Dipeptidyl nitrile inhibitors of Cathepsin L

Article abstract of DOI:10.1016/j.bmcl.2009.05.071

A series of potent Cathepsin L inhibitors with good selectivity with respect to other cysteine Cathepsins is described and SAR is discussed with reference to the crystal structure of a protein-ligand complex.

Full text of DOI:10.1016/j.bmcl.2009.05.071

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4280–4283
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Dipeptidyl nitrile inhibitors of Cathepsin L
Nabil Asaad a, Paul A. Bethel a, Michelle D. Coulson b, Jack E. Dawson a, Susannah J. Ford a, Stefan Gerhardt a,
Matthew Grist a, Gordon A. Hamlin a, Michael J. James a, Emma V. Jones a, Galith I. Karoutchi a,
a,
a
a
a
a
Peter W. Kenny a, , Andrew D. Morley , Keith Oldham , Neil Rankine , David Ryan , Stuart L. Wells ,
*
*
Linda Wood a, Martin Augustin c, Stephan Krapp c, Hannes Simader c, Stefan Steinbacher c
a Respiratory & Inflammation Research Area, AstraZeneca, Mereside, Alderley Park, Macclesfield SK10 4TG, UK
b Safety Assessment, AstraZeneca, Mereside, Alderley Park, Macclesfield SK10 4TG, UK
c Proteros Biostructures, Am Klopferspitz 19, D-82152 Martinsried, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of potent Cathepsin L inhibitors with good selectivity with respect to other cysteine Cathepsins is
described and SAR is discussed with reference to the crystal structure of a protein-ligand complex.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 20 April 2009
Revised 20 May 2009
Accepted 21 May 2009
Available online 27 May 2009
Keywords:
Cathepsin
Cathepsin B
Cathepsin K
Cathepsin L
Cathepsin L2
Cathepsin S
Cathepsin V
Cysteine protease
Nitrile
Covalent inhibitor
Selectivity
Protein structure
X-ray crystallography
Structure-based design
Molecular pincer
Conformational lock
Tautomeric lock
Osteoarthritis
Aggrecan
Collagen
Cartilage
Osteoarthritis is a chronic degenerative disease, resulting from
loss of articular cartilage and damage to underlying bone, leading
to joint instability and pain.1 The lysosomal cysteine protease2
Cathepsin L (CatL) is seen as a potential target for intervention in
treatment of this disease.3 The enzyme is secreted into the extra-
cellular matrix and has been shown in vitro that it can degrade
proteoglycans, including aggrecan4 and type II collagen,5 the major
components of articular cartilage.
Selectivity with respect to other Cathepsins is an important
consideration. Cathepsins B (CatB) and L2 (CatL2) are key anti-tar-
gets in optimization of CatL inhibitors. In mice the combined defi-
ciency of CatB and CatL has been shown to be lethal in the second
to fourth week of life.6 CatLÀ/À mice show abnormalities in skin
and hair differentiation.7 The human genome encodes a CatL-like
cysteine protease (CatL2), which shows a restricted tissue distribu-
tion and is not present in the mouse genome. Studies using CatLÀ/À
mice showed that transgenic, keratinocyte-specific expression of
human CatL2 largely rescued the skin and hair phenotype, indicat-
ing that this protease may play an important role in human skin
homeostasis.8 Both CatL and CatS have roles in MHC class II-med-
iated antigen processing and presentation and may compensate for
* Corresponding authors. Tel.: +44 1625 514396 (P.W.K.), +44 1509 644772
(A.D.M.).
E-mail addresses: pwk.pub.2008@gmail.com (P.W. Kenny), andy.morley@astra
zeneca.com (A.D. Morley).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.071

N. Asaad et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4280–4283
4281
Table 1
SAR for aromatic and cycloalkyl substituents
O
O
Ph
NH
H
N
CN
R
NH
H
N
CN
O
1
4
O
pIC50 = 6.6
ClogP = 2.1
Cl
1
Cl
5
6
Figure 1. Potential binding mode of the adduct of 1 with Cathepsin L showing
molecular surface of protein and locations of key residues. The covalent bond is
formed between the nitrile carbon atom and the sulfur atom of the catalytic
cysteine (Cys25; hidden by surface).
