Synthesis and SAR of arylaminoethyl amides as noncovalent inhibitors of cathepsin S: P3 cyclic ethers

Article abstract of DOI:10.1016/j.bmcl.2006.07.033

The synthesis and structure-activity relationship of a series of arylaminoethyl amide cathepsin S inhibitors are reported. Optimization of P3 and P2 groups to improve overall physicochemical properties resulted in significant improvements in oral bioavail

Full text of DOI:10.1016/j.bmcl.2006.07.033

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5112–5117
Synthesis and SAR of arylaminoethyl amides as noncovalent
inhibitors of cathepsin S: P3 cyclic ethers
David C. Tully,* Hong Liu, Arnab K. Chatterjee, Phil B. Alper, Robert Epple,
Jennifer A. Williams, Michael J. Roberts, David H. Woodmansee, Brian T. Masick,
Christine Tumanut, Jun Li, Glen Spraggon, Michael Hornsby, Jonathan Chang,
Tove Tuntland, Thomas Hollenbeck, Perry Gordon, Jennifer L. Harris and
Donald S. Karanewsky
Genomics Institute of the Novartis Research Foundation, 10675 John J. Hopkins Dr., San Diego, CA 92121, USA
Received 18 May 2006; revised 7 July 2006; accepted 11 July 2006
Available online 28 July 2006
Abstract—The synthesis and structure–activity relationship of a series of arylaminoethyl amide cathepsin S inhibitors are reported.
Optimization of P3 and P2 groups to improve overall physicochemical properties resulted in significant improvements in oral
bioavailability over early lead compounds. An X-ray structure of compound 37 bound to the active site of cathepsin S is also
reported.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Cathepsin S (Cat S) is a cysteine protease expressed in
antigen presenting cells such as B cells, dendritic cells,
and macrophages. Cat S mediates proteolysis of the
invariant chain that is associated with the major histo-
compatibility class II (MHC-II) complex.1–4 This prote-
olytic event is a prerequisite to productive loading of
antigen onto the MHC-II complex, rendering cathepsin
S an attractive therapeutic target for immunosuppres-
sion. The development of selective Cat S inhibitors for
the modulation and regulation of immune hyperrespon-
siveness may provide a novel treatment for chronic con-
ditions such as asthma, allergies, myasthenia gravis, and
rheumatoid arthritis.5–7
lead optimization of this series focused on improving
the physicochemical and PK parameters, while preserv-
ing the potency and selectivity over related members of
the cathepsin family.10 In this account, we describe the
continued optimization of the P3 aryl amide subunit
and the subsequent required modifications in P1 and P2.
1
R3
=
O
O
O
F3C
H
N
H
N
R3
N
H
Cathepsin S Ki = 3 nM
O
OMe
2
R3
=
We have recently detailed the synthesis and SAR of
arylaminoethyl amides that act as reversible, noncova-
lent inhibitors of Cat S.8–10 Compounds 1 and 2, while
potent and highly selective over related cathepsins K,
L, B, F, and V, exhibit poor PK properties in rats.
The lack of oral bioavailability of 1 and 2 is due in part
to low solubility but also to high clearance stemming
from poor metabolic stability of the compounds. Early
N
Cathepsin S Ki = 29 nM
Compounds 14–20 (Table 1), lacking a P1 alkyl substi-
tuent, were synthesized using the solid-phase procedure
previously described.8 The 5-phenyl-2-furanoic amide
motif in P3 generally confers excellent selectivity over
cathepsins K and L (Table 1). The distal region of
the S3 subsite of Cat S is defined by a flexible Lys64
residue which can re-orient its sidechain to accommo-
date the P3 moiety of the inhibitor.8,10,11 In contrast,
the analogous region of the Cat L S3 pocket is
Keywords: Cathepsin S; Cysteine protease inhibitor; Noncovalent
inhibitor; Peptidomimetic; X-ray structure.
