fluoroanilines can be
similarly metabolized.10 It is likely that this metabo-
lism proceeds via a homolytic process starting at
the aniline hydrogen. We were therefore pleased to
see that compounds 3f–h retained activity upon
replacement of the aniline with an indoline. The 5-
fluoroindoline analog 3h remained the most attrac-
tive, as it had an activity profile similar to that of
initial hit 1. In metabolite ID studies, there was no
product observed that lost a fluorine from the indo-
line in the analogues of 3h.11
a, b
O
H
OH
N
H2N
N
OH
H
O
O
2
O
c, d
H
N
R
N
N
H
H
O
3
Scheme 1. Reagents: (a) 1 M NaOH, m-toluoyl chloride; (b) ethanol-
amine, EDC, HOBt, CH2Cl2; (c) Dess–Martin periodinane, DCM; (d)
R-NH2, NaBH3CN, DMF, AcOH.
to afford the desired adducts 3a–h. The aniline was used
in a 3-fold excess to ensure that product 3 did not under-
go further reductive amination.
Exploration of the P1 position was initiated by installa-
tion of a side chain on the ethylenediamine linker. As
shown in Scheme 2, Boc protected amino acids were re-
duced to the alcohol, oxidized to the aldehyde, and then
reductively aminated with the appropriate aniline or
indoline to afford 5. The Boc group was removed and
the resulting amine then coupled to the appropriate
Compounds 3a and 3b indicated that there was addi-
tional space around the aniline for steric hindrance
(Table 1). Specifically, 3b was interesting because it
demonstrated that the interaction between the aniline
and the enzyme was not significantly perturbed by
the ortho-substituted methyl groups. para-Substituted
Table 1. Inhibition of the cathepsin S, K, and L—variation of the aniline on the hit structure
O
H
N
N
R
H
O
Compound
R
Cathepsin S, Ki (nM)
Cathepsin K, Ki (nM)
Cathepsin L ,Ki (nM)
1
68
91
26
HN
OMe
OMe
OMe
OCF3
Cl
3a
3b
3c
3d
3e
3f
107
132
104
458
563
230
196
98
125
131
125
408
478
310
139
80
20
14
HN
HN
HN
HN
120
92
62
HN
N
F
88
Cl
N
N
3g
3h
17
F
11

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P. B. Alper et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1486–1490
R1
Table 3. Inhibition of the cathepsin S, K, and L—substitution on P1
side chain
R1 R2
a, b, c
d, e
OH
N
R3
BocHN
BocHN
H
R5
N
CO2H
O
4
5
O
R4
6
O
F
H
N
O
R1 R2
N
MeO
H
R5
N
N
N
N
R3
H
O
R
H
O
R4
7: R4=iBu, R5=3-Me-Ph
8: R4=CH2CH2-cyclohexyl, R5=3-OMe-Ph
Compound
R
Cathepsin Cathepsin Cathepsin
L, Ki
(nM)
S, Ki
(nM)
K, Ki
(nM)
Scheme 2. Reagents and condition: (a) i—i-butyl chloroformate,
NMM, THF, ꢀ10 °C; ii—NaBH4, H2O; (b) Dess–Martin periodinane,
DCM; (c) R2R3-NH, NaBH3CN, DMF, AcOH; (d) TFA/DCM (1:1);
(e) HATU, DIEA, DMF.
8a
8b
8c
8d
8e
8f
H
CH(CH3)2
2130
14
>100,000 >33,750
>10,000 150
CH2CH(CH3)2
Ph
CH2Ph
28
68
>100,000 1800
>100,000 >11,000
>100,000 9200
133
23
CH2OBn
CH2SBn
>30,000 380
>100,000 5040
N-acylated amino acid 6 (prepared as in Scheme 1) using
standard peptide coupling conditions.
8g
149
8h
15
4320
2
OBn
First, we determined that the S-diastereomer 7a is more
active than the R-diastereomer 7b as shown in Table 2.
It should be noted that there was also a 5-fold increase
in activity versus 1 when an alanine side chain was intro-
duced at P1.
8i
8j
CH2OH
CH2CH2OH
50
208
>100,000 15,530
>100,000 1800
8k
8
2750
20
OH
8l
8m
CH2CH2SO2Mea 14
47
10,760
19
42
We then went on to do a more thorough investigation
of the nature of tolerated P1 substituent as shown in
Table 3. We noted that for intermediates 5 where R1
is larger than methyl racemization of the amino acid
stereocenter occurred during the coupling step. We
chose to analyze the mixture on the assumption that
the other diastereomer would have significantly less
activity (vide supra).
(CH2)3NH2
76
8n
8o
7
2150
5
N
O
96
>100,000 3410
OH
8p
8q
CH2CO2Bn
CH2CO2H
405
88
>37,250
>10,000
>100,000
>37,000
a P2 residue is t-butylalanine.
In the case of homocyclohexylalanine P2, it turned out
that the parent compound, 8a, did not have potent activ-
ity against cathepsin S, however, addition of a hydro-
phobic side chain in P1 afforded potent cathepsin S
inhibitors. In addition, the selectivity over cathepsin L
was highly dependent on the nature of the P1 side chain.
The valine derived isopropyl analog 8b gave only 10-fold
selectivity over cathepsin L, while substitution with
isobutyl 8c gave a 64-fold selectivity over cathepsin L.
in order to lower the lipophilicity of the inhibitors.
When hydroxyl containing side chains 8i, 8j, and 8k
were made, we found that these were not only tolerated
but had a roughly similar SAR to the aliphatics in that
chain substitution increases potency against cathepsin S
but decreases selectivity. With this observation in hand,
we pressed forward with hydrophilic substituents and
found that a sulfone 8l was accepted (although the P2
residue in this compound was t-Bu alanine, the differ-
ence in activity with homocyclohexyl P2 is one of abso-
lute potency and not so much selectivity—vide supra 8q
vs 11) and amines (8m and 8n) were tolerated but lost
selectivity against cathepsin L and K. We also wanted
to test whether a negative charge is accepted and 8o
implied that this might be the case (although it is not
certain whether the phenol is ionized under the assay
conditions). Finally, we tested both the ester 8p and
the acid 8q of the aspartate side chain and contrary to
what one might expect from substrate specificity study,12
these turned out not only to be tolerated but to confer a
great deal of selectivity to the molecule. The positively
charged guanidinium group at the S1 pocket emanating
from Arg141 represented a distinct feature of cathepsin
S that may account for the specificity of 8q. The same
region is occupied by Asn158 in cathepsin K and
Asp162 in cathepsin L.13
Compounds 8d–h showed that aromatic substituents
that were not directly attached to the side chain did
not add any potency beyond their aliphatic counter-
parts. We then went on to try hydrophilic substituents
Table 2. Inhibition of the cathepsin S, K, and L—substitution on P1
side chain
OMe
O
H
N
N
N
H
H
O
R
Compound
R
H
Cathepsin S, Cathepsin K, Cathepsin L,
Ki (nM)
Ki (nM)
Ki (nM)
1
68
91
55
26
1.6
75
7a
7b
(S)-Me 15
(R)-Me 212
337