Compd ClogPa CatL
CatL2
pIC50
CatS
pIC50
CatK
pIC50
CatB
pIC50
b
b
b
b
b
pIC50
1
4
5
6
2.1
2.5
2.3
3.0
6.6
5.9
6.3
6.5
6.1
5.2
6.4
5.2
6.9
5.6
5.7
5.6
5.5
<5
<5
5.3
<5
<5
<5
5.3
each other, suggesting that selectivity with respect to CatS is desir-
able in CatL inhibitors.9
a
See Ref. 14.
b
Values are means of at least three experiments; see Refs. 10,15–17.
Directed screening led to the identification of 1 as an inhibi-
10
tor of CatL with pIC50 of 6.6. This compound is >10-fold selec-
tive with respect to CatB and CatK but shows no selectivity with
respect to CatS or CatL2. Close structural analogues of 1 have
been described previously as CatL inhibitors and show similar
selectivities with respect to CatK and CatS.11 The binding of 1
to CatL was modeled (Fig. 1) with a covalent bond between ni-
trile carbon and active site cysteine sulfur.12,13 The 3-chloro-
phenyl substituent occupies the S2 pocket which is deeper in
CatL than in CatK. The phenyl ring of the capping group lies in
the shallow S3 pocket, making contact with Leu69, although
the interaction appears suboptimal. The corresponding residues
in other Cathepsins are tyrosine (CatK, CatB) or phenylalanine
(CatL2, CatS). Leu69 also makes contact with the chlorophenyl
ring and is held in what can be termed a ‘molecular pincer’.
The carbonyl oxygen of Gly68 accepts a hydrogen bond from
the benzamide donor.
Synthesis of compounds is summarised in Scheme 1. Esterifica-
tion of the commercial 3-chlorophenylalanine followed by stan-
dard amide coupling chemistry furnished 2. Ester hydrolysis
followed by concentration of the reaction mixture and subsequent
amide coupling of the crude lithium salts with aminoacetonitrile
provided good yields of the target compounds. The alternative, of
introducing diversity in the final step by acylating 3, resulted in
low yields and challenging purification.
Table 2
SAR for phenyl substitution
O
NH
H
N
R
CN
O
Cl
Compd
R
ClogPa CatL
CatL2
pIC50
CatS
pIC50
CatK
pIC50
CatB
pIC50
b
b
b
b
b
pIC50
1
7
8
9
10
11
H
2.1
2.6
2.6
2.3
3.0
2.8
6.6
6.6
6.6
6.1
7.1
7.1
6.1
6.0
6.1
5.3
6.7
5.8
6.9
6.2
6.4
5.8
6.7
5.9
5.5
5.9
5.2
5.3
5.2
5.2
5.3
5.6
5.3
<5
5.4
5.3
4-Me
3-Me
2-Me
3-Cl
2,5-
diMe
2,3-
diMe
3,5-
12
13
2.7
3.1
6.5
7.3
5.4
6.7
5.7
6.5
<5
<5
5.3
5.1
diMe
a
See Ref. 14.
b
Values are means of at least three experiments; see Refs. 10,15–17.
Structure activity relationships (SARs) for CatL inhibitors are
presented in Tables 1–5. Replacement of phenyl in 1 with the more
lipophilic cyclohexyl (4) leads to a 0.7 drop in pIC50 (Table 1). This
may reflect differences in dihedral angle preference for the bonds
linking substituent and carbonyl groups. The larger and more
Table 3
SAR for bicyclic substitiuents
O
Ar
NH
H
N
CN
O
O
14
NH2
O
15
O
R
NH
R
NH
OH
H
N
O
c, d
a, b
CN
Cl
N
O
O
16
17
Cl
Cl
Compd ClogPa CatL
CatL2
pIC50
CatS
pIC50
CatK
pIC50
CatB
pIC50
Cl
b
b
b
b
b
pIC50
2
14
15
16
17
3.3
3.7
2.7
4.3
6.6
6.4
7.0
7.7
5.4
<5
5.5
6.0
5.5
<5
5.5
6.0
<5
<5
<5
<5
5
BOCHN
<5
<5
<5
NH2
O
H
N
d, e
OH
CN
b
O
a
See Ref. 14.
b
Values are means of at least three experiments; see Refs. 10,15–17.
