Overcoming hERG issues for brain-penetrating cathepsin S inhibitors: 2-Cyanopyrimidines. Part 2

Article abstract of DOI:10.1016/j.bmcl.2008.08.067

We describe here orally active and brain-penetrant cathepsin S selective inhibitors, which are virtually devoid of hERG K⁺ channel affinity, yet exhibit nanomolar potency against cathepsin S and over 100-fold selectivity to cathepsin L. The new

Full text of DOI:10.1016/j.bmcl.2008.08.067

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5280–5284
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Overcoming hERG issues for brain-penetrating cathepsin S inhibitors:
2-Cyanopyrimidines. Part 2
a
a
a
a
a
Osamu Irie a, , Takatoshi Kosaka , Masashi Kishida , Junichi Sakaki , Keiichi Masuya , Kazuhide Konishi ,
Fumiaki Yokokawa a, Takeru Ehara a, Atsuko Iwasaki a, Yuki Iwaki a, Yuko Hitomi a, Atsushi Toyao a,
Hiroki Gunji a, Naoki Teno a, Genji Iwasaki a, Hajime Hirao a, Takanori Kanazawa a, Keiko Tanabe a,
*
Peter C. Hiestand b, Marzia Malcangio c, Alyson J. Fox c, , Stuart J. Bevan c, Mohammed Yaqoob c,
,
Andrew J. Culshaw c, , Terance W. Hart c, Allan Hallett c
a Global Discovery Chemistry, Novartis Institutes for BioMedical Research, Ohkubo 8, Tsukuba, Ibaraki 300-2611, Japan
b Novartis Institutes for BioMedical Research, Basel CH-4002, Switzerland
c Novartis Institutes for BioMedical Research, 5 Gower Place, London WC1E 6BS, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
We describe here orally active and brain-penetrant cathepsin S selective inhibitors, which are virtually
devoid of hERG K+ channel affinity, yet exhibit nanomolar potency against cathepsin S and over 100-fold
selectivity to cathepsin L. The new non-peptidic inhibitors are based on a 2-cyanopyrimidine scaffold
bearing a spiro[3.5]non-6-yl-methyl amine at the 4-position. The brain-penetrating cathepsin S inhibitors
demonstrate potential clinical utility for the treatment of multiple sclerosis and neuropathic pain.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 31 July 2008
Revised 15 August 2008
Accepted 16 August 2008
Available online 22 August 2008
Keywords:
Cathepsin S inhibitor
Brain-penetrating
hERG
Multiple sclerosis
Neuropathic pain
Cathepsin S (Cat S) is a cysteine protease predominantly ex-
pressed in dendritic cells, B cells, macrophages, and brain microg-
lia. In these antigen presenting cells Cat S plays an essential role in
the proteolytic events that lead to antigen presentation at the cells
surface for recognition by T cells.1 In addition, Cat S can be secreted
by activated microglia and might play a role in regulating extra cel-
lular matrix interaction.2 We have recently demonstrated that
inhibition of spinal microglial Cat S reversed neuropathic pain.3 It
was also reported that in Creutzfeldt–Jakob disease (CJD) infected
mice Cat S expression has been increased, which could result from
microglia cell activation.4 Cat S might be involved in multiple scle-
rosis (MS), myasthenia gravis (MG), Alzheimer’s disease (AD), and
Down disease.5 These findings suggest that a Cat S inhibitor which
penetrates blood–brain barrier (BBB) could be beneficial for vari-
ous brain diseases. Herein, we describe the discovery of non-pep-
tidic Cat S inhibitors having a balanced blood–brain penetration
after overcoming a human ether-a-go-go-related gene (hERG) K+
channel blocking issue which has often been observed in central
nervous system (CNS) drugs.6
We have recently reported a novel class of Cat S inhibitor 1, the
4,5,6-trisubstituted 2-cyanopyrimidine derivatives (Fig. 1).7 How-
ever, the selectivity against the off-target enzyme, Cat L was insuf-
ficient to reach the clinical phase. Cat L deficient mice develop
periodic hair loss and epidermal hyperplasia, indicating that Cat
L is essential for epidermal homeostasis and regular hair follicle
morphogenesis and cycling.8 Our next focus was thus on improv-
Solvent Space
N
H
N
N
O
N
P3
O
H
N
Cat S IC50 = 2nM
Cat L IC50 = 120nM
N
1
* Corresponding author. Tel.: +81 29 865 2384; fax: +81 29 865 2308.
