Synthesis of proline analogues as potent and selective cathepsin S inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.04.023

Cathepsin S is a potential target of autoimmune disease. A series of proline derived compounds were synthesized and evaluated as cathepsin S inhibitors. We discovered potent cathepsin S inhibitors through structure-activity relationship studies of proline analogues. In particular, compound 19-(S) showed promising in vitro/vivo pharmacological activities and properties as a selective cathepsin S inhibitor.

Full text of DOI:10.1016/j.bmcl.2013.04.023

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3140–3144
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of proline analogues as potent and selective cathepsin
S inhibitors
Mira Kim a,b, Jiyoung Jeon a, Jiyeon Song a, Kwee Hyun Suh a, Young Hoon Kim a, Kyung Hoon Min b,
,
⇑
Kwang-Ok Lee a,
⇑
a Department of Drug Discovery, Hanmi Research Center, 377-1 Yeongcheon-ri, Dongtan-myeon, Hwaseong, Gyeonggi-do 445-813, Republic of Korea
b College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Cathepsin S is a potential target of autoimmune disease. A series of proline derived compounds were syn-
thesized and evaluated as cathepsin S inhibitors. We discovered potent cathepsin S inhibitors through
structure–activity relationship studies of proline analogues. In particular, compound 19-(S) showed
promising in vitro/vivo pharmacological activities and properties as a selective cathepsin S inhibitor.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 16 March 2013
Revised 5 April 2013
Accepted 9 April 2013
Available online 16 April 2013
Keywords:
Cathepsin S
Proline analogues
Small molecules
Cathepsin S is a lysosomal cysteine protease in the papain
superfamily1,2 and is expressed predominantly in antigen present-
ing cells such as dendritic cells, macrophages, and B lymphocytes.3
Cathepsin S plays a key role initiating antigen presentation to CD4+
T-cells through degradation of the invariant peptide chain associ-
ated with major histocompatibility complex class II.4–7 Gene
knockout studies in mice have demonstrated that a deficiency of
cathepsin S diminishes its capacity to present antigens to immune
cells, and reduces susceptibility to collagen-induced arthritis8 and
impaired tumor angiogenesis and tumor growth.9 Furthermore,
extracellular cathepsin S is involved in the initiation and/or main-
tenance of neuropathic pain in peripheral nerve-injured mice and
rats.10 Thus, cathepsin S could be a therapeutic target of metastatic
cancer,11 and neuropathic pain,10,12 as well as autoimmune dis-
eases such as rheumatoid arthritis,13 psoriasis,14 multiple sclero-
sis,15 and atopic dermatitis.16 A number of cathepsin S inhibitors
have been developed18 (Fig. 1) and some are currently being inves-
tigated in clinical trials,11,17 in which therapeutic benefit of inhib-
iting cathepsin S will be validated. With regard to development of
cathepsin S inhibitors, besides the importance of potency for a
cathepsin S inhibitor, selectivity for cathepsin S may also be an
important issue to reduce potential side effects of non-selective
inhibitors because each of the subtypes of this family has different
functions. Herein, we describe potent and selective cathepsin S
inhibitors with a proline scaffold. We found a cathepsin S inhibitor,
compound 4, with an IC50 value of 156 nM through screening of
our in-house, proline-based focused chemical library. Our syn-
thetic approach focused on diversifying three parts (S1, S2, and
S3) of compound 4 to explore the structure–activity relationships
(SAR) as shown in Figure 2.
The general synthetic procedure for proline-derived analogues
is outlined in Scheme 1. Mesylation of commercially available
proline analogue 5 under Mitsunobu conditions and subsequent
nucleophilic substitution of thiol gave thioether 7, which was oxi-
dized to the corresponding sulfone 8 by mCPBA. The Boc group
was deprotected to provide amine 9. Derivatization of the S1 part
was achieved by introducing various substituents to the amino
group of compound 9. The acid 11 obtained from hydrolysis of
10 were coupled with amine 12 in the presence of EDCI, HOBt,
and DIPEA in dichloromethane to give 13, which was converted
to the desired analogues 14 by treatment with Dess–Martin
periodinane.
The synthetic route for compound 12 for the R3variation is
illustrated in Scheme 2. Commercially available amino-alcohol
15 as s racemate was protected with s tert-Boc group, followed
by oxidation with Dess–Martin periodinane that furnished the
aldehyde 17 in high yield (85% over two-steps). Coupling of 17
with 2-benzoxazole and Boc deprotection gave the desired frag-
ment 12.
