Diazinones as P2 replacements for pyrazole-based cathepsin S inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.05.086

A pyridazin-4-one fragment 4 (hCatS IC₅₀ = 170 μM) discovered through Tethering was modeled into cathepsin S and predicted to overlap in S2 with the tetrahydropyridinepyrazole core of a previously disclosed series of CatS inhibitors. This fragment served as a template to design pyridazin-3-one 12 (hCatS IC₅₀ = 430 nM), which also incorporates P3 and P5 binding elements. A crystal structure of 12 bound to Cys25Ser CatS led to the synthesis of the potent diazinone isomers 22 (hCatS IC₅₀ = 60 nM) and 27 (hCatS IC₅₀ = 40 nM).

Full text of DOI:10.1016/j.bmcl.2010.05.086

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4060–4064
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Diazinones as P2 replacements for pyrazole-based cathepsin S inhibitors
a
b
b
a
Michael K. Ameriks a, , Scott D. Bembenek , Matthew T. Burdett , Ingrid C. Choong , James P. Edwards ,
Damara Gebauer a, , Yin Gu a, , Lars Karlsson a, Hans E. Purkey b, Bart L. Staker c, Siquan Sun a,
Robin L. Thurmond a, Jian Zhu a
*
a Johnson & Johnson Pharmaceutical Research & Development, L.L.C., 3210 Merryfield Row, San Diego, CA 92121, USA
b Sunesis Pharmaceuticals, 341 Oyster Point Boulevard, South San Francisco, CA 94080, USA
c deCODE Biostructures, Inc., 7869 Northeast Day Road West, Bainbridge Island, WA 98110, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A pyridazin-4-one fragment 4 (hCatS IC50 = 170 lM) discovered through Tethering was modeled into
Received 1 May 2010
Accepted 20 May 2010
Available online 25 May 2010
cathepsin S and predicted to overlap in S2 with the tetrahydropyridinepyrazole core of a previously
disclosed series of CatS inhibitors. This fragment served as a template to design pyridazin-3-one 12
(hCatS IC50 = 430 nM), which also incorporates P3 and P5 binding elements. A crystal structure of 12
bound to Cys25Ser CatS led to the synthesis of the potent diazinone isomers 22 (hCatS IC50 = 60 nM)
and 27 (hCatS IC50 = 40 nM).
Keywords:
Cathepsin
Protease
Ó 2010 Elsevier Ltd. All rights reserved.
Tethering
X-ray crystallography
Fragment-based screening
The cathepsins comprise a large group of proteases whose gen-
eral function involves protein degradation, processing, and turn-
over.1 Cathepsin S (CatS), a cysteine protease of the papain
family, resides in the lysosome of certain antigen-presenting cells
and plays a key role in facilitating antigen presentation to CD4+
T-cells.2 CatS-deficient mice exhibit defects in antigen processing,
as well as a reduced susceptibility to collagen-induced arthritis
compared to wild-type animals.3 Recently, additional pharmaco-
logically relevant activities have also been attributed to CatS.4
We have previously reported a series of non-covalent CatS inhibi-
tors with a tetrahydropyridinepyrazole core.5 This scaffold was
originally discovered through a high-throughput screening (HTS)
campaign and further optimized to provide compounds such as
16 and 27 (Fig. 1).
As a complementary approach to the traditional HTS strategy
for lead generation, we also envisioned using fragment-based
screening protocols to identify low molecular weight CatS inhibi-
tors.8 Since proteases possess numerous extended binding sites
in which each amino acid of the peptide or protein substrate occu-
pies its own subsite, it was anticipated that cathepsin S would be
an ideal target for fragment-based drug discovery.9 To this end,
we employed a technique known as Tethering,10 in which site-di-
rected mutagenesis was used to introduce cysteine residues into
various subsites of a Cys25Ser mutant form of CatS.11 Each individ-
ual construct was then screened against a library of disulfide-con-
taining compounds under reducing conditions, and the resulting
fragments that formed stable protein–ligand disulfide adducts
were characterized by mass spectrometry. In this letter, we de-
scribe how
a low-affinity pyridazinone fragment 4 ( Fig. 2)
discovered through naïve Tethering was optimized for binding in
S2 and linked to P3 and P5 groups previously identified for a
CF3
Cl
N
N
N
N
N
N
O
SO2CH3
1, hCatS = 79 nM
Cl
H
N
N
N
N
O
N
SO2CH3
* Corresponding author.
