Azepanone-based inhibitors of human cathepsin S: Optimization of selectivity via the P2 substituent

Article abstract of DOI:10.1016/j.bmcl.2011.06.045

A series of azepanone inhibitors of cathepsin S is described. Selectivity over both cathepsin K and cathepsin L was achieved by varying the P2 substituent. Ultimately, a balanced potency and selectivity profile was achieved in compound 39 possessing a 1-methylcyclohexyl alanine at P2 and nicotinamide as the P′ substituent. The cellular potency of selected analogs is also described.

Full text of DOI:10.1016/j.bmcl.2011.06.045

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4409–4415
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Azepanone-based inhibitors of human cathepsin S: Optimization of selectivity
via the P2 substituent
Jeffrey K. Kerns a, , Hong Nie a, William Bondinell a,b, Katherine L. Widdowson b, Dennis S. Yamashita a,1
,
⇑
Attiq Rahman b, Patricia L. Podolin a, Donald C. Carpenter a, Qi Jin b, Benoit Riflade a,2, Xiaoyang Dong b,
Neysa Nevins b, Paul M. Keller b, Laura Mitchell b,3, Thaddeus Tomaszek b
a GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, United States
b GlaxoSmithKline, 1250 S. Collegeville Road, Collegeville, PA 19426, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 March 2011
Revised 7 June 2011
Accepted 10 June 2011
Available online 17 June 2011
A series of azepanone inhibitors of cathepsin S is described. Selectivity over both cathepsin K and cathep-
sin L was achieved by varying the P2 substituent. Ultimately, a balanced potency and selectivity profile
was achieved in compound 39 possessing a 1-methylcyclohexyl alanine at P2 and nicotinamide as the P0
substituent. The cellular potency of selected analogs is also described.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Cathepsin S
Cathepsin K
Cathepsin L
Azepanone
1-Methylcyclohexyl alanine
Cysteine protease
As part of our program to develop novel therapeutics for inflam-
matory diseases, we optimized a series of azepanone inhibitors of
cathepsin S. Cathepsin S is a lysosomal cysteine protease whose
primary function is in antigen presentation via invariant chain pro-
cessing. Intracellularly, invariant chain is associated with Major
Histocompatibility Complex class II (MHCII) molecules and acts
to block antigen presentation.1,2 By proteolytically processing
invariant chain, cathepsin S effectively unmasks MHC class II mol-
ecules in antigen presenting cells allowing free association of anti-
gen and MHCII proteins.3–5 Recognition of the cell surface antigen
peptide-MHCII complex by CD4+ T-cells initiates the antigen-
specific immune response. In addition to its role in antigen presen-
tation, cathepsin S also plays a role in degradation of extracellular
matrix proteins such as collagen, elastin, and laminin. Thus, inhibi-
tion of cathepsin S may be therapeutically relevant in antigen-
driven inflammatory diseases such as asthma and rheumatoid
arthritis and in diseases where extracellular matrix protein
degradation and remodeling are important such as asthma and
chronic obstructive pulmonary disease (COPD).
Given the high active site sequence homology among the
cathepsins S, K, and L, we anticipated that achieving selectivity
for cathepsin S over other members of the family would prove
challenging. Several groups have reported their discovery efforts
toward selective inhibitors.6–24 As our starting point, we used the
previously described series of azepanone based inhibitors of the
papain family.25–29 Modification of the P2 substituent in this series
has led to the identification of 1, a potent cathepsin L inhibitor with
exceptional selectivity over cathepsin K.28,30 In addition, the selec-
tive cathepsin K inhibitor 2 has been described in detail and dis-
plays good selectivity over cathepsin S (Fig. 1).25,30
Our cathepsin cross-screening efforts showed that the azepa-
none series was capable of producing potent inhibitors of cathep-
sin S; however, achieving high specificity versus cathepsins K
and L appeared challenging. We chose to focus on optimization
of the 7-methyl azepanone series as this series has better develo-
pability when compared to the 7-des-methyl series.31 Thus, we
aimed to improve the selectivity of the series for cathepsin S over
both cathepsins K and L. In the catalytic site, cathepsin S differs
⇑
Corresponding author. Tel.: +1 610 270 6579; fax: +1 610 270 4451.