The SAR study for the S1 moiety was initially investigated in an
in vitro assay for the human cathepsin S (hCat S) enzyme (Table 1).
Introducing substituents at the S1 position significantly increased
hCat S in vitro activities, compared to those of compound 4 without
any substituents (S1 = H). In particular, compound 19 (X = carbonyl)
⇑
Corresponding authors. Tel.: +82 2 820 5599; fax: +82 2 815 5262 (K.H.M.); tel.:
+82 31 371 5081; fax: +82 31 371 5068 (K.-O.L.).
E-mail addresses: khmin@cau.ac.kr (K.H. Min), kolee@hanmi.co.kr (K.-O. Lee).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.04.023

M. Kim et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3140–3144
3141
N
N
Cl
NH
O
N
O
N
H
N
O
O
N
H
N
O
N
H
S
N
N
H
N
N
HN
CN
Cl
N
O
O
NH2
S
O
O
2
3
1
Figure 1. Structures of representative cathepsin S inhibitors.18
S1
R2
R1
H
N
S3
X
O
O
N
R3
O
N
HN
O
N
S
HN
Y
O
S2
O
O
O
4
X = CO, SO2
Y = SO2, S
hCatS(IC50) = 156 nM
R1, R2 = alkyl, aryl, etc.
R3, R4 = H, alkyl, aryl, cyloalkyl, etc.
Figure 2. Structures of compound 4 and its derivatives for the structure–activity relationships study.
Boc
Boc
N
Boc
N
O
O
N
O
c
f
b
a
R2
O
O
S
O
MsO
HO
5
6
7
R1
Boc
N
X
N
H
N
O
O
O
O
O
O
d
e
R2
R2
R2
S
S
S
O
O
O
O
O
O
8
9
10
R1
R1
X
X
N
R3
O
O
N
H2N
HO
O
N
R3
g
+
R2
HN
R2
O
N
OH
S
O
S
O
O
O
HO
11
12
13
R1
X
O
N
R3
h
R2
HN
O
N
S
O
O
O
14
Scheme 1. Reagents and conditions: (a) MsOH, DIAD, PPh3,TEA, THF, 70 °C, 15 h; (b) R2SH, NaH, THF, 0 °C, 30 min then, 50 °C, 4 h; (c) mCPBA, DCM, 8 h; (d) 4 N HCl in 1,4-
dioxane, DCM, 2 h; (e) R1COOH, EDC, HOBT, DIPEA, DCM, 20 h or R1XCl, TEA, DCM, 2 h or (R1CO)2O, TEA, MeOH, 2 h; (f) LiOH in H2O/MeOH, THF, 0 °C–rt, 2 h; (g) EDCI, HOBt,
DIPEA, DCM, rt, 5–10 h; (h) Dess–Martin periodinane, DCM, 0 °C to rt, 2 h.
and 20 (X = sulfonyl) showed about 44-fold more potent activity
than compound 4. Compound 19 showed better microsomal stabil-
ity than compound 20 (data not shown). To explore the effects of
the S2 moiety on activity, thioether (23), sulfynyl (24), and ether
(25) derivatives were synthesized19 and compared to sulfonyl com-
pound 19. This replacement of a sulfonyl group decreased the
inhibitory activity for the hCat S enzyme. Therefore, substituents
at the S1 moiety and SO2 as a linker at the S2 moiety are crucial
for enhancing hCat S inhibitory activity.
Further optimization of the R1 and R2 groups was conducted as
shown in Table 2. Derivatization of R1 with various alkyl or aryl
moieties resulted in a subtle change in the inhibitory activity for

3142
M. Kim et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3140–3144
R3
R3
O
R3
O
a
b
O
OH
OH
O
N
H
O
N
H
H2N
H
17
15
16
R3
O
R3
O
H2N
HO
O
c
d
HN
HO
O
N
N
18
12
Scheme 2. Reagents and conditions: (a) Boc2O, DCM, rt, 2 h; (b) Dess–Martin periodinane, 0 °C to rt, 1 h; (c) benzoxazole, 2 M iso-PrMgCl, THF, ꢀ5 °C to rt, 2 h; (d) TFA, DCM,
rt, 1 h.
