Identification of a novel class of succinyl-nitrile-based Cathepsin S inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.02.046

The synthesis and in vitro activities of a series of succinyl-nitrile-based inhibitors of Cathepsin S are described. Several members of this class show nanomolar inhibition of the target enzyme as well as cellular potency. The inhibitors displaying the greatest potency contain N-alkyl substituted piperidine and pyrrolidine rings spiro-fused to the α-carbon of the P1 residue.

Full text of DOI:10.1016/j.bmcl.2007.02.046

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2465–2469
Identification of a novel class of succinyl-nitrile-based
Cathepsin S inhibitors
Younes Bekkali,* David S. Thomson, Raj Betageri, Michel J. Emmanuel, Ming-Hong Hao,
Eugene Hickey, Weimin Liu, Usha Patel, Yancey D. Ward, Erick R. R. Young,
Richard Nelson, Alison Kukulka, Maryanne L. Brown, Kathy Crane,
Della White, Dorothy M. Freeman, Mark E. Labadia,
Jessi Wildeson and Denice M. Spero
Department of Medicinal Chemistry, Boehringer Ingelheim Pharmaceuticals Inc., 900 Ridgebury Road, PO Box 368,
Ridgefield, CT 06877-0368, USA
Received 12 December 2006; revised 6 February 2007; accepted 9 February 2007
Available online 23 February 2007
Abstract—The synthesis and in vitro activities of a series of succinyl-nitrile-based inhibitors of Cathepsin S are described. Several
members of this class show nanomolar inhibition of the target enzyme as well as cellular potency. The inhibitors displaying the
greatest potency contain N-alkyl substituted piperidine and pyrrolidine rings spiro-fused to the a-carbon of the P1 residue.
Ó 2007 Elsevier Ltd. All rights reserved.
Antigen processing and presentation is a process during
which foreign proteins are degraded into smaller pep-
tides by antigen presenting cells (APCs) and then deliv-
ered to the cell surface in association with major
histocompatibility complex (MHC) class II molecules.1
The recognition of the MHC class II/peptide complex
by CD4+ T cells triggers an immune response.2 The
MHC class II molecules are associated in the endoplas-
mic reticulum with invariant chain (Ii) which, acting as a
chaperone peptide,3 mediates the delivery of the MHC
class II–Ii complexes into the endocytic compartment
where the antigenic peptides are generated. The invari-
ant chain must be proteolyzed prior to MHC binding
of the antigenic peptides. The proteolysis of the invari-
ant chain involves the participation of endosomal/lyso-
somal proteases, such as aspartate and cysteine
proteases.4
p10 oligomer (Iip10) to a small peptide called CLIP.
CLIP is subsequently released from the MHC class II
binding groove by association with a third protein called
HLA-DM.6,7 Due to its involvement in the antigen pro-
cessing and presentation process, Cat S became an
attractive target for medicinal chemists as inhibition
may modulate autoimmune diseases such as rheumatoid
arthritis (RA) and multiple sclerosis (MS).8
The catalytic site of Cat S contains a thiolate anion
formed by the Cys25 and His164 pair. Nucleophilic
addition of the thiolate anion at the carbonyl carbon
of the peptide bond followed by hydrolysis produces
the proteolyzed Ii. Most cysteine protease inhibitors
reported in the literature are peptide based and act
through the chemical interaction of an electrophilic
‘warhead’ with the thiolate anion of the active
site.9,10
Cathepsin S (Cat S) is a 24 kDa monomeric cysteine
protease, which is a member of the papain super-fam-
ily.5 Cat S is involved in the last step of the proteolysis
of Ii, where it degrades the remaining invariant chain
We previously reported the design and synthesis of such
inhibitors of Cat S (Fig. 1) and their inhibitory activities
as well as their cellular potencies. From this study we
ascertained that the nitrile ‘warhead’ is critical for po-
tency against Cat S.11 It was thought that such dipeptide
inhibitors may suffer pharmako-kinetic limitations com-
monly seen with peptidic compounds. With an attempt
to reduce peptide character of our first series our search
Keywords: Cathepsin S; Cat S; Nitrile; Succinyl.
*
Corresponding author. Tel.: +1 203 791 6746; fax: +1 203 837
5067; e-mail: ybekkali@rdg.boehringer-ingelheim.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.02.046