E-mail address: Jeffrey.K.Kerns@gsk.com (J.K. Kerns).
Present address: Trevena, Inc., 1018 W Eighth Ave., King of Prussia, PA 19406,
from cathepsin
K and L mainly in the S2 and S3 regions
1
(Fig. 2).32–34 In the S2 pocket, cathepsin S is lined with glycine
residues. This is in contrast to cathepsins K and L, both of which
contain the larger alanine in the S2 pocket (CatS:G137, CatK:A134,
CatL:A135). In addition, cathepsin K contains a bulkier Ala just
United States.
2
Present address: 4 Rue du Commandant Pilot, 92200, Neuilly sur Seine, France.
Present address: Drexel University iSchool, 3141 Chestnut Street, Philadelphia, PA
3
19104-2875, United States.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.045

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J. K. Kerns et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4409–4415
O
O
O
O
O
H
N
H
N
N
N
N
H
N
H
O
S
O
O
O
N
N
O
O
O
N
2
1
Cathepsin (Ki, app (nM))
1
2
hS
hK
hL
15.6
>10000
0.43
8.3
0.1
1.2
Figure 1. Comparison of cathepsin potency and selectivity for 1 and 2.
Figure 2. Cathepsin S structure (orange, refcode 2F1G17) displayed with key active site residues labeled in black. Cathepsin L (magenta, refcode 2XU539) and cathepsin K
(cyan, refcode 2FTD31) are superimposed and residues shown where different from cathepsin S in the S2 and S3 pockets. Crystal structure ligands, ions, and solvent have been
removed. Compound 30 has been modeled in the binding site combining the azepanone template from 2FTD and the P2 methylene-cyclohexyl substituent from the ligand of
Cathepsin S crystal structure 2R9M.A (unpublished). Hydrogens are shown at the 4,4-positions of the cyclohexyl alanine.
after the catalytic histidine instead of a Gly (CatS:G165, CatK:A163,
CatL:G164), as well as a Leu compared to a Val for cathepsin S
(CatS:V162, CatK:L160, CatL:M161). Taken together, the S2 pocket
of cathepsin S is slightly larger compared to cathepsin L and signif-
icantly larger compared to cathepsin K (see Fig. 2). This report
details our efforts at modulating the selectivity profile via modifi-
cation of the P2 substituent.
Preparation of the azepanone inhibitors followed largely from
previously reported work.25,27–29,31,35 The preparation of P2 amino
acid substituents which were not commercially available relied
heavily on a variant of the Strecker amino acid synthesis as
illustrated in Scheme 1 for 1-methylcyclohexyl alanine. As shown,
cyclohexanone is condensed with diethylmalonate to produce ole-
fin 5. Methylation via conjugate addition with methylmagnesium
iodide, followed by ester hydrolysis and subsequent decarboxyl-
ation, produces the intermediate acid 6 in moderate yield. Conver-
sion of acid 6 to the requisite aldehyde followed the two step
procedure of reduction with LAH to the primary alcohol followed
by TEMPO oxidation in the presence of trichloroisocyanuric acid
(TCIA) to give aldehyde 7. This was transformed to the amino
nitrile 8 via treatment with ammonium chloride and sodium
cyanide under basic conditions. The synthesis of racemic 1-meth-
ylcyclohexyl alanine (9) was completed by BOC protection of the
amine, acid hydrolysis of the nitrile to the carboxylic acid, followed

J. K. Kerns et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4409–4415
4411
COOH
O
COOEt
a
g
b,c,d
h,i
+
EtOOC
COOEt
COOEt
3
5
4
6
CN
O
COOH
NHBoc
e,f
NH2
9
8
7
Scheme 1. Synthesis of P2 amino acids. Reagents and conditions: (a) TiCl4, pyridine, THF/CCl4 (5:1), 0–25 °C, 72 h, 46%; (b) methylmagnesium iodide, THF, ꢀ5 to 0 °C, 57%; (c)
(i) 10% ethanolic KOH, reflux, 13 h, (ii) H+, 68%; (d) 160 °C, 6 h, 31%; (e) LiAlH4, THF, ꢀ5 to 25 °C, 49%; (f) TCIA, TEMPO, CH2Cl2, 0 °C, 3 h, 79%; (g) NH4Cl, NH4OH, NaCN, 25 °C,
12 h, 66%; (h) (Boc)2O, NaOH, THF/H2O; (i) 6 N HCl, (ii) (Boc)2O, NaOH, 59%.