*
Corresponding author. Tel.: +1 8588121559; fax: +1 8588121648;
e-mail: dtully@gnf.org
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.07.033

D. C. Tully et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5112–5117
Table 1. Inhibition of cathepsins S, K, and L—optimization of P3a
5113
O
R1
H
N
H
N
R3
N
H
O
OCF3
Compound
R3
R1
Ki (lM)
Cat S
Cat K
Cat L
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
5-(3-CF3phenyl)–2-furan
5-(3-fluorophenyl)–2-furan
2-Benzofuran
–H
–H
0.011
0.034
0.017
0.047
0.006
0.658
0.213
0.018
0.053
0.021
0.049
0.042
0.012
0.012
0.011
8.58
11.7
0.150
8.81
9.34
>30
>30
4.61
>30
>30
>30
>30
14.3
13.3
1.66
4.97
4.08
–H
–H
0.045
1.15
2-Furan
3-Furan
–H
–H
0.914
3.12
7.94
(R)-2-Tetrahydrofuran
3-Tetrahydrofuran
(R)-2-Tetrahydrofuran
(S)-2-Tetrahydrofuran
3-Tetrahydrofuran
–H
–Me
–Me
–Me
–Me
–Me
–Me
–Me
–Me
0.582
0.331
4.79
(S)-Tetrahydrofuran-3-yl-oxy
(R)-Tetrahydrofuran-3-yl-oxy
4-Tetrahydropyran
0.594
2.57
0.232
0.075
0.125
Tetrahydropyran-4-yl-oxy
1-Morpholine
a Details of the assay conditions can be found in the Supplementary data. Ki values are means of at least three experiments.
occupied by a comparatively less flexible Glu63, which
presumably sterically clashes with the distal phenyl ring
of the P3 moiety in compounds 1, 14, and 15.8,12 Sub-
stitutions on the distal phenyl ring such as small alkyl
groups (14) and halogens (15) are generally well toler-
ated, usually preserving both the potency and selectiv-
ity. While the biaryl amide in the P3 position confers
selectivity of the compounds over Cat L, this moiety
also contributes to suboptimal physicochemical proper-
ties such as low aqueous solubility, high logP, and
high molecular weight.
Commercially available N-Cbz amino acids 7 were
coupled to diamines 6a–d with HATU-activation and
deprotected under catalytic hydrogenation to provide 8.
Conversion to P3 amides 9 was achieved by HATU-
mediated condensation of 8 with the appropriate
carboxylic acid, while P3 carbamates 10 were prepared
by acylation of 8 with the corresponding nitrophenylcar-
bonate. Morpholine ureas, 13, were synthesized by acyl-
ation of the commercially available amino acids with
morpholinecarbonyl chloride under Schotten–Bauman
conditions, followed by peptide coupling with diamines
6a–d (Scheme 2).
The syntheses of analogs bearing a P1 alkyl substituent
are detailed in Schemes 1 and 2. Commercially available
(S)-2-amino-1-propanol 3 (or the corresponding butanol
for R1 = Me) was protected as the benzyl carbamate and
then oxidized to the aldehyde 4 with Dess–Martin
periodinane. Reductive amination with the appropriate
aniline or indoline afforded 5, which was then subjected
to hydrogenolysis to provide the diamines 6a–d.
Results from our efforts to optimize the P3 fragment of
this series are shown in Table 1. Replacement of the bia-
ryl amide with a fused bicyclic ring, such as benzofuran
16, results in a significant loss of selectivity over cathep-
sins K and L. A similar trend with this series has also
been observed with other fused heterocyclic P3 amides
such as 2-naphthyl.8 When the distal aryl ring was
R1
R1
R1
R1
d
c
a,b
NAr
Cbz
H
Cbz
NAr
OH
H2N
N
H
N
H
5
H2N
O
6a-d
3
4
F
OCF3
F
F
N
N
NAr =
N
N
H
d
c
b
a
Scheme 1. Reagents: (a) benzyl chloroformate, Et3N, CH2Cl2, 86%; (b) Dess–Martin periodinane, CH2Cl2; (c) HNAr (aniline or indoline),
NaB(CN)H3, AcOH, MeOH, 60–75% over two steps; (d) H2, Pd/C, EtOH, 90–95%.

