Synthesis and activity of phosphinic tripeptide inhibitors of cathepsin C

Article abstract of DOI:10.1016/j.bmcl.2004.04.028

Phosphinic tripeptide analogues Gly-Xaaψ[P(O)(OH)CH₂]-Gly have been developed as inhibitors of cathepsin C (DPP I), a lysosomal, papain-like cysteine protease. The target compounds were synthesised by addition of methyl acrylate to the appropri

Full text of DOI:10.1016/j.bmcl.2004.04.028

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3113–3116
Synthesis and activity of phosphinic tripeptide inhibitors
of cathepsin C
b
b
a,b
Artur Mucha,a, Małgorzata Pawełczak, Jozef Hurek and Paweł Kafarski
*
ꢀ
aInstitute of Organic Chemistry, Biochemistry and Biotechnology, Wrocław University of Technology,
Wybrzez_e Wyspiaꢀnskiego 27, 50-370 Wrocław, Poland
bInstitute of Chemistry, University of Opole, Oleska 48, 45-052 Opole, Poland
Received 11 March 2004; revised 6 April 2004; accepted 8 April 2004
Abstract—Phosphinic tripeptide analogues Gly-Xaaw[P(O)(OH)CH2]-Gly have been developed as inhibitors of cathepsin C (DPP
I), a lysosomal, papain-like cysteine protease. The target compounds were synthesised by addition of methyl acrylate to the
appropriate phosphinic acids followed by the N-terminus elongation using mixed anhydride procedure. The latter step has been
demonstrated to be a suitable method for N-terminal extension of the phosphinic pseudopeptide analogues without requirement of
hydroxyphosphinyl protection. The title compounds appeared to be moderate inhibitors of the cathepsin C. However, although
designed as transition state analogues, they surprisingly exhibited noncompetitive mode of binding to cathepsin C. Differences in
kinetics of C-terminal acids and esters have been additionally observed.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
been shown to result with autosomal-recessive pal-
ꢀ
moplantar keratoderma Papillon–Lefevre and Haim–
Munk syndromes.9;10
Cathepsin C or dipeptidyl peptidase I (DPP I, E.C.
3.4.14.1) is a lysosomal cysteine protease representing
the most numerous papain-like class of enzymes, that
are expressed throughout the animal and plant kingdom
as well as in viruses and bacteria. Cathepsins represent
promising drug targets as their key proteolytic activities
have been implicated in degenerative, invasive and im-
mune system related disorders.1–4 In mammals, multiple
functions have been associated to cathepsin C. It re-
moves dipeptides from the nonsubstituted N-terminus of
polypeptide substrates with broad specificity thus
exhibiting the exopeptidase activity and like other
lysosomal cysteine proteases, it is involved in nonspecific
intracellular protein degradation. Beyond this function,
DPP I is recognised to be crucial for activation of a
number of granular serine proteases, granzymes A and
B, cathepsin G, neutrophil elastase and chymase.5 It has
been also suggested to be involved in several patholo-
gical disorders, like Duchene muscular dystrophy6;7 and
basal cell carcinomas.8 Mutation in DPP I gene have
DPP I differs from the most other relatives of papain
family in its large molecular size, oligomeric structure,
retention of a large part of the propeptide in the mature
form, and requirements for halide ions for activity.11;12
Cathepsin C is inhibited by natural protein inhibitors
from the cystatine superfamily.13–15 Regarding low
molecular, irreversible, synthetic inhibitors, DPP I is
specifically inhibited by Gly-Phe-diazomethane16 and
weakly by E-64. Recently, novel, competitive inhibitors
based on oligoarginines were developed using peptide
combinatorial chemistry approach.17 Among phospho-
rus containing compounds C-terminal phosphono pep-
tides exhibited relatively low potency towards cathepsin
C acting additionally with the enzyme in quite complex
mode.18
The application of phosphinic analogues of peptides as
inhibitors of proteases is based on the concept of the
resemblance of a phosphorus moiety to the high-energy
tetrahedral transition state of the amide bond hydroly-
sis.19–23 This idea has been particularly attracting in
construction of a wide range of potent and selective
inactivators of metalloproteases that have been reviewed
recently.24–26 However, a phosphorus containing moiety
Keywords: Cathepsin C; Phosphinic tripeptides; Noncompetitive
inhibition.
* Corresponding author. Tel.: +48-71-320-3354; fax: +48-71-328-4064;
e-mail: artur.mucha@pwr.wroc.pl
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.04.028