H
O
H
N
O
N
a
b
+
10
11
12
OH
13
O
O
f
c,d,e
OH
OEt
NH2
15
16
14
Scheme 2. Synthesis of 4,4-dimethylcyclohexyl alanine. Reagents and conditions: (a) reflux, 76%; (b) (i) methylvinyl ketone 0 °C to rt; (ii) HCl, 0 °C to rt, 75% (c) H2, 7% Pd/C,
hexane, rt; (d) Ph3P@CHCO2Et, reflux, 85%; (e) H2, 10% Pd/C, EtOH, rt, 94%; (f) LiAlH4, THF, 0 °C to reflux, 85%.
OH
O
OH
H2N
H
N
O
S
O
N
H
a
O
N
O
O
N
S
O
N
N
18
17
b,c, d
e,f,b,g,d
O
N
N
(or)
(or)
R1
R2
=
=
O
O
S
O
O
O
O
O
H
N
O
H
N
R1
N
R1
N
H
N
O
H
O
O
N
O
N
S
O
N
N
20
19
Chiral HPLC
O
O
O
O
H
H
N
N
R1
N
H
R1
N
H
O
O
R2
N
R2
N
22
21
Scheme 3. Synthesis of azepanone analogs. Reagents and conditions: (a) HBTU, 4-methylmorpholine, 1-methylcyclohexyl alanine, DMF, (or) HATU, TEA, CH2Cl2, rt; (b) HCl,
MeOH, rt; (c) 2-furoic acid, HBTU, 4-methylmorpholine, DMF, (or) 2-furoic acid, HATU, TEA, CH2Cl2, rt; (d) Dess–Martin periodinane, CH2Cl2, rt; (e) Mg, MeOH, 0 °C to rt; (f)
HATU, TEA, picolinic acid, CH2Cl2, MeOH rt; (g) 4-morpholinecarbonyl chloride, TEA, CH2Cl2, 0 °C to rt.

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J. K. Kerns et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4409–4415
by re-protection of the amine as the tert-butylcarbamate. Prepara-
tion of 1-methycyclopentyl alanine followed a similar route start-
ing with cyclopentanone.
In all cases where amino acids were synthesized, the racemic
mixtures were used directly and separation of the resultant diaste-
reomers was performed at the final stage.
to the ketone to provide the desired target 19. In the case where
the P0 substituent was the nicotinamide, the sulfonamide was re-
moved with magnesium in methanol followed by coupling with
picolinic acid. The inhibitor synthesis was then completed as out-
lined above to yield ketone 20. Finally, as the P2 amino acid syn-
thesis produced racemic product, the diastereomeric mixture of
the final compound was purified via preparative chiral HPLC as
previously described, yielding 21 and 22.25,28
Adopting a selectivity cross-screening strategy allowed us to
rapidly assess potency and selectivity of our inhibitors for cathep-
sin S. The results showed, in part, that small alicyclic rings at P2
imparted excellent cathepsin S potency. In addition, it had been
previously shown that modifications at the P2 position allowed
modulation of the selectivity profile.28 As shown in Table 1, the
cyclopentene analog 23 demonstrated essentially equipotent inhi-
bition across human cathepsins S, K, and L. Progressively larger ali-
cyclic rings (C5–C7) (entries 24–26) produced sub-nanomolar
potency at cathepsin S, with 35- to 80-fold selectivity over cathep-
sin K, but little or no selectivity over cathepsin L. Alkyl substituents
(leucine (27) and tert-butyl alanine (28)) at P2 provided increased
potency at cathepsin K as previously described,28 at the expense of
cathepsin S potency thus reversing (27) or narrowing (28) the
selectivity window. While the selectivity window relative to
Preparation of the 4,4-dimethylcyclohexyl alanine, as shown in
Scheme 2, began with preparation of 4,4-dimethyl-2-cyclohexen-
1-one as previously described.36 Thus the enamine (12) arising
from condensation of pyrrolidine with isobutyraldehyde was sub-
jected to 4+2 cycloaddition using methylvinyl ketone. The result-
ing aminal was rearranged under acidic conditions to produce 13
in good overall yield. The olefin of 13 was reduced, the ketone sub-
jected to Wittig conditions and subsequently hydrogenated to pro-
duce ester 14, which was reduced to the resulting alcohol 15, and
the synthesis completed as described in Scheme 1.