E-mail addresses: osamu.irie@novartis.com, o.irie1@d4.dion.ne.jp (O. Irie).
Present address: Novartis Horsham Research Center, Wimblehurst Road, Hor-
sham RH12 5AB, UK.
P2
Figure 1. The 4,5,6-trisubstituted pyrimidine scaffold for Cat S inhibitors.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.067

O. Irie et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5280–5284
5281
O
O
HO
OH
O
O
TsO
ab
OTs
( )n
( )n
( )n
2a-c
3a-c
4a-c
OH
NH2
n=0-2
a, e, f
a; n=0
b; n=1
c; n=2
c, d
( )n
5a-c
( )n
6a-c
Scheme 1. Reagents and conditions: (a) TsCl, NMe3-HCl, NEt3, CH2Cl2, 0 °C, 1 h,
quant.; (b) CH2(CO2Et)2, NaH, THF, rt–60 °C, 3 h; (c) LiCl, H2O, DMSO, 185 °C, 10 h,
48–59% (3 steps); (d) LiAlH4, THF, 0 °C, 0.5 h, 82–84%; (e) NaN3, DMF, rt, 0.5 h; (f)
PPh3, THF–H2O, rt, 23 h, 73–75% (2 steps).
a slightly bulkier P2 group than the spiro[2.5]oct-6-yl-methyl
amine group of compound 1 (Fig. 2).
Figure 2. Compound 1 docked with human Cat S.
The spiro amine syntheses are shown in Scheme 1. Treatment of
2-[1-(2-hydroxy-ethyl)-cycloalkyl]-ethanol 2a–c11 with tosylchlo-
ride, triethylamine, and a catalytic amount of trimethylamine
hydrochloride12 in CH2Cl2 afforded ditosylate 3a–c. Cyclization of
3a–c with diethyl malonate under basic conditions in THF provided
spiro diesters 4a–c. Decarboxylation reaction of 4a–c with lithium
chloride and water in DMSO at 185 °C, followed by reduction of the
resulting esters by LiAlH4, gave spiro alcohols 5a–c which were
converted to amines 6a–c by treatment of the tosylates with so-
dium azide in DMF following by azide reduction with PPh3 in
THF–H2O.