3114
A. Mucha et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3113–3116
is additionally able to coordinate the zinc atom present
in the active sites of metalloproteases and to block its
function in the process of hydrolysis. This fact can be
considered as the major reason of such successful
applications for metallo-dependent enzymes. In cases of
the other protease families no similarly spectacular
achievements have been obtained. A single interesting
exception represents bis(a-aminoalkyl)phosphinic acids,
a modification of phosphinic peptidomimetics, which
formula offers a possibility of the transition state acting
core situated inside the symmetrical peptide environ-
ment. Nevertheless, the idea of such inhibitor geometry
was not inspired by the structure of the natural sub-
strates, but by the symmetry of enzymes, and it resulted
in strong inhibitors of the aspartic protease of the
human immunodeficiency virus type 1––the etiologic
agent of AIDS.27–30
base.36;37 In this work the first procedure have been
chosen (Scheme 1). An N-benzyloxycarbonylamino-
phosphinic acid 1 was activated by heating with hexa-
methyldisilazane and then methyl acrylate was added.
After workup and recrystallisation (ethyl acetate/me-
thanol, 95:5) the protected pseudopeptides 2 were sepa-
rated in good yields (65–80%). The benzyloxycarbonyl
group was removed by action of HBr in AcOH and the
products 3 were separated upon treatment with ether.
They were added as a solid to the solution of the N-
benzyloxycarbonylglycine preactivated into its mixed
anhydride by means of isobutyl chloroformate.
This procedure appeared to be a simple and satisfactory
method for the N-terminus elongation of the individual
phosphinic dipeptides in solution. Synthesis on solid
phase of 20member libraries of analogous tripeptides
has been reported in the literature.38 But combinatorial
chemistry on solid phase demands introduction of very
specific protection groups to the phosphinic dipeptide
building block (namely N-Fmoc and phosphinyl ada-
mantyl ester) to be compatible with the Fmoc SP
methodology and to suppress side reactions.34 Several
examples of successful application of this approach for
development of potent and selective inhibitors of zinc
metalloproteases has been reviewed recently.24–26 On the
contrary to C-terminal extension of phosphinic dipep-
tides by carboxylate activation, that can be assisted by
phosphonamidate side product formation, no necessity
of protection of the phosphinate function has been evi-
denced in our case. After standard work-up and purifi-
cation on column chromatography (chloroform/
methanol, 100:0 fi 90:10) the tripeptide analogues 4
were separated in satisfactory yields (50–60%).
Organophosphorus compounds studied so far appeared
to be surprisingly weak inhibitors of cysteine prote-
ases.31 In this paper we report the synthesis and evalu-
ation of phosphinic analogues of tripeptides designed as
inhibitors of cathepsin C. Although they appeared to be
moderate inhibitors of the enzyme, their binding mode
turned up to be somewhat surprising.
2. Chemistry
The starting phosphinic amino acid analogues 1 were
synthesised in the reaction of an appropriate aldehyde
with salt of diphenylmethylamine and hypophosphorous
acid, followed by hydrolysis of the adduct and N-ter-
minal protection with benzyl chloroformate, similarly as
described in the literature.32 Two typical synthetic pro-
cedures are usually applied to convert phosphinic acids
into pseudodipeptides: addition of an acrylate to the
aminophosphinic substrate preactivated into the form of
its trivalent silyl ester33–35 or corresponding addition of
to the appropriate ester in the presence of a strong
To obtain esters 5 the benzyloxycarbonyl group was
removed by action of HBr in AcOH. After evaporation
to dryness the residue was dissolved in 10% HCl and
benzyl bromide was washed out by extraction with
ether. Final evaporation gave compounds 5. The methyl
O
P
OH
H
O OH
P
O
P
OH
a
b
.
CbzHN
CbzHN
HBr H2N
COOMe
COOMe
COOMe
R
R
R
2a-d
3a-d
1a-d
O
OH
O
P
OH
H
N
H
N
c
d
P
.
HCl H2N
CbzHN
COOMe
O
R
O
R
4a-d
5a-d
O
P
OH
H
N
e
.
a R = CH2CH(CH3)2
b R = Ph
HCl H2N
COOH
O
R
cR = CH2Ph
6a-d
d R = CH2CH2Ph
Scheme 1. Reagents and conditions: (a) hexamethyldisilazane, 100–110 °C, 3 h, then H2C@CHCOOMe, 80–90 °C, 3 h, then MeOH; (b) 33% HBr/
AcOH, rt, 2 h; (c) CbzNHCH2COOH, isobutyl chloroformate, NEt3, 0 °C fi rt; (d) 33% HBr/AcOH, rt, 2 h; then evaporation, 10% HCl and washing
with ether; (e) bromotrimethylsilane, 10equiv, 3 d, rt, then H 2O, then concd HCl.

