Discovery of orally bioavailable cathepsin S inhibitors for the reversal of neuropathic pain

Article abstract of DOI:10.1021/jm800839j

Cathepsin S inhibitors are well-known to be an attractive target as immunological therapeutic agents. Recently, our gene expression analysis identified that cathepsin S inhibitors could also be effective for neuropathic pain. Herein, we describe the effic

Full text of DOI:10.1021/jm800839j

5502
J. Med. Chem. 2008, 51, 5502–5505
Chart 1
Discovery of Orally Bioavailable Cathepsin S
Inhibitors for the Reversal of Neuropathic
Pain
Osamu Irie,*,† Takatoshi Kosaka,† Takeru Ehara,†
Fumiaki Yokokawa,† Takanori Kanazawa,† Hajime Hirao,†
Astuko Iwasaki,† Junichi Sakaki,† Naoki Teno,†
Yuko Hitomi,† Genji Iwasaki,† Hiroaki Fukaya,†
Kazuhiko Nonomura,† Keiko Tanabe,† Shinichi Koizumi,†
Noriko Uchiyama,† Stuart J. Bevan,† Marzia Malcangio,§,‡
Clive Gentry,§,‡ Alyson J. Fox,§,‡ Mohammed Yaqoob,§,‡
Andrew J. Culshaw,§,‡ and Allan Hallett‡
Scheme 1a
NoVartis Institutes for BioMedical Research, Ohkubo 8, Tsukuba,
Ibaraki 300-2611, Japan, 5 Gower Place,
London WC1E 6BS, United Kingdom
ReceiVed July 9, 2008
a Reagents and conditions: R1NH2, MeOH, rt, 18 h; (b) NaCN, DABCO,
DMSO-H2O, 60 °C, 4 h; (c) R2-OCH2C′CH, Pd(PPh3)2Cl2, CuI, NEt3, DMF,
80 °C, 4 h; (d) DBU, DMF, 100 °C, 4 h.
Abstract: Cathepsin S inhibitors are well-known to be an attractive
target as immunological therapeutic agents. Recently, our gene expres-
sion analysis identified that cathepsin S inhibitors could also be effective
for neuropathic pain. Herein, we describe the efficacy of selective
cathepsin S inhibitors as antihyperalgesics in a model of neuropathic
pain in rats after oral administration.
Scheme 2a
There is a clear medical need for new classes of effective
analgesics, particularly in the treatment of chronic pain states.
Current marketed therapies are based largely on two well-
established classes of analgesics opiates and the nonsteroidal
anti-inflammatory drugs (NSAIDs). Both classes of drugs have
limited efficacy and evoke undesirable side effects. The lack of
suitable therapies has stimulated our research to identify novel
targets for neuropathic pain. We have recently discovered that
the mRNA encoding cathepsin S was up-regulated in rat dorsal
root ganglia following peripheral nerve injury by using a gene
expression analysis approach.1 We also published that LHVSa,
a potent nonspecific cathepsin S inhibitor, preferentially reversed
hyperalgesia in a rat model after subcutaneous injection.2 These
findings suggest that cathepsin S inhibitors would be applicable
for the reversal of neuropathic hyperalgesia. We thus sought to
discover reversible, nonpeptidic, selective, and orally active
cathepsin S inhibitors. Herein, we describe the efficacy of
selective cathepsin S inhibitors as antihyperalgesics in a model
of neuropathic pain in rats after oral administration.
a Reagents and conditions: (I) TsCl, NEt3, CH2Cl2, 0 °C rt, 15 h; (II)
NaCN, DMSO, rt, 15 h; (III) LiAlH4, H2SO4, Et2O, 0 °C-rt, 15 h.
in these cells is in the important proteolytic events that lead to
antigen presentation. Cathepsin S inhibitors are thus being
developed as immunological therapeutic agents.3 Eleven mem-
bers of the cysteine cathepsin family have been identified in
the human gene (cathepsins B, C, H, F, K, L, O, S, V, W, and
X).4 Recent cathepsin gene knock out studies in mice have
revealed that these lysosomal cysteine proteases have specific
and individual functions, which are important for the normal
functioning of an organism.5
We recently reported the discovery of the selective and orally
bioavailable cathepsin S inhibitor 2 after optimization of the
P2 and P3 substituents of the potent cathepsin K inhibitor 16
by using computer-assisted modeling studies7 (Chart 1). Com-
pound 2 exhibited potent inhibitory activity and selectivity
toward human cathepsin S. To avoid off-target effects due to
other cathepsins and to assess the efficacy of the cathepsin S
inhibition for the reversal of neuropathic pain, we initially
attempted to improve the selectivity of the compound 2 against
cathepsin K and L.