Cl
Cl
3
lipophilic cycloheptyl analogue (6), is nearly as active as 1 and
Scheme 1. Reagents: (a) 10 M HCl/MeOH; (b) Acid, HATU, DIPEA, DMF; (c) LiOH,
MeOH/H2O/THF; (d) H2NCH2CN, HATU, DIPEA, DMF; (e) Formic acid.
shows improved selectivity with respect to CatL2.

4282
N. Asaad et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4280–4283
Table 4
can lead to significant reductions in lipophilicity with minimal loss
SAR for heteroaryl substituents
of potency. Analogues of 19 have been described as Cathepsin
inhibitors in the patent literature.18
O
Increasing the size of the substituent at pyrazole C3 in 19 leads
to an increase in CatL potency (Table 5). The CatL pIC50 value of 25
exceeds that of 1 by 1.6 units. CatL2 (DpIC50 = 0.5) and CatS
Ar
NH
N
N
H
N
CN
N N
18
19
O
(
D
pIC50 = À0.1) are much less sensitive to this structural change
N
S
with the result of an improved selectivity profile relative to 1.
Compound 25 is as least as selective with respect to CatB and CatS
as previously described CatL inhibitors.3
O
S
22
Cl
N
21
20
Compd ClogPa CatL
CatL2
pIC50
CatS
pIC50
CatK
pIC50
CatB
pIC50
A weak correlation (
r = 0.41) was observed between CatL pIC50
b
b
b
b
b
and ClogP (Fig. 2) for these inhibitors. The correlations of CatL2 and
CatS inhibition with lipophilicity are much weaker, which reflects
the presence of CatL selective inhibitors in the data set. We believe
that Figure 2, in which selectivity is overlaid onto potency-lipo-
philicity relationships, represents a useful and general approach
to summarizing results from selectivity assays.
pIC50
18
19
20
21
22
0.6
0.9
0.7
1.9
2.6
5.9
6.3
6.2
6.4
7.1
5.9
5.9
5.6
6.6
6.3
5.9
6.1
6.1
6.7
6.1
5.6
5.2
5.0
5.9
5.3
<5
<5
<5
5.0
5.7
a
The crystal structure19–21 (Fig. 3) of 26 bound to CatL provides a
rationale for the SAR observed for 19, 23, 24 and 25. The 1-methyl
group of the pyrazole makes some contact with Leu69 and appears
to force the pyrazole ring out of co-planarity with the amide, prob-
ably functioning as a conformational lock.22,23 Less obviously, the
1-methyl group may also function as a tautomeric lock because
See Ref. 14.
b
Values are means of at least three experiments; see Refs. 10,15–17.
Table 5
3-Substituted pyrazole SAR analogues
O
N
NH
N
H
N
CN
R
O
X
Compd
R
X
ClogPa CatL
CatL2
pIC50
CatS
pIC50
CatK
pIC50
CatB
pIC50
b
b
b
b
b
pIC50
19
23
24
25
26
Me Cl
0.9
1.4
1.8
2.2
6.3
7.2
7.1
7.9
7.9
5.9
6.3
6.3
6.7
6.4
6.1
6.1
6.1
6.0
6.1
5.2
5.3
5.7
5.5
5.6
<5
Et
iPr
Cl
Cl
5.1
5.3
5.2
5.4
tBu Cl
tBu Me 2.0
a
See Ref. 14.
b
Values are means of at least three experiments; see Refs. 10,15–17.