Table 1
Optimization of the S1 moiety and linker Y from compound 4
Table 2
In vitro activities of human cathepsin S by modifying the R1 and R2 substitution
groups
R1
X
O
R1
N
O
HN
O
N
O
Y
N
R2
O
HN
O
N
S
O
O
Compound
X
Y
R1
—
Methyl
Methyl
Acetyl
—
Methyl
Methyl
Methyl
—
hCat S IC50 (nM)a
O
4
19
20
21
22
23
24
H
CO
SO2
CH2
Phenyl
CO
CO
CO
—
SO2
SO2
SO2
SO2
SO2
S
SO
O
—
156
3.3
3.5
45.4
12.4
20.7
83.7
>1,000
1.9
Compound R1
R2
hCat S IC50 (nM)
19
26
27
28
29
30
31
32
33
34
35
Metheyl
Phenyl
Ethyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
3.3
3.0
3.1
2.4
3.3
2.7
3.3
3.7
3.3
3.5
2.0
iso-Propyl
Cyclohexyl
CF3
Methoxyethyl
Pyrrolidinyl
4-Pyridyl
3-Pyridiyl
4-
Chlorophenyl
3-
Chlorophenyl
1,10-Biphenyl
Methyl
25
LHVSb,c
In vitro enzyme assay was done using a SensoLyteÒ 520 Cathepsin S Activity
Assay Kit from Anaspec, Inc.
a
b
LHVS (leucine homophenylalanine vinyl sulfone) was used as
a reference
compound, as an irreversible pan-cathepsin inhibitor.1,20
c
LHVS was synthesized using the synthetic protocol reported in WO95/23222.
36
Phenyl
Phenyl
1.1
37
38
39
40
4.1
2.3
3.2
3.2
2-Chloro-phenyl
4-Fluoro-phenyl
4-(1H-Pyrazol-1-
yl)phenyl
hCat S. (26–37). Additionally, adding a halogen onto the phenyl
ring at R2 (38 and 39) did not enhance activity for hCat S. Com-
pound 41 with an aliphatic group at R2 showed decreased cathep-
sin S inhibitory activity.
Methyl
Methyl
41
Methyl
Ethyl
>100
Finally, the SAR for the S3 moiety was examined by modifying
R3. Subsequent coupling of compound 12 with the acid 11, and oxi-
dation using Dess–Martin periodiane provided final compounds
43–46, whose activity is summarized in Table 3. Compound 46
(R1 = cyclohexyl, R3 = H) showed about an eightfold reduction in
activity. Introducing methyl (43), iso-Propyl (44), and benzyl (45)
groups to R1 resulted in decreased potency or loss of activity. In or-
der to scrutinize the effect of R3 stereochemistry, compounds 19-
(S)21 and 19-(R) were synthesized as a single diastereomer, using
(S)-2-aminobutan-1-ol and (R)-2-aminobutan-1-ol, respectively.
However, no significant difference in in vitro activity was observed,
indicating the R3 stereochemistry is not important for activity.
Some compounds (19-(S), 26, 29, 36 and 37) exhibiting potent
in vitro activity for hCat S were assayed for human cathepsin B
(hCat B) and K (hCat K) enzymes. As shown in Table 4, the com-
pounds tested showed high selectivity for hCat S but no activity
for hCat B and K. The pan-cathepsin inhibitor LHVS was used as a
positive control. Consequently, 19-(S) was chosen as the lead com-
pound, and further assessment for the inhibition of Lip10 cleavage
was carried out using Jiyoye cells (Human Berkitt’s lymphoma cell)
Table 3
Effect of substituents and stereochemistry at the R3 position on human cathepsin S
activity
R1
O
O
N
R3
HN
S
O
O
O
O
N
Compound
R1
R3
hCat S IC50 (nM)
43
44
45
46
19-(S)
19-(R)
Methyl
Methyl
Methyl
Methyl
iso-propyl
Benzyl
H
(S)-Ethyl
(R)-Ethyl
18.1
24.6
>100
36.6
3.3
Cyclohexyl
Methyl
Methyl
4.3

M. Kim et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3140–3144
3143
Table 4
Inhibitory activity of cathepsin S inhibitors for human cathepsin B and K
Table 6
Pharmacokinetic profile of compound 19-(S)a in mouse, rat, and dog
Compound
IC50(nM)
parameters
(po)
AUC0–24
(ng h/mL)
Cmax
(ng/mL)
T1/2
(h)
Bioavailability
(%)
hCat S
hCat Ba
hCat Ka
Mouseb
Ratc
1,027
7,247
5,047
1,910
3,523
3,817
1.6
2.1
3.7
28.7
64.8
48.6
19-(S)
26
29
36
37
3.3
3.0
3.3
1.1
4.1
1.9
>10,000
>10,000
>10,000
>1000
>10,000
3
>10,000
>10,000
>10,000
>1000
>10,000
4
Dogd
a
Compound was dissolved in a vehicle as a solution of 10% 2-hydroxypropyl
b-cyclodextrin in distilled water.
b
LHVS
The pharmacokinetic parameters were determined after a single oral admin-
istration (10 mg/kg, n = 3) and intravenous injection (5 mg/kg, n = 3) in ICR mice
a
Enzymatic assay for hCat B and hCat K was performed using SensoLyteÒ 520
(males, 7 weeks of age).
