Dipeptidyl nitriles as human dipeptidyl peptidase I inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.01.102

Using a dipeptide nitrile scaffold we have identified a potent and selective inhibitor of human dipeptidyl peptidase I.

Full text of DOI:10.1016/j.bmcl.2006.01.102

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3614–3617
Dipeptidyl nitriles as human dipeptidyl peptidase I inhibitors
Jon Bondebjerg,a,* Henrik Fuglsang,a Kirsten Rosendal Valeur,a
John Pedersenb and Lars Næruma
aArpida A/S, Vesterbrogade 188, DK-1800 Frederiksberg C, Denmark
bProzymex A/S, Dr. Neergaards Vej 17, DK-2970 Hørsholm, Denmark
Received 8 December 2005; revised 16 January 2006; accepted 18 January 2006
Available online 2 May 2006
Abstract—Using a dipeptide nitrile scaffold we have identified a potent and selective inhibitor of human dipeptidyl peptidase I.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Human dipeptidyl peptidase I (hDPPI, cathepsin C, EC
3.4.14.1) is a highly conserved lysosomal cysteine exo-
peptidase that belongs to the papain family.1–5 The en-
zyme is constitutively expressed in many tissues with
highest levels in lung, kidney, liver, and spleen. DPPI
is a 200 kDa homotetramer consisting of four identical
subunits, each composed of three different chains: a
heavy chain, a light chain, and an exclusion domain.6a,b
The S1 pocket is located near the surface, exposed to the
solvent. The S2 pocket is deep and hydrophobic, with a
chloride ion and two solvent molecules at the bottom,
and Asp 1 at the entrance to anchor the substrate via
an electrostatic interaction. The exclusion domain
blocks extension beyond the S2 pocket, and thus
prevents endopeptidase activity. Once activated,7 DPPI
catalyzes sequential removal of dipeptides from the N-
termini of peptide and protein substrates with broad
specificity.3,8,9 However, DPPI only cleaves substrates
with a free N-terminal amino group and does not cleave
substrates with P1 or P10 Pro, P1 Ile, and P2 ornithine,
Lys or Arg.
Dominant-negative mutations within the human DPPI
12
`
genes are linked to the Papillon-Lefevre syndrome,
a
disease characterized by early periodontitis, palmoplan-
tar hyperkeratosis, and a predisposition to bacterial
infections. This indicates that DPPI plays an important
role in the immune system. Recent data13 show that dis-
ruption of DPPI genes is beneficial for the survival from
sepsis and indicate that DPPI is potentially a new target
for the treatment of sepsis. Thus, in combination with
predictive animal models, inhibitors of DPPI will be
beneficial for further studies on the physiological role
of DPPI.
Inhibitors of DPPI include dipeptide diazomethyl keto-
nes,14a,b a dipeptide nitrile (1),15 dipeptide vinyl sulf-
ones,16 dipeptide acyloxymethyl and fluoromethyl
ketones,16 dipeptide O-acyl hydroxamic acids,17 argi-
nine-based peptides,18 and phosphinic tripeptides.19
The naturally occurring cysteine protease inhibitor
E-64 also inhibits DPPI at high concentrations.20
We have recently reported semicarbazide-derived inhib-
itors of hDPPI.21 Herein, we report a series of dipeptidyl
nitriles as inhibitors of hDPPI. Nitriles have previously
been reported as inhibitors of cathepsin B,22 cathepsin
K,23a,b cathepsin L,23b cathepsin S,23b,24 papain,23b and
dipeptidyl peptidase IV (DPPIV).25a
DPPI is an important enzyme in lysosomal protein deg-
radation and recent studies in DPPI knock-out mice
show that DPPI functions as a key enzyme in the activa-
tion of granule serine proteases in cytotoxic T lympho-
cytes and natural killer cells (granzymes A and B),3,10a
mast cells (chymase and tryptase),10b and neutrophils
(cathepsin G, proteinase 3, and elastase).11
The nitriles were synthesized by dehydration of the cor-
responding dipeptide amides, using POCl3 and imidaz-
ole in pyridine, as previously reported (Schemes 1 and
2).25a,b The protected dipeptide amides were either pre-
pared in solution (entries, 2,8–9, Scheme 1) or on solid
phase using the Rink amide linker (entries 3–7, Scheme
1). Solid phase yields (9–21%) were generally lower than
Keywords: Dipeptidyl nitriles; Cathepsin C; Dipeptidyl peptidase I;
DPPI; Inflammation; Sepsis.
*
Corresponding author. Tel.: +4533780018; fax: +4570222365;
e-mail: jbondebjerg@arpida.dk
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.01.102