Substitution of the phenyl ring of 1 with a single chloro or
methyl group led to increases in CatL pIC50 of no more than 0.5
(Table 2). The most interesting profile is that shown by 11 in which
the phenyl ring is substituted with methyl groups at the 2 and 5
positions. This compound is more than 10-fold selective against
both CatL2 and CatS. Contact between the 5-methyl substituent
and protein surface restricts the torsional space for the carbonyl–
phenyl bond in the bound state and effectively forces the 2-methyl
into contact with Leu69. The equivalents of Leu69 in CatL2 and
CatS are more sterically demanding aromatic amino acids and
the dimethyl-substitutions are deleterious for anti-target potency,
especially CatS.
Figure 2. Relationships between pIC50 and ClogP for Cathepsins L, L2 and
showing comparisons for 1 (red), 17 (blue) and 25 (green). Ellipses correspond to
probability of 90%.
S
The SAR of 2,3-disubstituted phenyl rings was explored further
using bicyclic aromatic systems (Table 3). Compounds 14 and 15
are equipotent with 12 although 15 shows a better selectivity pro-
file. Aza-substitution of 14 at C4 actually results in a small increase
in potency, which reflects likely exposure of this position to solvent
in the bound state. The 2,3 and 2,5 substitution patterns of 11 and
12 are brought together in 17. This compound is more potent than
analogues 11–14 and shows better than 50-fold selectivity with re-
spect to the other Cathepsins.
The improvements in potency and selectivity of 17 relative to 1
were achieved at the cost of increasing lipophilicity (ClogP) by >2
units. The results presented in Table 4 show that substituting
five-membered heteroaromatic rings (e.g., 19 and 20) for phenyl
Figure 3. Crystal structure of 26 bound to Cathepsin L showing molecular surface of
the protein and locations of key residues. The covalent bond is formed between the
nitrile carbon atom and the sulfur atom of the catalytic cysteine (Cys25; hidden by
surface).

N. Asaad et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4280–4283
9. Nakagawa, T. Y.; Rudensky, A. Y. Immunol. Rev. 1999, 172, 121.
4283
the more stable tautomer24 of the des-methyl analog is likely to be
10. pIC50 = Àlog(IC50/M).
the one with the imino nitrogen next to the amide linker.25
All three methyl groups of the t-butyl substituent make contact
with the protein. The similarity of the pIC50 values for 23 (7.2) and
24 (7.1) suggests that these three methyl groups do not contribute
equally to potency. One rationale for the observed trend in potency
is that the contribution to binding of one of the methyl groups is
primarily conformational.
11. Lser, R.; Schilling, K.; Dimmig, E.; Gtschow, M. J. Med. Chem. 2005, 48, 7688.
12. Guncar, G.; Pungercic, G.; Klemencic, I.; Turk, V.; Turk, D. EMBO J. 1999, 18, 793.
13. The model of the binding mode for
1 was generated from the RSB
(home.rcsb.org) Protein Data Bank (www.rcsb.org/pdb/home/home.do) entry
with reference code 1ICF (see Ref. 10) using the Maestro molecular modelling
program. The structure was energy-minimized using MacroModel (OPLS 2005
force field; water solvent model). Both Maestro and MacroModel were licensed
from Schrödinger, LLC (www.schrodinger.com).
14. ClogP, Version 4.3, BioByte (www.biobyte.com).
In conclusion, we have discussed selectivity requirements for
safe Cathepsin L inhibitors and shown how variation of the S3 sub-
stituent can be used to modulate potency and selectivity.
15. Values of pIC50 for inhibition of Cathepsins L, L2, S, K and B were determined
from dose dependent inhibition of cleavage of fluorogenic, AMC-tagged,
peptide substrates. See also Refs. 16,17.
16. Hulkower, K. I.; Butler, C. C.; Linebaugh, B. E.; Klaus, J. L.; Keppler, D.; Giranda,
V. L.; Sloane, B. F. Eur. J. Biochem. 2000, 267, 4165.
Acknowledgments
17. Werle, B.; Staib, A.; Jülke, B.; Ebert, W.; Zladoidsky, P.; Sekernik, A.; Kos, J.;
Speiss, E. Biol. Chem. 1999, 380, 1109.