The inhibitor syntheses are outlined in Schemes 1-3.
Treatment of 5-bromo-2,4-dichloropyrimidine 3 with a variety
of amines followed by addition of sodium cyanide, gave
compounds 5i-v (Scheme 1, Table 1). Sonogashira coupling
reaction of 5i-v with alkynes using a catalytic amount of
Pd(PPh3)2Cl2 and CuI followed by cyclization under basic
conditions provided 2-cyano-pyrropyrimidines 2 and 7a-g.
Preparation of the P2 amine for 7g is shown in Scheme 2.
Treatment of 4,4-difluorocyclohexylmethanol 8 with TsCl in
the presence of triethylamine followed by the addition of sodium
cyanide provided nitrile 9. Reduction of 9 with LiAlH4 in the
Cathepsin S is a lysosomal cysteine protease belonging to
the papain superfamily, and it is expressed in professional
antigen presentation cells (APC) such as dendritic cells, B
lymphocytes, and macrophages. The major role of cathepsin S
* To whom correspondence should be addressed. Phone: +81 29 865
2384. Fax: +81 29 865 2308. E-mail: osamu.irie@novartis.com.
†
Novartis Institutes for BioMedical Research, Ohkubo 8, Tsukuba,
Ibaraki 300-2611, Japan.
‡
Novartis Institutes for BioMedical Research, 5 Gower Place, London
WC1E 6BS, United Kingdom.
§
Present address: Novartis Horsham Research Center, Wimblehurst
Road, Horsham, RH12 5AB, United Kingdom.
a Abbreviations: LHVS, morpholineurea-leucine-homophenylalanine-
vinyl phenyl sulfone; hCat, human cathepsin; DABCO, 1,4-
diazabicyclo[2.2.2]octane; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; TsCl,
p-toluenesulfonyl chloride; MCA, 4-methylcoumaryl-7-amide; MHC II,
major histocompatability complex II; lIP10, leupeptin-induced polypeptide
10; CLIP, class II associated leupeptin induced peptide.
10.1021/jm800839j CCC: $40.75
2008 American Chemical Society
Published on Web 08/29/2008

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5503
Scheme 3a
tion with propargyl-bromide gave the fluoro-substituted alkynes
for 7a and b.
The IC50 values of the compounds are shown in Table 1.
Substitutions by fluorine on the phenyl ring of the P3 did not
significantly affect the selectivity while the potencies for
cathepsin S were maintained (compare 2, 7a, 7b). Expansion
of the piperidine ring led to a loss of selectivity toward cathepsin
K due to the structural differences of the S3 subsite between
cathepsins S and K (compare 2, 7c). The X-ray crystal structures
of human cathepsin S,10 K,6 and L11 suggest that the S2 subsite
is critical for both the potency and selectivity toward cathepsin
S because the S2 pocket in cathepsin S is larger than that of
cathepsin K and L (compare 2, 7d, 7e, 7f, 7g). Indeed, the 4,4-
difluorocyclohexylethyl group 7g is the optimal size for the P2
substituent based on the affinity to cathepsin S (IC50 ) 10 nM,
Ki ) 4.0 ( 0.3 nM), and selectivity against other cysteine
proteases, with >100-fold selectivity against human cathepsins
B (IC50 > 30 µM), C (Ki > 10 µM), H (Ki > 10 µM), and X
(Ki ) 0.65 µM).
a Reagents and conditions: (I) Pd2(dba)3, NaOtBu, 2-(di-tert-butylphos-
phino)biphenyl, toluene, 80 °C, 4 h; (II) 4N HCl in EtOAc, MeOH, rt,
0.5 h; (III) BrCH2C≡CH, K2CO3, DMF, 70 °C, 12 h.