J. Bondebjerg et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3614–3617
3615
O
R1
Table 1. IC50 values for 1–10
O
H
N
H
N
X
Entry Structure
IC50 (DPPI) (nM) Ratioa A:B
a
Boc
N
H
Boc
OH
R2
O
R2
H
O
R1
N
CN
CN
X = OH
X = NH2
d
H2N
H2N
b, c
1
2
4787 456
172 13
N.D.b
3:1
H2N
O
O
N
H
CN
R2
H
N
O
O
H
N
H2N
d, c
Boc
OH
NH2
R1
R1
N
CN
CN
H2N
H2N
Scheme 1. Reagents and conditions: (a) CDI, THF; then
H2NCH(R1)COOH, base or H2NCH(R1)CONH2ÆHCl, base; (b)
POCl3, imidazole, pyridine, ꢀ40 ꢁC to room temperature; (c) TFA;
(d) CDI, THF; then NH3 in 2-propanol.
3
4
94 4% remaining
enzymatic activity
at 10 lM
5:1
O
O
H
N
822 140
N.D.b
R2
O
H
N
a, b, c
d, e
Boc
H2N
N
H
N
H
H
N
O
R1
R2
CN
Rink-PEGA800
R2
H2N
O
5
6
137 27
7:1
6:1
O
H
H
N
f, d
Boc
N
CN
N
H
NH2
H2N
R1
O
R1
H
N
O
CN
CN
H2N
H2N
96
13
8
3
Scheme 2. Reagents and conditions: (a) FmocHNCH(R1)COOH,
TBTU, NEM, DMF; (b) 20% piperidine, DMF; (c) BocHNCH(R2)-
COOH, TBTU, NEM, DMF; (d) TFA; (e) Boc2O, NEM; (f) POCl3,
imidazole, pyridine, ꢀ40 ꢁC to room temperature.
O
O
Cl
H
N
7
8
11:1
those in solution (21–48%). In the former approach, the
amides were formed from the carboxylic acids via acti-
vation with CDI, followed by treatment with ammonia
in propanol. In the latter approach, it was necessary to
re-protect the amino group with Boc2O after cleavage
from the solid phase, prior to performing the dehydra-
tion. Entry 3 was prepared according to Scheme 2, start-
ing from Fmoc-N-Me-Phe-OH. All final products were
purified by preparative HPLC (Gilson HPLC system,
Zorbax 300SB RP-18 21.2 mm · 25 cm column, 0.1%
TFA in water/acetonitrile eluent system in a linear gra-
dient, flow 15 mL/min., UV/vis-155 detector at 215
and 254 nm) to >95% purity and characterized by LC–
H
N
294 16c
418 27d
CN
H2N
O
Isomers
6296 123e
separated
H
N
CN
N
H
9
9637 208
Trace of
isomer
O
O
H2N
N
CN
10
N.I.f
9:1
1
MS and H NMR. Several of the purified nitriles were
a Determined by 1H NMR.
b Not detectable.
c Isomer A.
d Isomer A + B (44:55 ratio by HPLC).
e Isomer B.
f No inhibition at 20 lM.
obtained as a mixture of isomers, presumably diastereo-
mers arising from racemization. Tripeptide nitriles have
been previously shown to be prone to racemization
due to the increased acidity of the nitrile a-position.25b
1
The A:B isomeric ratio was determined by H NMR
(Table 1), as distinct isomeric resonances were evident.
For entry 8 it was possible to isolate the isomers by
preparative HPLC.
group failed, and this was thus exchanged for the
more robust Cbz group in two steps (90% overall,
no purification). Activation of the carboxylic acid with
CDI and subsequent treatment with ammonia in 2-
propanol yielded 29% of the carboxamide after purifi-
cation. Dehydration with trifluoroacetic anhydride
(TFAA) in DCM afforded the protected nitrile in
48% yield after HPLC purification. Finally, the Cbz
group was removed by transfer hydrogenation, using
cyclohexene and Pd/C in ethanol at 80 ꢁC, to give
the desired product 10 in 38% yield after HPLC
purification (>99% purity). This compound was a 9:1
Entry 10 was prepared (Scheme 3) via Boc-methio-
nine, using CDI and phenylalanine methyl ester
hydrochloride to assemble the crude dipeptide in high
yield (96%) and purity (>95 % by HPLC). Methyla-
tion with methyl iodide afforded the sulfonium iodide
in quantitative yield. Cyclization to form the lactame
was achieved with NaH in DMF/DCM 1:1, giving
the crude carboxylic acid in 58% yield and >94%
HPLC purity directly after workup. Approximately
10% racemization of the Phe a-carbon was observed
1
in H NMR, as previously reported.26 Attempts to
1
form the nitrile in the presence of the Boc-protecting
mixture of isomers according to H NMR.