5114
D. C. Tully et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5112–5117
O
H
R1
R3
N
NAr
c
N
H
O
O
R2
O
O
R1
a,b
H
N
9
H2N
NAr
Cbz
OH
N
H
d
R2
7
R2
O
R1
H
N
8
R3O
NAr
NAr
N
H
R2
10
R1
O
O
O
O
O
a
H
e
H
N
H2N
N
N
N
OH
N
H
OH
R2
R2
13
O
R2
12
O
11
Scheme 2. Reagents: (a) 6a–d, HATU, DIEA, CH2Cl2, 75–87%; (b) H2, Pd/C, EtOH, 87–93%; (c) R3COOH, HATU, DIEA, CH2Cl2, 71–84%; (d)
para-NO2PhOCO2R3, DIEA, DMF, 44–63%; (e) morpholinecarbonylchloride, NaOH, H2O, 42–67%.
removed completely as in the 2-furyl analog, 17, a
potent cathepsin S inhibitor was obtained with modest
selectivity (25·) over cathepsin L. The 3-furyl analog,
18, not only improved the potency toward Cat S
(Ki = 6 nM) but also the selectivity over Cat L (150·).
We surmised that in the absence of a distal phenyl ring,
the furan oxygen may engage the positively charged
Lys64 sidechain to form a favorable electrostatic inter-
action. On the other hand, its affinity to Cat L may be
penalized by the repulsive interaction with Glu63.
(Table 1). The increased ring size of tetrahydropyran
26 resulted in a more potent cathepsin S inhibitor
(Ki = 12 nM) with modest (ꢀ20·) selectivity over
cathepsin L. The tetrahydropyranyl analog, 27, and
the morpholine analog, 28, each provided equally potent
cathepsin S inhibitors with solid selectivity over Cat K,
but narrowing windows of selectivity over Cat L.
Six-membered rings such as pyran or morpholine in the
P3 position of this series appeared to be the most
beneficial for improving the in vitro potency. We there-
fore opted to fix this position as the morpholine urea,
represented by 28, and investigated the SAR of the P2
substituent in an effort to improve selectivity. Several
analogs with different P2 and P1 substituents are listed
in Table 2. When the P1 alkyl group was increased to
ethyl (29) or 2-methanesulfonyl-ethyl (30), a consider-
able boost in inhibitory activity toward both Cat K
and Cat L resulted, significantly reducing the selectivity.
Alternatively, when the P1 alkyl group was removed
entirely (31) the activity dropped off proportionally
across each enzyme tested. Interestingly, while cathep-
sin S generally tolerates both aliphatic and aromatic
hydrophobic groups in the S2 binding pocket, the
benzyl analog, 32, was not particularly active
(Ki = 11.4 lM) when combined with morpholine in
P3. This is in contrast to previous SAR from this series
in which substituted phenylalanines in P2 in combina-
tion with a benzamide P3 gave potent Cat S inhibi-
tors.8 The tert-butyl alanine P2 was tolerated by Cat
S (33 and 34); however, the selectivity over Cat K
was only marginal. Cyclopentyl alanine 35 was four
times less potent than cyclohexyl alanine 28; however,
its selectivity over Cat L was significantly improved
(>50·). Compound 36, derived from benzylcysteine,
was equipotent to 28 and had an improved selectivity
profile. The potent sulfone analog 37 (Ki = 32 nM)
was completely inactive against cathepsins K, L, B,
F, H, V, and X in the concentration ranges tested
(>30 lM). Unfortunately, compared to amides 21 and
23, urea 37 had only modest oral bioavailability
(F = 16%) despite improved in vitro metabolic stability
and solubility at pH 6.8 (Table 3).
Saturation of the P3 furan ring to tetrahydrofurans 19
and 20 resulted in a considerable drop-off (>10·) in
activity, presumably due to the loss of the p–p interac-
tion between the furan ring and Phe70. Since the intro-
duction of a cyclic ether was considered to have a
greater potential for further improvement in solubility,
we sought to optimize further upon this motif. We have
previously shown that the introduction of an (S)-alkyl
substituent onto the diamine section of this series as a
P1 sidechain typically leads to an increase in potency.9,10
This was again observed to be the case, as the introduc-
tion of a methyl group (19 ! 21) improved the activity
by over 30-fold (Ki = 18 nM), while affording modest
selectivity over cathepsin L. In comparing the absolute
configurations in P3, the (R)-2-tetrahydrofuran diaste-
reomer 21 was determined to be preferred over the
(S)-diastereomer (22) by a factor of three. Tetrahydrofu-
ran 23 was equipotent with 21 and gave a significant
improvement in selectivity over both cathepsins K and
L. With the improvements in solubility and metabolic
stability over P3 aryl amides such as 1, the PK parame-
ters of tetrahydrofuranyl amides 21 and 23 were profiled
in male Wistar rats (Table 3). Both compounds gave sig-
nificant improvements in oral exposures over 1 and
related compounds, and had oral bioavailabilites of
36% and 19%, respectively. When dosed intravenously,
both compounds exhibited moderate clearance rates
and terminal half-lives.
