Azadipeptide nitriles: Highly potent and proteolytically stable inhibitors of papain-like cysteine proteases

Article abstract of DOI:10.1002/anie.200705858

(Chemical Presented) Nitrogen instead of carbon: Azadipeptide nitriles resulting from CH/N exchange in the P¹ position (see picture) are hitherto unknown. To access these compounds by conversion of amino acid-derived hydrazides with cyanogen bromide both nitrogen atoms of the hydrazide must be substituted. Despite a methylated P²-P¹ peptide bond, the azadipeptide nitriles show a strong inhibitory activity against cysteine proteases, and a high stability towards chymotryptic hydrolysis.

Full text of DOI:10.1002/anie.200705858

Angewandte
Chemie
DOI: 10.1002/anie.200705858
Enzyme Inhibitors
Azadipeptide Nitriles: Highly Potent and Proteolytically Stable
Inhibitors of Papain-Like Cysteine Proteases**
Reik Löser, Maxim Frizler, Klaus Schilling, and Michael Gütschow*
Cysteine proteases of the clan CA, the papain-like cysteine
proteases, are of outstanding medical interest owing to their
importance as virulence factors in different human patho-
genic parasites and their involvement in a number of different
systemic diseases. Apart from the active-site thiol, these
proteases share pronounced substrate specificity towards the
amino acid in the P2 position. Eleven human papain-like
lysosomal cysteine proteases with a high degree of homology,
the cathepsins, have been described.[1] Among them, the
cathepsins L, S, and K are involved in several pathological
conditions, such as tumor growth and invasion, autoimmune
diseases, and osteoporosis,[2] and are currently in the focus of
many drug discovery programs.[3] Inhibitors for papain-like
cysteine proteases are mainly derived from peptides and
contain electrophilic entities prone to covalent interactions
with the thiol at the active site. Among this generic type of
inhibitor, peptide nitriles have received much attention in
recent research.[4] Nitriles inhibit cysteine proteases by the
formation of a thioimidate adduct resulting from the attack of
cally closely related to nitriles, have been described as
powerful cathepsin K inhibitors.[8] We have recently found
that the replacement of the CaH group by a nitrogen atom in
the P2 position of a cathepsin-inhibiting dipeptide nitrile led
to the loss of inhibitory capacity.[9] As part of our ongoing
interest in cysteine protease inhibition, it was intended to
explore the impact of the CH/N exchange in the P1 position of
dipeptide nitriles on the enzyme–inhibitor interaction. Herein
we describe a synthetic entry to azadipeptide nitriles and their
inhibition kinetics and binding affinity towards papain and
the therapeutically important cathepsins L, S, and K.
It was envisaged that the synthesis of the aza-analogous
nitriles could be achieved by reaction of the corresponding
amino acid-derived hydrazides (Scheme 1) with cyanogen
bromide. This approach was initially unsuccessful: in the case
the active-site thiol at the C N-triple bond,[5] and, owing to a
À
soft–soft relationship according to the HSAB principle, they
are therefore potent and selective inhibitors for cysteine
proteases. Peptidic inhibitors however, generally exhibit low
bioavailability because of their susceptibility to degradation
by other proteases.
Scheme 1. Hydrazides 1–4 and carbadipeptide nitriles 11–14.
The isoelectronic replacement of the CaH group by a
nitrogen atom to give azapeptides is a common structural
modification in the chemistry of peptides and peptidomimet-
ics.[6] In contrast to other protease inhibitors,[7] this structural
modification has never been applied to the P1 position of
peptide nitriles. Nonpeptidic cyanamides, which are chemi-
of the unsubstituted and monomethylated hydrazides 1–3, the
formation of various cyclization products was observed. We
therefore considered N1,N2-dimethylation to prevent cycliza-
tion and, indeed, treatment of the hydrazide 4 with cyanogen
bromide led to the desired open-chain azadipeptide nitrile 6
(Scheme 2). Compound 6, the first representative of azadi-
peptide nitriles resulting from a CH/N replacement in the P1
position, showed inhibitory activity towards papain in the low
nanomolar range, and was even more potent towards the
therapeutically important cathepsins L, S, and K, with pico-
molar Ki values (Table 1). This unexpectedly strong inhibition
was time-dependent in terms of slow-binding inhibition,[10] as
shown in Figure 1. The linear plots of the pseudo-first-order
rate constants versus inhibitor concentration suggest that the
enzyme–inhibitor interaction follows a one-step mechanism
(see the Supporting Information). The second-order rate
[*] Dr. R. Löser,[+] M. Frizler, Prof. Dr. M. Gütschow
Pharmazeutisches Institut, Pharmazeutische Chemie I
Rheinische Friedrich-Wilhelms-Universität
An der Immenburg 4, 53121 Bonn (Germany)
Fax: (+49)228-73-2567
E-mail: guetschow@uni-bonn.de
Dr. K. Schilling
Institut für Biochemie I, Klinikum
Friedrich-Schiller-Universität
Nonnenplan 2, 07743 Jena (Germany)
[+] present address: Institute for Molecular Bioscience, The University
ofQueensland, 306 Carmody Road, Brisbane Qld 4072 (Australia).