Cathepsin Activity Assay Kit from Anaspec, Inc.
c
The pharmacokinetic parameters were determined after a single oral admin-
istration (10 mg/kg, n = 3) and intravenous injection (5 mg/kg, n = 3) in Sprague–
Dawley rats (males, 7 weeks of age).
d
The pharmacokinetic parameters were determined after a single oral admin-
istration (10 mg/kg, n = 3) and intravenous injection (1 mg/kg, n = 3) into beagle
dogs (males, 14 months of age).
IFN – γ (50 μM)
19-(S) (nM)
-
1000 100
10
1
-
Lip10
results of these in vivo studies are shown in Figure 4. 19-(S) treated
mice exhibited a significantly decreased mean clinical score com-
pared to that in vehicle-treated mice.
In summary, we discovered a novel cathepsin S inhibitor, com-
pound 19-(S), which showed not only excellent in vitro activity for
hCat S, but also high selectivity for other isozymes such as hCat B
and K. Moreover, 19-(S) showed a good DMPK profile as an orally
available compound, and significant in vivo efficacy in a mouse
CIA model. Taken together, compound 19-(S) surely deserves fur-
ther evaluation as a promising lead.
Figure 3. Lip10 accumulation assay of 19-(S) in Jiyoye cells.22
Table 5
In vitro microsomal stability and CYP inhibition assay of compound 19-(S)
b
Microsomal stabilitya, (%)
CYPs enzyme inhibition, IC50
(
lM)
Human Dog Rat Mouse CYP3A4 CYP1A2 CYP2C9 CYP2C19 CYP2D6
70 47 37 37 9.95 >20 5.00 >20 >20
Acknowledgments
a
Remaining percentage of metabolism by incubation of the parent molecule
(5
l
M) with liver microsomes of mouse, rat, dog, and human for 60 min (duplicate).
We thank Eun Young Kim, Hyo Jung Bang, Tae Hun Song, and
Dr. Hankyung Kim for their assistance in the pharmacokinetic
studies. This research was supported by a grant from the National
R&D Program for Cancer Control, Ministry for Health, Welfare and
Family Affairs, Republic of Korea (1020130), and partially by
the Institute for Biomedical and Pharmacological Research,
Chung-Ang University Health Care System in 2012.
b
Data were analyzed by fluorescence detection method.
10
Control
Compound 19-(S), 30 mg/kg
8
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F.; Ehara, T.; Iwasaki, A.; Iwaki, Y.; Hitomi, Y.; Toyao, A.; Gunji, H.; Teno, N.;
Iwasaki, G.; Hirao, H.; Kanazawa, T.; Tanabe, K.; Hiestand, P. C.; Malcangio, M.;
Fox, A. J.; Bevan, S. J.; Yaqoob, M.; Culshaw, A. J.; Hart, T. W.; Hallett, A. Bioorg.
Med. Chem. Lett. 2008, 18, 5280; (b) Kerns, J. K.; Nie, H.; Bondinell, W.;
Widdowson, K. L.; Yamashita, D. S.; Rahman, A.; Podolin, P. L.; Carpenter, D. C.;
23. Male DBA1/J mice (8 weeks old, n = 7 per group) were immunized with a
volume of 0.07 ml of emulsion of Complete Freund Adjuvant (CFA) and bovine
type II collagen (CII) by intradermal injection at the base of tail. Three weeks
later, the mice were given booster immunization with bovine type II collagen
emulsified in Incomplete Freund’s Adjuvant (IFA) in the same manner. The
compound 19-(S) was dissolved in
a vehicle as a solution of 10% 2-
Hydroxypropyl beta-cyclodextrin (HbCD) in distilled water. Animals were
orally treated with 19-(S) at dose of 30 mg/kg (Q1D) or vehicle for 14 days. The
severity of arthritis was assessed 2 weeks starting on 10 days after the booster
immunization, and was graded by arthritis score and degree of paw swelling.