ing the selectivity of Cat S inhibitors against Cat L by modifying the
P2 moiety of 1, which interacts with the S2 subsite of each cathep-
sin, a key pocket for controlling the selectivity of cathepsin
inhibitors.7
The X-ray crystal structures of human Cat S (PDB code 1MS6)
and Cat L (PDB code 3BC3) suggested that the S2 subsite of the
Cat S enzyme accepts a slightly larger group than that for Cat
L.9,10 The bottom of the S2 subsite in the Cat S enzyme has
Gly137 and Gly165, while the corresponding region in Cat L com-
prises of Ala135 and Gly164. Our computer-assisted modeling
studies concluded that the S2 pocket of Cat S would accommodate
We used a multi-parallel synthesis approach for the optimiza-
tion of the 4- and 6-substituents on the 2-cyanopyrimidine by
( )n
N
N
Boc
c, d, e
N
( )n
N
O
O
N
Boc
b
O
N
O
N
( )n
N
O
O
O
Cl
a
N
N
H
N
H
N
N
H
H
HN
Cl
N
Cl
N
8
S
N
Cl
N
7
S
R1
n=1 or 2
O
9
n=1 or 2
10a-d n=1 or 2
(See: Table 1)
O
O
O
Cl
N
Cl
N
a
cb
N
N
Boc
N
N
N
H
HN
N
H
H
HN
N
HN
S
N
N
N
11
12
14
d
f, g (for 13b)
R2
O
O
O
O
N
O
N
O
N
NH
N
R3
N
H
N
N
N
N
N
H
H
e or h
HN
HN
HN
N
N
N
13a,b
(See: Table 2)
15
16a-c
(See: Table 2)
Scheme 2. Reagents and conditions: (a) HO(CH2)n(4-piperidine-N-Boc), NaH, THF, 0–60 °C, 6 h, 52–97%; (b) i—mCPBA, NaHCO3, CH2Cl2, 0 °C–rt, 10 h; ii—KCN, nBu4N+Br-, 18-
Crown-6, CH2Cl2–H2O, rt, 3 h, 41–66% (2 steps); (c) R1-NH2, NEt3, THF, 0 °C–rt, 90–99%.; (d) 4 mol/L HCl–AcOEt, rt, 0.5 h, quant. or TFA, CH2Cl2 (for 10c, 10e), rt, 10 min, 30–
34%; (e) HCHO or acetone, NaBH3(CN), AcOH, THF, 0 °C–rt, 1.5 h, 38–58%; (f) HO(CH2)2OTHP, or 2,2,6,6-tetramethylpiperidine-4-ol, NaH, THF, 0–60 °C, 6 h, 82–99%; (g) PPTS,
EtOH, 70 °C, 6 h, 69–78%; (h) Br(CH2)2OH, K2CO3, DMF, 0 °C–rt, 12 h, 49%, or AcCl, NEt3, CH2Cl2, 0 °C–rt, 12 h, 71%.

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O. Irie et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5280–5284
using compounds 9 and 12 in Scheme 2 as key intermediates. Con-
densation of 4,6-dichloro-2-methylsulfanyl-pyrimidine-5-carbox-
ylic acid methylamide 77 with N-Boc-piperazine-4-methanol or
ethanol under the basic conditions in THF afforded 8a, b. Conver-
sion of the methyl sulfides to nitriles 9 was performed by oxidation
with mCPBA followed by treatment with potassium cyanide.
Treatment of the key intermediate 9 with amines 6a–c, the
commercially available cyclopentylethylamine, or C-(1,4-dioxa-
spiro[4.5]dec-8-yl)-methylamine13, followed by deprotection of
the Boc group and reductive amination with HCHO and NaBH3(CN),
provided the desired compounds 10a–d.
The synthesis for the optimization of the 6-substituent on the 2-
cyanopyrimidine is also described in Scheme 2. Condensation of 7
and C-spiro[3.5]non-7-yl-methylamine 6b gave compound 11
whichwasconverted to a 6-chloro-2-cyanopyrimidineintermediate
12. Addition of 2,2,6,6-tetramethylpiperidine-4-ol or 2-(tetrahydro-
pyran-2-yloxy)-ethanol with sodium hydride in THF, followed by
deprotection under acidic conditions provided the desired com-
pounds 13a, b. Condensation of 12 with N-Boc-piperidine 4-metha-
nol and subsequent treatment with 4 mol/L HCl solution in ethyl
acetate or trifluoroacetic acid afforded the secondary amine 15.
N-Substitution of the piperazine in 15 by acylation, reductive
amination, or by using alkyl halides provided tertiary amines 16a–c.
The results of the SAR study on the optimization of the P2 moi-
ety are described in Table 1. Expansion of the spiro ring at the end
of P2 by replacement of the spirocyclopropyl with cyclobutyl group
improved selectivity against Cat L (Table 1; 1 vs 10a). However,
further expansion of the spiro ring decreased both potency and
selectivity toward Cat S (10b vs 10d). Introduction of a polar func-
tional group on the P2 part was not tolerated because of the hydro-
phobic natures of the S2 subsites in both Cat S and Cat L (10b vs
10c).14 In summary of P2 moiety, the spiro[3.5]non-6-yl-methyl
amine 6a was an optimum size for P2 based on the balance of po-
tency and selectivity to Cat S.