2466
Y. Bekkali et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2465–2469
O
P2 side chain
O
O
i
ii
iii
EtO
R
OH
R
Br
OEt
R
OH
R
1
2
3
4
O
iv
H
N
N
O
N
N
P1 side chain
O
O
H
O
O
O
vi
v
Gly69
R
N
R
OH
R
Cl
P3 side chain
O
7
6
5
Figure 1. Dipeptide based inhibitor of Cathepsin S.
vii
R
O
O
O
O
O
viii
for new and novel Cat S inhibitors led us to a series of
succinate-based compounds (Fig. 2) which are reported
herein.
OH
N
N
N
O
O
O
9
R
8
From our earlier work on peptidic inhibitors,11,12 we
were aware of the importance of the hydrogen bond
established between the P2 amide hydrogen and the car-
bonyl of Gly69 of the protein. However, we envisioned
overcoming any loss in binding potency caused by the
removal of this key hydrogen bond interaction through
structural modifications of the P1 and P2 side chains.
x
R
O
O
R
O
ix
OMe
N
OMe
HO
O
O
10
11
Scheme 1. Reagents and conditions: (i) LiAlH4, Et2O (98%); (ii) HBr,
H2SO4 (82%); (iii) NaOEt, diethyl malonate (47%); (iv) KOH, MeOH,
180 °C (89%); (v) oxalyl chloride, CH2Cl2 (43%); (vi) (S)-(À)-4-
isopropyl-2-oxazolidinone, n-BuLi, THF (77%); (vii) NaHMDS (1 M
in THF), 2-bromo-1-morpholin-4-yl-ethanone, THF (17–47%); (viii)
LiOH, H2O2, THF, H2O (80%); (ix) morpholine, EDC, HOBt, DMF,
0 °C–rt (99%); (x) LiOH, THF, MeOH/H2O (76%). The yields reported
here were for R = 2-indanyl.
During the course of this study several structural models
and X-ray structures of Cat S complexed with inhibitors
were elucidated.11 The structural information was par-
ticularly helpful in guiding the design and optimization
of the side chains. We also took advantage of struc-
ture–activity relationships generated from the peptide
series.11,13
The absolute stereochemistry of the P2 side chain was
set using Evans’ chiral oxazolidine methodology.14,15
The chiral auxiliary was introduced by reacting acid
chloride 6 with the lithium anion of (S)-(À)-4-isopro-
pyl-2-oxazolidinone. The alkylation of 7 with 2-bro-
mo-1-morpholin-4-yl-ethanone, which was prepared
via the reaction of morpholine with bromoacetic anhy-
dride, provided the desired compound 8 which was iden-
To examine the optimal substitution in the P2 side
chain, several compounds were made using alkylated
succinic acid derivatives which were either commercially
available or synthesized using the general process de-
scribed in Scheme 1.
The requisite acid chlorides 6 were synthesized from
carboxylic acids 1 using a standard malonate ester
homologation sequence. Reduction of the carboxylic
acid 1 with lithium aluminum hydride gave the primary
alcohol 2. Compound 2 was converted to bromide 3
using hydrobromic acid. Reaction of 3 with the anion
of diethyl malonate gave malonic ester 4 which was
hydrolyzed and decarboxylated to the corresponding
carboxylic acid 5 using potassium hydroxide. Reaction
of 5 with oxalyl chloride gave the acid chloride 6.
1
tified by H NMR as a single diastereomer. Oxidative
hydrolysis of the chiral auxiliary gave the key carboxylic
acid intermediate 9. Compound 9 could also be obtained
through amide coupling of morpholine with commer-
cially available succinic acid derivatives 10 followed by
hydrolysis of the resulting methyl esters 11.
Several P1 amino amides 13 were prepared by treatment
of the commercially available N-Boc-amino acids 12
with EDC, HOBt, and NH4OH followed by Boc re-
moval with HCl (Scheme 2). Additional P1 residues,
amino nitriles 15 and 19 identified from our earlier work
P2 side chain
O
O
R
H
N
i, ii
H3N+
O
N
N
O
Cl
NH2
OH
O
N
R2
R1
O
R
R
O
12
13
P3 side chain
Figure 2. Succinate-based inhibitor of Cathepsin S.
Scheme 2. Reagents and condition: (i) EDC, HOBt, NH4OH, DMF,
0 °C–rt (90%); (ii) HCl (4 N in 1,4-dioxane) (>99%). The yields
reported here were for R = CH2CH2Ph.
P1 side chain