Table 1. Inhibitory Activity of Pyrropyrimidine Compounds 2, 7a-g
against Cathepsins S, K, and L
Cathepsin S is responsible for the final proteolytic cleavage
of the MHC II associated invariant chain (Ii) mediating the
cleavage of lip10 to CLIP in B cells and dendritic cells.12 We
then evaluated the cellular activities of the specific cathepsin S
inhibitors by measuring Ii processing in mouse splenocytes.
Compounds 7a and 7g induced the accumulation of the lip10
fragment in a concentration dependent manner beginning at 10
µM and 1 µM, respectively. For the comparison, in the same
assay, LHVS showed the cellular activity at 10 nM.
The pharmacokinetic profiles of compounds 2, 7a, and 7g
were determined after oral (3 mg/kg, suspension) and intrave-
nous (1 mg/kg, bolus) administration to Sprague-Dawley rats
by LC/MS quantification. The results are shown in Table 2.
The absolute bioavailability of 7g was 21% with modest
exposure level (AUCinf; 394 ( 3 nM· h). After intravenous
administration, the CLp value was 3.1 ( 0.4 L/h/kg and the
terminal elimination half-life was 0.7 ( 0.1 h.
With promising compounds for in vivo studies in our rodent
disease models in hand, we then evaluated our cathepsin S
specific compounds for their inhibition of rat and mouse
cathepsins. The inhibitory potencies to mouse cathepsin S
enzyme are similar to that of the human enzyme (Table 3).13
On the other hand, these selective cathepsin S inhibitors did
not show any efficacy against in-house recombinant rat cathepsin
S enzyme at 1 µM due to the 137Gly f Cys mutation (papain
numbering), which was confirmed by comparison of the cDNA
sequence compared to the Mason’s reported sequence.14 The
mutated sequence has already been reported in the rat EST
database (ACC nos. BQ196525 and BQ200106). Our modeling
studies suggest that the mutated Cys at 137 position is located
in the bottom of the S2 subsite, which is a key residue for
potency to the cathepsin S enzyme (Figure 1), thus causing
species specificity (Table 4). We next prepared the reported rat
137Gly cathepsin S enzyme, and then we remeasured the IC50
values to this cathepsin S (Table 3).14 All compounds maintained
inhibitory potencies to the rat 137Gly cathepsin S enzyme as
well as the selectivity to rat cathepsins K, L, and B (>100-fold
selectivity).
a Inhibition of recombinant human cathepsins K, L, and S in a
fluorescence assay, employing Z-Phe-Arg-MCA (Cat K and L) and Z-Leu-
Leu-Arg-MCA (Cat S) as synthetic substrates. Data represent means of two
experiments performed in duplicate. Individual data points in each experi-
ment were within a 2-fold range with each other.9
presence of sulfuric acid afforded the desired 4,4-difluorocy-
clohexylethylamine 10.
Acetylene derivatives for 2 and 7c-g were synthesized by
alkyation of commercially available phenol derivatives under
basic conditions (Scheme 3, conditions (III)). Buchwald-Hartwig
cross-coupling reactions8 with O-protected fluoro-substituted
bromophenol derivatives followed by deprotection and alkyla-
Before in vivo experiments with compounds 7a and 7g were
started, genotyping15 of the Wister rat strain commonly used
for our neuropathic models2,3 was carried out. After separation
of the animals having 137Gly or 137Cys cathepsin S enzymes,
the 137Gly cathepsin S animals were subsequently used in the
chronic neuropathic pain studies.