Incorporation of the P2 amino acid, for example, 1-meth-
ylcyclohexyl alanine, and completion of the inhibitor synthesis
was accomplished using a sequence analogous to the previous re-
ports and is shown in Scheme 3.25,28 Coupling of the P2 amino acid
to the aminoazepanol 17 to produce intermediate 18 was followed
in one instance by removal of the BOC protecting group, coupling
with the requisite P3 substituent, and oxidation of the azepanol
Table 1
Potency and selectivity of P2 analogsa
O
R1
O
H
N
N
H
O
N
O
O
S
O
N
Entry
R1
Cathepsin S IC50 (nM) (approx 95% CI)
4.9 (3.1, 7.7)
Cathepsin K IC50 (nM) (approx 95% CI)
9.0 (7.2, 11.3)
Cathepsin L IC50 (nM) (approx 95% CI)
3.8 (2.9, 5.0)
23
24
25
0.76 (0.72, 0.80)
0.54 (0.46, 0.63)
27.7 (25.7, 29.9)
40.5 (37.1, 44.2)
4.8 (3.4, 6.8)
1.2 (0.95, 1.5)
26
27
28
29
<0.4
28.2 (25.0, 31.9)
1.0 (0.84, 1.3)
4.7 (4.1, 5.4)
<0.4
2.9 (2.5, 3.3)
1.3 (1.3, 1.4)
<0.4
0.53 (0.48, 0.58)
11.5 (10.4, 12.8)
22.4 (19.2, 26.2)
11.2 (10.3, 12.2)
30
31
32
<0.4
44.5 (41.5, 47.6)
1900 (1800, 2000)
2300 (1700, 3100)
31.9 (19.7, 51.6)
4400 (4000, 4900)
1700 (1200, 2600)
101 (87.4, 117)
37 (28, 49)
a
Cathepsin IC50 values are the mean of n P2 determinations. Assay conditions are described in Ref. 37.

J. K. Kerns et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4409–4415
4413
cathepsin K was eroded with 28, compound 28 provided a small
improvement in selectivity over cathepsin L, albeit only 10-fold.
With these observations, we became intrigued with the idea that
a combination of the alicyclic and alkyl P2 substituents might pro-
vide a superior selectivity profile. As such, we produced com-
pounds 29 and 30 incorporating 1-methylcyclopentyl alanine and
1-methylcyclohexyl alanine, respectively, at P2. Both analogs dis-
played similar potency at cathepsin S relative to their correspond-
ing alicyclic parent (24 and 25). Like compound 28, both 29 and 30
were less potent at cathepsin L, and overall were greater than 25-
to 100-fold selective for cathepsin S over cathepsins K and L. This
result is consistent with the presence in the S2 pocket of an alanine
Table 2
Potency and selectivity of P2 analogsa
O
R1
O
H
N
N
H
O
N
O
O
S
O
N
Entry
R
Cathepsin S IC50 (nM) (approx 95% CI)
1.9 (1.5, 2.3)
Cathepsin K IC50 (nM) (approx 95% CI)
840 (790, 900)
Cathepsin L IC50 (nM) (approx 95% CI)
10.7 (9.7, 11.8)
33
34
35
0.5 (n = 1)
450 (425,470)
1.0 (0.74,1.4)
1.2 (1.0, 1.5)
6200 (3400, 11000)
58.5 (50.2, 68.1)
a
Cathepsin IC50 values are the mean of n P2 determinations. Assay conditions are described in Ref. 37.