Continued investigation led to carbamates 24 and 25,
which were equipotent with respect to cathepsin S,
with the R-isomer exhibiting a better selectivity profile

D. C. Tully et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5112–5117
Table 2. Inhibition of cathepsins S, K, and L—optimization of P2a
5115
O
O
R1
H
N
N
NAr
N
H
O
R2
Compound
R2
R1
NArb
Ki (lM)
Cat S
Cat K
Cat L
28
29
30
31
32
33
34
35
36
37
38
39
40
Cyclohexylmethyl
Cyclohexylmethyl
Cyclohexylmethyl
Cyclohexylmethyl
PhCH2–
–Me
–Et
a
a
a
a
a
a
a
a
a
a
b
c
0.011
0.011
0.005
0.099
11.4
1.66
0.130
0.139
18.8
>100
1.33
0.461
1.54
2.48
>100
5.13
13.1
16.9
0.125
0.006
0.004
2.11
–CH2CH2SO2Me
–H
–H
–H
>100
>30
14.6
tert-Butylmethyl
tert-Butylmethyl
Cyclopentylmethyl
PhCH2SCH2–
0.220
0.093
0.049
0.012
0.032
0.087
0.111
0.011
–Me
–Me
–Me
–Me
–Me
–Me
–Me
2.59
1.18
PhCH2SO2CH2–
Cyclohexylmethyl
Cyclohexylmethyl
Cyclohexylmethyl
>30
0.512
1.06
0.810
d
a Details of the assay conditions can be found in the Supplementary data. Ki values are means of at least three experiments.
b Structures of N-aryl moieties a–d are shown in Scheme 1.
Table 3. Pharmacokinetics of selected analogsa
Compound
clogP
In vitro
Single iv dose (3 mg/kg)
Single po dose (10 mg/kg)
Sol. pH = 6.8
(mg/mL)
RLM
(% remaining)
t1/2
(min)
Cl
(mL/ min/kg)
Vss
(L/kg)
AUC
(min lg/mL)
Cmax
(nM)
t1/2
(min)
F
(%)
1
2
7.2
5.8
5.3
5.0
3.2
6.3
<0.001
0.010
0.025
0.030
0.043
0.010
12
4
—
—
11
35
39
39
52
—
3
0.9
105
49
19
12
146
318
58
—
<1
36
19
16
4
288
66
1.63
1.78
2.48
0.87
1.76
21
23
37
40
66
56
1237
491
639
118
108
49
90
66
100
<1
42
8
43
51
a Pharmacokinetic data in male Wistar rats, where values are means of three individual experiments.
We have previously shown that the aniline moiety can
be replaced by 5-fluoroindoline and derivatives thereof
without significant loss in activity in the context of ana-
logs bearing benzamide9 or heteroaryl10 P3 units. In
combination with the morpholine P3 group, however,
replacement of the aniline with 5-fluoroindoline (38) or
5-fluoro-2,2-dimethylindoline (39) resulted in nearly an
order of magnitude loss in activity. The 5-fluoro-3,3-
dimethylindoline (40) did however retain potency to-
ward Cat S, as well as improve the selectivity over Cat
L. Despite the favorable shift in the biochemical selectiv-
ity profile, substitution of the aniline P10 moiety had a
deleterious effect on the PK parameters as demonstrated
by 40, which had low oral bioavailability and high clear-
ance, likely due to enhanced first-pass metabolism, as
suggested by poor in vitro metabolic stability.