18. Altmann, E.; Betschart, C.; Gohda, K.; Horiuchi, M.; Lattmann, R.; Missbach, M.;
Sakaki, J.; Takai, M.; Teno, N.; Cowen, S. D.; Greenspan, P. D.; McQuire, L. W.;
Tommasi, R. A.; Van Duzer, J. H. 1999, WO 9924460.
We thank Lyn Rosenbrier Ribeiro and Helen Sawney for sharing
assay development expertise.
19. Diffraction data for compound 26 were collected at 100 K on beamline PX at
the Swiss Light Source (SLS, Villigen, Switzerland). The structure was solved by
molecular replacement and refined to a final resolution of 1.27 Å and R-factor
of 11.6% using the CCP4 (Ref. 19) and Coot software (Ref. 20) packages. This
structure has been deposited in the RSB (home.rcsb.org) Protein Data Bank
(www.rcsb.org/pdb/home/home.do) with reference code 3HHA.
20. Collaborative Computatonal Project, Number 4; (http://www.ccp4.ac.uk) Acta
Crystallogr., Sect. D 1994, 50, 760.
References and notes
1. Goldring, M. B.; Goldring, S. R.. J. Cell Physiol. 2007, 213, 626.
2. Vasiljeva, O.; Reinheckel, T.; Peters, C.; Turk, D.; Turk, V.; Turk, B. Curr. Pharm.
Des. 2007, 13, 387.
3. Marquis, R. W.; James, I.; Zeng, J.; Trout, R. E. L.; Thompson, S.; Rahman, A.;
Yamashita, D. S.; Xie, R.; Ru, Y.; Gress, C. J.; Blake, S.; Lark, M. A.; Hwang, S.-M.;
Tomaszek, T.; Offen, P.; Head, M. S.; Cummings, M. D.; Veber, D. F. J. Med. Chem.
2005, 48, 6870.
4. Nguyen, Q.; Mort, J. S.; Roughley, P. J. Biochem. J. 1990, 266, 569.
5. Maciewicz, R. A.; Wotton, S. F.; Etherington, D. J.; Duance, V. C. FEBS Lett. 1990,
269, 189.
6. Felbor, U.; Kessler, B.; Mothes, W.; Ploegh, H. L.; Goebel, H. H.; Bronson, R. T.;
Olsen, B. R. Proc. Natl. Acad. Sci. 2002, 99, 7883.
21. Coot: http://www.ysbl.york.ac.uk/~emsley/coot.
22. Black, E.; Breed, J.; Breeze, A. L.; Embrey, K.; Garcia, R.; Gero, T. W.;
Godfrey, L.; Kenny, P. W.; Morley, A. D.; Minshull, C. A.; Pannifer, A. D.;
Read, J.; Rees, A.; Russell, D. J.; Toader, D.; Tucker, J. Bioorg. Med. Chem.
Lett. 2005, 15, 2503.
23. Boström, J.; Grant, J. A. Exploiting Ligand Conformations in Drug Design. In
Drug Properties: Measurement and Computation; Mannhold, R., Kubinyi, H.,
Folkers, G., Eds.; Wiley-VCH, 2007; p 183.
7. Reinheckel, T.; Hagemann, S.; Dollwet-Mack, S.; Martinez, E.; Lohmüller, T.;
Zlatkovic, G.; Tobin, D. J.; Maas-Szabowski, N.;Peters, C. J. CellSci. 2005, 118, 3387.
8. Hagemann, S.; Günther, T.; Dennemarker, J.; Lohmüller, T.; Brömme, D.; Schüle,
R.; Peters, C.; Reinheckel, T. Eur. J. Cell Biol. 2004, 83, 775.
24. Elguero, J.; Marzin, C.; Katritzky, A. R.; Linda, P. The Tautomerism of
Heterocycles; Academic Press: New York, 1976.
25. Claramunt, R. M.; García, M. A.; López, C.; Trofimenko, S.; Yap, G. P. A.; Alkorta,
I.; Elguero, J. Magn. Reson. Chem. 2005, 43, 89.