A. Mucha et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3113–3116
3115
esters were hydrolysed by use of bromotrimethylsilane
in dichloromethane. After evaporation of volatile com-
ponents the residue was treated consecutively with water
and then with concd HCl. Final evaporation gave the
target compounds as their hydrochlorides 6. Both steps
of deprotection proceeded practically quantitatively
(with yields exceeding 95%).39
Noncompetitive character of inhibition suggests that all
the studied compounds interact with the regulatory
rather than with the active site of the enzyme and thus
do not serve as transition-state analogues.
Cathepsin C moderately favours acids 6 against esters 5.
However, the differences in binding affinities are very
small suggesting that this part of the molecule may not
play significant role in inhibition. On the other hand this
is, most likely, not true since there is a significant dif-
ference in the mechanism of slow-binding process if
considering these two groups of compounds (Fig. 1).
Thus, the mechanism of binding of esters 5 is a slow one-
step process, as indicated from the linear dependence of
the apparent first-order rate constant for slow binding
versus inhibitor concentration.41 On the contrary,
binding of acids 6 was achieved accordingly to a two-
step mechanism, in which the enzyme upon binding of
inhibitor undergoes slow isomerisation to form slowly
dissociating complex. This is clearly seen from hyper-
bolic dependence of the apparent rate constant versus
inhibitor concentration.41 Thus, esters 5 and acids 6
seem to constitute two different classes of inhibitors of
cathepsin C.
3. Activity
As seen from Table 1 all the studied compounds ap-
peared to be reversible, noncompetitive and slow-bind-
ing inhibitors of cathepsin C. The equilibrium in the
inhibitor binding was reached within several minutes
after addition of the enzyme to the mixture of substrate
and inhibitor. Inhibition constants were determined
from Lineveawer–Burk plots of steady-state portion of
the reaction plots. Data presented in Table 1, although
consider only for eight compounds, enable to coil some
general conclusions.
Table 1. Inhibition of cathepsin C by phosphinic tripeptide ana-
logues40
Ester
Ki [mM]
Acid
Ki [mM]
5a
5b
5c
5d
0.040
0.188
0.514
0.312
6a
6b
6c
6d
0.039
0.176
0.429
0.187
Acknowledgements
This work has been supported by Komitet Badan
Naukowych.
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40. Assay of Inhibitory Activity: Cathepsin C was isolated
and purified according to literature procedure.42 Its
activity was equal 0.8 mU. Enzymatic reaction was
assayed at 25 °C in 0.2 M acetate buffer (pH 4.5) contain-
ing 2-mercaptoethanol (5 mM final concentration) and
sodium chloride (10mM final concentration). The assay
mixture contained 50 lL of cathepsin C, 2.0mL of
synthetic substrate (glycyl-L-phenylalanine-p-nitroanilide,
0.2–3.0 mM final concentration, KM ¼ 2:23 mM) and
0.4 mL of inhibitor (0.2–2.0 mM final concentration).
The total volume was adjusted to 2.45 mL with the assay
buffer. The course of reaction was monitored following the
change in absorbance at 400 nm with Specord M40 (Carl
Zeiss Jena).
Kinetic parameters were determined using the computer
programme worked-out for slow-binding kinetics. The
programme is available upon request.
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