In the past decade, a number of drugs failed to reach or were
withdrawn from the market due to cardiosafety issues. It is now
widely known that inhibition of hERG K+ channel is one of the ma-
jor causes of the observed cardiac side effects, which are drug-in-
duced QT prolongation and/or arrhythmia called Torsades de
Pointes. Our designed Cat S inhibitors 10a, b showed unacceptable
affinity to the hERG K+ channel as indicated by the binding assay
using [3H]dofetilide. We thus needed to decrease the hERG K+
channel binding affinity of compounds by modification of the 6-
substituent on the 2-cyanopyrimidine. Introduction of many func-
tional groups at the pyrimidine 6-position could be well tolerated
by the Cat S active site, as the 6-substituent orients itself toward
the solvent space in the modeling (Fig. 2). Indeed, all compounds
in Table 2 having a variety of substituents at the pyrimidine 6-po-
sition, showed excellent inhibitory activity and selectivity toward
Cat S. A SAR study of the 2-cyanopyrimidines on the hERG K+ chan-
nel binding affinity is shown in Table 2.
A typical 3D pharmacophore model for prediction of hERG K+
channel binding activity has been published.6,18 The model sug-
gests that the classic hERG K+ channel blocker motif contains a ba-
sic nitrogen center flanked by an aromatic or hydrophobic group.
We thus attempted to modulate the basicity of the piperidine ring
in the 6-substituent on the 2-cyanopyrimidine core. Decreasing the
basicity of the nitrogen atom by replacing the tertiary amine with a
secondary amine decreased the hERG binding activity (Table 2;
10b vs 15). An increase in basicity by replacement of N-methyl
piperidine by N-isopropyl piperidine showed the anticipated in-
crease in affinity to the hERG K+ channel (16a, hERG
Table 1
Table 2
Optimization of the P2 moiety
Optimization of the 6 position on the pyrimidine ring17
R
N
(CH )n
NH
O
2
NH
O
N
O
N
O
N
HN
N
N
HN
R
N
Compound
n
R
IC50 (nM)a
Cat L
Compound
R
IC50 (nM)a
Cat L
Cat S
2
hERGb
NTc
Cat S
hERGb
710
1
2
2
1
1
1
1
1
120
440
360
>1000
276
426
400
-CH2
-CH2
-CH2
-CH2
-CH2
-CH2
10b
16a
16b
15
3
360
-CH2
N
10a
10b
10c
10d
10e
4
420
3
330
540
-CH2
-CH2
N
N
3
710
3
300
890
O
O
OH
>1000
12
31
26
NTc
5
460
2340
-CH2
NH
1455
5770
360
13a
16c
NH
5
1390
2500
1200
5040
O
O
O
10
6
>30,000
>30,000
-CH2
N
10d
-(CH2)2
13b
–CH2CH2OH
a
Inhibition profiles were determined by a fluorometric assay with recombinant
human Cat L and Cat S, employing Z-Phe-Arg-AMC (Cat L) and L-Leu-Leu-Arg-AMC
(Cat S) as synthetic substrates.15 Data represent means of two experiments per-
formed in duplicate. Individual data points in each experiment were within a
a
Inhibition profiles were determined by a fluorometric assay with recombinant
human Cat K, L, and S, employing Z-Phe-Arg-AMC (Cat K and L) and L-Leu-Leu-Arg-
AMC (Cat S) as synthetic substrates.15 Data represent means of two experiments
performed in duplicate. Individual data points in each experiment were within a
twofold range with each other.
twofold range with each other.
b
Inhibition profiles were determined by
a radioligand binding assay with
[3H]dofetilide binding to a crude membrane preparation of HEK293 cell membranes
stably transfected with hERG channels.16
b
Inhibition profiles were determined by a radioligand assay with [3H]dofetilide
binding to
a crude membrane preparation of HEK293 cell membranes stably
c
transfected with hERG channels.16
NT, not tested.

