Development of peptidyl α-keto-β-aldehydes as new inhibitors of cathepsin L - Comparisons of potency and selectivity profiles with cathepsin B

Article abstract of DOI:10.1016/S0960-894X(00)00340-1

We have utilized previously known substrate and inhibitor specificity profiles for the lysosomal cysteine protease, cathepsin L, to design a new series of putative inhibitors of this enzyme, based on di- and tri-peptidyl α-keto-β-aldehydes. Kinetic evaluation of these compounds revealed Z-Phe-Tyr(OBut)-COCHO, with a K(i)=0.6 nM, to be the most potent, synthetic reversible inhibitor of cathepsin L reported to date. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00340-1

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1771±1773
Development of Peptidyl ꢀ-keto-ꢁ-aldehydes as New Inhibitors
of Cathepsin L Ð Comparisons of Potency and Selectivity Pro®les
with Cathepsin B
John F. Lynas,* Susan J. Hawthorne and Brian Walker
Division of Biomedicinal Chemistry, School of Pharmacy, Queen's University Belfast, Medical Biology Centre, 79 Lisburn Road,
Belfast BT9 7BL, Northern Ireland, UK
Received 15 March 2000; accepted 9 June 2000
AbstractÐWe have utilized previously known substrate and inhibitor speci®city pro®les for the lysosomal cysteine protease,
cathepsin L, to design a new series of putative inhibitors of this enzyme, based on di- and tri-peptidyl a-keto-b-aldehydes. Kinetic
evaluation of these compounds revealed Z-Phe-Tyr(OBut)-COCHO, with a Ki=0.6 nM, to be the most potent, synthetic reversible
inhibitor of cathepsin L reported to date. # 2000 Elsevier Science Ltd. All rights reserved.
Cathepsin L, a lysosomal cysteine protease has been
implicated in the progression of a number of important
pathophysiological processes, including rheumatoid
arthritis,1 tumour invasion and metastasis2 and bone
resorption and remodelling.3 In the latter process,
cathepsin L has been reported to degrade bone type I
collagen in tandem with another cysteine protease,
cathepsin K.4
aldehyde motif and evaluate such new compounds for
their selectivity for this protease versus cathepsin B. As
a result, we now wish to report the discovery of an
exceptionally potent, low molecular weight cathepsin L
inhibitor, Z-Phe-Tyr(OBut)-COCHO, which acts as a
slow, tight-binding reversible inhibitor of cathepsin L
with a Ki=0.6 nM. In addition, this compound shows
some 360-fold selectivity for cathepsin L compared with
cathepsin B.
A number of compounds have been shown to act as
inhibitors of cathepsin L, including diazomethyl
ketones,5 acyloxymethyl ketones,6 epoxysuccinyl deri-
vatives (such as E-64)7 and peptidyl aldehydes, such as
leupeptin.8 We have previously reported the inhibitory
activity of compounds analogous to peptidyl aldehydes,
termed peptidyl glyoxals (peptidyl a-keto-b-aldehydes)
which generally display enhanced potency and selectiv-
ity for the cysteine proteases.9
Chemistry
A representative synthetic protocol is outlined in Figure
1. It has been previously demonstrated that a-keto-b-
aldehydes can be easily obtained, in almost quantitative
yield, by the oxidative cleavage of the diazo-group of
peptidyl diazomethyl ketones, using dimethyldioxirane.10
The peptidyl diazoketones used in this study were pre-
pared, in turn, from their corresponding orthogonally
protected C-terminal free acids, using a rapid, combined
solid-/solution-phase methodology.
With this in mind, we decided to synthesize a number of
putative cathepsin L inhibitors based on the a-keto-b-
Inhibitor design was based on the simple use of known
peptide targeting sequences, previously shown to have
either selectivity for cathepsins L and B (Z-Phe-Tyr
(OBut)-11 and Ac-Arg-Arg-,12 respectively) or broader
spectrum speci®city for either protease (Z-Phe-Arg-12
and Ac-Leu-Leu-Arg-8). In brief, the desired target peptide
Abbreviations: Z-: benzyloxycarbonyl; -COCHO: a-keto-b-aldehyde;
DIPEA: N,N-diisopropylethylamine; Fmoc-: 9-¯uorenylmethoxycarbonyl;
HBTU: 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexa¯uoro-
phosphate; HOBt: 1-hydroxybenzotriazole; -NHMec: 7-amido-4-
methylcoumaryl; SASRIN: super acid sensitive resin.
*Corresponding author. Tel.: +44-028-9027-2047; fax: +44-028-
9024-7794; e-mail: j.lynas@qub.ac.uk
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00340-1