3616
J. Bondebjerg et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3614–3617
O
Boc
HN
Table 2. Selectivity profile for 7
O
H
N
N
a-c
Boc
OH
OH
Enzyme
IC50 (nM)
O
Cathepsin B
Cathepsin H
Cathepsin L
CYP1A2
> 10,000a
> 10,000a
> 10,000a
14896 556
> 20,000a
> 20,000a
12605 556
3604 3410
S
Cbz
O
HN
d-f
N
g, h
NH2
CYP2C9
O
CYP2C19
CYP2D6
CYP3A4
H2N
N
CN
a 10 lM or 20 lM cut-off determination (n = 2).
O
10
Table 3. Metabolic stabilitya
Scheme 3. Reagents and conditions: (a) CDI, H-Phe-OMeÆHCl,
DIPEA (96%); (b) MeI (100%); (c) NaH, DMF/DCM (58%, crude);
(d) TFA/DCM (100%); (e) CbzCl, DIPEA (90%, crude); (f) CDI, then
NH3 in propanol (29%); (g) TFAA/DCM (48%); (h) Pd/C, cyclohex-
ene/EtOH, 80 ꢁC (38%).
Entry
(%)b
2
4
6
7
8
9
80
74
70
3
Entry 1, previously reported as an inhibitor of
hDPPI with Ki = 2700 nM,15 was found to have
IC50 = 4787 456 nM in our assay. Using this as a start-
ing point, and having previously identified (S)-2-amino-
butyric acid as an optimal P2 residue for a series
of semicarbazide-based inhibitors of DPPI,21 led us to
45
39
a In rat liver microsomes (Cerep, Seattle). All values are means of two
determinations.
b % Remaining after 15 min. The values for reference compounds
Imipramine, Verapamil, and Terfenadine are 11, 63, and 60,
respectively.
prepare
2 with a >25-fold increase in potency
(IC50 = 172 13 nM). However, N-methylation of the
amide resulted in almost complete loss of activity at
10 lM (entry 3), presumably owing to unfavorable
geometry and disruption of the hydrogen bonding
network, as previously proposed to explain intolerance
toward P1 Pro.6a Subsequently, we focused on exploring
the P1 substituent with respect to potency. Attention
was turned to hydrophobic, aromatic residues, known
to be good P1 substrate residues.8 Homologation of
the P1 side chain gave a >4-fold reduction in potency
(entry 4), whereas 5 with a 3-phenyl-2-propenyl P1 side
chain was found to be slightly more potent compared to
2. However, when comparing the results it should be
kept in mind that 2 has the lowest A:B isomeric ratio
of the series. Interestingly, p-chloro substitution is well
tolerated (entry 6), and p-phenyl substitution of 2
resulted in the most potent inhibitor in the series (entry
7, IC50 = 13 3 nM). The results are consistent with the
S1 pocket being located at the surface of the enzyme,
exposed to the solvent, and tolerant to many different
residues.8
Acknowledgments
We gratefully acknowledge Anne-Lise R. Gudmundsson
and Jannie Rosendahl Christensen for their technical
support, and Conni Lauritzen and Gitte Petersen (Pro-
zymex A/S) for supplying purified recombinant human
DPPI.
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