O. Irie et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5280–5284
5283
affinity (16a vs 16c). Removal of the nitrogen atom by replacement
Table 3
Plasma and brain concentration profiles for several compounds in Sprague–Dawley
rats (po 10 mg/kg), where values are means of n = 3
of the piperidine ring with ethyl alcohol also showed significantly
attenuated hERG activity (IC50 = >30 lM) whilst retaining both po-
tency and selectivity to Cat S (13b, Cat S IC50 = 6 nM).
Compound
Plasma concentration (nM)
Brain concentration (nM)
We then turned our attention to the CNS penetration of the Cat
S inhibitors, which was assessed by following oral administration
in rats. The results of the CNS penetration assays are shown in
Table 3. The secondary amine 15 and N-acyl piperidine 16c did
not penetrate the BBB in rats, whilst the sterically hindered sec-
ondary amine 13a showed CNS penetration. A tertiary amine also
facilitated brain penetration (Table 3; 10b, 16a) although these
compounds also showed high hERG K+ channel binding affinity.
The 6-(2-hydroxy ethoxy) 2-cyanopyrimidine compound 13b rap-
1 h
3 h
1 h
3 h
10b
16a
15
13a
16c
13b
346
191
28
—
169
283
308
381
61
287
80
124
67
<13
—
27
692
718
455
<13
164
<13
849
170
Table 4
idly penetrated the BBB without hERG K+ channel affinity at 30
lM.
Pharmacokinetic parameters of 10b, 16a, and 13b in male Sprague–Dawley rats (iv
1 mg/kg; po 10 mg/kg), where values are means of n = 3
The pharmacokinetics (PK) parameters were determined for
selected compounds in Sprague–Dawley rats. Representative PK
results are shown in Table 4. After intravenous administration
compounds, 10b, 16a, and 13b were distributed with the Vdss of
1.2–9.8 L/kg and eliminated with the apparent half-lives of 1.5–
11.8 h. The maximum plasma concentration (Cmax) values of the
compounds after oral administration were 298–907 nM and the
estimated bioavailabilities (F) ranged from 14% to 40%.
Compound poCmax
Clp (L/h/
kg)
ivt1/2
(h)
F
(%)
AUC
(nM h)
Vdss (L/
kg)
po
(nM)
10b
16a
13b
461
298
907
1.1
0.6
1.5
6.7
11.8
1.5
40
24
14
8237
8083
2503
9.8
9.3
1.2
Having developed both orally active and brain-penetrating Cat S
inhibitors, we next turned to evaluate in vivo efficacy. Multiple
sclerosis (MS) is a chronic demyelinating disease of the CNS char-
acterized by scarring plaques distributed along the intracerebral
white matter and the spinal cord. The most suitable model of MS
is experimental autoimmune encephalomyelitis (EAE) that has
well-defined immunologic and genetic profiles for the murine sys-
tem.19 We thus evaluated the effect of 10b on the induction of the
acute phase of the chronic progressive form of EAE in SJL/J mice.20
Oral administration of 10b (10 mg/kg) twice a day for 21 days pre-
vented the onset of the acute phase of the chronic progressive form
of EAE (Fig. 3). These data suggest that brain-penetrating Cat S
inhibitors might be useful for the treatment of MS.
We also evaluated the anti-neuropathic pain effect of the devel-
oped Cat S inhibitors 10b and 13b in Wister rats following oral
administration.3 Both compounds reversed established mechanical
hyperalgesia in a dose dependent fashion. The activities of 10b and
13b were producing up to 50% reversal of the hyperalgesia (Fig. 4).