5504 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Letters
Table 2. Pharmacokinetic Parameters of 2, 7a, and 7g in Male Sprague-Dawley Rats (iv 1 mg/kg; po 3 mg/kg), Values are the Means of Three
Individual Experiments
a
compd
CLp (L/h/kg)
iv t1/2 (h)
F (%)
po AUCa (nM·h)
po Cmax (nM)
Vd (L /kg)
2
7a
7g
3.9 ( 0.7
2.7 ( 0.3
3.1 ( 0.4
1.1 ( 0.1
1.7 ( 0.1
0.7 ( 0.1
53
24
21
285 ( 139
174 ( 42
131 ( 1
61 ( 34
30 ( 6
37 ( 2
5.6 ( 0.8
5.7 ( 0.4
3.7 ( 0.2
a Dose normalized to 1 mg/kg, means ( SD.
Table 3. Inhibitory Activity of Compounds, 2, 7a, and 7g against Rat
Cathepsins S, K, L, B and Mouse Cathepsin
rat cathepsin IC50 (nM)a
mouse
compd
137Gly S
137Cys S
K
L
S IC50 (nM)a
2
7a
7g
10 ( 1
12 ( 1
28 ( 3
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
10 ( 1
14 ( 1
20 ( 2
a Inhibition of recombinant rat cathepsins K, L, S and mouse cathepsin
S14 in a fluorescence assay, employing Z-Phe-Arg-MCA (Cat K and L),
Z-Leu-Leu-Arg-MCA (Cat S) and Z-Arg-Arg-MCA (Cat B) as synthetic
substrates. Values represent means ( SD of three experiments.9
Figure 2. Oral activity of compound 7g against neuropathic mechanical
hyperalgesia in 137Gly-cathepsin S rats. Graph shows mean ( SEM
reversal of hyperalgesia from 6 animals per treatment group. ***p <
0.001, **p < 0.01, *p < 0.05 compared to vehicle by ANOVA followed
by Tukey’s HSD test carried out on withdrawal threshold data.
Figure 1. Compound 7g docked into the rat 137Gly (left) and rat
137Cys (right) cathepsin S. The compound covalently binds to the
catalytic residue, Cys25, which is located on the right side of the figures
(not shown for the sake of clarity). The P2 4,4-difluorocyclohexylethyl
group snugly fits in the S2 pocket with a number of hydrophobic
contacts when residue 137 is Gly, while the bulkier Cys residue rejects
the P2 group, which renders the compound basically inactive.
Table 4. Binding Site Residues for Human, Rat, and Mouse Cathepsin
S
S3
S2
S1
residue numbera
64
70
137 162 211 25
67
Figure 3. Effect of repeated administration of 7a and 7g on neuropathic
pain in rats. 7a and 7g were administered orally in 0.5% methylcel-
lulose/water twice daily for 5 days. Paw withdrawal thresholds were
measured 3 h following administration. Each point represents mean (
SEM from 6 animals/group. ***p < 0.001, **p < 0.01, *p < 0.05
compared to vehicle by ANOVA followed by Tukey’s HSD test.
human cathepsin S
Lys Phe Gly Val
Phe Cys Asn
rat 137Cys cathepsin S Lys Phe Cys Met Tyr Cys Gly
rat 137Gly cathepsin S Lys Phe Gly Met Tyr Cys Gly
mouse cathepsin S
a Papain numbering.
Lys Tyr Gly Val
Tyr Cys Gly
The effect of the specific cathepsin S inhibitor 7g was
examined in a disease model of chronic neuropathic pain in
rats following oral administration as a solution in 0.5%
methylcellulose/water. In this model, partial ligation of the
sciatic nerve induces a peripheral neuropathy that results in
mechanical hyperalgesia lasting for several weeks. Compound
7g reversed the established mechanical hyperalgesia in a dose
dependent fashion (Figure 2). On the other hand, 7g was not
active in rats having 137Cys cathepsin S, as 7g showed no
inhibition of 137Cys cathepsin S at 1 µM (Table 3). These
findings demonstrate that an inhibition of the cathepsin S
enzyme is efficacious in the reversal of neuropathic pain.
Compound 7a was also active in the rat having 137Gly cathepsin
S enzyme, producing a dose-related, maximum 54% reversal
of hyperalgesia (100 mg/kg po), with an effect that was rapid
in onset (1 h).
first dose of 7a produced 57% reversal of hyperalgesia, which
increased to 63% following the final dose on day 5 (Figure 3).