[**] This research was supported by the Deutsche Forschungsgemein-
schaft (GRK 804 “Analyse von Zellfunktionen durch kombinatori-
sche Chemie und Biochemie”). We wish to thank Prof. D. Brömme,
Vancouver, for providing cathepsin K, and Dr. J. Abbenante,
Brisbane, for critically reading the manuscript.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
Scheme 2. Synthesis ofazadipeptide nitriles 6 and 7. a) BrCN, NaOAc,
MeOH, RT. Bn=benzyl.
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Figure 1. Monitoring ofthe human cathepsin L-catalyzed hydrolysis of
Z-Phe-Arg-NHMec (10 mm; NHMec=4-methylcoumarin-7-yl) in the
presence ofincreasing concentrations ofcompound 6 (from top to
bottom: 0, 3, 4, 8, 9, 10, 20, 30, 50, 100 nm). The reaction (100 mm
sodium phosphate pH 6.0, 100 mm NaCl, 5 mm ethylenediamine
tetraacetate, 0.01% v/v polyoxyethylene(23) lauryl ether (Brij 35),
25 mm 1,4-dithiothreitol, 1% DMSO, 378C) was initiated by addition of
the enzyme (I=fluorescence intensity, FU=fluorescence units).
constants kon for the approach to the enzyme–inhibitor
equilibrium were in the range of 105–107 mÀ1 sÀ1, indicating a
rather fast association of the enzyme with the inhibitor
2
1
À
(Table 1). In spite of the N-methylated P P amide bond,
which is generally disadvantageous for the interaction of
proteases with substrates or inhibitors,[11] the azadipeptide
nitrile 6 showed affinities to the papain-like enzymes that are
higher by at least two orders of magnitude than those of the
corresponding carba-analogue 11[9] (Scheme 1, Table 1).
Although having high affinities to the enzymes, compound 6
is clearly a reversible inhibitor, as demonstrated by the
reactivation of cathepsin L and papain (see the Supporting
Information).
To comparatively assess the activity of the azadipeptide
nitrile 6 more precisely, the carba-analogues 12, 13, and 14
(Scheme 1) were synthesized by standard methods (see the
Supporting Information). The nitriles 12 and 14 were
prepared from the corresponding dipeptide amides by
dehydration with cyanuric chloride in DMF.[12] As shown in
Table 1, the N-methylated carba-analogues 13 and 14 exhib-
ited activities in the high micromolar range. Comparing the
direct analogues 6 and 14, it becomes obvious that the
isoelectronic CH/N replacement accounts for an increase in
the inhibitory activities by more than five orders of magni-
tude.
By applying the same synthetic methodology as for the
azadipeptide nitrile 6, the leucine derivative 7 was obtained
(Scheme 2), which showed selectivity for cathepsin K with a
Ki value of 29 pm (Table 1). To introduce substituents other
than methyl into the P1 position, it was intended to convert
the N1-methylated hydrazide 3 by reductive alkylation into
N1,N2-dialkylated hydrazides, followed by treatment with
cyanogen bromide (Scheme 3). By reacting 3 with different
aldehydes, hydrazones were obtained in good yields. To
À
reduce the C N double bond of these intermediates, several
established reducing agents were tried: sodium triacetoxy-
borohydride,[13] sodium cyanoborohydride,[14] sodium borohy-
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Chemie
dramatically reduced in comparison to the corresponding
values of their carba-analogues. This reduction can be under-
stood by considering that in the azadipeptide nitriles, the
cyano group is attached to a nitrogen atom having an electron
lone pair. Upon attack of the active-site thiol, a trigonal-
planar isothiosemicarbazide adduct is formed from the
cyanamide-like carbon atom. The enhanced stability of such
a covalent enzyme–inhibitor complex may arise from the
resonance of the nitrogen atom lone pair and the sp2-
hybridized carbon atom derived from the cyano group
(Scheme 4).