Following twice daily administration for 5 days, the anti-hyperal-
gesic activity of the first dose of 13b (30 mg/kg) against neuropath-
ic hyperalgesia was maintained without serious side effect (Fig. 5).
These findings suggest that Cat S inhibition has great promise as a
new therapeutic treatment for neuropathic pain.
Figure 3. Effect21 of compound 10b in acute experimental autoimmune enceph-
alomyelitis in SJL/J mice. Data presented are means SEM. Kruskal–Wallis non-
parametric ANOVA, followed by Dunn’s multiple comparison test *p < 0.05,
**p < 0.01 Disease incidence was analyzed using Fisher’s exact test (2 Â 2 contin-
gency table, 2-sided p value). n = 15. MBP, myelin basic protein.
In summary, we discovered brain-penetrating Cat S inhibitors
with nanomolar potency against Cat S, over 100-fold selectivity
against Cat L, and excellent PK profiles after overcoming a hERG
IC50 = 540 nM). Interestingly, a less basic substituent afforded by
N-acylation dramatically decreased the hERG K+ channel binding
Figure 4. Oral activity of compound 10b (left) and 13b (right) against neuropathic mechanical hyperalgesia in rats. Graph depicts mean SEM reversal of hyperalgesia from
six 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.

5284
O. Irie et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5280–5284
C. E.; Connolly, T. M.; Doran, S. M.; Fox, S. V.; Kraus, R. L.; Marino, M. J.;
Graufelds, V. K.; Vargas, Hu M.; Bunting, P. B.; Hasbun-Manning, M.; Evans, R.
M.; Koblan, K. S.; Renger, J. J. J. Med. Chem. 2008, 51, 3692; (b) Yoshizumi, T.;
Miyazoe, H.; Ito, H.; Tsujita, T.; Takahashi, H.; Asai, M.; Ozaki, S.; Ohta, H.;
Okamoto, O. Bioorg. Med. Chem. Lett. 2008, 18, 3778; (c) Yoshizumi, T.;
Takahashi, H.; Miyazoe, H.; Sugimoto, Y.; Tsujita, T.; Kato, T.; Ito, H.; Kawamoto,
H.; Hirayama, M.; Ichikawa, D.; Azuma-Kanoh, T.; Ozaki, S.; Shibata, Y.; Tani, T.;
Chiba, M.; Ishii, Y.; Okuda, S.; Tadano, K.; Fukuroda, T.; Okamoto, O.; Ohta, H. J.
Med. Chem. 2008, 51, 4021; (d) Kawai, M.; Ando, K.; Matsumoto, Y.; Sakurada,
I.; Hirota, M.; Nakamura, H.; Ohta, A.; Sudo, M.; Hattori, K.; Takashima, T.;
Hizue, M.; Watanabe, S.; Fujita, I.; Mizutani, M.; Kawamura, M. Bioorg. Med.
Chem. Lett. 2007, 17, 5558; For a recent review on hERG, see: (e) Thai, K.-M.;
Ecker, G. F. Curr. Med. Chem. 2007, 14, 3003.
7. Irie, O.; Yokokawa, F.; Ehara, T.; Iwasaki, A.; Iwaki, Y.; Hitomi, Y.; Konishi, K.;
Kishida, M.; Toyao, A.; Masuya, K.; Gunji, H.; Sakaki, J.; Iwasaki, G.; Hirao, H.;
Kanazawa, T.; Tanabe, K.; Kosaka, T.; Hart, W. T.; Hallett, A. Bioorg. Med. Chem.
Lett. 2008, 18, 4642.
8. Roth, W.; Deussing, J.; Botchkarev, V. A.; Pauly-Evers, M.; Saftig, P.; Hafner, A.;
Schmidt, P.; Schmahl, W.; Scherer, J.; Anton-Lamprecht, I.; Von Figura, K.; Paus,
R.; Peters, C. FASEB J. 2000, 14, 2075.