Y. Bekkali et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2465–2469
2467
O
N
15, 19 or amino-amide salts 13 (Scheme 4). In the latter
case the intermediate amide products were dehydrated
using cyanuric chloride. Further experimental details
of the preparation of these succinate inhibitors of Cat
S are available in the patent literature.16
H2N
i
N
R
N
R
14
15
The synthesized succinates were tested in a Cat S
binding assay as described in our previous published
studies11 and in a similar cellular assay to that developed
by Riese et al.17 in which the presence or absence of the
p10 invariant chain fragment (the natural Cathepsin S
substrate) was determined and the minimal inhibitory
concentrations (MICs) at which the p10 band can be de-
tected were reported.
OH
OH
O
N
H2N
iii
iv
ii
N
H
N
N
N
R1
R1
R1
16
17
18
19
Scheme 3. Reagents and conditions: (i) NH4Cl, KCN, H2O, 60 °C
(53%). The yield reported here was for R = Me; (ii) aldehydes or
ketones, AcOH, Na(OAc3)BH, THF (92%); (iii) oxalyl chloride,
DMSO, CH2Cl2, Et3N, À78 °C–rt (56%); (iv) NaCN, NH4Cl, NH3
(2.0 M in MeOH), MgSO4, 60 °C (51%). The yields reported here were
for R1 = 3,3-dimethyl-butyl.
Our optimization work began at the P1 site and the re-
sults are summarized in Tables 1 and 2. The P2 side
chain was kept as cyclohexylmethyl, which was previ-
ously determined to be a preferred P2 substituent.11
Table 1 shows several P1 substituents that were selected
and incorporated in the succinate series based on
previous dipeptides SAR.11,13 Compound 21b with ben-
zyloxy-methyl as P1 showed an improvement over 21a
in the binding activity and gave a hint of cellular po-
tency. Compound 21e with a piperidine based P1 group
showed potent cellular activity despite poorer binding
affinity; the boost in cellular activity could be explained
by the presence of the basic nitrogen in 21e as identified
in our previous investigations.13 Several N-substitution
variants, such as 21f, were prepared but they showed
no improvement over 21e.
on dipeptide inhibitors,13 were synthesized from com-
mercially available cyclic piperid-4-ones 14 and pyrroli-
din-3-ol 16, respectively (Scheme 3). In the first case,
Strecker reaction of commercially available ketone 14
with ammonium chloride and potassium cyanide in
water gave compound 15. In the second case, reductive
amination of the pyrrolidin-3-ol 16 with aldehydes or
ketones afforded 17 in high yields. Swern oxidation of
17 gave ketones 18. Strecker reaction of 18 with ammo-
nium chloride, ammonia, and potassium cyanide in
methanol gave amino-nitriles 19 in good yields.
Further investigations were performed using N-substi-
tuted pyrrolidines as P1 groups. These compounds are
generally more potent in both the binding and cellular
The targeted inhibitors 20 were then prepared via stan-
dard peptide coupling conditions of 9 and amino nitriles
R
R
O
O
H
i or i and ii
OH
N
CN
R2
H2N
N
Y
+
N
O
O
R1
O
O
R1 R2
13, 15 or 19
20
9
Y = CONH2, CN
Scheme 4. Reagents and conditions: (i) EDC, HOBt, Hunig’s base , DMF, 0 °C–rt (69%); (ii) cyanuric chloride, DMF, 0 °C (57%). The yields
reported here were for compound 22h.
Table 1. SAR of the P1 side chain
O
H
CN
R1 R2
N
N
O
O
21
Compound
R1
R2
IC50 (nM)
Kd (nM)
[p10 MIC]17(nM)
21a
21b
21c
21d
21e
21f
H
–CH2CH2Ph
–CH2OCH2Ph
–CH2OCH2(p-Cl)Ph
171–187
37–63
62
25
4.39
4.86
12
5000
1000
5000
5000
50
H
H
–CH2CH2–
135–154
251–641
207–347
–CH2CH2NMeCH2CH2–
–CH2CH2N(CH2Ph)CH2CH2–
177
52
1000