1772
J.F. Lynas et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1771±1773
Table 1. Comparison of the inhibition of cathepsins L and B by
peptidyl a-keto aldehydes
Enzyme
Cathepsin L
Cathepsin B
Ki (nM)a
Z-Phe-Tyr(OBut)-COCHO
Ac-Leu-Leu-Arg-COCHO
Ac-Arg-Arg-COCHO
Z-Phe-Arg-COCHO
0.6Æ0.054
970Æ85
N.I.b
90Æ8.7
214Æ22.5
1250Æ113
640Æ61
40Æ3.6
aValues are S.E.M for 4 determinations.
bN.I.=no inhibition (at 100 mM).
Figure 1. Solid-/solution-phase synthesis of Z-Phe-Tyr(OBut)-
COCHO. (i) 20% (v/v) piperidine/DMF, 45 min. (ii) Z-Phe-OH:HBTU:
HOBt:DIPEA (1:1:1:2), 1.5 h. (iii) 1% (v/v) TFA/DCM, 2Â30 min.
(iv) N-methylmorpholine (1.1 equiv), isobutyl chloroformate (1.1
equiv) at 0 ꢀC, 15 min, then add excess ethereal diazomethane, stir to
room temp. (v) dimethyldioxirane in moist acetone, 1.5 h.
Discussion
The peptidyl a-keto-b-aldehydes synthesized for the
present study contained previously described targeting
sequences which fell into three categories:
sequences were prepared (typically on a 0.25 mmol scale)
on a SASRIN1 polystyrene-based resin containing a super
acid-sensitive linker (2-methoxy-4-alkoxybenzyl alcohol)
employing standard Fmoc-/tBu-based solid phase pep-
tide synthesis methodologies.13 Upon completion of the
synthesis, the peptides were cleaved from the support by
treatment with tri¯uoroacetic acid in dichloromethane
(1%, v/v).
1. selective for cathepsin
COCHO)
2. selective for cathepsin B (Ac-Arg-Arg-COCHO)
3. exhibiting activity against both cathepsin B and L
L
(Z-Phe-Tyr(OBut)-
(Ac-Leu-Leu-Arg-COCHO
COCHO)
and
Z-Phe-Arg-
Such an approach was developed to examine if peptidyl
a-keto-b-aldehydes exhibited conventional binding
characteristics and as such could be utilized for the spe-
ci®c inhibition of cathepsin L.
After neutralization and standard work up, the peptide
free acids were then converted into the corresponding
diazomethyl ketone derivatives by reaction of their
unsymmetrical anhydrides (formed by reaction with
isobutyl chloroformate) with ethereal diazomethane.14
Finally, the peptidyl diazoketones were treated with a
solution of dimethyldioxirane in moist acetone10 (for
preparation of a-keto aldehydes). The identity and purity
of the ®nal products were con®rmed by a combination
A number of features are immediately apparent from
the ®nal Ki values presented in Table 1. First, with the
exception of Ac-Leu-Leu-Arg-COCHO, the remainder
of the peptidyl a-keto-b-aldehydes prepared in this
study exhibit sub-micromolar ®nal Ki values against
both cathepsins B and L. Secondly, the compounds
behaved as expected with regard to their substrate spe-
ci®city. Ac-Arg-Arg-COCHO had no activity against
cathepsin L, even at concentrations as high as 100 mM.
Ac-Leu-Leu-Arg-COCHO was the least potent and
selective inhibitor, which was somewhat surprising as
the analogous compound leupeptin is a good inhibitor
of both proteases. Initially, we believed this may be due
to cyclization of the C-terminal a-keto-b-aldehyde moi-
ety with the side chain guanidino group of Arg, however
subsequent NMR and mass spectral analysis have not
shown this to be the case (results not shown). In addi-
tion, the observation that, as expected, Ac-Arg-Arg-
possesses activity against cathepsin B (but not cathepsin
L) and Z-Phe-Arg- exhibits activity against both species
suggests that it is unlikely that cyclization occurs in
assay bu€ers, as this would render the inhibitors inac-
tive.
1
of TLC, H NMR and electrospray mass spectrometry.
Invariably, the peptidyl a-keto-b-aldehydes were
obtained as their hydrates.
Inhibition Studies
Recombinant human cathepsin L was a kind gift from
Dr Maurice Pagano (Faculte de Medecine, Universite
Pierre et Marie Curie, Paris, France). Bovine cathepsin B,
Z-Arg-Arg-NHMec and Z-Phe-Arg-NHMec were pur-
chased from Sigma Chemical Co. (Poole, Dorset, UK). All
other reagents were of analytical grade and were pur-
chased from either Sigma or Aldrich (Poole, Dorset, UK).
Compounds were examined for their ability to block
cathepsin B- or cathepsin L-catalysed hydrolysis of the
¯uorogenic substrates, Z-Arg-Arg-NHMec and Z-Phe-
Arg-NHMec, respectively, in the presence of varying
amounts of the peptidyl a-keto-b-aldehydes. Progress
curves obtained from these studies were typical of inhi-
bitors exhibiting reversible, slow-binding kinetics.15,16 A
comparison of the ®nal Ki values determined for both of
the peptidyl a-keto aldehyde sequences synthesized in
the present study, against cathepsins L and B are shown
in Table 1.
We believe that P0 hydrogen-bonding interactions,
between active site residues and the hydrated b-alde-
hyde hydroxyl groups of peptidyl a-keto-b-aldehydes,
are crucial for the e€ectiveness of these compounds. In
the absence of crystallographic/NMR data of cathepsin-
a-keto-b-aldehyde complexes, we would wish to draw
some tentative comparisons, for example, with vinyl
sulphone and vinyl ester-based inhibitors where the

J.F. Lynas et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1771±1773
1773
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Figure 2. Comparison of the structures of cysteine protease inhibitors.
importance of P0 hydrogen bonding interactions has
previously been demonstrated. Vinyl sulphone-based
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of which have been shown to interact with active site
residues close to the catalytic apparatus, are sub-
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esters.18 The fact that vinyl esters have only one car-
bonyl oxygen capable of interacting with P0 site residues
may explain the reduced potency of these compounds
(Fig. 2). In addition to hydrogen-bonding potential of
the b-hydroxyl groups of the hydrated b-aldehyde, the
high potency of a-keto-b-aldehydes may be attributable
to the presence of the a-carbonyl group, and its prob-
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the oxyanion binding site.
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