9. 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. Biochemistry 2003, 42, 3203.
10. Chowdhury, S. F.; Sivaraman, J.; Wang, J.; Devanathan, G.; Lachance, P.; Qi, H.;
Ménard, R.; Lefebvre, J.; Konishi, Y.; Cygler, M.; Sulea, T.; Purisima, E. O. J. Med.
Chem. 2002, 45, 5321.
Figure 5. Effect of repeated administration of 13b on neuropathic pain in rats.
Compound 13b was administered orally in 0.5% methylcellulose/water twice daily
for 5 days. Paw withdrawal thresholds were measured 3 h following administra-
tion. 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.
11. Foos, L.; Steel, F.; Rizvi, S. Q. A.; Fraenkel, G. J. Org. Chem. 1979, 44, 2522.
12. Wakasugi, K.; Nakamura, A.; Iida, A.; Nishii, Y.; Nakatani, N.; Fukushima, S.;
Tanabe, Y. Tetrahedron 2003, 59, 5337.
K+ channel binding issue by modeling the basicity of the 6-substi-
tuent on the 2-cyanopyrimidine core. The compounds demon-
strated in vivo activity for MS and neurophatic pain in rodents.
We believe that brain-penetrating Cat S inhibitors might be useful
for the treatment of these diseases and various other CNS disor-
ders, e.g., AD, CJD, and MG.
13. Becker, D. P.; Flynn, D. L. Synthesis 1992, 1080.
14. 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.
Bioorg. Med. Chem. Lett. 2008, 18, 3959.
15. Altmann, E.; Aichholz, R.; Betschart, C.; Buhl, T.; Green, J.; Irie, O.; Teno, N.;
Lattmann, R.; Tintelnot-Blomley, M.; Missbach, M. J. Med. Chem. 2007, 50,
591.
16. (a) Finlayson, K.; Turnbull, L.; January, C. T.; Sharkey, J.; Kelly, J. S. Eur. J.
Pharmacol. 2001, 430, 147; (b) Snyders, D. J.; Chaudhary, A. Mol. Pharmacol.
1996, 49, 949.
Acknowledgments
We thank Michie Kobayashi, Tomoko Ohkubo, Andrew McB-
ryde, Caroline Huntley, Prafula Copp, and Hendrikus Eggelete for
excellent technical assistance. The authors are grateful to Christo-
pher R. Snell, Pamposh Ganju, and Shinichi Koizumi for valuable
discussions. We acknowledge Steven Whitebread for the hERG K+
channel binding assay and Professor Dr. Masakatsu Shibasaki
(The University of Tokyo) for valuable discussions regarding chem-
istry part.
17. (a) All compounds were characterized by 1H NMR and LC–MS. The
experimental information is described in a patent application, see for details:
Hart, T. W.; Hallett, A.; Yokokawa, F.; Hirao, H.; Ehara, T.; Iwasaki, A.; Sakaki, J.;
Masuya, K.; Kishida, M.; Irie, O. WO 2006018284, 2006.(b) Spectral data of 10b
and 13b. Compound 10b: 1H NMR (400 MHz, CDCl3) d 10.13 (br s, 1H), 7.83 (br
s, 1H), 4.38 (d, J = 6.5 Hz, 2H), 3.32 (t, J = 6.3 Hz, 2H), 2.93 (d, J = 4.6 Hz, 3H),
2.97–2.87 (m, 2H), 2.31 (s, 3H), 1.99 (t, J = 11.0 Hz, 2H), 1.89–1.56 (m, 13 H),
1.55–1.36 (m, 3H), 1.29–1.17 (m, 2H), 1.09–0.95 (m, 2H). MS (ESI) m/z 441
(M+H)+. Compound 13b: 1H NMR (400 MHz, CDCl3) d 10.15 (br s, 1H), 8.03 (br
s, 1H), 4.64–4.59 (m, 2H), 4.03–3.97 (m, 2H), 3.33 (dd, J = 6.6 and 7.1 Hz, 2H),
2.92 (d, J = 5.0 Hz, 3H), 1.89–1.58 (m, 11H), 1.53–1.43 (m, 1H), 1.29–1.18 (m,
2H), 1.08–0.96 (m, 2H). MS (ESI) m/z 374 (M+H)+. Their purities were greater
than 99% by HPLC analysis. The HPLC was performed on analytical
References and notes
Phenomenex C18 Luna
3
l
M
column (30 Â 4.6 mm), gradient 10–100%
1. Riese, R. J.; Mitchell, R. N.; Villadangos, J. A.; Shi, G.-P.; Palmer, J. T.; Karp, E. R.;
De Sanctis, G. T.; Ploegh, H. L.; Chapman, H. A. J. Clin. Invest. 1998, 101, 2351.