2, hCatS = 310 nM
E-mail address: mameriks@its.jnj.com (M.K. Ameriks).
Former employee of Johnson & Johnson Pharmaceutical Research & Development,
L.L.C., 3210 Merryfield Row, San Diego, CA 92121, USA.
Figure 1. Representative pyrazole-based cathepsin S inhibitors.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.086

M. K. Ameriks et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4060–4064
4061
Rather than preparing direct analogs of pyridazin-4-one 4, we
initially focused on the regioisomeric pyridazin-3-ones (8–13,
Scheme 1) for two reasons: it was anticipated that this heterocycle
would provide better overlap with both ring nitrogens in the pyr-
azole core, and the synthetic route to this class of compounds was
well precedented.16 Thus, 6-arylpyridazin-3-one 6 was prepared in
five steps from 40-chloroacetophenone. This functionalized inter-
mediate allowed for late-stage incorporation of the P3 and P5
groups. The P5 amines could be installed via a two-step procedure
involving acetal hydrolysis and reductive amination, followed by
nitrile reduction and acylation to introduce the P3 amide. During
the optimization of this synthetic sequence, we observed that Ra-
ney-Ni was the only reducing agent capable of cleanly converting
E115C-S
Cl
Cl
NH
O
3 NH
N
N
O
N
O
N
O
4 (hCatS = 170 µM)
3 (FC 100%)
Cl
Br
Cl
d,e
N
a,b,c
O
HN
O
5
Cl
Cl
O
f,g
N
N
R2N
N
CN
O
N
CN
O
O
7
6
h,i
Cl
R'
H
N
N
R2N
N
O
O
Figure 2. Docking model of Tethering hit
structure of pyrazole-based CatS inhibitor
3
(yellow) overlayed with crystal
8-13
2 (orange). The residues at which
cysteine mutations were introduced individually to enable Tethering are shown in
cyan.
Scheme 1. Reagents and conditions: (a) AlCl3, Br2, DCE, rt (85%); (b) glyoxylic acid,
120 °C; (c) H2NNH2, aq NH4OH, 100 °C (38%, two steps); (d) 5% Pd2dba3, dppf,
Zn(CN)2, Zn(OAc)2, Zn, DMF, 95 °C (91%); (e) 2-(2-bromo-ethyl)-1,3-dioxolane, NaH,
DMF/DMSO, rt (61%); (f) 1 N HCl, acetone, 55 °C; (g) R2NH, Na(OAc)3BH, AcOH,
CH2Cl2 (50–60%, two steps); (h) Raney-Ni, H2 (30 psi), 2 M NH3/EtOH; (i) benzoyl
chloride or 4-fluorobenzoyl chloride, pyridine, CH2Cl2 (51–64%, two steps).
tetrahydropyridinepyrazole P2 core to provide a new series of po-
tent diazinone-based CatS inhibitors.