unable to resolve electron density for the P10 aniline
moiety.13 To further investigate this phenomenon and
to determine whether the prime-side aniline moiety
could be resolved, we endeavored to grow co-crystals
of Cat S in the presence of 37. Out of a broad screen
consisting of 480 different buffer conditions, co-crystals
of cathepsin S and 37 grew in a well containing 0.2M
Zn(OAc)2.13 The X-ray co-crystal structure was solved,
˚
and the crystals diffracted to 1.8 A resolution and be-
longed to space group R3. This co-crystal structure
clearly shows 37 bound to the active site of the enzyme
(Fig. 1). Clear electron density for all atoms in the inhib-
itor was evident for both molecules in the asymmetric
unit. It is apparent from this structure that Zn2+ has
co-crystallized with the inhibitor and protein. The
bound Zn2+ is tetrahedrally coordinated via the thiol
group of the catalytic cysteine (Cys25), the imidazole
sidechain of His164, the aniline nitrogen of 37, and a
chloride ion. It is unclear whether this interaction be-
tween the Cys25 thiolate, zinc dication, and the aniline
is necessary for the formation of a stable co-crystal com-
plex, or whether it is merely an artifact of the co-crystal-
lization conditions with the inhibitor in the presence of a
high concentration of Zn2+. We have further verified
We previously reported an X-ray structure of cathepsin
S produced by soaking compound 2 with crystals of Cat
S.10 This structure clearly showed the P1, P2, and P3
subsites of compound 2 bound to the active site of Cat
S, but the P10 aniline group was disordered. An X-ray
structure of Cat S with compound 37 was produced by
soaking in the same manner, but yet again we were

5116
D. C. Tully et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5112–5117
This report summarizes the SAR of a potent series of
noncovalent cathepsin S inhibitors and highlights
changes leading to improvements in oral bioavailability.
The hydrophobic biaryl amide P3 substituent of 1, an
early lead compound, was replaced by aliphatic cyclic
ethers, resulting in compounds with improved physico-
chemical properties and in vitro rodent microsomal sta-
bility. This led to major improvements in PK with
analogs such as 21 being 36% orally bioavailable in rats.
Acknowledgments
The authors acknowledge Kirk Clark and Raviraj
Kulathila (Novartis Institutes for Biomedical Research)
for valuable discussions.
Supplementary data
˚
Figure 1. Crystal structure of cathepsin S at 1.8 A co-crystallized with
compound 37 (RCSB PDB ID: 2HH5). Protein carbon atoms are
colored yellow whilst those of the inhibitor are green, oxygens are red,
nitrogens blue, sulfurs orange, and fluorine violet. The Zn2+ ion is
colored bronze whilst the chloride anion is colored green. Hydrogen
bonds are indicated by dashed black and white lines. Figure generated
using Pymol (http://www.pymol.org).
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2006.07.033.
References and notes
that Zn2+ is not required for in vitro inhibition of Cat S
in our biochemical assays.14 This was accomplished by
adding 2 lM ZnCl2 to the assay buffer in the presence
of and in the absence of 1 mM EDTA. In each case,
the measured Kis were unchanged relative to the stan-
dard assay buffer conditions.
1. (a) Riese, R. J.; Mitchell, R. N.; Villadangos, J. A.; Shi, G.
P.; Palmer, J. T.; Karp, E. R.; De Sanctis, G. T.; Ploegh,
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W.; Saftig, P.; Shi, G. P.; Chapman, H. A.; Peters, C.;
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S,11,15,16 particularly in the S3 and S2 subsites. The P3
morpholine urea assumes a chair conformation and
occupies the S3 pocket which is well defined by the back-
bone of Gly69 and the sidechains of Phe70 and Lys64.
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˚
´
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can adopt an open conformation to make room for
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clearly occupies the proximal S10 subsite lined by key
residues Ala140, Arg141, and Asn163. The indole ring
of Trp186 is situated at the base of the S10 pocket,
and it stacks edge-on with the aniline group of the inhib-
itor. This P10 aniline moiety essentially occupies the
analogous region of space as does the phenyl sulfone
moiety of the irreversible inhibitor LHVS in the co-crys-
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conditions for the structure shown in Figure 1 (RCSB
PDB ID: 2HH5) as well as a second solved X-ray structure
produced by soaking compound 37 with crystals of Cat S
in the absence of Zn2+ (P10 subsite of inhibitor is
disordered, RCSB PDB ID: 2HHN).
14. Experimental details of the biochemical assays are included
as Supplementary data.
15. Mcgrath, M. E.; Palmer, J. T.; Bromme, D.; Somoza, J. R.
Protein Sci. 1998, 7, 1294.
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McNeil, D.; Bekkali, Y.; Giradot, M.; Hrapchak,
M.; DeTuri, M.; Crane, K.; White, D.; Pav, S.;
Wang, Y.; Hao, M.-H.; Grygon, C. A.; Labadia, M.
E.; Freeman, D. M.; Davidson, W.; Hopkins, J. L.;
Brown, M. L.; Spero, D. M. J. Med. Chem. 2002, 45,
5471.