Compound 7g was slightly lower, producing approximately
40-45% reversal throughout the study, because of lower in vitro
potency to rat cathepsin S. The results suggested that there was
evidence for the development of tolerance to the antihyperalgesic
effect of 7a and 7g and no obvious side effect.
Compounds 7a and 7g were inactive at the maximum
concentration tested of 10 µM against a panel of cytochrome
P450 enzymes, including CYP1A2, CYP2C9, CYP2C19,
CYP2D6, and CYP3A4. They did not possess genotoxic
potential, as evidenced by the negative results obtained in the
standard Ames test by using liver S9 enzyme. Neither compound
induced increased numbers of cells containing in the in vitro
micronucleus test.
In conclusion, we demonstrate that cathepsin S specific
inhibitors, which have greater than 1000-fold selectivity against
rat cathepsin K, L and B, showed an antihyperalgesic effect in
a rat model of neuropathic pain after oral administration. These
Following twice daily administration for 5 days (30 mg/kg
po), the antihyperalgesic activity of compounds 7a and 7g
against neuropathic hyperalgesia was maintained (Figure 2). The

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5505
(6) 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.; Iwasaki, A.; Umemura, I.; Tada, S.; Kometani, M.;
Iwasaki, G.; Cowan-Jacob, S. W.; Missbach, M.; Lattmann, R.;
Betschart, C. Novel scaffold for cathepsin K inhibitors. Bioorg. Med.
Chem. Lett. 2007, 17, 6096–6100.
(7) Irie, O.; Ehara, T.; Iwasaki, A.; Yokokawa, F.; Sakaki, J.; Hirao, H.;
Kanazawa, T.; Teno, N.; Horiuchi, M.; Umemura, I.; Gunji, H.;
Masuya, K.; Hitomi, Y.; Iwasaki, G.; Nonomura, K.; Tanabe, K.;
Fukaya, H.; Kosaka, T.; Snell, C. R.; Hallett, A. Discovery of selective
and nonpeptidic cathepsin S inhibitors. Bioorg. Med. Chem. Lett. 2008,
18, 3959–3962.
results suggest that cathepsin S inhibition has great promise as
a new treatment for neuropathic pain. The cathepsin S inhibitors
discovered possess excellent inhibitory activity to the cathepsin
S enzyme, high selectivity against other cathepsins K, L, B, H,
X, and C, and show cellular activity and oral bioavailablity
without significant toxicological properties. We therefore believe
that they are useful compounds in the search for the mechanism
of neuropathic pain involving cathepsin S and constitute a novel
therapeutic approach for the treatment of chronic pain.
(8) Guram, A. S.; Rennels, R. A.; Buchwald, S. L. A Simple Catalytic
Method for the Conversion of Aryl Bromide to Arylamines. Angew.
Chem., Int. Ed. 1995, 34, 1348–1350.
(9) Altmann, E.; Aichholz, R.; Betschart, C.; Buhl, T.; Green, J.; Irie, O.;
Teno, N.; Lattmann, R.; Tintelnoy-Blomley, M.; Maissbach, M.
2-Cyano-pyrimidines: A New Chemotype for Inhibitors of the Cysteine
Protease Cathepsin K. J. Med. Chem. 2007, 50, 591–594.
(10) Pauly, T. A.; Sulea, T.; Ammirati, M.; Sivaraman, J.; Danley, D. E.;
Griffor, M. C.; Kamath, A. V.; Wang, I.-K.; Laird, E. R.; Seddon,
A. P.; Menard, R.; Cygler, M.; Rath, V. L. Specificity Determinants
of Human Cathepsin S Revealed by Crystal Structures of Complexes.
Biochemistry 2003, 42, 3203–3213.
Acknowledgment. We thank Andew McBryde, Caroline
Huntley, Michie Kobayashi, Nami Suzuki, and Tomoko Ohkubo
for excellent technical assistance. The authors are grateful to
Isobel Gonzales and Terance W. Hart for useful discussions.
We also thank Christopher R. Snell and Pamposh Ganju for
the initiation of this project. We acknowledge Jennifer L. Harris,
Christine Tumanut, and Jun Li for the in vitro cathepsin C, H,
and X assays, and Tim Wiltshire for genotyping assays (Novartis
Genomic Foundation, San Diego, CA).