In order to ensure adequate spatial coverage of the cathepsin S
topography, five individual mutant constructs of Cys25Ser cathep-
sin S were prepared for Tethering: Lys64Cys, Asn67Cys, Glu115Cys,
Phe211Cys, and Arg141Cys (Fig. 2).12 Approximately 16,500 com-
pounds from the Sunesis disulfide monophore library were
screened against each construct, and 352 monophores exhibited
>50% fractional conjugation (monophore-labeled CatS/total CatS)
with any of the proteins.13 Roughly a third of these hits bound to
the Glu115Cys mutant, including pyridazinone 3, which com-
pletely labeled the protein (100% fractional conjugation at 1 mM
b-ME). To confirm the viability of using this Tethering hit for sub-
sequent lead optimization, the corresponding des-sulfide mono-
phore fragment 4 was assayed against wild-type CatS (hCatS
Table 1
SAR of 6-arylpyridazinone CatS inhibitorsa
Cl
R'
H
N
N
R2N
N
O
O
Compound
NR2
R0
H
CatS IC50 (lM)
N
8
14
IC50 = 170 l
M).14
N
Although a X-ray crystal structure of the disulfide protein–li-
gand adduct 3 could not be obtained, the binding mode from our
docking model suggested that the pyridazinone fragment resides
in the S2 pocket of CatS (Fig. 2).15 Furthermore, when 3 was mod-
eled into a Cys25Ser CatS cocrystal structure of pyrazole-based
inhibitor 2, several key features overlapped between the two struc-
tures: (1) the 4-chlorophenyl rings in S2, (2) the nitrogen atoms at
N2 of the pyridazinone and pyrazole rings, and (3) the dihedral an-
gles between the 4-chlorophenyl ring and the attached heterocy-
clic S2 cores (/ = 29° for pyridazinone 3 and 36° for pyrazole 2).
Notably, the Tethering hit lacks the P3 amide and P5 pyrrolidine
groups found in pyrazole 2. Thus, despite the low affinity of frag-
ment 4 for CatS, it seemed possible that incorporating these addi-
tional binding elements would provide an opportunity to ‘grow’
the fragment into a potent CatS inhibitor.
F3C
O
N
9
H
1.6
10
11
H
F
2.3
1.5
N
N
12
13
Cl
H
F
0.43
0.32
N
N
N
O
a
CatS IC50 values are the mean of n ꢀ 2 runs and determined as described
previously.6a

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M. K. Ameriks et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4060–4064
ity, piperazine 9 and piperidines 10–11 exhibited a six- to ninefold
improvement in potency. The incorporation of a ketobenzimidaz-
ole-substituted piperidine yielded the most significant boost in
activity, as evidenced by the sub-micromolar IC50s for compounds
12 and 13. The effect of the P3 amide on CatS inhibition was more
subtle, with the 4-fluorobenzoyl group consistently providing a
minimal improvement in potency over the parent benzamide (cf.
10 vs 11 and 12 vs 13). Although the best inhibitor from this series,
pyridazinone 13, displayed only moderate enzymatic activity
(hCatS IC50 = 320 nM), it is important to note that this constitutes
more than a 500-fold improvement from the lead Tethering mono-
phore 4.
In order to glean insight into the binding mode of the 6-arylpy-
ridazin-3-one series, a crystal structure of 12 bound to Cys25Ser
CatS was obtained (Fig. 3). Gratifyingly, this compound binds in
an orientation consistent with the molecular modeling predictions
for Tethering hit 3. The bicyclic arylpyridazinone core occupies the
S2 pocket, while the ketobenzimidazole piperidine and benzamide
groups reside in S5 and S3, respectively. Notably, a hydrogen bond
is observed between the backbone carbonyl of Gly69 in S3 and the
P3 benzamide N–H. However, the most intriguing contacts be-
tween the protein and inhibitor 12 occur within an area of S2 that
spans Met71 and Thr72. In this region, two water molecules appear
to facilitate a network of bridging interactions between N1 of the
pyridazinone and the side chain hydroxyl group of Thr72, as well
as the backbone amide hydrogens of Met71 and Thr72.
Since the pyridazinone ring in 12 binds only indirectly to the
residues in S2, we anticipated that an alternate P2 group capable
of displacing one or both of the water molecules would bind dee-
per in the S2 pocket and also benefit from the entropic gain asso-
ciated with the release of these waters.17 The simplest analog of
pyridazinone 12 that fits this description is the isomeric 4-aryl-
pyridazin-3-one 22 (Scheme 2). This heterocycle retains the 1,3-
disposition of the aromatic group and aminopropyl linker required
to access S3 and S5, respectively. In addition, the polar carbonyl
Figure 3. Crystal structure of pyridazinone 12 bound to Cys25Ser cathepsin S (PDB
code: 3MPF). Two water molecules (red) are observed in the back of the S2 subsite.