P. B. Alper et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1486–1490
1489
Table 4. Inhibition of the cathepsin S, K, and L
Compound Cathepsin Cathepsin Cathepsin
S, Ki (nM) K, Ki (nM) L, Ki (nM) K/S
a
Selectivity
b
11
12
6
16
460
184
>15,000
1754
77
11.5
c
Compounds 11 and 12.
d
Table 5. PK parameters of 11 and 12 (Both dosed in Wistar rats at
10 mpk)
Scheme 3. Reagents and condition: (a) H2, Pd/C, MeOH; (b)
TFA:DCM (1:1); (c) i—i-butyl chloroformate, NMM, THF, ꢀ10 °C;
ii—NaBH4, H2O; (d) Dess–Martin periodinane, DCM; (e) Morpho-
line, NaBH3CN, DMF, AcOH.
Compound
PO AUC
(min lg/mL)
PO Cmax
(nM)
T1/2 (h)
%F
11
12a
6
18.5
45
293
0.56
N/A
2.0
8.0
a Dosing 12 and monitoring for 11.
CO2tBu
CO2tBu
O
a,b
CbzHN
N
N
N
H
CbzHN
for cathepsin S over cathepsins K and L. We also
showed that 11 is a reversible inhibitor through a dialy-
sis experiment.14 Table 5 shows the in vivo PK parame-
ters of 11 and 12.
F
F
10
9
OMe
CO2R
N
O
H
As is evident from Table 5, the use of the ethyl ester
prodrug leads to a 4-fold improvement in oral bioavail-
ability over the acid itself and generates a Cmax that is
50-fold greater than the Ki.
c, d, e
N
N
H
F
O
11R=H
12R=Et
f
In summary, we showed that there is a wide tolerance
for functional groups in the S1 pocket of cathepsin S
including carboxylic acid functionality which gave excel-
lent selectivity over other related cathepsins. This effort
represents the most thorough investigation of the P1 re-
gion reported to date. We have successfully replaced the
methoxyaniline toxicophore with a fluoroindoline while
maintaining potency and selectivity as exemplified by 11.
The synthesis of compound 12, the prodrug of 11, result-
ed in a modestly bioavailable, potent, and selective
inhibitor of cathepsin S. Further strategies based on this
work will be reported in due course.
Scheme 4. Reagents: (a) H2, 10% Pd/C, MeOH; (b) HATU, DIEA,
DMF, Cbz-t-Bu Ala (77% over 2 steps); (c) H2, 10% Pd/C, MeOH; (d)
HATU, DIEA, DMF, anisic acid (57% over 2 steps); (e) TFA/DCM
(1:1) (86%); (f) CSA, EtOH, triethylorthoformate (44%).
Scheme 3 shows the chemistry used to access the com-
pounds that required transformation of the side chain
after coupling in Scheme 2. In this scheme, R6 refers
to an alkyl, aryl or carbonyl group.
Scheme 4 shows the synthesis of the more optimized
compound 11 and an ethyl ester 12, synthesized as single
diastereomers by using N-carbamate protected amino
acids as building blocks.
Supplementary material
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2005.12.056.
The indoline conjugate 9 (prepared according to
Scheme 2, except using a Cbz protecting group) was
deprotected and coupled to Cbz-t-Bu Ala using stan-
dard conditions to afford 10. It was crucial to use the
t-Bu protecting group on the ester because once the
nitrogen is deprotected from 10, there is a strong tenden-
cy to cyclize. Indeed, when 10 is deprotected and cou-
pled to anisic acid, the conjugate is obtained in only
57% yield. Deprotection of the ester affords 11 in 86%
yield and the acid is esterified to 12 with CSA and TEOF
in ethanol. Table 4 shows the activities of these
compounds.
References and notes
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The inhibitory activity of 11 against cathepsin S proved
to be high with an acceptable selectivity window over
cathepsin K and complete selectivity over cathepsin L.
Compound 11 shows that the presence of the acid in
P1 increases the selectivity of this class of inhibitors

1490
P. B. Alper et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1486–1490
6. For a detailed description of the assay conditions, please
see supplementary material.
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