2. Liuzzo, J. P.; Petanceska, S. S.; Moscatelli, D.; Devi, L. A. Mol. Med. 1999, 5, 320.
3. (a) 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.
Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 10655; (b) 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. Pain 2007, 130, 225.
acetonitrile in water with 0.08% formic acid over 10 min at 3.0 mL/min as
the mobile phase. Purity was calculated by integration of UV at 254 nm.
18. (a) Morgan, T. K., Jr.; Sullivan, M. E. Prog. Med. Chem. 1992, 29, 65; (b) Cavalli,
A.; Poluzzi, E.; De Ponti, F.; Recanatini, M. J. Med. Chem. 2002, 45, 3844.
19. For a recent review, see: (a) Baxter, A. G. Nat. Rev. Immunol. 2007, 7, 904; (b)
Foster, C. A.; Howard, L. M.; Schweitzer, A.; Persohn, E.; Hiestand, P. C.;
Balatoni, B.; Reuschel, R.; Beerli, C.; Schwart, M.; Billich, A. J. Pharmacol. Exp.
Ther. 2007, 323, 469.
4. Baker, C. A.; Martin, D.; Manuelidis, L. J. Virol. 2002, 76, 10905.
20. Disease was induced on day 0 by sc injection to SJL/L mice with a mixture of
5. For recent reviews of cathepsin S, see: (a) Berdowska, I. Clin. Chim. Acta 2004,
342, 41; (b) Gupta, S.; Singh, R. K.; Dastidar, S.; Ray, A. Expert Opin. Ther. Targets
2008, 12, 291; (c) Maryanoff, B. E.; Costanzo, M. J. Bioorg. Med. Chem. 2008, 16,
1562; For increased expression of Cat S in hippocampal microglia following
kainite-induced seizures, see: (d) Akahoshi, N.; Murashima, Y. L.; Himi, T.;
Ishizaki, Y.; Ishii, I. Neurosci. Lett. 2007, 429, 136.
500 lg bovine MBP in 0.1 ml water emulsified in Freund’s complete adjuvant
DIFCO (1:1 v/v). On day 9, mice were boosted by re-injection of MBP and an
additional iv injection of adjuvant containing Bordetella pertussis toxin (200 ng/
200
ll saline). An additional injection of the pertussis toxin (125 ng/200 ll)
was given on day 11.
21. Clinical grades were as follows: 0, no disease symptoms; 1, loss of tail tonicity;
2, weakness of one of both hind legs and mild ataxia; 3, severe ataxia or
paralysis accompanied by urinary incontinence and/or death.
6. (a) Shipe, W. D.; Barrow, J. C.; Yang, Z.-Q.; Lindsley, C. W.; Yang, F. V.; Schlegel,
K.-A. S.; Shu, Y.; Rittle, K. E.; Bock, M. G.; Hartman, G. D.; Tang, C.; Ballard, J. E.;
Kuo, Y.; Adarayan, E. D.; Prueksaritanont, T.; Zrada, M. M.; Uebele, V. N.; Nuss,