Hydrogen bonds are shown in green.
the nitrile to a primary amine while leaving the arylchloride and
electron-poor pyridazinone ring intact. Furthermore, it was neces-
sary to use ammonia in ethanol as a solvent for this reaction in or-
der to suppress dimerization of the desired amine product. Based
on previously established SAR for pyrazole-based CatS inhibitors,
we surveyed several specific cyclic amines as P5 groups, and also
focused on benzamide and 4-fluorobenzamide as P3 moieties.
As shown in Table 1, the enzymatic activity of compounds 8–13
varied dramatically with the nature of the P5 amine. Although pyr-
rolidine 8 displayed only mid-micromolar inhibition of CatS activ-
O
O
O
Cl
F
Cl
F
Cl
d
c
Cl
Cl
a,b
H
N
H
N
Cl
Cl
+
F
HN
N
O
N
N
O
Br
B
O
Br
CN
O
O
16
15
14
e
O
O
Cl
Cl
F
Cl
O
O
O
H
O
O
N
N
O
H
N
N
f
O
N
N
H
N
N
N
H
O
O
Cl
Cl
g
Cl
F
18
19
17
Cl
NH
N
N
20
O
Cl
F
Cl
F
O
H
O
Cl
H
N
Cl
N
h
N
N
N
N
N
N
O
O
N
N
Cl
N
N
22
O
21
O
Scheme 2. Reagents and conditions: (a) BH3–THF, THF, rt; (b) 4-fluorobenzoyl chloride, pyr., CH2Cl2 (53%, two steps); (c) bis(pinacoloto)diboron, KOAc, 10% PdCl2(dppf), DMF,
90 °C (87%); (d) 2-(2-bromoethyl)-1,3-dioxolane, Cs2CO3, DMF (69%); (e) 10% PdCl2(dppf), 2 M Na2CO3/dioxane, 90 °C, (48%, 17:18 = 1:1.3); (f) 1 N HCl/acetone, 55 °C; (g)
Na(OAc)3BH, AcOH, CH2Cl2 (87%, two steps); (h) Et3N, HCO2H, Pd(OAc)2, PPh3, DMF, 60 °C (98%).

M. K. Ameriks et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4060–4064
4063
Table 2
group of the pyridazinone should be oriented directly towards the
SAR of diazinone-based CatS inhibitorsa
proximal water molecule situated in the back of the S2 pocket.
Based on the SAR from Table 1, 4-fluorobenzamide was selected
as a P3 group and incorporated into the key boronic ester interme-
diate 15. The subsequent Suzuki coupling with dichloropyridazi-
none 16 proceeded unselectively to afford a separable mixture of
arylpyridazinone isomers.18 The desired 4-aryl isomer 17 was ulti-
mately joined with the ketobenzimidazole piperidine P5 group 20
to provide pyridazinone 22.
Cl
F
O
Y
H
N
Cl
N
N
X
Z
O
N
N
O
Compound
R
X
Y
Z
CatS IC50
(
l
M)
JY Ii IC50 (lM)
In addition to pyridazinone 22, we also prepared the diazinone
isomers 27 (Scheme 3) and 32 (Scheme 4). These compounds pos-
sessed the structural features required for binding in S2, S3, and S5,
and also allowed us to evaluate the importance of the position of
the nitrogen atom within the diazine ring. Pyrimidinone 27 ulti-
mately derived from bromopyrimidine 23,19 boronic ester 15,
and ketobenzimidazole piperidine 20. A similar series of synthetic
transformations was used to assemble pyrazinone 32, with chloro-
pyrazine 28 functioning as the electrophile for the key Suzuki
coupling.