2468
Y. Bekkali et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2465–2469
Table 2. SAR of the P1 side chain
O
O
H
H
CN
CN
2
2
N
N
N
N
1
1
O
O
O
O
N
N
R
R
22 (1R, 2S)
22 (1R, 2R)
Compound
Configurationa
R
IC50 (nM)
Kd (nM)
[p10 MIC]17 (nM)
22a
22b
22c
22d
22e
22f
22g
22h
22i
(1R,2R)
(1R,2R)
(1R,2R)
(1R,2R)
(1R,2R)
(1R,2R)
(1R,2R)
(1R,2S)
(1R,2R)
(1R,2S)
(1R,2R)
H
141
123
161
60.6
55.3
20
100
1000
100
1000
100
100
50
Me
i-Pr
155–172
66–82
39–89
22–39
75–78
23–49
41–79
23–35
29–31
22.32
–CH2CH(CH3)2
–CH2CH(CH2CH2)
Cyclopentyl
Cy
n.d.
17.8
13.8
11
–CH2Cy
100
100
100
100
Cycloheptyl
–CH2Ph
–CH2CH2Ph
22j
22k
3.64
15.2
a The diastereomers were separated by reverse-phase HPLC. The assignment of relative stereochemistry was performed by analogy based on the
assignments of similar analogues in the dipeptide series.13 X-ray crystal structure determination of inhibitor 22h co-crystallized with Cathepsin S
confirmed our assignment of the relative stereochemistry.
assays. A variety of N-substituents investigated are well
tolerated. This exploration revealed 22g as the best
inhibitor in this series in terms of binding and cellular
potency Table 3.
Table 3. SAR of the P2 side chain
R
O
H
N
CN
Next, we sought to optimize compound 22g by fine-tuning
the cyclohexylmethyl P2 residue. Several substituted
cyclohexyl and related P2 side chains were incorporated
in an attempt to properly fill the lipophilic S2 pocket of
the enzyme and enhance the binding and cellular potency
of this series. This exercise demonstrated that a variety of
sterically demanding groups are tolerated at the P2
position and identified inhibitor 23c (Kd = 5 nM) as the
most potent Cat S inhibitor in the series in terms of
binding. However, we did not observe a corresponding
increase in cellular potency.
N
O
O
N
Compound
R
IC50
(nM)
Kd
(nM) (nM)
[p10 MIC]17
22g
23a
23b
23c
23d
23–49 17.8
47–62 77.3
14–17 13.8
50
100
100
100
nd
*
In conclusion, this study shows that the novel succinate
scaffold approach was successful in providing several
potent and cellularly active Cat S inhibitors such as
22g and 23c. These compounds are being evaluated in
other assays and the results will be reported in due
course.
*
*
1.9
5.4
*
References and notes
3136
nda
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*
*
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Physiol. 1997, 59, 63.
23e
23f
63–74 59
100
100
42
23.1
*
a nd, not determined.

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