(11) Chowdhury, S. F.; Sivaraman, J.; Wang, J.; Devanathan, G.; Lachance,
P.; Qi, H.; Me´nard, R.; Lefebvre, J.; Konishi, Y.; Cygler, M.; Sulea,
T.; Purisima, E. O. Design of Noncovalent Inhibitors of Human
Cathepsin L. From the 96-Residue Proregion to Optimized Tripeptides.
J. Med. Chem. 2002, 45, 5321–5329.
(12) Riese, R. J.; Wolf, P. R.; Bro¨mme, D.; Natkin, L. R.; Villadangos,
J. A.; Ploegh, H. L.; Chapman, H. A. Essential Role for Cathepsin S
in MHC Class II-Associated Invariant Chain Processing and Peptide
Loading. Immunity 1996, 4, 357–366.
(13) Gauthier, J. Y.; Black, W. C.; Courchesne, I.; Cromlish, W.; Desmarais,
S.; Houle, R.; Lamontagne, S.; Li, C. S.; Masse´, F.; McKay, D. J.;
Ouellet, M.; Robichaud, J.; Truchon, J.-F.; Truong, V.-L.; Wang, Q.;
Percival, M. D. The identification of potent, selective, and bioavailable
cathespin S inhibitors. Bioorg. Med. Chem. Lett. 2007, 17, 4929–4933.
(14) Mason, C. S.; Lamers, M. B. A. C.; Henderson, I. M. J.; Monk, T.;
Williams, D. H. Baculoviral Expression and Characterization of Rodent
Cathepsin S. Protein Expression Purif. 2001, 23, 45–54.
Supporting Information Available: Experimental procedures
for the synthesis of compounds and characterization (1H NMR and
MS) of compounds 2 and 7a-g. Description of in vitro and in vivo
tests including pharmacokinetic analysis. This material is available
free of charge via the Internet at http://pubs.acs.org.
References
(1) Barclay, J.; Clark, A. K.; Ganju, P.; Gentry, C.; Patel, S.; Wotherspoon,
G.; Buxton, F.; Song, C.; Ullah, J.; Winter, J.; Fox, A.; Bevan, S.;
Malcangio, M. Role of the cysteine protease cathepsin S in neuropathic
hyperalgesia. Pain 2007, 130, 225–234.
(2) Clark, A. K.; Yip, P. K.; Grist, J.; Gentry, C.; Staniland, A. A.;
Marchand, F.; Dehvari, M.; Wotherspoon, G.; Winter, J.; Ullah, J.;
Bevan, S.; Malcangio, M. Inhibition of spinal microglial cathepsin S
for the reversal of neuropathic pain. Proc. Natl. Acad. Sci. U.S.A. 2007,
104, 10655–10660.
(3) Gupta, S.; Singh, R. K.; Dastidar, S.; Ray, A. Cysteine cathepsin S as
an immunomodulatory target: present and future trends. Expert Opin.
Ther. Targets 2008, 12, 291–299.
(4) Turk, V.; Turk, B.; Guncˇar, G.; Turk, D.; Kos, J. Lysosomal cathepsins:
role in antigen processing and presentation, and cancer. AdV. Enzyme
Regul. 2002, 42, 285–303.
(15) Wiltshire, T.; Pletcher, M. T.; Batalov, S.; Barnes, S. W.; Tarantino,
L. M.; Cooke, M. P.; Wu, H.; Smylie, K.; Santrosyan, A.; Copeland,
N. G.; Jenkins, N. A.; Kalush, F.; Mural, R. J.; Glynne, R. J.; Kay,
S. A.; Adams, M. D.; Fletcher, C. F. Genome-wide single-nucleotide
polymorphism analysis defines haplotype patterns in mouse. Proc. Natl.
Acad. Sci. U.S.A. 2003, 100, 3380–3385.
(5) Berdwska, I. Cystein proteases as disease makers. Clin. Chim. Acta
2004, 342, 41–69.
JM800839J