As shown in Table 2, all three diazinone isomers demonstrated
improved CatS inhibition relative to pyridazinone 13, with pyri-
midinone 27 exhibiting the most dramatic difference (hCatS
IC50 = 40 nM vs 320 nM for 13). However, pyridazinone 22 dis-
played the best activity in a secondary cellular assay used to mea-
22
27
32
H
H
H
N
CH
CH
CH
N
CH
CH
CH
N
0.06
0.04
0.11
1.8
6.7
8.0
a
CatS IC50 values and JY Ii IC50 values are the mean of n ꢀ 2 runs and determined
as described previously.6a
sure inhibition of invariant chain (Ii) degradation in a human B-cell
line. In comparison, pyrimidinone 27 and pyrazinone 32 showed
markedly reduced cellular activity, and these trends correlated
only marginally well with cellular permeability.20
In order to discern the structural factors responsible for the im-
proved enzymatic activity of pyrimidinone 27, we obtained a crys-
tal structure of this compound bound to Cys25Ser CatS (Fig. 4). In
Cl
F
H
N
O
B
O
Cl
F
O
O
N
O
O
O
N
O
H
N
15
Br
Br
Br
Cl
b
a
N
O
O
N
HN
N
c
O
N
N
25
24
23
NH
N
e
Cl
F
O
Cl
F
H
N
Cl
O
O
N
O
H
20
N
d
N
N
N
H
N
O
N
N
O
N
26
27
O
Scheme 3. Reagents and conditions: (a) H3CO3H, H2SO4, acetone (63%); (b) 2-(2-bromoethyl)-1,3-dioxolane, Cs2CO3, DMF (93%); (c) PdCl2(dppf), K3PO4, dioxane, 90 °C (58%);
(d) 1 N HCl, acetone, 55 °C; (e) Na(OAc)3BH, AcOH, CH2Cl2 (53%, two steps).
Cl
F
H
N
O
B
O
Cl
F
Cl
F
Cl
O
OMe
OMe
N
H
N
H
N
O
Cl
15
c
Cl
a
N
HN
N
N
N
N
O
b
N
O
30
29
28
Cl
NH
N
f
Cl
F
O
Cl
F
H
N
Cl
20
O
O
O
H
N
N
d,e
N
N
H
N
N
O
N
N
O
N
31
32
O
Scheme 4. Reagents and conditions: (a) NaOMe, MeOH, 65 °C (71%); (b) PdCl2(dppf), 2 M Na2CO3, dioxane, 90 °C; (c) 4 N HCl, 100 °C (48%, two steps); (d) 2-(2-bromoethyl)-
1,3-dioxolane, Cs2CO3, DMF (87%); (e) 1 N HCl/acetone, 55 °C; (f) Na(OAc)3BH, AcOH, CH2Cl2 (46%, two steps).

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M. K. Ameriks et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4060–4064
References and notes
1. Roberts, R. Drug News Perspect. 2005, 18, 605.
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C. R.; Rennert, P.; Bailly, V.; Jarpe, M.; Dolinski, B.; Koteliansky, V.; de
Fougerolles, T.; Daeman, M. J. A. P. Circulation 2005, 111, 3443; (d) Wendt,
W.; Lubbert, H.; Stichel, C. C. Brain Res. 2008, 1232, 7; (e) Irie, O.; Kosaka, T.;
Ehara, T.; Yokokawa, F.; Kanazawa, T.; Hirao, H.; Iwasaki, A.; Skakai, J.; Teno, N.;
Hitomi, Y.; Iwasaki, G.; Fukaya, H.; Nonomura, K.; Tanabe, K.; Koizumi, S.;
Uchiyama, N.; Bevan, S. J.; Malcangio, M.; Gentry, C.; Fox, A. Y.; Yaqoob, M.;
Culshaw, A. J.; Hallett, A. J. Med. Chem. 2008, 51, 5502.