Table 3
Potency and selectivity of analogsa
O
R1
O
H
N
R2
N
H
O
N
R3
Entry
R1
R2
R3
Cathepsin S IC50 (nM) (approx 95% CI)
0.58 (0.48, 0.70)
Cathepsin K IC50 (nM) (approx 95% CI)
1100 (1040,1180)
Cathepsin L IC50 (nM) (approx 95% CI)
10.8 (9.8, 11.9)
O
N
N
N
S
S
S
O
O
O
36
O
N
O
37
38
<0.4
136 (135, 138)
174 (127, 238)
48.7 (29.5, 80.6)
139 (79.1, 240)
O
O
N
O
1.5 (1.1, 2.0)
N
O
N
O
N
O
39
40
1.5 (1.1, 2.0)
263 (236, 292)
10 (8.9, 13.3)
429 (425, 433)
10 (8.4, 14.8)
O
31.6 (19.1, 60.3)
a
Cathepsin IC50 values are the mean of n P2 determinations. Assay conditions are described in Ref. 37.

4414
J. K. Kerns et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4409–4415
for Cat L (A135) and Cat K (A134) compared to a glycine in Cat S
(G137), which will restrict the population of P2 rotamers (28–30)
that can be accommodated in this area of the S2 site (see Fig. 2).
Previous work with this series of inhibitors had defined the ste-
reochemical requirements at P2 for potent inhibitors, with the nat-
that the selectivity profile of 39 is due to the L-1-methylcyclohexyl
alanine at P2.
As mentioned previously, cathepsin S plays a key role in invari-
ant chain processing. To assess the cellular activity of the series,
key analogs were tested for their ability to block invariant chain
processing in Raji cells, a human cultured cell line derived from a
Burkitt’s lymphoma.30 As can be seen in Table 4, all the analogs
were effective at blocking intracellular cathepsin S. Importantly,
with the exception of 2, the rank order of cellular potency follows
the isolated enzyme potency. As mentioned previously, cathepsin S
resides in the lysosome, an acidic cellular compartment. Thus the
surprising potency of 2 is likely the result of protonation of the
weakly basic morpholine, thereby allowing it to concentrate in
the lysosome.15
Cathepsin S plays a key role in antigen presentation and as such
is anticipated to be therapeutically relevant in antigen-driven dis-
eases. While excellent potency against cathepsin S has been
achieved in a number of chemical series, obtaining good selectivity
over other members of the papain family has remained a focus of
several efforts. We have described a series of P2 modified azepa-
none inhibitors of cathepsin S with a favorable selectivity profile.
In particular, we have identified several P2 substituents, including
ural
preference for
for the 1-methylcyclopentyl and 1-methylcyclohexyl alanine com-
pounds, where the -amino acid analogs 31 and 32, obtained by
HPLC as previously described,25 were much less potent than the
corresponding -analogs, 29 and 30, at cathepsins K, L, and S.
L
-amino acid configuration being preferred.28
A
similar
L
stereochemistry at P2 was observed in this series
D
L
Given these initial results, we explored further modifications of
the P2 substituent (Table 2). Entry 33 extends the P2 ring system of
24 by a methylene unit. This resulted in decreased cathepsin K po-
tency with little to no effect on cathepsin S and L potency. The mix-
ture of cis and trans isomers, produced by introducing a methyl
group to the 4-position of 25 to produce the 4-methylcyclohexyl
alanine ring system (entry 34) was less potent at cathepsin K com-
pared to 25. This is consistent with the result that lengthening the
P2 substituent is detrimental to cathepsin K potency. No differen-
tiation between cathepsin S and L was seen for 34. Finally, we were
interested to see the recent report of Irie et al. showing that the
4,4-dimethylcyclohexyl system provided excellent selectivity
against both cathepsins K and L, albeit in a different chemical ser-
ies.10 We explored this system in the azepanone series. Given the
results seen with 34, we anticipated a similar profile with little
selectivity over cathepsin L. Similar to the report of Irie, the 4,4-di-
methyl cyclohexyl alanine analog 35 was less potent at cathepsins
K and L and exhibited improved cathepsin S selectivity, especially
with respect to cathepsin K. The selectivity against cathepsin L was
similar to that found in 30. Together, the results with 33, 34, and 35
are consistent with a smaller P2 pocket in cathepsin K compared to
cathepsin S, as described above.