Scheme 3. Synthesis ofazadipeptide nitriles 8–10. a) RCHO, THF, RT;
b) 1. (CH3)2NH”BH3, p-toluenesulfonic acid, CH2Cl2, 48C; 2. 1.5m
NaOH, RT; c) BrCN, NaOAc, MeOH, RT.
dride, and sodium borohydride in the presence of Ni2+ as
activating azaphilic Lewis acid.[15] All these attempts failed,
mostly leading to isolation of the unconverted hydrazones. By
applying an alternative reducing agent, dimethylamine–
borane complex, recently described by Casarini et al. for the
1,2-reduction of a,b-unsaturated hydrazones,[16] an almost
quantitative and smooth reduction of the hydrazones to the
N1-methyl-N2-alkylhydrazides was achieved. Upon treatment
with cyanogen bromide, the phenylalanine-derived N1-
methyl-N2-alkylhydrazides could be easily converted into
the desired azadipeptide nitriles 8–10 bearing different
residues in the P1 position. With the exception of inhibition
of cathepsin S by compound 8, all cathepsins were inhibited
by 8–10 with subnanomolar affinities, although these com-
pounds were less active than 6. The introduction of residues
other than methyl into the P1 position of the azadipeptide
nitriles did not lead to selective inhibition among the
cathepsins, and especially not between cathepsin L and S.
However, to draw conclusions regarding the impact of the P1
substituent on selectivity, a greater number of analogues need
to be made in future investigations. This approach is very
promising, as a large variety of aldehydes available by
different synthetic procedures can be applied.
The azadipeptide nitriles showed a time-dependent slow-
binding inhibition towards all investigated cysteine proteases
(Table 1, Figure 1). Unexpectedly, their binding affinity was
higher by five orders of magnitude in comparison to their
carba-analogues. This phenomenon can be understood from
the time-dependency of the inhibition by the azadipeptide
nitriles. Generally, slow-binding inhibition allows the deter-
mination of the kinetic constants kon and koff.[10] The second-
order rate constant kon governs the association of the enzyme
and the inhibitor to the enzyme–inhibitor complex, and koff
the decay of that complex. As the carba-analogous peptide
nitriles showed a time-independent inhibition (see the
Supporting Information), it can be concluded that they
exhibit higher second-order rate constants kon than their
nitrogen counterparts. In spite of this, the azadipeptide
nitriles had lower inhibition constants than the carbadipep-
tide nitriles. Because of the relation Ki = koff/kon, the first-
order rate constants koff of the azadipeptide nitriles have to be
Scheme 4. Reversible formation of isothiosemicarbazides from azadi-
peptide nitriles and cysteine proteases.
As the amide bond in these novel dipeptide derivatives is
N-methylated, an enhanced stability towards degradation by
proteolytic enzymes was assumed.[17] To test the stability, the
cleavage of the Phe-derived azadipeptide nitrile 6 and its
carba-analogues 12 and 14 catalyzed by chymotrypsin, a
serine protease which shows distinct specificity for phenyl-
alanine in the P1 position, was studied. Compounds 6, 12, and
14 were initially tested for inhibition of the chymotrypsin-
catalyzed conversion of a chromogenic peptide using an
enzyme concentration of 10 ngmLÀ1 (see the Supporting
Information). The azadipeptide nitrile 6 showed only poor
inhibition (IC50 = 1300 mm), and 12 and 14, at a concentration
of 600 mm, did not inhibit chymotrypsin at all. Next, the
stability of 6, 12, and 14 towards chymotrypsin was examined
by HPLC analysis (Figure 2). The nonmethylated carba-
analogue 12 was degraded with a half-life of 45 min at a rather
high chymotrypsin concentration of 100 mgmLÀ1. A similar
concentration of chymotrypsin is present in the human small
intestine.[18] At the same enzyme concentration, the N-
methylated carba-analogue 14 was cleaved at only a very
low rate. The azadipeptide nitrile 6 showed a similar stability
towards chymotryptic cleavage as the carba-analogue 14. The
degradation of the three dipeptide derivatives was due to a
2
1
À
chymotrypsin-catalyzed cleavage of the P P amide bond.
This was confirmed by comparing the rates of Z-Phe-OH
formation (Z = benzyloxycarbonyl) with the decay of the
inhibitors (see the Supporting Information). These results
lead to the conclusion that the azadipeptide nitriles exhibit a
considerable resistance to protease-catalyzed degradation.
Herein we have revealed azadipeptide nitriles to be novel,
highly potent, and proteolytically stable inhibitors for papain-
like cysteine proteases. Furthermore, the established syn-
thetic entry will allow the introduction of high structural
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*
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*
( ). Mixtures ofthe corresponding nitrile (600 mm) and chymotrypsin
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diversity into the P1 position of these peptide-derived
inhibitors, which provides a strategy to develop stable and
selective inhibitors for this important class of proteases.
Received: December 20, 2007
Published online: April 11, 2008
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Keywords: azapeptides · enzyme inhibitors · hydrazides · nitriles
.
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