5. Thurmond, R. L.; Beavers, M. P.; Cai, H.; Meduna, S. P.; Gustin, D. L.; Sun, S.;
Almond, H. J.; Karlsson, L.; Edwards, J. P. J. Med. Chem. 2004, 47, 4799.
6. (a) Thurmond, R. L.; Sun, S.; Sehon, C. A.; Baker, S. M.; Cai, H.; Gu, Y.; Jiang, W.;
Riley, J. P.; Williams, K. N.; Edwards, J. P.; Karlsson, L. J. Pharmacol. Exp. Ther.
2004, 308, 268; (b) Gustin, D. J.; Sehon, C. A.; Wei, J.; Cai, H.; Meduna, S. P.;
Khatuya, H.; Sun, S.; Gu, Y.; Jiang, W.; Thurmond, R. L.; Karlsson, L.; Edwards, J.
P. Bioorg. Med. Chem. Lett. 2005, 15, 1687.
7. (a) Allen, D.; Ameriks, M. K.; Axe, F. U.; Burdett, M.; Cai, H.; Choong, I.; Edwards,
James P.; Lew, W.; Meduna, S. P. PCT Int. Appl. WO2008100635A1, 2008.; (b)
Allen, D.; Ameriks, M. K.; Axe, F. U.; Burdett, M.; Cai, H.; Choong, I.; Edwards, J.
P.; Lew, W.; Meduna, S. P. PCT Int. Appl. WO2008100620A2, 2008.
8. For recent reviews on fragment-based drug discovery, see: (a) Congreve, M.;
Chessari, G.; Tisi, D.; Woodhead, A. J. J. Med. Chem. 2008, 51, 3661; (b) Jahnke,
W., Erlanson, D. A., Eds. Fragment-Based Approaches in Drug Discovery. In
Methods and Principles in Medicinal Chemistry, Mannhold, R., Kubinyi, H.,
Folkers, G., Eds.; 2006; Vol. 34, Wiley-VCH: Weinheim, Germany.
9. Fragment-based screening has been utilized to identify CatS inhibitors: Wood,
W. J.; Patterson, A. W.; Tsuruoka, H.; Jain, R. K.; Ellman, J. A. J. Am. Chem. Soc.
2005, 127, 15521.
10. The term Tethering is a service mark of Sunesis Pharmaceuticals Inc. for its
fragment-based drug discovery: (a) Oslob, J. D.; Erlanson, D. A. Drug Discovery
Today 2004, 3, 143; (b) Erlanson, D. A.; Braisted, A. C.; Raphael, D. R.; Randal,
M.; Stroud, R. M.; Gordon, E. M.; Wells, J. A. Proc. Natl. Acad. Sci. U.S.A. 2000, 97,
9367.
Figure 4. Crystal structure of pyrimidinone 27 bound to Cys25Ser cathepsin S (PDB
code: 3MPE). A bridging water molecule (red) is observed in the back of the S2
subsite. Hydrogen bonds are shown in green.
general, the binding modes for pyrimidinone 27 and pyridazinone
12 (Fig. 3) are very similar, with both inhibitors spanning S3, S2,
and S5 and interacting with the protein through only one direct
hydrogen bond (Gly69). However, although a conserved water
molecule is still present in the back of the S2 pocket for pyrimidi-
none 27, it appears that the carbonyl group of the inhibitor did in-
deed displace the second water molecule observed in the crystal
structure of pyridazinone 12. Thus, it is possible that the eightfold
improvement in activity for 27 relative to 12 can be attributed to
several factors: (1) the entropic gain associated with the release
of a bound water,17 (2) a stronger donor–acceptor interaction be-
tween the bound water and the pyrimidinone carbonyl (2.9 Å)
compared to the pyridazinone nitrogen (3.6 Å), and (3) the fact that
the water molecule bound to 27 interacts directly with the back-
bone amides of Met71 and Thr72, unlike the water molecule bound
to 12 which can only interact with these residues through another
bridging water. It is also important to note that pyridazinone 22,
pyrimidinone 27, and pyrazinone 32 all show similar enzymatic
activity, suggesting that the position of the second diazinone nitro-
gen atom is not essential. This may be attributed to the fact that
these diazinones are all electron-poor heterocycles which appear
11. Turkenberg, J. P.; Lamers, M. B. A. C.; Brzozowski, A. M.; Wright, L. M.; Hubbard,
R. E.; Sturt, S. L.; Williams, D. H. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2002,
D58, 451.