During the course of our optimization of the P2 position, we had
also explored variants at the P0 and P3 positions. In the P3 position,
we had identified morpholine urea as a suitable substituent. Table
3 displays the effects this substitution had on inhibitor potency
and selectivity. The P2 extended analog 36 shows similar activities
for the three cathepsins as compared to the P3 furan analog 33,
exhibiting >1000-fold selectivity for cathepsin S over cathepsin K.
Compound 37, possessing the P3 morpholine urea in combination
with the P2 1-methylcyclohexyl alanine, had improved selectivity
over both cathepsins K and L relative to 30. Combining 30 and 34
by tying back the cyclohexyl system into the 7,7-dimethylbicy-
clo[2.2.1]hept-1-yl alanine system, producing 38, resulted in de-
creased cathepsin S potency and a similar selectivity profile
relative to 37. In the P0 position, we had identified 2-nicotinamide
as a suitable substituent. Incorporating a P0 nicotinamide into 30,
producing 39 resulted in excellent overall selectivity over both
cathepsins K and L. To probe the effect the P0 nicotinamide has
on selectivity further, compound 40 was prepared possessing the
P2 leucine analogous to 27. Similar to 27, compound 40 was more
selective for cathepsins K and L than for cathepsin S suggesting
L-1-methylcyclohexyl alanine, which impart cathepsin S inhibitor
selectivity.
Acknowledgment
The authors acknowledge Edit Kurali for her assistance with the
statistical analysis.
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Table 4
Whole cell potency of selected analogsa
Entry
Cathepsin S IC50 (nM)
Whole cell IC50 (lM) SEM
14. Teno, N.; Miyake, T.; Ehara, T.; Irie, O.; Sakaki, J.; Ohmori, O.; Gunji, H.;
Matsuura, N.; Masuya, K.; Hitomi, Y.; Nonomura, K.; Horiuchi, M.; Gohda, K.;
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2
8.3
0.5
1.3
<0.4
<0.4
1.5
0.94 0.22
1.78 0.33
4.8 0.42
1.4 0.22
0.57 0.11
3.88 0.22
25
28
30
37
39
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a
Whole cell IC50 values are the mean of n P2 determinations. Assay conditions
are described in Ref. 38.

J. K. Kerns et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4409–4415
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assay. Cathepsin S concentration was 4 nM. The assay buffer contained 50 mM
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against human recombinant cathepsin K was tested in vitro using z-LR-AFC
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substrate at the substrate Km (5 lM). Cathepsin K concentration was 0.05 nM.
The assay the buffer contained 100 mM NaOAc, 150 mM NaCl, 5 mM EDTA,
1 mM CHAPS, 1 mM DTT, pH 5.5. Compound activity against human
recombinant cathepsin L was tested in vitro using peptide substrate Z-LR-
AMC substrate at the substrate Km (2 lM). Cathepsin L concentration was
0.01 nM. The assay buffer contained 100 mM NaOAc, 1.0 mM CHAPS, 1.0 mM
DTT, 5.0 mM EDTA, pH 5.5. The IC50 values obtained are the mean of at least
two experiments run in duplicate.
38. To determine cellular potency of the P2 modified azepanone inhibitors of
cathepsin S, human Raji cells were incubated with inhibitor in DMSO (vehicle),
or with DMSO only, and the inhibitor-induced decrease in cell surface
expression of MHC class II/CLIP (class II-associated invariant chain peptide)
complexes was measured by flow cytometric analysis using the CerCLIP
antibody (BD Pharmingen) as the detection reagent. This method has been
previously described in Ref. 30.
39. Hardegger, L. A.; Kuhn, B.; Spinnler, B.; Anselm, L.; Ecabert, R.; Stihle, M.; Gsell,
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