12. CatS cysteine mutants were expressed in SF9 cells as histidine-tagged proteins.
Following purification over
a Ni–NTA column, the CatS mutants were
processed by 0.4 mg/mL pepsin, 100 mM NaOAc, pH 4.5, 5 mM DTT, 2.5 mM
EDTA for 2 h at 40 °C. Pepsin enzymatic activity was stopped by raising the pH
of the digestion to 7.5. Further purification was not required for screening,
since the molecular weight of pepsin is larger than CatS and does not interfere
with deconvolution of the fragment hits.
13. Monophore screening conditions: 10–15
lM
CatS mutant, 1 mM b-
mercaptoethanol, 50 M monophore (500 M total in pools of 10), 50 mM
l
l
Tris, pH 7.5. Monophore hits were identified by visual inspection of the
deconvoluted masses of the monophore-protein conjugates and then ranked
by fractional conjugation (the fraction of protein that remains conjugated to
the discrete monophores in the presence of increasing concentrations of b-
mercaptoethanol).
14. For a detailed description of the enzymatic assay, see: Allen, D.; Choong, I.;
Lew, W. PCT Int. Appl. WO2008100622A2, 2008.
to interact with the protein through a face-to-face
interaction with Phe211 in S2.
p
-stacking
15. The monophore corresponding to adduct 3 was non-covalently docked to the
structure of CatS in the area surrounding the E115C residue. Only binding
conformations that placed the sulfur of the linker within 5 Å of the cysteine
residue were retained. The monophores were then covalently attached as
disulfides to the cysteine mutant and minimization was attempted to match
each conformation from the non-covalent docking. The conformations were
then scored and the lowest energy conformation for adduct 3 is shown in
Figure 2.
In conclusion, a pyridazin-4-one fragment 4 (hCatS = 170
l
M)
discovered through Tethering was modeled into cathepsin S and
predicted to overlap in S2 with the tetrahydropyridinepyrazole
core of a previously disclosed series of CatS inhibitors. This frag-
ment served as
a template to design pyridazin-3-one 12
16. Coates, W. J.; McKillop, A. Synthesis 1993, 25, 334.
(hCatS = 430 nM), which also incorporates P3 and P5 binding
elements. A crystal structure of 12 bound to Cys25Ser CatS re-
vealed a pair of bound water molecules in the back of the S2 pocket
that facilitate a network of bridging interactions between the
inhibitor and the protein. The oxygen atom of pyrimidinone 27
(hCatS = 40 nM) is able to displace one of these bound waters,
resulting in an eightfold improvement in enzymatic activity.
17. (a) Barillari, C.; Taylor, J.; Essex, J. W. J. Am. Chem. Soc. 2007, 129, 2577; (b) Lu,
Y.; Chao-Yie, Y.; Wang, S. J. Chem. Inf. Model. 2007, 47, 668; (c) Hamelberg, D.;
McCammon, J. A. J. Am. Chem. Soc. 2004, 126, 7683.
18. Gong, Y.; He, W. Heterocycles 2004, 62, 851.
19. Kress, T. J. J. Org. Chem. 1985, 50, 3073.
20. TC7 cellular permeability data (Cerep, Inc.), Papp (10ꢁ6 cm/s): compound 22, A–
B = 1.3/B–A = 1.5; compound 27, A–B = 2.5/B–A = 2.9; compound 32, A–B = 0.6/
B–A = 3.7.